vvEPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/AP-93/004C
December 1993
External Review Draft
Air Quality
Criteria for
Ozone and
Related
Photochemical
Oxidants
Review
Draft
(Do Not
Cite or
Quote)
Volume III of III
Notice
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
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DRAFT-DO NOT QUOTE OR CITE EPA/6007AP-93/004c
December 1993
External Review Draft
o
Air Quality Criteria for Ozone
and Related Photochemical Oxidants
Volume III of III
U.S. Environmental Protection Agency
Region 5, Library (PL-12J)
NOTICE
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on
its technical accuracy and policy implications.
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Printed on Recycled Paper
-------
DISCLAIMER
This document is an external draft for review purposes only and does not constitute
U.S. Environmental Protection Agency policy. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
December 1993 m-ii DRAFT-DO NOT QUOTE OR CITE
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PREFACE
In 1971, the U.S. Environmental Protection Agency (EPA) promulgated National
Ambient Air Quality Standards (NAAQS) to protect the public health and welfare from
adverse effects of photochemical oxidants. In 1979, the chemical designation of the
standards was changed from photochemical oxidants to ozone (Qj). This document,
therefore, focuses primarily on the scientific air quality criteria for O3 and, to a lesser extent,
for other photochemical oxidants like hydrogen peroxide and the peroxyacyl nitrates.
The EPA promulgates the NAAQS on the basis of scientific information contained in
air quality criteria documents. The previous O3 criteria document, Air Quality Criteria for
Ozone and Other Photochemical Oxidants, was released in August 1986 and a supplement,
Summary of Selected New Information on Effects of Ozone on Health and Vegetation, was
released in January 1992. These documents were used as the basis for a March 1993
decision by EPA that revision of the existing 1-h NAAQS for O3 was not appropriate at that
time. That decision, however, did not take into account some of the newer scientific data
that became available after completion of the 1986 criteria document. The purpose of this
revised air quality criteria document for O3 and related photochemical oxidants is to critically
evaluate and assess the latest scientific data associated with exposure to the concentrations of
these pollutants found in ambient air. Emphasis is placed on the presentation of health and
environmental effects data; however, other scientific data are presented and evaluated in
order to provide a better understanding of the nature, sources, distribution, measurement,
and concentrations of O3 and related photochemical oxidants and their precursors in the
environment. Although the document is not intended to be an exhaustive literature review, it
is intended to cover all pertinent literature available through the end of 1993.
This document was prepared and peer reviewed by experts from various state and
Federal governmental offices, academia, and private industry for use by EPA to support
decision making regarding potential risks to public health and welfare. The Environmental
Criteria and Assessment Office of EPA's Office of Health and Environmental Assessment
acknowledges with appreciation the contributions provided by these authors and reviewers as
well as the diligence of its staff and contractors in the preparation of this document at the
request of the Office of Air Quality Planning and Standards.
December 1993 ni-iii DRAFT-DO NOT QUOTE OR CITE
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Air Quality Criteria for Ozone
and Other Photochemical Oxidants
TABLE OF CONTENTS
Volume I
1. EXECUTIVE SUMMARY 1-1
2. INTRODUCTION 2-1
3. TROPOSPHERIC OZONE AND ITS PRECURSORS 3-1
4. ENVIRONMENTAL CONCENTRATIONS, PATTERNS, AND
EXPOSURE ESTIMATES 4-1
Volume n
5. ENVIRONMENTAL EFFECTS OF OZONE AND
RELATED PHOTOCHEMICAL OXIDANTS 5-1
APPENDIX 5A: COLLOQUIAL AND LATIN NAMES 5A-1
Volume m
6. TOXICOLOGICAL EFFECTS OF OZONE AND RELATED
PHOTOCHEMICAL OXIDANTS 6-1
7. HUMAN HEALTH EFFECTS OF OZONE AND RELATED
PHOTOCHEMICAL OXIDANTS 7-1
8. EXTRAPOLATION OF ANIMAL TOXICOLOGICAL DATA
TO HUMANS 8-1
9. INTEGRATIVE SUMMARY OF OZONE HEALTH EFFECTS 9-1
APPENDIX A: GLOSSARY OF TERMS AND SYMBOLS A-l
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TABLE OF CONTENTS
Page
LIST OF TABLES ffl-xiii
LIST OF FIGURES ffi-xvu
AUTHORS, CONTRIBUTORS, AND REVIEWERS m-xxi
U.S. ENVIRONMENTAL PROTECTION AGENCY PROJECT TEAM
FOR DEVELOPMENT OF AIR QUALITY CRITERIA FOR OZONE
AND RELATED PHOTOCHEMICAL OXEDANTS ffl-xxvii
6. TOXICOLOGICAL EFFECTS OF OZONE AND RELATED
PHOTOCHEMICAL OXIDANTS . 6-1
6.1 INTRODUCTION 6-1
6.2 RESPIRATORY TRACT EFFECTS OF OZONE 6-3
6.2.1 Biochemical Targets of Ozone Interaction 6-3
6.2.1.1 Introduction . 6-3
6.2.1.2 Polyunsaturated Fatty Acids 6-3
6.2.1.3 Antioxidants . 6-6
6.2.1.4 Proteins 6-6
6.2.2 Lung Inflammation and Permeability Changes 6-7
6.2.2.1 Introduction 6-7
6.2.2.2 Permeability Changes 6-9
6.2.2.3 Concomitant Changes in Permeability and
Inflammatory Cell Populations in the Lung ... 6-17
6.2.2.4 Sensitive Populations 6-21
6.2.2.5 Repeated Exposures 6-23
6.2.2.6 Mediators of Inflammation and Permeability . . 6-24
6.2.2.7 Summary 6-26
6.2.3 Effects on Host Defense Mechanisms 6-28
6.2.3.1 Introduction 6-28
6.2.3.2 Mucociliary Clearance 6-28
6.2.3.3 Alveolobronchiolar Transport Mechanism .... 6-31
6.2.3.4 Alveolar Macrophages 6-32
6.2.3.5 Immunology 6-38
6.2.3.6 Interaction with Infectious Agents 6-43
6.2.3.7 Summary 6-48
6.2.4 Morphological Effects 6-51
6.2.4.1 Introduction 6-51
6.2.4.2 Sites Affected 6-55
6.2.4.3 Considerations of Exposure Regimens
and Methods 6-76
6.2.4.4 Considerations of Degree of Susceptibility to
Morphological Changes 6-79
6.2.4.5 Summary 6-82
6.2.5 Effects on Pulmonary Function 6-87
December 1993 m-vii DRAFT-DO NOT QUOTE OR CITE
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TABLE OF CONTENTS (cont'd)
Page
6.2.5.1 Introduction 6-87
6.2.5.2 Brief Ozone Exposures (Less Than
30 Minutes) 6-88
6.2.5.3 Acute Ozone Exposures (Less Than
One Day) ....................... 6-95
6.2.5.4 Repeated Acute Exposure Experiments (More
Than Three Days) 6-107
6.2.5.5 Long-Term Exposure Studies 6-108
6.2.5.6 Summary 6-112
6.2.6 Biochemical Effects 6-113
6.2.6.1 Introduction 6-113
6.2.6.2 Effects of Ozone Exposure on Lung
Lipid Metabolism 6-114
6.2.6.3 Effects of Ozone on Lung Antioxidant
Systems 6-115
6.2.6.4 Effects of Ozone on Lung Protein
Metabolism 6-121
6.2.6.5 Effects of Ozone Exposure on Lung Xenobiotic
Metabolism 6-123
6.2.6.6 Summary 6-127
6.2.7 Genotoxicity and Carcinogenicity of Ozone 6-129
6.2.7.1 Introduction 6-129
6.2.7.2 Ozone-Induced Deoxyribonucleic Acid
Damage 6-130
6.2.7.3 Induction of Mutation by Ozone 6-132
6.2.7.4 Induction of Cytogenetic Damage by Ozone . . 6-135
6.2.7.5 Induction of Morphological Cell Transformation
by Ozone 6-137
6.2.7.6 Possible Direct Carcinogenic, Co-carcinogenic,
and Tumor-Promoting Effects of Ozone as
Studied in Whole Animal Carcinogenesis
Bioassays 6-139
6.2.7.7 Possible Effects of Ozone on Injected Tumor
Cells that Lodge in the Lung and Form Lung
Colonies 6-148
6.2.7.8 Epidemiology Studies on the Possible
Carcinogenicity of Ozone in Humans 6-149
6.2.7.9 Summary and Conclusions 6-149
6.3 SYSTEMIC EFFECTS OF OZONE 6-153
6.3.1 Introduction 6-153
6.3.2 Central Nervous System and Behavioral Effects 6-154
6.3.3 Cardiovascular 6-157
6.3.4 Hematological and Serum Chemistry Effects 6-159
6.3.5 Other Systemic Effects 6-160
December 1993
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TABLE OF CONTENTS (cont'd)
Page
6.3.6 Summary 6-162
6.4 INTERACTIONS OF OZONE WITH OTHER
CO-OCCURRING POLLUTANTS 6-163
6.4.1 Introduction 6-163
6.4.2 Simple (Binary) Mixtures Containing Ozone 6-165
6.4.2.1 Nitrogen Dioxide as Copollutant 6-165
6.4.2.2 Acidic Compounds as Copollutants 6-182
6.4.2.3 Other Copollutants 6-185
6.4.3 Complex (Multicomponent) Mixtures Containing
Ozone 6-186
6.4.4 Summary 6-190
6.5 SUMMARY AND CONCLUSIONS 6-192
6.5.1 Introduction 6-192
6.5.2 Molecular Mechanisms of Effects 6-192
6.5.3 Respiratory Tract Effects 6-194
6.5.3.1 Effects on Host Defenses 6-194
6.5.3.2 Effects on Inflammation and Permeability .... 6-196
6.5.3.3 Effects on Structure, Function, and
Biochemistry 6-197
6.5.3.4 Genotoxicity and Carcinogenicity of Ozone . . . 6-203
6.5.3.5 Risk Factors 6-205
6.5.4 Systemic Effects 6-206
6.5.4.1 Central Nervous System and Behavioral
Effects 6-206
6.5.4.2 Cardiovascular Effects 6-207
6.5.4.3 Reproductive and Developmental Effects .... 6-207
6.5.4.4 Other Systemic Effects 6-208
6.5.5 Effects of Mixtures 6-208
REFERENCES 6-211
7. HUMAN HEALTH EFFECTS OF OZONE AND RELATED
PHOTOCHEMICAL OXIDANTS 7-1
7.1 INTRODUCTION 7-1
7.2 CONTROLLED HUMAN EXPOSURE STUDIES 7-2
7.2.1 Pulmonary Function Effects of One- to Three-Hour
Ozone Exposures 7-2
7.2.1.1 Healthy Subjects 7-2
7.2.1.2 Subjects with Preexisting Disease 7-23
7.2.1.3 Influence of Gender, Age, Ethnic, and
Environmental Factors 7-33
7.2.1.4 Repeated Exposures to Ozone 7-54
7.2.1.5 Effects on Exercise Performance 7-64
7.2.2 Pulmonary Function Effects of Prolonged (Multihour)
Ozone Exposures 7-71
December 1993
m-ix DRAFT-DO NOT QUOTE OR CITE
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TABLE OF CONTENTS (cont'd)
Page
7.2.3 Increased Airway Responsiveness 7-80
7.2.4 Inflammation and Host Defense 7-89
7.2.4.1 Introduction 7-89
7.2.4.2 Inflammation Assessed by Bronchoalveolar
Lavage 7-95
7.2.4.3 Inflammation Induced by Ambient Levels
of Ozone 7-98
7.2.4.4 Time Course of Inflammatory Response 7-99
7.2.4.5 Effect of Anti-Inflammatory Agents on
Ozone-Induced Inflammation 7-99
7.2.4.6 Use of Nasal Lavage to Assess Ozone-
Induced Inflammation in the Upper
Respiratory Tract 7-100
7.2.4.7 Changes in Host Defense Capability Following
Ozone Exposure 7-101
7.2.5 Extrapulmonary Effects of Ozone 7-103
7.2.6 Ozone Mixed with Other Pollutants 7-104
7.2.6.1 Ozone and Sulfur-Containing Pollutants 7-105
7.2.6.2 Ozone and Nitrogen-Containing Pollutants ... 7-110
7.2.6.3 Ozone, Peroxyacetyl Nitrate, and More
Complex Mixtures 7-112
7.2.6.4 Summary 7-114
7.3 SYMPTOMS AND PULMONARY FUNCTION IN
CONTROLLED STUDIES OF AMBIENT AIR EXPOSURES . . 7-114
7.3.1 Mobile Laboratory Studies 7-115
7.3.2 High-Altitude Studies 7-118
7.4 FIELD AND EPIDEMIOLOGY STUDIES 7-119
7.4.1 Acute Effects of Ozone Exposure 7-119
7.4.1.1 Introduction 7-119
7.4.1.2 Individual-Level Studies 7-120
7.4.1.3 Aggregate Population Time Series Studies . . . . 7-155
7.4.1.4 Summary and Conclusions 7-178
7.4.2 Chronic Effects of Ozone Exposure 7-179
7.4.2.1 Introduction 7-179
7.4.2.2 Recent Epidemiological Studies of Effects of
Chronic Exposure 7-181
7.4.2.3 Conclusions 7-201
7.5 SUMMARY AND CONCLUSIONS 7-203
7.5.1 Controlled Human Studies of Ozone Exposure 7-203
7.5.1.1 Effects on Pulmonary Function 7-203
7.5.1.2 Effects on Exercise Performance 7-209
7.5.1.3 Effects on Airway Responsiveness 7-209
7.5.1.4 Inflammation and Host Defense Effects 7-211
7.5.1.5 Factors Modifying Responsiveness to Ozone .. 7-212
December 1993 m-x DRAFT-DO NOT QUOTE OR CITE
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TABLE OF CONTENTS (cont'd)
Page
7.5.1.6 Extrapulmonary Effects of Ozone 7-214
7.5.1.7 Effects of Ozone Mixed with Other
Pollutants 7-215
7.5.2 Field and Epidemiology Studies of Ozone Exposure . . . 7-216
REFERENCES 7-221
8. EXTRAPOLATION OF ANIMAL TOXICOLOGICAL DATA
TO HUMANS 8-1
8.1 INTRODUCTION 8-1
8.2 OZONE DOSIMETRY 8-2
8.2.1 Introduction 8-2
8.2.2 Summary of 1986 Review of Experimental and
Theoretical Dosimetry . . . . 8-3
8.2.3 Experimental Ozone Dosimetry Data 8-4
8.2.3.1 Introduction . . . . 8-4
8.2.3.2 Animal (In Vivo) Ozone Dosimetry Studies ... 8-5
8.2.3.3 Animal (In Vitro) Ozone Dosimetry Studies . . 8-10
8.2.3.4 Human Ozone Dosimetry Studies 8-13
8.2.3.5 Intercomparison of Ozone Dosimetry Studies . . 8-22
8.2.4 Dosimetry Modeling 8-32
8.2.4.1 Background 8-32
8.2.4.2 Dosimetry Model Predictions 8-38
8.3 SPECIES SENSITIVITY: LUNG FUNCTION AND
INFLAMMATORY ENDPOINTS EXEMPLIFYING
AN APPROACH 8-45
8.3.1 Introduction 8-45
8.3.1.1 Dosimetry 8-47
8.3.2 Homology of Response 8-47
8.3.2.1 Lung Function Endpoints as Homologous
Indicators 8-48
8.3.2.2 Inflammatory and Antioxidant Endpoints
as Homologous Indicators 8-48
8.3.3 Studies of Lung Function 8-49
8.3.3.1 Confounding Influences in Lung Function
Studies 8-49
8.3.3.2 Acute Exposure Data 8-53
8.3.3.3 Prolonged Exposure Studies 8-70
8.4 QUANTITATIVE EXTRAPOLATION 8-74
8.4.1 Introduction 8-74
8.4.2 Intraspecies Delivered Dose Response 8-76
8.4.3 Interspecies Delivered Dose Response 8-76
8.5 SUMMARY AND CONCLUSIONS 8-79
8.5.1 Ozone Dosimetry 8-80
8.5.2 Species Sensitivity 8-84
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TABLE OF CONTENTS (cont'd)
8.5.3 Quantitative Extrapolation 8-85
8.5.4 Conclusions 8-86
REFERENCES 8-88
9. INTEGRATIVE SUMMARY OF OZONE HEALTH EFFECTS 9-1
APPENDIX A: GLOSSARY OF TERMS AND SYMBOLS A-l
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LIST OF TABLES
Number
6-1
6-2
6-3
6-4
6-5
6-6
6-7
6-8
6-9
6-10
6-11
6-12
6-13
6-14
6-15
6-16
6-17
6-18
Lung Inflammation and Permeability Changes Associated
with Ozone Exposure
Effects of Ozone on Host Defense Mechanisms:
Physical Clearance
Effects of Ozone on Host Defense Mechanisms:
Macrophage Alterations
Effects of Ozone on Host Defense Mechanisms:
Immunology
Effects of Ozone on Host Defense Mechanisms:
Interactions with Infectious Agents
Effects of Ozone on Conducting Airways
Effects of Ozone on Lung Structure: Short-Term
Exposures (Less Than 2 Weeks)
Effects of Ozone on Lung Structure: Long-Term
Exposures (More Than 2 Weeks)
Effects of Ozone on Pulmonary Function
Effects of Ozone on Airway Reactivity
Effects of Ozone Exposure on Lung Lipids
Effects of Ozone Exposure on Lung Antioxidants
Effects of Ozone Exposure on Lung Proteins
Effects of Ozone Exposure on Lung Xenobiotic Metabolism ....
Effects of Ozone on Deoxyribonucleic Acid Damage
Summary of Findings on the Mutagenicity of Ozone
Effects of Ozone on Morphological Cell Transformation
Summary of Results on the Possible Carcinogenicity of Ozone . . .
Page
6-10
6-30
6-33
6-39
6-45
6-56
6-59
6-62
6-90
6-92
6-116
6-118
6-124
6-126
6-132
6-133
6-138
6-140
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LIST OF TABLES (cont'd)
6-19
6-20
6-21
6-22
6-23
6-24
6-25
6-26
7-1
7-2
7-3
7-4
7-5
7-6
7-7
7-8
7-9
Alveolar/Bronchiolar Tumor Incidence in B6C3F1 Mice in the
National Toxicology Program's Chronic Ozone Study
Effects of Inhaled Ozone on the Ability of Injected Tumor
Cells to Colonize the Lungs of Mice
Summary of Data on the Genotoxicity of Ozone
Effects of Ozone on Behavior
Effects of Ozone on the Cardiovascular System
Hematology and Serum Chemistry Effects
Toxicological Interactions of Ozone and Nitrogen Dioxide
Toxicological Interactions to Binary Mixtures of Ozone with
Acids and Other Pollutants
Controlled Human Exposure to Ozone
Ozone Exposure in Subjects with Preexisting Disease
Gender Differences in Pulmonary Function Responses to Ozone . .
Hormonal Influences on Pulmonary Function Responses to
Ozone
Age Differences in Pulmonary Function Responses to Ozone ....
Changes in Forced Expiratory Lung Volume After Repeated
Daily Exposure to Ozone
Pulmonary Function Effects with Repeated Exposures to Ozone . .
Ozone Effects on Exercise Performance
Pulmonary Function Effects After Prolonged Exposures to
Ozone
T ~PT
6-146
6-148
6-150
6-155
6-158
6-161
6-166
6-171
7-5
7-24
7-34
7-42
7-44
7-55
7-57
7-66
7-72
7-10 Increased Airway Responsiveness Following Ozone Exposures . . . 7-83
7-11 Inflammatory Effects of Controlled Human Exposure to Ozone . . . 7-90
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LIST OF TABLES (cont'd)
Number Page
7-12 Inflammatory and Host Defense Effects of Controlled Human
Exposure to Ozone 7-92
7-13 Ozone Mixed with Other Pollutants 7-106
7-14 Acute Effects of Ozone in Ambient Air in Field Studies with a
Mobile Laboratory 7-116
7-15 Acute Effects of Photochemical Oxidant Pollution: Lung Function
in Camp Studies 7-126
7-16 Slopes from Regressions of Forced Expiratory Volume in One
Second on Ozone for Six Camp Studies 7-133
7-17 Acute Effects of Photochemical Oxidant Pollution: Lung Function
in Exercising Subjects 7-135
7-18 Acute Effects of Photochemical Oxidant Pollution: Daily Life
Studies of Lung Function and Respiratory Symptoms 7-140
7-19 Acute Effects of Photochemical Oxidant Pollution: Symptom
Prevalance 7-144
7-20 Aggravation of Existing Respiratory Diseases by Photochemical
Oxidant Pollution 7-148
7-21 Hospital Admissions/Visits in Relation to Photochemical
Oxidant Pollution: Time Series Studies 7-159
7-22 Comparison of Regressions of Daily Summertime Respiratory
Admissions on Pollution and Temperature in Toronto, Ontario,
and Buffalo, New York, for the Summer of 1988 7-170
7-23 Daily Mortality Associated with Exposure to Photochemical
Oxidant Pollution 7-173
7-24 Pathologic and Immunologic Changes Associated with Chronic
Ozone Exposure 7-184
7-25 Effects of Chronic Ozone Exposure on Pulmonary Function,
Respiratory Symptoms, and Chronic Respiratory Disease 7-186
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LIST OF TABLES (cont'd)
Number Page
7-26 Effects of Chronic Ozone Exposure on the Incidence of
Cardiovascular and Malignant Diseases 7-202
8-1 Experimental Studies on Ozone Dosimetry 8-6
8-2 Total Respiratory Tract Uptake Data 8-24
8-3 Unidirectional Upper Respiratory Tract Uptake Efficiency Data . . 8-25
8-4 Lower Respiratory Tract Uptake Efficiency Data 8-26
8-5 Theoretical Ozone Dosimetry Investigations 8-33
8-6 Assumption for Application of Dosimetry Model to Breathing
Frequency Responses to Ozone 8-44
8-7 Comparison of Total Respiratory Tract Uptake Data with
Model Predictions 8-44
8-8 Pulmonary Antioxidant Substances in Various Laboratory Animal
Species and Humans 8-50
8-9 Polymorphonuclear Leukocyte and Protein Permeability Response
to Ozone by Species 8-66
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LIST OF FIGURES
Number Page
6-1 Major secondary and tertiary products of ozone interaction
with lung cells 6-5
6-2 Schematic representation of intrapulmonary conducting airways
and acini from animals with respiratory bronchioles (human
and nonhuman primates) and without respiratory bronchioles
(rats and most rodents) 6-52
6-3 Schematic comparison of the duration-response profiles for
epithelial hyperplasia, bronchioloalveolar exudation, and
interstitial fibrosis in the centriacinar region of lung
exposed to a constant low concentration of ozone 6-83
7-1 Individual dose response curves for five separate subjects
exposed to 0.10, 0.15, 0.20, and 0.25 ppm ozone for 2 hours
with moderate intermittent exercise 7-16
7-2 Mean percent change in post minus pre values of
forced expiratory volume in one second for each
gender-race group 7-51
7-3 The forced expiratory volume in one second is shown
in relation to exposure duration at four different ozone
concentrations 7-75
7-4 The distribution of response for 87 subjects exposed to
clean air and at least one of 0.08, 0.10, or 0.12 ppm is
shown here 7-77
7-5 The forced expiratory volume in one second is shown in
relation to exposure duration under three conditions 7-79
7-6 Airway function can be measured before and immediately after
the inhalation of an aerosolized bronchoconstrictor drug
like methacholine 7-82
8-1 Total respiratory tract uptake as a function of inspiratory
flow in humans 8-27
8-2 Unidirectional uptake efficiency in the upper respiratory
tract by the nasal pathway 8-29
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LIST OF FIGURES (cont'd)
Number Page
8-3 Unidirectional uptake efficiency in the upper respiratory
tract by the oral pathway 8-30
8-4 Uptake efficiency of the lower respiratory tract as a
function of inspiratory flow in humans 8-31
8-5 Net dose and tissue dose versus sequential segments along
anatomical model airway paths for human, rat, guinea pig,
and rabbit 8-39
8-6 Parallelogram paradigm for utilizing animal data for human
health predictions 8-46
8-7 Comparison of changes in frequency of breathing after ozone
exposure in humans and animals 8-55
8-8 Comparison of changes in resistance after ozone exposure in
humans and animals 8-59
8-9 Comparison of changes in forced vital capacity after ozone
exposure in humans and animals 8-64
8-10 Composite of data from Slade et al. (1989), Koren et al.
(1989b), and Crissman et al. (1993) comparing basal
bronchoalveolar lavage ascorbate levels to ozone-induced
changes in bronchoalveolar lavage protein 8-67
8-11 Composite of data comparing bronchoalveolar lavage
polymorphonuclear leukocytes 16 to 18 hours after ozone
exposure of humans (with exercise) to those of rats
exposed to ozone at rest or hyperventilated with carbon
dioxide 8-68
8-12 Comparison of changes in forced vital capacity in
humans and frequency of breathing in rats with up to
five consecutive ozone exposures 8-72
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LIST OF FIGURES (cont'd)
Number Page
8-13 Changes in forced expiratory volume in one second versus
pulmonary tissue dose 8-77
8-14 Relationship between change in alveolar wall thickness
and predicted ozone dose as a function of distance
from the bronchiole-alveolar duct junction 8-78
8-15 The maximum ratio of ozone-altered breathing frequency
to control breathing frequency at various ozone
concentrations versus predicted average dose rate to the
proximal alveolar region 8-80
8-16 Protein in the bronchoalveolar lavage for several laboratory
animal species and humans as related to the estimated
pulmonary dose 8-81
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHAPTER 6. TOXICOLOGICAL EFFECTS OF OZONE
AND RELATED PHOTOCHEMICAL OXIDANTS
Principal Authors
Dr. David J.P. Bassett—Department of Occupational and Environmental Health, College of
Pharmacy and Allied Health Professions, Shapero Hall, Wayne State University, Detroit, MI
48202
Dr. Deepak K. Bhalla—Department of Community and Environmental Medicine, University
of California, Irvine, CA 92717
Dr. Judith A. Graham—Environmental Criteria and Assessment Office (MD-52),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. George J. Jakab—Department of Environmental Health Sciences, The Johns Hopkins
University, School of Hygiene and Public Health, 615 N. Wolfe Street, Baltimore, MD
21209
Dr. Joseph R. Landolph—Department of Microbiology, USC Comprehensive Cancer Center,
USC School of Medicine, Norris Cancer Hospital and Research Institute,
1441 Eastlake Avenue, Los Angeles, CA 90033
Dr. Richard B. Schlesinger—Institute of Environmental Medicine, New York University
Medical Center, Long Meadow Road, Tuxedo, NY 10987
Dr. Jeffrey S. Tepper—ManTech Environmental Technology, P.O. Box 12313,
Research Triangle Park, NC 27709
Dr. Walter S. Tyler—Department of Anatomy, School of Veterinary Medicine, University of
California, Davis, CA 95616
Contributors and Reviewers
Dr. Gary A. Boorman—National Institute of Environmental Health Sciences,
P.O. Box 12233, Research Triangle Park, NC 27709
Dr. Daniel L. Costa—Health Effects Research Laboratory (MD-82), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Dr. Max Costa—Department of Environmental Medicine, New York University Medical
Center, 550 First Avenue, New York, NY 10016
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AUTHORS, CONTRIBUTORS, AND REVIEWERS (cont'd)
Dr. Donald L. Dungworth—Department of Veterinary Pathology, School of Veterinary
Medicine, University of California, Davis, CA 95616
Dr. Ian Gilmour—Center for Environmental Medicine and Lung Biology, U.S. Environmental
Protection Agency (MD-92), Research Triangle Park, NC 27711
Dr. Kenneth B. Gross—Biomedical Science Department, General Motors Research
Laboratory, Warren, MI 48090
Dr. Gary E. Hatch—Health Effects Research Laboratory (MD-82), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Dr. Rogene F. Henderson—Inhalation Toxicology Research Institute, Lovelace Biomedical
and Environmental Research Institute, P.O. Box 5890, Albuquerque, NM 87185
Dr. Dallas M. Hyde—Department of Anatomy, School of Veterinary Medicine, University of
California, Davis, CA 95616
Dr. Jerald A. Last—California Primate Research Center, University of California,
Davis, CA 95616
Dr. Daniel B. Menzel—Department of Community and Environmental Medicine, College of
Medicine, University of California, Irvine, CA 92715
Dr. Robert R. Mercer—Department of Medicine, Division of Allergy and Respiratory
Disease, P.O. Box 3177, Duke University Medical Center, Durham, NC 27710
Dr. Frederick J. Miller—Chemical Industry Institute of Toxicology, P.O. Box 12137,
Research Triangle Park, NC 27709
Dr. Kathleen M. Nauss—Health Effects Institute, 141 Portland St., Suite 7300,
Cambridge, MA 02139
Dr. Steven C. Nesnow—Health Effects Research Laboratory (MD-68), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Dr. Robert F. Phalen—Department of Community and Environmental Medicine, University of
California, Irvine, CA 92717
Dr. Charles G. Plopper—Department of Anatomy, School of Veterinary Medicine, University
of California, Davis, CA 95616
Dr. Edward M. Postlethwait—Pulmonary Division H-76, Department of Internal Medicine,
University of Texas Medical School, Galveston, TX 77550
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AUTHORS, CONTRIBUTORS, AND REVIEWERS (cont'd)
Mr. Mark E. Raizenne—Health and Welfare Canada, Environmental Health Center, Tunney's
Pasture, Ottawa, Ontario K1A OL2, CANADA
Dr. Edward S. Schelegle—Department of Human Physiology, School of Medicine,
Building 1-A, Room 4140, University of California, Davis, CA 95616
Dr. MaryJane Selgrade—Health Effects Research Laboratory (MD-92), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Dr. David Warshawsky—Kettering Laboratories, University of Cincinnati, School of
Medicine, Department of Environmental Health, 3223 Eden Avenue, Cincinnati, OH 45267
Dr. M. Jean Wiester—Health Effects Research Laboratory (MD-82), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Dr. Hanspeter R. Witschi—Toxic Substances Research and Teaching Program, University of
California, Davis, CA 95616
CHAPTER 7. HUMAN HEALTH EFFECTS OF OZONE
AND RELATED PHOTOCHEMICAL OXTOANTS
Principal Authors
Dr. Robert M. Aris—Lung Biology Center, San Francisco General Hospital, Bldg. 1,
Rm. 150, 1001 Potrero Ave., San Francisco, CA 94110
Dr. John R. Balmes—Center for Occupational and Environmental Heath, San Francisco
General Hospital, 1001 Potrero Avenue, Bldg. 30, 5th Floor, San Francisco, CA 94110
Dr. Robert B. Devlin—Health Effects Research Laboratory (MD-58), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Dr. Deborah M. Drechsler-Parks—Environmental Stress Laboratory, Neuroscience Research
Institute, University of California, Santa Barbara, CA 93106
Dr. Lawrence J. Folinsbee—Health Effects Research Laboratory (MD-58),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. Henry Gong, Jr.—Department of Medicine, UCLA Medical Center,
Los Angeles, CA 90024
Dr. Patrick L. Kinney—Institute of Environmental Medicine, New York University Medical
Center, Long Meadow Road, Tuxedo, NY 10987
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AUTHORS, CONTRIBUTORS, AND REVIEWERS (cont'd)
Dr. Edward S. Schelegle—Department of Human Physiology, School of Medicine,
Building 1-A, Room 4140, University of California, Davis, CA 95616
Dr. Ira B. Tager—School of Public Health, 140 Earl Warren Hall, University of California,
Berkeley, CA 94720
Dr. George D. Thurston—Institute of Environmental Medicine, New York University Medical
Center, Long Meadow Road, Tuxedo, NY 10987
Contributors and Reviewers
Dr. William C. Adams—Department of Physical Education, University of California,
Davis, CA 95616
Dr. Rebecca Bascom—The University of Maryland School of Medicine, 10 S. Pine St.,
MSTF-800, Baltimore, MD 21201
Dr. David V. Bates—4891 College Highroad, Vancouver, British Columbia,
CANADA V6T1C6
Dr. William S. Beckett—Yale School of Medicine, 333 Cedar St., New Haven, CT 06510
Dr. Philip A. Bromberg—Department of Medicine, The University of North Carolina,
724 Burnett-Womack Bldg., 229H, Chapel Hill, NC 27599
Dr. Douglas Dockery—Harvard School of Public Health, Environmental Epidemiology,
665 Huntingdon Avenue, Boston, MA 02115
Dr. William Eschenbacher—Methodist Hospital, 6565 Fannin, M.S., F980,
Houston, TX 77030
Dr. Mark Frampton—Pulmonary Disease Unit, Box 692, University of Rochester Medical
Center, 601 Elmwood Avenue, Rochester, NY 14642
Dr. Judith A. Graham—Environmental Criteria and Assessment Office (MD-52),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. Milan J. Hazucha—Department of Medicine, Center for Environmental Medicine and
Lung Biology, The University of North Carolina, Trailer #4, Medical Bldg. C 224H,
Chapel Hill, NC 27599
Dr. Steven M. Horvath—Environmental Stress Laboratory, Neuroscience Research Institute,
University of California, Santa Barbara, CA 93106
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AUTHORS, CONTRIBUTORS, AND REVIEWERS (cont'd)
Dr. Howard R. Kehrl—Health Effects Research Laboratory (MD-58), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Dr. Jane Q. Koenig—Department of Environmental Health, School of Public Health and
Community Medicine, University of Washington, SC-34, Seattle, WA 98195
Dr. Hillel S. Koren—Health Effects Research Laboratory (MD-58), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Dr. Debra L. Laskin—Department of Pharmacology and Toxicology, College of Pharmacy,
Rutgers University, Piscataway, NY 08855
Mr. William S. Linn—Rancho Los Amigos Medical Center, 51 Medical Science Building,
7601 East Imperial Highway, Downey, CA 90242
Dr. Fred Lipfert—23 Carll Ct., Northport, NY 11768
Dr. William F. McDonnell—Health Effects Research Laboratory (MD-58),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Mr. Mark E. Raizenne—Health and Welfare Canada, Environmental Health Center, Tunney's
Pasture, Ottawa, Ontario K1A OL2, CANADA
Dr. Jonathan M. Samet—Department of Medicine, University of New Mexico,
Albuquerque, NM 87131
Dr. Joel Schwartz—Office of Policy Planning and Evaluation (PM-221), U.S. Environmental
Protection Agency, 401 M Street, SW, Washington, DC 20460
Dr. Elston Seal—Health Effects Research Laboratory (MD-58), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Dr. Dalia M. Spektor—2832 Arizona Avenue, Santa Monica, CA 90404
CHAPTER 8. EXTRAPOLATION OF ANIMAL TOXICOLOGICAL DATA
TO HUMANS
Principal Authors
Dr. Daniel L. Costa—Health Effects Research Laboratory (MD-82), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
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Principal Authors (cont'd)
Dr. Timothy R. Gerrity—Health Effects Research Laboratory (MD-58), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Dr. John H. Overton—Health Effects Research Laboratory (MD-82), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
Reviewers
Dr. Daniel B. Menzel—Department of Community and Environmental Medicine, College of
Medicine, University of California, Irvine, CA 92715
Dr. Robert R. Mercer—Department of Medicine, Division of Allergy and Respiratory
Disease, P.O. Box 3177, Duke University Medical Center, Durham, NC 27710
Dr. Frederick J. Miller—Chemical Industry Institute of Toxicology, P.O. Box 12137,
Research Triangle Park, NC 27709
Dr. Kathleen M. Nauss—Health Effects Institute, 141 Portland St., Suite 7300,
Cambridge, MA 02139
Dr. Edward M. Posflethwait—Pulmonary Division H-76, Department of Internal Medicine,
University of Texas Medical School, Galveston, TX 77550
Mr. Mark E. Raizenne—Health and Welfare Canada, Environmental Health Center, Tunney's
Pasture, Ottawa, Ontario K1A OL2, CANADA
Dr. James S. Ultman—Department of Chemical Engineering, Pennsylvania State University,
University Park, PA 16802
Dr. M. Jean Wiester—Health Effects Research Laboratory (MD-82), U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711
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U.S. ENVIRONMENTAL PROTECTION AGENCY
PROJECT TEAM FOR DEVELOPMENT OF AIR QUALITY CRITERIA
FOR OZONE AND RELATED PHOTOCHEMICAL OXIDANTS
Scientific Staff
Mr. James A. Raub—Health Scientist, Environmental Criteria and Assessment Office
(MD-52), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. A. Paul Altshuller—Physical Scientist, Environmental Criteria and Assessment Office
(MD-52), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Mr. William G. Ewald—Health Scientist, Environmental Criteria and Assessment Office
(MD-52), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. J.H.B. Garner—Ecologist, Environmental Criteria and Assessment Office (MD-52),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Dr. Judith A. Graham—Associate Director, Environmental Criteria and Assessment Office
(MD-52), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Ms. Ellie R. Speh—Secretary, Environmental Criteria and Assessment Office (MD-52),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Ms. Beverly E. Tilton—Physical Scientist, Environmental Criteria and Assessment Office
(MD-52), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
Technical Support Staff
Mr. Douglas B. Fennell—Technical Information Specialist, Environmental Criteria and
Assessment Office (MD-52), U.S. Environmental Protection Agency, Research Triangle Park,
NC 27711
Mr. Allen G. Hoyt—Technical Editor and Graphic Artist, Environmental Criteria and
Assessment Office (MD-52), U.S. Environmental Protection Agency, Research Triangle Park,
NC 27711
Ms. Diane H. Ray—Technical Information Manager (Public Comments), Environmental
Criteria and Assessment Office (MD-52), U.S. Environmental Protection Agency, Research
Triangle Park, NC 27711
Mr. Richard N. Wilson—Clerk, Environmental Criteria and Assessment Office (MD-52),
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
December 1993 JH-xxvii DRAFT-DO NOT QUOTE OR CITE
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U.S. ENVIRONMENTAL PROTECTION AGENCY
PROJECT TEAM FOR DEVELOPMENT OF AIR QUALITY CRITERIA
FOR OZONE AND RELATED PHOTOCHEMICAL OXIDANTS
(cont'd)
Document Production Staff
Ms. Marianne Barrier—Graphic Artist, ManTech Environmental, P.O. Box 12313,
Research Triangle Park, NC 27709
Mr. John R. Barton—Document Production Coordinator, ManTech Environmental
Technology, Inc., P.O. Box 12313, Research Triangle Park, NC 27709
Ms. Lynette D. Cradle—Lead Word Processor, ManTech Environmental Technology, Inc.,
P.O. Box 12313, Research Triangle Park, NC 27709
Ms. Jorja R. Followill—Word Processor, ManTech Environmental Technology, Inc.,
P.O. Box 12313, Research Triangle Park, NC 27709
Ms. Wendy B. Lloyd—Word Processor, ManTech Environmental Technology, Inc.,
P.O. Box 12313, Research Triangle Park, NC 27709
Mr. Peter J. Winz—Technical Editor, Mantech Environmental Technology, Inc.,
P.O. Box 12313, Research Triangle Park, NC 27709
Technical Reference Staff
Mr. John A. Bennett—Bibliographic Editor, ManTech Environmental Technology, Inc.,
P.O. Box 12313, Research Triangle Park, NC 27709
Ms. S. Blythe Hatcher—Bibliographic Editor, Information Organizers, Inc., P.O. Box 14391,
Research Triangle Park, NC 27709
Ms. Susan L. McDonald—Bibliographic Editor, Information Organizers, Inc.,
P.O. Box 14391, Research Triangle Park, NC 27709
Ms. Carol J. Rankin—Bibliographic Editor, Information Organizers, Inc., P.O. Box 14391,
Research Triangle Park, NC 27709
Ms. Deborah L. Staves—Bibliographic Editor, Information Organizers, Inc.,
P.O. Box 14391, Research Triangle Park, NC 27709
Ms. Patricia R. Tierney—Bibliographic Editor, ManTech Environmental Technology, Inc.,
P.O. Box 12313, Research Triangle Park, NC 27709
December 1993 ffl-xxviii DRAFT-DO NOT QUOTE OR CITE
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i 6. TOXICOLOGICAL EFFECTS OF OZONE AND
2 RELATED PHOTOCHEMICAL OXIDANTS
3
4
5 6.1 INTRODUCTION
6 A wide range of effects of ozone (O3) has been demonstrated in laboratory animals (see
7 reviews by U.S. Environmental Protection Agency [1986], Lippmann [1989, 1993], and
8 Graham et al. [1990]). The major research findings are that environmentally relevant levels
9 of O3 cause lung inflammation; decreases in host defenses against infectious lung disease;
10 acute changes in lung function, structure, and metabolism; chronic changes in lung structure
11 and lung disease, some elements of which are irreversible; and systemic effects on targets
12 (e.g., liver, immune system) distant from the lung. The research has also served to expand
13 understanding of mechanisms of toxicity and the relationships between concentration and
14 duration of exposure. The framework for presenting the health studies of O3 in animals
15 begins with a discussion of respiratory tract effects and is followed by a presentation of
16 systemic effects and interaction of O3 with other common co-occurring pollutants.
17 Respiratory tract effects are often interrelated. For example, some types of structural
18 changes can affect pulmonary function. However, for the purposes of presentation, effects
19 on lung inflammation and permeability, host defenses, morphology, pulmonary function,
20 biochemistry, and mutagenic/carcinogenic potential are discussed separately in the main text,
21 drawing correlations where appropriate.
22 The purpose of this criteria document is also to describe effects of photochemical
23 oxidants other than O3. Nitrogen dioxide (NO?) and nitric oxide are the other two primary
24 photochemical oxidants and have been evaluated recently in another criteria document (U.S.
25 Environmental Protection Agency, 1993). Formaldehyde, which is formed photochemically
26 and can be toxic, has also been reviewed recently by the U.S. Environmental Protection
27 Agency (Grindstaff et al., 1991). Literature searches did not reveal any animal toxicology
28 inhalation studies of peroxyacetyl nitrate (PAN) since the last O3 document (U.S.
29 Environmental Protection Agency, 1986). A myriad of other individual photochemical
30 oxidants are formed in the ambient air (Chapter 3), but they have not been investigated in
31 animal inhalation toxicology. The very few publications on the effects of exposures to a
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1 mixture of oxidants are summarized in Section 6.4 which discusses pollutant interactions.
2 Therefore, other than in Section 6.4, this chapter does not address other photochemical
3 oxidants. Even so, considering the limited literature within the aforementioned documents,
4 the available evidence from animal toxicology studies shows that O3 is the most potent of the
5 oxidants for noncancer effects.
6 The animal toxicology database for O3 is extremely large, making it necessary to adopt
7 conventions for succinctly presenting the pertinent findings. Only research published after
8 the closure of the previous O3 criteria document (U.S. Environmental Protection Agency,
9 1986) is included here, but for the purposes of broader interpretation, the older literature is
10 very briefly summarized. Generally, only the highlights of the key recent studies and their
11 interpretation are provided here. Confirmatory recent studies are mentioned and presented in
12 the tables. Furthermore, only studies having concentrations < 1.0 ppm O3 are included with
13 rare exception. Such a cut point allows portrayal of the full array of the effects of O3 that
14 might occur from ambient air exposure, and also avoids the potential for confounding
15 mechanisms that can occur at very high, environmentally unrealistic concentrations. For
16 example, very high levels of O3 can cause severe pulmonary edema, resulting in types and
17 magnitudes of pulmonary function changes that would not occur in the real world.
18 In summarizing the literature, changes from control are described if they were statistically
19 significant at p < 0.05, rather than citing the probability values for each study. Where
20 appropriate, critique of a statistical procedure is mentioned. A probability value is provided
21 if it aids the understanding of trends observed in a study (e.g., p < 0.1).
22 Animal lexicological studies of O3 are of major interest because they illustrate a fuller
23 array of effects and exposure conditions than can be investigated in humans. Most experts
24 accept a qualitative animal-to-human extrapolation; that is O3 effects observed in several
25 animal species can occur in humans if causative exposure concentrations, durations, and
26 patterns also occur. However, there is less consensus on an approach to quantitative
27 extrapolation (e.g., the exposures at which effects in animals actually occur in humans).
28 Chapter 8, on extrapolation, provides more information on this topic and is necessary for full
29 interpretation of the animal studies.
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1 6.2 RESPIRATORY TRACT EFFECTS OF OZONE
2 6.2.1 Biochemical Targets of Ozone Interaction
3 6.2.1.1 Introduction
4 An understanding of the initial biochemical events by which O3 exposure causes
5 alterations in cellular function is a prerequisite to understanding how O3 affects the lung and
6 interpreting the potential health impacts of a variety of effects.
7 In vitro experiments have indicated that O3 has the potential to interact with a wide
8 range of different cellular components that include polyunsaturated lipids; various electron
9 donors (e.g., ascorbate and vitamin E); and the thiol, aldehyde, and amine groups of low
10 molecular weight compounds, and proteins (U.S. Environmental Protection Agency, 1986).
11 The mechanisms to explain the initial biochemical and physiological effects of O3 exposure
12 in vivo are therefore complex and most likely involve not only the direct action of Qj with
13 lung macromolecules, but also reaction of secondary biochemical products that result from
14 the generation of free radical-precursor molecules, the release of endogenous mediators of
15 physiological response, and the reactive oxygen intermediates and proteinases associated with
16 the activities of inflammatory cells that subsequently infiltrate into O3-damaged lungs (see
17 Section 6.2.2). Based on some theoretical calculations, Pryor (1992) has hypothesized that
18 because O3 is so reactive, it most likely does not penetrate beyond the surface lining fluids of
19 the lung except in those terminal airway regions of minimal lining thickness where epithelial
20 cells might well be relatively unprotected by either mucus or surfactant. In a review, Pryor
21 (1991) proposed that O3-induced cell damage more likely results from the reactions of more
22 stable but less reactive ozonide, aldehyde, and hydroperoxide products of O3 interaction with
23 surface-lining fluid components than direct interactions of O3 with intracellular components.
24 Although the surfactant lining is relatively rich in saturated phospholipids, it does contain
25 some lipids with unsaturated fatty acids, cholesterol, a protein A component, and small
26 molecular weight compounds (e.g., glutathione [GSH] and uric acid) that have all been
27 shown to react with O3 in both in vitro and in vivo studies.
28
29 6.2.1.2 Polyunsaturated Fatty Acids
30 Hitherto, the major products of O3-lipid interaction that account for cell membrane
31 damage have been assumed to be lipid hydroperoxides. However, evidence for the
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1 production of hydrogen peroxide and aldehydes has now been demonstrated. Pryor (1991)
2 proposed that although "Criegee ozonation" will ultimately lead to the production of ozonides
3 in a lipophilic environment, in the aqueous environment of lung airways, the carbonyl oxide
4 intermediate can form a hydroxyhydroperoxy compound, which on elimination of hydrogen
5 peroxide yields another aldehyde, or in the presence of iron ions can form an aldehyde and
6 the very reactive hydroxyl radical. Ozonation of aqueous emulsions of polyunsaturated fatty
7 acids (PUFAs), rat erythrocyte ghost membranes, and rat bronchoalveolar lavage fluid (BAL)
8 has shown hydrogen peroxide and aldehyde generation with a much smaller proportion of
9 ozonides and lipid hydroperoxides (Pryor et al., 1991). A mechanistic study by Santrock
10 et al. (1992) of the ozonation of l-paImitoyl-2-oleoyl-,y«-glycero-3-phosphocholine in
11 unilamellar phospholipids confirmed the generation of the hydroxyperoxy compounds, which
12 subsequently result in the generation of hydrogen peroxide and aldehydes with further
13 oxidation to carboxylic acids. Similar studies have demonstrated the production of secondary
14 ozonides (Lai et al., 1990). Madden et al. (1993) have recently demonstrated production of
15 arachidonate derived aldehydic substances and hydrogen peroxide on in vitro O3 exposure
16 (0.1 and 1.0 ppm for 1 h) of either arachidonate in a cell-free system or cultured human
17 bronchial epithelial cells. Ozonides, aldehydes, hydrogen and lipid peroxides, and related
18 reactive oxygen intermediates therefore represent the major secondary and tertiary products
19 of O3 interaction with lung cells that all have the potential to cause membrane damage
20 (Figure 6-1).
21 Evidence that interaction of O3 with PUFAs takes place in vivo has not been so easily
22 obtained. Goheen et al. (1986) investigated the effects of fat-free diets on rats exposed to air
23 or to 0.96 ppm O3 for 0, 1, 2, and 4 weeks and concluded that O3 does not oxidize
24 significant levels of the PUFAs linoleate (18:2) and arachidonate (20:4). However, cleavage
25 of lung fatty acid double bonds has been demonstrated in an in vivo study reported by
26 Rabinowitz and Bassett (1988) that involved rat exposures for 4 h to 2 ppm. These authors,
27 by using hydrogen peroxide treatment to convert ozonides and aldehydes to carboxylic acids,
28 were able to demonstrate O3-induced increases in glutaric and nonanoic acids that are the
29 ozonolysis breakdown products of lung tissue arachidonic and oleic acid, respectively. More
30 recent studies directed towards developing suitable biomarker and dosimeters for O3 exposure
31 have analyzed rat BAL lipids after a 12-h exposure to 1.3 ppm (Cueto et al., 1992) and
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A
o p
RHOCH- + Og —> RHC--CH > RHCO-O + RHC=0
PUFA ozone trtoxolane carbonyl oxide aldehyde
,
OH
— > RHCs CH- or RCH — > RHOO + H2O2
0 XOOH
Criegee ozonide hydroxyhydroperoxy cpd. aldehyde hydrogen peroxide
Figure 6-1. Major secondary and tertiary products of ozone interaction with lung cells.
1 demonstrated the appearance of the aldehydes nonanal and heptanal. Pryor et al. (1992) have
2 also been able to identify cholesterol ozonation products extracted from whole lung tissue
3 following the same in vivo exposure of rats to 1.3 ppm O$ for 12 h.
4 Evidence of the role of hydrogen peroxide in O3-induced lung damage has been
5 described by Warren et al. (1988), who demonstrated diminished O3-induced increased BAL
6 protein in rats after a day of exposure to 0.64 ppm O3 when treated with the hydrogen
7 peroxide scavenger dimethylthiourea before exposure. Hitherto, the exhalation of ethane and
8 pentane and tissue measurements of diene-conjugates and thiobarbituric acid reactive
9 substances (TEARS) have been used as evidence for O3-induced free radical autoxidation of
10 lipids (U.S. Environmental Protection Agency, 1986). However, these measurements have
1 1 been found to be relatively insensitive for use in inhalation experiments under conditions of
12 low O3 concentrations (<0.5 ppm). Ichinose and Sagai (1989) were unable to demonstrate
13 any changes in lung TEARS as a result of exposing rats for 2 weeks to 0.4 ppm Oj.
14 As noted by Pryor (1991), malondialdehyde and other thiobarbituric acid reacting aldehydes
15 can be produced by Criegee ozonation of di- and triolefinic fatty acids, as well as by free
16 radical peroxidative processes. In addition, malondialdehyde, being volatile as well as highly
17 reactive, might be readily lost from the lung or during sample preparation. However,
18 measurements of TEARS continue to be used for in vitro experiments designed to
19 demonstrate possible mechanisms by which such agents as taurine (Banks et al., 1991) and
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1 uric acid (Meadows and Smith, 1987) might protect against O3-induced lipid damage.
2 Rietjens et al. (1987b), by preincubating rat alveolar macrophages (AMs) with either
3 arachidonate (20:4) or phosphatidylcholine to respectively alter PUFA content and membrane
4 fluidity, demonstrated that PUFA content (not membrane fluidity) determined sensitivity to
5 O3 damage, measured as decreases in phagocytic activity.
6 Evidence for free radical mediated autoxidation comes indirectly from the
7 demonstration that vitamin E depletion increases O3 toxicity, as reported previously (U.S.
8 Environmental Protection Agency, 1986) and more recently (Elsayed, 1987; Elsayed et al.,
9 1988). More direct evidence for free radical generation has been obtained using electron
10 spin-trapping technology that correlated increased radical signals in isolated lung lipids from
11 rats exposed to increasing O3 concentrations (0 to 1.5 ppm, effect beginning at about
12 0.5 ppm; 2 h) under conditions of carbon dioxide (CO2)-stimulated respiration (Kennedy
13 etal., 1992).
14
15 6.2.1.3 Antioxidants
16 Although vitamin E directly reacts with O3 at the same rate as PUFAs, vitamin C
17 appears to react more effectively (Pryor, 1991), which together with intracellular taurine
18 (Banks et al., 1991) and BAL uric acid (Meadows and Smith, 1987) might act as direct
19 scavengers of O3. Glutathione represents another potential direct scavenger, but the
20 O3 would have to penetrate the lung lining fluid and cellular membrane without reaction, an
21 event that is considered unlikely (Pryor, 1991, 1992). Previously observed oxidation of GSH
22 and in some cases its loss from the lung more likely reflects its reaction with an O3-derived
23 oxidant such as a hydroperoxide or an ozonide, mediated by glutathione peroxidase (GSHPx)
24 and glutathione-S-transferases (GSTs), respectively (Rietjens et al., 1987a). Ozone-induced
25 increases in lung polyamine metabolism in vitamin E deficient rats has suggested their
26 possible role as antioxidants (Elsayed, 1987).
27
28 6.2.1.4 Proteins
29 Early studies reported that nonprotein sulfydryls (NPSHs) and the activities of various
30 cytosolic, microsomal, and mitochondria! enzymes are decreased immediately following
31 short-term exposures to relatively high levels (2 to 4 ppm) of O3 (U.S. Environmental
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GUIDELINES AND SPECIFICATIONS FOR REVIEWERS
AIR QUALITY CRITERIA FOR OZONE
AND RELATED PHOTOCHEMICAL OXIDANTS
Reviewers are encouraged to submit written comments regarding the material presented in the draft chapters
of the revised air quality criteria document for ozone and related photochemical oxidants. To facilitate the
revision process, the Environmental Criteria and Assessment Office in Research Triangle Park, NC, requests that
certain guidelines be followed. Both general and specific comments on each of the draft chapters will be useful.
However, we cannot emphasize enough the importance of making your comments specific, by indicating the
section, page, and line numbers of the material in question. This will greatly increase the likelihood that the
authors will be able to address your comments adequately within a reasonable period of time. We also hope your
comments will include your concurrence with the information as well as any disagreements. Please review
carefully the following guidelines prior to providing your written comments. We appreciate your assistance.
Guidelines to Reviewers
1. Chapters should be reviewed using a separate form for each.
2. General comments, impressions, or opinions of the chapter should be presented on the comment form,
separate from any specific comments.
3. Specific comments on the text within sections should be identified with the page and line numbers.
4. When commenting on specific sections, provide the appropriate details. Sometimes, reviewers' comments
note "this information is not correct!H You should be aware that unless specific evidence is provided
demonstrating that the information is, in fact, inaccurate, no action can be taken.
5. The following criteria are suggested as an aid in your review:
• Scientific Accuracy—Is the material presented accurate, irrespective of other shortcomings?
• Scientific Relevance—Is the material focused and pertinent, given the subject matter and scope of the
document?
• Regulatory Relevance—Does the material provide the U. S. Environmental Protection Agency with the kind
of critical review and sound data that could be used to set the National Ambient for Quality Standard for
Ozone?
• Literature Gted—Aie references current or outdated? Are there major or important omissions of pertinent
literature?
• Emphasis—Are the length and level of detail appropriate, given the relative importance of the topic in the
document?
• Coverage—Are all issues pertinent to a criteria document discussed? Are there any additional issues that
should be resolved or at least considered in the document?
• Editing—Although editorial suggestions are welcome, the above six points are most important at this stage
of the review process.
6. A copy of the review form is attached. This form should be copied and used for all written comments.
-------
DOCUMENT REVIEW FORM
Title/Draft
Air Quality Criteria for Ozone and Related
Photochemical Oxidants
Reviewer/Organization/Address
RECOMMENDATIONS
D (1) Acceptable as is
D (2) Acceptable after minor revision
D (3) Acceptable after major revision
D (4) Not acceptable
If you have checked either 3 or 4, please state
specific reason® in your comments.
Reviewer's Signature Date
Return To:
Project Manager for Ozone/Oxidants
Environmental Criteria and Assessment Office (MD-52)
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Chapter No./Title
SUMMARY RATING
Excel. Good Fair Poor
Scientific Accuracy
Scientific Relevance
Regulatory Relevance
Literature Cited
Emphasis
Coverage
Writing
GENERAL COMMENTS (Use extra pages if needed, but please be concise.)
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AIR QUALITY CRITERIA FOR OZONE
AND RELATED PHOTOCHEMICAL OXIDANTS
Section/Page/Line Specific Comments
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1 Protection Agency, 1986). However, although these early biochemical effects could not be
2 demonstrated after the first day of exposure to the lower O3 concentration of 0.8 ppm, the
3 methods employed might not have been sensitive enough to detect coenzyme and enzyme
4 changes in the centriacinar region, which is a primary target of O3 interaction. However,
5 together with surfactant lipids, the surfactant protein A has also been examined as a potential
6 target of O3 interaction (Costing et al., 1991c, 1992). In vitro studies by these authors have
7 suggested that either hydrogen peroxide- or O3-induced oxidation of methionine and
8 tryptophan residues of canine and human surfactant protein A account for the observed
9 changes in its physicochemical properties measured as an impairment of self-association and
10 a decreased ability to aggregate phospholipid vesicles and to bind mannose (Costing et al.,
11 199 Ic). Similar responses were indicated by in vivo experiments that demonstrated
12 surfactant isolated from rats exposed for 12 h to 0.4 ppm was less able to stimulate AM
13 superoxide anion generation than surfactant obtained from air-exposed control rats (Costing
14 et al., 1992). The previously reported presence of giant lamellar bodies in O3-exposed rat
15 lungs following exposure to 0.3 ppm for 3 h/day for 16 days is also consistent with the
16 hypothesis that O3 reacts with surfactant protein A (Shimura et al., 1984) and thereby
17 interferes with its homeostatic role in surfactant release from alveolar Type 2 cell lamellar
18 bodies and its subsequent reuptake by Type 2 cells and AMs.
19
20 6.2.2 Lung Inflammation and Permeability Changes
21 6.2.2.1 Introduction
22 The barrier functions of the airway epithelia have been investigated in the recent years
23 by isotope tracer techniques for detecting mucosal permeability and by analysis of the BAL
24 for total protein and albumin concentrations. Under normal conditions, the airway epithelia
25 restrict the penetration of exogenous particles and macromolecules from airway lumen into
26 airway interstitium and blood. The integrity of the zonula occludens (tight junctions) is
27 regarded as a major factor in providing barrier properties to the airway epithelia so that only
28 a small amount of intratracheally introduced tracers finds its way across the airway epithelia
29 into the blood. However, disruption of the epithelial barrier creates a leak across the airway
30 mucosa, resulting in increased permeability of serum proteins into the air spaces and of
31 intraluminal exogenous tracers into the blood. Therefore, permeability is generally detected
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1 either by the tracer transport from airway spaces to blood or by measurement of total protein
2 and albumin in the BAL. Both of these measures are, therefore, taken into account in
3 discussing permeability changes in this section. Although BAL protein measurement offers a
4 good marker for detecting permeability changes, it is important to note that the proteins in
5 the BAL can result from tissue injury and secretory activity in addition to leakage of the
6 serum proteins across the airway mucosa (Hatch et al., 1989; Hatch, 1992; Su et al., 1991).
7 Inflammatory cells in the lung constitute an important component of the pulmonary
8 defense system. In their unstimulated state, the inflammatory cells present no danger to
9 other cells or tissues, but upon activation, they are capable of generating proteolytic enzymes
10 such as elastase and reactive oxygen species such as superoxide, hydrogen peroxide (H2O2),
11 and hydroxyl radical. These oxidants can cause substantial injury to cell membranes and
12 intracellular components by their effects on membrane lipids and proteins (biochemical
13 effects of 63 are described in Section 6.2.6). Ozone exposure can also cause the epithelial
14 or activated inflammatory cells to liberate arachidonic acid, which is free to enter enzymatic
15 lipoxygenase or cyclooxygenase pathways that lead to the production of leukotrienes (LTs)
16 and prostaglandins (PGs), respectively. Although some of the studies indicate a lack of
17 change in the production and release of cellular mediators following O3 exposure, other
18 studies demonstrate an elevation in the levels of arachidonic acid and its metabolites in the
19 bronchial washings of rats as well as humans (see Chapter 7) exposed to 63 under controlled
20 conditions. These agents can cause a wide range of pathophysiological changes. For
21 example, LTB4 can cause polymorphonuclear leukocyte (PMN) aggregation and
22 degranulation in vitro and margination of circulating PMNs to capillary endothelium in vivo.
23 While LTC4 and LTD4 can cause contraction of vascular smooth muscle, PGEj has
24 bronchodilator activity, and LTD4 and PGF2a are regarded as bronchoconstrictors. Because
25 of the toxic potential of the products released by PMNs, AMs, mast cells, and other
26 inflammatory cells, it has been suggested that the recruitment of these cells into the
27 pulmonary interstitium is associated with lung injury and associated edema. Although an
28 inflammatory response in the lung and an elevation of transmucosal permeability are
29 observed after O3 exposure, the interdependence of these two events is a topic of debate.
30 Although AMs are involved in the cellular changes during the cource of inflammation, they
31 are only discussed in the host defense section (6.2.3.4) because this is their major function.
December 1993 6-8 DRAFT-DO NOT QUOTE OR CITE
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1 The previous O3 criteria document (U.S. Environmental Protection Agency, 1986)
2 discussed studies available at that time on the inflammatory and permeability effects of O3.
3 These studies recognized the increased thickness of the alveolar septa, presumably due to
4 their increased cellularity, after acute exposure, and due to excess collagen after chronic
5 exposure to O3. The inflammatory cell response was reported in rats and monkeys receiving
6 single or repeated exposures to O3 concentrations ranging from 0.2 to 0.8 ppm (Castleman
7 et al., 1980; Drummer et al., 1977; Moore and Schwartz, 1981; Crapo et al., 1984).
8 Exposures to O3 also resulted in increased mucosal permeability, as detected by the
9 nonspecific diffusion of phenol red from lung into the circulation (Williams et al., 1980) or
10 the appearance of serum proteins in the air spaces. Increased BAL levels of total protein,
11 albumin, and immunoglobulin (Ig) G were detected in rats, dogs, and guinea pigs exposed
12 acutely to O3 concentrations ranging from 0.1 to 2.5 ppm (Alpert et al., 1971; Reasor et al.,
13 1979; Hu et al., 1982). For example, Hu et al. (1982) found that a 72-h exposure of guinea
14 pigs to 5:0.26 ppm O3 increased BAL protein immediately after exposure and that when the
15 exposure duration was decreased to 3 h, protein increased 10 to 15 h postexposure (not
16 immediately).
17
18 6.2.2.2 Permeability Changes
19 A number of studies have demonstrated an increase in airway mucosal permeability
20 following inhalation exposure to O3 concentrations of 1.0 ppm or below (Table 6-1). In rats
21 exposed for 2 h to 0.8 ppm O3, labeled tracers, such as diethylenetriaminepentaacetate
22 (DTPA) and bovine serum albumin, introduced into the airway lumen were transferred to
23 blood to a greater extent than in the air-exposed rats (Bhalla et al., 1986; Bhalla and
24 Crocker, 1986). The rapidly rising concentration of the tracers in the blood during the initial
25 period of instillation of the tracers into the airways reflected both the accumulation, due to
26 slow instillation over a 5-min period, of the tracers in the airway lumen and then transfer
27 across the respiratory epithelium. The changes in permeability observed in this study were
28 transient in nature, returning to baseline value by 24 h postexposure in trachea and by
29 48 h in the distal airways. Reversible increases in airway epithelial permeability were also
30 observed in guinea pigs acutely exposed to 1 ppm O3 (Miller et al., 1986). The rate of
31 appearance of intratracheally administered horseradish peroxidase increased in blood at 2 and
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TABLE 6-1. LUNG INFLAMMATION AND PERMEABILITY CHANGES ASSOCIATED WITH OZONE EXPOSURE8
g Ozone
g. Concentration
i
i— >
O
O
6
o
g
H
I
g
ppm
0.1
1.2
0.1
0.3
1.0
0.1
0.3
1.2
0.1
0.3
1.0
0.1
to 10
0.1
0.2
0.4
0.8
0.1
0.3
1.2
0.1
0.2
1.2
4.0
0.12
0.30
/ig/m
196
2,352
196
588
1,960
196
588
2,352
196
588
1,960
196
to
19,600
196
392
784
1,568
196
588
2,352
196
392
2,352
7,840
235
588
Exposure
Duration
2h/dayfor 1,2,
6, and 13 days
2h
2h
In vitro and in vivo
2h
In vitro
2h
In vitro
2, 4, and 8 h
2h
In vitro
Continuous for
1 to 12 weeks
24 h,
48 h, and 72 h
Species, Sex
(Strain)
Rabbit, M
(NZW)
2-4 mo old
Rabbit, M
(NZW)
15-16 weeks old
Rabbit, M
(NZW) -
2-4 mo old
Rat (S-D)
12-18 weeks old
Cow
Rat(F344)
90 days old
Guinea pig
(Hartley)
60 days old
Rabbit, M
(NZW)
2-4 mo old
Rat, M
(Wistar)
16 weeks old for
1 week exposure;
21 weeks old for
longer exposures
Mice, M
(C57BL/
6J[B6]);
(C3H/
HeT[C3])
6-8 weeks old
Observed Effect(s)
Increase in AM number at 7 days following single exposure to 0.1 ppm and increase in
number of AMs and PMNs on 1-day after cessation of 6 or 13 days of exposure.
Increase in number of PMNs at 24 h after single exposure to 1 .2 ppm.
Increase in levels of PGE2 and PGF2ot in BAL immediately after exposure to 1 .0 ppm
03 only.
In vitro: increase in PGEj after 0.3 ppm and increase in PGF%a after 1 .2 ppm by AMs.
In vivo: increase in the release of POE2 and PGF2a by AMs after 1.2 ppm, but no
effect of 0.1 ppm.
Increased production of arachidonic acid metabolites by AMs at 1 .0 ppm only.
Increased production of PGE2 and PGF^ by tracheal epithelial cells after exposure to
0.1 and 0.3 ppm. Increased production of other arachidonic acid metabolites at
>1.0 ppm.
C X T exposure design; BAL 25 h after exposure started. PMNs measured in rats only;
no C and T interaction; effect dependent on C. Exponential and polynomial response
surface model used. Similar protein responses at low C X T products; generally, the
influence of T increased as C increased. Exponential model explained 86% of the data.
Exposure of AMs to >0.3 ppm 03 resulted in increased secretion of factors capable of
stimulating migration of inflammatory cells.
Number of AMs in BAL increased after exposure for 1 1 weeks to 0.2 ppm. Infiltration
of PMNs did not occur.
BAL immediately PE. Comparable increases in BAL protein, AMs, PMNs, and
lymphocytes in the two strains after exposure to 0. 12 ppm, but greater number of
inflammatory cells and protein concentration in 66 than in C3 mice after exposure to
0.30 ppm.
Reference
Driscoll et al. (1987)
Schlesinger et al. (1990)
Driscoll et al. (1988)
Madden et al. (1991)
Leikauf et al. (1988)
Highfill et al. (1992)
Driscoll and Schlesinger (1988)
Mochitate et al. (1992)
Kleeberger et al. (1993a)
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TABLE 6-1 (cont'd). LUNG INFLAMMATION AND PERMEABILrrY CHANGES
ASSOCIATED WITH OZONE EXPOSURE8
1
I—1
p— »
I— »
d
d
o
I
o
Ozone
Concentration
ppm
0.12
0.8
1.5
0.12
0.8
1.5
0.12 to
0.96
0.2
0.5
1.0
2.0
0.2
0.4
0.6
0.8
0.2
0.4
0.6
0.8
0.25
peak
over a
bkg of
0.06
Exposure
fjg/m Duration
235 6h
1,568
2,940
235 6h
1,568
2,940
255 6 h, 24 h,
to or 2 days
1,882
392 4h
980
1,960
3,920
392 6, 8, 12, and 24 h
784 for 3 days
1,178
1,568
392 7 h/day for
784 1, 2, or 4 days
1,176
1,568
490 13 h bkg, rose to
peak and returned to
bkg over 9 h
118
Species, Sex
(Strain)
Age
Rat, F
(F344/N)
12-18 weeks old
Rat, M
(F344/N)
12-18 weeks old
Rat, M
(S-D)
250 to 300 g
Mouse (Swiss Albino)
19-25 g
Guinea pig (Hartley)
3 14-522 g
Rat (S-D)
280-350 g
Rabbit (NZW)
1.7-2.5 kg
Hamster (Golden
Syrian)
94-107 g
Rat
(S-D)
10- 12 weeks old
Rat, M
(PVG)
12-16 weeks old
Rat, M
(F344)
60 days old
Observed Effect(s)
Increased number of PMNs in nasal Iavage, but not in BAL at 18 h after 0.12 ppm; increased
number of PMNs in BAL, but not in nose after 1 .5 ppm; number of PMNs decreased with time
in nose, with a concomitant increase in BAL PMNs after 0.8 ppm.
AMs and PMNs increased in number at various times PE at &0.8 ppm.
Total protein in BAL increased after exposure to >0.4 ppm for 6 h and >0.12 ppm for 1 or
2 days. Transport of radiolabeled albumin from blood to the airways increased after 6 or 24 h
exposure to 0.4 ppm or above and after 2 days exposure to 0.2 ppm.
Species differences in responsiveness. At 18-20 h PE, total protein in BAL increased in guinea
pigs exposed to 0.2 ppm, whereas mice, hamsters, and rats responded to 1.0 ppm and above,
and rabbits responded only to 2.0 ppm.
C and T matched such that all C x T = 14.4 ppm • h. BAL immediately after exposure
ceased. Increase in PMN's equivalent in all 03 groups. Increase in protein equivalent for 6-,
8-, and 12-h exposure groups, all of which are greater than protein in 24-h groups. Equivalent
results for BAL epithelial cells.
BAL approximately 17 h PE. The proportion of AMs in the BAL decreased, with a concomitant
increase in the proportion of PMNs after 1 or 2 days exposure to 50.6 ppm Oj.
Interstitial AMs increased in number in proximal alveolar region and terminal bronchioles at one
week of exposure, but the effects had subsided by 3 weeks of exposure.
Reference
Hotchkiss et al. (1989a)
Hotchkiss et al. (1989b)
Guth et al. (1986)
Hatch et al. (1986)
Gelzleichter et al. (1992b)
Donaldson et al. (1993)
Chang et al. (1992)
n
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TABLE 6-1 (cont'd). LUNG INFLAMMATION AND PERMEABILITY CHANGES
ASSOCIATED WITH OZONE EXPOSURE*
f
i
H- »
to
0
s
Ozone
Concentration
ppm
0.30
2.0
0.35
0.5
1.0
0.35
0.5
0.65
0.8
0.38
0.76
1.28
2.04
0.13
0.26
0.38
fig/m
588
3,920
686
980
1,960
686
980
1,274
1,568
750
1,500
2,500
4,000
250
500
750
Exposure
Duration
24, 48, or 72 h
0.30 ppm and 3
2.0 ppm
2.25 h/day for
5 days
2, 4, and 7 h
1, 2, 4, and 8 h
daytime
4, 8, and 12 h
nighttime
Species, Sex
(Strain)
Age
for Mice
h for (C57BL/
6J[B6]);
(C3H/
HeJ[C3])
DBA/2J (D2), hybrids
and recombinant
inbred strains (Rl):
BXD and BXH
6-8 weeks old
Rat, M
(F344)
3-4 mo old
Rat, M
(F344)
13 weeks old
Rat
(Wistar)
7-weeks old
Observed Effect(s) Reference
Inflammatory response was greater in B6 than in C3 or D2 mice. Fl progeny was categorized Kleeberger et al. (1990, 1993b)
as resistant; F2 generation segregated into 45:16 for resistant vs. susceptible phenotypes.
Among BXD Rl strains, 4 of 10 responded discordantly to the two exposures (0.30 and
2.0 ppm). Among BXD Rl, 4 of 16 were discordant.
Persistent increase in BAL protein and progressive inflammation at SO. 5 ppm. Tepper et al. (1989)
C x T exposure design. All exposure* included 45 min of CO% for 1 h to increase ventilation. Tepper et al. (1994, in draft)
BAL after pulmonary function tests completed. The quadratic model explained 92% of the
variance. The models suggest that C may have a more dominant influence than T.
C X T exposure design. BAL protein measured at various times PE. Rombout et al. (1989)
Daytime exposures: At 0.76 ppm, maximal increase 22 h after exposure started; after 4 and 8 h
of exposure, protein still elevated at 54 h from start of exposure.
Nighttime exposures: Temporal increase and decrease of protein more gradual, with maximal
response at 36 h after exposure started. Protein still elevated 72 h after start of 8- or 12-h
exposure to 0.26 or 0.38 ppm. Smallest tested C x T with effect was 0.13 ppm x 4 h.
3
(O °'4
cd °-6
0
a °-41
0
q
784 8 h/days for 90 days
1,176
800 12 h during day or
night
Monkey, M
(Bonnet)
5.2-8 years old
Rat, M
(Wistar)
Ouinea pig, M
(Hartley)
9 weeks old
influence as C increases.
Inflammatory response in respiratory bronchioles at 0.64 ppm.
Nighttime exposure of rats resulted in greater increase in BAL protein, albumin, and PMNs than
the daytime exposure. A similar difference was not observed in guinea pigs.
Moffatt et al. (1987)
Van Bree et al. (1992)
-------
I
f
S
2
December 1993
, ta>
1
-
-2
g
.3
t-5
oo
o 6
«
6-13 DRAFT-DO NOT QUOTE OR CITE
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TABLE 6-1 (cont'd). LUNG INFLAMMATION AND PERMEABILITY CHANGES
ASSOCIATED WITH OZONE EXPOSURE*
!
Ozone
Concentration
ppm
0.8
0.96
1.0
Exposure
fig/m Duration
1,568 3 h
1,882 8 h
1,960 4 to 24 h
Species, Sex
(Strain)
Age
Mouse, F
(CD-I)
5 and 9 weeks old
Monkey, M
(Rhesus)
2-8 .5 years old
Rat
(S-D)
63 to 70 days old
Observed Effect(s)
Increase in PGE2 in BAL in 5-week-old mice only; effect blunted by indomethacin pretreatment.
Number of labeled PMNs into lung tissue and BAL increased immediately after exposure, peaked at 12 h
PE, and returned to baseline by 24 h PE. Total labeled and unlabeled PMNs in BAL remained elevated
at 24 h, but returned to control levels by 72 h PE. Total protein in BAL was elevated only at 24 h PE.
Total protein and PMNs in BAL and PMNs in the centriacinar region of the lung increased with exposure
duration, but the number of AMs in BAL decreased. Treatment with anti-rat PMN serum resulted in
elimination of PMNs in BAL, but it did not affect the O3-induced increase in BAL protein.
Reference
Gilmour et al. (1993b)
Hydeetal. (1992)
Pino et al. (1992a, 1992b)
1.0 1,960 Smin Dog, M
03 delivered to a (Mongrel)
localized area of lung 21.2 ± 0.5 kg
via a teflon catheter
fitted to
bronchoscope
Number of PMNs in the subepithelial tissue increased at 1-3 h PE. Number of BAL PMNs increased at
24, but not at 1-3 h PE.
Kleeberger et al. (1989)
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TABLE 6-1 (cont'd). LUNG INFLAMMATION AND PERMEABILITY CHANGES
ASSOCIATED WITH OZONE EXPOSURE8
£5 Ozone
*"* Concentration
U> ppm
1.0
Exposure (Strain)
/tg/rn Duration Age
1,960 Ih Guinea pig, M
(Hartley)
250-300 g
Observed Effect(s)
The concentrations of PGEj, 6-keto PGFja, and TXB2 in BAL increased at various times following
exposure.
Reference
Miller et al. (1987)
1.0 1,960 3h Rat
isolated perfused (S-D)
lung 350 ± 42 g
No effect on BAL protein.
load et al. (1993)
Lft
1.8 3,528 2or4h Rat, M A decrease in number of AMs in BAL immediately after exposure. PMNs and albumin content of BAL Bassett et al. (1988b)
(Wistar) increased at 1 day PE. Increased albumin levels, but not the PMNs, persisted on Day 3 PE.
200-250 g
2.0 3,920 4h Guinea pig, M Interstitial PMNs increased in number immediately after exposure, but declined by 24 h PE. BAL PMNs Schultheis and Bassett (1991)
(Hartley) were maximal by 3-6 h and remained elevated by 3 days PE.
300-350 g
See glossary of terms and symbols for common abbreviations and acronyms.
Age or body weight at start of exposure.
d
o
o
-------
1 8 h after O3 exposure, as compared to the rats at 24 h postexposure to O3 and air-exposed
2 controls. When rats were exercised at a level that increased the minute ventilation twofold,
3 the effect of 0.8 ppm O3 was not only greater than in rats exposed at rest, but the increased
4 permeability persisted longer (Bhalla et al., 1987).
5 Guth et al. (1986) analyzed the permeability effects of O3 by injecting radiolabeled
6 albumin into the blood and measuring it in the BAL, as well as by measuring the total
7 protein concentration in the BAL. This study revealed an increase in permeability following
8 a 6-h exposure of rats to ^0.4 ppm or following 1 or 2 days of exposure to 0.12 ppm O3.
9 Tracer transport was also increased in rats exposed for 2 h to 0.8 or 2.0 ppm O3 (Crocker
10 and Bhalla, 1986; Bhalla and Crocker, 1987). Thus, these studies revealed increased
11 transport from airway lumen to blood as well as in the reverse direction.
12 The relative influence of concentration (C) and duration (T, tune) of O3 exposure was
13 evaluated by three laboratories using BAL protein as an indicator of effects. In the first
14 study, Rombout et al. (1989), exposed rats for 1, 2, 4, or 8 h to 0.38, 0.76, 1.28 or
15 2.04 ppm during the daytime (16 C x T products). A similar nighttime exposure study was
16 conducted using 0.13 to 0.38 ppm O3 and 4, 8, or 12 h of exposure (9 C X T products).
17 The smallest C x T products causing an increase in protein was 0.52 ppm • h (0.13 ppm X
18 4 h). A multivariate regression analysis accounted for 88.6% of the variance in the daytime
19 data and 73.2% of the nighttime data. Animals exposed during the night were more
20 responsive. A quadratic polynomial function showed that the influence of T increased with
21 increasing C and that the influence of T was still important at the lowest concentrated tested
22 (0.13 ppm). The second study employed rats and guinea pigs, each having 12 C x T cells
23 (0.1, 0.2, 0.4, and 0.8 ppm; 2, 4, and 8 h) (Highfffl et al., 1992). Using additional
24 modeling approaches, they obtained similar results to those of Rombout et al. (1989) . For
25 example, the exponential response surface model explained 86% of the variance in the data
26 and showed that the influence of T increased as C increased. However, at low C x T
27 products, similar BAL protein increases were observed. Further modeling of these data
28 (Highfill and Costa, 1994) again showed that C and T had interdependent influences. Tepper
29 et al. (1994, in draft) performed a similar C x T study with 12 C x T products (0.35 to
30 0.8 ppm; 2 to 7 h). However, rats were exposed to 8% CO2 for 45 min of each hour to
31 increase ventilation, and BAL was conducted on lungs that had been measured for pulmonary
December 1993 6-16 DRAFT-DO NOT QUOTE OR CITE
-------
1 function. The response surface predicted by the modeling again indicated that the influence
2 of T increased as C increased. Histopathological observations in the rats support the findings
3 that C had more influence than T. Tepper et al. (1994) compared their analysis of BAL
4 protein to that of Highfill et al. (1992) and found very good agreement, even though there
5 were experimental differences. However, in Tepper et al. (1994), there were larger
6 constants for C terms, indicating that C had a greater influence than in the Highfill et al.
7 (1992) study, probably because Tepper et al. increased ventilation (and hence O3 dose) by
8 using concurrent CO2 exposures. The Tepper et al. (1994) study also measured forced vital
9 capacity. Models showed that a decrease in forced vital capacity was a significant predictor
10 of increases in BAL protein.
11 Gelzleichter et al. (1992b) exposed rats to a single C x T (14.4 ppm • h) composed of
12 16 cells (0.2 to 0.8 ppm 03; 6 to 24 h/day for 3 days). They found that the 24 h/day
13 exposure groups had significantly less responses than the other groups which were all
14 equivalent. Thus, in this study, C and T had equivalent influences on the response, except
15 when T was 24 h/day. This study was well-conducted, but had some basic differences from
16 the Rombout et al. (1989) and Highfill et al. (1992) studies, in that the longer exposure
17 durations (i.e., 24 h/day) involved a mixture of daytime and nighttime exposure which likely
18 altered the dose-rate of 03. Also, Gelzleichter et al. used one C x T product, whereas the
19 other studies used several C X T products.
20
21 6.2.2.3 Concomitant Changes in Permeability and Inflammatory Cell Populations
22 in the Lung
23 Polymorphonuclear leukocyte infiltration in the lung following 63 exposure has been
24 investigated in a number of studies (Table 6-1), either by analyzing the cellular content of the
25 BAL or by counting PMNs in lung sections. Bassett et al. (1988a) found an increase in the
26 number of inflammatory cells in the BAL of rats continuously exposed for 3 days to
27 0.75 ppm O3. The inflammatory response was accompanied by elevated levels of albumin
28 and lactate dehydrogenase, suggesting increased permeability and cellular injury.
29 Comparable changes were also observed in rats acutely exposed to a higher O3 concentration
30 (Bassett et al., 1988b). In another study, a random count of PMNs in the lung sections at
31 4 h intervals following a 3-h exposure of rats to 0.8 ppm O3 revealed a gradual increase in
32 the number of PMNs, with a peak at 8 h postexposure and a return to the baseline value by
December 1993 6_17 DRAFT-DO NOT QUOTE OR CITE
-------
1 16 h postexposure (Bhalla and Young, 1992). The total protein and albumin concentrations
2 in the BAL also increased after the exposure, peaking at 8 h postexposure. Although the
3 protein concentrations returned to baseline by 16 h postexposure, the albumin levels
4 remained above the controls after 24 h. Alveolar lesions, having thickened septa,
5 parenchymal cellularity, and increased numbers of free cells, began to increase between
6 12 and 16 h postexposure and were still increasing at 24 h postexposure.
7 In trachea of rats exposed for 3 h to 0.8 ppm 63, a peak of PMN infiltration at
8 12 h postexposure was preceded by a decline in the number of PMNs in pulmonary
9 capillaries, suggesting exit of PMNs from the blood vessels and their migration across the
10 endothelial cells into the tracheal wall (Young and Bhalla, 1992). Although a significant
11 change in the tracheal population of PMNs did not occur until 12 h after the end of exposure,
12 tracheal permeability, as detected by DTPA transport, increased immediately following
13 O3 exposure. The results of this study suggest that the initial changes in tracheal
14 permeability may be independent of an inflammatory response, but the recruited PMNs may
15 serve to sustain the increased permeability and amplify O3 effects at later stages. This
16 conclusion was based on the observed shift of PMNs from the vascular compartment into the
17 tracheal wall and a concurrent peak of increased permeability. In comparable studies, Pino
18 et al. (1992a) exposed rats to 1.0 ppm O3 for periods ranging from 4 to 24 h. Total protein
19 and the number of PMNs in the BAL increased with time, with the maximum increase at the
20 end of 24 h of continuous exposure. The number of AMs was lower in the exposed animals
21 than in the controls. By morphometry, the peak PMN response in the terminal bronchioles
22 and alveolar ducts occurred at 4 h after an 8-h exposure. In dogs, local exposure of
23 peripheral airways to 1 ppm O3 for a short period (5 min) produced a recognizable
24 inflammatory response (Kleeberger et al., 1989). An increase in the number of PMNs was
25 detected in the subepithelial tissue within 3 h after 5 min of exposure of the dogs, but the
26 response had subsided 24 h later. In BAL, on the other hand, an increase in the number of
27 PMNs was not observed at 3 h postexposure; the PMNs increased in number at 24 h.
28 Hotchkiss et al. (1989a,b) have investigated the effects of a 6-h O3 exposure of rats to
29 0.12, 0.8, or 1.5 ppm O3 on AMs and PMNs and compared the inflammatory responses by
30 nasal lavage and BAL as well as morphometry in the nose and centriacinar region of the
31 lung, a site at which the lesions generally occur following O3 exposure. Animals were
December 1993 6-18 DRAFT-DO NOT QUOTE OR CITE
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1 examined 3, 18, 42, or 66 h after exposure ceased. From lavage data, 0.12 ppm O3 had no
2 effect. At 0.8 ppm, there was an increase in the number of nasal PMNs lavaged
3 immediately after exposure, which tapered off (no significant change at 42 h postexposure).
4 In contrast, BAL PMNs only increased later, beginning at 18 h postexposure and peaking at
5 42 h postexposure. From morphometric data, 0.12 ppm only caused an increase in nasal
6 PMNs 66 h postexposure. At 0.8 ppm, nasal PMNs increased to their greatest extent
7 immediately after exposure and were still increased at later time periods. However, PMNs
8 in the lung only increased at 18 and 66 h postexposure. The interpretation of these results
9 was based on the presence of potential competing mechanisms in nose and the lung.
10 Therefore, the attenuation of the nasal effects are matched by simultaneous enhancement of
11 the inflammatory response in the lung. Whether such a balance between nasal and alveolar
12 PMNs represents a specialization restricted to rats or if it is a more general phenomenon
13 remains to be investigated. A similar balance was not observed in humans exposed to
14 O3 (see Chapter 7). Subtle differences in species, O3 concentrations, and exposure
15 durations, however, need to be kept in mind when making interspecies comparisons.
16 Hyde et al. (1992) investigated the inflammatory response in monkeys exposed to
17 0.96 ppm O3 for 8 h. Polymorphonuclear leukocytes were isolated from peripheral blood,
18 labeled with indium-Ill labeled tropolonate and infused into the cephalic vein of monkeys
19 4 h before necropsy. Labeled PMNs in the lung tissue and the BAL peaked at 12 h and
20 returned to control values by 24 h postexposure. The total number of labeled and unlabeled
21 PMNs in the BAL, however, remained elevated at 24 h postexposure, but returned to
22 baseline by 72 h. Furthermore, the PMN peak at 24 h postexposure coincided with the
23 maximum increase in BAL protein at this time point. These studies suggest a strong
24 correlation between BAL protein concentration, epithelial necrosis, and inflammatory cells
25 (especially eosinophils) in bronchi, but not in the trachea or bronchioles. This observation
26 may represent a species-specific response. In rats, the inflammatory response in the terminal
27 airways involved an increase in the number of migratory cells, including PMNs, but not
28 eosinophils (Pino et al., 1992a). The available literature suggests that the precise time point
29 at which the maximum change in the number of inflammatory cells occurs is variable and
30 may be dependent upon several factors, including animal species, concentration, duration of
31 exposure, and mode of analysis (i.e., BAL versus morphometry of lung parenchyma).
December 1993 549 DRAFT-DO NOT QUOTE OR CITE
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1 Because the PMNs sampled by BAL represent only a small fraction of the cells shown to be
2 present in the air spaces by morphometry (Downey et al., 1993), the inflammatory response
3 detected by analyzing BAL may or may not match the response as obtained by microscopic
4 analysis of tissue sections. Even when the PMN response detected by the BAL analysis
5 accurately reflects the tissue PMNs (Hotchkiss et al., 1989a), the time at which the PMN
6 response peaks does not necessarily coincide when the analyses are made by the two
7 procedures. Therefore, the mode of analysis (BAL versus morphometry) and the time at
8 which this analysis is made needs to be taken into account when analyzing the inflammatory
9 response. This recommendation is consistent with the conclusion of Schultheis and Bassett
10 (1991) that BAL does not necessarily reflect cellular changes in lung interstitium.
11 Another approach to studying the inflammatory impact of 63 and its effects on airway
12 permeability is based on exposure of rats to drugs that destroy leukocytes or block the
13 activity of chemical mediators released by these cells. To determine whether the PMNs play
14 a role in O3-induced increased permeability, Pino et al. (1992b) studied O3 effects in PMN-
15 depleted rats. Although intraperitoneal injection of anti-PMN serum resulted in a nearly
16 complete depletion of PMNs in rats, it did not affect the increase in BAL protein following
17 an 8-h exposure to 1.0 ppm O3. In comparable studies, rats were rendered leukopenic by
18 intraperitoneal injection of cyclophosphamide (Bhalla et al., 1992). A 2-h exposure of
19 untreated rats to 0.8 ppm O3 caused a significant increase in the trachea! mucosal
20 permeability, as measured by enhanced trachea-to-blood transport of '"Tc-radiolabeled
21 DTPA immediately postexposure and accumulation of protein and albumin in BAL at
22 12 h postexposure. Pretreatment with cyclophosphamide did not change baseline values, but
23 did eliminate the O3 response. The reasons for the discrepancy between these results and the
24 results of the anti-PMN serum treatment study of Pino et al. (1992b) are not entirely clear,
25 but it is likely that the O3 effects are dependent, in part at least, on an interaction between
26 different inflammatory cell types. Therefore, it is not unreasonable to assume that in the
27 absence of PMNs, their role is taken up by another cell type. The attenuation of O3 effects
28 was also observed in rats pretreated with indomethacin, an inhibitor of cyclooxygenase
29 products, and FPL55712, which blocks LTD4 activity by preventing its binding to the
30 receptors (Bhalla et al., 1992). Based on these results, it was proposed that although O3 is
31 capable of producing direct injury to cells, inflammatory cells and their products may
December 1993 6-20 DRAFT-DO NOT QUOTE OR CITE
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1 contribute to the injury process (Bhalla et al., 1992). This conclusion is supported by the
2 recent studies of Joad et al. (1993). In the isolated perfused rat lung, PMNs but not O3,
3 increased BAL protein concentration. However, PMNs acted synergistically with O3 in the
4 induction of epithelial injury in the bronchioles. The recent demonstration of the effects of
5 O3 (0.8 ppm, 2 h) on some of the cellular activities of vascular PMNs (Bhalla et al., 1993)
6 further suggests potential mechanisms involved in the stimulation of PMNs and the induction
7 of inflammatory response. Polymorphonuclear leukocytes isolated from the blood of rats
8 exposed to O3 displayed shape changes, indicative of cell motility, and greater adhesion to
9 epithelial cells in culture than the PMNs from rats exposed to purified air.
10
11 6.2.2.4 Sensitive Populations
12 In addition to investigating the inflammatory response and permeability changes in
13 healthy adult animals, studies in recent years have analyzed the effects of 03 on lung
14 inflammation and airway permeability in different animal species, in potentially susceptible
15 subpopulations, and under special conditions (Table 6-1). Hatch et al. (1986) performed an
16 interspecies comparison to determine their relative responsiveness to O3. Although the
17 baseline BAL protein concentration of all the species was nearly the same, there were
18 noticeable differences in changes in BAL protein concentration among different species
19 following their exposure to O3. Significant changes were observed in guinea pigs exposed to
20 0.2 ppm O3. Mice, hamsters, and rats responded at O^ concentrations of 1 ppm and above,
21 but rabbits responded only to 2 ppm O3. In the case of rats, no differences were observed in
22 the sensitivity between males and females. When Slade et al. (1989) depleted guinea pigs of
23 lung ascorbic acid, they were more susceptible to an O3-induced increase in BAL protein
24 when the 4-h exposure was to 0.5 ppm, but not 1.0 ppm. Depletion of lung nonprotein
25 sulfhydryls did not enhance susceptibility. When Slade et al. (1989) depleted guinea pigs of
26 lung ascorbic acid, they were more susceptible to an O3-induced increase in BAL protein
27 when the 4-h exposure was to 0.5 ppm, but not 1.0 ppm. Depletion of lung nonprotein
28 sulfhydryls did not enhance susceptibility. Although Hu et al. (1982) report no elevation in
29 BAL protein in O3-exposed, vitamin C deficient guinea pigs, when their data were
30 statistically reanalyzed by Slade et al. (1989), vitamin C deficiency enhanced the effects of
31 0.5, but not 1.0, ppm O3.
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1 Kleeberger et al. (1990) found interstrain differences in inbred mice with regards to
2 inflammation and permeability changes following high-concentration (2 ppm) O3 exposure for
3 3 h that led them to propose that the PMN response to 63 may be controlled by a single
4 autosomal recessive gene at a chromosomal location designated as Inf (inflammation) locus.
5 In the followup studies, Kleeberger et al. (1993a) exposed the "susceptible" (C57BL/6J) and
6 the "resistant" (C3H/HeJ) strains of mice to lower concentrations of O3. Although changes
7 in inflammatory response and BAL protein were observed after exposure for 24 to 72 h to
8 0.12 as well as 0.3 ppm O3, the elevation in response in the susceptible strain over that in
9 the resistant strain was observed only at 0.3 ppm O3. Further studies with recombinant
10 inbred strains of mice suggested that genes at different loci may be responsible for responses
11 to 24-h (Inf locus) and 48-h O3 (Inf-2 locus) exposures (Kleeberger et al., 1993b).
12 Gunnison et al. (1992a) exposed rats aged 13 days, 18 days, 8 weeks, and 16 weeks old
13 to 1 ppm O3 for 2, 4, or 6 h. In the experiments to be discussed here, BAL was performed
14 immediately after exposure. Ozone exposure resulted in a decrease in the number of
15 leukocytes and an increase in protein in the BAL, but this decrease was not specific for a
16 certain age group. A weak relationship was observed between age and the number of
17 lavagable PMNs; a slightly greater influx of PMNs was observed in the younger rats.
18 A strong inverse relationship was, however, observed between age and leukocyte viability.
19 Approximately 50% of the total leukocytes recovered in the BAL from 13-day-old rats
20 exposed for 6 h were dead, as compared to about 10% dead in the 16-week-old rats.
21 Furthermore, 13-day-old animals were more responsive to a 2-h O3 exposure than the other
22 age groups of rats in terms of PGE^ levels in BAL; PGE levels were enhanced more in older
23 animals with the longer exposure durations. The authors attribute the increase in PGF-2 to an
24 increased release of arachidonic acid, rather than an effect on metabolism or formation on
25 PGE2- Gunnison et al. (1990) have also shown that levels of several eicosanoids in rabbits
26 show a similar pattern of age-responsiveness.
27 Factors such as physical activity and pregnancy of rats, in addition to their age, can
28 modify the airway sensitivity to O3. Van Bree et al. (1992) have reported circadian variation
29 in response to O3. In rats exposed to 0.4 ppm for 12 h, about 70% more PMNs were
30 recovered in the BAL after nighttime exposure than after daytime exposure. This increase
31 was attributed to greater physical activity and increased ventilation in the nocturnal animals.
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1 In guinea pigs, a similar difference between daytime and nighttime exposures was not
2 observed, but the variations appeared to be related to random physical activity. The
3 nighttime exposures also caused a greater increase in the BAL protein and albumin in rats,
4 but not in guinea pigs. Gunnison et al. (1992b) have found that pregnant rats are more
5 responsive to O3 (1 ppm for 6 h) than virgin females, as measured by an enhanced
6 inflammatory response, as detected by the analysis of protein, PMNs, leukocytes, and
7 enzyme activities in BAL at 18 h postexposure. When O3 exposure occurred on Day 17 of
8 pregnancy or Days 3, 13, and 20 of lactation, the magnitude of the increase in BAL protein
9 and number of PMNs was greater than the increase in virgin rats. No such increased
10 responsiveness was observed in rats at Day 10 to 12 of pregnancy or 14 days after lactation
11 ceased. Enzyme changes followed a similar pattern.
12
13 6.2.2.5 Repeated Exposures
14 The magnitudes of some of the effects of O3 observed after an acute exposure are less
15 upon repeated exposure. This phenomenon has been referred to as tolerance, adaptation, or
16 attenuation. Most of the older literature is on tolerance, which is classically defined as the
17 phenomenon in which a previous exposure to a nonlethal concentration O3 provides
18 protection against an otherwise lethal level. These studies and others at high 0^ levels are
19 discussed in the last O3 criteria document (U.S. Environmental Protection Agency, 1986).
20 Tepper et al. (1989) observed adaptation of pulmonary function changes hi rats exposed
21 for 2.25 h/day for 5 days, but a corresponding adaptation of lung inflammation did not
22 occur. Histologic examination of the lung sections revealed substantially more inflammatory
23 cells in alveoli after 5 days of exposure to 0.5 ppm O3 than after a single exposure to the
24 same O3 concentration. Increased protein concentration hi the BAL observed after a single
25 exposure also persisted after 5 days of repeated exposures. The morphologic studies of
26 Moffatt et al. (1987) identified an inflammatory response in the respiratory bronchioles of
27 bonnet monkeys exposed for 8 h/day for 90 days to 0.64 ppm O3. Significantly greater
28 numbers of AMs, mast cells, and PMNs reflected persistence of inflammation following
29 repeated exposures. Chang et al. (1992) exposed rats to an ambient pattern of 03. In this
30 morphometric study, the responses (epithelial inflammation in the proximal alveolar region
31 and terminal bronchioles, interstitial edema, and infiltration of AMs) to 1 week of
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1 O3 exposure had subsided after 3 and 13 weeks of exposure. Donaldson et al. (1993) did
2 not find a change in the total number of cells in BAL of rats exposed for 7 h/day for 4 days
3 to O3 concentrations ranging from 0.2 to 0.8 ppm. The number of PMNs, however,
4 increased after exposure to 0.6 and 0.8 ppm O3. This increase was greatest after the first
5 day of exposure, but it was resolved by Day 4. In the studies of Modulate et al. (1992), the
6 number of BAL AMs of rats exposed to 0.2 ppm 03 for 11 weeks was about 60% greater
7 than in the air-exposed controls. The preferential increase in the number of small AMs was
8 not dependent upon an enhancement of DNA synthesis. It was concluded that AMs adapt to
9 long-term exposures as a result of recruitment of immature AMs from an influx of
10 monocytes. No increase in the number of PMNs was observed in the BAL of exposed rats.
11 When analyzing the PMN data from different studies like the ones discussed above, it is
12 important to make a distinction between the PMN response in the lung interstitium versus
13 that observed in the BAL. It is possible that although the inflammatory response may persist
14 after repeated exposures, the PMNs do not necessarily continue to migrate from pulmonary
15 interstitium into the air spaces. As a result, the inflammatory response is detected in the
16 histological sections of the lung, but not in the BAL.
17
18 6.2.2.6 Mediators of Inflammation and Permeability
19 Although the presence of PMNs in the lung in large numbers is regarded as an evidence
20 of a morphological response to O3, the release of chemical mediators by inflammatory cells
21 indicates their state of activation and represents the functional modification as a consequence
22 of O3 exposure. Mediators with biological and chemotactic properties have been shown to
23 be released as a result of stimulation or injury of AMs, epithelial cells, and PMNs.
24 Arachidonic acid metabolites play an important role in a variety of processes, including
25 inflammatory response and permeability changes. Driscoll and Schlesinger (1988) found that
26 although AMs isolated from rabbit lungs continually released chemoattractant factors for
27 monocytes and PMNs, an in vitro exposure of AMs to 0.3 and 1.3 ppm Oj resulted in the
28 increased secretion of factors that stimulated the migration of PMNs. Driscoll et al. (1988)
29 also found increased eicosanoid biosynthesis following O3 exposure. In the latter studies,
30 elevated levels of PGE2 and PGF2a were detected in the supernatant following in vitro
31 exposure of rabbit AMs to 0.3 and 1.2 ppm O3 for 2 h. In a parallel in vivo study, an effect
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1 was seen only at the higher O3 concentration. In vitro exposure in a roller bottle system of
2 rat AMs to 1 ppm (but not 0.1 ppm) O3 causes stimulation of both cyclooxygenase and
3 lipoxygenase pathways of arachidonic acid metabolism, as shown by substantial increases in
4 the levels of 6-keto-PGFla, thromboxane B2 (TXB2), PGE^ LTB4, LTD4, and 15-HETE in
5 the supernatant of the AM culture (Madden et al., 1991). The authors attribute these effects
6 to both an increase in the availability of arachidonic acid as well as a stimulation of
7 cycloxygenase and lipoxygenase activities. Another in vitro study also demonstrated effects
8 on arachidonic acid metabolism (Leikauf et al., 1988) . Interpretation of these in vitro
9 studies is difficult. When Gunnison et al. (1990) compared the effect of in vitro and in vivo
10 exposures of AM to O3 on eicosanoid metabolism of AMs in culture, a disparity was found.
11 Cultured AMs from O3-exposed rabbits had a decrease in the elaboration of PGF2a; in vitro
12 exposure caused an increase.
13 Changes in the levels of eicosanoids have also been observed in in vivo studies.
14 Schlesinger et al. (1990) found elevation of PGE2 and PGFla in BAL of rabbits immediately
15 following a 2-h exposure to 1 ppm 03. Age may play a role. Five-, but not 9-week old
16 mice had increased levels of PGE2 in BAL (Gilmour et al., 1993b). Lower
17 O3 concentrations did not affect the levels of BAL eicosanoids. Hyde et al. (1992) found
18 anincrease in BAL concentrations of PGF2a, PGD2> and PGE2 following an 8-h exposure of
19 monkeys to 0.96 ppm O3. Prostaglandin concentrations in the BAL, detected using an
20 antibody that did not distinguish PGEX from PGE2, also increased with time following a
21 continuous exposure of mice to 0.5 ppm O3 (Canning et al., 1991). The peak levels of PGE
22 at 3 days were followed by a decline with time, but the levels remained higher than the
23 controls after 14 days of exposure. The time course of changes in the PGE levels was
24 matched by the time sequence of changes in BAL protein following exposure to 0.5 ppm Oj.
25 Plasma concentrations of 6-keto-PGFlo, and PGEl were also elevated in guinea pigs exposed
26 for 1 h to 1 ppm O3 (Miller et al., 1987). Kleeberger et al. (1989) delivered 1 ppm O3 for
27 5 min to a lobar bronchus of dogs using a wedged bronchoscope. An analysis of the lavage
28 fluid collected at 1 min postexposure revealed significant increases in the concentrations of
29 PGD2 and histamine. Although in this study the concentration of TXB2 did not change after
30 O3 exposure, significant increases in concentrations of TXB2 in the plasma and BAL were
31 observed following acute exposure of guinea pigs to 1 ppm 63 (Miller et al., 1987) and
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1 humans to lower levels of O3 (see Chapter 7). In addition, increased plasma concentrations
2 of PGFltt and PGEj were observed in the guinea pigs exposed to O3. Fouke et al. (1990,
3 1991) were unable to detect changes in the BAL concentrations of 6-keto-PGFla, PGF^,
4 TXB2, and PGF2a in baboons and mongrel dogs exposed to 0.5 ppm O3 for 2 h. The
5 reasons for the lack of this response in the baboon are not entirely clear, but the lower
6 O3 concentration used in this study, as compared to the exposure concentrations in dogs and
7 guinea pigs, and species differences offer possible explanations for the discrepancy.
8 Prostaglandin Et and £2 have been shown to influence the inflammatory processes in the
9 lung. Intrabronchial or intravenous administration of PGE^ was accompanied by increased
10 accumulation of the inflammatory cells in the lung and elevation of BAL protein (Downey
11 et al., 1988). A possible mechanism involved in the proinflammatory effects of the PGEs
12 included arteriolar vasodilation without venodilation, resulting in increased transfer of
13 proteins and cells from blood into the lung by hydrostatic pressure.
14
15 6.2.2.7 Summary
16 The airway epithelial lining serves as an efficient barrier against penetration of
17 exogenous particles and macromolecules into the lung tissue and circulation and against entry
18 of endogenous fluids, cells, and mediators into the air spaces. Disruption of this barrier
19 following O3 exposure represents a state of compromised epithelial defenses leading to
20 increased transmucosal permeability. Inflammatory cells represent another important
21 component of pulmonary defenses. The recruitment of these cells into the lung following
22 O3 exposure could result in the release of mediators capable of damaging other cells in the
23 lung.
24 Toxicological studies from several laboratories demonstrate alterations in epithelial
25 permeability and inflammatory responses in animals exposed to O3 concentrations of 1.0 ppm
26 and below. In these studies, an inflammatory response, as detected by an increase in the
27 number of PMNs in the BAL or in lung parenchyma, was accompanied by either an
28 increased tracer transport across the airway mucosa or an elevation in the levels of total
29 protein and/or albumin in the BAL. These changes were observed in animals exposed to
30 O3 concentrations as low as 0.1 ppm in rabbits (2 h/day for 6 days of exposure [Driscoll
31 et al., 1987]; 0.12 ppm in mice (24 h exposure [Kleeberger et al., 1993a]) and rats
December 1993 6-26 DRAFT-DO NOT QUOTE OR CITE
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1 (6 h exposure [Hotchkiss et al., 1989a] and 24-h exposure [Guth et al., 1986]); and 0.2 ppm
2 in guinea pigs (4-h exposure [Hatch et al., 1986]). Although monkeys also exhibit
3 inflammatory responses, concentrations this low have not been tested in this species. The
4 magnitude of response and the time at which it peaked appeared to vary with
5 O3 concentration, exposure duration, and the mode of analysis. Investigations of C X T
6 relationships for BAL protein in both rats and guinea pigs showed that T had increasing
7 influence as C increased (Rombout et al., 1989; Highffll et al., 1992; Highfill and Costa,
8 1994; Tepper et al., 1994, in draft). However, at low C x T products, similar increases
9 were observed (Highfill et al., 1992). The responsiveness to O3 also depended on the animal
10 species tested (Hatch et al., 1986) and increased under certain conditions, such as physical
11 activity (Van Bree et al., 1992) and pregnancy and lactation (Gunnison et al., 1992b).
12 To determine the impact of inflammatory cells on O3-induced airway permeability, rats
13 were exposed to drugs that either destroyed the inflammatory cells or blocked the activity of
14 their products. Treatment of rats with anti-PMN serum resulted in the depletion of PMNs,
15 but it did not affect the increase in BAL protein produced by O$ exposure (Pino et al.,
16 1992b). Depletion of all the leukocytes by cyclophosphamide or treatment of rats with PG
17 and LT antagonists resulted in an attenuation of the O3 effects on permeability (Bhalla et al.,
18 1992).
19 Inflammatory cells, when activated, are capable of releasing mediators with
20 pathophysiologic and a variety of modulating activities. An increase in the release of
21 arachidonic acid metabolites following O3 exposure has been shown after both in vitro
22 (Driscoll and Schlesinger, 1988; Driscoll et al., 1988; Madden et al., 1991; Leikauf et al.,
23 1988) and in vivo exposures (Schlesinger et al., 1990; Miller et al., 1987; Canning et al.,
24 1991).
25 Some of the effects seen after an acute exposure to Oj are modified upon repeated
26 exposures. The responses following repeated exposures included persistence or an increase
27 in the number of PMNs or AMs upon exposure of rats to 0.5 ppm (2.25 h/day) for 5 days
28 (Tepper et al., 1989), or exposure of monkeys to 0.64 ppm for 90 days (Moffatt et al.,
29 1987). Other studies report a reduced inflammatory response following repeated exposures
30 (Chang et al., 1992; Donaldson et al., 1993). The section on morphometry (Section 6.2.4)
31 has an extended discussion of microscopically evaluated inflammatory responses.
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1 In brief, these studies suggest that O3, at concentrations of 0.12 ppm and above, is
2 capable of producing inflammatory and permeability changes in laboratory animals. It is
3 clear that an assessment of the effects of O3 and interpretation of the results requires that
4 several factors be taken into consideration. These include O3 concentration, duration of
5 exposure, exposure conditions (e.g., repeated versus continuous exposure, day- versus
6 nighttime exposure, rest versus exercise during exposure), animal species, method of
7 evaluation, sensitive populations, and analysis time point postexposure.
8
9 6.2.3 Effects on Host Defense Mechanisms
10 6.2.3.1 Introduction
11 The mammalian respiratory tract has a number of closely integrated defense
12 mechanisms that, when functioning normally, provide protection from the adverse effects of
13 a wide variety of inhaled particles and microbes (Green et al., 1977; Kelley, 1990;
14 Schlesinger, 1989; Sibille and Reynolds, 1990). For simplicity, these interrelated defenses
15 can be divided into two major parts: the nonspecific (transport and phagocytosis) and the
16 specific (immunologic) defense mechanisms. A variety of sensitive and reliable methods
17 have been used to assess the effects of O3 on these components of the lung's defense system
18 to provide a better understanding of the health effects associated with the inhalation of this
19 pollutant.
20 The previous Air Quality Criteria Document for Ozone and Photochemical Oxidants
21 (U.S. Environmental Protection Agency, 1986) provided a review and evaluation of the
22 scientific literature published up to 1986 regarding the effects of O^ on host defenses. This
23 section briefly summarizes the existing database through 1986; describes the data generated
24 since 1986; and, where appropriate, provides interpretations of the data. This section also
25 discusses the various components of host defenses, such as the mucociliary escalator, the
26 phagocytic and regulatory role of the AMs, the immune system, and integrated mechanisms
27 that are studied by investigating the host's response to experimental pulmonary infections.
28
29 6.2.3.2 Mucociliary Clearance
30 This nonspecific defense mechanism removes particles deposited on the mucous layer of
31 the conducting airways by ciliary action. Ciliary movement directs particles trapped on the
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1 overlying mucous layer toward the pharynx, where it is swallowed or expectorated. The
2 effectiveness of the mucociliary transport system can be measured by the rate of transport of
3 deposited particles, the frequency of ciliary beating, and the structural integrity of the cells
4 that line the conducting airways. Impaired mucociliary clearance can result in an unwanted
5 accumulation of cellular secretions, increased infections, chronic bronchitis, and
6 complications associated with chronic obstructive lung disease.
7 Studies cited in the previous criteria document (U.S. Environmental Protection Agency,
8 1986) provided evidence on the effect of O3 on the morphologic integrity of the mucociliary
9 escalator and its ability to transport deposited particles from the respiratory tract. For
10 example, a number of studies with various animal species reported morphologic damage to
11 the cells of the tracheobronchial tree from exposures to 0$ (see Section 6.2.4). The cilia had
12 become noticeably shorter or were completely absent. Based on such morphologic
13 observations, related effects such as ciUostasis, increased mucous secretion, and a slowing of
14 mucociliary transport rates might be expected. Functional studies on mucociliary transport of
15 deposited particles from the respiratory tract have, in general, observed a delay in particle
16 clearance in early time periods following acute exposure. For example, a 4-h exposure of
17 rats to 0.8 ppm O3 slowed early clearance of inhaled latex spheres (Phalen et al., 1980).
18 Since the publication of the previous criteria document (U.S. Environmental Protection
19 Agency, 1986), several studies have been performed on the effects of acute O3 exposure on
20 the mucociliary transport apparatus (Table 6-2). Retarded mucociliary particle clearance was
21 observed following a 2-h exposure of rabbits to 0.6 ppm O3; extended exposures (up to
22 14 days) caused no effects (Schlesinger and Driscoll, 1987). Acute exposure of adult sheep
23 for 4 h/day for 2 days to 0.5 ppm O3 increased basal secretion of glycoproteins in sheep
24 trachea, whereas a longer exposure (4 h/day, 5 days/week for 6 weeks) to 0.5 ppm
25 O3 reduced trachea! glycoprotein secretions (Phipps et al., 1986). In a similar manner,
26 continuous exposure of ferrets to 1.0 ppm O3 for 3 days increased trachea! gland secretion of
27 glycoproteins, which remained elevated following 7 days of exposure (McBride et al., 1991).
28 Because the integrity of the periciliary space is vital for efficient mucociliary action,
29 O3-induced hyper- or hyposecretion by the mucous glands along the conducting airways can
30 alter the effectiveness of the mucociliary escalator.
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TABLE 6-2. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: PHYSICAL CLEARANCE8
g =— ===== —
jjjj Ozone
JJ* Concentration
i— >
vg ppm
W 0.06
base,
spike
rising to
0.25
0.1
0.6
1.2
0.1
0.25
0.6
0.5
O\
(1)
______
1.0
1.0
*p i.o
5? -
ftg/m
118 base,
spike rising
to 490
196
1,176
2,352
196
490
1,176
980
1,960
1,960
1,960
Exposure
Duration
13 h/day,
7 days/week base;
ramped spike
9 h/day,
5 days/week; 6 weeks
2 h/day for 1 or
13 days
2 h/day for 14 days
4 h/day,
5 days/week
for 6 weeks
24 h/day for 7 days
4 h/day for 5 days
2 h and 5 h/day for
4 days
Species, Sex
(Strain)
Age
Rat, M
(F344)
35 days old
Rabbit, M
(NZW)
2.5-3 .Okg
Rabbit, M
(NZW)
2.5-2.7 kg
Sheep, F
23-41 kg
Ferret
Sheep
1st week of
life
Sheep
26-41 kg
Observed Effect(s)c Reference
Increased retention of asbestos fibers in the lung parenchyma. Pinkerton et al. (1989)
Acceleration of early alveolar particle clearance at 0.1 and 0.6 ppm for 13 days. Driscoll et al. (1986)
After single exposure, increased clearance at 0.1 ppm and decrease at 1 .2 ppm
Retarded mucociliary particle clearance at 0.6 ppm only following a single 2-h Schlesinger and Driscoll (1987)
exposure; no effect of 14-day exposure.
Increase of tracheal glycoprotein secretion following acute exposure (4 h/day for Phipps et al. (1986)
2 days) with a decrease following ionger term exposure.
Increased secretion of glycoconjugates by tracheal glands. McBride et al. (1991)
Retardation of normal morphologic development of the tracheal epithelium. Mariassy et al. (1989, 1990)
Decreased tracheal mucous velocity. Decreased tracheal mucosa epithelial density.
Retardation of developmental decrease of tracheal mucous cells and their carbohydrate
composition.
Decreased tracheal mucous velocity. Allegra et al. (1991)
*See glossary of terms and symbols for common abbreviations and acronyms.
Q Age or body weight at start of exposure.
/Q
-------
1 Mariassy et al. (1990) exposed sheep during the first week of life to 1.0 ppm O3 for
2 4 h/day for 5 days and observed retardation of normal morphologic development of the
3 tracheal epithelium and a decrease in the tracheal mucous velocity. In a similar manner,
4 exposure of sheep during the first week of life for 4 h/day for 5 days to 1.0 ppm
5 O3 decreased epithelial mucosa density and retarded the developmental decrease of tracheal
6 mucous cells and their carbohydrate composition (Mariassy et al., 1989). Finally, exposure
7 of adult sheep for either 2 h or 5 h/day for 4 days to 1.0 ppm Oj decreased tracheal mucous
8 velocity (Allegra et al., 1991).
9
10 6.2.3.3 Alveolobronchiolar Transport Mechanism
11 In addition to the transporting of particles deposited on the mucous surface layer of the
12 conducting airways, particles deposited in the deep lung may be removed either up the
13 respiratory tract or through interstitial pathways to the lymphatic system (Green, 1973). The
14 pivotal mechanism of alveolobronchiolar transport involves the movement of AMs with
15 phagocytized particles to the bottom of the mucociliary escalator. Failure of the AMs to
16 phagocytize and sequester the deposited particles from the vulnerable respiratory membrane
17 can lead to particle entry into the interstitial spaces. Once lodged in the interstitium, particle
18 removal is more difficult and, depending on the toxic or infectious nature of the particle, its
19 interstitial location may allow the particle to set up a focus for pathologic processes.
20 Although Phalen et al. (1980) and Kenoyer et al. (1981) observed decreases in early
21 (tracheobronchial) clearance after acute O3 exposure of rats; late (alveolar) clearance was
22 accelerated.
23 Exposure of rabbits for 2 h/day for 13 days to 0.1 and 0.6 ppm O3 resulted in
24 acceleration of early alveolar clearance of polystyrene latex particles (Driscoll et al., 1986).
25 After a single exposure to 0.1 ppm, the greatest acceleration occurred over the period shortly
26 after exposure ceased (1 to 4 days), although the effect was still observed at Day 14
27 postexposure. A single exposure to 0.6 ppm caused no effect, whereas a higher
28 concentration (1.2 ppm) retarded clearance. To investigate the effects of longer term
29 O3 exposure on alveolobronchiolar clearance, rats were exposed to an urban pattern of
30 O3 (continuous 0.06 ppm, 7 days/week with a slow rise to a peak of 0.25 ppm and
31 subsequent decrease to 0.06 ppm over a 9-h period for 5 days/week) for 6 weeks and were
December 1993 6-31 DRAFT-DO NOT QUOTE OR CITE
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1 exposed 3 days later to chrysotile asbestos, which can cause pulmonary fibrosis and neoplasia
2 (Pinkerton et al., 1989). Ozone did not affect the deposition of asbestos at the first alveolar
3 duct bifurcation, the site of maximal asbestos and O3 deposition. However, 30 days later,
4 the lungs of the O3-exposed animals had twice the number and mass of asbestos fibers as the
5 air-exposed rats.
6
7 6.2.3.4 Alveolar Macrophages
8 Within the gaseous exchange region of the lung, the first line of defense against
9 microorganisms and nonviable particles that reach the alveolar surface is the AM. This
10 resident phagocyte is responsible for a variety of activities, including the detoxification and
11 removal of inhaled particles, maintenance of pulmonary sterility, and interaction with
12 lymphocytes for immunologic protection. Under normal conditions, AMs seek out particles
13 deposited on the alveolar surface and ingest them, thereby sequestering the particles from the
14 vulnerable respiratory membrane. If the particle is insoluble, the AMs serve as a repository
15 for the transport of the particle from the alveolus to the bottom of the mucociliary escalator
16 located at the far distal portion of the conducting airways. Degradable particles are
17 detoxified by powerful lysosomal enzymes, whereas microorganisms are killed by
18 biochemical mechanisms, such as superoxide anion radicals and lysosomal enzymes.
19 To adequately fulfill their defense function, the AMs must maintain active mobility, a high
20 degree of phagocytic activity, and an optimally functioning biochemical and enzyme system.
21 As discussed hi the previous criteria document (U.S. Environmental Protection Agency,
22 1986), short periods of O3 exposure can cause a reduction in the number of free AMs
23 available for pulmonary defense, and these AMs are more fragile, less phagocytic, and have
24 decreased lysosomal enzyme activities. The lowest O3 concentration showing AM effects in
25 this early work was 0.25 ppm; a 3-h exposure of rabbits decreased lysosomal enzyme
26 activities (Hurst et al., 1970).
27 Since the publication of the previous criteria document, the studies performed have
28 been, in general, confirmatory of previous observations (Table 6-3). Morphologic
29 observations showed that continuous exposure for 7 days to 0.13, 0.25, 0.5, and 0.77 ppm
30 O3 resulted in concentration-related increases in the number of rat AMs at 5 days
31 postexposure, as well as increased AM size and morphologic changes consisting of surface
December 1993 6-32 DRAFT-DO NOT QUOTE OR CITE
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TABLE 6-3. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: MACROPHAGE ALTERATIONS8
1
cf
1-1
h-*
%
u>
cr\
w
u>
g
§
z
9
|
3
8
n
Ozone
Concentration
ppm
0.05
0.1
0.2
0.4
0.1
0.2
0.1
1.2
0.1
1.2
0.11
to
3.6
0.12
0.8
1.5
0.12
0.27
0.8
0.13
to
0.77
0.13
0.26
0.51
0.77
0.2
0.4
ftg/m
98
196
392
784
196
392
196
2,352
196
2,352
216
to
7,056
235
1,568
2,940
235
529
1,568
250
to
1,500
250
500
1,000
1,500
392
784
Exposure
Duration
16 h
Continuous for
11 weeks
2h/dayforl,2,
6, and 13 days
2h
3h
6h
6h
Continuous for
7 days
Continuous for
7 days
Continuous for
14 days
3, 6, or 12 h;
12h/dayfor 1,
3, or 7 days
Species, Sex
(Strain)
Age
Rat, M
(Wistar)
210±10(SD)g
Rat, M
(Wistar)
16-21 weeks old
Rabbit, M
(NZW)
2-4 mo old
Rabbit
(NZW)
Mouse, M
(Swiss)
Rat, F
(S-D)
Rat, M
(F344)
12-18 weeks old
Rat, F
(F344)
12-14 weeks old
Rat, M
(Wistor)
8 weeks old
Rat, M
(Wistar)
8 weeks old
Rat, M
(Wistar)
10 weeks old
Mouse, M
(Nffl)
23-28 g
Rat, M
(Wistar)
1 80-200 g
Observed Effect(s)
Increased adherence to nylon fibers at 0.05 and 0.1 ppm, but not at 0.2 and 0.4 ppm.
Increases in enzyme activity.
Single exposure: decreased phagocytosis at 0.1 and 1.2 ppm immediately and 1 day after exposure;
recovery by 7 days after exposure in the 0.1-ppm group. Multiple exposure to 0.1 ppm only:
decreased phagocytosis 1 day after 2 and 6 days of exposure.
Increased release of PGE2 and PGF2a at 1 .2 ppm; no effect at 0.1 ppm.
Concentration-dependent decrease in superoxide anion radical production; mouse more sensitive. No
effect on murine AM phagocytosis at 0.42, 0.95, 1 .0, and 1 .2 ppm.
At 42 and 66 h PE, concentration-dependent increase in mitotic index beginning at 0.8 ppm; increased
size at 1 .5 ppm at 18 and 42 h PE.
Increase in mitotic index and chromosome damage at 0.27 and 0.8 ppm, no effect at 0.12 ppm.
Concentration-related effects on number, size, and surface morphology.
Decreased phagocytic ingestion (at all concentrations) and intracellular killing (at <0.26 ppm) of
Listeria monocytogenes .
Increases in enzyme activity at S3 days. Increased number of AMs by Day 3.
Decreased Fc-receptor mediated phagocytosis of AMs from mice at Days 1 and 7. Decreased
phagocytosis at 6 h, increased phagocytosis of AMs from rats on Day 1 . Decrease in superoxide
anion production at some exposures.
Reference
Veninga and Evelyn (1986)
Mochitate et al. (1992)
Driscoll et al. (1987)
Driscoll et al. (1988)
Ryer-Powder et al. (1988)
Amoruso et al. (1989)
Hotchkiss et al. (1989b)
Rithidech et al. (1990)
Dormans et al, (1990)
Van Loveren et al. (1988)
Mochitate and Miura (1989)
Costing et al. (1991a)
-------
I
-
8 S
'
re
n
Ex
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3
«»
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Mouse, F
(C3H/HeJ
C57B1/6)
30 days old
t 00
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6 6
December 1993
a
-------
1 microvilli and bleb formation (Dormans et al., 1990). Other morphological studies discussed
2 in Section 6.2.4 also show increased numbers of AMs. A 2-h exposure to 0.1 ppm O3 did
3 not affect the AM number in the BAL when the analyses were made immediately
4 postexposure, but the total number of AMs increased by about 70% 7 days later (Driscoll
5 et al., 1987). Upon repeated exposure for 6 or 13 days, the number of AMs increased on
6 the day after exposure. A single exposure to a higher concentration (1.2 ppm O3) did not
7 affect the number of AMs. It is assumed that although the lower O3 concentrations are
8 stimulatory for AMs activity, the higher concentrations are inhibitory because of their ability
9 to produce substantial cellular injury. The number of AMs in BAL is reduced after a single
10 exposure to 0.8 ppm or 1.8 ppm O3 (Bassett et al., 1988b; Bhalla and Young, 1992;
11 Donaldson et al., 1993). Hotchkiss et al. (1989b) also observed a slight decrease in the
12 number of AMs hi the BAL immediately after a 6-h exposure to 0.8 and 1.5 ppm O3, but the
13 number of AMs increased at 42 and 66 h after the exposure. The results of the studies on
14 AMs in general indicate that the immediate result of Q$ exposure is a reduction in the
15 number of AMs, but these cells increase in number over several days following exposure.
16 Exposure of rats to 0.12, 0.8, and 1.5 ppm O3 for 6 h also resulted in a concentration-
17 dependent increase in mitotic index at 0.8 ppm at 92 h after postexposure; AM size was only
18 increased at the highest concentration (18 and 48 h postexposure) (Hotchkiss et al., 1989b).
19 In a similar study, exposure of rats for 6 h to 0.12, 0.27, and 0.8 ppm O^ resulted in
20 AM chromosome damage at the two higher concentrations 28 h after exposure (Rithidech
21 etal., 1990).
22 Several studies have investigated the effect of O3 exposure on AM phagocytosis.
23 Exposure of C3H/HeJ and C57B1/6 mice for 3 h to 0.4 and 0.8 ppm O3 decreased AM
24 phagocytosis of Streptococcus zooepidemicus and latex beads (Gilmour et al., 1993a). In a
25 similar study, a 3-h exposure of 5- and 9-week-old CD-I mice decreased AM phagocytosis
26 of 5. zooepidemicus, but there was no effect of age (Gilmour et al., 1993b). Decreased
27 phagocytic ingestion of Listeria monocytogenes was also observed following continuous
28 exposure of rats to 0.13, 0.26, 0.51, and 0.77 ppm for 7 days; only the two lower
29 concentrations inhibited intracellular killing (Van Loveren et al., 1988). Although lower
30 O3 concentrations were not tested, rats exposed to 1.02 ppm 63 were unable to clear Listeria
31 from their lungs. Exposure of rabbits to 0.1 ppm O3 for 2 h/day resulted in decreased AM
December 1993 6-35 DRAFT-DO NOT QUOTE OR CITE
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1 phagocytosis of latex microspheres after 2 or 6 (but not 13) days of exposure (Driscoll et al.,
2 1987). In the same study, a single exposure to 0.1 or 1.2 ppm decreased AM phagocytosis
3 immediately after exposure; recovery occurred by 7 days postexposure in the 0.1-ppm group,
4 but not the 1.2-ppm group. That repeated exposures to O3 results in an initial suppression of
5 AM phagocytosis, which is followed by recovery of phagocytic potential while exposure
6 continues (Driscoll et al., 1987), was confirmed by the studies of Gilmour et al. (1991) and
7 Canning et al. (1991). Using identical exposure systems, it was observed that continuous
8 exposure of mice to 0.5 ppm O3 decreased AM Fc-receptor mediated phagocytosis of sheep
9 erythrocytes on Days 1, 3, 5, 7, and 8 of exposure, with return to control phagocytic levels
10 by Day 14. This temporal trend paralled the pattern of O3-induced reduction of lung
11 bactericidal activity against S. aureus.
12 Interspecies comparisons of AM phagocytic potential were made by Gilmour and
13 Selgrade (1994), who exposed C3H/HeJ and C57B1/6 mice and Fisher 344 rats to 0.4 and
14 0.8 ppm O3 for 3 h. Alveolar macrophage phagocytosis of latex beads was suppressed in all
15 animals immediately after 0.4 ppm O3 exposure, with the percent suppression greater in both
16 strains of mice as compared to similarly treated rats. No differences in phagocytic
17 suppression were observed between 0.4- and 0.8-ppm exposed rats or the C57B1/6 mice, but
18 phagocytosis by AMs from 0.8-ppm O3 exposed C3H/HeJ mice was more suppressed as
19 compared to the 0.4-ppm O3 exposed group. In a similar comparative study, Costing et al.
20 (1991a) exposed mice and rats to 0.4 ppm O3 for single (3, 6, and 12 h) and repeated
21 (12 h/day for 7 days) regimens. They observed a decrease in rat and mouse AM Fc-receptor
22 mediated phagocytosis following the single O3 exposure protocol. With the repeated
23 O3 exposure protocol, rat AM phagocytosis was increased a day after exposure with no
24 significant changes on Days 3 and 7. In contrast, phagocytosis by mouse AMs was
25 suppressed at Day 1 of exposure and still did not recover at Day 7. In the same study, when
26 mice were allowed to recover for 4 days following 3 O3 exposure days, phagocytosis by
27 AMs was increased. These interspecies comparisons on the effect of 0^ exposure on AM
28 phagocytic potential would indicate that mice may be more susceptible than rats.
29 A species comparison of superoxide anion radical production between mouse AMs and
30 rat AMs following a single 3-h exposure to O3 concentrations ranging from 0.11 to 3.6 ppm
31 showed the O3 concentration that inhibits superoxide anion radical production by 50% to be
December 1993 6-36 DRAFT-DO NOT QUOTE OR CITE
-------
1 0.41 ppm for mouse AMs and 3.0 ppm for rat AMs (Ryer-Powder et al., 1988). Oosting
2 et al. (1991a) also found a species difference in superoxide anion production using a more
3 varied exposure-duration protocol; mice appeared to be more responsive than rats. This
4 oxygen radical is important in antibacterial activity, and both sets of authors suggest that
5 O3-induced impairment of pulmonary antibacterial defenses may be related to decreases in
6 superoxide anion radical production. Decreased lysozyme enzyme levels in rat AMs were
7 also observed during chronic Pseudomonas aeruginosa bacterial infection following exposure
8 to 0.64 ppm O3 for 23 h/day for 27 days (Sherwood et al., 1986).
9 Exposure of rats for 16 h to 0.05, 0.1, 0.2, and 0.4 ppm O3 increased AM adherence
10 to nylon fibers at 0.05 and 0.1 ppm, but had no effect at 0.2 and 0.4 ppm (Veninga and
11 Evelyn, 1986). Increased metabolic activity of AMs retrieved from rats following continuous
12 O3 exposure for 14 weeks to 0.1 and 0.2 ppm was observed by Mochitate et al. (1992). In a
13 similar study, long-term O3 exposure of rats (continuous 0.2 ppm for 11 weeks) continued to
14 increase AM metabolic activity (Mochitate and Miura, 1989). Exposure of mice and rats for
15 14 h/day for 7 days to 0.4 ppm O3 also increased adenosine triphosphate (ATP) levels in the
16 mouse AMs, but had no effect on ATP levels in rat AMs (Oosting et al., 1991b).
17 In addition to their phagocytic function and particle removal, AMs also play several
18 other roles in host defense that include (1) a regulatory role through their release of
19 mediators (soluble substances secreted by the AMs that produce biologic effects on other
20 cells) such as tumor necrosis factor, interleukin-1, and PGs; (2) activities associated with
21 tumor surveillance; and (3) accessory cell function in antigen presentation to lymphocytes in
22 the initiation of the immune response. Investigating the effect of a single 2-h exposure of
23 rabbits to 0.1 and 1.2 ppm O3, Driscoll et al. (1988) observed an increased release of PGE2
24 and PGF2a by AMs following exposure to 1.2 ppm, but not to 0.1 ppm. Prostaglandin £2
25 can depress AM and natural killer cell cytotoxicity to tumor cells. Perhaps this is a
26 mechanism involved in the depression of AM-mediated cytotoxicity toward xenogeneic tumor
27 cells following exposure of rabbits for 2 h/day for 3 days to 1.0 ppm C^ (Zelikoff et al.,
28 1991). No studies were found on the effects of 05 on antigen presentation.
29
30
December 1993 6-37 DRAFT-DO NOT QUOTE OR CITE
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1 6.2.3.5 Immunology
2 In addition to the above nonspecific defense mechanisms, the respiratory system also
3 has specific immunologic mechanisms that can be initiated by inhaled antigens. There are
4 two types of immune mechanisms: antibody (humoral)-mediated and cell-mediated.
5 In general, humoral mechanisms neutralize viruses and microbial toxins, enhance the
6 ingestion of bacteria by phagocytes, and play an important role in defense of the lung against
7 fungal and parasitic infections. Cell-mediated mechanisms enhance the microbiocidal
8 capacity of AMs in defense to intracellular bacteria such as Mycobacteriwn tuberculosis and
9 Listeria monocytogenes, whereas another arm of the cellular immune response generates a
10 class of lymphocytes that are cytotoxic for virus-infected cells. Both the humoral and
11 cell-mediated responses protect the respiratory tract against infectious agents and operate in
12 three major temporal waves: (1) natural killer cells (nonspecific lymphocytes that can
13 destroy bacteria, viruses, and tumor cells), (2) cytotoxic T lymphocytes (lymphocytes that
14 lyse specifically recognized targets), and (3) by antigen-specific antibodies.
15 Little information was available in the previous criteria document (U.S. Environmental
16 Protection Agency, 1986) on the effects of O3 on immunologic defenses. However, the data
17 base indicated an immunotoxic effect of O3 exposure, especially on T cell populations. For
18 example, Aranyi et al. (1983) found that a 90-day (5 h/day, 5 days/week) exposure of mice
19 to 0.1 ppm O3 suppressed blastogenesis of splenic lymphocytes to T-cell mitogens, but not
20 B-cell mitogens; the ability of these cells to produce antibodies was not affected either.
21 As can be seen from Table 6-4, this database has greatly expanded and also has been recently
22 reviewed (Jakab et al., 1993). Many of the studies include both the pulmonary and systemic
23 immune system, which, to a degree, are compartmentalized; both systems are discussed here.
24 Studies on the effect of O3 exposure on the immune system can be divided into three
25 broad categories. These are (1) measurement of lymphoid organ weights and cellular
26 composition, (2) determination of the functional capacity of lymphocytes in the absence of
27 antigenic stimulation, and (3) measurement of the immune response following antigenic
28 stimulation.
29 Dziedzic and White (1986a) observed that exposure of mice to 0.3, 0.5, and 0.7 ppm
30 O3 for 20 h/day for 28 days resulted in a concentration-dependent initial depletion of cells in
31 the mediastinal lymph nodes (MLNs) (Days 1 and 2) that was followed by MLN T-cell
December 1993 6-38 DRAFT-DO NOT QUOTE OR CITE
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6-39 DRAFT-DO NOT QUOTE OR CITE
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TABLE 6-4 (cont'd). EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS: IMMUNOLOGY3
CT Ozone
5. Concentration
~
w
>_t Ppm P-gtm
io 0.7 1,372
U>
0.8 1,568
0.8 1,568
0.8 1,568
1.0 1,960
Exposure
Duration
20 h/day for
1 4 days
23 h/day for
14 days
24 h/day for
14 days
24 h/day for
4 weeks
24 h/day for
4 weeks
Species, Sex
(Strain)
Mouse, F
(BALB/c)
10-12 weeks old
Mouse, F
(B6C3Fi)
6-8 weeks old
Mouse, M
(BALB/c)
2-3 mo old
Mouse, M
(BALB/c)
7 weeks old
Mouse, M
(C57B/6 x
DBA/2 Fl)
11-14 weeks old
Observed Effect(s)
Decreased spleen and thymus weight at Day 4 with recovery at Day 14. Increased
infiltration of Thy-1 .2 lymphocytes and IgM cells into the O3-induced pulmonary
lesion.
Decreased blastogenesis of MLN and splenic lymphocytes to PHA mitogen on Day 1 ; no
effect on Days 3, 7, and 14. Decreased splenic NK cell activity on Days 1 and 3; no
effect on Days 7 and 14. Decreased pulmonary IgG and IgA response to ovalbumin
immunization.
Suppression of delayed-type hypersensitivity response to SRBCs on Day 7; no effect on
Days 1, 3, and 14. Thymic atrophy on Day 7 with decreased Thy-1 .2 cells in thymus.
Suppression of serum IgG following ovalbumin immunization.
Decreased ability of spleen cells to generate a primary antibody response to SRBCs
in vitro.
Reference
Bleavins and Dziedzic (1990)
Gilmour and Jakab (1991)
Fujimaki et al. (1987)
Ozawa (1986)
Wright et al. (1989)
1.0 1,960 8 h/day for 7 days Rat, M No effect on lung and splenic ADCC activity. Eskew et al. (1986)
(Long-Evans) Enhanced blastogenesis of splenic lymphocytes to PHA, ConA, and lipopolysacchande
7-8 weeks old mitogens at 1.0 ppm.
|
£
See glossary of terms and symbols for common abbreviations and acronyms.
Age or body weight at start of exposure.
8
-------
1 hyperplasia and an enhanced blastogenic response to the T-cell mitogen concanavalin A
2 (ConA). In a similar study, exposure of mice to 0.7 ppm 63 for 20 h/day for 28 days
3 resulted in an initial thymic atrophy, with return to normal thymus weights by Day 14 of
4 exposure (Dziedzic and White, 1986b). Exposure of rats to 0.5 ppm O3 for 20 h/day for
5 14 days also increased bronchus-associated lymph node and MLN cell proliferation at 3 days
6 of exposure, but not at 1, 2, 7, and 14 days of exposure (Dziedzic et al., 1990). Bleavins
7 and Dziedzic (1990) observed that exposure of BALB/c mice to 0.7 ppm Oj for 20 h/day for
8 14 days resulted in decreased spleen and thymus weights at Day 4 with recovery at Day 14.
9 The absolute number of thymocytes decreased following exposure of mice to 0.7 ppm O3 for
10 24 h/day for 7 days (Li and Richters, 199la) and to 0.3 ppm O3 for 24 h/day for 3 weeks
11 (Li and Richters, 1991 b). Although the latter exposure protocol (0.3 ppm for 24 h/day for
12 3 weeks) decreased the absolute number of thymocytes, an increase in the percentage of
13 thymocytes was observed in the absence of any changes in splenic T-cells (Li and Richters,
14 1991b). Continuous exposure of rats to ^0.26 ppm O3 for 7 days increased MLN T:B
15 lymphocyte ratios immediately and 5 days postexposure (Van Loveren et al., 1988).
16 Bleavins and Dziedzic (1990) observed an increased infiltration of Thy-1.2+ lymphocytes
17 and IgM cells into the O3-induced pulmonary lesion following exposure of mice to 0.7 ppm
18 O3 for 20 h/day for 14 days. Dziedzic amd White (1987c) further investigated these T cell
19 effects by exposing normal and athymic nude mice to 0.7 ppm O3 for 7 or 14 days
20 (20 h/day). After 7 days of exposure, the athymic nude mice did not have the MLN
21 hyperplasia seen in the euthymic mice. However, the athymic nude mice had a greater
22 inflammatory response and an increase in lung lesion volumes compared to the euthymic
23 mice. Thus, it appears that T cells have some involvement in protecting the lungs from the
24 morphological effect of O3.
25 The above longitudinal studies on the effects of O3 exposure on lymphoid organ cell
26 numbers provide information on cellular traffic and cell numbers, but provide few insights
27 into the functional capacity of the lymphocytes. A number of studies have investigated the
28 effect of O3 exposure on the blastogenic response of lymphocytes to nonspecific mitogens.
29 These assays measure nonspecific clonal expansion of the lymphocyte population, a critical
30 step during the amplification of the immune response. Exposure of mice to 0.7 ppm Oj for
31 20 h/day for 28 days enhanced the MLN cell blastogenic response to the T-cell mitogen
December 1993 6-41 DRAFT-DO NOT QUOTE OR CITE
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1 ConA at 4 and 7 days of exposure, with return to control levels by Day 14 (Dziedzic and
2 White, 1986a). In a similar manner, Gilmour and Jakab (1991) observed that continuous
3 exposure of mice to 0.8 ppm O3 decreased the MLN and splenic lymphocyte blastogenic
4 response to the T-cell mitogen phytohemagglutinin (PHA) on Day 1 of exposure, with the
5 effect abrogated after prolonged exposure. An enhanced blastogenic response of splenic
6 lymphocytes to PHA and Con A and a B-cell mitogen (Escherichia coli lipopolysaccharide)
7 was observed following exposure of rats to 1.0 ppm O3 for 8 h/day for 7 days (Eskew et al.,
8 1986).
9 Natural killer (NK) cell activity has also been studied. One such study in rats observed
10 a decreased lung NK cell activity following 1,5, and 7 days of exposure to 1.0 ppm for
11 23.5 h/day, with recovery by Day 10 (Burleson et al., 1989). In a similar experiment,
12 Van Loveren et al. (1990) observed decreased lung NK cell activity following 7 days of
13 continuous exposure of rats to 0.82 ppm O3. However, exposure to 0.2 and 0.4 ppm
14 enhanced lung NK cell activity. Exposure of mice for 23 h/day for 14 days to 0.8 ppm
15 O3 also decreased splenic NK cell activity on Days 1 and 3, with a return to control values
16 on Days 7 and 14 (Gilmour and Jakab, 1991). Finally, Selgrade et al. (1990) used an
17 experimental protocol designed to mimic diurnal urban O3 exposure patterns. Rats were
18 exposed to a background level of 0.06 ppm for 13 h (7 days/week), followed by a broad
19 exposure spike (5 days/week) rising from 0.06 ppm to 0.25 ppm and returning to 0.06 ppm
20 over 9 h, and then followed by a 2-h downtime. After 1, 3, 13, 52, or 78 weeks of
21 exposure, spleen cells were assessed for NK cell activity and responses to T-cell mitogens
22 (PHA and ConA) and a B-cell mitogen (Salmonella typhimurium glycoprotein). Ozone
23 exposure had no effect on NK cell activity, nor were there any O3-related changes in mitogen
24 responses in splenic or blood leukocytes. There were also no effects of a single 3-h exposure
25 to 1.0 ppm O3 on spleen cell responses to the mitogens immediately after exposure or at 24,
26 48, and 72 h thereafter.
27 Several studies have also investigated the effect of O3 exposure on the immune response
28 following antigenic stimulation. Fujimaki (1989) observed that exposure of mice to 0.8 ppm
29 O3 24 h/day for 56 days suppressed the primary splenic antibody response to sheep red blood
30 cells (SRBCs) (T-cell dependent antigen) but not to DNP-Ficoll (T-cell independent antigen).
31 In a similar study, exposure of mice to 0.8 ppm O3 for 24 h/day for 14 days suppressed the
December 1993 6-42 DRAFT-DO NOT QUOTE OR CITE
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1 delayed type hypersensitivity response to SRBCs on Day 7, but not on Days 1,3, and 14
2 (Fujimaki et al., 1987). Suppression of serum IgG levels upon ovalbumin immunization was
3 observed following exposure of mice to 0.8 ppm Qj for 24 h/day for 4 weeks (Ozawa,
4 1986). Decreased pulmonary IgG and IgA responses upon ovalbumin immunization were
5 also observed in mice during a 2-week O3 exposure for 23 h/day to 0.8 ppm (Gilmour and
6 Jakab, 1991). Exposure of mice to 0.5 ppm continuously for 14 days during the course of
7 influenza virus infection also decreased the serum hemagglutinin antiviral antibody response
8 (Jakab and Hmieleski, 1988).
9 Van Loveren et al. (1988) investigated antigen-specific responses following pulmonary
10 Listeria infection and observed no significant changes in the delayed-type hypersensitivity
11 response when O3 exposure (continuous, 0.77 ppm) was for 7 days prior to infection.
12 However, if the O3 exposure took place when an infection with Listeria was also present
13 (from Days 0 to 7 or from Days 7 to 14), the delayed-type hypersensitivity response was
14 significantly decreased. In a similar manner, no significant changes were observed in the
15 splenic lymphoproliferative response to Listeria antigen when the 0.77 ppm O3 exposure
16 preceded the infection, whereas the response was suppressed when the 0.77 ppm O3 exposure
17 occurred immediately after infection or from Days 7 to 14 after infection. In the same series
18 of experiments, Van Loveren et al. (1988) observed that continuous exposure to 0.26 ppm
19 O3 impaired the increase in T:B lymphocyte ratios that occurred in response to the Listeria
20 infection.
21
22 6.2.3.6 Interaction with Infectious Agents
23 Because respiratory infections remain one of the most common public health problems,
24 it is important to determine the environmental factors that may govern susceptibility.
25 Measurement of the competence of the host's antimicrobial mechanisms can best be tested by
26 challenging air pollutant-exposed animals and the clean-air exposed control animals to an
27 aerosol of viable organisms. If the test substance, such as O3, had an adverse influence on
28 the efficiency of the host's integrated protective mechanisms (i.e., physical clearance via the
29 mucociliary escalator, microbicidal activity of the AMs, and associated humoral and cellular
30 immunologic events), the microorganisms are less efficiently killed in the lungs or the
31 organisms may even multiply, resulting in the demise of the host.
December 1993 6-43 DRAFT-DO NOT QUOTE OR CITE
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1 The studies detailed in the previous criteria document (U.S. Environmental Protection
2 Agency, 1986) primarily used the mouse "infectivity model" (Gardner, 1982). Briefly,
3 animals are randomly selected to be exposed to either clean air or O3. After exposure, the
4 animals from both groups are combined and exposed to an aerosol of microorganisms. The
5 vast majority of these studies have been conducted with Streptococcus species. At the
6 termination of the infectious exposure period, the animals are housed in clean air and the
7 mortality rate in the two groups is determined during a 15-day holding period. In this
8 system, the concentrations of O3 used do not cause any mortality. The mortality in the
9 control group (clean air plus exposure to the microorganism) ranges from approximately
10 10 to 20% and reflects the natural resistance of the host to the infectious agents. The
11 difference in mortality between O3-exposure groups and the controls is concentration-related
12 (Gardner, 1982). These studies showed that, depending on the O3 exposure protocol, a 3-h
13 exposure to concentrations as low as 0.08 ppm O3 can enhance the increased mortality of
14 CD-I mice from Streptococcus infection (Coffin et al., 1967; Coffin and Gardner, 1972;
15 Miller et al., 1978). However, although a prolonged intermittent exposure (103 days) to
16 0.1 ppm increased mortality in this model system, the magnitude of the effect was not
17 substantially greater than that after acute exposure (Aranyi et al., 1983).
18 Another approach to assess the effect of air pollutants on host defenses is to quantitate
19 rates of pulmonary bacterial inactivation following aerosol infection with microorganisms.
20 In this system, the animals are exposed either to clean air or to the air pollutant and then are
21 exposed to an aerosol of microorganisms in a manner similar to the method used for the
22 infectivity model. However, instead of assessing enhancement of mortality, viable bacteria
23 are quantitated in lung homogenates at various times after inhalation of the microorganisms
24 (Goldstein et al., 1971a,b). In air-exposed control animals, there is a rapid inactivation of
25 the inhaled microorganisms that have been deposited in the respiratory tract. However,
26 O3 exposure alters the ability of the microbicidal mechanisms of the lungs to function
27 normally and bacterial inactivation proceeds at a slower rate, indicating impairment of host
28 defenses. For example, Goldstein et al. (1971b) showed that a 4-h exposure of mice to
29 ^0.6 ppm O, after infection with Staphylococcus aureus decreased lung bacterial activity.
30 Studies appearing in the literature since publication of the previous criteria document (U.S.
31 Environmental Protection Agency, 1986) are described below (also see Table 6-5).
December 1993 6-44 DRAFT-DO NOT QUOTE OR CITE
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TABLE 6-5. EFFECTS OF OZONE ON HOST DEFENSE MECHANISMS:
§
?
•— »
VO
vo
u>
o\
4
a
§
1
§
§
o
Ozone
Concentration
ppm
0.1
0.3
0.5
0.13
0.26
0.51
0.77
1.02
0.25
0.5
1.0
0.4
0.8
0.4
0.8
0.4
0.8
0.5
0.5
0.5
a
ftgfm
196
588
980
250
500
1,000
1,500
2,000
490
980
1,960
784
1,568
784
1,568
784
1,568
980
980
980
Exposure
Duration
24 h/day,
5 days/week for
3 weeks
Continuous for
7 days
3 h/day for
5 days
3h
3h
3h
24 h/day for
14 days
24 h/day for
15 days
24 h/day for
120 days
UVJ
Species, Sex
(Strain)
Age"
Mouse, F
(CD-I)
4-6 weeks old
Rat, M
(Wistar)
8 weeks old
Mouse, F
(CD-I)
3-4 weeks old
Mouse, F
(C3H/HeJ
C57B1/6)
30 days old
Mouse, F
(CD-I)
5 and 9 weeks old
Mouse, F
(C3H/HeJ
C57B1/6)
Rat
(F344)
30 days old
Mouse, F
(Swiss)
20-23 g
Mouse, F
(Swiss)
20-23 g
Mouse, F
(Swiss)
20-23 g
LJUtAlTlUJNS WITH UNJTOCTIUIIS AtrJfiJNTS
Observed Effect(s)
Increased mortality from Streptococcus zooepidemicus infection at 0.3 and 0.5 ppm.
Decreased bactericidal activity vs. Listeria monocytogenes at 0.77 ppm and 1 .02 ppm.
Increased mortality at 1.02 ppm.
Increased mortality from influenza virus infection and increased pulmonary virus liters at
1 .0 ppm when infection followed 2 days of exposure, but not at other time points.
Histopathologic and pulmonary function changes more severe with this regimen.
At >0.5 ppm, increased lung wet weight when virus given after 2 days of Oj exposure.
Decreased bactericidal activity vs. Streptococcus zooepidemicus. Increased mortality in
both strains, with greater mortality in the C3H/HeJ strain.
Decreased bactericidal activity vs. Streptococcus zooepidemicus. Increased mortality in
both age groups, with greater mortality in the 5-week-old mice.
Decreased intrapulmonary killing of Streptococcus zooepidemicus.
Decreased bactericidal activity vs. Staphylococcus aureus on Days 1 and 3; no effect on
Days 7 and 14. Enhanced bactericidal activity vs. Proteus mirabilis on Days 3, 7, and
14; no effect on Day 1 .
No effect on pulmonary virus tilers during influenza virus infection. 03 decreased lung
morphological injury due to virus (Day 9).
Ozone decreased acute lung influenza! injury, but increased pulmonary fibrosis during the
course of and period after influenza virus infection.
Reference
Graham et al. (1987)
Van Loveren et al. (1988)
Selgrade et al. (1988)
Gilmour et al. (1993a)
Gilmour et al. (1993b)
Gilmour and Selgrade (1994)
Gilmour et al. (1991)
Jakab and Hmieleski (1988)
Jakab and Bassett (1990)
^See glossary of terms and symbols for common abbreviations and acronyms.
Age or body weight at start of exposure.
-------
1 Gilmour et al. (1993a) observed that exposure of C3H/HeJ and C57B1/6 mice for 3 h to
2 0.4 and 0.8 ppm O3 resulted in decreased intrapulmonary killing of S. zooepidemicus in both
3 strains of mice. Although both strains were affected, the C3H/HeJ mice also appeared to be
4 more susceptible because bactericidal activity was decreased sooner and mortality was
5 enhanced more. Gilmour et al. (1993b) expanded these studies to CD-I mice of different
6 ages (5- and 9-weeks-old) exposed for 3 h to 0.4 and 0.8 ppm O3. The higher
7 concentration decreased intrapulmonary killing 4 h after infection with 5. zooepidemicus;
8 there was no effect of age. However, the 5-week-old mice were more susceptible to the
9 infection because mortalities were 9, 41, and 61% in the air, 0.4-, and 0.8-ppm exposure
10 groups, respectively, whereas only 4, 15, and 28% of the older animals died with analogous
11 exposure. Pretreatment of the mice with indomethacin reduced the O3-induced enhancement
12 of PGE2 levels in BAL as well as the enhanced mortality in the 5-week-old mice, suggesting
13 an involvement of arachidonic acid metabolites in antibacterial defenses.
14 Gilmour and Selgrade (1994) studied the interspecies response to experimental
15 S. zooepidemicus infection of rats and C3H/HeJ and C57B1/5 mice following a 3-h exposure
16 to 0.4 and 0.8 ppm O3. Exposure of rats to O3 suppressed intrapulmonary bacterial killing,
17 with no differences observed between the 0.4- and 0.8-ppm O3 exposure groups. Exposure
18 of C57B1/6 mice to 0.4 ppm O3 also resulted in a suppression of bactericidal activity,
19 whereas exposure to 0.8 ppm O3 led to bacterial proliferation in the lungs, resulting in 60%
20 mortality at Day 4. Exposure of C3H/HeJ mice to both 0.4 and 0.8 ppm O3 resulted in
21 bacterial proliferation with, respectively, 60 and 80% mortality at Day 4 after exposure.
22 Increased mortality from S. zooepidemicus infection following 24 h/day exposure
23 5 days/week for 3 weeks was also observed following 0.3 and 0.5 ppm, but not following
24 0.1 ppm, O3 exposure (Graham et al., 1987).
25 To investigate the effect of longer exposures and challenges with bacteria, Gilmour
26 et al. (1991) exposed mice continuously to 0.5 ppm O3 for 14 days. At 1, 3, 7, and
27 14 days, intrapulmonary killing was assessed by inhalation challenge with Staphylococcus
28 aureus and Proteus mirabilis. Ozone exposure impaired the intrapulmonary killing of
29 5. aureus at 1 and 3 days. However, with prolonged exposure, the bactericidal capacity of
30 the lungs returned to normal. In contrast to S. aureus, when P. mirabilis was the challenge
31 organism, O3 exposure had no suppressive effect on pulmonary bactericidal activity. The
December 1993 6-46 DRAFT-DO NOT QUOTE OR CITE
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1 authors attribute this difference to the defense mechanisms involved. Alveolar macrophages
2 are active against the Gram-positive 5. aureus; AMs and PMNs defend against the
3 Gram-negative P. mirabilis. The effects O3 on bactericidal activity against S. aureus
4 paralled the effects on AM phagocytosis (early decrease, then no change). With
5 P. mirabilis, there was more than a 1,000-fold increase in PMNs in the lung that was not
6 altered by O3, enabling bactericidal activity to occur. In a similar manner, exposure of rats
7 for 24 h/day for 7 days to 0.13, 0.26, 0.51, 0.77, and 1.02 ppm O3 decreased pulmonary
8 bactericidal activity against Listeria at 0.77 and 1.02 ppm, with increased mortality at
9 1.02 ppm (Van Loveren et al., 1988). These effects were associated with increased lung and
10 liver lesions in O3-exposed and Listeria infected animals as compared to Listeria infection
11 alone.
12 Fewer studies of viral infectivity have been conducted. Exposure for (15 days) to
13 0.5 ppm O3 during the course of murine influenza virus infection had no effect on pulmonary
14 virus tilers (Jakab and Hmieleski, 1988). A 2-day exposure for 3 h/day to 1.0 ppm
15 O3 followed by influenza virus infection had no effect on pulmonary virus titers, but did
16 show increased mortality, increased lung wet weight, and more severe nonsuppurative
17 pneumonitis and epithelial metaplasia and hyperplasia, with changes in lung function
18 consistent with that effect (Selgrade et al., 1988). Lung wet weight was also increased when
19 the mice were infected after the second day of exposure to 0.5 but not 0.25 ppm. When
20 infection followed other days of O3 exposure, up to 5 days, no such effects were found.
21 Typically, influenza virus infection causes a pneumonitis characterized by severe acute
22 lung damage that eventually resolves to a persistent alveolitis and changes in the parenchma
23 (focal interstitial pneumonia and collagen deposition). Jakab and Bassett (1990) investigated
24 the effect of long-term O3 exposure (24 h/day for 120 days to 0.5 ppm) on mice
25 administered influenza virus immediately before O3 exposure started. The authors observed
26 an increase in pulmonary fibrosis with the virus infection as compared to O3 exposure alone.
27 During the course of the viral infection, O3 exposure had no effect on pulmonary virus titers
28 and reduced the virus-induced acute lung injury. However, from Day 30 after infection,
29 increased numbers of AMs, lymphocytes, and PMNs were recovered from animals exposed
30 to virus plus O3 as compared to virus infection alone or O3 exposure alone. This increased
31 alveolitis correlated with increases in morphometrically determined lung damage and lung
December 1993 6-47 DRAFT-DO NOT QUOTE OR CITE
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1 hydroxyproline content, a biochemical marker indicative of pulmonary fibrosis. Ozone
2 exposure administered 10 days after viral infection enhanced lung hydroxyproline content at
3 Day 30, as compared to either virus infection or O3 exposure alone. Thus, O3 enhanced
4 postinfluenzal aveolitis and parenchymal changes. From these data, the authors speculated
5 that the mechanism for the postinfluenza lung damage may be related to O3 impairing the
6 repair process of the viral-induced acute lung injury.
7 In the studies reported to date, it is clear that the temporal relationships between
8 03 exposure and influenzal infection is important. This is not surprising because there are
9 several waves of different anti-viral defense mechanisms which might be affected differently
10 by O3. However, they have not been adequately studied for susceptibility to O3.
11 Apparently, O3 does not alter defenses responsible for clearing virus from the lungs, as
12 evidenced by the lack of effect of O3 on viral liters (Selgrade et al., 1988; Jakab and
13 Hmieleski, 1988). The interaction between virus and O3 on histological changes in lung
14 tissue can be damaging (Selgrade et al., 1988; Jakab and Bassett, 1990) or beneficial (Jakab
15 and Bassett, 1990), possibly depending on the time of observation relative to the stage of the
16 infectious process. The exact reasons are not known, but perhaps the induction of interferon
17 production by the virus plays a role. In noninfectious studies, Dziedzic and White (1987b)
18 observed that interferon induction mitigates O3-induced lung lesions and that anti-interferon
19 treatment exacaberates those lesions.
20
21 6.2.3.7 Summary
22 Exposure to O3 can result in alterations of all the defense mechanisms of the respiratory
23 tract, including mucociliary and alveolobronchiolar clearance, functional and biochemical
24 activities of AMs, immunologic competence, and susceptibility to respiratory infections.
25 Both structural (see Section 6.2.4), functional, and biochemical alterations in the mucociliary
26 escalator occur after O3 exposure. Mucociliary clearance is slowed in rabbits after a single
27 2-h exposure to 0.6 ppm, but repeated (up to 14 days) exposures have no such impact
28 (Schlesinger and Driscoll, 1987). Secretions of mucous components is affected by repeated
29 exposure (Phipps et al., 1986; McBride e( al., 1991). When lambs were exposed (1.0 ppm,
30 4 h/day, 5 days) shortly after birth, tracheal mucous components did not develop normally
31 (Mariassy et al., 1989, 1990). In contrast, alveolar clearance of rabbits after acute
December 1993 6-48 DRAFT-DO NOT QUOTE OR CITE
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1 O3 exposure (0.1 ppm, 2 h/day, 1 to 4 days) is accelerated (Driscoll et al., 1986). In the
2 same study, a 14-day exposure caused no effects, and a higher concentration (1.2 ppm)
3 slowed alveolar clearance. A similar pattern of slowed tracheobronchial clearance and
4 accelerated alveolar clearance occurs in rats (Phalen et al., 1980; Kenoyer et al., 1981).
5 A subchronic (6-week) exposure of rats to an urban pattern of 03 increased the retention of
6 asbestos fibers (Pinkerton et al, 1989).
7 Although AMs have numerous functions, one primary role is to clear the lung of
8 infections and non-infectious particles. Phagocytosis of bacteria, inert particles, and
9 antibody-coated RBCs is inhibited by acute exposure to O3. The lowest effective
10 concentration tested was 0.1 ppm O3 (2 h) in rabbits (Driscoll et al., 1987). If exposures are
11 repeated for several days, phagocytosis returns to control levels (Driscoll et al., 1987;
12 Gilmour et al., 1991; Canning et al., 1991). The ability of AMs to produce superoxide
13 anion radicals (important to bactericidal activity) is inhibited by acute exposure to O3,
14 especially in mice as compared to rats (Ryer-Powder et al., 1988; Costing et al., 1991a).
15 The effect is clearly evident after exposure for 3 h to 0.4 ppm, as observed by dysfunction in
16 AM phagocytosis and enhanced susceptibility to experimental respiratory infection (Gilmour
17 et al., 1993a, 1993b). Thus, the evidence indicates that the AM-dependent
18 alveolobronchiolar transport mechanisms are impaired, as are their phagocytic and
19 microbicidal activity, leading, to decreased resistance to respiratory infections.
20 The experimental database also shows the immunotoxic effects of O3 exposure. The
21 effects of O3 on the immune system are complex and not yet fully evaluated. It appears that
22 the T-cell dependent functions of the immune system are more affected that B-cell dependent
23 functions (U.S. Environmental Protection Agency, 1986; Fujimaki, 1989). Generally, there
24 is an early immunosuppressive effect that, with continued O3 exposure, results in either
25 return to normal responses or immunoenhancement. For example, in mice exposed for
26 28 days (20 h/day) to 0.3 to 0.7 ppm, there was an early (Days 1 and 2) depletion of cells in
27 the MLN, followed by MLN T-cell hyperplasia and increased blastogenic response to a
28 T-cell mitogen (Dziedzic and White, 1986a). Several investigations have found an initial
29 (Days 1 to 4) decrease in blastogenic response to T-cell mitogens in the MLN and spleen of
30 mice exposed for a few weeks to 0.7 or 0.8 ppm O3 that returned to control levels by the
31 end of the exposure (Dziedzic and White, 1986a; Gilmour and Jakab, 1991). There are also
December 1993 6-49 DRAFT-DO NOT QUOTE OR CITE
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1 changes in cell populations in lymphatic tissues. For example, T:B cell ratios in the MLN
2 increase when rats are exposed for 7 days to ^0.26 ppm O3 (Van Loveren et al., 1988).
3 Natural killer cells in the lung are affected under some circumstances. Van Loveren et al.
4 (1990) showed that a 1-week exposure to 0.2 or 0.4 ppm increased NK cell activity, but a
5 higher concentration (0.82 ppm) decreased it. Ozone also alters response to antigenic
6 stimulation. For example, antibody responses to a T-cell dependent antigen were suppressed
7 after a 56-day exposure of mice to 0.8 ppm O3, and a 14-day exposure to 0.5 ppm
8 O3 decreased the antiviral antibody response following influenza virus infection (Jakab and
9 Hmieleski, 1988). The temporal relationship between O3 exposure and antigenic stimulation
10 is important. When O3 exposure preceeded Listeria infection, there were no effects on
11 delayed-type hypersensitivity or splenic lymphoproliferative responses; when O3 exposure
12 was during or after Listeria infection was initiated, these immune responses were suppressed
13 (Van Loveren et al., 1988). With experimental viral infections, O3 exposure decreases the
14 T-lymphocyte responses and the antiviral antibody response (Jakab and Hmieleski, 1988); the
15 latter impairment may pave the way for lowered resistance to reinfection.
16 In addition to an immunotoxic effect on the pulmonary immunity, O3 exposure also can
17 affect systemic immunity. Although these systemic immunotoxic effects occur at
18 approximately twice the O3 exposure concentrations as those observed for pulmonary
19 immunotoxicity (with the exception of a study by Aranyi et al. [1983] at 0.1 ppm O3), the
20 observations are important because they show that the effects of O$ exposure on host
21 resistance is not limited to the lung alone, but may increase susceptibility to systemic
22 infections as well as pulmonary infections. However, Selgrade et al. (1990) found no effects
23 on selected systemic immune functions in rats exposed up to 78 weeks to an urban pattern
24 of O3.
25 Numerous studies have confirmed that acute or short-term exposure to O3 decreases
26 lung bactericidal activity and increases susceptibility to respiratory bacterial infections. The
27 lowest exposure showing such effects was 0.08 ppm (3 h) in the mouse streptococcal model
28 (Coffin et al., 1967; Coffin and Gardner; 1972; Miller et al., 1978). Further research has
29 indicated that changes in antibacterial defenses are dependent not only on exposure regimens,
30 but also on species and strain of animal, species of bacteria, and age of animal (young mice
31 more susceptible) (Gilmour et al., 1991, 1993a,b; Gilmour and Selgrade, 1994).
December 1993 6-50 DRAFT-DO NOT QUOTE OR CITE
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1 Furthermore, increasing the duration of an exposure to 0.1 ppm O3 from a few hours to
2 three weeks either causes no effect or does not enhance the streptococcal-induced mortality
3 observed after acute exposure (Graham et al., 1987; Aranyi et al., 1983). In general, the
4 effect of O3 exposure on antibacterial host defenses appears to be concentration and time
5 dependent. Acute exposures result in an impairment of host defenses, whereas the defense
6 parameters become reestablished with more prolonged exposures.
7 Effects of O3 on the course of viral infections are more complex and highly dependent
8 on the temporal relationship between O3 exposure and viral infection. For example, Belgrade
9 et al. (1988) only found increases in mortality and lung wet weight in mice infected with
10 influenza only after the second day of O3 exposure (1 ppm, 3h/day). Jakab and Bassett
11 (1990) found no detrimental effect of a 120-day exposure to 0.5 ppm O3 on acute lung injury
12 from influenza virus administered immediately before O3 exposure started. However,
13 O3 enhanced postinfluenzal alveolitis and lung parenchymal changes. Because O3 did not
14 affect lung influenza viral liters in any if these studies, it is unlikely that O3 has an impact on
15 anti-viral clearance mechanisms.
16 Ozone-induced susceptibility to experimental respiratory infections has been correlated
17 with the immunotoxic effects of O3 by the observation that O3 exposure increases the
18 severity of Listeria infection while concurrently suppressing the antigen-specific immune
19 responses (Van Loveren et al., 1988).
20
21 6.2.4 Morphological Effects
22 6.2.4.1 Introduction
23 All mammalian species studied react to inhaled concentrations of 65 less than 1.0 ppm
24 in a generally similar manner, with species variation in morphological responses depending
25 upon the distribution of sensitive cells and the type of junction between conducting and
26 exchange areas of the lung (U.S. Environmental Protection Agency, 1986). The cells most
27 damaged by O3 are the ciliated epithelial cells in airways and Type 1 cells in gas exchange
28 areas. Both of these cell types have very large surface areas exposed to inhaled gases
29 relative to their cell volume. The many factors that influence the distribution of inhaled
30 O3 within the respiratory system (see Chapter 8) result in some of the largest effective doses
31 to the epithelial cells lining the nose and to epithelial cells located at the junction of the
December 1993 6-51 DRAFT-DO NOT QUOTE OR CITE
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1 conducting and exchange areas, the centriacinar region (CAR), in lungs. The 1986 criteria
2 document did not contain studies of morphological effects of 03 on the nose. There are
3 species differences both in the basic structure of the CAR and in the epithelial cells that line
4 the CAR (Tyler, 1983; Plopper, 1983). The CAR of humans, other primates, dogs, cats,
5 and a few other domesticated species consists of the last conducting airway, the terminal
6 bronchiole (TB), several generations of respiratory bronchioles (RBs), alveoli that open
7 directly into RB lumens, and alveolar ducts (ADs) that branch from RBs (Figure 6-2).
8 In lungs from many other mammals, including those most commonly used for inhalation
9 toxicology (i.e., rats, mice, guinea pigs, and rabbits), RBs are poorly developed or absent
10 and the CAR consists of TBs that open directly into ADs.
11
Human and Nonhuman
Primates
Rats and Most
Other Rodents
Nonrespiratory
Bronchioles
Figure 6-2. Schematic representation of intrapulmonary conducting airways and acini
from animals with respiratory bronchioles (human and nonhuman
primates) and without respiratory bronchioles (rats and most rodents). For
simplicity, several generations of bronchi and nonrespiratory bronchioles
are not depicted.
Source: Redrawn from Weibel (1963) using information from Tyler and Julian (1991).
December 1993
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1 Epithelial degenerative changes in TBs and alveoli occur early, 2 to 4 h, in an
2 O3 exposure (Stephens et al., 1974a; Castleman et al., 1980). Depending on the dose to
3 individual ciliated cells, they may lose cilia; undergo degenerative changes; or become
4 necrotic and be sloughed into the lumen, leaving bare basement membrane until other cells
5 replace them (Stephens et al., 1974a; Castleman et al., 1980). In the TB of the CAR,
6 sloughed ciliated cells are replaced by nonciliated bronchiolar cells, which may become
7 hyperplastic following longer exposures. Although these changes in TB epithelial cells can
8 be readily studied by light microscopy (LM) or transmission electron microscopy (TEM), the
9 surface views provided by scanning electron microscopy (SEM) provide a more
10 comprehensive understanding of the three-dimensional aspects of CAR changes (Schwartz
11 et al., 1976; Castleman et al., 1980).
12 Changes in mucous secreting cells of conducting airways were considered minor in the
13 1986 criteria document (U.S. Environmental protection Agency, 1986). Schwartz et al.
14 (1976) did not find changes in mucous cells suggesting damage to cell organelles in rats
15 exposed to 0.2, 0.5, or 0.8 ppm for 7 days continuously or 8 h/day intermittently. Mellick
16 et al. (1977) reported similar negative findings in mucous cells of monkeys following
17 exposure to 0.5 or 0.8 ppm 8 h/day for 7 days. Wilson et al. (1984) reported more
18 prominent small-mucous-granule (SMG) cells in the tracheas from monkeys exposed to
19 0.64 ppm continuously for 3 or 7 days. They speculated that these SMG cells may be
20 related to repair processes.
21 Following O3 exposure, Type 1 cells lining alveoli in the CAR, especially those
22 opening into either RBs or ADs, may undergo vacuolization, fragmentation, or necrosis and
23 be sloughed from the surface, leaving bare basement membrane (Stephens et al., 1974a).
24 Although these degenerative changes can be seen by careful LM of thin plastic sections, they
25 are more reliably identified and the amount of damage can be estimated morphometrically
26 using TEM (Barry et al., 1983; Crapo et al., 1984; Fujinaka et al., 1985). In alveoli, the
27 bare basement membrane that follows O3 exposure is recovered by Type 2 alveolar epithelial
28 cells. Some Type 2 cells differentiate into Type 1 cells (Evans et al., 1975), but the
29 epithelium remains thickened (Barry et al., 1983; Crapo et al., 1984).
30 Epithelial replacement in both TBs and alveoli can be followed and the amount can be
31 estimated using radiolabeled thymidine and autoradiography (Evans et al., 1976a,b).
December 1993 6-53 DRAFT-DO NOT QUOTE OR CITE
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1 Although bare basement membrane in alveoli is usually recovered by multiplication of Type
2 2 cells, in chronic exposures, bronchiolar cells, especially nonciliated bronchiolar cells, may
3 cover part of the basement membrane formerly occupied by Type 1 or 2 cells. This process,
4 termed bronchiolization (Nettesheim and Szakal, 1972), results in remodeling of CAR
5 airways with the formation of new RBs. New RBs are readily identified by SEM or by LM
6 in lungs from species in which RBs are normally absent or are poorly developed (Boorman
7 et al., 1980; Moore and Schwartz, 1981). In animals whose lungs normally have well-
8 developed RBs, the extent of remodeling can be estimated using LM morphometry (Fujinaka
9 etal., 1985).
10 The above epithelial changes are accompanied by an inflammatory response in the CAR
11 characterized by increased numbers of PMNs in early stages, by increased numbers of AMs
12 in lumens and in tissue at later stages, by hyperemia and interstitial edema, and by a
13 fibrinous exudate (Stephens et al., 1974a; Schwartz et al., 1976; Boorman et al., 1980;
14 Castleman et al., 1980; Fujinaka et al., 1985). As exposure continues, alveolar septa in the
15 CAR thicken due to increased matrix, basement membrane, collagen, fibroblasts and other
16 cells, as well as by thickened alveolar epithelium (Boorman et al., 1980; Barry et al., 1983;
17 Crapo et al., 1984; Fujinaka et al., 1985).
18 Two morphometric studies cited in the 1986 criteria document reported thickened walls
19 in pulmonary arteries (P'an et al., 1972) and arterioles (Fujinaka et al., 1985).
20 Relative susceptibility to morphological change due to nutritional factors, age at start of
21 exposure, and pneumonectomy were also considered in the previous criteria document (U.S.
22 Environmental Protection Agency, 1986). Some investigators reported that rats on diets that
23 are deficient or have basal levels of vitamin E tended to develop more severe lesions at lower
24 concentrations (Plopper et al., 1979; Chow et al., 1981). Other studies concluded that the
25 extent of CAR lesions was independent of the level of vitamin E in lung tissue (Stephens
26 et al., 1983). The effects of age at start of exposure was also studied. Stephens et al.
27 (1978) reported that rats were not susceptible until weaning at 21 days and that CAR lesions
28 increased until they reached a plateau at 35 days of age. Barry et al. (1983) used TEM
29 morphometry to study the CAR lesions in lungs from 1-day-old rats exposed 12 h/day for
30 6 weeks to 0.25 ppm O3. They reported CAR alveoli had more Type 1 and 2 epithelial cells
31 and more AMs. The Type 1 cells were smaller in volume, covered less surface, and were
December 1993 6-54 DRAFT-DO NOT QUOTE OR CITE
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1 thicker. They were aware of the results of Stephens et al. (1978) and speculated that the
2 changes they described may have primarily occurred during the last 3 weeks of exposure.
3 Boatman et al. (1983) did not find effects of O3 on the lung growth that follows
4 pneumonectomy.
5 Several studies included both an exposure and a postexposure period during which the
6 animals breathed air without O3. Plopper et al. (1978) reported that CAR epithelial cells
7 returned to normal appearance 6 days after a 72-h exposure. Incomplete resolution was
8 reported 7 days after a 50-h exposure of monkeys (Castleman et al., 1980), 10 days after a
9 20-day exposure of mice (Ibrahim et al., 1980), and 62 days after a 180-day exposure of rats
10 (Moore and Schwartz, 1981).
11 The previous criteria document (U.S. Environmental Protection Agency, 1986)
12 comprehensively evaluated several citations reporting emphysema following O3 exposure
13 using current definitions of human emphysema (Snider et al., 1985). The lesions described
14 in those earlier publications did not meet the current criteria for emphysema of the type seen
15 in human lungs.
16
17 6.2.4.2 Sites Affected
18 The sites in the respiratory system that are affected will be discussed in the sequence
19 followed by inhaled air. The upper or extrapulmonary conducting airways (also referred to
20 as the nasopharyngeal region) include the nasal cavity, pharynx, larynx, and trachea.
21 Intrapulmonary conducting airways (also referred to as the bronchial region) include the
22 bronchi and nonterminal bronchioles. A summary of available information on these sites in
23 the respiratory system is in Table 6-6, Effects of Ozone on Conducting Airways. Summaries
24 of the information available concerning effects on the CAR, the gas exchange region (also
25 referred to as the pulmonary region), and other pulmonary structures are divided into effects
26 of short-term (< 2 weeks) exposures in Table 6-7 and effects of long-term exposures in
27 Table 6-8.
28
29 Nasal Cavity and Nasopharynx
30 The nasal cavity "conditions" inhaled air and in that process "scrubs" some reactive
31 pollutants from the inhaled air, thereby reducing the concentration to which other portions of
December 1993 6-55 DRAFT-DO NOT QUOTE OR CITE
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TABLE 6-6 (cont'd). EFFECTS OF OZONE ON CONDUCTING AIRWAYS8
oo
Ozone
Concentration
ppm ftg/m
0.96 1,882
0.96 1,882
1.0 1,960
1.0 1,960
1.0 1,960
Exposure
Duration
8 h/night,
7 nights/week for
3 or
60 nights
8h
4 h/day for
5 days
(Examined al
2 weeks)
4 h/day for
5 days
(Examined at
2 weeks)
96h
(In vitro tracheal
explants)
Species, Sex
(Strain)
Rat, M
(S-D)
234-263 g
Macaco mulata, M
2-8.5 years old
2.1-6.3 kg
Sheep
New-born
Sheep
New-born
Rat, M
(S-D)
250-270 g
Observed Effect(s)
LM morphometry, histochemistry, and autoradiography, SEM and TEM morphometry of tracheal
epithelium. Neither 3 nor 60 days exposure altered the cell density of ciliated, serous, basal, brush,
migratory, or unidentified cells in tracheal epithelium. 3 days: Damage to cilia and ciliated cells,
including necrosis. Thymidine labeling index increased. Serous cell histochemistry unchanged.
60 days: Less evidence of injury than at 3 days, but more damaged ciliated cells than in controls.
Complete recovery of the epithelial changes by 42 days PE.
LM and TEM morphometry of trachea, bronchi, and RBs. Also see Section 6.2.3 . Increased
necrotic cells in trachea and RBs at 1 h PE and in bronchi at 12 and 24 h PE. Decreased ciliated
and basal cells in bronchi at 1, 12, and 24 h PE. Basal cells in bronchi also decreased at 72 and
168 h PE. Nonciliated bronchiolar cells in RBs increased only at 24 h PE. In bronchi, smooth
muscle increased and amorphous matrix decreased at 24, 72, and 168 h PE. In RBs, smooth muscle
increased at 24 h, fibroblasts increased at 24 and 72 h, and amorphous matrix increased at 12 h PE.
LM morphometry of tracheal epithelium. Also see Section 6.2.3. Percentage of ciliated and
mucous cells remained at newborn levels, rather than ciliated cell percent increasing and mucous cell
percent decreasing as in control lambs.
LM morphometry of mucosubstances in tracheal epithelium. No evidence of damage or
inflammatory changes. Decreased epithelial cell density, decreased ciliated and basal cells. Lectin-
detectable intraepithelial mucosubstances did not undergo the maturation changes seen in control
lambs.
Tracheal organ cultures exposed in vitro. Filtered air + 03 resulted in extensive damage to cilia,
and intermediate cells were seen. Cultures exposed to 95% O2 + Oj had stratified thickened
epithelium with metaplastic cells in a middle zone and no ciliated celts at the surface.
Reference
Nikula et al. (1988a)
Hyde et al. (1992)
Mariassy et al. (1990)
Mariassy et al. (1989)
Nikula and Wilson (1990)
See glossary of terms and symbols for common abbreviations and acronyms.
Age or body weight at start of exposure.
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TABLE 6-8 (cont'd). EFFECTS OF OZONE ON LUNG STRUCTURE: LONG-TERM EXPOSURES (>2 WEEKS)8
1
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8
Ozone
Concentration
Exposure
ppm fig/m Duration
0.15 294 8 h/day for
0.30 588 6 or 90 days
0.25 490 12 h/day for
6 weeks
0.25 490 8 h/day,
7 days/week,
"daily" for
18 mo
or "seasonal"
Oj odd months,
filtered air even
months for
18 mo
(9 mo of 03)
0.25 490 8 h/night,
7 nights/week,
"daily" for
18 mo
or "seasonal"
03 odd months,
filtered air even
months for
18 mo
0.3 588 7 h/day,
5 days/week for
6 weeks
Species, Sex
(Strain)
Macaco radiata, F, M
2-6 years old
Rat, M
(F344)
1 day or
6 weeks old
Macaca fasciciilaris , M
6 mo old
Rat, M
(S-D)
22 days old
Mouse, M
(Swiss-Webster)
Newborn
Observed Effect(s)
TEM, SEM, and LM morphometry. First generation RBs had epithelial hyperplasia and alveoli
opening into these RBs had increased AMs. RB epithelium thickened, but no difference due to either
exposure time or concentration. RB interstitium was thickened in all exposed monkeys, but both
cellular and acellular compartments were only individually thickened after 90-days exposure to 0.3
ppm. No differences due to age or gender. No evidence of epithelial cell necrosis nor of inflammatory
cell infiltration other than the increased AMs.
TEM morphometry of terminal bronchioles. Luminal surface area covered by cilia decreased, as did
the luminal surface of Clara (nonciliated bronchiolar) cells. Number of brush cells decreased. No
differences due to age.
LM morphometry. Also see Sections 6.2.5 and 6.2.6. Low grade respiratory bronchiolitis in both
exposed groups. Compared with controls, both groups of exposed monkeys had increased Vv of tissue
other than parenchyma and Vv of RBs and their lumens. Both Vv and V of RB wall increased in the
"daily" group, but not in the "seasonal" group. The only significant morphometric difference between
the two exposed groups was the Vv of cells, mostly AMs, free in airspace lumens. This difference,
and the difference in significance of the RB wall thickness, was presumed due to the difference in time
after the last 03 exposure and necropsy. Daily group necropsied the day after the last exposure,
whereas seasonal group necropsied after a month of filtered air. Seasonal group had a similar amount
of morphological changes as daily group.
LM morphology and morphometry. Monkey data from Tyler et al. 1988 (above) compared with rats
exposed to a similar regimen. Rats: Estimated the extent of centriacinar remodeling by counting the
number of junctions of bronchioles with alveolar ducts per area of lung section (B/A J/crn). At the
end of the exposure, both exposed groups had more B/A J/cm than filtered air controls. Recovery by
30 days PE. No difference between the two exposed groups, even though the daily group was exposed
twice as many days as the seasonal group.
LM morphometry of histochemically identified Type 2 cells. Type 2 cells tended to be larger (longer
linear intercepts), and the number per microscope field tended to be greater, but the p values were
> 0.05 in final data in which the images were edited electronically. However, these values were
significant (p < 0.05) in unedited data. Exposed mice had larger (p < 0.05) body weights at both
3 and 6 weeks.
Reference
Harkema et al. (1993c)
Barry et al (1988)
Tyler et al. (1988)
Tyler et al. (1991a)
Sherwin and Richters
(1985)
-------
TABLE 6-8 (cont'd). EFFECTS OF OZONE ON LUNG STRUCTURE: LONG-TERM EXPOSURES (>2 WEEKS)8
Ozone
Concentration
M- ppm
/ig/m
Exposure
Duration
Species, Sex
(Strain)
Age
Observed Effect(s)
Reference
0.35 686 4.5 h/day, Mouse, M
5 days/week for (Swiss-Webster)
4 weeks, 32 g
380 mmHg
(5,400 m)
or sea level
Automated LM morphometry of stainable elastin in alveolar walls. Simulated high altitude (5400 m) with
03 (SHA-X) or without 03 (SHA-C) resulted in larger lung volumes than sea level controls (SL-C), but
not different from each other. Unlike most studies, sea level 03 exposed mice had the smallest lung
volumes. Alveolar wall areas, after adjustment to SL-C lung volumes, were increased only in the SHA-X
group. Alveolar wall elastin area, adjusted to the SL-C lung volumes, increased in both high altitude
groups compared to SL-C and were also different from each other with the largest amount of elastin area
in the SHA-X group. However, if the elastin areas were not adjusted for differences in lung volumes,
there were no differences between the groups.
Damji and Sherwin
(1989)
0.4
0.64
0.5
0.5
784 8 h/day,
1,254 7 days/week for
90 days
980 6 h/day,
6 days/week for
2, 3, 5, and
12 mo
980 20 h/day,
7 days/week for
52 weeks
Macaco radiata, M
5-8 years old
Rat, M
(Wistar)
100 g
Rat
(F344)
LM and TEM morphometry with emphasis on RBs. Respiratory bronchiolitis and peribronchiolar
inflammation. RB walls thicker with smaller lumens. Increased wall thickness due both to thicker
epithelium, significant only at 0.64 ppm, and interstitial components. Epithelial changes in both
03 groups include increased nonciliated bronchiolar epithelial cells and decreased Type 1 cells. Interstitial
changes in both 03 groups included increased smooth muscle cells, mast cells, and fibers. Components
increased at 0.64 ppm, but not at 0.4 ppm, included interstitial AMs, PMNs, and amorphous ground
substance.
LM, TEM, and LM morphometry of collagen fibers. Bronchitis, peribronchitis, CAR remodeling, and
increased stainable collagen in bronchioles. Rats apparently had intercurrent respiratory disease, as 10 of
44 exposed rats died of pneumonia or pulmonary edema. In addition, it appears that only one set of
12 rats maintained in room air served as controls for all exposed groups, even though controls and
exposed would be of significantly different age and size.
LM histopathology. Also see Section 6.2 .2. 6-mo exposure: inflammation, mononuclear cells, and
fibroblasts in alveolar duct walls and walls of adjacent CAR alveoli. TB not involved. 12-mo exposure:
similar to 6 mo with possibly a slight increase in AMs, some increased thickening of centnacinar alveolar
duct and alveolar walls, and a few foci of bronchiolization (CAR remodeling). 12-mo exposure + 6 mo
PE: slight dilation of alveolar ducts, minimal inflammatory reaction, slight thickening of alveolar duct and
CAR alveolar walls, and a few foci of bronchiolization.
Moffatt et al. (1987)
Hiroshima et al. (1989)
Gross and White
(1987)
0.5 980 Continuous for Mouse, F
120 days (Swiss N)
20-23 g
LM morphometry and histopathology. Also see Section 6.2.3. More tissue, primarily inflammatory cells, Jakab and Bassett
at Day 9. Little change from Days 10 to 120. Thickened airway walls with increased collagen. Increased (1990)
collagen in alveolar walls along the alveolar ducts.
0.64 1,254 8 h/night, Rat, M
0.96 1,881 7 nights/week for (S-D)
42 nights, 28 days old
"Pair" fed
LM morphometry and SEM. Also see Sections 6.2.5 and 6.2.6. End of 42-night exposure: No Tyler et al. (1987)
difference in BW, hemoglobin, or total serum proteins. At 0.96, but not 0.64 ppm, larger fixed- and
saline-filed lung volumes, lung volume/BW ratios, and volumes of parenchyma. At both ppm, increased
Vv and V of RB and RB walls and their ratios to BW. By SEM, remodeling of CAR airways with the
formation of RBs and thickened CAR septa at both ppm. After 42 days PE: Fixed lung volume at 0.96
ppm increased. Vv and V of RB walls and ratio to BW increased at 0.96 ppm. Ratios of volumes to BW
for parenchyma, alveoli, total RB, and RB wall increased at 0.96 ppm. SEM revealed persistence of CAR
remodeling and thickened septa.
0.64 1,254 8 h/day for Macaco faciscularis, M LM morphometry of CAR airway remodeling. Both Vv and V of RBs, and their walls and lumens,
12 mo 6-7 mo old increased at end of exposure and 6 mo PE. RB internal diameters were smaller at exposure end, but not
at 6 mo PE. Vv free cells, mostly AMs, increased only at exposure end. No differences in BW or fixed
lung volumes.
Tyler et al. (1991b)
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December 1993
6-65 DRAFT-DO NOT QUOTE OR CITE
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1 the respiratory system are exposed (Yokoyama and Frank, 1972; Miller et al., 1979).
2 Although this scrubbing process is protective of other portions of the respiratory system, it
3 results in a large dose of pollutant to the cells and tissues that line the nasal cavity.
4 There is a large range of variation in the structure of the nasal cavity among the
5 animals used for inhalation toxicology and in the structure of the nasal cavity between those
6 animals and humans (Schreider and Raabe, 1981). These investigators found a striking
7 similarity between the nasopharyngeal cavities of monkey and humans. They proposed that,
8 with appropriate scaling, the monkey could serve as a model for aerosol and gas deposition
9 in the nasopharyngeal region of humans. Thus, the studies on monkeys by Harkema et al.
10 (1987) provide useful information for extrapolation to humans as well as information
11 concerning cellular responses in monkeys.
12 Harkema et al. (1987) exposed bonnet monkeys to 0.15 or 0.30 ppm O3 8 h/day for
13 6 or 90 days. They sampled four regions of the nasal cavity and nasopharynx. Changes
14 were limited to the respiratory and transitional epithelium in the two most rostral (anterior)
15 of the four sections. No changes were reported in the caudal (posterior) two sections, the
16 last of which included the nasopharynx. The respiratory epithelium of the rostral nasal
17 cavity had both qualitative and quantitative changes. Quantitative changes included decreased
18 density of ciliated cells characterized qualitatively by multifocal loss of cilia, necrotic ciliated
19 cells, ciliated cells with attenuated cilia, and others with only microvillar surface. The
20 respiratory epithelium also had an increased density of SMG cells, presumably related to
21 repair processes. Monkeys exposed to 0.30 ppm for 90 days also had increased abnormal
22 cells with intracytoplasmic lumens containing both cilia and microvilli. Qualitative changes
23 were also seen in mucous (goblet) cells, which appeared to have fewer secretory granules
24 and dilated endoplasmic cisternae. Ozone exposure resulted in more nonciliated cells with
25 secretory granules and with dilated cisternae of the endoplasmic reticulum. Like the
26 respiratory epithelium, the transitional epithelium had an increased density of SMG cells.
27 In both epithelia, inflammatory cells were only increased in the monkeys exposed to 0.15 for
28 6 days. Most of the morphometric changes in the respiratory, but not the transitional,
29 epithelium were as large after 6 days of exposure to 0.15 as after 90 days of exposure to
30 either 0.15 or 0.30 ppm. The histochemistry and cytochemistry of the nasal epithelia from
31 these monkeys were studied by Dimitriadis (1992). He reported changes in the intraepithelial
December 1993 6-66 DRAFT-DO NOT QUOTE OR CITE
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1 mucosubstances and the presence of mucous cells with dilated cisternae in the granular
2 endoplasmic reticulum.
3 Acute changes in nasal epithelia from rats exposed to O3 concentrations of 0.12 to
4 1.0 ppm for 6 h to 7 days have been extensively studied (Table 6-6). In general, short-term
5 exposure to ^0.2 ppm results in either no changes detectable by LM or in mild hyperplasia.
6 Higher concentrations for up to 7 days can result in damaged cilia, hyperplasia, and
7 increased stored intraepithelial mucosubstances. Several studies document the hyperplasia
8 using morphometry or DNA synthesis and document the stored mucosubstance by
9 histochemistry and morphometry. In one study, the increased stored intraepithelial
10 mucosubstances reached their largest quantity 7 days postexposure (Harkema et al., 1989).
11 Details of individual studies follow.
12 Exposure to 0.12, 0.8, or 1.5 ppm for 6 h followed by postexposure periods up to
13 66 h resulted in inflammatory changes characterized by increased PMNs, but without LM
14 evidence of necrosis, ciliary loss, or hyperplasia (Hotchkiss et al., 1989a). Hotchkiss and
15 Harkema (1992) reported similar LM findings in rats exposed to 0.8 ppm for 6 h. They also
16 reported increased DNA synthesis by bromodeoxyuridine (BrdU) uptake in nasal nonciliated
17 transitional epithelium. Exposure to 0.8 ppm 6 h/day for 3 or 7 days, or for 3 days with
18 4 days postexposure, resulted in hyperplasia of the nasal nonciliated cuboidal (transitional)
19 epithelium with increased intraepithelial mucosubstances without significant changes in
20 histochemical staining characteristics (Hotchkiss et al., 1991). In that study, no changes
21 were reported for rats exposed for 3 days and examined 18 h postexposure.
22 Reuzel et al. (1990) exposed rats to 0.2, 0.4, or 0.8 ppm O3 22 h/day for 3 days.
23 They did not report changes in rats exposed to 0.2 ppm, but those exposed to 0.4 or 0.8 ppm
24 had loss of cilia and disarrangement of the epithelium with hyperplasia and metaplasia. Cell
25 proliferation, as measured by radiolabeled thymidine, was increased at the two higher
26 concentrations. The influence of O3 C XT on epithelial cell proliferation in the nasal anterior
27 maxilloturbinates was measured by BrdU uptake (Henderson et al., 1993). Rats were
28 exposed to 0.12, 0.24, and 0.48 ppm for 3, 6, 12, and 24 h, resulting in 6 C XT products.
29 Exposure to 0.12 ppm or C XT's of 0.72 or 1.44 ppnvh did not cause effects. For a given
30 CxT between 2.88 and 11.52 ppnvh, the increased DNA synthesis was similar; the response
31 did not linearly increase with increasing CxT's. Generally, above 0.12 ppm there was a
December 1993 6-67 DRAFT-DO NOT QUOTE OR CITE
-------
1 linear increase with increasing C, not T. Thus, apparently exposure duration was responsible
2 for the lack of CxT linearity. Johnson et al. (1990) also used BrdU to study DNA synthesis
3 in rats exposed to 0.12, 0.27, or 0.8 ppm O3 6 h/day for 3 or 7 days and examined 3 or
4 7 days postexposure. Rats exposed to 0.8 ppm, but not to the lower concentrations, had
5 increased DNA synthesis in the nonciliated cuboidal (transitional) epithelium at 3 and 7 days
6 and increased numbers of labeled cells in the ciliated respiratory epithelium and the olfactory
7 epithelium only at 3 days. No changes were found in squamous epithelia except a decrease
8 in labeled cells 7 days postexposnre to 0.8 ppm O3. They reported no LM changes in the
9 ciliated respiratory, olfactory, or squamous epithelia, but hyperplasia occurred in the
10 cuboidal transitional epithelium.
11 Epithelial mucosubstances were studied in rats exposed to 0.12 or 0.8 ppm O3 6 h/day
12 for 7 days or 7 days postexposure (Harkema et al., 1989). They reported no LM pathology
13 in the nasal or nasopharyngeal airways from rats exposed to 0.12 ppm, with the exception of
14 an increase in secretory cells in ciliated epithelium. Rats exposed to 0.8 ppm had attenuation'
15 of cilia in the lateral walls of the nasopharynx; 7 days postexposure, an increase in stored
16 intraepithelial mucosubstances was observed. The 0.8-ppm group also had hyperplasia of the
17 nonciliated transitional epithelium accompanied by an increase in PMNs in the lamina
18 propria. Seven days postexposure, rats in the 0.8-ppm exposure group had more stored
19 intraepithelial mucosubstances in some areas of ciliated respiratory and nonciliated
20 transitional epithelia.
21 In rats exposed to 0.12, 0.25, or 0.5 ppm 20 h/day for 2 years, Smiler et al. (1988)
22 reported hyperplasia, especially of mucous cells, in the respiratory epithelium over the rostral
23 portion of the nasoturbinate of rats in the 0.25- and 0.5-ppm groups. The respiratory
24 epithelium lining other parts of the nasal cavity were less affected, and no changes were
25 found in the squamous and olfactory epithelia.
26 In a preliminary report, Harkema et al. (1993b) reported no changes in the amount of
27 mucosubstances in conducting airways, including the nasal cavity, of rats exposed to
28 0.12 ppm for up to 20 mo. Stored intraepithelial mucosubstances were increased in the nasal
29 cavity of rats exposed to 0.5 or 1.0 ppm. (Note: More complete information from this
30 study should be available for the final O3 document.)
31
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1 Larynx
2 Leonard et al. (1991) reported disruption and thickening of the stratified squamous
3 epithelium over the vocal folds of bonnet monkeys (Macaca radiata) exposed 8 h/day for
4 7 days. The basement membrane appeared undulating rather than smooth. At 7 days
5 postexposure, the epithelium appeared thickened, but otherwise normal. The
6 O3 concentration to which the monkeys were exposed is not clear because different
7 concentrations appear in the summary and text sections of the publication. However, these
8 larynges were from bonnet monkeys that were also studied by Harkema et al. (1987) and
9 Dimitriadis (1992), and therefore were most likely exposed to 0.15 ppm.
10
11 Trachea and Bronchi
12 Several investigators studied effects of 0.96 or 1.0 ppm O3 on the tracheas of monkeys,
13 rats, and sheep during and after very brief, short-term, or long-term exposures. Hyde et al.
14 (1992) studied the trachea, bronchi, and RBs of rhesus monkeys exposed to 0.96 ppm for
15 8 h and examined them at 1, 12, 24, 72, and 168 h postexposure. Although the primary
16 objective of the study concerned inflammation (see Section 6.2.2), the study also provided
17 much new morphometric information concerning reactions of tracheal, bronchial, and RB
18 epithelia and their interstitiums to O3. Both epithelial and interstitial data were determined as
19 volume per surface area of epithelial basal lamina (Vg) . At 1 h postexposure, the major
20 change in the tracheal and RB epithelia was an increase in necrotic cells, whereas in the
21 bronchial epithelium, there were fewer ciliated and basal cells. There were no other changes
22 in tracheal epithelial cell Vs at any of the postexposure times examined. At 12 and
23 24 h postexposure, the Vs of necrotic cells was increased in bronchi, but not in the trachea or
24 RBs. The Vs of ciliated and basal cells was smaller in the bronchial epithelium, but not in
25 the trachea. Basal cells in bronchi were also increased at 72 and 168 h postexposure.
26 Respiratory bronchioles had smaller Vs of Type 1 alveolar epithelial cells at all times except
27 1 h postexposure. In RBs, nonciliated bronchiolar cells were only increased at
28 24 h postexposure. Epithelial cell DNA synthesis was studied in the filtered air controls and
29 at 1 and 12 h postexposure by radiolabeled thymidine incorporation. The only increase was
30 observed in the bronchial epithelium at 12 h postexposure. Changes in the interstitial
31 components of the trachea were minimal, with a decrease hi the amorphous matrix at
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1 24 h postexposure. Bronchi had increased Vg of smooth muscle and decreased amorphous
2 matrix at 24, 72, and 168 h postexposure. Collagen fibers in the bronchial interstitium were
3 decreased at 168 h. In RBs, the arithmetic mean thickness was increased at 12 and 24 h, but
4 not at other times. In RBs, smooth muscle Vs was increased at 24 h, Vs of fibroblasts was
5 increased at 24 and 72 h, and Vs of the amorphous matrix was increased at 12 h
6 postexposure.
7 Nikula et al. (1988a) exposed rats to 0.96 ppm O3 8 h/night for 3 or 60 nights or for
8 60 nights followed by 7 or 42 days postexposure and examined the tracheas using LM, TEM
9 morphometry, SEM, LM mucosubstance histochemistry, and DNA synthesis by radiolabeled
10 thymidine incorporation. Ciliated cells with short or damaged cilia were increased after
11 3 and 60 nights of exposure; cells with short cilia were increased after 60 nights of exposure
12 and 7 days postexposure. Intermediate cells, presumed to be immature ciliated cells, were
13 increased only after 3 nights of exposure. However, the numeric density of total ciliated
14 cells, basal cells, total serous cells, brush cells, and total migratory cells was not different
15 from controls. There were no changes in LM histochemistry for mucosubstances at any
16 time. The only increase in thymidine labeling occurred after 3 days of exposure. Recovery
17 was complete 42 days after 60 nights of exposure.
18 Mariassy et al. (1989, 1990) exposed newborn lambs to 1.0 ppm O3 4 h/day for 5 days
19 and studied controls at birth and controls and exposed lambs at 2 weeks of age. Tracheal
20 mucous velocity was decreased at 2 weeks and at several additional postexposure times (see
21 Section 6.2.3). In control lambs, the percent of ciliated cells increased and mucous cells
22 decreased in the tracheas from birth to 2 weeks of age. This normal change in cell
23 populations did not occur in the exposed lamb tracheas. In the more detailed morphological
24 study (Mariassy et al., 1989), epithelial cell density (cells/mm), rather than only differential
25 cell counts, was reported. In tracheas from control lambs, the density of mucous cells
26 decreased from birth to 2 weeks of age. Ozone exposure resulted in decreased total
27 epithelial cell density, with decreased densities of ciliated and basal cells. Mucous cell
28 density remained at newborn levels. Ozone exposure also prevented the normal maturational
29 changes of lectin-detectable, but not of tinctorially stained, mucosubstances.
30 The most comprehensive study of the effects of long-term O3 exposure on conducting
31 airways of rats is that by Plopper et al. (1993). They used LM morphometry and tinctorial
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1 histochemistry to study conducting airways from the trachea to centriacinar alveoli following
2 two "short" and one "long" pathway by airway dissection of fixed lungs. In rats exposed to
3 0.12, 0.5, or 1.0 ppm O3 6 h/day, 5 days/week for 20 mo, they did not find differences due
4 to O3 exposure in tracheal or bronchial epithelial thickness, cell populations, or stored
5 glycoconjugates. However, they did find a dose-dependent loss of stored glycoconjugates in
6 the tracheas and in the caudal "long-path" bronchi, but not in the cranial or central
7 "short-path" bronchi. Although not significantly different from controls, there was a
8 dose-dependent thinning of the epithelium in caudal long-path bronchi. Terminal bronchioles
9 from rats exposed to 0.5 and 1.0 ppm O3 had increased volume fraction (Vv) of nonciliated
10 bronchiolar (Clara) cells and the epithelium was thicker in TBs from rats exposed to
11 1.0 ppm. In all exposed rats, the mass (Vs) of nonciliated bronchiolar cells was increased in
12 TBs that had long pathways (caudal) but not in TBs with short pathways (cranial and
13 central).
14
15 Centriacinar Region
16 As described in the previous criteria document (U.S. Environmental Protection Agency,
17 1986) and in the summary of it above, the CAR varies with the species. By common usage
18 (Weibel, 1963; Schreider and Raabe, 1981; Weibel, 1983; Rodriguez et al., 1987; Haefeli-
19 Bleurer and Weibel, 1988), the acinus consists of a TB, RBs when present, and the AD and
20 alveoli supplied by that TB. In some species (e.g., humans, monkeys, dogs, and cats),
21 several generations of RBs are found between the TB and ADs. In other species (e.g., rats,
22 mice, guinea pigs, and rabbits), RBs are either very poorly developed and limited to a single
23 very short generation or are absent (Tyler, 1983; Tyler and Julian, 1991). The CAR consists
24 of the TB, RBs if present and alveoli that open directly into RBs, and the initial portions of
25 ADs. Acini that do not have RBs have a smaller volume than those that do. Rodriguez
26 et al. (1987) estimated the acinar volume in rat lungs to be 1.86 mm3, and Haefeli-Bleuer
27 and Weibel (1988), using the same methods, estimated the acinar volume in the human lungs
28 at 187.0 mm3. Mercer and Crapo (1989) and Mercer et al. (1991) found that variation in
29 acinar size within an individual lung is an important determinant of the intensity of lesions
30 due to inhaled reactive gases. Thus the intensity of CAR lesions may vary when animals
31 with differing size acini are compared (Plopper et al., 1991).
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1 The CAR lesion, both in animals with small acini (e.g., rats) and animals with large
2 acini (e.g., monkeys) has been well described, both in the original reports and in the 1986
3 document (U.S. Environmental Protection Agency, 1986). Some of the reports published
4 since that document contain additional details concerning cellular and interstitial responses in
5 the CAR to short- or long-term O3 exposure and are presented in this section (Chang et al.,
6 1992; Pino et al., 1992c; Harkema et al., 1993c). Most of these studies need TEM levels of
7 resolution and magnification and employ morphometric methods. In other reports, which are
8 presented in Sections 6.2.3.3 and 6.2.3.4, morphometric estimates of the volume of the CAR
9 lesion were used to study factors that might alter the intensity of the lesion or evaluate the
10 intensity of reaction to specific exposure regimens (Mautz et al., 1988; Warren et al., 1988;
11 Stiles and Tyler, 1988). Due to the size and definition of the CAR lesion, this approach can
12 use LM morphometry to estimate lesion volume. In other studies, the cumulative effect of
13 O3 on the CAR is estimated by LM morphometry of one of the components, distal airway
14 remodeling, which results hi the formation of new RBs (Barr et al., 1988; Tyler et al., 1988;
15 Pinkerton et al., 1993a). Examples of each type of study will be presented.
16 Pino et al. (1992c) exposed rats to 1.0 ppm O3 for 4, 6, 8, or 24 h followed by
17 postexposure periods of 4 h or more so the total exposure and postexposure period totaled
18 24 h. Some of the rats were used for BAL (see Section 6.2.2), others for TEM
19 morphometry. The morphometric data are expressed as Vs values. After a 4-h exposure,
20 necrosis was the dominant morphologic feature, with increases in Vs of necrotic cells in the
21 TB epithelium (ciliated cells) and in CAR alveoli (Type 1 cells). With increasing time of
22 exposure or postexposure, the volume of necrotic cells in TBs shifted from the epithelium to
23 the lumen, with this change being significant at 24 h. In CAR alveoli, increased Vs of total
24 necrotic cells occurred at 4, 6, and 24 h of exposure and in the epithelium at 24 h. Healing
25 in TBs, evidenced by increased Vs of undifferentiated cells, was underway 18 h after a 6-h
26 exposure, 16 h after an 8-h exposure, and after the 24 h of exposure. The only significant
27 change in viable alveolar cells was an increase in Vs of Type 1 cells after 24 h of exposure.
28 This increase appeared predominantly due to swelling of individual Type 1 cells. Increased
29 Vs of total TB interstitium occurred 4 h after an 8-h exposure. In CAR alveoli, total
30 interstitium was increased after 8 h of exposure, with much of the increase due to an increase
31 in capillary volume.
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1 Chang et al. (1992) used TEM morphometry to evaluate cellular and interstitial
2 responses in the CAR TB and alveoli (proximal alveoli) of rats exposed to a 9-h peak slowly
3 rising to 0.25 ppm superimposed on a 13-h background level of 0.06 ppm (the background
4 was 7 days/week, the peak was 5 days/week). They examined rats after 1, 3, 13, and
5 78 weeks of exposure; 6 weeks after a 13-week exposure; and 17 weeks after a 78-week
6 exposure. Centriacinar region alveoli had a larger volume of total tissue and total epithelium
7 per area of basement membrane (Vs) only after 13 or 78 weeks of exposure, and these values
8 were not different after postexposure periods. Type 1 cells had a larger volume only at
9 13 weeks of exposure, increased numbers at 78 weeks, and increased numbers after 13 weeks
10 of exposure plus 6 weeks postexposure. Type 2 cell Vs was increased only after 78 weeks of
11 exposure and 78 weeks of exposure plus 17 weeks postexposure. Macrophages in the CAR
12 alveoli were only increased after 1 week of exposure. In CAR alveoli, both interstitial cells
13 and matrix were increased after 1 week of exposure, and the matrix increased again after
14 13 and 78 weeks of exposure. This difference was no longer significant after either
15 postexposure period. Although the data are not in the tables or figures, the text indicates that
16 both epithelial and endothelial basement membranes were thickened after 13 and 78 weeks of
17 exposure and after the 17-week postexposure period. Crystalline deposits in the basement
18 membrane are demonstrated in a figure. In TBs, the luminal surface area of Clara cells was
19 reduced at 1 week of exposure, and both ciliated and Clara cells had smaller luminal surface
20 areas after 78 weeks of exposure. These returned to control values during the postexposure
21 period. However, increased Va of Type 2 cells persisted for the 17-week postexposure
22 period that followed the 78 weeks of exposure. Chang et al. did not find significant
23 bronchiolization of alveoli (i.e., distal airway remodeling). This observation may be due to
24 the way they sampled the CAR proximal alveoli and the strict orientation of the airways to
25 obtain exact cross sections. This study complements those of Barry et al. (1985, 1988), who
26 used similar TEM morphometric methods, by extending the exposure period and adding
27 postexposure periods.
28 Cellular and interstitial changes were studied in nonhuman primates exposed 8 h/day for
29 90 days by both Moffatt et al. (1987) and Harkema et al. (1993c), using TEM and
30 morphometry. The study by Harkema et al. (1993c) is reported here because the
31 concentrations used, 0.15 and 0.3 ppm O3, were lower than those used by used Moffatt et al.
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1 (1987), which were 0.4 and 0.64 ppm. Harkema et al. also studied reactions after only
2 6 days of exposure to 0.15 ppm. There were no major differences among the three exposed
3 groups (i.e., 6 days to 0.15 ppm, 90 days to 0.15 ppm, and 90 days to 0.3 ppm O3). All
4 exposed monkeys had thicker RB epithelium and thicker RB interstitium. There were more
5 nonciliated cuboidal epithelial cells per millimeter of basement membrane in all exposed
6 groups and increased squamous cells only in the 6-day group. The thickened total
7 interstitium was due to increases in both acellular (matrix) and cellular components, but both
8 compartments were individually increased only in the 90-day O3 group. There were no
9 differences in RB smooth muscle. Transmission electron microscopy and SEM observations,
10 which were not studied quantitatively, include increased AMs in alveoli opening into RBs
11 and increased "dome"-shaped nonciliated bronchiolar cells, which had more apical
12 cytoplasm, more agranular and granular endoplasmic reticulum, more mitochondria, and
13 more Golgi with secretory granules. With the exception of increased AMs, there was no
14 evidence of necrosis nor of inflammatory cells. No differences due to age or gender were
15 detected.
16 Harkema et al. (1993c) speculate that their finding of a larger percent increase in RB
17 cuboidal cells in monkeys exposed to a lower concentration of O3 for the same time than that
18 reported by Moffett et al. might be due to the difference hi sampling methods. Harkema
19 et al. studied only the first generation RBs, whereas Moffatt et al. (1987) studied a random
20 sample of all generations of RBs. The first generation tends to be more damaged than
21 succeeding generations (Mellick et al., 1977; Eustis et al., 1981). The Harkema et al.
22 sampling procedure also prevented examination for the increased volume density of RBs and
23 decreased RB diameter reported by Fujinaka et al. (1985) and Moffatt et al. (1987).
24
25 Remodeling of Centriacinar Region Airways. This is a less well known sequela of
26 long-term O3 exposure. Using SEM, Boorman et al. (1980) and later Moore and Schwartz
27 (1981) reported the development in rats of an airway with the appearance of RBs between the
28 TB and ADs. This new segment was longer than those occasionally seen in control rats.
29 Respiratory bronchioles in rats are either absent or developed to only a single very short
30 segment (Tyler, 1983; Tyler and Julian, 1991).
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1 Barr et al. (1988) examined the development of this new segment using LM and TEM
2 morphometry on lungs from rats exposed to 0.95 ppm O3, 8 h/day for 90 days. They
3 reported a significant increase in the total volume of RB and of RB lumen and wall. The
4 new RBs reached a maximum length of four alveolar opening rings. They also noted that, in
5 some of these RB segments, the capillary and epithelial basal laminae were fused as they are
6 in TBs, rather than separate as in alveoli. Most Type 1 cell necrosis was found at the tips of
7 alveolar septa immediately adjacent to the RB/AD junction. Thus, the most severe epithelial
8 damage did not occur at the most proximal alveolus in the CAR, but rather in the alveolus
9 immediately distal to the newly formed RB.
10 Recently Pinkerton et al. (1993a) developed a new LM morphometric method to
11 evaluate remodeling of CAR ADs. In rats exposed to 1.0 ppm O3 for 20 mo, they reported
12 well-differentiated ciliated and nonciliated bronchiolar epithelium lining CAR airways that
13 would otherwise be ADs. Some of this epithelium extended five alveoli from the TB. Thus,
14 the Type 1 and 2 cells characteristic of ADs were replaced by both types of bronchiolar cells
15 characteristic of RBs when they are present in control rats. In an abstract of information
16 soon to be published, Pinkerton et al. (1993b) used their new morphometric method to study
17 rats exposed to 0.12, 0.5, or 1.0 ppm O3 6 h/day, 5 days/week for 20 mo. They reported
18 significant thickening of alveolar septal tips 200 mm from the TB in rats exposed to
19 0.12 ppm, which increased with O3 concentration to 600 jwm in rats exposed to 1.0 ppm, but
20 did not describe the type of epithelium covering these thickened tips. Several studies that did
21 not find CAR remodeling also used a slightly different procedure (Barry et al., 1985; Chang
22 etal., 1992).
23 Plopper et al. (1993) examined CARs from rats exposed to the same regimen, but
24 studied CARs from one cranial "short" pathway and a caudal "long" pathway. They found
25 nonciliated bronchiolar epithelial cells in remodeled former alveolar ducts in short- and long-
26 pathway CARs from rats exposed to 1.0 ppm O3, but only in short-pathway CARs from rats
27 exposed to 0.5 ppm. Central short-pathway CARs were not examined. Using the airway
28 dissection method of selecting CARs to be studied, nonciliated bronchiolar cells were not
29 found in CARs from rats exposed to 0.12 ppm O3.
30 The same phenomena apparently occurs in animals with several generations of RBs as
31 increased Vvs and volumes (Vs) of RBs have been reported in all O3-exposed monkeys
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1 examined using morphometric methods to estimate Vv or V of RBs (Fujinaka et al., 1985;
2 Moffatt et al., 1987; Tyler et al., 1988, 19915). Inflammatory changes and CAR remodeling
3 occur concomitantly, and inflammatory changes in an airway may indicate future remodeling.
4 Mellick et al. (1977) noted that in monkeys exposed to 0.8 ppm 8 h/day for 7 days, the
5 inflammatory process extended throughout the RBs and into ADs. Eustis et al. (1981)
6 reported that in monkeys exposed to 0.8 ppm 8 h/day for 90 days, all generations of RBs
7 contained aggregates of inflammatory cells. Monkeys exposed to lower concentrations for
8 the same or longer time have increased Vv and V of RBs (Moffatt et al., 1987).
9
10 6.2.4.3 Considerations of Exposure Regimens and Methods
11 Recovery During Postexposure Periods
12 Evidence of healing occurs soon after short-term exposures cease. In the studies of
13 Pino et al. (1992c), evidence of healing is provided by the increased Vs of viable
14 undifferentiated cells in TBs detected 16 h after the end of an 8-h exposure to 1.0 ppm O3.
15 Chang et al. (1992) reported only an increased Vs of Type 2 cells 17 weeks after a
16 78-week exposure to a simulated urban exposure regimen with a peak O3 concentration of
17 0.25 ppm. There were no changes detected 6 weeks after a 13-week exposure. Gross and
18 White (1987) examined rats 3 and 6 mo after a 52-week exposure of rats (20 h/day,
19 7 days/week) to 0.5 ppm. Using LM pathology, the only changes visible 6 mo after a 12-mo
20 exposure were a few areas of bronchiolization, slight dilation of ADs, and a slight thickening
21 of AD walls and adjacent alveolar septa. In an earlier study, less complete healing was
22 reported by Gross and White (1986), who used LM pathology to study rats 4 and 9 weeks
23 after a 4-week exposure (20 h/day, 7 days/week) to 0.7 ppm. Four weeks postexposure,
24 they reported a slight, unevenly distributed inflammatory reaction with condensed
25 eosinophilic material, presumed to be collagen, in the interstitium. Nine weeks
26 postexposure, some alveolar duct walls and TBs were thickened. Rats exposed to 0.96 ppm,
27 8 h/night for 42 nights and examined 42 days later using LM morphometry had increased
28 Vv and V of the RB wall and SEM evidence of CAR remodeling (Tyler et al., 1987).
29 Collagen content of these lungs increased during the postexposure period (Last et al., 1984b).
30 Centriacinar region remodeling was more persistent in monkeys exposed to 0.64 ppm
31 8 h/day for 12 mo followed by 6 mo postexposure (Tyler et al., 1991b). By LM
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1 morphometry, the Vv and V of total RB, RB lumen, and RB walls were increased both at
2 exposure end and at 6 mo postexposure. At exposure end, but not at 6 mo postexposure,
3 RB internal diameters were smaller and AMs in the CAR increased.
4 One study concerned postexposure recovery of the trachea (Nikula et al., 1988a).
5 Complete recovery of the trachea (as evaluated by LM morphometry, SEM, and TEM) of
6 rats exposed to 60 nights (8 h/night, 7 days/week) to 0.96 ppm Qj occurred following a
7 42-day postexposure period.
8
9 Effects of Episodic and Seasonal Exposure Regimens
10 Many investigators have noted that lesions due to O3 reach a maximum intensity in a
11 very few days and that, with continued exposure, the intensity of the lesion decreases. Eustis
12 et al. (1981) reported half the number of inflammatory cells in the CAR of monkeys exposed
13 to 0.8 ppm for 90 days as found in monkeys exposed to the same concentration for 7 days.
14 Chang et al. (1992) noted that the acute reactions to the 0.06-ppm background (7 days/week)
15 with a 9-h peak (5 days/week) slowly rising to 0.25 ppm that they reported at 1 week of
16 exposure had subsided at 3 weeks of exposure, Harkema et al. (1993c) reported no
17 difference in first generation RB epithelial thickness or cell numbers among monkeys
18 exposed to 0.15 ppm for 6 days or to 0.15 or 0.3 ppm for 90 days.
19 These and other similar observations prompted Chang et al. (1991) to compare effects
20 of two exposure regimens, which were evaluated using the same TEM morphometric
21 approach. The first regimen was a "square wave" 12 h/day, 7 days/week exposure to
22 0.12 or 0.25 ppm. The second regimen simulated urban O3 exposures by exposing rats
23 7 days/week for 13 h to 0.06-ppm background and then raising that background 5 days/week
24 to a peak of 0.25 ppm over a 9-h period and then decreasing the concentration to 0.06 ppm.
25 They calculated cumulative O3 concentration (CxT) for each exposure regimen and
26 concluded that increases in volume of Type 1 and 2 alveolar epithelial cells were linearly
27 related to increasing CxT. The relationship for Type 1 cells was more robust.
28 Barr et al. (1990) used TEM and LM morphometry to compare effects of 90 days of
29 daily exposure of rats for 8 h/day to 0.95 ppm with a regimen that modeled 5-day episodes
30 of O3 exposure. Each 5-day episode was followed by 9 postexposure days of filtered air.
31 The cycle was repeated seven times so that the "episodic" group was exposed a total of
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1 35 days over an 89-day period and the "daily" group was exposed for 90 days to the same
2 O3 concentration. Both groups had CAR remodeling with the formation of RBs. The
3 volume of RBs formed was not different when the two exposure groups were compared. The
4 absolute volume of parenchymal lesion was the same in both groups. The RB epithelial
5 thickness was increased in the daily group, but not in the episodic group. Conversely, the
6 interstitium of both TBs and ADs was thickened in the episodic group, but not in the daily
7 group. Thus, rats exposed to the same concentration of O3 for 35 days over an 89-day
8 period in an episodic regimen had as severe lesions as those exposed daily for 90 days.
9 Effects of "seasonal" and "daily" exposure of young monkeys to 0.25 ppm O3 were
10 reported by Tyler et al. (1988). The daily group was exposed every day (8 h/day) for
11 18 mo, whereas the seasonal group was exposed only during odd months for the 18 mo.
12 Thus, the daily group was exposed twice as many days to the same concentration as the
13 seasonal group. By LM morphometry, both groups had increased Vv of total RB and RB
14 lumen, but RB wall was only increased in the daily group. The only significant
15 morphometric difference between the two groups was an increase in CAR AMs in the daily
16 group. This difference, and the difference in significance of the RB wall thickness in the
17 seasonal group, was presumed due to the daily group being exposed to O3 the day before
18 necropsy, whereas the seasonal group breathed filtered air the 30 days preceding necropsy.
19 This final 30 days of filtered air apparently allowed the more acute inflammatory changes in
20 the seasonal group to regress. The seasonal group, but not the daily group, had increased
21 lung collagen (Section 6.2.6) and increased chest wall compliance (Section 6.2.5). Exposure
22 to the same concentration of O3 for half as many days in a seasonal regimen resulted in
23 similar morphometric effects as daily exposure and in physiological and lung collagen
24 changes not found in the daily group.
25 Tyler et al. (199la) exposed rats to seasonal and daily regimens similar to those used
26 for the monkeys described above. The concentration used in both studies was 0.25 ppm and
27 the total length of exposure was 18 mo. Rats were exposed nights, during their natural
28 period of activity. Both groups of rats were studied at the end of the 18-mo exposure cycle
29 and 30 days postexposure. The lungs were evaluated using a simplified LM morphometric
30 method for CAR airway remodeling by estimating the number of junctions of bronchioles
31 (TB and RB) with alveolar ducts per surface area of section. At exposure end, the number
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1 of junctions of both exposure groups was increased compared to filtered-air controls; the
2 O3 groups were not different from each other. Neither group was different from the controls
3 30 days postexposure.
4
5 Ex Vivo and In Vitro Exposures
6 Results obtained from studies of isolated perfused lungs and organ culture explants were
7 consistent with some of the findings from in vivo studies (Pino et al., 1992a; Nikula et al.,
8 1988b; Nikula and Wilson, 1990).
9
10 6.2.4A Considerations of Degree of Susceptibility to Morphological Changes
11 Species Differences in Degree of Response
12 Plopper et al. (1991) reviewed data from nonhuman primates and rats that had been
13 exposed to O3 and evaluated using TEM morphometry. The data were generated in several
14 laboratories and the exposure and evaluation methods were somewhat different, but the data
15 were expressed in similar terms. In the CAR, the results were expressed as total epithelial
16 thickness or numbers of cells per mm of basal lamina. Exposure of rats to 0.25 ppm
17 03 8 h/day for 42 days resulted in less than 1 100% increase in either parameter compared
18 to controls (Barry et al., 1985, 1988). Exposure of monkeys to 0.15 ppm 8 h/day for 6 days
19 resulted in a 230% increase in thickness and a 700% increase in cell number compared to
20 controls (Harkema et al., 1993c). As noted earlier (Section 6.2.4.2), the CARs of rats and
21 monkeys are structurally different (Tyler, 1983) and the CAR cells are also different
22 (Plopper, 1983).
23 There was also a difference when Plopper et al. (1991) compared stored secretory
24 product per mm2 of basal lamina in the nasal septum and lateral wall of the nasal cavity of
25 03-exposed rats and monkeys. Data from the exposure of rats to 0.12 ppm 6 h/day for
26 7 days resulted in a < 10% increase in the nasal septum and a < 100% increase in the lateral
27 wall. Exposure of monkeys to 0.15 ppm 8 h/day for 6 days resulted in a 300% increase in
28 the nasal septum and a 125 % increase in lateral wall. As in the CAR, there are major
29 morphological differences in the nasal cavities of these two species (Schreider and Raabe,
30 1981).
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1 Plopper et al. (1991) also compared collagen metabolism in rats and monkeys exposed
2 to 1.5 ppm 23 h/day for 7 days using the uptake of tritium-labeled proline. In rats, there
3 was an increase of 200% above controls, whereas the increase was 800% in monkeys.
4 From these data, it appears that the respiratory system of monkeys responds much more
5 to near-ambient concentrations of O3 than does that of rats. The mechanisms responsible for
6 these species differences in response to O3 remain to be elucidated.
7
8 Effects of Age
9 Several studies published since the previous criteria document (U.S. Environmental
10 Protection Agency, 1986) have addressed the effects of age on the intensity of
11 O3 morphological changes. The study by Stephens et al. (1978) and the initial report by
12 Barry et al. (1983) were cited. Briefly, Stephens et al. exposed rats ranging in age from 1 to
13 40 days old to 0.85 ppm O3 for 24, 48, or 72 h and examined their lungs by LM and TEM.
14 They reported that prior to 20 days of age, they did not find damage to TB ciliated cells or
15 to CAR Type 1 cells and that the amount of injury increased from 21 days to 35 days when a
16 plateau in response was reached.
17 Barry et al. (1985, 1988) exposed 1-day-old and 6-week-old rats to 0.12 or 0.25 ppm
18 O3 12 h/day for 6 weeks. The 1985 study emphasized TEM morphometry of CAR
19 (proximal) alveoli. They did not find differences in response due to age. In both age
20 groups, they found Type 1 cells increased in number and thickness, but decreased in both
21 luminal and basement membrane surface area. They found bare basement membrane where
22 Type 1 cells had been sloughed, but the amount was not increased in exposed groups. In the
23 0.25-ppm groups, but not those exposed to 0.12 ppm, Type 2 cells were increased in density
24 per mm basement membrane but not in volume. Alveolar interstitium was increased only in
25 adults exposed to 0.25 ppm. Macrophages in alveoli were increased in both age groups
26 exposed to 0.25 ppm, but not adults exposed to 0.12 ppm. Interstitial AMs were increased
27 only in adults exposed to 0.25 ppm. The TEM morphometry of TBs from these rats did not
28 include adults exposed to 0.12 ppm (Barry et al., 1988). There were no differences due to
29 age at start of exposure. In both juvenile and adult rats exposed to 0.25 ppm, they found
30 that the luminal surface covered by cilia and by nonciliated bronchiolar (Clara) cells was
31 reduced. The number of brush cells was also decreased.
December 1993 6-80 DRAFT-DO NOT QUOTE OR CITE
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1 Stiles and Tyler (1988) studied effects in a wider range of ages using LM morphometry
2 and SEM. They exposed 60- and 444-day-old female rats to 0.35 or 0.8 ppm continuously
3 for 72 h. Body weights of the 444-day-old rats, but not those 60 days old, decreased during
4 exposure. Fixed lung volumes of 444-day-old rats exposed to 0.8 ppm, but not 0.35 ppm,
5 were smaller than same-age controls. The Vv of CAR lesions was larger in 60-day-old rats
6 exposed to either concentration than in the 444-day-old rats. The Vv of cells free in lumens
7 (AMs) was increased in young rats exposed to 0.35 ppm compared to the older rats, but was
8 not different for rats exposed to 0.8 ppm. Young rats exposed to either concentration had
9 larger CAR lesions than the older rats, and young rats exposed to the lower concentration
10 had more AMs. Older rats had greater changes in body weight and, in those exposed to the
11 higher concentration, in fixed lung volume.
12
13 Effects of Exercise
14 Exercise increases the dose of inhaled toxicants delivered to sensitive cells (see
15 Chapter 8). Mautz et al. (1985b) studied the effects of 0.2 and 0.38 ppm O3 on rats at rest
16 and during several treadmill exercise protocols. They found increased percent of lung
17 parenchyma! area containing free cells (AMs) in exercised rats exposed to both
18 concentrations compared to rats exposed at rest. At the higher concentration, there was also
19 an increase in the percent of parenchymal area with thickened ADs and alveolar septa.
20 Tyler et al. (1991c) exposed thoroughbred horses (trained to a treadmill) to 0.25 or
21 0.8 ppm O3 for 29 min on two consecutive days using a protocol that included 9 min of
22 graded exercise (3 min at maximum speed) and 20 min of "cool out". During maximal
23 exercise, horses increase their rate of oxygen (O2) consumption more than other athletes of
24 other species. Two of three horses exposed to 0.8 ppm had significant areas of hemorrhage
25 and edema, and one of them refused the second day's exercise and exposure. By TEM, all
26 horses exposed to 0.8 ppm had CAR lesions including necrosis of Type 1 cells. Lesions in
27 those exposed to 0.25 were limited to CAR ciliated cells. No horses were exposed at rest
28 for comparison.
29
30
December 1993 6-81 DRAFT-DO NOT QUOTE OR CITE
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1 Elastase-Induced Emphysema
2 Rats with elastase-induced emphysema and saline-instilled controls were exposed to
3 0.15 or 0.5 ppm for 3 or 7 days (Dormans et al., 1989). Mean linear intercepts, a measure
4 of alveolar size, were determined using LM. The incidence and severity of CAR
5 inflammatory changes were the same in O3-exposed elastase-treated and saline-control rats.
6 There were no changes in mean linear intercepts due to the O3 exposure.
7
8 6.2.4.5 Summary
9 Research since the previous O3 criteria document (U.S. Environmental Protection
10 Agency, 1986) continues to support the concept that all mammalian species respond to
11 O3 concentrations < 1.0 ppm in a similar manner, but with significant differences in intensity
12 of reactions among the species studied (Plopper et al., 1991). Dungworth (1989) provided a
13 schematic overview of morphological reactions of the CAR from mammalian lungs to
14 continuous exposure to low concentrations of O3 as a series of tune-response profiles
15 (Figure 6-3). Bronchioalveolar exudative processes are the predominate early response, but
16 the magnitude decreases rapidly with increasing duration of exposure and continues to decline
17 during postexposure periods. Epithelial hyperplasia also starts early and increases in
18 magnitude for several weeks, after which a plateau is reached until the exposure ends.
19 Epithelial hyperplasia declines slowly during postexposure periods. Interstitial fibrosis has a
20 later onset and may not be apparent for a month or more. The magnitude of this response,
21 however, continues to increase throughout the exposure and, at least in some cases (Last
22 et al., 1984b), continues to increase after exposure ends.
23 Nonhuman primates appear to respond more to O3 at concentrations < 1.0 ppm than do
24 rats. However, the mechanisms responsible for these differences in response have not been
25 elucidated. Differences in cell, tissue, and circulating levels of several antioxidants are being
26 studied, as are differences in in vitro responses to O3 by cultures of cells from the various
27 species. Basic morphological differences in the structure of the most injured portion of the
28 lung, the CAR, and the size (volume) of the basic structural unit, the acinus, may also be
29 factors in the greater response of monkeys to O3. Both human lungs and lungs from
30 nonhuman primates have CARs characterized by several generations of RBs, whereas rats
31 have no RBs, or only a single poorly developed generation (Tyler and Julian, 1991). Within
December 1993 6-82 DRAFT-DO NOT QUOTE OR CITE
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O
CL
0)
•O
=3
«-*
I
Epithelial hyperplasia
Bronchioloalveolar exudate
Interstitial fibrosis
Exposure
Post-
exposure
0361
days ' month
6
months
Time
Figure 6-3. Schematic comparison of the duration-response profiles for epithelial
hyperplasia, bronchioloalveolar exudation, and interstitial fibrosis in the
centriacinar region of lung exposed to a constant low concentration of
ozone.
Source: Dungworth (1989).
1 an individual lung, acinar volume is directly related to the intensity of CAR lesions (Mercer
2 and Crapo, 1989; Mercer et al., 1991). The volume of individual acini in human lungs is
3 100 times larger than individual acini in rat lungs (Rodriguez et al., 1987; Haefeli-Bleuer and
4 Weibel, 1988). Acinar volume of the monkeys used in O3 studies is not known, but on the
5 basis of the CAR structure, is assumed to be more like that of human lungs than rat lungs.
6 Another morphological factor that may be responsible in part for the greater response to
7 O3 by nonhuman primates than rats may be differences in the complexity of the nasal cavity.
8 Schreider and Raabe (1981) studied the cross-sectional morphology of the nasal-pharynx in
December 1993
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DRAFT-DO NOT QUOTE OR CITE
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1 rats, beagle dogs, and a rhesus monkey. They concluded that the complexity of the nasal
2 cavity, and therefore the "scrubbing" effect (Yokoyama and Frank, 1972; Miller et al., 1979)
3 that reduces the concentration of inhaled O3 delivered to the lower respiratory tract, would
4 be greater in rats than in monkeys. Schreider and Raabe proposed that, with appropriate
5 scaling, the monkey could serve as a model for aerosol and gas deposition in the
6 nasopharyngeal region of humans. However, the sensitivity of the nasopharyngeal epithelium
7 may be different because changes in the nasal epithelium that follow O3 inhalation, like those
8 in the CAR, are more severe in monkeys than in rats (Plopper et al., 1991).
9 The effect of age at start of exposure on O3-induced lung injury has not been resolved.
10 Barry et al. (1985, 1988) reported no differences in TEM morphometry of CAR and TB
11 lesions due to age at start of exposure. However, they studied a narrow range of ages, 1 and
12 42 days old at the start of a 42-day exposure. Barry et al. (1985, 1988) speculate that much
13 of the CAR response in the 1-day-old rats might have occurred from exposure Day 21 to
14 Day 42 because as the earlier studies of Stephens et al. (1978) found that rats were not
15 sensitive to morphological effects of O3 until weaning at 21 days of age. Thus, the rats that
16 were 1 day old at the exposure start may have developed the same intensity of lesions during
17 the last 21 days of exposure as the older rats did in 42 days of exposure. In studies using a
18 wider range of ages (60- and 444-day-old rats), Stiles and Tyler (1988) reported that
19 following a 3-day exposure, they found larger CAR lesions hi the younger rats, but greater
20 changes in body weight and fixed lung volume in the older rats.
21 The effects of exposure regimen and exposure duration were evaluated in more recently
22 published studies. Exposure of young monkeys to 0.25 ppm in a "seasonal" regimen (i.e.,
23 exposure in odd months and postexposure in even months) for 18 mo resulted in the same
24 quantity of CAR lesions as daily exposure to the same concentration for 18 mo (Tyler et al.,
25 1988). A similar quantity of CAR lesions was reported in rats exposed to 0.95 ppm in an
26 "episodic" regimen for 35 days, 5 days exposure and 9 days postexposure for 89 days, as
27 those exposed to the same concentration each day for 90 days (Barr et al., 1990). Chang
28 et al. (1991) calculated the cumulative O3 concentration for a "square wave" exposure to
29 0.12 or 0.25 ppm 12 h/day, 7 days/week with a simulated "urban" exposure regimen of
30 0.06 ppm 13 h/day, 7 days/week and then raising that background 5 days/week to a peak of
31 0.25 ppm over a 9-h period. Using TEM morphometry of the CAR, they found no
December 1993 6-84 DRAFT-DO NOT QUOTE OR CITE
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1 difference due to the pattern of exposure. Thus, it appears that the pattern of daily exposure
2 does not influence the intensity of CAR lesions, but that episodic and seasonal patterns of
3 exposure with multiple days of postexposure between days of exposure are equivalent to daily
4 exposure.
5 It has become clear that remodeling of centriacinar airways is cumulative. Using a
6 stereological approach, Barr et al. (1988) reported an increase hi the total volume of RB wall
7 and lumen in rats exposed to 0.95 ppm 8 h/day for 90 days. They also reported continuing
8 Type 1 cell necrosis at the tips of alveolar septa (alveolar opening rings) immediately distal
9 to the newly formed RB/AD junction rather than in the TB/AD junction. It appears that
10 some of the necrotic Type 1 cells were replaced by bronchiolar epithelium rather than by
11 Type 2 cells as previous studies indicated. This was confirmed by Pinkerton et al. (1993),
12 who reported fully differentiated ciliated and nonciliated bronchiolar epithelium lining
13 alveolar tips along a former AD up to 1 mm from the TB in lungs from rats exposed to
14 1.0 ppm 6 h/day, 5 days/week for 20 mo. Remodeling of centriacinar airways appears to be
15 a general phenomena, as increases in the Vv and V have been reported in lungs from all
16 exposed rats and monkeys examined using stereological or morphometric methods that could
17 detect this change (Fujinaka et al., 1985; Moffatt et al., 1987; Tyler et al., 1987; Tyler
18 et al., 1988; Barr et al., 1990; Pinkerton et al., 1992, 1993). Centriacinar region
19 remodeling has been demonstrated to persist in monkeys 6 mo after a 12-mo exposure to
20 0.64 ppm (Tyler et al., 1991) and in rats 42 days after a 42-night exposure to 0.96 ppm
21 (Tyler etal., 1987).
22 Several studies have confirmed and extended the earlier reports of epithelial
23 degenerative changes followed by sloughing, leaving bare basement membrane that is
24 recovered by other cell types, thus altering epithelial cell populations and increasing cell
25 density (hyperplasia) in TB and centriacinar alveoli (Barry et al., 1985; Moffatt et al., 1987;
26 Barry et al., 1988; Chang et al., 1988; Chang et al., 1992; Harkema et al., 1993c).
27 Epithelial replacement, a reparative process, occurs very early (Pino et al., 1992c) even
28 though degeneration and necrosis continues (Barr et al., 1988). In specific airways, these
29 processes appear to reach a maximum early in the exposure, as reported by Harkema et al.
30 (1993c), who found no difference in the intensity of lesions in first generation RBs, as
31 measured by RB epithelial cell thickness and numbers, among monkeys exposed to 0.15 ppm
December 1993 6-85 DRAFT-DO NOT QUOTE OR CITE
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1 for 6 days or to 0.15 or 0.3 ppm for 90 days. However, it is important to note that there
2 may have been differences in response if more distal generations of RBs or random
3 generation RBs had been selected for study. The interstitium in the CAR also thickens by
4 the addition of cells and matrix. Thickening of the basement membrane and the presence of
5 granular material in it was reported by Barr et al. (1988) and Chang et al. (1992). Chang
6 et al. (1992), using TEM morphometry, reported that some changes in epithelial cell
7 population changes persist in rats for 17 weeks after a 78-week exposure to a model urban
8 profile with a peak of 0.25 ppm. At the LM level, Gross and White (1987) reported that
9 6 mo following a 12-mo exposure to 0.5 ppm 20 h/day, CAR inflammation had all but
10 disappeared and only a slight dilation and thickening of some ADs and adjacent alveoli
11 remained.
12 The epithelia of the nasal cavity respond rapidly to O3. In ciliated regions, cilia are
13 attenuated and intraepithelial mucosubstances increase. Hyperplasia and increased
14 intraepithelial mucosubstances are reported in areas of nonciliated transitional epithelium
15 (Harkema et al., 1989). These effects persisted throughout a 20-mo exposure of rats to
16 0.5 or 1.0 ppm, but were not seen in rats exposed to 0.12 for that period (Harkema et al.,
17 1993a,b). After acute exposure, DNA synthesis of the epithelium of the anterior
18 maxilloturbinates of rats increases according to a given CxT product at S:2.88 ppm-h, but
19 the increase is not linear with increasing CxT (Henderson et al, 1993). Changes have not
20 been reported in the olfactory epithelium or in the squamous epithelium of the nasal cavity.
21 Respiratory epithelia in other conducting airways, especially the trachea, appear to react
22 in a similar manner with early necrosis of ciliated cells (Hyde et al., 1992). Cell
23 replacement starts early (Hyde et al., 1992) and after 60 nights of exposure of rats to
24 0.96 ppm, numeric density of specific cell types was not different from controls (Nikula
25 et al., 1988a). In newborn lambs exposed to 1.0 ppm 4 h/day for 5 days and examined
26 9 days later, the normal change in epithelial cell population that occurs by 2 weeks of age
27 did not occur (Mariassy et al., 1990).
28
29
December 1993 6-86 DRAFT-DO NOT QUOTE OR CITE
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1 6.2.5 Effects on Pulmonary Function
2 6.2.5.1 Introduction
3 Numerous studies have been published on the effects of O3 exposure on pulmonary
4 function in animal models. This work has been reviewed by the U.S. Environmental
5 Protection Agency (1986) and Tepper et al. (1993). The evaluation of pulmonary function
6 after exposure may help provide a more integrated assessment of the severity of health
7 effects by indicating the magnitude, location, and duration of functional disability. In an
8 attempt to summarize the literature here, only key studies employing multiple concentrations,
9 or studies demonstrating a particular functional effect, testing a different species or strain, or
10 showing the relationship with a unique variable such as age or sex will be discussed.
11 Because purely descriptive pulmonary function studies are now rarely reported, newer studies
12 will be discussed within the context of the study hypothesis.
13 This section is organized by duration of exposure (brief, acute, repeated, and long-term
14 exposures). Within each of these sections, subsections discussing different types of
15 pulmonary function measures are addressed. These subsections include a discussion of
16 ventilatory patterns, breathing mechanics, airway reactivity, and more extended
17 characterizations of lung function whenever such data are available.
18
19 Ventilation
20 Evaluation of the sinusoidal breathing pattern includes the measurement of tidal volume
21 (VT), frequency of breathing (fB), and their product, minute ventilation (VE). Such
22 measurements have proven to be sensitive indicators of O3 effects. Numerous animal and
23 human studies have shown that O3 exposure increases fB and decreases VT (tachypnea) (U.S.
24 Environmental Protection Agency, 1986).
25
26 Breathing Mechanics
27 Measurement of breathing mechanics (dynamic compliance [Cdyn] and lung resistance
28 [RJ) in animals has an advantage over simple measures of ventilation in that these
29 parameters can assess the mechanical effort required to breathe and can help localize airway
30 (resistance) versus parenchyma! (compliance) lesions. With sufficient O3 exposure, increases
December 1993 6-87 DRAFT-DO NOT QUOTE OR CITE
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1 in RL and decreases in Cdyn have been observed (U.S. Environmental Protection Agency,
2 1986).
3
4 Airway Reactivity
5 Increased airway reactivity, an exaggerated response of the lung to an exogenously
6 administered bronchoconstrictor, has been observed with O3 (U.S. Environmental Protection
7 Agency, 1986). Increased airway reactivity has been associated with lung injury and
8 inflammation, and thus, may increase an understanding of the potential role of O3 in allergy
9 and asthma. Typically in humans, heightened airway responsiveness is determined using
10 progressively increasing concentrations of aerosolized bronchoconstrictors, such as
11 methacholine or histamine (see Chapter 7, Section 7.2.3). Although bronchoprovocation
12 protocols employing doubling doses of inhaled bronchoconstrictors have been relatively
13 standardized for human experiments, no such standardization exists for animal studies,
14 making comparisons between animal and human studies difficult.
15
16 Extended Functional Characterizations
17 The most complete assessment for examining the extent and localization of chronic lung
18 injury related to O3 exposure includes an extended characterization of the lung using a
19 battery of human clinical pulmonary function test analogs. Such tests include measurement
20 of static lung volumes, volume-pressure and flow-volume relationships, as well as evaluation
21 of inhomogeneity of ventilation and problems associated with oxygen diffusion across the
22 epithelial barrier. Although these latter measurements require additional technical complexity
23 to perform, they can provide a more in-depth understanding of the nature, extent, and
24 localization of the lesion.
25
26 6.2.5.2 Brief Ozone Exposures (Less Than 30 Minutes)
27 Few experiments have evaluated the effects of brief exposures (<30 min) to O3. Most
28 of these brief exposure studies have examined changes in regional breathing mechanics using
29 exposures to the lower respiratory tract via a tracheal tube, thus, eliminating any scrubbing
30 by the nasal or oropharynx and thereby increasing the effective dose of O3 delivered to that
31 region of the lung. The relevance of this method of delivering the exposure, as compared to
December 1993 6-88 DRAFT-DO NOT QUOTE OR CITE
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1 the typical inhalation route, is uncertain. However, positive effects have been observed,
2 indicating that very rapid, reflex responses occur with brief, direct O3 exposure.
3
4 Ventilation and Breathing Mechanics
5 No studies have evaluated the effects of brief O3 exposure on ventilatory pattern;
6 however, breathing mechanics (Cdyn and RL) have been evaluated. The previous criteria
7 document (U.S. Environmental Protection Agency, 1986) described two experiments by
8 Gertner et al. (1983a,b,c), which demonstrated that anesthetized dogs exposed to O3 via a
9 fiber-optic bronchoscope wedged into a segmental airway caused increased collateral
10 resistance within 2 min of exposure to 0.1 ppm O3. The response rapidly attenuated with
11 0.1 ppm, but not to exposure to 1.0 ppm O3. Atropine or vagotomy blocked the increase in
12 collateral flow resistance to 0.1 ppm, indicating that it was elicited by vagal postganglionic
13 stimulation, but the response to 1.0 ppm O3 exposure was only partially blocked.
14 More recently, Kleeberger et al. (1988), using a technique similar to Gertner et al.
15 (1983a), exposed the segmental airways of mongrel dogs to 1.0 ppm O3 for 5 min through a
16 wedged bronchoscope (Table 6-9). As previously described, collateral resistance increased,
17 and this increase was reproducible even when four, 5-min exposures over a 3-h period were
18 performed. Thus, no immediate tolerance was observed. Furthermore, this response could
19 be partially blocked by administration of a cyclooxygenase inhibitor (indomethacin) and a
20 H^-receptor blocker (chlorpheniramine), whereas a thromboxane synthetase inhibitor was
21 ineffective. This study suggests that histamine or cyclooxygenase products released from
22 resident cells may directly, or via the parasympathetic nervous system, mediate the increase
23 in collateral resistance. However, because collateral resistance probably makes up only a
24 small proportion of pulmonary resistance, these results may not be generalizable to more
25 prolonged exposures and larger airway responses.
26
27 Airway Reactivity
28 Baboons were exposed via an endotracheal tube to 0.5 ppm O3 for 5 min after a
29 baseline methacholine inhalation challenge test (Fouke et al., 1988) (Table 6-10). Lung
30 resistance increased with O3 exposure, and the baboons showed an enhanced response to
31 methacholine. This enhanced methacholine response was almost exclusively due to the
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TABLE 6-9. EFFECTS OF OZONE ON PULMONARY FUNCTION3
i
y
>— >
*
u>
^
8
o
8
1
1
i
n
Ozone
Concentration
ppm
Base
0.06
Spike
0.25
0.13
0.22
0.45
0.2-
0.8
0.25
0.5
1.0
0.25
0.5
1.0
0.5
0.35
0.5
1.0
0.5
0.8
0.6
0.7
/tg/m
Base
118
Spike
490
255
431
882
392-
1,568
490
980
1,960
490
980
1,960
980
686
980
1,960
980
1,568
1,176
1,372
Exposure
Duration Drugs
Base 13 h/day,
7 days/week; ramped
spike 9-h/day,
5 days/week, for
1 week, 1, 3, 12, and
18 mo
3 h Pentobarbital
Through tracheal tube Gallamine
3 h
2h;
2, 4, 6, and 8% COj,
at alternating 15 min
3 h for Pentobarbital
5 days
2 h Chloralose
2 h/day for 5 days,
8% CO2 alternating
15 min
2 or 7 h, Halothane
8% CC>2 alternating
15 or 45 min/h
2h
exercise
muzzle
20 h/day for Halothane
28 days
Species, Sex
(Strain)
Age
Rat, M
(F344)
60 days old
Dog
(Foxhounds)
16-20 kg
Rat, M
(S-D)
7 weeks old
Rat,M
(F344)
90 days old
Mouse, F
(CD-I)
3-4 weeks old
Dog
(Mongrel)
15 ±0.9 kg
Rat, M
(F344)
110 days old
Rat, M
(F344)
90 days old
Dog.F
(Beagle)
2-7 years old
Rat, M
(F344)
14 weeks old
Observed Effect(s)
Increased expiratory resistance observed at all time points, but mostly at 78 weeks.
Positron camera indicated nonuniform distribution of ventilation in small airways;
no change in RL, Cdyn, or forced expiratory flow.
Maximum 02 consumption decreased tachypnea at 0.2 ppm, tachypnea observed at
0.4 ppm, and ventilation and core temperature decreased at 0.6 ppm.
Concentration response-related increase in fg and flow at zero pleural pressure,
decrease in Vf, no change in Vjj, Rg, or Cdyn.
Oj had no effect, but in combination with virus a decreased DL^-O, N2, and lung
volume were observed greater than virus alone 6, 9, and 14 days PE.
Increased RL, decreased Cdyn. No change in BAL prostanoids.
Attenuation of tachypnea with consecutive exposures; BAL antioxidants and protein
did not adapt with exposure, histopathology increased in severity.
FVC, DLCO, and N2 slope all decreased with increasing C x T products. The
magnitude of the decrement depended on the both the duration and concentration of
03 exposure and the measured parameter.
Tachypnea, VE, gas exchange, and RL increased; Cdyn decreased.
Decreased flow volume and DLcQ and increased FRC immediately PE, no effect at
4 weeks PE, decrease in forced expiratory flow at 9 weeks PE.
Reference
Tepperetal. (1991)
Morgan et al. (1986)
Mautz and Bufalino
(1989)
Tepper et al. (1990)
Belgrade et al. (1988)
Fouke et al. (1991)
Tepper et al. (1989)
Costa et al. (1989)
Mautz et al. (1985b)
Gross and White (1986)
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1 post-03 increase in RL and, thus, resulted in no change in the provocative dose that increased
2 RL by 50%. The experiment was repeated 5 to 14 days later, except that before
3 O3 exposure, cromolyn sodium was administered. In the presence of cromolyn, baseline RL
4 after O3 was less (not significant), but the response to methacholine challenge was
5 significantly lower post- O3 exposure. In a follow-up study (Fouke et al., 1990) using a
6 similar O3 exposure protocol (no methacholine challenge), cromolyn partially blocked the
7 O3-induced increase in RL; however, post-O3 exposure analysis of BAL indicated that
8 cromolyn did not affect the level of several measured prostanoids (6-keto PGFla, PGE2,
9 TXB2, or PGF2a), suggesting that these mediators were not related to the change in RL.
10 The response to antigen-induced bronchoconstriction, an animal model of allergy, has
11 also been recently evaluated. After a 5-min exposure to 1.0 ppm O3 via a wedged
12 bronchoscope, collateral resistance in dogs increased for 1 to 3 h (Kleeberger et al., 1989).
13 After the O3-induced resistance returned to baseline, the typical increase in collateral
14 resistance observed in dogs challenged with Ascaris suum antigen, for which they were
15 natively sensitive, was attenuated both 1 to 3 h and 24 h post-Oj exposure. The attenuated
16 antigen response appeared to be independent of PMNs in the airways. In a follow-up study,
17 the late phase response to antigen (bronchoconstriction 2- to 12-h postantigen challenge) was
18 also blocked in allergic dogs when O3 exposure (1.0 ppm, 5 mm, via a bronchoscope)
19 preceded antigen challenge (Turner et al., 1989). These studies suggest that, at least in the
20 dog, brief local administration of O3 to the airways may inhibit allergic responses.
21
22 6.2.5.3 Acute Ozone Exposures (Less Than One Day)
23 Ventilation
24 Alteration of the ventilatory pattern has long been established as a hallmark of acute
25 O3 exposure. Several animal studies evaluated tidal breathing changes during and after
26 O3 exposure (U.S. Environmental Protection Agency, 1986; Table 6-9). For most species, a
27 tachypneic response (rapid and shallow breathing) has been observed. For example, Murphy
28 et al. (1964) studied unanesthetized guinea pigs exposed for 2-h to 0.34, 0.68, 1.08, or
29 1.34 ppm O3 via nose cones and measured tidal breathing using a constant volume
30 plethysmograph. A similar experimental preparation was used by Amdur et al. (1978) to
31 evaluate the respiratory response of guinea pigs to 0.2, 0.4, and 0.8 ppm O3. In both
December 1993 6-95 DRAFT-DO NOT QUOTE OR CITE
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1 experiments, a monotonic increase in fB was observed. In the Amdur et al. (1978) study,
2 decreases in VT were not concomitantly observed.
3 Lee et al. (1979, 1980) showed that the tachypneic pattern observed in conscious dogs
4 exposed to 0.56 to 0.85 ppm was not altered by bronchodilator pretreatment, or by atropine
5 administration. These manipulations would suggest that the rapid shallow breathing was not
6 caused by bronchoconstriction. The response was, however, blocked by vagal cooling,
7 which was interpreted by the authors to suggest that vagal sensory afferent transmission had
8 been blocked. Thus, the authors suggested that increased vagal afferent impulses produced
9 tachypnea and that the response was independent of vagal efferents (increased smooth muscle
10 tone).
11 Several new studies evaluating ventilation after acute O3 exposure have appeared in the
12 literature. Mautz and Bufalino (1989) measured ventilation (VE) as well as oxygen
13 consumption and rectal temperature in awake rats exposed for 3 h to 0.2, 0.4, 0.6, and
14 0.8 ppm O3. Concentration-related increases in fB were significantly different from control
15 beginning at 0.4 ppm, with a maximal response observed up to 0.6 ppm. Tidal volume was
16 similarly reduced, whereas VE and rectal temperature were less sensitive to O3 exposure,
17 showing decreases at 0.6 and 0.8 ppm. Oxygen consumption was decreased at all
18 concentrations tested. The authors concluded that the O3-induced change in breathing pattern
19 did not cause a decrease in metabolic rate or impose a condition of hypoxia. The change in
20 ventilation and O2 consumption appeared coincident or possibly proceeded the irritant reflex
21 change in breathing pattern.
22 Tepper et al. (1990) exposed awake rats to 0.12, 0.25, 0.5, and 1.0 ppm O3 for
23 2.25 h in head-out pressure plethysmographs. During exposure, CO2-stimulated breathing
24 was incorporated to augment ventilation, similar to the use of exercise in human studies.
25 Frequency increased, whereas VT decreased monotonically between 0.25 and 1.0 ppm during
26 a 2.25-h exposure. A decrease in VE was not observed. This difference from the Mautz
27 and Bufalino (1989) study could be due to their restraining rats in a tightly fitting plastic flow
28 plethysmograph with the face sealed by an aluminum nose cone; in the Tepper et al. (1990)
29 study, the rats were exposed in oversized, steel, head-out plethysmographs and were
30 intermittently challenged with CO2, which may have overridden the metabolic depressant
31 effect.
December 1993 6-96 DRAFT-DO NOT QUOTE OR CITE
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1 Mautz et al. (1985a), using exercising dogs exposed to 0.6 ppm O3 for 140 min,
2 showed tachypnea, increased VE, and elevated ventilation equivalents for O2 and CO2
3 compared to exercised air-exposed control-day responses. Pulmonary resistance fell in air-
4 exposed exercising dogs, but climbed toward the end of the exposure in the O3-exposed
5 dogs.
6 In a follow-up study to Lee et al. (1979, 1980), Sasaki et al. (1987) performed similar
7 experiments on two awake dogs that were trained to run on a treadmill. Dogs were exposed
8 to 1.0 ppm O3 for 2 h and evaluated before O3 and at either 1 or 24 h postexposure. In all
9 studies with or without exercise, O3 increased fB and decreased VT, without affecting VE.
10 Vagal blockade diminished, but did not abolish, the tachypneic response, indicating that both
11 vagal and nonvagal mechanisms were important. The O3-induced change in fB rate was due
12 to an equal reduction in inspiratory time and expiratory time with no, or a small, diminution
13 (only during CO2 rebreathing experiments) of ventilatory drive (VT/inspiratory time).
14 Additionally, O3 did not affect functional residual capacity (FRC) or core temperature
15 measurements in resting, exercising, or vagally blockaded dogs. The authors speculate that
16 the change in fB is due to a vagally mediated lowering of the pulmonary stretch receptor's
17 volume threshold for inspiration and expiration with a concomitant increase in flow rate, thus
18 leaving FRC constant. Furthermore, the authors speculated that increased sensitization of
19 rapidly adapting receptors, C-fiber nerve endings, and nonvagal mechanisms may also be
20 contributors to the tachypneic response.
21 Two recent studies provide further insight into the mechanism of O3-induced changes in
22 ventilatory patterns. In the first study, Schelegle et al. (1993) showed that O^-induced
23 (3 ppm O3 for 40 to 70 min) tachypnea in anesthetized, spontaneously breathing dogs could
24 be largely abolished by cooling the cervical vagus to 0 °C, but not to 7 °C. This would
25 indicate that large myelinated fibers were not involved in this reflex response, but
26 nonmyelinated C fibers, whose activity is decreased only at the lower temperature, are
27 important. In a companion study, Coleridge et al. (1993) measured the responses of five
28 types of single vagal nerve fibers: bronchial C-fibers, pulmonary C-fibers, rapidly adapting
29 receptors, slowly adapting pulmonary stretch receptors, and unclassified fibers. During
30 exposure to O3, bronchial C-fibers were most affected. Because discharge of these fibers
31 was not immediate with the onset of exposure, but took time to develop, the authors
December 1993 6-97 DRAFT-DO NOT QUOTE OR CITE
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1 suggested that O3 may not directly stimulate these receptors and that autacoid mediators
2 released in the lung, which have previously been shown to stimulate these fibers, were
3 probably responsible for their activation. Rapidly adapting receptors were shown to play a
4 small part in this reflex response; although, surprisingly, pulmonary C-fibers and slowly
5 adapting receptors were found to be unimportant. Ozone also stimulated several unidentified
6 vagal fibers that may be responsible for residual effects not abolished by 0 °C cooling of the
7 vagus. The authors also conclude that tachypnea, inspiratory pain, and the reduction in
8 forced vital capacity in humans is likely to be due to the same mechanism (i.e., bronchial
9 C-fiber stimulation).
10
11 Breathing Mechanics
12 Although changes in breathing mechanics have been observed in laboratory animals,
13 they are not consistently observed and tend to be reported more frequently at higher exposure
14 concentrations (U.S. Environmental Protection Agency, 1986; Table 6-9).
15 The previously discussed studies by Murphy et al. (1964) and Amdur et al. (1978)
16 evaluated breathing mechanics in unanesthetized guinea pigs. The Murphy et al. (1964)
17 study showed an increase in flow resistance only at concentrations > 1 ppm O3. Pulmonary
18 compliance was not measured. Amdur et al. (1978) observed a decrease in Cdyn after
19 exposure to 0.4 and 0.8 ppm O3, but no significant change in RL was noted.
20 In more recent analogous studies in unanesthetized rats, Tepper et al. (1990) observed
21 no significant changes in RL or Cdyn after a 2.25-h exposure to 0.12, 0.25, 0.5, or 1.0 ppm
22 O3, in spite of intermittent 15-min periods of exercise-like hyperventilation induced by CC^.
23 Similarly, no changes in breathing mechanics were observed by Yokoyama et al. (1987)
24 when they evaluated anesthetized rats exposed to 1.0 ppm O3 for 24 h. However, when
25 Cdyn was normalized for differences in FRC, the resulting specific compliance was
26 decreased compared to air-exposed controls. Furthermore, when the animals were paralyzed
27 and ventilated between 40 and 200 breaths/min, O3-treated animals showed a frequency-
28 dependent decrease in Cdyn as fB increased above 120 breaths/min. The authors conclude
29 that because RL was not affected, the effect of O^ was to obstruct peripheral airways.
December 1993 6-98 DRAFT-DO NOT QUOTE OR CITE
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1 Pulmonary mechanics were evaluated in anesthetized, paralyzed dogs acutely exposed to
2 0.12, 0.22, and 0.45 ppm for 3 h via a stainless steel tracheal tube. No changes in RL or
3 Cdyn were observed at any concentration (Morgan et al., 1986).
4 In papers by Miller et al. (1987, 1988), the effect of a 1-h exposure to 1.0 ppm O3 was
5 evaluated in anesthetized, tracheostomized guinea pigs. A significant increase in RL was
6 noted at 2 h postexposure, but was resolved by 8 h postexposure. Both indomethacin and
7 cromolyn sodium partially blocked the increase in RL at 2 h postexposure (Miller et al.,
8 1988). These results suggest that eicosanoids produced from an inflammatory response in
9 the lung may be responsible for the increase in RL. However, plasma levels of PGF2a and
10 6-keto PGFa were not affected by O3 or drug treatment; PGE^ was not affected by O3.
11 In an attempt to understand the involvement of eicosanoids in the increase in RL observed
12 with O3 exposure, Fouke et al. (1991) showed that exposure to 0.5 ppm O3 for 2 h caused
13 an increase in R, and a decrease in Cdyn in anesthetized dogs. Bronchoalveolar lavage fluid
14 from these dogs did not have any increase in 6-keto PGFla, PGF^, TXB2, or PGF2oi,
15 suggesting that these cyclooxygenase products were not involved in the changes in breathing
16 mechanics. Similar findings were observed by these authors after brief exposures to baboons
17 (see Section 6.2.5.2).
18 Gas trapping in the excised guinea pig lung was evaluated by water displacement to see
19 if guinea pigs became hyperresponsive to acid aerosols after an in vivo 2 h, 0.8 ppm
20 O3 exposure (Silbaugh and Mauderly, 1986). Ozone exposure followed by an air exposure
21 increased gas trapping to roughly the same extent as O3 followed by a sulfuric acid exposure
22 (1 h, 12 mg/m3) when compared to the air-only control response. These data indicate that
23 O3 causes an acute peripheral airway obstruction, but no additive or synergistic effect of
24 sulfuric acid aerosol was observed.
25
26 Airway Reactivity
27 Probably, the most extensive amount of laboratory animal research has been conducted
28 concerning the role of O3 in producing acute airway injury resulting in an increase in airway
29 reactivity (U.S. Environmental Protection Agency, 1986; Table 6-10). Much of this research
30 has used O3 exposures that are never encountered in the ambient environment (5:3 ppm for
31 ^30 min); thus, their relevance may be questioned. However, the studies are the most
December 1993 5.99 DRAFT-DO NOT QUOTE OR CITE
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1 thorough mechanistic account of such 03 effects and have shown some agreement with
2 human O3 exposure studies (Chapter 7) and, therefore, are briefly summarized here. The
3 literature is focused around five primary issues that in recent years have been more
4 thoroughly evaluated.
5
6 Concentration and Peak Response Time. Easton and Murphy (1967) were the first to
7 demonstrate an increased responsiveness in unanesthetized guinea pigs post-O3 exposure (2 h,
8 0.5 to 7 ppm). In their study, responsiveness was assessed by increased mortality due to
9 severe histamine-induced bronchoconstriction, as well as by increased RL and decreased
10 Cdyn. Lee et al. (1977) examined anesthetized dogs exposed to O3 (0.7 to 1.2 ppm, 2 h) via
11 a trachea! tube and determined that increased airway reactivity to inhaled histamine occurred
12 24 h, but not 1 h, postexposure. A similar experiment, done in unanesthetized sheep by
13 Abraham et al. (1980), indicated that airway responsiveness was increased at 24 h, but not
14 immediately after a 2-h exposure to 0.5 ppm O3. After exposure to 1 ppm O3, an increase
15 in baseline RL was reported, as well as increased reactivity immediately and 24 h
16 postexposure. In apparent contradiction, Holtzman et al. (1983a) showed that airway
17 reactivity increased markedly 1 h after exposure to 2.2 ppm O3 for 2 h and was less evident
18 at 24 h postexposure in dogs. Gordon and Amdur (1980) also reported that airway reactivity
19 in guinea pigs was maximal 2 h after a 1-h exposure to 0.1, 0.2, 0.4, or 0.8 ppm O3, as
20 defined by a significant increase in RL or decrease in Cdyn after a single subcutaneous
21 challenge of histamine. The effect on RL was concentration-dependent, but was significant
22 only at 0.8 ppm. For Cdyn, there was no concentration-related response, but all
23 O3 exposures exacerbated the decrease in Cdyn after histamine relative to the air-exposed
24 group. The site of bronchoconstriction was suggested to be the conducting airways, rather
25 than the parenchyma, because dynamic compliance was affected and static compliance was
26 not (Gordon et al., 1984).
27 Recently, two studies have reported increased airway reactivity in rats to inhaled
28 methacholine after a 0.3-ppm (5-h) (Gross and Sargent, 1992) or 1.0-ppm (2-h) (Uchida
29 et al., 1992) O3 exposure. These conflict with the only other published study in rats also
30 using inhaled methacholine, which reported the inability to produce consistent increases in
31 airway reactivity after exposure to less than 4 ppm 63 (Evans et al., 1988). Tepper et al.
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1 (1993) reported that airway hyperresponsiveness in rats challenged with intravenous
2 acetylcholine only occurred around 1 ppm or higher. In these latter studies, exposure
3 durations ranged between 2 and 7 h, and in some tests, CO2 was added to the exposure to
4 increase ventilation. Similarly, no increased airway reactivity was observed in three strains
5 of rats (Fischer 344, Sprague-Dawley, Wistar) to inhaled methacholine after an 8-h nocturnal
6 exposure to 1.0 ppm O3 (Costa et al., 1993). Although guinea pigs are more responsive than
7 rats, they are not as responsive as humans to O3-induced increased airway reactivity, even
8 under optimal conditions (Tepper et al., 1993).
9 To examine the role of duration of exposure upon experimental outcome, Nishikawa
10 et al. (1990) exposed guinea pigs to concentration by time (C x T) products of 30 (1 ppm x
11 30 min), 90 (1 ppm x 90 min), 90 (3 ppm x 30 min), and 360 (3 ppm X 120 min)
12 ppm • min. After exposure, specific airway resistance (SRaw) during an inhaled
13 methacholine challenge was measured in unanesthetized animals at 5 min, 5 h, and 24 h.
14 In all but the 1 ppm-90 min exposure group, there was an increase in baseline SR^, at
15 5 min, but the response was neither concentration nor C x T dependent. At 5 min
16 postexposure, increased airway responsiveness was not observed at 30 ppm • min. Airway
17 hyperresponsiveness was observed at 90 ppm • min, using either exposure scenario (1 ppm
18 for 90 min or 3 ppm for 30 min), and the response to the 360 ppm • min was greater than
19 that observed with 90 ppm • min exposure. Significant increases in airway responsiveness at
20 both 5 and 24 h postexposure were only observed in the 360 ppm • min group. The authors
21 concluded that exposure duration was an important determinant of O3-induced airway
22 hyperresponsiveness.
23
24 Inhaled Versus Intravenous Challenge. In a follow-up study using a similar exposure
25 protocol as described above, Abraham et al. (1984) observed increased responsiveness to
26 intravenous (iv) carbachol 24 h postexposure; inhaled carbachol did not produce a similar
27 response. The authors interpreted this result to indicate a decreased penetration of the
28 carbachol aerosol in O3-exposed animals compared with the direct stimulation of smooth
29 muscle by the iv route. Similarly, Roum and Murlas (1984) observed that (^-induced
30 hyperresponsiveness was similar for inhaled versus iv acetylcholine or methacholine
31 challenge through 14 h postexposure, but after that time, only iv administration revealed a
December 1993 6_10j DRAFT-DO NOT QUOTE OR CITE
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1 persistent O3-related response. In contrast, Yeadon et al. (1992) reported that guinea pigs
2 exposed to 3 ppm for 30 min were hyperresponsive to inhaled histamine, serotonin,
3 acetylcholine, and substance P, but were not hyperresponsive to O3 after iv administration of
4 the same agonists. Similarly, Bethel and McClure (1990) showed that dogs were
5 hyperresponsive to inhaled acetylcholine, but not by parenteral administration. Tepper et al.
6 (1993) and Uchida et al. (1992) also showed that rats were more sensitive to inhaled
7 methacholine than to iv administration of the agonist.
8
9 Neurogenic Mediation. Lee et al. (1977) reported increased airway responsiveness to
10 histamine in dogs exposed to 0.7 or 1.2 ppm O3 for 2 h. Atropine and vagal-blockade were
11 effective in reducing the O3-induced hyperresponsiveness to histamine, suggesting that
12 heightened vagal activity was responsible. Katsumata et al. (1990) also showed that in the
13 cat, airway hyperresponsiveness to histamine could be attributed to cholinergic reflex. This
14 is in apparent contrast to the increased O3-induced (1.0 to 1.2 ppm O3, 2 h) responsiveness
15 to histamine (subcutaneous) that was not blocked by atropine or vagotomy, indicating
16 minimal vagal involvement in guinea pigs (Gordon et al., 1984). In agreement, Jones et al.
17 (1987) found that hexamethonium, a ganglionic blocker, did not prevent O3-induced
18 hyperresponsiveness in dogs exposed to 3 ppm for 0.5 h via an endotracheal tube. Similarly,
19 Yeadon et al. (1992) showed that atropine or bilateral vagotomy only partially reduced the
20 hyperresponsivenss in guinea pigs exposed to 3 ppm for 120 min, but did not block the
21 response in animals exposed for only 30 min.
22 A role for prejunctional muscarinic receptors has been demonstrated by Schulteis et al.
23 (1992). The M2 receptor, which is inhibitory for acetylcholine release, was shown to be
24 defective immediately after a 4-h, 2-ppm O3 exposure in guinea pigs. Thus, although species
25 difference may be important, the role of the cholinergic system in O3-induced airway
26 hyperresponsiveness has yet to be firmly established.
27 Peptidergic mediators have also recently been suggested as important modulators of this
28 response. Murlas et al. (1992) demonstrated that phosphoramidon, an inhibitor of neutral
29 endopeptidase (NEP), increased the responsiveness to substance P in air-exposed, but not
30 O3-exposed (3 ppm, 2 h), guinea pigs. Substance P-induced bronchoconstriction in
31 air-exposed animals was increased after phosphoramidon because NEP degrades substance P.
December 1993 6-102 DRAFT-DO NOT QUOTE OR CITE
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1 The finding was associated with a decrease in tracheal NEP in Qj-exposed animals.
2 Additionally, the increased airway responsiveness in ^-exposed animals was reversed by
3 inhalation of partially purified NEP. Taken together, these results suggest that O3 inactivates
4 NEP, thus increasing the response to endogenous tachykinin release. A similar result was
5 obtained by Yeadon et al. (1992) in guinea pigs exposed to 3 ppm Q$ (30 or 120 min) and
6 challenged with aerosolized substance P after pretreatment with the NEP inhibitors
7 phosphoramidon, thiorphan, and bestatin. Tepper et al. (1993) depleted guinea pigs of
8 substance P using multiple doses of capsaicin and found that airway reactivity after a
9 2-h exposure to 1 ppm O3 was partially blocked. However, although tracheal vascular
10 permeability was also blocked by capsaicin pretreatment, protein influx into the BAL and
11 tachypnea were not blocked. On the other hand, Evans et al. (1989) did not find increased
12 tracheal vascular permeability in rats exposed to 4 ppm O$ for 2 h. These studies suggest,
13 at least for the guinea pig, that enhancement of the substance P response, by inhibition of
14 NEP, may be important in O3-induced hyperresponsiveness.
15
16 Inflammation. Holtzman et al. (1983b) found a strong association between increased
17 airway responsiveness and increased PMNs present in the tracheal biopsy of dogs 1 h after a
18 2-h O3 exposure to 2.1 ppm. Fabbri et al. (1984) extended these findings, showing an
19 association between increased airway reactivity and increased lavageable inflammatory cells
20 from the distal airways of dogs. Further support for this hypothesis was engendered by the
21 demonstration that in PMN-depleted dogs (produced by administration of hydroxyurea),
22 O3-induced airway hyperresponsiveness was blocked. This is in contrast to Murlas and
23 Roum's (1985a) finding in guinea pigs exposed to 3 ppm for 2 h that indicate that increased
24 airway reactivity, mucosal injury, and mast cell infiltration occur before PMN influx. The
25 authors speculate that PMN influx is a response to the damage, not a cause of the increased
26 airway reactivity. Furthermore, Murlas and Roum (1985b) showed that PMN depletion in
27 the guinea pig with cyclophosphamide did not prevent O3-induced airway
28 hyperresponsiveness. Similar results were obtained by Evans et al. (1988), who reported that
29 airway hyperresponsiveness was not accompanied by airway PMN influx in rats, and by load
30 et al. (1993), who showed that adding human PMNs to the pulmonary circulation of the rat
31 lung during a 3-h, 1.0-ppm exposure to O3 did not further enhance O3-induced airway
December 1993 6-103 DRAFT-DO NOT QUOTE OR CITE
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1 reactivity. Beckett et al. (1988) evaluated dogs exposed for 2 h to 1 ppm O3 directly to the
2 peripheral airways via a wedged bronchoscope. Fifteen hours postexposure, the exposed
3 peripheral airway segments were hyperresponsive to aerosolized acetylcholine. However, at
4 the site of increased responsiveness, there was no association with increased PMNs, mast
5 cells, or mononuclear cells. Such studies agree with perhaps the most definitive study of this
6 hypothesis (Li et al., 1992), which used monoclonal antibodies (CDllb/CD18) to prevent
7 PMN influx into the airways. When PMNs were present in the circulation, but prevented
8 from entering the lung, the dogs were still hyperresponsive after a 30-min exposure to 3 ppm
9 O3. Thus, in three species, it appears that PMN influx may be associated with O$ exposure,
10 but is not necessary for producing airway hyperresponsiveness.
11 Several studies have suggested that arachidonic acid metabolites may be important in
12 O3-induced airway hyperresponsiveness. Although the primary source of arachidonic acid
13 metabolites is suspected to be inflammatory cells in the lung, cells other than PMNs could be
14 responsible for the liberation of arachidonic acid metabolites. Only one study in dogs
15 indicates that blockage of cyclooxygenase products with indomethacin can protect animals
16 from developing airway hyperresponsiveness (O'Byrne et al., 1984). However, several more
17 recent studies have found that cyclooxygenase inhibitors were ineffective in blocking this
18 response (Lee and Murlas, 1985; Holroyde and Norris, 1988; Yeadon et al., 1992). Two
19 papers indicated the importance of LTs, as demonstrated by the inhibition of the
20 hyperresponsiveness with the prior administration of 5-lipoxygenase inhibitors to guinea pigs
21 (Lee and Murlas, 1985; Murlas and Lee, 1985). In contrast, Yeadon et al. (1992) found that
22 a specific 5-lipoxygenase inhibitor did not block the response in guinea pigs. One study with
23 dogs showed that TX generation may be important in this phenomenon (Aizawa et al., 1985),
24 but more recently, two papers from the same group as the original paper have dispelled that
25 notion (Jones et al., 1990, 1992). Furthermore, exposure to 0.5 ppm O3 for 2 h caused a
26 decrease in the provocative dose of methacholine necessary to cause a 50% increase in RL in
27 anesthetized dogs (Fouke et al., 1991). Bronchoalveolar lavage on these dogs did not show
28 in any increase in 6-keto PGFla, PGF^, TXB2, or PGF2oi, suggesting that these
29 cyclooxygenase mediators of inflammation were not involved in the changes in airway
30 reactivity. In summary, the initial hypothesis of the role of PMNs or PMN-derived products
31 in O3-induced airway hyperresponsiveness is questionable because most newer studies using
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1 more specific inhibitors of PMNs and cyclooxygenase, 5-lipoxygenase, and TX receptors
2 indicate the lack of a protective effect.
3
4 Interactions with Biologies. In mice, Osebold et al. (1980) showed that there was an
5 increase in the percentage of animals sensitized to ovalbumin after 3 to 5 days of continuous
6 exposures to 0.5 and 0.8 ppm O3. Matsumura (1970a) and Yanai et al. (1990) made similar
7 findings in guinea pigs and dogs exposed acutely to higher O3 concentrations.
8 Ozone (1 ppm, 2 h) may also increase hyperreactivity associated with virus exposure.
9 Tepper et al. (1993) exposed rats to O3 either before or during an influenza virus infection.
10 Rats exposed to O3 before virus infection were more hyperresponsive to inhaled
11 methacholine 3 days later (at a time when there was no hyperresponsiveness to C^ alone)
12 than were virus-only exposed animals. An additive effect was observed in virus-infected rats
13 when O3 exposure was immediately before methacholine challenge.
14
15 Extended Functional Characterizations
16 Extended characterizations of pulmonary function in laboratory animals indicate that the
17 general pattern of functional impairment reported in human studies is also observed in animal
18 studies of acute O3 exposure. Anesthetized and ventilated cats showed a general decline in
19 vital capacity (VC), static lung compliance, or diffusing capacity for carbon monoxide
20 (DLco) ^h exposures up to 6.5 h of 0.26 to 1.0 ppm O3 (Watanabe et al., 1973). Inoue
21 et al. (1979) observed functional evidence of premature airway closure, as indicated by an
22 increase in closing capacity, residual volume, and closing volume, after rabbits were exposed
23 to 0.24 or 1.1 ppm for 12 h. The volume-pressure curve indicated increased lung volume at
24 low distending pressures; additionally, nonuniform distribution of ventilation was observed.
25 The effects were most prominent 1 day following exposure and had mostly subsided by
26 7 days postexposure.
27 Most studies of O3 in experimental animals make little effort to mimic human study
28 designs, thereby further confounding the extrapolation of their results to humans. Recently,
29 however, rat studies involving periods of intermittent CC^-induced hyperventilation to
30 enhance the delivered dose of O3, have attempted to capitalize on the qualitative similarity of
31 the rat and human maximum expiratory flow-volume curves as a potentially sensitive
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1 endpoint of toxicity (Costa et al., 1988; Tepper et al., 1989). In the rat, forced vital
2 capacity decreases acutely with O3 exposure, and this response has been mathmatically
3 modeled for O3 concentrations between 0.35 and 0.8 ppm with exposure durations between
4 2 and 7 h (Tepper et al., 1989). The magnitude of response is apparently less than that
5 observed in humans (Tepper et al., 1993), although the extent to which anesthesia mitigates
6 the rat response, or that there are inherent species differences in dosimetry or sensitivity, is
7 not clear from these studies.
8 Beside changes in the flow-volume curve, changes in lung diffusion can be observed.
9 In a study that examined concentration, duration, and ventilation factors, rats were exposed
10 for 2 or 7 h to 0.5 or 0.8 ppm O3 with intermittent 8% CO2 to hyperventilate (*2 to
11 3 times resting VE) the animals as an exercise analogue to human exposures (Costa et al.,
12 1988). The DLCO values were reduced by 10% at both 0.5 ppm time points and by
13 12% with a 2-h exposure to 0.8 ppm. Exposure to 0.8 ppm for 7 h, however, greatly
14 exacerbated the alveolar lesion, with a resultant 40% reduction in the DLcQ. Static
15 compliance was only affected at this latter exposure concentration and duration. This
16 O3-induced reduction in DLcO appeared to correlate with the degree of lung edema in
17 affected animals. Yokoyama et al. (1987) found decreases in rat lung volumes (functional
18 residual capacity [FRC] and residual volume [RV]), static compliance (from the volume-
19 pressure curve), and maximal flow at 50% of VC after a 24-h exposure to 1 ppm O3.
20 Flow-volume curves and measurements of regional distribution of ventilation, using a
21 positron camera, were evaluated in anesthetized, paralyzed dogs acutely exposed, via a
22 stainless steel tracheal tube, to 0.12, 0.22, and 0.45 ppm for 3 h (Morgan et al., 1986).
23 No changes in the flow-volume curve were observed at any concentration, but a less uniform
24 distribution of ventilation was noticed, with the greatest difference occurring between the
25 central and more peripheral regions. The authors conclude that the initial effect of
26 O3 appears to be obstruction of the small airways.
27 Miller et al. (1987, 1988) evaluated the effect of a 1-h exposure to 1.0 ppm C^ for
28 changes in a clinical-like battery of lung function tests in anesthetized, tracheostomized
29 guinea pigs. Decreases in lung volumes were noted at 2 h postexposure and were maximum
30 between 8 and 24 h postexposure, after which time they began to resolve. Alveolar
31 ventilation (VA) and DLcO were also decreased by exposure. The reduction in DLcO may
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1 have been initially (2 h postexposure) caused by a bronchoconstriction-related decrease in
2 VA. After this time, disproportionate ratios of DLco and VA suggest that different
3 mechanisms were responsible for the decreased DLCO- The authors speculate that this
4 probably involves the development of a peripheral inflammatory response (8 to 24 h
5 postexposure) because plasma concentrations of 6-keto PGFla and PGEj were also elevated
6 in guinea pigs exposed for 1 h to 1 ppm O3 (Miller et al., 1987). Significant increases in the
7 plasma and BAL concentrations of TXB2 were also observed following acute exposure of
8 guinea pigs to 1 ppm 03 (Miller et al., 1987) and humans to 0.4 or 0.6 ppm O3 (see Chapter
9 7). Both indomethacin and cromolyn sodium partially blocked the reduction in lung volumes
10 at 2 and 24 h postexposure (Miller et al., 1988). Indomethacin was ineffective in blocking
11 the O3-induced decrease in DLfjQ at either 2 or 24 h postexposure, but cromolyn sodium
12 blocked this O3 response. Both drugs were effective in blocking the O3-induced decrease in
13 VA at the same examination periods. These results suggest that eicosanoids produced from
14 an inflammatory response in the lung may be responsible for the observed changes in lung
15 function in guinea pigs. However, as noted above the role of eicosanoid mediators in
16 O3-induced lung injury is controversial.
17
18 6.2.5.4 Repeated Acute Exposure Experiments (More Than Three Days)
19 To date, few experimental animal studies have attempted to address the potential
20 significance of repeated (3 to 5 days) acute exposures to 0)3 (U.S. Environmental Protection
21 Agency, 1986; Table 6-9).
22
23 Ventilation
24 In the only laboratory animal study using a similar exposure protocol and analogous
25 experimental design as human repeated-exposure studies, Tepper et al. (1989) showed that
26 rats displayed an initial pulmonary irritant response (tachypnea) that attenuated after
27 5 consecutive days of exposure in a manner quite similar to the response pattern of humans
28 (see Section 7.2.1). Exposures were for 2.25 h and included challenge with CO2 during
29 alternate 15-min periods to augment ventilation (2 to 3 times VE — equivalent to light
30 exercise in humans). The functional changes were largest on Day 1 or 2, depending on the
31 parameter and the O3 concentration (0.35, 0.5, and 1.0 ppm were evaluated). Additionally,
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1 lung biochemical and structural consequences were examined at 0.5 ppm O3 and indicated
2 that several indices of lung damage increased (histopathology) or did not adapt (lavageable
3 protein), despite the loss of the functional response over the 5-day exposure period.
4 Functional attenuation, however, did not occur in the 1.0-ppm O3 group; such a nonreversing
5 effect has not been observed in humans. It is likely that this lack of reversal was attributable
6 to the high concentration of O3 and thus, may be predictive of the human response under
7 similar conditions.
8
9 Breathing Mechanics
10 In rats exposed to 1.0 ppm O3 for 6 h/day for 7 days, the only change in breathing
11 mechanics was an increase in RL (Yokoyama et al., 1989a). Whether attenuation occurred
12 cannot be ascertained because functional measurements were only obtained after the end of
13 exposure.
14
15 Extended Characterizations
16 Belgrade et al. (1988) evaluated mice exposed to 1.0 ppm O3 for 5 days (3 h/day), with
17 or without the inoculation of influenza virus on Day 2 of exposure. Ozone alone did not
18 cause an untoward effect on lung volumes, volume-pressure, and flow-volume relationships
19 when mice were evaluated 1, 4, and 9 days postexposure. Mice exposed to the combination
20 of virus and O3 showed a decrease in DLcO that persisted for 9 days.
21 A portion of the Tepper et al. (1989) study, discussed above, was conducted using
22 groups of animals that were exposed between 1 to 5 days to 0.5 ppm O3. Changes in the
23 shape of the flow-volume curve (as indicated by the change in forced expiratory flow at
24 25 % of VC) were maximal on Day 2 and then showed attenuation with further repeated
25 exposures.
26
27 6.2.5.5 Long-Term Exposure Studies
28 The question of degenerative or irreversible lung damage when exposure is extended
29 over periods of days to years remains paramount to the assessment of health risk. Several
30 new studies since the previous criteria document (U.S. Environmental Protection Agency,
31 1986) have been published using more integrated approaches (structure, function, and
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1 biochemical techniques) for understanding this problem. This is especially true for studies
2 evaluating near-lifetime exposures in rodent species.
3
4 Ventilation and Breathing Mechanics
5 Tepper et al. (1991) evaluated ventilation and breathing mechanics in rats exposed for
6 1,3, 13, 52, and 78 weeks to a simulated urban profile of 03 (Table 6-9). The exposure
7 consisted of a 5-day/week, 9-h "ramped spike" exposure that had an integrated average of
8 0.19 ppm and a maximum concentration of 0.25 ppm. During all other periods (13 h/day,
9 7 days/week), the exposure remained at a 0.06-ppm O3 background level. Pulmonary
10 function measurements were evaluated post-O3 exposure in response to a 0, 4, and 8 % CO2
11 challenge. Overall, there was a significant increase in expiratory resistance, but only at
12 78 weeks was resistance significantly different than the time-matched filtered-air control.
13 At all evaluation times, VT was reduced compared to control rats. This was especially true
14 during challenge with CO2. Frequency of breathing was significantly decreased when the
15 analysis included all evaluations times, but at no single evaluation time was the reduction
16 significant.
17 Other evidence of peripheral airflow abnormalities from extended exposures to O3 is
18 limited. Costa et al. (1983) exposed rats to 0.2 or 0.8 ppm O3 for 6 h/day, 5 days/week for
19 12 weeks and did not find a concentration-related increase in pulmonary resistance.
20 Yokoyama et al. (1984) measured increased central resistance in rats exposed for 30 days to
21 1.0 ppm, but found increased peripheral airway resistance when exposure was for 60 days to
22 0.5 ppm. These changes were consistent with morphological findings of mucus in the large
23 bronchi of rats exposed to 1.0 ppm compared to the rats exposed to 0.5 ppm. These data
24 also agree with a study by Wegner (1982) that suggests the occurrence of airflow
25 obstruction, revealed in terms of small increases in peripheral airways resistance
26 (as measured by oscillation harmonics) that were observed in monkeys after 1 year of
27 exposure to 0.64 ppm (8 h/day, 7 days/week).
28
29 Airway Reactivity
30 No studies of airway reactivity after long-term exposures were reported before 1985.
31 Since then, several studies have reported no increase in reactivity with daily O3 exposure
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1 (Table 6-10). Biagini et al. (1986) observed no changes in breathing mechanics, forced
2 expiratory flow parameters, or methacholine and platinum airway responsiveness in a group
3 of monkeys (cynomolgus) exposed to 1 ppm O3 6 h/day, 5 days/week for 12 weeks in a
4 study designed to examine the effects of combining O3 exposure with the respiratory
5 sensitizer platinum. Lee et al. (1989) also failed to observe an increase hi airway reactivity
6 after a 13-week, 6 h/day exposure to 0.25 ppm O3. Additionally, this exposure did not
7 potentiate airway responsiveness produced by a subsequent virus infection. Kagawa et al.
8 (1989) exposed guinea pigs 4 h/day, 5 days/week for 4 mo to 0.15 ppm O3. Baseline total
9 respiratory resistance and response to increasing concentrations of inhaled histamine were
10 assessed every 3 weeks, but did not change in response to O3. The only exception is the
11 Johnson et al. (1988) study that evaluated airway responsiveness in female rhesus monkeys
12 just before a 2-h single weekly exposure to 1 ppm O3 delivered via an endotracheal tube.
13 After 19 weeks of exposure, increased responsiveness to inhaled methacholine was observed
14 compared to the animal's historic control. The hyperresponsiveness persisted approximately
15 15 weeks after exposures were discontinued. Hyperresponsiveness to O3 was reinstated after
16 a similar 7-week exposure to the same animals. After this exposure regimen, animals
17 recovered in approximately 9 weeks, but hyperresponsiveness was again reinstated with four,
18 once-per-week exposures. The investigators described the effect of a 5-lipoxygenase
19 inhibitor on certain portions of this sequence; however, the descriptions of methods and
20 results were too scant to evaluate the effect of treatment on exposure.
21
22 Extended Functional Characterizations
23 The previous criteria document (U.S. Environmental Protection Agency, 1986)
24 cataloged several investigators that reported marginal increases in total lung capacity or its
25 component volumes in rats after intermittent or continuous exposures to ^0.25 ppm O3 for
26 4 to 12 weeks (Bartlett et al., 1974; Costa et al., 1983; Raub et al., 1983). In contrast to
27 these significant results, Yokoyama and Ichikawa (1974) previously had reported no effects
28 on rat static volume-pressure curves after a 6-week exposure to 0.45 ppm (6 h/day,
29 6 days/week). More recent studies are summarized in Table 6-9.
30 Exposures of rats to 0.7 ppm O3 for 28 days (20 h/day) showed an obstructive-type
31 lung lesion characterized by a significant reduction in forced expiratory flows, lung volumes,
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1 and DLcO, and a significant increase in FRC (Gross and White, 1986). These effects largely
2 reversed after an additional 9 weeks of clean air, but some airflow abnormalities persisted.
3 Tyler et al. (1988) exposed young monkeys to 0.25 ppm for 8 h/day repeatedly or
4 during alternate months of an 18-mo period and observed increased chest wall compliance
5 and inspiratory capacity. The authors speculated that perhaps this could occur through some
6 interference with lung maturation. This effect was greater than in animals exposed every
7 month of the 18-mo period; the morphometric assessment of the distal lung appeared to
8 corroborate these findings (see Section 6.2.4).
9 To address the issue of cumulative exposure over a near-lifetime, several rodent studies
10 have been performed using various exposure concentrations. With exposure of rats to
11 0.5 ppm O3 for 52 weeks (20 h/day, 7 days/week), increases in RV and FRC were apparent,
12 as was a fall in DL^O (Gross and White, 1987), suggesting substantial end-airway damage
13 and gas-trapping. After a 3-mo period in clean filtered air, these measurements were not
14 different than similarly treated, but air-exposed control rats. In partial contrast, 12 or 18 mo
15 of exposure to a daily urban profile of O3 (9-h time-weighted average of 0.19 ppm,
16 5 days/week; a background of 0.06 ppm 13 h/day, 7 days/week) resulted in small reduction
17 in lung volumes and an enhanced nitrogen washout pattern consistent with a stiffer, restricted
18 lung (Costa et al., 1993). Interestingly, in spite of mural remodeling of small airways
19 (which was concentration dependent), no evidence of airflow obstruction was apparent in this
20 study. However, in a cohort group of animals exposed at the same time, RL was increased
21 at all time points in unanesthetized animals, as described previously (Tepper et al., 1991).
22 Harkema (1993) exposed rats for 6 h/day, 5 days/week for 20 mo to either filtered air or
23 0.12, 0.5, or 1.0 ppm O3. Within 3 days of the end of exposure, an extended functional
24 evaluation was performed. Surprisingly, O3 caused little impact on respiratory function.
25 However, RV was decreased (between 21 and 36%), a finding similar, but of greater
26 magnitude, to the Costa et al. (1992b) study. The existing morphological data in monkeys at
27 the higher concentrations noted above appear consistent with end-airway remodeling, but no
28 clear functional evidence of obstruction has been described (Eustis et al., 1977; Wegner,
29 1982).
30
31
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1 6.2.5.6 Summary
2 Alterations in the pulmonary function of laboratory animals after exposure to O3 have
3 been reported by numerous investigators. These changes appear to be homologous with the
4 changes in pulmonary function observed in humans exposed acutely to O3 (Chapters 7 and
5 8). Although there are apparent differences in sensitivity among species, it is not clear
6 whether these differences are due to the use of anesthesia or restraint, or are differences in
7 tissue sensitivity or dosimetry.
8 Brief exposures to 03 of less than 30 min have been shown to produce reflex responses
9 (increased collateral resistance) and airway hyperresponsiveness. In the dog, these changes
10 appear to be, in part, related to parasympathetic stimulation and release of inflammatory
11 mediators. However, the relevance of these studies must be questioned because O3 was
12 delivered to a specific lung region via a bronchoscope and the contribution of collateral
13 resistance to total lung resistance was small.
14 With exposures lasting greater than an hour, a wide variety of effects have been
15 observed. Most notably, tachypnea (increased frequency of breathing and a decreased tidal
16 volume) has been noted in several species at exposures as low as 0.2 ppm for 3 h in rats
17 (Mautz and Bufalino, 1989). In addition to changes in breathing pattern, changes in
18 breathing mechanics (compliance and resistance) and increased airway reactivity have been
19 observed, but generally these effects have been reported at concentrations of 1 ppm or
20 greater. In dogs, RL increased and Cdyn decreased after a 2-h exposure to 0.5 or 0.6 ppm
21 O3 (Fouke et al., 1991; Mautz et al., 1985b). However, reactivity to bronchoconstrictors
22 has only been reported once at concentrations below 1 ppm O3 (guinea pig, 0.5 ppm, 2 h)
23 (Tepper et al., 1990). The mechanisms that may be responsible for the O3-induced increase
24 in airway reactivity have been extensively investigated; however, no firm conclusion can be
25 drawn. The most consistent evidence suggests a role for sensory afferent fibers, their
26 associated mediators (tachykinins) and the enzyme responsible for tachykinin degradation
27 (NEP). However, may studies suggest that the parasympathetic nervous system and
28 inflammatory cells and mediators may also play a role in O3-induced increase in airway
29 reactivity. Additionally, extended characterizations of pulmonary function indicate that the
30 general pattern of functional impairment seen in humans acutely exposed to high
31 concentrations of O3 (decreased lung volumes, diffusional disturbances, and inhomogeneity
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1 of ventilation) are also observed in animals exposed to high ambient (0.5 to 2.0 ppm) O3.
2 For example, FVC, DLcQ, and N2 slope decreased with increasing C x T products (0.5 and
3 0.8 ppm, 2 and 7 h) in rats (Costa et al., 1989).
4 With daily repeated exposure to O3, Tepper et al. (1989) showed attenuation of lung
5 function changes (tachypnea and flow volume curve) over 5 days (2 h/day to 0.35 to
6 1.0 ppm, with CO2 stimulation of breathing), similar to what is observed in repeatedly
7 exposed humans (Chapter 7). It is of interest that in this study, morphological changes
8 showed a progressive increase in severity, and other biochemical indicators of lung injury
9 (lavageable protein and antioxidants) did not show attenuation of the response over the same
10 time period. The findings from long-term exposures of O3 to laboratory animals are even
11 more difficult to summarize. Various findings in rats include no or minimal effects (Biagini
12 et al., 1986; Kagawa et al., 1989; Chang et al., 1992) or obstructive-like (Gross and White,
13 1986; Tepper et al., 1991) or restrictive-like lung lesions (Costa et al., 1992). However in
14 all cases, severe lung injury was not detected, and the physiological alterations that were
15 observed resolved several months after termination of exposure.
16
17 6.2.6 Biochemical Effects
18 6.2.6.1 Introduction
19 This section will summarize studies that describe acute and prolonged O3 exposures of
20 experimental animals where biochemical measurements of antioxidant and microsomal
21 enzyme activities, lipids, and proteins have been used to interpret and quantify some of the
22 functional effects of O3 discussed in other sections of this chapter. The identification of
23 these substances as molecular targets of O3 is discussed in Section 6.2.1. The ability to
24 extrapolate from in vitro to in vivo studies and from high to low levels of 03 are complicated
25 by an inability to detect biochemical changes in the whole lung when only a small proportion
26 of the lung might be affected by O3, especially at concentrations of O3 less than 1 ppm.
27 Interpretation of biochemical changes resulting from whole-lung measurements is also
28 complicated by the heterogeneity in cell types and functions present in lung tissue and the
29 changes in cell populations that result from (^-induced inflammatory cell infiltration and
30 epithelial cell and fibroblast proliferation.
31
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1 6.2.6.2 Effects of Ozone Exposure on Lung Lipid Metabolism
2 Arachidonate Metabolites
3 Ozone-induced damage to airway epithelia (Leikauf et al., 1988) and AMs (Madden
4 et al., 1991) in vitro has been associated with the production of arachidonic acid metabolites
5 by both cyclooxygenase and lipoxygenase pathways. These metabolites have been implicated
6 in a variety of different physiological processes that include changes in airway permeability,
7 in the infiltration of PMNs and eosinophils, and in airway smooth muscle reactivity discussed
8 elsewhere in this chapter (Sections 6.2.2 and 6.2.5). Leikauf et al. (1993) have examined
9 the effects of fatty acid O3-degradation products on human airway epithelial eicosanoid
10 metabolism and concluded that the stimulating effects were increased with product chain
11 length, with the 3-, 6-, 9-hydroxyhydroperoxides being more potent than their corresponding
12 aldehydes. Madden et al. (1993) concluded that aldehydic degradation products of
13 arachidonate and not hydrogen peroxide increased in vitro polarization of leukocytes, and
14 decreased peripheral blood T-cell mitogenesis and NK-cell cytotoxicity. In vivo experiments
15 of rabbits, guinea pigs, mice, and rats of different ages exposed to < 1.0 ppm 03 have
16 demonstrated increases in the products of arachidonic acid metabolics (see Section 6.2.2).
17
18 Surfactant
19 Surfactant having some PUFAs, cholesterol, and protein as possible targets of
20 O3 interaction might be expected to have an altered composition as a result of O3 inhalation.
21 However, surfactant isolated from rats following an 8-h exposure to 0.8 ppm retained its
22 ability to lower surface tension in spite of an increase in protein content (Nachtman et al.,
23 1986). In long-term exposure studies, monkeys were exposed for 8 h/day to 0.15 and
24 0.3 ppm O3 for 21 and 90 days (Rao et al., 1985a,b). After 21 days of exposure, there was
25 a relative increase in the proportion of PUFA in the percentage of BAL unsaturated fatty
26 acids (increases from 34% in air controls to 41, 42, and 45% in BAL lipids recovered from
27 monkeys exposed for 21 days to 0.15 ppm, 90 days to 0.15 ppm, and 90 days to 0.3 ppm
28 O3, respectively) (Rao et al., 1985b). The major increases were observed in linoleate (18:2)
29 and arachidonate (20:4). Because these PUFAs are potential targets for O3 interaction, their
30 increase rather than a decrease in BALF might best be explained by changes in surfactant
31 production associated with alveolar Type 2 epithelial proliferation (Section 6.2.4).
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1 Interestingly, a relative decrease in cholesterol ester with a concomitant increase in
2 phosphatidylcholine was observed that supports the hypothesis that cholesterol might
3 represent a major target of O3 interaction (Rao et al., 1985b; Pryor et al., 1992). The
4 observed O3-induced changes in BAL PUFA composition were consistent with those
5 previously reported by Roehm et al. (1972), but only for BAL lipids isolated from
6 vitamin E-depleted rats following 6 weeks of exposure to 0.5 ppm 03. Similarly, Wright
7 et al. (1990) were unable to detect changes in rat BAL lipid and fatty acid composition
8 following 0.12, 0.25, and 0.5 ppm O3 exposures for 20 h/day for 18 mo. Results from these
9 studies are summarized in Table 6-11.
10
11 Tissue Lipids
12 In vivo pulse-labeling with carbon-14 labeled acetate was used to estimate phospholipid
13 biosynthesis (Wright et al., 1990). Although found to be diminished at certain time points
14 (3 and 12 mo), no consistent trend could be demonstrated that would suggest that
15 O3 exposures of less than 0.5 ppm alter lung surfactant homeostasis. Bassett and Rabinowitz
16 (1985), using isolated perfused lungs taken from rats after 3 days of continuous exposure to
17 0.6 ppm O3, demonstrated an enhanced incorporation of glucose carbons into both fatty acid
18 and glycerol-glyceride moieties of lung lipids by 180 and 95%, respectively. The relative
19 increase in carbon incorporation into free fatty acids, phosphatidic acid, phosphatidyl
20 inositol, and sphingosine containing lipids was consistent with the needs of a dividing cell
21 population for increased lipids synthesis associated with alveolar epithelial proliferative
22 repair. It should be noted that in a separate study, under the same exposure conditions of
23 0.6 ppm O3 for 3 days, rat lungs demonstrated increased glycolytic activity and reduced
24 nicotinamide adenine dinucleotide phosphate (NADPH) generation consistent with the energy
25 and synthetic needs of a lung undergoing repair of (^-induced damage (Bassett and Bowen-
26 Kelly, 1986). Results from these studies are summarized in Table 6-11.
27
28 6.2.6.3 Effects of Ozone on Lung Antioxidant Systems
29 The O3-induced increased levels of the antioxidant NPSHs, mainly identified as GSH in
30 the lung, and the enzyme activities involved in its utilization, GSHPx and GST, and for
31 maintaining it in a reduced state, giutathione reductase (GR) and the NADPH-linked
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1 dehydrogenases of glucose-6-phosphate (G6PD) and 6-phosphogluconate (6PGD), have been
2 typically attributed to concurrent morphological changes rather than to any specific
3 biochemical response (U.S. Environmental Protection Agency, 1986). Numerous studies
4 conducted in mice, rats, and monkeys show increases in many of these enzyme activities at
5 exposures as low as 0.2 ppm O3 for 1 week (rat) (U.S. Environmental Protection Agency,
6 1986). The earlier research also included studies of age-dependent responsiveness of rats
7 (Tyson et al., 1982; Lunan et al., 1977, and Elsayed et al., 1982). Rats ranging in age from
8 5- to 90-days old were exposed to 0.8 or 0.9 ppm O3 for 3 or 4 days or for about 20 days,
9 depending on the experiment. Ozone altered activities of antioxidant enzymes in an age-
10 dependent manner. Generally, prior to wearing, enzyme activities decreased, and at older
11 ages, they increased. The reasons for these differences is not known, but differences in
12 (1) dose to the lung (due to differences in exposure concentrations in huddled neonates on
13 bedding prior to weaning or to differences in doses delivered to lung target sites), (2) basal
14 levels of antioxidants and antioxidant enzymes, or (3) cellular sensitivity. Increased lung
15 enzyme activities can result from either increased activity within a particular cell population
16 or increased numbers of cells with that activity. Age, nutritional, and species differences in
17 O3-mediated responses must therefore be interpreted with consideration of the underlying
18 morphological changes (Section 6.2.4). Relevant studies are summarized in Table 6-12.
19 An increase in lung alveolar Type 2 cells and hi infiltrating inflammatory cells
20 adequately explained the observed increases in succinate oxidase, G6PD, and 6PGD activities
21 observed after 3 days of continuous exposure of rats to 0.75 ppm O3 when represented on a
22 per milligram DNA basis (Bassett et al., 1988a). These cell types are enriched in
23 mitochondria and in NADPH-generating capacity needed for both lipid biosynthesis and GSH
24 maintenance. Similarly, no significant changes in these enzyme activities could be detected
25 after 3 days of exposure to the lower O3 concentration of 0.35 ppm, further illustrating the
26 need to take into account the concomitant changes in cell population and number when
27 interpreting whole-lung enzyme measurements. Increases of 150 and 108%, respectively,
28 were observed in the per milligram DNA activities of the ornithine carboxylase and
29 S-adenosyl-methionine decarboxylase enzymes involved in polyamine synthesis, which
30 together with enhanced tritiated thymidine incorporation into DNA, have been considered to
December 1993 6-117 DRAFT-DO NOT QUOTE OR CITE
-------
TABLE 6-12. EFFECTS OF OZONE EXPOSURE ON LUNG ANTIOXIDANTS4
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Ozone
Concentration
ppm
0.06
Base,
0.25
spike
0.12
0.20
0.64
0.35
0.75
0.4
0.41
0.45
0.5
0.5
0.5
0.64
ftg/m
118 Base,
490 spike
235
392
1,254
686
1,470
784
800
882
980
980
980
1,254
Exposure
Duration
Base 13 h/day,
7 days/week;
Ramped spike
9 h/day,
5 days/week for
12 mo
Continuous for
7 days
Continuous for
3 days
Continuous for
2 weeks
12 h during day or
night for 3 days or
continous for 72 h
Continuous for
2 days
Continuous for
5 days
Continuous for
5 days
2.25 h/day for
5 days
Continuous for
7 days
Species, Sex
(Strain)
Rat, M
(F344)
Rat, M
(S-D)
250-300 g
Rat, M
(Wistar)
200-250 g
Rat, M
(Wistar)
6 weeks old
Guinea pig
(Hartley)
6 weeks old
Rat, M
(Wistar)
Guinea pig, M
(Hartley)
9 weeks old
Rat, M
(S-D)
90 days old
Rat
(Long-Evans)
10 weeks old
Rat
(Long-Evans)
10 weeks old
Rat, M
(F344)
110 days old
Rat, M
(S-D)
3-5 weeks old
Observed Effect(s)
Whole lung increase in GSHPx and GSH reductase activities. SOD activity and NPSH content not affected.
Pretreatment with the H2O2 scavenger dimethylurea decreased Oj-induced tissue DNA and protein and BAL
protein, acid phosphatase, and Maceryl-iS-D-glucosaminidase. No effect of vitamin E or ^-carotene.
0.75 ppm Oj-induced whole lung increases in GSHPx and GR not significant when corrected for increases in
cell number. Increases in succinate oxidase, G6PD and 6PGD activities per DNA were consistent with
increased Type 2 and inflammatory cell content. No increases per DNA at 0.35 ppm 03.
Small increases in whole rat lung levels of NPSH, vitamin C, GSHPx. Guinea pig GSHPx and GSH
transferase activities decreased.
Rats: No effect of daytime exposure. Nighttime or continuous exposure increased activities of COH,
G6PDH, GR, and GSHPx. Guinea pig: No daytime-only exposure. No effect on GR or GSHPx, G6PDH
increased after nighttime or continuous exposure; lactate dehydrogenase activity increased only after
continuous exposure.
Large increase in omithine decarboxylase activity and DNA labeling reflecting polyamine metabolism and
DNA synthesis and/or repair, respectively.
Ozone increased lung putrescine in both vitamin E deficient or 1 ,000 lU/kg groups, but increases in
spermidine content and decarboxylase activities of ornithine and S-adenosy Imethionione only in vitamin E
deficient group.
Ozone increased lung vitamin E level in supplemented rats and remained unchanged in all other tissues
measured.
Lung glutathione initially enhanced, declining to control levels by Day 4. Lung ascorbate levels enhanced on
Days 3 and 5 only.
The whole lung 03-induced increase in ascorbate and GSH content unaffected by protein deficient diets.
Reference
Grose et al. (1989)
Warren et al. (1988)
Bassett et al. (1988a)
Ichinose and Sagai (1989)
vanBreeet al. (1992)
Elsayed et al. (1990)
Elsayed (1987)
Elsayed et al. (1990)
Tepper et al. (1989)
Dubick et al. (1985)
-------
TABLE 6-12 (cont'd). EFFECTS OF OZONE EXPOSURE ON LUNG ANTIOXTOANTS8
Ozone
Concentration
. 3
ppm fig/m
Exposure
Duration
Species, Sex
(Strain)
Age Observed Effect(s)
Reference
0.64 1,254 Continuous for
7 days
Rat, M Whole adult lung contents of Cu,Zn-SOD and GSHPx increased by 03 in all diet groups (ad libitum, 4-16%
(S-D) protein diets); GSHPx only increased in weanling rats fed 16% protein diet. Mn-SOD only increased in lungs
52 g and 295 g from 4 and 16% protein fed adult lungs.
Heng et al. (1987)
0.7
0.8
1,373 Continuous for
1-5 days
1,568 8h/dayfor
2 mo
Rat, M
(S-D)
45, 80, and
300 g
Rat, M
(S-D)
2 mo old
By 5 days, increased lung Cu,Zn-SOD, Mn-SOD, catalase, and GSHPx per DNA in all age groups. Adult
lungs: concomitant increases in mRNAs for Cu,Zn-SOD, catalase, and GSHPx without differences in mRNA
stability.
Absence of vitamin E exacerbates Oj-induced damage related to increases in whole lung levels of metabolic
enzymes. No additional amelioration by diet supplementation above 50 IU vitamin E.
Rahman et al. (1991)
Elsayed et al. (1988)
See glossary of terms and symbols for common abbreviations and acronyms.
Age or body weight at start of exposure.
-------
1 be more sensitive measures of biochemical changes in lungs from rats exposed continuously
2 for 3 days to 0.45 ppm O3 (Elsayed et al., 1990).
3 Microdissection techniques of lungs following short-term exposure of monkeys to
4 1.0 ppm for 2 h demonstrated a correlation between O3 dose, measured using 18O3,
5 decreased GSH, and the degree of epithelial cell injury of the respiratory bronchioles.
6 The potential role of superoxide dismutase (SOD) and catalase in protecting the lung
7 against O3 toxicity is not clear. Bassett et al. (1989), using a pretreatment with a phenyl-
8 urea compound (N[2-(2-oxo-l-imidazolindinyl)ethyl]-AT-phenylurea; EDU) that increased rat
9 lung SOD and catalase activities, failed to demonstrate any protection against acute lung
10 injury from a single 3-h exposure to 2.0 ppm. However, Zidenberg-Cherr et al. (1991) have
11 demonstrated that copper (Cu)- and manganese (Mn)-deprived mice might be more
12 susceptible to continuous O3 exposure of 1.2 ppm for 7 days. Rahman et al. (1991) have
13 demonstrated that lungs from O3-exposed rats had increased activities of Cu, zinc (Zn)-SOD,
14 Mn-SOD, catalase, and GSHPx 5 days following exposure to 0.7 ppm O3. These increases
15 were attributed to enhanced gene expression, indicated by higher mRNA concentrations,
16 rather than to the infiltration of cells enriched with these enzyme activities. Chronic
17 exposure of rats to an urban pattern of O3 did not affect SOD activity in rats, although
18 GSHPx and GR activities per lung were increased (Grose et al., 1989). In contrast, the use
19 of microdissection techniques following 20 mo of rat exposures to 0, 0.5, and 1.0 ppm have
20 shown increases in SOD, GST, and GSHPx per mg DNA in the distal trachea and distal
21 bronchioles with decreases in GST and GSHPx activities in major bronchi and increases of
22 GSHPx and catalase in minor bronchi (Duan et al., 1993).
23 Representing data on a per gram wet lung basis, Ichinose and Sagai (1989)
24 demonstrated increases in lung NPSH, vitamin C, and GSHPx, but no effect on vitamin E
25 levels, after continuous exposure of rats to 0.4 ppm O3 for 2 weeks. In contrast, guinea pig
26 lungs exhibited no changes in these antioxidant components when similarly exposed.
27 However, although using the higher concentration of 0.64 ppm for 7 days of continuous
28 O3 exposure, Dubick et al. (1985) demonstrated that whole lung content of ascorbate and
29 GSH was elevated, these changes were not significantly different when the data were
30 represented on a per 100 g wet tissue basis. Rat BAL analysis following a 12-mo exposure
December 1993 6-120 DRAFT-DO NOT QUOTE OR CITE
-------
1 to an urban pattern of O3 demonstrated decreased vitamin E and enhanced ascorbate and
2 protein levels (Grose et al., 1989).
3 In order to demonstrate that dietary vitamin E reduces the effects of O3 exposure on
4 lung biochemical parameters, comparisons between vitamin E depleted and supplemented
5 diets have been used (U.S. Environmental Protection Agency, 1986) and reviewed by Pryor
6 (1991). Elsayed et al. (1988) fed rats a test diet containing 0 or 50 International Units (IU)
7 of vitamin E/kg for 2 mo prior to exposure to 0.8 ppm 03 8 h/day for 7 days. Ozone
8 exposure increased the whole lung activities of mitochondria!, microsomal, and cytosolic
9 enzymes. Vitamin E deficiency alone had no significant effect on these lung enzyme
10 measurements measured on a per lung basis, but its addition to the diet (50 IU vitamin E/kg)
11 prior to O3 exposure diminished the observed O3-induced increases in mitochondrial
12 succinate cytochrome c reductase and GSHPx, microsomal NADPH cytochrome c reductase,
13 and cytosolic GSHPx and SOD observed in vitamin E-deficient rats by up to 50%.
14 Additional experiments using a relatively low range of vitamin E supplementation for short
15 time periods demonstrated that although absence of vitamin E in the diet exacerbates the
16 effects of O3 on lung injury, the magnitude of a protective effect does not increase
17 proportionately with increased dietary vitamin E. These data support the conclusion that any
18 supplementation beyond the normal recommended daily allowance for vitamin E might not
19 necessarily provide humans with any additional protection against the effects of ambient
20 O3 exposure (Pryor, 1991). However, possible failure in these animal experiments to reach
21 a steady-state tissue level of vitamin E might have obscured protective effects.
22
23 6.2.6.4 Effects of Ozone on Lung Protein Metabolism
24 Exposure of rodents to ^0.45 ppm &$ has been associated with increases in lung
25 collagen, collagen synthesis, and prolyl hydroxylase activity associated with fibrogenesis
26 (U.S. Environmental Protection Agency, 1986). These earlier studies showed an influence of
27 exposure pattern on the responses. When rats were exposed to 0.8 ppm for 7 days, prolyl
28 hydroxylase activity continued to increase, but hydroxyproline content plateaued about Day 3
29 of exposure and remained elevated 28 days after exposure ceased (Hussain et al., 1976 a,b).
30 Last et al. (1984b) employed 90-day exposure regimens of rats to 0.96 ppm O3 that included:
31 (1) a continuous 90-day exposure and (2) intermittent units of 5 days (8 h/day) of O3 and
December 1993 6-121 DRAFT-DO NOT QUOTE OR CITE
-------
1 9 days of air, repeated 7 times with a total of 35 03 exposure days over the 90-day period.
2 Both groups had equivalent increases in lung collagen content. When durations were
3 decreased to 3 weeks, the continuous and intermittent (1 week O3, then 2 weeks air)
4 regimens resulted in equivalent increases in lung collagen. In nonhuman primates receiving
5 0.25 ppm daily or seasonally (every other month only) for 18 mo, only the seasonal group
6 had an increase in collagen (Section 6.2.4, Tyler et al., 1988).
7 More recently, Choi et al. (1993) examined the earliest time points from the onset of
8 continuous O3 exposure of rats to 1.0 ppm that caused alterations in extracellular matrix
9 protein gene expression. These authors demonstrated an early increase in lung fibronectin
10 mRNA at 2 days, which preceded an increase in Type I collagen mRNA observed at 4 days.
11 However, increased collagen content indicated by lung hydroxyproline content was not
12 significantly enhanced until after 7 days of exposure. Pickrell et al. (1987a) demonstrated
13 concentration-dependent decreases in antiproteinase activities in serum and lung tissue of rats
14 exposed to concentrations of 0.5 and 1.0 ppm O3 for 48 h. Exposure to 1.0 ppm was
15 accompanied by a concomitant increase in inflammatory cell-derived proteinases. A second
16 study that examined lung collagen metabolism and proteinolysis in rat lungs exposed to
17 0.57 and 1.1 ppm O3 for 19 h/day for 11 days suggested that collagen accumulation in part
18 might result from decreased collagen degradation (Pickrell et al., 1987b).
19 Chronic exposures of monkeys to 0.61 ppm 8 h/day for 1 year demonstrated increased
20 lung collagen content even after 6 mo postexposure in air (Last et al., 1984b). Further
21 analysis has also demonstrated that the collagen isolated from these O3-exposed lungs
22 exhibited abnormalities as indicated by increased levels of the disfunctional crosslink
23 dehydrodihydroxylsinonorleucine (DHLNL) and the ratio of DHLNL to
24 dehydrohydroxylysinonorleucine (HLNL) (Reiser et al., 1987). Although collagen content
25 remained elevated, difunctional DHLNL and HLNL crosslink levels returned to normal by
26 6 mo postexposure, whereas trifunctional mature crosslinks (hydroxypyridinium) remained
27 elevated. These data suggest that structurally abnormal collagen is actively synthesized
28 during O3 exposure and that it becomes irreversibly deposited in the lungs.
29 Because O3-induced lung lesions are multifocal by nature, it is reasonable that changes
30 in collagen content within the lesions might not be easily detectable by measuring alterations
31 in whole lung hydroxyproline at earlier time points or in those experiments that have used
December 1993 6-122 DRAFT-DO NOT QUOTE OR CITE
-------
1 lower O3 concentrations. For example Wright et al. (1988), having calculated values for the
2 extent of collagen deposition using measured synthesis rates, concluded that 18 mo of
3 exposure (20 h/day) to concentrations up to 0.5 ppm O3 did not change either synthesis or
4 accumulation of lung collagen. On the other hand, Chang et al. (1992) demonstrated
5 sustained thickening of rat lung extracellular matrix on long-term exposure to a simulated
6 urban pattern of O3 exposure (baseline of 0.06 ppm, 7 days/week, with a slow rising peak
7 for 9 h/day, 5 days/week to 0.25 ppm) up to 38 weeks. Results from studies of lung protein
8 metabolism are summarized in Table 6-13.
9 Ozone exposure also affects airway secretion of mucous glycoproteins. After 2 days of
10 exposure of sheep to 0.5 ppm O3, with subsequent evaluation of trachea! sulfated
1 glycoprotein and ion fluxes in vitro, there was an increase in basal secretion that was
2 associated with a moderate hypertrophy of lower trachea! submucosal glands (Phipps et al.,
3 1986). Although 7 days of exposure resulted in hypertrophy of upper and lower trachea!
4 submucosal glands, glycoprotein secretion was reduced but chloride secretion was increased,
5 which can be explained by a relative decrease in gland mucus content.
6
7 6.2.6.5 Effects of Ozone Exposure on Lung Xenobiotic Metabolism
8 Previous studies have demonstrated that exposure to 0.75 to 1.0 ppm O3 for a few
9 hours diminishes microsomal cytochrome P-450 content and decreases the activities of
10 benzo[a]pyrene hydroxylase and benzphetamine Af-demethylase of lungs isolated from several
11 different experimental animal species (U.S. Environmental Protection Agency, 1986).
12 Because bronchiolar Clara cells and alveolar Type 2 cells are considered to be relatively
13 enriched with microsomal cytochrome P-450 enzyme systems, it is reasonable that damage
14 and subsequent proliferative repair of these cells types would be expected to change the
15 lung's capacity to conduct xenobiotic metabolism. In a series of rat studies, Takahashi et al.
16 (1985) and Takahashi and Miura (1985, 1987, 1989, 1990) have demonstrated that although
17 intermittent exposure of 0.4 ppm for 7 h/day for 14 days did not affect microsomal
18 metabolism, increasing the concentration to 0.8 ppm (Takahashi et al., 1985) or exposing the
19 rats continually to 0.2 and 0.4 ppm for 14 days (Takahashi and Miura, 1985) increased
20 cytochrome P-450 content and the activities of cytochrome P-450 reductase, benzo[a]pyrene
21 hydroxylase, and 7-ethoxycoumarin O-deethylase (see Table 6-14). These increased
December 1993 6-123 DRAFT-DO NOT QUOTE OR CITE
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Concentration-dependent increases in NADPH-cytochrome P-450 reductase ai
cytochrome P-450 content during 4-12 weeks exposure, reaching a maximum
with concomitant increases in benzofajpyrene hydroxylase and 7-ethoxycoum
O-deethylase activities. NADH-cytochrome b$ reductase activity unaffected .
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1 microsomal activities were observed to be sustained in rats exposed continuously for up to
2 12 weeks to 0.1 to 0.4 ppm O3, with a greater response being observed in the activity of
3 benzphetamine AT-demethylase, suggesting preferential increase in the associated P-450
4 cytochrome isozyme (Takahashi and Miura, 1987). Ozone-induced increases in cytochrome
5 P-450 have also been shown not to result in concomitant increases in all microsomal
6 xenobiotic metabolism (Rietjens et al., 1988). Rat lung microsomal benzo[a]pyrene
7 oxidation and benzphetamine demethylation were found to be enhanced after a 6-mo
8 continuous exposure to 0.5 ppm (Filipowicz and McCauley, 1986a). More recent studies
9 have explored O3-induced changes in cytochrome P-450 isozyme patterns and correlated
10 changes in lung xenobiotic metabolism with Clara cell enlargement and increased numbers
11 during a 14-day exposure of rats to 0.4 ppm O3 (Takahashi and Miura, 1990; Suzuki et al.,
12 1992). These authors also demonstrated by immuno-electron microscopy the presence of
13 cytochrome P-450b (HB1) in the Clara cell endoplasmic reticulum.
14 Changes in the extent and pattern of formation of benzo[fl]pyrene products were
15 investigated by Bassett et al. (1988c) in lungs from rats undergoing epithelial proliferative
16 repair resulting from 3 days of continuous exposure to 0.6 ppm O3. Although metabolism to
17 all benzo[a]pyrene metabolites was enhanced 4.7-fold, the relative proportion of metabolism
18 involving quinone formation was enhanced from 10 to 25%. The toxicity of other inhaled
19 pollutants that undergo lung xenobiotic metabolism might therefore be dependent not only
20 O3-induced changes in airway protective barrier function and clearance mechanisms, but also
21 on O3-induced changes in epithelial cell activation and detoxification reactions.
22
23 6.2.6.6 Summary
24 The biochemical mechanisms by which O3 causes lung toxicity at the onset of exposure
25 is a complex process that appears to involve a series of secondary reactive intermediates
26 resulting from an initial interaction with either lipids, enzymes, coenzymes, or proteins.
27 Acute exposures to relatively high concentrations of O3 (> 1 ppm) have been used to
28 demonstrate in vivo cleavage of PUFA double bonds (Rabinowitz and Bassett, 1988),
29 breakdown of cholesterol (Pryor et al., 1992), and production of aldehydes (Cueto
30 et al., 1992) and H2O2 (Warren et al., 1988) consistent with ozonation of lung fluid lining
31 components and cell membranes being initial targets of O3 interaction (Pryor, 1991). The
December 1993 5.127 DRAFT-DO NOT QUOTE OR CITE
-------
1 involvement of free radical intermediates and reactive oxygen intermediates in subsequent
2 cell damage resulting from autoxidation chain reactions has been illustrated in studies using
3 lower O3 concentrations (< 1 ppm) by the use of electron spin-trapping techniques (Kennedy
4 et al., 1992) and by demonstrating diminished lung damage as a result of either having the
5 lipophilic antioxidant vitamin E in the diet (Elsayed et al., 1988) or after pretreatment with
6 the H2O2 scavenger dimethylthiourea (Warren et al., 1988). The protein A component of
7 pulmonary surfactant has also been identified as a target of O3 interaction and has been
8 associated with changes in its ability to control surfactant homeostasis (Oosting et al., 1991c,
9 1992). However, 18 h of exposure of rats to 0.8 ppm did not affect the ability of the lavage
10 fluid to lower surface tension (Nachtman et al., 1986). Surfactant PUFA and cholesterol
11 ester composition have been shown to be altered by extended exposure of monkeys to levels
12 as low as 0.15 ppm (90 days) (Rao et al., 1985a, 1985b).
13 Studies of nonhuman primates exposed for a year to 0.61 ppm O3 have demonstrated
14 increased collagen content with an associated enhanced proportion of difunctional and mature
15 cross-links (Reiser et al., 1987). The abnormal collagen remained 6 mo postexposure.
16 In contrast, changes in total collagen content were not detected in rats exposed to <0.5 ppm
17 O3 for 18 mo (Wright et al., 1988), although extracellular matrix thickening (Section 6.2.4;
18 e.g., Chang et al., 1992) and increased collagen turnover have been detected (Filipowicz and
19 McCauley, 1986b). In rats, episodic exposure regimens, which had half or less of the
20 O3 exposure of "daily" regimens, had increases hi collagen content equivalent to the daily
21 exposure groups (Last et al., 1984b). In monkeys, only a "seasonal" exposure regimen
22 increased collagen content (Tyler et al., 1988).
23 Many studies have utilized whole-lung measurements of antioxidant enzyme changes as
24 indicators of biochemical responses to O3 exposure. The increased enzyme levels that have
25 been observed during the first week from the onset of exposure to O3 levels of 0.5 to
26 1.0 ppm are most likely a result of the epithelial proliferation and infiltration of inflammatory
27 cells taking place in this tune period (Bassett et al., 1988a; Rahman et al., 1991). Failure to
28 observe similar biochemical changes at lower O3 concentrations most likely reflects an
29 inability to detect focal changes of altered pathology when using whole lung tissue samples.
30 However, measurements of lung enzymes involved in polyamine biosynthesis have been
31 shown to provide a suitably sensitive measure for exposures to 0.45 ppm O3 for 2 days
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1 (Elsayed et al., 1990). Long-term exposure of rats to an urban pattern of O3 has
2 demonstrated increases in tissue GSHPx and GR, but not SOD, that could reflect either
3 changes in antioxidant capacity in response to chronic O3 exposure or changes in the steady-
4 state cell population (Grose et al., 1989). Interpretation of O3-induced changes in whole-
5 lung enzyme measurements can clearly not be done without consideration of the concomitant
6 alterations in cell populations.
7 Ozone-induced changes in the extent and pattern of lung microsomal metabolism of
8 xenobiotics have been investigated, which might in part reflect changes in the numbers and
9 function of bronchiolar epithelial Clara cell and alveolar Type 2 cells, cell types that are
10 relatively enriched with cytochrome P-450 dependent enzyme systems. Changes in both lung
11 activation and detoxification reactions represent an important area of investigation when
12 considering whether low-level O3 exposure alters the ability of the lung to adequately deal
13 with the coexposure with inhaled pollutants that undergo metabolic transformation in the
14 lung.
15
16 6.2.7 Genotoxicity and Carcinogenicity of Ozone
17 6.2.7.1 Introduction
18 Ozone is a very reactive molecule and a strong oxidizing agent that can dissolve in
19 aqueous solutions and generate hydroxyl radicals (reviewed in Menzel, 1970; Hoigne and
20 Bader, 1975; U.S. Environmental Protection Agency, 1986; Victorin, 1992). Early studies
21 of the effects of O3 upon purines, pyrimidines, nucleosides, nucleotides, and nucleic acids
22 showed that O3 rapidly degraded these compounds in vitro (Christensen and Giese, 1954;
23 reviewed in Menzel, 1984). Ozone-generated hydroxyl radicals can abstract hydrogen from
24 organic molecules, leading to further complex free-radical reactions (reviewed in Menzel,
25 1970; Menzel, 1984; Victorin, 1992). In addition, O3 initiates radical reactions resulting in
26 the ozonolysis of alkenes to form ozonides, which decompose upon reaction with water to
27 form peroxyl radicals, peroxides, and aldehydes. Ozone can also oxidize amines to amine
28 oxides and react with PUFA to form products of lipid peroxidation (reviewed in Menzel,
29 1970; Menzel, 1984; Pryor, 1978, 1991; Menzel, 1992). Ozone also has been shown to
30 cause a reduction in plaque formation by bacteriophage f2, release RNA from phage
31 particles, inactivate RNA, and degrade protein (Kim et al., 1980). Hence, because
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1 O3 generates hydroxyl radicals in aqueous solution and degrades DNA, RNA, protein, and
2 fatty acids in vitro, it poses a potential genotoxic hazard by virtue of its ability to generate
3 reactive intermediates that can oxidize nucleic acid bases (reviewed in Victorin, 1992).
4 However, the precise reactions that occur in living cells exposed to O3 have yet not been
5 completely defined. As the ensuing discussion shows, the genotoxic potential of O3 is, at
6 most, weak.
7 This section reviews the information available on the genotoxicity of O3 since the last
8 Air Quality Criteria Document (U.S. Environmental Protection Agency, 1986) was
9 published, although some earlier reports are cited in order to create a historical and scientific
10 perspective for the reader. The areas covered in this review are the ability of O3 to induce
11 DNA damage, mutagenesis, cell transformation, carcinogenesis, co-carcinogenesis, and
12 tumor promotion. Although modulation of the tumorigenic response by indirect effects of
13 O3 on the immune system is theoretically possible, no evidence for such modulation has been
14 reported (see Section 6.2.3). Unfortunately, experimental data to evaluate whether O3 is
15 genotoxic are limited. Hence, relevant data on genotoxic effects of O3 above 1 ppm have
16 also been included to ensure discussion of the full array of effects as they are currently
17 understood. Although data at points far above 1 ppm of O3 are not directly relevant to
18 human health, such high-concentration data serve to address concentration-response
19 relationships for the specific genotoxicity endpoint studied.
20 A further caveat is that in many experiments utilizing in vitro systems, O3 was added to
21 bacteria or to mammalian cells covered by culture medium. In all such experiments, the
22 reactivity of O3 makes it likely that the reaction products of O3 with culture fluid, not
23 O3 itself, actually reach and interact with the cells. This complicates interpretation of the
24 results and their extrapolation to potential genotoxicity in humans.
25
26 6.2.7.2 Ozone-Induced Deoxyribonucleic Acid Damage
27 Studies utilizing a wide range of O3 levels between 0.1 and 20 ppm have been
28 performed in an attempt to determine whether O3 is genotoxic. Hamelin (1985) showed by a
29 combination of agarose gel electrophoresis and electron microscopy that ozonation at 5 to
30 20 ppm caused single- and double-strand DNA breaks, nicking, relaxation, linearization, and
31 then degradation of double-stranded plasmid pAT153 DNA molecules in solution. He also
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1 showed that ozonation of plasmid DNA reduced the transforming ability of this plasmid, and
2 that Escherichia coli strains with mutations in DNA repair pathways (lexA, ozrA, and recA,
3 but not uvrA) were less able to support the transforming ability of the ozonated plasmid.
4 Hence, the lexA, ozrA, and recA gene products participate in repairing O3-induced DNA
5 breaks.
6 Similarly, Sawadaishi et al. (1985) showed that ozonolysis of supercoiled pBR322 DNA
7 resulted in conversion of closed-circular to open-circular DNA and caused single-strand
8 cleavage at specific sites. The concentrations of O3 employed were not listed. Sawadaishi
9 et al. (1986) further explored the specificity of O3-induced damage to supercoiled plasmid
10 pBR322 DNA by utilizing DNA sequencing techniques. Although the mechanistic data
11 obtained showing degradation of thymine bases are interesting, the O3 concentrations used
12 were too high (25,600 ppm) to be useful in assessing biologically relevant effects of Q$.
13 Mura and Chung (1990) studied the biological consequences of ozonation of DNA.
14 They exposed phage T7 DNA to 5 ppm O3 for periods of 5 to 15 min and found that
15 O3 decreased the template activity of the DNA. Both the rate of initiation of transcription
16 and the length of the RNA chains transcribed were reduced. They concluded that O3 induced
17 lesions in the structure of the phage T7 DNA and that these lesions interfered with the ability
18 of the DNA to be transcribed. In mammalian cells, Van der Zee et al. (1987) demonstrated
19 that ozonation of murine L929 fibroblasts caused DNA strand breaks, DNA inter-strand
20 cross-links, and DNA-protein cross-links, but the O3 concentrations used were too high
21 (615 ppm) to be relevant to ambient exposures.
22 Kozumbo and Agarwal (1990) conducted in vitro studies in which specific arylamines
23 contained in tobacco smoke (1-naphthylamine, 2-naphthylamine, aniline, p-toluidine,
24 o-toluidine, and m-toluidine) were ozonized with 0.1 to 1.0 ppm O3 for 1 h. When reaction
25 products were added to human lung fibroblasts and to transformed human Type 2 cells in
26 vitro, DNA damage occurred. This raises the possibility that smokers could incur DNA
27 damage in their lung cells due to the interaction of O3 with arylamines contained in tobacco
28 smoke, but there are no data on whether such reactions occur in vivo.
29 A logical consequence of these findings (Table 6-15) is that C^ could inhibit DNA
30 replication in mammalian cells and induce cytotoxicity to these cells, and this was found by
31 Rasmussen (1986). Rasmussen observed that DNA replication was inhibited in a
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TABLE 6-15. EFFECTS OF OZONE ON DEOXYRIBONUCLEIC ACID DAMAGE
Ozone
Concentration
ppm
0.1
1-10
5
5-20
exposure
/tg/m Duration
196 1 h
1,960- 1 h
19,600
9,800 5-15 min
9,800- 5-15 min
39,200
Exposure
Conditions
15 pM
1-napthylamine
Culture
DNA at
50 /tg/mL; Og at
0.5 L/min, room
temp.
10 mM
Tris/HCl
1 mM EDTA
Cells
Diploid human
lung fibre-
blasts and
transformed
Type 2 cells
Hamster
(Chinese V79
cells)
Ozonated T7
phage DNA
Plasmid
pAT153
Observed Effects
DNA breaks.
Inhibition of DNA
replication;
cytotoxicity.
Decreased hi vitro
transcription.
Single-/double- strand
breaks hi DNA.
Reference
Kozumbo and
Agarwal (1990)
Rasmussen (1986)
Mura and Chung
(1990)
Hamelin (1985)
1 concentration-dependent manner in Chinese hamster V79 cells by O3 concentrations from
2 1 to 10 ppm following a 1-h exposure. These exposure regimens also induced cytotoxicity.
3 Therefore, the available data show that O3 causes single- and double-strand breaks in
4 plasmid DNA in vitro, affects plasm id DNA so that its ability to serve as a template for
5 transcription is decreased, and inhibits DNA replication and causes cytotoxicity in Chinese
6 hamster V79 cells (Table 6-15).
7
8 6.2.7.3 Induction of Mutation by Ozone
9 Consistent with its ability to induce DNA damage, 50 ppm 03 also induced mutation to
10 streptomycin resistance in E. coli, via. both direct mechanisms and indirectly by the rec-lex
11 error-prone DNA repair system, by a factor from two- to 35-fold in an exposure-time
12 dependent manner (Table 6-16). However, no statistical analysis was performed on these
13 data (Hamelin and Chung, 1975a,b; L'Herault and Chung 1984). In assays designed to
14 detect base substitution mutations by gases in Salmonella typhimurium, Victorin and
15 Stahlberg (1988a) showed that O3 alone at concentrations of 0.1 to 3.5 ppm did not induce
16 mutation to histidine auxotrophy in Ames' strains TA100, TA102, or TA104. Ozone
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TABLE 6-16. SUMMARY OF FINDINGS ON THE MUTAGENICITY OF OZONE
Ozone
Concentration
Exposure Exposure
ppm /tg/m3 Duration Conditions
Cells
Observed Effects
Reference
0.024 47
35 min Culture
0.039 76 35 min
0.39 764 35 min
5. typhimurium At 0.024 ppm: 2.4-fold Dillon et al. (1992)
TA102 increase in mutation frequency.
At 0.039 ppm: 1.6-fold
increase in mutation frequency.
At 0.39 ppm: 1.3-fold
increase in mutation frequency.
No effects seen in
5. typhimurium TA98,
TA1535, TA100, or TA104.
0.1-0.3
0.1-3.5
0.5
1.0
1.0
50
50
196-
588
196-
6,860
980-
1,960
1,960
98,000
98,000
5 or
llh/day
for
1-15 or
18 days
6h
6h
6h
1-20 min
30-90 min
Culture
Culture 1 %
vinyl
chloride
Culture 0.1
or 1.0%
butadiene
Culture
Culture
Nicotiana
tabacam
Tradescantia
S. typhimurium
TA100,
TA102, or
TA104
5. typhimurium
TA100
5. typhimurium
TA100
E. coli
Saccharomyces
cerevisiae
No mutation at color locus.
No mutation with or without
metabolic activation.
170% increase in mutation.
Statistical analysis not
conducted.
170% increase in mutation.
Statistical analysis not
conducted.
Exposure-time-dependent
mutation to streptomycin
resistance, up to 35-fold
increases in mutation
frequency.
Forward mutations, reversions,
gene conversion, mitotic
crossing over. No statistical
analysis conducted.
Gichner et al.
(1992)
Victorin and
Stahlberg (1988a)
Victorin and
Stahlberg (1988b)
Victorin and
Stahlberg (1988b)
L'Herault and
Chung (1984)
Dubeau and Chung
(1982)
1 concentrations ^2 ppm were cytotoxic. Victorin and Stahlberg (1988b) showed that 0.5 and
2 1.0 ppm O3 in combination with 1 % vinyl chloride and 1 ppm O3 plus 0.1 or 1 % butadiene
3 gave rise to a slight (approximately twofold) increase in mutation frequency. In these
4 in vitro studies, as in many studies reviewed in this section, it must be pointed out that
5 because O3 is so reactive, placing both chemicals in the experiments would result in exposure
6 of the bacteria to reactive intermediates and reaction products resulting from the mixture, not
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1 exposure of the bacteria to O3 alone. Further, these increases in mutation were small
2 (^twofold) and not statistically analyzed, and the authors did not test strictly for
3 concentration-dependent effects (Table 6-16). Thus, it is not clear that these small effects are
4 real.
5 More recently, Dillon et al. (1992) studied the ability of O3 to induce mutation in the
6 Ames' strains of Salmonella (Table 6-16). Ozone caused no mutation in Salmonella strains
7 TA1535, TA98, TA100, and TA104. These authors found that O3 exposure caused small
8 increases in the mutation frequency in Salmonella tester strain TA102, which is uniquely
9 sensitive to detecting mutation induced by oxygen radicals, at concentrations of 0.024 and
10 0.039 ppm O3. These increases in mutation frequency were significant. However, the
11 authors did not observe consistent concentration-dependent increases in the mutation
12 frequency. At the higher concentrations of O3, there appeared to be an inverse dependence
13 for induction of mutation by O3. The authors indicated that the cytotoxicity of
14 O3 complicated attempts to obtain a clear concentration response for mutagenicity. The
15 presence of Arochlor 1254-induced rat liver S-9 metabolic activation did not affect the
16 mutational responses in any of the strains tested. These authors did not observe reproducible
17 increases in mutation frequency in Salmonella strains TA98, TA100, TA104, or TA1535.
18 Dillon et al. (1992) concluded that O3 is a weak bacterial mutagen only under specific
19 conditions utilizing noncytotoxic concentrations in TA102. However, because clear
20 concentration-dependent responses for mutation could not be achieved, it is not clear that
21 O3 is definitively mutagenic in these studies.
22 Gichner et al. (1992) investigated whether O3 could induce mutation in two
23 mutagenicity assays in plants, and found no induction of mutation in the Nicotiana tabacum
24 leaf color reversion assay or in the Tradescantia stamen hair assay at 0.1 to 0.3 ppm
25 O3 (Table 6-16).
26 Dubeau and Chung (1979) showed that mutants of the yeast of Saccharomyces
27 cerevisiae deficient in repair of single- and double-strand DNA breaks were more sensitive to
28 the cytotoxicity of O3 than wild-type cells, indicating that O3 kills cells partly by generating
29 these types of breaks. Dubeau and Chung (1982) also showed that treatment of S. cerevisiae
30 with O3 at 50 ppm for 30 to 90 min resulted in (1) an 11- to 14-fold increased frequency of
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1 forward mutations, (2) an increase in reversions at six different loci by two- to threefold,
2 (3) an increase in gene conversions by two- to threefold, and (4) an increase in mitotic
3 crossing-over by 1.3-fold. No statistical analysis was performed on these data, nor were
4 concentration-response curves generated by the authors. These authors, therefore,
5 demonstrated that O3 was a mutagen and recombination-inducing agent in S. cerevisiae.
6 However, they also showed that its genotoxic activity was weak (20- to 200-fold less activity
7 in terms of the frequency of mutants or recombinants induced) compared to the known
8 mutagens ultraviolet light, X rays, and N-methyl-N'-nitro-N-nitrosoguanidine.
9 In this section, the inclusion of data on bacterial mutation utilizing exposures up to
10 50 ppm O3 can be justified in order to help determine whether O3 was genotoxic at all. The
11 available information shows that O3 is not mutagenic in four Salmonella tester strains and
12 may cause weak mutagenicity in Salmonella strain TA102, but these positive results are
13 rendered ambiguous by the lack of a concentration-response effect. The data also show O3 is
14 mutagenic in E. coli, is weakly mutagenic in S. cerevisiae, but is not mutagenic in
15 N. tabacam or Tradescantia. Hence, because O3 is mutagenic in three assays but not in six
16 others, and is weakly mutagenic in assays where the results are positive, it should be
17 considered, at best, a weak mutagen (also reviewed in Victorin, 1992).
18
19 6.2.7.4 Induction of Cytogenetic Damage by Ozone
20 A number of investigators have studied whether O3 induces cytogenetic damage. The
21 previous air quality criteria document (U.S. Environmental Protection Agency, 1986)
22 described a number of in vitro and in vivo studies in which O3 exposure produced toxic
23 effects on cells and cellular components, including genetic material; very few newer studies
24 have been reported. Cytogenetic and mutational effects of O3 have been previously reported
25 in isolated cultured cell lines, human lymphocytes, and microorganisms (Fetner, 1962;
26 Hamelin et al., 1977a,b; Hamelin and Chung, 1975a,b; Scott and Lesher, 1963; Erdman and
27 Hernandez, 1982; Guerrero et al., 1979; Dubeau and Chung, 1979, 1982). One of the
28 earliest studies by Fetner (1962) demonstrated that in vitro exposure of human KB cells to
29 8 ppm O3 for 5 and 10 min induced two- and sixfold increases in the number of chromatid
30 deletions. Shiraishi et al. (1986) found that treatment of Chinese hamster V79 cells with
31 0.1 to 1.0 ppm O3 inhibited growth of V79 cells by 10 to 70% and also induced a
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1 concentration-dependent increase in the number of sister chromatic exchanges (SCEs)/cell, up
2 to a maximum of fourfold of that in control, untreated cells. The results indicate that if cells
3 in culture are exposed to sufficiently high concentrations of Oj for sufficiently long periods,
4 chromosome damage will result.
5 In vivo exposure studies are of greater potential interest. Cytogenetic and mutational
6 effects of O3 in laboratory animals and humans are controversial. Lymphocytes isolated
7 from animals exposed to O3 were found to have significant increases in the numbers of
8 chromosome (Zelac et al., 1971a,b) and chromatid (Tice et al., 1978) aberrations, after 4- to
9 5-h exposures to 0.2 and 0.43 ppm 03, respectively. Single-strand breaks in DNA of mouse
10 peritoneal exudate cells were measurable after a 24-h exposure to 1 ppm O3 (Chaney, 1981).
11 Gooch et al. (1976) analyzed the bone marrow samples from Chinese hamsters exposed to
12 0.23 ppm O3 for 5 h and the leukocytes and spermatocytes from mice exposed for up to
13 2 weeks to 0.21 ppm O3. No effect was found on either the frequency of chromosome
14 aberrations, nor were there any reciprocal translocations in the primary spermatocytes.
15 These authors did show that there was a slight but significant increase in the frequency of
16 chromatid aberrations in human peripheral leukocytes exposed in vitro to 7.2 and 7.9 ppm of
17 O3. The small increases observed in chromatid lesions in peripheral blood lymphocytes from
18 humans exposed to 0.5 ppm O3 for 6 to 10 h were not significant, possibly because of the
19 small number (n = 6) of subjects studied (Merz et al., 1975). Subsequent investigations
20 with improved experimental design and more human subjects, however, have not shown that
21 exposure to O3 at various concentrations and for various times causes cytogenetic effects
22 (McKenzie et al., 1977; McKenzie, 1982; Guerrero et al., 1979). Guerrero et al. (1979)
23 showed that there was no elevation in the frequency of SCEs in the circulating lymphocytes
24 of humans exposed to 0.5 ppm of O3 for 2 h. However, these authors did find that exposure
25 of diploid human fetal lung (WI38) cells to 0.25, 0.50, 0.75, and 1.0 ppm 03 for 1 h in
26 vitro led to a concentration-dependent increase in SCEs in these cells. In addition,
27 epidemiological studies have not shown any evidence of chromosome changes in peripheral
28 lymphocytes of humans exposed to O3 in the ambient environment (Scott and Burkart, 1978;
29 Magie et al., 1982). Evidence now available, however, fails to demonstrate any cytogenetic
30 or mutagenic effects of O3 in humans when exposure regimens are used that are
31 representative of exposures that the population might actually experience.
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1 A study conducted by Erdman and Hernandez (1982) showed that treatment of
2 Drosophila virilis with 30 ppm O3 for 2 to 6 h resulted in an exposure time-dependent
3 accrual of dominant lethals.
4 Therefore, O3 does induce chromosomal aberrations in cultured cells, but the results in
5 animals exposed to O3 for chromosomal breakage are, at most, weak and their biological
6 significance is controversial.
7
8 6.2.7.5 Induction of Morphological Cell Transformation by Ozone
9 Ozone has been studied in a number of mammalian cell culture systems to determine
10 whether it can induce cell transformation (Table 6-17). The cell transformation assay in
11 C3H/10T1/2 (10T1/2) mouse embryo cells is a standard assay that has been used by many
12 investigators (reviewed in Landolph, 1985, 1989, 1990) to detect and study mechanisms of
13 cell transformation induced by organic chemicals, carcinogenic metals, and radiation. Syrian
14 hamster embryo (SHE) cells are also widely used to detect cell transformation by many
15 classes of chemical carcinogens and radiation (Borek et al., 1986, 1989a,b). Borek et al.
16 (1986) demonstrated that exposure of SHE cells and 10T1/2 mouse embryo cells to 5 ppm
17 O3 for 5 min inducted morphological transformation in both cell types. In both cell types,
18 there was also a synergistic induction of morphological transformation when the cells were
19 treated with 3 Grays of gamma radiation and 5 ppm O3. These authors therefore concluded
20 that O3 acts as a direct cell transforming agent and as a co-cell transforming agent in the
21 presence of gamma radiation. Borek et al. (1989a) also observed an additive amount of
2
22 transformation when these cell types were treated with 6 ppm O3 and 4J/m of ultraviolet
23 light. A further study by Borek et al. (1989b) showed that exposure of 10T1/2 mouse
24 embryo cells to 1 ppm O3 for 5 min did not result in morphological transformation, but that
25 increasing exposure to 5 ppm increased the transformation frequency by a factor of 15.
26 Ozone and gamma radiation caused a synergistic increase in morphological transformation
27 when O3 was added to cells after the gamma radiation. When O3 was added to cells before
28 the gamma radiation, the transformation was not increased over that due to gamma radiation.
29 These authors also showed that three O3-induced transformed cell lines possessed dominantly
30 acting transforming genes, as shown by DNA transfection experiments. In these studies,
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TABLE 6-17. EFFECTS OF OZONE ON MORPHOLOGICAL
CELL TRANSFORMATION
Ozone
Concentration
ppm
0.14,
0.6,
1.2
0.7
0.7
10
1.0
5.0
5.0
-~^~~—^~ exposure
/ig/m Duration
274, 6 h/day,
1,176, 5 days/week
2,352 for 1, 2, or
4 weeks
1,372 40 min twice
weekly for
5 weeks
(in vitro)
1,372 40 min
19,600 (in vitro)
1,960 5 min
9,800 (in vitro)
9,800 5 min
(in vitro)
Cells/
Species, Sex
(Strain)
Rat, M
(F344/N)
7-9 weeks old
Rat
(Tracheal
epithelial cells)
Rat
(Tracheal
epithelial cells)
Mouse
(C3H/10T1/2
embryo cells)
Hamster
(Primary diploid
cells)
Observed Effects
No induction of preneoplastic
variants in cultured trachea!
epithelial cells.
Twofold increase in frequency of
pre-neoplastic variants; additive
effects with MNNG.
No induction of preneoplastic
variants
At 1.0 ppm: no morphological
transformation alone; increased
transformation by 0.4 Gray
radiation by 1.7 -fold like a
co -carcinogen.
At 5.0 ppm: 15-fold increase hi
morphological transformation and
syngergism with 4 Gray gamma
radiation transformation.
In both cell lines, induction of
morphological transformation and
synergism with gamma rays.
Reference
Thomassen et al. (1991)
Thomassen et al. (1991)
Thomassen et al. (1991)
Borek et al. (1989b)
Borek et al. (1986)
Mouse
(C3H/10T1/2
embryo cells)
6.0 11,760 10 min
(in vitro)
Hamster In both cell lines: induction of
(Syrian primary morphological transformation;
embryo cells) additive transformation with UV
light.
Mouse
(C3H/10T1/2
embryo cells)
Borek et al. (1989a)
1 cells were incubated in phosphate-buffered saline during O3 treatment, and hence were likely
2 exposed to reaction products of O3 rather than O3 itself.
3 Thomassen et al. (1991) exposed rats by inhalation to 0.14, 0.6, or 1.2 ppm O3 for
4 6 h/day, 5 days/week, for a total of 1, 2, or 4 weeks. There was no increase in the
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1 frequency of preneoplastic transformation in cells removed from the tracheas and
2 subsequently cultured. Cells incubated in serum-free medium and exposed to 0.7 or 10 ppm
3 O3 for 40 min in vitro also did not show an increased frequency of preneoplastic
4 transformation. When rat tracheal epithelial cells were exposed in vitro to 0.7 ppm O3 for
5 2 times/week for 5 weeks, there was approximately a twofold increase in the frequency of
6 preneoplastic variants detected. These authors also showed that treatment of rat tracheal
7 epithelial cells with AT-methyl AP-nitro-W-nitrosoguanidine (MNNG) followed by exposure of
8 cells to 0.7 ppm O3 twice weekly for 5 weeks resulted in an approximately additive increase
9 in the frequency of preneoplastic variants of the cells.
10 In all these cell transformation experiments, the reactivity of O3 makes it likely that
11 secondary reaction products of O3 formed in the aqueous medium, not O3 itself, induced the
12 cell transformation. Therefore, O3 is able to induce morphological transformation in
13 C3H/10T1/2 mouse embryo cells and in SHE cells but causes no significant effects in rat
14 tracheal epithelial cells in vitro or in vivo.
15
16 6.2.7.6 Possible Direct Carcinogenic, Co-carcinogenic, and Tumor-Promoting Effects
17 of Ozone as Studied in Whole Animal Carcinogenesis Bioassays
18 To investigate whether O3 has carcinogenic, co-carcinogenic, or tumor-promoting
19 effects, a number of investigators have conducted in vivo carcinogenesis bioassays with
20 O3 (Table 6-18). Some studies have used strain A mice. The advantages and disadvantages
21 in using strain A mice as a general screen for carcinogens by the intraperitoneal route have
22 been discussed in the literature (Stoner and Shimkin, 1985; Maronpot et al., 1986; Stoner,
23 1991; Maronpot, 1991). Strain A mice have rarely been used in proper inhalation
24 carcinogenesis assays. In particular, the A/J strain of mice have a high spontaneous
25 incidence of benign pulmonary tumors (adenomas). This strain of mice has been shown to
26 be very sensitive to tumor induction by polycyclic aromatic hydrocarbons, carbamates, and
27 aziridines and insensitive to aromatic amines, metal salts, and halogenated organic
28 compounds administered by the intraperitoneal route (Maronpot et al., 1986). In addition,
29 carcinogenicity results in strain A mice do not correlate well with 2-year mouse and rat
30 carcinogenicity results where the results of chemical testing in strain A/St mice (59 chemicals
31 tested) were compared with strain A/J mice (30 chemicals tested) in a 2-year chronic
32 bioassay (Maronpot et al., 1986). The chemicals chosen were heavily weighted with
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TABLE 6-18. SUMMARY OF RESULTS ON THE POSSIBLE
CARCINOGENICITY OF OZONE
Ozone
Concentration
ppm
0.05
(sine wave
from 0 to
0.1)
0.31
0.50
0.40
0.80
0.80
0.12
0.50
1.0
0.5
1.0
0.12
0.50
1.0
0.5
1.0
Exposure
Uglm Duration
98 10 h/day for
(sine 13 mo
wave
from 0 to
96)
608 103 h/
week, every
other week for
6 mo
980 102 h/first
week of each
month for
6 mo
784 8 h/day,
1 ,568 7 days/week
for 1 8 weeks
1,568 23 h/day
7 days/week
for 6 mo
6 h/day
5 days/week
104 weeks
(2 years)
6 h/day
5 days/week
124 weeks
(lifetime)
6 h/day
5 days/week
105 weeks
(2 years)
6 h/day
5 days/week
130 weeks
(lifetime)
Species, Sex
(Strain)
Rat, M
(Wistar)
4 weeks old
Mouse, F
(A/J)
7 weeks old
Mouse, F
(A/J)
7 weeks old
Mouse, M
(Swiss Webster
and A/J)
8 weeks old
Hamster, M
(LVG Syrian
Golden)
7-11 weeks old
Rat, M, F
(F344/N)
Rat, M, F
(F344/N)
Mouse, M, F
(B6C3Fj)
Mouse, M, F
(B6C3F,)
Observed Effects
Lung tumor response increased from 0%
in BHPN- or C^-treated animals, to 8.3%
in animals treated with 0.5 g/kg BHPN +
0.05 ppm Oj (not significant).
1 .33-fold increase in percent of mice with
adenomas, 1 .42-fold increase in number of
tumors/mouse. No promotion of
carcinogeniciry of urethane (2 mg/mouse
before 03).
2.1 1-fold increase in percent of mice with
tumors, 3 .42-fold increase in number of
tumors/mouse, interaction between Oj and
urethane (2 mg/mouse after each Oj
week).
Urethane treatment before 03 started. In
Swiss Webster mice: no increase in lung
tumor incidence; nonsignificant decrease in
tumors/lung in urethane-treated animals.
In A/J strain: No effect at 0.4 ppm.
At 0.8 ppm: threefold increase in percent
mice with tumors, and 4.2-fold increase in
number of tumors/mouse. Both 0.4 and
0.8 ppm 03 decreased yield of
tumors/mouse in urethane-treated mice,
but had no effect on tumor incidence.
No tumors observed in animals treated
with 0.8 ppm 03 only. In animals treated
with 20 mg/kg DEN S.C. twice/week,
0.8 ppm O3 did not increase tumors of
lung, bronchus, trachea, nasal cavity.
Tumors of lung were decreased 50%
(N.S.)
No increase in neoplasms at any ppm
tested.
No increase in neoplasms at any ppm
tested.
No effects in males. In females:
Increase in number of mice with
neoplasms at 1 .0 ppm (combined
alveolar/bronchiolar adenoma or
carcinoma in lung).
In males: increase in number of mice with
carcinoma at 0.5 and 1.0 ppm, but not
significant for change in number of mice
with total neoplasms. In females:
increase in number of mice with
adenomas, but not carcinomas or total
neoplasms.
Reference
Ichinose and Sagai (1992)
Hassett et al. (1985)
Hassett et al. (1985)
Last et al. (1987)
Witschi et al. (1993)
National Toxicology
Program (1993)
National Toxicology
Program (1993)
National Toxicology
Program (1993)
National Toxicology
Program (1993)
December 1993
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1 aromatic amines. The author concluded that "carcinogenicity test data are relevant only to
2 the test model employed since there is no absolute reference for carcinogenicity." Maronpot
3 (1991) also demonstrated that there was a poor concordance between the results of testing
4 chemicals in the strain A assay and testing them in 2-year rat and mouse carcinogenicity
5 assays at the National Cancer Institute. Stoner (1991), using the strain A mouse pulmonary
6 assay, indicated that intraperitoneal injection of polycyclic aromatic hydrocarbons,
7' nitrosamines, nitrosureas, carbamates, aflatoxin, metals, hydrazines induces tumors, but that
8 the assay is not responsive to aromatic amines, aliphatic halides, and certain compounds
9 carcinogenic in rodent liver and/or bladder. In this assay, an increase in lung tumor
10 multiplicity (average number of lung tumors per mouse) caused by a chemical is considered
11 as evidence for the carcinogenicity of a chemical.
12 Hassett et al. (1985) used inbred strain A/J mice, which are very sensitive to induction
13 of pulmonary adenomas by chemical carcinogens. Exposure of A/J mice to 0.31 ppm 03 for
14 103 h per week, every other week, for 6 mo, resulted in a 1.3-fold increase (not statistically
15 significant) in the percent of mice with tumors (tumor incidence) and a statistically significant
16 1.4-fold increase in the number of tumors per mouse (tumor multiplicity). In this
17 experiment, O3 did not promote the carcinogenicity of urethane when 63 exposure began
18 1 week after a single injection of animals with a total dose of 2 mg urethane/mouse.
19 In a second experiment, exposure to 0.50 ppm O3 (102 h during first week of every
20 month for 6 mo) caused a nonsignificant 2.1-fold increase in tumor incidence and a 3.2-fold
21 increase in tumor multiplicity (statistics not shown) (Hassett et al., 1985). These authors
22 reported that exposure to 0.5 ppm O3, followed by urethane treatment (2 mg after each
23 O3 exposure set), resulted in an interaction between O3 and urethane, such that there were
24 more animals with greater than 16 lung tumors/mouse. These authors concluded that
25 exposure to 0.31 and 0.5 ppm of O3 increased the yield of pulmonary adenomas in A/J mice
26 and that O3 interacted with urethane to produce more lung tumors than urethane alone when
27 O3 was added before urethane.
28 The study of Hassett et al. (1985) was extensively reviewed by scientists from EPA and
29 the National Institutes of Environmental Health Sciences in 1985 and 1986. The consensus
30 of these extensive reviews was that (1) the tumor yields in O3-exposed mice were not
31 statistically significantly different from the control animals, (2) any effects were marginally
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1 different from the control values, and (3) the strain A mouse has a high spontaneous
2 incidence of tumors, making it difficult to interpret the effects of Oj. Chemical induction of
3 tumors in this assay system did not correlate well with the 2-year National Cancer Institute
4 carcinogenesis bioassay results. In addition, because Hassett et al. (1985) did not
5 demonstrate a concentration-response effect in animals exposed to O3, the consensus among
6 the reviewers was that one could not conclude from these experiments that O3 was a
7 significant carcinogen or tumor promoter, and that rigorous inhalation carcinogenesis
8 bioassays needed to be carried out with O3-exposed animals to address this issue properly.
9 Last et al. (1987) also studied whether O3 exposure could influence the yield of
10 urethane-induced lung tumors in A/J and Swiss-Webster mice. Urethane treatment consisted
11 of a single ip injection (1,000 mg/kg) 1 day before O3 exposure started hi Swiss Webster
12 mice, exposure to 0.4 or 0.8 ppm O3 alone did not increase the tumor yield but actually
13 decreased the yield of urethane-induced lung tumors/mouse, although the differences were
14 not statistically significant. In A/J mice, exposure to 0.4 ppm 63 did not increase the lung
15 tumor yield, but exposure to 0.8 ppm O3 caused a threefold increase in tumor incidence and
16 a 4.2-fold increase in lung tumor multiplicity. Exposure of urethane-treated mice to 0.4 or
17 0.8 ppm O3 decreased lung tumor multiplicity, but had no effect on tumor incidence. These
18 differences in the strain A mouse were significant. The authors concluded that O3 was not a
19 tumor promoter or tumor-enhancing agent.
20 Last et al. (1987) also studied whether O3 exposure could influence the yield of
21 urethane-induced lung tumors in A/J and Swiss-Webster mice. Urethane treatment consisted
22 of a single ip injection (1,000 mg/kg) 1 day before O3 exposure started. In Swiss Webster
23 mice, exposure to 0.4 or 0.8 ppm O3 alone did not increase the tumor yield but actually
24 decreased the yield of urethane-induced lung tumors/mouse, although the differences were
25 not statistically significant. In A/J mice, exposure to 0.4 ppm O3 did not increase the lung
26 tumor yield, but exposure to 0.8 ppm O3 caused a threefold increase in tumor incidence and
27 a 4.2-fold increase in lung tumor multiplicity. Exposure of urethane-treated mice to 0.4 or
28 0.8 ppm O3 decreased lung tumor multiplicity but had no effect on tumor incidence. These
29 differences in the strain A mouse were significant. The authors concluded that O3 was not a
30 tumor promoter or tumor-enhancing agent.
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1 Ichinose and Sagai (1992) studied the ability of O3 to interact with
2 N-£«(2-hydroxypropyl) nitrosamine (BHPN) in the induction of lung tumors in Wistar rats.
3 A single intraperitoneal injection of BHPN (0.5 g/kg) did not cause any tumors in the rats.
4 Rats were exposed for 10 h/day for 13 mo (examination time 11 mo later) to a pattern of
5 O3 consisting of a sine curve from 0 to 0.1 ppm, with a mean concentration of 0.05 ppm.
6 No tumors were observed in the O3 alone or control groups. However, when the rats were
7 exposed to 0.5 g/kg BHPN plus 0.05 ppm O3, the lung tumor incidence of 8.3% (3/36) was
8 not statistically significantly increased. The tumors observed in this study cannot be stated
9 definitively to have been induced by the treatment agent. The 13-mo O3 exposure started the
10 day after BHPN injection. It therefore appeared that O3 might have acted like a tumor
11 promoter in this study.
12 Witschi (1988) reviewed the available data on O3 and lung carcinogenesis up until
13 1988. The chemical reactivity of O3 and, in particular, its radiomimetic activity, also make
14 it a potential risk factor for human lung cancer. Nevertheless, as of 1988, there were no
15 experimental studies conclusively linking lifelong exposure to O3 with lung tumor induction
16 in any animal species, nor was there conclusive epidemiological evidence to associate
17 O3 exposure with the development of lung cancer in humans. Witschi (1988) also pointed
18 out that the only data implicating O3 as a possible tumorigenic agent were from studies
19 carried out in mice, where the tumors are adenomas derived from Type 2 alveolar cells or
20 from Clara cells. In the A and Swiss-Webster mouse strains used to assay the
21 carcinogenicity of O3, the spontaneous incidence of lung tumors is very high. Hence, results
22 of carcinogenicity experiments conducted on O3 to date that utilize tumor incidence as an
23 endpoint are not strongly positive, due to this high background. In strain A mice, in which
24 the spontaneous multiplicity is usually less than one tumor per mouse, the tumor multiplicity
25 is considered by many investigators to be a sensitive indicator of a carcinogenic effect.
26 However, even using this indicator, the increase in tumor multiplicity after O3 exposure is
27 small, raising questions about the biological significance of the effects. In addition, the assay
28 for inhalation carcinogenesis in strain A mice had not been fully validated at this time.
29 Although Hassett et al. (1985) concluded that O3 increased the number of pulmonary
30 adenomas in strain A mice, Witschi (1988) concluded that O3 was not yet implicated
31 unequivocally as a carcinogen in strain A/J mice, that no classical carcinogen bioassays had
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1 been conducted on O3, and that a definitive judgment could not be made on the
2 carcinogenicity of O3.
3 Witschi (1991) recently reviewed the then available data on the carcinogenicity of
4 O3 and oxygen in mouse lungs and indicated that oxidants can enhance or inhibit mouse lung
5 tumorigenesis, depending on the experimental protocol employed. In his view, the
6 carcinogenicity of O3 in mouse lung had not been unequivocally established. Witschi pointed
7 out that strain A mice treated only with O^ induced hyperplasia of Type 2 alveolar cells,
8 leading to expansion of the target cell population and spontaneous transformation of these
9 cells. In lungs of animals treated with a carcinogen such as urethane, and then exposed to
10 O3 before or after carcinogen administration, O3 may cause cell proliferation and result in
11 fixation of DNA damage (Witschi, 1991). The addition of O3 after carcinogen exposure
12 leads to a decreased tumor incidence compared to treatment with carcinogen alone; the
13 reasons for this decrease with late O3 exposure are not clear (Witschi, 1991).
14 Witschi et al. (1993) (Table 6-18) studied the effects of treating male Syrian Golden
15 hamsters with dimethylnitrosamine (DEN) (20 mg/kg twice per week) during the course of a
16 6-mo exposure to 0.8 ppm O3. After exposure ceased, the animals were maintained in air
17 for about 1 mo. Ozone exposure did not increase the incidence of lung, bronchus, trachea,
18 or nasal cavity tumors in the DEN-treated hamsters. There was a 50% decrease in the
19 percent of animals with lung tumors in the DEN plus O3-exposed animals compared to the
20 DEN plus air-exposed animals, but this was not statistically significant. Ozone did not affect
21 the incidence of DEN-induced liver tumors. Witschi et al. (1993) concluded that O3 did not
22 increase the number of DEN-induced respiratory tumors in hamsters and that O3 exposure
23 might have inhibited or delayed tumor development. Although reduction in tumor incidence
24 caused by O3 was not significant in this particular study alone, overall analysis of these data
25 with other data from a 4-mo exposure of similar design (Witschi et al., in press) was
26 significant.
27 A definitive study of the carcinogenicity of O3 and of its ability to act as a
28 co-carcinogen or tumor promoter was conducted by the U. S. National Toxicology Program
29 (NTP) (National Toxicology Program, 1993). At the time this external review draft of the
30 O3 criteria document was written, the NTP report was in draft form and had recently
31 undergone external peer review. The peer reviewers agreed with the substantive matters in
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1 the draft report, and thus the final report (expected in 1994) will be quite similar to the draft;
2 the tumor incidences will be identical in the draft and final versions. Animals were exposed
3 to air or 03 for 6 h/day, 5 days/week for the number of weeks described below. Male and
4 female F344/N rats were exposed to 0.12, 0.5, and 1.0 ppm O3 for 104 weeks (about
5 2 years) and to 0.5 ppm and 1.0 ppm 124 weeks ("lifetime") of the animals. Similar
6 protocols were used for B6C3FJ male and female mice, with the exception that the "2-year"
7 study was 105 weeks and the "lifetime" study was 130 weeks. This study did not find any
8 evidence of carcinogenic activity in rats. There was a negative trend for mammary gland
9 neoplasms in the female rats in the 2-year study, an effect that was not seen in the lifetime
10 study. The NTP found "equivocal evidence"1 of carcinogenic activity" in O3-exposed male
11 mice and "some evidence" of carcinogenic activity of O3 in female mice exposed to O3.
12 In co-carcinogenesis experiments, male rats were treated with a known pulmonary
13 carcinogen, 4-(A/Lmethyl-A^-nitrosomino)-l-(3-pyridyl)-l-butanone(NNK) (0.1 and 1.0 mg/kg,
14 subcutaneous injection 3 times a week for first 20 weeks) and exposed to 0.5 ppm O3 for
15 6 h/day, 5 days/week for 105 weeks. The NTP found "no evidence" that O3 enhanced the
16 incidence of NNK-induced pulmonary neoplasms. Table 6-19 shows the tumor incidences hi
17 mice. In the discussion to follow, all tumors described were at lung alveolar/bronchiolar
18 sites. There was a decrease in the number of hepatocellular adenomas or carcinomas in
19 female mice exposed to 1.0 ppm O3 for 2 years and for hepatocellular carcinomas in the
20 lifetime study. There was no statistically significant increase in tumors at any site other than
21 the lung.
22 In male mice exposed to O3 for 2 years, there were no statistically significant increases
23 in the mice with alveolar/bronchiolar carcinomas or a combination of adenomas or
24 carcinomas; at 0.05 ppm O3, there was small twofold increase in the incidence of adenomas.
25 In the lifetime studies of male mice, the incidence of mice with carcinomas increased
26 1.9-fold at 0.5 ppm and 2.3-fold at 1.0 ppm. The incidence of adenomas in male mice did
27 not change significantly. In female mice exposed for 2 years, there were no statistically
28 The NTP evaluates the strength of the evidence for conclusions regarding each carcinogenicity study, under the
29 conditions of that particular study. They have five categories: two for positive results ("clear evidence" and
30 "some evidence"), one for uncertain findings ("equivocal evidence"), one for no observable effects ("no
31 evidence"), and one for experiments that cannot be judged because of major flaws ("inadequate study")
32 (National Toxicology Program, 1993). This approach is very different from the weight-of-evidence approach
33 used by EPA for cancer classification because the EPA approach considers all the available studies.
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TABLE 6-19. ALVEOLAR/BRONCHIOLAR TUMOR INCIDENCE IN B6C3Fj MICE
IN THE NATIONAL TOXICOLOGY PROGRAM'S CHRONIC OZONE STUDY
Males
ppm
2-year
exposure
0.0
0.12
0.50
1.0
Lifetime
exposure
0.0
0.5
1.0
Combined
0.0
0.5
1.0
Adenomas
6/50
9/50 (p =
12/50 (p =
ll/50(p =
8/49
8/49 (p =
9/50 (p =
14/99
20/99 (p =
20/100 (p
0.3)
= 0.06)
= 0.11)
0.61)b
0.47)
= 0.16)
= 0.14)
Carcinomas
8/50
4/50 (p =
8/50 (p =
10/50 (p =
8/49
15/49 (p =
18/50(p =
16/99
23/99 (p =
28/100 (p
0.15)
0.45)
= 0.27)
= 0.05)
= 0.007)
Both
14/50
13/50 (p
18/50 (p
19/50 (p
16/49
22/49 (p
21/50 (p
30/99
= 0.44)
=0.12)
= 0.10)
= 0.14)
= 0.15)
= 0.08) 40/99 (p = 0.06)
= 0.009) 40/100 (p =0.04)
Adenomas
4/50
5/50 (p = 0.55)
5/49 (p = 0.52)
8/50 (p = 0.24)
3/50
3/49 (p = 0.63)
ll/50(p = 0.02)
7/100
8/98 (p = 0.48)
19/100 (p = 0.01)
Females
Carcinomas
2/50
2/50 (p =
5/49 (p =
8/50 (p =
3/50
5/49 (p =
2/50 (p =
5/100
0.65)b
0.26)
0.053)
0.33)
0.50)b
Both
6/50
7/50 (p =
9/49 (p =
16/50 (p =
6/50
8/49 (p =
12/50 (p =
12/100
10/98 (p = 0.14) 17/98 (p =
10/100 (p = 0.14) 28/100 (p
0.57)
0.33)
= 0.02)
0.34)
= 0.10)
= 0.20)
= 0.004)
Number of animals with neoplasm/number of animals necropsied (p value, logistic regression test).
Lower incidence.
Source: National Toxicology Program (1993).
1 significant changes at 0.12 or 0.5 ppm O3. However, at 1.0 ppm O3, there was a fourfold
2 increase in the frequency of female mice with carcinoma and a 2.7-fold increase in combined
3 adenomas plus carcinomas. In female mice exposed for their lifetimes to O3, 0.5 ppm
4 O3 caused no significant effects. At 1.0 ppm O3, there was a 3.7-fold increase in the
5 incidence of mice bearing pulmonary adenomas, a nonsignificant change in the frequency of
6 mice with carcinomas and a twofold (p = 0.1) increase in the incidence of combined
7 adenomas and carcinomas.
8 When the results of the 2-year and lifetime O3 carcinogenesis studies were combined
9 and analyzed, for male mice there was no statistically significant increase in the incidence of
10 animals bearing adenomas. For carcinomas, there was a marginally signifcant increase at
11 0.5 ppm (1.4-fold increase, p = 0.08) and a significant 1.7-fold increase at 1.0 ppm O3.
12 The incidence of male mice bearing adenomas or carcinomas showed a marginally significant
13 increase (1.3-fold, p = 0.06) at 0.5 ppm and a 1.3-fold increase at 1.0 ppm O3 (p = 0.045).
14 In the combined analysis of the 2-year and lifetime exposure of female mice, there were no
15 statistically significant changes at 0.5 ppm O3. At 1.0 ppm O3, there was a 2.7-fold increase
16 in the percent of mice bearing adenomas and a 2.3-fold increase hi the frequency of mice
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1 with adenomas or carcinomas. There was no statistically significant increase in the
2 carcinoma incidence.
3 The overall conclusions of the authors of the NTP O3 inhalation carcinogenesis study
4 were (1) there was no increased pulmonary tumor incidence in male or female F344/N rats
5 exposed to 0.12, 0.5, or 1.0 ppm O3; (2) male F344/N rats treated with the tobacco
6 carcinogen NNK and exposed to 0.5 ppm O3 did not have an increase in the pulmonary
7 tumor incidence above that caused by NNK alone; (3) 03 caused a slightly increased
8 incidence of alveolar/bronchiolar adenoma or carcinoma that yielded equivocal evidence of
9 carcinogenicity of 03 in male B6C3F] mice; and (4) O3 increased the incidence of
10 alveolar/bronchiolar adenoma or carcinoma in female B6C3FJ mice, yielding some evidence
11 of carcinogenic activity of C^ in female mice.
12 Generally, in mice, adenomas appear to progress into carcinomas with time, and thus,
13 the incidence of mice having both adenomas and carcinomas is probably the more useful
14 indicator of effects. The incidence of tumor bearing mice was only significantly elevated in
15 female mice exposed for 2 years to 1.0 ppm O3. When both the 2-year and lifetime
16 exposure studies were combined, there was a increased incidence of tumors in the males at
17 0.5 and 1.0 ppm and in females at 1.0 ppm. The NTP designated the data for male mice as
18 equivocal for carcinogenesis because the combined tumor incidence in the 2-year study was
19 within historical range and the combined incidence for the lifetime study was not significant,
20 even though the carcinoma incidence was significant in the lifetime study. The evaluation of
21 female mice resulted in NTP's finding of "some evidence of carcinogenic activity" because
22 the combined pulmonary adenoma/carcinoma incidence was significantly increased and
23 outside the range of the historical control tumor rates. When the lifetime and 2-year studies
24 were combined, there were 28/100 adenomas plus carcinomas in the 1.0 ppm-exposure group
25 versus 12/100 in the controls (p = 0.004).
26 In summary, the strongest data on the carcinogenicity come from the NTP study, which
27 was only positive in female mice at high concentrations of 0^ (i.e., 1.0 ppm). Because the
28 carcinogenicity data are equivocal in male mice, negative in F344/N male and female rats,
29 and negative for co-carcinogenesis in male rats, it is not justified to extrapolate these data to
30 humans at the present time in the absence of further mechanistic studies justifying
31 extrapolation on a mechanistic basis.
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1 6.2.7.7 Possible Effects of Ozone on Injected Tumor Cells That Lodge in the Lung
2 and Form Lung Colonies
3 To date, no rigorous studies have been conducted to examine the effects of O3 on true
4 lung tumors that would have metastasized. No studies have been conducted in which lung
5 tumor cells detach themselves from primary lung or other tumors growing in organs and
6 invade adjacent tissue, blood vessels, or lymphatics. A few studies have been conducted in
7 which tumor cells are injected intravenously into animals and then lodge in the lung, forming
8 lung colonies (Table 6-20). It must be stressed, however, that this experimental model is not
9 a proper or ideal model for metastasis.
10
TABLE 6-20. EFFECTS OF INHALED OZONE ON THE ABILITY OF INJECTED
TUMOR CELLS TO COLONIZE THE LUNGS OF MICE
Ozone
Concentration
ppm
0.15
0.30
0.1
0.2
0.4
0.8
jttg/m
294
588
196
392
784
1,568
• exposure species
Duration (Strain)
60 days Mouse, M
(C57B/6J)
5 weeks old
1-14 days Mouse, M
(C3H/He)
8-12 weeks old
Observed Effects Reference
No increase in lung metastases from iv Richters (1988)
injected B16 melanoma cells.
Pulmonary metastases in of IV injected Kobayashi et al. (1987)
NR-FS fibrosarcoma cells. After 1 and
14 days of 0.1 ppm, 1.3-fold increases.
After 5 and 7 days of 0.2 ppm, 1.3 and
2.3-fold increases. After 1 and 5 days
of 0.4 ppm, 2.3-fold and 2.2-fold
increases. After 1 day of 0.8 ppm,
4.6-fold increase.
1 Kobayashi et al. (1987) showed that exposure of C3H/He mice for 1 or 14 (but not
2 other) days to ^0.1 ppm O3 after mice were injected in the tail vein with the fibrosarcoma
3 line (NR-FS) increased the number of metastatic lung tumors. Animals were exposed to
4 O3 for 14 days, then fibrosarcoma cells were injected into the tail vein of the animals, and
5 pulmonary metastases were scored 14 days later. One day of exposure to 0.8 ppm O3 gave
6 the maximal enhancement of pulmonary metastases. This enhancement of pulmonary
7 metastasis was concentration-dependent, in the range from 0.4 to 0.8 ppm O$ from 1 to
8 14 days, but increases were small. Witschi (1988) reviewed and interpreted this effect as
9 arising in two possible ways, (1) by damage to the microvasculature and (2) by the
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1 differential sensitivity of various tumor cells to O3 cytotoxicity. Richters (1988) reported
2 that exposure of mice to 0.15 or 0.30 ppm O3 for 60 days did not increase colonization of
3 the lungs of mice injected iv with B16 melanoma cells.
4
5 6.2.7.8 Epidemiology Studies on the Possible Carcinogenicity of Ozone in Humans
6 As reviewed in Speizer (1986), cigarette smoking is the major cause of lung cancer in
7 humans, and may account for as much as 90% of human lung cancer. Hence, any attribution
8 of another agent's causation of lung cancer must be proved against the high frequency of
9 cigarette-induced human lung cancer, which is very difficult. This would be particularly true
10 in the case of attempts to assign a role to O3 in human lung cancer causation. Mills and
11 Abbey (1991) found an increased incidence of respiratory cancers in Seventh Day Adventists
12 who were nonsmokers at only one exposure-time index of 0$. The relative risk was only
13 2.25, and this was of borderline statistical significance, at exposures to 0.01 ppm O3 for an
14 exposure time of 500 h/year. Therefore, no firm data on human lung cancer causation
15 following exposure to O3 has yet been shown (see Chapter 7 for details).
16
17 6.2.7.9 Summary and Conclusions
18 In summary, there are some weakly positive data and some negative data on the
19 genotoxicity of O3 (summarized in Table 6-21). Ozone at very high concentrations (5 to
20 20 ppm) causes DNA strand breakage in plasmid DNA (Hamelin, 1985). Ozone is, at most,
21 weakly mutagenic in some assays and negative in others. Ozone is not mutagenic in
22 Tradescantia or N. tabacam (Gichner et al., 1992), is weakly mutagenic in E. coli and
23 S. cerevisiae (L'Herault and Chung, 1984; Dubeau and Chung, 1982), and is nonmutagenic
24 in three strains of Salmonella and, at most, marginally mutagenic in Salmonella strain TA102
25 (Victorin and Stahlberg, 1988a,b; Dillon et al., 1992). Despite extensive studies by Dillon
26 et al. (1992), the mutagenicity of O3 in Salmonella TA102 is not conclusive because
27 convincing concentration-dependent mutagenic effects have not yet been demonstrated.
28 Ozone causes cytogenetic damage in cultured cells in vitro (e.g., Hamelin et al., 1977 a,b;
29 Dubeau and Chung, 1979, 1982), but no effects or small and conflicting effects when
30 animals are exposed in vivo (Zelac et al., 1971 a,b; Tice et al, 1978). Cell transformation
31 studies have shown positive results upon exposure of cells to O3, but these studies were
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TABLE 6-21. SUMMARY OF DATA ON THE GENQTOXICITY OF OZONE
Assay System in Which Ozone Was Tested Result Comments
Mutation to histidine prototrophy in
Salmonella TA100
Mutation to histidine prototrophy in
Salmonella TA102
Mutation to streptomycin resistance in
Eshcherichia coli
Mutation in Saccliaromyces cerevisiae
Small effects obtained, less than
twofold, dose-response effect was
not shown.
'— Small effects obtained, and there
was no direct dose response.
Ozone caused mutation and
recombination, but this was a weak
response compared to known strong
mutagens (20-to 200-fold less
mutagenic than UV light, X rays,
and MNNG).
Mutation in Nicotiana tabacam in a
leaf-color reversion assay
Mutation in Tradescantia in a stamen-hair
assay
Chromosomal breakage in cultured
mammalian cells
Chromosomal breakage in animals
Morphological transformation in
C3H/10T1/2 mouse embryo cells and in
Syrian hamster embryo cells
Induction of preneoplastic variants in rat
trachea! epithelial cells
Results are at best weak and
controversial; results in this assay
are considered ambiguous and not
definitively positive at present.
Experiments need to be conducted
without or with only minimal
amounts of fluid bathing the cells.
Concentrations giving positive
results are high (5 and 6 ppm).
Both in vitro and in vivo exposures
give negative or at most only
twofold increases.
December 1993
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TABLE 6-21 (cont'd). SUMMARY OF DATA ON THE GENOTOXICFTY OF OZONE
Assay System in Which Ozone Was Tested Result Comments
Lung tumor induction in whole animals
a) Strain A/J mice, Swiss-Webster mice,
Wistar rats
b) National Toxicology Program Studies
Male and female F344/N rats
Male B6C3F! mice
(2-year study)
Male B6C3FJ
(Lifetime studies)
Female B6C3FJ mice
(2-year study)
Female B6C3FJ
(Lifetime studies)
+/- a) Positive results marginal, not
statistically significant; experiments
not designed to determine whether
a concentration-response exists.
- No increased incidence of
pulmonary adenomas or carcinomas
in rats exposed 0.12, 0.5, or
1.0 ppm for 2 years or with 0.5 or
1.0 ppm for animals' lifetimes.
+/- No effect at 0.12 ppm O3, slight
increases in the total pulmonary
neoplasms at 0.5 and 1.0 ppm O3,
but they were not statistically
significant.
+/— Alveolar/bronchiolar carcinoma
incidence increased twofold, at
0.5 ppm O3 (p = 0.05) and
1.0 ppm O3 (p = 0.007); no
increases in total pulmonary
neoplasms.
+ Fourfold increase in
alveolar/bronchiolar carcinoma at
1.0 ppm O3 (p = 0.053).
+ Fourfold increase in
alveolar/bronchiolar adenomas and
carcinomas at 1.0 ppm O3
(p = 0.02).
+ Threefold increase in
alveolar/bronchiolar adenomas at
1.0 ppm O3 (p = 0.02).
+ Twofold increase in
bronchiolar/alveolar adenomas and
carcinomas at 1.0 ppm O3, but not
statistically significant.
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1 conducted with a fluid barrier above the cells that may have resulted in artifacts compared to
2 an in vivo exposure (Borek et al., 1986, 1989 a,b).
3 The in vitro studies are mechanistically interesting, but their relevance and predictive
4 value to human health is questionable for the following reasons. First, the concentrations
5 used in these in vitro studies were typically orders of magnitude greater than what is found in
6 ambient air. Second, extrapolation of in vitro exposure concentrations to human exposure
7 dose requires special methods that were not used in these studies. Third, direct exposure of
8 isolated cells to O3 is somewhat artifactual because it bypasses all the host defenses that
9 would normally be functioning to protect the individual from the inhaled dose.
10 Furthermore, direct exposure of isolated cells in vitro to O3 likely results in chemical
11 reactions between O3 and culture media to generate chemical species that might not be
12 produced in vivo. The most relevant data on the genotoxicity of O3 should therefore be
13 obtained from in vivo studies.
14 The earlier studies in whole animal carcinogenesis bioassays must be considered
15 ambiguous at this time (Witschi, 1988, 1991). The NTP study utilized an appropriate
16 inhalation model and assayed the carcinogenicity of O3 in male and female F344/N rats and
17 B6C3FJ mice, and also tested whether O3 could enhance the tumorigenicity of the tobacco-
18 specific pulmonary carcinogen, NNK (National Toxicology Program, 1993). This study
19 clearly showed that O3 was not carcinogenic in female and male rats at 0.12, 0.5, and
20 1.0 ppm O3 (6 h/day, 5 days/week, 2 years) or at 0.5 and 1.0 ppm O3 (6 h/day,
21 5 days/week, lifetime). Exposure to 0.5 ppm O3 did not enhance the carcinogenicity of
22 NNK in male rats, leading to conclusion that O3 does not act as a co-carcinogen or tumor
23 promoter in these animals. In the male mice, O3 had equivocal effects at 0.5 and 1.0 ppm
24 O3 in the 2-year and lifetime inhalation studies. In the female mice, there was some
25 evidence for the carcinogenicity of O3 at 1.0 ppm (2.7-fold increase in total pulmonary
26 neoplasms fp = 0.02] in the 2-year study; twofold increase in total pulmonary neoplasms
27 [p = 0.1] in the lifetime study; 2.3-fold increase in total pulmonary neoplasms when the
28 2-year and lifetime study were combined [p = 0.004]).
29 Therefore, the earlier negative animal carcinogenesis studies, the negative
30 carcinogenicity results in inhalation carcinogenesis studies in F344/N male and female rats,
31 the ambiguous data in male B6C3Fj mice, and the weak carcinogenicity of O3 in female
December 1993 6-152 DRAFT-DO NOT QUOTE OR CITE
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1 B6C3FJ mice indicate that O3 is only carcinogenic in female B6C3Fj mice at high
2 concentrations (1.0 ppm). Because the weak carcinogenicity of O3 in female mice is
3 inconsistent with ambiguous results in male mice and negative results in male and female
4 F344/N rats, it is not possible to extrapolate these results to humans without further
5 mechanistic studies justifying such an extrapolation. Ozone also does not function as a tumor
6 promoter in NNK-treated male rats, so there is also no basis for extrapolating tumor-
7 promoting activity to humans. The weak genotoxicity of O3 in a few systems and negative
8 results in other assay systems at very high concentrations, well beyond those that humans
9 would be exposed to, also do not lend themselves to extrapolation to humans.
10
11
12 6.3 SYSTEMIC EFFECTS OF OZONE
13 6.3.1 Introduction
14 Ozone has long been known to cause effects in organs/tissues outside the respiratory
15 tract. The mechanisms are not known, but it is quite unlikely that O3 itself enters the
16 circulation (Pryor, 1992). Another possibility it that transported reaction products cause
17 distant effects. Some effects may be secondary to effects on the lung (e.g., aversive
18 behaviors that may result from lung irritation). The relatively few systemic studies reported
19 since the last O3 criteria document (U.S. Environmental Protection Agency, 1986) are
20 discussed below. Some classes of effects (i.e., reproduction/development, endocrine system)
21 were only studied earlier) and hence are briefly cited here in the introduction.
22 No reproductive toxicity studies of O3 were found. Only two developmental studies
23 provided sufficient details in the report to determine the exposures used. The only effect
24 observed by Kavlock et al. (1979) in pregnant rats exposed to 0.44 to 1.97 ppm for the
25 entire period of organogenesis or 3 stages of gestation was an increased resorption of fetuses
26 in rats exposed to 1.49 ppm in midgestation; no terata were found. A follow-up study
27 revealed that pups from dams exposed to 1 ppm 0$ during mid- or late gestation showed
28 lower body weights 6 days after birth (Kavlock et al., 1980). A higher concentration
29 (1.5 ppm) delivered during late gestation permanently runted 14% of the male pups.
30 Studies on the effects of O3 on the endocrine system date back to 1959. Generally, the
31 body of work indicates that O3 can affect the pituitary-thyroid-adrenal axis
December 1993 6-153 DRAFT-DO NOT QUOTE OR CITE
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1 (U.S. Environmental Protection Agency, 1986). For example, a 1-day exposure to 1 ppm
2 decreased serum levels of thyroid-stimulating hormone, thyroid hormones, and protein-bound
3 iodine; prolactin levels increased (demons and Garcia, 1980 a,b). Structural changes
4 occurring tin the parathyroid glands after a 4 to 8-h exposure to 0.75 ppm included chief
5 cells hyperplasia of chief cells, but circulating hormone levels were not measured (Atwal and
6 Wilson, 1974).
7
8 6.3.2 Central Nervous System and Behavioral Effects
9 Reports of headache, dizziness, and irritation of the nose, throat, and chest are common
10 complaints that are associated with O3 exposure in humans (see Chapter 7). Laboratory
11 animal studies have been performed that demonstrate behavioral effects over a wide range of
12 O3 concentrations (0.08 to 1.0 ppm) and suggest that these behavioral changes may be
13 analogous to the symptoms reported in humans. Although these behavioral changes may be
14 indicative of O3-induced symptoms, they are not indicative of neurotoxicity. Most of the
15 studies prior to 1986 indicated that behavior could be suppressed with O3 exposure. For
16 example, Murphy et al. (1964) and Tepper et al. (1982) showed that running wheel behavior
17 was suppressed, and Peterson and Andrews (1963) and Tepper et al. (1983) showed that
18 mice would alter their behavior to avoid O3 exposure. Furthermore, Weiss et al. (1981)
19 showed that bar-pressing responses for food reinforcement were suppressed, but greater
20 O3 concentrations were required to decrease this behavior than the concentrations needed to
21 decrease running wheel behavior.
22 Since 1986, several reports have extended the previous findings (Table 6-22). Tepper
23 et al. (1985) compared the effects of a 6-h exposure to O3 on the suppression of running
24 wheel behavior in rats and mice. The study indicated that the lowest effective concentration
25 was about 0.12 ppm O3 in the rat and about 0.2 ppm in the mouse. It was also observed that
26 with exposure to 0.5 ppm, recovery from O3 required at least 3 h. In a follow-up study,
27 Tepper et al. (1985) required mice to make a response that turned off the brief delivery
28 (60 s) of O3 at concentrations between 0.25 to 16 ppm. Mice learned to terminate
29 O3 exposures at 0.5 ppm. With each of three determinations of the concentration-response
30 curve, mice got better at terminating O3 exposure rather than showing an adaptation to
31 exposure. The authors suggest that mice may have learned to use the odor as a conditioned
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TABLE 6-22. EFFECTS OF OZONE ON BEHAVIOR3
1
h- '
Ul
o
I
I
§
Ozone
Concentration
ppm
0.08
0.12
0.25
0.5
0.08
0.12
0.25
0.5
0.08
0.12
0.25
0.5
0.12-
1.5
0.1-
0.8
0.25-
16
0.4
1.2
0.5
2.0
/tg/m3
157
980
157
980
157
980
235-
2,940
196-
1,568
490-
31,360
784
2,352
980
3,920
Duration Conditions
6 h Free-access wheel
running
6 h Wheel running for food
6 h Lever pressing for
access to the running
wheel
6 h Nose poke response for
food
7 days Drinking, eating
continuous
60-s Nose poking terminated
maximum O3 exposure
13 days Home cage behavior
continuous
3 h Lever pressing to avoid
electric shock
Species, Sex
(Strain)
Age
Rat, M
(Long-Evans)
10 weeks old
Mouse, M
(Swiss-Webster)
5 weeks old
Rat, M
(Long-Evans)
300 g
Rat, M
(Long-Evans)
300 g
Rat, M
(Long-Evans)
275 g
Mice, M
(ICR)
8-26 weeks old
Mouse, M
(Swiss-Webster)
30 g
Mice
Rat
(Wistar)
300 g
Observed Effect(s)
Mice less responsive than rats. Reduction in free-access wheel running at
approximately 0.12 ppm in rats and 0.2 ppm in mice. Recovery from
exposure to 0.5 ppm did not occur by 5 h postexposure in either mice or
rats.
0.12 ppm Oj decreased wheel running for food reinforcement.
0.12 ppm 03 decreased bar press for access to the running wheel. Two of
the four animals were affected at 0.08 ppm.
0.5 ppm 03 decreased nose poking for food reinforcement. Effects were
enhanced postexposure.
Drinking, food consumption, and body weight initially decreased, but adapted
with continued exposure, starting at 0.2 ppm.
At 0.5 ppm, mice learned to terminate Oj exposure.
During first hour, rearing, grooming, sniffing, and social interactions
increased, crossings and wall climbing decreased. These behaviors did not
adapt with continued exposure.
Suppression of lever pressing began after 45 min of 2.0-ppm exposure and
after 90 min of 0.5- or 1.0-ppm exposures.
Reference
Tepper et al. (1985)
Tepper and Weiss (1986)
Tepper and Weiss (1986)
Tepper and Weiss (1986)
Umezu et al. (1993)
Tepper and Wood (1985)
Musi et al. (1993)
Ichikawa et al. (1988)
*See glossary of terms and symbols for abbreviations and acronyms.
Age (or body weight) at start of exposure.
-------
1 stimulus to initiate termination of exposure rather than respond directly to the irritant
2 properties of O3.
3 Because free-access wheel running behavior was suppressed at 0.12 ppm (Tepper et al.,
4 1982) and lever pressing for food reinforcement was reduced only at 0.5 ppm (Weiss et al.,
5 1981), a series of experiments was performed to evaluate the behavioral determinants of the
6 O3 response (Tepper and Weiss, 1986). Food deprivation and response contingencies
7 (having to perform a certain response to get a reward) were found to be relatively
8 unimportant determinants of behavior because rats that had to run rather than lever press to
9 obtain food reinforcement showed behavioral suppression of running at 0.12 ppm. However,
10 in another experiment, suppression of lever pressing was shown to be equally sensitive to
11 O3 exposure when pressing the lever allowed rats to have access to the running wheel. The
12 authors concluded that increased physical activity, either used as the response to obtain
13 reward, or as the reward, was an important behavioral variable in determining sensitivity to
14 O3 exposure. Ichikawa et al. (1988) demonstrated that behavior (lever pressing) maintained
15 by the avoidance of electric shock, was even less sensitive to Oj exposure (3 h, 1.0 ppm)
16 than behaviors maintained by food reinforcement, as described above. Furthermore, the
17 animals recovered quickly after O3 exposure was terminated (60 to 120 min).
18 In mice exposed to O3 continuously for 13 days (0.4 to 1.2 ppm), both increases and
19 decreases in measured behaviors were observed (Musi et al., 1993). During the first hour of
20 exposure to 0.8 or 1.2 ppm, but not 0.4 ppm, increases in rearing, grooming, sniffing, and
21 social interactions were observed, but locomotion and bar-holding declined. With continued
22 exposure (measurements on Days 3, 7, and 10), grooming and rearing were still increased
23 but crossings and wall climbing remained depressed. The affected behaviors did not show
24 adaptation. However, drinking, food consumption, and body weight were initially depressed,
25 but abated with continued exposure; a finding previously reported in mice at
26 O3 concentrations as low as 0.2 ppm (Umezu et al., 1993).
27 In summary, the behavioral data indicate that transient changes in behavior occur in
28 rodent models that are dependent on a complex interaction of factors such as (1) the type of
29 behavior being measured, with some behaviors increased and others suppressed; (2) the
30 factors motivating that behavior (differences in reinforcement); and (3) the sensitivity of the
31 particular behavior (e.g., active behaviors are more affected than more sedentary behaviors).
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1 6.3.3 Cardiovascular Effects
2 Several reports have demonstrated that O3 exposure causes dramatic effects to the
3 cardiovascular system in the rat (Table 6-23). Uchiyama et al. (1986) initially reported that
4 heart rate (HR) and mean arterial blood pressure (MAP) were decreased by 53 and 29%,
5 respectively, during a 3-h exposure to 1.0 ppm 03. Arrhythmias, including A-V block and
6 premature atrial contractions, were also frequently observed. The effects appeared to be
7 age-, but not sex-, dependent, with 11-week-old rats showing a greater response than did
8 8- or 4-week-old rats. Yokoyama et al. (1989) showed that recovery from the effects of the
9 3-h, 1.0-ppm O3 exposure were not complete by 5 h and that with three daily consecutive
10 exposures, both the HR and MAP responses were attenuated. Further investigations by the
11 same group of authors (Uchiyama and Yokoyama, 1989) showed that with exposures to
12 0.5 ppm O3 for 6 h, HR and MAP decreased by 32 and 18%, respectively. A 4-week
13 continuous exposure to 0.2 ppm initially resulted in a 12% decrease in HR, but this response
14 was attenuated on Day 2 and was almost eliminated by Day 3. No further effects were
15 observed during the rest of the 4-week exposure period. When these same animals were
16 subsequently challenged with 0.8 ppm O3 for 1.5 h, they also had an attenuated response
17 when compared to rats that were O3 naive. Additionally, some rats were intratracheally
18 instilled with elastase to create an animal model of emphysema. This pretreatment, however,
19 did not affect outcome of either the HR or MAP responses to Oj in any of the experiments,
20 except in the 0.8-ppm challenge experiment. In this experiment, elastase-treated, Oyexposed
21 rats challenged with O3 had a similar response to O3 challenge as did O3 naive rats,
22 suggesting that the elastase treatment affected the ability of the rats to develop an adaptive
23 lung response. In contrast, Tepper et al. (1990) did not observe an alteration in blood
24 pressure of rats exposed to 1.0 ppm for 135 min, even though their ventilation was increased
25 by CO2.
26 Arito et al. (1990) demonstrated bradycardic responses at 0.2 ppm O3 during the first
27 2 days of a continuous 5-day exposure; bradyarrhythmia occurred during the first 3 days of a
28 0.1 ppm exposure. Simultaneously, these authors measured the sleep-wakefulness of the rats
29 during exposure and found that more bradyarrhythmias occurred during wakefulness than
30 during slow-wave sleep or paradoxical sleep. Sleep-wakefulness patterns were not altered by
31 this O3 exposure. At high O3 concentrations (1 ppm, 3 h), wakefulness and paradoxical
December 1993 6-157 DRAFT-DO NOT QUOTE OR CITE
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1 sleep were suppressed, the amplitude of the electroencephalogram (EEG) was lowered, and
2 slow-wave sleep was increased (Arito et al., 1992). These EEG changes appear to be
3 temporally associated with the decrease in behavioral activity previously discussed (Tepper
4 et al., 1982). Atropine sulfate blocked the suppression of wakefulness and bradycardia in a
5 concentration-related manner, and decreased slow-wave sleep, suggesting that some of the
6 O3 effects are parasympathetically mediated. The effects of O3 on paradoxical sleep and the
7 EEG amplitude were not affected by atropine administration. Watkinson et al. (1993)
8 extended these findings by showing that the core temperature of rats was also reduced when
9 HR fell at 03 exposure concentrations between 0.37 and 1.0 ppm (2 h). Increasing ambient
10 temperature to 30 to 32 °C attenuated the 1.0 ppm O3-induced reduction in HR and core
11 temperature.
12 In an attempt to synthesize the results from these studies, Watkinson and Gordon (1993)
13 questioned the relevance of these parameters in the rat as compared to the human. Rats have
14 different thermoregulatory responses than humans and typically respond to toxic insult by
15 lowering core temperature. This response has been shown to increase survival value
16 (Watkinson et al., 1989). Similar changes in core temperature and HR have not been
17 reported in humans. This may be because of the large, and thus stable, thermal mass of
18 humans, or alternatively, these effects have not been observed because they weren't
19 measured and because most O3 exposure experiments are done using exercise, which may
20 mask these responses. In support of this latter idea, Coleridge et al. (1993) reported that
21 stimulation of bronchial C-fibers produces bradycardia. Ozone preferentially stimulates
22 bronchial C-fibers and, as a result, induces bradycardia and tachypnea in the anesthetized,
23 open chest dog model. Furthermore, the tachypnea produced by O3 exposure is inhibited by
24 atropine administration (the effect on HR was not reported).
25
26 6.3.4 Hematological and Serum Chemistry Effects
27 Hematological effects reported in laboratory animals and humans after inhalation of
28 O3 indicate that the gas or (more likely) some reaction product can cross the blood-gas
29 barrier. The effects of in vivo O3 exposure in animals were summarized in the previous
30 O3 criteria document (U.S. Environmental Protection Agency, 1986). The hematologic
31 parameters most frequently used to evaluate O3 toxicity were morphologic and biochemical
December 1993 6-159 DRAFT-DO NOT QUOTE OR CITE
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1 effects on erythrocytes (red blood cells, RBCs). These studies reported alterations in RBC
2 morphology, increased RBC fragility, increased hemolysis, and decreased survival. The
3 biochemical studies reported variable results, depending on the O3 exposure concentration
4 and the RBC enzyme under investigation.
5 More recent studies have stressed serum effects of O3 exposure (Table 6-24). Exposure
6 of rats for 2 h to 0.1 ppm O3 increased plasma creatinine kinase activity, whereas no such
7 effect was observed when exposure was to 0.05 and 0.25 ppm O3 (Veninga and Fidler,
8 1986). Decreased serum retinol concentrations were observed following continuous exposure
9 of rats for 14 days to 0.4 ppm (Takahashi et al., 1990), but no changes in plasma retinol,
10 ascorbic acid, and a-tocopherol were observed following exposure of rabbits for 3 h to
11 O3 ranging from 0.1 to 0.6 ppm (Canada et al., 1987). In similar studies, a decrease in
12 plasma lactic dehydrogenase isoenzyme activity was also observed following exposure of rats
13 for 18 h to 0.8 ppm O3 (Nachtman et al., 1988).
14 Miller et al. (1987, 1988) investigated the effect of a 1-h exposure of guinea pigs to
15 1.0 ppm O3 on plasma eicosanoid levels and observed increases in TXB2, 6-keto-PGFla, and
16 PGEj. These data suggest that some of the systemic effects of O3 exposure, such as
17 impairment of peritoneal AM phagocytosis (Canning et al., 1991), may be mediated by the
18 immunosuppressive effects of the prostanoids (Oropeza-Rendon et al., 1979).
19 Heat-inactivated plasma from rats exposed for 23 h/day for 2 weeks to 1.0 ppm O3 also
20 increases DNA synthesis by lung fibroblasts (Tanswell et al., 1989) and lung pneumocytes
21 (Tanswell etal., 1990).
22
23 6.3.5 Other Systemic Effects
24 Previous studies suggest that O3 has effects on the zenobiotic metabolism of the liver
25 (U.S. Environmental Protection Agency, 1986). This effect has been observed in mice, rats,
26 and hamsters as a prolongation of pentobarbital sleeping time (Graham et al., 1981). The
27 effect appears to be sex dependent, with females having greater responses than males.
28 Canada and Calabrese (1985) performed a similar experiment in both young (3 to 4 mo) and
29 older (2 years) rabbits exposed for 3.75 h/day to 0.3 ppm O3 for 5 consecutive days. They
30 observed significant prolongation of the elimination of theophylline in older, but not young
31 rabbits, and the effect was more pronounced in females than in males. In a follow-up study,
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TABLE 6-24. HEMATOLOGY AND SERUM CHEMISTRY EFFECTS8
§
y
, .
S
CTN
h— »
ON
g
M
>
»"l
1
0
o
8
o
Ozone
Concentration
ppm
0.05
0.1
0.25
0.1
0.2
0.4
0.6
0.4
0.8
1.0
1.0
1.0
1.0
exposure
Duration
Mg/m
100 2h
200
500
196 3h
392
784
1,176
784 Continuous
for 14 days
1,568 18 h
1,960 1 h
1,960 Ih
1,960 23h/dayfor
2 weeks
1,960 4h
Species, Sex
(Strain)
Age
Rat, M
(Wistar)
200 g
Rabbit, F
(NZW)
2.5-3 .5 years old
Rat, M
(Wistar)
20 weeks
Rat, M
(F344)
Guinea Pig. M
(Hartley)
250-300 g
Guinea Pig, M
(Hartley)
250-300 g
Rat, M
(CD)
400±25g
Mouse, M
(CD-I)
8 weeks old
Observed Effect(s)
Increased plasma creatine kinase activity at 0.1 ppm, but not at 0.05 and 0.25
ppm.
No change in plasma retinol, ascorbic acid, and a-tocopherol concentrations.
Decrease in serum retinol concentration.
Decrease in plasma lactic dehy drogenase isoenzyme activity.
Increases in plasma concentrations of TXB^, 6-keto-PGFj^, and PGEj .
Increases in plasma concentrations of TXRj, 6-keto-PGFia, and PGEj .
Heat-inactivated plasma increases DNA synthesis by lung fibroblasts and
pneurnocytes.
Inhibition of RBC deformiability.
Reference
Veninga and Fidler (1986)
Canada et al. (1987)
Takahashietal. (1990)
Nachtman et al. (1988)
Miller et al. (1987)
Miller et al. (1988)
Tanswell et al. (1989, 1990)
Morgan et al. (1988)
"See glossary of terms and symbols for abbreviations and acronyms.
Age (or body weight) at start of exposure.
-------
1 Canada et al. (1986) could not demonstrate increased pentobarbital sleeping in young
2 (2.5 mo) mice or rats of comparable age to the study by Graham et al. (1981). However,
3 effects were observed in older (18 mo) female mice and rats. Two other studies (Heng
4 et al., 1987; Zidenberg-Cherr et al., 1991), from the same group of investigators, indicate
5 that liver antioxidant enzymes (Cu/Zn- and Mn- SOD and GSHPx) are decreased
6 commensurate with the increase in these enzymes that is observed in the lung.
7
8 6.3.6 Summary
9 Several reports have recently appeared that extend previous observations in laboratory
10 animals that indicate that ambient levels of O3 can affect animal behavior. These effects are
11 most likely interpreted as analogous to O3-induced symptoms in humans and not as evidence
12 of neurotoxicity. The behavioral changes are transient, but may persist several hours after
13 acute exposure. Different types of behaviors appear to be variably sensitive to O3 exposure,
14 with active behaviors showing suppression at lower O3 concentrations that do more sedentary
15 behaviors or behaviors maintained by electric shock (Ichikawa et al., 1988; Tepper et al.,
16 1985; Tepper and Weiss, 1986). For example, a 6-h exposure of rats to 0.12 ppm
17 suppressed running wheel behavior (Tepper et al., 1985). Furthermore, animals will respond
18 to terminate a 1-min exposure to 0.5 ppm O3, thus directly implicating the irritant properties
19 of O3 (Tepper and Wood, 1985). It appears that with additional training, animals can learn
20 to terminate exposure using conditioned stimuli rather than relying directly on the aversive
21 properties of O3 (Tepper et al., 1985).
22 Ozone has been found to profoundly decrease HR, MAP, and core temperature in rats
23 (Watkinson et al., 1993; Arito et al., 1990; Uchiyama and Yokoyama, 1989). During
24 exposure, arrhythmias frequently occur. After a 3-h exposure to 1.0 ppm, these effects
25 appear to occur more in adult rats (11 weeks) than in younger animals (4 and 8 weeks),
26 especially when the rats were awake (as measured by EEG) (Uchiyama et al., 1986). The
27 lowest exposures causing bradycardia in rats was 0.2 ppm for 48 h; 0.1 ppm for 24 h caused
28 bradyarrhythmia (Arito et al., 1990). Similar effects have not been observed in humans or
29 other species. In part, this may be because they have not been systemically examined or that
30 human studies have been carried out during concurrent exercise, which may mask these
December 1993 6-162 DRAFT-DO NOT QUOTE OR CITE
-------
1 effects. More likely, these effects represent a species differences related to the magnitude
2 and localization of reflex responses and differences in thermal mass.
3
4
5 6.4 INTERACTIONS OF OZONE WITH OTHER CO-OCCURRING
6 POLLUTANTS
7 6.4.1 Introduction
8 Most of the toxicological data for O3 are derived from studies using O3 alone.
9 However, it is also important to evaluate responses to inhalation of typical pollutant
10 combinations because ambient exposures involve mixtures. Such mixtures provide a basis
11 for toxicological interactions, whereby combinations of chemicals may behave differently
12 than would be expected from consideration of the action of each separate constituent. This
13 section discusses toxicological studies of pollutant mixtures in which O3 is one component.
14 Discussions of many of these studies addressing the effect of 03 alone on various organs or
15 systems appear elsewhere in this chapter.
16 Evaluating the role of O3 in observed responses to inhaled mixtures is not easy.
17 In spite of the myriad of interpretative difficulties, it is essential to attempt to understand the
18 potential for interactions because O3 does not exist alone. One of the problems involves
19 definitions of terms. In general, and for the purposes of this document, an interaction is
20 considered to occur when the response to a mixture is statistically significantly different from
21 the sum of the responses to the individual pollutants (i.e., not additive). A less than additive
22 interaction is antagonism, whereas synergism is an interaction that is more than additive.
23 A subclassification of synergism, termed potentiation, is often used to describe an interaction
24 in which response to a mixture is greater than the sum of the responses to the individual
25 components, but where only one component produced a response different from control when
26 administered alone. In many instances, however, potentiation and synergism have been used
27 interchangeably. Although some synergistic interactions may actually serve to stimulate
28 repair processes, or otherwise reduce the harmful effects of O3, and some antagonistic
29 interactions may eventually increase the risk of disease development, synergism as currently
30 used generally implies greater risk and antagonism implies lesser risk. However, such
31 assumptions may eventually be proven to be invalid in some instances. Also, interactions
December 1993 6-163 DRAFT-DO NOT QUOTE OR CITE
-------
1 with the large number of natural air ppllutants, such as microbes, spores, and dusts, that can
2 produce considerable responses alone are not included in this section.
3 In most cases, the interaction of O3 with other pollutants has been studied using
4 mixtures that contained only one other copollutant (i.e., simple or binary mixtures). In such
5 studies, the role played by each pollutant in eliciting measured responses can be elucidated
6 with the appropriate experimental design, but a substantial database involves exposures to the
7 mixture and O3 only, with no exposure to the copollutant alone. Although the
8 O3 concentration may have been varied among exposure groups or was present in one group
9 and not in another, so its relative influence could be assessed to some extent, it cannot be
10 determined in such cases whether the response to the mixture involved actual interaction or
11 was merely additive.
12 The ambient atmosphere in most environments is generally a mixture of a number of
13 pollutants, and assessing effects of such multicomponent atmospheres may serve to provide
14 some indication of biological responses under conditions that better mimic ambient exposure.
15 However, the ability to discern the contribution of O3 to observed responses becomes even
16 more difficult when such complex mixtures are studied. Even when binary mixtures are
17 used, they often do not mimic the ambient pattern (e.g., NO2 levels peak before O3 levels
18 do) or ambient concentrations (as absolute values or as ratios). Rarely are concentration-
19 response mixture studies performed. This raises the possibility that an unrealistic
20 experimental design may lead to masking the effect of a copollutant or identifying a response
21 that may not occur in the real world.
22 Another problem in assessing responses to mixtures involves the statistical basis for
23 conclusion of significant interaction. For example, a number of studies determined
24 interaction by comparison of the response from exposure to only one component of the
25 mixture with that from exposure to the complete mixture. On the other hand, some studies
26 used statistical approaches specifically designed to indicate interactions. As another example,
27 it may be relatively straightforward to study interactions when one exposure concentration of
28 each of two pollutants is used, but it becomes much more difficult when there are multiple
29 concentrations used, and even more difficult still when more than two pollutants are
30 involved. Because variable criteria for conclusions of interaction have been used, the
December 1993 6-164 DRAFT-DO NOT QUOTE OR CITE
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1 available database is one in which the statistical significance for determination of interaction
2 varies in terms of its robustness.
3
4 6.4.2 Simple (Binary) Mixtures Containing Ozone
5 Tables 6-25 and 6-26 outline studies performed since publication of the last O3 criteria
6 document (U.S. Environmental Protection Agency, 1986) in which experimental animals
7 were exposed to atmospheres containing O3 with only one other copollutant. These tables
8 provide the experimental details for the discussion that follows.
9
10 6.4.2.1 Nitrogen Dioxide as Copollutant
11 The most commonly studied copollutant in binary mixtures with O3 is NO2. Studies
12 discussed in the previous O3 criteria document indicated that although interaction may occur
13 between these two pollutants, in general O3 often masked the effects of the NO2 or accounted
14 for most of the response. This is because on a mole to mole basis, O3 is considerably more
15 toxic than NO2, and the relative contribution of O3 and NO2 to pulmonary injury is driven
16 by the exposure ratio of the two pollutants. Commonly studied endpoints for assessing
17 effects of these mixtures were lung morphology, biochemistry, and resistance to bacterial
18 infection.
19 To put the exposure concentrations of NO2 into some perspective, short term,
20 24-h averages are generally ^0.17 ppm and 1-h averages are generally ^0.4 ppm in major
21 metropolitan areas. However, hourly averages in most regions often exceed 0.2 ppm at least
22 once during the year (Schlesinger, 1992).
23 An earlier study noted that the morphological response of the rat lung alveolar
24 epithelium following 60 days of exposure to O3/NO2 mixtures (0.25 ppm O3 + 2.5 ppm
25 NO2, or 0.9 ppm O3 + 0.9 ppm NO2) was due to the O3 (Freeman et al., 1974). However,
26 the duration of exposure may affect the contributory role of the copollutant. Thus, for
27 example, Terada et al. (1986) exposed rats to O3 alone, or to a mixture of 0.1 ppm
28 O3 + 0.3 ppm NO2, with O3 administered 8 h/day and NO2 administered 24 h/day for up to
29 18 mo. Following 1 mo of exposure, observed lesions in the group exposed to the mixture
30 were similar in severity to those noted with exposure to Oj alone, but as the duration of
31 exposure increased, the morphological changes in interstitial tissue appeared more marked in
December 1993 6-165 DRAFT-DO NOT QUOTE OR CITE
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TABLE 6-25. TOXICOLOGICAL INTERACTIONS OF OZONE AND NITROGEN DIOXIDE11
Sf
~
5
^o
U)
ON
(— *
£
o>
5
t-rt
i
8
z
Q
H
0
d
Q
3
o
s
Concentration
ppm
0.05
0.04
0.05
0.4
0.05
0.4
0.1
0.5
1.2
4.0
0.1
0.3
1.2
2.5
0.05
0.1
0.5
1.0
0.05
0.1
0.05
0.1
0.1
0.3
fig/m
98
75
98
752
98
752
196
980
2,256
7,520
196
588
2,256
4,700
98
196
940
1,880
98
196
94
188
196
564
Pollutant
°3
NO2
°3
N02
°3
N02
03 (baseline)
03 (peak)
NC>2 (baseline)
N02 (peak)
Oj (baseline)
03 (peak)
NO2 (baseline)
NOjteeak)
03 (baseline)
03 (peak)
NO2 (baseline)
N02(peak)
Oj (baseline)
03 (peak)
NO2 (baseline)
N02(peak)
°3
NO2
Exposure Duration
NO2: 24 h/day ,
03: intermittent during
hours 9-19/day following
sine curve from
0-0.1 ppm (0.05 avg),
total duration: 5-22 mo
03: concentration ranged
from 0 to 0.1 with sine
curve over 9-19 h
(0.05 avg), NO2:
24 h/day, both 13 mo,
11 mo recovery
15 day, continuous
exposure to basal level;
peaks: 1 h, twice daily,
5 days/week beginning
after 64 h of continuous
exposure
NCty 24 h/day;
03: 8 h/day;
1, 3, 6, 18 mo
s
Species, Sex
(Strain)
Age0
Rat, M
(Wistar)
7 weeks old
Rat, M
(Wistar)
6 weeks old
Mouse, F
(CD-I)
4-6 weeks old
Rat, M
(F344)
5 weeks old
Time of
Endpoint
Endpoints Measurement
Lung protein content; lung 0 PE
lipid peroxides; antioxidant
enzymes (G6PD, 6PGD, GR,
GST, GSHPx, SOD)
Development of lung tumors 0 PE
from exposure (ingestion) of
carcinogen,
JV-bis(2-hydroxypropyl)
nitrosamine prior to 03 and
NOj
Bacterial infectivity Bacterial
(to Streptococcus challenge
zooepidemicus given after given 0 or
pollutant exposure) 18 h PE
Histopathology (LM, TEM) 0 PE
Response to
Mixture
Protein: no change;
peroxide: increase
between 5 and 9 mo,
return to control at
>9 mo (greater effect
with 0.4 ppm NO^J
enzymes: no change.
Increased tumor
incidence (compared to
air-exposed control).
No effect at low level;
increased mortality at
other levels.
Connective tissue
edema, Type 2 cell
hypertrophy and
enlarged lamellar bodies.
Interaction
Protein: none (no
effect of 03 or NO2
alone), peroxide:
synergism (no change
with 03 or NO2 alone
for 9 mo), enzyme:
none (no effect of 03
or NO2 alone).
Suggested synergism:
no increase with 03
alone (NOj alone not
done).
Synergism: at 0.05 03
+ 0.5(1.0)NO2,
0.1 03 + 1.2(2.5)
NO2; marginal
synergism at 0.1 03 +
1.2(4)NO2- BothO3
and NO2 increased
mortality at two highest
levels; only NC>2
increased at two lowest
levels.
Changes more marked
than with 03 alone,
NO2 affected response
to 63 (no quantitation
performed).
Reference
Sagai and Ichinose
(1991)
Ichinose and Sagai
(1992)
Graham et al. (1987)
Terada et al. (1986)
-------
TABLE 6-25 (cont'd). TOXICOLOGICAL INTERACTIONS OF OZONE AND NITROGEN DIOXIDE*
CD
1
sf
—
\§
U)
^
Os
"-1
P
CH
i
Q
o
s
o
H
O
^^
s
s
0
!^
0
g
Concentration
3
ppm jig/m
0.15 294
0.35 564
0.2 392
4.0 7,520
0.2 392
3.6 6,768
0.4 784
7.2 13,536
0.6 1,176
10.8 20,304
0.8 1,568
14.4 27,072
0.2 392
3.6 6,768
0.4 784
7.2 13,536
0.6 1,176
10.8 20,304
0.2 392
3.6 6,768
0.4 784
7.2 13,536
0.6 1,176
10.8 20,304
0.8 1,568
14.4 27,072
0.3 588
1.2 2,256
Pollutant
°3
NO2
°3
N02
°3
NO2
°3
NO2
°3
NO2
°3
NO2
03
N02
°3
NO2
03
NO2
03
NO2
°3
NO2
°3
NO2
°3
NO2
03
N02
Exposure Duration
7 h/day, 5 days/week
for 12 weeks
Continuous 1-2 mo
24 h/day for 3 days
12 h/day for 3 days
8 h/day for 3 days
6 h/day for 3 days
6 h/day for 3 days
24 h/day for 3 days
12 h/day for 3 days
8 h/day for 3 days
6 h/day for 3 days
Continuous for 3 days
Species, Sex
(Strain)
c
Age
Mouse, M
(C57B1/6J)
5 weeks
Rat, M
(Wistar)
22 weeks old
Rat, M
(S-D)
10-12 weeks
old
Rat, M
(S-D)
10-12 weeks
old
Rat, M
(S-D)
250-275 g
Rat, M
(S-D)
3 mo old
Endpoints
Colonization of lung by
melanoma cells (injected
after pollutant exposure)
Pulmonary xenobiotic
metabolism, lung protein
(homogenate)
Protein (lavage), lavaged
cells (epithelial PMN)
Protein (lavage), lavaged cell
counts (epithelial, PMN,
AM), DNA content of cell
pellet
Airway labeling index
Lung enzymes (G6PD,
6PGD, ICD, GSHPx, OR,
DR, GOT, NADPH-CR)
Time of
Enpoint Response to
Measurement Mixture
Inject Increase in number of
melanoma cells colonies/lung (compared
0 PE and to air control) .
sacrifice
3 weeks PE
0 PE No effect on protein
content, increase in
selected enzymes.
0 PE Increased protein and
cells.
0 PE Increased protein and
cells, depending on
concentration.
4 days PE Increased index in
peripheral airways
(terminal bronchioles
opening into alveolar
ducts) and large airways
at 3 highest doses,
increased alveolar index
at 0.8 + 14.4 ppm only.
0 PE Increased activity.
Interaction
Not specified: no
change with 03, but
previous study showed
effect with NO2.
Suggested antagonism
for xenobiotic enzymes:
03 induced increase in
selected enzymes is
lowered by addition of
N02.
Synergism: protein at
0.6 and 0.8 ppm 03
mixtures, lavaged cells
at 0.4-0.8 ppm 03;
others additive.
Synergism: cell counts
at S:0.4 ppm 03
mixture, protein
additive.
Synergism for
peripheral airways at
highest dose and large
airways at three highest
doses only.
Synergism: 6PGD,
ICD, OR, SOD;
additive: GP, DR;
others: effect same as
03 only.
Reference
Richters (1988)
Takahashi and Miura
(1989)
Gelzleichter et al.
(1992a)
Gelzleichter et al.
(1992b)
Rajini et al. (1993)
Lee et al. (1990)
-------
a ~z c
00
Z o "3
< ™* H
-Isl
o «- s
CO O
C) en
II!
i Hi
#11-8
* |f.?
S d "
8f|
H <~ h
s
p
December 1993
6-168 DRAFT-DO NOT QUOTE OR CITE
-------
TABLE 6-25 (cont'd). TOXICOLOGICAL INTERACTIONS OF OZONE AND NITROGEN DIOXIDE3
«
5T
$
vS
\o
u>
o\
I—*
s
d
§*
M
1
o
g
2
M
o
a
2
3
o
»
n
g
Concentration
ppm
0.4
0.4
0.45
4.8
0.6
2.5
0.35
0.6
0.6
2.5
0.8
4.0
3
fig/m
784
752
882
7,520
1,176
4,700
686
1,128
1,176
4,700
1,568
7,520
Pollutant
°3
NO2
03
NO2
°3
N02
°3
NO2
03
N02
°3
NO2
Species, Sex
(Strain)
Exposure Duration Agec
Continuous for 2 weeks Rat, M
(Wistar)
10 weeks old;
Guinea pig, M
(Hartley)
10 weeks old
8 h/day for 7 days Mouse, M
(Swiss
Webster)
2 mo old
4 h (rest) Rat, M
(S-D)
3 h (exercise) 7 weeks old
2 h Rat, M
(rest and exercise) (S-D)
47-52 days old
Continuous for 3-56 days Mouse, M
(BALB/c)
Endpoints
Lipid peroxides; lung
antioxidants, and antioxidant
enzymes
Lung protein, DNA,
sulfhydryl and nonsulfhydryl
content; GR, GST, G6PD,
6PGD, ICD activities
Parenchymal histopathology
Epithelial permeability
(tracheal, bronchoalveolar)
(measured 1 and 24 h PE)
Antibody response to T-cell
dependent and independent
8-10 weeks old antigens in spleen
0.8
1,568
14.4 27,072
03
NO2
6 h/day for 3 days Rat, M
(concurrent) or sequential (S-D)
O3 pre NO2; NO2 pre 10-12 weeks
O3; 6 h each old
Protein (lavage); lavaged cell
counts (epithelial, PMN,
AM), DNA content of cell
pellet
Time of
Endpoint Response to
Measurement Mixture
0 PE Increased peroxides in
guinea pig but not rat;
increased antioxidants in
rat but not guinea pig;
enzymes increased or
decreased in guinea pigs,
increased to no change in
rat.
0 PE No change in protein,
DNA; increase in
activity of ICD, G6PD,
6PGD.
2 days PE Increased focal lesions.
0, 1, 2 days Rest: increased
PE bronchoalveolar
permeability at 1 and
24 h PE; exercise:
increased
bronchoalveolar
permeability at 1 and
24 h PE (effects greater
than with O$ alone, no
effect of NC>2).
0 PE Inconsistent pattern of
increases and decreases
of lung weight, thymus
weight, or plaque
formation.
0 PE Increased protein for
concurrent or sequential,
increase in cell counts
for concurrent.
Interaction
Synergism for some
endpoints, additive to
no interaction for
others; species
dependent.
Synergism: enzyme
activity.
Synergism: ascribed to
production of HNO3 in
exposure atmosphere.
Enhanced magnitude
and duration of
response (suggested
potentiation).
Most responses similar
to O3 only; mixture
affected some time
points not affected by
O3 alone: implied
nonadditive interaction,
but specifics not
determinable.
Reference
Ichinose and Sagai
(1989)
Mustafa et al. (1985)
Mautz et al. (1988)
Bhalla et al. (1987)
Fujimaki (1989)
Synergism: protein and Oelzleichter et al.
cell counts for
concurrent, protein
additive or antagonistic
for sequential.
(1992b)
-------
TABLE 6-25 (cont'd). TOXICOLOGICAL INTERACTIONS OF OZONE AND NITROGEN DIOXIDE*
Concentration
ppm pg'm
0.8 1.568
14.4 27,072
Species, Sex Time of
(Strain) Endpoint Response to
Pollutant
°3
NO2
Exposure Duration Agec Endpoints Measurement Mixture Interaction
6 h/day for 45-79 days Rat, M Various biochemic and 0 PE
(S-D) histological endpoints
10/12 weeks
old
Increased lung DMA, Suggested synergism
protein, collagen, elastin; for hydroxyproline and
some deaths with hydroxypyridinium.
mixture only at
£ 55 days; decreased
hydroxypyridinium .
Reference
Last et al. (1993)
See glossary of terms and symbols for abbreviations and acronyms.
Grouped by pollutant mixture.
cAge or body weight at start of exposure.
-------
TABLE 6-26. TOXICOLOGICAL INTERACTIONS TO BINARY MIXTURES OF OZONE
WITH ACIDS AND OTHER POLLUTANTS3
VO
OJ
Concentration
3
ppm ng/m Polluter
0.1 196 03
125 H2S04 (0.3
Species, Sex
(Strain) Time of Endpoint Response to
it Exposure Duration Age° Endpoints Measurment Mixture
2 h/day, 5 days/week Rabbit, M Tracheobronchial Mucociliary Normal to accelerated
fan) for up to 1 year (NZW) mucociliary transport, transport during clearance, increase in
Nose-only 4.5 mo old bronchial tree epithelial exposure; secretory cell numbers
secretory cell numbers secretory cells at early time points
3 days after 4, 8, (4-mo exposure).
and 12 mo of
exposure
0.1 196 03 3h Rabbit, M Lavage cell counts; lavage 0 PE
0.3 588 Oj Nose-only (NZW) LDH, PGEj, PGF2«; AM
0.6 1,176 03 4.5 mo old phagocytosis; superoxide
50 H2SO4 (0.3 ion) production; TNF activity
75 H2SO4 (0.3 ion)
125 H2S04 (0.3 tan)
O\
i— »
O
8
§
0.12- 235- Oj
0.64 1,254
40- H2SO4
1,000
0.12- 235- 03
0.64 1,254
40- H2SO4
1,000
0.12- 235- 03
0.64 1,254
40- H2S04
1,000
6 h-7 days Rat, M Lavageable protein 0 PE
(23 .5 h/day) (S-D)
250-300 g
23 .5 h/day for Rat, M Lung tissue protein 0 PE
5-9 days (S-D)
250-300 g
23 .5 h/day for 7 days Rat, M Rate of collagen synthesis 0 PE
(S-D)
250-300 g
Interaction Reference
Clearance: no interaction; Schlesinger et al.
secretory cell numbers: (1992a)
synergism at 4 mo,
antagonism at 8 and 12 mo.
No effects on lavage cell Antagonism: phagocytosis, Schlesinger et al.
counts or LDH, PGE2, at all combinations; (1992b)
PGF2a; or increase or antagonism: superoxide at
decrease in TNF and 0.1, 0.3 pom 03 and 75,
phagocytosis depending 125 jug/m H2SO4;
on exposure synergism: TNF at
concentration; no 125 figlm H2SO4 and 0.3,
change in superoxide. 0.6 ppm 03.
Increase (compared to
air control) .
Increase (compared to
air control).
Increase (compared to
air control).
Synergism at > 100 fig/m Warren and Last
H2SO4 and 0.2 ppm O3 for (1987)
3 days.
Synergism at 1,000 fig/m Warren and Last
H2SO4 and Q.64 ppm 03, (1987)
S:100ftg/m H2SO4
and 0.20 ppm Oj.
Synergism at a 200 ftg/m Warren and Last
H2S04 and 0.64 ppm Oj, (1987)
&500 fig/m H2S04
and 0.2 ppm 03 (with
o
d 0.15
g
294 03
300 H2SO4
(0.09 urn)
1 h to H2SO4, 2 h
rest, then 1 h
to 03
Head-only (acid)
whole-body (03)
Guinea pig, M Pulmonary function 0 PE
(Hartley)
260-325 g
H2SO,4).
Acid-induced decrease None: 03 did not alter
in DI*£Q not affected acid effect.
by03.
Chen et al. (1991)
n
-------
TABLE 6-26 (cont'd). TOXICOLOGICAL INTERACTIONS TO BINARY MIXTURES OF OZONE
WITH ACIDS AND OTHER POLLUTANTS8
•". . b
yf Concentration
1
o
1
g
a
ppm ng/m
0.15 294
84
0.15 294
24
0.2 392
1,000
0.2 392
1,000
0.64 1,254
1,000
0.64 1,254
1,000
0.64 1,254
1,000
0.8 1,568
1,200
0.2 392
5
0.2 392
5
0.2 392
5
Pollutant
Exposure Duration
03 1 h to H2SO4,
H2SO4 (layered 2 h rest, then 1 h
on ZnO) to Og
Head-only (acid)
whole-body (O3)
03
H2SO4 (layered
on ZnO)
H2S04
H2SO4
H2SO4
3«,
03
H2SO4
03
H2SO4
(0.63 fun)
93
03
(NH4)2SO4
H2SO4 3 h/day for
7 days, Oj on Day 9
Head-only (acid)
whole-body (03)
23.5 h/day for 7 days
15 or 30 days
23 .5 h/day for 7 days
23 .5 h/day for 7 days
23 .5 h/day for 7 days
O3 for 2 h, followed
by H2SO4 for 1 h
23. 5 h/day for 7 days
23 .5 h/day for 2 days
23.5 h/day for 3 days
Species, Sex
(Strain)
Agec
Guinea pig, M
(Hartley)
260-325 g
Guinea pig, M
(Hartley)
260-325 g
Rat,M
(S-D)
250-300 g
Rat, M
(S-D)
250-300 g
Rat, M
(S-D)
250-300 g
Rat, M
(S-D)
250-300 g
Rat, M
(S-D)
225-275 g
Guinea pig, F, M
(Hartley)
1.5-2 mo old
Rat, M
(S-D)
250-300 g
Rat, M
(S-D)
250-300 g
Rat, M
(S-D)
250-300 g
Endpoints
Pulmonary function
Pulmonary function
Rate of collagen synthesis
Lung protein content
Protein content (lavage)
Proximal acinar lesion
volume
Lung protein and free
proline content
Airway constriction
(measured by trapped gas
volume)
Rate of collagen synthesis
Lavageable protein
Lavageable protein
Time of
Endpoint Response to
Measurement Mixture
0 PE Greater decrease in DLCQ,
VC after Oj; no change in
alveolar volume, TLC with
mixture.
0 PE Decrease in TLC, VC,
DLcQ enhanced by Oj.
0 PE Increase.
0 PE Increase only at 15 days.
0 PE Increase.
0 PE Increase.
0 PE Increase with H2SO4 and
03 only.
0 PE Increase compared to air.
0 PE Increase.
ft PE Increase.
0 PE Increase.
Interaction
Suggested synergism
(> additive) for
DLcQ, but not VC.
Suggested synergism
(> additive).
Possibly synergism:
effect different from
O3 alone.
Suggested synergism.
None: effect same as
03 alone.
None: effect same as
03 alone.
Synergism.
No interaction: effect
same as O3 alone,
H2SO4 had no effect.
Synergism: effect
greater than O3,
sulfate had no effect.
Synergism: effect
greater than O3,
sulfate had no effect.
No interaction: effect
same as O3 alone.
Reference
Chenetal. (1991)
Chen et al. (1991)
Warren et al. (1988)
Last (1991)
Warren et al. (1988)
Warren et al. (1988)
Last et al. (1986)
Silbaugh and Mauderly
(1986)
Warren et al. (1986)
Warren et al. (1986)
Warren et al. (1986)
-------
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December 1993
6-174 DRAFT-DO NOT QUOTE OR CITE
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1 those animals exposed to the mixture. Edema of pulmonary connective tissue was more
2 pronounced and alveolar Type 2 cells became swollen. Although this study was not
3 quantitative, qualitative observations led the authors to conclude that the lesions were not
4 due to O3 alone but that NO2 played some, albiet undefined, contributory role.
5 The effect of exposure duration on interaction was also noted in studies of Schlesinger
6 et al. (1990, 1991). Rabbits exposed to 0.3 ppm O3 + 3 ppm NO2 for 2 h showed
7 synergistic increases in certain BAL eicosanoids obtained immediately after exposure,
8 whereas animals exposed to the same mixture for 2 h/day for 14 days showed no interaction
9 for the same parameters.
10 A number of studies examined other biochemical responses to O3/NO2 mixtures (e.g.,
11 sulfhydryl metabolism and the activity of certain enzymes). Some of these discussed in the
12 previous O3 criteria document were found to involve synergism (e.g., Mustafa et al., 1984).
13 More recent studies of lung biochemistry also suggest that O3 and NO2 interact
14 synergistically. Ichinose and Sagai (1989) exposed rats and guinea pigs to 0.4 ppm
15 O3, 0,4 ppm NO2, or a mixture of the two pollutants continuously for 2 weeks. No change
16 in lung peroxide production was observed in rats, but the mixture synergistically increased
17 peroxide levels in guinea pigs. The guinea pig showed no change in lung antioxidant content
18 following any exposure, whereas the mixture synergistically increased antioxidant levels hi
19 rat lung. The conclusion of a significant interaction was based upon relative changes from
20 air controls following exposure to the mixture compared to changes following exposure to
21 each pollutant alone using the t-test, with synergism defined as a change greater than the sum
22 of the responses to individual pollutants; no specific test for interaction was performed.
23 Ichinose and Sagai (1989) also noted that levels of antioxidant enzymes in rat or guinea
24 pig lungs were variously affected by exposure to the above mixture. For example, GST was
25 decreased in both species exposed only to O3, but the mixture produced a reduction of this
26 enzyme in guinea pigs and no change in rats. Thus, the occurrence of interaction was
27 dependent on endpoint as well as species. This latter finding likely reflected interspecies
28 differences in biochemical defenses against oxidant pollutants, given the results of a study by
29 Sagai et al. (1987) with four animal species. This study suggested that observed species
30 differences in lipid peroxide formation following exposure were related to the relative content
December 1993 6-175 DRAFT-DO NOT QUOTE OR CITE
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1 of antioxidants and the specific composition of phospholipids and their fatty acids. The
2 guinea pig was the most sensitive animal, and the hamster was the most resistant.
3 The effects of exposures to O3/NO2 mixtures on lung lipid peroxides and antioxidant
4 activity have been examined in a number of other studies (Ichinose et al., 1988; Lee et al.,
5 1990; Sagai and Ichinose, 1991) and the results generally confirm those noted above, namely
6 that such mixtures tend to produce synergistic interaction. However, there is also some
7 evidence for antagonism. Takahashi and Miura (1989) examined effects on the pulmonary
8 xenobiotic system of rats exposed 1 or 2 mo to a mixture of 0.2 ppm O3 + 4 ppm NO2, as
9 well as to each pollutant alone. Ozone induced an increase in lung cytochrome P-450
10 content, but the activity of these enzymes was reduced by the addition of NO2 to the
11 exposure atmosphere; that is, the mixture resulted in levels intermediate between those found
12 with O3 or NO2 when given alone. However, the reduction in enzyme activity induced by
13 NO2 was restricted to those systems that had been increased by exposure to O3 alone. The
14 authors suggested that antagonism was due to the production of undefined secondary reaction
15 products in the exposure atmosphere. A similar explanation was proposed to explain
16 observed synergism of lung antioxidant activity in another study (Lee et al., 1990). Thus,
17 the response to any secondary product likely depends upon the endpoint examined, assuming
18 that the same reaction products were formed in these two studies.
19 The role of exposure parameters in producing an interaction between simultaneously
20 inhaled O3 and NO2 was examined by Gelzleichter et al. (1992a). Rats were exposed to
21 various combinations of O3 and NO2 for various durations (6, 8, 12, and 24 h), such that the
22 C x T products were identical for each of four exposure sets. As indicated in Table 6-24,
23 as the exposure duration increased, the exposure concentration of each component of the
24 mixture decreased. Lavaged protein levels and recovered cells were the endpoints. For each
25 exposure combination, the additive response was predicted from results of exposure to each
26 pollutant alone, and then synergism was indicated when there was deviation from additivity.
27 Responses to exposure to either O3 or NO2 alone for 6, 8, or 12 h showed that the product
28 of C x T was a constant for the observed biological effects. However, less severe changes
29 occurred when delivery was at the lowest dose rate (i.e., when the lowest concentration of
30 each pollutant was delivered over the 24-h exposure duration). Exposure at higher dose rates
31 (i.e., 6 to 12 h) increased the magnitude of the response. Thus, the degree of response to
December 1993 6-176 DRAFT-DO NOT QUOTE OR CITE
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1 each pollutant alone was not a constant function of C x T throughout the entire range of
2 dose rates, but was concentration driven, and was not identical at the highest and lowest
3 rates. Responses following exposure to the mixture did not follow C x T even over the
4 range of dose rates in which it was constant following exposure to the pollutants individually.
5 Thus, interaction, in this case synergism, appeared to be concentration dependent, in that the
6 response was disproportionately greater at the higher concentrations (higher dose rates) of the
7 constituent pollutants in the mixture. The response following exposure to the mixtures
8 appeared to be a function of peak concentration, rather than of cumulative dose. More
9 recently, Rajini et al. (1993) noted that analysis of all kinetics following similar exposure to
10 mixtures also did not follow a C x T relationship.
11 All of the studies described above involved simultaneous exposure to O3 and NO2.
12 However, ambient exposure to these pollutants has temporal patterns, and exposure to one
13 agent may then alter the response to another subsequently inhaled agent. The realism of
14 these studies is somewhat dependent upon their relationship to actual temporal patterns of
15 pollutants in ambient air (i.e., whether one material is the precursor of the other, as is the
16 case for O3 and NO^. As described in the previous O3 criteria document (U.S.
17 Environmental Protection Agency, 1986), Fukase et al. (1978) exposed mice for 7 days to
18 3 to 15 ppm NO2 for 3 h/day, followed by 1 ppm O3 for 3 h/day, and noted an additive
19 effect on the level of lung GSH.
20 Yokoyama et al. (1980) exposed rats to 5 ppm NGj or 1 ppm O3 for 3 h/day, or to
21 NO2 for 3 h followed by O3 for 3 h/day, for various total durations up to 30 days, and
22 assessed lung mechanics in postmortem lungs, lung histology, and enzyme activity in
23 subcellular fractions of lung tissue. The activity of phospholipase A2 in the mitochondria!
24 fraction was increased in those animals exposed to O3 only or to O3 after NO2, and the
25 response in the latter was significantly greater than that in the former. A decrease in activity
26 of lysolecithin acyltransferase in the supernatent fraction was found only in those animals
27 exposed to both NO2 and O3. Pulmonary mechanics showed a change in pulmonary
28 resistance (as a function of elastic recoil pressure) in the O3-only and NO2/O3-exposed
29 animals. Histologically, the lungs of the animals exposed to both NC^ and O3 appeared
30 similar to those exposed to O3 alone. However, a slight degree of epithelial necrosis in
31 medium bronchi not found with either NO2 or O3 alone was seen in the animals exposed to
December 1993 6-177 DRAFT-DO NOT QUOTE OR CITE
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1 both pollutants. In addition, damage at the bronchoalveolar junction appeared to be
2 somewhat more marked in animals exposed to both gases than in those exposed to O3 alone.
3 This study suggested that sequential exposures produced responses that were, in most cases,
4 not greatly different from those due to O3 alone.
5 Aside from sequential exposures, simulation of ambient exposure scenarios involving
6 NO2 and O3 has been performed by examining the effects of a continuous baseline exposure
7 to one concentration of both pollutants, with superimposed short-term peaks to a higher level
8 of one or both. The endpoint generally examined in this regard has been bacterial resistance.
9 Studies reported in the previous criteria document (e.g., Ehrlich et al., 1979; Ehrlich, 1983)
10 in which mice were exposed to O3 under various scenarios of baseline concentrations of NO2
11 upon which were superimposed daily "peak" exposures to NO2, or a combination of NO2
12 and O3, suggested that exposure with peaks can enhance response to pollutant mixtures, and
13 that the sequence of peak exposures was important in producing reduced resistance to
14 infection that was different from that due to exposure to the baseline concentration only.
15 As a comparison, toxicologic interactions for infectivity involving simultaneous
16 exposure to NO2 and O3 discussed in the previous O3 criteria document were found generally
17 to be additive following acute exposures, with each pollutant contributing to the observed
18 response when its concentration reached the threshold at which it would have affected
19 bacterial resistance when administered alone (Goldstein et al., 1974). If the exposure level
20 of either NO2 or O3 was below this threshold, then the response was due solely to the
21 constituent inhaled at the more toxic concentration (Ehrlich et al., 1977).
22 More recently, Graham et al. (1987) examined resistance to respiratory infection
23 (as measured by bacterial-induced mortality) in mice continuously exposed (for 15 days,
24 24 h/day) to baseline levels of an NO2/O3 mixture with two daily, 1-h peaks of the mixture
25 at very high, high, intermediate, and low exposure concentrations (see Table 6-25 for
26 concentrations). Animals were also exposed to the same baseline levels of either NO2 or
27 O3 onto which were superimposed two daily, 1-h peaks of the same single gas in
28 concentrations as above. At the low concentration, only NC^ increased mortality. At the
29 intermediate exposure level, the mixture was synergistic, whereas NO2 alone increased
30 mortality and O3 had no effect. At the high exposure level, the combined exposure was
31 again synergistic, even though exposure to each gas separately also increased mortality.
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1 A similar effect was seen at the very high level, although the combined exposure just missed
2 statistical significance for synergism. These results are consistent with those of the earlier
3 studies reported in the previous O3 criteria document and support the conclusion that
4 response depends upon the specific exposure pattern. The results of Graham et al. (1987)
5 are also consistent with those from the earlier studies with simultaneous exposures, in that
6 the mixture did not produce any interaction when exposure to either gas alone was
7 ineffective.
8 The relationship between exposure and response is very complex, and seems to depend
9 upon exposure duration, the ratio of O3 and NO2 concentrations, and other factors that may
10 include the production of secondary reaction products within the exposure atmosphere. This
11 complexity was highlighted by the study of Gelzleichter et al. (1992b), who examined effects
12 of combined or sequential exposures of rats to mixtures of Qj and NO2 at various
13 concentrations ranging from 0.2 to 0.8 ppm O3 and 3.6 to 14.4 ppm NO2. Sequential
14 exposures consisted of 6 h of O3 at night, followed by 6 h of NO2 during the day, or vice
15 versa; concurrent exposures were for 6 h/day for 3 days. Various endpoints were examined,
16 and it was noted that sequential and concurrent exposures did not result in the same response.
17 Thus, lavage protein levels were increased additively with sequential exposure (in any
18 pollutant order), but were found to be greater than additive with concurrent exposure.
19 An increase in the number of lavaged epithelial cells was additive for the 03 night/NO2 day
20 sequence, antagonistic for the NO2 night/O3 day sequence, and additive for concurrent
21 exposure. An increase in the number of lavaged PMNs was additive for both sequential
22 conditions and was synergistic for concurrent exposure. It was concluded that production of
23 synergism depended upon the concentration of each pollutant within the mixture, and
24 additivity would result for any endpoint when the concentration of each component of the
25 mixture fell below a certain threshold level. However, these threshold concentrations were
26 endpoint specific, with some being more sensitive than others; it was speculated that the least
27 sensitive assays were based upon changes that were reversible, whereas the most sensitive
28 ones were irreversible. The authors also noted that the extent of chemical reaction within the
29 O3/NO2 mixture atmosphere was related to the extent of lexicological interaction, suggesting
30 that interaction was due to production of some secondary reaction product, which in this case
31 was suggested to be nitrogen pentoxide. This particular chemical had also been suggested in
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1 earlier studies to be responsible for interactions following exposure to NO2/O3 atmospheres
2 (e.g., Diggle and Gage, 1955).
3 The concentrations at which synergism occurred in the study of Gelzleichter et al.
4 (1992b) discussed above (namely, ^0.4 ppm O3 and ^7.2 ppm NO2) were higher than
5 those that are generally found in ambient air. However, the threshold concentration for
6 interaction was dependent upon exposure dose rate, with higher rates leading to lower
7 threshold concentrations for synergism. This suggests that a threshold may occur at ambient
8 concentrations with longer term exposures.
9 Although interaction is clearly modulated by environmental exposure factors, such as
10 concentration, duration of exposure, or specific exposure regime, host factors may also play
11 a role. Mautz et al. (1988) examined the effect of exercise on rats exposed to mixtures of
12 O3 and NO2. Exercise modified the toxic interactions of combined pollutants, resulting in
13 synergistic interaction occurring at lower exposure concentrations of the constituent pollutants
14 than with exposure at rest. Thus, a similar magnitude of response, namely an increase in the
15 extent of focal lesions in lung parenchyma, was noted 2 days following a 4-h exposure to
16 0.6 ppm O3 + 2.5 ppm NO2 at rest, or with a shorter (3-h) exposure to lower
17 concentrations, 0.35 ppm O3 + 0.6 ppm NO2, with exercise. In both cases, the response
18 was different from that due to either pollutant given alone. Furthermore, a greater response
19 was noted with a 3-h exposure to 0.6 ppm O3 + 2.5 ppm NO2 with exercise than to the
20 same mixture for the same exposure duration at rest. Thus, exercise also increased the
21 response at similar concentrations compared to rest. The effect of exercise was ascribed to
22 an increase in delivered dose or dose rate, due to increased VE. The ability of exercise to
23 enhance response to a pollutant mixture was also noted by Bhalla et al. (1987).
24 The study of Mautz et al. (1988) above also provided further evidence suggesting that
25 chemical reactions within the exposure atmosphere may play some role in toxicologic
26 interaction. In this case, nitric acid (HNO3) vapor was noted at concentrations ranging from
27 0.02 to 0.73 ppm, depending upon the concentrations of the primary constituents.
28 As discussed below, acids have been found to interact with O3. This study also found
29 interaction to occur at a concentration of one of the components, namely NO2, that had no
30 effect when administered alone. Although this appears to contrast with the conclusions of
31 Graham et al. (1987) above, the endpoints in these two studies were quite different.
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1 Another aspect of pollutant interaction involves the ability of O3/NO2 mixtures to affect
2 the course of other lung lesions (e.g., malignant tumor colonization). Richters (1988)
3 exposed mice to a mixture of 0.15 ppm O3 + 0.35 ppm NO2 for 7 h/day, 5 days/week for
4 12 weeks, following which they were injected (iv) with viable melanoma cells. The mice
5 were sacrificed 3 weeks later and the lungs were examined for melanoma colonies. Although
6 exposure to O3 alone produced no change in the percentage of animals with colonies or in
7 the average number of colonies per lung (compared to air control), exposure to the mixture
8 produced an increase in the latter, suggesting to the authors that it faciliated cancer cell
9 colonization. However, the exact role played by O3 in the mixture is not clear because a
10 previous study had indicated that NO2 alone facilitates blood-borne cancer cell spread to the
11 lungs (Richters and Kuraitis, 1981). Furthermore, the experimental model used is not
12 generally accepted as representing metastatic mechanisms.
13 Ichinose and Sagai (1992) also examined the ability of an O3/NO2 mixture to promote
14 primary lung tumor development. Rats were injected (intraperitoneally) with
15 AMns(2-hydroxypropyl) nitrosamine (BHPN) and then were exposed for 13 mo to a mixture
16 of 0.05 ppm O3 + 0.4 ppm NO2, O3 alone, or to clean air (chamber control). Although the
17 NO2 exposure was continuous, the O3 exposure was intermittent, with the concentration
18 altered between 0 and 0.1 ppm following a sine curve from 9 to 19 h of each day (resulting
19 in a daily mean concentration of 0.05 ppm). One other group of rats served as a room
20 control, maintained in a clean room for 24 mo following injection of BHPN. After an 11-mo
21 recovery period, all animals were autopsied. Compared to clean air-exposed animals, lung
22 tumor incidence was increased in mice exposed to the mixture; O3 alone did not increase the
23 tumor incidence. However, the authors noted that tumor incidence in the room control group
24 was not different from that in the group exposed to the mixture, and suggested that the clean
25 air (chamber control) group should be used as a control in interpreting the data from the
26 pollutant-exposed animals. The enhanced incidence in mixture-exposed animals was
27 suggested to be due to synergistic increases in lipid peroxidation, which was noted in other
28 studies (see Table 6-25). A complication in interpreting this study is that a previous study
29 (Sagai and Ichinose, 1991) had suggested tumor development in animals exposed to an
30 O3 and NO2 mixture without BHPN, although this latter study involved a longer exposure
31 duration and somewhat higher pollutant concentrations.
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1 6.4.2.2 Acidic Compounds as Copollutants
2 Binary mixtures containing acids comprise another type of commonly examined
3 exposure atmosphere. Most of these involved acidic sulfate aerosols as the copollutant.
3
4 Peak (1-h) ambient levels of sulfuric acid (H2SO4) are estimated at 75 /tg/m , with longer
5 (12-h) averages about one-third of this (Spengler et al., 1989).
6 Earlier studies that employed simultaneous single, repeated, or continuous exposures of
7 various animal species to mixtures of acid sulfates and O3 found responses for several
8 endpoints, including tracheobronchial mucociliary clearance, alveolar clearance, pulmonary
9 mechanics, and lung morphology, to be due solely to the O3 (U.S. Environmental Protection
10 Agency, 1986; Cavender et al., 1977; Moore and Schwartz, 1981; Phalen et al., 1980; Juhos
11 et al., 1978). However, synergism was noted for bacterial infectivity in mice (Grose et al.,
12 1982), for response to antigen in mice (Osebold et al., 1980), and for effects on lung protein
13 content and the rate of collagen synthesis in rats (Last et al., 1983, 1984a; Last and Cross,
14 1978). More recent studies performed since publication of the previous O3 criteria document
15 support the earlier finding of synergism between O3 and acid sulfates on lung biochemistry,
16 while providing possible explanations for underlying mechanisms.
17 Last et al. (1986) exposed rats for 7 days to O3 alone (at 0.96 ppm) and to mixtures of
18 O3 with one of three aerosols, namely sodium chloride, sodium sulfate, or ammonium sulfate
19 [(NH4)2SO4] (all at 5 mg/m3); only the (NH4)2SO4 was acidic. Lung protein content,
20 proline content, collagen synthesis rate, fibroblast numbers hi parechymal lesions, and the
21 volume of these lesions were examined following exposure. Mixtures of O3 with sodium
22 chloride or sodium sulfate produced changes that did not differ from those found with
23 O3 alone. On the other hand, mixtures of (NIL^SC^ with O3 resulted in increases in all of
24 the measured parameters, and the increases were greater in magnitude than those due to
25 O3 alone; synergism was concluded. These results suggested that acidity was necessary for
26 synergism of the aerosols with O3. This was further supported by demonstrating that
27 significant interaction of O3 with H2SO4, which is much more acidic than (NH4)2SO4,
28 occurred at lower concentrations than was noted for mixtures of O3 and (NH4)2SO4 (Warren
29 and Last, 1987); interaction was suggested with H2SO4 concentrations as low as 40 jig/m
30 (with 0.2 ppm O3) for some lung biochemical endpoints. The studies above did not use any
31 specific statistical test for interaction, and conclusions of interaction were based upon
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1 findings of significant differences from responses following exposure to 63 alone and the
2 absence of detectable responses following exposure to H2SO4 aerosols at the same and higher
3 concentrations.
4 Warren et al. (1986) found synergistic interaction with the above endpoints following
^
5 7 days of exposure to 0.2 ppm O3 plus 5 mg/m (NH4)2SO4. However, exposure for only
6 3 days produced responses that were not different from those noted with O3 alone. This
7 seems to indicate that the duration of exposure is a factor affecting the occurrence of any
8 interaction. However, exposure duration may also affect the type of interaction. In a study
9 by Schlesinger et al. (1992a) in which rabbits were exposed to a mixture of 0.1 ppm O3 plus
q
10 125 /tg/m H2SO4 for 2 h/day, 5 days/week, a synergistic increase in bronchial epithelial
11 secretory cell number was noted after 4 mo of exposure, whereas antagonism was noted
12 following 8 mo of continued exposure.
13 The mechanism underlying interaction between acid sulfates and O3 is not known. Last
14 et al. (1986) noted that similar sites of deposition for O3 and acid aerosols favored
15 synergism. A synergistic response of biochemical indices in rat lung with exposure to
16 1,000 /ig/m3 H2SO4 with 0.6 ppm O3 was found when the acid droplet diameter was
17 0.5 /*m, whereas no increase compared to the O3-only response was noted when the droplet
18 diameter was 0.02 /*m. Apparently, the larger particles that deposited to a greater extent
19 within the bronchioalveolar junction, the major target site for O3, were most interactive.
20 Observed synergism between O3 and acid sulfates in rats was also suggested to be due
21 to a shift in the local microenvironmental pH of the lung following deposition of acid,
22 enhancing effects of O3 by producing a change in the reactivity or residence time of
23 reactants, such as free radicals, involved in O3-induced tissue injury (Last et al., 1984a).
24 If this was the only explanation, then the effects of O^ should be consistently enhanced by
25 the presence of acid in an exposure atmosphere. However, in the study of Schlesinger et al.
26 (1992b), in which rabbits were exposed for 3 h to combinations of O3 at 0.1, 0.3, and
27 0.6 ppm with H2SO4 (0.3 /*m) at 50, 75, and 125 jDtg/m3, antagonism was noted when
28 evaluating stimulated production of superoxide anion by AMs harvested by lavage
29 immediately after exposure to 0.1 or 0.3 ppm O3 in combination with 75 or 125 jttg/m3
30 H2SO4, and also for AM phagocytic activity at all of the O3/acid combinations. Mixtures of
31 O3 (0.6 ppm) and another acid, namely HNO3 vapor (1,000 /ig/m3), (Nadziejko et al., 1992)
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1 also produced antagonism for certain aspects of the function of AMs harvested from acutely
2 exposed rats. Although the deposition sites of both acid and O3 should be comparable in
3 these two studies, perhaps the particular cellular endpoints examined are subject to this type
4 of interaction.
5 Last (1989) observed an apparent all-or-none response in rats exposed to the acid
6 sulfate/O3 mixtures. That is, there was no concentration-response relationship between the
7 concentration of acid in the mixture and the extent of change in various endpoints, compared
8 to effects observed with O3 alone. In the study of Schlesinger et al. (1992b), a similar
9 phenomenon was noted, but in this case, the concentration of O3 in the mixture did not
10 always influence the response compared to that seen with acid alone. Thus, exposure-
11 concentration-response relationships noted with individual pollutants may not necessarily
12 reflect responses following exposure to their mixtures. This is consistent with the results of
13 Gelzleichter et al. (1992a) for mixtures of O3 and NO2.
14 The above studies involved simultaneous exposures to O3 and acidic pollutants, but
15 some studies involving sequential exposures to O3 and acid sulfate aerosols were described in
16 the previous O3 criteria document. For example, Gardner et al. (1977) found an additive
17 increase in infectivity when mice were exposed to 0.1 ppm O3 for 3 h prior to a
18 2-h exposure to 900 jwg/m3 H2SO4, whereas no difference from air control was noted when
19 the acid was administered prior to O3. Grose et al. (1980) noted a reduction in ciliary
20 activity in isolated trachea! sections obtained from hamsters exposed to 0.1 ppm O3 for
21 3 h followed by exposure to 1,090 jwg/m3 H2SO4 for 2 h that was less in magnitude than that
22 found with exposure to acid alone (O3 alone had no effect).
23 More recently, Silbaugh and Mauderly (1986) examined the ability of O3 to increase
24 susceptibility to a subsequent exposure to H2SO4 in terms of producing airway constriction.
25 Guinea pigs were exposed to 0.8 ppm O3 for 2 h followed by H2SO4 (12 mg/m for 1 h).
26 An increased volume of trapped gas in the lungs (the metric of constriction) was seen with
27 both O3 alone and with the mixture, but the response to the latter did not differ from that due
28 to the former, and acid alone had no effect. Thus, preexposure to O3 in this case did not
29 affect response to a subsequent exposure to acid.
30 Chen et al. (1991) examined the reverse exposure scenario, namely whether exposure to
31 H2SO4 affected subsequent response to O3. Guinea pigs were exposed to H2SO4 or H2SO4
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1 coated on ultrafine zinc oxide particles. A 1-h exposure to 0. 15 ppm O3 following a
2 1-h exposure to acid (300 /*g/m , 0.09 fim) did not alter the response seen with acid alone,
3 namely a decline in DLCO- However, when single (1-h) or multiple (3-h/day, 7-day)
4 exposures to acid-coated zinc oxide (24 or 84 jug/m equivalent H2SO4) were followed by a
5 1-h exposure to 0.15 ppm O3, the effect on DLco appeared to be greater than additive,
6 although no specific statistical test for interaction was performed. This study suggested that
7 prior exposure to acid increased the susceptibility of the guinea pig to subsequent exposure to
8 O3, but it also showed that the manner in which the acid was delivered affected whether or
9 not any interaction occurred. It is likely that the number concentration of particles was
10 greater in the zinc oxide-H2SO4 aerosol than in the H2SO4 aerosol, and the interaction may
1 1 reflect this greater particle number.
12
13 6.4.2.3 Other Copollutants
14 Although the bulk of the database for binary mixtures of O3 involves NO2 or acids, a
15 few studies examined responses to combinations of O^ with other pollutants.
16 Reuzel et al. (1990) exposed rats to mixtures of O3 (0.2, 0.4, 0.6 ppm) +
17 formaldehyde (HCHO) (0.3 to 3.0 ppm). Although exposure to the mixtures did not alter the
18 nature or extent of histological lesions, namely cilia loss and epithelial hyperplasia, compared
19 to exposure to each pollutant alone, a site-specific synergistic increase in turnover of nasal
20 epithelial cells was found with all concentrations of HCHO together with 0.4 ppm Oj.
21 A lack of such response with 0.8 ppm O3 was ascribed to an O3 -induced alteration in
22 breathing pattern, which reduced the delivered dose. It was, however, noted that interaction
23 occured only when one constituent of the mixture was administered at cytotoxic
24 concentrations, an exposure scenario that rarely occurs in ambient air. In any case, the
25 authors concluded that because cell proliferation likely plays a role in carcinogenesis, and
26 that if mixtures potentiate cell proliferation, then exposure to pollutant mixtures may increase
27 cancer risk.
28 Mautz et al. (1988) exposed rats for 3 h, at both rest and exercise, to a mixture of
29 0.6 ppm O3 + 10 ppm HCHO. A synergistic increase in nasal epithelial cell turnover
30 followed exposure with exercise, whereas exposure at rest resulted in no difference from that
31 seen with HCHO alone. Likewise, exposure to the mixture with exercise resulted in an
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1 increase in the number of focal lesions in lung parenchyma compared to either O3 or HCHO
2 alone, but exposure at rest resulted in a lower incidence of lesions than seen with O3 alone.
3 This latter observation was ascribed to an effect of the HCHO upon breathing pattern,
4 producing a change in inhaled dose of O3 that did not occur with exercise.
5 Nishikawa et al. (1992) examined the effect of sequential exposure to cigarette smoke
6 and O3 in altering airway responsivity to inhaled brochoconstrictor challenge and trachea!
7 vascular permeability in guinea pigs. Animals were exposed to 1 ppm Oj for 0.5 h followed
8 by 5 puffs of cigarette smoke, or to 1 ppm O3 for 1.5 h followed by 10 puffs. Exposure to
9 O3 and five puffs increased responsivity and vascular permeability immediately after
10 exposure, whereas no effect on either endpoint was noted with either pollutant given alone.
11 Exposure to O3 and 10 puffs also increased responsivity and permeability, but to the same
12 extent as did the lower concentration mixture or exposure to O3 alone, whereas exposure to
13 10 puffs of smoke only increased responsivity. Thus, sequential exposure to O3 and cigarette
14 smoke enhanced the magnitude of response compared to either pollutant alone, but the
15 duration of response was not altered.
16 The potential role of O3 in enhancing fibrotic lung disease by interaction with silica was
17 examined by Shiotsuka et al. (1986), who exposed rats with developing silica-induced
18 fibrosis to O3 at 0.8 ppm for 6 h/day, 5 day/week for 37 exposure days. Silica had been
19 instilled (2, 12, or 50 mg) on Day 1 of the study and exposure to O3 began on Days 3 or
20 4 postinstillation. There was found to be no interaction between silica and O3 in
21 development of fibrosis, as assessed biochemically (lung content of hydroxyproline) or
22 histopathologically. Although an increase was found in the ratio of hydroxyproline to total
23 protein in the group exposed to the mixture and instilled with the highest amount of silica,
24 this was not considered by the authors to be biologically significant.
25
26 6.4.3 Complex (Multicomponent) Mixtures Containing Ozone
27 Ambient pollution in most areas is a complex mix of more than two chemicals, and a
28 number of studies have examined the effects of exposure to multicomponent atmospheres
29 containing O3. Some of these attempted to simulate photochemical reaction products
30 occurring under actual atmospheric conditions. However, the results of these studies are
31 often difficult to interpret due to chemical interactions between the components and the
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1 resultant production of variable amounts of numerous secondary reaction products, and a lack
2 of precise control over the ultimate composition of the exposure environment. In addition,
3 the role of O3 in the observed biological responses is often obscure.
4 One type of experimental multicomponent atmosphere that has been examined is
5 ultraviolet-irradiated and nonirradiated automobile exhaust mixtures. Irradiation leads to the
6 formation of photochemical reaction products that are biologically more active than those in
7 nonirradiated mixtures. Such mixtures are characterized by total oxidant concentrations
8 (expressed as O3) in the range of 0.2 to 1.0 ppm. Although the effects described following
9 exposure were not necessarily uniquely characteristic of O3 and although O3 could have been
10 responsible for some or even most of them, the biological effects have been difficult to
11 associate with any one particular component in most cases. Effects of exhaust mixtures on
12 different species have been discussed in the previous O3 criteria document (U.S.
13 Environmental Protection Agency, 1986). Pulmonary function changes were demonstrated in
14 guinea pigs after short-term exposures to irradiated exhaust and in dogs after long-term
15 exposure to both irradiated and nonirradiated exhaust mixtures.
16 Additional studies of complex mixtures have been performed since publication of the
17 previous O3 criteria document. Kleinman et al. (1985) exposed rats (Sprague-Dawley, male,
18 7 weeks, nose-only) for 4 h to atmospheres, designed to represent photochemical pollution,
19 consisting of 0.6 ppm (1,180 ng/m3) O3 + 2.5 ppm (4,700 jttg/m3) NO2 + 5 ppm
20 (13,100 /ig/m3) sulfur dioxide (SO2) + particles. The paniculate phase consisted of
21 1,000 /tg/m3 of either H2SO4 or (NH4)2SO4, laced with iron sulfate [Fe^SO^] and
22 manganese sulfate. The metallic salts act as catalysts for the conversion of sulfur IV into
23 sulfur VI, and the incorporation of gases into the aerosol droplets. The respiratory region
24 was examined for morphological effects. A confounding factor in these studies was the
25 production of HNO3 vapor in atmospheres that contained O3 and NO2, a phenomenon
26 discussed previously, and nitrate in those that contained O3 and (NH4)2SO4, but not NO2.
27 Nevertheless, a significant enhancement of tissue damage was noted with exposure to
28 atmospheres containing H2SO4, or secondarily produced HNO3, compared to those
29 containing (NH4)2SO4, a less acidic compound. In addition, there was some suggestion that
30 the stronger acidic atmospheres resulted in a greater area of the parenchyma becoming
31 involved in lesions, which were characterized by a thickening of alveolar walls, cellular
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1 infiltration in the interstitium, and an increase in free cells within alveolar spaces.
2 An increased rate of nasal epithelial cell turnover was noted following exposure to
3 atmospheres containing paniculate acids compared with exposure to either O3 alone or to a
4 binary mixture of O3 + NO2. Furthermore, exercise seemed to potentiate the nasal and
5 parenchymal responses to the complex mixtures containing strong acids (Kleinman et al. ,
6 1989), a finding similar to that with binary mixtures of O3 and NO2, or O3 and HCHO,
7 previously discussed.
8 Bhalla et al. (1987) examined the effects of a seven-component atmosphere, similar to
9 that above, on epithelial permeability of rat lungs (Sprague-Dawley, male, 47 to 52 days
10 old). The animals were exposed for 2 h (chambers, relative humidity [RH] = 85%) to the
11 following: O3 (0.6 ppm) + NO2 (2.5 ppm) + SO2 (5 ppm) + ferric oxide
12 (Fe2O3) (241 /ig/m3) + (NH4)2SO4 (308 to 364 jig/m3) 4- Fe^SO^ (411 to 571 /*g/m3) +
13 MnSO4 (7 to 9 /ig/m3). The response to this mixture was compared to that following
14 exposure to O3 (0.6 ppm) + NO2 (2.5 ppm); O3 alone (0.6 or 0.8 ppm); or NO2 alone (6 or
15 12 ppm). As above, the complex mixture was found to result in production of HNC^, in this
16 case at measured concentrations of 1,179 to 2,558 /ig/m3 (0.46 to 1.02 ppm); the O3 + NO2
17 atmosphere also resulted in some HNO3 vapor formation. Epithelial permeability was found
18 to increase immediately following exposure to O3, O3 + NO2, or to the complex mixture.
19 Although the magnitude of this change was similar following exposure to O3 alone or in
20 combination with other pollutants, there was increased persistence of effect after exposure to
21 either the binary or complex mixture.
22 Prasad et al. (1988) used a similar multicomponent atmosphere and examined effects on
23 AM surface receptors. Rats (Sprague-Dawley, male, 200 g) were exposed for 4 h/day, for
24 7 or 21 days to a mixture of O3 (0.3 ppm) + NO2 (1.2 ppm) + SO2 (2.5 ppm) +
25 (NH4)2S04 (270 /xg/m3) + Fe^SO^ (220 ptg/m3) + MnSO4 (4 ^g/m3) +
26 Fe2O3 (150 jug/m3), or to O3 alone. Both the mixture and O3 alone resulted in a decrease in
27 Fc receptor activity beginning immediately after the last exposure. Exposure to the complex
28 atmosphere for 7 days resulted in a response similar to that seen with C^ alone, but
29 continued exposure to this mixture for up to 21 days resulted in an even greater reduction in
30 receptor function compared to O3 alone. However, as with most studies of complex
31 mixtures, although the response to the mixture was different from that found with O3, the
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1 role of other constituents was not clear. Phagocytic function of AMs was also examined
2 following exposure to the mixture, but there were no O^-only controls for comparison.
3 Mautz et al. (1985a) examined the effects of a complex mixture upon pulmonary
4 mechanics in exercising dogs. Exposures (nose-only) were for 200 min to a mixture of
5 O3 (0.45 to 0.7 ppm) + SO2 (4.8 to 5.2 ppm) + H2SO4 (800 to 1,200 /*g/m3, 0.2 /mi) +
6 catalytic salts of Fez (SO4)3 and MnSO4. A greater increase in resistance and decrease in
7 compliance was found with the complex atmosphere than with 63 alone, but the effect was
8 ascribed to the presence of the H2SO4. Although synergism was implied, it could not be
9 definitively concluded because the mixture was not tested without Oj.
10 Mautz et al. (1991) further examined the ability of components of acidic fogs to alter
11 the response to O3. Rats (Sprague-Dawley, male, 7 weeks, n = 12/group) were exposed for
12 4 h (nose-only; temperature = 22 to 23 °C, RH = 82 to 83%) to 0.4 ppm 03 or to a
13 mixture of 0.4 ppm O3 + 670 /tg/m3 HNO3 vapor + 610 /ig/m3 H2SO4 particles (0.32 /*m).
14 Exposure to either O3 or the mixture resulted in comparable changes, namely development of
15 a rapid, shallow breathing pattern, a decrease in fatty acid composition of pulmonary
16 surfactant, and focal parenchymal lesions with thickened alveolar septa and cellular
17 infiltration. The lack of any modulation of the O3-induced effects by acids prompted the
18 authors to raise the question of the sensitivity of rats to inhaled acids. Although responses to
19 any pollutant are somewhat species dependent, there is some evidence that rats are not the
20 most sensitive species to acidic aerosols (U.S. Environmental Protection Agency, 1989).
21 As discussed previously, the extent of interaction within any one species of animal is
22 endpoint dependent, and it is likely that the sensitivity of various endpoints is species
23 dependent. Thus, rats do show biochemical changes (e.g., in collagen metabolism) with
24 exposure to fairly low levels of acidic aerosols in combination with 03 (see Table 6-10),
25 although these involved longer duration exposures. In any case, the underlying reasons for
26 the lack of interaction in the complex mixture study above remain unclear.
27 Kleinman et al. (1989) exposed rats (Sprague-Dawley, male, 7 weeks, nose-only) to a
28 mixture of O3 (0.8 ppm) + SO2 (5 ppm) + H2SO4 or (NH^SC^ (1,000 /ig/m3) at high RH
29 (85%), and noted a delay in early clearance of inert particles from the lungs, compared to
30 air-exposed controls. However, it is difficult to relate any effects to the 03 because
31 responses to O3 alone were not examined.
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1 The ideal complex mixture is one that actually exists in the ambient environment.
2 Saldiva et al. (1992) exposed rats (Wistar, male, 2 mo) for 6 mo to actual atmospheres of
3 Sao Paulo, Brazil, with controls maintained for the same period of time in a clean, rural
4 environment. The mean pollution levels over the exposure period were as follows:
5 0.011 ppm O3, 1.25 ppm CO, 29.05 /xg/m3 SO2, and 35.18 /xg/m3 particles. The animals
6 exposed to the urban air showed evidence of bronchial secretory cell hyperplasia, ciliary
7 structural changes, increased viscosity of mucus, and impaired mucociliary clearance.
8 Although chronic exposure to air pollution may result in pulmonary dysfunction, the specific
9 components producing the response could not be determined.
10 Inhalation exposures to air pollutants are, of course, the ideal way to assess interaction,
11 but in vitro exposures may provide indications of potential interactions. Shiraishi and
12 Bandow (1985) exposed Chinese hamster V79 cells for 2 h to photochemical reaction
13 products produced from the reaction of propylene and NO2 in a smog chamber. The
14 resultant exposure atmospheres consisted of various proportions of propylene (0.07 to
15 0.16 ppm), N02 (0.22 to 0.28 ppm), O3 (0.09 to 0.38), PAN (0.04 to 0.41 ppm), and
16 HCHO (0.23 to 1.50 ppm). Exposures to NO2 and O3 alone were also performed. All of
17 the complex mixtures resulted in an increased frequency of sister chromatid exchange and
18 growth inhibition. The effects of the mixture were greater than those due to either O3 or
19 NO2 alone for sister chromatid exhange, but growth inhibition was similar to that induced
20 solely by O3. The authors concluded that the observed effects were not due to any single
21 compound within the mixture, but rather to various compounds producing multiple effects.
22
23 6.4.4 Summary
24 It is difficult to summarize the role that O3 plays in response to exposure to binary
25 mixtures, and it is even harder to determine its role in response to multicomponent
26 atmospheres. One of the problems in understanding interactions is that although the specific
27 mechanisms of action of the individual pollutants within a mixture may be known, the exact
28 bases for toxic interactions have not been clearly elucidated. There are, however, certain
29 generic mechanisms that may underlie pollutant interactions. One is physical, involving
30 adsorption of one pollutant onto another and subsequent transport to more or less sensitive
31 sites, or sites where one of the components of the mixture normally would not deposit in
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1 concentrated amounts. This, however, probably does not play a major role in O3-related
2 interactions. A second mechanism involves production of secondary product(s) that may be
3 more lexicologically active than the primary materials. This has been demonstrated or
4 suggested in a number of studies as a basis for interaction between O3 and NO2. A third
5 mechanism involves biological or chemical alterations at target sites that affect response to
6 the O3 or the copollutant. This has been suggested to underly interactions with mixtures of
7 O3 and acid sulfates. A related mechanism is an O3- or copollutant-induced physiological
8 change, such as alteration in ventilation pattern, resulting in changes in the penetration or
9 deposition of one pollutant when another is present. This has been implicated in enhanced
10 responses to various O3-containing mixtures with exercise.
11 Evaluation of interactions between O3 and other copollutants is a complex procedure.
12 Responses are dependent upon a number of host and environmental factors, such that
13 different studies using the same copollutants may show different types or magnitudes of
14 interactions. The occurrence and nature of any interaction is dependent upon the endpoint
15 being examined, and is also highly related to the specific conditions of each study, such as
16 animal species, health status, exposure method, dose, exposure sequence, and the
17 physiocochemical characteristics of the copollutants. Because of this, it is difficult to
18 compare studies, even those examining similar endpoints, that were performed under
19 different exposure conditions. Thus, any description of interactions is really valid only for
20 the specific conditions of the study in question and cannot be generalized to all conditions of
21 exposure to a particular chemical mixture. Furthermore, it is generally not possible to
22 extrapolate the effect of pollutant mixtures from studies on the effects of each component
23 when given separately. In any case, what can be concluded from the database is that
24 interactions of O3-containing mixtures are generally synergistic, but antagonism has been
25 noted in a few studies, depending upon the various factors noted above, and O3 may produce
26 more significant biological responses as a component of a mixture than when inhaled alone.
27 Furthermore, although most studies have shown that interaction occurs only at higher than
28 ambient concentrations with acute exposure, some have demonstrated interaction at more
29 environmentally relevant levels (e.g., 0.05 to 0.1 ppm O3 with NO^ with repeated
30 exposures.
31
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1 6.5 SUMMARY AND CONCLUSIONS
2 6.5.1 Introduction
3 In the past 30 years, thousands of research studies on the effects of Qj in laboratory
4 animals have been reported in the literature. This body of evidence presents a clear picture
5 of the types of alterations O3 can cause on respiratory tract host defense mechanisms,
6 biochemistry, structure, and lung function. Less is known about carcinogenic potential and
7 effects on organs distant from the lungs. These types of effects are observed in many animal
8 species from mice to nonhuman primates, lending credence to the qualitative extrapolation of
9 these effects to humans. The major issue is what levels, durations, and patterns of exposure
10 are capable of causing these effects in humans. Extrapolation is discussed in Chapter 8.
11 Suffice it to say here that the animal lexicological studies both assist in interpreting
12 observations made in O3-exposed humans and extend our knowledge of potential human
13 hazards that can never be studied adequately in humans.
14 This summary and conclusion section deals exclusively with the effects of O3, alone
15 and in mixture. Other photochemical oxidants either have been evaluated elsewhere (NO2
16 and formaldehyde; U.S. Environmental Protection Agency, 1993; Grindstaff et al., 1991) or
17 in an earlier O3 criteria document (U.S. Environmental Protection Agency, 1986). This
18 section is first organized by respiratory tract effects, systemic effects, and effects of
19 mixtures. Between this chapter and the animal lexicological chapter in the 1986 document
20 (U.S. Environmental Protection Agency, 1986), over 1,000 references are cited. Although
21 all of them contribute to choosing and understanding the key issues to be summarized here,
22 there must obviously be a highly selective choice which references to include here.
23 Generally, the papers discussed here were selected either because they represent lowest
24 effective concentration for an endpoint or they significantly influenced a particular
25 conclusion.
26
27 6.5.2 Molecular Mechanisms of Effects
28 Molecular mechanisms (the manner in which chemical reactions of O3 are translated
29 into biological effects) are alluded to in different sections of this document. Studies which
30 link O3 chemistry with O3 effect measurements would greatly strengthen the theoretical basis
31 for understanding the biological effects of O3. They would also allow examination of the
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1 similarity between animals and humans, thus strengthening interspecies extrapolations.
2 Ozone has been shown to react directly with a variety of biomolecules that are present in
3 both animals and humans. Most of the attention has been centered on polyunsaturated fatty
4 acids and carbon-carbon double bonds, although reactions with sulfhydryl, amino, and some
5 electron-rich compounds may be equally important. Free radicals may be involved, and
6 antioxidant defenses appear to lessen the effect of these reactions. A "molecular target" for
7 03—the biomolecules most affected by reaction with O3 or most crucial in mediating the
8 observed responses, has not been identified for any of the endpoints studied. In fact, the
9 target may be different for different endpoints.
10 An important concept in evaluating molecular targets was recently elucidated by Pryor
11 (1992) who suggests (based on reaction and diffusion rate data) that the 03 molecule does not
12 penetrate through cell membranes or even the surfactant layer of the lung. Instead, a
13 "reaction cascade" forms intermediates (organic or oxygen free radicals, lipid
14 hydroperoxides, aldehydes, hydrogen peroxide, etc.), which penetrate into the cells, causing
15 the biological effects observed (Pryor et al., 1991). Confirmation of such O3-induced free
16 radical autoxidation of lipids has been sought in vivo, but the indirect nature of the
17 measurement methods produced equivocal results. More direct evidence has been obtained
18 by Kennedy et al. (1992) who used electron spin-trapping methods to measure a
19 concentration-related increase in radical adducts of the lipid fraction of lungs from
20 O3-exposed rats. Increased radical signals were detected after a 2-h exposure to >0.5 ppm
21 O3, but because the rats' respiration was stimulated by CO2, the effective dose would be
22 greater than it appears. Oxidized (oxygenated) biomolecules that result from reaction with
23 O3 may also mediate the effects of O3. Studies by Hatch et al. (1994) show that crude
24 fractions of the lung lining layer become labeled with oxygen-18 after exposure to oxygen-
25 18-labeled O3. The label is concentrated in the airway lining layers, and the amount of
26 oxygen-18 incorporation in this layer appears to be correlated with effects of
27 O3 (permeability and inflammation) in both rats and humans. These findings are consistent
28 with the hypothesis that O3 reacts with the lining of the lung, that the same types of
29 interactions occur in both animals and humans, and that these reactions lead to similar
30 effects.
31
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1 6.5.3 Respiratory Tract Effects
2 6.5.3.1 Effects on Host Defenses
3 Several systems defend the respiratory tract of the host against infectious and neoplastic
4 disease as well as nonviable inhaled particles. All these systems can be affected by O3. The
5 mucociliary clearance system moves particles deposited on the mucous layer (either through
6 deposition from the air stream or entry of cells/cellular debris from the alveoli) upwards and
7 out of the lower respiratory tract. The nasal passages also have an effective clearance
8 system. Concentrations as low as 0.15 ppm O3 (8 h/day, 6 days) caused structural changes
9 in the nasal respiratory epithelium (e.g., ciliated cell necrosis, shortened cilia) of monkeys
10 (Harkema et al., 1987). Ciliated cells are also lost or damaged in the conducting airways of
11 the lower respiratory tract after short exposures (e.g., 0.96 ppm, 8 h, monkeys; Hyde et al.,
12 1992). Mucous chemistry also is changed (McBride et al., 1991). Sufficient morphologic
13 damage would be expected to have functional consequences. Acute exposures (0.6 ppm, 2 h)
14 slow mucociliary particle clearance in rabbits, but repeated exposures (up to 14 days) caused
15 no effects. Alveolar clearance is slower and involves clearance of particles through
16 interstitial pathways to the lymphatic system or movement of particle-laden AMs up to the
17 bottom of the mucociliary escalator. Effects on alveolar clearance are concentration-
18 dependent. A single 2-h exposure of rabbits to 0.1 ppm accelerated clearance up to 14 days
19 postexposure, exposure to 0.6 ppm caused no effect, and a higher concentration (1.2 ppm)
20 slowed alveolar clearance (Driscoll et al., 1986). Alveolar clearance of asbestos particles
21 was slowed by a 6-week exposure to an urban pattern of O3 (Pinkerton et al., 1989).
22 Alveolar macrophages are the first line of defense against microbes and nonviable
23 particles that reach the pulmonary region of the lung. They phagocytize particles, kill
24 microbes, and interact with lymphocytes in the development of an immune response. Thus,
25 their proper functioning is critical. Alveolar macrophages from several species of animals
26 exposed acutely to O3 can exhibit decreased phagocytosis; decreased lysosomal enzyme
27 activities and superoxide anion radical production, both of which function in killing bacteria;
28 alterations in membrane morphology; chromosomal damage; decreased cytotoxicity to tumor
29 cells; increased release of PGE2 and PGF2a; and alterations in the number of AMs.
30 Phagocytic changes are the most investigated. Exposure of rabbits to level as low as
31 0.1 ppm (2 h/day) decreased non-specific phagocytosis (of latex microspheres) after 2 or 6,
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1 but not 13, days of exposure (Driscoll et al., 1987). Recovery from the single 2-h exposure
2 to 0.1 ppm was complete by 7 days postexposure. This pattern of response was confirmed in
3 mice for Fc-receptor mediated phagocytosis (Gilmour et al., 1991; Canning et al., 1991).
4 The humoral- and cell-mediated immune system of the lung is also affected by O3.
5 Generally, T-cell dependent immunity is more susceptible than B-cell dependent immunity,
6 but most immune functions examined have exhibited effects. However, because relatively
7 few studies have been conducted, it is not currently possible to adequately interpret the
8 impacts of O3-induced alterations on the immune system (e.g., decreases in mitogenic
9 responses of T cells, alterations in T:B cell ratios in the MLN). Only a few studies have
10 attempted to correlate immunological changes and infectious disease outcome. Van Loveren
11 et al. (1988) infected rats with Listeria, exposed them to 03 for 1 week (0.26 to 1.02 ppm)
12 and measured several endpoints. Ozone increased Listeria-induced mortality and severity of
13 lesions in the lung and liver. They interpreted these findings as due to O3-induced impaired
14 clearance of the bacteria caused by decreased AM function and decreased cellular immunity
15 (e.g., decreased delayed-type hypersensitity and decreased T/B cell ratios in MLN).
16 A reasonably large body of evidence indicates that the impact of 03 on one or several
17 host defense mechanisms leads to the inability of animals to fight bacterial infection and
18 alters the course of viral infection. Antibacterial models are more commonly used. Mice
19 exposed for 3 h to 0.4 ppm O3 have decreased intrapulmonary killing of S. zooepidemicus
20 (Gilmour et al., 1993a; Gilmour and Selgrade, 1994). Similar results have been obtained for
21 S. aureus at a slightly higher concentration (Goldstein et al., 1971b). Correlations have been
22 made between O3 exposure and decreases in AM phagocytosis, decreases in bactericidal
23 activity, growth of bacteria in the lungs, presence of bacteria in the blood, and mortality in
24 mice (Coffin and Gardner, 1972b; Gilmour and Selgrade, 1994). The lowest O3 exposure
25 causing increased streptococcal-induced mortality is 0.08 ppm for 3 h in mice (Coffin et al.,
26 1967; Coffin and Gardner, 1972b; Miller et al., 1978). However, prolonged intermittent
27 exposure to 0.1 ppm for 15 weeks only slightly increased the mortality (Aranyi et al., 1983),
28 and continuous exposure for 15 days to 0.1 ppm with two daily 1-h peaks (5 days/week) to
29 either 0.3 or 0.5 ppm did not enhance mortality in the same model system (Graham et al.,
30 1987). Prolonged exposure (1 to 2 weeks) also did not affect bactericidal activity to
31 S. aureus (Gilmour et al., 1991).
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1 Generally, short-term exposure to O3 does not affect viral titers in the lungs of mice
2 infected with influenza virus, but reduced numbers of lung tissue T and B cells reduced
3 antibody titers to the virus, mortality, lung pathology, and increased lung wet weight do
4 occur (Selgrade et al., 1988; Jakab and Hmieleski, 1988). Ozone also enhances
5 postinfluenzal alveolitis and structural changes that begin at 30 days postinfection (Jakab and
6 Bassett, 1990). The complexity of the interaction of viral infection and O3 exposure is
7 further illustrated by Selgrade et al. (1988) who found that the effects of O3 on influenza
8 virus infection was dependent on the temporal relationship of O3 exposure and day of
9 infectious challenge. Also, interferon, which can be induced by viral infection, mitigates the
10 O3-induced lung lessons in mice, raising the possibility that certain stages of viral infection
11 may have interactions with the lung that are different from other stages (Dziedzic and White,
12 1987b).
13
14 6.5.3.2 Effects on Inflammation and Permeability
15 The barrier function of the respiratory tract is disrupted by O3, allowing cellular and
16 fluid components from the blood to enter the lung and allowing certain types of substances in
17 the lung to enter the blood. Markers of inflammation generally included increased proteins
18 and PMNs in BAL. Concurrent with these events, but not necessarily interdependently,
19 AMs liberate more arachidonic acid, which results in the production of biologically active
20 LTs and PGs. Similar responses are observed in mice, rats, rabbits, guinea pigs, hamsters,
21 and nonhuman primates. After acute exposure, the lowest effective concentration that
22 increases BAL protein and number of PMNs is 0.12 ppm (mice, 24 h of exposure, BAL
23 immediately after exposure) (Kleeberger et al., 1993a). However, typically the increase in
24 BAL protein is maximal roughly 16 to 24 h postexposure. In rats exposed to 0.8 ppm for
25 6-h and examined by lavage and morphometry at several times postexposure, the increase in
26 nasal PMNs occurs sooner and wanes about the time that these cells are increasing in number
27 in the lungs (Hotchkiss et al., 1989a,b). It should be recognized that BAL can measure the
28 protein and cells accessible by lavage, including the resident material (i.e., may include
29 protein from O3-induced cellular destruction) and the material entering from the
30 tissue/circulation. Thus, interstitial inflammation, which has been observed in several
31 species microscopically, is not detectable by BAL.
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1 Several C x T studies have been conducted in mice using BAL protein as an endpoint.
2 In two studies, there was combination of various C's (0.1 to 2.04 ppm) and T's (1 to 12 h),
3 resulting in a number of different C x T products (Rombout et al., 1989; Highfill et al.,
4 1992). Both of these studies showed that the influence of T increased as C increased. That
5 is, there was no simple relationship of Ca X T* = constant product. However, at the lowest
6 C x T products, there was a more equivalent influence of C and T. Gelzleichter et al.
7 (1992b) used a single C x T product composed of a variety of Cs and Ts for up to 3 days of
8 exposure. The 24 h/day exposure group had less response than the other groups which
9 responded equivalently. Effects of longer term exposure on permeability and inflammation
10 are more complex to interpret (also see subsequent discussion on lung structure).
11 Histological examination of rat lungs exposed to 0.5 ppm O3 for 5 days showed more
12 inflammatory cells in the alveoli after 5 days (2.25 h/day) of exposure compared to 1 day of
13 exposure (Tepper et al., 1989). In contrast, the increase in BAL PMNs that occurred after
14 Day 1 of exposure of rats had resolved by Day 4 (7 h/day) (Donaldson et al., 1993).
15 Some studies suggest that although protein and PMN increases are observed
16 concurrently, this may be more of a function of the experimental design than the biological
17 sequence of events. For example, in rats depleted of PMNs with anti-PMN serum, Oj did
18 not increase BAL PMNs, but BAL protein was still increased (Pino et al., 1992b). Also
19 Young and Bhalla (1992) observed an increase in trachea! protein earlier than increased
20 tracheal PMNs. They interpreted this and other related results to suggest that the recruited
21 PMNs may serve to sustain an increase in permeability.
22
23 6.5.3.3 Effects on Structure, Function, and Biochemistry
24 Theoretically, and in some cases empirically, lung structure, function, and biochemistry
25 are linked. Correlations are not exact because of differences in available measurement
26 methods (e.g., most lung function tests used do not sensitively measure the function of the
27 smallest airways, where the "classical" O3 lesion is observed) and some independence of
28 effects (e.g., a transient change in breathing frequency would not be morphologically
29 detectable). Also, most biochemical measurements are made of whole lung, rather than focal
30 areas of damage, and only some enzyme activities measured would be expected to be
31 correlated to structure and/or function (e.g., collagen metabolism, antioxidant metabolism).
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1 After acute exposure to O3, the most commonly observed effect in several species is
2 tachypnea (increased fB and decreased VT) with little (if any) change in VE; the lowest
3 exposure causing tachypnea was 0.2 ppm for 3 h in rats (Mautz and Bufalino, 1989). Other
4 effects reported after acute exposure to < 1 ppm include increased RL and decreased Cdyn,
5 TLC, VC, FRC, RV, FVC, DLCO, and N2 slope (e.g., Fouke et al., 1991; Mautz et al.,
6 1985b; Miller et al., 1988). However, these changes are not observed in all studies,
7 probably due to differences in animal species, measure method, and exposure protocols.
8 With rare exception, concentrations well in excess of 1 ppm O3 are required to increase
9 airway reactivity.
10 Two C x T studies of pulmonary function using acute exposure periods have been
11 performed. Costa et al. (1989) found that FVC, DLcO, and N2 slope decreased with
12 increasing C x T products in rats and that the influence of T is greater at higher Cs.
13 In guinea pigs, Nishikawa et al. (1990) observed that airway responsiveness to methacholine
14 increased at higher C x T products (e.g., at 90 ppm • min, but not at 30 ppm • min). The
15 authors concluded that T was an important factor in the 63 response.
16 When rats were exposed for 5 days (2.25 h/day, with CO2 to stimulate ventilation
17 equivalent to light exercise in humans) to 0.35, 0.5, and 1 ppm 03, the change in shape of
18 the flow-volume curve and tachypnea peaked occurred on Days 1 and 2, but by Day 5, there
19 was no difference from control (except at 1 ppm) (Tepper et al., 1989). This attenuation is
20 similar to that observed in humans. However, in other, similar groups of animals,
21 histological changes in the lung progressed and BAL protein remained elevated. Other
22 similarities between laboratory animals and humans in their pulmonary function responses to
23 short-term O3 exposure are explored in Chapter 8.
24 Ozone causes similar types of alterations in lung morphology in all laboratory animal
25 species studied. The most affected cells are the ciliated epithelial cells of the airways and
26 Type 1 cells in the gas exchange region. Within the nasal cavity, anterior portions of the
27 respiratory and transitional epithelium are affected. Cilia are lost or damaged; some ciliated
28 cells become necrotic, are lost, and are replaced with nonciliated cells. Mucous secreting
29 cells are affected.
30 The centriacinar region (CAR, the junction of the conducting airways and the gas
31 exchange region) is a primary target, possibly because it receives the greatest dose of
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1 O3 delivered to the lower respiratory tract (see Chapter 8) and has Type 1 epithelial cells
2 covering a large surface area. Even though there are significant interspecies differences in
3 the structure of the CAR (e.g., primates, including humans, have respiratory bronchioles,
4 which are rudimentary or absent in laboratory animals such as rats or mice), it is the target
5 in all species studied. Exposure to O3 causes loss of cilia or necrosis of the ciliated cells,
6 leaving a bare basement membrane that is replaced by nonciliated bronchiolar cells, which
7 may become hyperplastic after longer exposures. Mucous secreting cells can be affected, but
8 not as significantly as ciliated cells. Type 1 cells are also damaged and can be sloughed
9 from the surface; Type 2 cells, which are thicker, replace them. Sometimes Type 2 cells
10 differentiate into Type 1 cells. This epithelial remodeling is accompanied by an
11 inflammatory response in the CAR, primarily consisting of an increase in numbers PMNs in
12 the earlier stages and increase in numbers of AMs in later stages; interstitial edema occurs.
13 With increased duration of exposure, alveolar septa in the CAR thicken due to increased
14 matrix, basement membrane, collagen, fibroblasts, and a thickened alveolar epithelium.
15 These patterns of change have different relationships to duration of exposure, as
16 illustrated by Dungworth (1989) (see Figure 6-3; Section 6.2.4.5). Inflammatory changes
17 peak after a few days of O3 exposure; are still observable, but to a much lesser degree, in
18 tissue during months of exposure; and begin to return to control values after exposure ceases.
19 In contrast, epithelial hyperplasia rapidly increases during about the first week of exposure,
20 plateaus as exposure continues, and begins to decrease slowly when exposure stops.
21 Interstitial fibrosis requires months of exposure to be observed microscopically and increases
22 slowly, but when exposure ceases, it can still persist or continue to increase. Numerous
23 studies using several different species and experimental approaches support these findings.
24 Only a few of the studies (primarily those using more sensitive morphometric measurements)
25 are selected here to illustrate key points and show correlations with pulmonary function and
26 lung biochemistry. Only rat and nonhuman primate studies are discussed because most
27 investigations were conducted on them. At equivalent exposures, nonhuman primates appear
28 to be more responsive than rats (Section 6.2.4).
29 Generally short-term exposures to concentrations ^0.2 ppm O3 do not cause changes in
30 the nasal cavities of rats or nonhuman primates detectable by LM, except for inflammation
31 and an occasional delayed postexposure finding of mild hyperplasia. For example, Hotchkiss
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1 et al. (1989a) reported inflammation in the nasal epithelium of rats up to 66 h after a 6-h
2 exposure to levels as low as 0.12 ppm O3; there was no necrosis, loss of cilia, or hyperplasia
3 even at 1.5 ppm. After 3 days (22 h/day) of exposure, >0.4 ppm (but not 0.2 ppm) caused
4 loss of cilia and hyperplasia and metaplasia of the nasal epithelium of rats (Reuzel et al.,
5 1990). Nonhuman primates appeared to be more responsive. Harkema et al. (1987)
6 observed that exposure to 0.15 or 0.3 ppm for 6 or 90 days (8 h/day) caused necrosis of
7 ciliated cells, shortened cilia, and increased mucous granule cells in the respiratory
8 epithelium; alterations in cell numbers were also found in the transitional epithelium.
9 Within the CAR, a number of alterations occur. In rats and monkeys ciliated and
10 Type 1 cells become necrotic and are sloughed from the epithelium as soon as the first 2 to
11 4 h of an exposure to about 0.5 ppm (Stephens et al., 1974a,b). Repair, as shown by
12 increased DNA synthesis by nonciliated bronchiolar and Type 2 cells, begins by about 18 to
13 24 h of exposure (Evans et al., 1976; Stephens et al., 1974a; Castleman et al., 1980),
14 although cell damage continues (Castleman et al., 1980). The lesion is fully developed by
15 about 3 days of continuous exposure, after which the rate or repair exceeds the rate of
16 damage. The increase in antioxidant enzyme activities (e.g., succinate oxidase, G6PD, and
17 6PGD) parallels the increase in Type 2 cells, which are rich in these enzymes; the increase
18 in the Type 2 cell population is probably responsible for these biochemical changes (Bassett
19 et al., 1988a; U.S. Environmental Protection Agency, 1986).
20 Lesions in the CAR are one of the hallmarks of O3 toxicity, having been well
21 established. The study by Chang et al. (1992) provides examples of some of the patterns of
22 cellular alterations. Chang et al. (1992) exposed rats to an urban pattern of O3 (0.06 ppm
23 background, 7 days/week on which were superimposed 9-h peaks [5 days/week] slowly rising
24 to 0.25 ppm) for 78 weeks and made periodic examinations of the CAR TB and proximal
25 alveoli by TEM morphometry during and after exposure. Type 1 cells had a larger volume
26 at Week 13 and increased numbers at Weeks 13 and 78; there was no such changes at
27 17 weeks after exposure ceased. Type 2 cell volume per area of basement membrane
28 increased immediately after Week 78 and was still increased 17 weeks after exposure ceased.
29 Interstitial cells and matrix were increased after Weeks 1, 13, and 78, but returned to control
30 by 17 weeks after exposure ceased. However, epithelial and endothelial basement membrane
31 were thickened and accompanied by increased collagen fibers at the later examination times
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1 and 17 weeks after the 78-week exposure ended. In TBs, surface areas of ciliated and
2 nonciliated cells decreased during exposure. Pulmonary function studies conducted in
3 identically exposed groups of rats were consistent with the morphometric findings (Tepper
4 et al., 1991). Generally expiratory resistance was increased (suggesting central airway
5 narrowing), but it was only significantly different from control at 78 weeks. Tidal volume
6 was reduced at all evaluation times. Overall, breathing frequency was reduced, but no single
7 evaluation time was significant. Monkeys exposed to a higher concentration of
8 O3 (0.64 ppm, 1 year) also showed increased resistance and decreased flows, which were
9 interpreted as central and peripheral airway narrowing; during a 3-mo postexposure period,
10 decreases in static lung compliance persisted (Wegner, 1982).
11 Several studies have demonstrated distal airway remodeling. This bronchiolization of
12 the alveoli is so named because bronchial epithelium replaces the Type 1 and 2 cells typical
13 of alveolar ducts, resulting in the appearance of respiratory bronchioles in rats and an
14 increase volume fraction and volume of respiratory bronchioles in monkeys. This has been
15 observed at exposures as low as 0.5 ppm O3 (50 days) in rats (Moore and Schwartz, 1981)
16 and as low as 0.25 ppm (8 h/day, 18 mo) in monkeys (Tyler et al., 1988). Inflammation
17 occurs concurrently, perhaps indicating an influence on remodeling. In monkeys, such
18 bronchiolization can persist 6 mo after a 1-year (8 h/day) exposure to 0.64 ppm ends (Tyler
19 etal., 199 Ib).
20 Exposure regimens can have unexpected impacts on experimental outcomes. Several
21 investigations of combinations of O3 "episodes" or O3 "seasons" with clean-air periods have
22 been examined. In the first of these, Last et al. (1984b) compared air control rats to
23 2 groups of rats exposed to 0.96 ppm. One group received a 90-day (8 h/day) exposure
24 ("daily") the other had intermittent units of 5 days of O3 (8 h/day) and 9 days of air, such
25 that there were 35 O3 exposure days over the 90-day period ("episodic"). Both groups had
26 equivalent increases in lung collagen. Using a similar exposure regimen, Barr et al. (1990)
27 found equivalent CAR remodeling and volumes of CAR lesions in both groups. In contrast,
28 respiratory bronchiole thickness increased in the daily group only and the CAR interstitium
29 increased in thickness only in the episodic group. Monkeys were studied more extensively
30 after a daily (8 h/day) exposure to 0.25 ppm for 18 mo and a "seasonal" exposure only
31 during the odd months of the 18-mo period (Tyler et al., 1988). Most morphometric
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1 measurements were similar between the two groups (e.g., both had respiratory bronchiolitis).
2 However, only the daily group had an increased number of AMs in the lumen and
3 interstitium. Only the seasonal group had increased lung collagen content; increased chest
4 wall compliance, suggesting delayed lung maturation; and increased inspiratory capacity.
5 This body of work indicates that under these types of exposure circumstances, the simple
6 product of C x T does not predict the outcome. Indeed half the O3 (on a C XT basis)
7 caused equivalent or more effects than a "full" O3 exposure.
8 The complexity of understanding C X T relationships is further illustrated by Chang
9 et al. (1991). She and her colleagues compared two different exposure regimens (one a
10 square wave and the other an urban pattern O3) on the basis of C x T products. There was
11 a linear relationship between C x T products and the increase in Type 1 cell volume in the
12 CAR; a similar observation on Type 2 cell volumes was less robust. There was not such
13 relationship for other morphometric endpoints in the same animals. Cell proliferation in the
14 nasal epithelium does not increase linearly with increasing C x T, but does increase linearly
15 with increasing C (Henderson et al., 1993).
16 Long-term exposure also thickens CAR alveolar septa, due to an increase in
17 inflammatory cells, fibroblasts, and amorphous extracellular matrix (Fujinaka et al., 1985;
18 Barry et al., 1985; Zitnik et al., 1978). There is some morphological evidence of mild
19 fibrosis (i.e., local increase in collagen) in CAR interalveolar septa (Last et al., 1979;
20 Boorman et al., 1986; Chang et al., 1992; Pickrell et al., 1987b; Freeman et al., 1974;
21 Moore and Schwartz, 1981). Biochemical evidence supports these findings, even though
22 biochemical approaches would be expected to be less sensitive because the whole lung (rather
23 than focal lesions) is examined. Last et al. (1979) directly demonstrated the correlation by
24 observing increased collagen histologically and biochemically (collagen synthesis rate) in rats
25 similarly exposed to 0.5 to 2.0 ppm for 7 to 21 days. The increase became greater with
26 increasing concentration and duration of exposure. Similar correlations were observed at a
27 higher concentration by Pickrell et al. (1987b). The increased collagen content can persist
28 after exposure ceases (Chang et al., 1992; Hussain et al., 1976a,b; Last et al., 1984b), but
29 some, not all, studies suggest that higher concentrations (>0.5 ppm) may be required for
30 such persistence (Last and Greenberg, 1980; Pickrell et al., 1987b). Collagen cross-links
31 were studied in monkeys exposed to 0.61 ppm O3 for 1 year (8 h/day) (Reiser et al., 1987).
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1 Earlier examination of these same monkeys revealed that collagen content was increased
2 (Last et al., 1984b). When specific collagen crosslinks were measured, the increase in
3 "abnormal" cross-links observed immediately after exposure remained in the lungs at 6 mo
4 postexposure.
5 As discussed above, O3 can cause fibrotic changes. However, there is no evidence that
6 O3 causes emphysema, using the currently accepted morphological definition of emphysema.
7 These morphologic/morphometric and biochemical findings of fibrotic changes are
8 supported by some pulmonary function studies. For example, rats exposed for up to
9 78 weeks using the same urban exposure protocol as Chang et al. (1992) exhibited reduced
10 lung volume and hastened N2 washout patterns, consistent with a "stiffer" lung (i.e.,
11 restrictive lung disease) (Costa et al., 1994).
12
13 6.5.3.4 Genotoxicity and Carcinogenicity of Ozone
14 At this point in time, a significant amount of research has been conducted to determine
15 whether O3 is genotoxic or carcinogenic. Many of the earlier experiments have flaws in
16 experimental design or have used O3 concentrations far above levels that could occur in the
17 ambient air. In evaluating the data, a number of conclusions can be made. In vitro exposure
18 of naked plasmid DNA to very high O3 concentrations results in single and double-strand
19 breaks in the DNA, as confirmed by gel electrophoresis and electron microscopy studies
20 (Hamelin, 1985). Testing of O3 in various mutagenesis assays has led to marginal or small
21 results in a number of assays and negative results in other assay systems. Ozone is not
22 mutagenic in Salmonella strains TA98, TA100, TA104, and TA1535 and causes, at most,
23 weak effects in strains TA102 that are not strictly concentration-dependent (Dillon et al.,
24 1992; Victorin and Stahlberg, 1988a,b). Extremely high concentrations of 03 (50 ppm)
25 caused mutation to streptomycin resistance in E. coli and caused various types of mutations
26 in the yeast, S. cerevisiae, but it was a weak mutagen compared to known strong mutagens
27 in the yeast system (L' Herault and Chung, 1984; Dubeau and Chung, 1982). Ozone was
28 not mutagenic in the AT. tabacam or Tradescantia mutation assay systems (Gichner et al.,
29 1992). Hence, overall, the data on the mutagenicity of O3 are mixed: negative in six
30 assays, marginally positive in one system, and weakly positive in two systems. The present
31 data indicate that O3 is, at most, a weak mutagen, but further data are needed in mammalian
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1 cell systems to draw definitive conclusions regarding this point. There are some data
2 indicating that O3 may cause chromosome breakage in cultured cells, but in vivo animal
3 studies are conflicting (Zelac et al., 1971a,b; Tice et al., 1978). A human study with an
4 appropriate experimental design was negative (McKenzie et al., 1977; McKenzie, 1982).
5 Regarding carcinogenicity, O3 has been shown to induce morphological transformation
6 in cultured C3H/10T1/2 mouse embryo cells and in SHE cells, and to cause a synergistic
7 transformation in cells treated also with gamma radiation (Borek et al., 1986, 1989b).
8 However, the results could be due to interactions of O3 with the culture medium that
9 generate chemical species different from those produced in vivo. Whole animal
10 carcinogenesis assays performed in strain A mice have demonstrated marginal increases in
11 tumor yield that were not statistically significant or concentration-dependent (Hassett et al.,
12 1986; Last et al., 1987). The NTP study demonstrated that O3 was not a tumor promoter or
13 a co-carcinogen when NNK-treated male F344/N rats were exposed for 2 years to 0.5 ppm
14 O3 (National Toxicology Program, 1993). This NTP study showed no evidence of
15 carcinogenic activity in male or female F344/N rats, equivocal evidence of carcinogenic
16 activity in male B6C3Fi mice, and some evidence of carcinogenic activity in female B6C3FJ
17 mice at a high concentration (1.0 ppm). Hence, O3 may be a weak pulmonary carcinogen
18 only in female B6C3FJ mice at toxic concentrations.
19 At present, O3 is shown to be nonmutagenic in some assay systems; at most, weakly
20 mutagenic in a few assay systems; and clastogenic in vitro but not in vivo. Ozone can
21 transform cells in vitro. Ozone does not cause concentration-dependent tumor induction that
22 is statistically significant in hamsters, Wister male rats, F344/N male or female rats, male or
23 female A/J mice, or Swiss-Webster male mice. There are ambiguous data for pulmonary
24 carcinogenesis in male B6C3Fj mice and weak carcinogenesis data in female B6C3Fj mice
25 from chronic exposure to 1.0 ppm O3. Therefore, O3 has been shown to be a carcinogen
26 only in female E6C3Fl mice in one experiment. Ozone needs to be studied further in well-
27 designed experiments to determine definitively whether it can significantly induce tumors
28 reproducibly in B6C3Fj mice and other species of rats and mice in a concentration-dependent
29 manner. Because the rat and mice inhalation carcinogenesis experiments do not agree and
30 mechanistic data to assist in interpretation are either not useful or not available, these
31 pulmonary carcinogenesis results in female B6C3Fi mice cannot be extrapolated to humans.
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1 Hence, there is no justification to consider O3 a human carcinogen at this time. Further,
2 since it required a chronic exposure to 1 ppm O3 to induce pulmonary tumors in mice, it is
3 possible that pulmonary toxicity that does not occur at lower levels contributed to the tumor
4 development.
5
6 6.5.3.5 Risk Factors
7 Factors that increase the delivered dose of O3> decrease biochemical defense
8 mechanisms, or increase cellular sensitivity can increase the impact of a given O3 exposure.
9 The most commonly studied factors include exercise, age, and nutrition.
10 As discussed in Chapter 8, exercise increases the dose of O3 delivered to the
11 respiratory tract and alters its distribution. As would be expected, exercise during exposure
12 enhances the effect of O3. This has been demonstrated by Mautz et al. (1985b) who showed
13 that exercising rats had more extensive lung lesions than those at rest. Similarly, Tepper
14 et al. (1990, 1994) found that rats were more responsive to O3 when coexposed to CO2 to
15 increase ventilation, simulating exercise.
16 A number of studies have been conducted to compare the effects of O3 on various ages
17 of mice and rats, from 1 day old to older adults. Interpretation of these studies is difficult
18 because prior to weaning, the huddling behavior of the neonates with their dams as well as
19 the bedding material (present in some studies) may have affected the concentration of O3 in
20 the breathing zone and hence the subsequent delivered dose. Generally, in short-term
21 exposure biochemical studies of antioxidant metabolism, there was a decrease or no change
22 in enzyme activity in neonates. As age increased after weaning, the typical increase in
23 antioxidant metabolism became greater with age (Elsayed et al., 1982; Tyson et al., 1982;
24 Lunan et al., 1977; Mustafa et al., 1985). Stephens et al (1978) found that morphological
25 effects did not occur in animals exposed prior to weaning at 21 days of age. This may
26 explain the results of Barry et al. (1985, 1988) who found no morphometric differences in
27 the CAR and TB in rats that started a 42-day exposure at ages of 1 day and 42 days.
28 In identically exposed rats, however, Raub et al. (1983) found more, though admittedly
29 subtle, pulmonary function changes in the youngest group of animals. Yokoyama et al.
30 (1984) did not detect any age-related differences in lung function of rats at 4-, 7-, and
31 10-weeks of age. Although O3-induced increases in BAL protein and PMNs do not show an
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1 age-dependence, BAL prostaglandins increased sooner and more leukocytes were dead in
2 younger (13-days old) rats, compared to adults (e.g., 16- weeks old) (Gunnison et al., 1990,
3 1992a). Age (5- versus 9-weeks old) did not influence the O3-induced decrease in lung
4 bactericidal activity (Gilmour et al., 1993a).
5 The literature on O3-exposed pregnant animals is extremely sparse. Exposure of rats
6 (1 ppm 6 h) on day 17 of pregnancy or days 3, 13, and 20 of lactation caused a greater
7 increase in lung permeability and inflammation than that observed in nonpregnant rats
8 (Gunnison etal., 1992b).
9 Numerous reports document that animals made vitamin E deficient are more susceptible
10 to the biochemically detected effects of O3 (e.g., lipid changes, antioxidant metabolism
11 changes) (U.S. Environmental Protection Agency, 1986; Pryor, 1991). Generally, the
12 research shows that although vitamin E deficiency enhances susceptibility to lung biochemical
13 changes, there is not a proportionate relationship between vitamin E supplementation (above
14 normal levels) and protection from O3. Also, vitamin E deficiency did not alter the impact
15 of O3 on lung structure (Chow et al., 1981). Vitamin C deficiency also has an influence.
16 Guinea pigs deficient in vitamin C had a greater (compared to vitamin C-normal animals)
17 when exposed acutely to 0.5, but not 1.0 ppm O3 (Slade et al., 1989).
18
19 6.5.4 Systemic Effects
20 Theoretical studies (Pryor, 1992) indicate that the O3 molecule does not penetrate to the
21 blood, yet there are numerous reports of systemic effects (i.e., effects on lymphocytes,
22 erythrocytes, serum, CNS, parathyroid gland, circulatory system, and liver). Possibly one or
23 several of the reaction products of O3 (see Section 6.2.1) penetrates the lung tissue, or
24 perhaps some systemic responses are secondary to pulmonary effects. Although a variety of
25 clinical chemistry changes occur after O3 exposure, they cannot be interpreted and will not
26 be discussed here (see U.S. Environmental Protection Agency, 1986 and Section 6.3).
27 Effects on systemic immunity are discussed earlier (6.5.3.1).
28
29 6.5.4.1 Central Nervous System and Behavioral Effects
30 Acute exposure to O3 caused transient changes in behavior. The lowest exposure
31 causing effects was 0.12 ppm for 6 h in rats; wheel-running activity decreased (Tepper et al.,
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1 1985; Tepper and Weiss, 1986). Because exercising animals were exposed in these studies
2 (i.e., they received a higher dose of O3), it is not surprising that higher 03 concentrations
3 (0.5 ppm, 6 h) are required to affect sedentary behavior (e.g., operant behaviors such as
4 lever pressing for food reinforcement) (Weiss et al., 1981). Mice show aversive responses
5 to O3 (60s, 0.5 ppm) by terminating O3 exposure (Tepper et al., 1985). The lowest
6 exposures causing effects are impacted by the type of reward. For example, 03 had less
7 effect on behaviors to avoid electric shock (Ichikawa et al., 1988) than on behaviors to obtain
8 food or access exercise (Tepper et al., 1982, 1985; Weiss et al., 1981).
9
10 6.5.4.2 Cardiovascular Effects
11 In rats, O3 can cause bradyarrhythmia at exposures as low as 0.1 ppm for 3 days;
12 bradycardia, at exposures as low as 0.2 ppm for 2 days; and decreased mean arterial blood
13 pressure, at exposures as low as 0.5 ppm for 6 h (Arito et al., 1990, 1992; Uchiyama and
14 Yokoyama, 1989; Watkinson et al., 1993; Yokoyama et al., 1989; Uchiyama et al., 1986).
15 There is an interaction between some of these responses and thermoregulation in the rat. For
16 example, when heart rate decreased, the core temperature of the exposed rats also decreased;
17 and when exposures were conducted at higher ambient temperature, there was no change in
18 core temperature or heart rate (Watkinson et al., 1993). Such interactions add to the
19 complexity of extrapolating this type of response to humans, and therefore, without more
20 information, qualitative extrapolation would be highly speculative.
21
22 6.5.4.3 Reproductive and Developmental Effects
23 No reports of "classical" (e.g., 2-generation studies) reproductive assays with O3 were
24 found. Kavlock et al. (1979, 1980) performed several developmental toxicity experiments in
25 rats. Pregnant rats exposed intermittently (8 h/day) from 0.44 to 1.97 ppm (ppm during
26 early, mid-, or late- gestation or the entire period of organgensis (days 6 to 15) had no
27 significant teratogenic effects. Continuous exposure during mid gestation increased the
28 resorption of embryos. Postnatal growth and behavioral development were also investigated.
29 There was no effect on neonatal mortality (up to 1.5 ppm). Pups from dams exposed
30 continuously to 1 ppm during mid- or late gestation weighed less 6 days after birth. Pups
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1 from pregnant rats exposed continuously to 1 ppm during late gestation had delays in
2 behavioral development (e.g., righting, eye opening).
3
4 6.5.4.4 Other Systemic Effects
5 A number of investigations have shown the effects of O3 on the pituitary-thyroid-
6 adrenal axis, as evidenced by changes in circulating hormones and morphological changes in
7 the thyroid and parathyroid glands. (U.S. Environmental Protection Agency, 1986).
8 No newer studies could be found.
9 Several approaches have been used to study the effects of O3 on the liver: increase in
10 sleeping time following the injection of drugs (e.g., pentobarbital) metabolized by the liver,
11 drug pharmacokinetics, and changes in liver enzymes. The lowest exposure causing
12 increased sleeping time from pentobarbital was 0.1 ppm for at least 15 or 16 days (3 h/day)
13 in female mice (Graham et al., 1981). In three species of animals, only females were
14 affected (Graham et al., 1981). Pentobarbital pharmacokinetics was marginally (p = 0.06)
15 slowed in mice exposed to 1 ppm O3 for 3 h (Graham et al., 1985); theophylline clearance
16 was slowed in older rabbits exposed to 0.3 ppm O3 for 5 days (3.75 h/day) (Canada and
17 Calabrese, 1985). Ozone has caused both increases, decreases, and no changes in liver
18 xenobiotic metabolism, depending upon the exposure and enzyme being measured (U.S.
19 Environmental Protection Agency, 1986).
20
21 6.5.5 Effects of Mixtures
22 Humans in the real world are exposed to complex mixtures of gases and particles.
23 Sufficient evidence exists to know that the health outcome is dependent on the mixture, but
24 the relative role (or even the exact identity) of the "major" components is not known.
25 Because of this, it is crucial to evaluate the health effects of O^ in light of epidemiological,
26 human clinical, and animal lexicological studies. For the purposes of this document, an
27 interaction is considered to occur when the response to the mixture is statistically
28 significantly higher (synergism) or lower (antagonism) than the sum of the individual
29 pollutants. Most animal lexicological studies of interactions have been conducted with binary
30 mixtures (predominantly NO2 and H2SO4). The rarer reports on complex mixtures are
31 interesting, but less helpful because often they did not include a group exposed only to 03,
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1 and therefore, knowledge of the role of O3 is confounded. Thus, only the binary mixture
2 studies will be summarized here. This research has demonstrated that exposure to O3 in
3 combination with another chemical can result in antagonism, additivity, or synergism,
4 depending on the animal species, exposure regimen, and endpoint studied. Interpretation is
5 further complicated by the fact that most studies used exposure regimens unlike the real
6 world in terms of ratios of pollutant concentrations, "natural" sequencing of exposure
7 patterns, and other factors. For example, when O3 and NO2 exposures were sequential
8 (in any order), there was an additive increase in BAL protein, as compared to a synergistic
9 increase when the exposures were concurrent (Gelzleichter et al., 1992a).
10 A range of interactions has been shown with O3 and NO2 combinations. For example,
11 a 2-week exposure to an O3-NO2 mixture (0.4 ppm of both) synergistically increased
12 antioxidants in the lungs of rats, but not guinea pigs; peroxide levels were synergistically
13 increased in guinea pigs, but not rats; GST activity was decreased in guinea pigs and
14 unchanged in rats (Ichinose and Sagai, 1989). Most of the interaction studies using lung
15 biochemical endpoints display synergism. A rare exception was the antagonism to the
16 increase in lung cytochrome P-450 content caused by 0.2 ppm O3 (1 to 2 mo) when the rats
17 were coexposed to 4 ppm NO2 (Takahashi and Miura, 1989). Combinations of various acute
18 exposure durations and O3 and NO2 concentrations did not follow a C x T relationship for
19 increased lung permeability, but were synergistic at higher C x T products (Gelzleichter
20 et al., 1992b). For pulmonary host defenses against bacterial infection, the interaction is
21 dependent on the exposure pattern. Graham et al. (1987) showed that a 15-day exposure of
22 mice to mixtures of O3 and NO2, each having a baseline level with two daily 1-h peaks of
23 the pollutant, resulted hi synergism when exposure to either gas alone caused an increase in
24 bacterial-induced mortality.
25 Both synergistic and antagonistic interactions have been found with combinations of
26 O3 and acidic sulfates. Warren et al. (1986) reported that with 3 days of exposure to
'j
27 0.2 ppm O3 plus 5 mg/m (NH4)2SO4, O3 alone was responsible for increasing BAL protein,
28 collagen synthesis rate, and other parameters; but, by 7 days of exposure, synergism
29 occurred. When rabbits were exposed for 4 mo (2 h/day, 5 days/week) to 0.1 ppm O3 plus
•5
30 125 /*g/m H2SO4, there was a synergistic increase in epithelial secretory cell number,
31 whereas 8 mo of exposure resulted in antagonism (Schlesinger et al., 1992a). Antagonism
December 1993 6-209 DRAFT-DO NOT QUOTE OR CITE
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1 was also observed for effects or certain AM functions after acute exposures to O3-H2SO4
2 mixtures (Schlesinger et al., 1992b). Sequential exposures to O3 and H2SO4 have also been
3 examined. Exposure to O3 did not influence the subsequent effects of H2SO4 on
4 bronchoconstriction in guinea pigs (Silbaugh and Mauderly, 1986). Gardner et al. (1977)
5 found an additive increase in bacterial infectivity when mice were exposed acutely to
6 0.1 ppm O3 before, but not after, H2SO4.
7 In summary, the animal lexicological studies clearly demonstrate the major complexities
8 and potential importance of interactions, but do not provide a scientific basis for predicting
9 the results of interactions under untested ambient exposure scenarios.
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31
32
December 1993 6-245 DRAFT-DO NOT QUOTE OR CITE
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i 7. HUMAN HEALTH EFFECTS OF OZONE AND
2 RELATED PHOTOCHEMICAL OXIDANTS
3
4
5 7.1 INTRODUCTION
6 In the previous chapter, results of ozone (O3) studies in laboratory animals were
7 presented in order to understand the wide range of potential effects that might occur in
8 exposed human populations and to expand our understanding of the mechanisms of
9 O3 toxicity and the basic exposure-response relationships for O3. The concept of
10 quantitatively extrapolating results from laboratory animals to humans is further explored in
11 Chapter 8. Whenever possible, however, risk assessment and risk management of pollutants
12 should be based on direct evidence of their health effects in human populations. Information
13 on human health responses to O3 can be obtained through controlled human exposure studies
14 on volunteer subjects or through field and epidemiological studies of populations that are
15 exposed to ambient air containing O3. Controlled human studies typically use fixed
16 concentrations of O3 under carefully regulated environmental conditions, whereas realistic
17 O3 exposure conditions occur in field and epidemiology studies, but are more variable. The
18 primary purpose of all these studies, however, is to obtain exposure-response data for O3.
19 This chapter will summarize the results of controlled human, field, and epidemiologic studies
20 on the health effects of exposure to O3 that have been published in the peer-reviewed
21 literature. Further evaluation of the most important key information from this chapter, as it
22 relates to the rest of the document, will be provided in the remaining chapters.
23 Most of the scientific information selected for review and comment in this chapter
24 comes from the literature published since completion of the previous O3 criteria document
25 (U.S. Environmental Protection Agency, 1986). Some of these newer studies were briefly
26 reviewed in the supplement to that document (U.S. Environmental Protection Agency, 1992),
27 but more intense evaluation of these studies is included here. In order to give a broader
28 overview of the known human health effects of O3, the older literature is summarized and
29 specific studies whose data were judged to be significant because of their usefulness in
December 1993 7-1 DRAFT-DO NOT QUOTE OR CITE
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1 deriving the current National Ambient Air Quality Standards (NAAQS) are briefly discussed.
2 The reader should, however, be referred to the more extensive discussion of these "key"
3 studies in the previous document. Other, older studies are also briefly discussed in this
4 chapter if they are (1) open to reinterpretation because of newer data, or (2) potentially
5 useful in deriving revised NAAQS for O3. To further aid in the development of this chapter,
6 summary tables of the relevant O3 literature are included for each of the major subsections.
7 In summarizing the human health effects literature, changes from control are described if
8 they were statistically significant at a probability (p) value less than 0.05. A specific p value
9 is provided, however, if it aids understanding of the data, particularly trends toward
10 significance, or if major effects need to be emphasized. Where appropriate, critique of a
11 statistical procedure also is mentioned.
12
13
14 7.2 CONTROLLED HUMAN EXPOSURE STUDIES
15 7.2.1 Pulmonary Function Effects of One- to Three-Hour Ozone
16 Exposures
17 7.2.1.1 Healthy Subjects
18 Introduction
19 The pulmonary responses observed in healthy human subjects exposed to ambient
20 O3 concentrations consist of a decreased inspiratory capacity, a mild bronchoconstriction, a
21 rapid shallow breathing pattern during exercise, and subjective symptoms of cough and pain
22 on deep inspiration. In addition, O3 has been shown to result hi airway hyperresponsiveness
23 as demonstrated by an increased physiological response to a nonspecific stimulus. The
24 decrease in inspiratory capacity results in a decrease in forced vital capacity (FVC) and total
25 lung capacity (TLC) and in combination with bronchoconstriction contributes to a decrease in
26 the forced expiratory volume in one second (EEVj). However, it is important to stress that
27 in many of the studies reporting the effects of O3 concentrations critical to the standard-
28 setting process (i.e., <0.30 ppm), the observed decrements in FEV! to a large extent reflect
29 decrements in FVC of a similar magnitude (i.e., a decreased inspiratory capacity) and, to a
30 lesser extent, increases in central and/or peripheral airway resistance (RaW).
December 1993 7-2 DRAFT-DO NOT QUOTE OR CITE
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1 The majority of controlled human studies have been concerned with the effects of
2 various O3 concentrations in healthy subjects performing continuous exercise (CE) or
3 intermittent exercise (IE) for variable periods of time. Controlled human exposure studies of
4 this type have provided the strongest and most quantifiable concentration-response data on
5 the health effects of O3. As a result of these studies, a large body of data regarding the
6 interaction of O3 concentration, minute ventilation, and duration of exposure is available.
7 The most salient observations from these studies are (1) O3 concentration is more important
8 than either ventilation or exposure duration in determining pulmonary responses; and
9 (2) normal healthy subjects exposed to O3 concentrations <0.12 ppm (the level of the
10 current NAAQS) develop significant reversible, transient decrements in pulmonary function if
11 ventilation and/or duration of exposure are increased sufficiently. There is typically a large
12 intersubject varability in physiologic and symptomatic responses to O3; however, with most
13 individuals these responses tend to be reproducible. The relationship among response
14 variables such as spirometry, resistance measurements, symptoms, and nonspecific bronchial
15 responsiveness is yet to be fully determined, but the generally weak associations suggest that
16 several response mechanisms may be operant. In addition, a growing number of studies are
17 beginning to provide insight into the relationship between regional dosimetry (see Chapter 8),
18 mechanisms of pulmonary responses elicited by acute O3 exposure, and tissue level events
19 within the airways. This type of information promises to provide further insight into the
20 health effects relevance of O3-induced pulmonary responses with regard to determining which
21 individuals are at greatest risk from ambient O3 exposure.
22 In this section, the effects of acute single 1- to 3-h O3 exposures on pulmonary
23 function in healthy subjects are examined by reviewing studies that investigate (1) the
24 O3 dose-response relationship; (2) intersubject variability, individual sensitivity, and the
25 association between responses; and (3) mechanisms of pulmonary function responses and the
26 relationship between tissue-level events and functional responses. Recent single O3 exposure
27 studies of greater than 3 h duration are reviewed in Section 7.2.2. These single-exposure,
28 long-duration studies are beginning to provide important insights into the O3 concentration X
29 ventilation X time interaction and the minimum O3 concentration required to elicit a
30 significant pulmonary response. Important key studies of less than 3 h duration that have
31 contributed to the dose-response database and other studies that have contributed to a better
December 1993 7.3 DRAFT-DO NOT QUOTE OR CITE
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1 understanding of O3-induced pulmonary responses in healthy individuals are summarized in
2 Table 7-1. Table 7-1 summarizes several studies reviewed in the previous air quality criteria
3 document (U.S. Environmental Protection Agency, 1986) and its supplement (U.S.
4 Environmental Protection Agency, 1992), as well as studies published since completion of
5 these earlier documents. Not reviewed in this section are studies that examine changes in
6 airway responsiveness induced by O3 inhalation (see Section 7.2.3).
7
8 The Ozone Concentration-Response Relationship
9 At-Rest Exposures. No new studies examining the acute effects of a single exposure to
10 O3 concentrations below 1 ppm in resting humans have been published since the 1986 U.S.
11 Environmental Protection Agency (EPA) criteria document (U.S. Environmental Protection
12 Agency, 1986). Seven studies (Young et al., 1964; Bates et al., 1972; Silverman et al.,
13 1976; Folinsbee et al., 1978; Horvath et al., 1979; Kagawa and Tsuru, 1979; Konig et al.,
14 1980) examining 2-h at-rest exposures were discussed in the 1986 EPA criteria document
15 (U.S. Environmental Protection Agency, 1986) involving 91 healthy subjects (74 males,
16 17 females) exposed to O3 concentrations ranging from 0.10 to 1.00 ppm. The lowest
17 concentration at which significant reductions in FVC and ¥EVl were reported was 0.50 ppm
18 (Folinsbee et al., 1978; Horvath et al., 1979). Reports of increases in airway resistance are
19 inconsistent in resting human subjects exposed to O3 concentrations below 1.00 ppm.
20
21 Exposure with Exercise. Bates et al. (1972) and Hazucha et al. (1973) were the first
22 investigators to examine the effect of increasing ventilation via exercise during O3 inhalation
23 on pulmonary function responses. The intermittent exercise protocol used consisted of the
24 subjects alternating rest and light exercise on a cycle ergometer at a rate sufficient to double
25 resting minute ventilation for 15 min during a period of 2 h.
26 Hazucha et al. (1973) observed significant decreases in forced expiratory endpoints at
27 0.37 ppm O3 (p < 0.05) and 0.75 ppm O3 (p < 0.001), with subjects exposed to 0.75 ppm
28 O3 having the greatest decrements. After exposures, all subjects complained to varying
29 degrees of substernal soreness, chest tightness, and cough. The important findings from
December 1993 7-4 DRAFT-DO NOT QUOTE OR CITE
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TABLE 7-1. CONTROLLED HUMAN EXPOSURE TO OZONE3
r:
n>
1
5
u>
•^J
U)
O
§
1
I
3
g
Ozone
Concentration
ppm
Hg/m
Exposure
Duration and Exposure
Activity Conditions
Number
and
Gender of
Subjects
Subjects
Characteristics6
Observed Effect(s)
References
Healthy Adult Subjects at Rest
0.00
0.25
0.50
0.75
0.00
0.37
0.50
0.75
0.00
0.50
Healthy
0.00
0.08
0.10
0.12
0.14
0.16
0.00
0.10
0.15
0.20
0.25
0.00
0.12
0.18
0.24
0.00
0.12
0.18
0.24
0.30
0.40
0
490
980
1,470
0
726
980
1,470
0
980
2h NA
2h NA
2h NA
8M
5 F
20 M
8F
40 M
Young, healthy
adults
FVC decreased with 0.50- and 0.75-ppm 03 exposure compared
with FA; 4% nonsignificant decrease in mean VO2inax following
0.75 ppm 03 compared with FA exposure.
Decrease in FEVj, ^2S%VC' an(^ ^50%VC w*"1 0-75 ppm
03 exposure compared to FA.
Decrease in forced expiratory volume and flow.
Horvath et al. (1979)
Silverman et al. (1976)
Folinsbee et al. (1978)
Exercising Adult Subjects
0
157
196
235
274
314
0
196
294
392
490
0
235
353
470
0
235
353
470
588
784
2hIE Tdb = 32 ;C
(4xl5minat RH = 38%
VE =
68 L/min)
2 h IE Tdb = 22 °C
(4 x 14min RH = 50%
treadmill at
mean Vg =
70.2 L/min)
1 h competitive Tdb = 23-26 °C
simulation RH = 45-60%
exposures at
mean VE =
87 L/min
2.5 h IE Tdb = 22 CC
(4xl5min RH = 40*
treadmill
exercise
[VE =
65 L/min])
24 M
20 M
10 M
20 M
22M
20 M
21 M
20 M
29 M
Young, healthy
adults
Young, healthy
NS
10 highly
trained
competitive
cyclists
Young, healthy
adults
No significant changes in pulmonary function measurements.
FVC, FEVj, FEF25.755J, SGaw, 1C, and TLC all decreased with
(1) increasing 03 concentration, and (2) increasing time of
exposure; threshold for response was above 0. 10 ppm but below
0.15 ppm 03.
Decrease in FVC and FEV1-0 for 0.18- and 0.24-ppm
O3 exposure compared with FA exposure; decrease in exercise
time for subjects unable to complete the competitive simulation at
0.18 and 0.24 ppm 03, respectively.
Significant decrease in FVC, FEVj , and FEF25_75% at 0.12 ppm
63; decrease in VT and increase in f and SR,W at 0.24 ppm 03.
Linn et al. (1986)
Kulle et al. (1985)
Schelegle and Adams (1986)
McDonnell et al. (1983)
-------
TABLE 7-1 (cont'd). CONTROLLED HUMAN EXPOSURE TO OZONE3
0
1
cr
n
—
i
-j
ON
O
C
§'
M
O
H
0
Q
M
O
O
Ozone
Concentration
ppm
Healthy
0.12
0.18
0.24
0.30
0.40
0.12
0.18
0.24
0.30
0.40
0.00
0.12
0.18
0.24
0.30
0.40
0.00
0.12
0.20
fig/m
Exposure
Duration and Exposure
Activity Conditions
Number
and
Gender of
Subjects
Subjects
Characteristics6 Observed Effect(s)
References
Exercising Adult Subjects (cont'd)
235
353
470
588
784
235
353
470
588
784
0
235
353
470
588
784
0
235
392
2 x 2.5 h IE Tdb = 22 °C
(4 x 15 min RH = 40%
treadmill
exercise
[ VE =
35 L/min/m
BSA]).
Exposure
separated by
48 ± 30 days
and 301 ±
77 days
2 x 2.5 h IE Tdb = 22 °C
(4 X 15 min RH = 40%
treadmill
exercise
[ ^P ~
35 L/min/m2
BSA])
2.5 h IE Tdb = 22 °C
(4 X 15 min RH = 40%
treadmill
exercise
[ VE =
25 L/min/m2
BSA])
1 h CE (mean Tdb = 31 °C
VE = 89
L/min)
8M
8M
5M
5M
6M
290 M
17 WM/15 BM/15 WF/15
BF
15 WM/15 BM/15 WF/16
BF
15 WM/17BM/17 WF/15
BF
16 WM/15 BM/17 WF/16
BF
15 WM/15 BM/15 WF/15
BF
15 WM/15 BM/15 WF/15
BF
15 M
2F
Young, healthy Pulmonary function variables SRaw and Vg were not significantly
adults different in repeat exposures, indicating that the response to
0.18 ppm 03 or higher is reproducible.
Young, healthy 03 concentration and age predicted FEVj decrements; it was
adults concluded that age is a significant predictor of response (older
subjects being less responsive to 03).
Young, healthy Decreases in FEVj for all levels of 03 as compared with FA;
whites and increase in SRaw with 0.18 ppm O3 and greater compared with FA;
blacks black men and women had larger FEV] decrements than white men,
and black men had larger FEVj decrements than white women.
Highly trained Decrease in Vgmax, VO2inax, VTmax, work load, ride time,
competitive FVC, and FEVj with 0.20 ppm Oj exposure, but not significant
cyclists with 0.12 ppm 03 exposure, as compared to FA exposure.
McDonnell et al.
(1985b)
McDonnell et al. (1993)
Seal et al. (1993b)
Gong et al. (1986)
-------
TABLE 7-1 (cont'd). CONTROLLED HUMAN EXPOSURE TO OZONE3
o
I
Ozone
Concentration Exnogure
ppm
Duration and
Hg/m Activity
Exposure
Conditions
Number
and
Gender of
Subjects
Subjects
Characteristics
Observed Effect(s)
References
^O Healthy Exercising Adult Subjects (cont'd)
i
o
6
o
1
i
0.00
0.16
0.24
0.32
0.00
0.20
0.00
0.20
0.30
0.00
0.20
0.35
0.00
0.21
0.00
0.21
0.00
0.25
0
314
470
627
0
392
0
392
588
0
392
686
0
412
0
412
0
490
1 h CE (mean
57 L/min)
4hffi
(4 x 50 min
cycle
ergometry or
treadmill
running [ VE
= 40 L/min])
30-80 min
CE cycle
ergometry
(VE = 33or
66 L/min)
1 h CE or
competitive
simulation
(mean VE =
77.5 L/min)
1 h CE (75%
VCynax)
1 h CE cycle
ergometry
(mean VE =
80 L/min)
IhCE
(mean VE =
63 L/min)
Tdb =
RH =
Tdb =
RH =
Tdb =
•c
RH =
Tdb =
"C
RH =
Tdb =
°C
RH =
Tdb =
RH =
Tdb =
RH =
32 °C
42-46%
20 °C
50%
20-24
40-60%
23-26
45-60%
19-21
60-70%
22.5 °C
58.8%
20 °C
70%
42 M
8F
11 M
3F(FA
exposure);
9M
exposure)
8M
10 M
6M
1 F
14 M
1 F
19 M
7F
Competitive
bicyclists
Adult, healthy
NS
Aerobically fit
Well-trained
distance
runners
Well-trained
cyclists
Highly fit
endurance
cyclists
Active
nonathletes
Small decrements in FEVj at 0.16 ppm with larger decrements at
0.24 ppm 03.
Decrease in FVC, FEVj, VT, and SRaw and increase in f with
Oj exposure compared with FA; total cell count and LDH increased in
isolated left main bronchus lavage and inflammatory cell influx occurred
with 03 exposure compared to FA exposure.
03 effective dose was significantly related to pulmonary function
decrements and exercise ventilatory pattern changes; multiple regression
analysis showed that 03 concentration accounted for the majority of the
pulmonary function variance.
Decrease in FVC, FEVj, and FEF25_75% with 0.20 and 0.35 ppm 03
exposure compared with FA; Vj decreased and f increased with
continuous 50-min 03 exposures; three subjects unable to complete
continuous and competitive protocols at 0.35 ppm 03.
Decrease in FVC, FEVj, FEF25_75%, and MW with 0.21 ppm 03
compared with FA exposure.
No significant differences in the effects of albuterol on metabolic data,
pulmonary function, airway reactivity, and exercise performance vs.
placebo; decrease in VEmax during 03 conditions.
FVC, FEVlt and MW all decreased with 0.25 ppm 03 exposure
compared with FA.
Avol et al. (1984)
Aris et af. (1993a)
Adams et al. (1981)
Adams and Schelegle
(1983)
Folinsbee et al. (1984)
Gong et al. (1988)
Folinsbee et al. (1986)
-------
TABLE 7-1 (cont'd). CONTROLLED HUMAN EXPOSURE TO OZONE3
CT Ozone
5. Concentration
1
VO
VO
U)
-J
00
d
6
o
1
$
ppm
Healthy
0.00
0.25
0.00
0.30
0.00
0.35
0.00
0.37
0.50
0.75
0.00
0.40
0.00
0.40
Hg/m
Exposure
Duration and Exposure
Activity Conditions
Number
and
Gender of Subjects
Subjects Characteristics
Observed Effect(s)
References
Exercising Adult Subjects (cont'd)
0
490
0
588
0
686
0
726
980
1,470
0
784
0
784
1 h CE NA
cycle
ergometer
(VE= 2
30 L/min/m
BSA)
1 h CE cycle NA
ergometry
(mean Vg =
60L/min)
1 h CE cycle Tdb = 21-25
ergometry °C
(mean VE = RH = 45-60%
60L/min)
2 h IE cycle
ergometry
(VE = 2.5x
rest)
2 h IE Tdb = 22 °C
treadmill RH = 40%
exercise
( VE = 50 to
75 L/min)
1 h CE NA
treadmill
exercise;
( VE =
20 L/min/m2
BSA)
5 M Young, healthy
2F NS
5 M Normal
14 M Moderately fit,
young, healthy
adults
20 M Young, healthy
8 F adults
8 M Young, healthy
NS
20 M Young, healthy
NS
12 A % decrease in FEVj . Significant elevation of sub stance P and
8-epi-PGFj in segmental airway washing, but not bronchoalveolar
lavage fluid.
Decrease in FVC and FEVj, and increase in SR,,W 1 h post-Oj
exposure; increase in percent neutrophils at 1, 6, and 24 h post-Oj
exposure compared with FA in first aliquot "bronchial" sample.
Neutrophils peaked at 6 h post-Oj in "bronchial" sample. Percent
neutrophils elevated at 6 and 24 h post-Oj in pooled aliquots.
Significant decreases in FVC and FEVj with 03 exposure compared to
FA exposure; FVC and FEVj decreases with 03 exposure were
significantly attenuated with indomethacin compared to no drug and
placebo; SRgW increases were not effected by indomethacin.
Decrease in FVC with 0.50 ppm and FEVj with 0.50 and 0.75 ppm
03 compared to FA; decrease in ^25% VC ""^ "-37 and 0.75 and
VjQ^vc with 0.37, 0.50, and 0.75 ppm Oj exposure compared to
FA.
Decreases in FVC, FEVj, VT, and TLC, and increases in SR,,W and f
with 03 exposure compared with FA. Atropine pretreatment abolished
O3-induced increase in SRaw and attenuated FEVj and Ff-F25-7S%
response.
VT fell by 25 % , and O3 uptake efficiency in the lower respiratory
tract fell by 9% during 03 exposure.
Hazbunetal. (1993)
Schelegleetal. (1991)
Schelegleet al. (1987)
Silverman et al. (1976)
Beckett et al. (1985)
Gerrity et al. (1993a)
-------
TABLE 7-1 (cont'd). CONTROLLED HUMAN EXPOSURE TO OZONE8
g Ozone
g. Concentration11 Exoosure
Cl>
t£ PPm
Duration and
fig/m Activity
Exposure
Conditions
Number
and
Gender of Subjects
Subjects Characteristics0 Observed Effect(s)
References
£O Healthy Exercising Adult Subjects (cont'd)
0.00
0.40
0 2hIE
784 (4 x 15 min
heavy treadmill
exercise
NA
1 1 M Young, healthy No correlation between pulmonary function and inflammatory endpoints
NS measured in BAL fluid obtained 18 h after exposure; increase in percentage of
PMNs, total protein, albumin, IgG, and neutrophil elastase; decrease in
percentage of macrophages with Oj exposure compared to FA exposure.
Koren et al. (1989a)
35 L/min/m
BSA])
0.00 0
0.40 784
O 0.00 0
vb 0.40 784
0
j> 0.00 0
2 0.40 784
i
5 0.00 0
^ 0.40 784
.- 0.60 1,176
1
a
g
2 h IE Tdb = 22 °C
(4 x 15 min RH = 40%
heavy treadmill
exercise
35 L/min/m
BSA])
2 h IE Tdb = 22 °C
(4 X 15 min RH = 50%
bicycle
ergometry
[VE =
30 L/min/m
BSA])
1 h CE Tdb = 22 °C
(treadmill RH = 40%
exercise; Vc =
20 L/min/m
BSA)
2hIE Tdb = 71.5 "C
(4 x 15 min RH = 55%
cycle ergometry
at 100 W for
males and 83 W
for females)
10 M Young, healthy PMN, PGE2, and IL-6 were higher in BAL fluid obtained 1 h post-Oj exposure
NS than 18 h; fibronectin and urokinase-type plasminogen activator were higher 18
h post-Oj exposure than 1 h.
13 M NS Indomethacin pretreatment and Oj exposure resulted in a significantly smaller
decrease in FVC and FEVj than 03 exposure alone; airway hyperresponsiveness
was not significantly affected by indomethacin pretreatment.
22 M Young, healthy Significant decreases in FVC, FEV,, FEVj/FVC, and FEF25_75%. The half-
NS width of an expired aerosol bolus was significantly increased, suggesting an
ozone-induced change in small airway function.
7 M Healthy Increase in airway responsiveness to methacholine challenge, in mean percentage
3 F NS of neutrophils, and in PGF2a, TXB2, and PGE2 concentrations measured in
BALF 3 h after 0.40- and 0.60-ppm 03 exposure compared with FA exposure.
Koren et al. (1991)
Ying et al. (1990)
Keefe et al. (1991)
Seltzer et al. (1986)
-------
TABLE 7-1 (cont'd). CONTROLLED HUMAN EXPOSURE TO OZONE3
o>
u>
Ozone
Concentration1" Exoosure
Duration and Exposure
ppm fig/m Activity Conditions
Number
and
Gender of Subjects
Subjects Characteristics0 Observed Effect(s)
References
Healthy Exercising Adult Subjects (cont'd)
0.00 0 2 h IE Tdb = 21 °C
0.50 980 (4 x 15 min RH = 40%
treadmill
exercise;
40L/min)
0.00 0 2 h IE NA
0.75 1,470 (4 x 15 min
light [50 W]
cycle
ergometry)
1 8 M Healthy, young Decrease in VC, VT, and maximal transpulmonary pressure, and increase
adults in SR^ and f with O3 exposure compared to FA exposure; lidocaine
inhalation partially reversed the decrease in VC.
13 M 4 light S, Decrease in FVC, FEV^ ERV, 1C, and FEF50% after 1 h exposure to
9 NS 0.75 ppm 03; decrease in VO2max, VTmax, VEmax, maximal
workload, and heart rate following 0.75-ppm 03 exposure compared with
FA.
Hazucha et al. (1989)
Folinsbeeet al. (1977b)
aSee glossary of terms and symbols for abbreviations and acronyms.
Grouped by rest and exercise; within groups listed from lowest to highest 03 concentration.
Age range in years or as mean + SEM.
-------
1 these early studies were that the exercise-induced increase in minute ventilation accentuated
2 the observed pulmonary response at any given O3 concentration and lowered the minimum
3 O3 concentration at which significant pulmonary responses were observed. Subsequently, the
4 interaction between O3 concentration and minute ventilation was examined by using similar
5 intermittent exercise protocols in which both O3 concentration and level of minute ventilation
6 were varied.
7 Silverman et al. (1976) and Folinsbee et al. (1975) exposed a group of 20 males and
8 8 females to 0.37, 0.50, or 0.75 ppm for 2 h while resting or exercising intermittently. The
9 IE protocol used alternated 15 min of rest with 15 min of exercise, sufficient to increase the
10 minute ventilation (VE) by a factor of 2.5 over the value at rest. The submaximal exercise
11 responses of the subjects were tested postexposure using a three-stage cycle ergometer test,
12 with loads adjusted to 45, 60, and 75% of maximum oxygen uptake (VO2max) (Folinsbee
13 et al., 1975). Pulmonary function responses were related to the total inhaled dose or the
14 "effective dose" of O3 calculated as the product of concentration, exposure time, and
15 ventilation. Neither submaximal exercise oxygen uptake (VO2) nor VE were significantly
16 affected by any level of O3 exposure. However, a significant increase in respiratory
17 frequency (f), and a significant decrease in tidal volume (VT) at the 75% VO2max workload,
18 was observed. The relationship between the effective dose of O3 and the mean percent
19 change in selected measures of lung function was analyzed using linear regression. Forced
20 vital capacity, maximum expiratory flow at 25 and 50% of FVC (Vmax25% and Vmax50%.
21 respectively), and FEVj were found to have a significant linear correlation with the effective
22 dose. The description of the relationship between O3 pulmonary function decrements and
23 effective dose was apparently improved by the use of a second-order polynomial model in
24 which effective dose was used as the independent variable.
25 Though the investigations of Silverman et al. (1976) and others (Bates et al., 1972;
26 Hackney et al., 1975; Hazucha et al., 1973) clearly demonstrate {he potentiating effects of
27 exercise on O3 responses, the level of exercise used in these studj|$5 was low, requiring
28 increases in VE of only 2 to 2.5 times resting, a level of exercisp lower than walking at
29 5.5 km/h (DeLucJa and Adams, 1977). In order to address this concern, DeLucia and
30 Adams (1977) exposed six healthy nonsmoking male subjects on 12 separate occasions to
31 FA, 0.15, and 0.30 ppm O3 for 1 h, while at rest, and while exercising continuously at
December 1993 7_n DRAFT-DO NOT QUOTE OR CITE
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1 workloads that required 25, 45, and 65% of the subjects' VO2max. They observed a
2 significant time-dependent increase in f during the 65% VO2max, 0.30-ppm O3 exposure and
3 a significant decrease in FEVj and forced expiratory flow at 25 to 75 % of FVC (FEF25_75%)
4 immediately following this same exposure.
5 These initial studies, which clearly demonstrated the potentiating effects of exercise on
6 human responses to acute O3 exposure, provided the impetus for a series of studies (Adams
7 et al., 1981; Folinsbee et al., 1978; McDonnell et al., 1983; Kulle et al., 1985; Linn et al.,
8 1986) designed to more accurately define O3 dose-response relationships. These
9 investigations utilized both IE (Folinsbee et al., 1978; McDonnell et al., 1983) and CE
10 (Adams et al., 1981) of varying intensity. Folinsbee et al. (1978) exposed four groups of
11 10 subjects each to FA, 0.10, 0.30, and 0.50 ppm O3 for 2 h. One group was exposed
12 while at rest and the other three groups were exposed while performing IE at levels requiring
13 a ventilation of 30, 50, or 70 L/min. These combinations of ventilation and O3 concentration
14 resulted in a range of total inhaled dose of 0.00 to 4.41 mg O3. Adams et al. (1981)
15 exposed eight trained male subjects to FA, 0.20, 0.30, and 0.40 ppm O3 while exercising
16 continuously at two different workloads (35 and 62% of VO2max) for durations ranging
17 from 30 to 80 min. Each subject completed all 18 protocols with at least 3 days between
18 each. The findings from these two studies confirmed that significant pulmonary responses
19 occurred at 0.30 ppm when subjects exercised at moderately heavy workloads. It was further
20 demonstrated, by multiple regression analysis, that the O3 effective dose was a better
21 predictor of response than O3 concentration, VE, or duration of exposure, alone. Multiple
22 regression analysis also revealed that the majority of variance for pulmonary function
23 responses was accounted for by O3 concentration, followed by VE. In the Adams et al.
24 (1981) study, in which both workload and duration of exposure were varied, duration of
25 exposure was observed to be the poorest predictor of response for all parameters analyzed.
26 However, the minor impact of changes in exposure duration could have been an artifact of
27 the limited combinations of ventilation and durations of exposure used by these investigators.
28 McDonnell et al. (1983) conducted a study with the primary purpose of discerning the
29 lowest concentration of O3 at which group mean decrements in pulmonary function occur in
30 heavily exercising healthy men. In order to determine a concentration-response relationship,
31 six groups of subjects (n = 20 to 29) were exposed to either an FA control or one of five
December 1993 7-12 DRAFT-DO NOT QUOTE OR CITE
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1 03 concentrations (0.12, 0.18, 0.24, 0.30, or 0.40 ppm) at a VE of 67 L/min and exposure
2 duration of 2.5 h (15-min rest, 15-min exercise. These investigators observed small
3 significant changes in FVC, FEVb FEF25_75%, and cough at 0.12 ppm O3 and concentration-
4 dependent responses in all variables measured (FVC, FEV^ FEF25_75%, specific airway
5 resistance [SR^], f, VT, and subjective symptoms) at O3 concentrations > 0.24 ppm.
6 Kulle et al. (1985) also conducted a similar study on healthy, nonsmoking men
7 performing intermittent exercise at a VE of 70 L/min for an exposure duration of 2 h,
8 (16-min rest, 14-min exercise). Twenty subjects were exposed to an FA control or one of
9 four O3 concentrations (0.10, 0.15, 0.20, or 0.25 ppm). These investigators observed a
10 significant concentration x time interaction at 0.15 ppm O3 for FVC, FEVls FEF25_75%, and
11 in all variables measured (FVC, FEVj, FEF25_75%, SR^, f, VT, and subjective symptoms)
12 at O3 concentrations greater than 0.15 ppm.
13 Linn et al. (1986) exposed 24 healthy, well-conditioned male subjects (18 to 33 years of
14 age) for 2 h to 0.00, 0.08, 0.10, 0.12, 0.14, or 0.16 ppm O3, using an intermittent exercise
15 protocol (15-min rest, 15-min exercise; VE = 68 L/min) combined with an ambient heat
16 stress (32 °C and 38% relative humidity). They observed no statistically significant changes
17 in forced expiratory endpoints and symptoms after exposure to O3 concentrations from
18 0.08 to 0.14 ppm. These authors observed a small (-2.3%) but significant (p < 0.05)
19 reduction in FEV, following the 2-h 0.16-ppm O3 exposure that was not associated with
20 symptoms of respiratory discomfort.
21 More recently, Seal et al. (1993b) examined whether gender or race differences in
22 responsiveness to O3 exist. The authors exposed 372 white and black males and females
23 (n > 90 in each gender-race group) once for 2.33 h to 0.0, 0.12, 0.18, 0.24, 0.30, or
24 0.40 ppm O3 using an intermittent exercise protocol (15-min rest, 15-min exercise;
* 9
25 VE = 25 L/min/m BSA). Statistical analysis (nonparametric two-factor analysis of
26 variance) of the percent changes from baseline for FEVj, SRaw, and cough responses
27 demonstrated no significant differences in responsiveness to O3 between the race-gender
28 groups studied. Changes in FEVl5 SR^, and cough were first noted at 0.12, 0.18, and
29 0.18 ppm O3, respectively, for the group as a whole. It is difficult to compare the results
30 from this study with others that have examined the 0$ concentration-response relationship in
31 healthy adult males because the authors did not present a separate analysis of male responses.
December 1993 7_13 DRAFT-DO NOT QUOTE OR CITE
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1 For further evaluation of the influence of gender and race on O3 responsiveness, see
2 Section 7.2.1.3.
3 The observation of significant decrements in pulmonary function in heavily exercising
4 healthy subjects at O3 concentrations of 0.20 ppm and lower has been confirmed by
5 numerous investigators (Adams and Schelegle, 1983; Avol et al., 1984; Folinsbee et al.,
6 1984; Gong et al., 1986) who utilized 1-h continuous heavy exercise exposure protocols.
7 Adams and Schelegle (1983) and Folinsbee et al. (1984) observed significant decrements in
8 FVC and FEVj in well-trained subjects exposed to 0.20 ppm O3 while exercising with a
9 VE of approximately 80 L/min. Avol et al. (1984) observed small but significant decrements
10 in FVC and FEVt in a group of 50 competitive cyclists (42 males, 8 females) exposed to
11 0.16 ppm O3 while exercising with a VE of 57 L/min in combination with added heat stress
12 (32 °C). Similarly, Gong et al. (1986) observed modest but significant decrements in FVC
13 and FEVj in a group of 17 top-caliber endurance cyclists exposed to 0.12 ppm O3 while
14 exercising at approximately 70% of their VO2max (mean VE = 89 L/min) with an added
15 heat stress (32 °C). In addition to the above studies that used continuous exercise, Schelegle
16 and Adams (1986) observed significant reductions in FVC and FEVj and increased
17 symptoms of respiratory discomfort following exposure to 0.18 ppm O3, but not 0.12 ppm
18 O3, in a group of competitive endurance athletes exposed while performing a competitive
19 simulation consisting of a 30-min warm-up followed by a 30-min competitive bout (mean
20 VE over entire protocol = 87 L/min).
21 The results of the studies reviewed above demonstrate that the minimum
22 O3 concentrations between 0.12 to 0.18 ppm are required to elicit significant pulmonary
23 function decrements and increased symptoms in healthy young adult subjects exposed while
24 performing moderate to severe IE and CE of from 1 to 3 h duration.
25 Retrospective analysis by Hazucha (1987) confirmed the previously reported (Adams
26 et al., 1981; Folinsbee et al., 1978) dominant role that O3 concentration plays in determining
27 O3-induced responses. Hazucha (1987) analyzed data from studies that utilized intermittent
28 exercise protocols of 2 h in duration. While controlling for ventilation, he found that the
29 data best fit a model that was a quadratic function of O3 concentration. Based on his
30 analysis, Hazucha (1987) also concluded that he could not define an O3 concentration below
31 which no pulmonary function response would be elicited.
December 1993 7-14 DRAFT-DO NOT QUOTE OR CITE
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1 Intersubject Variability, Individual Sensitivity, and the Association Between Responses
2 Bates et al. (1972) noted that variation in sensitivity and response was evident for
3 various symptoms and pulmonary functions they assessed following O3 exposure. This
4 observation of large intersubject variability in response to O3 has also been reported by
5 numerous other investigators (Adams et al., 1981; Folinsbee et al., 1978; McDonnell et al.,
6 1983; Kulle et al., 1985) and is illustrated by data from Kulle et al. (1985) plotted in
7 Figure 7-1. The description of the factors that contribute to intersubject variability is
8 important for the understanding of individual responses, mechanisms of response, and health
9 risks associated with acute exposures. The effect of this large intersubject variability on the
10 ability to predict individual responsiveness to O3 was recently demonstrated by McDonnell
11 et al. (1993). These investigators analyzed the data of 290 white male subjects (18 to
12 32 years of age) who inhaled either 0.00, 0.12, 0.18, 0.24, 0.30, or 0.40 ppm O3 for
13 2 h while performing an IE protocol (VE = 35 L/min/m2 BSA) to identify personal
14 characteristics (i.e., age, height, baseline pulmonary functions, presence of allergies, and
15 past smoking history) that might predict individual differences in FEVj response. Of the
16 personal characteristics studied, only age contributed significantly to intersubject
17 responsiveness (younger subjects being more responsive), accounting for 4% of the observed
18 variance. Interestingly, O3 concentration accounted for only 31 % of the variance, clearly
19 demonstrating the importance of as yet undefined intrinsic individual characteristics that
20 determine responsiveness to O3.
21 McDonnell et al. (1985b) examined the reproducibility of individual responses to
22 O3 exposure in healthy human subjects exposed twice, with from 21 to 385 days separating
23 exposures (mean = 88 days). This investigation was conducted in order to determine
24 whether the observed intersubject variability is primarily due to real differences in
25 O3 responsiveness among subjects, or whether it can be accounted for by other sources of
26 variability. The authors examined the reproducibility of FVC, FEV], FEF25.75%, SR^,
27 cough, shortness of breath (SB), pain on deep inspiration (PDI), VT, and f responses induced
28 by O3 exposure to concentrations ranging from 0.12 to 0.40 ppm. Reproducibility was
29 assessed using the intraclass correlation coefficient (R), which directly measures the
30 reproducibility of responses by incorporating into a single measure all the information
31 contained in the correlation coefficient, slope, and intercept obtained in linear
December 1993 7.15 DRAFT-DO NOT QUOTE OR CITE
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22-
20-
18
16 -|
J4
12-1
k*H
C
'c 10
o
1 M
*
DC 6-
4 _
2-
0-
-2-
0.10 0.15 0.20
Ozone (ppm)
0.25
Figure 7-1. Individual dose response curves for five separate subjects exposed to 0.10,
0.15, 0.20, and 0.25 ppm O3 for 2 h with moderate intermittent exercise.
Illustrates the wide variability in responsiveness to O3 from individual to
individual.
Source: Kulle et al. (1985).
1 regression analysis. Similar to the more routinely used correlation coefficient, R is equal to
2 one when two identical measurements occur in the same subject; and the "worst" possible
3 coefficient is equal to l/(n — 1), which approaches zero for a large n. The ranking of most
4 to least reproducible for the responses studied was FVC (R = 0.92), FEV^ (R = 0.91),
5 FEF25.75% (R = 0.83), cough (R = 0.77), SB (R = 0.60), SRaw (R = 0.54), PDI
6 (R = 0.37), f (R = -0.20), and VT (R = -0.03). The value of R was significantly
7 different from zero for FVC, FEV,, FEF25_75%, cough, SB, and SR^. McDonnell et al.
8 (1985b) concluded that the reported large intersubject variability in magnitude of response
December 1993
7-16
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1 was due to large differences in the intrinsic responsiveness of individual subjects to
2 O3 exposure. However, the factors that contribute to this large intersubject variability
3 remain undefined.
4 The examination of intersubject variability is complicated by a poor association between
5 the various O3 responses. In their study investigating O3 dose-response relationships,
6 McDonnell et al. (1983) observed very low correlation between changes in SRaw and FVC
7 (r = -0.16) for 135 subjects exposed to O3 concentrations ranging from 0.12 to 0.40 ppm
8 for 2.5 h.
9
10 Mechanisms of Acute Pulmonary Responses
11 The pulmonary responses observed during and following acute exposure to O3 at
12 concentrations between 0.10 and 0.50 ppm in normal healthy human subjects include
13 decreases in TLC, 1C, FVC, FEVl5 FEF25_75%, and VT; and increases in SR^, f, and
14 airway responsiveness. Ozone exposure has also been shown to result in the symptoms of
15 cough, PDI, SB, throat irritation, and wheezing. When viewed as a whole, changes in these
16 specific parameters can be categorized into four general responses, including alterations in
17 lung volumes, airway caliber, bronchomotor responsiveness, and symptoms. The absence of
18 consistent associations between the various responses from individual to individual suggests
19 that the functional responses observed are the result of multiple interactions within the
20 respiratory tract. These interactions may be the result of O3 action on the biochemical,
21 anatomical, and physiological systems of the respiratory tract. In turn, these factors
22 determine O3 dose distribution and the resulting cellular and reflex responses.
23 Bates et al. (1972) observed that the most significant decrement in pulmonary function
24 was the reduction in the transpulmonary pressure at maximal inspiratory volume without a
25 concomitant decrease in static compliance. This would suggest an inhibition of maximal
26 inspiratory effort after O3 exposure and result in reductions in 1C. These authors speculated
27 that this inhibition is an early result of stimulation of rapidly adapting pulmonary stretch
28 receptors, or "irritant receptors", located hi the major bronchi. Since 1972, when this initial
29 hypothesis was published, numerous studies have examined the underlying mechanisms
30 leading to the functional responses observed hi human subjects. These mechanistic studies
31 have used both animal models and human subjects. This discussion of mechanisms will
December 1993 747 DRAFT-DO NOT QUOTE OR CITE
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1 focus on studies that used human subjects, but also will cover those animal studies that have
2 direct relevance to O3-induced functional responses.
3 The acute inhalation of ambient concentrations of O3 by healthy human subjects has
4 been shown to result in a dose-dependent increase in airway resistance (Folinsbee et al.,
5 1978; McDonnell et al., 1983; Kulle et al., 1985; Seal et al., 1993b). This O3-induced
6 increase in airway resistance has been shown to be poorly correlated with changes in forced
7 expiratory endpoints (McDonnell et al., 1983). Ozone-induced increases in airway resistance
8 have a rapid onset (Beckett et al., 1985) compared with the gradual development of
9 decrements in forced expiratory endpoints (Kulle et al., 1985). Ozone-induced increases in
10 airway resistance also appear to be greater in atopic subjects as a group (Kreit et al., 1989;
11 McDonnell et al., 1987), although this does not appear to be the case for O3-induced
12 decrements in FVC and symptoms. Taken together, these observations suggest that different
13 pathways lead to O3-induced decrements in 1C and to O3-induced increments in airway
14 resistance.
15 Increases in airway resistance induced by O3 have been shown to be blocked by
16 atropine sulfate pretreatment in human subjects (Beckett et al., 1985; Adams, 1986). This
17 inhibition suggests that the release of acetylcholine from parasympathetic postganglionic
18 fibers that innervate airway smooth muscle plays a role in this response. However, the
19 observation that acute O3 inhalation also results in a hyperresponsiveness to methacholine, a
20 cholinergic agent (Holtzman et al., 1979), suggests the possibility that acute O3 exposure can
21 also increase the sensitivity of airway smooth muscle to acetylcholine independent of a reflex
22 mechanism involving cholinergic postganglionic nerves. The role that an increase in airway
23 smooth muscle sensitivity to the endogenous release of acetylcholine might play in
24 O3-induced increases in airway resistance has not been studied.
25 Analyses by Colucci (1983) have suggested that the increase in airway resistance is not
26 as large as would be expected when O3 exposure is combined with moderate to heavy
27 exercise. However, the observation that circulating epinephrine levels increase as a function
28 of the relative workload in exercising human subjects (Galbo, 1983; Warren and Dalton,
29 1983) suggests that stimulation of airway smooth muscle beta-adrenoreceptors may counteract
30 airway smooth muscle contraction induced by O3 exposure. The observations by Beckett
December 1993 7-18 DRAFT-DO NOT QUOTE OR CITE
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1 et al. (1985) that the beta-agonists abolish O3-induced bronchoconstriction is consistent with
2 this possibility.
3 Another question to be addressed with regard to O3-induced increases in airway
4 resistance is where along the airway (central versus peripheral airways) is the increase in
5 resistance produced? Studies of acutely and subchronically exposed animals have
6 demonstrated tissue damage in the central acinar region (Castleman et al. , 1977; Mellick
7 et al., 1977), as well as increases in peripheral resistance and reactivity (Gertner et al.,
8 1983a,b,c; Beckett et al., 1988). The observations by Kreit et al. (1989) and Keefe et al.
9 (1991) of small significant decreases in the ratio of FEVj/FVC in normal subjects exposed to
10 0.40 ppm O3 suggest that O3 exerts an effect on the small peripheral airways of humans.
11 Keefe et al. (1991) examined this possibility using an inhaled aerosol bolus dispersion
12 technique in 22 healthy, nonsmoking male subjects exposed to 0.40 ppm O3 for 1 h using a
13 CE protocol ( VE = 20 L/min/m BSA). The bolus dispersion technique is not dependent
14 upon vital capacity maneuvers and compares the profile of a bolus of small (0.5 to 1.0
15 aerosol particles injected into the inspired airstream (at a fixed lung volume) with the profile
16 of the bolus during expiration. Dispersion of the bolus during expiration can be affected by
17 increases in turbulence within the airway, the development of asymmetries in ventilation due
18 to unequal regional time constants within the lung, and an increase in aerosol deposition in
19 the small airways. Keefe et al. (1991) observed that O3 exposure in their subjects resulted in
20 a significant increase in dispersion of an aerosol bolus (without an increased aerosol
21 deposition) that was not correlated with changes in SRaw. These findings suggest that
22 exposure to 0.40 ppm O3 under the conditions of this experiment results in changes in small
23 airway function that are not detectable by more conventional techniques.
24 Ozone-induced alterations in ventilatory pattern have been observed in exercising dogs
25 (Lee et al., 1979) and humans (Adams et al., 1981; Folinsbee et al., 1978; McDonnell et al.,
26 1983; Kulle et al., 1985). In exercising humans, O3 exposure has been shown to result in a
27 decrease in VT and an increase in f in the absence of any change in VE. A rapid shallow
28 breathing pattern is consistent with the maintenance of an appropriate ventilation with a
29 reduced tidal volume. Reduction of tidal volume is probably related to the reduction of
30 inspiratory capacity and is anecdotally related to reduction in breathing discomfort caused by
31 pain on deep inspiration.
December 1993 749 DRAFT-DO NOT QUOTE OR CITE
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1 Lee et al. (1979), who produced a reversible vagotomy by cooling the vagus nerves to
2 0 °C, abolished the rapid shallow breathing induced by O3 inhalation in conscious dogs.
3 More recently, Schelegle et al. (1993) have shown in anesthetized dogs exposed to O3 that
4 cooling the cervical vagus nerves to 7 °C did not abolish the observed O3-induced rapid
5 shallow breathing pattern and bronchoconstriction, but cooling the vagus nerves to 0 °C did
6 abolish both the rapid shallow breathing and the bronchoconstriction. These findings suggest
7 that O3 stimulates nonmyelinated C fiber afferents arising from the lung, whose conduction is
8 not totally blocked at 7 °C, but is totally blocked at 0 °C. This conclusion is consistent with
9 the findings of Coleridge et al. (1993) that bronchial C fibers are the only receptors that are
10 stimulated directly during O3 inhalation in anesthetized dogs. If similar bronchial C fibers
11 were stimulated or sensitized in humans exposed to O3, this could explain the O3-induced
12 rapid shallow breathing observed during exercise, as well as the subjective symptoms
13 associated with taking a deep inspiration.
14 Hazucha et al. (1989) exposed 11 healthy normal volunteers to FA and 0.50 ppm
15 O3 for 2 h while performing moderate intermittent exercise. Ozone exposure induced a
16 significant decrement in FVC, which was associated with a marked fall in inspiratory
17 capacity without an increase in residual volume. Spraying of the upper airway with lidocaine
18 aerosol by these subjects was immediately followed by restitution of FVC toward control
19 values. Hazucha et al. (1989) concluded that O3 inhalation stimulates lidocaine-sensitive
20 tracheal and laryngeal airway receptors, which leads to an involuntary inhibition of full
21 inspiration, a reduction in FVC, and a concomitant decrease in maximal expiratory flow rates
22 in humans.
23 The airway afferents blocked by lidocaine in the Hazucha et al. (1989) investigation
24 remain undefined. However, it seems likely that the lung afferents involved are the same
25 ones that result in O3-induced rapid shallow breathing in dogs (i.e., bronchial C fibers).
26 When stimulated with exogenous chemicals in animal experiments, bronchial C fibers induce
27 a reflex apnea (Coleridge and Coleridge, 1986). In dogs, this reflex apnea involves the
28 inhibition of inspiratory neurons, expiratory neurons, and y- and a-motoneurons in the
29 intercostal nerves (Koepchen et al., 1977; Schmidt and Wellhoner, 1970). Such a reflex
30 response in humans would explain the reflex inhibition of maximal inspiration consequent to
31 acute O3 exposure.
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1 Data consistent with an O3-induced stimulation of bronchial C fibers in human subjects
2 has recently been published by Hazbun et al. (1993). These investigators observed a
3 significant increase in substance P, the neurotransmitter released from the afferent endings of
4 bronchial C fiber during excitation, in segmental airway washings of seven (2 female/5 male)
5 healthy, nonsmoking subjects after a 1 h CE (VE = 30 L/min/m BSA) exposure to
6 0.25 ppm O3. Substance P was not elevated in bronchoalveolar lavage (BAL) fluid after air
7 exposure. In addition, the segmental airway substance p levels were significantly correlated
8 (r = 0.89; p < 0.05) with an elevated airway concentration of 8-epi-prostaglandin F2a, a
9 marker of oxidative free radical reactions. These results are consistent with (1) an increased
10 release of substance P secondary to an increased discharge of bronchial C fibers induce by
11 O3 inhalation, and/or (2) an O3-induced inhibition of neutral endopeptidase, the enzyme that
12 degrades substance P, within the airways.
13 Lung C fibers have been shown to be stimulated by prostaglandin £2 and other lung
14 autacoids (Coleridge et al., 1978, 1976). Interestingly, Schelegle et al. (1987),
15 Eschenbacher et al. (1989), and Ying et al. (1990) have shown that pretreatment with the
16 cyclooxygenase inhibitor indomethacin reduces—and in some subjects totally abolishes—
17 O3-induced pulmonary function decrements in human subjects. Schelegle et al. (1987)
18 examined whether O3-induced pulmonary function decrements could be inhibited by the
19 prostaglandin synthetase inhibitor indomethacin in healthy human subjects. Fourteen college-
20 age males completed six 1-h exposure protocols consisting of no drug, placebo, and
21 indomethacin (Indocin SR, 75 mg every 12 h for 5 days) pretreatments, with filtered air and
22 O3 (0.35 ppm) exposure within each pretreatment. Pretreatments were delivered weekly in
23 random order in a double-blind fashion. Exposures consisted of 1 h exercise on a cycle
24 ergometer with work loads set to elicit a mean VE of 60 L/min. Statistical analysis revealed
25 significant (p < 0.05) effects for FVC and FEVj across pretreatment, with no drug versus
26 indomethacin and placebo versus indomethacin comparisons being significant. These
27 findings suggest that cyclooxygenase products of arachidonic acid, which are reduced by
28 indomethacin inhibition of cyclooxygenase, play a role in the development of pulmonary
29 function decrements. These and similar findings by Eschenbacher et al. (1989) and Ying
30 et al. (1990) suggest that the release of some cyclooxygenase product consequent to
31 O3 inhalation plays a role in O3-induced pulmonary function decrements. This idea is
December 1993 7_2i DRAFT-DO NOT QUOTE OR CITE
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1 supported by the findings of Koren et al. (1991), who obtained a positive correlation between
2 O3-induced pulmonary function decrements and the level of prostaglandin £2 in BAL fluid
3 collected within 1 h after the end of exposure in human subjects who varied greatly in
4 O3 responsiveness.
5 The release of cyclooxygenase products of arachidonic acid from injured airway
6 epithelium can thus be viewed as a link in a cascade of events, which begins with the initial
7 reaction of O3 with the tissues and ends with the observed pulmonary function responses.
8 The apparent components of this chain of events include factors that influence (1) O3 delivery
9 to the tissue (i.e., the inhaled concentration, breathing pattern, and airway geometry);
10 (2) O3 reactions with components in airway surface liquid and/or epithelial cell membranes;
11 (3) local tissue responses, including injury and inflammation; and (4) stimulation of neural
12 afferents (bronchial C fibers) and the resulting reflex responses. More studies need to be
13 conducted to determine how each event in this cascade contributes to the pulmonary
14 responses induced by acute O3 inhalation.
15 The influence that individual responsiveness has on this cascade of events has not been
16 determined; however, recent data suggest that individual O3 responsiveness may feed back
17 and influence the distribution of O3 dose within the lung. Gerrity et al. (1993a) tested the
18 hypothesis that O3-induced rapid shallow breathing helps to limit the dose of O3 reaching the
19 lower respiratory tract. They found that the degree of O3-induced rapid shallow breathing
20 (25% decrease in VT) was significantly correlated (p < 0.004) with a decrease in 03 uptake
21 efficiency of the lower respiratory tract measured using a fast responding O3 analyzer. This
22 observation may explain the recent data of Schelegle et al. (1991) and Aris et al. (1993a) that
23 suggest individual responsiveness to O3 as measured by FEVl decrements may be negatively
24 correlated with the number of neutrophils present in bronchoalveolar lavage samples. These
25 limited findings indicate that more studies are needed that examine the interrelationship
26 between the responsiveness to O3, the distribution of dose within the airway, and resulting
27 airway inflammation.
28
29
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1 7.2.1.2 Subjects with Preexisting Disease
2 Introduction
3 Ten studies (Konig et al., 1980; Linn et al., 1982a; Koenig et al., 1985; Linn et al.,
4 1983a; Linn et al., 1978; Solic et al., 1982; Kehrl et al., 1985; Superko et al., 1984;
5 Silverman, 1979; Kulle et al., 1984) examining the pulmonary responses to acute
6 O3 exposures of less than 3 h in patients with preexisting disease were discussed in the 1986
7 EPA criteria document (U.S. Environmental Protection Agency, 1986). This section
8 examines the effects of O3 exposure on pulmonary function in subjects with preexisting
9 disease by reviewing O3 exposure studies that utilized subjects with (1) chronic obstructive
10 pulmonary disease (COPD), (2) asthma, (3) allergic rhinitis, and (4) ischemic heart disease.
11 Due to their important health implications, all of the available studies are reviewed and
12 summarized in Table 7-2.
13
14 Subjects with Chronic Obstructive Pulmonary Disease
15 In five of the studies cited above, the C^-induced pulmonary function responses of
16 patients with mild to moderate COPD were examined (Konig et al., 1980; Linn et al., 1982a;
17 Linn et al., 1983a; Solic et al., 1982; Kehrl et al., 1985). No significant changes in
18 pulmonary function or symptoms were reported in any of the studies of the effects of O3 in
19 patients with COPD. Four of these studies (Linn et al., 1982a; Linn et al., 1983a; Solic
20 et al., 1982; Kehrl et al., 1985) examined the effects of O3 concentrations between 0.10 and
21 0.30 ppm O3 in 66 mild to moderate COPD patients using mild IE exposure protocols of 1 to
22 2 h duration. The total exercise time in all three of these studies was 30 min with intensity
23 being variable (exercise VE approximately 14 to 28 L/min). Linn et al. (1982a) observed a
24 small but significant reduction in arterial oxygen saturation in 25 mild to moderate COPD
25 patients at the end of the 0.12 ppm O3 exposure for 1 h (absolute mean difference = 1.3%,
26 p < 0.05). Similarly, Solic et al. (1982) observed a small reduction in arterial oxygen
27 saturation in 13 mild to moderate COPD patients at the end of a 0.20-ppm O3 exposure for
28 2 h (absolute mean difference = 0.48%, p < 0.008). In contrast, Kehrl et al. (1985) did
29 not find a significant effect on arterial oxygen saturation in 13 mild to moderate COPD
30 patients after exposure to 0.30 ppm O3 using the same IE exposure protocol used by Solic
31 et al. (1982). Similarly, Linn et al. (1983a) found no significant effect on arterial oxygen
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TABLE 7-2. OZONE EXPOSURE IN SUBJECTS WITH PREEXISTING DISEASE8
2 Ozone
§• Concentration ExpOsure
1
g
i
o
d
ppm
Duration and Exposure
ftg/m Activity Condition
Subjects with
0.00
0.12
0.00
0.18
0.25
0.00
0.20
0.00
0.30
0.00
0.41
0
236
0
353
490
0
392
0
588
0
804
Chronic Obstructive Pulmonary Disease
1 h IE Tdb = 25 "C
(2 x 15 min RH = 50%
light bicycle
ergometry)
1 h IE Tdb = 25 °C
(2 x 15 min RH = 50%
light bicycle
ergometry)
2 h IE Tdb = 22 °C
(4 x 7.5 min RH = 40%
light
treadmill
running)
2 h IE Tdb = 22 °C
(4 x 7.5 min RH = 40%
light
treadmill
running)
3 h daily Tdb = 22 °C
(1 X 15 min RH = 50%
light bicycle
ergometry
during each
exposure) for
5 days
Number
and
Gender of Subjects
Subjects Characteristics
18 M 8 smokers,
7 F 14 ex-smokers,
3 nonsmokers;
FEV^FVC =
32-66%
15 M 15 smokers,
13 F 11 ex-smokers,
2 nonsmokers;
FEVj/FVC =
36-75%
13 M 8 smokers,
4 ex-smokers,
1 nonsmoker;
productive
cough;
FEVj/FVC =
46-70%
13 M 9 smokers,
4 nonsmokers;
FEV^FVC =
37-65%
17 M All smokers;
3 F productive
cough;
FEVj/FVC =
56-82% and/or
FEV3/FVC =
75-93%
Observed Effect(s)
No significant changes in pulmonary function measurements;
small significant decrease in arterial O^ saturation.
No significant changes in pulmonary function measurements;
no significant change in arterial O^ saturation.
No significant changes in pulmonary function measurements;
small significant decrease in arterial 0% saturation.
No significant changes in pulmonary function measurements or
arterial 02 saturation.
Decrease in FVC and FEVj with 0.41 ppm 03 compared with
FA exposure.
References
Linn et al. (1982a)
Linn et al. (1983a)
Solic et al. (1982)
Kehrl et al. (1985)
Kulle et al. (1984)
Subjects with Heart Disease
0.00
0.20
0.30
0
392
588
40 min CE NA
treadmill
walking
6 M Coronary heart
disease with
angina pectoris
threshold
No significant changes in pulmonary function measurements,
exercise ventilatory pattern, oxygen uptake, or cardiovascular
parameters.
Superko et al. (1984)
-------
TABLE 7-2 (cont'd). OZONE EXPOSURE IN SUBJECTS WITH PREEXISTING DISEASE3
o
§
sr
8
t— *
VO
vO
U)
Ozone
h
Concentration"
ppm
Aig/m
Exposure
Duration and
Activity
Number
and
Exposure Gender of Subjects
Conditions Subjects Characteristics
Observed Effect(s)
References
Subjects with Allergic Rhinitis
0.00
0.18
0.00
0.50
0
353
0
980
2hffi
(4 X 15 min)
4h rest
NA 26 M History of
allergic rhinitis
Tdb = 20-24 °C 6 M History of
RH = 40-48% 6 F seasonal allergic
rhinitis; acute
response to nasal
challenge with
antigen
Increased respiratory symptoms, SRj,,,, and reactivity to histamine
with Oj exposure and decreased FVC, FEVj, and FEF25_75%
with Oj exposure compared to FA.
Increase in upper and lower respiratory symptom scores, cell
influx, epithelial cells with O3 exposure compared to FA; no
effect on acute allergic response to nasal antigen challenge
between 03 and FA exposure.
McDonnell et al.
(1987)
Bascom et al. (1990)
Adult Subjects with Asthma
-jj
N)
<-^i
O
§>
M
I
O
o
25
0
H
O
c
o
m
i
0.00
0.10
0.25
0.00
0.12
0.00
0.12
0
196
490
0
236
0
236
2 h light IE
(2 x 15 min
on treadmill,
vE =
10-20 L/min)
1 h rest
0.75 h m
vE =
30 L/min
(15 min rest,
15 min
exercise,
15 min rest)
followed by
15 min
exercise
inhaling
0.10 ppm
SO2
Tdb = 21 ° C; 7 M Stable mild
RH = 40% asthmatics with
FEVj > 70%
and
methacholine
responsiveness
NA 7 M, 8 F Never smoked,
asthmatics mild stable
asthmatics with
exercise-induced
asthma
Tdb = 22° C; 8 M, 5 F Asthmatics
RH = 75 % asthmatics, ages classified on
12 to 18 years basis of positive
clinical history
and
methacholine
challenge.
Asymptomatic at
time of study.
No significant differences in FEVj observed for Oj-filtered air
exposures or postexposure exercise challenge.
Exposure to 0.12 ppm Oj did not effect pulmonary function.
Preexposure to 0.12 ppm 03 at rest did not effect the magnitude
or time course of exercise-induced bronchoconstriction.
Filtered air followed by SOj and 03 alone did not cause
significant changes in pulmonary function. Ozone followed by
SOj resulted in significant decrease in FEVj (8%) and V^^Q^
(15%) and a significant increase in RT (19%).
Weymer et al. (1993)
Femandeset al.
(1993)
Koenig et al. (1990)
-------
TABLE 7-2 (cont'd). OZONE EXPOSURE IN SUBJECTS WITH PREEXISTING DISEASE8
«
8
3
H
I—*
v§
LtJ
^J
1
M
M
%^
J>
H
bL
O
2
^-«
o
O
d
g
a
IAJ
/~\
8
Ozone
Concentration Exposure
Duration and Exposure
ppm fig/m Activity Conditions
Adult Subjects with Asthma (cont'd)
0.00 0 1.5 h IE, Tdb = 22°C
0.12 236 VE = RH = 65%
0.24 472 25 L/min
0.00 0 6.5h/dayIE NA
0.12 236 (6 x 50 min)
(2 days of
exposure),
VE =
31 L/min
(asthmatic),
VE =
34 L/min
(healthy)
0.00 0 1 h rest NA
0.12 236
0.00 0 2 h IE Tdb = 31 °C
0.20 392 (4 x 15 min RH = 35%
at 2x rest
VE cycle
ergometry)
0.00 0 2 h rest NA
0.25 490
Number
and
Gender of
Subjects
4M, 4F
(nonasthmatics);
5M.5F
(asthmatics)
8M.7F
(nonasthmatics);
13 M, 17 F
(asthmatics)
4M
3F
20 M
2F
5M
12 F
Subjects
Characteristics
Physician-
diagnosed
asthma
confirmed with
methacholine
challenge test.
All nonsmokers
and
asymptomatic at
time of study.
Nine were
atopic.
Asthmatics
classified on
basis of positive
clinical history,
previous
physician
diagnosis, and
low PDjO' Mild
to severe
asthmatics.
Mild, stable
asthma
Physician
diagnosed
asthma;
6 smokers,
9 exsmokers,
7 nonsmokers
Nonsmoking
asthmatics
selected from a
clinical practice
Observed Effect(s)
No significant changes in pulmonary and nasal function
measurements in either asthmatics or nonasthmatics. Significant
increase in nasal lavage white cell count and epithelial cell
following 03 exposure in asthmatics only.
Significant increase in bronchial reactivity to methacholine in both
asthmatics and nonasthmatics. FEVj decreased 9.6% in
asthmatics and 1.7% in nonasthmatics, with difference not being
significant.
Increase in bronchial responsiveness to allergen; no change in
baseline airway function.
No significant changes in pulmonary function measurements;
significant blood biochemical changes.
No significant changes in pulmonary function measurements.
References
McBride et al. (1993)
Linn et al. (1993)
Molfino et al. (1991)
Linn et al. (1978)
Silverman (1979)
-------
TABLE 7-2 (cont'd). OZONE EXPOSURE IN SUBJECTS WITH PREEXISTING DISEASE3
0
1
cr
Ozone
Concentration1 Exoosure
ppm
Duration and Exposure
ftg/m Activity Conditions
Number
and
Gender of
Subjects
Subjects
Characteristics0 Observed Effect(s)
References
£O Adult Subjects with Asthma (cont'd)
0.00
0.40
0 2 h ffi Tdb = 22 °C
784 (4 x 15 min RH = 50%
cycle
ergometry)
4M, 5F
(normals);
4M, 5 F
(asthmatics)
Asthmatics as Decrease in FVC and 1C with 03 in asthmatics; increase in airway
diagnosed by a responsiveness to methacholine in asthmatics with 03 and FA;
physician; asthmatic subjects had significantly greater decreases in FEVj and
history of chest f^25-75% w*m ®3 exposure than did normal subjects.
tightness and
wheezing
Kreit et al. (1989)
Eschenbacher et al.
(1989)
Adolescent Subjects with Asthma
to
0
d
o
1
0.00
0.12
0.00
0.12
0.00
0.12
0.18
0 1 h rest Tdb = 22 °C
235 RH ^ 75%
0 1 h IE Tdb = 22 °C
235 (2 x 15 min RH a 75%
treadmill
walking at
mean Vg =
32.5 L/min)
0 40 min IE NA
235 (1 X 10 min
353 treadmill
walking at
mean VE =
32.5 1/min)
4M, 6F
(normals);
4M, 6F
(asthmatics)
5M, 8F
(normals);
9M, 3 F
(asthmatics)
4M, 9F
(normals);
8M, 8F
(asthmatics)
Asthmatics had a Decrease in FRC with 03 exposure in asthmatics; no consistent
history of atopic significant changes in pulmonary functional parameters in either
extrinsic asthma group or between groups.
and exercise-
induced
bronchospasm
Asthmatics Decrease in maximal flow at 50% of FVC in asthmatics with
selected from a 03 exposure compared to FA; no significant changes with
clinical practice combined O3-NO2 exposure.
and had
exercise-
induced
bronchospasm
Asthmatics had Decrease in FEVj and increase in Rj in normals and asthmatics
allergic asthma, with 0.12 and 0.18 ppm 03 exposure compared to FA; no
positive consistent differences between normals and asthmatics.
responses to
methacholine,
and exercise-
induced
bronchospasm
Koenig et al. (1985)
Koenig et al. (1988)
Koenig et al. (1987)
See glossary of terms and symbols for abbreviations and acronyms.
Grouped by rest and exercise; within groups listed from lowest to highest 03 concentration.
°Age range in years or as mean ± SEM.
-------
1 saturation in 28 mild to moderate COPD patients exposed to 0.18 and 0.25 ppm O3 for 1 h.
2 The combined observations of these studies indicate that persons with COPD are not
3 responsive to O3 concentrations of 0.30 ppm and lower in combination with mild exercise.
4 However, this conclusion should be viewed within the context of the low total inhaled dose
5 of O3 involved in the above studies, in that studies in healthy subjects using similar total
6 inhaled doses have also not shown significant pulmonary function effects. Interpretation of
7 these studies is also complicated by the wide range of the pulmonary function impairment of
8 the patients studied (FEV^FVC from 0.30 to 0.70), their variable smoking history, and the
9 fact that these patients are older (» 60 years of age). The inconsistency of the observed
10 small decreases in arterial oxygen saturation makes the interpretation of the clinical
11 significance of this data difficult and uncertain.
12 Despite similar limitations, Kulle et al. (1984) observed small (<4%) significant
13 (p < 0.05) decreases in FVC and forced expiratory volume in 3 s (FEV3) in 20 smokers
14 (age range 31 ot 51 years) diagnosed with mild chronic bronchitis exposed to 0.40 ppm
15 O3 for 3 h using an IE protocol (one 15-min exercise period beginning 1 h prior to end of
16 exposure, VE approximately 29 to 38 L/min). In addition, Kulle et al. (1984) observed that
17 repeated daily exposure over a 5-day period led to an adaptation of these forced expiratory
18 endpoints, and that this adaptive response did not last longer than 4 days. The pulmonary
19 responses induced by O3 exposure in this study were associated with only mild symptoms.
20
21 Subjects with Asthma
22 Three studies examining the pulmonary responses to acute O3 exposures in adult (Linn
23 et al., 1978; Silverman, 1979) and adolescent (Koenig et al., 1985) asthmatics were
24 discussed in the 1986 EPA criteria document (U.S. Environmental Protection Agency, 1986).
25 Significant decrements in group mean pulmonary function were not observed for adult
26 asthmatics exposed for 2 h at rest (Silverman, 1979) or with light IE (Linn et al., 1978) to
27 O3 concentrations of 0.25 ppm or less. However, it should be noted that although group
28 mean pulmonary function responses were not significantly affected in these studies, there
29 were responsive asthmatic subjects who had obvious decrements in pulmonary function.
30 Koenig and coworkers (Koenig et al., 1985, 1987, 1988) conducted a series of studies
31 examining the pulmonary responses of adolescent asthmatics and nonasthmatics (11 to
December 1993 7-28 DRAFT-DO NOT QUOTE OR CITE
-------
1 19 years of age) exposed to low levels of O3. Koenig et al. (1985) found no significant
2 changes in pulmonary function or symptoms in 10 adolescent normal and asthmatic subjects
3 (four male, six female) who inhaled 0.12 ppm O3 for 1 h at rest. The asthmatic subjects in
4 this study were characterized as having histories of atopic (Type I, immunoglobulin E PgE]
5 mediated) asthma and exercise-induced bronchospasm. Subsequently, in two separate studies
6 of similar groups of adolescent asthmatics and nonasthmatics, Koenig and coworkers (Koenig
7 et al., 1987, 1988) observed no significant changes in pulmonary function or symptoms
8 following exposure to 0.12 and 0.18 ppm O3 with intermittent moderate exercise up to 1 h,
9 although a small significant decrease in flow at 50% of FVC was observed in the adolescent
10 asthmatics exposed to 0.12 ppm O3.
11 Kreit et al. (1989) and Eschenbacher et al. (1989) have demonstrated that exposure to
12 0.40 ppm 03 with heavy IE (exercise VE = 30 L/min/m2 BSA) for 2 h elicits a significant
13 decrease in FVC, FEVj, FEVyFVC, and FEF25_75% in both normal and asthmatic subjects.
14 In these studies, O3 exposure elicited significantly greater decrements in FEV19 FEVYFVC,
15 and FEF25_75% in asthmatic subjects. In contrast, Kreit et al. (1989) and Eschenbacher et al.
16 (1989) found no significant difference between asthmatic and normal subjects in FVC and
17 subjective symptoms. In addition, the effect of O3 exposure on bronchial responsiveness as
18 measured by the concentration of methacholine needed to increase SRaw 100% (PC^SR^)
19 was also studied. The asthmatic subjects had a significant decrease in PCi00SRaw following
20 filtered air and O3 exposure. In comparison, the normal subjects had a significant decrease
21 in PCjQgSR^ following O3 exposure, with the percent decrease in mean PC10oSRaw after
22 O3 exposure being similar in normal and asthmatic subjects, although the asthmatic patients'
23 baseline PC100SRaw was significantly lower than that of the normal subjects. The findings
24 from this study indicate that if the total inhaled dose is increased sufficiently by either
25 increasing VE during exposure or O3 concentration, mild to moderate asthmatics will respond
26 with a greater obstructive response than will normal subjects.
27 Linn et al. (1993) have reported responses of healthy (n = 15) and asthmatic (n = 30)
28 subjects to 0.12 ppm O3 and 100 /*g/m3 of respirable sulfuric acid (H2SO4) aerosol, alone
29 and in combination using the EPA prolonged-exposure protocol (see Section 7.2.2). They
30 observed a significant O3-induced reduction in FEVl that was statistically significant and an
31 increase in airway responsiveness to methacholine for all subjects combined. The asthmatic
December 1993 7-29 DRAFT-DO NOT QUOTE OR CITE
-------
1 subjects demonstrated a statistically significant decrease in FEVj as a function of exposure
2 duration regardless of pollutant exposure. In addition, Linn et al. (1993) observed a greater
3 reduction in FEV] following O3 alone in the asthmatics as compared to the nonasthmatics
4 (-8.6% versus -1.7%), although this difference was not statistically significant. Despite
5 the lack of a significant difference between asthmatics' and nonasthmatics' group mean FEV]
6 responses with O3 exposure, the responses observed in the asthmatics may be considered
7 more important because their average was already significantly depressed by the underlying
8 illness.
9 The findings from the above studies comparing the pulmonary function responses
10 following O3 exposure in asthmatic and nonasthmatic subjects suggest that asthmatics are at
11 least as sensitive, if not more sensitive to the acute effects of O3 inhalation. The underlying
12 mechanism that would explain a possible increased responsiveness of asthmatic subjects to
13 O3 is undefined. One possible mechanism could be that asthmatic subjects have an
14 exaggerated airway inflammatory response to acute O3 exposure. A recent study conducted
15 by McBride et al. (1993) would support this hypothesis. McBride et al. (1993) exposed
16 10 asymptomatic asthmatic subjects with a history of allergic rhinitis and eight nonallergic
17 healthy subjects to filtered air and 0.12 and 0.24 ppm O3 for 90 min using a light IE
18 protocol (VE = approximately 25 L/min). Pulmonary function tests, posterior
19 rhinomanometry, and nasal lavage were performed before exposure and 10 min, 6 h, and
20 24 h after exposure. No significant changes in pulmonary or nasal function were found in
21 either the allergic asthmatic or nonallergic nonasthmatic subjects. The allergic asthmatic
22 subjects had a significant increase in the number of white blood cells in nasal lavage fluid
23 10 min and 24 h following the 0.24-ppm Oj exposure. In addition, a significant increase in
24 epithelial cells was present 10 min after exposure to 0.24 ppm O3 in the asthmatic subjects.
25 No significant cellular changes were observed in the nonasthmatic subjects. These data
26 indicate that the upper airways of asthmatic individuals are more sensitive to the acute
27 inflammatory effects of O3 than those of nonallergic nonasthmatic subjects.
28 The above studies compared the effects of O3 inhalation on pulmonary function in
29 asthmatic and normal subjects, but do not address the effect of preexposure to ambient
30 concentrations of O3 on the responsiveness of asthmatic subjects to other respiratory
31 challenges, including other irritant gases, allergens, and exercise. Koenig et al. (1990)
December 1993 7-30 DRAFT-DO NOT QUOTE OR CITE
-------
1 reported an increase in the bronchial response to an SO2 challenge in a group of
2 13 asymptomatic adolescent asthmatic subjects following inhalation of 0.12 ppm O3 for
3 45 min using a light to moderate intermittent exercise protocol (VE = approximately
4 30 L/min).
5 In a recent study, Molfino et al. (1991) investigated whether resting exposure to
6 0.12 ppm O3 for 1 h potentiates the airway response to inhaled allergen in seven mild
7 asthmatic patients with seasonal symptoms of asthma and positive skin tests for ragweed or
8 grass. This study was conducted during 4 separate weeks during the winter when ambient
9 allergen levels were low. In each week, there were 3 consecutive study days. On Days 1
10 and 2, subjects underwent methacholine challenges, whereas on Day 2, the subjects received
11 one of four combined challenges in a single blind design: air breathing followed by
12 inhalation of allergen diluent, O3 exposure followed by inhalation of allergen diluent, air
13 breathing followed by inhalation of allergen, and O3 exposure followed by inhalation of
14 allergen. Molfino et al. (1991) observed no significant differences in baseline FEVj after
15 O3 exposure, but did observe a significant reduction in the provocative concentration of
16 allergen required to reduce FEVj 15 %. This study was limited by its small subject number
17 and the results were confounded by possible ordering effects with the "O3 exposure followed
18 by allergen protocol" being the last protocol for all but one subject. Despite these
19 limitations, the findings suggest that O3 concentrations as low as 0.12 ppm may increase the
20 bronchial responsiveness to allergen in atopic subjects and clearly indicates the need for more
21 studies that address this potentially significant health problem.
22 In order to examine whether preexposure to O3 results in exacerbation of
23 exercise-induced asthma, two studies were recently conducted (Fernandes et al., 1993;
24 Weymer et al., 1993). Fernandes et al. (1993) preexposed 15 stable mild asthmatics who
25 had never smoked with exercise-induced asthma to 0.12 ppm O3 for 1 h at rest followed by
26 6 min exercise challenge test and found no significant effect on either the magnitude or time
27 course of exercise-induced bronchoconstriction. Similarly, Weymer et al. (1993) observed
28 that preexposure to either 0.10 or 0.25 ppm O3 for 60 min while performing light IE did not
29 enhance or produce exercise-induced asthma in seven otherwise healthy adult subjects with
30 stable mild asthma. Although the results of these studies would suggest that preexposure to
31 O3 neither enhances or produces exercise-induced asthma in asthmatic subjects, the small
December 1993 7_31 DRAFT-DO NOT QUOTE OR CITE
-------
1 sample size and the relatively low total inhaled doses used in the above studies limit coir
2 ability to draw any conclusions.
3
4 Subjects with Allergic Rhinitis
5 McDonnell et al. (1987) exposed 26 adults (18 to 30 years of age) with allergic rhinitis
6 to clean air and 0.18 ppm O3 for 2 h using an IE protocol (VE = 64 L/min at 15-min
7 intervals). The study subjects with allergic rhinitis did not have a history of asthma-like
8 symptoms. Following O3 exposure, the subjects with allergic rhinitis exhibited significant
9 (p < 0.01) increases in respiratory symptoms, airway reactivity to histamine, and SRaw and
10 significant decreases in FVC, FEVj, and FEF25_75% when compared to clean air exposure.
11 When compared to normal subjects without allergic rhinitis similarly exposed to 0.18 ppm
12 O3, the subjects with allergic rhinitis were no more responsive to O3 based on symptoms,
13 forced expiratory parameters, or airway reactivity to histamine aerosols, although subjects
14 with allergic rhinitis did have a small but significantly greater increase in SR^. The data on
15 subjects with allergic rhinitis and asthmatic subjects suggest that both of these groups have a
16 greater rise in airway resistance to O3 with a relative order of airway responsiveness to
17 O3 being normal < allergic < asthmatic.
18 Bascom et al. (1990) conducted a study to characterize the upper respiratory response
19 to acute O3 inhalation, nasal challenge with antigen, and the combination of the two.
20 Bascom et al. (1990) exposed 12 resting asymptomatic subjects with a history of allergic
21 rhinitis in a randomized, crossover design on each of 2 days, separated by 2 weeks, to clean
22 air or 0.5 ppm O3 for 4 h. Following exposure, subjects underwent nasal challenge with
23 four doses of antigen (1, 10, 100, and 1,000 PNU ragweed or grass). Upper and lower
24 airway symptoms were rated and nasal lavage was performed before and after clean air and
25 0.5 ppm O3 exposure, and following each antigen challenge. Exposure to O3 caused
26 significant increases in upper and lower airway symptoms, a mixed inflammatory cell influx
27 with a sevenfold increase in nasal lavage neutrophils, a 20-fold increase in eosinophils and a
28 10-fold increase in mononuclear cells as well as an apparent sloughing of epithelial cells.
29 There was a significant increase in nasal lavage albumin concentration following
30 O3 exposure. When expressed as a change from the postexposure values, there was no
31 significant difference between O3 and clean air exposure in antigen-induced upper and lower
December 1993 7-32 DRAFT-DO NOT QUOTE OR CITE
-------
1 airway symptoms, cells, albumin and mediators (histamine and TAME-esterase activity).
2 These results suggest that acute exposure to O3 does not alter the acute response to nasal
3 challenge with antigen.
4
5 Subjects with Ischemic Heart Disease
6 One study has been conducted examining the cardiopulmonary effects of acute
7 O3 inhalation in patients with ischemic heart disease. Superko et al. (1984) exposed six
8 middle-aged males with angina-symptom-limited exercise tolerance for 40 min to filtered air
9 and to 0.20 and 0.30 O3 while exercising continuously according to a protocol simulating
10 their angina-symptom-limited exercise training prescription (mean VE = 35 L/min).
11 No significant pulmonary function impairment or evidence of cardiovascular strain induced
12 by O3 inhalation was observed. The low workloads were dictated by the patient's angina-
13 symptom-limited exercise tolerance and these low workloads acted to "protect" them from
14 O3-induced effects by limiting the total inhaled dose.
15
16 7.2.1.3 Influence of Gender, Age, Ethnic, and Environmental Factors
17 Gender Differences
18 As was noted in the previous O3 criteria document (U.S. Environmental Protection
19 Agency, 1986), the pulmonary function responses to O3 of only a small number of female
20 subjects have been evaluated under controlled laboratory conditions. Although the database
21 on females has expanded (see Table 7-3), there are still fewer data than for males. Most
22 studies involving mixed groups of male and female subjects include too few female subjects
23 to allow for meaningful comparisons between the responses of the sexes, or fail to consider
24 the question at all. There are, however, a few studies including only female subjects.
25 Several studies cited in the 1986 O3 criteria document suggested that females might be more
26 responsive to O3 than males (Horvath et al., 1979; Gliner et al., 1983; Gibbons and Adams,
27 1984; Lauritzen and Adams, 1985). De Lucia et al. (1983), on the other hand, did not find
28 significant differences in the responses of young men and young women to 63 exposure.
29 Messineo and Adams (1990) hypothesized that differences previously observed between
30 the responses of males and females exposed to O3 were related to differences in lung size
31 between the sexes. They addressed this issue by selecting two groups of 14 women each.
!
December 1993 7.33 DRAFT-DO NOT QUOTE OR CITE
-------
TABLE 7-3. GENDER DIFFERENCES IN PULMONARY FUNCTION RESPONSES TO OZONE3
1
O"
rt>
vO
VO
W
-j
OJ
•£>•
o
x^
55
H
O
t— ^
Z
o
H
1
O
90
Ozone
Concentration
Exposure Duration
ppm
0.00
0.12
0.18
0.24
0.30
0.40
0.18
0.30
0.20
0.30
0.30
/ig/m and Activity
0
235
353
470
588
784
353
588
392
588
588
2.33 h
, O
VE = 25 L/min/ni
BSA
(one
exposure/subject)
1 h (mouthpiece)
CE
VE » 47 L/min
1 h (mouthpiece)
IE (20 min exercise)
VE * 28 L/min for
men
VE » 23 L/min for
women
1 h (mouthpiece)
CE
VE « 70 L/min for
men
VE « 50 L/min for
women
Exposure
Conditions'"
Mean T = 22 °C
MeanRH = 4%
treadmill
T = 21-25 °C
RH = 45-60%
cycle
T « 22 °C
RH > 75%
treadmill
T = 21-25 °C
RH = 45-60%
cycle
Number and
Gender of
Subjects
30-33 F and
30-33 M in
each
concentration
group; total of
372 individuals
participated
14 F
14 F
9M
10 F
20 M
20 F
Subject
Characteristics
Healthy NS, 18-35 years,
blacks and whites
Mean FVC =
5.11 ± 0.53 L,
NS, 20-24 years
Mean FVC =
3.74 ± 0.30 L,
NS, 19-23 years
55-74 years, NS
56-74 years, NS
18-30 years, NS
19-25 years, NS
Observed Effect(s)
Decrements in FEVj,
increases in SRaw and cough,
correlated with 03 dose.
There were no significant
differences between the
responses of males and
females.
Significant dose/response
effect on FVC and FEVj;
lung size had no effect on
percentage decrements in
FVCorFEV].
No changes in spirometry in
men or women. Women had
significant 13% increase in
Rj following exposure,
which was sustained at
20 min postexposure.
Significant decrements in
FVC, FEV1; and FEF25_75%
following 03 exposure.
No significant differences
between men and women for
spirometry or SRaw.
Reference
Seal et al. (1993b)
Messineo and Adams
(1990)
Reisenauer et al. (1988)
Adams et al. (1987)
-------
TABLE 7-3 (cont'd). GENDER DIFFERENCES IN PULMONARY FUNCTION RESPONSES TO OZONE3
o
1
1-J
>J
-n
Ozone
Concentration
Exposure Duration
ppm fj,g/m and Activity
0.45 882 2 h
IE
VE « 27.9 L/min
for men
VE « 25.4 L/min
for women
0.45 882 2 h
IE
Mean VE =
28.5 L/min for men
Mean VE =
26. 1 L/min for
women
0.48 941 2 h
IE
VE « 25 L/min
Exposure
Conditions0
T = 24 °C
RH = 58%
cycle
MeanT = 23.1
MeanRH =
46.1%
cycle/treadmill
T = 21 °C
WBGT
cycle
Number and
Gender of
Subjects
8M
8F
°C 10 M
6F
10 F
Subject
Characteristics
Healthy NS,
51-69 years
Healthy NS,
56-76 years
Healthy NS,
60-89 years
Healthy NS,
64-71 years
Healthy NS,
19-36 years
Observed Effect(s)
Range of responses in FEVj :
0 to -12% (mean = -5.6%).
No significant difference in
responses of men and women.
Tendency for women to have
greater effects.
Mean decrement in FEVj =
5.7%. Decrements in FVC and
FEVj were the only pulmonary
functions significantly altered by
03 exposure. No significant
differences between responses
of men and women.
Mean decrement in
FEVj = 22.4%.
Significant decrements in all
Reference
Drechsler-Parks et al.
(1987a,b)
Bedi et al. (1989)
Horvath et al. (1986)
spirometry measurements.
Results not significantly
different from a similar study
on males (Drechsler-Parks
etal., 1984).
See glossary of terms and symbols for abbreviations and acronyms.
Listed from lowest to highest O3 concentration.
^WBGT = 0.7 Twetbulb + 0.3 Tdry bulb or globe.
Age range in years or as mean ± SEM.
-------
1 One group had a mean FVC of 5.11 L, and the other group had a mean FVC of 3.74 L. All
2 subjects were 19 to 24 years of age and were healthy nonsmokers who had not lived in a
3 high air pollution area for at least 6 mo. The subjects completed three 1-h CE (VE =
4 47 L/min) exposures: (1) FA, (2) 0.18 ppm O3, and (3) 0.30 ppm O3. The mouthpiece
5 exposures were presented in random order, at least 4 days apart, and all were performed
6 when the subject was in the follicular phase of her menstrual cycle. Two subjects in the
7 small lung group and one in the large lung group were unable to complete the 0.30 ppm
8 O3 exposure. Both groups had similar O3-induced percentage decrements in all measures of
9 lung function, regardless of lung size, leading to the conclusion that lung size, per se, is not
10 systematically related to percentage decrements in FEV1 consequent to O3 exposure.
11 Horvath et al. (1986) exposed 10 healthy, young, nonsmoking females, 19 to 36 years
12 of age (mean age 23.6 years) to 0.48 ppm O3 or FA for 2 h in an environmental chamber
13 while they exercised intermittently at a target ventilation of 25 L/min. The subjects engaged
14 in three 20-min cycle ergometer exercise periods alternated with four 15-min rest periods.
15 The exposures were a minimum of 1 week apart. The responses of these subjects were
16 compared with those of a group of 10 young males who had earlier completed the same
17 protocol (Drechsler-Parks et al., 1984). There were no statistically significant differences in
18 the responses based on gender. The female subjects had decrements of 18.8, 22.4, and
19 30.8% in FVC, FEVl5 and FEF25.75%, respectively, compared to 19.8, 25.0, and 31.9% for
20 the male subjects. On an individual basis, 4 of the 10 males and 3 of the 10 females had
21 decrements of 30% or more in FEV]^ following the exposure to 0.48 ppm O3. One male
22 subject did not respond to the O3 exposure. It was noted, however, that the female subjects
23 inhaled an absolute dose of O3 about 22% less than the male subjects due to a slightly lower
24 exercise VE and the inherently lower resting VE of females compared to males. However,
25 if O3 dose was related to BSA or to FVC, the females inhaled slightly higher relative doses
26 of O3 than the males.
27 Adams et al. (1987) compared the responses of 20 young men (18 to 30 years of age)
28 and 20 young women (19 to 35 years of age) to exposure to 0.30 ppm 05 via mouthpiece.
29 All subjects were healthy nonsmokers with clinically normal pulmonary function. None had
30 a history of significant allergies, and none had resided in a high air pollution area for at least
31 3 mo. The subjects completed 1-h CE exposures (mean VE » 70 L/min for males and
December 1993 7-36 DRAFT-DO NOT QUOTE OR CITE
-------
1 50 L/min for females) to FA and 0.30 ppm O3. The exposures were given in random order
2 and were separated by a minimum of 5 days. Ozone exposure induced significant
3 decrements in FVC, FEVj, and FEF25_75% compared to FA exposure. Three females and
4 four males were unable to complete the O3 exposure. Females experienced mean decrements
5 of 14.2, 20.3, and 24.5% in FVC, FEVl5 and FEF25.75%, respectively, compared to mean
6 decrements of 15.8% in FVC, 23.8% in FEV1? and 35.7% in FEF25.75% for males. There
7 were no statistically significant differences between the spirometry or SRaw responses related
8 to gender. Because the female subjects inhaled a substantially smaller absolute dose of
9 O3 due to the considerably lower exercise VE, yet had similar decrements in pulmonary
10 function compared to men, the authors concluded that females are more responsive to
11 O3 than males. In this study, the female subjects inhaled a lower relative dose of
12 O3 compared to males, if expressed on the basis of BSA, but a similar relative dose if
13 expressed on the basis of FVC.
14 Seal et al. (1993b) reported on 372 healthy black and white men and women between
15 18 and 35 years of age who each were assigned to complete one 2.33-h exposure to FA,
16 0.12, 0.18, 0.24, 0.30, or 0.40 ppm O3. Subjects exercised intermittently on a motor-driven
• 0
17 treadmill at a work load inducing a VE of about 23 L/min/m BSA for women and about
18 24.5 L/min/m2 BSA for men. Although female subjects inhaled about 22% less total dose of
19 O3 than males in each exposure-concentration group, there were no significant differences in
20 the changes in FEVj 0, SRaw, or cough ratings between males and females among either
21 blacks or whites. Women also inhaled a lower absolute dose of O3 than men.
22 Drechsler-Parks et al. (1987a,b) compared the responses of eight men and eight women
23 between 51 and 76 years of age to FA and 0.45 ppm O3. The subjects were all healthy
24 nonsmokers who were long-term residents of a relatively low pollution area. The subjects
25 participated in 2-h intermittent exercise (20 min rest/20 min exercise at VE = 25 L/min)
26 environmental chamber exposures that were presented in random order and were separated
27 by at least 1 week. Except for FEV3, there were no statistically significant differences
28 between the responses of the men and women subjects, although women had slightly larger
29 mean decrements in FVC and FEVj 0 than men. Individual decrements in FVC and FEVj 0
30 ranged from 0 to about 12% for both male and female subjects. Based on FEVj, two
31 females and three males had no response to the O3 exposure. Male subjects inhaled a
December 1993 7.37 DRAFT-DO NOT QUOTE OR CITE
-------
1 somewhat larger absolute effective dose of O3 due to a higher exercise and resting VE.
2 If VE was normalized to BSA, females inhaled a larger dose of O3 than males. If VE was
3 normalized to FVC, the relative inhaled doses of O3 were similar.
4 Reisenauer et al. (1988) reported on the pulmonary function responses of 9 men and
5 10 women between 55 and 74 years of age who were exposed to 0.0, 0.20, and 0.30 ppm
6 O3. The three exposures were presented in random order and at the same time of day for
7 each subject. The subjects were exposed via mouthpiece for 1 h, during which seven men
8 exercised for 10 min and rested for 50 min, and the other two men and all of the women
9 alternated two 20-min rest periods and two 10-min exercise periods. Ventilation rates were
10 about 28 L/min for men and 23 L/min for women, although if VE is normalized to BSA, the
11 relative VE for males and females was similar. All data were pooled, regardless of the total
12 exercise tune. There were no significant changes in any parameter of pulmonary function in
13 the males. Females had no significant changes in any spirometric parameter, but did have a
14 small (13%) increase in total respiratory resistance (RT) following the 0.30 ppm
15 O3 exposure, which remained at this level 20 min postexposure.
16 Bedi et al. (1989) reported on the responses of 10 men and 6 women (60 to 89 years of
17 age) exposed for 2 h to FA or 0.45 ppm O3 for 3 consecutive days. Only the first O3 day
18 results will be discussed in this section. The issue of repeated exposures is addressed in
19 Section 7.2.1.4. Exposures were conducted at the same time of day, on consecutive days,
20 with the FA exposure always conducted first. The subjects alternated 20-min exercise
21 periods (mean VE = 28.5 L/min for men and 26.1 L/min for women) and 20-min rest
22 periods throughout the 2-h chamber exposures. If VE is normalized to BSA, women inhaled
23 slightly higher relative doses of O3; but if normalized to FVC, women inhaled a slightly
24 lower relative dose of O3 than men. There were no statistically significant group mean
25 differences between the responses of men and women subjects. The mean decrements in
26 FVC and FEVj following the O3 exposure for the 16 subjects were 2.8 and 5.7%,
27 respectively. In an exploratory analysis, the subjects were divided into two groups based on
28 whether their decrement in FEV^^ following the first O3 exposure compared to the FA
29 exposure was >5% or <5%. There were eight subjects in each group, with the sensitive
30 group consisting of two females and six males. The mean post-O3 exposure decrement in
31 FEVt was 320 mL for the sensitive group, versus 21 mL for the nonresponsive group.
December 1993 7-38 DRAFT-DO NOT QUOTE OR CITE
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1 Similar patterns of response were evident in FVC and FEV3 0. There were no significant
2 changes in any flow parameter, maximum voluntary ventilation (MVV), expiratory reserve
3 volume, or functional residual capacity.
4 The question as to whether there is a difference hi responsiveness to O3 between men
5 and women remains unresolved. Different conclusions can be arrived at depending on
6 whether VE is normalized to body or lung size when the inhaled doses of O3 are calculated.
7 The sensitive subgroup studied by Bedi et al. (1989) included six males and two females,
8 suggesting that older males may be more responsive to O3 than females. However,
9 Reisenauer et al. (1988) found a significant increase in RT only in women. Horvath et al.
10 (1986), Adams et al. (1987), and Drechsler-Parks et al. (1987a,b) suggested that because
11 their female subjects had similar pulmonary function responses to their male subjects, even
12 though the females inhaled less O3, females were more responsive than males. Messineo and
13 Adams (1990) suggested that some factor other than absolute lung size accounted for
14 observed differences between males and females. Their two groups of females with widely
15 different lung sizes experienced similar decrements in pulmonary function following
16 equivalent exposures. Although the currently available literature suggests that females may
17 be somewhat more responsive to O3 than males, the question is not settled. Further,
18 comparative studies have included only small subject groups, except for Seal et al. (1993b),
19 and often only group mean data are presented, with little information about individual
20 responses. Small subject groups with wide differences among the inherent responsiveness of
21 the individuals in the groups can have disproportionate effects on group mean data and on
22 conclusions reached.
23
24 Hormonal Influences
25 Seal et al. (1993a) compared the pulmonary function responses of 48 white and
26 55 black women (18 to 35 years of age) whose menstrual phase was known at the time of a
27 single 2.3-h exposure to 0.18, 0.24, 0.30, or 0.40 ppm O3. Subjects performed intermittent
28 treadmill exercise (VE = 20 L/min/m2 BSA) during the first 2 h of exposure. There were
29 no significant effects for SR^ or cough that could be related to menstrual cycle. There was
30 a race x menstrual phase interaction for FEVj. However, when the groups of black and
31 white women were analyzed separately, there was no significant main effect for menstrual
December 1993 7.39 DRAFT-DO NOT QUOTE OR CITE
-------
1 cycle phase. The significance of the observed interaction between race and menstrual cycle
2 phase is unknown.
3 Gerbase et al. (1993) compared the pulmonary function responses of six healthy,
4 nonsmoking women to a 130-min exposure to 0.35 ppm O3 4 to 8 days after the onset of
5 menses and 4 to 8 days after ovulation. Subjects performed intermittent exercise at a
6 workload that induced a VE of 10 x FVC. Ovulation was confirmed by a blood
7 progesterone test. Spirometry was performed pre- and 25-min post-O3 exposure. Although
8 resting VE was the same during both exposures, exercise load had to be reduced 30% during
9 the luteal phase in order to match the ventilatory response to exercise during the follicular
10 phase. There were no significant effects related to phase of the menstrual cycle. The
11 authors concluded that menstrual phase does not need to be considered in experimental
12 design. One problem with the study is that the postexposure measurements were made
13 25 min after the conclusion of the exposure. Typically, pulmonary function decrements
14 begin to reverse once exposure ends; thus, any pulmonary function changes that did occur
15 could be expected to be reduced at 25-min post-O3 exposure compared to immediately after
16 exposure.
17 Acute O3 exposure has been shown to induce short-term airway inflammation (see
18 Section 7.2.4) induced by prostaglandins, among other inflammatory substances. It has also
19 been demonstrated that progesterone inhibits prostaglandin production in the uterine
20 endometrium, which fluctuates as the progesterone concentration varies throughout the
21 menstrual cycle. Fox et al. (1993) investigated the hypothesis that O3 exposure during the
22 follicular phase, when progesterone concentration is lowest, might result in greater
23 pulmonary function responses due to reduced anti-inflammatory influences of progesterone.
24 Nine nonsmoking women completed 1-h mouthpiece exposures to FA and 0.30 ppm O3 while
25 exercising continuously (VE about 50 L/min) during both the follicular and mid-luteal phases
26 of two to four ovulatory menstrual cycles. There were no differences in any pulmonary
27 function responses to FA related to menstrual phase, nor was there a difference in the mean
28 FVC decrements following the follicular or mid-luteal phase O3 exposures. The O3-induced
29 decrements hi FEVj and FEF25_75% were significantly larger during the follicular phase than
30 during the mid-luteal phase. The authors speculated that the difference between the FEVj
December 1993 7-40 DRAFT-DO NOT QUOTE OR CITE
-------
1 and FEF25_75% responses to the two O3 exposures could be due to differences in circulating
2 progesterone, and the effect of progesterone on prostaglandin activity.
3 Available data (see Table 7-4) do not permit a conclusion regarding the influence of the
4 menstrual cycle on responses to O3 exposure. Two of the three studies available, Fox et al.
5 (1993) and Gerbase et al. (1993), were performed with small groups of subjects and resulted
6 in opposite conclusions. Seal et al. (1993a) compared race (black versus white) and
7 menstrual phase, obtaining a significant interaction between race and phase, but post-hoc
8 analysis failed to establish a basis for the interaction, leaving the implications of the study
9 unclear.
10
11 Age Differences
12 It has been hypothesized that age may be a factor in responsiveness to O3. Although
13 children make up a large proportion of the population, few controlled laboratory studies of
14 the pulmonary function effects of any air pollutant have been reported on subjects under
15 age 18. Field and epidemiological studies (see Section 7.4) attempting to relate ambient air
16 pollutant exposure to pulmonary function in children have suggested that children may be
17 more responsive to ambient air pollution than young adults.
18 The previous O3 criteria document (U.S. Environmental Protection Agency, 1986)
19 included only one laboratory exposure study in which children were the subjects. McDonnell
20 et al. (1985a) evaluated the pulmonary function responses of 23 boys between 8 and 11 years
21 of age to 0.00 and 0.12 ppm O3 in random order. The boys alternated 15-min rest and
. o
22 exercise periods (VE = 35 L/min/m BSA) for the first 120 min of the 150-min exposure.
23 Forced expiratory spirometry and respiratory symptoms were measured before exposure and
24 at 125 min of exposure, whereas airway resistance was measured before exposure began and
25 after 145 min of exposure. The group mean decrement in FEVj following the O3 exposure
26 was 3.4%, compared to 4.3% for a group of young adult males who had earlier completed
27 the same protocol (McDonnell et al., 1983). It should be noted that the absolute VE for the
28 children (39.4 L/min) and adults (65.0 L/min) was similar if normalized for BSA (about
29 35 L/min/m BSA). Assuming that adjusting ventilation for differences in BSA is an
30 appropriate normalizing technique, these children appeared to experience similar O3-induced
December 1993 7.41 DRAFT-DO NOT QUOTE OR CITE
-------
D
TABLE 7-4. HORMONAL INFLUENCES ON PULMONARY FUNCTION RESPONSES TO OZONE3
3 Ozone
g- Concentration5 ETOMim
H^
^O
\O ppm
0.12
0.24
0.30
0.40
0.30
-j
^
0.35
d
Duration and Exposure Number and Subjects
Atg/m Activity Conditions Gender of Subjects Characteristics0
235 2.3 h IE NA 48 WF Healthy NS,
470 VE = 55 BF 18-35 years
588 20 L/min/m2 BSA
784
588 1 h CE NA 9 F Healthy NS,
VE - 50 L/min ^£Ll
cycles,
20-34 years
686 130 min NA 6F Healthy NS,
regular
menstrual cycles
(age not stated)
Observed Effect(s) References
Significant menstrual cycle phase X race Seal et al. (1993a)
interaction for FEVj. No significant
menstrual cycle phase effect when blacks
and whites were analyzed separately.
No significant menstrual phase effects
for SRaw or cough score.
FEV! decreased 13. 1 % during the mid- Fox et al. (1993)
luteal phase and 18.1% during the
follicular phase. Decrement in
FEF25_75% was significantly larger
during the follicular phase than the mid-
luteal phase. Changes in FVC were
similar in both phases.
Changes in FVC, FEVj, FEF25.75%, Gerbase et al.
Vmax50%> *&& ^msx25% were similar (1993)
during both the follicular and luteal
phases.
See glossary of terms and symbols for abbreviations and acronyms.
Listed from lowest to highest O3 concentration.
cAge range in years or as mean ± SEM.
-------
1 pulmonary effects as adults. The children reported no symptoms, although the adults
2 reported a small but statistically significant increase in cough following O3 exposure.
3 It continues to be the case that controlled laboratory studies of the effects of exposure
4 to air pollutants are rarely performed with children as subjects (see Table 7-5). Avol et al.
5 (1987) have reported on the pulmonary function responses of 33 healthy boys and 33 healthy
6 girls having a mean age of 9.4 years. The children completed exposures to purified air and
7 outdoor ambient air that was drawn into an environmental chamber. Ambient temperature
8 averaged about 33 °C. Exposures were 1 h in duration, were separated by a minimum of
9 2 weeks, and were conducted from June through September, beginning in the early afternoon
10 when ambient air pollutant concentrations generally peak. The subjects performed
11 continuous exercise throughout the hour of exposure. Boys and girls exercised at similar
12 VE, 22 to 23 L/min. It should be noted that the ambient exposure included the full range of
13 air pollutants present in the outdoor air mix on the days of the exposures, except for small
14 fractions of O3 and paniculate lost in the inlet duct. Concentrations of O3, nitrogen dioxide
15 (NO2), total suspended particulate (TSP), paniculate nitrate, paniculate sulfate, paniculate
16 sodium, and particulate ammonium were measured throughout the exposures. The
17 O3 concentration during the ambient air exposures averaged 0.113 ± 0.033 ppm, whereas it
18 averaged 0.003 + 0.002 ppm during the purified air exposures. The children consistently
19 had a decline in pulmonary function with time, following both FA and ambient air
20 exposures. Typical mean decrements in FVC and FEV1 were 50 mL or less. A correlation
21 analysis between inhaled O3 dose and the mean net pre- to postexposure volume change
22 (in liters) in FEVl5 including all valid pulmonary tests, indicated an r of -0.27 (p < 0.05).
23 The investigators have also published similar studies on adolescents and adults (Avol et al.,
24 1984, 1985a). The responses of the adolescents and adults to both exposures were not
25 substantially different from those of the children whose results are reported here. The
26 authors further noted that the children seemed to have difficulty performing consistent,
27 reproducible pulmonary function tests, a factor which could have impacted on these results.
Several studies comparing the pulmonary function responses of healthy and asthmatic
adolescents to O3 exposure have appeared in the literature. The responses of the asthmatics
are presented in Section 7.2.1.2; only data on normal adolescent subjects will be discussed in
this section.
December 1993 7.43 DRAFT-DO NOT QUOTE OR CITE
-------
TABLE 7-5. AGE DIFFERENCES IN PULMONARY FUNCTION RESPONSES TO OZONE8
8
I
Co
VO
u>
-J
£
0
§
2,
S
1
i
Ozone
Concentration Exposure Duration
3
ppm jug/m and Activity
0.113
+
other
ambient
pollutants
0.12
0.12
0.18
0.18
0.24
0.30
0.40
0.20
0.30
221
235
235
353
353
470
588
784
392
588
In
CE
VE « 22 L/min
1 h (mouthpiece)
IE
VE = 4-5 X resting
40 min (mouthpiece)
IE
10 min exercise at
VE = 32.6 L/min;
40 min (mouthpiece)
IE
10 min exercise at
VE = 41.3 L/min
2.3 h
IE
VE = 20 L/min/m2
BSA
1 h (mouthpiece)
IE (20 min)
VE « 28 L/min for
men
VE « 23 L/min for
women
Exposure
Conditions
T = 32.7 °C
RH « 43%
cycle
T = 22 °C
RH = 75%
treadmill
NA
treadmill
NA
T » 22 °C
RH S: 75%
treadmill
Number and
Gender of Subject
Subjects Characteristics6
33 M
33 F
5 M
7 F
3 M
7 F
4M
6F
48 WF
55 BF
9M
10 F
NS for both groups,
mean age = 9.4 years
Healthy NS,
12-17 years
Healthy NS,
14-19 years
Healthy NS,
18-35 years
Black and white
Healthy NS,
55-74 years
Observed Effect(s)
No differences in responses
of boys and girls. Similar
decrements (<5% on
average) following both
purified air and ambient air
(03 at 0.11 ppm) exposures.
No significant changes in any
pulmonary function in
healthy subjects.
No significant change in
FEVj; increased Rf with
exposure to 0.18 ppm O3.
Some subjects responded to
5-10 mg/mL methacholine
after 0. 18-ppm 03 exposure,
whereas none responded to
25 mg/mL methacholine at
baseline bronchochallenge.
Older women had smaller
changes in FEV1 than
younger women. No age-
related differences in SRaw
or cough score.
No change in any spirometry
measure. Women had 13 %
increase in RT after
0.30-ppm exposure.
Reference
Avol et al. (1987)
Koenig et al. (1988)
Koenig et al. (1987)
Seal et al. (1993b)
Reisenauer et al. (1988)
-------
TABLE 7-5 (cont'd). AGE DIFFERENCES IN PULMONARY FUNCTION RESPONSES TO OZONE3
o
CD
cr
UJ
d
6
o
Ozone
Concentration Exposure Duration
O
ppm jig/m and Activity
0.45 882 2 h
IE
VE ~ 26 L/min
0.45 882 2 h
IE
Mean VE =
28.5 L/min for men
Mean VE =
26.1 L/min for
women
0.45 882 2 h
IE
VE « 26 L/min
0.45 882 1 h
CE
VE « 26 L/min
2h
IE
VE « 26 L/min
Exposure
Conditions
T « 23 °C
RH = 53%
cycle
T = 23 °C
RH = 46%
cycle/treadmill
T « 24 °C
RH = 63%
cycle
T « 23 °C
RH = 58%
cycle/treadmill
Number and
Gender of
Subjects
8M
8F
10 M
6F
8M
8F
7M
5F
Subject
Characteristics0
Healthy NS
51-76 years
Healthy NS
60-89 years
Healthy NS
51-69 years
Healthy NS
56-76 years
Healthy NS
60-79 years
(all hi 60s
except one
79-year-old)
Observed Effect(s) Reference
Mean decrement hi Drechsler-Parks et al.
FEV! = 5.6 ± 13%; range of (1987a,b)
decrements = 0-12%.
Mean decrement in Bedi et al. (1989)
FEVj = 5.7%; eight subjects had a 5%
or greater difference between their
response to 03 and FA, and the other
eight had less than a 5% difference
between their responses to FA and
0.45 ppm 03.
13 subjects had decrements in FEVj Bedi et al. (1988)
on three separate exposures to 0.45 ppm
within 5% of their mean response to the
three exposures. The other
three subjects were not reproducible.
Symptom reports did not correlate well
with pulmonary function changes.
Comparison of 1-h CE protocol and 2 h Drechsler-Parks et al.
IE protocol indicated no difference (1990)
between the changes hi pulmonary
function following the two protocols.
See glossary of terms and symbols for abbreviations and acronyms.
Listed from lowest to highest O3 concentration.
Age range hi years or as mean ± SEM.
-------
1 Koenig et al. (1987) reported on 20 adolescents, 14 to 19 years of age, who were
2 exposed for 40 min to air, 0.12 ppm O3, or 0.18 ppm O3 via a mouthpiece system. Ten
3 subjects were exposed to each O3 concentration, but not all subjects were exposed to both
4 concentrations. None of the healthy subjects had a history of asthma or allergies, and all had
5 pulmonary function within the predicted range, based on age, sex, and height. There was a
6 5- to 7-min break in exposure for pulmonary function test performance following 30 min of
7 resting exposure, followed by a 10-min exercise period (VE = 32.6 ± 6.4 L/min for the
8 0.12-ppm O3 exposure and 41.3 ± 9.3 L/min for the 0.18-ppm O3 exposure). Changes in
9 FEV1 were not significant following any exposure. After exposure to 0.18 ppm O3, RT was
10 increased 15%.
11 Koenig et al. (1988) have also reported on the pulmonary function responses of another
12 group of 12 healthy adolescents (12 to 17 years of age) to 1-h exposures to air and 0.12 ppm
13 03. The subjects were exposed by mouthpiece to air and 0.12 ppm O3 while alternating
14 15 min periods of exercise (VE = 32.8 ± 6.0 L/min) with 15 min periods of rest (VE =
15 8.8 ± 1.2 L/min). Tests of pulmonary function included forced expiratory spirometry and
16 RT. Healthy subjects had no significant alterations in any parameter of pulmonary function
17 consequent to either air or 0.12-ppm O3 exposure.
18 Although few data are available on the responses of healthy adolescents exposed to 03,
19 the limited existing data do not identify adolescents as being either more or less responsive
20 than young adults.
21 At the time the 1986 O3 criteria document was released, no studies specifically
22 evaluating the pulmonary function responses of older adults had been reported. Several
23 studies (Folinsbee et al., 1985; Adams et al., 1981) that included a few middle-aged
24 individuals among the subjects were suggestive that there might be a decrease in
25 O3 responsiveness with advancing age. Several reports have since appeared, collectively
26 suggesting that healthy older adults (i.e., over 50 years of age) are generally minimally
27 responsive to O3, although some individuals remain responsive to O3.
28 Drechsler-Parks et al. (1987a) reported on eight men and eight women between 51 and
29 76 years of age who were exposed for 2 h to FA or 0.45 ppm O3. The subjects were
30 healthy nonsmokers with normal baseline pulmonary function. The chamber exposures
31 involved alternating 20-min rest and exercise periods (VE averaged 27.9 ± 0.29 L/min for
December 1993 7-46 DRAFT-DO NOT QUOTE OR CITE
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1 men and 25.4 ± 0.80 L/min for women). Exposures were presented in random order at
2 least 1 week apart. The only significant difference related to sex was in FEV3, in which
3 women had larger decrements than men. There were no significant decrements in any other
4 parameter of pulmonary function related to O3 exposure, except when the data of all
5 16 subjects were pooled, resulting in significant mean decrements of 5.3 ± 1.3% in FVC,
6 and 5.6 ± 1.3% in FEVj. The range of individual decrements in FEVj was from 0 to 12%.
7 Two women and three men had no response following the O3 exposure. The subjects
8 reported more symptoms following the O3 exposure than the FA exposure. Seven subjects
9 reported cough, nine reported sore throat, and six reported chest tightness. The authors
10 compared their results on older adults with a group of young adults who had completed the
11 same protocol. Both the older men and women inhaled slightly higher doses of O3 than the
12 young men and women (10.23 x 104 L for older men versus 10.12 x 10"4 L for young
13 men, and 8.48 x 10"4 L for older women versus 7.94 X 10"4 L for young women).
14 However, older men had a mean decrement in FEVj of 4.2% versus 23.7% for young men,
15 whereas older women had a mean decrement in FEVj of 7.0% versus 14.7% for young
16 women. The decrements of the older subjects were also compared to published values for
17 young adults, with the older subjects studied consistently showing smaller changes in
18 pulmonary functions than young adults. These comparisons indicated that these older adults
19 were less responsive to O3 exposure than typical young adults, in terms of pulmonary
20 function changes and symptom reports.
21 Reisenauer et al. (1988) reported on the pulmonary function responses of 19 healthy
22 adults between 55 and 74 years of age. All were nonsmokers with baseline pulmonary
23 function within the predicted normal range. None had a history of asthma, atopy, or
24 cardiovascular disease, and none responded to a baseline methacholine bronchochallenge
25 (up to 25 mg/mL). Subjects were exposed by mouthpiece to 0.0, 0.20, or 0.30 ppm O3 for
26 1 h. Seven men rested for 50 min and exercised for 10 min, whereas the other two men and
27 all women alternated two 20-min rest periods with two 10-min exercise periods; VE was
28 approximately 28 L/min for men and 23 L/min for women. The three exposures were
29 presented in random order at the same time of day for each individual, but the separation
30 between exposures is not stated. The only significant change in pulmonary function was a
31 13% increase in RT with exposure to 0.30 ppm O3 in women only, which was sustained for
December 1993 7.47 DRAFT-DO NOT QUOTE OR CITE
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1 at least 20 min postexposure. The authors concluded, based on the increase in RT in the
2 older women, that older adults were at increased risk for pulmonary function changes with
3 near ambient O3 exposure. However, RT is a highly variable parameter, and no other
4 changes were significant. Given the large number of variables tested, this isolated result is
5 possibly related to the large number of statistical tests performed. In contrast, the results of
6 Koenig et al. (1987), from 10 healthy adolescents exposed to 0.18 ppm O3 using a similar
7 protocol, reported a mean decrement in FEVj 0 of 2%, and an increase of 10.5% in RT at
8 2 to 3 min postexposure. At 7 to 8 min postexposure, the increase in RT was 15.3%.
9 Comparison of these results to those of Reisenauer et al. (1988) at 0.20 ppm O3 supports the
10 contention that younger individuals are more responsive to O3 than older individuals, in that
11 no changes in spirometry were noted in the older adults exposed to 0.30 ppm C^, although
12 older women showed increased RT with 0.30 ppm O3 exposure. Adolescents had alterations
13 in both spirometry and RT with exposure to 0.18 ppm O3.
14 Bedi et al. (1988) reported that older men and women 51 to 76 years of age who
15 completed three exposures to 0.45 ppm O3 did not respond equivalently to each of three
16 exposures. The subjects were healthy nonsmokers with baseline pulmonary function within
17 predicted normal limits. The subjects alternated 20-min exercise (VE was approximately
18 26 L/min) and 20-min rest periods throughout the 2-h chamber exposures. There was a
19 minimum of 1 week between exposures, although separations ranged from 1 to 4 weeks
20 between exposures 1 and 2, and between 1 and 7 weeks between exposures 2 and 3.
21 Analysis of variance indicated no difference between the group mean responses to the three
22 exposures. The data were then subjected to a correlation analysis, which led to the
23 conclusion that the responses within an individual subject were not reproducible. McDonnell
24 et al. (1985b), on the other hand, found good reproducibility of pulmonary function
25 responses after exposure to various concentrations of O3 between 0.12 and 0.40 ppm in
26 young adult males between 18 and 30 years of age.
27 Seal et al. (1993a) compared the pulmonary function responses of 48 white and
28 55 black women (18 to 35 years) who each completed a 2.3-h exposure to 0.18, 0.24, 0.30,
29 or 0.40 ppm O3. The subjects participated in only one exposure each while intermittently
30 exercising (VE = 20 L/min/m2 BSA) during the first 2 h of the exposure. Older subjects
31 within the age range tested had smaller decrements in I!EVl than younger subjects.
December 1993 7-48 DRAFT-DO NOT QUOTE OR CITE
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1 One simple method to estimate the O3 exposure dose is to calculate the product of
2 O3 concentration (parts per million), VE (liters per minute), and exposure duration
3 (minutes). Research on young adults (Folinsbee et al., 1978; Adams et al., 1981) has
4 demonstrated that the order of relative importance of the three factors is O3 concentration,
5 VE, and exposure duration. Drechsler-Parks et al. (1990) investigated the relative role of
6 the three components of effective dose in 12 healthy, nonsmoking adults between 61 and
7 79 years of age. The subjects were exposed to both FA and 0.45 ppm, once while they
8 performed a 1-h continuous exercise protocol and once while they performed a 2-h
9 intermittent exercise protocol in which they alternated 20-min exercise periods and 20-min
10 rest periods. Mean VE ranged from 25.2 to 27.3 L/min among the four exposures.
11 Exposures were separated by at least 1 week. Regardless of protocol, O3 exposure induced
12 significant decrements in FEV0 5, FEVj, FEV3, and peak flow rate compared to FA
13 exposure. There were significant decrements in FEF25-75%, FEF50%, FEF25%, and MVV
14 following all four exposures. The only significant difference between the responses to the
15 1- and 2-h O3 protocols was in FEV0 5. The total number of symptoms reported was 10 for
16 the 1-h FA exposure, 6 for the 1-h O3 exposure, and 12 for both the 2-h FA and 2-h
17 O3 exposures. It appears that resting ventilation during the 2 h protocol had a lesser effect
18 compared to exercise ventilation. This supports earlier reports that the O3 concentration is
19 the most significant factor among the three factors that contribute to effective dose (Adams
20 et al., 1981; Folinsbee et al., 1978; Hackney et al., 1975).
21 Available data, though on a limited number of subjects, consistently indicate that
22 responsiveness to O3 is decreased in persons over 50 years of age compared to young adults.
23 Although there are few data available on adults in their thirties and forties, the statistical
24 modeling study of McDonnell et al. (1993) on subjects from 18 to 32 years of age suggests
25 that responsiveness to O3 is already diminishing by age 30, and that the most responsive
26 individuals are likely to be less than 25 years of age. The results of Bedi et al. (1988)
27 suggest that older adults may be less reproducible in their responses to O3 than young adult
28 males (McDonnell et al., 1985b); however, this finding is based on only 16 subjects and
29 should be confirmed before being considered conclusive.
30
31
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1 Ethnic and Racial Factors
2 Young white males have been the most frequently studied population in published
3 reports on pulmonary function responses to O3. There is concern, however, that responses
4 to O3 may be influenced by ethnic differences based on the observation that blacks have
5 smaller lungs than whites for a given standing or sitting height (Rossiter and Weill, 1974;
6 McDonnell and Seal, 1991). Thus, an equivalent inhaled volume of O3 could result in a
7 larger O3 dose per unit of lung tissue in blacks compared to whites, potentially inducing
8 greater effects in blacks than whites exposed to O3 under the same conditions. Seal et al.
9 (1993b) evaluated the pulmonary function responses of 372 individuals, black, white, male,
10 and female (n > 90 per group), between 18 and 35 years of age who were exposed to 0.00,
11 0.12, 0.18, 0.24, 0.30, or 0.40 ppm O3. Each subject was randomly assigned to an
12 exposure group and participated in only one experimental session. The protocol involved
13 environmental chamber exposure for 2.33 h to the assigned condition. During the first
14 2 h of exposure, the subjects alternated 15-min rest periods and 15-min exercise periods
2
15 (VE = 25 L/min/m BSA). Spirometric and plethysmographic measurements were made at
16 5 and 20 min following the final exercise period. The initial nonparametric analysis of the
17 percentage changes in FEVj indicated that FEVj responses increased with increasing
18 O3 concentration, and a group effect occurred that was independent of O3 concentration.
19 There was an O3 effect, but no group effect or group x O3 interaction for SR^, indicating
20 an increase in SR^ with increasing O3 concentration. Both group and O3 effects were
21 significant for cough, but the interaction was not significant. A post hoc analysis, using a
22 different statistical method on the absolute changes in FEVl5 indicated that the black males
23 experienced significant decrements in FEVj following exposure to 0.12 ppm O3, whereas
24 black women and white men and women did not have significant decrements in PEVl at
25 O3 concentrations below 0.18 ppm. These results are not easily explained because there was
26 no gender difference among whites and no racial difference among women. Furthermore,
27 the black men had significantly greater decrements in FEV^ at only some of the
28 O3 concentrations studied (see Figure 7-2), Although the results can be considered
29 suggestive of an ethnic difference, more subjects must be studied before the issue of ethnic
30 difference in O3 responsiveness can be considered settled. It should be noted that although
31 this study included a large number of subjects, each subject participated in only one
December 1993 7-50 DRAFT-DO NOT QUOTE OR CITE
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0.00 0.12 0.18 0.24
Ozone Concentrator) (ppm)
0.30
0.40
Figure 7-2. Mean percent change (± SFJVQ in post minus pre values of FEV! for each
gender-race group. Open bars = white women; cross-hatched bars =
black women; hatched bars = white men; solid bars = black men.
Source: Seal et al. (1993).
1
2
3
4
5
6
7
8
9
10
11
12
experiment. Thus, the range of individual responsiveness could have been different between
groups.
Environmental Factors
A number of environmental factors, such as ambient temperature and humidity, season
of the year, route of inhalation, and smoking history have been hypothesized to potentially
impact on responses to O3 exposure in additive or synergistic ways. None of these
potentially interacting agents has been adequately addressed in the extant O3 literature.
Although O3 concentrations in Los Angeles, for example, generally are highest on hot, dry
days, most research on responses to O3 exposure has been conducted under temperature and
humidity conditions not substantially different from those typical of indoor environments.
The few studies that included temperature and humidity as experimental factors have
December 1993
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1 produced equivocal results (see p. 10-44 in the 1986 O3 criteria document). No new reports
2 of temperature or humidity effects have appeared since the 1986 O3 criteria document.
3 It has been suggested that the effects induced by oral inhalation of O3 may be greater
4 than those induced when subjects breathe nasally or oronasally because the scrubbing
5 function of the nasal passages and upper airways is bypassed. This issue has potential
6 importance in comparing results from different laboratories because some groups utilize
7 environmental chambers with the subjects breathing freely, whereas others use obligatory
8 mouthpiece exposure systems. [For additional information on this subject, see Chapter 8.]
9 Hynes et al. (1988) had 17 nonsmoking, nonallergic individuals (18 to 31 years of age)
10 exercise continuously for 30 min (VE about 30 L/min) while exposed to 0.40 ppm O3. The
11 subjects completed the protocol once while breathing orally and once while breathing nasally.
12 There was no baseline FA exposure. No significant difference in any pulmonary function
13 was evident when the oral and nasal exposure responses were compared.
14 Adams et al. (1989) compared the pulmonary function responses to oral and oronasal
15 inhalation of O3. Six healthy young males (23 to 30 years of age) completed five exposures
16 to 0.40 ppm O3. There was no FA baseline exposure. Exposures lasted 30 or 75 min,
17 during which the subjects exercised continuously at 75 or 30 L/min (effective dose for all
18 exposures was about 900 ppm • L). The exposures were presented in random order with at
19 least 4 days between exposures. Subjects breathed on one occasion through a mouthpiece
20 exposure system while wearing a noseclip and through a facemask system configured for
21 either oral breathing only, or oronasal breathing, on the other four occasions. There were no
22 significant differences in the pulmonary function changes related to the two facemask
23 configurations. Group mean decrements in FEVj ranged between 0.67 and 0.92 L (about
24 15 to 17%) among the four exposures. The only significant differences in the FVC and
25 FEVj decrements between the mouthpiece system and the 30 min, 75 L/min facemask
26 configured for oral breathing only was that the decrements with the mouthpiece system were
27 larger. With the facemask, FEV! decreased 0.70 ± 0.32 L compared to 0.92 ± 0.44 L
28 with the mouthpiece. The subjects did not report any differences in symptomatology among
29 the five exposures. A potential confounding factor in the facemask studies compared to the
30 mouthpiece exposure is whether or not O3 degrades inside the mask either on the mask
31 surface or by contact with the facial skin, thereby altering the O3 concentration of the inhaled
December 1993 7-52 DRAFT-DO NOT QUOTE OR CITE
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1 air, at least with short-term exposures. The authors concluded that the lack of significant
2 difference in FEVj responses between the four facemask exposures supports the contention
3 that breathing route during moderate and heavy continuous exercise does not alter O3-induced
4 pulmonary function and subjective symptoms effects in humans.
5 Earlier studies (see p. 10-38 in the 1986 O3 criteria document) have suggested that
6 cigarette smokers are less responsive to O3 than nonsmokers. Frampton et al. (1993)
7 compared the pulmonary function responses of 18 current smokers (^3 pack-years smoking
8 history) and 18 never smokers to 0.22 ppm O3. All subjects had normal spirometry and no
9 evidence of exercise-induced bronchospasm. Exposures were 4 h in duration, during which
10 subjects exercised for 20 min out of each 30 min of exposure (VE = 25 L/min/m2 BSA).
11 Spirometry and specific airway conductance were measured after 2 h of exposure and at the
12 conclusion of the 4 h exposure period. Smokers had smaller responses in all measures of
13 pulmonary function tested compared to nonsmokers.
14 The question as to whether reactivity to O3 returns with cessation of cigarette smoking
15 has been addressed by Emmons and Foster (1991). Thirty-four individuals with no history of
16 asthma or obvious respiratory disease who enrolled in a smoking cessation program were
17 randomly assigned to an O3 group (n = 18) or an FA group (n = 16). The subjects ranged
18 from 24 to 58 years of age, and had a group mean smoking history of 33.9 ± 13 pack-years.
19 Most of the subjects had baseline pulmonary functions somewhat below predicted values,
20 based on age, height, and sex. Subjects completed 2 h exposures to 0.42 ppm O3 or FA, as
21 assigned, prior to beginning the smoking cessation program. The subjects rested during the
22 exposures, except for 5 min of exercise at 150 kg • m/min (no VE given) at the beginning of
23 the last half-hour of exposure. Nine subjects in the O3 group and six in the FA group
24 completed the 6-mo smoking cessation program and repeated their assigned exposures at the
25 end of the program. Prior to beginning the smoking cessation program, both the FA and
26 O3 groups had pre- to postexposure changes in FVC, FEVj, and FEF25_75% within the
27 variability of repeated tests. The O3 group had a significant mean change in FEF25.75% of
28 -22.5%, comparing postexposure to preexposure, whereas the FA group had a
29 nonsignificant -12% change. Changes in FVC and FEVj were not significant in either
30 group. It should be noted that smoking cessation led to a group mean improvement in
31 baseline FEF25_75% of 22.9%. The post-O3 exposure values for FEF25_75% were similar
December 1993 7.53 DRAFT-DO NOT QUOTE OR CITE
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1 following the initial and the post-smoking cessation exposures. Thus, the difference in the
2 FEF25_75% decrement with O3 exposure post-smoking cessation was largely due to the
3 improvement that ensued from 6 mo of abstinence from smoking.
4 The results of Emmons and Foster (1991) and Frampton et al. (1993) suggest that
5 active smoking blunts responsiveness to O3 and that cessation of smoking for 6 mo leads to
6 improved baseline pulmonary function and possibly the reemergence of O3 responsiveness.
7
8 7.2.1.4 Repeated Exposures to Ozone
9 Repeated daily exposure to O3 hi the laboratory setting leads to attenuated changes in
10 spirometry and symptom responses that were initially termed "adaptation" (Hackney et al.,
11 1977a). A series of repeated exposure studies, performed in various laboratories, was
12 reviewed in the previous criteria document (U.S. Environmental Protection Agency, 1986).
13 The spirometric responses to repeated O3 exposure typically showed that the response was
14 increased on the second exposure day to concentrations in the range of 0.40 to 0.50 ppm
15 O3 in exposures accompanied by moderate exercise (see Table 7-6). Thus, the response was
16 enhanced on the second consecutive day. Mechanisms for enhanced responses had not been
17 established, although it was hypothesized that persistence of O3-induced damage for greater
18 than 24 h may have contributed to the larger Day 2 response. An enhanced Day 2 response
19 was less obvious or absent in exposures that were repeated at lower concentrations or that
20 caused relatively small group mean O3-induced decrements in spirometry. Two reports (Bedi
21 et al., 1985; Folinsbee et al., 1986) indicated that enhanced spirometric responsiveness was
22 present within 12 h and lasted for at least 24 and possibly 48 h, but was clearly absent after
23 72 h. After 3 to 5 days of consecutive daily exposures to O3, responses were markedly
24 diminished or absent. One study (Horvath et al., 1981) suggested that the rapidity of this
25 decline in response was related to the magnitude of the subjects' initial responses to O3 or
26 their "sensitivity". Finally, the persistence of the attenuation of spirometric and symptom
27 responses has been studied (Horvath et al., 1981; Linn et al., 1982b; Kulle et al., 1982).
28 These studies indicate that the attenuation of response is relatively short-lived, being partially
29 reversed within 3 to 7 days and typically abolished within 1 to 2 weeks. Repeated exposures
30 separated by 1 week (for up to 6 weeks) do not apparently cause any lessening of the
31 spirometric response (Linn et al., 1982b).
December 1993 7-54 DRAFT-DO NOT QUOTE OR CITE
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TABLE 7-6. CHANGES IN FORCED EXPIRATORY LUNG VOLUME AFTER REPEATED
DAILY EXPOSURE TO OZONE3
O" Ozone
*"* Concentration
£
M^*
U>
-------
1 Folinsbee et al. (1993a) (also see Devlin et al., 1993b) exposed a group of healthy
2 males (preliminary n = 8 or 9) to 0.40 ppm O3 for 2 h/day on 5 consecutive days. Subjects
3 performed heavy intermittent exercise ( VE = 60 to 70 L/min, 15 min rest/15 min exercise).
4 Decrements in FEVj averaged 21.8, 28.6, 24.6, 9.1, and 5.5% on the 5 exposure days.
5 Baseline preexposure FEY^ decreased from the first day's preexposure measurement and was
6 depressed by an average of about 7% on the third day. This study illustrates that with high
7 concentration and heavy exercise exposures, spirometry and symptom responses are not
8 completely recovered within 24 h.
9 Besides the absence of pulmonary function responses after several days of O3 exposure,
10 symptoms of cough and chest discomfort usually associated with O3 exposure are generally
11 absent (Folinsbee et al., 1993a,b; Linn et al., 1982; Foxcroft and Adams, 1986; Folinsbee
12 et al., 1980). In addition, airway responsiveness to methacholine is increased with an initial
13 O3 exposure (Holtzman et al., 1979; Folinsbee et al., 1988), may be further increased with
14 subsequent exposures (Folinsbee et al., 1993b), and shows a tendency for the increased
15 response to diminish with repeated exposure (Kulle et al., 1982; Dimeo et al., 1981).
16 A number of possible explanations for the initially enhanced and then lessened response may
17 be related to changes that are occurring in pulmonary epithelia as a consequence of
18 O3 exposure. Inflammatory responses (Koren et al., 1989a), epithelial damage, and changes
19 in permeability (Kehrl et al., 1987) could be invoked to explain at least a portion of these
20 responses. By blocking spirometric and symptom responses with indomethacin pretreatment,
21 Schonfeld et al. (1989) demonstrated that these responses were not enhanced by repeated
22 exposure. However, the mechanisms of these responses with regard to repeated exposures in
23 humans remains to be elucidated.
24 Recent studies of repeated O3 exposures have addressed some other features of the
25 responses (see Table 7-7). A series of reports from the Rancho Los Amigos group have
26 examined changes in response to O3 as a result of the season of the year in the
27 Los Angeles/South Coast Air Basin. The purpose of this study (Linn et al., 1988; also
28 Hackney et al., 1989; Avol et al., 1988) was to determine whether responsive subjects
29 (n = 12), identified during an initial screening following a period of low ambient O3
30 exposure, would remain responsive after regular ambient exposure during the "smog season".
31 Responses of so-called "nonresponsive" subjects (n = 13) were also examined across the
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TABLE 7-7. PULMONARY FUNCTION EFFECTS WITH REPEATED EXPOSURES TO OZONE
,a
Ut
Ozone Concentration
Exposure Exposure
ppm /ig/m Duration and Activity Conditions
6~l2 235 6.6 h 18~°C
50-min exercise/ 1 0-min 40 % RH
rest, 30-min lunch 5 consecutive
VE = 38.8 L/min daily exposures
018 353 2h JT^C
IE (heavy) 35% RH
VE = 60-70 L/min (Screen exposures
(35 L/min/m BSA) in spring 1986;
second exposures
in summer/fall
1986 and winter
1987 and spring
1987 for
responders and
nonresponders
only)
0.20/0.20 392/392 1 h 21 to 25 °C
0.35/0.20 686/392 CE at 60 L/min 40-60% RH
0.35/0.35 686/686 (three 2-day pairs
of exposures)
Number and Subject
Gender of Subjects Characteristics
17 M Healthy NS
59 adult Responders:
Los Angeles Age =
residents 19-40 years
12 responsive 6 atopic
13 nonresponsive 2 asthmatic
4 normal
Nonresponders:
Age =
18-39 years
13 normal
15 M Healthy
aerobically
trained NS
FVC = 4.24 -
6.98 L
Observed Effect(s) Reference
FEVj responses were maximal on first day Folinsbee et al. (1993b)
of exposure (- 13 %), less on second day (also see Table 7-9)
(—9%), absent thereafter. Symptom
responses only the first 2 days.
Methacholine airway responsiveness was at
least doubled on all exposure days, but was
highest on the second day of ozone. Airway
responsiveness was still higher than air
control after 5 days of ozone exposure.
Trend to lessened response, but it was not
achieved after 5 days.
Responders had ApEVj = 12.4% after Linn et al. (1988)
initial screening, nonresponders had no (also see Hackney et al., 1989)
change. Responders had nonsignificant
response in late summer or early winter, but
were responsive again in early spring (spring
1986, -385 mL; Autumn 1986, -17 mL;
winter 1987, +16mL; spring 1987,
—347 mL). Nonresponders did not change
with season. Suggests that responders
responses may vary with ambient exposure,
but nonresponders generally remain
nonresponsive.
Consecutive days of exposure to 0.20 ppm Brookes et al. (1989)
produced similar responses on each day
(-5.02, -7.80); 0.35/0.20 ppm pair caused
increased response to 0.20 ppm on second
day (-8.74); 0.35/0.35 ppm caused much
increased response on day 2 (—15.9,
-24.6). Symptom responses were worse on
the second exposure to 0.35 ppm, but not
with second exposure to 0.20 ppm.
-------
d
8
n>
TABLE 7-7 (cont'd). PULMONARY FUNCTION EFFECTS WITH REPEATED
EXPOSURES TO OZONE3
Sg Ozone Concentration
S PPm ^
6 mo
Healthy NS
61 years for M
and 65 years for
F
FVC = 4.97 L
for M and
3.11 LforF
Healthy NS
60-89 years
(median age =
65 years; mean
FVC = 3.99 L;
mean FEVj =
3. OIL;
FEVj/FVC range
= 61-85%)
Observed Effect(s) Reference
Largest FEVj decrease on second of 4 days Foxcroft and Adams (1986)
Oj exposure (—40% mean 1). Trend for
adaptation not complete in 4 days.
VO2tnax decreased with single acute 03
exposure (—6%), but was not significant
after 4 days of 03 exposure (-4%).
Performance time was less after acute 03
(211 s) exposure than after FA (253 s).
No differences between responses to Schonfeld et al . (1989)
exposures separated by 72 or 120 h.
Enhanced FEVj response at 24 h (- 16.1 %
versus —30.4%). Possible enhanced
response at 48 h (—14.4% versus
—20.6%). Similar trends observed for
respiratory pattern and SRaw.
Spirometric changes were not reproducible Bedi et al. (1988)
from time to time after ozone exposure
(r < 0.50). Repeat exposures to air
yielded consistent responses.
Overall increase in symptoms, but no single Bedi et al. (1989)
symptom increased significantly. FVC
decreased 111 mL and 104 mL on Days 1
and 2, respectively. FEVj fell by 171 and
164 mL and FEV3 fell by 185 and
172 mL. No significant changes on Days 3
and 4 or with FA. FEVj changes were
-5.8%, -5.6%, -1.9%, and -1.7% on
the four 03 days.
-------
TABLE 7-7 (cont'd). PULMONARY FUNCTION EFFECTS WITH REPEATED
EXPOSURES TO OZONEa
§, Ozone Concentration
I^Q ppm Mg/m Duration and Activity
U> 0.45 882 2h
(+ 0.30 IE (20 min rest, 20 min
PAN) exercise)
VE = 27 L/min
Exposure
Conditions
22 °C
60% RH
Five days
consecutive
exposure to PAN
+ 0,
Number and
Gender of Subject
Subjects Characteristics Observed Effect(s) Reference
3 M Healthy NS
5 F Mean age =
24 years
FEVj decreased =19% with 03 alone, Drechsler-Parks et al. (1987b)
* 15% on Day 1 of 03 + PAN, =5% on (also see Table 7-13)
Day 5 of Oj + PAN, =7% 3 days after
5 days of 03 + PAN, =15% after 5 days
of 03 + PAN. Similar to O3 adaptation
studies, 03 responses peaked after 2 days,
were depressed 3 days later, and responses
returned 7 days later. PAN probably had
no effect on adaptation to 03.
See glossary of terms and symbols for abbreviations and acronyms.
Listed from lowest to highest 03 concentration.
Age range in years or as mean + SEM.
71
-------
1 year. The subjects were exposed to 0.18 ppm O3 on four occasions, spring, fall, winter, and
2 the following spring. Only 17 subjects (8 responders) participated hi the final spring
3 exposures. The marked difference in FEVj response seen initially (-12.4% versus +1%)
4 was no longer present after the summer smog season (fall test) or 3 to 5 mo later (whiter
5 test). However, when the reduced subset of subjects was exposed during the following
6 spring, the responsive subjects again had significantly larger changes in FEVj. Seasonal
7 changes in FEVj response to O3 in the responsive and nonresponsive subjects are shown
8 below.
9
10 AFEV^ Spring AFEVj Fall AFEX^ Winter AFEVj Spring
(mL) (mL) (mL) (mL)
11 Responders -385 -17 +16 -347
12 Nonresponders +28 +90 +34 +81
13 =^^^======^^=====^^
14
15 These results suggest a seasonal variability hi response that may be attributed to
16 increased ambient O3 exposure during the summer months. It must be noted that the
17 "responders" included subjects who had a history of complaints from ambient air pollution.
18 Furthermore, this group included a significant proportion of allergic individuals whose
19 seasonal allergies could have contributed to their varying responses. Historically, however,
20 studies with the subjects drawn from the population of Los Angeles residents have reported
21 reduced responses to O3 exposure in the laboratory compared to nonresidents (Hackney
22 etal., 1976, 19775).
23 Brookes et al. (1989) recently reexamined a hypothesis previously tested 5y Gliner
24 et al. (1983), namely that repeated exposure to one concentration can alter response to
25 subsequent exposure to a different O3 concentration. Gliner et al. (1983) had previously
26 shown that the response to 0.40 ppm O3 was not influenced by previously being exposed to
27 0.20 ppm O3 for 2 h on 3 consecutive days. Brookes et al. (1989) tested whether exposure
28 to 0.20 or 0.35 ppm O3 would change subsequent response to 0.20 or 0.35 ppm O3. They
29 found increased responses to 0.20 ppm for both preexposures (AFEVj = -5.02%, -7.80%,
30 and -8.74% foF 0.20 ppm acutely, 0.20 ppm after 0.20 ppm, and 0.20 ppm after 0.35 ppm,
31 respectively), but this trend was significant only for the higher concentration. Although not
December 1993 7-60 DRAFT-DO NOT QUOTE OR CITE
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1 statistically significant, the response increase seen on the second exposure day at 0.20 ppm is
2 similar to that seem by Gliner et al. (1983). These observations suggest that although
3 preexposure to low concentrations of O3 may not influence response to higher concentrations,
4 preexposure to a high concentration of O3 may significantly increase response to a lower
5 concentration on the following day.
6 Schonfeld et al. (1989) confirmed previous observations of Bedi et al. (1985) and
7 Folinsbee et al. (1986) that the period of enhanced responsiveness to Oj following an initial
8 exposure persists for about 24 to 48 h, but is absent by 72 h after the initial exposure. In a
9 series of paired exposures to 0.35 ppm with continuous heavy exercise separated by intervals
10 of 1, 2, 3, or 4 days, they found that the responses to the second exposure were clearly
11 increased at 24 h (AFEVj = —16.1% and —30.4% for the first and second exposures,
12 respectively) and possibly also at 48 h (^FEVl = -14.4% and -20.6%). Similar trends
13 were observed for other physiological variables such as SRaw and respiratory pattern during
14 exercise. With a 3- or 4-day interval between exposures, the responses to the two exposures
15 were similar.
16 Foxcroft and Adams (1986) demonstrated that decrements in exercise performance seen
17 after a 1-h exposure to 0.35 ppm (continuous heavy exercise) were less after 4 consecutive
18 days of O3 exposure than they were after a single acute exposure. Maximal aerobic power
19 and performance time on a progressive bicycle exercise test were reduced 6% and
20 42 s respectively, from FA control, after a single 0.35 ppm exposure. After 4 consecutive
21 days of 1-h exposures, the maximal aerobic power was reduced only 4% and the
22 performance time by only 14 s; these differences from FA control were not statistically
23 significant. Despite the change in exercise performance, Foxcroft and Adams (1986) did not
24 show the attenuation of FEVj response seen in many previous studies (Folinsbee et al., 1980;
25 Linn et al., 1982b). However, these investigators selected known O3-sensitive subjects
26 whose FEY^ decrements exceeded 30% on the first 3 days of exposure. The large
27 magnitude of these responses, the trend for the responses to decrease on the third and fourth
28 day, the decreased symptom responses, and the observations of Horvath et al. (1981) that
29 O3-sensitive subjects adapt more slowly, suggest that attenuation of response would have
30 occurred if the exposure series had been continued for another 1 or 2 days. These
31 observations support the contention advanced by Horvath et al. (1981) that the progression of
December 1993 7-61 DRAFT-DO NOT QUOTE OR CITE
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1 attenuation of response is a function of "O3 sensitivity." Furthermore, these results suggest
2 that exercise responses after O3 exposure may be limited, either voluntarily or involuntarily,
3 more by subjective symptoms than by alterations in gas exchange consequent to changes in
4 ventilatory function.
5 Bedi et al. (1989) examined the responses of elderly subjects (median age, 65 years) to
6 four exposures to 0.45 ppm O3 for 2 h with mild intermittent exercise. The first three
7 exposures were on consecutive days, with the fourth exposure following the third by 3 days.
8 Changes in FEVj on the first two exposure days averaged —5.8% and —5.6%, about half
9 the response expected in a group of healthy young males (-12.7%; Folinsbee et al., 1978).
10 There were no significant changes in FEY^ on the third (-1.9%) and fourth (-1.7%)
11 exposure days. Symptom responses were negligible, although there was an overall increase
12 in symptoms on the first day of O3 exposure compared to air exposure. Despite the high
13 concentration of the exposure, there was no enhancement of the spirometry response on the
14 second day of exposure. Although similar observations have been made in previous studies
15 producing small changes in spirometry (Folinsbee et al., 1980, 1993a) with repeated
16 exposures, the responses of older subjects are not sufficiently understood to explain these
17 responses. Bedi et al. (1988) had previously reported that responses to O3 in the older
18 subjects tended to be less reproducible, although this observation alone could not explain
19 these responses.
20 Drechsler-Parks et al. (1987b) examined response to repeated exposures to 0.45 ppm
21 O3 plus 0.30 ppm peroxyacetyl nitrate (PAN). Exposures to O3 and O3 plus PAN yielded
22 similar changes in spirometry (AFEVj = —19% and —15%, respectively). Thus, PAN did
23 not increase responses to O3. Repeated exposure to the PAN plus O3 mixture resulted in
24 similar changes as those that would be seen with Oj exposure alone. Responses in FEVj
25 exceeded -30% on the second exposure and fell to less than -5% after the fifth day. The
26 attenuation of response persisted 3 days after the repeated exposures, but was absent after
27 7 days. These observations suggest that PAN does not influence the attenuation response to
28 repeated O3 exposure. If the PAN responses are considered negligible, this study confirms
29 the observation that the attenuation of O3 responses with chamber exposures lasts no longer
30 than 1 week.
December 1993 7-62 DRAFT-DO NOT QUOTE OR CITE
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1 Repeated prolonged exposure to low concentrations of O3 has recently been examined
2 (Horvath et al., 1991; Folinsbee et al., 1993b; Linn et al., 1991, 1992). Horvath et al.
3 (1991) exposed subjects for 2 consecutive days to 0.08 ppm using the 6.6-h prolonged-
4 exposure protocol (see Section 7.2.2). They observed small pre- to postexposure changes in
5 FEVj (—2.5%) on the first exposure, but no change on the second day. Linn et al. (1993)
6 observed a 1.7% decrease in FEVj after a 6.5-h exposure to 0.12 ppm in healthy subjects.
7 A second consecutive exposure yielded even smaller responses. With exposure to a mixture
8 of O3 plus 100 jitg/m3 of sulfuric acid aerosol, there was a 4.2% decrease in FEVl on the
9 first exposure day. In a group of asthmatics exposed under similar conditions, the FEVj
10 response on the first day was -8.6% (O3) and -11.6% (O3 plus acid). After adjustment for
11 the exercise effect (-4.6%), the responses (-4% and -7%) were still greater than those of
12 nonasthmatics. Responses were slightly reduced on the second day of exposure.
13 Folinsbee et al. (1993b) exposed 17 subjects to 0.12 ppm O3 for 6.6 h on 5 consecutive
14 days. Spirometry responses were typified by changes in FEV1 that reached -13% on the
15 first day and -9% on the second day of exposure. No significant differences in spirometry
16 responses between FA and subsequent O3 exposures were observed. Symptom responses
17 were also greatest on the first exposure day and were largely absent from the third day on.
18 Methacholine responsiveness was tested using a single dose of methacholine and then
19 comparing changes (as a ratio) in airway resistance between methacholine and saline
20 aerosols. The responses to FEVj and methacholine testing are shown below.
21 Methacholine responsiveness was increased (over the clean air response) throughout the
22 5 days of O3 exposure, although it reached a peak on the second day and there was a trend
23 for responsiveness to decrease after 5 days in some subjects. These results suggest that
24 repeated exposure to low levels of O3, despite the attenuation of symptoms and pulmonary
25 function changes, is not without hazard. It is likely that some epithelial damage persists that
26 contributes to the enhanced response to methacholine throughout the exposure series.
27 However, it must be noted that, in this study, subjects were initially selected based on their
28 FEVj response to 0.16 ppm O3 for 4 h. This may in part explain the greater FEVt
29 responses seen in this study, but there was no correlation between individual FEVj
30 decrements and changes in methacholine responsiveness. Furthermore, the Horvath et al.
31 (1991) subjects were exposed to only 0.08 ppm and they were somewhat older than the
December 1993 7-63 DRAFT-DO NOT QUOTE OR CITE
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1 Folinsbee et al. (1993b) subjects; the Linn et al. (1991) subjects, on the other hand, had
2 lower ventilation during exercise and were residents of Los Angeles accustomed to exposure
3 to these levels of O3 (see Chapter 4 for typical O3 concentrations).
4
5
6
7
8
9
10
A%FEV,
SRaw Ratio
Based on
Day
-12.
3.
1
79
67
studies cited
Day 2
-8.73
4.55
here and in
Day 3
-2.54
3.99
the previous
Day
-0.
3.
4
6
24
criteria document
Day
+0.
3.
5
Clean
2 +1.
74 2.
(U.S.
Air
1
22
Environmental
11 Protection Agency, 1986), several conclusions can be drawn about repeated 1- to 2-h
12 O3 exposures. Repeated exposures to O3 can cause an enhanced (i.e., greater) response on
13 the second day of exposure. This enhancement of response appears to be dependent on the
14 interval between the exposures (24 h causes the greatest increase) and is absent with intervals
15 >3 days. An enhanced response also appears to depend to some extent upon the magnitude
16 of the initial response. Small responses to the first O3 exposure are less likely to result in an
17 enhanced response on the second day of O3 exposure. Repeated daily exposure also results
18 in attenuation of spirometric responses, typically after 3 to 5 days of exposure. This
19 attenuated response persists for less than 1 or possibly 2 weeks. In temporal conjunction
20 with the spirometry changes, symptoms induced by O3, such as cough and chest discomfort,
21 are also attenuated with repeated exposure. Ozone-induced changes in airway responsiveness
22 attenuate somewhat more slowly than spirometric and symptom responses. Attenuation of
23 the changes in airway responsiveness may also persist longer than changes in spirometry,
24 although this has only been studied on a limited basis. In longer duration-lower
25 concentration studies that do not cause an enhanced second-day response, the attenuation of
26 response to O3 appears to proceed more rapidly.
27
28 7.2.1.5 Effects on Exercise Performance
29 Introduction
30 An early epidemiological study examining race performances in high school cross-
31 country runners (Wayne et al., 1967) suggested that exercise performance is depressed by
32 inhalation of ambient oxidant air pollutants. Wayne et al. (1967) suggested that the
December 1993 7-64 DRAFT-DO NOT QUOTE OR CITE
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1 detrimental effects of oxidant air pollutants on race performance may have been related to
2 increased airway resistance or to the associated discomfort in breathing, thus limiting
3 runners' motivation to perform at high levels. The effects of acute O3 inhalation on exercise
4 performance have been evaluated in numerous controlled human studies. These studies can
5 be divided into two categories, those that examine the effects of acute O3 inhalation on
6 maximal oxygen uptake and those that examine the effects of acute O3 inhalation on the
7 ability to complete strenuous continuous exercise protocols up to 1 h in duration. Five
8 studies (Folinsbee et al., 1977b; Horvath et al., 1979; Folinsbee et al., 1984; Adams and
9 Schelegle, 1983; Savin and Adams, 1979) examining the affects of acute O3 exposures on
10 exercise performance were discussed in the 1986 EPA criteria document (U.S.
11 Environmental Protection Agency, 1986). This section summarizes the studies reviewed in
12 that document and reviews newer studies published since that examine the effect of acute
13 O3 inhalation on (1) maximal oxygen uptake and (2) endurance performance. Studies are
14 also summarized in Table 7-8.
15
16 Effect on Maximal Oxygen Uptake
17 Three studies (Folinsbee et al., 1977b; Horvath et al., 1979; Savin and Adams, 1979)
18 examining the effects of acute O3 exposures on VO2max were discussed in the 1986 EPA
19 criteria document (U.S. Environmental Protection Agency, 1986). Of these studies, only one
20 showed a reduction in VO2max. Folinsbee et al. (1977b) observed that VO2max was
21 significantly (p < 0.01) decreased 10.5% following a 2-h exposure to 0.75 ppm O3 with
22 light IE. Reductions in VO2max were accompanied by a 9.5% decrease in maximum
23 attained workload (p < 0.01), a 16% decrease in maximum ventilation (p < 0.01), and a
24 6% decrease in maximum heart rate (p < 0.05). The 16% decrease in maximum ventilation
25 was associated with a 21 % decrease in VT. In addition, the O3 exposure resulted in a
26 22.3% decrease in FEVj and subjective symptoms of cough and chest discomfort.
27 In contrast, Horvath et al. (1979) did not observe a change in VO2max or other maximum
28 cardiopulmonary endpoints in male and female subjects exposed at rest to 0.75 ppm O3 for
29 2 h, although FVC was significantly decreased 10% (p < 0.05). Similarly, Savin and
30 Adams (1979) observed no effect on maximum attained workload or VO2max in nine
31 subjects exposed to 0.30 ppm O3 while performing a progressively incremented exercise test
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TABLE 7-8. OZONE EFFECTS ON EXERCISE PERFORMANCE3
g Ozone
g. Concentration
cs
>— ppm
S 0.00
0.06-
0.07
0.12-
0.13
0.00
0.12
0.18
0.24
0.00
0.12
0.20
ON 0.00
°^ 0.20
0.35
O
S 0.00
jri °-2i
i
y o.oo
U 0.25
§ 0.00
H 0-25
o °-50
C °'75
Exposure
Duration and Exposure
fig/m Activity Conditions
0
120-
140
245-
260
0
235
353
470
0
235
392
0
392
686
0
412
0
490
0
490
980
1,470
CE ( VE = Tdb = 23-24.5 °C;
30-120 L/min) RH = 50-53%
16-28 min
progressive
maximum
exercise protocol
1 h competitive Tdb = 23-26 °C
simulation RH = 45-60%
exposures at
mean VE =
87 L/min
1 h CE Tdb = 31 °C
VE = 89 L/min
1 h CE or Tdb = 23-26 °C
competitive RH = 45-60%
simulation at
mean VE = 77.5
L/min
1 h CE at 75% Tdb = 19-21 °C
VO2max RH = 60-70%
1 h CE Tdb = 20 °C
VE = 63 L/min RH = 70%
2 h rest NA
Number
and
Gender of Subjects
Subjects Characteristics
12 M Athletic
12 F
10 M Highly trained
competitive
cyclists
15 M Highly trained
2 F competitive
cyclists
10 M Well-trained
distance runners
6 M Well-trained
1 F cyclists
19 M Active
7 F nonathletes
8M
5F
Observed Effect(s) References
Reduced maximum performance time and increased Linder et al. (1988)
respiratory symptoms during 03 exposure.
Decrease in exercise time of 7.7 min and 10.1 min Schelegle and Adams (1986)
for subjects unable to complete the competitive
simulation at 0.18 and 0.24 ppm 03, respectively;
decrease in FVC and FEVj for 0.18- and 0.24-ppm
03 exposure compared with FA exposure.
Decrease in VEmax, VO2max, VTmax, workload, Gong et al. (1986)
ride time, FVC, and FEVj with 0.20 ppm 03
exposure, but not significant with 0.12-ppm 63
exposure, as compared to FA exposure.
VT decreased and f increased with continuous 50- Adams and Schelegle (1983)
min 03 exposures; decrease in FVC, FEVj, and
FEFy.75% from FA to 0.20 ppm and FA to 0.35
ppm O3 exposure in all conditions; three subjects
unable to complete continuous and competitive
protocol at 0.35 ppm 03.
Decrease in FVC, FEVj, FEF25.75sj, and MW Folinsbee et al. (1984)
with 0.21 ppm Oj compared with FA exposure.
FVC, FEVj, and MW all decreased with 0.25 ppm Folinsbee et al. (1986)
Oj exposure compared with FA.
FVC decreased with 0.50- and 0.75-ppm 03 Horvath et al. (1979)
exposure compared with FA; 4% nonsignificant
decrease in mean VO2max following 0.75 ppm 03
compared with FA exposure.
-------
TABLE 7-8 (cont'd). OZONE EFFECTS ON EXERCISE PERFORMANCE8
g Ozone
1" Concentration1" Exoosure
JJj
f£ ppm
S °-00
0.35
0.00
0.75
Duration and Exposure
jUg/m Activity Conditions
0 50 min CE NA
686 VE = 60 L/min
0 2 h IE NA
1,470 (4 X 15 min light
[50 W] bicycle
ergometry)
Number
and
Gender of Subjects
Subjects Characteristics
8 M Trained
nonathletes
13 M 4 light S,
9NS
Observed Effect(s)
VT decreased, f increased with 50-min O3
exposures; decrease in FVC, FEVj, FEF25-75%>
performance time, VO2max, VEmax, and HR,,^
from FA to 0.35 ppm Oj exposure.
Decrease in FVC, FEVj, ERV, 1C, and FEF50%
after 1-h 0.75 ppm 03 exposure; decrease in
VO2inax, Vijinax, Vgmax, maximal workload
and heart rate following 0.75 ppm 03 exposure
compared with FA.
References
Foxcroft and Adams (1986)
Folinsbee et al. (1977b)
See glossary of terms and symbols for abbreviations and acronyms.
Listed from lowest to highest 03 concentration.
<1
-0
-------
1 to volitional fatigue lasting 30 min. In addition, Savin and Adams (1979) observed no
2 significant effect on pulmonary function, performance time, maximum heart rate, or
3 anaerobic threshold, although maximum ventilation was significantly reduced 7%.
4 More recent findings of Foxcroft and Adams (1986) and Gong et al. (1986) support the
5 earlier observations of Folinsbee et al. (1977b). Foxcroft and Adams (1986) observed
6 significant (p < 0.05) reductions in performance time (16.7%), VO2max (6.0%), maximum
7 ventilation (15.0%), and maximum heart rate (5.6%) in eight aerobically trained males
8 during a rapidly incremented VO2max test following 50 min exposure to 0.35 ppm O3 with
9 CE (exercise VE = 60 L/min). Similarly, Gong et al. (1986) found significant (p < 0.05)
10 reductions in performance tune (29.7%), VO2max (16.4%), maximum ventilation (18.5%)
11 and maximum workload (7.8%) in 17 top-caliber endurance cyclists during a rapidly
12 incremented VO2max test following 1-h exposure to 0.20 ppm O3 with very heavy
13 CE (VE = 90 L/min) and the addition of ambient heat stress (31 °C). In both studies
14 (Foxcroft and Adams, 1986; Gong et al., 1986), the reductions in maximal exercise
15 endpoints were accompanied by significant decrements in pulmonary function and marked
16 subjective symptoms of respiratory discomfort. More recently, Linder et al. (1988) observed
17 small decrements in performance time during a progessive maximal exercise test at
18 O3 concentrations as low as 0.06 ppm. These small effects were associated with increased
19 respiratory symptoms and small, inconsistent changes in FEVj. Hence, it appears that
20 maximal oxygen uptake is reduced if it is preceded by an O3 exposure entailing a sufficient
21 total inhaled dose of O3 to result in significant pulmonary function decrements and/or
22 subjective symptoms of respiratory discomfort.
23
24 Effect on Endurance Exercise Performance
25 Two studies (Adams and Schelegle, 1983; Folinsbee et al., 1984) that addressed the
26 effects of acute O3 exposures on the ability of highly trained subjects to complete strenuous
27 continuous exercise protocols were discussed in the 1986 EPA criteria document (U.S.
28 Environmental Protection Agency, 1986).
29 Adams and Schelegle (1983) exposed 10 well-trained distance runners to filtered air,
30 0.20 ppm O3, and 0.35 ppm O3 while the runners exercised on a bicycle ergometer at
31 workloads simulating either a 1-h steady-state "training" bout or a 30-min warm-up followed
December 1993 7-68 DRAFT-DO NOT QUOTE OR CITE
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1 immediately by a 30-min "competitive bout". The exercise levels in the steady-state training
2 bout were of sufficient magnitude (68% of their VO2max) to increase mean VE to 80 L/min.
3 The VE averaged over the entire competitive simulation was also 80 L/min, whereas the
4 mean VE during the 30-min competitive bout was 105 L/min. Subjective symptoms
5 increased as a function of O3 concentration for both training and competitive protocols.
6 In the competitive protocol, four runners exposed to 0.20 ppm O3 and nine exposed to
7 0.35 ppm O3 indicated that they could not have performed maximally. Three subjects were
8 unable to complete both the training and competitive protocols at 0.35 ppm O3, and a fourth
9 failed to complete only the competitive ride.
10 Folinsbee et al. (1984) exposed six well-trained men and one well-trained woman to
11 0.21 ppm O3 while they exercised continuously on a bicycle ergometer for 1 h at 75% of
12 their VO2max (VE = 81 L/min). Following O3 exposure, FVC and FEVl were
13 significantly reduced and the subjects reported symptoms of laryngeal and/or trachea!
14 irritation and chest soreness and tightness upon taking a deep breath. Anecdotal reports
15 obtained from the cyclists suggested that their performance would have been limited if they
16 experienced similar symptoms during competition.
17 Avol et al. (1984) exposed 50 well-conditioned cyclists to 0.0, 0.08, 0.16, 0.24, and
18 0.32 ppm O3 for 1 h in ambient heat (32 °C) while exercising continuously
19 (VE = 57 L/min). Reductions in FEVj and symptoms increased in a concentration-
20 dependent manner, being initially detected at 0.16 ppm O3. Three and 16 cyclists could not
21 complete the 1-h exposure to 0.16 and 0.24 ppm O3, respectively, without a reduction in
22 workload. Similarly, in their study of the effects of O3 exposure on VO2max, Gong et al.
23 (1986) reported that 6 of 17 highly trained endurance cyclists were not able to complete 1-h
24 exposure to 0.20 ppm O3 with very heavy CE (VE = 90 L/min) and the addition of ambient
25 heat stress (31 °C).
26 In a study designed to determine the effects of the inhalation of low ambient
27 O3 concentrations on simulated competitive endurance performance, Schelegle and Adams
28 (1986) exposed 10 highly trained endurance athletes to 0.12, 0.18, and 0.24 ppm O3 while
29 performing a 1 h "competitive" protocol. The competitive protocol used in this study was
30 similar to that used by Adams and Schelegle (1983) except that the workload during the final
31 30 min competitive bout was more intense, being selected based on the maximum workload
December 1993 7.59 DRAFT-DO NOT QUOTE OR CITE
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1 (approximately 86% of their VO2max, mean VE = 120 L/min) each subject could maintain
2 for 30 min while breathing FA. All subjects completed the FA exposure, whereas one, five,
3 and seven subjects could not complete the 0.12-, 0.18-, and 0.24-ppm Oj exposures,
4 respectively. Following 0.18- and 0.24-ppm O3 exposures, FVC and FEVj were
5 significantly reduced (p < 0.05) and subjective symptoms were significantly elevated
6 (p < 0.05). No significant effect of O3 was found for metabolic or ventilatory pattern
7 responses. Similarly, Folinsbee et al. (1986) found that highly trained runners experienced a
8 reduced run time on a treadmill (speed and grade set at approximately 80% of their subjects
9 VO2max) when exposed to 0.18 ppm O3 compared with filtered air. These subjects did
10 have significantly elevated symptoms of respiratory discomfort and significantly decreased
11 FVC and FEVl5 whereas arterial oxygen saturation at the end of the run was not affected by
12 O3 exposure.
13 Determining the mechanisms leading to the observed decrements in maximal oxygen
14 uptake and the inability to complete strenuous exercise protocols is problematic. As stated
15 by Astrand and Rodahl (1977) "the capacity for prolonged rhythmic muscular exercise is
16 limited by an interrelated composite of cardiorespiratory, metabolic, environmental, and
17 psychological factors." Many investigators cited above have concluded that the observed
18 reductions in exercise performance appeared to be due to symptoms limiting the ability of
19 their subjects to perform. However, in every case, this is a conclusion achieved by
20 exclusion and not by the demonstration of a causal relationship. Other factors could also
21 contribute to O3-induced decrements in exercise performance. One possibility is that
22 stimulation of neural receptors in the airways may result in an inhibition of alpha-motor
23 nerve activity to respiratory muscles during inspiration (Koepchen et al., 1977; Schmidt and
24 Wellhoner, 1970), resulting in the observed decrease in VT and at the same time increasing
25 the subject's sensation of respiratory effort. This mechanism would not be directly related to
26 symptoms of discomfort, but because of the common role of airway neural afferents, may be
27 difficult to discern from the affects of symptoms of respiratory discomfort. Indeed, a reflex
28 inhibition of the ability to inspire would be consistent with the reduced VTs following
29 O3 exposure in subjects performing maximal exercise and would be consistent with the
30 development of a physiologically induced ventilatory limitation to maximal oxygen uptake.
31
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1 7.2.2 Pulmonary Function Effects of Prolonged (Multihour) Ozone
2 Exposures
3 Since 1988, a series of studies have described the responses of subjects exposed to
4 relatively low O3 concentrations from 0.08 to 0.16 ppm for durations of 4 to 8 h (see
5 Table 7-9). These studies have demonstrated statistically significant changes in spirometry,
6 airway resistance, symptoms, and airway responsiveness during and after such exposures.
7 The only related study to be cited in the previous criteria document (U.S.
8 Environmental Protection Agency, 1986) was that of Kerr et al. (1975), who exposed
9 subjects for 6 h to 0.50 ppm, but with only two brief 15-min periods of moderate exercise
10 during the exposure. Small changes in spirometry were observed. Because of the minimal
11 exercise level and the high O3 concentration, these results cannot be compared to the more
12 recent studies.
13 The first prolonged exposure study involving low concentrations and a substantial
14 amount of moderate exercise was reported by Folinsbee et al. (1988). The basic protocol
15 used by these investigators has been used in a number of subsequent investigations and is
16 worth describing in some detail. The exposures lasted 6 h and 35 min (i.e., 6.6 h). Except
17 for a 35-min lunch break (during which 03 exposure continued at rest) after 3 h, the subjects
18 exercised at a moderate level (with a ventilation of about 40 L/min) for 50 min of each hour.
19 Pulmonary function tests were conducted during the 10-min rest period and at the beginning
20 and end of exposure. The exposure was intended to simulate a day of heavy outdoor work
21 or play. For convenience, this protocol is referred to as the EPA prolonged-exposure
22 protocol.
23 In this study (Folinsbee et al., 1988), a group of 10 subjects was exposed to clean air
24 and 0.12 ppm O3 for 6.6 h. Forced vital capacity and FEVl decreased in a roughly linear
25 fashion throughout the exposure and had fallen by 8.3 and 13%, respectively, by the end of
26 the exposure. Symptoms of cough and chest discomfort were increased and airway
27 responsiveness to methacholine was approximately doubled after O3 exposure. There was
28 a wide range of response and three subjects had FEVj decrements of 25% or greater,
29 whereas the three least sensitive subjects had less than 5% change in FEVj.
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2
I
§
TABLE 7-9. PULMONARY FUNCTION EFFECTS AFTER PROLONGED EXPOSURES TO OZONE3
Ozone Concentration
ppm
0.08
0.10
0.12
Hg/m Duration and Activity Conditions
157 6.6 h 18 °C
196 IE (6 x 50 min) 40% RH
235 VE « 39 L/min
Number and
Subjects Characteristics Observed Effect(s)
22 M Healthy NS FVC and FEVj decreased throughout the
exposure; FEVj decrease at end exposure
was 7.0, 7.0, and 12.3%, respectively.
FEVj change > 15% occurred in 3, 5, and
9 subjects at 0.08, 0.10, and 0.12 ppm,
respectively. Methacholine responsiveness
increased by 56, 89, and 121 %, respectively.
Reference
Horstman et al. (1990)
See Horstman et al. (1990)
and Folinsbee et al. (1988)
A lognormal model was fitted to FEVj data. Larsen et al. (1991)
Model parameters indicate Oj concentration
had greater effect than VE or duration
(estimated exponent for [Oj] = 4/3).
See Folinsbee et al. (1993b)
See Folinsbee et al. (1993b). Slight
increases in clearance of ^T"c-DTPA
occurred after 03 exposure (t = 10%).
Suggests possible increase in permeability.
Kehrl et al. (1991)
0.08
0.10
0.08
0.12
(a) 0.12
(b) Varied from 0.0
to 0.24 (increased
by 0.06 ppm/h then
decreased by
0.06 ppm/h)
See McDonnell et al. (1991a)
157 6.6 h
196 IE (6 x 50 min)
VE = 40 L/min
157 6.6 h
IE (6 x 50 min)
VE = 35-38 L/min
(1 day of air, 2 days of Oj)
235 6.6 h
IE (6 x 50 min)
VE = 42.6 L/min
235 8h
IE (8 x 30 min)
VB = 40 L/min
18 °C
40% RH
25 °C
48% RH
18 °C
40% RH
(1 exp. to clean
air; 1 exp to 03)
22 °C
40% RH
<3 ftg/m TSP
38 M Healthy NS
Mean age =
25 years
5 F Healthy NS
6 M Age = 30-45 years
10 M Healthy NS
23 M Healthy NS
No change in ySmTc-DTPA clearance. Kehrl et al. (1989b)
Possible masking of response by exercise.
FEVj, decreased 8.4% at 0.08 ppm and McDonnell et al. (1991a)
11.4% at 0.10 ppm. Symptoms of cough,
PDI, and SB increased with Oj exposure.
FVC decreased 2.1%, FEVj decreased 2.2% Horvath et al. (1991)
on first day of Oj exposure; no change on
second Oj day.
FEVj decreased by 13 % after 6.6 h. FVC Folinsbee et al. (1988)
dropped 8.3 % . Cough and PDI increased
with Oj exposure. Airway responsiveness to
methacholine doubled after 03 exposure
(t 100%).
(a) FEVj decreased 5% by 6 h and remained Hazucha et al. (1992)
at this level through 8 h.
(b) FEVj change mirrored 03 concentration
change with a lag time of ~ 2 h. Max
decrease of 10.2% after 6 h. FEVj change
was reduced in last 2 h of exposure.
-------
O TABLE 7-9 (cont'd). PULMONARY FUNCTION EFFECTS AFTER PROLONGED EXPOSURES TO OZONE4
3 Ozone Concentration
£5 PPm lig/m Duration and Activity
^ 0.12 235 6.5h/day
'O IE (6 x 50 min)
(2 days of exposure)
VE = 3 1 L/min (asthmatic)
VE = 34 L/min (healthy)
0.12 235 6.6 h
IE (6 x 50 min)
VE = 38.8 L/min
0.16 314 4h
IE (4 x 50 min)
VE = 38.9 L/min
Exposure
Conditions
21 °C
50% RH
18 °C
40% RH
(5 consecutive
days of exposure
to 03, 1 day
exposure to C A) .
18 °C
40% RH (one
exposure to 63,
no control
exposure).
Number and
Gender of Subject
Subjects Characteristics Observed Effect(s)
15
(8M,
7F)
30
(13 M,
17 F)
17 M
15 M
Healthy NS Bronchial reactivity to methacholine increased with
Age 22-41 03 exposure in healthy subjects. FEVj decreased 2%
(pre- to postexposure) in healthy subjects and 7.8% in
Asthmatic NS asthmatics. Responses were generally less on the
Age 18-50 second day. Two healthy and four asthmatics had
FEVj decreases >10%.
Healthy NS FEVj decreased by 12.8, 8.7, 2.5, 0.6, and +0.2 on
Days 1-5 of 03 exposure. Methacholine airway
responsiveness increased by > 100% on all exposure
days. Symptoms increased on the first O3 day, but
were absent on the last three exposure days.
Healthy NS FVC decreased 9.5% and FEVt decreased 16.6% .
FEVj/FVC ratio decreased from 0.79 to 0.73.
Reference
Linn et al. (1993)
Folinsbeeetal. (1993b)
Folinsbee et al. (1993b)
-J a
U See glossary of terms and symbols for abbreviations and acronyms.
Age range in years or as mean + SEM.
-------
1 In order to extend these initial observations, Horstman et al. (1990) used the same
2 protocol to expose a group (n = 22) of subjects to clean air and three different
3 O3 concentrations (0.08, 0.10, and 0.12 ppm). Similar responses were shown at 0.12 ppm
4 as in the previous study, with the exception that the symptom responses were less in the new
5 group of subjects. A similar pattern of response in spirometry, airway resistance, and
6 airway responsiveness, but of smaller magnitude, was seen at the two lower concentrations.
7 The mean FEVi responses during the four exposures are shown in Figure 7-3. The
8 responses were dependent on concentration and exposure duration (ventilation was not
9 varied) and averaged 7, 8, and 13% at the three O3 concentrations. Larsen et al. (1991)
10 have used these data (Horstman et al., 1990) to develop a "dose-response" relationship for
11 percent change in FEV^ as a function of O3 concentration and exposure duration. The
12 lognormal multiple linear regression model suggested that FEVj responses were
13 approximately linear with duration of exposure but that O3 concentration plays a more
14 important role. The exponent of approximately 4/3 suggests that doubling O3 concentration
15 would be similar to increasing exposure duration by about 2'/2 times.
16 A series of additional studies were conducted at 0.08 and 0.10 ppm. The primary aim
17 of these studies was to study changes in cells and inflammatory mediators from BAL (see
18 Section 7.2.4). McDonnell et al. (1991a) reported an 8.4% decrease in FEVl at 0.08 ppm
19 and an 11.4% decrease at 0.10 ppm. These responses were slightly larger than seen in the
20 previous Horstman et al. (1990) study. The duration—FEVj response data were fit to a
21 three parameter logistic model, which significantly improved the amount of variance
22 explained by the model compared to a linear model. This is consistent with exploratory
23 analyses in the Folinsbee et al. (1988) report. The reasonably good fit to the logistic model
24 suggests that the O3-pulmonary function response relationship may have a sigmoid shape.
25 The main importance of this observation is that it suggests there is a response plateau. That
26 is, there is some level of response (i.e., plateau) to a given O3 concentration and exercise
27 ventilation level (i.e., dose rate) beyond which the FEV^ response tends not to increase with
28 increasing duration of exposure.
29 In the fourth study in this series (Folinsbee et al., 1993b), 17 subjects were exposed to
30 0.12 ppm for 6.6 h on 5 consecutive days. Subjects who were not responsive to O3 were not
31 selected to participate in this study. Responses in FEV] on the first of these exposures
December 1993 7.74 DRAFT-DO NOT QUOTE OR CITE
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FEV, During Air and Ozone Exposure
4,500
4,400
4,300
^ 4,200
:>" 4,100
UJ
LJ.
4,000
3,900
3,800
Air
0.08 ppm
O.IOppm
0.12 ppm
12345
Exposure Duration (h)
Figure 7-3. The forced expiratory volume in one second (FEV}) is shown hi relation to
exposure duration (hours) at four different O3 concentrations:
(1) — <0.01 ppm, (2) • = 0.08 ppm, (3) • = 0.10 ppm, and (4) A =
0.12 ppm. A 35-min resting exposure period was interposed between the
end of third and the beginning of the fourth hour. There were six 50-min
exercise periods (minute ventilation « 39 L/min) during the exposure; these
measurements were made 5 min after the end of each exercise. The total
exposure duration was 6.6 h. The standard error of the mean (not shown)
for these FEV} averages ranged from 120 to 160 mL.
Source: Horstman et al. (1990).
1 averaged -12.8%. Again, symptom responses were modest with a significant increase in a
2 lower respiratory symptom score on the first exposure day. A significant increase in airway
3 responsiveness to methacholine was also shown. The response to the repeated exposures is
4 discussed in Section 7.2.1.4 on repeated exposures to 03. In addition, 15 subjects were
December 1993
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1 exposed to 0.16 ppm for 4 h using the same hourly exposure protocol as described above.
2 In these subjects, FVC decreased -9.5% and FEVj declined -16.6%.
3 Folinsbee et al. (1991) took the FEVl response data from all four studies conducted at
4 the EPA Health Effects Research Laboratory, using the same prolonged exposure protocol,
5 and examined the distribution of responses among the subjects at the three concentrations.
6 This response distribution is illustrated graphically in Figure 7-4. This figure illustrates that
7 FEVj decrements as large as 30 to 50% have been observed with prolonged exposure to
8 O3 concentrations equal to or less than 0.12 ppm. This response distribution also allows one
9 to determine the number or percentage of subjects with responses in excess of a certain level.
10 The proportion of subjects with an FEVj decrease in excess of 10% is shown. With air
11 exposure, no one exceeded this response level; however, 46% of the subjects exposed to
12 0.12 ppm using this exposure protocol had greater than a 10% drop hi FEVt after 6.6 h.
13 This response distribution also illustrates the wide range of response to O3 under these
14 exposure conditions and reinforces the observation by others (McDonnell et al., 1983;
15 Horvath et al., 1981) of a substantial range of individual response to O3.
16 Kehrl et al. (1989b) reported that clearance of radiolabeled diethylene triamine
17 pentacetic acid ( "Tc-DTPA) was not altered by the 0.08-ppm exposure (see McDonnell
18 et al., 1991a for exposure details). Subsequent studies (Kehrl et al., 1991) suggested that a
19 small acceleration of clearance may have occurred with exposure to 0.12 ppm for 6.6 h.
20 Increased clearance of labeled 99mTc-DTPA from the lung is thought to indicate increased
21 permeability of the respiratory epithelium (Kehrl et al., 1987). These preliminary data
22 (Kehrl et al., 1991) have not been published. Other summaries of the four studies from the
23 EPA laboratory, however, have been published (McDonnell et al., 1991b; Horstman et al.,
24 1988a,b; Horstman et al., 1991).
25 Horvath et al. (1991) examined the responses of healthy middle aged (ages 30 to 45)
26 men and women to 0.08 ppm O3 for 6.6 h using the EPA prolonged-exposure protocol.
27 When compared with the clean air exposure, FEVj decreased about 5 % with O3 exposure.
28 However, the variability of this response among this small heterogeneous group of subjects
29 was enough to preclude statistical significance of this observation. No significant changes
30 were observed with a second exposure on the next day. On the first day of Oj exposure,
December 1993 7-76 DRAFT-DO NOT QUOTE OR CITE
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Distribution of Percent Change in FEVj
= 87
AIR
i 10 15 20 2S 30 38 «
N-60
0.08
0510152025903543
Percent of Subjects
> 10% Drop
inFB< °%
26%
31%
46%
Figure 7-4. The distribution of response for 87 subjects exposed to clean air and at least
one of 0.08, 0.10, or 0.12 ppm is shown here. The O3 exposures lasted
6.6 h, during which time the subjects exercised for 50 min of each hour
with a 35-min rest period at the end of the third hour. The abscissa
indicates the number of subjects. The bar labels on the ordinate indicate
decreases in FEVj, expressed as percent change from baseline. For
example, the bar labeled 10 indicates the number of subjects with a
decrease in FEV, of >5% but ^10%; the bar labeled -5 indicates
improvement in FEVj of >0% but <5%. The rectangle across the bottom
of the graph indicates the percentage of subjects at each O3 concentration
with a decrease of FEVj hi excess of 10%.
Source: Folinsbee et al. (1991).
1 7 of the 11 subjects reported chest tightness. The authors point out that the range of
2 variability in response in their study was similar to that reported by Folinsbee et al. (1988)
3 and Horstman et al. (1990), although fewer of their subjects experienced large negative
4 changes in FEV!. One possible explanation for the differences between the findings of
5 Horvath et al. (1991) and Horstman et al. (1990) may be that the subjects in Horvath's study
6 were significantly older, which may result in reduced responses to O3, as Drechsler-Parks
7 et al. (1987a) have shown (see Section 7.2.1.3). The ventilation during exercise (37 to
8 39 L/min) was similar to that reported by Horstman et al. (1990). An additional FEVi
9 measurement was made in this study at the end of the lunch period (i.e., after 40 min of
10 rest). At this time, the small decrements in FEVj seen after the third exercise were reversed
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1 and the FEV^ was similar to the response in FA at the same time point. Although
2 spirometry was not measured at this time in the other prolonged-exposure studies (Folinsbee
3 et al., 1988; Horstman et al., 1990), it was noted that the decline in FEV^ was attenuated
4 between the third and fourth postexercise measurement. These observations suggest that
5 subject's lung function may indeed have improved during the lunch rest period.
6 Linn et al. (1991, 1992) have reported responses of healthy and asthmatic subjects to
7 0.12 ppm O3 using the EPA prolonged-exposure protocol. [These results are presently
8 available as a pair of abstracts and a preliminary project report.] They observed a small
9 (2.0%) decrease in FEV^ which was statistically significant, and an increase in airway
10 responsiveness to methacholine. The functional responses in asthmatics were greater than
11 those of the healthy subjects, a decrease in FEV^ of 7.9%. They observed smaller responses
12 on a second consecutive day of exposure, as did Horvath et al. (1991) and Folinsbee et al.
13 (1993b). The ventilation averaged about 34 and 31 L/min in the healthy and asthmatic
14 subjects, respectively. The FEVj responses observed in this study, although statistically
15 significant, are much lower than those observed by EPA investigators (Folinsbee et al.,
16 1988; Horstman et al., 1990; Folinsbee et al., 1993b). The smaller responses may be due to
17 previous ambient exposures, lower ventilations, or a larger proportion of O3-insensitive
18 subjects in Los Angeles. Only 1 of 15 healthy subjects experienced an FEVj decrement in
19 excess of 10%, whereas 9 of 30 asthmatics had an FEVj decrement in excess of 10%.
20 Asthmatic responses ranged from +12% to —35%.
21 To further explore the factors that determine responses to O3, Hazucha et al. (1992)
22 designed a protocol to examine the effect of a varying, rather than a constant,
23 O3 concentration. In this study, subjects were exposed to both a constant level of 0.12 ppm
24 O3 for 8 h and to an O3 level that ramped linearly from 0 to 0.24 ppm for the first 4 h and
25 then ramped linearly from 0.24 to 0 over the second 4 of the 8 h exposure (triangular
26 concentration profile). Subjects performed moderate exercise for the first 30 min of each
27 hour. The overall exposure dose for these two exposures, calculated as the O3 concentration
28 x exposure duration X ventilation, was almost identical (difference <1%). With exposure
29 to the constant 0.12 ppm O3, the FEVj declined approximately 5% by the fifth hour of
30 exposure and remained at that level for the remainder of the exposure. These responses are
31 illustrated in Figure 7-5. This observation clearly indicates a response plateau, suggested in
December 1993 7-78 DRAFT-DO NOT QUOTE OR CITE
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8T
cc
Steady vs. Variable Ozone Concentration
FEV, Responses
4.5
4.4
4.3
4.2
4.1
3.9
246
Exposure Duration (h)
8
Figure 7-5. The forced expiratory volume in one second (FEVj) is shown in relation to
exposure duration (hours) under three conditions: (1) • clean air exposure,
(2) • a constant concentration of 0.12 ppm O3, and (3) A a variable
O3 concentration increasing from 0.0 to 0.24 ppm over the first 4 h and
then decreasing from 0.24 to 0.0 ppm over the second 4 h. Duration of
exposure (hours) is shown on the abscissa. Mean FEVj (liters) is shown on
the ordinate. Subjects exercised (minute ventilation * 40 L/min) for
30 min during each hour; FEVX was measured at the end of the intervening
rest period. Total exposure duration was 8 h. Standard error of the mean
for these FEVt averages (not shown) ranged from 120 to 150 mL.
Source: Hazucha et al. (1992).
1 other studies (Horstman et al., 1990), with an exposure regimen that produces relatively
2 small changes in lung function.
3 With the triangular O3 concentration profile, the ¥EVV decreased almost twice as much
4 after 6 h of exposure. The initial response over the first 3 h was minimal and then there was
5 a substantial decrease in FEVj, corresponding to the higher average O3 concentration, that
December 1993
7.79 DRAFT-DO NOT QUOTE OR CITE
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1 reached a nadir after 6 h. Despite continued exposure to a lower O3 concentration
2 (<0.12 ppm), the FEVj began to improve and was reduced by only 5.9% at the end of the
3 8-h exposure. (However, note that the average O3 concentration in the eighth hour was
4 0.03 ppm). This study illustrates two important points for those planning to model responses
5 to O3 exposure. First, the model should include a response plateau. It is intuitively obvious
6 that there must be a limit to the acute decrease that can occur in FEV^ However, from this
7 study, it is clear that the response plateau must be dependent upon the O3 concentration
8 because much larger decreases in FEVl occur with exposure to higher concentrations than
9 0.12 ppm. Secondly, the response to O3 exposure is dependent on the dose rate (some
10 function of O3 concentration and ventilation) as well as the cumulative dose (some function
11 of dose rate and exposure duration), at least when O3 concentration is varied. This study
12 also affirms the observation (Folinsbee et al., 1978; Adams et al., 1981; Hazucha, 1987;
13 Larsen et al., 1991) that O3 concentration is a more important factor in determining
14 O3 responses than either exposure duration or the volume of air breathed during the
15 exposure.
16
17 7.2.3 Increased Airway Responsiveness
18 Increases in airway responsiveness are an important consequence of exposure to 0^.
19 Increased airway responsiveness indicates that the airways are predisposed to
20 bronchoconstriction induced by a variety of stimuli (e.g., specific allergens, sulfur dioxide,
21 cold air, etc.). Airway responses are usually measured by having the individual forcefully
22 exhale into a spirometer designed to measure expiratory flow rates (e.g., FEVj) or, less
23 commonly, by measuring airway resistance in a body plethysmograph. In order to determine
24 the level of airway responsiveness, airway function is measured before and immediately after
25 the inhalation of small amounts of an aerosolized bronchoconstrictor drug (e.g., methacholine
26 or histamine). The dose of the bronchoconstrictor drug is increased in a step-wise fashion
27 until a predetermined degree of airway response (e.g., a 20% drop in FEVj or a 100%
28 increase in airway resistance) has occurred. The dose of the bronchoconstrictor drug that
29 produced the aforementioned response is often referred to as the PD20 (i.e., the provocative
30 dose that produced a 20% drop in FEVj) or the PD10o (i.e., the provocative dose that
31 produced a 100% increase in airway resistance).
December 1993 7-80 DRAFT-DO NOT QUOTE OR CITE
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1 A high level of bronchial responsiveness is a hallmark of asthma. However, varying
2 degrees of increased airway responsiveness may occur in other lung disease (e.g., chronic
3 bronchitis or viral respiratory infections) or in healthy asymptomatic individuals. The range
4 of nonspecific bronchial responsiveness, as expressed by the PD20 for example, is at least
5 1,000-fold from the most sensitive asthmatics to the least sensitive healthy subjects (see
6 Figure 7-6). The average PD20 for healthy subjects is 10 to 100 times that of mild to
7 moderate asthmatics (Chatham et al., 1982; Cockcroft et al., 1977). Atopic or allergic
8 individuals without asthma typically have a lower PD2o than healthy individuals (Townley
9 et al., 1975; Cockcroft et al., 1977), being intermediate in responsiveness between healthy
10 subjects and mild asthmatics. Increasing severity of asthma, as indicated by increased
11 symptoms or medication usage, is associated with a decreased PD20. Mild asthmatics may
12 have a PD20 that is 10 times higher than that of moderate/severe asthmatics (Cockcroft et al.,
13 1977). A low PD20 in nonasthmatics is also associated with increased symptoms and a
14 reduced baseline FEV1 (Kennedy et al., 1990). The average changes in airway
15 responsiveness induced by O3 range from 150 to 500%. This means that a healthy subject
16 with a PD20 of 20 units would decrease to a PD20 between 13 and 4 units. Therefore, with a
17 pronounced O3-induced change in airway responsiveness, a healthy subject could move from
18 the normal range into the upper half of the mild asthmatic range of airway responsiveness.
19 Results of studies reporting changes in airway responsiveness following 03 exposure
20 are summarized in Table 7-10. Increased airway responsiveness associated with O3 exposure
21 was first reported by Golden et al. (1978), who studied histamine-responsiveness in eight
22 healthy men after exposure to 0.6 ppm O3 for 2 h at rest and found that the histamine-
23 induced ARaw for the group was 300% greater 5 min after O3 exposure than at baseline.
24 Two of their subjects, however, had an increased response to histamine 1 week or greater
25 after exposure, raising the possibility that high O3 levels can result in more persistent
26 increases in airway responsiveness. Later, Holtzman et al. (1979) found in 16 nonasthmatic
27 subjects that a 10-breath methacholine or histamine challenge increased SR^ almost twice as
28 much after O3 as after air exposure, but this effect resolved after 24 h. Atopic subjects
29 showed similar increases in responsiveness to histamine after O3 exposure. The authors
30 concluded that the increased nonspecific bronchial responsiveness after O3 exposure was not
December 1993 7-81 DRAFT-DO NOT QUOTE OR CITE
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Arbitrary Logarithmic Scale
0.01 0.10 1.00 10.0 100
*— severe asthma +
•*— moderate asthma—>
* mild asthma »
allergy only—+
+ healthy
Figure 7-6. Airway function can be measured before and immediately after the
inhalation of an aerosolized bronchoconstrictor drug like methacholine.
The provocative dose that produces a 20% drop in FEVj (PD^) has been
used to express the range of nonspecific bronchial responsiveness.
1 related to atopy. Konig et al. (1980) found increased responsiveness to inhaled acetylcholine
2 after a 1-h exposure to 627 and 1,960 jwg/m3 (0.32 and 1.0 ppm, respectively). Folinsbee
3 and Hazucha (1989) found increased airway responsiveness in 18 females 1 and 18 h after a
4 70-min exposure to 0.35 ppm O3 when compared to air. Taken together, these studies
5 suggest that O3-induced increases in airway responsiveness usually resolve 18 to 24 h after
6 exposure, but may persist in some individuals for longer periods.
7 Dimeo et al. (1981) were the first to investigate "adaptation" to the increases in airway
8 responsiveness following O3 exposure. Over 3 days of a 2 h/day exposure to 0.40 ppm O3,
9 they found progressive attenuation of the increases in airway responsiveness such that after
10 the third day of O3 exposure histamine airway responsiveness was no longer different from
11 the sham exposure levels. Kulle et al. (1982) extended these findings by exposing two
12 groups of healthy volunteers (n = 48) to 0.40 ppm O3 for 3 h/day for 5 days in a row in an
13 experiment designed to study the process of adaptation to O3 and found that there was a
14 significantly enhanced response to methacholine after the first 3 days of exposure, but this
15 response slowly normalized by the end of the fifth day. Thus, the attenuation of C^-induced
16 increases in airway responsiveness followed the same tune course as attenuation of other
17 pulmonary function changes.
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TABLE 7-10. INCREASED AIRWAY RESPONSIVENESS FOLLOWING
OZONE EXPOSURES3
^f Ozone
*"* • Concentration
"— '
vo
i
oo
o
K
i
5
2
1
O
g
§
M
g
n
ppm
0.00
0.08
0.10
0.12
0.00
0.10
0.32
1.00
0.00
0.12
0.20
0.00
0.12
0. 12 ppm O
0. 12 ppm O
air- 100 ppb
air-antigen
/j
0
157
196
235
0
196
627
1,960
0
235
392
0
235
3-100ppbSO2
3-0.12ppm O3
SO2
Number and
Exposure Exposure Gender of
Duration and Activity Conditions Subjects
6.6 h 18 °C 22 M
IE at =39 L/min 40% RH
2 h NA 14
1 h at VE = 89 L/min 31 °C 15 M
followed by 3-4 min at 35% RH 2 F
» 150 L/min
6.6 h with IE at NA 10 M
«25 L/min/m2 BSA
45 min in first 75% RH 8 M
atmosphere and 15 min in 22 °C 5 F
second.
IE
1 h at rest NA 4 M, 3 F
0. 12 ppm O3-antigen
0.00
0.35
0
686
70 min with IE at NA 18 F
40 L/min
auDjeci
Characteristics
Healthy NS,
18-32 years
old
NS
Elite
cyclists
NS
18-33 years
old
Asthmatic
12-18 years
old
Asthmatic
21-64 years
old
19-28 years
old
Observed Effect(s)
33, 47, and 55% decreases in cumulative
dose of methacholine required to produce
a 100% increase in SRaw after exposure to
O3 at 0.08, 0.10, and 0.12 ppm,
respectively.
Increased airway responsiveness to
methacholine immediately after exposure
at the two highest concentrations of O3.
Greater than 20% increase in histamine
responsiveness in one subject at 0. 12 ppm
O3 and in 9 subjects at 0.20 ppm C^.
Approximate doubling of mean
methacholine responsiveness after
exposure. On an individual basis, no
relationship between O3-induced changes
in airway responsiveness and FEVj or
FVC.
Greater declines in FEVj and Vmax50%
and greater increase hi respiratory
resistance after O3-SO2 then after O3-O3
or air-SO2.
Increased bronchoconstrictor response to
inhaled ragweed or grass after O3
exposure compared to air.
PDjoo decreased from 59 CIU after air
exposure to 41 CIU and 45 CIU, 1 and
18 h after 63 exposure, respectively.
Reference
Horstman
et al. (1990)
Konig et al.
(1980)
Gong et al.
(1986)
Folinsbee
et al. (1988)
Koenig et al.
Q990)
Molfino
et al. (1991)
Folinsbee
and Hazucha
H989)
-------
TABLE 7-10 (cont'd). INCREASED AIRWAY RESPONSIVENESS FOLLOWING
OZONE EXPOSURES3
Ozone
Concentration
ppm
0.20
0.40
0.40
0.00
0.40
0.00
0.40
0.00
0.60
0.00
0.60
fig/m Duration and Activity
392
784
784
0
784
0
784
0
1,176
0
1,176
2 h with IE at 2 X resting
2 h with IE at 2 X resting
2 h/day for 3 days
3 h/day for 5 days in a row
2 h with IE at
VE = 53-55 L/min
2 h at rest
2 h with IE at 2 X resting
Number and
Exposure Gender of Subject
Conditions Subjects
22 °C 12 M
55% RH 7 F
13 M
11F
22 °C 8 M
50% RH 10 F
NA 5M
3 F
22 °C 11 M
55% RH 5 F
Characteristics Observed Effect(s)
NS
21-32 years
old
NS
9 asthmatics
(5 F, 4 M)
9 healthy
(5 F, 4 M)
18-34 years
old
NS
22-30 years
old
9 atopic
7 nontopic
NS
110% increase in ASR^ to a 10-breath histamine
(1.6%) aerosol challenge after exposure to O3 at
0.40 ppm, but no change at 0.20 ppm. Progressive
adaptation of this effect over 3-day exposure.
Enhanced response to methacholine after first 3 days,
but this response normalized by Day 5.
Decreased PC100SR from 33 mg/mL to 8.5 mg/mL in
healthy subjects afterwO3. PC100Sn fell from
0.52 mg/mL to 0.19 mg/mL in asffimatic subjects after
exposure to O3 and from 0.48 mg/mL to 0.27 mg/mL
after exposure to air.
300% increase hi histamine-induced ARaw 5 min after
O3 exposure; 84% increase and 50% increase 24 h and
1 week after exposure (p > 0.05). Two subjects had an
increased response to histamine 1 week after exposure.
Ten-breath methacholine or histamine challenge
increased SRaw > 150% in 16 nonasthmatics after O3.
On average, the atopic subjects had greater responses
than the nontopic subjects. The increased
responsiveness resolved after 24 h. Atropine
premedication blocked the O3-induced increase hi airway
responsiveness .
Reference
Dimeo
et al.
(1981)
Kulle et al.
(1982)
Kreit et al.
(1989)
Golden
et al.
(1978)
Holtzman
et al.
(1979)
See glossary of terms and symbols for abbreviations and acronyms.
Listed from lowest to highest O3 concentration.
Age range hi years or as mean ± SEM.
-------
1 Gong et al. (1986) demonstrated increased airway responsiveness to histamine at
2 0.20 ppm O3 in 17 vigorously exercising elite cyclists who were exposed for 1 h. Folinsbee
3 et al. (1988) found an approximate doubling of the mean methacholine responsiveness in a
4 group of healthy volunteers exposed for 6.6 h to 0.12 ppm O3. However, on an individual
5 basis, they found no relationship between O3-induced changes in airway responsiveness and
6 those in FVC and FEV^ suggesting that these two processes occurred by different
7 mechanisms. Horstman et al. (1990) corroborated and extended Folinsbee's observations by
8 demonstrating significant decreases in the PD100 in 22 healthy subjects immediately after a
9 6.6-h exposure to concentrations of 03 as low as 0.08 ppm. Because methacholine
10 challenges were not conducted at later time points in any of these studies, the duration of the
11 increased airway responsiveness after ambient-level O3 exposure could not be determined.
12 Kreit et al. (1989) were first to investigate the change in airway responsiveness that
13 occurs after O3 exposure in individuals with asthma. They exposed nine mild asthmatics
14 (baseline PC100SRaW < 1.5 mg/mL) for 2 h to 0.40 ppm O3 with IE and found that the
15 baseline PC^SR^ declined from 0.52 to 0.19 mg/mL after O3 as compared to 0.48 to
16 0.27 mg/mL after air.
17 No doubt exists that O3, even at ambient concentrations, produces acute increases in
18 airway responsiveness. Whether O3 exposure causes protacted increases in airway
19 responsiveness in healthy individuals or even induces or predisposes to asthma is a more
20 difficult question to answer (see Section 7.4.2). However, the increases in airway
21 responsiveness following O3 exposure, even if short in duration, may have important clinical
22 implications. Several studies have been conducted specifically to determine the significance
23 of acute increases in airway responsiveness after O3 exposure. These studies, designed to
24 test the hypothesis that an O3 exposure heightens the response to a subsequent
25 bronchoconstrictor challenge, have exposed asthmatics to O3 or air and, then, to a known
26 bronchoconstrictor agent to compare the pulmonary function changes after O3 to those after
27 air. Koenig et al. (1990) demonstrated that a 45-min exposure to 0.12 ppm O3 followed by a
28 15-min exposure to 120 ppb sulfur dioxide (SO2) caused greater changes in FEV^
29 respiratory resistance, and Vmax50% in 14 adolescent asthmatics than an air-SO2 exposure
30 combination.
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1 Molfmo et al. (1991) examined the effects of a 1-h resting exposure to 0.12 ppm O3 on
2 the response to a ragweed or grass allergen inhalation challenge. Asthmatic subjects were
3 exposed twice to air and twice to ozone, once per week over a period of 4 weeks. Two
4 allergen challenges were performed, once after air and once after ozone exposure. The other
5 air and ozone exposures were followed by a placebo challenge. A ragweed allergen extract
6 was used for six of the seven subjects. The order of experiments was not randomized; six of
7 the seven subjects were exposed to the ozone/allergen condition last and five of the seven
8 were exposed to the air/placebo condition first. Allergen responsiveness was expressed as
9 the allergen concentration needed to cause a 15% reduction in FEVj or PC15. The PC15 was
10 lower after the O3 exposure than after the air exposure (p = 0.04). These observations
11 suggest that allergen-specific airway responsiveness is increased after O3 exposure. Although
12 it is expected that specific bronchial reactivity will be increased by O3 exposure based on the
13 marked increases in nonspecific bronchial responsiveness induced by O3 exposure, such a
14 response would not have been anticipated under these mild exposure conditions where lung
15 function or symptomatic responses have not been observed. The experimental design flaw in
16 this study makes it difficult to assess the validity of conclusions based on the statistical
17 analysis. These results are provocative but should be considered preliminary until this
18 experiment can be repeated.
19 Contrary to these studies, Bascom et al. (1990) found no increase in responses to
20 antigen nasal challenge in a group of allergic subjects exposed to 0.5 ppm O3 for 4 h when
21 compared to the air-antigen challenge (see also Section 7.2.4.6). Because more than half of
22 the population in the United States lives in regions that experience ambient C^ concentrations
23 at the levels used in these studies, O3 may be a clinically important cofactor in the response
24 to airborne bronchoconstrictor substances in individuals with asthma. It is plausible that this
25 phenomenon could contribute to increased asthma exacerbations and, even, increased hospital
26 admissions for asthma exacerbations (see Section 7.4.1). Whether the increased airway
27 responsiveness following O3 exposure produces an accentuated bronchoconstrictor response
28 to inhaled allergens or SO2 in healthy individuals or those with other lung diseases is
29 unknown.
30 Several studies have been undertaken to determine the mechanism of O3-induced
31 increases in airway responsiveness. Early experiments in dogs (Lee et al., 1977) and
December 1993 7-86 DRAFT-DO NOT QUOTE OR CITE
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1 humans (Golden et al., 1978) suggested an important role for vagal reflexes because vagal
2 nerve cooling and atropine inhibited the increase in histamine-induced bronchoconstriction
3 caused by O3. Subsequent studies, however, revealed that O3 exposure increased
4 bronchomotor responses to cholinergic stimuli (e.g., acetylcholine and methacholine) in dogs
5 (Holtzman et al., 1983) and humans (Seltzer et al., 1986) and that bilateral vagotomy did not
6 inhibit O3-induced hyperresponsiveness to subcutaneous histamine in guinea pigs (Gordon
7 et al., 1984). These data provide strong evidence that O3-induced increased airway
8 responsiveness is mediated, at least in part, by cholinergic receptors on airway smooth
9 muscle cells. Interestingly, Gordon et al. also noted that isometric tension in guinea pig
10 tracheal smooth muscle and lung parenchymal strips in response to histamine and carbachol
11 was not affected by exposure to Oj, suggesting that O3 affected the hi vivo milieu
12 surrounding the smooth muscle rather than producing direct effects on the smooth muscle
13 itself.
14 Osebold et al. (1980), having shown that ovalbumin-sensitized mice were much more
15 sensitive to inhaled albumin after O3 exposure (compared to air), hypothesized that the
16 increased epithelial permeability caused by O3 may allow greater penetration of
17 bronchoconstrictor substances, including methacholine and histamine, and that this would
18 lead to increased airway responsiveness. However, Roum and Murlas (1984) suggested that
19 the increased epithelial permeability after O3 could not totally explain this phenomenon
20 because parenteral cholinergic challenge after O3 more reproducibly caused bronchospasm
21 than did inhalation challenge with methacholine. The increased responsiveness to parenteral
22 compared to inhaled cholinergic challenge may, however, have been due to increased
23 bronchial blood flow after O3 exposure. Therefore, their findings do not exclude increased
24 epithelial permeability as the cause of increased airway responsiveness after 63 exposure.
25 Holtzman et al. (1983) first pointed out that O3-induced acute inflammation may be
26 important in the induction of the increased airway responsiveness. In mongrel dogs exposed
27 to O3, they found bronchial wall neutrophil infiltration in those animals that developed
28 increased airway responsiveness to acetylcholine, but not in animals that failed to develop
29 increased airway responsiveness. O'Byrne et al. (1984) later demonstrated that hydroxyurea
30 simultaneously decreased peripheral blood leukocyte counts, decreased neutrophil influx into
31 bronchial tissue, and increased airway responsiveness in dogs exposed to O3. Both
December 1993 7.37 DRAFT-DO NOT QUOTE OR CITE
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1 O3-induced increased airway responsiveness and bronchial tissue neutrophil influx returned
2 6 weeks after treatment was discontinued when peripheral leukocyte counts had normalized.
3 Seltzer et al. (1986) found a larger percentage of neutrophils (30.8 versus 8.0%) in BAL
4 fluid after O3 exposure in their subjects that had a greater than threefold decrease in the
5 provocative concentration that caused a > 8 L x cm H2O/L/s increase in SRaw for
6 methacholine compared to those that had less than a twofold decrease. These data suggest a
7 possible association between inflammation and increased airway responsiveness after
8 O3 exposure. However, McDonnell et al. (1990a,b) found no significant relationship
9 between changes in airway responsiveness or FEVj and inflammatory response as judged by
10 changes in BAL neutrophil levels. The concentration of PGE2 was associated with changes
11 in both FEVj and changes in airway responsiveness. Folinsbee et al. (1993) found no
12 association between either symptoms or FEVt responses and changes in airway
13 responsiveness in subjects exposed to 0.12 ppm O3 for 6.6 h. Furthermore, FEVi responses
14 were attenuated after three exposures, whereas changes in airway responsiveness persisted
15 for a longer period (see also Section 7.2.1.4). Schelegle et al. (1991) examined the time
16 course of O3-induced airway neutrophilia and found no correlation between FEVj changes
17 and individual increases in neutrophil levels. If anything, the least sensitive FEVX
18 responders tended to have fewer neutrophils in their airways.
19 However, Evans et al. (1988) found that the increased airway responsiveness in rats
20 caused by O3 was not associated with an increase in tracheal mucosal neutrophils or vascular
21 permeability. In the first of several studies with a prostaglandin inhibitor, indomethacin did
22 not attenuate the increase in airway responsiveness in humans exposed to 0.4 ppm O3 for
23 2 h (Ying et al., 1990), but did ameliorate the effect of O3 on spirometric endpoints.
24 Kleeberger and Hudak (1992) observed a marked reduction in PMN influx in O3-exposed
25 mice given indomethacin without any change in O3-induced increases in permeability, as
26 indicated by BAL protein. However, Hazucha et al. (1993) found no effect of ibuprofen on
27 PMN levels or protein in the BAL fluid of O3-exposed humans (also see Section 7.2.4.5).
28 Seltzer et al. (1986) and Koren et al. (1989a,b) found that O3 increases a large number of
29 BAL inflammatory mediators (including prostaglandin E^ [PGEj], prostaglandin F2 [PGF2a],
30 and thromboxane B2 [TXB2]), one or more of which may play a role in the increase in
December 1993 7-88 DRAFT-DO NOT QUOTE OR CITE
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1 airway responsiveness after O3 exposure. To date, only PGE2 has been shown to be
2 associated with the increased airway responsiveness (McDonnell et al., 1990a,b).
3 The role of reactive oxygen metabolites or neuropeptide mediators in the increase in
4 airway responsiveness after O3 has not been investigated. Furthermore, there has been no
5 direct assessment of alterations in nerve afferents, changes in neurotransmitter
6 concentrations, changes in smooth muscle postsynaptic receptors, or modulation of nerve
7 signal transmission by inflammatory mediators as these pertain to the increase in airway
8 responsiveness after O3. In conclusion, although the mechanism of O3-induced increases in
9 airway responsiveness is not completely understood, it appears to be a consequence of
10 cellular or biochemical changes in the airway (see Section 7.2.4 and Tables 7-11 and 7-12).
11 Because these alterations are part of a complex process, it comes as no surprise that the
12 mechanistic studies on O3-induced increases in airway responsiveness have not pinpointed an
13 isolated derangement.
14
15 7.2.4 Inflammation and Host Defense
16 7.2.4.1 Introduction
17 In general, inflammation can be considered as the host response to injury, and the
18 induction of inflammation can be accepted as evidence that injury has occurred.
19 Inflammation induced by exposure of humans to O3 can have several outcomes:
20 (1) inflammation induced by a single exposure (or several exposures over the course of a
21 summer) can resolve entirely; (2) continued acute inflammation can evolve into a chronic
22 inflammatory state; (3) continued inflammation can alter the structure or function of other
23 pulmonary tissue, leading to diseases such as fibrosis or emphysema; (4) inflammation can
24 alter the body's host defense response to inhaled microorganisms, particularly in potentially
25 vulnerable populations such as the very young and old; and (5) inflammation can alter the
26 lung's response to other agents such as allergens or toxins. It is also possible that the profile
27 of response can be altered in persons with preexisting pulmonary disease (e.g., asthma or
28 COPD) or smokers.
29 The previous O3 criteria document (U.S. Environmental Protection Agency, 1986)
30 contained no studies in which inflammation was measured in humans exposed to 03. Since
31 then, the use of fiberoptic bronchoscopy has made possible the sampling of cells and fluids
December 1993 7.39 DRAFT-DO NOT QUOTE OR CITE
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TABLE 7-11. INFLAMMATORY EFFECTS OF CONTROLLED HUMAN
EXPOSURE TO OZONE (BRONCHOALVEOLAR LAVAGE STUDIES)3
Sr
<,
O
O
I
3
8
R
Ozone Concentration
ppm
0.00
0.08
0.10
0.00
0.20
0.00
0.30
0.00
0.40
0.00
0.40
0.00
0.40
0.00
0.40
3
0
157
196
0
392
0
588
0
784
0
784
0
784
0
784
- Exposure Activity Level
Duration ( Vg)
6.6 h IE (40 L/min)
six 50-min
exercise periods
+ 10 min rest;
35 min lunch
4 h IE (50 min at
40 L/min, 10 min
rest)
1 h (mouth- CE (60 L/min)
piece)
2 h IE (70 L/min) at
15-min intervals
2 h IE (70 L/min) at
15-min intervals
2 h IE (70 L/min) at
15-min intervals
2 h IE (70 L/min) at
15-min intervals
Number and
Gender of
Subjects0
18 M
(18-35 years)
15 M
13 F
(21-39 years)
5M
11 M
11 M
11 M
10 M
(18-35 years)
Observed Effect(s)
BAL fluid 18 h after exposure to 0.1 ppm O3 had
significant increases in PMNs, protein, PG^, fibronectin,
IL-6, lactate dehydrogenase, and a-1 antitrypsin compared
with the same subjects exposed to filtered air. Similar but
smaller increases in all mediators after exposure to
0.08 ppm 03 except for protein and fibronectin.
Decreased phagocytosis of yeast by alveolar macrophages
was noted at both concentrations.
Bronchial lavage, bronchial biopsies, and BAL done 18 h
after exposure. BAL shows changes similar to other
studies. Airway lavage shows increased cells, LDH,
IL-8. Biopsies show increased number of PMNs.
Significantly elevated PMNs in the BAL fluid 1,6, and
24 h after exposure, with peak increases at 6 h.
BAL fluid 18 h after exposure had significant increases hi
PMNs, protein, albumin, IgG, PGE2, plasminogen
activator, elastase, Complement C3a, and fibronectin.
Macrophages removed 1 8 h after exposure had changes in
the rate of synthesis of 123 different proteins as assayed
by computerized densitometry of two-dimensional gel
protein profiles.
BAL fluid 18 h after exposure contained increased levels
of the coagulation factors Tissue Factor and Factor VH.
Macrophages in the BAL fluid had elevated Tissue Factor
mRNA.
BAL fluid 1 h after exposure to 0.4 ppm O3 had
significant increases hi PMNs, protein, PGE2, TXB2,
IL-6, LDH, a-1 antitrypsin, and Tissue Factor compared
with the same subjects exposed to FA. Decreased
phagocytosis of yeast by alveolar macrophages.
Reference
Devlin et al.
(1990, 1991)
Koren et al.
(1991)
Aris et al.
(1993a)
Schelegle et al.
(1991)
Koren et al.
(1989a,b)
Devlin and
Koren (1990)
McGee et al.
(1990)
Koren et al.
(1991)
Devlin et al.
(1993)
-------
TABLE 7-11 (cont'd). INFLAMMATORY EFFECTS OF CONTROLLED HUMAN
TO OZONE (BRONCHOALVEOLAR LAVAGE STUDIES)8
n> Ozone Concentration
,
VO
u>
-------
TABLE 7-12. INFLAMMATORY AND HOST DEFENSE EFFECTS OF CONTROLLED
3
y
H-*
*?
Ui
-jj
vb
K>
o
6
o
1
-------
TABLE 7-12 (cont'd). INFLAMMATORY AND HOST DEFENSE EFFECTS OF CONTROLLED
HUMAN EXPOSURE TO OZONE (CLEARANCE STUDIES)3
if
VO
O
I
O
1
0
1
o
Ozone Concentration11 Number and
ppm
0.00
0.40
0.00
0.50
0.00
0.25
0.50
0.00
0.25
0.50
1.00
0.00
0.25
0.50
1.00
0.00
0.30
1.00
bxposure Activity Level uenaer or
fig/m Duration ( Vg) Subjects0
0
784
0
784
0
490
980
0
490
980
1,960
0
490
980
1,960
0
588
1,960
See glossary of terms
Listed from lowest to
cAge range in years or
2 h IE (70 L/min) at 8 M
15 min intervals (20-30
years)
2.25 h IE (70 L/min) at 16 M
15 min intervals (20-30
years)
6 h Human nasal
epithelial cells
1 h Airway epithelial
cell line
1 h Airway epithelial
cell line and
alveolar
macrophages
1 h Alveolar
macrophages
and symbols for abbreviations and acronyms.
highest O3 concentration.
as mean ± SEM.
Observed Effect(s)
Subjects inhaled "^Tc-DTPA 75 min after exposure.
Significantly increased clearance of ""Tc-DTPA from the
lung hi O3-exposed subjects. Subjects had expected
changes in FVC and SRaw.
Similar design and results as earlier study (Kehrl et al. ,
1987). For the combined studies the average rate of
clearance was 60% faster in O3-exposed subjects.
Increased in ICAM-1, IL-6, IL-1, and TNF expression at
0.5 ppm. No increase hi IL-8 expression. No increases
at 0.25 ppm.
Dose-dependent increased secretion of PGE2, TXB2,
PGF2Q,, LTB4, and LTD4. More secretion basolaterally
than apically.
Increased secretion of IL-6, IL-8, and fibronectin by
epithelial cells, even at lowest O3 concentration. No
O3-induced secretion of these compounds by macrophages.
Dose-dependent increases hi PGE2 production, and
decreases hi phagocytosis of sheep erythrocytes. No
O3-induced secretion of IL-1, TNF, or IL-6.
Reference
Kehrl et al.
(1987)
Kehrl et al.
(1989a)
Beck et al.
(1993)
McKinnon
et al. (1993)
Devlin et al.
(1993a)
Becker et
al. (1991)
n
-------
1 lining the respiratory tract of humans for many markers (Reynolds, 1987). Bronchoalveolar
2 lavage primarily samples the alveolar region of the lung; however, the use of small volume
3 lavages (Rennard et al., 1990) or balloon catheters also allows sampling of the airways.
4 Nasal lavage (NL) allows sampling of cells and fluid removed from the nasal passages.
5 In the past 6 years, several studies have analyzed BAL and NL cells and fluid from
6 humans exposed to O3 for markers of inflammation and lung damage (see Tables 7-11 and
7 7-12). The presence of neutrophils (polymorphonuclear leukocytes [PMNs]) in the lung has
8 long been accepted as a hallmark of inflammation and has been taken as the major indicator
9 that O3 causes inflammation in the lungs of humans. Soluble mediators of inflammation (or
10 its resolution) such as cytokines and arachidonic acid metabolites have also been measured in
11 the BAL fluid of humans exposed to O3. Cytokines that have been reported most often are
12 interleukin (IL)-6 and IL-8, although IL-1 and tumor necrosis factor (TNF) have also been
13 studied. Soluble metabolites of arachidonic acid involved in inflammation and host defense
14 such (e.g., PGE2 and PGF2a, thromboxane, and leukotrienes [LTs] such as LTB4) have also
15 been reported hi the BAL fluid of humans exposed to O3. In addition to their role in
16 inflammation, many of these compounds have bronchoconstrictive properties, and may be
17 involved in increased airway hyperreactivity observed following O3 exposure.
18 Under normal circumstances, the epithelia lining the large and small airways develop
19 tight junctions and restrict the penetration of exogenous particles and macromolecules from
20 the airway lumen into the interstitium and blood, as well as restricting the flow of plasma
21 components into the airway lumen. However, several studies (see Table 7-12) report that
22 O3 disrupts the integrity of the epithelial cell barrier in human airways, as measured by
23 increased passage of radiolabeled compounds out of the airways, as well as passage of
24 markers of plasma influx such as albumin, immunoglobulin, and total BAL fluid protein into
25 the airways. In addition, markers of epithelial cell damage such as lactate dehydrogenase
26 (LDH) have also been measured hi the BAL fluid of humans exposed to O3.
27 Inflammatory cells of the lung such as macrophages, monocytes, and PMNs also
28 constitute an important component of the pulmonary host defense system. In their
29 unstimulated state, they present no danger to surrounding pulmonary cells and tissues, but
30 upon activation, they are capable of generating free radicals and enzymes with microbicidal
December 1993 7.94 DRAFT-DO NOT QUOTE OR CITE
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1 activities, but also with the potential of damaging nearby cells. Animal studies have assessed
2 the effect of O3 on host defense system function by measuring the lethality to an inhaled
3 bacteria (see Chapter 6, Section 6.2.3). Although these experiments are not directly
4 comparable to human studies, the ability of macrophages removed from O3-exposed
5 individuals to phagocytose and kill microorganisms in culture has been assessed.
6 Other soluble factors that have been studied include those involved with fibrin
7 deposition and degradation (Tissue Factor, Factor Vn, and plasminogen activator), potential
8 markers of fibrogenesis (fibronectin, platelet derived growth factor), and components of the
9 complement cascade (C3a).
10
11 7.2.4.2 Inflammation Assessed by Bronchoalveolar Lavage
12 Seltzer et al. (1986) were the first to demonstrate that exposure of humans to
13 O3 resulted in inflammation in the lung. In this study, 10 volunteers were exposed to
14 0.4 ppm or 0.6 ppm O3 for 2 h while undergoing exercise, and BAL was performed
15 3 h later. Bronchoalveolar lavage fluid from subjects exposed to O3 contained 7.8-fold more
16 neutrophils compared with BAL fluid from the same subjects exposed to filtered air.
17 Additionally, BAL fluid from O3-exposed subjects contained increased levels of PGE2,
18 pGF2Q;, and TXB2 compared to fluid from air-exposed subjects. A report by Koren et al.
19 (1989a,b) also described inflammatory changes in the lungs of humans exposed to Oj.
20 Eleven subjects were exposed to 0.4 ppm O3 for 2 h while undergoing IE at 70 L/min in a
21 study designed to simulate adults working outdoors or children actively playing.
22 Bronchoalveolar lavage was performed 18 h after O3 exposure. Subjects exposed to O3 had
23 an eightfold increase in neutrophils in the BAL fluid, confirming the observations of Seltzer
24 et al. In addition, Koren et al. reported a twofold increase in BAL fluid protein, albumin,
25 and immunoglobulin G (IgG) levels, suggestive of increased epithelial cell permeability as a
26 result of O3 exposure. There was also a 12-fold increase in IL-6 levels in the BAL fluid.
27 Interleukin-1 and TNF were not present in detectable levels in the BAL fluid of any subject.
28 There was, however, a twofold increase in the proinflammatory eicosanoid PGE2, as well as
29 a twofold increase in the complement component C3a. This study also provided evidence for
30 stimulation of fibrogenic processes in the lung by demonstrating significant increases in two
31 components of the coagulation pathway, Tissue Factor and Factor VH (McGee et al., 1990),
December 1993 7.95 DRAFT-DO NOT QUOTE OR CITE
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1 as well as urokinase plasminogen activator and fibronectin (Koren et al., 1989a). Taken
2 together, these two studies demonstrate that exposure of humans to moderate levels of
3 O3 results in an inflammatory reaction in the lung, as evidenced by substantial increases in
4 neutrophils and proinflammatory compounds. Furthermore, these studies demonstrate that
5 both cells and mediators capable of damaging pulmonary tissue are increased after
6 O3 exposure, as are compounds that play a role in fibrotic and fibrinolytic processes.
7 Although animal studies have shown that the airway is a major site of O3-induced
8 inflammation, few human studies have confirmed this finding because BAL primarily
9 samples cells and fluid in the terminal bronchioles and alveoli. However, isolated lavage of
10 the mainstream bronchus using balloon catheters or the more traditional BAL using small
11 volumes of saline have the ability to preferentially measure O3-induced changes in the large
12 airways. In one study, isolated airway lavage was performed on 14 subjects 18 h after
13 exposure to 0.2 ppm O3 while undergoing moderate exercise (Aris et al., 1993a). Increases
14 in total lavagable cells, LDH, and IL-8 were reported. In contrast with Schelegle et al.
15 (1991), there was no increase in PMNs found in the bronchial fluid; however, bronchial
16 biopsies showed increased numbers of PMNs in airway tissue.
17 The data suggestive of O3-induced changes in epithelial cell permeability described by
18 Koren et al. (1989a), Devlin et al. (1991), and Koren et al. (1991) support earlier work in
19 which epithelial cell permeability as measured by increased clearance of "'Tc-DTPA from
20 the lung of humans exposed to O3 was demonstrated (Kehrl et al., 1987). In that study,
21 eight healthy subjects who inhaled '"Tc-DTPA just prior to exposure to air or 0.4 ppm
22 O3 for 2 h while undergoing heavy exercise (65 L/min) had increased clearance of the
23 compound. Kehrl et al. (1989a) reported similar observations on an additional 16 subjects.
24 For the combined group of 24 subjects exposed for 2 h to 0.4 ppm O3, the average clearance
25 rate was 60% faster than that observed after air exposure, strongly suggesting increased
26 permeability from the airway lumen and alveolar space to the blood and interstitial spaces.
27 The average O3-induced decrement in FVC in these subjects was —10%. These changes in
28 permeability are most likely associated with acute inflammation and could potentially allow
29 better access of inhaled antigens and other substances to the submucosa.
30 Studies in which human alveolar macrophages and airway epithelial cells were exposed
31 to O3 in vitro suggest that most of the components found in increased levels in the BAL fluid
December 1993 7-96 DRAFT-DO NOT QUOTE OR CITE
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1 of O3-exposed humans are produced by epithelial cells. Macrophages exposed to 0.1, 0.3,
2 and 1.0 ppm O3 for 1 h showed small increases in PGE2 in cells exposed to the latter two
3 concentrations, but no change in superoxide anion or cytokine production (Becker et al.,
4 1991). In contrast, airway epithelial cells exposed to 0.1, 0.25, 0.5, and 1.0 ppm O3 for
5 1 h showed large dose-dependent increases in PGE^, TXB2, LTB4, LTC4, and LTD4
6 (McKinnon et al., 1993). These cells also showed increases in IL-6, IL-8, and fibronectin at
7 O3 concentrations as low as 0.1 ppm (Devlin et al., 1993a). Interestingly, macrophages
8 removed 18 h later from subjects exposed to 0.4 ppm O3 for 2 h while undergoing
9 intermittent heavy exercise (Koren et al., 1989a) showed changes in the rate of synthesis of
10 123 different proteins as measured by quantitative computerized densitometry of two-
11 dimensional gel protein profiles. However, macrophages exposed to O3 in vitro only showed
12 changes in the rate of synthesis of six proteins, suggesting that most of the changes seen in
13 the in vivo exposed macrophages were due to actions resulting from mediators released by
14 other cells following O3 exposure, which then altered macrophage function.
15 Numerous controlled human studies have shown that humans exposed to O3 for
16 5 consecutive days experience decrements in pulmonary function on the first and second
17 days, but the decrements diminish with each succeeding day so that by the fifth day, O3 no
18 longer induces decrements in lung function (see Section 7.2.1). This phenomenon has been
19 termed "adaptation". These studies did not address the question of whether repeated
20 exposure to O3 also induced "adaptation" to inflammation or lung damage, although two
21 animal studies suggest that although some markers of inflammation may be diminished,
22 underlying damage to lung epithelial cells continues (Tepper et al., 1989; VanBree et al.,
23 1993). In a recent study (Devlin et al., 1993b), humans were exposed to 0.4 ppm O3 for
24 5 consecutive days (2 h/day while undergoing IE) and then were exposed to Oj a single time
25 either 10 or 20 days laters. The results show that numerous indicators of inflammation (e.g.,
26 PMN influx, IL-6, IL-8, PGI^, BAL protein, fibronectin, macrophage phagocytosis) show
27 "adaptation" (i.e., there is a complete disappearance of response and values are different
28 from those observed in the same individual after 5 days of exposure to FA). These
29 attenuated responses show a gradual recovery. Ten days later, some of these markers have
30 regained full susceptibility, but others have not regained susceptibility even after 20 days.
December 1993 7.97 DRAFT-DO NOT QUOTE OR CITE
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1 In agreement with animal studies, some markers (LDH, elastase) never show adaptation,
2 indicating that tissue damage may continue to occur during repeated exposure.
3
4 7.2.4.3 Inflammation Induced by Ambient Levels of Ozone
5 Devlin et al. (1991) reported an inflammatory response in humans exposed to levels of
6 O3 at or below 0.12 ppm. In this study, 10 volunteers were exposed to FA, 0.10 ppm O3,
7 and 0.08 O3 ppm for 6.6 h while undergoing moderate exercise (40 L/min), and underwent
8 BAL 18 h later. An additional eight subjects were exposed to filtered air and 0.08 ppm O3.
9 Increased levels of neutrophils and IL-6 were found in BAL fluid of subjects exposed to
10 0.10 and 0.08 ppm O3. There were also increases in most of the other compounds reported
11 by Koren et al. (1989a,b), including fibronectin and PGE^. In this study, alveolar
12 macrophage phagocytic capability was also monitored, and it was reported that macrophages
13 removed from humans exposed to both O3 concentrations had decreased ability to
14 phagocytose Candida albicans opsonized with complement. Comparison of the magnitude of
15 inflammatory changes observed in this study and by Koren et al. (1989a,b), when normalized
16 for differences in concentration, duration of exposure, and ventilation, suggest that lung
17 inflammation from O3 may occur as a consequence of exposure to ambient levels while
18 exercising. Although the mean changes in IL-6, PGE2, and neutrophils reported by Devlin
19 et al. (1991) were low, there was a considerable range of response among the individuals
20 participating in the study. Thus, although some of the study population showed little or no
21 response to O3, others had increases in IL-6 or PMNs that were as large or larger as those
22 reported by Koren et al. (1989a,b) when subjects were exposed to 0.4 ppm O3.
23 Interestingly, those individuals who had the largest increases in inflammatory mediators in
24 this study did not necessarily have the largest decrements in pulmonary function, suggesting
25 separate mechanisms underlying these two responses to O3. These data suggest that,
26 although the population as a whole may have a small inflammatory response to low levels of
27 O3, there may be a significant subpopulation that is very sensitive to these low levels of O3.
28 Furthermore, even a small inflammatory response (if it recurs) in the population as a whole
29 should not be discounted.
30
31
December 1993 7-98 DRAFT-DO NOT QUOTE OR CITE
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1 7.2.4.4 Time Course of Inflammatory Response
2 The time course of the inflammatory response to O3 in humans has not been fully
3 explored. Studies in which BAL was performed 1 h (Devlin et al., 1990; Koren et al.,
4 1991) or 3 h (Seltzer et al., 1986) after exposure to 0.4 ppm O3 demonstrate that the
5 inflammatory response is quickly initiated, and the data reported by Koren et al. (1989a,b)
6 indicate that even 18 h after exposure, inflammatory mediators such as IL-6 and PMNs are
7 still substantially elevated. However, a comparison of these studies shows there are
8 differences in the magnitude of response of some indicators depending on when BAL is
9 performed after O3 exposure. Ozone-induced increases in PMNs, IL-6, and PGE^ are
10 greater 1 h after O3 exposure, whereas BAL levels of fibronectin and plasminogen activator
11 are greater 18 h after exposure. Still other compounds (protein, Tissue Factor) are equally
12 elevated both 1 and 18 h after O3 exposure. Schelegle et al. (1991) exposed five subjects to
13 filtered air or 0.3 ppm O3 for 1 h with a ventilation of 60 L/min. Each subject was exposed
14 to O3 on three separate occasions and BAL was performed either 1, 6, or 24 h after
15 exposure. In addition, the BAL was separated into two fractions: the first 60 mL wash was
16 designated the "proximal airways" fraction (PA), and the remaining three 60 mL washes
17 were pooled and designated the "distal airways and alveolar surface" fraction (DAAS). The
18 percent of PMNs in the PA sample was statistically elevated at 1, 6, and 24 h after
19 O3 exposure, with a peak response at 6 h. The percent of PMNs in the DAAS sample was
20 elevated at only the 6 and 24 h time points, with equivalent elevations at each time.
21 However, more extensive studies will be needed to better define the kinetics of appearance
22 and disappearance of the various inflammatory mediators observed in the lungs of humans
23 exposed to O3.
24
25 7.2.4.5 Effect of Anti-Inflammatory Agents on Ozone-Induced Inflammation
26 Previous studies (Schelegle et al., 1987; Eschenbacher et al., 1989) have shown that
27 indomethacin, an anti-inflammatory agent that inhibits cyclooxygenase products of
28 arachidonic acid metabolism, is capable of blunting the well-documented decrements in
29 pulmonary function observed in humans exposed to O3. In a recent study, 10 healthy male
30 volunteers were given 800 mg ibuprofen, another anti-inflammatory agent that blocks
31 cyclooxygenase metabolism, or a placebo 90 min prior to a 2-h exposure to 0.4 ppm O3.
December 1993 7.99 DRAFT-DO NOT QUOTE OR CITE
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1 An additional 200 mg was administered following the first hour of exposure.
2 Bronchoalveolar lavage was performed 1 h after the exposure. As expected, subjects given
3 ibuprofen had blunted decrements in lung function following O3 exposure compared to the
4 same subjects given a placebo (Hazucha et al., 1993). Bronchoalveolar lavage fluid from
5 subjects given ibuprofen also had reduced levels of the cyclooxygenase product PGEz as well
6 as IL-6, but no decreases were observed in PMNs, fibronectin, permeability, LDH, or
7 macrophage phagocytic function (Hazucha et al., 1993). These data suggest that although
8 anti-inflammatory agents may blunt O3-induced decrements in FEVj and increases in PGI^,
9 most inflammatory mediators are elevated in the BAL of these subjects. Additional studies
10 will be needed to determine the role, if any, played by PGE2 and IL-6 in the pulmonary
11 function decrements and increases in PMNs, fibronectin, and other components in the BAL
12 fluid of humans after O3 exposure.
13
14 7.2.4.6 Use of Nasal Lavage to Assess Ozone-Induced Inflammation in the Upper
15 Respiratory Tract
16 Bronchoalveolar lavage has proven to be a powerful research tool to analyze changes in
17 the lung following exposure of humans to xenobiotics. However, because BAL is expensive,
18 somewhat invasive, and requires specialized personnel and facilities, it is usually done only
19 with small numbers of subjects and in selected medical centers. Therefore, there is
20 increasing interest in the use of NL as a tool in assessing C^-induced inflammation in the
21 upper respiratory tract, which is the primary portal for inspired air, and therefore the first
22 region of the respiratory tract to come in contact with airborne xenobiotics. Nasal lavage is
23 simple and rapid to perform, is noninvasive, and allows collection of multiple sequential
24 samples from the same person. Graham et al. (1988) reported increased levels of PMNs in
25 the NL fluid of 21 humans exposed to 0.5 ppm O3 at rest for 4 h on 2 consecutive days,
26 with NL performed immediately before and immediately after each exposure as well as
27 22 h after the second exposure. Nasal lavage fluid contained elevated PMN levels at all
28 postexposure times tested, with peak values occurring immediately prior to the second day of
29 exposure. There were no changes in PMN levels at any time in 20 subjects exposed to clean
30 air for 2 consecutive days. Bascom et al. (1990) exposed 12 subjects with allergic rhinitis to
31 filtered air or 0.5 ppm O3 at rest for 4 h, followed immediately by NL. They reported a
32 7-fold increase in PMNs, a 20-fold increase in eosinophils, and a 10-fold increase in
December 1993 7-100 DRAFT-DO NOT QUOTE OR CITE
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1 mononuclear cells following 03 exposure, as well as a 2.5-fold increase in albumin. Graham
2 and Koren compared inflammatory mediators present in both the NL and BAL fluids of
3 humans exposed to O3. The same 11 subjects who were exposed to 0.4 ppm O3 for 2 h with
4 BAL performed 18 h later, as described earlier (Koren et al., 1989a,b), also underwent NL
5 immediately before, immediately after, and 18 h after each exposure (Graham and Koren,
6 1990). There were significant increases in PMNs in the NL fluid taken both immediately
7 after exposure as well as the next day. Increases in NL and BAL PMNs were similar
8 (6.6- and 8-fold, respectively), demonstrating a qualitative correlation between changes in the
9 lower airways as assessed by BAL and the upper respiratory tract as assessed by BAL.
10 Furthermore, all individuals who had increased PMNs in BAL fluid also had increased
11 PMNs in NL fluid, although the NL PMN increase could not quantitatively predict the BAL
12 PMN increase. Albumin, a marker of epithelial cell permeability, was increased 18 h later,
13 but not immediately after exposure. There were no changes in PGE^, plasminogen activator,
14 or LTC4/D4/E4 (Graham and Koren, 1990). However, tryptase, a constituent of mast cells
15 and contained in the same granules as histamine, was found in elevated levels immediately
16 after O3 exposure, but not 18 h later (Koren et al., 1990). These studies suggest that NL
17 may serve as a sensitive and reliable tool to detect inflammation in the upper airways of
18 humans exposed to xenobiotics.
19
20 7.2.4.7 Changes in Host Defense Capability Following Ozone Exposure
21 Concern about the effect of O3 on human host defense capability derives from
22 numerous animal studies demonstrating that exposure to as little as 0.08 ppm O3 increases
23 mortality in rodents subsequently challenged with aerosolized or instilled bacteria (Gardner
24 et al., 1982; Van Loveren et al., 1988, see Chapter 6, Section 6.2.3). Because of the
25 difficulty in repeating these types of studies with humans, there are little data available on the
26 direct effects of O3 on human host defense systems. A study of experimental rhinovirus
27 infection in susceptible volunteers failed to show any effect of 5 consecutive days of
28 O3 exposure on the clinical evolution or host response to a viral challenge (Henderson et al.,
29 1988). In this study, 24 young males were inoculated with type 39 rhinovirus
30 (1,000 TCID-50) administered as nose drops. Half were then exposed to 0.3 ppm O3
31 (6 h per day) for 5 consecutive days while undergoing intermittent light exercise, and half
December 1993 7-101 DRAFT-DO NOT QUOTE OR CITE
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1 were exposed to clean air under the same regimen. There was no difference in rhinovirus
2 tilers in nasal secretions between the O3-exposed and control groups, nor were there any
3 differences in levels of interferon gamma or PMNs in NL fluid and blood lymphocyte
4 proliferative response to rhinovirus antigen. However, recent findings that rhinovirus can
5 attach to the intracellular adhesion molecule (ICAM) receptor on respiratory tract epithelial
6 cells (Greve et al., 1989) and that O3 can up-regulate the ICAM receptor on nasal epithelial
7 cells (Beck et al., 1994) suggest that more studies are needed to explore more fully the
8 potential interaction between O3 exposure and viral infectivity.
9 In a single study, human macrophage host defense capacity was measured in vitro in
10 macrophages removed from subjects exposed to low levels of O3 (0.08 and 0.10 ppm) for
11 6.6 h while undergoing moderate exercise. Macrophages from O3-exposed subjects had
12 significant decrements in complement receptor (but not Fc receptor) mediated phagocytosis of
13 Candida albicans compared with macrophages removed from air-exposed individuals (Devlin
14 et al., 1991). These data show that acute in vivo exposure of humans to 63 results in
15 impairment of alveolar macrophage host defense capability, potentially resulting in decreased
16 ability to phagocytose and kill inhaled microorganisms in vivo. Human alveolar
17 macrophages have also been exposed to O3 in vitro to investigate whether changes in
18 macrophage host defense functions are due to a direct effect of O3 on macrophages or
19 secondary effects resulting from lung injury and inflammation. Becker et al. (1991) exposed
20 macrophages to 0.1 to 1.0 ppm O3 for 1 h and showed a dose-dependent decrease in
21 phagocytosis of antibody-coated sheep erythrocytes; a small increase in PG!^; and
22 production of significantly lower levels of IL-1, TNF, and IL-6 upon stimulation with
23 lipopolysaccharide when compared with air-exposed cells (Becker et al., 1991). Although
24 there are several studies in which animals have been exposed to bacteria or virus in
25 conjunction with O3 exposure, which provide little evidence to suggest that O3 impairs the
26 immune system's ability to fight viral infections, there is insufficient human data to know
27 whether O3 exposure affects viral infectivity. However, there is potential cause for concern
28 that O3 may render humans and animals more susceptible to a subsequent bacterial challenge.
29 There are two studies that have investigated the effect of O3 exposure on mucociliary
30 clearance of inhaled particles, with conflicting results. In one study (Foster et al., 1987),
31 seven male volunteers inhaled radiolabeled ferric oxide (99mTc-Fe2O3) particles and were
December 1993 7-102 DRAFT-DO NOT QUOTE OR CITE
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1 then exposed to FA, 0.2 ppm O3, and 0.4 ppm O3 for 2 h while undergoing light IE. They
2 observed a concentration-dependent increase in rate of particle clearance 2 h after exposure,
3 although increased clearance was primarily confined to the peripheral airways in subjects
4 exposed to 0.2 ppm O3. In the second study (Gerrity et al., 1993), 15 male or female
5 subjects were exposed to clean air or 0.4 ppm O3 for 1 h while undergoing CE (40 L/min).
6 Two hours after exposure, subjects inhaled 99mTc-Fe2O3 particles and clearance was
7 measured with a gamma camera for the next 3 h and then the next morning. There was no
8 difference in the clearance rate of particles in air and O3-exposed subjects. The discrepancy
9 between these studies may be explained by differences in exposure protocol, time of particle
10 inhalation, or time of clearance measurement, or the presence of cough immediately
11 following O3 exposure, which may have accelerated clearance in the first study. Further
12 studies will be needed to better define the relationship between O3 exposure and mucociliary
13 clearance.
14
15 7.2.5 Extrapulmonary Effects of Ozone
16 It is still believed that O3 immediately reacts on contact with respiratory system tissue
17 and is not absorbed or transported to extrapulmonary sites to any significant degree (see
18 Chapter 8). A number of laboratory animal studies presented hi Chapter 6, however, suggest
19 that reaction products formed by the interaction of O3 with respiratory system fluids or
20 tissues may produce effects measured outside the respiratory tract—either in the blood, as
21 changes in circulating blood lymphocytes, erythrocytes, and serum, or as changes in the
22 structure or function of other organs, such as the parathyroid gland, the heart, the liver, and
23 the central nervous system (see Section 6.3). Very little is known, however, about the
24 mechanisms by which O3 could cause these extrapulmonary effects.
25 The results from human exposure studies discussed in the previous criteria document
26 (U.S. Environmental Protection Agency, 1986) were not able to demonstrate any consistent
27 extrapulmonary effects (see Section 10.6 of the 1986 document). Early studies on peripheral
28 blood lymphocytes collected from human volunteers did not find any significant genotoxic or
29 functional changes at O3 exposures of 0.4 to 0.6 ppm for up to 4 h/day. Limited data on
30 human subjects available at the tune the 1986 criteria document was published also indicated
31 that 0.5 ppm O3 exposure for over 2 h caused transient changes in blood erythrocytes and
December 1993 7-103 DRAFT-DO NOT QUOTE OR CITE
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1 sera (e.g., erythrocyte fragility and enzyme activities), but the physiological significance of
2 these studies remains questionable. The conclusions drawn from these early studies raises
3 doubt that cellular damage or altered function is occurring to circulating cells at
4 O3 exposures under 0.5 ppm.
5 New studies on the potential extrapulrnonary effects of in vivo O3 exposure of human
6 subjects published since the publication of the previous criteria document (U.S.
7 Environmental Protection Agency, 1986) have not been very definitive. Johnson et al.
8 (1986) exposed 11 male nonsmokers to 0.5 ppm O3 for 4 h on 2 consecutive days. When
9 compared to air controls, O3 exposure did not result in any significant change in the activity
10 of blood plasma a-1-proteinase inhibitor. Schelegle et al. (1989) exposed 20 O3-sensitive,
11 healthy young men to 0.20 and 0.35 ppm with heavy exercise (VE = 50 L/min). Plasma
12 concentrations of PGF2a were elevated after 40 and 80 min of exposure to the higher
13 O3 level (0.35 ppm). It is likely, however, that the elevation of this ecosanoid in the blood
14 was due to either increased production or decreased metabolism of PGF2a in the lung.
15 The demonstration in the previous section (Section 7.2.4) of an array of inflammatory
16 mediators and immune modulators released at the airway surface provides a possible
17 mechanism for effects to occur elsewhere in the body. Additional in vivo studies are needed,
18 therefore, in order to determine if there are significant extrapulmonary effects of
19 O3 exposure in humans and at what levels of exposure they might occur.
20
21 7.2.6 Ozone Mixed with Other Pollutants
22 Although it is well-known that polluted air contains a large number of chemical species,
23 the most common approach to evaluating air pollution effects under laboratory conditions has
24 been assessment of responses consequent to exposure to single pollutants. This has been the
25 case for a variety of reasons, not the least of which is the problem inherent in adequately
26 controlling the concentrations of multiple pollutants simultaneously. Further, atmospheric
27 chemistry is very complicated, and it is difficult to adequately assess the exposure mixture as
28 the number of constituent pollutants increases. Observed effects may be related to unknown
29 reaction products, the monitored pollutants being only surrogates. Other problems inherent
30 in mixture studies involve considerations such as whether pollutants are presented
31 simultaneously, or in sequential or overlapping patterns. Ideally, the selected pattern should
December 1993 7-104 DRAFT-DO NOT QUOTE OR CITE
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1 at least approximate one that occurs in the ambient environment. In spite of these
2 difficulties, information from mixture studies is important from the standpoint of attempts to
3 better understand responses of humans to the complex mixture of ambient air.
4 The previous O3 criteria document (U.S Environmental Protection Agency, 1986)
5 evaluated the limited data base of information available on mixtures of O3 with one or more
6 pollutants, concluding that pulmonary function changes were no more than additive, and in
7 most cases, attributable to O3 alone. Several new studies have since appeared in which
8 human subjects were exposed to mixtures of two or more pollutants, or to individual
9 pollutants sequentially (Table 7-13), extending the database for controlled studies.
10 Epidemiological studies have also investigated mixtures of pollutants and have found
11 evidence suggestive of synergistic effects (see Section 7-4).
12
13 7.2.6.1 Ozone and Sulfur-Containing Pollutants
14 Horvath et al. (1987) compared the pulmonary function responses of male subjects
15 (19 to 29 years of age) with normal baseline pulmonary function to four experimental
16 conditions: (1) FA, (2) 0.25 ppm O3, (3) 1,200 to 1,600 /*g/m3 H2SO4 aerosol, and
2
17 (4) 0.25 ppm O3 + 1,200 to 1,600 /xg/m H2SO4 aerosol. Exposures were completed in
18 random sequence, a minimum of 1 week apart, and were conducted at 35 °C and 83%
19 relative humidity. Subjects alternated 20-min rest and exercise (VE = 30 to 32 L/min)
20 periods throughout the 2-h exposures. The results indicated that neither O3 alone nor
21 O3 mixed with H2SO4 aerosol had significant effects on any pulmonary function, metabolic,
22 or ventilatory parameter.
23 Koenig et al. (1990) evaluated sequential O3 (0.12 ppm) and SO2 (0.10 ppm) exposures
24 in 13 allergic, asthmatic adolescents (12 to 18 years of age). Three subjects used no regular
25 medications, the other 10 used one or more of beta-adrenergic agents, theophylline, and
26 antihistamines. All subjects had a PC20 for methacholine of 10 mg/mL or less. Subjects
27 took their morning medication on experiment days if needed, but at least 4 h elapsed between
28 any medication use and the start of the experiment. The subjects participated in three
29 exposures (22 °C and 75% relative humidity), which were presented in random order and at
30 least 1 week apart. The three exposures were (1) air - SO2, (2) O3 - O3, and
31 (3) O3 - SO2. The mouthpiece exposures were 1 h in duration, during which the subjects
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TABLE 7-13. OZONE MIXED WITH OTHER POLLUTANTS8
-jj
t—*
o
Concentration
ppm
Exposure Number and
Duration and Exposure Gender of
3 c
/tg/m Pollutant Activity Conditions Subjects
Subject
Characteristics
Observed Effect(s)
Reference
Peroxyacetyl Nitrate
0.485
0.27
0.45
0.30
0.45
0.60
0.13
951 O3
1,337 PAN
882 O3
1,485 PAN
882 O3
1,128 N02
644 PAN
Nitrogen-Containing
0.12
0.30
0.20
235 O3
564 N02
392 O3
500 HNO3
H2O
2 h T = 21 °C 10 F
IE WBGT
VE « 25 L/min
2 h T « 22 °C 3 M
IE RH « 60% 5 F
VE « 27 L/min
2 h T = 24 °C 16 M
IE RH = 55-58% 16 F
VE = 25 L/min
Pollutants
1 h (mouthpiece) T = 22 °C 5 M
IE RH = 75% 7 F
VE = 4-5 times
resting
5 h T = 20 °C 6 M
IE (50 min/h RH = 5% 4 F
exercise)
VE « 40 L/min
2 h HNO3 or
H2O fog or air,
followed by 1-h
break, followed
Healthy NS
19-36 years
Healthy NS
Mean age = 24 years
Healthy NS
16 subjects,
19-26 years;
16 subjects,
51-76 years
Healthy NS
12-17 years
Healthy NS
Minimum of 10%
decrement in FEVj
after 3 h exposure to
0.20 ppm O3 with
50 min exercise/h
Exposure to the mixture of
PAN + O3 induced decrements
in FVC and FEVt averaging 10%
greater than observed following
exposure to O3 alone.
No differences between
responses to exposure to O3
alone and Oj + PAN.
No differences between
responses to O3 alone,
QI + NO2, 03 + PAN, or
03 + NO2 + PAN.
No significant changes hi any
pulmonary function with O3
alone or O3 -1- NO2.
Exposure to HNO3 or H2O fog
followed by O3 induced smaller
pulmonary function decrements
than air followed by O3 .
Horvath et al.
(1986)
Drechsler-Parks
et al. (1987b)
Drechsler-Parks
et al. (1989)
Koenig et al.
(1988)
Aris et al. (1991)
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December 1993
7-107
DRAFT-DO NOT QUOTE OR CITE
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TABLE 7-13 (cont'd). OZONE MIXED WITH OTHER POLLUTANTS8
I
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Exposure
Concentrationb Duration and Exposure
ppm £ig/m Pollutant Activity Conditions0
Sulfur-Containing Pollutants (cont'd)
0.12 235 O3 6.5 h T = 21 °C
100 H2SO4 2 consecutive RH = 50%
days
50 min exercise/h
VE = 29 L/min
0.12 235 O3 1.5 h with IE for T = 22 °C
0.30 564 NO2 two consecutive RH = 65%
70 H2SO4 days; VE »
0.050 HNO3 23.2 L/min
0.25 490 O3 2 h T = 35 °C
1,200- H2S04 IE RH = 83%
1,600 aerosol VE = 30-
32 L/min
Number and
Gender of Subject
Subjects Characteristics
Nonasthmatic 22-41 years
8M NS
7F
Asthmatic 1 8-50 years
13 M NS
17 F
Asthmatic 12-19 years
adolescents NS
22 completed
study
15 M
7F
9 M Healthy NS
19-29 years
Observed Effect(s) Reference
Exposure to O3 or O3 -t- H2SO4 Linn et al.
induced significant decrements hi (1993)
forced expiratory function.
Differences between O3 and O3 +
H2SO4 were at best marginally
significant. O3 is the more important
pollutant for inducing respiratory
effects. A few asthmatic and
nonasthmatic subjects were more
responsive to O3 -1- H2SO4 than to
O3 alone.
No significant pulmonary function Koenig et al.
changes following any exposure (1993)
compared to response to clean air.
Six additional subjects started the
study, but dropped out due to
uncomfortable symptoms.
No significant effects of exposure to Horvath et al.
O3 alone or combined with H2SO4 (1987)
aerosol.
aSee glossary of terms and symbols for abbreviations and acronyms.
Grouped by pollutant mixture.
°WBGT = 0.7 Twet bulb + 0.3 Tdiy bulb or globe.
Age range in years or as mean ± SEM.
-------
1 breathed one test gas for 45 min, followed by a second gas for the final 15 min. Subjects
2 exercised at a VE of about 30 L/min during the second and fourth 15-min segments of the
3 exposure. Pulmonary functions were measured 2 to 3 and 7 to 8 min postexposure.
4 Changes in ¥EVl and RT were significantly greater following the O3 - SO2 exposure than
5 following the other two exposures. Although the subject group was small, the results
6 indicate that O3 exposure may potentiate responses to SC^ exposure in asthmatic adolescents.
7 It should be noted that the SO2 concentration (0.100 ppm) used in this study is a subthreshold
8 level.
9 Linn et al. (1993) evaluated the pulmonary function and symptom responses of
10 15 atopic or normal subjects and 30 asthmatic subjects exposed to FA, 0.12 ppm O3,
11 100 /xg/m respirable H2SO4 aerosol, and a mixture of the two pollutants. The chamber
12 exposures were 6.5 h in duration, during which the subjects walked on a treadmill (VE about
13 29 L/min) for 50 min of each hour. There was a 30 min lunch period following the third
14 hour. Pulmonary function and symptom responses were measured preexposure and during
15 the hourly 10 min breaks, and a methacholine bronchochallenge test was performed following
16 each exposure. Relative to responses to the FA exposure, H2SO4 aerosol exposure alone
17 induced no significant alteration in pulmonary function, symptoms, or bronchial reactivity to
18 methacholine. Exposure to O3 alone, or mixed with H2SO4 aerosol, induced significant
19 decrements in forced expiratory function and increased bronchial reactivity. Both effects
20 were greater on the first of two consecutive days of exposure. Group mean lung function
21 and methacholine reactivity changes were somewhat larger following O3 + H2SO4 aerosol
22 compared to exposure to O3 alone, but the differences were at best marginally significant,
23 and usually nonsignificant, depending on the function tested. However, there were a few
24 individual subjects who showed significantly larger pulmonary function decrements following
25 the exposure to O3 + H2SO4 than following exposure to O3 alone. The authors concluded
26 that O3 is more important than H2SO4 aerosol in inducing pulmonary dysfunction in normal,
27 atopic and asthmatic adults. There does, however, appear to be a more sensitive
28 subpopulation that does respond to O3 + H2SO4 aerosol more strongly than the average
29 adult.
30 Kagawa (1986) exposed Japanese men to three mixtures (1) O3 (0.30 ppm) + NO2
31 (0.30 ppm) + H2S04 (200 /ig/m3), (2) O3 (0.15 ppm) + NO2 (0.15 ppm) + H2SO4
December 1993 7409 DRAFT-DO NOT QUOTE OR CITE
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1 (200 /ig/m3), or (3) O3 (0.15 ppm) + N02 (0.15 ppm) + SO2 (0.15 ppm)+ H2SO4
2 (200 j«g/m ). Exposures were 2 h in duration, and subjects exercised for a total of 20 min
3 during exposure 1 or 60 min during exposures 2 and 3. Some of the subjects were smokers.
4 Reported symptoms were attributed to O3 exposure, whereas small decrements in airway
5 conductance (^10%) were observed following exposures to mixtures 1 and 2. Although the
6 magnitude of the FEVj decrement is not stated, a possible decrease was observed after
7 exposure 3. The responses observed with these mixed exposure conditions were no different
8 than responses reported for exposures to similar concentrations of O3, indicating no enhanced
9 response due to the presence of the other pollutants in the mixtures.
10
11 7.2.6.2 Ozone and Nitrogen-Containing Pollutants
12 Adams et al. (1987) reported on the responses of 20 males and 20 females (18 to
13 30 years of age), all healthy nonsmokers, exposed to (1) FA, (2) 0.30 ppm O3, (3) 0.60 ppm
14 NO2, and (4) 0.30 ppm O3 + 0.60 ppm NO2. Subjects were exposed via mouthpiece for
15 1 h, during which they exercised continuously at VE of about 70 L/min for males and
16 50 L/min for females. The exposures were presented in random order, a minimum of 5 days
17 apart. There were no differences in any pulmonary function between the O3 and
18 NO2 + O3 exposures, except for SR^, which was lower following NO2 + O3 than
19 following O3 alone for both men and women.
20 Koenig et al. (1988) exposed 14 male and 10 female adolescents to FA, 0.30 ppm NO^
21 0.12 ppm O3, and 0.30 ppm NO2 + 0.12 ppm O3. Twelve of the subjects were healthy
22 normals, and the other 12 were allergic asthmatics. The asthmatics, except for one who took
23 no regular medications, used one or more of beta-adrenergic agents, theophylline, and
24 antihistamines. Asthmatic subjects took their morning medications if needed, but refrained
25 from medication use for at least 4 h prior to the exposures. The mouthpiece exposures were
26 1 h in duration, during which the subjects exercised hi 15-min periods (mean VE = 32.8 ±
27 6.0 L/min), alternated with 15-min rest periods. Neither normal nor asthmatic subjects
28 evidenced changes in any measure of pulmonary function following O3 or NO2 +
29 O3 exposure.
30 Aris et al. (1991) examined pulmonary function responses to a 3-h exposure to
31 0.20 ppm O3 following a 2-h exposure to nitric acid (HNO3) or water (H2O) fog. This is a
December 1993 7410 DRAFT-DO NOT QUOTE OR CITE
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1 common pattern of pollutant exposure in coastal California areas. Subjects were 10 healthy
2 adults 21 to 31 years of age. They were prescreened for a decrement of 10% or greater in
3 FEVj following 3 h of exposure to 0.20 ppm 63, during which they exercised for 50 min of
4 each hour (VE = 40 L/min). Decrements in FEVt following the screening O3 exposure
5 ranged from 15 to 49%. The three exposures (FA + O3, H2O fog + O3, and HNO3
6 fog + O3) were presented in random order, and were separated by a minimum of 2 weeks.
7 The authors hypothesized that exposure to acidic fog, followed by O3 exposure, would
8 induce greater decrements in FVC and FEVt than H2O fog or air followed by O3 exposure.
9 In fact, both HNO3 and H2O fog exposure seemed to ameliorate the effect of subsequent
10 O3 exposure on FEVj and FVC, although only the difference between the FEVj responses to
11 FA + O3 and H2O fog + O3 was significant. The results of comparisons between baseline
12 methacholine response and that following the exposures indicated that the O3 sensitive
13 subjects were more sensitive to methacholine than O3 nonsensitive subjects, who participated
14 only in the sensitivity screening O3 exposure, suggesting that O3 responsiveness may be
15 related to nonspecific airway responsiveness.
16 Aris et al. (1993b) further examined pulmonary responses to combined 0^ and HNO3
17 exposures. Ten healthy, nonsmoking adults, 19 to 41 years of age, were exposed to filtered
18 air, 500 /xg/m of HNO3 gas plus 0.20 ppm O3, or to 0.20 ppm O3 alone. The exposure
19 protocol was 4 h in duration, with 50 min IE at 40 L/min alternating with 10-min rest
20 periods each hour. Pulmonary function was measured during each rest period, whereas
21 BAL, proximal airway lavage, and bronchial biopsies were performed 18 h after completion
22 of each exposure. Mean FEVj and FVC decreased, and mean SR^ and respiratory
23 symptom scores increased across both the HNO3 + O3 and O3 exposures. The results
24 indicated, however, that HNO3 combined with O3 did not exacerbate the pulmonary function
25 decrements or respiratory symptoms caused by O3 alone. Similarly, there were no
26 statistically significant differences in the cellular or biochemical constituents in either the
27 BAL or proximal airway lavage fluids or in the bronchial biopsy specimens between the
28 HNO3 + O3 and the O3 exposures. The authors concluded that HNO3 does not potentiate
29 the inflammatory response produced by O3 in healthy individuals.
30 The objective of a study by Koenig et al. (1993) was to investigate possible interactions
31 between oxidants (0.12 ppm O3 + 0.30 ppm NO^ and H2SO4 aerosol (70 /*g/m3) or HNO3
December 1993 7-111 DRAFT-DO NOT QUOTE OR CITE
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1 aerosol (0.05 ppm). Twenty-two adolescent allergic asthmatics who also had exercise-
2 induced bronchospasm and a positive response to a standardized methacholine
3 bronchochallenge test completed all exposures. Subjects inhaled FA, O3 + NO2, O3 + NO2
4 + H2SO4, or O3 + NO2 + HNO3 through a mouthpiece for 90 min on 2 consecutive days.
5 Each pair of exposures was separated by at least 1 week. Subjects exercised (VE about
6 10 times FVC) and rested in alternating 15-min periods. Pulmonary function (FVC, FEV1?
7 Vmax50%, Vmax75%, and RT) were measured before and after each exposure and on the day
8 following the second consecutive exposure, at which time only pulmonary function was
9 evaluated and a methacholine bronchochallenge was performed. Six additional subjects
10 began the study, but dropped out before completion due to uncomfortable symptoms
11 associated with the exposures. None dropped out following an FA exposure. There were no
12 statistically significant changes in any measured parameter of pulmonary function following
13 the three pollutant-containing exposures, compared to following the FA exposure, contrary to
14 expectations. (See also Section 7.4 for related epidemiological studies.)
15
16 7.2.6.3 Ozone, Peroxyacetyl Nitrate, and More Complex Mixtures
17 Horvath et al. (1986) exposed 10 healthy young women (19 to 36 years of age) to
18 (1) FA, (2) 0.48 ppm O3, (3) 0.27 ppm PAN, and (4) 0.48 ppm O3 + 0.27 ppm PAN. The
19 chamber exposures were 2 h in duration, during which subjects alternated 20-min exercise
20 periods (VE = 25 L/min) and 15-min rest periods. Exposures were completed in random
21 order, and were at least 1 week apart. Exposure to PAN alone did not induce any significant
22 changes in pulmonary function. Both O3 and PAN + O3 exposure induced significant
23 decrements in FVC, FEVj, and FEF25.75%; however, the decrements following the
24 PAN + O3 exposure were significantly larger (average of about 10%), suggesting interaction
25 between PAN and O3. It should be noted that typical peak ambient PAN concentrations are
26 about 0.050 ppm. Symptom reports indicated that O3 + PAN exposure induced greater
27 subjective stress than exposure to O3 alone.
28 Drechsler-Parks et al. (1987b) exposed eight healthy young adults (mean age 24 years)
29 to a mixture of 0.30 ppm PAN + 0.45 ppm O3 on 5 consecutive days to evaluate possible
30 desensitization. Subjects were reexposed to the PAN + O3 mixture on the third and seventh
31 days following the last consecutive day of exposure to evaluate the persistence of the
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1 desensitization response. Desensitization occurred with the same pattern and time sequence
2 as has been reported for O3 alone. The largest group mean decrements occurred following
3 the second exposure, and the subjects became progressively less responsive with subsequent
4 exposures. Two subjects failed to desensitize fully with 5 consecutive days of exposure.
5 Puhnonary function changes after the follow-up exposures indicated that the desensitization
6 response is relatively short lived, in that the desensitization began to abate between 3 and
7 7 days following the fifth consecutive day of exposure. These results are consistent with
8 those of similar studies using exposure to O3 only (Horvath et al., 1981; Kulle et al., 1982),
9 suggesting that PAN had no additional effect on the desensitization response to O3.
10 A greater number of symptoms was reported following all PAN + Oj exposures than
11 following exposure to O3 alone.
12 Drechsler-Parks et al. (1989) studied 16 older men and women (51 to 76 years of age)
13 and 16 young men and women (19 to 26 years of age) who each completed 2-h chamber
14 exposures to FA, 0.45 ppm O3, and mixtures of 0.45 ppm O3 with 0.60 ppm NO2 and/or
15 0.13 ppm PAN. Subjects alternated 20-min exercise (VE about 25 L/min) and rest periods.
16 Exposure to O3 alone and in all combinations induced significant decrements in FVC, FEVj,
17 and FEF25_75% in the young group. In the older group, these same three variables were
18 significantly decreased only with NO2 + O3 exposure. Exposure of the older subjects to
19 PAN + O3 induced significant decrements only in FVC and FEVj. The PAN + NO2 +
20 O3 exposure induced a significant decrement only in FVC in the older subjects. Both young
21 and older subjects reported more symptoms following the mixture exposures than following
22 exposure to O3 alone. These puhnonary function results following the exposure to
23 O3 + PAN are in contrast to those reported by Drechsler-Parks et al. (1984) and Horvath
24 et al. (1986) on young adults exposed to 0.45 ppm O3 -I- 0.30 ppm PAN. The results of
25 both earlier studies suggested an interaction between O3 and PAN, in that puhnonary
26 function decrements following the mixture exposure were approximately 10% larger than
27 following exposure to O3 alone, whereas there were no significant puhnonary function effects
28 with exposure to PAN alone. A likely explanation for this discrepancy is that the PAN
29 concentration used by Drechsler-Parks et al. (1989) was slightly less than half that used by
30 Drechsler-Parks et al. (1984) and Horvath et al. (1986). Thus, if the additional effect of
31 PAN is linear, an additional effect of PAN + O3 would be expected to be less than 5%,
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1 which would probably not be detected because it is within the variability of the pulmonary
2 function measurements. In any case, ambient PAN concentrations are considerably less than
3 even 0.13 ppm. This indicates that even if PAN and O3 do interact in some way in their
4 effects on pulmonary function, at typical ambient concentrations of Oj and PAN, effects can
5 be attributed to O3 alone.
6
7 7.2.6.4 Summary
8 Information on interactive effects between O3 and other pollutants remains sparse at this
9 time. However, several comments can be made when the material discussed above is
10 summarized collectively. It is clear that O3 is responsible for the largest share of observed
11 effects when subjects are exposed to the mixtures of O3 and other pollutants that have been
12 studied to date. There is no evidence that simultaneous exposure of healthy individuals to
13 ambient concentrations of O3 plus NO2, PAN, H2SO4, HNO3, or SO2 results in significant
14 interaction. However, Aris et al. (1991) have reported that HNOg and H2O fog exposure
15 ameliorates the pulmonary function effects of a subsequent O3 exposure. Koenig et al.
16 (1990) found that preexposure to O3 induced significant pulmonary function decrements in
17 allergic asthmatic adolescents following a sequential SO2 exposure. Both the O3 and SO2
18 concentrations were at subthreshold levels.
19 Both studies that have reported potentially significant effects have involved sequential
20 exposure protocols, in contrast to the simultaneous exposure protocols, which have not
21 generally shown effects beyond those that would be expected at the O3 concentration used.
22 It may be that certain preexposures predispose an individual to responses following a
23 subsequent exposure; however, this question remains far from being resolved. Further, these
24 results are related only to spirometry and plethysmography and may not be applicable to
25 other possible endpoints.
26
27
28 7.3 SYMPTOMS AND PULMONARY FUNCTION IN CONTROLLED
29 STUDIES OF AMBIENT AIR EXPOSURES
30 Controlled O3 exposure studies under a variety of different experimental conditions
31 have generated a large amount of informative exposure-effects data. However, complete
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1 laboratory simulation of the pollutant mix present in ambient air is impossible on practical
2 grounds. Thus, the exposure effects of one or several artificially generated pollutants (i.e.,
3 a simple mixture) on symptoms and lung function may not be comparable to those in ambient
4 air where complex mixtures of pollutants likely exist. This section reviews two types of
5 studies that utilize a mobile laboratory or a hypobaric chamber to investigate the acute effects
6 of 63 during exposures to ambient air or altitude, respectively. These studies can be
7 designed to determine the independent effects of O3 as well as possible interactions among
8 many pollutants and other conditions present in typical ambient air.
9
10 7.3.1 Mobile Laboratory Studies
11 Quantitatively useful information on the effects of acute exposure to photochemical
12 oxidants on symptoms and pulmonary function originated from field studies using a mobile
13 laboratory, as presented in the previous criteria document (U.S. Environmental Protection
14 Agency, 1986). These studies offer the advantage of studying the effects of ambient air on a
15 local subject population by combining the experimental methods of both epidemiology and
16 controlled-exposure studies. Field studies using mobile exposure chambers can expose
17 subjects to ambient air, FA without pollutants, or FA containing artificially generated
18 concentrations of O3 that are comparable to those measured in the ambient environment.
19 The exposure air can also be conditioned to a desired temperature and humidity. As a result,
20 measured health responses in ambient air can be compared to those found in more artificial
21 or controlled conditions. The mobile laboratory shares many of the same limitations of
22 stationary exposure laboratories (e.g., limited number of both subjects and artificially
23 generated pollutants for testing). Ambient air studies in the mobile laboratory are dependent
24 on ambient conditions, which can be unpredictable, uncontrollable, and not completely
25 characterizable. Logistical problems (space, power, and locations with local interfering
26 outdoor conditions) limit access to many ambient pollution sites of interest.
27 As summarized in Table 7-14, investigators at the Rancho Los Amigos Medical Center
28 in California used a mobile laboratory and demonstrated that respiratory effects in
29 Los Angeles residents are related to O3 concentration and level of exercise (Linn et al.,
30 1980, 1983b; Avol et al., 1983, 1984, 1985a,b,c, 1987). Such effects include pulmonary
31 function decrements at O3 concentrations of 0.144 ppm in exercising healthy adolescents
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TABLE 7-14. ACUTE EFFECTS OF OZONE IN AMBIENT AIR IN
FIELD STUDDZS WITH A MOBILE LABORATORY8
Mean uzone
Concentration
ppm pg/m
0.113 22T"
± .033 ± 65
Amoient
Temperature Exposure
(°C) Duration
Th—
Activity
Level ( Vg) Number of Subjects
CE (22 L/min) 66 hei
children
(8-11 years)
Observed Effects) Reference
No significant changes in Avol et al. (1987)
forced expiratory
function and respiratory
symptoms after
exposure to 0.113 ppm O3 in
ambient air.
33 ± 1
0.144 282 32 ± 1 TE CE (32 L/min) 59 healthy adolescents
± .04S ±84 (12-15 years)
"Olll300 32 ± 2 Tl CE (53 L/min) 50 healthy adults
± .025 ± 49 (competitive bicyclists)
Small significant decreases in Avol et al. (1985a,b)
FVC(-2.1%),FEV075
(-4.0%), FEV! (-4.2%), and
PEFR (-4.4%) relative to
control with ao recovery during
a 1 -h postexposure rest; no
significant increases in
symptoms.
0.156 306 33 ± 4 Th CE (38 L/min) 48 healthy adults
± .055 ± 107 50 asthmatic adults
Mild increases in lower
respiratory symptom
scores and significant decreases
in FEV, (-5.3%)andFVC;
mean changes in ambient air
were not statistically different
from those in purified air
containing 0.16 ppm 03.
Avol et al. (1984, 1985c)
No significant changes for total Linn et al. (1983b)
symptom score or forced Avol et al. (1983)
expiratory performance in
normals or asthmatics; however,
FEVj remained low or
decreased further (-3%) 3 h
after ambient air exposure in
asthmatics.
-------
TABLE 7-14 (cont'd). ACUTE EFFECTS OF OZONE IN AMBIENT AIR IN
FIELD STUDIES WITH A MOBILE LABORATORY8
S3
f
K-»
^o
vo
LO
-J
i»i
*— *
•J
Mean Ozone
Concentration
ppm ng/m
0.165 323
± .059 ± 115
0.174 341
± .068 ± 133
Ambient
Temperature0
(°C)
33 ± 3
33 ± 2
Exposure
Duration
1 h
2h
Activity
Level ( Y£) Number of Subjects
CE (42 L/rnin) 60
(7
IE (2 X resting) 34
at 15-min 30
intervals
"healthy" adults
were asthmatic)
"healthy" adults
asthmatic adults
Observed Effect(s)
Small significant decreases in FEV i
(-3.3%) and FVC with no recovery
during a 1-h postexposure rest; TLC
decreased and AN2 increased slightly.
Increased symptom scores and small
significant decreases in FEVj
(-2.4%), FVC, PEFR, and
TLC in both asthmatic and healthy
subjects; however, 25/34 healthy
subjects were allergic and "atypically"
reactive to polluted ambient air.
Reference
Linn et al. (1983b)
Avol et al. (1983)
Linn et al. (1980, 1983b)
See glossary of terms and symbols for abbreviations and acronyms.
Ranked by lowest level of 63 in ambient air, presented as the mean ± SD.
cMean ± SD.
-------
1 (Avol et al., 1985a,b) and increased respiratory symptoms and pulmonary function
2 decrements at 0.153 ppm in heavily exercising athletes (Avol et al., 1984, 1985c) and at
3 0.174 ppm in lightly exercising normal and asthmatic subjects (Linn et al., 1980, 1983b).
4 The observed effects were typically mild, and generally no substantial differences were seen
5 between asthmatic and nonasthmatic subjects. Postexposure pulmonary function decrements
6 appeared to last several hours longer in the asthmatics, but no significance test was reported
7 for this difference (Avol et al., 1983; Linn et al., 1983b). The medication status of the
8 asthmatic subjects during the studies was not reported, although medications were
9 temporarily withheld prior to exposures. The subjects' clinical severity was likely mild,
10 based upon their baseline lung function and exercise capability. Many of the normal subjects
11 with a history of allergy appeared to be more responsive to O3 than "nonallergic" normal
12 subjects (Linn et al., 1980, 1983b), although a standardized evaluation of atopic status was
13 not performed. Direct comparative studies of exercising athletes (Avol et al., 1984, 1985c)
14 with chamber exposures to oxidant-polluted ambient air (mean O3 concentration of 0.15 ppm)
15 and purified air containing a controlled concentration of generated O3 at 0.16 ppm showed no
16 significant differences in lung function and symptoms, suggesting that coexisting ambient
17 pollutants had minimal contribution to the measured responses under the typical summer
18 ambient conditions in Southern California. Effects of copollutants in other regions of the
19 country remain to be investigated with the mobile laboratory. These field studies emphasize
20 the importance of adequate characterization of subjects and the ambient air, exercise levels,
21 duration of exposure, and individual variations hi sensitivity in interpreting observed
22 exposure effects. Although these factors need to be investigated over a wider range of
23 experimental conditions, the results from these field studies are, so far, consistent with those
24 from controlled human exposure studies. Short-term respiratory effects of summer ambient
25 oxidant pollution in Southern California are predominantly, if not entirely, caused by ambient
26 O3 in typical healthy or asthmatic residents, according to mobile laboratory studies (Avol
27 etal., 1984, 1985c).
28
29 7.3.2 High-Altitude Studies
30 Symptoms and pulmonary function resulting from exposure to O3 in commercial aircraft
31 flying at high altitudes and in altitude-simulation studies were reviewed in the previous
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1 criteria document (U.S. Environmental Protection Agency, 1986). Much attention has
2 focused on the health effects in flight crew and, specifically, flight attendants because of their
3 physical activities at altitude and exposure patterns to peak levels of cabin 03. The most
4 quantitatively useful information was based on a series of hypobaric studies of normal
5 nonsmoking subjects who were exposed to 1,829 m (6,000 ft) and O3 at concentrations of
6 0.2 and 0.3 ppm for 3 or 4 h (Lategola et al., 1980a,b). Increased symptoms and pulmonary
7 function decrements occurred at 0.30 ppm but not at 0.2 ppm under light exercise conditions.
8 However, the exposure conditions did not reflect higher (peak) O3 concentrations reported to
9 occur in certain aircraft at high altitudes or the higher cabin altitudes attained by new-
10 generation commercial aircraft.
11 No reports have subsjequently appeared in the literature that specifically study the health
12 effects of aircraft cabin O3. However, O3 levels were reported to be very low (average
13 concentration 0.01 to 0.02 ppm) during 92 randomly selected smoking and nonsmoking
14 flights in 1989 (Nagda et al., 1991). None of the flights exceeded the time-weighted average
15 standard of 0.10 ppm (during any 3-h interval) promulgated by the U.S. Federal Aviation
16 Administration, perhaps related to the use of O3-scrubbing catalytic filters (Melton, 1990).
17 However, in-flight O3 exposure is possible because catalytic filters are not necessarily in
18 continuous use during flight.
19
20
21 7.4 FIELD AND EPIDEMIOLOGY STUDIES
22 7.4.1 Acute Effects of Ozone Exposure
23 7.4.1.1 Introduction
24 Field and epidemiology studies addressing the acute effects of O3 on lung function
25 decrements and increased morbidity and mortality in human populations involve those
26 combinations of environmental conditions, copollutant levels, and activity levels present
27 under real-world conditions of O3 exposure. This real-world relevance is an advantage over
28 animal or human chamber studies. Thus, results of such studies are essential components of
29 an understanding of overall effects of O3. However, the conditions under which
30 epidemiologic studies are carried out cannot be controlled in the same way that they can in
31 experimental studies. Parameters that may be difficult or impossible to estimate or control
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1 outside the laboratory include actual O3 exposures, levels of temperature, relative humidity,
2 allergens, correlated pollutants other than O3, and breathing rates and activity patterns of
3 subjects. Variations in these factors can be important sources of variability in data and
4 results, and may, under certain conditions, lead to biases (e.g., confounding) in results.
5 These and other issues of importance in the interpretation of epidemiology study results are
6 discussed in each relevant section below.
7 The above noted weaknesses of epidemiologic studies of O3 health effects were
8 highlighted in the previous O3 criteria document (U.S. Environmental Protection Agency,
9 1986), which reached the conclusion that, because of such factors, epidemiologic studies on
10 the acute effects of O3 on lung function do not provide information that is quantitatively
11 useful in the standard-setting process. Since publication of 1986 O3 criteria document,
12 however, results have become available from a substantial number of well-conducted,
13 individual-level field studies and aggregate-level time-series studies. In the following
14 section, these studies will be collectively evaluated to determine to what extent epidemiologic
15 studies may now play a quantitative role in the evaluation of the NAAQS for O3.
16
17 7.4.1.2 Individual-Level Studies
18 The studies discussed in this section fall into three main categories: summer camp
19 studies, exercise studies, and daily life studies. Summer camp studies involve collection of
20 sequential (usually daily) data on lung function, respiratory symptoms, and environmental
21 conditions over the course of 1 or 2-week camps. Exercise studies are unique in that lung
22 function and respiratory symptoms are measured before and after each of a series of discrete
23 exercise events in the presence of ambient air pollution. Daily life studies measure lung
24 function, respiratory symptoms, and/or exacerbation of existing respiratory diseases, along
25 with environmental variables at regular intervals in the course of normal daily activities of a
26 population. These include studies in healthy adults and school-aged children as well as
27 studies in individuals with preexisting disease (e.g., asthma). Medication use may also be
28 monitored in asthmatics.
29 The important differences among the three study types relate mainly to issues of
30 exposure assessment. Because subjects are mainly out-of-doors or in well-ventilated cabins,
31 exposure estimation errors are usually minimized in camp and exercise studies. In contrast,
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1 larger exposure estimation errors may occur in daily life studies. Camp and daily life studies
2 enable assessment of effects of cumulative O3 exposures, whereas exercise studies limit
3 attention to rather brief exposures. Exercise studies offer the potential of assessing individual
4 VE values and O3 doses during the relevant exposure period.
5 Although the study designs differ in some ways, the central design feature of all of
6 these study types is the collection of repeated measurements on individuals. This feature is
7 exploited in data analysis by having each subject serve as his or her own control. For
8 continuous outcomes such as lung function, subject-specific linear regressions are usually
9 performed with lung function (or change in lung function) as the outcome variable and 63 or
10 other environmental factors as the explanatory variable(s). The regression slope is a measure
11 of individual lung function response to O3. The mean slope across individuals is often used
12 as a measure of the average population response. A more statistically valid approach
13 involves computing the mean slope with weighting proportional to the inverse variances of
14 the individual slopes. An alternative approach has been to use analysis of covariance
15 methods to fit a population-pooled slope and separate, subject-specific y-intercepts. To date,
16 no studies have used nonlinear (e.g., quadratic) models, which in chamber studies have been
17 shown to better describe the functional relationship between low-level O3 exposures and lung
18 function decrements (Hazucha, 1987).
19
20 Issues in the Interpretation of Individual Level Studies
21 The most basic question affecting the interpretation of acute O3 epidemiology studies is
22 whether, and to what extent, the associations observed between O3 and decreased pulmonary
23 function are causally related to O3 and not merely due to confounding by some other
24 factor(s) (e.g., temperature, allergens, time trends in spirometry, or other pollutants).
25 By definition, a confounder is an unmeasured, or unaccounted-for, variable that both has an
26 effect on lung function and is also correlated with O3 concentrations. Variables that satisfy
27 only one of these latter two conditions are not confounders. For example, a variable that
28 affects lung function, but is independent of O3, would add variation to lung function
29 measurements, but would not confound an O3/lung function analysis. A variable that
30 correlates with O3, but does not directly affect lung function (in the range of measurements),
31 would not confound an analysis of O3 effects. Other variables might modify the effect of
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1 O3 on lung function, thereby increasing or decreasing the O3 effect under the conditions of
2 study. Epidemiologists refer to this latter phenomenon as "effect modification". The
3 presence of effect modification does not bias the results of a study, and also can provide
4 insights into the range of effect magnitudes (e.g., slope of lung function development on
5 O3 level) that occur under varying environmental conditions.
6 Ambient air temperature often exhibits a moderate to high correlation over time with
7 O3 in acute epidemiology studies due, in part, to the dependence of O3 formation rate on
8 light intensity. Among the studies reviewed in this section, correlations ranging from
9 -0.06 to 0.90 (mean = 0.51) have been reported. Correlations between O3 and relative
10 humidity, when reported, have been in the range -0.4 to -0.6. Several human chamber
11 studies have examined the possible effects of temperature and relative humidity on lung
12 function independent of O3, with somewhat mixed results (Stacy et al., 1982; Folinsbee
13 et al., 1985; Eschenbacher et al., 1992). Two studies have reported increases in FEVl at
14 high temperature (30 and 37 °C) and 60% relative humidity (Stacy et al., 1982;
15 Eschenbacher et al., 1992), whereas another reported no effect on FEVj at 35 °C, but a drop
16 at 40 °C (Folinsbee et al., 1985). Referring to results of acute Oj epidemiology studies,
17 Eschenbacher and colleagues (1992) concluded based on their results that "the associations
18 found between ambient O3 and daily changes in ventilatory function cannot be attributed to
19 the heat and humidity stress often associated with high O3 concentrations." Temperatures
20 observed in the epidemiology studies reviewed in the present section have mainly been below
21 30 °C, with occasional peaks as high as 35 °C. It should be noted that subjects studied
22 epidemiologically will usually have had an opportunity to acclimate to ambient temperatures
23 prior to, or soon after, the start of the study. In any event, given the laboratory findings, a
24 significant confounding role for temperature in these studies is unlikely. The possibility
25 exists, however, that temperature may semetimes act as an effect modifier. Folinsbee et al.
26 (1977a) reported that high temperature potentiated O3 effects on lung function, perhaps by
27 increasing VE for a given workload. Effect modification in the opposite direction is also
28 possible due to curtailment of activity on very hot days.
29 Exposure to specific allergens can influence lung function in individuals who have
30 diseases characterized by IgE-mediated antigen-antibody interactions (i.e., atopy) and may
31 also affect individuals who have an atopic tendency (e.g., as assessed by positive prick skin
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1 test or serum levels of total IgE) without diagnosed clinical disease. Raizenne and colleagues
2 (1989) detected positive reactions to one or more allergens by skin prick in 49% of 96 young
3 nonasthmatic females enrolled in a summer camp study. Few data are available on the
4 correlation between O3 and allergen levels during acute epidemiology studies. However,
5 because both variables are to some extent influenced by weather patterns, some correlation
6 seems likely. Thus, a possible confounding role of airborne allergens in such studies cannot
7 be ruled out. Because of the specific nature of individual antigen sensitization, and
8 uncertainty regarding the full set of relevant allergens in a given setting, attempts to measure
9 and statistically control for allergen levels on a group level in epidemiology studies may not
10 be very effective.
11 The potential effects of time trends in spirometry due to training effects are also of
12 concern. There have been several recent studies that have looked at time trends in serial
13 lung function measurements (mainly FEVj and peak expiratory flow rate [PEFR])
14 independent of air pollution effects (Raizenne et al., 1989; Avol et al., 1990; Hoek and
15 Brunekreef, 1992). In each case, average FEV, measurements have been observed to decline
16 steadily over the first few measurements and then to stabilize or recover slightly to a flat
17 pattern. Average FVC measurements follow a similar pattern. In contrast, PEFR often has
18 been observed to increase steadily over successive measurements. Similar patterns have been
19 observed in studies with intervals between lung function measurements ranging from half a
20 day to 1 week. The consistency of these observations across studies suggests that they
21 represent real phenomena that should be recognized in designing and analyzing studies
22 involving repeated lung function measurements. However, time trends will result in
23 confounding of O3 effects only if, by chance, the trend correlates with temporal variations in
24 O3 concentrations. Such chance correlations could be either positive or negative, and, if
25 present, would have a larger impact (i.e., produce an undesirable degree of confounding) on
26 studies in which all subjects begin the study simultaneously and have few follow-up
27 measurements. Studies that focus on daily changes in lung function may be less impacted by
28 this phenomenon.
29 It is also important to consider the roles of other pollutants as possible confounders or
30 effect modifiers. In the studies to be reviewed in this section, levels of measured
31 copollutants (e.g., SO2, NO2, sulfate and acid aerosols) were present at levels well below
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1 those that have produced lung function decrements in healthy subjects following short-term
2 exposures in chamber studies (see Section 7.2.6). In contrast, an extensive and growing
3 database is available from chamber studies documenting the independent acute effects of
4 ambient-level O3 on lung function (see Section 7.2.1). Although direct lung function effects
5 of other pollutants at typical ambient concentrations seems unlikely, it has been suggested
6 that the effects of O3 may be potentiated by coexposure or previous exposure to other
7 pollutants, most notably acid aerosols (Spektor et al., 1988b). Some data from animal
8 studies suggest interactive effects of O3 and acid exposures for certain pulmonary outcomes
9 (see Chapter 6). However, to date, analyses directed towards this phenomenon in field
10 studies of human lung function (via analysis of the relationship between acid aerosol levels
11 and residuals from regressions of lung function on C^) have proven negative (Spektor et al.,
12 1988a,b). That is, after controlling for the influence of O3, no significant association
13 between acid aerosol peaks and lung function decrements has been observed. Acid aerosol
14 episodes, which often occur coincident with high O3 levels in the summer in the northeastern
15 United States, may extend for several hours or days. The possible potentiating effects of
16 prolonged acid peaks on O3 effects require further study under controlled conditions before
17 this issue can be further resolved.
18 In epidemiologic studies, activity levels are difficult to control and to measure, although
19 this varies with study type (see below). Chamber studies have shown clearly that lung
20 O3 doses and associated functional effects increase as a function of physical activity level
21 (Hazucha, 1987). Epidemiologic study designs have often been chosen that result in
22 relatively high subject activity levels (exercise studies and camp studies), but generally the
23 studies have been carried out without quantitative information on VE distributions across
24 subjects and across time.
25 Variations in activity levels will introduce variability in the relationship between
26 personal exposure and personal dose. If this variability occurs primarily between subjects, it
27 will result in differing O3 doses to people exposed to the same O3 level and yield differences
28 in response that may be misinterpreted as O3 sensitivity variations. If variability in activity
29 levels occurs over time for a given subject, it will add error to the functional relationship
30 linking lung function and O3 exposure. In either case, the influence of activity level
31 variability is to add dose estimation error (or misclassification). Estimates of VE based on
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1 heart rate measurements can be derived using subject-specific calibrations under
2 representative ranges of exercise levels and types (Samet et al., 1993; Raizenne and
3 Spengler, 1989). However, the utility of such VE estimates for reducing dose-
4 misclassification errors in acute O3 epidemiology studies has not yet been demonstrated
5 (Kinney, 1986; Spektor et al., 1988b; Raizenne and Spengler, 1989). This is partly due to
6 the logistical difficulties associated with collecting accurate data, and may also be due to the
7 fact that, for a given subject, VE variations across days are usually small in comparison to
8 O3 concentration variations. The same issues arise in the context of O3 exposure
9 misclassification in "daily life" studies (see below), where outdoor O3 concentrations are
10 used to estimate exposures of subjects who spend substantial amounts of time indoors during
11 the period over which lung function measurements take place.
12
13 Camp Studies of Lung Function in Children
14 Summer camp studies provide the most extensive and reliable information on the acute
15 pulmonary effects of O3 under natural conditions. Camp studies involve the collection of
16 sequential (usually daily) data on lung function on each of a large number of children, along
17 with concurrent measurements of O3 exposures and other environmental factors over the
18 course of a single-week or multiweek summer camp. Data analyses usually consist of
19 estimating the linear association between lung function and environmental variables on an
20 individual basis (allowing each subject to serve as his/her own control) and then testing the
21 mean population association for statistical significance. As noted, summer camps offer the
22 significant advantage that subject exposures are especially well estimated based upon on-site,
23 outdoor O3 monitoring. In addition, these studies assess the pulmonary effects of natural
24 diurnal patterns of O3 exposures, which often involve broad daytime peaks.
25 Since the last O3 criteria document (U.S. Environmental Protection Agency, 1986),
26 eight camp studies have been reported. Design characteristics and results are summarized in
27 Table 7-15. Six of these studies have focused just on normal (i.e., nonasthmatic) children,
28 one focused on asthmatics exclusively (Thurston et al., 1993c), and one looked at both
29 normal and asthmatic children (Raizenne et al., 1987). Although methods and results varied
30 somewhat across studies, this group of studies collectively provides substantial evidence for
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TABLE 7-15. ACUTE EFFECTS OF PHOTOCHEMICAL OXTOANT POLLUTION:
LUNG FUNCTION IN CAMP STUDIES8
Pollutants/Environmental Variables
Study Description
Results and Comments
Reference
Hourly O3 ranged from = 10 to 110 ppb. SOj,
NOX, 03, S04~, H2S04, pH, PM)0, PM2.5, RH,
T, barometric pressure, wind speed, and direction
were also measured.
Effects of pollutants and other environmental
variables on symptoms and lung function were
examined in children attending a summer camp at
Lake Couchiching, about 100 km norm of
Toronto, ON. Study was conducted 6/30-7/8/83;
n = 52, 23 nonasthmatic (11 males, 12 females)
and 29 asthmatics (16 males, 13 females), avg.
age = 12.1 years. Symptom questionnaire and
function tests given twice daily to each child
between 7:30-9:30a.m. and 4:30-6:3* p.m.
Children's activity levels net estimated.
Strongest association between lung function
and environmental variables was in
nonasthmatics, with FVC decrements
significantly correlated (p < 0.01) with
lagged-avg. 804, PM2.5, and T. Unlagged
PEFR significantly correlated with 1 h Oj.
Also, significant association of T with all
lung function indices in nonasthmatics, but
not in asthmatics. Coefficient of variation
stable across morning and evening tests.
Raizenne et al. (1987)
1-h Oj ranged from < It to 143 ppb; max 1-h Oj
> 10» ppb on 14 days of total study (6 weeks).
For other pollutants and variables measured, see
Raizenne et al. (1987) because same protocol used
here as in *at study.
(a) Effects of pollutants and other environmental
variables on lung function were examined in girls
attending one of three 2-week Girl Guide camp
sessions on north shore of Lake Erie. Cohort (n
= 194) screened by MC and skin prick testa for
!• common respiratory allergens; 5 asthmatics
withdrawn from the study (n = 99). Lung
fiinction tests administered twice daily. Children's
activity levels net estimated.
(») Subset of 12 girls (7 MC + , 5 MC-) studied
pre- and postexercise on 1 low-pollution (control)
day and 1 peak pollution dav (episode, 1 h Oj >
139 ppb, S04~ > 80pg/m ).
(a) Associations between aerometric data
and lung function measurements were not
reported by pollutant in this reference.
Aggregate analysis for full study not
reported. Lung function changes reported
for 5 episode days only. FEVj decrements
statistically significant on 2 episode days for
methacholine nonrespensive subjects.
(b) Group mean FVC increased postexercise
in the n = 12 subset by 46 mL. 71 mL in
MC- and 17 mL in MC+. Pollution effect
not statistically significant
Raizenne et al. (1917; 1989)
Continuous 1-h Oj, S(>2, N(>2, and acid aerosols
(as r^SOjj); 1-h 03 range = 40-143 ppb; max
12-h acid particle concentration = 28 fig/m in
one episode; FP SO4 100 /zg/m for peak hour.
Time-activity model used to evaluate likely
cumulative (6 h) Oj and H2SO4 exposures/doses
experienced by children in above Lake Erie Girl
Guide camp study, summer 1986. See Raizenne
et al. (1987, 1989) for protocol and related
information. Dosimetry model was developed for
relating heart rate (from a 12-min, graded cycle
ergometer test) to ventilation and then to Oj and
H2SO4 dose. Also, five randomly selected
children wore portable heart-rate monitors,
providing data for use in the dosimetric model.
Application of the dosimetry model used to
estimate individual 6-h cumulative doses for
03 and H2SO4 exposures on one control
and one episode day indicated negative
trend in lung function (PEFR) as cumulative
dose increased for both 03 and HjSC^,
although slopes for each did not differ
significantly from zero (p > 0.10).
Raizenne and Spengler (1989)
-------
1
if
Co
TABLE 7-15 (cont'd). ACUTE EFFECTS OF PHOTOCHEMICAL OXIDANT POLLUTION:
LUNG FUNCTION IN CAMP STUDIES3
Pollutants/Environmental Variables
Study Description
Results and Comments
Reference
•^J
I—*
to
Max 1 h 03 ranged from 40 to »100 ppb, with
max 1 h > 80 ppb on 9 of 27 days of Oj
recorded O3, SO4 H2SO4, PM15, PM2.5, T,
humidity, and wind speed and direction measured.
Levels not reported for SC>2, pH, NC^, and
Effects of pollutants and other environmental
variables on respiratory functions in 91 children
(53 boys, 38 girls; ages 8-15) attending 2 to
4 weeks of summer camp at Fairview Lake, NJ.
Subsets were n = 37 for all 4 weeks, n = 34 for
first 2 weeks only, n = 20 for last 2 weeks only.
Symptom questionnaire; FVC, FEVj, MMEF by
spirometry; and PEFR by mini-Wright flow
meter were measured once/test day (most of days
in camp) sometime between 11:00 a.m. and
6:30 p.m. All children had validated spirometric
data for >7 days of their 2- or 4-week camp
stay. Activity levels of the children were not
estimated. Respiratory health status determined
by parental questionnaire only. Children slept in
screened-in shelters but otherwise were exposed
to ambient air 24 h/day. Average regression
slopes for respiratory function vs. max. 1-h Oj
concentration reported for the full cohort, for
boys and girls separately, and for subsets in
attendance for all 4 weeks and for respective
2-week sessions. Regressions also repeated for
data below 80 and 60 ppb 1-h 03, and for data
with THI < 78 °F.
Average regression slopes (±SE) were
-1.03 ± 0.24 and -1.42 + 0.17 mL/ppb for
FVC and FEVj, respectively; and -6.78 ±
0.73 and -2.48 ± 0.26 mL/s/ppb for PEFR
and MMEF, respectively. Most slopes of
regression significant at p < 0.05
(differences from zero). Not clear if slopes
for data subsets significantly different from
each other (e.g., function vs. 03 < 60 ppb
and function vs. 03 < 80 ppb). No formal
analysis performed for possible concentration
threshold.
Spektor et al. (1988a)
-------
TABLE 7-15 (cont'd). ACUTE EFFECTS OF PHOTOCHEMICAL OXTOANT POLLUTION:
LUNG FUNCTION IN CAMP STUDIES3
Pollutants/Environmental Variables
Study Description
Results and Comments
Reference
NJ
09
Maximal I h 03 concentrations ranged from
approximately 40 to 150 ppb over the course of
the study. 12-h average aerosol acidity
measurements ranged from near 0 to 18.6 fig/m
(H2SC>4 equivalent). T and RH measured but
levels not reported. THI reached a maximum of
81 °F. All environmental measurements were
made on site.
Effects of 03 and other environmental variables
on lung function studied in a group of
46 children (13 girls, 33 boys; ages 8-14) at a
4-week, 1988 summer camp in southwestern
New Jersey (Fair-view Lake). Same location
used in previous camp study by same
investigators. Subjects had no history of lung
diseases or atopy. Two lung function
measurement periods each day (a.m. and p.m.)
along with collection of respiratory symptom
data. Datt collected during or after periods of
rain were excluded from analysis. Results for
FVC, FEVt, FEVi/FVC, FEF25.75%, and
PEFR reported. Linear day-of-study trends were
examined for lung function. Subject-specific
linear regressions were performed relating lung
function in a.m., p.m., and p.m.-a.m.
differences to 03 averaged over various periods.
Average slopes across subjects were tested for
significant differences from zero. Regressions
were repeated after excluding days with 03 at or
above 120 ppb. Regression residuals were tested
for correlation with THI and H concentrations.
No significant linear day-of-study effect seen
for any of the lung function variables tested,
but the linear model may not have been
optimal for testing this effect. In a subset of
35 subjects with at least 2 consecutive days of
lung function measurements, mean regression
slopes of a.m. lung function variables on
previous-day mean or 1-h maximum 03 were
all significantly negative (e.g., mean slope of
FEVi on 1-h maximum 03 was -0.50
±0.12 mL/ppb). These results suggest a
possible carry-over effect from previous-day
03 exposures. In the full set of 46 subjects,
regressions of p.m. lung function on previous
hour 03, maximum 1-h 03 for same day, or
average 03 for day were significantly
negative in most cases (e.g., mean slope of
FEVj on previous hour 03 was -1.60
±0.30 mL/ppb). All regressions of the
p.m.-a.m. lung function differences on
intervening 03 concentrations were
significantly negative (e.g., mean slope of
FEVj on mean 03 between a.m. and p.m.
measurements was -0.63 {+ 0.09] mL/ppb).
No correlation seen between regression
residuals and THI or H concentrations,
indicating there was no remaining effect of
these variables with lung function after
accounting for O3. However, no models
were fit that included 03 and these variables
simultaneously, nor were interaction effects
tested for. The strong and consistent
associations between lung function
decrements and 03 concentrations in this
study contrast with results reported from
studies in Canada and California at similar
levels of 03.
Spektoret al. (1991);
Spektor and Lippmann (1991)
-------
I
U>
TABLE 7-15 (cont'd). ACUTE EFFECTS OF PHOTOCHEMICAL OXTOANT POLLUTION:
LUNG FUNCTION IN CAMP STUDIES3
Pollutants/Environmental Variables
Study Description
Results and Comments
Reference
-J
to
1 h O3 preceding lung function measurements
ranged from 25 to 245 ppb. Pollutants measured
on site included O3; NO2 (range: 0 to 40 ppb),
SO2 (range. 1 to 8 ppb); and fine (mean =
23.9 ng/m ), coarse (mean = 36.6 fig/m ), and
total (mean = 59 jug/m ) PMjg mass. T averaged
21.5 °C (range: 13.5 to 25.5 °C) and RH
averaged 43.3%.
Effects of O3 and other environmental factors on
lung function examined in 43 children
(24 female, 19 male; ages 7-13) attending one of
3 sequential weeks (three subjects stayed an
additional week) of summer camp in the
San Bernardino Mountains of California. Camp
was at 5,710 ft above sea level. Lung function
measured by spirometry up to three times daily
on each subject; analytical measures included
FVC, FEV^ and PEFR. No report of
respiratory data derived from questionnaires.
Subject activity levels prior to lung function
testing were not characterized. Campers slept in
well-ventilated cabins. Subjects came mostly
from homes in the Los Angeles Basin, and were
thus likely to have been exposed to high
O3 levels prior to camp. Simple linear
regression models were fit on an individual basis
(subject-specific slopes) and by pooling across
individuals (common population slope) to
determine the linear relations between the three
lung function variables and various O3 metrics
(1-h average preceding hour of spirometry, 1-h
average 2 h previous to hour of spirometry, or
6-h average preceding spirometry). The
common slope model was repeated separately for
morning, noon, and evening lung function
measurements, and separately for data with 1-h
O3 levels above and below 120 ppb. Multiple
regression models were fit that included O3
along with T, RH, and coarse and fine
paniculate matter mass.
The population-pooled regression slopes
(+SE) of FVC and FEVj on previous hour
O3 were -0.40 (+ 0.10) and -0.38
(± 0.09) mL/ppb, respectively (p < 0.0001
in both cases); for PEFR, the regression
slope was -0.13 (+ 0.36) mL/s/ppb (not
significant). Similar, though slightly more
negative, slopes were obtained using 2-h and
6-h average O3. Interpretation of differences
across the three O3 metrics is substantially
hampered by the high correlations among
them (r > 0.90). When T, RH, and coarse
and fine paniculate matter mass were
included with O3 in multiple regression
models, the O3 slopes increased in absolute
magnitude to -0.68 (+ 0.16) and -0.76 (±
0.15) mL/ppb for FVC and FEVj,
respectively, and to -1.91 (± 0.63) for
PEFR. Technical problems with the
temperature sensor in the first week of the
study did not appear to influence these
results. Data were split on the basis of
whether or not the maximum 1-h O3
concentration in the 6 h preceding spirometry
was above 120 ppb. Regression slopes
relating lung function and previous 1-h, 3-h,
and 6-h average O3 were more negative, and
only statistically significant, in the high
concentration stratum. This result is
consistent with the nonlinear (e.g., quadratic)
relationships between lung function and 03
exposure observed in chamber studies.
Because levels of pollutants other than
O3 were quite low (NOj and SO^, and/or
were uncorrelated with 03 levels (paniculate
matter), the regression results reported from
this well-conducted study are likely to
represent real influences of 03 on lung
function.
Higgins et al. (1990);
Gross etal. (1991)
-------
TABLE 7-15 (cont'd). ACUTE EFFECTS OF PHOTOCHEMICAL OXTOANT POLLUTION:
LUNG FUNCTION IN CAMP STUDIES8
Pollutants/Environmental Variables
Study Description
Results and Comments
Reference
Daily maximum Oj concentrations ranged from
approximately 60 to 160 ppb (derived visually
from figure presented in paper). Other pollutants
measured on site included SOj, NO2, CO, total
hydrocarbons, and size-segregated paniculate
matter mass. Aside from 03, all gaseous pollutant
levels reported to be very low (data not
presented). 24-h TSP concentrations ranged from
H to 54 ng/m . Airborne allergen data collected.
T ranged from 1« t» 15 °C at night and from
25 to 35 °C during day. RH ranged from 39 to
45% at night and from 5 t» 29% during day.
Effects of 03 and other environmental variables
on lung iunction examined in 293 children
(139 girls, 154 boys; ages 8-17) attending one of
six 1-week camp sessions at a summer camp
located in the mountains near Idyllwild, CA,
190 km southeast of Los Angeles (altitude:
1,570 m). Lung function measured twice daily
on each camper (a.m.: 0730 to 0930; p.m.:
16OB to 1930). Analyses presented for FVC,
FEVj, PEFR, and fEe^_i^%. Symptom
questionnaires completed prior to each test.
Used repeated measures analysis of variance
model to test for day-of-study and a.m./p.m.
effects on lung function independent of pollution
concentrations. Linear regressions of morning,
afternoon, and p.m.-a.m. difference of lung
function on 03 were performed with
simultaneous control of day and a.m./p.m.
effects. Upper and lower quartiles of
distribution of individual FEV|/Oj regression
slopes were examined with respect to subject
characteristics. Changes in FEVj over several
days analyzed in relation to intervening
integrated 03 concentrations.
Significant day-of-study effect observed for
FVC and FEVj characterized by steady drop
over first few days of measurement, followed
by partial reversal later in week. For PEFR,
p.m. measurements were significantly higher
than a.m. measurements. Controlling for day
and a.m./p.m. effects, the authors reported
that no consistent 03 effects on lung function
were observed. The a.m. lung function
measurements had a significant positive
correlation with 03 averaged over the
previous 1, 8, or 24 h. The p.m.
measurements reported to have no correlation
with 03. p.m.-a.m. lung function differences
were negatively correlated with previous 8-h
average 03 concentrations, but not with
previous 1-h 03 concentrations. No
quantitative results reported for the above
lung function/03 findings. There were no
discernable differences between subjects in
the upper and lower quartiles of the
distribution of individual regression slopes of
a.m.-p.m. FEVj difference on previous 1 h
03. Regressions of change in FEVj over
several days (four separate intervals ranging
from approximately 8 h to approximately
80 h) with integrated 03 concentrations
yielded negative slopes ranging from -0.41 to
-1.46 mL/ppb, one of which was statistically
significant. The time-trends in FVC and
FEVj measurements observed in this study
are qualitatively consistent with those seen in
some other summer camp studies. The lack
of consistent negative slopes relating lung
function with 03 concentrations contrasts
with other, eastern U.S., summer camp
studies at similar 03 levels.
Avol et al. (1990);
Avol et al. (1991)
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I
VO
vO
U>
TABLE 7-15 (cont'd). ACUTE EFFECTS OF PHOTOCHEMICAL OXIDANT POLLUTION:
LUNG FUNCTION IN CAMP STUDIES3
Pollutants/Environmental Variables
Study Description
Results and Comments
Reference
O3 data collected at a site 8 mi from camps
Daily 1-h O3 maxima ranged from approximately
40 ppb to approximately 200 ppb. 12-h aerosol
acidity concentrations ranged between 14 and
360 neq/m . T and RH data obtained from a
nearby site.
Report of data collected during two simultaneous
summer camps located 2 mi apart in central New
Jersey in 1988. 34 subjects were studied,
including 20 camp counselors (ages 14-35) and
14 campers (ages 9-13). Study spanned 19 days.
Spirometry and respiratory symptom data
collected each afternoon. Analysis of FVC,
FEV], and PEFR in relation to Oj and T using
linear regression within camps and subject types
(i.e., counselors vs. campers).
Regressions of lung function on 1-h and 8-h
average O3 within several subject subsets
yielded inconsistent results, with some mean
slopes apparently significantly positive, and
one negative mean slope, highlighted by
authors, of borderline significance
(p < 0.10).
Berry et al. (1991)
Daily 1-h maximum O3 concentrations ranged
from 70 to 160 ppb in 1991 and from 10 to
63 ppb in 1992 On-site measurements also made
for acid aerosols (approximately 20 to
110 nmoles>/m in 1991 and 15 to 55 nmoles/m in
1992) and temperature (between 21 and 32 °C
over 2 years)
Effects of O3 and acid aerosols on peak flow,
respiratory symptoms, and medication usage in
asthmatic children evaluated at two 1-week
summer camps (June of 1991 and 1992) in the
Connecticut River Valley. 52 and 55 subjects
were studied in 1991 and 1992, respectively,
ranging in age from 7-13. Peak flow measured
twice daily (approximately 9:00 a.m. and
5:00 p.m.). Combining data from the two
studies, individual regressions of daily change in
FEVj on O3 or H concentrations were
performed.
In subjects without asthma exacerbations
during the camps, statistically significant,
negative mean slopes were found relating
APEFR and O3 or H concentrations. The
correlation between these two pollutants was
not reported. The mean slopes were
-2.3 (± 0.7) mL/s/ppb for O3, and
-1.2 (± 0.6) mL/s/nmol/m for H+. In the
case of O3, a scatter-plot with APEFR
demonstrated an apparently linear trend.
In contrast, the H regression results
appeared to be driven entirely by one data
point.
Thurstonet al. (1993c)
See glossary of terms and symbols for abbreviations and acronyms.
Cited in U.S. Environmental Protection Agency (1992).
-------
1 associations between ambient O3 exposures, together with other pollutants, and acute
2 decrements in lung function. Interpretation of these associations as causal is supported by
3 evidence of biological plausibility. For example, the well-documented direct effects of O3 on
4 lung function in human chamber studies; the evidence, also from chamber studies, indicating
5 a lack of direct effects of other collinear environmental factors (e.g., temperature and acid
6 aerosols) at the levels these factors occur in the camp studies reported to date; exposure-
7 response relationships; and consistency across studies all provide strong support. Camp
8 studies involving asthmatic children have generally yielded lung function/O3 associations that
9 are similar in absolute magnitude to those observed in nonasthmatics (Raizenne et al., 1987;
10 Thurston et al., 1993c); however, the health significance of a given drop in FEVj may be
11 greater for those with preexisting, compromised respiratory function.
12 Although similar study designs have been employed in most of the camp studies
13 summarized in Table 7-15, differences in analytical methods have made quantitative
14 comparisons between studies difficult to interpret. In particular, it has not been clear to what
15 extent differences in results across studies may be due to differences in study characteristics
16 (e.g., O3 effect potentiation by other pollutants and activity levels) as opposed to differences
17 in data analysis methods.
18 Recently, Kinney et al. (1993) reported a reanalysis of data from six of the camp
19 studies summarized in Table 7-15 using uniform analytical methods. For each study,
20 afternoon lung function data (FEV,) were regressed on concurrent 1-h O3 concentrations
21 using an analysis of covariance model that included subject-specific intercepts and a single,
22 pooled slope on O3. The study-specific slopes computed with this model ranged from
23 -0.29 to -1.34 mL/ppb across the six studies (Table 7-16). All but one of these slopes
24 were statistically significant (p < 0.02). The inverse standard-error weighted mean slope for
25 the six studies was -0.64 mL/ppb (p = 0.008). Slopes from the two Fairview Lake, NJ,
26 studies (about -1.3 mL/ppb) were consistently greater in absolute magnitude than the slopes
27 from the other four studies (which ranged from about -0.3 to -0.5 mL/ppb). Overall,
28 however, these results indicate a quantitative consistency among studies that is not as readily
29 apparent in the absence of the combined analysis.
30 It is not clear why the New Jersey studies have yielded larger slopes than the other
31 studies. Possible explanations include greater subject activity levels (resulting in higher
December 1993 7-132 DRAFT-DO NOT QUOTE OR CITE
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TABLE 7-16. SLOPES FROM REGRESSIONS OF FORCED EXPIRATORY
VOLUME IN ONE SECOND ON OZONE FOR SIX CAMP STUDIES8
Study Name
Fairview Lake, 1984
Fairview Lake, 1988
Lake Couchiching
Lake Erie
San Bernardino Mountains
Pine Springs Ranch
All studies
Slope ± SE (mL/ppb)
-1.34 ± 0.13
-1.29 ± 0.28
-0.44 ± 0.51
-0.29 ± 0.10
-0.47 ± 0.13
-0.32 ± 0.13
-0.64 ± 0.18
p-Value
<0.0001
0.0001
0.39
0.003
0.0006
0.013
0.008
Reference
Spektor et al. (1988a)
Spektor et al. (1991)
Spektor and Lippmann (1991)
Raizenne et al. (1987)
Raizenne et al. (1987, 1989)
Higgins et al. (1990)
Gross et al. (1991a)
Avol et al. (1990, 1991)
b
lFor each study, data were analyzed in one model that fit subject-specific intercepts, but one, pooled,
O3 slope. See glossary of terms and symbols for abbreviations and acronyms.
'Slope is the weighted mean of six study-specific slopes. The SE is the weighted SE of mean slope.
Source: Kinney et al. (1993).
1 O3 doses at a given exposure level), potentiation of the O3 effect by other pollutants (such as
2 acid aerosols), the relative absence of O3 tolerance in the New Jersey studies, or confounding
3 by airborne allergens. There are no firm data on activity levels across the six studies. Thus,
4 whereas this factor surely contributes to the random variability within and between studies, it
5 is not known whether activity levels were substantially and systematically higher in the
6 New Jersey studies. Potentiation of the O3 effects on lung function in asthmatics by acid
7 aerosols has been demonstrated in a recent chamber study in which O3 exposure was
8 administered 1 day following a 3-h exposure to 100 jwg/m3 H2SO4 (Frampton et al., 1992).
9 Although the relevance of these data to the nonasthmatic subjects who experienced much
10 lower acid levels at northeastern summer-camps is not clear, they do demonstrate that
11 potentiation can occur between these pollutants. However, this factor cannot, alone, explain
12 the observed differences across camp results, because a camp study in southern Ontario
13 (Raizenne et al., 1989), which yielded relatively low FEVl slopes on O3, experienced sulfate
14 aerosol levels that were comparable to those seen in New Jersey. Similarly, whereas
15 tolerance due to prior exposures to high O3 levels has been suggested as an explanation for
16 the smaller slopes seen in the California studies, it cannot explain the smaller slopes in
17 southern Ontario. Data have not been reported on comparative levels of airborne allergens
December 1993 7-133 DRAFT-DO NOT QUOTE OR CITE
-------
1 during the various camp studies. None of the subjects in the two New Jersey studies
2 reported a history of asthma or atopy, minimizing the likelihood of confounding by airborne
3 allergens. However, given the lack of allergen data, and the potential for substantial
4 numbers of "silent hyper-responders" (Raizenne et al., 1989), this possibility cannot be
5 completely discounted. Thus, no one factor seems adequate to explain the differences in
6 results across studies. Quite possibly these differences reflect the combined influence of
7 several of the factors discussed above. Indeed, given the many possible sources of camp-to-
8 camp variability, it is surprising that results are as consistent as they are across six studies by
9 three investigative groups.
10 Several investigators have reported regression results both for 1-h average O3 and for
11 longer averaging times (e.g., 6 to 8 h) (Higgins et al., 1990; Avol et al., 1990, 1991;
12 Spektor et al., 1988a; Spektor et al., 1991). In general, similar results have been obtained
13 regardless of the averaging time. Attempts to draw conclusions regarding the relative
14 importance of short-term peaks and longer term averages from such analyses have been
15 hampered by the high degree of correlation between 1-h and multihour averages. Until
16 better analytical methods are found for dealing with this problem, comparative results will
17 remain difficult to interpret.
18
19 Lung Function in Exercising Subjects
20 This subsection discusses studies involving lung function measurement immediately
21 before and after a series of discrete outdoor exercise activities in the presence of air
22 pollution. This design is similar in principle to the ambient chamber studies conducted in the
23 early 1980's (see Section 7.3), in which subjects exercised under a specified protocol in a
24 chamber ventilated with ambient air. Here, however, there is typically less control imposed
25 over exercise duration and intensity, and less assessment of achieved VE. Compensating to
26 some extent for this diminished control is the relative ease of collecting numerous repeated
27 measurements at varying ambient O3 levels on the same subjects, improving the precision of
28 dose-response estimation. In contrast to camp studies, duration of relevant 03 exposure is
29 assumed to be known—as defined by the length of each exercise event.
30 Results from three exercise studies (Selwyn et al., 1985; Spektor et al., 1988b; Hoek
31 and Brunekreef, 1992; Hoek et al., 1993a) are summarized in Table 7-17. One of the
December 1993 7-134 DRAFT-DO NOT QUOTE OR CITE
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TABLE 7-17. ACUTE EFFECTS OF PHOTOCHEMICAL OXIDANT POLLUTION:
LUNG FUNCTION IN EXERCISING SUBJECTS3
Pollutants/Environmental Variables
Study Description
Results and Comments
Reference
vj
OJ
1-h 03 concentration ranged from 21-124 ppb,
max. THI =78 °C: max acidic aerosol (as
H2SO4) = 9 /tg/m _during study. SO2, NOX,
PM,5> PM2.5, S04~, N03, NH4+, T, and RH
measured but not reported.
Effects of Og on respiratory function and
symptoms examined in 30 nonsmoking adults
(2 of 10 females non-Caucasian) exercising
almost daily outdoors (Tuxedo, NY) for 15 to
55 min (average ca. 30 min) from July to early
August, 1985. Pre- and postexercise lung
function measured and questionnaire answered
postexercise. Pulse rate, calibrated to VE
indoors, taken postexercise. Exercise regimen
self-selected but constant for each subject over
the course of study. Dosimetry estimated and
linear regressions done for pulmonary function
changes vs. (1) mean O3 concentration during
exercise, and (2) inhaled Oj dose. Persistence
of effects tested by linear regressions of before-
exercise lung function on previous-day O3 during
exercise. Subjects screened only by
questionnaire; two with previous history of
asthma but asymptomatic.
Significant (p < 0.01) decrements in FVC,
FEV^ PEFR, FEF25_75%, and FEV,/FVC
associated with O3. For example, the mean
slope of AFEVj on O3 across all subjects
was -1.35 mL/ppb (+ 0.35). No
persistence of effects seen. No symptoms
reported by subjects. Mean decrements
showed unexpected inverse relationship with
calculated Vg levels, as indicated by
regressing pulmonary function changes and
postexercise function against inhaled O3
during exercise. VE ranges given, but not
group or subset means. Subjects not
screened for atopy. Exercise done in Sterling
Forest, wooded research park, on paved
roads or trails.
Spektoret al. (1988b)
15-min peak O3 measured during runs averaged
47 ppb (range: 4 to 135 ppb). Ambient T
averaged 29.4 °C (range: 18.0 to 37.8 °C).
RH averaged 62.6% (range: 37.0 to 88.0%).
Levels of other pollutants were low, median values
were SO2, 3 ppb; NO2, 6 ppb; FP, 10 jig/m3.
Median of subject-specific correlations of O3 and
RH correlated was -0.42.
Effects of O3 on lung function change during
running outdoors was examined in
24 conditioned, recreational runners (6 women,
18 men, ages 29-47) at a track 30 mi southeast
of Houston, TX, from May to October, 1981.
All runs were 3 mi in length, and each subject
performed at a near-constant heart rate for the
duration of the study. An average of 28 runs
completed by each subject during the study.
Spirometry carried out before and after each run,
with analysis of FVC, FEVj, FEF2j_75^, and
FEFfj 2.j 2L- Chang6 in each lung function
variable was regressed, for each subject, on
15-min maximum O3 measured during the run.
The mean slope across subjects was tested for
significance. The regression was repeated with
T and RH in the model.
Mean slope of FEVj on O3 alone was -
0.4 mL/ppb (p = 0.03). In regressions that
included T and RH, the 05 slope dropped to
-0.07 (not significant). Although T reached
high levels during study, a substantial direct
effect of T or RH on lung function, relative
to that of O3, seems unlikely. A possible
potentiating role of high T and RH on VE,
and corresponding O3 dose, cannot be ruled
out. Lung function effect observed in simple
O3 model seem likely to be a valid reflection
of O3 effects under varying environmental
conditions.
Selwyn et al. (1985)
-------
TABLE 7-17 (cont'd). ACUTE EFFECTS OF PHOTOCHEMICAL OXIDANT POLLUTION:
LUNG FUNCTION IN EXERCISING SUBJECTS3
Pollutants/ Environmental Variables
Study Description
Remits and Comments
Reference
1-h maximum Oj concentrations during study
ranged from 50 to 240 fig/m (25 to 120 ppb).
The highest 4-h average PM2.5 level was
70 fig/m , the highest 4-h average sulfate
concentration was 21 /ig/m , the highest 24-h
average NO2 concentration was 51 /ig/m . T data
were collected but levels were not reported.
The relationship between lung function change
and 03 exposures during outdoor exercise
examined in a population of 83 children
(43 girls, 40 boys; ages not given) in
Wageningen, The Netherlands. Study covered
the period from late May to mid-July, 1989.
Lung function assessed using hand-held peak
flow meters before and after various outdoor
sports training exercises listing approximately
1 h. Change in PEFR regressed on 03, 03 X
exercise duration, and T for each subject and
distribution of slopes examined. Postexercise
PEFR analyzed in relation to same and previous
day 1-h O3 maximum, and T. Analyses repeated
in subsets of subjects with varying levels of
correlation between 03 and T during their series
of exercise events.
For 55 children with at least four sets of
before and after exercise peak flow
measurements, the mean slope of the PEFR
change on Oj during exercise was 0.035
(± 0.030) mL/s/jtg/m . For 65 subjects with
at least four postexercise measurements, the
mean slope of PEFR on previous hour Oj
was 0.080 (± 0.023), which is statistically
significant, but in the nonplausible direction.
Adjustment for T resulted in negative mean
slopes, but these are difficult to interpret
because of the high statistical correlation
between same-day O3 and T (r = 0.86).
Exercise events were of low intensity as
compared with chamber studies and with the
Tuxedo runners study (Spektor et al., 1988b).
Significant exposures may have occurred
prior to the exercise period. H levels were
low (<5 ng/m ) as measured simultaneously
at three other nonurban sites in The
Netherlands. The possibility of a physical
effect of temperature on mini-Wright peak
flow meter measurements was noted by
authors.
Hoekand Brunekreef (1992)
Hoeket al. (1993a)
See glossary of terms and symbols for abbreviations and acronyms.
-------
1 studies (Selwyn et al., 1985) was discussed in the previous O3 criteria document (U.S.
2 Environmental Protection Agency, 1986), but is reviewed again here because of its apparent
3 consistency with the more recent runners study of Spektor et al. (1988b).
4 Certain design variations across studies are worth noting. In the Houston runners
5 study, each of 24 recreational runners performed spirometry before and after a series of
6 approximately 28 runs on a track from late spring to early fall (Selwyn et al., 1985). Each
7 run was 3 mi long and each subject attempted to maintain a similar heart rate across all runs.
8 Minute ventilation was not assessed. In the adult runner/walker study carried out in Tuxedo,
9 NY, subjects were allowed to choose their own exercise level and duration, but again were
10 encouraged to maintain a steady heart rate for the duration of the study (Spektor et al.,
11 1988b). Minute ventilation of each subject while running was estimated by measurement of
12 VE during a treadmill test that achieved a heart rate typical of that subject's experience while
13 running.
14 In contrast to these two studies, the study in Wageningen, The Netherlands, involved
15 lower and more variable exercise levels, without any specific attempt to control exercise
16 intensity (Hoek and Brunekreef, 1992; Hoek et al., 1993a). Here, subjects engaged in sports
17 training and skills development activities that were characterized by the investigators as low
18 to moderate in intensity. Lung function change after exercise was assessed using peak flow
19 meters.
20 Although the designs varied somewhat, O3 exposure levels were similar in the three
21 studies: in Houston, 15-min peaks while running varied from 4 to 135 ppb; in Tuxedo, 1-h
22 O3 levels ranged from 21 to 124 ppb; in Wageningen, 1-h maxima on study days ranged
23 from 30 to 120 ppb.
24 The two studies involving fairly intense exercise yielded significant mean slopes of
25 AFEVj (i.e., FEVj after exercise minus FEVj before exercise) regressed on O3 levels
26 measured during exercise, whereas the Wageningen study did not. The mean slope observed
27 in the Tuxedo study across all subjects was -1.35 mL/ppb (± 0.35), but was reduced to
28 -0.55 (± 0.45) in the group of 10 runners who achieved the highest VE values during
29 exercise. The mean slope reported from the Houston study was similar to the latter number:
30 -0.4 mL/ppb (± 0.16). The large effect level observed in the Tuxedo study led Spektor
31 et al. (1988b) to speculate that 65 effects may have been potentiated by other pollutants such
December 1993 7_137 DRAFT-DO NOT QUOTE OR CITE
-------
1 as acid aerosols; however, this phenomenon was not demonstrated analytically from the
2 available acid monitoring data. In the Houston study, the O3 effect became small and
3 nonsignificant when temperature and relative humidity were added to the model. In spite of
4 this observation, and given the available knowledge base on the independent effects of
5 O3 and temperature on lung function, it seems reasonable to interpret both of these well-
6 conducted studies as demonstrating acute effects of O3 on lung function with moderate to
7 heavy exercise. The negative findings of the Wageningen study are difficult to interpret, but
8 may be related to the low exercise intensities achieved, exposures (and, perhaps, associated
9 O3 tolerance) that occurred prior to the exercise period under study, or some subtle physical
10 effect of ambient temperature on the peak flow measurements.
11
12 Lung Function in Daily Lafe Studies
13 This set of studies is characterized by the assessment of lung function, respiratory
14 symptoms, and environmental factor associations in the course of people's daily lives. This
15 section discusses only the lung function data from these studies. For logistical reasons,
16 studies of this kind have usually involved either spirometry conducted at regular intervals
17 (every 1 to 3 weeks) in schools (Kinney et al., 1989; Castillejos et al., 1992; Hoek et al.,
18 1993b), or self-administered peak flow measurements in subjects of various ages over various
19 periods (Vedal et al., 1987; Krzyzanowski et al., 1989). Although daily life studies have the
20 worthwhile goal of characterizing air pollution effects on respiratory health in the real world,
21 they may suffer from significant exposure assessment uncertainties owing to the use of
22 outdoor O3 monitoring, the incomplete and variable penetration of O3 indoors, and the
23 preponderance of time spent indoors by study subjects. This problem is probably less severe
24 for the studies involving schoolchildren, who often spend substantial time outdoors after
25 school, when O3 levels may be elevated. Indeed, three of the school-based studies have
26 found statistically significant associations between lung function and previous-day O3 levels
27 (Castillejos et al., 1992; Kinney et al., 1989; Hoek et al., 1993b). Another difficulty in
28 interpreting the results of these studies is the possible role of seasonal factors (e.g., pollens,
29 epidemics of respiratory infection, changes in activity patterns) as potential confounders of
30 the analyses.
December 1993 7-138 DRAFT-DO NOT QUOTE OR CITE
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1 In addition to these general limitations inherent in the study design, several of the
2 studies summarized in Table 7-18 have other problems that limit their utility for assessing
3 O3 effects on lung function. The study of Vedal and colleagues (1987), though well
4 conducted, took place from September through May, a period when O3 levels are generally
5 low and other potential respiratory insults may dominate. The statistical significance of
6 results from a study carried out in Tucson, AZ, is difficult to interpret because of the
7 multiple statistical tests performed (Krzyzanowski et al., 1989).
8 The remaining studies, though subject to the general criticisms noted previously,
9 provide suggestive evidence that ambient O3 may play a role in short-term lung function
10 declines among children engaged in their normal daily routines (Kinney et al., 1989;
11 Castillejos et al., 1992; Hoek et al., 1993b). The Mexico City study of Castillejos and
12 colleagues (1992) is especially noteworthy because of the novel observation of FEVj and
13 FEF25_75% decrements that were strongly related to O^ levels averaged over 24 to
14 168 h previous to spirometry, but not to previous-hour O3 levels. The strength of these
15 associations (measured by the ratio of the regression slope to its standard error) increased
16 steadily as averaging time increased. As noted by the authors, these results may reflect an
17 inflammatory response in the airways rather than the well-known acute physiological
18 response. Ozone levels observed throughout this 6-mo study were high by U.S. standards;
19 1-h average O3 concentrations in the hour preceding lung function measurements ranged from
20 14 to 287 ppb, with a mean of 99 ppb.
21
22 Panel Studies of Symptom Prevalence
23 Many field and epidemiological studies reviewed both in the last criteria document
24 (U.S. Environmental Protection Agency, 1986) and in the previous section of this document
25 reported results that indicated associations between ambient oxidant exposures and various
26 measures of respiratory effects (e.g., irritative respiratory symptoms and acute pulmonary
27 function decrements) in children and adults. The aggregation of individual studies provides
28 reasonably good evidence for an association between ambient photochemical oxidants and
29 acute respiratory effects and a database that is generally coherent, consistent, and biologically
30 plausible. In addition, other studies of irritative symptoms in children and adults were also
31 reported in the 1986 document. For example, Hammer et al. (1974) reported qualitative
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TABLE 7-18. ACUTE EFFECTS OF PHOTOCHEMICAL OXTOANT POLLUTION:
DAILY LIFE STUDDZS OF LUNG FUNCTION AND RESPDXATORY SYMPTOMS3
PolIutants/EnvironmentHl Variables
Study Description
Results and Comments
Reference
Max 03 (1-h) concentrations ranged from 3 to
63 ppb. Other ambient pollutants measured_were
N02, TSP, IP, RSP. FP, SC>2, and FP SQf.
Lung function measured by spirometry for
154 children ages 10-12 years (90 males,
64 females) in Kingston and Harriman, TN
Spirometry done between 10 a.m. and 1 p.m. on
up to 6 days at least 1 week apart during February
to April, 1981. Child-specific linear regression
models of FVC, FEV0 75, MMEF, and V75% fit
on 1-h O3 max. and 24-h FP and FP SO£".
Means ± SD of distributions of estimated child-
specific slopes computed and tested for
significance by <-test.
Significantly negative mean slopes on 03 for all lung
function variables. For example, mean slope of
FEV0 75 on O3 was -0.99 mL/ppb (±0.36).
Among regressions on FP and FP 804 , only one
statistically significant mean slope (i.e., positive mean
slope of MMEF on FP). Results insensitive to outlier
audits and inconclusive for sensitivity variation.
Association between fitted slopes and individual
characteristics not significant. Low O3 levels raise
plausibility questions.
Kinney (1986)
Kinney et al. (1989)b
1-h average O3 concentrations in hour preceding
spirometry ranged from 14 to 287 ppb, with mean
of 99 ppb. No other pollutants measured.
T ranged from 3 9 to 27.8 °C. RH ranged from
18.9 to 92.3%
Effects of O3 on lung function examined during
regular school hours in a group of 148 children
(65 girls, 83 boys; ages 7-9) from three schools in
Mexico City. Spirometry and symptom data
(cough/phlegm) collected between 0800 and
1400 houri every 2 weeks over the period January
through June, 1988. To account for lung growth
over the study period, residuals from lung
function prediction equations were used in
analyses. Analyses limited to 143 subjects with at
least seven valid measurements. Schools were not
air conditioned, and windows were usually open.
Schools and subject residences all within 5 km of
O3 monitoring site. Associations between O3 and
lung function (FVC, FEVj, and fEF2S-75lt)
examined by computing the weighted mean of
subject-specific regression slopes relating these
variables. Various O3 averaging times (from 1 h
to 168 h) were tested. After analyzing population
as a whole, regressions were repeated in subject
subsets defined by sex, report of chronic
symptoms, and maternal smoking. Overall
regressions repeated with T and RH in model with
Only FVC had a statistically significant negative mean
slope in relation to previous hour O3 concentration
(-0.059 + 0.23 mL/ppb). This slope is
approximately 1 order of magnitude lower than those
observed in some camp studies. Both FEVj and
FEF25_75% had significant negative associations with
O3 averaged over the previous 24, 48, and 168 h.
For example, the mean slope of FEVj on 48-h
average O3 was -0.592 + 0.109 mL/ppb. The
authors speculated that the FVC result reflects the
acute, reversible effects of O3 on one's ability to take
a deep breath, whereas the FEVj and FEF2S-75%
observations may reflect inflammatory effects of more
prolonged O3 exposures. It should be noted that both
FVC and FEVj had significant negative slopes on 1-h
maximum O3 measured in the previous 24 h.
Adjustment for T and RH diminished somewhat the
associations between lung function and O3.
Associations between lung function decrements and
O3 exposure often appeared larger in children with
chronic respiratory symptoms than in those without,
and in children of mothers who were current smokers;
however, these results were not statistically
confirmed.
Castillejos et al. (1992)
-------
o
8
I
\o
VO
U)
TABLE 7-18 (cont'd). ACUTE EFFECTS OF PHOTOCHEMICAL OXTOANT POLLUTION:
DAILY LIFE STUDIES OF LUNG FUNCTION AND RESPIRATORY SYMPTOMS3
Pollutants/Environmental Variables
Study Description
Results and Comments,
Reference
Daily maximum 1-h Oj concentrations on days
prior to lung function testing ranged from 7 to
206 fjig/m (3.5 to 103 pph). Levels of other
pollutants measured (SO2, NO2, PMjrj, and
aerosol H ) were reported to he low during study.
Ambient T (range: 5 to 31 °C) and some pollen
data were also collected
Associations between morning lung function and
previous day Og examined during school in
533 children (ages 7-11) from seven schools in
three towns in the Netherlands. Towns were
selected without local pollution sources and with
low levels of pollutants other than Oj. Study
spanned the period from March through July,
with lung function measurements collected every
2-3 weeks. An overall time trend was fit to the
lung function data to account for lung growth
Data on FVC, FEV^ PEFR, and FEF25_755{
analyzed in relation to previous-day 1-h
maximum O-j concentrations using subject-
specific linear regressions followed by analysis
of mean slopes. Intersubject variations in
responsiveness to 03 were tested via an F-test.
The influence of chronic respiratory symptoms
and other subject characteristics (e.g., age, sex)
on O3 responsiveness was examined. Models
that included other pollutants were also
considered.
Negative, usually statistically significant
mean slopes seen for lung function regressed
on previous-day 1-h maximum 03 for the
seven individual schools. Over all
533 subjects, mean regression slopes for FVC
and FEVj were -0.20 ± 0.05 and -0.21 ±
0.04 mL//ig/m , respectively; and for PEFR
and FEF25_7j% were -0.72 + 0.22 and
-0 45 + 0.12 mL/s/jig/m , respectively.
These coefficients may be doubled to convert
to slopes in terms of ppb. The authors report
that adding SO2, NO2, or PM10 did not
materially change the Oj slopes. There was
evidence for inter-subject variation in Oj
responsiveness, but this variation was not
statistically related to available subject
characteristics data. T data not included in
models, perhaps due to high correlation with
Oj. The lung function/O3 relationships noted
above are qualitatively similar to those
reported in the 1988 Fairview Lake camp
study and the Mexico City school children's
study
Hoek and Brunekreef (1992)
Hoek et al. (1993b)
-------
I
«
U)
TABLE 7-18 (cont'd). ACUTE EFFECTS OF PHOTOCHEMICAL OXIDANT POLLUTION:
DAILY LIFE STUDIES OF LUNG FUNCTION AND RESPIRATORY SYMPTOMS3
Pollutants/Environmental Variables
Study Description
Results and Comments
Reference
-J
NJ
1-h maximum O3 concentrations on PEFR
measurement days ranged from 20 to 103 ppb.
No other pollutants assessed, but ambient T data
included.
Relationship between daily peak flow
measurements and ambient O3 concentrations in a
population sample of 732 subjects (both adults
and children) over 2-week periods during normal
daily activities in Tucson, AZ. Peak flow
assessed using hand-held peak flow meters up to
four times per day. PEFR measurements on
initial 2 days for each subject dropped to avoid
possible learning effects, leaving a series of up to
12 measurement days per subject. Population
pooled regression slopes computed for PEFR on
1-h and 8-h avenge Oj for children (ages < 15)
and adults (ages > 15), controlling for residual
auto-correlations. Outcome measures included
PEFR diurnal variability and afternoon PEFR
levels. Besides O3, potential explanatory
variables included T, average time outdoors, acute
respiratory infections, asthma, and environmental
tobacco smoke exposure.
Significant positive associations observed
between 03 concentrations and PEFR diurnal
variability; the effect magnitude was greatest
in asthmatic subjects. In children only, noon
PEFR was suppressed on days with higher O3
levels. The uncertain relationship between
central site O3 levels and personal exposures
in this southwestern community was not
addressed. Although the statistical models
employed were appropriate and well chosen, it
appears that a substantial amount of
exploratory data analysis was performed prior
to selection of results to present in paper,
leading to uncertainties regarding the statistical
validity of the hypothesis tests presented.
Krzyzanowski et al. (1989)
Means and range of max. daily 1-h values:
O3 mean = 32.4 /ig/m .range = 0-129 ng/m ;
SO2 mean = 51.2 ^g/m . range = 18-176 jug/m ;
NC>2 mean = 40.5 fig/m , range = 12-79 fig/m ;
CoH mean = 0.38 CoH units, range =
0.1-1.3 CoH units; T mean = 1.3 °C, range =
-22° to +22 °C.
Follow-up study (September, 1980-April, 1981)
of pollutant-respiratory symptom relationships in
subsets of children from 1979 Chestnut Ridge
cross-sectional study of > 4,000 elementary
school children. Subsamples selected from six
schools in study area with consistently higher
levels of air pollution during previous 4 years.
Subsamples (three) stratified by reported
symptoms. One or more of following measures
taken for 144 children: diaries, symptom
questionnaire, spirometry. Telephone follow-up
each 2 weeks on diaries, spirometry done at
school, pollutants (including O3) measured at one
monitor (data from 17 monitors for SO2 generally
reflected in data at single monitor). Diary panel
study covered 8 mo; successive PEFR spirometry
studies of 9 weeks each done in respective groups
of the three Subsamples.
Relationships of maximum hourly SO2>
O3, and CoH and minimum. T for each 24-h
period to daily upper and lower respiratory
illness, wheeze, and PEFR were evaluated
using multiple regression models adjusted for
illness occurrence or levels of PEFR on
preceding day. No air pollutant was strongly
associated with respiratory illness or with
PEFR. Authors concluded that this study can
best be interpreted as showing no acute effects
of studied pollutants on respiratory symptoms
or PEFR in children at levels lower than the
current NAAQS, but also noted that
conclusion must be tempered by relatively low
levels of pollutants encountered and possibility
of exposure misclassiftcation.
Vedal et al. (1987)
aSee glossary of terms and symbols for abbreviations and acronyms.
Cited in U.S Environmental Protection Agency (1992).
-------
1 associations between ambient oxidant levels and symptoms such as eye and throat irritation,
2 chest discomfort, cough, and headache at total oxidant levels greater than 0.15 ppm in young
3 adults (nursing students). Wayne et al. (1967) reported a high correlation (R2 = 0.89)
4 between ambient total oxidant levels (1 h prior to competition) and impaired exercise
5 performance (running time) in high school students during cross-country track meets in
6 Los Angeles, CA. Symptoms were not measured, but Wayne speculated that chest
7 discomfort from oxidant inhalation impaired exercise performance. Although results such as
8 these are consistent with evidence from controlled human exposure studies, precise
9 characterization of ambient pollutants and environmental conditions and rigorous statistical
10 analyses were lacking in the studies. Thus, the primarily qualitative data from these and
11 other studies were not satisfactory to provide quantitative conclusions about the relationship
12 of ambient O3 concentrations and acute respiratory illness.
13 Schwartz (1992), Schwartz and Zeger (1990), and Schwartz et al. (1988) reanalyzed the
14 original diary data of student nurses reported earlier by Hammer et al. (1974) (Table 7-19).
15 The nurses were told that the diaries were part of a prospective study of viral infections.
16 Logistic regression models including time series analyses were used to control for
17 autocorrelation effects that are frequently present in time series data. The reanalysis for
18 daily prevalence rates of symptoms (Schwartz et al., 1988) confirmed that ambient oxidants
19 were significantly associated with cough and eye discomfort. However, earlier reported
20 associations between oxidants and headache or chest discomfort were not confirmed. Cough
21 was the one symptom that showed an apparent threshold near 0.20 ppm total oxidants, which
22 approximates the threshold value reported by Hammer et al. (1974). Further reanalysis of
23 the diary data (Hammer et al., 1974) by Schwartz (1992) and by Schwartz and Zeger (1990)
24 for the effects of air pollutants on the risk of new episodes of respiratory and other
25 symptoms and on their durations revealed interesting findings. The mean plus or minus
26 standard deviation (SD) level of oxidants was 0.102 ± 0.074 ppm. In logistic regression
27 models, an increase in oxidant concentration by one SD (0.074 ppm) was associated with a
28 17% increased risk of chest discomfort and a 20% increased risk of eye irritation. These
29 associations were highly significant (p < 0.001). In addition, photochemical oxidants were
30 significantly (p < 0.0001) associated with the duration of episodes of cough, phlegm, and
31 sore throat.
December 1993 7-143 DRAFT-DO NOT QUOTE OR CITE
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TABLE 7-19. ACUTE EFFECTS OF PHOTOCHEMICAL OXIDANT POLLUTION:
SYMPTOM PREVALENCE3
Pollutants and Environmental Variables
Study Description
Results and Comments
Reference
Total oxidant, CO, SO2, NO, and NO2
measured; total oxidant concentrations
reached episodic levels (maximum
1-h/day < 0.4-0.5 ppm); mean daily
temperature was 71.8 °C.
Reanalysis of daily diary study of
student nurses working and living at
schools in Los Angeles (see U.S.
Environmental Protection Agency,
1986, for details of Hammer et al.,
1974). This series of papers
reexamines the nurses' data using
logistic regression models and time-
series methods to account for serial
correlation (autocorrelation) of
symptoms on successive days. The
effects of total oxidants on daily
prevalence rates of symptoms, risks
of developing new symptoms
(episodes), and duration of episodes
were analyzed.
Associations found between total
oxidants and prevalence of cough and
eye irritation, confirming part of
findings of original study.
Association with cough only at
oxidant concentrations above
approximately 0.20 ppm. Previously
reported associations between
oxidants and chest discomfort and
headache (Hammer et al., 1974) not
confirmed. Oxidants associated with
increased risk (incidence) of chest
discomfort and eye irritation and
duration of episodes of cough,
phlegm, and sore throat. Duration of
symptoms showed dose-response
relationships even below current
NAAQS for O$. Findings suggest
different effects of oxidants on
symptom characteristics. Lack of
daily paniculate measurements, small
number of subjects, and
heterogeneous individual responses
restrict quantitative interpretation of
results. Lung function was not
measured.
Schwartz (1992)
Schwartz and Zeger (1990)
Schwartz et al. (1988)
-------
TABLE 7-19 (cont'd). ACUTE EFFECTS OF PHOTOCHEMICAL OXTOANT POLLUTION:
SYMPTOM PREVALENCE3
Pollutants and Environmental Variables
Study Description
Results and Comments
Reference
Daily O3, NO2, SO2, and CH and every
sixth-day sulfates measured at one site
(Azusa). 1-h daily maximum O3
0.1 ppm, 7-h average O3 0.07 ppm;
sulfates 8.43 /tg/m ; maximum
temperature was 22.4 °C.
Reanalysis of daily diaries
completed during 181-day survey
period (September 1978 to March
1979) by 756 children and
572 adults (Krupnick et al., 1990)
and 321 nonsmoking adults (Ostro
et al., 1993) living in Glendora,
Covina, or Azusa, CA (see Flesh
et al., 1982 for details). Presence
or absence of 19 (upper and lower)
respiratory and 2 nonrespiratory
symptoms recorded daily. Presence
or absence of symptoms analyzed in
a pooled cross-sectional time-series
model. Nonpollution factors,
including sex, gas stove use, day of
study, and a chronic disease
indicator were included in final
regression models used to measure
effects of ambient air pollution.
Logistic regression analyses for
entire sample to determine effect of
each pollutant on health endpoints.
Lagged effects of each pollutant and
effects hi individuals (n = 74)
without air conditioners and those
with preexisting respiratory infection
were analyzed.
Logistic regression model indicated
significant associations between incidence
of lower respiratory symptoms and healthy
nonsmoking adults (but not among
smokers, children, or patients with chronic
respiratory disease); 1-h daily maximum O3
levels OR = 1.22, 95% CI of 1.11-1.34,
for a 0.1 ppm change; 7-h average O3 level
(OR = 1.32, 95% CI of 1.14-1.52); and
ambient sulfates (OR = 1.30, 95% CI of
1.09-1.54, for a 10 /tg/m3 change). CH
was significantly related to daily symptoms
in children. Gas stove in the home was
associated with lower respiratory tract
symptoms (OR = 1.23, 95% CI of
1.03-1.47), as were the effects of O^ in
subgroups without residential air
conditioner (OR = 1.24) and with
preexisting respiratory infection
(OR =1.24). All the above increased risks
were statistically significant (p < 0.05).
Interpretation of results limited by selection
of sample; undersampling of young adults;
aggregation of symptoms of all severity
levels into one measure; possible reporting
bias; and absence of indoor exposure,
aeroallergen, and lung function data.
Krupnick et al. (1990)
Ostro et al. (1993)
See glossary of terms and symbols for abbreviations and acronyms.
-------
1 Krupnick et al. (1990) and Ostro et al. (1993) reanalyzed daily health data from over
2 5,000 children and adults living in the Los Angeles area during a 6-mo period (September
3 1978 to March 1979) (Table 7-19). The original study was reported by Flesh et al. (1982) of
4 the U.S. Environmental Protection Agency. The presence or absence of daily respiratory
5 symptoms associated with daily exposure to ambient O3 and other air pollutants was analyzed
6 in a pooled cross-sectional time-series model. Krupnick et al. (1990) reported statistically
7 significant effects of O3 levels on daily reported respiratory symptoms in healthy nonsmoking
8 adults, but not among smokers, children, and patients with chronic respiratory disease.
9 Ostro et al. (1993) evaluated the daily reports of 321 nonsmoking adults and, using a logistic
10 regression model, found a statistically significant association between the incidence of lower
11 respiratory tract symptoms and 1-h daily maximum and 7-h average O3 levels (22 and 32%
12 increased risk, respectively, with 0.10 ppm increase in O3) and ambient sulfates (30%
13 increased risk with 10-^g/m3 change). The lower respiratory tract effects of O3 were greater
14 in the subgroups with gas stoves, without residential air conditioners, and with preexisting
15 respiratory infection. Interpretation of the results is limited by the selection of the sample
16 for analysis; undersampling of young adults; aggregation of symptoms of all severity levels
17 into one measure; possible reporting bias; and absence of indoor exposure, outdoor
18 aeroallergen, and lung function data.
19 The results from the above panel studies suggest a modest but biologically plausible
20 relationship between short-term exposure to ambient oxidants/O3 and respiratory symptoms.
21 The interpretation of these recent reanalyses is limited by several factors. Heterogeneous
22 individual responses occur and analyses of grouped data may possibly miss susceptible
23 subgroups. The lack of specific measurements of O3 and/or other pollutants (especially
24 particulates) and of personal exposure or risk variables weaken the assessment of confounders
25 and effect modifiers. In addition, the overall data analysis pertains to small and very
26 selected samples that have uncertain representativeness to the general population.
27
28 Aggravation of Existing Respiratory Diseases
29 Prior epidemiological data on the effects of ambient O3 levels in subjects with existing
30 respiratory disease have been difficult to interpret due to methodological limitations (U.S.
31 Environmental Protection Agency, 1986). Exacerbation of asthma and other health endpoints
December 1993 7-146 DRAFT-DO NOT QUOTE OR CITE
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1 has been subsequently evaluated, and more recent studies have observed possible increases in
2 symptom aggravation or changes in lung function of asthmatic subjects in relation to
3 increased O3 or total oxidant levels, as well as interactions between O3 concentrations and
4 temperature. However, no consistent pattern of findings for aggravation of symptoms or
5 lung function changes has been reported for patients with other types of chronic lung disease.
6 Some of the major issues in interpreting results from studies of respiratory exacerbations
7 have been inadequate sample size and characterization of the study subjects, lack of
8 information on the possible effects of medications, the absence of records for all days on
9 which symptoms could have occurred, inadequate interpretation of the clinical significance of
10 measured changes, the role of confounders and effect modifiers (e.g., temperature, humidity,
11 particulates, and aeroallergens), and personal or group characterization of indoor-outdoor
12 exposures. For example, Whittemore and Korn (1980) and Holguin et al. (1985) found small
13 increases in the probability of asthma attacks associated with previous attacks, decreased
14 temperature, and incremental increases in oxidant and O3 concentrations. Lebowitz et al.
15 (1982, 1983, 1985) and Lebowitz (1984) reported effects in asthmatics, such as decreased
16 PEFR and increased respiratory symptoms, that were related to the interaction of O3 and
17 temperature. None of these studies adequately assessed possible effect modification by other
18 pollutants, particularly inhalable particulates, which may have independent effects.
19 Epidemiological studies published since the 1986 criteria document (U.S.
20 Environmental Protection Agency, 1986) have attempted to control for many methodological
21 issues (e.g., with [1] better estimates of exposure to pollutants [as well as O3] and
22 environmental variables that can confound or modify responses, [2] serial measurements of
23 pulmonary function for determining correlations with pollutants and other environmental
24 variables, [3] better biomedical characterization of cohorts, and [4] more robust analytical
25 approaches that control for autocorrelation of environmental variables and health responses).
26 Recent studies have generally provided further evidence that supports a relationship between
27 ambient O3/oxidant concentrations and respiratory morbidity in asthmatic subjects
28 (Table 7-20).
29 Gong (1987) studied the relationship between air quality and respiratory status of
30 83 asthmatic subjects living in a high-oxidant area of Los Angeles County. The study
31 covered February to December 1983, but data analyses were limited to a 230-day period
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TABLE 7-20. AGGRAVATION OF EXISTING RESPIRATORY DISEASES BY
PHOTOCHEMICAL OXIDANT POLLUTION8
Pollutants and Environmental Variables
Study Description
Results and Comments
Reference
oo
Air pollutant measurements for April to
November 1983 used in statistical analyses.
Daily maximum of NOX, SO2, CO, THC
less than California standards or NAAQS.
SO4= >25 fig/m on 4 days;
TSP> 100 /ig/m3 on 78% of days with
data. Daily maximum 1-h average 03
concentrations (from continuous
monitoring) = 0.01-0.11 ppm on
103 days, 0.12-0.19 ppm on 65 days,
0.2-O.34 ppm on 60 days, and
0.35-0.38 ppm on 3 days. Outdoor
aeroallergens sampled with Roto-Rod:
spores, pollens, grasses, molds,
miscellaneous debris; all generally low
except for group of common molds (rusts,
smuts, mushroom) present in
thousands/cm on sampler. Mean (±SD)
daily temperature at 1 p.m. during
200 days: 26 ± 11 °C, range 13-41 °C;
128 days with £ 24 °C.
Effects of pollutants and other
environmental variables on respiratory
symptoms and PEFR evaluated in 11-mo
population study of asthmatics living in
high-O3 area (Glendora) of Los Angeles
County, CA. Detailed questionnaires
given at outset on medical/occupational
histories and personal factors, including
general activity patterns; psychological
testing (Asthma Symptom Checklist,
State-Trait Anxiety Inventory, etc.) also
given, once during good air period and
once during smoggy period. Lung
function (spirometry) and bronchodilator
responses measured at outset in all
subjects. Daily diaries (checked
2 X/week), mini-Wright peak flow
meters, and Nebulizer Chronolog
attached to metered-dose inhaler used to
record symptoms, day and night PEFR,
and medication use, respectively.
Multiple regression analyses for overall
group; then subsets (two groups of
"responders") analyzed separately and
compared with rest of cohort.
Eight of 91 subjects completing study (of
109 recruited) showed no variability in asthma
status during the 230-day study. Respiratory
status of final study population (n = 83 with
generally mild or stable asthma) as a whole not
related, either clinically or statistically, to
maximum 1-h average O3 on Days t, t-1, t-2,
t-3 for any respiratory variable even when
adjusting for medication use, symptoms, and
PEFR on Day t-1. Subset analyses showed
association of O3 with symptoms and with day and
night PEFR hi subjects in top quartile for
respiratory measures, but association did not
follow a consistent relationship with ambient
O3 concentrations. VE levels during outdoor time
not estimated. Outcomes not related to time
outdoors vs. indoors or to outdoor time on "clean"
versus "smoggy" days. Subsets ("responders")
differed from rest of cohort mainly hi scores of
Asthma Symptom Checklist for factors
representing fatigue, hyperventilation, and rapid
breathing, but there was no difference in
responders between clean and and smoggy
periods. Aeroallergens from maple, oak, beech,
and elm trees showed significant (and clinically
relevant) relationships to respiratory variables.
Exposure assessment limited by outdoors-only
monitoring and lack of time-activity data.
Gong (1987)
Gong et al. (1985)
-------
g
U)
TABLE 7-20 (cont'd). AGGRAVATION OF EXISTING RESPIRATORY DISEASES BY
PHOTOCHEMICAL OXTOANT POLLUTION8
Pollutants and Environmental Variables
Study Description
Results and Comments
Reference
Hourly outdoor O3, CO, NO2, and TSP
measured from three stations. Hourly
maxima used for O3 and CO; daily CO
and NO2 derived as weighted measures for
each cluster sampling site and daily values
were used. Sample of homes monitored
inside and outside for particulates and
gases and evaluated for housing
characteristics (e.g., gas stove usage).
Meteorological variables measured daily.
Temperature data not reported.
Effects of outdoor and indoor air
pollutants and aeroallergens evaluated in
a 2-year study of 22 subjects with
asthma, 33 with airway obstructive
disease, 30 atopies, and 14 normals
living in a dry arid environment
(Tucson, Arizona). Subjects part of a
community population sample of
117 families (see U.S. Environmental
Protection Agency, 1986, for details of
Lebowitzetal., 1982, 1983, 1985;
Lebowitz, 1984) and had well-
characterized symptoms, medication use,
lung function, methacholine response (in
a subsample), and immunological status.
Daily diaries (acute symptoms,
medication use, and doctors' visits) and
daily PEFR (2x/day) performed for
3 mo, 2-4 X /study period. Duration
spent outdoors recorded. Spectral time
series analyses used to evaluate each
respiratory response variable for
periodic tendencies and covariance
(dependent and independent) functions as
processes in time in the different
groups.
Asthmatics had greatest number of respiratory
complaints, which were related to the presence of
gas stoves, active smoking, humidity, and
temperature. Ozone was associated with peak
flow (late spring and with temperature), wheeze
(Day t-3 with humidity), and productive cough
(Day t-2). O3 (Day t-3) was related to productive
cough during the summer in allergic subjects.
Outdoor gases and meteorological variables
significantly related to symptoms and PEFR both
independently and as effect modifiers.
No significant O3 effect in patients with
obstructive disease or in normals. Small number
of subjects and study days and lack of indoor NO2
and PM10 measurements, measured pollutant
values, and effect estimates limit quantitative
interpretation of study.
Lebowitz et al.
(1987)
-------
TABLE 7-20 (cont'd). AGGRAVATION OF EXISTING RESPIRATORY
DISEASES BY PHOTOCHEMICAL OXTOANT POLLUTION3
Pollutants and Environmental Variables
Study Description
Results and Comments
Reference
-J
o
Outdoor O3 levels measured hourly by
three stations and maximum 1-h and 8-h
average values were used to represent
O3 levels for all subjects on a given day.
For each day of the study, the mean of the
maximum 8-h O3 average for the
4 preceding days was calculated to be an
index of cumulative exposure. PMjQwas
measured daily at one station. Mean
± SD of maximum 1-h O3 concentrations
was 0.055 ± 0.014 ppm (range: 0.015-
0.092 ppm), moving average maximum 8-h
O3 levels were 0.046 + 0.013 ppm (0.09-
0.082 ppm). Maximum daily outdoor
temperature was 87 °F (30 °C) per person-
day, maximum PM10 was 187 j«g/m
(mean 42 /ig/m ).
Temporal effect of ambient O$
concentration on PEFR during 30-mo
study period in 287 children (13% with
physician-diagnosed asthmatics) and
523 nonsmoking adults (9% asthmatics)
in the Tucson community population
sample. Mini-Wright peak flow meters
used four or fewer times/day but for
only 2-week periods and only one meter
was assigned/household. Children's
tests were supervised by adult and initial
2 days of observation were eliminated
from analysis. Symptoms from daily
diaries were also used in analysis.
Random-effects longitudinal model was
used for analyses to account for
autocorrelation of PEFR values.
Multifactorial ANCOVA was used to
analyze day-to-day changes in daily
average PEFR and symptom prevalence
rates (the dependent variables) in
relation to 8-h O3 values on the same
day and previous days (lags of 0 and 1).
Analyzed PEFR data limited to at least
12 measurements for at least 6 days in 78 % of
children and 74% of adults. Noon PEFR in
nonasthmatic and asthmatic children was lower
with higher 1-h maximum O3 levels:
-11.9 L/min/0.1 ppm €5 (p < 0.05) and
-31.0 L/min/0.1 ppm 0$ (p < 0.1), respectively.
Effect of 8-h O3 mean on evening PEFR only seen
in asthmatic children, possibly reflecting a
cumulative Oj response during course of day.
Among adults, evening PEFR was decreased in
asthmatics who spent more time outdoors on days
with higher Oj concentrations (time X
concentration effect). The ANCOVA model
showed significant interactive effects of Oj X
temperature x PMjQ on daily average PEFR.
Daily rates of allergic-irritant symptoms increased
with the maximum 8-h O3 average (> 0.056 ppm)
on the previous day and increased more with
interactions of O3 X temperature X PM10.
Missing PEFR data, possible overestimation of
outdoor O3 exposure, large variability of
responses in asthmatics, medication use on days
with high O3 levels, relatively low O3 levels, and
uncertain effects of indoor and outdoor allergens
and respiratory infections limit interpretation.
Lebowitz et al.
(1991)
Krzyzanowski et al.
(1992)
-------
p-t
>— Hourly O3, twice daily (9:00 a.m. and
^O 9:00 p.m.) acidic aerosols (sulfates, SO4,
and H ), and pollen counts were measured
on site. Hourly T, RH, and O3 measured
from nearby monitors. In 1991, pollution
levels increased daily until Day 5, when
maximum 1-h O3 reached 0.154 ppm and
daytime H and sulfate levels were
245 nm/m and 26.7 /xg/m , respectively.
In 1992, air quality was better (e.g., the
highest daily 1 -h maximum O3 was
0.063 ppm). Temperature data not
reported.
TABLE 7-20 (cont'd). AGGRAVATION OF EXISTING RESPIRATORY
DISEASES BY PHOTOCHEMICAL OXTOANT POLLUTION3
Pollutants and Environmental Variables
Study Description
Results and Comments
Reference
Effects of ambient summertime haze air
pollution on asthmatic children
(ages 7-13) attending 1-week asthma
camp in Connecticut River Valley were
evaluated during June 1991 (n = 50)
and 1992 (n = 55). PEFR and
symptoms (2x/day) and number of
as-needed (p.r.n.) inhaled bronchodilator
treatments given by on-site physician
during each study day were recorded.
Correlations between health outcomes
and air pollutants were performed.
In 1991, daily total number of p.r.n. treatments Thurston et al.
highly correlated (r > 0.80) with maximum O3, (1992a, 1993c)
804, daytime H , and maximum temperature, but
only SO4 (r = 0.97) and H+ (r = 0.985) were
significant (p < 0.05) and remained so after
temperature was included in the analysis. Daily
pollen counts were not associated with as-needed
treatments. Afternoon chest symptoms (cough,
phlegm, and wheeze) and changes in morning-
afternoon PEFR values (excluding children given
medication) were significantly correlated
(p < 0.05) with O3 and H+, respectively.
Scheduled medications did not apparently provide
a protective effect (X2 = 3.25, p = 0.067),
although the failure to achieve statistical
significance is not unexpected given the small
sample size. In 1992, change in PEFR
(magnitude not reported), chest symptoms, and the
fewer daily exacerbations (maximum 27 vs. 37 in
1991) were not significantly correlated with
pollution, pollen, or temperature. Only sore
throat, runny nose, and eye irritation were
correlated with pollen counts. Although the data
are only hi preliminary form, the 1991 results
appear consistent with an effect of summertime
haze air pollution on PEFR, chest symptoms, and
asthma exacerbations. The 1992 results are
consistent with less health effects owing to cleaner
ambient conditions. Small number of subjects and
study days and lack of results for other pollutants
limit the interpretation of the studies.
See glossary of terms and symbols for abbreviations and acronyms.
-------
1 (April 15 through November 30) because of staggered entry of subjects into the study and the
2 high frequency of missing or incomplete data encountered in the earlier part of the study
3 period. Regression and correlation analyses between 03 and average symptom scores,
4 asthma medication index (AMI), and day and night PEFR across subjects showed weak,
5 nonsignificant relationships. These daily outcome variables were compared across days with
6 maximum 1-h-average O3 in three ranges: <0.12 ppm, 0.12 to 0.19 ppm, and >0.20 ppm;
7 "no statistical or clinical significance was detected." Individual exposures and activity
8 patterns were not estimated in these two analyses. Multiple regression analyses also
9 indicated the lack of a significant overall relationship between O3 (and their independent
10 variables) and respiratory status, despite the use of lagged variables and the inclusion of
11 other pollutants, meteorological variables, aeroallergens, and AMI. Total suspended
12 particulates directly affected PEFR, but the relationship was not consistent in the analysis.
13 Aeroallergens showed significantly negative relationships to respiratory variables, but only
14 the effect of certain molds was considered clinically relevant. Temperature and humidity
15 showed no significant effect on the respiratory variables on this study.
16 Although there was no significant overall effect of O3 on respiratory variables in the
17 83 asthmatic subjects, multiple regression analysis of subjects whose O3 coefficients on
18 various days were in the top quartile for dependent variables (respiratory measures) showed
19 significant and consistent effects of O3 on Day t and the previous day (Day t-1). Multiple
20 regression testing of subsets for associations of symptom score or day or night PEFR on the
21 same day's O3 and the previous day's value of the same responses showed highly significant
22 O3 coefficients for all three respiratory measures.
23 The clinical significance of responses in symptom scores and day and night peak flow
24 was evaluated for all subjects by individual regression analyses. No subject had evidence of
25 significant worsening of symptoms attributable to O3 during the study. Adult subjects with
26 high scores in fatigue, hyperventilation, dyspnea, congestion, and rapid breathing in the
27 Asthma Symptom Checklist had more negative slope coefficients for O3 than subjects with
28 low-to-moderate scores on the checklist. "Re&ponders" (statistically identified by multiple
29 regression analysis) scored consistently higher in the factors representing fatigue,
30 hyperventilation, and rapid breathing. The higher scores of these responders, however,
31 "were apparently not associated with differences in ambient O3 concentrations since the test
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1 scores were similar during relatively low (first test) and high (second test) O3 days. The
2 significance of the psychological results is unclear at this time."
3 Lebowitz et al. (1987) performed time series analysis to evaluate daily respiratory
4 responses to outdoor and indoor air pollutant and aeroallergen exposures in potentially
5 sensitive adults living in a dry climate (Tucson, Arizona). Daily symptoms and PEFR were
6 recorded in well-characterized groups of asthmatics, allergic subjects, patients with chronic
7 airways obstruction, and asymptomatic healthy controls (total sample size of 204) over
8 2 years. Daily diaries included acute symptoms, medication use, and doctors' visits.
9 A sample of homes was evaluated for environmental characteristics and was monitored
10 indoors and outdoors for gases and particulates, in addition to stationary outdoor monitors.
11 Asthmatics showed the most respiratory responses. Outdoor O3 levels were significantly
12 (p < 0.05) related to wheeze, productive cough, and peak flow (late spring) in the asthmatic
13 group. Statistical interactions between O3 and smoking, presence of a gas stove, maximum
14 temperature, and minimum humidity (R = 0.49) were found. The other groups did not
15 demonstrate an O3 effect, except for the atopic group, which had increased summertime
16 productive cough related to O3 levels. Thus, these results indicate an O3 effect on asthmatics
17 and that statistical interactions between O3 and other environmental factors are significantly
18 related to symptoms and peak flow. On the other hand, the results are largely descriptive
19 and qualitative without adequate effect estimators.
20 A subsequent analysis of the same community population sample in Tucson (Lebowitz
21 et al., 1991; Krzyzanowski et al., 1992) evaluated the temporal relationship between PEFR
22 and ambient O3 in 287 children and 523 nonsmoking adults. During part of the study
23 period, ambient particulates (paniculate matter of mass median aerodynamic diameter 10 jum
24 or less [PM10]) were collected daily at one monitoring station. A random-effects longitudinal
25 model and multifactorial analysis of covariance were used for analyses. During the study
26 period, the maximum ambient O3 concentrations were relatively low (i.e., the 1-h maximum
27 never exceeded 0.092 ppm). In children, noon peak flows were decreased on days when
28 there was a high O3 concentration. Children with physician-confirmed asthma experienced
29 the greatest decrease in noon peak flow. Evening peak flow was also significantly related to
30 O3 in children, especially asthmatic children, suggesting a cumulative O3 response during the
31 course of the day. Among adults, evening peak flows were decreased in asthmatics who
December 1993 7-153 DRAFT-DO NOT QUOTE OR CITE
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1 spent more time outdoors on days when O3 levels were high. After adjustment for
2 covariates, significant statistical interactions of 8-h O3 levels with participate matter (PM10)
3 and temperature on daily PEFR were found. There was a significant increase in allergic-
4 irritant symptom rates related to prolonged exposure to O3 (maximum 8-h average on the
5 previous day and the interactions of O3, temperature, and humidity). The study had some
6 methodologic problems (e.g., missing daily PEFR data in many subjects, lack of information
7 about specific hours spent outdoors, medication usage, and relatively low O3 levels during
8 the study period). Nonetheless, the data analyses, control of confounders, and overall
9 exposure assessment strengthen the conclusions of the study (i.e., that the respiratory
10 response to O3 is acute, occurs more often in asthmatics, and increases as temperature and
11 PMjo increase).
12 The respiratory effects of ambient O3 and other coexisting pollutants were evaluated
13 during a 1-week asthma camp located in the Connecticut River Valley during June of 1991
14 and 1992 (Thurston et al., 1992a, 1993c). Each child (age 7 to 13 years) participated in the
15 same daily activities all week. Peak flow and symptoms were recorded twice a day, as well
16 as the number of as-needed (p.r.n.) treatments of inhaled bronchodilator administered by an
17 on-site physician during each day (each representing an exacerbation of asthma). Hourly
18 measurements of O3 and twice daily samples of acidic aerosols (sulfates, SO4, and hydrogen
19 ions, H+) were collected. The preliminary results (manuscript in preparation) indicate a
20 strong association between the ambient air pollution mix and the occurrence of asthmatic
21 exacerbations in children. During 1991, pollution levels progressively increased until Day 5,
22 when the 1-h maximum O3 concentration reached 0.154 ppm, and the daytime (9:00 a.m. to
23 9:00 p.m.) H+ and SO4 concentrations were 254 nm/m3 and 26.7 jig/™3, respectively. The
24 correlations of the daily total number of p.r.n. treatments required with daily maximum O3,
25 daytime SO4 and H+, and maximum temperature were all high (r > 0.8), but only SO4
26 (r = 0.97) and H+ (r = 0.98) were significant (p < 0.05) given the small number of days
27 involved. Afternoon symptoms (cough, phlegm, and wheeze) and morning-afternoon change
28 in PEFR (without medication) were significantly correlated (p < 0.05) with 03 and H ,
29 respectively. During 1992, the local air quality was better (e.g., the daily 1-h maximum
30 O3 concentration was only 0.063 ppm). There were fewer asthmatic exacerbations
31 (maximum of 27 versus 37 in 1991), and they were not significantly correlated with
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1 pollution, pollen, or temperature. Pollutants were not significantly correlated with symptoms
2 or PEFR. Overall, the 1991 data indicate a coherence in the associations of summertime
3 haze air pollution with peak flow, chest symptoms, and asthma exacerbations hi children.
4 The lack of correlation during 1992 was likely due to the improved air quality and indirectly
5 supports the results of the previous year. An adequate interpretation of these preliminary
6 results is limited by the small number of subjects and study days and the lack of results for
7 other pollutants. These camp studies remain to be reported in more detail.
8 The above epidemiological studies have generally supported a direct association
9 between ambient O3/oxidant concentrations and acute respiratory morbidity in asthmatics.
10 The recent studies have strengthened their conclusions by improvements or new approaches
11 in the estimations of O3 exposure, confounders, and effect modifiers, characterization of the
12 subjects and serial measurements of their responses; and analytical approaches. Thus, the
13 aggregate results can be viewed as biologically and temporally plausible, consistent, and
14 coherent to some extent. However, the studies share certain deficiencies such as small
15 numbers of subjects (which may reduce statistical power) and the lack of significant data
16 about individual responses and their distribution. The independent effect of ambient 63, as
17 estimated by statistical models in epidemiological studies, is difficult, at best, to clearly
18 differentiate from those of copollutants because O3 (or another pollutant such as H+) may
19 only be acting as an indicator of the toxic potency of the ambient mixture of pollutants.
20 This, in combination with measurement error and uncontrolled associations with other
21 factors, complicates analytical findings about the relationships among components of an
22 ambient mixture and may not accurately disentangle the effects of O3 in a biologically
23 appropriate fashion.
24
25 7.4.1.3 Aggregate Population Time Series Studies
26 Aggregate population, or "ecological", time series studies are epidemiological
27 investigations in which the associations between air pollution and human health outcomes are
28 evaluated over time in the population as a whole (e.g., with respect to deaths per day in a
29 given city) and for which outcomes and exposures are not matched for the individuals within
30 the population. Indeed, aggregate population time series studies of extreme air pollution
31 episodes have provided some of the clearest evidence of the adverse effects of air pollution
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1 on humans. For example, during the historic December 1952 London Fog episode, in which
2 extremely high sulfur oxide and paniculate matter air pollution levels were experienced, total
3 mortality in Greater London rose from roughly 300 to 900 deaths/day, and acute respiratory
4 hospital admissions rose from 175 to 460/day (Ministry of Health of Great Britain, 1954).
5 At more routine levels of air pollution, any effects of air pollution are necessarily less
6 obvious, and, as shall be discussed below, methodological issues exist as to the proper
7 analysis and interpretation of such aggregate population time series data.
8 The previous criteria document (U.S. Environmental Protection Agency, 1986)
9 discussed several methodological issues with regard to the epidemiological studies of O3 and
10 photochemical oxidants available at that time. Limitations identified included interferences
11 by, or interactions with, other pollutants and meteorological factors in the ambient
12 environment; lack of comprehensive exposure issue assessments, such as individual activity
13 patterns and evaluation of pollutant monitor appropriateness; difficulty in identifying the
14 responsible oxidant species; and inadequate characterization of the study population.
15 However, most of these criticisms are not relevant to time series studies. For example,
16 because the same population is being followed from day-to-day, the study population acts as
17 its own "control", obviating the need for a detailed population characterization. Also, the
18 use of central site monitoring data can be appropriate in these studies for two reasons:
19 (1) although O3 concentrations can vary spatially within an airshed, they are usually highly
20 correlated across sites over time, so that correlational time series studies are not as dependent
21 on detailed exposure assessments as are, for example, cross-sectional studies; and (2) if the
22 ultimate use of these studies is to be included as criteria for ambient standards, the attainment
23 of which is evaluated at central monitoring stations, then these are the data most relevant for
24 analysis. Of the concerns raised by the previous criteria document regarding epidemiological
25 studies, then, the most important to these studies is the potential for other correlated
26 environmental factors to confound the unique identification of O3 as a critical causal factor in
27 any health effects found via time series analyses of aggregate population data.
28 One aspect of evaluating tune series epidemiologic studies of the health effects of air
29 pollution that was not directly raised by the previous criteria document, but which can be
30 crucial to their proper interpretation, is the statistical question as to how each study has
31 addressed the potentially confounding influences of long-wave (e.g., seasonal) variations in
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1 the health outcome data. The seasonality of morbidity and mortality was explicitly
2 mentioned in Hippocrates' treatise on "Airs, Waters, and Places" and has itself been much
3 studied over the years (Hechter and Goldsmith, 1961). In respiratory diseases such as
4 asthma, this seasonality of admissions is very common, due in part to the multifactorial
5 nature of these diseases. For example, spring and fall rises in pollen and winter influenza
6 epidemics superimpose long-wave cycles on the day-to-day variations in respiratory hospital
7 admission rates. Such long-wave cycles need to be addressed as part of any time series
8 analysis for two reasons: (1) they result in strong autocorrelations, which violate the
9 underlying assumptions of most statistical approaches used to analyze such data; and (2) their
10 inclusion can lead to misleading conclusions, in that the long-wave relationships would likely
11 obscure the acute (i.e., short-wave) effects being evaluated. The need to address seasonal
12 cycles in respiratory disease time series data in order to avoid spurious long-wave dominated
13 correlations has been long recognized (e.g., Ipsen et al., 1969), but has all too often been
14 ignored or inadequately addressed in the published literature. Lipfert (1993), in his literature
15 review of hospitalization and air pollution, also noted the need for such studies to take into
16 account both weekly and seasonal temporal patterns in the data. Thus, an important criterion
17 for the evaluation of aggregate population time series studies of the acute morbidity and
18 mortality effects of O3 is whether or not the authors have appropriately addressed long-wave
19 periodicities in the data as part of their analysis.
20 There are a variety of statistical approaches available to address long-wave confounding
21 in time series analyses, each having advantages and disadvantages. The primary goal in
22 invoking such procedures is to eliminate the long-wave autocorrelation "noise" in the data
23 without inadvertently removing any O3-related health effects "signal" at the same time.
24 In particular, steps that address autocorrelation in the model but also remove or explain
25 short-wave variance in the health outcome variable of interest (e.g., by applying prefilters to
26 the series that affect periodicities down to a few days, or by analyzing the residuals from
27 prior regressions of the outcome variable on "control" variables that are correlated with 03,
28 such as temperature) carry with them the risk of also removing short-wave associations of
29 interest before the actual analysis has begun. Similarly, the inclusion of autoregressive terms
30 to remove all autocorrelation has the advantage of being a very conservative approach, but
31 may also alter any short-wave O3-related health effects signals present in the process. Less
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1 severe methods of autocorrelation removal (e.g., the selection of more limited study time
2 periods during that the series is quasi-stationary, applying longer wave prefilters that do not
3 affect cycles less than a week in periodicity, calculating deviations from annual cycle sine-
4 cosine fits of the data series, or using longer term averages of control variables, such as
5 temperature, in simultaneous regressions) may not remove autocorrelation as completely as
6 the previously mentioned methods, but have the advantage that they are less likely to affect
7 the short-wave data variations of interest. Furthermore, although there are standard
8 regression diagnostics available to determine whether autocorrelation remains a significant
9 problem (e.g., the Durbin-Watson statistic), no such check exists to determine whether the
10 more severe autocorrelation removal methods have also inadvertently removed an O3-health
11 effects association of interest. Thus, although steps must be taken in time series analyses to
12 address the potentially large biases resulting from long-wave (e.g., seasonal)
13 autocorrelations, care must be taken not to also remove the signal of interest when dealing
14 with the autocorrelation problem.
15
16 Emergency Room Visits and Hospital Admissions
17 Many investigators have evaluated the associations between hospital emergency room
18 visits or hospital admissions and air pollution. Hospital admissions are far more common (as
19 counts per day) than, for example, mortality, thereby providing greater statistical reliability
20 and avoiding the distributional complications that may be presented by low counts. Also,
21 admission to the hospital is a well defined endpoint, having the desirable feature that every
22 patient must have been seen by a physician and deemed sick enough to require
23 hospitalization. Emergency room (ER) visits provide even larger daily counts, but are not as
24 well defined an endpoint because the patient usually decides whether or not to attend the ER.
25 Furthermore, in a well designed study in Quebec, Canada, hospital admission diagnosis at
26 discharge was found to be very reliable, with the study confirming the classification of
27 respiratory admissions in general 92% of the time, and asthma admissions 95% of the time
28 (Delfino et al., 1993). Daily series of hospital admissions thus represent an especially useful
29 research resource for the investigation of the human health consequences of O3 exposure.
30 Hospital admission and emergency room visit studies that have considered
31 O3 associations are summarized in Table 7-21. In the previous criteria documents (U.S.
December 1993 7-158 DRAFT-DO NOT QUOTE OR CITE
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TABLE 7-21. HOSPITAL ADMISSIONS/VISITS IN RELATION TO PHOTOCHEMICAL
OXIDANT POLLUTION: TIME SERIES STUDIES8
Concentration(s)
ppm
0.11 -0.28 avg
max 1 h during
low and high
periods,
respectively
(Not reported)
Pollutant Study Description
Oxidant Comparison of admissions to Los Angeles
County Hospital for respiratory and cardiac
conditions during smog and smog-free
periods from August to November, 1954.
Oxidant Respiratory and cardiovascular admissions to
Los Angeles County Hospital for residents
living within 8 mi of downtown Los Angeles
between August and December, 1954.
Results and Comments
No consistent relationship between admissions and high
smog periods; however, statistical analyses were not
reported. Clear seasonal trends in admissions
(increasing from summer to winter) not addressed.
Inconclusive results; partial correlation coefficients
between total oxidants and admissions were variable.
Method of patient selection was not given. Other
pollutants were not considered. Seasonal trend not
Reference
California Dept. of
Public Health
(1955b, 1956b,
1957b)
Brant and Hill
(1964b)
Brant (1965)
addressed.
(Not reported) Oxidant
Admissions of Blue Cross patients to Los
Angeles hospitals with > 100 beds between
March and October, 1961; daily average
concentrations of oxidant, 03, CO, SO2,
NO2, NO, and PM by Los Angeles air
pollution control districts.
1967b)
Correlation coefficients between admissions for Sterling et aL
allergies, eye inflammation, and acute upper and lower (1966
respiratory infections and all pollutants were statistically
significant; correlations between cardiovascular and
other respiratory diseases were significant for oxidant,
Og, and SO2; significant positive correlations were
noted with length of hospital stay for SO2, NO2, and
NOX. Correlations were not significant for T and RH
or for pollutants with other disease categories. Reported
seasonal variations in admissions and pollution not
addressed.
(Not reported) Oxidant
Admissions for all adults and children with
acute respiratory illness in four Hamilton,
Ontario hospitals during the 12 mo from
July 1, 1970, to June 30, 1971; city-average
pollution monitoring for OX(KI), SC^, PM,
CoH, CO, NOX, HC, T, wind direction and
velocity, RH, and pollen.
Correlation found between admissions and an air
pollution index for SO2 and CoH; negative correlation
between T and admissions; and nonsignificant negative
correlations found with concentrations of OX(K1).
However, clear, long-wave trends (e.g., seasonality) not
addressed.
Levy et al. (1977°)
-------
TABLE 7-21 (cont'd). HOSPITAL ADMISSIONS/VISITS IN RELATION TO PHOTOCHEMICAL
^ OXIDANT POLLUTION; TIME SERIES STUDIES8
Concentration(s)
ppm
Pollutant
Study Description
Results and Comments
Reference
(Not reported) O3 Emergency room visits for cardiac and
respiratory disease in two major hospitals in
the city of Chicago during April 1977 to
April 1978; 1-h concentrations of O3, SO2,
NO2, NO, and CO from an EPA site close to
the hospital, 24-h concentrations of TSP,
SO2, and NO2 from the Chicago Air
Sampling Network.
No significant association between admissions for any Namekata et al.
disease groups and O3, CO, or TSP; SO2 and NO (1979C)
accounted for part of the variation of ER visits for
respiratory and cardiovascular admissions. Questionable
analysis, including lack of control for confounding,
possible seasonality in admissions (time series not
shown), and model overspecification (e.g., use of wind
speed).
0.07 and 0.39 O3 Emergency room visits and hospital
avg max 1 h admissions for children with asthma
during low and symptoms during periods of high and low air
high periods, pollution in Los Angeles from August 1979 to
respectively January 1980; daily maximum hourly
concentrations of 03, SO2> NO, N02, HC,
and CoH; weekly maximum hourly
concentrations of SO4~ and TSP; biweekly
allergens and daily meteorological variables
from regional monitoring stations.
Asthma positively correlated with CoH, HC, NC^, and Richards et al.
allergens on same day and negatively correlated with O3 (1981°)
and SO2; asthma positively correlated with NO2 on
Days 2 and 3 after exposure; correlations were stronger
on Day 2 for most variables; nonsignificant correlation
for SO4~ and TSP. Monthly admissions and pollution
data indicate strong seasonality, which is not accounted
for. This questionable analysis results in (likely
spurious) positive correlations with CoH, HC, and NO2
and negative correlations with O3.
0.03 and 0.11 Oxidant
avg max 1 h for
low and high
areas,
respectively
Daily hospital emergency room admissions in
four Southern California communities during
1974-1975. Maximum hourly average
concentrations of oxidant, NO2, NO, CO,
SO2, CoH; 24-h average concentrations of
PM and SO4~; and daily meteorological
conditions from monitoring sites 8 km from
the hospitals.
Admissions significantly associated with oxidant and
temperature in all locations. Long-term trends and
day-of-week effects appropriately controlled, but not
seasonality. Path analysis guided regression used to
discriminate among the intercorrelated pollutant and
meteorological factors, indicating O3 to be most
important only at the highest O3 site. However, lack of
catchment area population figures and inadequate
seasonality adjustments prevent quantitative use of
results.
Goldsmith et al.
(1983C)
-------
2!
i—>
u>
OS
TABLE 7-21 (cont'd). HOSPITAL ADMISSIONS/VISITS IN RELATION TO PHOTOCHEMICAL
OXTOANT POLLUTION: TIME SERIES STUDIES3
Concentration(s)
ppm
0.03-0.12
avg of max
1 -h/day for
15 stations
0.025-0.075
3 -mo avg of
monthly means
from all city
sites
0.001-0.085
mean 1-h max
O3 (avg of 1 1
sites)
0-0. 13 avg of
max 1 h/day for
two stations
Pollutant Study Description
O3 Admissions to 79 acute-care hospitals in
Southern Ontario for the months of January,
February, July, and August in 1974, 1976-
1983. Hourly average concentrations of
particulate (CoH), O3, SO2, NO2, and daily
temperature from 15 air sampling stations
within the region.
O3 Analysis of quarterly hospital admission rates
for childhood asthma in Hong Kong during
1983-1987 (n = 19). Quarterly means of
SO2, NO2, NO, O3, TSP, and RSP
considered.
O3 Analysis of emergency room visits, by cause,
to acute care hospitals in the Vancouver, BC,
area July 1984-October 1986. SO2, NO2,
O3, SO4, and temperature considered.
O3 Emergency room admissions for COPD in
Barcelona, Spain during 1985-1986. 24-h
avg SO2 and BS city wide averages. 1-h max
SO2, CO, NO2, and O3 obtained from two
stations.
Results and Comments
Excess respiratory admissions most strongly associated
(p<.001) with O3, sulfate, and T during
July and August with 24- and 48-h lag. No such
associations exist for nonrespiratory (control)
diseases. Seasonality minimized by selection of
narrow study period, and day-of-week effects controlled.
SO4~ and O3 highly intercorrelated (r = 0.65), making
effect discrimination difficult. A lack of independent
regression coefficients prevents quantitative application of
results.
Concludes that asthma is negatively correlated with SO2,
but not with O3. Questionable analysis, including use of
quarterly means and lack of seasonality controls.
Summer (May-October) total emergency (but not
respiratory) visits significantly correlated with temperature
and O3. Day-of-week effects addressed. Seasonality
reduced by study period selection, but opposing within
season cycles in asthma visits and O3 not addressed, which
may have weakened reported O3 -respiratory visit
relationship. Also, O3 levels much lower than in previously
studied Southern Ontario.
A weak but statistically significant association found between
COPD admissions and levels of SO2, BS, and CO, after
accounting for seasonality and autocorrelation and during
eliminated from the analysis based on its Initial overall
Reference
Bates and Sizto
(1983, 1987,
1989)
Bates (1985)
Tseng and Li
(1990)
Bates et al.
(1990)
Sunyer et al.
(1991)
negative correlation with admissions (prior to long-wave
controls). Thus, no conclusions regarding Q$ can be made
from this work.
-------
TABLE 7-21 (cont'd). HOSPITAL ADMISSIONS/VISITS IN RELATION TO PHOTOCHEMICAL
OXIDANT POLLUTION; TIME SERIES STUDIES3
Concentration(s)
ppm
Pollutant
Study Description
Results and Comments
Reference
0-0.04 daily
mean
Hospital admissions for asthma in Helsinki,
Finland during 1987 to 1989; 24 h average
SO2, NO2, TSP, and 03 city wide averages.
After accounting for daily minimum temperature, NO2 Ponka (1991)
and O3 were significantly correlated on the same day as
admissions, while O3 was most significant on the prior
day (p = 0.006). However, long wave peaks (e.g., in
April for asthma) were not addressed and
autocorrelation was not assessed.
0.06-0.13 mean
of 1000 to
1500 hours O3
(0.12 1-hmax
was exceeded on
42 of 226 total
study days.
whereas 0.08
was exceeded on
102 days)
O3 Emergency room visits for asthma,
bronchitis, and finger wounds
(a nonrespiratory control) at nine hospitals in
central New Jersey were analyzed for the
period May-August 1988 and 1989. Daily
values of 03 and SO2 obtained from nearest
of five monitoring sites. Barometric
pressure, T, RH, and visibility (as an index
of sulfate) obtained from a Newark net
station.
Bivariate correlations indicated asthma visits to be
strongly negatively correlated with T and weakly
negatively correlated with O3, suggesting a seasonality
influence, despite limitation to the O3 season.
However, simultaneous regression of asthma visits on
all environmental variables yielded significant (positive)
03 and (negative) T coefficients only, suggesting that T
acted as a long-wave control variable, revealing the
short-wave O3 relationship with asthma. Day-of-week
effects on visits found unimportant. No environmental
associations seen with bronchitis or control cases (finger
cuts).
Cody et al. (1992)
0.00-0.05 avg of
daily means
from 22 stations
O3 Admissions to 79 acute-care hospitals in
Southern Ontario for the months of January,
February, July, and August in 1979-1985.
Hourly average O3, SO2, NO2, T, RH, wind,
speed, barometric pressure, and daily average
TSPandSO4=.
An elaborate reanalysis of the Bates and Sizto (1989)
data set augmented to 1985. Long-wave influences
controlled using time period subsets and AR modeling.
Despite possible overspecification of models (e.g., use
of wind speed) and AR filtering of the short wave,
results confirm Bates and Sizto's overall conclusions
regarding significant O3 associations. Response to air
pollution estimated to be 19-24% of summer respiratory
admissions, although the exact contribution by O3 to the
total was not estimated.
Lipfert and
Hammerstrom
(1992)
0.01-0.05 3-mo
avg of daily
means from all
city sites
O3 Age-specific quarterly asthmatic hospital
discharge rates in Hong Kong during
1983-1989 examined in relation to quarterly
mean levels of TSP, RSP, NOj, NOX, and
03 (n = 27).
Concludes that asthma morbidity is correlated with
paniculate matter, but not O3. Questionable analysis,
including use of quarterly means and lack of seasonality
controls.
Tseng et al. (1992)
-------
8
I
I
vo
U)
TABLE 7-21 (cont'd). HOSPITAL ADMISSIONS/VISITS IN RELATION TO PHOTOCHEMICAL
OXIDANT POLLUTION; TIME SERIES STUDIES3
Concentration(s)
ppm
Pollutant
Study Description
Results and Comments
Reference
0.03-0.21
1-h daily max at
central site in
each area
O3 Daily emergency admissions to acute care
hospitals for asthma, total respiratory, and
control disease categories in the New York
City, Albany, and Buffalo, NY, metropolitan
areas during June-August 1988 and 1989;
daily 1-h maximum 03 and temperature and
daily average sulfate and acid aerosols (H )
considered.
Significant positive associations found for O3, SO4 ,
and H with asthma and total respiratory admissions,
but not for control categories. Long-wave and
day-of-week effects removed, and T effects controlled.
Strongest O3 associations in higher pollution year (1988)
and in more urban population centers (Buffalo and New
York, NY).
Thurston et al.
(1992b)
0.01-0.16
1-h daily max at
central site
O3 Daily admissions to 22 acute care hospitals in
Toronto, Ontario, for asthma, total
respiratory, and control disease categories
during July and August 1986, 1987, and
1988; daily 1-h maximum O3, SO2, NO^, T,
and daytime (9:00 a.m.-5:00 p.m.) SO4~~ and
H considered.
Significant positive correlations found for O3, H ,
SO4 = , PM10, and TSP with asthma and for total
respiratory admissions, but not for SO2 or NO2, and not
with control admissions. Long-wave and day-of-week
effects removed. Multivariate regressions and
sensitivity analyses suggested that O3 was the pollutant
of primary importance, but H may potentiate O3
effects. Except for H , all PM metrics considered
became nonsignificant when entered into regressions
simultaneously with O3. Ozone significant even after
dropping days >0.12 ppm.
Thurston et al.
(1993)
0.01-0.15 O3 Daily emergency respiratory admissions to
1-h daily max 168 acute care hospitals in Ontario, Canada,
during May-August 1983-1988 were related
to daily levels of O3 and SO4 at the nearest
of 22 and 9 monitoring sites, respectively.
Admissions broken into 0-1, 2-34, 35-64,
and 65 4- age groups, and by geographical
subregion.
Ozone and SO4 positively and significantly associated
with admissions for asthma and COPD in all age
groups. Associations consistent across regions.
Seasonal and day of week effects addressed prior to
analysis. Analyses also controlled for individual
hospital influences. No pollutant associations found for
nonrespiratory control admissions. Simultaneous
regressions suggest O3 to be more important than
SO4=.
Burnett et al.
(1993)
-------
TABLE 7-21 (cont'd). HOSPITAL ADMISSIONS/VISITS IN RELATION TO PHOTOCHEMICAL
OXIDANT POLLUTION; TIME SERIES STUDIES3
Concentrations)
ppm
Pollutant
Study Description
Results and Comments
Reference
0.02-0.16
1-h avg daily
max 0.01-0. 12
8-h avg daily
max
O
Daily numbers of emergency asthma visits by
patients 1-16 years old to an inner city
hospital in Atlanta, GA, during June-August
1990 were related to daily levels of O3, SO2,
, pollen, and T.
Hospital visits were found to be significantly higher on White et al. (1993)
days when the previous day's 1-h max O3 exceeded
0.11. No relationship was found below 0.11, or with
8-h avg daily maximum O3. Day-of-week effects were
accounted for. Seasonality effect reduced by study
period selection, but probable long-wave seasonal cycles
superimposed on the day-to-day fluctuations were not
directly addressed, which likely weakened the reported
O3-admissions associations.
0.02-0.09 O3 Daily respiratory admissions for patients
1-h avg daily >65 years of age in Detroit, MI during 1986
max (10th to to 1989 were related to daily levels of O3,
90th percentile) PM10, and temperature.
Pneumonia and COPD respiratory admissions were
found to be significantly associated with both PM10 and
O3, even after eliminating non-compliance days.
Monthly dummy variables were employed to account for
seasonal variations, but day of week effects were not
addressed. Asthma admissions were not associated with
pollution, but this was attributed to the very low counts
in this category for the elderly.
Schwartz (1993)
aSee glossary of terms and symbols for abbreviations and acronyms.
Reviewed in U.S. Environmental Protection Agency (1978).
°Reviewed in U.S. Environmental Protection Agency (1986).
-------
1 Environmental Protection Agency, 1978, 1986), such studies were found to give inconsistent
2 results for reasons that were not apparent. A common weakness of many of those studies,
3 however, was a failure to control adequately for seasonal differences in hospital usage and
4 O3 concentration. Therefore, each of the updated critiques in this table now includes an
5 evaluation as to how the data were (or were not) controlled for long-wave influences (e.g.,
6 seasonally). With this factor taken into account, the older studies' varying results are now
7 more understandable. In a number of these studies, documented long-wave periodicities in
8 the data had been ignored, resulting in either nonsignificant associations (i.e., California
9 Department of Public Health, 1955, 1956, 1957; Brant and Hill, 1964; Brant, 1965; Levy
10 et al., 1977; and Namekata et al., 1979) or even significant negative correlations between
11 O3 and hospital visits/admissions (Richards et al., 1981) as a result of the generally higher
12 respiratory admission rates in the colder months, when O3 levels are at their lowest. Two
13 studies that did not control for seasonably still reported significant positive correlations
14 between hospital admissions and oxidants (Sterling et al., 1966, 1967; Goldsmith et al.,
15 1983), though Sterling et al. excluded the winter months from the analysis and Goldsmith
16 et al. did detrend the data. Also, unlike any of the previously cited studies, both of these
17 analyses controlled for day of week effects on hospital admission rates (e.g., due to
18 consistently lower admissions on weekends), an important factor in hospital admissions
19 variations that must also be addressed (see Sterling et al., 1966). Moreover, the one
20 previously reviewed study that adequately controlled for both long-wave and day-of-week
21 influences (Bates and Sizto, 1983, 1987, 1989) reported very significant associations
22 (p < 0.001) between O3 levels and summertime (July and August) respiratory hospital
23 admissions. However, other intercorrelated environmental variables (e.g., acidic sulfates)
24 may also have been cofactors in this association. Overall, a review of these older studies
25 suggests that, if the data are appropriately analyzed, a significant association may be found
26 between elevated ambient O3 concentrations and acute increases in daily respiratory hospital
27 admissions.
28 Since the last criteria document (U.S. Environmental Protection Agency, 1986),
29 a number of new ER visit and hospital admissions studies have been completed, a few of
30 which share some of the same statistical flaws found in many of the older studies. For
31 example, Tseng and Li (1990) and Tseng et al. (1992) failed to control for the seasonally of
December 1993 7-165 DRAFT-DO NOT QUOTE OR CITE
-------
1 the admissions and of the pollutants in their statistical analyses of quarterly hospital
2 admissions in Hong Kong, causing them to report no associations with O3, but a significant
3 (and likely spurious) negative correlation of age-specific asthma admissions with quarterly
4 mean SO2 in the first of these papers, and a significant (and also likely spurious) positive
5 association with TSP in the second paper, but no association with O3. Sunyer et al. (1991)
6 failed to consider seasonality in their initial evaluation of an O3 relationship with COPD
7 hospital admissions in Barcelona, Spain; causing them to eliminate O3 from consideration in
8 the study and any evaluation of possible health effects. Also, Bates et al. (1990), using a
9 largely descriptive approach, characterized the seasonal periodicities of Vancouver, BC,
10 respiratory ER visits. Their subanalysis of the warm season (May through October) included
11 a dominant fall asthma peak, which would obscure any summertime O3 associations and,
12 therefore, little can be inferred from this data analysis about the existence or nonexistence of
13 an acute relationship between O3 and Vancouver hospital visits for respiratory causes. Ponka
14 (1991) showed significant O3 associations with asthma hospital admissions in Helsinki over a
15 3-year period. The model also included temperature, but did not directly address noted long-
16 wave variations in both admissions and pollution. Thus, whether a study has adequately
17 addressed statistical confounding by the prominent long-wave cycles in respiratory hospital
18 admissions series, which are clearly dominated by other causes (e.g., the spring pollen, fall
19 respiratory infection, and winter influenza seasons), continues to be a crucial criterion in
20 evaluating the usefulness of a study's results.
21 Fortunately, there are also a number of new studies that have addressed both long-wave
22 and day-of-week influences in their analyses. Cody et al. (1992) did not directly control for
23 seasonality, but they did narrow their analysis of central New Jersey hospital ER visits to the
24 high O3 season (May through August). Even so, their initial correlational analysis yielded
25 negative associations between hospital visits and both temperature and O3, which suggests
26 that within season long-wave effects existed (e.g., generally higher asthma visits in May, at
27 the end of the pollen season, when O3 and temperature are lower on average than in July or
28 August). However, the authors did conduct subsequent regressions of respiratory visits on
29 both temperature and O3 simultaneously, yielding a significant positive coefficient for O3 and
30 a negative coefficient for temperature, which suggests that the inclusion of temperature may
31 have indirectly accounted for the long wave cycle, allowing the positive short wave O3-visit
December 1993 7-166 DRAFT-DO NOT QUOTE OR CITE
-------
1 relationship to be seen. Day of week influences were considered, but found to be
2 unimportant for these emergency room visit data. No such pollution-hospital visit
3 relationship was found for finger cut (i.e., control disease) visits.
4 Thurston et al. (1992b) analyzed unscheduled (emergency) admissions to acute care
5 hospitals in three New York State metropolitan areas during the summers of 1988 and 1989.
6 Environmental variables considered included daily 1-h maximum O3 and 24-h average SO4
7 and acid aerosol (H+) concentrations, as well as daily maximum temperature recorded at
8 central sites in each community. Long-wave periodicities in the data were reduced by
9 selecting a June through August study period. However, because of remaining within-season
10 long-wave cycles in the data series (i.e., day-to-day fluctuations superimposed on an annual
11 cycle in admissions), they were prefiltered using sine and cosine waves with annual
12 periodicities. Day of week effects were also controlled via regression. These adjustments
13 resulted in nonsignificant autocorrelations in the data series and also improved the pollution
14 correlations with admissions. For example, in New York City, the same day O3-asthma
15 correlation rose from a nonsignificant r = 0.04 in the raw data to a significant
16 r = 0.24 after prefiltering. This shows the importance of addressing long-wave cycles in
17 such data, even when these data come from within a single season. In contrast, correlations
18 between the pollution data and hospital admissions for nonrespiratory control diseases were
19 nonsignificant both before and after prefiltering. The strongest O3-respiratory admissions
20 associations were found during the high pollution 1988 summer and in the most urbanized
21 communities considered (i.e., Buffalo and New York City). After controlling for
22 temperature effects via simultaneous regression, the summer haze pollutants (i.e., 804",
23 H , and O3) remained significantly related to total respiratory and asthma admissions.
24 However, these pollutants' high intercorrelation prevented the clear discrimination of a single
25 pollutant as the causal agent. Depending on the index pollutant, the admission category, and
26 the city considered, it was found that summer haze pollutants accounted for roughly 5 to
27 20% of June through August total respiratory and asthma admissions, on average, and that
28 these admissions increased approximately 30% above average on the highest pollution days.
29 Based on these models' regression coefficients, daily respiratory admissions were estimated
30 to rise 1.4 (± 0.5) and 3.1 (± 1.6) admissions per hundred ppb O3 per million persons in
31 New York City and Buffalo, NY, respectively.
December 1993 7-167 DRAFT-DO NOT QUOTE OR CITE
-------
1 Lipfert and Hammerstrom (1992) reanalyzed the Bates and Sizto (1989) hospital
2 admissions data set for 79 acute-care hospitals in southern Ontario, incorporating more
3 elaborate statistical methods and extending the data set through 1985. Long-wave influences
4 were once again reduced by using the short study periods previously employed by Bates and
5 Sizto (e.g., July and August only for summer), as well as by employing prewhitening and
6 autoregressive procedures to the data. Day of week effects were also controlled. In
7 addition, the models were much more extensively specified, including a variety of new
8 meteorological variables which may have caused some confounding with the pollutant
9 variables (e.g., wind speed correlated at r = -0.55 with NO2). Despite the application of
10 prefiltering (which may have inadvertently removed some of the short-wave signal of
11 interest) and possible model overspecification (e.g., the inclusion of wind speed), summer
12 haze pollutants (i.e., O3, SO4, and SO2) were still found to have significant effects on
13 hospital admissions in southern Ontario. In contrast, pollution associations with hospital
14 admissions for accidental causes became nonsignificant in these models. Although air
15 pollution concentrations were generally within U.S. air quality standards, the pollutant mean
16 effect accounted for 19 to 24% of all summer respiratory admissions, though the
17 "responsible" pollutant(s) could not be selected by the authors with certainty.
18 Burnett et al. (1993) also employed the Ontario acute care hospital database to analyze
19 the effects of air pollution on hospital admissions, but their analysis considered all of Ontario
20 and analyzed the data from each individual hospital, rather than aggregating the counts by
21 region. Slow moving temporal cycles, including seasonal and yearly effects, were removed
22 (via an 19-day moving average equivalent high pass filter) and day-of-week effects were
23 controlled prior to the analysis. Poisson regression techniques were employed because of the
24 low daily admission counts at individual hospitals. Ozone displayed a positive association
25 with respiratory admissions in 91 % of the 168 hospitals, and 5% of summertime (May
26 through August) respiratory admissions were attributed to O3. Positive associations were
27 found in all age groups (0 to 1, 2 to 34, 35 to 64, and 65+). A parallel analysis of
28 nonrespiratory admissions showed no such associations, which indicates the association
29 specificity. Ozone was found to be a stronger predictor of admissions than SO4, which
30 accounted for an additional 1 % of summertime respiratory admissions. Temperature had no
31 effect on the pollution-respiratory admission relationship. Based upon the reported relative
December 1993 7-168 DRAFT-DO NOT QUOTE OR CITE
-------
1 risks, this analysis indicates that O3 is associated with 1.3 (± 0.2) respiratory admissions/day
2 per hundred ppb O3 per million persons in the province of Ontario.
3 Thurston et al. (1993) focused their analysis of respiratory hospital admissions in the
4 Toronto metropolitan area during the summers (July through August) of 1986 to 1988, when
5 they directly monitored for strong paniculate acidity (H+) pollution on a daily basis at
6 several sites in that city. Long-wave cycles, and their associated autocorrelations, were
7 removed by first applying an annual periodicity sine-cosine fit to the data (as well as day-of-
8 week dummy variables) and analyzing the resulting residuals. Strong and significant positive
9 associations with asthma and respiratory admissions were found for both O3 and H , and
10 somewhat weaker significant associations with SO4~, PM2 5, PM10, and TSP, as measured
11 at a central site in downtown Toronto. No such associations were found for SO2 or NO2,
12 nor for any pollutant with nonrespiratory control admissions. Temperature was only weakly
13 correlated with respiratory admissions and became nonsignificant when entered in regressions
14 with air pollution indices. Simultaneous regressions and sensitivity analyses indicated that
15 O3 was the summertime haze constituent of greatest importance to respiratory and asthma
16 admissions, though elevated H was suggested as a possible potentiator of this effect.
17 During multipollutant, simultaneous regressions on admissions, O3 was consistently the most
18 significant. Of the particle metrics, only H+ remained statistically significant when entered
19 into the admissions regressions simultaneously with O3. Sensitivity analyses also showed that
20 dropping all days above the current U.S. O3 standard of 0.12 ppm (2 of a total 117 days) did
21 not significantly change the O3 coefficients. The simultaneous O3, H+, and temperature
22 model indicated that 21 ± 8% of all respiratory admissions during the three summers were
23 associated with O3 air pollution, on average, and that admissions rose an estimated
24 37 + 15% above otherwise expected on the highest O3 day (0.159 ppm). Regression results
25 for the three summers also indicated that daily respiratory admissions increased an estimated
26 2.1 ± 0.8 admissions per day per hundred ppb O3 per million people in the city of Toronto.
27 Moreover, despite differing health care systems, the Toronto regression results for the
28 summer of 1988 were remarkably consistent with previously reported results for that same
29 summer in Buffalo, NY, (see Table 7-22).
30 White et al. (1993) abstracted daily emergency room visit records from June through
31 August, 1990, at a large inner city hospital in Atlanta, GA. Daily counts of visits for asthma
December 1993 7-169 DRAFT-DO NOT QUOTE OR CITE
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TABLE 7-22. COMPARISON OF REGRESSIONS OF DAILY SUMMERTIME RESPIRATORY
ADMISSIONS ON POLLUTION AND TEMPERATURE IN TORONTO, ONTARIO,
BUFFALO, NEW YORK, FOR THE SUMMER OF 1988a
5?
1— *
^D
^D
^j
i
h- »
-J
0
1
g
u
1
3
O
City and Year
Toronto
1988 summer
Toronto
1988 summer
Buffalo
1988 summer
Buffalo
1988 summer
Respiratory Admissions
Category
Total Respiratory
(mean = 14. I/day)
Total Asthma
(mean = 9.5/day)
Total Respiratory (mean
= 25.0/day)
Total Asthma
(mean = 7. I/day)
aSee glossary of terms and symbols for abbreviations
Temperature, Pollutant
Model Specification
T(LG2),
T(LG2),
T(LG2),
T(LG2),
T(LG2),
T(LG2),
T(LG2),
T(LG2),
T(LG2),
T(LG2),
T(LG2),
T(LG2),
S04~(LGl)b
H+(LG1)
O3 (LG1)
S04=(LG1)
H+(LGO)
03 (LG1)
S04=(LGO)
H+(LGO)
03 (LG2)
S04=(LG1)
H+(LG1)
03 (LG3)
Pollutant
Regression
Coefficient
(adm//tg/m 710
persons
0.07
0.18
0.011
0.04
0.13
0.007
0.11
0.35
0.015
0.03
0.09
0.006
±
±
±
±
±
±
±
±
±
±
±
±
± SE)
0.03C
0.09d
A
0.005°
0.02d
0.07d
A
0.004C
0.04C
0.12C
J
0.008
0.02d
0.05d
0.002C
Pollutant Mean
Effect (% ± SE)
13.3 ± 5.3
7.7 ± 3.9
26.4 ± 11.8
13.0 ± 6.8
8.1 ± 4.5
25.3 ± 14.9
8.0 ± 2.7
6.4 ± 2.2
18.4 ± 9.9
7.0 ± 3.9
5.6 ± 3.3
23.9 ± 10.1
Max/Mean Pollutant
Relative Risk
(
1.41
1.50
1.34
1.40
1.53
1.32
1.22
1.47
1.25
1.29
1.43
1.25
±
±
±
±
±
±
±
±
±
±
±
±
±
SE)
0.16
0.25
0.15
0.21
0.29
0.19
0.12
0.16
0.09
0.12
0.26
0.14
and acronyms.
bLG = lag between exposure and admission, in days.
cp < 0.01 (one-way
p < 0.05 (one-way
Source: Thurston et
test).
test).
al. (1993).
-------
1 or reactive airway disease by patients 1 to 16 years of age were related to daily levels of 03,
2 SO2, PM10, pollen, and temperature. Seasonality was likely reduced by the study period
3 selection, though no effort was made to address possible within-season long-wave cycles in
4 the data. Day-of-week and temperature effects were controlled as part of a Poisson model
5 employed so as to address the small admission numbers at a single hospital. This model
6 yielded a 1.42 admissions rate ratio (p = 0.057) for the number of asthma visits following
7 days with O3 levels equal to or exceeding a 1-h maximum of 0.11 ppm, which is consistent
8 with the relative risk values reported by Thurston et al. (1992b, 1993). No admissions
9 relationship with O3 was seen below 0.11 ppm, or with 8-h average O3.
10 Schwartz (1993) analyzed O3 and PM10 air pollution relationships with daily hospital
11 admissions of persons 65 years or older in the Detroit, MI metropolitan statistical area during
12 1986 to 1989. Daily counts for pneumonia (mean = 15.7/day), asthma (mean = 0.75/day),
13 and all other chronic obstructive pulmonary diseases (mean = 5.8/day) were regressed on the
14 pollution variables and various seasonal, trend and temperature dummy variables, using
15 Poisson modeling. However, day-of-week effects were not addressed. Ozone was analyzed
16 with respect to both its daily 24 h average and 1 h maximum. Autoregressive analyses and
17 residuals plots indicated no remaining autocorrelation in the model. Both O3 and PM10 were
18 significant in simultaneous pollutant models for pneumonia and COPD, but not asthma
19 (which was ascribed to the low daily counts for this category). These simultaneous
20 coefficients were reportedly similar to those from the single pollutant models, though the
21 correlations of the coefficients were not provided. Dropping all days exceeding the 1 h
22 maximum O3 standard did not change the size of the O3 coefficients, which remained
23 significant (p<0.01). Based on the regression coefficients and data presented, it can be
24 estimated that the mean effect for O3 (11.6%) was double that for PM10 (5.7%) in the
25 pneumonia model, but comparable for COPD (12.2% for O3 vs. 10.2% for PM10). On an
26 absolute scale, these results imply that O3 was associated with 1.9 (±0.2) respiratory
27 admissions by the elderly/day per hundred ppb (as a 1 h max) per million persons in the
28 Detroit metropolitan area. This estimate does not include admissions by persons less than
29 65 years of age (which would likely included higher asthma admissions, for example), so that
30 the total respiratory admissions associated with O3 in the entire population would likely be
31 higher than estimated from this work.
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1 Daily Mortality
2 Past studies of the possible association of O3 (oxidants) with human mortality
3 summarized in prior O3 criteria documents (U.S. Environmental Protection Agency, 1978,
4 1986) were sometimes suggestive of an association, but were each flawed in some way.
5 These studies are included in Table 7-23, with annotation as to the document in which they
6 were reported. Most of these studies considered daily mortality in Los Angeles, CA, during
7 the 1950s and 1960s. Unlike most historical hospital admissions studies, many of these
8 studies did recognize and attempt to control for seasonality in the data series. Notable
9 exceptions are the California Department of Public Health studies (1955, 1956, and 1957),
10 which were further weakened by their qualitative treatment of the air pollution data. The
11 Mills (1957a,b) analyses also employed a questionable exposure assessment method (the
12 Standard Research Institute smog index), which diminishes its usefulness. Massey et al.
13 (1961) reported no significant correlations between community differences in mortality and
14 differences in oxidant levels over time, but compared two communities having very different
15 populations (e.g., age distributions), a likely confounder in such cross-sectional comparisons.
16 Mills (1960), while reporting mortality-oxidant associations and effects (370 respiratory and
17 cardiovascular deaths/year), did not control for potential temperature influences on mortality
18 below 96 °F daily maximum. Hechter and Goldsmith (1961) reanalyzed the Mills (1960)
19 data using a simple sine wave seasonality correction and obtained significant oxidant
20 correlations until they applied an autocorrelation adjustment, which reportedly caused the
21 pollutant-mortality correlations to drop to nonsignificance (results not presented). Biersteker
22 and Evendijk (1976) conducted a t-test of difference analysis of two summers of time series
23 data from Rotterdam during 1974 to 1975. Although significant mortality differences could
24 be seen during 1975 heat-pollution episodes (0.05 < O3 < 0.125 ppm), no significant
25 mortality increase could be seen during the cleaner and cooler summer's episodes
26 (0.05 < O3 < 0.08 ppm). Statistical time series methods to address probable confounding
27 by temperature effects were needed. Overall, the various exposure assessment and statistical
28 analyses weaknesses in the studies reported in previous O3 criteria documents have prevented
29 the drawing of definitive conclusions in those past documents as to whether or not there is a
30 significant association between O3 and human mortality.
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3
TABLE 7-23. DAILY MORTALITY ASSOCIATED WITH EXPOSURE
TO PHOTOCHEMICAL OXIDANT POLLUTION3
•jj
^—'
-J
Concentration(s)
ppm
< 1 .0 peak
(undefined)
<0.38 max
1-h (?)/day
(Not reported)
0.10-0.42
(undefined)
for 1 48 days
of 1949
(Not reported)
0.02-0.37
average of 1-h
daily max from
all Los Angeles
sites
Pollutant Study Description
Oxidant Relationship between daily concentrations of
photochemical oxidants and daily mortality
among residents of Los Angeles County aged
65 years and over the periods August through
November, 1954, and July through
November, 1955.
Oxidant Data extended to include the period from
1956 through the end of 1959.
Oxidant Relationship between daily maximum oxidant
concentrations and daily cardiac and
03 respiratory mortaility in Los Angeles for the
periods 1947 to 1949; August 1953 through
December 1954; and January through
September, 1955.
Oxidant Comparison of daily mortality in two
Los Angeles County areas similar in T, but
with different levels of daily maximum and
mean oxidant levels (KI); SO2 and CO
concentrations were also measured.
Oxidant Daily respiratory and cardiac death counts for
Los Angeles County during 1956 to 1958
related to daily maximum oxidant
concentrations. All days above 96 °F daily
maximum T eliminated from analysis. Each
day's average of daily oxidant maxima was
related to that day's deviation from monthly
mean mortality.
Results and Comments
Heat had a significant effect on mortality; no consistent
association between mortality and high oxidant
concentrations in the absence of high temperature.
However, seasonal trends were not addressed, and
pollution data treatment was qualitative.
Positive relationship between daily maximum oxidant
concentrations and mean daily death rates on high-smog
versus low-smog days. Questionable exposure analysis,
including use of the "SRI smog index".
No significant correlations between differences hi
mortality and differences in pollutant levels. However,
the populations differed in socioeconomic and age
distribution characteristics.
A stratification of the mortality deviations versus oxidant
concentration revealed increasing mortality with
increasing oxidant concentration, even in the cooler
months. The use of deviations addresses data
seasonal! ty. It is estimated that over 300 deaths/year in
Los Angeles are associated with oxidants. However,
the lack of T controls below 96 °F is a major weakness.
Reference
California
Department of
Public Health
(1955b, 1956b,
1957b)
Tucker (1962)
Mills (1957ab,bb)
Massey et al.
(1961)
Mills (1960)
-------
TABLE 7-23 (cont'd). DAILY MORTALITY ASSOCIATED WITH EXPOSURE
TO PHOTOCHEMICAL OXIDANT POLLUTION8
Concentrations)
ppm
Pollutant
Study Description
Results and Comments
Reference
0.05-0.21
monthly
avg
Oxidant Reanalysis of the relationship between daily
maximum oxidant concentrations (KI) and
daily mortality from cardiac and respiratory
diseases in Los Angeles for the years 1956
through 1958.
Used deviations from sine wave fit to reduce seasonal! ty Hechter and
of pollution and mortality, but fit of monthly variations Goldsmith (1961)
was inadequate. Significant correlations found between
pollutants and mortality for cardiorespiratory diseases, but
(poorly documented) autocorrelation adjustments by
authors reportedly reduced these associations to
nonsignificance.
0.003-0.128
max 1-h/day
O3 Relationship between daily mortality and daily Mortality significantly higher during relatively high
1 -h maximum concentrations of 03 in
Rotterdam, The Netherlands during the
months of July and August, 1974 and 1975.
pollution (0.05 < O3 < 0.125) and heat episodes in
1975. However, no significant mortality difference due
to moderate O3 episodes (0.05 < O3 < 0.08) in 1974, in
the absence of high T. Such simplistic analyses of time
series data makes interpretation difficult.
Biersteker and
Evendijk (1976)c
0.02-0.29 03 Total, respiratory, and cardiovascular Frequency domain analysis indicated a significant short Shumway et al.
six site mean mortality in Los Angeles County during 1970 wave O3-mortality association, but this was not (1988)
of daily 1-h to 1979 related to O3, CO, SO2, NO2, HC, investigated further. The filtered (i.e., long-wave) data
max particulate matter, daily max T, and RH. analysis indicated O3 to be a nonsignificant contributor to
Low-pass filter used to eliminate short-wave seasonal variations in mortality.
associations so that only seasonal associations
could be studied.
0.02-0.29 O3 Shumway et al. (1988) 1970 to 1979
six site mean Los Angeles mortality dataset reanalyzed
of daily 1-h using a high-pass filter to allow investigation
max of short-wave (acute) associations with
environmental variables, after removing
seasonably effects. Environmental variables
considered included temperature, relative
humidity, extinction coefficient, carbonaceous
particulate matter, SO2, NO2, CO, and O3.
Filtered environmental and mortality data analyses
demonstrated significant associations between short-term
variations in total mortality and pollution, controlling for
temperature. Day-of-week effects found not to affect the
relationships. Of the pollutants considered, O3 had the
strongest association with total mortality. Similar results
found for cardiovascular deaths, but not for respiratory
deaths (for which only T was significant),
Kinney and
Ozkaynak (1991)
-------
cr
VO
TABLE 7-23 (cont'd). DAILY MORTALITY ASSOCIATED WITH EXPOSURE
TO PHOTOCHEMICAL OXTOANT POLLUTION3
Concentration(s)
ppm
0.000-0.064
24-h avg
^ TN, no
exceedances of
0.12 ppm 1-h
max; in MO,
5 exceedances
with max =
0.15 ppm)
0.00-0.21
daily 1 -h max
See glossary of
Reviewed in U.
Pollutant Study Description
Oj Associations between total daily mortality and
air pollution were investigated in St. Louis,
MO, and Kingston-Harriman, TN, during
September 1985 through August 1986.
Environmental variables considered include
T, RH, PM]0, PM2 5, sulfate, aerosol
acidity, SO2, NO2, and O3.
O3 Associations between total and cause-specific
daily mortality and air pollution were
investigated for New York City during April
through September, 1971 to 1976.
Environmental variables considered included
T, RH, CoH, particulate matter, and O3.
terms and symbols for acronyms and abbreviations.
S. Environmental Protection Agency (1978).
Results and Comments
Statistically significant daily mortality associations were
found with PMjQ, but not with 03. Autocorrelation
removed via season indicators, multiple T/climate
variables, and AR modeling. The nonsignificant O3
coefficient may have been contributed to by the more
conservative autocorrelation removal measures taken,
lower O3 concentrations, and shorter study period,
relative to other recent mortality studies.
Seasonality controlled using high pass filter, as in the
prior Los Angeles analysis. Controlling for T, RH, and
CoH, the previous day's O3 (lag 1) was significant
(p < 0.01) in the total mortality regression. The
elasticity of the resulting C^ regression coefficient is
consistent with the Los Angeles elasticity value.
Reference
Dockery et al.
(1992)
Kinney and
Ozkaynak (1992)
-------
1 Although there have been relatively few O3 mortality studies conducted and published
2 since the last criteria document (U.S. Environmental Protection Agency, 1986), the statistical
3 methods and pollution data employed in these studies have improved, as compared with the
4 older studies discussed above. Shumway et al. (1988) focused on long-wave variations in
5 mortality, finding that O3 was a nonsignificant contributor to seasonal variations in
6 Los Angeles mortality during 1970 to 1979. As might have been expected, temperature was
7 found to be the principal environmental factor influencing seasonal mortality fluctuations.
8 This paper's exploratory frequency domain analysis did indicate a significant short-wave
9 (i.e., cycles on the order of a few days in period) association between O3 and mortality, but
10 this result was not pursued in the subsequent regression analyses.
11 Kinney and Ozkaynak (1991) reanalyzed the 1970 to 1979 Los Angeles County
12 mortality and environmental data set for short-wave pollution-mortality associations using
13 seasonal and day-of-week controls. After prefiltering the environmental and mortality time
14 series using a high-pass filter, significant associations were demonstrated between air
15 pollution and short-wave (acute) variations in total mortality, even after controlling for
16 temperature influences. Day-of-week effects were also accounted for, but were found not to
17 affect pollutant-mortality associations. In the regression models considered, the 1-day lag of
18 O3 concentration gave the strongest pollutant associations with total mortality. This
19 O3 coefficient was statistically separable from the other significant pollutants in the analysis
20 (CO, NO2, and paniculate matter), although these other three pollutants were too
21 intercorrelated to separate from each other. Expressed as an elasticity, the O3 regression
22 coefficient (0.03 ± 0.01 deaths/ppb) indicated that a 1 % increase in O3 concentration was
23 associated with a 0.015% increase in total mortality. This result would imply an O3 mean
24 effect of on the order of 1.5% of total mortality throughout the year (i.e., 830 total
25 deaths/year). Regression results for cardiovascular deaths (average = 87/day) were
26 qualitatively similar to those for total mortality (average = 152/day), but only temperature
27 was significant for respiratory deaths (average = 8/day), probably due to low count number
28 effects for this category (i.e., Poisson models may have been required). Overall, although
29 the Shumway et al. (1988) analysis of these 1970 to 1979 Los Angeles data indicates that
30 disease factors and other pollutants dominate the overall seasonal cycles in mortality in Los
31 Angeles, the Kinney and Ozkaynak (1991) short-wave analysis documents that O3 explained a
December 1993 7-176 DRAFT-DO NOT QUOTE OR CITE
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1 small but statistically significant portion of day-to-day variations in total mortality in that
2 city.
«
3 Dockery et al. (1992) conducted an analysis of total daily human mortality in St. Louis,
4 MO, and Kingston-Harriman, TN, during a 1-year period from September 1985 through
5 August 1986 aimed primarily at assessing the effects of paniculate matter on mortality. One
6 of the strengths of this study is the fact that multiple air pollutants were measured and
7 considered. Thus, as part of the analysis, O3 and other gaseous pollutants were also
8 considered and found to have nonsignificant associations with mortality in these cities. The
9 statistical analysis addressed autocorrelation in the mortality data through the use of multiple
10 climate indices (i.e., daily mean temperature, hot day, cold day, humid day, hot and humid
11 day, season, and interactive terms) and through the incorporation of autoregressive modeling.
12 This approach is more conservative than that employed by Kinney and Ozkaynak (1991) and
13 others, and the lack of a significant O3 coefficient in this analysis may be due in part to the
14 statistical modeling, which may or may not have affected an O3 mortality relationship in the
15 data in the process of conservatively addressing autocorrelation and controlling for
16 temperature (which is usually correlated with O3 over time). Also, the lack of any
17 03 associations with total mortality may in part be due to the relatively low O3 levels found
18 in these particular communities (especially in Kingston-Harriman, where no O3 exceedances
19 occurred) during the study year (maximum 24-h mean O3 <0.065 ppm). Overall, this study
20 did not show an association between O3 and mortality, but this may be a product of the
21 particular methodological and exposure characteristics of this study vis-a-vis the identification
22 of O3 health effects.
23 Kinney and Ozkaynak (1992) analyzed the associations between daily air pollution
24 concentrations and total and cause-specific mortality in New York City, NY, during April
25 through September, 1971 to 1976, in a manner similar to their prior Los Angeles analysis
26 (Kinney and Ozkaynak, 1991). Environmental variables considered included the daily
27 concentrations of SO2, paniculate matter (coefficient of haze [CoH]), O3, temperature,
28 relative humidity, and visibility. Long-wave cycles were removed prior to the analysis using
29 a high-pass filter. Prior to regression analyses, cross correlations between mortality and each
30 environmental variable were used to determine the optimal lag for each variable. A multiple
31 simultaneous regression yielded significant coefficients for 1-day lagged O3 (p = 0.004), and
December 1993 7-177 DRAFT-DO NOT QUOTE OR CITE
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1 same day CoH (p = 0.003), temperature (p = 0.0001), and relative humidity (p = 0.0002).
2 Expressed as an elasticity, the O3 coefficient (0.058 ± 0.03 deaths/ppb) indicated that
3 a 1 % increase above the mean O3 concentration was associated with a 0.019% increase in
4 mortality, which is very close to the value found by these authors for Los Angeles, CA.
5
6 7.4.1.4 Summary and Conclusions
7 Recent epidemiology studies addressing the acute effects of ambient O3 have yielded
8 significant associations with a wide range of health outcomes, including lung function
9 decrements, aggravation of preexisting respiratory disease, and increases in daily hospital
10 admissions and mortality. Individual-level camp and exercise studies clearly indicate that
11 lung function can decrease in an exposure-related manner in response to O3 exposures
12 occurring in ambient air. The combined results of these studies provide useful, quantitative
13 information on the pulmonary effects of ambient O3 exposures. Results from daily life
14 studies, although more difficult to interpret quantitatively due to exposure assessment
15 uncertainties, are qualitatively consistent with camp and exercise studies. There is limited
16 evidence from several studies suggesting that ambient O3-induced lung function decrements
17 may persist for up to 24 h. Results from lung function epidemiology studies are generally
18 consistent with those of human chamber studies. An O3-related worsening of symptoms in
19 selected groups of healthy individuals and adverse changes in symptoms, lung function, and
20 medication use in asthmatics have been observed qualitatively and, to a lesser extent,
21 quantitatively. The relationship is consistent, temporally plausible, and moderately coherent.
22 Subsequent research should add more quantitative characterization of individual exposure and
23 speciation of copollutants and their clinical effects, as well as larger numbers of subjects.
24 Emergency room visit and hospital admission studies considered in this document
25 collectively indicate that, when the major confounders to such analyses are addressed (e.g.,
26 seasonally and day of week effects), consistent associations are seen between acute
27 occurrences of respiratory morbidity and O3 exposure. The evidence is especially strong for
28 hospital admissions, as the association has been seen by numerous researchers at a variety of
29 localities using a wide range of appropriate statistical approaches. Although the absolute size
30 of the effect varied somewhat across localities and statistical approaches, these analyses
31 suggest that, in the summertime (when many other respiratory illness causes have abated),
December 1993 7-178 DRAFT-DO NOT QUOTE OR CITE
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1 O3 air pollution is associated with a substantial portion (on the order of 10 to 20%) of all
2 respiratory hospital visits and admissions. Moreover, certain of these analyses also indicate
3 that, on the highest O3 days, this pollutant's estimated contribution can increase to the point
4 where it is associated with nearly half of all respiratory hospital admissions. Moreover,
5 significant associations are also seen between O3 and hospital visits/admissions below the
6 U.S. standard of 0.12 ppm 1-h daily maximum O3.
7 As was also the case for the O3-hospital admissions time series studies, many of the
8 older O3-mortality studies had methodological or statistical weaknesses that prevented clear
9 conclusions. However, since the previous Criteria Documents, two out of the three relevant
10 new studies (Kinney and Ozkaynak, 1991, 1992) indicated consistent and statistically
11 significant effects by O3 on short-term (acute) human mortality. These studies considered
12 two very different locales (Los Angeles, CA, and New York City, NY), though the statistical
13 methods employed were similar. The one relevant new study that did not show any
14 O3 association (Dockery et al., 1992) employed more conservative climate and
15 autocorrelation control methods and was conducted over a much shorter time period than the
16 other two studies. Also, the two studies that showed O3-mortality associations considered
17 urban areas experiencing 1-h maximum O3 concentrations above 0.15 ppm, whereas the
18 other study areas (eastern Tennessee and St. Louis, MO) did not. Thus, although the
19 analysis of daily series of human mortality and air pollution has yielded small but statistically
20 significant associations with O3, the sensitivity of these associations to statistical modeling
21 methods and to O3 concentration level needs further investigation.
22
23 7.4.2 Chronic Effects of Ozone Exposure
24 7.4.2.1 Introduction
25 At the time of the publication of the previous EPA air quality criteria document (U.S.
26 Environmental Protection Agency, 1986), little useful data were available on the chronic
27 effects of O3 exposure. Table 11-10 of that document summarized the limited number of
28 studies available at that time and concluded "...it is unlikely that any of these studies can be
29 used to develop quantitative exposure-response relationships for ambient oxidant exposures.
30 Further study of well-defined populations over long periods of time is required before any
31 relationship between photochemical oxidants and the progression of chronic diseases can be
December 1993 7_179 DRAFT-DO NOT QUOTE OR CITE
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1 conclusively demonstrated from population studies." The document noted that existing
2 studies failed to demonstrate any consistent relationship between chronic oxidant exposure
3 and changes in pulmonary function, chronic symptoms, chromosomal abnormalities, or
4 chronic disease mortality.
5 The largest study that had been performed at the time of the 1986 criteria document
6 was that of Detels et al. at the University of California at Los Angeles (UCLA) (Detels
7 et al., 1979, 1981; Rokaw et al., 1980). This study employed a population based sample of
8 households in selected communities in the Los Angeles Basin. A standardized interview was
9 administered, and individuals underwent various tests of lung function. Air pollution data
10 were derived from a network of monitoring stations maintained by the South Coast Air
11 Quality Management District (California Air Resources Board). The usefulness of the
12 findings of this study was considered to be limited due to a number of factors: (1) testing in
13 the several study communities occurred at different times over a 4-year period, (2) little data
14 were presented on self-selection (completion rates between 70 to 79%) and migration in and
15 out of the study communities, (3) reproducibility of the pulmonary function measurements
16 could not be demonstrated consistently, (4) mixed ethnicity of the study population,
17 (5) inadequate data on individual exposure and the failure to adjust exposure estimates for
18 migration in and out of the study areas, and (6) the methods employed for the comparisons
19 of health effects.
20 The 1986 criteria document also summarized the first of the Adventist Health Smog
21 (AHSMOG) studies (Hodgkin et al., 1984) on the occurrence of COPD in relation to chronic
22 air pollution exposures. However, the data from this first publication were felt to be of
23 limited value because only symptom data were reported and the exposure assessment was
24 insufficient.
25 The 1992 supplement to the 1986 criteria document (U.S. Environmental Protection
26 Agency, 1992) focused most of its attention on more acute health effects attributed to
27 O3 exposure and provided no new data on chronic effects in humans. The document did
28 summarize important laboratory animal data derived from multiday exposure studies (c.f.,
29 Table 3-5; U.S. Environmental Protection Agency, 1992) that provide a pathologic
30 framework for the occurrence of chronic health effects in humans. Data from the primate
31 studies are particularly useful (Tyler et al., 1988; Reiser et al., 1987; Rao et al., 1985a,b;
December 1993 7-180 DRAFT-DO NOT QUOTE OR CITE
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1 Moffatt et al., 1987; Hyde et al., 1989; Harkema et al., 1987a,b). Respiratory bronchiolar
2 changes (bronchiolitis, cell hypertrophy, and shift to more cuboidal cells), alterations of
3 collagen cross linking and inflammatory cells in the interstitium and airway lumen have been
4 observed with prolonged exposure of monkeys to levels as low as 0.25 ppm for up to 18 mo.
5 Of importance for the pathogenesis of chronic effects is the observation by Tyler et al.
6 (1988) that intermittent exposures were associated with continued injury during nonexposure
7 periods. See Chapter 6, Section 6.2.4 for a more detailed discussion of the laboratory
8 animal data.
9
10 7.4.2.2 Recent Epidemiological Studies of Effects of Chronic Exposure
11 By the very nature of the problem of the establishment of a link between chronic
12 exposure to O3 and the occurrence of chronic health effects, epidemiological studies remain
13 the principal, if not the only, studies from which data can be sought. As has been noted in
14 the 1986 document, principal problems for such studies relate to (1) the specification of
15 individual exposures over the relevant periods of life of the study subjects; (2) the coincident
16 effects of other oxidant species (e.g., NOX and derivative acid species) and other air
17 pollutants (acid aerosols, paniculate species); (3) seasonal effects that relate to pollutant and
18 meteorologic factors that effect specific pulmonary function measurements relevant during the
19 course of longitudinal studies or over studies that utilize multiple cross-sectional samples; and
20 (4) control for effects of factors such as occupational exposures, cigarette smoking, etc.
21 In addition, past epidemiologic studies have not had access to any human histologic
22 specimens in relation to the exposure groups under study nor have specific mechanisms been
23 investigated to explain any of the symptom or functional outcomes observed.
24
25 Histologic and Immunologic Effects
26 Sherwin has presented some provocative preliminary, histologic data that he uses to
27 offer a hypothesis on the importance of pathologic changes in the centriacinar region (CAR)
28 of the lung in relation to chronic pulmonary effects of oxidant air pollution (Sherwin, 1991;
29 Sherwin and Richters, 1991). Only the publication that presents the primary data is reviewed
30 (Sherwin, 1991) because there is some redundancy in the two available publications.
31 Sherwin and Richters obtained lungs from 107 subjects, 15 to 25 years of age, who died of a
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1 sudden death without evidence of overt disease, lived in Los Angeles County, had no autopsy
2 evidence or history of drug use, and had no lung trauma. Abnormalities of the CAR were
3 evaluated by a pathologist who was "blinded" to basic demographic data. Centriacinar
4 region disease was defined as the extension of a respiratory bronchiolitis into the proximal
5 acinar structures (i.e., chronic inflammatory cells and histiocytes into alveolar ducts, sacs
6 and alveoli immediately adjacent to a respiratory bronchiole). The odds ratio for severe
7 CAR disease in subjects who lived in metropolitan Los Angeles versus those who lived in
8 other cities in Los Angeles County was 4.0 (95% confidence limit [CL], 1.4 to 11.3; this
9 author's calculation based on data in Sherwin Tables 2 and 3).
10 Unfortunately, no exposure data (or lifetime residence data) were available for the
11 subjects in the Sherwin study, nor were smoking histories, cotinine results, or occupational
12 histories available. The smoking history data is of critical importance because a respiratory
13 bronchiolitis has been shown to be an early lesion found in the pulmonary airways of young
14 smokers (Niewoehner et al., 1974). Additional problems for this study were the fact that
15 most subjects were of low socioeconomic status and only 10 of the subjects were female.
16 Furthermore, the study is limited by a lack of quantitative morphometry on the lung
17 specimens and by the lack of a control group from an ambient environment with low oxidant
18 pollution. Therefore, although Sherwin and Richter's data are of considerable interest,
19 particularly in relation to the primate exposure data cited previously, they currently are not
20 of value in the determination of appropriate human exposure levels for O3, nor do they even
21 establish the fact that the oxidant environment found in metropolitan Los Angeles, indeed, is
22 responsible for the observed lesions.
23 Zwick et al. (1991) carried out a study of allergic sensitization and cellular immune
24 responses in children from four schools in two Austrian cities. Subjects were median
25 11 years of age. Two years of meteorologic data and continuously measured levels of SO2,
26 NO2, and O3 were available for both cities. Monitors were within 2 km of the study
27 schools, except for one O3 monitor that was 13 km from a school in the "high" 03 area.
28 "Allergic diseases" (rhinitis, conjunctivitis, and asthma), response to prick test antigens, total
29 IgE concentration, number of subjects with IgE > 100 kU/L, and total IgG concentration did
30 not differ between the subjects in the two cities. Adjustment for sex, age, active and passive
31 smoking, and types of cooking and home heating did not alter the results. Children from the
December 1993 7-182 DRAFT-DO NOT QUOTE OR CITE
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1 "high O3" environment had small, but statistically significant, decreases in the absolute and
2 relative numbers of OKT4+ (helper/inducer) T-cells and OKNK+ (natural killer) cells and
3 increases in OKT8+ (suppressor) T-cells. Adjustment for active and passive smoking and
4 recent respiratory illness did not alter the results. The frequency of subjects with a
5 measurable PD2o to histamine also was increased in the high O3 area. No relationship
6 between the T-cell findings and PD20 or any of the other immunologic markers are provided.
7 The Zwick et al. (1991) results are limited by lack of any exposure data and by lack of
8 detail for the O3 and other ambient air pollution data. Except for data on the average
9 percentage time above specific levels of O3, there are no useful data that can be applied to
10 the observations reported. Moreover, the differences observed in the various T-cell subsets
11 were relatively small and of questionable biological significance. There are no analyses that
12 relate the T-cell findings to the clinical and functional data (see Table 7-24) that are reported.
13 Finally, although the communities were said to be similar on all meteorologic and other
14 ambient pollution data, inspection of the author's Table 1 (Zwick et al., 1991) indicates that
15 the mean (averaging time not given) NO2 levels in the "low" O3 community were fourfold
16 greater than those in the "high" O3 community (42 ng/m3 versus 11 jwg/m3). No data on
17 acid species or particulates are provided, although both study cities were free of heavy
18 industry and heavy traffic.
19 Calderon-Garciduenas et al. (1992) have studied chronic exposure to the ambient air of
20 southwestern Metropolitan Mexico City in relation to histologic abnormalities of the nasal
21 mucosa. The exposed group consisted of subjects who spent at least 8 h per day while
22 working at a Naval hospital in southwestern Metropolitan Mexico City. Ninety-two percent
23 of the group lived in same area as the hospital and all had lived in southwestern Metropolitan
24 Mexico City for >2 mo (n = 47). Controls consisted of (1) subjects who lived in Veracruz
25 and who had not left this area over a period of at least 5 years before the onset of the study
26 (n = 12); and (2) new arrivals (<30 days residence in southwestern Metropolitan Mexico
27 City) at the Naval hospital who came from low-O3, "non-polluted" ports (n = 17). Nasal
28 biopsies were obtained for all subjects in May through June, 1990, as were histories on
29 residence, smoking, occupation, allergies, etc. All three groups were matched for age, sex,
30 and occupation. There were no difference in familial allergy history or personal smoking
31 (specific data not given in paper). There was a progressive increase in both nasal symptoms
December 1993 7_183 DRAFT-DO NOT QUOTE OR CITE
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TABLE 7-24. PATHOLOGIC AND IMMUNOLOGIC CHANGES ASSOCIATED WITH CHRONIC OZONE EXPOSURE8
Concentrations(s) Pollutants and
ppm
iiii v 11 uiiiiiciiuti
/ig/m Variables
Study Description
Results and Comments Reference
Not Provided
Not provided,
Los Angeles County,
not further specified
Autopsy study of lungs from sudden
death victims 15 to 25 years whose
residence was Los Angeles County;
examination of lungs for inflammatory
changes in CAR of lungs.
Most severe CAR disease in residents of Sherwin and
metropolitan Los Angeles County versus Richters (1991)
other county areas; data limited by lack
of smoking history, personal exposure
and occupational data; interesting
hypothesis—not useful for standards.
•^J
00
0.095-0.188
time metric
not given
186-368
Study of allergic sensitization and
cellular immune responses in children
median age 11 years in two Austrian
cities, 1989.
Small increases in OKT4 +
(helper/inducer) and OKT8 +
(suppressor) T-cells and small decrease
in natural killer cells in "high" ozone
community; t number of subjects with
measureable PD2Q histamine in "high"
ozone area; no relationship between
T-cell findings and any clinical
immunologic measure, lung function, or
PD2o; meaning of results unclear.
Zwick et al.
(1991)
0.150-0.275
monthly
average
Approx.
294-539
03
Study of nasal histology in persons
living in southwestern Mexico City and
Veracruz; subjects matched on age,
sex, occupation; similar allergy and
smoking histories.
Increased occurrence of nasal dysplasia
in southwestern Mexico City residents,
especially those with more than 5 years
residence; no data on other air
pollutants; data not relevant for U.S.
conditions because Mexico City residents
are exposed to O3 levels between 0.100
and 0.400 ppm for several hours each
day all year long, with relatively few
days below 0.100 ppm.
Calderon-
Garciduenas et al.
(1992)
aSee glossary of terms and symbols for acronyms and abbreviations.
-------
1 and nasal histologic abnormalities in relation to presumed O3 exposure (Veracruz < new
2 arrivals < chronic residents of southwestern Metropolitan Mexico City). The principal
3 histologic lesion was basal cell hyperplasia, with squamous cell metaplasia and mucosal
4 atrophy occurring less frequently. Only 11 % of those with greater than 60 days residence in
5 Southwestern Metropolitan Mexico City showed normal mucosa.
6 Unfortunately, no ambient air data were presented for SO2 or particles, which are said
7 to be low relative to other parts of the city. In addition, because the monthly average
8 maximal O3 concentrations are (and have been since late 1986) well above the current U.S.
9 1-h standard of 120 ppb, the Calderon-Garciduenas et al. (1992) data are of limited value to
10 understanding low ambient O3 exposures. (This conclusion probably applies even if one
11 considers the different concentrations represented by a given parts-per-billion value at
12 different altitudes.) Subjects in southwestern Metropolitan Mexico City are subjected to
13 O3 levels of between 100 and 400 ppb for several hours per day in the winter and spring.
14 Despite the lack of data on other air pollutants and specific exposure data for individual
15 subjects, this study does provide useful evidence to suggest upper respiratory damage as a
16 consequence of prolonged exposure to ambient air containing high levels of O3.
17
18 Pulmonary Function, Respiratory Symptoms, and Chronic Respiratory Disease
19 The Adventist Health Smog Study. Since the publication of the 1986 criteria document
20 and its 1992 supplement (U.S. Environmental Protection Agency, 1986, 1992), a number of
21 studies have been published that attempt to define chronic respiratory system health effects in
22 relationship to ambient O3 concentrations (Hodgkin et al., 1984; Euler et al., 1987, 1988;
23 Abbey et al., 1991a,b) (see Table 7-25). Among these, the series of publications from the
24 AHSMOG study (Hodgkin et al., 1984; Euler et al., 1987, 1988; Abbey et al., 1991a,b) are,
25 perhaps, the most important and the most useful for issues that are related to O3 standards.
26 Therefore, these studies will be discussed first and as a set.
27 The basic population for these studies represents California-resident, Seventh-Day
28 Adventists aged >25 years of age who had lived 11 years or longer (as of August 1976) in
29 either a high-oxidant polluted area (South Coast Air Basin [Los Angeles and vicinity], and a
30 portion of the nearby Southeast Desert Air Basin) and two low-pollution areas (San Francisco
31 and San Diego). This sample was supplemented by an additional group of subjects who met
December 1993 7_185 DRAFT-DO NOT QUOTE OR CITE
-------
I
u>
TABLE 7-25. EFFECTS OF CHRONIC OZONE EXPOSURE ON PULMONARY FUNCTION, RESPIRATORY
SYMPTOMS, AND CHRONIC RESPIRATORY DISEASE3
Concentrations(s)
ppm
Pollutants and
Environmental
Variables
Study Description
Results and Comments
Reference
0.033 median
average annual
hourly value
65
Study of relationship of air pollution to
levels of FVC, FEV1; and PEFR based on
1976-80 supplement to NHANES and data
from EPA SAROAD monitoring system;
subjects 6-24 years of age; exposure
values based on hourly O3 values for
previous 365 days; data for TSP, NO2,
and SO2; and data for important
demographic, smoking, and health
covariates.
Nonlinear relationship between annual
average O3 and function measurements
with threshold at approximately
0.040 ppm; findings limited by inability
to control for multiple pollutants effects,
relatively crude assignment of exposure;
data consistent with effect on forced
flow at O3 levels at or below 0.12 ppm.
Schwartz
(1989)
~J
oo
0.034-0.050
90th percentile
annual mean
1 h daily max.
67-98
1983-1984 cross-sectional study of 2nd-6th
grade students in Ontario and Manitoba,
Canada; data on SO2, NO2, nitrates, and
sulfates; respiratory health, demographic,
smoking, and home cooking fuel data; and
spirometry.
Ontario town had more O3 days
> 0.080 ppm; small decrements (<2%)
in FVC and FEVj were found in the
Ontario town compared to the Manitoba
town; any O3 possible effects were
completely confounded with SO4 effects.
Stern et al.
(1989)
0.024-0.031
Annual mean
1 h daily max.
47-61
1985-1986 cross-sectional study of 3,945
7- to 11-year-old children from five rural
towns in Ontario and five towns in
Saskatchewan, Canada; data on SO2,
sulfates, NO3, NO2, and PM10;
respiratory health multiple covariates;
spirometry including flow at mid lung
volumes.
Ontario towns had higher levels of O3
and SO4 in summer months and for
90 and 99 percentiles of distributions;
90 percentile mean 1-h maxima were
80 ppb versus 47 ppb for Oa and
11.5 /tg/m versus 3.1 /ig/m for SO4;
magnitude of FEV, and FVC effects
was similar to Stern et al. (1989); no
effect for raid volume flows, except for
subjects with asthma; coincidence of
increased O3 and SO4 preclude definite
statements concerning O3 effects.
Stern et al.
(1993)
-------
n>
I
Ui
TABLE 7-25 (cont'd). EFFECTS OF CHRONIC OZONE EXPOSURE ON PULMONARY FUNCTION, RESPIRATORY
SYMPTOMS, AND CHRONIC RESPIRATORY DISEASE3
Concentrations(s)
ppm
/ig/m
Pollutants and
Environmental
Variables
Study Description
Results and Comments
Reference
0.008-0.118
average
hourly
concen-
trations
1974-1979
16-231
Study of chronic respiratory symptoms in
adults with use of 1979 National Health
Interview Survey data and 1974-1979 EPA
SAROAD data; data on respiratory health,
demography, and smoking; and data for
TSP.
Data for only 29% of those eligible
could be used; average hourly
03 concentration over period 1973-1979
associated with report of sinusitis and
hayfever after control for covariates and
TSP; no association with asthma or
emphysema; large amount of data
reduction, lack of adequate exposure
assignment, lack of occupational
exposure histories, and lack of adequate
data on other pollutant make results very
difficult to interpret.
Portney and
Mullahy (1990)
oo
-J
0.015-0.052 29-102
average
HMV
Cross-sectional study of children ages
6-15 years in a community in Austrian alps
divided into three zones based on SC^, NC>2,
and O^; respiratory health, demographic, and
spirometry data.
Only difference in respiratory history
was increased adjusted prevalence of
asthma in zone with highest O3 (6.4%;
0.052 ppm HMV) versus the zones with
lower 63 concentrations (4.8%;
0.015 ppm HMV; 2.7%, 0.026 ppm
HMV); no meaningful differences in
spirometry indices; data limited by use
of single monitoring site for 1,200 km
area; effects of SO2 and NC>2 on asthma
prevalence not well studied.
Schmitzberger
et al. (1993)
-------
TABLE 7-25 (cont'd).
oo
00
EFFECTS OF CHRONIC OZONE EXPOSURE ON PULMONARY FUNCTION, RESPIRATORY
SYMPTOMS, AND CHRONIC RESPIRATORY DISEASE3
Concentrations(s)
3
ppm ^g/m
0.10-0.20 196-392
3-mo mean
daily peak
hourly
values for
Lancaster
and
Glendora,
respectively
0.04-0.07 78-137
mean peak
daily peak
hourly
values 1972-
1981; Long
Beach and
Lancester,
respectively
(Not reported)
Pollutants and
Environmental
Variables Study Description
Oxidants 5 year follow-up of Lancaster and
Glendora, CA, cohorts; from UCLA
population study of CORD restricted to
nonsmoking, non-Hispanic whites,
7-59 years.
Oxidants 5-6 year follow-up of Lancaster and Long
Beach, CA, cohorts from UCLA CORD
study; Long Beach with higher NO2, SO4,
and TSP then Lancaster.
Oxidant Prevalence of respiratory symptoms in
nonsmoking Seventh Day Adventists
Results and Comments
No difference in respiratory symptoms over
follow-up for either community; across all age
groups, slope of Phase III of N2— washout
deteriorated more rapidly in Glendora; in
subjects ^ 14 years of age, more rapid
decrease in spirometric indices in Glendora;
interpretation hampered by large losses to
follow-up, inability to disentangle multiple
pollutant effects.
All reported excess functional decline for
Long Beach likely due to bias in decline
estimates between locations; data not useful
with regard to possible O3 effects.
Slightly increased prevalence of respiratory
symptoms in high pollution area; after
Reference
Detels et al.
(1987)
Detels et al.
(1991)
Hodgkin
etal. (1984)
residing for at least 11 years in high (South
Coast) and low (San Francisco, San Diego)
photochemical air pollution areas of
California; ARB regional air basin
monitoring data for oxidants, NO2, SO2,
CO, TSP, and SO4 from 1973 to 1976.
adjusting for covariables, 15% greater risk for
COPD due to air pollution (not specific to
oxidants); past smokers had greater risk than
never smokers; when past smokers were
excluded, risk factors were similar. In
addition, insufficient exposure assessment and
confounding by environmental conditions limit
the quantitative use of this study.
-------
1-t
VO
u>
TABLE 7-25 (cont'd). EFFECTS OF CHRONIC OZONE EXPOSURE ON PULMONARY, FUNCTION
RESPIRATORY SYMPTOMS, AND CHRONIC RESPIRATORY DISEASE3
Concentrations(s)
ppm
/ig/m
Pollutants and
Environmental
Variables
Study Description
Results and Comments
Reference
Not reported
Oxidant
Cross-sectional analysis of above
populations; uses hours above various
"threshold" values for oxidants, TSP, SO2
based upon California, EPA, and World
Health Organization max. levels; period
covered 1966-1976; data available for
important covariates (sex, occupation,
environmental tobacco smoke, race, age,
education, past smoking).
OX{10) most significantly associated with COPD
after adjustment for covariates; number of hours
above higher thresholds less significant; when TSP,
SO2> and OX(10) entered in same regression,
TSP(200) only pollutant associated with COPD; high
correlation between OX(10), TSP(200), and SO2
(hours more than 4 pphm). Improved exposure
assessment over previous paper; however, no clear
statement possible about effects of oxidants due to
colinearity with TSP and SO2.
Euler et al.
(1988)
-jj
t—*
00
Not reported
03
Same as Abbey et al, (199la) but analysis
applied to COPD severity and a "multi-
pollutant" analysis performed; also
evaluated effect of using data for different
time periods of ambient air monitoring.
Cumulative incidence of COPD symptoms when each Abbey et al.
pollutant entered separately similar to above study;
joint effects of OZ(10) and TSP(200) and mean
concentrations of each pollutant evaluated only for
cumulative asthma incidence; TSP(200) entered
logistic regression in preference to OZ(10) but mean
03 concentration entered in preference to mean TSP;
change in asthma severity associated with mean
O3 concentration (1977-87) and with exceedance
frequency for OZ(10), OZ(12), and TSP(200)
considered separately; findings for asthma severity
similar to cumulative incidence when TSP and
03 evaluated together; in no analysis did TSP and
O3 both remain jointly significant, nor were there any
interactions; data unable unequivocally to disentangle
effects of individual pollutants.
(1993)
See glossary of terms and symbols for acronyms and abbreviations.
-------
1 the 11-year residence requirement but who came from low-exposure rural areas in California,
2 The total, baseline sample (March 1977) comprised 8,572 individuals, of whom
3 7,267 enrolled. From this group, 109 current smokers and 492 subjects who had lived
4 outside of the designated areas for a portion of the previous 11 years were excluded.
5 Detailed respiratory illness and occupational histories were obtained. In these studies,
6 "COPD" refers to "definite chronic bronchitis", "definite emphysema", and "definite asthma"
7 as defined by the study questionnaire. Measures of pulmonary function are not included.
8 Air monitoring data were obtained from the California Air Resources Board (ARE)
9 monitoring system. Ninety-nine percent of the subjects (excluding the rural supplement)
10 lived at a distance from the nearest ARE monitoring site that was considered to provide
11 relatively reliable concentration estimates for the outdoor, ambient environment at their
12 residence. Concentrations at the monitors were interpolated to the centroid of each
2
13 residential zip code from the three nearest monitoring sites with the use of a 1/R
14 interpolation. Subsequent development of exposure indices took account of the improvements
15 in ARE data after 1973. Data were available for total oxidants, O3, TSP, SO2, NO2, carbon
16 monoxide, and SO4 (excluding 1973 to 1975).
17 The initial report from this study was summarized in Table 11-10 of the 1986 Criteria
18 document (U.S. Environmental Protection Agency, 1986). Based upon a multiple logistic
19 regression that adjusted for smoking, occupation, race, sex, age, and education, it was
20 estimated that residence in the South Coast Air Basin conferred a 15 % increase in risk for
21 prevalent COPD. No estimates of exposure were provided and the data were considered to
22 be of limited utility.
23 In their 1988 publication, Euler et al. provided exposure estimates based upon the
24 cumulative number of hours, over 11 years prior to the baseline, that individuals lived in
25 environments at various oxidant thresholds, beginning at 10 pphm (OX[10]) (196 /ig/m ) and
26 the total dosage to which they would be exposed. The estimates in this report did not correct
27 for time spent indoors. When the OX(10) was the only pollutant considered, each 750 h/year
28 increment in exposure was associated with a 20% increase in risk for COPD in a multiple
29 logistic regression analysis that adjusted for effects of occupation, passive exposure to
30 tobacco smoke, personal smoking, sex, age, race, and education (baseline data only).
31 Moreover, the data were compatible with a threshold effect at 10 pphm. However, when
December 1993 7-190 DRAFT-DO NOT QUOTE OR CITE
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1 hours above a TSP concentration of 200 /xg/m3 (TSP[200]) and SO2 concentration of 4 pphm
2 were included in the logistic regression model, only TSP(200) was associated with the
3 occurrence of COPD. No significant interactions were found between the various pollutant
4 thresholds. The authors noted that their failure to control for time spent indoors may have
5 led to an underestimation of the oxidant effect. Moreover, the fact that 74% of the variance
6 of OX(10) was explained by the other pollutants certainly reduced the power of this study to
7 detect an independent effect of oxidants on the occurrence of COPD. The authors also noted
8 the limitations imposed by the cross-sectional nature of the data that were used in this
9 analysis. Thus, on the basis of this study, no clear statement could be made about the
10 chronic respiratory system effects of oxidant exposure.
11 A major improvement in the methods for assessment of exposure was presented in
12 Abbey et al (1991a). Previous exposure estimates were refined by the computation of
13 "excess concentrations" (concentration minus cutoff, summed over all relevant time periods
14 and corrected for missing data). Exposures also were corrected for time spent at work and
15 time away from residence, with estimates provided for the environments where work
16 occurred and for geographic areas away from residence. The quality of the interpolations
17 (in terms of distance of monitor from residence zip codes) also was evaluated and
18 incorporated into the estimates. Adjustments were made for the time spent indoors by
19 individuals. New indices were developed that were based upon O3, rather than on total
20 oxidants. The investigators demonstrated correlation coefficients of 0.98 between monthly
21 mean total oxidants and O3 at concentrations < 12 pphm. (It should be noted that a more
22 appropriate comparison would have been between the mean and the differences of the two
23 measurements.)
24 The above estimates were applied to data that included 6 years of follow-up of the
25 study population (Abbey et al., 1991b). This analysis focused on incident occurrence of
26 obstructive airways disease (AOD—same definition as for COPD above). Incident symptoms
27 of AOD were significantly associated with hours above several TSP thresholds, but not with
28 hours above any O3 threshold. There was a suggestion of an association between hours
29 above 10 pphm O3 (OZ[10]) and the 6-year cumulative incidence of asthma (relative risk
30 [RR] for 500 h/year above OZ[10] = 1.40 [95% CL = 0.90 to 2.34]) and definite bronchitis
31 (RR 1.20 [95% CL = 0.97 to 1.53]). Approximately 43% of the study population
December 1993 7-191 DRAFT-DO NOT QUOTE OR CITE
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1 experienced at least 500 h in excess of the OZ(10) standard. Cumulative incidence estimates
2 were adjusted with the use of Cox proportional hazard models for the same variables noted in
3 the original publication of Hodgkin et al. (1984) as well as the presence of possible
4 symptoms in 1977 and childhood respiratory illness history. None of the analyses included
5 both O3 and TSP thresholds. No data are provided on the details of the subjects available for
6 the prospective analysis and their representativeness versus the entire base population.
7 Therefore, assuming no bias due to selective loss to follow-up, these data are consistent with
8 a small O3 effect and are limited by the same considerations of colinearity and subsequent
9 reduction of power noted above.
10 Another analysis by Abbey et al. (1993) evaluated changes in respiratory symptom
11 severity with the TSP and O3 thresholds noted above. In this analysis, logistic regression,
12 rather than Cox proportional hazard modeling, was used to assess cumulative incidence of
13 components of the COPD/AOD complex; and multiple, linear regression was used to
14 evaluate changes in symptom severity. When O3 was considered by itself, there was a trend
15 toward an increased risk of asthma for a 1,000 h average annual increment in the OZ(10)
16 standard (RR = 2.07, 95% CL = 0.98 to 4.89). In this analysis, there was a suggestion
17 that recent ambient O3 concentrations were more related to cumulative incidence than past
18 concentrations. Change in asthma severity score was significantly associated with the 1977
19 to 1987 average annual exceedance frequency for O3 thresholds of 10 and 12 pphm.
20 No significant effects were found for COPD or bronchitis alone. In contrast to the above
21 study of cumulative incidence, the investigators carried out an analysis in which TSP(200)
22 and OZ(10) were allowed to compete for entry into a model to evaluate asthma cumulative
23 incidence and changes in severity. In the cumulative incidence model that employed
24 exceedance frequencies (number of hours above threshold), TSP(200) entered before OZ(10);
25 when average annual mean concentrations were used, O3 entered before TSP. From this, the
26 authors concluded that both TSP and O3 were relevant to asthma cumulative incidence.
27 In no case did both pollutants remain significant simultaneously hi the same regression.
28 No interactions were observed between TSP and O3 for either metric. A similar result was
29 observed for change in asthma severity. As in previous analyses, there was a high
30 correlation between TSP(200) and OZ(10) exceedance frequencies (0.72) and their respective
31 average annual mean concentrations (0.74).
December 1993 7-192 DRAFT-DO NOT QUOTE OR CITE
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1 The AHSMOG study represents the most extensive effort to date to provide realistic
2 exposure estimates within the constraints of a large, population-based study. Moreover, the
3 exposure estimates for oxidants/O3 have been tied to current standards and have taken into
4 account many of the sources of inaccuracy and imprecision in the assignment of exposures to
5 individuals (short of detailed personal monitoring). As such, they do represent a
6 considerable improvement over all other studies to date. Nonetheless, these data do not
7 provide unequivocal evidence of an effect of O3 on the outcomes that were studied. This
8 largely is due to the difficulty of partitioning effects between O3 and particulates. However,
9 it fairly can be concluded that, if there is an effect of exposure to O3 on the occurrence of
10 the COPD/AOD symptom/disease complex as defined in these studies, such an effect is
11 occurring at exposure levels at or below 0.12 ppm.
12
13 Other Studies. Subsequent to the publication of the 1986 criteria document, two
14 additional publications have emerged from the UCLA study (Detels et al., 1987, 1991). The
15 data presented are derived from the same population bases that were used in previous
16 publications, and they are therefore subject to the same limitations that were cited hi the
17 introduction to this section.
18 In 1987, Detels et al. reported a 5-year follow-up study of white, non-Hispanic subjects
19 from the Lancaster and Glendora study areas. The 12- and 3-mo mean peak hourly total
20 oxidant values from 1972 to 1982 for Lancaster and Glendora were 7 and 10 pphm and
21 11 and 20 pphm, respectively. Only 47 and 58% of subjects, respectively, were retested
22 with both the questionnaire and measures of lung function. Effects of air pollution on the
23 days of testing were evaluated by comparing lung function test results hi a subgroup of
24 individuals who were tested three times at 3- to 4-mo intervals. No effect was observed, but
25 the power to find differences was low. Over the follow-up period, there were no changes in
26 reported respiratory symptoms for either community. In adults (^19 years of age) who
27 never smoked, all spirometric and nitrogen-washout results showed more rapid deterioration
28 in Glendora. Differences were significant only for mid-expiratory flows and for slope of
29 Phase ffl from the nitrogen-washout curve. The effects were greater in females, in whom
30 changes in FEV1 also were significant. In subjects less than 19 years of age, only changes in
December 1993 7.193 DRAFT-DO NOT QUOTE OR CITE
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1 slope of Phase in were significant, although FVC in Glendora females was lower than that
2 observed in Lancaster.
3 The results of this study remain limited by the lack of adequate exposure data and the
4 failure to control for the possible effects of other ambient pollutant differences between the
5 communities. Problems with loss-to-follow-up represent a significant issue, especially for the
6 pulmonary function measurements, given that approximately 50% of the original subjects
7 were not available for repeated testing. Baseline comparability is also of concern because
8 subjects who were retested in Lancaster had a better slope of Phase HI than those not
9 retested. Because this measure most consistently differed between the two study
10 communities, the possibility of selection bias is very real. Overall, these results do not
11 strengthen the usefulness of this study for the attribution of an effect of oxidant exposure on
12 respiratory health.
13 The 1991 report from the UCLA group compared Lancaster with Long Beach, the latter
14 area with relatively high levels of SO2, sulfates, nitrogen oxides, and hydrocarbons as well
15 as increased total oxidant levels (mean daily hourly peak values, 1972 to 1982, 30 pphm
16 versus 110 pphm, respectively) (Detels et al., 1991). As above, the analysis was restricted
17 to non-Hispanic whites who never smoked cigarettes and with 5 years of follow-up. Only
18 47% of the Lancaster cohort and 44% of the Long Beach cohort had pulmonary function
19 retested on two occasions. Over the age range 25 through 59 years, changes in slope of
20 Phase m of the nitrogen-washout curve and most spirometric indices were significantly
21 worse in Long Beach, compared to Lancaster. In subjects under 25 years, there were
22 significant differences in slope of Phase ffl, especially in subjects 7 to 10 years of age.
23 All of the limitations identified for the 1989 report apply to this report as well.
24 Moreover, comparison between the two communities of the interlaboratory differences
25 (mobile laboratory versus UCLA reference laboratory; 3% sample) indicated that average
26 annual decrements in FEVj were exaggerated by -13 mL/year (standard error ±7 mL/year)
27 in Long Beach versus -2 mL/year (±7 mL/year) in Lancaster. Application of this
28 difference to the data in their Table 6A would suggest that the "significant" difference in
29 FEVj for both males and females may be largely, if not entirely, due to bias. Thus, all of
30 the functional difference reported in this study are suspect on this basis alone. This, of
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1 course, ignores any additional biases that may have been due to the large losses to follow-up
2 in both communities.
3 A number of additional studies have addressed data relevant to the chronic effects of
4 O3 on respiratory health (see Table 7-24).
5 Schwartz (1989) evaluated the effect of air pollution on children and young adults ages
6 6 to 24 years with the use of data derived from NHANES (National Health and Nutrition
7 Examination Survey, February 1976 to 1980). All individuals in each census tract were
8 assigned average pollutant values derived from monitors located within 10 miles of the
9 centroid of the census tract. Average hourly values for the 365 days preceding spirometry
10 were used, and an annual average was created for O3 (EPA Storage and Retrieval of
11 Aerometric Data [SAROAD] database). For O3 (chemiluminescence and ultraviolet [UV]
12 spectroscopy), six of the seven hourly readings between 11:00 a.m. and 5:00 p.m. were
13 required to include a day's data. Only 1,005 of the 3,922 (25.6%) of the subjects lived close
14 enough to a monitor to have O3 exposures assigned to them. Data for TSP, NO2, and SO2
15 were assigned to 47.1, 13.6, and 21.2%, respectively. Analyses were restricted to
16 consideration of single pollutants because the author reported that there was insufficient
17 overlap between the locations where data were available for all or any combination of
18 pollutants. Data for a variety of relevant personal and demographic covariates were
19 available. Statistical analyses appropriate to the correlation structure of the data (induced by
20 the sampling design of NHANES) were utilized. There was an nonlinear relationship
21 between the annual hourly average O3 concentration and FVC, FEVl5 and PEFR.
22 A threshold of effect around 0.04 ppm was observed, above which there appeared to be a
23 linear decline in FVC (only data shown graphically). The effect persisted after control for
24 sex, race, age, family income, educational level, chronic respiratory symptoms, and smoking
25 history. Results were little affected by region or use of a 2-year averaging time. Ozone
26 levels above the threshold were significantly associated with an FVC <70%, a result not
27 seen for TSP but observed for NO2.
28 The major limitation of the Schwartz (1989) analysis is the inability to distinguish
29 between the effects of O3, TSP, and NO2 and the choice of only a single metric for
30 O3 (hourly average). Support for the former concern can be seen in the similarity of the
31 effects of NO2 and O3 in the logistic regression analyses, which suggests that the results
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1 could reflect the joint effect of a number of species in a complex oxidant environment. The
2 operating assumption that near-term (1 to 2 years) exposure reasonably reflects a "lifetime"
3 of exposure is highly suspect in the mobile U.S. population. In fact, restriction of the
4 analysis to subjects who still resided in the state in which they were born led to slight
5 reductions of O3 effect, especially for FEV, and PEFR. Despite these limitations, the data
6 do suggest that, for children and young adults, if there is a chronic O3 effect (or, more
7 accurately for these data, a subacute effect) on lung function, it could occur at levels at or
8 below 120 ppb. However, the particular pattern of exposure (peaks, season, etc.) that may
9 be relevant cannot be discerned from these data.
10 In 1989, Stern et al. reported a cross-sectional study, conducted in 1983 to 1984, of the
11 relationship between respiratory health effects of second through sixth grade children in
12 two Canadian communities (one in southern Ontario and one in southern Manitoba). The
13 Ontario region was characterized by low levels of gaseous pollutants (SC^ and NO2) and
14 moderately elevated levels of paniculate sulfate, fine particles, and O3. Frequent episodes of
15 elevated sulfate and O3 concentrations occurred in the summer and early fall. The Manitoba
16 community was not subject to the same pattern of transported air pollutants. Gases and
17 O3 (measured by chemiluminescence) were sampled continuously, and TSP, sulfates, and
18 total nitrates were sampled every sixth day. Fixed monitoring stations were established at
19 the center of each community, and monitoring was carried out from October 1983 to April
20 1984. Ozone measurements in Ontario were derived from sites between 35 to 45 km from
21 the study area. Average annual maximum O3 concentrations were similar in the two
22 communities (0.136 ppm and 0.130 ppm; Ontario and Manitoba, respectively), but the
23 frequency of elevated O3 events (>0.080 ppm, Canadian standard for 1-h max) was more
24 frequent in Ontario (30 days) than in Manitoba (3 days) in 1983. Ninety-two percent of
25 subjects (n = 1,317) provided data from detailed questionnaires, but only 70% (1,010)
26 provided spirometric data (tested in fall and winter months). There were no meaningful
27 differences in the prevalence of all of the respiratory health outcomes studied after
28 adjustment for parental smoking, gas cooking, sex, length of residence, parental education,
29 and past respiratory illness history. Ontario children had a 2% lower FVC (adjusted for age,
30 sex, height, and parental smoking) and a 1.7% lower FEV); both differences were
31 statistically significant. The differences were somewhat greater when children with
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1 underlying respiratory illness or symptoms were excluded from the analyses. These data are
2 very difficult to interpret in relation to O3 due to the marked colinearity between the O3 and
3 sulfate levels in Ontario. Moreover, the differences observed in lung function are very small
4 (an average of 50 mL and 40 mL for FVC and FEV1? respectively) and of questionable
5 importance without further follow-up data on the subjects. Such follow-up data would need
6 to attempt to identify whether the small decrements observed are "across the board" with
7 respect to the overall population or the result of decrements in a susceptible subset of the
8 population, particularly a set of children at the lower end of the pulmonary function
9 distribution. In these later children, small decrements might be associated with adverse
10 respiratory effects as a result of their already lowered (absolute or relative) levels of lung
11 function.
12 Stern and colleagues (Stern et al., 1993) extended the 1983 to 1984 Stern study to
13 10 rural Canadian communities. Five towns in southwestern Ontario and five towns in
14 Saskatchewan were selected and studied between September 1985 and March 1986. Children
15 7 to 11 years of age were studied (n = 3,945) with techniques similar to the previous study.
16 In 1986, SO2, NO2, and O3 were continuously monitored through a 10-site network; one site
17 in each town. Particulates were sampled every 3 days for 24 h in Saskatchewan and every
18 6 days in Ontario. Annual mean 1-h maximum O3 concentrations were slightly higher in
19 Ontario, but the 90th and 99th percentile values were much greater (90th: 80 ppb versus
20 47 ppb, 99th: 115 ppb versus 57 ppb). This was particularly true for the months of June to
21 August. The levels of PM10 and nitrate did not vary between the areas and were well within
22 the Canadian Ambient Air Quality Objectives. Annual mean SO4 levels were 3 to 4 tunes
23 greater in Ontario communities (6.6 /mi/m3 versus 1.9 /*g/m3).
24 The adjusted (age, sex, parental education, gas cooking, parental smoking) prevalence
25 of respiratory symptoms did not differ between the 10 communities. Adjusted (height,
26 weight, plus above adjustment factors) FVC and FEV, averaged 1.7% and 1.3% less,
27 respectively, in the five Ontario towns. No differences were observed for PEFR,
28 FEF25_75%, or Vmax50%. The results did not change when the analysis was restricted to life-
29 long residents or to children without respiratory symptoms. Although not statistically
30 significant, Ontario children with doctor-diagnosed current asthma had FEF25_75% and
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1 Vmax50% levels that were 6.6% and 6.5%, respectively, lower than similar children in
2 Saskatchewan. Overall, the prevalence of asthma was 4% for the entire sample.
3 These results are consistent, in terms of the magnitude of the FEVj and FVC effects,
4 with those of the previous Stern study (Stern et al., 1989). In addition, these data provide
5 suggestive evidence of enhanced effects for children with current asthma. The two major
6 limitations of the study are recognized by the authors: (1) the effects observed cannot be
7 attributed to O3 or to SO4 (or acid) aerosols and could be due to either part of the pollutant
8 mixture or attributed to the combination of the component, and (2) the differences in the
9 mean values that reported do not take into account the variability in the pulmonary function
10 distribution and the variability of responses across the distribution (see above). It would
11 have been helpful, in this latter regard, had the investigators provided data on the distribution
12 of the lung function measurements in the asthmatic subject, in whom there appeared to be a
13 larger effect than in the nonasthmatics.
14 Portney and Mullahy (1990) used the 1979 U.S. National Health Interview Survey and
15 EPA SAROAD data to explore the relationship between O3 and TSP and chronic respiratory
16 disease. Average hourly O3 concentrations from 1974 to 1979 were used; data from 1974 to
17 1979 and data from 1979 alone were evaluated. Individuals were matched to the nearest
18 centroid of the census tract in which they lived in 1979. Individuals were excluded if they
19 lived >20 miles from the nearest monitor. Only 29.3% of the 4,500 adults surveyed who
20 participated in the smoking and respiratory disease supplemental interview and for whom
21 residential data were available could be included. Seven different model specifications
22 (probit analysis) evaluated cumulative (5-year) and 1-year effects of O3 on various
23 respiratory diseases. Hourly average O3 concentrations, but not TSP concentrations, over
24 1974 to 1979 were significantly associated with the report of sinusitis and hay fever after
25 control for smoking, sex, income, race, education, temperature, and stability of residence.
26 In contrast, neither O3 nor TSP were associated with reported asthma and emphysema.
27 An enormous amount of data reduction, the lack of individual exposure data, lack of
28 specification of the age and sex distribution of the study population, lack of data on
29 occupational exposures, the use of a single O3 metric, and the limit formulation of the
30 paniculate data all severely limited the usefulness of these data.
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1 Kilburn et al. (1992) studied the effect of "air pollution" on expiratory flows and vital
2 capacity in Mexican-American children in Los Angeles. In 1984, 556 second and fifth
3 graders were studied, and 251 of these were studied again in 1987. The analytical strategy,
4 the losses to follow-up, and the lack of reasonable exposure data make the data from this
5 study virtually uninterpretable.
6 A study by Castillejos et al. (1992) evaluated the effects of acute exposures to ambient
7 O3 concentrations on pulmonary function and respiratory symptoms. One-hundred and forty-
8 eight 9-year-old children in the southwest part of Mexico City were studied between January
9 and June 1988. Weekly spirometric measurements were made over 10 weeks. Ambient air
10 data were obtained from the monitoring system maintained by Mexican government and
11 included hourly values for temperature, relative humidity, and O3 concentration. Ozone
12 concentration exceeded 120 ppb on 74% of days and "frequently" exceeded 240 ppb.
13 No data are presented for SO2 or particulates, which are said to be low relative to other parts
14 of the city. All subjects had to live within 5 km of a monitoring station. The study
15 demonstrated that levels of FEVj and FEF25_75% were associated with mean hourly O3 levels
16 in the preceding 24, 48, and 168 h. The authors interpreted their data as consistent with an
17 subacute effect of O3 on measures derived from spirometry that may be due to an
18 "inflammatory process". However, this interpretation seems at odds with the statement in
19 the paper that the initial FEVj measurements for the group did not differ from those
20 observed in a comparable age group in the Harvard Six Cities Study who were not exposed
21 to O3 concentrations as high as those reported in this study. If the overall level of
22 pulmonary function of this group does not differ from those children who live in ambient
23 environments with far lower O3 concentrations, the data would suggest that the subacute
24 effects observed are not translated into persistent abnormalities, at least as can be observed
25 with spirometry.
26 In a brief letter to the Lancet, Austrian investigators (Schmitzberger et al., 1992)
27 described a cross-sectional study of the effects of O3 on the respiratory health of
28 1,156 children, ages 6 to 15 years. Pulmonary function in two different areas with differing
29 "annual" O3 concentrations (actual metric on which "annual" based not given) were
30 compared (52 ppb versus 26 ppb). No differences were observed for FVC. All flow
31 measures (FEVj, FEF50, and FEF75) were significantly lower in the children in the
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1 "high"-O3 area. These data are of limited value for a variety of reasons, the most important
2 being lack of individual exposure data, lack of data on other pollutants, the uninterpretable
3 specification of "annual" O3 concentration, lack of data on chronic respiratory illness
4 (especially asthma) and the lack of data on smoking for the teenage members of the subject
5 group.
6 Schmitzberger et al. followed up their preliminary data (Schmitzberger et al., 1992)
7 with a full report of their study that included additional subjects (Schmitzberger et al., 1993)
8 An area in the Austrian Tyrolian alps was studied. Three zones were identified based upon
9 ambient air conditions: (1) Zone 1 was characterized by annual mean SO2 (UV fluorescence)
10 of 20 /xg/m3, monthly mean NO2 (Palmes tubes) of 17 ppb, and annual mean
11 O3 (chemiluminescence) of 15 ppb (max. half-hour mean =102 ppb); (2) Zone 2 was
12 characterized by values of 14 /*g/m3, 13 ppb, and 26 ppb (112 ppb), respectively; and
13 (3) Zone 3 was characterized by 12 /xg/m3, 8 ppb, and 52 ppb (146 ppb), respectively,
14 Children ages 6 to 15 years who lived in the study areas for ^3 years were enrolled.
15 Respiratory health questionnaire data and forced expiratory flows were obtained. Full data
16 were available from 81% of the enrolled subjects. Adjusted (age, sex, environmental
17 tobacco smoke, socioeconomic status, and home heating) levels of FVC and forced flows did
18 not follow the gradient in O3 concentrations. Although Zone 3 differed significantly from
19 Zone 2 on several measures, there were no meaningful differences with Zone 1. Adjusted
20 asthma prevalence was highest in Zone 3 (6.4% versus 4.8 and 2.7% for Zones 1 and 2,
21 respectively). There were no differences for other respiratory symptoms. Although the
22 authors conclude that "residence in the area of elevated O3 increases the risk...of low small
23 airway-related lung function", careful inspection of the data does not support this conclusion.
24 This conclusion is based upon the supposed increased frequency of FEVj of less than 70% in
25 Zone 3 relative to the other zones, although the specific data are not provided. Moreover,
26 the mean levels for all functional measurements are lowest in Zone 1, the zone with the
27 lowest O3 concentrations and the highest SO2 and NO2 concentrations. This study is
28 handicapped by the lack of any information that could be used to access individual exposures.
29 Moreover, only a single monitoring station was employed that was placed at the center of
30 Zone 1, which itself was at the center of the study area (1,200 km2). No information is
31 provided as to how the concentrations of the various pollutants were estimated for Zones 2
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1 and 3. Therefore, these data virtually are of no quantitative value for standard-setting
2 purposes.
3
4 Other Chronic Disease Morbidity and Mortality
5 Only the Adventist Health Study has provided any data on possible O3-related health
6 effects other than those related to the respiratory system or malignant disease of the
7 respiratory system (Abbey et al., 1991b; Mills et al., 1991) (see Table 7-26). The
8 population studied and the assignment of exposures has been presented previously (Hodgkin
9 et al., 1984; Euler et al., 1988; Abbey et al., 1991a).
10 In their initial study based on 6 years of follow-up, Mills et al. (1991) found that for
11 500 h in excess of the O3 10-pphm threshold, there was a relative risk of 2.24 for respiratory
12 cancer incidence after adjustment for a number of factors listed previously. When the TSP
13 200 yug/m and O3 10 pphm thresholds were allowed to compete for entry into a Cox
14 proportional hazards model for respiratory cancer incidence, the O3 threshold entered in
15 preference to TSP. Ozone exposure was not associated with excess respiratory cancer
16 mortality or incidence of nonrespiratory cancer over the 6-year follow-up period.
17 A second chronic disease study from the Adventist population extended the above
18 observations to include myocardial infarction and all-cause mortality (Abbey et al., 1993).
19 Incident chronic respiratory disease also was included in this analysis. Ambient levels of
20 O3 were not associated with incidence of myocardial infarction at any of the threshold indices
21 that were tested. Neither the mean concentration of O3 nor any of the thresholds were
22 associated with incidence of chronic respiratory diseases, as previously defined. However,
23 there was a trend toward an association between 6-year cumulative incidence of asthma and
24 500-h exceedance of the OZ(10) threshold (RR = 1.40, 95% CL = 0.99 to 2.34).
25
26 7.4.2.3 Conclusions
27 The body of data that has accumulated since publication of the previous air quality
28 criteria document for O3 (U.S. Environmental Protection Agency, 1986) provides largely
29 suggestive evidence for health effects of chronic O3 exposure. Most of the studies suffer
30 from one or another of the following limitations: (1) simplistic assignment of exposure in
31 terms of choice of O3 metrics, assignment of exposure, or adequate adjustment for relevant
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I
CT
CD
VO
VO
TABLE 7-26. EFFECTS OF CHRONIC OZONE EXPOSURE ON THE INCIDENCE OF
CARDIOVASCULAR AND MALIGNANT DISEASES
Concentrations(s)
ppm
/ig/m
Pollutants and
Environmental
Variables
Study Description
Results and Comments
Reference
S
Not reported
Hodgkin et al. Hodgkin et al. (1984) and Abbey et al.
(1984) (199la); analysis based upon exceedance
frequencies 1973-1977 and cancer
cumulative incidence 1977-1982.
Exceedance of OZ(10) threshold
borderline associated with respiratory
cancer; no association with mean
concentration; multipollutant analysis with
TSP(200) and OZ(10), only QZ(10)
entered the logistic regression for
respiratory malignancy; TSP(200) was
significant for females for all malignancy;
no association between 03 and of any
measure of cancer mortality; overall
results suggestive of O3 effect on
respiratory cancer morbidity at level of
exposure within range experienced by
large percentage of study population.
Mills et al. (1991)
Not reported
See above
See above
No association between any O3 threshold
and all cause mortality or incidence of
myocardial infarction.
Abbey et al. (1991b)
-------
1 covariates; and (2) lack of ability to isolate effects related to O3 from those of other
2 pollutants, especially the paniculate fraction. The Adventist Health Study has made
3 substantive progress in the problem of the assignment of individual exposures (Abbey et al.,
4 1991a). Unfortunately, the results from this study cannot disentangle the effects of chronic
5 O3 exposure from those due to chronic exposure to the paniculate fraction of ambient
6 pollution. The study also lacks sufficient power to evaluate the possibility of interactions
7 between O3 and paniculate pollution in relation to health effects. Thus, the overall data are
8 not conclusive, but current evidence is suggestive of possible health effects from chronic
9 exposure to O3, and research efforts should continue to look for responses in populations
10 exposed to levels of O3 at or below 0.12 ppm.
11
12
13 7.5 SUMMARY AND CONCLUSIONS
14 7.5.1 Controlled Human Studies of Ozone Exposure
15 7.5.1.1 Effects on Pulmonary Function
16 Healthy Subjects
17 Controlled human O3 exposure studies have provided the strongest and most
18 quantifiable exposure-response data on the health effects of O3. This chapter reviews the
19 results of studies involving healthy, young adult subjects exposed to O3 concentrations
20 ranging from 0.08 to 0.75 ppm O3 while at rest or during CE or IE of varying intensity for
21 periods of up to 8 h. In many of these studies, small sample size and suboptimal
22 experimental design limit the ability to generalize from a small sample to the larger
23 population. Of particular concern in considering studies with small sample sizes is the risk
24 of making a beta (Type H) error, the incorrect conclusion that no difference exists between
25 treatments when comparisons are not significantly different. The likelihood of making a
26 Type H error greatly limits the ability to determine the minimum O3 concentration that
27 results in a significant pulmonary response in the larger population. As a result, the
28 conclusions drawn from many of the studies cited in this chapter may underestimate the
29 presence of responses at low O3 concentrations in healthy, young adults.
30 Results from studies of at-rest exposures to O3 for 2 h in healthy adult subjects have
31 demonstrated decrements in forced expiratory volumes and flows occurring at and above
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1 0.50 ppm O3 (Folinsbee et al., 1978; Horvath et al., 1979). Airway resistance is not clearly
2 affected during at-rest exposure to these O3 concentrations.
3 With moderate IE for 2 h, eliciting a VE of 30 to 50 L/min, decrements in forced
4 expiratory volumes and flows, secondary to decreases in 1C, have been observed in healthy
5 adult subjects at and above 0.30 ppm O3 (Folinsbee et al., 1978; Seal et al., 1993b). With
6 IE (VE ^ 65 L/min), pulmonary symptoms and decrements in forced expiratory volumes
7 and flows are present following 2 h exposures to 0.12 ppm O3 (McDonnell et al., 1983).
8 Symptoms are present and decrements in forced expiratory volumes and flows occur at
9 0.16 to 0.24 ppm O3 following 1 h of continuous heavy exercise (VE = 55 to 90 L/min)
10 (Adams and Schelegle, 1983; Folinsbee et al., 1984; Avol et al., 1984; Gong et al., 1986)
11 and following 2 h intermittent heavy exercise (VE « 65 to 68 L/min) (McDonnell et al.,
12 1983; Kulle et al., 1985; Linn et al., 1986). With longer exposures of 4 to 8 h duration,
13 responses have been observed at lower O3 concentrations and lower ventilation rates. In the
14 range of concentrations between 0.08 and 0.16 ppm, a number of studies using moderate IE
15 and durations between 4 and 8 h have shown significant responses under the following
16 conditions: 0.16 ppm for 4 h of IE at VE « 40 L/min (Folinsbee et al., 1993), 0.08 to
17 0.12 ppm for 6.6 h of IE at VE « 35 to 40 L/min (Folinsbee et al., 1988; Horstman et al.,
18 1990), and 0.12 ppm for 8 h of IE at VE « 40 L/min (Hazucha et al., 1992). Symptom and
19 spirometry responses were increased with increased duration of exposure, increased
20 O3 concentration, and increased total ventilation. Airway resistance is only modestly
21 affected with moderate or even heavy exercise combined with O3 exposure to concentrations
22 as high as 0.50 ppm O3 (Folinsbee et al., 1978; McDonnell et al., 1983; Seal et al., 1993).
23 Increased fB and decreased VT, while maintaining VE, occur with exposure to 0.20 to
24 0.24 ppm O3 when combined with heavy exercise for 1 to 2.5 h (McDonnell et al., 1983;
25 Adams and Schelegle, 1983). Differences in response to a given O3 concentration among
26 individuals have been shown to be reproducible (Gliner et al., 1983; McDonnell et al.,
27 1985b), indicating some individuals are consistently more responsive to O3 than others.
28 Group mean decrements in pulmonary function can be roughly estimated when
29 expressed as a nonlinear function of effective (i.e., exposure) dose of O3, the simple product
30 of O3 concentration, mean ventilation, and exposure duration (Silverman et al., 1976;
31 Folinsbee et al., 1978; Adams et al., 1981). The O3 concentration ([O3]) appears to make a
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1 greater impact on the pulmonary function response than does VE or exposure duration
2 (Folinsbee et al., 1978; Adams et al., 1981) and indeed, Larsen et al. (1991) suggest an
3 exponent of approximately 4/3 for [O3]. Another way of expressing this relationship is that
4 doubling [O3] under any given exposure scenario will have a greater impact on spirometry
5 responses than doubling either VE or exposure duration. However, at any given
6 O3 concentration, the major external determinants of response are VE and exposure duration.
7 Because of the broad range of intersubject variability, and the inability to identify
8 characteristics that influence this variability (other than age), efforts to estimate or model
9 individual responses have so far been fruitless (McDonnell et al., 1993). Nevertheless,
10 prediction of group mean FEVj responses using the variables of [O3], VE, and exposure
11 duration can be successful (Adams et al., 1981; Folinsbee et al., 1978, 1988; Hazucha,
12 1987; Hazucha et al., 1992; Larsen et al., 1991; McDonnell et al., 1993).
13 In acute O3 exposure studies of 3 h or less in duration, the responses observed during
14 and following acute exposure to O3 at concentrations between 0.12 and 0.50 ppm in normal
15 healthy human subjects include decreases in TLC, 1C, FVC, FEVl5 FFJF25_75%, and VT and
16 increases in SR^, fB, and airway responsiveness. Ozone exposure has also been shown to
17 result in the symptoms of cough, PDI, SB, throat irritation, and wheezing. Similar responses
18 are seen with 4- to 8-h exposures in the O3 concentration range between 0.08 and 0.16 ppm.
19 When viewed collectively, these physiological and symptom responses may be separated
20 into four general categories, including (1) symptoms, (2) changes in lung volume or
21 spirometry, (3) changes in airway resistance, and (4) changes in airway responsiveness.
22 These categories are based on the absence of correlation between spirometry responses and
23 change in airway resistance or airway responsiveness. The attenuation by atropine of Raw,
24 but not spirometry, responses supports the notion of independent mechanisms. The
25 attenuation by indomethacin or ibuprofen of spirometry responses but not changes in airway
26 resistance or airway responsiveness also supports this categorization. A bronchodilator,
27 albuterol, given to healthy subjects prior to O3 exposure did not prevent changes in
28 spirometry, symptoms, or airway responsiveness. Symptoms ratings represent reflex
29 responses (e.g., cough) or a perceptual evaluation of consciously appreciated afferent
30 information (e.g., chest tightness, pain on deep inspiration) and it is therefore somewhat
31 difficult to separate these responses from the more objective physiological responses.
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1 However, cough and pain on deep inspiration are related temporally to spirometry and
2 breathing pattern responses (i.e., volume-related changes). In repeated exposure studies,
3 changes in spirometry and breathing pattern become attenuated with the same time course as
4 the changes in symptom responses.
5 Recent multihour O3 exposure studies indicate that spirometry and symptom responses
6 to concentrations as low as 0.08 ppm occur in healthy subjects with exposures lasting 6 to
7 8 h. Prolonged exposures (8 h) at lower Oj concentrations (0.12 ppm) also indicate that
8 there is a plateau of response to O3 (Hazucha et al., 1992). Although suggested in previous
9 studies (Gliner et al., 1983), such a plateau is difficult to verify with the typical duration of
10 less than 2 h and the large responses seen with higher concentrations. The level of the
11 response plateau (i.e., the spirometry decrement at which the response no longer changes)
12 must be dependant upon the dose rate of exposure (i.e., the product of [03] and VE) because
13 the magnitude of response at a higher dose rate may greatly exceed the response plateau seen
14 at a lower dose rate. Prolonged exposure studies also suggest that O3-induced spirometry
15 responses depend upon the immediate exposure history. With relatively low dose rates (e.g.,
16 Hazucha et al., 1992), responses to exposure that occurred 2 to 4 h previously may influence
17 the current response. The cumulative effect of exposures has not been studied at higher dose
18 rates, but greater persistence of effects may be expected based on the longer recovery period
19 at higher doses rates.
20 Recovery from O3 exposure has not been systematically investigated in a large group of
21 subjects, but available information indicates that an initial phase of recovery proceeds
22 relatively rapidly and some 40 to 65 % of the acute response appears to be recovered within
23 about 2 h (Folinsbee and Hazucha, 1989). However, there is some indication that the
24 spirometric responses, at least to higher O3 concentrations, are not fully recovered within
25 24 h (Folinsbee and Horvath, 1986; Folinsbee et al., 1993a). Collectively, these
26 observations suggest that there is a rapid recovery of O3 induced spirometry and symptom
27 responses, which may occur during resting exposure to O3 (Folinsbee et al., 1977b) or as
28 O3 concentration is reduced during exposure (Hazucha et al., 1992), and a slower phase,
29 which in some cases may take at least 24 h to complete. Repeated exposure studies at higher
30 concentrations typically show that the response to O3 is enhanced on the second of several
31 days of exposure. This enhanced response suggests a residual effect of the previous
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1 exposure, about 22 h earlier, even though the preexposure spirometry may be the same as on
2 the previous day. The absence of the enhanced response with repeated exposure at lower
3 O3 concentrations may be the result of a more complete recovery and/or less damage to
4 pulmonary tissues.
5 Studies of repeated daily exposure to O3 have shown that O3-induced changes in
6 spirometry, symptoms, airway resistance, airway responsiveness, and airway inflammation
7 are attenuated with repetitive exposure. At higher dose rates, symptom and spirometry
8 responses may be enhanced on the second exposure. Attenuation of response within 3 to
9 5 days is a consistent finding in repeated exposure studies, regardless of O3 exposure dose
10 rate, although attenuation of response occurs after fewer exposures at the lower dose rates.
11 The attenuation of response appears to occur more rapidly in less responsive individuals
12 (Horvath et al., 1981) or in responsive subjects exposed to lower O3 dose rates (Folinsbee
13 et al., 1979, 1993b). Loss of attenuation is relatively rapid with responsiveness being
14 partially restored within 4 to 7 days (Kulle et al., 1982; Linn et al., 1982) and normal
15 responsiveness within 1 to 2 weeks after a series of 4 or 5 daily O3 exposures. The
16 attenuation of airway responsiveness may occur somewhat more slowly than for symptom
17 and spirometry responses. Airway inflammation also appears to attenuate, but less
18 completely than the spirometry responses and with a more gradual recovery (Devlin et al.,
19 1993b; Folinsbee et al., 1993b). Some markers of inflammation (e.g., LDH and elastase)
20 have not demonstrated attenuation.
21 The mechanisms leading to the observed pulmonary responses induced by O3 are
22 beginning to be better understood. The available descriptive data suggest several possible
23 mechanisms, some leading to alterations in lung volumes, symptoms, and exercise breathing
24 pattern, and others leading to increases in central and peripheral airway resistance. These
25 mechanisms appear to involve (1) O3 reactions with the airway lining fluid and/or epithelial
26 cell membranes; (2) local tissue responses, including injury and inflammation; and
27 (3) stimulation of neural afferents (bronchial C fibers) and the resulting reflex responses and
28 symptoms. More studies need to be conducted to determine how each event in this cascade
29 contributes to the pulmonary responses induced by acute ozone inhalation in human subjects.
30
31
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1 Subjects with Preexisting Disease
2 Of the subpopulations studied, those with preexisting limitations in pulmonary function
3 and exercise capacity are of primary concern in evaluating the health effects of 03. Studies
4 on the acute effects of O3 inhalation in subjects with various preexisting diseases were
5 reviewed. Inherent in these studies are several limitations that, at present, hamper our ability
6 to make a definitive conclusions regarding the relative O3 responsiveness of subjects studied.
7 Furthermore, it is necessary to clarify whether their responses are representative of the larger
8 population with preexisting disease. These limitations include subject selection, the absence
9 of standardized methods of characterization, and the limited range of exposure doses utilized.
10 These limitations are evident in studies on subjects with COPD, chronic bronchitis, and
11 ischemic heart disease. For patients with COPD performing light to moderate IE, no
12 decrements in pulmonary function were observed for 1- and 2-h exposures to
13 O3 concentrations of 0.30 ppm and less (Linn et al., 1982a, 1983a; Solic et al., 1982; Kehrl
14 et al., 1985) and only small decreases in forced expiratory volume were observed for
15 3 h exposures of chronic bronchitics to 0.41 ppm O3 (Kulle et al., 1984). Small decreases in
16 arterial blood oxygen saturation have also been observed in some of these studies, but the
17 interpretation of these results and their clinical significance is uncertain.
18 Similar limitations also apply to the early studies examining O3 effects in adult and
19 adolescent asthmatics. Decrements in pulmonary function were not observed for adult
20 asthmatics exposed for 2 h at rest (Silverman, 1979) or with intermittent light exercise (Linn
21 et al., 1978) to O3 concentrations of 0.25 and less. Similarly, no significant changes in
22 pulmonary function or symptoms were found in adolescent asthmatics exposed for 1 h at rest
23 to 0.12 ppm O3 (Koenig et al., 1985) and in adolescent asthmatics and nonasthmatics
24 exposed to 0.12 and 0.18 ppm O3 with intermittent moderate exercise up to 1 h (Koenig
25 et al., 1987, 1988), although a small decrease in forced expiratory flow at 50% of FVC was
26 observed in asthmatics after exposure to 0.12 ppm O3. More recent observations by Kreit
27 et al. (1989), Eschenbacher et al. (1989), and Linn et al. (1993) suggest that mild to
28 moderate asthmatics are at least as sensitive to the acute effects of O3 inhalation as healthy
29 subjects when they are exposed to O3 under conditions that elicit a significant response in
30 healthy subjects. Kreit et al. (1989) and Eschenbacher et al. (1989) exposed adult asthmatic
31 and nonasthmatic subjects to 0.40 ppm O3 with intermittent moderate exercise for 2 h and
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1 observed a greater response in airway resistance, FEVj, and FEF25_75% in the asthmatic
2 subjects, although changes in FVC and symptoms were similar in both groups. Ozone
3 exposure also resulted in a marked increase in airway responsiveness to methacholine in both
4 the asthmatic and nonasthmatic subjects. These responses take on greater importance when it
5 is considered that the observed O3-induced pulmonary effects were superimposed upon
6 preexisting impairment of pulmonary function and airway responsiveness. In addition, the
7 observations of Koenig et al. (1990) and Molfino et al. (1991) suggest the possibility that
8 acute exposure to O3 at doses that do not produce measurable pulmonary function decrements
9 may increase the responsiveness of asthmatics to inhaled SO2 or antigens.
10
11 7.5.1.2 Effects on Exercise Performance
12 Endurance exercise performance and VO2max may be limited by acute exposure to
13 O3 (Adams and Schelegle, 1983; Schelegle and Adams, 1986; Gong et al., 1986; Foxcroft
14 and Adams, 1986; Folinsbee et al., 1977b; Under et al., 1988). Gong et al. (1986) and
15 Schelegle and Adams (1986) found that significant reductions in maximal endurance exercise
16 performance may occur in well conditioned athletes while performing continuous exercise
17 (VE > 80 L/min) for 1 h at O3 concentrations >0.18 ppm. Whereas, data from Linder
18 et al. (1988) suggest that small decrements in maximal exercise performance may occur at
19 O3 concentrations less than 0.18 ppm. The mechanisms that lead to these responses and the
20 minimum O3 concentration at which these effects occur have not yet been clearly defined.
21 Reports from studies of exposure to O3 during high-intensity exercise indicate that breathing
22 discomfort associated with maximal ventilation may be an important factor in limiting
23 exercise performance. However, these studies do not exclude the possibility that some as yet
24 undefined physiological mechanism may limit exercise performance.
25
26 7.5.1.3 Effects on Airway Responsiveness
27 Ozone exposure causes an increase in nonspecific airway responsiveness as indicated by
28 a reduction in the concentration of methacholine or histamine required to produce a given
29 reduction in FEVj or increase in SRaw. Increased airway responsiveness is an important
30 consequence of exposure to O3 because its presence means that the airways are predisposed
31 to narrowing upon inhalation of a variety of stimuli (e.g., specific allergens, SO2, cold air).
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1 Markedly increased airway responsiveness is a classical feature of asthma and may also be
2 present with other respiratory diseases (e.g., chronic bronchitis and acute viral infections)
3 and even in a sizeable percentage of the healthy asymptomatic population. Many studies
4 have demonstrated O3-induced increases in nonspecific airway responsiveness in healthy
5 subjects after 1 to 2 h exposure to concentrations in the range of 0.2 to 0.6 ppm (Golden
6 et al., 1976; Holtzman et al., 1979; Konig et al., 1980; Dimeo et al., 1981; Gong et al.,
7 1986; Folinsbee et al., 1989) and after 6.6 h exposure to concentrations in the range of
8 0.08 to 0.12 ppm (Folinsbee et al., 1988; Horstman et al., 1990). Ozone-induced increases
9 in airway responsiveness tend to resolve within 24 h after exposure but may persist in
10 selected individuals for longer periods (Golden et al., 1978).
11 Ozone exposure of asthmatic subjects, who characteristically have increased airway
12 responsiveness at baseline, can cause further increases in responsiveness (Rreit et al., 1989).
13 The difference in baseline airway responsiveness between healthy and mild asthmatic subjects
14 may be as much as 100-fold, whereas the changes in airway responsiveness induced by
15 O3 are typically two- to fourfold. Similar relative changes in airway responsiveness are seen
16 in asthmatics exposed to O3 despite their markedly different baseline airway responsiveness.
17 One study (Molfino et al., 1991) has been published suggesting an increase in specific (i.e.,
18 allergen-induced) airway reactivity. This response was observed after a 1-h resting exposure
19 of atopic asthmatics to 0.12 ppm O3. One of the important aspects of this observation of
20 increased airway responsiveness after O3 exposure is that this represents a plausible link
21 between ambient O3 exposure and increased hospital admissions for asthma. However,
22 experimental design flaws preclude the use of this study in the determination of a LOEL.
23 Changes in airway responsiveness after O3 exposure appear to be resolved more slowly
24 than changes in FEVj or respiratory symptoms. Furthermore, in studies of repeated
25 exposure to O3, changes in airway responsiveness tend to be somewhat less susceptible to
26 attenuation with consecutive exposures than changes in FEVj (Dimeo et al., 1981; Kulle
27 et al., 1982a; Folinsbee et al., 1993). The question of whether chronic O3 exposure can
28 induce a persistent increase (or decrease) in airways responsiveness has not been adequately
29 studied.
30 Increases in airway responsiveness do not appear to be strongly associated with
31 decrements in lung function or increases in symptoms. A number of observations support
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1 this conclusion. First, asthmatics who have widely different baseline airway responsiveness
2 show similar FEVj changes after O3 exposure to those seen in healthy subjects (Kreit et al.,
3 1989). The changes in airway responsiveness are not correlated with changes in FEVl5
4 although a weak association has been seen with increased airway responsiveness and PGE2
5 levels in BAL fluid (McDonnell et al., 1990).
6 The mechanism of O3-induced increases in airway responsiveness is only partially
7 understood, but it appears to be a consequence of cellular and biochemical changes in airway
8 tissue. Airway inflammation may be temporally associated with the presence of increased
9 airway responsiveness (Holtzman et al., 1983; O'Byrne et al., 1984; Seltzer et al., 1986),
10 but at least one study in rats demonstrated that increased airway responsiveness could be
11 induced by O3 exposure in the absence of an influx of neutrophils (i.e., airway inflammation)
12 into the airway mucosa (Evans et al., 1988). Interestingly, indomethacin treatment prior to
13 exposure in one human study blocked the effect of O3 on FEVj and FVC but not on airway
14 responsiveness (Ying et al., 1990). Because cyclooxygenase inhibitors have not been
15 effective at blocking the O3-induced influx of neutrophils into BAL fluid in other studies
16 (Hazucha, 1991; Kleeberger and Hudak, 1992), this result provides further evidence that
17 inflammation is not required for the induction of increased airway responsiveness.
18
19 7.5.1.4 Inflammation and Host Defense Effects
20 A number of studies clearly show that a single acute exposure (1 to 4 h) of humans to
21 moderate concentrations of O3 (0.2 to 0.6 ppm) while exercising at moderate to heavy levels
22 results in a number of cellular and biochemical changes in the lung, as assessed by
23 measurement of bronchoalveolar lavage constituents (Seltzer et al., 1986; Kehrl et al., 1987,
24 1989; Koren et al., 1989a,b, 1991; Schelegle et al., 1991; McGee et al., 1990; Aris et al.,
25 1993; Devlin et al., 1993b). These exposures result in an inflammatory response
26 characterized by increased numbers of PMNs, increased permeability of the epithelial cells
27 lining the respiratory tract, cell damage, and production of proinflammatory cytokines and
28 prostaglandins. This response can be detected as early as 1 h after exposure (Koren et al.,
29 1991; Schelegle et al., 1991) and persists for at least 18 h (Koren et al., 1989; Aris et al.,
30 1994). The response profile of these mediators is not adequately defined, although it is clear
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1 that the time course of response varies for different mediators and cells (Schelegle et al.,
2 1991, Koren et al., 1989, 1991).
3 A single study (Devlin et al., 1991) provides evidence that many of these changes also
4 occur in humans exposed to near-ambient levels of O3 (0.08 to 0.10 ppm) with moderate
5 exercise for 6.6 h. Decrements in the ability of alveolar macrophages to phagocytose
6 microorganisms were also reported in this study.
7 Ozone also causes inflammatory changes in the nose, as indicated by increased levels of
8 PMNs and albumin, a marker for increased epithelial cell permeability. Increases in tryptase
9 levels immediately after O3 exposure, suggested the release of mast cell products.
10 There appears to be no strong correlation between any of the measured cellular and
11 biochemical changes and changes in lung function measurements, suggesting that different
12 mechanisms may be responsible for these processes. Alternatively, the absence of a
13 correlation may reflect either the temporal misalignment of these measurements, the fact that
14 changes detected in the lavage fluid do not quantitatively reflect events occurring in tissues
15 where functional or symptomatic events originate, or that lavage fluid may not be collected
16 from the same lung region primarily implicated in pulmonary function responses. The idea
17 of different mechanisms is supported by a study in which ibuprofen, a cyclooxygenase
18 inhibitor, blunted the O3-induced decrements in lung function without altering the O3-induced
19 increase in PMNs or epithelial cell permeability, although ibuprofen did change the
20 concentration of a number of mediators, some of which may be related to changes in
21 function (Hazucha et al., 1993).
22 In vitro studies suggest that epithelial cells are the primary target of O3 in the lung and
23 that O3 induces them to produce many of the mediators found in the BAL fluid of humans
24 exposed to O3. Although O3 does not induce alveolar macrophages to produce these
25 compounds in large quantities, it does directly impair their ability to phagocytose and kill
26 microorganisms.
27
28 7.5.1.5 Factors Modifying Responsiveness to Ozone
29 Many variables that at least have potential for influencing response to O3 remain
30 inadequately addressed in the available clinical data. Factors such as smoking status, age,
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1 gender, race or ethnic group, season, and mode of breathing during exposure have been
2 evaluated for their potential influence on responses to O3 exposure.
3 Information derived from O3 exposure of smokers is limited. Some degree of
4 attenuation appears to occur in active smokers (Frampton et al., 1993), which may be
5 reversed following smoking cessation (Emmons and Foster, 1991), but available results
6 should be interpreted with caution. The possibility of age-related differences in response to
7 O3 has been explored to some extent since the publication of the previous O3 criteria
8 document (U.S. Environmental Protection Agency, 1986). Young adults historically have
9 provided the subject population for air pollutant exposure studies. Pulmonary function
10 responsiveness appears to decrease with age, although symptom rates remain similar to
11 young adults (Drechsler-Parks et al., 1987b, 1989, 1990; Bedi et al., 1988; Reisenauer
12 et al., 1988; McDonnell et al., 1993). The limited information available on the responses of
13 children and adolescents to O3 (McDonnell et al., 1985a; Avol et al., 1985a, 1987; Koenig
14 et al., 1987, 1988) does not indicate that children and adolescents are either more or less
15 responsive than young adults. Of the studies that have investigated gender differences in
16 responsiveness to O3, some (Lauritzen and Adams, 1985; Horvath et al., 1986; Adams
17 et al., 1987; Drechsler-Parks et al., 1987a,b; Messineo and Adams, 1990) have suggested
18 that women are more responsive to O3 than men. However, the absence of consistent
19 findings with respect to gender differences indicates that it cannot be concluded that men and
20 women respond differently to O3. Comparison of responses across gender, racial, ethnic,
21 and age groups is complicated by the determination of equivalent exposures. For example,
22 women and children have smaller lungs than adult men. Thus with a given exposure
23 concentration, duration, and ventilation, humans with smaller lungs will presumably receive a
24 large relative intrapulmonary exposure. Some attempts have been made to normalize
25 responses according to BSA or lung capacity (e.g., FVC). The only study in which this
26 factor has been systematically investigated (Messineo and Adams, 1990) found no influence
27 of lung size on the spirometry responses under identical exposure ([Qj], VE, and T)
28 conditions. Three studies (Fox et al., 1993; Gerbase et al., 1993; Seal et al., 1993a) have
29 compared pulmonary function responses of women during different phases of the menstrual
30 cycle, but the results are conflicting. The responses of black and white young adults to
31 various concentrations of O3 have been compared in one study (Seal et al., 1993b). The data
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1 suggested that black males experienced significant decrements in pulmonary function at a
2 lower concentration of O3 than white males, but there were no differences among the
3 responses of white males and black and white females. Thus, the question of ethnic or racial
4 differences in responsiveness to O3 is inadequately answered, and the available results should
5 be interpreted with caution. No new studies are available on the effects of heat stress (i.e.,
6 increased temperature or relative humidity) on O3 responses. One study (Linn et al., 1988)
7 suggests that sensitivity to 03 may be related to seasonal variations in ambient
8 O3 concentrations. This finding needs to be confirmed if seasonal variations in
9 responsiveness are to be considered both in design of future studies and in the risk
10 assessment process. Two studies (Hynes et al., 1988; Adams et al., 1989) have reported that
11 differences in the inhalation route (e.g., oral versus nasal or oronasal) appears to be of
12 negligible importance in the responses of exercising adults to O3 exposure. Studies of
13 O3 uptake in the upper airway (Gerrity et al., 1988) confirm the negligible differences
14 between oral and nasal inhalation (also see Chapter 8). None of these potential influences on
15 O3 responsiveness (age, gender, race, hormonal fluctuations, smoking, seasonal variations in
16 responsiveness, and ambient environmental factors) has been thoroughly investigated.
17 However, the observation that healthy older adults appear to be less responsive to
18 O3 exposure than young adults has been confirmed to the point that it can be considered in
19 risk assessment. Nevertheless, this does not fully address the question of age differences
20 because children and adolescents remain inadequately studied.
21
22 7.5.1.6 Extrapulmonary Effects of Ozone
23 It is still believed that O3 immediately reacts on contact with respiratory system tissue
24 and is not absorbed or transported to extrapulmonary sites to any significant degree.
25 A number of laboratory animal studies reported in the previous chapter (Chapter 6) and early
26 studies on human subjects reported in this chapter suggest that reaction products formed by
27 the interaction of O3 with respiratory system fluids or tissues may produce effects measured
28 outside the respiratory tract—either in the blood, as changes in circulating blood
29 lymphocytes, erythrocytes, or serum, or as changes in the structure or function of other
30 organs, such as the parathyroid, the heart, the liver, and the central nervous system.
31 No extrapulmonary effects have been reported to date in other organ systems of O3-exposed
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1 human subjects, except for limited data indicating that acute (1- to 2-h) exposures with
2 exercise at concentrations ^0.35 ppm O3 caused transient changes in blood cells and plasma.
3 The interpretation of all these effects in regards to potential human health risk at ambient
4 levels of exposure (<0.35 ppm O3) is not clear. However, the demonstration in this chapter
5 of an array of inflammatory mediators and immune modulators released at the airway surface
6 in response to O3 provides a possible mechanism for effects to occur outside of the lung.
7 Additional studies are needed, therefore, in order to determine if there are any significant
8 extrapulmonary effects of O3 exposure and at what levels of exposure they might occur.
9
10 7.5.1.7 Effects of Ozone Mixed with Other Pollutants
11 No significant enhancement of respiratory effects (i.e., more than additive) has been
12 consistently demonstrated for mixtures of O3 with SO2, NO2, H2SO4, HNO3, or paniculate
13 aerosols, or with multiple combinations of these pollutants. There is general agreement
14 among studies of simultaneous exposure of healthy adults and asthmatic adolescents to
15 mixtures of O3 and NO2, SC>2, H2SO4, or HNO3 that pulmonary function responses are not
16 significantly different from those following exposure to O3 alone when compared to studies
17 conducted at the same O3 concentration. Exposure to high PAN concentrations (i.e.,
18 0.30 ppm) combined with O3 has been reported to induce greater pulmonary function
19 responses than exposure to O3 alone (Horvath et al., 1986), but when the PAN concentration
20 is reduced to the ambient range, any additional effect of PAN in the mixture appears to be
21 negligible (Drechsler-Parks et al., 1989).
22 In addition to simultaneous exposures to pollutant mixtures, studies of the responses to
23 O3 exposure either preceded or followed by another pollutant have been performed. To the
24 extent that these exposure sequences mimic real ambient conditions, the results could be
25 useful in the risk assessment process. Koenig et al. (1990) demonstrated that exposure of
26 allergic (and probably asthmatic) adolescents to O3 and then to SO2 resulted in significant
27 pulmonary function decrements not seen with an O3-O3 sequence or filtered air-SO2
28 sequence. These results can also be interpreted in light of the fact that O3 increases
29 nonspecific bronchial responsiveness and that the increased SO2 responses may simply reflect
30 this increased responsiveness. Such responses would be unlikely in nonatopic healthy
31 adolescents. Other studies (Aris et al., 1991; Hazucha et al., 1994) have assessed the
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1 responses to O3 after previous exposure to another pollutant. Aris et al. (1991) found that
2 preexposure to water or HNO3 fog appeared to attenuate responses to O3 whereas Hazucha
3 et al. (1994) observed an increased airway responsiveness after O3 exposure preceded by
4 NO2 exposure relative to O3 exposure alone. These findings are intriguing, but must be
5 replicated before they can be useful for quantitative risk assessment. Much remains unknown
6 with reference to responses to air pollutant mixtures. Only a limited number of pollutant
7 combinations and exposure protocols have been investigated, subject groups are small and
8 may not be representative of the general population. Few studies have included more than
9 two pollutants, and most combinations have been evaluated in single studies. Furthermore,
10 only rarely are endpoints other than pulmonary function and plethysmography measured.
11 Additional studies are, therefore, required to evaluate the relationships between O3 and the
12 complex mix of pollutants found in the ambient environment.
13
14 7.5.2 Field and Epidemiology Studies of Ozone Exposure
15 Individual-level camp and exercise studies provide useful, quantitative information on
16 the exposure-response relationships linking human lung function declines with O3 exposure
17 occurring in ambient air. Their utility derives largely from the reliability with which
18 individual exposures can be estimated using outdoor measurements in studies of these kind.
19 Although it usually has not been possible to isolate O3 exposures from other copollutants
20 (e.g., acid aerosols) and environmental factors (e.g., temperature) in the design of such
21 studies, the available body of evidence now strongly supports a dominant role of O3 in the
22 observed lung function decrements.
23 The most extensive epidemiologic database on pulmonary function responses to ambient
24 O3 comes from camp studies. Six recent key studies from three separate research groups
25 provide a combined database on individual exposure-response relationships for 616 children
26 ranging in age from 7 to 17 years, each with at least 6 sequential measurements of FEVj and
27 previous-hour O3 exposures while attending summer camps (Avol et at., 1990; Higgins
28 et al., 1990; Raizenne et al., 1987; Raizenne et al., 1989; Spektor et al., 1988a; Spektor
29 et al., 1991). When analyzed together using consistent methods, these data yielded an
30 average relationship between FEVj and previous-hour O3 concentration of -0.64 mL/ppb
31 (Kinney et al., 1993). The highest 1-h O3 levels measured in 5 of the 6 studies ranged from
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1 100 to 160 ppb, with one study reporting concentrations as high as 245 ppb. Minimum
2 O3 values ranged from 10 to 60 ppb. Although the regression results noted above were
3 based on 1-h O3 levels, exposure in camp studies usually extended for multiple hours.
4 Because of the high level of correlation between single- and multiple-hour averages in the
5 studies, these results may therefore, represent, to some extent, the influence of multihour
6 exposures. In addition to the camp study results, two key studies involving lung function
7 measurements before and after well-defined exercise events in adults have yielded exposure-
8 response slopes of -0.40 and -1.35 mL/ppb (Spektor et al., 1988b; Selwyn et al., 1985).
9 Ozone concentrations during exercise events of approximately 0.5 h duration ranged from
10 4 to 135 ppb in these studies. Consistent with chamber studies, there is no clear evidence
11 from individual-level studies for a response threshold for the average population effects of
12 O3 on pulmonary function decline. However, as with chamber studies, there is evidence that
13 responsivity varies across individuals. Thus, pulmonary function decline as a function of
14 ambient O3 exposure for an individual may be either greater than or less than the mean
15 responses noted above.
16 Recent results of daily-life studies also support a consistent relationship between
17 ambient O3/oxidant exposure and acute respiratory morbidity in the population. Respiratory
18 symptoms (or exacerbation of asthma) and decrements in PEFR occur with increasing
19 ambient O3, especially in asthmatic children (Lebowitz et al., 1991; Krzyzanowski et al.,
20 1992; Thurston et al., 1992a, 1993b). Concurrent temperature, particulates, H+,
21 aeroallergens, and asthma severity or medication status may also contribute as independent or
22 modifying factors. The aggregate results show greater responses in asthmatic individuals
23 than in nonasthmatics (Lebowitz et al., 1991; Krzyzanowski et al., 1992), indicating that
24 asthmatics constitute a sensitive group in epidemiologic studies of oxidant air pollution.
25 Recent aggregate population time series studies of O3-related health effects provide
26 relevant evidence of acute responses, even below a 1-h maximum of 0.12 ppm. Emergency
27 room visits, hospital admissions, and mortality have all been examined as possible outcomes
28 of exposure to O3. In the case of ER visits, the evidence is limited and somewhat mixed
29 (e.g., Bates et al., 1990; Cody et al., 1992; White et al., 1993), probably due to the fact that
30 ER visits are not a very well defined endpoint because they are dependent upon the patient to
31 decide whether or not to attend the ER. Mortality is, however, a well-defined endpoint
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1 (though cause of death is not nearly so certain), and two new, well-designed studies indicate
2 a significant association between O3 and total morality in both Los Angeles, CA, and
3 New York City, NY, even after controlling for the potentially confounding effects of
4 temperature and paniculate matter (Kinney and Ozkaynak, 1991, 1992). These studies
5 suggest an effect on the order of 0.4 to 0.8 total deaths/day per 100 ppb O3 per million
6 persons. Both cities experienced peak 1-h maximum O3 concentrations above 0.2 ppm
7 during their respective study periods. At lower concentrations, over a shorter time span, and
8 using more conservative statistical methods, a third recent study (Dockery et al., 1992) did
9 not detect a significant O3 association with mortality. The strongest and most consistent
10 evidence of O3 effects, both above and below 0.12 ppm O3, however, is provided by the
11 multiple studies that have been conducted over the last decade on summertime daily hospital
12 admission for respiratory causes in various locales in eastern North America (Bates and
13 Sizto, 1983, 1987, 1989; Thurston et al., 1992b, 1993a,b; Lipfert and Hammerstrom, 1992;
14 Burnett et al., 1993). These studies have consistently shown that O3 air pollution is
15 associated with an increased incidence of admissions, accounting for roughly one to three
16 excess respiratory hospital admissions per hundred ppb O3 per million persons. This
17 association has been shown to remain even after statistically controlling for the possible
18 confounding effects of temperature and copollutants (e.g., H , SO4, and PM10), as well as
19 when considering only concentrations below 0.12 ppm O3. Furthermore, these results imply
20 that O3 air pollution can account for a substantial portion of summertime hospital admissions
21 for respiratory causes on the most polluted days. Overall, the aggregate population time
22 series studies considered in this chapter provide strong evidence that ambient exposures to
23 O3 can cause significant exacerbations of preexisting respiratory disease in the general public
24 at concentrations below 0.12 ppm O3, and suggest that higher O3 levels may also cause a
25 much smaller but statistically significant increase in total daily mortality.
26 Studies of chronic health effects that may relate to long-term exposure to ambient
27 pollutants still have not provided unequivocal evidence in support of respiratory or other
28 health effects that result directly from chronic O3 exposure. However, the aggregate
29 evidence to date suggests that chronic O3 exposure could be responsible for health effects in
30 exposed populations, and research efforts should continue to look for effects at O3 levels
31 <£0.12 ppm.
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1 The most useful set of data has been provided by the AHSMOG studies (Hodgkin
2 et al., 1984; Euler et al., 1987, 1988; Abbey et al., 1991a,b, 1993). These studies have
3 provided the most refined measures of chronic exposure to date (including adjustment for
4 quality of the monitoring data as determined by distance of monitoring sites from subject
5 residences, topography, time spent indoors, and time spent at work). The most consistent
6 effects that can be attributable, in part, to O3 relate to an increase in 10-year cumulative
7 incidence of asthma (KR = 2.07 for each 1,000 h above 10 pphm) and an increase in asthma
8 severity. Unfortunately, for the entire set of studies, the colinearity between O3 and TSP
9 reduces the confidence that effects can be attributed to O3 alone, O3 in combination with the
10 paniculate fraction of ambient pollution, or the combination of the two. Some support for an
11 effect on persons with asthma also can be derived from a recent Canadian study (Stern et al.,
12 1993) that demonstrated non-statistically significant 6.6 and 6.5% reductions in FEF25-75%
13 and Vmax50% for people living in Ontario relative to those in Saskatchewan. Again,
14 however, the effects of O3 are impossible to disentangle from the other contributors such as
15 the acid summer haze that characterizes the United States east of the Mississippi River.
16 Most of the other studies that purport to demonstrate effects that relate to O3 exposure
17 are sufficiently limited in one or another of their design or analysis features to minimize their
18 usefulness for the setting of standards. However, most of the studies are compatible with
19 regard to low-level effects on the upper and lower respiratory tract. In this latter regard, the
20 overall body of studies of health effects that relate to chronic exposure to ambient 0)3 are
21 quite consistent with observations summarized in the previous section.
22 A major issue that virtually has been ignored in epidemiologic studies of chronic
23 O3-related health effects relates to the effect of measurement error (both in terms of ozone
24 exposure and important covariates) on the estimates of the magnitude of the effects that have
25 been attributed to O3. Unfortunately, given current uncertainties about the relevant metrics
26 by which chronic O3 exposure should be evaluated and the lack of an accepted methodology
27 for the assignment of "lifetime" exposures and the highly variable measurement-error
28 structure that is likely to be associated with the important covariates (e.g., cigarette smoking,
29 competing exposures in the work environment, lifetime activity patterns), it is difficult to
30 provide estimates either for the direction or magnitude of any biases that might exist for
31 current estimates of effects on lung function of specific respiratory disease or symptom
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1 outcomes. This issue should be addressed specifically in future studies, with specific
2 attention given as to how data derived from personnel monitoring of O3 exposure can be
3 utilized for the assignment of more long-term exposures.
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1 REFERENCES
2 Abbey, D. E.; Moore, J.; Petersen, F.; Beeson, L. (1991a) Estimating cumulative ambient concentrations of air
3 pollutants: description and precision of methods used for an epidemiological study. Arch. Environ. Health
4 46: 281-287.
5
6 Abbey, D. E.; Mills, P. K.; Petersen, F. F.; Beeson, W. L. (1991b) Long-term ambient concentrations of total
7 suspended particulates and oxidants as related to incidence of chronic disease in California Seventh-Day
8 Adventists. Environ. Health Perspect. 94: 43-50.
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36 Sherwin, R. P. (1991) Air pollution: the pathobiologic issues. J. Toxicol. Clin. Toxicol. 29: 385-400.
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9
10 Spektor, D. M.; Lippmann, M.; Thurston, G. D.; Lioy, P. J.; Stecko, J.; O'Connor, G.; Garshick, E.; Speizer,
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14 Spektor, D. M.; Thurston, G. D.; Mao, J.; He, D.; Hayes, C.; Lippmann, M. (1991) Effects of single- and
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17 Stacy, R. W.; Seal, E., Jr.; Green, J.; House, D. (1982) Pulmonary function in normal humans with exercise
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20 Sterling, T. D.; Phair, J. J.; Pollack, S. V.; Schumsky, D. A.; DeGroot, I. (1966) Urban morbidity and air
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23 Sterling, T. D.; Pollack, S. V.; Phair, J. J. (1967) Urban hospital morbidity and air pollution: a second report.
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26 Stem, B.; Jones, L.; Raizenne, M.; Burnett, R.; Meranger, J. C.; Franklin, C. A. (1989) Respiratory health
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30 Stern, B. R.; Raizenne, M. E.; Burnett, R. T.; Jones, L.; Kearney, J.; Franklin, C. A. (1993) Air pollution and
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34 Sunyer, J.; Anto, J. M.; Murillo, C.; Saez, M. (1991) Effects of urban air pollution on emergency room
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36
37 Superko, H. R.; Adams, W. C.; Daly, P. W. (1984) Effects of ozone inhalation during exercise in selected
38 patients with heart disease. Am. J. Med. 77: 463-470.
39
40 Tepper, J. S.; Costa, D. L.; Lehmann, J. R.; Weber, M. F.; Hatch, G. E. (1989) Unattenuated structural and
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43
44 Thurston, G.; D'Souza, N.; Lippmann, M.; Bartoszek, M.; Fine, J. (1992a) Associations between summer haze
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46
47 Thurston, G. D.; Ito, K.; Kinney, P. L.; Lippmann, M. (1992b) A multi-year study of air pollution and
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7 Am. Rev. Respir. Dis. 147: A633.
8
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15
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18
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21
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28 PB80-124753.
29
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34
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40
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47 Toxicol. Appl. Pharmacol. 94: 374-393.
48
49 Vedal, S.; Schenker, M. B.; Munoz, A.; Samet, J. M.; Batterman, S.; Speizer, F. E. (1987) Daily air pollution
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52 Warren, J. B.; Dalton, N. (1983) A comparison of the bronchodilator and vasopressor effects of exercise levels
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3
4 Weymer, A.; Paulo, M.; Lyness, A.; Gong, H., Jr. (1993) Pre-exposure to ozone does not enhance or produce
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6
7 White, M. C.; Etzel, R. A.; Wilcox, W. D.; Lloyd, C. (1993) Childhood asthma and ozone pollution in Atlanta.
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9
10 Whittemore, A. S.; Korn, E. L. (1980) Asthma and air pollution in the Los Angeles area. Am. J. Public Health
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12
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15
16 Ying, R. L.; Gross, K. B.; Terzo, T. S.; Eschenbacher, W. L. (1990) ladomethacin does not udtiMtthe
17 ozone-induced increase in bronchial responsiveness in human subjects. Am. Rev. Respir. Dis.
18 142: 817-821.
19
20 Young, W. A.; Shaw, D. B.; Bates, D. V. (1964) Effect of low concentrations of ozone on pulmonary function
21 in man. J. Appl. Physiol. 19: 765-768.
22
23 Zwick, H.; Popp, W.; Wagner, C; Reiser, K.; Schmoger, J.; Bock, A.; Herkner, K.; Radunsky, K. (1991)
24 Effects of ozone on the respiratory health, allergic sensitization, and cellular immune system in children.
25 Am. Rev. Respir. Dis. 144: 1075-1079.
26
27
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i 8. EXTRAPOLATION OF ANIMAL TOXICOLOGICAL
2 DATA TO HUMANS
3
4
5 8.1 INTRODUCTION
6 A full evaluation of the health effects of ozone (03) requires an integrated interpretation
7 of human clinical, epidemiological, and animal lexicological studies. Each of these three
8 research approaches has inherent strengths and limitations. Animal toxicological data are
9 valuable because they provide concentration- and duration-response information on a fuller
10 array of effects and exposures than can be studied in humans. However, historically, use of
11 animal toxicological data have been limited because of difficulties in quantitative
12 extrapolation to humans. Recent advances in the state-of-the-art of extrapolation have
13 reduced several uncertainties, as will be discussed in this chapter.
14 Qualitative animal-to-human extrapolation is generally accepted because O3 causes
15 similar types of effects in several animal species, from mouse to nonhuman primate
16 (Chapter 6). Also when similar endpoints (e.g., inflammation and pulmonary function) have
17 been examined in O3-exposed animals and humans, similar effects are observed. However,
18 quantitative extrapolation is the goal and is more controversial. That is, if a certain exposure
19 causes a specific effect in animals, what exposure is likely to cause that same effect in
20 humans? Such an extrapolation requires an integration of dosimetry and species sensitivity.
21 Dosimetry is defined as the dose delivered to a site in the respiratory tract. As can be seen
22 in Section 8.2, substantial information is available on dosimetry in several species, including
23 humans. Dosimetric studies that are referenced in the earlier O3 criteria document (U.S.
24 Environmental Protection Agency, 1986) are only briefly summarized here; newer research is
25 the focus. Species sensitivity, discussed in Section 8.3, refers to the sensitivity of a specific
26 species to the delivered dose. For example, even if the same dose of O3 were delivered to a
27 specific respiratory tract site in rats and humans, differences in species sensitivity to that
28 dose are likely because of variations in defense mechanisms and perhaps other factors.
29 Section 8.3 also provides a more holistic approach to extrapolation by quantitatively
30 comparing exposure-response data obtained in animals and humans. The final Section 8.4 is
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1 intended to draw the forgoing information together, reaching conclusions about the potential
2 for human health effects based on animal studies.
3 Although this chapter focuses on animal-to-human extrapolation, dosimetric studies can
4 also be used to elucidate interpretations of the human studies described in Chapter 7. For
5 example, knowledge of dosimetry in humans as related to age and exercise can enhance
6 understanding of human susceptibility factors.
7
8
9 8.2 OZONE DOSIMETRY
10 8.2.1 Introduction
11 Dosimetry refers to measuring or estimating the quantity or rate of a chemical absorbed
12 by target sites within the respiratory tract. The compound most directly responsible for toxic
13 effects may be the inhaled gas, 03, or its chemical reaction products. Complete
14 identification of the actual toxic agents and their integration into dosimetry is a complex issue
15 which has not been resolved. Thus, most dosimetry investigations are concerned with the
16 dose of the primary inhaled chemical. In this context, a further confounding aspect can be
17 the units of dose (e.g., mass retained per breath, mass retained per breath per body weight,
18 mass retained per breath per respiratory tract surface area). That is, when comparing dose
19 between species, what is the relevant measure of dose? This question has not been
20 answered; units are often dictated by the type of experiment and/or by a choice made by the
21 investigators.
22 Experimental and theoretical (dosimetry modeling) studies are used to obtain
23 information on dose. Experiments have been carried out to obtain direct measurements of
24 absorbed O3 in the total respiratory tract (RT), in the upper respiratory tract (URT; region
25 proximal to the tracheal entrance), and in the lower respiratory tract (LRT; region distal to
26 tracheal entrance); however, experimentally obtaining dosimetry data in smaller regions or
27 locations, such as specific airways or in the centriacinar region (junction of conducting
28 airways and gas exchange region) where lesions due to O3 occur (see Section 6.2.4), is
29 extremely difficult. Nevertheless, experimentation is important for determining dose, making
30 dose comparisons between subpopulations and between different species, assessing
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1 hypotheses and concepts, and in validating mathematical models that can be used to predict
2 dose at specific respiratory tract sites and under more general conditions.
3 Theoretical studies are based on the use of mathematical models developed for the
4 purposes of simulating the uptake and distribution of absorbed gases in the tissues and fluids
5 of the respiratory tract. Because the factors affecting the transport and absorption of gases
6 are general to all mammals, a model that uses appropriate species and/or disease-specific
7 anatomical and ventilatory parameters can be used to describe absorption in the species and
8 in different-sized, aged, or diseased members of the same species. Importantly, models may
9 also be used to make interspecies and intraspecies dose comparisons, to compare and
10 reconcile data from different experiments, to predict dose in conditions not possible or
11 feasible experimentally, and to better understand the processes involved in toxic effects.
12
13 8.2.2 Summary of 1986 Review of Experimental and Theoretical
14 Dosimetry
15 Presented is a summary of the more relevant experimental and theoretical dosimetry
16 studies contained in the previous O3 criteria document (U.S. Environmental Protection
17 Agency, 1986). The reader is referred to this document for completeness.
18 Experiments on the nasopharyngeal removal of O3 in laboratory animals suggested that:
19 (1) the fraction of O3 uptake depends inversely on flow rate (Yokoyoma and Frank, 1972),
20 (2) uptake was greater for nose than mouth breathing (Yokoyoma and Frank, 1972), and
21 (3) tracheal and chamber concentrations were linearly related (Yokoyoma and Frank, 1972;
22 Miller et al., 1979). Only one investigation measured uptake by the LRT, finding 80 to 87%
23 uptake by the LRT of dogs (Yokoyoma and Frank, 1972). At the time, there were no
24 reported results for human URT or LRT uptake. With the exception of two relatively crude
25 studies by Clamann and Bancroft (1959) and Hallett (1965), there were no data on O3 uptake
26 in humans at the time of the earlier Criteria Document (U.S. Environmental Protection
27 Agency, 1986).
28 Several mathematical dosimetry models had been developed to simulate the processes
29 involved in O3 uptake and to predict O3 uptake by various regions and sites within the
30 respiratory tract. The model of Aharonson et al. (1974) had been used to analyze
31 nasopharyngeal uptake data. Applied to O3 data, the model indicated that the average mass
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1 transfer coefficient of this region and the mass retained increased with increasing air flow,
2 but the percent uptake decreased.
3 Models had been developed to simulate LRT uptake (Miller et al., 1978, 1985). The
4 models were very similar in their treatment of O3 in the airways and airspaces and hi their
5 use of morphometric data to define the dimensions of the air compartments and liquid lining.
6 Both of the 1978 and the 1985 models of Miller and co-workers took into account reactions
7 of O3 with constituents of the liquid lining. However, the models of Miller et al. differed in
8 their treatment of chemical reactions in the liquid lining, and the later model included
9 transport and chemical reactions within tissue and blood, whereas the first Miller et al. model
10 did not (an instantaneous reaction at the liquid-tissue interface was assumed, so that the
11 O3 concentration here was defined as zero). In both models, tissue dose was defined as the
12 O3 flux to the liquid-tissue interface. The models of Miller et al. predicted O3 tissue dose to
13 be relatively low in the trachea, to increase to a maximum in or near the centriacinar region,
14 and then to decrease distally. This was characteristic for both the animal and the human
15 simulations (Miller et al., 1978, 1985).
16 Prior to 1986, there were no experimental results that were useful in judging the
17 validity of the modeling efforts. However, a comparison of the results of Miller and
18 co-workers with morphological data (that show the centriacinar region to be most effected by
19 O3; see Section 6.2.4) indicated qualitative agreement between the site of predicted maximum
20 tissue dose and the site of observed maximum morphological damage in the pulmonary
21 region.
22
23 8.2.3 Experimental Ozone Dosimetry Data
24 8.2.3.1 Introduction
25 Models of Oj uptake in the respiratory tract have reached a scale of sophistication that
26 provide some highly specific predictions regarding the location and magnitude of O3 dose.
27 However, before these models can be exploited to their fullest degree in extrapolating dose
28 within and between species, validation of the models with experimental data is essential.
29 This section will review the experimental data base upon which the modeling of
30 O3 dosimetry is both based and validated. This will help facilitate discussion of the models
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1 themselves in subsequent sections. Table 8-1 provides a summary of all post-1986
2 experimental O3 dosimetry studies.
3
4 8.2.3.2 Animal (In Vivo) Ozone Dosimetry Studies
5 The model predictions of Overton et al. (1987) based on the original model of Miller
6 et al. (1985) provided specific predictions about the regional and total uptake efficiencies of
7 O3 in laboratory rats. It was therefore necessary to test these predictions with actual data.
8 The first data on total RT uptake of O3 in rats were obtained by Wiester et al. (1987).
9 Ozone uptake was measured in 30 awake unanesthetized Sprague-Dawley rats receiving a
10 nose-only exposure. A rat was situated within a plethysmograph that could continuously
11 monitor the animal's breathing pattern. Air with O3 flowed by rats' noses at 1,200 mL/min
12 for 1 h at a concentration of 0.3, 0.6, or 1.0 ppm O3. Determination of RT 03 uptake was
13 determined by mass balance. The uptake was the difference in mass in the upstream air and
14 the air downstream of the rat. Total RT O3 uptake efficiency of approximately 40% was
15 measured and was independent of O3 concentration. During data acquisition, the animals had
16 an average tidal volume (VT) of about 2.8 mL, an average breathing frequency (fB) of about
17 150 breaths per minute (bpm), and an average minute ventilation (Vg) of about 400 mL/min.
18 This study was followed by another (Wiester et al., 1988), in which total RT uptake was
19 measured in three strains of rats and in the guinea pig. Specifically, Fischer 344, Sprague-
20 Dawley, Long Evans rats, and Hartley guinea pigs were all exposed for 1 h to 0.3 ppm O3;
21 F-344 rats also received a 0.6 ppm exposure. Uptake was measured as in the previous
22 experiment (Wiester et al., 1987). Total RT uptake of O3 was species-independent and
23 averaged 47%. This was higher than in the previous study because a different calculation
24 method for fractional uptake was used. Wiester et al. (1987) corrected all flows used in
25 uptake calculations for body temperature and relative humidity. In their later work,
26 however, they found that this correction was not warranted, resulting in slightly higher
27 computed O3 uptake efficiency (Wiester et al., 1988).
28 All rats in the Wiester et al. (1988) study had fBs between 112 to 132 bpm, VTs
29 between 2.4 to 2.8 mL, and VEs between 299 and 364 mL/min. The guinea pigs had a
30 VT of 2.4 mL (not different from rats), an fB of 77 bpm, and a VE of 188 mL/min. As in
31 the first experiment, exposure concentration did not affect uptake.
December 1993 8-5 DRAFT-DO NOT QUOTE OR CITE
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TABLE 8-1 (cont'd). EXPERIMENTAL STUDIES ON OZONE DOSIMETRY8
Species
In Vivo/In Vitro (Strain)
Uptake
Breathing Patterns
Results
Reference
In vitro
Pig
Sheep
Trachea
Vjjjj = 50 mL/s to Unidirectional 03 uptake efficiencies of trachea decreased with
200 mL/s increasing flow from 0.5 to 0.15 for the sheep and 0.12 for the pig.
Mass transfer coefficients were generally independent of flow.
Ben-Jebria et al. (1991)
In vitro
N/A
N/A
N/A
Ozonolysis studies on various unsaturated fatty acids, rat
erythrocyte ghost membranes and rat BAL. Dominant processes are
the production of aldehydes and peroxides due to reactions between
03 and olefms.
Pryoretal. (1991)
In vivo
Human
20-200 mL depth VT = 500 mL
into respiratory
tract
Vtas = 250 mL/s
Uptake efficiencies by measuring recovery of 03 boluses delivered
at 20 mL increments into lung to depth of 200 mL. At deepest
depth, only 6% of 03 could be recovered. Ozone uptake by
conducting airways larger than predicted by Miller et al. (1985).
Hu et al. (1992b)
00
In vivo
Human
20-200 mL depth
into respiratory
tract
VT = 500 mL
Vj^ = 150, 250, 500,
750, 1,000 mL/s
Same technique as Hu et al. (1992b), but investigating flow effects.
Increasing flow caused marked shift of delivered Oj toward the
periphery of the conducting airways (i.e. , the greater the inspiratory
flow the greater the amount of 03 delivered to the lung periphery),
where it is available for absorption. Mass transfer coefficients in
upper airways independent of flow, but in conducting airways they
increase proportional to flow. Lung liquid lining mass transfer
coefficient computed to be 1 .4 cm/s in the URT, falling to
0.17 cm/s in the respiratory airways. Reaction rate constant
between 03 and the lung liquid lining was computed as 7.3 X
10 s" in the URT, falling to 8.2 X 10 s" in the distal conducting
airways.
Hu et al. (1993)
In vivo
Human
20-200 mL depth
into respiratory
tract
VT = 500 mL
Vj^ = 250 mL/s
Comparison of 03 bolus uptake between oral and nasal routes.
Nose was found to be 30% more efficient at removing 03 from air
stream than the mouth.
Ben-Jebria et al. (1993)
In vitro
Rat
(Sprague-Dawley)
Lung
VT = 2.71 mL
fB = 50 - 103 bpm
FRC = 4 and 8 mL
Perfused and non-perfused rat lungs ventilated with 1 ppm 03.
Uptake efficiency of lungs dropped from 95% at 50 bpm to about
50% at 103 bpm. No change in uptake efficiency when lungs
inflated from FRC = 4 mL to 8 mL.
Postlethwait et al. (1993)
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1 These data are in disagreement with the model predictions of Overton et al. (1987) who
2 made predictions of O3 uptake in two different rat anatomical lung models (Kliment, 1973
3 versus Yeh et al., 1979). They conducted simulations in both anatomical models, varying
4 fB and VT at fixed VE; they also varied functional residual capacity (FRC) as a fraction of
5 total lung capacity (TLC). The Kliment anatomical lung model gave consistently high
6 predictions for uptake when compared with actual data. The predictions using the Yeh et al.
7 (1979) model came closer. Total RT uptakes (not including the head) for the Yeh et al.
8 (1979) model were predicted to range between 46 and 60% at fB = 154 bpm and
9 VT = 1.25 mL and between 70 and 80% for fB = 81 bpm and VT = 2.4 L. However,
10 when the fact that these predictions do not include the head of the animal is considered, it is
11 evident that the model predictions overestimate the total RT uptake in rats. The question is
12 whether the measurements are accurate or whether there is a problem with the model
13 formulation. The data of Postlethwait et al. (1993) (see below) suggested that the data of
14 Wiester et al. (1987, 1988) may be reasonable. Their data in the excised rat lung suggested
15 a clear inverse dependence of lung uptake on fB. At a VT of 2.51 mL, the O3 uptake
16 efficiency of the excised lung fell from nearly unity at fB = 50 bpm to almost 50% at
17 fB = 103 bpm. For extrapolation purposes, a key question here is what fB should be
18 considered as the normal resting fB of a rat. Although Wiester et al. (1987, 1988) allowed
19 their rats to acclimate to the plethysmograph by monitoring fB and only began uptake
20 measurements after fB had plateaued at a minimum, it is still uncertain whether frequencies
21 of 120 to 150 bpm are reasonable. In a summary of studies of the pulmonary function of
22 rats in response to O3, Tepper et al. (1993) found typical breathing frequencies of 100 bpm.
23 Although the models appear to overestimate the O3 uptake efficiency of the rat respiratory
24 tract, the discrepancy is not large, and the near agreement indicates that the O3 dosimetry
25 models have predictive capability.
26 In addition to data on the total RT O3 uptake efficiency, in vivo data on regional
27 O3 dosimetry in animal models have begun to emerge. Hatch and Aissa (1987) and Aissa
28 and Hatch (1988) first described a method to measure O3 uptake in animals by exposing them
29 nose-only, while in a plethysmograph, to O3 enriched with 18O, a stable isotope of oxygen.
30 After exposure, bronchoalveolar lavage fluid (BAL) and respiratory tract tissue were assayed
1 R
31 for excess O using isotope ratio mass spectrometry. One problem with this technique is
December 1993 g_9 DRAFT-DO NOT QUOTE OR CITE
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1 that all of the absorbed 18O cannot be accounted for. The technique used by Hatch and
_ * O | Q
2 Aissa (1987) involves the detection of excess O in O3 reaction products after tissue
3 pyrolysis. Thus, 18O3 that is degraded to H218O or 18O2 is lost and cannot be detected in
4 dry tissue. Eight male F-344 rats (four previously exposed chronically to O3 for 1 year:
1 C
5 0.06 ppm baseline with 1 h spikes of 0.25 ppm) were exposed for 2 h to 1 ppm of O-
6 enriched O3 (Hatch et al., 1989). During exposure, the rats breathed at 150 bpm, a VT of
7 2.05 mL, and a VE of 290 mL/min. After exposure, the lung, trachea, and head were
8 separately analyzed for O. Overall, the animals took up 54.3% of inspired O3. Although
9 this value of O3 uptake efficiency is higher than that found by Wiester et al. (1987, 1988),
10 considering the fact that the coefficient of variation for O3 uptake efficiency measurements is
11 around 20% in all studies, the result of Hatch and Aissa (1987) is consistent with the data of
12 Wiester et al. (1987, 1988). Of the O3 taken up by the animals, 49.6% was taken up by the
13 head, 6.7% by the larynx/trachea, and 43.6% by the lungs. By assuming equal uptake
14 efficiencies by compartments on inspiration and expiration, inspiratory uptake of O3 by these
15 regions was computed. It was determined that the rat nasopharynx had an inspiratory
16 efficiency of 17.4% and that the larynx/trachea removed 2.7% of the remaining O3.
17 This technique has recently been extended to humans. Hatch et al. (1993) showed that
18 when human subjects were exposed to 0.4 ppm 18O3 while intermittently exercising at VE =
19 60 L/min for 2 h, the amount of recovered 18O in lavagable cells indicated that the humans
20 received 3-4 times the O3 dose than rats exposed to 0.4 ppm for 2 h at rest. This ratio was
21 derived from the concentration of 18O in the BAL constituents of both rats and humans.
22 Consequently, to compare absorbed 18O3 doses between rats and humans using BAL requires
18
23 the assumption that the amount of lavagable cell membrane available to react with O3 is
24 comparable between the two species. The difference between rats and humans could be
25 accounted for by the fact that the humans were exercising whereas the rats were not.
1 R
26 However, as was noted above, not all absorbed O3 can be accounted for.
27
28 8.2.3.3 Animal (In Vitro) Ozone Dosimetry Studies
29 The use of whole, intact animals to study O3 uptake is needed to ascertain the actual
30 amounts of O3 absorbed. However, it is also important to understand some of the more
31 fundamental processes governing O3 uptake, such as the biochemistry of Oyiiquid and
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1 O3/tissue interactions to determine chemical reaction rates essential to O3 dosimetry models.
2 Furthermore, the use of intact animals does not allow more precise detenninations of the role
3 of physiological parameters on O3 uptake. For this reason, there have been some limited
4 attempts at utilizing animal tissue explants and whole lungs to study O3 uptake.
5 Ben-Jebria et al. (1991) studied O3 uptake by the trachea of sheep and pigs to
6 investigate mass transfer coefficients. Ozone boluses of 1 ppm were passed through excised
7 tracheae. Tracheae were obtained from a slaughter house 0.5 to 2 h after slaughter, and
8 although they were kept coated with physiologic saline, they were not maintained at body
9 temperature, possibly resulting in underestimation of in vivo uptake. The lengths and
10 diameters of the pig tracheae were not too different from human tracheal dimensions. The
11 flow dependence of mass uptake and the mass transfer coefficient K were determined for
12 flows between 50 mL/s and 200 mL/s. Uptake efficiencies in the pig decreased with
13 increasing flow from about 0.5 to 0.12 and in the sheep decreased from about 0.5 to 0.15.
14 Mass transfer coefficients were generally independent of flow (K = 0.5 cm/s in pigs and
15 0.35 cm/s in sheep), indicating the lack of dependence of uptake on gas-phase diffusion
16 processes. This contrasts with the conclusion of Aharonson et al. (1974) for the nasopharynx
17 of dogs where they observed that the slight inverse dependence of uptake on flow observed
18 by Yokoyama and Frank (1972) leads one to conclude that the mass transfer coefficient for
19 the nasopharynx of the dog should increase with flow, suggesting a role of the boundary
20 layer in limiting diffusion of O3 to the wall of the nasopharynx. The different geometries of
21 a trachea and a nasopharynx may account for the differing observations.
22 A significant feature of the Ben-Jebria et al. (1991) study was the use of a rapidly
23 responding O3 analyzer. In order to conduct their O3 uptake studies, Ben-Jebria and Ultman
24 (1989) and Ben-Jebria et al. (1990) developed a rapidly responding O3 analyzer. The
25 analyzer relies on reaction of O3 with alkenes such as ethylene, propylene, cyclohexane, etc.
26 Ten alkenes were tested. Ninety percent step-response times of 130 to 540 ms were achieved
27 with varying degrees of linear response with O3 concentration. The authors concluded that
28 the best alkene was 2-methyl-2-butene, with optimum 10 to 90% responses of 110 ms and
29 minimum detectable limits of O3 of 18 ppb. Interference with CO2, however, was found,
30 requiring measurements of CO2 to correct the analyzer response.
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1 Postlethwait et al. (1993) used an isolated rat lung preparation to investigate the effects
2 of vascular perfusion, inspired dose, temperature, and distal lung surface area on
3 O3 absorption by the LRT. Vascular perfusion had little or no effect on uptake efficiency of
4 O3. When the lung was exposed to 1 ppm O3 and ventilated with a VT of 2.71 mL, FRC of
5 4 mL, and fB of 50 bpm, uptake efficiency was 95%. As fB increased with fixed VT, uptake
6 efficiency began to drop, reaching nearly 50% at fB of 103 bpm. When the lung temperature
7 was dropped from 37 to 25 °C, uptake efficiency dropped from 95 to 85% at 50 bpm. This
8 drop was exploited to investigate other factors (such as flow, volume, and lung surface area)
9 governing uptake because it moved RT uptake further below 100%. The observation of a
10 dependence of uptake on temperature indicates that uptake efficiency is chemical reaction-
11 dependent, thus possibly coupling uptake to reaction product formation.
12 Another interesting result from this study was the lack of dependence of uptake on
13 FRC. When FRC was doubled from 4 to 8 mL at 25 °C, fractional O3 uptake was
14 unchanged. This latter result suggests that O3 uptake is virtually complete by the time
15 O3 reaches the alveolar spaces of the lung. Otherwise it would have been expected that the
16 uptake efficiency would have risen with increased FRC.
17 To further investigate the reactions of 03 with the lung, Pryor et al. (1991) performed
18 ozonolysis studies of various unsaturated fatty acids (UFAs), rat erythrocyte ghost
19 membranes, and rat BAL. These studies demonstrated significant production of hydrogen
20 peroxide and aldehydes and that production of hydrogen peroxide was primarily due to
21 reactions between O3 and olefins. The authors concluded that the reaction of O3 with UFAs
22 hi the lung fluid lining and cell membranes produce hydrogen peroxide and aldehydes that
23 may be important mediators in the toxicity of O3. The quantitative results of these studies
24 led Pryor (1992) to hypothesize about the degree to which O3 reacts with the liquid lining of
25 the lung and with lung tissue. A simple model calculation was performed using the Einstein-
26 Smoluchowski equation to estimate the half-life of O3 in bilayers and cell membranes. Pryor
27 (1992) concluded that a substantial fraction of O3 reacts in the bilayer and that only in
28 regions of the lung where the lung lining fluid layer is less than 0.1 jum will O3 penetrate to
29 tissue and only then will O3 react in cell membranes before penetrating further. The overall
30 conclusion is that the toxic effects of O3 may be mediated not just by O3 directly but by
31 reactive intermediates such as aldehydes and hydrogen peroxide. This would bring into
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1 question what the relevant dose of O3 is. Is it the total dose, the dose to the liquid lining,
2 the tissue dose, or the dose of reactive intermediates delivered to tissue?
3
4 8.2.3.4 Human Ozone Dosimetry Studies
5 Significant progress has been made since the previous criteria document (U.S.
6 Environmental Protection Agency, 1986) in the area of human Oj dosimetry. Studies have
7 been conducted defining total and regional RT uptake; the dependence of uptake on
8 physiological parameters; and the role of uptake in modulating response.
9 Gerrity et al. (1988) reported on measurements of O3 uptake by the extrathoracic
10 airways (airways proximal to the posterior oropharynx) and intrathoracic airways (airways
11 distal to the posterior oropharynx) in 18 healthy, young male volunteers. Ozone uptake was
12 measured by placing a small polyethylene catheter through the nose and positioning the distal
13 tip in the posterior oropharynx. Breath-by-breath samples of O3 were collected, and the peak
14 plateau concentrations were compared with chamber concentrations. The effects on uptake of
15 O3 concentration (0.1, 0.2, 0.4 ppm), fB (12 and 24 bpm at fixed VT), and mode of
16 breathing (oral, nasal, and oronasal) were tested. The O3 analyzer had a moderately rapid
17 response with a 90% response time of 700 ms. Inspiratory VT ranged between 754 mL and
18 848 mL; mean inspiratory flow at 12 bpm was 350 mL/s; at 24 bpm it was 634 mL/s. The
19 authors measured extrathoracic uptake efficiency of O3 on inspiration to be approximately
20 40%, with intrathoracic uptake efficiency (inspiration plus expiration) being approximately
21 92%. They essentially found no effect of O3 concentration on uptake (intrathoracic uptake
22 was significantly higher at 0.4 ppm, but the difference was very small). They did find that
23 both intrathoracic and extrathoracic uptake decreased with increasing fB (at fixed VT), falling
24 by about 7 % for extrathoracic uptake and about 3 % for intrathoracic uptake when
25 fB increased from 12 bpm to 24 bpm. The finite response time of the analyzer may have
26 affected the results at the 24 bpm fB by overestimating extrathoracic uptake and
27 underestimating intrathoracic uptake. However, because uptake was defined relative to
28 plateaus of concentration, the response time of the analyzer was adequate to reach a plateau
29 in the 1.2 s inspiratory time at 24 bpm. It is important to note here that when utilizing the
30 data from this study to compare with other studies and models, the uptake efficiencies
31 measured here are comparable to steady-state unidirectional measurements of uptake.
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1 Another feature is that Gerrity et al. (1988) consistently measured a small, non-zero plateau
2 of O3 on expiration. This plateau is not consistent with the suggestion from models of
3 O3 uptake and the data of Postlethwait et al. (1993) (nor with later work of Gerrity et al.
4 [1993b] as presented below) that no O3 should be washed out from lung volumes beyond the
5 conducting airways. This observation of Gerrity et al. (1988) may have been an artifact of
6 how O3 was measured by sampling from the posterior oropharynx. There may have been
7 entrainment of O3 in the pharyngeal airspaces that was washed out after expiration of dead
8 space air. Regardless, the concentration of O3 exhaled from the alveolar phase of washout
9 was very low.
10 One of the most startling results from the work of Gerrity et al. (1988) was the finding
11 that there was only a small, but statistically significant, difference between uptake by the
12 nose and the mouth. The mouth had about a 10% (in relative terms) greater uptake
13 efficiency than the nose. The combined oronasal passage had an uptake efficiency greater
14 than the nose by another 8%. This suggests that persons who nasally breathe are at no less
15 risk than persons who oro-nasafly breathe. Adams et al. (1989) investigated this possibility
16 by comparing functional responses in subjects acutely exposed to O3 while either orally or
17 oro-nasally breathing. Healthy subjects were exposed on five separate days to 0.4 ppm O3.
18 In the first four exposures, subjects were exposed by facemask (with or without noseclip) for
19 30 min at an exercise level of 75 L/min or for 75 min at exercise level of 30 L/min. The
20 fifth exposure was for 30 min at 75 L/min, with exposure through a mouthpiece. There
21 were no differences in pulmonary functional response (forced expiratory volume at 1 s
22 [FEVJ or forced vital capacity [FVC] or forced expiratory flow [FEF25_75%]) with facemask
23 exposure among all experimental groups (i.e., no noseclip, VE, or time effect). Pulmonary
24 function response was, however, greater with a mouthpiece. Adams et al. (1989) speculated
25 that the greater response with the mouthpiece was due to C^ scrubbing by the facemask or by
26 facial hair. It may also have been due to different oral configurations imposed by a
27 mouthpiece. Hynes et al. (1988) also investigated whether functional responses were affected
28 by the mode of breathing. Healthy subjects were exposed to 0.4 ppm O3 for 30 min in an
29 exposure chamber. On two different occasions, each subject breathed either through the nose
30 or the mouth exclusively. There was no difference in pulmonary function response between
31 these two routes of exposure. Taken together, the studies of Adams et al. (1989) and Hynes
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1 et al. (1988) are consistent with the observations of Gerrity et al. (1988) on the equal
2 efficiency of all routes of breathing for extrathoracic O3 scrubbing.
3 This study was followed by another study (Gerrity and McDonnell, 1989; Gerrity
4 et al., 1989, 1993a) in which the relationship between O3 uptake and functional response was
5 investigated. Healthy subjects were exposed to 0.4 ppm O3 for 1 h while continuously
6 exercising at 40 L/min. Ozone uptake was measured at the beginning and at the end of
7 exposure while the subjects were still exercising, using the technique of Gerrity et al. (1988).
8 In contrast to the work of Gerrity et al. (1988), uptake was computed in this study by
9 integrating concentration times flow instead of using peak plateau measurements. Also, in
10 this work, the 90% response time of the analyzer was 1.2 s (compared with 0.7 s in the
11 previous work). The authors found that about 40% of the inspired O3 was taken up by the
12 URT (i.e., the same as the extrathoracic airways described in Gerrity et al., 1988) during
13 inspiration and that this did not change during exposure. Total RT uptake efficiency was
14 approximately 80%, and it did not change during exposure. However, LRT (i.e., the
15 intrathoracic airways described in Gerrity et al., 1988) uptake efficiency fell during exposure
16 from 68 to 62% and was correlated with the O3-induced fall in VT (VT fell from 1,650 to
17 1,239 mL; fB increased from 25.2 to 34.8 bpm; inspiratory flow fell from 1,506 to
18 1,357 mL/s; VE slightly increased from 40.8 to 40.9 L/min), suggesting that the
19 VT reduction may have a protective effect on dose delivered to the periphery of the lung.
20 It is not likely that the finite analyzer response time affected uptake measurements. Evidence
21 of this is the lack of dependence of URT uptake on changes in VT or f. The low values for
22 uptake in the LRT may have been due to artifact from the relatively slow response time of
23 the analyzer which was approximately equal to inspiratory and expiratory times. As a check
24 on their results, the authors compared their data with the previous work of Gerrity et al.
25 (1988) by computing uptake by the original technique using peak plateau concentrations.
26 When that was done, the URT uptake efficiencies were 17% and 22% at the beginning and
27 end of exercise, respectively; the LRT efficiencies were 96% and 92% at the beginning and
28 end of exercise. The URT change computed this way was not significant, but the LRT
29 efficiency drop was. When looked at in this manner, the data from this experiment are
30 consistent with those from the previous experiment.
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1 Because there is a need to compare human O3 uptake data with rat O3 uptake data, it is
2 essential that there be confidence in the reliability of the different approaches. To help
3 establish the comparability of techniques, Wiester et al. (1993) measured total RT uptake in
4 humans using a similar, though obviously scaled-up, system to that used for rats (Wiester
5 et al., 1987, 1988). Healthy subjects breathed 0.3 ppm O3 while seated in an exposure
6 chamber. Their faces were placed in a sealed facemask. The facemask was attached to a
7 large tube through which chamber air was circulated with a pump at a rate of «40 L/min.
8 Upstream and downstream O3 concentrations were measured continuously, as was ventilation
9 with an induction plethysmograph. Subjects breathed at rest either through their nose or
10 their mouth: average fB = 16 bpm, VT = 598 to 642 mL, VE = 9.2 to 9.8 L/min. While
11 nose breathing, 73 % of inspired O3 was taken up by the total RT; and while mouth
12 breathing, 76% of inspired O3 was taken up, which was significantly higher than what was
13 found with nose breathing. This difference is probably not, however, biologically
14 significant. Significant negative correlations between fB and uptake in both mouth and nose
15 breathers were found. Similar correlations were found with VE. No correlations were found
16 between uptake and VT or any other measure of breathing pattern or pulmonary function.
17 The observations in Wiester et al. (1993) of a slight increase of total RT uptake
18 efficiency with oral breathing, and the inverse correlation of total RT uptake efficiency with
19 fB are consistent with those of Gerrity et al. (1988). Furthermore, the data on total RT
20 uptake are consistent overall with that of Gerrity et al. (1988, 1993a). The data from Gerrity
21 et al. (1988) reporting total RT uptake efficiencies of about 95 % were based on minimum
22 plateau measurements, thus reflecting uptake during steady-state flow conditions, as opposed
23 to the cyclical conditions of actual breathing. The data of Genity et al. (1993a), on the other
24 hand (in which total RT efficiencies of 80% were reported), were obtained by integrating the
25 product of concentration and flow, thus more accurately reflecting the acutal mass uptake of
26 O3 during cyclical breathing. As was reported by Gerrity et al. (1993a), when they
27 computed uptake using the methodology from Gerrity et al. (1988), they found that the
28 total RT uptake measurements were comparable. Thus total RT mass uptake efficiencies at
29 rest of 80% are not unreasonable.
30 Hu (1991), Hu et al. (1992b, 1993), and Ultman et al. (1993) took a different approach
31 to measuring respiratory tract uptake of O3. They exploited the development of a rapid
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1 responding O3 analyzer (Ben-Jebria and Ultman, 1989 and Ben-Jebria et al., 1990) to
2 measure the recovery of small boluses of O3 delivered to different volumetric depths of the
3 RT. Ozone uptake was measured in a set of four experiments. In the baseline experiments,
4 absorption of O3 boluses was measured in healthy male subjects at rest. The O3 boluses
5 were 10 mL in size, with a peak concentration of 3 ppm. The O3 analyzer characteristics
6 were: sample flow of 400 mL/min, 2-methyl-2-butene as reactive alkene, 10 to 90% step-
7 response time of 110 ms, and lower detectable detection limit of 18 ppb. In the baseline
8 experiments, the VT was 500 mL and the inspiratory and expiratory flow rates were
9 250 mL/s. In a complete set of measurements, bolus recovery was examined for
10 penetrations of 10 to 200 mL in 10 mL increments. In the second set of experiments, the
11 effects of flow were measured by measuring bolus recovery as a function of penetration
12 depth for flows of 150, 250, 500, 750, and 1,000 mL/s at a fixed VT of 500 mL. In a third
13 set of experiments, bolus recovery was measured as a function of penetration depth at a flow
14 of 250 mL/s and VT of 500 mL, comparing the bolus delivered to a rubber mouthpiece or to
15 a nasal cannula and thereby examining potential uptake differences between the two
16 pathways. In the fourth set of experiments, the effects of Oj concentration on uptake were
17 determined by delivering boluses with peak concentrations of 0.5, 1.0, 2.0 and 4.0 ppm.
18 The latter experiments were conducted because acute studies in isolated dog airways showed
19 that absorption efficiency was inversely related to inhaled concentration between 0.1 to
20 20 ppm (Vaughan et al., 1969; Yokoyama and Frank, 1972). However, later experiments in
21 guinea pigs, rabbits (Miller et al., 1979), and humans (Gerrity et al., 1988) showed a lack of
22 concentration dependence. The dependence or lack of dependence of uptake efficiency on
23 O3 concentration provides information on the order of reactions of O3 with lung fluid lining
24 and tissue. Under steady-state conditions, concentration independence of uptake efficiency
25 suggests that first-order processes are playing a role. However, because O3 absorption is
26 coupled to both interfacial transfer (gas-phase to solute O3) and subsequent reaction, at face
27 value one cannot conclude that saturated absorption rates are solely due to saturation of the
28 reaction components.
29 In all four experiments, Hu and colleagues computed the first three moments of the
30 inspired and expired bolus distributions with respect to volume. The zeroth moment of a
31 bolus is the O3 mass contained in the bolus. Thus the zeroth moments of the inspired and
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1 expired boluses were used to compute O3 uptake efficiency (or absorbed fraction) A,
2 breakthrough volume VB (the mean volume of the exhaled bolus), and bolus dispersion a2.
3 The first moment of a bolus is its mean volumetric position. The first moment on inspiration
4 gives the penetration volume (Vp), and the first moment on expiration gives the breakthrough
5 volume (VB). In the absence of any O3 uptake, a longitudinally mixed bolus should have
6 Vg=Vp. The second moment of a bolus is its variance. The difference in variance between
7 the expired and inspired bolus (a ) is a measure of gas mixing, or dispersion, in the lung.
8 In the baseline experiments, the breathing pattern was a resting pattern with a VT of
9 500 mL and an average inspiratory flow of 250 mL/s. These experiments were performed
10 on 9 male subjects and showed that almost all O3 was absorbed beyond a penetration depth
11 of 180 mL. Only about 6% of inhaled 63 was recovered at the 180 mL penetration depth,
12 and beyond that depth, it was very difficult to obtain an accurate measurement of recovery.
13 The investigators also found that VB was greater than V at penetration depths less than
14 100 mL, after which VB leveled out at a constant value. Dispersion was insensitive to
15 penetration depth. An important finding of the baseline experiment was that at quiet resting
16 ventilation, about 50% of the O3 mass in a bolus inhaled through the upper airways is taken
17 up by the upper airways. To compare these data with results of Gerrity et al. (1988), it is
18 necessary to assume that inspiratory and expiratory uptake efficiencies are equal. Then the
19 unidirectional uptake efficiency of the upper airways to a depth comparable to that at which
20 Gerrity et al. (1988) positioned their sampling catheter is about 30%, which is approximately
21 25% less than the 40% results of Gerrity et al. (1988). This difference might be due to the
22 presence of a mouthpiece in the experiments of Hu and colleagues which could reduce the
23 uptake efficiency of the oral pathway. The functional response data of Adams et al. (1989)
24 suggest that this might be the case.
25 The flow experiments showed that there was a general right shifting of the A-Vp curves
26 with increasing flow (i.e., increasing flow causes a deeper penetration of O3 into the lung
27 with lower fractional uptake by the conducting airways). Eventually, all of the O3 is still
28 absorbed. Breakthrough volume showed a similar pattern at all flows (i.e., greater than
29 penetration volume at small Vp but flattening out at larger Vp). As flow increased, the level
30 of the plateau increased. Dispersion, though constant as a function of Vp at all flows,
31 increased linearly with increasing flow.
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1 The studies of Hu and colleagues investigating the role of exposure route in modulating
2 O3 uptake efficiencies reported that the nose absorbed approximately 30 % more than the
3 mouth. This result is at variance with the findings of Gerrity et al. (1988) indicating that
4 there was only a slightly higher uptake by the oral pathway when compared to the nasal
5 pathway. Gerrity et al. (1988) studied uptake by the two pathways without the use of a
6 mouthpiece or any other delivery system. Subjects were free to breathe naturally. It is
7 possible that the use of mouthpieces and nasal canulas in the studies of Hu and colleagues
8 caused artifacts, resulting in their findings for nasal and oral uptake efficiencies. The study
9 of Adams et al. (1989) showing enhanced pulmonary function response to O3 during a
10 mouthpiece exposure compared to facemask/oral exposure supports this. Finally, the
11 concentration-dependence studies showed that uptake efficiency was not affected by the
12 concentration of inspired O3 between 0.3 to 4 ppm, implying that O3 uptake is governed by
13 linear processes.
14 One of the very unique features of the approach to measuring uptake efficiency taken
15 by Hu and colleagues is that the O3 bolus recovery data can be used to derive local mass
16 transfer coefficients for the conducting airways. Regional mass transfer coefficients derived
17 experimentally in this way can then be used as input into mathematical model simulations,
18 thereby potentially leading to more accurate models of O3 dose to the respiratory tract.
19 Hu and colleagues define the parameter Ka (s"1) as one that is suitable to characterize
20 local O3 absorption. It is the product of the overall mass transfer coefficient K (cm/s) that
21 reflects the combined contribution of diffusion and chemical reaction to uptake, and the local
22 surface/volume ratio a (cm" ). From the A-Vp curves, these investigators derived values for
23 Ka. Thus, the mass transfer coefficients that were experimentally derived depended upon
24 assumptions about airway anatomy and morphology. As a result of the various experiments,
25 these investigators found a number of important properties for Ka:
26
27 • The proximal subcompartment of the nose has a Ka that is 70% larger than
28 Ka for the proximal mouth compartment (see, however, the comment made
29 above regarding the nose/mouth differences).
30
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1 • Ka's in the upper airway compartment were between 1.20 and 2.24 s"1 and
2 relatively insensitive to flow, indicating that diffusion resistance of
3 O3 through the gas boundary layer is much less than through the mucus
4 film. These data also are consistent with the pig and sheep tracheae
5 experiments of Ben-Jebria et al. (1991).
6
7 • In the proximal and distal conducting airway subcompartments, (Ka)"1 was
8 linearly related to (flow)"1, suggesting that the gas phase absorption rate
9 constant is directly proportional to flow. In lower airways, therefore,
10 diffusion resistance of the gas boundary layer is important. Hu and
11 colleagues concluded that the gas boundary layer contributes 80 to 90% of
12 the overall diffusion resistance in the central airway compartment.
13
14 • The mass transfer coefficient in the lung liquid lining (kj) is estimated to
15 fall from 1.4 cm/s in the URT to 0.17 cm/s in the respiratory airways.
16
17 • The chemical reaction rate between O3 and the lung liquid lining was
18 estimated to be 7.3 x 106, 2.3 x 106, and 8.2 x 105 s'1 in the upper
19 airways, proximal conducting airways, and distal conducting airways
20 respectively.
21
22 • The overall mass uptake coefficients determined in the work of Hu (1991)
23 are significantly higher than those used in the model of Miller et al. (1985).
24 If the mass transfer coefficients in the model are adjusted upward, the total
25 uptake efficiency would be higher than measurements have shown,
26 requiring a downward adjustment of pulmonary region mass transfer
27 coefficients.
28
29 Gerrity et al. (1993b) took a somewhat more conventional approach in an attempt to
30 acquire regional information on O3 absorption in the human respiratory tract. Healthy
31 subjects underwent transnasal bronchoscopy while in an exposure chamber in which 0.4 ppm
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1 O3 was present. Subjects were asked to breathe at 12 bpm. Inspired and expired air was
2 sampled through a teflon catheter that had been passed through the biopsy channel of the
3 bronchoscope and positioned approximately in the center of the airway lumen. The air was
4 drawn into a rapid response O3 analyzer (Ben-Jebria and Ultman, 1989 and Ben-Jebria et al.,
5 1990) with a 90% response time of 250 mL/s while using ethylene as the reactive alkene.
6 Average VT was 810 mL, and average inspiratory flow was 320 mL/s. Air was sampled for
7 5 breaths from above the vocal cords, at the entrance to the trachea, above the main
8 bifurcation carina, and midway through bronchus intermedius. Flow was measured
9 simultaneously by a pneumotach attached to a simple cylindrical mouthpiece. Before and
10 after each measurement, a set of samples from the mouth was collected for reference.
11 Uptake was defined as the fraction of O3 mass lost across any anatomical segment; mass was
12 determined by integrating the product of O3 concentration and flow. By way of comparison
13 with the other human O3 uptake studies, Gerrity et al. (1993a) found that total RT uptake of
14 O3 measured in this fashion was 91 %. This is higher than the resting data of Wiester et al.
15 (1993); however, the average VT in this study of 810 mL compared with the 600 mL VT
16 reported by Wiester et al. (1993) may account for this difference. When Gerrity et al.
17 (1993b) compute the unidirectional uptake efficiencies between the mouth and the various
18 sampling sites, they find that 17.6%, 27.0%, 35.5% and 32.5% of O3 passing into the mouth
19 is taken up by structures up to the vocal cords, upper trachea, main bifurcation carina, and
20 bronchus intermedius, respectively. They also computed the unidirectional uptake
21 efficiencies across individual airway segments which were: 17.6% between the mouth and
22 just above the vocal cords, 12.8% from above the vocal cords to the upper trachea, 11.5%
23 from the upper trachea to the main bifurcation carina, and essentially zero between the carina
24 and bronchus intermedius. The uptake between the mouth and just above the vocal cords is
25 considerably lower than what was measured earlier by Gerrity et al. (1988), even considering
26 the fact that in the earlier study peak plateaus were used. As has been noted earlier, it is
27 possible that the mouthpiece played a role in reducing the uptake efficiency of the mouth.
28 The uptake efficiency of O3 across the trachea is in line with the data from sheep and porcine
29 tracheae at the higher flow rates (Ben-Jebria et al., 1991). The present data are also
30 consistent with the bolus uptake data of Hu (1991) and colleagues, which were also acquired
31 with a mouthpiece. When the O3 bolus data are used to compute unidirectional uptake
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1 efficiencies (assuming that the segmental efficiencies are the same on inspiration and
2 expiration), the Hu et al. (1992b) data yield uptake efficiencies of 21, 36, 44, and 46%
3 between the mouth and the vocal cords, upper trachea, main bifurcation carina, and bronchus
4 intermedius, respectively. The O3 bolus data are, therefore, in good accord with the data of
5 Gerrity et al. (1993b). The measured uptake efficiencies across airway segments clearly
6 appear to be higher than those predicted by the model of Miller et al. (1985). If higher
7 uptake coefficients in the conducting airways were used in the model of Miller et al. (1985),
8 the model would overestimate total RT O3 uptake. To adjust for this, pulmonary uptake
9 coefficients would have to be reduced. Unfortunately , the data of Hu and associates cannot
10 provide information beyond the conducting airways.
11 Gerrity et al. (1993b) also measured O3 washout volumes (i.e., the expired volumes
12 required to cause a specified drop in O3 concentration). These type of data provide insight
13 into the location of major sites of O3 uptake. At the mouth, the 90% washout volume was
14 142 mL; at the upper trachea, the 90% washout volume was 62 mL. By the time the entire
15 anatomical dead space of the lungs was washed out, the Oj concentration had fallen to zero
16 (Gerrity et al., 1993b). It is unclear whether the absence of recovered O3 after washout of
17 the conducting airways was due to O3 not penetrating beyond the conducting airways or all
18 of the O3 penetrating beyond the conducting airways being absorbed. The latter possibility is
19 more likely based on the observations of Hu (1991).
20 In assessing the work of Gerrity et al. (1993b), it is significant to note that these
21 investigators measured expired plateaus of O3 concentration that were zero. This contrasts
22 with the earlier work of Gerrity et al. (1988, 1993a) in which a non-zero expiratory plateau
23 was observed. The non-zero expiratory plateau may have been due to a number of factors
24 that are unclear. Since ethylene was the reacting alkene in all cases, it is unlikely that
25 interference with other gases such as CO2 was responsible. Another possibility is that O3 in
26 the early expiratory phase became entrained in the posterior oropharynx and persisted for the
27 duration of expiration.
28
29 8.2.3.5 Intel-comparison of Ozone Dosimetry Studies
30 The previous sections emphasized the methods and results of individual experimental
31 studies on O3 dosimetry. This section will focus on comparisons of the in vivo studies with
December 1993 8-22 DRAFT-DO NOT QUOTE OR CITE
-------
1 each other and will draw upon these comparisons to arrive at conclusions regarding the utility
2 of these data for extrapolation purposes. The discussion will be divided into three sections,
3 focusing on total RT uptake efficiency, unidirectional URT uptake efficiency, and LRT
4 uptake efficiency.
5 There are two categories that distinguish various data sets among each other. The first
6 category is the mouth/nose category listed in the Tables 8-2 to 8-4. Studies indicated by
7 M or N were performed with unencumbered breathing by either oral (M) or nasal (N)
8 breathing. Unencumbered in the present context means the absence of a mouthpiece or
9 canula. Data listed as M/N are pooled from data encompassing oral and nasal breathing.
10 Data shown as mouthpiece or nasal canula are data acquired using these devices to deliver
11 the O3 to the animal or human subject, and/or to measure flow.
12 The second category is the method used to compute O3 uptake efficiency. There are
13 essentially two methods. One method, refered to as the steady-state method, relies on
14 measuring the loss of O3 from a steady air flow moving across an anatomical structure.
15 An example is the data of Yokoyama and Frank (1972) in which a constant flow of ozonated
16 air through the URT of a dog was maintained by a trachea! canula attached to a pump.
17 Uptake efficiency was measured by changes in equilibrium O3 concentration. Another
18 example is the study of Gerrity et al. (1988) which used the steady-state method by
19 measuring the peak inspiratory and minimum expiratory O3 concentrations through a catheter
20 in the posterior pharynx. These measurements were compared to the ambient chamber
21 concentration to obtain uptake efficiencies of the URT and LRT. The second method is
22 refered to as the non-steady state method. This method uses the integration of the product of
23 flow and O3 concentration to compute O3 masses that are, in turn, used to compute uptake.
24 The studies of Wiester et al. (1988) in rodents, and Wiester et al. (1993) in humans are an
25 example of this technique, as is the study of Gerrity et al. (1993a).
26
27 Total Respiratory Tract Uptake Efficiency
28 Table 8-2 provides a summary of in vivo data in all animal species of RT O3 uptake
29 efficiency (F^. The data reported for the studies of Gerrity et al. (1988, 1993a) have been
30 adjusted from the published values to account for the fact the Ft cited in those papers did not
31 include uptake in the URT on expiration. To make the adjustment, the URT uptake
December 1993 g-23 DRAFT-DO NOT QUOTE OR CITE
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TABLE 8-2. TOTAL RESPIRATORY TRACT UPTAKE DATA8
Species
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Rat (F-344)
Rat (SD)
Rat (LE)
Rat (F-344)
Guinea Kg
Mouth/Note
M
N
M/N
M/N
M
M
M
M
Mouthpiece
M
N
Mouthpiece
Mouthpiece
N
N
N
N
N
Method
Steady
Steady
Steady
Steady
Non-Steady
Non-Steady
Steady
Steady
Non-Steady
Non-Steady
Non-Steady
Non-Steady
Non-Steady
Non-Steady
Non-Steady
Non-Steady
Non-Steady
Non-Steady
VT(mL)
832
754
832
778
1,650
1,239
1,650
1,239
825
631
642
500
1,000
2.8
2.4
2.7
2.6
2.4
Inspiratory
Flow (mL/s)
509
456
350
634
1,360
1,360
1,350
1,360
330
539
514
250
250
12.2
9.6
12.3
11.3
7.5
fB (bpm)b
18
18
12
24
25
35
25
35
12
16
16
15
7.5
118
123
132
113
77
Ft
0.97
0.96
0.97
0.96
0.88
0.87
0.97
0.95
0.91
0.76
0.73
0.86
0.93
0.44
0.46
0.48
0.54
0.53
Reference
Gefrity et al. (1988)°
Gerrity et al. (1988)
Gerrity et al. (1988)
Gerrity et al. (1988)
Gerrity et al. (1993a)b
Gerrity et al. (1993a)
Gerrity et al. (1993a)
Gerrity et al. (1993a)
Gerrity et al. (1993b)
Wiester et al. (1993)
Wiester et al. (1993)
Hu et al. (1992b)
Hu et al. (1992b)
Wiester et al. (1988)
Wiester et al. (1988)
Wiester et al. (1988)
Hatch et al. (1989)
Wiester et al. (1988)
°M=mouth exposure by natural breathing; N=nasal exposure by natural breathing; M/N=pooled data from mouth and nasal exposure;
Mouthpiece=exposureby mouthpiece; Nasal Canula=exposure by nasal canula; Steady=uptake computed during constant unidirectional
flow; Non-Steady=uptake computed by integration during cyclic breathing; Ft=total RT uptake.
fB is either the measured frequency or is computed based on reported flows and volumes.
'Total RT uptake reported by Gerrity et al. (1988) and Gerrity et al. (1993a) did not include the contribution from URT uptake efficiency
during expiration. The data as listed here include an expiratory URT contribution assuming it equals inspiratory URT uptake efficiency.
1
2
3
4
5
6
7
efficiency upon expiration was assumed to equal to the inspiratory uptake efficiency. The
Ft data listed for the study of Hu et al. (1992b) were derived from their bolus recovery data
by integrating the data over the desired VT. Because Hu et al. (1992b) could not recover
boluses from a depth greater than 220 mL, it was assumed that any bolus delivered to a
depth greater than 220 mL was absorbed completely. The derivation of Ft from the bolus
data was done for VTs of 500 mL and 1,000 mL.
December 1993
8-24
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TABLE 8-3. UNIDIRECTIONAL UPPER RESPIRATORY TRACT
UPTAKE EFFICIENCY DATA8
Species
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Dog (Beagle)
Dog (Beagle)
Dog (Beagle)
Dog (Beagle)
Rat (F-344)
Guinea Pig
Rabbit
Mouth/Nose
M
N
M/N
M/N
M
M
M
M
Mouthpiece
Mouthpiece
Mouthpiece
Nasal Canula
Nasal Canula
Mouthpiece
Mouthpiece
N
N
N
Method
Steady
Steady
Steady
Steady
Non-Steady
Non-Steady
Steady
Steady
Non-Steady
Non-Steady
Non-Steady
Steady
Steady
Steady
Steady
Non-Steady
Steady
Steady
Inspiratory
Flow (mL/s)
509
456
350
634
1,360
1,360
1,360
1,360
337
250
250
83.3
667
83.3
667
11.3
2.7
16.7
fB (bpm)
18
18
12
24
25
35
25
35
12
15
15
N/AC
M/A
N/A
N/A
113
N/A
N/A
FU«
0.40
0.43
0.41
0.38
0.37
0.41
0.16
0.22
0.18
0.30
0.47
0.72
0.37
0.34
0.12
0.17
0.62
0.41
Reference
Gerrity et al. (1988)
Gerrity et al. (1988)
Gerrity et al. (1988)
Gerrity et al. (1988)
Gerrity et al. (1993a)
Gerrity et al. (1993a)
Gerrity et al. (1993a)
Gerrity et al. (1993a)
Gerrity et al. (1993b)
Ultman et al. (1993)
Ultman et al. (1993)
Yokoyama and Frank (1972)
Yokoyama and Frank (1972)
Yokoyama and Frank (1972)
Yokoyama and Frank (1972)
Hatch et al. (1989)
Miller et al. (1979)
Miller et al. (1979)
aM=mouth exposure by natural breathing; N = nasal exposure by natural breathing; M/N=pooled data from mouth and nasal exposure;
Mouthpiece=exposure by mouthpiece; Nasal Canula=exposure by nasal canula; Steady=uptake computed during constant unidirectional
flow; Non-Steady=uptake computed by integration during cyclic breathing; Ft=total RT uptake.
fg is either the measured breathing frequency or is computed from flows and VT.
N/A=not applicable.
1 To assess the consistency of the data, it is useful to examine it as a function of flow.
2 Figure 8-1 shows Ft as a function of inspiratory flow for all human studies. The Ft from the
3 bolus recovery data of Hu et al. (1993) are shown here for VTs of 500 mL, 1,000 mL, and
4 1,500 mL. The constitency of the data suggests that with respect to Ft there is good
5 agreement among the various experimental methods for humans. The data clearly show that
6 Ft decreases with increasing flow and increases with increasing VT, all of which is
7 qualitatively consistent with model predictions.
December 1993
8-25
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TABLE 8-4. LOWER RESPIRATORY TRACT UPTAKE EFFICIENCY DATA8
Species
Human
Human
Human
Human
Human
Human
Human
Human
Mouth/Nose
M/N
M/N
M
M
M
M
Mouthpiece
Mouthpiece
Method
Steady
Steady
Non-Steady
Non-Steady
Steady
Steady
Non-Steady
Non-Steady
VT (mL/8)
832
778
1,650
1,239
1,650
1,239
844
500
Inspiratory
Flow (mUs)
350
634
1,360
1,360
1,360
1,360
337
250
fB (bpm)b
12
24
25
35
25
35
12
15
Fjft Reference
0
0
0
0
0
0.
.93
.89
.68
.62
.96
.92
0.95
0.
.78
Oerrity et al.
Gerrity et al.
Oerrity et al.
Oerrity et al.
Gerrity et al.
Oerrity et al.
Gerrity et al.
(1988)
(1988)
(1993a)
(1993a)
(1993a)
(1993a)
(1993b)
Hu et al. (1992b)
Human Mouthpiece Non-Steady 1,000
Dog (Beagle) N/AC Non-Steady 168
Dog (Beagle) N/A
Rat (F-344) N
Non-Steady
Non-Steady
168
2.6
250
112
168
11.3
7.5 0.89 Hu et al. (1992b)
20 0.87 Yokoyama and Frank
(1972)
30 0.83 Yokoyama and Frank
(1972)
113
0.33 Hatch et al. (1989)
*M=mouth exposure by natural breathing; N=nasal exposure by natural breaming; M/N=pooled data from mouth and nasal exposure;
Mouthpiece=exposure by mouthpiece; Nasal CanuU=exposure by nasal canula; Steady=uptake computed during constant unidirectional
flow; Non-Steady=uptake computed by integration during cyclic breathing; Ft=total RT uptake.
fjj is either the measured breathing frequency or is computed from flows and Vj.
cN/A=snot applicable.
1 One observation is quite prominent. The rat data of Wiester et al. (1988) and Hatch
2 et al. (1989) (not shown in Figure 8-1) are considerably lower than the human data. Even if
3 it is assumed that the rats were breathing up to three times resting ventilation (equivalent to
4 an inspiratory flow in humans of approximately 1,000 mL/s), the rat data would still be
5 significantly lower than what was measured in humans. The consistency of the human data
6 of Wiester et al. (1993) (in which the same methodology was used to measure Ft in humans
7 as was used for rats) with the other human data strongly suggests that the low Ft in rats is
8 not a function of the methodology employed. Overall, the evidence reasonably points to the
9 conclusion that Ft in a rat is smaller than in a human.
10
December 1993
8-26
DRAFT-DO NOT QUOTE OR CITE
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Vj-SOOml
Hu9tal.,(1993)
,-1500ml VT-g26ml
Q«rrity«tal, (19936)
V,-1239nl V,-1850nl VI-631ml
Q«rrily«(l.,(1993a) Q«rrtty»tal.,(1993) WNMtwet«l,(19fl3)
»DO
1
0.9
0.8
UJ
0.7
(0
Q.
D
0>
0 0.6
N
0
0.5
i 1,1,1,1,1,1,
0 200 400 600 800 1,000 1,200 1,400 1.600
Inspiratory Flow (mL/s)
Figure 8-1. Total respiratory tract uptake as a function of inspiratory flow in humans.
1 Unidirectional Upper Respiratory Tract Uptake
2 Table 8-3 summarizes the data on the unidirectional O3 uptake efficiency of the URT
3 (FUrt)- *n general, Furt describes the uptake efficiency of anatomical structures proximal to
4 the larynx. A possible exception to this are the data of Hu et al. (1992b). In that study, and
5 other bolus studies, it was assumed that the URT is the volume of the respiratory tract
6 50 mL distal to the lips. This may or may not include the larynx.
7 The wide variety of URT uptake data in different species and under different flow
8 conditions allows one to make some intra- and interspecies comparisons. To make
9 comparisons among different species, however, requires assumptions about the scaling of
10 breathing patterns among species. Gerrity (1989) examined nasopharyngeal uptake data
11 available in different species at that time by examining the data as a function of the ratio of
December 1993
8-27
DRAFT-DO NOT QUOTE OR CITE
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1 predicted resting flow to measured flow (scaled flow). The predicted flows were obtained
2 using the allometric equations of Guyton (1947). Ozone uptake efficiency of the URT was
3 then described as a single function of scaled flow and species weight. When newer data
4 become available, this approach can be extended to URT data acquired by both mouth and
5 nasal breathing.
6 Figure 8-2 shows l-Furt for nasal breathing plotted as a function of the ratio between
7 predicted and measured flow. As with the previous work of Gerrity (1989), all of the data,
8 except the Beagle dog data, are roughly consistent with each other. The body weight
9 dependence of the Gerrity (1989) analysis is illustrated by the two dashed lines representing
10 the predicted range of flow dependences between rats (lower line) and humans (upper line).
11 All species, except the Beagle dogs, fall within these boundaries.
12 Figure 8-3 shows the data for l-Furt by the oral route plotted hi the same manner. All
13 of the data are for humans except for the two Beagle dog data points. Several observations
14 are worth noting. First, the data for the Beagle dog appear to be consistent with the human
15 data. The analysis of Gerrity (1989) predicts that the human data would generally lie above
16 the dog data, though not to a large degree. This suggests that the oral passage of the dog
17 may have similar O3 scrubbing properties as the human oral passage. Second, within the
18 human data, use of a mouthpiece appears to reduce the uptake efficiency of the oral
19 passageway. The closed diamonds are the data from the study of Gerrity et al. (1993a)
20 which involved unencumbered breathing. These data are generally lower than the data of
21 Hu et al. (1993) and the data of Gerrity et al. (1993b). The fact that the Hu et al. (1993)
22 and Gerrity et al. (1993b) data are consistent with each other supports this speculation. This
23 observation may account for the result of Ultman et al. (1993) that the uptake efficiency of
24 the URT is greater by the nasal pathway than by the oral pathway, which is counter to the
25 observations of Gerrity et al. (1988).
26
27 Lower Respiratory Tract Uptake
28 Table 8-4 summarizes the data on the uptake efficiency of the LRT tract (Flrt). In the
29 current discussion, F,rt is the uptake efficiency of the LRT relative to the concentration of
30 O3 entering the LRT. The human data of Gerrity et al. (1988, 1993a, 1993b) and the rat
31 data of Hatch et al. (1989) include the larynx in the LRT. The Beagle dog data of
December 1993 8-28 DRAFT-DO NOT QUOTE OR CITE
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Natural Natural Canute Canute
Steady State Non-Steady State Steady State Non-Steady State
0.7
0.5
LU
i
-------
Natural Natural Canute Canute
Steady State Non-Steady State Steady State Non-Steady State
• » D 0
UJ
0.7
0.5
0.3
0.2
0.1
D
Bragl*
I
Boagto
0.1 0.2 Q.3 0.5 1 23
Predicted, Resting Flow/Measured Row
Figure 8-3. Unidirectional uptake efficiency in the upper respiratory tract by the oral
pathway. The ratio of predicted resting flow to measured flow scales the
flow to allow for interspecies comparison. The closed squares and
diamonds at a scaled flow of 0.25 are from Gerrity et al. (1993a); the closed
square at a scaled flow of 0.65 is from Gerrity et al. (1988); the open
diamond at a scaled flow of 0.97 is from Gerrity et al. (1993b); the
remaining open diamonds are from Hu et al. (1993).
1 observation is that the single data point of Gerrity et al. (1993b) is approximately 20% higher
2 than the data of Hu et al. (1993) at a comparable flow and VT. Second, the data of Gerrity
3 et al. (1993a) are markedly lower than what the data of Hu et al. (1993) suggest for
4 comparable flows and VTs. In assessing this discrepancy, it is important to keep in mind
5 that the URT is defined in the work of Hu et al. (1993) as a fixed volume of 50 mL distal to
6 the lips. This is an inaccurate definition that could influence greatly the estimation of LRT
7 uptake efficiency. The coherence of all of the data on Ft and Furt by the oral pathway
December 1993
8-30
DRAFT-DO NOT QUOTE OR CITE
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1 ,-
-£0.9
Ul
0.8
q> ._
§ °'7
0.6
\ft-500n4
VHESml
\ft-1000mt
Hu •(•!., (1883)
Vt-1500ml
VM239mi wiesoml
G«rt*y«tal.,(1983a) Qwr«y«tal.,(1M3)
* D
Q
J I L.
I I I I 1 1 1_
0 200 400 600 800 1,000 1,2001,4001,600
Inspiratory Flow (mL/s)
Figure 8-4. Uptake efficiency of the lower respiratory tract as a function of inspiratory
flow in humans.
1 suggests that F^ should also be consistent among studies. If it is assumed that the data
2 represented by the Gerrity et al. (1993a, 1993b) studies more accurately reflect human LRT
3 uptake efficiency, then the flow dependence for F^ would be considerably steeper than
4 suggested by the data of Hu et al. (1993).
5 Finally, the F^ data of Yokoyama and Frank (1972) in Beagle dogs were acquired with
6 flows that were about 100% and 150% of resting flow rates and VTs approximately 150% of
7 normal (Guyton, 1947). When flow is scaled in the dogs, the F^s of 0.87 and 0.83 at the
8 two flow rates are consistent with the human data of Gerrity et al. (1993b) but higher than
9 the Hu et al. (1993) data.
10
December 1993
8-31
DRAFT-DO NOT QUOTE OR CITE
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1 8.2.4 Dosimetry Modeling
2 8.2.4.1 Background
3 Table 8-5 presents a summary of theoretical studies of the uptake of O3 by the
4 respiratory tracts (or regions) of humans and laboratory animals that have become available
5 since the 1986 review. Although there are ten investigations listed, there are only five
6 distinct dosimetry models (with respect to groups of co-workers and independent model
7 formulation): The models of (1) Overton et al. (1987) and Miller et al. (1985), (2) Hanna
8 et al. (1989), (3) Grotberg et al. (1990)—although they consider two reaction schemes,
9 (4) Mercer et al. (1991), and (5) Hu et al. (1992a). In some cases, several references have
10 been grouped into one investigation. This is because the multiple studies came from the
11 same co-workers or laboratory, but added to or were complementary to the original or
12 common dosimetry modeling theme.
13 Major factors affecting the local uptake of reactive gases in the respiratory tract are
14 respiratory tract morphology and anatomy; the route of breathing (nose or mouth); the depth
15 and rate of breathing (VT and fB); the physicochemical properties of the gases; the processes
16 of gas transport; and the physicochemical properties of the liquid lining of the respiratory
17 tract, respiratory tract tissue, and capillary blood. A detailed discussion of these factors can
18 be found in Overton (1984), Ultman (1988), and Overton and Miller (1988),
19 Because all of the dosimetry models listed in Table 8-5 were developed to simulate
20 uptake in the LRT or the total RT, they have some aspects in common. This includes the
21 formulation of O3 transport and wall loss in the air compartments of the RT, the use of
22 species-dependent morphometric models or data to define air and liquid lining compartment
23 dimensions, and a description of the transport and loss of O3 in the liquid lining and tissue.
24 In all the dosimetry models that have become available since 1986, except for Ultman
25 and Anjilvel (1990) and Grotberg et al. (1990) which are discussed later, O3 transport and
26 loss processes in air compartments are approximated in terms of a one-dimensional, time
27 dependent, partial differential equation of continuity. This type of equation accounts for
28 axial convection, axial dispersion or diffusion, and the loss of O3 by absorption at the gas-
29 liquid interface. The use of this approximation is very common in modeling the transport in
30 the LRT of gases such as oxygen, nitrogen, helium, carbon dioxide (COj), etc. (e.g.,
31 Scherer et al., 1972; Paiva, 1973; Chang and Fartii, 1973; Yu, 1975; Pack et al., 1977) and
December 1993 8-32 DRAFT-DO NOT QUOTE OR CITE
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TABLE 8-5. THEORETICAL OZONE DOSIMETRY INVESTIGATIONS8
Species and Region
Modeled/ Anatomical
Model
Liquid Lining and Tissue
Transport and Chemical
Reactions
Dosimetry Model /Subject of Investigation
Results, Predictions
Reference
Guinea pig
LRT/Kliment
(1973), Schreider
and Hutchens
(1980); rat LRT/
Kliment (1973), Yeh
etal. (1979)
Miller et al. (1985) Enhanced Miller et al. (1985). Investigates the
effect on predictions of anatomical models,
FRC, ventilation, and TB liquid lining rate
constant. Simulates 03 uptake in anatomical
models of rat lobes.
With respect to different anatomical models for the same and
different species: qualitative similarity in the shape of net
and tissue dose versus airway number curves, but significant
differences in regional fractional uptakes. Maximum tissue
dose in vicinity of PAR. PAR dose decreases with
increasing time of flight to this region. Maximum tissue
dose in the vicinity of the first PUL region segment of
anatomical models.
Overton et al.
(1987)
OO
Human, rat, rabbit,
and guinea pig
LRT/Not specified
Miller et al. (1985) and
Overton et al. (1987)
Miller et al. (1985) and Overton et al. (1987).
Compares 03 dose profiles of human, rat,
guinea pig, and rabbit. Uses model and
experimental data to estimate 03 dose-response
curves for decrements in FEVj (humans) and
for BAL proteins in rat, guinea pig, and rabbit.
Compares LRT uptake predictions to the human
experimental data of Gerrity et al. (1988).
Similarity among species in the shape of the airway segment
curve: tissue dose increases distally in the TB region,
reaches a maximum in the in the first PUL region segment
for human, rat, and guinea pig and in the last TB region
segment of the rabbit, and then decreases distally in the PUL
region. Predictions of uptake distal to the oropharynx are in
agreement with Gerrity et al. (1988).
Miller et al. (1988)
Rat total RT/URT:
Schreider and Raabe
(1981); TB: Uses
rat cast data (Raabe
etal., 1976) to
define TB region
paths; PUL: Yeh
et al. (1979) for
model of the acinus
TB and PUL region mass
transfer coefficients based
on Overton et al. (1987)
Enhanced Overton et al. (1987). Illustrates
dose distribution along the longest and shortest
(as defined by time of flight) paths from the
trachea to the most distal alveoli.
Three-fold difference in PAR doses of the shortest and
longest paths from trachea to PAR. Dose distributions along
the longest or shortest path were qualitatively similar to
Overton et al. (1987) with maximum tissue dose in the first
PUL region generation.
Overton et al.
(1989)
Human LRT
(newborn to
adult)/TB: based on
Yeh and Schum
(1980); PUL: based
on Hansen and
Ampaya (1975)
Miller et al. (1985) Enhanced Overton et al. (1987). From various
sources, develops age-dependent LRT
anatomical models. For quiet and maximal
exercise breathing, applies the dosimetry model
of Miller et al. (1985) to several ages from birth
to adult, illustrating the LRT distribution of
absorbed 03.
For quiet breathing, the LRT distribution of dose, the
percent uptake, and the PAR dose are not very sensitive to
age; but are more sensitive during exercise. Regardless of
age and breathing state, the largest 03 dose occurs in the
PAR. No uptake in the URT.
Overton and Graham
(1989); Miller and
Overton (1989)
-------
TABLE 8-5 (cont'd). THEORETICAL OZONE DOSBMETRY INVESTIGATIONS1
Species and Region
Modeled/ Anatomical
Model
Liquid Lining and Tissue
Transport and Chemical
Reactions
Dosimetry Model /Subject of Investigation
Results, Predictions
Reference
Human total RT/URT:
Hanna and Scherer
(1986); LRT: Weibel
(1963)
Time-dependent molecular
diffusion and first order
reactions in liquid lining
and interstitium; transfer
through epithelium
modeled as a permeability
process—no reactions in
this layer. URT and LRT
liquid lining rate
constants: 50 and 1 times
that of Miller et at.
(1985), respectively
Model development. Lung dimensions scaled to
those of a young male and a young female.
Contrasts LRT airphase concentrations during
exercise and rest. Compares male and female
airphase 03 concentrations and male and female
subepithelial concentrations.
URT uptake may be greater in cold than in warmer air.
For the ventilatory parameters used: (1) Subepithelial
Oj concentrations are a maximum in the terminal
bronchioles or the first respiratory bronchioles, and
(2) These concentrations are greater in the female than
male for most of the RT.
Hanna et al. (1989)
oo
u>
*>•
Human, distal
segment of a lobe/is
based on Horsfield
etal. (1971)
Similar to Miller et al.
(1985)
Monte Carlo simulation; transport processes
defined in terms of probabilities based on
physical and chemical principles. The effect of
lung asymmetries on the distribution of uptake
in the pulmonary region.
Tissue dose in the PAR along the shortest path is
approximately 50% larger than that along the longest path.
Ultman and Anjilvel
(1990)
Human LRT/Weibel
(1963)
Investigates two
formulations:
Miller et al. (1978) and
Miller et al. (1985)
Model development; assumes quasi-steady
conditions. Air phase concentration and tissue
dose profiles for the two reaction schemes, for
various ventilatory parameters and various
liquid lining transport and chemical parameters.
Compares predictions of first order reaction
scheme to results of Miller et al. (1985).
This model does not conserve mass. Predications should Orotberg et al.
only be considered qualitatively. Maximum tissue dose in (1990);
respiratory bronchioles for both chemical reaction schemes. [Grotberg (1990)]
Rat LRT/based on
serial reconstruction
of a set of
intrapulmonary
airways and their
ventilatory units
combined with a
single path from the
larynx to the
reconstructed set
based on Yeh et al.
(1979)
TB and PUL region mass
transfer coefficients based
Overton etal. (1987)
Model development. Illustrates the influence of
ventilatory unit size and proximal anatomic dead
space and on the uptake and distribution of
inhaled 03 in ventilatory units.
[Illustrates the influence of ventilatory unit
volume on the distribution of inhaled 03 within
ventilatory units.]
Ventilatory unit uptake is significantly influenced by both
proximal airway deadspace and ventilatory unit volume.
Flux of 03 to air-liquid interface in the proximal portions
of larger ventilatory units are significantly greater man in
smaller units.
Mercer et al. (1991)
[Mercer and Crapo
(1993)]
-------
TABLE 8-5 (cont'd). THEORETICAL OZONE DOSIMETRY INVESTIGATIONS'
Species and Region
Modeled/Anatomical
Model
Liquid Lining and Tissue
Transport and Chemical
Reactions
Dosimetry Model /Subject of Investigation
Results, Predictions
Reference
Rat ventilatory unit/
Mercer et al. (1991)
Mercer et al. (1991)
Mercer et al. (1991). Along a path distally
from a bronchiolar-alveolar duct junction ,
compares experimentally determined changes in
ventilatory unit wall thickness due to an O3
exposure to dosimetry model predictions of flux
to the air-liquid interface.
As a function of distance from the broncho-alveolar duct Pinkerton et al.
junction, experimentally determined relative changes in (1992);
ventilatory unit wall thickness due to Oj exposure are very Miller and Conolly
similar to predicted relative fluxes to the air-liquid (1993)
interface.
Human total RT/URT:
Fredberg et al.
(1980);
LRT: Weibel (1963)
Pseudo steady-state
diffusion and first order
reactions combined with
biochemical data of Miller
et al. (1985)
Model development. Illustrates LRT
distribution of (1) airphase concentration at
various times during the breathing cycle and
(2) Oj flux (dose) to liquid lining and to tissue.
Flux of 03 to air-liquid interface decreases distally; flux to
tissue increases along the conducting airways, reaches a
maximum in the terminal bronchioles then decreases
rapidly in the gas exchange region.
Hu et al. (1992a)
oo
URT = upper respiratory tract; total RT = total respiratory tract; LRT = lower respiratory tract; PAR = proximal alveolar region; generally, for modeling purposes, the first pulmonary
region
generation or model segment; PUL = pulmonary region; FRC = functional residual capacity; TB = tracheobronchial region; FEVj = forced expiratory volume at 1 s.
Refers to the theoretical or mathematical formulation aspects of gas transport and reactions without the specification of morphometric and physiological parameter values.
-------
1 has been assumed to be applicable to O3. Ultman and Anjilvel (1990) use a Monte Carlo
2 method to simulate O3 uptake. Based on the physical and chemical principles of mass
3 transport in the respiratory tract, probabilities are assigned to the fate of a molecule in a way
4 so as to account for convection, dispersion, and loss to the liquid lining.
5 Dosimetry models published since 1986 can be grouped according to how transport and
6 chemical reactions are modeled in respiratory tract fluids and tissues: those based on the
7 formulation of (1) Miller (1977) and Miller et al. (1978) who used an instantaneous reaction
8 scheme and (2) Miller et al. (1985) who used a quasi-steady first order reaction scheme.
9 These two approaches are discussed in the earlier criteria document (U.S. Environmental
10 Protection Agency, 1986). In addition to the use of similar formulations for liquid and tissue
11 transport/reactions, all of the post 1986 studies use essentially the same biochemical data of
12 either Miller et al. (1985) for humans or Overton et al. (1987) for laboratory animals. The
13 implication is that most of the studies are expected to predict qualitatively similar results.
14 There are minor variations on the second chemical reaction formulation. Hanna et al.
15 (1989) use a time-dependent diffusion reaction equation, instead of time-independent (quasi-
16 steady) equation as do Miller et al. (1985). Based on the rate constants used by Hanna et al.
17 (1989) and on discussions in Miller et al. (1985) and in Grotberg et al. (1990), one can infer
18 that the modeled transport processes in the liquids and tissues are essentially quasi-steady,
19 which is equivalent to the second formulation. Another variation uses mass transfer
20 coefficients determined by the second formulation and the biochemical assumptions of Miller
21 et al. (1985) or Overton et al. (1987). The liquid and tissue transport/reaction formulation
22 for specific investigations is indicated in column 3 of Table 8-5.
23 In addition to the assumptions and the formulation of equations that describe the
24 transport and loss of O3 in the respiratory tracts of humans or laboratory animals, it is
25 important to evaluate whether simulation results reflect accurate solutions to the mathematical
26 dosimetry model formulation. Of the five distinct model formulations listed in Table 8-5,
27 Overton et al. (1987), Mercer et al. (1991), and Hu et al. (1992a) discuss most or all of the
28 relevant issues of stability, solution convergence, and mass conservation. In addition, using
29 steady unidirectional flow in a straight tube as a test case, they report successfully simulating
30 analytical solutions to their equations of transport and uptake. Neither Hanna et al. (1989),
31 Ultman and Anjilvel (1990), nor Grotberg et al. (1990) address the issue of accuracy. There
December 1993 8-36 DRAFT-DO NOT QUOTE OR CITE
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1 is no reasonable way to judge whether or not the solutions of Hanna et al. (1989) or of
2 Ultman and Anjilvel (1990) accurately represent solutions to their dosimetry model
3 assumptions and formulations; however, with the exception of Grotberg et al. (1990), there
4 are no reasons to assume that solutions of these models are not accurate.
5 Because the Grotberg et al. (1990) model formulation is different than the others, an
6 explanation is needed. Based on the smallness of relevant parameters, Grotberg et al. (1990)
7 assume quasi-steady conditions for O3 concentration and air velocity in the air compartments
8 and obtain approximate analytical solutions to the tune-independent three-dimensional
9 equation of continuity for a model airway, and apply the results to the morphometric model
10 of Weibel (1963). One advantage of analytical solutions is that they account naturally for
11 parameters (such as dispersion and gas phase mass transfer coefficients) or local processes
12 (e.g., possibility of high uptake at airway entrances) that must be known and estimated for,
13 or incorporated into, the one-dimensional approach. Grotberg et al. (1990) carry out
14 simulations using anatomical and physiological conditions based on Miller et al. (1985) and
15 compare their results to the results of the latter. Although their predictions are qualitatively
16 similar to Miller et al. (1985), they predict significantly larger pulmonary tissue doses (up to
17 as much as 10 times) than do Miller et al. (1985). A comparison of the pulmonary region
18 doses predicted by Grotberg et al. (1990) to those of Miller et al. (1985) indicates that the
19 Grotberg et al. (1990) model does not conserve mass: it predicts that the pulmonary region
20 absorbs over 3.4 times the amount of O3 inhaled. The overprotection may be an artifact of
21 the quasi-steady approximation, because effects due to differences in the time of flights from
22 the trachea to different LRT locations are not taken into account. In any case, the
23 quantitative predictions reported by Grotberg et al. (1990) are questionable.
24 Chemical data more recent than used for the dosimetry models in Table 8-5 show that
25 compounds other than unsaturated fatty acids (the only compound with which O3 is assumed
26 to react in the models using the second chemical reaction formulation) are as reactive or
27 more reactive with O3 (Pryor, 1992). Using these data, estimates of O3 diffusion
28 coefficients in the liquid lining and lipid bilayers, layer thicknesses, and data on the
29 concentrations of biocompounds in these layers, Pryor (1992) estimates that (1) most of any
30 O3 that penetrates into a cell bilayer reacts within the layer, very little if any penetrates to
31 the cell interior, and (2) O3 will not penetrate the liquid lining where it is greater than
December 1993 8-37 DRAFT-DO NOT QUOTE OR CITE
-------
1 0.1/*m thick. Several relevant comments are given by Pryor (1992): (1) the calculations
2 are considered a crude first approximation; (2) the possibility that a small fraction of 63 may
3 penetrate the bilayer and reach the cell interior can not be excluded; and (3) surfactant layers
4 can be very thin, and some cells may not be protected very well or at all.
5 If the conclusions of Pryor (1992) are essentially correct, they have implications for
6 past and future dosimetry modeling studies because: (1) past investigations have
7 underestimated the reactivity of O3 with biocompounds; (2) with respect to cellular damage,
8 products of O3 reactions in the liquid lining may be the main toxic compound; (3) increasing
9 the value of rate constants would have no effect on predictions of dosimetry models using the
10 instantaneous reaction scheme, because rate constants in this approach are assumed to be
11 infinite, however, increasing the concentration of reacting biocompounds would increase
12 uptake; (4) use of only unsaturated fatty acid data (with a first order reaction scheme) results
13 in an underestimate of the reactivity of O3 in the liquid lining and an overestimate of the
14 O3 tissue dose, but a possible underestimation of the toxic dose that is due to reaction
15 products; and (5) with higher O3 reaction rates, the first order chemical reaction formulation
16 would result in larger predicted uptakes.
17
18 8.2.4.2 Dosimetry Model Predictions
19 Similarity of Model Predictions
20 A survey of dosimetry modeling results shows that in some areas of investigation there
21 is a qualitative similarity in predictions by models of different groups of investigators for
22 different species or sub-populations.
23 (1) Distribution ofLRTdose (dose profiles or dose versus generation). As shown in
24 Figure 8-5, beginning at the trachea, net dose (O3 flux to air-liquid interface)
25 slowly decreases distally in the tracheobronchial region and rapidly decreases
26 distally in the pulmonary region. Tissue dose (O3 flux to liquid-tissue interface)
27 is very low in the trachea, increases to a maximum in the terminal bronchioles or
28 first generation in the pulmonary region, and decreases rapidly distally from this
29 location, (e.g., Miller et al., 1978, 1985, 1988; Overton et al., 1987, 1989;
30 Overton and Graham, 1989; Grotberg et al., 1990; Hu et al., 1992a).
December 1993 8-38 DRAFT-DO NOT QUOTE OR CITE
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10"
cr>
10~8-
101
10
Zone
Order
Generation
A Generation
1 ^
O
O
O
0
Human -
Rat
Guinea Pig
Rabbit
(No
•.
2 3
2 3
2 4
2 4
•~.u
VT (mL)
• 8OO.O
-• — -•- 1 .88
2.63
1 3.20
absorption In tha
4
4 s e
e a 10
e 8 10
f (bpm)
1S.O
ee.o
60. »
38.8
URT)
**~l
•" 1
s e
7 8 » 1
12 14 16 1
12 14 181
i . 1.1 .1
^^ N
\ \.
s\ v
v. \ '» ' .
X'\ s *
N\ •»
^>>
P
^•I ^^ 1
7 8
311 12 13 14
S 17 18 21 23
7 18 2O 22 23
-
-
Rabbit
Guinea Pig
Rat
Human
10-
<&-'•'
•^,.:-*
•" Human ^~
Rat --
Guinea Pig ——-
Rabbit
VT (mL) f (bpm)
8OO.O 1S.O
1.88 86.O
2.63 8O.8
13.2O
(No absorption In the URT)
Order
Generation
B Generation
O
O
O
O
2
2
2
2
3
3
4
4
4
e
e
s
a
8
e
10
10
B
7
12
12
8
14
14
e 7
0 10 11
1B 18 17
1« 17 18
12
18
SO
13
21
22
8 Rabbtt
14 Guinea Pig
23 Rat
23 Human
Figure 8-5. Net dose (A) and tissue dose (B) versus sequential segments along
anatomical model airway paths for human, rat, guinea pig, and rabbit.
In general, each segment represents a group of airways or ducts, with
common features as defined by the designers of the anatomical model
(human and rat: generation; guinea pig: order; rabbit: zone). For a
given species the plotted dots represent a predicted dose that corresponds to
a given segment. The dots have been joined by lines for ease of
interpreting the plots; these lines do not represent predicted values except
where they intercept the dots. TB = tracheobronchial region.
P = pulmonary region.
Source: Overtoil and Miller (1988).
1 If O3 were the only toxic agent and all the tissues of the LRT were equally sensitive to
2 the same dose, the models predict that the greatest morphological damage would occur in the
3 vicinity of the junction of the conducting airways and the pulmonary region and decrease
4 rapidly (distally) from this area, which is consistent with observations in laboratory animals
December 1993
8-39
DRAFT-DO NOT QUOTE OR CITE
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1 (see Section 6.2.4). On the other hand, using the best estimates of morphometric and
2 physiologically based biochemical parameters of Miller et al. (1978, 1985) and Overton et al.
3 (1987), the models predict extremely (relatively) low tissue doses in the trachea and large
4 bronchi; this suggests very little or no tissue damage should occur there, which is contrary to
5 observations (see Section 6.2.4). However, this is moot, if as suggested by Pryor (1992),
6 the toxic substances are primarily reaction products of and not O3 itself. In this case, the
7 O3 net local dose, not the local O3 tissue dose, may be a better estimator of local toxic tissue
8 dose, since the rate of production of products would be related to the rate of O3 uptake.
9 (2) Effect of exercise or increased ventilation. The effect of exercise is to slightly
10 increase the tracheobronchial region dose and significantly increase the
11 pulmonary region total dose (mass of O3) and the centriacinar region dose (mass
12 per unit surface area), (e.g., Miller et al., 1979, 1985; Overton et al., 1987,
13 1989; Overton and Graham, 1989; Hanna et al., 1989; Grotberg et al., 1990.)
14
15 (3) Effect of respiratory tract inhomogeneity. Models have predicted that the further
16 the proximal alveolar region is from the trachea, the less the O3 tissue dose
17 (mass of O3 absorbed per unit surface area) to the proximal alveolar region.
18 (For modeling purposes, the proximal alveolar region has been defined as the
19 first pulmonary generation or the first pulmonary region model segment along a
20 path; this region is a part of the centriacinar region.) Overton et al. (1989)
21 predicted a threefold greater proximal alveolar region dose for the shortest path
22 relative to the longest path in rats. Ultman and Anjilvel (1990) simulated
23 O3 distribution in a small segment (< 1 %) of the distal airways of an asymmetric
24 anatomic model of the human lung. They found that the O3 tissue dose
25 (mass/cm2) in the proximal alveolar region along the shortest path was
26 approximately 50% greater than that along the longest path. Mercer et al.
27 (1991) found that path distance and ventilatory unit size affect dose: predicted
28 dose in the proximal segments (essentially, the proximal alveolar region) of the
29 larger ventilatory units (with the smallest proximal dead space) are significantly
30 larger than the average proximal segment doses. Further, for the small sample
31 of ventilatory units modeled (43), Mercer et al. (1991) predicted a range of
December 1993 8-40 DRAFT-DO NOT QUOTE OR CITE
-------
1 proximal segment doses of greater than a factor of 6. Because the proximal
2 alveolar regions of Ultman and Anjilvel (1990) and of Mercer et al. (1991)
3 belonged to a "local cluster" and there are many clusters with varying distances
4 from the trachea, a greater variability than 50% and a factor of 6, respectively,
5 in proximal alveolar region doses is to be expected. Mercer and Crapo (1993)
6 illustrateed the effect of ventilatory unit volume alone on the distribution of dose,
7 predicting that a 2.3 times bigger unit receives 1.9 times the dose (mass per
8 surface area) of the smaller unit at the entrance of the unit.
9 The variability of predicted proximal alveolar region and, by inference, centriacinar
10 region doses suggests the magnitude of lexicological effects for different centriacinar regions
11 are different. This prediction is consistent with the observations of Schwartz et al. (1976)
12 and Boorman et al. (1980) of variation of damage among different centriacinar regions of the
13 same rat. It is reasonable to assume that variable damage at equivalent but different
14 morphological locations also occur in humans.
15
16 Specific Topics
17 Effect of Assumptions About Anatomical Dimensions. For rats and guinea pigs,
18 Overton et al. (1987) used two morphometrically based anatomical models (rat anatomical
19 models: Kliment, 1973, and Yeh et al., 1979; guinea pig anatomical models: Kliment,
20 1973, and Schreider and Hutchens, 1980) to investigate the influence of anatomical model
21 formulation on predicted uptake. Results with all four anatomical models in combination
22 with different ventilatory parameters showed a qualitative similarity in the shapes of the dose
23 profiles, but the two anatomical models for the same species resulted in considerable
24 differences in predicted percent total and pulmonary region uptakes.
25
26 Respiratory Tract Uptake in Human Adults and Children. Overton and Graham
27 (1989) used several sources of data on age-dependent LRT dimensions and structure to
28 construct theoretical LRT anatomical models for humans from birth to adult. The
29 O3 dosimetry model of Miller et al. (1985) was used to estimate the regional and local
30 uptake of O3. For the percent uptake (84 to 88%) during quiet breathing, the LRT
31 distribution of absorbed O3 and the centriacinar O3 tissue dose are not very sensitive to age.
December 1993 8-41 DRAFT-DO NOT QUOTE OR CITE
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1 For heavy exercise or work, regional percent uptakes are more dependent on age than during
2 quiet breathing, and total uptakes range from 87 to 93%. Generally, the total quantity of
3 O3 absorbed per minute increases with age. For all conditions simulated, the largest
4 O3 tissue dose is predicted to occur in the centriacinar region. Miller and Overton (1989)
5 present similar results. Because uptake by the URT was not simulated and because this
6 region can be assumed to have an important effect on LRT uptake, a comparison of
7 predictions of LRT uptakes in children and adults should be viewed with caution. On the
8 other hand, URT uptake probably has little effect on the shape of the dose curves.
9
10 Dosimetry Modeling Results Compared to Dosimetry Data
11 Based on the experimental conditions discussed in Gerrity et al. (1988) and using the
12 model and parameters of Miller et al. (1985), Miller et al. (1988) simulated the uptake of
13 O3 distal to the oropharynx of human subjects. For the target fBs of 12 and 24 bpm and VTs
14 ranging from 0.4 to over 1.6 L, the simulation results were in good agreement with the
15 breath-by-breath experimental data. The average experimental LRT uptake efficiency was
16 «0.91 as compared to the 0.89 prediction given by Miller et al. (1985) for the region distal
17 to the oropharynx. It should be kept in mind, however, that values for uptake efficiency
18 from the Gerrity et al. (1988) study were derived from the raw data using a steady-state
19 method, whereas the models of Miller et al. (1985) and Miller et al. (1988) utilize cyclic
20 flow, thus making the predictions more appropriate for comparison with uptake data from
21 non-steady-state methods. From an analysis hi Gerrity et al. (1993a), it appears that total RT
22 uptake computed by either steady-state or non-steady state methods differ by only about 10%
23 in relative terms.
24 A further refinement of the models of Miller et al. (1985) and Overton et al. (1987) has
25 been made in which an URT has been added, thus allowing comparisons with total RT
26 uptake efficiency data. The refined model utilizes assumptions shown in Table 8-6.
27 Based on Table 8-6, the mass transfer coefficients for the nasopharyngeal and
28 tracheobronchial regions of the rat were computed using two sets of data. Firstly,
29 calculations were based on the total uptake and the 18O3 nasopharyngeal uptake data of Hatch
30 et al. (1989), which were the averages for 8 rats. The second set of data was that of the
31 8 rats. Both approaches gave essentially the same set of parameters. The first method
December 1993 8-42 DRAFT-DO NOT QUOTE OR CITE
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1 reproduced the two uptakes exactly; for the second, the simulation results deviated from the
2 experimental values by an average of «23%. Given the assumptions and results, predictions
3 of total uptake are expected to be realistic, but predictions of respiratory tract dose
4 distribution should be considered speculative.
5 Using the human assumptions (Table 8-6), the bolus-response experiment of Hu et al.
6 (1992b) was simulated. Results for the experimental bolus fractional uptakes were good,
7 over-predicting by «5%; the results for the remaining two experimental variables deviated
8 from the experimental values by »30%. As a consequence of the assumptions and results,
9 simulations with flow rates of «250 mL/sec are expected to overpredict by no more than
10 « 5 %, and predictions of the distribution of dose within the respiratory are speculative.
11 Given the assumptions of Table 8-6, Table 8-7 shows a comparison between
12 experimental total RT uptake efficiency data and model predictions for humans. Use of the
13 rat assumptions in conjunction with the model will be discussed later (Section 8.4.3).
14 The model predictions show good agreement with the total RT uptake efficiency data of
15 Gerrity et al. (1988), Gerrity et al. (1993a), and Hu et al. (1992b). In all cases, the
16 predictions are within 10% of the measured values. The agreement with the data of Hu
17 et al. (1992b) is even better.
18 The model prediction for the data of Wiester et al. (1993) is less accurate. Comparison
19 of the Wiester et al. (1993) data with the Hu et al. (1993) data (Figure 8-1) show, however,
20 that they are in good agreement with each other. It would thus appear the VT dependence of
21 the model does not necessarily reflect the real world. However, the general agreement
22 between the model predictions and data are quite good.
23 Although the models are capable of making reasonable predictions of total RT uptake
24 efficiency, their accuracy on the regional level remains uncertain. The O3 bolus data (Hu
25 et al., 1992b, 1993; Ultman et al., 1993); and the airway uptake efficiency data (Gerrity
26 et al., 1993b) bring into question the model predictions about uptake in the conducting
27 airways. These latter data sets suggest that the models of Miller et al. (1985, 1988) may
28 underestimate the O3 uptake coefficients in the conducting airways. Ultman et al. (1993)
29 show in their analysis of the bolus data that the reactivity of O3 with the lung liquid lining
30 decreases with increasing depth into the lung. This could imply that more O3 is taken up in
31 central airways than had previously been thought. The predictions presented in Table 8-7
December 1993 8-43 DRAFT-DO NOT QUOTE OR CITE
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TABLE 8-6. ASSUMPTION FOR APPLICATION OF DOSIMETRY MODEL
TO BREATHING FREQUENCY RESPONSES TO OZONE
Species
Mode of
Breathing
Respiratory Tract Morphology
Mass Transfer Coefficients
Human Oral
Rat
Nasal
LRT structure from Weibel (1963)
Volume of oral cavity through
larynx and surface to volume
ratio (S/V) from Hu et al.
(1992a,b)
Dead space volume (Vj) and FRC
from Hart et al. (1963); TB
region volume at PRC equals V^
minus oropharyngeal volume
Proximal alveolar region defined as
first respiratory bronchiole
Pulmonary region expands;
tracheobronchial (TB)
region does not expand
• Nasopharyngeal (NP) dimensions
from Schreider and Raabe (1981)
TB region from Yeh et al. (1979)
Volumes and surface areas of LRT
isotropically scaled to FRC
• Pulmonary region from Mercer
et al. (1991)
• TB and pulmonary regions
uniformly expand during
breathing
- Proximal alveolar region is first
generation of pulmonary region
Mass transfer coefficients for each
oropharyngeal segment and each TB
generation defined as Ka/(S/V) where
S/V for the TB region is from Weibel
(1963) dimensions reduced to FRC.
Ka from Hu et al. (1992b)
Pulmonary mass transfer coefficient is
0.10 cm/a (Miller et al. [1985])
NP and TB mass transfer coefficients
estimated using data of Hatch et al.
(1989)
Mass transfer coefficient of pulmonary
region = 0.137 cm/s; inferred from
Pinkerton et al. (1992)
TABLE 8-7. COMPARISON OF TOTAL RESPIRATORY TRACT UPTAKE
DATA WITH MODEL PREDICTIONS
VT(mL)
832
832
500
1,000
1,650
1,239
631
fB (bpm)
12
24
15
7.5
1,360
1,360
16
Measured Ft
0.97
0.96
0.86
0.93
0.88
0.87
0.76
Predicted Ft
0.96
0.93
0.89
0.94
0.95
0.93
0.94
Data Source
Gerrity et al. (1988)
Gerrity et al. (1988)
Hu et al. (1992b)
Hu et al. (1992b)
Gerrity et al. (1993a)
Gerrity et al. (1993a)
Wiester et al. (1993)
December 1993
8-44
DRAFT-DO NOT QUOTE OR CITE
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1 based on the assumptions of Table 8-6, however, represent a revision of the Miller et al.
2 (1985) model in that the mass transfer coefficients are derived from the actual human data of
3 Hu et al. (1992b).
4 Because the utility of dosimetry models is in their ability to facilitate interspecies
5 extrapolation, it is important to compare predictions with animal data as well as human data.
6 Using the Overton et al. (1987) model formulation and parameters, Overton et al. (1989)
7 developed a formula that can be used to calculate respiratory tract uptakes in rats, given their
8 VTs, and fBs. For comparison purposes, the data of Wiester et al. (1987, 1988) and Hatch
9 et al. (1989) can be used. The average uptake efficiency for the rat from these data is 0.45.
10 Based on the VTs and fBs of these animals, an average LRT uptake of 0.61 is computed
11 using Overton et al. (1989). If Overton et al. (1989) had included the effects of URT
12 uptake, their model would have predicted more than 0.61.
13
14
15 8.3 SPECIES SENSITIVITY: LUNG FUNCTION AND
16 INFLAMMATORY ENDPOINTS EXEMPLIFYING AN APPROACH
17 8.3.1 Introduction
18 Quantitative extrapolation of animal-based O3 toxicity data to the human circumstance
19 requires a paradigm that includes both an estimate of target tissue dose (dosimetry) and an
20 algorithm which relates the responsiveness of the test species to that of the human (species
21 sensitivity). This paradigm can be depicted as an extrapolation parallelogram (Figure 8-6)
22 which conceptualizes a relationship between chronic animal study data and long-term human
23 health effects based upon understanding of acute effects in both species (Graham and Hatch,
24 1984). Though recent studies have begun to elucidate the underlying mechanisms
25 determining response, the bulk of the present O3 toxicity data base in animals and humans
26 remains largely descriptive. Hence, only a simplified application of this paradigm is feasible
27 at this time. The following section will attempt to harmonize a selective literature on acute
28 human and animal responses to O3 exposure (already reviewed in detail in Chapters 6 and 7)
29 with what is known about the dosimetry of O3 in an effort to discern relative species
30 sensitivity. To construct an argument that is plausible for this test application, focus is on
31 endpoints for which there are sufficient data in both humans and test animal species and for
December 1993 g-45 DRAFT-DO NOT QUOTE OR CITE
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Acute Effect
Human
Chronic Effect
Human
Acute Effect
Animal
Chronic Effect
Animal
— Experimental correlation
Extrapolation
Figure 8-6. Parallelogram paradigm for utilizing animal data for human health
predictions. Acute homologous endpoints serve as the basis for
extrapolating chronic effects hi humans from animal data.
1
2
3
4
5
6
7
8
9
10
11
12
13
which exposure scenarios are similar. The endpoints compared include measures of
pulmonary function and markers of lung inflammation, most notably bronchoalveolar (BAL)
protein and cells. When possible, other influencing parameters, such as ventilation
augmentation and antioxidants within the lung will also be discussed. The body of in vitro
cell studies has not been included because of the difficulties in interpretation associated with
dosimetry and culturing systems. The reader is referred to Section 7.2.5 and the recent
review by Koren et al. (1993). The goal here is to develop a hypothetical model for the
assessment of species specific sensitivity with acute O3 exposure that can serve as a
framework to better predict human responses, especially with regard to chronic effects. The
complex issue of whether controlled human clinical studies accurately reflect population-
based responses also will not be considered in this discussion.
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1 8.3.1.1 Dosimetry
2 This topic has been discussed in detail in Section 8.2. Recent studies by Hatch et al.
3 (1993) utilizing the nonradioactive isotope of oxygen, 18O, to label O3, have shown that
4 exposure of exercising humans (60 L/min) and resting rats to 0.4 ppm for 2 h resulted in
18
5 3 or 4 times the O dose (as adduct) to the BAL constituents of humans as compared to
6 those of F344 male rats. This 3- to 4-fold difference appeared to be due to the exercise-
7 stimulated hyperventilation of the humans when compared to the rat and compared favorably
8 with indices of effect (i.e., BAL cells and protein at 24 h). Only when the rats were
9 exposed to 2 ppm for 2 h did their 18O3 labeling of BAL constituents approximate that of the
10 human. Thus, on the basis of this study of cellular and protein influx due to O3 injury, the
11 rat and human appear to have similar sensitivity to O3 when exercise is considered.
1 8
12 Additional related studies with O3 indicate that deposition in the respiratory tract is a
13 cumulative function of ventilation over the initial period of exposure which would lend
14 support to these findings (Santrock et al., 1989). Attempts to compare animal data obtained
15 without exercise to human study data with exercise would thus underestimate the dose to the
16 lung and presumably the resultant risk of effect. Studies of O3 effects (but without
17 assessment of dose) in exercising rodents have confirmed this conclusion (Mautz et al.,
18 1985).
19 Exercise is only one factor that can alter dose and effect. Studies in laboratory animals
20 which incorporate other factors, such as time of day or diurnal rhythms (Van Bree et al.,
21 1992), animal strain (Pino et al., 1991; Costa et al., 1993a), or nutrition (Section 6.2.5),
22 also show substantial modification of response to O3, and thus emphasize the need for careful
23 consideration of exogenous factors when attempting to compare or extrapolate study findings.
24 It is likely that a similar range of factor-dependent variability exists within human test
25 subjects.
26
27 8.3.2 Homology of Response
28 The concept of species sensitivity actually consists of two integrated components. The
29 first, homology of response, indicates whether the outcome seen in the animal test species
30 represents the same biological response in the human. In many cases, a measurement of the
31 same endpoint in both species can be presumed to reflect the same toxic phenomenon and/or
December 1993 3.47 DRAFT-DO NOT QUOTE OR CITE
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1 mechanism (i.e., the pulmonary irritant-induced tachypnea) (see below). On the other hand,
2 there may be endpoints which, while homologous, are not expressed similarly. One such
3 example would be the burning discomfort of sensory irritation in the human and the pause on
4 tidal expiration seen in rodents. The second component of species sensitivity relates the
5 dose-response curve for given homologous responses. Alterations in permeability of the air-
6 blood barrier of the lung appear to reflect true species differences in sensitivity to pulmonary
7 irritants such as O3 (Hatch et al., 1986). Ideally, these elements of species sensitivity should
8 flow directly into extrapolation formulae developed to integrate animal and human research
9 data.
10
11 8.3.2.1 Lung Function Endpoints as Homologous Indicators
12 Lung function studies of small mammals have provided basic physiological information
13 important to the understanding of both normal and diseased lungs (Snider and Sherter, 1977;
14 Harkema et al., 1982; Raub et al., 1982; Mauderly, 1984; Costa, 1985). Animal lung
15 function tests, adapted from those used clinically, have proven useful in describing the nature
16 and severity of lung injury as well as distinguishing toxicant-induced effects in the central or
17 peripheral airways from those that exist in the parenchyma. In practice, the interpretation of
18 functional changes detected in animals derives from knowledge and experience in human
19 pulmonary medicine. Supporting this view in theory is the allometric data base in normal
20 mammals in which the lung function variables associated with ventilation and aerobic
21 metabolism scale systematically to body mass over nearly seven orders of magnitude (Stahl,
22 1967; Leith, 1976). The lung function studies of O3 toxicity hi animals and humans,
23 considered in the present discussion, are described in detail in Sections 6.2.5 and 7.2 of the
24 current document or in the previous O3 criteria document (U.S. Environmental Protection
25 Agency, 1986).
26
27 8.3.2.2 Inflammatory and Antioxidant Endpoints as Homologous Indicators
28 Inflammation of pulmonary airways and airspaces is best described as a cascade of
29 events networking infiltrating leukocytes, plasma proteins, and cell-derived mediators which
30 function presumably to defend or repair (but may further damage) the injured lung (see
31 Sections 6.2.2 and 7.2.5; Koren et al., 1993). Key markers of the basic inflammatory
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1 process include plasma-derived proteins (including, albumin, globulins, plasmin, etc.) and a
2 primary inflammatory cell, the polymorphonuclear leukocyte (PMN). Of these two markers,
3 plasma-derived proteins in the acute phase are generally thought to represent a "leak" from
4 the vasculature to the airspace lumen. Hence, under controlled temporal conditions, plasma
5 protein residing in the airspace and accessible by BAL can be used as a proportional marker
6 of effect, which in turn should be related to dose. The presence of PMNs in the airspaces is
7 a bit more complex because of the signals involved in recruiting these cells into the lung
8 lumen after injury and the cascade of events apparently involved in their poesis from the
9 vasculature to the lung lumen. For the purposes of species comparison, plasma-derived
10 protein (nonspecific) and the proportion of PMNs among total cells as sampled by BAL will
11 be emphasized as primary indices of damage and inflammation within the lung.
12 Antioxidant substances in lung tissue (Slade et al., 1985) and BAL fluid and cells
13 (Slade et al., 1993) have been identified and quantified for humans and several laboratory
14 animal species. The species profiles of these antioxidants in the lung tissue and their
15 respective BAL cells and fluid can differ appreciably (Table 8-8), but collectively they
16 appear to play a significant role in defense of the lung against both endogenous and
17 exogenous oxidant challenge. Ascorbate and vitamin E in particular appear to have major
18 functions in protecting the lung from O3 challenge (Section 6.2.6; Slade et al., 1989;
19 Crissman et al., 1993; Koren et al., 1989b; Elsayed et al., 1988) and when their levels are
20 manipulated in vivo, either can influence the degree of toxic outcome. Hence, the
21 measurement of basal and O3 response levels of these antioxidants in BAL cells and fluids is
22 useful in assessing the qualitative and quantitative responses among humans and laboratory
23 test species.
24
25 8.3.3 Studies of Lung Function
26 8.3.3.1 Confounding Influences in Lung Function Studies
27 Ideally, a system for measuring pulmonary function in small animals would
28 approximate that used in humans with cooperative, unrestrained subjects. However, in
29 animal studies, this is usually not possible. Fortunately, certain measures (e.g., static lung
30 volumes, diffusion capacity) appear to be minimally influenced by sensory reflex or muscular
31 activity in spite of unnatural stresses or blunting of responses caused by anesthetic or
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TABLE 8-8. PULMONARY ANTIOXIDANT SUBSTANCES IN VARIOUS
LABORATORY ANIMAL SPECIES AND HUMANS8
Mouse
Ascorbate
tissue 41 ±4
BAL cells0 —
BAL fluid0 -
Glutathione
tissueb 62 ±3
BAL cells0 -
BAL fluidc —
Tocopherol
tissue 1.0 ±0.1
BAL cellsd -
BAL fluidd -
Uric Acid
tissue —
BAL cells0 -
BAL fluid0 -
Hamster Rat
26±2 34±2
— 50.3 ±5.4
— 199.4±9.1
61 ±2 50±2
— 14.8±2.7
- 12.1 ±5.0
1.0±0.1 2.1±0.1
- 577.7 ±83.1
- 0.6 ±0.2
— 0.35 ±0.05
- <0.01
- 4.3 ±0.6
Guinea Pig Rabbit
39±1 27 ±3
17.9±1.4 —
28.8±2.2 —
83 ±3 83 ±3
14.6±2.4 —
li.2±1.9 —
2.0±0.2 1.4±0.2
454.5 ±58.2 —
1.4±0.5 —
4-.14±0.24 -
0.8 ±0.1 —
2.7 ±0.4 -
Human
22 ±7
3.5±0.1
21.4±2.8
7±1
2.9±0.5
20.4±3.8
0.8±0.1
95.1 ±23.4
47.2±3.8
_
0.07 ±0.03
15.9±2.5
aData (mean ± SE) extracted and summarized from Slade et al. (1985, 1993).
Data expressed as mg/100 g wet tissue.
Data expresses as nmol/mg protein.
Data expressed as nmol/mg lipid phosphorus.
1 physical immobilization. On the other hand, some measurements, typically those involved in
2 the assessment of ventilatory mechanics, can be profoundly influenced by these and other
3 factors, such as ambient and toxicant-altered body core temperature, thus confounding
4 cross-species comparisons. Because a major emphasis of this section is the comparison of
5 lung function data of animals and humans, it is important that the reader realize potentially
6 confounding influences borne by studies of lung function in rodents when compared to
7 analogous measurements in humans. These are discussed briefly below.
8
9 Anesthesia
10 Anesthesia alters pulmonary function measurements in both humans (Rehder et al.,
11 1975) and laboratory animals (Skornick and Brain, 1990; Lamm et al., 1982; Rich et al.,
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1 1979). In general, ventilation is reduced and changes in ventilatory patterns occur (Pavlin
2 and Hornbein, 1986; Belville et al., I960; Hunter et al., 1968; Siafakas et al., 1983).
3 In humans, anesthesia can decrease compliance and functional residual capacity (FRC), and it
4 can also increase airway resistance (Rehder et al., 1974, 1975). In small laboratory
5 mammals, an analogous decrease in FRC occurs, though apparently via a different
6 physiological mechanism (Lamm et al., 1982). Additional anesthesia-related effects include a
7 blockade of irritant reflexes (Weissberg et al., 1976) and alteration of ventilatory patterns in
8 response to carbon dioxide (CO2) (Martin-Body and Sinclair, 1985). Hence, while not
9 invalidating experimental results, choice of anesthetic agent may affect the measured
10 response and may confound cross-species comparison.
11
12 Restraint
13 To enable collection of small animal pulmonary function data without the use of
14 anesthesia, some type of physical immobilization is usually required. Restraint may range
15 from minimally restrictive, allowing turning and some locomotion, to extremely confining,
16 as occurs when animals are inserted into nose-only exposure tubes. Although restraint
17 reduces movement artifacts and permits attachment of delicate probes or sensors,
18 immobilization can also produce undesirable physiological disturbances such as changes in
19 body core temperature (Nagasaka et al., 1979), hypermetabolism (Nagasaka et al., 1980),
20 increased expiratory CO2 (Jaeger and Gearhart, 1982), changes in ventilation and ventilatory
21 pattern (Lai et al., 1978; Mauderly, 1986) and gastric lesions (Toraason et al., 1980). Such
22 stress-related responses are poorly understood and their influence on toxicologic responses
23 may well pass unnoticed unless specifically examined.
24
25 Temperature
26 Although not widely appreciated, toxicant-induced changes in thermoregulatory function
27 can modify the results of toxicological studies (Gordon et al., 1988; Gordon, 1991). Recent
28 studies indicate that exposure to 0.37, 0.5 and 1.0 ppm O3 also can decrease core
29 temperature (Tco), heart rate, and blood pressure over 2 or more hours in unrestrained,
30 unanesthetized rodents maintained at normal room temperature (Uchiyama et al., 1986;
31 Watkinson et al., 1992). On the other hand, when rats were restrained in a head-out body
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1 plethysmograph and exposed to the same concentration of O3 (1.0 ppm), as in the Uchiyama
2 et al. (1986) and Watkinson et al. (1992) studies, no change in blood pressure was observed
3 (Tepper et al., 1990). The discordance between these findings may be the result of restraint
4 stress which has been shown to increase Tco (Nagasaka et al., 1979) and in this circumstance
5 could have blunted the decrease in Tco associated with O3 exposure.
6 While O3-induced changes in heart rate and Tco may be unique to rodents, this
7 phenomenon has not been well studied in humans. It is possible that because of their larger
8 thermal mass and different thermoregulatory mechanisms, humans do not exhibit similar
9 changes in these parameters upon exposure. For example, rectal temperature increased by
10 the same amount in both air and 0.4 ppm O3 groups of humans during a 2-h exposure at
11 35 °C (Bedi et al., 1982). The effects on Tco may have been confounded because the
12 subjects performed moderate exercise during alternate 15 min periods during exposure.
13 On the other hand, women intermittently exercising in moderate (24 °C) and hot (35 °C)
14 ambient conditions showed no change in Tco related to O3 exposure, but did show less of an
15 increase in heart rate (2.7%) than did air-exposed (8.1 %) subjects at 35 °C (Gibbons and
16 Adams, 1984). It should be noted, however, that several studies have shown potentiation of
17 human lung function responses associated with increased ambient temperature and
18 O3 exposure (Folinsbee et al., 1977; Gibbons and Adams, 1984). The full importance of
19 temperature in relating rodent and human responsiveness to O3 remains to be understood.
20
21 Exercise and Ventilation
22 Exercise has long been employed in human studies to enhance the effects of air
23 pollutants, especially O3 (Folinsbee and Raven, 1984). Exercise appears to exacerbate
24 functional effects by increasing the inhaled dose (Hatch et al., 1993) and possibly by shifting
25 the deposition of the pollutant to more sensitive pulmonary sites (Gerrity and Wiester, 1987).
26 While exercise can be used in laboratory animals to enhance deposition of O3, no direct
27 methods for measuring ventilation or breathing mechanics are available for small animals
28 during exercise. Alternatively in an attempt to mimic the increase in ventilation produced by
29 exercise in humans, studies employing restrained animals have used CO2 as a ventilatory
30 stimulant. Carbon dioxide (8-10%) maximally increases minute ventilation (VE) 3 to 5 times
31 in rodent species; CO2 in excess of 10% will result in a reduction in ventilation (Wong and
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1 Alarie, 1982; Tepper et al., 1988). This increase in VE is equivalent to light (2 to 3 x
2 resting VE) or moderate exercise (4 to 6 x VE) in humans (U.S. Environmental Protection
3 Agency, 1986). In many O3 studies in humans, both heavy (7 to 8 x VE) or very heavy
4 (>9 x VE) exercise has been used. Similar increases in ventilation cannot be attained in
5 small animals using the CO2 challenge technique, thus posing a limitation for animal studies
6 attempting to make direct comparisons with human studies.
7 The application of this CO2-challenge methodology in O3-exposed rats (0.25 to 1.0 ppm
8 for 2.25 h with 15 min alternating hyperventilation) clearly demonstrates enhanced
9 pulmonary irritation as reflected in breathing pattern changes during exposure (Tepper et al.,
10 1988, 1990). The breathing pattern alterations typical of O3 exposure appeared to be larger
11 than would be predicted based solely on increased dose, suggesting that CO2 challenge
12 during O3 exposure may have enhanced deposition at critical lung sites (Tepper et al., 1989).
13 This augmented response was clearly reflected in the large increases in protein observed in
14 the BAL (Costa et al., 1988b). In postmortem studies, rats exercised during exposure have
15 been found to have exacerbated lung pathology, thus appearing to confirm this hypothesis
16 (Mautz et al., 1985) and suggesting that exercise may, in fact, enhance toxicity
17 disproportionate to the apparent dose of toxicant.
18
19 8.3.3.2 Acute Exposure Data
20 Two corners of the parallelogram paradigm can be readily constructed from data
21 gathered in empirical studies of acute O3 exposure in humans and laboratory animals. These
22 studies have the bulk of the data with the highest frequency of common endpoints which can
23 be compared. Hence, the following discussion will focus on several categories of
24 homologous lung function and BAL study-data which have been obtained from humans and
25 animals exposed similarly to O3. The human studies were drawn from the large existing data
26 base on lung function and represent typical responses. The corresponding BAL data are
27 more limited and are used to the extent possible. In contrast, the animal studies selected for
28 comparison are highly selective and represent a rather small data base involving similar
29 exposure scenarios and homologous endpoints. This approach, of necessity, excludes the
30 large majority of animal studies, not because they do not contain important toxicologic data
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1 on O3, but rather, they are disparate in their exposure parameters or biologic endpoints
2 which can be readily tied to those available in humans.
3
4 Tidal Breathing
5 In humans, O3 produces pulmonary irritation, a response associated with cough and
6 substernal soreness (Chapter 7). While these symptoms are difficult to assess in animals,
7 exposure to sufficient concentrations of O3 produces reflex alterations in tidal breathing that
8 can be objectively measured. Most notably, the response is an increase in fB that is usually
9 accompanied by a decrease in VT (tachypnea), whereas VE may not be altered. Although
10 the magnitude of the tachypneic response is variable depending on the species and exposure
11 conditions, this endpoint is quite sensitive and consistent across many species (e.g., guinea
12 pigs, cats, dogs, rats, monkeys, and humans).
13 To examine the cross-species response to O3, data from human and animal studies
14 reporting immediate postexposure alterations in fB were evaluated. Most human studies
15 employed an exercise regimen during O3 exposure to increase dose. On the other hand, few
16 animal studies have used exercise, relying rather on high exposure concentrations or
17 CO2-induced hyperventilation. Three representative human studies were selected because
18 they used a large range of concentrations and ventilation rates. Selected data from these
19 three human studies are compared to the available animal data in Figure 8-7 and discussed
20 further below.
21 Folinsbee et al. (1975) examined the upper limits of the O3 concentration-response
22 curve in human subjects exposed either at rest or while performing light/moderate
23 intermittent exercise (29 L/min; »5x resting VE). Measurements of fB were obtained during
24 exercise following a 2-h exposure to 0.37, 0.5, or 0.75 ppm O3. Concentration-dependent
25 increases in fB were observed in both the resting and exercise group, although the magnitude
26 of the change in the exercise group was greater. The role of exercise in altering the
27 O3-induced changes in fB was examined further by DeLucia and Adams (1977) who exposed
28 humans to 0.15 or 0.3 ppm for 1 h while the subjects exercised at one of four ventilation
29 levels (1 x, 3x, 4x and 6x resting VE). The magnitude of fB response increased with
30 concentration and exercise level, but was only significant in the 6x exercise group at
31 0.15 and 0.3 ppm. Lastly, the lower limits of the concentration-response were explored by
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160
150
<§ 140
e 130
o>
I120
•B 110
|
100
LL
90
80
0.2 0.4 0.6 0.8 1
Ozone (ppm)
1.2
Human, Rest, 2
Folinsbeeetal. (1975)
Human, Light, 2
Folinsbeeetal. (1975)
H
Human, Heavy, 1
DeLucia and Adams (1977)
Human, Very Heavy, 2.5
McDonnell etal. (1983)
Guinea Pig, Rest, 2
Amdur etal. (1978)
Guinea Pig, Rest, 2
Murphy etal. (1964)
-A-
Rat, Light, 2.25
Tapper etal. (1990)
Rat, Rest, 3
Mautz and Bufalino (1989)
1.4
Figure 8-7. Comparison of changes in frequency of breathing after O3 exposure in
humans and animals. Data are expressed as percent of the control
response. Right hand legend indicates species, exercise level, exposure
duration in hours, and on the second line, the reference. Human data are
plotted with solid lines and open symbols, while animal data are plotted
with broken lines (differentiated by species) and closed symbols.
1 McDonnell et al. (1983) where subjects performing very heavy exercise (65 L/min; »10 x
2 resting VE), were exposed to 0.12, 0.18, 0.24, 0.30 or 0.40 ppm O3 for 2.5 h. Significant
3 changes in fB were observed at all exposure levels.
4 Although several animal studies have evaluated tidal breathing changes during and after
5 O3 exposure, only four studies have examined multiple concentrations such that comparisons
6 to human data can be made. Unanesthetized, restrained guinea pigs were exposed for 2 h to
7 0.34, 0.68, 1.08, or 1.34 ppm O3 via nose cones, while tidal breathing was measured using
8 a constant volume plethysmograph (Murphy et al., 1964). A similar experimental
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1 preparation was used by Amdur et al. (1978) to evaluate the respiratory response of guinea
2 pigs to 0.2, 0.4 and 0.8 ppm O3. In both of these experiments, a monotonic increase in
3 fB was observed; however, the animals studied by Murphy et al. (1964) were uniformly more
4 sensitive to O3 than those of Amdur et al. (1978). Mautz and Bufalino (1989) measured
5 breathing patterns in awake restrained rats exposed for 3-h to 0.2, 0.4, 0.6, and 0.8 ppm O3.
6 Concentration-related increases in fB were observed up to 0.6 ppm, but the responses to
7 0.6 and 0.8 were the same. In another study, awake rats were exposed to 0.12, 0.25, 0.5,
8 and 1.0 ppm O3 for 2.25 h in head-out pressure plethysmographs where CO2-stimulated
9 breathing was incorporated to augment ventilation (Tepper et al., 1990). With the added
10 CO2, rats appeared to be of similar responsiveness to O3 as were guinea pigs. In general, as
11 depicted in Figure 8-7, restrained guinea pigs and rats appeared to be as responsive as the
12 lightly exercising humans, and clearly more responsive than the humans exposed at rest.
13 Only with strenuous exercise does the response of humans appear to exceed that of rodents.
14 In addition to similar concentration-related effects in humans and animals, the time-
15 related effects of O3 exposure appear to be similar. To demonstrate this homology,
16 Mauderly (1984) compared the time course of response to O3 in humans and guinea pigs
17 exposed under somewhat similar conditions. Humans were exposed to 0.75 ppm O3 for
18 2 h while engaging in nonstrenuous, intermittent exercise at 15 min intervals (Folinsbee
19 et al., 1975). In another study, respiratory parameters were measured at 30 min intervals
20 during and for 4 h postexposure (Bates and Hazucha, 1973). Similarly, unanesthetized,
21 restrained guinea pigs were exposed to 0.68 ppm O3 for 2 h as part of a concentration-
22 response study (described above), with respiratory function assessed at 15 min intervals
23 during and for 3.5 h postexposure (Murphy et al., 1964). In both guinea pigs and humans,
24 fB increased and VT decreased; both parameters then returned toward control values during
25 the postexposure period. The percent change from control in fB and VT was nearly the
26 same, throughout the exposure and postexposure periods, indicating that a similar
27 concentration of O3 («0.7 ppm) produced similar temporal alterations in ventilation. Again,
28 the guinea pigs would appear to be slightly more responsive than humans since they were
29 exposed to a lower concentration (0.68 ppm) at rest, while the humans were exposed to
30 0.75 ppm with light intermittent exercise.
31
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1 Mechanics
2 Breathing mechanics have been examined in several animal and human-O3 exposure
3 studies, but there is little similarity between the data bases in the concentrations or specific
4 techniques used. Bates et al. (1972) examined breathing mechanics in resting and in lightly
5 exercised (2 x resting VE) humans exposed for 2 h to 0.75 ppm O3. Although no
6 concentration-response data were obtained, increased lung resistance (R^ and decreased
7 dynamic compliance (Cdyn) were found for both resting (+22 and -12%) and exercising
8 (+67 and -51%) subjects exposed to O3. In a similar study, Hazucha et al. (1989) exposed
9 men for 2 h to 0.5 ppm O3 using intermittent moderate (40 L/min) exercise, and found a
10 significant 12.5% increase in airway resistance (RaW), although no concomitant change in
11 Cdya was detected. McDonnell et al. (1983), using a broad range of O^ concentrations
12 (0.12, 0.18, 0.24, 0.3 and 0.4 ppm; 2 h) and very heavy exercise (65 L/min), reported
13 concentration-dependent increases in Raw. In humans at rest or performing light exercise,
14 however, a 2-h exposure at near-ambient O3 concentrations would be expected to induce only
15 modest increases in R^ and no changes in Cdyn (Hazucha, 1987).
16 While relatively high O3 concentrations (> 1.0 ppm) showed effects on RL and Cdyn in
17 animals (Murphy et al., 1964), only three studies in animals have evaluated these parameters
18 at lower, more relevant O3 concentrations. Watanabe et al. (1973) studied anesthetized,
19 paralyzed, and mechanically ventilated cats exposed via a steel tracheal tube to either 0.25,
20 0.5 or 1.0 ppm O3 for between 2 and 6.5 h. Measurements of breathing mechanics were
21 recorded every 30 min. With increasing O3 concentration and exposure duration, RL
22 increased and, to a lesser extent, Cdyn decreased. Bronchoconstriction at 0.25 ppm O3 was
23 reversed following atropine (a parasympathetic receptor blocker), but only partially reversed
24 at the two higher concentrations, suggesting the involvement of more than
25 bronchoconstriction in the increase in RL at these levels. Unfortunately, relating the
26 concentrations used in this study to other animal or human studies is difficult because
27 exposure through a tracheal tube would eliminate scrubbing of O^ by the nasal and
28 oropharynx and would likely exaggerate the pulmonary O3 dose (Gerrity et al., 1988).
29 Other studies have attempted to examine breathing mechanics in unanesthetized animals
30 with natural nasal breathing and avoidance of potential anesthesia-related blunting of reflex
31 responses. Murphy et al. (1964) exposed unanesthetized guinea pigs to several
December 1993 8-57 DRAFT-DO NOT QUOTE OR CITE
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1 concentrations of O3 for 2 h and measured ventilation, as previously discussed, and RL.
2 At concentrations less than 1 ppm, O3 had no effect, but RL increased 48% and 147% at
3 1.08 and 1.34 ppm, respectively. Using a similar test system, Amdur et al. (1978) observed
4 no significant alteration of RL in unanesthetized guinea pigs during a 2-h exposure to 0.2,
5 0.4, or 0.8 ppm O3. However, Cdyn decreased significantly at 0.4 and 0.8 ppm O3.
6 In analogous studies in unanesthetized rats, Tepper et al. (1990) observed no significant
7 changes in RL or Cdyn after a 2.25 h exposure to 0.12, 0.25, 0.5, or 1.0 ppm O3, in spite of
8 intermittent 15 min periods of exercise-like hyperventilation induced by CO^.
9 Although increased resistance is demonstrable in guinea pigs, cats, dogs and humans, a
10 comparison of percent change in resistance from control measurements after an acute («2 h)
11 O3 exposure (Figure 8-8) suggests that humans are more likely to bronchoconstrict due to
12 O3 exposure than rodents. Neither of the guinea pig studies (Murphy et al., 1964; Amdur
13 et al., 1978) nor the rat study (Tepper et al., 1990) showed a significant increase in RL at
14 less than 1 ppm. However, closer examination of the human data reveals that the McDonnell
15 et al. (1983) study employed very heavy exercise, and most of the studies included in the
16 Hazucha (1987) model used moderate to heavy exercise. Thus, again the inhaled dose would
17 likely be greater than in spontaneously breathing animals. A more comparable study in
18 humans that employed only light exercise reported that 0.25, 0.37 and 0.5 ppm for
19 2 h resulted in minimal, nonsignificant 118%, 124%, and 104% increase in R^ (Hackney
20 et al., 1975). Likewise, the Bates et al. (1972) data obtained in subjects at rest and with
21 light exercise (0.75 ppm O3) also argue against an unusually high O3 responsiveness in
22 humans relative to test animals for this endpoint when exercise-related dose is considered.
23 In general, similar findings have been observed using the measurement of Cdyn;
24 however, the response decrements were more variable and of smaller magnitude. Given the
25 distal deposition of O3, as indicated by morphological studies (Section 6.2.4), it is surprising
26 that so little attention has been given to this parameter. Available data suggest that these
27 changes in Cdyn are of little biological significance for ambient exposures.
28
29 Airway Responsiveness
30 The ability of O3 to increase airway responsiveness to nonspecific bronchoconstricting
31 stimuli in both humans and other mammalian species has been known for at least a decade
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200
180
o 160
E
o
O
140
to
'(5
120
100
80
60
0.2 0.4 0.6 0.8
Ozone (ppm)
Human, Very Heavy, 2.5
McDonnell at a). (1983)
Human, Rest, 2
Bates at al. (1972)
A
Human, Light, 2
Bates etal. (1972)
#•
Human, Combined, 2
Hazucha (1987)
Human, Light, 2
Hackney etal. (1975)
H
Cat, Rest, 2
Watanabe etal. (1973)
Rat, Light, 2.25
Tepperetal. (1990)
Guinea Pig, Rest, 2
Amduretal. (1978)
Figure 8-8. Comparison of changes in resistance after O3 exposure in humans and
animals. Data are expressed as percent of the control response. Right
hand legend indicates species, exercise level, exposure duration in hours,
and on the second line, the reference. Human data are plotted with solid
lines and open symbols. The line labelled, Hazucha (1987), is a model of
predicted response. Animal data are plotted with dashed lines
(differentiated by species) and closed symbols.
1 (Sections 6.2.5 and 7.2.4). However, airway responsiveness is perhaps the least understood
2 response to O3, particularly in the context of species comparisons. Humans clearly exhibit
3 increases in airway responsiveness at environmental 03 exposure levels (Gong et al., 1986;
4 McDonnell et al., 1987; Folinsbee et al., 1988; Horstman et al., 1990), while analogous
5 responses in animals at O3 concentrations below 1 ppm are controversial. Most studies of
6 airway responsiveness in laboratory animals focus on the development of asthma-like models
7 to elucidate generic mechanisms of airway responsiveness and utilize concentrations as high
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1 as 3 ppm for brief periods of time to injure the airways. Hence, anything more than a
2 qualitative comparison between animal species and humans is tenuous and thus will not be
3 further discussed in this section. Details of the methodologies of the laboratory animal and
4 human bronchoreactivity studies can be obtained elsewhere in the reviews of pulmonary
5 function found in Sections 6.2.6 and 7.2.3, respectively.
6
7 Elasticity and Diffusion
8 The integrity of the pulmonary air-blood barrier is essential for efficient exchange of
9 oxygen (O^ and CO2. This fragile epithelial interface with matrixed interstitial connective
10 tissues and capillaries possesses inherent elastic properties and presents a finite resistance to
11 O2 diffusion to the blood. Although the elastic and diffusionary properties of the blood-air
12 barrier are not implicitly linked to one another functionally, both properties can be readily
13 quantified and compared between humans and laboratory animals (Costa, 1985). When
14 combined, assessment of these functional properties is often sufficient to evaluate pathologic
15 or toxic events in the distal reaches of the lung. For this reason and the fact that O3 deposits
16 in the deep lung, the effects of O3 on these parameters will be discussed together.
17 Inhaled O3 is known to penetrate to the depths of the lung and preferentially deposit in
18 the smallest airways and its proximal acini (Section 8.2). Somewhat surprisingly, relatively
19 few studies in humans have sought to characterize potential functional impairments at the air-
20 blood interface. The reasons for this are likely two-fold: First, in the early studies of the
21 health effects of O3 on humans, static compliance and diffusing capacity of CO (DLcO) were
22 affected at only very high concentrations, well above what would be considered
23 environmentally relevant. Second, from a practical perspective, these measurements proved
24 to be considerably more tedious to perform than the forced expiratory measurement, which
25 sensitively detects O3-induced alterations (discussed below). Nevertheless, there are
26 sufficient data in humans exposed acutely to O3 to allow a reasonable comparison of these
27 endpoints with their more abundant animal homologs.
28 The earliest studies leave little doubt that O3 is edemagenic at high concentrations in
29 virtually all mammalian species. In the past, occupational exposures of 2 to 3 ppm C^ were
30 not uncommon, and a 9 ppm peak exposure has been reported (Kleinfeld et al., 1957;
31 Challen et al., 1958). The resultant worker symptoms and signs, including chest radiograms,
December 1993 8-60 DRAFT-DO NOT QUOTE OR CITE
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1 were consistent with the manifestations of edema reported in experimental animals
2 (i.e., increased lung weight and stainable edema in the airspaces) (Stokinger, 1965). Lung
3 function, however, was not typically measured in these work-related exposures. In a later
4 study of arc welders exposed to 0.2 to 0.3 ppm O3, little, if any, convincing evidence of
5 functional impairment, in terms of altered lung volumes, maximal expiratory flow rates, and
6 DLco was obtained (Young et al., 1963). To further explore the possible effects of O3,
7 Young and coworkers (1964) subjected 11 human volunteers at rest to controlled atmospheres
8 of 0.6 to 0.8 ppm O3 via mouthpiece for 2 h. Small reductions in vital capacity (VC),
9 forced expiratory volume in 0.75 s, dynamic and static lung compliance, and intrapulmonary
10 gas distribution were observed, but only the 25 % fall in Dl^o proved to be statistically
11 significant. Similar, but considerably more variable effects on lung function were reported
12 by Hallett (1965) in 10 subjects exposed to 1 to 4 ppm for 30 min. Nonetheless, of
13 10 exposed subjects, 7 showed at least a 20% drop in DLCO. Like Young and coworkers,
14 Hallett interpreted these changes to indicate lung edema, in agreement with the hypothesis
15 that the deep lung irritant O3 was having its effect at the alveolar level. Interestingly,
16 additional work from the same laboratory of the Young study (Bates et al., 1972) found that
17 resting subjects receiving nasal exposure to 0.75 ppm O3 for 2 h resulted in a nonsignificant
18 3% reduction in DLCO. However, in a limited test group, the co-imposition of light
19 exercise, which doubled ventilation, enhanced this response («12%). It appears that the
20 nasal (Bates et al., 1972) versus mouthpiece (Young et al., 1964) routes of exposure were
21 instrumental in the differential response, because it is likely that the mouthpiece diminished
22 what scrubbing occurs when exposure is via the unencumbered mouth in human test subjects
23 as reported by Gerrity et al. (1988). Since these early studies, there have been no additional
24 controlled human acute studies that have examined alterations in DI^o at O3 concentrations
25 below 0.6 ppm.
26 Analogous animal studies of acute O3 exposure indicate that the general pattern of
27 functional impairment is similar to that reported in human studies. Anesthetized and
28 ventilated cats showed a general decline in VC, static lung compliance, or DI^o with
29 exposures up to 6.5 h of 0.26 to 1.0 ppm O3 (Watanabe et al., 1973). The responses of the
30 20 animals were variable, and these declines, which did not achieve overall statistical
31 significance, were thought to be largely secondary to the substantial (36 to 200%) increases
December 1993 8_61 DRAFT-DO NOT QUOTE OR CITE
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1 in R^. In a more complex study design, rats were exposed for 2 or 7 h to 0.5 or 0.8 ppm
2 O3 with intermittent 8% CO2 to hyperventilate (~2 to 3 X resting VE) the animals as an
3 exercise analogue to human exposures (Costa et al., 1988a). The DLcO values were reduced
4 by about 10% at both 0.5 ppm time-points and by about 12% with a 2-h exposure to
5 0.8 ppm. Exposure to 0.8 ppm for 7 h, however, greatly exacerbated the alveolar lesion
6 with a resultant 40% reduction in DL^. Static compliance, unaffected by the other
7 exposure conditions, was affected only at this latter exposure duration. These O3-induced
8 effects, particularly the reductions in DLCO, appeared to correlate with the degree of lung
9 edema in affected animals as had been surmised for the acutely exposed humans. With the
10 multitude of more recent studies of O3 at ambient levels, alterations in static lung compliance
11 or DLCO are rarely reported in either humans or animals.
12
13 Forced Expiration
14 Reductions in FVC and FEVj have become the hallmarks of acute lung dysfunction in
15 humans after O3 exposure (Chapter 7). These measures are sensitive to O3 levels as low as
16 0.12 ppm for as little as 2 h when intermittent heavy exercise is included during the exposure
17 (McDonnell et al., 1983) and show cumulative dysfunction resulting from 6.6 h of lower
18 levels of this oxidant (0.08 and 0.10 ppm) when nearly continuous, moderate exercise is
19 employed (Horstman et al., 1990). Reductions in the FEV1 and FVC induced by O3 are
20 believed to be partly the result of pain-mediated interruption of maximal inspiration (Hazucha
21 et al., 1989). Exactly what level of tissue injury or inflammation correlates with these
22 functional deficits is unclear and is an active area of research (Section 7.2.4).
23 Most studies of O3 in experimental animals make little effort to mimic human study
24 designs, thereby impeding the extrapolation of their results to humans. Recently, however,
25 rat studies involving periods of intermittent CO2-induced hyperventilation to enhance
26 dosimetry, have attempted to capitalize on the qualitative similarity of the rat and human
27 maximum expiratory flow volume (MEFV) curves as a potentially sensitive endpoint of
28 toxicity (Costa et al., 1988a; Tepper et al., 1989). In the rat, FVC does indeed decrease
29 with O3 exposure, though the magnitude of response is apparently less than that observed in
30 humans. As in the human, the reduced FVC in the animal appears not to be the result of a
31 change in residual volume (RV). Total lung capacity (TLC) may be reduced slightly, but
December 1993 8-62 DRAFT-DO NOT QUOTE OR CITE
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1 lung compliance does not change. However, it is premature to assume a common
2 mechanism for the FVC reductions in the rat and human. Unlike the human, pain on
3 inspiration in the animal model is likely not an issue since the animal is anesthetized during
4 the procedure and is brought to TLC by a defined airway pressure (»30 cm H2O). Since
5 general anesthesia is known to diminish sensory afferent stimuli, an analogous O3-induced
6 fall in rat FVC should expectedly have been blunted, if not totally eliminated. To what
7 extent anesthesia mitigates the rat response or that there are inherent species differences in
8 dosimetry or sensitivity is not clear from these studies. Nevertheless, comparison of model-
9 predicted FVC changes in humans (Hazucha, 1987) with analogous rat data (Costa et al.,
10 1988a) would suggest that this response in the anesthetized rat is about half that of the human
11 (Figure 8-9)
12
13 Studies of Inflammation and Antioxidant Content of Bronchoalveolar Lavage Fluid
14 Both humans and animals exhibit a PMN inflammatory response with associated
15 changes in lung permeability after acute exposure to O3. Recent studies indicate that humans
16 exposed to concentrations as low as 0.08 ppm for 6.6 h with moderate exercise (40 L/min)
17 exhibit a fourfold increase in the percentage PMNs when BAL is obtained 18-h postexposure
18 (Devlin et al., 1993). To date, animal studies at comparable exposure levels are rare,
19 (Hotchkiss et al., 1989) and exercise enhancement of exposure dose has yet to be
20 incorporated. As noted above, the issue of dosimetry is critical if extrapolation at such levels
21 is to be attempted. Nevertheless, in the one acute rat study at 0.12 ppm for 6 h, an increase
22 in nasal lavage derived PMNs was noted 18 h postexposure with no similar change in PMN
23 number in the BAL (Hotchkiss et al., 1989). In contrast, in the same study when higher
24 concentrations of O3 (0.8 and 1.5 ppm) were used, BAL PMNs were elevated, but no
25 changes were observed in the nose washings. Such a "competetive" nasal-pulmonary
26 response has yet to be directly studied in humans. Nevertheless, the data support the general
27 hypothesis that there is comparability between inflammatory responsiveness of rats and
28 humans. More direct comparison of laboratory animal with human inflammatory responses
29 can be drawn from studies at higher concentrations when the nasal/lung competetive response
30 in the rat is skewed to the lung and, like in the lung function comparison, analogous
December 1993 3.53 DRAFT-DO NOT QUOTE OR CITE
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••-•A
Human, R»tt, 2 h
Hazucha(1987)
Huron, V«y Heavy, 2 h
Hazucha (19
Rat,Ught,4h
Costa etaL(1988a)
Rat,Ught,7h
Costa etaL(1988a)
Rat,Reit,8h
OMM*tal.(1988a)
0.4
0.6 0.8
Ozone (ppm)
1.2
Figure 8-9. Comparison of changes in forced vital capacity after O3 exposure in
humans and animals. Data are expressed as percent of the control
response. Right hand legend indicates species, exercise level, exposure
duration, and on the second line, the reference. Human data are plotted
with solid lines from the equation of Hazucha (1987), with the shaded area
representing the predicted range of decrements expected between light
exercise (top line) and very heavy exercise (bottom line). Rat data are
plotted with dashed lines and closed symbols.
1 exposure conditions can be more directly compared. These studies are tabulated in
2 Table 8-9 and discussed in more detail below.
3 Four representative human studies of lung inflammatory responses after acute
4 O3 exposure can be compared with existing acute animal data from studies of analogous
5 design. Seltzer et al. (1986) exposed moderately exercising (83 to 100 watts) subjects to
6 0.4 or 0.6 ppm O3 for 2 h with BAL obtained 3 h postexposure. The BAL (combined
7 0.4 and 0.6 ppm groups) contained an average 7.8-fold more PMNs than after filtered air.
8 Protein levels in the BAL were not assayed in this study. In separate studies, humans were
9 exposed to 0.4 ppm for 2 h with heavy exercise (60 L/min) with BAL samples collected at
10 1 and 18 h postexposure (Devlin et al., 1993a; Koren et al., 1989b). These two studies
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1 showed that the inflammatory response is quickly initiated postexposure. The PMN and
2 protein content were elevated at both times (1 h: PMN 18.2X; protein 1.2 x) with the
3 18 h timepoint (PMN 8x; protein 2.2x) being higher for protein, but somewhat less for
4 PMNs. Schelegle et al. (1991) followed the time course of the the inflammatory response
5 (1-, 6-, and 24-h postexposure) after an exposure of 0.3 ppm for 1 h with exercise
6 (60 L/min). The PMN content of the combined airway and alveolar BAL was elevated at
7 6 h (3X) and 24 h (2.5 x) only, with the 6-h point representing the peak. Protein content of
8 the BAL did not change significantly at any point.
9 The many studies of O3-induced inflammation in laboratory animals are reviewed in
10 Section 6.2.3. For the purpose of species comparison, only selected studies (Table 8-9) will
11 be considered. The spectrum of exposure conditions used in the various animal studies
12 makes difficult the direct comparison among laboratory test species. However, one study by
13 Hatch and coworkers (1986) specifically addressed this question by exposing mice, guinea
14 pigs, rats, rabbits, and hamsters under identical conditions (0.2, 0.5, 1.0, and 2.0 ppm for
15 4 h), followed at 18 h postexposure with BAL and assay for protein. Guinea pigs were most
16 responsive, responding at 0.2 ppm, whereas mice, hamsters and rats began responding at
17 1.0 ppm and rabbits responded only to 2.0 ppm. Only one study involving BAL assessment
18 of PMNs and protein in exposed monkeys has been published (Hyde et al., 1992).
19 At 0.96 ppm (8 h), the monkeys had a significant inflammatory response, but it is difficult to
20 assess monkey responsiveness relative to the human for this endoint. Assuming a linear
21 C x T relationship, the monkey data appear on the order of the guinea pig response.
22 However, none of these species showed BAL protein increases approximating those reported
23 in human studies.
24 In recent studies (0.4 ppm for 2 h, with BAL 16 to 18 h postexposure) (Slade et al.,
25 1989; Crissman et al., 1993), guinea pigs made vitamin C-deficient exhibited enhanced
26 responsiveness to O3, making them comparable to that of the exercised humans of Koren
27 et al. (1989b) (Figure 8-10). Similarly, when rats were exposed to 0.5 ppm O3 for 2 h with
28 intermittent CO2-induced hyperventilation (Tepper et al., 1993) to mimic mild-moderate
29 exercise (three- to fivefold VE), the BAL protein, as well as PMN responses at 18-h
30 postexposure compared favorably with those data of Koren and coworkers (1989a,b)
31 (Figure 8-11).
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TABLE 8-9. POLYMORPHONUCLEAR LEUKOCYTE AND PROTEIN
PERMEABILITY RESPONSE TO OZONE BY SPECIES
Exposure
Parameters
0.4-0.6
2h
0.4
2h
0.4
2h
0.3
Ih
0.96
8h
0.2
0.5
1.0
2.0
4h
0.5
1.0
4h
0.5
0.8
2or7h
Exposure
Conditions
Exercise
(15 min/alt;
83-100 W)
Exercise
(15 min/alt;
60 L/min)
Exercise
(15 min/alt;
60 L/min)
Exercise
(15 min/alt;
60 L/min)
Daytime;
Rest
Daytime;
Rest
Daytime;
Rest;
Vit. C
(AH2+/-)
Daytime;
(15 min/alt
for 2 h;
45 min/alt
for 7 h);
3-5 X VE
Species (Strain)
Human
Human
Human
Human
Rhesus monkey
(a) Mouse
(Swiss Albino)
(b) Guinea Pig
(Harley)
(c) Rat
(Sprague-Dawley)
(d) Hamster
(Golden Syrian)
(e) Rabbit
(NZW)
M
Guinea Pig
(Hartley)
M
Rat
(F344)
M
PMN Protein
Fold8 Folda
BAL Time Increase Increase
3 h 7.8 Not done
18 h 8.0 2.2
1 h 18.2 1.2
1,6, 24 h 3.0 at 6 h No change
2.5 at 24 h
1,12,24 27atlh 3 at 1 h
72, 168 h 19 at 12 h 3 at 12 h
24 at 24 h 8 at 24 h*
6 at 72 h 3 at 72 h
3 at 168 h 1 at 168 h
16 h Not done (a) 1.8 at 1.0 ppm
3.2 at 2.0 ppm
(b) 1.4 at 0.2 ppm
2.0 at 0.5 ppm
4.1 at 1.0 ppm
4.5 at 2.0 ppm
(c) 2.1 at 1.0 ppm
3.6 at 2.0 ppm
(d) 1.5 at 1.0 ppm
2.6 at 2.0 ppm
(e) 2.7 at 2.0 ppm
16 h Not done For 0.5 ppm;
1.1 for AH2+,
2.1 for AH2".
For 1.0 ppm;
2.4 for AH2+,
2.7 for AH2"
1 h Not done For 0.5 ppm;
1.2at6h,
2.1 at7h.
For 0.8 ppm;
1.5 at 2 h,
3.3 at 7 h
Reference
Seltzer et al.
(1986)
Koren et al.
(1989a,b)
Devlin et al.
(1990)
Schelegle
et al. (1991)
Hyde et al.
(1992)
Hatch et al.
(1986)
Slade et al.
(1989)
Costa et al.
(1988a)
"Fold represents (03 response/air response).
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Basal Levels of Ascorbic Acid
3500
3000
12500
en
:§ 2000
| 1500
«: 1000
500
Humans
Normal
Guinea Pigs
AH2-def
Effect of Ozone Inhalation (0.4 ppm, 2 hr)
on BAL Protein
B
p- 0.004
p - 0.025
Humans
Rats
Normal AH2-def
Guinea Pigs
Figure 8-10. Composite of data from Slade et al. (1989), Koren et al. (1989b), and
Crissman et al. (1993) comparing basal BAL ascorbate levels (A) to
O3-induced changes in BAL protein (B). Ozone-exposures (0.4 ppm; 2 h;
16 to 18 h BAL) of humans (exposed with exercise), rats (exposed resting),
and guinea pigs (exposed resting) with (AH2-def) and without (normal)
ascorbic acid deficiency.
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1 1.5
Ozone (ppm)
Figure 8-11. Composite of data comparing BAL PMNs 16 to 18 h after O3 exposure
(0.4 ppm; 2 h) of humans (with exercise) (Korea et al., 1989b) to those of
rats exposed to O3 (0.5 to 2.0 ppm) at rest or hyperventilated with CO2
(Tepper et al., 1993).
1 Within a given laboratory animal species, responses among strains can also differ
2 appreciably, as recently demonstrated in rats by Pino et al. (1991) and Costa et al. (1993a).
3 These studies indicated that Wistar rats exhibit greater inflammatory responses (protein and
4 PMN) to O3 than Sprague-Dawley and Fischer-344 rats after an 8-h exposure to 0.5, 1.0,
5 and 1.5 ppm with BAL sampled at 2 or 24 h later. Similarly, mice strains (C3H/HeJ and
6 B6C57/6) (Kleeberger et al., 1990) and Sprague-Dawley rat substrains (Costa et al., 1985)
7 have been shown to possess specific genetic susceptibility to high levels of O3 (2 ppm).
8 In the case of the mice, the responsive strain is 7 times (at the 6-h postexposure peak) as
9 susceptible as the resistant strain for the PMN response to 2 ppm; the protein response is
10 twice (at the 24-h postexposure) that of the resistant strain. In the Sprague-Dawley substrain,
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1 protein extravasation into the alveolar lumen immediately postexposure is 40% higher in the
2 responsive strain than the resistant (no other time points were examined).
3 Humans have an order of magnitude less ascorbate in BAL fluid as compared to the rat,
4 but they have nearly twice the glutathione, 4 times the uric acid, and 80 times the vitamin E
5 (as normalized to lipid P - surfactant) (Slade et al., 1993) (Table 8-8). However, on a BAL
6 cell protein basis, the ratios clearly favor the rat for all of these antioxidants, with the
7 exception of uric acid which is generally not high in rats because of species differences in
8 protein and prime nucleotide catabolism (urea being the major N-by-product with rat). The
9 guinea pig most closely resembles the human for ascorbate. Since these antioxidants are
10 thought to function in the defense against oxidant challenge, it would appear critical to
11 appreciate their presence and function when attempting to interpret data for extrapolation.
12 Unfortunately, the data base in both animals and, particularly, humans with regard to these
13 antioxidants is quite limited. Supplementation and deprivation studies with vitamins C and E
14 have shown that these antioxidants have some role in protecting against the effects of O3 in
15 both animals (Elsayed et al., 1988; Slade et al., 1989; Crissman et al., 1993) and in humans
16 (Chatham et al., 1987). Of the animal models, ascorbate-deprived guinea pigs appear to
17 have BAL ascorbate levels most like humans, with a protein permeability response (without
18 exercise in the animal) very similar to the human exposed to the same concentration
19 (0.4 ppm for 2 h) with exercise. However, Crissman et al. (1993) also reported that
20 reduced-ascobate levels in BAL (18 h postexposure) increase in the human, while BAL
21 reduced-ascorbate levels in the rat decrease. Whether this relates to the distinctly different
22 basal levels of this vitamin and is associated with the disparate protein responsiveness
23 (ignoring exercise) is unclear because deficiency in animals (guinea pigs) appears more
24 critical to the responsiveness at low ambient-like concentrations than at higher concentrations
25 (1.0 ppm). While it would appear that vitamin C is involved in the interplay between O3 and
26 the exposed subject (human and animal), there is not full coherence of the data. For
27 example, Hatch et al. (1986) showed that the rabbit was the least responsive to 03 in terms
28 of BAL protein, but this species has among the lowest tissue levels of vitamin C (Slade
29 et al., 1985). However, rabbits apparently have a low propensity to form lipid peroxides
30 (Arakawa et al., 1986), an expected product of lung lipid-O3 interaction (Pryor, 1992).
December 1993 g_69 DRAFT-DO NOT QUOTE OR CITE
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1 Thus, to interpret interspecies sensitivity only in terms of basal antioxidant levels, however
2 templing this may be, would be premature and overly simplistic at this time.
3
4 8.3.3.3 Prolonged Exposure Studies
5 The previous sections of this text have utilized lung function responses to acute
6 O3 exposure in an attempt to elucidate the relative sensitivity among laboratory test species
7 and humans and thereby complete two corners of the parallelogram paradigm. Because acute
8 responses represent only part of the extrapolation paradigm, temporal-based exposure
9 responsiveness will also be considered, despite the relative paucity of comparative data
10 between laboratory animals and humans in that context. Again, the criteria for selection of
11 studies was similarity in exposure scenario in an effort to best extact species-specific
12 response differences. The discussion will focus on relative adaptability of acute functional
13 changes and associated BAL-derived findings after repeated exposures and the coherence of
14 the findings from prolonged human and animal exposure studies and epidemiological results.
15
16 Lung Function Studies
17 Reversal ("attenuation") of pulmonary function decrements using a scenario of repeated
18 exposure to O3 has been reported for both humans and laboratory animals. At least 9 studies
19 between 1977 and 1984 have documented that, for repeated exposures between 0.2 and
20 0.5 ppm O3 (2 h/day, up to 5 days), spirometric changes were most severe on the first or
21 second day of exposure, waned over the next 3 days of exposure and by the fifth day, had
22 returned to control pre-exposure levels (Section 7.2.1.4). In the only animal study using a
23 similar exposure protocol and analogous experimental design, Tepper et al. (1989) showed
24 that rats initially displayed a tachyneic response to O3 that attenuated after 5 consecutive days
25 of exposure, a pattern quite similar to that of humans. Exposures were for 2.25 h and
26 included challenge with CO2 during alternate 15 min periods to augment ventilation (2 to
27 3 X resting VE - equivalent to light exercise in humans). As in the human studies, the
28 functional changes were largest on Day 1 or 2, depending on the parameter and the
29 O3 concentration (0.35, 0.5 and 1.0 ppm). Attenuation of the shape constant of the flow-
30 volume curve of the rats was also observed over this period. Thus, under analogous
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1 conditions of exposure, both the humans and the rats exhibited similar initial functional
2 responses to O3 with full and kinetically similar reversal of effects.
3 More difficult is the direct comparison between human and animal lung function
4 responses to prolonged (several weeks) O3 exposure, largely because of the limited
5 availability of controlled human study data. In the only study of its kind, Bennett (1962)
6 exposed 12 human subjects at rest to 0.2 or 0.5 ppm for 3 h/day, 6 days/week for
7 12 consecutive weeks. Although no effects were discernable early in the exposure, there
8 appeared to be a small, but significant O3-induced reductions in FEVj (and small,
9 nonsignificant reductions in FVC), particularly in the last weeks of the study. This reduction
10 in FEVj would more likely reflect obstructive changes within the lung at these points late in
11 time rather than the pain-mediated reductions that are seen with acute O3, which adapt away
12 after a few days of exposure. The lack of concentration-related decrements in FEVl and
13 FVC is somewhat unsettling, but regardless, after 9 postexposure weeks in clean air, all
14 measured effects had reversed.
15 Unfortunately, there are no directly parallel animal studies to compare to this limited
16 data base. But, if the 2-fold sensitivity difference between the rat to human FVC response
17 (see Figure 8-12) is assumed, a number of animal studies may be considered comparable for
18 the purposes of this discussion. On the one hand, rats exposed to 0.2 or 0.8 ppm O3 for
19 6 h/day, 5 days/week for 12 weeks were reported to exhibit some degree of small airway
20 obstruction based on the MEFV curves, but little, if any reduction in FVC or Dl^o was
21 observed (Costa et al., 1983). Others have reported analogous marginal increases in rat TLC
22 or its component volumes (Bartlett et al., 1974; Costa et al., 1983; Raub et al., 1983) or in
23 regional airway resistances (Yokoyama et al., 1984) after intermittent or continuous
24 exposures to >0.25 ppm O3 for 4 to 12 weeks, which would not be unexpected with distal
25 airway or lung damage. Actual pathology in the distal lung tends to be focal and difficult to
26 correlate precisely with the marginal functional impairment.
27 The limited functional data available in monkeys generally agree with the pattern of
28 distal lung pathophysiology reported in rats. When exposed to 0.5 ppm for 90 days
29 (8 h/day), monkeys exhibited a slight increase in lung distensibility (Eustis et al., 1981).
30 Likewise, monkeys exposed to 0.25 ppm (8 h/day for 18 months) exhibited increased chest
-------
-? i
* I
a
re
CD
cr
v>
IX)
S
o
I f
5" ?
•s
O
cr
-------
1 conditions of exposure, both the humans and the rats exhibited similar initial functional
2 responses to O3 with full and kinetically similar reversal of effects.
3 More difficult is the direct comparison between human and animal lung function
4 responses to prolonged (several weeks) O3 exposure, largely because of the limited
5 availability of controlled human study data. In the only study of its kind, Bennett (1962)
6 exposed 12 human subjects at rest to 0.2 or 0.5 ppm for 3 h/day, 6 days/week for
7 12 consecutive weeks. Although no effects were discernable early in the exposure, there
8 appeared to be a small, but significant O3-induced reductions in FEV1 (and small,
9 nonsignificant reductions in FVC), particularly in the last weeks of the study. This reduction
10 in FEVi would more likely reflect obstructive changes within the lung at these points late in
11 time rather than the pain-mediated reductions that are seen with acute O3, which adapt away
12 after a few days of exposure. The lack of concentration-related decrements in FEVl and
13 FVC is somewhat unsettling, but regardless, after 9 postexposure weeks in clean air, all
14 measured effects had reversed.
15 Unfortunately, there are no directly parallel animal studies to compare to this limited
16 data base. But, if the 2-fold sensitivity difference between the rat to human FVC response
17 (see Figure 8-12) is assumed, a number of animal studies may be considered comparable for
18 the purposes of this discussion. On the one hand, rats exposed to 0.2 or 0.8 ppm O3 for
19 6 h/day, 5 days/week for 12 weeks were reported to exhibit some degree of small airway
20 obstruction based on the MEFV curves, but little, if any reduction in FVC or DLgQ was
21 observed (Costa et al., 1983). Others have reported analogous marginal increases in rat TLC
22 or its component volumes (Bartlett et al., 1974; Costa et al., 1983; Raub et al., 1983) or in
23 regional airway resistances (Yokoyama et al., 1984) after intermittent or continuous
24 exposures to >0.25 ppm O3 for 4 to 12 weeks, which would not be unexpected with distal
25 airway or lung damage. Actual pathology in the distal lung tends to be focal and difficult to
26 correlate precisely with the marginal functional impairment.
27 The limited functional data available in monkeys generally agree with the pattern of
28 distal lung pathophysiology reported in rats. When exposed to 0.5 ppm for 90 days
29 (8 h/day), monkeys exhibited a slight increase in lung distensibility (Eustis et al., 1981).
30 Likewise, monkeys exposed to 0.25 ppm (8 h/day for 18 months) exhibited increased chest
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1 Morphometric analyses of the end-airways and distal lung regions of O3 exposed monkeys
2 consistently show altered cell profiles and interstitial restructuring, even when functional
3 changes are marginal which, like in the rat, likely reflects the large functional reserve of the
4 integrated lung. Thus, while these collective data from subchronic animal studies suggest a
5 reasonably homologous distal lung response to O3, many of these linkages in functional
6 outcomes remain uncertain in terms of what to anticipate in the human response.
7 Clearly, the question of potential lung impairment resulting from a near-lifetime
8 exposure to O3 ranks among the most pressing concerns of this toxicant. The animal data,
9 while demonstrating that chronic O3 can induce changes in the structure and function of the
10 lung, have yet to provide definitive evidence of potential disease in disability in humans
11 exposed to O3 over prolonged periods of their lives. The existing epidemiologic studies
12 (Chapter 7; Section 7.4.2), too, merely provide suggestive evidence that persistent or
13 progressive deterioration in lung function may be associated with long-term oxidant pollutant
14 exposure (Detels et al., 1981, 1987). Most recently, Detels and coworkers (1991) reported
15 decrements in FEV] and nitrogen washout across all age groups in areas where oxidant
16 pollution is high. Similarly, analysis of the pulmonary function data from the National
17 Health and Nutrition Examination Survey (NHANES H) showed loss of lung function when
18 annual averages of ambient O3 exceeded 0.04 ppm (Schwartz, 1989). This pattern of
19 impairment is consistent qualitatively with the chronic animal studies (Costa et al., 1993b).
20
21 Studies of Inflammation and Antioxidant Content of the Bronchoalveolar Lavage Fluid
22 The virtual absence of human BAL study data after repeated or prolonged exposures to
23 O3 hinders the comparison of non acute human and animal inflammatory responses.
24 However, the recent study of Devlin et al. (1993b) suggests that the PMN and protein
25 responses to repeated daily exposures to 0.4 ppm (2 h with intermittent exercise for
26 5 consecutive days) attenuate, much as do the functional responses. Hence, for these BAL
27 parameters (exceptions - e.g., lactate dehydrogenase activity - a marker of cell injury), there
28 is indeed an apparent reversal phenomenon for several of the components of acute
29 inflammation when exposures are continued over the 5-day exposure period. Rat studies
30 largely appear to show similar attenuation to O3, but this response seems to be influenced by
31 exposure patterns or conditions (Bassett et al., 1988; Tepper et al., 1989; Van Bree et al.,
December 1993 8.73 DRAFT-DO NOT QUOTE OR CITE
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1 1989). The study with the most similar design to the human protocol (Tepper et al., 1989)
2 showed some reduction in BAL protein with repeated exposure involving intermittent
3 hyperventilation with CO2, but over the 5-day period, the protein levels remained
4 significantly elevated^ cells were not evaluated in this study. Interestingly, vitamin C and
5 glutathione levels in the BAL fluid increased over the 5-day course of exposure, a response
6 consistent with an upregulated antioxidant role in the adaptative mechanism. Van Bree and
7 coworkers (1992), on the other hand, reported that 5 consecutive days (12 h/day) of 0.4 ppm
8 O3 resulted in complete reversal of both BAL albumin and PMN measures to the control
9 values. It should be noted, however, that other biomarkers and mediators within the BAL
10 were not fully recovered which might suggest a slower reversed time frame or continued
11 O3-induced pathogenesis, a conclusion of the Tepper et al. (1989) study.
12 In guinea pigs made deficient in vitamin C and exposed to O3 (0.2, 0.4, or 0.8 ppm)
13 continuously for 7 days, attenuation of the functional and inflammatory endpoints appeared
14 nearly complete in spite of the deficiency (Kodavanti et al., 1993). Other antioxidants, not
15 altered basally, were upregulated more by the O3 challenge; the small residual reservoirs of
16 ascorbate, which persisted in the nearly 98% deficiency state of the animals, were apparently
17 mobilized to the site of injury allowing repair to proceed. Likewise, chronically exposed rats
18 have elevated BAL ascorbate indicative of the oxidant burden and the ongoing repair (Grose
19 et al., 1988). Prolonged exposures up to 18 mo appear to sustain a low-grade interseptal
20 inflammation and evidence of lung matrix remodeling in both rats and monkeys, suggesting
21 that humans would behave similarly. However, such data are not presently available from
22 humans.
23
24 8.4 QUANTITATIVE EXTRAPOLATION
25 8.4.1 Introduction
26 Advances in dosimetry since the previous O3 criteria document (U.S. Environmental
27 Protection Agency, 1986) fall into five major areas: (1) greater sophistication of model
28 applications (e.g., Overton et al., 1989; Mercer et al., 1991), (2) appearance of experimental
29 uptake data that can be compared to model predictions (Wiester et al., 1987, 1988, 1993;
30 Hatch et al., 1989, Gerrity et al., 1988, 1993a,b), (3) experiments specifically designed to
31 estimate model parameters (Hu et al., 1992b), (4) a better understanding of the role of O3 in
December 1993 8-74 DRAFT-DO NOT QUOTE OR CITE
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1 the liquid linings and tissues of the respiratory tract (Pryor, 1992), and (5) better
2 understanding of anatomical aspects (Mercer et al., 1991). The role of these advances in
3 interspecies dosimetric extrapolation follows.
4 With the information available for rats, reasonably reliable predictions of the flux of
5 O3 to the air-liquid lining interface of lexicologically important regions, such as the
6 centriacinar region, is possible. There are two main investigations that make this feasible:
7 (1) Hatch et al. (1989), who estimated the percent uptake and the fraction of the retained
8 O3 that is in the URT, trachea, and lung of rats, and (2) Pinkerton et al. (1992) (with
9 elaboration by Miller et al., 1993) who illustrate the basic correctness of modeling
10 assumptions for ventilatory units. (The judgement of basic correctness is based on the
11 assumption that the dose causing the response is proportional to the flux of O3 to the air-
12 liquid lining interface.) Using this information, regional mass transfer coefficients could be
13 estimated which would allow the prediction of local respiratory tract O3 doses in rats exposed
14 under general conditions.
15 The results of the investigation of Hu et al. (1992b) can be used to estimate URT and
16 tracheobronchial region model parameters, but may not be sensitive enough to determine
17 pulmonary region parameters. If uptake is not confined to the URT and tracheobronchial
18 regions, then their mass transfer coefficients alone would not be sufficient to account for total
19 RT uptake (e.g., as measured by Wiester et al., 1993 or Gerrity et al., 1988), and the
20 difference in predicted (without pulmonary region uptake) and experimental uptakes could be
21 used to estimate the pulmonary region mass transfer coefficient. Unfortunately, no dosimetry
22 data for the human pulmonary region are available.
23 Despite limitations, the O3 dosimetry data that have been obtained over the past several
24 years, coupled with the advances in modeling, suggest that there has been continual
25 convergence between the model predictions and experimental observations. Given the many
26 areas of consistency between models and experiments, it is valuable to begin to employ these
27 models to provide the dosimetric basis for animal-to-human extrapolation. One of the
28 greatest sources of uncertainty in such an application of dosimetry models is the lack of full
29 understanding of the appropriate O3 target site (e.g., upper/lower airways, airways,
30 pulmonary region) from which a particular response is initiated (especially for functional
31 endpoints). However, reasonable assumptions can be made to narrow the target site.
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1 The first application of dosimetry models given in Section 8.4.2 is an examination of
2 delivered dose versus response within a given species. This is followed in Section 8.4.3 by
3 some interspecies comparisons of delivered dose versus response.
4
5 8.4.2 Intraspecies Delivered Dose Response
6 Assuming that changes in FEVj in humans exposed to O3 are mainly the result of
7 O3 pulmonary tissue dose, Miller et al. (1988) constructed a dose-response curve. They
8 plotted decrements in FEV| versus predicted cumulative pulmonary region O3 tissue dose
9 scaled to body mass (Figure 8-13). The concentration-response data are from McDonnell
10 et al. (1983), in which 135 healthy subjects were exposed to 0.0, 0.12, 0.18, 0.24, 0.3, and
11 0.4 ppm O3. Exposure was for 2.5 h with intermittent heavy exercise. Miller et al. (1988)
12 used the average weight and height of the subjects to estimate FRC that was used in the
13 model to simulate O3 dose. The exercise breathing parameter data were used along with an
14 estimate of resting breathing parameters. Figure 8-13 is similar in shape to the
15 concentration-response curve of McDonnell et al. (1983). Differences between these two
16 curves, however, are accounted for by the translation between exposure concentration and
17 O3 dose.
18 Another example of the use of computed delivered dose to interpret intraspecies
19 response is provided by the work of Pinkerton et al. (1992) and Miller and Conolly (1993).
20 Pinkerton et al. (1992) examined the relation between actual tissue response of rats
21 chronically exposed to O3 and a prediction of O3 dose as a function of distance from the
22 bronchiole-alveolar duct junction to ventilatory units. Using these data, Miller and Conolly
23 (1993) plotted the predicted O3 dose and the observed change in wall thickness due to the
24 exposure versus distance from the bronchiole-alveolar duct junction (Figure 8-14). Even
25 though considerable variability in the thickness change can be inferred from the data, the two
26 curves, scaled to their values at the junction, show a remarkable similarity and suggest a
27 basic correctness in regards to the ventilatory unit model parameters.
28
29 8.4.3 Interspecies Delivered Dose Response
30 The illustrations presented in this section are based on dosimetric estimates for humans
31 and rats based on the existing or currently modified theoretical models (Miller et al., 1985;
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1
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24
Figure 8-13. Changes in FEVt versus pulmonary tissue dose. Plotted are decrements in
FEVj (liters) for human subjects against predicted pulmonary tissue dose
normalized to body weight. In order, from left to right, the dose values
correspond to 0, 0.12, 0.18, 0.24, 0.3, and 0.4 ppm O3 exposure
concentrations. The continuous curve was an "eye fit".
Source: Miller et al. (1988)
1 Miller et al., 1988; Overton et al., 1987). One functional and one inflammatory endpoint
2 will be provided drawing from the fB and BAL protein data described in Section 8.3.
3 Because the diversity of exposure scenarios across species is so great, the window of
4 exposure parameters has been narrowed to minimize exposure-based differences in relating
5 species responsiveness.
6 Since the theoretical models developed by Miller et al. (1985, 1988) and Overton et al.
7 (1987) estimate the dose distribution to the respiratory tract on a per breath basis, a
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Figure 8-14. Relationship between change hi alveolar wall thickness and predicted
O3 dose as a function of distance from the bronchiole-alveolar duct
junction (BADJ). Rats were exposed to 0.98 ppm for 8 h/day for 90 days.
Source: Miller and Conolly (1993).
1 minimum quantitative description of tidal breathing (VT and fB) is needed to utilize the
2 theoretical models of O3 deposition. The need for detailed breathing parameters, therefore,
3 severely restricts the application of the model to studies providing such data. Unfortunately,
4 breath-by-breath parameters over the the course of an exposure are not normally measured or
5 reported in most publications. Two studies, each in humans and rats, providing adequate
6 detail over the course of the exposure allowed the model computations to proceed for this
7 illustration (Figure 8-15). The human studies examined were DeLucia and Adams (1977),
8 who exposed the subjects to 0.15 and 0,3 ppm O3 for 1 h with continuous exercise (65%
9 Vo max) periods and Beckett et al. (1985), who exposed the subjects to 0.4 ppm O3 with
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1 alternating 15 min exercise (70 L/min). The rat studies evaluated were Tepper et al. (1989),
2 who exposed the rats to 0.35, 0.5, and 1.0 ppm O3 for 2.25 h with alternating 15-min
3 periods of CO2-induced hyperventilation (2 to 3 X resting VE) and Mautz and Bufalino
4 (1989), who exposed the rats to 0.8 ppm O3 for 3 h (at rest). The response parameter was
5 the ratio of O3-altered fB to control fB (fO3 / fcnt). Dose rate was the average dose to the
6 proximal alveolar region (PAR) computed from time 0 to the time of the maximum fo3 / fcnt.
7 The PAR was chosen as the target based on the perception that O3 acts on deep lung stretch
8 receptors directly or indirectly (via tissue fluid pressure—edema) as the stimulus for
9 tachypnea. A number of assumptions were necessary to implement the deposition model for
10 comparison of the human and rat in this context. These are noted in Table 8-6. Though
11 admittedly not validated, Figure 8-15 would suggest that the response of the rats not only
12 exceeded that of the human, but was initiated at a lower dose rate to the targeted lung
13 region. This conclusion is in general agreement with that emanating from Figure 8-7, but
14 the dose-to-target approach appears to accentuate the apparent difference in sensitivity for
15 this endpoint in favor of the rat.
16 Using an analysis similar to that applied above for fB, Miller et al. (1988) related
17 pulmonary tissue dose (normalized to body weight) to BAL protein from rats, guinea pigs,
18 and rabbits (Hatch et al., 1986) for O3 concentrations of 0 to 2.0 ppm for 4 h. These values
19 have been supplemented in Figure 8-16 to include the results of the human study of Koren
20 et al. (1989). As can be seen in the illustration, there appears to be a log-normal relationship
21 between BAL protein (/-eg/ml) and dose to the pulmonary region, the purported site of plasma
22 leakage to the airspace lumen. This relationship would support the contention that there is a
23 mechanistic consistency in response across species which may exhibit a quantitative
24 sensitivity factor for use in further quantitative interspecies extrapolation. This sensitivity
25 factor is evident from the clustering together of data from different animal species.
26
27
28 8.5 SUMMARY AND CONCLUSIONS
29 8.5.1 Ozone Dosimetry
30 There have been significant advances in O3 dosimetry since publication of the previous
31 O3 criteria document (U.S. Environmental Protection Agency, 1986) that better enable
December 1993 g-79 DRAFT-DO NOT QUOTE OR CITE
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D Tepperetal., (1989)
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0.0 0.2 0.4 0.6 0.8 1.0 1.2
Predicted average PAR dose rate (ng Qj/crrrVmin)
1.4
Figure 8-15. The maximum ratio of O3-altered breathing frequency to control breathing
frequency (fO3 / fcnt) at various O3 concentrations versus predicted
average dose rate to the proximal alveolar region (PAR first generation
distal to terminal bronchioles). The average is for the tune from the
initiation of exposure to the time of occurrence of the maximum ratio.
Beckett et al. (1985) and Mautz and Bufalino (1989) provided sufficient
ventilatory data for only one O3 concentration.
1 quantitative extrapolation with marked reductions in uncertainty. Prior to 1986, there were
2 limited data on O3 uptake in laboratory animals (Yokoyama and Frank, 1972; Miller et al.,
3 1979), essentially no reliable data in humans (Clamann and Bancroft, 1959; Hallett, 1965),
4 only one realistic model of Oj dose (Miller et al., 1978, 1985), and no data on O3 reaction
5 kinetics in lung lining fluids. At the present time, data gaps in all of these areas have begun
6 to fill in. Experiments and models describing the uptake efficiency and delivered dose of
December 1993
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Pulmonary Tissue Dose (ng Qj /g body weight)
Figure 8-16. Protein in the BAL for several laboratory animal species and humans as
related to the estimated pulmonary dose (normalized per g body weight).
Source: Modified from Miller et al. (1988).
1 O3 in the RT of animals and humans are beginning to present a clearer picture than has
2 previously existed.
3 The total RT uptake efficiency of rats at rest is approximately 0.50 (Wiester et al.,
4 1987, 1988; Hatch et al., 1989). Data in excised rat lungs support these in vivo findings,
5 and further indicate that O3 uptake efficiency is chemical reaction-dependent (Postlethwait
6 et al., 1993). Within the total RT of the rat, 0.50 of the O3 taken up by the total RT is
7 removed in the head, 0.07 in the larynx/trachea, and 0.43 in the lungs (Hatch et al., 1989).
8 The regional uptake efficiency data in the rat have been useful in estimating O3 mass transfer
9 coefficients for the rat.
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1 Ozone dosimetry models require input of regional mass transfer coefficients. Limited
2 studies have been conducted to quantitate the mass transfer coefficients of lung tissue directly
3 using excised animal tissue. In pig and sheep tracheae, mass transfer coefficients were
4 determined for unidirectional flow conditions and were found to be independent of flow,
5 suggesting a lack of dependence of O3 uptake on gas-diffusion processes (Ben-Jebria et al.,
6 1991) . These findings contrast with Aharonson et al. (1974) who found that the mass
7 transfer coefficient in dog nasopharynx increased as a function of increasing flow.
8 In humans at rest, the total RT uptake efficiency is between 0.80 and 0.95 (Gerrity
9 et al., 1988; Hu et al., 1992; Wiester et al, 1993). At VTs around 500 mL, total RT uptake
10 efficiency falls from about 0.9 to 0.75 as flow increases from 250 to 1,000 mL/s. As VT
11 increases, uptake efficiency increases and flow dependence lessens, suggesting that at high
12 VT, uptake may be gas diffusion limited. At a VT around 1500 mL, total RT uptake falls
13 from 0.96 at a flow of 250 mL/s to 0.92 at a flow of 1,000 mL/s. The studies of Gerrity
14 et al., (1988) and Wiester et al., (1993) indicate that the mode of breathing (oral versus nasal
15 versus oro-nasal) has little effect on URT or on total RT uptake efficiency. This observation
16 is supported by experiments comparing pulmonary function response as a function of mode
17 of breathing (Adams et al., 1989; Hynes et al., 1988). Uftman et al. (1993), however, found
18 that URT uptake efficiency was lower when mouthpiece breathing was compared with nasal
19 breathing. One possible explanation of the discrepancy among the studies is that a
20 mouthpiece may decrease URT uptake efficiency in comparison with unencumbered
21 breathing. The enhanced physiologic response to O3 shown by Adams et al. (1989) with
22 mouthpiece breathing supports this concept.
23 To obtain data on regional O3 uptake efficiency in humans, Gerrity et al. (1993b)
24 measured O3 concentrations at various anatomical sites (from the mouth to bronchus
25 intermedius) in spontaneously breathing humans. They found that the unidirectional uptake
26 efficiency of the trachea was similar to that of the sheep and pig trachea (Ben-Jebria et al.,
27 1991), suggesting a similar mass transfer coefficient behavior in the human trachea. Gerrity
28 et al. (1993b) also found that the uptake efficiencies between the mouth and various
29 anatomical sites in the total RT agreed well with the O3 bolus data of Hu et al. (1992). Both
30 the Hu et al. (1992) and Gerrity et al. (1993b) data indicate that the mass transfer
31 coefficients of the large conducting airways are larger than had been previously thought.
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1 When all of the animal and human in vivo O3 uptake efficiency data are compared,
2 there is a good degree of consistency across data sets. This agreement raises the level of
3 confidence with which these data sets can be used to support dosimetric model formulations
4 In the area of mathematical model formulation, there have been several models
5 developed since 1986. They can be grouped according to how transport and chemical
6 reactions are modeled: instantaneous reactions or quasi-steady first order reactions. The
7 models (Overton et al., 1987; Miller et al., 1988; Overton et al., 1989; Hanna et al., 1989;
8 Grotberg et al., 1990) predict that net O3 dose to lung lining fluid plus tissue gradually
9 decreases distally from the trachea toward the end of the tracheobronchial region, and then
10 rapidly decreases in the pulmonary region. When the dose of O3 to lung tissue is computed
11 theoretically, it is low in the trachea, increases to a maximum in the terminal bronchioles of
12 the first generation of the pulmonary region, and then decreases rapidly distally into the
13 pulmonary region. The models also provide insight into the role that increased ventilation
14 plays in enhancing O3-induced responses. The increased VT and flow, associated with
15 exercise in humans or CO2-stimulated ventilation increases in rats, shifts O3 dose further into
16 the periphery of the lung, causing a disproportionate increase in distal lung dose. This
17 prediction is supported by the data of Postlethwait et al. (1993) in excised rat lungs, and Hu
18 et al. (1992) and Gerrity et al. (1993b) in human lungs.
19 Ozone dosimetry models have also enabled examination of regional dosimetry among
20 parallel and serial anatomical structures. When an asymmetric lung morphology is used in
21 dosimetric models, the variation of O3 dose among anatomically equivalent ventilatory units
22 as a function of path length from the trachea varies as much as six-fold (Overton et al.,
23 1989; Mercer et al., 1991; Mercer and Crapo, 1993). This could have significant
24 implications for regional or localized damage to lung tissue. Whereas the average lung dose
25 might be at a level that would be considered insignificant, local regions of the lung may
26 receive significantly higher than average doses and therefore be at greater risk for chronic
27 effects.
28 Theoretical models have also been applied to make predictions about delivered doses
29 from exposure scenarios that are not necessarily achievable experimentally. Overton and
30 Graham (1989) and Miller and Overton (1989) have scaled the human lung dimensions to
31 account for age variations. They predicted that LRT uptake efficiency is not sensitive to age
December 1993 g_83 DRAFT-DO NOT QUOTE OR CITE
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1 at resting ventilation, but is age-dependent when exercise conditions are invoked. The total
2 quantity of O3 absorbed/minute is predicted to increase with age during heavy work or
3 exercise.
4
5 8.5.2 Species Sensitivity
6 Examining functional parameters measured analogously in humans and various animal
7 species discloses remarkable similarity in acute O3-induced effects and dysfunctions. The
8 tachypneic response to this oxidant is clearly concentration-dependent in both humans and
9 animals and shows parallel exacerbation when hyperventilation (e.g., exercise or CO2) is
10 superimposed. Indeed, rodents appear to be slightly more responsive than humans in this
11 regard. What is not known is whether this is evidence of pulmonary irritant sensitivity,
12 perhaps as a prelude to toxicity, or whether tachypnea is a defensive posture taken by the
13 respiratory system to minimize distal lung O3 deposition. Airway or lung resistance in
14 humans is not appreciably affected by acute exposure to O3 except under conditions of heavy
15 exercise; animals appear to need high level exposures or special preparations which bypass
16 nasal scrubbing. Dynamic lung compliance, on the other hand, tends to decrease across
17 species. However, the evidence in both animals and humans is not as strong as one might
18 expect, given the distal lung deposition of this poorly soluble oxidant.
19 Ozone-induced spirometric changes, the hallmark of response in humans, also occur in
20 exposed rats, though the relative responsiveness of these alterations in the rodent appears to
21 be about half that of the human. It is unclear, however, the degree to which anesthesia (rat)
22 and the comparability of hyperventilation induced by CO2 (rat) or exercise (human) may
23 influence this difference in responsiveness. Collectively, the acute functional response of
24 laboratory animals to O3 appears quite homologous to that of the human. Likewise, the
25 studies of BAL constituents indicate that the influx of inflammatory cells and protein from
26 the serum are influenced by species, but perhaps to a less extent than by ventilation and
27 antioxidant status, since adjustment for these factors can modulate responses to approximate
28 animal responses to those of humans. Unfortunately, these influential factors are rarely
29 measured and even less often, controlled.
30 When humans are exposed repeatedly for several consecutive days, lung function
31 decrements subside, and normal spirometric parameters are regained. This phenomenon of
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1 functional attenuation has also been demonstrated in rats, not only in terms of spirometry,
2 but also in terms of the classic tachypneic ventilatory response. Full or partial attenuation of
3 the BAL parameters appears to also to occur in both rats and humans, but exposure scenario
4 appears to play a role. This leads to the question of potential lung impairment resulting from
5 a near-lifetime exposure to O3. Existing epidemiologic studies provide only suggestive
6 evidence that persistent or progressive deterioration in lung function is associated with long-
7 term oxidant pollutant exposure. These long-term effects are thought to be expressed in the
8 form of maximum airflow or spirometric abnormalities, but the foundation for this
9 conclusion remains weak and hypothetical. Animal study data, while suggesting that Oj has
10 effects on lung function at near-ambient levels, present a variable picture of response which
/
11 may or may not relate to technical conditions of exposure or some other yet undiscovered
12 variable of response. Thus, a cogent interpretation of the animal findings as definitive
13 evidence of chronic deterioration of lung function would be difficult at this time. However,
14 the subtle functional defects apparent after 12 to 18 mo of exposure and the detailed
15 morphometric assessments of the O3-induced lesions do appear consistent with the modicum
16 of studies focusing on long-term effects in human populations. Based on the apparent
17 homology of these responses between humans and laboratory animals, animal studies provide
18 a means to more directly assess such chronic health concerns.
19
20 8.5.3 Quantitative Extrapolation
21 The agreement between theoretical models of O3 uptake and experimental
22 determinations of O3 uptake efficiency now provide a basis upon which responses may be
23 examined as a function of delivered O3 dose instead of exposure concentration of O3.
24 By examining responses as a function of delivered dose, the goal of quantitative extrapolation
25 between species can be approached.
26 The use of delivered dose to investigate responses has been examined in two contexts:
27 intraspecies comparisons and interspecies comparisons. With respect to intraspecies
28 comparisons, Miller et al. (1988) assumed that the relevant dose mediating the human
29 pulmonary function response was the pulmonary tissue dose. They then utilized the
30 breathing patterns, exposure concentrations, and pulmonary function responses from the
31 human studies of McDonnell et al. (1983) to predict the dose-response. They found that
December 1993 g-85 DRAFT-DO NOT QUOTE OR CITE
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1 there was general agreement between the shapes of the concentration-response curves and the
2 dose-response curves and that differences could be accounted for by the translation between
3 exposure concentration and O3 dose. In another example dealing with intraspecies
4 comparisons, Miller and Conolly (1993) compared the distribution of predicted O3 tissue
5 dose to a ventilatory unit in a rat as a function of distance from the bronchoalveolar duct
6 junction with the distribution of alveolar wall thickening as a function of the same distance
7 measure. Miller and Conolly (1993) found remarkable consistency between the predicted
8 dose distribution and the response distribution, (i.e., as predicted delivered dose declined,
9 response declined).
10 In an attempt to make an interspecies comparison of dose and response, existing or
11 currently modified models of Miller et al. (1985), Miller et al. (1988), and Overton et al.
12 (1987) were used to predict doses among species for two difference types of responses.
13 In the first case, the tachypneic response to O3 as a function of dose was analyzed. The
14 maximum ratio of O3-altered fB to control fB was plotted as a function of the average
15 centriacinar dose over the period from the beginning of exposure to the point of maximum
16 fB ratio. Rat and human data were used for this comparison. It was found that at
17 comparable O3 doses, the responses of rats greatly exceeded that of humans and were
18 initiated at lower doses. By examining the dose-response instead of the concentration-
19 response, the difference in tachypneic response between rats and humans is magnified.
20 In another example, an analysis similar to Miller et al. (1988) was performed to examine
21 recovered BAL protein as a function of O3 dose to the pulmonary region. The species
22 considered were the rat, guinea pig, rabbit, and human. In all cases, the BAL protein
23 response followed a log-linear relationship, suggesting a consistency of response across
24 species. Yet the data from different species tended to cluster together, suggesting species-
25 specific sensitivity factors.
26
27 8.5.4 Conclusions
28 There is an emerging consistency among a variety of O3 dosimetry data sets and
29 between the experimental data and theoretical predictions of O3 dose. The convergence of
30 experimental data with theoretical predictions lends a degree of confidence to the use of
31 theoretical models to predict total and regional O3 dose. Data have been acquired in animals
December 1993 8-86 DRAFT-DO NOT QUOTE OR CITE
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1 and humans that provide confirmation of total RT uptake efficiency predictions and provide
2 regional mass transfer coefficients that enhance the predictive capacity of theoretical models.
3 It is clear that the total RT uptake efficiency of rat is considerably less than that of humans
4 on a per breath basis. This difference is still one that is not totally accounted for in
5 theoretical models, but further refinements may narrow this discrepancy. Both the
6 theoretical models, and experimental data indicate that regional dose distribution and the
7 dependence of this distribution on breathing pattern can account for the role exercise plays in
8 enhancing acute O3-induced effects.
9 Comparison of O3-induced responses among animal species show that the acute
10 functional and inflammatory responses of laboratory animals to O3 is quite homologous to
11 human responses, although the detailed regional dose-response relationship appears to be
12 species dependent. At equivalent delivered doses to the centriacinar region, rat tachypneic
13 responses are considerably larger than humans. Inflammatory responses (influx of cells and
14 protein) may be less species dependent. On an exposure concentration basis, the relative
15 spirometric response of rats is less than humans by about one-half. A detailed dose-response
16 comparison has not been done, but differences in regional delivered dose could account for
17 the difference.
18 Laboratory animals and humans demonstrate similar attenuation of functional and
19 inflammatory responses with repeated exposure to O3, raising the question of potential lung
20 impairment in humans resulting from near life-time exposure.
21 There are still uncertainties that remain to be resolved before precise quantitative
22 extrapolation can be used to accurately predict human chronic O3 response. Now that the
23 technology for accurate regional dose determinations in animals and humans is available,
24 more fully integrated studies examining dose-response are required.
December 1993 g.g7 DRAFT-DO NOT QUOTE OR CITE
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APPENDIX A
GLOSSARY OF TERMS AND SYMBOLS
99mTc-DTPA
AD
AHSMOG
AM
AMI
ANOCOVA
AOD
AR
ARE
ATP
BAL
BALF
BHNP
BF
bkg
BM
Brdu
BS
BSA
BW
CA
CAR
CC10
Cdyn
CE
CI
CI
CIU
CL
Radiolabeled diethylene triamine pentaacetic acid
Radiolabeled ferric oxide
Alveolar duct
Adventist Health Smog studies
Alveolar macrophage
Asthma medication index
Analysis of covariance
Obstructive airways disease
Autoregressive
California Air Resources Board
Adenosine triphosphate
Bronchoalveolar lavage
Bronchoalveolar lavage fluid
JV-bis (2-hydroxypropyl) nitrosamine
Black female
Background
Black male
Bromodeoxyuridine
Black smoke
Body surface area
Body weight
Clean air
Centriacinar region
Dynamic compliance
Constant exercise
Cardiac index
Confidence interval
Cumulative methacholine inhalation units
Confidence limit
December 1993
A-l
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CO
CO2
CoH
ConA
COPD
CORD
CXT
Cu
DAAS
DR
DTPA
EEG
EPA
ER
ERV
ESC
f
F
F344
FA
FEF
FEF
25-75%
FEV3
FEV0.75
FP
FRC
FVC
GDT
G6PD
GR
Carbon monoxide
Carbon dioxide
Coefficient of haze
Concanavalin A
Chronic obstructive pulmonary disease
Chronic obstructive respiratory disease
Concentration times duration of exposure
Copper
Distal airways and alveolar surface fraction
Diffusing capacity for CO
Disulfide reductase
Diethylenetriaminapentaacetate
Electroencephalogram
U.S. Environmental Protection Agency
Emergency room
Expiratory reserve volume
Epithelial secretory cell
Respiratory frequency
Female
Fischer 344
Filtered air
Breathing frequency (also fR)
Forced expiratory flow
Forced expiratory flow between 25 and 75 % of vital capacity
Ferric oxide
Iron sulfate
Forced expiratory volume in 1 s
Forced expiratory volume in 3 s
Forced expiratory volume in 0.75 s
Fine particle
Functional residual capacity
Forced vital capacity
Glutathione-disulfide transhydrogenase
Glucose-6-phosphate
Glutathione reductase
December 1993
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GSH
GSHPx
GST
H+
HC
HCHO
HMV
HNO3
H2O
HR
HRmax
H2SO4
1C
ICAM
ICD
IE
Ig
IL
ip
IP
IU
iv
LDH
LM
LT
M
MAP
MC
MLN
MMEF
Mn
MnSO4
MVV
N2
Glulathione
Glutathione peroxidase
Glutathione-S-transferase
Hydrogen ion
Hydrocarbon
Formaldehyde
Half-hour mean value
Nitric acid
Water
Heart rate
Maximum heart rate
Sulfuric acid
Inspiratory capacity
Intracellular adhesion molecule
Nicotinamide adenine diphsophate-specific isocitrate dehydrogenase
Intermittent exercise
Immunoglobulin (IgA, IgE, IgG, IgM )
Interleukin (IL-1, IL-6, IL-8)
Intraperitoneal
Inhalable particles
International Units
Intravenous
Lactate dehydrogenase
Light microscopy
Leukotriene (LTB4, LTC4, LTD4, LTE4)
Male
Mean arterial blood pressure
Methacholine challenge
Mediastinal lymph modes
Maximum mid-expiratory flow
Manganese
Manganese sulfate
Maximum voluntary ventilation
Nitrogen
December 1993
A-3
DRAFT—DO NOT QUOTE OR CITE
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AN2
NA
NAAQS
NADH
NADPH
NADPH-CR
NEP
NH4+
NHANES
(NH4)2S04
Nffl
NK
NL
NO
NO2
N03
NOX
NPSH
NS
NZW
02
03
OR
OX (10)
OX
-------
PDI
PE
PEFR
PG
6PGD
PHA
PM2.5
PM
10
PM15
PMN
p.r.n.
PUFA
R
Raw
RB
RH
RL
RR
RSP
RT
RV
S
SAROAD
SB
SD
S-D
SE
SEM
SEM
Provocative dose that produces a 100% decrease in forced expiratory
volume in 1 s
Pain on deep inspiration
Postexposure
Peak expiratory flow rate
Prostaglandin (PGD2, PGE^ PGE2 PGFla, PGF2a)
6-Phosphogluconate
Phytochemagglutinin
Particulate matter of mass median aerodynamic diameter 2.5
or less
Particulate matter of mass median aerodynamic diameter 10
or less
Particulate matter of matter of mass median aerodynamic diameter
15/um or less
Polymorphonuclear leukocyte
As needed (pro re nata)
Polyunsaturated fatty acid
Intraclass correlation coefficeint
Airway resistance
Respiratory bronchiole
Relative humidity
Lung resistance
Relative risk
Respirable suspended particulates
Total respiratory resistance
Residual volume
Smoker
Storage and Retrieval of Aerometric Data, U.S. EPA
centralized data base; superseded by AIRS (q.v.)
Shortness of breath
Standard deviation
Sprague-Dawley
Standard error
Standard error of the mean
Scanning electron microscopy
December 1993
A-5 DRAFT—DO NOT QUOTE OR CITE
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SHAPE
SMG
S02
S04
S04=
SOD
SRBC
T
TB
TEARS
Tdb
THC
Tffl
TLC
TNF
TSP
TSP (200)
TX
UCLA
UV
VC
vs
VT
VTmax
V
25%VC
VEmax
Specific airway conductance
Simulation of Human Activity and Pollutant Exposure
Small-mucous-granule
Sulfur dioxide
Sulfate
Sulfate ion
Superoxide dismutase
Specific airway resistance
Sheep red blood cell
Temperature
Terminal bronchiole
Thiobarbituric acid reactive substances
Dry bulb temperature
Total hydrocarbon content
Temperature-humidity index
Total lung capacity
Tumor necrosis factor
Total suspended particulates
Number of hours above a total suspended paniculate concentration of
200 Atg/m3
Thromboxane (A2, B2)
University of California at Los Angeles
Ultraviolet
Vital capacity
Volume per surface area
Tidal volume (also TV)
Maximum tidal volume
Volume fraction
Lung volume at 25 % of the vital capacity
Lung volume at 50 % of the vital capacity
Alveolar ventilation
Minute ventilation; expired volume per minute
Maximum minute ventilation
Maximum expiratory flow at 25 % of the vital capacity
Maximum expiratory flow at 50% of the vital capacity
December 1993
A-6
DRAFT-DO NOT QUOTE OR CITE
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VO2 Oxygen uptake by the body
VO2max Maximal oxygen uptake (maximal aerobic capacity)
W Watt
WBGT Wet bulb globe temperature
WF White female
WM White male
Zn Zinc
ZnO Zinc oxide
December 1993 A-7 DRAFT—DO NOT QUOTE OR CITE
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