&EPA
                United States
                Environmental Protection
                Agency
                 Office of
                 Research and Development
                 Washington, DC 20460
EPA/600/8-91/049cA
August 1991
External Review Draft
Air Quality
Criteria for
Oxides  of
Nitrogen
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Do not
Cite or Quote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.

-------

-------
(Do not Cite or Quote)
EPA 600/8-91/049cA
August 1991
External Review Draft
                          Air Quality Criteria for
                            Oxides of Nitrogen

                               Volume HI 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.
                      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.
August 1991
m-ii      DRAFT-DO NOT QUOTE OR CITE

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              Air Quality Criteria for Oxides of Nitrogen
                       TABLE OF CONTENTS

                             Volume I

 1. SUMMARY OF EFFECTS OF OXIDES OF NITROGEN AND
   RELATED COMPOUNDS ON HUMAN HEALTH AND
   WELFARE	

 2. INTRODUCTION	

 3. GENERAL CHEMICAL AND PHYSICAL PROPERTIES OF NOX
   AND NOX-DERIVED POLLUTANTS  	

 4. SOURCES OF NITROGEN OXIDES INFLUENCING AMBIENT
   AND INDOOR AIR QUALITY  	

 5. TRANSPORT AND TRANSFORMATION OF NITROGEN
   OXIDES	

 6. SAMPLING AND ANALYSIS FOR OXIDES OF NITROGEN
   AND RELATED SPECIES 	

 7. AMBIENT AND INDOOR CONCENTRATIONS OF NITROGEN
   DIOXIDE	

 8. ASSESSING TOTAL HUMAN EXPOSURE TO NITROGEN
   DIOXIDE	


                             Volume II

 9. EFFECTS OF NITROGEN OXIDES ON VEGETATION	

10. THE EFFECTS OF NITROGEN OXIDES ON NATURAL
   ECOSYSTEMS AND THEIR COMPONENTS   	

11. EFFECTS OF NITROGEN OXIDES ON VISIBILITY	

12. EFFECTS OF NITROGEN OXIDES ON MATERIALS	
                      'age
                      1-1

                      2-1


                      3-1


                      4-1


                      5-1


                      6-1


                      7-1


                      8-1
                      9-1


                     10-1

                     11-1

                     12-1
August 1991
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                  Air Quality Criteria for Oxides of Nitrogen



                      TABLE OF CONTENTS (cont'd)


                              Volume in


 13. STUDIES OF THE EFFECTS OF NITROGEN COMPOUNDS
    ON ANIMALS
 14. EPIDEMIOLOGY STUDIES OF OXIDES OF NITROGEN .......       14-1


 15. CONTROLLED HUMAN EXPOSURE STUDIES OF OXIDES
    OF NITROGEN  .......... . ............... ........       15-1


 16. HEALTH EFFECTS ASSOCIATED WITH EXPOSURE TO
    NITROGEN DIOXIDE ..............................       16_j


 APPENDIX A:  GLOSSARY OF TERMS AND SYMBOLS  ..... .....       A-l
August 1991                      m_iv     DRAFT-DO NOT QUOTE OR CITE

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                                   CONTENTS
                                                                          Pas
TABLES	
FIGURES	
AUTHORS 	
CONTRIBUTORS AND REVIEWERS  	

13. STUDIES OF THE EFFECTS OF NITROGEN COMPOUNDS
    ON ANIMALS	.  . .	
    13.1  INTRODUCTION  	
    13.2  NITROGEN DIOXIDE	
          13.2.1  Respiratory Tract Transport and Absorption  ...
                  13.2.1.1   Introduction	
                  13.2.1.2   Principles of Gas Uptake and
                            Dosimetry Models	
                  13.2.1.3   Dosimetry of Nitrogen Dioxide ....
          13.2.2  Mortality	
          13.2.3  Respiratory Effects  	
                  13.2.3.1   Host Defense Mechanisms  .......
                  13.2.3.2   Lung Biochemistry	
                  13.2.3.3   Pulmonary Function	
                  13.2.3.4   Morphologic Studies	
          13.2.4  Extrapulmonary Effects	
                  13.2.4.1   Body Weight  	
                  13.2.4.2   Hematologic Changes	
                  13.2.4.3   Cardiovascular Effects	
                  13.2.4.4   Hepatic Function	
                  13.2.4.5   Effects on the Kidney and on Urine
                            Content	
                  13.2.4.6   Effects on the Central  Nervous System
                            and Behavioral Effects   	
                  13.2.4.7   Reproductive, Developmental, and
                            Heritable Mutagenic Effects	
                  13.2.4.8   Potential Carcinogenic or
                            Co-Carcinogenic Effects  	
    13.3  EFFECTS OF MIXTURES CONTAINING NO2	
    13.4  NITRIC OXIDE	
    13.5  NITRIC ACID AND NITRATES	
          13.5.1  Nitric Acid	
          13.5.2  Nitrates	
    13.6  SUMMARY	
          13.6.1  Animal-to-Human Dosimetric Estimates	
          13.6.2  Biochemical and Cellular Mechanisms	
          13.6.3  Effects on Host Defenses	
                                   Ill-ix
                                   Ill-xvi
                                    13-1
                                    13-1
                                    13-1
                                    13-1
                                    13-1

                                    13-3
                                    13-6
                                    13-12
                                    13-17
                                    13-17
                                    13-59
                                    13-76
                                    13-85
                                    13-120
                                    13-120
                                    13-124
                                    13-131
                                    13-133

                                    13-137

                                    13-139

                                    13-141

                                    13-144
                                    13-153
                                    13-168
                                    13-175
                                    13-175
                                    13-176
                                    13-177
                                    13-179
                                    13-180
                                    13-180
August 1991
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                              CONTENTS (cont'd)
                                                                       Page

          13.6.4  Effects of Chronic Exposure on the Development
                 of Chronic Lung Disease	        13-183
          13.6.5  Potential Carcinogenic or            '
                 Co-Carcinogenic Effects	        13-184
          13.6.6  Extrapulmonary Effects  ......	        13-185
          13.6.7  Susceptibility of Subpopulations  . .	        13-186
          13.6.8  Influence of Exposure Patterns	        13-186
          13.6.9  Interactions with Other Pollutants	        13-187
    REFERENCES	 .  .	        13-189

 14. EPIDEMIOLOGY STUDIES OF OXIDES OF NITROGEN  .......        14-1
    14.1  INTRODUCTION  ..........		        14-1
    14.2  STUDIES OF RESPIRATORY ILLNESS	        14-5
          14.2.1  United Kingdom Studies	        14-8
          14.2.2  United States Six Cities Studies	        14-14
          14.2.3  Tayside Study	 .        14-18
          14.2.4  Iowa Study .	        14-19
          14.2.5  Dutch Studies	        14-19
          14.2.6  Ohio Study   . ,	 •.	 .        14-22
          14.2.7  Swiss Study  . .,	•....,.	        14-23
          14.2.8  Connecticut Study	,       14-24
          14.2.9  Chestnut Ridge Study	 .        14-26
          14.2.10  California Seventh-Day Adventist Study  .:........        14-27
          14.2.11  Maryland Study	        14-28
          14.2.12  Glendora, California Study ......	        14-28
          14.2.13  Chattanooga Studies	        14-29
          14.2.14  United States and Canadian Skating
                 Rink Exposures	        14-31
    14.3 STUDIES OF PULMONARY FUNCTION • •  •	        14-31
          14.3.1   United States Six City Studies	        14-32
          14.3.2  Tucson Study	 .        14-32
          14.3.3   New York Study  .  . . .-	,	        14-33
          14.3.4   Chestnut Ridge Study	  ..     14-33
    14.4 OUTCOMES RESULTING FROM OCCUPATIONAL
         EXPOSURES	        14-34
    14.5 SYNTHESIS  OF THE EVIDENCE		        14-35
          14.5.1   Health Outcome Measures	        14-35
          14.5.2   Biological Basis	        14-43
         14.5.3   Quantitative Analysis	        14-47
    14.6 CONCLUSIONS	        14-55
    14.7 SUMMARY	           14-56
    REFERENCES	        14-61
    APPENDIX 14A	 .        14A-1
August 1991                          III-vi      DRAFT-DO NOT QUOTE OR CITE

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                             CONTENTS (cont'd)

15.  CONTROLLED HUMAN EXPOSURE STUDIES OF OXIDES
    OF NITROGEN	
    15.1  INTRODUCTION	
    15.2  EFFECTS OF NOX IN HEALTHY NORMAL SUBJECTS  .
         15.2.1   Lung Function Effects of NO2	
                15.2.1.1 Concentrations Above 1.0 ppm	;
 , .             15.2.1.2 Concentrations Below 1.0 ppm	
                15.2.1.3 Respiratory Symptom and Sensory Effects
                        of NO2 Exposure  .-...-	
                15.2.1.4 Mucociliary Clearance after NO2
                        Exposure	
         15.2.2   Effects of Nitric Oxide .	
         15.2.3   Effects of NO2-Gas or Gas/Aerosol Mixtures on
                Lung Function in Normal Subjects  ..........
         15.2.4   Summary	
    15.3  THE EFFECTS OF NOX EXPOSURE IN SENSITIVE
         SUBJECTS	
         15.3.1   The Effects of NO2 on Asthmatics  	
                15.3.1.1 Effects of HNO3 Vapor on Asthmatics  . .
         15.3.2   Effects of NO2 on Patients with Chronic
                Obstructive Lung Disease	
         15.3.3   Summary	'...'..
    15.4  EFFECTS OF NO2 EXPOSURE ON AIRWAYS
         RESPONSIVENESS	
         15.4.1   Healthy Normal Subjects	 .
         15.4.2   Asthmatic Subjects	
    15.5  EFFECTS OF NO2 OR HNO3 EXPOSURE ON BLOOD,
         URINE, AND BRONCHOALVEOLAR LAVAGE
         FLUID BIOCHEMISTRY  .  .	
         15.5.1   Biochemical Effects in Blood	 . .  . .
         15.5.2   Bronchoalveolar Lavage Fluid Biochemistry . . .  . .
         15.5.3   Urine Biochemistry	: .  . .
    15.6  EFFECTS OF NO2 OR HNO3 VAPOR EXPOSURE ON
         HUMAN PULMONARY HOST DEFENSE RESPONSES  . .
    15.7  EFFECTS OF NITRATES ON HUMAN LUNG
         FUNCTION  . . .	
    15.8  CONCLUSIONS  AND  DISCUSSION	
    REFERENCES	
    APPENDIX 15A	

16.  HEALTH EFFECTS ASSOCIATED WITH EXPOSURE TO
    NITROGEN DIOXIDE	
    16.1  INTRODUCTION	
    16.2  AMBIENT AND  INDOOR NITROGEN DIOXIDE LEVELS
    16.3  KEY HEALTH EFFECTS OF NO2	
                        Page

                        15-1
                        15-1
                        15-9
                        15-9
                        15-9
                        15-21

                        15-24

                        15-24
                        15-24

                        15-25
                        15-32

                        15-32
                        15-33
                        15-51

                        15-52
                        15-56

                        15-61
                        15-61
                        15-62
                        15-71
                        15-71
                        15-72
                        15-73

                        15-74

                        15-79
                        15-82
                        15-88
                        15 A-1
                       ' 16-1
                        16-1
                       .16-1
                        16-3
August 1991
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                              CONTENTS (cont'd)
          16.3.1  Airway Reactivity in Asthmatics and Short-Term
                 (1-3 h) Exposure to NO2	       16-3
          16.3.2  Respiratory Morbidity in Children Associated with
                 Exposure to NO2	       16-6
          16.3.3  Biological Bases Relating NO2 Exposure to
                 Respiratory Morbidity: Effects of NO2 on
                 the Respiratory Host Defense System  ............       16-12
          16.3.4  Emphysema and Exposure to NO2	       16-16
          16.3.5  Subpopulations Potentially Susceptible to
                 NO2 Exposure	       16-18
    REFERENCES	.  .'.	       16-21

 APPENDIX A:  GLOSSARY OF TERMS AND SYMBOLS .	 .       A-l
August 1991                         Ill-viii     DRAFT-DO NOT QUOTE OR CITE

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                                     TABLES
Number
13-1      Mortality from Acute (<24 hr) Exposure to NO2	> .

13-2      Mortality from Repeated or Chronic Exposure to NO2  ... . .

13-3      Mucociliary Activity	 ,

13-4      Alveolar Macrophages	  . . .

13-5      Immunological Effects	,

13-6      Interaction of NO2 with Infectious Agents	,

13-7      Effects of NO2 on Lipid Metabolism	,

13-8      Effects of NO2 on Lung Amino Acids, Proteins, and Enzymes

13-9      Effects of NO2 on Antioxidant Metabolism and Influence
          of Antioxidants	,

13-10     Effects of NO2 on Pulmonary Function  	,

13-11     Effects of Acute Exposure to NO2 on Lung Morphology  . . .

13-12     Effects of Subchronic Exposure to NO2 on Lung Morphology  ,

13-13     Effects of Chronic Exposure to NO2 on Lung Morphology . . ,

13-14     Effects of NO2 on the Development of Emphysema	,

13-15     Extrapulmonary Effects of NO2: Body Weight	

13-16     Effects of NO2 on Red Blood Cells and Hemoglobin  	

13-17     Effects of NO2 on Leukocytes and Platelets	

13-18     Effects of NO2 on Red Blood Cell Membranes	

13-19     Effects on NO2 on Serum Proteins and Clinical Chemistries .

13-20     Effects of NO2 on the Liver  . .	

13-21     Effects of NO2 on the Kidney and on Urine Contents  . .  . . .
                                      Page

                                     13-13

                                     13-15

                                     13-21

                                     13-29

                                     13-38

                                     13-43

                                     13-60

                                     13-62


                                     13-66

                                     13-77

                                     13-86

                                     13-89

                                     13-93

                                     13-118

                                     13-121

                                     13-125

                                     13-126

                                     13-129

                                     13-132

                                     13-134

                                     13-138
August 1991
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                                 TABLES (cont'd)
 Number                                                                     Page

 13-22     Effects of NO2 on the Central Nervous System and Behavior  . . .        13-140

 13-23     Effects of NO2 on Reproduction, Development, and Heritable
          Mutagenesis	        13-142

 13-24     Effects of NO2 on Carcinogenesis or Co-carcinogenesis .	        13-145

 13-25     Toxicologic Interactions to Simple Mixtures Containing NO2  . . .        13-154

 13-26     Effect of NO on Respiratory Tract Morphology  	        13-170

 14-1      Symptom Rates of United Kingdom Children by Gender,
          Social Class,  and Cooking Type	        14-9

 14-2      U.S. Environmental Protection Agency Multiple Logistic
          Analysis of Data from the Melia et al. (1977) Study	        14-9

 14-3      Unadjusted Rates of One or More Symptoms Among United
          Kingdom Children by Gender, Social Class,
          and Cooking Type	        14-11

 14-4      U.S. Environmental Protection Agency Multiple Logistic
          Analysis of Data from Melia et al. (1979) Study	        14-11

 14-5      Unadjusted Rates of One or More Symptoms Among United
          Kingdom Boys and Girls by Bedroom Levels of NO2	        14-13

 14-6      Unadjusted Rates of One or More Symptoms Among United
          Kingdom Boys and Girls by Bedroom Levels of NO2	  .        14-14

 14-7      NO2 Concentrations (ppm) by Season and Stove Type in
          Portage, WI	        14-15

 14-8      Odds Ratios and 95% Confidence Intervals for the Effect
          of an Additional 17.3 ppb NO2 on the Symptom Prevalence ....        14-17

 14-9      Regression  Coefficients for Multiple Logistic Analyses of
          Respiratory Illness in Tayside Children	        14-18

 14-10     Analysis of Iowa City School Children Respiratory
          Symptoms by Gas Stove Type and Parental Smoking  	        14-20

 14-11     Dutch Study Estimated and Measured Personal NO2                           '•
          Exposure Oug/m3)  for a Single Weekly Average  	.  „        14-20

August 1991                           HI-x      DRAFT-DO NOT QUOTE OR CITE

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                                TABLES (cont'd)
Number                              .             .    .

14-12,    Frequency and Prevalence of Reported Respiratory
         Symptoms in Different Categories of Mean Indoor NO2
         Concentrations in a Population of 775 Dutch Children of
    ';.    6 to 12 Years Old  	        14-21

14-13.;    Adjusted Annual Symptom Incidence Rates and NO2   •   ; •  :
         Exposure Levels for Four Communities in Switzerland	        14-24

14-14    Health Outcome Measures in Selected NO2 Epidemiology Studies         14-37

14-15    Classification of Illnesses in Surveillance Studies	        14-42

14-16    Summary of Odds Ratios from Studies of the Effects of
         Nitrogen Dioxide	        14-51

14-17    U.S. Environmental Protection Agency Combined Analyses
         of Studies on Respiratory Illness Effects of Nitrogen Dioxide  . . .        14-53

14A-1    Summary of the Effects of NO2 on Respiratory Illness  .......        14A-2

14A-2    Summary of Effects of NO2 on Pulmonary Function  	        14A-9

15-1     Responses of Healthy Normal Subjects to NO2 Exposure	        15-10

15-2     Exposure of Healthy Normal Subjects to NO2 Mixtures . ......        15-26

15-3     Subject  Characteristics of Asthmatics Exposed to NO2  	        15-34

15-4     Exposure Conditions and Responses in Asthmatics     :
         Exposed to NO2   	        15-37

15-5     Subject  Characteristics for COPD Patients Exposed to NO2  ....        15-53

15-6     Exposure Conditions and Responses in COPD Patients
         Exposed to NO2   .	....;..	        15-54

15-7     FEV^ and Resistance Changes in Asthmatics
         Exposed to NO2	 . .	        15-57

15-8     Changes in Airway Responsiveness Associated with
         NO2 Exposure	....-........;.....	        15-63

15-9     Fraction of NO2-Exposed Subjects with Increased
         Airway Responsiveness  ............................        15-70

August 1991                           IH-xi     DRAFT-DO NOT QUOTE OR CITE

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 Number
 15-10
 16-1
 16-2
                                  TABLES (cont'd)
Exposure Conditions and Responses in Subjects Exposed
to Nitrates	
 15A-1    Diagnostic Features of Clinical Categories of Asthma

 15A-2    Clinical Severity of Asthma . .	

 15A-3    Temporal Course of Asthma	

 15A-4    Questionnaire and Survey	
U.S. Environmental Protection Agency Analysis of Variability
in 2-week Ambient Averages of 1-h NO2 Data at Ten
Selected Locations	
Estimates of the Resident Population of Children and Young
Adults of the United States, by Age and Sex, July 1, 1989 .
                           Page


                           15-80

                           15A-3

                           15A-4

                           15A-4

                           15A-7



                           16-12


                           16-20
August 1991
                            IH-xii
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                                      FIGURES
Number
                                       'age
13-1      Mortality enhancement for mice exposed to NO2 at various
          concentrations and for various durations prior to challenge
          with streptococci	

14-1      Total personal exposure to NO2 vs. NO2 levels in Connecticut
          residences  	;.....

14-2      For the Melia et al. (1979) study, a graph  of the marginal
          likelihood function of the odds ratios for combined gender
          (boys and girls) of the outcome measures colds to  chest
          and any respiratory illness developed by EPA	

14-3      U.S. Environmental Protection Agency meta-analysis of
          epidemiologic studies of NO2 exposure effects on respiratory
          disease in children <12 years old  	

15-1      Percent change  (post-air vs. post NO2) in FEV1 0  vs. NO2 dose
          in ppm X liters in asthmatics	

15-2      Percent change  (post-NO2-post air/post-air) in resistance
          (Raw, SRaw, or RT) vs. NO2 dose in ppm  X liters in asthmatics

16-1      U.S. Environmental Protection Agency meta-analysis of
          epidemiologic studies of NO2 exposure effects on  respiratory
          disease in children <12 years old   	
                                      13-51
                                      14-25
                                      14-40



                                      14-50


                                      15-59


                                      15-60



                                      16-9
 August 1991
IH-xiii     DRAFT-DO NOT QUOTE OR CITE

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                                      AUTHORS
                            Chapter 13:  Animal Toxicology
 Ms. Beverly Comfort
 Environmental Criteria and Assessment Office
 U.S. Environmental Protection Agency
 Research Triangle Park, NC 27711

 Dr. Donald Gardner
 ManTech Environmental Technology, Inc.
 P.O. Box 12313
 Research Triangle Park, NC 27709

 Dr. Judith A. Graham
 Environmental Criteria and Assessment Office
 U.S. Environmental Protection Agency
 Research Triangle Park,  NC 27711

 Dr. Jerry Last
 California Primate Research Center
 University of California
 Davis, CA 95616

 Dr. Susan Loscutoff
 2594 W. Ellery Street
 Fresno, CA  93711
           Dr. Richard Schlesinger
           New York'University
           Long Meadow Road
           Tuxedo, NY  10987

           Dr. Jeffrey L.  Tepper            .—'
           ManTech Environmental Technology, Inc.
           P.O. Box  12313
           Research Triangle Park, NC 27709

           Dr. Walter S. Tyler
           V.M. Anatomy
           University of California
           Davis, CA 95616           •

          Dr. John Overton
          Health Effects Research Laboratory
          U.S. Environmental Protection Agency
          Research Triangle Park, NC 27711
                              Chapter 14: Epidemiology
Dr. Victor Hasselblad
Center for Health Policy Research
Duke University
Durham, NC 27713
          Dr. Dennis J. Kotchmar
          Environmental Criteria and Assessment Office
          Office of Health and Environmental
           Assessment
          U.S. Environmental Protection Agency
          Research Triangle Park, NC 27711
                             Chapter 15:  Clincal Studies
Dr. Lawrence J. Folinsbee
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
August 1991
IH-xiv     DRAFT-DO NOT QUOTE OR CITE

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                                AUTHORS (cont'd)

      Chapter 16:  Health Effects Issue Associated with Exposure to Nitrogen Dioxide
Dr. Donald Gardner
ManTech Environmental Technology, Inc.
P.O. Box 12313
Research Triangle Park, NC 27709

Dr. Victor Hasselblad
Center for Health Policy Research
Duke University
Durham, NC  27713

Dr. Dennis J. Kotchmar
Environmental Criteria and Assessment Office
Office of Health and Environmental
  Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
        Dr. Lawrence J. Folinsbee
        Health Effects Research Laboratory
        U.S. Environmental Protection Agency
        Research Triangle Park, NC  27711

        Dr. Judith A. Graham
        Environmental Criteria and Assessment Office
        U.S. Environmental Protection Agency
        Research-Triangle Park, NC  27711
 August 1991
HI-xv
DRAFT-DO NOT QUOTE OR CITE

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                          CONTRIBUTORS AND REVIEWERS
                                     Chapters 13-15
  Dr. Ursula Ackermann-Liebrich
  Department of Social and Preventative
   Medicine
  University of Basel
  St. Albanvorstadt 19
  4052 Basel
  Switzerland
  41-61-21-60-67

  Dr. Ester Azoulay-Dupuis
  INSERM Unit 13-Hospital Claude Bernard
  10 Avenue de la Porte
  D'Aubervilleis
  75019 Paris
  France

  Dr. Michael A. Berry
  Environmental Criteria and Assessment Office
  U.S. Environmental Protection Agency
  Research Triangle Park, NC 27711

  Dr. Irwin H. Billick
  Gas Research Institute
  8600 West Bryn Mawr Avenue
 Chicago, EL 60631

 Dr.  Gary R. Burleson
 Health Effects Research Laboratory
 U.S. Environmental Protection Agency
 Research Triangle Park, NC 27711

 Dr. Robert Chapman
 Health Effects Research Laboratory
 U.S. Environmental Protection Agency
 Research Triangle Park, NC  27711

 Mr. David Coffin
 Health Effects Research Laboratory
 U.S. Environmental Protection Agency
 Research Triangle Park, NC  27711
           Dr. Albert Collier
           535 Clinical Sciences Bldg. C#7220
           University of North Carolina
           Chapel Hill, NC  27599-7220

           Dr. Steven D. Colome
           Integrated Environmental Services
           University Towers, Suite 1090
           4199 Campus Drive
           Irvine, CA  92715

           Ms. Beverly Comfort
           Environmental Criteria and Assessment Office
           U.S. Environmental Protection Agency
           Research Triangle Park, NC 27711

           Dr. James Crapo
           Division of Allegy, Critical Care and
            Respiratory Medicine
           Box 3.177
           Duke University Medical Center
           Durham, NC 27710

          Dr. Douglas Dockery
          Harvard School of Public Health
          Department of Environmental Science and
           Physiology
          665 Huntington Avenue
          Boston, MA  02115

          Dr. D. L.  Dungworth
          Department of Veterinary Pathology
          University  of California
          Davis, CA 95616

          Dr. Lawrence J. Folinsbee
          Health Effects Research Laboratory
          U.S. Environmental Protection Agency
          Research Triangle Park, NC  27711
August 1991
m-xvi     DRAFT-DO NOT QUOTE OR CITE

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                    CONTRIBUTORS AND REVIEWERS (cont'd)
Dr. Donald Gardner
ManTech Environmental Technology, Inc.
P.O. Box 12313
Research Triangle Park, NC 27709

Dr. Judith A. Graham
Environmental Criteria and Assessment Office
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711

Dr. Lester D. Grant
Environmental Criteria and Assessment Office
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711

Dr. Elaine Grose
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711

Dr. Jack Hackney
Environmental Health Services-Room 51
Rancho Los Amigos Hospital
7601 Imperial Highway
Downey, CA  90242

Dr. Victor Hasselblad
Center for Health Policy Research
Duke University
Durham, NC  27713

Dr. Gary Hatch
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park,  NC  27711

Dr. Carl Hayes
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC  27711
        Dr. Milan Hazucha
        Center for Environmental Medicine and
          Lung Biology
        CB#7310 Trailer#4 Medical Research
          Building C
        University of North Carolina .
        Chapel Hill, NC  27599

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                      CONTRIBUTORS AND REVIEWERS (cont'd)
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                    CONTRIBUTORS AND REVIEWERS (cont'd)
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                                     Chapter 16
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Center for Health Policy Research
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Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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                     CONTRIBUTORS AND REVIEWERS (cont'd)
  Dr. Dennis J. Kotchmar
  Environmental Criteria and Assessment Office
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  Mr. Tom McMullen
  Environmental Criteria and Assessment Office
  U.S. Environmental Protection Agency
  Research Triangle Park, NC  27711
          Dr. Maryjane Selgrade
          Health Effects Research Laboratory
          U.S. Environmental Protection Agency
          Research Triangle Park, NC 27711
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     13.  STUDIES OF  THE EFFECTS  OF NITROGEN
                    COMPOUNDS ON ANIMALS
13.1  INTRODUCTION
     This chapter discusses the effects of the oxides of nitrogen (NOX) in experimental
animals. Previous reviews of the literature have appeared in a criteria document (U.S.
Environmental Protection Agency, 1982). A World Health Organization summary has also
been published (World Health Organization, 1987).
     Most of the data presented in this chapter relate to the  effects of nitrogen dioxide (NO2)
on experimental  animals because the vast majority of the NOX literature is on NO2.  The
results of the few comparative NOX studies show that NO2 appears to be the most toxic.  The
majority of the literature describes the effects of NO2 on the respiratory tract; however,
extrapulmonary effects also have been observed and are included here.  A broad range of
NO2 concentrations have been evaluated, but emphasis has primarily been placed on those
studies at exposure concentrations of 9,400 jwg/m3 (5 ppm) or less, with the exception of
studies on dosimetry, mortality, and emphysema.  Related studies demonstrating the potential
of NO2 to produce structural, functional, and biochemical changes in mammalian species at
higher concentrations are incorporated in a series of tables.
     Discussions of the available literature on the effects of other NOX compounds and
mixtures containing NO2 also are included in this chapter.  These sections are short because
of the general lack of information in these areas.
13.2 NITROGEN DIOXIDE
13.2.1  Respiratory Tract Transport and Absorption
13.2.1.1  Introduction
     Dosimetry refers to measuring or estimating the quantity of a chemical absorbed by
target sites such as the pulmonary region tissue or more locally, the tissue of the centriacinar
region. Dosimetry allows exposure-response data to be transformed to dose-response
relationships.  However, to extrapolate animal data to humans, knowledge of dosimetry and
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  1     species sensitivity must both be considered.  Even when two species receive an identical local
  2     tissue/cellular dose, cellular sensitivity and repair/defense mechanisms determine the extent
  3     and type of injury produced.  These mechanisms are likely to vary among humans and
  4     animals because of dissimilarities in pharmacokinetics, genetic make up, metabolic rates,
  5     detoxification systems, and/or other factors.  At present, our knowledge of dosimetry is more
  6     advanced than that of species sensitivity, inhibiting quantitative animal-to-human extrapolation
  7     of effective NO2 concentrations. Nevertheless, knowledge of interspecies differences and
  8     similarities in dosimetry alone is crucial to the process of risk assessment, as will be
  9     discussed.                                                             .
 10          The compound most directly responsible for toxic effects may be the inhaled gas, here
 11     NO2, or chemical reaction products or metabolites. Complete identification of the actual
 12     toxic agents and their integration into dosimetry is a complex issue that has not been fully
 13     resolved.  Thus, most dosimetry investigations, which are difficult enough,, are concerned
 14     with the dose of the primary inhaled chemical. In the context of inter- or intraspecies
 15     dosimetric extrapolation, a further confounding aspect can be the units of dose (e.g., mass
 16     retained per breath,  mass retained per breath per body weight, mass retained per breath per
 17     respiratory tract surface area). That is, when comparing dose between species, what is the
 18     relevant measure of dose?  This question, like the previous issue, has not been answered;
 19     units are often dictated by the type of experiment and/or by the choice of the investigators.
20          Theoretical (modeling) and experimental studies  are  used to obtain information on dose.
21     Experiments have been carried out to obtain direct measurements of absorbed NO2 in the
22     total respiratory tract, the upper respiratory tract (region proximal to the tracheal entrance),
23     and in the lower respiratory tract (region distal to tracheal entrance). However, obtaining
24     experimental  absorption data for smaller regions or locations, such as specific airways  or in
25     the centriacinar region where  lesions due to NO2 occur (see Section 13.2.3.4), is extremely
26     difficult, and  may not be possible in the near future due to technical limitations.
27     Nevertheless, experimentation is important for determining dose, assessing hypotheses  and
28     concepts, and validating mathematical models that may be of use in predicting  dose to
29     specific sites.
30          Theoretical studies are based  on the use of mathematical models developed for the
31      purposes of simulating the uptake and distribution of gases that are absorbed in the tissues

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 1      and fluids of the respiratory tract, usually at the generational level.  Because the factors
 2      affecting  the transport and absorption of gases are general to all mammals, a model that uses
 3      appropriate species and/or disease-specific anatomical and ventilatory parameters can be used
 4      to describe  absorption by different-sized, aged, or diseased members of the species.  Models
 5      may also be used to identify areas needing additional research,  to make inter- and intraspecies
 6      dose comparisons, to compare and reconcile data from different experiments, to predict dose
 7      in conditions not possible or feasible experimentally, to better understand the processes
 8      involved  in toxic effects, and to design experiments.
 9           The amount of a gas acting at a given site in the lung is related to the airway luminal or
10      airspace concentration at that site.  As a result, NO2 does not immediately interact with
11      cellular components of the respiratory tract.  Instead,  NO2 first conies into contact with the
12      liquid or  fluid that lines the cells or tissues of the respiratory tract (mucus plus periciliary
13      fluid in the upper respiratory tract and tracheobronGhial regions; surfactant film plus serous
14      fluid layer in the pulmonary region). Nitrogen dioxide reacts chemically with the constituents
15      of fluids  and tissues.  The reactions with the chemical components of the liquid lining is
16      likely to reduce the  quantity of the inhaled gas reaching the tissue.  However,  reactions in the
17      lining may  also produce products that in turn may increase toxicity above that produced by
18      the  direct action of NO2 alone.
19                               .
20      13.2.1.2  Principles of Gas Uptake and Dosimetry Models
21           To  further our understanding of the absorption of gases, mathematical models have been
22      developed to simulate the processes involved and to predict absorption by various regions and
23      sites within the respiratory tract.
24           Species-specific information :that characterizes respiratory  tract morphology and anatomy
25      and transport processes, including chemical reactions, is needed for mathematical modeling.
26      Anatomical information needed includes data about the physical .dimensions and geometry of
27      the structural elements of the .respiratory- tract (e.g., airways, alveoli), the liquid linings, the
28      underlying  tissues, and the capilliary blood system. In the air phase or air compartment
29      (airway lumens and airspaces) the processes of convection, molecular diffusion, turbulence,
30      dispersion, and the  loss or gain of gaseous species to  and from the respiratory tract walls
31      must be taken into account.  Factors to be considered in lung fluids and tissues include the
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  1     biochemical constituents, chemical reactions, solubility, molecular diffusion, convection due
  2     to mucociUary action, and capillary blood flow.  A detailed discussion of these factors can be
  3     found in Overton (1984) and Ultman (1988).
  4          Although physiological processes and structural variations occur between species,
  5     laboratory animals and humans are very similar.  So much so that dosimetry models can be
  6     and are developed based on the valid concept of a general mammalian respiratory tract
  7     structure and physiological processes that apply to both humans and laboratory animals.  As a
  8     result,  only one dosimetry model is needed for the simulation of uptake in several species or
  9     subjects. However, species- or subject-dependent structural and physiological data in a form
 10     useable by such a model is required  to simulate the absorption of NO2 by a specific animal or
 11     human.
 12          There is only one reported theoretical investigation of NO2 dosimetry; it was discussed
 13     originally in Miller et al.  (1982) and later in Overton (1984).  The dosimetry model used for
 14     this investigation was one developed for modeling of ozone (O3) uptake; however, since
 15     NO2, like O3, is highly reactive in the respiratory tract tissues and fluids and is not very
 16     soluble, this model is considered to be valid for NO2 (Miller et al., 1982).  Because there is
 17     very little NO2 dosimetry modeling results and the principles of O3 uptake aretgeneral and
 18     apply to NO2, the following is a discussion of the general aspects and the factors that have
 19     been considered for dosimetry models of a gas similar to NO2, namely O3.
20          For dosimetry models, lung dimensions and form usually are accounted for  by using
21     airway or anatomical models based on upper respiratory tract, tracheobronchial, and
22     pulmonary region data. The upper respiratory tract can be modeled as a series of segments
23     along the path through the upper respiratory tract airways; segment dimensions (length,
24     diameter, surface areas, etc.) are often based on cast data (e.g., Schreider and Raabe [1981]
25     for the rat and Patra et al. [1986] for a human child).   In the lower respiratory tract, the
26     complex and numerous branching airways are represented by a sequence of sets of right
27     circular cylinders (e.g., Weibel [1963] for humans and Yeh et al.  [1979] for rats).  Each set
28     corresponds to a generation and each cylinder represents an airway or alveolar duct.  This
29     type of lower respiratory tract model simplifies the development of dosimetry models in that
30     all paths from the trachea to the last generation airway or duct are identical.  Thus,  a
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 1     description of transport and absorption in one airway in a given generation is the same as any
 2     other airway in that generation (Overton, 1984).
 3          Although the transport of gases in the airways is a three-dimensional problem, the use
 4     of the stylized anatomical models have lead to the development of methods that allow the
 5     description of transport in terms of a one-dimensional, time-dependent partial differential
 6     equation (Overton, 1984).  The change with respect to time of the average cross-sectional
 7     concentration of the inhaled gas in an airway is related to axial dispersion, convection, and
 8     wall loses due to physical absorption and chemical reactions.  Transfer to the liquid lining is
 9     often described in terms of mass transfer coefficients and the liquid-phase and gas-phase
10     interfacial concentrations, related by Henry's law constant.  In respiratory tract fluids and
11     tissues, only chemical reactions and transport perpendicular to the interfaces between the air,
12     liquid, and tissue compartments have been taken into account.  Chemical reactions in the
13     tissue and fluids have been modeled in several ways, depending on assumptions  about the
14     type and nature of the reactions that effectively describe the reaction process.  Reactions have
15     been modeled' as instantaneous or first-order and time-dependent or steady state, depending on
16     the investigators and the state of understanding.  Dosimetry models also account for
17     ventilation which plays an important role in delivered dose; some models include the effects
18     of expansion and contraction to account for the fact that dimensions vary during the breathing
19     cycle (Overton etal., 1987).
20           For the NO2 dosimetry investigation of Miller et al. (1982), reactions were modeled as
21     instantaneous and only in the liquid lining.  (The NO2 concentration at the air-liquid lining
22     interface was set to zero, and the tissue dose was defined in terms of the flux of NO2 to the
23     tissue at the interface.) Mucociliary processes have been considered too slow to be effective
24     and are usually ignored.  Because NO2 is highly reactive, only a small fraction of the NO2
25     would be assumed to enter the capilliary blood, where it would not survive to be recirculated
26     to the lung.  Thus, the blood compartment would be a sink for NO2.  However, if the
27     products of chemical reactions and their distribution in the rest of the body are important, as
28     is possible with NO2,  then relevant systemic processes would have to be taken into account.
29           The word  "uptake" is often used in conjunction with gas dosimetry.  Generally, the
30     meaning of this word depends on context and should be defined to reduce ambiguity, if the
31     meaning is not obvious.  Uptake can denote a measure of the quantity of gas absorbed (e.g.,
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  1      100 g) in a region or at a specific location.  Unless otherwise stated, the terms "fractional
  2      uptake" and "percent uptake" refer, respectively, to the fraction or the percent of the inhaled
  3      NO2 retained by the specified respiratory tract region.
  4
  5      13.2.1.3 Dosimetry of Nitrogen Dioxide
  6          Morphological studies on animals suggest that NO2 is absorbed along the entire
  7      respiratory tract.
  8
  9      Upper Respiratory Tract Absorption
 10          The upper respiratory tract uptake of NO2 has been experimentally estimated in dogs,
 11     rats, and rabbits.  Using unidirectional flow, Yokoyama (1968) measured NO2 uptake in the
 12     isolated upper airways of dogs and rabbits exposed to .7,520 to 77,080- jug/m3 (4 to 41 ppm).
 13     The upper respiratory tract of the two species was observed to remove 42.1 % (standard
 14     deviation =  14.9%) of the NO2 drawn through the noses.  The authors did not discuss the
 15     relative humidity of the air.  If it were.not sufficiently high, the continuous airflow would
 16     dehydrate the mucous membrane, possibly affecting the uptake properties of the upper
 17     respiratory tract.  Cavanagh and Morris (1987) exposed the isolated upper respiratory tract of
 18     naive and previously exposed rats (76,000 jug/m3; 40.4 ppm NO2) under unidirectional flow
 19     and found the uptakes to be 28 and 25%, respectively.  The relative humidity of the
 20     "inhaled" air was maintained so as to be equivalent to 92% at 37°  C.  The reported uptake
 21      difference between the naive  and previously exposed rats may not be significant since a
 22     MANOVA, and not an ANOVA, test should have been used to analyze the data.  Kleinman
 23      and Mautz (1987)  exposed dogs to 1,880  or 9?400 jig/m3 (1 or 5 ppm) NO2 in air at 85%
 24      relative humidity.  Uptake was measured  only during inhalation. They found that more NO2
 25      was absorbed in the upper respiratory tract of dogs with nasal breathing than with oral
 26      breathing. They also found that the percent uptake of NO2 by the  upper respiratory tract of
 27      dogs decreased with increasing ventilation rates (during exercise for both nasal and oral
28      breathing).  Specifically, nasal uptakes ranged from approximately  85% to less than 80%  and
29      oral breathing ranged from about 60% to  approximately 45%  as the ventilation increased
30      from resting values to four times this value.  The negative correlation between percent uptake
31      and ventilation is what would be expected based on the work of Aharonson et al. (1974).

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 1      Lower Respiratory Tract Absorption
 2           Corn et al. (1976) estimated the average mass transfer coefficient (K) of NO2 in the
 3      lower respiratory tract of cats. The values of K measured were associated with the region
 4      between the trachea and a portion of the lung associated with the "right lateral thoracic region
 5      between the fourth and fifth rib". No dependence of K on NO2 concentration changes or
 6      ventilatory rates was observed.  According to Postlethwait and Bidani (1990), the estimated
 7      value of K is unreasonably  large. Also, a comparison of the Corn et al.  (1976) value of K to
 8      theoretical values inferred from Overton et al. (1987) for O3 indicate that the experimentally
 9      determined K for NO2 is over 240 times as large as those used for modeling ozone uptake.
10      Further work is needed to determine mass transfer coefficients of NO2.
11            Postlethwait and Mustafa (1981) measured the uptake of NO2 by an isolated ventilated
12     perfused rat lung.  The isolated lungs  were exposed for 90 min to'0,400 jig/m3  (5 ppm)
13     NOo at a ventilation rate of 45 mL/min.  Thirty-six percent of the ventilated NO2 was
           2j
14     retained.  In a  later similar experiment with the exposure concentration ranging from 7,520 to
15     37,600 fig/m3  (4 to 20 ppm) and the minute volume from 45 to  130 mL/min for different
16     groups of lungs, Postlethwait and Mustafa (1989) found that the quantity of NO2 retained was
17     related linearly to the inhaled quantity of NO2;  the percent uptake ranged from 60 to 72%
18     with an average of 65%.  These results differ considerably from the first experiment and no
 19     reasons for the differences in results were given. 'Although the tidal volumes in these
20     experiments are realistic, the breathing frequencies are generally much lower than for rats
21     breathing normally. Similar experiments should be performed using more realistic ventilatory
22     parameters.
 23           In addition to measuring upper respiratory tract uptakes in dogs, Kleinman and Mautz
 24      (1987) also measured  lower  respiratory tract uptake.  They found that at ventilatory
 25      parameters corresponding  to rest about 85% of the inhaled NO2 entering the lungs was
 26     absorbed by this region; this increased to 100% with high ventilation rates.
 27          Results of simulating the uptake of'NO2 in the lower respiratory tract were described by
 28     Miller et al. (1982) and Overton (1984).  The model used for this investigation was the same
 29     as the dosimetry model described in Miller et al. (1978) for O3, but with the diffusion
 30     coefficient and Henry's law constant appropriate to NO2;  however, values of the latter
 31     constant and me chemistry of reaction were considered uncertain.  The  investigation was
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  mainly a sensitivity study of the effects of Henry's law constant and reaction rates for which
  the upper limit of the latter was assumed to be the reaction rate of O3. For humans, the
  results indicate that NO2 is absorbed throughout the lower respiratory tract, but the major
  dose to tissue would be delivered in the centriacinar region, that is, the junction between the
  conducting and respiratory airways.  Simulations also predicted that peak tissue levels would
  occur in this same anatomical region of the rat, guinea pig, and rabbit. These findings are
  consistent with  the site of morphological effects (Section  13.2.3.4). Beyond this region there
 is a rapid fall in the NO2 dose delivered to tissue. Depending on the tracheal concentration
 and tidal volume, the model predicted that 75 to 95% of  the NO2 entering the trachea could
 be retained in the lower respiratory tract tissues and fluids.  However, these predictions are
 dependent on the investigator's choices of values for the uncertain parameters.  The results
 also predict that exercise will increase the amount of NO2 delivered to and absorbed in the
 pulmonary region over that at rest,  and wiU reduce percent  uptake in the tracheobronchial
 region (Miller etal., 1982; Overton,  1984).                 .

 Total Respiratory Tract Absorption
     Total respiratory tract uptake has been measured in healthy and diseased humans.
 Healthy humans were exposed by Wagner (1970) to a  NO/NO2 mixture containing 550 to
 13,500 jig/m3 (0.29 to  7.2 ppm) NO2 for brief (but unspecified) periods.  Of the inhaled
 NO2, 81 to 90% was absorbed during normal respiration; this increased to 91 to 92% with
 maximal ventilation.  For 30 min Bauer et al. (1984) exposed adult asthmatics to 560 ^g/m3
 (0.3 ppm) NO2.  The exposed subjects inhaled NO2 by mouthpiece for 20 min at rest, then
 exercised for 10 min on a bicycle ergometer.  The inspired and expired NO2 concentrations
 were measured showing that at rest the average uptake was 73%; whereas, during exercise
 the average uptake was  88%, a statistically significant increase.  The effects of NO2 exposure
 on pulmonary function in humans following this exposure regime are reported in Chapter  15.

Effect of 'Exercise on Respiratory Tract Dosimetry
     Experiments indicate that increased ventilation decreases percent uptake in the upper
respiratory tract, and increases the percent uptake in the lower and  total respiratory tract.  In
all cases, the effect of increased ventilation is an increase in  the quantity of NO2 absorbed by
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 1      the individual regions.  The reduction of percent uptake in the upper respiratory tract due to
 2      increased ventilation results in a greater proportion of inhaled NO2 being delivered to the
 3      lower respiratory tract. Also, the switch from oronasal to oral breathing at high exercise
 4      levels, is expected to increase the delivery of NO2  to the lower respiratory tract because the
 5      percent of NO2 removed by the mouth is less than that removed by the nasal cavity.
 6           Aharonson et al (1974) predicted a negative correlation between percent upper
 7      respiratory tract uptake and ventilation using a model. The model analyzed data from
 8      experiments on the uptake of vapors by the nose.  The model was based on the assumption of
 9      quasi-steady-state flow, mass balance, that the flux of a trace gas at the air-mucus interface is
10      proportional to the gas-phase concentration of the  trace gas and a local mass trace gas, and a
11      local mass transfer coefficient. Miller et al. (1985) and Overton et al. (1987) illustrated the
12      effects of ventilation on the lower respiratory tract uptake of O3, a reactive gas whose uptake
13      processes are  similar to those of NO2. The theoretical work of Miller et al. (1985) on uptake
14      in humans shows that exercise has a minimal effect in the tracheobronchial region on tissue
15      dose (i.e., quantity of O3 absorbed by unit area of tissue per unit time) and a pronounced
16      increase in the dose to the pulmonary region tissues.  As the exercise level was increased, the
17      maximum .tissue dose (at the first respiratory bronchiole for the resting state) shifted distally
18      several generations and increased by a factor of 19 (over that of the resting state and at the
19      generation of the shifted maximum) for the highest simulated exercise level.  Furthermore,
20      this dose increase was more than twice the ratio of the exercise to rest minute volumes.  On
21      the other hand, the maximal increase in mass absorbed by the tracheobronchial region which
22      was by a factor of  1.4 as compared to the  increase of absorbed O3  in the pulmonary region
23      which was by a factor greater than 13 (1.5 times the  ratio of exercise to rest minute
24   ,   volumes). The simulation results for rats (Overton et al., 1987) are in agreement with these
25      predictions.  Since the upper respiratory tract delivers a greater proportion of inhaled NO2 to
26      the lower respiratory tract during exercise  than at  rest^ the factors quoted above are expected
27      to be conservative.  Thus, the modeling results predict that exercise (with respect to the
28      resting state) delivers a disproportionately  greater  quantity of the inhaled mass to the
29      pulmonary region and an even greater disproportionate quantity to the more distal pulmonary
30      region surfaces.  Qualitatively, similar conclusions for NO2 are reasonable.
31
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  1     Systemic Dosimetry
  2          Once deposited, NO2 dissolves in lung fluids, and various chemical reactions occur
  3     giving rise to products that are found in the blood and other body fluids. Svorcova and Kaut
  4     (1971) suggested that inhaled NO2 entered the bloodstream based upon estimates of
  5     radiolabelled nitrate (NO3~) and nitrite (NO2~) levels in the blood and urine of rabbits
  6     following exposure to 45,120 ^cg/m3 (24 ppm) NO2 for 4 h.  The initial transformation
  7     products following NO2 absorption have been the subject of some speculation, however.
  8          The distribution of 13NO2 (560 to  1,710 /*g/m3  [0.3 to 0.91 ppm] inhaled for 7 to
  9     9 min) in rhesus monkeys was investigated by Goldstein  et al. (1977b).  They concluded that
 10     inhaled NO2 was distributed throughout the lungs, and that it probably  reacts  with the water
 11     molecules in the fluids of the respiratory tract to form nitrous acid (HNO2)  and  nitric acid
 12     (HNO3); the authors suggested that the acids were responsible for subsequent  toxic effects.
 13     Based upon the absorption of NO2 by isolated ventilated  perfused rat lungs, Postlethwait and
 14     Mustafa (1981) proposed that the main reaction of NO2 was not with lung water but with
 15     readily oxidizable tissue components  (e.g., proteins and lipids) to produce NO2". These
 16     investigators found that 70% of absorbed NO2 appeared as NO2" in perfusate and lung tissue,
 17     and that the concentration of NO2" produced increased with time during exposure. They also
 18     hypothesized that NO2" in the blood may then be oxidized to NO3~ by interaction with
 19     hemoglobin in red blood cells.  Saul  and Archer (1983) provided support for this pathway
20     using an in  vivo system in which rats were exposed for 24 h to NO2 at 2,260  to
21      16,540 /tg/rn3 (1.2 to 8.8 ppm). They also concluded that the main reaction pathway of
22     absorption in the lungs was the reaction of NO2 with oxidizable tissue components to produce'
23      NO2". This product may then serve as a precursor for other chemical reactions at
24     extrarespiratory sites.
25          The current data base indicates that once NO2 is absorbed in lung  fluids, the subsequent
26      reaction products are rapidly taken up and then translocated via the bloodstream.  For
27      example, intratracheally instilled NO2" has been shown to be rapidly absorbed  into blood
28      from the lungs (Parks et al., 1981).   Oda et al. (1979, 1980b), after exposing  rats to 20,870
29      to 112,610 jtg/m3  (11.1 to 59.9 ppm) 15NO2 for 0.5 to 53.9 h, found increased levels of
30      labelled nitrogen in the lungs, kidneys, plasma, and urine, as well as an increased level of
31      NO2" in plasma.  In a later study, Oda et al. (1981) noted a dose-dependent increase in both

        August 1991                             13_10     DRAFT-DO NOT QUOTE OR CITE

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 1     NO2" and NO3" levels in the blood of mice during 1-h exposures to 9,400 to 75,200 jug/m3
 2     (5 to 40 ppm) NO2.  The blood levels of NO2" and NO3" declined rapidly after exposures
 3     ended, with the decay half-times of a few minutes for NO2~-and about 1 h for NO3".  The
 4     shorter time for the former was ascribed to its rapid reaction (oxidation) with hemoglobin,
 5     producing NO3" and methemoglobin (Oda et al.,  1981; Kosaka et al.,  1979;  Case et al.,
 6     1979), although such measurements were not made.  Free NO3" in blood is generally
 7     excreted in urine (Green and Hiatt, 1954; Hawksworth and Hill, 1971).
 8
 9     Summary
10          The important physical, chemical, and biological factors involved in the uptake of NO2
11     by the respiratory tract were reviewed. These factors must be taken into account in order to
12     interpret and understand experimental dosimetry results and to develop models that simulate
13     NO2 uptake for extrapolation purposes.  With respect to dosimetry, the following has been
14     observed.  Total respiratory tract uptake in humans ranged from 73 to 92% depending on the
15     investigation and the breathing state.  The percent total uptake was found to  increase with
16   •  increasing exercise level.  Upper respiratory tract uptakes have been measured in dogs,
17     rabbits, and rats.  Uptake values ranged from as low as 25% to as high as 85%  depending on
18     the study, species, air flow rate, and mode of breathing (nasal or oral).  Percent upper
19     respiratory tract uptakes were found to decrease-with increasing ventilation; uptakes via nasal
20     breathing were determined to be significantly greater than oral breathing uptakes.  For the
21     lower respiratory tract,  uptake values of 36 and 65% have been reported for isolated,
22     ventilated, perfused rat  lungs. Experimental evidence indicated that NO2 chemically reacted
23     with lung tissue, but did not penetrate directly to blood.  However, the reaction products,
24     NO2" and NO3", were found throughout the body of experimental animals.
25          -There has been very little modeling of the uptake of NO2.  The results of  the only
26     simulation study predicted that the maximum NO2 tissue dose in humans, rats, guinea pigs,
27     and rabbits occurred in  the vicinity of the centriacinar region where morphological lesions are
28     commonly observed.  Modeling has been used to estimate the effect of increasing ventilation
29     on the distribution of absorbed O3, which is similar to NO2, in humans.  These simulations,
30     the qualitative results of which were expected to apply to NO2, predicted that increasing
        August 1991
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DRAFT-DO NOT QUOTE OR CITE

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   1
   2
   3
   4
   5
   6
   7
   8
   9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
 20
 21
 22
 23
 24
 25
 26
 27
 28
29
 ventilation had little effect on uptake in the tracheobronchial region but greatly enhanced
 pulmonary region uptake.

 13.2.2  Mortality
      Studies with various species of experimental animals (Table 13-1) have shown that
 single exposures to NO2 for <24 h are lethal only at exposure concentrations far exceeding
 those routinely found in ambient air (i.e., >37.6 mg/m3 [20 ppm]).  The acute lethal effect
 of NO2 is due primarily to pulmonary edema; however, laryngeal spasm has been reported in
 guinea pigs. Delayed deaths are often the result of bronchiolitis and pneumonia.  Continuous
 or repeated exposures to NO2 result in little or no mortality in .most species, with the possible
 exception of the guinea pig, until concentrations exceed ~ 18.8 mg/m3 (10 ppm)
 (Table  13-2).  Furthermore, the data base suggests that brief exposures to high concentrations
 are more lethal than equivalent exposures to lower levels for longer periods of time.
      There is evidence for differences in sensitivity between animal species.  This is seen in
 the series of acute exposures reported by Steadman et al. (1966), and  summarized in
 Table 13-1.  Species sensitivity was also mathematically assessed by Book (1982), who used
 the data of Hine et al.  (1970) and Steadman et al. (1966) to calculate  estimated concentrations
 (LC50)  of NO2 which would result in  50% lethality after a 1-h exposure; the values obtained
 were 186.12 mg/m3  (99 ppm) for mice, 206.8 mg/m3  (110 ppm) for rats, 171.08 mg/m3
 (91 ppm)  for guinea pigs,  263.2 mg/m3 (140 ppm) for rabbits, and 244.4 mg/m3 (130 ppm)
 for dogs.
     There are, however,  reported variations in interspecies and strain sensitivity to NO2
 exposure (e.g., different LC50 values for similar exposures).  These may be due to a number
 of factors, which include differences in age, sex, or strain of animals,  genetic differences
 within different strains-of animals, differences in analytical procedures for measuring NO2
 and even failure to distinguish between NO2 and mixed oxides of nitrogen in some studies,
 and differences in environmental conditions (e.g., temperature, humidity, and diet).  The
influence of diet (e.g., Vitamin E) on NO2 toxicity is discussed more fully in the section on
lung biochemistry (Section 13.2.3.2).
       August 1991
                                        13-12      DRAFT-DO NOT QUOTE OR CITE

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August 1991
13-16
DRAFT-DO NOT QUOTE OR CITE

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  1      13.2,3  Respiratory Effects
  2      13.2.3.1  Host Defense Mechanisms
  3           The respiratory tract defenses encompass many interrelated responses; however, for
  4      simplicity, they can be divided into two major parts: the physical defense mechanisms and
  5      the cellular defense mechanisms.  The first part, the physical defense mechanisms, includes
  6      structural and physicochemical adaptations in the proximal upper and lower airways and their
  7      lining layers.  The pulmonary defense mechanisms (mucociliary system) in this part begins
  8      with aerodynamics (Newhouse et al.,  1976).  There is a large amount of turbulence
  9      experienced by the incoming airstream, causing the nasopharyngeal removal of many of the
10      large particles (>10 ^m).  Smaller particles can also be deposited on the mucociliary escalator
11      of the tracheobronchial region. Particles deposited on the mucus layer are removed by ciliary
12      action or coughing or both (Breeze and Wheeldon, 1977; Green, 1970;  Newhouse et al.,
13      1976; Proctor, 1977).  The majority of airways have an epithelial lining of ciliated columnar
14      cells and mucus-secreting goblet cells and mucous glands.  The number of mucus-secreting
15      cells increases in disease states or after inhalation of irritants, while the number of ciliated
16      cells decreases.  Ciliary movement directs overlying mucus, particles, and absorbed gases
17      toward the pharynx where they are swallowed or expectorated.  The trapping of potential
18      pathogens on the mucous layer followed by the ciliary escalator action and expulsion from the
19      airways is the lung's first line of  defense.
20           The second part of the pulmonary defense system is the cellular defense mechanisms
21      (phagocytic and immunologic reactions) which operate mostly in the pulmonary region of the
22      lung.  Large mononuclear cells, alveolar macrophages (AMs), are the first line of cellular
23      defense (Hocking and Golde, 1979).   The role of AMs in host defense is diverse and varied,
24      including such important activities as detoxifying and/or removing inhaled particles;
25      maintaining sterility against inhaled microorganisms; interacting with lymphoid cells in a
26      variety immunologic reactions; and removing damaged  or dying cells from the alveoli.
27      through phagocytosis (Pels and Cohn, 1986). Alveolar macrophages migrate throughout the
28      pulmonary region and it is  this mechanism that.allows contact with foreign material  entering
29      the lungs.  Once phagocytosis has occurred, the particle is encased in a  phagocytic vesicle
30      and fuses with lysosomes to form; phagolysosomes, the prime subcellular compartment for
       August 1991
13-17
DRAFT-DO NOT QUOTE OR CITE

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  1     digestion of particles and the killing of engulfed bacteria (Nathan et al.,  1980; Silverstein
  2     etal., 1977).
  3          During the phagocytosis process, AMs release oxygen radicals and enzymes either into
  4     the phagosome or into the external milieu (Johnston et al., 1978).   Oxygen radicals and
  5     enzymes are believed to be essential for bacterial killing by phagocytes.  Both oxidation and
  6     decarboxylation of bacterial membranes appear to be the major mechanisms by which oxygen
  7     radicals induce bacterial killing (Klebanoff, 1968; Strauss etal., 1970).
  8          Polymorphonuclear leukocytes  (PMNs), another phagocytic cell, are present in a small
  9     percentage in normal lungs.  In response to a variety of insults, there can be an influx of
 10     PMNs from blood into the lung by chemotaxis.  Once recruited to the lung, PMNs then
 11     ingest and Mil opsonized microbes and other foreign substances by  mechanisms that are
 12     similar to those described for AMs (Sibille and Reynolds, 1990).  In contrast to AMs, PMNs
 13     contain substantial amounts of myeloperoxidase (MPO) stored in the primary granules and,
 14     therefore, are an important source of OH" radicals, considered to be major bactericidal agents
 15     (Klebanoff,  1982).
 16          In most cases, optimal phagocytosis of microorganisms by PMNs and AMs requires the
 17     presence of opsonins.  Opsonins are  immunoglobulins which have the capacity to enhance
 18     phagocytosis of microorganisms.  When the phagocytic response is  not sufficient to
 19     effectively remove particles from the respiratory tract, immunologic (humoral and cell-
20     mediated immunity) responses are provided by the lymphocytes. The humoral part of this
21     system primarily involves the B cells that function in the synthesis and secretion of antibodies
22     (IgG, IgA, IgM) into the blood and body fluids. Secretory immunoglobulin A (IgA) is found
23     in the upper respiratory tract and tracheobronchial region and its primary role is to inhibit
24     microbial attachment to the conducting airway surfaces.  Immunoglobulin G (IgG)  is the
25     predominant class of immunoglobulins of the lower respiratory tract.  These immunoglobulins
26     act to enhance macrophage functioning. Immunoglobulin M (IgM)  is also present in the
27     lower respiratory tract but in low concentrations.  The interactions of these defense
28     mechanisms  are complicated and not completely understood.
29          The cell-mediated component primarily involves T lymphocytes which, are the effectors
30     of cellular defense.  These cells are responsible for delayed hypersensitivity and defense
31      against viral, fungal, bacterial, and neoplastic disease. There is little replacement of T cells
       August 1991
13-18
DRAFT-DO NOT QUOTE OR CITE

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 1      in the adult human.  Alveolar macrophages may be responsible for the initiation of humoral
 2      and cellular immune responses by the ingestion of particles or soluble antigens and the
 3      "processing" and "presenting" of these antigens for specific antigen-reactive B and T cells.
 4      This process stimulates the B and T cells to proliferate and differentiate into effectors of
 5      humoral (antibody production) and cell-mediated  (cytotoxic lymphocytes) immunity.  It is
 6      these responses that are important in acute and chronic infections and form the basis for
 7      antimicrobial immunity (Harada and Repine,  1985).
 8           Alveolar macrophages also secrete alpha, beta, and gamma interferon and platelet
 9      activating factor (PAF).  The interferons have potentially important implications in
10      modulating the antiviral and immune activities of AMs.  Recently,  AMs were found to
11      release a factor first thought to be related to the interferon family and therefore initially
12      named interferon beta 2.  Interferons are protein substances produced by virus-invaded cells
13      that prevent the replication of the virus (Lefkowitz et al.,' 1984; Lefkowitz et al.,  1983).  It
14      was later determined to be a cytoMne (IL6) (Wong and Clark,  1988).  Cytokines are believed
15      to play an immunoregulatory role in the respiratory tract defenses,  affecting inflammatory
16      responses and tumbricidal activities (Dinafello et al.,  1986).  Platelet activating factors may
17      play an important role in the inflammatory response and are capable; of PMN aggregation and
18      of the release of arachidonic acid metabolites from PMNs (Braquet and Rola-Pleszczynski,
19      1987).
20           Although antiviral pulmonary immune functions have not been adequately evaluated
21      after NO2 exposure,  their proper functioning is essential in humoral and cell-mediated
22      immunity against viral disease.  These functions include:   (a) interferon production in
23      bronchoalveolar lavage fluid; (b) AM function; (c) natural killer activity; (d) cytotoxic
24      T lymphocyte activity; and (e) antibody activity (Burleson, 1987).  A host infected with virus
25      manifests a cascade of immune responses, both specific and nonspecific.   Local and
26      circulating interferons (alpha,  beta, and gamma) are produced in the first 24 h after viral
27      exposure. Interferons have antiviral, antitumor, and immunoregulatory activities.  Interferons
28      serve as an immunoregulator to stimulate the immunological activity of both AMs and
29      lymphocytes exhibiting natural killer activity.  Interferon, AMs, and natural killer cells all
30      contribute to the nonspecific antiviral immunity of the host, The first specific immunological
31      activity important for defense against viral disease is cytotoxic T lymphocytes. These cells
        August 1991
13-19
DRAFT-DO NOT QUOTE OR CITE

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  1      can be activated by interferons and therefore, are most relevant in defense against viral
  2      infections and tumors.
  3
  4      Mucodliary Clearance
  5           The effectiveness of the mucociliary system is dependent upon the integrity of the cilia
  6      and respiratory epithelia, the chemicophysical properties of the mucus, and the rate of mucus
  7      transport.  Viral and bacterial infections and various chemicals can lead to over or under
  8      secretion of mucus and to loss or paralysis of the cilia. The cilia can respond to insults in a
  9      number of ways: changes in beat frequency, cessation of ciliary beating, and/or development
10      of abnormal forms of cilia.  Substances that produce such disruption or impairment of this
11      defense system are known to result in an unwanted accumulation of cellular secretions,
12      increased acute bacterial and viral infections, chronic bronchitis, and prolonged pulmonary
13      complications possibly associated with the pathogenesis of chronic obstructive lung disease or
14      bronchial cancer through the unwanted accumulation of inhaled carcinogens (Schlesinger
15      et al., 1987).  Continual loss of these ciliated cells will eventually result in replacement of the
16      normal ciliated epithelium with squamous metaplasia of other nonciliated cell types.
17           Most evident are the reports of significant loss of cilia in the bronchiolar epithelium.
18      These effects, as reported in earlier studies, were at NO2 concentrations greatly exceeding
19      those encountered in the environment.  When lower levels of NO2 (3,760 jttg/m3 [2 ppm])
20      were studied, the loss of cilia was not detected until after 6 weeks of exposure when small
21      areas devoid of cilia became evident (Azoulay et al., 1978).  The loss of cilia may be
22      accompanied by a prolonged  aberration of ciliogenesis. A description of histopathological
23      changes of the epithelial cells of the conducting airways can be found in Section 13.2.3.4,
24      addressing the morphological effects of NO2 exposure. This section and Table 13-3  below
25      review those studies that have shown that the mucociliary clearance defenses are vulnerable to
26      the effects of NO2.
27           Rombout et al. (1986),  in a series of experiments covering both continuous (24 h/day)
28      and intermittent (6 h/day) exposure regimens, reported that a loss of cilia was evident in rats
29      after 2 days of exposure to 5,000 jtg/m3  (2.7 ppm) NO2.  No effects were observed  in rats
30      exposed to  1,000 and 2,500 jtg/m3 (0.5 and 1.3 ppm) NO2.  Reduction in number, evidence
31      of swelling, hypertrophy,  and focal hyperplasia of ciliated epithelial cells in rats exposed to

        August 1991                             13-20      DRAFT-DO NOT  QUOTE OR CITE

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                         13-22
DRAFT-DO NOT QUOTE OR CITE

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  1
 2
 3
 4
 5
 6
 7
 8
 9
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3,760 jwg/m3 (2.0 ppm) NO2 for 72 h was reported by Stephens et al. (1972).  The effects
became more pronounced as the length of exposure increased up to 14 days, at which time
the recovery process began.  In another study, there was a reduction in the number of cilia
and the cilia appeared swollen in rats continuously exposed to 940 /ig/m3 (0.5 ppm) NO2 for
7 months (Yamamoto  and Takahashi, 1984). When the exposure concentration was increased
to 1,800 and 7,520 jug/m3 (1.0 and 4.0 ppm), the effects became more pronounced and
additional morphological changes were evident, indicating a concentration-response
relationship.
     Clearance of marker substances deposited in the airways has been used to assess the
effects of NO2 on mucociliary clearance.  This method has merit as an index of overall
efficiency of mucociliary clearance.  The mucocilary clearance of inhaled tracer particles
deposited into the tracheobronchial tree of rabbits exposed for 2 h/day for 14 days to 0, 560,
and 1,880 jitg/m3 (0, 0.3 and 1.0 ppm) NO2 did not alter the normal  clearance rate
(Schlesingeretal., 1987).
     Giordano and Morrow (1972) demonstrated significant impairment of tracheobronchial
clearance rates in rats  following exposure to 11,280 jug/m3 (6.0 ppm) NO2 for 6 weeks.  This
decrease in ciliary clearance was not accompanied by any observable abnormality of the
airways. A 2-h exposure to 14,100 jig/m3 (7.5 ppm) failed to alter the trachea! mucus
velocity in sheep, but  exposure to twice that concentration for 2 h produced a significant
slowing of mucus movement (Abraham et al., 1980).
     Thus, exposures  to NO2 found to compromise this protective function are not only
dependent upon the concentration of the NO2, but the duration of, the exposure.  It appears
that it would take prolonged exposure to higher concentrations to induce alterations that
would have detrimental health effects. Data would indicate that eyen a severely damaged
airway epithelium has  the ability to maintain mucus transport (Abraham, 1984).
     A few in vitro experiments have examined the effect of NO2 on isolated ciliated
epithelium cells and tracheal rings. Kita and Omichi (1974) reported that exposure to NO2 at
concentrations greater  than 9,400 /ig/m3 (5.0 ppm) resulted in a decreased rate of ciliary
beating.  Schiff (1977) isolated and exposed hamster tracheal  ring cultures to 3,760 jwg/m3
(2.0 ppm) NO2 for 1.5 h/day, 5 days/week for 1 to 4 weeks. After 14 days of exposure, the
NO2 produced decreased ciliary beating and various morphological changes.
       August 1991
                                        13-23
DRAFT-DO NOT QUOTE OR CITE

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  1     Alveolar Macrophages
  2          Besides altering the protective function of the epithelial cells of the conducting airways,
  3     NO2 may also produce structural, biochemical, and functional changes in AMs.  The
  4     effectiveness of AMs depends on the type, number, and viability of the cells.  The cells must
  5     maintain an intact membrane, mobility, phagocytic activity, and have functioning enzyme
  6     systems. Evidence from both in vivo and in vitro studies indicate that NO2 exposure alters
  7     many of these functions, thereby increasing the risk of the host to disease.
  8
  9          In Vivo Exposure. Alveolar macrophages isolated from mice continuously exposed to
 10     3,760 ftg/m3 (2.0 ppm) NO2 for 33 weeks or exposed to 940 /*g/m3 (0.5 ppm) NO2.
 11     continuously with a 1-h peak to 3,760 jig/m3 (2.0 ppm) for 5 days/week showed distinctive
 12     morphological changes as compared to controls after 21 weeks of exposure (Aranyi et al.,
 13     1976).  Examples of structural changes observed included the loss of surface processes,
 14     appearance of fenestrae, bleb formation, and denuded surface areas.   Continuous exposure to
 15     a lower level (i.e., 940 jwg/m3 [0.5 ppm] or 188 /*g/m3 [0.1 ppm] with a 1-h peak to
 16     1,880 ftg/m3 [1.0 ppm]) for periods of up to 24 weeks did  not result in any significant
 17     identifiable morphological or biochemical changes.  Such morphological changes would be
 18     expected to interfere with a number of functional  changes such as chemotaxis, phagocytosis,
 19     and bactericidal  ability.
20          Numerous  studies have shown that NO2 exposure at concentrations greater than
                  A
21     9,400 jtig/nr (5.0 ppm) increases, in a concentration-related manner, the number of AMs
22     (Foster et al., 1985; Kleinerman et al., 1982; DeNicola et al.,  1981; Sherwin et al., 1968;
23     Busey et al., 1974; Wright et al., 1982).  Alveolar macrophage accumulation has also been
24     reported after 15 weeks exposure at lower concentrations such as to either 9,400 jug/m3
25     (5.0 ppm) NO2 or to 1,880 /ig/m3 (1.0 ppm) with two 1.5-h spikes to 9,400 /xg/m3
26     (5.0 ppm)/day (Gregory et al., 1983). After a 1-day exposure to 5,000 /j.g/m3 (2.7 ppm)
27     NO2, Rombout et al. (1986) reported an increase  in the number of AMs in the terminal
28     bronchioles and proximal alveoli of rats.  The accumulation of AMs was accompanied by an
29     increase in mitosis. This accumulation of AMs reached a peak after 8 days of exposure.
30     Two days after the cessation of this exposure, the accumulation was  no longer evident.  No
31      effect was seen at 1,000 or 2,500 /zg/m3 (0.5 or 1.3 ppm).
                                                                    i
        August  1991                              13-24      DRAFT-DO NOT QUOTE OR CITE

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 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
     Mochitate et al. (1986) reported a significant increase in the total number of AMs
                                                       o
isolated from rats during 10 days of exposure to 7,520 |wg/nr (4 ppm) NO2. At this
exposure concentration, AMs accounted for 97% of the total lavaged cells.  The number of
PMNs did not show any significant increase over this exposure period.  The AMs from
exposed animals also exhibited increased metabolic activity as measured by the activity of
glucose-6-phosphate dehydrogenase, glutathione peroxidase, and pyruvate kinase, all of which
were significantly increased by 1.29-fold, 1.17-fold,  and 1.2-fold, respectively.
Simultaneously with these effects, the exposed AMs exhibited a significant increase in the
rate of synthesis of protein and DNA.  All responses peaked on Day 4 and all values had
returned to control levels by the tenth day.
     Suzuki et al. (1986) found that NO2 exposure significantly increased the number of
AMs that could be lavaged from rats exposed to either 7,520 jug/m3 (4 ppm) or
15,040 fig/m3 (8 ppm).  In this study the exposure continued for 10 days, but the effect
became most significant between the third and fifth days of exposure. The viability of these
isolated cells was decreased on Day 1 and remained depressed for the remainder of the
exposure period.  There was no evidence that either exposure caused an increase of PMNs.
Schlesinger et al. (1987) failed to find any significant changes in the number or the viability
of AMs in lung lavage from rabbits exposed to 1,880 jwg/m3 (1 ppm) NO2.
     The morphometric methods used to examine for altered lung structure failed to find any
changes in the number of AMs per meter squared or the average cell volume (^m3) in rats
following exposure for 23 h/day to 3,700>tg/m3 (2.0 ppm) NO2 for 6 weeks with two daily
spikes of 0.5 h each to  11,280 /-cg/m3 (6.0 ppm) NO2 (Crapo et al., 1984).  Chang et al.
(1986) studied the response of 1-day-old and  6-week-old rats following exposure to 0.5 ppm
(940 |Ug/m3) NO2, 23 h/day for 6 weeks.  Another group of adult rats was exposed to
                    "3                                '   '
2.0 ppm (3,760 ^g/nr) for the same duration.  Two daily 1-h spikes that were three times
the baseline level (i.e.,  2,820 jttg/m3 [1.5 ppm] and 11,280 ^g/m3 [6.0 ppm]) were applied
Monday-Friday. In these studies, the adult rats were more sensitive to NO2 than young
animals, showing a significant increase in both  number and volume of AMs. The studies
conducted by Azoulay-Dupuis et al. (1983) also demonstrated that in both the rat and the
guinea pig, newborns were less affected by a 3-day exposure to 3,760 and 18,800
        August 1991
                                         13-25
DRAFT-DO NOT QUOTE OR CITE

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  1     (2.0 ppm and 10.0 ppm) NO2 than adults based on changes in lung structure and superoxide
  2     dismutase activity in isolated AMs.
  3          There have been numerous reports demonstrating a decrease in phagocytic activity of
  4     AMs from experimental animals following exposure to NO2 concentrations greater than
  5     18,800 /ig/m3 (10 ppm) (Gardner et al.,  1969;  Acton and Myrvik, 1972; and Foster et al.,
  6     1985); however, few studies evaluated the effect of NO2 on phagocytosis at lower exposure
  7     levels. Suzuki et al. (1986) proposed that the suppression of phagocytosis might be due to
  8     the ability of NO2 to mediate membrane lipid peroxidation.  The study by Dowell et al.
  9     (1971) adds support to this hypothesis. They reported seeing evidence of swelling of AMs
 10     exposed to 5,640 /jg/m3 (3 ppm), which became most evident when exposed to 13,160 ^g/m3
 11     (7 ppm) NO2.  The authors described such swelling as a manifestation of damage to the
 12     membrane function. The apparent recovery of the AMs after  10 days of exposure is thought
 13     to be due to the influx of new AMs into the alveoli.
 14          The phagocytic activity of rat AMs was significantly depressed after 5 days of exposure
 15     to 15,040 jig/m3 (8 ppm) (Suzuki et al., 1986).  A similar suppression was noted following
 16     exposure to 7,520 /ig/m3 (4 ppm) but only after 7 days of exposure. In all cases, the
 17     phagocytic activity of these affected cells recovered to the control value at 10 days of
 18     exposure. Lefkowitz et al. (1986) failed to find any depression in phagocytosis after mice
 19     were exposed for 7 days to 9,400 /ig/m3 (5 ppm) NO2.                       !
20          Macrophages isolated from humans exposed for 3 h to 1,120 /Kg/m3 (0:6 ppm) NO2
21     were less able to inactivate influenza virus than  controls cells.  However, when the
22     concentration was reduced to 94>g/m3 (0.05 ppm) NO2 with three  15 min spikes to
23     3,760 ^g/m3 (2.0 ppm), the rate of viral inactivation  was similar to controls (Frampton
24     etal., 1987; Smeglin et al., 1986).
25          Two independent studies have reported that NO2 exposure significantly decreases the
26     ability of rat AMs to produce superoxide anion radical. Oxygen radicals are believed to be
27     essential for phagocytosis.  Failure of the AM to produce this free radical anion limits  the
28     antibacterial activity of these cells, making them less able to defend the lung from infectious
29     agents. Amoruso et al. (1981) presented evidence of such an effect at NO2 concentrations
30     ranging from 2,444 to 31,960 jig/m3 (1.3  to 17.0 ppm).  The duration of the NO2 exposure
31      was not given.  In these studies, the authors' objective was to compare the measured NO2

        August 1991                              13_26     DRAFT-DO NOT QUOTE OR CITE

-------
 1     response with O3 effects, and thus all data were expressed in terms of parts per million-hours
 2     (ppm-h) making it impossible to determine the specific concentration and duration of
 3     exposure that elicited this effect.  A decrease of superoxide anion radical production began
 4     after exposure to 18.3 ppm-h NO2.  Production was reduced by 50%  after 29.1 ppm-h and at
 5     51 ppm-h, the highest exposure tested,  the production was decreased by 85%. Similar
 6     responses were reported by Suzuki et al. (1986), who reported a marked decrease in the
 7     ability of rat AMs, to produce superoxide anion radical following a 10-day exposure to either
 8     7,520 or 15,040 jwg/m3 (4.0 or 8.0 ppm) NO2. At the highest concentration, the effect was
 9     significant each day, but at the lower concentration the depression was significant only on  ,
10     exposure Days 3, 5, and 10.
                                                                                      o
11          Alveolar macrophages obtained by lavage from baboons exposed to 3,760 /*g/m
12     (2.0 ppm) NO2 for 8 h/day, 5 days/week for 6 months had significantly impaired
13     responsiveness to migration inhibitory factor produced by sensitized lymphocytes (Greene and
14     Schneider, 1978). This substance affects the behavior of AMs by inhibiting free migration
15     which in turn,  interferes with the functional capacity of these defense cells.  The random
16     mobility was significantly depressed  in rabbits following a 2 h/day exposure for 13 days to
17     560 fjig/m3 (0.3 ppm) but not at 1,880 /jg/m3 (1.0 ppm) (Schlesinger et al., 1987), Such
18     effects are important in the mediation of local immunologic responses in the lung and would
19     be expected  to prolong the residence time of the multitude of inhaled deposited particles in
20    , the deep lung.  ,
21           Vollmuth et al. (1986) studied  the clearance of 85Sr-tagged polystyrene latex spheres
22     from the lungs of rabbits following a single 2-h exposure to 560,  1,880, 5,600,  or
23      18,800 Mg/m3  (0.3, 1.0, 3.0,  or  10.0 ppm) NO2.  An acceleration in clearance  (decreased
24     daily retention) was evident immediately after exposure to the two lowest NO2
25     concentrations; at the higher levels of NO2, an acceleration in clearance was not evident until
26  ,   midway through the 14-day postexposure period.  Repeated exposure for 14  days for 2 h/day
27     to 1,880  or  18,800 /ig/m3 NO2 produced a response similar to a single exposure at the same
28    .. concentration,  indicating that some attenuation (tolerance) may be produced after, the  initial
29     exposure providing some degree  of protection during subsequent exposures.
30,,         A number of animal studies have been performed to induce, various structural,
31      functional, and chemical changes in  AMs by exposing the test animals to exceedingly high
      ,  August 1991
13-27
DRAFT-DO NOT QUOTE OR CITE

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  1
  2
  3
  4
  5
  6
  7
  8
  9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
20
21
22
23
24
25
26
27
28
29
30
31
 concentrations (i.e, greater than 9,400 /*g/m3 [5 ppm] NO^. Some of these studies, along
 with the studies conducted at lower exposure concentrations, are listed in Table 13-4 to
 familiarize the reader with the broad spectrum of biological responses that AMs can exhibit
 following NO2 exposure.

      In Vitro Exposure. A number of investigators have utilized in vitro models to determine
 the direct effects of NO2 on specific target cells.  Voisin et al. (1977) have shown
 concentration-related effects after exposure for 30 min to NO2 concentrations as low as
         ij
 188 /ig/nr (0.1 ppm).  In this system, the AMs, attached to cellulose acetate fibers, were
 floated on top of a nutrient medium which diffused through the filter, maintaining cell
 viability without submerging the celln.  These floating cells were then exposed to NO2.  In
 these studies, guinea pig AMs seemed to be sensitive to NO2, exhibiting a reduction in
 phagocytic and normal bactericidal activity, reduced ATP content, and major morphological
 changes.  The severity of these changes was related to the NO2 concentration (Voisin et al.,
 1977).  Increasing either the exposure concentration to 9,400 ji*g/m3 (5.0 ppm) or the
 exposure duration to 24 h resulted in complete destruction of the cell  (Voisin et al.,  1984).
 Normal human AMs exposed in vitro to high levels of NO2 for 3 h did not exhibit any
 change in cell viability or the release of either neutrophil chemotactic  factor or interleukin-1
 (Pinkston et al., 1988).

Humoral and Cell-Mediated Immunuity
     It is most relevant to assess the effects of inhaled compounds on cell-mediated and
antibody responses in the lung itself, because this is the primary site of defense against
respiratory infections.  However, because of technical obstacles, which in some cases have
only recently been overcome, many studies have assessed the responses to inhaled pollutants
or NO2 in the spleen or peripheral blood.  These studies are more difficult to interpret in
terms of enhanced risk of respiratory infection.  However, the immune system is one of the
many potential targets of inhaled pollutants, such as NO2. In some cases, the immune system
can be affected immediately, whereas under different conditions the changes may be
secondary to the injury of other organs or to a general deterioration of the host's health. In
either case, these changes can often seriously compromise the health and well being of the
       August 1991
                                         13-28      DRAFT-DO NOT QUOTE OR CITE

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  1
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 16
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 30
31
 exposed host.  Such changes can result in a decrease in immune function (suppression)
 following exposure to toxic chemicals and may lead to a partial or complete deficiency.
 Enhanced immune activity may result in an exaggerated response to an antigenic stimulus
 causing an unwanted hypersensitivity.  There are many examples of such undesirable
 immunological changes that have been associated with chemical exposure both in laboratory
 animals and humans.  These changes can, under certain circumstances, trigger a specific
 response in the immune system, possibly leading to a subsequent pathological state that is far
 more serious than the original lesions.
      Because the immune system is a multicomponent, carefully regulated system, it offers
 numerous target sites for NO2 action and there are some studies that indicate that high
 concentrations of NO2 (>9,400 jwg/m3; [5.0 ppm]) can significantly depress the immune
 response, as determined by one or more tests  available to assess the functional integrity of the
 specific components.  Unfortunately, there are only a very few studies conducted at near
 ambient concentrations, and, independent of the concentrations tested, only a few of the
 numerous immune parameters have been evaluated.
     Exposing sheep  to 9,400 ^g/m3 (5 ppm) NO2, 1.5 h/day for 10  to 11 days showed that
 such intermittent, short-term exposure may temporarily alter their pulmonary immune
 responsiveness (Joel et al.,  1982).  One technique commonly .used in determining the
 production of antibody forming cells is to measure the number of plaque-forming cells
 (PFCs) in the spleen.  In this study, the authors assessed immunological response by
 monitoring the daily output of PFC in the efferent lymph of caudal mediastinal lymph nodes.
 Although the number  of animals used was small and the data were not analyzed statistically,.'
 it would appear that in the animals that were immunized (with sheep red blood cells, SRBC)
 (a T cell dependent antigen) 2 days after NO2 exposure the output of PFC was consistently
 below controls.
     Hillam et al.  (1983) examined the effects of a 24-h exposure to 9,400, 18,800, and
 48,900 /tg/m3 (5, 10,  and 26 ppm) NO2 on cellular immunity  in rats after intratracheal
 immunization of the lung with SRBC.  Cellular immunity was  evaluated by antigen-specific
 lymphocyte stimulation assays of pooled lymphoid cell suspensions from either the thoracic
lymph nodes or the spleen.  A concentration-response effect with elevated cellular immunity
was observed.
       August 1991
                                        13-34
DRAFT-DO NOT QUOTE OR CITE

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 1           Studies conducted by Fenters et al. (1971, 1973) and Ehrlich and Fenters (1973) using
 2    . squirrel monkeys showed an impact on NO2 on the humoral immune response to
 3     intratracheally delivered influenza vaccine.  In monkeys exposed for 493 days to 1,880 jug/m3
 4     (1 ppm) NO2 and immunized with monkey-adapted virus  (A/PR/8/34), the serum neutralizing
 5     antibody titers were significantly increased earlier and to a greater degree than controls
 6     (Fenters et al., 1973; Ehrlich and Fenters, 1973). In monkeys exposed to 9,400 ^cg/m3
 7     (5 ppm NO^ for a total of 169 days and immunized with mouse-adapted influenza virus
 8     (A/PR/8), serum neutralization titers were lower than controls initially; no  significant
 9     difference was observed by 133 days of exposure (Fenters et al., 1971; Ehrlich and Fenters,
10     1973). In all of these studies, the hemagglutination inhibition antibody titers were not
11     affected. The authors discussed these differences, suggesting that the difference in the viral
12     strain used for immunization played a role, along with exposure differences.  The authors
13     also hypothesized that exposure to 1,880 >g/m3 (1 ppm) NO2 improved the establishment and
14     survival of the monkey-adapted virus within the respiratory tract, resulting  in an increase in
15     antibody production.  The results of Holt et al. (1979) also  suggest that both  the nature of
16     and rate of change in immunological function from NO2 exposure can be both concentration-
17     and time-dependent.  At 7-week intervals, tests were conducted to assess the  functioning
18     ability of the immune system of mice exposed to 18,800 ^g/m3 (10 ppm) NO2, 2 h/day for
19     periods of up to 30 weeks (Holt et al., 1979). Chronic exposure exhibited a general
20     suppression in antibody titers and ability of the T cells to function in a graft vs. host-reaction.
21     However, the more acute exposures resulted in an enhancement of immunological
22     responsiveness.
23           A series of immunological studies designed to  examine the effects of NO2 on the
24     humoral antibody response to SRBC was reported by Fujimaki and Shimizu (1981).  Initially
25     they exposed mice for 12 h to 9,400, 37,600, and 75,200 ^g/m3 (5, 20, and 40 ppm) NO2
26     and reported a significant suppression in primary antibody response to SRBC at the two
27     highest levels of exposure. Expanding on these studies, they later demonstrated that NO2,  in
28     addition to reducing the primary antibody response,  also significantly altered  the host's
29     secondary response to this antigen.  The data led the authors to speculate that the suppression
30     of the primary response was due to a reduction in the functioning of the B  cells, whereas the
31     depression of the secondary response was related to  a T cell defect (Fujimaki et al., 1981).
        August 1991
13-35
DRAFT-DO NOT QUOTE OR CITE

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   1
   2
   3
   4
   5
   6
   7
   S
   9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
 20
 21
 22
 23
 24
 25
 26
 27
28
29
30
      Using this same model, FujimaH et al. (1982) reported a similar effect in mice (i.e.,
 suppression of primary antibody response in the spleen) after a 4-week exposure to 752 and
 3,000 /ig/m3 (0.4 and 1.6 ppm) NO2.  At the higher concentration, there was no significant
 difference observed in the activities of the T and B lymphocytes.  Secondary antibody
 response was not affected at 752 ^g/m3 (0.4 ppm) but was slightly enhanced at 3,000 /tg/m3
 (1.6 ppm) NO2.  However, Maigetter et al. (1978) found that the normal transformation
 response of mouse splenic T and B ceUs to phytohemagglutinin and bacterial
 lipopolysaccharide, respectively, was suppressed following a 3-month exposure to 940 jug/m3
 (0.5ppm)NO2.
      Lefkowitz et al. (1986) employed several methods to measure immunoactivity of mice
 exposed to 9,400 jttg/m3 (5.0 ppm) NO2 for 24 h/day for  6 days and injected with SRBC
 after the first day of exposure.  Nitrogen dioxide did not affect hemagglutination antibody
 liters or cell-mediated immunity (blastogenesis of splenic T cells), but did significantly reduce
 the number of splenic PFC to SRBC.  The authors stated (data were not shown) that mice
 exposed to 2,820 ^g/m3 (1.5 ppm) NO2 for 14 or 21  days also showed a 33 and 50%
 decrease, respectively, in the number of PFCs.
     Kosmider et al.  (19735) exposed guinea pigs to 1,880 /xg/m3 (1 ppm) NO2 for 6 months
 and reported a significant reduction in all serum immunoglobulin fractions and complement.
 Decreased levels of these substances may lead to an increase in the frequency, duration, and
 severity of an infectious disease. Mice exposed to NO2 had decreased  serum levels of IgA
 and exhibited nonspecific increases in serum IgM, IgG, and IgG2 (Ehrlich et al., 1975).
     Extrapulmonary  effects on the functional activity of the immune system was tested in
 mice by Richters and Damji  (1988). The percentage of the total T lymphocyte population
 was lower in the spleens of AKR/cum mice exposed for 7  weeks (7 h/day, 5 days/week) to
 470 /xg/m3 (0.25 ppm) NO2. The percentages of mature helper/inducer T and
 T cytotoxic/suppressor lymphocytes were also lower in the spleen of exposed animals. There
 were no statistically significant changes in the percentages of natural killer cells  or mature T
 cells. C57BL/6J mice exposed to 670 jwg/m3  (0.35 ppm) for 7 h/day, 5 days/week for
 12 weeks also  showed a  suppression in the percentage of total  matured T lymphocytes but no
statistically significant effect on any specific subpopulation,
       August 1991
                                        13-36      DRAFT-DO NOT QUOTE OR CITE

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 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
     Richters and Damji (1990) reported similar findings in AKR/cum mice exposed for up
to 181 days, 7 h/day, 5 days/week to 470 jug/m3 (0.25 ppm) NO2.  The T-helper/inducer
(CD4+) lymphocytes (spleen) were reduced; no effects were observed on T cytotoxic/
suppressor cells. Spontaneously developing lymphomas in NO2-exposed animals had
progressed more slowly than those in control animals.  This was attributed to the NO2-
induced reduction in the T-helper/inducer lymphocytes.
     Mice that were vaccinated with influenza virus (A-2/Taiwan/l/64) after 3 months of
continuous exposure to 3,760 /tg/m3 (2.0 ppm) or to 940 jug/m3 (0.5 ppm) NO2 with a 1-h
daily 5 days/week spike exposure to 3,760 /ig/m3 (2.0 ppm) had mean serum neutralizing
antibody liters that were fourfold lower than clean air controls (Ehrlich et al., 1975).  The
hemagglutination inhibition titers in these animals were unchanged.  This agrees with  the
Fenters et al. (1973) findings in monkeys exposed to 1,880 /*g/m3 (1.0 ppm) for over 1 year.
     Few studies have been undertaken to  assess the effects of NO2 on interferon production.
Mice exposed to either 9,400 or 47,000 ^g/m3 (5 or 25 ppm) NO2 for 3 to 7 days had serum
levels of interferon  similar to controls (Lefkowitz et al., 1984; Lefkowitz et al., 1983).
     An increase in certain immunological functions may also be detrimental to, the host's
health by stimulating the immune system to react against the host's own tissue.  Balchum
                                                                             o
et al.  (1965) identified such an effect when guinea pigs were exposed to  9,400  /tcg/m
(5 ppm) and 28,200 jig/m3 (15 ppm) NO2. There was a noticeable increase in the titer of
serum antibodies against lung tissue in all test animals exposed, starting after 160 h of
exposure. These antibody titers continued  to increase with the concentration and duration of
exposure to NO2.
     The effects of NO2 exposure on the immune system appear to  be concentration- and
time-dependent. Some studies suggest little effect,  while others suggest suppression or
activation, depending not only on concentration, but also on length of exposure, species
tested, and specific  end points measured.  Although we lack a thorough understanding of the
potential effects of NO2 on the immune system, it is evident that the immune system is
sensitive to NO2. Table 13-5 summarizes  the reported immunological effects following
exposure to NO2.
        August 1991
                                         13-37
DRAFT-DO NOT QUOTE OR CITE

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August 1991
13-38     DRAFT-DO NOT QUOTE OR CITE

-------









CVIUNOLOGICAL EFFECTS '
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August 1991
13-40     DRAFT-DO NOT QUOTE OR CITE

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 1     Interaction With Infectious Agents
 2          Different experimental approaches using intact animals have been employed in an effort
 3     to determine the functional efficiency of the host's pulmonary defenses following NO2
 4     exposure.  Animals are randomly selected for exposure to either a pollutant, in this case
 5     NO2, or filtered air.  After exposure, the treatment groups are combined and exposed for
 6     approximately 15 min to an aerosol of a viable agent, such as Streptococcus sp., Klebsiella
 7     pneumoniae, Diplococcus pneumoniae, influenza A2/Taiwan virus, or A/PR/8 influenza
 8     virus.  The animals are then returned to clean air for a holding  period (usually 15 days) and
 9     the mortality rates in the NO2-exposed and the control groups are compared.  If the normal
10     pulmonary defenses are functioning properly, the deposited viable microorganisms will be
11     quickly killed and the lungs will remain sterile, and only a small percentage (typically
12     between 5 to 15%) of the control animals will succumb to the laboratory infection.
13     However, if host defenses are compromised by the chemical exposure, mortality rates will be
14     higher (Ehrlich, 1963, 1966, 1980; Gardner, 1982; Coffin and  Gardner, 1972; Henry et al.,
15     1970).
16          A wide variety of mammalian species, including humans,  share an array of defensive
17     mechanisms that are anatomically and physiologically integrated in the respiratory tract to
18     prevent and control most invading infectious organisms.  The infectious disease model is an
19     excellent indicator of a weakened host defense system.  The effects seen in laboratory animals
20     represent alterations in host defenses.  Studies have shown that  these responses are valid
21     across species, sensitive  to a variety of chemicals, supported by mechanistic studies, and are
22     capable of epidemiological confirmation.  One can believe that  similar alterations in these
23     basic defense  mechanisms that occur in animals could also occur in humans, since they have
24     equivalent pulmonary defenses.  In the animal model frequently used, mortality is the
25     sensitive response indicator for alterations in host defense functioning.  However, with
26     today's medical care, few people die of bacterial pneumonia so a better comparison in
27     humans  would be the prevalence of respiratory illness in the community as discussed in
28     Chapter 14 (epidemiological studies).  Such a comparison is proper, since both  mortality
29     (animals) and morbidity  (humans) result from a loss of pulmonary defenses. However, one
30     should understand that different exposure levels may be required to produce a similar
        August 1991
13-41
DRAFT-DO NOT QUOTE OR CITE

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  1     response in humans.  Table 13-6 summarizes effects of exposure to NO2 and an infectious
  2     agent.
  3          An enhancement in mortality following exposure to NO2 and a pathogenic
  4     microorganism could be due to several factors.  Studies by Goldstein et al. (1973) showed
  5     decreases in pulmonary bactericidal activity following NO2 exposure. In their first
  6     experiments, mice breathed aerosols of Staphylococcus aureus (S. aureus) labelled with
  7     radioactive phosphorus and then were exposed to NO2 for 4 h.  Physical removal of the
  8     bacteria was not affected by any of the NO2 concentrations used up to 27,800 ftg/rn3
  9     (14.8 ppm). Concentrations of 13,200, 17,200, and 27,800 /ig/m3  (7.0, 9.2, and 14.8 ppm)
 10     NO2 did lower the bactericidal activity by 7,  14, and 50%, respectively, when compared to
 11     controls. Lower concentrations (3,570 and 7,140 /ig/m3 [1.9 and 3.8 ppm]) had no
 12     significant effect.  The cause of the alteration was an impairment of the AM's bactericidal
 13     activity and not an impairment of the host's mechanical clearance system. In another
 14     experiment (Goldstein et al., 1974), mice breathed 1,800, 4,320, and 12,400 (1.0, 2.3, and
 15     6.6 ppm) NO2 for 17 h and then were exposed to an aerosol of S. aureus.  Four hours later
 16     the animals were examined for the number of organisms present in the lungs. No difference
 17     in the number of bacteria inhaled was found in the NO2-exposed animals. Concentrations of
 18     4,320 and 12,400 jug/m3 NO2 decreased pulmonary bactericidal activity by 6 and 35%,
 19     respectively, compared to controls.  Exposure to  1,800 /*g/m3 NO2  had no significant effect.
20     Goldstein et al.  (1974) hypothesized that the decreased bactericidal activity was due to defects
21      in AM function.  Research conducted by Jakab (1987) indicate that the  concentration of NO2
22     required to suppress normal pulmonary bactericidal activity in mice probably depends on the
23      specific invading organism.  For example, exposure to > 7,520 j«g/m3 (4 ppm) NO2 for 4 h
24      significantly depressed normal bactericidal defenses of the lungs against deposited S. aureus,
25      but it required a concentration of 19,000 jig/m3 (10 ppm) before the lung's ability to kill
26      deposited Pasteurella and Proteus was impaired.
27           A 4-h exposure to 9,400 jtg/m3 (5.0 ppm) NO2 significantly impaired the ability of
28      mice to Mil Staphylococcus organisms.  This effect became more significant with increasing
29      concentrations.  A similar exposure to 4,700 and  7,500 ^g/m3 (2.5 and 4.0 ppm) produced
30      no  significant response (Jakab, 1988).  The combination of corticosteroid (subcutaneous
31      injection) and NO2 exposure (4 h) significantly impaired the intrapulmonary killing of

        August 1991                             13_42     DRAFT-DO NOT QUOTE OR CITE

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                 13-44      DRAFT-DO NOT QUOTE OR CITE

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 1     staphylococci at concentrations between 1,880 and 4,700 jiig/m3 (1.0 and 2.5 ppm) (Jakab,
 2     1988). These results would demonstrate that such a pretreated host is more susceptible to the
 3     effects of NO2.  The implication of this finding is the probable existence of a high-risk
 4     population (because of immunosuppression, chronic lung disease, or old age) whose altered
 5     host status makes them more susceptible to infection following NO2 exposure (Green,  1970).
 6          Differences in species sensitivity to NO2 or to a pathogen may play a role in the
 7     enhancement in mortality seen in experimental animals.  An enhancement in mortality was
 8     noted in mice, hamsters, and monkeys exposed to NO2 for 2 h followed by a challenge of
 9     K. pneumonia (Ehrlich, 1975).  However, differences in sensitivity were noted between the
10     species.  Concentrations of 9,400 to 65,830 ^g/m3 (5.0 to 35.0 ppm) NO2 had no effect on
                                                                                           o
11     monkeys. Squirrel monkeys exposed continuously to NO2 levels of 9,400 and 18,800 jug/m
12     (5.0 and 10.0 ppm) for 2 and 1 months, respectively, showed increased susceptibility to a
13     challenge with K. pneumoniae and reduced lung clearance of viable bacteria (Henry et al.,
14     1970). Two of seven monkeys exposed to 9,400 jug/m3  (5.0 ppm) for 2 months died and the
15     rest had bacteria in the lungs on autopsy. Hamsters exhibited enhanced mortality at
16     concentrations of > 65,830 ^g/m3  (35 ppm) but not at 9,400 to 47,000 /ig/m3 (5.0 to
17     25.0 ppm) NO2.  The mouse model was the most sensitive to NO2 exposure, as evidenced by
18     enhanced mortality following exposure to 6,580 //.g/m3 (3.5 ppm) but not to 2,820 to
19     4,700 jug/m3 (1.5 to 2.5 ppm) NO2 for 2 h (Ehrlich, 1975).  Purvis and Ehrlich (1963) also
20     reported no effect on mortality in mice exposed for 2 h to 2,820 or 4,700 /xg/m3 (1.5 or
21     2.5 ppm); effects occurred at 6,600 jug/m3 (3.5 ppm) and higher.  However, when
                                                                          o
22     Streptococcus sp. was the infectious agent, a 3-h exposure to 3,760 ^g/m  (2.0 ppm) NO2
23     caused an increased in mortality in mice (Ehrlich et al.,  1977). This suggests that the normal
24     pulmonary defenses had not recovered sufficiently to successfully clear the lung of these
25     viable microbes even after several weeks in clean air.
26           Squirrel monkeys exposed to 9,400 or 18,800 jtg/m3 (5 or 10 ppm) NO2 for 2 or
27      1 month, respectively, also showed increased susceptibility to a laboratory induced viral
28     influenza infection (Henry et al., 1970).  All  six animals exposed  to the highest concentration
29     died within 2 to 3 days of infection with the influenza virus.  At the lower concentration, one
30     of three monkeys died. Susceptibility to viral infection was enhanced when the NO2
31      exposure occurred 24 h after the infectious challenge.
        August 1991
13-49
DRAFT-DO NOT QUOTE OR CITE

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   1           McGrath and Smith (1984) investigated whether changes in susceptibility to bacterial
   2      infections in mice were related causally to the effects of NO2 on respiration.  Because no
   3      spontaneous changes in respiratory patterns were produced following a 3-day exposure to
   4      9,400 Atg/m3 (5 ppm) NO2, the authors concluded that any such relationship was unlikely.
   5           The importance of the test organism used with the infectivity models was demonstrated
   6      by Sherwood et al. (1981).  These researchers illustrated that exposure to 1,880 ^cg/m3
   7      (1 ppm) NO2 for 48 h increased the propensity of virulent group C streptococci, but not
   8      S. aureus, to proliferate within the murine lungs and cause earlier mortality.
  9           In a series of investigations,  the relationships of concentration and time to susceptibility
 10      to respiratory infection and to subsequent mortality in infections with Streptococcus sp. were
 11      examined by Gardner et al. (1977a,b), Gardner et al. (1982), and Coffin et al. (1977).  The
 12      concentrations of NO2 varied from 1,880 to 26,320 ^g/m3 (1 to 14 ppm), and the duration of
 13      exposure ranged from 0.5 to 7 h so that the product of concentration and time (C x T)
 14      equalled a value of 7,  In these studies exposure to high concentrations of NO2 for brief
 15     periods of time resulted in greater mortality than did prolonged  exposures to lower
 16     concentrations of NO2.  This indicated that susceptibility to infection was influenced more by
 17     the concentration of NO2 than by the duration of the exposure.
 18          Using the same model, Gardner et al. (1977b) examined the effect of varying durations
 19     of continuous exposure on the mortality of mice exposed to 6 concentrations of NO2 (940 to
 20     52,670 /*g/m3 [0.5 to 28.0 ppm]).   Streptococcus sp. was used for all concentrations, except
 21     940 jig/m (0.5 ppm), in which case K. pneumoniae was used.  A linear concentration-
 22     response indicated that mortality increases with increasing length of exposure to a given
 23     concentration of NO2 (Figure 13-1). Mortality also increased with increasing concentration
 24     of NO2 as indicated by  the steeper slopes with higher concentrations.  When C  X T was held
 25     constant, the relationship between concentration and time produced significantly different
 26     mortality responses. At a constant C X T of approximately 21 (ppm x h), a 14-h-exposure
 27     to 2,800 /tg/m3  (1,5 ppm) NO2 increased mortality by 12.5% whereas  a 1.5-h  exposure to
28     27,300 ^g/m3 (14.0 ppm) NO2 enhanced mortality by 58.5%. These studies confirmed the
29     previous conclusion that concentration is more important than time in determining the.degree
30     of injury induced by NO2 in this model.  According to Larsen et al. (1979),  NO2 modeling


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            LU
            o
            LU
            CC
            LU

            LU
            LU
            CL
                        SYMBOL ppmNO2
                          O     28.0
                          O     14.0
                          •      7.0
                          V      3.5
                          A      1-5
                          •      0.5
                                                               7   1416  30   23   6  9 12
                                                                   days	+\<	months-
                                                    TIME
       Figure 13-1. Mortality enhancement for mice exposed to NO2 at various concentrations
                    and for various durations prior to challenge with streptococci.  At all
                    concentrations, prolonged exposure results in enhanced mortality, but the
                    severity of resistance reduction is more directly related to concentration.
       Source: Gardner et al. (1977b).     .                                          .     ,
 1     studies have shown that the concentration (c) of NO2 expected to cause a certain mortality
 2     level (z) as a function of the hours of exposure (t) can be expressed as c  = 9.55 (2.42)zt~°-33.
 3          Gardner et al. (1979) also compared the effect of continuous versus intermittent
 4     exposure to NO2 followed by bacterial challenge with Streptococcus sp.   Mice were exposed
 5     either continuously or intermittently (7 h/day, 7 days/week) to 2,800 or 6,600 jug/m3 (1.5 or
     ,                  '          ,                       ,                 •   -         o
 6     3.5 ppm) NO2.  The results of'continuous and intermittent exposure to 6,600 £tg/m
 7     (3.5 ppm) for periods up to 15 days indicated that there was a significant increase in
 8     mortality for each of the experimental groups with increasing duration of exposure. When
 9     the data were adjusted for the difference in C X T, the mortality was essentially the same for
                                                                                          o
10     the continuous and intermittent groups.  The continuous exposure of mice to 2,800 /*g/m
11     (1.5 ppm) NO2  increased mortality after 24 h of exposure. During the first week of
12     exposure, the mortality was significantly higher in mice exposed continuously to NO2 than in
13     those exposed intermittently.  By the  14th day of exposure, the difference between
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   9
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28
29
30
  intermittent and continuous exposure became indistinguishable.  This suggests that fluctuating
  levels of NO2 may ultimately be as toxic as sustained high levels (Gardner et al., 1979).
       Mice were exposed continuously or intermittently (6 or 18 h/day) to 940 /ig/m3
  (0.5 ppm) NO2 for up to 12 months (Ehrlich and Henry, 1968).  Neither exposure regimen
  affected murine resistance to K. pneumoniae infection during the first month.  Those exposed
  continuously exhibited decreased resistance to the infectious agent as demonstrated by a
  significant enhancement in mortality at 3, 6, 9, and 12 months.  In another  experiment, a
  significant enhancement did not occur at 3 months but was observed after 6 months of
 exposure.  After 6 months, mice exposed intermittently (6 or 18 h/day) to NO2 showed
 significant increases in mortality over controls (18%). Only the continuously exposed
 animals showed increased mortality  (23%) over controls  following 12 months of exposure.
 After 12 months of exposure, mice in the three experimental groups showed a reduced
 capacity to clear viable bacteria from their lung.  This was  first observed after 6 months in
 the continuously  exposed groups and after 9 months in the intermittently exposed groups.
 These changes, however, were not statistically tested for significance. Therefore,  while it is
 not possible to directly compare the  results of the studies using Streptococcus sp. to those
 using K. pneumoniae, the data suggest that as the concentration of NO2 is decreased, a, longer
 exposure time is necessary for the intermittent exposure regimen to produce a level of  effect
 equivalent to that of a continuous exposure.
      Mice exposed continuously to 9,400 ^g/m3 (5.0 ppm) for 3 days had both a  significant
 increase in percent mortality and a decrease in relative mean survival time (McGrath and
 Oyervides, 1985). However, similar continuous exposure for 24 h/day, 7 days/week to 940,
 1,880, and 2,820 /ig/m3 (0.5, 1.0, and 1.5 ppm) NO2 for 3 months did not demonstrate a
 difference in either of these parameters.  These subchronic exposure results do not agree with
 Ehrlich and Henry (1968) who found excess mortality after continuous exposure to
 2,280 ^g/m3 (1.5 ppm) for 3 months, but the short-term exposure results  do agree with those
 of Gardner et al.  (1979) and Ehrlich  (1980).  The inconsistency may also  be attributed  to the
 fact that the McGrath and Oyervides  study had 95% mortality in the control groups, making
it virtually impossible to detect a NO2-induced enhancement in mortality.
     Gardner (1980), Gardner et al.  (1982), and Graham et al. (1987) reported further
investigations on the response of mice to airborne infections during or following intermittent
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 1      exposure to NO2. These studies investigated the toxicity of NO2 spike exposures
 2      superimposed on a lower continuous background level of NO2.  Such a regimen approximates
 3      the pattern of exposure which humans receive in the urban environment.  Mice were exposed
 4      to spikes of 8,100 jug/m3 (4.5 ppm) for 1, 3.5, or 7 h and then  challenged with Streptococcus
 5      sp. either immediately or 18 h postexposure.  Mortality was proportional to the duration of
 6      the spike when the mice were exposed to bacteria immediately postexposure, but mice had
 7      recovered from the exposure by 18 h.  Similar findings were reported by Purvis and Ehrlich
 8      (1963) who found a significant increase in excess mortality in mice exposed for 2 h to
 9      9,40oVg/m3  (5.0 ppm)  NO2, followed by a challenge with K. pneumoniae. He reported that
10     the increased mortality due to the infectious challenge was present at 6 h postexposure but
11      was no longer present if the infectious challenge was given 27 h after the animals were
12     removed from the inhalation chambers. When a spike of 8,100 ^g/m3 (4.5 ppm) was
13     superimposed on a continuous background of 2,800 /xg/m3 (1.5 ppm) for 62 h preceding and
14     18 h following the spike, mortality was significantly enhanced by a spike lasting 3.5 or 7 h
15     when the infectious agent was administered 18 h after the spike (Gardner, 1980; Gardner
16     et al., 1982;  Graham et al., 1987).  Possible explanations for these differences due to the
17     presence or absence of a background exposure are that mice continuously exposed are not
 18     capable of recovery or that new AMs or PMNs recruited to the site of infection are impaired
 19     by the continuous exposure to NO2.  The effect of multiple spikes was examined by exposing
20     mice for 2 weeks to two daily 1-h spikes (morning and afternoon)  of 8,100 ^g/m3  (4.5 ppm)
 21      superimposed on a continuous background of 2,800 jig/m3 (1.5 ppm) NO2.  Spikes were not
 22      superimposed on the continuous background during the weekends.   Mice were exposed to the
 23      infectious agent either immediately before or after the morning spike. When  the infectious
 24      agent was given before the morning spike, the increase in mortality did not closely approach
 25      that of a continuous exposure to 2,800 ^g/m3 (1.5 ppm) NO2.  However, in  mice exposed
 26     after the morning spike, by 2 weeks of exposure, the increased mortality over controls
 27     approached that equivalent to continuous exposure to 2,800 /tig/m3  (1.5 ppm) NO2. These
 28     findings demonstrate that the pattern of exposure determines the response and that the
 29     response is not predictable based on a simple C  X T relationship.
 30           Further investigations into the effects of NO2 spikes on murine antibacterial lung
 31      defenses  have been conducted using a spike to baseline ratio of 4:1, which is not uncommon
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30
  in the urban environment (Miller et al., 1987).  For 1 year, mice were continuously exposed
  23 h/day, 7 days/week to a baseline of 370 /^g/m3 (0.2 ppm) or to this baseline level on
  which was superimposed two times a day a 1-h spike of 1,500 /*g/m3 (0.8 ppm) NO2,
  5 days/week. The animals exposed to the baseline level did not reveal any significant
  treatment-related effects; however, the infectivity mortality of the mice exposed to the
  baseline plus spike regimen was significantly greater than that of either the NO2 background
  exposed mice or the control mice.  This chronic study indicates that short-term spikes of NO2
  can cause detectable effects on antibacterial lung defenses.  It is of interest that such spikes
  also may have induced a subtle lung lesion as indicated by pulmonary function analyses of
  these exposed animals (see Section 13.2.3.3) (Miller et al., 1987).
      The persistence of the effects of NO2 was investigated by Purvis and Ehrlieh (1963)
  who exposed mice for 2 h to NO2 before or after an aerosol challenge with K. pneumoniae.
  At 9,400, 18,800, and 28,200 ^g/m3 (5.0, 10.0, and 15.0 ppm)  NO2 there was a significant
  enhancement of mortality in mice challenged with bacteria 1 h and 6 h after the NO,
                                                                             Zj
 exposure.  When bacteria challenge was delayed for 27 h, there was  an effect only in the
 group exposed to the highest concentration.
      Mice exposed continuously for 3 months to 560 to 940 /*g/m3 (0.3 to 0.5 ppm) NO2
 followed by a challenge with A/PR/8 influenza virus caused significant pulmonary
 pathological responses (Motomiya et al.,  1973). A greater incidence of adenomatous
 proliferation of bronchial epithelial cells resulted from the combined exposures of virus plus
 N02 than with either the viral or NO2 exposures alone.  Continuous NO2 exposure for an
 additional 3 months did not enhance the effect of NO2 or the subsequent virus challenge.
     Ito (1971) challenged mice with influenza A/PR/8 virus after continuous exposure to
 940 to 1,880 ^g/m3 (0.5 to 1.0 ppm) NO2 for 39 days and to 18,800 ^g/m3 (10.0 ppm)
 NO2, 2 h daily for 1, 3, and 5 days. Acute and intermittent exposure to 18,880 ^g/m3
 (10.0 ppm) NO2 as well as continuous exposure to 940 to 1,880 /ig/m3 (0.5 to 1.0 ppm)
 NO2 increased the susceptibility of mice to influenza virus as demonstrated by increased
 mortality. Further, when isolated hamster tracheal organ explants were exposed for 1, 2, and
3 weeks to 3,760 ^g/m3 (2.0 ppm), 1.5 h/day for 5 days/week and then immediately infected
with influenza virus (A/PR/8/34), the maximum virus titer reached.was the same for both the
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 1     exposed and unexposed explants.  However, NO2 exposure caused the peak virus production
 2     to occur earlier (Schiff,  1977).
 3          The lower respiratory tract of mice became significantly more susceptible to murine
 4     cytomegalovirus infection after 6-h exposures for 6 days to 9,400 jitg/m3 (5.0 ppm) NO2
 5     (Rose et-al., 1988).  The data would indicate no effects at exposures to levels of 4,700 jwg/m3
 6     (2.5 ppm) and below.
,1.  -       Exposure to a NO2 concentration of 9,400 jig/m3 (5.0 ppm) did not significantly alter
 8     the course of a parainfluenza (murine sendai virus) infection in mice as measured by the
 9     infectious pulmonary virus titers in the lungs.  However, this concentration of NO2, when
10     combined with the virus exposure, did increase the severity of the pulmonary disease process
11     (viral pneumonitis)  (Jakab,  1988).
12          Environmental stress,  in addition to influencing the lethality of a particular exposure
13     concentration, as discussed in the section on mortality, has been shown to enhance the toxic
14     effect of NO2.  Mice placed on continuously moving exercise wheels during exposure to
15     5,600 jig/m3 (3.0 ppm) NO2, but not 1,880 /xg/m3 (1.0 ppm), for 3 h showed enhanced
16     mortality over nonexercised NO2-exposed mice using the streptococcal infectivity model
17 .    (Illing et al., 1980). The presence of other environmental factors, such as ozone (Ehrlich
18     et al.,  1977; Gardner, 1980; Gardner et al., 1982;  Graham et al., 1987), elevated
19     temperatures (Gardner et al., 1982), or tobacco smoke (Henry et al., 1971), also augments
20     the effect of NO2 on host resistance to infection. When mice were stressed by elevated
21     temperature (32 °C) for 7 days, but not for 4 days, and exposed to 2,800 jtg/m3 (1.5 ppm)
22     NO2, there was a significant enhancement in mortality rate (Gardner et al., 1982).
23                                                                    '
24     Summary
25          A wide variety of mammalian species, including humans, depend on a similar array of
26     host defense mechanisms necessary  to protect or reduce the risk to infectious disease.  Since
27   , animals and humans share similar host defense systems, generally it is assumed that if a
28     particular seffect has been identified in a number of animal species then it is likely that a
29    >  similar effect also may occur in humans.
30         The host defense system is one of the many potential targets whose function has been
31      shown to be altered significantly by exposure to NO2.  Evidence would indicate that any
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30
  breach in these defenses should be considered as a possible indicator of an increased risk of
  infectious pulmonary and/or systemic disease.  The host defense parameters which have been
  most widely used to examine the association between NO2 exposure and pulmonary host
  defenses include the functional activity of AMs, mucociliary clearance, immunological
  competence, and susceptibility  to experimentally induced respiratory infectious disease.
       The ciliated cells in the conducting airways seem to be the most vulnerable cells of the
  tracheobronchial region to the effects of NO2 exposure.  In animals, NO2 causes structural"
  alterations in the ciliated cells of the airways.  The lowest concentration and exposure
  duration that showed a reduction in total number of cilia was a 7-month exposure of rats to
  940 ^g/m3 (0.5 ppm) NO2.  This study and others have shown that when the concentrations
  of N02 were increased, the effects became significantly more pronounced.  Clearance of
  marker substances have been used to determine if the number and structural effects on cilia
  also resulted in a significant reduction in the rate of mucociliary clearance.  Significant
 impairment of tracheobronchial clearance rates were not seen at levels below 9,400 >g/m3
  (5.0 ppm) NO2.  This would indicate that even a severely damaged airway epithelium (i.e.,
 loss of cilia) still has the ability to maintain mucus transport at a normal rate, and that the
 exposure of animals to NO2 would have to be greater than 9,400 Mg/m3 to induce any     '
 significant alterations that would have detrimental health effects.  These effects may be due to
 the direct cytotoxic action of NO2 on the ciliated cells, the alteration in amount and viscosity
 of the mucus layer, or to the chemical paralysis of the cilia.
      Within  the pulmonary region of the lung,  the primary cellular defense affected by both
 acute and long-term exposure to NO2 is the AM.  A common functional assay evaluates the
 capacity of these ceUs to conduct phagocytic and bactericidal activities.  Nitrogen dioxide
 causes a marked depression of phagocytic activity, reduces cell viability, disrupts  macrophage
 membrane integrity, reduces the total number of available cells, produces morphological
 changes, and decreases bactericidal activity.  While a few of these effects were seen
 following exposure to concentrations < 1,880 ^g/m3 (1.0 ppm), most of the studies showed
 effects at concentrations between 1,880 and 9,400 /xg/m3 (1.0 and 5.0 ppm) NO2.  Evidence
clearly indicates that these ceUs are no longer capable of isolating, transporting, detoxifying^
or clearing inhaled substances, and, because of the effects on host defenses, animals exposed
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 1      to NO, could be expected to succumb to a bacterial or viral infection when compared with
        • •    £j
 2      clean air control animals.
 3           The systemic cell-mediated and humoral immune system is also a target for NO2, as
 4      evidenced by animal studies.  The immunological effects reported seem to be variable.  Some
 5      studies show effects while others do not.  It has been suggested that long-term exposure may
 6      result in a suppression of the various humoral and cell-mediated functions, while shorter
 7      exposures may cause an enhancement of immunological reactivity. The response seems to be
 8      dependent not only on concentration and duration of exposure but also on animal species and
 9      the specific immunological end point measured. Nevertheless, the data suggest that NO2 or
10     one of its reactive products may penetrate the lung epithelial and endothelial layers to enter
11      the blood and produce some response in the systemic immune system.  Research on the
12     systemic immune system in mice, guinea pigs, and monkeys indicate that subchronic and
13     chronic exposure at or below 1,880 jug/m3 (1.0 ppm) can suppress T and B cell
14     responsiveness to mitogens and decrease the number of T cells.  Nitrogen dioxide influences
15     the production of serum-neutralizing antibodies to viruses and humoral primary antibody
16     response to red blood cells. Other immunological effects attributed to NO2 include an
17     increase in IgM and IgG, and a decrease in IgA serum levels.
18           The significance of many of these changes is uncertain, and studies conducted at lower
19     levels of exposure and for longer periods are needed to improve our understanding of these
20     immunological responses.  Future studies need to take into account the kinetics of the
21      immune response vis-a-vis toxicokinetics and dose-rate influences on toxicity.  Unfortunately,
22      being able to apply state-of-the-art methods for measurement of the pulmonary immune
23      system have not been reported for NO2.   In the absence of adequate data, one can only
 24      speculate that if NO2 affects the systemic immune system, it is likely that it also would affect
 25      the pulmonary immune system.                          .  ..        ,..  .
 26           A great amount of evidence exists in the literature that illustrates the relationship
 27      between an increased susceptibility to viral, fungal, or bacterial infections and the suppression
 28      of various host defenses.  Research with NO2 clearly indicates that exposure to NO2 can
 29      impair a number of the body's defense mechanisms.  The consequence of such a suppression
 30     would lead ultimately to increased  microbial proliferation within the lung, resulting in an
 31      increase in the incidence and severity of pulmonary infections.
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30
31
      Animal studies have demonstrated that both acute and chronic exposures to,NO2 can
 significantly increase susceptibility to viral and bacterial infections.  The exact exposures
 producing such effects are dependent upon the animal species, the microbial species/strain,
 and the model used. The infectivity model in which air- and NO2-exposed mice are
 challenged with a viable microbial aerosol and mortality measured is the, most sensitive.  For
 example, a 39-day exposure to 940 jtg/m3 (0.5 ppm) NO2 increased influenza-induced
 mortality, and a 6-month exposure to 940 /*g/m3 (0.5 ppm) NO2 increased bacterial-induced
 mortality. After an acute exposure, 3,760 /*g/m3 (2.0 ppm) is the lowest effective level
 tested for the bacterial model.
      The mouse streptococcal infectivity model has been applied extensively to elucidate
 C  X T relationships.  Concentration has a predominant influence over time of exposure. In
 the urban air, the typical pattern of NO2 is a low-level baseline exposure on which are
 superimposed peaks corresponding to peaks  of NOX mobile source emissions.  When the
 relationship of the peak to baseline exposure and of enhanced susceptibility to bacterial
 infection was  investigated, the results indicated that no simplistic C  X T relationship ,,was
 present, and that peaks had a major influence on the outcome.  When one compares the effect
 of a subchronic continuous one-level exposure to an exposure consisting of baseline and peaks
 having a lower C x T, the effect was roughly equivalent.   In a 1-year chronic study with the
 infectivity model, the effect of a 376 ^g/m3  (0.2 ppm)  NO2 baseline exposure (21 h/day,
 7 days/week)  was compared to baseline plus two daily 1-h spikes of 1,504 ^g/m3 (0.8 ppm)
 NO2 for (5 days/week).  Only the baseline plus spike group exhibited significant increased
 susceptibility to bacterial infection.
     The effects associated with NO2 exposure on the host defense system .are dependent on
 the concentration of the gas, the duration of  the exposure, the animal species tested, and the
 specific end point of toxicity measured.  As  stated earlier, basic defense mechanisms are
 common across mammalian species.  Thus, the summation of the effects on a number of host
 defense systems may make the mammalian host more vulnerable to infectious disease.
 Although the outcome measured in animals is mortality, morbidity would be expected to
 occur first or occur at exposures too low to induce mortality. In humans, especially those
 under medical treatment, such a loss in pulmonary defenses would  be expected to result in an
increased incidence  of morbidity, especially in that segment of the population that may be
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 1     more susceptible, such as young children or the elderly.  In assessing or predicting such
 2     human risk from animal data, it is understood that in humans, different exposure levels of
 3     NO2 may be required to produce effects similar to those seen in animals.
 4                         •  • .   '   '      •-.:•-.,
 5     13.2.3.2  Lung Biochemistry
 6          Studies of lung biochemistry in animals exposed to NO2 have focussed on either the
 7     putative mechanisms of toxic action of NO2 or on detection of indicators of tissue and cell
 8     damage.  The currently popular theory to explain NO2 toxicity is that NO2 initiates lipid
 9     peroxidation in unsaturated fatty acids in membranes of target cells. These changes' are
10     thought to cause cell'injury or death and the symptoms associated with NO2 inhalation. An
11     alternative theory is that NO2 oxidizes water-soluble, low molecular weight reducing
12     substances and proteins, resulting in a metabolic dysfunction that evidences itself as the toxic
13     symptom. Nitrogen dioxide  may, in fact, act by both means and,- as a consequence,  may
14     affect the intermediary metabolism of animals and their growth and maturation.  Nitrogen
15     dioxide dissolves in water to produce nitrous (HNO2) and nitric (HNO3) acids; thus,  the
16     possibility of an acid or a pH effect as a primary or secondary mechanism of injury should
17     also be considered.  In addition, several potential biochemical mechanisms related to
18     detoxification of NO2 or to responses to NO2 intoxication have been proposed and are
19     outlined in Tables 13-7, 13-8, and 13-9.
20                '   '  •                         ''-•'••           '            '
21     Lipid Metabolism
22           Various investigators have performed in vitro  experiments to evaluate the effects of NO2
23     on isolated lung cells or subcellular components to examine the possible relevance of these
24     effects  as they relate to the NO2 oxidation of unsaturated fatty acids. In a recent series of
25     studies, Patel and Block (1986a, 1986b,  1987, 1988) examined the effects of NO2 exposure
26     on cultured endothelial cells  from either pulmonary arteries or aortae of pigs.  After  exposing
27     the lung cells to 9,400 jug/m3 (5 ppm) for 24 h, they observed changes  in membrane fluidity,
28     lipid peroxide formation, 5-hydroxytryptamine uptake, and release of lactate dehydrogenase
29      (a marker for cell membrane abnormalities). The authors concluded that injury of endothelial
30      cells by NO2 can be ascribed to decreased membrane fluidity secondary to lipid peroxidation,
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S OF NO2 ON LIEOD METABOLISM
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13-63
DRAFT-DO NOT QUOTE OR CITE

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but the optimum pH was acidic (3.0); not active al
physiological pH of 7.2; attributed to cathepsins, i
C, D, and E.


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NPSH kcreased at the 2 higher NO2 levels after 9 or
18 mo; GSH peroxidase activity decreased at 0.4 ppm
after 18 mo and at 4.0 ppm after 9 and 18 mo; GSH
reductase activity kcreased after a 9 mo exposure to
4.0 ppm; G-6-PD was kcreased after a 9- and 18-mo
exposure to 4.0 ppm; no effects on 6-phosphogluconate
dehydrogenase, superoxide dismutase, or disulfide
reductase; some GSH S-transferase had decreased
activities after 18-mo exposure to 0.4 and 4.0 ppm.

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improved growth.
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supplemented (30 ppm); no change k tissue weight
with, exception of kcreased kidney weight k 1 ppm
exposed animals with vitamk E-deficient diet; slightly
decreased survival rate.

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August 1991
                                   13-67
                                             DRAFT-DO NOT QUOTE OR CITE

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  1     and that the altered physical state of the cell membrane causes impaired functionality of the
  2     membrane, leading to cellular abnormalities of metabolism and biochemistry.  In a more
  3     recent study, Sekharam et al. (1991) found that NO2-induced oxidative damage in epithelial
  4     cells (from the pulmonary artery of pigs) exposed to 9,400 /xg/m3 (5 ppm) for 48 h activates
  5     phospholipase Aj and increases degradation of phosphatidylethanolamine to lyso-
  6     phosphatidylethanolamine.  The plasma membrane concentration of phosphatidylserine was
  7     also increased in these cells.
  8          Rietjens and co-workers (1986, 1987) exposed cultured rat AMs to NO2 or O3 by gas
  9     diffusion through a Teflon® film.  Ozone appeared to be about 10 times more toxic than NO2
 10     in the system. Based on various experiments using different radical scavengers,  the authors
 11     concluded that O3 and NO2 acted by different mechanisms, where NO2 exerted its toxicity
 12     via a free radical-mediated peroxidative pathway and O3 via a pathway involving the
 13     formation of lipid ozonides.  Both O3 and NO2 appear to act at the level of lipid oxidation in
 14     causing  AM toxicity.  However, is should be noted that the chemical reactions of O3 or NO2
 15     with organic compounds in aqueous solutions can be very complex (Glaze, 1986), and
 16     prediction of which pathway(s) may predominate in complex biological systems is by no
 17     means straightforward. For example, NO2 or O3 may  react with unsaturated fatty acids, but
 18     also with water-soluble reducing substances of low molecular weight or reducible groups on
 19     proteins. A discussion of the functional and structural effects of NO2 on AMs appears in the
20     sections on host defense mechanisms and morphological effects, respectively.
21          Severe malnutrition of animals can drastically affect their response to toxicants,
22     including NO2. Experimental interest in this area has mainly  focussed on dietary lipids,
23     vitamin  E and other lipid-soluble antioxidants, and vitamin C and other water-soluble
24     antioxidants.  Roehm et al. (1971) studied the in vitro oxidation of unsaturated fatty acids by
25      O3 and NO2. Both NO2 and O3 initiated the oxidation of unsaturated fatty acids through free
26     radicals.  Typically, an induction period was noted with either anhydrous thin  films or
27      aqueous emulsions of linolenic acid exposed to 2,800 fj-g/m3 (1.5 ppm) NO2.  The addition
28      of free radical-scavenging agents such as vitamin E, butylated hydroxytoluene, or butylated
29      hydroxyanisol delayed the onset of oxidation in vitro.  The rate of oxidation of linolenic acid
30      in thin films was proportional to concentrations of NO2 from 940 to  10,200 /xg/m3 (0.5 to
31      5.4 ppm).  Thin-layer chromatography of the oxidation products of linolenic acid showed a
        August 1991
13-68
DRAFT-DO NOT QUOTE OR CITE

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 1     conversion to polar nitrogen-containing compounds and to peroxides.  A suggested
 2     mechanism of formation of these products (Menzel, 1976) involves addition of NO2 across a
 3     double bond between two carbon atoms in an unsaturated fatty acid to form a nitro compound
 4     and a carbon-centered free radical.  Such a radical can extract an electron from various
 5     potential electron donors, thereby initiating the chain reaction.  Nitrohydroperoxides and fatty
 6     acid hydroperoxides are produced in vitro from the oxidation of unsaturated fatty acids by
 7     NO2.  Phenolic antioxidants can prevent the autoxidation of unsaturated fatty acids by NO2
 8     by reacting with both fatty acid hydroperoxyl free radicals and nitrohydroperoxyl free radicals
 9     generated by the addition of NO2 to unsaturated fatty acids.  It is not known whether this
10     sequence of reactions is important in the lung in vivo.
11          Sagai et al.  (1984) reported  an increase in thiobarbituric acid  (TEA) reactants in rats
12     exposed to 7,520 /xg/m3 (4.0 ppm)  NO2 continuously for 9 months. When exposure was
13     increased to 18 months, an exposure-related increase in TEA reactants was seen in rats
14     exposed to 75, 752, and 7,520 /xg/m3 (0.04, 0.4, and 4.0 ppm) NO2, but the increase was
15     only significant in animals exposed  to the two highest concentrations.  Total lung protein
16     content was not affected by the NO2 exposure.  The exhalation of ethane in the breath was
17     measured in an assay of in vivo lipid peroxidation.  In the first series of studies, excess
18     mortality in chamber control rats  forced the use of room control rats in the statistical
19     analyses; however, there was not a  major difference between room and chamber control
20     values.  At 75, 750, and 7,500 jug/m3, 9 and 18 months of exposure increased the exhalation
21     of ethane. The two lower NO2 concentrations also increased ethane exhalation after
22     27 months of exposure, but at 7,500 jug/m3, ethane was within the control range.  Pentane
23     exhalation was measured to determine if the lipid peroxidation was bacterial in origin.
24     Pentane was only increased after  18 mo of exposure to 75 and 750 ,wg/m3 NO2, supporting
25     the interpretation of the ethane results.  In a second series of experiments, chamber control
26     rats were used and rats were exposed to 75, 225, and 750 j-ig/m3 (0.04, 0.12, and 0.4 ppm)
                                                                                         o
27     for 6, 9, and 18  months.  After 6 months, ethane exhalation only increased at 750 /ug/m .
28     All NO2 concentrations increased ethane exhalation after 9 and  18  months of exposure.
29     These studies show that N02 increased lipid peroxidation in a concentration- and exposure
30      duration-related manner.  An inverse relationship with lung antioxidant metabolism was also
31      found (see later subsection on antioxidant metabolism).  Based on their body of work and
        August 1991
13-69
DRAFT-DO NOT QUOTE OR CITE

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  1
  2
  3
  4
  5
  6
  7
  8
  9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
 20
 21
 22
 23
 24
 25
 26
 27
28
29
30
31
 other related studies, Sagai et al. (1984) hypothesize that the cause of the increased lipid
 peroxidation is also related to NO2-induced thickening of alveolar walls and decreased O2
 tension in arterial blood.
      Arner and Rhoades  (1973) exposed rats for 9 months to 5,450 /xg/rn3 (2.9 ppm) NO2
 for 24 h/day, 5 days/week.  The lung wet weight increased by 13% compared to that of
 controls.  The lipid content of the lung was significantly depressed by about 9%.  The total
 saturated fatty acid content of the lungs was decreased.  Values for specific unsaturated fatty
 acids of biological importance were not reported.  The surface tension of extracts of the lung
 increased, and the authors suggested that the increased surface tension corresponded to;a
 decrease in the lung surfactant concentration.
      Trzeciak et al. (1977) exposed guinea pigs to 940 /*g/m3 (0.8 ppm) NO2 plus 61 ^g/m3
 (0.05 ppm) NO or this NOX mixture plus an equal amount  of ammonia, for 8 h/day for a
 total of 122 days and analyzed lung phospholipids.  No differences were found in the total
 weight of phospholipids of exposed vs. control lungs. It should be noted that the vast
 majority of lung phospholipids contain saturated fatty acids.  Significant alterations were
 found in the  individual phospholipid classes.  Decreases were noted in
 phophatidylethanolamine,  sphingomyelin, phosphatidyl serine, phosphatidyl
 glycerol-3-phosphate, and phosphatidic acid.  Increases were noted in the lysophosphatidyl
 ethanolamine content, while the phosphatidyl choline (lecithin) content remained constant or
 was slightly depressed.  Such changes could be indicative of changes in cell content of the
 lung. The presence of ammonia did not significantly influence the results.
     Lecithin synthesis appeared to be depressed in the lungs of rabbits exposed to
 1,880 Atg/m3 (1 ppm) of NO2 for 2 weeks (Seto et al., 1975). The most marked effect was
 observed after 1 week of exposure and appeared to decline after the second week of
 exposure.
     Csallany (1975) exposed mice continuously for 1.5 years to 750 to 940 /xg/m3 (0.4 to
 0.5 ppm) or  1,790 to 1,880 jig/m3 (0.95 to 1.0 ppm) NO2.  Animals were fed a basal diet
 that was  either deficient in, or supplemented with, vitamin E at 30 or 300 mg/kg of diet.
 Nitrogen dioxide reduced the growth rate in all four diet groups, but the vitamin E-
 supplemented groups grew faster than  the nonsupplemented  groups. The higher level  of
vitamin E in  the diet gave the same growth rate as the intermediate vitamin E content  diet.
       August 1991
                                         13-70
DRAFT-DO NOT QUOTE OR CITE

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 1     In other studies from this group, (Ayaz and Csallany, 1977, 1978; Csallany and Ayaz, 1978)
 2     female weanling mice were exposed to 940 or 1,880 j-tg/m3 (0.5 or LO ppm) NO2
 3     continuously for 17 months.  Animals were divided into three groups receiving the basal diet
 4  ..  with either a normal supplement of vitamin E (30 mg/kg), or 300, mg/kg, or 30 mg/kg of the
 5     synthetic antioxidant N,N-diphenylphenylenediamine. After, 17 months of exposure, the
 6  :   presence of lipofuscin pigment in the liver, lungs, spleen, heart, brain, kidney, and uterus
 7     was determined. No effect could be ascribed to NO2 exposure.
 8          Exposure of vitamin A-deficient hamsters to 18,800 /ig/m3 (10 ppm) NO2 for 5 h once
 9     a week for 4 to 8 weeks reportedly caused lung damage as compared to NO2-exposed
10     hamsters with  sufficient vitamin A intake (Kim 1977, 1978).
11  .        Normal or vitamin C-depleted guinea pigs were exposed to 752, 1,880, 5,460, or
12     9,400 jtig/m3 (0.4, 1,0, 3.0,  or 5.0 ppm) NO2 for 72 h and the,lung lavage protein and lipid
13     content were determined (Selgradeetal.,  1981).  No effect was observed in guinea pigs
14     having normal blood contents of vitamin C, but vitamin,C-depleted guinea pigs, having an
15     average of 25 %  of the normal blood vitamin C content, had greater lavagable protein and
16     lipid content, except for those guinea pigs exposed to 752 'j«g/m3 (0.4 ppm) NO2. In animals
17     exposed  to 9,400 ^g/m3 (5.0 ppm) NO2, the changes in lavage fluid composition were
18     correlated with mortality (50%) and alveolar edema as observed by conventional light
19     microscopic histopathology.
20
21     Effects on Suljhydryl Compounds and Pyridine Nucleotides
22          Oxidation of sulfhydryl compounds and pyridine nucleotides in the lung is well-
23   ,  established for O3 exposures (Evans et al., 1974), but little evidence has been reported for
24     NO2 (Guth and  Mavis, 1985).                          .
25
26     Effects on Lung Amino Acids, Proteins, and Enzymes
27          Nitrogen dioxide can oxidize various reducible amino acids or side chains of proteins in
28     aqueous solution (Freeman and Mudd, 1981).  For example, Prutz et al. (1985) showed that
29     NO2 could oxidize tyrosine  in peptides or proteins in pulse radiolysis experiments.  Suzuki
30     et al. (1988) have reported increased amounts of trytophan metabolites in  the urine of rats
31     exposed for 2 weeks to 9,400 jug/m3 (5 ppm) NO2.  Concentrations of NO2 above
        August 1991
13-71
DRAFT-DO NOT QUOTE OR CITE

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 10
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20
21
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23
24
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30
31
 9,400 #g/m3 produce lung edema with concomitant infiltration of serum protein and
 enzymes.  Influx of inflammatory cells (predominantly leukocytes) from blood may occur.
 Alterations in the epithelial cell types of the lung also can occur.  Thus, some reports of
 changes in lung enzyme activity and protein content may reflect either edema, altered
 inflammatory cell populations, and/or changes in cell types, rather than  direct effects of NO0
                                                                               \      &*,
 on lung cell enzymes.
      Nitrogen dioxide also increased the protein content of lung lavage  in Vitamin C-depleted
 guinea pigs (Selgrade et al., 1981; Sherwin and Carlson, 1973).  Selgrade et al. (1981) found
 effects as low as 1,880 fig/m3 (1.0 ppm) after a 72-h exposure, but a 1-week exposure to
          sj
 750 /*g/nr (0.4 ppm) did not increase protein in lung lavage.  The results of the 1-week
 exposure apparently conflict with those of Sherwin and Carlson (1973),  who found increased
 protein content of lavage fluid from vitamin C-deficient guinea pigs exposed to 752 jug/m3
 NO2 for 1 week. Differences in exposure techniques, protein measurement methods, and/or
 degree of Vitamin C deficiencies may explain the difference between the two studies.
      Sherwin et al. (1972) exposed guinea pigs to 3,760 jug/m3 (2 ppm) NO2 for  1, 2, or
 3 weeks. They  examined lung sections histochemically for lactic acid dehydrogenase (LDH).
 With this technique, LDH is suggested to be primarily an indicator of Type 2 cells rather
 than Type 1 cells. The number of Type 2 cells per alveolus was determined. In control lung
 sections, a mean value of 1.9 Type 2 cells per alveolus was found, with a range of 1.5 to
 3.4.  Exposure to NO2 significantly increased the LDH content of the lower lobes of the lung
 by increasing the number of Type 2 cells per alveolus. The increase was progressive over the
 3-week exposure period. The authors suggested that the  increase in lung LDH content was
 due to the replacement of Type 1 cells by Type 2 cells, as shown in morphological studies
 (see Section 13.2.3.4).
     Benzo(a)pyrene hydroxylase activity of the tracheobronchial region of the lung was
 studied by Palmer et al. (1972) in rabbits that had been exposed to 9,400, 37,600, or
 94,000 jig/m3 (5, 20, or 50 ppm) NO2 for 3 h. No effect was observed on the
benzo(a)pyrene hydroxylase activities with  NO2 exposure. Law et al. (1975) studied the
effect of NO2 on benzo(a)pyrene hydroxylase, microsomal O-methyl transferase, catechol
O-methyl transferase, and supernatant catechol O-methyl  transferase activities of rat lungs.
While benzo(a)pyrene hydroxylase activity  of the lung could be induced  by treatment with the
       August 1991
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 1     carcinogen 3-methylcholanthrene, exposure to 75,200 or 132,000 j«g/m3 (40 to 70 ppm) NO2
 2     for 2 h had no effect. Thus, the studies of Palmer et al. (1972) and Law et al. (1975) agree
 3     that NO2 exposure does not effect total benzo(a)pyrene hydroxylase activity of the lung.  The
 4     O-methyl transferase activity studied by Law et al. (1975) relates to the ability of the lung to
 5     metabolize catecholamine hormones, and does not appear to be affected by NO2 treatment.
 6          A major concern has been the effect of NO2 on the structural proteins of the lungs,
 7     since elastic recoil is lost after exposure. Bils (1976) reported a thickening of the collagen
 8     fibrils in squirrel monkeys exposed to 5,640 jug/m3 (3 ppm) NO2, 4 h/day for 4 days.
 9     Kosmider et al. (1973a) reported that the urinary hydroxyproline and acid
10     mucopolysaccharide content of guinea pigs exposed to 1,880 /*g/m3 (1 ppm) NO2 for
11     6 months were significantly increased.   Since the remodeling of bone is the major source
12     (>90%) of urinary hydroxyproline in normal animals and dietary ascorbate status would
13     affect hydroxyproline homeostasis in guinea pigs, the significance of these observations to
14     lung structure and function remains to be shown.
15          Last and co-workers have examined the response of rats to exposure to O3 or NO2 by
16     quantitative analysis of apparent collagen synthesis rates by lung minces from animals
17     exposed in vivo for 7 days. In one study  (Last et al., 1983), rats were continuously exposed
18     to 9,400 to 47,000 jug/m3 (5 to 25 ppm) NO2 for 7 days.  The authors found a linear
19     exposure concentration-response curve for plots of collagen synthesis rate vs.  NO2
20     concentration over this range of NO2 exposures, with a correlation coefficient (least squares
21     analysis) for fit of the data to a straight line of 0.92.  Interestingly, the ratio of the slope of
22     this line to a similar exposure concentration-response curve for O3, a measure of the relative
23     toxicity of O3 to NO2, was about 18:1—in good agreement with calculations of their relative
24     toxicity based upon histological  indices of damage (Freeman et al., 1974a). Extrapolation
25     (linear) of the line to an estimated no-observable-effect level (NOEL) gave a value of about
26      1,880 to 5,640 /*g/m3 (1 to 3 ppm) NO2, which is in good agreement with results presented
27     in a subsequent paper by this group (Last and Warren, 1987). It was assumed by these
28     workers,  although not proven, that the increases in lung collagen synthesis rate observed after
29      acute exposure regimens are predictive of increases in total lung collagen (pulmonary fibrosis)
30      after longer periods of exposure.
 31
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   1
   2
   3
   4
   5
   6
   7
   8
   9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
 20
 21
 22
 23
 24
 25
 26
 27
 28
29
30
31
  Effects on Antioxidant Metabolism and Influence of Antioxidants
      Menzel (1970) proposed that antioxidants might protect the lung from NO2  damage by
  inhibiting lipid peroxidation.  Data related to this hypothesis have been reported Ayaz and
  Csallany (1978), Csallany (1975), Fletcher and Tappel (1973), Menzel et al. (1972), and
  Thomas et al. (1968).  Many laboratories have observed changes in the activity of enzymes in
  the lungs of NO2-exposed animals that regulate levels of glutathione (GSH), the major water-
  soluble reductant in the lung's armamentarium (Tyson et al., 1982) or in lung content of
  GSH in rodents exposed to NO2. Buthionine sulfoxime, an inhibitor of GSH synthesis, has
 also been shown to cause increased lung damage in mice exposed to 1.0 ppm O3,  suggesting
 a role for GSH as a protective agent against oxidant gases in vivo (Sun et al., 1988).  Chow
 and Tappel (1972) proposed an enzymatic mechanism for the protection of the lung against
 lipid peroxidation damage by O3, involving coupled reactions of glucose-6-phosphate
 dehydrogenase (to produce NADPH), GSH reductase (to regenerate NADP), and GSH
 peroxidase (to regenerate GSH).  Chow et al. (1974) exposed rats to 1,880, 4,330, or
 11,560 /ig/m3 (1.0, 2.3, or 6.2 ppm) NO2 continuously for 4 days to examine the effect on
 the GSH peroxidase system.  They determined the activity of GSH reductase, glucose-6-
 phophate dehydrogenase, and GSH peroxidase in the soluble fraction of exposed rat lungs.
 Linear regression analysis of the correlation between the NO2 concentration and enzymatic
 activity showed a significant positive correlation coefficient of 0.63 for GSH reductase and of
 0.84 for glucose-6-phosphate dehydrogenase.  No correlation was found between the GSH
 peroxidase activity and the NO2 exposure concentration.  The activities of GSH reductase and
 glucose-6-phosphate dehydrogenase were significantly increased during exposure to
 11,560 fig/m  NO2.  The possible role of edema and cellular inflammation in these findings
 was not examined.  These researchers concluded:  "Since exposure of rats to NO2 has
 insignificant effect on lung GSH peroxidase activity,  but had significantly increased the
 activities of GSH reductase and G-6-P dehydrogenase, it appears that this oxidant attacks
 mainly glutathione and NADPH while O3 not only initiates lipid peroxidation but also
 directly attacks these reducing substances."
     Sagai et al. (1984) studied the effects of prolonged (9 and 18 months) exposures to 75,
750, and 7,500 jig/m3 (0.04, 0.4, and 4.0 ppm) NO2 on rats. Non-protein sulfhydryl levels
were increased by 750 and 7,500 jig/m3 after both exposure durations. Nine- and 18-month
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 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
exposures to 7,500 jwg/m3 caused a decrease in the activity of GSH peroxidase and an
increase in glucose-6-phosphate dehydrogenase activity.  Glutathione peroxidase activity was
also decreased in rats exposed to 750 jitg/m3 NO2 for 18 months.  Three GSH S-transferases
were also studied, two of which (aryl S-transferase and aralkyl S-transferase) exhibited
decreased activities after  18 months of exposure to 750 and 7,500 jttg/m3 NO2.  No effects
were observed on the activities of 6-phosphogluconate dehydrogenase,  superoxide dismutase,
or disulfide reductase.  When effects were observed, they followed a concentration- and
exposure duration-response.  The decreases in antioxidant metabolism were inversely related
to the formation of lipid peroxides (see lipid subsection).
     Ayaz and Csallany (1978) exposed female mice continuously for  17 months to 940 or
1,880 /ig/m3 (0.5 or 1.0  ppm) NO2 and fed the animals a basal diet that was either deficient
in vitamin E or supplemented with 30 or 300 mg/kg of diet.   Blood, lung, and liver tissues
were assayed for GSH peroxidase activity.  Exposure to 940 jwg/m3 NO2 did not alter blood
or lung GSH peroxidase; however, exposure to 1,880 jug/m3 NO2 suppressed enzyme
                                                             fj
activity.  A  combination  of vitamin E deficiency and 1,880 /ug/m NO2 exposure resulted in
the lowest GSH peroxidase activity in blood and lung.  Liver GSH peroxidase was unaffected
by either vitamin E deficiency or NO2 exposure.
     Selgrade et al. (1981) expanded earlier studies of Sherwin and Carlson (1973) on the
effects of vitamin C deficiency on NO2 toxieity. Taken together, these investigations support
a role for dietary vitamin C in influencing the  susceptibility of NO2-exposed animals to
increased protein and lipids in lung lavage.  Since vitamin C is readily oxidized and reduced,
it could serve to detoxify oxidative products formed by NO2 or to maintain the intracellular
redox potential.

Summary
     The most popular theory describing NO2 toxieity is that of lipid peroxidation of
unsaturated  fatty acids in target cell membranes. An alternate theory is that NO2 oxidizes
water-soluble low molecular weight reducing substances and proteins.  Significant alterations
in individual phospholipid classes have been reported in Vitamin C-deficient guinea pigs
exposed  to 750 jig/m3 (0.4 ppm) for 72 h and Vitamin C-normal guinea pigs exposed for
8 h/day, for 122  days to 940 jig/m3 (0.5 ppm) NO2.  Exposure to 75  (0.04 ppm) NO2
        August 1991
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  1     increased lipid peroxidation (as indicated by increased ethane exhalation) in the lungs of rats
  2     exposed for 9 months or longer.  No changes in blood and lung GSH peroxidase activity
  3     were reported in mice exposed to 940 jug/m3 (0.5 ppm) NO2 continuously for up to
  4     17 months; however, when the exposure concentration was increased, a suppression of GSH
  5     peroxidase activity was noted.  This enzyme activity was decreased in rats exposed to
  6     750 /tg/m3 for 18 months; other antioxidant enzymes were also affected.
  7
  8     13.2.3.3  Pulmonary Function
  9          The key issues addressed by investigators evaluating the effects of NO2 on pulmonary
 10     function in experimental animals were (1) the effects of low-level, long-term exposures to an
 11     urban pattern of NO2, the lowest concentrations which stimulated respiratory reflexes and
 12     impaired gas exchange in  the lung, and (2)  differences in responses between very young and
 13     mature animals.  Compared with humans, rats and hamsters used in experimental studies of
 14     NO2 have very immature  lungs at birth.  Humans have approximately 50 million alveoli at
 15     birth which multiply rapidly until age 3 years and slowly until about age 8 years when
 16     alveolar development is complete.  Growth  continues until maturity, 16 to 18  years, through
 17     alveolar enlargement.  Rats and hamsters are born with no true alveoli.  Alveolar
 18     proliferation is most rapid between 4 and 30 days of age and is essentially complete by
 19     40 days.  While hamster lungs at 40 days have reached adult volumes and elasticity, lung
20     growth through alveolar enlargement continues in rats to 5 months of age (Mauderly,  1989).
21           Results of exposures to NO2 are shown in Table  13-10. Although data in Table 13-10
22     are presented  in order of increasing concentrations, results will be discussed in a slightly
23      different order so that, where appropriate, papers using similar exposure patterns or by the
24      same investigators can be  discussed together.
25           Nitrogen dioxide concentrations in urban areas are not constant but consist of spikes
26      superimposed  on a relatively constant background level. Miller et al. (1987) evaluated this
27      urban pattern of NO2 exposure in mice using continuous 7 days/week, 23 h/day exposures to
28      380 jLtg/m3 (0.2 ppm) NO2 with twice daily (5 days/week), one-hour spike exposures to
29      1,500 /Kg/m3 (0.8 ppm) NO2 for 32 and 52  weeks.  Mice exposed to clean air and to the
30      constant background concentration of 380 /wg/m3 (0.2 ppm) served as controls. Data from
       August 1991                            13_76      DRAFT-DO NOT QUOTE OR CITE

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                                 13-80     DRAFT-DO NOT QUOTE OR CITE

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 1     animals examined immediately and 30 days following both exposure durations were combined
 2     for analysis since there was no statistical difference between the groups (i.e., immediately and
 3     30-days postexposure). Most of the differences in pulmonary function were measured
 4     between groups exposed to background concentrations with diurnal spikes and those exposed
 5     to constant background NO2 levels, although the same pattern of effects was found when
 6     comparing spike and air-exposed animals.  Both end-expiratory volume and vital capacity, the
 7     difference in lung volume between maximum inflation and deflation,  were significantly lower
 8     in mice exposed to NO2 with diurnal spikes than in mice exposed to the constant level of
 9     NO2.  Lung distensibility, measured as respiratory system compliance,  also tended to be
10     lower (p = 0.072) in mice exposed to diurnal spikes of NO2 compared with constant NO2
IT    exposure.  These changes suggest that up to 52 weeks of low-level NO2 exposure  with
12   "  diurnal spikes produces some decrease in lung distensibility resulting in decreased
13     respiratory-system compliance and vital capacity.   Lung morphology in these mice was
14     evaluated only by light microscopy and showed no exposure-related lesions.  The absence of
15     observable morphologic lesions using light microscopy does not preclude the presence of
16     subtle morphologic changes detectable by more sensitive techniques of electron microscopy
17     and qualitative morphometry  (see Section 13.2.3.4).  The decrease in lung distensibility
18     measured in this study is consistent with the thickening of collagen fibrils in squirrel monkeys
19     exposed to 5,640 /ig/m3  (3.0 ppm) NO2, 4 h/day for 4 days (Bils, 1976)  and the increase in
20     in vitro lung collagen synthesis rates measured in lung minces from rats exposed in vivo for
21     7 days to 9,400 to 47,000 ptg/m3  (5.0 to 25.0 ppm) NO2 (Last et al., 1983).
22           Winsett et al. (1990) exposed 60-day-old rats to 940 ^g/m3  (0.5 ppm) NO2, 23 h/day,
23     7 days/week,  with a 2-h spike of 2,820 ^g/m3 (1.5 ppm) NO2, 5 days/week for up to
24     78 weeks. Evaluations of pulmonary function conducted at 1, 3,  12, and 52 weeks showed
25     no significant changes.  Following 78 weeks of exposure, delta flow at 25 % forced vital
26     capacity was 71% lower in NO2-exposed animals compared with  air-exposed animals. The
27     lower delta flow in NO2-exposed animals may indicate airway obstruction or premature
28     airway closure in the lung periphery.  This slowing of the terminal portion of forced
29     expiration may or may not be related to progressive increases in expiratory resistance and
30    expiratory time, and decreases in frequency of breathing between NO2- and  air-exposed rats
31     during a 7-min challenge with 4 and 8% CO2. Taken together, these results suggest that the
       .August 1991
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   1     ability of the lung to empty rapidly, particularly at low lung volumes, has been compromised
   2     by exposure to NO2.  Morphometric data showing a loss of elastic tissue in the proximal
   3     alveolar region provides morphologic correlation to these functional changes.    ,.- -
   4          Effects of exposure to diurnal spikes of NO2 were also studied by Stevens et al. (1988).
   5     These investigators exposed 1-day and 7-week old rats to 940, 1,880, and 3,760 /xg/m3
   6     (0.5, 1.0, and 2.0 ppm) NO2 with twice daily 1-h spikes at three-times the baseline
   7     concentration for 1, 3, and 7 weeks.  The effects on rats beginning exposures at 1 day and
   8     7 weeks of age were substantially different.  In rats beginning exposure at 1-day-old, vital
   9     capacity and respiratory system compliance were increased following 3 weeks but not
 10     6 weeks of exposure to the 1,880 to 3,760 ^g/m3 NO2 baselines with spikes.  In rats
 11      beginning exposure at 7 weeks of age,  respiratory system compliance was decreased
 12      following 6 weeks of exposure and body weight was decreased following 3- and 6-weeks of
 13      exposure to the 3,760 jig/m3 baseline with spike.  The decreased compliance in adult rats
 14      exposed to NO2 was associated with a thickening of the alveolar interstitium, particularly in
 15      the proximal alveolar region (Chang et al.,  1986).  A more detailed description of the
 16      morphometric findings of this study is reported in Section 13.2.3.4.  In the adult rats,
 17      pulmonary function changes,returned to normal values by 3 weeks after exposure ceased.
 18          Mauderly et al. (1987) measured no substantive effects on pulmonary.function in rats
 19      exposed to 17,860 ^g/m3  (9.5 ppm) NO2, 7 h/days, 5 days/week either beginning exposure
 20      at 6 months of age or beginning from the time of conception until 6 months after birth.
 21     Differences in the observed effects of NO2 between this study and that of Stevens et al.
 22      (1988) may possibly be attributed to differences  in exposure schedules (7 h/day, 5 days/week
 23     vs. 23 h/day with diurnal spikes), to differences in the stage of lung development at the time
 24     of sacrifice (alveolar expansion essentially complete at 6 months, but still continuing at
 25     13 weeks), or to some other factor.
 26          Lafuma et al. (1987) exposed 12-week-old  hamsters to 3,760 ,ug/m3 (2.0 ppm) NO2,
 27     8 h/day, 5 days/week for 8 weeks. Half the animals had been pretreated intratracheally with
 28     elastase to produce a condition of experimental emphysema.  Fixed lung volumes (20 cm
29     H2O with 2.5% glutaraldehyde) were significantly higher in  NO2-exposed animals than in
30     air-exposed controls independent of elastase treatment. Vital capacity and pulmonary
31      compliance were not affected by NO2 exposure.  Mauderly et al. (1990) exposed rats to

        August 1991                             13_82      DRAFT-DO NOT QUOTE OR CITE

-------
 1     higher concentrations of NO2 (17,860 jug/m3 [9.5 ppm]) for longer times (7 h/day,
 2     5 days/week for 24 months) and also found no increased susceptibility to NO2 in elastase-
 3     treated animals.
 4          Suzuki and Tsubone, along with their colleagues, have conducted extensive studies on
 5     the effects of NO2 on respiratory and cardiac function in mice and rats.  Since many
 6     cardiovascular effects observed following exposure to NO2 are most likely secondary to
 7     pulmonary edema and/or stimulation of sensory receptors in the respiratory tract, these
 8     changes will be discussed together.
 9       > ,  Suzuki et[al. (1984) reported that the heart rate in'unanesthetized mice was lower
10     following 1-month exposure to 2,250 and 7,520  jwg/m3 (1.2 and 4.0 ppm) NO2, but not
11     following 2 and 3 months exposure. Arterial O2 tension (PaO2) was decreased following
12  ,   3 months exposure to 7,520 jwg/m3 NO2.  Respiratory rate was  not affected by NO2
13     exposures.  Suzuki et al. (1981) also exposed rats for up to 3 months to between 752 and
14     7,520 jiig/m3 (0.4 and 4.0 ppm) NO2. After 3 months of exposure to 7,520 jig/m3 NO2,
15     anesthetized rats, artifically ventilated at high frequencies, had a significant reduction in
16     PaO2.
17     .     Effects of 24-h exposures to 9,400, 18,800, 37,600, and 75,200 jug/m3 (5, 10, 20, and
18     .40 ppm) NO2 on swimming performance in mice were evaluated by Suzuki et al. (1982a).
19     Blood lactic-acid levels measured in mice after a 4-min forced swim were approximately 50%
20     higher following exposure to 9,400 /ig/m3 NO2  compared with  controls;  Exposures to higher
21     concentrations of NO2 (> 18,800 pig/m3) resulted in a concentration-related decrease in
22     maximum forced swimming time.  These results are similar to those of Campbell (1976) who
23     measured decreased forced swimming times in rats exposed to 37,600 and 75,200  ^tg/m3 NO2
24     for 24 and 5 h, respectively, but not in rats exposed to 15,000 jug/m3 (8 ppm) NO2 for
25      19 days. Changes in blood lactic acid levels in  the Suzuki et al. study indicate that although
26    . mice exposed to 9,400 ^cg/m3 NO2 were able to swim  as long as control mice, the
27     cardiorespiratory system was not able to supply  sufficient  O2 to meet the metabolic demands
28     of swimming, and anaerobic pathways were activated producing lactic acid.
29          Suzuki et al. (1982b) evaluated breathing pattern and gas exchange in mice following
30     exposure to 9,400, 18,800, and 37,600 ^g/m3 (5, 10,  and 20 ppm) NO2 for 24 h.  The
31     irritant effect of exposure to 9,400 £ig/m3 NO2 resulted in increased respiratory  rate and an
        August 1991
13-83
DRAFT-DO NOT QUOTE OR CITE

-------
   1
  2
  3
  4
  5
  6
  7
  8
  9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
 20
 21
 22
 23
 24
 25
 26
 27
 28
29
30
31
 associated decrease in arterial carbon dioxide tension (PaCO2), but no effect on PaO2.
 Respiratory rates were increased at the two highest NO2 exposure concentrations, but because
 of impaired gas exchange associated with increased lung wet weight and lung water content,
 PaCO2 was unchanged and PaO2 was decreased following exposure.  The studies of Suzuki
 and Tsubone together have shown that 30 min to 3 months exposures of mice and rats to NO0
                                                                    1                 At
 concentrations of 9,400 jtg/m3 NO2 and greater stimulated respiratory reflexes  which slow
 the heart rate and produce concentration-related pulmonary edema which decreases blood
 oxygenation and impairs maximum exercise performance.
      McGrath and Smith (1984) found that the irritant response in mice to an 8 min exposure
 to 188,000 /xg/m3 (100 ppm) NO2 was lessened by a 3-day continuous preexposure to
 9,400 ^g/m3 (5 ppm) NO2.  Considering both 188,000 jug/m3 NO2 exposures and phenyl
 diguanide injections as irritant challenges, 3 to 7 days exposure to 7,520 to 9,400 jwg/m3  (4 to
 5 ppm) NO2 lessened the response to 188,000 /tg/m3 NO2 exposure, suggesting the
 development of a tolerance or  attenuated response to NO2  (McGrath and Smith, 1984), but
 heightened the response to phenyl diguanide injections (Tsubone and Suzuki,  1984).  Several
 other studies have reported the development of a tolerance or attenuated response to
 subsequent exposures to NO2 in experimental animals and  humans and are discussed in the
 section on mortality and controlled human clinical studies.   Certainly different processes are
 involved to account for these different responses.
     Yoshida et al.  (1980b) exposed guinea pigs to 5,898 ^g/m3  (5.02 ppm)  NO or
 9,400 /tg/m3 NO2 (5.0 ppm).  After four preliminary 30-min NO or NO2 exposures, spread
 over 2 weeks, animals were exposed twice a week for 10 weeks to either 30 min of NO or
 NO2, followed 20 min later by a 10-min exposure to aerosolized albumin.  After 5 weeks of
 NO or NO2 plus albumin exposures (two 30-min exposures/week), animals were exposed  to
 aerosolized acetylcholine for 10 min.  Results were evaluated by grading the animals
 breathing pattern on a scale of 1 to 7, with 1 representing normal breathing and 7 almost total
 apnea with only rare respiratory efforts.  Using this relatively subjective measure, the authors
 state that dyspneic breathing patterns were significantly more severe in animals exposed to
NO or NO2 followed by exposure to aerosolized albumin than in animals exposed to albumin
alone, with the greatest differences occurring between the fourth and seventh week of
exposure to albumin. Animals exposed to NO compared with NO2 appeared to  be slightly
       August 1991
                                        13-84      DRAFT-DO NOT QUOTE OR CITE

-------
 1      more affected by subsequent albumin exposures, but differences between NO- and
 2      NO2-exposed animals were not significant.  Animals previously exposed to NO or NO2 plus
 3      albumin were also more affected by the final exposure to acetylcholine.  Although the authors
 4      state that the effects they observed are statistically significant, quantitative measures of
 5      pulmonary function would allow for much more thorough statistical evaluation and better
 6     .definition of the functional changes occurring in the lungs.
 7    '••"••
 8      Summary
 9           Pulmonary function following NO2 exposures in experimental animals has shown
10     consistent patterns among  different treatment conditions and animal species.  Exposures to
11      diurnal spikes of NO2 superimposed on a constant background level, simulating NO2 patterns
12     in the urban environment, produced a decrease in lung distensibility in both mice and rats.
                                                                                    o
13     These changes were very subtle and in mice occurred at concentrations of 380 jig/m
14     (0.2 ppm) NO2 with spikes of 1,500 jig/m3 (0.8 ppm) after 1 year of exposure.  Impaired
15     gas exchange  was a predominate feature following several months of exposure to
16     7,520 jug/m3 (4.0 ppm) and was reflected in decreased PaO2, impaired physical  performance,
17     and increased anaerobic metabolism.  Newborn and mature animals are affected  differently by
 18     NO2 exposures, particularly rats exposed subchronically to continuous background NO2
 19     concentrations with  diurnal spikes.  Lung  distensibility was increased transiently in rats
20     exposed as  newborns, but was decreased in rats exposed as young adults. Hamsters exposed
21     to high NO2 concentrations as newborns had functional changes 1 year later indicative of
22     mild pulmonary emphysema, which were  not found in hamsters exposed when older.  All
 23     these studies taken together demonstrate that NO2 produces subtle to dramatic changes in
 24     pulmonary  function, depending on the concentration  and  duration of exposure.   Lung
 25      distensibility and gas exchange are the parameters most consistently affected by  exposure.
 26                                       '
 27      13.2.3.4 Morphologic Studies
 28           Inhalation of NO2 produces morphological alterations in the respiratory tract.
 29      Tables 13rll, 13-12, and 13-13 provide an overview of results of studies using  NO2 levels
 30      >9,400 jwg/m3 (5 ppm).   This discussion is generally limited to those studies using NO2
                                       I                         i
        August 1991
13_85      DRAFT-DO NOT QUOTE OR CITE

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13-96     DRAFT-DO NOT QUOTE OR CITE

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   1      levels < 9,400 /zg/m3; although results of studies with higher concentrations are discussed if
  2      they reveal critical findings.                                               ,
  3           Examination of the tables shows variability in responses at similar exposure levels in
  4      different studies.  This may be due to differences in animal species or strain, age, diet,
  5      microbiological status of the animals, or aspects of experimental protocol.  This latter
  6      includes the methodology used to evaluate the morphologic response.  For example, simple
  7      light microscopic examination may reveal no effect, while other techniques, such as
  8      quantitative morphologic (morphometric) procedures, can detect more subtle structural
  9      changes which may occur in the absence of overt lesions.  It should also be kept in mind that
 10      conclusions as to resolution of lesions with continued exposure or after the end of-exposures
 11      are highly dependent upon the method of morphologic assessment being used.
 12           There is a large degree of interspecies variability in responsiveness to NO2;  this is
 13      clearly evident from those few studies  (Table 13-11 and  13-12) where different species  were
 14      exposed under identical conditions (Wagner et al., 1965; Furiosi et al., 1973; Foster et al.,
 15      1985; Azoulay-Dupuis et al., 1983).  Such response differences, which may be due to
 16      differences in effective dose of NO2 reaching target sites (due to anatomical and ventilatory
 17      differences) and/or to inherent differences in sensitivity of these sites, imply that the choice
 18      of experimental animal affects the end result observed.  In  direct comparisons, the guinea
 19      pig,  hamster, and monkey all appear to be more severely affected by equivalent exposure to
 20      NO2 than is the rat, which is the  most commonly used experimental animal. However, in
 21       most cases, the types of histological lesions produced are similar when appropriate effective
 22      concentrations are used.
 23
 24      Sites Affected
 25           The anatomic region most sensitive to NO2, and within which injury is first noted,  is
 26      the area that includes the terminal conducting airways (terminal bronchioles), and adjacent
 27      alveolar ducts and alveoli.  Within this region, those cells that are most sensitive to
 28      NO2-induced injury are the ciliated cells of the bronchiolar epithelium and the Type 1 cells of
29      the alveolar epithelium.
30           In the alveolar region,  short-term  (acute) exposure to NO2 results in hypertrophy of
31      Type 1 cells, followed by death and desquamation of these cells, and proliferation of and

        August 1991                              13_98      DRAFT-DO NOT QUOTE OR CITE

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 1     replacement by Type 2 cells.  This may result in a thickened air-blood barrier.  In the
 2     bronchioles, there is hypertrophy and hyperplasia of epithelial cells, Clara cells lose their
 3     secretory granules and surface protrusions, and there may also be loss of cilia or ciliated
                                                                                        o
 4     cells. These effects are generally seen, however, with exposure to levels >9,400 ^g/m3
 5     (5 ppm) NO2.  With chronic exposure, many of these same changes are observed, but there
 6     is increased loss of cilia over larger areas of epithelium and in more proximal airways, and
 7     the structure of the remaining cilia may be altered.  Furthermore, intraepithelial cilia have
 8     been noted between cells near the basement membrane, or elsewhere.
 9          Permanent alterations in lung architecture resembling emphysema-like disease may result
10     from chronic exposure.  A detailed discussion of emphysema in relation to NO2 exposure
11     follows the discussion on susceptibility to NO2-induced morphological changes.
12          The temporal progression of early events due to NO2 exposure has best been described
13     in the rat (e.g.,  Cabral-Anderson et al., 1977; Evans et al., 1972, 1973a,b,  1974, 1975,
14     1976, 1977; Freeman et al., 1966, 1968c, 1972; Stephens et al., 1971a, 1972).  The earliest
15     alterations resulting from exposure to concentrations of >3,760 /*g/m3 (2 ppm) are seen
16     within 24 to 72 h of continuous exposure. These include increased macrophage aggregation,
17     desquamation of the Type 1 cells and ciliated bronchiolar cells, and accumulation of fibrin in
18     small airways.  However, repair of injured tissue and replacement of  destroyed cells can
19     begin within 24 to 48 h of continuous exposure. The new cells in the bronchioli are derived
20     from nonciliated cells, while in the alveoli the damaged Type 1 cells are replaced with
21     Type 2 cells.  These new cells are relatively resistant to effects of any continued NO2
22     exposure.  Incorporation of 3H-thymidine by Type 2 cells is observed within 12 h after the
23     initial NO2 exposure, with the number of labeled cells becoming maximal by about 48 h and
24     decreasing to  preexposure levels by about 6  days, despite continued exposure (Evans et al.,
25      1975).  If exposure levels are very high (> 18,800 /ig/m3 [10 ppm]), however, resolution and
26     return to normal may be delayed or prevented,  and  the presence of increased numbers  of
27     Type 2 cells may be prolonged or permanent.
28          The resolution of NO2-induced morphologic changes may be complete after the
29     exposure ends.  On the other hand, some lesions may remain, depending upon exposure
30     duration and concentration. For example, hamsters showed normal bronchiolar and alveolar
31     epithelium by 6 days after 48 h of exposure to 22,600 /ig/m3 (12.0 ppm) or 32,000
        August 1991
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DRAFT-DO NOT QUOTE OR CITE

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  1      (17.0 ppm), or 19 days after 6 days of exposure to 41,400 /*g/m3 (22.0 ppm) NO2 (DeNicola
  2      et al., 1981).  Rombout et al. (1986) exposed rats to 20,000 ^g/m3 (10.6 ppm) continuously
  3      for 28 days.  Bronchiolar epithelial hyperplasia was totally resolved beyond 4 days after
  4      cessation of exposure, while hypertrophy was totally resolved after 16 days of recovery; on
  5      the other hand, some abnormal cilia remained after 4 weeks.  Finally, Kubota et al. (1987)
  6      examined the time course of alveolar lesions in small groups of rats exposed to 7,520 /ig/m3
  7      (4.0 ppm) NO2, 24 h/day for up to 27 months.  One phase, which lasted for 9 to 18 months
  8      of exposure, consisted of a decrease in number and an increase in cell volume of Type 1
  9      epithelium, an increase in the relative ratio of Type 2 to Type 1 cells, and an increase in the
 10      number and volume of Type 2 cells.  A second phase, which occurred at 18 to 27 months of
 11      exposure, showed some recovery of alveolar epithelium, but a decrease in total volume of
 12      interstitial tissue and an increase in collagen fibers in the interstitium. Thus, some lesions
 13      resolved with continued exposure, while others progressed. Furthermore, the temporal
 14     pattern of recovery may differ in different regions of the lungs (Kawakami et al., 1989).  In
 15     general the largest percentage of NO2-induced lesions will resolve following a recovery
 16     period.  This period may be as short as 30 days for exposures at  <9,400 /xg/m3  (5.0 ppm).
 17     With continuous exposure, morphologic damage induced early in the exposure regime may
 18     also be resolved. For example, rats exposed continuously  for 7 months to 940 ^g/m3
 19     (0.5 ppm) NO2 showed resolution of epithelial lesions between 4 to 6 months after exposure
 20     began (Yamamoto and Takahashi, 1984).
 21           In spite of the fact that there is a fairly extensive animal toxicologic data base
 22     concerning morphologic effects of NO2, it is still quite difficult to establish a "no-observed-
 23      effect" level based upon these data. This is due to the great complexity of changes occurring
 24      with NO2 exposure, as well as to the large interspecies differences in response. In general,
 25      morphologic alterations, some of which may be persistent,  are found with chronic exposure
 26      to concentrations < 1,880 /xg/m3 (1 ppm). However, long-term exposure to levels equal to
 27      or above 3,760 /tg/m3 (2 ppm) are needed for more extensive and permanent changes.
28
29      Effects of Nitrogen Dioxide as Function of Exposure Pattern
30          Although the extent and degree of morphologic alterations appear to correspond to NO2
31      exposure concentrations, little is actually known about effects of other modifying  factors,

        August 1991                            13-100     DRAFT-DO NOT QUOTE OR CITE

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 1     such as the exposure duration- and concentration-relationship or the effect of temporal
 2     patterns of exposure.
 3          The relative roles of concentration and time in response to acute exposure were
                                                                                            o
 4     examined by Rombout et al. (1986).  These researchers exposed rats  to 1,000 to 5,000 jug/m
 5     (0.53 to 2.66 ppm) for up to 28 days, or to 20,000 jug/m3 (10.6 ppm) for either 6 h, 6 h/day
 6     for 28 days, or 24 h/day for 28 days and concluded that concentration played a more
 7     important role in inducing lesions than did exposure duration, as long as the product of
 8     concentration x time was constant.  These findings were consistent with Stavert and Lehnert
 9     (1988) who  exposed rats to much higher concentrations of NO2 (47,000 to 470,000 ^g/m3
10     [25 to 250 ppm] for 2 to 30 min).  They also noted that the effect of concentration was
11     stronger with intermittent exposure than with continuous exposure, and that the onset of
12     response was delayed with intermittent compared to continuous exposure. Furthermore,
13     continuous exposure resulted in an increase in AM number not seen with intermittent
14     exposure at  the same concentration (Rombout et al.,  1986).  These findings are similar to
15     those using the infectivity model discussed in Section 13.2.3.1.
16          Most of the morphological studies with NO2 involved a constant exposure level.
17     However, actual ambient exposures are often characterized by  transient peaks, which are
18     superimposed upon a lower and relatively constant baseline level.  The morphological effects
19     of exposure patterns involving transient spikes was examined in a number of studies.  The
20     findings of these studies are equivocal in that it is not clear whether these transient peaks
21     contribute to morphologic damage in excess of that due to exposure to a constant baseline
22     level.  In some cases, there was no group run at the constant level for comparison to those
23     tested with transient peaks.  Gregory et al. (1983) exposed rats (14 to 16 weeks old) for
24     7 h/day, 5 days/week for up to 15 weeks to atmospheres consisting of the following
25     concentrations of NO2:  (1)  1,880 /xg/m3 (1 ppm); (2) 9,400 /ig/m3  (5 ppm); or
26  .   (3) 1,880 jug/m3 (1 ppm) with two, 1.5-h spikes of 9,400 jwg/m3  (5 ppm) per day (i.e.,
27     animals were exposed to NO2 at 1,880 jug/m3 for 1.5 h, 9,400 ^g/m3 for 1.5 h,  1,880 Aig/m3
28     for 3 h, 9,400 jug/m3 for 1.5 h, and  1,880 /*g/m3 for 0.5 h).  No change in lung weight was
29     found in any exposure group.  After 15 weeks of exposure, histopathology was minimal, with
30     focal hyperinflation and areas of subpleural accumulation of macrophages found in some of
        August 1991
13-101     DRAFT-DO NOT QUOTE OR CITE

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  1      the animals exposed either to a constant level of 9,400 ^g/m3 (5 ppm), or to 1,880 jug/m3
  2      (1 ppm) with the 9,400 /*g/m3 (5 ppm) spikes.                                          ;
  3           Miller et al. (1987) exposed mice (6 to 8 weeks old) for 1 year (23 h/day, 7 days/week)
  4      either to a continuous baseline of 380 #g/m3 (0.2 ppm) NO2, or to this baseline onto which
  5      was superimposed, for 5 days/week, two, 1-h spikes (given in the morning and afternoon) of
  6      1,470 /Ltg/m3 (0.78 ppm).  Morphologic examination (light microscopy) performed after
  7      32 and 52 weeks of exposure, and then 1 month after all exposures had ended,  revealed no
  8      treatment-related lesions in either exposure group, although host defense and functional
  9      changes were noted (Sections 13.2.3.1 and 13.2.3.3).  Furthermore, the type of fixation and
10      analysis would not allow for detection of emphysema even if it were present.
11           Port et al. (1977) studied mice exposed to 190 /xg/m3 (0.1 ppm) NO2 with daily,  2 h
12      peaks to 1,880 jwg/m3 (1.0 ppm), for 6 months; dilated respiratory bronchioles and alveolar
13      ducts were noted.
14           Crapo et al.  (1984) and Chang et al. (1986)  reported on the NO2-induced changes in the
15      proximal alveolar region of rats exposed for 6 weeks to a baseline concentration of 940 or
16      3,760 ^tg/m3 (0.5 or 2.0 ppm), 23 h/day for 7 days/week into which was superimposed two
17      daily 0.5-h spikes of three times the baseline concentration for 5 days/week.  They used
18      quantitative morphometric analyses.  At the lower exposure level,  the volumes of the Type 2
19      epithelium, interstitial matrix, and AMs  increased, while the volume of the fibroblasts
20      decreased.  The surface area of Type 2 cells increased.  The arithmetic mean of the
21      interstitium increased.  Most of these changes also occurred at the higher exposure level, and
22      in some cases the change was greater than that at the lower level (i.e., increase in Type 1 and
23      Type 2 epithelial volume).  Other cellular changes occurred. At both levels exposures, the
24      volume of Type 2 cells and interstitial fibroblasts increased, with no significant changes in  .
25      their numbers. The number of Type 1 cells decreased and their average surface area
26      increased in the highest exposure group.  The number of AMs decreased at both exposure
27      levels. Generally, there was a spreading and hypertrophy of Type 2 cells.  The terminal
28      bronchiolar region of these rats was also examined morphometrically (Chang et al., 1988).
29      The lower exposure level caused no effects.  At the  higher level, there was a 19% decrease in
30      ciliated cells per unit area of the epithelial basement membrane and a reduction in the mean
31      surface area of 29% in the remaining cilia in the high exposure group. In the Clara cells,  the

        August 1991                            13-102     DRAFT-DO NOT QUOTE OR CITE

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 1      size of the dome protrusions were decreased, giving the bronchial epithelium a flattened
 2      appearance.  Pulmonary function changes in similiarily exposured were assessed by Stevens
 3v.   .etal. (1988) (Section 13.2.3.3).                   •
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Susceptibility to Nitrogen Dioxide-Induced Morphological Changes
     The morphologic effects of NO2 may depend upon the age of the animals at the time of
exposure; this could account for some of the variability in response seen for the same species
exposed to comparable concentrations.
     Azoulay-Dupuis et al. (1983) exposed both rats and guinea pigs aged 5 to >60 days old
(adult) to 3,760 or 18,800 /ig/m3  (2 or  10 ppm) for 3 days. In both species, adult animals
showed greater effects of exposure than did the newborns.  At 3,760 /ig/m3 (2 ppm), rats
showed no effects. Exposure of guinea pigs <45 days old to this level also produced no
effect. The 45-day-old animals showed thickening of alveolar walls, alveolar edema, and
inflammation, while animals older than  45 days showed similar, but more frequent,
alterations which seemed to increase with age.  Adults  also had focal loss of cilia in
bronchioli.  At 18,800 /ig/m3 (10 ppm), only rats >45  days old showed any response, which
included fibrinous deposits in alveoli and focal loss of cilia in bronchioli. On the other hand,
guinea pigs of all ages were affected, although the adults showed the most severe alterations,
namely focal  emphysema-like changes and inflammation in the bronchi and bronchioles.  All
guinea pigs showed cilia loss in bronchioli and trachea, edema, increase  in number of Type  2
cells, and alveolar inflammation.  Similar findings were reported by Stephens et al. (1982)
after exposing rats (adults and nursing pups) to high concentrations of NO2.  (See section on
host defense mechanisms.)
     Chang et al. (1986, 1988) exposed 1 day or 6-week-old rats for 6 weeks to a baseline of
940 jug/m3  (0.5 ppm) NO2 for 23 h/days, 7 days/week,' with two, 1-h spikes (given in the
                                   o
morning and  afternoon) of 2,820 /*g/m  (1.5 ppm) 5 days/week, and examined the proximal
alveolar region.  Age clearly influenced morphometric  response. The  older animals showed
an increase in the surface density of the alveolar basement membrane which -was not found in
the younger animals. Although both age groups showed an increase in the mean cellular
volume of Type 2 cells,  this increase was also greater in the 6-week-old  animals.  Thus, the
responses differed with age during exposure, but the 6-week-old animals seemed to be
        August 1991
                                        13-103
DRAFT-DO NOT QUOTE OR CITE

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  1
  2
  3
  4
  5
  6
  7
  8
  9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
20
21
22
23
24
25
26
27
28
29
30
31
 generally more susceptible to injury than were the 1-day-old animals. While there was no
 qualitative evidence of morphological injury in the terminal bronchioles of the juvenile rats,  '
 there a 19% increase in the average ciliated cell surface that was not evident in the adult rats.
 The authors also reported a 13% increase of the mean luminal surface area of Clara cells in ,.,
 the juvenile animals vs. control animals of the same age.
      An extensive series of exposures designed to relate morphometric changes to age was
 performed by Kyono and Kawai (1982).  Rats at 1, 3, 12 and 21 months of age were exposed
 continuously for 1 month to NO2 at 207 jwg/m3 (0.11 ppm), 865 jig/m3 (0.46 ppm),  5,264
     A                          n
 /tg/nr (2.8 ppm), or 16,544 fj.g/nr (8.8 ppm).  Light and electron microscopic analyses
 followed exposure.  Various morphometric parameters were assessed, including arithmetic
 mean thickness of the air blood barrier (i.e., the thickness of the tissue between the surfaces
 of the alveolar and capillary lumens) and the volume density of various alveolar wall
 components.  This study is difficult to evaluate, and quantitative estimations deliberately
 excluded the site of main damage. Analysis of individual results was complex, but depending
 upon the animal's age, exposure levels  as low as 207 jwg/m3 did change specific
 morphometric parameters.  There was a trend towards  a concentration-dependent increase in
 air-blood barrier thickness in all age groups, with evidence of age-related differences in
 response.  At any concentration, the response of this end point decreased from 1 to
 12 months, but increased again in 21-month-old animals.  Type  1 and 2 cells showed various
 degrees of response, depending on both age at onset of exposure and exposure concentration.
 In general, the response of each lung component did not always  show a simple concentration-
 dependent increase or decrease, but suggested a multiphasic reaction pattern.  The
 investigators suggested that part of this  observation may have been due to varying stages of
 impairment and repair.  Thus,  although age does play a role in modulating morphometric
 response, it may be a complex one.
     In general, it seems that neonates, specifically prior to weaning, are relatively resistant
 to NO2, and that responsiveness increases with age until weaning.  Furthermore, the
 responsivenss of mature animals appears to decline somewhat with age, until an increase in
responsiveness occurs at some point in  senescence.  However, the morphological response to   '
NO2 in animals of different ages involves similarities in the cell  types affected and in the
nature of the damage incurred.  Age-related differences occur in the extent of damage and in
       August 1991
                                        13-104     DRAFT-DO NOT QUOTE OR CITE

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 1     the time required for repair, the latter taking longer in older animals.  The reasons for age
 2     differences in sensitivity are not known, but may involve differences in diet and variable
 3     sensitivity of target cells during different growth phases (Stephens efal.,  1982; Halm, 1979)
 4     or perhaps dosimetry.
 5          A possible concern in assessing NO2 toxicity is the effect on adults  from exposure
 6     during early life, especially during the period of lung development.  Unfortunately, there are
 7     few data to allow evaluation of this.  Mauderly et al.  (1987) exposed developing rats (0 to
 8     6 months of age) and adult animals (6 to 12 months of age) to 17,900 jitg/m3  (9.5 ppm) NO2
 9     for 7 h/day, 5 days/week for 6 months.  Lung development, as determined at young
10     adulthood, was not significantly affected by earlier exposures. There was no  significant light
11     microscopic evidence of lung injury in either group of animals, and there were no exposure-
12     related differences in mean linear  intercept of the alveoli or the internal  surface area of the
13     .lungs.   Thus, the available data base does not provide an answer to  the question concerning a
14     possible role of NO2 exposure during development upon adult lung  structure.  The potential
15     for early exposure to result in long-term effects  was suggested by Lam et al. (1983) who
16     reported finding emphysema-like changes in rats exposed to high concentrations of NO2 as
17     neonates.
18          Of importance in evaluating the effects of NO2 is consideration of individuals with
19     compromised lung function.  One such group comprises those with  respiratory disease.
20     There is a very limited data base concerning morphologic effects of NO2 in experimental
21     animals with pre-existing chronic disease.  Lafuma et al. (1987) exposed both normal and
22     elastase-induced emphysematous hamsters (2 month old) to 3,760 jttg/m3  (2 ppm) NO2 for
23     8 h/day, 5 days/week for 8 weeks and analyzed tissue with morphometric assays.   Sacrifice
24     was performed immediately after exposures had ended. The emphysematous lesions produced
25     by elastase appeared to be aggravated by subsequent exposure to NO2; those exposed to NO2
26     showed increases in mean linear intercept and pulmonary volume (volume of  fixed lung as
27     measured by saline displacement) and a decrease in internal alveolar surface area, compared
28     to those treated with elastase and exposed to clean air. The investigators suggested that these
29     results may imply a role for NO2 in  enhancing pre-existing emphysema.
30          In contrast, Stavert et al. (1986) reported that NO2 exposure did not potentiate pre-
31     existing emphysema in the elastase-induced emphysema animal model. Normal and diseased
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   1     rats (12 week old) were exposed to 65,800 ^g/m3 (35 ppm) NO2 for 6 h/day, 5 days/week
   2     for 5 weeks.  The animals were allowed a 10-week recovery period prior to sacrifice.
   3     Morphologic analysis by light microscopy showed there to be no difference between animals
   4     treated with elastase and exposed to clean air or those treated with elastase and exposed to
   5     NO2.
   6         The above studies examined the effect of NO2 exposure on pre-existing chronic lung
   7     disease. It is also possible that acute lung disease may affect morphologic response to NO2.
   8     Fenters et al. (1973) challenged squirrel monkeys with an influenza virus at various times
   9     during continuous exposure to 1,880 jwg/m3 (1  ppm) NO2 for 16 months, and compared the
 10     response to that seen in animals not challenged but exposed to NO2.  Only the virus-
 11      challenged animals showed a response to NO2, which included thickening of bronchial and
 12      bronchiolar epithelium.  This suggests that acute lung disease may affect NO2 toxicity. The
 13      effects of NO2  on infectivity due to challenges  with microorganisms are discussed in
 14      Section 13.2.3.1.
 15
 16     Emphysema Following Nitrogen Dioxide Exposure
 17          In evaluating the reports of emphysema following NO2 exposure, it is necessary to
 18     consider both the current and previous definitions of emphysema and  to try to determine the
 19     morphological lesions the investigators observed which led them to the diagnosis of
 20     emphysema.  Several professional groups have presented definitions of emphysema.
 21          In 1959> a group of British physicians meet under the auspices of Ciba to clarify the
 22     terminology, definition, and classification of emphysema and related disorders (Fletcher
 23     et al., 1959). Their definition was:  "Emphysema is a condition of the lung characterized by
 24     increase beyond the normal in size of air spaces distal  to the terminal  bronchiole either  from
 25     dilatation or from destruction of their walls." They continued: "Emphysema can be
 26     diagnosed and classified consistently only on preparations from lungs distended and fixed
 27     before they are cut.  The simplest technique is intrabronchial infusion  of fixative."  They also
 28     recognized the necessity of both appropriate aged controls and of morphometry to establish
29     upper limits of normal size. One of the  lasting benefits of their efforts was the development
30     of the concept that emphysema should be defined in terms of morbid anatomy rather than
31      altered physiology or clinical observations (Fletcher and Pride, 1984).

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 1           In 1961, an expert committee of the World Health Organization defined a series of lung
 2      diseases including emphysema.  They defined emphysema in anatomical terms as follows:
 3      "Emphysema is a condition of the lung characterized by increase beyond normal in the size of
 4      air spaces distal to the terminal bronchiole, with destructive changes in their walls."
 5           In 1962, an American Thoracic Society committee developed the following definition:
 6      "Emphysema is an anatomic alteration of the lung characterized by abnormal enlargement of
 7      the air spaces distal to the terminal, nonrespiratory bronchiole, accompanied by destructive
 8      changes of the alveolar walls."  The American Thoracic Society Committee also .defined
 9      pulmonary overinflation as a "pathologic condition of the lung characterized by abnormal
10     enlargement of the air spaces distal to the terminal, nonrespiratory bronchiole, without
11      destructive changes in the alveolar walls". Following their definition of emphysema, the
12     American Thoracic Committee also noted that "Determination of these characteristics in tissue
13     obtained by surgical resection or at autopsy requires proper inflation prior to fixation and
14     gross and microscopic examination.  For this purpose, the use of the stereomicroscope is of •
15     real value." While the definitions of both the World Health Organization and the American
16     Thoracic Society emphasized the presence of destruction of air space wall in emphysema,
17     they did not define destruction.
18           The most recent definition is in the report of a National Heart, Lung and Blood Institute
19     (NHLBI), Division of Lung Diseases Workshop (National Institutes, of Health, 1985).  This
20     document first defines respiratory airspace enlargement.   "Respiratory airspace enlargement is
21     defined as an increase in airspace size  as compared with the airspace size of normal lungs.
22     The term applies to all varieties of airspace enlargement distal to the terminal bronchioles,
23      whether occurring with or without fibrosis or destruction." Emphysema is one of several
 24     forms of airspace enlargement. In human lungs,  "Emphysema is defined as a condition of
 25      the lung characterized by abnormal, permanent enlargement of airspaces distal to the terminal
 26      bronchiole, accompanied by destruction of their walls, and without obvious fibrosis."
 27      Destruction is further defined:  "Destruction in emphysema is defined as nonuniformity in the
 28      pattern of respiratory airspace enlargement so that the orderly appearance of the acinus and its
 29      components is disturbed and may be lost."  The report further indicates "Destruction...may
 30     be recognized by subgross examination of an inflation-fixed lung slice....".  Emphysema in
 31      animal models was defined differently.  The stated reason for this  difference in  the definitions
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   1     of emphysema in humans and in animal models was "In order to foster the development of
   2     new knowledge, animal models of emphysema are defined as nonrestrictively as possible:  An
   3     animal model of emphysema is defined as an abnormal state of the lungs in which there is
   4     enlargement of the airspaces distal to the terminal bronchiole.  Airspace enlargement should
   5     be determined qualitatively in appropriate specimens and quantitatively by  stereologic
   6     methods."  Thus, in animal models of emphysema, airspace wall destruction need not be
   7     present.  "Appropriate specimens" presumably refers to lungs fixed in the inflated state and is
   8     similar to the American Thoracic  Society Committee's requirement for tissue fixation.  This
   9     document further states "It is still not clear whether the airspace enlargement of age is due to
 10     age alone or to the combination of age and environmental history, but the occurrence of these
 11      changes in nearly all subjects suggests that the changes are normal."  Control animals of the
 12      same age as the experimental animals appear necessary to avoid potential confusion due to
 13      age. This National Institutes of Health committee also noted that to date animal models of
 14      emphysema faU into two general classes.  "The first class centers on testing the pathogenicity
 15      of agents suspected of being relevant to the genesis of emphysema; models produced by NO2,
 16      cadmium, and tobacco smoke are examples of this type. The second class of models is
 17      analytical, for testing specific hypotheses of the pathogenesis of emphysema."  Both classes
 18      of studies are in this review.
 19          Thus, in reviewing reports of emphysema foUowing experimental NO2 exposure
 20     important considerations include:   (1) whether the tissue was fixed in an inflated state,
 21     (2) whether air spaces distal to the terminal bronchiole were enlarged beyond normal and
 22     whether that enlargement was  determined quantitatively by stereologic methods, and
 23     (3) whether or not air space wall destruction was present.  Control animals  of the identical
 24     age as the exposed animals should  be used for stereologic studies to exclude the possibility
 25     that the airspace enlargement was due to age or the combination of age and environmental
 26     history.  The importance of each of these factors must be weighed, depending upon the
 27     specific purpose of the study.  Several of the papers reviewed in this section concerned the
28     pathogenesis of emphysema or the  sequence of events in the NO2-injured  respiratory systems
29     rather than only addressing the presence or absence of emphysema following a specific NO2
30     exposure regimen.
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 1           Presumably, both investigators who wrote manuscripts and reviewers for journals that
 2      published manuscripts concerning NO2-exposed animals were aware of and used the latest
 3      definition at the time the manuscripts were written and reviewed. Thus, the date of
 4      publication of the reports of effects of NO2 inhalation relative to the dates of publication of
 5      the definitions of emphysema must be considered in this document.  Because of these changes
 6      in the definitions of emphysema and in the methods used for evaluation of results of NO2
 7      exposures,  the studies were reviewed chronologically. It is necessary to recall that many of
 8      the methods required by the National Institutes of Health workshop report were neither well
 9      developed nor widely used when these early manuscripts were written.
10          When reports of emphysema following NO2 exposures of animals are to be extrapolated
11      to potential hazards for humans, the definition of human emphysema, rather than that for
12     emphysema in experimental animals, must be used.  Thus, the presence of airspace wall
13     destruction is critical.  In published reports of emphysema following NO2 exposure, evidence
14     of airspace wall destruction can only be obtained by careful review of the authors' description
15     of the lesions or by examining the micrographs the author selected for publication.  In
16     reviewing the research reports, the authors'  descriptions of tissue changes relative to the
 17     definition of destruction in the 1985 NHLBI definition of emphysema are quoted, and all
 18     published micrographs were examined for evidence of destruction.
 19           Freeman et al. (1964) reported emphysema in rats exposed to 47,000 /*g/m3  (25 ppm)
20     NO2 for 32 to 65 days.  Control rats of the same age were maintained in an identical
21     chamber.  They reported that "alveolar ducts and alveoli in experimental rats are more
 22     variable in size and many are much larger than in  controls".  The methods for fixation of the
 23      lungs are not mentioned nor are the methods for evaluating size of airspaces.  While the size
 24      of the lungs from exposed rats was stated to be increased, lung weights rather than lung
 25      volumes were reported.  The conclusion that emphysema was present was presumably based
 26      on the large size of the lungs and the observation of enlarged airspaces which were variable
 27      in size, as no evidence of destruction of airspace walls was presented. In terms of the 1985
 28      NHLBI definitions, these lesions appear to  represent airspace enlargement rather than
 29      emphysema of the type seen in human lungs.
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   1           Wagner et al. (1965) exposed dogs, rabbits, guinea pigs, rats, hamsters,, and mice to
   2     9,400 Aig/m3 (5 ppm) NO2 for up to 18 months.  They did not observe morphological; effects
   3     due to the exposure.
   4           Haydon et al. (1965) reported .emphysema in rats exposed for varying periods; from
   5     51 to 813 days to 22,560 ^g/m3 (12.0 ppm) NO2, but not in other rats exposed to 7,520 or
   6     1,504 Atg/m3 (4.0 or 0.8 ppm) NO2.  The lungs were fixed via the trachea in the inflated
   7     state. The alveoli  were described as "quite variable in size, being either dilated or
   8     collapsed".  The most striking microscopic abnormality was the "persistent variation in size .,
   9     of alveoli".  The diagnosis of emphysema was based on alveolar size and variation in size and
 10     on the "grossly distended,  air-filled lungs that fail to collapse when removed from the
 11      thorax".  They reported "occasional rupture of alveolar walls", but did not report destruction
 12      of airspace walls. In terms of the 1985 NHLBI definitions, these lesions appear to be
 13      primarily airspace enlargement rather than emphysema of the type seen in human lungs.
 14           Haydon et al.  (1967)  also reported emphysema in rabbits exposed continuously
 15      (presumably  24  h per day)  for 3 to 4 months for to 15,040 or 22,560 ^g/m3 (8 or .12 ppm)
 16      NO2.  The lungs were fixed via the trachea in an expanded state.  They reported enlarged
 17      lungs which failed to collapse when the thorax was opened.  In lOO-^m thick sections from
 18      formaldehyde fixed dried lungs they reported "dilated" airspaces with "distorted architecture".
 19      In those and other tissue preparations they reported that the airspaces .appeared "grossly
 20      enlarged and  irregular, which appears to be due to disrupted alveoli.. .and the absence of
 21     adjacent alveolar collapse". Thus, in appropriately fixed lungs they reported evidence of    ;
 22     enlarged airspaces with destructive changes in alveolar walls.  While no  stereology was done,
 23     this appears to be emphysema of the type seen in human lungs. Davidson et al. (1967)
 24     reported physiologic changes in these rabbits  but no .new observations related to the criteria
 25     for emphysema.
 26          Unlike their previous reports of emphysema in rats exposed to higher concentrations of
 27     NO2, Freeman et al. (1968c) reported that rats exposed continuously (24 h/day) to
 28     3,760 jig/m3 (2 ppm) NO2 for 112 to 763 days had only equivocal increases in lung weight
 29      and distension of airspaces.  These lungs were fixed via the trachea in a distended state.
30      These NO2 exposures did not result in emphysema.
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 1           Freeman et al. (1968a) reviewed some of their earlier studies and new observations on
 2      rats exposed to 32,400 ftg/m3 (18 ppm) NO2 24 h/day for 1 to 6 days or for 4 weeks.  The
 3      authors did not present new data relative to enlarged airspaces or destruction of airspace
 4      walls, but rather appear to have based their diagnosis of emphysema on the gross size of the
 5  •:    lungs.  They again referred to the Ciba Symposium definition of emphysema.  This review
 6      did not present relevant new information.
 7           Freeman et al. (1968b) summarized many of their previously published NO2 exposures
 8    •  in a manuscript resulting from a meeting  (symposium) presentation.  They again followed the
 9      definition of emphysema proposed by the Ciba Symposium and did not present new data
10     relative to enlarged airspaces or destruction of alveolar walls.  No new information relevant
11      to emphysema following NO2 exposure was presented.
12          At the same  meeting (symposium), Kleinerman and Cowdrey (1968), reported results of
13     exposures of hamsters to 84,600 to 103,400 ^g/m3 (45 to 55 ppm) NO2,  22 to 23 h daily for
14     10 continuous weeks.  Some of the hamsters were held in room air for a 4-week postexposure
15     period.  The lungs were fixed in a distended state by  formaldehyde fumes via the trachea.
16     Fixed lung volumes were estimated by water displacement. Fixed lung volumes of exposed
17     and postexposed hamsters were statistically larger than similar-aged controls.  While the size
18     of alveolar spaces appeared enlarged in the exposed animals, as compared to the controls
 19     similarly fixed, there was no evidence of destruction of alveolar septal tissue.  The authors
20     concluded that  "emphysema had not  been produced in this experiment." These authors also
21     reported previously unpublished observations that 21 to 23 h/day exposure of guinea pigs,
22    rabbits, and rats to concentrations of NO2, which results in a mortality of approximately 35%
 23    does not result in emphysema due to the nondestructive character of the tissue response.
 24    They based their  conclusions on the  definition of emphysema proposed by the American
 25    Thoracic Society  and discussed the necessity for inflation fixation of lungs using standard
 26  •   techniques. The  conclusion that emphysema of the type seen in human lungs was not
 27     produced appears appropriate.
 28          Gross et al. (1968) studied the effects of NO2 on control and pneumoconiotic lungs
 29     using hamsters and guinea pigs.  Most exposures were for 2 h/day for 5  days/week. The
 30     N02 concentration appears to have been planned for 41,360 ^g/m3 (22 ppm), but varied with
 31     an initial range of 94,000 to 169,200 ^g/m3  (50 to 90 ppm) the first 4 weeks, which was
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    1      then reduced to ranges of 56,400 to 94,000 /.g/m3 (30 to 50 ppm) for a total exposure period
   2      of 12 months.  The lungs were fixed via the trachea with formaldehyde fumes in a distended
   3      state.  No morphometry qf airspaces is reported.  The complex experimental design and
   4      deaths of animals during  exposure made interpretation difficult. In hamsters, "since more
   5      animals with emphysema were found in the group not exposed to NO2 it can 'also be
   6      concluded that long-term  exposure of hamsters to NO2 did not cause emphysema".   In guinea
   7     pigs this exposure resulted in "multiple small foci of emphysema with a prevalence of only
   8      15%.   More animals (guinea pigs) without pneumoconiosis developed this emphysema than
   9     did animals with pneumoconiosis".  Both enlarged airspaces and destruction of alveolar walls
  10     can be seen in some of the published micrographs.  While emphysema with alveolar wall
  11     destruction was  present, the relationship of the emphysema to NO2 exposure is not clear.
  12         Blair et al. (1969) exposed mice to 940 jig/m3 (0.5 ppm)  NO2, 6,  18, or 24 h/day for
  13     1 to 12 months. The method of fixation of the lungs is not entirely clear.  They were'fixed
  14     by immersion, presumably by immersion of slices of lung, which would be collapsed rather
  15      ^a11 fixed in a distended state as recommended by the American Thoracic Society.  It is
  16      possible that the lungs might have been distended with air, the trachea tied and then
  17      immersed in  fixative which would diffuse into the lung through the thin pleura.  Control
 18      unexposed mice  had pneumonitis. While these investigators made an attempt to measure
 19      alveolar size, they  properly conclude that their measurements "did not represent quantitatively
 20      whole lung structure...". Thus, the data concerning enlarged airspaces is not reliable due to
 21      the type of lung  fixation and of alveolar morphometry. They also mentioned alveolar "septal
 22     breakage", but not destruction, and the septal breakage was not a factor in the increased size
 23     of alveoli. No evidence of emphysema was presented in this publication.
 24          Buckley and Loosli (1969) studied the effects of 75,200 ^g/m3 (40 ppm) NO2 for 6 or
 25     8 weeks on germ-free mice.  Following exposure, the mice were either killed and the lungs
 26     examined, or the mice were infected with microorganisms for additional studies.  The lungs
 27     were fixed by inflation via the trachea.  They made no mention of lung or alveolar size or of
 28     the presence or absence of  alveolar destruction, even though they cited two of the earlier
 29     studies of Freeman  and Haydon.  In the published micrographs of control-and NO2-exposed
 30     mice the airspaces appear to be the same size and evidence of alveolar wall destruction was
31      not seen. No evidence of emphysema was presented in this publication.

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 1          Freeman et al. (1972) exposed rats to 37,600 jttg/m3 (20 ppm) NO2, which was reduced
 2     during the exposure to 28,200 jwg/m3  (15 ppm) or to 18,800 j^g/m3 (10 ppm) NO2 for
 3     varying periods up to 33 months.  Following removal at necropsy, the lungs were fixed via
 4     the trachea at 25 cm of fixative pressure.  Morphometry of lung and alveolar size was
 5     performed in a suitable, although unconventional, manner.  The morphometry indicated
 6     enlargement of alveoli and reduction in alveolar surface area. They .also  both reported
 7     alveolar destruction and illustrated alveolar destruction in their figures. They correctly
 8     conclude that they have demonstrated emphysema in their NO2-exposed rats.  However, it is
 9     not entirely clear whether both experimental groups or only the group exposed to
10     28,200 jug/m3 NO2 had emphysema.
11          Ehrlich and Fenters (1973) exposed squirrel monkeys to 9,400 or 18,800 ^g/m3 (5 or
12     10 ppm) NO2  for 3 months or to 1,880 fig/m3 (1 ppm) NO2 for 16 months and then
13     challenged  the monkeys with influenza virus.  Pieces of lung were probably fixed in a
14     collapsed condition by immersion as they state:  "At autopsy representative lung tissues were
15     obtained for histopathological examination."  Neither alveolar wall destruction nor
16     morphometry are  mentioned in the article.  They concluded that "slight emphysema" was
17     present in the monkeys exposed to the two highest exposure levels and to the virus and
18     "slight to moderate emphysema" in those exposed to 1,880 jug/m3 NO2 and to the virus.
                                                                                   o
19     They also reported emphysema was not present in monkeys exposed to 1,880 i«g/nr NO2
20     without the viral challenge. The morphological methods used preclude useful information
21     concerning emphysema following NO2 exposure.
22          Stephens et al. (1976) in a long  abstract of papers presented at that  years Aspen
                                                                                     o
23     conference, stated that "Rats exposed for long periods (3 to 5 months) to 28,200 /ig/m
24     (15 ppm) NO2 or 0.8 ppm O3 develop a disease which closely resembles emphysema...".
25     Because it is only a long abstract and the emphasis of the paper was cellular injury, there
26     were no data relative to alveolar size, nor was there information concerning the presence or
27     absence of alveolar destruction. Thus, this paper does not provide new data relative to the
28     presence of emphysema of the type seen in human lungs.
29          Port et al. (1977) studied experimental and spontaneous emphysema in the rat, mouse,
30     hamster, horse, and human lungs and compared them  with normal control lungs from the
31     same species.  Only six mice and one rat had been exposed to NO2.   The mice were exposed
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  1     to 188 |Kg/m3 (0.1 ppm) NO2 with a 2-h peak of 1,880jtg/m3 (1.0 ppm) NO2 daily for
  2     6 months.  Control mice of the same age were also examined.  Only one NO2-exposed and
  3     one control rat were studied.  The exposed rat breathed 28,200 jtig/m3 (15.0 ppm) NO2 from
  4     35 days to approximately 5  months of age, when clinical illness became,apparent.  The NO2
  5     was then administered intermittently, based on the clinical signs, to permit survival  for at
  6     least 2 years. The control rat was approximately the same age as the exposed one.  The
  7     lungs were fixed via the trachea at a constant pressure in a distended condition.  The lungs
  8     were examined by light and scanning electron microscopy.  The NO2-exposed rat had
  9     distended alveoli and evidence of alveolar wall destruction. Both dilated airspaces and
 10     evidence of alveolar wall destruction were reported in the NO2-exposed mice lungs. While
 11     alveolar wall destruction was demonstrated, the small number of animals studied severely
 12     limits the value of this study.
 13          Hyde et al.  (1978) studied beagle dogs that had been exposed 16 h daily for 68 months
 14     to either filtered air or to 1,210 j«g/m3 (0.6 ppm) NO2 with 310 jtg/m3.(046 ppm)  NO or to
 15     270 jtg/ni3 (0.14 ppm) NO2 with  2,050 )«g/m3 (1.1 ppm) NO.  The dogs then breathed clean
 16     air during a 32- to 36-month postexposure period.  The right lungs were fixed via the  trachea
 17     at 30 cm fixative pressure in a distended state.  Semiautomated image analysis was used for
 18     morphometry of air spaces.  The dogs exposed to 1,210 jwg/m3 NO2 with 310 jug/m3 NO had
 19     statistically significantly larger lungs with enlarged airspaces and evidence of destruction of
 20     alveolar waUs.  These effects were not observed in dogs exposed to 270 /xg/m3 NO2 with
 21      2,050 /ig/m3 NO, implying a significant role of the NO2 in the production of the lesions.
 22     The lesions  in dogs exposed to the higher NO2 concentration meet the criteria of the 1985
 23      NHLBI workshop for emphysema of the type seen in human lungs.
 24          Lam et al. (1983) exposed 3- and 21-day-old hamsters to 56,400 to 65,800  /ig/m3
 25      (30 to 35 ppm) NO2, 23 hours/day for 7 days.  The hamsters  then breathed room air until
 26      they were 1-year-old when they were killed and examined. The lungs were fixed via the
 27      trachea at 25 cm of fixative pressure and fixed lung volumes were determined by
28      displacement. Appropriate stereologic methods were used to determine the mean linear
29      intercept and internal surface area.  The authors did not report emphysema or evidence of
30      alveolar destruction nor was  emphysema demonstrated in the published micrographs of
31      exposure-related lesions.  The group exposed  starting at 3 days old, but not those exposed

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1      starting at 21 days old, had longer mean linear intercepts indicating larger alveoli.  While
2      they indicated that these changes were "compatible" with "early emphysema" they did not
y     specifically conclude that the hamsters had emphysema.  This conclusion is appropriate as no
4   •'  evidence of emphysema of the type seen in human lungs was presented.
5     '      Kleinerman et al. (1985b) describe the effects following exposure of hamsters to
6      56,400 M/m3  (30 ppm) NO2, 22 hours/day for 12 months.  The'authors did not describe the
7    .  methodology used in detail, for example,  the methods of lung fixation and of morphometry.
8      Mean linear intercept was statistically significantly longer in the exposed hamsters  indicating
9      larger alveoli.  However, whether there was alveolar destruction was not indicated.  The
10     authors conclude that "a small definable degree of emphysema developed" using the  1985
11      National Institutes of Health workgroup's definition of emphysema in animal models.
12     Evidence of emphysema of the type seen  in humans was not presented.
13          Stavert et al. (1986)  exposed rats  which had received a single intratracheal instillation of
14     sterile normal saline to either 65,800 /*g/m3 (35 ppm) NO2 6 h/day or to filtered air for
15     25 days.  The rats were held an additional 10 weeks and then examined.  The lungs were
16     fixed by intratracheal formalin at 25 cm water pressure and morphometry performed
17     according  to standard, acceptable techniques.  They reported that microscopically, the lungs
18     from these two groups appeared identical.  The mean linear intercepts of these two groups
19   '  were nearly identical indicating similar-sized alveoli. They did not report alveolar wall
20      destruction nor is it evident in their published micrographs. They concluded that  regimen
21      "does not bring about irreversible changes in the lungs of rats which are consistent with either
22      centrilobular or panlobular emphysema."  This conclusion is appropriate.
23           Glasgow et al. (1987) exposed rats to 56,400 /*g/m3  (30 ppm) NO2, 24 h/day for up to
24     140 days. The primary objective of this study was to evaluate neutrophil recruitment and
25     degranulation from NO2-induced emphysema.  The lungs were fixed via the trachea in a
 26     appropriate manner and mean linear intercept was determined by semiautomatic image
 27     analysis which was compared to manual methods. Exposed rats had statistically longer mean
 28     linear intercepts,  indicating larger alveoli and reported alveolar wall destruction.  The authors
 29     concluded that the exposed rats had emphysema based on  the 1985 National Institutes of
 30     Health's workgroup definition of emphysema in animal models. However, it appears that
 31     they- either pooled data from several ages of control rats or terminated all of the control rats
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   1     at or before the day the exposures were started.  Thus, controls may have been younger than
   2     the exposed rats.  In addition, there may have been differences in the methods by which the ;
   3     lung tissues were processed; exposed and control rats may not have been processed at the
   4     same time.  For example, in their Figure 1 (the example of alveolar wall destruction), the  -.-
   5     alveoli in the control lung do not appear to be as fully distended, as the alveolar walls are
   6     less straight than those of the lungs from the exposed rat.  The figure legend does not
   7     indicate the ages  of either the control or exposed rat. While these problems may not ;
   8     influence the investigators' main objectives, they are troublesome with respect to the presence
   9     or absence of emphysema.  The results are inconclusive with respect to the production of
 10     emphysema of the type seen in human lungs.                               .
 11           Blank et al. (1978) used NO2 to produce an animal model of emphysema based on the
 12      1985 National Institutes of Health's workgroup definition.  The objective of this study was to
 13      determine the effect of beta-aminopropionitrile, which inhibits cross-linking of collagen and
 14      elastin, on that animal model. They exposed rats to 56,400 jtg/m3 (30 ppm) NO2, 24 h/day
 15      for up to 8 weeks.  The lungs were fixed by appropriate methods, and the mean linear
 16      intercept was determined by semiautomatic image analysis. Control and exposed rats fed the
 17      usual rat chow were terminated at the same time and age. They report longer mean linear
 18      intercepts in the exposed rats, indicating larger alveoli.  They mention that the lungs were
 19      examined for alveolar wall destruction, but do not clearly indicate whether destruction was
 20     present.  They properly refer to the NO2-induced lesions as "emphysema-like" rather than
 21     emphysema of the type seen in human lungs.
 22          Lafuma et al. (1987) studied the effect of 3,760 /*g/m3 (2 ppm) NO2,  8 h/day,
 23     5 days/week for 8 weeks on control hamsters and on hamsters with elastase-induced
 24     emphysema.  The lungs were fixed via the trachea at a constant pressure.  Relative alveolar
 25     sizes were estimated using standard stereological methods. They found  statistically
 26     significantly larger lung volumes and mean linear intercepts and smaller internal surface
 27     areas, indicating larger alveoli in hamsters exposed to NO2 alone. They did not state whether
28     alveolar wall destruction  was or was not found. There is no evidence of alveolar wall
29     destruction in the published micrographs of lungs from that group of hamsters. There is no
30     evidence from this publication that NO2 alone produced emphysema of the type seen in
       August 1991               '             13-116      DRAFT-DO NOT QUOTE OR CITE

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 1      human lungs.  Table 13-14 provides an overview of the results of the studies discussed in this
 2      section.
 3      •  •           •  •             -         ,    •     •     "  '                         '
 4      Summary
 5    •;<:,     The anatomic region most sensitive to NO2 and within which injury is generally first
 6      noted is the area that encompasses the terminal conducting airways and adjacent alveolar
 7      ducts and alveoli. Within this region, those cells most sensitive to NO2-induced injury are
 8      the ciliated .cells of the bronchiolar epithelium and the Type 1 cells of the alveolar epithelium.
 9      Hypertrophy of Type 1 cells, followed by proliferation of and replacement  by Type 2 cells, is
10      the general lesion in the alveoli, while hypertrophy and hyperplasia of ciliated cells  occurs in
11      the bronchioli,  Such  effects are found with acute exposures to levels > 9,400 ^g/m3 (5 ppm)
12     NO2.  Chronic exposure results in similar changes, but in this case effective levels may be
                                                                                o
13       < 1,880 jug/m3 (1 ppm); in general, repeated exposures  to levels > 3,760 /xg/m   (2 ppm) are
14     needed for extensive and permanent morphologic changes. Both exposure concentration and
15     duration are important, with concentration perhaps  playing a more significant role.  Age may
16     also be a factor affecting response, with neonates being more resistant to the morphological
17     effects of NO2, and, responsiveness increasing with age until weaning.  Mature animals show
18     somewhat of a decline in responsiveness with age,  until  an increase occurs  at some  point in
19     senescence:.
20           Several groups of investigators who experimentally exposed different  species of
21     laboratory animals to NO2 have reported emphysema of the type seen in human lungs as
22      defined by the 1985 NHLBI workgroup. In human lungs, the workgroup defined emphysema
23      as "a condition of the lung characterized by abnormal, permanent enlargement of airspaces
24      distal to. the terminal bronchiole, accompanied by destruction of their walls, and without
25      obvious fibrosis".  Studies in this group include those by Haydon et al. (1967), Freeman
26      et al. (1972), and Hyde  et al. (1978).  Results of studies by several additional groups of
27      investigators are inconclusive because although they demonstrated enlargement of airspaces,
28      they did not document or report whether or not destruction of alveolar walls occurred. The
29      latter group includes several studies testing specific hypotheses of the pathogenesis  of
 30      emphysema which, as appropriate to their  research objectives, used  the 1985 NHLBI
 31      workgroup's definition of emphysema in experimental animals. That definition requires
        August 1991
13-117     DRAFT-DO NOT QUOTE OR CITE

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13-119    DRAFT-DO NOT QUOTE OR CITE

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   1     airspace enlargement but not alveolar wall destruction. Several studies were considered
   2     inconclusive due to problems with control animals or to insufficient numbers of animals.
   3
   4     13.2.4  Extrapulmonary Effects
   5          Exposure to NO2 produces a wide array of health effects beyond the confines of the
   6     lung.  Although the aggregate data are inconclusive and do not portray any single issue as
   7     paramount, the evidence suggests that NO2 and/or some of it's reactive products penetrate the
   8     lung epithelial and endothelial layers to enter the blood and produce alterations in blood and
   9     various other organs.
 10
 11      13.2.4.1 Body Weight
 12          Traditionally, the measurement of body weight in animal toxicology studies has been
 13      considered a primary and sensitive end point. However, its biological significance and
 14      extrapolation to humans are still generally uninterpretable.  The measurement of body weight
 15      may be useful in examining questions related to differences in species sensitivity, age, and
 16      different exposure scenarios. A compilation of the effects of NO2 on body weight can be
 17     found in Table 13-15.
 18          The most comprehensive study was performed by Wagner et al.  (1965) who exposed
 19     rabbits, guinea pigs, rats, hamsters, and 4 strains of mice to 1,880,  9,400, and 47,000 ^g/m3
 20     (1, 5, and 25 ppm) and dogs to 1,880 and 9,400 /*g/m3 NO2. For all species examined, no
 21     significant differences in body weight were observed after 6,  12, and 18 months of exposure.
 22         The possibility that newborn mice were more sensitive to ambient levels of NO2 than
 23     similarly exposed adults was investigated by Richters et al.  (1987) who exposed  7-day gravid
 24     mice to 470 /*g/m3  (0.25 ppm) NO2.  Examination of the male offspring born during NO2
 25     exposure and exposed for 12 additional  weeks showed less of an increase in body weight after
 26     3 and  12 weeks of exposure than did air-exposed rats.  However, at the 6 week measurement
 27     interval the body weights of air- and NO2 fetally-exposed rats were not different. Neonates
 28     similarly exposed to 564 /tg/m3 (0.3 ppm)  NO2 did not show a reduction in body weight gain
29     at 3 weeks, but did  after 6 weeks of exposure; whereas, adult mice exposed to 320 to
30     1,504 pg/m3 (0.17 to 0.8 ppm) NO2 for periods ranging from 1 to 12 weeks  showed no
31      significant body weight changes.

        August 1991                             13-120      DRAFT-DO NOT QUOTE OR CITE

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 1
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     Kuraitis et al. (1981) also reported that the body weights of newborn mice exposed to
658 Atg/m3 (0.35 ppm) NO2 for 6 weeks were significantly less than age-matched controls.
No decrease in body weight was observed when adult mice were exposed.  Similarly,
Nakajima et al.  (1969) found no effects in adult female mice exposed 30 days to 1,316 or
1,504 jug/m3 (0.7 or 0.8 ppm) NO2.
     Only very high exposure concentrations of NO2 have been found to cause reduced body
weights in rats.  No exposure-related effects were found in rats exposed for 90 days to
94 /ig/m3 (0.05 ppm)  NO2 (Shalamberidze,  1969).  Freeman et al. (1966,  1968c) found no
effects on body weight after lifetime exposure to 1,504 or 3,760 jug/m3 (0.8 or 2.0 ppm)
NO2.  Freeman and Haydon (1964) did observe body weight changes after continuously
exposing rats to 24,000 /ig/m3 (12.5 ppm) NO2.
     Although the data in mice suggest that newborns may be more sensitive to NO2
exposure than adults, other evidence (see Section  13.2.3.4),  including body weight data, do
not support this contention for rats.  In contrast to the effects on body weight observed in
newborn mice, Stevens et al.  (1988) reported that juvenile rats were less sensitive to NO2
than young adult rats. Stevens and coauthors exposed young adult (7-week old)  and juvenile
(1-day old) Fischer-344 rats to 940,  1,880, and 3,760 /ig/m3 (0.5, 1.0, and 2.0 ppm) NO2
for 6 weeks. To  simulate ambient exposure patterns, twice daily the NO2 exposure was
increased to three times the baseline concentration for 1 h. ; The body weight of young adult
rats were less than the control rats after 1 week and significantly less at 3 and 6 weeks when
the baseline exposure concentration was 1,880 or 3,760 Atg/m3. Rats exposed to 940 ^g/m3
NO2  showed a transitory decrease at 3 weeks that was no longer evident at 6 weeks.  Juvenile
rats, exposed within 36 h after birth to the same NO2 concentrations, showed no changes in
body weight after 1,  3, or 6 weeks of exposure.
      Kosmider et al.  (1973) reported a decline in body weight gain in guinea pigs exposed
continuously to 1,880 ^g/m3  (1.0 ppm) NO2 for 6 months.  Mitina (1962) exposed rabbits to
2,400 and 5,680 jug/m3  (1.3 and 3.0 ppm) NO2 for  15 and  17 weeks and found significant
decreases in body weight gain that persisted 5 to 7 weeks after the exposure ended.
        August 1991
                                        13-123      DRAFT-DO NOT QUOTE OR CITE

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   1      13.2.4.2  Hematologic Changes
  2          Alterations of blood constituents as a result of NO2 exposure may be due to a variety of
  3      causes. Direct effects of NO2, formation of NO2" and NO3~, or secondary effects emanating
  4      from other organs such as the lung, liver, heart, Mdneys, and spleen could all result in
  5      alterations of blood content and chemistry. However, the significance of many of these
  6      hematological changes is uncertain.
  7
  8     Effects on Blood Cell Counts and Hemoglobin
  9          Several authors have measured and shown effects on the number of erythrocytes (red
 10     blood cells [RBCs]) and hemoglobin concentration, although the results have been
 11     inconsistent.  A summary of these studies can be found in Table 13-16.  In some of these
 12     studies, leukocytes (white blood cells) and platelet counts were also examined.  Table 13-17
 13     separates these results for easy comparison.
 14          Shalamberidze (1969) exposed rats continuously to 94 j«g/m3  (0.05 ppm) NO2 for
 15     90 days with no change in blood hemoglobin or RBC counts. YaMmchuk and Chelikanov
 16     (1972)  reported that during a 3-month continuous exposure to 600 jug/m3 (0.32 ppm) NO2,
 17     rats showed a significant'increase in the number of leukocytes, and a tendency toward
 18     decreased hemoglobin and RBCs.   However, by the end of exposure, these changes returned
 19     to within the control range.  Plasma cholinesterase (ChE) was measured, but no significant
 20     effects were found.
 21           Fenters et  al. (1973) showed that exposing male squirrel monkeys to 1,880 /-cg/m3
 22     (1 ppm) NO2 continuously for 493 days had no significant effect on hematocrit, hemoglobin,
 23      total protein, globulins, chloride, sodium, calcium, potassium, glucose, blood urea  nitrogen,
 24      glutamicpyruvic transaminase, lactate dehydrogenase, and lactate dehydrogenase isoenzymes.
 25      Challenge with influenza A/PR/8/34 increased the leukocyte number in NO2-exposed animals
 26      above the levels in similarly challenged controls.
 27          Dogs exposed to 1,880 and 9,400 jug/m3 (1 and 5 ppm) NO2 for 18 months showed no
 28      significant change in hemoglobin, hematocrit, white cell count, or serum alkaline phosphatase
29      and magnesium-activated phosphatase activity (Wagner etal., 1965).  Rabbits exposed to
30      2,400 to 5,680 fig/m3 (1.3 to 3.0 ppm) NO2 for 15 and 17 weeks had a decrease in the
31      number of RBCs and a significant increase in the number of leukocytes (Mitina, 1962).

        August  1991                             13-124     DRAFT-DO NOT QUOTE OR CITE

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13-126    DRAFT-DO NOT QUOTE OR CITE

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As discussed in the section on host defenses, a decrease in the phagocytic activity of the
leukocytes was also reported.
     Furiosi et al. (1973) exposed rats to 3,760 ± 1,880 jig/m3 (2+1 ppm) NO2 for
between 12 and 21 months and found polycythemia with reduced mean corpuscular volume,
but normal mean corpuscular hemoglobin concentrations.  Exposure to NO2 also increased
the ratio of polymorphonuclear leukocytes to lymphocytes.  Because exposures occurred while
the rats were in plastic cages inside the exposure chamber, the actual concentration of NO2
would probably be less than the stated concentration.  However, the reported observations are
supported by similar findings in monkeys that were simultaneously exposed in wire cages.
     Kobayashi et al. (1983) reported a reduction in rat platelets after 1, 3, 5, and 7, but not
14 days of continuous exposure to 18,800 jig/m3 (10 ppm) NO2. Because platelet
thromboxane A2 increased on Day 3, corresponding with a decrease in lung PGI2, and the
balance between these two arachidonic acid metabolites is thought to regulate platelet
aggregation, the authors speculate that NO2 may induce increased platelet aggregation.
     Although not a direct measure of RBC content, the number of RBCs in the red pulp of
                                                                                  o
the spleen of mice was increased  by a 6-week, 5-day/week,  8-h/day exposure to 658 jug/nr
(0.35 ppm) NO2 (Kuraitis et al.,  1981).  This finding could be interpreted as  supporting the
polycythemia that was observed in NO2-exposed animals. Spleen weights and the size of
spleen lymphoid nodules were also increased.
     Three studies examined the  production of physiologically inactive hemoglobin
(methemoglobin) that might be produced if NO3" or NO2" reacted with hemoglobin.
Nakajima and Kusumoto (1968) found that the amount of methemoglobin was not increased
when mice exposed to 1,500 pg/m3 (0.8 ppm) NO2 for 5 days.  Oda et al. (1981) also found
no increase in methemoglobin, but NO3" and especially NO2" were elevated in the blood of
mice exposed for 1 h to between  9,400 and 75,200 jug/m3 (5 to 40 ppm)  NO2.  In contrast,
Case et al. (1979) showed that mice exposed to 1,880 to 56,400 jug/m3 NO2 (1 to 30 ppm)
exhibited a concentration-related  increase in methemoglobin and nitrosylhemoglobin and
decreased ferric catalase and iron transferrin activities.
        August 1991
                                        13-127     DRAFT-DO NOT QUOTE OR CITE

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  1     Effects on Red Blood Cell Membranes
  2          Several studies have examined changes in RBC membranes of rats after NO2 exposure
  3     (see Table 13-18).  In a preliminary report, Mersch et al. (1973) showed that RBC
  4     D-2,3-diphosphoglycerate,  a measure of tissue deoxygenation, was increased  in all four
  5     guinea pigs continuously exposed to 677 ^g/m3 (0.36 ppm) NO2 for 1 week.             '
  6          Changes in the contents of RBC membranes were detected after exposure to
  7     7,520 jig/m3 (4 ppm) NO2 (Kaya et al., 1980).  Increased amounts of sialic acid were noted
  8     in rats exposed between 1 and 10 days to 7,520 ^g/m3 (4 ppm)  NO2.  Increased sialic acid, a
  9     glycosidic residue distributed on the outer surface of the RBC, is found in younger RBCs
 10     (Durocher et al., 1975), suggesting that NO2 inhalation may have stimulated renewal of the
 11     RBC population (increased population of immature cells).  An increased amount of
 12     lysophosphatidyl-ethanolamine, known to increase cell membrane fragility, was  found on
 13     Days 5, 7, and 10 after exposure to 7,520 /-tg/m3 and after. 1, 5, and 7 days of exposure to
 14     18,800 jig/m3 (10 ppm) NO2.  Protein content of RGBs was slightly decreased at
 15     18,800 jig/m3 after 1, 5, and 7 days of exposure, but not after 3 days.
 16          The possibility of a younger circulating RBC population was investigated by Kunimoto
 17     et al. (1984a) who showed  that after 1 and 4 days exposure to 7,520 /ng/m3 NO2, the activity
 18     and content of Na+, K+ -ATPase, and the amount of sialic acid were increased in RBC
 19     membranes.  These changes have previously been associated with a younger population of
 20     RBCs (Cohen etal., 1976).
 21          In contrast, Mochitate and Miura (1984)  found that after 7  days of continuous exposure
 22     to 7,520 jug/m3 (4 ppm) NO2, there was a decreased population  of younger RBCs.
 23      However, the activity of two glycolytic enzymes (pyruvate Mnase and phosphofructokinase)
 24     was elevated in NO2-exposed animals on Days 5 and 7, but returned to control levels on
 25      Day 10.  The authors concluded that there was not a corresponding reduction  in the activity
 26      of the glycolytic pathway with the NO2-induced increase in the apparent aging of the RBCs.
 27          Kaya and Miura (1982) investigated the effects of NO2 on fatty acids in  RBCs, sera,
28      and liver.  They found that in RBC membranes a net increase in  unsaturated fatty acids
29      (predominately arachidonic  acid) occurred after exposure to 7,520 Atg/m3 (4 ppm) NO2 for.
30      10 days.
       August 1991                             13_128      DRAFT-DO NOT QUOTE OR CITE

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13-129     DRAFT-DO NOT QUOTE OR CITE

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  1      Effects on Serum and Plasma
  2           In the previous section, Kaya and Miura (1982) showed that arachidonic acid was
  3      increased in the RBC membrane after exposure to NO2.  Since de novo synthesis of fatty
  4      acids is not possible in the mature RBC, composition changes in the membrane usually reflect
  5      exchange with serum fatty acids, probably originating in the liver.  In fact, Kaya and Miura
  6      (1982) found that after exposure to 7,520 ^g/m3 (4 ppm) NO2 for 10 days, arachidonic acid
  7      was elevated in both the serum and in liver homogenates.  However, exposure to
  8      18,800 jtg/m3 (10 ppm) NO2 for 7 days increased RBC and serum arachidonic acid, but liver
  9      concentrations were decreased.  The authors suggested that the rat cannot completely
 10      overcome the consequences of a 18,800-^g/m3 NO2 exposure, but could metabolically
 11      compensate for the effects of exposure to 7,520 ^tg/m3 NO2.
 12          Menzel et al. (1977) demonstrated that acute effects do not necessarily predict chronic
 13     injury by contrasting the serum changes of guinea pigs after short-term (7 day) and long-term
 14      (4 months)  continuous exposure to 940 ^g/m3 (0.5 ppm) NO2.  Plasma eholinesterase was
 15     significantly elevated after a 7-day exposure but was significantly decreased compared to
 16     control values with a long-term exposure (4 months).  This depression in ChE suggests an
 17     hepatic lesion (Moore etal., 1957).  A statistically significant depression in RBC GSH
 18     peroxidase activity was also initially observed, but the effect did not persist after 4 months of
 19     exposure.  Similarly, several indices of nonspecific tissue damage (serum CPK, lactate
 20     dehydrogenase,  SCOT, and SGPT) were also increased after 7 days, but were not altered on
 21      long-term exposure.
 22           The following studies all indicate a general decrease in serum proteins and lipoproteins
 23      and an increase in serum globulins,  thus suggesting hepatic damage.  Drozdz et al. (1976)
 24      reported decreased serum total protein, albumin, and seromucoid concentrations in guinea
 25      pigs exposed to  2,000 ^g/m3 (1.05 ppm) NO2, 8 h/day for  180 days. However, serum levels
 26      of ar and j3-globulins were increased.  These authors also found that serum alanine and
 27      aspartate aminotransferase activity was increased in the mitochondrial fraction but decreased
28      in the cytoplasmic fraction.  In agreement with the Menzel et al. (1977) subchronic data,
29      Drozdz et al. (1976) also observed decreased plasma ChE levels.  However, the meaning of
30      cytoplasmic and mitochondrial fraction of serum is not clear from the translation of the
31      article.

        August 1991               '        .    13-130     DRAFT-DO  NOT QUOTE OR CITE


-------
 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
     Kosmider et al. (1973) reported a general decrease in protein synthesis evidenced by
                                                                          /j
decreased serum proteins in guinea pigs after continuous exposure to 1,880 /xg/m  (1 ppm)
NO2 for 6 months.  Following exposure to 1,000 /xg/m3 NOX (mainly NO2, —0.5 ppm),
8 h/day for 120 days, Kosmider (1975) reported decreased serum cholesterol, total lipids,
/J and gamma lipoproteins and sodium, and increased serum a-lipoproteins in guinea pigs.
Similarly, Mitina  (1962), after exposing  rabbits to 2,400 to 5,680 /ig/m3 (1.3 to 3.0 ppm)
NO2 for 15 and 17 weeks, found reduced amounts of albumin, but increased serum  globulins.
Table 13-19 summaries the data on NO2-induced changes in serum proteins and clinical
chemistries.

13.2.4.3 Cardiovascular Effects
     Few papers have reported the effects of NO2 exposure on the heart.  Potential  changes
in hemoglobin and RBCs as well as lung edema could reduce oxygen uptake and affect
cardiovascular performance. Because many of the NO2-induced cardiovascular effects are
secondary to pulmonary edema or stimulation of sensory receptors in the respiratory tract,
some of the studies addressing effects on the cardiovascular system are addressed in  the
discussion on pulmonary function (see Section 13.2.3.3).
     Suzuki et al. (1981) exposed rats for up to 3 months to between 752 and 7,520 jig/m3
                                                               <3
(0.4 and 4.0 ppm) NO2. After 3 months of exposure to 7,520 /^g/m  NO2, anesthetized rats,
artificially ventilated at high frequencies, had  a significant reduction in PaO2.  A reduction in
heart rate was reported in unanesthetized mice exposed to 2,250 and 7,520 jug/m  (1.2 and
4.0 ppm) NO2 for 1 month (Suzuki et al., 1984).
     Tsubone et al. (1982) reported that rats developed bradycardia that progressed to
significant electrocardiographio (ECG) abnormalities including atrioventricular blocks,
                                                                           *2
premature beats, and wandering pacemakers at a 30 min exposure to 37,600 jitg/m
(20.0 ppm) NO2.  Intravenously administered blood NO2" and NO3" did not affect the ECG,
while injection of atropine sulfate during NO2 exposure immediately reversed the
bradycardia, suggesting that NO2 alters parasympathetic activity. Eleetrocardiographic effects
were not observed after exposure to 18,800 /ig/m3  (10.0 ppm) NO2.  Comparisons between
tracheostomized and nontracheostomized rats suggested that irritation to the upper airways
(trigeminal nerve)  was not involved in the cardiovascular response (Tsubone et al., 1984).
August 1991
                                                13-131
DRAFT-DO NOT QUOTE OR CITE

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August 1991
13-132    DRAFT-DO NOT QUOTE OR CITE

-------
 1     In a follow-up study, the cardiovascular effects of phenyl guanide were enhanced by
 2     preexposure to 18,800 jwg/m3 NO2 for 24 h, but not by 7,520 jiig/m3 (4.0 ppm) for 1 week
 3     or 752 jug/m3 (0.4 ppm) for 4 weeks (Tsubone and Suzuki, 1984).
 4          Conscious sheep exppsed to 28,200 /ng/m3 (15 ppm) for 4 h showed no significant
 5     changes in arterial blood gases, pulmonary vascular pressure or resistance,  cardiac output, or
 6     pulmonary and capillary blood volumes (Abraham et al.,  1980).  This is in contrast to the
 7     findings of Dowell et al. (1971) who showed decreased cardiac output, blood pressure, PaO2,
 8     and pH in dogs after a 1-h exposure to between 13,160 and 30,080 jwg/m3  (7 and 16 ppm)
 9     NO2.  However, exposures in the Dowell et al. (1971) study were delivered via an
10     endotracheal tube in anesthetized dogs, thereby bypassing any scrubbing effects of the upper
11     airways.
12
13     13.2.4.4  Hepatic Function
14          As previously described (Section 13.2.4.2), changes in serum chemistries suggest that
15     NO2 exposure may affect the liver.  Several studies have examined hepatic function either
16     directly or by indirect means. These studies are cataloged in Table  13-20.
17          One important function of the liver is detoxification of xenobiotic compounds.
18     Measurement of the duration of barbiturate-induced sleeping time has been used as an indirect
19     measurement of hepatic mixed function oxidase activity, the enzymes responsible for
20     xenobiotic metabolism.  Nitrogen dioxide has been shown to increase pentobarbital-induced
21     sleeping times in mice (Miller et al., 1980; Graham et al., 1982).  The effect was observed
22     in female mice but not in males, occurred only at specified time intervals after exposure, and
23     usually did not persist beyond 1 day.  However, the effects reliably occurred after a 3-h
24     exposure to concentrations as low as 470  /xg/m3 (0.25  ppm) NO2. .No significant effects
25     were detected after 1 or 2 days exposure to 235 jLcg/m3 (0.125 ppm).  In an attempt to
26     examine the mechanism of this response,  the level of hepatic cytochrome P-450 and the
27     activities of aminopyrine N-demethylase,  p-nitroanisole O-demethylase, and aniline
                                                                  .               «2
28     hydroxylase were measured  in the livers of mice exposed for 3 h to 9,400 jwg/m (5.0 ppm)
29     NO2; however, no NO2-related effects were found (Graham et al., 1982).
30          Increased hexobarbital-induced sleeping times were also reported in the progeny of
31     maternally exposed rats (Tabacova et al., 1985).  This effect was measured in the offspring
        August 1991
13-133     DRAFT-DO NOT QUOTE OR CITE

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DRAFT-DO NOT QUOTE OR CITE

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    1      on Days 7, 14, and 21 postexposure (10,000 ^g/m3 [5.3 ppm] NO2).  Additionally, lipid
   2      peroxides increased and O2 consumption decreased in liver homogenates.  Cytochrome P-450
   3      content and aminopyrine-N-demethylase activity were decreased on postnatal Day 30. In the
   4      animals exposed to 1,000 /*g/m3 (0.5 ppm) NO2, increased hexobarbital-induced sleeping
   5      times occurred on Days 7 and 21, but not on Day 14.  Liver lipid peroxides were increased
   6      on postnatal Day 30 in this exposure group.  No exposure monitoring method was cited and
   7      the details of the biological methods used were not available.
   8          Components of the rat microsomal electron-transport system, especially cytochrome
   9     P-450, were generally decreased after continuous exposure to 752 to 7,520 ^g/m3 (0.4 to
  10     4.0 ppm) N02 during the first 12 weeks of exposure, but with continued exposure the levels  ,
  11      returned to control values (Takahashi et al., 1986). Takano and Miyazaki (1984) exposed
  12     rats to 7,520 jig/m3 NO2 for 1,  14, or 30 days.  After 14 days, cytochrome  P-450 levels  and
  13      aminopyrine N-demethylase activity were increased, while aniline hydroxylase activity was
  14      decreased.  Aminopyrine N-demethylase activity was also increased at 30 days, but none of
  15      the other measures were affected at Day 1 or 30.
  16          Drozdz et al. (1976) found decreased total liver protein and sialic acid,  but increased
  17      protein-bound hexoses in guinea  pigs exposed  to 2,000 /.g/m3 (1.05 ppm) NO2, 8 h/day for
  18      180 days.  Liver alanine and aspartate aminotransferase activity was increased in the
 19      initochondiial fraction.   In contrast to the effect seen in the cytoplasmic fraction of the
 20      serum, aspartate aminotransferase activity was decreased in the cytoplasmic fraction of the
 21     liver. Electron micrographs of the liver  showed intracellular edema and inflammatory and
 22     parenchymal degenerative changes.
 23          Kosmider (1975) reported liver magnesium and zinc .stores were depleted in guinea pigs
 24     following exposure to 1,000 ^g/m3 NOX  (mainly NO2,  -0.5 ppm), 8 h/day for 120  days.
 25     Swollen liver mitochondria were also  observed. Significant reductions of succinate-
 26     cytochrome C reductase activity in rat liver homogenates were found during the third  and
 27     fifth day of an 18,800 /.g/m3 (10.0 ppm) NO2  exposure, but not during the first and seventh
28     day, or with exposure to 7,520 ^g/m3 (4.0 ppm) NO2 (Mochitate et al., 1984).  Significant
29     decreases in cytochrome P-450 from liver microsomes were also found after 7 days of
30     exposure to 752 or 7,520 jig/m3 (0.4 or 4.0 ppm) NO2, but not after exposure to
       August 1991                             13_136     DRAFT-DO NOT QUOTE OR CITE

-------
 1
 2
 3
 4
 5
 6 ...
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20   .
21
22
23
24
25
26
27
28
29
30
31
 2,256 /ig/m3 (1.2 ppm) NO2.  NADPH-cytochrome C reductase was reduced with 5 days of
 exposure to 7,520 and 18,800 /ig/m3 NO2.

 13.2.4.5 Effects on the Kidney and on Urine Content
      The direct effects of NO2 exposure on the kidney and spleen have been described, and
 several studies have explored the composition of urine during and after exposure.  These
 studies are summarized in Table 13-21 and discussed below.                    •
      Takahashi et al. (1986) found that continuous exposure to 2,256 and 9,400 pcg/m3
 (1.2 and 4.0 ppm) NO2 increased the amount of cytochrome P-450 and cytochrdme b5 in the
 kidney after 8 weeks of exposure. Continued exposure for 12 weeks resulted in less
• .substantial increases in the amount and activity of the microsomal electron-transport enzymes.
 This is in contrast to the decreased activity these authors reported for the liver discussed in
 Section 13.2.4.4 on hepatic effects.                            .     ,
      YaMmchuk and Chelikanov (1972) reported that during a 3-month continuous exposure
 to 600 jug/m3 (0.32 ppm) NO2,  rats showed a significant increase in the urinary excretion of
 coproporphyrins.  Kosmider (1975) also reported increased levels of urinary coproporphyrin
 in guinea pigs exposed to 1,000 /ug/m3 NOX (mainly NO2, -0.5 ppm), 8 h/day for
 120 days;  Increased coproporphyrins can indicate increased heme synthesis which might
 occur if an increased number of RBCs were synthesized.  As discussed in Section 13.2.4.2,
 NO2 exposure has been reported to cause polycythemia and an increase in the number of
 RBCs in the red pulp of the spleen.
      Increases in urinary protein and specific gravity of the urine were reported by Sherwin
 and Layfield (1974) in guinea pigs exposed continuously to 940 /*g/m3 (0.5 ppm) NO2 for
 14 days. Proteinuria was detected in another group of animals when the  exposure was
 reduced to 750 jwg/m3 (0.4 ppm) NO2 for 4 h/day.  Disc electrophoresis  of the  urinary
 proteins demonstrated the presence of albumin and alpha, beta, and gamma globulins. The
 presence of high molecular weight proteins in urine is characteristic of the nephrotic
 syndrome.  Differences in water consumption or in the histology of the kidney were not
 found.
      In a more comprehensive study  of the relationship between inhaled  NO2 and urinary
 NO2~ and NO3", Saul  and Archer (1983) exposed  rats for 24 h to 2,256 to 16,544 /*g/m3
        August 1991
                                         13-137     DRAFT-DO NOT QUOTE OR CITE

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August 1991
                              13-138     DRAFT-DO NOT QUOTE OR CITE

-------
 1     (1.2 to 8.8 ppm) NO2. They demonstrated that mostly NO3~, with very little NO2", was
 2     excreted in the urine.  The small amount of urinary NO2" appeared to be an artifact that
 3     originated from an in vitro reaction with urine. The rate and linearity of the conversion of
 4     NO2 to urinary NO3" suggested that NO2 does not form NO3" by reacting with respiratory
 5     water, but reacts with oxidizable tissue to form NO2".  Nitrite is then further oxidized in the
 6     blood by oxyhemoglobin (Kosaka et al., 1979) to form NO3~ which is excreted in the urine.
 7     Nitrite and NO3" was also found in the urine of guinea pigs exposed to  1,000 jug/m3 NOX
 8     (mainly NO2,  -0.5 ppm), 8 h/day for  120 days (Kosmider, 1975).
 9
10     13.2.4.6 Effects on the Central Nervous System and Behavioral Effects
11          Information regarding the effects of NO2 on development and animal behavior is limited
12     to a few studies (see Table 13-22),  most of which have uncertain relationships to humans.
13     Shalamberidze (1969) exposed rats  to 100 ^g/m3 (0.05 ppm) NO2  for 3 months with no
14     demonstrated effects on the central  nervous system. YaMmchuk and  Chelikanov (1972)
                                                                 i^t
15     reported that during 3 months continuous exposure to  600 jug/m  (0.32  ppm) NO2, rats
16     developed an increased latency of response to conditioned sound and  light stimuli.
17          Exposure of guinea pigs to 1,000 jug/m3 (0.53 ppm) NO2, 8 h/day for  180 days
18     affected brain enzyme activity levels (Drozdz et al., 1975).  Decreased  activity in brain
19     protein metabolism enzymes were seen in brain malate dehydrogenase,  alanine
20     aminotransferase, sorbitol dehydrogenase, lactate dehydrogenase, adenosine triphosphatase,
21     5'-nucleotidase, and asparagine aminotransferase.  Increases in brain  glycolytic enzyme
22     activity were seen in 1,6-diphosphofructose aldolase, isocitrate dehydrogenase, alpha-
23     hydroxybutyrate dehydrogenase, phosphocreatine kinase, and cholinesterase.
24           A recent study (Sherwin et al., 1986) indicated that the brain content of serotonin
25     (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA, the primary metabolite of 5-HT) increased
26     in mice exposed to 846 ^g/m3 (0.45 ppm) NO2, 7 h/day for 4 weeks.  The ratio of
27     5-HIAA:5-HT was also increased.  The authors did not speculate as to  what  these
28     observations mean; however, they noted that increased turnover, as reflected in the increased
29     ratio of 5-HIAA: 5-HT, have also been observed in trimethyltin and chlordecone exposure.
30           Vyskocil et al. (1985) measured a variety of hormone levels and organ weights after
31     continuous exposure to 6,580 ^g/m3 (3.5 ppm) NO2 for 1 or 2 months. The only significant
        August 1991
13-139
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1      effect reported was a decrease in the hypothalamic concentration of noradrenaline at both
2      exposure durations.
3           As discussed in the section on reproductive, developmental, and heritable mutagenic
4      effects (Section 13.2.4.7), Tabacova et al. (1985) also reported significant postnatal deficits
5      in the onset of normal neuromotor development and reduced open field activity in the
6      progeny of maternally exposed rats 2 months after the dams were exposed to 1,000 or
7      10,000 |ug/m3 (0.5 or 5.3 ppm)  NO2 for 6 h/day, 7 days/week. Postural and gait defects
8      were also reported at 0.1 ^g/m3 (0.05 ppm).  No monitoring method was specified.
9           Tusl et al. (1973)  exposed rats to 9,400 jug/m3 (5  ppm) NO2 for 8 weeks.  The
10      influence of NO2 on forced swimming endurance time was measured.  By the fifth and sixth
11      weeks of exposure, swimming performance had decreased 25 %.  In rats exposed to
12     1,880 /*g/m3  (1 ppm) NO2, performance was maintained with a slight tendency toward
13     deterioration.
14          A concentration-dependent decrease in forced swimming endurance time after a single
15     24-h exposure to between 9,400 to 75,200 /xg/m3 (5  to 40 ppm) NO2 was reported by Suzuki
16     etal. (1982a).  Significant decrements in performance were reported at exposure
17     concentrations as low as 18,000 ^g/m3 (10 ppm).  Recovery from exposure required 5 to
18     6 days, 7 to  8 days,  and over 9 days for the 9,400, 18,800, and  37,600 ^g/m3 (5, 10, and
19     20 ppm) groups, respectively.   In an attempt to examine the mechanism that produced the
20     decrement in performance, it was observed that as forced swimming endurance time
21      decreased, lung edema increased. Furthermore, compared to similarly exercised control rats,
22      blood lactate concentration was increased in rats exposed to 9,400 /xg/m3 (5 ppm)  both
23      immediately and 24  h after exposure.  These two findings suggest that lung edema prevented
24      sufficient O2 from entering the blood during exercise to meet aerobic demands.  See section
25      discussing NO2-induced effects on pulmonary function.
 26
 27     13.2.4.7 Reproductive, Developmental, and Heritable Mutagenic Effects
 28          As summarized in Table  13-23, few studies have examined the effects of NO2 on
 29     reproduction and development  or the heritable mutagenic potential of NO2 in vivo.  Exposure
 30     to  1,800 Mg/m3 (1 ppm) NO2 for 7 h/day, 5 days/week for 21 days resulted in no alterations
         August 1991
13_141     DRAFT-DO NOT QUOTE OR CITE

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13-142    DRAFT-DO NOT QUOTE OR CITE

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 1      in spermatogenesis, germinal cells, or interstitial cells of the testes in 6 rats (Kripke and
 2      Sherwin, 1984). Additionally, the level of vitamin B12, a coenzyme in folate metabolism
 3      that is used for DNA synthesis, was not affected by NO2 exposure. Similarly, breeding
 4      studies by Shalamberidze and Tsereteli  (1971) found that long-term NO2 exposure had no
 5      effect on fertility.  However, there was a statistically significant decrease in litter size and
 6      neonatal weight when male and female rats exposed to 2,360 jtig/m3 (1.3 ppm) NO2,
 7      12 h/day for 3 months were bred.  In utero death due to NO2 exposure resulted in smaller
 8      litter sizes, but no direct teratogenic effects were observed in the offspring.  In fact, after
 9      several  weeks, NO2-exposed litters approached weights similar to controls.
10          In the only study that has examined post-natal development, a significant delay in eye
11      opening and incisor eruption was observed in the progeny of maternally exposed Wistar rats
12     (Tabacova et al., 1985). The dams were exposed to 50,  100, 1,000, or  10,000 ^g/m3  (0.03,
13     0.05, 0.5, or  5.3 ppm) NO2 for 6 h/day, 7 days/week throughout gestation and the offspring
14     were studied for 2 months postexposure. Significant effects were detected in the offspring of
15     dams exposed to 1,000 and  10,000 jug/m3 NO2. There were also concentration-related
16     increases  in neurobehavioral development reported in the offspring of the maternally exposed
17     animals.  These findings are discussed  in the section on NO2 effects on the central nervous
18     system  and behavior (Section 13.2.4.6).  The method of monitoring was not reported.
19           Balabaeva and Tabacova (1985) exposed pregnant and nonpregnant albino rats to  1,000
20     or 10,000 jug/m3 (0.5 or 5.3 ppm) NO2, 5 h/day for 21 days and examined lipid peroxidation
21     in lung, liver, and placenta.  Nonpregnant rats  had greater lipid peroxidation in  the liver than
22     in the lung, while the opposite was true in pregnant rats. Even more surprising was a four-
23     fold increase  in lipid peroxidation in the placenta of 10,000 jug/m3 NO2-exposed rats
24     compared to unexposed pregnant controls.  The authors then examined the offspring of the
25     pregnant  rats. The 1-month-old F1 nonpregnant rats, exposed to air or the same
26      concentrations of NO2, showed similar changes as was observed in their mothers.  However,
27      pregnant Fx rats, exposed to 1,000 or  10,000 fig/m3, had a 9- or 17-fold increase in placental
 28      lipid peroxides, respectively.  The authors report that increased placental formation of toxic
 29      lipid peroxides in the F1 rats could be due to decreased blood glutathione (no measurements
 30      presented) and that such levels of lipid peroxides could be fetotoxic.  However, the method
 31      of monitoring NO2 and lipid peroxides was not reported, nor were the statistical methods.
        August 1991
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  1           Potential mutagenic effects were investigated by Gooch et al. (1977) who reported that
  2      exposure to 188, 1,880, 9,400, and 18,800 ^g/m3 (0.1, 1.0, 5.0, and 10.0 ppm) NO2 for
  3      6 h did not increase either chromatid- or chromosome-aberrations in the leukocytes of C3H
  4      mice. Blood samples were obtained immediately after exposure and 1 and 2 weeks
  5      postexposure.  Similarly, no increase in the number of translations in primary
  6      spermatocytes was detected. Therefore, in these experiments the authors concluded that N02
  7      exposure did not induce mutagenesis.                                               '   ;
  8                                                                '            -..         S'V
  9      13.2.4.8 Potential Carcinogenic or Co-Carcinogenic Effects                         ^
 10           No direct evidence indicates that tumors may be produced by NO2 exposure alone.
 11       Several studies have evaluated the issue of carcinogenesis and co-carcinogenesis, but results
 12      are often unclear or conflicting.  Insofar as we are aware, there are no published reports on
 13       studies using classical carcinogenesis whole animal bioassays.  An excellent critical review
 14       and discussion of some of the important theoretical issues in interpreting these types of
 15       studies has recently been published (Witschi, 1988).  Table 13-24  summarizes the available
 16      information on the carcinogenic or co-carcinogenic potential of NO2.
 17
 18      Studies of Hyperplasia and Enhanced Retrovirus Expression
 19           Hyperplasia of the lung epithelium, although a common response to lung injury, could
 20      be construed as suggesting a potential carcinogenic or co-carcinogenic effect of NO2.
 21      However, the relatively frequent reports of hyperplasia,  as discussed in the section on
 22      morphological  effects, did not report the observation of any tumors. It should be noted  that
 23      these studies were not designed to detect tumors, so it is not surprising that none were found.
 24      Nakajima et al. (1972) found hyperplastic foci due to proliferation of epithelial cells of the
 25      terminal bronchioles and alveoli in mice exposed to 940 to 1,504 ^g/m3 (0.5 to 0.8 ppm) '•-
 26      NO2 for 30 days. The authors reported that these lesions were completely identical to early
 27      changes  that appeared in the development of pulmonary adenomas  after administration of
 28      known carcinogenic chemicals such as INH, urethane, and 4-nitroquinoline-l-oxide.
29      However, no adenomas were detected.
30          Rejthar and Rejthar (1975) exposed rats to 9,400 ^g/m3 (5 ppm) NO2 continuously for
31      periods of 3, 5, 7, 9, and 11 weeks. After 3 weeks of exposure, the bronchioles contained

        August 1991                             13-144      DRAFT-DO NOT QUOTE OR CITE

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 1     uniform cuboidal one-layer epithelium composed of nonciliated cells.  The cells showed
 2     vacuolization, and hyperplastic foci appeared in the bronchiolar epithelium. The foci
 3     were 2- to 4- layer pyramidal formations.  By 5 weeks, extensive hyperplasia composed of
 4     three to four layers of epithelial cells was apparent.  Centers of cuboidal metaplasia were
 5     found in adjacent alveoli.  By 7 weeks, hyperplasia was apparent in all bronchioles, thereby
 6     narrowing the bronchiolar lumen. The polymorphous epithelium was extensive with a few
 7     ciliated cells in hyperplastic areas.  After 9 weeks, the terminal bronchiolar epithelium
 8     generally showed two or three irregular layers. The number of ciliated cells increased, but
 9     cilia were often located atypically in intercellular spaces.  A return to a single layer of
10     epithelium without cilia was observed after 11 weeks.  Seven weeks after exposure to NO2,
11     the lungs appeared to be in a state of repair moving towards reversal of the lesions.
12          The possibility that NO2 may facilitate the production of tumors has been suggested and
13     examined by several authors.  Endogenous retrovirus expression was enhanced in the spleen
14     of low expressor Swiss Webster mice after exposure to 1,504 jttg/m3 (0.8 ppm) NO2,
15     8 h/day, 5 days/week for  1 or 18 weeks (Roy-Burman et al., 1982).  However,
16     measurements taken at intermediate time points were not different from controls.  High
17     expressor AKR mice also  showed an increase in the concentration of virus-specific RNA in
18     the spleen after 8, 12, and 15 weeks of exposure to 564 ^g/m3 (0.3 ppm) NO2. The authors
19     suggest that such data may indicate inappropriate or inordinate expression  of genes that could
20     potentially influence genetically controlled diseases, such as cancer.
21
22      Studies With NO2 Plus Known Carcinogens
23           Ide and Otsu (1973) showed tumor production in conventional mice receiving 5 weekly
24      injections of 0.25 mg of 4-nitroquinoline-l-oxide (4NQO, a lung tumor-specific carcinogen)
25      during exposure from birth to somewhere between 9,400 and  18,800 jug/m3 (5 and 10 ppm)
26      NO2 for 2 h/day, 5  days/week for 50 weeks. There was no difference in the number of
27      tumors produced by 4NQO alone (6 of 10) and the number produced in combination with
28      NO2 (6  of  13). Mice exposed to NO2 alone had a similar number of tumors as the air
29      controls. Thus, NO2 did not facilitate the production  of tumors.
J30          One of the goals of a study by Benemanskii et al. (1981) was to evaluate the potential
31      of NO2 to influence the production of tumors' during co-exposure to a known carcinogen,
        August 1991
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   1
   2
   3
   4
   5
   6
   7
   8
   9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
 20
 21
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 23
 24
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 29
 30
31
  nitrosodimethylamine (NDMA) or its precursor dimethylamine (DMA).  No excess tumors
  were observed in rats during a continuous lifetime exposure to the combination 0.07 mg/m3
  DMA + 2,000 Atg/m3 (1.1 ppm) NO2. This suggested that NO2 did not convert DMA to
  NDMA, which alone was shown to produce tumors. However, when the rats were exposed
  to NDMA at a concentration that alone did not produce tumors (0.06 mg/m3), an excess of
  tumors, especially in males, was observed when DMA  (0.05 mg/m3) plus NQ2
  (3,000 Atg/m3; 1.6 ppm) was added to the exposure. Appropriate statistical techniques and
  control groups were not incorporated into the design, and the methods of exposure and
  monitoring of NO2 were not reported,  making the study difficult to evaluate.

 Facilitation ofMetastases
      Richters and Kuraitis (1981) performed two experiments in which mice were exposed to
 either 720 or 1,504 /*g/m3 (0.4 or 0.8 ppm) NO2 for 8 h/day, 5 days/week for 10 or
 12 weeks, respectively. After exposures were terminated, the mice were injected
 intravenously with a cultured-derived melanoma cell line (B16).  The first experiment
 suggested that there was an increased tumor yield if they were counted at 21 days
 postinjection; however, they did not observe a significant interaction or main time effect in
 the analysis of variance. For the second experiment, they chose a 3-week time point to count
 tumors.  The results indicated an increased number of tumors in the NO2 group compared to
 filtered chamber and room air control groups.  The authors concluded that NO2 might
 facilitate the production of tumors; again, these conclusions were based on inappropriate
 statistics.  In more recent experiments, consistent effects have not been observed. For
 example, tumor facilitation was observed when mice were exposed to 564 or 752 ^g/m3
 (0.3 or 0.4 ppm) NO2 for 12 weeks (Richters and Kuraitis, 1983).  However,  at similar
 concentrations 940 pg/m3 (0.5 ppm) for 8 weeks (Richters and Kuraitis,  1983) and
 752 pg/m  (0.4 ppm)  for 12 weeks with intermittent air exposures (Richters and Richters,
 1983) facilitation was not observed.  Richters et al. (1985), attempted to  extend their findings
by showing that, if allowed, the increased metastases from exposure  to 752 jig/m3 (0.4  ppm)
for 12 weeks lead to increased mortality in the mice. However, their post-hoc analysis  of the
data precludes this conclusion.  Furthermore, the actual experimental design used in these
studies probably did not evaluate metastases formation, as the term is generally understood,
       August 1991
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 1
 2
 3
 4
 5
 6
 1
 8
 9
10
11
12
13
14
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16
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 18
 19
20
 21
 22
 23
 24
 25
 26
 27
 28:
 29 .••:
 30
 31
 but more correctly, colonization of the lung by tumor cells. Studies in a true tumor
 metastases model such as the Lobin Wistar rat, should be performed.
•--. Recently, an abstract by Weinbaum et al. (1987) indicated that NO2 could inhibit
 metastases formation if exposure occurred after injection of the the B16F10 tumor cell
 suspension.  Thus suggests caution for the interpretation of NO2 as a facilitator because NO2
 could inhibit metastases as well as facilitate their colonization or cause no change.

 Studies in Animals With Spontaneously High Tumor Rates
      A study by Wagner et al. (1965) suggested that NO2 may accelerate the production of
 tumors in CAF^Jax mice (a strain that is genetically susceptible to pulmonary tumors) after
 continuous exposure to 9,400 ^g/m3 (5 ppm) NO2. At the 12-month evaluation, 7 of
  10 mice had tumors in the exposed group compared to 4 out of 10 in the controls. The
 number of tumors per animal was not reported.  At the 14- and 16-month evaluation, no
 differences in tumor production were observed.  A statistical evaluation of the data was  not
 presented.
       The frequency and incidence of spontaneously occurring pulmonary adenomas was
  found to increase in strain A/J mice after exposure to 18,800 ^g/m3 (10 ppm) NO2 for
  6 h/day, 5 days/week for 6 months (Adkins et al., 1986). These small, but  statistically
  significant increases, were only detectable when the control response from nine groups
  (N=400) were pooled. Exposure to 1,880 and 9,400 ^g/m3 (1 and 5 ppm)  NO2 did not
  increase the number of spontaneous adenomas in this in vivo short-term model for predicting
  carcinogenicity,

  Production ofN-Mtroso Compounds
        Because of evidence that NO2 could produce NO2" and NO3' in the blood, and NO2~ is
  known to  react with amines to produce' animal carcinogens (nitrosamines), the possibility that
  NO2  could produce cancer via nitrosoamine formation has been investigated.
        Iqbal et al. (1980) was the first to demonstrate  a linear time-dependent and
  concentration-dependent relationship between the amount of N-nitrosomorpholine (NMOR)
 . found in whole-mouse homogenates after the mice were gavaged with 2 mg of morpholine
  (an exogenous amine that is rapidly nitrosated) and exposed for between 1 and 4 h to 28,200
   August 1991
                                                13-149     DRAFT-DO NOT QUOTE OR CITE

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   1     to 94,000 Atg/m3 (15 to 50 ppm) NO2.  Thus, since NMOR (a nitrosamine) is an animal
   2     carcinogen, these studies are sometimes used to suggest that NO2 exposure could theoretically
   3     react with amines in the body to produce tumors.
   4          An extension  of the previous study was performed (Iqbal et al., 1981) using DMA, an .
   5     amine that is slowly nitrosated to dimethylnitrosamine (DMN).  A concentration-related
   6     increase in biosynthesis of DMN was found to occur at concentrations as low as 190, ^g/m3
   7     (0.1 ppm) NO2; however,  the rate was significantly greater at concentrations above
   8     18,800 pg/m3 (10 ppm) NO2.  Increased length of exposure also increased DMN formation
   9     between 0.5 and 2 h, but synthesis of DMN was less after 3 and 4 h of exposure than after
 10     0.5 h.                              ,
 11          Mirvish et al.  (1981)  concluded that the results of Iqbal et al. (1980) were technically
 12      flawed.  The Iqbal et al. method,- which involved homogenization of the whole frozen mouse,
 13      did not use an adequate stopping solution to prevent in vitro production of nitrosamines.
 14      Mirvish et al.  (1981) could verify the results of these researchers by .eliminating the use of
 15      the stopping solution, but found no in vivo  production of NMOR when in vitro production
 16      was eliminated.  However,  they did find that in vivo exposure .to;NO2 could produce a
 17      nitrosating agent (NSA) that would nitrosate morpholine when morpholine was added
 18      in vitro. Additional experiments showed that NSA was localized in the skin (Mirvish et al.,
 19      1983) and that mouse skin cholesterol was a likely nitrosating agent (Mirvish et al.,  1986).  It
 20     has also been reported that  only very lipid soluble amines, which can penetrate the skin,
 21     would be available to the NSA.  Compounds such as morpholine, which is not lipid-soluble
 22     could only react with NO2 when it was painted directly on the skin (Mirvish et al., 1988).
 23          Iqbal (1984), responding to the  criticisms of Mirvish et  al.  (1981), concluded after the
 24     completion of several control experiments, that in  vitro nitrosation could only account for
 25     between 1 to 2% of the total amount  of NMOR collected using his previous technique (Iqbal
 26     et al., 1980). Several control experiments further suggested that the effects in the original
 27     experiments were due to in  vivo nitrosation.  One  experiment showed that nitrosamine
 28     biosynthesis could be inhibited in vivo with  the addition of sulfamate, ascorbate, or
29     a-tocopherol prior to NO2 exposure.  Another experiment indicated tfiat the rapid half-life of
30     morpholine (48 to 54 min) might explain why significant levels were not found by Mirvish
31      et al. (1981) since they transferred the NO2-exposed  rats to room air for 30 min prior to

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 1      sacrifice.  In vivo nitrosation was also demonstrated by Norkus et al. (1984) after morpholine
 2      administration and a 2 h exposure to 84,600 |i*g/m3 (45 ppm) NO2.
 3           Postiethwait and Mustafa (1983) examined this problem of in vivo production of
 4      nitrosamines using an isolated perfused rat lung.  Rat lungs were ventilated with
 5      37,400 j«g/m3 (19.9 ppm) NO2 and the perfusion media was supplemented with 10 mM of
 6      morpholine.  An excess of NMOR was found in the NO2-exposed group when lung tissue
 7      and perfusate were combined. Control experiments could not exclude the possibility that
 8      NMOR was produced in the perfusate.
 9           Another study (Van Stee et al., 1983) reported that mice gavaged with 1 gm of
10     morpholine/kg of body weight/day and then exposed 5 to 6 h for 5  days to 31,020 to
11      38,540  jLcg/m3- (16.5 to 20.5 ppm) NO2 revealed that NMOR could be produced in vivo. The
12     single site containing the greatest amount of NMOR was in the gastrointestinal tract. Upon
13     replicating this study in hamsters and rats at 18,800 jwg/m3 (10 ppm) NO2:for 1 or 2 years,
14     respectively,  during chronic ingestion to  various doses of morpholine in the drinking water,
15     no excess incidence of cancer was found (Adkins, personal communication).  Regardless of
16     whether in vivo nitrosation can occur, the relative significance of NO2" from NO2 compared
17     to NO2" resulting from food,  tobacco, and  NO3"-reducing oral bacteria is questionable
18     (Murdiaetal., 1982).
19          Aside from nitrosamines, recent evidence suggests the possibility that inhaled NO2 may
20     be involved in the production of other potentially hazardous N-nitroso compounds.  Protein
21     and peptides may undergo nitrosation to  produce diazo derivatives, most of which are
22     mutagenic and/or carcinogenic.  Challis  et al. (1987)  suggested, based upon in vivo studies,
23     that diazopeptides could be produced from inhaled NO2 that is absorbed into the blood.
 24     These diazopeptides would be relatively  stable at blood pH so as to allow them to act as
 25      circulating carcinogens.
 26                                    ' '            •             ••'•'•
 27      Summary
 28           Exposure to NO2 produce a wide array of health effects beyond  the confines of the
 29  •:*•   lung.   Evidence suggests that NO2 and/or  some of its reactive products penetrate the lung
 30      epithelial layers and enter the blood, producing alterations in the blood and other organs.
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   1          Conflicting results have been reported on whether NO2 affects body weight gain in
   2     experimental animals.  One study reported that NO2 did not affect body weight gain in
   3     rabbits, guinea pigs, rats, hamsters, and mice at exposure concentrations of up to
   4     47,000 /ig/m3 (25 ppm) and dogs at 9,400 ^g/m3 (5 ppm) for up to 18 months.  However, a
   5     decline in body weight in guinea pigs exposed continuously to 1,880 jwg/m3 (1 ppm) for
   6     6 months, in rabbits exposed to 2,400 j«g/m3 (1.3 ppm), and in rats exposed to 24,000 /zg/m3
   7     (12.5 ppm) NO2 for 213 days have been reported.  Newborn mice appear to be more
   8     sensitive to NO2 exposure than adult mice but, based on limited data, juvenile rats appear to
   9     be less sensitive to the effects of NO2 exposure than young adult rats.
 10         Nitrogen dioxide-induced changes in blood constituents may result from the direct effect
 11      of NO2, formation of NO3" and NO2", or secondary effects emanating from other organs such
 12      as the lung, liver, hear, kidneys, and spleen.  No effect on hematocrit and hemoglobin have
 13      been reported in squirrel monkeys exposed to 1,880 ^g/m3 (1  ppm) NO2 for 493 days and in
 14      dogs exposed to up to 9,400 /*g/m3  (5 ppm) for 18 months. There was, however,
 15     polycythemia and an increase ratio of polymorphonuclear leukocytes to lymphocytes found in
 16     rats exposed to 3,760±1,880 ^g/m3 (2±1 ppm) NO2 for 21 months.  There have also been
 17     reported changes in RBC membranes of experimental animals following NO2 exposure.  Red
 18     blood cell D-2, 3-diphosphoglycerate was reportedly increased in guinea pigs exposed to
 19     677 /ig/m (0.36 ppm) NO2 for 1 week.  An increase in RBC  sialic acid, indicative of a
 20     younger population of RBC, was reported in rats exposed to 7,520 ^g/m3 (4 ppm)
 21     continuously for 1 to 10 days, but in another study, exposure to the same concentration of
 22     NO2 produced a decrease in RBC.
 23          Only limited information is available on the effect of NO2 on the heart.  However,
 24     cardiac performance  may be affected by lung edema and changes in hemoglobin.
 25     A significant reduction in PaO2 has been reported  in rats exposed to 7,520 /ig/m3 (4 ppm) for
 26     3 months.  No affect on PaO2 was reported in rats exposed to 752 ^g/m3  (0.4 ppm) over the
 27     same exposure period.
28         Decreases in serum proteins and lipoproteins and increases in serum globulins,
29     indicating NO2-induced hepatic damage have also been reported.  Nitrogen dioxide increased
30     pentobarbital-induced sleeping times in female mice after a 3 h exposure to 470 ^g/m3
31      (0.25 ppm), suggesting effects on hepatic xenobiotic  metabolism.  The effects only occurred

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at specified time periods after exposure ended and did not exceed 1 day.  Similar effects
(increased hexobarital-induced sleeping time) were reported in the progeny of maternally
exposed rats on postexposure Day 7 and 21 but not on Day 14 after being exposed to
1,000 /ig/m3  (0.5 ppm) NO2. Decreases in cytochrome P-450 from liver microsomes have
been found in rat liver homogenates after 7 days of exposure to 752 ^g/m3  (0.4 ppm) but not
                           ^
after exposure to 2,256 jug/m (1.2 ppm) NO2.
     The available data do not support the possibility that NO2 is a direct acting carcinogen.
The data that suggest that NO2 may act as a promoter or facilitator of neoplastic disease is
fraught with methodological and interpretive problems. The evidence suggests that further
study may be warranted.
13.3 EFFECTS OF MIXTURES CONTAINING NO2
     Exposure to pollutant mixtures in ambient air provides a basis for possible toxicologic
interactions, whereby combinations of pollutants may behave differently than would be
expected from consideration of the action of each constituent separately.  In many cases, the
study of mixtures containing NO2 involved exposures to only two pollutants, in which case
the role played by each can be elucidated with the appropriate experimental design.
However, there is a fairly large data base that involves mixtures of more than two
components, often with no single pollutant control, so the contribution of each individual
agent to overall response is often obscure.  In some cases, the NO2 (or NOX)  may have
varied between exposure groups, or NO2 was present in one group and not in another, so its
relative  influence could be assessed.  This section discusses only those studies where the role
of NO2  (or NOX) can be elucidated.  In addition, a discussion of many of these studies
addressing the effect of NO2 alone on various organs and systems appears elsewhere in this
chapter.
     Table 13-25 outlines those studies in which experimental animals were exposed to
constant levels of an atmosphere containing NO2 with only one other material (binary
mixtures).  By far, the largest data base is for NO2 plus O3.. Examination of  these studies
indicates that various degrees of interaction may occur.  The morphologic response to an
NO2/O3 mixture, as reported by Freeman et al. (1974b), was primarily that due to O3 alone,
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 1     although in this study the level of NO2 used would have produced only small changes that
 2     would easily be obscured by the more potent O3.  Acute lethality and some biochemical
 3     responses to NO2/O3  mixtures involve synergism (Diggle and Gage, 1955; Mustafa et al.,
 4     1984); some have ascribed this interaction to the production of new reaction products in the
 5     exposure atmosphere.
 6          In terms of host antimicrobial defense (discussed in Section 13.2.3.1), toxicologic
 7     interactions involving NO2 and O3 are generally additive after acute exposures.  It seems that
 8     each pollutant contributes to the observed response when its concentration reaches the
 9     threshold at which it would have affected bacterial resistance when administered alone
10     (Goldstein et al., 1974).  The subchronic exposure conducted by Ehrlich et al. (1977) did
11     provide some evidence of synergism with 3,760 /tg/m3 (2.0 ppm) NO2 and 97.5 jug/m3
12     (0.05 ppm) O3.   Graham et al.  (1987) exposed mice to various combinations and found
13     synergism using bacterial infectivity as the end point.
14          Sagai et al. (1987) exposed mice, hamsters,  guinea pigs, and rats to a mixture of
15     750 jwg/m3 (0.4  ppm) NO2 and 780 ,ug/m3 (0.4 ppm)  O3,  24 h/day for 2 weeks, to assess
16     effects on lipid peroxidation in the lungs.  Although the two gases were not also administered
17     singly to allow assessment of effects due to each alone, the study showed there to be species
18     differences in lipid peroxide formation following exposure that were related to the relative
19     content of antioxidants and the specific composition of phospholipids and their fatty acids.
20     The guinea pig was the most sensitive animal and the  hamster the most resistant.
21          The studies described above involved simultaneous exposure to both NO2 and another
22     gas.  However,  "real world"  exposures to these pollutants typically have temporal patterns,
23     and exposure to one agent may then alter the response to another subsequently inhaled.
24     Thus,  order of exposure to inhaled NO2 may be important in toxic interactions.   Yokoyama
25     et al.  (1980) exposed rats to either NO2 or O3 for 3 h or to NO2 for 3 h followed by O3  for
26     3 h, and assessed lung mechanics in postmortem lungs, lung histology, and enzyme activity
27     in subcellular fractions of lung  tissue.  In  one series of exposures, rats were exposed for 7 or
28      14 days to NO2 and O3 at concentrations of 10,340 jttg/m3 (5.5 ppm)  and 2,120 jug/m3
29     (1.1 ppm), respectively.  The activity of phospholipase A2 in the mitochondrial  fraction was
30     increased in those animals exposed to O3-only, or to O3 after NO2,  for 14 days; however, the
31     response in the latter was significantly greater than that in the former.  A decrease in activity
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 of lysolecithin acyltransferase in the supernatant fraction was found after 14 days only in
 those animals exposed sequentially to both NO2 and O3.  In a second study, rats were
 exposed for 14 or 30 consecutive days to  10,150 jig/m3 (5.4 ppm) NO2 followed by
 1,960 /zg/m3 (1.0 ppm) O3.  Pulmonary mechanics performed on the postmortem lung
 indicated a change in pulmonary resistance in the O3- and sequential NO2/O3-exposed
 animals. Histologically, the lungs of the animals exposed to both NO2 and O3 appeared
 similar to those exposed to O3 alone. However, a slight degree of epithelial necrosis in the
 medium bronchi, not found with either NO2 or O3 alone, was seen in the animals exposed to
 both pollutants.  In addition, damage at the bronchioloalveolar junction appeared to be
 somewhat more marked in animals exposed to both gases than in those exposed to O3 alone.
 These studies suggest that sequential exposures produced responses which were, in  most
 cases, not greatly different from those due to O3 alone (i.e., NO2 preexposure did not
 markedly alter the response to subsequent  exposure to O3).
     Aside from sequential exposures, simulation of actual mixed'pollutant exposure
 scenarios involving NO2 and O3 have also been performed by examining the effects, on
 bacterial resistance, of a continuous baseline exposure to one concentration, with
 superimposed short-term peaks to a higher level.  Ehrlich et al. (1979) exposed mice for 1 to
 6 months (24 h/day, 7 days/week) to a baseline concentration of 0 (air) or 190 /ig/m3
 (0.1 ppm) NO2, upon which was superimposed 3  h/day, 5 days/week peak exposures of
 940 Atg/m3  (0.5 ppm) NO2, or a combination of 940 fig/m3 NO2 and 200 /tg/m3 O3;
 bacterial exposure followed pollutant exposure, and animals were then observed for 14 days.
 A significant and similar increase in percentage mortality was found by 6 months in all
groups,  with no evidence that exposure to  the NO2/O3 peaks altered the response; there was
also no change in survival time.  In another experiment, mice were re-exposed for 14 days
 (after 1  to 3 months of initial pollutant exposure and bacterial challenge) to the same pollutant
concentrations as above,  and mortality was examined during this time. Animals pre-exposed
for a least 2 months either to NO2/O3 peaks over  the air baseline, or to NO2/O3 peaks over
            />
the 190  /*g/m  NO2 baseline, showed significant reductions in survival time.  Although no
conclusions were drawn as to the efficacy  of the mixture, the investigators concluded that the
sequence of peak exposure was important in altering resistance to infection.
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     Ehrlich et al. (1973) also examined the effect of the same baseline and peak exposures
(for 1 to 3 months) on AMs.  Cell viability was decreased after 3 months of exposure only
when the NO2/O3 peaks were superimposed on continuous exposure to clean air.  There was
also a general increase in blood enzyme activity, but continuous exposure to 190 ^g/m3
(0.1 ppm) NO2 with superimposed peaks of NO2 and O3 was the most effective in this
regard.
     In another study, Ehrlich (1983) examined the effects of continuous  exposure (24 h/day,
5 days/week) to a baseline level of 380 jug/m3 (0.2 ppm) NO2, with two daily peaks given
5 days/week as follows:  1,880 fig/m3 (1.0 ppm) NO2 for 1 h in the morning, and a mixture
            *5                             *3
of 200 jwg/m  (0.1 ppm) O3 plus 1,880 jwg/m NO2 given for 1 h in the afternoon.
Exposures lasted for 9 months, followed by bacterial challenge.  Other groups were exposed,
continuously, to 380 pcg/m3  NO2 with either no peak, or to 1,880 j«g/m3 given for 1 h in
both the morning and afternoon. The only group that showed a significant increase in
mortality was that exposed for 9 months to 380 jug/m3 NO2 with daily peaks of NO2 in the
morning,'and NO2 and O3 in the afternoon.  In addition, only this group  showed a change
(increase) in cellular ATP levels in AMs.  By 8 months of exposure, this  group also showed
an increase in counts of RBCs, leukocytes, and lymphocytes,  and a decrease in mean
hemoglobin concentration.  The other pollutant exposure groups showed increases only in
leukocyte count.
     Gardner et al. (1982),  Gardner (1980), and Graham et al. (1987) examined bacterial
resistance in mice continuously exposed (15 days, 24 h/day) to a baseline  level of an NO2/O3
mixture with two daily 1-h peaks of the mixture, as follows:  (1) high exposure level:
2,760 ,ug/m3 (1.2 ppm) NO2 plus 200 jWg/m3 (0.1 ppm) O3 baseline; 4,700 ,ug/m3 (2.5 ppm)
NO2 plus 590 /ig/m3 (0.3 ppm) O3  peak; (2) intermediate exposure level:  940 ^cg/m3
(0.5 ppm) NO2 plus 100 ngfm3 (0.05 ppm) O3 baseline; 1,880 /«g/m3 (1.0 ppm) NO2 plus
200 jwg/m3 (0.1 ppm) O3 peak; or (3) low exposure level:  90 ju.g/m3 (0.05 ppm)  NO2 plus
100 /ig/m3 (0.05 ppm) O3 baseline; 190 jug/m3 (0.1 ppm) NO2 plus 200 /*g/m3 (0.1 ppm) O3
peak. Animals were also exposed to the same baseline levels of either NO2 or O3 onto which
were superimposed twice daily, 1-h peaks  of either NO2 or O3 at concentrations as above.
The low concentrations of either given alone, or in combination, did not significantly increase
mortality.  At the intermediate exposure levels, the mixture was synergistic, while NO2-alone
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30
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 increased mortality and O3 had no effect.  At the high exposure level, the combined exposure
 was again synergistic, even though exposure to each given separately also increased mortality.
      Some limited data exist for combinations of NO2 with gases other than O3
 (Table 13-25). In the two reported studies with SO2, neither SO2 nor NO2 given alone, or
 together, produced any response.  The concentrations used by Azoulay et al. (1980) were
 quite low, while the respiratory mechanical end points assessed by Antweiler and, Brockhaus
 (1976) were likely not very sensitive to pollutant-induced change.
      One major interaction that may occur in ambient air is that between NO2 and particles.
 Particle contact may result in gas adsorption, and subsequent  transport to,target sites where
 the gas normally would not deposit in concentrated amounts.  Boren (1964) adsorbed NO2
 onto carbon to determine whether this carrier changed the toxicity of the NO2. Mice were
 exposed  6 h/day, 5 days/week for 3 months to carbon (38% of particles were <2 jum;
 16,000 particles/cm3) onto which 553 mg NO2 was adsorbed  per gram; the exposure air also
 contained 47,000 to 56,400 /zg/m3 (25 to 30 ppm) free NO2 as well. The exposed animals
 showed focal changes in the lung parenchyma.  These lesions contained carbon particles, and
 were characterized by enlarged airspaces and loss of alveolar walls. Exposure solely to NO2
 resulted in edema and inflammation, but no parenchyma! lesions, while no lesions were found
 due to carbon-only exposure.  Thus, Boren concluded that the carbon particles served as a
 carrier for NO2, delivering high concentrations of NO2 to localized areas in the lungs where
 the carbon deposited.
     Other aerosols, while not necessarily acting as carriers, may potentiate response to NO2
 by producing local changes in the lungs that enhance the toxic action of co-inhaled NO2.
 Last et al. (1983) and Last and Warren (1987) have .examined the effects of inhalation of
 acidic  sulfate aerosols plus NO2 on biochemical end points, using minces prepared from the
 lungs of rats after various  exposure regimes.  Last  et al. (1983) exposed rats to 9,400 to
 47,000 jtig/m3 (5 to 25 ppm) NO2 alone, or in combination with 5,000 jwg/m3 (NH4)2SO4
 (1 jtm, MMAD), for up to 7 days, and examined the rate of collagen synthesis by lung
 minces.  Ammonium sulfate alone caused no effects.   Analysis of the slope of the exposure
concentration-response curve for NO2 indicated there to be an approximate doubling of
synthesis rate when the mixture was employed compared to NO2 alone; examination of
responses at individual NO2 concentrations showed that the mixture clearly began to increase
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synthesis rate (above NO2 alone) when NO2 was > 18,800 pg/m3 (10 ppm).  The
investigators also noted that there was a tendency towards a reduction in lethal concentration
for 75% of the animals (LC75) when exposures were to (NH4)2SO4 plus NO2, compared to
that for NO2 alone.  On the other hand, there was no difference in the level of pulmonary
edema between animals exposed to NO2 alone, or NO2 in combination with (NH4)2SO4. In
a later study,  Last and Warren (1987) exposed rats to 9,400 /^g/m3 (5 ppm) NO2 alone, or in
combination with either 1,000 jwg/m3 H2SO4 (0.4 pm) or NaCl (0,4 jicm), for up to 7 days.
A synergistic  interaction for collagen synthesis rate was found when either aerosol was used
with NO2.  Reduction of the NO2 level to 3,760 /*g/m3 (2 ppm) also resulted in a synergistic
increase in collagen  synthesis rate when combined with 1,000 jwg/m3 H2SO4 (Last, 1989).
Changes in protein content of the lavage fluid (an index of lung edema) showed evidence of
synergism at 1 day with H2SO4, or 3 days with NaCl-.  The investigators suggested that the
interaction with NaCl was due to the formation of acids (e.g., HC1, HNO2, HNO3) from
nitrosyl chloride (NOC1) following its hydrolysis after deposition in the deep lung; the latter
may be formed from a chemical reaction between NO2 and NaCl.  Similarly,  potentiation
with the acid  sulfate aerosols was likely due to localized effects following their deposition.  It
has been proposed that the acid aerosols would produce a shift in local pH within the alveolar
milieu.  This  shift would result in a change in the reactivity or residence time of reactants
involved in oxidant-induced pulmonary effects  (Last et al., 1984).
     The effects of exposure to mixed atmospheres of NO2 and H2SO4 on lung clearance
mechanisms have been examined by Schlesinger and Gearhart (1987) and Schlesinger (1987).
In the former study, rabbits were exposed for 2 h/day  for 14  days to either 560 /tg/m3
(0.3  ppm) or  1,880 ^g/m3 (1.0 ppm) NO2, or 500 jwg/m3 H2SO4 (0.3 pm) alone, or to
mixtures of the low  and high NO2 concentrations with acid.  After the first exposure, an inert
tracer aerosol was administered to assess clearance from the respiratory region of the lungs.
In the single pollutant groups, both concentrations of NO2 accelerated clearance, while
H2SO4 retarded clearance, compared to air-exposed controls.   Exposure to the combination of
         f^t
560 /ig/m NO2 plus H2SO4 resulted in a response that was not different from that due to the
acid  alone.  However, exposure to 1,800 jwg/m3 NO2 plus H2SO4 resulted in a clearance
pattern that differed  from that of both NO2 and H2SO4, but was more similar to that of the
latter.
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  1          Schlesinger (1987) exposed rabbits to the same NO2/H2SO4 atmospheres as above, but
  2     then sacrificed animals 24 h after 2, 6, or 13 exposures and recovered cells from the lungs by
  3     bronchopulmonary lavage.  Exposure to 1,800 jtig/m3 (1 ppm) NO2 with acid resulted in an
  4     increase in PMNs at all time points (not seen with either pollutant alone), and an increase in
  5     phagocytic capacity of AMs after two or six exposures.  In contrast, exposure to 560 jwg/m3
  6     (0.3 ppm) NO2 with acid resulted in depressed phagocytic capacity and mobility. A
  7     comparison of responses due to exposure to the NO2/H2SO4 mixture with those due to either
  8     pollutant alone showed that the effects of the combined atmospheres were generally either
  9     additive or synergistic, depending on the specific cellular end point being examined.
 10          Furiosi et al.  (1973) exposed rats and monkeys continuously to a combination of
 11     3,760 ptg/rn3 (2 ppm) NO2 and 330 jiig/m3 NaCl.  Histological response after 14 months of
 12     exposure in monkeys (respiratory bronchiolar epithelial hypertrophy) was similar in groups
 13     exposed to NO2 alone, or with NaCl. Hematologic changes (polycythemia) in both monkeys,
 14     after 18 months, and rats, after 6 months, were similar for groups exposed to NO2 with or
 15     without NaCl.  Thus, in this study, the NaCl did not potentiate response to NO2. Perhaps
 16     the end points were not sensitive to the effects of any reaction products between NO2 and
 17     NaCl, or the concentration of NaCl was too low to allow production of significant  amounts  of
 18     such products.
 19          The role of adsorbed NO2 in the toxicity of mineral dusts was addressed by Robertson
20     et al. (1982).  They examined the effects of NO2 adsorption on the cytotoxicity of  coal,
21     quartz, or  kaolinite on PSSSDj cells exposed in vitro to the dusts for 48 h; viability and
22     enzyme release (e.g., LDH) were used as end points.  The amount of NO2 absorbed was 5 to
23     10 /zg/mg  dust.  Although a small decrease in cytotoxicity was found after adsorption of
24     NO2, the investigators concluded  that there was no systematic or significant difference in
25     biochemical measures of toxicity from cells exposed to dust with or without NO2.  On the
26     other hand, Shevchenko (1971) noted an increase in the fibrogenicity of quartz dust in rats
27     following adsorption of 0.36 jug NO2/mg dust, a lower level than that used by Robertson
28     et al. (1982).  These different results may be due to differences in particle residence time:
29     Robertson  et al. (1982) exposed the cells for only 48 h, while dust was present in the lungs  in
30     the Shevchenko study for months, allowing a  greater time for gas desorption.
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 1          Although many studies have examined the response to NO2 with only one additional
 2     pollutant, the atmosphere in most environments is a complex mixture of more than two
 3     materials., A number of studies have attempted to examine the effects of multicomponent
 4     atmospheres containing NO2.  But, as mentioned, in many cases, the exact role played by
 5     NO2 in the observed responses is not always clear.
 6          Kleinman et al. (1985a,b) exposed rats for 4 h to atmospheres consisting of various
 7     combinations of O3 (1,180 jug/m3 [0.6 ppm]), NO2 (4,700 jug/m3 [2.5 ppm]), SO2
 8     (13,100 jttg/m3 [5.0 ppm]), and particles.  The particles consisted of  1 mg/m3 (0.2 ^m) of
 9     either H2SO4 or (NH4)2SO4, laced with iron and manganese sulfates. The metallic salts act
10     as catalysts for the conversion of sulfur IV into sulfur VI, and the incorporation of gases into
11 ,    the aerosol droplets. The respiratory region was examined for morphological effects.
12     A confounding factor in these studies was the production of nitric acid (HNO3) in
13     atmospheres that contained O3  and NO2, and NO3" in those that contained O3 and
14     (NH4)2SO4, but not NO2.  Nevertheless, a significant enhancement of tissue damage was
15     produced by exposure to atmospheres containing H2SO4 or HNO3, compared to those
16     containing (NH4)2SO4.  In addition, there was a suggestion that the former atmospheres
17     resulted in a greater area of the lung becoming involved in lesions, which were characterized
18     by a thickening of alveolar walls, cellular infiltration in the interstitium, and an increase in
19     free cells within alveolar spaces.  Exercise seemed  to potentiate the histological response to
20     the complex mixtures containing acids (Kleinman et al., 1980).
21'          One of the more common complex mixtures studied is that of automobile exhaust.  In
22     many cases, the exhaust is irradiated to produce a reactive mixture that is a model for
23     photochemical smog. Coffin and Bloomer (1967) exposed mice for 4 h to irradiated
24     automobile exhaust to assess effects on bacterial resistance.  Levels of NOX in the atmosphere
25     were as follows:  NO2, 200 to 1,600 )«g/m3 (0.1 to 0.85 ppm) and NO,  20 to 180 /*g/m3
26      (0.02 to 0.15 ppm). Exposure was found  to result in an increase in  bacterial-induced
27      mortality, but the investigators were not able to clearly ascribe the results to any one
28     pollutant.  However, they noted that the exposure levels of NO2 were less than those that
29      were known to alter resistance when NO2  was given alone and, thus, they suggested that the
 30      effect of the exhaust mixture was due to other oxidants, such as, O3.
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  1          Stupfel et al.  (1973) exposed rats for 6 h/day, 5 days/week for 2.5 months to 2 years to
  2     exhaust mixtures for morphologic analysis.  The atmosphere contained carbon dioxide (CO2),
  3     aldehydes, carbon monoxide (CO), and either 0.2 or 23 ppm NOX. Only the mixture with
  4     the higher NOX concentration produced any significant toxic response, namely a decrease in
  5     body weight and increase in spontaneous tumors.  However, the latter was ascribed to the
  6     hydrocarbon component of the exhaust mixture.
  7          Cooper et al.  (1977) exposed rats continuously for 38 or 88 days to three auto exhaust
  8     atmospheres that differed in their component concentrations; all contained H2SO4, SO2, and
  9     CO, as weU as NO (8,700 to 13,300 jig/m3 [7.1 to 10.8 ppm]) and NO2 (560 to
 10     9,600 jig/m3 [0.3 to 5.1 ppm]).  All exposures resulted in a significant depression of
 11     spontaneous locomotor activity not seen with exposure to either H2SO4 or CO alone;  the
 12     investigators concluded that this response was due to either the hydrocarbon or NOX
 13     components of the mixture.
 14          The results of a long-term exposure of dogs to automobile exhaust have been described
 15     by several investigators (Stara et al.,  1980).  Animals were exposed for 68 months (16 h/day)
 16     to various atmospheres, which included raw auto exhaust, irradiated auto exhaust, or two
 17     mixtures of NOX—one with a high NO2 level and low NO  level (1,210 ^g/m3 [0.64 ppm]
 18     NO2, 310 /ig/m3 [0.25 ppm] NO),  and one with low NO2  and  high NO (270 /-ig/m3
 19     [0.14 ppm] NO2, 2,050 jug/m3 [1.67 ppm] NO). Following the end of exposure, the animals
 20     were maintained for about 3 years in normal indoor air. Numerous pulmonary function,
 21      hematologic, and histologic end points were examined after various times of exposure (Lewis
 22     et al., 1974; Vaughan et al., 1969; Stara et al., 1980; Block et al., 1973).  Only results
 23      related to NOX will be described.  Vaughan et al. (1969) reported no alterations in
 24      CO-diffusing capacity, dynamic compliance, or total expiratory resistance to flow after
 25      18 months of exposure.  However,  by 36 months, a significant number of animals exposed to
 26      high NO2/low NO had an abnormally low CO diffusing capacity (as a ratio of total lung
 27      capacity) (Lewis et al., 1974).  Additional changes  were observed after 61 months of
 28      exposure; in the dogs breathing low NO2/high NO or raw auto  exhaust, residual volume was
29      increased compared to animals exposed to control or high NO2/low NO.  The common
30      treatment factor causing this effect appeared to be the higher concentration of NO.
31      A significant number of dogs exposed to high NO2/low NO had a lower mean CO diffusing

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 1      capacity/total lung capacity ratio, and a lower peak flow rate, compared to controls.  The
 2      investigators attributed the change in diffusing capacity to an alteration in the alveolo-
 3      capillary membrane.  Block et al.  (1973) reported no significant change in hematcrit, blood
 4      viscosity, or level of metHb due to any of the exposure atmospheres after 48 months of
 5      exposure.                           ,
 6           After all exposures were terminated, the animals were allowed to recover ,for 2 years
 7      before pulmonary function measurements,were made again (Stara et al., 1980).  In all
 8      pollutant-exposed dogs, total lung  capacity was increased relative  to the control group of
 9      animals.  Those animals that received the NO2/NO mixtures experienced modest increases in
10      inspiratory volume, vital capacity, and total  lung  capacity.
11           Orthoefer et al. (1976) evaluated biochemical  alterations 2.5 to 3 years after the end of
12      all exposures.  In groups exposed to irradiated auto exhaust or high NO2/low NO, there was
13      a rise in lung prolylhydroxylase, an enzyme involved in collagen synthesis.  In addition, a
14      correlation was found between lung weight and hydroxyproline content in animals exposed to
15      the NOX atmospheres,
16           Lung morphology of the dogs was evaluated by Hyde et al.  (1978) 32 to 36 months
17      after 68 months of exposure.  In the high NO2/low NO group,  there were increases in total
18      lung capacity and lung volume, and decreases in the surface density of the alveoli and the
19      volumetric density of parenchyma! tissue. Alveoli were enlarged  in both the high NO2 and
20      high NO groups.  In the high NO2, but not  the high NO group, there was cilia loss and
21      hyperplasia of nonciliated bronchiolar cells.  In the high NO group, there were lesions in the
22      interalveolar pores.  In the most severely affected dogs, in the high NO? group, morphological
23      changes considered to be analogous to centrilobular emphysema were present.  Since these
24      morphologic measurements, were made after a 2.5 to 3 years holding period in clean air, it
25      cannot be determined with certainty whether these disease processes abated or progressed
26      during, this time. However, indications were that the long-term exposures produced persistent
27      damage that was indeed progressive even after exposures ended.
28
29      Summary
30           Exposures to mixtures containing NO2 are quite common and provide a basis for
31      toxicological interactions whereby  combinations of pollutants may behave differently than
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  1     would be expected from consideration of the action of each constituent separately.  The
  2     largest data base exists for the combination of NO2 and O3.  Morphologic response to
  3     exposure to this mixture is generally due to O3, but biochemical effects may involve
  4     synergism.  Reactions of host defenses, specifically antibacterial activity, may be additive or
  5     synergistic. Mixtures of NO2 and acid sulfates result in additive to synergistic effects.
  6     Although many studies examined response to simple mixtures of NO2 with one other
  7     material, the atmosphere in most environments is a complex mix of more than two pollutants.
  8     Although effects of complex mixtures has been examined to some extent, the exact role
  9     played by NO2 in  the observed responses is not always clear.
 10
 11
 12     13.4  NITRIC OXIDE
 13         The toxicologic data base for nitric oxide (NO)  is not extensive, except for its
 14     interaction with blood.  One problem is that it is often difficult to obtain pure NO in air
 15     without some contamination with NO2.  Little is actually known about NO absorption in the
 16     respiratory tract, and nothing on its subsequent intrapulmonary  distribution.  Since NO is less
 17     water soluble and less reactive than NO2, it follows that its absorption from inhaled air
 18     should be less.   Yoshida et al.  (1981) found that < 10%  of the NO  "inhaled" by isolated,
 19     perfused lungs  of rabbits was absorbed.  On the other hand, absorption in normal breathing
20     humans in vivo was  85 to 92% for NO concentrations ranging from 400 to 6,100 jug/m3
21      (0.33 to  5.0 ppm)  (Wagner,  1970; Yoshida and Kasama,  1987); values for NO2 were 81 to
22     90% (Wagner,  1970).  Absorption of NO with exercise was 91 to 93% in humans (Wagner,
23     1970).  Yoshida et al. (1980a) found the percentage absorption in rats acutely exposed to
24     169,300  jig/m3 (138 ppm), 331,300 ^g/m3 (270 ppm) and 1,079,800 jug/m3 (880 ppm) to be
25     90%, 60%, and 20%, respectively. The lower absorption at the two highest concentrations
26     was ascribed to an exposure-induced decrease in ventilation.  Vaughan et al.  (1969) exposed
27     dogs to auto exhaust mixtures and found that 73% of the constituent NO was removed when
28      the mixture was passed in through the nose and out through a tracheostomy tube; this
29      compared to 90% removal for  NO2. Thus, respiratory tract absorption of NO is quite similar
30     to that for NO2 in  spite of solubility differences.  The lower solubility of NO may, however,
31      result in  greater amounts reaching the pulmonary region, where it then diffuses into blood

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and reacts with hemoglobin (Yoshida and Kasama, 1987).  In fact, exposures in vivo do seem
to indicate that NO has a faster rate of diffusion through tissue than does NO2 (Chiodi and
Mohler, 1985).
     High exposure levels of NO are apparently needed to be lethal.  Pflesser (1935)
reported that mice exposed to 380,400  ^g/m3 (310 ppm) NO for 8 h showed no mortality,
                                                                  
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August 1991
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 1          The effects of NO on defense function of the lungs has been examined in two studies.
                                                                                       S)
 2     Holt et al. (1979) examined immunological end points in mice exposed to 12,270 jug/m
 3     (10 ppm) NO for 2 h/day, 5 days/week up to 30 weeks. Leukocytosis (i.e., an increased
 4     number of leukocytes) was evident by 5 weeks of exposure, while a decrease in mean
 5     hemoglobin content of RBCs was found by 30 weeks.  A decrease in RBC count at Week  15
 6     was not found at 30 weeks.  An enhancement  of the humoral immune response to sheep
 7     RBCs was seen at 10 weeks, but this was not evident at the end of the exposure series.
 8     Spleen cell phytohemagglutinin response (a measure of the incorporation of tritrated in
 9     T lymphocyte cultures) was decreased after 15 weeks of exposure, but levels then recovered
10     and became greater than control.  The ability of spleen cells to mount a graft vs. host
11     reaction was stimulated by 20 weeks of exposure, but suppressed by 26 weeks. Finally, the
12     ability of mice to reject virus-induced tumors was assessed; only 40% of the NO-exposed
13     animals survived tumor challenge, compared with 66% for control animals. This study
14     suggests that NO exposure may have affected  the immunologic competence of exposed
15     animals. A discussion of the effect of NO2 on immunological end points appears in the
16     section on host defense mechanisms.
17          Effects of NO on bacterial defenses were examined by Azoulay et al. (1981). Male and
18     female mice were exposed continuously to 2,450 pcg/m3 (2 ppm) NO for 6 h to 4 weeks, to
19     assess the effect on resistance to infection induced by a bacterial aerosol administered after
20     each NO exposure. Although there appeared  to be somewhat of an increase in mortality in
21     each group of females exposed to NO for at least 1 week compared to control (bacterial
22     inoculation only), there was no statistically significant difference for either sex.  Likewise,
23     each group of females exposed to NO for at least 1 week showed a slight decrease in mean
24     survival time, but this change was not statistically significant, nor was there any observable
25     difference in males exposed to NO. When the data for those groups exposed for 1 to
26     4 weeks were combined, NO-exposed females showed a significant increase in percentage
27     mortality and a significant decrease in survival time; this was not seen for males.  Thus, this
28     study  suggests some gender related difference in response, at least to the one level of NO
29     examined.
 30           One possible mechanism of toxic  action of NO is lipid peroxidation. The GSH
 31     transferase system serves to protect vital molecules from peroxidative damage. Thus,
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  1      changes in constituents of this system may serve as a marker of effect from inhaled NO.
  2      However, mice exposed to 12,300 to 25,800 /*g/m3 (10 to 21 ppm) NO, 3 h/day for 7 days
  3      showed no change in levels of reduced GSH, a co-factor for GSH peroxidase, in their lungs
  4      (Watanabe et al., 1980).
  5          There is some evidence that NO may alter the activity of other enzymes.  A number of
  6     in vitro studies (Arnold et al., 1977; Braughler, 1982; Katsuki et al., 1977) have indicated
  7     that NO may affect guanylate cyclase, the enzyme that catalyzes  the formation of cyclic-GMP
  8     from GTP.  They have shown,  based upon exposure of purified enzymes or tissue minces
  9     from various organs, that NO increases enzyme activity in a concentration-dependent fashion,
 10     and that the activation is reversible when NO is removed from the preparation.  Although
 11     variable degrees of activation were seen in different tissues, lung tissue showed one of the
 12     highest. It is, however, not known whether NO would alter guanylate cyclase activity with
 13     in vivo exposure; this would suggest possible enzyme regulation by an environmental agent.
 14          The bulk of the toxicologic data base for NO biochemistry concerns its reaction with
 15     hemoglobin (Hb). Inhaled NO  that enters the bloodstream through the lungs binds to
 16     hemoglobin, producing nitrosylhemoglobin (NOHb) (Oda et al., 1975, 1980a,  1980b; Case
 17     et al., 1979; Nakajima et al., 1980). This may, in fact, be its major route of action, and
 18      studies in vitro have suggested that NO may severely reduce  the ability of RBCs to carry O2.
 19      These studies have shown that the affinity of Hb for NO is very high, much higher even than
 20      that for O2 (Gibson and Roughton, 1957; Moore and Gidson, 1976).  In addition, in vitro
 21      measurement of O2-dissociation curves for partially NO-liganded  human Hb have shown that
 22      NO binding tends to reduce dissociation of bound O2 on the molecule (Kon et al., 1977).
 23      Finally, NOHb is easily and rapidly oxidized to methemoglobin (MetHb) in the presence of
 24      O2 (Chiodi and Mohler, 1985; Kon et al., 1977), further reducing the ability of RBCs to
 25      transport O2.
 26          Following in vivo exposures, a linear relationship was found between the exposure
 27      concentration of NO  (24,500 to  98,200 ^g/m3 [20 to 80 ppm], 1  h) in mice and blood
 28      content of NOHb; however, levels of MetHb were found to increase exponentially with  NO
 29      concentration, resulting in greater blood levels of MetHb than NOHb (Oda et al., 1980b).
30      After exposure of mice to 49,100 ^g/m3 (40 ppm) for 1 h, concentrations of both MetHb and
31      NOHb decreased rapidly, with half-times of only a few minutes (Oda et al., 1980b). Thus,

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the steady-state concentration of NOHb during NO exposure would be fairly low, while that
for MetHb would be somewhat higher (Maeda et al., 1987).
     Studies of animals exposed to NO in vivo have shown that the amount of NOHb in
blood was much less than would be expected from in vitro exposure data (Oda et al., 1980a,
1975), and that any reduction of O2 transport was not lethal.  Lifetime (23  to 29 months)
exposures of mice to 2,940 /xg/m3 (2.4 ppm) NO resulted in the blood content of NOHb
remaining relatively steady at 0.01%, while the maximum amount of MetHb was 0.3%  (Oda
et al., 1980b). Mice exposed to 12,300 ^g/m3 (10.0 ppm)  NO for 6.5 months showed
NOHb at 0.13% and MetHb at 0.2% (Oda et al., 1976).  Thus, although the results of
various studies have shown that the final product of NO reaction with hemoglobin is MetHb,
with some persistent NOHb, this effect of NO is not generally lethal because of a number of
factors; these include the conversion of inhaled NO to NO2 in the airways,  the rapid
oxidation of NOHb into MetHb, and the subsequent reduction of MetHb into ferrous Hb by
MetHb reductase, an enzyme present in RBCs (Kon et al., 1980;  Maeda et  al., 1984b, 1987).
As long as the activity of MetHb reductase is maintained, the  conversion of NOHb to MetHb
should mitigate any potentially toxic effect on Hb due to NO inhalation (Kon et al., 1980).
In long-term exposure  studies, Oda et al. (1976, 1980a) exposed mice to 4,512 or
18,800 /u,g/m3 (2.4 or  10.0 ppm) NO2, and after examination  of organs sensitive to O2
depletion (e.g., brain and heart) found no evidence of hypoxic damage.
     Azoulay et al. (1977) exposed rats to 2,450 ftg/m3 (2 ppm)  NO continuously for
6 weeks to examine various hematologic parameters, including blood-O2 affinity.  No
exposure-related changes were found in Hb content, hematocrit, RBC count, red cell glucose
metabolism, or in the oxyhemoglobin dissociation curve.  In addition, no MetHb was
detected in either exposed or control animals. This showed that low level NO exposure did
not alter the blood-O2  affinity.  On the other hand, the same investigation reported that
in vitro studies had shown that blood-O2 transport was altered by high levels of NO
              o
(> 12,300 j«g/m  [10 ppm]) in both human and rat blood.
     In addition to interaction with Hb, exposure to NO may  alter other aspects of blood.
Case et al. (1979) exposed mice to 11,070 jug/m3 (9 ppm) NO for 16 h and found a decrease
in the level of iron transferrin, suggesting a reduction in the activity of this enzyme. Mice
                      o
exposed to 12,300 ng/m (10 ppm) NO for 6.5 months showed increased white blood cell
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   1      counts and an increase in the ratio of polymorphonuclear leukocytes to lymphocytes (Oda
  2      et al., 1976).  These investigators noted that 11% of the RBCs obtained from NO-exposed
  3      mice contained Heinz bodies, while the control group showed none.  Coupled with an
  4      increase in spleen weight and bilirubin, the investigators suggested that this indicated that NO
  5      facilitated the destruction of RBCs.
  6           A slight increase in RBC hemolysis was seen in mice exposed to 2,940 /xg/m3
  7      (2.4 ppm) NO for their lifetime (Oda et al., 1980a).  In vitro, rat RBCs exposed to NO
  8      showed oxidative cross-linking between cell membrane proteins and hemoglobin (Maeda
  9      et al., 1984a), an alteration which could change the cells' rheological properties.  However,
 10      in an in vivo exposure study (30.7 to 245.4 mg/m3 [25  to 200 ppm] for  1 h) of rats, no
 11      cross-linking of membrane proteins was detected (Maeda et al., 1987); the investigators
 12      suggested that this may have been due to rapid repair mechanisms operating in vivo.
 13          The pH of blood has been shown  to be reduced by NO, but only with very high
 14      exposure levels (e.g., 0.5-2.0% NO [5,000 to 20,000 ppm]) (Toothill, 1967; Greenbaum
 15     et al., 1967). Rats exposed to 2,450 /Kg/m3 (2 ppm) NO continuously for 6 weeks showed
 16     no change in blood pH (Azoulay et al., 1977).
 I*7          A few studies have examined the response to inhalation of mixtures of NO plus one
 18     other component.  Watanabe et al. (1980) exposed mice for 3 h/day, for 7 days to  NO
 19     (12,300 /ig/m3 [10 ppm]) plus O3 (1,960 ^g/m3 [1 ppm]); they observed an increase in the
 20     level of lung GSH,  but this was due solely to the O3.  Azoulay et al.  (1980) exposed rats to
 21      NO (2,460 A«g/m3 [2 ppm]) with SO2 (5,240 ^g/m3 [2 ppm]) for 13 weeks; no change in
 22     blood-O2 affinity, MetHb level, RBC count or lung histology was seen with the mixture, or
 23      with either pollutant given above. Finally, Robertson et al. (1982) adsorbed NO onto
 24      mineral dusts (2 to 5 ^g NO/mg dust); no change in the cytotoxicity of coal, quartz or
 25      kaolinite to PSSSDj cells was found, compared to dust without adsorbed NO.
 26           McFaul and McGrath (1985) examined the effect of inhalation exposure to NO (at
 27      levels of 18.4 to 78.6 mg/m3 [15 to 64  ppm] for up to 38 h) on the reduction of MetHb
 28      produced initially in the blood of rats by injection of sodium nitrate.  They found the MetHb
29      reduction was impaired at all of the NO levels used, and suggested that exposure to NO may
30      modulate certain repair processes following exposure to other oxidant pollutants.
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Summary
     The toxicologic data base for NO is not extensive, except for its interaction with blood.
Fairly high levels 2,460 fcg/m3 (2 ppm) or greater, are needed for morphologic changes in
the lungs following chronic inhalation.  Inhaled NO that is absorbed into the bloodstream
results in production of NOHb which, in turn,  is oxidized to MetHb.  This has the potential
to reduce the ability of RBCs to transport O2.  But as with other effects, high exposure levels
are needed for significant changes.
13.5  NITRIC ACID AND NITRATES
13.5.1  Nitric Acid
     There are only a few toxicologic studies of nitric acid (HNO3) which exists in ambient
air generally as a highly water soluble vapor. In an early study, Diggle and Gage (1954)
                                                       o
noted that a single exposure to HNO3 vapor at 63,000 /*g/m  (25 ppm) had no "obvious
effect on rats"; exposure duration and end points examined were unspecified.
     More recent studies have examined the histological response to instilled HNO3 (usually
   *                     ,                        •            -
1%), a procedure used in developing models of bronchiolitis obliterans in various animals,
namely dog, rabbit, and rat (Totten and Moran, 1961; Greenberg et aL, 1971; Mink et al.,
1984). The major changes noted were  degeneration of alveolar Type 2 cells and alveolar cell
hyperplasia.  In a somewhat similar study, Peters and Hyatt (1986) delivered 1% HNO3 into
a catheter positioned in the main bronchi of the dog; however, in this case, the acid was
delivered via nebulization, alternately (every other day) as either a coarse spray or as a fine
mist, for 2 h/day for 4 weeks.  Pulmonary  function testing after 4 weeks of exposure
indicated decreases in expiratory flow rate,  dynamic compliance, total lung capacity, and vital
capacity; and increases in pulmonary resistance, closing capacity, the ratio of functional
residual capacity to total lung capacity, and in phase III of the single breath nitrogen washout
curve. Histologically, there was widespread chronic inflammation of conducting airways,
especially medium and small ones, peribronchiolar  fibrosis, focal hemorrhage, edema, and
hyperplasia of goblet cells in the trachea and bronchi.
     Gardiner and Schanker (1976) examined the effect of HNO3-induced damage on drug
absorption from the lungs of rats.  Instillation of 1% HNO3 produced bronchiolitis and
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  1     alveolitis, and also increased the rate of pulmonary absorption of various drugs up to
  2     1.6 times control values. This change was ascribed to an increase in the permeability of the
  3     alveolocapillary barrier.                                                 .       ;
  4          The only study which was designed specifically to examine the pulmonary response to
  5     pure HNO3 vapor was that of Abraham et al. (1982), who exposed both normal sheep and
  6     allergic sheep (i.e., those having airway responses similar to that occurring in humans with
  7     allergic airway disease) for 4 h to 4,120 ^g/m3 (1.6 ppm) HNO3 vapor. The exposure,
  8     which was performed using a "head-only" chamber, resulted in a decrease in specific
  9     pulmonary flow resistance,  compared to pre-exposure control values,  in both groups of sheep;
 10     this indicated the absence of any bronchoconstriction.  To assess  airway reactivity, pulmonary
 11     resistance was also measured after challenge with a brorichoconstrictor aerosol (carbachol).
 12     Allergic sheep showed increased reactivity, both immediately and 24 h after HNO3 exposure.
 13     Although there was no significant change in reactivity in the normal groups as a whole, two
 14     of the animals showed an increase in reactivity to carbachol after HNO3 exposure; according
 15     to the investigators, this suggested that some individuals in the normal population may be
 16     more sensitive than others.
 17
 18     13.5.2 Nitrates
 19          The toxicologic data base for inhaled NO3" is quite sparse.  Sackner et al.  (1976)
 20     exposed anesthetized dogs to sodium nitrate (< 1 /mi diameter particles) at 740 or
 21      4,000 /ig/m3 for 7.5 min.  No effects on pulmonary function  (i.e., static compliance,
 22      functional residual capacity, total respiratory resistance) were found.  An injection of 100 mg"
 23      NaNO3 resulted in no change in pulmonary mechanics, arterial blood gases,  cardiac output,
 24      or pulmonary blood pressure.  Ehrlich (1979) examined the effect of NO3" on resistance to
 25      respiratory infection.  Mice were exposed for 3 h to various NO3" salts at maximal
 26      concentrations as follows: lead nitrate (Pb[NO3]2),  2,000 jug/m3; calcium nitrate
 27      (Ca[NO3]2), 2,800 jig/m3; sodium nitrate (NaNO3), 3,100 j«g/m3; potassium nitrate (KNO3),
28      4,300 /*g/m3; ammonium nitrate (NH4NO3), 4,500 /xg/m3; and zinc nitrate (Zn[NO3]2),
29      1,250 /zg/m3. Following exposure, the animals were challenged with a bacterial aerosol, and
30      mortality determined after 14 days.  Only the Zn(NO3)2 exposure resulted in any significant
31      mortality increase, the extent of which seemed to be exposure concentration related; the peak

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exposure level increased mortality by -20%.  However, since the response was similar to
that seen with zinc sulfate (ZnSO4), the investigator ascribed the effect to the zinc ion (Zn+2)
rather than to the NO3".
     Busch et al. (1986) exposed rats and guinea pigs with either normal lungs or elastase-
induced emphysema to NH4NO3 aerosols at 1 mg/m3 for 6 h/day, 5 day/week for 4 weeks.
Using both light and electron microscopy, the investigators concluded that there were no
significant effects of exposure on lung structure due to nitrate exposure.
     Charles and Menzel (1975) examined  the effects of NO3~ on the release of histamine by
guinea pig lung fragments; response to some pollutants may be a function of their ability to
elicit histamine  release. Lung fragments were incubated for 30 min with 20-200  mM
NH4NO3. Histamine was released in proportion to the concentration of salt present.
However, the response was not totally due  to NO3"; NH4" was also a possible contributor.
     Kunimoto et al. (1984b) studied the effects of NO3" upon components of rat RBC
membranes.  Cells were incubated with NaNO3 for 1 h at levels up to 1 mM.  A decrease in
sialic acid content of the cell membrane was found at concentrations >0.1 mM,  and a
concentration dependent decrease in Ca2+,  Mg2+-ATPase activity of the membrane, with
little change in  Na+, K+-ATPase activity,  was noted. In vivo testing, injection  of >50 mM
of NaNO3 also  resulted in depressed Ca2+, Mg2+-ATPase activity.  The investigators
concluded that NO3" may affect RBCs by altering the transport of Ca2+ across the cell
membrane.

Summary
      Inhalation studies with nitric acid are limited and no conclusions can be reached.
Likewise, the toxicologic data base for inhaled nitrates is sparse, with no conclusions
possible.
 13.6  SUMMARY
      A large number of experiments, designed to evaluate the health effects of NOX
 (primarily NO2) on various animal species, have been conducted; and NO2 appears to be the
 most toxic.  Many of the experiments were performed at very high concentrations of NO2
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 (above 9,400 /xg/m3 [5.0 ppm] for days, weeks, or months) to ensure eliciting the effects
 being studied and thus provide only limited or no information directly applicable to standard
 setting.  Therefore, this document focuses on studies conducted at less than 9,400 /*g/m3
 (5.0 ppm).  Even so, ambient levels are much lower.  For example, except for the Los
 Angeles Basin, annual averages are  less than the NAAQS of 100 ^g/m3 (0.053 ppm).  How
 then are animal studies at levels considerably above ambient to be interpreted in.terms of
 human health risk?  Extrapolating animal data to humans has both qualitative and quantitative
 components.  As has been discussed and will be summarized below, NO2 causes a
 constellation of effects in several animal species, most notably, effects on host defenses
 against infectious pulmonary disease, lung metabolism/biochemistry, lung function, and lung
 structure.  Because of basic physiological, metabolic, and structural similarities in all
 mammals (laboratory animals and humans), the commonality of the observations in several
 animal species leads to a reasonable conclusion that NO2 could cause similar types of effects
 in humans. However,  because of the differences between mammalian species, we do  not
 know exactly what exposures  would actually cause these effects in humans.  That is the topic
 of quantitative extrapolation.  Limited research on the dosimetric aspect (i.e., the dose to the
 target tissue/cell that actually  causes toxicity) of quantitative extrapolation suggests that dose
 patterns within the respiratory tract of animals and humans are similar, without yet providing
 adequate values to use for extrapolation. Unfortunately, very little information is available
 on the other key aspect of extrapolation, species sensitivity (i.e., the response of the tissues
 of different species to a given dose).  Thus, from currently available animal studies we know
 what human health  effects NO2 could cause, rather than what effects NO2 actually causes.
 Such knowledge is still quite valuable because it can enable:  (1) identification of potential
 hazards for humans that are either unmeasured or unmeasurable in humans; (2)  estimation of
 the biological plausibility of effects observed in epidemiological studies, which, by their very
 nature, cannot provide definitive cause-effect relationships;  (3) identification of mechanisms
 of effects that can enable enhanced interpretation of the potential severity of effects in  human
 studies; and (4) generation of hypotheses for testing in human studies, thereby advancing the
human data base.
     With the above issues in  mind,  the animal toxicology data base for NO2 will be
summarized according to major classes of effects and topics of special interest.  These
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 1     include:  ariimal-to-human dosimetric estimates, biochemical and cellular mechanisms, effects
 2    > on host defenses, effects of chronic exposure on the development of chronic lung disease,
 3     potential carcinogenic or co-carcinogenic effects, extrapulmonary effects, susceptibility of
 4     subpopulations, influence of exposure patterns, and interactions with other co-occurring
 5     pollutants.  Several other topics or issues are not summarized here because of the paucity of
 6     the data or lack of current interpretability.  For more information on these topics (e.g.,
 7     mortality from NO2 and effects of other NOX compounds, such as NO and HNO3), see the
 8     section summaries within this chapter.
 9      ,..•:.-•--
10     13.6.1  Animal-to-Human Dosimetric Estimates
11          The number of experiments in which NO2 uptake values is reported are few, two for
12     the upper respiratory tract, one for the lower respiratory tract, and two for the total
13     respiratory tract.  The upper respiratory tract uptake efficiencies were 25 to 28 % for rats and
14     45 to 85%  for dogs, depending on the mode of breathing and ventilation.  Thirty-six percent
15     uptake was obtained in the lower respiratory tract of isolated ventilated rat lungs; however,
16    ' because the low ventilation used is not representative of living rats, the measured uptake most
17     probably is not representative.  The two total respiratory tract uptake experiments involved
18     normal humans and asthmatics and resulted in uptakes of 81 to 92% and 73 to 88%,
19     respectively.   In both experiments percent uptake increased as ventilation increased.  Thus,
20     the available data on in vivo uptake of NO2 by various species indicate that a large
21     percentage of inhaled gases is removed in the respiratory tract, depending on the species,
22     mode  of breathing, ventilation, and possibly just as important, the experimental  procedures.
23          A comparison of the results from  the one NO2 dosimetry modeling study to
24     morphological data that shows the centriacinar region to be most affected by NO2 indicates
25     qualitative agreement between predicted maximum tissue doses and observed effects in the
26     pulmonary region.  Comparisons in the tracheobronchial region, however, indicate that dose-
27     effect  correlations may be improved by considering other expressions of dose such as total
28     absorption by an airway.  Further research is needed to define toxic mechanisms, to refine
29     our knowledge of important physical, chemical, and morphological parameters,  to develop
30     NO2 dosimetry models using this information, and to perform dosimetry experiments with a
31     variety of species.
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   1      13.6.2  Biochemical and Cellular Mechanisms
   2           Acute exposure to NO2 at concentrations at or below 9,400 /*g/m3 (5 ppm) can oxidize
   3      polyunsaturated fatty acids in cell membranes as well as functional groups of proteins (either
   4      soluble proteins in the cell, such as enzymes,  or structural proteins, such as components of
   5      cell membranes).  Such oxidation (free radical-mediated) reactions may well be the
   6      mechanisms by which NO2 elicits direct toxicity on lung cells.  Such a proposed mechanism
  7      of action suggests the importance of lung antioxidant defenses, both endogenous (e.g.,
  8      maintenance of lung glutathione levels) and exogenous (e.g., dietary vitamin C and E), in
  9      identification of potential susceptible populations at risk of NO2 inhalation.  The direct
 10      cytotoxic effects of NO2 on epithelial cell membranes may be the  fundamental mechanism of
 11      edemagenesis in response to NO2 exposure, while the direct cytotoxicity of NO2 to
 12      membranes of alveolar macrophages could  well be the mechanism underlying increased
 13      infectivity of bacteria and viruses in lungs of animals and humans  exposed to NO2.  A large
 14      number of studies have suggested that various  enzymes in the lung, including glutathione
 15     peroxidase, superoxide dismutase, and  catalase, may also serve to  defend the lung against
 16     oxidant attack.  One may speculate that were there to be a threshold level for NO2 toxicity to
 17     the lung, it would be that concentration of NO2 that was able to overwhelm  these endogenous
 18     defense systems of the lung.
 19
 20     13.6.3 Effects on Host Defenses
 21          Although the primary function of the respiratory tract is to insure an efficient exchange
 22     of gases, this organ system must also provide the body with a first line protective barrier
 23     against inhaled viable and nonviable airborne agents.  Thus, any breach in this defense
 24     system might increase the risk of disease in the host.  During the past several years numerous
 25     studies that have contributed to our understanding  of these various  defense systems have been
 26     reported.  From this literature it becomes readily evident that exposure to NO2 can  result in
 27     the dysfunction of these host defenses, increasing susceptibility to infectious respiratory
28     disease. The typical host defense parameters affected by NO2 include the rate of mucociliary
29     clearance, functional and biochemical activity of alveolar macrophages, immunological
30     competence,, and susceptibility to experimentally induced respiratory infections.


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 1          The distribution of cellular injury from NO2 extends throughout the lower respiratory
 2     tract. In the conducting airways, NO2 reacts with the ciliated epithelium,  causing a decrease
 3 ,    in. ciliary beating rates and, as a consequence, a significant reduction in mucus transport.
 4     The first of these.effects begins to appear after several weeks of exposure  to around
 5     1,880 jwg/m3 (1 ppm) NO2 and  becomes clearly evident at 3,760 /xg/m3 (2 ppm). Shorter
 6     periods  (several days) of exposure to levels as low as 3,760 /Ltg/m3 are also effective.  Such
 7     impairment could render this physiological line of defense less effective in the process of lung
 8     clearance.
 9          At the alveolar level the host defense system that receives direct exposure to the inhaled
10     pollutant is the alveolar macrophage, which is responsible for maintaining sterility of the
11     pulmonary region, clearing particles from this region, and participating in immunological
12     functions.  Morphological appearance of these defense cells changes after  a 6-month exposure
13     to as little  as 940 /^g/ni3 (0.5 ppm) with a 1-h spike to 3,760 ^g/m3 (ppm 2.0 ppm).
14     Measurements of the functional integrity of macrophages isolated from the lungs of
15     NO2-exposed animals indicate that macrophages can be depressed to such  a degree that the
16     host can no longer effectively maintain pulmonary sterility.  Functional changes that have
17     been reported are:  at 560 jug/m3 (0.3 ppm), intermittent for 13 to 14 days,  the suppression
18     in phagocytic ability and stimulation of lung clearance; at 4,320 jug/m3 (2.3 ppm) for 17 h,
19     a decrease in bactericidal activity; and at 3,760 jtig/m3 (2.0 ppm) for 6 months, a decreased
20     response to migration inhibition factor.  Macrophages isolated from humans exposed for 3 h
21     to  1,120 /ig/m3 (0.6 ppm) NO2 were less able to inactivate influenza virus than controls.
22          The importance of these defenses in maintaining pulmonary sterility  against invading
23     microorganisms becomes evident when these "impaired" animals have to cope with a
24     laboratory-induced  pulmonary infection.  Animals exposed to NO2 succumb to the bacterial
25     or  viral infection in a concentration-response manner. Mortality also increases with
26     increasing length of exposure to a given concentration of NO2.  After acute exposure,
27     3,760 jug/m3 (2 ppm) is the lowest-observed-effect level.  Exposure to concentrations as low
28     as  940  jtig/m3 (0.5  ppm) will cause effects in the infectivity model after 6 months, but as the
29     concentration increases, the effect becomes evident sooner and is more significant.  Thus,
30     evidence indicates that these cells are no longer capable of isolating, transporting, or
31     detoxifying these deposited microbes due to their reduced level of phagocytosis, lytic
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   1     potential, ability to produce interferon, and other responses.  Impacts of different exposure
   2     patterns on infectivity are described later (Section 13.6.8).
   3          Since NO2 has been shown to cause an increase in susceptibility to both bacterial and
   4     viral infections, a few investigators have examined the immune system to explain this change
   5     in susceptibility. In the cases in which lung humoral and cell-mediated immunity have been
   6     investigated, effects have been observed after acute exposure to concentrations
   7     > 9,400 fj.g/m3 (5 ppm).  These findings illustrate the complexity of pulmonary
   8     immunotoxicity, since the direction of the effect (i.e., increase or decrease) was dependent
   9     upon NO2 concentration and the length of exposure.  Furthermore, systemic and pulmonary
 10     immune responses sometimes differed.
 11           Subchronic exposure to NO2 has also been found to affect the production of serum
 12      neutralizing antibody to viruses and humoral primary antibody responses at concentrations as
 13      low as 752 /*g/m3( 0.4 ppm) and 940  ,Kg/m3 (0.5 ppm) with 1-h daily spikes to 3,760 jwg/m3
 14      (2.0 ppm).  Other significant immunological effects attributed to subchronic NO2 exposure
 15      include alterations in serum immunoglobulin levels at 1,880 ^g/m3 (1.0 ppm).
 16          Acute exposure to high concentrations of NO2 significantly decreased the number of
 17      spleen and thymus cells; however, at a lower concentration, the number of spleen cells
 18     actually increased. Others have shown that the reduction in primary antibody response of
 19     spleen cells against sheep red blood cells was more influenced by a suppression of B cell
 20     function, while for the secondary antibody response,  the T cells were more affected. Levels
 21     of NO2  (intermittent exposure, 7 weeks) that affect the percentages of subpopulations of
 22     T and B cells have been as low as 470 ^g/m3 (0.25 ppm).  Chronic intermittent exposure to
 23     high levels of NO2 results in a biphasic response. Antibody titers to sheep red blood cells
 24     were increased after 10 weeks of exposure, unchanged at  20 weeks, and decreased at
 25     30 weeks. There were no effects when a T-independent antigen was used.  A similar
 26     biphasic response was observed for a graft versus host reaction.  Response of lymphocytes to
27     a T-cell mitogen (phytohemagglutin) was depressed at all  exposure times tested.
28
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 1     13.6.4  Effects of Chronic Exposure on the Development of Chronic Lung
 2              Disease
 3          Since humans can be exposed chronically to NO2, such exposures in animals have been
 4     studied rather extensively, typically using morphologic and/or morphometric methods.  Less
 5     focus has been on pulmonary function effects.  Generally, this research has shown that a
 6     variety of structural and correlated functional alterations occur, many of which are reversible
 7     when exposure ceases; however, in some cases, emphysema has been observed.
 8          Pulmonary function following NO2 exposures in experimental animals showed, consistent
 9     patterns among different treatment conditions and animal species.   Exposures to diurnal
10     spikes of NO2 superimposed on a constant background level, simulating NO2 patterns in the
11     urban environment, produced a decrease in lung distensibility in both mice and rats. These
12     changes were very subtle, occurring in mice after  1 year of exposure to very low
13     concentrations, 380 jag/m3 (0.2 ppm) NO2 with spikes of 1,500 jug/m3 (0.8 ppm) NO2.  The
14     sensitivity of the lung to irritants was generally increased following NO2 exposures, except
15     when the irritant was higher concentrations of NO2. Impaired gas exchange occurred
16     following 3 months of exposure to 7,520 jug/m3 (4.0 ppm) NO2 and was reflected in
17     decreased arterial O2 tensions, impaired physical performance, and increased anaerobic
18     metabolism.  All these studies taken together demonstrate that NO2 produces subtle to
19     dramatic changes in pulmonary function depending on the concentration and duration of
20     exposure.  Lung distensibility and gas exchange are the parameters most consistently affected
21     by exposure.
22           Although NO2 produces morphological changes in the respiratory tract, the data base is
23     sometimes confusing due to quantitative and qualitative variability in responsiveness between,
24     and even within, species.  Thus, for example,  the rat appears to be relatively resistant to
25     NO2,  although this is the most commonly used experimental animal involved in
26     morphological assessments of exposure.  In any case, when effective levels are used, the
27     target site is the region  that includes the terminal and/or respiratory bronchioles, alveolar
28     ducts, and alveoli. Sensitive cells are the ciliated epithelial cells of the bronchioli and the
29     Type  1 epithelial cells of the alveoli.
30           Acute exposures to concentrations of 9,400 jwg/m3  (5 ppm) or less generally produce
31      minimal to no lesions in the rat; however, similar exposures in the guinea pig  may result in
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  1     some epithelial damage.  Higher exposure concentrations result in changes typified by
  2     hypertrophy and hyperplasia of bronchiolar and alveolar Type  1 epithelium and proliferation
  3     of Type 2 alveolar cells.
  4          Longer-term exposures result in lesions in some species with concentrations as low as
  5     560 to 940 jtig/m3 (0.3 to 0.5 ppm).  These are characterized by epithelial damage similar to
  6     that described above but with the involvement of more proximal airways.  Many of these
  7     changes,  however, will resolve even with continued exposure,  and long-term exposures to
  8     levels above about 3,760 ^g/m3 (2.0 ppm) are required for more extensive and permanent
  9     changes in the lungs.  Some effects are relatively persistent, for example bronchiolitis; while
 10     others tend to be reversible and limiting even with continued exposure, for example,
 11     epithelial cell hyperplasia.  In any case, it seems that for both acute or longer term exposure
 12     regimens, the response is more dependent upon concentration than exposure duration.
 13     Although ambient exposures are generally to a baseline level with superimposed spikes
 14     reaching some higher level, the contribution of these spikes to  morphologic damage over that
 15     due to baseline exposure is not as yet certain.
 16          There is very substantial evidence that long-term exposure of several species of
 17     laboratory animals to high concentrations of NO2 results in morphologic lung lesions that
 18     meet the current NIH Workshop criteria for  an animal model of emphysema. Those criteria
 19     are:  "An animal model of emphysema is defined as an abnormal state Of the lungs in which
20     there is enlargement of the airspaces distal to the terminal bronchiole. Airspace enlargement
21      should be determined qualitatively in appropriate specimens and quantitatively by stereologic
22     methods." Destruction of alveolar walls, an essential additional criterion for human
23      emphysema, has been reliably reported in lungs from animals in a limited  number of studies.
24      While the lowest NO2 concentration for the shortest exposure duration which will result in
25      emphysematous lung lesions can not be determined from these published studies, the required
26      N02 concentrations for such long-exposure durations are far greater than those currently
27      reported in ambient air.
28
29      13.6.5  Potential Carcinogenic or Co-Carcinogenic Effects
30          Literature searches revealed no published reports on NO2  studies using  classical whole
31      animal bioassays for carcinogenesis.  Research with mice having spontaneously high tumor

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rates was equivocal.  Although several co-carcinogenesis investigations have been undertaken,
methodological and interpretative problems prevent drawing adequate conclusions.  Reports
on whether NO2 facilitates the metastases of tumors to the lung are also inadequate to form
conclusions, since the most appropriate models and statistical methods were not used.  Other
investigations have centered on whether NO2 could produce nitrates and nitrites that, by
reacting with amines in the body, could produce animal carcinogens (nitrosamines).  A few
studies found that in  animals treated with high doses of amines and exposed to NO2,
nitrosoamines were formed.  However, in a related study in two species, a 1- or  2-year
exposure to  18,800 jwg/m3 (10 ppm) did not cause an excess incidence of cancer. In
summary, the evidence suggests that further study may be warranted.

13.6.6  Extrapulmonary Effects
     Although it is clear that the effects of NO2 exposure extend beyond the confines of the
lung, the interpretation of these effects relative to pptential human risk is not clear.
     After acute exposure,  there appears to. be a transient rise in WBCs.  Red blood cell
count has been shown to increase and decrease, but changes in hematocrit and hemoglobin do
not occur. Although NO2"  and especially NO3" do appear in  the blood, increased MetHb is
not typically reported at near ambient levels of exposure.  The evidence does suggest that
there is a change in composition of the RBC membrane constituents; however,  whether this
indicates.an  increased younger population of erythrocytes or an apparent aging has not been
resolved.  Several of the traditional serum markers of tissue injury are elevated with acute
near ambient exposure to concentrations as low as 470 ^g/m3 (0.25 ppm), but this effect is
also transient.  Chronic exposure (at least 3 months)  seems to decrease serum proteins,
lipoproteins, and plasma cholinesterase; all of which suggest hepatic damage.  Although
changes in the microsomal oxidative system are observed during acute exposure to
concentrations  as low as 470 /tig/m3 (0.25 ppm), little evidence of chronic hepatic damage has
been reported.  Swollen liver mitochondria, edema, and inflammatory parenchymal changes
after chronic exposure to between 940 and 2,000 fig/m3 (0.5 and 1.05 ppm) NO2 have been
reported in two studies.  Studies of NO2 effects on the kidney are too few to justify any
conclusions.
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  1          Nitrogen dioxide can produce effects on the cardiovascular system, but these effects
  2     seem to occur only at levels 20 to 100 times ambient concentrations. The changes that have
  3     been observed in the central nervous system require further study before any kind of
  4     interpretation is possible.  The behavioral changes seen, especially the changes in forced
  5     swim and running wheel performance, are reminiscent of the acute performance decrements ,
  6     seen in humans with ozone exposure.
  7
  8     13.6.7  Susceptibility of Subpopulations
  9          Two subpopulations have been examined for susceptibility, young animals and animals
 10     with elastase-induced emphysema. Neither were more susceptible and in some studies were
 11     found to be more resistant than their appropriate NO2-exposed controls.
 12          Susceptibility to NO2 exposures was not affected  in animals treated intratracheally with
 13     elastase to produce a condition of experimental emphysema,  but sensitivity was increased in
 14     mice with a genetic defect in connective-tissue metabolism that results in pulmonary
 15     emphysema.   Newborn and mature animals are affected differently by NO2 exposures,
 16     particularly rats exposed to continuous background NO2 concentrations of 1,880 or
 17     3,760 ^g/m3 (1 or 2 ppm) with diurnal spikes to three  times the baseline. A 6-week
 18     exposure of the adult rats  caused an increase in the thickness of the alveolar basement
 19     membrane and a greater increase in the volume of Type 2 cells, compared to the neonatally-
20     exposed rats.  Pulmonary  function effects were correlated to these changes.  Lung
21     distensibility was increased transiently (after 3 but not 6 weeks of exposure) in rats exposed
22     as newborns, but was decreased in rats exposed for 6 weeks  as young adults.  Hamsters
23     exposed to high NO2 concentrations as newborns had pulmonary function changes one year
24     later indicative of mild pulmonary emphysema, but these changes were not found in hamsters
25     exposed when older.
26
27     13.6.8  Influence of Exposure Patterns
28         Several animal  lexicological studies have elucidated the relationships between
29     concentration (C) and duration (T, time) of exposure, clearly indicating that the relationship
30     is quite complex, rather than the simple formula, C1 x T1 = Effect. Most of this research
31      used the infectivity model in which air- and NO2-exposed mice are exposed to bacteria, and

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 1     decreases in host defenses are measured as increases in mortality.  Early C X" T studies
 2     demonstrated that concentration had more impact on mortality than did exposure duration.
 3     More recent work evaluated ambient exposure patterns consisting of a continuous, low
 4     baseline of NO2 on which are superimposed brief peaks of higher concentrations of NO2.
 5     When components of this pattern were tested and compared to the entire exposure pattern, the
 6     decrease in host defenses was dependent upon the specific exposure pattern; that is, the
 7     outcome could not be predicted based upon knowledge of the components. Few studies of
 8     ambient patterns of NO2 have been investigated for other classes of effects (e.g., pulmonary
 9     function, morphology); they generally support the conclusion that the pattern, rather than the
10     simple product of C x T, is responsible for the results.  Therefore, this body of work does
11     not provide a mathematical model to relate various exposure patterns to specific effects.
12     However, by illustrating the importance of concentration, duration, .and exposure patterns, it
13     does demonstrate the need for rather advanced exposure assessments for more precise
14     estimates of health effects and the need to understand the G  x  T;relationship far better to
15     enable extrapolations of effects from one exposure scenario to another.
16                   ,                                                :
17     13.6.9  Interactions  with Other Pollutants
18           Humans are exposed to complex pollutant mixtures, not NO2 alone, making it important
19     to understand the interaction of NO2 with these other pollutants. Unfortunately, such studies
20     are rare and most were conducted with binary mixtures containing O3.  For information on
21  ,   other interactions, see Section 13.3.  Studies  of O3 plus NO2,  the focus of this summary, are
22     especially pertinent because: (1) they both are photochemical oxidants, (2) NO2, being a
23     primary precursor of O3, is temporally and spatially related to O3, and (3) O3 and NO2
24     generally cause most of the same classes of effects, with O3 being significantly more potent.
25     Numerous studies of lung morphology, antioxidant metabolism, and host  defenses against
26     infection have found that the effects were primarily due to O3  (typically in cases using low,
27     noneffective levels of NO2), were additive, or were synergistic, depending on the   -
28     concentrations, exposure durations, exposure patterns, and end point examined. The findings
29     of either additivity or synergism are of concern because of the ubiquitous, co-occurring
30      nature of O3 and NO2 and the type of effects observed. For example, if one of these
31      pollutants is causing a decrease in host defenses, even an additive response to the other
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 1      pollutant would increase the incidence or severity of the effect.  Precise interpretation of
2      these findings to ambient scenarios is confounded, however. In the ambient air, the common
3      diurnal pattern is a series of peaks of the photochemical oxidants and their precursors  (e.g.,
4      NO, NO2, O3); there is some mixing between the peaks.  Such a "real-world" pattern has not
5      been tested under controlled conditions allowing estimations of the relative lexicological role
6      of the compounds.  Nevertheless, studies with NO2 and NO2-O3 mixtures illustrate the
7      importance of exposure patterns, so extrapolating the mixture study results to ambient
8      patterns raises concern, but does not allow precise conclusions.
9
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                   14.  EPIDEMIOLOGY STUDIES
                     OF OXIDES OF NITROGEN
14.1  INTRODUCTION
     This chapter discusses the epidemiological evidence for the effects of oxides of nitrogen
(NOX) on human health. The major emphasis is on the effects of nitrogen dioxide (NO^
because it is the NOX compound measured in most epidemiological studies and because it is
the NOX compound currently of greatest concern from a public health perspective.  Human
health effects associated with exposure to NO2 have been the subject of several literature
reviews since 1970:  National Research Council (1971, 1977), World Health Organization
(1977),  Samet et al. (1987, 1988) and Graham et al. (1990). Oxides of nitrogen have also
been reviewed previously by the U.S. Environmental Protection  Agency (1982),  which
presented a comprehensive review of studies conducted up  to 1980.  This chapter focuses
mainly on studies conducted since 1980, while also utilizing some key information from
earlier literature.
     Studies discussed in the text of the chapter are those studies which provide useful
quantitative information on exposure-effect relationships for health effects associated with
ambient air levels of NO2 likely to  be encountered in the United States. In addition, some
studies that do not provide quantitative information are briefly discussed in the main text as
appropriate to help elucidate particular points concerning the health effects of NO2.  The
studies discussed in the main text and additional studies, evaluated by the author but found to
be of limited usefulness for present criteria development purposes, are summarized in
Appendix 14 A.
     The chapter is organized as follows.  First respiratory illness studies are discussed to
include studies that meet criteria  (see  Section 14.5.3) for use in  a quantitative analysis
followed by studies that provide qualitative information. Next,  studies are described that
examine effects of ambient NO2  exposure on pulmonary function. Then, a short discussion
of occupational studies is provided. Finally a quantitative  analysis is presented that
synthesizes the available evidence on  respiratory illness.
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  1          In U.S. Environmental Protection Agency (1982), a group of studies examining the
  2     relationship between respiratory illness and exposure in the home to gas combustion products
  3     (cooking fuel) were evaluated. At that time, those studies inferred the presence of NO2 by
  4     the presence of gas combustion emission sources.  Since then, new studies and updates of
  5     earlier ones have been conducted which provide data on NO2 concentrations ,and estimates of
  6     exposure. Most studies of NO2 exposure show more variation in indoor air than in outdoor
  7     air (see Chapter 7).
  8          The studies assessed in this chapter are evaluated for several factors of importance for
  9     interpreting epidemiological studies of the health effects of NO9.  These factors are
                                                                  £-t
 10     (1) measurement error in exposure, (2) misclassification of the health outcome,  (3) selection
 11     bias, (4)  adjustment for covariates, (5) publication bias, (6) internal consistency, and
 12     (7) plausibility of the effect based on other evidence. Because these factors are common to
 13     all studies,  a brief discussion follows.
 14          Measurement error in exposure is potentially one of the most important methodological
 15     problems in epidemiological studies of NO2. Ideally, personal monitors would be placed on
 16     all subjects for the entire period of a study. Even then, some error associated with the
 17     monitoring device itself would remain.  Such intensive personal monitoring is not feasible.
 18     Instead, NO2 exposure may be estimated by source description, personal monitors,  in-home
 19     monitors, and fixed-site outdoor monitors.  In most of the early studies, gas stove use was
20     related to health outcomes without any direct exposure estimates.
21          The effect of measurement error on estimation has been studied by several authors
22     including Shy et al. (1978), Gladen and Rogan (1979), StephansM and  Carroll (1985),
23     Walker and Blettner (1985), Fuller (1987), Lebret (1987), Schafer (1987), Whittemore and
24     Keller (1988), Samet and Utell (1990), and Yoshimura (1990). In general, measurement
25     error that is independent of the health outcome will result in estimated effects biased towards
26     the null.  Whittemore and Keller (1988) specifically consider the data of Melia et al. (1980)
27     as described by Florey et al.  (1979) and show that a 20%  misclassification rate of the
28     exposure  category will result in an underestimate of the logistic regression coefficient by as
29     much as 50%. StefansM and Carroll (1985) have shown that even without the independence
30     of error related to outcome, the bias is towards the null in situations where the risks are not
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 1     extremely close to 0 or 1.  The use of the presence of a gas stove as a surrogate for an actual
 2     NO2 exposure introduces misclassification.
 3          If the observed health effects (see Chapter 13 and Section 14.2) result from peaks
 4     generated during source use rather than long-term averages, then the use of estimated
 5     averages creates another source of measurement error. However, inadequate data are
 6     available to adequately evaluate the relative contributions of personal exposures of peak vs.
 7     average values to health effects studied in epidemiology studies.  Peak levels in bedrooms and
 8     other locations probably are not as high as in kitchens (see Chapter 7), and most indoor
 9     activity probably occurs in locations other than the kitchens (see Chapter 8). Harlos et al.
10     (1987) state that NO2 concentrations in the kitchen are different for each cooking  event in a
11     12- or 24-h period.  To improve the measure of exposure, the NO2 concentration  during
12     room occupancy is needed. The average bedroom NO2 concentration already contains most
13     of the time-location information by virtue of being a primary daily location, especially for
14     infants.  In  most homes, peak values may be related to average values such that reducing
15     peaks reduces the average concentration.  Average values may serve as surrogates for the
16     peaks; however, if effects are associated with the peaks,  then the use of averages will
17     increase measurement error.
18          Misclassification of the health outcome can occur whether the outcome is continuous,
19     such as a measure of pulmonary function, or dichotomous, such as the presence or absence of
20     respiratory symptoms.  Lung function is typically measured with spirometry, a well
21     standardized (Ferris, 1978) technique.  The measurement errors of the instruments collecting
22     the data have also been carefully estimated, and random errors will simply add to the error
23     variance.  On the other hand, respiratory symptoms, illness, and infections are usually
24     measured by a questionnaire.  Obviously, questionnaire measurements that depend on recent
25     recall are better than one those based on recall of events that occurred several years in the
26     past.  Questionnaires for cough and phlegm production have been standardized, such as the
27     British Medical Research Council (BMRC) questionnaire (American Thoracic Society, 1969)
28     and revisions of the BMRC questionnaire (Ferris, 1978; Samet, 1978). These questionnaires
29     and modifications of them have been used extensively.  The effect of misclassification of the
30     health outcome is an area in need of additional research.
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 1           The possibility of selection bias, although a concern of every study, seems very low for
 2      the epidemiologic studies of NO2.  Most studies are population-based with little selection by
 3      the individuals themselves.  The selection of an area based on previous demonstration of an
 4      association of symptoms with gas stove use could potentially represent a serious flaw.
 5      However,  we are unaware of the selection of any area for inclusion in the studies discussed
 6      based on existing health response data.
 7           Most studies of respiratory disease and NO2 exposures discussed here measured the
 8      important  covariates of age, socioeconomic level of the parents, gender, and parental smoking
 9      habits. The estimated effect (regression coefficient of disease on NO2 exposure) will be an
10      overestimate when a missing covariate is either positively or negatively correlated with both
11      the exposure variable and the health outcome.  The estimated effect will be an underestimate
12      in the other  two situations.  Ware et al.  (1984) found that parents with some college
13      education  were more likely to report respiratory symptoms and were less likely to use a gas
14      stove, leading to an underestimate of the health effect if education were to be left out of the
15      analysis.
16           Publication bias is the result of the increased likelihood of publication of studies that
17      have positive results, leading  to a bias in the literature reviewed towards positive results.
18      There are  two factors that make this bias less likely for epidemiological studies. First, the
19      studies are expensive, well  publicized, and the results are usually published in order to give
20      credit to the researchers involved.  Second, many of the studies included in this section did
21      not produce statistically significant findings, indicating that  there was not a problem in
22      publishing negative studies.
23           Internal consistency is always a check on the validity of a study, but often the authors
24      do not report sufficient detail to check for such consistency. For known risk factors for
25      respiratory effects, a study  should provide the  anticipated associations (e.g., passive smoking
26      with increased respiratory illness or with more wheeze in asthmatic children) and certain
27      patterns of age  or gender effects might be predicted. Results that differ dramatically by  age
28      or gender  should be viewed with caution.  If studies report  results suggesting a significant
29      beneficial  effect of NO2 exposure, then  there may be a problem with the design or  the
30      analysis.
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 1          Finally, the health outcome should be an outcome for which there is a plausible basis to
 2     suspect that NO2 exposure was associated with the effect. Two health outcome measures
 3     have been most extensively considered in the epidemiologic studies:  lung function
 4     measurements and respiratory illness occurrence.  Human clinical and animal lexicological
 5     studies have not indicated a demonstrated effect on lung function at ambient levels in normal
 6     subjects (see Chapters 13 and 15).  The human clinical and animal toxicological studies (see
 7     Chapters 13, 15 and Section 14.5.2) provide a data base demonstrating that NO2,affects
 8     aspects of the respiratory host defense system—thus providing a biologically plausible basis
 9     for epidemiologic observations of associations between NO2 exposure and an increase in
10     respiratory symptoms and infections, especially in children.
11          Each study is subsequently reviewed with special attention given  to the factors just
12     discussed.  Those studies that address these factors more appropriately provide a stronger
13     basis for the conclusions that they draw.  Consistency between studies indicates the level of
14     the strength of the total data base.
15
16
17     14.2  STUDIES OF RESPIRATORY ILLNESS
18          Respiratory illness and the factors  determining occurrence and severity are important
19     public health concerns.  This chapter discusses epidemiological findings relating NO2
20     exposure to respiratory  illness.  This effect is of public health importance because of the
21     potential for exposure to NO2 and since childhood respiratory illness is common (Samet
22     et al., 1983; Samet and Utell, 1990). This takes on added importance since recurrent
23     .childhood respiratory illness (independent of NO2) may be a risk factor for later susceptibility
24     to lung damage (Glezen, 1989; Samet et al., 1983; Gold et al., 1989).
25           A brief discussion of the epidemiology of lower respiratory illness (LRI) in children
26     provides a background for studies examining NO2 exposure in relation to LRI.   Lower
27     respiratory illnesses are generally classified into one of four clinical syndromes:. croup
28     (laryngotracheobronchitis), tracheobronchitis, bronchiolitis,  and pneumonia (Glezen and
29     Denny, 1973; Wright et al.,  1989; McConnochie et al., 1988) (see Section 14.5.1 for further
30     discussion).  In a study in Tucson during the first year of life the most common diagnosis
31     was bronchiolitis, which accounts for 60% of all LRI identified (Wright et al.,  1989).  The
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  1     most common signs and symptoms associated with LRI were wet cough (85%), wheeze
  2     (77%), tachypnea (48%), fever (54%), and croupy cough (38%) (Wright et al., 1989).
  3     A few infectious agents are presumed to cause the majority of childhood LRI.  Bacteria are
  4     not thought to be common causes of LRI in nonhospitalized infants in the United States
  5     (Wright et al., 1989). Seventy-five percent of the isolated microbes were one of four types:
  6     respiratory syncytial virus (RSV), para influenza virus type 1 and 3, and Mycoplasma
  7     pneumoniae (Glezen and Denny,  1973; McConnochie et al., 1988).  Respiratory syncytial
  8     virus is particularly likely to cause LRI in the first 2 years of life.  More than half of all
  9     illnesses diagnosed as bronchiolitis, for which an agent was identified, were positive for RSV
10     (Wright et al., 1989). Denny et  al. (1986) noted that studies that rely on parental reports of
11     symptoms may underestimate illness.  Asking parents about illnesses at the end of the first
12     year of life revealed that one-third of them failed to report illnesses diagnosed by
13     pediatricians and evaluated by study nurses.
14          In  summary, LRI remains one of the major causes of childhood morbidity in the United
15     States (McConnochie et al., 1988).  A large number of factors affect the susceptibility of
16     children and, thus, the subsequential occurrence of respiratory symptoms.  Special attention is
17     directed at viral LRI in the first 2 years of life because the highest incidence and rate of
18     hospitalization for LRI are found at this time and because of the risk of chronic sequelae from
19     LRI in early childhood.  The occurrence of LRI in early childhood may be associated with
20     impaired lung function and growth that appears to persist through adolescence.   Early insult
21     from virus infection in the lower  respiratory tract may be an essential element in the
22     development of chronic and persistent impairment (Glezen, 1989; Gold et al., 1989).  Britten
23     et al. (1987) reported that the extension to age 36 of the earlier work of the Medical Research
24     Council's national survey of health  and development of the 1946 Great Britain Cohort
25     indicates that there can be little doubt in this cohort  of the existence of an association between
26     childhood respiratory experience and adult respiratory morbidity.  They comment that their
27     study, coupled with evidence from Colley et al. (1976), lends support to the  model of
28     acquired lung damage predisposing  to adult disease with genetic susceptibility to respiratory
29     disease being less of a factor.  Denny and Clyde (1986) stated that it is now recognized that
30     infections, reactive airways, and inhaled pollutants (mostly cigarette smoke) are the most
31      important risk factors in the development of chronic lung disease. Thus, factors such as the

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 1     presence of NO2, which increases the risk for LRI, are important because of the associated
 2     public health concern and the potential for increase in the development of chronic lung
 3     disease.
 4           Various studies of LRI have reported rates based on visits to physicians ranging from
 5     about 20 to 30 illnesses per 100 children in the first year of life (Glezen and Denny,  1973;
 6     Wright et al., 1989;  Denny and Clyde,  1986; McConnochie et al., 1988).  Glezen and Denny
 7     (1973)  reported that the rate for LRI ranged from 24 per 100 person-years in infants under
 8      1 year of age and decreased steadily each year through the preschool years, tending to level
 9     off in school children (age 12-14 years) to about 7.5 illnesses per  100 person-years.   Several
10     factors  affect the rate of LRI in children, including: age, immunologic status, prior viral
11     infections, level of health, socioeconomic status (Chanock et al., 1989),  day care attendance,
12     environmental tobacco smoke, NO2 and other pollutants.  Rates also depend on method of
13     illness ascertainment. Studies in the United States (Wright et al.,  1989;  Denny and Clyde,
14      1986; McConnochie et al., 1988) indicated that the overall pattern and incidence of LRI is
15     consistent in different geographic regions during the 2 decades covered by the studies,
16     suggesting that diagnosis and infectious agents have changed little in that time period.
17           The biologic basis underlying increased susceptibility for respiratory illness reflects the
18     status of functioning respiratory host defense mechanisms against the specific infectious
19     organism (e.g., species, strain, virulence).  The host defense system provides protection
20     against inhaled infectious agents through physical and immunologic mechanisms.
21     Additionally, there is an immunologic basis for increased susceptibility of the neonate to
22     infection (Wilson, 1986).  Full-term infants are immune-deficient  (as compared with older
23     children and adults) in essentially all measured immunologic parameters  due to lack of prior
24     exposure and subsequent development of immunity, thus rendering them susceptible to serious
25     infections (Bernbaum et al., 1984; Kibler et al., 1986).
26           The respiratory illness studies in this section are organized as follows.  First, eleven
27      studies that meet criteria (see Section 14.5.3) for use in a quantitative analysis are presented.
28     Within these studies  five related studies conducted by Melia and colleagues in Great Britain
29     are discussed first.  Next two large studies conducted in six United States cities are examined.
30     Then four other quantitative studies are presented that were conducted by different authors in
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  1      different locations. The remainder of the section reviews several other studies that provide
  2      qualitative information concerning respiratory illness.
  3
  4      14.2.1  United  Kingdom Studies
  5           Results of five British studies have been reported by Melia et al. (1977; 1978; 1979; ;
  6      1980; 1982;  1985), Goldstein et al.  (1979; 1981), and Florey et al. (1979; 1982). Parts of
  7      these studies were reviewed previously (U.S. Environmental Protection Agency, 1982), but
  8      their importance requires a more complete discussion of them.
  9           The initial study, reported by Melia et al. (1977), was based on a survey of 5,658
10      children (excludes asthmatics, thus 100 less than the number reported), aged 6 to 11 years,
11      with sufficient questionnaire information in 28 randomly  selected areas of England and
12      Scotland.   The study included a self-administered questionnaire, completed by a parent, that
13      obtained information on the presence of morning  cough,  day or night cough,  colds going to
14      chest, chest sounds of wheezing or whistling,  and attacks of bronchitis. The questionnaire
15      was distributed in  1973 and asked about  symptoms during the previous 12 months.  Colds
16      going to the chest accounted for the majority of the symptoms reported. Information about
17      cooking fuel (gas or electric), age, sex, and social class (manual vs. nonmanual  labor) was
18      obtained,  but information on parental smoking was not.  No measurements of NO2, either
19      indoors or outdoors, were given.
20           The authors presented the results in the form of a contingency table for nonasthmatics
21      with complete covariate information. Table 14-1 is a summary  of that data for nonasthmatic
22      children.  The authors indicated that there was a trend  for increased symptoms in homes with
23      gas stoves, but the increase was only significant for girls  in urban areas. The authors gave
24      no measures  of increased risk.
25           A U.S. Environmental Protection Agency (EPA)  reanalysis of the data in Table 14-1
26      using a multiple logistic model is given in Table 14-2.  Because it had been suggested that
27      gender had an effect on the relationship with "gas cooker",  interaction terms for gender were
28      included in the original model.  None of these proved to  be significant, and they were  ,
29      subsequently dropped  from the model. When separate terms for each gender were used for
30      the effect of  "gas cooker", an estimated odds ratio of 1.25 was obtained for boys and an odds
31      ratio of 1.39 was obtained for girls. The combined odds ratio for both genders was 1.31

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      TABLE 14-1.  SYMPTOM RATES OF UNITED KINGDOM CHILDREN
             BY GENDER, SOCIAL CLASS, AND COOKING TYPE
Age < 8 years
  Social Classes I-EH
     (Nonmanual)

Electric         Gas
       Social Classes IE-V
           (Manual)

    Electric          Gas
Boys
Girls
25.6%
(203)a
22.2%
(171)
26.1%
(88)
30.4%
(112)
29.9%
(375)
31.8%
(393)
37.5% ,
(309)
33.5%
(337)
Age >8 years
Boys
Girls
20.8%
(365)
18.1%
(303)
23.3%
(189)
19.2%
(187)
25.0%
(675)
17.8%
(674)
29.0%
(654)
27.8%
(623)
'Numbers in parentheses refer to number of subjects.

Source: Melia et al. (1977).
         TABLE 14-2. U.S. ENVIRONMENTAL PROTECTION AGENCY
            MULTIPLE LOGISTIC ANALYSIS OF DATA FROM THE
                        MELIA ET AL. (1977) STUDY
Factor81
SES and age
by gender interactions (2 d.f.)
Gas by gender interaction (1 d.f.)
Gas cooker
Gender (female)
SES (Manual)
Age ( < 8 years)
Regression
Coefficient


0.2733
-0.1531
0.2730
0.3864
Standard Likelihood Ratio
Error Chi-Square p-Value


0.0616
0.0612
0.0702
0.0626
2.46
0.72
19.78
6.29
15.48
37.77
0.2922
0.3953
< 0.0001
0.0121
0.0001
< 0.0001
aSES = Socioeconomic status.
d.f. = Degrees of freedom.
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 1      (95% confidence limits of 1.16 to 1.48) and was statistically significant (p< .0001).  The
 2      other main effects of gender, socioeconomic status, and age were all statistically significant.
 3      This reanalysis suggests that gas stove use in this study for children under age eight is
 4      associated with an estimated 31% increase in the odds of having respiratory illness symptoms.
 5          Melia et al. (1979) report further results of the national survey covering two groups:
 6      (1) a new cohort of 4,827 boys and girls, aged 5 to 10 years, from 27 randomly selected
 7      areas that were examined in 1977, and (2) 2,408 children first examined in 1973 who were
 8      followed-up for at least 1 year and whose mothers reported the use of the same cooking fuel
 9      in each year the child was studied.  The 1977 study collected information on the number of
10      smokers in the home.  In the 1977 cross-sectional study, only the prevalence of day or night
11      cough in boys (p ~ 0.02) and colds going to the chest in girls (p<0.05) were found to be
12      significantly higher in children from homes where gas was used for cooking compared with
13      children from homes where electricity was used. Grouping responses according to the six
14      respiratory questions into (1) none or (2) one or more symptoms or diseases yielded a
15      prevalence higher in children from homes where gas was used for cooking than in those from
16      homes where electricity was used (p ~ 0.01 in boys, p=0.07 in girls).  The results of this
17      analysis are presented in Table 14-3. The effect of gender, social class, use of pilot lights,
18      and number of smokers in the house were examined.
19          An EPA reanalysis of the data in Table 14-3 applying a multiple logistic model is given
20      in Table 14-4.  This model contained the same terms as the analysis in Table 14-2.  As in the
21      previous analysis, none of the interaction terms proved to be significant, and they were
22      subsequently dropped from the model.  When separate terms for each gender were used for
23      the effect of "gas cooker" an estimated odds ratio of 1.29 was obtained for boys and an odds
24      ratio of 1.19 was obtained for girls.  The combined odds ratio for both genders was 1.24
25      (95% confidence limits of 1.09 to 1.42). This effect was statistically significant (p< .0001).
26      The other main effects of gender, socioeconomic status, and age were all statistically
27      significant.  This reanalysis suggests that gas stove use in this study is associated with an
28      estimated 24% increase in the odds of having symptoms.
29          The longitudinal results seem to indicate that the relative risk may decline as the
30      children grow older.  The prevalence tended to be higher in 1973 than in  1977 for children of
31      the same age, and the effect of gas cooking seemed to be smaller in 1977 than in 1973.

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  TABLE 14-3.  UNADJUSTED RATES OF ONE OR MORE SYMPTOMS AMONG
        UNITED KINGDOM CHILDREN BY GENDER, SOCIAL CLASS,
                          AND COOKING TYPE
                      Social Classes I-III
                        (Nonmanual)
                  Social Classes III-V
                      (Manual)
Age < 8 years
Boys
Girls
Electric
27.4%
(277)a
. 24.4%
(291)
Gas
31.7%
(145)
27.6%
(134)
Electric
32.8%
(485)
27.8%
(497)
Gas
36.7%
(313)
36.3%
(336)
.
Age >8 years
Boys
Girls
19.2%
(286)
14.8%
(243)
28.3%
(113)
18.6%
(118)
23.6%
(501)
21.5%
(437)
26.9%
(338)
18.5%
(313)
"Numbers in parentheses refer to number of subjects.

Source: Melia et al. (1979).
    TABLE 14-4.  U.S. ENVIRONMENTAL PROTECTION AGENCY MULTIPLE
      LOGISTIC ANALYSIS OF DATA FROM MELIA ET AL. (1979) STUDY
Regression
Factora Coefficient
SES and Age
by gender interactions (2 d.f.)
Gas by gender interaction (1 d.f.)
Gas cooker
Gender (female)
SES (Manual)
Age (<8 years)


.2183
-.1970
.2225
.5253
Standard Likelihood Ratio
Error Chi-Square p- Value


.0674
.0664
.0764
.0675
1.11
.35
10.43
8.81 -
8.60
61.48
.5749
.5566
.0012
.0030
.0034
< .0001
aSES = Socioeconomic status.
 d.f. = Degrees of freedom.
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  1     However, overall the study observed an association between respiratory illness and the use of
  2     gas for cooking in two separate groups of children, seen 4 years apart in this national study.
  3          This study was followed by a study in 1978 of 808 schoolchildren (Melia et al., 1980),
  4     aged 6 to 7 years, in Middlesborough, an urban area of northern England. Respiratory
  5     illness was defined in the same manner as in the previous study. Weekly indoor NO2
  6     measurements were collected from 66% of the homes with the remaining  34% refusing to
  7     participate.  Nitrogen dioxide was measured weekly by triethanlamine (TEA) diffusion tubes
  8     (Palmes tubes) attached to walls  in the kitchen area and in the children's bedrooms.  In
  9     homes with gas stoves, weekly levels of NO2 in kitchens ranged from 0.005 to 0.317 ppni
 10     (10 to 596 /Kg/m3) with a mean of 0.112 ppm (211 j«g/m3), and levels in  bedrooms ranged
 11     from 0.004 to 0.169 ppm (8 to 318 /*g/m3) with a mean of 0.031 ppm (56 ^g/m3).  In
 12     homes with electric stoves, weekly levels of NO2 in kitchens ranged from 0.006 to
 13     0.188 ppm (11 to 353 /ig/m3) with a mean of 0.018 ppm (34 jwg/m3), and in bedrooms NO2
 14     levels ranged from 0.003 to 0.037 ppm (6 to 70 /ig/m3) with a  mean of 0.014 ppm
 15     (26 jig/m3).  Outdoor levels of NO2 were determined using diffusion tubes systematically
 16     located throughout the area, and  the weekly average ranged from 26 to 45 jtcg/m3 (0.014  to
 17     0.024 ppm).
 18          One analysis by the authors was restricted to those 103 children in homes where gas
 19     stoves were present and where bedroom NO2 exposure was measured; the data are shown in
20     Table 14-5. A linear regression  model was fit to the logistic transformation of the rates.
21     Cooking fuel was found to be associated with respiratory illness, independent of social class,
22     age, sex, or presence of a smoker in the house (p=0.06).  However, when social class was
23     excluded from the regression, the association was weaker (p=0.11).  For  the 6 to 7-year-old
24     children  living in gas stove homes, there appeared to be an increase of respiratory illness  with
25     increasing levels of NO2 in their bedrooms (p=0.10), but no significant relationship was
26     found between respiratory symptoms in those children or their siblings or  parents and levels
27     of NO2 in kitchens.
28          Since no concentration-response estimates were given by the authors, a multiple logistic
29     model was fitted by EPA to the data in Table  14-5 with a linear slope for NO2 and  separate
30     intercepts for boys and girls.  Nitrogen dioxide levels for the groups were estimated by fitting
31      a lognormal distribution to the grouped NO2 data and the average exposures within each

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         TABLE 14-5. UNADJUSTED RATES OF ONE OR MORE SYMPTOMS AMONG
            UNITED KINGDOM BOYS AND GIRLS BY BEDROOM LEVELS OF NO2

Boys
Girls
TOTAL

<0.020
43.5%
(23)a
44.0%
(25)
43.7%
(48)
Bedroom Levels of
0.020-0.039
57.9%-
(19).
60.0%
(15)
58.8%
(34)
NO2 (ppm)
> 0.039
69.2%
.- (13)
75.0%
(8)
71.4%
(21)

Total
54.5%
(55)
. 54.2%
(48)
54.4%
(103)
       "Numbers in parentheses refer to number of subjects.
       Source:  Melia et al. (1980).
 1     interval were estimated (see Hasselblad et al., 1980).  The estimated logistic regression
 2     coefficient for NO2 (in |ug/m3) was 0.015 with a standard error of 0.007. The likelihood
 3     ratio test for NO2 gave a chi-square of 4.94 with one degree of freedom, with a
 4     corresponding p-value of 0.03.  This result suggests that an increase of 30 /ig/m3
 5     (0.016 ppm) in bedroom NO2 levels  (well within the range of the data) would result in a
 6     53% increase in the odds of respiratory illness.  The 95% confidence limits for  the odds
 7     ratio were 1.04 to 2.24.
 8          The study was repeated in January to March of 1980 by Melia et al. (1982).  This time
 9     children aged 5 to 6 years were sampled from the same neighborhood as the previous study,
10     but only  families with gas stoves were recruited.  Environmental measurements were made
11     and covariate data were collected in a manner similar to the previous study (Melia et al.,
12     1980). Measurements of NO2 were available for 54%  of the homes. The unadjusted rates of
13     one or more symptoms by gender and exposure level are shown in Table 14-6. The authors
14     concluded that "... no relation was found between the prevalence of respiratory illness and
15     levels of NO2."  An EPA reanalysis  of the data in Table 14-6 was made using a multiple
16     logistic model similar to the one used for the previous  study (Melia et al., 1980). The model
17     included a linear slope for NO2 and separate intercepts for boys and girls.  Nitrogen dioxide
18     levels for the groups  were estimated  by fitting a lognormal distribution to the grouped
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           TABLE 14-6. UNADJUSTED RATES OF ONE OR MORE SYMPTOMS AMONG
             UNITED KINGDOM BOYS AND GIRLS BY BEDROOM LEVELS OF NO2


Boys

Girls


< 0.020
56.4%
(39)a
60.0%
(25)
Bedroom Levels of
0.020-0.039
67.6%
(37)
41.0%
(39)
NO2 (ppm)
> 0.039
72.0%
(25)
52.2%
(23)

Total
64.4%
(101)
49.4%
(87)
        "Numbers in parentheses refer to number of subjects.
        Source:  Melia et al. (1982).


  1      bedroom NO2 data. The estimated logistic regression coefficient for NO2 (in /*g/m3) was
  2      0.004 with a standard error of 0.005.  The likelihood ratio test for the effect of NO2 gave a
  3      chi-square of 0.51 with one degree of freedom (p=0.48).  This reanalysis suggests that an
  4      increase of 30 jwg/m3 (0.016 ppm) in bedroom NO2 levels would result in a 11% increase in
  5      the odds of respiratory illness.
  6           Melia et al. (1983) investigated the association between gas cooking in the home and
  7      respiratory illness in a study of 390 infants born between 1975 and 1978. When the child
  8      reached 1 year of age, the mother was interviewed by a trained field worker to complete a
  9      questionnaire. The mother was asked whether the child usually experienced morning cough,
10      day or night cough, wheeze on colds going to the chest, and whether the child had
11      experienced bronchitis, asthma, or pneumonia during the past 12 months. No relation was
12      found between type of fuel used for cooking at home and the prevalence of respiratory
13      symptoms and diseases recalled by the mother after allowing for  the effects of gender, social
14      class, and parental smoking.  The authors gave prevalence rates of children having at least
15      one symptom by gas stove use and gender.  The combined odds ratio for presence at
16      symptoms by gas stove use was 0.63 with 95% confidence limits of 0.36 to  1.10.
17
18      14.2.2  United States Six Cities Studies
19          Several authors (Spengler et al., 1979; Speizer et al.,  1980; Ferris et al., 1983;
20      Spengler et al., 1986; Berkey et al., 1986; Ware et al.,  1984; Quackenboss et al,, 1986;

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 1     Dockery et al., 1989; Neas et al., 1990; Neas et al.,  1991) have reported on a series of
 2     studies conducted in six different U.S. cities.  The six cities were selected to represent a
 3     range of air quality based on their historic levels of outdoor pollution and included:
 4	 Watertown, MA; Kingston and Harriman, TN; southeast St. Louis, MO; Steubenville, OH;
 5     Portage, WI; and Topeka, KS.  Approximately  1,500 grade school children were enrolled in
 6    ' each community and were followed for several years.  Families reported the number of
 7     persons living in the home and their smoking habits, parental occupation and educational
 8     background, and the fuels used for cooking and heating.  Outdoor pollution was measured at
 9     fixed sites in the communities as well as at selected households. Indoor pollution including
10     NO2 was measured in several rooms of selected households.  Results of monitoring in
11     Portage, WI, verify the fact that the presence of a gas stove contributes dramatically to the
12     indoor NO2 levels. Table 14-7 is taken from Quackenboss et al. (1986) based on data
13     collected from 1981-82.  These results clearly show the effect of a gas stove not only on the
14     indoor concentrations but also on the personal exposure of the individual.  The study was
15     conducted very carefully with excellent quality control. It gives a good estimate of the
16     exposure resulting from the use of gas stoves in the United States  (see Chapter 7).
17
                    TABLE 14-7. NO2 CONCENTRATIONS (ppm) BY SEASON
                               AND STOVE TYPE IN PORTAGE, WI
Indoor
Season
Summer
Winter
Stove
Gas
Electric
Gas
Electric
Mean
0.016
0.007
0.027
0.005
Std.
Dev.
0.006
0.003
0.013
0.003
Outdoor
Mean
0.006
0.008
0.008
0.009
Std.
Dev.
0.003
0.003
0.003
0.003
Personal
Mean
0.014
0.009
0.023
0,008
Std.
Dev.
0.004
0.003
0.009
0.003
        Source:  Quackenboss et al. (1986).


  1           Speizer et al.  (1980) reported results from the six cities studies based on 8,120 children,
  2      aged 6 to 10 years, who were followed from 1974-77. Health end points were measured by
  3      a standard respiratory questionnaire, which was completed by the parents of the children.
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  1      The authors used log-linear models to estimate the effect of current gas stoves vs. electric
  2      stoves on the rates of serious respiratory illness before age 2.  The analysis gave an odds
  3      ratio of 1.12 (95% confidence limits of 1.00 to 1.26) for gas stove use.  The results were
  4      adjusted for the presence of adult smokers, presence of air conditioning, and socioeconomic
  5      status of the  family. Ware et al. (1984) reported results from the same six communities over
  6      a longer period (1974-79).  Directly standardized rates of reported illnesses and symptoms did
  7      not show any consistent pattern of increased risk for children from homes with gas stoves.
  8      Logistic regression analyses controlling for age, sex, city, and maternal smoking level gave
  9      estimated odds ratios for the effect of gas stoves ranging from 0.93 to 1.07 for bronchitis,
10      cough,  wheeze, LRI index, and illness for the last year.  The index for LRI indicated the
11      presence of either bronchitis, respiratory illness, or persistent cough during the past year.
12      None of these odds ratios were statistically different from 1.  Only two odds ratios
13      approached statistical significance:  (1) history  of bronchitis (odds ratio =  0.86, 95%
14      confidence interval 0.74 to 1.00) and (2) respiratory illness before age 2 (odds ratio —  1.13,
15      95% confidence interval 0.99 to  1.28).  When  the odds ratio for respiratory illness before
16      age 2 was adjusted for parental education, the odds ratio was 1.11 with 95% confidence
17      limits of 0.97 to 1.27 (p=0.14).  Thus, the study suggests an increase in respiratory illness
18      of about 11%, although the increase was not statistically significant at the 0.05 level.  The
19      end point in the Ware et al. study most similar to that of the Melia studies was the LRI
20      index.  The authors gave the unadjusted rates, and from those an  estimated odds ratio of 1.08
21      with 95%  confidence limits of 0.97 to 1.19 were calculated.  Although this rate was not
22      adjusted for other covariates, the effect of those adjustments on other end points was
23      minimal.
24           Dockery et al. (1989) studied a different sample of 5,338 white children in the same six
25      cities, but enrolled during  the period 1983-86.  The children were aged 7 to 11  years at the
26      time of the study.  The end points of chronic cough, bronchitis, chest illness, persistent
27      wheeze, and nonrespkatory illness were not associated with gas stove use in the home.  The
28      health end point of doctor-diagnosed respiratory illness prior to age 2 was marginally
29      significant. Multiple logistic regressions, including use of a gas stove for cooking, gave an
30      odds ratio of 1.15 with 95% confidence limits of 0.96 to 1.37. The odds ratio was adjusted
31      for age, sex, parental education,  city of residence, and use of unvented kerosene heaters.

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 1          Neas et al. (1990, 1991) studied a cohort of 6,273 children from the same six cities.
 2     This cohort included children that were part of the Dockery et al. (1989) analysis, but was
 3     restricted to white children 7 to 11 years of age with complete covariate information and at
 4     least one valid indoor measurement of both NO2 and respirable particles.  This resulted in
 5     1,286 children being included in the analysis.  Methods for measuring indoor pollutants were
 6     described by Spengler et al. (1986).  Indoor pollutants were measured in each child's home
 7     for 2 weeks during the heating season and 2 weeks during the cooling season.  Nitrogen
 8     dioxide was measured by Palmes passive diffusion tubes at three locations.
 9          The analysis of the Neas et al. (1990, 1991)  study was based on the third symptom
10     questionnaire, which was completed by parents following the indoor measurements. The
11     questionnaire reported symptoms during the previous year, including  shortness of breath,
12     chronic wheeze, chronic cough,  chronic phlegm, and bronchitis.  The authors used a multiple
13     logistic model with separate city intercepts, indicator variables for gender and age, parental
14     history of chronic obstructive pulmonary disease, parental history of asthma, parental
15     education, and single parent family status. The increases in symptoms were estimated for an
16     additional 17.3 ppb (31 jug/m3) NO2 exposure. This corresponded to the average effect of a
17     gas stove with a pilot light, based on exposure information from the study.  Table 14-8 shows
18     the odds ratios for the five separate symptoms associated with the increase in NO2 exposure.
19
20
           TABLE 14-8.  ODDS RATIOS AND 95% CONFIDENCE INTERVALS FOR THE
         EFFECT OF AN ADDITIONAL 17.3 ppb NO2 ON THE SYMPTOM  PREVALENCE
Symptom
Shortness of breath
Chronic wheeze
Chronic cough
Chronic phlegm
Bronchitis
Odds Ratio
1.27
1.19
1.21
1.29
1.05
95% Confidence Interval
0.92 to 1.73
0.87 to 1.61
0.86 to 1.71
0.93 to 1.79
0.71 to 1.56
        Source: Neas et al. (1990).
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 1          All of these odds ratios are consistent with the size of effect seen in the other analyses
 2     of the Six City data and the analyses of the British studies.  The authors defined a combined
 3     symptom that was the presence of any of the symptoms just reported. A similar analysis of
 4     this combined lower respiratory symptom gave an estimated odds ratio of 1.47 with a 95%
 5     confidence interval of 1.17 to 1.86.
 6          When split by gender, the odds ratio was higher in girls, and when split by smoking vs.
 7     nonsmoking homes, was higher in smoking homes.  The odds ratio for the combined
 8     symptom score is slightly  higher than the results of the other analyses, but is not inconsistent
 9     with those results.
10
11     14.2.3  Tayside Study
12          Ogston et al.  (1985) studied infant  mortality and morbidity in the Tayside region of
13     northern Scotland.  The subjects were 1,565 infants born to mothers who were living  in
14     Tayside in  1980. Episodes of respiratory illness were recorded during the first year of life.
15     The information  was  supplemented by observations made by a health visitor and scrutinized
16     by a pediatrician who checked diagnostic criteria and validity.  One health end point assessed
17     was defined as the presence of any respiratory disease during the year. This end point was
18     analyzed using a multiple  logistic regression model that included terms for parental smoking,
19     age of mother, and presence of a gas stove.  The results of this analysis are shown in
20     Table 14-9.
21
            TABLE 14-9.  REGRESSION COEFFICIENTS FOR MULTIPLE LOGISTIC
                ANALYSES OF RESPIRATORY ILLNESS IN TAYSIDE CHILDREN
Factor
Parental smoking
Age (in 5-year groups)
Presence of gas stove
Regres. Coeff.a
0.429
-0.094
0.130
Odds Ratio
1.54
(NA)b
1.14
95% Confidence Limits


(0.86 to 1.50)
       •Regres. Coeff. = Regression coefficient.
       bNA = Not available.
       Source:  Ogston et al. (1985).
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 1          Only the coefficient for parental smoMng was statistically significant (p<0.01).  The
 2     coefficient for gas stove use would have been significant only at the 0.14 level. The odds
 3     ratio of 1.14 suggests an increase in episodes of respiratory illness of about 14%.  The study
 4     did not give measured NO2 exposure values, but referenced the other Melia studies conducted
 5     elsewhere in the United Kingdom for exposure estimates.
 6
 7     14.2.4  Iowa Study
 8     -.     Ekwo et al. (1983) surveyed 1,355 children 6 to 12 years of age for respiratory
 9     symptoms and lung function in the Iowa City School District.  Parents of the school children
10     completed a questionnaire that was a modification of the questionnaire developed by the
11     American Thoracic Society. Pulmonary function measurements were obtained from
12     89 children whose parents did not smoke and 94 children whose parents smoked.  The
13     children were a random sample from those families whose parents had completed the
14     questionnaire. Eight different measures of respiratory illness were reported by the authors,
15     but only two of those were similar to the end points used in the British studies and the Six
16     City studies. Parental smoking was also measured and used a covariate in the analyses. The
17     results of the analyses are presented in  Table 14-10, and are based on 1,138 children.  No
18     measurements of NQ2 exposure, either inside or outside the homes, were reported.  No
19     differences in lung  function were associated  with gas stove use. The very small sample size
20     used in the lung function study precludes any meaningful interpretation of the lung function
21     results.
22
23     14.2.5  Dutch Studies
24          In  the Netherlands, Houthuijs et al. (1987), Brunekreef et al. (1987), and Dijkstra et al.
25     (1990) studied the effect of indoor factors on respiratory health in children. The population
26     consisted of 6- to 9-year-old children from 10 primary schools in five nonindustrial
27     communities in the southeast region of the Netherlands.  Personal exposure to NO2 and home
28     concentrations were measured. An important NO2 emission and exposure source in these
29     homes are geysers, which are unvented, gas-fired, hot water sources at the water tap.
30     Exposure to tobacco smoke was assessed with a  questionnaire that also reported symptom
31     information.  Pulmonary function was  measured at school.  The study used Palmes diffusion
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   TABLE 14-10.  ANALYSIS OF IOWA CITY SCHOOL CHILDREN RESPIRATORY
  	SYMPTOMS BY GAS STOVE TYPE AND PARENTAL SMOKING
                            Hospitalization for Chest
                              Illness before Age 2
                     Chest Congestion and
                      Phlegm with Colds
Factor
Gas stove use
Smoking effects
Father alone smokes
Mother alone smokes
Both smoke
Odds Ratio
2.4b

2.3b
2.9b
1.6
SEa
0.684

0.856
1.239
0.859
Odds Ratio
1.1

1.0
1.3
1.2
SEa
0.188

0.213
0.363
0.383
 "SB = Standard error of the odds ratio.
 blndicates statistical significance at the 0.05 probability level.

 Source: Ekwo et al. (1983).
 tubes to measure a single weekly average personal NO2 exposure. In January and February

 of 1985, the homes of 593 children who had not moved in the last 4 years were measured for
 1 week for NO2. Personal exposure was also estimated from time budgets and  room

 monitoring.  Estimated and measured exposures to NO2 are given in Table 14-11.


     TABLE 14-11.  DUTCH STUDY ESTIMATED AND MEASURED PERSONAL
          NO2 EXPOSURE Qtg/m3) FOR A SINGLE WEEKLY AVERAGE
Estimated
Source
No geyser
Vented geyser
Unvented geyser
Number
370
112
111
Arith. Meana
22
29
40
S.D.b
7
9
9
Measured
Arith. Mean
22
31
42
S.D.
9
12
11
*Arith. Mean = Arithmatic mean.
"S.D. « Standard deviation.

Source:  Brunekreef et al. (1987).
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 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
     Three measures of health were obtained from the questionnaire that was a modified
form of the World Health Organization questionnaire. The different items were combined to
create three categories: cough, wheeze, and asthma. Asthma was defined as attacks of
shortness of breath with wheezing in the last year.  The presence of any of the three
symptoms was used as a combination variable.  The results are presented in Table 14-12.
A logistic regression model was used to fit the combination variable.  Exposure was estimated
by fitting a lognormal distribution to the grouped data and the mean exposure values for each
group were estimated by a maximum likelihood technique (Hasselblad et al., 1980). The
estimated logistic regression coefficient was -0.002, corresponding to an odds ratio of 0.94
for an increase of 30 jitg/m3 (0.016 ppm) in NO2, with 95 % confidence limits of 0.66 to
1.33.  Thus,  the Dutch studies did not demonstrate an increase in respiratory disease with
increasing NO2 exposure, but the range of uncertainty is quite large and the rates were not
adjusted for covariates such as parental smoking and age of the child.
               TABLE 14-12. FREQUENCY AND PREVALENCE OF REPORTED
             RESPIRATORY SYMPTOMS IN DIFFERENT CATEGORIES OF MEAN
                 INDOOR NO2 CONCENTRATIONS IN A POPULATION OF 775
                         DUTCH CHILDREN OF 6 TO 12 YEARS OLD
Symptom
Cough
Wheeze
Asthma
One or more
symptoms

Frequency
0-20 )«g/m3
. n=336
16
30
22

36
4.8%
8.9%
6.6%

10.7%
and
prevalence
21-40 jug/m3
n=267
12
18
12

24
4.5%
6.7%
• 4.5%

9.0%
in category of
41-60 /zg/m3
n=93
7
3
2

8
7.5%
3.2%
2.2%

8.6%
indoor
>
3
7
3

8
N02

60 /zg/m3
n=79
3.
8.
3.

10
8%
9%
8%

.1%
       Source:  Dijkstra et al. (1990).


 1          Of several potential explanations for the negative findings of the study with respect to  ,
 2     NO2 exposure offered by the authors, one consideration was that the power of the study to
 3     detect health effects may have been reduced by the smaller sample size of the measured NO2
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 1      data compared to the categorical data (e.g., gas stove vs. electric stove use). They could not
 2      estimate whether they gained more precision by measured NO2 than was lost by the reduction
 3      in the sample size.
 4
 5      14.2.6  Ohio Study
 6           Keller et al.  (1979a) and Mitchell et al. (1974) conducted a 12-month  study of
 7      respiratory illness and pulmonary function in families in Columbus,  OH, prior to 1978. The
 8      sample included 441 families divided into two groups:  those using gas and  those using
 9      electric cooking. Participating households were given diaries to record respiratory illnesses
10      for 2-week periods.  Respiratory illnesses included colds, sore  throat, hoarseness, earache,
11      phlegm, and cough.   Only one incident of illness per person per 2-week period was recorded.
12           The study measured NO2 exposure, but it included both Jacobs-Hochheiser and
13      continuous chemiluminescence methods.  The electric stove users averaged 20 ppb
14      (38 /tg/m3)  NO2 exposure, whereas the gas stove users averaged 50 ppb (94 /-cg/m3).  The
15      paper does not report which rooms were  measured in order to get this average.  Thus, the
                                                                                      «3
16      estimated average difference between gas and electric stove use was 30 ppb  (58 jug/m ),
17           The analysis of incidence rates was done using the "Automatic Interaction Detector."
18      No differences were found in any of the illness rates for fathers, mothers, or children. No
19      analyses were done using multiple logistic regression or Poisson regression (these methods
20      were relatively new at the time).  No estimates were made that can be considered comparable
21      to the odds  ratios reported in the other studies.  The authors did show a bar graph of all
22      respiratory illness for children under 12.  The rates were 389 (per 100 person-years) for
23      electric stove use and 377 for gas stove use.  These rates were not significantly different even
24      after adjustment for covariates, including family size, age, gender, length of residence, and
25      fathers education.  No mention was made of adjustments  for smoking status or smoking
26      exposure for the children.
27           In a second related study (Keller et al. 1979b), 580 persons drawn from households that
28      participated in the earlier study were examined to confirm the reports and to determine the
29      frequency distribution of reported symptoms among parents and children in  gas or electric
30      cooking homes. A nurse-epidemiologist examined selected  persons reported ill and obtained
31      throat cultures.  Unfortunately these rates were not adjusted for other covariates.  The percent

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 1      of children haying lower respiratory symptoms in homes with a gas stove was 53.2%
 2     : (n=267) vs. 50.7% (n=286) in homes with electric stoves.  Although the difference is not
 3      statistically significant, these rates give an estimated odds ratio of 1.10 with 95% confidence
 4      limits of 0.74 to 1.54. Neas (1990, personal communication) observed that Keller's model
 5      controls for a series of variables that specify the child's prior illness history and that if
 6      chronic exposure to NO2 is a risk factor for prior illnesses, controlling for the child's illness
 7      history would substantially reduce the estimated effect of current NO2 exposure.
 8
 9      14.2.7  Swiss Study
10          Braun-Fahrlaender et al. (1989) and Rutishauser et al. (1990a,b) studied the frequency
11  ..-:..• of common airway symptoms in children up to 5-years old.  The study was conducted for six
12     periods in two rural and two urban areas of Switzerland.  Nitrogen dioxide was measured
13     weekly using Palmes  tubes both inside and outside the home of the participants during a
14     6-week period.  Additional air quality and meteorological data were obtained from local fixed
15     monitoring stations.   Parents recorded their child's respiratory symptoms daily by means of a
16     diary. .The symptoms recorded included cough during the day, cough at night, sore throat,
17     running nose, fever, and earache.  Physician visits and drug use also were recorded.
18     Additionally, covariates, including respiratory symptoms of other family members, family
19     social situation, size,  living conditions, smoking habits of the family, age, and nationality,
20     were recorded. • A total of 1,225 families were included, of which 1,063 were Swiss
21     nationals.
22           The incidence of upper respiratory  illness, cough, breathing difficulties, and total
23     respiratory illness was analyzed using Poisson regression.  The model included age, social
24      class, frequency of colds, and gas stove  use as covariates, as well as a term for the number of
25      days the individual was in the study.  The adjusted annual symptom incidence rates and NO2
26      exposure levels are given in Table 14-13.
27           The results show a pattern suggestive of increased incidence as a function of NO2
 28      exposure. Regression coefficients for NO2 exposure were calculated by Schwartz  (1990) but
 29      were not reported in  the original paper.  The incidence of any respiratory disease was used as
 30      the dependent variable, and annual indoor and outdoor NO2 levels in micrograms per meter
 31      cubed were used as independent variables.  The coefficient for indoor NO2-was 0.0013 and
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                TABLE 14-13. ADJUSTED ANNUAL SYMPTOM INCIDENCE RATES
          AND NO2 EXPOSURE LEVELS FOR FOUR COMMUNITIES IN SWITZERLAND81
Regions and NO2
Concentrations in the
Outside Air
Basel
Zurich
Wetzikon
Rafzerfeld
£30 /Kg/m3
30 - 49 Atg/m3
£50 /ig/m3
Number of
Symptoms per Child
and Day (average)
0.40
0.39
0.38
0.32
0.31
0.39
0.41


Standard Error
0.02
0.03
0.03
• 0.02
0.02
0.02
0.02


Number of Children
507
245
219
254
310
'492
412
        "Statistics: t - test regions were Basel vs. Rafzerfeld, t = 2.51, p<0.01; Zurich vs. Rafzerfeld, t = 1.99,
        p<0.05; Variance analysis; NO2 concentration: F-5.4, p<0.05.

        Source: Braun-Fahrlaender et al. (1989).
  1
  2
  3
  4
  5
  6
  7
  8
  9
10
11
12
13
14
15
 for outdoor NO2 it was 0.0002.  The standard errors were 0.0064 and 0.0088, respectively.
 The indoor coefficient suggests that an increase of 30 ^g/m3 (0.016 ppm) in NO2 would
 result in a 4% increase in the respiratory disease rate.

 14.2.8 Connecticut Study
     Berwick (1987), Berwick et al, (1984, 1987, 1989), and Leaderer et al. (1986) reported
 on a 12-week study (six 2-week time periods) of lower and upper respiratory symptoms in
 159 women and 121 children, aged 12 or less, living in Connecticut.  Nitrogen dioxide levels
 were measured in 91% of the homes,  57 of which had kerosene heaters and 62 which did
 not.  Ambient NO2 levels ranged from 9 to 19 jttg/m3 (0.005 to 0.01 ppm) for the six 2-week
 time periods.  Two-week average indoor NO2 levels in homes of monitored Children were
 highest for homes with kerosene heaters and gas stoves (91 j«g/m3; 0.05 ppm, n=8), second
 highest for kerosene only (36 jig/m3; 0.02 ppm, n=45), third highest for gas stoves only
 (32 jtg/m3; 0.02 ppm, n=13), and lowest for no sources (6 /^g/m3; 0.003 ppm, n=43).
Indoor levels did not fluctuate greatly  over time, as indicated by the 2 wk averages.
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1
2
3
4
5
A comparison of personal NO2 exposures, as measured by Palmes' diffusion tubes, and NO2
exposures measured in residences had a correlation of 0.94 for a subsample of 23 individuals.
Results of this comparison are depicted in Figure 14-1 and show an excellent correlation
between average household exposure and measured personal exposure.
                   130
              UJ
              tx.
              CO
              o
              CL
              X
              UJ
              o
              If)
              GtL
              Ul
              CL
             120 -
             110 -
             100 -
              90-
              80 -
            •  70 -
              60 -
              50-
              40 -
              30-
              20 -
              10 -
               0
                       0
                            •  KEROSENE HEATERS & GAS STOVES
                            •  NO SOURCE
                            A  GAS STOVES
                            T  KEROSENE HEATERS
                         —i—
                         20
40
60
80
100
                                                                            120
140
                                         AVERAGE N02/HOUSE, ug/m3
       Figure 14-1.  Total personal exposure to NO2 vs. NO2 levels in Connecticut residences.
       Source:  Leaderer et al. (1986).-

            The study defined LRI as the presence of at least two of the following:  fever, chest
       pain, productive cough, wheeze, chest cold, physician-diagnosed bronchitis, physician-
       diagnosed pneumonia, or asthma.  This LRI definition may not be consistent with that
       defined by studies in Section 14.5.  Upper respiratory illness was defined as the presence of
       two of the following:  fever, sore throat, nasal congestion, dry cough, croup, or head cold.
       Although both upper and lower respiratory  illness were investigated, the major outcome of
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        interest was lower respiratory symptoms.  The study obtained information of many potential
        covariates, which included socioeconomic status, age, gender, and exposure to environmental
        tobacco smoke.  The ones having the largest effect were: age of the child, socioeconomic
        status of the family, and history of respiratory illness.  Multiple logistic analysis was used to '
  5     allow for the various factors.
             When controlling for socioeconomic status and history of respiratory illness, children
        under the age of 7 exposed to 30 /*g/m3 (0.016 ppm) NO2 or more were found to have an
        increased risk of lower respiratory symptoms 2.25 times children who were not exposed
        (95% confidence limits of 1.69 to 4.79).  They also had an increased risk of upper
 10     respiratory symptoms of 1.33 (95%  confidence limits of 1.19 to 1.49). .Older children and
        adults showed no increased risk.
             Although the Berwick study had relatively extensive information on exposure, several
        problems are evident. The 3 year age-specific relative risks for lower respiratory disease are
        very unstable, possibly due to the small sample sizes. The rates do not appear to be
 15     consistent with the rates for ages 0 to 6 and 7 and above, and it is not clear why a cut-off of
        7 years of age was used. The analyses may be sensitive to the adjustment for socioeconomic
        status, which can be correlated with exposure.  This is less of a problem in studies with
        larger sample sizes (e.g., Melia et al. 1977, 1979), but may be critical in the Berwick study.
        Also, Neas (1990, personal communication) notes that the Berwick study controls for prior
20     illnesses, as did the Keller study, which would reduce the estimated effect of current NO2
        exposure.

        14.2.9  Chestnut Ridge Study
             Vedal et al. (1987) conducted a panel study on 351 children selected  from  the 1979
25     Chestnut Ridge, Pennsylvania cross-sectional study of elementary school-aged children (mean
        age = 9.5 years).  Parents completed a daily diary on respiratory symptoms.  Most pollutants
        were measured at a single site, but sulfur dioxide (SO2)  was measured at .17 -sites and then
        averaged.  No air pollutant was strongly associated with respiratory symptoms.  All pollutant
        levels were relatively low:   maximum hourly levels for NO2 levels ranged from  12 to
30     79 /*g/m3 (0.006 to 0.04 ppm).  No  indoor measurements were made nor were any
        surrogates for indoor pollution included in the analysis.

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 1
 2
 3
 4
 5
 6
 7
 8
 9  ,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
 28
 29
 30
 31
     Schenker et al. (1983) reported a respiratory disease study of 4,071 children aged
5 to 14 in the same Pennsylvania community that utilized the ATS-DLD-78-C standardized
questionnaire that was completed by the parents.  The presence of a gas stove was reported to
be used as a surrogate for NO2 exposure.  No significant association was found between use
of gas stove use and any respiratory symptom or illness variable. No data were reported.

14.2.10  California  Seventh-Day Adventist Study
:     In a California study, Euler et al. (1988) assessed the risk of chronic respiratory disease
symptoms due to long-term exposure to ambient levels of total suspended particulate (TSP),
oxidants,  SO2,  and NO2. Symptoms were ascertained using the National Heart, Lung, and
Blood Institute  (NHLBI) questions on 8,572 Southern California Seventh-Day Adventists
(nonsmokers—25 years  and older)  who had lived 11 years or longer in their 1977 residential
area.  Tobacco smoke (active and passive) and occupational exposures were assessed by
questionnaires,  as  well as lifestyle characteristics relative to pollution exposure as time spent
outside and residence history.   For each of the 7,336 participants who responded and
qualified  for analysis, cumulative exposures to each pollutant were estimated using monthly
residence zip code histories and interpolated exposures from state air monitoring stations.
      Multiple logistic regression analyses were conducted for pollutants individually and
together with eight covariables, including environmental tobacco smoke exposure at home and
at work, past smoking,  occupational exposure, sex, age, race,  and  education. Statistically
significant associations  with chronic respiratory symptoms were seen for: (1) SO2 exposure
above 0.04 ppm,  (p=0.03), relative risk 1.18 for 13% of the study population with
500 h/year of exposure; (2) oxidants above 0.1 ppm, p<0.004, relative risk of 1.20 for 18%
with 750 h/year; and (3) TSP above 200 ^g/m3, (p<0.00001), relative risk of 1.22 for 25%
 with 750 h/year.  When these pollutant exposures were analyzed together, TSP was the only
 one showing statistical  significance (p<0.01).  Nitrogen Dioxide exposure levels in this
 population were not linked to chronic respiratory disease symptoms.  Individuals working
 with smokers for  10 years had relative risks of 1.11 and those living with a smoker for
 10 years had relative risks of 1.07.
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  1      14.2.11  Maryland Study
  2          Helsing et al. (1982) analyzed the records of 708 nonsmoking white adult residents of
  3      Maryland to evaluate the effects of exposure to environmental tobacco smoke at home and
  4      use of gas as a cooking fuel.  The frequency of cough and/or phlegm among these
  5      nonsmokers showed a nonsignificant association with the presence of cigarette smokers in the
  6      household. Persons whose households had gas as a cooking fuel reported significantly more
  7      chronic cough, and chronic cough and phlegm, than those in households using electricity for
  8      cooking.  The author noted that while gas cooking has been considered by some as simply
  9      another indicator of poor social conditions, the multiple adjustments for factors such as years
 10      of schooling and persons per room should fully compensate for variations in socioeconomic
 11     level.  They also noted that all the independent'variables combined in the analysis accounted
 12     for only 5 to  10% of the variations in symptomatology.  Respiratory ventilation function tests
 13     gave consistent results with symptom reporting with those using gas cooking showing
 14     impaired pulmonary function.
 15
                                                                              . ,:.
 16     14.2.12  Glendora, California Study
 17          In another California  study,  Detels et al. (1979, 198la,  198Ib) and Rokaw et al, (1980)
 18     studied chronic respiratory  disease symptoms and lung function in two areas of Los Angeles
 19     County. The low exposure area was Lancaster, a city in the high desert country about
 20     113 km from downtown Los Angeles, which was studied from November 1973 to October
 21      1974. The high exposure area was Glendora, an area in the Los Angeles basin, which was
 22      studied from April 1977 to  March 1978.  The aerometric exposures for Glendora were
 23      estimated from a station in  Azuza, about 5 km away.  Pollutants measured included total
 24      oxidant (ultraviolet absorption method), NOX (Saltzman method), carbon monoxide
 25      (nondispersive infrared spectroscopy method), SO2 (conductimetric method), hydrocarbons
 26      (flame ionization detection method), and particles (high-volume method). The 5-year
 27      averages were computed for those pollutants with sufficient  data. Comparing Lancaster to
28      Glendora, the  NO2 levels were 3.2 vs. 11.4 jwg/m3 (0.002-0.006 ppm);  the total oxidant
29      levels were 6.5 vs. 11.6 /*g/m3, and the hydrocarbons were 2.9 vs.  4.8 jwg/m3.  Comparable
30      differences existed for SO2, particles, and sulfate fraction of particles, but the data were only
31      complete for the year 1977.

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 1          The authors looked at symptom prevalence of cough, sputum production, wheezing, and
 2     frequent chest illness.  All symptoms except frequent chest illness showed significantly higher
 3     rates in Glendora for both sexes and all three smoking categories.  The authors also found
 4     significantly lower peak flow values iri Glendora when compared with Lancaster, and these
 5     differences were significant across all  smoking categories and both sexes.  The percent of
 6     subjects with forced expiratory volume (FEVj) or forced vital capacity (FVC) below 50%  of
 7     expected was also significantly higher in Glendora.  Other lung function measurements
 8     showed less significant differences, but the trend was always towards lower values in
 9     Glendora.
10          The two primary weaknesses of the study are:  (1) the two areas (Lancaster and
11     Glendora) were measured at different times (1974 vs. 1977) and (2)  the areas are quite
12     different with respect to climate, commuting patterns, altitude, socioeconomic status, season,
13     and general lifestyle.  No specific analysis related to NO2 levels were discussed. The effects
14     of smoking habits were carefully controlled and should not be considered as a serious
15     confounder.  The authors did attempt to  control variability in measurement methods and
16     technicians, and results of this are reported by Tashkin et al. (1979).
17          '                                                                 '
18     14.2.13  Chattanooga Studies
19          Several studies were conducted in the greater Chattanooga area during the late 1960s
20     and early 1970s.  Although these studies were discussed in detail previously (U.S.
21     Environmental Protection Agency, 1982),  there are at least two additional points that need to
22     be  made.  First, there were many measurements made in the area by methods other than the
23     Jacobs-Hochheiser method (e.g., chemiluminescence).  Reevaluation of the Jacobs-Hochheiser
24     method at a later time questioned its accuracy for use in the studies.  Second,  much of the
25     pollution may have been in the form of nitric acid (HNO3), and possible health effects may
26     be  related to HNO3 exposure rather than NO2 itself. The source of  pollution was a large
27     trinitrotoluene (TNT) plant,  located northeast of Chattanooga, which produced a substantial
28     proportion of all TNT made in the United States during World War  II and the Korean War.
29     The plant was reopened in April, 1966,  to supply munitions  for use  in Vietnam. Annual
30     averages of NO2 reached 286 ^g/m3 (0.152  ppm) near the arsenal (as measured by the
31     Saltzman method),  and nitrate fraction levels reached 4.1 jug/m3 at the downtown post office.
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 1      It is likely that the elevated NO2 levels were accompanied by elevated HNO3 levels, although
 2      no direct measurements were made.  The U.S. Environmental Protection Agency (1971)
 3      measured several factors related to ambient air pollution including corrosion of zinc, steel,
 4      and nylon.  The corrosion levels in Chattanooga in 1967 and  1968 were among the highest,
 5      and in the case of nylon, were 10 to 100 times the levels of most other  cities.  According to
 6      the report, the arsenal was known to emit acid gases.  Additionally, Warner and Stevens
 7      (1973, 1975) give other evidence suggesting the presence of sulfuric acid and HNO3. Thus,
 8      it is possible that any adverse  health effects seen in Chattanooga during  this time period were
 9      associated with HNO3 and NO2 rather than with NO2 alone.  However, no conclusion is
10      possible since the health effects of HNO3 are poorly understood (see Chapter 13).
11           Pearlman et al.  (1971) reported the results  of a respiratory disease survey conducted in
12      the Chattanooga area in 1969.  The study reported illness rates in children for the period June
13      1966 to June 1969.  Higher rates of bronchitis in school-aged children were found in both the
14      intermediate and  high exposure areas as compared with the low exposure area.  The results
15      were not completely consistent with the exposure gradient since the rate in the intermediate
16      area was just as high as the high pollution area.
17           Shy and co-workers (Shy, 1970;-Shy et al., 1970a,b; 1973) studied the effects of
18      community exposure to NO2 in residential areas of Chattanooga on respiratory illness rates in
19      families.  The incidence of acute respiratory disease was assessed at 2-week intervals during
20      the 1968-69 school year and the respiratory illness rates adjusted for group differences in
21      family size and composition were reported to be significantly  higher for each family segment
22      (mothers, fathers, children) in the high-NO2 exposure neighborhood than in the intermediate-
23      and low-NO2 areas. Although individual area pollution estimates are not available, one part
                                                                          tj
24      of the high pollution area had  an annual average NO2 level of 286 j«g/m (0.152 ppm).
25      Areas further from the major NO2 source (TNT plant) had lower levels.
26           Love et al.  (1982) studied acute respiratory disease in the same area during the years
27      1972-73. Fathers, mothers, school children, and preschool children all  showed significantly
28      higher illness  rates in the area designated as high pollution area during the beginning of 1972.
29      There were almost no significant differences in illness rates during the periods September to
30      December 1972,  and January to April 1973. During the period January to June 1972, NO2
                                                                                          sy
31      levels (as measured by the continuous chemiluminescent method) ranged from 60.2 [j.g/m

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(0.032 ppm) in the high area to 28.9 jug/m3 (0.015 ppm) in the low area.  However, by the
second half of 1972, the exposures in all areas were quite comparable because of reduced
emissions.  Thus, the results of the study tend to confirm the effect of NO2 or its by-products
on acute respiratory disease.

14.2.14  United States and Canadian  Skating Rink Exposures
     Hedberg et al. (1989) reported cough, shortness of breath, and other symptoms among
players and spectators of two high school hockey games played at an indoor ice arena in
Minnesota related to emissions from a malfunctioning engine of the ice resurfacer. Although
the exact levels of NO2 were not known at the time of the hockey game, levels of 4 ppm
were detected 2 days later with the ventilation system working, suggesting that levels during
the games were higher.  Hedberg et al. (1989, 1990) reported that pulmonary function testing
performed on members of one hockey team with a single exposure demonstrated no decrease
in lung function parameters at either 10 days or 2 months after exposure.  Dewailly et al.
(1988) reported another incident in a skating rink in Quebec, Canada, in 1988 involving
referees and employees reporting respiratory symptoms such as coughing, dyspnea, and a
suffocating feeling.  Five days after the incident, NO2 levels had come down to 3 ppm,
suggesting much higher levels during the incident..
14.3 STUDIES OF PULMONARY FUNCTION
     Pulmonary function studies are part of any comprehensive investigation of the possible
effects of any air pollutant.  The measurements can be done in the field, they are
noninvasive, and their reproducibility has been well documented. Age, height, and gender
are important determinants pf lung function. In addition, changes in pulmonary function
have been associated with environmental tobacco smoke (Hasselblad et al.,  1981) and
particulate matter in combination with SO2 (Dockery et al., 1982 and Dassen et al.,  1986)
and other factors.
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 1      14.3.1  United States Six City Studies
 2          Several of the pulmonary function studies also included respiratory disease symptoms as
 3      described earlier. Ware et al. (1984), reporting on the Six City study, described analysis of
 4      lung function values using multiple linear regression on the logarithm of the lung function
 5      measures.  Covariates included sex, height, age,  weight, smoking status of each parent, and
 6      educational attainment of the parents. Forced expiratory volume in 1 second (FEVj) values
 7      were significantly lower for children of current smokers than for children of nonsmokers at
 8      both examinations and highest for children of ex-smokers.  Forced vital capacity (FVC)
 9      values were lower for children of nonsmokers than for children of current smokers at both
10      examinations, but the difference was statistically  significant only at the first examination.
11      Both the increase in mean FVC and the decrease in mean FEVj among children of current
12      smokers were linearly related to daily cigarette consumption.  Exposure to,gas stoves, was
13      associated with reductions of 0.7% in mean FEVj and 0.6%  in mean FVC at the first
14      examination (p<0.01), and reductions of 0.3% at the second examination (not significant).
15      The estimated effect of exposure to gas stoves was reduced by approximately 30% after
16      adjustment for parental education.  The authors state that the adjustment for parental
17      education may be an over-adjustment, and may partially represent gas stove use because of
18      association between parental education and type of stove.  Hasabelnaby et al. (1989) give
19      estimation formulas for linear regression models  that incorporate errors in exposure variables
20      using this data set as an example.
21          Berkey et al.  (1986) used the Six City study data from children seen at two to five
22      annual visits to study factors affecting pulmonary function growth.  Children whose mothers
23      smoked one pack of cigarettes per day had levels of FEVl at age 8 that were approximately
24      0.81% lower than children of nonsmoking mothers (p<0.0001), and FEVj growth rates
25      approximately 0.17% per year lower (p=0.05).  The same data provided no evidence for an
26      effect of gas stove exposure on growth rate.
27
                                                                              , $
28      14.3.2  Tucson  Study
29          Lebowitz et al. (1985) studied a cluster sample of 117 middle-class households in
30      Tucson, AZ. Symptom diaries and peak flows were obtained over a 2-year period.  Outdoor
31      sampling of ozone (O3), TSP, carbon monoxide  (CO), and NO2 was done in or near the

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clusters.  Indoor sampling of O3, TSP, respirable suspended particles (RSP), and CO was
done in a subsample of the homes.  Additional information such as the presence of a gas
stove or smoking also was obtained. The presence of a gas stove was used as  a surrogate for
indoor NOX exposure, because it was not measured directly.                 -
     The relationship of children's  peak flow and gas stove use was of borderline
significance (p=0.066)  for an analysis excluding TSP, and was not close to significance with
TSP included in the analysis.  In asthmatics, gas stove use was significantly associated with
peak flow decrements (p< 0.001).  This was true across smoking groups, but the difference
was greatest for smokers. Peak flow in adults was also related to gas stove use, but the level
of significance was not given.

14.3.3 New  York Study
     Goldstein  et al. (1987) reported preliminary data examining acute exposure to NO2 and
pulmonary function tests. Eleven asthmatic and 12 nonasthmatic women and children were  "
monitored for 5 days with a portable continuous NO2 monitoring'instrument held at breathing
level that provided 5-min average NO2 levels.  Pulmonary function (FEVl5 FEV25_75) ^
peak flow as weir as tracings of the entire flow curve were monitored at several different
points during the exposure.  While the data are limited, it seemed that at average NO2
exposures below 0.3 ppm (564 jug/m3) pulmonary function was as likely to be decreased as
increased, whereas at exposure above 0.3 ppm there was mainly -a decrease.

14.3.4  Chestnut Ridge Study
     Vedal et al. (1987) conducted a panel study on 351 children  selected from the 1979
Chestnut Ridge cross-sectional study of elementary school-aged children (mean age =
9.5 years).  Peak expiratory flow rate (PEFR) was measured daily for 9 consecutive weeks.
Most pollutants were measured at a single  site, but SO2 was measured at 17 sites and then
averaged.  No  air pollutant was strongly associated with level of PEFR. All pollutant levels
were relatively low;  NO2 levels ranged from 0.006 to 0.042 ppm  (12 to 79 fcg/m3): No
indoor measurements were made, nor were any surrogates for indoor pollution included in the
analysis.
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  1     14.4 OUTCOMES RESULTING FROM OCCUPATIONAL EXPOSURES
  2          Gamble et al. (1987) studied 232 workers in four diesel bus garages for the effects of
  3     NO2 on acute respiratory illness and pulmonary function.  Response was assessed by an acute
  4     respiratory questionnaire and before and after shift spirometry. Measurements over the shift
  5     of NO2 (using passive Palme tube samplers) were made on each worker and collected on the
  6     same day as the pulmonary function tests and questionnaires.  Other irritant gases were
  7     measured and were well below federal standards.  Mean NO2 levels over the shift ranged
  8     from 0.56 (standard deviation [SD] = 0.38) ppm NO2 in the highest garage to
  9     0.13 (SD = 0.06) ppm NO2 in the lowest garage. Short-term NO2 measurements indicated
 10     levels above 1 ppm as being not uncommon.  The authors report that the prevalence of acute
 11     respiratory symptoms were elevated above expected in the high-exposure (>0.3 ppm) group
 12     only. No reduction in pulmonary function was associated with exposure.
 13          Gamble et al. (1983) examined chronic respiratory effects in 259 sodium chloride
 14     (NaCl) miners. The Medical Research Council respiratory symptom questionnaire containing
 15     smoking history was administered by trained interviewers. A chest X-ray and spirometry was
 16     also conducted. Personal samples of NO2 and respirable particles for jobs in each mine were
 17     used to estimate cumulative exposure.   Mean exposure ranged from a low of
 18     0.2 (SD = 0.1) ppm NO2 to a high of 2.5 (SD = 1.3) ppm  NO2.  Diesel emissions were the
 19     principal NO2  source.  The author reported that while cough was associated with age and
20     smoking and dyspnea was associated with age, neither symptom was associated with exposure
21      (i.e., years worked, estimated cumulative NO2 or respiratory particle exposure.) Reduced
22     pulmonary function showed no association with NO2 exposure.
23          Robertson et al. (1984) reported on a 4-year study of lung function in 560 British coal
24      miners.  Overall average NO2 levels at nine coal mine sites ranged from 0.02 to 0.06 ppm
25      (38 to 113 Aig/m3), and NO levels ranged from 0.13 to 1.19 ppm.  No relationship was found
26      between exposure and decline in FEVj  or respiratory symptoms. Jacobsen et al. (1988)
27      conducted a more extensive investigation. Jacobsen et al. (1988) studied nearly
28      20,000 miners  at the same nine British coal  mines to examine whether long-term exposure to
29      low concentration of NO2 and NO were associated with increased susceptibility to respiratory
30      infections.  The NOX source consisted of diesel emissions and blasting.  Shift median levels
31      were 0.2 ppm NO and 0.03 ppm NO2.  This complete and intensive study had problems with

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misclassification of exposure and outcome that are not uncommon when existing data are used
for purposes that were not foreseen when the data were collected. The authors concluded that
the long-term exposure to the levels above do not detectably increase the chance that miners
will absent themselves from work because of a chest infection.
     Douglas et al.  (1989) report data between 1955-87 on 17 patients examined at the Mayo
Clinic for Silo-Fillers disease shortly after exposure to silo gas (NO2 levels from 200 to
2,000 ppm).  Health outcome ranged from death to hypoxemia and transient obstruction of
the airways.  Epler (1989) notes that prevention is essential for elimination of silo-fillers
disease.  Other studies also examine high exposures (Lowry  and  Schuman, 1956; Grayson,
1956; Gregory et al., 1969; Yokey et al., 1980).
14.5  SYNTHESIS OF THE EVIDENCE
     The weight of the evidence does not indicate that NO2 exposure at the levels reported in
the studies  in this chapter has any effect on pulmonary function of biologically significant
magnitude.  Several of the studies, however, suggest an increase in respiratory morbidity in
children from exposure to levels seen with gas stove use as compared to electric stove use.
The effects in the majority of the studies do not reach statistical significance.  The
consistency of these studies is examined and the evidence synthesized in a quantitative
analysis in  the following section.

14.5.1  Health Outcome Measures
     A concern in interpreting these studies (see Section 14.5.3 for criteria for including a
study in the quantitative analysis) is that the LRI variable measured in these studies may
represent differing outcomes that may have different mechanisms  of causation related to NO2
exposure.  The origin of cough may be different than that of wheeze. The agents that cause
them may be different.  Different disease syndromes are being studied:  croup, bronchitis,
bronchiolitis, asthma, and pneumonia.  The instruments measuring LRI in the studies are
different.  If the similarity between the outcome measures between and within  the studies is
not adequate, the potential interpretation of a  quantitative analysis may be limited.  Is LRI in
children a  relatively similar outcome measure in both a statistical  sense and as  a biological
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  1     end point across the studies?  This discussion considers this question by evaluating the
  2     outcome measures in the NO2 studies, similarities in outcome measures in other LRI studies
  3     in children, the use of questionnaires as instruments for LRI, and, in general, the use of LRI
  4     as an outcome measure.
  5          The studies in the quantitative analysis that follows use health outcome measures that
  6     provide an indication of the state of respiratory health of the various samples of children
  7     12-years-old or younger. The NO2 studies utilized standard questionnaires to evaluate LRI in
  8     children. As discussed below, some studies used a specific symptom while others used an
  9     index consisting of symptoms and disease. Diagnoses of specific respiratory diseases such as
 10     bronchiolitis or asthma were not made. The important factor is an attempt to measure some
 11     aspect of LRI.  Table 14-14 lists the health outcome measures for each study considered.
 12     While specific measures such as colds going to chest (Melia et al., 1977),  chest congestion,
 13     and phlegm with colds (Ekwo et al., 1983) are used to provide measures of LRI, other
 14     measures use indexes, grouped responses, or combined indicators of LRI, some of which
 15     include measures such as colds going to chest.
 16          In the Melia et al.  (1977, 1979, 1980, 1982, 1983) studies, a self-administered
 17     questionnaire was completed by parents of children in the study.   Questions were asked about
 18     each child's respiratory disease episodes and symptoms during the previous 12 months.
 19     Respiratory symptoms and diseases surveyed include asthma in the last year, wheeze,
20     bronchitis in the last year, cough (night or day), and colds going to chest.  Irwig et al. (1975)
21      examined the association of these reported questions with reduced peak flow rates on a
22     sample of the children examined in 1973.  Since the answers to these questions were related
23     to the reduced adjusted mean peak flow rates, an indicator of decreased respiratory function,
24     this suggested that these  questions may be indicators of LRI (while others such as earache  or
25     hospitalization for upper respiratory disease in the last 12 months may not).  Thus, the more
26     subjective instrument,  the questionnaires, was supported to  some extent by the more direct
27     objective lung function measurements.  Melia et al. (1977)  indicated that the highest
28      prevalence was for colds going to the chest (approximately  25%) followed  by wheeze
29      (approximately 10%).  Bronchitis and asthma episodes in the past year had respective
30      prevalences of less than 6% and 3%.  The 1977 studies showed very similar prevalences
31      (Melia et al.,  1979) to the 1973  data with the prevalence of asthma and bronchitis episodes

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 1      both under 4% and colds going to chest at approximately 25% in 1977.  For these Melia
 2      studies, two indicators, colds going to chest and wheeze, provide the major contribution to
 3      the combined indicator for LRI.  Disease indicators such as asthma and bronchitis episodes in
 4      the past year, while measures of lower respiratory disease, appear to play a substantially
 5      smaller role.  The Melia et al. (1979) study provides data that allow development of graphs
 6      of the marginal likelihood functions (see Fig. 14-2) of the odds ratios for colds going to the
 7      chest and any respiratory illness for the combined indicator of boys  and girls. The odds ratio
 8      for colds to chest is 1.21 (SD of the log [OR] = 0.0675) and for any respiratory illness is
 9      1.24 (SD of the log [OR] =  0.0703).  This demonstrates the similarity of these two outcome
10      measures.  More specifically, it shows how  colds going to the chest represent an important
11      component of Melia's respiratory index.  In the quantitative analysis the outcome measure
12      used is colds going to chest for the Melia et al. (1977) study and the LRI for the other Melia
13      studies.
14          Other NO2 studies use different indexes or combinations  that include symptoms such as
15      chronic phlegm and wheeze,  persistent cough, respiratory illness, and asthma and bronchitis
16      (Ware et al., 1984; Neas et al.,  1990, 1991; Ogston et al., 1985; Dijkstra et al., 1990;
17      Keller et al., 1979b).   The measure of effect on the lower respiratory tract varied among the
18      studies; the indicators, however, are conventional symptom and illness outcomes.  The
19      symptoms are tabulated from similar standardized questionnaires (Ferris, 1978) and are
20      directed at eliciting the same basic data—an  indication of the presence of infection or illness
21      in the  lower respiratory tract. These symptoms are all indicators of lower respiratory illness
22      and as such form a measure of respiratory health.  In most cases chest colds and wheeze,  or
23      similar indicators, are the prominent symptoms in the index.  Use of the same index in
24      several of the NO2 studies and similar indexes in the other NO2 studies provides consistency
25      in the  outcome measurements of the studies.
26          How do the outcome measures in the NO2 studies  compare with indicators of lower
27      respiratory health status done in other studies?  The studies of LRI (unrelated to NO2
28      exposures) discussed in Section 14.2 were based on visits to physicians.  The signs  and
29      symptoms that were most common in those studies, wet cough and wheeze, are  similar to the
30      most common end points in the NO2 studies, that is, colds going to  chest and wheeze. Thus,
31      to an extent, the NO2 respiratory studies are providing a measure of lower respiratory health
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                                   Odds  ratios -  combined
                               Colds to Chest
                                                            Combined  Indicator
                                                            for any Respiratory
                                                            Illness
                                                            (Lower Respiratory
                                                            Index)
                                                1
                                           odds  ratio
       Figure 14-2. For the Melia et al. (1979) study, a graph of the marginal likelihood
                   function of the odds ratios for combined gender (boys and girls) of the
                   outcome measures colds to chest and any respiratory illness developed by
                   EPA.
1
2
3
4
5
6
7
somewhat equivalent to these other studies. While specific pediatric diseases and agents such
as bronchiolitis and RSV are investigated in these other studies and not in the NO2
respiratory studies, the overall pattern and incidence of LRI are considered to be consistent in
different geographic regions over time (Wright et al.,  1989; Denny and Clyde 1986;
McConnochie et al., 1988).  As such, lack of direct evidence for disease and agents should
not limit conclusions that could be drawn from the quantitative analysis of the NO2
respiratory studies.
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 1           Lower respiratory illness is an entity commonly defined by criteria for clinical diagnosis
 2      (Wright et al., 1989).  The instrument to determine LRI in these studies is substantially
 3      uniform—a standardized respiratory disease questionnaire mainly filled out by the parent for
 4      illness and symptoms during the past 12 months.  How comparable are the measures of LRI
 5      in the studies?  As background, consider the classification of lower respiratory disease and
 6      surveillance methods.  Samet (1990) reviews key studies that include Monto et al. (1971),
 7      Taussig et al. (1989), Glezen et al.  (1971), Gardner et al. (1984), and Maletzky et al.
 8      (1971).  Standardized and uniformly accepted clinical criteria have not been developed for the
 9      illnesses considered to make up LRI; that is, for croup, tracheobronchitis, bronchiolitis,  and
10      pneumonia.  The extent to which the diagnostic criteria for LRI were documented varies in
11      these studies (see Table 14-15, Samet, 1990).  Diagnosis of  pneumonia usually requires
12     evidence on chest X-rays.  Characteristic signs and symptoms are wheeze, phlegm from
13     chest,  and deep cough. These are basic respiratory symptoms indicative of lower respiratory
14     involvement.
15          For the purpose of analysis  of illness rates, children with LRI may be considered to
16     belong to a single population with a similar illness. While different diseases are grouped
17     together as LRI (such  as croup, bronchiolitis, and pneumonia), the LRI syndrome designation
18     (Monto et al.,  1971) is a means of grouping these illnesses of similar pattern for analytic
19     purposes.  It is relatively easy to classify illnesses on the basis of anatomic area of
20     involvement.  Graham (1990) states that classification by anatomic site remains the preferred
21     system for most physicians and is compatible with the International Classification of Diseases
22     system.  Such a classification is often a good indication of the severity of illness.
23           The respiratory questionnaires used in the NO2 studies determined the presence of LRI
24     in the subjects by tabulating responses to questions on respiratory symptoms such as cough
25     and wheeze, and respiratory illnesses such as asthma and bronchitis. Symptom reporting
26     provides subjective evidence of respiratory infection and diseases such as bronchiolitis.
27      Diseases are characterized by groups of symptoms. Disease end points such as asthma and
28      bronchiolitis may have similar defining symptoms. Lower respiratory illness reflects  a
29      broader grouping, within which the lack of the ability to differentiate between the wheeze of
 30      asthma and wheeze related to other infections in the pediatric age group may not be
 31      important.  Wright et al. (1989)  note that wheeze is recorded as occurring for several
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          TABLE 14-15. CLASSIFICATION OF ILLNESSES IN SURVEILLANCE STUDIES
          Study
Reference
                         Classification
         Tecumseh
         Seattle
         North
         Carolina
Monto et al.
(1971)
Maletzky et al.
(1971)
Glezen et al.
(1971)
         Houston

         Tucson
Gardner et al.
(1984)
Taussig et al.
(1989)
Lower respiratory illness (LRI) = Pain on breathing, wheezy breathing,
or phlegm from chest
Upper respiratory illness (URI) = Coryza with/without other symptoms
Laryngopharyngeal illness = Sore throat or hoarseness
Cough = Cough with/without earache
Earache  = Earache
Pneumonia = Fine inspiratory rales and/or infiltrate on chest X-ray
Croup = Presence of inspiratory stridor
Bronchiolitis = Presence of wheezing and air-trapping on exam or chest
X-ray
Epiglottitis = Marked edema and erythema of the epiglottis
Croup = Hoarseness and barking cough with/without inspiratory stridor
Tracheobronchitis = Deep cough and ronchi audible in larger air
passages
Bronchiolitis = Expiratory wheezing, often with evidence of air
trapping
Pneumonia = Fine rales or evidence of pulmonary consolidation
Not given

Criteria for LRI:  (1) a history of acute cough and/or wheeze and
positive physical findings, or (2) acute inspiratory stridor, or
(3) positive chest X-ray findings including hyperaeration, and
(4) for infants under 6 months, a history of prolonged cough and/or
wheeze and positive findings as in 1-3. No specific criteria for
individual syndromes.
        Source:  Samet (1990).



 1      diagnoses to include croup, bronchiolitis, bronchitis, and pneumonia.  Questions of asthma in
 2      the past year may not represent a disease-defining measure as much as a measure of wheeze.
 3      Additionally, asthma may be related to the occurrence of infection as a precipitating cause,
 4      thus further confusing differences between symptoms and disease.  Mitchell et al. (1976),
 5      note that the information obtained from the subjects in a respiratory health study that used a
 6      questionnaire are probably an accurate reflection of the lung disease experienced by the total
 7      population of these communities.
 8           The studies of LRI are implemented by use of standardized questionnaires.  The purpose
 9      of a respiratory symptom questionnaire is to compare prevalence of symptoms (Fairbairn
10      et al.,  1959) and to follow their rate of progression in populations.  This facilitates the
11      quantitative comparison between groups.  This,  however, contrasts fundamentally with the
12      usual objective of clinical medicine,  that is, a decision about an individual providing an
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 1     accurate diagnosis of the patient's condition to determine the correct treatment or prognosis.
 2     In surveys of prevalence, valid comparisons can be made with less accurate data, providing
 3     that a sufficient population is studied and that inaccuracies of reporting are randomly
 4     distributed between the groups being compared. Standardized questionnaires permit
 5     meaningful comparisons  of the results of prevalence surveys (Samet, 1978).
 6          In perspective, a question of similarity exists for other potential outcomes usually
 7     considered similar.  FEVj  changes may be caused by different diseases and may have
 8     different mechanisms of  causation.  But a study of FEVl changes in children would probably
 9     be considered as a homogenous outcome.  Thus, LRI may be a comparatively similar end
10     point.
11          Childhood LRI is a grouping of symptoms and diseases that reflect changes located
12     anatomically in the lower respiratory tract.  This characterization represents an indication of
13     severity, that has been used to characterize the respiratory health status of children.  The LRI
14     syndrome designation presents a means of grouping illnesses of similar patterns. Lower
15     respiratory illness is a multifaceted approach to respiratory health in a population living under
16     natural conditions. The  classification is the combination of different respiratory effects that
17     have in common an evaluation of the morbidity status of the lower respiratory tract. While
18     the use of identical health outcome measures would be most desirable, the level of similarity
19     and common elements between the outcome measures in the NO2 studies provide confidence
20     in their use in the qualitative analysis. Also, the similarity to other non-NO2 studies of LRI,
21     while again less than perfect,  provide confidence as to LRI  use as an outcome measure.
22
23     14.5.2 Biological Basis
24           The biological basis relating NO2 exposure to respiratory symptoms and infection shown
25     in epidemiological studies are presented in discussions in the animal toxicology and clinical
26      studies (Chapters  13 and 15 respectively) and in a recent review (Kotchmar et al.,  1990).
27     A brief summary is presented here.
28           The  lung is  one of "the sites of microbial infection. While many  types of
29      microorganisms are implicated in  respiratory infection, viruses represent a major cause of
30      respiratory disease particularly for infants and children.  In a viral respiratory infection, viral
31      replication produces injury and, thus, the signs and symptoms of respiratory illness (Douglas,
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 1      1986).  The respiratory system has several defense mechanisms against inhaled infectious and
 2      chemical agents.  Host defense mechanisms consist of nonspecific and specific components.
 3      The nonspecific aspects include mucociliary clearance and alveolar macrophages, while
 4      humoral and cellular immunity offer specific defenses. The immune system functions
 5      through a sequence of events, starting with the nonspecific components followed by responses
 6      by the specific components.  The immune system is a principal factor in the host's interaction
 7      with infectious agents such as viruses and in the host's ability to contain and/or eradicate the
 8      establishment of infection.  Specific T cell-mediated immune mechanisms are important in
 9      augmenting host defenses of the lung (Kaltreider, 1986).  T lymphocytes augment resistance
10      to intracellular microbial organisms and lyse virally infected cells.  To an extent, an increase .
11      of reported respiratory symptoms in some epidemiology studies may be an indication of the
12      ability of the respiratory host-defense mechanism to either overcome an infection or limit its
13      severity. Nitrogen dioxide may affect the immune system in such a way that one or several
14      aspects of the immune system do not function at a level sufficient to limit the extent or
15      occurrence of infection.  Nitrogen dioxide may to some degree influence respiratory symptom
16      rates by direct toxic mechanisms. Meulenbelt and Sangster (1990) note that NO2 may cause
17      direct epithelial damage that could increase susceptibility to infection.
18          Based on available information, exposure to NO2 impairs one or more defense
19      mechanisms, leaving the host potentially more susceptible to respiratory  infections and
20      diseases. One of the first lines of defense against foreign agents entering the respiratory tract
21      is the mucociliary clearance system, responsible for the removal of particles from the
22      conducting airways of the lungs (Proctor et al., 1977). Studies on the effects of NO2
23      exposure on mucociliary transport show a reduction in the number and activity of the cilia,
24      morphological changes in the cilia, and  decreased mucociliary  velocity (Stephens et al.,  1972;
25      Azoulay et al., 1978; Schiff, 1977;  Abraham et al., 1980; Yamamoto and Takahashi, 1984).
26      As a foreign agent moves beyond the mucociliary region, and enters the gaseous exchange
27      region of the lung, host defenses are provided by the alveolar macrophage, which acts to
28      remove or kill viable particles and thereby maintain sterility against infectious agents and
29      remove damaged and dead cells.  Exposure to NO2 has produced a variety of effects on
30      alveolar macrophages including decreased viability (Rombout et al., 1986; Suzuki et al.,
31      1986; Chang et al., 1986; Mochitate et  al., 1986).  Decreases  in the phagocytic ability of

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  1 -.
  2
  3
  4
  5
  6
  7
  8
  9
10
11..-,
12
13
14
15
16  ,
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
 alveolar macrophages have been reported in rats, hamsters, and rabbits (Suzuki et al.,  1986;
 Gardner et al., 1969; Acton and Myrvik, 1972;  Foster et al., 1985;, Schlesinger et al., 1987).
 In addition to functional changes to alveolar macrophages,' NO2 exposure has also been
 reported to produce structural changes in the macrophages (Aranyi et al., 1976).  Controlled
 human exposure studies have examined alveolar  macrophage function and show that alveolar
 macrophages exposed to NO2 tended to inactivate influenza virus in vitro less effectively than
 cells collected after air exposure (Frampton et al., 1989a).
    . Few studies are available that evaluate the effects of.NO2 on  the immune system;
r however, there is some indication that exposure,to NO2 suppresses the immune response and
 that the effect is concentration- and time-dependent.  For example, a significant suppression
 of serum humoral antibody response has  been reported in experimental animals acutely
 exposed to NO2 (Lefkowitz et al., 1986;  Fujimaki and Shimizu, 1981).  Exposure to NO2
 suppresses the number of mature splenic  helper T and cytotoxic T lymphocytes (Richters and
 Damji, 1988).  The cause of the suppression is not clear. Exposure to NO2 resulted in
 decreased numbers  of T lymphocyte, T-helper/induced lymphocytes, and
 T-cytotoxic/suppressor lymphocytes in mice (Damji and Richters,  1989).
     Recent controlled human exposure studies examining the effects of NO2 on pulmonary
 host defense systems report a trend toward an elevated rate of infection, though not
 statistically significant, in a study examining the  effect of NO2 exposure on the infectivity
 rate of live attenuated influenza A/Korea/reassortment virus (Goings et al., 1989).  Also,
 exposure" to NO2 may transiently increase levels  of antiprotease alpha-2-macroglobulin  (a2M)
 in bronchoalveolar  lavage (BAL) fluids presumably through local release.  While serving as
 an indicator of changes in protease-antiprotease balance, alterations in a2M in the alveolus
 may have significance for local immunoregulation.  Changes in a2M may alter alveolar
 macrophage defenses against infection (Frampton et al.; 1989a,b).  These findings suggest,
 but do not prove, that NO2 may play a role in increasing the susceptibility of adults to
 respiratory virus infections.                                 ,  .
     Animal'infectivity-studies present key data relating exposure to NO2 and effects on host
 defense mechanisms.  In these studies, animals were exposed to varying concentrations and
 durations of NO2 followed by exposure to an aerosol of an infectious agent.  Mortality was
 used as the end point.  Exposure to NO2  increased bacterial-induced mortality and
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 1      influenza-induced mortality.  The studies indicate that NO2 increased microbial-induced
 2      mortality by impairing the host's ability to defend the respiratory tract from infections agents
 3      (Ehrlich and Henry, 1968; Ito, 1971; Ehrlich et al.,  1977).  Recently, an infectivity model
 4      designed to evaluate the effects of NO2 on nonfatal respiratory disease indicated that NO2
 5      decreased the intrapulmonary removal (killing) of the microorganisms in mice in a
 6      concentration-related manner without a decrease in the number of alveolar macrophages.
 7      Exposure to NO2 was found to increase the severity of the mycoplasma-induced lesions put
 8      not found to increase the susceptibility of the mice to the infection (Parker et al.,  1989).
 9      These animal studies clearly demonstrate that acute and chronic exposures to NO2 increase
10      susceptibility to viral, mycoplasma, and bacterial  infections and the effects were concentration-
11      and time-dependent, species-dependent, and infectious agent-dependent (Ehrlich and Henry,
12      1968; Ito, 1971; Ehrlich et  al.,  1977; Parker et al., 1989; Gardner et al.,  1977a,b, 1979,
13      1980, 1982; Graham et al., 1987; Jakab,  1987a,b; Motomiya et al.,  1973; Miller et al.,
14      1987).
15           The evidence from animal toxicological and human clinical studies of host defenses
16      provide a rationale for investigating the relation between exposure to NO2 and an, increase in
17      frequency and severity of respiratory symptoms and/or infections in humans.  The strength of
18      various parts of the data base help substantiate the biological basis for the relationship seen in
19      epidemiology studies between respiratory  disease  and exposure to NO2.  The above discussion
20      provides a plausible biological basis  for similarity in the pattern of biological mechanisms for
21      the various outcome measures studied in the epidemiology studies. When microorganisms
22      attack a lung that has been exposed to NO2, host defense mechanisms altered by the NO2,
23      exposure may result in increased severity  or rate of respiratory illness.  While  the host
24      defense system reacts both very specifically and generally to the challenge, the overall
25      response in humans is expressed as a generalized  demonstration of signs and symptoms that
26      may be associated with a site such as the  lower respiratory tract and also be reported or
27      objectively discerned as a general outcome such as a chest cold, a cough, or an incident of
28      asthma or bronchitis.
29
30
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 1     14.5.3  Quantitative Analysis
 2          In order to compare available studies on respiratory effects of NO2, a common end
 3     point was defined, and then each study was compared with this standard end point.  For
 4     purposes of discussion, the end point chosen was the presence of LRI in children aged 12 or
 5     under.  Assume that the relative odds of developing LRI is similar across this age range as a
 6     function of NO2 exposure,  even though the actual rates may not be.  (This is a common
 7     assumption in many analyses.)  The goal was to estimate the odds ratio corresponding to an
 8     increase in the household average of 30 /ig/m3 (0.016 ppm) in NO2 exposure for a chronic
 9     exposure such as a year.  This is approximately the increase seen as a result of gas stove use
10     as compared  with electric stove use in both the United States and England. Studies with NO2
11     exposure measures used 1 to 2 week integrated indoor measurements by Palmes passive
12     diffusion tubes that provide an estimate for chronic exposure such as a year.
13          An attempt was  made to include as many studies as possible.  The requirements for
14     inclusion were:  (1) the health end point measured must be reasonably close to the standard
15     end point; (2) exposure differences must exist and some estimate of exposure must be
16     available; and (3) an odds ratio for a specified exposure must have been calculated, or data
17     presented so  that it can be  calculated.
18          As discussed earlier in this chapter, the exposure estimates used in these studies are
19     either  a surrogate (gas vs. electric) or a 2-week integrated NO2 average measured by Palmes
20     tubes.  The effects studied may be related to peak exposures, average exposures, or a
21     combination  of the two.  To the extent that health effects depend on peak exposures rather
22     than average exposures, the above exposure estimates introduce measurement error.  These
23     studies can not distinguish between the relative contributions of peak and average exposures
24     and their relationship with the observed health effects.
25     ' L   .  The British studies give us several estimates of this odds ratio.  Melia et al. (1977)
  -»*-•*,•••/*-
26      studied children aged 6 to  11 years old, and developed an indicator of the presence of at least
27     one of a group of symptoms including cough, colds going to the chest, and bronchitis.  The
28      symptom reported the majority of the time was colds going to the chest, which was used as
29      an indicator  of LRI.  This study did not measure NO2 exposure, and so the assumption was
30      made  that the increase in NO2 exposure from gas stove use in England was reasonably similar
31      to that in the other British studies which measured NO2.  The average increment in NO2
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  1     resulting from the presence of a gas stove was 31 jig/m3 (0.016 ppm) (see Chapter 7).  This
  2     value is extremely close to the specified value of 30 jug/m3 (0.016 ppm), and so no
  3     adjustment to the estimated odds ratio was made.  The estimated odds ratio was 1.31, with
  4     95% confidence limits of 1.16 to 1.48.  No adjustment was made for parental smoking in this
  5     study.
  6          The cross-sectional data reported by Melia et al. (1979) on children aged 5 to 10 also
  7     was employed to estimate an odds ratio, although no exposure estimates were made. The
  8     presence or absence of a gas stove was used as a surrogate as in the Melia et al. (1977)
  9     study. The estimated odds ratio was 1.24, with 95% confidence limits of 1.09 to 1.42.
 10          Melia et al. (1980) studied children aged 6 to 7 and measured bedroom NO2 levels for
 11     the exposure estimate.  This study applied the same combined health end point as the
 12     previous study. The estimated odds ratio for an increase of 30 /ig/m3 (0.0159 ppm) was
 13     1.53,  with 95% confidence limits of 1.04 to 2.24.
 14          Melia et al. (1982) studied children aged 5 to 6 and also measured NO2 exposure in the
 15     bedroom and also applied the same combined  health  end point.  The estimated odds ratio for
 16     an increase of 30 jtg/m3 (0.0159 ppm) was 1.11, with 95% confidence limits of ?0.83 to 1.49.
 17          Melia et al. (1983) studied children aged 1 or less and used the presence or absence of a
 18     gas stove as a surrogate for NO2 exposure, as in the other Melia studies.  The same
 19     respiratory index was applied as in the other Melia studies, with the addition of episodes of
20     pneumonia during the last 12 months.  The estimated odds ratio for an increase of 30 jwg/ni2
21      (0.0159 ppm) was 0.63, with 95% confidence limits  of 0.36 to 1.10.
22         In the first Six City study cohort, Ware et al. (1984) reported an index of respiratory
23      illness.  Exposure to NO2 with and without gas stove use was measured for only one of the
24     six cities (Portage, WI).  The analysis was based on  the presence or absence of a gas stove.
25      The estimated odds  ratio was 1.08 with 95% confidence limits of 0.97 to 1.19.
26          A second cohort of subjects in the Six City study was initially reported by Dockery
27      et al. (1989).  This  cohort of children aged 7 to 11 years was then reinterviewed after indoor
28      NO2 measurements  were made,  and the results of this analysis were reported by Neas et al.
29      (1990, 1991).  The  estimated odds ratio for an increase in the presence of any respiratory
30      symptom resulting from an increase in exposure of 31 j«g/m3 was 1.47, with 95% confidence
31      limits of 1.17 to 1.86.

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 1          Ogston et al. (1985) studied the presence of any respiratory disease in children of age
 2     one in Tayside, Scotland. No NO2 exposure estimates were obtained, and exposure
 3     classification was based on the presence or absence of a gas stove.  The estimated odds ratio
 4     was  1.14 with 95% confidence limits of 0.86 to 1.50.
 5          Ekwo et al. (1983) studied several respiratory illness end points from children surveyed
 6     at aged 6 to 12. No exposure measurements were obtained, and the exposure was based on
 7     the presence or absence of a gas stove.  None of the end points matched the end point of
 8     interest closely.  The two most similar were hospitalization for chest illness before age 2 and
 9     chest congestion and phlegm with colds. The estimated odds ratio for hospitalization was
10     2.40.  The estimated confidence limits for cough and phlegm with colds was 1.10, with 95%
11     confidence limits of 0.79 to 1.53.  This last symptom appears to be most similar to the end
12     point of interest, and so it was included in the synthesis.                       ,
13          The data presented by Dijkstra et al. (1990) on the study in the Netherlands were
14     analyzed and gave an estimated odds ratio of 0.94 for an increase of 30 ^g/m3 (0.0159 ppm)
15     in NO2 exposure.  The 95% confidence limits were 0.66 to 1.33.  The  study had measured
16     NQ2 exposure data, but the EPA analysis did not adjust for covariates since the covariates
17     were not included in the tables that included NO2 exposure.  Keller et al. (1979b) did not
18     find any statistically significant changes in respiratory disease associated with gas stove use,
19     but the unadjusted estimated odds ratio  for LRI was  1.10, with 95% confidence limits of 0.74
20     to 1.54.                        *
21           Only two studies with sufficient information for analysis were excluded in the synthesis.
22     The Schwartz (1990) analysis of the Swiss study gave Poisson regression coefficients that
23     could  have been converted to  odds ratios, but these computations were not published and
24     available for peer review. The Berwick et al. (1989) analysis has  been  criticized for its lack
25     of consistency across age groups, which may have resulted from the very small sample sizes.
26     For these  reasons, the above two studies were not included in the synthesis.
27           Graphs of the odds ratio from  each study are depicted in Figure 14-3.  Each curve can
28     be given one of three interpretations:  (1) the normal approximation to the marginal
29     likelihood of the logarithm  of the odds  ratio, (2) a distribution such that the 2.5  percentile
30     and the 97.5 percentile points of the distribution are the 95% confidence limits of the
31      estimated  odds ratio, and (3) the posterior for the odds ratio for a particular study given a flat
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                                                                   COMBINED fflxed)
                                                                   COMBINED (random)
                                                                       MeJiaotal. (1979)

                                                                       Mellaotal. (1977)
                                Wametal. (19S4)
                                                                Qgstonatal. (19S5)
                                                                Mellaotal. (19S2)
                                                                      Neasetal.(1990)
                                                                      M0IIa«rtal.{1980)
                                   K»lleretaJ. fI979b)

                        Dijksfra eta], (1990)
                                               Odds Ratio
1

2

3

4

5

6
Figure 14-3.  U.S. Environmental Protection Agency meta-analysis of epidemiologic
              studies of NO2 exposure effects on respiratory disease in children
              <12 years old.  Each curve can be treated as a likelihood function or
              posterior probability distribution.  If treated as a likelihood function, the
              95% confidence limits for the odds ratio can be calculated as those two
              points on the horizontal axis for which 95% of the area under the curve is
              contained between the two points.  If treated as a posterior probability
              distribution, then the area under the curve between any two points is the
              probability that the odds ratio lies between those two points.


prior on the log-odds ratio.  The basic information for each curve are provided in
Table 14-16.

     Methods for combining or synthesizing evidence,  often referred to as meta-analyses, are
being used more frequently as indicated by Mann (1990).  The National Research Council

(1986) combined evidence on the effect of environmental tobacco smoke on lung  cancer using
Peto's method as described by Yusuf et al. (1985).  Several methods for combining clinical
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               TABLE 14-16.  SUMMARY OF ODDS RATIOS FROM STUDIES OF
                            THE EFFECTS OF NITROGEN DIOXIDE
Author
Melia et al. (1977)
Melia et al. (1979)
Melia et al. (1980)
Melia et al. (1982)
Melia et al. (1983)
Ware et al. (1984)
Neas et al. (1990)
Ogston et al. (1985)
Ekwo et al. (1983)
Dijkstra et al. (1990)
Keller et al. (1979b)
Estimated Odds Ratio
1.31
1.24
1.53
1.11
0.63
1.08
1.47
1.14
1.10
0.94
1.10
2.5 and 97.5 Percentiles
(Confidence Interval)
(1.16, 1.48)
(1.09, 1.42)
(1.04, 2.24)
(0.83, 1.49)
(0.36, 1.10)
(0.97, 1.19)
(1.17, L86)
(0.86, 1.50)
(0.79, 1.53)
(0.66, 1.33)
(0.79, 1.54)
1     trials were discussed by Laird and Mosteller (1990). The evidence to be combined in this
2     section comes from epidemiological studies and, as a result, some of the methods used for
3     clinical trials are not appropriate for this section.
4          Two basic models are employed in order to combine evidence. The first model assumes
5     that each study estimates the  same fixed, but unknown, parameter. Most methods of
6     combining evidence make this assumption.  One of the earliest classical methods was the
7     variance weighted method (Hald,  1962).  This method weights the estimates inversely by
8     their variances and produces  a combined estimate and associated confidence limits.  Other
9     methods include the Mantel-Haenszel method (Mantel and Haenszel, 1959), which is used to
0     combine contingency tables.  Recently, Bayesian methods have been used to combine
1     evidence,  and methods particularly appropriate to these kinds of studies were described by
2     Eddy (1989) and Eddy et al.  (1990a,b). Bayesian analyses require the choice of a prior
3     distribution for the parameter of interest, which is often a noninformative prior.
4     A noninformative prior is one that prior to seeing the evidence favors no value of the
5     parameter over any other.  The interesting fact about use of these methods is that, for the
6     data sets considered in Table 14-16, the results of the computations were nearly identical.
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  1     This is because the (marginal) likelihood for the odds ratio is closely approximated by a
  2     lognormal curve.  The interpretations of these curves are different, as described earlier.
  3          The second basic model assumes that the parameter of interest is not fixed, but is itself
  4     a random variable from a distribution.  The value of this random variable is different for each
  5     study,  but each study gives some information about the mean of the distribution.  These
  6     models go by several names, including random effects models, mixed models, two-stage
  7     models, or hierarchical models.  The purpose of a random effects model is to relax the
  8     assumption that each study is estimating exactly the same parameter.  For a discussion of the
  9     interpretation of random effects  models in clinical trials and several methods of estimating the
 10     parameters of these models, see DerSimonian and Laird (1986).  If the studies being
 11     combined tend to estimate the same parameter,  then the results using a random effects model
 12     will approach the results using a fixed-effects model.  On the other hand, if the studies are
 13     estimating vary different parameters, then the variances of the estimates will more closely
 14     reflect  the variance of the unknown distribution being sampled.
 15          The 11 studies described earlier (Tables 14-14,  14-16) were combined using both kinds
 16     of models. The results using a fixed-effects model are labeled "fixed", and results of the
 17     random effects model are labeled "random" (see Figure 14-3). Two different estimates of
 18     random effects models were used.  The first was estimated using the noniterative estimates
 19     assuming unequal variances as described by DerSimonian and Laird (1986) and is labeled
20     "D & L." The second is a hierarchical Bayesian model using a flat prior for  the unknown
21      variance and this model is labeled "H-B". The results of the analyses are provided in Table
22     14-17.
23           The first line of Table 14-17 shows the results of combining all 11 studies using a fixed
24      model.  The estimated odds ratio is 1.18 and the 95% confidence limits are 1.11 and 1.25.
25      The analysis was made assuming that the parameters were the same (homogeneous), and this
26      can be  tested.  The chi-square for homogeneity for the 11 studies was 18.75 for 10 degrees of
27      freedom,  which has a p-value of 0.0436. Thus, there is some evidence that the parameters
28      from each study are not identical. The estimates for the two random effects models give
29      similar  estimates to the fixed model,  but the confidence limits are slightly broader. The
30      conclusion from all three models is the same, namely that the odds ratio is estimated to  be
31      about 1.2 with 95% confidence limits ranging from about 1.1 to 1.3.

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         TABLE 14-17.  U.S. ENVIRONMENTAL PROTECTION AGENCY COMBINED
                    ANALYSES OF STUDIES ON RESPIRATORY ILLNESS
                              EFFECTS OF NITROGEN DIOXIDE
Study group
All
All
All
Children > 1
Children > 1
Children >1
Children <1
Measured NO2, age > 1
Gas stove, age > 1
British, age > 1
Six City
Other studies
Number
of
Studies
11
11
11
9
9
9
2
4
5
4
2
3
Modela
fixed
H-B
D&L
fixed
H-B
D&L
fixed
fixed
fixed
fixed
fixed
fixed
Odds ratio
1.18
1.18
1.18
1.19
1.20
1.19
1.01
1.27
. 1.18
1.27
1.13
1.05
Percentiles
(Conf. Int.)
(1.11, 1.25)
(1.08, 1.29)
(1.08, 1.30)
(1.12,1.27)
(1.10, 1.31)
(1.09, 1.30)
(0.79, 1.30)
(1.09, 1.47)
(1.10, 1.26)
(1.17, 1.39)
(1.03, 1.24)
(0.86, 1.27)
      "Fixed = Fixed-effects model; H-B = Hierarchial Bayesian model; D-L = DerSimonian and Laird model.


 1          Two of the 11 studies were on children under 1 year of age, whereas the others were on
 2     children of elementary school age. Since the end point of wheeze is more predominant in the
 3     children less than 1 year old as opposed to the older children and the outcome measure in
 4     Ogston et al. (1985) includes upper respiratory illness, making it dissimilar to the others,
 5     there may be reason to separate these studies from the others.  These analyses are shown on
 6     lines four through seven of Table 14-17. Note that the odds ratio for the two studies of
 7     children under age 1 gives an odds ratio of 1.01, which is well below the estimate of 1.19 for
 8     the older children.  The estimate for the younger children has much greater uncertainty due to
 9     the fewer number of studies. The nine studies of older children have a chi-square for
10      homogeneity of 13.55 for  8 degrees of freedom (p=0.0942).  Although this value is no
11      longer statistically significant at the 0.05 level, it still  suggests some randomness in  the
12     parameter values. In spite of this, the estimates from all three models are reasonably similar
13     to each other and are similar to the results of all 11 studies combined.
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   1           Four of the nine studies of older children used measured NO2 values, whereas the other
  2      five did not.  The use of a surrogate for exposure should tend to reduce the estimate of the
  3      effect (see Samet and Utell, 1990).  The measured NO2 studies gave an estimated odds ratio
  4      of 1.27, whereas the others gave an estimate of 1.18, which is consistent with a measurement
  5      error effect. The chi-square tests for homogeneity were not significant at the 0.1 level for
  6      either group of studies.
  7           Finally, the studies were compared by similarity of subjects.  Four of them were done
  8      in Great Britain (Melia studies), two were based on the Harvard Six City studies (Ware et al.
  9      and Neas et al.), and the other three had little in common.  The British studies provide the
 10      highest estimated odds ratio, 1.27, the Six City Studies give a combined estimate of
 11      1.13, and the other three studies give an estimate of 1.05.  The estimates from the British and
 12      Six City studies have confidence intervals that do not include 1.0.
 13          Although there may be reasons to weight certain studies or groups of studies more
 14      heavily than others, the final conclusion has to be that there is an increase in the odds of
 15      respiratory disease of children, especially those of elementary school age; The estimates are
 16      generally centered about an odds ratio of 1.2 with 95% confidence limits of 1.1 to 1.3,
 17      although the studies using measured NO2 give a slightly higher estimate of the odds ratio.
 18      The estimates are not sensitive to the assumption that each study is estimating the same
 19     parameter as indicated by the hierarchical analyses.  Thus, any lack of homogeneity is not a
 20      major concern.
 21           These results are not sensitive to the inclusion or exclusion of any one study. It would
 22     have been possible to include the hospitalization results of Ekwo et al.  (1983), the Schwartz
 23      (1990) analysis of the Swiss study, or the Berwick et al. (1989) study.  None of these studies
 24      would have made any real change in the estimated odds ratios or their 95% confidence limits.
 25      It is also possible to delete any one study from the analysis, and still obtain nearly the same
 26      results.  In fact, any two studies can be deleted from the analysis, and the estimated odds
 27      ratio will have a confidence interval that does not include  1.0.
 28           There is always the concern that the studies described in this document are not the
29      complete list of studies,  but contain primarily the positive studies, since these are the studies
30      most likely to get published. This is referred to as "publication bias."  There are two reasons
31      to be less concerned with publication bias in this particular situation. First, epidemiological

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 1
 2
 3
 4.,
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
studies are very expensive and require the work of many individuals.  The studies are usually
described to the scientific community before the results are even known.  Second, most of the
studies cited in this section were reported as negative studies by the authors themselves,
indicating that there was no difficulty publishing negative results. In spite of this, it is of
interest to contemplate,an undiscovered study with results so negative that, when combined
with the other studies, produces a confidence interval for the odds ratio that includes the
value 1. If we assume that the hypothetical study would be the size of the Ware et  al. study,
then its odds ratio for increased respiratory symptoms as the result of a 30 jug/m3 exposure
would have to be 0.766.
 14.6  CONCLUSIONS
      The evidence from individual studies of the effect of NO2 on respiratory disease is
 somewhat mixed.  All but two of the studies used in the synthesis showed increased
 respiratory disease rates associated with increased exposure.  A few of the individual studies
 were statistically significant.  Combining the studies giving quantitative estimates of effects
 tend to show increases of respiratory illness in children associated with long-term exposure to
 NO2.  Combining the studies as if the end points were similar gives an estimated odds ratio
 of 1.20 (95% confidence limits of 1.1 and 1.3) for the effect of gas stove use on respiratory
 illness. When combined, the studies indicated that an increase of 30 Atg/m3 in NO2 exposure
 would result in an increase of about 20%  in the odds of respiratory disease, subject to the
 assumptions made for the synthesis. Although several assumptions were made to combine the
 studies, the consistency between the individual studies is demonstrated, indicating greater
 strength to the data base that suggests  that the effect is real.  To the extent that health effects
 depend on peak exposures rather than  average exposures, the exposure estimates used
 introduce measurement error.  These studies can  not distinguish between the relative
 contributions of peak and average exposures and  their relationship with the observed health
 effects.  However, the estimated effect is almost  surely an underestimate, given the problems
 of misclassification of exposures and outcomes.  The effect was not dependent on any one or
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  1
  2
  3
  4
  5
  6
  7
  8
  9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
 20
 21
 22
 23
 24
 25
 26
 27
28
29
30
 two studies.  Thus, the combined evidence is supportive for the effects of exposure to NO2
 on respiratory disease in children under 12 years of age.
      Only the Harvard Six City study attempted to relate some measure of indoor and
 outdoor NO2 exposure to long-term changes in pulmonary function.  These changes were
 marginally significant.  No short-term studies had indoor exposures.  Most studies did not
 find any effects, which is consistent with results from controlled human exposure studies (see
 Chapter 15).  However, the basic conclusion is that there is insufficient epidemiological
 evidence to make any conclusion about the long- or short-term effects of NO2 on pulmonary
 function.
 14.7  SUMMARY
     This chapter discusses the epidemiological evidence for the effects of NO  on human
                                                                         X
 health. The major emphasis is on the effects of NO2 because it is the NOX compound studied
 in most epidemiological studies and because it is the NOX compound currently of greatest
 concern from a public health perspective.  The results from the various epidemiological
 studies of effect of NO2 exposure on human health outcomes are summarized in
 Appendix 14A.                                                 ......
     The studies considered in this chapter were evaluated for several key factors, including:
 (1) measurement error in exposure, (2) misclassification of the health outcome, (3) selection
 bias, (4) adjustment for covariates, (5) publication bias,  (6)  internal consistency, and
 (7) plausibility of the effect based on other evidence.  The health outcome should be an
 outcome for which there is good reason to suspect that NO2 exposure has an effect.  Two
 health  outcome measures are generally considered:  lung function measurements and
 respiratory illness. Each study is reviewed with special attention given to the factors just
 discussed. Those studies which address these factors more appropriately  provide a stronger
basis for the conclusions that they draw.  Consistency between studies  indicates the level of
the strength of the total data base.
     Respiratory illness and factors that affect its rate and/or severity are important public
health concerns.   This chapter  discussed epidemiological findings relating NO2 exposure to
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 1     respiratory illness.  This effect is of public health importance because of the potential for
 2     exposure to NO2 and since childhood respiratory illness is common (Samet etal., 1983;
 3     Samet and Utell, 1990).  This takes on added importance since recurrent childhood
 4     respiratory illness may be a risk factor for later susceptibility to damage to the lungs (Glezen,
 5     1989).                  -
 6          A biologic basis for an increased susceptibility for respiratory illness with exposure to
 7     NO2 is found in studies of the respiratory host defense system. The host defense system
 8  ,   provides protection against inhaled infectious and chemical agents.  The available
 9     information, discussed in the animal toxicology and clinical studies chapter, indicates that
10     exposure to NO2 impairs one or more defense mechanisms, leaving the host susceptible to
11     respiratory illness.
12           Several of the epidemiological studies gave some evidence that repeated NO2 exposure
13     increases respiratory illness in children, although many were not statistically significant.
14     Melia-et al. (1977) first  reported on a survey of children in randomly selected areas of
15     England and Scotland using the presence of a gas stove as a measure of'NO2 exposure.
16     A reanalysis of that data yields an estimated  odds ratio of 1.31 for the presence of respiratory
17     symptoms.  The cross-sectional study of Melia et al. (1979) also found that the presence of a
18     gas stove was associated with increased risk of respiratory disease. The odds ratio was
19      1.24 with 95% confidence limits of 1.09 to 1.42.   Melia et al. (1980) described on the  results
20     of a third,study of respiratory symptoms in children aged six to seven in northern England.
21     Multiple logistic regression analysis of the data presented by Melia et al. (1980) showed a
22      significant increase in symptoms as a function of bedroom  NO2 levels. Melia et al.  (1982)
23      reported on :a fourth  study of children in England.  Multiple logistic regression analysis of
24      this data was not statistically significant, although the symptoms were positively related to
25      NO2 exposure.  An EPA reanalysis suggests that an increase of 30 /ig/m3 in bedroom NO2
26      levels yields an 11 % increase in  the odds of respiratory illness. In a final Melia et al. (1983)
27      study, infants under  1 year of age were examined.  No relation was found between type of
28      fuel used for cooking and the prevalence of respiratory symptoms.
29           The analysis  of the Six  City studies by Ware et al. (1984) estimated an unadjusted odds
30      ratio of 1.08 (95% confidence limits of 0.97 to 1.19) for a LRI index associated with gas
31      stove use.  Other indicators such as bronchitis, cough, and  wheeze did not show any
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   1     increased incidence. Neas et al. (1990, 1991) analyzed a different cohort enrolled later, used
   2     a different symptom questionnaire and made indoor NO2 measurements for all subjects.
   3     They found increased respiratory disease which gave an estimated odds ratio of 1.47 (95%
   4     confidence limits of 1.17 to 1.86)  at an exposure of 31 jtg/m3.
   5          Ogston et al. (1985) studied respiratory disease in  1-year-olds in the Tayside region of
   6     northern Scotland.  The presence of a gas stove yielded  an odds ratio of 1.14, with 95%
   7     confidence limits of 0.86 to 1.50.  Ekwo et al. (1983) studied respiratory symptoms in
   8     relation to gas stove use in Iowa City, IA.  Gas stove use provided in an  odds ratio of 2.4 for
   9     hospitalization for chest illness before age 2, and 1.1 for chest congestion and phlegm  with
 10     colds.  Dijkstra et al. (1990) studied the effect of indoor factors on respiratory health in
 11      children in The Netherlands.  A logistic regression analysis yielded an odds ratio of 0.94 with
 12      95% confidence limits of 0.66 to 1.33, thus showing no evidence of an increase in
 13      respiratory disease with increasing  NO2 exposure.  Keller et al. (1979b) did not find any
 14      statistically significant changes in respiratory disease associated with gas stove use,  but the
 15      unadjusted estimated odds ratio for lower respiratory illness was 1.10,  with 95% confidence
 16      limits of 0.74 to 1.54.
 17           Other studies did not provide  sufficient information to derive any quantitative estimates
 18      of the effect of NO2 or gas stove use on respiratory disease.  Several other studies contain
 19      information about the effects of NO2 on respiratory illness, but most of the studies either
 20      used very different health end points or did not provide quantitative estimates of the effects.
 21      The study  of Berwick (1987) showed increased relative risk of respiratory  disease in some
 22      age groups, but not in others.  Euler et al. (1988) did not find increases in chronic respiratory
 23      symptoms  associated with changes in NO2 levels. Vedal et al. (1987) studied children in the
 24      Chestnut Ridge in Pennsylvania, but did not obtain any relationship between daily respiratory
 25      symptoms and NO2 levels.
 26          Several of the studies suggest an increase in respiratory symptoms in  children from
 27     exposure to levels  seen with gas stove use as compared to electric stove use.  The associations
 28     in the majority of the studies do not reach statistical significance. The consistency of these
 29     studies was examined and the evidence synthesized in the following quantitative analysis.
30     The studies described used different indicators to study health endpoints. In order to compare
31      these studies, a standard end point was defined, and then  each study will be compared with

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 1     this standard end point.  For purposes of discussion, the end point chosen was the presence of
 2     lower respiratory disease in children aged 12 or under.  It was assumed that the relative odds
 3     of developing lower respiratory disease is similar across this age range as a function of NO2
 4     exposure, even though the actual rates may not be.  (This is a common assumption  in many
 5     .analyses.)  The goal was to estimate the.odds ratio corresponding to an increase of 30 fig/m3
 6     in NO2 exposure.  This is approximately the increase seen as a result of gas stove use as
 7     cpmpared with electric stove use in both the U.S and England.          .    .     -
 8          An attempt was  made to include as many studies as possible.  The requirements for
 9     inclusion were (1)  the health end point measured must be reasonably close to the  standard end
10     point,  (2) exposure differences must exist and some estimate of exposure must be available,
11     and (3) an odds ratio  for a specified exposure must have been calculated,  or data  presented so
12     that it  can be calculated.
13          Graphs of the odds ratio from each study are presented in Figure 14-3. Each curve can
14     be given one of three interpretations:  (1) the normal approximation to the likelihood of the
15     logarithm of the odds ratio, (2) a distribution such that the 0.025 percentile  and the 0.975
16     percentile points of the distribution are the 95% confidence limits of the estimated odds ratio,
17     and (3) the posterior for the odds ratio for a particular study given  a flat prior on the log-
18     odds ratio.
19          Two methods of combining evidence are employed here. The first of these is a
20     Bayesian method,  and is described by Eddy (1989) and Eddy et al, (1990a,b) as it is used
21     here.  The result of the analysis is a distribution for our belief about the location  of the true
22     value of the odds ratio.  This can be done separately for each study or for the combination of
23     all studies, and the results are shown in Figure 14-3.  The second basic model  assumed that
24     the parameter of interest is not fixed,  but is itself a random variable from'a distribution.
25     These models are  designated by several names including random effects models or
26     hierarchical models.  The purpose of a random effects model is to  relax the assumption that
27     each study is estimating exactly the same parameter. DerSimonian and Laird (1986) discuss
28     the random effect model.
29          The conclusion  from the several models was the same, namely that the odds ratio is
30     estimated to be about 1.2 with 95% confidence limits ranging from about 1.1 to  1.3.  The
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  1     measured NO2 studies gave an estimated odds ratio of 1.27, whereas the others yield an
  2     estimate of 1.18, which is consistent with a measurement error effect.
  3          The individual evidence of the effect of NO2 on respiratory disease is somewhat mixed.
  4     All but one of the studies used in the synthesis showed increased respiratory disease rates
  5     associated with increased exposure.  A few of the individual studies were statistically
  6     significant.  Combining the studies giving quantitative estimates of effects tend to show
  7     increases of respiratory illness in children associated with long-term exposure to NO2.  When
  8     combined, the studies indicated  that of increase of 30 jug/m3 in NO2 exposure would result in
  9     an increase of about 20% in respiratory disease, subject to the assumptions  made for the
10     synthesis.  Although  several assumptions were  made to combine the studies, the consistency
11     between the individual studies is demonstrated, indicating greater strength to the data base
12     which suggests that the effect is real.  To the extent that health effects depend on peak
13     exposures rather than average exposures, the exposure estimates used introduce measurement
14     error. These studies  can not distinguish between the relative contributions of peak and
15     average exposures and their relationship with the observed health effects. However, the
16     estimated effect is almost surely an underestimate, given the problems of misclassification of
17     exposures and outcomes. The effect was.not dependent on any one or two  studies.  The
18     results of this analysis are not sensitive to the inclusion (or exclusion) of any one study.  In
19     fact, any two studies  can be eliminated, and the 95% confidence limits will exclude the no
20     effect odds ratio of 1.0. Thus, the combined evidence is supportive for  the effects of
21     exposure to NO2 on respiratory  disease in children under 12 years of age.
22          Only the Harvard Six City study attempted to relate some measure of indoor and
23     outdoor NO2 exposure to long-term changes in pulmonary function. These changes were
24     marginally significant.  No short-term studies had indoor exposures.  Most studies did not
25     find any effects, which is consistent with controlled human exposure study data (see
26     Chapter 15).  However, the basic conclusion is that there is insufficient epidemiological
27     evidence to make any conclusion about the long- or short-term effects of NO2 on pulmonary
28     function.
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31            respiratory study: I. design and implementation of a prospective study of acute and chronic respiratory
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33
34     Thielebeule, U.; Huelsse, C.  (1989) Epidemiologische Untersuchung zur Formaldehyd- und Stickoxidbelastung in
35            Innenraeumen [Epidemiological examinations  on formaldehyde and nitrogenous oxide indoor air
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37
38     Tominaga, S.; Ono,  M. (1985) A plan of the comprehensive study on indoor pollution and its  health effects by
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40
41     Tsunetoshi, Y.; Fukutomi, K.; Yoshida, K.;  Doi, M. (1987) [Epidemiological study of respiratory symptoms in
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43
44     U. S. Environmental Protection Agency. (1971) Air quality criteria for nitrogen oxides.  Washington, DC: U. S.
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46            NTIS, Springfield, VA; PB-197333/BE.
47
48     U. S. Environmental Protection Agency. (1982) Review of the national ambient air quality standards for nitrogen
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51            NTIS, Springfield, VA; PB83-132829.
52
53
         August 1991                                   14-74       DRAFT-DO NOT QUOTE OR CITE

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  1
  2
  3
  4
  5
  6
  7
  8
  9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
Vedal, S.; Schenker, M. B.; Munoz, A.; Samet, J. M.; Batterman, S.; Speizer, F. E. (1987) Daily air pollution
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Ware, J.  H.; Dockery, D. W.; Spiro, A., Ill; Speizer, F. E.; Ferris, B. G., Jr. (1984) Passive smoking, gas
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       respiratory study: II. lower respiratory tract illness in the first year of life. Am. J. Epidemiol.  129:
       1232-1246.


Yamamoto, I.; Takahashi, M. (1984) Ultrastructural observations of rat lung exposed to nitrogen dioxide for
       7 months. Kitasato Arch. Exp. Med. 57: 57-65.

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       dioxide exposure. JAMA J. Am. Med.  Assoc. 244: 1221-1223.
         August 1991
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1
2
3
4
5
6
Yoshimura, I. (1990) The effect of measurement error on the dose-response curve. Environ. Health Perspect. 87:
       173-178.


Yusuf, S.; Peto, R.; Lewis, J.; Collins, R.; Sleight, P. (1985) Beta blockade during and after myocardial
       infarction: an overview of the randomized trials. Prog. Cardiovasc. Dis. 27: 335-371.
       August 1991
                                                14-76
DRAFT-DO NOT QUOTE OR CITE

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APPENDIX 14A. SUMMARY OF HEALTH EFFECTS OF NO,
                      14A-1

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                            14A-12    DRAFT-DO NOT QUOTE OR CITE

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 1
 2
    15.  CONTROLLED  HUMAN EXPOSURE STUDIES
                     OF OXIDES OF NITROGEN
 4
 5'
 6
 7
 8
 9
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15.1  INTRODUCTION
'•    This chapter discusses the effects of nitrogen oxides (NOX) on human volunteers
exposed under controlled conditions.  The NOX species of major concern is nitrogen dioxide
(NO2).  Nitric oxide (NO), and nitrates  (XNO3) have also been evaluated in controlled
human exposures and nitric acid (HNO3) effects have only recently been studied.  The 1982
Air Quality Criteria for Oxides of Nitrogen Document (U.S. Environmental Protection
Agency,  1982a) presents  a.comprehensive review of studies conducted up to about 1980.
The present chapter provides summaries and critiques of studies conducted since then, while
also including the earlier literature.
     Controlled human exposure studies deal with relatively brief exposures to higher
concentrations compared  to the annual arithmetic mean standard (0.053 ppm).  One of the
purposes in reviewing these studies is to evaluate the data base for short-term (typically
<4h), NO2-related health effects; thus,  consideration of the time course of responses and the
pattern of exposure in controlled human exposure studies is important.
     Because of the prevalence of NO2  in both outdoor and indoor environments, there are
major concerns regarding the potential impact of NO2 exposure on human health particularly
with regard to the lung.  Dosimetry modeling and animal histological studies indicate that
NO2's impact should be primarily seen in  the small airways and gas exchange regions of the
lung;  thus, tests that specifically evaluate responses in this region are of particular interest in
evaluating the effects  of NO2.  These concerns are presented in the following list of critical
questions. Some of these questions are of a "generic" nature, applying to many ambient air
pollutants and some are specific to NO2 inhalation.  Several of the questions may be
answered only partially by controlled human exposure studies.
       August 1991
                                        15-1
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  1           NO2 Exposure and Human Health:  Critical Questions.

  2

  3           1.    Does short-term NO2 exposure cause acute changes in lung function, increased
  4                 respiratory symptoms, or increased airways responsiveness in normal, healthy
  5                 subjects at levels that may be expected in the ambient (or indoor) environment?
  6                 Are these effects reproducible?  If so, what is the possibility that such acute
  7                 responses may contribute to chronic changes in lung function, promote the
  8                 development of respiratory disease, cause acceleration of normal age-related
  9                 declines in lung function or aggravate existing respiratory disease?
10
11           2.    Are there groups within the population who exhibit greater responses to NO2
12                 exposure than the average healthy subject? The possible groups may include
13                 young children, adolescents, elderly subjects, patients of all ages with asthma,
14                 chronic obstructive lung disease, or other lung diseases.  If there are subject
15                 groups who are more responsive, can they be identified prospectively (i.e.,
16                 without exposing them to NO2 first)?
17
18           3.    Does NO2 cause an inflammatory response in the lungs of healthy individuals or
19                 patients with lung disease?  Specifically, does NO2 exposure cause increased
20                 capillary  permeability, increased local blood flow, extravasation of fluid or  influx
21                 of leukocytes—especially neutrophils and eosinophils—into the interstitium and
22                 the airways, secretion of pro-inflammatory mediators, mast cell degranulation,  or
23                 epithelial desquamation?
24
25           4.    Does NO2 exposure cause increased responses of the lung (including airways
26                 responsiveness, lung function, inflammation, cell damage, etc.) to other
27                 pollutants such as ozone (O3), sulfur dioxide (SO^, acid aerosols; to
28                 bronchoconstrictors  such as histamine or methacholine; to other agents such as
29                 cold-dry air or exercise; or to specific antigenic substances?
30
31           5.    Does NO2 exposure alter respiratory tract host defenses? Does NO2 alter airway
32                 epithelial permeability or mucociliary clearance?  Does NO2 exposure alter local
33                 or systemic immune response to infection? Is killing or removal of
34                 microorganisms impaired by NO2 exposure?  Is inflammatory response or tissue
35                 injury caused by microorganisms worsened by coincident NO2 exposure?
36
37           6.    What is the time course of response to acute exposures?  Are there both
38                 immediate and delayed  responses? Do responses  increase or decrease with
39                 increased exposure duration?  What is the time course of response to repeated
40                ° exposures? Do responses increase or decrease with increased frequency of
41                 exposure?
42
43
44
        August 1991                              15-2      DRAFT-DO NOT QUOTE OR CITE

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 1           Controlled human exposure studies serve as an important source of inhalation toxicology
 2      data, particularly for the criteria pollutants such as NOX.  Methodological and experimental
 3      design considerations for controlled human exposure studies have recently been reviewed
 4      (Folinsbee,  1988).  These studies are typically conducted on volunteers who have been
 5      informed of the possible risks of such studies and who have given their "informed consent" to
 6      participate.  The subject group that most often participates in such studies are young adult
 7      males with no history of respiratory disease, allergies, smoking, and no contraindication to
 8      exercise. In addition to young men,  participants, of either gender and of different racial
 9      groups,  may include children, adolescents, elderly persons, rand adults. Other subject groups
10     that have been specifically studied include healthy subjects with allergies, asthmatics,
11      smokers, patients with chronic obstructive pulmonary disease (COPD), or otherwise healthy
12     persons  with upper respiratory infections.  These latter subject groups may  be considered
13     potentially "sensitive subjects," especially asthmatics, COPD patients, children, and the
14     elderly.  Characterization of asthma is discussed in more detail in Appendix A.  For
15     individuals with existing lung disease and/or hyperresponsive airways, special consideration
16     of the potential impact of pollutant exposure is  required.  These individuals, including the
17   '  healthy  elderly population,  often have limited pulmonary reserves and, therefore, a given
18     insult (increased airways resistance, restriction of lung volume) has a greater
19     physiological/pathological consequence.  Children are of special concern because their lungs
20      are still growing and developing and, hence, the possibility of a long-term  impact on lung
21      health may be greater than for the mature adult lung.
22           "Controlled" exposures, by definition, occur in a laboratory setting.  The most "natural"
23      mode of. exposure is unencumbered breathing within an exposure chamber. Other modes of
24      exposure include facemask, hood,  or mouthpiece exposures.  A "controlled"  exposure implies
 25      that the environmental variables such as the concentration of the pollutant,  temperature, and
 26      humidity are monitored and maintained at some specified level.  In addition, the duration of
 27      the exposure and amount of activity during the exposure are closely regulated.  The activity
 28      level is closely correlated with the volume of air breathed into the lung.  In order to  simulate
 29      an outdoor exposure where the subject is active, many exposure studies include some form of
 30     controlled exercise.  However, exercise alone may  have some important confounding effects,
 31      particularly in the case of exercise-induced bronchoconstriction in asthmatics.  Exercise alone
         August 1991
15-3
DRAFT-DO NOT QUOTE OR CITE

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  1     may induce significant decrements in spirometric variables or significant increments in airway
  2     resistance. Exercise-induced bronchoconstriction is followed by a refractory period of several
  3     hours during which asthmatics are less susceptible to bronchoconstriction (Edmunds et al.,
  4     1978).  This period of refractoriness could alter the subject's responsiveness to NO2 or other
  5     inhaled substances.  The major external determinants of the exposure "dose" of a pollutant
  6     are the concentration of pollutant, the duration of the exposure, and the volume of air
  7     breathed (specifically, the route, depth, and frequency of breathing) during the exposure.
  8     Further information is, of course, necessary to determine the actual dose delivered to the
  9     various "target" regions of the respiratory tract  (i.e., total respiratory uptake) and are
 10     presented in Chapter 13. Many of these considerations have been discussed in greater detail
 11     by Folinsbee (1988).
 12          In human exposure studies, the methods used for assessment of effects involve primarily
 13     "noninvasive" procedures.  Lung function tests such as  spirometric measures of lung
 14     volumes, measures of resistance of lung or nasal airways, ventilation volume (volume  of air
 15     inhaled into the lung), breathing pattern (frequency and depth  of breathing), and a variety of
 16     other "breathing" tests have been utilized (Bouhuys, 1974).  These tests provide information
 17     about some of the basic physiological functions of the lung. Dynamic spirometry tests
 18     (forced expiratory tests such as forced expiratory volume in 1  second  [FEVj], maximal and
 19     partial  flow-volume curves [including those using gases of different densities such as helium],
20     peak flow measurements, etc.)  and specific airway resistance/conductance measurements
21      (SRaw, SGaw) provide information primarily about large airway function.  The reader should
22     refer to the glossary for more specific descriptions of various tests. These "standard
23      pulmonary function" tests are relatively simple to administer, provide a good overall index of
24      lung function, and have a relatively low coefficient of variation (CV)  (for FEV1? the CV is
25      about 3% and for SRaw the CV is about 10 to 20%).  However, because NO2 deposits
26      primarily in peripheral airways, many of the above tests may not provide the necessary
27      information to fully evaluate the effects of NO2.  Other tests purported to provide evidence of
28      small airway function include multiple breath nitrogen washout tests, closing volume tests,
29      aerosol deposition/distribution tests, density dependence of flow-volume curves (using gases
30      of different densities such as helium),  and frequency dependence of dynamic compliance, but
       August 1991                             15_4      DRAFT-DO NOT QUOTE OR CITE

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 1     none are used routinely and use of these procedures  to assess "small airways function" is not
 2     widely accepted.                                              ......
 3          Somewhat more invasive procedures have been more utilized in recent years to
 4     determine response to pollutants including pharmacologic airway inhalation challenge tests,
 5     measurements of pulmonary clearance of inhaled aerosols, bronchoalveolar lavage, nasal
 6     lavage, and arterial blood gas measurements.
 7          Airway inhalation challenge tests are used to evaluate the "responsiveness" of a
 8     subject's airways to inhaled materials. Airway responsiveness (AR) may change as a result
 9     of alterations in a disease state, such as inflammation associated with asthma, viral
10     respiratory infection, or as a result of damage to the airway caused by disease or insult from
11     inhaled toxic or allergenic materials.  Thus, one of the problems in evaluating changes in
12     airway responsiveness with respect to inhalation of air pollutants is that the baseline
13     responsiveness can be changed by other factors not associated with pollutant exposure.  In
14    . order to test for the degree of airway responsiveness, a chemical that causes constriction of
15     .the airways, such as histamine, carbachol, or methacholine, is typically used.  Other
16     challenge tests involve the use  of allergenic substances, exercise, hypertonic saline, or cold-.
17     dry air. Responses are usually measured by evaluating changes in airway resistance or
18     spirometry after each dose of the challenge is administered.  Usually, the test will proceed
19     until some target effect level is achieved (e.g., doubling of airway resistance) and the "airway
20     responsiveness" is then characterized by the dose required to achieve that level.  The
21     procedures for administering and interpreting inhalation challenges are discussed in detail
22     elsewhere (Cropp et al.,  1980; Cropp,  1979; Chai et al.,  1975; Fish  and Kelly, 1979;
23      O'Byrneetal., 1982).
24           Asthmatics, as a group are significantly more responsive than healthy normal subjects to
25     ,a variety of airway challenges. The differences in airway responsiveness may span several
26      orders of magnitude (at least 100 fold) between normals and asthmatics (O'Connor et al.,
27      1987).  Nevertheless, there is  considerable overlap between the more responsive healthy
28      subjects and the less responsive (histamine) asthmatics (Pattemore et al., 1990).  Airway
29      responsiveness to methacholine appears to be somewhat better than airway responsiveness to
 30      histamine at differentiating normals and asthmatics, although responses to the two
 31      bronchoconstrictors are well correlated (r=0.70) (Chatham et al., 1982).  Unfortunately,
        August 1991
15-5
DRAFT-DO NOT QUOTE OR CITE

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  1     because of the large variety of methods and provocative agents used in the airway challenges
  2     and the absence of adequate standardization, it is difficult to compare responses between
  3     laboratories in a quantitative manner.  Thus, it is not useful to suggest standard ranges of
  4     responsiveness for normals and asthmatics.
  5          Tests of pulmonary clearance of inhaled aerosols are used to assess the efficiency of the
  6     mucociliary clearance mechanism and to estimate pulmonary epithelial permeability.
  7     Typically, a radioactively labeled test aerosol, of a specific size range that will deposit in the
  8     lung region of interest, is deposited in the lung by inhalation. External detectors are then
  9     used to assess the amount of remaining test aerosol at various times after the initial deposition
 10     of the aerosol. This methodology is discussed in Clarke and Pavia (1980) and Raabe (1982)
 11     and in Section 13.2.2.1.  A particular application of clearance of radiolabeled aerosols is for
 12     the estimation of epithelial permeability, typically using technetium (99mTc)-labeled
 13     diethylene triamine penta-acetate or pentetate (DTPA).  This methodology is discussed in
 14     Nolop  et al. (1987).
 15          In the past several years, bronchoalveolar lavage techniques have been used in clinical
 16     exposure studies of several  different pollutants.  In this procedure, a fiberoptic bronchoscope
 17     is passed into the airways and wedged in a subsegmental bronchus where sterile buffered
 18     saline is used to wash free cells and airway secretions from the segment (Reynolds, 1987).
 19     The resulting lavage fluid may be analyzed for various chemical mediators or reaction
 20     products, numbers and types of cells, and the functions of some lung cell  types. Another less
 21      invasive procedure, known  as nasal lavage, may be used to  obtain nasal secretions and cells
 22     (Graham etal., 1988).
 23           There are a number of limitations of controlled human exposure or "clinical" studies.
 24      In contrast to many animal  models, humans have a wide range of response to a variety of
 25      physiological and pathological stimuli.  This variability and  the small sample numbers limit
 26      the  extent to which the data can be generalized to the population as a whole or to certain
 27      defined segments of the population (e.g., asthmatics).  The  small sample size may limit the
28      interpretation of the study, especially when the results are negative (i.e., the null hypothesis
29      is not rejected).  One cannot have great confidence in a study that finds no effect of a
30      treatment (in this case NO2  exposure) if the sample size (i.e., the number  of subjects) is too
31      small to statistically detect an effect, if present.  This must be kept in mind in interpreting the

        August 1991                              15_6      DRAFT-DO NOT QUOTE OR CITE

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  1
  2
  3
  4
  5
  6
  7
  8
  9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
20
21
22
23
24
25
26
27
28
29
30
results of human exposure studies with a small number of subjects.  Investigators have
reported a wide variation in responsiveness of asthmatics to NO2 which may be partially
attributed to intrinsic variation in response as well as variation in exposure variables.  In
addition, place of residence, season of the year, and indoor home environment may all be
determinants of the asthmatic's response to NO2.  Controlled human exposure studies are
ethically limited to acute or subchronic fully reversible functional and/or symptomatic
responses. This may in many cases limit the magnitude of expected responses and, hence,
the statistical significance of responses in studies with small numbers of subjects. Exposures
seldom last longer than 1 to 2 weeks for up to 8 h per day.   These data, therefore, are
primarily useful in evaluation of short-term, NO2-induced health effects.
     True simulation of ambient conditions, given the number of potential pollutants and the
variety of possible combinations, is not a realistic goal for controlled human exposure studies.
For example, the typical  temporal pattern of ambient concentrations is seldom duplicated in
controlled exposure studies.  However, simple mixtures of two or three pollutants can be
evaluated to determine the potential for either additive or synergistic effects. Further
discussion of the design considerations for human clinical studies are presented by Bates et al.
(1970), Hackney et al. (1975a), and Folinsbee (1988)  and were the subject of a symposium
proceedings (Frank et al., 1985).  Since controlled exposure studies of humans deal
exclusively with acute or subchronic exposures, the applicability of these data are limited to
short-term exposure effects and of limited usefulness in the evaluation  of the effects of
chronic NO2 exposure.
     More than 25 additional studies have become available since the  1982  Air Quality
Criteria for Oxides of Nitrogen Document (U.S.  Environmental Protection Agency, 1982a)
on the effects of NO2 on healthy, normal subjects. Several new studies of the effects of NO2
on individuals with pulmonary disease (asthma and COPD) have been published, helping to
alleviate a critical information deficit of the earlier review (U.S. Environmental Protection
Agency,  1982a,b).  Although more than 10 new reports have been published, the data base
concerning NO2 effects in sensitive subjects still requires concentration-response studies in
moderately sensitive asthmatics, information concerning the inflammatory response to NO2
inhalation, further examination of the effects of NO2 on infectivity in humans, and further
        August 1991
                                         15-7
DRAFT-DO NOT QUOTE OR CITE

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 1     evaluation of patients with COPD.  Only one study is available on pulmonary epithelial
 2     permeability or mucociliary clearance effects of NO2 in humans.
 3          One of the more important observations in studies of NO2-exposed animals is that NO2
 4     exposure is associated with increased susceptibility to viral and bacterial infections due to
 5     impairment of host-defense mechanisms (see Section  13.2.2.1).  Epidemiology studies clearly
 6     suggest a link between NO2 exposure and increased rates of respiratory illness
 7     (Section 14.2.1). These studies have provided a basis for several recent investigations of
 8     human immune host defenses after NO2 exposure. Studies have utilized both in vitro
 9     exposure of cultured human cells (e.g., macrophages) and in vivo exposures of human
10     subjects.
11          Recently published reports generally support prior conclusions regarding the effects of
12     NO2 exposure on healthy young adults. There were  several new studies that examined  the  .
13     specific effect of NO2 on cardiopulmonary function in normal adults (see Section 15.2). The
14     NO2 concentrations ranged from 0.2 to 4.0 ppm.  In another group of studies, the effects of
15     pollutant mixtures or ambient air, of which NO2 was a component, were examined. These
16     studies are summarized in Section 15.2.3.
17          Studies examining the specific effects of NO2 in normal subjects supported the
18     conclusions reached in the Air  Quality Criteria for Oxides of Nitrogen Document (U.S.
19     Environmental Protection Agency,  1982a) in that they consistently demonstrated the absence
20     of effect of NO2 on lung function at concentrations between 0.3 and 0.6 ppm (Adams et al.,
21     1987; Drechsler-Parksetal., 1987;  Drechsler-Parks, 1987; Kagawa,  1986).
22          Four studies (Avol et al., 1983; 1985a; 1987; Linn et al., 1980a) were published  in
23     which NO2 was a component of an ambient oxidant air mixture.  The effects of ambient air
24     exposures, if any, were attributed to O3.  There was no apparent influence of the very low
25     (0.04 to 0.07 ppm) concentrations of NO2 present in the ambient air.
26          In addition, several controlled exposure studies used pollutant mixtures containing NO2
27     in concentrations from 0.16 to 5.0 ppm (Kagawa, 1983a,b; Folinsbee et al., 1981; Islam and
28     Ulmer, 1979a,b; Kagawa and Tsuru, 1979; Kagawa, 1986; Kleinman et al., 1985; Linn
29     et al., 1980b;  Toyama et al., 1981; von Nieding  et al.,  1979; Stacy et al., 1983; Drechsler-
30     Parks et al., 1987).  At concentrations less than 1.0 ppm these studies demonstrated no
        August 1991                             15-8       DRAFT-DO NOT QUOTE OR CITE

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 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
obvious effects of NO2 in pollutant mixtures, which, in addition to NO2, contained O3, SO2,
and/or particles.                                                    •        '

15.2  EFFECTS OF NOX IN HEALTHY NORMAL SUBJECTS
     Early studies indicated that the effect of NO2 on airway resistance was noted at
concentrations above 1.5 ppm in healthy volunteers (Aber 1967; von Nieding'et al., 1970,
1973a). Other studies have indicated no significant lung function effects of NO2 in healthy
normal subjects at concentrations below 1.0 ppm (Folinsbee et al., 1978; Hackney et al.,
1978; Beil and Ulmer, 1976; Kerr et al., 1979).  Also discussed are studies on NO and
NO2-mixtures.

15.2.1 Lung Function Effects of NO2
     This section is divided according to the concentrations of NO2 used in the study.  The
first subsection deals with subjects exposed to greater than 1.0 ppm,  the second with
exposures to less than 1.0 ppm. All studies  dealing with NO2 exposure in healthy subjects
are summarized in Table 15-1. (The details  of the exposure conditions, number of subjects,
ventilation levels, temperature, relative humidity, etc.'are presented in Table 15-1.
Occasional reference to this information is made in the text when necessary, but the reader
should refer to the table for these details.)

15.2.1.1  Concentrations Above 1.0 ppm
     The  effects of NO2 levels greater than  1.0 ppm have been examined in several studies.
In two studies, von  Nieding and Wagner (1977) and von Nieding et al. (1979) studied
11 males exposed to 5.0 ppm NO2 for 2  h while performing light, intermittent exercise.
Airway resistance increased from 1.51  to 2.41 cm H2O/L/s after 2 h of exposure.  There was
also an apparent decrease of arterial oxygen  partial pressure (PaO2) from 90 to  82 torr.
(These samples were taken from "arterialized venous" blood drawn from the ear lobe.) The
statistical  analysis of this data is impacted slightly due to an adjustment in the PaO2 data prior
to testing  for significance,  von Nieding and Wagner, 1977 state:  "To increase the power of
the tests PaO2-differences <5 mm Hg and Rt-increases <0.5 cm H2O/L/s were regarded as
zero." This transformation would increase the likelihood of finding  a significant effect.
        August 1991
                                         15-9
DRAFT-DO NOT QUOTE OR CITE

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DRAFT-DO NOT QUOTE OR CITE

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  1           In a subsequent synopsis of several studies, von Nieding et al. (1980) discussed the
  2      results of two experimental exposures that were previously published (in German). In the
  3      first study,  14 normal patients were exposed to 5 to 8 ppm NO2 for up to 5 min on
  4      4 separate days.  The airway resistance (Raw) increased by an average of 0.58 cm H2O/L/s
  5      (range 0.39 to 1.03 for the individual four exposure mean) after the NO2 exposures.  It was
  6      noted that there were no differences in response between smokers and nonsmokers.
  7           Beil and Ulmer (1976) studied the effects of 2 h exposures to 0.0, 1.0 (n=8),
  8      2.5 (n=8),  5.0, and 7.5 ppm NO2 in 16 healthy resting subjects.  An additional group of
  9      8 healthy resting subjects were exposed for 14 h to 5.0 ppm NO2 for 2 consecutive days.
10      They found a small significant increase in total respiratory resistance (RT) after exposure to
11      2.5 ppm NO2 or greater. The main response, no more than 1 cm H2O/L/s above a baseline
12      of 2.6 cm H2O/L/s, occurred during the first 30 min of exposure and the response was not
13      appreciably increased by raising the NO2 concentration to 5.0 or 7.5 ppm NO2. The increase
14      in RT following NO2 exposure was related to the baseline airway responsiveness to
15      acetylcholine. Airway responsiveness to acetylcholine was increased after exposure to
16      7.5 ppm for 2 h or to 5.0 ppm for 14 h but not after the 2-h exposures to 5.0 ppm or less.
17      The pattern of response in  the 14-h exposure indicated an initial increase in resistance during
18      the first 30  min  (~30%), a slight decline in resistance over the subsequent 90 min, and then
19      a modest further increase over the next 14 h (a total increase of ~60%). Resistance returned
20      to baseline during the subsequent 10 h and this response pattern was repeated on the second
21      exposure day. This study was cited in the 1982 Air Quality Criteria for Nitrogen Oxides   *
22      Document as indicating that responses were "clearly demonstrated to occur in healthy adults
23      with single 2 h exposures to NO2 ranging from 4,700 to 14,000 Mg/m3 (2.5 to 7.5 ppm)."
24           Linn et al. (1985b) exposed 25 healthy,  nonsmoking subjects (9  female,  16 male) for
25      75 min to 4.0 ppm of NO2 or purified air. Subjects were exposed to each condition twice, a
26      total of four exposures.  The authors reported that approximately 11 jug/m3 of particulate
27      nitrate was present during NO2 exposures.  During the exposures, subjects performed 15 min
28      of light (25 L/min) and 15 min of heavy (50 L/min) exercise.  There were no significant
29      effects of NO2 on Raw or symptoms.  Although heart rate and skin conductance were
30      similarly unaffected, there  was a slight but statistically significant reduction in systolic blood ,'
31      pressure associated with NO2 exposure.  Although inhalation of NO2 can result in increased

        August 1991                             15-18      DRAFT-DO NOT QUOTE OR CITE

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 1      blood levels of nitrite and nitrate ion, the mechanism for this small change in systolic
 2      pressure has not been established (see Section 13.2.1).  Blood pressure was obtained while
 3      the subject was seated quietly in the body plethysmograph using an automated procedure.
 4           Mohsenin (1987b) studied the responses to 1 h resting exposure to 2.0 ppm NO2 on
 5      11 normal subjects to determine the effect of ascorbic acid administration prior to NO2
 6      exposure.  The author hypothesized that the antioxidant properties of ascorbic acid would
 7      modify the effect of NO2.  There were a total of four exposures.  In the first set of clean air
 8      and NO2 exposures, the subjects received a placebo for 3 days prior to the exposures. In the
 9      second  air/NO2 exposure pair, the subjects received vitamin C.  In both cases, the order of
10      the NO2 and air exposures were randomized. The blood ascorbic acid levels  were increased
11      from 0.76 mg/dL after placebo to  1.90 mg/dL after vitamin C supplementation.  Neither
12      plethysmography nor  spirometry tests indicated a significant effect of NO2  in  these subjects
13      under placebo or vitamin C conditions.  There was a significant increase in airway
14      responsiveness to methacholine (bromide) after NO2 exposure. Responsiveness to
15      methacholine was quantified by the dose required to reduce SGaw by 40% (PD40); this
16      corresponds to a 67% increase in SRaw. (A 50% decrease in  SGaw corresponds to a doubling
17      of SRaw.)  After the two air exposures, PD40 averaged about  64 mg/mL but  was reduced to
18      53 mg/mL after NO2  exposure (placebo).  When the  subjects were given ascorbic acid prior
19      to exposure, methacholine responsiveness after NO2 exposure  was unchanged. Ascorbic acid
20      pretreatment apparently blocked the airway responsiveness increase, which  had previously
21      been  observed with NO2 exposure, although it had no effect on baseline methacholine
22      responsiveness.  Ascorbic acid has been previously shown to cause a decrease in
23      methacholine responsiveness in both normals and asthmatics (Mohsenin et al., 1983; Ogilvy
24      et al-., 1981).  Thus it is unclear whether this apparent block of NO2 effects could be
25      explained by a decreased methacholine response after ascorbic acid supplementation.
26           Mohsenin (1988) studied the  response of 18 normal adults exposed to 2  ppm NO2 for
27      1  h at rest.  There were no symptoms, no  changes  in lung volume, no change in flow-volume
28      characteristics on either full or partial expiratory flow-volume (PEFV) curves, and no change
29      in specific airway conductance.  However, airway responsiveness to methacholine was
30      increased following exposure in 13 of 18 subjects and decreased in only 2 of  the
       August 1991
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DRAFT-DO NOT QUOTE OR CITE

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  1      18 (p=0.003). The dose of methacholine needed to cause a 40% reduction in SGaw was
  2      101 ± 44 mg/mL after air and 81 ±45 mg/mL after NO2.
  3           Kulle and Clements (1988) studied the effects of NO2 exposure on infectivity of live
  4      attenuated influenza A/Korea/reassortment virus in healthy nonsmoking adults (see Goings
  5      et al., 1989).  Independent control and exposure groups were exposed  to clean air for 1 day
  6      and then either clean air or NO2 (1, 2, or 3 ppm) for the next 3 consecutive days.  Included
  7      in this evaluation were measurements of respiratory symptoms, lung function and airway
  8      reactivity to methacholine hi the 2- and 3-ppm studies.  There were no significant changes in
  9      respiratory or other symptoms as a result of a 3-ppm NO2 exposure.  The only apparently
10      significant changes in spirometry were observed in the control group who showed slightly less
11      decrease in forced vital capacity  (FVC) or forced expiratory flow at 25-75% of VC
12      (FEF25_75%) during the last of four consecutive clean air exposures. Methacholine  airway
13      responsiveness was measured following exposure to 2 and 3  ppm.  The clean air control
14      groups showed a small significant decrease in airway responsiveness on the  second,  third, and
15      fourth days, but airway responsiveness remained unchanged  in the NO2-exposed subjects.
16      Influenza virus infection did not  alter airway responsiveness  in either air or  NO2 exposure
17      groups.  Reactivity returned  to control at 2 and 4 weeks after the exposure series.  The
18      infectivity portion of this study is discussed hi Section 15.4.
19           Frampton et al. (1991) studied a group  of 39 healthy nonsmokers exposed for 3 h to
20      either 0.60 ppm (n=9), 1.5 ppm (n=15), or a variable concentration protocol where three
21      15 mm "peaks" of 2.0 ppm were added to a background level of 0.05  ppm.  (See Frampton
22      et al., 1989b,  in Section  15.4.2 for details).  There were no direct effects on airway
23      mechanics (FVC, FEV, SGaw) after any of these exposures.   However, there was a small
24      statistically significant increase in FVC response to carbachol challenge after the 1.5 ppm
25      exposure, indicating an increase in airway responsiveness. There was  no increase in airway
26      responsiveness after the 0.6 ppm or the "peaks" protocol.  However, one subject  had a 20%
27      greater drop in FEV after the "peaks" NO2 exposure than after the air exposure.  This
28      observation suggests the possibility that some subjects may be affected by NO2 to a
29      considerably greater extent than others.
30
31

        August 1991                             15-20       DRAFT-DO NOT QUOTE OR CITE

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  1
  2
  3
  4
  5
  6
  7
  8
  9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
 15.2.1.2 Concentrations Below 1.0 ppm
   -   In NO2 exposure studies conducted at concentrations below 1.0 ppm, the findings have
 been generally negative.  Although some authors have indicated occasional findings, there
 does not appear to be a consistent pattern of response at these low NO2 concentrations that
 would be indicative of short-term health effects. Kagawa and Tsuru (1979) studied six
 healthy men exposed to 0.15 ppm NO2 for 2 h while performing light, intermittent exercise.
 There were no symptoms reported during NO2 exposure. Although the authors suggested
 that there might be some responses to NO2 exposure, the overall pattern of response does not
 support the conclusion that changes in lung function were iriduced by NO2: These authors
 reported "significance" for individual subjects, although the precise technique for making this
 judgment is unclear.  Two mean differences were reportedly "significant" (multiple t-test
 unadjusted for  multiple comparisons), a 0.5% decrease in vital capacity (VC)  and a 16%
 decrease in the ratio of FEF 75%(He)/FEF 75%(air). However, nonsignificant responses of
 greater magnitude were observed under other exposure conditions (e.g., air control).  It
 appears that these "significant" observations may only be chance occurrences but of nearly
 100 t-tests, 6 of which showed "significance."  Furthermore,  a temporary 0.5% decrease in
 VC is of little, if any, physiological significance.
      Kagawa (1983a) reported the results of exposing an additional 7 subjects to 0.15 ppm
 NO2 (also to other pollutants, separately and  in combination)  for 2 h with light, intermittent
 exercise.  Using the same protocol and exposure conditions (i.e., Kagawa and Tsuru, 1979)
 in this new data set, there were no statistically significant mean changes in any of the
 plethysmographic or spirometric tests associated with NO2 exposure.
• •     Toyama and colleagues (1981) exposed  five healthy subjects (two were smokers; three
 were investigators) to 0.7 ppm NO2.  The exposures lasted 60 min at rest. They observed no
 responses to this  NO2 exposure that altered airway conductance or flow-volume tests.
      Kulle (1982) presented a reanalysis of the data previously published by Kerr et al.
 (1979).  In this study,  10 normal, 13 asthmatic,  and 7 chronic bronchitic subjects were
 exposed to 0.5 ppm NO2 for 2 h; several of the subjects were smokers (3 normals,
 3 asthmatics, and 5 bronchitics).  There were no significant effects of NO2 exposure on
 pulmonary function, but  it was unclear whether the change in quasistatic compliance was due
 to NO2 exposure or was  a statistical artifact, as the authors (Kerr et al., 1979) suggested.
        August 1991
                                         15-21
DRAFT-DO NOT QUOTE OR CITE

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  1      Rather than compare the data across the postexposure measurements obtained with clean air
 2      and NO2 respectively, in the reanalysis (Kulle, 1982) a difference score (post-pre) was
 3      determined for each condition and the differences were tested for significance. All subjects
 4      perceived the odor of NO2 upon entering the exposure chamber.  The author reported a
 5      significant increase in the normal subjects in the phase IV of the single breath nitrogen
 6      washout test.  However, the data suggest that the difference was probably due to a reduced
 7      pre-exposure value on the NO2 exposure day, an effect that could not be attributed to NO2.
 8      Quasistatic lung compliance was decreased after NO2 exposure in the normal group.  The
 9      absence of a change in dynamic compliance suggests that the original authors (Kerr et al.,
10      1979) may have been correct in concluding that "significance" was probably due to chance
11      alone. No other spirometric or plethysmographie measurements were significantly altered by
12      NO2 exposure.  With the exception of the apparently artifactual change in "closing volume".,
13      no new conclusions can be drawn from the reanalysis of this data.
14           Stacy et al. (1983), as part of a large multipollutant exposure study, exposed a group of
15      10 men to 0.5 ppm NO2 for 4 h, including two 15-min periods of moderately heavy exercise.
16      None of the plethysmographie or spirometric tests showed a significant effect of NO2.  The
17      experimental design  of this study was complex, having a total of 20 treatment cells. The data
18      were analyzed by both a multivariate analysis of variance with an adjusted p value (a level)
19      of 0.0026  and by individual t-tests with a less conservative p value of 0.05.  Neither analysis
20      indicated significant effects of NO2.
21           Hazucha et al.  (1982, 1983) studied a group of 15 healthy adult males  exposed to either
22      air or 0.1 ppm NO2 for 1 h.  Control measurements were performed on the  day before and
23      the day after the exposure.  The subjects did not  detect the odor of NO2 nor was there an
24      increase of symptoms related to the NO2 exposure.  There were no  effects of N02 on
25      spirometry, airway resistance (SRaw or RT),  or methacholine responsiveness.
26           Rehn et al. (1982) reported a small (17%) increase in SRaw after exposure of eight
27      healthy men to 0.27 ppm (500 /Jg/m3) for 1 h. However, no response was seen at 1.06 ppm
28      (2,000 /ig/m3).  This was reported hi a technical  paper  (in Swedish) and has not yet been
29      published in a peer-reviewed journal.
30           Bylin et al. (1985) exposed eight normal subjects to 230, 460, and 910 jwg/m3 (0.12,
31      0.24, and 0.48 ppm) for 20 min. An analysis of variance did not reveal any significant

        August 1991                             15-22      DRAFT-DO NOT QUOTE OR CITE

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  1
  2
  3
  4
  5
  6
  7
  8
  9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
20
21
22
23
24
25
26
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28
29
30
 effects of NO2 on changes in SRaw, but specific comparisons indicated a significant 11%
 increase in SRaw at 0.24 ppm and a 9% decrease in SRaw at 0.48 ppm.  Even though
 statistically significant, such small changes (±15%) in airway resistance are well within the
 normal variation of 10 to 20% (Pelzer and Thomson, 1966; Skoogh, 1973).  Histamine
 bronchial responsiveness was tested after the 0.48 ppm exposure but, there were no changes.
      Koenig et al. (1987a) exposed normal subjects to (1) 0.12 ppm NO2 for two 30-min
 periods at rest, (2) 0.12 ppm for 30 min at rest plus 10 min with exercise, and (3) 0.18 ppm
 for 30 min at rest and 10 min during exercise.  In the (1) rest exposures, there were no
 significant changes in lung function, symptoms, or  arterial oxygen saturation.  In the mild
 exercise exposures to 0.12 (2) and 0.18 (3) ppm NO2,  there were no significant effects
 related to NO2 exposure.
      Morrow and Utell (1989) studied both young  (20 to 48) healthy subjects and elderly
 (49-69) healthy subjects exposed to 0.3 ppm NO2 for 3.75 h.  The young subjects performed
 a total of 30 min of moderate exercise during exposure and the older subjects exercised for
 21 min.  There were no differences between air exposure and NO2 exposure for symptom
 responses, changes in lung function, or in airway responsiveness to carbachol in either young
 or older subjects.
      Adams et al. (1987) studied the effects of 0.60 ppm NO2 on young men and women
 during 1 h of continuous heavy exercise.  There was no significant effect of NO2 exposure on
 airway resistance, symptoms, spirometry, or exercise responses.
      Kim et al.  (1991) studied nine athletes exposed to filtered air, 0.18, and 0.30 ppm NO2
 for 30 min while exercising (running and walking).  Sixteen minutes were spent running at a
ventilation of about 72 L/min; 10 of the remaining  14 minutes were spent walking.  Overall
ventilation is  estimated to have averaged about 50 L/min.  There were no significant changes
in respiratory symptoms, FEVl5 RT, peak expiratory flow rate (PEFR), or ventilation
 (^50%vc) as a result of NO2 exposure in this group of athletic male subjects.
      Young (18  to 26) and older (51 to 76) men and women were exposed to 0.60 ppm NO2
by Drechsler-Parks et al. (1987) and Drechsler-Parks (1987).  Subjects performed light,
intermittent exercise during the 2-h exposures.  There were no effects on spirometry or
symptoms.  None of the individual pre-post exposure differences in NO2 (as compared to air)
       August 1991
                                        15-23      DRAFT-DO NOT QUOTE OR CITE

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  1     for FVC or FEVj were outside of the normal range (i.e., there were no individual subjects
  2     who appeared reactive to NO^.
  3
  4     15.2.1.3 Respiratory Symptom and Sensory Effects of NO3 Exposure
  5          Several studies reported in the previous section examined symptomatic responses of
  6     subjects exposed to NO2.  None of the studies of NO2 exposure in normal subjects, including
  7     exposure for as long as 75 min to 4.0 ppm NO2 resulted in a significant increase in
  8     respiratory symptoms. Sensory effects were examined in at least two studies (Bylin et al.,
  9     1985; Hazucha et al., 1983).  Hazucha et al.'s (1983) subjects were unable to detect the odor
10     of 0.1 ppm NO2.  Bylin et al. (1985) reported an odor threshold of 0.04 ppm for normals
11     and 0.08 for asthmatics.
12
13     15.2.1.4 Mucociliary Clearance after NO2 Exposure
14          Rehn et al. (1982) examined the effects of NO2 exposure on mucociliary clearance in
15     both the nose and lung. Nasal clearance was determined using the rate of saccharin
16     transport.  Tracheobronchial clearance was determined by monitoring the rate of
17     disappearance of radiolabeled teflon aerosol.  After a 1-h exposure to either 0.27 or
18     1.06 ppm (500 or 2,000 jug/m3) NO2, there were no changes in either nasal or tracheo-
19     bronchial clearance rates.
20
21      15.2.2  Effects of Nitric Oxide
22          In addition to NO2, Kagawa (1982) examined the effects of 1 ppm NO  exposure for 2 h
23     in eight normal  subjects.  The data were analyzed by multiple t-tests using  individual data.
24     All changes were referenced to the mean baseline (i.e., mean of the pre-exposure
25     measurement for the air and the NO exposure) value rather than the corresponding
26     measurement time from the clean air exposure. Three  "significant" individual changes in
27     SGaw were reported at 1 h of exposure: one increase and two decreases. After 2 h of
28     exposure there were three decreases and one increase and in the postexposure period there
29     were two increases and two decreases, all reported to be  "significant."  These statistical
30     analyses may not be the most appropriate.  Analysis of the mean data using similar
31      procedures (i.e., multiple t-test referenced to the mean  baseline level) produced only one

        August 1991                             15-24      DRAFT-DO NOT  QUOTE OR CITE

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 1     significant change:  an 11% decrease in the ratio HeV5Q/air/V5Q. Given that this effect
 2     (1) occurred only at one of the three measurement points for the NO exposure,  (2) was one
 3     of 98 paired t-tests, and (3) was significant at only the p < 0.05 level, it is reasonable to
 4     suggest that this effect may have occurred by chance.                       '
 5          A study of the effects of a mixture of NO2 (0.3 ppm) and NO (0.6 ppm) was recently
 6     reported by Kagawa (1990).  Exposures lasted 120 min and included mild (50 w) intermittent
 7     exercise.  There were no significant changes in pulmonary function (airway conductance
 8     t^wl'  V50%vc, slope of alveolar nitrogen concentration), symptoms, or airway
 9     responsiveness to acetylcholine.
10          von Nieding et al. (1973b) exposed healthy subjects and smokers to 15 to 39 ppm NO
11     for 15 min. Total respiratory resistance increased significantly (~ 10 to  12%) after exposure
12     to >20 ppm NO.  Diffusing capacity was not changed, but a small decrease (7 to  8 torr) in
13     PaO2 was noted.
14
15     15.2.3  Effects of NO2-Gas or Gas/Aerosol Mixtures on Lung Function
16              in Normal Subjects
17          Several studies of NO2-containing pollutant mixtures were included in Air Quality
18     Criteria for Oxides  of Nitrogen Document (U.S. Environmental Protection Agency, 1982a).
19     The general finding in these studies was that NO2 did not enhance the effects caused by other
20     oxidants, notably O3 (Hackney et al., 1975a,b,c; von Nieding et al.,  1977; Horvath and
21     Folinsbee, 1979 [preliminary report of Folinsbee et al.,  1981]; Suzuki and Ishikawa, 1965).
22     Abe (1967) studied NO2-SO2 mixtures (4 to 5 ppm) and reported that their effects were
23     additive, with both  gases causing bronchoconstriction. Independently, the effect of SO2 was
24     immediate and short-lasting while the effect of NO2 was delayed and  more persistent. The
25     effect of mixed gas was intermediate between the two responses. Other reports suggesting
26     possible interactions of NO2, principally with some type of particle, included Schlipkoeter
27     and Brockhaus (1963) and Nakamura (1964; cited in U.S. Environmental Protection Agency,
28     1982a.)  These studies are reviewed extensively in Air Quality Criteria for Oxides of
29     Nitrogen Document (U.S. Environmental Protection Agency, 1982a).  Table 15-2
30     summarizes studies of healthy subjects exposed to NO2-containing pollutant mixtures.
        August 1991
15-25
DRAFT-DO NOT QUOTE OR CITE

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                                                    DRAFT-DO NOT QUOTE OR CITE

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 1          The pollutant mixture of greatest interest, in terms of research effort, was the
 2     combination of NO2 and ozone (Adams et al., 1987; Drechsler-Parks, 1987; Drechsler-Parks
 3     et al.,  1989; Folinsbee et al., 1981; Kagawa and Tsuru, 1979; Kagawa,  1983a,b; Toyama
 4     et al.,  1981).  Also reported were several studies of NO2-SO2 combination
 5     exposures(Kagawa,  1983a,b; Kleinman et al.,  1985; Linn et al.,  1980a).  Other pollutant
 6     mixtures containing NO2 were also studied (Kagawa, 1986; Islam and Ulmer, 1979a,b; Stacy
 7     etal.,  1983; Kleinman etal., 1985).
 8          Several recent studies evaluated the effects of  O3 and NO2 in combination.  Three
 9     studies investigated  the effects of 0.5 to 0.6 ppm NO2 in combination with 0.3 to 0.5 ppm
10     O3 (Adams et al., 1987; Folinsbee et al., 1981; Drechsler-Parks,  1987).  In these studies,
11     NO2 alone had no effect on measured health end points and the significant effects of O3 on
12     lung function were not altered by the presence of NO2.  Kagawa and Tsuru (1979) and
13     Kagawa (1983a) reported conflicting results. In their first study, it was suggested that the
14     effects of NO2 and  O3 were "more than additive."  However, in  the second study, they
15     reported no significant enhancement of the effect of O3 by the mixture of other pollutants.
16     (All pollutants—NO2, O3, and SO2—were at a concentration of 0.15 ppm).  All of the above
17     studies included exercise during the 1- to 2-h exposures.  Toyama et al. (1981) also studied
18     subjects exposed to  NO2-O3 mixtures (0.5 ppm of each gas).  Because the concentration of
19     each gas was 0.7 ppm when the exposures were to a single pollutant, it is impossible to
20     determine if there was an additive effect.  The above studies taken as a whole suggest
21     strongly that there is no interaction between NO2 and O3 that would result in enhancement of
22     acute O3-induced changes in lung function in normal subjects following short duration
23     exposures at NO2 concentrations less than 1 ppm.  However,  this conclusion is not
24     necessarily applicable to other measured end points.
25          Linn et al. (1980a) exposed a group of normal subjects to a mixture of 0.5 ppm NO2
26     and 0.5 ppm SO2 during which light, intermittent exercise was performed for the 2-h period.
27     There  were no lung function (spirometry, closing volume, RT) responses. There was an
28     overall increase in symptoms as a result of the exposure but no significant increase in any
29     specific symptom category.  Using the same 2-h intermittent,  light exercise exposure
30     protocol, Kleinman et al. (1985) examined the effects of similar NO2-SO2 levels in
31     combination with sodium chloride (NaCl) aerosol (330 jwg/m3) and zinc ammonium sulfate
        August 1991
15-29
DRAFT-DO NOT QUOTE OR CITE

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  1      (20 /ig/m3).  They found a slight increase in symptoms in the aerosol plus gas exposure
  2      compared to the aerosol alone, suggesting that the mixture was slightly more irritating.
  3      There were no pulmonary function effects (spirometry, closing volume, RT) of this exposure
  4      regimen.  Kagawa (1983a,b) reported results of SO2-NO2 (0.15 pphi each) exposures in
  5      normal subjects for 2 h with light, intermittent exercise.  Unfortunately, the analysis of
  6      differences (using repeated t-tests) is confusing.  If a reasonable alpha level (<0.01) is used
  7      to determine significance (based on the large number of comparisons),  then there were no
  8      statistically significant changes in Gaw in response to the NO2-SO2 mixture.
  9           Islam and Ulmer (1979a) examined the effects of a mixture of 5 ppm NO2, 5 ppm SO2
10      and 0.1 ppm O3 on a group of 24 healthy subjects divided into three groups according to age.
11      There were two series of 2-h exposures:  one at rest, the other including exercise.  In
12      subjects <30 years,  Raw increased 48%, FVC decreased  5%, and FEVj decreased 11.7%;
13      ^a^2' determined from ear lobe blood samples, was unchanged. Similar effects occurred in
14      the older subjects, but the changes were of a smaller magnitude. PaO2 fell (6.8 and 8.3 torr)
15      during the exposures in the older subjects, but it also decreased (4.5 and 4.3 torr) during the
16      control exposures.  The methods of data analysis were not presented in the paper so that the
17      statistical significance of the observed changes cannot be evaluated.  Furthermore, the
18      additivity of effects due to the different pollutants cannot be determined.
19           Islam and Ulmer (1979b) also studied 15 healthy subjects exposed to 0.34 ppm SO2,
20      0.16 ppm NO2, and  0.08 ppm O3 for 8 h at rest on 4 successive days.  This mixture did not
21      cause any changes in lung function, blood gases, or blood chemistry.
22           Studies using several gas and/or aerosol mixtures were conducted by  Stacy et al. (1983)
23      and Kagawa (1986).  Stacy et al. (1983) exposed healthy, young males to mixtures of NO2
24      (0.5 ppm) and aerosols of either sulfuric acid (H2SO4, 100 jig/m3), ammonium sulfate
25      ((NH4)2SO4, 133 jug/m3), ammonium bisulfate (NH4HSO4, 116 /ig/m3), ammonium nitrate
26      (NH4NO3, 80 /zg/m3).  There were no effects of any of the pollutant mixtures on spirometry,
27      plethysmography, or symptoms. Kagawa  (1986) studied the effects of a mixture of (A) NO2
28      (0.30 ppm), O3 (0.30 ppm), and H2SO4 (200 /*g/m3);  (B) NO2 (0.15 ppm),  O3  (0.15 ppm),
29      and H2SO4 (200 pig/m3); or (C) NO2 (0.15 ppm), O3 (0.15 ppm), SO2 (0.15 ppm), and
30      H2SO4  (200 Atg/m3). Exposure A included 20 min of exercise (total) and B and C included
31      60 min of exercise over 2 h.  Symptoms were attributed to O3 exposure. Small,  possibly

        August 1991                            15-30      DRAFT-DO NOT QUOTE OR CITE

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 1     significant decreases (<-10%) in Gaw were observed after exposure to mixtures A and B.
 2     A possible decrease in FEV1 (unknown magnitude) was observed after exposure C. The
 3     differences observed with these mixtures were no different than responses observed with O3
 4     alone, indicating no enhanced response due to the presence of NO2 in the mixture.
 5          Several reports of exposure to NO2-containing ambient air mixtures have been published
 6     by the Rancho Los Amigos group (Linn et al., 1980b; Linn and Hackney, 1983; Avol et al.,
 7     1983; Avol et al., 1985a; Avol et al.,  1987).  The mean NO2 level in the ambient air (from
 8     the Los Angeles Air Basin) ranged from 0.04  to 0.07 ppm during the approximately 2-h
 9     exposure periods. These studies  were  conducted during the summer smog seasons of
10     1978-84.  In the Linn et al. (1980b) study, there was no association between NO2 levels and
11     symptom or lung function effects either in normal or asthmatic subjects. In the Linn et al.
12     (1982) study, the O3 levels were only  0.03 ppm, and there were no significant effects
13     associated with ambient exposure in normals or asthmatics. There was a relationship between
14     O3  concentration and change in FEVj  in the Avol et al. (1983) report.  There were few
15     differences between the responses of asthmatics and normal subjects in these studies and no
16     apparent influence of NO2 level.   Ambient air exposure of adolescents (Avol et al., 1985a)
17     was associated with decreased FEVj, in male and female adolescents with a somewhat larger
18     response in female subjects (-7.5% [female] vs.  -3.4% [male]). The responses tended to be
19     associated with the O3 levels.  A similar study (Avol et al., 1987) was conducted with
20     exercising children who  showed a trend to larger pulmonary function decrements with
21     increasing O3 levels.  Although these ambient air exposure studies were not designed to test
22     for the interaction of NO2 with other pollutants,.even though Los Angeles has the highest
23     NO2 levels in the United States,  they do illustrate that the lung function effects of ambient air
24     exposure (in Los Angeles) appear to be primarily accounted for by the presence of O3.
25          There has been one study of the  effects of HNO3 vapor in combination with O3 (Aris
26     et al., 1991).  Ten healthy  men were exposed to 500 /xg/m3 HNO3 vapor or clean air.
27     Another six subjects were exposed to either 0.20 ppm O3 or the combination of HNO3 vapor
28     and O3.  Exposures lasted 4 h and were accompanied by moderate exercise  (ventilation
29     during exposure [VE] = 40 L/min).  No changes were observed in FVC, FEVj, or SRa
30     after HNO3 exposure.  Ozone exposure caused increased SRaw and decreased FVC and
31     FEVj, which was increased slightly by the presence of HNO3  vapor.
                                       aw
        August 1991
15-31
DRAFT-DO NOT QUOTE OR CITE

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  1          There appears to be no obvious synergistic or more than additive interactions between
  2     NO2 and other pollutant gases or particles that have been evaluated to date.  Pulmonary
  3     function responses to O3, for example, do not appear to be increased by the addition of low
  4     levels (<0.6 ppm) of NO2. The variety of physiologic end points used to evaluate
  5     combination exposures have, however, been limited primarily to spirometry and
  6     plethysmography.
  7                                                ...      .    .           '    ..'.-':
  8     15.2.4 Summary
  9          In the studies summarized in this section, several observations indicated that, at
 10     concentrations in excess of 2.0 ppm, there were functional changes in the lungs of healthy
 11     normal volunteers that could be attributed to NO2 exposure.  Increases in Raw were reported
 12     at concentrations of 5 to 8 ppm from both short (5 min) and longer (1 to 2 h) exposures
 13     (1-2 h) (von Nieding et al., 1979; von Nieding and Wagner,  1977; von Nieding et al., 1980;
 14     Islam and Ulmer, 1979a,b). At slightly lower concentrations (2 to 4 ppm) no changes were
 15     seen in resistance or spirometry (Linn et al., 1985b; Mohsenin, 1987b, 1988).  However,
 16     Mohsenin reported an increase in airway responsiveness to  methacholine (bromide) after
 17     exposure to 2 ppm, and Frampton et al. (1991) reported increased carbachol response after
 18     exposure to 1.5 ppm.  None of the seven studies of exposure to less than 1.0 ppm in normal
 19     subjects demonstrated clear responses to NO2.  There were several instances of isolated
20     observations indicating statistical significance, but there was no consistent pattern of response.
21      For example, Kagawa and Tsuru (1979) reported small changes in VC at 0.15 ppm but did
22     not verify this observation in two subsequent studies (Kagawa, 1983a,b,  1986), even at
23      0.30 ppm.  Furthermore, other studies (Folinsbee et al.,  1978; Toyama et al., 1981; Stacy
24      et al.,  1983; Adams et al., 1987; and Drechsler-Parks et al.,  1987), conducted at higher
25      concentrations (0.5 to 0.7 ppm), found no evidence of lung function effects.
26
27
28      15.3  THE EFFECTS OF NOX EXPOSURE IN SENSITIVE SUBJECTS
29          Certain groups within the population may be more responsive to the effects of NO2
30      exposure including  persons with respiratory disease, children,  the elderly, and other
31      individuals not readily identified as members of a specific group. The reasons for paying

        August 1991                            15_32     DRAFT-DO NOT QUOTE OR CITE

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 1      special attention to these, groups is that potential for NO2-induced responses or exacerbation
 2      of disease may be much higher than in healthy young adults.  Studies on other NOX gases and
 3      NOx-mixtures are also discussed.
 4          The airways of asthmatics may be hyperresponsive to a variety of inhaled materials,
 5      including pollens, cold-dry air, allergens, and air pollutants.  Asthmatics have the potential to
 6      be among the most susceptible members of the population with regard to respiratory
 7      responses to NO2 (Section 15.3.1). On the average, asthmatics are much more sensitive to
 8      inhaled bronchoconstrictors such as histamine, methacholine, or carbachol. The potential
 9      addition of an NO2-induced increase in airway response to the already heightened
10      responsiveness to other substances raises the possibility of exacerbation of this pulmonary
11      disease by NO2.  One of the potential mechanisms by which NO2 could affect asthmatics is
12      via a change in airways responsiveness. This is discussed in Section 15.4.
13          Other potentially susceptible groups.include patients with COPD; these subjects are
14      discussed in Section 15.3.2. A major concern with COPD patients is the absence of an
15      adequate pulmonary reserve.  Any alteration in lung function in these patients can potentially
16      cause serious problems.    ,
17             >:.                     .         .....",                 •
18      15.3.1  The Effects of NO2  on Asthmatics
19          Studies of the exposure of asthmatics to NO2 are summarized in two tables that describe
20      characteristics of the asthmatic subjects (Table 15-3) and the exposure conditions and  .
21      responses to NO2 (Table 15-4).
22      ,    In many cases of chamber exposures of asthmatics, the exposures are accompanied by
23      moderate exercise. The potential for an increase in airway resistance or decline in lung
24      volumes or. forced expiratory flow caused by exercise alone is an important covariate in these
25      studies.  Exercise, even of moderate intensity,, can induce some increase in airway resistance
26      even in clean air at normal room  temperature and relative humidity (RH) (20 °C, 50% RH).
27      In order to determine the true  effect of an air pollutant in exercising asthmatics, the response
28      to exercise must be considered. However, in all studies summarized in this section, a control
29      exposure to clean air was performed including exercise when appropriate.
30          A major issue in the evaluation of human clinical studies involving asthmatics is the
31      variability in response between and even within laboratories.  In the absence of a clear reason
        August 1991
15-33
DRAFT-DO NOT QUOTE OR CITE

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 1     based on exposure protocol or exposure regimen, the explanation most often invoked to
 2     explain differences in response is that the severity of disease in one subject group may have
 3     been different than the other.  Appendix 15-A (Asthma Severity) discusses a possible
 4     approach that can be used to evaluate physiological and clinical differences among asthmatics.
 5     These "asthma severity" descriptors will be applied to the key asthma studies discussed,
 6     insofar as possible based on the data available.  Where the authors' evaluation of asthma
 7     severity disagrees with the scheme in Appendix 15-A, this will be noted.
 8          Symptomatic effects were observed in asthmatics exposed to 0.5 ppm for 2 h
 9     (Kerr et al.,  1979).  However only four of the subjects reported symptoms of
10     respiratory discomfort and the authors concluded  that:  "The symptoms reported were
11     minimal, did not correlate with functional changes, and are of doubtful significance."
12          Avol et al. (1988) studied a group of 59 moderate-to-severe asthmatics exposed to clean
13     air,  0.3, and 0.6 ppm NO2 for 2 h while performing moderate (VE = 41 L/min), intermittent
14     (6 X 10 min) exercise.  Each subject was exposed once each to clean air,  0.30 ppm, and
15     0.6  ppm. There were significant changes in SRaw and FEVj as a function of exposure
16     duration for all exposure conditions, but there was no significant, effect of NO2 exposure on
17     these measures of pulmonary function.  Cold air bronchial reactivity  (assessed by the decrease
18     in FEV1 after breathing cold-dry air) was measured 1 h postexposure and then again the
19     following day. There was a significant interaction between response  and time of testing (i.e.,
20     1 h  post and 24 h post), suggesting a slightly increased response after exposure to 0.30 ppm,
21     but  not after 0.6 ppm. There were no respiratory symptom responses attributable to NO2
22     exposure.  A post hoc analysis of a subgroup of subjects  with the most abnormal lung
23     function (i.e., FEVj/FVC ratios  <0.65) revealed no statistically significant effects of  NO2.
24     In addition to the controlled exposures, 36 subjects also were exposed to ambient air ,.
25     containing 0.09 ppm NO2 and low levels of other pollutants.  Neither lung function, cold-air
26     reactivity, nor symptom responses were significantly different in ambient air than in clean air.
27           Bauer et al. (1986a) reported a statistically significant spirometric response to NO2 in a
28     group of 15 asthmatics exposed to 0.3 ppm NO2  by mouthpiece for 20 min at rest followed
29     by 10 min of exercise (30 L/min).  These subjects were characterized as having "mild
30     obstructive lung disease [asthma]." All subjects had elevated response to cold air
31     bronchoprovocation.  NO2 deposition studies indicated that 72%, at rest, and 87%,  during
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  1      exercise, of the inhaled NO2 was deposited within the respiratory tract.  According to the
  2      authors, the measurements of NO2 deposition were in general agreement with the model
  3      predictions of Miller et al. (1982) (see also Section 13.2.1).  After NO2 exposure, 9 of
  4      15 asthmatics had a decrease in FEV1 relative to their postexercise FEVl in clean air.  The
  5      postexercise FEV1 was 4.1% lower after NO2 (mean = 2,788 mL) than after air
  6      (mean = 2,906 mL) exposure; the pre-post exposure difference on the NO2 day (10.1 %) and
  7      the pre-post NO2 minus the pre-post air (i.e., delta-delta) differences (6%) were significant
  8      using a paired t-test. These differences were no longer present by 60 min after the exposure.
  9      Maximum expiratory flow (MEF) 60% total lung capacity (TLC)  (PEFV curve) was also
 10      decreased more after NO2 exposure than after air exposure.  Changes in FVC and SGaw were
 11     not different between air and NO2 exposures. Airway responsiveness to cold air in this study
 12     was determined as follows.  At each ventilation rate of cold air breathing, the respiratory heat
 13     exchange (RHDB)  was calculated.  From the relationship of the log RHE vs. the percentage
 14     decrease in FEV^ the RHE, which caused a 10% decrease in FEVl5 was linearly interpolated
 15     and is referred to as PD10RHE (provocative dose in RHE units needed to  decrease FEV1 by
 16      10%).  Of the 12 subjects for whom the PD10RHE could be determined, 9 showed an
 17     increased response to cold air after the NO2 exposure. The average PD10RHE decreased
 18      from 0.83, after air exposure to 0.54 kcal/min after NO2 exposure.
 19           One of the factors that may have led to the demonstration of increased response after
 20      exposure to a low concentration of NO2 in this group of asthmatics could be the fact that a
 21      mouthpiece exposure system containing relatively dry air (RH of 9 to 14% at 20 °C)  was
 22      used, and that there was possibly some  interaction between the NO2 effect and airway drying.
 23      It is well known that breathing dry (cold) air will induce bronchoconstriction in asthmatics,
 24      and that the effect of SO2 on asthmatics is exacerbated by cold-dry air breathed via the mouth
 25      (U.S. Environmental Protection Agency, 1986; SO2 document addendum). Concern over
 26      this possible confounding effect is tempered by the fact that Bauer et al. (1986a) controlled
 27      for the airway drying effect by exposing subjects to clean air at the same temperature and
28      RH.  However, if the formation of nitric or nitrous acid (HNO2 or HNO3) is potentially
29      involved in the observed responses, the  air chemistry could be strongly influenced by  RH.
30      (Sequestration of HNO3 on surfaces is increased with increased ambient water vapor content.)
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 1          Eight asthmatics exposed to 0.0> 0.1, 0.25, and 0.5 ppm NO2 for 20 min were studied
 2     by Bylin et al. (1985). Exposures were conducted in a body plethysmograph and the range of
 3     concentrations was +18% to -26% of the target concentration.  Changes in SRaw, during the
 4     four exposures averaged +3%, +9%, -2%, and -14%, respectively; the coefficient of
 5     variation for the SRaw measurements was 19% for these subjects. A three-way analysis of
 6     variance (ANOVA) revealed no significant differences in SRaw due to NO2 exposure.  There
 7     was  a tendency for the pre-post exposure difference for thoracic gas volume (TGV) to be
 8     larger for the NO2 exposures (9 to 10%).  However, the absolute volume of TGV was at
 9     most 3 to 4% lower than at comparable times in other NO2 exposures and only 2% less than
10     the air exposure.  The significance of this difference was in the higher pre-exposure values
11     for the 0.1 - and the 0.5-ppm NO2 exposures; such an effect, if real, should not be attributed
12     to NO2.  There were no significant changes in tidal volume or respiratory rate, which would
13     have been suggestive of an irritant response.  At the highest concentration tested (0.5 ppm),
14     histamine bronchial responsiveness was also evaluated after exposure.  The authors reported a
15     significant increase in histamine responsiveness due to NO2 exposure.  Significance was
16     evaluated by a sign test (p<0.04; responsiveness increased in five subjects  and was
17     unchanged in three).  However, this finding should be interpreted cautiously since the sham
18     (air) exposure histamine challenge had to be discontinued in two subjects, one of whom was
19     later classified as having increased responsiveness.  Five of the eight asthmatics had several
20     months previously been hyperreactive to histamine but were not at the time of the NO2
21     exposures.  This paper suggested possible increased histamine reactivity after 0.50 ppm NO2
22     exposure of asthmatics but no direct effect of NO2 on Raw at  concentrations up to 0.5 ppm
23     for 20 min.
24          Bylin et al.  (1988) also reported the effects of 260, 510,  and 1,000 jwg/m3 (0.14, 0.27,
25     0.53 ppm) on a group of 20 mild asthmatics.  There were no significant changes in SRaw,
26     although there was a general trend for SRaw to fall throughout the period of exposure
27     regardless of the pollutant level. There was, however, a significant increase (p=0.03) in
28     airway responsiveness to histamine after 30 min exposure to the middle concentration (i.e.,
29     510  £tg/m3) but not at the lowest and highest concentration (see note below).  The absence of
30     a concentration-related increase in responsiveness,  and the fact that the significance of these
31     findings is based on repeated application of a nonparametric pair comparison test using an
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 1      alpha level (p value) that was not adjusted for multiple comparisons, suggests that these
 2      results should be interpreted with some reservation.  This observation contrasts with the
 3      earlier observation (Bylin et al., 1985) that suggested a possible increased responsiveness
 4      after exposure to 910 /ttg/m3.  (Note, however, the discussion above regarding the statistical
 5      approach used in the 1985 study).  The raw data presented in the paper were subjected to
 6      reanalysis (data available on request to EPA) using a Friedman nonparametric analogue of an
 7      F test, which is probably more appropriate for these data than a series of Wilcoxon matched
 8      pairs signed rank tests.  The Friedman test showed no difference across treatment groups
 9      (i.e., there was no statistically significant increase in histamine responsiveness as a result of
10      NO2 exposure).
11          In a study that was an important precedent to a number of studies,  Orehek et al. (1976)
12      studied the effects of low levels of NO2 exposure on the bronchial sensitivity of minimal to
13      mild asthmatic patients to carbachol, a bronchoconstricting agent.  Exposures took place in an
                                                                    n
14      airtight room.  Nitrogen dioxide concentration started at  246 jwg/mr (0.13 ppm) and declined
                   o                                                           o
15      to 169 /tg/m  (.09 ppm) over 60 min; the average concentration was 210 jug/m  (0.11 ppm).
16      For 20 asthmatics, dose-response curves were developed for changes in SRaw as a result of
17      the subjects inhaling carbachol after a 1-h exposure to either clean air or NO2. Following
18      NO2 exposure, increases in SRaw were observed in only  3 of 20 asthmatic test subjects.
19      However, NO2 exposure was associated with increased airway responsiveness in 13 of
20      20 subjects.  The mean dose of carbachol producing a twofold (100%) increase in SRaw in
21      the 13 sensitive subjects was significantly decreased from 0.66 mg to 0.36 mg as a result of
22      NO2 exposure.  Seven of the asthmatic subjects showed neither an increase in Raw in
23      response to the exposure to NO2 alone nor an enhanced effect of NO2 on carbachol-ihduced
24      bronchoconstriction.
25          The results of this study are of interest because they are suggestive of possible
26      bronchoconstrictive responses being produced in some asthmatics by very low concentrations
27      of NO2. However, the mean of measurements of SRaw in 13 responders to the carbachol
28      treatment was significantly higher after the NO2 exposure than it had been prior to exposure.
29      The criticism of this reported change was that the comparisons of SRaw were made in
30      subjects who were selected, not at the time of NO2 exposure, but after the fact,  following the
31      carbachol exposure.  An important criticism of this study, discussed by Hazucha et al.

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 1      (1983), is that, in addition to the retrospective stratification of subjects into responders and
 2      nonresponders, other statistical methods may have been more appropriate than the selected
 3      group of paired t-tests used in this study.
 4           Orehek et al. (1981) also studied seven allergic subjects, three of whom had asthma,
 5      who were exposed to 0.11 ppm NO2 for 1 h.  The major hypothesis to be  tested  was that
 6      NO2 may alter bronchial responsiveness to an inhaled allergen (grass pollen).  Studies were
 7      conducted outside the typical pollen season.  The exposure technique was somewhat primitive
 8      in that NO2 was added to an exposure room with a starting concentration of 0.16 ppm, which
 9   .   was allowed to decay over the exposure to a concentration of 0.07 ppm.  There was no
10      change in Raw or symptoms as a result of the NO2 exposure.  There was also no  difference in
11      the Raw response to allergen challenge in these subjects (i.e.,  NO2 did not  act synergistically
12      with allergen challenge).  Furthermore, there was no difference between the responses of the
13      three asthmatics and the other four subjects.
14           Hazucha et al. (1982, 1983) published  two reports that contain complementary data
15      from a study in which the Orehek protocol was repeated.  In contrast to  the report of Orehek
16      et al. (1976), Hazucha et al. (1982, 1983) found no statistically significant change in airway
17      reactivity to methacholine (another parasympathomimetic drug) following 1 h resting
18      exposure to 0.1 ppm NO2 in a well characterized group of 20 methacholine-reactive mild
19      asthmatics.  A small (8%) increase in SRaw  (p=0.23) was observed after NO2 exposure.
20      Three of the 15 subjects had a greater than 20% decrease in the dose of methacholine
21      required to double SRaw (PD100).  However, at least three of the subjects had a change of
22      similar magnitude in the opposite direction, judging from the graphical presentation of the
23      methacholine dose-response curves. Respiratory system resistance measured by the forced
24      oscillation method was not changed by NO2  exposure.  Hazucha et al. (1983) suggested that
25      the difference in the conclusions regarding statistical significance reached by Orehek et al.
26      (1976), despite similar findings, was because "the statistical approach used  by Orehek was
27      not appropriate."   Hazucha et al.  (1983) discussed the factors that led to  their conclusions
28      that, had they analyzed their data in a similar manner to Orehek et al. (1976), the findings
29      would have been comparable.
30           The hypothesis that NO2 exposure may cause airway hyperresponsiveness was also
31      examined by Kleinman et al.  (1983) who employed a different experimental design than
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 1     Orehek et al. (1976) and Hazucha et al. (1983).  They studied 31 mild to moderate
 2     asthmatics who were exposed to 0.2 ppm NO2 for 2 h while performing light, intermittent
 3     exercise.  There were no significant effects of NO2 exposure on forced expired spirometry.
 4     Total RT (forced oscillation) tended to increase (9%) after NO2 exposure, but the difference
 5     was not significant (p=0.11).  Symptom responses tended to be slightly higher after air
 6     exposures. A number of different methods were used to evaluate the methacholine challenge
 7     data.  The general tendency was for greater responsiveness to methacholine after NO2
 8     exposure. The determination of the dose which would cause a 10% decrease in FEVj (DIG)
 9     was the most "conventional" approach (O'Connor et al., 1987) to assessing methacholine
10     responsiveness.  In the 21  subjects in which this dose could be ascertained, D10 was
11     8.6 ± 16.2 /tg on the air day and 3.0 + 6.2  /j.g on the NO2 days (p<0.05 by t-test and
12     Wilcoxon test).  Thus, it appears that the results of this study suggest a possible increase in
13     airway responsiveness after exposure to 0.20  ppm NO2 for 2 h.
14           Koenig et al. (1985)  have studied the effects of 1 h resting exposure of asthmatic
15     adolescents to 0.12 ppm NO2. There were no "consistent significant changes in pulmonary
16     functional parameters" after NO2 exposure.  Although symptom data were not presented, the
17     authors indicated that  subjects had more symptoms after NO2 exposure but that the trend was
18     not significant                                                   .  . ..
19           Subsequent studies by Koenig et al. (1987a,b) of mouthpiece exposures to 0.12  ppm
20     NO2, which incorporated exercise (30 min rest followed by 10 min exercise), indicated
21     increases in RT and decreases in FEY^ after both  air and NO2 exposure. These changes were
22     apparently due to exercise alone (RT increased 8.1% with air and 10.4% with NO2;
23     postexercise FEV1 was decreased 7.4% with  air and 4.1% with NO2).   In the final phase of
24     the study, subjects were exposed to 0.18 ppm NO2 using the same exercise protocol.  In this
25     case, no differences in RT were seen  and FEVl decreases were -1.3 and -3.3% for air and
26     NO2, respectively.  This difference (p=0.06) may indicate a possible response trend.  There
27     were no differences in symptoms between exposure conditions in either the 0.12 or 0.18 ppm
28     NO2 exercise exposure studies.
29           Morrow and Utell (1989) studied a group of 20 asthmatics exposed to 0.30 ppm NO2
30     for 3.75 h.  The exposure included three 10-min periods of moderate exercise.  There were
31     no statistically significant group changes in symptoms, spirometry, plethysmography, or

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  1      airway reactivity to carbachol as a result of the NO2 exposure.  Some of the subjects (n=7)
 2      had participated in the Bauer et al. (1986a) study.  The 13 remaining (new) subjects were
 3      judged to have more severe asthma than the "repeaters."  Although the "repeaters"  tended to
 4      have responses that were similar to those in the previous study (larger FEVj decrements in
 5      NO2 than in air), the new subjects had significantly greater FEVj decrements during the air
 6      exposures.
 7       '    Linn et ali  (1985b) and Linn and Hackney (1984) exposed a group of 23 mild
 8      asthmatics to 4 ppm NO2.  Subjects completed a total of four exposures (two each to NO2
 9      and clean air) separated by 1 week.  Exposures lasted for 75 min and included two 15-min
10      exercise periods separated by a 25-min rest period.  The first exercise was light  (25 L/min)
11      and the second was heavy (49 L/min).  All subjects were responsive to inhaling  0.75 ppm
12      SO2 during exercise.  Mean baseline preexposure SRaw measurements varied from 5.48 and
13      5.59 on the air exposure days to 6.14 and 6.44 on the  NO2 exposure days,  although it is
14      unlikely that the  slightly higher baseline values on the NO2 exposure days affected the
15      subjects' responses.  Airway resistance increased after exercise and more so after the heavy
16      (57.2%) than after the light (17.6%) exercise (percentages represent mean values collapsed
17      across all exposure conditions).  There was no significant difference in lung function that
18      could be attributed to NO2; if anything, SRaw tended to be slightly lower with the NO2
19      exposures.  Other physiological tests, such as skin conductance and heart rate, were not
20      different between exposure conditions.  As with  the group of normal subjects studied under
21      similar conditions, these asthmatics had a slightly, but  significantly, lower systolic blood
22      pressure towards the end of the NO2 exposure.  The authors suggested the possibility that
23      NO2 deposited in the respiratory tract may form a vasoactive substance such as an organic or
24      inorganic nitrate.  Nitrate formation after NO2 inhalation has been observed in animal studies
25      (Postlethwait and Mustafa, 1981).  However, measurements of blood levels of nitrate were
26      not performed by Linn et al. (1985b).  Both symptoms and state-trait anxiety scores were
27      evaluated during  and after exposure; there were no significant variations which could be
28      attributed to NO2 exposure.
29 '          It is difficult to explain the differences between this group of asthmatics exposed to
30      4 ppm for 75 min (with exercise) compared to the group exposed to 0.30 ppm for 30 min
31      with exercise studied by  Bauer et al. (1986a).  The subjects of Bauer et al. were exposed to
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  1      NO2 in dry air through a mouthpiece which could have caused some "drying" of the upper
 2      airways.  This would not be a factor in the Linn et al, (1985b) study where a chamber
 3      exposure was used.  Another possible explanation is that the asthmatics studied by Linn et al.
 4      were accustomed to NO2 exposure because of their place of residence (although the ambient
 5      levels in Los Angeles are, of course, much lower tnan 4 ppm).  However, the indoor
 6      environment can be an important avenue of NO2 exposure but is not known for either group.
 7      Secondly, the asthmatics in the Linn et al. study, although reactive to SO2, tended to have
 8      milder disease; none used regular asthma medications and all but three subjects had an
 9      FEVj/FVC ratio in excess of 75% (i.e., minimal asthma as per Appendix 15-A).  All of the
10      subjects in the Bauer et al. study used some form of bronchodilator (oral or inhaled) and 9 of
11      15 subjects had a baseline FEVyPVC ratio less than 75% (i.e., mild to  moderate asthma). It
12      is not clear whether the effects of NO2 could have been confounded by exposure to an
13      ambient aeroallergen.  All subjects in the Linn et al. study were exposed in March at a time
14      when outdoor aeroaUergens would have tended to be minimal for several months (because of
15      the mild climate, allergens are present during the winter months).  Also, increased bronchial
16      reactivity to cold air was an important finding in the Bauer et al. study; it was not measured
17      by Linn et al.                                                    .    ,
18           Further studies were conducted by Linn et al. (1986) on 21 (minimal to mild)
19      asthmatics exposed to 0,  0.30, 1.0,  and 3.0 ppm NO2 for 1 h.  The exposures included
20      intermittent, moderate exercise (VE= 41 L/min).  This group was characterized as "clinically
21      mild extrinsic (allergic)"  asthmatics who required infrequent,  if any,  medication. As in the
22      previous study with 4.0 ppm NO2 exposures, there were no significant effects of NO2 on
23      spirometry, SRaw, or symptoms.  Furthermore, there was no  significant effect on airway
24      reactivity as measured by cold-air challenge (see Section 15.4).  In order to examine the
25      suggestion that the severity of response to NO2 may be related to the clinical severity of
26      asthma, the authors selected three subjects whom they characterized as having more severe
27      illness.  There was no indication that the responses of these subjects were related to NO2
28      exposure, although they experienced markedly  larger changes in resistance than other milder
29      asthmatics under all exposure conditions.  Heart rate or minute ventilation did not vary
30      significantly with NO2 exposure.  The previously observed decrease in systolic pressure,
31      associated with 4.0 ppm NO2 exposure, was not examined in these subjects.

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 1          Mohsenin (1987a) studied 10 mild asthmatics exposed to 0.5 ppm NO2 for 1 h at rest in
 2     an environmental chamber.  There were no changes in symptoms, spirometry,  or
 3     plethysmography that could be attributed to NO2 exposure.  The response to methacholine
 4  '   was evaluated with partial expiratory flow at 40% VC (PEF40%VC), rather than changes in
 5     SRaw or FEVj, to test for "small airway abnormality" without the influence of prior deep
 6     breaths.  There was a significant increase in airway responsiveness to methacholine (bromide)
 7     after the NO2 exposure.  The dose of methacholine required to decrease PEF40%VC by 40%
 8     was 9.2  + 15 after air and 4.6 ± 8.2 after NO2 (p=0.042).
 9     ' *   Roger et at. (1990) reported the results of NO2 exposure in mild asthmatics.  The first
10     was a pilot study of 12 mild asthmatics exposed to 0.30 ppm for  110 min, including three
11     10-min periods of exercise.  After the first 10 min of exercise in  NO2, they found an  11 %
12     decrement in FEVj, which was significantly larger than the 7% decrease seen  after the clean
13     air exposure.  These differences between air and NO2 exposure persisted for the remainder of
14     the exposure period, although the overall responses were progressively less with successive
15     periods of exercise, as is common with exercise-induced asthma when the exercise stimulus is
16     intermittent.
17          A concentration-response study was subsequently conducted (Roger et al., 1990) with
18     21  mild asthmatics, including 6 subjects from the pilot study,  who were exposed to 0.0,
19     0.15, 0.30,  and 0.60 ppm NO2. The 75-min exposures included three 10-min exercise
20     periods.  In contrast to the pilot study,  there were no differences  in response between  the air
21     and NO2 exposure at any exposure concentration or time during the exposure.  Bronchial
22    - reactivity to methacholine, tested 2 h after the exposures, was similar for air and NO2
23     exposures.  There were no significant differences in symptom scores across the four exposure
24     conditions.  The authors were unable to specifically identify factors that could  have caused
25     the difference in response between the pilot study and the larger,  more comprehensive
26     concentration-response study.  They suggested that the pilot study asthmatics may have had
27     more reactive airways, based on their poorer baseline lung function and greater airway
28     responsiveness to methacholine compared to the subjects in the concentration-response study.
29     Furthermore, the studies were conducted during different seasons which may account for
30     some of the variability in response.
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  1          Rasmussen et al. (1990) presented a preliminary report of a concentration-response
  2     study of healthy asthmatic subjects exposed to 0.1, 0.2, and 0.8 ppm NO2.  Exposures lasted
  3     120 min and included 10 min of exercise. There were no significant changes in lung function
  4     (SRaw, FEVj) or airway responsiveness to histamine resulting from NO2 exposure at any
  5     concentration in either normal or asthmatic subjects.  Also assessed were acoustic rhinometry,
  6     nasal mucociliary clearance, and alveolar epithelial permeability; these results were not
  7     reported.
  8          A series of abstracts have been presented by investigators from Mt. Sinai Medical
  9     Center in Miami (Sackner et al., 1980; Ahmed et al., 1983a,b); these reports have hot
 10     appeared in the peer-reviewed literature but are available as technical reports (Ahmed et al.,
 11     1983a,b).  The latter report presents data that are qualitatively similar to Orehek et al. (1981)
 12     and Hazucha et al.  (1983) in that some subjects (13 out of 20) showed increased airways
 13     responsiveness to carbachol after NO2 exposure and some (7 out of 20) did not.   Even with
 14     the post hoc separation of subjects into "reactive" and "nonreactive"  groups, the increase in
 15     airway responsiveness in the reactive group (n=13) was not statistically significant. There
 16     were no significant changes in lung function.  Adequate characterization of the exposure
 17     conditions was not presented.  The former report (Ahmed et al., 1983a) dealt with effects of
 18     NO2 on nine ragweed-sensitive asthmatics.  There were no group mean changes in Gaw or
 19     FEVj after NO2 exposure. There was also no change in bronchial responsiveness to a
20     ragweed antigen inhalation challenge either immediately or 24 h after exposure to 0.1 ppm
21     NO2.
22          The effects of prior NO2 exposure on SO2-induced bronchoconstriction has been
23     examined in two studies (Jorres and Magnussen,  1990; Rubinstein  et al., 1990).  Torres and
24     Magnussen (1990) exposed 14 mild-to-moderate asthmatic subjects to 0.30 ppm NO2 for
25     30 min while breathing through a mouthpiece at rest.  There were  no changes in SRaw as a
26     result of the exposure. After the exposure, airways responsiveness to SO2 was assessed by
27     isocapnic hyperventilation of 0.75 ppm SO2 using stepwise increases in ventilation; the initial
28     level was 15 L/min with subsequent increases to  30, 45, 60, L/min and such. After each
29     3-min period of hyperventilation, SRaw was determined.  The ventilation of SO2 required to
30     produce a 100% increase in SRaw (PV100SRaw[SO2]) was estimated using interpolation of
31      ventilation vs. SRaw (dose-response)  curves.  The PV100SRaw(SO2) was significantly reduced

        August 1991                             15-50      DRAFT-DO NOT QUOTE OR CITE

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 1      after NO2 exposure compared to after filtered air exposure suggesting that the airways were
 2      more responsive to SO2 as a result of the prior NO2 exposure.
 3          Rubinstein et al. (1990) exposed nine asthmatics to 0.30 ppm NO2 for 30 min
 4      (including 20 min light exercise). There were no significant effects of NO2 exposure on lung
 5      function (single breath nitrogen washout, SRaw, FVC, FEVj 0) or respiratory symptoms,
 6      although a slight increase in SRaw was observed as a result of exercise.  An SO2-
 7      bronchoprovocation test was administered after exercise, but using a different technique than
 8      Jorres and Magnussen (1990). Increasing amounts of SO2 were administered by successive
 9      doubling of the SO2 concentration (0.25, 0.5, 1.0, 2.0,  4.0) at a constant, isocapnic
10      ventilation of 20 L/min, maintained for 4 min. SRaw was measured  after each step increase in
1.1      SO2 concentration. The concentration of SO2 required to increase SRaw by 8 units
12      (PD8uSO2) was interpolated from a dose-response curve of SO2 concentration vs. SRaw. The
13      PD8uSO2 was 1.25 + 0.70 ppm after air exposure and  1.31 ± 0.75 after NO2 exposure,
14      indicating no mean change in responsiveness to SO2.  Only one subject showed a tendency
15      toward increased responsiveness to SO2 after NO2 exposure (see also Section 15.4).
16          The contrasting findings in these two studies is somewhat puzzling because the subjects
17      of  Rubinstein et al. (1990) were exposed to a higher NO2 concentration and exercised during
18      exposure.  However, Jorres and Magnussen's subjects appeared to have had slightly more
19      severe asthma and were somewhat older. The modest increase  in  SRaw induced by exercise
20      in the  Rubinstein et al. study may have interfered with the response to SO2 (i.e., the subjects
21      may have been in  a refractory state). Finally, the different method of administering the SO2
22      bronchoprovocation test (i.e., increased  VE at constant  SO2 vs. increasing SO2 at constant
23      VE) may produce a different response, since hyperventilation alone could contribute to the
24      increase in SRaw (Deal et al., 1979; Eschenbacher and Sheppard,  1985).  Thus, although
25      similar, the two SO2 challenges are not necessarily comparable.
26                                               '
27      15.3.1.1 Effects of HNO3 Vapor on Asthmatics
28          Koenig and associates (1988;  1989a,b) have recently reported preliminary results of a
29      study of nine adolescent asthmatics exposed to HNO3 vapor.  Subjects were exposed to
30      50  and 100 ppb HNO3 and to 50 ppb HNO3 plus 68 ^g/m3 H2SO4.  FEV1 decreased
31      following exposure (30 min rest followed by 10 min mild, exercise) under all three conditions.
       August 1991
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  1           Koenig et al. (1989a) and Koenig (1989) have examined the responses of adolescent
  2      asthmatics to a 40-min exposure to 50 ppb (2 jttM/m3) HNO3 vapor exposure via a
  3      mouthpiece exposure system.  In the initial study, after 30 min of rest and 10 min of exercise
  4      while breathing HNO3 vapor, there was a 4.4% decrease in FEV1 compared to 1.7%
  5      decrease after air breathing.  A 22.5% increase in total respiratory resistance was also
  6      observed after HNO3 compared to a 7.4% increase after air.
  7           In the second study (Koenig et al., 1989a),  subjects were exposed for 45 min including
  8      two 15-min mild  (VE = ~25  1/min) exercise periods. The subjects were exposed to air and
  9      57±16 ppb HNO3 twice: once without and once with a preliminary gargle of lemonade,
10      intended to reduce oral ammonia (NH3) levels.  Baseline oral NH3 of 318+84 ppb was
11      reduced to 113 ±98 ppb  after lemonade gargle.  There were small, but not statistically
12      significant, decrements in FEVj after all exposures, -3.3%  after HNO3 alone, and -1.7%
13      after both air and HNO3 plus  lemonade.  Similar trends (-9.4%, HNO3; -5.5%, HNO3 plus
14      lemonade; -5.1%, air) were observed for V50%vc-  The author suggested that the HNO3
15      vapor was likely to have been neutralized by oral NH3 and that this potential reaction may
16      minimize the entry of HNO3 to the respiratory tract.  This led to the  interesting suggestion
17      that,  in mixtures of HNO3 vapor and H2SO4 aerosol, NH3  may react preferentially with the
18      gaseous HNO3, thus reducing the potential neutralization of H2SO4 aerosol.
19
20      15.3.2  Effects of NO2 on Patients with Chronic Obstructive Lung Disease
21           Patients with COPD represent an important potentially sensitive population group.
22      Many of these patients have airways hyperresponsiveness to physical and chemical stimuli.
23      In addition, because of their already compromised lung function, they have much less reserve
24      than people with normal lung  function.  The poor distribution of ventilation in COPD may
25      lead to a greater delivery of NO2 to the segment of the lung which is well ventilated, thus
26      resulting in a greater regional  tissue dose.  Tables 15-5 and 15-6 summarize these studies.
27           In a review of their studies, von Nieding and Wagner (1979) summarized previously
28      reported findings. The main observations were that Raw increased in chronic bronchitics
29      exposed to 2.0 ppm NO2 or greater and that,  after exposure to 4 to 5 ppm NO2, arterial
30      partial pressure of oxygen (PO2) was decreased and the alveolar-arterial O2 gradient was
31      widened.

        August 1991                             15-52     DRAFT-DO NOT QUOTE OR CITE

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 1          The results of two NO2 exposure studies were discussed in von Nieding et al. (1980).
 2     In the first study, 14 normal and 14 bronchitic patients were exposed to 5 to 8 ppm NO2 for
 3     up to 5  min on 4 separate days.  The mean increase in Raw was 1.07 cm H2O/L/s. Except
 4     for three subjects with an increase in Raw of greater than 2.0 cm H2O/L/s, the responses of
 5     the bronchitics were similar to  the normal subjects.    .      .    '
 6          In the second study (von Nieding et al., 1980), 30 normal subjects and 40 bronchitic
 7     subjects were exposed to 5 ppm NO2 for 5 min. The subjects were divided into clusters
 8     according to their pre-exposure Raw's, which ranged from less than 1  cm H2O/L/s to greater
 9     than 4.0 cm H2O/L/s.  There was a tendency for the response to NO2 to be greater in the
10     subjects with the highest baseline Raw.  In subjects with baseline Raw  >4.0 cm H2O/L/s, the
11     increase in Raw averaged just less than 1.5 cm H2O/L/s; it was less than 0.5 cm H2O/L/s in
12     subjects with baseline Raw < 1cm H2O/L/s.  Percentage changes ranged from approximately
13     25 to 50%. Unfortunately, this synopsis does not provide a more comprehensive review of
14     the data.
15          More recently, Linn and co-workers (1985a) studied  a diverse group of 22 COPD
16     patients, including men and women emphysematics and chronic bronchitics, exposed, while
17     exercising  intermittently, for 1  h to 0.5, 1.0, and 2.0 ppm NO2.  In agreement with the
18     previous von Nieding and Wagner  (1979) study, no changes in arterial oxygenation (ear
19     oximetry measurements of hemoglobin saturation) were observed.  Also, no changes in lung
20     function (spirometry, plethysmography) were observed that could be attributed to NO2. The
21     only exception was the tendency (not statistically significant) for peak flow to be slightly
22     lower (about 5%) during the 2.0-ppm exposures. No increase in symptoms was reported.
23          Morrow and Utell (1989) examined the responses of 20 patients with COPD who were
24     exposed to 0.3 ppm NO2 for 3.75 h during which time they performed  mild exercise for
25     three 7-min periods.  FVC showed progressive and  significant decreases during and following
26     NO2 exposure with the largest  change of -9.6% occurring after 3.75 h of exposure.  Smaller
27     decrements were seen in FEVj (-5.2%) at the end of exposure.  There was no effect on SGaw
28     or diffusing capacity as a result of  NO2 exposure.  The differences between subjects with
29     more severe disease (FEVj <60% predicted) and those with milder disease (FEVj >60%) in
30     terms of their relative responses to NO2 were generally not significant,  except for a possible
31     slightly greater decrease in FEVl in the milder COPD group.  When the COPD patients were
        August 1991
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  1     compared with healthy, elderly nonsmokers, there was an apparently significant difference
  2     between the two groups in their response to NO2; the COPD patients showed a decrement
  3     while the healthy nonsmokers showed an improvement in FEY^  There were also apparent
  4     differences in NO2 response between healthy, elderly smokers and healthy, elderly
  5     nonsmokers.  These exploratory post-hoc analyses generate interesting hypotheses, but they
  6     do not explain whether the COPD patients responded to NO2 because of their current or
  7     previous smoking habit or because of some predisposition to NO2 effects caused by their lung
  8     disease. The reasons for the marked difference in response between these subjects and those
  9     of Linn et al. (1985a) are unclear.  Possible explanations include differences in ambient
 10     concentrations due to the place of residence (Los Angeles vs. Rochester; but see discussion in
 11     Section 15.3 regarding this factor) and, more importantly,  duration of exposure (4 h vs. 1 h).
 12     However,  the higher concentrations used in the Linn et al. (1985a)  study could be expected to
 13     produce greater effects in equally reactive subjects.  Differences in  the severity of COPD
 14     could be related to the differences in response, although Linn et al.'s subjects had similar or
 15     worse lung function than Morrow  and Utell's subjects.  Note also that Morrow and Utell's
 16     mild COPD subjects had greater responses.
 17
 18     15.3.3 Summary
 19          Although findings in asthmatics have been mixed, the pulmonary function responses to
20     NO2, within the ambient range, are relatively small  when compared to SO2 exposure (see
21      Table 15-7 and Figures 15-1 and 15-2). Several studies (Bauer et al., 1986b; Roger et al.,
22     1985; Koenig et al., 1987a,b;  Avol et al., 1986) suggest possible small changes in spirometry
23      or plethysmography at concentrations in the range of 0.1 to 0.5 ppm.  However, the absence
24      of changes at higher NO2 concentrations, indicative  of a concentration-response relationship
25      (Avol et al., 1986; Bylin et al., 1985; Linn et al., 1985b; Linn et al., 1986) is problematic.
                                                                                          \
26      Patients with COPD experience pulmonary function  changes with brief exposure to high
27      concentrations (5 to 8 ppm for 5 min) or with more  prolonged exposed to lower
28      concentrations (0.3 ppm for 3.75 h). In both asthmatic and COPD  populations there remain
29      several unanswered questions regarding the interaction of disease state and exposure
30      variables.
       August 1991                             15_56      DRAFT-DO NOT QUOTE OR CITE

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 1     15.4  EFFECTS OF NO2 EXPOSURE ON AIRWAYS RESPONSIVENESS
 2          Physiological changes in the airways induced by a variety of inhaled substances have
 3     been used to assess their "responsiveness" or "reactivity."  Comparing results across studies is
 4     difficult because of the variety of types of airway challenges, methods used to administer the
 5     tests, physiological end points used to quantify the responses, waiting period after the
 6     exposure to determine reactivity, and whether or not the exposure involved exercise.
 7     A variety of stimuli have been used to challenge the airways, including (1) chemical
 8     mediators such as histamine, methacholine, carbachol, or hypertonic saline; (2) physical
 9     methods  such as exercise or isocapnic hyperpnea with cold air; (3) other pollutants such as
10     SO2; or (4) specific antigenic substances such as ragweed or grass pollen. In this section, the
11     effects of NO2 on measures of airway responsiveness are discussed.  A more detailed
12     discussion of many of the studies including exposure conditions and measurement of other
13     variables are presented in Sections  15.2 and 15.3 and the tables associated with those
14     sections.
15          Despite the absence of bronchial or airways hyperresponsiveness (BHR) in some
16     asthmatics and the presence of BHR in some nonasthmatics (Pattemore et al.,  1990),  there is
17     a correlation between increased asthma symptoms or increased medication usage and
18     increased airway responsiveness (Britton et al.,  1988). Alterations in airway responsiveness
19     may also occur as a result of repeated challenges with histamine (Hamielec et al., 1988),
20     hypertonic saline (Belcher  et al., 1987), or exercise (Stearns et al., 1981), or in some cases
21     by interaction  between two different challenges;  either histamine  (Hamielec et al.,  1988) or
22     hypertonic saline challenge (Belcher et al., 1987) administered before an exercise challenge
23     can reduce the airway response to exercise.  Prior exercise-induced bronchoconstriction can
24     reduce responsiveness to hypertonic saline (Belcher et al., 1987) but not, apparently, to
25     histamine (Belcher et al., 1987; Hamielec et al., 1988). Thus, while the responses to a
26     number of airway challenges may be correlated (Chatham et al.,  1982), they cannot be
27     considered equivalent.
28
29     15.4.1  Healthy Normal Subjects
30   .       In a small  number of recent studies, the effects of NO2 on airway responsiveness in
31     healthy,  normal subjects have been reported. Airway responsiveness has been shown to
        August 1991
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 1     increase in normal subjects after exposure to NO2 concentrations in excess of 1.0 ppm. Beil
 2     and Ulmer (1976) found increased responsiveness to acetylcholine in subjects exposed to
 3     either 7.5 ppm for 2 h or to 5.0 ppm for 14 h.  Mohsenin (1987b,  1988) found increased   ,
 4     responsiveness to methacholine after 1-h exposures to 2.0 ppm and  Frampton et al. (1991)  ,
 5     reported increased carbachol responsiveness after 3-h exposures to  1.5 ppm.  Mohsenin   .
 6     (1987b) also reported that the increased airway responsiveness post-NO2 exposure could be
 7     blocked by elevation of serum ascorbate levels through vitamin C pretreatment.  In contrast, :
 8     using subjects exposed to 0.1 ppm at rest, Hazucha et  al. (1982) and Ahmed et al. (1983b)   ,
 9     found no significant change in airway responsiveness to cholinergic agonists (methacholine
10     and carbachol, respectively).  A 20-min exposure to 0.48 ppm similarly had no significant
11     effect on airway responsiveness to histamine (Bylin et  al., 1985). Kulle and Clements (1988)
12     examined airway responsiveness after 2-h exposures to 2.0 and 3.0  ppm NO2 on
13     3 consecutive days.  Nasal inoculation with influenza virus occurred on the second day of
14     NO2 exposure.  Although there was a significant trend for airway responsiveness to decline in
15     one group of subjects exposed to clean air, there was no trend for airway responsiveness to
16     increase after exposure to either NO2 concentration. Responses were not altered after virus
17     inoculation.  In two studies, NO2 concentrations in excess of 1.5 ppm NO2 inhaled over at   ,
18     least 60 min were associated with increased responsiveness to  either cholinergic or          :
19     histaminergic agonists.  The mechanism for this increase in responsiveness  needs to. be
20     established before the clinical implications of this finding can be ascertained.
21
22     15.4.2 Asthmatic Subjects
23          A change in airway responsiveness  appears to be one of the more sensitive indicators of
24     response to NO2 exposure in asthmatics.  The findings from the various studies reported here
25     are summarized in Table 15-8.  See Section 15.3.1 for a more detailed description of the
26     exposure and measurement methodology.
27          There have been several studies of NO2-exposed asthmatics in which  the airway
28     responsiveness was evaluated using  cholinergic agonists  (carbachol, acetylcholine,
29     methacholine).  Subjects were exposed to 0.1 to 0.2 ppm NO2 in five such  studies.  Of these,
30     both Hazucha et al., 1983 and Roger et al.,  1990 found no significant change in group mean
31     response to methacholine challenge. Ahmed et al. (1983b)  reported a trend for airway

       August 1991                              15-62      DRAFT-DO NOT QUOTE OR CITE

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   1      responsiveness to carbachol to increase after a 1 h exposure to 0.1 ppm NO2, but the trend
  2      was not significant (p=0.07).  Nevertheless,  some subjects appeared to be more responsive
  3      than others.  Orehek et al. (1976) reported that 13 of 20 subjects exposed to 0.1 ppm NO2
  4      experienced an increased airway responsiveness to carbachol.  In these 13 subjects, the mean
  5      PD100 decreased from 0.66 to 0.36 mg.  However, in the seven "nonresponders," the PD100
  6      of 0.36 mg remained unchanged.  A number  of questions have been raised about the
  7      analytical approach used in this study, and these are discussed in more detail in Section
  8      15.3.1. Kleinman et al. (1983) also evaluated airway responsiveness to methacholine after a
  9      2-h exposure to 0.2 ppm NO2.  The dose of methacholine required to cause a 10% drop in
 10      FEVj decreased from 8.6 to 3.0 /zg.  As a group, these studies appear to suggest that some
 11     individuals, if not a subgroup of asthmatics, may experience increased airway responsiveness
 12     after NO2 exposure.            •                                             ^
 13          It might be anticipated, when there is a trend for a response at a low concentration, that
 14     exposure to increased concentrations would tend to confirm the trend by producing a  less
 15     equivocal response. Mohsenin (1987a) found a significant decrease in the dose of
 16     memacholine required to produce a 40% decrease in flow at 40% of VC on a partial  flow
 17     volume curve; the PD40 decreased from 9.2 after air to 4.6 after exposure to 0.5 NO2 for
 18     1 h.  On the other hand, at both 0.3 and 0.6 ppm for 110 min, Roger et al. (1990) found no
 19     difference in airway responsiveness to methacholine.  Morrow and Utell (1989) also found no
 20     change in airway responsiveness to carbachol after a 3.75-h exposure to 0.3 ppm. These
 21     differences cannot be explained either on the basis of NO9  concentration or total NO, dose
                                                           &                        &    '
 22     since the total dose in the Mohsenin (1987a) study was lower than either of the other  two
 23     studies.
 24          Histamine airway challenges have been used in three studies following NO2 exposure.
 25     Two studies by Bylin et al. (1985,  1988) at NO2 concentrations ranging from 0.14 to
 26     0.53 ppm. suggest possible increased responsiveness to histamine after 20 to 30 min resting
 27     NO2 exposure. In the first study, 5 of 8 subjects showed an increase in response after
 28      0.48 ppm exposure and in the second study 14 of 20 subjects  showed an increase in response
29      after 0.27 ppm exposure.  However, the second larger study (n=2Q) did not confirm the
30      observations (at 0.53 ppm) of the first study and a somewhat more conservative statistical
31      approach (Friedman nonparametric test) failed to confirm the significance of these findings.

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 1     In a preliminary report, Rasmussen et al. (1990) examined the effects of 3-h exposures to
 2     0.1, 0.2, and 0.8 ppm NO2 on airway responsiveness to histamine. They found no
 3     significant group mean change in airway responsiveness.  Again, these results are suggestive
 4     that some asthmatics may experience increased airway responsiveness after NO2 exposure,
 5     but the inconsistent nature of the findings from study to study and  the absence of a
 6     dose-response relationship is problematic.
 7          Bauer et al. (1986a), Linn et al. (1986), and Avol et al. (1988, 1989) have examined
 8     the effects of NO2 exposure on airway responsiveness to cold air inhalation. Bauer et al.
 9     (1986a) found a decrease in cold air airway responsiveness after a  30-mih exposure to
10     0.30 ppm NO2. The airway responsiveness was expressed as the quanitity of respiratory heat
11     loss reuired to produce a 10% drop in FEV^; this averaged 0.83 kcal/min after air exposure
12     and 0.54 kcal/min (p< 0.05) after NO2 exposure, indicating an increase in airway
13     responsiveness.  Linn et al. (1986) found no change in airway responsiveness to cold air after
14     1-h exposures to 0.3, 1.0, or 3.0  ppm NO2 in a group of 21 asthmatics.  Avol et al. (1988)
15     found  a trend'for a group mean increase in airway responsiveness to cold  air after 0.3 ppm,
16     but not after 0.60 ppm, NO2 exposure;  this increased response was observed in only 11 of
17     the 29 subjects at 0.30 ppm.  In a study of young asthmatics, also exposed to 0.30 ppm NO2
18     for 1 h, Avol et al. (1989) found  no mean change in cold air airway responsiveness. Indeed,
19     only 12 of 33 subjects demonstrated a change in cold air airway responsiveness in the
20     direction indicative of increased responsiveness.  Again, these cannot be explained on the
21     basis of NO2 concentration or total NO2 exposure dose since both  were lower in the Bauer
22     et al. (1986a) study where a significant change in the airway responsiveness was observed.
23     Comparison of the studies in Table 15-8 and 15-4 indicates that the Bauer et al. (1986a) study
24     was shorter, included less exercise, and utilized a mouthpiece exposure system.  Additional
25     discussion is presented in Section 15.3.1.
26          Airway responsiveness to SO2 has been evaluated after 30 min of exposure to 0.25 to
27     0.30 ppm NO2 in two studies.  Jorres and Magnussen (1990) found an increased airway
28     responsiveness  to SO2 after a resting exposure to 0.25 ppm N02, but Rubinstein et al. (1990)
29     found  no change in airway responsiveness to SO2 after a 0.30-ppm NO2 exposure of similar
30     duration that included 20 min of exercise. The SO2 challenges were administered by
                          t             i
31     different techniques; Jorres and Magnussen used a series of increasing levels of ventilation at
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  1     a constant SO2 concentration whereas Rubinstein et al. (1990) used increasing concentrations
  2     of SO2 at a constant ventilation.                              .    ;
  3          The effects of NO2 on airway responsiveness to a specific antigen have been examined
  4     in only two studies.  Ahmed et al. (1983a) reported no increase in airway responsiveness to
  5     ragweed antigen in a group of allergic asthmatics following 60-min of exposure to 0.1 ppm
  6     NO2.  Orehek et al.  (1981) found no change in airway responsiveness  to grass pollen in a
  7     group of allergic subjects (including three asthmatics) after a 60-min exposure to 0.11 ppm
  8     NO2.                               .
  9          For the studies for which individual data were readily available, the number of subjects
 10     whose airway responsiveness increased and whose airway responsiveness decreased is listed in
 11     Table 15-8. Tabulation of data from this table provided information regarding the' direction
 12     of the change (i.e., increase or decrease) in airway responsiveness following NO2 exposure.
 13     One of the  problems in this kind of analysis  is that it is often difficult to distinguish between
 14     a negative and no-change situation (i.e., it is less likely that airway responsiveness would
 15     decrease from its baseline level than increase).
 16          For studies of exposure to <0.20 ppm, the overall data indicated 67 subjects with
 17     airway responsiveness increased and 38 with airway responsiveness decreased.  Further
 18     breakdown  of this data into exercise/ no-exercise or exposure duration  was not fruitful.
 19          For the  studies  (n=ll) of exposure to 0.20 to 0.30 ppm,'using all types of challenges,
20     airway responsiveness increased in 96 subjects and  decreased in 73.  For studies involving
21     exercise during the exposure, airway responsiveness increased in 71 and decreased in 65.
22     However, both  studies involving resting  exposure showed significant increases in airway
23     responsiveness whereas only two of nine studies using  exercise  exposures were significant.
24     In the resting studies airway responsiveness increased in 25 subjects and decreased in 8.
25     These studies also were of shorter duration (—30 min) than many of the exercise studies.
26     Airway responsiveness increased in 29 and decreased in 13 subjects in  studies of 30-min
27     duration, whereas there were 41 increases and 53 decreases in exposures lasting 60 min or
28     longer.
29          At concentrations greater than 0.30 ppm, the overall total  indicated 47 increases and
30     33 decreases in airway responsiveness.  For resting studies, 24  subjects had increased airway
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 1     responsiveness while only 9 showed decreased airway responsiveness (5 did not change),
 2     whereas 23 increased and 24 decreased in the exercise studies.
 3.':         These data are summarized in Table 15-9.  The  studies in which the change in airway
 4     responsiveness was assessed after NO2 exposure are presented in three concentration ranges
 5     and divided according to whether or not exercise was  involved in the exposure.  The data are
 6     presented as the fraction of the total number, of subjects with increased airway responsiveness.
 7     The increase in airway responsiveness does not appear to be associated with any particular
 8     type of airway challenge. The overall percentage of increased airway responsiveness in
 9     NQ2-exposed subjects was 59%.  This is accounted for almost entirely by the resting studies
10     with an overall percentage of 69% (106 increased and 48 decreased) since, in the exercising
11     studies, only 52% (104 increased and 96 decreased) had an increase in airway responsiveness
12     (i.e., no effect).  There was a trend for a slightly larger percentage ( — 75%) of subjects to
13     have increased airway responsiveness after NO2 exposure when the exposure is performed
14     both under resting conditions and at concentrations above 0.20 ppm.  In fact,-of the six
15     studies reporting a significant response (Kleinman et aL, 1983; Bauer  et al., 1986a; Bylin et
16     al., 1988; Torres and Magnussen, 1990; Mohsenin,  1987a; Bylin et al., 1985), four were
17     resting exposures and in four the exposure duration was 30-min or less.
18          The implication of this trend in unclear since the brief duration and low ventilation
19     during exposure indicate that the,NO2 exposure dose in these studies is relatively low.  If this
20    ., trend is real, some interesting hypotheses could be generated.  Is it possible that exercise
21     during exposure somehow interferes with the mechanism causing increased airway
22     responsiveness?  It is known, for example, that repeated exercise induces,a refractory state
23     such that the subject is less sensitive to exercise-induced bronchoconstriction (Edmunds et al.,
24     1978;  Ben-Dov et al.,  1982). In many cases of NO2 exposures involving exercise, repeated
25     bouts  of exercise were performed during exposure, which could possibly have made the
26     subjects refractory to the effects of NO2.  During exercise, the responsiveness to
27     methacholine is reduced substantially (Inman et al., 1990) and exercise causes a more rapid
28     reversal of methacholine-induced bronchoconstriction  than occurs at rest (Freedman et al.,
29      1988). On the other hand, is there a biphasic response of NO2 causing increased airway
30     responsiveness at a low exposure doses (for example causing  mast cell degranulation, vis a
31     vis Sandstrom et al. (1990a), with a reversal of this response at higher exposure doses
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 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
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14
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16
17
18
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possibly through a direct relaxing effect on airway smooth muscle?  For example, nitrites
formed in the lungs of NO2-exposed animals (Postlethwait and Mustafa, 1981) may have a
direct relaxing effect on smooth muscle, including bronchial smooth muscle.
     In healthy normal subjects, an increase in airway responsiveness clearly occurs at higher
NO2 exposure doses (Beil and Ulmer, 1976; Frampton et al., 1991; Mohsenin, 1988).  In the
normal subjects at all concentrations, there were 37 airway responsiveness increases,and
23 airway responsiveness decreases.  At greater than 1.0 ppm, there were 23 increases and
6 decreases, that is, a ratio of 0.79.
15.5 EFFECTS OF NO2 OR HNO3 EXPOSURE ON BLOOD, URINE,
      AND BRONCHOALVEOLAR LAVAGE FLUID BIOCHEMISTRY
     The effects of NO2 on the constituents of bronchoalveolar lavage (BAL) fluid, blood,
and urine have been examined, both in vivo and in vitro.  The general purpose of these
studies has been to examine mechanisms of pulmonary effects or to determine NO2-induced
alterations in body fluids that could potentially result in systemic effects. Investigations have
been aimed at determing the effects of NO2 on levels of serum enzymes and antioxidants as
well as direct effects on red blood cells and hemoglobin.  Studies of the effects of NO2 on
airway lining fluids have focused on changes in alphaj-antitrypsin levels. Potential effects of
NO2 on collagen metabolism have been investigated by examining urinary excretion of
collagen metabolites.

15.5.1 Biochemical Effects in Blood
     Chaney et al. (1981) examined the effects of 0.20 ppm NO2 on various blood
parameters in 19 healthy subjects exposed for 2 h while exercising intermittently.  A control
group of 15  subjects was exposed to clean air.  They observed a significant increase in
glutathione (GSH) levels after exposure. None of the other blood parameters (red blood cell
GSH reductase, 2-3 disphosphoglycerate, methemoglobin, vitamin E, immunoglobulin, and
complement  C3)  were changed significantly.  The significance of the response reported in this
study appears to be the result of a difference between the control group and the exposure
group in general  (different subjects were used in each group).  The changes in GSH were
       August 1991
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  1     small and were within the normal range.  The average baseline level of GSH was
  2     approximately 38.5 mg/dL. The postexposure average of the air group was
  3     36.4 mg/dL ± 1.35 (standard error of the mean [SEM]) and of the NO2 groups was 40.3 +
  4     1.19 (SEM) mg/dL.  The authors suggested that the increased level of GSH may be in
  5     response to oxidation of hemoglobin to methemoglobin by NO2. However, Gohil et al.
  6     (1988) have recently demonstrated substantial decreases in GSH levels during prolonged
  7     submaximal exercise, which was followed by elevated GSH levels in the postexercise period.
  8     GSH levels varied from 0.15 mM during exercise to 0.6 mM 3 days postexercise,, varying
  9     about a baseline level of approximately 0.4 mM.  It is not clear to what extent the
 10     observations of Chaney et al. (1981) may have been confounded by this exercise effect.
 11          It should be noted that Posin et al. (1978)  found no association between NO2 exposure
 12     (1 ppm for 2.5 h) and GSH levels, although there were apparent changes in blood
 13     biochemistry including increased levels of GSH  reductase.  However, it is not clear from the
 14     Posin et al. (1978) study that any of the observed  "effects" can  be attributed to NO2
 15     exposure; there was no concentration-response relationship, effects were, not reproducible
 16     from concentration to concentration and similar  effects were seen with clean air exposures.
 17          In vitro exposure of human blood to high levels of NO2 (6 and 45 ppm) resulted in
 18     methemoglobin formation (Chiodi et al., 1983).  However, Borland et al.  (1985) were unable
 19     to demonstrate increased methemoglobin levels in smokers exposed to high NO levels from
20     cigarette smoke.  Methemoglobin is also formed during in vitro exposure to NO  (1,000 ppm)
21      (Chiodi and Mohler, 1985). These observations appear to have no relevance to the potential
22     effects of ambient NO2.
23
24     15.5.2 Bronchoalveolar Lavage Fluid Biochemistry
25          Mohsenin and Gee (1987) have reported that subjects exposed to 3 to 4 ppm NO2 for
26      3  h had a 45% decrease in the activity of alpha-1-protease inhibitor, the major lung protease
27      inhibitor of the enzyme, elastase.  These levels were measured in BAL fluid obtained 3.5 to
28      4  h after exposure.  Alpha-1-protease inhibitor is "important in protecting the lung from
29      proteolytic damage, particularly from the elastase of neutrophils."  The mean elastase
30      inhibitory capacity decreased from 95 ± 12%  in the air group to 55% in the NO2-exposed
31      group.  (Due to analytical impurities in. the standard, the 95% inhibition measured in the

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  1
  2
  3
  4
  5
  6
  7
  8
  9
 10
 11
 12
 13
 14
 15
 16
 17
 18
 19
20
21
22
23
24
25
26
27'
28
29
30
31
 air-exposed group was presumed equivalent to 100%, thus the 45% difference).  The authors
 noted that even a 50% reduction in alpha-1-protease inhibitor activity is not associated with
 an increased risk of emphysema (Kabiraj  et al., 1982).  However, reduction in protease
 inhibition could result in connective tissue damage and could conceivably be important in
 individuals with  an a-1-antitrypsin deficiency.
      Johnson et al.  (1990) also examined the response of alpha-1 protease inhibitor (a^PI) to
' in vivo NO2 exposure in a group of 24 healthy nonsmokers.  The subjects were exposed to
 either 1.5 ppm NO2 for 3 h or to a variable concentration consisting of a baseline level of
 0.05 ppm NO2 with three 15-min "peaks" of 2.0 ppm.  The details of the exposure protocol
 and the subject characteristics are provided in Frampton et al.  (1989b); (Table 15-1,
 Section 15.2).  BAL was performed 3.5 h after exposure and the fluid was frozen for
 subsequent analysis.  The functional activity of oijPI was taken to be the elastase inhibitory
 activity corrected for the concentration of c^PI determined by immunoassay.  Neither the
 levels of ttjPI, as determined by immunbreactivity, or its  functional activity were
 significantly changed by NO2 exposure.
      The different findings by Johnson et al. (1990) and Mohsenin and Gee (1987)  with
 regard to oijPI activity may be accounted for by the considerably larger (about two- to
 threefold) exposure levels in the latter study.  Furthermore, different methods were  used to
 handle the BAL fluid and to quantify a{Pl concentrations in the two studies. This issue is
 also discussed in Section 13.2.2.2.6.  As discussed by Mohsenin and Gee (1987), there
 appears to be a large range of o^PI activity that is compatible with lung health, and there is
 broad range of activity of ttjPI in relation to its concentration. The importance of small
 changes in a{Pl is not clearly established, and therefore, the usefulness of changes in o^PI
 activity as a marker  of NO2 exposure will require additional research.

 15.5.3  Urine  Biochemistry
      Muelenaer et al.  (1987) studied normal males exposed to 0.6 ppm NO2 for 4 h/day on
 3  consecutive days to examine the possibility that NO2 exposure  caused diffuse pulmonary
 injury. They used hydroxyproline excretion as a marker of increased collagen  catabolism or
 connective tissue injury.  Subjects had no  residential NO2 exposure, no allergies or infections
 that might have produced inflammatory responses, and were minimally exposed to
        August 1991
                                         15-73
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 1     environmental tobacco smoke.  Despite controlling for these potentially confounding
• 2     variables, the authors observed no significant changes in hydroxyproline excretion as a result
 3     of NO2 exposure, either immediately or for up to 9 days after exposure.
 4
 5
 6     15.6 EFFECTS OF NO2 OR HNO3 VAPOR EXPOSURE ON HUMAN
 7           PULMONARY HOST DEFENSE RESPONSES
 8          From the epidemiological (Sections 14.2.1 and 14.3.1) and animal toxicology
 9     (Section 13.2.2.1) literature, it is clear that there is considerable concern regarding  the role
10     NO2 exposure may play in potentiating susceptibility to both bacterial and viral infections.
11     Important host defenses that may be affected by NO2 exposure include the mucociliary
12     clearance system, alveolar macrophages (e.g., altered viral inactivation), and humoral and
13     cell-mediated immune responses (e.g., changes in antibodies and changes in cell populations
14     and their activities in the lung)  (see also Section 13.2.2.1.3).  In human volunteers, the
15     effects of NO2 exposure on viral infectivity have been studied.  The effects of NO2 on
16     macrophage functions has been examined both using macrophages from NO2-exposed subjects
17     or macrophages exposed to NO2 in vitro.   The effects of NO2 on mucociliary clearance in
18     humans is discussed in Section  15.2.1.4.
19          Kulle and Clements (1988) and Goings et al (1989) [two reports of the same study]
20     examined the effect of NO2 exposure on infectivity rate of live attenuated influenza
21     A/Korea/reassortment virus in healthy, nonsmoking adults exposed to NO2. Seven  separate
22     groups were exposed to either clean air (n=23;21;21) or to NO2 at 1.0 ppm (n=22),
23     2.0 ppm (n=21;22), or 3.0 ppm (n=22).  The exposures consisted of one preliminary day  of
24     clean air exposure and then 3 consecutive days of the treatment (i.e., either NO2 or clean
25     air). The virus was administered intranasally after the second exposure day (i.e., the third of
26     the four days).  Infectivity was defined as evidence of virus recovery or a rise in either nasal
27     wash or serum antibody titers after virus inoculation.  Infectivity rates in the three clean air
28     groups were 65%, 71%, and 71%, and in the 3.0, 2.0, 2.0, and 1.0 ppm NO2 exposure
29     groups were 77%, 57%, 91%, and 91%.  Although the rates of infection were elevated  after
30     NO2 exposure in three of four NO2-exposed groups, these changes were not significant. The
31     investigators and an expert review committee (Kulle and Clements,  1988) concluded that the

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 1      results of the study were inconclusive rather than negative; this implies that the hypothesis
 2      that NO2 exposure may alter the frequency or severity of viral infections was neither
 3      confirmed nor denied by the results of this study.
 4           Goings et al.  (1989) have further elaborated on the results of the above study.  They
 5      made the point that the experimental design had a low power to detect a 20% difference in
 6      infection rate of influenza A/Korea (i.e., 71% vs. 91%) and, thus, the lack of statistical
 7      significance is not unexpected.  At least 70 subjects would be required to detect such a
 8      difference using this virus, which has a relatively high rate of infectivity.  Finally, the
 9      influenza A/Korea/reassortment virus is  not likely to infect the lower respiratory tract where
10      most of the NO2 deposition occurs.  There is also the possibility that the results may have
11      been confounded by an influenza epidemic, which occurred concurrently with this study,
12      although caused by a different but related virus.  The epidemic occurred between year 1 and
13      year 2.
14           Frampton et al. (1989a) studied two groups of normal subjects exposed to NO2 under
15      two different protocols that had the same concentration X time (C X T) product.  One  group
16      was exposed continuously for 3 h to 0.60 ppm while the other was exposed to a background
17      level of 0.05 ppm with three "spikes" of 2.0 ppm for 15 min each.  The C  X T product for
18      each of these two protocols was the same.  The major aims of this study were to test the
19      hypothesis that the ability of alveolar macrophages to inactivate  influenza virus was reduced
20      by NO2 exposure, and to examine the possibility that a series of peak exposures would cause
21      more impairment than a constant concentration (see also 13.2.2.1).  Healthy, normal
22      nonsmokers with no history of airway hyperresponsiveness or of recent upper respiratory
23      infection were exposed to both air and NO2 in random sequence.  Exposures included six
24      10-min exercise periods, coinciding with the "spikes" in the  second protocol.  There were no
25      significant effects of these exposures on spirometry or plethysmography under either protocol.
26      Alveolar macrophages obtained by BAL were tested in vitro  for their ability to inactivate
27      influenza (A/AA/Marton/43 H1N1) virus and for the in vitro production of Interleukin-1
28      (IL-1) by virus exposed macrophages. IL-1 is an important proinflammatory protein
29      produced by macrophages which performs a number of functions, including induction of
30      fibroblast proliferation, activation of lymphocytes, and is chemotactic for monocytes during
31      the immune response to infection.  There were no differences in total cell recovery,  viability,
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 1     or differential cell counts between air and NO2 exposed samples for either protocol.  There
 2     was a trend (p<0.07) for less effective inactivation of virus by macrophages obtained from
 3     subjects exposed continuously to 0.60 ppm NO2. This trend was due to the responses of only
 4     four of the nine subjects. The macrophages harvested from these four subjects also showed
 5     an increase in EL-1 production not seen in macrophages from the other subjects. No effects
 6     of virus inactivation were seen in the subjects exposed to the 2.0-ppm spikes. Although the
 7     results of this study were not statistically significant, the study had relatively low power to
 8     detect an effect.  The findings are provocative and suggest that further work is necessary to
 9     test the hypothesis that NO2 may influence host defense mechanisms in humans.
10          Frampton et al. (1989b) also analyzed the protein content of BAL fluid obtained from
11     NO2-exposed subjects at either 3.5 or  18 h postexposure.  Three different exposure protocols
12     were used:  3-h exposure to 0.60 ppm or 1.5 ppm NO2 or a 3-h variable concentration
13     exposure where three 15-min "peaks" of 2.0 ppm were superimposed on a background of
14     0.05 ppm NO2.  Exposures included 10 min of exercise during each half-hour of exposure.
15     There were no  significant changes in pulmonary function, airways reactivity, or respiratory
16     symptoms observed after NO2 exposure.  Two groups of subjects were exposed to 0.60 ppm
17     so that BAL could be obtained either at 3.5 or 18 h postexposure.  Analysis of BAL fluid
18     obtained 3.5 h  after 0.60 ppm exposure indicated an increase in alpha-2- macroglobulin
19     (<*2"JM)> a regulatory protein that has antiprotease activity and immunoregulatory effects.  The
20     observed increase in a2-M appears to be transient (no change was seen at 18 h postexposure)
21     and was not observed at a higher NO2 concentration (1.5 ppm). Further information appears
22     to be necessary to establish the implications of this finding.
23          The effects  of NO2 on macrophage function in NO2-exposed animals is discussed in
24     Section 13.2.2.1.2.  NO2-induced changes have been noted in macrophages harvested from
25     animals exposed to NO2 concentrations less than 1.0 ppm.  These studies are discussed in
26     detail in the animal toxicology chapter.
27          The effect of in vitro exposure to NO2 on alveolar macrophages harvested by BAL was
28     examined by Pinkston  et al. (1988).  Fifteen healthy adults underwent BAL to provide
29     macrophages for  culture.  After 18 h incubation, the cells were exposed to 5, 10, or 15 ppm
30     NO2 or 5% CO2 as a control for an additional 3 h.  Following the exposure, some of the
31     cells were incubated for an additional 24 h and cell-free supernatants were then obtained for

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 analysis of neutrophil chemotactic factor (NCF).  Other macrophage cultures were incubated
 for 24 h with influenza virus and the supernatant was then obtained for analysis of IL-1.
 There were no changes in macrophage viability, determined by trypan blue exclusion, in cells
 exposed to any of the three NO2 concentrations.  There were no changes in release of NCF
 in any of the NO2-exposed cell cultures.  Furthermore, NO2 exposure did not impair the
 ability of cells to release NCF after stimulation with activated zymosan.  Nitrogen dioxide
 exposure did not stimulate release of IL-1 from exposed macrophages.  Influenza virus
 stimulated the release of IL-1,  but there were no  significant  differences between NO2-exposed
 and air-exposed  macrophage cultures.  Therefore, NO2 exposure triggered neither the release
 of NCF, which would attract neutrophils to the airways, nor the release of IL-1, which
 activates lymphocytes (among other functions). Equally important, NO2 exposure did not
 impair the ability of macrophages to produce either IL-1 or NCF, in response to conventional
 stimuli.
     Sandstrom  et al. (1989) exposed a group of 18 healthy nonsmokers to 2.25,  4.0, and/or
 5.5 ppm (n=8 in each concentration group) for 20 min of moderate exercise (VE
 -35 L/min) in an exposure chamber.  BAL was  performed  at least 3 weeks before and 24 h
 after each exposure.  Increased levels of mast cells in BAL fluid were observed  after all NO2
 exposures.  Increased levels of lymphocytes were observed only at the two higher
 concentrations.
     In order to  determine the time course of this response,  Sandstrom et al. (1990a) exposed
 32 subjects to 4 ppm NO2 for 20 min, including 15 min of mild exercise, and then performed
 BAL at 4, 8, 24, or 72 h postexposure (in four different groups of eight subjects). Increased
 levels of mast cells and lymphocytes were observed at 4, 8,  and 24 but not at 72 h
 postexposure.  There was no change in macrophage numbers nor in albumin concentration in
 BAL fluid.  Eosinophils, neutrophils, and epithelial cell counts were not altered  as a result of
 NO2 exposure.  Unpleasant odor and mild nasopharyngeal irritation were typical symptoms.
There were no changes in spirometry.  The observation of increased numbers of mast cells
appears to be unique to this study; other investigators (Frampton et al.,  1989a,b) did not,
apparently,  observe changes in mast cell numbers. The authors considered the increased
numbers of mast cells and lymphocytes to represent an unspecific inflammatory response.
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  1           Boushey et al. (1988) studied five healthy volunteers exposed to 0.60 ppm NO2 on
  2      4 days over a 6-day period.  Exposures each lasted 2 h and included alternating 15-min
  3      periods of rest and exercise (VE ~ 30-40 L/min).  On the final (fourth) day of NO2
  4      exposure, venous blood samples were obtained and a BAL was performed. Baseline BAL
  5      and pulmonary function data were obtained on a separate occasion. There were no effects of
  6      repeated NO2 exposure on pulmonary function (SRaw, FVC, FEV1>0) or respiratory
  7      symptoms.  Following the fourth day of NO2 exposure,  a slight increase in circulating
  8      (venous blood) lymphocytes was observed (1792 + 544/mm3 post-NO2 vs. 1598 ± 549/mm3
  9      baseline). The only change observed in  BAL cells was an apparent increase (p<0.04) in
10      natural killer (NK) cells from 4.2  ± 2.4% (baseline) to  7.2 ±3.1% (post-NO2).  The
11      authors expressed reservations that the apparent increase in NK cells may have been an
12      artifact of the cell separation process.  DL-1 and tumor necrosis factor (TNF) levels in BAL
13      fluid  were not detectable.  TNF is another proinflammatory protein that, among other
14      activities, promotes aherence of polymorphonuclear leukocytes to endothelial cells and
15      enhances their phagocytic activity.
16           Sandstrom et al. (1990b) studied a  group of eight healthy nonsmokers exposed to
17      4.0 ppm  NO2 for 20 min per day (moderate exercise, VE ~ 35 L/min) on alternate days
18      over a 12-day period (seven exposures total).  BAL was performed 2 weeks before the first
19      exposure and 24 h after the last exposure.  The first 20 mL of BAL fluid was treated
20      separately and presumed to represent primarily bronchial cells and secretions.  After NO2
21      exposure, there was a reduction in numbers of macrophages in the bronchoalveolar portion
22      although on a per cell basis alveolar macrophage phagocytic activity was increased.  There
23      were decreased numbers of mast cells in the bronchial portion of lavage fluid.  In addition,
24      there were reduced numbers of T-suppressor, B lymphocytes, and NK cells in the alveolar
25      portion of the BAL fluid compared to the baseline lavage.  These observations contrast with
26      those seen by Sandstrom et al. (1989) after single NO2 exposures,  suggesting some alteration
27      in bronchial and alveolar cell  populations after repeated NO2 exposure.  The most obvious
28      difference between Sandstrom et al. (1990b) and Boushey et al. (1988) is the higher NO2
29      concentration and the longer duration of  the former study.  Further work is necessary to
30      confirm these observations, to  determine the time course of response to repeated exposure
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and to determine the NO2 exposure dose necessary to invoke modification of bronchoalveolar
cell populations.
     The effects of HNO3 vapor exposure (in vivo) have been examined in two recent
studies.  Becker et al.  (1991) exposed five healthy subjects (4 males/1 female) to 200 ftg/m3
of HNO3 vapor for 120 min, including 100 min of moderate exercise.  Bronchoalveolar
lavage performed 18 h postexposure indicated an increase in number of lymphocytes, an
increased concentration of protein in the lavage fluid, and increased phagocytic activity of
macrophages harvested from the exposed lung. These results suggest that HNO3 may cause
lymphocyte infiltration into the lung, stimulate macrophage phagocytosis, and increase
pulmonary epithelial permeability. The absence of markers of tissue damage, (lactate
dehydrogenase [LDH]) suggest that under these exposure conditions HNO3 did  not cause
frank tissue damage.
     Aris et al. (1991) exposed 10 healthy subjects to  500 /ug/m3 HNO3 vapor for 4 h,-
including moderate exercise. Lavage fluid was obtained from bronchial as well as broncho
alveolar washings.  No change in LDH levels were observed as a result of HNO3 exposure.
These investigators found no differences in differential cell counts in the lavage fluid. They
also exposed a different group of subjects to 500  jug/m3 HNO3 plus 0.20 ppm O3 and found
no potentiation of the O3-induced inflammatory response by the addition of HNO3 vapor to
the exposure. Their data suggest that HNO3 does not cause tissue injury, nor does HNO3
alter the inflammatory response typical of O3 exposure.
15.7 EFFECTS OF NITRATES ON HUMAN LUNG FUNCTION
     Five studies have been conducted on human exposure to nitrate aerosols since 1979
(see Table 15-10).  These studies have been discussed in the Acid Aerosol Issues Paper (U.S.
Environmental Protection Agency, 1989).  The only obvious effect was a decrease in Gaw
and in PEFV curves in normal subjects with influenza exposed to 7,000 ^g/m3 of sodium
nitrate (NaNO3) aerosol. This is probably three orders of magnitude (i.e., approximately
1,000 times) above the nitrate concentration that could exist in the ambient air. These studies
indicate that, at least as far as lung function is concerned, there is no present concern for
adverse  effects from current ambient levels of nitrate aerosols.
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     Sackner et al. (1979) studied a diverse group of normal and asthmatic subjects exposed
                                   n
to concentrations reaching 1,000 /*g/m  of NaNO3 for 10 min at rest.., There were no
significant effects on an extensive battery of pulmonary function tests.
     Utell et al.  (1979) studied both normal and asthmatic volunteers exposed to
7,000 jitg/m3 of 0.46 pm  NaNO3 aerosol for 16 min via mouthpiece.  The major health effect
end points measured in their study included Raw, both full and PEFV curves, airway
reactivity to carbachol, and aerosol deposition.  Aerosol deposition as a percentage of inhaled
aerosol averaged about 50% for normals and about 56% for asthmatics; the group differences
were not significant.  The exposure to NaNO3 aerosol was indistinguishable from the control
NaCl exposure in normals. Similarly, there were no effects of NaNO3 exposure in
asthmatics.
     Utell et al.  (1980) subsequently studied 11 subjects with influenza exposed to the same
NaNO3 regimen  as above.  The subjects were initially exposed at the time of illness and then
re-exposed 1,3,  and 6 weeks later.  Aerosol deposition ranged from 45 to 50% over the,four
exposure sessions. All subjects had cough and  fever, and 10 of 11 had viral or immunologic
evidence of acute influenza. Baseline measurements of FVC and FEVj were within normal
limits and did not change throughout the 6-week period. There were small but significant
decreases in Gaw following NaNO3 inhalation but not after NaCl exposure.  This difference
was present during,acute illness and 1 week later, but was not seen at  3 and 6 weeks after
illness.  The decrease in SGaw seen on the initial exposure was accompanied by a decrease in
partial expiratory flow at 40%TLC; this was also observed at the 1 week follow-up exposure.
This study suggests that the presence of an acute viral respiratory tract infection may render
humans more susceptible  to the acute effects of nitrate aerosols. Nevertheless, the
concentration of nitrates used in this exposure study exceed maximum  ambient levels by more
than 100-fold.
     In addition to NaNO3 aerosols,  ammonium nitrate (NH4NO3) exposure has been studied
by Kleinman and associates (1980).  Twenty normal and 19 asthmatic  subjects were exposed
to a nominal 200 jwg/m3 of 1.1 /*m NH4NO3 aerosol. The 2-h exposures included mild,  -
intermittent exercise and were conducted under warm conditions (31 °C, 40% RH).  There
were no significant physiologically meaningful  effects of the NH4NO3 exposure in either
subject group.
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  1           Stacy et al. (1983) also studied the effects of 80 jug/m3 of NH4NO3 in a group of
 2      healthy male adults.  As in the Kleinman et al. (1980) study, there were no changes in lung
 3      function or symptoms.
 4
 5
 6      15.8  CONCLUSIONS AND DISCUSSION
 7           At the beginning of this chapter, a series of questions were posed concerning the
 8      potential biological responses to NO2 exposure in humans.  Some of these questions can be
 9      answered in part using the data presented in this section, others will clearly require additional
10      research.
11           NO2 exposure at sufficiently high  concentrations produces changes in lung function in
12      healthy subjects.  A number of investigators have reported increased airway resistance after
13      exposure to NO2 concentrations exceeding  1 ppm (Beil and Ulmer,  1976; von Nieding et al.,
14      1979; von Nieding and Wagner,  1977; von Nieding et al., 1980).  However, at
15      concentrations of NO2 between 2 and 4 ppm, some investigators have not observed any NO2-
16      induced changes in airway resistance or spirometry (Linn et al.,  1985b; Mohsenin,  1987b;
17      Mohsenin,  1988; Sandstrom et al., 1990a). At NO2 exposure concentrations below 1.0 ppm,
18      there is little if any convincing evidence of change in lung volumes, flow-volume
19      characteristics of the lung, or airways resistance  in healthy subjects.  Nitrogen dioxide is
20      believed to have its primary effect on small airways. However, routine spirometry and
21      airway resistance measurements are not sensitive indicators of small airways function.  Thus,
22      the absence of change in these physiological indicators of large airways function at low NO2
23      concentrations should not be viewed as evidence that NO2 has no effects on lung function.
24      Further developments will be necessary to permit sensitive, reproducible, noninvasive
25      evaluation of small airways, the primary site of NO2 deposition in the lung.
26           Symptoms associated with NO2 exposure in healthy subjects have been limited to
27      detection of the odor of NO2, in some cases at surprisingly low concentrations, less than
28      0.1 ppm (Bylin et  al., 1985).  Few of the studies examined in this review noted a significant
29      increase in respiratory symptoms. Sandstrom et  al. (1990a) noted mild nasopharyngeal
30      irritation after exposure to 4 ppm for 20 min.
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  1           Nitrogen dioxide exposure does result in increased airway responsiveness in normal
  2      subjects exposed to concentrations in excess of 1.0 ppm.  Mohsenin (1987b) and Frampton
  3      et al. (1991) reported an increase in airway responsiveness after exposure to 2.0 and 1.5 ppm
  4      respectively.  Increased airway responsiveness may be associated with airway inflammation.
  5      Repeated bouts of airway inflammation could promote deleterious long-term changes in the
  6      lung such as loss of elasticity and acceleration of age-related changes in lung function.
  7      However, the development of such responses is only speculative, given the present level of
  8      scientific evidence.
  9           Potentially sensitive subjects in the population include children, older adults, patients
10      with asthma or COPD, or individuals who may be unusually sensitive to NO2 for other
11      reasons.  There are insufficient data on children, adolescents or older adults, either healthy or
12      with asthma, to determine their NO2 responsiveness relative to healthy young adults.
13           At the concentrations that may fall within the ambient range (e.g.,  < 1.0 ppm), the
14      effects of NO2 on lung function (i.e., spirometry, airway resistance) in asthmatics have
15      tended to be small. For example,  Bauer et al. (1986a) observed a 4 to 6% decline in FEVj
16      in asthmatics exposed to 0.3 ppm NO2 for 30 min.  Koenig et al. (1988) reported a 4%
17      decrease in  FVC, but no significant change in other spirometry variables, after exposure of
18      adolescent asthmatics to 0.30 ppm NO2.  On the other hand, several other investigators (Avol
19      et al., 1988; Bylin et al., 1985;  Hazucha et al., 1982, 1983; Kleinman et al., 1983; Koenig
20      et al., 1985; Linn et al., 1985b, 1986; Mohsenin, 1987a; Roger et al,, 1990) have not found
21      any significant changes in spirometry or airway resistance of asthmatics exposed to
22      concentrations  < 1.0 ppm. Again, spirometry and airway resistance are not sensitive
23      measures of small airways function, where NO2 is known to be primarily deposited.
24          A second important category of sensitive subjects includes patients with COPD who
25      have shown increased airway resistance after brief exposures to greater than 1.6 ppm NO2
26      (von Nieding et al., 1970, 1971, 1973a) (see Table 4.3-54). In addition, during a longer
27      (4-h) exposure, Morrow  and Utell  (1989) reported decreased (approx.  5%)  FVC in COPD
28      patients exposed to 0.30 ppm. Other investigators (Linn et al., 1985a; Kerr et al., 1979) did
29      not find responses in COPD patients even with exposures to levels as high as 2.0 ppm.  It
30      appears that brief acute exposure to relatively high concentrations of NO2 (> 2 ppm) will
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  1      cause bronchoconstriction in some COPD patients and that these responses may also be
 2      observed with longer exposures to lower concentrations.
 3           An unresolved issue with the current data base is the existence of NO2-induced
 4      pulmonary responses in asthmatics that have been reported at low but not at high
 5      NO2 exposures.  Although small functional responses have been observed in studies from
 6      various laboratories, effects are not consistently present and demonstrating reproducibility of
 7      responses has been difficult, even within the same laboratory.  Furthermore, all responses to
 8      NO2 that have been observed in asthmatics have occurred at concentrations between 0.2 and
 9      0.5 ppm. Changes in lung function or airway reactivity have not been seen even at much
10      higher concentrations (i.e., up to 4 ppm).  There is, at present, no plausible explanation for
11      this apparent lack of a concentration-response relationship. There is a possibility that a
12      portion of the variability in response to NO2 may be attributed to differences in the severity
13      of asthma.  This is a complex issue and is discussed in Appendix A.  In patients with chronic
14      obstructive lung disease, Bauer  et al. (1987) and Morrow  and Utell (1989) have observed
15      decreased lung function (FVC, FEVj) after exposure to 0.30 ppm for  4 h but Linn et al.
16      (1985a) and von Nieding and Wagner (1979) found no effects in COPD patients below 2.0
17      ppm for short duration exposures. It appears that further  work will be necessary  to provide
18      enough information to estimate  the concentration-response relationships for NO2 exposure of
19      asthmatics and COPD patients,  who appear to be the sensitive subpopulations.
20           Li several studies of asthmatics exposed to NO2,. airway responsiveness to a variety of
21      agents has been demonstrated.  However, in many other studies using  similar experimental
22      exposures, there was no significant change in airway responsiveness.  In order to evaluate
23      this apparent dilemma, a meta-analysis was utilized; the approach is described in
24      Section 15.4.  Without regard to the type of airway challenge, NO2 concentration, exposure
25      duration, or other variables, the overall trend was for airway responsiveness to increase (59%
26      of 354 subjects increased). This trend was somewhat more convincing for exposures
27      conducted under nonexercising conditions (69% of 154 subjects increased); indeed the excess
28      positive responses were almost entirely accounted for by exposures conducted under resting
29      conditions.  The implications of this overall trend are unclear and will require further
30      investigations to verify if there is an interaction with exercise-induced  changes in  lung
31      function that may possibly obscure changes in airway  responsiveness due to NO2  exposure.

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  1      Increased airway responsiveness could potentially lead to temporary exacerbation of asthma
  2      leading possibly to increased medication usage or even increased hospital admissions.  The
  3      lowest observed effect level for this response appears to be in the 0.2- to 0.3-ppm range.
  4           Several recent studies have examined the possibility that NO2 could induce a pulmonary
  5    ,  inflammatory response and/or alter immune  system host defenses.  These studies typically
  6    ,  include collection of cells and airways fluids washing from the lung using BAL.  In contrast
  7      to O3 exposure, NO2 does not, at the concentrations studied, induce an increase in
  8      neutrophils or eosinophils, the typical markers of inflammation following O3 exposure.
  9      However,  Sandstrom et al. (1990a) have observed an increase in mast cells and lymphocytes
10      in BAL fluid, which they attribute to an unspecific inflammatory response.  Boushey et al.
11      (1988) have reported an increase in natural killer lymphocytes in BAL fluid.: Macrophage
12      numbers have not been increased by NO2  exposure nor did their ability to,kill virus appear to
13      have been  altered by exposure, although Frampton et al. (1989a) suggested that, in some
14      subjects, macrophage responses may have been impaired.  At present there is no evidence of
15      increased pulmonary epithelial permeability, although this possibility has not been examined
16    ;  systematically. Mucociliary clearance was not altered after NO2 exposure in the one study in
17      which it was measured (Rehn et al., 1982),  Nitrogen dioxide was found to cause a reduction
18      in alpha-1-antiprotease activity in one study  (Mohsem'n and Gee, 1987) but not in another
19      (Johnson et al., 1990).  Following NO2 exposure, Frampton et al. (1989b) found an increase
20      in alpha-2-macroglobulin, a molecule  that has immunoregulatory as well as antiprotease
21      activity.  Immunological responses to  NO2 exposure are just beginning to be elucidated and
22      additional research  will be required to determine whether these responses  have any
23      implications for epidemiologically determined associations between NO2 exposure and
24,   •  increased respiratory tract infections.               -
25           The effects  of repeated NO2 exposure have been examined in two studies (Sandstrom
26      et al., 1990b; Boushey et al., 1988).  Boushey et al. (1988) reported only a slight increase
27      (12%) in circulating lymphocytes and  a possible  increase in natural killer  lymphocytes  after
28      four 2-h exposures  to 0.60 ppm.  There were no detectable changes in inflammatory
29      mediators.  Sandstrom et al.  (1990b),  on the other hand, found decreased numbers of mast
30      cells, macrophages, and lymphocytes in the BAL fluid.  Despite the decreased numbers, the
31      phagocytic activity  of alveolar macrophages  was  enhanced.  These observations suggest that
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 1     host defense responses are different after repeated exposure than after a single acute exposure.
 2     More research appears to be necessary to confirm and expand these observations because of
 3     the important potential connection between altered host defense responses and increased
 4     respiratory infectivity.
 5          In healthy adults, a variety of mixtures of other pollutants with NO2 have been
 6     examined, primarily using spirometry and airway resistance measurements as end points.
 7     In general, NO2 does not cause responses  to other pollutants,  such as O3,  SO2, or particulate
 8     matter, to be increased significantly.  In other words, there is no more than an additive
 9     response when NO2 is included in the pollutant mixture.  However, further investigation of
10     N02 mixtures appears warranted using other biological markers, including measures of
11     epithelial permeability, clearance, airway responsiveness, airway inflammation, 'and measures
12     that are sensitive to changes in small airways function.  In asthmatics, there is a tendency for
13     increased responsiveness to cold air, methacholine, carbachol, and histamine after NO2
14     exposure (see previous discussion).  In one study, asthmatics were also  more responsive to
15     SO2 after a previous exposure to NO2 (Torres and Magnussen, 1990).  In addition to other
16     pollutants, NO2 exposure could potentially enhance (or inhibit) responses to other substances,
17     particularly airborne antigens. In two studies (Ahmed et al.,  1983a; Orehek et al.,  1981) the
18     response to grass pollen inhalation was examined in sensitive subjects after exposure to
19     0.1 ppm NO2 but no significant difference in the response after air and NO2 exposures was
20     observed.  Given the increase in responsiveness to nonantigenic substances such as
21     methacholine, histamine, SO2, or cold air discussed previously, it may  be worthwhile to
22     re-examine this hypothesis using higher  NO2 concentrations or more prolonged exposures.
23          Responses to other NOX species have also been studied.  Nitric oxide does not appear to
24     cause any lung function effects at low concentrations (< 1.0 ppm) either alone (Kagawa,
25     1982)  or combined with NO2 (Kagawa,  1990).  von Nieding et al., (1973b) reported
26     increased airways resistance in subjects exposed to excessively high concentrations
27     (>20  ppm). Responses to HNO3 vapor have been studied in adolescent asthmatics (Koenig
28     et al., 1989a,b).  The results are suggestive of small changes  in lung function but further
29     investigation is needed to confirm these apparent responses to HNO3 vapor.  Nitrates (e.g.,
30     sodium nitrate) have not been found to cause any deleterious effects (Utell et al., 1979,  1980;
31     Kleinman et al., 1980; Stacy et al.,  1983) at levels that might be expected in the atmosphere.

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27
28
29
30
31
Conclusions:
1.   Nitrogen dioxide causes decrements in lung function, particularly increased airway
     resistance in healthy subjects at concentrations exceeding 1.0 ppm.
2.


3.
4.
5.
Nitrogen dioxide exposure results in increased airway responsiveness in healthy subjects
exposed to concentrations exceeding 1.0 ppm for exposure durations of 1 h or longer.

Nitrogen dioxide exposure is associated with cellular inflammatory responses in the
airways that may include increased levels of mast cells and lymphocytes but not
neutrophils and eosinophils.  Changes in some biochemical mediators of inflammation
or enzymes may be altered by NO2 exposure.

Nitrogen dioxide exposure of asthmatics causes in some subjects increased airway
responsiveness to a variety of provocative mediators, including cholinergic and
histaminergic chemicals, SO2, and  cold air. However, the presence of these responses
appears to be influenced by the exposure protocol, particularly whether or not the
exposure includes exercise. However, NO2 concentration-response relationships are not
evident.

Modest decrements in spirometric measures of lung function (3 to 8%) may occur in
asthmatics and COPD patients exposed to NO2 concentrations (0.30 ppm).

Nitric acid exposure may cause some pulmonary  function responses in asthmatics but
other commonly occurring nitrogen oxide species do not appear to cause any function
responses at concentrations expected, even at higher levels than in worst-case scenarios,
in the ambient environment.
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   1      REFERENCES

  2      Abe, M. (1967) Effects of mixed NO2-SO2 gas on human pulmonary functions: effects of air pollution on the
  3             human body. Bull. Tokyo Med. Dent. Univ. 14: 415-433.
  4
  5      Adams, W. C.; Brookes, K. A.; Schelegle,  E. S. (1987) Effects of NO2 alone and in combination with O3 on
  6             young men and women. J. Appl. Physiol. 62: 1698-1704.
  7
  8      Ahmed, T.; Marchette, B.; Danta, I.; Birch, S.; Dougherty, R. L.; Schreck, R.; Sackner, M. A. (1982) Effect
  9             of 0.1 ppm NO2 on bronchial reactivity in normals and subjects with bronchial asthma. Am. Rev. Respir.
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54


         August 1991                                  15_94       DRAFT-DO NOT QUOTE OR CITE

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       August 1991
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                  APPENDIX ISA. SEVERITY OF ASTHMA

     A major issue in the evaluation of human clinical studies involving asthmatics is the
variability in response between, and even within, laboratories.  In the absence of significant
differences in exposure protocol or exposure dose, the explanation most often invoked to
explain differences in response is that the severity of disease and/or disease characteristics in
one subject group may have been different than the other.  There are a large number of
physiological tests which may be used to characterize the severity of asthma, although no
single test appears to be definitive.  There  are also numerous clinical distinctions between the
various categories of asthma that are discussed below. The premise presented here suggests
that since asthma is a clinically defined disease, the factors used to characterize subjects
should be based on clinical and physiological information.
     Asthma is a disease "characterized by increased responsiveness of the tracheobronchial
tree to a variety of stimuli" (American Thoracic Society, 1987; McFadden, 1988, 1991).
A characteristic feature of asthma is widespread narrowing of the airways that changes in
severity either spontaneously or with treatment. There is a broad range of severity, from
"mild and almost undetectable to severe and unremitting",  and  considerable heterogeneity of
clinical features that  include, most commonly, wheezing, dyspnea, and cough.  Fatal asthma
is also characterized  by mucosal edema, inflammation of the airways, and mucus plugging of
the peripheral airways.  Even mild asthma is associated with eosinophilic inflammation,
epithelial damage,  degranulation of mast cells, and thickening of the subepithelial basement
membrane (Beasley et al.,  1989; Laitinen et al.,  1985; Wardlaw et al., 1988).  Although
there have been numerous attempts to precisely define asthma,  Bates (1990) suggests that
"endless attempts to  refine linguistic definition are misplaced."
      Prevalence:  The prevalence of asthma in the U.S. adult population ranges from 2 to
4% (McWhorter et al., 1989); a point prevalence rate of 2.6% was estimated from the
National Health and  Nutrition Examination Survey I  (NHANES) survey.  Based on data from
National Health and  Nutrition Examination Survey II, Schwartz et al. (1990) reported a
prevalence rate for asthma in children less than 12 years of age at 4%; 3% among whites and
7.2%  among blacks.  Prevalence was higher among males, older children (age  8-11), and
urban residents.
                                         15A-1

-------
      Categorization:  Scadding (1985) discussed extensively the categorization and degree of
 severity of asthma.  He defined two basic classes of asthmatics which he referred to as
 extrinsic and cryptogenic.  The term extrinsic, or allergic, implies that an external agent (such
 as ragweed pollen) is responsible for the antigen-antibody reaction which triggers the cascade
 of events responsible for airway  narrowing.  The term cryptogenic,  or intrinsic,  implies .there
 is no known environmental or antigen-antibody reaction that has been identified to have
 precipitated the disease. Scadding (1985)  proposed further subdivision of these two basic
 qualitative classifications into extrinsic atopic (the largest category), extrinsic nonatopic,
 cryptogenic intrinsic, and cryptogenic unspecified.  Diagnostic features of the various
 categories of asthma suggested by Scadding (1985) are presented in  Table 15A-1.
      Classes of asthmatics:
            a.  Extrinsic
               i.  Atopic (IgE)
              ii.  Nonatopic (non-IgE)
            b.  Cryptogenic
               i.  Intrinsic
              ii.  Unspecified

      Severity of Disease:  Each qualitative class is then graded in severity (Table 15A-2)
 according to the clinical course of the disease: mild (controlled by bronchodilators and
 avoidance of precipitating factors, does not interfere with normal activities); moderate
 (requires steroids, occasionally interferes with activities);  and severe (history of life-
 threatening episodes, seriously interferes with activities).  The temporal course of the asthma
 is also described (Table 15A-3) as episodic (episodes of asthma interspersed with symptom-
 free periods) or persistent (persistent symptoms with periodic exacerbations).
      There is a group of mild extrinsic atopic asthmatics  (often participants in clinical
 exposure studies) who rarely use  medication; have infrequent episodes of mild bronchospasm
 that usually do  not require medical intervention or medication, although they may
 occasionally use a bronchodilator; and have normal lung function. Perhaps these individuals
 with near normal function should be classified as minimal asthmatics.  There appears also to
be some difference of opinion as  to what precise criteria are required before an individual is
classified as asthmatic.  For example, many (about 40%) individuals with allergic rhinitis
                                          15A-2

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15A-3
DRAFT-DO NOT QUOTE OR CITE

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                 TABLE 15A-2. CLINICAL SEVERITY OF ASTHMA
 Mild                                    Controlled by bronchodilators and
                                          avoidance of known precipitating
                                          factors; does not interfere with
                                          normal activities

 Moderate                                Occasionally interferes with normal
                                          activities; requires use of systemic
                                          corticosteroids in treatment

 Severe                                   Seriously interferes with normal
                                          activities; life-threatening episodes
                                          (status asthmaticus)
                 TABLE 15A-3. TEMPORAL COURSE OF ASTHMA
 Episodic                                     Episodes of wheezy dyspnea with
                                              symptom-free intervals; frequency
                                              and severity of episodes may be
                                              indicated

 Persistent                                    Persistent symptoms with episodic
                                              exacerbations; severity both of
                                              persistent symptoms and of episodes
                                              may be indicated
 Other forms of chronic bronchopulmonary disease may be present, notably bronchial
 hypersecretion, persistent airways obstruction, and emphysema and may be partly
 responsible for persistent symptoms; if so, additional diagnostic terms, as appropriate,
 should be added
may experience exercise-induced bronchospasm (Voy,  1986).  Furthermore, allergic subjects

may be more sensitive than normal subjects to SO2-induced bronchoconstriction (Koenig

et al., 1987). However, the nonspecific airway reactivity of these individuals may be within

the normal range (McDonnell et al.,  1987).  (Note, however, that McDonnell et al. [1987]

screened their subjects so that only subjects with allergic rhinitis were selected; none of the
subjects had exercise-induced bronchospasm.)
                                        15A-4

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      In general, the asthmatics who participate in human clinical studies come from the
group of asthmatics who would fall into the category of mild extrinsic atopic asthma with an
episodic time course. In most cases the disease is sufficiently mild or outside the normal
allergy  season that the patient may go without medication altogether or can discontinue it for
brief periods.
      Two clinical features which can easily be ascertained are the temporal nature of the
disease  (i.e., frequency of episodes of bronchospasm) and the type, dosage and frequency of
medication use (i.e., frequency of use of inhaled or oral bronchodilators or steroids). These
could be documented by a diary and intermittent measurement of peak flow.  In order to
differentiate between the minimal and mild severity group, several criteria could be used
including  frequency of episodes (bronchospasm and/or medication) and normality of function
(FEVj/FVC ratio, SRaw, Peak Flow). An alternative solution may be to provide a detailed
clinical  and physiological characterization of individual subjects and allow the reader to
judge.
      From the standpoint of interpreting responses to a specific pollutant, it may be useful
to work with a more homogeneous group of asthmatics, defined as indicated above.  Though
this might result in increased sensitivity to detection of adverse responses, it would,  of
course,  reduce the size of the population to  which the findings could be extrapolated.
Nevertheless,  more precise definition of study populations would facilitate comparisons
between different studies.
      Certain basic anthropomorphic information is necessary to characterize any subject
population, minimally including height, weight, age, and gender.  However, in the case of
asthmatics, other useful quantitative information can be valuable in comparing subjects in
various studies.
      In addition to evaluation of baseline lung function and frequency of episodes (as
medication use, frequency of attacks, age at onset of disease, family history of atopy,
frequency of emergency treatment, hospitalization, or all of the above, possibly using a diary
and peak  flow meter), the following information may be useful in evaluating studies of
asthmatics exposed to inhaled materials:
                                          15A-5

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       1.    Nonspecific bronchial reactivity (methacholine or histamine)
       2.    Evaluation of reversibility of obstruction with beta agonist
       3.    General classes of medication and frequency of use
       4.    Levels of serum Immunoglobulin E (IgE)
       5.    Number of positive skin tests to regionally/seasonally common allergens
       6.    Known precipitating factors; seasonal variability
       7.    Response to standard exercise challenge
       8.    Duration of disease

Many of these factors and suggested categories to quantitate them are included in a
questionnaire and survey (Table 15A-4) developed by Howard Kehrl, M.D., of the Clinical
Research Branch, Health Effects  Research Laboratory of the U.S. Environmental Protection
Agency. It is recognized that it may,  at times, be difficult to persuade journal editors to
include an extensive list of subject  (patient) characteristics; but that does not preclude
obtaining the information and making  it available to interested parties or submitting the data
to a data repository.
      Many of the more recently published studies have carefully evaluated the clinical and
physiological characteristics of asthmatics (and others) used as subjects. However, the
subjects have not been adequately characterized in some studies and the basis of the patient
classifications is unclear.  Table  15-A-4 provides a format for collecting some of the
important clinical information. The absence of adequate subject characterization significantly
inhibits cross-study comparisons  that are needed  to evaluate differences between  studies.
                                          15A-6

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                                    TABLE 15A-4.
 ASTHMA SYMPTOMS

 1.  Spontaneous Wheezing or Chest Tightness or Shortness of Breath
    a.  never - none in last year
    b.  rare - less than once a month
       infrequent - less than 5 times a month
c.
    d.  frequent - more than once a week but not every day
    e.  daily
    f.  nocturnal or early a.m.
2.  Cough - frequency
   a.  never
   b.  with chest infections
       usually only with wheezing or chest tightness
       occasional, usually upon awakening
       frequent, often nocturnal and interferes with sleep
      daily, interferes with sleep and activities of everyday living
c.
d.
e.
f.
    Cough - productive of sputum
   a. never
   b. only with chest infection
      rare - 1 to 5 times per month
      occasionally - 6 to 15 times per month
      frequently or almost daily
c.
d.
e.
FACTOR PROVOKED ASTHMA

1. Exercise-induced Wheezing or Chest Tightness
   a.  all seasons with
      1.  mild exercise
      2.  moderate exercise
      3.  heavy exercise
   b.  only with cold air
      1.  mild exercise
      2.  modest exercise
      3.  rigorous exercise
   c.  abates with sustained exercise
      yes	   no	
                                       15A-7

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                              TABLE 15A-4.  (cont'd)
2. Avoidance of
   a. aspirin -   (i)  rhinitis
                (ii)   facial flushing
               (iii)   wheezing
   b. NSAID's
   c. sulfites
   d. pets
   e. environmental tobacco smoke
   f. other	(foods, perfumes, odors, etc.)


3. Seasonal or Allergen Provoked Asthma
   yes	  no	
   a. concommittant allergen-induced hay fever
   yes	  no	
   b. percent component of symptoms due to allergen exposure
      1.  100%
      2.  50 to 100%
      3.  20 to 49%
      4.  <20%
   c. allergen exposure increases asthma activity
      yes	  no	
   d.  allergen  exposure increases susceptibility to chest colds
      yes	  no	


4.  Infection Induced Asthma Flare
   a. colds/URI - nasal congestion or discharge, sore throat sinusitis
      1.  cause wheezing
      2.  frequency
          a.  almost never; less than once a year
          b.  infrequent; no more than twice a year
          c.  occasional; 3-6 times per year
          d.  frequent; more than 6 times a year
   b. LRI - chest cold, bronchitis, pneumonia
      1. usually preceded by URI
         yes	  no	
      2. cause wheezing
         yes	  no	
      3. frequency
         a.  none in last 5 years
         b. almost never; less than once a year
         c.  infrequent; no more than twice a year
         d. occasional; 3 to 6 times per year
         e.  frequent; more than 6 times a year	
                                        15A-8

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                              TABLE 15A-4.  (cont'd)
MEDICATION

1.  Steroid Burst and Taper within Past 3 Years (oral corticosteroids)
   a. short, tapering course
   b. chronic use

2.  Theophyline Usage
   a. daily
   b. intermittent

3.  Beta Agonist Inhaler
   a. routine daily usage
   b. usage more than 5 times per week
   c. from 1 to 5 times per week
   d. occasional usage
   e. only for EIB (exercise-induced bronchoconstriction)

4.  Inhaled Corticosteroids
   a. intermittent use
   b. chronic use

5.  Other Medications (e.g., chromolyn sodium)
PHYSICIAN DIRECTED TREATMENT

1.  Insured or Student Health
   yes	  no	

2.  Hospitalization within Last 2 Years

3.  Emergency Treatments (ER or doctor's office)
   a.  more than once a month
   b.  more than once a year but less than once a month
   c.  less than once a year
   d.  none in last 3 years

4.  Routine physician contacts (doctor's office)
   a.  visits more than once a month
   b.  more than once a year but less than once a month
   c.  less than once a year
   d.  none in last 3 years
                                       15A-9

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                                                                                        1
                            TABLE 15A-4. (cont'd)
IMMUNOLOGY PARAMETERS

1. Eosinophil Count (cells x 104/nl)
  a. less than 200
  b. 200-400
  c. 401-600
  d. 600-900
  e. more than 900
  f. Note: Allergy season? (Y/N)  Recent steroid use? (Y/N)

2. Serum IgE (lU/ml)
   a.  less than 10
   b.  10-100
   c.  101-400
   d.  401-800
   e.  more than 800

3. Skin Tests (number positive)
   a.  less than 3
   b.  3-5
   c.  6-10
   d.  more than 10
PULMONARY FUNCTION

1. Spirometry - FEV^FVC
   a.  more than 80%
   b.  72-79%
   c.  64-71%
   d.  55-63%
   e.  less than 55%
                                   -i.
   Plethysmography - SRaw (cm H2O»L
     Male             Female
   a.  <4.0                  <5.0
   b.  4.0 to 7.5              5.0 to 8.0
   c.  7.5 to 12.0            8.0 to 12.0
   d.  >12.0                >12.0
                                   15A-10

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                                TABLE 15A-4. (cont'd)
 AIRWAY RESPONSIVENESS

 1.  Airway Narrowing Triggered by (Note provocative dose, if known)
    a. Methacholine
    b. Histamine
    c. Cold/Dry Air
    d. Exercise
    e. Sulfur Dioxide

 2.  Nonspecific Airway Reactivity
    a. Normal range
    b.  < 10th percentile of normal range
    c. 75th-100th percentile for asthmatic range
    d. 25th-50th percentile for asthmatic range
    e. < 25th percentile for asthmatic range

NSAID =  Non-steroidal anti-inflammatory drugs.
URI   =  Upper respiratory infection.
LRI   =  Lower respiratory infection.
                                        ISA-11

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REFERENCES

American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary
       disease and asthma. Chapter 2. Asthma. Am. Rev.  Respir.  Dis. 136:228.

Bates, D. V. Workshop Summary. Chest 98(#5, Suppl): 251S, 1990.

Koenig, J. Q.; Marshall, S. G.; Horike, M.; Shapiro, G. G.; Furukawa, C. T.; Bierman, C. W.; Pierson,
       W. E. (1987) The effects of albuterol on sulfur dioxide-induced bronchoconstriction in allergic
       adolescents. J. Allergy Clin. Immunol. 79: 54-58.

Koenig, J. Q.; Morgan, M. S.; Horike, M.; Pierson, W. E. (1985) The effects of sulfur oxides on nasal and
       lung function in adolescents with extrinsic asthma. J. Allergy Clin. Immunol. 76: 813-818.

McDonnell, W. F.;  Horstman, D. H.; Abdul-Salaam, S.; Raggio, L. J.; Green, J. A. (1987) The respiratory
       responses of subjects with allergic rhinitis  to ozone exposure and their relationship to nonspecific airway
       reactivity. Toxicol. Ind. Health 3: 507-517.

McFadden, E. R. Asthma:  General features, pathogenesis and pathophysiology. Chap 79 in Fishman, A. P.
       Pulmonary Diseases and Disorders,  New York: McGraw-Hill, 19,88. p. 1295.

McFadden, E. R. Airway responsivity and chronic obstructive lung disease. Chap 10 in Cherniack, N.S.'
       Chronic Obstructive Pulmonary Disease, Philadelphia:  Saunders,  1991. pp. 90-96.

McWhorter, W. P.; Polis,  M. A.; Kaslow,  R. A.  (1989) Occurrence, predictors,  and consequences of adult
       asthma in NHANESI and follow-up survey. Am. Rev.  Respir. Dis. 139: 721-724.

Scadding, J. G. (1985) Definition and clinical categorization. In: Weiss, E. B.; Segal, M. S.;  Stein, M., eds.
       Bronchial asthma: mechanisms and therapeutics. 2nd ed.  Boston, MA: Little, Brown and Company;
       pp. 3-13.                 ,

Schwartz, J.; Gold,  D.; Dockery, D. W.; Weiss, S. T.; Speizer, F. E. (1990) Predictors of asthma and
       persistent wheeze in a national sample of children in the United States: association with social class,
       perinatal events, and race. Am. Rev. Respir. Dis.  142: 555-562.

Voy, R. O. (1986) The U. S.  Olympic committee  experience with exercise-induced bronchospasm, 1984. Med.
       Sci. Sports Exercise 18: 328-330.
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             16.   HEALTH EFFECTS ASSOCIATED
        WITH EXPOSURE TO NITROGEN DIOXIDE
16.1  INTRODUCTION
     This chapter concisely summarizes and integrates key information and conclusions from
preceding chapters into a coherent framework or perspective upon which to base
interpretations concerning human health risks posed by ambient or near-ambient levels of
NO2 in the United States.  Toward this end, the chapter is organized into several sections,
each of which discusses one or more major components of an overall health risk evaluation:
(1) ambient and indoor NO2 levels and related exposure aspects; (2) qualitative and
quantitative characterization of key health effects of NO2 and their biological bases; and
(3) identification of population groups potentially at enhanced risk for health effects
associated with NO2 exposure.
16.2  AMBIENT AND INDOOR NITROGEN DIOXIDE LEVELS
     In urban areas, hourly NO2 patterns at fixed-site, ambient air monitors often show a
bimodal pattern of morning and evening peaks, related to motor vehicular traffic patterns,
superimposed on a lower baseline level. Sites affected by large stationary sources of NO2
(or NO that rapidly  converts to NO2) are often characterized by short episodes at relatively
high concentrations.
     The highest hourly and annual ambient NO2 levels are reported from stations in
California.  The seasonal patterns at California stations are usually quite marked and reach
their highest levels through the fall and winter months, whereas stations elsewhere in the
U.S. usually have less prominent seasonal patterns and may peak in the winter, in the
summer, or contain  little discernable variation. One-hour NO2 values can exceed 0.2 ppm;
but, in 1988, only 16 stations (12 in California) reported an apparently credible second high
1-h value greater than 0.2 ppm.  Since at least 98% of 1-h values at most stations are below
0.1 ppm,  such values above 0.2 ppm are quite rare excursions.
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 1          Since 1980, the U.S. nationwide mean annual-average level among reporting NO2
 2     stations has been consistently below 0.03 ppm, with no significant trend evident. For
 3     103 Metropolitan Statistical Areas reporting a valid year's data for at least one station in 1988
 4     and/or 1989, annual averages ranged from 0.007 to 0.061 ppm.  The only recently measured
 5     exceedances of the current annual standard, 0.053 ppm, have occurred at stations in southern
 6     California.
 7          Most people, however, spend a significant portion of their  time indoors.  This can result
 8     in increased exposure, depending on the presence and use of indoor sources (e;g., gas stoves,
 9     kerosene heaters, and unvented gas space heaters) or reduced exposure, depending on the
10     absence of such sources and on the tightness of home construction and the use of air
11     conditioning and other building features which affect the degree  of penetration of outdoor
12     NO2 into buildings. Although the data base on peak hourly concentrations in homes With and
13     without indoor sources  is much smaller than for 24-h and weekly averages, several studies
14     (Wade et al., 1975; Hollowell and Traynor, 1978; Hollowell et  al., 1980) report peak hourly
15     NO2 values in gas stove kitchens in the range of 0.1  to 1.2 ppm.  Modeling efforts  suggest
16     that 1-h NO2 concentrations are commonly 0.15 ppm or greater  during cooking periods in gas
17     stove homes (Sexton etal., 1983).
18          Several studies have examined the issue of human exposure to NO2 and the relationship
19     of indoor/outdoor air quality for occupants of homes with and without significant indoor
20     sources of NO2 (Quackenboss et al., 1986; Sexton et al., 1983;  Colome et al.,  1987; and
21     Leaderer et al., 1987).  Quackenboss et al. (1986) found that in the winter in Portage, WI,
22     indoor weekly average  NO2 concentrations were 3.2  times higher that outdoor NO2 levels in
23     gas stove homes, while indoor levels were 0.6 times outdoor NO2 levels in electric stove
24     homes during the same period.  The fact that indoor  NO2 levels in electric stove homes were
25     below measured outdoor levels may be due to chemical reactions of NO2 with indoor
26     surfaces.  Given the large amount of time spent at home by most subjects, Quackenboss et al.
27     found relatively high correlations between measurements of indoor NO2 concentrations and
28     total personal exposure in gas stove homes (r = 0.85 summer and 0.87 winter) and to a
29     lesser extent in electric stove homes (r = 0.68 summer and 0.61 winter).  Correlation
30     between outdoor NO2 levels and total personal exposure is less in the summer (r =  0.55 for
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gas stove homes and r = 0.68 for electric stove homes) and much lower in the winter
(r = 0.20 and 0.28, respectively).
     In contrast, Colome et al. (1987), in a study of over 600 randomly sampled residences
in southern, California, report that outdoor concentrations of NO2 .are found to be the single
most important determinant of average indoor levels of NO2 in southern California. Outdoor
NO2 levels account for between 15 to 40% of the variation in indoor concentrations in this
study.  Based on the regression analysis of data from multiple homes, indoor/outdoor ratios
varied  from 0.46 to 1.00, depending on the season of the year.
     In most regions of the country, except southern California, total personal exposure to
elevated NO2 concentrations appears to be dominated by the existence of indoor sources of
NO2 (i.e., gas stoves, kerosene heaters, etc.). Total personal exposure for residents of
electric stove homes (approximately 50% of the U.S. population) is expected to be
significantly lower than levels observed at outdoor fixed monitoring stations  (Sexton et al.,
1983).
16.3  KEY HEALTH EFFECTS OF NO2
     This section concisely discusses below two key types of health effects that are of most
concern at ambient or near-ambient concentrations of NO2: (1) increases in airway
responsiveness in response to acute, short-term exposures of asthmatic individuals; and
(2) increased occurrence of respiratory illness among children associated with longer-term
exposures to NO2. A third category of NO2 effects,  emphysema, is also noted below but
appears to be of major concern with exposures to  much higher  than ambient levels of NO2.

16.3.1  Airway Reactivity in Asthmatics and Short-Term  (1-3 h) Exposure to
         N03
    . Subjects with asthma who as a group have airway hyperresponsiveness to a variety of
chemical and physical stimuli are considered one of the potentially most NO2-responsive
groups in the population.  The physiological end point which, to date, appears to be the most
sensitive indicator of response to NO2 in asthmatics is a change in airway responsiveness or
reactivity. Airway inhalation challenge tests are used to evaluate the "responsiveness" of a
       August 1991.
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 1     subject's airways to inhaled materials.  To test for the degree of airway responsiveness, a
 2     pharmacologically active chemical (such as histamine, methachloine, or carbachol) that causes
 3     constriction of the airways is used. Responses are usually measured by evaluating changes in
 4     airway resistance or spirometry after each dose of the challenge is administered.  Airway
 5     hyperresponsiveness is an abnormal degree of airway narrowing,  caused primarily by airway
 6     smooth muscle shortening in response to nonspecific stimuli. An extensive discussion of such
 7     responses is presented in Chapter 15.
 8          Asthma is defined  as a disease "characterized by increased responsiveness of the
 9     tracheobronchial tree to  a variety of stimuli"  (American Thoracic Society, 1987;  McFadden,
10     1988).  A characteristic  feature of asthma is widespread narrowing of the airways that
11     changes in severity either spontaneously or with treatment.  There is a broad range of
12     severity, from "mild and almost undetectable to severe and unremitting," and considerable
13     heterogeneity of clinical features that most commonly include wheezing, dyspnea, and cough.
14     Even rnUd asthma is associated with eosinophilic inflammation, epithelial damage,
15     degranulation of mast cells, and thickening of the subepithelial basement membrane (Beasley
16     et al., 1989; LaMnen et al., 1985; Wardlaw et al.,  1988). Asthmatics as a group are
17     significantly more responsive than healthy normal subjects to a variety of airway challenges.
18     The differences in airway responsiveness may span  several orders of magnitude (at least
19     100 fold) between normal and  asthmatic individuals (O'Connor et al.,  1987).  Despite the
20     absence of airway hyperresponsiveness in some asthmatics and the presence  of airway
21     hyperresponsiveness in some non-asthmatics (Pattemore et al.,  1990), there is  a correlation
22     between increased asthma symptoms or increased medication usage and increased airways
23     responsiveness (Britton et al.,  1988).
24           The prevalence of asthma in the U.S. adult population  ranges from 2 to 4%
25     (McWhorter et al.,  1989); a point prevalence rate of 2.6% was estimated from the
26     NHANES I survey.  Based on data from the more recent NHANES II survey, Schwartz et al.
27     (1990)  reported a prevalence rate for asthma in children less than 12 years of age at 4%; 3%
28     among whites and 7.2% among blacks. Prevalence was higher among males,  older children
29     (age 8-11), and urban residents.
30           At concentrations below  1.0 ppm NO2, there is little (if any) convincing evidence of
31     lung function decrements or changes in airway responsiveness in healthy individuals.  There

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29
30
31
 is some evidence that acute exposure to NO2 may cause an increase in airway responsiveness
 in asthmatics. This response has been observed only at relatively low NO2 concentrations,
 mostly within the range of 0.20 to 0.30 ppm NO2, the concentration range of concern within
 the ambient environment.  A meta-analysis of data on more than 300 asthmatics
 experimentally exposed to NO2 indicates a slight excess increase of airway responsiveness
 following NO2 exposure (see Chapter 15).  In the concentration range between 0.20 and 0.30
 ppm, the excess  increase in airway responsiveness was attributable to subjects exposed to
 NO2 ,at rest.  The change in nonspecific (i.e., not to specific allergens) airway responsiveness
 may reflect increased NO2-induced permeability of the airway epithelium leading to increased
 access of the provocative agent to airway receptors, release of local mediators of
 inflammation, or alterations in airway smooth muscle tone.  Since NO2 does not appear to
 cause airway inflammation  at these levels and the increase in airway responsiveness appears
 to be fully reversible,  the implications of the observed increase in responsiveness are unclear.
 Although it is conceivable that increased nonspecific airway responsiveness caused by NO2
 could lead to increased responses to a specific antigen, there is presently  no plausible
 evidence to support this hypothesis.  It is also possible that persistence of airway
 hyperresponsiveness may be associated with ah accelerated rate of decline in pulmonary
 function with age (O'Connor et al., 1987).
      An unresolved issue with the current data base is the existence of NO2-induced
 pulmonary responses in asthmatics that have been reported at low, but not at high, NO2
 concentrations. Although small changes in spirometry or airway resistance  have been
 observed in studies from various laboratories,  effects are not consistently  present and
 demonstrating reproducibility of responses has been difficult, even within the same
 laboratory.  Furthermore, most responses to NO2 that have been observed in asthmatics have
 occurred at concentrations between 0.2 and 0.5 ppm.  Changes in lung function or airway
 responsiveness have not been seen even at much higher concentrations (i.e., up to 4 ppm).
 There is, at present, no plausible explanation for this apparent lack of a concentration-
response relationship for both airway responsiveness and pulmonary  function changes.
     Controlled human exposure studies are limited to  acute fully reversible functional and/or
symptomatic responses.  This may in many cases limit  the magnitude of expected responses
and hence the statistical significance of responses in studies with small numbers of subjects.
       August 1991
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 1      Exposures seldom last longer than one to two weeks and rarely longer than 8 h/day.  These
 2      data, therefore, are primarily useful in evaluation of short-term NO2-induced. health, effects.
 3                                                        .,-.•,..',-•
 4      16.3.2  Respiratory Morbidity in Children Associated with Exposure to NO2
 5           The effects of NO2 on  respiratory illness and the factors determining occurrence and
 6      severity are important public health concerns because of the potential for exposure to NO2
 7      and because childhood respiratory illness is common (Samet et al., 1983; Samet and Utell,
 8      1990).  This takes on added  importance  since recurrent childhood respiratory illness may be a
 9      risk factor for later susceptibility to lung damage (Glezen, 1989; Samet et al., 1983; Gold
10      etal., 1989).
11           The discussion of epidemiological findings in Chapter  14 indicates that the combined
12      evidence is supportive for the effects  of  exposure to NO2 on respiratory disease in children
13      under 12 years of age.  The  studies were evaluated for several key factors, including:
14      (1) measurement error in exposure, (2) misclassification of the health outcome, (3) selection
15      bias, (4) adjustment for covariates, (5) publication bias, (6)  internal consistency, and
16      (7) plausibility of the effect based on other evidence. The health outcome should be an
17      outcome for which there is good reason  to suspect that NO2 exposure has an effect. The
18      health outcome measure considered was  lower respiratory illness, which is typically attributed
19      to infectious disease, probably of viral origin.  Symptoms evaluated commonly consisted of
20      cough, wheeze, colds going  to chest, chronic phlegm and bronchitis. Each study was
21      reviewed with special attention given to  the factors just discussed.  Those studies which most
22      appropriately address these factors provide stronger bases for conclusions to be drawn.
23      Consistency between studies  indicates the level of the strength of the total data base.
24           The epidemiological studies generally provide some evidence that repeated NO2
25      exposure increases respiratory illness in  children, although many reported non-statistically
26      significant results.  Melia et  al.  (1977) first reported on a survey of children in randomly
27      selected areas of England and Scotland,  using the presence of a gas stove as a measure of
28      NO2 exposure.  An EPA reanalysis of that data yields an estimated odds ratio of 1.31 for the
29      presence of symptoms of respiratory illness.  The cross-sectional study of Melia et al. (1979)
30      also found that the presence  of a gas  stove was associated with increased risk of respiratory
31      disease.  The odds ratio was 1.24 with 95%  confidence limits of 1.09 to 1.42.  Melia et al.

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 1  '--•  (1980) described the results of a third study of respiratory symptoms in children aged six to
 2      seven in northern England.  Multiple logistic regression analysis of the data presented by
 3      Melia et al. (1980) showed a significant increase in symptoms as a function of bedroom NO2
 4      levels.  Melia et al. (1982), reporting on a fourth study of children in England, found that a
 5      multiple logistic regression analysis of these data was not statistically significant, although the
 6    .  symptoms were positively related to NO2 exposure.  However, an EPA reanalysis of the
 7      Melia et al. (1982) results suggest that an increase of 30 /xg/m3 (0.016 ppm) in bedroom NO2
 8      levels yields an 11 % increase in the odds of respiratory illness.  In a final Melia et al.  (1983)
 9      study, infants under 1  year of age were examined, but no relationship was found between
10      type of fuel used for cooking and the prevalence of respiratory symptoms.
11           An analysis of the Six City studies reported by Ware et al. (1984) estimated an
12      unadjusted odds ratio of 1.08  (95% confidence limits of 0.97 to 1.19) for an index of lower
13      respiratory illness associated with gas stove use.  Other indicators such as bronchitis, cough,
14      and wheeze did not show any increased incidence.  Neas et al. (1990, 1991) analyzed a
15      different cohort enrolled later in the Six City studies, used a different symptom questionnaire,
16      and made indoor NO2 measurements for all subjects. They found evidence for increased
17      respiratory disease, with an estimated odds ratio of 1.47 (95% confidence limits of 1.17 to
18      1,86) at an exposure of 31 jug/m3  (0.016 ppm).
19           Ogston et al.  (1985) studied respiratory disease in 1-year-olds in the Tayside region of
20      northern Scotland.  The presence of a gas stove yielded an odds ratio of 1.14, with 95%
21      confidence limits of 0.86 to 1.50.  Ekwo et al. (1983) studied respiratory symptoms  in
22      relation to gas stove use in Iowa City, I A. Gas stove use provided an odds ratio of  2.4 for
23      hospitalization for chest illness before age 2, and 1.1 for chest congestion and phlegm with
24      colds. Dijkstra et al. (1990) studied the effects of indoor factors on respiratory health of
25      children in The Netherlands.  A logistic regression analysis yielded an odds ratio of  0.94 with
26      95%, confidence limits of 0.66 to 1.33, thus showing no evidence of an increase in
27      respiratory disease  with increasing NO2 exposure. Keller et al. (1979) also did not find any
28 ,     statistically  significant changes in respiratory disease associated with gas stove use, but the
29      unadjusted estimated odds ratio for lower respiratory illness was 1.10, with  95% confidence
30      limits of 0.74 to 1.54.
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   1           Many of the above studies suggest an increase in respiratory symptoms in children from
  2      exposure to levels of NO2 occurring in homes with gas stoves as compared to homes with
  3      electric stoves, but the reported associations in the majority of the studies did not reach
  4      statistical significance. The overall consistency of these studies was examined and the
  5      evidence synthesized in a quantitative meta-analysis carried out be EPA (Hasselblad  and
  6      Kotchmar, submitted).
  7           The studies used in the EPA meta-analysis employed different health end point
  8      indicators.   In order to compare these studies, a standard end point was defined, and then
  9      each study was compared with this standard end point.  For purposes of discussion,  the end
 10      point chosen was the presence of lower respiratory illness in children aged 12 or under.  It
 11      was assumed that the relative odds of developing lower respiratory illness is similar across
 12      this age range as a function of NO2 exposure, even though the actual rates may  not be
 13      (a common assumption in many analyses). The goal was to estimate the odds ratio
 14      corresponding to an increase of 30 jug/m3 (0.016 ppm) in NO2 exposure. This is
 15      approximately the increase seen as a result of gas stove use as compared with electric stove
 16     use in both the United States and England.
 17          An attempt was made to include as many studies as possible. The criteria  for inclusion
 18     were:  (1) the health end  point measured must be reasonably close to the standard end point;
 19     (2) exposure differences must exist and some estimate of exposure must be available; and
20     (3) an odds ratio for a specified exposure must have been calculated, or data presented so that
21     it can be calculated.
22          Graphs of the odds ratio from each study are presented in Figure 16-1.  Each curve can
23     be given one of three interpretations: (1) the normal approximation to the likelihood of the
24     logarithm of the odds ratio; (2) a distribution such that the 0.025 percentile and the
25     0.975 percentile points of the distribution are the 95% confidence limits of the estimated odds
26     ratio;  and (3) the posterior for the odds ratio for a particular study given a flat prior on the
27     log-odds ratio.
28           Two methods of combining evidence were employed in the EPA meta-analysis,  one
29      being  the Bayesian method described by Eddy (1989) and Eddy et al. (1990a,b).   The result
30      of the analysis is a distribution for estimates of the location of the true value of the odds
31      ratio.  This  can be done separately for each study or for the combination of all studies, and

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           li
           s-1
§.4
                                                                COMBINED (fixed)
                                                                 COMBINED (random)
                                                Mefiaetal. (1979)

                                                Meaaetal.fl977)
                                     Ware etal. (1984)
                                  EkwoetaI.(19S3)
                                  Keller etal. (1979)
                                                Ogsstonetal. (19S5)
                                                Mela etal. (1982)
   D)jkstraetal.(1990)

Uellaetal.(1983)
                                                                          Mala et si. (1960)
                                             Odds Radio
      Figure 16-1.  U.S. Environmental Protection Agency meta-analysis of epidemiologic
                    studies of NO2 exposure effects on respiratory disease in children
                    <12 years old. Each curve can be treated as a likelihood function or
                    posterior probability distribution. K treated as a likelihood function, the
                    95% confidence limits for the odds ratio can be calculated as those two
                    points on the horizontal axis for which 95% of the area under the curve is
                    contained between the two points. If treated as a posterior probability
                    distribution, then the area under the curve between any two points is the
                    probability that the odds ratio lies between those two points.
1      the results are shown in Figure 16-1. The second basic model assumed that the parameter of

2      interest is not fixed, but is itself a random variable from a distribution.  Such models are

3      designated by several names including random effects models or hierarchical models.  The

4      purpose of a random effects model is to relax the assumption that each study is estimating

5      exactly the same parameter. DerSimonian and Laird (1986)  discuss the random effects
6      model.  The results from using the different models were basically  the same, namely  that the
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  1     odds ratio is estimated to be about 1.2 with 95% confidence limits ranging from about 1.1 to
  2     1.3. The studies in which NO2 was actually measured gave an estimated odds ratio of 1.27,
  3     whereas the others yielded an estimate of 1.18 (which is consistent with a measurement error
  4     effect).
  5          The individual evidence for an effect of NO2 on respiratory disease is somewhat mixed.
  6     All but one of the studies used in the synthesis showed increased respiratory disease rates
  7     associated with increased exposure.  A few of the individual studies were statistically
  8     significant.  Combining the eleven studies giving quantitative estimates of effects tend to
  9     show increases of respiratory illness in children associated with exposure to NO2.  The EPA
 10     meta-analysis indicates that, when combined, the studies collectively provide evidence for an
 11     increase of 30 /*g/m3 (0.016 ppm) in NO2 exposure being associated with an increase of
 12     about 20% in the odds of respiratory illness, subject to the assumptions made for the
 13     synthesis. Although several assumptions were made to combine the studies, the consistency
 14     between the individual studies is demonstrated, indicating greater collective strength in the
 15     data base and suggesting that the effect is real.
 16          The exposure estimates used in these studies are either a surrogate (gas vs. electric) or a
 17     2-week integrated NO2 average measured by Palmes tubes.  The effects studied may be
 18     related to peak exposures,  average exposures, or a combination of the two.  To the extent
 19     that the observed health effects depend  on peak exposures rather than average exposures, the
 20     above exposure estimates introduce measurement error. These studies can not distinguish
 21      between the relative contributions of short-term peak exposures and longer-term average
 22     exposures to the observed health effects. However, the estimated effect is almost surely an
 23      underestimate, given the problems of misclassification of exposures and outcomes.  The effect
 24      was not dependent on any one or two studies.  The results of this analysis are not sensitive to
 25      the inclusion (or exclusion) of any one study.  In fact,  any two studies can be eliminated, and
 26      the 95% confidence limits will exclude  the no effect odds ratio of 1.0.  Thus, the combined
 27      evidence is supportive for the effects of exposure to NO2 on respiratory disease in children
28      under 12 years of age.
29           Several of the epidemiology studies in the quantitative analysis in Chapter 14 used a
30      single 2-week NO2 average or used two 2-week NO2 averages to characterize chronic
31      exposure.  The representativeness of such estimates of chronic exposure (e.g., 1 year) is a

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 1      consideration in generalizing the results of these studies to the long-term ambient situation.
 2      To roughly estimate the possible divergence from an actual annual ambient mean resulting
 3      from selecting one or two 2-week averages, data from the U.S.  Environmental Protection
 4      Agency's Aerometric Information and Retrieval System data bank (AIRS, 1991) for ten
 5      stations, selected from among those having fairly complete records for 1988 or 1989 (see
 6      Chapter 7), have been analyzed for the variability in their 2-week averages (Table 16-1).
 7      These stations represent various regions of the United States  but emphasize California where
 8      high levels most frequently occur.  For each station, 2-week averages were calculated week
 9      by week; these were then ranked and the 10th and 90th percentiles determined.  These two
10      percentiles were then divided by the station's annual mean to provide a common frame of
11      reference.  For example, the 10th and 90th percentile fractions for a single 2-week average
12      for Los Angeles in  1988 were 0.77 and 1.27 times the annual mean, respectively. This
13      would indicate that 80%  of the 2-week averages were between 77 and 127%  of the annual
14      average of 0.061 ppm ( 0.047 to 0.078 ppm respectively). In a similar manner, percentiles
15      were calculated from  two 2-week averages, using the means  of 2-week averages that were
16      26 weeks apart,  thus providing data for two opposite seasons such as winter and summer.
17      The 26 possible averages were ranked as  before.  Since these averages represent more weeks
18      of data taken at  two different times,  it is  expected that they would come closer to the true
19      annual average;  this is generally the case.
20           The results suggest that most (80%) of the single 2-week averages are within 80 and
21      125% of the mean, except for possibly cities with very low means such as Dallas. The two
22      2-week averages produce a better estimate,  with most lying between 85 and  120% of the
23      mean.  The reader is  cautioned that these numbers and analysis  may not be representative of
24      actual exposure  situations.  These selected data and  analyses  are offered only to describe
25      potential relationships between ambient NO2 annual averages and 2-week data periods.
26           The plausibility  of respiratory illness in children being increased by NO2 exposure (as
27      demonstrated by the epidemiologic studies and EPA meta-analysis discussed above) is
28      supported by findings from numerous animal studies evaluating  NO2 impacts on host defense
29      mechanisms.  Key findings from such studies are  summarized next.
30
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            TABLE 16-1. U.S. ENVIRONMENTAL PROTECTION AGENCY ANALYSIS
                OF VARIABILITY IN 2-WEEK AMBIENT AVERAGES OF 1-H NO2
                            DATA AT TEN SELECTED LOCATIONS
Fraction of Annual Average
Two 2-week


City
Los Angeles, CA
Azusa, CA
Upland, CA
Anaheim, CA
New York, NY
Chicago, IL
Cincinnati, OH
Worchester, MA
Miami, FL
Dallas, TX


Year
1988
1989
1988
1988
1988
1989
1989
1988
1989
1989
Annual
Average
(ppm)
0.061
0.051
0.047
0.046
0.041
0.034
0.030
0.029
0.018
0.011
2-week Average
10th
Percentile
0.77
0.81
0.79
0.71
0.84
0.86
0.84
0.81
0.77
0.45
90th

1.27 •
1.27
1.38
1.23
1.19
1.18
1.23
1.19
1.26
1.46
10th

0.87
0.83
0.87
0.86
0.91
0.93
0.88
0.83
0.90
0.75
Average
90th
Percentile
1.20
1.17
1.22
1.10
1.16
1.10
1.11
1.23
1.18
1.31
 1     16.3.3  Biological Bases Relating NO2 Exposure to Respiratory Morbidity:
 2             Effects of NO2 on the Respiratory Host Defense System
 3          The lung is one of the common sites of attack of microorganisms.  While many types of
 4     microorganisms are implicated in respiratory infection, viruses represent a major cause,
 5     particularly for infants and children.  In a viral respiratory infection, viral replication and
 6     altered immune responses to viral infections produce signs and symptoms of respiratory
 7     illness (Douglas, 1986).  The respiratory system has several defense mechanisms against
 8     inhaled infectious and chemical agents.  Host defense mechanisms comprise a complex,
 9     cooperative response system of several cell types, cell products,  tissues, and organs in, the
10     body. Two major approaches have been used to demonstrate the effects of NO2 on host
11     defenses: (1) evaluation of effects  on selected mechanisms of host defenses; and (2) use of
12     infectivity models, which reflect the overall functioning of all host defense mechanisms
13     against the infectious agent used. These two approaches are discussed below.
14          Nitrogen dioxide exposure can impair one or more components of this important defense
15     system, resulting in  the host being more susceptible to respiratory infection.  Epidemiological
16     studies have reported an association between an increase in symptoms of respiratory disease
17     of infectious origin and NO2 exposure (Chapter 14). Animal studies provide important

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 1      evidence indicating that several defense systems are a target organ for inhaled NO2. Nitrogen
 2      dioxide affects all of the host defenses studied (e.g.,  mucociliary clearance, alveolar
 3      macrophages, and  the humoral and cell-mediated immune system.) The biological basis
 4      relating NO2 exposure to respiratory symptoms and infection shown in epidemiological
 5      studies (Chapter 14) are presented in extensive discussions in Chapter 13 (animal toxicology).
 6           Studies on the effects of NO2 exposure on mucociliary transport in the conducting
 7      airways show a reduction in the number and activity of the cilia, morphological changes in
 8      the cilia, and decreased mucociliary velocity at concentrations  as low as 0.5 ppm for 7 mo of
 9      exposure (Yamamoto and Takahashi, 1984).  Others have observed similar effects  at higher
10     levels for shorter exposure durations. Several short-term (days to weeks) studies with
11      concentrations at or above  2 ppm demonstrated structural changes in cilia and ciliated cells,
12     decreases in numbers of ciliated cells, and decreases in ciliary beating.  As a foreign agent
13     deposits below the mucociliary region, in the gaseous exchange region of the lung, host
14     defenses are provided by the alveolar macrophage which acts to remove or kill viable
15     particles, to remove nonviable particles,  and to process and present antigens to lymphocytes
16     for antibody production. Exposure to NO2 has produced a variety of effects on alveolar
17     macrophages in several animal species after several weeks of exposure to levels as low as
 18     0.3 ppm; however,  most effects were observed at higher levels.  These effects included
 19      decreased phagocytosis and bactericidal activity, altered metabolism, increases in numbers of
20      macrophages and  morphological changes (Rombout et al.,  1986; Aranyi et al., 1976;
 21      Goldstein et al., 1974; Suzuki et al., 1986; Chang et al., 1986; Suzuki et al., 1986;
 22      Schlesinger et al., 1987; Mochitate et al., 1986).  Decreases in the ability of alveolar
 23      macrophages to engulf foreign particles  (phagocytosis) and bactericidal  activity are likely
 24      highly related  to increased susceptibility to pulmonary infections.  Controlled human exposure
 25      studies have also  examined macrophage function and show  that these cells, when exposed to
 26     NO2, tended to inactivate influenza virus in vitro less effectively than cells collected after air
 27     exposure (Frampton etal., 1989a).
 28           An example of the alteration of host defenses  against tumor cells  following exposure to
 29     NO0  is seen in studies examining the colonization of the lung by B16 melanoma tumor cells.
            Zj
 30     Richters and Kuraitis (1981, 1983)  and  Richters et al. (1985) reported  that when mice were
 31     exposed to 0.4 or 0.8 ppm NO2 for 10-12 weeks and then injected  with transplantable tumor
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   1     cells, colonization of these cells increased in the lung, indicating decreased resistance to such
   2     challenge.  Questions have been raised regarding the appropriateness of the statistical analysis
   3     used in evaluating these data and the interpretation of the model used.  Although the authors
   4     attribute effects to increased metastases, an alternative interpretation is an NO2-induced
   5     change in lung permeability to cells.  Another study reported that NO2 inhibits formation of
   6     B16F10 tumors in the lung (Weinbaum etal.,  1987).
   7          The humoral and cell-mediated immune systems together are essential for antibody
   8     production and the secretion of cellular products that are lethal to  certain invading organisms
   9     and also regulate the normal host's defense response. There is some indication that exposure
 10     to NO2 suppresses some of these specific immune responses and that the effect is both
 11      concentration- and time-dependent.  For example, a significant suppression of antibody
 12      production by spleen cells has been reported in experimental animals exposed for 1 week to
 13      1 mo to NO2 concentrations as low as 0.4 - 0.5 ppm (Lefkowitz et al., 1986;  Fujimaki et al.,
 14      1982).  Subchronic exposure to NO2 also resulted in decreased numbers of circulating
 15      T lymphocytes, T-helper/inducer lymphocytes, and T-cytotoxic/suppressor lymphocytes in
 16      mice (Damji and Richters, 1989). The cause of this suppression is not clear.
 17          Animal infectivity  studies present key data relating exposure to NO2 and  effects on the
 18      overall functioning of host defense mechanisms.  In these studies, animals were exposed to
 19      varying concentrations and durations of NO2 followed by exposure to an aerosol of an
 20     infectious agent. Microbial-induced mortality was used as the end point.  Exposure to NO2
 21     increased both bacterial- and influenza-induced mortality after subchronic exposures to levels
 22     as low as 0.5 - 1.0 ppm NO2 (Ehrlich and Henry, 1968; Ito, 1971; Ehrlich et al.,  1977).
 23     After acute (2 h) exposure, 2.0 ppm NO2 is the lowest effective concentration measured
 24     (Ehrlich et al., 1977). Nitrogen dioxide increases microbial-induced mortality  by impairing
 25     the host's ability to defend the respiratory tract from infectious agents, thereby increasing
 26     susceptibility to viral, mycoplasma, and bacterial infections (Ehrlich and Henry, 1968; Ito,
 27     1971; Ehrlich et al.,  1977; Parker et al.,  1989; Gardner et al., 1977a,b, 1979, 1980,  1982;
 28     Graham et al., 1987;  Jakab, 1987; Motomiya et al., 1973; Miller et al., 1987).  Using an
29     animal model designed to evaluate the effects of NO2 on non-fatal respiratory infection, NO2
30     decreased the intrapulmonary bactericidal activity in mice in a concentration-related manner,
31      without a decrease in  the number of alveolar macrophages (Goldstein et al., 1973).  Exposure

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 1     to NO2 was found to increase the severity of mycoplasma-induced lesions within the lung,
 2     but did not increase the susceptibility of the mice to the infection (Parker et al., 1989).
 3     Animal studies have also shown that influenza infection is exacerbated with NO2 exposure
 4     (Ito, 1971). In studies with cytomegalovirus and paramyxovirus (Jacob, 1987; Rose et al.,
 5     1988), the pathogenesis of these infections are enhanced.
 6          The toxicology literature also provides evidence that the host's response to inhaled NO2
 7     can be significantly influenced by the exposure duration, concentration, and temporal pattern
 8     of exposure. The relationship of concentration (C) times duration (T, time) to susceptibility
 9     to respiratory infections indicates that when the product of C XT is held constant and the
10     individual C's and T's are varied, a difference in response occurs.  The incidence of
11     mortality was  significantly more influenced by the concentration of NO2 than by the duration
12     of the exposure (Gardner et al., 1977a,b).  The exposure pattern of NO2 is also important
13     when comparing and determining the effects of continuous versus intermittent exposure.
14     When such  data were adjusted for differences  in C XT,  the incidence of respiratory infection
15     was essentially the same for both groups (Gardner et al., 1979). When animal studies were
16     designed to mimic a typical urban outdoor exposure environment having periodic spikes of
17     NO2 superimposed on a lower continuous background level of NO2,  the evidence indicates
18     that the animals exposed to the baseline plus short-term spikes were significantly more
19     susceptible to  a laboratory-induced infection than either the control or the background-NO2-
20     exposed mice  (Miller et al.,  1987; Gardner et al., 1982; Graham et al., 1987).  It should be
21     noted that the exposure patterns tested  in animals are likely to be different from indoor NO2
22     exposure patters.  This body of work for host defenses in mice shows that an average
23     exposure value (CxT) is not an exact  index or predictor of effects; rather, actual patterns of
24     exposure represent the causative exposure.
25           It is of interest that morphological studies have also indicated that the NO2
26     concentration  played a more important role in inducing lung lesions than did exposure
27     duration when the product of CXT was constant. The measured effect of concentration was
28     greater with intermittent exposure than with continuous (Chapter 13).
29           Recent controlled human exposure studies examining the effects of NO2 on pulmonary
30     host defense systems report a trend (not statistically significant) toward an elevated rate of
31     infection to a laboratory-induced live attenuated influenza, A/Korea/reassortment virus
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   1      (Goings et al., 1989).  Frampton et al. (1989a) report a trend (p<0.07) for less effective
   2      inactivation of virus by macrophages obtained from subjects exposed continuously to
   3      0.60 ppm NO2, but no effects of virus inactivation were seen in subjects exposed to 2.0 ppm
   4      spikes. Exposure to NO2 may transiently increase levels of antiprotease alpha-2-
   5      macroglobulin (a2M) in lavaged fluids isolated from the respiratory system' through local
   6      release.  While serving as an indicator of changes in protease-antiprotease balance, alterations
   7      in a2M in alveoli may  have significance for local immunoregulation and may alter alveolar
   8      macrophage defenses against infection (Frampton et al., 1989b). These findings suggest, but
  9      do not prove, that NO2 may play a role in increasing the susceptibility of adults to respiratory
 10      virus infections.
 11          The strength of the animal lexicological, human clinical, and epidemiological studies on
 12      host defenses provides  the rationale and plausible biological basis to explain the relationship
 13      seen in population studies showing increased frequency and severity of respiratory symptoms
 14      and/or infections in humans exposed to NO2.  The human body must be able to defend itself
 15      against a wide variety of inhaled foreign substances.  When these defenses are overcome,
 16      serious consequences can occur.  The significance of the observations from relevant animal
 17     models is clear.  With minor variations, the mammalian species, including humans, share in
 18     common an array of defensive mechanisms that are anatomically, functionally, and
 19     physiologically integrated in the respiratory tract to prevent and control infectious disease.
 20     All information available to date would indicate that qualitative extrapolation of data  observed
 21     in animals to humans is valid.  The accumulated evidence that NO2 causes dysfunction in
 22     several defenses provides the plausible biological mechanism necessary to link NO2 exposure
 23     to increased morbidity and respiratory symptomatology in children indicative of respiratory
 24     infection.
 25                                                       -...-...,
 26     16.3.4 Emphysema and  Exposure to NO2                                  -
 27          Studies in several  species of animals have shown that chronic exposure to high levels of
28     NO2 (relative to ambient) can  cause emphysema.  Since emphysema is an irreversible disease,
29     representing important public health concerns, whether NO2 creates a risk for this disease in
30     humans is a major question. Although this question cannot be definitely answered yet, the
31      potential for risk requires discussion. The definition of emphysema as used in the United

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 1     States is an anatomic one best characterized by National Institutes of Health (NIH) (1985)
 2     criteria.  These criteria are:  "An animal model of emphysema is defined as an abnormal state
 3     of the lungs in which there is enlargement of the airspace distal to the terminal bronchiole.
 4     Airspace enlargement should be determined qualitatively in appropriate specimens and
 5     quantitatively by stereologic methods."  An additional essential criterion for human
 6     emphysema is the destruction of alveolar walls.
 7          Several studies (Haydon et al., 1967; Freeman et al.-, 1977; Port et al., 1977) relate
 8     long-term (1 to over 30 months) exposure of rats and rabbits to high concentrations of
 9 .    NO2 (>  8 ppm, much greater than ambient levels) with morphologic  lung lesions which
10     meet the 1985 NIH workshop criteria for a human model of emphysema (i.e., alveolar wall
11     destruction occurred in addition to other characteristic changes),  One study (Hyde et al.,
12     1978) reported on dogs exposed to a mixture of 0.64 ppm NO2 and 0.25 ppm NO for
13     68 months.  Upon examination 32 to 36 months after exposure ceased, the dogs had
14     morphologic lesions that meet the 1985 NIH workshop criteria for human emphysema.  In the
15     same dogs, pulmonary function  was also measured.  Pulmonary  function decrements observed
16     at the end of exposure progressed post-exposure.  This suggests that the morphological effects
17     may also have been progressive. Another group of dogs in the same study was exposed to a
18     mixture of "low" NO2 (0.14 ppm) and  "high"  NO (1.1 ppm), but emphysema was not
19     observed.  Since the study did not include an NO2-only group, it is not possible to discern
20    , the effects of NO2 in the mixture.  However, the presence of emphysema in the "high"
21     NO2-"low"  NO group and its absence in the "low" NO2 - "high" NO group implies that NO2
22     was a significant etiologic factor.
23          Emphysema was reportedly observed in numerous other NO2 studies with several
24     species of animals, but either the reports lacked sufficient detail  for independent conclusions
25     or only the criteria for animal (not human) emphysema were met.  Several other studies
26     discussed in Chapter 13 were negative for emphysema.  Various factors such as the exposure
27     protocol may also play a role in the outcome of studies.  Potential differences may relate to
28     age of the animals during exposure, concentration and duration of exposure, and the duration
29     after exposure ceases before the animals are evaluated for emphysematous pathology.
30          In spite of the fact that there is a fairly extensive toxicologic data base concerning
31     morphologic effects of NO2, it is still not possible to establish a reasonably accurate
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  1      "no-observed-effect" level for emphysema.  This is likely due to a combination of factors:
  2      the complexity of changes occurring with NO2 exposure, the lack of published papers
  3      utilizing highly sensitive morphometric techniques, interspecies difference in response, and
  4      inadequate description of methods and findings in some published reports.  Qualitatively, it is
  5      clear that NO2 can cause emphysema in animals. While the lowest NO2 concentration for the
  6      shortest exposure duration that will result in emphysematous lung lesions pan not be reliably
  7      determined from the published studies, the exposures that did cause emphysema, according to
  8      the NIH criteria, are far higher than those currently reported in ambient air.       .
  9                                                       '••;.,
10      16.3.5 Subpopulations Potentially Susceptible to NO2 Exposure
11           Certain groups within the population may be more susceptible to the effects of NO2
12      exposure, including persons with preexisting respiratory disease, children, -and the elderly.
13      The reasons for paying special attention.to these groups is that they may be.affected by lower
14      levels of NO2 than other subpopulations or the impact of a given response may be greater.
15      Some causes of heightened susceptibility are better understood than others. Subpopulations
16      that already have reduced lung functions and reserves (e.g., the elderly, asthmatics,
17      emphysemics, chronic bronchitics) will be more impacted than other groups by decrements in
18      pulmonary function.  For example, a young healthy person may not even  nqtice a small
19      percentage change in pulmonary function, but a person whose activities are already limited by
20      reduced lung function may not have the reserve to compensate for the same percentage
21      change.
22           The airways of asthmatics may be hyperresponsive to a variety of inhaled materials
23      including pollens, cold-dry air, allergens, and  air pollutants.  Asthmatics have the potential to
24      be among the most susceptible members of the population with regard to respiratory
25      responses to NO2 (Section 15.3.1).  On the average, asthmatics are much  more sensitive to
26      inhaled bronchoconstrictors such as histamine, methacholine, or carbachol. The potential
27      addition of an NO2-induced increase in airway response to the already heightened
28      responsiveness to other substances raises the possibility of exacerbation of this pulmonary
29      disease by N02.  This is discussed in Section  15.4.
30           Other potentially susceptible groups include patients with chronic obstructive pulmonary
31      disease (COPD), such as emphysema and chronic bronchitis.  Many of these patients have
                                                                   t
        August 1991   ,           , ,              16-18      DRAFT-DO NOT QUOTE OR CITE

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 1     airway hyperresponsiveness to physical and chemical stimuli.  A major concern with COPD
 2     patients is the absence of an adequate pulmonary reserve, a susceptibility factor described
 3     , above.;  The poor distribution of ventilation in COPD may lead to a greater delivery of NO2
 4     -to the segment of the lung that is well ventilated, thus resulting in a greater regional tissue
 5  '  • dose.    :                                                   ,       ,-'••''
 6           Since over 2 million Americans have emphysema, it would be important to know
 7     whether NO2 has the potential to excaberate the disease.  Lafuma et al.  (1987) exposed both
 8     normal hamsters and hamsters with laboratory-induced (with elastase) emphysema to
 9     3,760 /ig/m3 (2 ppm) NO2 for 8 h/day, 5 days/week for 8 weeks.  The emphysematous
10     lesions produced by elastase and NO2 showed increases in mean linear intercepts and
11     pulmonary volumes and a decrease in internal alveolar surface areas, compared to those
12     treated with elastase and exposed to clean air.  The NO2-exposed animals developed a
13  -•-.   different type of emphysematous lesions than those caused by elastase.  The investigators
14     . suggested that the results may imply a role for NO2 in enhancing preexisting  emphysema.
15     Stavert et al. (1986) state that whereas NO2 inhalation alone may not cause emphysema, it is
16     conceivable that this oxidant gas may act as a co-determinant to allow the progression or
17     amplification of emphysema caused by other means.  However, it is not clear what the
18     potential would be for exacaberation of emphysema in humans at ambient concentrations.
19       .    Based upon epidemiology studies, children  12 years or younger  constitute a
20     subpopulation potentially susceptible to an increase in respiratory morbidity associated with
21     NO2 exposure (Section  14.5). Children may be more susceptible due to an immature host
22     defense system.  Data on the resident population of the United States  provides information on
23,     the numbers of children in various age ranges (Table 16-2). Approximately 37 million
24     children are in the age group 9 years and younger, while around 54 million children are in
25     the age group 14 years and younger. ,It is also possible that the increase in respiratory
26     morbidity may be more detectible in this age group compared to adults due to the greater
27     frequency of lower respiratory illness in this age group of children (Glezen and Denny,
28      1973).                      '
29
        August 1991
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TABLE 16-2. ESTIMATES OF THE RESIDENT POPULATION OF CHILDREN AND
  YOUNG ADULTS OF THE UNITED STATES, BY AGE AND SEX, JULY 1, 1989
Age
All Ages
<1
1-4
5-9
10-14
15-19

Total
248,239,000
3,945,000
14,808,000
18,212,000
16,950,000
17,812,000
Population
Male
120,982,000
2,020,000
7,578,000
9,321,000
8,689,000
9,091,000

Female
127,258,000,
1,925,000
7,229,000
8,891,000
.8,260,000
8,721,000
Source: Centers for Disease Control (1990).
August 1991
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20     Quackenboss, J. J.; Spengler, J. D.; Kanarek, M. S.; Letz, R.; Duffy, C. P.  (1986) Personal exposure to
21            nitrogen dioxide: relationship to indoor/outdoor air quality and activity patterns. Environ. Sci. Technol.
22            20: 775-783.
23
24     Richters, A.; Damji, K.  S. (1988) Changes in T-lymphocyte subpopulations and natural killer cells following
25            exposure to ambient levels of nitrogen dioxide. J. Toxicol. Environ. Health 25: 247-256.
26
27     Richters, A.; Kuraitis, K. (1981) Inhalation of NO2 and blood borne cancer cell spread to the lungs. Arch.
28            Environ. Health 36: 36-39.
29
30     Richters, A.; Kuraitis, K. (1983) Air pollutants and the facilitation of cancer metastasis. Environ. Health
31            Perspect. 52: 165-168.
32
33     Richters, A.; Richters, V.; Alley, W.  P. (1985) The mortality rate from lung metastases in animals inhaling
34            nitrogen dioxide (NOj). J.  Surg. Oncol. 28: 63-66.
35
36     Rombout, P. J. A.; Dormans, J. A. M. A.; Marra, M.; van Esch, G. J. (1986) Influence of exposure regimen
37            on nitrogen dioxide-induced morphological changes in the rat lung. Environ. Res. 41: 466-480.
38
39     Rose, R. M.; Fuglestad, J. M.; Skornik, W. A.; Hammer, S. M.; Wolfthal, S. F.; Beck, B. D.; Brain, J. D.
40            (1988) The pathophysiology of enhanced susceptibility to murine cytomegalovirus respiratory infection
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42
43     Samet, J. M.; Utell, M. J. (1990) The risk of nitrogen dioxide: what have we learned from epidemiological and
44            clinical studies? Toxicol. Ind. Health 6: 247-262.
45
46     Samet, J. M.; Tager, I. B.; Speizer, F. E. (1983) The relationship between respiratory illness in childhood and
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48
49     Schiff, L. J. (1977) Effect of nitrogen dioxide on influenza virus infection in hamster trachea organ culture. Proc.
50            Soc. Exp. Biol. Med. 156: 546-549.
51
52     Schlesinger, R. B.; Driscoll, K. E.; Vollmuth, T. A. (1987) Effect of repeated exposures  to nitrogen dioxide and
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54            Res. 44:  294-301.


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       dioxide. Am. Rev. Respir. Dis. 135(suppl.): A141.


Yamamoto, L; Takahashi, M. (1984) Ultrastructural observations  of rat lung exposed to nitrogen dioxide for
       7 months. Kitasato Arch. Exp.  Med. 57: 57-65.
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                             APPENDIX A:
           GLOSSARY OF TERMS AND SYMBOLS

     ABBREVIATIONS,  ACRONYMS, AND SYMBOLS
A           Angstrom (10~10 meter)
"A" strain    A particular type of influenza virus
AaDO2      Difference between alveolar and arterialized partial pressure of oxygen
AAS        Atomic absorption spectroscopy
AATCC     American Association of Textile Chemists and Colorists
Ad          A particular strain of laboratory mouse
AICHE      American Institute of Chemical Engineers
AM         Alveolar macrophage
AMP        Adenosine monophosphate; adenosine 5' phosphate
ANSA       8-anilino-l-naphthalene-sulfonic acid
APCD       Air Pollution Control District
APHA       American Public Health Association
A/PR/8      A particular strain of influenza virus
A/PR/8/34   A particular strain of influenza virus
AQCR       Air Quality Control Region
AQSM       Air Quality Simulation Model
ASTM       American Society for Testing and Materials
atm         One atmosphere, a unit of pressure
ATP        Adenosine triphosphate
avg         Average
BAKI       Potassium iodide solution acidified with boric acid
BHA        Butylated hydroxyanisole
BHT        Butylated hydroxytoluene
BP          Blood pressure
                                     A-l

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 "scat
 C3H
 C57BL
 C57BL/6
 cAMP
 CAMP
 CD-I
 cGMP
 °C
 14C
 CHE
 CL
 CLdyn
cm
CNS
CO
C02
CoA
COH
CPK
CR-1
CRD
CV
CxT

d
DEN
DIFKIN
DLCO
DMN
DNA
 Extinction coefficient due to scatter by aerosols
 A particular strain of laboratory mouse
 A particular strain of laboratory mouse
 A particular strain of laboratory mouse
 Cyclic adenosine monophosphate; adenosine 5'-phosphate
 Community Air Monitoring Program
 A particular strain of laboratory mouse
 Cyclic guanosine monophosphate; guanosine 5'-phosphate
 Degrees Celsius (Centigrade)
 A radioactive form of carbon
 Cholinesterase
 Lung compliance
 Dynamic lung compliance
 Static lung compliance                                  ,
 Centimeter
 Central nervous system; the brain and spinal cord
 Carbon monoxide
 Carbon dioxide
 Coenzyme A
 Coefficient of haze
 Creatine phosphokinase
 A particular strain of laboratory mouse
 Chronic respiratory disease
 Closing volume
Exposure concentration in ppm multiplied by time of exposure in hours or
other time measurement
Day
Diethylnitrosamine (also DENA)
Diffusion Kinetics Model
Diffusion capacity of the lung for carbon monoxide
Dimethylnitrosamine
Deoxyribonucleic acid
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 D = CT
 DPPD
 EC
 EKG
 EPA
 °F
 FEF
 FET
 FEV
 FEV,
 FEV,
1.0
0.75
 FRM
 ft
 FT
 FVC
 g
 G6P
 GC
 GL
 GC-MS
 GM
 GMP
 GSH
 GSSG
 H*
 3H
 ha
 HbO2
 HNO2
 HNO3
HO*
 Dose equals concentration multiplied by time
 N,N diphenylphenylenediamine
 Prefix of International Commission on Enzymes' identification numbers
 Electrocardiogram
 U.S. Environmental Protection Agency
 Degrees Fahrenheit
 Forced expiratory flow
 First-edge time
 Forced expiratory volume
 One-second forced expiratory volume
 0.75-second forced expiratory volume
 Federal Reference Method for air quality measurement
 Foot
 Fourier transform spectroscopy (also FS)
 Forced vital capacity
 Gram
 Glucose-6-phosphate
 Guanylate cyclase
 Gas chromatography
 Gas chromatograph in combination with mass spectrometry
 General Motors Corporation
 Guanosine 5'-phosphate;  guanosine monophosphate
 A tripeptide, glutathione  (reduced form)
 The disulfide (oxidized) form of GSH
 Hydrogen (free radical)
 Tritium; a radioactive form of hydrogen
 Hectare
 Oxyhemoglobin
Nitrous acid (also HONO)
Nitric acid (also HONO^
Hydroxyl free radical (also OH)
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H02*        Hydroperoxyl free radical
HO2NO2     Pernitrous acid
HO2NO2     Pernitric acid (also
hr           Hour                                            ......
HR          Heart rate                                          .    .
hv           Planck's constant (h) times the frequency of radiated energy (v) = Quanta of
             energy (E)
H2O2        Hydrogen peroxide
H2S         Hydrogen sulfide
H2SO4       Sulfuricacid
IARC        International Agency for Research on Cancer
Ig           Immunoglobulins
IgA         Imniunoglobulin A fraction
IgG         Imniunoglobulin G fraction
IgGj         Imniunoglobulin Gj fraction
IgG2         Imniunoglobulin G2 fraction
IgM         Imniunoglobulin M fraction
in           Inch
IR           Infrared
k            Rate constant or dissociation constants
kg           Kilograms
km          Kilometer
1            Liter (also £)
LC50        Lethal concentration 50%; that concentration which is lethal to 50% of test
             subjects         '      .','••.
LD50        Lethal dose 50%; dose which is lethal to 50% of the subjects
LT50        The time required for 50% of the test animals to die when given a lethal dose,
LDH         Lactic acid (lactate) dehydrogenase
LPS         Bacterial lipopolysaccharide                ,      -,•••,         .
m           Meter                     •            ..
M           Molar
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M
MAK
max
MFR
     •
mg/m
Mg
ml
mM
MMD
MMFR
mo
MFC
MT
N
N
1 "%
  N
N-6-MI
NA
NAAQS
NaCl
NAD+
NADB
NADH
NADPH
NaOH
NAS
NASN
NDIR
NEDA
NEDS
             Third body (in a reaction)
             Maximum permissible concentration (in Germany)
             Maximum
             Maximal flow rate
             Micrograms per cubic meter
             Milligrams per cubic meter
             Magnesium
             Milliliter
             Millimoles
             Mass median diameter
             Mid-maximal flow rate
             Month
             Maximum permissible concentration (in the U.S.S.R.)
             Metric Ton
             Nitrogen
             Normal
             A radioactive form of nitrogen
             N-nitrosoheptamethyleneimine
             Not applicable
             National Ambient Air Quality Standard
             Sodium chloride; common  table salt
             Nicotinamide-adenine dinucleotide (+ indicates oxidized form)
             National Air Data Bank
             Nicotinamide-adenine dinucleotide (reduced form)
             Nicotinamide-adenine dinucleotide phosphate (reduced form)
             Sodium hydroxide
             National Academy of Sciences
             National Air Surveillance Network
             Nondispersive infrared
             N-(l-Naphthyl)-ethylenediamine dihydrochloride
             National Emissions Data System
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NEIC
ng
NH4
nm
NO
NOHb
NOX
N20
NO2
N203
N204
NSF
O
O^D)
03
OH
0(3P)
32P
PaCO2
PaCO2
PAH
PAN
Pa02
PA02
pH
PHA
PO2
ppb
pphm
ppm
ppt
National Enforcement Investigations Center
Nanogram
Ammonium ion or radial
Nanometer
Nitric oxide
Nitrosylhemoglobin
Nitrogen oxides
Nitrous oxide
Nitrogen dioxide
Dinitrogen trioxide
Dinitrogen tetroxide
National Science Foundation
Atomic oxygen
Excited atomic oxygen
Ozone
Hydroxyl group
Ground state atomic oxygen
A radioactive form of phosphorus
Alveolar partial pressure of carbon dioxide
Arterial partial pressure of carbon dioxide
p-Ammiohippuric acid
Peroxyacetyl nitrate
Arterial partial pressure of oxygen
Alveolar partial pressure of oxygen
Log of the reciprocal of the hydrogen ion concentration
Phytohemagglutinin
Partial oxygen pressure
Parts per billion
Parts per hundred million
Parts per million
Parts per trillion
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 Q
 QRS

 RAMS
 RAPS
 Raw
 RBC
 RM
 RNA
 RV
 SAI
 SD
 SCOT
 SGPT
 SH-
 SMSA
 SN
 S02
 SPF
 SRaw
 SRM
 ss
 STP
 TEA
 Tg
 TGS-ANSA
 TLC
 TPTT
 TSP
 USEPA
 UV
vc
 Cardiac output
 A complex of three distinct electrocardiogram waves which represent the
 beginning of ventricular contraction
 Regional Air Monitoring System
 Regional Air Pollution Study
 Airway resistance
 Red blood cell; erythrocyte
 Reference method for air quality measurement
 Ribonucleic acid
 Residual volume
 Science Applications, Inc.
 Standard deviation
 Serum glutamic-oxaloacetic transaminase
 Serum glutamic-pyruvic transaminase
 Sulfhydryl group
 Standard Metropolitan Statistical Area
 Suspended nitrates
 Sulfur dioxide
 Specific pathogen free
 Specific airway resistance
 Standard reference material
 Suspended sulfates
 Standard temperature and pressure
 Triethanolamine
 Terragram; 106 metric tons or 1012 grams
 A 24-hour method for the detection of analysis of NO2 in ambient air
 Total lung capacity
 20% transport time
 Total suspended particulate
 U.S. Environmental Protection Agency
Ultraviolet radiation
Vital capacity
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VE
VEE
Vmax
VT
V/V
WBC
wk
yr
Zn
>
<
             Ventilatory volume
             Venezuelan equine encephalomyelitis (virus)
             Maximum expiratory flow rate
             Total volume
             Volume per volume
             White blood ceUs
             Week
             Year
             Zinc
             Microgram
             Microliter
             Micrometer
             Greater than
             Less than
             Approximately
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                                   GLOSSARY
AaDO2:  Alveolar-arterial difference or gradient of the partial pressure of oxygen.  An overall
      measure of the efficiency of the lung as a gas exchanger.  In healthy subjects, the
      gradient is 5 to 15 mm Hg (torr).

A/PR/8 virus: A type of virus capable of causing influenza in laboratory animals; also,
      A/PR/8/34.

Abscission:  The process whereby leaves, leaflets, fruits, or other plant parts become detached
      from the plant.

Absorption coefficient:  A quantity which characterizes the attenuation with distance of a beam
      of electromagnetic radiation (like light) in a substance.

Absorption spectrum:  The spectrum that results after any radiation has passed through an
      absorbing substance.

Abstraction:  Removal of some constituent of a substance or molecule.

Acetaldehyde: CH3CHO; an intermediate in yeast fermentation of carbohydrate and in alcohol
      metabolism; also  called acetic aldehyde, ethaldehyde, ethanal.

Acetate rayon:  A staple or filament fiber made by extrusion of cellulose acetate.  It is
      saponified by dilute alkali whereas viscose rayon remains unchanged.

Acetylcholine:  A naturally-occurring substance in the body which can cause constriction of
      the bronchi in the lungs.

Acid:  A substance that  can donate hydrogen ions.

Acid dyes:  A large group of synthetic coal tar-derived dyes which produce bright shades in a
      wide color range.  Low cost and ease of application are features which make them the
      most widely used dyes for wool.  Also used on nylon.  The term acid dye is derived
      from their precipitation in an acid bath.

Acid mucopolysaccharide:  A class of compounds composed of protein and polysaccharide.
      Mucopolysaccharides comprise much of the substance of connective tissue.

Acid phosphatase: An enzyme (EC 3.1.3.2) which catalyzes the disassociation of phosphate
      (PO4) from a wide range of monoesters of orthophosphoric acid.  Acid phosphatase  is
      active in an acidic pH range.

Acid rain: Rain having a pH less than 5.6, the minimum expected from atmospheric CO2.
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 Acrolein:  CH2=CHCHO; a volatile, flammable, oily liquid giving off irritant vapor. Strong
       irritant of skin and mucuous membranes.  Also called acrylic aldehyde, 2-propenal..

 Acrylics (plastics):  Plastics which are made from acrylic acid and are light in weight, have • ••••
       great breakage resistance, and a lack of odor and taste.  Not resistant to scratching,
       burns, hot water, alcohol or cleaning fluids.  Examples include Lucite and Plexiglass. ;
       Acrylics are thermoplastics and are softened by heat and hardened into, definite shapes
       by cooling.

 Acrylic fiber: The generic name of man-made fibers derived from acrylic resins (minimum of
       85% acrylonitrite units).

 Actinic:  A term applied to wavelengths of light too small to affect one's sense of sight,  such
       as ultraviolet.                                                    ., <,

 Actinomycetes:  Members of the genus Actinomyces; nonmotile, nonsporeforming, anaerobic
       bacteria, including  both soil-dwelling saprophytes and disease-producing parasites.

 Activation energy: The energy required to bring about a chemical reaction.

 Acute respiratory disease: Respiratory infection, usually with rapid onset and of short
       duration.

 Acute toxicity:  Any poisonous effect produced by a single short-term exposure, that results in
       severe biological harm or death.

 Acyl:  Any organic radical or group that remains intact when an organic acid forms an ester.

 Adenoma: An ordinarily  benign neoplasm (tumor) of epithelial tissue; usually well
       circumscribed, tending to compress adjacent tissue rather than infiltrating or invading.

 Adenosine monophosphate (AMP):  A nucleotide found among the hydrolysis products of all
       nucleic acids;  also called adenylic acid.

 Adenosine triphosphatase  (ATPase):  An enzyme (EC 3.6.1.3) in muscle and elsewhere that
       catalyzes the release of the high-energy, terminal phosphate group of adenosine
       triphosphate.

Adrenalectomy:  Removal of an adrenal gland.  This gland is located near or upon the kidney
       and is the site of origin of a number of hormones.                                  . \

Adsorption: Adhesion of a thin layer of molecules to a liquid or solid surface.

Advection: Horizontal flow of air at the surface or aloft; one of the means by which heat is
       transferred from one region  of the earth to another.
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Aerodynamic diameter: The diameter of a unit density sphere having the same settling speed
       (under gravity) as the particle in question of whatever shape and density.

Aerosol: Solid particles or liquid droplets which are dispersed or suspended in a gas.

Agglutination:  The process by which suspended bacteria, cells or similar particles adhere and
       form into clamps.

Airborne pathogen:  A disease-causing microorganism  which travels in the air or on particles
       iri the air.                                                                   '

Air pollutant:  A substance present in the ambient atmosphere, resulting from the activity of
       man or from natural processes, which may cause damage to human health or welfare,
       the natural environment, or materials or objects.

Air spaces:  All alveolar ducts, alveolar sacs,  and alveoli.  To be contrasted with airways.

Airway conductance (Gaw): Reciprocal of airway resistance. Gaw = (l/Raw).

Airway resistance (Raw):  The (frictional) resistance to airflow afforded by the airways
       between the airway opening at the mouth and the alveoli.

Airways:  All passageways of the respiratory tract from mouth or nares down to and including
       respiratory bronchioles.  To be contrasted with air spaces.

Alanine aminotransferase:  An enzyme (EC  2.6.1.2) transferring amino groups from L-alanine
       to 2-ketoglutarate.  Also known as alanine transaminase.

Albumin:  A type of simple,  water-soluble protein widely distributed throughout animal tissues
       and fluids, particularly serum.
                                                          9
Aldehyde:  An organic compound characterized by the group -C-H.

Aldolase:  An enzyme (EC 4.1.2.7) involved in metabolism of fructose which catalyzes the
       formation of two 3-carbon intermediates in the major pathway of carbohydrate
       metabolism.

Algal bloom:  Sudden spurt in growth of algae which can affect water quality adversely.

Alkali:  A salt of sodium or potassium capable of neutralizing acids.

Alkaline phosphatase:  A phosphatase (EC 3.1.3.1) with an optimum pH of 8.6, present
       ubiquitously.

Allergen:  A material that, as a result of coming into contact with appropriate tissues of an
      animal body, induces a state of sensitivity resulting in various reactions; generally
      associated with idiosyncratic hypersensitivities.

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 Alpha-hydroxybutyrate dehydrogenase:  An enzyme (EC 1.1.1.30), present mainly in
       mitochondria, which catalyzes the conversion of hydroxybutyrate to acetoacetate
       intermediate biochemical pathways.

 Alpha rhythm: A rhythmic pulsation obtained in brain waves exhibited in the sleeping state of
       an individual.

 Alveolar capillary membrane:  Finest portion of alveolar capillaries, where gas transfer to and
       from blood takes place.
         ,                                                       -     .      .   'i
 Alveolar macrophage (AM):  A large, mononuclear, phagocytic cell found on the alveolar
       surface, responsible for particle clearance from the deep lung and for viral and bacterial
       killing.

 Alveolar oxygen partial pressure (PAO2):  Partial pressure of oxygen in the air contained in  -<
       the air sacs of the lungs.

 Alveolar septa: The tissue between two adjacent pulmonary alveoli, consisting of a
       close-meshed capillary network covered on both surfaces by thin alveolar epithelial
       cells.

 Alveolus:  An air cell; a terminal, sac-like dilation in the lung.  Gas exchange
       occurs here.

 Ambient: The atmosphere to which the general population may be exposed.  Construed here
       not to include atmospheric conditions indoors, or in the workplace.

 Amine:  A substance that may be derived from ammonia (NH3) by the replacement of one,
       two  or three of the hydrogen  (H)  atoms by hydrocarbons or other radicals (primary,
       secondary or tertiary amines,  respectively).

 Amino acids:  Molecules consisting  of a carboxyl group, a basic amino group, and a residue
       group attached to a central carbon atom. Serve as the building blocks of proteins.

p-Aminohippuric  acid (PAH):  A compound used to determine renal plasma flow.

Aminotriazole: A systemic herbicide,  C2H4N4, used in areas other than croplands, that also
      possesses some antithyroid activity; also called amitrole.

Ammonification:  Decomposition with production of ammonia or ammonium compounds, esp.
      by the action of bacteria on nitrogenous organic matter.

Ammonium: Anion (NH4+) or radical (NH4) derived from  ammonia by combination with
      hydrogen.  Present in rainwater,  soils and many commercial fertilizers.

Amnestic:  Pertains to immunologic memory:  upon receiving a second dose of antigen, the
      host "remembers" the first dose and responds faster to the challenge.

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Anaerobic:  Living, active or occurring in the absence of free oxygen.

Anaerobic bacteria: A type of microscopic organism which can live in an environment not
       containing free oxygen.

Anaphylactic dyspneic attack:  Difficulty in breathing associated with a systemic allergic
       response.

Anaphylaxis:  A term commonly used to denote the immediate, transient kind of
       immunological  (allergic) reaction characterized by contraction of smooth muscle and
       dilation of capillaries due to release of pharmacologically active substances.

Angiosperm:  A plant having seeds enclosed in an ovary; a flowering plant.

Angina pectoris:  Severe constricting pain in the chest which may be caused by depletion of
       oxygen delivery to the heart muscle; usually caused by coronary disease.

Angstrom (A): A unit (10~8 cm) used in the measurement of the wavelength of light.

Anhydride:  A compound resulting from removal of water from two molecules  of a carboxylic
       (-COOH) acid.  Also, may refer to those substances (anhydrous) which do  not contain
       water in chemical  combination.              .    ,

Anion: A negatively charged atom, radical, or ion.

Anorexia: Diminished appetite; aversion to food.

Anoxic:  Without or deprived of oxygen.            ,

Antagonism:  When the effects of a mixture are less than the sum of the effects of each
       individual chemical.

Anthraquinone:  A yellow crystalline ketbne, C14H8O2 derived from anthracene and used in
       the manufacture of dyes.

Anthropogenic:  Of, relating to or influenced by man.  An anthropogenic source of pollution
       is one caused by man's actions.

Antibody: Any body or substance evoked by the stimulus of an antigen and which reacts
       specifically with antigen in some demonstrable way.

Antigen:   A material such as a foreign protein that, as a result of coming in contact with
       appropriate tissues of an animal,  after a latent period, induces a state of sensitivity
       and/or the production of antibody.

Antistatic  agent:  A chemical compound applied to fabrics to  reduce or eliminate accumulation
       of static electricity.
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 Arachidonic acid:  Long-chain fatty-acid which serves as a precursor of prostaglandins.

 Area source:  In air pollution, any small individual fuel combustion or other pollutant source;
      also, all such sources grouped over a specific area.

 Aromatic:  Belonging to that series of carbon-hydrogen compounds in which the carbon atoms
      form closed rings containing unsaturated bonds (as in benzene).

 Arterial partial pressure of oxygen (PaO2): Portion of total pressure of dissolved gases in
      arterial blood as measured directly from arterial blood.

 Arterialized partial pressure of oxygen:  The portion of total pressure of dissolved gases in
      arterial blood attributed to oxygen, as measured from non-arterial (e.g., ear-prick)
      blood.

 Arteriosclerosis: Commonly called hardening of the arteries.  A condition that exists when the
      walls of the blood vessels thicken and become infiltrated with excessive amounts of
      minerals and fatty materials.

 Artifact: A spurious measurement produced by the sampling or analysis process.

 Ascorbic acid:  Vitamin C, a strong reducing agent with antioxidant properties.

 Aspartate transaminase:  Also known as aspartate aminotransferase (EC 2.6.1.1). An enzyme
      catalyzing the transfer of an amine group from glutamic acid to oxaloacetic, forming
      aspartic acid in the process.  Serum level of the enzyme is increased in myocardial
      infarction and in diseases involving  destruction of liver cells.

 Asphyxia:  Impaired exchange of oxygen and carbon dioxide, excess of carbon dioxide and/or
      lack of oxygen, usually caused by ventilatory problems.

 Asthma:  A disease characterized by an increased responsiveness of the airways to various
      stimuli and manifested by slowing of forced expiration which changes in severity either
      spontaneously or as a result of therapy.  The term asthma may be modified by words or
      phrases indicating its etiology, factors provoking attacks, or duration.

 Asymptomatic: Presenting no subjective evidence of disease.

 Atmosphere:  The body of air surrounding the earth.  Also, a measure of pressure (atm.) equal
      to the pressure of air at sea level, 14.7 pounds per square inch.

Atmospheric deposition: Removal  of pollutants from the atmosphere onto land, vegetation,
      water bodies or other  objects, by absorption, sedimentation, Brownian diffusion,
      impaction, or precipitation in rain.
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Atomic absorption spectrometry:  A measurement method based on the absorption of radiant
      energy by gaseous ground-state atoms. The amount of absorption depends on the
      population of the ground state which is related to the concentration of the sample being
      analyzed.

Atopic: Clinical hyperreactivity of the airways associated with asthma and allergies.

Atropine:  A poisonous, white crystalline alkaloid (C17H23NO3) derived from belladonna and
      related plants, used to relieve spasms of smooth  muscles. It is an anticholinergic agent
      that blocks the parasympathetic actions of acetylcholine and other cholinergic agents.

Autocorrelation:  Statistical interdependence of variables being analyzed; produces problems,
      for example, when observations may be related to previous measurements or other
      conditions.

Autoimmune disease: A condition in which antibodies are produced against the subject's own
      tissues.

Autologous:  A term referring to cellular elements, such as red blood cells and alveolar
      macrophage, from title same organism; also, something natually and normally occurring
      in some part of the body.

Autotrophic:  A term applied to those microorganisms which are able to maintain life without
      an exogenous organic supply of energy, or which only need carbon dioxide or
      carbonates and simple inorganic nitrogen.

Autotrophic bacteria: A class of microorganisms which require only carbon dioxide or
      carbonates and a simple inorganic nitrogen compound for carrying on life processes.

Auxin:  An organic substance that causes lengthening of the stem when applied in low
      concentrations to shoots of growing plants.

Awn:  One of the slender bristles that terminate the glumes of the spikelet in some cereals and
      other grasses.

Azo dye:  Dyes in which the azo group is the chromophore and joins benzene or naphthalene
       rings.

Background measurement:  A measurement of pollutants in ambient air due to natural sources;
       usually taken in remote areas.

Bactericidal activity:  The process of killing bacteria.

Barre:  Bars or stripes in a fabric, caused by uneven weaving, irregular yarn or uneven dye
       distribution.

Basal cell:  One of the innermost cells of the deeper epidermis of the skin.

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 Base cation:  CA2+, Mg2+, K+, orNa+.

 Base saturation:  The degree to which soil cation exchange capacity is occupied by base
       cations. This expressed as a percent, use charge-equivalents.

 Benzenethiol:  A compound of benzene and a hydrosulfide group.

 Beta (j8)-lipoprotein:  A biochemical complex or compound containing both lipid and protein
       and characterized by having a large molecular weight, rich in cholesterol.  Found in
       certain fractions  of human plasma.

 Bilateral renal sclerosis: A hardening of both kidneys of chronic inflammatory origin.

 Biomass: That part of  a given habitat consisting of living matter.

 Biosphere:  The part of the earth's crust, waters and atmosphere where living organisms can
       subsist.

 Biphasic: Having two distinct successive stages.

 Bleb:  A collection of fluid beneath the  skin; usually smaller than bullae or blisters.

 Blood urea:  The chief  end product of nitrogen  metabolism in mammals, excreted in human
       urine in the amount of about 32 grams (1 oz.) a day.

 Bloom:  A greenish-gray appearance imparted to silk and pile fabrics either by nature of the
       weave or by the finish; also, the creamy  white color observed on some good cottons.

 Blue-green algae:  A group of simple plants which are the only N2-fixing organisms  which
       photosynthesize as do higher plants.

 Brightener:  A compound such as a dye, which  adheres to fabrics in order to provide better
       brightness or whiteness by converting ultraviolet radiation to visible light.  Sometimes
       called optical bleach or whitening agent.  The dyes used are of the florescent type.

 Broad bean:  The large  flat edible seed of  an Old World upright vetch (Vicia faba), or the
       plant itself, widely grown for its seeds and for fodder.

 Bronchi: The first subdivisions of the trachea which conduct air to and from the bronchioles
       of the lungs.

Bronchiole:  One of the finer subdivisions  of the bronchial (trachea) tubes, less than  1 mm in
       diameter, and having no cartilage in its wall.

Bronchiolitis:  Inflammation of the bronchioles,  which may be acute or chronic.  If the
       etiology is known, it should be stated.  If permanent occlusion of the lumens is present,
       the term bronchiolitis obliterans may be used.

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Bronchiolitis fibrosa obliterans syndrome:  Obstruction of the bronchioles by fibrous
      granulation arising from an ulcerated mucosa; the condition may follow inhalation of
      irritant gases.

Bronchitis:  A non-neoplastic disorder of structure or function of the bronchi resulting from
      infectious or noninfectious irritation.  The term bronchitis should be modified by
      appropriate words or phrases to indicate its etiology, its chronicity, the presence of
     ' associated  airways dysfunction, or the type of anatomic change. The term chronic
      bronchitis, when unqualified, refers to a condition associated with prolonged exposure
      to nonspecific bronchial irritants and accompanied by mucus hypersecretion and certain
      structural alterations in the bronchi.  Anatomic changes may include hypertrophy of the
      mucous-secreting apparatus and epithelial metaplasia, as well as more classic evidences
      of inflammation.  In epidemiologic studies,  the presence of cough or sputum production
      on most days for at least three months of the year for at least two consecutive years has
      sometimes been accepted as a criterion for the diagnosis.

Bronchoconstrictor:  An agent that causes a reduction in the caliber (diameter) of a bronchial
      tube.

Bronchodilator:  An agent which causes  an increase in the caliber  (diameter) of a bronchus or
      bronchial tube.

Bronchopneumonia: Acute inflammation of the walls of the smaller bronchial tubes, with
      irregular area of consolidation due to spread of the inflammation into peribronchiolar
      alveoli and the alveolar ducts.

Bronchospasm:  Temporary narrowing of the bronchi due to a violent, involuntary contraction
      of the smooth muscle of the bronchi.

Bronchus:  One of the  subdivisions of the trachea serving to convey air to and from the lungs.
      The trachea divides into right and left main bronchi which in turn form lobar,
       segmental, and subsegmental bronchi.

Brownian diffusion: Diffusion by random movement of particles  suspended in liquid or gas,
       resulting from the impact  of molecules of the fluid surrounding the particles.

BTPS conditions (BTPS):   Body temperature, barometric pressure, and saturated with water
       vapor.  These are the conditions existing in the gas phase of the lungs.  For humans the
       normal temperature is 37°C, the pressure is based on the barometric pressure, and the
       partial pressure of water vapor is 47 torr.

Buffer:  A  substance in solution  capable of neutralizing both acids and bases and thereby
       maintaining the  original pH of the solution.

 Buffering:  In reference to soil acidification,  this is resistance to change resulting from
       reserves of acid or base cations on the soil cation exchange sites.
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 Buffering capacity:  Ability of a body of water and its watershed to neutralize introduced acid.

 Butanol: A four-carbon, straight-chain alcohol, C4H9OH, also known as butyl alcohol.

 Butylated hydroxytoluene (BHT):  A crystalline phenolic antioxidant.

 Butylated hydroxyanisol (BHA):  An antioxidant.

 14C labeling: Use of a radioactive form of carbon as a tracer, often in metabolic studies.

   C-proline:  An amino acid which has been labeled with radioactive carbon.

 Calcareous:  Resembling or consisting of calcium carbonate (lime), or growing on limestone or,
       lime-containing soils.

 Calorie:  Amount of heat required to raise temperature of 1 gram of water at  15°C by
       1 degree.

 Cannula: A tube that is inserted into a body cavity, or other tube or vessel, usually to remove
       fluid.

 Capillary: The smallest type of vessel; resembles a hair.  Usually in reference to a blood or
       lymphatic capillary vessel.                                                     <

 Carbachol:  A cholinergic parasympathetic stimulant, carbamoylcholine chloride
       (^6H15CIN2O2)' mat produces constriction of the bronchial smooth muscles similar to
       acetylcholine.

 Carbon monoxide:  An odorless, colorless,  toxic gas with a strong affinity for hemoglobin and
       cytochrome; it reduces oxygen absorption capacity, transport and utilization.

 Carboxyhemoglobin:  A fairly stable union  of carbon monoxide with hemoglobin  which
       interferes with the normal transfer of carbon dioxide and oxygen during circulation of
       blood.  Increasing levels of carboxyhemoglobin result in various degrees of
       asphyxiation, including death.

 Carcinogen:  Any agent producing or playing a stimulatory role in the formation of a
       malignancy.

 Carcinoma: Malignant new growth made up of epithelial cells tending to infiltrate the
       surrounding tissues and giving rise to metastases.

 Cardiac output: The volume of blood passing through the heart per unit time:

Cardiovascular: Relating to the heart and the blood vessels or the circulation.
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Carotene:  Lipid-soluble yellow-to-orange-red pigments universally present the photosynthetic
      tissues of higher plants, algae, and the photosynthetic bacteria.

Cascade impactor:  A device for measuring the size distribution-of particulates and/or aerosols,
      consisting of a series of plates with orifices of graduated size which separate the sample
      into a number of fractions of decreasing aerodynamic diameter.

Catabolism: Destructive metabolism involving the release of energy and resulting in
      breakdown of complex materials in the organism.

Catalase:  An enzyme (EC 1.11.1.6) catalyzing the decomposition of hydrogen peroxide to
      water and oxygen.

Catalysis:  A modification of the rate of a chemical reaction by some material which is
      unchanged at the end of the reaction.

Catalytic converter:  An air pollution abatement device that removes organic contaminants by
      oxidizing them into carbon dioxide and water.

Catecholamine:  A pyrocatechol with an alkalamine side chain, functioning as a hormone or
      neurotransmitter, such as  epinephrine,  morepinephrine, or dopamine.

Cathepsins: Enzymes which have the ability to hydrolyze certain proteins and peptides; occur
      in cellular structures known as lysosomes.

Cation:  A positively charged ion.

Cation exchange capacity:  The  ability of a soil to absorb positively-charged ions by
      electostatic forces.  This absorption occurs on negatively-charged sites on clays and
      organic matter in soils.

Cellular permeability:  Ability of gases to enter and leave cells; a sensitive indicator of injury
      to deep-lung cells.

Cellulose:  The basic substance which is contained in all vegetable fibers and in certain
      man-made fibers.  It is a  carbohydrate and constitutes the major substance in plant life.
      Used to make cellulose acetate and rayon.

Cellulose acetate:  Commonly refers to fibers or fabrics in which the cellulose is  only partially
      acetylated with acetate groups. An ester made by reacting cellulose with acetic
      anhydride with SO4 as a catalyst.

Cellulose rayon:  A regenerated  cellulose which is chemically the same as cellulose except for
      physical differences in molecular weight and crystallinity.
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 Cellulose triacetate:  A cellulose fiber which is completely acetylated.  Fabrics of triacetate
       have higher heat resistance than acetate and may be safely ironed at higher temperature.
       Such fabrics have improved ease-of-care characteristics because after heat treatment
       during manufacture, a change in the crystalline structure of the fiber occurs.      •

 Cellulosics:  Cotton, viscose rayon and other fibers made of natural fiber raw materials.

 Celsius scale:  The thermometric scale in which freezing point of water is ,0 and boiling point
       is 100.

 Central hepatic necrosis:  The pathologic death of one or more cells, or of a portion of the
       liver, involving the cells adjacent to the central veins.

 Central nervous system (CNS):  The brain and the spinal cord.

 Centroacinar area:  The center portion of a grape-shaped gland.

 Cerebellum:  The large posterior brain-mass lying above the pons and medulla and beneath the
       posterior portion of the cerebrum.                                          ,

 Cerebral cortex:  The layer of gray matter covering the entire surface of the cerebral
       hemisphere of mammals.

 Chain reaction: A reaction that stimulates its own repetition.

 Challenge: Exposure of a test organism to a virus, bacteria, or other stress-causing agent,
       used in conjunction with exposure to a pollutant of interest, to explore possible
       susceptibility brought on by the pollutant.                       :

 Chamber study: Research conducted using a closed vessel in which pollutants are reacted or
       substances exposed to pollutants.

 Chemiluminescence:  A measurement technique in which radiation is produced as a result of
       chemical reaction.

 Chemotactic:  Relating to attraction or repulsion of living protoplasm by chemical stimuli.

 Chlorophyll:  A group of closely related green photosynthetic pigments occurring in leaves,
       bacteria, and organisms.                                                   '

 Chloroplast:  A plant cell inclusion body containing chlorophyll.

Chlorosis:  Discoloration of normally green plant parts that can be caused by disease, lack of
       nutrients, or  various air pollutants, resulting in  the failure of chlorophyll to develop.

Cholesterol:  A steroid alcohol C2H45OH; the most abundant steroid in animal cells and body
       j-» • i                     ** ^^            -                                      J
       fluids.

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Cholinesterase (CHE):  One (EC 3.1.1.8) of a family of enzymes capable of catalyzing the
     ' hydrolysis of acylcholines.

Chondrosarcoma: A malignant neoplasm derived from cartilage cells, occurring most
      frequently near the ends of long bones.

Chromatid:  Each of the two strands formed by longitudinal duplication of a chromosome that
      becomes visible during an early stage of cell division.

Chromophore: A chemical group that produces color in a molecule by absorbing near
      ultraviolet or visible radiation when bonded to a nonabsorbing, saturated residue which
      possesses no unshared, nonbonding valence electrons.

Chromosome: One of the bodies (46 in  man) in the cell nucleus that is the bearer and carrier
      of genetic information.

Chronic obstructive pulmonary disease (COPD):  This term refers to diseases of uncertain
      etiology characterized by persistent slowing of airflow during forced expiration. It is
      recommended  that a more specific term,  such as chronic obstructive bronchitis or
      chronic obstructive emphysema, be used whenever possible.  Synonymous with  chronic
      obstructive lung disease (COLD).               "•  '

Cilia:  Motile, often  hairlike extensions of a cell surface.

Ciliary action: Movements of cilia in the upper respiratory tract, which move mucus and
      foreign material upward.

Ciliogenesis:  The formation of cilia.

Citric acid (Krebs) cycle:  A major biochemical pathway in cells, involving terminal oxidation
      of fatty acids and carbohydrates. It yields a major portion of energy needed for essential
      body functions and  is the major source of CO2. It couples the glycolytic breakdown of
      sugar in the cytoplasm with those reactions producing ATP in the mitochondria. It also
      serves to regulate the synthesis of a number of compounds required by a cell.

Clara cell: A nonciliated cell in the epithelium of the respiratory tract.

Closing capacity (CC):  Closing volume plus residual volume, often expressed as a ratio of
      TLC (i.e., CC/TLC%).

Closing volume  (CV):  The volume exhaled after the expired gas concentration is inflected
      from an alveolar plateau during a controlled breathing maneuver.  (Most commonly
      obtained during a single-breath nitrogen washout test.)  Since the value obtained is
      dependent on  the specific test technique, the method used must be designated in the
      text, and when necessary, specified by a qualifying symbol.  Closing volume is often
      expressed as a ratio of the vital capacity (i.e., CV/VC%).
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Codon:  A sequence of three nucleotides which encodes information required to direct the
       synthesis of one or more amino acids.                               .,...,"

Coefficient of haze (COH):  A measurement of visibility interference in the atmosphere.

Cohort:  A group of individuals or vital statistics about them having a statistical factor in
       common in a demographic study (e.g., year of birth, sex, level of exposure to a'
       pollutant, etc.).                                                  .    .-,.•..,

Collagen: The major protein of the white fibers of connective tissue, cartilage,  and bond^
       Comprises over half the protein of the mammal.

Collisional deactivation:  Reduction in energy of excited molecules caused by collision with
       other molecules or other objects such as the walls of a container.

Colorimetric:  A chemical analysis method relying on measurement of the degree of color
       produced in a solution by reaction with the pollutant of interest.

Community  exposure:  A situation in which people in a sizeable area are subjected to ambient
       pollutant concentrations.

Compliance (CL,Cst): A measure of distensibility.  Pulmonary compliance is given by the
       slope of a static volume-pressure curve at a point, or the linear approximation of a
       nearly straight portion of such a curve, expressed as the change in volume per unit
       change in distending pressure in L/cm H2O or mL/cm H2O.  Since the static volume-  '
       pressure characteristics of lungs are nonlinear (static compliance decreases as lung
       volume increases) and vary according to the previous volume history (static compliance
       at a given volume increases immediately after full inflation and decreases following  '
       deflation), careful specification of the conditions of measurement are necessary.
       Absolute values also depend  on organ size.  See also Dynamic compliance.

Complement:  Thermolabile substance present in serum that is destructive to certain bacteria
       and other cells which  have been sensitized by specific complement-fixing  antibody-.

Compound:  A substance with its own distinct properties, formed by the chemical combination
       of two or more elements in fixed proportion.

Concanavalin-A:  One of two crystalline globulins occurring in the jack bean; a  potent
       hemagglutinin.

Conductance (G):  The reciprocal of resistance.  See Airway conductance.

Conifer:  A  plant, generally  evergreen,  needle-leafed, bearing naked seeds singly or in cones.

Converter: See catalytic converter.

Coordination number: The number of bonds formed by the central atom in a complex.

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Copolymer:  The product of the process of polymerization in which two or more monomeric
      substances are mixed prior to polymerization.  Nylon is a copolymer.

Coproporphyrin: 'One of two porphyrin compounds found normally in feces as a
      decomposition product of bilirubin (a bile pigment).  Porphyrin is a widely-distributed
      pigment consisting of four pyrrole nuclei joined in a ring.

Cordage:  A general term which includes banding, cable, cord, rope, string, and twine made
      from fibers.   Synthetic fibers used in making cordage include nylon and dacron.

Corrosion: -Destruction or deterioration of a material because of reaction with its environment.

Corticosterone:  A  steroid obtained from the adrenal cortex. It induces some deposition of
      glycogen in the liver, sodium conservation, and potassium excretion.

Cosmopolitan:  In the biological sciences, a term denoting worldwide distribution.

Coulometric:  Chemical  analysis performed by determining the amount of a substance released
      in electrolysis by measuring the number of coulombs used.

Coumarin: A toxic white crystalline lactone (C9H6O2) found in plants.

Coupler:  A chemical used to combine two others in a reaction (e.g., to produce the azo dye
      in the Griess-Saltzman method for NO2).

Crevice corrosion:   Localized corrosion occurring within crevices on metal 'surfaces exposed to
      corrosives.                        '

Critical Load: A quantitative estimate of an exposure to one or more pollutants below which
      significant harmful effects on specified sensitive elements of the ecosystem do not occur
      according to present knowledge.

Crosslink:  To connect, by  an atom or molecule, parallel chains in a complex chemical
      molecule, such as a polymer.

Cryogenic trap: A pollutant sampling method in which  a gaseous pollutant is condensed out of
      sampled air by cooling (e.g., traps in'one method for nitrosamines are maintained below
      -79°C, usirig solvents maintained at their  freezing points).

Cuboidal:  Resembling a cube in shape.

Cultivar:  An organism produced by parents belonging to different species or to different
      strains of the same species, originating and persisting under cultivation.'

Cuticle: A thin outer layer, such as the thin continuous fatty film on the surface of many
      higher plants.
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 Cyanosis:  A dark bluish or purplish coloration of the skin and mucous membrane due to
       deficient oxygenation of the blood.

 Cyclic GMP: Guanoshie 5'-phosphoric acid.

 Cytochrome:  A class of hemoprotein whose principal biological function is electron and/or
       hydrogen transport.                                                               ;

 Cytology:  The anatomy, physiology, pathology and chemistry of the cell.

 Cytoplasm: The substance of a cell exclusive of the nucleus.

 Dacron:  The trade name for polyester fibers made by E.I. du Pont de Nemours and Co.,
       Inc., made from dimethyl terephthalate and ethylene glycol.

 Dark adaptation:  The process by which the eye adjusts under reduced illumination and the
       sensitivity of the eye to light is' greatly increased.             . .

 Dark respiration: Metabolic activity of plants at night; consuming oxygen to use stored sugars
      and  releasing carbon dioxide.

 Deciduous  plants:  Plants which drop their leaves at the end of the growing  season.

 Degradation (textiles):  The decomposition of fabric or its components or characteristics
      (color, strength, elasticity) by means of light, heat, or air pollution.

 Denitrification: A bacterial process occurring in soils, or water, in which nitrate is used as the
      terminal electron acceptor and is reduced primarily to N2.  It is essentially an anaerobic
      process, it can occur in the presence of low levels of oxygen only 'if the microorganisms
      are metabolizing in an anoxic microzone.

De novo:   Over again.

Deoxyribonucleic acid (DMA):  A nucleic acid considered to be the carrier of genetic
      information coded in the  sequence of purine and pyrimidine bases (organic bases).
      It has the form of a double-stranded helix of a linear polymer.

Depauperate: Falling short of natural development or  size.

Deposition:

      Acidic: Removal of acidic pollutants from the atmosphere by  dry and wet deposition.

      Dry: Removal of pollutants from the atmosphere through interactions with various
      surfaces of plants, land, and water.
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      Respiratory tract: . The depositing of inhaled pollutants within the respiratory tract-which
      depends on breathing patterns, airway geometry, and the physical and chemical
      properties of the inhaled pollutants.

      Wet:  Removal of pollutants from the atmosphere by precipitation (e.g., rain or snow).

Derivative spectrophotometer:  An instrument with an increased capability for detecting
      overlapping spectral  lines and bands and also for suppressing instrumentally scattered
      light.                                              .,.'.."..•-.•

Desorb:  To release a substance which has been taken into another substance or held on its
      surface; the opposite of absorption or adsorption.

Desquamation:  The sheading of the outer layer of any surface.

Detection limit:  A level below which an element or chemical compound cannot be reliably
      detected by the method or measurement being used for analysis.  :

Detritus: Loose material that results directly from disintegration.

DeVarda alloy:  An alloy of 50 percent Cu, 45 percent Al, 5 percent Zn.

Diastolic blood pressure: The blood pressure as measured during the period of filling the
      cavities of the heart with blood.                  ,                                :

Diazonium salt:  A chemical compound (usually colored) of the general structure ArN2+Cl",
      where Ar refers to an aromatic  group.

Diazotizer:  A chemical which, when  reacted with amines  (RNH2, for example), produces a
      diazonium salt (usually a colored compound).                    ,    ,   ;

Dichotomous sampler:  A device used to collect separately fine and coarse particles from an
      aerosol and to measure gravimetrically the concentration of such different-sized particles
      in the ambient air.

Differentiation:  The process by which a cell, such as a fertilized egg, divides into specialized
      cells, such as the embryonic types that eventually develop into an entire organism.

Diffusion:  The process by which molecules or other particles intermingle as a result of their
      random thermal motion.                                                      ,     ,
                                                          f
Diffusing capacity of the lung  (DL, DLO2, DLCO2, DLCO): Amount of gas (O2,  CO, CO2)
      commonly expressed as milliliters of gas (STPD) diffusing between alveolar gas and
      pulmonary capillary  blood per torr mean gas pressure difference per minute, such as
      mL O2/(min-torr).  Synonymous with transfer  factor and diffusion factor.

Dimer:  A compound formed by the union of two like radicals or molecules.

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 Dimerize:  Formation of dimers.                                            •  ,

 1,6-diphosphofructose aldolase: An enzyme (EC 4.1.1.13) cleaving fructose 1,6-bisphosphate
       to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate;

 D-2,3-diphosphoglycerate:  A salt or ester of 2,3-diphosphoglyceric acid, a major component
       of certain mammalian erythrocytes involved in the release of O2 from HbO2.  Also a
       postulated intermediate in the biochemical pathway involving the conversion of 3- to
       2-phosphoglyceric acid.

 Diplococcus pneumonias:  A species of spherical-shaped bacteria belonging to the genus
       Streptococcus. May be a causal agent in pneumonia.

 Direct dye: A dye with an affinity for most fibers; used  mainly when color resistance to
       washing is not important.

 Disperse dyes:  Also known as acetate dyes; these dyes were developed for use on acetate
       fabrics, and are now also used on synthetic fibers.

 Distal:  Far from some reference point such as median line of the body, point of attachment or
       origin.

 Diurnal:  Having a repeating pattern or cycle 24 hours long.

 DLCO: The diffusing capacity of the lungs for carbon monoxide.  The ability of the lungs to
       transfer carbon monoxide from the alveolar air into the pulmonary capillary blood.

 Dorsal hyphosis: Abnormal curvative of the spine; hunch-back.

 Dose:  The quantity of a substance to betaken all at one time or in fractional amounts within a
      given period;  also the total amount of a pollutant delivered or concentration per unit
      time times time.

 Dose-response curve: A curve on a graph based on responses occurring in a system as a result
      of a series of stimuli intensities or doses.

 Dry deposition:  The processes by which.matter is transferred to ground from the atmosphere,
      other than precipitation; includes surface absorption of gases and sedimentation,    "
      Brownian diffusion and impaction of particles.

Dyeing:  A process of coloring fibers, yarns, or fabrics with either natural or synthetic dyes.

Dynamic calibration: Testing of a monitoring system using a continuous sample stream of
      known concentration.                                                            '
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 Dynamic compliance (Cd  ):  the ratio of the tidal volume to the change in intrapleural
       pressure between the points of zero flow at the extremes of tidal volume in L/cm H2O
       or mL/cm H2O.  Since at the points of zero airflow at the extremes of tidal volume,
       volume acceleration is  usually other than zero, and since, particularly in abnormal
       states, flow may still be taking place within lungs between regions which are
       exchanging volume.  Dynamic compliance may differ from static compliance, the latter
       pertaining to condition of zero volume acceleration and zero gas flow throughout the
       lungs. In normal lungs at ordinary volumes and respiratory frequencies, static and
       dynamic compliance are the same.

 Dynel:  A trademark for a modacrylic staple fiber spun from a copolymer of acrylonitrile and
       vinyl chloride.  It has high strength, quick-drying properties, and resistance to alkalies
       and acids.

 Dyspepsia:   Indigestion, upset stomach.

 Dyspnea:  Shortness of breath; difficulty or distress in breathing; rapid breathing.

 Ecosystem:  The interacting system of a biological community and its environment.

 Eddy:  A current of water or  air running contrary to the main current.

 Edema:  Pressure of excess fluid in cells, intercellular tissue or cavities of the body.

 Elastance (E): The reciprocal of compliance; expressed in cm H2O/L or cm H2O/mL.

 Elastomer:  A synthetic rubber product which has the physical properties of natural rubber.

 Electrocardiogram: The graphic record of the electrical currents that initiate the heart's
    ;   contraction.                                     .

 Electrode:  One of the two extremities of an electric circuit.

.Electrolyte:  A non-metallic electric  conductor in which-current is carried by the movement of
       ions; also a substance which displays these qualities when dissolved in water or another
       solvent.

 Electronegativity: Measure of affinity for negative charges or electrons.

 Electron microscopy:  A technique which utilizes a focused beam of electrons to produce a
       high-resolution image of minute objects such as paniculate matter, bacteria, viruses, and
       DNA.

 Electronic excitation energy:   Energy associated in the transition of electrons from their normal
       low-energy orbitals or orbitals of higher energy.

 Electrophilic: Having an affinity for electrons.

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Electrophoresis:  A technique by which compounds can be separated from a complex, .mixture
      by their attraction to the positive or negative pole of an applied electric potential.

Eluant:  A liquid used in the process of elution.

Elute:  To perform an elution.

Elution:  Separation of one material from another by washing or by dissolving one in a solvent
      in which the other is not soluble.

Elutriate:  To separate a coarse, insoluble powder from a finer one by suspending them
      in water and pouring off the finer powder from the upper part of the fluid.

Emission spectrometry:  A rapid analytical technique based on measurement of the   .
      characteristic radiation emitted by thermally or electrically excited  atoms or ions.

Emphysema: A condition of the lung characterized by abnormal, permanent enlargement
      of airspaces distal to the terminal bronchiole, accompanied by the destruction of
      their walls, and without obvious fibrosis.

Emphysematous lesions:  A wound or injury to the lung as a result  of emphysema.

Empirical modeling: Characterization  and description of a phenomena based on experience or
      observation.

Encephalitis: Inflammation of the brain.

Endoplasmic reticulum:  An elaborate membrane structure extending from the nuclear
      membrane or eucaryotic cells  to the cytoplasmic membrane.

Endothelium:  A layer of flat cells lining especially blood and lymphatic  vessels.

Entropy:  A measure of disorder or randomness in a system.  Low  entropy is associated  with
      highly ordered systems.                                      ,            .

Enzyme:  Any of numerous  proteins produced by living cells which catalyze biological
      reactions.                                                                ,  ,

Enzyme Commission (EC):  The International Commission on Enzymes,  established in 1956,
      developed a scheme of classification and nomenclature under which each enzyme is
      assigned an EC number which identifies it by function.

Eosinophils: Leukocytes (white blood cells) which stain readily with the dye, eosin.

Epidemiology: A study of the distribution and determinants of disease in human population
      groups.


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 Epidermis: The outermost living layer of cells of any organism.        '

 Epididymal fat pads:  The fatty tissue located near the epididymis.  The epididymis is the first
       convoluted portion of the excretory duct of the testis.                    ;

 Epiphyte:  A plant growing on another plant but obtaining food from the atmosphere.

 Epithelial:  Relating to epithelium, the membranous cellular layer which covers free surfaces
       or lines tubes or cavities of an animal body, which encloses, protects, secretes, excretes
       and/or assimilates.

 Erosion corrosion: Acceleration or increase in rate of deterioration or attack on a metal
       because of relative movement between a corrosive fluid and the metal surface.
       Characterized by grooves, gullies, or waves in  the metal surface.

 Erythrocyte: A mature red blood cell.

 Escherichia coll:  A short, gram-negative, rod-shaped bacteria common to the human intestinal
       tract.  A frequent cause of infections in  the urogenital tract.

 Esophageal:  Relating to the portion of the digestive tract between the pharynx and the
       stomach.

 Estrus:  That portion or phase of the sexual cycle of female animals characterized by
       willingness to permit coitus.

 Estrus cycle:  The series of physiologic uterine, ovarian and other changes that occur in higher
       animals.                      •

 Etiolation:  Paleness and/or altered development resulting from the absence of light.

 Etiology:  The causes of a disease or condition; also, the study of causes.

 Eucaryotic: Pertaining to those cells having a well-defined nucleus surrounded by a
       double-layered membrane.

 Euthrophication:  Elevation of the level of nutrients in a body of water, which can contribute
       to accelerated plant growth and filling.

 Excited state:  A state of higher electronic energy than the ground state, usually a less stable
       one.

Expiratory  (maximum) flow rate:  The maximum rate  at which air can be expelled from the
       lungs.

Exposure level:  Concentration of a contaminant to which an individual or a population is
       exposed.

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Extinction coefficient:  A measure of the space rate of diminution, or extinction, of any
      transmitted light, thus, it is the attenuation coefficient applied to visible radiation.

ExtrameduUary hematopoiesis:  The process of formation and development of the various types
      of blood cells and other formed elements not including that occurring  in bone marrow.

Extravasate:  To exclude from or pass out of a vessel into the tissues; applies to urine,  lymph,
      blood and similar fluids.

Far ultraviolet: Radiation in the range of wavelengths from 100 to 190 nanometers.

Federal Reference Method (FRM):  For NO2, the EPA-approved analyzers based on the gas-
      phase chemiluminescent measurement principle and associated calibration procedures;
      regulatory specifications prescribed in Title 40, Code of Federal Regulations, Part 50,
      Appendix F.

Fenestrae:  Anatomical aperatures often  closed by a membrane.

FEVt/FVC:  A ratio of timed (t = 0.5,  1, 2,  3 s) forced expiratory volume  (FEVt) to forced
      vital capacity (FVC).  The ratio is often expressed in percent 100 x FEVt/FVC.  It is an
      index of airway obstruction.

Fiber:  A fine, threadlike piece, as of cotton, jute, or asbestos.

Fiber-reactive dye:  A water-soluble dyestuff which reacts chemically with the cellulose in
      fibers under alkaline conditions; the dye contains two chlorine atoms which combine
      with the hydroxyl groups of the cellulose.

Fibrin:  A white insoluble elastic filamentous protein derived from fibrihogen by the action of
      thrombin, especially in the clotting of blood.

Fibroadenoma: A benign neoplasm derived from  glandular epithelium, involving proliferating
      fibroblasts, cells found in connective tissue.

Fibroblast: An elongated cell with cytoplasmic processes present in connective tissue, capable
      of forming collagen fibers.

Fibrosis: The formation of  fibrous tissue, usually as a reparative or reactive process and  not
      as a normal constituent of an organ or tissue.

Fine particles:  Airborne particles smaller than 2 to 3 ^m in aerodynamic diameter.
Flocculation:  Separation of material from a solution or suspension by reaction with a
      flocculant to create fluffy masses containing the material to be removed.
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 Flow volume curve:  Graph of instantaneous forced expiratory flow recorded at the mouth,
       against corresponding lung volume. When recorded over the full vital capacity, the
       curve includes maximum expiratory flow rates at all lung volumes in the vital capacity
       range and is called a maximum expiratory flow-volume curve (MEFV).  A partial
       expiratory flow-volume curve (PEFV) is one which describes maximum expiratory flow
       rate over a portion of the vital capacity only.

 Fly ash:  Fine, solid particles of noncqmbustible ash carried out of a bed of solid fuel by a
       draft.

 Fogs: Suspension of liquid droplets formed by condensation of vapor or atomization; the
       concentration of particles is sufficiently high to obscure visibility.

 Folded-path optical system:  A long (e.g., 8-22 m) chamber with multiple mirrors at the ends
       which can be used to reflect an infrared beam through an ambient air sample many
       times; a spectrometer can be used with such a system to detect trace pollutants at very
       low levels.

 Forced expiratory flow (FEF): Related to some portion of the forced vital capacity (FVC)
       curve.  Modifiers refer to the amount of the FVC already exhaled when  the
       measurement is made.  For example:

       FEF75% = Instantaneous forced exhaled flow after 75% of the forced vital capacity has
       been exhaled.

       FEF200-1200  = Mean forced expiratory flow between 200 mL and 1200 mL of the
       forced vital capacity (formerly called the maximum expiratory flow rate [MEFR]).

       FEF25-75% = Mean forced expiratory flow during the middle half of the forced vital
       capacity (formerly called the maximum mid-expiratory flow rate [MMFR]).
      FEF
      capacity.
max = The maximal forced expiratory flow achieved during an forced vital
Forced expiratory volume (FEV):  Denotes the volume of gas which is exhaled in a given time
      interval from the beginning of the execution of a forced vital capacity. Conventionally,
      the times used are 0.5, 0.75, or 1 sec, symbolized FEV0 5, FEV0 75, FEVj 0. These '
      values are often expressed as a percent of the forced vital capacity; for example
      (FEVj o/FVC) X 100.

Forced inspiratory vital capacity (FIVC):  The maximal volume of air inspired with  a
      maximally forced effort from a position of maximal expiration.

Forced vital capacity (FVC):  The maximum volume of air that can be forcibly expelled from
      the lungs after the deepest inspiration.
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Fractional threshold concentration:  The portion of the concentration at which an event or a
      response begins to occur, expressed as a fraction.

Free radical:  Any of a variety of highly-reactive atoms or molecules characterized by having
      an unpaired electron.

Fritted bubbler:  A porous glass device used in air pollutant sampling systems to introduce
      small bubbles into solution.

Functional residual capacity (FRC):  The volume of gas remaining in the lungs at the end of a
      normal expiration. It is the sum of expiratory reserve volume and residual volume (see
      Pulmonary measurements).

Gas chromatography (GC):  A method of separating and analyzing mixtures of chemical
      substances.  A flow of gas causes the components of a mixture to migrate differentially
      from a narrow starting zone in a special porous,  insoluble sorptive medium.  The
      pattern formed by zones of separated pigments and of colorless substances in this
      process is called a chromatogram,  and can be analyzed to obtain the concentration of
      identified pollutants.

Gas exchange:  Movement of oxygen from the alveoli into the pulmonary capillary blood as
      carbon dioxide enters the alveoli from the blood. In broader terms, the exchange of
      gases between alveoli and lung capillaries.

Gas-liquid chromatography:  A method of separating and analyzing volatile organic
      compounds, in which a sample is vaporized and  swept through a column filled with
      solid support material covered with a nonvolatile liquid.   Components of the sample can
      be identified and their concentrations determined by analysis of the characteristics of
      their retention in the column, since compounds have varying degrees of solubility in the
      liquid medium.

Gas trapping:  Trapping of gas behind small airways that were opened during inspiration but
      closed during forceful expiration.  It is a volume difference between forced vital
      capacity and vital capacity.

Gastric juice:  A thin watery digestive fluid secreted by glands in the mucous membrane of the
      stomach.

Gastroenteritis:  Inflammation of the mucous membrane of stomach  and intestine.

Genotype: The type of genes possessed by an organism.

Geometric mean:  An estimate of the average of a distribution.  Specifically, the nth root of
      the product of n observations.

Geometric standard deviation:  A measure of variability of a distribution.   It is the
      antilogarithm of the standard deviation of the logarithms of the observations.

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Globulins (a, b, q):  A family of proteins precipitated from plasma (or serum) by
      half-saturation with ammonium sulfate, or separable by electrophoresis. The main
      groups are the a, b, q fractions, differing with respect to associated lipids and
      carbohydrates and in their content of antibodies (immunoglobulins).

Glomular nephrotic syndrome:  Dysfunction of the kidneys characterized by excessive protein
      loss in the urine, accumulation of body fluids and alteration in albumin/globulin ratio.

Glucose:  A sugar which is a principal source of energy for man and other organisms.

Glucose-6-phosphatedehydrogenase:  An enzyme (EC 1.1.1.49) catalyzing the
      dehydrogenation of glucose-6-phosphate to 6-phospnogluconolactone.

Glutamic-oxaloacetic transaminase (SGOT): An enzyme (EC 2.6.1.1) whose  serum level
      increases in myocardial infarction and in diseases involving destruction of liver cells.
      Also known as aspartate aminotransferase.

Glutamic-pyruvic transaminase (SGPT):  Now known as alanine aminotransferase
      (EC 2.6.1.2), the serum levels of this enzyme are used in liver function tests.

Glutathione (GSH):  A tripeptide composed of glycine, cystine, and glutamic  acid.

Glutathione peroxidase: An enzyme (EC 1.11.1) which catalyzes the destruction of
      hydroperoxides formed from fatty acids and other substances.  Protects tissues from
      oxidative damage. It is a selenium-containing protein.                         ,

Glutathione reductase:  The enzyme (EC 1.6.4.2) which reduces the oxidized  form of
      glutathione.

Glycolytic pathway:  The biochemical pathway by which glucose is converted to lactic acid in
      various tissues, yielding energy as a result.

Glycoside: A type of chemical compound formed from the condensation of a sugar with
      another chemical radical via a hemiacetal linkage.

Goblet cells:  Epithelial cells that have been distended with mucin and when this is discharged
      as mucus, a goblet-shaped shell remains.

Golgi apparatus:  A membrane system involved with secretory functions and transport in a
      cell. Also known as a dictyosome.

Grana:  The lamellar stacks of chlorophyll-containing material in plant chloroplasts.

Griege carpet:   A carpet in its unfinished state (i.e., before it has been scoured and dyed).
      The term also is used for woven fabrics in the unbleached and unfinished state.

Ground state:  The state of minimum electronic energy of a molecule or atom.

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Guanylate cyclase (GC):  An enzyme (EC 4.6.2.1) catalyzing the transformation of guanosine
      triphosphate to guanosine 3':5'-cyclic phosphate.

H-Thymidine: Thymine deoxyribonucleoside:  One of the four major nucleosides in DNA.
      3H-thyrnidine has been uniformly labeled with tritium, a radioactive form of hydrogen.

Haze:  Fine dust, smoke or fine vapor reducing transparency of air.

Hemagglutination:  The agglutination of red blood cells.  Can be used as a measurement of
      antibody concentration.                                                        ^

Hematocrit: The percentage of the volume of a blood sample occupied by cells.

Hematology: The medical specialty that pertains  to the blood and blood-forming tissues.

Hemochromatosis: A disease characterized by pigmentation of the skin possibly due to
      inherited excessive absorption of iron.

Hemoglobin (Hb): The red, respiratory protein of the red blood cells, hemoglobin transports
      oxygen from the lungs to the tissues as oxyhemoglobin (HbO^ and returns carbon
      dioxide to the lungs as hemoglobin carbamate, completing the respiratory cycle.

Hemolysis:  Alteration or destruction of red blood cells, causing hemoglobin to be released
      into the medium in which the cells are suspended.

Hepatectomy: Complete removal of the liver in an experimental animal.     ......

Hepatic:  Relating to the liver.

Hepatocyte:  A liver cell.

Heterogeneous process: A chemical reaction involving reactants of more than one phase or
      state, such as one in which gases are absorbed into aerosol droplets, where the reaction
      takes place.

Heterologous: A term referring to donor and recipient cellular elements from different
      organisms, such as red blood cells from sheep and alveolar macrophage from rabbits.

Heterotrophs: Fungi and bacteria that rely on organic matter for their energy source.

Hexose monophosphate shunt:  Also called the phosphogluconate oxidative pathway of glucose
      metabolism which affords a total combustion of glucose independent of the  citric acid
      cycle. It is the important generator of NADPH necessary for synthesis of fatty acids
      and the operation of various enzymes.  It serves as a source of ribose and 4- and
      7-carbon sugars.
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High volume (hi-vol) sampler: A high flow-rate device used to collect particles from the
       atmosphere and to measure gravimetrically the concentration of particles across a broad
       range of sizes in ambient air.

Histamine:  A  depressor amine derived from the amino acid histidine and found in all body
       tissues, with the highest concentration in the lung; a powerful stimulant of gastric
       secretion, a constrictor of bronchial smooth muscle, and a vasodilator that causes a fall
       in blood pressure.

Homogenate: Commonly refers to tissue ground into a creamy consistency in which the cell
       structure is disintegrated.

Host defense mechanism: Inherent means by which a biologic organism protects itself against
       infection, such as antibody formation, macrophage action, ciliary action, etc.

Host resistance: The resistance exhibited by an organism, such as man, to an infecting agent,
       such as a virus or bacteria.

Humoral: Relating to the extracellular fluids of the body, blood and lymph.

Hybrid:  An organism descended from parents belonging to different varieties or species.

Hydrocarbons:  A vast family of compounds containing carbon and hydrogen in various
       combinations; found especially in fossil fuels.   Some contribute to  photochemical smog.

Hydrolysis:   Decomposition involving splitting of a bond and addition of the H and OH parts
       of water to the two sides of the split bond.

Hydrometeor:  A product of the condensation of atmospheric water vapor (e.g., fog, rain,
       hail, snow).

Hydroxyproline:  An amino acid found among the hydrolysis products of collagen.

Hygroscopic: Pertaining to a marked ability to accelerate the condensation of water vapor.

Hygroscopic growth:  Growth induced by moisture; often applied in reference to the growth in
       size of inhaled particles within the respiratory tract in combination with resident
       moisture.

Hyperplasia:  Increase in the number of cells in a tissue or organ excluding tumor formation.

Hyperplastic:  Relating to hyperplasia; an increase in the number of cells.

Hypertrophy: Increase in the size of a tissue element, excluding tumor formation.

Hypertension:  Abnormally elevated blood pressure.
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Hypolimnia:  Portions of a lake below the thermocline, in which water is stagnant and uniform
      in temperature.

Hypoxia:  A lower than normal amount of oxygen in the air, blood or tissues.

Immunoglobulin (Ig): A class of structurally related proteins consisting of two pairs of
      polypeptide chains. Antibodies are Ig's and all Is's probably function as antibodies.

Immunoglobulin A (IgA): A type of antibody which comprises approximately 10 to 15% of
      the total amount of antibodies present in normal serum.

Immunoglobulin G (IgG): A type of antibody which comprises approximately 80% of the
      total amount of antibodies present in normal serum.  Subtractions of IgG are fractions
      Gi, and G2-                                                       ...-..,

Immunoglobulin M (IgM):  A type of antibody which comprises approximately 5 to 10% of
      the total amount of antibodies present in normal serum.

Impaction: An impinging or striking of one object against another; also, the force transmitted
      by this act.

Impactor:  An instrument which collects samples of suspended particulates by directing a
      stream of the suspension against a surface, or into a liquid or a void.

Index of proliferation:  Ratio of promonocytes to polymorphic monocytes in the blood.

Infarction: Sudden insufficiency of arterial or venous blood supply due to emboli, thrombi, or
      pressure.

Infectivity model: A testing system in which the susceptibility of animals to airborne
      infectious agents with and without exposure to air pollutants is investigated to produce
      information related to the possible effects of the pollutant on man.

Inflorescence:  The arrangement and development of flowers on an axis; also, a flower cluster
      or a single flower.

Influenza A2/Taiwan Virus:  An infectious-viral disease, believed to have originated in
      Taiwan, characterized by sudden  onset, chills, fevers, headache, and cough.
                                                                        1        *3
Infrared: Light invisible to the human eye, between the wavelengths of 7 x 10"' and 10" m
       (7,000 and 10,000,000 A).

Infrared laser: A device that utilizes the natural oscillations of atoms or molecules to generate
       coherent electromagnetic radiation in the infrared region of the spectrum.

Infrared spectrometer:  An instrument for measuring the relative amounts of radiant energy in
       the infrared region of the spectrum as a function  of wavelength.

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Ingestion:  To take in for digestion.

In situ: In the natural or original position.

Instrumental averaging time:  The time over which a single example or measurement is taken,
       resulting in a measurement which is an average of the actual concentrations over that
       period.

Insult:  An injury or trauma.

Intercostal:  Between the ribs, especially of a leaf.

Interferant:  A substance which a measurement method cannot distinguish completely from the
       one being measured, which therefore can cause some degree of false response or error.

Interferon:  A macromolecular substance produced in response to infection with active or
       inactivated virus, capable of inducing a state of resistance.

Intergranular corrosion:  A type of corrosion which takes place at and adjacent to grain
       boundaries, with relatively little corrosion of the grains.

Interstitial edema: An accumulation of an excessive amount of fluids in a space within tissues.

Interstitial pneumonia:  A chronic inflammation of the interstitial tissue of the lung, resulting
       in compression of air cells.

Intraluminal mucus:  Mucus that collects within any tubule.

Intraperitoneal injection:  An injection of material into the serous sac that lines the abdominal
       cavity.

In utero:  Within the womb; not yet born.

In vitro: Refers to experiments conducted outside the living organism.

In vivo:  Refers to experiments conducted within the living organism.

Irradiation:  Exposure to any form of radiation.

Ischemia:  Local anemia due to mechanical obstruction (mainly arterial narrowing)  of the
       blood supply.

Isoenzymes: Also called isozymes.  One of a group of enzymes that are very similar in
       catalytic properties, but may be differentiated by variations in physical properties, such
       as isoelectric point or electrophoretic mobility.  Lactic acid dehydrogenase is an
       example of an enzyme having many isomeric forms.
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Isopleth: A line on a map or chart connecting points of equal value.

Jacobs-Hochheiser method:  The original Federal Reference Method for NO2 currently
      unacceptable for air pollution work.

Klebsiella pneumoniae:  A species of rod-shaped bacteria found in soil,  water, and in  the
      intestinal tract or man and other animals. Certain types may be causative agents in
      pneumonia.                                          '

Kyphosis:  An abnormal curvature of the spine, with convexity backward.               .   ..,, ,

Lactate:  A salt or ester of lactic acid.

Lactic acid (lactate) dehydrogenase (LDH): An enzyme (EC 1.1.1.27) with many isomeric
      forms which catalyzes the oxidation of lactate to pyruvate via transfer of H to NAD.
      Isomeric forms of LDH in the blood are indicators of heart damage.

Lamellar bodies:  Arranged  in plates or scales.  One of the characteristics of Type II alveolar
      cells.                                                                      :

Lavage fluid:  Any fluid used to wash out hollow  organs, such as the lung.

Leaching:  The removal of elements from soil, litter, or plant foliage by water.

Lecithin:  Any of several waxy hygroscopic phosphatides that are widely distributed in animals
      and plants; they form colloidal solutions in  water and have emulsifying, wetting and
      hygroscopic properties.

Legume:  A plant with root  nodules containing nitrogen fixing bacteria.

Lesion: A wound, injury or other more or less circumscribed pathologic change in  the tissues.

Leukocyte:  Any of the white blood cells.

Lewis base: A base, defined hi the Lewis acid-base concept, is a substance that can donate an
      electron pair.

Lichens: Perennial plants which are a combination of two plants, an  alga and a fungus,
      growing together in an association so intimate that they appear as one.

Ligand:  Those molecules or anions attached to the central atom in a  complex.

Light-fastness:  The ability of a dye to maintain its original color under natural or indoor light.

Linolenic acid: An unsaturated fatty acid essential in nutrition.
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 Lipase:  An enzyme that accelerates the hydrolysis or synthesis of fats or the breakdown of
       lipoproteins.

 Lipids:  A heterogeneous group of substances which occur widely in biological materials. They
       are characterized as a group by their extractability in nonpolar organic solvents.

 Lipofuscin: Brown pigment granules representing lipid-containing residues of lysosomal
       digestion.  Proposed to be an end product of lipid oxidation which accumulates in
       tissue.

 Lipoprotein: Complex or protein Containing lipid and protein.

 Loading rate:  The amount of a nutrient available to a unit area of body of water over a given
       period of time.

 Locomotor activity:  Movement of an organism from one place to another of its own volition.

 Long-pathlength infrared absorption: A measurement technique in which a system of mirrors
       in a chamber is used to direct an infrared beam through a sample of air for a long
       distance (up to 2 km);  the amount of infrared absorbed is measured to obtain the
       concentrations of pollutants  present.

 Lung compliance (CI):  The volume change produced by an increase in a unit change in
       pressure across the lung (i.e., between the pleura! surface and the mouth).

 Lycra:  A spandex textile fiber created by E.  I. du Pont de Nemours & Co., Inc., with
       excellent tensile strength, a long flex life and high resistance to abrasion and heat
       degradation.  Used in brassieres, foundation garments, surgical hosiery, swim suits, and
       military and industrial uses.

 Lymphocytes:  White blood cells formed in lymphoid tissue throughout the body, they
       comprise about 22 to 28% of the total number of leukocytes in the circulating blood and
       function in immunity.

 Lymphocytogram: The ratio, in the blood, of lymphocyte with narrow  cytoplasm to those
       with broad cytoplasm.

 Lysosomes:  Organelles found in cells of higher organisms that contain high concentrations of
       degradative enzymes and are known to destroy foreign substances that cells engulf by
      pinocytosis and phyocytosis.  Believed to be a major site where proteins are broken
      down.

 Lysozymes:  Lytic enzymes destructive to cell walls of certain bacteria.  Present in some body
      fluids, including tears and serum.

Macaca speciosa:  A species of monkeys used in research.
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Macrophage:  Any large, ameboid, phagocytic cell having a nucleus without many lobes,
      regardless of origin.

Malaise:  A feeling of general discomfort or uneasiness, often the first indication of an
      infection or disease.

Malate dehydrogenase:  An enzyme (EC 1.1.1.37) with at least six isomeric forms that
      catalyze the  dehydrogenation of malate to oxaloacetate or its decarboxylation (removal
      of a CO2, group) to pyruvate. Malate, oxaloacetate, and pyruvate are intermediate
      components  of biochemical pathways.

Mannitol: An alcohol derived from reduction of the sugar, fructose.  Used in renal function
      testing to measure glomerular (capillary) filtration.

Manometer:  An instrument for the measurement of pressure of gases or vapors.

Mass median diameter (MMD):  Geometric median size of a distribution of particles based on
      weight.

Mass spectrometry (MS):  A procedure for identifying the various kinds of particles present in
      a given substance, by ionizing the particles and subjecting a beam of the ionized
      particles to an electric or magnetic field such that the field deflects the particles in
      angles directly proportional to the masses of the particles.

Maximal expiratory flow Cv^^ x):  Forced expiratory flow, related to the total lung capacity
      or the actual volume of the lung at which the measurement is made.  Modifiers refer to
      the amount of lung volume remaining when the measurement is made.  For example:

      ^max75% =  Instantaneous forced expiratory flow when the lung is at 75% of its total
      lung capacity.

      'v'maxS 0 =  Instantaneous forced expiratory flow when the lung volume is 3.0 L

Maximal expiratory flow rate (MEFR):  Obsolete terminology.  See FEF200_i2oo under
      Forced expiratory flow.

Maximal mid-expiratory flow rate (MMFR or MMEF): Synonymous with FEF25_75%.
Maximal ventilation (max Vg):  The volume of air breathed in 1 min during repetitive
      maximal respiratory effort.  Synonymous with maximum ventilatory minute volume.

Maximal voluntary ventilation (MW):  The volume (L/min, DTPS) of air breathed by a
      subject during voluntary maximum hyperventilation (rapid deep breathing) lasting a
      specific period of time.  Replaces maximal breathing capacity.

Mean (arithmetic):  The sum of observations divided by sample size.


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Mechanical clearance: See Mucociliary action.

Median:  A value in a collection of data values which is exceeded in magnitude by one-half
      the entries in the collection.

MEFR: See FEF2oo-i200 under Forced.expiratory flow.

Mesoscale:  Of or relating to meteorological phenomena from 1 to 100 kilometers in
      horizontal extent.

Messenger RNA:  A  type of RNA which conveys genetic information encoded in the DNA to
      'direct protein synthesis.     :

Metaplasia:  The abnormal transformation of an adult, fully differentiated tissue of one kind
      into a differentiated tissue of another kind.

Metaproterenol:  A bronchodilator used for the treatment of bronchial asthma.               :

Metastases:  The shifting of a disease from one part of the body to another; the appearance of
      neoplasms in parts of the body remote from the seat of the primary tumor.'

Meteorology:  The science that deals with the atmosphere and its phenomena.    '

Methacholine: A parasympathomimetic bronchoconstrictor drug with similarities to carbachol
      and acetylcholine.       •"'                "'             ,   •   •

Methemoglobin:  A form of hemoglobin in which the normal reduced state of iron (Fe2+) has
      been oxidized to Fe3+.  It contains oxygen in firm union with ferric (Fe3+) iron and is
      not capable of exchanging oxygen in normal respiratory processes.

Methimazole:  An anti-thyroid drug similar in action to propylthiouracil.

Methyltransferase: Any enzyme transferring methyl groups from one compound to another.

Microcoulometric: Capable of measuring millionths of coulombs used in electrolysis of a
      substance, to determine the amount of a substance in a sample.

Microflora:  A small or strictly localized plant.

Micron:  One-millionth of a meter.

Microphage:  A small phagocyte; a polymorphonuclear leukocyte that is phagocytic.

Millimolar:  One-thousandth of a molar solution.  A solution of one^thousandth of a mole  (in
      grams) per liter.
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Mineral acid anion:  An anion associated with strong, or mineral acids such as sulfuric, nitric,
      or hydrochloric.  These anions include NO3", SO42", and Cl1.

Minute ventilation C^E):  See Pulmonary measurements.
Minute volume:  The minute volume of breathing; a product of tidal volume times .the
      respiratory frequency in one minute; synonymous with minute ventilation.

Mitochondria: Organelles of the cell cytoplasm which contain enzymes active in the
      conservation of energy obtained in the aerobic part of the breakdown of carbohydrates
      and fats, in a process called respiration.                            ,

MMFR:  Maximal midexpiratory flow. See FEF25.75% under Forced expiratory flow.

Mobile sources:  Automobiles, trucks and other pollution sources which are not fixed in one
      location.

Modacrylic fiber:  A manufactured fiber in which the fiber-forming substance is any long
      chain synthetic polymer composed of less than  85% but at least 35% by weight of
      acrylonitrite units.

Moeity:  One of two or more parts into which something is divided.

Mole: The mass, in grams,  numerically equal to the  molecular weight of a substance.

Molecular correlation spectrometry:  A spectrophotometric technique which is used to identify
      unknown absorbing materials and measure their concentrations by using preset
      wavelengths.

Molecular weight: The weight of one molecule of a substance obtained by adding the
      gram-atomic weights of each  of the individual atoms in the substance.

Monocyte:  A relatively large mononuclear leukocyte, normally constituting 3 to 7%  of the
      leukocytes of the circulating blood.

Morbidity:  The quantity or  state of being diseased; also, used in reference to the ratio of the
      number of sick individuals to the total population of a community (i.e., morbidity rate).

Mordant: A substance which acts to bind dyes to a textile fiber of fabric.

Morphological:  Relating to  the form and structure of an organism or any  of its parts.

Morphology:  Structure and form of an organism at any stage of its life history.

Morphometry:  The quantitative measurement of structure (morphology).
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Mortality rate:  For a given period of time, the ratio of the number of deaths occurring per
      1,000 population.  Also known as death rate.

Moving average:  A procedure involving taking averages over a specific period prior to and
      including a year in question, so that successive averaging periods overlap (e.g., a
      three-year moving average would include data from 1967 through 1969 for the 1969
      average and from 1968 through 1970 for 1970).

MSA:  Metropolitan statistical area.                     .

Mucociliary action: Ciliary action of the mucous membranes lining respiratory tract airways
      that aids in removing particles from the lungs.

Mucociliary clearance: Removal of materials from the upper respiratory tract via ciliary
      action.

Mucociliary transport:  The process by which mucus is transported, by ciliary action, from the
      lungs.

Mucosa: The mucous membrane; it consists of epithelium, lamina propria and,  in the
      digestive tract, a layer of smooth muscle.

Mucous membrane: A membrane secreting mucus which lines passages and cavities
      communicating with the exterior of the body.
              i.  .  .    -
Mucus:  The clear, viscid  secretion of mucous membranes, consisting of rrtucin, epithelial
      cells, leukocytes, and various inorganic salts suspended in water.

Murine:  Relating to mice.

Mutagen:  A substance capable of causing, within an organism, biological changes that affect
      potential offspring through genetic mutation.

Mutagenic:  Having the power to cause mutations.  A mutation is  a change in the character of
      a gene (a sequence of base pairs in DNA) that is perpetuated in  subsequent divisions of
      the cell in which it occurs.

Myocardial infarction:  Infarction of any area of the heart muscle usually as a result of
      occlusion of a coronary artery.

Mycorrhizae:  Fungi that live in  association with plant roots and assist in the uptake of water
      and nutrients in exchange  for carbohydrates.

Nares:  The nostrils.

Nasopharyngeal:  Relating to the nasal cavity and the pharynx (throat).
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National Air Surveillance Network (NASN):  Network of monitoring stations for sampling air
      to determine extent of air pollution; established jointly by federal and state
      governments.                                                                    ..

Near ultraviolet:  Radiation of the wavelengths 2,000-4,000 Angstroms.

Necrosis: Death of cells that can discolor areas of a plant or kill the entire plant.

Necrotic: Pertaining to the pathologic death of one or more cells, or of a portion of tissue or
      organ, resulting from irreversible damage.

Neonate:  A newborn.

Neoplasm:  An abnormal tissue that grows more rapidly than normal; synonymous with tumor.

Neoplasia:  The pathologic process that results in the formation and growth of a tumor.

Neutrophil:  A mature white blood cell formed in bone marrow and  released into the
      circulating blood, where it  normally accounts for 54 to 65 % of the total number of
      leukocytes.

Ninhydrin:  An organic reagent used to identify amino acids.

Nitramine:  A compound consisting of a nitrogen attached to the nitrogen of amine.
                                                   ' '                      . ;)
Nitrate:  A salt or ester of nitric acid (NO3~).

Nitrification:  The principal  natural source of nitrate in which ammonium (NH4+) ions are
      oxidized to nitrites by specialized microorganisms. Other organisms oxidize nitrites to
      nitrates.

Nitrifiers: Soil microorganisms that convert NH4+ or organic  N to NO3",  a process referred
      to as  nitrification. Organisms that convert NH4+ to NO3" are referred to as autotrophic
      nitrifiers, and organisms that convert organic N to NO3" are referred to as heterotrophic
      nitrifiers.

Nitrite:   A salt or ester of nitrous acid (NO2=).

Nitrocellulose: Any of several esters of nitric acid formed by its action on cellulose, used in
      explosives, plastics, varnishes and rayon; also called cellulose nitrate.
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Nitrogen cycle:  Refers to the complex pathways by which nitrogen-containing compounds are
      moved from the atmosphere into organic life, into the soil, and back to the atmosphere.

Nitrogen fixation:  The metabolic assimilation of atmospheric nitrogen by soil microorganisms,
      which becomes available for plant use when the microorganisms die; also, industrial
      conversion of free nitrogen into combined forms used in production of fertilizers and
      other products.

Nitrogen oxide:  A compound composed of only nitrogen and oxygen. Components of
      photochemical smog.
Nitrogen saturation:  A condition in which ecosystems are unable to accumulate anymore
      nitrogen.

Nitrogen washout: The multiple breath curve obtained by plotting the fractional concentration
      of nitrogen in  expired alveolar gas vs. time, for a subject switched from breathing
      ambient air to  an inspired mixture of pure oxygen.  A progressive decrease of nitrogen
      concentration ensues which may be analyzed into two or more exponential components.

Nitrosamine: A compound consisting of a nitrosyl group connected to the nitrogen of an
      amine.

Nitrosation: Addition of a nitrosyl group.

N-Nitroso compounds:   Compounds carrying the functional nitrosyl group.

Nitrosyl:  A group composed of one oxygen and one nitrogen atom  (~N=0).

Nitrosylhemoglobin (NOHb): The red, respiratory protein of erythrocytes to which a nitrosyl
      group is attached.

N/P Ratio:  Ratio of nitrogen to phosphorous dissolved in lake water, important due to its
      effect on plant growth.

Nucleolus: A small  spherical mass of material within the  substance of the nucleus of a cell.

Nucleophilic: Having an affinity for atomic nuclei; electron-donating.

Nucleoside: A  compound that consists of a purine or pyrimidine base combined with
      deoxyribose or ribose and found in RNA and DNA.

5'-Nucleotidase:  An enzyme (EC 3.1.3.5) which hydrolyzes nucleoside 5'-phosphates into
      phosphoric acid (H3PO4)  and nucleosides.

Nucleotide: A compound consisting of a sugar (ribose or  deoxyribose),  a base (a purine or a
      pyrimidine), and a phosphate; a basic structural unit of RNA  and  DNA.

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 Nylon: A generic name chosen by E. I. du Pont de Nemours & Co., Inc. for a group of
       protein-like chemical products classed as synthetic linear polymers; two main types are
       Nylon 6 and Nylon 66.

 Occlusion:  A point which an opening is closed or obstructed.

 Olefin: An open-chain hydrocarbon having at least one double bond.

 Olfactory:  Relating to the sense of smell.

 Olfactory epithelium:  The inner lining of the nose and mouth which contains neural tissue
       sensitive to smell.

 Oligotrophic:  A body of water deficient in plant nutrients; also generally having abundant
       dissolved oxygen and no marked stratification.

 Oribitals:  Areas of high electron density in an atom or molecule.

 Orion:  An acrylic fiber produced by E. I. du Pont de Nemours and Co., Inc., based on a
       polymer of acrylonitrite;.used extensively for outdoor uses, it is resistant to chemicals
       and withstands  high temperatures.

 Oronasal breathing: Breathing through the nose and mouth simultaneously; typical human
       breathing pattern at moderate to high levels of exercise versus normally predominant
       nasal breathing while at rest.

 Osteogenic osteosarcoma: The most common and malignant of bone sarcomas (tumors). It
       arises from bone-forming cells and affects chiefly the ends of long bones.

 Ovarian primordial follicle: A spheroidal cell aggregation in the ovary in which the
      primordial oocyte (immature female sex cell) is  surrounded by a single layer of flattened
       follicular cells.

 Oxidant: A chemical  compound which has  the ability to remove electrons from another
      chemical species, thereby oxidizing it; also, a substance containing oxygen which reacts
      in air to produce a new substance, or one formed by the action of sunlight on oxides of
      nitrogen  and hydrocarbons.

Oxidation:  An ion or molecule undergoes oxidation by donating electrons.

Oxidative deamination:  Removal of the NH2 group from an amino  compound by reaction
      with oxygen.

Oxidative phosphorylation:  The mitochondrial process by which "high-energy" phosphate
      bonds form from the energy released as a result  of the  oxidation of various substrates.
      Principally occurs in the tricarboxylic acid pathway.


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Oxyhemoglobin:  Hemoglobin in combination with oxygen. It is the form of hemoglobin
      present in arterial blood.

Ozone layer:  A layer of the stratosphere from 20 to 50 km above the earth's surface
      characterized by high ozone content produced by ultraviolet radiation.

Ozone scavenging:  Removal  of O3 from ambient air or plumes by reaction with NO,
      producing NO2 and O2.

Paired electrons:  Electrons having opposite intrinsic spins about their own axes.

Parenchyma:  The essential and distinctive tissue of an organ or an abnormal growth, as
      distinguished from its supportive framework.

Parenchymal:  Referring to the distinguishing or specific cells of a gland or organ.

Partial pressure:  The pressure exerted by a single component in a mixture of gases.

Particle:  Any object, solid or liquid, having definite physical boundaries in all directions;
      includes, for example,  fine solid particles such as dust, smoke, fumes, or smog, found
      in the air or in emissions.

Particulate matter (PMX):  Matter in the form of small airborne liquid or solid particles,
      subscript "x" indicates  the particulate mean aerodynamic diameter.

Particulates:  Fine liquid or solid particles such as dust, smoke, mist, fumes or smog, found in
      the air or in emissions.

Pascal:  A unit of pressure in the International System of Units.  One pascal is equal to
      7.4 x 10"3 torr. The pascal is equivalent to one newton per square meter.

Pathogen:  Any virus, microorganism, or other substance causing disease.

Pathophysiological:  Derangement of function seen in disease; alteration in function as
      distinguished from structural defects.

Peak expiratory flow (PEF):  The highest forced expiratory flow  measured with a peak flow
      meter.

Peptide bond: The bond formed when two amino acids react with each other.

Percentiles:  The percentage of all observations exceeding or preceding some point; thus, 90th
      percentile is a level below which will fall 90% of the observations.

Perennial:  Trees and other plants that live more than one year are called perennials.
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 Perfusate:  A liquid, solution or colloidal suspension that has been passed over a special
       surface or through an appropriate structure.

 Perfusion:  Artificial passage of fluid through blood vessels.

 Permanent-press fabrics: Fabrics in which applied resins contribute to the easy care and
       appearance of the fabric and to the crease and seam flatness by reacting with the
       cellulose on pressing after garment manufacture.

 Permeation tube: A tube which is selectively porous to specific gases.

 Peroxidation: Refers to the process by which certain organic compounds are converted to  '
       peroxides.

 Peroxyacetyl nitrate (PAN): Pollutant created by action of sunlight on hydrocarbons and NOX
       in the air; an ingredient of photochemical smog.

 pH: A measure of the effective acidity or alkalinity of a solution.  It is expressed as the
       negative logarithm of the hydrogen-ion concentration. Pure water has a hydrogen ion
       concentration equal to 10~' M/L at standard conditions (25°C).  The negative
       logarithm of this quantity is 7. Thus, pure water has a pH value of 7 (neutral).  The
       pH scale is usually considered as extending from 0 to 14. A pH less than 7 denotes
       acidity; more than 7 denotes alkalinity.

 Phagocytosis: A mechanism by  which alveolar  macrophages and polymorphonuclear
       leukocytes engulf particles; one of several lung defense mechanisms by which foreign
       agents (biological and nonbiological) are removed from the respiratory tract.

 Phenotype:  The observable characteristics of an organism, resulting from the interaction
       between an individual genetic structure and the environment in which  development takes
       place.

Phenylthiourea:  A  crystalline compound, C7H8N2S,  that is bitter or tasteless depending on a
       single  dominant gene in the  tester.

Phlegm: Viscid mucus  secreted in abnormal quantity in the respiratory passages.

Phosphatase:  Any of a  group of enzymes that liberate inorganic phosphate from phosphoric
       esters (E.G. sub-subclass 3.1.3).

Phosphocreatine Mnase:  An enzyme (EC 2.7.3.2) catalyzing the formation of creatine and
       ATP, its breakdown  is a source of energy in the contraction of muscle; also called
       creatine phosphate.

Phospholipid: A molecule  consisting of lipid and phosphoric acid group(s).  An example is
       lecithin.  Serves as an important structural factor in biological membranes.


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Photochemical oxidants:  Primary ozone, NO2, PAN with lesser amounts of, other compounds
      formed as products of atmospheric reactions involving organic pollutants, nitrogen
      oxides, oxygen, and sunlight.

Photochemical smog:  Air pollution caused by chemical reaction of various airborne chemicals
      in sunlight,             ,

Photodissociation:  The process by which a chemical compound breaks down into simpler
      components under the influence of sunlight or other radiant energy.

Photolysis:  Decomposition upon irradiation by sunlight.

Photomultiplier tube:  An electron multiplier in which electrons released by photoelectric
      emission are multiplied in successive stages by dynodes that produce secondary
      emissions.

Photon:  A quantum of electromagnetic energy.

Photostationary:  A substance or  reaction which  reaches and maintains a steady state in the
      presence of light.     , .    ,                       .

Photosynthesis: The process in which green parts of plants, when exposed to light under
      suitable conditions of temperature and  water supply,  produce carbohydrates using
      atmospheric carbon dioxide and releasing oxygen.
  :           > v            .-             >                      •
Phyllosphere:  Usually refers to the leaf surface of plants.

Phytotoxic:  Poisonous to plants.

Phytoplankton:  Minute aquatic plant life.

P,i (II) bonds: Bonds in which electron density is not symmetrical about a line joining the
      bonded atoms.

Pinocytotic:  Refers to the cellular process (pinocytosis) in which the cytoplasmic membrane '
      forms imaginations in the form of narrow  channels leading into the cell. Liquids can
      flow into these channels and the membrane pinches off pockets that are incorporated
      into the cytoplasm and digested.

Pitting: A form of extremely localized corrosion that results in holes in the metal. One of the
      most  destructive forms of corrosion.

Pituary:  A  stalk-like gland near the base of the brain which is  attached to the hypothalmus.
      The anterior portion is a major repository for  hormones that control growth, stimulate
      other glands, and regulate the reproductive cycle.
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Placenta:  The organ in the uterus that provides metabolic interchange between the fetus and
      mother.

Plasmid:  Replicating unit, other than a nucleus gene, that contains nucieoprotein and is
      involved in various aspects of metabolism in organisms; also called paragenes.

Plasmolysis:  The dissolution of cellular components, or the shrinking of plant cells by osmotic
      loss of cytoplasmic water.

Plastic:  A plastic is one of a large group of organic compounds synthesized from cellulose,
      hydrocarbons, proteins or resins and capable of being cast, extruded, or molded into   ;
      various shapes.

Plasticizer:  A chemical added to plastics to soften, increase malleability or to make more
      readily deformable.

Platelet (blood):  An irregularly-shaped disk with no definite nucleus; about one-third to one-
      half the size of an erythrocyte and containing no hemoglobin.  Platelets are more
      numerous than leukocytes, numbering from 200,000 to  300,000 per cu. mm. of blood.

Plethysmograph:  A device for measuring and recording  changes in volume of a part, organ or
      the whole body; a body plethysmograph is a chamber apparatus surrounding the entire
      body.

Pleura:  The serous membrane enveloping the lungs an lining the walls of the chest cavity.

Plume:  Emission from a flue or chimney, usually distributed  stream-like downwind of the
      source, which can be distinguished from the surrounding air by appearance or chemical
      characteristics.

Pneumonia (interstitial): A chronic inflammation of the interstitial tissue of the lung, resulting
      in compression of the air cells. An acute, infectious disease.

Pneumonocytes:  A nonspecific term sometimes used in referring to types of cells
      characteristic of the respiratory part of the lung.

Podzol: Any of a group of zonal soils that develop in a  moist climate, especially under
      coniferous or mixed forest.

Point source:  A single stationary location  of pollutant discharge.

Polarography: A method of quantitative or qualitative analysis based  on current-voltage curves
      obtained by electrolysis of a  solution with steadily increasing voltage.

Pollution gradient:  A series of exposure situations in which pollutant concentrations range
      from high to low.
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Polyacrylonitrile:  A polymer made by reacting ethylene oxide and hydrocyanic acid.  Dynel
      and Orion are examples.

Polyamides:  Polymerization products of chemical compounds which contain amino (-NH2)
      and carboxyl (-COOH) groups.  Condensation reactions between the groups form
      amides (-CONH2).  Nylon is an example of a polyamide.

Polycarbonate:  Any of various tough transparent thermoplastics characterized by high impact
      strength and high softening temperature.

Polycythemia: An increase above the normal in the number of red cells in the blood.

Polyester fiber: A man-made or manufactured fiber in which the fiber-forming substance is
      any long-chain,synthetic polymer composed of at least 85% by weight of an ester of a
      dihydric alcohol and terephthalic acid.  Dacron is an example.

Polymer:  A large molecule produced by linking together many like molecules.

Polymerization:  In fiber manufacture, converting a chemical monomer (simple molecule) into
      a fiber-forming material by joining many like molecules into a stable, long-chain
      structure.            •     .     -    .               .

Polymorphic monocyte:   Type of leukocyte with a multi-lobed nucleus.

Polymorphonuclear leukocytes:  Cells which represent a secondary nonspecific cellular defense
      mechanism.  They are transported to the lungs from the bloodstream when the burden
      handled by the alveolar macrophages is too large.

Polysaccharides:  Polymers made up of sugars.  An example is glycogen which consists of
      repeating units of glucose.

Polystyrene: A thermoplastic plastic which may be transparent, opaque, or translucent.  It is
      light in weight, tasteless and odorless, it also is resistant to ordinary chemicals.

Polyurethane: Any of various polymers that contain NHCOO linkages and are used especially
      in flexible and rigid foams, elastomers and resins.

Pores of Kohn:  Also known as interalveolar pores; pores between air cells. Assumed to be
      pathways for collateral ventilation.

Precipitation:  Any of the various forms of water particles that fall from the atmosphere to the
      ground, rain, snow, etc.

Precursor:  A substance from which another substance is formed; specifically, one of the
      anthropogenic or natural emissions or atmospheric constituents which reacts under
      sunlight to form secondary pollutants comprising photochemical smog.
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Probe:  In air pollution sampling, the tube or other conduit extending into the atmosphere to
      be sampled, through which the sample passes to treatment, storage and/or analytical
      equipment.

Proline:  An amino acid, C5H9NO2, that can be synthesized from glutamate by animals.

Promonocyte:  An immature monocyte not normally seen in the circulating blood.

Proteinuria:  The presence of more than 0.3 gm of urinary protein in a 24-hour urine
      collection.

Pulmonary:  Relating to the lungs.

Pulmonary edema: An accumulation of excessive amounts of fluid in the lungs.

Pulmonary lumen:  The spaces in the interior of the tubular elements of the lung (bronchioles
      and alveolar ducts).

Pulmonary measurements: Measurements of the volume of air  moved during a normal or
      forced inspiration or expiration.  Specific lung volume measurements are defined
      independently.

      Lung volume measurements = Tidal volume, inspiratory reserve volume, expiratory
      reserve volume, residual volume (four basic independent volumes).

      Capacities = Combinations of basic volumes.

      Total lung capacity (TLC)  = Tidal volume + inspiratory reserve volume + expiratory
      reserve volume + residual volume;  the volume of gas in the lungs at the time of
      maximal inspiration or the sum of all volume compartments.  The method of
      measurement should be indicated, as with residual volume.

      Vital capacity (VC) = Tidal volume + inspiratory reserve volume + expiratory reserve
      volume; the greatest volume of gas that can be expelled by voluntary effort after
      maximal inspiration.  Also forced vital capacity  and forced inspiratory vital capacity.

      Functional residual capacity (FRC) = Residual volume + expiratory reserve volume;
      the volume of gas remaining in the lungs at the resting, end-tidal expiratory position.
      Equivalent to the sum of residual volume and expiratory  reserve volume.  The method
      of measurement should be indicated as with residual volume.

      Inspiratory capacity (1C)  = Tidal volume + inspiratory reserve volume.

      Inspiratory vital capacity (IVC) = The maximal, volume that can be inspired from the
      resting end-expiratory position; also forced inspiratory vital capacity.
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      Expiratory reserve volume (ERV) = The maximal that can be exhaled from the resting
      end-tidal expiratory position. See also Functional residual capacity.

      Residual volume (RV) = That volume of air remaining in the lungs after maximal
      exhalation.  The method of measurement should be indicated in the text or, when
      necessary, by appropriate qualifying symbols.

      Residual volume (RV) = Total lung capacity ratio (RV/TLC) = expresses the
      percentage of the total lung capacity occupied by residual volume;  varies somewhat with
      age but ordinarily should be no more than 20 to 30%.

      Tidal volume = That volume of air inhaled or exhaled with each breath during quiet
      breathing, used only to indicate a subdivision of lung volume. When tidal volume is
      used in gas exchange formulations, the symbol VT should be used.

      Minute ventilation (MV) = The volume of gas exchanged per minute at rest or during
      any stated activity; it is the tidal volume times the number of respirations per  minute.
      See Ventilation.

Pulmonary resistance: Sum of airway resistance and viscous tissue resistance.

Purine bases:  Organic bases which are constituents of DNA and RNA, including adenine and
      guanine.                   ,

Purulent:  Containing or forming pus.

Pyrimidine bases:  Organic bases found in DNA and RNA.  Cytosine and thymine occur in
      DNA and cytosine and uracil are found in RNA.

QRS:  Graphical representation on the electrocardiogram of a complex of three distinct waves
      which represent the beginning of ventricular contraction.

Quasistatic compliance:  Time dependent component of elasticity; compliance is the reciprocal
      of elasticity.              -                  :

Rainout:  Removal of particles and/or gases from the atmosphere by their involvement in
      cloud formation (particles act as condensation nuclei, gases are absorbed by cloud
      droplets), with subsequent precipitation.

Rayleigh scattering: Coherent scattering in which the intensity of the light of wavelength X,
      scattered in any direction making an angle with the incident direction, is directly
      proportional to  1 + cos2 and inversely proportional to X4.

Reactive dyes: Dyes which react chemically with cellulose in fibers under alkaline conditions.
      Also called fiber reactive or chemically reactive dyes.

Reduction:  Acceptance of electrons by an ion or molecule.

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Reference method (RM): For NO2, an EPA-approved gas-phase chemiluminescent analyzer
      and associated calibration techniques; regulatory specifications are described in Title 40,
      Code of Federal Regulations, Part 50, Appendix F.  Formerly,  Federal Reference
      Method.                                                                   ...-.-

Residual capacity: The volume of air remaining in the lungs after a maximum expiratory
      effort; same as residual volume.                                     -,         •...-.•

Residual volume (RV):  The volume of air remaining in the lungs after a maximal expiration.
      RV = TLC-VC

Resin: Any of various solid or semi-solid amorphous natural organic substances, usually
      derived from plant secretions, which are soluble in organic solvents but not in water;
      also any of many synthetic substances with similar properties used in finishing fabrics,
      for permanent press shrinkage control or water repellency.

Resistance flow (R):  The ratio of the flow-resistive components of pressure to simultaneous
      flow, in centimeters of H^O/L per sec.  Flow-resistive components of pressure are
      obtained by subtracting any elastic or inertial components, proportional respectively to
      volume and volume acceleration. Most flow resistances in the respiratory system are
      nonlinear, varying with the magnitude and direction of flow, with lung volume and lung
      volume history, and possibly with volume acceleration.  Accordingly, careful ,
      specification of the conditions of measurement is necessary; see Airway resistance and
      Total pulmonary resistance.

Ribosomal RNA:  The most abundant RNA in a cell and an integral constituent of ribosomes.,

Ribosomes:  Discrete units of RNA and protein which are instrumental in the synthesis of
      proteins in  a cell.  Aggregates are called polysomes.

Runoff:  Water from precipitation, irrigation or other sources that flows over the ground
      surface to streams.

Sclerosis: Pathological hardening of tissue, especially from overgrowth of fibrous tissue or
      increase in  interstitial tissue.

Secondary particles (or secondary aerosols): Dispersion aerosols that form in the atmosphere
      as a result of chemical reactions, often involving gases.

Selective leaching: The removal of one element from a solid alloy by  corrosion processes.

Septa: A thin wall dividing two cavities or masses of softer tissue.

Seromucoid:  Pertaining to a mixture of watery and mucinous material such as that of certain
      glands.
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Serum antiprotease:  A substance, present in serum, that inhibits the activity of prbteinases
      (enzymes which' destroy proteins).

Sigma (s) bonds:  Bonds in which electron density is symmetrical about a line joining the
      bonded atoms.

Silo-filler's disease:  Pulmonary lesion produced by oxides of nitrogen produced by fresh
      silage.

Single breath nitrogen elimination rate: Percentage rise in nitrogen fraction per unit of volume
      expired.

Single breath nitrogen technique:  A procedure in which a vital capacity inspiration, of
      ' 100% oxygen is followed by examination of nitrogen in the vital capacity expired.

Singlet state: The highly-reactive energy state of an atom in which certain electrons have
      unpaired spins.       .

Sink: A reactant with or absorber of a substance.                            •

Sodium'arsenite:  Na3AsO3, used with sodium hydroxide in the absorbing solution of a
      24-hour integrated manual method  for NO2.

Sodium dithionite:  A strong reducing agent (a supplier of electrons).

Sodium metabisulfite: Na2S2O5, used in  absorbing solutions of NO2 analysis methods.

Sorb: To take up and hold by absorption or adsorption.
             '     '    .
Sorbent:  A substance that takes up and holds another by absorption or adsorption.

Sorbitol dehydrogenase:  An enzyme that interconverts the sugars, sorbitol and fructose.

Sorption:  The process of being sorbed.

Spandex:  A manufactured fiber in which the  fiber forming substance is a long chain synthetic
      elastomer composed of at least 85% of a segmented polyurethane.

Specific airway conductance (SGaw):  Airway conductance divided by the lung volume at
      which it was measured; that is,  normalized airway conductance.  Airway conductance
      (Gaw)/thoracic gas volume (TGV).
            I                             •  '         L
Specific airway resistance (SRaw): Airway resistance multiplied by the volume at which it was
      measured.  SRaw  = airway conductance (Raw) x thoracic gas volume (TGV); liter (L) x
      centimeter  of water per liter per. second (cm H2O/L/S).
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 Spectrometer:  An instrument used to measure radiation spectra or to determine wavelengths of
       the various radiations.

 Spectrophotometry: A technique in which visible, UV, or infrared radiation is passed through
       a substance or solution and the intensity of light transmitted at various wavelengths is
       measured to determine the spectrum of light absorbed.

 Spectroscopy:  Use of the spectrometer to determine concentrations of an air pollutant.

 Spermatocytes:  A cell destined to give rise to spermatozoa (sperm).

 Sphingomyelins:  A group of phospholipids found  in brain, spinal cord, kidney and egg yolk.

 Sphygomenometer:  An apparatus, consisting of a  cuff and a pressure gauge, which is used to
       measure blood pressure.

 Spirometer:  A mechanical device, including bellows or other sealed, moving parts, which
       collects and stores gases and provides a graphical record of lung volume changes over
       time.  See Breathing pattern and Respiratory cycle.

 Spirometry:  The measurement, by a form of gas meter (spirometer), of volumes of air that
       can be moved in and out of the lungs.                             •

 Spleen:  A large vascular organ located on the upper left side of the abdominal cavity. It is a
       blood-forming organ in early life. It is a  storage organ for red corpuscles and because
       of the large number of macrophages, acts  as a blood filter.               '

 Sputum:  Expectorated matter,  especially mucus  or mucopurulent matter expectorated in
       diseases of the air passages.                                 '

 Squamous:  Scale-like, scaly.

 Standard deviation: Measure of the dispersion of values about a mean value.  It is calculated
       as the positive square root of the average of the squares of the individual deviations
       from  the mean.

 Standard temperature and pressure:  0°C, 760  mm  mercury.

 Staphylococcus aureus:  A spherically-shaped,  infectious species of bacteria found especially
       on nasal mucous membrane and skin.

Static lung compliance (0^^):  Measure of lung's elastic recoil (volume change resulting
       from change in pressure) with no or insignificant airflow.

Steady state  exposure:  Exposure to air pollutants whose concentration remains constant for a
      period of time.                                                                 'IT'*•


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Steroids:  A large family of chemical substances comprising many hormones .and vitamins and
      having large ring structures.

Stilbene:  An aromatic hydrocarbon C14H12 used as a phosphor and in making dyes.

Stoichiometric factor:  Used to express the conversion efficiency of a nonquantitative reaction,
      such as the reaction of NO2 with azo dyes in air monitoring  methods.

Stoma:  A minute opening or pore (plural is stomata).

STPD conditions: Standard temperature and pressure, dry. These  are the conditions of a
      volume of gas at 0°C, at 760 torr, without water vapor.  A STPD volume of a given
      gas contains a known number of moles of that gas.

Stratosphere: That region of the atmosphere extending from 11 km above the surface of the
      earth to 50 km.  At 50 km above the earth temperature rises to a maximum of 0°C.

Streptococcus pyogenes:  A species of bacteria found.in the human  mouth, throat and-
      respiratory tract and in inflammatory exudates, blood stream, and lesions in human
      diseases. It causes formation of pus or even fatal septicemias.

Stress corrosion cracking:  Cracking caused by  simultaneous presence of tensile stress and a
      specific corrosive medium. The metal or alloy is virtually unattached over most of its
      surface, while fine cracks progress through it.                 •'.•..

Strong interactions:  Forces or bond energies holding molecules together.  Thermal energy will
      not disrupt the formed bonds.

Sublobular hepatic necrosis:  The pathologic death of one or more cells, or of a portion of the
      liver, beneath one or more lobes.

Succession:  The progressive natural development of vegetation towards a climax,  during
      which one community is gradually replaced by others.          ,               .

Succinate:  A salt of succinic acid involved in energy production in the citric acid  cycle.

Sulfadiazine: One of a group of sulfa drugs. Highly effective against pneumococcal,
      staphlococcal, and streptococcal infections.

Sulfamethazine:  An antibacterial agent of the sulfonamide group, active against homolytic
      streptococci,  staphytococci, pneumococci and meningococci.

Sulfanilimide: A crystalline sulfonamide (C6H8N2O2S), the amide of sulfanilic acid and
      parent compound of most sulfa drugs.

Sulfhydryl group: A chemical radical consisting of sulfur and hydrogen which confers
      reducing potential to the chemical compound to which it is attached (-SH).
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Sulfur dioxide (SO^:  Colorless gas with pungent odor released primarily from burning of
      fossil fuels, such as coal, containing sulfur.

Sulfur dyes:  Used only on vegetable fibers, such as cottons.  They are insoluble in water and
      must be converted chemically in order to be soluble.  They are resistant (fast) to alkalies
      and washing and fairly fast to sunlight.

Supernatant:  The clear or partially clear liquid layer which separates from the homogenate
      upon centrifugation or standing.

Surfactant: A substance capable of altering the physiochemical nature of surfaces, such as one
      used to reduce surface tension of a liquid.

Symbiotic: A close association between two organisms of different species in which at least
      one of the two benefits.

Synergistic: A relationship in which the combined action or effect of two or more components
      is greater than that of the components acting separately.

Systolic: Relating to the rhythmical contraction of the heart.

Tachypnea:  Very rapid breathing.

Terragram (Tg):  One million  metric tons, 1012 grams.

Teratogenesis:  The disturbed growth processes resulting in  a deformed fetus.

Teratogenic: Causing or relating to abnormal development of the fetus.

Threshold: The level at which a physiological or psychological effect begins to be produced.

Thylakoid: A membranous lamella of protein and lipid in plant chloroplasts where the
      photochemical reactions of photosynthesis take place.           :
Thymidine:  A nucleoside (CjQHj^^C^) that is composed of thy mine and deoxyribose; occurs
      as a structural part of DNA.                               :..-.'-.>,

Tidal volume (VT):  The volume of air that is inspired or expired in a single breath during
      regular breathing.

Titer:  The standard of strength of a volumetric test solution.  For example, the titration of a
      volume of antibody-containing serum with another volume containing virus.

Tocopherol:  a-d-Tocopherol is one form of Vitamin E prepared synthetically.  The a form
      exhibits the most biological activity.  It is an antioxidant and retards rancidity of fats.
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Torn A unit of pressure sufficient to support a 1 mm column of mercury; 760 torr =
      1 atmosphere.                                               -

Total lung capacity (TLC): The sum of all the compartments of the lung, or the volume of air
      in the lungs at maximum inspiration.
Total pulmonary resistance (RL):  Resistance measured by relating flow-dependent
      transpulmonary pressure to airflow at the mouth.  Represents the total (factional)
      resistance of the lung tissue (R^) and the airways (Raw).  RL = R   +
                                                                   a\v
Total suspended particulates (TSP):  Solid and liquid particles present in the atmosphere.

Trachea:  Commonly known as the windpipe, a cartilaginous air tube extending from the
      larnyx (voice box) into the thorax (chest) where it divides, serving as the entrance to
      each of the lungs.

Tracheobronchial region:  The area encompassed by the trachea to the gas exchange region of
      the lung; the conducting airways.                                        ..'-•..

Transaminase:  Aminotransferase; an enzyme transferring an amino,group from an a-amino
      acid to the carbonyl carbon atom of an a-keto acid.

Transmissivity (UV): The percent of ultraviolet radiation passing through a medium.

Transmittance:  The  fraction of the radiant energy entering an absorbing layer which reaches
      the layer's further boundary.

Transpiration: The process  of the loss  of water vapor from plants.

Triethanolamine:  An amine, (HOCH2CH2)3N,  used in the absorbing solution of one analytical
      method for NO2.

Troposphere:  That portion of the atmosphere in which  temperature decreases rapidly with
      altitude, clouds form, and mixing of air masses by convection takes place. Generally
      extends to about 7 to 10 miles above the  earth's surface.

Type 1  cells:  Thin,  alveolar surface, epithelial  cells across which gas exchange occurs.

Type 2  cells:  Thicker, alveolar surface, epithelial cells that produce surfactant and serve as
      progenitor cells for Type 1 cell replacement.

Ultraviolet:  Light invisible to the human eye of wavelengths between 4 x 1(T7 and 5 x 10~9 m
      (4,000 to '50A).

Urea-formaldehyde resin: A compound composed of urea and formaldehyde in an     '
      arrangement that conveys thermosetting properties.           ;
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 Urobilinogen: One of the products of destruction of blood cells; found in the liver, intestines
      and urine.                                                               .

 Uterus: The womb; the hollow muscular organ in which the impregnated ovum (egg)
      develops into the fetus.

 Vacuole:  A minute space in any tissue.

 Vagal:  Refers to the vagus nerve.  This mixed nerve arises near the medulla oblongata and
      passes down from the cranial cavity to supply the larynx, lungs, heart, esophagus,
      stomach, and most of the abdominal viscera.

 Valence:  The number of electrons capable of being bonded or donated by an atom during
      bonding.

 Van Slyke reactions: Reaction of primary amines, including amino acids, with nitrous acid,
      yielding molecular nitrogen.

 Variance:  A measure of dispersion or variation of a sample from its expected value; it is
      usually calculated as the square root a sum of squared deviations about a mean divided
      by the sample size.

 Vat dyes:  Dyes which have a high degree of resistance to fading by light, NOX and washing.
      Widely used on cotton and viscose rayon. Colors are brilliant and of almost any shade.
      The name was originally derived from their application in a vat.

Venezuelan equine encephalomyelitis:  A form of equine encephalomyelitis found in parts of
      South America, Panama, Trinidad, and the United States, and caused by a virus.
      Fever, diarrhea, and depression are common.  In man, there is fever and severe
      headache after an incubation period of 2 to 5 days.

Ventilation:  Physiological process by which gas is exchanged between the outside air and the
      lungs.  The word ventilation sometimes designates ventilatory flow rate (or ventilatory
      minute volume), which is the product of the tidal volume by the ventilatory frequency.
      Conditions  are usually indicated as modifiers; for example:                   ,

      VE  = Expired volume per minute  (L/min, BTPS),

      Vj = Inspired volume per minute  (L/min, BTPS).

      Ventilation  is often referred to as "total ventilation" to distinguish it from "alveolar
      ventilation" (see Ventilation, alveolar).                                         :  .
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Ventilation, alveolar (V^)1 The portion of the total ventilation that is involved in gas
      exchange with the blood; alveolar ventilation is less than total ventilation because when
      a tidal volume of gas leaves the alveolar spaces, the last part does not get expelled from
      the body but occupies the dead space, to be reinspired with the next inspiration.  Thus
      the volume of alveolar gas actually expelled completely is equal to the tidal volume
      minus the volume of the dead space.  This truly complete expiration volume times the
      ventilatory frequency constitutes the alveolar ventilation.

Ventilation, dead-space (VD):  Ventilation per minute of the physiologic dead space (volume
      of gas not involved in gas exchange with the blood), BTPS, defined by the following
      equation:                                         :

      VD = VE(PaCO2 - PECO2)/(PaCO2 - PTCO2)

Ventilation/perfusion ratio (^A/Q):  Ratio of the alveolar ventilation to the blood perfusion
      volume flow through the pulmonary parenchyma, such as, pulmonary blood flow or
      right heart cardia output; this ratio is a fundamental determinant of the oxygen and
      carbon dioxide pressure of the alveolar gas and of the end-capillary blood.  Throughout
      the lungs the local ventilation/perfusion ratios vary, and, consequently, the local
      alveolar gas and end-capillary blood compositions also vary.              ,

Villus:  A projection from the surface, especially of a mucous  membrane.

Vinyl chloride: A gaseous chemical suspected of causing at least one type of cancer.  It is
      used primarily in the manufacture of polyvinyl chloride, a plastic.

Viscose ,rayon: Filaments of regenerated cellulose coagulated from a solution of cellulose
      xanthate.  Raw materials can be cotton linters or chips of spruce, pine, or hemlock.

Visible region: Light between the wavelengths of 4,000-8,000 A.

Visual range: The distance at which an object can be distinguished from background.

Vital capacity (VC): The greatest volume of air that can be exhaled from the lungs after a
      maximum inspiration (see Pulmonary measurements).

Vitamin E:  Any of several fat-soluble vitamins (tocopherols), essential in nutrition of various
      vertebrates.

Washout: The capture of gases and particles by  falling raindrops.

Weak interactions:  Forces, electrostatic in nature, which bind  atoms and/or molecules to each
      other. Thermal energy will disrupt the interaction.  Also called van der Waal's forces.
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Weathering:  In this context, weathering refers to the releases of base cations from soil
      minerals to cationic forms which can be taken up by plants, leached, or absorbed to
      cation exchange sites.

Wet deposition:  The process by which atmospheric substances are returned to earth in the
      form of rain or other precipitation.

Wheat germ lipase:  An enzyme, obtained from wheat germ, which is capable of cleaving a
      fatty acid from a neutral fat; a lipolytic enzyme.

X-ray fluorescence  spectrometry:  A nondestructive technique which utilizes the principle that
      every element emits characteristic x-ray emissions when excited by high-energy
      radiation.

Zeolites:  Hydrous  silicates analogous to feldspars, occurring in lavas and various soils.

Zooplankton:  Minute animal life floating or swimming  weakly in a body of water.
                                                   *U.S. GOVERNMENT PRINTING OFFICE: 1W2-64S-003/40672
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