Un red States
Environmental Protection
Agency
OTdce of
Washington. DC 20460
EPA 560-6 84 004
Toxic Substances
Scientific Rationale for the Selection of
Toxicity Testing Methods
II. Teratology, Immunotoxicology,
and Inhalation Toxicology
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ORNL-6094
EPA-560/6-84-004
SCIENTIFIC RATIONALE FOR THE SELECTION
OF TOXICTTY TESTING METHODS
II. TERATOLOGY, IMMUNOTOXICOLOGY,
AND INHALATION TOXICOLOGY
Editors
Michael G. Ryon
Daljit S. Sawhney
Contributors
Mary Lou Daugherty
Robert H. Ross
Michael G. Ryon
Chemical Effects Information Group
Information Research and Analysis Section
Information Resources Organization
Oak Ridge National Laboratory
Project Officers
Daljit S. Sawhney
William H. Farland
Diane D. Beal
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, DC 20460
Date of Issue - September 1985
OAK RIDGE NATIONAL LABORATORY
Oak Ridge, Tennessee 37831
operated by
MARTIN MARIETTA ENERGY SYSTEMS, INC.
for the
U.S. DEPARTMENT OF ENERGY
under Contract No. DE-AC05-84OR21400
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DISCLAIMER
This report was prepared as an account of work sponsored by an agency of
the United States Government. Neither the United States Government
nor any agency thereof, nor any of their employees, contractors, subcon-
tractors, or their employees, nor the publisher, makes any warranty,
express or implied, nor assumes any legal liability or responsibility for any
third party's use or the results of such use of any information, apparatus,
product or process disclosed in this report, nor represents that its use by
such third party would not infringe privately owned rights.
This report has been reviewed by the Office of Pesticides and Toxic Sub-
stances, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency or the
publisher, nor does mention of trade names or commercial products consti-
tute endorsement or recommendation of use.
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DALJIT S. SAWHNEV
CONTENTS
FIGURES vii
TABLES ix
ACKNOWLEDGMENTS xi
ABSTRACT xiii
1. EXECUTIVE SUMMARY
1.1 TERATOGENICITY 1
1.2 IMMUNOTOXICOLOGY 2
1.3 INHALATION TOXICOLOGY 4
1.3.1 General Test Design 4
1.3.2 Exposure Chamber Design 5
1.3.3 Generation of Test Atmospheres 7
2. TERATOGENICITY
2.1 INTRODUCTION 9
2.2 GENERAL EXPERIMENTAL CONSIDERATIONS ... 9
2.2.1 Dosage—Number and Level 10
2.2.2 Dosage—Duration 10
2.2.3 Positive Controls 16
2.2.4 Number of Species 16
2.2.5 Number of Test Animals Per Dose Group 24
2.2.6 Administration Route 25
2.2.7 Fetal Examination 25
2.3 STRUCTURE-ACTIVITY RELATIONSHIPS 25
2.4 TERATOGENESIS AND TIME OF
ADMINISTRATION 26
2.4.1 Preimplantation 26
2.4.2 Organogenesis 27
2.4.3 Histogenesis and Fetal Period 30
2.5 BEHAVIORAL TERATOGENESIS 30
2.5.1 Historical Perspective 32
2.5.2 Behavioral Testing Methodologies 32
m
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2.6 SPECIES COMPARISONS 33
2.6.1 General Aspects 37
2.6.2 Rat 37
2.6.3 Mouse ... .... ... • 38
2.6.4 Rabbit 39
2.6.5 Hamster 40
2.6.6 Nonhuman Primates 41
2.6.7 Other Species 42
2.6.7.1 Dog 42
2.6.7.2 Cat 42
2.6.7.3 Pig 43
2.6.8 Results of Testing Some Human Teratogenic
Chemicals in Animal Models .... 43
2.6.8.1 Aminopterin 44
2.6.8.2 Methotrexate 44
2.6.8.3 Thalidomide 44
2.7 RECENT APPROACHES IN TERATOLOGY TESTING 46
2.7.1 In Vivo Methods 46
2.7.2 In Vitro Methods 47
2.7.2.1 Whole mammalian embryo culture .... 47
2.7.2.2 Embryonic limb bud organ culture .... 48
2.7.2.3 Avian embryonic cell 48
2.7.2.4 Ascites tumor cell assay 48
2.7.2.5 Drosophila embryo cell assay 49
2.7.2.6 Poxvirus morphogenesis 49
2.7.2.7 Neuroblastoma cells .. 49
2.7.2.8 Hydra attenuata system 50
2.7.2.9 Planarian assay 50
2.7.2.10 Frog embryo assay 51
2.8 CONCLUSIONS AND RECOMMENDATIONS
FOR FURTHER RESEARCH . ... 51
2.8.1 Conclusions .. 51
. 1 General experimental considerations ... 5 ]
.2 Structure-activity relationships 51
.3 Teratogenesis and time of administration 52
.4 Behavioral teratogenesis 52
.5 Species comparisons . .... . . 52
.6 Recent approaches in teratology testing 53
2.8.2 Recommendations for Further Research. ... 53
2.9 LITERATURE CITED... .... . 54
2.8.
2.8.
2.8.
2.8.
2.8.
2.8.
IV
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3. IMMUNOTOXICOLOGY
3.1 INTRODUCTION 69
3.2 TESTS TO DETECT ALTERATIONS IN THE
IMMUNE RESPONSE 71
3.2.1 Assessment of Cell-Mediated Immunity 72
3.2.1.1 In vivo tests 75
3.2.1.2 In vitro tests 82
3.2.2 Assessment of Humoral Immunity 87
3.2.2.1 Assays for local production of antibody ... 88
3.2.2.2 Measurement of circulating antibody and
immunoglobulins 93
3.2.2.3 Other tests of B-cell function 99
3.2.3 Assessment of Indirect Parameters of Immunity ...100
3.2.3.1 Macrophage functions 100
3.2.3.2 Host resistance to infection 101
3.3 ALLERGIC REACTIONS TO ENVIRONMENTAL
CHEMICALS 102
3.3.1 Allergic Response to Inhalants 106
3.3.2 Allergic Response to Dermal Sensitizers 106
3.3.3 Tests to Detect Sensitizing Potential
of Chemicals 106
3.3.3.1 Skin sensitization 107
3.3.3.2 Respiratory sensitization 109
3.4 TIER TESTING 110
3.4.1 Moore and Faith (1976) 110
3.4.2 Luster and Faith (1979) Ill
3.4.3 Speirs and Speirs (1979) 112
3.4.4 Deanet al. (1979b) 113
3.4.5 White et al. (Unpublished Observations) 113
3.4.6 Luster et al. (1982b) 116
3.5 SUMMARY 117
3.6 GLOSSARY 118
3.7 LITERATURE CITED 134
3.8 GENERAL REFERENCES 148
4. INHALATION TOXICOLOGY 149
4.1 INTRODUCTION 149
4.2 GENERAL TEST DESIGN 150
4.2.1 Introduction 150
4.2.2 Test Species 150
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4.2.3 Duration and Mode of Exposure ...
4.2.4 Dosage ^
4.2.5 Observations for Effects
4.2.5.1 Clinical observations ^
4.2.5.2 Biochemical and hematological tests 158
4.2.5.3 Pathological observations ^9
4.2.5.4 Respiratory function tests 162
4.3 EXPOSURE CHAMBER DESIGN 167
4.3.1 Introduction 167
4.3.2 Types of Exposure Systems 167
4.3.2.1 Static systems 167
4.3.2.2 Dynamic systems 169
4.3.2.3 Whole-body exposure systems 172
4.3.2.4 Nose- and head-only exposure systems .. .. 173
4.3.3 Chamber Design 174
4.3.3.1 Chamber shape 175
4.3.3.2 Chamber size 178
4.3.3.3 Chamber materials and construction 179
4.3.4 Airflow Systems 182
4.3.5 Sampling and Monitoring of Chamber Conditions ... 188
4.3.6 Maintenance of Sanitary Conditions 202
4.3.7 Specialized Equipment 203
4.4 GENERATION OF TEST ATMOSPHERES 204
4.4.1 Introduction 204
4.4.2 Generation of Gas and Vapor Atmospheres 206
4.4.3 Generation of Aerosol Atmospheres 208
4.4.3.1 Introduction 208
4.4.3.2 Generation of aerosols from solids 210
4.4.3.3 Generation of aerosols from liquids,
solutions, and liquid suspensions 212
4.5 CONCLUSIONS AND RESEARCH
RECOMMENDATIONS 223
4.5.1 Conclusions 223
4.5.2 Research Recommendations 224
4.6 LITERATURE CITED 225
vi
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FIGURES
2.1 Representation of the susceptibility of the human
embryo to teratogenesis, beginning with ferti-
lization and continuing throughout intrauterine
development 31
3.1 Hypothetical mechanisms for cellular and humoral
immune reactions 77
3.2 Direct fluorescent antibody test and "sandwich" test
for staining antibody-producing cells 92
3.3 The four types of allergic reaction 104
4.1 Concentration-time relationships in a chamber
operated for a long period of time 170
4.2 Time-concentration curve for exposure to constant
concentration using air-lock mechanism 170
4.3 Schematic diagram of the Rochester exposure
chamber 176
4.4 Schematic diagram of the New York University
exposure chamber 177
4.5 Small-scale exposure chamber based on a bell
jar design 179
4.6 Whole body dynamic exposure chamber of
1.3-m side 182
4.7 Cage arrangement and chamber design of Moss and
Brown showing airflow pattern 183
4.8 Schematic diagram of airflow system 184
4.9 Sketch of chamber indicating the eight corner and
one reference sampling positions 190
4.10 Limits of particle size measuring equipment 193
¥11
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4.11 Hexhlet two-stage aerosol sampler . . ^
4.12. Conicycle aerosol sampler • 195
4.13 Performance curves for the hexhlet and conicycle
samplers 196
4.14 Schematic diagram showing operation of cascade
impactor 197
4.15 Schematic of Anderson Mark HI cascade impactor 198
4.16 Calibration of an Anderson Mark III impactor 199
4.17 Working principle of the inertial spectrometer 200
4.18 Electrostatic precipitator using radioactive tritium
(H3) as an ion source 201
4.19 Improved counter-current vaporizing apparatus, with
thermostating jacket 207
4.20 J-tube vaporization assembly 209
4.21 The Wright dust feed mechanism ... 212
4.22 Schematic view of Ettinger's modification of
TimbrelFs fibrous aerosol generator 213
4.23 Diagram of the DeVilbiss nebulizer 215
4.24 Schematic view of ultrasonic nebulizer 218
4.25 Schematic drawing of a spinning disk generator
used to produce monodisperse aerosols of both
soluble and insoluble forms from solutions of
suspensions 220
4.26 Schematic view of Sinclair-LaMer monodisperse
aerosol generator .... T-I
Vlll
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TABLES
2.1 Literature Survey of Experimental Parameters in
Testing of Chemicals for Teratogenicity 11
2.2 Thalidomide Action in Various Species 17
2.3 Aminopterin Action in Various Species 22
2.4 Methotrexate Action in Various Species 23
2.5 Preimplantation Developmental Stages in Man and
Laboratory Animals 27
2.6 Critical Periods of Organogenesis in Animals 29
2.7 Behavioral Testing Procedures 34
2.8 Behaviors Tested and Methods Used in Teratologic
Evaluation 35
2.9 Species Susceptibility to Drugs 38
2.10 Thaliomide Teratogenesis in Primates 39
2.11 Comparative Teratogenicity of Thalidomide 40
3.1 Tests for Immunotoxicity 73
3.2 A Classification of Toxicity-Influencing Factors 74
3.3 Products of Activated Lymphocytes (PALS) In Vitro 76
3.4 PFC Response in Experimental Animals Following Exposure
to Environmental Pollutants 91
3.5 Definition of the Four Types of Allergic Reaction 103
3.6 Respiratory Allergens 107
3.7 Testing Approaches for Evaluating the Immunobiologic
Effects of Food Additives, Drugs, and
Environmental Chemicals 114
4.1 Some Physiological Indices of Man and Animals 151
4.2 Parameters Measured in Lavage Fluid 160
4.3 Organs Suggested for Microscopic Examination 161
4.4 Respiratory Function Tests to Evaluate Breathing
Pattern, Lung Volumes, Pulmonary Pressures,
and Lung Mechanics 163
4.5 Inhalation Exposure Apparatus: Basic Requirements 168
IX
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4.6 Measurement Principles Used in Continuous Monitoring
Instruments for Gases and Vapors
4.7 Particulate Sampling and Characterization Apparatus 192
4.8 Output Characteristics of Some Compressed Air
Nebulizers 216
4.9 Output Characteristics of Some Ultrasonic Nebulizers 219
4.10 Operating Conditions at Which Model I Spinning Disk
Was Used 222
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ACKNOWLEDGMENTS
The authors would like to thank Tim Ensminger, manager of the Infor-
mation Research and Analysis Section (IR&A), Information Resources
Organization, Oak Ridge National Laboratory (ORNL), for his support
during the preparation of this document. The advice and support of Drs.
Daljit Sawhney, William Farland, and Diane Beal, U.S. Environmental
Protection Agency (EPA) project officers, and the assistance of Helen
Warren, Jan Pruett, and other members of the Toxicology Information
Response Center are gratefully acknowledged. Most helpful in the
preparation of the immunotoxicity section was the bibliography The
Effects of Environmental Chemicals on the Immune System: A Selected
Bibliography With Abstracts, 1969-1980, by S. G. Winslow
(NLM/TIRC-81/2).
Special thanks are extended to Drs. W. E. Dalbey, N. Gengozian, and
H. R. Witschi of the ORNL Biology Division; to Drs. Harold Grice and
Clifford Chappel of FDC Consultants, Inc.; to Dr. Carl Wust of the
University of Tennessee at Knoxville; and to Drs. David Anderson, Larry
Chitlik, Ernest Falke, Elaine Francis, Stan Gross, Carole Kimmel, James
Murphy, Daljit Sawhney, and John Whalan of the U.S. EPA for their
technical review and helpful suggestions. The authors are also greatly
indebted to Judy Crutcher, Pat Hartman, Sherry Hawthorne, Frances Lit-
tleton, and Donna Stokes of the IR&A Publications Office for their assis-
tance in document preparation and to Carolyn Seaborn and Lois Thurston
of the Chemical Effects Information Group for their assistance in the col-
lection and organization of reference materials.
XI
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ABSTRACT
This document is the second of a two-part literature analysis of param-
eters associated with the various toxicity testing methods (test animal
selection, pathology, etc.). Acute, subchronic, chronic, and carcinogenic
testing methods are covered in ORNL/EIS-151. Testing methods for
developmental toxicity, immunotoxicology, and inhalation toxicology and
research needs associated with these areas are covered in this volume,
ORNL-6094. These reports were prepared for the purpose of assisting and
supporting the U.S. Environmental Protection Agency in its efforts to
develop guidelines for more efficient and economical testing procedures.
Xlll
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1. EXECUTIVE SUMMARY
1.1 TERATOGENICITY
Proper attention to the design of a teratogenicity experiment is essen-
tial. Parameters such as the number and level of doses, the number of
species, the number of test animals per dose group, and the time during
pregnancy for the administration of the test agent are important considera-
tions.
At least three dose levels should be tested; the low dose should permit
normal embryonic development, the intermediate dose(s) should produce
malformed offspring if the test chemical is teratogenic, and the high dose
should be maternally toxic or produce embryotoxicity or fetotoxicity. At
least two species of test animals with a sufficient number of animals per
dose group to permit meaningful statistical evaluation of the results should
be used. Current guidelines recommend the use of at least 20 pregnant
animals of a rodent species and 12 pregnant rabbits. Generally, the test
agent is administered during the period of organogenesis because this is
considered to be the developmental stage most sensitive to the action of
teratogenic chemicals. The period of prenatal development that slightly
overlaps organogenesis but extends primarily into the fetal period is known
as histogenesis. Although teratogenic agents that come into contact with
the developing fetus during this time can cause minor structural deviations,
the abnormalities that are more likely to occur during the fetal period are
those involving growth or functional aspects of development.
The term behavioral teratogenesis is used to describe the branch of
teratology devoted to studying behavioral modifications that result from
prenatally administered agents. In addition to the histogenesis period,
administration of chemicals during organogenesis or pregestationally has
resulted in behavioral alterations in offspring.
Although many different animal species have been used as test animals
in teratogenicity testing experiments, no ideal species has been identified.
The rat and mouse are the most commonly used rodent species, and the
rabbit is the most commonly used nonrodent species. The nonhuman pri-
mate has received considerable attention as a test animal because of its
anatomical and physiological similarity to man; however, the cost associ-
ated with the use of nonhuman primates will likely restrict their use to
special situations (e.g., testing a drug designed for use during pregnancy).
In recent years alternative approaches to the standard methods of test-
ing agents for teratogenicity have been investigated, primarily due to
economic and practical considerations. These include an in vivo method
where determinations of teratogenic potential are made on the basis of
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litter size and weight and several in vitro methods, such as whole mam-
malian embryo culture, a Hydra attenuata system, and a frog embryo
assay.
Possible areas for future research include further investigation of post-
natal behavioral changes as they relate to pregestational and gestational
chemical exposure, further research directed toward standardization of the
methodology for screening chemicals for behavioral teratogenicity, and
determination of an acceptable battery of tests for in vitro screening of
compounds for teratogenicity.
1.2 IMMUNOTOXICOLOGY
Investigations into the toxic effects of industrial and environmental
chemicals have revealed that some of these chemicals have distinct, and
usually very specific, effects on the immune system. This complex
organization of organs and cells plays a critical role in the protection of
man against infection and neoplastic diseases; thus dysfunction of the sys-
tem can have serious health effects. These issues are the basis for the evo-
lution of the field of immunotoxicology (the study of immunologic
alterations caused by industrial and environmental chemicals), and they
have stimulated interest in test methods originally designed for use in
immunopharmacology (to assess the therapeutic potential of immu-
nosuppressant or immunopotentiating drugs). Several toxicology groups
have suggested the use of immunotoxicology assays as an adjunct to toxi-
cology studies.
The protective responses of the body against foreign entities (antigens)
are carried out by the immune system, which is composed of cells and
organs of the lymphoreticular system. Immune responses may be specific
or nonspecific.
The specific immune response is characterized by the highly specific
recognition of (and response to) antigen by lymphocytes and by the induc-
tion of immunological memory. Specific immunity includes cell-mediated
immunity (an expression of the activities of T-Iymphocytes), humoral
immunity (an expression of the activities of B-lymphocytes), and immuno-
logical tolerance.
When a chemical compound is suspected of altering the immune
response, both the cellular and humoral responses must be tested Tests of
cell-mediated immunity usually measure T-cell activities, either in vivo or
in vitro. Basic in vivo procedures for evaluating the cell-mediated immune
response include tests of delayed hypersensitivity, allograft reject' H
graft vs host reactions. These are well-established, valuable procedures but
are generally cumbersome and time consuming, requiring large nu b
animals. Newer tests have been developed, some utilizing isotone th ^ °
more efficient, quantitative, and sensitive. Some of these h ^ *K
radiometric ear test and the footpad assay, have been recomme &*A f
inclusion in toxicity screening protocols.
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In vitro tests of cell-mediated immunity are versatile and convenient
methods for the large-scale screening of chemicals. Tests recommended for
screening protocols include lymphocyte transformation by mitogens, mixed
lymphocyte cultures, and assays for the migration inhibiton factor. For
these assays, the test substance is generally administered to the test
animal, appropriate cells are removed, and immunocompetence of these
cells is measured in vitro. In some assays, however, the test chemical can
be added to the test system in vitro, thus bypassing the animal treatment
step.
Tests of humoral immunity measure, in some cases, both T- and B-cell
functions (T-cell dependent humoral response) and, in others, only B-cell
functions (T-cell independent humoral response), depending on the antigen
used. Humoral immunity is generally evaluated on the basis of circulating
antibody or local production of antibody, the identification and quantita-
tion of the various immunoglobulins, and the colony-forming and blasto-
genic capabilities of B-cells. Several of these tests have been suggested for
use in toxicity screening protocols. These include assays for circulating
antibody, the classic hemagglutination and hemolysin titrations, and more
modern techniques, such as single radial immunodiffusion (SRID) assay
and the enzyme-linked immunosorbent assay (ELISA). Tests designed to
detect the local production of antibody include the plaque-forming assay
(localized hemolysis in gel), immunofluorescence techniques, and
erythrocyte-antibody-complement (EAC) rosette assay.
The nonspecific immune response does not involve the recognition of
antigen or the mounting of an immune response but, instead, involves the
generalized activities of phagocytic cells, lysozymes, the interferon system,
the complement system, and the kinin system. Tests for macrophage func-
tion, hormone and complement activities, host resistance to infection, resis-
tance to tumor challenge, and endotoxin hypersensitivity are typical pro-
cedures used to assess this system.
Another facet of immunology test procedures, also important in assess-
ing the toxic properties of industrial/environmental chemicals, are tests for
allergencity, which, in contrast to immunotoxicity procedures that measure
immunocompetence, are designed to measure the sensitizing potential of a
chemical. Chemical allergens found in industrial environments, thought to
be highly protein-reactive chemicals, usually affect a small subset of
workers who are hypersusceptible to the low-dose exposure legally permit-
ted in chemical plants. Sensitization can occur via dermal or respiratory
routes, and, in either case, the resulting allergic reaction can have serious
ramifications for the worker involved. Predictive tests in animals, usually
guinea pigs, have been designed to assess the allergenic potential of chemi-
cals. Skin procedures include the Draize test, Freund's complete adjuvant
test, the guinea pig maximization test, the "split adjuvant" test, and the
open cutaneous test. Respiratory sensitization in animals, although difficult
to attain, has been demonstrated in limited studies.
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If assays to evaluate the immunotoxic effects and allergic potential of
chemicals are to become incorporated in routine toxicity test protocols, the
selection of the proper combination of tests that would be most efficient,
economical, and informative becomes an important consideration. Experts
in the field of immunotoxicology have made their independent recommen-
dations for the types of immunology procedures that should be included in
routine toxicity studies and have designed tier-testing schemes to demon-
strate how this can be accomplished.
1.3 INHALATION TOXICOLOGY
1.3.1 General Test Design
Toxicity tests designed to evaluate effects from inhalation exposure
involve many of the same variables as tests utilizing other exposure routes.
The selection of test species should be based on the anatomical and physio-
logical similarity of the respiratory tracts of humans and the test species,
as well as metabolic similarities and economic/practicality considerations.
Larger animals (e.g., horses, monkeys) are generally more similar to
humans, but practical restraints favor the use of smaller animals (e.g.,
rats).
Test durations for inhalation studies include acute (one exposure with
14 days of observation), subchronic (repeated exposures for 14, 28, or 90
days), and chronic (repeated exposures for 1 to 3 years). Due to the large
costs involved in a chronic study, these should be undertaken only when
preliminary studies indicate the need for an evaluation of lifetime effects.
Exposure to the test agent can be intermittent (6 to 8 h per day, 5 days
per week), based on occupational exposure, or continuous (22 to 24 h per
day, 7 days per week) to simulate environmental exposure. Intermittent
exposure allows recovery from the test agent (which can affect toxicity)
but is generally simpler to maintain and operate.
Inhalation studies with repeated exposures should include at least three
dose levels and suitable controls. The determination of the actual dose
delivered to the test animal is a major difficulty in inhalation studies. The
common method is to calculate the dose based on the concentration (C) in
the test chamber multiplied by the length of exposure (T). However this
indicates only the maximum possible dose because not all of this quantity
is inhaled by the animal or, once inhaled, reaches the lungs. Dosimetric
formulas have been developed that consider the volume intake of the lun&s
and the percentage of the agent retained in the lungs. Unfortunately, these
variables are difficult to determine under actual use conditions and th
CXT estimation of dose is still widely used.
The evaluation of toxic effects resulting from inhalation exposure
includes many of the procedures used in other toxicity studies as 11
procedures specific for inhalation-caused effects. Clinical observations of
the animals should be made daily, before, during, and after
These include signs of irritation, behavioral changes, and changes n
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functions. Biochemical and hematological tests should be performed on
blood samples (taken before and after exposure) to aid in evaluating sys-
temic effects. Biochemical tests for evaluating lung damage are currently
being modified from similar tests used to evaluate hepatotoxicity. Patho-
logical techniques are the most useful evaluations for assessing toxic
effects from inhalation exposure. A thorough gross examination of all test
animals is necessary with special attention given to the respiratory system.
Microscopic evaluations should be performed on animals from the high
dose and control groups as well as any lesion-bearing animals found in the
gross examination. The organs to be examined should cover any potential
targets for systemic toxicity. Respiratory tissues should be examined in
detail, using special techniques such as histochemistry, when indicated.
Respiratory function tests are techniques for evaluating inhalation expo-
sure effects that require special equipment and training. They can quantify
changes at low-dose levels and can produce dose-response data. However,
they are not conclusive by themselves and should be correlated with other
toxicity evaluations.
1.3.2 Exposure Chamber Design
The design of the inhalation exposure chamber is an important con-
sideration in an inhalation study. It can be operated in a static mode,
where a predetermined dose is introduced into a closed chamber, or in a
dynamic mode, where a continuous flow of air and test agent is vented
through the chamber. Static exposures are limited by problems associated
with the finite volume of air used without replacement and are generally
most useful for short-term exposures or when the test agent is available
only in limited quantities. Dynamic exposures are used for the majority of
inhalation tests and require accurate calculations of airflow and test agent
feed rates to achieve the desired doses. The chambers are most frequently
designed to house the whole animal within the exposure area, particularly
for long-term studies. Nose- and head-only systems can be used when der-
mal or accidental oral exposure must be avoided. These chamber designs
require special equipment to restrain the animals and seals to prevent the
loss of test agent.
The shape of most large, dynamic, whole-body exposure chambers is
based on a cubical or hexagonal design. Pyramidal or conical additions to
the top and bottom of the chamber increase the distribution efficiency of
air within the chamber, making the design closer in operational charac-
teristics to the most efficient shape, the sphere. The most widely used
chambers of this design are the Rochester (hexagonal) and New York
University (cubical) designs. For carcinogenesis studies, more isolated
chambers or exposure rooms are often used because of the long duration of
the tests and the need to limit exposure to the test compound. Small-scale
exposure chambers designed for pilot or acute studies occur in more varia-
tions including bell jars, Lucite cylinders, and scaled-down versions of
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larger units. Flexibility is important in these designs since the unit will
usually be used for many types of chemical exposures.
Chamber size is governed primarily by air distribution and animal
volume considerations. The maximum animal volume should rarely be
more than 5% of the total chamber volume, and chambers of 1 to 8 cubic
meters usually provide the best air distribution.
The chambers are usually constructed of smooth, nonabsorbent materi-
als that are resistant to a wide range of chemicals. Stainless steel walls
and glass or Plexiglass® observation ports and doors are the most common
materials. Rubber or plastic seals are necessary to prevent air loss and
should be resistant to the test chemical. Animal cages are generally made
of stainless steel mesh without any solid surfaces. Their arrangement
should facilitate exposure with rotation of the individual positions for
long-term studies.
Airflow systems for the chamber should provide units to condition,
filter, meter, and control the flow of the air, test agent, and their mixture.
The rate of airflow is determined by the animal loading and the test agent
feed rates. Various meters and valve arrangements are used to provide the
necessary control, with valves located downstream from filter units to
prevent clogging. An air pump is usually located in the exhaust line to pro-
vide the negative operational pressure (0.1 to 0.5 in. of water) for the
chamber. Air enters the chamber through the top pyramid and usually
exits through an arm of a Y-joint at the bottom pyramid. The air needs to
be conditioned to control temperature and humidity within an acceptable
range (75 to 82°F and <55% humidity). Automatic control systems for
this are available. Filter banks should be placed in the main supply line
and in the chamber and main exhaust lines. Usually several types of filters
are included in each bank to ensure satisfactory removal of contaminants.
During operation of the chamber, samples must be taken to monitor
the exposure conditions, including temperature, humidity, and test agent
concentrations (also particle sizes for aerosol atmospheres). Sampling
probes should monitor all areas of the chamber distribution with added
emphasis on conditions at the breathing zone of the animals. Sampling and
monitoring techniques for vapor and gas atmospheres usually utilize filters
with standard analytic techniques. Aerosol atmospheres are more difficult
to sample, and a whole array of samplers have been developed for this
including elutriators, cascade impactors, centrifugal force samplers precip-
itators, and optical monitors. These usually include some me'ans for
separating samples into size classes to determine particle size numbe d
concentration.
To maintain sanitary conditions within the chamber, techniques and
equipment have been developed to remove animal wastes Us 11
manent spray ring is used to wash down the chamber walls with^
appropriate cleaning solution. Cleaning procedures must be followed trf^
prevent cross contamination of different chambers and that t
animal technicians. ° e
-------
Occasional use of specialized chambers or equipment may be neces-
sary. Examples of such equipment include chambers that operate under
reduced or increased pressures and apparatus to evaluate effects of animal
activity during exposure to test agents.
1.3.3 Generation of Test Atmospheres
Inhalation testing requires that the test agent be put into an atmos-
pheric form for exposure. Atmospheres of gas or vapor test agents are
relatively simple to generate, compared to substances that must be in an
aerosol form.
Gases and vapors can be classified as irritants or asphyxiants. One
common method for generating gas and vapor atmospheres is to store the
chemical in high pressure cylinders. For batch introduction, air can be
mixed into the cylinder, while for dynamic systems the gas can be released
into the airstream. Porous diffusion plugs or containers can also be used to
release gases. If the substance is not volatile or chemically stable enough
to store in cylinders, it can be vaporized through controlled heating or
counter-current techniques.
The generation of aerosols must include techniques for many types of
aerosols including dusts, fumes, smoke, mists, and fogs using solid or fluid
feed materials. Aerosols occur in monodisperse forms where the particles
are all within a narrow size range or polydisperse forms that include a
wide range of particle sizes. Monodisperse aerosols are more useful for
studying particle size effects since they simplify the experimental condi-
tions, but polydisperse aerosols are more typical of actual exposure condi-
tions. The division of the parent material into particles or droplets of a
small enough size to remain airborne intensifies the chemical and physical
activities of the test agent because of the increased surface area and total
space occupied.
The generation of aerosols from solid test substances has been per-
formed using loose and compacted forms of the particles. Loose particles
must be kept in suspension by vibrating, rotating, or stirring techniques
usually combined with an aspirator to distribute the particles. Such tech-
niques are used more often for handling fibrous dusts or to produce
polydisperse aerosols. The most common technique for generating dust
aerosols is to use a compacted plug of powder and then scrape off particles
to redistribute. The Wright dust feed generator is a typical apparatus of
this design and uses a horizontal, rotating scraper blade and a packed plug
of powder in a brass cup. By controlling the humidity of the distributing
air stream, plus the packing density and particle sizes of the plug, there
should be no problem with redistributing the particles.
The generation of aerosols from liquids, solutions, or suspensions can
be accomplished with various techniques. Aspirators, which use a pressure
drop across an orifice and a stream of pressurized air to create droplets,
have been widely used for generating liquid aerosols. These are usually
combined with an impaction surface to limit the particle sizes produced. A
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8
typical example of such an air pressure-driven nebulizer is the Vaponeph-
rin nebulizer, which uses a glass sphere directly opposite from an air-
stream jet passing over a capillary tube to create monodisperse aerosols.
Ultrasonic vibrations from a piezoelectric crystal have also been used to
fractionate liquids in a nebulizer apparatus. The most common liquid
aerosol-generating technique is the spinning disk or top. These use centri-
fugal force to separate particles from a rotating flat surface upon which
the test liquid is applied. Such apparatus are capable of producing highly
monodisperse aerosols. Another technique for creating monodisperse aero-
sols is based on the controlled condensation of heated vapors on suitable
nuclei. Most of the above methods can also produce dry particles by
adding dry or heated air to the droplets after they are generated.
-------
2. TERATOGENICITY
Robert H. Ross
2.1 INTRODUCTION
The evaluation of agents for teratogenic effects and their impact on
human health is an area that only recently has become a significant regu-
latory concern. As Wilson (1979) states, "The notion that chemical and
physical agents in the environment need to be tested for their potential to
cause teratogenic effects when there is likelihood of exposure during preg-
nancy is a relatively new concept." It was not until the thalidomide
tragedy in the early 1960s that the world's attention was focused on the
fact that the human embryo is not isolated in an impervious maternal body
where it is shielded from all but genetic harm. Wilson points out that the
year 1966 probably deserves special recognition because of the significant
efforts initiated at that time to minimize the risks to the unborn popula-
tion from radiation (by the International Committee for Radiation Protec-
tion Recommendations) and to minimize such risks from drugs (by the
Food and Drug Administration Guidelines).
This chapter will be primarily limited to a discussion of chemically
induced congenital malformations and functional impairment. The first
section will discuss some basic test parameters, the second section will
examine the influence of the time of administration with regard to induc-
ing teratogenicity, the third will explore the science of behavioral terato-
genicity, the fourth will discuss the choice of species for teratological test-
ing, and the last section will address some of the recent approaches in
teratogenicity testing. The terms embryolethality or fetolethality will refer
to the death of the embryo or fetus, teratogenicity will refer only to mal-
formed offspring (both morphological and behavioral), and embryotoxicity
or fetotoxicity will refer to teratogenic, growth retardation, abortifacient,
or intrauterine death responses of offspring to a chemical.
2.2 GENERAL EXPERIMENTAL CONSIDERATIONS
Some basic parameters of testing chemicals for teratogenicity include
dosage (number, levels, and duration), positive controls, number of species,
number of test animals per dose group, administration route, and fetal
examination.
In reviewing teratogenicity studies with thest parameters in mind, it is
important to distinguish between screening studi -u d those aimed at the
further delineation of a teratogenic effect or thos^; lesigned to investigate
the toxicology of a clinically used dose. The protocols used in the latter
two instances will often be different from those for the screening study.
-------
10
Most of the comments in the following subsections will be directed to the
screening study. Table 2.1 presents a brief insight into the protocols used
by different researchers when conducting screening studies. The selection
of studies in this table was determined by searching the recent issues of
(1) Teratology, (2) Teratogenesis, Carcinogenesis, and Mutagenesis, and
(3) Toxicology and Applied Pharmacology.
2.2.1 Dosage—Number and Level
The Environmental Protection Agency (EPA) (USEPA 1982a, 1982b),
the Organization for Economic Cooperation and Development (OECD)
(OECD 1982), and the Food and Drug Administration (FDA) (USFDA
1982) each recommends the use of at least three doses. An examination of
the data in Table 2.1 (from 20 relatively recent research efforts) indicates
that most researchers are apparently testing at least three concentration/
dose levels.
Ideally, the high dose level should produce either maternal toxicity (but
not lethality), embryotoxicity, or fetotoxicity [Wilson 1975b; World
Health Organization (WHO) 1967]. The low dose should permit normal
embryonic development, and the intermediate dose(s) should produce mal-
formed offspring if the test chemical is teratogenic (Collins and Collins
1976). Extrapolation between the low dose and the intermediate dose or
doses would provide an indication of the effect/no effect range of the test
chemical.
2.2.2 Dosage—Duration
Schardein (1976) states that, in general, acute dosing of a chemical
results in a greater teratogenic insult than does prolonged dosing. One
example is that of the anticancer and antibiotic drug, actinomycin D, in
rats. Malformations were induced in 27.9% of the surviving offspring with
a single dose of 200 Mg/kg given on day 9 of gestation, but when the drug
was given as ten daily injections of 25 ng/kg on each of days 0 to 9 of ges-
tation, only 8.7% of the surviving offspring was malformed (Wilson 1966).
Comparable results were seen with the number of dead and resorbed
fetuses—32.5% were either dead or resorbed with the single 200-^g/kg
dose but only 9.7% were dead or resorbed when 25 /tg/kg was given on
each of days 0 to 9 of gestation. Further experimentation demonstrated
that the dam was sensitized by prolonged treatment with actinomycin D at
high doses so that for the dam or her offspring even moderately terato-
genic doses become lethal. The stimulation of drug-metabolizing enzymes
in the liver microsomes by prolonged dosing was suggested as the possible
mechanism of this phenomenon, which has been observed by Koppanyi and
A very in experiments with more than 100 drugs (Koppanyi and Avery
1966, as reported by Schardein 1976).
Similar results were demonstrated with the administration of the benz-
hydrylpiperazine antihistamine drug chlorcyclizine (King et al 1965^
-------
Table 2.1. Literature Survey of Experimental Parameters in Testing of Chemicals for Teratogenicity
Chemical
Species—number
per group
Number of doses/concentrations,
duration, and route'
Extent of
fetal examination
Positive
control
Reference
Concanavalin A
Rabbit—7 to 11 1 dose level—160 fig intracoelomically
Indole-3-acetic acid
Octoxynol-9
Chlordiazepoxide
(Cdz) and
amitriptyline (Amt)
Tridemorph
Tetrachlorobenzene
isomers
ONO-802"
Rat—26 to 40; 4 dose levels—5, 50, 200, or 500
mouse—25 to 38 mg/kg by gavage on gestation days 7
Rat—25 2 dose levels—0.5 or 5 mg/kg/day intra-
vaginally on days 6 to 15 of gestation
Hamster—11 to 1 dose level—28.5 mg/kg Cdz HCL or
18 70.3 mg/kg Amt HCL or combination
intraperitoneally on day 8 of gestation
Rats—21 to 27; 3 dose levels—20.6, 60.2, or 189.2 mg/kg
mice—22 to 27 for rats and 27.5, 81.7, or 245.1 mg/kg
for mice on days 6 to 15 of gestation
Rat—10 3 dose levels—50, 100, or 200 mg/kg by
gavage on days 6 to 15 of gestation
Rabbit—19 to 23 3 dose levels—0.0125, 0.0625, or 0.25
mg/kg intravaginally on gestation days
6 to 18
External (all live fetuses), skeletal No DeSesso 1979
(all live fetuses), soft tissue (internal
organs of all live fetuses examined
grossly and selected fetuses prepared
for visualization)
External (all fetuses), skeletal (all No John et al. 1979
fetuses), soft tissue (1/3 of fetuses)
External, (all fetuses), skeletal (2/3 of No Saad et al. 1984
litter), soft tissue (1/3 of litter)
External, skeletal, and soft tissue (all
fetuses)
No Beyer et al. 1984
External (all fetuses), skeletal and soft No Merkle et al. 1984
tissue (proportions not specifically stated)
External (all fetuses) skeletal (2/3 of No Kacew et al. 1984
litter), soft tissue (1/3 of litter)
Skeletal (1/2 of fetuses), soft tissue No Petrere et al. 1984
(1/2 of fetuses)
-------
Table 2.1 (continued)
Chemical
Species — number
per group
Number of doses/concentrations,
duration, and route*
Extent of
fetal examination
Positive
control
Reference
Cyproheptadine Rat—7 to 61C 2 dose levels in one series (25 or 50
chlorhydrate mg/kg) and 3 in another (15, 25, or 35
mg/kg); all given by oral intubation on
gestation days 6 to 15
Hexamethylmelamine Rat—15 to 30;
Rabbit—15
Trimethyl phosphite Rat—25
Rat: 3 dose levels—10, 20, or 40
mg/kg by gavage on gestation days
6 to 15 or for 4-day periods during
organogenesis; rabbit: 3 dose levels—20,
40, or 60 mg/kg by gavage on gestation
days 6 to 18
3 dose levels—16, 49, or 164 mg/kg by
gavage on gestation days 6 to 15
Triamcinolone Rat—3 3 dose levels—0.125, 0.25, or 0.5 mg/kg
intramuscularly on gestation days 9 to
11, 12 to 14, or 15 to 17
Methyl chloride Rat—25; Rat: 3 concentrations - 100, 500, or 1500
mouse—33 ppm by inhalation on gestation days 7 to
19 (6 h/day); mouse: 3 concentrations—
100, 500, or 1500 ppm by inhalation on
gestation days 6 to 17 (6 h/day)
External (all fetuses); fetuses with
abnormal appearance studied for soft
tissue and skeletal defects, other
fetuses randomly selected for soft
tissue or skeletal examination
Rat—external (all fetuses), skeletal
(~2/3 of fetuses), soft tissue (—1/3
of fetuses); rabbit—all fetuses exam-
ined for external skeletal and soft
tissue malformations
External (all fetuses), skeletal (2/3 of
fetuses), soft tissue (1/3 of fetuses)
External (all fetuses), skeletal (1/2 of
fetuses), soft tissue (1/2 of fetuses)
Skeletal (1/2 of fetuses), soft tissue (1/2
for head and fetal trunk examination)
No
No
Yes
No
No
Rodriguez-Gonzalez
et al. 1983
Thompson et al.
1984
Mehlman et al.
1984
Rowland and
Hendrickx 1983
Wolkowski-Tyl
et al. 1983
-------
Table 2.1. (continued)
Chemical
Acetonitrile
Species - number
per group
Hamster — 6 to 12
Number of doses concentrations,
duration, and route*
4 concentrations/dose levels — 1800,
Extent of
fetal examination
External (all living fetuses), ratio of
Positive
control
No
Reference
Willhite 1983
Delalutin
Cocaine hydro-
chloride
Valproic acid
Mouse—11 to 14d
Rat—8 to 12";
mouse—8d
Mouse—5 to 21
2-Nitro-p-phenylene Mouse—25 to 39
diamine
3800, 5000, or 8000 ppm by inhala-
tion on gestation day 8; 100, 200, 300,
at 400 mg/kg by intubation or intra-
peritoneally on gestation day 8
3 dose levels—42, 416, or 833 mg/kg sc
on gestation days 6 to 15
Rat: 3 dose levels—50, 60, or 75 mg/kg
mg/kg intraperitoneally on gestation days
8 to 12; Mouse: 1 dose level—60 mg/kg
intraperitoneally on gestation days
7 to 16
5 dose levels—1.0, 1.2, 1.4, 1.65, or 2.7
mmole/kg intraperitoneally on days 8 to 10
of gestation; 2 dose levels - 1.0 or 4.4
mmole/kg on days 11 to 13 of gestation
7 dose levels—32, 64, 128, 160, 192,
224, or 256 mg/kg sc on gestation
days 6 to 15
fetuses examined for skeletal and soft
tissue defects not given
External (all fetuses), skeletal (—1/2 of Yes Seegmiller et aL
fetuses), soft tissue (-1/2 of fetuses) 1983
External (all fetuses), 3:1 ratio for fetal No Fantel and
examination of soft tissue to skeletal Macphail 1982
All live fetuses examined for external, No Kaoetal. 1981
skeletal, and soft tissue abnormalities
External (all live fetuses), skeletal (all No Marks et al. 1981
live fetuses), soft tissue (at least 1/3
of each litter, as well as all stunted
fetuses and those with external
malformations)
-------
Table 2.1. (continued)
Chemical
Phencyclidine
Species - number
per group
Mouse — 18 to 45
Number of doses concentrations,
duration, and route*
4 dose levels— 60, 80, 100, or 120 mg/kg
Extent of
fetal examination
External (all live fetuses), skeletal (all
/ , i -* 1 /i
Positive
control
No
Reference
Marks et al. 1980
by gavage on gestation days 6 to 15
o-Toluenediamine Rat—22 to 25; Rat: 4 dose levels—10, 30, 100, or 300
rabbit—14 to 16 mg/kg orally on gestation days 6 to 15;
rabbit: 4 dose levels—3, 10, 30, or 100
mg/kg orally on gestation days 6 to 18
Acrylonitrile Hamster—NGC 4 dose levels—0.09, 0.19, 0.47, or 1.23
mmole/kg intraperitoneally on day 8 of
gestation
L-Azetidine-2- Hamster—5 to 16" 4 dose levels—100 mg/kg on gestation
carboxylic acid days 7 to 12; 200 mg/kg on gestation days
7 to 9, 7 to 12, or 10 to 12; 300 mg/kg on
gestation days 9, 10, or 11; 600 mg/kg on
gestation days 7, 8, 9, 10, 11, or 12; all
doses administered intraperitoneally
live fetuses), soft tissue (at least 1/3
of each litter, as well as all stunted
fetuses and those with external
malformations)
Skeletal (1/2 of each litter), soft tissue
(1/2 of each litter)
Skeletal (all live fetuses)
External (all live fetuses), skeletal (1/3
of each litter), soft tissue (10 fetuses
from remaining specimens in each
group)
Yes Becci et al. 1983
No Willhite et al. 1981
No Joneja 1981
"Although negative controls not specified, each experiment used them.
bSynthetic E, postaglandin.
'All but the high dose group had at least 20 animals.
dNumber of litters examined.
°NG = not given.
-------
15
Chlorcyclizine hydrochloride administered to pregnant rats in 25-mg/kg
doses over a 4-day period (days 12 to 15 of gestation) produced 16% mal-
formed young; the same dosage given over a 15-day period (days 1 to 15)
resulted in malformations in only 2% of the offspring. The results were
even more striking when a 50-mg/kg dose was administered. Treatment on
days 10 to 15 of gestation resulted in 82% malformed individuals, but only
0.1% malformed offspring was produced when a dosage of 50 mg/kg was
given over days 1 to 15 (implantation was not inhibited). A subsequent
paper (King et al. 1972) showed that the explanation behind this lack of
teratogenic activity from prolonged treatment (defined in this instance as a
15-day period) of chlorcyclizine was the drug's self-stimulation of its own
metabolism.
Wilson (1975b) and the WHO (1967) also report that the repetitive
administration of certain chemicals during pregnancy can alter or mask a
teratogenic action because of the ability of these chemicals to change their
own metabolism. Wilson states three ways that some chemicals can change
the rate of their own metabolism: (1) the induction of catabolizing
enzymes (microsomes) in the liver or other tissues, which increases meta-
bolism (also mentioned above); (2) the inhibition of naturally present
enzymes which degrade chemicals; and (3) the induction of impaired func-
tion or overt pathology in important homeostatic organs such as the liver
or kidneys. He further states that it is assumed these changes can occur
within three or four days of the beginning of repeated treatments.
What then is the optimum dosing period for testing chemicals for tera-
togenicity? The foregoing paragraphs suggest that dosing for only one day
or for short periods of three to four days during organogenesis would
enable detection of chemical teratogens whose teratogenicity might go
undetected if dosing duration was increased. Wilson (1975b) recommends
both short-term and repeated tests. His recommended procedure consists
of dividing the period of organogenesis into shorter dosage periods of three
to four days but also dosing some animals throughout organogenesis. He
states that fewer animals would be needed in each dose group than if
dosage were given throughout organogenesis because the range of likely
developmental defects, and hence overall variability, would be reduced by
the shorter treatment span. As mentioned, some animals would still be
dosed throughout organogenesis to detect any teratogenic effects that
would result from the cumulative action of the chemical. Collins and Col-
lins (1976) list three reasons why the single-dose technique—which, they
report, is recognized as the most successful in producing abnormalities—is
not used in place of the repetitive dosing method in screening studies.
These are: (1) the necessity of treating different animals for each day of
organogenesis increases the cost of the experiment, (2) without prior
knowledge of the compound's effects, there is no way of predicting
whether a specific organ or the entire developmental sequence will be
affected, and (3) compounds that are likely to be tested for teratogenic
-------
16
action are usually encountered by humans as repetitive doses, and with sin-
gle dosing cumulative effects cannot be measured.
Current guidelines (OECD 1981, USEPA 1982a, 1982b, USFDA
1982) recommend dosing during the period of organogenesis. The critical
exposure periods for induction of behavioral changes in the postnatal
animal are at present not well defined, and it is conceivable that existing
guidelines will be amended as more information becomes available.
2.2.3 Positive Controls
Although positive controls are not recommended in current guidelines
for teratogenicity testing (OECD 1981, USEPA 1982a, 1982b, USFDA
1982), they have been used to demonstrate that the test animal will pro-
duce malformed offspring after exposure to an established teratogenic
chemical as well as to validate the soundness of a specific laboratory's or
researcher's methodology. This use of positive controls in teratology has
not been completely abandoned (Table 2.1).
The data generated from positive controls must be evaluated with some
caution because an animal species can be very sensitive to one teratogen
but only mildly or not at all sensitive to another. Table 2.2 shows the tera-
togenic effects from thalidomide in several animals, including man. Man,
monkeys, and rabbits are shown to be susceptible to the teratogenic action
of thalidomide, but several strains of rats, mice, and hamsters are not (at
least by conventional testing protocols). The action of the human teratogen
aminopterin (Table 2.3) also shows apparent species variability. Terato-
genic effects have been seen in man and mice but not in rats and monkeys
(Table 2.3). The data in these tables clearly demonstrate that an animal
may be susceptible to the teratogenic action of one chemical but not neces-
sarily to that of another chemical. Therefore, if a positive control is esta-
blished in the animal species being tested and the test chemical does not
produce teratogenic effects, it cannot be assumed that the test chemical
would not induce teratogenic effects in another animal species or even
another strain of the same species. Conversely, if an established teratogen
such as thalidomide fails to induce teratogenicity in an animal being used
as a positive control, it cannot be assumed that the test chemical will not
produce teratogenic effects in the same species.
2.2.4 Number of Species
As Tables 2.2, 2.3, and 2.4 indicate, the choice of species or strain can
determine whether or not a chemical is identified as a teratogen. Species
variability to potential teratogenic agents would thus dictate that at least
two species should be used. Current EPA guidelines recommend the use of
at least two species, with the rat and rabbit being the preferred species
when only two species are used (USEPA 1982a, 1982b). The FDA guide-
lines (USFDA 1982) also state that these are the preferred species.
-------
Table 2.2. Thalidomide Action in Various Species
Animal
Species
strain
Dose, route
and duration
Effects
Reference
Man
Mouse Schofield
A, C3H, Swiss
CF,, ICR, C57,
CBA, SJL
Rat
Dose not specified, oral adminis-
tration during first trimester
Dose and duration not known; oral
administration
400 mg/kg orally throughout
pregnancy
A-31 to 625 mg/kg/da/; C3H,
Swiss - 62 mg/kg/day; admin-
istered orally on days 6 to 7 of
gestation and continued for 4 to
6 days
200 mg/kg total given orally for
gestation days 2 to 12
2 g/kg total ipb on gestation day
12, days 11 and 12, or days 12
and 13
Missing limbs; auricle or pinna of ear decreased
in size; lesser deficiencies
Reports of several cases—hypoplasia and aplasia
of the extremity or individual bones, absence
and malformation of limbs; malformations of
other organs such as stenoses and malrota-
tion of the gastrointestinal tract and dysplasias
of the external ears and eyes often associated
No malformed fetuses in albino mice
Skeletal malformation; open eye; enlarged skull;
curvature of back; kinky tail; phocomelia;
micromelia
No malformed offspring but increased resorptions
in CF, strain
16 litters - two had abnormalities, complete
resorption in one, more than 50% resorption
in three, and normal offspring in ten; abnor-
malities consisted of stunting of extremities
and tails, absence of dorsal and lumbar
vertebrae and ribs, absence of digital bones,
and curved bones of the fore and rear extremities
Smithells 1962
Pfeiffer and Kosenow 1962,
Lenz and Knapp 1962
Somers 1963
DiPaolo et al. 1964
Fratta et al. 1965
Murphy 1962
-------
Table 2.2 (continued)
Animal Species
strain
Wistar
Wistar
Dose, duration,
and route
200 or 400 mg/kg total given
orally throughout pregnancy
45 mg/kg ivgon day 10, 11, or 12
of gestation
Effects
Fetal resorptions but no abnormalities
Skeletal deformities of thoracic ribs and of
spinal column in 33 and 56% of fetuses on
Reference
Somers 1963
Parkhie and Webb 1983
Sprague-Dawley 10 (ip), 20 (orally), 50 mg
(orally or ip) per day at
various dosing schedules be-
tween days 7 and 17 of gestation
Charles River 100 mg/kg 6 to 12 total given orally
on gestation days 6 to 12
Long Evans and 150 mg/kg total given orally on
Dunning Fischer gestation days 3 to 12
Holtzmann 25-500 mg/day orally or ip at
various dosing schedules
during organogenesis
day 11 and 12, respectively; deformities of
eyeball as a result of administration on
day 10 or 12
520 young examined—36 grossly malformed (6.9%),
40 showed malformations after clearing of fetuses,
76 total malformations (14.6%); malformations
included malrotation of hind limbs, hamartoma of
the palate with accessory incisors, lack
of a tail in one instance, and a subcutaneous
cartilaginous-type mass of tissue from the
middorsal region to the tail
Resorptions but no malformations
No apparent injury
More resorptions than controls; only one fetus
showed serious limb malformation: sig-
nificant teratogenic effect shown by missing
sternebrae and delayed ossification of
the sternum; malformations different
from humans
King and Kendrick 1962
oo
Delahunt et al. 1966
Fratta et al. 1965
Moore 1965
-------
Table 2.2 (continued)
Animal
Species
strain
Dose, duration,
and route
Effects
Reference
Hamster Not given
Not given
Inbred and random
Monkey Rhesus and
stump-tailed macaque
Bonnet
Baboon, bonnet,
and cynomolgus
Marmoset
Up to 8000 mg/kg throughout
pregnancy
150 mg total given orally for
gestation days 3 to 12
350 mg/kg/day orally throughout
pregnancy
5 mg/kg/day orally on
gestation days 26 to 28
or 24 to 30 or 10 mg/kg on days
25 to 27, 24 to 30, or 23 to 29
10 mg/kg orally on day 24, 25,
26, 27, 28, or 29 of gestation;
30 mg/kg orally on day 25 or 28
17.5-300 mg/kg total given
orally at various dosing
schedules on gestation days
18 to 43 (both single and
multiple doses)
25 mg/kg/day orally on
gestation days 38 to 52
Not teratogenic
Somers 1963
Fratta et al. 1965
Homburger et al. 1965
Not teratogenic
Inbred lines showed 6.2% incidence of grossly
malformed fetuses—acrania or split cranium,
abnormal positioning of legs, kinking of
tail, and cleft palate; random bred lines
showed no significant teratogenicity
Severe deformities in most species—
missing digits, missing radii and ulnae,
shortened humeri, kinking and in some
cases shortening of tail
Temporal and mandibular bones malformed Hendrickx and Newman 1972
Vondruska et al. 1971
Malformed fetuses - deformed limbs, spina
bifida, kinked tail, etc
Reduction in size of spinal cord and
dosal root ganglia; pattern of deformities
said to be similar to man
Hendrickx 1970
McBride and Vardy 1983
-------
Table 2.2 (continued)
Animal
Species
strain
Dose, duration,
and route
Effects
Reference
Rabbit New Zealand
white and
Dutch-Belted
New Zealand white
New Zealand white
Japanese white
Not given
150 mg/kg/day orally
on days 8 to 16 of
pregnancy
150 mg/kg/day orally on
gestation days 8 to
16 or 2 to 9
50, 150, or 300 mg/kg/day
oralJy on gestation days
6 to 18
Low incidence of deformed appendages in
New Zealand whites; increased incidence
of limb defects in Dutch-Belted—
agenesis or hypogenesis of the ulna,
radius, tibia, and/or fibula; absence
of some bones in limbs
Limb deformities in almost every litter
Sixty-seven percent of kits deformed
when administered on days 8 to 16—most
predominant effects seen in limbs
(arthrogryposis, micromelia, absence of
digits), but developmental failure of
visceral organs (kidneys, adrenals) and
in bony structures also observed; only
17% abnormalities in the one litter
exposed on days 2 to 9
External skeletal and internal anomalies
significantly (.P<0.01) different from
controls at 150 and 300 mg/kg day;
external anomalies included contracture
of forearm and club foot
Delahunt 1965
Somers 1963
Drobeck et al. 1965
Matsubara et al. 1983
-------
Table 2.2 (continued)
. . . Species Dose, duration, _., _ ,
Animal . , Effects Reference
strain and route
Japanese white— 50, 150, or 300 mg/kg/day External, skeletal, and internal Matsubara et al. 1983
NIBS orally on gestation days anomalies significantly different at all
6 to 18 dose levels (P<0.01); external anomalies
of highest frequency were holoprosen-
cephaly, anencephaly, hypoplasia of
ala nasi, and club foot
"625 mg/kg dose resulted in 55% abortion rate and thus a minimum number of malformations resulted.
blntraperitoneally
'Intravenously
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Table 2.3. Aminopterin Action in Various Species
Animal Spedes/
strain
Dose, route,
and duration
Effects
Reference
Man
29 mg orally over 10 days at ~6.5
to 8 weeks of gestation
20 mg orally over several weeks during
second trimester of pregnancy
12 mg orally over 12 days during early
pregnancy
Mouse ICR-DUB 25 mg ip'on day 11 or 12 of gestation
Rat
Wistar and
others
Wistar
Monkey Rhesus
Rhesus and
cynomolgus
0.05, 0.075, 0.1, or 0.2 mg/kg single ip
injections on various gestation days
0.1 mg/kg ip on gestation day 11 or 12
0.12 mg/kg orally on days 24, 25, and
26 of gestation
0.1-0.2 kg/day on gestation days 21 to 33;
1.0 kg/day on gestation days 38 to 39
Excessively large head; nasal bridge broad and flattened;
eyes widely separated; malformed ears; mandibular
hyoplasia; posterior cleft palate; absence of parietal
bones in skull
Multiple skull anomalies; left talipes equinovarus
Numerous head abnormalities—soft skull, no ossification of
parietal bones, eyes wide apart, broad nasal bridge,
posterior cleft palate, and others; large hands, other
anomalies also described
Short or small limbs; hemimelia; severe ectrodactly
(absence of one or more fingers or toes)
Injections on days 7 and 10 effective—embryolethality
No malformed offspring—fetuses resorbed with high doses
No injury to offspring
Two aborted and one normal fetus, but no malformed
offspring
Rabbit Not given 2 mg/kg by injection to six-day-old blastocyst No injury
Emerson 1962
Meltzer 1956
Warkany et al. 1959
Kochhar 1975
Baranov 1965
Murphy 1962
Tanimura 1972
Wilson 1969
Hay 1964
"Intraperitoneally.
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Table 2.4. Methotrexate Action in Various Species
. . . Species/
Animal . '
strain
Dose, route,
and duration
Effects
Reference
Man
Mouse ICR
Rat
Monkey Rhesus
Rhesus
Rabbit New Zealand
white
New Zealand
white
5 mg/day orally through second
month of pregnancy
2.5 mg/day orally for five days ~
between 8th and 10th week of preg-
nancy
0.3 to 50.0 mg/kg ip'on day 10 of
gestation
0.2 mg/kg on day 9 of gestation
2.5 to 4 mg/kg various regimes on
gestation days 17 to 45
3 mg/kg ivb on gestation days 29 to
32
9.6 mg/kg iv on day 10 of gestation
19.2 mg/kg iv during days 11 to 14
of gestation
Major abnormalities of skull of infant
Major anomalies—absence of frontal bone, absent lambdoid
and coronal sutures, multiple anomalous ribs, unusual facies,
and absence of all digits on right foot and all but one on left
foot
25 and 50 mg/kg produced congenital defects, primarily cleft
palate and reduction of digits
103 embryos exposed—64% resorptions and 30% malformations
Most fetuses normal, three aborted and one showed moderate
gut rotation; only 13 embryos exposed, so trivial teratogenicity
might reflect small sample size
Mildly embryolethal, nonteratogenic, transitory growth retar-
dation
50% fetal mortality and a 25% malformation rate in surviving
fetuses
Multiple anomalies—facial clefts, cleft palates, defects of fore
and hind limbs, etc.
Powell and Ekert 1971
Milunsky et al. 1968
Skalko and Gold 1974
Wilson 1971b
Wilson 1971b
Wilson et al. 1979
Jordan et al. 1970
Jordan 1973
"Intraperitoneally.
blntravenously.
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24
An economical alternative would be to test the two species separately
instead of concurrently and test the second species only when the first
species has given negative or ambiguous results. In the interest of human
safety, the test chemical would have to be considered potentially terato-
genic to humans if one animal species produces malformed offspring.
However, the more animal species that demonstrate a chemical to be tera-
togenic, the greater the possibility that the chemical will be teratogenic in
humans, and by testing two species data would be available for species
comparisons. Such information as the difference or similarity in dose or
anatomical defects would become available and would be beneficial in
other teratogenic studies with these same species. In addition, the use of
two species would give a better understanding of the effect/no effect level
of the test chemical.
The use of only two species (a rodent and rabbit) appears to be justi-
fied in view of the fact, stated by the WHO in 1967, that all substances
shown to be teratogenic in man have also demonstrated teratogenic
activity in the mouse, rat, and rabbit. The WHO cautions, however, that
negative results obtained by testing chemicals in these species provide no
absolute assurance that the chemical will not induce teratogenic effects in
man. This situation would particularly be of concern when the chemical
tested is a drug intended for use during human pregnancy or a chemical to
which heavy exposure is likely for pregnant women. In these instances,
when the rabbit or rodent does not demonstrate teratogenicity, a third
species closer to man physiologically probably should also be used as a test
animal. This is similar to the testing scheme proposed by Wilson (1975),
who recommends the use of the rat, mouse, hamster, or rabbit as screening
species to establish the embryotoxic dose range and not as an end point for
teratogenic studies unless appreciable embryotoxicity is demonstrated at
appropriate multiples of the anticipated human dose.
2.2.5 Number of Test Animals Per Dose Group
The number of pregnant animals per dose group is an important con-
sideration. As Weil (1970) points out, for a statistical analysis of the
results of an experiment designed to assess teratogenicity, the number of
independent sampling units, N, is the number of dams or litters. Using the
number of pups as N will produce invalid conclusions.
The number of animals used to test chemicals for teratogenicity is vari-
able. The results of some of the studies cited in Table 2.1 might be ques-
tioned because too few animals in some dose groups were used to permit
reliable statistical analysis. The WHO (1967), although not specifying
animal numbers, states that the number of rodents used must be large
enough to satisfy statistical requirements. For species more closely related
to man, the WHO recommends that the number of animals be as large as
practicable, in order to obtain reproducible results. Current guidelines
recommend 20 pregnant rats, 20 pregnant mice, 20 pregnant hamsters
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25
and 12 pregnant rabbits per dose group (OECD 1981, USEPA 1982a,
1982b, USFDA 1982). The FDA guidelines add that these are minimum
numbers of animals at or near term.
2.2.6 Administration Route
The choice of administration route is usually dictated by the present or
expected human exposure to the test chemical (Collins and Collins 1976).
In most instances, the chemical should be administered orally, but
administration by other methods such as dermal or inhalation may be
necessary in order to simulate human exposure. The EPA (USEPA 198la,
1982b), the FDA (USFDA 1982), and the OECD (OECD 1981) guide-
lines recommend oral administration by gavage unless the chemical and
physical characteristics of the test chemical or the use pattern would sug-
gest another route.
2.2.7 Fetal Examination
Current guidelines (OECD 1981, USEPA 1982a, 1982b, USFDA
1982) are in agreement concerning the extent of fetal examination. All
fetuses should be examined externally, and one-third to one-half of all rat,
mouse, and hamster fetuses should be examined for skeletal defects with
the remainder examined for soft tissue malformations. All rabbit fetuses
should be examined for both skeletal and soft tissue malformations. The
data in Table 2.1 indicate that, while many researchers are following this
protocol, some are not (e.g., Mehlman et al. 1984).
2.3 STRUCTURE-ACnvrTY RELATIONSHIPS
As Schardein (1983) states "One of the most potentially promising
measures of teratologic risk is the structure-activity relation of chemicals."
Schuler et al. (1984) in subjecting 15 glycol ethers to an in vivo
reproductive toxicity assay found that the 5 ethers with terminal methyl
groups and 2 with terminal ethyl groups produced few viable CD-I mice
litters whereas 3 butyl ethers, 3 glycol ethers with terminal hydroxy
groups, and 2 ethyl ethers did not produce such profound fetotoxicity.
The evidence generated by Willhite et al. (1984) in their study of the
structure-activity relationships of retinoids suggested that the changes in
teratogenic activity associated with structural modification of vitamin A at
carbon 15 were primarily dependent upon the presence of or biotransfor-
mation to a free carboxyl or a moiety with an equivalent pKa at carbon
15. The molecular size of the substitutent or the sterochemical position
about carbon 13 was considered of secondary importance for the induction
of terata.
A series of papers (Keeler 1970, Brown 1978, Brown and Keeler 1978)
have discussed the teratogenic structural requirements of naturally occur-
ring steroids. Keeler (1970) studied jervine, 11-deoxojervine, and
3-glucosyl-ll-deoxojervin, three compounds teratogenic to sheep (causing
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26
clyclopia) which structurally differ only in substitution at the 3 and 11
positions. Keeler tentatively concluded that the nature of the substitution
of these two positions has little influence on teratogenicity. Testing with
more than 18 additional compounds including rubijervine, veratramine,
and testosterone in sheep did not produce cyclopia, although veratramine
did cause occasional bowing of limbs, joint flexure, and lack of muscular
control. None of these compounds possessed the fused oxide and piperidine
rings present in the active compounds (i.e., jervine, 11-deoxojervine, and
3-glucosyl-l 1-deoxojervine).
Brown (1978) investigated the teratogenic activity in the hamster of
several derivatives of jervine and 11-deoxojervine. His findings showed that
highly teratogenic compounds present a negatively charged center accessi-
ble to the steroid a face. Similarly, Brown and Keeler (1978) stated that
"conventional steroids with a secondary basic nitrogen bonded in a position
analogous to hormone binding sites (accessible to the a face of the steroid)
would be predicted to be substantially teratogenic."
Freese et al. (1979) studied the correlation between the growth inhibi-
tory effects, partition coefficients, and teratogenic effects of a number of
lipophilic acids and found many of the acids that were potent inhibitors of
mammalian cell replication to be also teratogenic.
Schumacher (1975) reported that very strict structural requirements
govern the teratogenic effects of thalidomide (in particular an intact
phthalimide or phthalimidine moiety) and, although investigations with
thalidomide have revealed a number of interesting and rather unique pro-
perties of this chemical, no conclusive evidence connecting any special pro-
perty of thalidomide with its teratogenicity has been presented.
2.4 TERATOGENESIS AND TIME OF ADMINISTRATION
A chemical should be considered an unlikely teratogen only after
proper teratological experimental procedures have been performed. One of
the most critical aspects of testing chemicals for teratogenicity is determin-
ing the most susceptible time during gestation for chemical administration.
2.4.1 Preimplantation
In both rat and man, there is an interval of approximately six days
between the fertilization of the oocyte and the implantation in the
endometrium known as the preimplantation or predifferentiation stage
(Leonard 1983; also see Table 2.5). In this largely undifferentiated state,
the ovum is generally considered to not be at risk from teratogenic agents.
With respect to drugs Leonard (1983) states, "It appears that drugs are
either toxic to the entire embryo, thereby resulting in its death, or affect
only a relatively small number of cells, that can be repaired without caus-
ing any obvious physical damage." However, consideration must be given
to the possibility of chemicals with long half-lives whose effects may be
manifested days after exposure. An example is the progestin cyproterone
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27
Table 2.5 Preimplantation Developmental Stages in Man and Laboratory Animals
Figures as Postconceptional Days
Stage
Fertilzed eggs in the
fallopian tubes
2 cells in oviduct
4 cells in oviduct
8 to 12 cells in oviduct
Blastocele begins to
develop - oviduct
Free blastocyst in uterus
Implanting blastocyst
Implantation in progress
Post implantation
Delivery
Human
1
2
2 1/2
3
4
5
6
6 to 13
19 to 260
260 to
280
Monkey &
baboon
1
2
3
4
5 to 6
7 to 8
9
9 to 15
18 to 160
164 to
168
Rabbit
1/4
1/2
1
2
3
4
7
7to8
8 to 29
30 to
32
Rat
1
2
3
3 1/4
4
5
6
6 to 8
9 to 21
22
Mouse
1/2
1
2 1/4
21/2
3
4
4 1/2
4 1/2 to 6
7 1/2 to 19
20
The following days are those of high teratogenic risk: human, 19 to 80; monkey and
baboon, 18 to 50; rabbit, 8 to 18; rat, 9 to 17; mouse, 7 1/2 to 15.
Source: Adapted from Leone 1977, Fig. 1, p. 19.
acetate whose long biological half-life (60 h) was responsible for abnormal
morphological development when administered in high doses on day 2 of
gestation in the mouse (Eibs et al. 1982).
With respect to behavioral teratogens, limited evidence suggests that
exposure during the predifferentiation period may result in behavioral
abnormalities. Werboff and Havlena (1962) found that tranquilizers
administered to the gravid rat induced behavioral changes in the offspring
independent of the trimester of gestation during which these drugs were
given. In this study, the tranquilizers reserpine, chlorpromazine, and
meprobamate (0.1, 6.0, and 60.0 mg/kg/day subcutaneously, respectively)
were administered on days 5 to 8, 1 to 14, or 17 to 20 of gestation with
each daily dose divided into three equal injections. Because day 8 (Leo-
nard 1982) or day 9 (see Table 2.5) in the rat is considered the first day
of the differentation phase of development, administration on days 5 to 8
would be during the predifferentiation phase.
2.4.2 Organogenesis
The period of embryological differentiation (organogenesis) is con-
sidered to be the most sensitive developmental stage to the action of tera-
togenic chemicals (Wilson 1975, Schardein 1976). Schardein (1976) lists
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28
what he considers to be the critical periods for a number of animal species
(see Table 2.6); the period of greatest susceptibility in man is similar to
that of the baboon and the rhesus monkey. As indicated in Table 2.1, dos-
ing periods used by researchers vary. Current regulatory guidelines
(OECD 1982, USEPA 1982a, 1982b, USFDA 1982) recommend
appropriate dosing periods for the mouse, rat, hamster, and rabbit. These
guidelines are consistent with respect to the mouse and rat (gestation days
6 to 15). The USFDA guidelines are slightly different with respect to the
hamster and rabbit; the OECD and EPA recommend dosing during gesta-
tion days 6 to 14 for the hamster and 6 to 18 for the rabbit, whereas the
FDA recommends days 4 to 14 for the hamster and 7 to 18 for the rabbit.
Although Table 2.6 indicates several days categorized as in the critical
period, the embryologic differentiation of the various organs proceeds at
varying rates; thus, the time of teratogenic insult is important with regard
to which tissues are affected. This is illustrated in the research of King et
al. (1972), Kajii et al. (1973), Sadler and Kochhar (1975), Inouye and
Murakami (1977), and Eibs et al. (1982). Inouye and Murakami studied
the teratogenicity in mice of the hair dye constituent 2,5-diaminotoluene
and found that subcutaneous or intraperitoneal injections of 50 mg/kg
body weight caused a low incidence of exencephaly (cranial malformation)
and prosoposchisis (fissure of the face, e.g., hairlip) and a high incidence
of skeletal malformations in those animals treated on day 8 of gestation.
No such malformed fetuses, however, were found in those animals treated
on days 10 to 14 of pregnancy, and only a very low incidence of vertebral
and rib anomalies was observed in the fetuses treated on gestation day 7
or 9.
In studying the teratogenic effects of a single oral injection (intuba-
tion) of chlorambucil (14.2 or 20 mg/kg) on the 10th, llth, 12th, or 13th
day, Sadler and Kochhar (1975) observed limb defects from treatments on
the llth or 12th days of gestation and tail defects from treatment on all
days. Treatment on the 13th day resulted in digital defects but without the
long-bone defects observed with day 11 and 12 treatments; defects were
observed only at the highest dose level. Sadler and Kochhar also found
that in vitro limb bud response to chlorambucil was similar whether the
limb buds were taken from pregnant mice after chlorambucil treatment or
the chlorambucil was applied to limb bud cultures after removal from non-
treated pregnant mice.
Eibs et al. (1982) administered cyproterone acetate (30 mg/kg subcu-
taneously) to mice on one of days 1 to 12 of gestation. The most sensitive
gestation days for abnormalities were days 5 and 6 for the urinary tract,
days 8 and 9 for the respiratory tract, and days 10 and 11 for cleft palate.
The significance of the critical period is also shown by the human tera-
togen thalidomide. One study identified 113 pregnant women who took
thalidomide between August 1959 and December 1961, but only 7 women
took the drug during the critical period (reported here to be between 34
and 50 days after the last menstrual period) (Kajii et al. 1973). Of these
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29
Table 2.6. Critical Periods of Organogenesis in Animals
Species
Hamster, golden
Mouse
Rat
Rabbit
Ferret
Cat
Dog
Guinea pig
Pig
Sheep
Monkey, rhesus
Monkey, baboon
Armadillo
Human
Cow
Horse
Mean duration
of gestation
(days)
16
19
21
31
43
63
63
68
114
150
168
175
225
278
284
336
Gestation
Days"
4 to 14
7 to 16
9 to 17
8 to 21
8 to 28
5 to 58; 5 to 15
most favorable
1 to 48; 8 to 20
estimated
1 1 to 20
12 to 34
14 to 36
20 to 45; 22 to 30
most susceptible
22 to 47
1 to 30
20 to 55
8 to 25
?
aP*»ri/t/1 f\f r*mhr\jn\ri{rir*ti] nro5mnoenp.sic fir nftrind nf
known susceptibility.
Source: Adapted from Schardein 1976, Table 2-5, p. 17.
Data collected from several sources. Reprinted with per-
mission of the publisher.
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30
seven women, three delivered malformed babies, and four delivered babies
without apparent malformations. Possible explanations for the four normal
babies include inaccurate pregnancy diagnosis time, so that thalidomide
possibly was not taken during the critical period, or actual resistance to
thalidomide. Two additional women took the drug before the critical
period, on day 19 and day 23, respectively; both women aborted (induced
and spontaneous abortions, respectively). The remaining 104 women took
thalidomide after the critical period with all offspring apparently normal,
except for one reported case of anal stenosis.
One other example of the importance of the critical period is shown in
rats administered |8-aminopropionitrile, a chemical that induces cleft palate
(King et al. 1972). In this instance, there were a significant number of
offspring with cleft palate only when day 15 of the gestation period was
included in the dosing schedule. All of the viable fetuses had cleft palate
when 875 mg/kg was given on day 15, or over days 13 to 15 or 14 to 15,
but when a total dose of 4320 mg/kg was administered over days 12 to 14,
only 8% of the young were malformed.
2.4.3 Histogenesis and Fetal Period
The period of prenatal development that slightly overlaps organo-
genesis but extends primarily into the fetal period is known as histogenesis
(Fig. 2.1). Teratogenic agents that come into contact with the developing
fetus during this time of tissue formation and development can cause
minor structural deviations, but the abnormalities that are more likely to
occur during the fetal period are those involving growth or functional
aspects of development (Wilson 1975b). An example is the administration
of vitamin A during the early- and mid-fetal period (Hutchings et al.
1973, Hutchings and Gaston 1974). Pregnant rats administered a terato-
genic dose of vitamin A during the early fetal period (days 14 and 15 of
gestation) produced offspring that showed a behavioral deficit indicating
nonreinforcement (characterized by a decreased ability to inhibit respond-
ing to an auditory signal). In addition to behavioral effects, a generalized
retardation in growth was induced, as evidenced by delayed onset of fur
growth and eye-opening and by reduced body weight as well as reduced
brain size with obvious microcephaly in one animal. Pregnant rats treated
with vitamin A during the mid-fetal period (days 17 and 18 of gestation)
produced offspring with no retardation in growth or in brain size, but with
a possible motor deficit affecting coordination that resulted in slower rates
of response to auditory stimuli than control animals.
2.5 BEHAVIORAL TERATOGENESIS
As described by Hutchings (1983), behavioral teratology [Vorhees
(1983) uses the synonym psychoteratology] is an integration of teratology
with experimental psychology. The primary concern of behavioral teratol-
ogy is the study of neurobehavioral changes that occur when germ cells,
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31
ORNL-DWG 79-13670
TERATOGENIC SUSCEPTIBILITY IS GREATEST
DURING EARLY ORGANOGENESIS
I-FUNCTIONAL MATURATION-
EMBRYONIC PERIOD FETAL PERIOD
ENTIRE DEVELOPMENTAL SPAN
Figure 2.1. Representation of the susceptibility of the human embryo to
teratogenesis, beginning with fertilization and continuing throughout intra-
uterine development. (Source: Adapted from Wilson 1975b, Figure 4, p.
662).
embryos, fetuses, and immature postnatal organisms are exposed to a
variety of environmental disturbances and events. Regarding the state of
the art of behavioral teratology, Hutchings (1983) writes, "I should
emphasize, however, the current state of behavioral teratology as a fled-
gling discipline; its subject matter remains largely unexplored and
uncharted, its methodology has been uneven and occasionally wanting, and
its accomplishments strike a tenuous balance between false starts and pal-
pable inroads made by a few pioneering researchers. But despite its imma-
turity and tentative beginnings, there should be no doubt that it is emerg-
ing as a new scientific speciality."
Although the period of organogenesis is generally considered the time
the embryo is at greatest risk to morphological alteration, the exposure
period is not as well defined with respect to the induction of behavioral
changes. As discussed in Section 2.4.3, the histogenesis period can be a
sensitive period for behavioral teratogens. However, as Coyle et al. (1976)
reported, behavioral anomalies can also be induced in the offspring as a
result of exposure during organogenesis. They cite Hoffeld and Webster
(1965) and Murai (1966), who found that when chlorpromazine was
administered during early pregnancy maze learning was impaired, but no
effects were observable when it was applied late in pregnancy. In addition,
Werboff and Havlena (1962) demonstrated that behavioral changes
induced by the administration of tranquilizing drugs are independent of
the trimester of gestation. Compounding the situation even further are
studies by Gauron and Rowley (1969) and Friedler (1978) which show
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32
that the behavior of offspring can be affected even by pregestational expo-
sure to certain chemicals.
An investigation of the holdings of the Environmental Teratology
Information Center (ETIC) of the Oak Ridge National Laboratory
indicates that of the over 25,000 citations indexed concerning various
aspects of teratogenicity testing, 2645 are related to behavioral teratology.
By species this breaks down as follows: rat (1078), mouse (297), hamster
(11), rabbit (69), monkey (38), and human (1152).
2.5.1 Historical Perspective
Werboff and his coworkers are credited with establishing the branch of
teratology known as behavioral teratology. They published several papers
(e.g., Werboff et al. 1961a, 1961b; Werboff and Havlena 1962; Werboff
and Kesner 1963) detailing the postnatal effects of prenatally administered
psychotropic drugs. As indicated by Leonard (1983) and Hutchings
(1983), it was not until the early 1970s that the work of Werboff and col-
leagues was extended. The realization that prenatal exposure can cause
behavioral alterations has resulted in both Japan and Britain mandating
that behavioral evaluations be conducted on all new drugs during
premarket reproductive testing (Vorhees and Butcher 1982, as reported in
Vorhees 1983). Although similar regulations have not been established in
the United States, Vorhees (1983) cites several events which suggest that
U.S. regulatory authorities are giving serious consideration to establishing
guidelines for behavioral teratogenicity testing.
2.5.2 Behavioral Testing Methodologies
Because no single test gives a comprehensive behavioral assessment, a
multitesting procedure is usually employed. Jensh (1983) in his review on
behavioral testing procedures cites several authors (Spyker and Avery
1976, Kimmel 1977, Rodier 1978, Silverman 1978, Buelke-Sam and
Kimmel 1979, Lasagna 1979, Vorhees et al. 1979) who have suggested cri-
teria for an effective multitest procedure. These criteria are listed by Jensh
(1983):
1. The tests should be simple.
2. The tests should be comprehensive, examining a variety of behavioral
functions (global).
3. The tests should be sensitive to slight alterations in the organism's
functions, as well as to alterations produced by a number of different
agents.
4. The behavioral schedule should be simple, to minimize the training
time of personnel.
5. The tests should take a minimum amount of time.
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33
6. The tests should be reliable, reproducible, and valid in the same
laboratory as well as in the laboratories of other investigators.
7. The tests should have a proven history of positive results.
8. The tests should be economical in terms of financial expense and utili-
zation of animals.
9. The tests should be within the framework of existing guidelines.
10. The protocol should move in graded steps from elementary to more
sophisticated tests.
11. Data should be quantifiable.
12. The tests should be predictive of effects in humans.
Table 2.7 lists what Jensh (1983) considers the more commonly used
tests either singly or as part of a behavioral test battery. Earlier, Buelke-
Sam and Kimmel (1979) reported on a survey they conducted concerning
behavioral methods in teratologic evaluation used by those researchers who
responded to their survey (Table 2.8). As both Tables 2.6 and 2.7 indicate,
a variety of behavioral test methods are being used to assess behavioral
alteration. This variety of test methods as well as the varying exposure
regimens and postnatal environmental conductions used by researchers
emphasizes the need for standardization of screening methods for
behavioral teratology (Buelke-Sam and Kimmel 1979). Jensh (1983) states
that "standardization of test procedures is of paramount importance" and
adds that "the investigator needs to have access to a standardized pro-
cedure for each of the chosen tests, including experimental protocols, con-
struction designs, techniques, past results, and interpretative implications
and limitations." Standardization procedures are in fact under way (Kim-
mel et al. 1982, 1983; Kimmel 1984), and a workshop to present the data
from a six-laboratory study was held on September 3-6, 1985, in Cincin-
nati, Ohio.
2.6 SPECIES COMPARISONS
Proper selection of a test species is important. The large number of
animals necessary for toxicological research usually dictates that the test
species be easily available and economical. The following subsections will
consider some general aspects of species selection; will briefly discuss the
advantages and disadvantages of the rat, mouse, hamster, rabbit, monkey,
and other less commonly used species as models for extrapolation to man;
and will compare the response of three known human
teratogens—aminopterin, methotrexate, and thalidomide—with the
response in test animals.
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34
Table 2.7. Behavioral Testing Procedures'
I. Physical Development
Pinna detachment Testes descent
Incisor eruption Vaginal opening
Eye opening Development of fur
Ear unfolding and opening
II. Behavioral Analyses
A. Reflex development tests'*
Crossed extensor Tail pinch
Flexor dominance Tremors
Roofing reflex Surface righting
Limb withdrawal Air righting
Forelimb placing Auditory startle
Hindlimb placing Negative geotaxis
Forelimb and hindlimb grasp Visual placing
Suckling ability Vibrissae placing
Tail hang Cliff avoidance
Vibrissae stroking Pain reflex reaction
Ear twitch Olfactory reflex
B. Motor and coordination tests0
Bar holding
Forelimb hanging
Visual orientation
Inclined plane
Rotarods
Parallel bars
C. Spontaneous or nonforced behavior tests'1
Spontaneous motor behavior
Head elevation
Head pointing
Hindlimb elevation
Rearing
Pivoting
Gait
Crawl
Walk
Backward walking
Grooming
Circling behavior
Sleeping and somnolence
D. Forced behavior tests'
1. Mazes
T-maze
Y-maze
Hebb-Williams maze
Lashley III maze
Olton maze
Ascending wire mesh
Ascending a vertical rod
Clinging to and descending
a vertical rope
Swimming
Hopping
Homecage emergence
Resistance to handling
Feeding
Undisturbed behavior
Spontaneous and general activity
Nose poke
Head dip
Step-down test
Thirst test
Maternal behavior
Open-field
Activity wheel
2. Operant conditioning tests
Bar-(lever-) press
Avoidance testing
Discrimination reversal
learning
"Data compiled from Jensh 1983.
bSome of these include complex motor activity.
'Primarily include forced, complex activities.
dMay include some motor evaluations.
'Primarily include learning skills.
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35
Table 2.8. Behaviors Tested and Methods Used
in Teratologic Evaluation"
Observation (23 )b
18C
3
2
1
Reflex
17
15
11
10
6
6
6
5
4
3
3
2
1
Motor
18
10
8
7
5
4
3
2
2
Sexual
12
general activity and condition
modified Irwin method
photography /video taping
residential unit
development (34)
surface righting
auditor startle
pupillary contraction
air righting
cliff avoidance
touch-escape
visual placing
Fox method
nonspecified, animal
negative geotaxis
Smart & Dobbing method
nonspecified, human
stabilimeter
coordination (35)
rotating rod/drum
swimming
hanging/grasping
gait
inclined plane
traverse rods
pivoting
ambulation times
nonspecified, human
behavior (15)
mating success
Activity (39)
23 open field
10 activity wheels
6 photoactomers
3 behavioral sequencing
2 hole board
2 inductance monitors
2 nonspecified human
2 stabilimeter
1 automated plus-maze
1 exploratory
1 home cage
1 motron
1 residential unit
7 some monitoring of activity
for ^ 24-h period
Aggression (6)
3 shock-induced
1 muricide
1 nonspecified, human
1 tail-rattling
Classical conditioning (2)
1 nonspecified, human
1 tone-shock pairing
Avoidance (26)
12 passive
12 2-way active
7 1-way active
2 nonspecified CAR
2 Sidman
2 Y-maze
5 male performance
2 female receptivity signs
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36
Table 2.8 (continued)
Operant behavior (17)
13 simple schedules
9 FR
7 DRL
4 CRF
4 VI
3 FI
2 punishment
9 discrimination procedures
6 acquisition/extinction
4 multiple schedules
3 progressive/titrating schedules
2 various nonspecified types
1 discrete trial
Maze/instrumental behavior (30)
15 water mazes
9 discrimination procedures
4 T-maze
3 neonatal home-cage seeking maze
3 nonspecified types
3 straight alley
3 Y-maze
2 Hebb-Williams
2 Lashley III
7 reversal learning
2 delayed responding
Sensory function (29)
4 thresholds
3 auditory
2 taste/olfactory
1 shock
3 photophobia
22 assessed as part of
reflex development
9 assessed as part of a
discrimination paradigm
Pharmacologic/environmental
challenge (12)
3 amphetamine
2 barbiturate sleep time
1 alcohol preference
1 audiogenic seizure
1 drug discrimination
1 LD50
1 metrazol seizure
1 morphine analgesia
1 neonatal CNS lesion
1 neonatal 6-OH-dopamine
1 nonspecified
1 shock stress
Social/group behavior (4)
2 competitive situation
1 group homing response (4)
1 nonspecified
1 residential unit
Other (7)
4 spontaneous alteration
3 consummary patterns
1 psychological evaluation,
children
"Tabulated summary of methods used by those researchers who
responded to the questionnaire.
bNumber of researchers who reported using one or more methods
to evaluate behavior falling into each major category.
°Number of researchers who reported using the specific method or
monitoring the specific behavior.
Source: Buelke-Sam and Kimmel (1979), Table 2, p. 24.
Reprinted with permission of the publisher.
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37
2.6.1 General Aspects
No ideal species has been identified with regard to extrapolating exper-
imental results from teratogenic tests in animals to man (NAS 1977;
Schardein 1976, 1983; Palmer 1978). That is, there is no one species that
fits all the criteria for an ideal test animal as listed by Wilson (1975):
(1) absorbs, metabolizes, and eliminates test substances as does man,
(2) transmits test substances and their metabolites across the placenta as
does man, (3) has embryos and fetuses with developmental and metabolic
patterns similar to those of man, (4) is easily bred and has large litters
and a short gestation period, (5) is inexpensively maintained under labora-
tory conditions, and (6) does not bite, scratch, kick, howl, or squeal. The
problem of choosing the most appropriate species is further complicated
when the test chemical has no record of human administration from which
to draw a comparison. Thus, with little scientific data available, the choice
of species is often based on availability, economic feasibility, and ease of
management (Brown 1963, as reported in Palmer 1978).
Kalter (1968) writes that there are three different types of inter-
specific and intraspecific susceptibility: (1) an agent teratogenic in some
species or groups of animals may be completely or almost completely
without teratogenic effect in others, (2) a teratogen may produce similar
defects in various species, stocks, or strains of animals but with varying
susceptibility (frequency) between one strain and another or between one
species and another, and (3) a teratogen may induce one or more abnor-
malities in some species or groups but have entirely different or only some-
what different effects in others. Kalter indicates that these are not mutu-
ally exclusive categories and certain situations may contain features of
more than one category. These differences are illustrated in Tables
2.9-2.11 and also in the following sections.
The rat followed by the mouse and the rabbit are the most commonly
used laboratory animals for the teratogenic screening of chemical agents
(World Health Organization 1967, Palmer 1978). Further substantiation
of this fact comes by examination of the holdings of ETIC, whose files
contain over 25,000 papers indexed on various aspects of teratogenic test-
ing. Of these, approximately 8350 cite the rat as a test animal, 4640 cite
the mouse, 600, the hamster, 1700, the rabbit, and 460, the nonhuman pri-
mate. In addition, nearly 9550 papers discuss teratogens and teratogenicity
in relation to humans.
2.6.2 Rat
Advantages of using the rat as a test animal for teratogenic testing
include (1) short duration of pregnancy (21 to 22 days), (2) high fertility
rate, (3) large litters and a relatively good resistance to the toxic effects of
most drugs, (4) a fairly good developmental stability, and (5) a low spon-
taneous rate of major malformations (1 per 1000 fetuses, 0.001%)
(Tuchmann-Duplessis 1972, Palmer 1978). The rat is also easily handled
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38
Table 2.9. Species Susceptibility to Drugs
Drug
Azathioprine
Cortisone
Demecolcine
Ethyndiol
Meclizine
Methylprednisolone
Perphenazine
Dosage
(mg/kg)
and route
10POa
5 or 10 IMb
1.5 IPcorSCd
1 PO or SC
125 PO
1 IM or SC
25 PO
Malformed species
(percent incidence)
Mouse
0
79
23
30
0
92
100
Rat
0
0
0
0
92
0
98
Rabbit
57
50
3
0
0
0
0
Others
0 (monkey)
aPO—per os (oral).
bIM—intramuscular.
°IP—intraperitoneal.
dSC—subcutaneous.
Source: Adapted from Schardein, 1976, Table 2-8, p. 21. Data collected
from several sources. Reprinted with permission of the publisher.
and economically maintained, and test materials can be administered by a
wide variety of routes (Palmer 1978). The main limitation of the rat is its
poor teratogenic susceptibility to drugs such as cortisone (Thompson and
Schweisthal 1969, Hansson and Angervall 1966), azathioprine
(Tuchmann-Duplessis and Mercier-Parot 1966), and thalidomide (Somers
1963). Thus, definite conclusions based only on tests with rats could be
misleading (Tuchmann-Duplessis 1972).
2.6.3 Mouse
The advantages listed for the use of the rat in testing (Section 2.6.2)
also apply to the mouse, with the addition that the mouse is even more
economical, has a shorter gestation period (18 to 19 days), has a wide
variety of defined inbred strains for special studies, and is more susceptible
to some teratogens (Tuchmann-Duplessis 1972, Palmer 1978). Disadvan-
tages include: (1) small size of the fetus and consequent difficulty in
examination of malformations; (2) arrangement of the malformations in
clusters, which creates difficulty in assessment; (3) higher spontaneous
malformation rate than the rat (0.5% in the Swiss albino colony of
Tuchmann-Duplessis); and (4) resorption rates much higher than in the
rat, necessitating a large control group (Tuchmann-Duplessis 1972, Palmer
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39
Table 2.10. Thalidomide Teratogenesis in Primates
Species
Man
Cynomolgous
monkey
Baboon
Rhesus monkey
Bushbaby
Japanese
monkey
Stump-tailed
monkey
Marmoset
Bonnet monkey
Teratogenic
dose
(mg/kg,
oral route)
0.5 to 1.0
10
5
12 to 19
20
20
5 to 10
45
5 to 30
Gestation
days
treated
20 to 36
22 to 32
18 to 44
24 to 26,
27, or 30
16 to 30
24 to 26
24 to 30
25 to 35
24 to 29 or
41 to 44
Defect
Limbs (80%), ear (20%)
Limbs (67%), teratomas (33%)
Limbs and tail (40%)
Limbs (100%)
Limbs (100%), tail (17%),
central nervous system (17%)
Limbs (100%), tail (20%)
Limbs, ear, and jaw (100%)
Limbs (45%), visceral (52%)
Source: Adapted from Schardein, 1976, Table 2-11, p. 25. Data collected from
several sources. Reprinted with permission of the publisher.
1978). The mouse is also known to respond differently than the rat to
some drugs such as cortisone.
With regard to the human teratogen thalidomide (see also Section
2.6.8.3), mice embryos are relatively insensitive, as is evident in a study by
Somers (1963) in which oral administration of doses up to 400 mg/kg
throughout pregnancy did not reduce the number of newborn mice or their
ability to survive to weaning. Even when the doses were increased to 4000
mg/kg only a small increase in resorption sites was observed.
2.6.4 Rabbit
The rabbit was the first laboratory animal shown to be susceptible to
the human teratogen thalidomide (Somers 1962) and has since been con-
sidered by many biologists to be one of the most favorable animals for
teratogenic studies (Tuchmann-Duplessis 1972). The susceptibility of the
rabbit to thalidomide was also reported by Somers (1963), who produced
limb defects in the offspring by administering 150 mg/kg to pregnant rab-
bits on days 8 to 16 of gestation, and by Shepard (1976), who states that
limb defects in New Zealand rabbits were observed after administration of
250 mg/kg on days 8 to 10 of pregnancy.
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40
Table 2.11. Comparative Teratogenicity of Thalidomide
Organ system
affected
Limbs
Ears
Eyes
Cardiovascular
Urinary
Genital
Gastrointestinal
Pulmonary
Prenatal and/or
postnatal death
Human
X
X
X
X
X
X
X
X
X
Nonhuman
primate
X
X
X
X
X
X
X
X
Rabbit
X
X
X
X
X
X
Rat Mouse
X (low%) X (low%)
X
X X
Source: Hendrickx et al. 1983, Table II, p. 153. Reprinted with permission of
the publisher.
Advantages of using the rabbit include the larger size of the rabbit
fetus compared to the rat or mouse (Palmer 1978); the ability of the rab-
bit to show a variety of spontaneous malformations, which suggests that
this species would be more susceptible than some other species to a variety
of teratogenic actions (Palmer 1968, as reported in Palmer 1978); and, for
comparison purposes, the rabbit's physiological difference from the rat and
the mouse (Tuchmann-Duplessis 1972). The necessity of having large con-
trol groups, a higher spontaneous malformation rate (1.7% for a 5-year
period) (Tuchmann-Duplessis 1972), and the rabbit's dependence on gut
flora for nutrition, which prevents the testing of antibiotics and makes
incorporation into the diet a hazardous and inaccurate means of adminis-
tering test compounds (Palmer 1978), are some disadvantages of using the
rabbit as a model for testing compounds for teratogenicity. More practical
disadvantages are problems associated with handling and intubation.
2.6.5 Hamster
Palmer (1978) reports that the hamster is mainly a competitor for the
mouse in animal studies; it is almost as economical to maintain and is
reputed to be more stable genetically. The hamster has several advantages
that make it a potentially valuable animal for developmental and terato-
genic studies: unique reproductive features such as large litter size and
short gestation period (16 days) (Perm 1967). Palmer (1978), however,
lists several disadvantages of using the hamster: (1) aggressive behavior
generally necessitates individual caging; (2) achieving precisely timed mat-
ing, although not difficult, is not as convenient as with the mouse;
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41
(3) intravenous injection is more difficult than in the mouse [Perm (1967)
does demonstrate a method requiring anesthesia whereby the injection is
made utilizing the lingual vein]; and (4) as with the rabbit, the hamster's
gut flora renders it susceptible to change in the diet and to antibiotics.
Also, the hamster has a poor historical data base. Tuchmann-Duplessis
(1972) concludes that from available data the hamster does not have a
definite advantage as a test animal in the teratogenic screening of drugs.
2.6.6 Nonhuman Primates
Much has been written concerning the use of nonhuman primates as
animal models in the teratological testing of chemicals (Wilson et al. 1968;
Wilson 1969, 1971a, 1971b, 1978; Hendrickx 1972; Poswillo et al. 1972;
Tanimura 1972; Siddall 1978). The rhesus monkey (Macaco mulatto) is
the most popular nonhuman primate test species according to Wilson
(1978), but teratological studies have been performed using other nonhu-
man primates such as the cotton-eared marmoset (Poswillo et al. 1972;
Siddall 1978) and the baboon (Hendrickx 1972).
One of the primary advantages of using nonhuman primates in the
teratological screening of chemicals is their similarity in many respects to
man. Wilson (1978) reports that: (1) in some instances the metabolism of
drugs in man approximates that of other higher primates; (2) the repro-
ductive physiology of Old World monkeys (rhesus monkeys and baboons)
closely resembles that of man, particularly the menstrual cycle, spermato-
genesis, and parturition; (3) the placental structure of man and nonhuman
primates appears to be quite similar, although the similarities of function
during the critical periods of gestation have yet to be fully established;
(4) the anatomical and temporal aspects of embryonic development
between the higher nonhuman primates and man are strikingly similar;
and (5) similarity in response to thalidomide is well established [(the com-
parative response to other agents, however, has been variable (Section
2.6.8)].
Similarly, Leone (1977) states in an excellent review of the develop-
mental stages in mammals used for teratogenetic tests that "Significant
differences are detectable between rodents and primates in the early stages
of development, which especially affect the structure and functions of the
embryonic membranes and consequently influence the teratogenic process."
In his opinion, the morphological and comparative aspects of development
deserve as much attention as do other parameters (i.e., metabolic path-
ways, dose threshold, etc.).
In a discussion favoring greater diversity in test species used for terato-
genicity testing, Wilson (1975a) states that rodents and rabbit embryos are
dependent during the initial days of organogenesis on the inverted yolk-sac
placenta, which is structurally and functionally different from the chorioal-
lantoic placenta of other mammals (e.g., primates) at corresponding times
in development.
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42
Wilson (1978) cites scarcity; difficulties in handling, especially the
unpredictability of behavior and general susceptibility to infections; and
low fecundity as disadvantages to using nonhuman primates for teratologi-
cal testing of drugs. Two other disadvantages of nonhuman primates are
the expense (high cost usually necessitates a test group size that is not
adequate) and the long gestation period.
It should be mentioned that prosimians, and to a certain extent New
World monkeys such as marmosets, have not been used extensively for
teratological evaluations of chemicals. The greater bush baby (Galago
crassicaudata), a prosimian, was found by Hendrickx (1972) to give a
negative response to thalidomide and has since received little attention.
The marmoset has shown similar responses as man to thalidomide
(Poswillo et al. 1972, Siddall 1978), but the fact that marmosets do not
menstruate causes difficulty in following the reproductive cycle and possi-
bly accounts for their sparse use (Wilson 1978). Because of the advantages
afforded by their smaller size, however, Wilson (1978) recommends
further testing of the suitability of New World monkeys and prosimians as
animal models in teratology.
2.6.7 Other Species
Other animals such as dogs, cats, and swine have been used for terato-
logical testing but not to the extent of the previously mentioned species.
2.6.7.1 Dog
Palmer (1978) writes that the primary advantage of the dog is its con-
current use as a nonrodent species for other toxicity tests that may provide
further information of the test material such as pharmacokinetics. Earl et
al. (1973) tested the beagle with the known human teratogens thalidomide
and aminopterin as well as with methyl mercuric chloride, hydroxyzine,
and hydroxyurea. They found that the dog (1) responds to known human
teratogens, but not in a classical manner, (2) does not appear to be as sen-
sitive an indicator of the above-mentioned compounds as some primates,
and (3) offers little advantage over other laboratory animals. In addition,
the frequency of estrus (twice a year), a high cost of maintenance, and a
poor historical data base are also disadvantages.
2.6.7.2 Cat
The cat has been used infrequently in teratology studies for such rea-
sons as the difficulty in exact determination of the time of ovulation and
the time required to establish a stable colony (Palmer 1968). In addition,
the cat has a poor historical data base from which to make comparisons.
Khera (1975) did show that thalidomide (10 to 480 mg/kg per day)
induced a wide variety of fetal cardiovascular defects including ventricular
septal defect, malpositioned great vessels, and narrowed left ventricular
chamber with hypertrophied walls.
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43
Other research by Khera and coworkers (Khera and Iverson 1978,
Iverson et al. 1980), however, demonstrated that the cat is not as suscepti-
ble to the teratogenic effects of some chemicals as are other animal
species. Khera and Iverson (1978) administered ethylenethiourea (a degra-
dation product of the ethylenebisdithiocarbamate group of fungicides)
orally in single daily doses of 0, 5, 10, 30, 60, or 120 mg/kg to 14, 9, 7, 8,
7, and 10 female cats, respectively, beginning on day 16 of gestation and
continuing through pregnancy unless maternal toxicity was exhibited.
Although ethylenethiourea proved to be toxic to pregnant cats (several cats
died or were killed in a moribund condition in the 30-, 60- and 120-mg/kg
dose groups) and had caused malformations in some of the dead fetuses
taken from those cats killed in the moribund state, no clear evidence for
teratogenicity of ethylenethiourea in live fetuses was seen. This result is in
contrast to the ethylenethiourea-induced teratogenesis observed in rats
(Khera 1973; Khera and Tryphonas 1977, as reported by Khera and Iver-
son 1978). In a follow-up study, Iverson et al. (1980) suggest that the rea-
son for the absence of teratogenic effects in the cat was the ability of the
cat to extensively metabolize ethylenethiourea to its S-methyl derivative;
examination of rat urine showed no evidence of the S-methyl derivative.
2.6.7.3 Pig
Tuchmann-Duplessis (1972) and Palmer (1978) list several advantages
of using the pig: (1) it is easily available and highly prolific compared to
other nonrodent species, (2) its embryology and genetics are well known,
(3) pregnancy can be obtained year round, and (4) incorporation of the
test material into the diet is easily and accurately accomplished because of
the pig's voracious eating habits. These same authors cite disadvantages of
using the pig as the large amounts of the test material required due to the
size of the pig and the excessive floor space required. Earl et al. (1973)
observed malformed offspring in miniature swine that had been exposed to
thalidomide, hydroxyurea, and aminopterin and conclude that, although
the reaction to thalidomide was not the classical response, miniature swine
offer certain advantages such as low incidence of spontaneous malforma-
tions. These researchers recognize that more data are necessary before the
role of the pig in teratogenic studies can be determined.
2.6.8 Results of Testing Some Human Teratogenic Chemicals in Animal
Models
Three known human teratogens, aminopterin, methotrexate, and thali-
domide, will be used to compare the teratogenic response between humans
and several animal species; the data are summarized in Tables 2.2, 2.3,
and 2.4.
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44
2.6.8.1 Aminopterin
In addition to man, the folic acid antagonist aminopterin (Table 2.3)
induced malformations in the mouse but not in the rat, where
embryolethality and resorptions were observed, or in the rhesus (Macaco
mulatto) and cynomolgus (Macaco irus) monkeys. The one reference con-
cerning aminopterin toxicity in the rabbit reports no injury to a six-day-old
blastocyst, but the assumption from these data that no injury would result
to the fetus probably would not be reliable.
2.6.8.2 Methotrexate
Methotrexate, also a folic acid antagonist and a methyl derivative of
aminopterin, is teratogenic to humans, mice, rats, and rabbits but
apparently not to monkeys, although abortions were induced (Table 2.4).
The data in Table 2.4 indicate that (1) teratogenic defects in the man,
mouse, rat, and rabbit are somewhat similar, especially with regard to rib
and limb abnormalities; (2) the rat shows a teratogenic response to metho-
trexate but not to aminopterin (Table 2.3); and (3) the monkey seems to
be resistant to the teratogenic action of both methotrexate and aminopte-
rin (Table 2.3). Skalko and Gold (1974) point out, however, that no tera-
togenic effects are produced in mice at doses that are teratogenic in
humans, rats, and rabbits and abortifacient in rhesus monkeys (0.3 to 10
mg/kg), thus suggesting that the mouse embryo is more resistant to the
embryotoxic effects of methotrexate than any other mammalian species yet
studied. The minimum dose required to produce teratogenicity in the
mouse was 25 mg/kg and is the same as that necessary for aminopterin to
be teratogenic (Table 2.3). In rabbits, methotrexate-induced abnormalities
increase as the dose increases, a fact that is related to metabolism by the
maternal system (Jordan 1974). Jordan discovered that when methotrexate
is present in low concentrations, it is rapidly converted by the maternal
enzyme aldehyde oxidase to a relatively inactive metabolite, 7-hydroxy-
methotrexate; at higher levels of methotrexate, the enzyme becomes sat-
urated and, consequently, more unmetabolized drug reaches the embryo.
2.6.8.3 Thalidomide
Table 2.2 indicates that human, monkey, and rabbit embryos are
highly susceptible to thalidomide but that mouse, rat, and hamster
embryos are not nearly as sensitive and show apparent strain differences.
In the study by Parkhie and Webb (1983) thalidomide teratogenicity was
demonstrated in Wistar rats by the administration of single intravenous
doses. This is in contrast to the data of Somers (1963) (see Table 2.2),
who, however, administered the compound orally. Parkhie and Webb state
that the intravenous administration of thalidomide may circumvent the
factors involved in its loss of teratogenicity when given orally.
Although both the monkey and the rabbit embryos exhibit malforma-
tions similar to those observed in the human fetus, especially with regard
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45
to limb defects, Delahunt (1965) concludes that the thalidomide-induced
abnormalities in man are much more similar to those in the monkey than
they are to those in the rabbit. Although Drobeck et al. (1965) do not
disagree with Delahunt, they do believe that the results observed in the
rabbit with thalidomide generally confirm what has been reported in
humans. They continue by stating that micromelia and phocomelia in the
human cases have been so popularized as to overshadow the wide range of
effects that has actually been observed including developmental failure of
visceral organs such as the kidneys and adrenals as well as developmental
failure in bony structures. According to Drobeck and coworkers, these
effects are well characterized in the rabbit.
Detailed examination of the data on rats, hamsters, and mice in
Table 2.2 illustrates the apparent strain differences in susceptibility to
thalidomide. Fratta et al. (1965) found no teratogenicity in mice strains
CFb ICR, C57, CBA, and SJL; DiPaolo et al. (1964) induced
malformations in strain A, C3H, and Swiss. Similar results are seen in
hamsters, where inbred lines are susceptible and random bred lines are
not, and in rats, where the Sprague-Dawley and Holtzmann (descended
from Sprague-Dawley) strains and one unidentified strain produced mal-
formed young, whereas the Wistar (by oral administration), Charles River,
Long Evans, and Dunning Fischer strains did not.
Although many explanations have been offered for species or strain
susceptibility to the teratogenicity of thalidomide, the issue has not been
resolved. Gordon et al. (1981) postulated that thalidomide is metabolized
to a toxic electrophilic intermediate that is not produced in vitro by liver
microsomes from Sprague-Dawley rats, which, they state are not a sensi-
tive species. This intermediate was produced by hepatic preparations from
rabbits, monkeys, and humans, species regarded by Gordon et al. (1981)
as sensitive. However, Table 2.2 shows that some strains of rats, including
Sprague-Dawley, have demonstrated the teratogenicity of thalidomide.
Vaisman et al. (1983) have postulated that the mechanism of the tera-
togenic action of thalidomide is the induction of a deficit of ascorbic acid
in species such as nonhuman primates and man that do not have the abil-
ity to synthesize this vitamin. In their tests with the guinea pig, which also
cannot synthesize ascorbic acid, thalidomide was shown to induce a deficit
of ascorbic acid in both adults and fetuses. Their premise is based on the
fact that a deficiency of ascorbic acid leads to marked inhibition of col-
lagen synthesis (Matusis 1975, as reported in Vaisman et al. 1983) which
may damage the anlagen of the limbs. However, teratogenicity has been
demonstrated in rabbits (including limb malformations) and, although
perhaps not as convincingly, in rats, mice, and hamsters (see Table 2.2),
which can synthesize ascorbic acid.
Thus, as stated by Fabro (1981), "We must accept the facts that a
clear understanding of the mechanism by which thalidomide exerts its
teratogenic effects escapes us after almost two decades of intense scrutiny
and that the teratogen thalidomide still remains an embryologic enigma."
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46
2.7 RECENT APPROACHES IN TERATOLOGY TESTING
In recent years alternative approaches to the standard methods of test-
ing agents for teratogenicity have been investigated, primarily due to
economic and practical considerations. Most, although not all, of the
recently proposed methods have been in vitro test procedures. An investi-
gation of the current holdings of the ETIC concerning mammalian studies
reveals 889 indexed reference citations pertaining to various aspects of in
vitro teratogenicity. Analysis of these with respect to species indicates that
322 are associated with rats, 409 with mice, 14 with hamsters, 48 with
rabbits, 1 with monkeys, and 105 with humans. The total is small com-
pared to the approximately 25,000 citations recorded by ETIC for in vivo
testing, but, as many of the citations for in vitro testing are within the last
5 to 8 years, the current interest in alternatives to whole mammal testing
is evident.
2.7.1 In Vivo Methods
An alternative to current embryo toxicity screening techniques was pro-
posed by Martian and Jelinek (1979) using a caudal morphogenetic sys-
tem. In this proposed teratology screen, which the authors describe as a
compromise approach, the test substance is administered to rats on days
10 and 11 of pregnancy, and the effects on the activity of the caudal mor-
phogenetic system are determined by measurements of those parts of the
trunk arising through activity of that system of 13-day-old embryos. The
authors concluded that "the caudal morphogenetic system of rat embryos
reflected some of the changes induced in the maternal-embryonic com-
plex."
Chernoff and Kavlock (1982) have proposed a teratology screen that
differs from previously employed in vivo testing methods in that it does not
require the labor-intensive examination of fetuses for soft-tissue and skele-
tal anomalies. Rather, the dams (CD-I mice were used in the 1982 study)
are allowed to give birth, and determinations of teratogenic potential are
made on the basis of litter size and weight on postpartum days 1 and 3 as
compared with concurrent controls. Also, because the intent is priority-
setting and not testing, only one dose level (at or near the maximally toxic
dose) is used, in comparison with at least three levels in conventional
teratology test methods.
In the 1982 study, Chernoff and Kavlock tested 28 compounds of
known teratogenic potential—15 that were teratogenic by standard test
criteria did exhibit some form of developmental toxicity; 2 chemicals
known to produce only reduced weight caused reduced weight (endrin) or
reduced litter size (sodium selenite) in the proposed assay; 2 compounds
which in standard tests caused only an increased incidence of super-
numerary ribs produced no effect in the proposed screen; and of 9 com-
pounds for which no effects had been reported in conventional test pro-
cedures, 6 produced no effects in the screen and 3 produced positive effects
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47
(2 compounds caused reduction in litter weight and 1 caused reduced litter
size).
Chernoff and Kavlock (1982) state that their teratology screen can be
used to divide chemicals into three categories: (1) those that induce peri-
natal death should be tested as rapidly as possible, (2) those that induce
only perinatal weight changes would be given a lower testing priority, and
(3) those producing no effect would be given the lowest priority. In
October 1984, the EPA released guidelines for the use of this preliminary
developmental toxicity screen. Personal communication with Dr. Kavlock
has indicated that approximately 10 to 12 laboratories are currently using
this proposed method. Results of using this teratology screen in four of
these laboratories have been reported by Schuler et al. (1984). Fifteen
glycol ethers were tested using Charles River CD-I mice (no two labora-
tories tested the same chemical). Schular et al. provided results from con-
ventional testing for five of these. Of the five, three were known terato-
gens, and a correlation with results of the proposed screen was seen in that
these three compounds were designated either in a high priority or middle
to high priority group. Two of the five compounds showed no reproductive
toxicity in conventional tests; one was assigned to the middle priority
group and the other to the low priority group in the in vivo screen.
2.7.2 In Vitro Methods
Over the last few years several in vitro methods for screening chemicals
for teratogenicity have been proposed. Interest in testing for teratogenic
chemicals in vitro was evident as early as 1975, when the International
Conference on Tests of Teratogenicity In Vitro was held (Kimmel et al.
1982b). In August 1981 the consensus workshop on In Vitro Teratogenesis
Testing was convened in Arkadelphia, Arkansas, and the proceedings were
published in the Journal of Teratogenesis, Carcinogenesis, and
Mutagenesis (Vol. 2, 1982). Much of the information presented below is
taken from those proceedings. As defined at the workshop and used in this
section, the term in vitro refers to any system that uses test subjects other
than the intact pregnant mammal.
2.7.2.1 Whole mammalian embryo culture
Sadler et al. (1982) tested the potential teratogenicity of several com-
pounds using a whole mouse embryo procedure. Their approach was to
administer the test compound at the 4- and 5-somite stages, which accord-
ing to them showed survival and a high percentage of malformations. They
found that early stages (primitive streak to 0-1 somites) were often
severely affected and in some cases failed to survive. Sadler and coworkers
showed that glucose, ketone bodies, vitamin A, and hyperthermia produced
malformations (neural tube and/or cardiac effects) in vivo. Although stat-
ing that the whole-embryo culture system may be useful as a screening
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technique for potentially teratogenic substances, they recognize that
unanswered questions, such as the appropriate exposure time, point to the
need for further investigation before standardized protocols can be
developed.
The reader is referred to a paper by Kochhar (1975) for a description
of early attempts to culture whole mammalian embryos.
2.7.2.2 Embryonic limb bud organ culture
As described by Kochhar (1982), this method involves the removal of
limb buds from mouse embryos of known ages (usually of the 12th day of
gestation). Limb buds are placed on top of ultrathin Millipore or Nucleo-
pore Filters and incubated in a nutrient medium in the presence or absence
of test chemicals. End points that may be monitored include cell
proliferation, differential growth, morphogenetic cell death, size and shape
of limb parts, chondrogenesis, and collagen or proteoglycan biosynthesis.
Kochhar stated that validation was still in the early stages; to achieve full
potential the system should be combined with an efficient drug-
metabolizing preparation.
2.7.2.3 Avian embryonic cells
Wilk et al. (1980) describe two in vitro systems using chick embryos,
one using neural crest cells obtained from very early chick embryos and
the other, limb bud mesenchyme cells obtained at a later stage of develop-
ment. Tests were performed both with and without S-9 added for meta-
bolic activation. The S-9 fraction was toxic to cells when added directly to
the medium, and thus the S-9 plus a NADPH-generating system and test
compound was combined in autoclaved dialysis bags and added to the cul-
tures. A combination of both the neural crest and limb mesenchyme sys-
tem correctly predicted the teratogenicity of the known teratogens tested
with the exception of thalidomide. Isoniazid and glutethimide, which are
not teratogenic in vivo, were also inactive in the Wilk et al. system. Wilk
et al. (1980) state that a modification of their system by incorporating the
use of postmitochondrial fractions from placental and fetal tissues may
improve its sensitivity and enable the detection of the teratogenic activity
of thalidomide.
2.7.2.4 Ascites tumor cell assay
An in vitro teratogenicity assay that identifies teratogens by their abil-
ity to inhibit the attachment of ascitic mouse ovarian tumor cells to plastic
surfaces coated with concanavalin A has been proposed by Braun et al.
(1982). This system correctly identified 60 known teratogens, including
thalidomide, but failed to predict the teratogenicity of 14 known terato-
gens, including X irradiation and methotrexate. With respect to predicting
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49
nonteratogenicity, 21 nonteratogens were correctly identified but 7 com-
pounds were false positives. Recognizing the limitations of any single in
vitro system, the authors state that "attachment inhibition in concert with
other, complementary, in vitro assay systems can become a useful method
for all assessment of the teratogenic potential of environmental agents."
[According to the National Toxicology Program's annual plan for 1984
(NTP 1984), a complementary in vitro test measuring the chemical inhibi-
tion of growth potential of human embryonic mesenchymal cells is
currently being conducted by two separate laboratories. ]
2.7.2.5 Drosophila embryo cell assay
Bournias-Vardiabasis and Teplitz (1982) report the development of an
in vitro assay that detects teratogens by adding them to primary cultures
of embryonic Drosophila cells and analyzing the degree of change in cell
differentiation and tissue formation. In validation tests of over 100 com-
monly used industrial chemicals, drugs, and food additives, only two false
negatives (ethyl alcohol and cyclophosphamide) and two false positives (p-
aminobenzoic acid and dimethylamino antipyrine) were observed. When
the serum of animals fed the test agent is added to the differentiating cul-
ture, metabolic products of ingested compounds can be tested for terato-
genicity. The authors note that further testing, validation, and incorpora-
tion of a metabolic activation system are needed.
2.7.2.6 Poxvirus morphogenesis
Because the poxvirus has a rapid, simple morphogenetic pathway that
is dependent upon cell proliferation, the effect on the growth of poxvirons
in cell culture can be used as an indicator of teratogenic potential (Keller
and Smith 1982). In this assay, vaccinia WR-infected BSC 40 monolayers
are exposed to the test agent for 24 h; the number of functional virons is
then determined by plaque assay. In tests with 42 known teratogens,
Keller and Smith observed that 33 inhibited the number of virons
produced and 3 stimulated the production virons and were thus considered
teratogenic by this assay. Of nine nonteratogens tested, all but one (ascor-
bic acid) were negative in the poxvirus assay. Thus six teratogens were
false negatives and one was a false positive.
2.7.2.7 Neuroblastoma cells
Mummery et al. (1984) investigated the ability of 39 teratogenic and
18 nonteratogenic compounds to interfere with normal growth and dif-
ferentiation of murine neuroblastoma cells (clone NIE-115). This system
correctly predicted the teratogenicity of 35 of the 39 and the nonteratogen-
icity of 14 of the 18. The authors state that substances such as thali-
domide that require metabolic activation for teratogenicity are unlikely to
be detected by this system.
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2.7.2.8 Hydra attenuate system
Johnson et al. (1982) describe a teratogenic screen that uses the fresh-
water Hydra attenuata. The first of a two-part experiment involves expos-
ing adult hydra to a test substance over a broad range of concentrations
and determining the minimal toxic concentration to within one-tenth log.
In the second experiment, cells of dissociated hydra are manipulated into a
configuration that permits them to achieve the developmental events
characteristic of any embryo and undergo total whole-body regeneration;
these cells are exposed during a 4-day ontogenesis period to the test sub-
stance in the same manner as the adult, and the minimally developmental
toxic concentration is determined to one-tenth log. Then, the ratio of the
adult (A) to the developmentally (D) toxic concentration is calculated. A
small A/D ratio indicates that the agent affects adults and developmental
events at generally the same dosage and, provided an adult toxic dose level
is not attained, the embryo will not be at risk. A large A/D ratio indicates
the agent is a primary developmental hazard with the conceptus as the
most vulnerable target, and at risk even at sub-adult toxic exposure levels.
This system is also described by Johnson and Gabel (1982) and by John-
son (1984).
The Hydra attenuata system has been validated by comparing the
A/D ratios for chemicals tested using this system with the A/D ratios for
chemicals tested using conventional developmental toxicity test procedures.
Data supplied by Dr. Johnson of comparison of the A/D ratios of approxi-
mately 40 test substances indicate a good correlation in most instances.
The A/D ratios of mammals and hydra seldom varied by as much as a
factor of 2. One exception was 6-aminonicotinamide with A/D ratios of
over 4 for mammals and 26 for hydra. Personal communication with
Dr. Johnson indicates that the use of the bioactivation in the hydra system
is currently being investigated under the aegis of the March of Dunes.
2.7.2.9 Planarian assay
Best and Morita (1982) describe the results of experiments using fresh-
water planarians as indicator organisms in a teratogenicity assay. Either
surgical fragments of planarians that undergo regeneration or intact
planarians can be used as test organisms. When fragments are used, dis-
tortion of the regeneration is the measure of teratogenicity, and with intact
planarians the development of morphologic or other observable abnormali-
ties is the indicator. Citing the results of testing several chemicals for tera-
togenicity, Best and Morita state that "both regeneration of surgical frag-
ments and aberrant remodeling of whole planarians model important
features of embryogenesis and are potentially useful for assaying terato-
gens." The authors suggest that the planarian system might also be useful
in screening for chemicals that cause behavioral teratogenicity.
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2.7.2.10 Frog embryo assay
In this method described by Dumont et al. (1982) and Dumont and
Epler (1984), early frog embryos are exposed for 96 h to concentration
ranges of suspect agents. The following end points may be observed: LC50,
EC50 (concentration required to produce 50% terata among survivors),
growth and developmental stages attained, anatomical abnormalities,
motility (behavior), and pigmentation. Personal communication with
Dr. Dumont indicates that 7 of 41 compounds (17%) tested were false
negatives and 5 of 41 (12%) were false positives.
2.8 CONCLUSIONS AND RECOMMENDATIONS FOR FURTHER
RESEARCH
2.8.1 Conclusions
2.8.1.1 General experimental considerations
1. At least three dose levels of a chemical should be tested. The low dose
should allow normal development, the intermediate dose should show
teratogenicity if the chemical tested is a teratogen, and the high dose
should produce either maternal toxicity, embryotoxicity, or fetotoxi-
city.
2. Positive controls cannot provide certainty that the test chemical is not
teratogenic if tests indicate no teratogenicity.
3. For each chemical at least two species should be tested for teratogeni-
city.
4. The number of pregnant test animals per group must be sufficient to
permit reliable statistical analysis. A minimum of 20 pregnant
animals of a rodent species and 12 pregnant rabbits has been recom-
mended by several regulatory groups.
5. The administration route should be oral intubation unless the physical
and chemical properties of the chemical or human use conditions dic-
tate otherwise.
6. All fetuses should be examined for external malformations. Approxi-
mately one-third to one-half of the fetuses of rats, mice, and hamsters
should be examined for skeletal defects and the remainder for soft tis-
sue malformation. All rabbit fetuses should be examined for both
skeletal and soft tissue anomalies.
2.8.1.2 Structure-activity relationships
Although selected teratogenicity studies seem to illustrate certain
structure-activity relationships, generalizations regarding the prediction of
human risks from chemical structure are not yet feasible. This conclusion
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52
is supported by the statement of Zimmerman (1975) that "Prediction of
teratogenesis by chemical structure will have to await a future time" and
of that by Schumacher (1975) that "Structure-activity studies, as per-
formed until now, will not, we feel, provide the needed information and are
therefore of no predictive value with regard to teratogenicity in animals,
much less in humans."
2.8.1.3 Teratogenesis and time of administration
When a chemical is tested for teratogenicity, observations for
behavioral as well as physical defects should be performed. Although it is
generally recognized that organogenesis is the most sensitive prenatal
developmental period to the malforming action of teratogenic chemicals,
the possibility of a test chemical inducing behavioral teratogenic effects
when administered in each of the three trimesters (and even pregestation-
ally, see Section 2.5) must be considered.
2.8.1.4 Behavioral teratogenesis
After a slow start, interest in behavioral teratogenicity increased during
the early 1970s, and today it is a widely recognized and respected science.
Although some countries (the United States excluded) have proposed
guidelines for the testing of agents for behavioral teratogenicity, standardi-
zation of test methodologies is still needed.
2.8.1.5 Species comparisons
There is no ideal animal model for use in teratological research. The
monkey (primarily the higher primates) has received considerable attention
in recent years, especially since the thalidomide syndrome appears to be
mimicked to a high degree in the monkey. This animal offers the
advantage of having a chorioallantoic placenta as does man, in contrast to
the inverted yolk-sac placenta of rodents and rabbits, and having other
anatomical and physiological similarities to man. Yet, studies using the
folic acid antagonists methotrexate and aminopterin, known human terato-
gens, have been unable to demonstrate any significant teratogenicity in the
monkey, although abortions have been induced. However, if a compound
whose embryotoxicity is being tested was shown to have abortifacient
action but not necessarily teratogenic potential (inducing malformed
offspring), the use of the compound would probably be restricted for
women of child-bearing age. Thus, the decision on potential use of this
compound by pregnant women would probably be the same whether it
induced malformations or abortions. Using the criteria of embryotoxicity
(embryolethality, malformations, or growth retardation), Wilson (197la)
has shown that the effects induced by several agents in man and in the
rhesus monkey (chemical and viral) are comparable. Wilson acknowledges
that before the rhesus monkey can be considered more reliable than other
laboratory animals for anticipating embryotoxicity in man, more detailed
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comparative information on drug metabolism and distribution between
laboratory animals must be obtained. However, even if the rhesus monkey
is shown to be more reliable than other laboratory animals for predicting
embryotoxicity in man, the expense will likely prohibit serious considera-
tion as a routine test animal for embryotoxicity.
Rats, mice, and rabbits have been and will probably continue to be
used more frequently than the monkey as laboratory test animals primarily
due to availability, ease of handling, and economy. Rats and mice have
been the most commonly used, but use of the rabbit has increased since
the early 1960s because the rabbit was the first laboratory animal shown
to be susceptible to thalidomide teratogenicity.
All chemicals do not require the same degree of teratogenicity testing.
Compounds for which human exposure, especially to pregnant women, is
considered a probability should receive more intensive teratological testing
than those chemicals for which human exposure is a remote possibility. In
the same vein, Wilson (1978) believes that despite the limited availability
of higher primates, testing with macaques and baboons is essential for
those drugs needed for therapeutic purposes during human pregnancy and,
perhaps, for the environmental chemicals to which women may be inadver-
tently heavily exposed prior to the diagnosis of pregnancy. Wilson contin-
ues by stating that potential teratogenic effects to the central nervous sys-
tem can be adequately tested only in those higher primates that have, at
least roughly, a range of mental and nervous activities comparable to man.
2.8.1.6 Recent approaches in teratology testing
Investigations of practical alternatives to standard in vivo testing
methods for teratogenicity screening are needed because of the cost and
labor-intensive efforts. Although a few recent approaches have used in vivo
procedures, most have used systems other than the whole mammal. At
present, although validations of some of these in vitro methods are still
being conducted, no single test can be considered as a screen; a battery of
tests is more appropriate. For further insights into in vitro screening of
agents for teratogenicity the reader is referred to the overview of the 1981
proceedings of the consensus workshop on In Vitro Teratogenesis Testing
(Kimmel et al. 1982b).
2.8.2 Recommendations for Further Research
The following are identified as possible research areas for the terato-
genicity testing of chemicals:
• An extensive analysis, using existing data, of the teratogenic action of a
select group of chemicals representing different chemical classes. Such
an analysis should enhance our understanding of species specificity,
mechanisms of action, and structure-activity relationships.
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• Further investigation of postnatal behavioral changes as they relate to
pregestational and gestational chemical exposure.
• Further research directed toward standardization of the methodology
for screening chemicals for behavioral teratogenicity.
• Further research on variations on present in vivo test procedures, espe-
cially with regard to economy and simplicity.
• Continued validation of existing in vitro methods for screening com-
pounds for teratogenicity as well as further research on the identifica-
tion of additional in vitro test systems.
• In conjunction with the above, determination of an acceptable battery
of tests for in vitro screening of compounds for teratogenicity.
2.9 LITERATURE CITED
Baranov VS. 1965. Injurious effect of aminopterin at different stages in
the embryonic development in rats. Embryology 16:448-450.
Becci PJ, Reagan EL, Knickerbocker MJ, Barbee SJ, Wedig JH. 1983.
Teratogenesis study of o-toluenediamine in rats and rabbits. Toxicol Appl
Pharmacol 71:323-329.
Best JB, Morita M. 1982. Planarians as a model system for in vitro terato-
genesis studies. Teratog Carcinog Mutagen 2:277-291.
Beyer BK, Guram MS, Geber WF. 1984. Incidence and potentiation of
external and internal fetal anomalies resulting from chlorodiazepoxide and
amitriptyline alone and in combination. Teratology 30:39-45.
Bournias-Vardiabasis N, Tepliz RL. 1982. Use of Drosophila embryo cell
cultures as an in vitro teratogen assay. Teratog Carcinog Mutagen
2:333-341.
Braun AG, Buckner CA, Emerson DJ, Nichinson BB. 1982. Quantitative
correspondence between the in vivo and in vitro activity of teratogenic
agents. Proc Nat Acad Sci USA 79:2056-2060.
Brown AM. 1963. Matching the animal with the experiment. In: Animals
for research. Lana-Petter, ed. London: Academic, pp. 261-685. (As
reported in Palmer 1978).
Brown D. 1978. Structure-activity relation of steroidal amine teratogens.
In: Effects of poisonous plants on livestock. Keeler RF, VanKampen KR,
James LF, eds. New York: Academic, pp. 409-418.
-------
55
Brown D, Keeler RF. 1978. Structure-activity relation of steroid terato-
gens. I. Jervine ring system. J Agric Food Chem 26(3):561-569.
Brummett ES, Johnson EM. 1979. Morphological alterations in the
developing fetal rat limb due to maternal injection of chlorambucil. Tera-
tology 20(2):279-288.
Buelke-Sam J, Kimmel CA. 1979. Development and standardization of
screening methods for behavioral teratology. Teratology 20:17-30.
Chernoff N, Kavlock RJ. 1982. An in vivo teratology screen utilizing preg-
nant mice. J Toxicol Environ Health 10:541-550.
Collins, TFX, Collins EV. 1976. Current methodology in teratology
research. In: Advances in modern toxicology. Mehlman MA, Shapiro RE,
Blumenthal H, eds. New York: John Wiley, pp. 155-174.
Coyle, I, Wayner MJ, Singer G. 1976. Behavioral teratogenesis: a critical
evaluation. Pharmacol Biochem Behav 4:191-200.
Delahunt CS. 1965. Teratogenic effects of thalidomide in the rabbit, mon-
key, and man. In: Proceedings of second teratology workshop, Berkeley,
Calif, pp. 51-81.
Delahunt, CS, Lassen LJ, Rieser N. 1966. Some comparative teratogenic
studies with thalidomide. Proc Eur Soc Study Drug Toxic. 7:229-240.
DeSesso JM. 1979. Lectin teratogenesis: defects produced by Concanavalin
A in fetal rabbits. Teratology 19:15-26.
DiPaolo, JA, Gatzek H, Pickren J. 1964. Malformations induced in the
mouse by thalidomide. Anat Rec 149:149-156.
Drobeck HP, Coulston F, Cornelius D. 1965. Effects of thalidomide on
fetal development in rabbits and on establishment of pregnancy in mon-
keys. Toxicol Appl Pharmacol 7:165-178.
Dumont JN, Schultz TW, Newman SM. 1982. A frog embryo terato-
genesis assay: Xenopus (FETAX)—a model for teratogen screening. Tera-
tology 25:37A.
Dumont JN, Schulz TW, Elper RG. 1983. The response of the FETAX
model to mammalian teratogens. Teratology 27:39A.
Dumont JN, Elper RG. 1984. Validation studies on the FETAX terato-
genesis assay (frog embryos). Teratology 29(2):27A.
-------
56
Dwornik JJ, Moore KL. 1965. Skeletal malformations in the Holzman rat
embryo following the administration of thaliomide. J Embryol Exp Mor-
phol 13(2):181-193.
Earl FL, Miller E, Van Loon EJ. 1973. Teratogenic research in beagle
dogs and miniature swine. In: The laboratory animal in drug testing.
Spiegel A, ed. Stuttgart: Gustav Fischer Verlag, pp. 233-247.
Eibs HG, Spielmann H, Hagele M. 1982. Teratogenic effects of cypro-
terone acetate and medroprogesterone treatment during the pre- and post-
implantation period of mouse embryos. I. Teratology 25:27-36.
Eibs HG, Spielmann, Jacob-Muller U, Klose J. 1982. Teratogenic effects
of cyproterone acetate and medroxyprogesterone treatment during the pre-
and postimplantation period of mouse embryos. II. Cyproterone acetate
and medroxyprogesterone acetate treatment before implantation in vivo
and in vitro. Teratology 25:221-299.
Emerson DJ. 1962. Congenital malformation due to attempted abortion
with aminopterin. Am J Obstet Gynecol ll(3):356-357.
Fabro S. 1981. Biochemical basis of thalidomide teratogenicity. In:
Biochemical basis of chemical teratogenesis. Juchau MR, ed. New York:
Elsevier, pp. 159-178.
Fantel AG, Macphail BJ. 1982. The teratogenicity of cocaine. Teratology
6:17-19.
Perm VH. 1967. The use of the golden hamster in experimental teratol-
ogy. Lab Anim Care 17(5):452-462.
Perm VH, Hanlon DP. 1974. Toxicity of copper salts in hamster
embryonic development. Toxicol Reproduction 11:97-101.
Fratta ID, Sigg EB, Maiorana K. 1965. Teratogenic effects of thalidomide
in rabbits, rats, hamsters, and mice. Toxicol Appl Pharmacol 7:268-286.
Freese E, Levin BC, Pearce R, et al. 1979. Correlation between the growth
inhibitory effects, partition coefficients and teratogenic effects of lipophilic
acids. Teratology 20:413-440.
Friedler G. 1978. Pregestational administration of morphine sulfate to
female mice: long-term effects on the development of subsequent progeny.
J Pharmacol Exp Ther 205( 1 ):33-39.
Gauron EF, Rowley VN. 1969. Effects on offspring behavior of mothers'
early chronic drug experience. Psychopharmacologia (Berl.) 16:5-15.
-------
57
Gordon GB, Spielberg SP, Blake DA, Balasubramanian V. 1981.
Thalidomide teratogenesis: evidence for a toxic arene oxide metabolite.
Proc Natl Acad Sci USA 78(4):2545-2548.
Hansson CG, Angervall L. 1966. The parathyroids in corticosteroid-
treated pregnant rats and their offspring. Acta Endocrinol 53:547-552.
Hay MF. 1964. Effect of maternal treatment with various agents on early
embryonic development in the rabbit. In: Proceedings of 5th International
Congress of Animal Reproduction and Artificial Insemination, Trento,
Italy, pp. 309-312.
Hendrickx AG. 1970. Teratogenicity of thalidomide in the baboon (Papio
cynocephalus), bonnet monkey (Macaco radiatd) and cynomolgus monkey
(Macaca irus). Proc 3rd Int Congr Primat 2:230-237.
Hendrickx AG. 1972. A comparison of temporal factors in the embryologi-
cal development of man, old world monkeys, and galagos, and craniofacial
malformations induced by thalidomide and triamcindone. Medical Prima-
tology (Switzerland), Proc 3rd Conf Exp Med Surg Primates, Part III,
pp. 259-269.
Hendrickx AG, Binkerd PE, Rowland JM. 1983. Developmental toxicity
and nonhuman primates. In: Issues and reviews in teratology. Kalter H,
ed. New York: Plenum Press, pp. 181-214.
Hendrickx AG, Newman L. 1972. Temporal and mandibular bone malfor-
mations induced by thalidomide in the bonnet monkey (M. radiata). Tera-
tology 5(2):257.
Hoffeld DR, Webster RL. 1965. Effect of injection of tranquilizing drugs
during pregnancy on the offspring. Nature 295: 1070-1072. (As reported
in Coyle et al. 1976).
Homburger F, Chaube S, Eppenberger M, Bogdonoff PD, Nixon CW.
1965. Susceptibility of certain inbred strains of hamsters to teratogenic
effects of thalidomide. Toxicol Appl Pharmacol 7:686-693.
Hutchings DE. 1983. Behavoral teratology: a new frontier in neuro-
behavioral research. In: Handbook of experimental pharmacology. Vol. 65.
Born GVR, Farah A, Herken H, Welch AD, eds, pp. 207-235.
Hutchings DE, Gibbon J, Kaufman MA. 1973. Maternal Vitamin A
excess during the early fetal period: effects on learning and development in
the offspring. Dev Psychobiol 6(5):445-457.
-------
58
Hutchings DE, Gaston J. 1974. The effects of Vitamin A excess admin-
istered during the mid-fetal period on learning and development in rat
offspring. Dev Psychobiol 7(3):225-233.
Inouye M, Murakami U. 1977. Teratogenicity of 2,5-diaminotoluene, a
hair-dye constituent, in mice. Food Cosmet Toxicol 15:447-451.
Iverson F, Khera KS, Hierlihy SL. 1980. In vivo and In vitro metabolism
of ethylenethiourea in the rat and the cat. Toxicol Appl Pharmacol
52:16-21.
Jensh RP. 1983. Behavioral testing procedures: a review. In: Teratogenesis
and reproductive toxicology. Johnson EM and Kochhar DM, eds. New
York: Springer-Verlag, pp. 171-206.
John JA, Blog DD, Murray FJ, Schwetz BA, Gehring PJ. 1979. Terato-
genic effects of the plant hormone indole-3-acetic acid in mice and rats.
Teratology 19(3):321-326.
Johnson EM. 1984. A prioritization and biological decision tree for
developmental toxicity safety evaluations. J Am Coll Tox 3(2):141-147.
Johnson EM, Gabel BEG. 1982. Application of the Hydra assay for rapid
detection of developmental hazards. J Am Coll Tox 1(3):57-71.
Johnson EM, Gorman RM, Gabel BE, George ME. 1982. The Hydra
attenuata system for detection of teratogenic hazards. Teratog Carcinog
Mutagen 2:263-276.
Joneja MG. 1981. Teratogenic effects of proline analogue L-azetidine-
2-carboxylic acid in hamster fetuses. Teratology 23:365-372.
Jordan RL. 1973. Response of the rabbit embryo to methotrexate. Teratol-
ogy 7(3): 19 A.
Jordan RL. 1974. Studies on the relationship between methotrexate meta-
bolism and teratogenicity. Excerpta Med:89.
Jordan RL, Terapane JF, Schumacher JH. 1970. Studies on the teratogen-
icity of methotrexate in rabbits. Teratology 3:198.
Kacew S, Ruddick JA, Parulekar M, Valli VE, Chu I, Villeneuve DC.
1984. A teratological evaluation and analysis of fetal tissue levels following
administration of tetrachlorobenzene isomers to the rat. Teratology
29:21-27.
-------
59
Kajii TM, Kida M, Takahashi K. 1973. The effect of thalidomide intake
during 113 human pregnancies. Toxicology 8:163-166.
Kalter H. 1968. Teratology of the central nervous system. Chicago: The
Univesity of Chicago Press, 483 pp.
Kao J, Brown NA, Schmid B, Goulding EH, Fabro S. 1981. Teratogeni-
city of valproic acid: in vivo and in vitro investigations. Teratog Carcinog
Mutagen 1:367-382.
Keeler RF. 1970. Teratogenic compounds in Veratrum caifornicum
(Durand). IX. Structure-activity relation. Teratology 3:169-174.
Keller SJ, Smith MK. 1982. Animal virus screens for potential teratogens.
I. Poxvirus morphogenesis. Teratog Carcinog Mutagen 2:361-374.
Khera KS. 1973. Ethylenethiourea: teratogenicity study in rats and rab-
bits. Teratology 7:243-252. (As reported in Khera and Iverson 1978).
Khera KS. 1975. Fetal cardiovascular and other defects induced by tha-
lidomide in cats. Teratology 11:65-72.
Khera KS, Tryphonas L. 1977. Ethylenethiourea-induced hydrocephalus:
pre- and post-natal pathogenesis in offspring from rats given a single oral
dose during pregnancy. Toxicol Appl Pharmacol 42:85-97. (As reported in
Khera and Iverson 1978).
Khera KS, Iverson F. 1978. Toxicity of ethylenethiourea in pregnant cats.
Teratology 18(3):311-314.
Kimmel CA. 1984. Behavioral teratology in experimental animals. Paper
presented at the European Teratology Society Meeting, The Netherlands,
September 1984.
Kimmel CA, ed. 1977. Final report of the committee on postnatal evalua-
tion of animals subjected to insult during development. Research Triangle
Park, North Carolina: National Institute of Health. (As reported in Jensh
1983).
Kimmel CA, Buelke-Sam J, Adams J, Reiter LW, Sobotka TJ, Tilson
HA. 1982a. Design considerations in the evaluation of standardized
methods in a behavioral teratology protocol. Teratology 25:54A.
Kimmel CA, Buelke-Sam J, Adams J, Reiter LW, Sobotka TJ, Tilson
HA. 1983. An intralaboratory comparison of selected methods in a
behavioral teratology protocol. Teratology 27:57A.
-------
60
Kimmel GL, Smith K, Kochhar DM, Pratt RM. 1982b. Proceedings of
the consensus workshop on in vitro teratogenesis testing. Teratog Carcinog
Mutagen 2:(34) i-ii.
King, CTG, Horigan E, Wilk AL. 1972. Fetal outcome from prolonged
versus acute drug administration in the pregnant rat. Drugs Fetal Dev,
pp. 61-75.
King CTG, Kendrick FJ. 1962. Teratogenic effects of thalidomide in the
Spague Dawley rat. Lancet 2:1116
King CTG, Weaver SA, Narrod SA. 1965. Antihistamines and teratogeni-
city in the rat. J Pharmacol Exp Ther 147:391-398.
Kochhar DM. 1975. The use of in vitro procedures in teratology. Teratol-
ogy 11:273-288.
Kochhar DM. 1982. Embryonic limb bud organ culture in assessment of
teratogenicity of environmental agents. Teratog Carcinog Mutagen
2:303-312.
Koppanyi T, Avery MA. 1966. Species differences and the clinical trial of
new drugs: a review. Clin Pharmacol Ther 7:250-270. (As reported in
Schardein 1976).
Kuroki T, Drevon C. Montesano R. 1977. Microsome-mediated
mutagenesis in V79 Chinese hamster cells by various nitrosamines. Cancer
Res 37(4): 1044-1050.
Lasagna L. 1979. Toxicological barriers to providing better drugs. Arch
Toxicol 43:27-33. (As reported in Jensh 1983).
Leonard BE. 1982. Behavioural teratology: post-natal consequences of
drug exposure in utero. Arch Toxicol Suppl 5, 48-58.
Leonard BE. 1983. Behavioral teratology and toxicology. Psychophar-
macology (Amsterdam): l(l):248-299.
Leone VG. 1977. Comparative aspects of developmental stages in mam-
mals used for teratogenetic tests. In: Methods in prenatal toxicology. Neu-
bert D, Merker H-J, Kwasigroch TE, eds. Massachusetts: PSG, pp. 14-24.
Lenz W, Knapp K. 1962. Thalidomide embryopathy. Arch Environ Health
5:110-105.
-------
61
Marhan O, Jelinek R. 1979. Efficiency of embryotoxicity testing pro-
cedures. I. A compromise approach. Toxicol Lett 4:385-388.
Marks TA, Worthy WC, Staples RE. 1980. Teratogenic potential of phen-
cyclidine in the mouse. Teratology 21:241-246.
Marks TA, Gupta BN, Ledoux TA, Staples RE. 1981. Teratogenic evalua-
tion of 2-nitro-p-phenylenediamine, 4-nitro-o-phenylenediamine, and
2,5-toluenediamine sulfate in the mouse. Teratology 24:253-265.
Matsubara Y, Goto M, Mikami T, Suzuki Y, Chiba T. 1983. Teratogenic
effects of thalidomide in the rabbit: difference in susceptibility between
two breeds. Cong Anom 23:223-229.
Matusis II. 1975. Vitamins and antivitamins. Moscow. (As reported in
Vaisman et al. 1983).
McBride WG, Vardy PH. 1983. Pathogenesis of thalidomide teratogenesis
in the marmoset (Callithrix jacchus): evidence suggesting a possible
trophic influence of cholinergic nerves in limb morphogenesis. Develop
Growth Differ 25(4):361-373.
Mehlman MA, Craig PH, Gallo MA. 1984. Teratological evaluation of
trimethyl phosphite in the rat. Toxicol Appl Pharmacol 72:119-123.
Meltzer HJ. 1956. Congenital anomalies due to attempted abortion with
4-aminoteroglutamic acid. J Am Med Assoc 161(13):1253.
Merkle J, Schulz V, Gelbke HP. 1984. An embryotoxicity study of the
fungicide tridemorph and its commercial formulation Calixin®. Teratology
29:259-269.
Milunsky AJ, Graef W, Gaynor MF Jr. 1968. Methotrexate-induced
congenital malformations. J Pediatr 72(6):790-795.
Mummery CL, Van Den Brink CE, Van Der Saag PT, De Laat SW.
1984. A short-term screening test for teratogens using differentiating neu-
roblastoma cells in vitro. Teratology 29:271-279.
Murai N. 1966. Effect of maternal medication during pregnancy upon
behavioral development of offspring. Tohokv J Exp Med 89:265-272. (As
reported in Coyle et al. 1976).
Murphy ML. 1962. Teratogenic effects in rats of growth inhibiting chemi-
cals including studies of thalidomide. Clin Proc Child Hosp 18:307-322.
-------
62
NAS. 1977. National Academy of Sciences. Principles and procedures for
evaluating the toxicity of household substances. Washington, D.C.: Print-
ing and Publishing Office. 130 pp.
NTP. 1984. National Toxicology Program. Fiscal year 1984 annual plan.
U.S. Department of Health and Human Services, p. 174.
OECD. 1981. Office of Economic Cooperation and Development. OECD
guidelines for testing of chemicals. Section 4, No. 414.
Palmer AK. 1968. Spontaneous malformations of the New Zealand white
rabbit: the background to safety evaluation tests. Lab Anim 2:195-206.
(As reported in Palmer 1978).
Palmer AK. 1978. The design of subprimate animal studies. In: Handbook
of teratology, Vol. 4. Research procedures and data analysis. Wilson JG,
Fraser FC, eds. New York: Plenum, pp. 215-253.
Parkhie M, Webb M. 1983. Embryotoxicity and teratogenicity of thali-
domide in rats. Teratology 27(3):327-332.
Petrere JA, Humphrey RR, Sakowsky R, Fitzgerald JE, de la Iglesia FA.
1984. Teragology study with the synthetic prostaglandin ONO-802 given
intravaginally to rabbits. Terato Carcio Mutagen 4:225-231.
Pfeiffer RA, Kasenow W. 1962. Thalidomide and congenital abnormali-
ties. Lancet 1:45-46.
Poswillo DC, Hamilton WJ, Sopher D. 1972. The marmoset as an animal
model for teratological research. Nature (London) 239(5366):460-412.
Powell HR, Ekert H. 1971. Methotrexate-induced congenital malfor-
mations. Med J Aust 2:1076-1077.
Randall CL, Taylor WJ. 1979. Prenatal ethanol exposure in mice: terato-
genic effects. Teratology 19(3):305-312.
Rodier PM. 1978. Behavioral teratology. In: Handbook of teratology, Vol.
4. Research procedures and data analysis. Wilson JG, Fraser FC, eds.
New York: Plenum, pp. 397-428. (As reported in Jensh 1983).
Rodriguez-Gonzalez MD, Lima-Perez MT, Sanabria-Negri JG. 1983. The
effect of cypropeptadine chlorhydrate on rat embryonic development.
Terato Carcino Mutagen 3:439-446.
-------
63
Rowland JM, Hendrickx AG. 1983. Teratogenicity of triamcinolone
acetonide in rats. Teratology 27:13-18.
Saad DJC, Kirsch RM, Kaplan, LL, Rodwell DE. 1984. Teratology of
intravaginally administered contraceptive jelly containing octoxynol-9 in
rats. Teratology 30:25-30.
Sadler TW, Horton WE, Warner CW. 1982. Whole embryo culture: a
screening technique for teratogens? Teratog Carcinog Mutagen 2:243-253.
Sadler TW, Kochhar DM. 1975. Teratogenic effects of chlorambucil on in
vivo and in vitro organogenesis in mice. Teratology 2:71-78.
Schardein JL. 1976. Drugs as teratogens. Cleveland, Ohio: CRC, 291 pp.
Schardein JL. 1983. Teratogenic risk assessment. In: Issues and reviews in
teratology. Kalter H, ed. New York: Plenum, pp. 181-214.
Schuler RL, Hardin BD, Niemeier RW et al. 1984. Results of testing fif-
teen glycol ethers in a short-term in vivo reproductive toxicity assay.
Environ Health Perspect 57:141-146.
Schumacher HJ. 1975. Chemical structure and teratogenic properties. In:
Methods for detection of environmental agents that produce congenital
defects. Shepard TH, Miller JR, Marois M, eds. New York: American
Elseiver, Inc., pp. 65-77.
Seegmiller RE, Nelson GW, Johnson CK. 1983. Evaluation of the terato-
genic potential of delalutin (17a-hydroxyprogesterone caproate) in mice.
Teratology 28:201-208.
Shepard TH. 1976. Catalog of teratogenic agents. Johns Hopkins Univer-
sity. 291 pp.
Siddall RA. 1978. The use of marmosets (Callithrix jacchus) in teratolog-
ical and toxicological research. Primates Med 10:215-224.
Silverman P. 1978. Animal behavior in the laboratory. New York: Pica,
pp. xi-409. (As reported in Jensh 1983).
Skalko RG, Gold MP. 1974. Teratogenicity of methotrexate in mice. Tera-
tology 9:159-164.
Smithells RW, Bond MB. 1962. Thalidomide and malformations. Lancet
1:1270-1273.
-------
64
Somers GF. 1963. The foetal toxicity of thalidomide. Proc Eur Soc Study
Drug Toxic 1:49-58.
Somers GF. 1962. Thalidomide and congenital abnormalities. Lancet
1:912-913.
Spyker JM, Avery DL. 1976. Behavioral toxicology and the developing
organism. In: Proceedings, workshop on behavioral toxicology. DHEW
Publication No. (NIH)76-1189. Washington, B.C.: U.S. Dept. Health,
Education, and Welfare, pp. 95-109. (As reported in Jensh 1983).
Sullivan FM. 1970. Mechanisms of action of teratogenic drugs. Proc R
Soc Med 63(12):42-43.
Tanimura T. 1972. Effects of macaque embryos of drugs reported or
suspected to be teratogenic to humans: discussion paper. Acta Endocrinol
(Copenhagen) Suppl. 16:293-308.
Thompson DJ, Dyke IL, Molello JA. 1984. Reproduction and teratology
studies on hexamethylmelamine in the rat and rabbit. Toxicol Appl Phar-
macol 72:245-254.
Thompson JF, Schweisthal MR. 1969. Study of closure of the embryonic
rat palate in vitro with the effects of certain chemicals. J Dent Res
48(4):568-572.
Tuchmann-Duplessis H. 1972. Teratogenic drug screening, present pro-
cedures and requirements. Teratology 5:271-286.
Tuchmann-Duplessis H, Mercier-Parot L. 1966. Effect of two chemically
related antimetabolites on the embryo. Bull Schweiz Akad Med Wiss
22:153-165.
USEPA. 1982a. U.S. Environmental Protection Agency. Health effects
test guidelines. Organ/tissue teratogenicity study. Washington, DC: Office
of Toxic Substances. EPA 560/6-82-001.
USEPA. 1982b. U.S. Environmental Protection Agency. Subdivision F:
Pesticide assessment guidelines. Washington, D.C.: Office of Pesticide
Programs, pp. 126-130.
USEPA. 1984. U.S. Environmental Protection Agency. Health Effects
Test Guidelines. Specific organ/tissue toxicity—primary developmental
toxicity screen. Washington, D.C.: Office of Toxic Substances, 10 pp.
-------
65
USFDA. 1982. U.S. Food and Drug Administration. Toxicological princi-
ples for the safety assessment of direct food additives and color additives
used in food. Washington, D.C.: U.S. Food and Drug Administration,
pp. 108-118.
Vaisman BL, Popov VP, Ignat'eva TV. 1983. Reduction in the tissue
ascorbic acid level in guinea pigs by thalidomide. Byull Eksp Biol Med
96(7):27-29.
Vondruska JF, Fancher OE, Calandra JC. 1971. An investigation into the
teratogenic potential of captan, folpet, and difolation in nonhuman pri-
mates. Toxicol Appl Pharmacol 18:619-624.
Vorhees CV. 1983. Behavioral teratogenicity testing as a method of
screening for hazards to human health: a methodological proposal. Neuro-
behav Toxicol Teratol 5:469-474.
Vorhees CV, Brunner RL, McDaniel CR, Butcher RE. 1978. The relation-
ship of gestational age to vitamin A induced postnatal dysfunction. Tera-
tology 17:271-276.
Vorhees CV, Butcher RE. 1982. Behavioral teratogenicity. In: Develop-
mental toxicology. Snell K, ed. London: Croom-Helm, pp. 247-298. (As
reported in Vorhees 1983).
Vorhees CV, Butcher RE, Brunner RL, Sobotka TJ. 1979. A developmen-
tal test battery for neurobehavioral toxicity in rats: a preliminary analysis
using monosodium glutamate, calcium carrageenan and hydroxyurea.
Toxicol Appl Pharmacol 50:267-282. (As reported in Jensh 1983).
Warkany J, Beaudry PH, Hornstein S. 1959. Attempted abortion with
aminopterin (4-aminopteroylglutamic acid). AMA Am J Dis Child
97:274-281.
Weil CS. 1970. Selection of the valid number of sampling units and a con-
sideration of their combination in toxicological studies involving reproduc-
tion, teratogenesis or carcinogenesis. Fd Cosmet Toxicol 8:117-182.
Werboff J, Gottlieb JS, Dembicki EL, Havlena J. 1961b. Postnatal effect
of antidepressant drugs administered during gestation. Exptl Neurol
3(6):542-555.
Werboff J, Gottlieb JS, Havlena J, Word TJ. 196la. Behavioral effects of
prenatal drug administration in the white rat. Pediatrics 27:318-324.
-------
66
Werboff J, Havlena J. 1962. Postnatal behavioral effects of tranquilizers
administered to the gravid rat. Exptl Neurol 6:263-269.
Werboff J, Kesner R. 1963. Learning deficits of offspring after adminis-
tration of tranquillizing drugs to the mothers. Nature 197:106-107.
Wilk AL, Greenberg JH, Horigan EA, Pratt RM, Martin GR. 1980.
Detection of teratogenic compounds using differentiating embryonic cells
in culture. In Vitro 16(4):269-276.
Willhite CC, Perm VH, Smith RP. 1981. Teratogenic effects of alphatic
nitriles. Teratology 23:317-323.
Willhite CC. 1983. Developmental toxicology of acetonitrile in the Syrian
golden hamster. Teratology 27:313-325.
Willhite CC, Dawson MI, Williams KJ. 1984. Structure-activity relation-
ships of retinoids in developmental toxicology. Toxicol Appl Pharmacol
74:397-410.
Wilson JG. 1966. Effects of acute and chronic treatment with actinomycin
D on pregnancy and the fetus in the rat. Harper Hosp Bull 24:109-118.
Wilson JG. 1969. Teratological and reproductive studies on nonhuman pri-
mates. In: Methods for teratological studies in experimental animals and
man, II. Tokyo, Japan, pp. 6-31.
Wilson JG. 197la. Use of primates in teratological research and testing.
In: Malformations congenitales des mammiferes, Paris, pp. 273-292.
Wilson JG. 1971b. Use of rhesus monkeys in teratological studies. Fed
Proc 30(1): 104-109.
Wilson JG. 1975a. Critique of current methods for teratogenicity testing.
In: Methods of environmental agents that produce congenital defects.
Shepard TH, Miller JR, Marois M, eds. New York: American Elseiver,
pp. 29-48.
Wilson JG. 1975b. Reproduction and teratogenesis: current methods and
suggested improvements. J Assoc Off Anal Chem 58(4):657-667.
Wilson JG. 1977. Current status of teratology general principles and
mechanisms derived from animal studies. In: Handbook of teratology, Vol.
I. General principles in etiology. Wilson JG, Fraser FC, eds. New York:
Plenum, pp. 47-74.
-------
67
Wilson JG. 1978. Feasibilty and design of subhuman primate studies. In:
Handbook of teratology, Vol. 4. Research procedures and data analysis.
Wilson JG, Fraser FC, eds. New York: Plenum, pp. 255-273.
Wilson JG. 1979. The evolution of teratological testing. Teratology
29(2):205-212.
Wilson JG. Fradkin R, Hardman AA. 1968. Progress report on
teratological testing of drugs in rhesus monkeys. Teratology 1:223.
Wilson JG, Scott WJ, Ritter EJ, Fradkin R. 1979. Comparative distribu-
tion and embryotoxicity of methotrexate in pregnant rats and rhesus mon-
keys. Teratology 19:71-79.
WHO. 1967. World Health Organization. Principles for the testing of
drugs for teratogenicity. 20 pp.
Wolkowski-Tyl R, Phelps M, Davis JK. 1983. Structural teratogenicity
evaluation of methyl chloride in rats and mice after inhalation exposure.
Teratology 27:181-195.
Zimmerman, EF. 1975. Chemical structure and teratogenic mechanism of
action. In: Methods for detection of environmental agents that produce
congenital defects. Shepard TH, Miller JR, Marois M, eds. New York:
American Elseiver, pp. 79-91.
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3. IMMUNOTOXICOLOGY
Mary Lou Daugherty
3.1 INTRODUCTION
The science of immunology, once limited to studies of host-defense
mechanisms against bacterial, fungal, viral, protozoal, and helminth
diseases, has expanded into a complex but well-studied discipline that
includes allergy, tumor immunity, host-graft reactions, tolerance, and the
immune mechanisms involved in the pathogenesis of various occupational
disorders of the lung and skin.
These protective responses of the body against foreign entities (or
antigens) are carried out by the immune system, a complex and
widespread organization of organs and cells. Immune responses are of two
types: nonspecific (or innate) and specific (or acquired) (De Bruin 1976).
Nonspecific immunity is characterized by mechanisms for the disposal
of foreign and potentially harmful macromolecules, microorganisms, or
metazoa which do not involve the recognition of antigen or the mounting
of an immune response. Nonspecific immunity is generalized and includes
the activities of phagocytic cells, certain microbial inhibitors (lysozymes),
the interferon system, the complement system, and the kinin system.
Specific immunity is characterized by (1) the highly specific recogni-
tion of and response to antigen by lymphocytes and (2) the induction of
immunological memory in long-lived lymphocytes (memory cells), which
are then capable of producing an enhanced immune response upon second
contact with an antigen (Luster et al. 1982b). Specific immunity includes:
cell-mediated immunity, humoral immunity, and immunological tolerance.
The major organs of the specific immune system in man are the thymus
and bursa-equivalent, which are the primary lymphoid tissues, and the
lymph nodes, the spleen, the bone marrow, the gut-associated lymphoid tis-
sue (GALT) (e.g. Peyer's patches), and the bronchus-associated lymphoid
tissue (BALT), which are the peripheral lymphoid tissues (Luster et al.
1982b). The major cells are the circulating lymphocytes. These cells play a
central role in both cellular and humoral immunity and and can be divided
into two distinct populations, T-cells and B-cells, with different functions
and properties (Vos 1977). Also important are the macrophages, both
fixed and free, which are involved in and seem to be necessary for the ini-
tiation and the effector phases of humoral and cell-mediated immune reac-
tions (Faith et al. 1980). Unlike the lymphocytes, however, the macro-
phages do not clonally divide and are not antigen specific. Other cell types,
such as monocytes, granulocytes, mast cells, and reticuloendothelial cells,
facilitate in various ways the expression of the immune response even
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though they are not equipped ab initio to recognize and interact with
antigen or to participate in the induction of the immune response (Miller
1975).
Because the immune system plays a critical role in the protection of
man against infectious and neoplastic disease, dysfunction of the system
can have serious health consequences (Gardner et al. 1979); it therefore
becomes important to be able to identify immunotoxic agents that may be
released into the workplace or environment.
Although the concept of immunotoxicology is relatively new, it has
been known for some time that certain drugs can cause suppression of the
immune system.
In recent years, chemicals other than those used clinically for immune
suppression have been recognized as agents capable of inducing alterations
in the immune response. These chemicals are of environmental concern
and include polychlorinated biphenyls, 2,3,7,8-tetrachlorodibenzo-/>-dioxin,
hexachlorobenzene, polybrominated biphenyl, chlorinated dibenzofuran,
NO2, SO2, benzo[a]pyrene, tobacco smoke, heavy metals, organotins,
butylated hydroxytoluene, ortho-phenyl phenol, and pesticides such as
DDT, Dieldrin, organochlorines, and organophosphates (Vos 1977, Moore
1979, Silkworth and Loose 1981, Luster et al. 1982b). Information from a
variety of human studies (reviewed by Holt and Keast 1977) indicates that
increased prevalence of infections, and perhaps neoplastic diseases, result-
ing from chronic exposure to air contaminants is associated with impaired
immunological control mechanisms. These human data are supported by
the results of animal studies that demonstrate the impairment of host
resistance by atmospheric contaminants such as pesticides, metals, and
cigarette smoke [reviewed by Koller (1979a, 1979b), Treagan (1975), Vos
(1977), Moore and Faith (1976), Holt and Keast (1977), and Faith et al.
(1980)]. In addition, Loose et al. (1977, 1978, 1979) demonstrated that
polychlorinated biphenyl and hexachlorobenzene were profoundly immu-
nosuppressive at clinically subtoxic levels and suggested that the evaluation
of immune parameters may prove to be a sensitive indicator of general
toxicity.
Procedures for testing the immunotoxicity of chemicals are generally
not adequately represented in current routine toxicity testing schemes, and,
as a result, only a limited number of chemicals have been shown to have
immunosuppressive properties in such tests. Vos (1977) suggested that
these chemicals may be representative of a much larger group of immuno-
toxic chemicals and advocated the inclusion of techniques for the evalua-
tion of the function and morphology of the immune system in routine test
protocols. In order to accomplish this, it is necessary to incorporate
knowledge from the expansive, growing immunology discipline into the
field of toxicology.
The development of tests to evaluate the immune response during toxi-
cological testing is gaining interest. Older in vivo test methods, which can
be expensive and cumbersome, are undergoing revaluation, and in vitro
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correlates that are cheaper, easier to handle, and more reproducible are
being developed.
The primary purpose of this section is to review those tests of immune
function, immunocompetence in particular, that appear to be suitable for
inclusion in routine toxicity testing batteries. In addition to tests for immu-
nocompetence, tests for the induction of clinical allergy (those diseases in
which hypersensitivity plays a part, such as contact hypersensitivity, hay
fever, and asthma) will be mentioned briefly. Recommendations for tier
testing schemes for immunotoxicity will be presented.
An in-depth review of the organ systems and the complex cellular and
humoral interactions that are necessary for, and ultimately result in, an
immune response is not within the scope of this document. A glossary of
immunological terms, prepared for the nonimmunologist, is provided at the
end of the summary. For a more comprehensive survey of basic immunol-
ogy, the references listed in Section 3.8 are recommended.
3.2 TESTS TO DETECT ALTERATIONS IN THE IMMUNE RESPONSE
Chemicals capable of inducing alterations in the immune response may
alter both specific and nonspecific responses, and the response may be
enhanced or suppressed, depending on a variety of circumstances that
include chemical and dosage level and the species and age of the test
animal (Luster and Faith 1979). For example, inorganic metals have been
shown to affect primarily humoral immune responses (Koller 1979a) while
halogenated aromatic hydrocarbons affect cell-mediated immunity; low-
level exposure to certain chemicals may lead to immunoenhancement while
higher levels are immunosuppressive (Koller et al. 1977, Holt and Keast
1977); hexachlorobenzene is reported to produce opposite responses in the
rat and the mouse (Moore 1979); and the developing immune system may
be more sensitive to the effects of environmental chemicals than the adult
immune system (Vos and Moore 1974, Moore and Faith 1976).
It has generally been thought that no agent selectively suppresses the
antibody-forming mechanism but that any agent that suppresses cell
proliferation or interferes with protein or nucleic acid formation will,
under proper circumstances, suppress the antigen-antibody mechanism
(Loomis 1974). However, there is evidence to suggest that this is not
necessarily the case, but that the various cell types that participate in the
immune response can be specifically depressed (Nicolin et al. 1980).
When a chemical compound is suspected of altering the immune
response, both the cellular and humoral systems must be tested (Koller
1979a). Tests of cell-mediated immunity generally measure the specific
activities of the T-lymphocyte populations and may be performed using in
vivo or in vitro techniques. Tests of humoral immunity generally measure
cellular synthesis of antibody and circulating antibody, primarily B-
lymphocyte properties. There are also tests to measure macrophage proper-
ties and other indirect parameters of the immune system. Thus, tests are
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available to help determine which of the following steps in the immune
response are altered by a particular chemical (Vos 1977):
1. antigen processing (phagocytosis),
2. antigen recognition by lymphocytes,
3. adaptive phase (differentiation and proliferation of T- and B-
lymphocytes), or
4. effector phase.
Representative tests, primarily the original techniques or the modifica-
tions of those techniques that seem suitable for toxicity screening, are
listed in Table 3.1 and are reviewed in the following sections.
As in any discussion of toxicity testing procedures, the importance of
proper test conditions should be recognized. Factors that may affect the
outcome of the tests are listed in Table 3.2 and are discussed in detail by
Doull (1975).
3.2.1 Assessment of Cell-Mediated Immunity
The main categories of cell-mediated immunity include: classical cell-
mediated protective immunity, which is mainly effective against protozoal,
viral, fungal, and some bacterial infections; delayed hypersensitivity skin
reactions to extracts or whole suspensions of organisms, also identified as
tuberculin hypersensitivity; chemical contact sensitivity; allograft rejection;
immunological surveillance to tumors; and certain organ-specific autoaller-
gic diseases, such as thyroiditis, encephalomyelitis (following rabies vacci-
nation), adrenalitis, and orchitis (Turk 1975, Faith et al. 1980).
T-cells are required for the induction of the cell-mediated immune
response. When stimulated by antigen, T-cells form a pool of sensitized,
antigen-specific lymphocytes which then can function as memory cells
(which produce the secondary immune response), effector cells, killer cells,
helper cells, or suppressor cells (Faith et al. 1980). T-cells do not produce
antibody (Roller 1979a).
Effector cells, in delayed-type hypersensitivity, produce biologically
active products called lymphokines. Lymphokines are soluble nonantibody
products of lymphocyte activation by antigen and mitogens which are
thought to act as molecular mediators of cellular immune responses
(Merely et al. 1978). Lymphokines exhibit the following characteristics:
(1) produce increased vascular permeability following intradermal injec-
tion; (2) increase tritiated thymidine uptake by lymphocytes in culture;
and (3) inhibit macrophage migration in vitro. Lymphokines retain biologi-
cal activity after removal of the antigen that stimulated them. They may
also be involved in both expression and regulation of lymphoid cell activity,
and they may represent a pathway for the expression of T-cell function.
Some lymphokines, such as the migration inhibition factor, the chemotac-
tic factor, and the macrophage-activating factor, influence macrophage
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Table 3.1 Tests for Immunotoxicity
Assessment of cell-mediated immunity
In vivo tests
Tests for delayed hypersensitivity
Cutaneous hypersensitivity to tuberculin
Radiometric ear test
Footpad assay
Allograft rejection
Graft-vs-host reaction
In vitro tests
Transformation of lymphocytes by mitogens
Mixed lymphocyte culture
Macrophage migration inhibition factor (MIF)
Macrophage aggregation
Leukocyte adherence inhibition (LAI)
Assessment of humoral immunity
Assays for local production of antibody
Plaque-forming assays
Immunofluorescence
Measurement of circulation antibody and immunoglobulins
Precipitation
Hemagglutination and hemolysis
Passive hemagglutination
Enzyme-linked immunosorbent assay (ELISA)
Other tests of B-cell function
EAC rosette technique
Transformation of lymphocytes by B-cell mitogens
Assessment of indirect parameters of immunity
Macrophage functions
Ingestion
Intracellular killing
Host resistance to infection
Tests to detect the sensitizing potential of chemicals
Skin sensitization
Draize test
Freund's complete adjuvant test
Guinea pig maximization test
Split adjuvant technique
Buhler test
Open epicutaneous test
Respiratory sensitization
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74
Table 3.2 A Classification of Toxicity-Influencing Factors
1. Factors Related to the Toxic Agent
Chemical composition (pH, choice of anion, etc.)
Physical characteristics (particle size, method of formulation, etc.)
Presence of impurities or contaminants
Stability and storage characteristics of toxic agents
Solubility of the toxic agent in biologic fluids
Choice of the vehicle
Presence of excipients: adjuvants, emulsifiers, surfactants, binding
agents, coating agents, coloring agents, flavoring agents, preser-
vatives, antioxidants, and other intentional and nonintentional
additives
2. Factors Related to the Exposure Situation
Dose, concentration, and volume of administration
Route, rate, and site of administration
Duration and frequency of exposure
Time of administration (time of day, season of the year, etc.)
3. Inherent Factors Related to the Subject
Species and strain (taxanomic classification)
Genetic status (littermate, siblings, multigeneration effects, etc.)
Immunologic status
Nutritional status (diet factors, state of hydration, etc.)
Hormonal status (pregnancy, etc.)
Age, sex, body weight, and maturity
Central nervous system status (activity, crowding, handling, presence
of other species, etc.)
Presence of disease or specific organ pathology
4. Environmental Factors Related to the Subject
Temperature and humidity
Barometric pressure (hyper- and hypobaric effects)
Ambient atmospheric composition
Light and other forms of radiation
Housing and caging effects
Social factors
Chemical factors
Source: Adapted from Doull (1975).
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75
functions (Faith et al. 1980). The properties of lymphokines or products of
activated lymphocytes (PALs) have been summarized by Bloom et al
(1974) in Table 3.3.
Killer cells are cytotoxic and participate in the rejection of tumor cells
and transplants.
Helper cells interact with B cells in antibody response against many
antigens, while suppressor T-cells function to suppress the functions of B-
cells or effector T-cells. [There is also evidence for the existence of B-cells
that modulate T-cells (Turk 1975).]
The induction and manifestation of the cell-mediated immune response,
illustrated in Figure 3.1, proceeds as follows (Park and Good 1974, Miller
1975, Luster et al. 1982b):
1. Antigen interacts with T-cells, usually outside the lymphoid tissues (at
the site of antigen accumulation or within a graft).
2. Lymph nodes draining the site of sensitization become enlarged, and
on day two of the response the small T-lymphocytes in the thymus-
dependent areas differentiate to large pyroninophilic cells and divide
to produce a progeny of effector cells (small lymphocytes), which
enter the circulation after 3 to 4 days. The onset of delayed hypersen-
sitivity coincides roughly with the appearance of these cells in the cir-
culation.
3. The small sensitized lymphocytes (effector cells) now in the circula-
tion come in contact with the antigen and are stimulated to produce
lymphokines or effector molecules, which recruit nonspecific cells,
such as monocytes, to the site of the reaction.
4. Those monocytes that accumulate are then transformed to macro-
phages, which through the release of lysozymal enzymes produce vari-
ous effects leading to the ultimate expression of the cellular
response—a delayed hypersensitivity lesion, a graft rejection, or a
microbiocidal effect.
In general, in vivo assays of cell-mediated immunity mainly measure
the effector mechanism, while in vitro assays may measure cellular com-
ponents (Luster et al. 1982a). In vivo and in vitro assays that are com-
monly employed to measure T-lymphocyte functions will be described.
These will be discussed with respect to preferred test species, advantages
and disadvantages, usefulness in detecting the effects of environmental
chemicals, and, when possible, applicability of the test to particular chemi-
cal classes.
3.2.1.1 In vivo tests
Basic in vivo procedures for evaluating the cell-mediated immune
response include tests of delayed hypersensitivity, allograft rejection, and
graft-vs-host reactions. These are well-established and valuable procedures,
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76
Table 3.3 Products of Activated Lymphocytes (PALs) In Vitro
A. Affecting Macrophages
Migration Inhibitory Factor (MIF)
Macrophage Aggregation Factor
Macrophage Spreading Factor
Macrophage Activating Factor (MAP)
Macrophage Chemotactic Factor
Cytophilic Antibodies
B. Affecting Lymphocytes
Blastogenic or Mitogenic Factors (BF, MF)
Thymus Replacing Factor (TRF) or
Helper Factor (HF)
Suppressor Factor
Transfer Factor (TF)
C. Affecting Neutrophils
Leucocyte Inhibitory Factor (LIF)
Leucocyte Chemotactic Factor
D. Affecting Eosinophils
Eosinophil Chemotactic Factor (ECF)
Eosinophil Promoting Factor
E. Affecting Other Cell Types
Lymphotoxin (LT)
Proliferation Inhibitory Factor (PIF)
Cloning Inhibitory Factor (C1IF)
Osteoclast Activating Factor (OAF)
Colony Stimulating Factor (CSF)
Interferon (IF)
Inhibits migration of normal macrophages
Agglutinates macrophages in suspension
Increases adherence and surface area of
macrophages
Increases glucose-C-1 oxidation and phagocytosis
Causes macrophages to migrate along gradient
through micropore filter
Confer some specific reactivity to antigens
Induce blast cell transformation and thymidine
uptake by normal lymphocytes
Permits differentiation of B cells to
Ab secreting cells
Inhibits activation of, or antibody production
by, B cells
Converts nonreactive cells to cells capable
of reacting to specific antigens
Inhibits migration of polymorphs
Causes polymorphs to migrate through
micropore filter
Together with Ab-Ag complex, causes migration
through micropore filter
Increases migration from agarose drops
Cytotoxic, especially for L-cells
Inhibits proliferation without killing cells
Causes release of 45Ca from embryonic bone
cultures
Stimulates differentiation of bone marrow
stem cells into granulocytic and/or
monocytic cells
Renders cells resistant to virus infection
Source: Adapted from Bloom et al. 1974.
-------
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78
but they are generally cumbersome and time consuming and require large
numbers of animals. Newer tests have been introduced, some utilizing iso-
topes, that are quantitative and more sensitive. Some of these have been
recommended for inclusion in toxicity screening protocols.
Tests of delayed-type faypersensitivity. Cell-mediated immunity is often
measured by delayed hypersensitivity reactions (Koller 1979a). The
evaluation of delayed hypersensitivity is generally based on dermal reac-
tions that lead to erythema, induration, and increase in skin thickness.
More recent tests have been introduced that are based on the radioactive
labeling of bone marrow precursors of monocytes that subsequently
migrate into sites of inflammation (Lefford 1974) or on the extravasation
of human serum albumin into the area of hypersensitivity response
(Paranjpe and Boone 1972). All of these tests are important to the assess-
ment of immunocompetence. Examples are described in the following
paragraphs.
Skin Reactions (Useful in Immunotoxicological Studies)
An important test for measuring cell-mediated immunity is the delayed
cutaneous hypersensitivity reaction to tuberculin as described by Flax and
Waksman (1962). In the original experiment rats were injected in one
hind footpad with a suspension of heat-killed tubercle bacilli and then were
challenged with a purified protein derivative of tuberculin (FDD), which
was injected intradermally into the flank. The animals were observed rou-
tinely at 4 to 6, 24, 48, and 72 h, and the diameter and degree of indura-
tion of the skin reactions were determined. The reaction was maximal at
24 to 48 h.
In addition to the rat, the guinea pig is often used in this system, and
in the past was considered to be the species of choice.
The usefulness of this skin test in the evaluation of immunotoxicologic
properties of chemicals, as well as species specificity of the test, was
demonstrated by Vos et al. (1973) and by Vos et al. (1979b). The pro-
cedure used was similar to that of Flax and Waksman (1962); for a higher
degree of quantitation, the thickness of the reaction was measured as an
end point, in addition to diameter and degree of induration. In the study
by Vos et al. (1973) the skin test reaction was evaluated in guinea pigs
and rats following administration of 2,3,7,8-tetrachlorodibenzo-/?-dioxin
(TCDD). Guinea pigs were dosed for 8 weeks with 0.008, 0.04, 0.2, or 1.0
jig/kg (body weight). Skin reactions were significantly reduced at the 0.2-
and 0.04-jtg/kg levels. All guinea pigs treated with 1.0 Mg/kg died or
displayed overt signs of toxicity. Thymus atrophy and lymphopenia were
observed. The rats were dosed for 6 weeks with higher doses of 0, 0.2, 1.0,
and 5.0 ng/kg, and, although animals at the 5-/*g/kg level had lower body,
thymus, and adrenal weights, the skin reactions were not altered.
In the second study, Vos et al. (1979b) included the skin test in a bat-
tery of immunological assays to test the effects of hexachlorobenzene in
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79
the rat. The cell-mediated immune response was not altered by the chemi-
cal, but the antibody response was stimulated.
The cutaneous hypersensitivity reaction to tuberculin is not a very sen-
sitive test for measuring cell-mediated immunity (Vos 1977), and measure-
ments of the reaction are extremely subjective and vary considerably
between observers (Carter and Bazin 1980). However, the skin test offers
several test sites per animal, is quick and easy to perform (Crowle 1975),
and can yield reproducible results with little variation if careful measure-
ments are made. Lefford (1974) introduced a new test using radiolabeling
techniques, which is more quantitative and less subjective than the more
traditional skin test. This procedure is described in the following section.
Radiometric Ear Test
The Lefford (1974) technique for measuring delayed-type hypersensi-
tivity (DTH) is based on the radioactive labeling of bone marrow precur-
sors of monocytes, which subsequently migrate to sites of inflammation
and accumulate in DTH reactions. Rats were sensitized intravenously or
subcutaneously with viable bacillus Calmette-Guerin (BCG), then injected
subcutaneously with thymidine methyl-3H. Twenty-four hours later the
animals were injected in the left ear with tuberculin purified protein
derivative (PPD). The right ear, injected with diluent, served as a control.
Twenty-four hours after the injection of antigen the central portion of each
ear was removed, and radioactivity of the tissue was measured in a liquid
scintillation spectrometer. Counts of the test ear were then compared with
those of the control ear, and it was possible to calculate the left ear/right
ear ratio as the parameter of delayed hypersensitivity.
Mice and guinea pigs may also be tested by this method, and various
protein antigens, other than PPD, can be used; however, PPD is recom-
mended for rats and guinea pigs while other protein antigens, including
bovine serum albumin, are generally used in mice (Luster and Faith
1979).
Moore and Faith (1976) introduced a modification of the Lefford
method in which rats were sensitized to oxozalone by skin-painting the
ears. Tritiated thymidine was injected intraperitoneally 10 days later, and
24 h after thymidine injection a challenging dose of oxozalone in olive oil
was painted on one ear. Standard procedures were then used to determine
the ratio of radioactivity in the treated and untreated ears. Moore and
Faith (1976), by using this method, were able to detect suppression of
cell-mediated immunity in Fischer rat pups exposed to TCDD on gestation
day 18 and on postnatal days 0, 7, and 14.
In a later study, Fraker (1980) used a similar technique to test the
effects of polybrominated biphenyls (PBB) in mice. The animals (fed
PBB) were sensitized percutaneously and challenged on one ear lobe with
dinitrofluorobenzene. After intravenous injection of 125I-deoxyuridine, the
ratio of radioisotope incorporated in the challenged versus the unchal-
lenged ear lobe served as a measure of T-cell-dependent delayed
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80
hypersensitivity. No effect was seen on this response. This was of
particular interest because, in the same study, the IgG and IgM plaque-
forming cells were impaired by PBB, indicating that both helper T- and
B-cells were affected. T-cells involved in delayed-type hypersensitiviy and
those involved in humoral immunity are of the same phenotype and
probably follow the same developmental pathway; thus the differential
effects of PBB were thought to be due to differences in susceptibility to
the chemical of the various subclasses of T-cells (Fraker 1980).
There are certain disadvantages to this method: first, each ear can be
tested only once, so each animal can receive a maximum of two tests;
furthermore, if the left ear/right ear ratio is calculated the animal can be
used only once. Advantages of the technique are objectivity, reproducibil-
ity, and sensitivity, and these far outweigh the disadvantages. It appears to
be particularly useful as a general screen for chemical effects because the
participation of many cell types is required for a normal reaction.
Footpad Assay
The footpad assay is based on the observation that 125I-human serum
albumin (125I-HSA) will extravasate into the edematous area produced by
a delayed hypersensitivity response (Paranjpe and Boone 1972). In the
procedure described by Kauffmann et al. (1982), Sanders et al. (1982),
and Munson et al. (1982), recommended for testing the effects of chemi-
cals on cell-mediated immunity, mice are immunized in the left hind foot-
pad with sheep red blood cells (SRBC). Four days later the mice are chal-
lenged in the same footpad with SRBC and 17 hours later are injected
intravenously with 125I-HSA. The animals are killed and both feet are
removed and measured for radioactivity in a gamma scintillation counter.
Control animals are not sensitized but are challenged in order to determine
nonspecific swelling. The stimulation index (S.I.) is calculated:
_ Left footpad - _ Left footpad unsensitized
Right footpad Right footpad unsensitized
Minor aspects of the technique, such as the number and route of
immunizing injections, the timing of the challenge, and timing of the iso-
tope injection, may vary between laboratories. Also, footpad swelling
instead of extravasation of isotope may be the end point of the assay
(Muller et al. 1977). Mice are generally used in this assay. However, it
has been noted that the strain of mouse may influence detectability of the
response (Crowle 1975).
Muller et al. (1977) used the footpad assay to demonstrate the
suppressive effects of lead on DTH in mice. Alterations of both primary
and secondary responses to SRBC were correlated with the concentrations
of lead in the blood. In another study, DTH, assayed by footpad injection,
was also shown to be suppressed in male mice by trichloromethane and in
males and females by bromodichloromethane (but not by bromoform and
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81
dibromochloromethane) (Schuller et al. 1978). Smith et al. (1978)
reported a similar inhibition of the footpad response with poly chlorinated
biphenyls (Aroclor 1254). Sanders et al. (1982), using the footpad assay,
observed a suppression of the stimulation index by subchronic administra-
tion of trichloroethylene in female (but not male) mice.
According to Dean et al. (1979a), the footpad assay is probably the
best single parameter for immunologic competence because it measures
both the recognition and effector phases of the cell-mediated immune
response and requires the cooperation of the macrophage/monocyte series.
The isotope system is sensitive and quantitative, and the reaction
appears to be of the pure delayed type without the humoral component of
the immediate anaphylactic or arthus type, which often accompanies the
delayed hypersensitivity reaction (Paranjpe and Boone 1972).
The assay can be used to determine the effects of chemicals on the
ability of the host to be immunized or to respond to challenge after
immunization (Dean et al. 1979a).
Allograft Rejection. Skin grafting is a useful technique that has been
applied to the study of various biological problems. The assay is dependent
upon the ability of an experimental host animal to reject a tissue trans-
planted from a donor that differs from the host at an important
histocompatibility locus. Allograft rejection time is used as the end point
for detecting alterations in the cell-mediated immune response of experi-
mental animals.
Standard techniques for skin grafting in rabbits, guinea pigs, and mice
were described by Billingham and Medawar (1951). These classic methods
can be laborious and time consuming and are not practical for situations
that require large numbers of grafts.
A rapid method of grafting skin on tails of mice was described by
Baily and Usama (1962). This widely used procedure consists of slicing an
approximately 5- by 2-mm section of skin from the tail and transferring it
to a raw area of the host tail from which the reciprocal graft has just been
removed. Grafts are placed on the host so that the direction of hair growth
is opposite to that of the host in order that "takes" can be distinguished
from possible host replacement. The grafts adhere well and do not require
extraneous pressure or dressing. Also, they are protected by, and can be
observed through, a length of glass tubing fitted over the tail.
This technique can also be successfully performed on rats. Using allo-
graft rejection as one of the parameters, Vos and Moore (1974) detected
impairment of cell-mediated immunity in the offspring of rats treated, dur-
ing gestation and postnatally, with TCDD. In a more recent study, Vos et
al. (1979b) incorporated allograft rejection into a battery of function tests
used to ascertain the effects of hexachlorobenzene on the immune system
of the rat. The chemical had no effect on the in vivo cell-mediated immune
response but did enhance humoral immunity.
The tail skin-grafting technique is simple and rapid, and a particular
advantage is that the graft is in plain view at all times; however, the assay
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82
is not applicable to studies requiring the use of large grafts or host areas
other than skin, and it is not particularly quantitative.
Graft-vs-host reaction. In the graft-vs-host reaction (a classic assay of
cell-mediated immunity), immunocompetent cells are injected into hybrids
of parental strains of animals that differ from each other at the major his-
tocompatibility locus. The donor cells do not possess any antigens foreign
to the host and thus are tolerated by the hybrids. However, the immuno-
competent donor cells do react against the foreign antigen in the host and,
therefore, induce a host reaction that can be measured by several methods,
including the spleen weight assay and the renal enlargement assay. These
methods have been reviewed by Ford (1978). In immunotoxicology studies
the test compound is given to the donor animals.
A particularly useful technique was described by Ford et al. (1970) in
which donor spleen or thoracic duct lymphocytes were injected into the
feet of young F[ hybrid rats. The recipients were killed seven days later,
and the weights of their popliteal lymph nodes were plotted against the
dose of cells injected, the degree of enlargement of the draining lymph
nodes being a measure of the graft-vs-host activity of the donor cells.
Differences in the effects of various chemicals on the immune response
have been demonstrated with the graft-vs-host popliteal lymph node assay.
For example, TCDD suppressed the response in rats and mice (Vos et al.
1973, Vos and Moore 1974), while Aroclor 1016 and hexachlorobenzene
enhanced the response.
The popliteal lymph node assay is a sensitive and quantitative measure
of cell-mediated immunity. Its in vitro analog, the mixed lymphocyte cul-
ture reaction, is discussed in a later section.
3.2.1.2 In vitro tests
In vitro tests of cell-mediated immunity were initially developed for the
purpose of analyzing the mechanisms of the response (Bloom et al. 1974),
but it soon became evident that these methods for testing cell-mediated
immunity also offered possibilities, at the clinical level, to objectively
assess the status or level of delayed hypersensitivity without risk to the
patient. These assays are also useful in the detection of alterations in the
immune response of experimental animals following exposure to potential
immunotoxicants. Those tests that seem most suitable for this purpose are
described.
Transformation of lymphocytes by mitogens. The ability of cultured
lymphocytes to undergo transformation and to incorporate labeled thymi-
dine (3H-TdR) into DNA following nonspecific stimulation with mitogens
is the basis of one of the most valuable tools of immunotoxicology. The
mitogenic response of lymphocytes is a parameter of the adaptive phase of
the immune response (Vos et al. 1979c). Some mitogens selectively stimu-
late the cellular immune (T-cell) components while others stimulate the
humoral immune (B-cell) components. Phytohemagglutinin (PHA) and
concanavalin A (Con A) are the T-cell mitogens of choice (Luster and
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83
Faith 1979), while lipopolysaccharides seem to stimulate primarily B-cells
(Smith 1972). On the other hand, pokeweed mitogen stimulates both B-
and T-cells and can be used to assay peripheral blood lymphocytes that do
not respond to other mitogens (Luster and Faith 1979). The response of
lymphoid cells to Con A and PHA is discussed in greater detail by Stobo
et al. (1972).
Thorpe and Knight (1974) describe a microplate culture technique for
lymphocyte transformation and define optimal conditions for performing
the assay. Essentially, various concentrations of mouse mesenteric lymph
node cells were cultured in microplate wells in the presence of mitogens.
The plates were incubated at 37°C in 5% CO2. Twenty-four hours prior to
harvesting, 3H-TdR was added to each culture. Cultures were usually har-
vested after 72 h and then processed for scintillation counting to measure
3H-TdR incorporation. An alternative test that measures mitogen- (and
alloantigen-) induced lymphocyte proliferation in a one-way mixed leuko-
cyte culture was described by Dean et al. (1979a), who advocate its use
for defining changes in host immunocompetence.
Examples of the use of mitogen stimulation in immunotoxicology stu-
dies are numerous. Mitogen stimulation is usually performed on mouse
lymphocytes, because Fischer rats, which are commonly used in toxicology
studies, respond poorly to the usual mitogens (Tada et al. 1974, as
reported in Luster and Faith 1979). The response to T-cell mitogens corre-
lates well with cell-mediated immunity and can be applied to cells that
have been exposed to chemicals in vivo or in vitro. Vos and Moore (1974)
tested the mitogen response of lymphoid cells from mice and rats that had
been exposed to TCDD at different ages. In rats whose mothers were
exposed during gestation and postnatally, the PHA response was reduced
on a cell-for-cell basis, but both the PHA and Con A responses recover-
able from the whole thymus were depressed. In mice treated with TCDD
at 1 month of age, reduced responsiveness of spleen cells to PHA was seen
only at a clearly toxic dose level; in 4-month-old mice PHA responsiveness
was not depressed. The thymus was the main organ affected by TCDD,
and other parameters of cell-mediated immunity were depressed in the
very young animals. The data suggest that TCDD-suppression of cell-
mediated immunity may be age related. In a later study, Sharma et al.
(1978) similarly reported that PHA and pokeweed mitogen responses in
mice and rabbits were suppressed with toxic levels of TCDD and, further-
more, that low levels of the chemical caused enhancement of immune
reponsiveness similar to that produced by antigenic substances.
A related chemical, 2,3,7,8-tetrachlorodibenzofuran (TCDF), also pro-
duced a moderate suppression of the lymphoproliferative response (to PHA
and lipopolysaccharides, but not to Con A) of splenic lymphocytes taken
from guinea pigs treated orally with the chemical (Luster et al. 1979).
Other industrial chemicals that have been shown to alter the mitogenic
response of lymphocytes include polybrominated biphenyl (Firemaster
FF-1), which decreased the responsiveness of spleen cells from rats and
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84
mice (Moore et al. 1978); dioxane, which suppressed mouse T-cell
responses while augmenting B-cell responses (Thurman et al. 1978); and
toluene di-isocyanate, which stimulated the response of both human and
mouse lymphocytes (Thurman et al. 1978).
Chronic exposure of mice to fresh cigarette smoke resulted in a marked
depression of the PHA response of lymphocytes taken from the regional
lymph nodes of the respiratory system and from the blood (Thomas et al.
1974a).
The sensitivity of the mitogen response to the effects of heavy metals
has been tested by several investigators. In one study, Gaworski and
Sharma (1978) observed depressed responses to PHA and pokeweed mito-
gen in splenic lymphocytes taken from mice fed high, but subtoxic, levels
of lead, cadmium, or mercury. Similar effects were observed in rat lym-
phocyte responses to PHA and Con A following chronic prenatal and post-
natal exposure to low levels of lead (Faith et al. 1979). However, in a
third study (Roller et al. 1979), lead and cadmium did not significantly
affect the response of mouse lymphocyte proliferation by Con A. Koller et
al. (1979) suggested that the conflicting results of the two mouse studies
could be due to variation in amount and length of exposure to the com-
pounds, strain differences, or differences in the response of lymphocyte
subpopulations to PHA and Con A.
Lymphocyte transformation is a semiquantitative and reproducible
technique, and the microplate method is practical and labor saving (Vos
1977; Luster and Faith 1979). The procedure has been adapted for use
with human cells in clinical laboratories to determine the immune status of
patients (Thurman et al. 1978). Luster and Faith (1979) point out, how-
ever, that it does not measure effector function nor does it represent a nor-
mal response.
Mixed Lymphocyte Culture. The mixed lymphocyte culture (MLC)
reaction is an in vitro analog of the allograft rejection response (Thurman
et al. 1978, Luster and Faith 1979). Similar to the mitogen stimulation
assay, lymphocytes in culture are normally stimulated by allogeneic lym-
phocytes to undergo blastogenesis, and lymphoproliferation is then meas-
ured by the amount of tritiated thymidine incorporated by the test cells.
The response, probably a measure of both B- and T-cell proliferation, is
stimulated by cell surface antigens and requires macrophages for the
response to be initiated (Thurman et al. 1978).
The MLC assay has not been used extensively in immunotoxicology
studies. However, the system was employed by Thurman et al. (1978) as
one of several parameters examined in the evaluation of the immunotoxi-
cologic effects of toluene diisocyanate, dioxane, and vinyl chloride in mice.
Their procedure was as follows: spleens were removed from untreated
CBA/J and C57B1/6J mice and cell suspensions were prepared; blasto-
genesis of these cells was blocked with mitomycin. For both control and
test groups, responding (unblocked) thymus, spleen, or lymph node cell
suspensions were prepared from CBA/J mice that had been treated with
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85
the different chemicals, and those cells were cultured in microtiter plates
for 72 or 96 h. Control cultures received 0.1 mL of syngeneic, blocked
CBA/J lymphocytes, and the test cultures received allogeneic, blocked
C57B1/6J lymphocytes. The cultures were pulse-labeled and prepared for
scintillation counting to measure lymphoproliferation of the unblocked
CBA/J cells.
The proliferation response of the spleen, thymus, and lymphoid cells
was generally inhibited by concentrations of 0.001 to 0.05% toluene diiso-
cyanate but apparently was stimulated by 0.1% toluene diisocyanate. The
response was lowered slightly by 10% dioxane and was not affected by
vinyl chloride (500 ppm for 7 h).
The assay is fairly simple to perform; spleen, thymus, and lymph node
cells can be tested; and treatment with chemicals can be in vitro as well as
in vivo, which qualifies the test for use with human cells. However,
Oehler et al. (1977) found that it is sometimes difficult to demonstrate an
MLC response in spleens from rats because of the presence of suppressor
macrophages. Therefore, it has been suggested that blood lymphocytes
from most species can be substituted for spleen lymphocytes, following
Hypaque-Ficol density gradient centrifugation (Luster and Faith 1979).
Macrophage Migration Inhibition Factor (MIF). Bloom and Bennett
(1966) observed that sensitized peritoneal lymphocytes, upon interaction
with specific antigen in vitro, elaborated into the medium a soluble
material that could inhibit migration of normal exudate cells. In their
experiment, peritoneal macrophages, from guinea pigs sensitized to tuber-
culin with Freund's adjuvant, were drawn into capillary tubes and centri-
fuged. The capillary tubes were cut at the cell-medium interface and were
placed in special chambers containing various supernatants from sensitized
peritoneal lymphocyte cultures. Macrophages, exposed to the supernatants
from cultures of lymphocytes growing in medium alone, migrated out of
the tube onto a specially placed coverslip. Macrophages that were exposed
to the supernatant from cultures of lymphocytes growing in purified pro-
tein derivative (PPD) did not migrate. The soluble material elaborated into
the supernatant by the PPD-stimulated lymphocytes has been identified as
the lymphokine MIF. This type of cellular interaction can be detected in
experiments typified by the following (David and David 1971):
1. In the simplest migration assay, sensitized peritoneal exudate cells
(macrophages and lymphocytes) are assayed in a capillary tube
against the appropriate antigen.
2. In another assay, lymphocytes from lymph nodes or other organs are
mixed with normal peritoneal exudate cells in order to detect sensitive
cells in the various organs.
3. In the "indirect" assay, lymphocytes are incubated with antigen, and
the cell-free supernatants are assayed for MIF activity using normal
peritoneal exudate cells.
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86
The in vitro migration system has been used primarily in guinea pigs,
but also in mice, rats, hamsters, and monkeys (David and David 1971);
and it has been adapted to the assay of human cells (Thor 1971).
The principle of this reaction has been applied to the measurement of
the effector stage of cell-mediated immunity in immunotoxicological stu-
dies (Luster and Faith 1979). For example, Luster et al. (1979) used an in
vitro MIF assay to demonstrate a modest, but dose-related suppression of
cell-mediated immunity in TCDF-exposed lymphocytes. These results were
in agreement with the mitogen stimulation assay, another in vitro test of
cell-mediated immunity.
The migration assay is semiquantitative and not extremely expensive;
however, it does require attention to technical detail (Luster and Faith
1979, Morley et al. 1978). Assay (1), above, is thought to be more sensi-
tive than other in vitro methods of assessing delayed hypersensitivity pro-
vided high concentrations of antigens are used (Carter and Bazin 1980).
Macrophage Aggregation. Macrophage aggregation, another in vitro
correlate of DTH, may become more widely used than MIF tests because
of its relative simplicity (Crowle 1975).
During investigations of the interaction of antigen and sensitive lym-
phoid populations, aggregation of sensitive peritoneal exudate cells was
observed in tubes in the presence of antigen. Lolekha et al. (1970) studied
this reaction using BCG and egg albumin as antigens. Guinea pigs were
immunized via footpad injections, and the test was performed 4 to 8 weeks
following BCG immunization and 10 to 14 days after egg albumin. The
animals were injected intraperitoneally with light mineral oil and 5 days
later were exsanguinated. Peritoneal exudate cells were removed by
conventional methods and were washed, counted, and resuspended in
sterile tubes containing PPD or egg albumin. After 24 h at 37°C and 5%
CC>2 the tubes were shaken to resuspend the cells for observation of aggre-
gates. The reaction was antigen-specific, and it was also found that super-
natant fluids from lymphoid cells cultured from antigen-sensitive animals
caused aggregation of nonsensitive peritoneal exudate cells in the presence
of antigen. The investigators concluded that exposure to antigen leads to
the release of a macrophage aggregation factor (MAP) from sensitive lym-
phoid cells in culture.
The soluble material MAP, which causes aggregation of peritoneal
exudate cells may be the same as MIF (Lolekha et al. 1970). The MAP
assay is simpler and less time consuming than the MIF assay (Lolekha et
al. 1970, Crowle 1975) and lends itself to semiquantitation by routine
titration.
No examples were found for the use of this test in immunotoxicology
studies.
Leukocyte Adherence Inhibition (LAI). This assay, originally described
by Halliday and Miller (1972), measures the release of a diffusable factor
from sensitized T-lymphocytes following contact with antigen. The factor,
which inhibits the adherence of leukocytes to glass or plastic surfaces, has
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87
been used to assess cell-mediated immunity. A modified microtest, adapted
from Halliday and Miller by Holt et al. (1974), was used in the following
study to detect immunological effects of cigarette smoke.
In this study (Holt et al. 1976) mice were exposed to high- or low-tar
cigarette smoke for 36 or 31 weeks, respectively, then inoculated with
BCG. Purified protein derivative of BCG was dispensed into microtest tis-
sue culture plates, and splenic lymphocytes from the BCG-treated mice
were added to the plates. After a 2-h incubation period the plates were
washed and stained, and adherent cells were counted. The numbers of
adherent cells in the wells containing antigen were compared with the
numbers in wells containing no antigen. Exposure to high- and low-tar
cigarette smoke resulted in suppression of the LAI, indicating suppression
of cell-mediated immunity.
The reaction of LAI is similar to that of macrophage migration inhibi-
tion, but the procedure is much simpler to perform (Halliday and Miller
1972).
3.2.2 Assessment of Humoral Immunity
Humoral immunity is defined as that specific immunity which is medi-
ated by antibodies (immunoglobulins) present in the plasma, lymph, and
tissue fluids of the body and which may also become attached to cells
(Herbert and Wilkinson 1971). The cells ultimately responsible for the
secretion of antibody are the B- (bursa-derived; bone marrow-derived)
lymphocytes. When stimulated by antigen, B-cells, specific for that
antigen, either proliferate and develop into plasma cells (antibody-secreting
cells) or serve as memory cells.
The introduction of antigen is usually followed by a lag phase when no
antibody can be detected in the circulation (Park and Good 1974). During
this period (the first 24 to 48 h after antigen) the following occur:
antigen-processing by macrophages, active synthesis of mRNA and rRNA,
transformation of the lymphocytes, and proliferation of these cells, which
differentiate into antibody producers. Morphologic changes occur in the
red pulp or at the edges of the white pulp of the spleen or in the medullary
cords or zones surrounding the germinal centers in the cortical areas of the
lymph nodes.
The lag phase is followed by a logarithmic phase during which the rate
of production of 19S IgM antibody, the first detected, follows a loga-
rithmic curve. After IgM production, 7S IgG antibody appears and enters
the log phase. In a typical antibody response the plateau of antibody pro-
duction is reached about 8 days after introduction of antigen, and the
decay period which then follows is short. The primary response is weak,
sluggish, and short lived.
In contrast to the primary response, the secondary response (antibody
production after the second immunization) is characterized by faster
development and higher levels of antibody titers and a predominance of
IgG early in the response.
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88
It has been determined (Robertson 1981) that immunoglobulin M is
synthesized by the B-cells at early stages of maturation and that IgG, IgA,
or IgE are synthesized at later stages.
For most antigens, cooperation between both T- and B-cells is neces-
sary for the induction of antibody-formation (this is the T-cell-dependent
humoral response) (Claman et al. 1966, Miller and Mitchell 1968). How-
ever, certain antigens, such as type III pneumococcal polysaccharide and
E. coli lipopolysaccharide, can stimulate a good immune response in
athymic animals (e.g., nude mice) (this is the T-cell-independent humoral
response) (Moller and Michael 1971, Andersson and Blomgren 1971).
Tests of humoral immunity include those which measure circulating anti-
body or local production of antibody to both thymus-dependent and
thymus-independent antigen; those which identify and quantitate the vari-
ous immunoglobulins; and those which assess the colony-forming and blas-
togenic capabilities of B-cells. Although numerous useful tests (and
modifications of these tests) have been developed for the purpose of
evaluating the humoral immune response, the following discussion will be
limited to those which, because of convenience, speed, sensitivity, etc.,
appear to be likely candidates for inclusion in toxicity-screening protocols.
3.2.2.1 Assays for local production of antibody
Many methods have been developed for the enumeration of individual
cells producing antibody. Dresser (1978) defined three approaches to the
study of these cells with respect to localization of antibody at various sites:
(1) detection of antibody as it is secreted from the cell; (2) detection of
intracellular antibody; and (3) detection of antibody on the cell surface.
Tests designed to identify antibody at these sites include plaque-forming
assays (localized hemolysis in gel), immunofluorescence techniques, and
the "erythrocyte-antibody-complement" (EAC) rosette assay, respectively.
Plaque-forming assays (localized hemolysis in gel). Jerne and Nordin
(1963) first described this method for enumerating antibody-producing
cells, and it has become one of the most widely used tests in experimental
immunology.
According to the original assay, cells are obtained from the spleen or
other lymphoid organs of an animal that has been immunized against
SRBC. These cells are mixed with SRBC and molten agar, the mixture is
poured into a petri dish and allowed to set, and the dish is incubated at
37°C. After incubation the release by each cell of hemolysins (antibodies
capable of lysing red blood cells in the presence of complement) is revealed
with the addition of complement to the dish. A clear zone of hemolysis
appears around each antibody-forming cell.
The technical aspects of the plaque-forming cell assay have been
described in detail by Dresser (1978).
The PFC technique of Jerne and Nordin (1963) detected only those
cells producing antibody, primarily IgM, which could fix complement and
lyse cells directly. These cells are referred to as direct plaque-forming
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89
cells. Other classes of immune-globulins (IgG, IgA) are either secreted in
insufficient amounts or are incapable of fixing complement, unless they are
complexed with antiglobulin antibody. Cells that produce these antibodies
are referred to as indirect plaque-forming cells and are revealed when
developing serum is added to the plates. The PFC technique can be used
to measure both primary and secondary responses.
Modifications of the original technique have been introduced that are
more convenient, sensitive, and economical than the original procedure.
Methods have been described that utilize agarose instead of agar as the
support medium (Plotz et al. 1968, Yamada and Yamada 1969, Duke and
Harshman 1971, Urso and Gengozian 1973) or that use no support
medium at all (Cunningham and Szenberg 1968, Kennedy and Axelrad
1971). Some of these procedures employ microscope slides instead of petri
dishes (Plotz et al. 1968, Urso and Gengozian 1973). Three of these assays
were compared for sensitivity by Peterman and Wust (1975), who also
defined optimal conditions for the assay by testing spleen cells from rats
immunized with sheep or mouse erythrocytes in combination with human
or guinea pig complement. They found that the most sensitive system was
one using SRBC, no agar or agarose, and either human or guinea pig com-
plement.
Other modifications of particular importance to immunotoxicology
have been introduced by: (1) Mishell and Dutton (1967), who performed
the plaque-forming cell assay following in vitro immunization of normal
spleen cells; (2) Archer et al. (1978), who used the Mishell-Dutton modifi-
cation to develop an assay for the in vitro immunosuppresive effects of
water soluble and insoluble chemicals; and (3) Tucker et al. (1982), who
adapted the Mishell-Dutton assay for testing chemicals that require meta-
bolic activation.
The plaque-forming cell assay can be performed with a variety of
antigens such as polysaccharides, synthetic polymers, proteins, and hap-
tens, which can be bound covalently to indicator erythrocytes (Peterman
and Wust 1975). Thus, both T-cell dependent and T-cell independent
antigens can be tested with this procedure. Protein antigens are particu-
larly recommended when Fischer rats or guinea pigs are used because
these species do not respond well to SRBC (Luster and Faith 1979).
The plaque-forming cell assay and its modifications have been used fre-
quently in immunotoxicology studies. It has been shown to be a sensitive
indicator of alterations in the immune response. Table 3.4 lists examples of
the plaque-forming cell response in experimental animals following expo-
sure to environmental pollutants.
It is noteworthy that certain of these chemicals were reported to pro-
duce suppression of the plaque-forming cell response at dose levels which
produce no signs of overt clinical illness [i.e., hexachlorobenzene,
polychlorinated biphenyl (Loose et al. 1977, 1978, 1979), polybrominated
biphenyl (Fraker 1980), cadmium chloride (Roller et al. 1975)]. In addi-
tion, the plaque-forming cell response was depressed by nickel chloride at
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90
levels falling below the threshold limit value for humans (Graham et al.
1978).
The plaque-forming cell response was suppressed by all the chemicals
listed in Table 3.4 except for nickel oxide (Graham et al. 1978) and the
platinum ethylene diamines (Berenbaum 1971). The lack of effect of
nickel oxide could be attributed to insolubility of the compound; lack of
effect of the platinum ethylene diamines could be attributed to their chem-
ical nature because other platinum diamines were highly toxic to mice at
the doses given.
The advantages of the plaque-forming cell assay include the following:
(1) it is sensitive to the immunosuppressive effects of industrial chemicals;
(2) it is versatile and convenient and can be performed with a variety of
antigens; (3) it lends itself well to the analysis of multiple samples; and
(4) the rate of antibody secretion can be determined mathematically by
measuring the direct plaque size (Luster and Faith 1979).
Immunofluorescence. Antigens and antibodies can be made fluorescent
by chemically binding them to a fluorescent dye, most often isocyanate or
isothiocyanate of fluorescein (Gell and Coombs 1975). This fluorochrome
labeling procedure can provide rapid, accurate localization of the site of
antigen-antibody interaction when one of the reactants forms part of a
cell, tissue, or other biological structure.
Fluorochromes are substances that will absorb radiation (ultraviolet
light) and become excited. The excited molecules emit observable radiation
which ceases almost immediately after the exciting radiation is withdrawn.
There are several approaches to fluorescent labeling. The basic pro-
cedures for these have been described in detail by Johnson and Holborow
(1973). These microscopic methods are especially applicable to localization
of 7-globulin and specific antibody. 7-Globulin in cells can be demon-
strated with a fluorescent conjugate prepared from antiglobulin serum
(direct method) while specific antibody can be shown with the "sandwich"
(or indirect) technique, where the antibody-containing cells are first
treated with the antigen and then free valencies of the antigen are used to
combine a fluorescent specific antiserum (Figure 3.2).
The application of immunofluorescence in the detection of cellular anti-
bodies using the "sandwich" technique involves these basic procedures
(Johnson and Holborow 1973):
1. The preparation of the substrate material (cells or tissue) for micros-
copic examination. (Care must be taken to avoid fixatives that destroy
reactivity of immunoglobulins as antibodies. Cryostat or liquid nitro-
gen methods may be used for tissue sections; dried smears may be
used for cell suspensions.)
2. The preparation and immunological characterization of the fluores-
cent reagents.
3. Staining with the fluorochrome dye. (Care must be taken to keep the
slides moist and to remove nonspecifically bound reactants.)
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91
Table 3.4 PFC Response in Experimental Animals Following
Exposure to Environmental Pollutants
Chemical
Hexachlorobenzene
Polychlorinated biphenyl
Polybrominated biphenyl mixture
Trichloroethylene
Cigarette smoke
Benzo[a]pyrene
Carbon and sulfur dioxide
Sulfur Dioxide
Carbon
Carbon dust
Cadmium chloride
Chromium chloride
Nickel chloride
Nickel sulfate
Nickel oxide
Platinum diamine dichloride
Platinum diamine tetrachloride
Platinum ethylene diamines
Methyl mercury
Lead
Species
Mouse
Mouse
Mouse
Mouse, rat
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse"
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Rat
Mouse
"Some doses produced immunosuppression without
bExposed in utero.
"Doses below threshold limit value.
PFC
Response
Suppressed
Suppressed
Suppressed
No effect
Suppressed
Suppressed
Suppressed
Suppressed
Suppressed
Suppressed
Suppressed
Suppressed
Suppressed
Suppressed
Suppressed
Suppressed
No effect
Suppressed
Suppressed
Suppressed
No effect
Suppressed
Suppressed
No effect
Suppressed
Suppressed
Suppressed
Suppressed
signs of overt
Reference
Loose et al. 1977'
Loose et al. 1978"
Loose et al. 1979"
Moore et al. 1978
Fraker 1980"
Sanders et al. 1982
Thomas et al. 1973
Nulsen et al. 1974
Thomas et al. 1974b
Urso and Gengozian 1980
Zarkower 1972
Zarkower 1972
Zarkower 1972
Zarkower and Merges 1972
Koller et al. 1975"
Graham et al. 1978
Graham et al. 1978
Graham et al. 1975
Graham et al. 1978C
Graham et al. 1978
Graham et al. 1978
Berenbaum 1971
Berenbaum 1971
Berenbaum 1971
Koller et al. 1977
Ohi et al. 1976
Luster et al. 1978
Koller and Kovacic 1974
clinical illness.
4. Microscopy under conditions suitable for observing fluorescence. (To
achieve maximal fluorescence, a careful choice of filters is necessary,
based on the excitation and emission characteristics of the fluoro-
chrome in question.)
Fluorescent techniques have been used successfully in immunotoxicol-
ogy studies, and their versatility is illustrated by the following examples.
Verschuuren et al. (1970) used a fluorescent-antibody technique to
demonstrate a decrease in immunologically active cells in the popliteal
lymph nodes of guinea pigs which were fed triphenyltin acetate (TPTA)
for 90 days and then injected with tetanus toxoid. The popliteal lymph
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92
ORNL-DWG 85-10740
Direct fluorescent antibody test
(for nuclear antigen and antibody)
Nucleated cells
on slide
Treated with
fluorescent
nuclear antibody
Washed
M^MMMMt
JH AiA
Key
A Fluorescent nuclear antibody
| Fluorescent globulin
V Antibody in ceil to egg albumin
O Egg albumin
1 Fluorescent antibody to egg albumin
Sandwich test
(for specific onhbody- producing cells)
Single cell
producing antibody
( V ) t° egg albumin
Treated with
specific antigen
O (egg albumin)
Washed
Treated with
fluorescent
antibody^'
to egg albumin
Washed
rAsassf^
i — iftft — i
r±Vt&^
r=>§Ki 1
Figure 3.2. Direct fluorescent antibody test and "sandwich test" for
staining antibody-producing cells. (Source: Gell and Coombs 1975. Used
with permission of Blackwell Scientific Publications, Ltd.)
nodes were subsequently removed and quick-frozen. For detection of all
gamma-globulin-containing cells, the cryostat sections were treated with
fluoresceinated rabbit anti-guinea pig gamma globulin. For the specific
detection of tetanus antitoxin-producing cells, serial sections were treated
first with tetanus toxoid, then with rabbit tetanus antitoxin serum, and
finally with fluoresceinated goat anti-rabbit serum.
In a later study, Vos and de Roij (1972) used a similar technique to
determine the effect of PCB on gamma globulin synthesis in popliteal
lymph nodes of guinea pigs stimulated with tetanus toxoid. Photographs
were taken of the fluorescein-labeled tissue sections using a fluorescence
microscope, and the photographs were ranged in order of increasing
number of 7-globulin-containing cells to make statistical evaluation possi-
ble. The investigators made the observation that this technique is a sensi-
tive parameter for the detection of immunosuppressive activity of PCB in
tetanus toxoid-stimulated animals.
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93
Bozelka et al. (1977) used a direct immunofluorescence method to elu-
cidate the mechanism of cadmium-induced splenomegaly in mice. Follow-
ing SRBC injection, increased numbers of cells labeled with goat
antimouse IgM and IgG were observed in both the white and red pulp of
the enlarged spleens of cadmium-intoxicated animals. This led the authors
to conclude that the B lymphocyte was responsible for the hyperplastic
changes induced by cadmium.
Fluorescent techniques require special equipment, which may be expen-
sive. Also, unwanted fluorescein staining may occur with both the direct
and indirect methods. However, the procedure is rapid and specific and
can be performed on tissue sections so that antibody-producing cells can be
observed in situ. In toxicity testing the procedure would be especially valu-
able in answering specific questions regarding antibody synthesis once
immunotoxicologic effects have been established.
3.2.2.2 Measurement of circulating antibody and immunoglobulins
The production and secretion of antibody is a highly specific response
of plasma cells that have been exposed to antigen. Analysis of serum anti-
body provides a direct and quantitative measurement of the production of
specific antibody by these cells. Most procedures require the interaction, in
vitro, of antigen and antibody; these procedures can be applied to measure-
ment of the primary and secondary responses to both thymus-dependent
and thymus-independent antigens. These procedures, such as routine
hemagglutination and hemolysin titrations, are simple to perform and are
widely used. More sophisticated methods, such as radial immunodiffusion
and the enzyme-linked immunosorbent assay, have been applied to the
identification and quantitation of the individual immunoglobulins present
in the antisera.
Several of these procedures, sensitive to alterations in humoral immune
functions, will be described in the following sections.
Precipitation. When soluble antigen is mixed with antiserum specific
for that antigen a precipitate is formed in a few minutes that can be
observed and measured (Gell and Coombs 1975). The interfacial or ring
technique, a qualitative test for precipitating antibodies, was developed by
Ascoli in 1902 (as cited in Gell and Coombs 1975): A drop or two of
antigen is layered above a similar volume of undiluted antiserum, and in a
positive reaction a ring of precipitate forms at the interface. This type of
test is more sensitive for the detection of antigen than for antibody.
Precipitation techniques have been standardized and quantitated. A
sensitive and reproducible method for the detection of antibody, which can
be completed in less than 2 h, was described by Raney and McLennan
(1979). To test their procedure, samples of rabbit antisera produced
against a nucleoside-protein conjugate were added to tubes containing
increasing amounts of this antigen in a buffered solution of 2%
polyethylene glycol. The tubes were incubated at 37°C for 30 or 60 min
and stored at 4°C for 15 minutes to 1 week with daily resuspension of the
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94
precipitates. The precipitate was washed and dried, and the protein content
was determined at various time points. These results were compared with
those of duplicate samples that were assayed by the standard procedure
that does not incorporate the use of polyethylene glycol. In comparison
with the standard procedure, the method of Raney and McLennan requires
less time for complete precipitation (2 h vs 2 to 10 days); it is also more
sensitive and more reliable.
Another precipitation technique, gel diffusion, has become widely used
for the qualitative detection of antibodies to soluble antigens, and, though
slightly slower than the ring test, it is more versatile. In single diffusion
one reagent is incorporated in the supporting gel while the other, in liquid
form, diffuses into it.
A quantitative modification of this procedure, single radial diffusion
(SRID). was developed by Mancini et al. (1965). The technique, useful for
quantitative determination of different immunoglobulins in serum, is per-
formed as follows (Ouchterlony and Nillson 1973): a melted 3% agar solu-
tion is mixed with an equal volume of antiserum that has been properly
diluted. The agar mixture is then poured as a 1-mm thick layer onto a
glass plate. Holes 2 mm wide are punched in the gel and filled with accu-
rately measured volumes of the antigen solutions to be tested along with
references of known concentration of antigen. The plate is incubated in a
humid atmosphere until the resulting precipitate becomes stationary. The
area of the halo of precipitate is determined. Quantitation is possible since
the area of the halo is directly proportional to the concentration of
antigen. The "reversed" SRID technique allows quantitative determination
of specific antibody when the antigen constitutes the internal reactant and
the antiserum to be tested constitutes the external reactant.
The SRID technique is not very sensitive but can be used satisfactorily
when high antibody titers are present, as in the secondary response to an
antigen (Vos 1977).
Immunodiffusion methods have been employed in immunotoxicology
studies for the assessment of antibody formation and for the measurement
of immunoglobulins. Vos et al. (1973) measured serum tetanus-antitoxin
levels in immunized guinea pigs using a RID procedure in which the
tetanus toxoid was incorporated into the agar and the antiserum was
allowed to diffuse out from the well. A slight suppression of the humoral
immune response by sublethal doses of TCDD was detected with this
method.
In a similar study, Luster et al. (1979), using a RID assay, quantified
serum IgG in the serum of guinea pigs that had been exposed to TCDF. A
minimal depression in serum IgG was observed, which correlated with
antibody production to bovine gamma globulin.
The RID assay was used by Loose et al. (1979) to measure serum IgG,
IgA, and IgM immunoglobulin concentrations in mice that had been
treated with polychlorinated biphenyls or hexachlorobenzene, then immun-
ized with SRBC. There was a reduction in serum IgA, IgG, and IgM that
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95
was concomitant with a decrease in the spleen plaque formation response.
In the response to a secondary challenge only IgA was depressed. The
chemicals were, however, administered at subtoxic levels.
Precipitation methods are simple and convenient, especially since com-
mercial plates have been introduced. There seems to be a good correlation
between antibody and Ig levels measured by SRID and antibody produc-
tion measured by agglutination or plaque formation.
Hemagglutination and hemolysis. The direct agglutination test is
another classical serological reaction which involves, simply, the clumping
of a cell suspension by specific antibody (Gell and Coombs 1975). The test
is performed by mixing dilutions of antiserum with the cell suspension and
observing agglutination of the cells. Antibody combines rapidly with
antigen, but no agglutination occurs until the cells come in contact with
each other. Early agglutination tests were performed in tubes. A more effi-
cient micromethod of the test was described by Stavitsky (1954) for use
with tannic acid and protein-treated cells (see below); the test is also quite
useful with untreated red blood cells. A modification of the method, one of
several procedures recommended by White et al. (in press) for assessing
the effects of toxicants or immunocompetence, will be outlined below. By
adding complement to the system, the hemagglutinin reaction can be con-
verted to a hemolytic reaction (Vos 1977). There is generally good correla-
tion between the hemagglutination and hemolysis titrations.
In the method described by White et al. (unpublished observations),
mice are exposed to the test chemical for the desired length of time and on
day 0 are immunized intraperitoneally with 1 x 109 SRBC. On day 7,
blood is collected in an anticoagulant (sodium citrate), and following cen-
trifugation an aliquot of the plasma is removed and heat inactivated at
56°C for 30 minutes. Serial 1:1 dilutions are made in phosphate-buffered
saline in conical microtiter plates to a final volume of 0.1 mL per well.
Eight samples can be diluted at once using a semiautomatic technique.
One-tenth milliliter of a 0.5% SRBC suspension is added to each well, and
the plates are covered and placed in a 37°C humidified incubator. After 2
h the plates are observed for agglutination using a magnifying mirror. The
end point is the serum dilution at which no agglutination occurs. (The titer
is expressed as the reciprocal of the highest dilution that gives a positive
reaction.) The secondary antibody response can also be tested in this way,
and estimation of the proportions of the IgM and IgG antibodies can be
achieved by degradation of IgM with 2-mercaptoethanol.
The sheep erythrocyte hemagglutination and/or hemolysin techniques
have been used frequently to assess the humoral immune response follow-
ing exposure of experimental animals to chemicals, such as tri-
chloroethylene in mice (Sanders et al. 1982); PCB in monkeys (Thomas
and Hinsdill 1978), carbon dust in mice (Zarkower and Morges 1972),
and cigarette smoke in mice (Esber et al. 1973; Nulsen et al. 1974, Holt et
al. 1976).
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96
Sanders et al. (1982) detected slight suppression in the antibody
response to SRBC following exposure of mice to subtoxic levels of tri-
chloroethylene. Also, Thomas and Hinsdill (1978) observed suppression of
the SRBC, but not the tetanus typhoid, antibody response of monkeys
after exposure to PCB. They speculated that the SRBC response is more
sensitive to damage than the tetanus toxoid response.
The hemagglutination and hemolysin assays are thus sensitive, easy to
perform, economical, and quantitative. They can be used to titrate primary
and secondary antibody responses and to measure the relative amounts of
IgG and IgM produced in response to an antigen.
Passive hemagglutination. The chief difference in the precipitation reac-
tion and the hemagglutination reaction is that in the former case the
antigen molecules are free in solution and in the latter they are fixed on
the surface (membrane) of the cell (Coombs and Gell 1975). Agglutina-
tion is far more sensitive than precipitation in detecting the antigen-
antibody reaction (Herbert 1973, Coombs and Gell 1975). Boyden (1951)
thus developed a technique for the adsorption of various proteins on the
surface of tannic acid-treated (tanned cell technique) SRBC and demon-
strated the agglutination of such cells by highly diluted specific antisera.
The term "passive" agglutination is used because the red cells act as
passive carriers of the protein antigens. Stavitsky (1954) adapted the
technique to the titration of antitoxin and other protein sera using the
microtiter assay previously discussed.
Technical details for the preparation of the antigen-coated cells for use
in agglutination procedures have been outlined by Herbert (1973). These
will be briefly summarized here:
Choice of Cells Although it would seem logical to use cells of the
same species for agglutination as that from which the antiserum is derived,
most investigators use SRBC or human O cells. Both of these are fairly
easy to obtain and have been satisfactory in the tanned cell technique,
human cells being the more sensitive of the two. The cells should be fresh.
Preservation of Cells Unless the fresh cells can be used right away
they must be preserved (or fixed). This can be done before or after coating
but usually takes place prior to coating because fixation may alter the
coating antigen. The most commonly used fixative is formalin. Preserved
cells are, however, considerably less sensitive than fresh ones.
Adsorption of Antigen by Red Cells Coating of the red cells with
antigen may be accomplished in three ways: by direct adsorption of
antigen by the cells, by the tanned cell technique, and by chemical bond-
ing of antigen to the cell.
Direct Adsorption Direct coating of fresh cells can be done by simply
incubating the antigen and cells at 37°C for approximately 2 h. Such cells
are used in both hemagglutination and, with the addition of complement,
hemolytic tests. These cells are, however, very insensitive to antibody in
comparison to tanned cells. Preserved (fixed) cells can also be coated this
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97
way but with a longer incubation period, with constant mixing, and at the
pH that is appropriate for the particular antigen.
Certain haptens can also become directly attached to red blood cells,
and hemagglutination tests with such cells appear to be quite sensitive.
Tanned Cell Technique It is generally assumed that tannic acid acts
on red cells in such a way to cause them to take up protein antigens, but
Herbert (1973) points out that the chief function may well be to increase
the instability of the cells, making a normally nonagglutinating reaction
result in agglutination. Cells that have been treated with tannic acid will
show nonspecific agglutination settling patterns, but the tendency to agglu-
tinate may be balanced by adding normal serum as a stabilizer. The
coated cells are thus highly sensitive to agglutination, in the presence of a
very small amount of antibody.
The tanning procedure itself is easy to perform and involves simply
incubating the fresh or fixed cells with tannic acid in buffer for a brief
period of time. The cells can then be stored for subsequent coating with
antigen, which can be accomplished by incubating the cells and antigen at
room temperature for a very short time. During the tanning and coating
procedures the cells must be carefully washed at each step, the pH must
be carefully monitored, and optimal concentrations of the reactants must
be used. The cells thus prepared are ready for use in conventional titration
procedures.
Attachment of Antigen to Red Cells by Use of an Intermediate Com-
pound Bis-diazotized benzidine is a suitable chemical for use in this pro-
cedure. Coupling the antigen to the cells is accomplished by simply incu-
bating cells (usually formalinized), bis-diazotized benzidine, and antigen
together for a short time and then washing the sensitized cells. Other
chemicals recommended for this include chromic chloride, difluorodinitro-
benzene, and carbodiimide.
Passive hemagglutination techniques have been employed to test the
humoral response to the following: (1) E. coli lipopolysaccharide following
exposure of rats to lead acetate (Luster et al. 1978) and of mice to carbon
(Zarkower 1972; Zarkower and Morges 1972); (2) human gamma globulin
following exposure of rats to cadmium chloride (Jones et al. 1971);
(3) diphtheria toxoid following exposure of guinea pigs to DDT (Gabliks
et al. 1973); (4) Brucella abortus antigen following exposure of mice to
methyl mercury (Spyker 1975); and (5) tetanus toxoid following exposure
of monkeys to PCB (Thomas and Hinsdill 1978).
Luster et al. (1978) tested the humoral immune response to both sheep
erythrocytes (SRBC), a thymus-dependent antigen, and E. coli
lipopolysaccharide, a thymus-independent antigen, in mice exposed to lead
acetate. The SRBC response was suppressed by lead while the lipopolysac-
charide response was not, indicating an alteration of the T-, rather than
the B-, lymphocyte population.
The reciprocal effect was demonstrated in the study reported by
Spyker (1975). Mice were exposed to methyl mercury in utero and were
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challenged 14 months later with Brucella abortus antigen, a thymus-
independent antigen, and SRBC. In this case B-cell function was impaired
while T-cell function was unaffected. (No impairment was observed in the
response to either antigen when mice were challenged at 4 months of age,
indicating that impaired immune function may be a delayed effect of pre-
natal exposure to methyl mercury.)
In summary, agglutination methods are objective, quantitative, sensi-
tive, and versatile. Passive hemagglutination techniques are a bit compli-
cated, but they are valuable tools for the study of responses to soluble
antigens. Micromethods simplify the procedures and allow the use of small
quantities of reagents, thereby lowering the cost of the tests.
Enzyme-linked immunosorbent assay. Another test that, similar to the
radial immunodiffusion technique, has the capability for the measurement
of both antigen and antibody in sera is the enzyme-linked immunosorbent
assay (ELISA), which was introduced by Engvall and Perlmann in 1971.
The technique was adapted by Vos et al. (1979a, 1979b, 1979c) for use in
studies on the humoral immune response of rats. The following "sandwich"
ELISA was used by them for the measurement of rat IgG and IgM:
Polystyrene tubes were coated (by rotation for 18 h) with antimmuno-
globulin solution; test and reference serum were then diluted and added to
the appropriate tube and the tubes were incubated under rotation for 1 h.
After washing, 1 mL of a conjugate of horseradish peroxidase and antiim-
munoglobulin (IgG or IgM) was added. The tubes were then incubated
under rotation for 2 h. Excess conjugate was removed, and 1 mL of
specific hydrogen peroxide substrate was added. Extinction was measured
at 490 nm following incubation for one hour at room temperature. Vos et
al. (1979a) reported that the measurements of IgM and IgG correlated
well with results obtained by SRID. ELISA is less time-consuming than
the SRID technique, and the macro-ELISA used here required less serum.
Vos et al. (1979a) also utilized ELISA for determining antibody titers
to E. coll lipopolysaccharide and tetanus toxoid. The method performed
and described by them was that of Ruitenberg et al. (1975):
Polystyrene microplates were coated with the antigens for 2 h at 37°C
(LPS did not attach well and special procedures were required here). The
microplate trays were incubated with 100 pL of test or control serum dilu-
tions for 1 h at 37°C. After washing, 100 juL of the horseradish
peroxidase-antiimmunoglobulin conjugate was added, and the microplates
were incubated and rotated for 2 h. The plates were washed and substrate
was added. After incubation for 1 h at room temperature the reaction pro-
duct was evaluated visually and expressed as 2log of the highest dilution
giving a positive reaction. The ELISA results were compared with those
obtained with passive hemagglutination, and ELISA was found to be more
sensitive except when secondary response titers to tetanus toxoid were
compared.
Vos et al. (1979b, 1979c) included the ELISA with routine studies to
assess immune function in rats exposed prenatally and postnatally, or as
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adults, to hexachlorobenzene. The procedure appeared to provide a satis-
factory assessment of the humoral immune response in the rats.
According to Vos et al. (1979a) and Roller (1982) the procedure is a
reliable, sensitive, specific, and economic method for detecting IgM and
IgG antibodies and antigens. It has the potential for large-scale screening
because mechanization of all steps of the assay is feasible. In the labora-
tory of Roller (1982), ELISA has been accepted as the test procedure of
choice for assessing humoral immunity in animals exposed to chemicals.
3.2.2.3 Other tests of B-ceU function
B-cell function can be evaluated by procedures that measure parame-
ters other than direct antibody formation. Two of these, the EAC rosette
technique and transformation by B-cell mitogens, are discussed.
EAC rosette technique. There is a population of lymphoid cells that is
capable of binding "erythrocyte-antibody-complement" (EAC) to form
rosettes. This is possible because B-cell membranes possess a receptor for
the third component of complement (C3).
Erythrocytes are prepared for this test by first reacting them with rab-
bit antierythrocyte antibody (forming the complex EA) (Bianco et al.
1970). The EA complex can then be treated either with serum from mice
genetically deficient in C3 or with purified human complement com-
ponents. The complex thus formed by antibodies, erythrocytes, and com-
plement is the EAC complex.
Roller and Brauner (1977) applied the EAC rosette assay to the
enumeration of B-cells from mice that had been exposed to lead acetate or
cadmium chloride. In their experiment, spleen lymphoid cells were incu-
bated with EAC for 5 min at 37°C; the cells were then centrifuged for 5
min and incubated on ice for 1 to 2 h. After gentle resuspension, the cells
were stained with crystal violet and examined microscopically in a hema-
cytometer. Cells with three or more bound erythrocytes were counted as
EAC rosettes. Fewer rosettes were formed by the B-cells from mice
exposed to the chemicals than by B-cells from control mice. Cell viability
tests indicated that this effect was not due to toxicity of the lead and cad-
mium.
The rosette test is a simple assay for the enumeration of B-cells and is
one of the tests routinely employed by Luster and Faith (1979) for the
detection of alterations in the humoral immune response.
Transformation of lymphocytes by B-ceU mitogens. The technical
aspects of the mitogen-stimulated lymphocyte transformation assay have
been discussed in Section 3.2.1.2. The procedure is the same for both T-
and B-cell transformation with the exception of the mitogens used. Bac-
terial lipopolysaccharides (LPS) are generally used to stimulate the mito-
gen response of B-cells, while concanavalin A and phytohemagglutinin are
primarily T-cell mitogens. Pokeweed mitogen, which stimulates both T-
and B-cells, is also used frequently.
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The B-cell response has been shown to be sensitive to the immu-
nosuppressive effects of metals (Gaworski and Sharma 1978, Koller et al.
1979), TCDD (Sharma et al. 1978), and TCDF (Luster et al. 1979), but
the response has been either enhanced or unaffected by hexachlorobenzene
(Vos et al. 1979b, I979c).
3.2.3 Assessment of Indirect Parameters of Immunity
A number of indirect factors must be considered when evaluating the
immune response. Indirect parameters commonly assessed include macro-
phage functions, hormones, complement, host resistance to infection, resis-
tance to tumor challenge, and endotoxin hypersensitivity (Luster and Faith
1979). Of these, macrophage functions and host resistance to infection are
often assayed in immunotoxicology studies. Some procedures for evaluat-
ing these parameters are discussed in the following sections.
3.2.3.1 Macrophage functions
Polymorphonuclear and mononuclear phagocytes are the two major
classes of cells that are characterized by the ability to ingest and destroy
invading microorganisms (van Furth et al. 1978). This process of phago-
cytosis is completed in the following stages: opsonization of the particles
(viable and nonviable) by serum factors, attachment of the opsonized par-
ticles to the cell surface, engulfment of such particles, intracellular killing
of microorganisms, and digestion of microorganisms and other ingested
matter. Of these stages, ingestion and intracellular killing are most fre-
quently tested.
Experiments for the study of macrophage functions can be performed
in vivo or in vitro. In vitro methods are preferred by some investigators
(Vos 1977, van Furth et al. 1978, Luster and Faith 1979). The advantages
of these methods are that they allow the use of homogeneous cell popula-
tions so that the functions of individual cell types (e.g., monocytes, granu-
locytes, and macroc-hages) can be studied and they eliminate the effects of
serum and other factors on the macrophage function.
The main disadvantage is that phagocytosis and intracellular killing do
not appear to be extremely sensitive to the chemicals administered
systemically in studies on the immune response. For example, in a few of
the experiments reviewed, even though other immune parameters were
altered by exposure to trichloroethylene (Sanders et al. 1982) or
hexachlorobenzene (Vos et al. 1979b, 1979c) macrophage functions were
not affected. Methods for obtaining macrophages for study have been
reviewed in detail by Stuart et al. (1978) and van Furth et al. (1978).
Ingestion. Peritoneal macrophages of mice are frequently used in the
study of phagocytosis. In the procedure described by van Furth et al.
(1978) phagocytes and an equal number of bacteria are mixed together in
a tube containing serum. The mixture is incubated at 37°C under continu-
ous rotation. At various time points (0, 30, 60, 90, and 120 min) samples
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are taken from the suspension and centrifuged, and supernatants of the
samples are plated on agar for subsequent bacterial colony counts; or the
cell suspensions are examined microscopically after 15 minutes incubation
for morphological assessment of phagocytosis. If colony counts are made,
phagocytosis is measured by the decrease in the number of extracellular
organisms in the supernatants. If morphological assessment is made, cells
are dried on a slide and stained, and cells that have ingested bacteria are
counted.
Sanders et al. (1982) describe an alternative method for assessing
phagocytic activity in which peritoneal exudate cells are collected and
counted and allowed to adhere to a 24-well Costar culture dish. The
adhered cells are washed, and chromated SRBC (opsonized with IgG) is
added. The plates are incubated and removed at 10 to 45 min. The cells
are washed and scraped from the dishes and placed in tubes for gamma
counting.
Intracellular Killing. Intracellular killing by peritoneal macrophages
from the mouse can be assessed by the following method (van Furth et al.
1978). The cells can be removed and incubated with bacteria in vitro for
30 min, or the mouse can be injected intraperitoneally with the bacteria.
Macrophages can ingest bacteria in vivo in 3 min. After the initial period
of phagocytosis, the macrophages are washed, suspended in medium, and
counted. The suspension is divided into aliquots, which are incubated at
37°C (one at 4°C). After 15, 30, or 60 min of incubation, aliquots are
removed, the cells are disrupted by freezing and thawing, and the mix is
plated on agar. The number of viable bacteria is determined from the
number of colonies.
The assays just described are performed with peritoneal macrophages
from mice. However, similar tests can be performed with alveolar macro-
phages. In addition, macrophages can be obtained from other species such
as the rat and guinea pig. Clearance and intracellular killing by alveolar
macrophages appear to be sensitive indicators of injury by inhaled gases
and particles such as nitrous oxide (Acton and Myrvik 1972, Vassallo et
al. 1973); cigarette smoke (Rylander 1971), ozone (Goldstein et al. 1971),
and carbon black (Rylander 1969).
3.2.3.2 Host resistance to infection
The assessment of host resistance to infection following exposure to
chemicals represents a practical approach to defining immunosuppression.
For testing host resistance Listeria monocytogenes and Streptococcus
pneumoniae are recommended because resistance to these organisms
appears to be dependent primarily upon normal T-cell and B-cell func-
tions, respectively (Vos 1977, Luster and Faith 1979). All phases of the
cell-mediated immune response, including macrophage killing in the effec-
tor stage, participate in the resistance to Listeria (Vos 1977).
Tripathy and Mackaness (1969) first described the Listeria procedure
and applied it to testing the effects of several drugs on the immune
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response. The assay can be performed on mice and is based on the princi-
ple that effective suppression of the immune response by drugs or other
chemicals (which can be administered at the onset of infection or during
the course of the infection) would result in continued multiplication of
Listeria, particularly in the liver and spleen. The animals are injected
intravenously with the live bacteria, and resistance can be measured in two
ways: (1) mortality rates of treated animals are compared with those of
the controls or (2) bacterial growth in the spleen is enumerated at days 4
and 6 after inoculation, when cell-mediated immunity interrupts the
growth of the organism in vivo. The spleen is homogenized, and the homo-
genate is plated for the subsequent enumeration of colonies formed by
viable bacteria.
The advantages of the technique as listed by Tripathy and Mackaness
(1969) are as follows:
1. the immunosuppressive effect is quickly revealed
2. the method provides an accurate and quantitative assessment of the
effect of chemicals.
When Streptococcus pneumoniae is the infectious agent, resistance to
infection is simply assessed by comparing mortality rates of controls to
those of treated animals (Vos 1977).
Numerous alternatives to the use of these organisms include the use of
viruses and other bacteria and parasitic expulsion. Increased mortality due
to impaired host resistance to infectious organisms has been demonstrated
in various laboratory animals treated with chemicals such as PCBs (Friend
and Trainer 1970, Thomas and Hinsdill 1978, Loose et al. 1979), metals
(Exon et al. 1975, Gainer 1977, Roller 1979b, Exon et al. 1979), and
TCDD (Thigpin et al. 1975).
3.3 ALLERGIC REACTIONS TO ENVIRONMENTAL CHEMICALS
Coombs and Gell classified allergic reactions in 1963. A detailed dis-
cussion of these reactions can also be found in Coombs and Gell (1975).
The four classifications are briefly described in Table 3.5 and are illus-
trated in Figure 3.3. Coombs and Gell stress that the circumstances in
which these four basic types of reactions may be studied in an uncompli-
cated form are limited and that the pattern seen in any one human disease
is often complex, perhaps involving several of the responses listed in Table
3.5.
The Type I reaction in man includes both general and local anaphy-
laxis. In generalized anaphylaxis if the patient does not die rapidly, local
symptoms develop such as bronchial asthma, hay fever, pulmonary edema,
and urticaria. An example of local anaphylaxis is the appearance of a
wheal at the site of antigen application, as in the prick or scratch diagnos-
tic skin tests.
The Type II reaction is manifest as: complement-dependent antibody
cytotoxicity (transfusion reactions, hemolytic disease of the newborn, or
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Table 3.5. Definition of the Four Types of Allergic Reaction
I (Anaphylactic, reagin-dependent)
Initiated by allergen or antigen reactng with tissue cells (basophils and
mast cells) passively sensitized (allergized) by antibody produced else-
where, leading to the release of pharmacologically active substances
(vasoactive amines). An example of local anaphylaxis is the appearance of
a wheal at the site of antigen application, as in the prick or scratch diag-
nostic skin tests
II (Cytotoxic or cell stimulating)
Initiated by antibody reacting with either (1) an antigenic component of a
cell or tissue element or (2) an antigen or hapten which has become inti-
mately associated with these; damage may then occur in the presence of
complement or of certain kinds of mononuclear cells. Stimulation of secre-
tory organs may also occur (e.g., the thyroid). The Type II reaction is
manifest as transfusion reactions, hemolytic disease of the newborn, or
lesions produced in tissues by action of antibody and complement
in (Damage by antigen-antibody complexes)
Initiated when antigen reacts in the tissue spaces with potentially precipi-
tating antibody, forming microprecipitates in and around the small vessels,
causing damage to cells secondarily, or being precipitated in and interfer-
ing with the function of membranes, or when antigen in excess reacts in
the blood stream with potentially precipitating antibody, forming soluble
circulating complexes which are deposited in the blood-vessel walls or in
the basement membrane and cause local inflammation or massive comple-
ment activation. Antigen excess complexes mediate allergic damage of
three general kinds: (1) the Arthus reaction (situations in which antigen is
in excess locally in the body); (2) serum sickness (antigen-antibody union
takes place in the bloodstream and complexes are deposited at sites
throughout the body); (3) "massive complement activation" (especially
associated with endotoxin shock)
IV (Delayed, tuberculin-type, cell-mediated)
Initiated essentially by the reaction of actively allergized lymphocytes,
probably of the T (thymus-derived) population responding specifically to
allergen by the release of lymphokines, and/or the development of cytotox-
icity without the participation of free antibody. Locally, it is manifested by
the infiltration of cells at the site where the antigen is injected
Source: Adapted from Coombs and Gell (1975).
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ORNL-DWG 85-10741
Key.
• A * Antigens
1— Liberation of histamine and other
, pharmacologically active substances
Antibody
--^Sites of involvement of complement or
non-allergized lymphocytes
- Specific antigen-combining receptors on
membrane of specifically allergized lymphocytes
Figure 3.3. The four types of allergic reaction. (Source. Coombs and
Gell 1975.)
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lesions produced in tissues by action of antibody and complement); cell
dependent antibody cytotoxicity; antibody reacting with hapten or antigen
combined with or adsorbed on tissue cells to cause cytotoxicity (drug sensi-
tivity, e.g., purpura, hemolytic anemia, and granulocytopenia); antibody
reacting with tissue cells to cause stimulation (thyroid stimulator,
"enhancement" of malignant and other grafts).
The Type III reactions are characterized by antigen-antibody com-
plexes formed in moderate antigen excess so that they do not precipitate
but remain soluble, thus becoming toxic to the surrounding tissue (precipi-
tated complexes are normally nonirritating and are easily cleared by
phagocytes). Antigen excess complexes mediate allergic damage of three
general kinds: (1) the Arthus reaction (situations in which antigen is in
excess locally in the body); (2) serum sickness (antigen-antibody union
takes place in the bloodstream, and complexes are deposited at sites
throughout the body); and (3) "massive complement activation" (especially
associated with endotoxin shock).
Type IV reactions are referred to as delayed hypersensitivity, cellular
hypersensitivity, cell-mediated allergy, and others. Nomenclature is con-
stantly under discussion. An essential characteristic of delayed reactivity is
that the state can be transferred by cells although not by serum. It is
currently thought that the cell-mediated response is a property of T-cells
and has no need for B-cells. The development of the delayed lesion was
described in Section 3.2.1.
Chemical allergens found in industrial environments usually affect a
small subset of workers who are hypersusceptible to the low-dose exposure
legally permitted in chemical plants (Adkinson 1977). For example, from
1 to 10% of industrial workers exposed to legally permissible levels of
organic isocyanates will develop hypersensitivity reactions following low-
dose exposure to the chemicals (Pepys 1976). Similarly, a small group of
highly susceptible workers becomes immunologically sensitive when
exposed to numerous other industrial chemicals, and in almost all cases the
principal allergen has been identified as a chemical that is highly protein-
reactive (Adkinson 1977).
Landsteiner and Jacobs (1935, 1936) were the first to demonstrate the
antigenicity of chemicals. They induced reproducible immunological effects
in guinea pigs with simple compounds, halogenated dinitrobenzene deriva-
tives. They discovered, however, that if the halogen was replaced with a
hydrogen, hydroxyl, methane, or an amino group the compound was
immunologically inactive. The ability to sensitize seemed to be related to
the lability of the Cl ion when treated with alkali. The investigators con-
cluded that this property facilitated the binding of a simple nonantigenic
chemical with an endogenous protein carrier, creating an antigenic com-
plex.
This concept led to the "hapten theory," named for the chemical or
"hapten" or "fastening agent" involved (Loomis 1974). A hapten is, there-
fore, a compound of low molecular weight that combines with a protein
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carrier to form a product having antigenic properties that can elicit antibo-
dies in the animal. Although many chemicals do not appear to react with
protein in vitro, these complexes are nonetheless formed in the intact
animal, probably through metabolic alteration of the chemical.
It is now known that simple chemicals of molecular weight less than
1000 cannot elicit an immune response unless they are capable of covalent
linkage to carrier molecules (Adkinson 1977).
3.3.1 Allergic Response to Inhalants
The lung is constantly exposed to biologically active materials in
inhaled gases and particulate matter. The immune response to inhaled
physical and chemical agents and the injurious consequences of this
response have been reviewed in detail by Burrell (1977) and De Bruin
(1976).
Burrell (1977) recognized the respiratory route as an excellent means
of immunization, particularly in the stimulation of the IgA and IgE
classes. He also recognized that these immune responses are not always
beneficial to the host.
Inhalants thought to evoke allergic responses include physical and
chemical agents and antigens of plant and animal origin. Some of these
are listed in Table 3.6.
Illnesses due to inhalation of organic dusts include farmer's lung,
bagassosis, byssinosis, ptilosis, tabacosis, malt worker's lung, and suberosis
(De Bruin 1976). The organic dusts set up toxic asthma and allergic
alveloitis, responses thought to be of the Arthus type. Illness due to inhala-
tion of inorganic particles include inorganic pneumoconiosis and chronic
pneumoitis. (The latter disorder is a consequence of a delayed hypersensi-
tive reaction induced by exposure to beryllium.)
3.3.2 Allergic Response to Dermal Sensitizers
Many chemicals have been demonstrated to evoke allergic reactions of
the skin. The most common response is allergic contact dermatitis, a
delayed-type hypersensitivity reaction. Among these allergenic chemicals
are: 2,4-dinitrochlorobenzene, p-nitrodimethylaniline, p-phenylenediamine,
hexavalent chromium, nickel, beryllium, and cobalt.
For the induction of contact sensitization the eliciting contact allergen
must be applied to the skin, penetrate the epidermis and become combined
with protein, with which a complex antigen is formed (De Bruin 1976).
Contact sensitivity is apparently a complex hypersensitivity to
numerous conjugates (formed in situ) of a chemical with various analogous
proteins.
3.3.3 Tests to Detect the Sensitizing Potential of Chemicals
Marzulli and Maibach (1977) stated that three types of tests are
needed to evaluate skin sensitization potential:
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Table 3.6. Respiratory Allergens
Physical agents
Asbestos
Coal dust
Silica
Chemical agents
Beryllium
Detergent enzymes
Isocyanates
Miscellaneous chemical incitants
Antigens of plant origin
Aspergilli
Cotton and other textile dusts
Miscellaneous hypersensitivity pneumonitis incitants
Thermophilic actinomycetes
Antigens of animal origin
Source: Adapted from Burrell (1977).
1. the predictive test to identify allergenic substances
2. the diagnostic test to identify a substance that may be actually pro-
ducing skin reactions, and
3. the use test to provide information regarding the safety of ingredients
in combinations before they enter the marketplace.
Some of the predictive tests of skin allergy will be described in the fol-
lowing sections. These are useful for testing chemicals to which occupa-
tional exposure may occur via the skin. Also, a new test for the detection
of allergy induced via respiratory exposure will be described.
3.3.3.1 Skin sensitization
According to the OECD (1981), skin sensitization tests require an ini-
tial exposure to a test substance, followed by a challenge exposure which is
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administered no less than a week later. Sensitization is evaluated from the
reaction to the challenge exposure, usually 24 and 48 h after the pertinent
exposure, based on the proportion of each group that became sensitized
and the severity of the reaction in each animal. The recommended species
for animal testing is the guinea pig, with the number and sex determined
by the method used. For the induction exposure, the concentration used
should produce a skin reaction; for the challenge exposure the concentra-
tion should be nonirritating.
Skin predictive test procedures in guinea pigs have been identified by
Klecak (1977) for ascertaining the allergenic potential for chemicals.
These include the Draize test, Freund's complete adjuvant test, the guinea
pig maximization test, the "split adjuvant" test, the Buhler test, and the
open cutaneous test.
The Draize Test In this test, guinea pigs are sensitized intradermally
on the flank every other day for 18 days, and skin reactions are read 24 h
after each injection. The challenging dose is administered intradermally on
the opposite flank on day 35. Controls are treated only with the challeng-
ing dose. On days 36 to 37 the test areas are shaved, and the intensity of
erythema and occurrence and size of edema of the test reaction are
recorded. The test and control reactions are compared. The test is difficult
to quantitate but it is easy to perform, and material and operational
expenditures are minimal.
Freund's Complete Adjuvant (FCA) Test - In the FCA test, guinea
pigs are sensitized every other day for 10 days intradermally in the flank
with a mixture of test material and FCA (killed mycobacteria). Skin reac-
tions are read 24 h after each injection. The challenging doses of appropri-
ate concentrations are applied topically to the opposite flank. The chal-
lenging dose is not mixed with Freund's adjuvant. Skin reactions are
evaluated as for the Draize test.
The technique is simple and inexpensive, but Klecak (1977) believes
that it is more useful for identifying sensitizers than as a predictive test.
The Guinea Pig Maximization Test For this test, guinea pigs are
immunized on the shoulder both intradermally with the test agent in FCA
and topically (test agent in petrolatum) 7 days later. The challenging dose
is applied topically to the flank on day 21, and the reactions are read on
days 23 and 24. This test combines the factors that favor sensitization in
the guinea pig and is considered to be the most sensitive procedure for
detecting allergenic potential in these animals. There is also a high degree
of correlation between the results of this test and the human maximization
test (Klecak 1977).
The Split Adjuvant Technique This test makes use of the finding
that intradermal challenge of FCA beneath the site of topical application
of allergen potentiates sensitization. Sensitizing doses are administered to
the back (to which dry ice has been applied) of the guinea pig on days 0,
2, 4, and 7. On day 4 FCA is injected intradermally twice into the sensiti-
zation site. The challenging dose is applied to the back on day 20, and the
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reaction is read on days 22, 23, and 24. Klecak (1977) believes this
method is particularly effective with weak sensitizers, but it is time-
consuming.
The Buhler Test In this test, the chemical is applied to the flank and
held there with an occlusive patch for 6 h on days 0, 7, and 14. On day 28
the challenging dose is applied to the same site for 24 h. The skin reaction
is read on days 29, 30, and 31, and the results are compared with those of
negative and/or vehicle controls. The test is fairly expensive, but it
corresponds to the practical needs of a predictive test method (Klecak
1977).
The Open Epicutaneous Test In the open epicutaneous test, guinea
pigs are immunized on the flank three or five times weekly for four weeks,
always using the same site, with undiluted compound and/or its progres-
sive dilutions. The reactions are read once a week or 24 h after each appli-
cation. On days 21 and 35 the minimal irritant (previously determined)
and some lower concentrations are applied to the contralateral flank. A
concentration is considered to be allergenic when at least one animal in a
group shows a positive reaction with nonirritant concentrations. The test is
suitable for testing simple chemicals as well as finished products, and the
topical application methods used are similar to common use (Klecak
1977).
Technical details and original references for the skin sensitization tests
can be found in Klecak (1977).
3.3.3.2 Respiratory sensitization
Although experimental induction of respiratory hypersensitivity has
been readily accomplished with natural antigens (such as animal danders
and pollen) and with purified proteins, the induction of such a response
with hapten-protein complexes has been more difficult (Karol et al. 1978).
Karol et al. (1978), however, were successful in producing respiratory
hypersensitivity in guinea pigs with repeated exposure to aerosols of
hapten-ovalbumin conjugates. The animals were exposed to aerosols of
p-azobenzenearsonate ovalbumin and p-tolylureido ovalbumin in a Plexig-
las inhalation chamber. Respiratory hypersensitivity was evaluated on the
basis of changes in respiratory rate, a sensitive indicator of the response.
The hypersensitivity response was hapten-specific, as were most of the
antibodies produced during the course of the study. The guinea pig model
was used in a later study in which respiratory hypersensitivity was induced
with hexyl isocyanate-ovalbumin aerosol (Karol et al. 1979). Hapten-
specific antibodies were detected as well as hapten-specific IgE antibodies.
Karol et al. (1978) recommend their method for screening industrial
chemicals for their potential induction of respiratory hypersensitivity. In
more recent studies using the guinea pig model, Karol (1983) and Karol et
al. (1981) demonstrated that the immunologic response to toluene diiso-
cyanate following inhalation exposure is dose dependent and that respira-
tory tract hypersensitivity can be induced by dermal contact.
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3.4 TIER TESTING
Tests suitable for assessing the various cellular and humoral functions
of the immune system have been discussed in the previous sections. If the
evaluation of immunocompetence is to become a permanent fixture in rou-
tine toxicity test protocols, then the selection of the proper combination of
tests for use in these protocols becomes an important consideration. First,
it seems obvious that both humoral and cellular functions should be tested.
Second, it is important that tests be chosen that will efficiently and
economically provide maximum information.
Investigators in the field of immunotoxicology have made their
independent recommendations for the types of immunology procedures to
be included in routine toxicity studies or in tier testing protocols. Sugges-
tions of Moore and Faith (1976), Luster and Faith (1979), Speirs and
Speirs (1979), Dean et al. (1979b), White et al. (unpublished observa-
tions), and Luster et al. (1982b) are presented in the following sections.
3.4.1 Moore and Faith (1976)
Moore and Faith, interested in the developing immune system, recom-
mended the rat and mouse as the species to be tested, initially at weaning.
The following methods were selected because they were generally accepted
by immunologists and were within the capabilities of many laboratories.
A. Tests for humoral (or B-cell) immune function
1. Hemagglutination techniques using
a. lipopolysaccharide (thymus independent)
b. SRBC (thymus dependent)
In a minimal experimental design where only one antigen can be
tested, one should select the SRBC test because both T- and B-
cells can be evaluated with this procedure.
2. Mitogen stimulation of thymus and spleen cells with lipopolysac-
charide (a B-cell stimulator)
B. Tests of cellular (or T-cell) immune function
1. Radiometric ear test
2. Mitogen stimulation of thymus and spleen cells with phy-
tohemagglutinin and Concanovalin A, T-cell stimulators.
Moore and Faith further recommended that:
1. Chemicals that depress immune function in adults should be evaluated
in the fetus/neonate.
2. Routine teratology studies should include an evaluation of the thymus
and spleen (organ weights and histologic examination). If effects are
observed on maturation of the tissue, then one should consider subse-
quent experiments that specifically assess the immune response.
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3.4.2 Luster and Faith (1979)
These investigators outlined the immune tests used in their laboratory
at the National Institute of Environmental Health Sciences. The initial
assessment of the effects of a particular chemical on the immune system is
generally made in a routine toxicity study. At necropsy Luster and Faith
perform the following procedures:
A. Examination of lymphoid organs (thymus, spleen, lymph nodes, and
bursa of Fabricius) and perhaps the adrenals
1. Gross examination
2. Histopathology
3. Lymphoid organ to body weight ratio
4. Quantitation of viability for the determination of cytotoxicity
B. Hematology
1. Differential
2. Leukocyte count
C. Immunology
1. Serum immunoglobulin concentrations—electrophoresis
2. Lymphocyte transformation (PHA and LPS)
3. T-cell rosettes (not practical in mice or rats)
4. Surface antigens on lymphocytes (theta antigen in mice)
D. Clinical signs, infections, tumors
Further assessment of the immune system can be accomplished with the
following:
A. Tests of cell-mediated immunity
1. In vitro transformation of lymphocytes by concanavalin A
2. In vitro mixed lymphocyte cultures
3. Radiometric ear test
4. Assay for migration inhibition factor
B. Tests of humoral-mediated immunity
1. In vitro transformation of lymphocytes by dextran sulfate or
pokeweed mitogen
2. B-cell surface antigens
a. EAC rosettes
b. Fluorescent labeling of B-cell immunoglobulins
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3. Quantitation of serum immunoglobulins by radial immunodiffu-
sion
4. Antibody response to T-dependent and T-independent antigen
using the plaque assay
C. Tests of indirect immunity
1. Macrophage
a. In vivo uptake of aggregated 125I-BSA or colloidal carbon
b. In vitro or in vivo determination of phagocytosis and intra-
cellular killing of bacteria
c. Cytotoxicity and proliferation
2. Hormone concentrations
a. Cortisol/cortisone
b. Estrogens or androgens
3. Complement (measurement of C-dependent hemolysis)
4. Host resistance to infection
5. Resistance to tumor challenge
6. Endotoxin hypersensitivity
The tests were selected by Luster and Faith for their reliability and for
their ability to detect subtle differences in immune status.
3.4.3 Speirs and Speirs (1979)
The Immunotoxicology Program at the National Center for Toxicologi-
cal Research has devised a model for in vivo assessment of the effects of
toxic agents on immunocompetence. This procedure is summarized as fol-
lows:
1. Immunize mice with an antigenic mix consisting of:
Diphtheria toxoid
Pertussis vaccine
Polyvalent pneumococcal polysaccharide antigens
Tetanus toxoid
2. Determine levels of antibody and other plasma proteins using radio-
immunoassay procedures.
3. Assess functional capabilities of macrophages, lymphocytes, and other
blood cells using cytochemical analysis.
4. Develop an immune profile for each toxicant tested. Compare with
profles of known immunosuppressants and other toxic agents.
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5. Compare the immune profiles of mouse and man with respect to a
particular toxicant.
In their model Speirs and Speirs (1979) use licensed vaccines for
immunization, and commonly used pharmaceutical agents as prototype
immunosuppressants so that animal and human data can be more readily
compared. Risk assessment is based on comparison of immune profiles of
animals treated with known immunotoxicants with those of animals treated
with a potential toxicant.
3.4.4 Dean et al. (1979b)
This group at Litton Bionetics has been involved for the last few years
in developing and refining assays of humoral and cell-mediated immuno-
competence. They have proposed a tier approach for screening for immu-
nological effects (tier I) and to help determine which mechanisms are
responsible for the effects observed (tier II). The assays were selected on
the basis of relevance to the human experience, cost, reproducibility, ease
of performance, and application to routine toxicology studies. The tests
included in tiers I and II are listed in Table 3.7.
After 90 days of a chronic or subchronic toxicity study, 15 animals are
removed for immunology studies. Testing can then proceed in either a
hierarchical or nonhierarchical fashion. Hierarchical testing assesses chem-
icals in a stepwise procedure following administration of a dose of the test
agent with minimal or no overt toxicity. The testing begins with the sim-
plest or least expensive assays and continues through the more time-
consuming and expensive in vitro tests. It is assumed that those chemicals
with deleterious effects would be detected with the initial assays and would
require further testing only when information regarding mechanism is
required.
In nonhierarchical testing the assays (tier I) would all be performed at
once. This procedure is useful when the number of compounds to be tested
is small or when the data are needed quickly.
If no immunological effect is observed in tier I, no further studies are
performed. If a positive effect (suppression or enhancement) is observed,
the tier II assays are performed.
3.4.5 White et al. (Unpublished Observations)
This group at the Medical College of Virginia has developed a pro-
cedure to investigate the effects of a given chemical on the immune system
against a background of standard toxicological procedures.
An acute study to determine the LD50 is performed first. Next, a sub-
chronic 14-day range-finding study is performed, usually at 1/10 and
1/100 the LD50. The parameters measured in the 14-day subchronic study
are:
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Table 3.7. Testing Approaches for Evaluating the Immunobiologic Effects of
Food Additives, Drugs, and Environmental Chemicals
Tier I: Screening
Tier II: Mechanism
Clinical laboratory
Pathology
Cell-mediated
immunity
Humoral immunity
Susceptibility
Immunepotentiation
Tumor challenge
Hematology
Blood chemistry
Urine
Lymphoid organs
• Relative weight
• Cell viability
« Histology
Delayed hypersensitivity (DHS)
• T-dependent antigen (keyhole
limpet hemocyanin, bovine
gammaglobulin, tetanus toxoid)
toxoid study using the radio-
metric assay
Lymphocyte proliferation
• T- and B-cell mitogens
• Mixed leukocyte culture
Ig levels
Specific antibody tier
• T-dependent antigen
Jerne plaque assay
• T-dependent antigen
Tumor challenge - TO,^
Specific blood or tissue levels
of compound
Hormone levels
Cell surface markers
• T-cells
- B-cells
• Null cells
• Fc-cells
DHS
• T-independent antigen
Lymphocyte proliferation antigen
Helper cell function
Macrophage function
Mishell-Dutton assay
Local product of antibody
Specific antibody tier
• T-independent antigen (SIII)
Cytotoxicity
Bacterial challenge
Virus challenge
Source: Adapted from Dean et al. (1979b).
A. Standard toxicology
1. Body weights6
2. Necropsy—gross pathology*5
3. Organ weights'" (brain, liver, spleen, lungs, thymus, kidneys, and
testes)
4. Hernatologyb (leukocyte counts, hematocrit, hemoglobin)
5. Clinical chemistries3 (glutamic pyruvic transaminase, lactic dehy-
drogenase, blood urea nitrogen)
6. Blood coagulation15
B. Immunotoxicology
1. Cell-mediated immunity0—delayed-type hypersensitivity response
to sheep erythrocytes (footpad assay)
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2. Humoral immunity*—spleen antibody-forming cell response to
sheep erythrocytes (IgM plaque-forming cells)
Three sets of mice are used; the superscripts a, b, and c designate the
group on which the test is performed.
The 90-day subchronic study is then performed to demonstrate the tar-
get organ. The following variables are measured during and at the end of
the 90-day study:
A. Standard toxicology
1. Body weights, twice weekly
2. Fluid consumption
3. Necropsy3—gross pathology
4. Organ weights8—brain, liver, spleen, lungs, thymus, kidneys, and
testes
5. Urinalysis—pH, glucose, protein, bilirubin, blood, and urobilino-
gen
6. Hematology"—hematocrit, erythrocytes, leucocytes, differential,
platelets, and hemoglobin
7. Coagulation3—prothrombin time, activated partial thromboplas-
tin time, and fibrinogen
8. Clinical chemistries'"—sodium, calcium, potassium, serum glu-
tamic pyruvic transaminase, serum glutamic oxaloacetic transam-
inase, lactic dehydrogenase, total protein, albumin, creatinine,
and blood urea nitrogen
B. Hepatic microsomal mixed functional oxidase parameters
1. Liver weight
2. Microsomal protein
3. Glutathione levels
4. P-450 content
5. Cytochrome b5
6. Aminopyrine N-demethylase
7. Aniline hydroxylase
C. Cell-mediated immunity
1. Delayed-type hypersensitivity response to sheep erythrocytesb
2. Spleen cell response to Concanavalin Ab
D. Humoral immunity
1. Spleen antibody-forming cell response to sheep erythrocytesb
(footpad assay)
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2. Spleen cell response to lipopolysaccharide
3. Basal levels of IgM, IgG, and IgA
E. Functional activity of the reticuloendothelial system
1. Vascular clearance rate of sheep erythrocytesd
2. Organ uptake of sheep erythrocytesd
3. Chemotaxis, adherence, and phagocytosis of peritoneal exudate
cells6
F. Bone Marrow3
1. DNA synthesis
2. Granulocyte—monocyte stem cells
Five sets of mice are used; superscripts a, b, c, d, and e designate the
group on which a test is performed.
White et al. demonstrated that these assays, used to evaluate immune
status, are quantifiable and reproducible and that such assays can be easily
incorporated into a routine toxicology study.
3.4.6 Luster et al. (1982b)
In November 1979, the National Institute of Environmental Health
Sciences held a Consensus Meeting to develop a list of relevant immunolo-
gic parameters suitable for evaluating chemically induced immunotoxici-
city. The group of more than 35 immunologists and toxicologists of varied
experience and expertise prioritized the following scheme of immunologic
parameters and methods considered necessary to measure immunotoxicity.
These tests are to be performed in the order listed.
Chemical for immunological evaluation —>• Chronic or Subchronic Study
I I
Immunology Screening Panel
1. Pathotoxicology-hematology, liver, chemistry, serum proteins, lym-
phoid organ weights, and histology.
2. Host resistance-tumor challenge and infectious agent challenge.
3. Radiometric delayed hypersensitivity.
4. Lymphoproliferative responses-PHA, Con A, LPS, and MLC.
5. Humoral immunity-Ig levels, specific antibody titer, plaque-forming
cell assay.
6. Macrophage function assays.
7. Bone marrow progenitor cell assays.
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Data Evaluated
I I
No immuno- Positive immune- —>• Study mechanism of
logical effect logical effect immunological effect
I I
No further study
3.5. SUMMARY
Reports of immunotoxic effects of environmental chemicals, some at
clinically subtoxic levels, have stimulated interest in tests of immune func-
tion and in the inclusion of such tests in routine toxicity test protocols.
These procedures provide information related to direct injury to the
immune system and, equally important, are capable of detecting subtle
alterations in the immune response. This system could be an extremely
sensitive indicator of the general toxicity of certain chemicals.
The preceding review was undertaken to examine some of the test
methods capable of detecting chemically induced alterations in the specific,
as well as the nonspecific, immune response.
Tests of cell-mediated, humoral, and indirect immune functions were
described, and examples of the use of these tests in the assessment of the
potential immunotoxic effects of chemical were presented. A few tests that
are useful for detecting the sensitizing effects of chemicals were also
included.
There was no evidence to indicate that any particular test or battery of
tests would be more suitable for evaluating a particular class of chemicals
than others. However, there are indications that certain chemical classes
may be selectively toxic to the humoral immune system, while others may
affect cellular immunity. For example, lead, cadmium, mercury,
polychlorinated biphenyls, and DDT impair primarily the humoral immune
response, while organochlorines impair primarily the cell-mediated
response. These observations, however, should not preclude the assessment,
in toxicity testing, of both humoral and cell-mediated immunity.
Information related to the costs of these tests was not found in the
literature. Generally, immunology procedures are regarded as being fairly
expensive; but the introduction of in vitro techniques may enhance the pos-
sibility of large-scale testing at more reasonable costs.
Recommendations for the types of immunology tests that should be
included in routine toxicity studies or in tier-testing schemes were
presented.
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3.6 GLOSSARY
ADJUVANT. A substance administered with or before an antigen that
nonspecifically enhances or alters the cell-mediated or humoral
immune response to that antigen (Herbert and Wilkinson 1971,
Sharma 1981b).
ALLERGEN. An antigenic substance capable of eliciting an allergic
response or an allergic state (Herbert and Wilkinson 1971, Sharma
1981b).
ALLERGENIC. Causing allergy (Sharma 198 Ib).
ALLERGY. (1) Hypersensitivity; (2) heightened reactivity to antigen,
according to Gell and Coombs, which is synonomous with immunity
(Herbert and Wilkinson 1971); (3) abnormal or harmful reaction
which includes delayed, immediate, or contact hypersensitivity and
may also include a number of reactions that have no immunological
basis (Platts-Mills 1982).
ALLOANTIGEN. Cell-surface antigens in different strains of the same
species (Wust, unpublished observations).
ALLOANTISERA. Mouse or rat antisera prepared by immunizing one
inbred strain with cells from another strain (Wust, unpublished obser-
vations).
ALLOGRAFT. A syngeneic graft, or one that has been exchanged
between two genetically dissimilar members of the same species, such
as of two different inbred strains (Herbert and Wilkinson 1971).
ANAPHYLAXIS. An immediate hypersensitivity or Type I reaction
resulting from the administration of antigen to a primed subject. The
reaction is caused by the release of vasoactive substances, such as his-
tamine and leukotrienes, when antigen combines with antibody on cell
surfaces. The reaction may be generalized or localized. The systemic
reaction can be violent and can cause death in minutes (Herbert and
Wilkinson 1971; Wust, unpublished observations).
ANTIBODIES. Immunoglobulins that are produced in the body by cells
of the lymphoid series, particularly plasma cells, in response to stimu-
lation by antigen that are capable of specific combination with
antigen (Herbert and Wilkinson 1971, Sharma 1981b).
ANTIBODY TITER. In serological reactions, a measure of units of anti-
body in an antiserum per unit volume of the undiluted serum. To
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determine the titer, the serum is serially diluted, antigen is added, and
the end point is determined (Herbert and Wilkinson 1971).
ANTIGEN. A substance that elicits a specific immune response, either
cell-mediated or humoral, when introduced into the tissues of an
animal. An antigen may also induce specific immunological tolerance
(Herbert and Wilkinson 1971). Can be a protein, nucleic acid, car-
bohydrate, or lipid (Wust, unpublished observations).
ANTIGEN PROCESSING. Macrophage-antigen interaction in which the
macrophage localizes the antigen and brings it closer to the
antibody-forming cell. This interaction may also result in enhanced
immunogenicity of the antigen (Koller 1981).
ANTIGEN RECOGNITION SITE. The site on the surface of a lympho-
cyte which may be identical to an antibody-combining site. Can react
specifically with antigen and thus initiate humoral or cell-mediated
immune resposes (Herbert and Wilkinson 1971).
ANTIGEN-ANTIBODY REACTION. The interaction between an
antigen and the antigen-binding receptor on the surface of a small
lymphocyte which stimulates the differentiation and proliferation of
the cell, leading to the production of a progeny of effector cells
(which execute the immune response) and of memory cells (which are
responsible for the enhanced or secondary response following a subse-
quent encounter with the same antigen) (Miller 1975). The immune
system responds to an unlimited variety of antigens, some of them
normally not present in nature (Polak 1977).
There is now evidence for the existence, on the cell membrane of
small lymphocytes, of antigen recognition units that can bind
antigenic determinants and of Ig molecules with specificity similar or
identical to that of secreted antibody (Miller 1975). There is also evi-
dence to show that the Ig determinants on the cell membrane are the
antigen-binding receptors. Apparently both T- and B-cells have
antigen-binding receptors on their surface, but they are of such low
density on nonimmune cells that they cannot be detected by routine
antigen-binding techniques.
ANTISERA. Serum, from an animal, that contains antibodies specific to
a given antigen [e.g. anti-ovalbumin (Herbert and Wilkinson 1971)].
ARTHUS REACTION. An inflammatory reaction that follows the
administration of antigen to an animal that already possesses precipi-
tating antibody to that antigen. The reaction is caused by the forma-
tion, in the presence of complement, of antigen-antibody complexes
that adhere to the endothelium of vessels and become surrounded by
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fibrin, platelets and neutrophils. This is followed by plugging of the
vessels with thrombi and exudation of neutrophil-rich fluid into the
surrounding tissues. The reaction is manifested as an ulcer that
appears several hours after intradermal injection of antigen into a
primed animal (Herbert and Wilkinson 1971).
B-LYMPHOCYTE. B-cells arise from stem cells in the bursa of Fabricius
in birds and in the bursal equivalent in mammals (thought to be gut-
associated lymphoid tissue or bone marrow) (Faith et al. 1980). B-
cells mature independently of thymic influence (Koller 1979a). They
circulate and rest temporarily in the thymus-independent areas of the
spleen (follicles and peripheral regions of the white pulp) and lymph
nodes (follicles and medulla) (Faith et al. 1980), where they consti-
tute 10 to 20% of the nucleated free cells. They are not present in the
thymus (Wust, unpublished observations).
B-cells are stimulated by antigen to become antigen-specific cells that
proliferate and differentiate into plasma cells, which secrete antibody
or become memory B-cells.
BACILLE CALMETTE-GUERIN (BCG). An attenuated, living bovine
strain of Mycobacterium tuberculosis used to vaccinate against tuber-
culosis and leprosy (Herbert and Wilkinson 1971).
BLASTOGENESIS. Stimulation of cell (lymphocyte) blast formation by
an antigenic substance (Sharma 1981b).
BLASTOGENIC. Capable of producing cell blasts. In immunology,
antigenic substances capable of producing cell blasts (Sharma 1981b).
BONE MARROW. The principal source of the lymphoid stem cell in
postnatal life and the only general hemopoetic tissue in the healthy
adult. The vascular compartment of the bone marrow is occupied by
the vessels that distribute nutrients throughout the marrow cavity and
the hematopoetic compartments (red marrow), which contain
erythrocytes, granulocytes, lymphocytes, megakaryocytes, monocytes,
macrophages, plasma cells, mast cells, and their precursor stem cells.
In adult animals much of the red marrow is replaced by fatty tissue
and becomes yellow marrow. The bone marrow does not contain signi-
ficant numbers of fully competent thymus-dependent lymphocytes.
However, theta-positive incompletely developed T-cells and more
mature T-cells are demonstrable. The marrow also contains significant
numbers of B-lymphocytes (fully differentiated immunoglobulin-
producing lymphocytes) and plasma cells (Bloom and Fawcett 1969,
Herbert and Wilkinson 1971, Park and Good 1974).
BURSA-EQUIVALENT. A poorly identified tissue in mammals, con-
sidered to be equivalent to the bursa of Fabricius in birds, which gives
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rise to B-cells (Luster et al. 1982b). The bursa is a saclike lymphoep-
ithelial structure arising as a dorsal diverticulum from the cloaca of
young birds that contains many lymphoid follicles in which lympho-
poiesis proceeds until the structure involutes about the time of sexual
maturity (Herbert and Wilkinson 1971). The lymphocytes are B-cells
that migrate from the bursa to colonize the secondary lymphoid
organs (Wust, unpublished observations). The bursa is associated with
humoral immunity, demonstrated by the fact that bursectomized
chickens fail to make antibodies to a variety of antigens and lack
plasma cells and germinal centers in their lymphoid tissues (Herbert
and Wilkinson 1971).
CELL-MEDIATED IMMUNITY (CMI). The type of specific immunity
that is effected by specifically sensitized lymphocytes and can be
transferred with those cells. (Vos 1977, Faith et al. 1980). The main
categories of cell-mediated immunity include: classical cell-mediated
protective immunity, which is mainly effective against protozoal, viral,
fungal, and some bacterial infections; delayed hypersensitivity skin
reactions to extracts or whole suspensions of organisms, also identified
as tuberculin hypersensitivity; chemical contact sensitivity; allograft
rejection; immunological surveillance to tumors; and certain organ-
specific autoallergic diseases, such as thyroiditis, encephalomyelitis
(following rabies vaccination), adrenalitis, and orchitis (Turk 1975,
Faith et al. 1980).
CELL SURFACE MARKERS. Antigens or alloantigens on the surface of
lymphocytes (IgD and IgM on B-cells; theta and TL antigens on T-
cells; and Ly alloantigens on both T- and B- cells) (Luster et al.
1982b).
CF. Chemotactic factor.
CHEMOTACTIC FACTOR. A product of sensitized T-lymphocytes
exposed to antigens that direct the migration of leucocytes to sites of
inflammation (Allison 1982).
CLINICAL ALLERGY. A disease or diseases in which hypersensitivity
plays a part, such as contact hypersensitivity, hay fever, and asthma.
CLONAL THEORY. The theory of F. M. Burnet (1959), who believes
that if a cell comes in contact with an antigen, and if any part of that
antigen happens to fit the antigen-binding site of the cell, the cell is
stimulated to divide and increase its production of that particular glo-
bulin. Because each region of a given antigen may more or less fit a
number of different antigen binding sites, a considerable array of dif-
ferent antibodies is likely to be produced, directed toward the several
antigenic determinants of the antigen molecule.
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COMPLEMENT. An enzymatic system of 11 serum proteins, 9
components, which act in a specific sequence, or cascade. The
complement system is activated by many antigen-antibody reactions
and is essential for antibody-mediated immune hemolysis and bac-
teriolysis. Complement participates in other biological reactions such
as phagocytosis, opsonization, chemotaxis, and immune cytolysis
(Park and Good 1974, Herbert and Wilkinson 1971).
The complement proteins have been named according to the numeri-
cal order of their role in the immune reaction, except for C4, which
was named before the sequence was described (Roller 1981).
CONCANAVALIN A. A mitogen derived from Canavalis ensiformis,
selective to immature T-cells (Sharma 1981e).
CONTACT DERMATITIS. Delayed hypersensitivity reaction of the skin
resulting from exposure to chemical allergens, including environmen-
tal and industrial chemicals, drugs and cosmetics, and the catechols of
poison oak and poison ivy (Ligo and James 1974, as reported in Gigli
1982).
CYTOTOXIC. Capable of producing cell lysis (Sharma 198le).
CYTOTOXIC FACTOR. A lymphokine, released by a sensitized lympho-
cyte upon contact with antigen, that is capable of producing cell lysis
(Sharma 1981c).
DELAYED HYPERSENSITIVITY. The state mediated by lymphocytes,
which is evident only when lesions appear about 24 h after contact of
the primed subject with antigen. This reaction is a manifestation of
cell-mediated immunity and can be transferred to another animal with
cells (Herbert and Wilkinson 1971).
DIRECT PFC. Plaque-forming cells producing antibody, primarily IgM,
that can fix complement and lyse cells directly (Jerne and Nordin
1963).
EFFECTOR T-CELLS. Cells that, in delayed-type hypersensitivity, pro-
duce biologically active products called lymphokines, following contact
with specific antigen (Luster et al. 1982b).
FREUND'S COMPLETE ADJUVANT (FCA). A water-in-oil emulsion
adjuvant in which killed, dried mycobacteria (usually M. tubercu-
losis) are suspended in the oil phase (Herbert and Wilkinson 1971).
GRAFT-VS-HOST REACTION. The reaction of a graft containing
immunologically competent cells against the tissues of a genetically
different host (Herbert and Wilkinson 1971).
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HAPTEN. A compound of low molecular weight, not antigenic itself, that
can be combined with a protein carrier to form a product having
antigenic properties and can thus elicit antibody formation (Sharma
1981c).
HELPER T-CELLS. T-lymphocytes that, through the release of soluble
factors, activate B-cells and amplify their responses against many
antigens (Sharma 1981b, Roller 1981).
HEMAGGLUTINATION. The clumping of a cell suspension by specific
antibody (Gell and Coombs 1975).
HEMOLYSIS. The bursting of red blood cells. In immunology, this
occurs when complement is added to the hemagglutinin reaction (Vos
1977).
HISTOCOMPATIBILITY ANTIGEN. An isoantigen carried on the sur-
face of nucleated cells of many tissues which may induce an immune
response that causes the rejection of a graft from one individual to
another of the same species whose tissues do not carry that antigen
(Herbert and Wilkinson 1971).
HISTOCOMPATIBILITY LOCUS. The locus on a chromosome at which
the genes that determine the histocompatibility antigens are located
(Herbert and Wilkinson 1971).
HUMORAL IMMUNE RESPONSE. The type of specific immunity that
operates through antibody-producing cells and is transferable by
serum (Vos 1977, Faith et al. 1980). The antibodies (immunoglobu-
lins) are present in the plasma, lymph, and tissue fluids of the body
and may become attached to cells (Herbert and Winkinson 1971).
Upon antigenic stimulation, a small number of B-lymphocytes,
specific for that antigen, proliferate and differentiate into plasma cells
that secrete antibody in a primary response or serve as memory cells
for the secondary response (Faith et al. 1980).
HYPERSENSITIVITY. State of abnormal susceptibility or sensitivity,
usually in reference to allergy (Sharma 1981b).
IMMEDIATE HYPERSENSITIVITY. Antibody-mediated immune reac-
tion that results from the release of histamine and other vasoactive
substances that can be transferred from one individual to another by
serum alone. The antibody is usually of the IgE type, which fixes par-
ticularly to mast cells. The reaction usually appears within a few
seconds to 30 minutes after contact with antigen (Herbert and
Wilkinson 1971).
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IMMUNE RESPONSE. The manifestation of the interaction of the
organs and cells of the immune system, which is expressed through
protective mechanisms such as rejection of allografts or delayed
hypersensitivity (cellular immunity) or production of antibodies
(humoral immunity). The responses of organisms to antigens are of
two types: innate (or nonspecific) immunity and acquired (or specific)
immunity (De Bruin 1976).
IMMUNE SUPPRESSION. Suppression of the immune response
(Sharma 1981c).
IMMUNE SYSTEM. The organization of organs and cells which interact
at various levels to effect the responses of the body to foreign entities
(or antigens) (Miller 1975).
IMMUNIZATION. Administration of antigen to produce an immune
response to that antigen. Administration of either antigen to produce
active immunity or antibody to produce passive immunity to protect
against the harmful effects of antigenic substances or organisms
(Herbert and Wilkinson 1971).
IMMUNOASSAY. Analytical techniques that utilize antigen-antibody
type reactions (Sharma 198 Ib).
IMMUNOCOMPETENCE. The capacity of the body to cope with infec-
tion and malignant growths. Immunocompetence has been character-
ized as (1) the establishment of barriers to maintain integrity and
protect the body from the environment; (2) the capacity to recognize,
neutralize, isolate, and reject foreign agents; (3) diverse reactions such
as inflammation, granulomatous reactions, cell-mediated and humoral
responses, and healing and repair processes; and (4) a process of
"adoptive immunization" in which cells react in a greatly augmented
or more effective manner to sequential exposures to a particular
pathogen (Speirs and Speirs 1979).
IMMUNOGLOBULINS. Proteins, each of which is made up of two light
and two heavy chains that are usually linked together by disulfide
bonds. The various immunoglobulins differ from each other in
primary structure even though some amino acid sequences are
remarkably constant throughout phylogeny. All antibodies are immu-
noglobulins although all immunoglobulins may not be antibodies.
Antibodies are produced in the body by plasma cells in response to
stimulation by antigen, and they are capable of specific combination
with antigen. Five major classes of antibody proteins, or immunoglo-
bulins, have been identified in man: IgG, IgA, IgM, IgD, and IgE
(Herbert and Wilkinson 1971).
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The heavy and light chains of the immunoglobulins are symmetrically
arranged, each consisting of a variable and a constant region. Accord-
ing to Goldstein et al. (1974):
In the constant region of a given immunoglobulin class, the polypep-
tide chain is the same (or nearly the same) in its amino acid
sequence. This similarity is common among various species and is
found even in primitive organisms. In contrast, the variable region is
composed of a variety of different amino acid sequences, while the
total number of amino acid residues remains the same. The antigen
binding sites are apparently located in the variable portions of the
light and heavy chain. Generally, lymphoid cells differentiate in such
a way that a given cell produces only a single immunoglobulin while
all lymphoid cells together produce thousands of different immunoglo-
bulins.
IMMUNOLOGICAL TOLERANCE. An acquired state of specific
unresponsiveness to an antigen (Moreno 1982).
IMMUNOSUPPRESSION. Inhibition of the stimulation of an immune
response by an antigen by a physical, chemical, or biological agent
(Herbert and Wilkinson 1971).
INDIRECT PFC. Plaque-forming cells that produce antibody that either
is insufficient for or is incapable of fixing complement unless it is
complexed with antiglobulin antibody. Revealed in the PFC assay
when developing serum is added to the plate (Dresser 1978).
INTERFERON. A protein (lymphokine) released by cells in response to
virus infection. When taken up by other cells, interferon inhibits the
replication of viruses within them. Interferon is also released after the
injection of bacterial endotoxin (Park and Good 1974, Herbert and
Wilkinson 1971, Sharma 198la).
IgA. The major immunoglobulin component of secretions in the respiratory
and gastrointestinal tracts and the second most abundant immunoglo-
bulin class (10% of total) (Park and Good 1974, Rowe 1975), but
present in relatively low concentrations in the serum. The molecule is
synthesized locally in plasma cells of the submucosa; the secretory
component is synthesized in the epithelial cells (Roller 1981). The
IgA molecules of external secretions are primarily 11S, while those of
the serum are mainly 7S. Although IgA does not fix complement, it
may activate C3 by alternate pathways. IgA is active against viral
and bacterial antigens at the sites of secretion, but the role of the
antibodies in protection is not clear. It has been suggested that IgA
antibodies may activate the alternate complement pathway and that
they may be active in promoting phagocytosis and intracellular killing
of organisms by peritoneal macrophages.
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126
IgD. Prominent surface-bound immunoglobulin in the newborn; constitutes
only 0 to 0.2% of immunoglobulins of normal serum (Roller 1981).
Half-life about 3 days. Antibody activity against penicillin, insulin,
milk proteins, diphtheria toxoid, and nuclear and thyroid antigens has
been demonstrated in IgD molecules (Park and Good 1974).
IgE. The immunoglubulin that is associated with the reaginic antibodies
active in Type I (see Section 3.4) allergic reactions (Bennich and
Johansson 1971, Ishizaka and Ishizaka 1971). IgE is produced by the
plasma cells of the regional lymph nodes and mucous membranes of
the respiratory and gastrointestinal tracts (Roller 1981). The concen-
tration of IgE in the serum is very small. It binds to tissue mast cells
and blood basophils, and exposure of these cells to a specific allergen
(the antigen to which the IgE is directed) leads to the release of his-
tamine and other mediators. No protective role has been demonstrated
for IgE antibodies. Rowe (1975) postulates that IgE antibodies may
assist in protection against certain parasites either directly through
pharmacological mediators or by the induction of an increase of vas-
cular permeability allowing freer access of antibodies and cells from
the blood.
IgG. The most abundant (85% of total) immunoglobulin in man, syn-
thesized primarily during the secondary immune response. Has half-
life of about 23 days (Park and Good 1974, Rowe 1975, Faith et al.
1980). It diffuses readily into extravascular tissue, where it can react
with bacteria, parasites, and viruses (Park and Good 1974), and is
responsible for long-lasting protection to these agents (Roller 1981).
IgG is selectively transported across the placenta, and at birth is the
predominant immunoglobulin in the circulation (Park and Good
1974).
IgM. Often the first immunoglobulin to be detected following antigenic
challenge and the most effective specific first line of defense (Rowe
1975, Faith et al. 1980). The IgM antibodies constitute 5 to 10% of
the total immunoglobulins in the blood (Park and Good 1974). One of
the characteristic properties of IgM is its high molecular weight
(900,000). It has a serum half-life 5 to 6 days.
RILLER LYMPHOCYTES. Special cytotoxic T-lymphocytes that partici-
pate in the delayed hypersensitivity reactions (Sharma 198la).
RININS. Small polylpeptides that are released from a plasma alpha-
globulin, kininogen, by proteolytic enzymes such as kallikrein. These
enzymes are released when polymorophonuclear cells phagocytize
immune complexes. Rinins are active as vasodilators and facilitate the
increase of vascular permeability. They also cause contraction or
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relaxation of smooth muscle (Park and Good 1974, Herbert and
Wilkinson 1971, Sharma 198la).
LIPOPOLYSACCHARIDES. Compounds in which lipid is linked to
polysaccharide. A common component of cell membranes. In immu-
nology usually refers to components of O-antigen (endotoxin) complex
of gram-negative bacilli such as E. coli, Salmonella, and Bordetalla
pertussis. E. coli lipopolysaccharide is a thymus-independent antigen
(Luster et al. 1978, Herbert and Wilkinson 1971).
LYMPH NODE. A kidney-shaped accumulation of reticuloendothelial
cells and lymphoid tissue, connected to other lymph nodes by lym-
phatic vessels. These vessels enter the node at various places but leave
it only at the hilus (a slight indention on the side of the node) on their
way to the thoracic or right lymphatic ducts. The lymph nodes act as
filters through which foreign materials must pass. These materials
thus must come in contact with macrophages and lymphocytes; the
lymph nodes are important centers for phagocytosis and for the initia-
tion and development of the cellular and humoral immune responses.
The lymph node is divided into the outer cortical and inner medullary
parts. The cortex consists primarily of dense lymphatic tissue which
continues into the medulla as medullary cords. In the mid-cortex are
thymus-dependent areas, which appear selectively depleted of cells in
neonatally thymectomized animals. The cortex contains lymphatic
nodules about 1 mm in diameter, which develop and disappear in
response to various stimuli. These nodules contain dendritic macro-
phages which are capable of fixing antigens at their surface. In the
embryo and the immediately postnatal stage they lack the central
"germinal" areas which are thought to develop in response to
antigenic stimulation. In old age the germinal areas may disappear.
The medulla consists of the same cellular constituents as the cortex,
but the medullary cords rarely contain nodules (Bloom and Fawcett
1969, Park and Good 1974, Herbert and Wilkinson 1971).
LYMPHOCYTES. During embryological development, lymphocyte pro-
genitor cells migrate in the prenatal yolk sac through the liver to the
bone marrow, with lymphocyte maturation occurring outside the bone
marrow (Faith et al. 1980). Some cells enter the thymus, where they
proliferate and mature under the influence of thymic epithelial cells.
A small number of lymphocytes (T-cells) are released from the
thymus, circulate through the body, and rest temporarily in thymus-
dependent areas of the spleen and lymph nodes. Lymphocytes play a
central role in both cellular and humoral immunity, but these lympho-
cytes consist of two distinct populations, T-cells and B-cells, with dif-
ferent functions and properties (Vos 1977).
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LYMPHOCYTE TRANSFORMATION. The change in lymphocyte
morphology when the cells are cultured in the presence of phy-
tohaemagglutinin (or other mitogen) or of an antigen to which they
are primed. The changes include: increased size, increased cytoplasm,
and increased visibility of the nucleoli. After about 72 h the altered
cells resemble blast cells (Herbert and Wilkinson 1971).
LYMPHOID ELEMENTS. Cells of the circulating blood that originate in
the lymphatic tissue (e.g., lymphoctes and probably monocytes)
(Bloom and Fawcett 1969).
LYMPHOKINES. Soluble nonantibody products of lymphocyte activation
by antigen and mitogens that are thought to act as molecular media-
tors of cellular immune responses (Morely et al. 1978). Lymphokines
exhibit the following characteristics: (1) produce increased vascular
permeability following intradermal injection; (2) increase tritiated
thymidine uptake by lymphocytes in culture; and (3) inhibit macro-
phage migration in vitro. Lymphokines retain biological activity after
removal of the antigen that stimulated them. They may also be
involved in both expression and regulation of lymphoid cell activity,
and they may represent a pathway for the expression of T-cell func-
tion. Some lymphokines, such as the migration inhibition factor
(MIF), the chemotactic factor (CF), and the macrophage-activating
factor (MAP), influence macrophage functions (Faith et al. 1980).
LYMPHOPROLIFERATIVE RESPONSE. Lymphocyte proliferation
induced by mitogens; the basis for in vitro assays of T- and B-cell
function (Luster et al. 1982a).
LYSOZYMES. Enzymes found in many body fluids which lyse certain
bacteria, primarily gram-positive cocci, and enhance the bactericidal
action of antibody and complement for gram-negative bacteria (Park
and Good 1974, Herbert and Wilkinson 1971).
MACROPHAGE. Nucleated cells that have been classified as part of the
mononuclear phagocyte system, which replaces the concept of the reti-
culoendothelial system (Vos 1977). Macrophages, both fixed and free,
are involved in and seem to be necessary for the initiation and the
effector phases of humoral and cell-mediated immune reactions (Faith
et al. 1980). The mechanisms of these activities are not clear. Macro-
phages do not clonally divide, as do lymphocytes, and they are not
antigen specific (Pierce and Kapps 1976, as reported in Faith et al.
1980). They do, however, process the antigen, either breaking it down
internally or retaining some material on the cell surface. It is this
surface-bound antigen and perhaps a soluble factor from the macro-
phage which interact with lymphocytes in immune induction. Interac-
tions of macrophages and sensitized lymphocytes in the effector phase
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129
of the cell-mediated immune response are probably mediated by lym-
phokines (Faith et al. 1980).
MACROPHAGE AGGREGATION FACTOR (MAP). Soluble material
released by sensitized lymphocytes in culture (lymphokine), following
exposure to antigen, that causes the aggregation of peritoneal exudate
cells (Crowle 1975). MAF may be the same as MIF (Lolekha et al.
1970). Basis for in vitro test for delayed hypersensitivity.
MACROPHAGE MIGRATION INHIBITION FACTOR (MIF). Solu-
ble material (lymphokine) elaborated by T-cells, and perhaps other
cell types, following interaction with specific antigen in vitro. MIF
inhibits the in vitro migration of normal exudate cells. (Bloom and
Bennett 1966, Sharma 198la). Can be used to test for delayed hyper-
sensitivity in vitro.
MAF. Macrophage aggregation factor.
MEMORY CELLS. Long-lived small lymphocytes (B-cells), which are
differentiated from virgin B-cells that have been stimulated by
antigen. These cells are concentrated in the thoracic duct and are
responsible for secondary antibody responses (Koller 1981).
MIF. Macrophage Migration Inhibition Factor.
MITOGENS. Molecules of certain lectins derived from plants or similar
macromolecules that stimulate lymphocyte transformation (blast cell
formation) in vitro. This is analogous to the in vivo formation of blast
cells that precedes clonal division of B or T lymphocytes when there is
an antigen challenge (Sharma 1981d).
MITOMYCIN C. Antibiotic obtained from Streptomyces. Has antitumor
activity (Hawley 1981).
MYELOID ELEMENTS. Cells of the circulating blood that originate in
the myeloid tissue (e.g., erythrocytes and granular leukocytes) (Bloom
and Fawcett 1969).
NATURAL KILLER CELLS. A population of lymphocytes having
natural cell-mediated cytotoxicity against various tumor cell lines
(Luster et al. 1982b).
NONSPECIFIC IMMUNE RESPONSE. The means of disposal of
foreign and potentially harmful macromolecules, microorganisms, or
metazoa that, does not involve the recognition of antigen or the induc-
tion of an immune response. Nonspecific immunity is generalized and
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130
includes the activities of phagocytic cells, microbial inhibitors (lyso-
zymes), the interferon system, the complement system, and the kinin
system (Herbert and Wilkinson 1971, Vos 1981).
NUDE MOUSE. Born without a thymus and unable to make T-
lymphocytes. Responds poorly to thymus-dependent antigens, but
mounts a good antibody response to thymus-independent antigens.
Can serve as a laboratory model for a congenital immunodeficiency
syndrome and may be useful in testing B-cell responses (Hayward
1982).
NULL CELLS. Lymphocytes that have characteristics of neither B- or
T-cells. Useful in cellular cytoxicity assay (Luster et al. 1982b).
OPSONIN. (1) An antibody that, combined with antigen, facilitates the
phagocytosis of that antigen by a macrophage or polymorphonuclear
leucocyte. (2) Heat labile substances (nonantibody) found in blood
plasma that facilitate phagocytosis. Probably activated components of
complement, C3 in particular (Herbert and Wilkinson 1971).
PERIARTERIOLAR LYMPHOID SHEATH. T-lymphocyte-dependent
lymphoid masses arranged around arteries in the white pulp of the
spleen (Kociba 1981).
PEYER'S PATCHES. Gut-associated lymphoid tissue, histologically simi-
lar to secondary (peripheral) lymphoid tissue (Kociba 1981). T-cells
of Peyer's patches are reported to have specific suppressor activity fol-
lowing oral antigen administration (Ngan and Kind 1978, as reported
in Doe 1982).
PFC. Plaque-forming cells.
PHAGOCYTES. Cells, both fixed and free, that ingest and often digest
large particles such as effete blood cells, bacteria, protozoa, and dead
tissue cells. These include macrophages, Kupffer cells, and neutro-
phils. They are also active in ingesting foreign materials such as finely
divided carbon (Park and Good 1974, Herbert and Wilkinson 1971).
PHYTOHEMAGGLUTININS. Lectins extracted from the beans of
Phaseolus vulgaris or P. communis that stimulate lymphocyte
transformation and causes agglutination of certain red blood cells
(Herbert and Wilkinson 1971).
PLAQUE-FORMING CELLS (PFC). Cells obtained from the spleen or
other lymphoid organs of an animal, immunized against SRBC or a
protein antigen, which are mixed with sheep red blood cells (or
antigen-coated SRBC if protein antigen was used) and molten agar,
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131
poured into a petri dish, and allowed to set. The dish is then incu-
bated at 37°C. After incubation the release by each cell of hemolysins
(antibodies capable of lysing red blood cells in the presence of com-
plement) is revealed with the addition of complement to the dish. A
clear zone of hemolysis appears around each antibody-forming cell.
The PFC technique can be used to measure both primary and secon-
dary response (Jerne and Nordin 1963).
PLASMA CELLS. Antibody-forming cells, differentiated from B-
lymphocytes, that have a short half-life and secrete specific antibody
(Sharma 1981b).
PPD. Purified protein derivative.
PRECIPITATION. The formation of a visible complex on mixing soluble
antigen with antiserum specific for that antigen (Gell and Coombs
1975).
PRIMARY IMMUNE RESPONSE. The response of the body to the ini-
tial encounter with antigen. The response is weak, sluggish, and
short-lived (Herbert and Wilkinson 1971).
PRIMARY LYMPHOID ORGANS. The thymus and bursa-equivalent
(Luster et al. 1982b). The characteristics that distinguish primary
organs from other lymphoreticular tissues are as follows: (1) the lack
of cellular responses to antigen, such as those leading to antibody for-
mation; most cell-mediated immune responses; and the induction of
immunological memory (the medullary cells of the thymus, however,
can respond to mitogens and participate in graft-vs-host reactions);
(2) the intensive production of lymphoid cells in these organs,
independent of antigenic stimulation; and (3) a reticular framework of
epithelial cells that is not present in other lymphoreticular tissues
(White 1975).
PRIMED. (1) Of a whole animal: activation of appropriate cells in lym-
phoid tissue by exposure to antigen so that further contact of a
primed host with the same antigen usually results in a rapid and
vigorous secondary immune response. (2) Of cells: activation of a
given cell with antigen so that the cell can either produce more cells
that have been so activated, or synthesize immunoglobulin, or mediate
the reactions of cell-mediated immunity (Herbert and Wilkinson
1971).
PURIFIED PROTEIN DERIVATIVE (PPD). A soluble protein fraction,
precipitated from the medium in which M. tuberculosis has been
grown (Herbert and Wilkinson 1971).
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RED PULP (SPLENIC). Consists of large numbers of blood-filled
sinusoids in which phagocytosis of effete red cells takes place. Many
phagocytes and plasma cells are found in the cords of Billroth, a
modified lymphatic tissue that merges into the white pulp. In many
mammals the red pulp of the spleen contains various sized groups of
myelocytes, erythroblasts, megakaryocytes, and hemocytoblasts. The
red pulp is also heavily populated with all the cells of the circulating
blood (Bloom and Fawcett 1969, Herbert and Wilkinson 1971, Park
and Good 1974).
SECONDARY IMMUNE RESPONSE. Antibody production after the
second immunization. This response is characterized by faster
development, higher levels, and greater persistence of antibody titers
and a predominance of IgG (Roller 1981).
SECONDARY LYMPHOID ORGANS. The spleen, lymph nodes, cir-
cuiting lymphocytes, GALT, and BALT (Luster et al. 1982b).
SPECIFIC IMMUNE RESPONSE. The result of the recognition of
antigen in such a way that antibody or primed lymphocytes can react
specifically with that antigen. Specific immunity includes cell-
mediated immunity, humoral immunity, and immunologic tolerance
(Herbert and Wilkinson 1971). A major characteristic of specific
immunity is the establishment of immunological memory (Vos 1981).
SPLEEN. Situated in the bloodstream, unlike the lymphatic tissue, which
is scattered in the lymph stream. The spleen, as does the lymph node,
has a collagenous framework within which is suspended a reticular
framework. There is also a capsule which is thickened at the hilus
where the veins enter the organ and the arteries leave it. Continua-
tions of the capsule, the trabeculae, penetrate the organ and form part
of its framework. The reticular framework and the cells form the
splenic tissue, which is composed of typical lymphatic tissue, the white
pulp, and an atypical lymphatic tissue, the red pulp.
The spleen functions to (1) provide a site of differentiation for the
lymphocytes and hematopoetic stem cells, (2) trap the blood-borne
foreign and altered endogenous particles, (3) provide reservoir space
for the circulating blood, and (4) form antibodies, particularly IgM,
after stimulation by antigen in the circulating blood (Bloom and
Fawcett 1969, Herbert and Wilkinson 1971, Park and Good 1974).
SRBC. Sheep red blood cells. Thymus-dependent antigen, commonly used
in testing the humoral immune response (Vos 1981).
SUPPRESSOR T-CELLS. Lymphocytes that suppress antibody synthesis
by inhibiting B-cells (Koller 1981).
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T-LYMPHOCYTES. T-cells are stimulated by antigen to form a pool of
sensitized, antigen-specific lymphocytes which then can function as
memory cells, effector cells, killer cells, helper cells, or suppressor
cells (Faith et al. 1980). T-cells do not produce antibody (Roller
1979a).
THYMUS-DEPENDENT ANTIGEN. Antigen, such as SRBC or tetanus
toxoid, that elicit an immune response only when the thymus or
thymus cells are present (Vos 1981).
THYMUS-INDEPENDENT ANTIGEN. Antigens, such as lipopolysac-
charide, that can elicit an immune response by B-cells in the absence
of the thymus (Moreno 1982).
THYMUS. In mammals the thymus is considered to be the primary
lymphoid organ. In most mammals the thymus consists of two lobes
located in the anterior part of the thorax, ventral to the trachea
(Herbert and Wilkinson, 1971). Histologically, it consists mainly of
lymphocytes distributed throughout distinct cortical and medullary
areas on a network of reticular cells. These lymphocytes are
continually being produced in the gland, but following the neonatal
period only about 5% seem to leave. Hassal's corpuscles, which are
groups of flattened, concentrically arranged cells, are present in the
medulla.
During the neonatal period the thymus supplies small lymphocytes,
those responsible for cell-mediated immunity, to the blood, lymph and
thymus-dependent areas of the peripheral lymphoid organs.
The thymus also plays a part in humoral immunity. Neonatally thy-
mectonized animals can mount only a poor antibody response to cer-
tain antigens such as SRBC and tetanus toxoid.
Investigations of thymic functions have established that the thymus is
essential for the development of adaptive immunity. It appears to pro-
vide the microenvironment for the differentiation and proliferation of
primitive stem cells migrating from the bone marrow; to elaborate a
humoral factor that influences immunocompetency of lymphocytes
elsewhere; and to foster cell-mediated immunity (Bloom and Fawcett
1969, Herbert and Wilkinson 1971, Park and Good 1974).
WHITE PULP (SPLENIC). Contains a stroma composed of reticular
filters closely joined to primitive reticular cells and fixed macro-
phages. As in the lymph nodes, the meshes of the framework are filled
with free lymphocytes of various sizes which form both diffuse and
nodular lymphatic tissue. The amounts of dense and nodular lym-
phatic tissue vary in response to various stimuli. The lymphatic nodule
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134
contains a corona or peripheral zone and a central germinal center
that may be involved in the immune response (Bloom and Fawcett
1969, Herbert and Wilkinson 1971, Park and Good 1974).
3.7 LITERATURE CITED
Acton J, Myrvik QN. 1972. Nitrogen dioxide effects on alveolar macro-
phages. Arch Environ Health 24:48-52.
Adkinson NF, Jr. 1977. Environmental influences on the immune system.
Environ Health Perspect 20:97-103.
Allison AC. 1982. Macrophages and neutrophils. In: Clinical aspects of
immunology, 4th ed. Lachmann PJ, Peters DK, eds. Boston: Blackwell
Scientific Publications, pp. 101-129.
Andersson B, Blomgren H. 1971. Evidence for thymus-independent
humoral antibody production in mice against polyvinylphenolidone and E.
coli Lipopolysaccharide. Cell Immunol 2:411-424.
Archer DL, Smith BG, Bukovic-Wess JA. 1978. Use of an in vitro
antibody-producing system for recognizing potentially immunosuppressive
compounds. Int Arch Allergy Appl Immunol 56:90-93.
Ascoli M. 1902. Ueber den Mechanismus der Albuminurie durch
Eireiweiss. Munch med Wschr 49:398. (As reported in Gell and Coombs
1975).
Bailey DW, Usama B. 1962. A rapid method of grafting skin on tails of
mice. Transplant Bull 7:424-425.
Bennich H, Johansson SG. 1971. Structure and function of human immu-
noglobulin E. Advan Immunol 31:1.
Berenbaum MC. 1971. Immunosuppression by platinum diamines. Br J
Cancer 25:208-211.
Bianco C, Patrick R, Nussenzweig V. 1970. A population of lymphocytes
bearing a membrane receptor for antigen-antibody-complement complexes.
I. Separation and characterization. J Exp Med 132.
Billingham RE, Medawar PB. 1951. The technique of free skin grafting in
mammals. J Exp Biol 28:385-405.
Bloom BR, Bennett B. 1966. Mechanism of reaction in vitro associated
with delayed-type hypersensitivity. Science 153:80-82.
-------
135
Bloom BR, Stoner G, Fischetti V, Nowakowski M, Muschel R, Rubinstein
A. 1974. Products of activated lymphocytes (PALs) and the virus plaque
assay. In: Progress in immunology II, Vol. 3. Brent L, Holborow J, eds.
Amsterdam: North-Holland, pp. 133-144.
Bloom W, Fawcett DW. 1969. A textbook of histology, 9th ed. Philadel-
phia: W.B. Saunders Co., 858 pp.
Boyden SV. 1951. The adsorption of proteins on erythrocytes treated with
tannic acid and subsequent hemagglutination by antiprotein sera. J Exp
Med 93:107-120.
Bozelka B, Burkholder PM, Chang LW. 1977. Cadmium induced
immunosuppression and splenomegaly in mice. Am J Pathol
86(2):21a-22a.
Burnet M. 1959. The clonal selection theory of acquired immunity.
Nashville, TN: Vanderbilt Press, 209 pp.
Burrell R. 1977. Immunological reactions to inhaled physical and chemical
agents. In: Handbook of physiology. Section 9: Reactions to environmental
agents. Lee D, Falk H, Murphey S, Geiger S, eds. Bethesda, MD: Ameri-
can Physiological Society, pp. 285-298.
Carter PB, Bazin H. 1980. Immunology. In: The laboratory rat, Vol. II,
Research applications. Baker HJ, Lindsey JR, Weisbroth SH, eds. Ameri-
can College of Laboratory Animal Medicine Series.
Claman HN, Chaperon EA, Triplett RF. 1966. Thymus-marrow cell com-
binations. Synergism in antibody production. Proc Soc Exp Biol Med
122:1167.
Coombs RRA, Gell PGH. 1963. The classification of allergic reactions
underlying disease. In: Clinical aspects of immunology, Gell PGH, Coombs
RRA, eds. Oxford: Blackwell, pp. 317-359.
Coombs RRA, Gell PGH. 1975. Classification of allergic reactions respon-
sible for clinical hypersensitivity and disease. In: Clinical aspects of immu-
nology, 3rd ed. Gell PGH, Coombs RRA, Lachman P, eds. Oxford:
Blackwell, pp. 761-781.
Crowle AJ. 1975. Delayed hypersensitivity in the mouse. Adv Immunol
20:197-264.
Cunningham AJ, Szenberg A. 1968. Further improvements in the plaque
technique for detecting single antibody-forming cells. Immunology
14:599-601.
-------
136
David JR, David R. 1971. Assay for inhibition of macrophage migration.
In: In vitro methods in cell-mediated immunity. Bloom BR, Glade PR,
eds. New York: Academic Press, pp. 249-261.
Dean JH, Padarathsingh ML, Jerrells TR. 1979a. Application of immuno-
competence assays for defining immunosuppression. Ann NY Acad Sci
320:579-590.
Dean JH, Padarathsingh ML, Jerrells TR. 1979b. Assessment of immuno-
logical effects induced by chemicals, drugs, or food additives. I. Tier test-
ing and screening approach. Drug Chem Toxicol 2(1/2): 5-17.
De Bruin A. 1976. Biochemical toxicology of environmental agents. New
York: Elsevier Publishing Company, 1544 pp.
Doe WF. 1982. Immunological aspects of the gut. In: Clinical aspects of
immunology, 4th ed. Lachmann PJ, Peters DK, eds. Boston: Blackwell
Scientific Publications, pp. 985-1010.
Doull J. 1975. Factors influencing toxicology. In: Toxicology: the basic sci-
ence of poisons. Casarett LJ, Doull J, eds. New York: MacMillan Publish-
ing Co., Inc., pp. 133-147.
Dresser DW. 1978. Assays for immunoglobulin-secreting cells. In: Hand-
book of experimental immunology, Vol. 2, 3rd ed. Weir DM, ed. Oxford:
Blackwell Scientific Publications, pp. 28.1-28.25.
Duke L, Harshman S. 1971. In vitro induction of anti-DNP antibody by
immunogenic RNA from rabbit liver. Immunochemistry 8:431-445.
Engvall E, Perlmann P. 1971. Enzyme-linked immunosorbent assay
(ELISA). Quantitative assay of immunoglobulin G. Immunochemistry
8:871-874.
Esber H, Menninger F, Bogden A. 1973. Immunological deficiency
associated with cigarette smoke inhalation by mice: primary and secondary
hemagglutinin response. Arch Environ Health 27:99-104.
Exon JH, Koller LD, Kerkvliet NI. 1979. Lead-cadmium interaction:
effects on viral-induced mortality and tissue residues in mice. Arch
Environ Health 34:469-475.
Exon JH, Patton NM, Koller LD. 1975. Hexamitiasis in cadmium-exposed
mice. Arch Environ Health 31:463-464.
-------
137
Faith RE, Luster MI, Kimmel CA. 1979. Effect of combined pre- and
postnatal lead exposure on cell-mediated immune function. Clin Exp
Immunol 35:413-420.
Faith RE, Luster MI, Vos JG. 1980. Effects on immunocompetence by
chemicals of environmental concern. In: Reviews in biochemical toxicology
2. Hodgson E, Bend JR, Philpot RM, eds. New York: Elsevier/North Hol-
land, pp. 173-211.
Flax MH, Waksman BH. 1962. Delayed cutaneous reactions in the rat. J
Immunol 89:496-504.
Ford WL. 1978. Measurement of graft-versus-host activity. In: Handbook
of experimental immunology, Vol. 2, 3rd ed. Weir DM, ed. Oxford:
Blackwell Scientific Publications, pp. 30.1-30.12.
Ford WL, Burr W, Simonsen M. 1970. A lymph node weight assay for the
graft-versus-host activity of rat lymphoid cells. Transplantation
10:258-266.
Fraker PJ. 1980. The antibody-mediated and delayed type hypersensitivity
response of mice exposed to polybrominated biphenyls. Toxicol Appl Phar-
macol 53(l):l-7.
Friend M, Trainer DO. 1970. Polychlorinated biphenyl: interaction with
duck hepatitis virus. Science 170:1314-1316.
Gabliks J, Askari EM, Yolen N. 1973. DDT and immunological responses.
I. Serum antibodies and anaphylactic shock in guinea pigs. Arch Environ
Health 26(6):305-308.
Gainer JH. 1977. Effects of heavy metals and of deficiency of zinc on
mortality rates in mice infected with encephalomyocarditis virus. Am J
Vet Res 38:869-872.
Gardner DE, Hu EPC, Graham JA, Chernoff N, and Hueter FG. 1979.
Review of some toxicology models to assess health effects of environmental
pollutants. In: In vitro toxicity testing of environmental agents, current and
future possibilities. Part B: Development of risk assessment guidelines.
Kolber AR, Wong TK, Grant LD, DeWoskin RS, Hughes TJ, eds. New
York: Plenum Press, pp. 443-460.
Gaworski CL, Sharma RP. 1978. The effects of heavy metals on
3H-thymidine uptake in lymphocytes. Toxicol Appl Pharmacol 46:305-313.
-------
138
Gell PGH, Coombs RRA. 1975. Basic immunological methods. In: Clini-
cal aspects of immunology, 3rd ed. Gell PGH, Coombs RRA, Lachmann
PJ, eds. Oxford: Blackwell Scientific Publications, pp. 3-53.
Gigli I. 1982. Immunological aspects of skin diseases. In: Clinical aspects
of immunology, 4th ed. Lachmann PJ, Peters DK, eds. Boston: Blackwell
Scientific Publications, pp.790-821.
Goldstein AL, Aronow, Kalman SM. 1974. Drug allergy. In: Principles of
drug action: the basis of pharmacology, 2nd ed. New York: John Wiley
and Sons, pp. 489-515.
Goldstein L, Tyler WS, Hoeprich PD, Eagle C. 1971. Ozone and antibac-
terial defense mechanisms of the murine lung. Arch Intern Med
127:1099-1102.
Graham JA, Gardner DE, Miller FJ, Daniels MJ, and Coffin DL. 1975.
Effect of nickel chloride on primary antibody production in the spleen.
Environ Health Perspect 12:109-113.
Graham JA, Miller FJ, Daniels MJ, Payne EA, and Gardner DE. 1978.
Influence of cadmium, nickel and chromium on primary immunity in mice.
Environ Res 16(l-3):77-87.
Halliday WJ, Miller S. 1972. Leukocyte adherence inhibition: a simple
test for cell-mediated immunity and serum blocking factors. Int J Cancer
9:477-483.
Hawley GG. 1981. The condensed chemical dictionary, 10th ed. New
York: Van Nostrand Reinhold Co., p. 697.
Hayward A. 1982. Immunodeficiency. In: Clinical aspects of immunology.
Lachmann PJ, Peters DK, eds. Boston: Blackwell Scientific Publications,
pp. 1658-1712.
Herbert WJ. 1973. Passive hemagglutination with special reference to the
tanned cell technique. In: Handbook of experimental immunology, Vol. 1,
2nd ed. Weir DM, ed. Oxford: Blackwell Scientific Publications,
pp. 20.1-20.20.
Herbert WJ, Wilkinson PC. 1971. A dictionary of immunology. Oxford:
Blackwell Scientific Publications, 195 pp.
Holt PG, Chalmer J, Roberts LM, Papadimitriou JM, Thomas WR, Keast
D. 1976. Low-tar and high-tar cigarettes: comparison of effects in mice.
Arch Environ Health 31:258-265.
-------
139
Holt PG, Keast D. 1977. Environmentally induced changes in immunologi-
cal function: acute and chronic effects of inhalation of tobacco smoke and
other atmospheric contaminants in man and experimental animals. Bac-
teriol Rev 41(1):205-216.
Holt PG, Roberts LM, Keast D. 1974. The MLA test: a rapid micropro-
cedure for the demonstration of delayed type hypersensitivity in vitro. J Int
Res Comm 2:1416.
Ishizaka K, Ishizaka T. 1971. Mechanisms of reaginic hypersensitivity: a
review. Clinical Allergy 1:9.
Jerne NK, Nordin AA. 1963. Plaque formation in agar by single
antibody-producing cells. Science 140:405.
Johnson GD, Holborow EJ. 1973. Immunofluorescence. In: Handbook of
experimental immunology, Vol. 1, 2nd ed. Weir DM, ed. Oxford:
Blackwell Scientific Publications, pp. 18.1-18.20.
Jones RH, Williams RL, Jones AM. 1971. Effects of heavy metal on the
immune response. Proc Soc Exp Biol Med 137:1231-1236.
Karol MH. 1983. Concentration-dependent immunologic response to
toluene diisocyanate (TDI) following inhalation exposure. Toxicol Appl
Pharmacol 68:229-241.
Karol MH, Hauth BA, Alarie Y. 1979. Pulmonary hypersensitivity to
hexyl isocyanate-ovalbumin aerosol in guinea pigs. Toxicol Appl
Pharmacol 51(1):73-80.
Karol M H, Hauth BA, Riley EJ, Magreni CM. 1981. Dermal contact
with toluene diisocyanate (TDI) produces respiratory tract hypersensitivity
in guinea pigs. Toxicol Appl Pharmacol 58:221-230.
Karol MH, loset HH, Riley EJ, Alarie YC. 1978. Hapten-specific respira-
tory hypersensitivity in guinea pigs. Am Ind Hyg Assoc J 39(7):546-556.
Kauffmann BM, White KL, Sanders VM, et al. 1982. Humoral and cell-
mediated immune status in mice exposed to chloral hydrate. Environ
Health Perspect 44:147-151.
Kennedy J, Axelrad M. 1971. An improved assay for hemolytic plaque-
forming cells. Immunology 20:253-257.
-------
140
Klecak G. 1977. Identification of contact allergens: predictive tests in
animals. In: Advances in modern toxicology, Vol. 4: Dermatotoxicology
and pharmacology. Marzulli FN, Maibach HI, eds. Washington, DC:
Hemisphere Publishing Corporation, pp. 305-339.
Kociba RJ. 1981. Morphologic considerations in the detection of immune
suppression in routine toxicity studies. In: Immunologic considerations in
toxicology, Vol. II. Sharma RP, ed. Boca Raton, FL: CRC Press,
pp. 123-131.
Roller LD. 1979a. Effects of environmental contaminants on the immune
system. Adv Vet Sci Comp Med 23:267-295.
Koller LD. 1979b. Some immunological effects of lead, cadmium, and
methylmercury. Drug Chem Toxicol 2(l/2):99-110.
Koller LD. 1981. Humoral immunity. In: Immunologic considerations in
toxicology. Sharma RP, ed. Boca Raton, FL: CRC Press, pp. 19-30.
Koller L D. 1982. In vitro assessment of humoral immunity following
exposure to heavy metals. Environ Health Perspect 43:37-39.
Koller LD, Brauner JA. 1977. Decreased B-lymphocyte response after
exposure to lead and cadmium. Toxicol Appl Pharmacol 42:621-624.
Koller LD, Exon JH, Brauner JA. 1977. Methylmercury: decreased anti-
body formation in mice. Proc Soc Exp Biol Med 155:602-604.
Koller LD, Exon JH, Roan JG. 1975. Antibody suppression by cadmium.
Arch Environ Health 30(12):598-601.
Koller LD, Kovacic S. 1974. Decreased antibody formation in mice
exposed to lead. Nature 250:148-150.
Koller LD, Roan JG, Kerkvliet N. 1979. Mitogen stimulation of lympho-
cytes in CBA mice exposed to lead and cadmium. Environ Res 19:177-188.
Landsteiner K, Jacobs J. 1935. Studies on the sensitization of animals with
simple chemical compounds. I. J Exp Med 61:643-656.
Landsteiner K, Jacobs J. 1936. Studies on sensitization of animals with
simple chemical compounds. II. J Exp Med 64:625-639.
Lefford MJ. 1974. The measurement of tuberculin hypersensitivity in rats.
Int Arch Allergy 47:570-585.
-------
141
Ligo RN, James RB. 1974. Occupational skin disease: the scope of the
problem. Cutis 13:527. (As reported in Gigli 1982).
Lolekha S, Dray S, Gotoff SP. 1970. Macrophage aggregation In vitro: A
correlate of delayed hypersensitivity. J Immunol 104:296-304.
Loomis TA. 1974. Abnormal response to chemicals. In: essentials of toxi-
city, 2nd ed. Philadelphia: Lea and Febiger, pp. 105-117.
Loose LD, Pittman KA, Benitz KF, and Silkworth JB. 1977. Polychlori-
nated biphenyl and hexachlorobenzene induced humoral immunosuppres-
sion. J Reticuloendothel Soc 22(3):255-272.
Loose LD, Pittman KA, Benitz KF, Silkworth JB, Mueller W, Coulston F.
1978. Environmental chemical-induced immune dysfunction. Ecotoxicol
Environ Saf 2(2):173-198.
Loose LD, Silkworth JB, Mudzinski SP, Pittman KA, Benitz KF, Mueller
W. 1979. Modification of the immune response by organochlorine xenobi-
otics. Drug Chem Toxicol 2(1-2): 111-132.
Luster M I, Dean JH, Boorman GA. 1982a. Cell-mediated immunity and
its application in toxicology. Environ Health Perspect 43:31-36.
Luster MI, Dean JH, Moore JA. 1982b. Evaluation of immune functions
toxicology. In: Principles and methods of toxicology. Hayes AW, ed. New
York: Raven Press, pp. 561-586.
Luster MI, Faith RE. 1979. Assessment of immunological alterations
caused by halogenated aromatic hydrocarbons. Ann NY Acad Sci
320:572-578.
Luster MI, Faith RE, Kimmel CA. 1978. Depression of humoral immun-
ity in rats following chronic developmental lead exposure. J Environ Pathol
Toxicol 1:379-402.
Luster MI, Faith RE, Lawson LD. 1979. Effects of 2,3,7,8-tetrachlorodi-
benzofuran (TCDF) on the immune system in guinea pigs. Drug Chem
Toxicol 2(l-2):49-60.
Mancini G, Carbonara AO, Heremans JE. 1965. Immunochemical quanti-
tation of antigens by single radial immunodiffusion. Immunochem 2:235.
Marzulli FN, Maibach HI. 1977. Contact allergy: predictive testing in
humans. In: Advances in modern toxicology, Vol. 4, Dermatology and
pharmacology. Marzulli FN, Maibach HI, eds. Washington, DC: Hemi-
sphere Publishing Corporation, pp. 353-372.
-------
142
Miller JFAP. 1975. Induction of the immune (allergic) response. In: Clini-
cal aspects of immunology, 3rd ed., Gell PGH, Coombs RRA, Lachmann
PJ, eds. Oxford: Blackwell Scientific Publications, pp. 447-469.
Miller JFAP, Mitchell GF. 1968. Cell to cell interaction in the immune
response. I. Hemolysin-forming cells in neonatally thymectomized mice
reconstituted with thymus of thoracic duct lymphocytes. J Exp Med
128:801-820.
Mishell RI, Dutton RW. 1967. Immunization of dissociated mouse spleen
cell cultures from normal mice. J Exp Med 126:423-442.
Moller G, Michael G. 1971. Frequency of antigen-sensitive cells to
thymus-independent antigens. Cell Immunol 2:309-316.
Moore JA. 1979. The immunotoxicology phenomenon (polychlorinated
biphenyls, halogenated hydrocarbons). Drug Chem Toxicol 2(1/2): 1-4.
Moore JA, Faith RE. 1976. Immunologic response and factors affecting its
assessment. Environ Health Perspect 18:125-131.
Moore JA, Luster MI, Gupta BN, McConnell EE. 1978. lexicological
and immunological effects of a commercial polybrominated biphenyl
mixture (Firemaster FF-1). Toxicol Appl Pharmacol 45(l):295-296.
Moreno C. 1982. Tolerance. In: Clinical aspects of immunology, 4th ed.
Lachmann PJ, Peters DK, eds. Boston: Blackwell Scientific Publications,
pp. 199-242.
Morley J, Wolstencroft RA, Dumonde DC. 1978. Measurement of lym-
phokines. In: Handbook of experimental immunology, Vol. 2, 3rd ed. Weir
DM, ed. Oxford: Blackwell Scientific Publications, pp. 27.1-27.28.
Munson AE, Sanders VM, Douglas KA, et al. 1982. In vivo assessment of
immunotoxicity. Environ Health Perspect 43:41-52.
Muller S, Gilbert KE, Krause C, Gross U, Age-Stehr JK, and Diamant-
stein T. 1977. Suppression of delayed-type hypersensitivity of mice by
lead. Experintia 33(5):667-668.
Ngan J, Kind LS. 1978. Suppressor T cells for IgE and IgG in Peyer's
patches of mice made tolerant by the oral administration of ovalbumin. J
Immunol 120:861. (As reported in Doe 1982).
Nicolin A., Cosco A, Marelli O, Sarra F. 1980. Toxicology of immunity.
In: The principles and methods in modern toxicology. Galli CL, Murphy
SD, Paoletti R, eds. Amsterdam: Elsevier/North Holland, pp. 295-310.
-------
143
Nulsen A, Holt PG, Keast . 1974. Cigarette smoking, air pollution and
immunity: A model system. Infect. Immun. 10:1226-1229.
OECD. 1981. Organisation for Econonic Co-operation and Development.
Skin sensitization. In: OECD guidelines for testing of chemicals. Paris,
France: OECD, Section 406, pp. 1-9.
Oehler JR, Herberman RB, Campbell DA, and Deu JK. 1977. Inhibition
of rat mixed lymphocyte cultures by suppressor macrophages. Cell Immu-
nol 29:r38-m50.
Ohi G, Fukuda M, Seto H, Yagyu H. 1976. Effect of methylmercury on
humoral immune responses in mice under conditions simulated to practical
situations. Bull Environ Contam Toxicol 15:175-180.
Ouchterlony O, Nillson LA. 1973. Immunodiffusion and immunoelectro-
phoresis. In: Handbook of experimental immunology, Vol. 1, 2nd ed. Weir
DM, ed. Oxford: Blackwell Scientific Publications, pp. 19.1-19.39.
Paranjpe MS, Boone CW. 1972. Delayed hypersensitivity to Simian virus
40 tumor cells in BALB/c mice demonstrated by a radioisotopic footpad
assay. J Nat Cancer Inst 48:563-566.
Park BH, Good RA. 1974. Principles of modern immunobiology: basic and
clinical. Philadelphia: Lea and Febiger, 618 pp.
Pepys J. 1976. The role of industrial agents in the etiology of asthma. Adv
Asthma Allergy (Fisons Labs.) 3:1.
Peterman GM, Wust CJ. 1975. Comparison of three hemolytic plaque
assays using sheep and mouse erythrocytes as antigens in rats. Proc Soc
Exp Biol Med 150:289-294.
Pierce CW, Kapp JA. 1976. The role of macrophages in antibody
responses in vitro. In: Immunobiology of the macrophage. Nelson DS, ed.
New York: Academic Press, p. 2. (As reported in Faith et al. 1980).
Platts-Mills TAE. 1982. Type I or immediate hypersensitivity: Hay fever
and asthma. In: Clinical aspects of immunology. Lachmann PJ, Peters
DK, eds. Boston: Blackwell Scientific Publications, pp. 580-686.
Plotz P, Talal N, Asofsky R. 1968. Assignment of direct and facilitated
hemolytic plaques in mice to specific immunoglobulin classes. J Immunol
100:744-751.
-------
144
Polak L. 1977. Immunological aspects of contact sensitivity. In: Advances
in modern toxicology, Vol. 4, Dermatotoxicology and pharmacology. Mar-
zulli FN, Maibach HI, eds. Washington: Hemisphere Publishing Corpora-
tion, pp. 225-288.
Raney JP, McLennan BD. 1979. A modified quantitative precipitin test
which is rapid, sensitive and reproducible. J Immunol Methods
29(1):65-70.
Robertson M. 1981. Genes of lymphocytes. Diverse means to antibody
diversity. Nature 290:625-627.
Rowe DS. 1975. Immunoglobulins. In: Clinical aspects of immunology, 3rd
ed. Gell PGH, Coombs RRA, Lachmann PJ, eds. Oxford: Blackwell
Scientific Publications, pp. 285-306.
Ruitenberg EJ, Steerenberg PA, Brosi BJM. 1975. Micro-system for the
application of ELISA (enzyme-linked immunosorbent assay) in the serodi-
agnosis of Trichinella spiralis infections. Medikon Nederland 4:30-31.
Rylander R. 1969. Alterations of lung defense mechanisms against air-
borne bacteria. Arch Environ Health 18:551-555.
Rylander R. 1971. Lung clearance of particles and bacteria. Effects of
cigarette smoke exposure. Arch Environ Health 23:321-326.
Sanders VM, Tucker AN, White KL, et al. 1982. Humoral and cell-
mediated immune status in mice exposed to trichloroethylene in the drink-
ing water. Toxicol Appl Pharmacol 62:358-368.
Schuller GB, Kaufmann BM, Borzelleca JF, Sanders VM, Munson AE.
1978. Effect of four haloalkanes on humoral and cell-mediated immunity
in mice. Toxicol Appl Pharmacol 45(1):329.
Sharma RP. 198la. Cellular immunity. In: Immunologic considerations in
toxicology, Vol. 1. Sharma RP, ed. Boca Raton, FL: CRC Press, pp.
31-36.
Sharma RP. 198 Ib. Glossary. In: Immunologic considerations in toxicol-
ogy, Vol. 2. Sharma RP, ed. Boca Raton, FL: CRC Press, pp. 157-160.
Sharma RP. 1981c. Immunity and the immune system. In: Immunologic
considerations in toxicology, Vol. 1. Sharma RP, ed. Boca Raton, FL:
CRC Press, pp. 9-18.
-------
145
Sharma RP. 198Id. Splenic lymphocyte transformation in culture as a tool
for immunotoxicologic evaluation of chemicals. In: Immunologic considera-
tions in toxicology, Vol. 2. Sharma RP, ed. Boca Raton, FL: CRC Press,
pp. 133-145.
Sharma RP. 1981e. Tissue or cell injury in immune mechanisms. In:
Immunologic considerations in toxicology, Vol. 1. Sharma RP, ed. Boca
Raton, FL: CRC Press, pp. 37-44.
Sharma RP, Kociba RJ, Gehring PJ. 1978. Immunotoxicologic effects of
2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Toxicol Appl
Pharmacol45(l):333.
Silkworth JB, Loose LD. 1981. Assessment of environmental
contaminant-induced lymphocyte dysfunction. Environ Health Perspect
39:105-128.
Smith RT. 1972. Specific recognition reactions at the cellular level in
mouse lymphoreticular cell subpopulations. Transplant Rev 11:178-216.
Smith SH, Sanders VM, Barrett BA, Borzelleca JF, Munson AE. 1978.
Immunotoxicological evaluation on mice exposed to polychlorinated
biphenyls. Toxicol Appl Pharmacol 45(1):330.
Speirs RS, Speirs EE. 1979. In vivo models for assessing effects of drugs
and toxicants on immunocompetence. Drug Chem Tox 2(1-2): 19-33.
Spyker JM. 1975. Assessing the impact of low level chemicals on develop-
ment: behavioral and latent effects. Fed Proc Fed Am Soc Exp Biol
34:1835-1844.
Stavitsky AB. 1954. Micromethods for the study of proteins and antibo-
dies. I. Procedure and general applications of hemagglutination and
hemagglutination inhibition reactions with tannic acid and protein-treated
red blood cells. J Immunol 72:360-367.
Stobo JD, Rosenthal AS, Paul WE. 1972. Functional heterogeneity of
murine lymphoid cells. I. Responsiveness to and surface binding of con-
canavalin A and phytohaemagglutinin. J Immunol 108:1-17.
Stuart AE, Habeshaw JA, Davidson AE. 1978. Phagocytes in vitro. In:
Handbook of experimental immunology, Vol. 2, 3rd ed. Weir DM, ed.
Oxford: Blackwell Scientific Publications, pp. 31.1-31.30.
Tada NK, Itakura K, Aizawa M. 1974. Genetic control of the antibody
response in inbred rats. J Immunogenet 1:265-273. (As reported in Luster
and Faith 1979).
-------
146
Thigpin JE, Faith RE, McConnell EE, and Moore JA. 1975. Increased
susceptibility to bacterial infection as a sequela of exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin. Infect Immun 12:1319-1324.
Thomas PT, Hinsdill RD. 1978. Effect of polychlorinated biphenyls on the
immune responses of Rhesus monkeys and mice. Toxicol Appl Pharmacol
44(l):41-52.
Thomas WR, Holt PG, Keast D. 1973. Effect of cigarette smoking on pri-
mary and secondary immune response in mice. Nature (London)
243:240-241.
Thomas WR, Holt PG, Keast D. 1974a. Alterations in the primary
immune response of mice by exposure to fresh cigarette smoke. Int Arch
Allergy Appl Immunol 46:481-486.
Thomas WR, Holt PG, Keast D. 1974b. Mice chronically exposed to fresh
cigarette smoke. Arch Environ Health 27:372-375.
Thor DE. 1971. The capillary tube migration inhibition technique applied
to human peripheral lymphocytes using the guinea pig peritoneal exudate
as the indicator cell population. In: In vitro methods in cell-mediated
immunity. Bloom B, Glade P, eds. New York: Academic Press,
pp. 273-280.
Thorpe PE, Knight SC. 1974. Microplate culture of mouse lymphoid cells.
I. Quantitation of responses to allogeneic lymphocytes, endotoxins and
phytomitogens. J Immunol Methods 5:387-404.
Thurman GR, Goldstein AL, Kilian DJ. 1978. The effects of organic com-
pounds used in the manufacture of plastics on the responsivity of murine
and human lymphocytes. Toxicol Appl Pharmacol 44(3):617-641.
Treagan L. 1975. Metals and the immune response: a review. Res Com-
mun Chem Pathol 12(2): 189-220.
Tripathy SP, Mackaness GB. 1969. The effect of cytotoxic agents on the
primary immune response to Listeria monocytogenes. J Exp Med 130:1-16.
Tucker AN, Sanders VM, Hallett P, Kauffmann B, Munson AE. 1982. In
vitro immunotoxicological assays for detection of compounds requiring
metabolic activation. Environ Health Perspect 43:123-127.
Turk JL. 1975. The mechanism and mediators of cellular hypersensitivity.
In: Clinical aspects of immunology, 3rd ed., Gell PGH, Coombs RRA,
Lachmann PJ, eds. Oxford: Blackwell Scientific Publications,
pp. 1019-1042.
-------
147
Urso P, Gengozian N. 1973. T cell deficiency in mouse allogeneic radia-
tion chimeras. Immunol 111:712-719.
Urso P, Gengozian N. 1980. Depressed humoral immunity and increased
tumor incidence in mice following in utero exposure to benzo[a]pyrene. J
Toxicol Environ Health 6:569-576.
van Furth R, van Zwet TL, Leijh PCJ. 1978. In vitro determination of
phagocytosis and intracellular killing by polymorpho-nuclear and mononu-
clear phagocytes. In: Handbook of experimental immunology, Vol. 2, 3rd
ed. Weir DM, ed. Oxford: Blackwell Scientific Publications,
pp. 32.1-32.19.
Vassallo CL, Domm BM, Poe RH, Duncombe ML, Gee JBL. 1973. NO2
Gas and NO2 effects on alveolar macrophage phagocytosis and metabol-
ism. Arch Environ Health 26:270-274.
Verschuuren HG, Ruitenberg EJ, Peetoom F, Hellman PW, van Esch GJ.
1970. Influence of triphenyltin acetate on lymphatic tissue and immune
responses in guinea pigs. Toxicol Appl Pharmacol 16:400-410.
Vos JG. 1977. Immune suppression as related to toxicology. Crit Rev Tox-
icol 5(1):67-101.
Vos JG. 1981. Screening and function tests to detect immune suppression
in toxicity studies. In: Immunologic considerations in toxicology, Vol. II.
Sharma RP, ed. Boca Raton, FL: CRC Press, pp. 109-122.
Vos JG, Buys J, Beekhof P, Hagenaars AM. 1979a. Quantification of total
IgM and IgG and specific IgM and IgG to a thymus-independent (LPS)
and a thymus dependent (tetanus toxoid) antigen in the rat by Enzyme-
linked Immunosorbent Assay (ELISA). Ann NY Acad Sci 320:518-534.
Vos JG, De Roij T. 1972. Immunosuppressive activity of a polychlorinated
biphenyl preparation on the humoral immune response in guinea pigs. Tox-
icol Appl Pharmacol 21:549-555.
Vos JG, Moore JA. 1974. Suppression of cellular immunity in rats and
mice by maternal treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int
Arch Allergy Appl Immunol 47:777-794.
Vos JG, Moore JA, Zinkl JG. 1973. Effect of 2,3,7,8-tetrachlorodi-
benzo-p-dioxin on the immune system of laboratory animals. Environ
Health Perspect 5:149-162.
-------
148
Vos JG, Van Logten MJ, Kreeftenberg JG, and Kruizinga W. 1979b.
Hexachlorobenzene-induced stimulation of the humoral immune response
in rats. Ann NY Acad Sci 320:535-550.
Vos JG, Van Logten MJ, Kreeftenberg JG, Steerenberg PA, Kruizinga W.
1979c. Effect of hexachlorobenzene on the immune system of rats follow-
ing combined pre- and postnatal exposure. Drug Chem Toxicol
2(l-2):61-76.
White RG. 1975. Immunological functions of lymphoreticular tissues. In:
Clinical aspects of immunology, 3rd ed. Gell PGH, Coombs RRA,
Lachmann PJ, eds. Oxford: Blackwell Scientific Publications, pp. 411-445.
White KL, Jr., Sanders VM, Kauffmann BM et al. Immunotoxicological
investigations in the mouse I. General approach and methods. (In press).
Yamada H. Yamada A. 1969. Antibody formation against
2,4-dinitrophenyl-hapten at the cellular level. J Immunol 103:357-363.
Zarkower A. 1972. Alterations in antibody response induced by chronic
inhalation of SO2 and carbon. Arch Environ Health 25:45-50.
Zarkower A, Morges M. 1972. Alteration in antibody responses induced
by carbon inhalation: a model system. Infect Immun 5:915-920.
3.8 GENERAL REFERENCES
Lachmann PJ, Peters DK, eds. 1982. Clinical aspects of immunology, 4th
ed., Vols. I and II. Boston: Blackwell Scientific Publications, 1751 pp.
Weir DM, ed. 1978. Handbook of experimental immunology, 3rd ed.
Oxford: Blackwell Scientific Publications.
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4. INHALATION TOXICOLOGY
Michael G. Ryon
4.1. INTRODUCTION
Inhalation toxicology is the branch of toxicology that deals with expo-
sure by the inhalation or respiratory route and with the resulting effects
(Campbell 1976). Inhalation toxicology exists as a special subset of toxi-
cology because of the unique exposure situation of the lungs and the spe-
cial technique and equipment needs of the inhalation toxicity test.
The lung is the organ with the greatest direct contact between the
environmental air (and the contaminants therein) and its vital, functional
elements as a direct unavoidable consequence of living (Casarett 1975).
This contact of the lung with the air is even more significant because of
the tremendous surface area and number of structures involved. There are
several hundred million alevoli in the human lung, with an approximate
surface area of 100 m2 when distended (Hatch and Gross 1964, Cam-
bridge 1973). These are serviced by 2000 km of lung capillaries that are
separated by only 1 fj,m from the gas in the alevoli (Hatch and Gross
1964, USDHEW 1977). Due to this large surface area/blood contact, even
the rate of intake of highly soluble compounds can be ten times greater for
the inhalation route than for the oral route. Added to these structural con-
siderations is the tremendous amount of air (10 m3) processed each day by
the lungs (Hatch and Gross 1964). Thus, the contact with a potential lung
toxicant can be extremely hazardous. The concern about the impact of air
pollution exposure on man (Stokinger 1953) and the tremendous death
rate from lung cancer [in 1967 in the United States over 50,000 deaths or
one every 10 minutes (Saffiotti 1969)] underscore the importance of inha-
lation toxicity studies.
Inhalation toxicity also receives special emphasis because of the unique
equipment and techniques required for its evaluation. Because the lungs
not only are responsible for maintaining the oxygen supply and the excre-
tion of carbon dioxide, but also have a role in several body defense sys-
tems, inhalation toxicity studies must deal with the systemic as well as
specific lung damage (Casarett 1975). Thus, there is no way that only one
standard and accepted protocol can be used for all inhalation studies
(MacFarland 1975). However, base guidelines can be and have been pro-
posed to ensure that minimum requirements of toxicity testing are being
met; these guidelines should still allow the flexibility and freedom to sub-
stitute more stringent or comprehensive procedures (Gross 198la, USEPA
1982). Inhalation studies are faced with unique problems with regard to
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determining dosage parameters and designing exposure conditions. A spe-
cial technology has been developed to duplicate man's environment for
study in the laboratory, including exposure chamber designs, generating
and monitoring equipment for the various types of test atmospheres, and
procedures for the evaluation of effects (Drew and Laskin 1973). These
special requirements increase the average cost of a toxicity study using
inhalation exposure to two or three times that of a study done by oral
exposure (WHO 1978).
It is for these reasons that inhalation toxicology studies have received
special emphasis. This report will discuss further the special needs of the
inhalation study and in particular will cover basic equipment considera-
tions and unique additions to test designs. Current guidelines for inhalation
testing are not emphasized in this report; for a discussion of these, see
Gross (198la) and USEPA (1982).
4.2 GENERAL TEST DESIGN
4.2.1 Introduction
Toxicity tests designed to evaluate the effects of a chemical after inha-
lation exposure involve many of the same variables as tests using other
exposure routes. All toxicity tests require the selection of test species, test
duration, exposure regimen, dosage levels, and procedures to evaluate toxic
effects. These variables will be discussed briefly in this section with
emphasis on those aspects restricted to inhalation exposure.
4.2.2 Test Species
In selecting the most suitable species for inhalation tests, the anatomi-
cal and physiological similarity between human and test animal respiratory
systems must be considered along with metabolic similarity and
economic/practicality restrictions. The anatomical and physiological differ-
ences include variations in the structure of nasal cavities, trachea, and
lower respiratory tree, in the site of particle deposition, and in the clear-
ance and defense mechanisms (Giovacchini 1972). Table 4.1 lists some of
the physiological variations between man and various test species. Because
no single animal species is anatomically similar to man in all respects,
researchers have used mice, monkeys, rats, rabbits, hamsters, guinea pigs,
cats, and dogs for inhalation studies (Drew and Laskin 1973, WHO 1978).
McLaughlin, Tyler, and Canada studied the respiratory system of vari-
ous animals, examining such subgross anatomical variables as the lobular-
ity, pleura and interlobular septa, arterial supply to the pleura, and termi-
nal bronchioles. They classified species according to their similarity to
humans and concluded that the horse is anatomically the most similar to
man (McLaughlin et al. 196la, 1961b). The rat, rabbit, guinea pig, dog,
cat, and monkey were considered to have basically the same type of lung
structure and degree of relative similarity to man (McLaughlin et al.
1966). Among these species, Roe (1968) found that the smaller species
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Table 4.1. Some Physiological Indices of Man and Animals*
Physiological index
Body surface (m2)
Relation body surface to body
weight (m2/kg)
Basal metabolism (kJ/kg)
Frequency of respiration (min)
Size of alveoli (nm)
Surface of lungs (m2)
Relation of lung surface to
body weight (m2/kg)
Inhaled air (mL)
Lung ventilation (mL/min)
Relation of lung ventilation
to body weight (mL/min/g)
Consumption of oxygen (mL/kg/h)
Elimination of CO2 (mL/kg/h)
Coefficient of respiration
Pulse frequency for 1 min
Man
1.8
0.0257
105
14-18
150
50
0.7
616
8732
0.13
203.1
168.8
0.82
70-72
Dog
0.528
0.044
222
10-30
100
100
8.3
40-60
NG
NG
3600
NG
NG
90-120
Cat
0.2
0.066
NGb
20-30
100
7.2
2.8
NG
1000
0.30
9420
NG
NG
120-180
Rabbit
0.18
0.072
188
50-100
NG
5.21
2.5
NG
600
0.29
522.7
NG
0.83
150-240
Guinea
Pig
0.040
0.12
360
80-135
NG
1.47
3.2
1.75
155
0.33
2180
NG
NG
206-280
Rat
0.030
0.15
615
110-135
50
0.56
3.3
0.865
73
0.05
2199
2650
0.82
300-500
Mouse
0.006
0.3
711
140-210
30
0.12
5.4
0.154
25
1.24
3910
4240
0.85-1.33
520-780
aAdapted from Sanockij (1970a) as reported in
bNG = not given.
WHO (1978).
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were markedly different from man, especially in the factors that affect
particle deposition. The mouse, hamster, and rabbit lack the extensive tra-
cheal or bronchial mucus-secreting glands that humans possess, while the
rat possesses such glands only in the trachea. Although the cat does have
these glands, its pattern of secretion is so unlike man as to make its use
generally unsuitable. The guinea pig, which also has these glands, is lim-
ited in its applicability because of a predisposition to asthmatic-type
attacks (Roe 1968). Roe felt that the dog and monkey were closer to man
in gross anatomy and response but that their size reduced their applicabil-
ity. Additionally, the monkey is useful only when laboratory-bred speci-
mens are available. In contrast to some other researchers, Roe considered
the mouse to be the preferred species for inhalation carcinogenesis studies
due to its high susceptibility, which enhances the chances for tumor forma-
tion.
In an evaluation of dust retention in small animals, Palm et al. (1956)
found that the retention pattern for the monkey was more similar to man
than that of the guinea pig. In general, the smaller the animal the lower
the rate of lung deposition for particles above 1 jim in diameter with less
alveolar deposition. Also, the overall retention is greater in small animals,
most likely due to greater impingement in the upper respiratory tract.
However, Palm et al. (1956) did find that the most favorable particle size
(1 /im) for alveolar deposition was similar for man and small animals.
Another anatomical variable that influences particle deposition is the hor-
izontal layout of respiratory tracts of quadrupeds, which is totally different
from the vertical tract of man and other primates (Palm et al. 1956,
WHO 1978). Carney (1979) found that, for evaluations of immunological
sensitivity to particles, the rat was totally unsuitable due to sensitivity
problems and that the guinea pig was a more suitable model. Brain and
Mensah (1983) have reviewed many aspects of anatomical differences of
species that can affect inhalation testing. They discussed variations in
deposition, clearance, and biological response and concluded the following:
(1) the fraction of aerosol actually deposited in the respiratory tract
appears independent of body size; (2) different species breathing the same
aerosols do not necessarily receive the same lung doses, and thus exposure
concentration is not an adequate description of lung dose; (3) factors that
affect the lung dose include changes in ventilation, collection efficiency,
lung anatomy, and clearance mechanisms; and (4) interspecies variations
in substance metabolism and innate biological responsiveness make it
unlikely that lung damage will be the same even if the lung dose is
equivalent.
The available literature clearly indicates that there is no species ana-
tomically identical to man in all respiratory aspects. The most similar test
species are generally the larger animals (horse, dog, and monkey), which
are impractical for the majority of inhalation chambers and tests. It is
therefore not surprising to find guidelines that recommend the rat (OECD
1979, Gross 198la) or other rodent species, especially for acute tests
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153
(Drew and Laskin 1973, NAS 1977). Other guidelines suggest that meta-
bolic similarity should also be stressed, because systemic effects are impor-
tant (Troy 1974), that a multiple species system is better (Hammond
1970, Drew and Laskin 1973), or that a rodent and nonrodent are needed
for chronic studies (NAS 1977). A recent anatomical and physiological
evaluation of the ferret indicates that its airways are more similar to
humans than the dog's and other favorable aspects of its lung anatomy
make it a more useful and less expensive choice for a nonrodent test
species (Vinegar et al. 1981). As with other types of toxicity tests, no sin-
gle species can be found to satisfy all the needs of inhalation tests, and the
specific species chosen should be the one judged to be best suited for that
particular chemical and exposure condition (Troy 1974).
4.2.3 Duration and Mode of Exposure
Similar to tests using other routes of exposure, there are three principal
test durations for inhalation studies (acute, subchronic, and chronic). The
initial evaluation of toxicity is often obtained through an acute test. This
test is defined as a single uninterrupted exposure of high concentration,
usually with a duration of less than 8 h (Drew and Laskin 1973, NAS
1977, WHO 1978). An acute test can provide various information, includ-
ing approximate comparative toxicity of the chemical, the range of doses
needed for studies of longer durations, and the nature of possible toxic
effects (Drew and Laskin 1973, Troy 1974, NAS 1977). This information
is provided primarily by determination of an LC50 value, which is the
atmospheric concentration that will kill 50% of the test animals within a
predetermined period of time (Drew and Laskin 1973, Carney 1979).
Often the LC50 is a statistically derived value (OECD 1979) and is given
in units of test substance weight per volume of air (mg/L, mg/m3, or
ppm) (Troy 1974, OECD 1979). Guidelines have suggested an exposure
period of 1 to 4 h with a 14-day observation period (NAS 1977, Gross
1981a). The length of the observation period is critical, because a short
period might miss delayed mortality and a long period could allow repair
of any tissue damage (Carney 1979). EPA guidelines state that the obser-
vation period should be flexible; a length beyond 14 days could be justified
by the toxic reactions, rate of onset, and length of recovery (USEPA
1982). Evaluations of acute toxic effects can include clinical observations
(NAS 1977), airway irritancy responses (Giovacchini 1972, Cambridge
1973, Wells 1979), gross necropsy, and microscopic examination of the
lungs or other target organs (NAS 1977, OECD 1979). Occasionally, no
acute effects can be obtained even with the maximum concentration that
can be generated (Carney 1979). In this case testing may be halted
(OECD 1979) unless potential acute human exposure is expected (Carney
1979).
To initially evaluate toxicity resulting from repeated exposure to a sub-
stance, a subchronic test is usually performed. The duration of a sub-
chronic inhalation test is no more than 10% < the lifespan of the test
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154
animal, with typical lengths of 14, 28, or 90 days (OECD 1979). An expo-
sure time of 6 h/day is often used for these tests (Wells 1979, OECD
1979). Information on the nature of chronic toxic effects, detailed
exposure-effect relationships, and range of doses for further chronic studies
represents the principal results of a subchronic test (Drew and Laskin
1973). The degree of information obtained depends on the type of evalua-
tions in the test design. For maximum information from the subchronic
inhalation test, evaluations could include clinical observations, biochemical
tests, hematology tests, gross and microscopic pathology, and pulmonary
function tests (Giovacchini 1972, Carney 1979, OECD 1979, Page et al.
1980).
A chronic inhalation test is necessary to fully evaluate the effects of
lifetime exposure to a substance. To simulate lifetime exposure in humans,
the duration of the chronic test should be greater than 10% of the test
animal's lifespan, which for most animals would be 1 to 5 years (NAS
1977, OECD 1979). Additionally, the exposure period should be patterned
either after human occupational exposure (6 to 8 h/day, 5 days/week) or
possible environmental exposure (22 to 24 h/day, 7 days/week) depending
on the intended use of the product (Drew and Laskin 1973, WHO 1978).
Procedures to evaluate the chronic effects could include clinical observa-
tions, biochemical tests, hematology tests, gross necropsy, microscopic
pathology, and pulmonary function tests (NAS 1977, OECD 1979). To
use these evaluations for the duration of a chronic test, large numbers of
test animals (e.g., a minimum of 400 rodents) and exposure chambers are
required (NAS 1977). Therefore the chronic test should be undertaken
only after extensive preliminary testing has indicated the need for an
evaluation of lifetime exposure effects.
As mentioned in discussion of chronic tests, exposure to the test sub-
stance can be scheduled on a continuous or an intermittent basis. Although
this allows the substance to be evaluated in the potential mode of human
exposure, there are certain factors of the two methods that must be con-
sidered.
With intermittent exposure (6 to 8 h/day, 5 days/week), the dose is
pulsed with substantial periods of time, both daily and over the weekend,
for recovery from the test substance. These periods of recovery can affect
the toxicity of the chemical (Haun 1972, Drew and Laskin 1973, WHO
1978). Wright (1957) studied the effects of continuous versus intermittent
exposure on dust retention in rats in a 3-month subchronic study and
found that rats exposed for 2 h at a high concentration retained less dust
(20 to 30%) than rats exposed for 20 h at a low concentration, even
though both groups had the same daily concentration X time (CXT)
dose. Sidorenko and Pinigin (1976) examined the two exposure modes in
short-term studies with rats and mice and found that, generally, intermit-
tent exposure is less harmful (in terms of mortality and time to onset of
effects) than continuous exposure. As the intervals between exposures were
increased in duration, the toxic effect of a substance in mice declined
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155
exponentially or proportionally, depending on the specific substance. In
rats, the interrupted exposure had a smaller effect on the central nervous
system and blood than did continuous exposure. However, Sidorenko and
Pinigin did find some contradictory evidence in the rate of onset of effects
with increasing time intervals between exposures. The varying response to
discontinuous exposure is related to the quantity of test substance that can
cross lung membranes and/or be absorbed by the aveolar blood supply
during a given time and is perhaps related to the processes of adaptation
and accumulation in the organism (Sidorenko and Pinigin 1976). Gardner
et al. (1979) studied the variation between the two exposure modes using
NO2 and concluded that the concentration factor is more important than
the length of exposure. When a constant CXT dose was employed, a short
duration at a high concentration produced a greater effect than a long
duration at a low concentration.
Intermittent exposure has some practical advantages that makes it
easier and cheaper to use than continuous exposure. The chamber design
can be much simpler, as there is no need to feed or water the animals dur-
ing exposure (Drew and Laskin 1973, WHO 1978). Similarly, the atmo-
sphere generating equipment and monitoring devices can be simpler
because they operate only for 6 to 8 h/day (Drew and Laskin 1973,
OECD 1979). The shorter period actually spent in the chamber also allows
more animals to be exposed per cage (Drew and Laskin 1973). In large
studies, these advantages can be substantial.
The advisability of initially evaluating a substance using actual expo-
sure conditions has been questioned. The National Academy of Sciences
(NAS 1977) has suggested that controlled studies can often give more
informative data, especially if the actual use conditions are unique, and
standard exposures also make comparisons of relative toxicity easier. Such
tests basically evaluate the toxicity of the test substance (which is an
inherent property of the compound) and do not fully address the hazard of
the compound (which is a combination of toxicity and exposure conditions)
(Gross 198la). Additionally, even though intermittent exposure may simu-
late the time span of potential human exposure, actual concentrations in
the environment vary, with peaks higher than the average concentration
value. Thus, intermittent exposures may not be realistic or representative
of the actual use conditions (Drew and Laskin 1973, WHO 1978).
4.2.4 Dosage
The design of inhalation exposure studies considers two factors of
dosage: first, the appropriate number of levels (including controls) and,
second, how to accurately determine the dosage.
The number of concentrations varies with the duration and goals of the
test. Most guidelines recommend a minimum of three dose levels of the
test substance for all studies (WHO 1978, OECD 1979, Page et al. 1980).
Acute tests require that the dose levels be designed to produce a range of
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responses to permit construction of a mortality-concentration curve for cal-
culation of an LC50 value. Subchronic and chronic tests should use a dose
range with the high dose producing toxic effects, but not necessarily mor-
tality, and the low dose producing minimal or no effects and being roughly
equivalent to the No-Observable-Effect Level (NOEL) (WHO 1978,
Gross 198la). At least one intermediate dose is necessary if information
on the dose-response relationship is desired (Page et al. 1980, Gross
1981a).
Incorporation of control groups is more extensive in chronic and sub-
chronic tests. There should be at least one control group that undergoes
the same procedures, except for actual exposure to the test substance, as
the treatment groups. In addition, a control group is necessary to evaluate
the administration vehicle, if one is employed. The use of a vehicle control
group is also recommended for acute studies (OECD 1979).
The principal uncertainty associated with dosage in inhalation studies
is the determination of the actual effective dose. For example, there may
be variations in the dose to the lung, dose to the blood, and dose to the
target organ. The factors that determine the dose delivered to the lung
include, of course, length of exposure and atmospheric concentration of the
test agent in the chamber (Roe 1968, MacFarland 1976). However, these
factors describe, at best, only the amount of test agent available to the test
animals. The critical value is the dose that reaches the sites of action in
the lung (Hatch and Gross 1964, MacFarland 1975, Clark 1977). Many
factors can affect this parameter, particularly test species variables such as
minute volume of ventilation; frequency of respiration; bronchial dimen-
sions, pulmonary and bronchial arterial bloodflow; rate of elimination
including natural clearance mechanisms; metabolic conversion; activity lev-
els of the animals; and percentage of retention (DuBois and Rogers 1968,
Drew and Laskin 1973, NAS 1977, USDHEW 1977). Physical and chem-
ical properties of the test agent also determine the effective dose, especially
the solubility, diffusion coefficient, and, for aerosols, particle size (DuBois
and Rogers 1968, NAS 1977). The degree that these factors affect the
dose can also vary. It has been shown, at least theoretically, that the pul-
monary factors may be less important in determining dose for tests of
chronic duration than those of shorter exposures (DuBois and Rogers
1968). Further, defense mechanisms can concentrate, via phagocytic
macrophages, a relatively diffuse and even exposure to create "hot spots"
of high test agent concentration (USDHEW 1977). This effect may be
especially important if thresholds for effects apply to the test agent.
In addition to these variables, there is often interference from other
accidental routes of exposure. Clearance of the test agent from the lung
via the mucociliary escalator can result in unintended gastrointestinal
exposure (Clark 1977). Similarly, animals can ingest the test agent during
grooming behavior or experience dermal exposure if the test is performed
in a whole-body exposure chamber (Drew and Laskin 1973, Kawai and
Nozaki 1978).
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157
Because one of the primary objectives of the inhalation test is to eluci-
date upon the dose-response relationship, the variables and interferences
discussed above have influenced how the inhalation toxicologist calculates
dose and relates it to effects. The product of the atmospheric concentration
(C) and the duration of exposure (T) is often used to determine
dose-response relationships. This calculation does not produce a dose per
se, as a dose by definition is a mass quantity (MacFarland 1976). How-
ever, it is still used to estimate the dose on the theory that the CXT dose
is proportional to the inhaled dose, which is proportional to the absorbed
or effective dose at the site of action (MacFarland 1976, Carney 1979).
The principles behind this theory were revealed by Haber (1924) in his
studies of chemical weapons and is often known as Haber's rule. This rela-
tionship holds true at most exposures, but at the high and low extremes
the observed values deviate from the predicted values. Because the CXT
dose is proportional to the real dose, it has been used to calculate
L(CXT)50 data in acute tests and to develop dose-response data.
The main problem with the use of the CXT dose and Haber's rule to
calculate dose-response relationships is that they represent a simplification
of the real situation by ignoring interspecies and individual variations in
minute volume and retention percentage (Weston and Karel 1946, Carney
1979). These factors are especially important because animal data are
used as a basis for extrapolation to human exposure conditions. The large
variation in the animal data could produce human exposure levels that are
still within hazardous concentrations (MacFarland 1976).
Recognizing this potential problem, Weston and Karel (1946)
developed a dose formula incorporating the minute volume (MV) and the
percentage of inhaled agent that is retained (a). This Dosimetric Formula,
dose = aCTrMV, can be considered a true dose because it results in a
value with dimensions of a mass quantity. However, the Dosimetric For-
mula has proved to be difficult to use in practical situations. Values for
the minute volume of animals at rest for different species are available in
the literature, but because it is the values under the conditions of exposure
that are important, studies should allow for measurement of the MV dur-
ing actual exposure (MacFarland 1976). The retention percentage is even
more difficult to quantify as the values vary depending on the chemical
(particularly its solubility and particle size), duration of exposure, test
species, and exposure chamber conditions (e.g., humidity). Despite these
drawbacks, the Dosimetric Formula represents a better approach than does
the CXT dose, especially for evaluating comparative toxicities or differ-
ences between routes of exposure (MacFarland 1976).
The difficulties associated with the CXT dose and the Dosimetric For-
mula have prompted researchers to experiment with alternative methods of
evaluating inhalation doses, particularly in regard to the retained dose.
Radioactive isotopes have been used to measure the actual amount of the
test substance retained in the lung (Albert et al. 1967, Roe 1968). Griffis
et al. (1981b) reported a method using acid-insoluble sodium to determine
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amounts of glass fiber retained in lungs after exposure to aerosols. Magne-
tometric techniques for measuring lung burdens of ferrimagnetic dust have
also been reported (Oberdoerster and Freedman 1982). Circulating blood
levels or rates of excretion have been used to estimate the body burden
and are in some cases better estimates of dose than the CXT product
(MacFarland 1976, Clark 1977). Such techniques still require additional
development and validation before they can be incorporated into a routine
inhalation protocol.
4.2.5 Observations for Effects
In inhalation studies, many methods have been used to observe and
determine the toxic effects of the test agent. To detect systemic effects,
most of the tests and observations are the same as those used in other toxi-
city studies (Poynter 1977). However, special procedures are used in
observing toxic effects on the portal of entry, the lung. The observation of
systemic effects will be only briefly discussed, with the majority of the dis-
cussion focusing on observations unique to inhalation studies. No attempt
is made to suggest a rigid or standard protocol because the flexibility to
choose between a battery of basic, simple tests and an array of special
tests for inhalation effects can often be the most efficient and informative
procedure (MacFarland 1975). With the vast array of special tests that
are being developed and the many potential effects that could be moni-
tored, the difficult task is to decide which aspects of inhalation toxicity to
examine, as a truly comprehensive evaluation for all agents is impractical
(Campbell 1976, Clark 1977).
4.2.5.1 Clinical observations
During and following exposure, observation of the test animals for clin-
ical signs can often indicate systemic or lung damage. All animals should
be observed at least daily and preferably twice a day (NAS 1977, OECD
1979, Gross 198la). Observations should include any signs of irritation;
changes in skin, fur, eyes, or mucous membranes; tremors; convulsions;
salivation; bleeding; coughing; frothing at the mouth; presence and condi-
tion of urine and/or feces; lethargy; and changes in respiratory rate or pat-
tern (NAS 1977, OECD 1979, Page et al. 1980). The observer should be
particularly alert for moribund animals, in order to prevent loss from
autolysis or cannibalism. In long-term studies, the animals can also be
weighed once a week and the food consumption calculated.
4.2.5.2 Biochemical and hematological tests
Hematological and biochemical tests, including enzyme assays, should
be performed on all animals before and after exposure (or at any interim
sacrifice during exposure). Frequently performed hematology tests include
hematocrit, hemoglobin concentration, erythrocyte count, total and dif-
ferential leucocyte counts, prothrombin time, partial thromboplastin time,
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159
platelet count, and a blood smear examination (OECD 1979, Gross
198la). Many biochemical tests have been used in inhalation studies,
including alkaline phosphatase, serum glutamic-pyruvic transaminase,
serum glutamic-oxaloacetic transaminase, blood urea nitrogen, total pro-
tein, albumin, globulin, cholesterol, fasting glucose, calcium, sodium,
chloride, and potassium (OECD 1979). Such tests can give information on
systemic effects or suggest additional special tests. The use of special
biochemical evaluations of lung tissue has also been suggested, particularly
for intermediate metabolism (Tierney 1974), and pulmonary surfactants
(King 1974). The majority of biochemical tests indicating lung damage are
currently in the developmental stage with many methods being modified
from established hepatotoxicity evaluations (Witschi 1975). Although
these studies have not yet identified a biochemical parameter that clearly
indicates toxic effects in the lung, an encouraging beginning has been
made (Witschi 1975). Biochemical analysis of pulmonary lavage fluid has
also shown promise as an evaluative tool of inhalation toxicity. Henderson
and coworkers (Henderson et al. 1978, 1979b, 1980) have used the lavage
techniques developed by Mauderly (1977) as a short-term in vivo screen of
acute toxicity in the rat and hamster. By measuring levels of various
enzymes and components in the fluid (Table 4.2) after exposure to toxic
agents, they determined that lavage was a more sensitive and earlier indi-
cator of initial lung injury than was analysis of lung tissue. With continued
development, it may be a useful technique for evaluation of chronic pathol-
ogy. Other in vivo and in vitro assays may find some applications as alter-
natives to traditional inhalation toxicity studies (Brown and Poole 1983).
These alternatives include in vitro studies using immunocompetent cells of
the lungs, cytoxicity and cell morphology of cell cultures, and specific cel-
lular biochemical activities as end points (Brown and Poole 1983).
4.2.5.3 Pathological observations
Evaluation of effects by pathology studies is the main method for
determining inhalation toxicity. As in other types of toxicity tests, it is
essential to have pathological observations that include both a thorough
gross examination of all animals and a detailed microscopic examination
of, at least, the high-dose, control, and lesion-bearing animals and the
suspected target organs of animals at the intermediate levels (Gross
1981a).
The gross examination or necropsy is often more involved for inhala-
tion studies than for other routes of exposure (Dungworth et al. 1976),
with examination of additional respiratory tissues (nasopharynx, larynx,
tracheobronchial tree, and parenchyma). The gross necropsy typically
includes an examination of the external surfaces of the body; all orifices;
and the cranial, abdominal, and thoracic cavities, including the organs and
tissues contained within them (OECD 1979). Improved evaluations of the
morphology of the nasal turbinates and mucosa have been recommended
by Jersey and Kociba (1979), who suggest that grading the extent and
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Table 4.2. Parameters Measured in Lavage Fluid
Parameter Indication
Cytoplasmic enzymes: Membrane damage
lactate dehydrogenase;
glucose-6P-dehydrogenase
organ specific enzymes;
isoenzymes
Lysosomal enzymes: Phagocytic activity
acid phosphatase;
beta-glucuronidase
Membrane enzyme: Broken membranes
alkaline phosphatase
Mucin component: Increased mucin secretion
sialic acid
Cell count: Inflammatory response
total; differential
Source: Adapted from Henderson et al. (1980).
severity of the lesions can give dose-response relationships. During the
necropsy, examination of the structures within the lung depends on the
extent of gross damage; when edema or exudative lesions are absent, the
airways and parenchyma are best examined after fixation (Dungworth et
al. 1976).
After the necropsy, samples of all organs and tissues are preserved by a
fixative solution. The lung requires special attention to ensure correct
preservation. The preferred technique is to perfuse the lung via the air-
ways, because this maintains the correct dimensions and configuration of
the tissues while permiting rapid fixation (Dungworth et al. 1976, NAS
1977). The fixative (10% neutral buffered formalin or Karnovsky's
formaldehyde/glutaraldehyde formula) should be perfused at a pressure of
25 to 30 cm of water for 1 to 4 h (Roe 1968, Dungworth et al. 1976).
The microscopic examination should include a wide array of body tis-
sues and organs in addition to respiratory structures in order to assess both
the systemic effects and the specific effects from inhalation. The exact
organs to be sampled vary with the chemical and its potential effects; some
suggested lists are given in Table 4.3. The assessment of specific lung
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Table 4.3. Organs Suggested for Microscopic Examination*
NASb
WHOC
OECD"
Bone
Bone marrow
Brain
Eye
Gonads
Heart
Intestine, large
Intestine, small
Kidneys
Liver
Lung
Lymph node, mesenteric
Lymph node, peribronchial
Muscle, skeletal
Nasal cavity
Nerve, peripheral
Pancreas
Pituitary
Prostrate
Salivary gland
Skin
Spleen
Stomach
Thyroids
Trachea
Urinary bladder
Uterus
Accessory genital organs
Adrenals
Aorta
Bone marrow, sternum
Brain
Caecum
Colon
Duodenum
Gall bladder (if present)
Gonads
Heart
Ileum
Jejunum
Kidneys
Liver
Lung
Lymph nodes, axillary
Lymph nodes, mesenteric
Mammary glands
Muscle, thigh
Nerve, peripheral
Oesophagus
Pancreas
Pituitary
Rectum
Salivary gland
Spinal cord (at three levels)
Spleen
Stomach
Thymus
Thyroid
Urinary bladder
Accessory genital organs
Adrenals
Aorta
Brain
Caecum
Colon
Duodenum
Gall bladder (if present)
Gonads
Heart
Ileum
Jejunum
Kidneys
Liver
Lung
Lymph nodes, axillary
Lymph nodes, mesenteric
Mammary glands
Nerve, peripheral
Oesophagus
Pancreas
Pituitary
Rectum
Salivary gland
Spleen
Stomach
Thymus
Thyroid
Urinary bladder
"Gross abnormalities or lesion-bearing tissues detected in gross necropsy should also be
examined.
bNAS = National Academy of Sciences (1977).
CWHO = World Health Organization (1978, Chapter 5).
dOECD = Organization for Economic Cooperation and Development (1979).
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effects requires sampling that is wide in distribution but specific in ana-
tomic location because the size, diversity, and variable responses of the
respiratory tissue are so great (Dungworth et al. 1976). Generally, samples
to be taken include: (1) a longitudinal section of the distal trachea and the
bifurcation into bronchi; (2) vertical sections of one lung and the cranial,
middle, and caudal lobes of the other lung; (3) the proximal trachea;
(4) the lobar bronchus; (5) the pharynx; (6) the larynx; and (7) the prox-
imal and distal regions of the nasal sinuses and turbinates (Dungworth et
al. 1976). These samples should be initially evaluated by light microscopy
and/or scanning electron microscopy. If additional detail is desired,
transmission electron microscopy can be used (Dungworth et al. 1976).
Occasionally, to fully elucidate the toxic effects, special pathology tech-
niques may be employed, including autoradiography, histochemistry, and
morphometry studies. Autoradiography is useful in determining cytokinet-
ics of damaged pulmonary cells (Dungworth et al. 1976) and the correla-
tion between deposition of particles and effect (Lisco 1959). Histochemis-
try is useful in localizing biochemical changes to specific cells or tissue
types (Dungworth et al. 1976), whereas morphometic analysis is useful for
accurately measuring the degree of damage and for statistically confirming
subtle effects (Dungworth et al. 1976; WHO 1978). Although these pro-
cedures are not routine, they may serve a useful function in toxicity
evaluations.
4.2.5.4 Respiratory function tests
The respiratory function tests are observation methods for assessing
toxic effects that are uniquely used in inhalation studies. These tests allow
the inhalation toxicologist to observe the effects of the test agent on
breathing pattern, lung volumes, pulmonary pressures, flow volumes, and
lung mechanics (Table 4.4). Other tests can be performed to determine
changes in gas exchange and gas levels in the blood (Likens and Mauderly
1979). Generally, the respiratory function tests can be applied to intact
animals, can be performed rapidly and repeatedly without affecting the
animal, and will produce dose-response relationships, often with low varia-
bility (Swann et al. 1965, Alarie 1966, Hiett 1974, MacFarland 1976,
WHO 1978, Wong and Alarie 1982, Juhos et al. 1985). One key to these
methods is the understanding of baseline or mean values to allow interpre-
tation of responses as toxic or nontoxic (Kennedy and Trochimowicz
1982). Because respiratory function can vary greatly between test animals,
it is vital to have a series of baseline data for each animal prior to dosing.
Concurrent evaluations of control animals can demonstrate any changes in
respiratory function over time due to aging and repeated testing. The
respiratory function tests are useful in acute studies for delineating effects
and in chronic tests for following the development of effects (MacFarland
1976). They can also provide information on the mechanisms of action
(Amdur 1958, MacFarland 1976), although such data are not always
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Table 4.4. Respiratory Function Tests to Evaluate Breathing Patterns, Lung Volumes, Pulmonary Pressures, and Lung Mechanics
Test
Definition
Method of measurement"
Reference
Frequency (Respiratory rate)
Number of respirations per
minute
Tidal volume (VT)
Minute volume (MV)
Vital capacity (VC)
Inspiratory capacity (1C)
Expiratory reserve volume
(ERV)
Volume of air inspired during
a quiet breath
Volume of air expired per
minute
Change in volume between
maximal inspiration and maxi-
mal expiration
Maximum volume that can be
inhaled after a quiet expiration
Volume that can be exhaled
after a quiet inspiration
Visual count; volume pneumograph
(detects pressure changes in thorax);
impedance pneumograph (uses electrical
circuit to measure impedance change
resulting from thorax movement); tem-
perature change across nostrils; non-
rebreathing valve
Respirometer; plethysmograph used with
a volume transducer; non-rebreathing
valve (calculated value); pneumotachc-
graph
Non-rebreathing valve; respirometer (cal-
culated value); plethysmograph with vol-
ume transducer (calculated value); pneu-
motachograph
Plethysmograph (used with applied pres-
sure); air injected into lung via syringe
Plethysmograph (used with applied pres-
sure); air injected into lung via syringe
Plethysmograph (used with applied pres-
sure); air injected into lung via syringe;
pneumotachograph
McCutcheon 1951, Alarie 1966, Barrow
et al. 1971, Mauderly and Tesarek 1975,
Crossland et al. 1977
McCutcheon 1951, Leong et al. 1964,
Thomas and Morgan 1969, Diamond and
Lipscomb 1970, Barrow et al. 1971,
Barer et al. 1976
Truog and Standaert 1978, Leong et al.
1964, Diamond and Lipscomb 1970,
Thomas and Morgan 1969
Comroe et al. 1954, Drorbaugh 1960
Comroe et al. 1954, Drorbaugh 1960
Comroe et al. 1954, Drorbaugh 1960
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Table 4.4 (continued)
Test
Definition
Method of measurement"
Reference
Total lung capacity (TLC)
Residual volume (RV)
Functional residual capacity
(FRC)
Intrapleural pressure
Esophageal pressure
Transpulmonary pressure
Volume of gas contained in
lungs at maximum inspiration
Volume of gas remaining in
the lung after maximal expira-
tion
Air remaining in lungs at end
of quiet expiration
Pressure within the pleural
space
Pressure within the esophagus
(approximates intrapleural
pressure)
Pressure difference between
pleural pressure and tracheal
opening
O2 absorption technique (used with
plethysmograph) calculated by adding
residual volume and vital capacity values
Calculated by subtracting expiratory
residual volume from functional residual
capacity values; determining amount of
inhaled inert gas retained in lungs after
exhalation
Inert gas dilution (uses tracheal cannula,
O2 chamber and percent N2 to calculate
value): FRC = ERV + RV
Intrapleural catheter
Fluid-filled or balloon-tipped esophageal
catheter
Differential pressure transducer con-
nected to pleural or esophageal catheter
and mask or breathing chamber
Cavagna et al. 1967
King 1966a, Caldwell and Fry 1969,
Koo et al. 1976, Koen et al. 1977,
Diamond and O'Donnell 1977
Amdur and Mead 1958
Cavagna et al. 1967, Caldwell and Fry
1969, Koo et al. 1976
Amdur and Mead 1958, Palecek 1969
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Table 4.4 (continued)
Test
Definition
Method of measurement"
Reference
Compliance
Resistance
Forced vital capacity; forced
expiration volume; maximum
expiratory flow-volume
Distensibility of the lungs and
thorax (relation of transpul-
monary pressure to lung vol-
lume changes, C = AV/AP)
Represents the frictional and
viscous forces that occur with
respiration (relation between
transpulmonary pressure and
R = AP/AV)
Dynamic forced respiratory
characteristics useful for
detecting changes in upper and
lower airway resistance
Dynamic: plethysmograph (for volume)
and pleural or eosphageal pressure (cal-
culated on analog computer, from oscillo-
scope displays, or by manual calcula-
tions); static: pressure volume curves gen-
erated by plethysmograph and air
injected in lungs; forced oscillation of res-
piratory movements in a plethysmograph
using volume changes measured by a
pressure transducer
Dynamic: plethysmograph (for volume),
pleural or esophageal pressure, and pneu-
motachograph (for flow) (calculated
using same techniques as compliance);
static: change in trans-pulmonary pres-
sure at end expiration (using a pneumo-
tachograph); forced oscillation of respira-
tory movements in plethysmograph
Whole-body plethysmograph used with
negative and positive pressure chamber;
whole-body respirator and pneumotacho-
graph
Davis and Morris 1953, Radford 1957,
Amdur and Mead 1958, Dennis et al.
1969, Nattie 1977, Diamond et al. 1973,
Hiett 1974, Decker et al. 1979
Amdur and Mead 1958, Mead 1960,
Murphey and Ulrich 1964, Frank and
Speizer 1965, King 1966b, Palecek 1969,
Giles et al. 1971, Diamond et al. 1973
Diamond and O'Donnell 1977, Moorman
et al., 1977
"Note: pneumotachograph can be used to measure all lung volumes except for RV. It is generally the most precise and easy to use means for measuring
flow and volume (by intergration).
Source: Adapted from Likens and Mauderly (1979).
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166
necessary in toxicological studies. One important advantage that respira-
tory function tests have over other evaluations of toxic effects is that they
can quantify certain changes (e.g., reflex bronchoconstriction) at concen-
trations below the levels that produce morphological damage (Amdur
1958, WHO 1978). Therefore, they may allow measurement of such
effects at levels of reversible rather than irreversible changes. Another
advantage, particularly for chronic tests, is that the same animal's
responses can be followed over the entire exposure period, thus avoiding
the variability introduced by testing different groups at each point in time
(Gross 1981b). Finally, similar data can often be generated for humans
and test animals, which facilitates comparisons (WHO 1978).
The respiratory function tests do have disadvantages. First, they
require special equipment and training for proper use (NAS 1977).
Second, many of the tests (e.g., vital capacity or residual volume) used on
humans cannot be used on test species without some modification or
anesthesia because, as currently administered, the tests require the under-
standing and cooperation of the test subject (Roe 1968, MacFarland
1976). Third, some tests can be used only with anesthetized animals under
highly unnatural physiological conditions (Roe 1968). Finally, the informa-
tion gathered by these tests is rarely conclusive regarding the total effects
of a test substance and must be supplemented by the other evaluation
methods discussed in earlier sections, especially histopathology (Roe 1968,
MacFarland 1976).
In addition to the tests of the mechanics, volumes, and pressures associ-
ated with respiratory function, tests to evaluate ventilation distribution,
diffusion, and mucociliary action have been developed. A test that can be
used to evaluate effects on the ventilation distribution is the nitrogen
wash-out technique (MacFarland 1976). Generally, the technique meas-
ures the decline in nitrogen concentration of expired air after switching to
pure oxygen inspiration. A rapid, exponential decline indicates normal ven-
tilation distribution, but an uneven or less steep slope indicates an irregu-
lar ventilation distribution. Effects on the diffusion capabilities can be
tested by determining the rate at which carbon monoxide or oxygen
transfers from the alveolus to the hemoglobin (MacFarland 1976, Likens
and Mauderly 1979). One way to test this process is a closed-circuit
rebreathing technique using carbon monoxide. The rate of decline of the
carbon monoxide indicates the diffusing capabilities. Diffusion capabilities
can also be determined during a single vital capacity inspiration and
expiration. Measurement of blood gas (oxygen, carbon dioxide) levels is
another technique suggested for evaluating respiratory function. However,
these tests can be difficult to perform in small animals and often require
surgical approaches (Likens and Mauderly 1979). To determine effects on
the mucociliary capacity of the lungs, a tracheal preparation can be taken
from an exposed animal, test particles (e.g., pollen grains) applied, and
their progress between two points observed. Another test uses radiolabeled
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167
particles in a live test animal and monitors their progress with a scintilla-
tion detector (Troy 1974). This latter approach was used by Kenoyer et al.
(1982) to determine the toxic effects of ozone on short- and long-term
clearance rates. They found that ozone exposure delays the initial clear-
ance (perhaps by affecting ciliary beat rate or mucus changes), but the
long-term clearance is accelerated (perhaps due to greater activity of
deep-lung macrophages). These tests would be most useful in a study
designed to evaluate only effects on the lung, because they are too compli-
cated for use in a routine test of systemic toxicity.
4.3. EXPOSURE CHAMBER DESIGN
4.3.1 Introduction
The design of the exposure chamber should be one of the first factors
considered in an inhalation test to ensure that the requirements of the
exposure protocol are met. Specifically designed buildings and facilities are
preferred for such tests. The building should have a high ceiling to accom-
modate chambers and necessary air ducts, have plenty of floor space to
allow access to at least two and preferably all sides of the chamber, pro-
vide good illumination without allowing direct sunlight exposure of the
chambers, and have an adequate air supply system with appropriate con-
trols for humidity, temperature, and flow (Roe 1968, Drew and Laskin
1973, WHO 1978). The exposure chamber units should be designed to
meet the majority of the basic requirements listed in Table 4.5. This
chapter will deal with the necessary equipment to meet such requirements,
including discussions of the various types of exposure systems, chamber
design, air supply and exhaust systems, monitoring of the chamber, and
use of specialized equipment. Consideration of test agent delivery and
atmosphere generation equipment are covered in Section 4.4.
4.3.2 Types of Exposure Systems
This section will focus on the various operation modes possible for
inhalation chambers. The administration of the test agent can be in a
static or dynamic mode of operation. Also the animals can be exposed to
the test agent via whole-body, nose-only or head-only exposure systems.
The advantages, disadvantages, and uses of each system will be discussed.
4.3.2.1 Static systems
A static exposure system consists of a closed chamber into which a
predetermined dose of the test agent is introduced and mixed (Lodge
1968). The test animals remain in this closed system for the duration of
the test without any replacement of the test atmosphere. Obviously the
lack of replacement air will limit the duration of exposure, because it
allows depletion of the oxygen concentration, increased carbon dioxide lev-
els, increased temperature (from buildup of animal body heat), and
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Table 4.5. Inhalation Exposure Apparatus: Basic Requirements
Exposure chamber
Adequate size for exposure of sufficient animals for statistical evaluation
Even distribution of test gases, aerosols, or particle clouds
Temperature control
Humidity control
Easily cleaned and decontaminated
No hazard to personnel
Identical chambers available for untreated or vehicle-only treated controls
Test material
Gases — reliable control of concentration in chamber
Aerosols and particle clouds — reliable monodisperse system
as well as control of concentration; vehicles, if used, should
be without effect on respiratory system
Monitoring of exposure
Apparatus should be available for monitoring the concentration
of gases, particle cloud density, and distribution of particle
size at breathing zone, continuously during exposure
Safety
The absence of leaks in the chambers or
connecting pipe systems should be ascertained
repeatedly, preferably continuously, by a suitably designed
monitoring system where hazardous agents are under test
The atmospheric pressure within the inhalation exposure system
should be slightly below the ambient atmospheric pressure
Suitable traps and filters should guard both ends of all pipeflow systems
Source: Adapted from Hinners et al. (1966), as reported in Roe (1968).
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169
increased humidity (MacFarland 1976, Clark 1977, NAS 1977). An expo-
sure duration of 0.5 to 1 h has been suggested for smaller static systems
(MacFarland 1976).
In addition to the problems associated with a finite volume, there is
also a loss of test agent by deposition on the chamber walls or by removal
for sampling purposes (Lodge 1968, Drew and Laskin 1973). Although
many of these problems can be eliminated or reduced by use of a large-
volume chamber (e.g., a minimum of 500 L/kg test animal weight for
every hour of exposure, Clark 1977), nonintrusive sampling techniques, or
nonreactive wall materials, there is still one major disadvantage. Because
the total amount of test agent must be introduced at the beginning of the
exposure, the concentration declines with time from an initial peak higher
than the desired exposure to a final level below the desired exposure. Even
though the average concentration value of the entire exposure may be at
the desired level, the variations introduce uncertainty and prevent the uni-
form exposure concentration that is preferred for inhalation studies
(MacFarland 1976).
Despite these drawbacks, there are several situations for which the
static system is the best choice. Static systems are useful for assessing
acute toxicity and in performing preliminary pilot studies (WHO 1978).
Also, if the test agent is available only in limited quantities, a static expo-
sure should be used (MacFarland 1976). A static system is likewise
preferred in the testing of biological aerosols as these are difficult to gen-
erate continuously (Drew and Laskin 1973). The majority of inhalation
tests, however, will use the dynamic system.
4.3.2.2 Dynamic systems
A dynamic exposure system consists of a chamber that has a continu-
ous flow through of air and test agent at a constant rate and in a predeter-
mined ratio (Silver 1946). The constant airflow avoids many of the prob-
lems that limit the use of static systems and is usually generated by plac-
ing an air pump in the exhaust line of the system. Such a placement
creates a negative pressure in the chamber, which will aid in preventing
loss of the test agent through leakage (Silver 1946, MacFarland 1976).
Occasionally, to ensure an even airflow, buffer tanks are used to eliminate
pump-induced flow fluctuations (Silver 1946). Usually the test animals are
inserted in the chamber prior to exposure and then the gas flow is started
(MacFarland 1976). This creates an exposure curve of the form shown in
Figure 4.1. If immediate exposure to a constant concentration is desired,
an airlock device, usually built into the chamber door, can be used (Silver
1946). The animals are placed in the airlock, the outer door is closed, and
the airlock is opened to the chamber atmosphere. This produces the expo-
sure curve shown in Figure 4.2.
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170
ORNL-DWG 81-11341
O
QC
LU
O
2
O
O
0
'99
TIME
Figure 4.1. Concentration-time relationships in a chamber operated for
a long period of time. ta = Start of flow through chamber; t99 = time at
which equilibrium concentration is reached; tb = time at which test agent
is no longer added to airflow; and tc = end of airflow through chamber.
(Source: Adapted from MacFarland 1976. Used with permission of
Academic Press.)
ORNL-DWG 81-11340
CC
LJ
O
2
O
O
'0
TIME
Figure 4.2. Time-concentration curve for exposure to constant concen-
tration using air-lock mechanism. t0 = Animals introduced into chamber
and i\ = animals removed from chamber. (Source: Adapted from MacFar-
land 1976. Used with permission of Academic Press.)
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171
In normal use situations, it takes a certain amount of time for the
chamber concentration to reach the desired level (see Figure 4.1). Silver
(1946) developed the following equation to calculate this time lag:
^w-|
C = -11 -
where
C = the concentration (mg/L) at time t,
w = mg agent introduced per minute,
a = the volume of chamber (L),
b = the volume of air passing through the chamber each minute,
e = natural logarithm
Two assumptions, constant flow and complete mixing, which are generally
satisfied, are required for this equation (MacFarland 1976). Because this
is an exponential rate, Silver assumed a value of 99% to be sufficiently
close to the desired equilibrium value for testing; based on that value the
following equation can be used to determine when the chamber has
reached equilibrium:
T99=4.6052-f- .
b
MacFarland (1981) points out that, after the T99 concentration level is
reached, the aerosol distribution should be uniform within the chamber, as
inlet air would not differ by more than 1% from the chamber concentra-
tions in most chamber arrangements. Other numerical values for the con-
stant (4.6052) are given by Silver (1946) for percentages lower than 99%.
Because the T99 value depends on chamber size and flow rate, one could
reduce the time to equilibrium by increasing the flow (for both test agent
and total flow) above that required for the desired exposure (MacFarland
1976). Once the T99 equilibrium has been reached, the flow rate can be
reduced. Another technique to avoid the delay would be to use dual rate
supply systems. With the latter method, one must be careful that the con-
centration does not exceed the desired level (MacFarland 1976). The T99
equilibrium is a theoretical value, and, although the chamber should be
uniformly mixed, the actual chamber concentrations may be lower due to
losses of test agent from adsorption on walls or animal fur and uptake by
the animals. Therefore, monitoring of the actual concentration in the
chamber is always necessary to ensure proper exposure levels (Lodge 1968,
Drew and Laskin 1973).
The appropriate flow rate to be used in dynamic chambers must fall
within a limited range if the chamber is to avoid the heat buildup and oxy-
gen loss problems that occur with static chambers. MacFarland (1976)
discusses the ranges of flow rates commonly used and recommends the
following equations:
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172
F = 0.1 to 0.2V for gases,
F = 0.2 to 0.5V for volatile vapors,
F = 0.5 to 0.7V for light aerosols, and
F = 0.7 to 1.0V for heavy aerosols,
where
F = flow rate in liters per minute
V = chamber volume in liters.
For example, if the chamber size is 1000 L, the flow rate should be in the
range of 100 to 200 L/min for a gas, but 700 to 1000 L/min for a heavy
aerosol. If flow rates are calculated in this manner and test animals loaded
in the chamber at a density (animal volume per chamber volume) of 5% or
less, the use of special air conditioning systems are not necessary. EPA
guidelines require a dynamic air flow of 12 to 15 air changes per hour to
ensure an oxygen content of 19% (USEPA 1982). The flexibility shown by
the dynamic exposure system makes it the preferred choice for chronic,
subchronic, or any repeated exposure studies where test agent availability
is not a limiting factor. Dynamic chambers are required for testing within
the EPA guidelines (USEPA 1982).
4.3.2.3 Whole-body exposure systems
Whole-body exposure systems, especially when operated dynamically,
are the most frequently used exposure systems in inhalation testing. The
animals are placed in cages within the exposure chamber and exposed to
the test agent atmosphere. Whole-body exposure requires no surgical or
anesthetic preparation prior to testing (MacFarland 1976). The animals
are free to move within their cages, and the use of exercise devices can
provide information on the effects in active animals. With whole-body
exposure systems, large numbers of animals or several different species can
be exposed simultaneously in one chamber (MacFarland 1976). Uniform
exposure conditions for all animals are obtained even when large numbers
of animals are exposed. The whole-body system does, however, have some
disadvantages. Deposition of the test agent (especially aerosols) on animal
fur with the potential for ingestion during grooming behavior is a major
problem (MacFarland 1976, Nettesheim and Griesemer 1978). The
animals should be housed in individual cages to reduce such ingestion and
to avoid dosage interference resulting from filtration through the fur of
huddling animals (MacFarland 1976, Clark 1977). When whole-body
exposure systems are operated in the usual dynamic mode, large quantities
of the test agent are required, which can be a disadvantage (MacFarland
1976), and, if the test agent is extremely hazardous, special handling tech-
niques and equipment, such as glove ports, must be used to protect the
laboratory technicians (Nettesheim and Griesemer 1978). These draw-
backs have stimulated the development of the alternative exposure systems
discussed in Section 4.3.2.4.
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4.3.2.4 Nose- and head-only exposure systems
In some inhalation studies, exposure of the whole-body is not accept-
able; for these situations nose- or head-only exposure systems have been
developed. Basically, these systems consist of a central chamber into which
the test agent is introduced and separate holding devices that position the
animal so that only the nose or head is protruding into the test atmo-
sphere. The design of the central chamber may be circular, hexagonal, or
linear and be constructed of Plexiglass® or stainless steel (Thomas and Lie
1963, MacFarland 1976). The nose, muzzle, or neck of the exposed
animals must be sealed to prevent loss or dilution of the test agent. The
holding devices include cylindrical Plexiglass® containers (e.g.,
plethysmograph) used mainly for rodents, sling arrangements for dogs, and
adjustable chairs for primates (MacFarland 1976). If exposure of many
animals is desired, cylindrical or hexagonal chambers can be stacked to
provide a larger central chamber for exposure (Drew and Laskin 1973). If
a linear arrangement is used to repeatedly expose large numbers of
animals, the positions of the animals must be rotated between exposures
because the linear arrangement can affect test agent concentrations. An
alternative method involves using a molded face mask to deliver the test
atmosphere. This system also requires some type of restraining device and
an airtight seal between mask and animal to prevent air loss and eliminate
dead space. Latex molding techniques have proved satisfactory for main-
taining a tight seal and have been fitted to dogs, rodents, and primates
(Dubin and Morrison 1969, MacFarland 1976).
The nose- and head-only systems are most useful for studies where no
or little dermal or oral exposure can be tolerated (Drew and Laskin 1973,
MacFarland 1976). Both systems reduce the total area exposed, with the
face mask system exposing the least surface area. A comparison of a
whole-body exposure system and two nose-only exposure systems (a
cylindrical polycarbonate tube and a stocklike restrainer) using radioactive
tracers indicated that the nose-only exposures were much more accurate
for delivery of inhalation doses (Henry et al. 1983). Data show that only
13% of the total dose was deposited in the lungs of mice during a 130-min
whole-body exposure, with 60 to 80% of the dose eventually reaching the
gastrointestinal tract. The nose-only exposure systems delivered approxi-
mately threefold to fivefold more test material to the lungs and threefold
less material to the gastrointestinal tract under the same test conditions.
These systems are also advantageous when the test agent is extremely
hazardous (e.g., radioactive aerosols) or when it is available only in limited
quanities (Drew and Laskin 1973, WHO 1978). For extremely hazardous
substances, the entire exposure apparatus can be set up within an enclosed
inhalation chamber with glove boxes or a rolling drawer (Moorman 1978).
This provides a double measure of safety, enables animals to be manipu-
lated, and allows rapid reestablishment of exposure conditions. Other
advantages include the potential for slower air flow through the chamber,
because body heat buildup is not a problem inside the chamber, and the
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174
capability for rapid exposure of the animals to the test atmosphere (espe-
cially for aerosols) due to the small chamber size (Wells 1979). One
unique application of these systems is a design that exposes animals either
dermally and/or by inhalation, depending on how the animal holder tubes
are inserted into the chamber (Francovitch et al. 1982). Most applications
of these systems are for acute testing, as such tests do not require feeding
or watering of the animals during exposure (Drew and Laskin 1973).
Long-term testing is difficult due to the need to restrain the animals.
One of the major disadvantages of these exposure systems is the diffi-
culty of exposing large numbers of animals (MacFarland 1976). Often a
large crew of technicians is needed if many animals are to be exposed
(Nettesheim and Griesemer 1978). Other disadvantages are caused by the
extra handling and restraints required. The physical stress created or the
anesthetic required to avoid this stress can affect the outcome of the expo-
sure (Drew and Laskin 1973). One aspect of physical stress is the buildup
of body heat within the restraining tube. Perforations in the tube or
other means of ventilation can offset this disadvantage (Kennedy and
Trochimowicz 1982). Nettesheim and Griesemer (1978) found reduced
body weight gain and increased adrenal gland weight in animals exposed
by these systems. However, Smith et al. (1980) reported on a nose-
exposure system using a polycarbonate tube for chronic aerosol exposures
of fibers that did not affect stress as measured by body weight, tempera-
ture, blood counts, and plasma corticosterone levels. Thus, some of the
nose- and head-only exposure systems may be useful for long-term,
repeated exposure tests but are best utilized for the specialized exposure
conditions described.
4.3.3 Chamber Design
As mentioned previously, the majority of inhalation chambers are
designed for dynamic, whole-body exposures. The discussions in this sec-
tion will pertain to this type of chamber unless otherwise specified. In
designing an inhalation chamber the principal objectives are performance
and ease of operation (Fraser et al. 1959). Performance refers to the abil-
ity of the chamber to meet the requirements of the particular inhalation
study being undertaken. Ease of operation includes ready accessibility of
animals for sampling and handling, and maintenance and sanitation
considerations. Due to the wide variety of potential test substances and
their different properties, many modifications of a few basic chamber
designs have been developed. This section will deal with the main design
considerations of shape, size, construction materials, and some operational
features of the basic chambers. Modifications of these basic chambers will
be discussed, depending on their potential utility.
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4.3.3.1 Chamber shape
The shape of inhalation chambers is determined by requirements for
uniformly distributing the test agent and minimizing the internal surface
area in order to reduce adsorption and deposition of the test agent (Silver
1946). Theoretically, a large volume with a small surface area is the
desired condition. This is achieved best by a chamber of spherical shape
(Fraser et al. 1959); however, such a chamber is difficult to build and fit
with doors or windows (MacFarland 1976). The practical solution is to use
a chamber with a cubical shape, but this results in 25% more surface area
than does the sphere (Frazer et al. 1959, Hinners et al. 1966, MacFarland
1976). An intermediate design is a hexagonally shaped chamber, but the
gain in efficiency is usually not worth the increased cost of construction
(MacFarland 1976). By adding pyramid (or cone) structures to the top
and bottom with air entering at the top and exhausting at the bottom, and
by avoiding animal placement in the corners, the air distribution efficiency
can be increased and a cubical chamber used for most applications (Fraser
et al. 1959, Roe 1968).
Large-scale exposure chambers, incorporating most of the shape con-
siderations discussed above, are best represented by the Rochester and the
New York University chambers. The Rochester chamber (Figure 4.3) is a
hexagonal-shaped chamber with conical additions to the top and bottom
(Fraser et al. 1959). The New York University chamber (Figure 4.4) is
based on a cubical shape with pyramid additions (Drew and Laskin 1973)
and is considered the system of choice for repeated exposure studies (NAS
1977).
Other large-scale designs have been used for specialized exposures or
when unique equipment is available. Spherical chambers composed of clear
Lucite® plastic have been used for exposures to carcinogens at low flow
rates and can be built at a lower cost than steel chambers (Stuart et al.
1970). The Longley chamber is a modification of the standard rectangular
design and is subdivided into two or four separate sections (MacEwen
1978). This allows simultaneous exposure of control and three dose levels
of test animals. Also, the design is flexible enough to permit testing of
multiple dose levels for acute studies.
Carcinogenic studies often require specialized chamber designs because
of the long duration of the studies, the large number of animals, and need
to isolate the carcinogens. Laskin et al. (1970) designed a system of three
box chambers that are connected by internal sliding doors. One chamber is
used for the inhalation exposure while the other two chambers house the
control and experimental animals. Pass boxes and neoprene gloves built
into the system allow the animals, food, and wastes to be introduced into,
moved within, or removed from the system. Thus, the animals never have
to be removed from the unit and can be totally isolated. Entire rooms have
also been designed as large-scale exposure units for exposures of many
animals. Chambers with an 2.4 m X 2.4 m floor area and a submarinelike
door hatch at each end were designed at Dow Chemical Corporation for
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ORNL-DWG 85-10742
CONTAMINANT
SUPPLY
SUPPLY
AIR
EXHAUST
AIR'
Figure 4.3. Schematic diagram of the Rochester exposure chamber.
(Source: Adapted from Drew and Laskin 1973. Used with permission of
Academic Press.)
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EXHAUST AIR
SUPPLY AIR
ORNL-DWG 85-10743
CONTAMINANT
SUPPLY
DRAIN
Figure 4.4. Schematic diagram of the New York University exposure
chamber. (Source: Adapted from Drew and Laskin 1973. Used with per-
mission of Academic Press.)
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178
exposure of large numbers of test animals (MacEwen 1978). The doors
were connected to a clean corridor and a "dirty" corridor to control the
spread of the test materials via the movement of animal racks in and out
of the chamber. The current state of large chamber design is "one
chamber-one room-one project," which achieves the performance of indivi-
dual experiments in an integrated framework (MacEwen 1978).
Smaller-scale exposure chambers offer more variations in design than
the larger units because they are used for single exposures or short test
durations. One of the first and simplest small-scale designs (Figure 4.5) is
a glass bell jar mounted and sealed to a wooden or metal base with ports
for air inlet and exhaust (Fraser et al. 1959). These can be operated in a
dynamic or static mode and are usually enclosed in a fume hood for added
safety. Similar to this approach, Laskin and Drew (1970) designed a
Lucite cylinder with injection molded domes at each end, supported on a
wooden frame. The top dome can be removed for access and is sealed with
rubber O-rings. This system is relatively inexpensive and its size (36 cm in
diameter and 60 cm in length) makes it ideal for acute or pilot studies.
Another approach to small-scale units is to scale down a larger design.
Fraser et al. (1959) describe a dynamic, cubical unit with two stainless
steel walls, one Plexiglass® wall, a removable Plexiglass® door, and a max-
imum size of 25 ft3 (7.6 m3). Drew and Laskin (1973) described another
scaled-down unit, called a California Hood, that utilizes the New York
University chamber design mounted within a square exhaust chamber.
Besides these small-scale exposure chambers, a unique chamber design of a
converted, cylindrical human respirator with a horizontal layout and a
once-through airflow from end to end has been described for short-term
exposure of monkeys (Thiede et al. 1974). An important factor that should
be incorporated into any of the small chamber designs is adaptability of
the components (Fraser et al. 1959). Because the purpose of these designs
is to perform acute, pilot, or other limited studies, component adaptability
will allow the unit to be used repeatedly for different purposes or test
chemicals and consequently will reduce cost.
4.3.3.2 Chamber size
As discussed in the previous section, exposure chambers occur in a
wide range of sizes. The most important consideration for size is the
volume occupied by the animals in relation to the total chamber volume.
The animal volume should never be greater than 5% of the total volume to
prevent heat and carbon dioxide buildup and to avoid interfering with air
circulation (Silver 1946, Fraser et al. 1959). In this regard, it is better to
use a chamber that is too large rather than one that may be too small
(MacFarland 1976). The size range of large-scale chambers that maintain
good air distribution (especially for dusts and aerosols) is 1 to 8 m3
(MacFarland 1976). Chambers of a size outside of this range can be used
if alternative methods for mixing and distributing the test atmosphere are
used or if a static system is employed. For example, Schreck et al. (1981)
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179
Mixing flask
Vapour generator
rotameter
Vapour
generator
Door -
Chamber exhaust outlet
Compressed air inlet
ORNL-DWG 85-10744
Diluting air rotameter
- 7 gallon battery jar
Wooden
clamping bar
Wing nut
Battery jar
support
Wooden frame
Figure 4.5. Small-scale exposure chamber based on a bell jar design.
(Source: Adapted from Leach 1963, as reported in Drew and Laskin 1973.
Used with permission of Academic Press.)
describe a large Rochester-type chamber (~12.6 m3) that uses an exhaust
manifold to aid in maintaining an acceptable air distribution. The large
system was designed to reduce maintenance time and allow large numbers
of test animals to be chronically exposed. However, it is generally recom-
mended that the chamber size be within the l-to-8-ft3 range and that more
chambers be used if space is insufficient for the test purposes.
4.3.3.3 Chamber materials and construction
Ideally, materials used in constructing the inhalation exposure chamber
should be determined by the nature of the test agent (Eraser et al. 1959).
However, most chambers will be used repeatedly for tests of different sub-
stances and they are generally constructed of materials that are highly
resistant to a wide range of substances. The interior of the chamber should
be simple in design and have smooth finished surfaces with no sharp edges.
The walls are usually constructed of, or lined with, a smooth nonabsor-
bent material (Fraser et al. 1959). Common wall materials include alumi-
num, stainless steel, safety glass, and plastic. If less resistant materials are
used (or if the test agent reacts with one of the above materials), the walls
can be lined with glass, enameled, or lacquered (Silver 1946). Galvanized
iron riveted together has been suggested for the pyramid funnels, depend-
ing on the test substance (Timbrell et al. 1970). Stainless steel is the most
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180
commonly used metal, but it is susceptible to crevice corrosion after weld-
ing. If welding is used, Type 304 stainless steel is preferred due to its resis-
tant properties (Hinners et al. 1968). In large chambers, it may be neces-
sary to reinforce the walls with 3/4-in. pipe to provide support (Hinners et
al. 1968).
The door and observation ports are made of plate glass or Plexiglass-
type plastics (Fraser et al. 1959). The door should permit easy access and
be as large as practically possible. If a large glass door is used, stainless
steel framing and cross members may be necessary for support. The door
can be of a sliding design (either vertically or horizontally) or hinged with
full-length piano hinges (Fraser et al. 1959, Hinners et al. 1966). Toggle
clamps, construction clamps, or magnetic devices have been used to main-
tain the tight seal for the long periods that is necessary for subchronic and
chronic inhalation studies (Hinners et al. 1968, Timbrell et al. 1970). A
stainless steel angle around the interior edge of the door will prevent the
test animals from clawing or chewing the door gaskets (Hinners et al.
1968). Seals for the door and removable ports can be made of neoprene,
PVC plastic, or rubber but must be resistant to the test substance. Reversi-
ble neoprene gloves attached to the door have been used in some studies to
allow handling of the test animals in the chamber without opening the
door (Timbrell et al. 1970). At least one observation port should be
included, either at the back of the chamber or in the front of the top
pyramid.
In addition to observation ports, access is needed for sampling and
monitoring of the test atmosphere. These sampling ports may be specially
designed inserts or merely holes cut into the observation port or door; they
should be sealed with rubber or PVC stoppers. Hinners et al. (1968)
recommend specially designed ports located at the back of the chamber at
various heights. These are 1.3-cm diameter stainless steel full couplings
with PVC plugs through which steel sampling probes or other monitoring
equipment can be inserted into the test atmosphere. Further discussion on
sampling equipment is presented in Section 4.3.5.
An important part of the chamber design is the construction and
arrangement of animal cages within the chamber. In general the following
conditions for cages should be followed whenever possible: (1) for exposure
to aerosols, animals should be housed individually in separate cages;
(2) larger animals should be placed in the bottom tier of cages; (3) no
pans or solid surfaces should be used in cage designs; (4) a layer of open
space should separate each cage layer; and (5) cage positions should be
rotated to maintain equal exposures for all animals (Fraser et al. 1959).
The individual cages should be made of a mesh material, most frequently
stainless steel. Some recommended sizes and materials for the cages are:
46 X 46 X 46 cm of stainless steel mesh for dogs and monkeys, 23 X 23
X 38 cm of 1.3-cm mesh hardware cloth for rabbits, and 7.5 X 7.5 X 15
cm of 0.6-cm hardware cloth for small rodents (Fraser et al. 1959). If the
test animals are to be continuously exposed, larger cage sizes may be
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181
necessary, such as those recommended by the U.S. Department of Health,
Education, and Welfare (1974). The small rodent cages can be combined
into a single tray of 20 or so components to aid in handling (Fraser et al.
1959). If the chamber door is large enough, the cages can be mounted on
a cart, which can then be rolled in and out of the chamber as needed. If
this is not possible, cage supports should be added to the chamber walls.
These can be metal pins welded into the corners at six levels 25 cm apart
or metal tracts mounted on the chamber sides (Hinners et al. 1966). Often
metal grates or mesh partitions are used to support the smaller rodent
cages. An interesting modification of these standard cage arrangements
has been reported by Wells (1979) and Carney (1979). This consists of a
"ferris wheel" type arrangement (Figure 4.6) that allows the cages to be
rotated automatically during exposure. Such a cage arrangement can help
stabilize and equalize exposure, especially for dust exposures, by eliminat-
ing shadow effects from the cages. It can also be used to increase the
number of animals per chamber (Carney 1979). A variation of this ferris
wheel design was discussed by Doe and Tinston (1981); air enters the
chamber at the top front, flows in a cyclical pattern concurrent with the
rotation of the cage wheel, and is exhausted at the top rear of the
chamber. Moss and Brown at Battelle Pacific Northwest Laboratory
designed a chamber modification with another alternative cage arrange-
ment (Beethe et al. 1979). This design provides constant concentrations
and size distributions of aerosols within the chamber as well as reduced
handling time for the animals. The cages are constructed in compartmen-
tal units that occupy one-half of the cross section of the chamber. The
units are arranged at six different heights and are designed to operate with
waste catch pans in place (Figure 4.7). Air enters the top of the chamber,
flows down the sides, and is diverted into distributing eddies by the catch
pans. However, verifying studies showed that lung deposition in rats varied
somewhat for different locations in the chamber. Griffis et al. (1981b)
tested the chamber design and found that animals on tiers 2, 4, and 6
(Figure 4.7) had lung burdens 8 to 11% greater than animals on the other
tiers. Also, animals on the lower tiers had burdens 5 to 6% greater than
animals on the upper tiers. Griffis et al. recommended that animals be
rotated among the tiers for chronic tests.
Other design considerations include heating and cooling of the
chamber, which is usually accomplished through the air supply (see Sec-
tion 4.4). Light can be provided by indirect sunlight or preferably by
artificial illumination. If artificial illumination is used, light fixtures should
be placed outside the chamber to avoid the necessity of specially sealed
fixtures (Fraser et al. 1959). Equipment and techniques for maintaining
the sanitary conditions in the chamber are discussed in Section 4.3.6.
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ORNL-DWG 85-10745
Figure 4.6. Whole-body dynamic exposure chamber of 1.3-m side. The
animals are accommodated in 16 mesh cages that are supported on the
ferris wheel. (Source: Adapted from Wells 1979. Used with permission of
Blackwell Scientific Publishers, Ltd.)
4.3.4 Airflow Systems
The airflow system of an inhalation exposure chamber should include
the following components: a source of air, several banks of filters (for both
supply and exhaust lines), air conditioning units, a system of ducts, lines,
or other containers for distribution, and devices to control and measure the
flux and/or amount of air, test substance, and final mixtures (Lodge
1968). There are many ways of incorporating these features into the air-
flow system. Use of common air supply and exhaust lines is recommended
to minimize costs (Fraser et al. 1959). The system should be designed with
flexibility of use in mind, since individual components of the system may
have to be modified or moved to allow testing of varied substances. A
schematic diagram of a "typical" airflow system for dynamic, whole-body
exposure is presented in Figure 4.8.
Control of the airflow within the system relies on the proper position-
ing of values, meters, and pumps to determine its rate, distribution, and
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183
ORNL-DWG 85-10746
Figure 4.7. Cage arrangement and chamber design of Moss and Brown
showing airflow pattern. (Source: Adapted from Beethe et al. 1979. Used
with permission of the publisher.)
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184
ORNL-DWG 81-H342
AIR INTAKE
^ LJ LJ
CONDITIONING - COMPRESSOR FILTERS
n n
COMMON SUPPLY LINE
VENTURI
FLOW
METER
INJECTION PORT FOR
TEST SUBSTANCE
COMMON EXHAUST LINE
EXHAUST
I PUMP |
FILTERS
ANIMAL
EXPOSURE
CHAMBER —
AIR
RELEASE
WASTE DRAIN
Figure 4.8. Schematic diagram of airflow system. (Source: Adapted
from Wright 1957 and Wehner et al. 1972.)
operational pressure. An integrated airflow control system was designed by
Koizumi and Ikeda (1981) that automatically controlled exposure concen-
trations, flow rates, and sampling for vapor atmospheres. Their system
consisted of four exposure chambers with vapor generators, a tank of
methane gas for calibration, a gas liquid chromatograph, a computer with
a printer, and a flow controller. Depending on the computer program, a
variety of atmosphere types can be generated, including continuous expo-
sure at a given and constant concentration; mixed vapors exposure; expo-
sures with commanded geometric means and standard deviations; wave
patterns; and repeated short-term peaks. This variety allows the researcher
to duplicate real-life exposure situations, such as variable concentrations of
workplace atmospheres, with low variability (coefficients of variation of
3.7 to 6.1%) and with greater reproducibility.
The air pumped into the exposure chamber is generally taken from the
room atmosphere (Hinners et al. 1968) or from a central pressurized
laboratory supply line (Fraser et al. 1959, Timbrell et al. 1970). These
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185
sources provide "pure air," which can be operationally defined as air free
of anything known to interfere with the study (Lodge 1968). Of course, to
do this, the internal supplies of air must process air from the environment
through filtration, compression, and conditioning units (Figure 4.8.).
The rate that the air is delivered through the system is limited at the
lower end by the amount of air needed to overcome heat buildup generated
by the animals and on the upper end by the atmosphere generation or feed
rate (Fraser et al. 1959). For most exposure situations, a rate for small
chambers of 2 to 10 ft3/min (0.6 to 3 m3/min) or for large chambers of 2
to 20 ft3/min (0.6 to 6 m3/min) is recommended (Roe 1968). A venturi-
type flow meter, placed in the supply duct and hooked to a magnehelic
pressure gauge, is used to monitor the rate (Fraser et al. 1959). Various
types of valves have been used to control the rate, including sliding gates
mounted on, or geared cones inserted into, orifice plates in the supply and
exhaust lines (Fraser et al. 1959); a butterfly damper in the supply line
and a globe valve in the exhaust line (Hinners et al. 1966); and flexible
pinch valves in the exhaust line (Fraser et al. 1959). The valves should be
placed downstream from filter units to prevent clogging (Drew and Laskin
1973). It is necessary to verify the accuracy of the construction and the
calibration of valves and meters after actual placement in the system
(NAS 1977). Alarms can also be installed in the airflow system to signal
when valves, pumps, or other apparatuses have malfunctioned and dis-
rupted the airflow system (Schreck et al. 1981).
The distribution of the air (and test substance) in the exposure
chamber must avoid a direct flow from inlet to outlet and areas of stag-
nant air (Fraser et al. 1959). Criteria for an acceptable chamber exposure
were proposed by Carpenter and Beethe (1978) and include: (1)
nonrelational uniform flow from inlet to outlet, (2) no aerosol recirculation
from below to above animal levels, and (3) uniform aerosol size distribu-
tion and concentration distribution. The pyramidal top provides an area
for mixing of the atmosphere and works quite well when the air enters
tangentially to the short cylinder at the top of the pyramid (Hinners et al.
1968). The test substance is injected similarly (Figure 4.8). However,
there is a tendency for the inlet air to flow down the sides of the chamber
instead of uniformly. A venturi-type mixing section attached to the top of
the pyramid has been suggested as a remedy (Fraser et al. 1959). The air
is usually exhausted from the bottom center of the chamber through one
arm of a Y joint (Roe 1968). It is just as important to maintain uniform
flow for air leaving the chamber as for air entering. A distribution plenum
inserted below the exposure zone of the chamber has been recommended
to provide this uniform exhaust flow (Fraser et al. 1959, Wehner et al.
1972). Another exhaust alternative producing uniform exhaust flow uses
several exhaust ports spread over the bottom of the chamber leading to
one central exhaust (Carpenter and Beethe 1978). Modifications of the
standard chambers often include airflow alterations. For example, Holm-
berg and associates modified the Rochester chamber to handle dense diesel
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186
aerosols by adding a dispersing cone and screens to produce a more lam-
inar airflow (Holmberg and Moneyhun 1980, Holmberg et al. 1981).
Another variation in airflow design involved a switch to horizontal, nearly
laminar airflow (Hemenway and MacAskill 1982). This design used a
standard rectangular tank with a unique inlet and outlet baffle/plenum to
create an evenly distributed flow through the chamber. A prechamber mix-
ing tube allowed the test substance to be blended at the proper concentra-
tion prior to injection into the chamber. Fecal pans set below the cages
prevented the flow from being totally laminar, but particle size samples
indicated no variation in size distribution. The horizontal laminar flow was
also used in a chamber design by Ferin (1978). This design used four
modules, with one module for filters, the second for injecting the test
atmosphere (using perforated tubes), the third containing the animal cages
and a perforated plate (diffuser) to convert the airflow from turbulent to
laminar, and the fourth containing an exhaust manifold.
Among the various designs mentioned, one study was found that com-
pared the efficiency of their airflow systems. This study used a model to
calculate from test data the amount of dead space and turbulence in the
design (Hemenway et al. 1982). The model was derived from the mass
balance of the system and incorporated a dispersion coefficient as
representative of the amount of turbulence. The results showed that a con-
ventional design had 84.5 to 89.5% dead space and a negative dispersion
coefficient (often the tracer profile appeared at the outlet point before it
showed at the inlet point). The horizontal flow design had dead space
areas of 34 to 37% and a dispersion coefficient of 0.14 m2/s. The multi-
tiered chamber (Figure 4.7) showed almost no dead space (0 to 6%) and
had a dispersion coefficient of 0.12 m2/s. These tests were done without
the presence of animal cages. Dead space increases time to concentration
equilibration at start-up and shutdown, decreasing the utility for
pharmacokinetic studies.
The operational pressure of the exposure chamber is controlled by the
valves and meters placed in the airflow system. It is generally recom-
mended that the chamber be operated at a negative pressure (relative to
the external atmosphere) of 0.25 to 1.3 cm of water (Hinners et al. 1966,
Roe 1968, USEPA 1982). This pressure is maintained by exhausting air
through a pump in the exhaust line at a faster rate than the rate of incom-
ing air (Fraser et al. 1959). A pressure of 15 to 28 cm of water at an air-
flow rate of 1000 to 2000 ft3/min (305 to 610 m3/min) in the exhaust line
will provide the necessary suction for a negatively pressurized chamber
(Roe 1968). As a result of the negative chamber pressure, precautions
must be built into the valve control system to prevent asphyxiation of the
test animals if the air supply in interrupted (Wright 1957).
Conditioning of the air in the exposure chamber is a primary function
of the airflow system. This includes maintaining proper temperature and
humidity levels and removing any impurities by filtration. Temperature of
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187
air flowing through the exposure chamber is affected by temperature of air
from the external source, temperature of air inside the exposure building,
and heat released by the animals. Bernstein and Drew (1980) have shown
that, when intake air is at room temperature, the majority (95%) of excess
heat is radiated through the chamber walls. Cooling of the air stream
allowed more heat to be removed. With low animal loading of the
chamber (eight rats in a 380-L chamber) 40 to 80% of the excess heat is
removed by the cooled airstream, but as more animals are used, only 17 to
30% of the animal heat is removed by the airflow. Thus, if high animal
loading is necessary and high flow rates cannot be used, then the exposure
room temperature should be lowered to provide the necessary cooling to
maintain proper chamber temperatures. By maintaining the exposure room
and chambers in the same temperature range, heat transfer between these
two areas, and radiant heat exchange between the chamber walls and
nearby animals are avoided (Fraser et al. 1959). The recommended tem-
perature range for exposure chambers and rooms is based upon preferred
temperatures of the most frequently used test species and is 75 to 82° F
(24 to 28°C) (Fraser et al. 1959, Hinners et al. 1966). The proper tem-
perature is even more important in inhalation studies because it can affect
respiration rate and pattern and thereby the extent of exposure. Fraser et
al. (1959) discussed the calculations necessary to select the proper cooling
unit and recommended a 1/2-ton refrigeration unit for chambers of 145
ft3/min (44 m3/min). If extremely high flow rates (>50 ft3/min or >15
m3/min) are used, a 3/4-ton unit is suggested. If the local climate does
not require such a large cooling capacity, then copper cooling coils can be
wrapped around the incoming air ducts to provide the proper temperature
(Hinners et al. 1966). In either case the thermostat should be located in
the exhaust line.
Similarly to the detrimental effects on test animals of high tempera-
ture, excessive humidity can affect inhalation exposure and, depending on
the test substance, test agent concentration. It is necessary to know the
heat load and the moisture generation rate of the animals in order to
determine the proper controls for humidity. Fraser et al. (1959) discuss the
necessary calculations to determine these factors. Frequently, the cooling
process will reduce the humidity to the desired levels; however, it may also
be necessary to cool the incoming air to below the desired temperature and
then heat it to remove excess humidity and provide the correct tempera-
ture level. The desired humidity levels are generally accepted as equal to
or lower than 55% relative humidity (Fraser et al. 1959). EPA guidelines
require a humidity of 40 to 60% (USEPA 1982), but exceptions can occur
if the test agent makes this difficult.
Another element of chamber atmospheres that can be a confounding
factor is the presence of ammonia. This ammonia comes from the expired
air of the test animals (Barrow and Steinhagen 1980) or arises from the
action of bacteria on urine and feces of test animals. Elevated ammonia
concentrations can react with test chemicals (Barrow 1978) and affect the
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188
health of test animals (Weissman et al. 1980). Several studies evaluated
the potential impact of ammonia in whole-body chambers and nose-only
exposure systems. As expected, ammonia concentrations increased with
time. Several mechanisms could be used to acceptably control the levels,
including use of bacteriostatic cageboard (impregnated with neomycin); an
increase in airflow rates; frequent changing of cageboard; and frequent
removal of animal wastes (Barrow 1978, Weissman et al. 1980). A recom-
mended procedure would be to use an airflow rate equivalent to 14
chamber volumes per hour, use bacteriostatic cageboard, change the cage-
board daily, and clean the chambers and cages weekly; this should keep
levels below 2.7 ppm for mice (Weissman et al. 1980).
To remove potentially interferring impurities and achieve the "pure air"
necessary for inhalation studies, it is widely recommended that several
banks of filters be employed in both supply and exhaust stages (Fraser et
al. 1959, Hinners et al. 1968, Roe 1968). The most common impurities
are water vapor and oil droplets (Wright 1957). However, potentially more
harmful substances can be found in most air supply sources, and filtration
should be designed to handle a wide range of pollutants. Lodge (1968)
recommends the use of several filters of increasing efficiency, including
charcoal beds to remove organics and polar gases, a furnace for combus-
tion of trace organics, and a final filter to remove particulates from the
furnace. Drew and Laskin (1973) recommend filtration through an
absolute (HEPA) filter and charcoal beds. Most recommendations require
at least two different filters for incoming air, with the first filter to handle
large materials (Hinners et al. 1968, NAS 1977). The air with traces of
test agent leaving the exposure chamber must also be thoroughly filtered,
preferably at both the individual chamber exhaust and the centralized
exhaust line (Roe 1968, Drew and Laskin 1973). Timbrell et al. (1970)
recommend the use of two primary filters followed by one absolute filter
for each chamber exhaust. Drew and Laskin (1973) recommend filtration
of chamber exhaust through a roughing filter (to remove animal hair and
debris), an absolute filter, and a charcoal filter. They also recommend a
similar filter composition for the central exhaust. In special circumstances
it may be necessary to use scrubbers, electrostatic precipitators, or
cyclones (Drew and Laskin 1973). As an added precaution, Timbrell et al.
(1970) recommend high release stacks to help dilute the released exhaust
air in the environment.
4.3.5 Sampling and Monitoring of Chamber Conditions
During operation of the exposure chamber, it is necessary to sample
the atmosphere inside the chamber to verify that correct exposure condi-
tions are being maintained. Environmental factors that should be moni-
tored include temperature, humidity, pressure, and certain gas concentra-
tions. In addition, the test agent distribution and concentration and the
airflow rates must be monitored (USEPA 1982).
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189
The temperature of the chamber can be monitored by mounting a ther-
mometer inside the chamber or by installing, in the chamber wall, thermo-
couples that are connected to dial thermometers outside the chamber
(Fraser et al. 1959, Timbrell et al. 1970). The latter method is preferred
when large chambers are used with samples taken from various locations
in the air system and exposure chamber (Figure 4.9.). This sampling pat-
tern can also be used to determine humidity and to verify adequate air
mixing and distribution within the chamber. Humidity determinations can
be obtained by remote probes connected to a hygrometer or by a hair
hygrometer placed inside the chamber (Timbrell et al. 1970). Nelson and
Taylor (1980) have described an integrated, automatic system that moni-
tors and controls chamber temperature, humidity, and airflow rate. The
system uses a commercially built temperature-humidity indicator with a
specially designed airflow-temperature-humidity control module. The mon-
itors provide feedback control for the system and, once calibrated, the sys-
tem provides flow rates with variations of 0.2%, temperatures with varia-
tions of 0.1°C, and humidities with variations of 0.5%. This type of
automated system requires less operator time and produces excellent con-
trol. Correct operational pressures can be monitored by magnehelic gauges
connected to the chamber, exhaust line, and supply line (Drew and Laskin
1973). The concentrations of certain gases (e.g., oxygen, carbon dioxide)
may need to be monitored occasionally; standard chemical analyses have
proved satisfactory for this monitoring (Fraser et al. 1959). For all these
monitoring techniques, the accuracy of the procedure and equipment
should be periodically verified using reference standards (Fraser et al.
1959).
The main monitoring effort is normally focused on test agent concen-
tration and distribution, using different techniques for gases and aerosols.
In general, to determine concentrations accurately, you must sample the
actual concentrations in the chamber and at the breathing zone of the
animals (Figure 4.9.) and not rely on theoretical concentrations calculated
from rate of airflow and test substance feed rate (Fraser et al. 1959). Even
if concentrations are measured accurately, there will be variation in the
dose calculations as a result of variations in the precision of the analytical
method. Also, with most monitoring techniques, there is a delay between
sampling and analysis that can limit its applicability (Fraser et al. 1959).
In systems that use a blower-powered chamber exhaust, a potential prob-
lem is dilution of the test atmosphere by withdrawing air for the sample. If
a blower is used to withdraw the sample, the test agent concentration can
be affected; to avoid this dilution the discharge from the sampling blower
should be routed into the chamber discharge in front of the exhaust
blower. This will ensure that the total amount of air withdrawn from the
chamber remains constant (MacFarland 1981). Automatic sampling
techniques can be used, especially where chemical or photoelectronic
methods are employed. These are usually cheaper in the long run, produce
a permanent record of exposure, and if rapid responses are possible can be
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ORNL-OWG 85-10747
w reference
corner
Figure 4.9. Sketch of chamber indicating the eight corner and one refer-
ence sampling positions. Letters indicate positions of thermocouples used
for measurement of temperature distribution within chamber. (Source:
Adapted from Fraser et al. 1959.)
linked to a feedback method for controlling chamber feed rates (Fraser et
al. 1959). Currently, the number of automated procedures is increasing
(Hinners 1978, Van Stee and Moorman 1978, Kennedy and Trochimowicz
1982), but those in operation should still be verified by calibration or by
nonautomated procedures to ensure representative sampling and instru-
mental accuracy (Lodge 1968, Tillery et al. 1976, WHO 1978).
Sampling techniques are comparatively simple when the test agent is a
gas or vapor. One technique is to obtain a definite volume of the test
atmosphere at a known temperature and pressure in a container (e.g., eva-
cuated flask). An alternative method is to pass a known volume of test
atmosphere through a suitable collective material (e.g., impregnated
paper). The samples obtained can then be analyzed by standard analytical
techniques (Drew and Laskin 1973). Direct methods for measuring gase-
ous concentrations can also be used and adapted for automatic analysis
(Table 4.6) depending on the test agent. Automatic techniques for sample
analysis should be selected based on their specificity, sensitivity, reliability,
response time, and cost (Bryan 1970). Nader (1971) reviewed the instru-
mentation aspects of these continuous monitors.
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191
Table 4.6. Measurement Principles Used in Continuous Monitoring
Instruments for Gases and Vapors
Absorption spectrometry
Visible and ultraviolet O3, NO2, SO2, CHO'
Infrared CO, hydrocarbons
Chemiluminescence O3
Electrometric
Conductometry SO2, acid gases
Coulometry O3, SO2, and other electroreducible
and oxidizable gases
lonization (flame) Organics
Electron capture* Peroxyacylnitrates, halogenated
organics
aSemiautomatic sequential analysis with gas chromatography.
Source: Adapted from Bryan (1970).
Sampling and monitoring of aerosol and dust exposure studies are more
difficult, because both mass concentration and particle size must be deter-
mined. Some of the varied sampling methods used for aerosols are listed in
Table 4.7 with the particle size range for which they are appropriate
shown in Figure 4.10. Sampling methods and equipment for mass concen-
tration and particle size determinations are essentially of three types. One
type of sampler roughly separates particles into two groups representative
of the upper and lower respiratory tract, another type is designed to
separate the test particles into several classes of different sizes or aero-
dynamic behaviors; and some samplers collect or analyze the particles
without any physical separation based on size.
The first aerosol sampling devices were designed to mimic the natural
separation of particles that occurs between the upper and lower respiratory
tract. Generally, particles of 7 to 10 pm diam are considered to be non-
respirable and represent the particle size found in the upper respiratory
tract (Hatch and Gross 1964, Roe 1968). Gravity sedimentation was one
technique used to simulate this separation pattern. With laminar flow of
air through a horizontal channel, particles will settle out depending on
their size and settling velocities (Hatch and Gross 1964). Wright (1954)
designed a collector (Hexhlet sampler) using this principle with an elutria-
tor composed of two banks of horizontal aluminum plates separated by a
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Table 4.7. Participate Sampling and Characterization Apparatus
Apparatus
Quantification
Remarks
Dust jars, screens, plates, boxes
Cascade jet impactors
Single jet impactors
Fine-fiber impinger
Greensburg-Smith impinger
Glass, asbestos, metal cellulose, and
other esters in porous membranes
Paper tape high-volume
Cyclones
Light scattering devices
Direct microscopy
Precipitators
Sedimentation
Count, weight, chemical analysis
Impingement
Count, chemical weight, micrurgic
Count, chemical weight, micrurgic
Count, chemical, micrurgic
Count, chemical
Filtration
Weight, chemical, visual, etc.
Chemical, color, light
transmission
Centifugation
Weight, chemical, count
Optical
Particle count concentration and size fractiona-
tion by light scattering, tyndallometry
Count, visual examination, sizing
Electrostatic thermal
Count, micrurgic, visual
Simple, inexpensive, readily available. Accepted method for
pollen. Long settling time for submicron particles. Not isomatric
Rapid aerosol fractionation. If to count, not good for high
concentrationa. Can calibrate for BAD
Efficient to 1 pm, then less efficient. Distinguish solid from
liquid aerosols
Simple, compact. Solid-liquid distinctions
Soluble gases and particulates. Commercially available, portable
Wide range of aerosols and applications. Inexpensive, readily
available (e.g., Millipore, Gelman)
Smoke and aerosol density
Wide range of sizes, materials, utilities. Available, inexpensive
Commerially available. Expensive. Need careful calibration
and operation. Automatic, rapid, small sample requirements
On impaction, filter, or electrostatically collected samples
Electrocharge, thermal gradient principles. Submicron particles
Source: Adapted from Campbell (1976).
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ORNL-DWG 81-11343
—
ULTRA
ULn
CENTF
CRAMICROSCC
PE
ELECTRON MICROSCOPE
ULTRAFILTRATION
1IFUGE
X-RAY DIFFRACTION
—
M
CENTRIFl
CROSCOPE
GAS ELUTRIATION
JGE
SEDIMENTATION & GRAVITY SETTLING
'
PERMEABILITY
TURBIDIMETRY
LIGHT SCATTERING
SIEVING
VISIBLE TO EYE
MAC
MINE TOOLS
(MICROMETER, VERNIER CALIPER, etc.)
0.0001
0.0005 0.001
0.005 0.01
0.05 0.1 0.5 1 5
PARTICLE SIZE (microns)
10
50 100
500 1000
5000 10,000
-*- PARTICLE SIZE LIMITS UNDER AVERAGE CONDITIONS.
•»- STATED METHOD IS OF DOUBTFUL UTILITY IN THESE SIZE RANGES.
Figure 4.10. Limits of particle size measuring equipment. [Source: Sheehy et al. 1967, as reported in Campbell 1976.
Reprinted from Clinical Toxicology 9(6):863 (1976) by courtesy of Marcel Dekker Inc.]
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194
vertical partition into 120 inlet ducts for the first separation stage and a
soxhlet filter to collect the "respirable" particles in the second stage (Fig-
ure 4.11). This unit will collect about 1 g of particles when operated at
100 L/min for 8 h (Hatch and Gross 1964).
Another two-stage collector, the conicycle, uses centrifugal force to
both reject the coarse upper respiratory tract particles and collect the fine
respirable particles (Figure 4.12). In Figure 4.12, large particles are
rejected at A and small particles enter B with deposition on surface C.
Very small particles may be partially deposited or exit through orifices O.
The sampler operates at a rotational speed of 8000 rpm and a sampling
rate of 10 L/min (Wolff and Roach 1961 as cited in Hatch and Gross
1964). Performance curves for the conicycle and hexhlet samplers are
shown in Figure 4.13. A two-stage continuous flow cascade impactor that
produces a separation window between 4 and 1 nm from a polydisperse
aerosol has also been developed (Gat 1980). Other two-stage samplers
include the cyclone separator, which uses centrifugal force to separate
large particles and a filter to trap small particles, and the preimpinger,
which uses changes in air direction and water filled-glass flasks to separate
and collect the different particle sizes (Fraser et al. 1959, Hatch and
Gross 1964). Generally, there is little difference in collection efficiency
between these methods, except for the potential for disaggregation of large
particles or aggregations with the cyclone or preimpinger (Hatch and
Gross 1964). These differences can be important, because the particles
must be collected in the form occurring in the aerosol cloud to be valid
particle size determinations.
Aerosol samplers have also been designed to produce multiple stages of
particle separation based on size or aerodynamic behavior. The cascade
impactor (Figure 4.14) was the first sampler designed with this capability
(Hatch and Gross 1964). It consists of a series of impingement slots (usu-
ally 5 to 10) arranged in order of decreasing width and distance followed
by a high-efficiency filter. It works on the principle that progressively finer
particles are deposited at each stage of impingement as a result of increas-
ing jet velocities and decreasing jet dimensions (Hatch and Gross 1964). A
commercial model and its particle size separation series are shown in Fig-
ures 4.15 and 4.16. The separation series compares rather well to lung
separation patterns with, stages 1 and 2 corresponding to nasal chamber
deposition, stages 3 to 5 corresponding to upper respiratory airways and
pulmonary air spaces deposition, and stages 6 to 8 corresponding to lung
deposition. Thus it can be used to identify different sites of deposition for
different size particles. The cascade impactor does have two main draw-
backs. First, it tends to break up aggregations of particles that would
deposit as large particles, and, second, only a limited amount of material
can be safely collected on each surface before the risk of dislodgement
becomes a factor (Hatch and Gross 1964).
A variation of this sampler is the Electronic Cascade Impactor (ECI).
It was designed by Tropp et al. (1980) to provide the following desired
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195
ORNL-DWG 85-10748
Inches
Figure 4.11. Hexhlet two-stage aerosol sampler. A = First stage of
gravity elutriator with size separation based on the equation P = 1 — d2/50,
where P = percentage penetrating and d = diameter of unit density
sphere in microns; B = tapered critical orifice; C = ejector to maintain a
constant airflow rate; and D = soxhlet filter. (Source: Adapted from
Wright 1954. Used with permission of the publisher.)
ORNL-DWG 85-10749
1 cm
Figure 4.12. Conicycle aerosol sampler. A = Inlet for atmosphere;
B = inner rotational space; C = deposition surface (removable) for smaller
particles; O = air outlet; Z = rotational axis; and / = radial distance
(only particles coarse enough to travel this distance in the time required for
the air to pass through the chamber will be deposited). (Source: Adapted
from Wolff and Roach 1961.)
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ORNL-DWG 85-10750
o
CL
02468
EQUIVALENT PARTICLE DIA, MICRONS
Figure 4.13. Performance curves for the hexhlet and conicycle samplers.
1 = Experimental pulmonary deposition curve of Brown et al.; 2 =
Findersen's theoretical curve for human alveolar deposition; 3 = hexhlet
calculated performance curve; and 4 = conicycle calculated performance
curve. (Source: Adapted from Wolff and Roach 1961.)
characteristics: (1) a response time of one second or less for the entire size
spectrum; (2) capability to be calibrated absolutely; (3) capability to
measure very dense (without a dilution step) as well as very dilute aero-
sols; (4) ability to classify (in time sequence, if desired) aerosol particles
for subsequent chemical or physical analyses; (5) a large volumetric sam-
pling rate to minimize errors introduced when sampling a spatially hetero-
geneous aerosol; (6) measurement of the aerodynamic particle diameter
rather than such quantities as optical diameter and electrical mobility
diameter; (7) low cost in comparison with available instruments; (8) real-
time output of data that is easily interfaced to recording or data acquisi-
tion equipment. Generally, the ECI operates as does a standard cascade
impactor, except that the aerosol particles are given a unipolar electrical
charge as they enter, each collection stage is isolated electrically from the
other stages, and each stage is connected to an electrometer detector. As
particles strike the collection surfaces, they give rise to a current that is
used to measure and record the particles. Because each collection stage
captures only a certain size range of particles, both the number and size
distribution can be determined. Other automatic analysis techniques have
also been combined with cascade impactors including piezoelectric sensors
and sensors that detect decreases in beta radiation resulting from particle
deposition (Smith et al. 1978).
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197
ORNL-DWG 85-10751
-• SMALLER PARTICLES
1 \ PATH OF
\ SMALL PARTICLE
Figure 4.14. Schematic diagram showing operation of cascade impactor.
(Source: Adapted from Smith et al. 1978).
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198
ORNL-DWG 85-10752
JET STAGE (9 TOTAL!
Figure 4.15. Schematic of Anderson Mark in cascade impactor.
(Source: Adapted from Smith et al. 1978.)
The collection performance of two cascade impactors and a new instru-
ment, the inertial spectrometer, are compared by De Zaiacomo et al.
(1983). The inertial spectrometer was designed by Prodi et al. (1979) and
works by injecting the aerosol into a clean air stream above a 90° bend.
The bend separates the particles by inertia, and they leave the original air
stream at distances as a function of their aerodynamic size (Figure 4.17).
The particles then impact on a filter that is the outer edge of the bend.
This design allows the particles to be separated while they are airborne, in
a continuous distribution, and avoids the problems of bouncing and re-
entrainment (De Zaiacomo et al. 1983). The two cascade impactors were a
multislot design and a multiorifice design with both having eight stages
and a backup filter. The comparison found that for smaller size particles
the three designs performed equally well. For larger aerosols, the multislot
impactor overestimates the median size and geometric standard deviation
in comparison with the other two designs.
Another multistage sampler design is the conifuge developed by Sawyer
and Walton (1950). It uses gravitational force (similar to the elutriator)
with an increase in force supplied by centrifugation. Air is introduced at
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199
ORNL-DWG 85-10753
100
.3 .4 .5 .6 .7 .8.91.0 2 3 4 5 6 7 8 9 10
PARTICLE DIAMETER, micrometers
Figure 4.16. Calibration of an Anderson Mark III impactor. Collection
efficiency is plotted vs particle size for stages 1 through 8. (Source:
Adapted from Smith et al. 1978.)
the apex of a double-walled, hollow, rotating cone, and the particles are
deposited on microscopic slides on the outer wall in decreasing order of
size from top to bottom (Roe 1968). This design produces excellent size
separation without breaking up large or aggregated particles but is limited
by a slow sampling rate (Hatch and Gross 1964).
Samplers have also been designed to collect particles without any dif-
ferential separation. Simple filters and membrane filters are occasionally
operated without separators. Precipitator devices can be fitted with some
type of separator but are usually operated without them. Optical detection
devices are designed to operate without the need for a separator.
Precipitators have been designed that collect particles by thermal or
electrostatic methods. The thermal precipitator consists of a narrow chan-
nel with a heated wire or surface opposed by a cool collection plate or
slide. The particles are struck by molecules with a higher velocity from the
heated side and are forced toward the cool deposition surface (Tillery et
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200
ORNL-DWG 85-10754
Figure 4.17. Working principle of the inertial spectrometer. (Source:
Adapted from DeZaiacomo et al. 1983. Used with permission of the pub-
lisher.)
al. 1976). Electrostatic precipitators are usually operated on a point to
plane discharge design, with a corona discharge from a needle point charg-
ing particles for deposition on a conductive carbon-coated surface (Tillery
et al. 1976). An electrostatic precipitator that uses a radioactive tritium
source to provide ions for charging particles is shown in Figure 4.18. Pre-
cipitators generally operate at low flow rates of 5 to 10 cm3/min and may
occasionally show some size separation.
Optical devices may be used to analyze particle size, mass, and
numbers. In general, they operate by using photomultiplier tubes to detect
absorbtion or refraction of projected light by particles (Drew and Laskin
1973). The photomultiplier produces a pulse proportional to the size of the
particle. One optical monitor uses a laser Doppler velocimeter (LDV) to
measure particulate velocity in an acoustic field (Mazumder and Kirsch
1977). A particle entering the sensing field of the LDV is excited by an
acoustic wave, and the particle oscillations are monitored. By measuring
the phase lag between the oscillation signal and the acoustic wave signal
(measured by a microphone) the aerodynamic diameter and number of
particles can be determined. A prototype instrument using this technique,
the single particle aerodynamic relaxation time (SPART) analyzer, has
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201
ORNL-DWG 85-10755
0-RING
DIRECTION
Of FLOW
_» DIRECTION
OF FLOW
MEMBRANE FILTER
ELECTRON
MICROSCOPE
GRID
TOGROUNO
Figure 4.18. Electrostatic precipitator using radioactive tritium (H3) as
an ion source. (Source: Adapted from Tillery et al. 1976. Used with per-
mission of the publisher.)
been developed (Mazumder et al. 1979). It can analyze particulate sam-
ples in the size range of 0.2 to 10 MHI at a rate of 200 particles per second.
Another laser (He:Ne) device for continuously monitoring the mass con-
centration of monodisperse aerosols was reported by Carlon et al. (1980).
Optical devices are most useful when nondestructive or noninvasive tech-
niques for monitoring particle size and number are necessary. However,
caution must be used with these devices, because light scattering is not
simply a function of size (Tillery et al. 1976); many variables can affect
the results (Drew and Laskin 1973). The use of optical techniques to mon-
itor particulates in industrial process streams has been reviewed by Smith
et al. (1978). Potential problems with other nonoptical aerosol samplers
have also been reviewed. Heindryckx and Dams (1981) investigated some
errors associated with cascade impactors used in field situations, and some
of their information also applies to inhalation study sampling. Lund et al.
(1979) reviewed practical problems arising from the relationship between
dust generators and collectors. Formignani et al. (1982) discussed calibra-
tion apparatuses for filter media of aerosol samplers.
Except for the optical techniques just discussed, the above sampling
techniques must be followed by analysis of the sample. If the sample is
obtained on a filter media, various routine gravimetric or analytic tech-
niques (e.g., chemical, UV absorption, chromatography) can be used to
determine mass concentration. The filter media to be used is often deter-
mined by the analytical technique employed. Microscopic techniques (both
light and electron) can be used to determine particle number and size and
are most frequently used with samplers that deposit the particles on a
removable surface (e.g., an electron microscopic grid in an electrostatic
precipitator). Computerized scanning devices can be used to automate
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202
analysis of microscopic samples and simplify counting and sizing of parti-
cles (Smith et al. 1978). In general, the various sampling and monitoring
methods produce comparable results. Halbert et al. (1981) compared par-
ticle size distribution analysis using three techniques. They found little
difference in analysis results between an electrostatic
precipitation-microscopic technique, a cascade impactor-gravimetric
technique, and a laser-acoustic relaxation-time technique.
4.3.6 Maintenance of Sanitary Conditions
It is important to maintain sanitary conditions in the exposure room
and chambers to avoid potential interference from disease or illness. This
maintenance includes both design features of the chamber and cleaning
techniques.
To aid collection of test animal wastes, the chamber should have a
pitched floor, smooth interior walls, and a centrally located drain (Drew
and Laskin 1973). If the chamber is used for continuous exposures, a
spray ring is usually installed to wash down wastes. The preferred location
is at the top of the bottom pyramid below the metal floor grate (Hinners
et al. 1966). The ring is a l/2-in.-diam stainless steel pipe with four to
eight wide-angle nozzles and is controlled by an external hand valve
(Hinners et al. 1968). At the bottom of the chamber a 3-in.-diam gate
operated by a quick release is used to drain the wash water. This is usually
connected to a sewer line, unless the test substance is dangerous enough to
require collection in containers (Drew and Laskin 1973). If the chamber is
used for intermittent or acute exposures that allow removal of the animals,
a spray ring can be installed at the lower edge of the top pyramid to wash
down the sides or an external hose can be used (Hinners et al. 1966, Drew
and Laskin 1973). To avoid problems with the airflow systems (especially
clogging with animal hair), the lines should be free from rough surfaces,
restrictions, or sharp bends (Hinners et al. 1968).
During cleaning, it is important to prevent cross contamination between
chambers and to protect the health of the technicians. Thus only one
chamber at a time should be opened and cleaned (Timbrell et al. 1970).
While cleaning, the operators should wear protective clothing and respira-
tors (Timbrell et al. 1970). If a hose is used to wash down the chambers,
the water pressure should be low to avoid creating respirable mists of the
water-chemical mixture (Smith and Spurling 1974). For chambers with
spray rings, the water temperature should be no more than 102°F (39°C),
to prevent steaming within the chamber (Hinners et al. 1968). Chambers
with stainless steel components must be cleaned frequently to maintain the
useful life of the chamber. Cleaning solutions should not remain in contact
very long. A cleaning solution of acid detergent with wetting, penetrating,
and low-foaming agents has been used successfully for cleaning deposits on
stainless steel (Hinners et al. 1968). After all chambers have been cleaned,
the exposure room should also be washed down or vacuumed (not swept)
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203
to ensure that no contaminants or waste materials remain (Timbrell et al.
1970).
One aspect of maintaining sanitary conditions is the protection of
workers associated with the testing procedures. In addition to the steps
mentioned above, efforts are needed to ensure that workers are not
accidentally exposed to remnant or leaking test atmosphere. Such exposure
could occur through loading of the vapor generation system during start-
up, leakage during the test procedure, and release during shut-down pro-
cedures (Klein and Geary 1982). Automatic sampling of the chamber and
test room will assist in determining when hazardous or safe conditions
exist. A special problem occurs when particulate substances are being
tested, especially particulate carcinogens (Drew 1978). In these cases,
workers can be exposed to particulates on the cages, animals, and walls of
the test chamber. In addition to dermal contact, these particulates may
reenter the atmosphere and be breathed by the workers (Drew 1978). Use
of a service module or glove and pass boxes will reduce this potential for
exposure. Also, use of and monitoring for an inert indicator substance
(such as sulfurhexafluoride) in the aerosol airstream can indicate when
small leaks occur. This approach is especially useful when rapid monitor-
ing techniques are not available for the test substance (Moorman 1978).
These procedures along with a disease control program will avoid interfer-
ence with the exposure studies.
4.3.7 Specialized Equipment
In addition to the standard equipment described in earlier sections, spe-
cialized equipment is often used in inhalation studies. This includes whole
chambers designed for unique exposure conditions as well as equipment
designed to test nonroutine conditions of inhalation.
Specialized exposure chambers have been developed by research
laboratories of the armed services. These include chambers that operate at
pressures higher or lower than normal atmospheric pressure, and systems
for studying biological aerosols at low temperatures (Drew and Laskin
1973). Outer space and underwater exploration programs have prompted
many of these special chamber designs.
Another special inhalation exposure chamber design for nose- or head-
only exposure was discussed in Section 4.3.2.4. A part of this system, the
body plethysmograph, is also used for measuring functional alterations in
respiration (see Section 4.2.5.4). The body plethysmograph consists of an
airtight metal or plastic (Lucite® or Plexiglass®) cylinder designed to con-
tain the animal with its nose and head inserted in the test atmosphere and
to allow measurement of pressure changes resulting from breathing move-
ments. For rodents, the cylinder varies in size from 9 to 23 cm in length
and from 3 to 12 cm in width (Swann et all965, Alarie 1966). The front
is usually a combination of straight or curved rigid plastic plates and flexi-
ble sheets of sealing material (neoprene rubber, latex, etc.) with a cen-
trally located hole for the head or nose (Thomas and Lie 1963, Murphy
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204
and Ulrich 1964). Occasionally, a sealed face mask is used to deliver the
test agent and provide the necessary front seal. The cylinder is connected
to three tubing lines, one a transducer pressure line to monitor pressure
changes, another to conduct the pressure wave from the pump, and a third
to bleed off excessive pressure (Murphy and Ulrich 1964, Swann et al.
1965). Dorato et al. (1983) discuss a modification of the body plethysmo-
graph that uses an intraesophageal catheter and an analog computer to
produce real-time calculations of pulmonary parameters in rats. This pro-
cedure did not require surgery or prolonged anesthesia and was capable of
providing multiple evaluations over extended time periods (40 weeks). A
body plethysmograph for use with dogs has been described by Boecker et
al. (1964) and consists of a large rectangular Plexiglass chamber with an
internal sling for restraining the animal and a latex face mask for delivery
of the test agent. Although the test animals are usually kept in the
plethysmograph for only a few hours at a time, it is best to acclimate them
prior to the study by placing them in the container for increasing periods
(Smith and Spurling 1974). This will avoid unwanted stress.
Specialized equipment has also been designed to be inserted into the
chamber for use. One such type of equipment allows evaluation of inhala-
tion effects on animals that are exercising. For rodents, this consists of a
revolving cylindrical wire cage (46 cm diam and 61 cm long) that rotates
at a speed of 6.5 rpm and can hold 10 to 15 animals (Fraser et al. 1959).
Another version designed by Illing et al. (1975), consists of a wheel 15 cm
in diameter and 3.8 cm wide that is open on one side and closed by the
converging wires on the other. The cages are mounted on a steel rod run
through the center and separated by Teflon® sheets. A small motor rotates
the cages via the rod at speeds of 1 to 6 rpm. Mice or hamsters can be
tested individually in this design. To test dogs, a rubber treadmill with an
effective area of 56 cm by 1.4 m was designed with operational speeds of
0.9 to 7.5 mph (Fraser et al. 1959). Such equipment is not part of a rou-
tine study, but would be useful if the expected exposure of humans would
involve activity.
4.4. GENERATION OF TEST ATMOSPHERES
4.4.1 Introduction
Testing of chemicals by the inhalation exposure route requires the gen-
eration of many types of test atmospheres. The production of an atmo-
sphere of a precisely known composition is one of the most difficult tasks
the toxicologist faces because the ratio of agent to air can be one to
several million (Lodge 1968). The wide variety of potential atmospheres
precludes a universal apparatus suitable for generating atmospheres for all
inhalation tests (Clark 1977). Each type of atmosphere must be treated
differently, and, even within each type, chemicals with different properties
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205
can require unique generation techniques or equipment. The desired objec-
tive is to produce the required atmosphere with the most efficient tech-
nique available (Carney 1979).
The types of atmospheres generated can be grouped into two broad
categories, gases or vapors, and aerosols. Gases and vapors were classified
according to their mode of physiological action rather than their structure
by Casarett (1975). Asphyxiants are one class of gases and vapors and
include both simple and chemical forms. An asphyxiant is any substance
that has the capacity to deprive tissues of oxygen by any means other than
impairment of respiration mechanics (Casarett 1975). Simple asphyxiants
are usually physiologically inert gases (e.g., nitrogen) that prevent oxygen
from reaching the respiratory surfaces, while chemical asphyxiants (e.g.,
carbon monoxide) prevent the body from utilizing the oxygen (Casarett
1975). Irritants are the other major type of gases or vapors and produce
inflammation in tissues with which they are in contact (Casarett 1975).
Aerosols include any system of liquid droplets or solid particles
dispersed in air with small particle sizes (usually <10 p.m) and conse-
quently low settling velocities to possess considerable stability as an aerial
suspension (Hatch and Gross 1964). Even more than gases or vapors, aero-
sols exhibit a high interdependence between physical characteristics and
toxicity (Casarett 1975). There are many types of aerosols; some of the
more common are discussed below.
1. Dusts are fine particles (diameter range of angstrom to 100 /tm) of
solid material formed by mechanical disintegration of matter and
dispersion into air (Hatch and Gross 1964). They are chemically and
constitutionally the same as their parent material (Casarett 1975).
2. Fumes are clouds of particles (diameter <0.1 /mi) formed by combus-
tion, sublimation, or condensation and are usually accompanied by a
chemical change (Casarett 1975). Often this term is restricted to
descriptions of droplets from metal oxides of zinc, magnesium, iron,
etc. (Hatch and Gross 1964).
3. Smoke refers to a mixture of fine liquid droplets and solid particles
(<0.5 /um) with liquid and vapor phases produced by combustion
(usually of organic material) (Hatch and Gross 1964, Casarett 1975).
4. Mists and fogs also occur in liquid and vapor phases with liquid dro-
plets of any size range formed by condensation of vapors on suitable
aerial nuclei or by uptake of water by hydroscopic particles (Hatch
and Gross 1964, Casarett 1975).
Each of these aerosol types and the gases and vapors require a unique gen-
eration methodology; the following sections will discuss the equipment used
to produce such atmospheres.
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206
4.4.2 Generation of Gas and Vapor Atmospheres
The generation of test atmospheres for gases and vapors is relatively
simple, involving fewer potential complications than the generation of
aerosol atmospheres. Gases and vapors can be tested in a static or dynamic
mode. The only requirement is that the method of introducing the contam-
inant must be compatible with the mode of operation (batch introduction
for static systems and continuous feed for dynamic systems) (Lodge 1968).
A commonly used batch method for generating an atmosphere is to
mix and store the contaminant in a high-pressure cylinder (Cotabish et al.
1961, Tillery et al. 1976). The gas or volatile liquid is introduced into the
empty cylinder by syringe injections or manometer measurements and
diluted with air to the desired concentration before release into the expo-
sure chamber. This technique can be used with stable contaminants and, if
the release rate is slow, can also be used with a dynamic system. Another
simple technique uses high-pressure cylinders containing only the contam-
inant. The contaminant is metered into a flowing stream of pure air by
valves and a calibrated rotometer, which delivers the proper concentration
to the exposure chamber (Amdur 1957, Saltzmann 1961). This technique
is also limited to stable gases or highly volatile liquids during dynamic
exposures. An expansion chamber or reservoir is often used in conjunction
with such a system (Carson et al. 1962).
When the gas or vapor is not highly volatile or chemically stable
enough to store in high-pressure cylinders, other methods exist to introduce
contaminants into a flowing air stream. One of the earliest used techniques
for liquids with high boiling points is to inject the liquid through a motor-
ized syringe onto a heated block or into a heated tube which vaporizes it
(Carpenter et al. 1949, Tillery et al. 1976). The vaporized contaminant is
then introduced into the air stream by a pump or aspiration needle.
A problem with using such direct heating systems to vaporize liquids is
the potential for charring at the heat source or thermal decomposition of
the sample (NAS 1977). To avoid this problem, two techniques have been
recently reported. Potts and Steiner (1980) redesigned a counter-current
system to use a perforated-plate distillation column (Figure 4.19). The
liquid contaminant is introduced at the top of the column and flows down-
ward. Air is introduced at the bottom of the column and flows upward,
becoming saturated with the vapor as it passes over the distillation plates.
This system provides a vapor source of constant concentration, without
thermal decomposition or condensation resulting from cooling of a heated,
saturated vapor. However, the concentration must be measured in the
exposure chamber because no direct measurement of the amount of liquid
vaporized is possible. Also, since the vapor pressure of low volatile liquids
is not accurately known at room temperatures, the experimenter must use
trial and error to determine the correct parameters necessary to achieve
the desired concentrations. A second new technique to avoid thermal
decomposition is designed to handle liquids that are neither highly or
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ORNL-DWG 85-10756
Ambient Air
Inlet
Figure 4.19. Improved counter-current vaporizing apparatus, with ther-
mostating jacket. (Source: Adapted from Potts and Steiner 1980. Used
with permission of the publisher.)
-------
208
minimally volatile (Miller et al. 1980). A J-tube design vapori/er (Figure
4.20) is filled with 1/4-in. glass beads to increase the vaporization surface.
The liquid is introduced at the bottom of the J through a capillary tube.
Heated air flows up through the system, vaporizing the liquid as it passes
over the glass beads. By preheating the air, chances for charring and ther-
mal decomposition are greatly reduced. This design also provides a greater
safety margin when potentially explosive liquids are vaporized.
Another technique for vaporizing liquids is based on two saturation
chambers and is similar in principle to the counter-current column of Potts
and Steiner (1980). An inert carrier gas is passed through two gas washing
bottles containing test liquid, with the first bottle at a higher temperature
than the second (Saltzmann 1961, Drew and Laskin 1973). The use of two
bottles ensures saturation, but a filtering step following the chambers may
be necessary to trap unwanted liquid droplets.
Some unique methods for dispersing vapors and gases are based on dif-
fusion through permeable materials. In one system vapor diffuses at a con-
stant rate through a porous asbestos plug (Lodge 1968). Certain plastics
(e.g., Teflon®) have also been used (O'Keeffe and Ortman 1966, Drew and
Laskin 1973). These systems require constant flow and temperature. The
rate of diffusion can be controlled by varying wall or plug thickness and
area (Drew and Laskin 1973, Tillery et al. 1976).
4.4.3 Generation of Aerosol Atmospheres
4.4.3.1 Introduction
The generation of aerosol atmospheres for inhalation testing involves
more variables than the generation of gas or vapor atmospheres. The divi-
sion of solids or liquids into small particles or droplets and their dispersal
in air produces two major changes, increased surface area and total space
occupied, that affect the toxicity (Drew and Laskin 1973). These changes
intensify the basic chemical and physical activity of the test agent, includ-
ing its rate of oxidation, solubility, evaporation, and deposition. Also, the
particle size that the parent material is divided into determines the depth
of penetration and deposition site in the respiratory tract. Therefore, it is
necessary to determine the particle-size capability as well as the concentra-
tion capability of the aerosol generation equipment in order to select the
proper unit for each test. The equipment must produce and maintain the
desired mass concentration and the desired mean effective particle size
with an acceptable dispersion around the mean (Lodge 1968). Size fre-
quency information is generally not useful for dosage evaluations, so most
discussions refer to mass frequency because the mass quanity (mass
median diameter) is generally a cube function of the easily measured par-
ticle diameter (Drew and Laskin 1973).
Aerosols can be produced in two forms, monodisperse and polydisperse.
Polydisperse aerosols include a wide range of particle diameters with the
mass median diameter having a large geometric standard deviation and are
-------
209
ORNL-DWG 85-10757
1 IH HOHUSILICAH GLASS
WITH Jb/?«, SHHtHICAL JOINTS
FILL WITH
1/4" GLASS BEADS
Figure 4.20. J-tube vaporization assembly. (Source: Adapted from
Miller et al. 1980. Used with permission of the publisher.)
-------
210
considered the normal form for aerosols occurring in actual exposure con-
ditions (Drew and Laskin 1973, Casarett 1975). Monodisperse aerosols are
composed of particles with a narrow range of sizes resulting in a mass
median diameter having such a small geometric standard deviation that no
significant error is introduced into the experiment (Wilson and LaMer
1948, Mercer 1973). Monodispersity of an aerosol thus varies depending
on the experimental design or requirements and is an arbitary determina-
tion of the experimenter (Mercer 1973). Fuchs and Sutugin (1966)
defined it as those aerosols having a geometric standard deviation of 1.22
or less when the particles have a log normal distribution; this definition is
reasonable for most applications (Mercer 1973). The advantages of a
monodisperse aerosol relate to simplification for the study of factors
affecting toxicity and handling of data (Wilson and LaMer 1948). Mono-
disperse aerosols are also useful for studying deposition and clearance pat-
terns and for calibrating the monitoring equipment (Mercer 1973).
Many techniques and types of equipment for generating aerosols,
whether monodisperse or polydisperse, have been developed. The following
sections will discuss the major designs for generating aerosols using solid
feed materials and liquid, solution, or liquid-solid suspension feed
materials.
4.4.3.2 Generation of aerosols from solids
The earliest generating designs for solid or dust aerosols involved
dispersion of loose noncompacted particles. Air was blown through a con-
taining device in which the loose dust particles were being kept in suspen-
sion by vibrations, rotation of the container, or stirring. These devices pro-
duced polydisperse aerosols unless the loose dusts had been previously
sorted and sifted to make a monodisperse aerosol. Unfortunately, these
techniques did not produce aerosol clouds with very consistent characteris-
tics, due in part to the inability to predict the quantitative effects of
changes in their methodology (Wright 1950, Roe 1968). Details of the
early (1885-1940) designs can be found in Wright (1950). Later dust aero-
sol generators of this type have been designed to avoid many of these
problems.
Drew and Laskin (1971) designed a dust generator to use with
material that is not readily dispersed from the preferred prepacked plug
(e.g., polyester-fiberglass dusts). For the fluidizing chamber, they used an
inverted 1-gallon glass jar with a 3-in., four-blade fan mounted in the
screw cap. Two 3/4-in. access holes were provided in the top of the
chamber for an air inlet and an aspirator baffle chamber connection. The
dust (either polydisperse or monodisperse) is kept in suspension by the fan
and removed via an aspirator. Upon leaving the aspirator, the particles
impact on the walls of the Lucite® baffle chamber (mounted directly above
the glass fluidizing chamber), which removes the larger particles. The
remaining particles are piped into the exposure chamber. The chamber
concentrations depend on the initial dust charge, the overall airflow
-------
211
through the chamber, and the aspirator airflow. In addition to providing a
generation method for noncompactible dusts, this device also avoids
agglomeration resulting from electric charges normally associated with
dusts.
Another alternative design for generating aerosols from loose dusts is
the fluidized bed generator (FBG). The fluidized bed uses air blown
through a porous bottom of a container to put dry particles in suspension.
The FBG can be combined with a vertical aspirator, an inverted funnel in
the bed (Tillery et al. 1976), or a horizontal eductor to remove the dust
particles (Guichard 1976). To produce fine particles, the bed can be con-
structed from 100 to 200-Min-diam glass beads (Guichard 1976) or
100-jtm diam nickel spheres (Willeke et al. 1974). The concentration of
the aerosol and the particle size distribution are dependent on the operat-
ing conditions of the bed (Carpenter and Yerkes 1980). Bed loading and
superficial air velocity influence the mass median diameter and the con-
centration (bed volume also affects concentration) in such a manner that
high bed loadings and volumes and low superficial velocities produce max-
imum concentrations while moderate bed loadings and high velocities pro-
duce the smallest particle sizes. Thus, a trade-off must be made between
size and concentration in selecting operating conditions (Carpenter and
Yerkes 1980). The FBG has been used to generate fibrous dusts (Spurney
et al. 1976, Marple et al. 1978) and shows good potential in this area.
The two systems just described are typical of the recent loose dust
dispensing devices. The majority of these use an aspirator to remove the
loose particles from various types of fluidizing chambers. In general, this
type of aerosol generator is most useful for processing dusts that cannot be
successfully compacted and redistributed.
The most successful and widely used technique for generating dust
aerosols utilizes compressed plugs of powder that are ground or abraded to
release the particles. The Wright dust feed mechanism (Figure 4.21) was
one of the first of these designs and is still widely used today. Basically,
the design consists of a gear-train assembly powered by a synchronous
motor which drives a brass cup packed with dust down a spindle shaft in a
rotating motion and against a scraper blade that continuously abrades off
a uniform amount of dust (Wright 1950, Fraser et al. 1959). The brass
cup is slightly larger than the scraper blade, which is a piston with a
spring top having a radial cut edge turned up to form a scraper. Air is
blown into the lower part of the brass cup; passes through an air channel
under the scraper blade, where it picks up the dust; goes down a hollow
shaft that supports the blade; and after leaving the shaft, strikes an impac-
tor plate that breaks up any aggregated particles (Wright 1950, Tillery et
al. 1976). If the dust plug is compacted with a consistent density and if
90% of the particles have a diameter of 10 pm or less, there should be no
problem in redistributing the dust at the proper size, provided the relative
humidity of the airstream is controlled (Wright 1950, Roe 1968, Drew and
Laskin 1973). Any fluctuations in dust concentration (e.g., due to buildup
-------
212
Scraper Blade
ORNL-OWG 85-10758
Dust T'.oe
Scraper Blade
Scraper Head
- Scraper Head
Outlet Nozzle
mpactor Plate
Figure 4.21. The Wright dust feed mechanism. (Source: Adapted from
Leach et al. 1959.)
on the scraper blade) can be smoothed out by adding a large reservoir
after the impactor.
Another dust generator using a horizontal feed of a compacted powder
plug has been designed to handle fibrous dusts (Timbrell et al. 1968). The
horizontal feed mechanism consists of a piston-cylinder arrangement with
a threaded rod controlling the extent that the powder plug is introduced
into the scraper (Figure 4.22). A specially designed rotor driven by an
electric motor shaves off a layer of dust inside the dispersing chamber. The
particles are carried away by air entering through a side or bottom port
and exiting through a port in the top. As with the Wright mechanism,
proper packing of the plug and control of relative humidity are needed to
produce the desired concentrations. The plug should be compacted all at
once to prevent the formation of layers. The Timbrell generator was
specifically designed to process asbestos and is capable of separating indi-
vidual fibers or fiber bundles for distribution. Compacted powder plugs
have also been abraded to produce dust particles by using air jets
(Dimmick 1959, Hounam 1971).
4.4.3.3 Generation of aerosols from liquids, solutions, and liquid suspensions
More types of aerosol generators exist to create particles from liquids,
solutions, and suspensions of solids in liquids than from dust or solids.
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213
ORNL-DWG 85-10759
-AEROSOL EXHAUST
VIEWING PORT
ROTOR
COMPACT FIBROUS
PLUG
AIR
INLET
Figure 4.22. Schematic view of Ettinger's modification of Timbrell's
fibrous aerosol generator. (Source: Adapted from Tillery et al. 1976. Used
with permission of the publisher.)
Depending on the design, these can produce either liquid droplet or solid
particle aerosols.
One type of liquid aerosol generator designed from some of the same
principles used in dust dispensers is based on the aspirator. Basically the
aspirator functions by creating a pressure drop across an orifice or capil-
lary tube, which draws the liquid test agent up the tube where it is sheared
into particles by the air stream. Although it is used with dusts, the aspira-
tor can function more efficiently with liquids because the flow characteris-
tics are more favorable and easily achieved. Thus, many types of liquid
aerosol generators use the aspirator to atomize the liquid into particles.
A typical atomizer based on the above principle is the Laskin atomizer
(Fraser et al. 1959). Pressurized air is pumped up a central, hollow tube
and out four radially drilled holes at the top. Directly beneath these holes
is a collar with four vertical capillary tubes lined up with the radial holes.
As air passes over the capillaries, liquid is drawn up and broken into dro-
plets. The test agent feed rate in this system is dependent on the venturi
-------
214
effect at the top of the capillaries and the air flow rate through the atom-
izer. Because the most efficient atomization of liquids occurs near the
maximum air flow rate (Gage 1953), only a limited range of output varia-
tion is possible by changing the air flow rate. A motor-driven syringe
delivery system for the test liquid can be used to avoid this limiting factor
(Gage 1953). Although this system is used primarily with liquids, solid
particles can be created by mixing in dry or hot air with the droplets to
evaporate the liquid (Gage 1968), leaving crystals (when using a solution)
or solid particles (when using a solid-liquid suspension).
A special category of aspiration atomizers is often referred to as nebu-
lizers. Nebulizers atomize liquids, suspensions, and solutions, usually with
compressed air to form particles, and also include size-selective devices,
most frequently impaction surfaces.
The Vaponephrin nebulizer is an all-glass design consisting of a nozzle
directing a stream of air across the top of a capillary tube, with the bot-
tom of the tube resting in the test agent liquid. Directly opposite from the
nozzle is a glass sphere that alters the direction of the air stream. This
change in direction impacts larger particles on the sphere and allows
smaller particles to remain airborne (Fraser et al. 1959). The whole com-
plex is contained in a glass sphere to control loss of liquid. The critical
dimensions of the design are the diameter of the nozzle orifice, which lim-
its the air volume and determines the air pressure needed; the distance
from the nozzle to the capillary; and the distance from the capillary to the
impaction sphere.
Another simple nebulizer design (Figure 4.23) is the DeVilbiss nebu-
lizer. This is also a glass design with a vertical aspirator (Raabe 1976).
The impaction surface is the curved area of the exhaust tube, which res-
trains about 99% of the particles produced (Mercer et al. 1968b). Other
impaction surfaces include: a series of baffle holes that require the parti-
cles to follow a tortuous path (the Dautrebande nebulizer), the curved
walls of the generator (the Lauterbach nebulizer), a hollow cylinder
around the aspirator jet (the Collison nebulizer), and a sharp right angle
with a flat surface or a rounded curve baffle (Wright, Retec, and Lovelace
nebulizers) (Whitby et al. 1965, Mercer et al. 1968, Mercer 1973, Raabe
1976). Some of the output characteristics of the common nebulizers are
given in Table 4.8 (Tillery et al. 1976). In general, the differences in out-
put characteristics are due to the internal geometry of the chambers and
the flow rates through them (Tillery et al. 1976). For specific applications,
these factors, and the critical dimensions mentioned above, can be modi-
fied to produce the desired output concentrations and particle sizes.
A unique variation of the nebulizer design is fractionation of the liquid
stream by ultrasonic vibrations rather than by air pressure. Piezoelectric
crystals are typically used to create the vibrations, which are transmitted
through a coupling fluid (Figure 4.24) to the test liquid (Mercer 1973,
Tillery et al. 1976). The test liquid reacts to the vibrations by forming a
fountain above the crystal with droplets breaking off at the top. The size
-------
215
ORNL-DWG 85-10760
VENT
LIQUID INLET
TUBE
COMPRESSED AIR
IN
Figure 4.23. Diagram of the DeVilbiss nebulizer. (Source: Adapted
from Mercer et al. 1968b. Used with permission of the publisher.)
-------
Table 4.8. Output Characteristics of Some Compressed Air Nebulizers
Nebulizer
Vaponefrin
DeVilbiss #40
DeVilbiss #40
DeVilbiss #40
Bennett Twin (2814)
Puritan (R6-051)
Lauterbach
Lauterbach
Lauterbach
Dautrebande D-30
Dautrebande D-30
Dautrebande D-30
Lovelace
Lovelace
Lovelace
Collison
Jet
pressure
(psi)
12
10
20
30
7.5
23
10
20
30
10
20
30
10
20
30
15
Mass
median
diameter
(^m)
5.6
4.1
3.2
2.8
6.8
6.5
3.8
2.4
2.4
1.7
1.4
1.3
NGd
5.4
NG
NG
Geometric
standard
deviation
1.80
-1.85
-1.85
-1.85
1.80
1.90
-2.05
-2.05
-2.05
-1.65
-1.65
-1.65
NG
1.90
NG
NG
Specific output
(liL solution/L
jet air)
-29
16.0*
13.8*
12.8*
23.8
26.6
3.9
5.7
6.0
1.42
2.3
2.4
15.3
30
35
8.7
Output (fiL
solution/rain")
117
155
229
270
119
266
30C
67C
91
21
49
65
14e
39e
58.3e
53
Reference
Mercer et al. 1965
Mercer et al. 1968b
Mercer et al. 1968b
Mercer et al. 1968b
Mercer et al. 1965
Mercer et al. 1965
Mercer et al. 1968b
Mercer et al. 1968b
Mercer et al. 1968b
Mercer et al. 1968b
Mercer et al. 1968b
Mercer et al. 1968b
Mercer et al. 1968b
Mercer et al. 1968b
Mercer et al. 1968b
May 1973
-------
Table 4.8. (continued)
Nebulizer
Collison
Collison
Retec
Retec
Retec
Jet
pressure
(psi)
25
30
20
30
50
Nlass
median
diameter
(Mm)
1.9
NG
5.7
3.6
3.2
Geometric
standard
deviation
2.5
NG
1.8
2.0
2.2
Specific output
(nL solution/L
jet air)
6.7
5.8
35.2
35.9
31.9
Output (/*L
solution/mina)
55
55
208
284
376
Reference
May 1973
May 1973
Raabe 1972
Raabe 1972
Raabe 1972
"Dioctyl sebacate is the nebulized liquid for the Collison, while water containing salt or a fluorescent dye is the nebu-
lized liquid for the other nebulizers.
bZero auxiliary air flow.
cOrifice diameter, 0.032 in.
dNG = not given.
'Orifice diameter, 0.001 in.
Source: Adapted from Tillery et al. (1976).
-------
218
ORNL-OWG 85-10761
AEROSOL OUT
AIR IN
J
COUPLING FLUID
TRANSDUCER
Figure 4.24. Schematic view of ultrasonic nebulizer. (Source: Adapted
from Tillery et al. 1976. Used with permission of the publisher.)
of the droplets depends on the frequency of the acoustic field, the proper-
ties of the test chemical, and the rate at which they are removed from the
fountain (Raabe 1976). A stream of air is routed through the chamber to
disperse the accumulating aerosol. Some output characteristics of various
ultrasonic nebulizers are given in Table 4.9. In general, ultrasonic nebuliz-
ers produce more monodispersed aerosols than do typical compressed air
nebulizers (Mercer 1973). Kreyling and Perron (1983) describe an ultra-
sonic nebulizer design that produces aerosols from low concentrated parti-
cle solutions, can be optimized to produce specific particle sizes by varying
air pressure and impaction plate angle, and can deliver a constant concen-
tration for more than one hour. Another form of ultrasonic nebulizer (the
Hartman Whistle) utilizes compressed air shot into a resonant chamber to
produce sound waves of 25 kilocycles, which immediately disperse any
introduced liquid into a fine aerosol (Fraser et al. 1959).
The most commonly used technique for generating liquid aerosols,
especially monodisperse aerosols, is the spinning disk generator first
developed by Walton and Prewett (1949). The generator consists of a flat
horizontal disk surface, which is rotated at high speeds (up to 100,000
rpm) by a direct drive air or electric motor (Raabe 1976). The test liquid
is fed onto the center of the disk from a fixed-height reservoir or a syringe
pump and is spread out in a thin layer by centrifugal force (Figure 4.25).
-------
Table 4.9. Output Characteristics of Some Ultrasonic Nebulizers
Nebulizer
Mist O2 Gen,
50-mL cup'
Mist O2 Gen,
10-mL cup"
DeVilbiss,
gain setting 2b
DeVilbiss,
gain setting 3b
DeVilbiss,
gain setting 4b
Denton-Swartz
Denton-Swartz
Frequency
(MHz)
1.40
1.40
1.35
1.35
1.35
1.00
3.00
MMD
(/.m)
6.5
6.5
5.7
6.9
6.9
NGC
NG
CMD 0.34X Output
(
-------
220
ORNL-DWG 85-10762
Liquid Feed
Spinning Disk
/('IOO.OOO rpm)
Primary
'Droplets
Satellite
Droplets
Satellite
Air Flow
Figure 4.25. Schematic drawing of a spinning disk generator used to
produce monodisperse aerosols of both soluble and insoluble forms from
solutions of suspensions. Air flow into the satellite collector is adjusted so
that the inertia of the primary particles allows them to enter the. main air
flow. (Source: Adapted from Raabe 1976. Used with permission of
Academic Press.)
-------
221
As the liquid layer reaches and accumulates at the edge of the disk, dro-
plets are thrown off when the centrifugal force exceeds the restraining
capillary action (Mercer 1973). The majority of the liquid mass forms par-
ticles of the desired size, but smaller satellite droplets are also formed,
usually four for every primary droplet (May 1949). A stream of air, insuf-
ficient to overcome the inertia of the primary droplets, is used to control
the satellite droplets.
A similar generator, the spinning top, also was designed by Walton and
Prewett (1949) and modified by May (1949, 1966). It used a flat disk
with a conical bottom cut into turbine blades. Pressurized air directed at
the blades provided support and rotation for the disk. Generally, spinning
tops are smaller and operate at higher speeds (up to 240,000 rpm) than
the spinning disk (Mercer 1973, Tillery et al. 1976). Using the spinning
top, May was able to generate monodisperse particles with coefficients of
variation as low as 0.05 when the disk surface was properly treated. The
May spinning top generator is usually mounted within the exposure
chamber, as droplets are thrown off in a 360° area around the top. A
recent modification incorporates an encapsulating plenum to control the
aerosol generated, allowing it to be used outside the chamber (Gussman
1981).
The droplets created by spinning disks and tops are mostly mono-
disperse when the liquid feed rate is slow with particle size varying with
the rotational speed (Roe 1968). Table 4.10 shows the change in particle
size with the rotational speed of a spinning disk that used an air-driven
tool grinder motor (Whitby et al. 1965). The range of particle sizes possi-
ble with the spinning disks is usually above 0.5 ^m and can be up to 50
iim (Raabe 1976). Dry particles can also be created with the spinning disk
by evaporation of the liquid solvent. Hurford (1980) describes a modifica-
tion of the May generator that produces a monodisperse, solid aerosol with
a size range of 1 to 6 microns and a standard deviation of 1.1. The sizes
were verified by optical and scanning electron microscopy. The modifica-
tion to the May generator, by Gussman (1981), also provides the smooth,
nonturbulent column of rising air needed to evaporate the liquid solvent
and produce particles of 5 pm or less in diameter.
The condensation of vapors on nuclei has also been used to create
monodisperse aerosols. Sinclair and LaMer (1949) used the apparatus
shown in Figure 4.26 to form particles with a narrow size range. The
liquid test agent is first vaporized in the boiler, then nuclei (from an elec-
tric spark or NaCl-coated wire source) are added, and the mixture is car-
ried by a stream of air to the reheater. Because the reheater is at a higher
temperature, complete vaporization of the liquid is assured. The mixture
then enters the chimmey, where it is cooled to allow condensation of the
vapor on the nuceli. By carefully controlling the nucleus size and the
cooling process, particles of uniform size are created (Mercer 1973, Tilley
et al 1976) Modifications in the basic design were made by Rapaport and
Weinstock (1955), Muir (1965), and Liu et al. (1966). These modified
-------
Table 4.10. Operating Conditions at Which Model I Spinning Disk Was Used*
Operating
conditions
Main droplet diameter (pm ± 10%)
Dye concentration by weight (g/g)
Solid particle diameter (jim)
Discharge air flow (cfm)
Orifice diameter (in.)
Satellite air flow (cfm)
Satellite funnel diameter (in.)
Liquid feed rate (mL/min ± 20%)
Feed needle size (number)
Particle generation rate
(particles/min ± 20%)
Projection of the hypodermic needle
below the satellite funnel (in.)
70,000
19
5 X 10-5to
3 X Iff3
0.6-2.4
100
6
15
2.5
2.5
22
350 X 106
1/16
Air
45,000
28
1 X 10-5to
3 X 10-3
0.6-3.5
100
6
15
3
3
22
130 X 106
3/32
motor speed (rev/n
30,000
41
5 X ia5to 5
3 X 1(T3
1.3-5.1
70
7.5
15
4
3
22
40 X 106
1/8
nin)
20,000
60
X 10-5to
3 X 10°
1.9-7.5
50
9
16
5
M
19
15 X 10'
3/16
12,000
100
5 X iasto
3 X Iff3
3.2-12
40-50
15
18
6
4
19
3 X 106
3/16
"Data obtained using methylene blue dye in alcohol. Disk diameter = 4.65 cm.
Source: Adapted from Whitby et al. (1965).
-------
223
FILTERED
AIR
ORNL-DWG 85-10763
ELECTRIC
HEATER
;\ ,
-INSULATION
CONDENSATION
NUCLEI GENERATOR
AEROSOL
MATERIAL
VAPORIZER
AEROSOL
OUT
ELECTRIC
HEATER
Figure 4.26. Schematic view of Sinclair-LaMer monodisperse aerosol
generator. (Source: Adapted from Tillery et al. 1976. Used with permis-
sion of the publisher.)
designs are capable of producing monodisperse aerosols in the range of 0.3
to 1.4 /im (Mercer 1973, Tillery et al. 1976).
4.5. CONCLUSIONS AND RESEARCH RECOMMENDATIONS
4.5.1 Conclusions
Toxicity tests designed to evaluate effects from inhalation exposure
involve many of the same variables as tests using other exposure routes.
Exceptions to the standard protocols include: (1) selection of test species
should consider anatomical and physiological similarity of respiratory tract
to that of humans; (2) exposure modes can be intermittent (6 to 8 h/day
for 5 days/week) or continuous; (3) determination of actual. effective dose
is more complicated — generally the chamber concentration times length of
exposure is used; and (4) observations for effects include more extensive
pathological examination of respiratory structures and use of respiratory
function tests.
An important aspect of inhalation testing is the design of the test
chamber of equipment for generating the exposure atmosphere, and of
equipment to monitor chamber atmospheric conditions and test material
-------
224
concentrations. In most tests, operation of the test chamber is in a
dynamic mode, where a continuous stream of test agent and air is vented
through the chamber. The test chamber is usually designed for whole-body
exposure, although nose- and head-only exposure systems are also used.
The shape of most large chambers is cubical or hexagonal with pyramidal
or conical additions at top and bottom to increase distribution efficiency of
air within the chamber. Chamber size is governed by air distribution and
animal volume considerations. Construction materials need to be smooth-
surfaced, nonabsorbant materials that are resistant to a wide range of
chemicals. Airfow systems for the chamber should contain units to condi-
tion, filter, meter, and control the flow of air and test agent to and from
the chamber. The chamber should be maintained at a negative operational
pressure and within narrow temperature and humidity ranges.
Sampling and monitoring of the test atmosphere and chamber condi-
tions should be performed during operation. Sampling techniques for moni-
toring gas and vapor atmospheres usually rely on filters and automated
analytical procedures. Aerosol atmospheres are more difficult to sample,
particularly at the breathing zones, and a whole array of equipment has
been developed for this, including elutriators, cascade impactors, centrifu-
gal force samplers, and optical monitors. Some type of size separation for
the particles is usually part of the procedure.
Generation of the test atmospheres also differs based on the type of
test material. Gas and vapor generation is usually achieved by release into
the airstream from high pressure cylinders or through vaporization using
controlled heating or counter-current techniques. Aerosol atmospheres can
be generated in monodisperse or polydisperse forms. Generation of aerosols
from solid materials can be accomplished from loose materials by use of
aspirators and a vibrating, rotating, or stirring technique to create a source
suspension. Compacted forms of test materials can be put into an aerosol
suspension by abrading the surface with a scraper blade in the path of an
airstream. Generating aerosols from liquid test materials can be done in
more varied methods. Aspirators with an impaction surface can produce
particles of limited size ranges. This technique can be combined with a
fractionating device to produce aerosols. Applying the liquid to a spinning
disk or top is another technique frequently used to produce liquid aerosols.
4.5.2 Research Recommendations
More research is needed on:
• Applicability of test species, particularly regarding metabolic similarity
and particle deposition sites in the respiratory tract.
• Determination of actual effective doses in inhalation testing. This could
include actual dose delivered to animal lungs, dose delivered to blood
of test animals, and dose reaching target organs.
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225
• The use of alternative testing methods for inhalation toxicity, perhaps
more in vivo tests such as lavage fluid tests or in vitro tests that could
be used to determine acute or target organ (lung) effects.
• The use of respiratory function tests to widen their applicability,
increase knowledge as to the significance of their data, and provide
data for comparisons between test animals and humans.
• The various types of atmosphere generating equipment, particularly
aerosol generators, including comparisons of efficiency, ability to main-
tain desired particle size and distribution ranges, and avoidance of
operating problems (e.g., particle clumping on size separators in aerosol
generators).
• The potential for extrapolation of results from oral or intravenous
exposure studies to inhalation studies for non-respiratory-tract effects
or for certain chemical classes. This might allow reduction in the total
number of inhalation studies or in those studies aimed at systemic
effects of inhalation exposure.
• The extrapolation of inhalation toxicity data gained from animal stu-
dies to human exposure. Although this problem is not specific to inha-
lation studies, it is one of the most important areas in which existing
data are insufficient to determine the true relationships between animal
and human studies.
• The relationship between toxicity evaluation of a chemical and hazard
evaluation of a chemical, particularly as applied to inhalation studies.
The regulatory guidelines require toxicity data applicable to general
situations, but the real life situation requires hazard (toxicity X expo-
sure situation) data for determining the impact of a chemical in a
specific situation. How to satisfy both requirements without duplication
of testing efforts needs more study.
4.6. LITERATURE CITED
Alarie Y. 1966. Irritating properties of airborne materials to the upper
respiratory tract. Arch Environ Health 13:433-449.
Albert RE, Lippmann M, Spiegelman J, Luzzi A, Nelson N. 1967. The
deposition and clearance of radioactive particles in the human lung. Arch
Environ Health 14:10-15.
Amdur MO. 1957- The influence of aerosols upon the respiratory response
of guinea pigs to sulfur dioxide. Am Ind Hyg Assoc Q 18:149-155.
Amdur MO. 1958. The respiratory response of guinea pigs to sulfuric acid
mist. Arch Ind Health 18:407-414.
-------
226
Amdur MO, Mead J. 1958. Mechanics of respiration in unanesthetized
guinea pigs. Am J Physiol 192:364-368.
Barer GR, Edwards CW, Jolly AI. ^1976. Changes in the carotid body and
the ventilatory response to hypoxia in chronically hypoxic rats. Clin Sci
Mol Med 50:3111313.
Barrow C. 1978. Is ammonia a problem? In: Proceedings, Workshop on
Inhalation Chamber Toxicology, Drew RT, ed. Brookhaven National
Laboratory Associated Universities, Inc. BNL 51318. pp. 55-58.
Barrow CS, Steinhagen WH. 1980. NH3 concentration in the expired air
of the rat: importance to inhalation toxicology. Toxicol Appl Pharmacol
53:116-121.
Barrow RE, Vorwald AJ, Domier E. 1971. The measurement of small
animal respiratory volumes by capacitance respirometry. Am Ind Hyg
Assoc J 32:593-598.
Beethe RL, Wolff RK, Griffis LC, Hobbs CH, McClellan RO. 1979.
Evaluation of a recently designed multi-tiered exposure chamber. Lovelace
Biomedical and Environmental Research Institute, LF-67. 23 pp.
Bernstein DM, Drew RT. 1980. The major parameters affecting tempera-
ture inside inhalation chambers. Am Ind Hyg Assoc J 41:420-426.
Boecker BB, Aguilar FL, Mercer TT. 1964. A canine inhalation exposure
apparatus utilizing a whole body plethysmograph. Health Phys
10:1077-1089.
Brain JD, Mensah GA. 1983. Comparative toxicology of the respiratory
tract. Am Rev Respir Dis 128:S87-S90.
Brown RC, Poole A. 1983. Inhalation toxicology. In: Animals and alterna-
tives in toxicity testing, Balls M, Riddell RJ, Worden AN, eds. Academic
Press, New York, pp. 300-309.
Bryan RJ. 1970. Generation and monitoring of gases for inhalation stu-
dies. In: Inhalation Carcinogenesis, Hanna Jr. M, Nettesheim P, Gilbert J,
eds. U.S. Atomic Energy Commission Symposium Series 18. pp. 193-205.
Caldwell EJ, Fry DL. 1969. Pulmonary mechanics in the rabbit. J Appl
Physiol 27:280-285.
Cambridge GW. 1973. An examination and evaluation of inhalation
toxicity studies. Aerosol Age. 18:32-35, 67-68.
-------
227
Campbell KI. 1976. Inhalation toxicology. Clin Toxicol 9(6):849-921.
Carlon HR, Kimball DV, Wright RJ. 1980. Determination of aspirated
aerosol sampler efficiencies using laser reference techniques. Army Arma-
ment Research and Development Command. Aberdeen Proving Ground,
MD. ARCSL-TR-80003; AD-E410266. 21 pp. (abstract only).
Carney IF. 1979. Biological problems in defining hygiene standards for
particulates. Ann Occup Hyg 22:163-173.
Carpenter CP, Smyth HF, Pozzani UC. 1949. The assay of acute vapor
toxicity, and the grading and interpretation of results on 96 chemical com-
pounds. J Ind Hyg Toxicol 31:343-346.
Carpenter RL, Beethe RL. 1978. Cones, cone angles, plenums, and mani-
folds. In: Proceedings, Workshop on inhalation chamber toxicology, Drew
RT, ed. Brookhaven National Laboratory Associated Universities, Inc.
BNL 51318. pp. 21-30.
Carpenter RL, Yerkes K. 1980. Relationship between fluid bed aerosol
generator operation and the aerosol produced. Am Ind Hyg Assoc J
41:888-894.
Carson TR, Rosenholtz MS, Wilinski FT, Weeks MH. 1962. The response
of animals inhaling nitrogen dioxide for single, short-term exposures. Am
Ind Hyg Assoc J 23:457-462.
Casarett LJ. 1975. Toxicology of the respiratory system. In: Toxicology,
the basic science of poisons, Casarett LJ, Doull J, eds. MacMillan Publish-
ing Co., Inc., New York. pp. 201-224.
Cavagna GA, Stemmler EJ, DuBois AB. 1967. Alveolar resistance to
atelectasis. J Appl Physiol 22:441-452.
Clark DB. 1977. Long-term inhalation toxicology studies. In: Current
approaches in toxicology, Ballantyne B, ed. John Wright and Sons Ltd.,
Bristol, pp. 105-115.
Comroe JH Jr, Nisell OI, Nims RG. 1954. A simple method for con-
current measurement of compliance and resistance to breathing in anesthe-
tized animals and man. J Appl Physiol 7:225-228.
Cotabish HN, McConnaughey PW, Messer HC. 1961. Making known
concentrations for instrument calibration. Am Ind Hyg Assoc J
22:392-402.
-------
228
Crossland NJ, Horsfall GB, Oxenham ST, Shaw JS, Turnbull MJ. 1977.
A simple device for measurement of respiratory rate in the mouse. Br J
Pharmacol 61:490P-491P.
Davis J, Morris B. 1953. Factors affecting the intrapleural pressure and
pulmonary ventilation in rats. Austral J Exp Biol 31:201-214.
Decker JR, Park JF, Craig DK. 1979. A method for measuring respiratory
volume parameters of large animals during exposure to aerosols. Am Ind
Hyg Assoc J 40:567-577.
Dennis MW, Douglas JS, Casby JU, Stolwijk JAJ, Bouhuys A. 1969. On-
line analog computer for dynamic lung compliance and pulmonary resis-
tance. J Appl Physiol 26:248-252.
Denton MB, Schwartz DB. 1974. An improved ultrasonic nebulizer system
for the generation of high density aerosol dispersions. Rev Sci Instrum
45:81-83.
De Zaiacomo T, Tarroni G, Prodi V, Melandri C, Formignani M, Lom-
bardi CC, Barilli L, Olivieri P, Niccolai I. 1983. An intercomparison
among aerodynamic sizing instruments. J Aerosol Sci 14:314-317.
Diamond L, Lipscomb W. 1970. Design of a small animal pneumotacho-
graph. J Appl Physiol 29:720-722.
Diamond L, Adams GK, Bleidt B, Williams B. 1973. Experimental study
of a potential anti-asthmatic agent: SCH 15280. J. Pharmacol Exp Therap
193:256-263.
Diamond L, O'Donnell M. 1977. Pulmonary mechanics in normal rats. J
Appl Physiol: Respirat Environ Exer Physiol 43:942-949.
Dimmick RL. 1959. Jet dispenser for compacted powders in the one-to-
ten-micron range. Arch Ind Health 20:8-14.
Doe JE, Tinston DJ. 1981. Novel chambers for long term inhalation stu-
dies. In: Proceedings of the inhalation toxicology and technology sympo-
sium, Kalamazoo, MI, October 23-24, 1980. Ann Arbor Science Publish-
ers, Inc., Ann Arbor, MI. pp. 77-88.
Dorato MA, Carlson KH, Copple DL. 1983. Pulmonary mechanics in
conscious Fischer 344 rats: multiple evaluations using nonsurgical tech-
niques. Toxicol Appl Pharmacol 68:344-353.
-------
229
Drew RT. 1978. Exposure of rodents to participate carcinogens, first
presentation. In: Proceedings, workshop on inhalation chamber toxicology,
Drew RT, ed. Brookhaven National Laboratory Associated Universities,
Inc. BNL51318. pp. 63-64.
Drew RT, Laskin S. 1971. A new dust-generating system for inhalation
studies. Am Ind Hyg Assoc J 32:327-330.
Drew RT, Laskin S. 1973. Environmental inhalation chambers. In:
Methods of animal experimentation. Vol. IV. Environment and the special
senses, Gay WI, ed. Academic Press, New York. pp. 1-41.
Drorbaugh JE. 1960. Pulmonary function in different animals. J Appl
Physiol 15:1069-1072.
Dubin ED, Morrison G. 1969. A face mask and mouthpiece for respiratory
studies in unanaesthetised beagle dogs. J Appl Physiol 27:104-105.
DuBois AB, Rogers RM. 1968. Respiratory factors determining the tissue
concentrations of inhaled toxic substances. Respir Physiol 1968(5):34-52.
Dungworth DL, Schwartz W, Tyler WS, Phalen RF. 1976. Morphological
methods for evaluation of pulmonary toxicity in animals. Ann Rev Phar-
macol Toxicol 16:381-399.
Ferin J. 1978. Can laminar flow systems be used? In: Proceedings,
workshop on inhalation chamber toxicology, Drew RT, ed. Brookhaven
National Laboratory Associated Universities, Inc. BNL 51318. pp. 39-42.
Formignani M, Melandri C, Tarroni G, Prodi V, De Zaiacomo T. 1982.
Calibration apparatus for aerosol samplers. Comitato Nazionale per
1'Energia Nucleare Bologna (Italy). CNEN-RT/PROT-(82)4. 32 pp.
(abstract only).
Francovitch RJ, Raitano L, Lee T, George WJ. 1982. Comparison of der-
mal and inhalation toxicities of volatile agents employing a dual function
chamber. Fed Proc 41 abstract 7583.
Frank NR, Speizer FE. 1965. SO2 effects on the respiratory system in
dogs. Arch Environ Health 11:624-634.
Fraser DA, Bales RE, Lippmann M, Stokinger HE. 1959. Exposure
chambers for research in animal inhalation. Public Health Monograph No.
157. 54pp.
-------
230
Fuchs NA, Sutugin SG. 1966. Generation and use of monodisperse aero-
sols. In: Aerosol science, Davis CN, ed. Academic Press, London.
Gage JC. 1953. A controlled fluid-feed atomizer. J Sci Instrum 30:25.
Gage JC. 1968. Toxicity of paraquat and diquat aerosols generated by a
size-selective cyclone: effect of particle size distribution. Br J Ind Med
25:304-314.
Gardner DE, Miller FJ, Blommer EJ, Coffin DL. 1979. Influence of expo-
sure mode on the toxicity of NO2. Environ Health Perspec 30:23-29.
Gat N. 1980. Development study of a two-stage continuous flow impactor.
Army Armament Research and Development Command. Aberdeen Prov-
ing Ground, MD. TRW-33437-6001-RU-00; ARCSL-CR-80009; AD-
E410241. 88 pp. (abstract only).
Giles RE, Finkel MP, Mazurowski J. 1971. Use of an online computer for
the evaluation of pulmonary resistance and dynamic compliance in the
anaesthetised dog. Arch Pharmacodyn 194:213-222.
Giovacchini RP. 1972. Old and new issues in the safety evaluation of
cosmetics and toiletries. Crit Rev Toxicol l(4):361-378.
Griffis LC, Henderson JH, Pickrell JA. 198la. A method for determining
glass in rat lung after exposure to a glass fiber aerosol. Am Ind Hyg Assoc
J 42:566-569.
Griffis LC, Wolff RK, Beethe RL, Hobbs RF, McClellan RO. 198Ib.
Evaluation of a multitiered inhalation exposure chamber. Fund Appl Toxi-
col 1:8-12.
Gross SB. 198la. Regulatory guidelines for inhalation toxicity testing. In:
Proceedings of the inhalation toxicology and technology symposium, Leong
BKJ, ed. Ann Arbor Science Publishers, Inc. pp. 279-298.
Gross KB. 1981b. Pulmonary function testing of animals chronically
exposed to diluted diesel exhaust for 267 days. Environ Int 5:331-338.
Guichard JC. 1976. Aerosol generation using fluidized beds. In: Fine par-
ticles, Liu BYH, ed. Academic Press, New York. pp. 173-193.
Gussman RA. 1981. A further modification of the May spinning top aero-
sol generator. Am Ind Hyg Assoc J 42:208-212.
-------
231
Haber R. 1924. "Funf vortrage aus den jahren 1920-1923." No. 3. Die
Chemie in Kriege, No. 5. Zur Geschichte des Gas Kampfes. Julius
Springer, Berlin.
Halbert MK, Mazumder MK, Bond RL. 1981. Size-distribution analysis
of respirable particulates in cosmetic aerosols: a methodological compari-
son. Food Cosmet Toxicol 19:85-88.
Hammond PB. 1970. The use of animals in toxicological research
(prepared for the National Institute of Environmental Health Sciences
Task Force on Research Planning in Environmental Health Sciences)
Natl. Library of Medicine, Bethesda, MD. (As reported in Drew and
Laskin 1973).
Hatch TF, Gross P. 1964. Pulmonary deposition and retention of inhaled
aerosols. Academic Press, New York. 189 pp.
Haun CC. 1972. Continuous animal exposure to methylene chloride. In:
Proceedings of the 2nd annual conference on environmental toxicology,
Dayton, Ohio, 1971. U.S. Government Printing Office, pp. 309-326.
Heindryckx R, Dams R. 1981. Investigation of errors associated with the
use of cascade impactors. Sci Total Environ 19:167-178.
Hemenway DR, MacAskill SM. 1982. Design, development and test
results of a horizontal flow inhalation toxicology facility. Am Ind Hyg
Assoc J 43:874-879.
Hemenway DR, Carpenter RL, Moss OR. 1982. Inhalation toxicology
chamber performance: a quantitative model. Am Ind Hyg Assoc J
43:120-127.
Henderson RF, Damon EG, Henderson TR. 1978. Early damage indica-
tors in the lung. I. Lactate dehydrogenase activity in the airways. Toxicol
Appl Pharmacol 44:291-297.
Henderson RF, Rebar AH, DeNicola DB. 1979a. Early damage indicators
in the lung. IV. Biochemical and cytologic response of the lung to lavage
with metal salts. Toxicol Appl Pharmacol 51:129-135.
Henderson RF, Rebar AH, Pickrell JA, Newton GJ. 1979b. Early damage
indicators in the lung. III. Biochemical and cytological response of the
lung to inhaled metal salts. Toxicol Appl Pharmacol 50:123-136.
-------
232
Henderson RF, Rebar AH, DeNicola DB, Henderson TR. 1980. The use
of pulmonary lavage fluid in screening for early indicators of lung injury:
In: Pulmonary toxicology of respirable particles, Saunders CL, Cross FT,
Dagle GE, Mahaffey JA, eds. U.S. Department of Energy, pp. 378-391.
Henry CJ, Hwang KK, Kanagalingam KK, Kouri RE. 1983. Recent
developments in inhalation toxicology: evaluation of selected short-term
endpoints following 'nose-only' exposure of rodents. In: Safety evaluation
and regulation of chemicals, Homburger F, ed. Karger, NY. pp. 233-242.
Hiett DM. 1974. Tests of ventilatory function for use in long-term animal
studies. Br J Ind Med 31:53-58.
Hinners RG. 1978. A system for automatically monitoring chamber tem-
perature, humidity, and pollutant concentrations. In: Proceedings,
workshop on inhalation chamber toxicology, Drew RT, ed. Brookhaven
National Laboratory Associated Universities, Inc. BNL 51318. pp. 89-92.
Hinners RG, Burkart JK, Contner GL. 1966. Animal exposure chambers
in air pollution studies. Arch Environ Health 13:609-615.
Hinners RG, Burkart JK, Punte CL. 1968. Animal inhalation exposure
chambers. Arch Environ Health 16:194-206.
Holmberg RW, Moneyhun JH. 1980. Inhalation exposure system for toxi-
cological studies of diesel oil aerosols. Oak Ridge National Laboratory.
CONF-801156-2. 25 pp. (abstract only).
Holmberg RW, Moneyhun JH, Dalbey WE. 1981. An exposure system for
toxicological studies of concentrated oil aerosols. In: Proceedings of the
inhalation toxicology and technology symposium, Kalamazoo, MI, October
23-24, 1980. Ann Arbor Science Publishers, Inc., Ann Arbor, MI.
pp. 53-64.
Hounam RF. 1971. The konimiser—a dispenser for the continuous genera-
tion of dust clouds from milligram quantities of asbestos. Ann Occup Hyg
14:329-335.
Hurford MJ. 1980. Production of monodisperse soild aerosols with the
May spinning-top generator. United Kingdom Atomic Energy Establish-
ment, Winifrith, England. AEEW-M-1816. 26 pp. (abstract only).
Illing JW, Gardner DE, Coffin DL. 1975. Motorized exercise wheel for
exposure of small animals to environmental pollutants. Lab Animal Sci
25:103-105.
-------
233
Jersey GC, Kociba RJ. 1979. The importance and results of proper mor-
phological evaluation of the nasal turbinates in inhalation toxicity studies.
Toxicol Appl Pharmacol 48:A1. (abstract only).
Juhos LT, Sasmore DP, Falconer DG, Tyson CA. 1985. Tests for pul-
monary function. In: Organ function tests in toxicity evaluation, Tyson
CA, Sawhney DS, eds. Noyes Publications, Park Ridge, NJ. pp. 123-167.
Kawai K, Nozaki K. 1978. Inhalation techniques for developmental toxi-
city testing. Teratology 18:13la.
Kennedy GL, Trochimowicz HJ. 1982. Inhalation toxicology. In: Princi-
ples and methods of toxicology, Hayes AW, ed. Raven Press, New York.
pp. 185-207.
Kenoyer JL, Phalen RE, Davis JR. 1981. Particle clearance from the
respiratory tract as a test of toxicity: effect of ozone on short and long
term clearance. Exper Lung Res 2:111-120.
King RJ. 1974. The surfactant system of the lung. Fed Proc Fed Am Soc
Exp Biol 33:2238-2247.
King TKC. 1966a. Measurement of functional residual capacity in the rat.
J Appl Physiol 21:233-236.
King TKC. 1966b. Mechanical properties of the lungs in the rat. J Appl
Pharmacol 21:259-264.
Klein GW, Geary DL. 1982. Analytical monitoring for inadvertent test
chemical release during the conduct of inhalation toxicity studies. Abstr
Pap Am Chem Soc 183:CHAS-23. (abstract only).
Koen PA, Moskowitz GD, Shaller TH. 1977. Instrumentation for measur-
ing functional residual capacity in small animals. J Appl Physiol: Respirat
Environ Exercise Physiol 43:755-758.
Koizumi A, Ikeda M. 1981. A servomechanism for vapor concentration
control in experimental exposure chambers. Am Ind Hyg Assoc J
42:417-425.
Koo KW, Leith DE, Sherter CB, Snider GL. 1976. Respiratory mechanics
in normal hamsters. J Appl Physiol 40:936-942.
Kreyling WG, Perron GA. 1983. An aerosol nebulizer for low concen-
trated particle suspensions. J Aerosol Sci 14:264-267.
-------
234
Laskin S, Drew RT. 1970. An inexpensive portable inhalation chamber.
Am Ind Hyg Assoc J 31:645-646.
Laskin S, Kuschner M, Drew RT. 1970. Studies in pulmonary carcino-
genesis. In: Inhalation carcinogenesis, Hanna, Jr. M, Nettesheim P, Gil-
bert J, eds. U.S. Atomic Energy Commission Symposium Series 18.
pp. 321-351.
Leach LJ. 1963. A laboratory test chamber for studying airborne
materials. AEC Progress Report UR-629. University of Rochester,
Rochester, NY. pp. 1-12.
Leach LJ, Spiegl CJ, Wilson RH, Sylvester GE, Lauterbach KE. 1959. A
multiple chamber exposure unit designed for chronic inhalation.
Leong KJ, Dowd GF, MacFarland HN. 1964. A new technique for tidal
volume measurement in unanesthetized small animals. Can J Physiol Phar-
maco!42:189-194.
Likens SA, Mauderly JL. 1979. Respiratory measurements in small
laboratory mammals: a literature review. Lovelace Biomedical and
Environmental Research Institute Publication LF-68. 13 pp.
Lippmann M. 1961. A compact cascade impactor for field survey sam-
pling. Am Ind Hyg Assoc J 22:348-353.
Lisco H. 1959. Autoradiographic and histopathologic studies in radiation
carcinogenesis of the lung. Lab Invest 8:162-168.
Liu BYH, Whitby KT, Yu HYS. 1966. A condensation aerosol generator
for producing monodisperse aerosols in the size range, 0.036um to 1.3um.
J Recherches Atmos 3:397.
Lodge JP. 1968. Production of controlled test atmospheres. In: Air pollu-
tion Vol. II. Analysis, monitoring, and surveying, Stern AC, ed. Academic
Press, New York. pp. 465-483.
Lund IE, Mills B, Moyes AJ. 1979. Review of practical problems arising
from the relationship between dust generators and dust collectors. J Inst
Energy 52:32-43. (abstract only).
MacEwen JD. 1978. Nonconventional systems. In: Proceedings, workshop
on inhalation chamber toxicology, Drew RT, ed. Brookhaven National
Laboratory Associated Universities, Inc. BNL 51318. pp. 9-16.
-------
235
MacFarland HN. 1975. Inhalation toxicology. J Assoc Off Anal Chem
58:689-691.
MacFarland HN. 1976. Respiratory toxicology. Essays in Toxicol
7:121-154.
MacFarland HN. 1981. A problem and a non-problem in chamber inhala-
tion studies. In: Proceedings of the inhalation toxicology and technology
symposium, Kalamazoo, MI, October 23-24, 1980. Ann Arbor Science
Publishers, Inc., Ann Arbor, MI. pp. 11-18.
Marple VA, Liu BYH, Rubow KL. 1978. A dust generator for laboratory
use. Am Ind Hyg Assoc J 39:26-32.
Mauderly JL. 1977. Bronchopulmonary lavage of small laboratory
animals. Lab Anim Sc. 27:255-261.
Mauderly JL, Tesarek JL. 1975. Nonrebreathing valve for respiratory
measurements in unsedated small mammals. J Appl Physiol 38:369-371.
May KR. 1949. An improved spinning top homogeneous spray apparatus.
J Appl Phys 20:932-938.
May KR. 1966. Spinning-top homogeneous aerosol generator with shock-
proof mounting. J Sci Instrum 43:841-842.
May KR. 1973. The collison nebulizer: description, performance, and
application. Aerosol Sci 4:235.
Mazumder MK, Kirsch KJ. 1977. Single particle aerodynamic relaxation
time analyzer. Res Sci Instrum 48:622-624.
Mazumder MK, Ware RE, Wilson JD, Renninger RG, Hiller FC,
McLeod PC, Raible RW, Testerman RG. 1979. SPART analyzer: its
application to aerodynamic size distribution measurement. J Aerosol Sci
10:561-569.
McCutcheon FH. 1951. The mammalian breathing mechanism. J Cell
Comp Physiol 37:447-476.
McLaughlin RF, Tyler WS, Canada RO. 196la. A study of the subgross
pulmonary anatomy in various mammals. Am J Anat 108:149-164.
McLaughlin RF, Tyler WS, Canada RO. 1961b. Subgross pulmonary ana-
tomy in various mammals and man. J Am Med Assoc 175:694-697.
-------
236
McLaughlin RF, Tyler WS, Canada RO. 1966. Subgross pulmonary ana-
tomy of the rabbit, rat, and guinea pig with additional notes on the human
lung. Am Rev Respir Dis 94:380-387.
Mead J. 1960. Control of respiratory frequency. J Appl Physiol
15:325-336.
Mercer XT. 1973. Production and characterization of aerosols. Arch
Intern Med 131:39-50.
Mercer TT, Goddard RF, Flores RL. 1965, Output characteristics of
several commercial nebulizers. Ann Allergy 23:314-326.
Mercer TT, Goddard RF, Flores RL. 1968a. Output characteristics of
three ultrasonic nebulizers. Ann Allergy 26:18-27.
Mercer TT, Tillery M, Chow HY. 1968b. Operating characteristics of
some compressed air nebulizers. Am Ind Hyg Assoc J 29:66-78.
Miller RR, Letts RL, Potts WJ, McKenna MJ. 1980. Improved methodol-
ogy for generating controlled test atmospheres. Am Ind Hyg Assoc J
41:844-846.
Moorman WJ, Hornung RW, Wagner WD. 1977. Ventilatory functions in
germ-free and conventional rats exposed to coal dusts. Proc Soc Exp Biol
Med 155:424-428.
Moorman WJ. 1978. Exposure of rodents to particulate carcinogens,
second presentation. In: Proceedings, workshop on inhalation chamber tox-
icology, Drew RT, ed. Brookhaven National Laboratory Associated
Universities, Inc. BNL 51318. pp. 64-74.
Muir DCF. 1965. The production of monodisperse aerosols by a LaMer
Sinclair generator. Ann Occup Hyg 8:223-228.
Murphy SD, Ulrich CE. 1964. Multi-animal test system for measuring
effects of irritant gases and vapors on respiratory function of guinea pigs.
Am Ind Hyg Assoc J 25:28-36.
Nader JS. 1971. Direct-reading instruments for analysing airborne gases
and vapors. In: Air sampling instruments, 4th ed., Lippmann M, ed.
American Conference of Governmental Industrial Hygiene, p. J-l.
NAS. 1977. National Academy of Sciences. Inhalation exposure. In: Prin-
ciples and procedures for evaluating the toxicity of household substances.
Washington, DC. pp. 60-73.
-------
237
Nattie EE. 1977. Breathing patterns in the awake potassium-depleted rat.
J Appl Physiol: Respirat Environ Exercise Physiol 43:1063-1074.
Nelson GO, Taylor RD. 1980. An automated flow-temperature-humidity
control system. Am Ind Hyg Assoc J 41:769-771.
Nettesheim P, Griesemer RA. 1978. Experimental models for studies of
respiratory tract carcinogenesis. In: Pathogenesis and therapy of lung
cancer: lung biology in health and disease, Vol. 10, Lenfant C, Harris C,
eds. Marcel Dekker, Inc., New York. pp. 75-188.
Oberdoerster G, Freedman AP. 1982. Lung retention of iron-59 labeled
magnetite in rats comparing magnetometric and radioactive measurement
techniques. Am Rev Respir Dis 125:165.
O'Keeffe AE, Ortman GC. 1966. Primary standards for trace gas analysis.
Anal Chem 38:760-763.
OECD. 1979. Organization for Economic Cooperation and Development.
Final report of the OECD short-term and long-term toxicity groups.
unpublished. 185 pp.
Page N, Sawhney DS, Ryon MG, eds. 1980. Proceedings of the workshop
on subchronic toxicity testing. Denver, Colorado, May 20-24, 1979. Oak
Ridge National Laboratory Publication ORNL/EIS-189. 63 pp.
Palecek F. 1969. Measurement of ventilatory mechanics in the rat. J Appl
Physiol 27:149-156.
Palm PE, McNerney JM, Hatch T. 1956. Respiratory dust retention in
small animals, a comparison with man. Arch Ind Health 13:355-365.
Potts WJ, Steiner EC. 1980. An apparatus for generation of vapors from
liquids of low volatility for use in inhalation studies. Am Ind Hyg Assoc J
41:141-145.
Poynter D. 1977. Inhalation toxicology: the questions which must be
answered. In: Current approaches in toxicology. John Wright and Sons
Ltd., Bristol, pp. 86-104.
Prodi V, De Zaiacomo T, Melandri C, Tarroni G, Formignani M,
Hochrainer D. 1979. An inertial spectrometer for aerosol particles. J Aero-
sol Sci 14:411-419.
-------
238
Raabe OG. 1972. Operating characteristics of two compressed air nebuliz-
ers used in inhalation experiments. Fission Product Inhalation Program
Annual Report (1971-1972). LF-45.
Raabe OG. 1976. Generation of aerosols of fine particles. In: Fine parti-
cles, Liu BYH, ed. Academic Press, New York. pp. 57-110.
Radford EP, Jr. 1957. Recent studies of mechanical properties of mam-
malian lungs. In: Tissue elasticity, Remington JW, ed. American Physio-
logical Society, Washington, DC. pp. 177-190.
Rapaport E, Weinstock SE. 1955. A generator for homogeneous aerosols.
Experientia 11: 363-364.
Roe FJC. 1968. Inhalation tests. In: Modern trends in toxicology, Vol. I,
Boyland E, Goulding R, eds. Appleton-Century-Crofts, New York.
pp. 39-74.
Saltzmann BE. 1961. Preparation and analysis of calibrated low concen-
trations of sixteen toxic gases. Anal Chem 33:1100-1112.
Saffiotti U. 1969. Experimental respiratory tract carcinogenesis. Progr Exp
Tumor Res 11:302-333.
Sanockij IV. 1970. Complete toxicometry. In: Methods for determining the
toxicity and hazards of chemicals. Moscow. Medicina:50-54. (In Russian).
Sawyer KF, Walton WH. 1950. The "conifuge", a size separating sam-
pling device for airborne particles. J Sci Instrum 27:272-275.
Schreck RM, Chan TL, Soderholm SC. 1981. Design, operation and
characterization of large volume exposure chambers. In: Proceedings of
the inhalation toxicology and technology symposium, Kalamazoo, MI,
October 23-24, 1980. Ann Arbor Science Publishers, Inc., Ann Arbor, MI.
pp. 29-52.
Sheehy JP, Archinger WC, Simon RA. 1967. Handbook of air pollution.
U.S. Department of Health, Education, and Welfare, pp. 9-10.
Sidorenko GI, Pinigin MA. 1976. Concentration—time relationship for
various regimens of inhalation of organic compounds. Environ Health Per-
spect 13:17-21.
Silver SD. 1946. Constant flow gassing chambers: principles influencing
design and operation. J Lab Clin Med 31:1153-1161.
-------
239
Sinclair D, LaMer V. 1949. Light scattering as a measure of particle size
in aerosols. Chem Rev 44:245-267.
Smith SW, Spurling NW. 1974. Device for exposing the respiratory tract
of the rat to medicinal aerosols. Lab Practice 23(12):717-721.
Smith WB, Cavanaugh PR, Wilson RR. 1978. Technical manual, a survey
of equipment and methods for particulate sampling in industrial process
streams. U.S. Environmental Protection Agency Report No. 600/7-78-043.
217 pp.
Smith DW, Ortiz LW, Archuleta RF, Spalding JF, Ettinger HJ. 1980.
Method for chronic nose-only exposures of laboratory animals to inhaled
fibrous aerosols. Los Alamos National Laboratory, NM. LA-UR-81 963;
CONF-8010202-1. 34 pp. (abstract only).
Spurney KR, Boose C, Hockrainer D, Monig FJ. 1976. A note on the
dispersing of fibrous powders. Ann Occup Hyg 19:85-87.
Stokinger HE. 1953. Toxicologic perspective in planning air pollution stu-
dies. Am J Public Health 43:742-751.
Stuart BO, Willard DH, Howard EB. 1970. Uranium mine contaminants
in dogs and hamsters. In: Inhalation carcinogenesis, Hanna Jr M, Net-
tesheim P, Gilbert J, eds. U.S. Atomic Energy Commission Symposium
Series, No. 18. pp. 413-428.
Swann HE, Brunol D, Balchum OJ. 1965. Pulmonary resistance measure-
ments of guinea pigs. Arch Environ Health 10:24-32.
Thiede FC, Hackney JD, Linn WS, Spier C, House W. 1974. Animal
atmospheric exposure chamber system using a modified tank respirator.
Am Ind Hyg Assoc J 35:370-375.
Thomas RG, Lie R. 1963. Procedures and equipment used in inhalation
studies on small animals. AEC Research and Development Report LF-11.
30pp.
Thomas RG, Morgan BN. 1969. The effects of age, weight, strain and
anesthesia upon the breathing patterns of rats. Matrix 69:13-18.
Tierney DG. 1974. Intermediary metabolism of the lung. Fed Proc Fed
Am Soc Exp Biol 33:2232-2237.
-------
240
Tillery MI, Wood GO, Ettinger HJ. 1976. Generation and characteriza-
tion of aerosols and vapors for inhalation experiments. Environ Health
Perspect 16:25-40.
Timbrell V, Hyett AW, Skidmore JW. 1968. A simple dispenser for gen-
erating dust clouds from standard reference samples of asbestos. Ann
OccupHyg 11:273-281.
Timbrell V, Skidmore JW, Hyett AW, Wagner JC. 1970. Exposure
chambers for inhalation experiments with standard reference samples of
asbestos of the international union against cancer (UICC). Aerosol Sci
1:215-233.
Tropp RJ, Kuhn PJ, Brock JR. 1980. A new method for measuring the
particle size distribution of aerosols. Rev Sci Instrum 51(4):516-520.
Troy WR. 1974. Testing for inhalation toxicity. J Soc Cosmet Chem
25:283-296.
Truog WE, Standaert TA. 1978. Nonrebreathing valve for spontaneously
breathing or mechanically ventilated animals. J Appl Physiol: Respirat
Environ Exercise Physiol 44:974-976.
USDHEW. 1974. U.S. Department of Health, Education, and Welfare.
Guide for the care and use of laboratory animals, 4th ed. National Insti-
tutes of Health. DHEW Publication Number (NIH)74-23, Washington,
DC. 56 pp.
USEPA. 1982. U.S Environmental Protection Agency. Health effects test
guidelines. Washington, DC. EPA 560/6-82-001.
Van Stee EW, Moorman MP. 1978. Monitoring for temperature, humi-
dity, concentration. In: Proceedings, workshop on inhalation chamber toxi-
cology, Drew RT, ed. Brookhaven National Laboratory Associated Univer-
sities, Inc. BNL 51318. pp. 81-88.
Vinegar A, Stinett EE, Kosch PC, Miller ML. 1981. Pulmonary physiol-
ogy of the ferret and its potential as a model for inhalation toxicology.
Teratology 24:17A. (abstract only).
Walton WH, Prewett WC. 1949. The production of sprays and mists of
uniform drop size by means of spinning disc type sprayers. Proc Phys Soc
London Sec B 62(6):342-350.
Wehner AP, Craig DK, Stuart BO. 1972. An aerosol exposure system for
chronic inhalation studies with rodents. Am Ind Hyg Assoc J 33:483-487.
-------
241
Weissman SH, Beethe RL, Redman HC. 1980. Ammonia concentrations
in an animal inhalation exposure chamber. Lab Animal Sci 30:974-980.
Wells AB. 1979. Inhalation testing of toiletries in animals. Int J Cosmet
Sci 1:135-146.
Weston RE, Karel L. 1946. An application of the dosimetric method for
biologically assaying inhaled substances. J Pharmacol Exp Ther
88:195-207.
Whitby KT, Lundgren DA, Peterson CM. 1965. Homogenous aerosol gen-
erators. Int J Air Water Pollut 9:263-277.
WHO 1978. World Health Organization. Inhalation exposure. In: Princi-
ples and methods for evaluating the toxicity of chemicals. Geneva.
pp. 199-235.
Willeke K, Lo CSK, Whitby KT. 1974. Dispersion characteristics of a
fluidized bed. Aerosol Sci 5:449-455.
Wilson IB, LaMer VK. 1948. The retention of aerosol particles in the
human respiratory tract as a function of particle radius. J Ind Hyg Toxicol
30(5):265-280.
Witschi H. 1975. Exploitable biochemical approaches for the evaluation of
toxic lung damage. Essays Toxicol 6:125-191.
Wolff HS, Roach SA. 1961. The conicycle selective sampling system. In:
Inhaled particles and vapors, Davis CN, ed. Oxford: Pergamon Press.
Wong K, Alarie Y. 1982. A method for repeated evaluation of pulmonary
performance in unanesthetized, unrestrained guinea pigs and its applica-
tion to detect effects of sulfuric acid mist inhalation. Toxicol Appl Phar-
macol 63:72-90.
Wright BM. 1950. A new dust feed mechanism. J Sci Instrum 27:12-15.
Wright BM. 1954. A size selecting sampler for airborne dust. Br J Ind
Med 11:284-288.
Wright BM. 1957. Experimental studies on the relative importance of con-
centration and duration of exposure to dust inhalation. Br J Med
14:219-228.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-560/6-84-004
o
3. RECIPIENT'S ACCESSION NO.
4. TITLE ANOSUBTITLE /
Scientific Rationale for the Selection of Toxicity
Testing Methods II. Teratology, Immunotoxicology,
and Inhalation Toxicology
5 REPORT DATE
September 1985
6. PERFORMING ORGANIZATION CODE
M. G. Ryon, D. S. Sawhney, M. L. Daugherty, and
R. H. Ross
3. PERFORMING ORGANIZATION REPORT NO.
ORNL-6094
9, PERFORMING ORGANIZATION NAME AND ADDRESS
Information Research and Analysis
Information Resources Organization
Oak Ridge National Laboratory
Oak Ridge, TM 37831
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Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, DC 20460
13. TYPE OP REPORT AND PERIOD COVERED
Final
14. SPONSORING AG6NCY CODE
16. ABSTRACT
This document is the second of a two-part lite
associated with the various toxicity testing method
pathology, etc.). Acute, subchronic, chronic, and
are covered in ORNL/EIS-151. Testing methods for d
immuno toxicology, and inhalation toxicology and res
these areas are covered in this volume, ORNL-6094.
for the purpose of assisting and supporting the U.S
Agency in its efforts to develop guidelines for mor
testing procedures.
rature analysis of parameters
(test animal selection,
carcinogenic testing methods
evelopmental toxicity,
earch needs associated with
These reports were prepared
Environmental Protection
efficient and economical
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Reviews
Toxicity
Tests
Toxicity Testing
Methodology
06
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