ORNL
Oak Ridge
National
Laboratory
Operated by
Union Carbide Corporation
for the Department of Energy
Oak Ridge, Tennessee 37830
ORNL/EIS-151
&EPA
United States
Environmental Protection
Agency
Office of Pesticides and
Toxic Substances
Washington, DC 20460
EPA-560/1 -80-001
December 1980
Pesticides and Toxic Substances
SCIENTIFIC RATIONALE FOR THE
SELECTION OF TOXICITY TESTING
METHODS: HUMAN HEALTH ASSESSMENT
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ORNL/EIS-151
EPA-560/1-80-001
Contract No. W-7405-eng-26
Information Division
SCIENTIFIC RATIONALE FOR THE SELECTION OF TOXICITY
TESTING METHODS: HUMAN HEALTH ASSESSMENT
Robert H. Ross, Michael G. Ryon, Mary W. Daugherty,
John S. Drury, John T. Ensminger, and M. Virginia Cone
Health and Environmental Studies Program
Information Center Complex
Work sponsored by the Office of Pesticides and Toxic Substances,
U.S. Environmental Protection Agency, Washington, D.C., under
Interagency Agreement No. 80-D-X0856.
Project Officers
Norbert Page, D.V.M.
Daljit Sawhney, D.V.M., Ph.D.
Date Published - December 1980
OAK RIDGE NATIONAL LABORATORY
Oak Ridge, Tennessee 37830
operated by
UNION CARBIDE CORPORATION
for the
DEPARTMENT OF ENERGY
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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, subcontrac-
tors, or their employees, 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
Substances, U.S. Environmental Protection Agency, and approved for publi-
cation. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor
does mention of trade names or commercial products constitute endorsement
or recommendation of use.
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CONTENTS
Figures vii
Tables ix
Foreword xiii
Acknowledgments xv
Abstract xvii
1. Summary 1
1.1 Basic Experimental Considerations 1
1.2 Acute Toxicity Testing 2
1.3 Subchronic Toxicity Testing ..... 2
1.4 Chronic Toxicity and Carcinogenicity Testing 4
2. Basic Experimental Considerations 7
2.1 Introduction 7
2.2 Test Materials 7
2.2.1 Introduction 7
2.2.2 Chemical Stability 8
2.2.3 Chemical Impurities 8
2.2.4 Vehicles 9
2.2.5 Homogeneity in Vehicle 10
2.2.6 Conclusions 10
2.3 Husbandry 10
2.3.1 Introduction 10
2.3.2 Animal Selection 11
2.3.3 Transportation, Quarantine, and Disease
Control 12
2.3.4 Design of Testing Facility 13
2.3.5 Caging 14
2.3.6 Bedding 15
2.3.7 Temperature and Humidity 18
2.3.8 Light 19
2.3.9 Ventilation 19
2.3.10 Noise and Handling 21
2.3.11 Personnel 22
2.3.12 Conclusions 22
2.4 Diet 23
2.4.1 Introduction 23
2.4.2 Dietary Requirements for Laboratory Animals ... 24
2.4.3 Types of Diets for Laboratory Animals 26
2.4.4 Analysis for Nutrients and Contaminants 31
2.4.5 Effects of Diet on Toxicity Test Results .... 33
2.4.6 Conclusions 35
2.5 Pathology 36
2.5.1 Introduction 36
2.5.2 Gross Examination 36
2.5.3 Tissue Preservation and Storage 40
2.5.4 Trimming, Staining, and Embedding of Tissue ... 41
2.5.5 Microscopic Examination 42
2.5.6 Conclusions 45
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2.6 Pharmacokinetics 45
2.6.1 Introduction 45
2.6.2 Basic Concepts 46
2.6.3 Utility of Pharmacokinetics 48
2.6.4 Conclusions 50
3. Acute Toxicity 59
3.1 Introduction 59
3.2 LD5Q Determinations 59
3.2.1 Introduction 59
3.2.2 Difference Between Species 63
3.2.3 Difference Between Administration Routes .... 67
3.2.4 Sex Differences in the Laboratory Rat 70
3.2.5 Test-Limiting Criteria 71
3.2.6 Conclusions 71
3.3 Human vs Animal Response 72
3.3.1 Introduction 72
3.3.2 Comparison of Lowest Published Lethal Doses
0V 72
3.3.3 Comparison of Acute Toxicity Response 75
3.3.4 Conclusions 76
3.4 Pathology 77
3.4.1 Introduction 77
3.4.2 Histopathology 77
3.4.3 Conclusions 80
3.5 Observation Period 80
3.5.1 Introduction 80
3.5.2 Purpose of Observation Period 84
3.5.3 Length of Observation Period 84
3.5.4 Conclusions 86
4. Subchronic Test Design 93
4.1 Introduction 93
4.2 Species 95
4.2.1 Introduction 95
4.2.2 Discussions in Literature Reviews Concerning
Species Suitability 95
4.2.3 Species Comparison Studies 101
4.2.4 Conclusions 121
4.3 Duration 123
4.3.1 Introduction 123
4.3.2 Duration Reviews 124
4.3.3 Comparison Summaries 130
4.3.4 Conclusions 150
4.4 Route of Exposure 151
4.4.1 Introduction 151
4.4.2 Route Discussions 152
4.4.3 Route Comparisons 157
4.4.4 Conclusions 168
4.5 Pathology 168
4.5.1 Introduction 168
4.5.2 Use of Pathology 170
4.5.3 Basis for a Minimum Pathology Screen 180
4.5.4 Conclusions 182
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4.6 Clinical Laboratory Tests 184
4.6.1 Introduction 184
4.6.2 General Clinical Testing 185
4.6.3 Hematology 188
4.6.4 Biochemical and Organ Function 192
4.6.5 Urinalysis 198
4.6.6 Conclusions 202
Appendix A 217
Appendix B 229
Appendix C 237
5. Chronic Toxicity and Carcinogenicity Testing 265
5.1 Introduction 265
5.2 Test Animals 266
5.2.1 Species 266
5.2.2 Strain 288
5.2.3 Spontaneous Tumors 294
5.2.4 Number 300
5.2.5 Controls 306
5.2.6 Age 308
5.2.7 Sex 311
5.2.8 Conclusions 320
5.3 Routes of Administration 323
5.3.1 Exposure Routes 324
5.3.2 Carcinogen Exposure by Different Routes 329
5.3.3 Conclusions 340
5.4 Dose and Duration 340
5.5 Interim Sacrifice 349
5.6 Data Collection and Evaluation 350
5.6.1 Food Consumption and Body Weight 350
5.6.2 Clinical and Laboratory Examinations 351
5.6.3 Pathological Examinations 352
5.6.4 Conclusions 354
5.7 Short-Term Tests for Carcinogenicity 356
5.7.1 Embryo Homograft 356
5.7.2 Site Transfer 357
5.7.3 Partial Hepatectomy 358
5.7.4 Alkaline Elution 360
5.7.5 a-Fetoprotein in Serum 361
5.7.6 Strain Susceptibility 362
5.7.7 The Sebaceous Gland Test 364
5.7.8 Host-Mediated In Vivo-In Vitro Assay 364
5.7.9 Conclusions 365
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FIGURES
2.1 Hydraulic analogy of a three-compartment body model .... 47
4.1 Changes in the mean body weight of rats fed Sumithion in
the diet for 90 days 133
4.2 Changes in cholinesterase activity in the kidney, liver,
red blood cells, and brain cortex of rats fed Sumithion
for 90 days 135
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TABLES
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
3.1
3.2
3.3
3.4
3.5
3.6
3.7
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
Space recommendations for laboratory animals
Cage space requirements for rodents . .
Requirements for essential amino acids expressed as
percentage of protein
NIH-7 open formula rat and mouse ration
AIM-76® purified diet (for rats and mice)
AIH-7^8 vitamin mixture
MH-76® mineral mixture
Tissue to be included in a gross examination
Correlation between gross and microscopic lesions in
carcinogenic studies in mice ....
Organs and tissues to be examined in routine toxic ity
tests ...
Compounds examined for LD50 differences between species
and between routes of administration
Percent LDsg differences between species
Percent U>5o differences between routes of
administration .
Compounds selected for human vs animal LD
comparisons . .
LD_ differences between humans and animals
Lo
Examples of histopathological changes from acute exposures
to chemicals .....
LDso observation periods
Experimental design for recently proposed subchronic oral
toxic ity tests
Ranking of various animal models as predictors of
metabolic fate in man
Occurrence of 39 physical signs from six drugs in three
species
Organ system toxic it ies
Comparison of significant variations in the response of
four species to an oral analgesic
Predictive abilities of the dog, monkey, and
combination
Summary of dioxin oral administration data
Summary of biological effects of TCDD
16
17
25
28
29
30
31
38
39
43
61
64
68
73
74
78
81
96
98
103
104
108
110
114
116
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4.9 Summary of treatment-related effects in hamsters, rats,
and rabbits repeatedly exposed to acrolein (ppm) for
13 weeks 120
4.10 Relationship of dosage levels of short-term and 2-year
feeding of materials in the diets of rats 125
4.11 Parameters of ratios of acute peroral LDsgs, 7- or
90-day and 2-year minimum effect (MiE) dosage levels . . . 126
4.12 Prediction formulas 127
4.13 Approximate duration of drug administration required to
define toxicity in animals 128
4.14 Prediction of long-term no-effect doses 129
4.15 Urinary ascorbic acid excretion and relative liver and
adrenal weights of rats fed butylated hydroxyanisole
(BHA) or butylated hydroxytoluene (BHT) at 0.1% of
the diet for 16 weeks 131
4.16 Mean values of body weights, food consumption, and
dibutyl(diethylene glycol bisphthalate) (DDGB) intake
of rats fed DDGB at 0% to 2.5% of the diet for 11
weeks 132
4.17 Mean testes and brain weights, as percentage of body
weight, of control rats and rats fed a dietary level
of 500 ppm of Sumithion for various periods of time . . . 134
4.18 Cumulative toxicity of epichlorohydrin: body weight
gain in grams (mean ± statistical error) 137
4.19 Subacute toxicity of epichlorohydrin: body weight gain
in grams (mean ± statistical error) 138
4.20 Cumulative toxicity of epichlorohydrin: percent organ
weight to body weight of rats (mean ± statistical
error) 139
4.21 Subacute toxicity of epichlorohydrin: percent organ
weight to body weight of rats (mean ± statistical
error) 139
4.22 Comparison of 13-week and 2-year 2,3,7,8-tetrachloro-
dibenzo-p-dioxin (TCDD) studies 140
4.23 Weight gain of female rats receiving 31 daily doses of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 141
4.24 Liver and thymus weights of female rats receiving daily
doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) . . . 142
4.25 Mean body weights and water intake of rats fed diets
containing 0% to 2.0% di-(2-ethylhexyl)phthalate (DEHP)
for up to 17 weeks 147
4.26 Mean hematological values for rats fed diets containing
0% to 2.0% di-(2-ethylhexyl)phthalate (DEHP) for 2, 6,
or 17 weeks 148
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4.27 Relative organ weights of rats fed diets containing
0% to 2.0% di-(2-ethylhexyl)phthalate (DEHP) for 2,
6, or 17 weeks 149
4.28 Summary of chemical examples tested for toxicity by
various routes of administration 158
4.29 Main toxic effects produced by oral or intravenous
administration of A9-tetrahydrocannabinol 166
4.30 Postmortem studies 169
4.31 Range-finding data on subacute oral toxicity 171
4.32 Summary of observations of effects detected in short-
term and two-year oral studies 173
4.33 Incidence (in percent) of drug-induced pathological
changes encountered over a ten-year period in
approximately 14 000 animals 176
4.34 Summary of histological quantification methods and
their applications for chemically induced lesions .... 178
4.35 Comparison of the usefulness of individual organs to
indicate toxicity when pathologically examined in a
subchronic study 181
4.36 Hematological studies during toxicity tests 190
4.37 Relative sensitivity of liver enzyme tests 197
5.1 Some factors in the experiments with MH 101 upon a
variety of species 272
5.2 Induction of skin carcinomas in mice, hamsters, and
rats with weekly applications of 9,10-dimethy1-
1,2-benzanthracene 274
5.3 Tumor induction in various species with aromatic
amines 277
5.4 Compounds, not previously listed, which have produced
bladder cancer in the dog 280
5.5 The carcinogenic action of diethylnitrosamine in
different animal species 281
5.6 Tumor induction of various species with diethylnitro-
samine and dimethylnitrosamine , 283
5.7 Carcinogenicity of different forms of asbestos in
various species 287
5.8 Chemicals or industrial processes associated with cancer
induction in humans: comparison of target organs and
main routes of exposure in animals and humans 289
5.9 Spontaneous tumor incidence in animals used in the
National Cancer Institute Bioassay Program 296
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5.10 Sex and age incidence of characteristic tumors of inbred
strains 297
5.11 Spontaneous tumors in laboratory animals 301
5.12 Incidence of tumors in treated groups required for
significance (P = 0.05) depending on experimental
group size and spontaneous tumors in controls 307
5.13 Systemic tumor induction in mice with 3-methylcho-
lanthrene by the intragastric route 313
5.14 Influence of sex on response to carcinogens 314
5.15 Comparison of continuous versus intermittent adminis-
tration of BZ»2HC1 on development of liver, Harderian
gland, and lung 319
5.16 Induction of tumors in the Syrian hamster with diethyl-
nitrosamine 330
5.17 Local tumor induction in mice with 3-methylcholanthrene
by various routes of administration 332
5.18 Incidence of pulmonary tumors in strain A mice after
intravenous injection of methylcholanthrene 335
5.19 Incidence of pulmonary tumors in strain A mice after
intravenous injection of 0.5 mg of methylcholanthrene
or 0.5 mg of dibenzanthracene 336
5.20 Incidence of pulmonary tumors in strain A mice given
subcutaneously 0.5 mg of methylcholanthrene or 0.5 mg
of dibenzanthracene dispersed in 0.5 cc of horse serum
and cholesterol 336
5.21 Tumor induction with aromatic amines by various routes
of exposure 338
5.22 Summary of recommendations on dose selection 342
5.23 Time relationships among drug exposure, life span, and
time equivalents in man 347
xii
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FOREWORD
This report was prepared by the Health and Environmental Studies
Program (HESP), Information Center Complex, Information Division, Oak
Ridge National Laboratory, under an interagency agreement between the
U.S. Department of Energy and the U.S. Environmental Protection Agency
(EPA). Under this agreement HESP is providing a variety of technical
information support services to the EPA Office of Testing and Evaluation,
including: performing technical review and follow-up on chemical studies,
arranging workshops, writing reviews on health effects of specific
chemicals, and conducting structure-activity oriented reviews of chemicals
associated with mutagenic or teratogenic effects.
This document is part one of a two-part literature analysis of
parameters associated with the various toxicity testing methods. The
information contained herein has been compiled for the purpose of assisting
and supporting EPA in its efforts to develop guidelines for more efficient
and more economical toxicity testing procedures.
xiii
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ACKNOWLEDGMENTS
The authors would like to thank Halgo Cerstner, manager of the
Information Center Complex (ICC), Information Division, Oak Ridge National
Laboratory (ORNL), for her support during the preparation of this docu-
ment. The advice and support of Drs. Norbert Page and Daljit Sawhney,
U.S. Environmental Protection Agency (EPA) project officers, and the
assistance of Helen Warren and other members of the Toxicology Information
Response Center of the ICC are gratefully acknowledged. Special thanks
are extended to Dr. Peter Witschi of the ORNL Biology Division, to Dr.
Guy Griffin of the ORNL Health and Safety Research Division, to Drs.
Harold Grlce and Clifford Chappel, FDC Consultants, Inc., and to Drs.
David Anderson, Salvatore Biacardi, William Butler, Larry Chltlik,
William Dykstra, Richard Fournier, Wayne Galbraith, Stan Gross, Robert
Jaeger, Steve Johnson, Raymond Landolf, Carl Morris, and James Murphy of
EPA for their technical review and helpful suggestions. The authors are
also greatly indebted to Judy Crutcher, Sherry Hawthorne, Pat Hartman,
and Donna Stokes, of the ICC Publications Office, and to Carolyn Seaborn
for her assistance in the collection and organisation of data.
xv
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ABSTRACT
This document is the first of a two-part literature analysis of
parameters associated with the various toxicity testing methods (test
animal selection, pathology requirements, etc.)- Acute, subchronic,
chronic, and carcinogenic testing methods are covered; a discussion of
some basic experimental considerations is also included. This report
was prepared for the purpose of assisting and supporting the U.S. Environ-
mental Protection Agency in its efforts to develop guidelines for more
efficient and economical testing procedures.
xvii
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1. SUMMARY
1.1 BASIC EXPERIMENTAL CONSIDERATIONS
To assist in the development of test standards, this section
addresses certain aspects of test materials, husbandry, diet, patho-
logical examination, and pharmacokinetics.
The chemical and physical properties of the test chemical should be
known prior to conducting a toxicity test of a chemical. The chemical
and physical properties will influence laboratory storage practices
and will permit the researcher to determine the stability of the chemical
under the conditions of the experiment. In addition, the chemical
purity of the test material and possible interactions with the vehicle
may be investigated.
Husbandry conditions associated with toxicity testing can affect
the test results and should be controlled or standardized. Included
among the husbandry factors are animal selection, transportation,
quarantine, disease control, cage design, test facility design, regula-
tion of macroclimate and microclimate variables, and experienced
technical personnel.
Nutrients required by laboratory animals are supplied through
various types of diets. Variations in the concentration of essential
nutrients and the presence of contaminants in the diet may affect the
response of test animals to toxic substances. Periodic testing of diets
for nutrient concentrations and contaminants has been proposed.
Pathology is a very essential part of the evaluation of a chemical's
toxicity, providing information on the nature of the lesions and indicat-
ing dose-effect relationships. A successful pathology evaluation should
include the following: (1) the pathologist should perform or supervise
all steps in the protocol; (2) the gross necropsy should be extremely
thorough and include every animal in the study; (3) the tissues should
be properly preserved in an accepted fixative immediately after the
necropsy; (4) the staining, embedding, and sectioning of the tissues
must be well planned and coordinated, especially if special stains are
utilized; and (5) the microscopic examination should be as thorough as
practical limitations will allow.
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The pharmacokineti.es of a chemical, which is influenced by four
major rate processes — absorption, distribution, metabolism, and elimin-
ation — can provide valuable information to the researcher. The results
of pharmacokinetic studies can help decide the future testing protocol
for a chemical as well as explain why a chemical can be teratogenic when
administered during one part of the gestation period but not teratogenic
when administered in another part of the gestation period.
1.2 ACUTE TOXICITY TESTING
Acute toxicity studies are very useful because they provide base-
line information such as the LDsg (the most frequently determined index
of toxicity), the relative sensitivity of the various species, and the
comparative toxicity of various chemicals. They also identify the
nature of the toxicity or the mode of the toxic action and provide
guidance on doses to be used in subsequent experiments. Administration
may be a single oral or single parenteral injection or may be given in
food or drinking water over a 24-h period. When the laboratory rat is
used as the test animal, both sexes should be tested because of the
possibility of sex-related differences in LDsg values.
The time following dosing, during which deaths are recorded for the
purpose of determining an LD5Q, is the observation period. This period
must be of sufficient duration for manifestation of delayed effects but
not so long that there is doubt whether the animal died as a direct
result of the test substance. The 7- and 14-day observation periods,
particularly the 14-day period, seem to be the most common lengths for
observation periods among researchers in the United States. Researchers
in other countries use observation periods of various lengths. Close
clinical observation of the animal is necessary to assist in the assess-
ment of the toxic effects.
1.3 SUBCHRONIC TOXICITY TESTING
Subchronic tests are designed to provide two types of toxicity data.
As a precursor to a chronic study, subchronic tests should identify target
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organs and define appropriate dose ranges for long-term exposures. When
used as the primary evaluation of the effects of chronic exposure,
subchronic tests can provide detailed information on toxicity and the
potential hazard of the chemical to man. The parameters to be employed
to assess toxicity vary with the purpose of the test; they are usually
more complex when the subchronic test is to be the primary evaluation of
long-term effects.
The number of species employed in a subchronic test is an area
under discussion in the protocol design. Most information suggests that
in a subchronic test both a rodent and nonrodent species should be used.
The overlap provided by the use of two mammals increases the efficiency
of hazard assessment for human exposure. In general the rat and dog
appear to be the species most often employed in these hazard assessments.
However, whenever possible the choice of species should be governed by
information on pharmacokinetics and metabolism of the compound. Under
such circumstances other species might also be considered.
The duration of exposure for a subchronic test is another area under
discussion. The literature suggests that a 90-day exposure can provide
essentially the same toxicity data as would be provided by a long-term
test, excluding carcinogenicity and teratogenicity. This information
could be the basis for regulatory decisions. However, there is addi-
tional information in the literature suggesting that a 30-day duration
might be acceptable. The comparisons available indicate that in many
cases the effects can be predicted qualitatively but not quantitatively.
This question needs more data for analysis. Comparison studies or
access to unpublished information in industry files is needed to fully
resolve the debate over the minimum duration necessary for a subchronic
test.
The routes of exposure for a subchronic test should ideally represent
the expected routes of human exposure. Most often this is by ingestion
or inhalation, which are the principal exposure routes utilized in test
designs. Oral exposure can be achieved relatively easily, but inhalation
exposure is more complicated, requiring special test facilities. Extrap-
olation of data obtained from one route to evaluate the hazard with
exposure to another route could provide a solution to the problems of
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inhalation tests. However, there is little information on the criteria
necessary for or the hazards involved in such an extrapolation. Much
more research is needed to assess the predictability and feasibility of
route-to-route extrapolations.
In subchronic testing much use is made of biochemical tests and
pathology as parameters to evaluate toxicity. Both biochemical tests
and pathology can provide data to assess toxicity.
Pathological evaluations should include a gross examination of all
organs of all test animals at all dose levels. Organ weights should
also be recorded for the major tissues. Microscopic examination, because
of its higher costs, may be limited to the evaluation of selected
tissues at high and control dose levels. Additionally, any lesion-
bearing organs, detected in the gross examination, should be examined
microscopically. Further microscopic examination at other dose levels
should be based on the pathology results from the high dose level
examination.
Biochemical tests should be employed to provide information on
subtle changes and to fully assess the extent of toxic effects. A
well-designed subchronic study should include a basic hematology examina-
tion (evaluating cellular damage and hemorrhaging), a set of enzyme and
organ function tests (primarily evaluating liver and kidney damage), and
a urinalysis examination (primarily for kidney damage). Changes indi-
cated by these tests can be investigated further by more complex bio-
chemical tests.
1.4 CHRONIC TOXICITY AND CARCINOGENICITY TESTING
Many factors influence the results of chronic toxicity and carcino-
genicity tests in animals, including route of administration, dosage and
frequency of exposure, species, strain, sex, age of the animal when the
test is initiated, dietary constituents, immunologic status of the animal,
duration of the experiment, and other factors.
The principal objective for conducting chronic toxicity tests is to
determine the toxic potential of a chemical when it is administered for
the greater part of the life span of the animal. In testing of chemicals
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for their carcinogenic potential the major objective may be simply to
determine whether or not the chemical is capable of inducing tumors in a
particular strain or species of animals.
Because most chronic toxicity and carcinogenicity tests are con-
ducted over the major portion of the life span of the animal, short-
lived rodents are the choice of most investigators. However, the dog
is occasionally used, particularly in tests for chronic toxicity.
Test duration is an issue under discussion. Attempts to standard-
ize this parameter have been unsuccessful. Generally, when mice and
rats are used for chronic toxicity and carcinogenicity tests, 24 to
27 months and approximately 24 months are recommended, respectively.
Typically, dogs are treated for 1 to 2 years in chronic toxicity studies.
New strains of animals that are especially susceptible to tumor
induction and genetic factors that influence the spontaneous tumor
incidences in laboratory animals have been exploited in the development
of certain short-term assays. These and other in vivo short-term bio-
assays could be considered as intermediate between in vitro short-
term studies and lifetime studies and may become valuable tools in
screening potential carcinogens.
The number of test animals used is a compromise between requirements
for good statistical precision and reasonable costs and work loads.
Most authorities now recommend 50 rodents or four to eight nonrodents
per test group. Concurrent control groups comprising animals of the
same species, age, sex, weight, and number are also required.
In tests for chronic toxicity at least three dose levels should be
used, and both treatment and observation of animals should be conducted
7 days per week. In tests for carcinogenicity in which induction of
tumors is the primary end point, two or three doses of the test chemical
may be employed. If information on dose-response relationships is re-
quired, then several doses are necessary to provide data for appropriate
statistical analysis.
Interim sacrifices may be performed to provide information on the
progression of toxic events. Macroscopic examination of animals and
microscopic examination of tissues are necessary in evaluating the hazard
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of chemical exposure. As a minimum, all major organs and tissues of
high dose and control animals are recommended for histological examina-
tion. This study should also include any other tissues exhibiting gross
lesions.
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2. BASIC EXPERIMENTAL CONSIDERATIONS
2.1 INTRODUCTION
This section will briefly discuss some basic experimental parameters,
such as the purity and chemical stability of the test chemical; the
importance of selecting the appropriate vehicle; the diet, husbandry,
and pathological examination of the test animal; and the importance of
pharmacokinetics in the toxicological evaluation of chemicals. The
discussions will illustrate why careful attention to each of these is
essential to accurate and reliable experimental results.
2.2 TEST MATERIALS
2.2.1 Introduction
The chemical and physical properties of the test material must be
known prior to the initiation of a toxicity experiment. This is essen-
tial because the toxicologist must know that the results obtained from
his experiment are caused by the compound under investigation and not
by a degradation or contamination product. The scientific committee of
the Food Safety Council in agreement with this states that the "lack or
disregard of information on the chemical nature of the material to be
tested not only limits the usefulness of toxicological data for regula-
tory purposes but can, in some instances, provide an erroneous impres-
sion of its toxicity" (Food Safety Council, 1978). A document published
by the authority of the Ministry of Health and Welfare Canada (1975)
further states that, before a long-term study is initiated, batch-
to-batch variations of the compound, chemical synthetic processes,
packaging and handling procedures, storage requirements, and inter-
actions with other chemicals must be considered. The following subsec-
tions will briefly describe some aspects of the pretesting examination
of the test material.
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2.2.2 Chemical Stability
As soon as a substance arrives in the toxicology laboratory, the
stability of the chemical at various pH values and its photochemical
properties must be determined (World Health Organization, 1978). These
parameters may determine the manner in which the chemical should be
stored prior to use. The stability of the test material under the same
conditions in which it will be administered must also be determined
(Sontag, Page, and Saffiotti, 1976). If the chemical nature of the
test substance changes during an experiment as a result of the experi-
mental conditions, the results will not be reliable. In a worst-case
situation, the chemical stability of the test material could be altered
without the researcher's knowledge, thus permitting incorrect conclusions
concerning the action of the test substance. Therefore, prior to the
start of the toxicity test the stability of the test chemical in the
feed and/or vehicle, at the temperature and/or pH to be used in the test,
must be investigated to ensure that the chemical stability is not altered
by the experimental conditions. The stability of the test material
under its conditions of storage and/or administration will influence the
frequency at which fresh treatment mixtures are prepared (National
Academy of Sciences, 1975; Sontag, Page, and Saffiotti, 1976).
When the test material is a chemical used in food processing, the
possibility exists that under conditions of use the chemical may be
transformed or degraded in foods, with the resulting products being
either more or less toxic than the original chemical (Food Safety Council,
1978). The knowledge of potential chemical transformation would be
essential for the safety assessments of chemicals in foods.
2.2.3 Chemical Impurities
The chemical purity of the test material should be established
prior to toxicity testing (Sontag, Page, and Saffiotti, 1976). This is
important because of the possiblity that trace contaminants could be
responsible for, or at least modify, the observed biological effects
attributed to the test material under investigation. As pointed out by
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9
the World Health Organization (1978), if the contaminants are unknown or
their biological activity unsuspected, toxicity tests may lead to
erroneous conclusions concerning the test material.
A related issue that confronts the toxicologist and is one of the
earliest and most difficult decisions to be made is the selection of the
desired purity of the sample to be studied (technical grade, highly
purified, etc.) (World Health Organization, 1978). If the test material
is a commercial product that is known to contain impurities, toxicity
testing of a highly purified sample could result in erroneous conclu-
sions. If the impurities are standardized, then testing of the commer-
cial product would be the answer; however, for those commercial products,
chemicals used in manufacturing, or chemicals incidentally released into
the environment for which the impurities are not standardized, the
toxicologist must make a decision concerning test sample purity.
2.2.4 Vehicles
The choice of the appropriate vehicle is important because the
toxic activity of the test chemical can be altered, especially if the
test chemical reacts chemically with the vehicle (also mentioned in
Sect. 2.2.2). However, the toxic activity of the test chemical can be
altered without a change in chemical structure. One example is that of
cottonseed oil, which, unless refined by superheated-steam processing,
may contain cyclopropenoid fatty acids, which can enhance the activity
of some carcinogens (Lee et al., 1968, as cited by National Academy of
Sciences, 1975). Other examples of vehicle interference in tests for
carcinogenicity are described by Pott, Brockhaus, and Huth (1973),
who observed that when a subcutaneous dose of benzo(a)pyrene was kept
constant, increasing the dose of the vehicle, tricoprylin, increased the
rate of tumor production, and by Mori (1965), who reported an increase
in organs affected for tumor induction when 4-nitroquinoline-l-oxide
was administered in olive oil and lecithin instead of in a solvent
mixture containing cholesterol.
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2.2.5 Homogeneity in Vehicle
If a text mixture is prepared in sufficient quantity for the dosing
of several animals that are to receive the same concentration of the
test material, the homogeneity in the vehicle is essential. Sontag,
Page, and Saffiotti (1976) state that in a long-term study where the test
material is incorporated into the feed, the homogeneity and concentra-
tion in the diet mix should be determined before the start of the study
and periodic analysis of random samples from freshly mixed batches
should be performed to ensure that proper mixing and formulation pro-
cedures are being used.
2.2.6 Conclusions
Prior to the initiation of an experiment designed to evaluate the
toxicity of a chemical, consideration should be given to the character-
istics of the test chemical and the vehicle. Specifically, the stability
and the purity of the test chemical should be known, and the possibility
of the vehicle altering the test results must be considered.
2.3 HUSBANDRY
2.3.1 Introduction
In all types of toxicity tests, the results can be influenced by
the husbandry conditions under which the testing is performed. Husbandry
factors that should be managed include selection, transportation, and
quarantine of the test animals; control of diseases; proper design of
the cages and testing facility; regulation of the temperature, humidity,
light, ventilation, noise, and handling; and certification of personnel.
Unless these factors are standardized or controlled, the data obtained
cannot be used with any certainty to evaluate the potential harmful
effects of the chemical to man. The following subsections will briefly
discuss these factors.
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2.3.2 Animal Selection
In addition to the consideration of species and human metabolic
similarity discussed elsewhere in the text, several important factors
should be considered or controlled when selecting animals for use in
toxicity studies. First, all animals should have standardized life
cycle variables, such as age, sexual maturity, and mating status, as
these factors can affect toxicity (Hurni, 1970). Next, the genetic
composition should be uniform, because this ensures consistency of
response and reproducibility of results (Food Safety Council, 1978;
Zbinden, 1963). The current breeding methods for maintaining a constant
genotype include inbred, outbred, and hybrid strains. Inbred animals
are crosses of brother and sister or parent and offspring that have
been maintained for at least 20 generations (National Academy of Sciences,
1969). This results in a specific homozygous genotype where all animals
in the colony are as similar as identical twins (Hurni, 1970). Outbred
or random bred strains maintain an unaltered, heterozygous pool of
genetic material on a population level (Hurni, 1970) but are rarely used
in toxicity studies (National Academy of Sciences, 1969). Because both
of these options have problems, hybrids from crosses of inbred strains
are often utilized. These animals are genetically uniform but are not
homozygous (Food Safety Council, 1978; Hurni, 1970). Festing (1979)
reviews, in more detail, the pros and cons of inbred, outbred, and
hybrid animals in toxicity testing.
The need for reduction of animal health as a factor stimulated the
development and use of specific pathogen-free (SPF) strains. These are
specially bred animals (best obtained by aseptic hysterectomy and arti-
ficial rearing of germ-free derived animals) that have accepted levels
of health and are known to be free of certain standard diseases and
parasites (Hurni, 1970; Meister, Hobik, and Metzger, 1967). However,
these animals require special isolated colonies with "clean zone"
barriers and can be difficult to maintain (Hurni, 1970). Therefore,
as with other factors, the nature of the experiment and the risks
involved will decide the animal type and quality to be used (Page,
1977).
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2.3.3 Transportation, Quarantine, and Disease Control
After selection of the appropriate test animals, transportation to
the testing facility or animal room is necessary. This can affect the
response of the animals by subjecting them to unusual stresses, and
they should be given time to recover (National Academy of Sciences,
1969). Heinecke (1967) has shown that even transport between buildings
(e.g., from an in-house breeding colony to the testing facility) has
affected the blood status of mice for 3 to 4 days afterwards. More
extensive transportation can disrupt the normal state for up to 6 weeks
(Hurni, 1970). Therefore, it is necessary to allow the animals to
readjust and acclimate before initiating testing.
While the animals are acclimating, they can be kept isolated from
the rest of the facility for quarantine purposes (Page, 1977). The
quarantine is necessary to prevent the introduction of diseases into the
existing facility population. The duration of quarantine varies with
the species, its source, and the testing purpose (U.S. Department of
Health, Education, and Welfare, 1974), but should be at least 2 weeks
(National Academy of Sciences, 1969). As part of the quarantine process,
it is a good practice to include: (1) a physical examination upon
arrival; (2) veterinary care to check for parasites and disease and to
allow for immunization; (3) general grooming; and (4) a pathological
examination of a small number of the test animals (Sontag, Page, and
Saffiotti, 1976; U.S. Department of Health, Education, and Welfare,
1974). If any animals in quarantine are found to have a communicable
disease, the whole group should be destroyed (Sontag, Page, and Saffiotti,
1976).
The quarantine of animals upon arrival is just part of a general
need to control disease in the test facility. The presence of disease
in the test facility can invalidate the test results, especially if the
disease produces subtle, nonspecific, or long-term effects (van der
Waaij and van Bekkum, 1967). To control disease, several steps are
necessary: (1) acquire and include (after quarantine) only healthy
animals; (2) prevent introduction of pathogens into test facility;
(3) maintain sanitary conditions; and (4) utilize a well-planned disease
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13
control program (Flynn, 1967; National Academy of Sciences, 1969). The
prevention of pathogen introduction should include monitoring and/or
control of water supply, air supply, feed and bedding quality, and
personnel contact (National Academy of Sciences, 1969). The use of
filters and filter caps is particularly helpful to control airborne
diseases (Clough, 1976). Sanitary control should include facility and
personnel cleanliness, proper waste disposal practices, and vermin
control (U.S. Department of Health, Education, and Welfare, 1974). The
disease control program can be the most vital part of the process and
should (1) be specific for that situation; (2) be aimed at the most
common or expected diseases; and (3) include frequent observation by
appropriately trained personnel (Flynn, 1967; U.S. Department of Health,
Education, and Welfare, 1974). A detailed example of a disease control
program is discussed by Flynn (1967). Fox (1977) reviewed some of the
most common diseases, for each organ system, that occur in testing
facilities.
2.3.4 Design of Testing Facility
The design, scope, and size of a toxicity testing facility is the
product of many compromises, depending on the nature of the research,
the number of test animals to be housed, and the types of species used
(U.S. Department of Health, Education, and Welfare, 1974). The facility
should be designed to provide maximum comfort and safety for the test
animals and personnel, to minimize disease factors, and to minimize costs
(National Academy of Sciences, 1969). It is essential that a facility
design include a building, wing, floor, or room for the animals that is
separate from human work areas; specialized laboratories for maintenance
and assessment of the animals; a supply-receiving area; a quarantine
area; and an incinerator (U.S. Department of Health, Education, and
Welfare, 1974). Specific construction details are discussed in several
reviews (Grange, 1976; National Academy of Sciences, 1969; U.S. Department
of Health, Education, and Welfare, 1974).
In general the rooms should be arranged to provide barriers between
individual rooms and the outside environment (National Academy of Sciences,
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14
1969; Sontag, Page, and Saffiotti, 1976). There are three common types
of control systems used in test facilities: open, closed, and isolated
(Shaw, 1976). The open system does not have hermetically sealed rooms,
and the entering of materials and personnel is not controlled. The
closed system is hermetically sealed with sterilization controls over
entering materials and personnel. An isolated system is similar to a
closed one, but does not allow personnel to enter. One special type of
control system is the "clean-dirty" system, which utilizes a unidirec-
tional corridor flow of materials and personnel (Sontag, Page, and
Saffiotti, 1976). Each room has a separate entrance and exit door,
allowing personnel and materials to be decontaminated before entering
and to leave only through the exit door (Page, 1977). This system is
especially useful for minimizing disease contamination (Sontag, Page,
and Saffiotti, 1976). If possible, there should also be individual
rooms for each species and each test treatment. A separate room for
controls, to evaluate the influence of various factors of the test
facility, has also been suggested (Food Safety Council, 1978). The
overall goal of the facility design is to reduce or standardize the
environmental factors affecting test results in the most efficient
manner.
2.3.5 Caging
The cage is the single most important element of the physical envi-
ronment for laboratory animals since it represents the immediate barrier
between the microenvironment and macroenvironment of the testing facility
(Clough, 1976; U.S. Department of Health, Education, and Welfare, 1974).
The criteria to evaluate a good cage design include: (1) it should meet
the investigator's research requirements; (2) it should be designed with
the animal's physical comfort as a prime consideration and; (3) it
should be compatible with proper maintenance requirements, especially
health and sanitary procedures (U.S. Department of Health, Education,
and Welfare, 1974). Some important features that a good cage design
incorporates are: (1) it should be stackable or fit on racks; (2) it
should have smooth solid walls and be accessible for cleaning; (3) it
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should be constructed of stainless steel or plastic; (4) it should have
a perforated bottom (depending on species or experiment requirements);
and (5) it should have accessible feed and water sources (usually built
into a removable lid) (National Academy of Sciences, 1969). Stainless
steel and especially hard plastics are used for cage designs, because
they are lighter and resistant to corrosion (Clough, 1976). A solid
cage design offers more control over the microclimate (usually increasing
humidity, NH3, C02, and temperature levels) and affords more protection
from microbial agents than wire mesh designs (Fox, 1977; Hurni, 1970).
However, animals in solid cages require transfer to a clean cage at
least every week, while animals in wire mesh cages can last 2 weeks
(Sontag, Page, and Saffiotti, 1976). Also Winter and Flataker (1962)
found that rats in solid cages developed more resistance to chemicals
than those in wire mesh. Therefore, the choice of exact cage design is
dependent on the goals and circumstances of the specific study.
The number of animals per cage and the amount of space for each are
also determined by practical and experimental considerations. Hatch et
al. (1965) found that a single mouse per cage was more susceptible to
certain cancers than groups of 1 to 5 mice. Hurni (1970) discussed
several studies in which isolated animals showed evidence of an increase
in toxicity. However, grouping of animals in cages can indirectly
affect toxicity, for example, by increasing food competition or fecal/
urinary contamination (Food Safety Council, 1978). Table 2.1 gives some
recommended space standards for routine housing of laboratory animals
(U.S. Department of Health, Education, and Welfare, 1974). Table 2.2
lists the minimum number of square inches necessary per rodent and the
maximum number of animals per cage (National Academy of Sciences, 1969).
These guidelines address practical considerations, but the nature of the
experiment often dictates the actual numbers of animals per cage.
2.3.6 Bedding
Bedding material is frequently used in housing test animals, espe-
cially rodents, and has been shown to affect toxicity. In general,
bedding serves several purposes: (1) it provides thermal insulation;
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Table 2.1. Space recommendations for laboratory animals
Species
Mouse
Rat
Hamster
Guinea pig
Rabbit
Cat
Dog*
Primates0' d
Group 1
Group 2
Group 3
Group 4
Group 5
Weight
Up to 10 g
10 to 15 g
16 to 25 g
Over 25 g
Up to 100 g
100 to 200 g
201 to 300 g
Over 300 g
Up to 60 g
60 to 80 g
81 to 100 g
Over 100 g
Up to 250 g
250 to 350 g
Over 350 g
Up to 2 kg
2 to 4 kg
Over 4 kg
Up to 4 kg
Over 4 kg
Up to 15 kg
15 to 30 kg
Over 30 kg
Up to 15 kg
15 to 30 kg
Over 30 kg
Up to 1 kg
Up to 3 kg
Up to 15 kg
Over 15 kg
Over 25 kg
Type of
housing
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Pen or run
Pen or run
Pen or run
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Cage
Floor area per
animal (squared)
39 cm (6 in.)
52 cm (8 in.)
77 cm (12 in.)
97 cm (15 in.)
110 cm (17 in.)
148 cm (23 in.)
187 cm (29 in.)
258 cm (40 in.)
64.5 cm (10.0 in.)
83.9 cm (13.0 in.)
103.2 cm (16.0 in.)
122.6 cm (19.0 in.)
277 cm (43 in.)
374 cm (58 in.)
652 cm (101 in.)
0.14 m (1.5 ft)
0.28 m (3.0 ft)
0.37 m (4.0 ft)
0.28 m (3.0 ft)
0.37 m (4.0 ft)
0.74 m (8.0 ft)
1.12 m (12.0 ft)
2.23 m (24.0 ft)
0.74 m (8.0 ft)
1.12 m (12.0 ft)
6
0.15 m (1.6 ft)
0.28 m (3.0 ft)
0.40 m (4.3 ft)
0.74 m (8.0 ft)
2.33 m (25.0 ft)
Heighta
12.7 cm (5 in.)
12.7 cm (5 in.)
12.7 cm (5 in.)
12.7 cm (5 in.)
17.8 cm (7 in.)
17.8 cm (7 in.)
17.8 cm (7 in.)
17.8 cm (7 in.)
15.2 cm (6 in.)
15.2 cm (6 in.)
15.2 cm (6 in.)
15.2 cm (6 in.)
17.8 cm (7 in.)
17.8 cm (7 in.)
17.8 cm (7 in.)
35.6 cm (14 in.)
35.6 cm (14 in.)
35.6 cm (14 in.)
61.0 cm (24 in.)
61.0 cm (24 in.)
81.3 cm (32 in.)
91.4 cm (36 in.)
b
50.8 cm (20 in.)
76.2 cm (30 in.)
76.2 cm (30 in.)
91.4 cm (36 in.)
213.4 cm (84 in.)
Height means from the resting floor to the cage top.
These recommendations may require modifications according to the body con-
formations of particular breeds. As a further general guide, the height of a dog
cage should be equal to the height of the dog over the shoulders (at the withers),
plus at least 6 inches, and the width and depth of the cage should be equal to
the length of the dog from the tip of the nose to the base of the tail, plus at
least 6 inches.
°The primates are grouped according to approximate size with examples of
species that may be included in each group: Group 1 — marmosets, tupaias, and
infants of various species; Group 2 — cebus and similar species; Group 3 —
macaques and large African species; Group 4 — baboons, monkeys larger than 15 kg,
and adult members of brachiating species such as gibbons, spider monkeys and
woolly monkeys; Group 5 — great apes.
'where primates are housed in groups in pens, only compatible animals should
be kept. Minimum height of pens should be 6 feet. Resting perches, nesting
boxes, and escape barriers necessary for the well-being of the particular animals
should also be provided.
Source: Adapted from U.S. Department of Health, Education, and Welfare, 1974.
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Table 2.2. Cage space requirements for rodents
Minimum square Maximum number
inches per animala of animals per cage
Mice
Weaning to 5 weeks
5 to 8 weeks
8 to 12 weeks
Over 12 weeks
Hamsters
Weaning to 5 weeks
5 to 10 weeks
Over 10 weeks
Rats
Up to 50 g
50 to 100 g
100 to 150 g
150 to 200 g
200 to 300 g
Over 300 g
Guinea pigs
Weaning to 350 g
350 g and over
Breeders
6
8
12
15
10
12.5
15
15
17
19
23
29
40
60
90
180
40
30
20
20
20
16
13
50
50
40
40
30
25
15
10
5
In many circumstances, more space per animal may be needed.
Source: Adapted from National Academy of Sciences, 1969.
(2) it absorbs fecal and urinary wastes and water spillage; (3) it is
used to build nests; and (4) it reduces stress by providing a protective,
isolating cover (National Academy of Sciences, 1969). The material
selected should therefore be absorbant, nonedible, and innocuous but not
dusty or highly resinous (Clough, 1976; Hurni, 1970; National Academy of
Sciences, 1969). Some of the bedding materials rated as acceptable by
the National Academy of Sciences (1969) include: coarse pine sawdust;
pine, cedar, basswood, or poplar shavings; crushed corn cobs; and
hardwood chips. However, other researchers have found problems with
some of these materials. Porter (1967) found that sawdust often contains
many possible contaminants, and he preferred sterilized pine shavings.
In contrast, Clough (1976) and Fox (1977) warned that pine, cedar, and
other softwoods, if fresh, can induce biological variations in the
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microsomal metabolizing enzymes and alter chemical toxicity. Hurni
(1970) warned that hardwood shavings, which are high in tannic acid
content, can lead to constipation in rodents. However, Sontag, Page,
and Saffiotti (1976) preferred hardwood shavings for long-term carcino-
genicity studies. In any case, the material must be sterilized, pref-
erably by autoclaving, and be changed frequently.
2.3.7 Temperature and Humidity
Temperature and humidity are two environmental factors that should
be controlled by the experimenter, since they can influence the test
results. The effect of changes in these factors will be influenced by
the magnitude, frequency, and duration of the changes, by whether or not
the test animals have behavioral modifications to adapt to these, and
by the current physical and health status of the animals (Weihe, 1976a).
The easiest solution is to control temperature and humidity by air
conditioning each animal room (U.S. Department of Health, Education, and
Welfare, 1974).
Weihe (1964, as cited by Heinecke, 1967), by varying the environ-
mental temperature, was able to affect the LDsgS of chemicals. Heinecke
(1967) found that temperature changes also affected blood values. Hurni
(1970) reviewed several studies in which cold temperatures or increases
in ambient temperature affected both the resistance of test animals and
the toxic effects of chemicals. Changes in ambient temperature can
affect the metabolism of chemicals, and the effects produced can mimic
chemical effects, making diagnosis difficult (World Health Organization,
1978). Humidity levels can also affect toxicity and were found to be a
principal factor of ringtail in young rats (Clough, 1976; Flynn, 1967).
Thus it is very important to control these factors.
The exact limits of temperature and humidity beyond which signifi-
cant metabolic adaptation will occur are not known, but if the changes
are moderate (±5°K or 20% to 30% humidity) the animals should be able to
adapt behaviorally without damage (Weihe, 1976a). The National Academy
of Sciences (1969) recommends a fluctuation of no more than ±2°F in a
range of 70° to 80°F and 40% to 70% humidity for rats and mice. The
U.S. Department of Health, Education, and Welfare (1974) suggests levels
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of 65° to 85°F and 30% to 70%, depending on the test species. Sontag,
Page, and Saffiotti (1976) recommend more precise levels of 74°F ± 2°
and 40% ± 5% for rats and mice. In any case, the investigator should
define the levels used, along with the population size and cage type, so
that the study can be properly evaluated.
2.3.8 Light
Light is another environmental variable that should be controlled.
The damaging effects of light are dependent on the intensity and duration
(Weihe, 19762?)- All common laboratory animals are sensitive to light,
and by using low-level, artificial light sources, with exclusion from
alternate sources such as windows, the proper control can be maintained
(Weihe, 19762?).
Strong light has proved to decrease the reproductive ability of
mice (Porter, 1967) and to damage the retina and indirectly the endocrine
system (Fox, 1977; Weihe, 19762?). Therefore, the light should be
diffused through the animal room, with minimum levels of 100 lux at the
level of the cage racks (U.S. Department of Health, Education, and
Welfare, 1974). Hurni (1970) suggests room levels of 300 to 500 lux for
rodents. In the computation of light levels, the effect of opaque or
translucent cage construction should be considered, since this can
greatly affect the level of light reaching the test animal (Clough,
1976).
The duration of the photoperiod should also be controlled, prefer-
ably in a constant pattern (U.S. Department of Health, Education, and
Welfare, 1974; Weihe, 19762?). The daylight cycle should be designed to
suit the test species, for example, 10 h for rats and mice, but 14 h for
cats (Hurni, 1970). In addition to controlling the diurnal pattern, it
is necessary to standardize the time of "day" when blood samples and
sacrifices are made, in order to reduce variation (Fouts, 1976).
2.3.9 Ventilation
Proper ventilation of the testing facility and particularly the
animal room is necessary to maintain low concentrations of atmospheric
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contaminants (e.g., particulates, C02, NHs, moisture, and microorganisms),
reduce odors, regulate temperatures, and promote comfort (Clough, 1976;
U.S. Department of Health, Education, and Welfare, 197A). The ability
to limit odor depends on the species used and the population densities,
as well as the proper design of the facility (U.S. Department of Health,
Education, and Welfare, 1974). The precise pattern of air movements in
each room results from the interaction of convection currents arising
from the test animals, from the air input by the ventilation system,
and from the deflections caused by the cages, racks, and equipment
(Clough, 1976). Nevins (1971) discusses the design criteria needed to
optimize the ventilation patterns. Rooms used by humans and animals
should each be ventilated separately (National Academy of Sciences, 1969;
U.S. Department of Health, Education, and Welfare, 1974). The air
should be changed frequently without causing drafts, and incoming or
recirculated air must be filtered (National Academy of Sciences, 1969;
Hurni, 1970; Sontag, Page, and Saffiotti, 1976). The recommended rates
of air changes per hour include: 6 to 15 (National Academy of Sciences,
1969); 15 to 23 (Hurni, 1970); and 10 to 15 (Shaw, 1976; Sontag, Page,
and Saffiotti, 1976; U.S. Department of Health, Education, and Welfare,
1974). The exact rate will depend on the population density, refuse
removal schedule, test design, and degree of desired comfort (Hurni,
1970; National Academy of Sciences, 1969). Hurni (1970) also suggests
that the air should be negatively charged (about 2000 negative ions per
cubic centimeter) to increase the oxidation of odors and microorganisms.
The use of special ventilation systems for testing facilities
requires some specific precautions. If a "clean-dirty" corridor system
is used, the air pressure in each room should be positive to the "dirty"
corridor and negative to the "clean" one (Sontag, Page, and Saffiotti,
1976). This will help reduce the possibility of backflow contamination.
For a laminar flow system where air is introduced at slow rates of speed
from large wall-sized vents, the room design and equipment placement
must be carefully considered, because turbulence and eddy effects could
disrupt the flow (Shaw, 1976). Other design considerations affecting
air movements and filtration are reviewed by Shaw (1976) and Dymet
(1976).
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2.3.10 Noise and Handling
Noise can be undesirable because of its effects, often underrated,
on test animals and personnel. Because there is always some unavoidable
background noise, it should be considered in designing test facilities
and toxicity studies (Hurni, 1970; U.S. Department of Health, Education,
and Welfare, 1974). The sources of noise are derived from the feeding
and cleaning operations, ventilation equipment, animal vocalizations
(especially dogs and monkeys), and animal/cage contacts (Fletcher,
1976). The damaging effects of noise include the expected auditory
(damage to ear structures and tissues) and nonauditory (stress) effects,
which have been demonstrated in special acute studies (Fletcher, 1976).
However, extrapolation of these effects to chronic laboratory situations
is uncertain because many critical factors such as species auditory
levels and frequency, intensity, and temporal patterns of common noises
are unknown. In general, effects of noise should be minimized, especially
where the exposure could be lengthy or where studies are very delicate
(Fletcher, 1976). Noise can be minimized by the separation of animal
and human occupancy zones, use of soundproofing materials, padding of
equipment, proper personnel training, transferring cleaning chores
outside the housing area, and acclimating the animals to unavoidable
background noises (Fletcher, 1976; Hurni, 1970; U.S. Department of
Health, Education, and Welfare, 1974). If these procedures are followed,
the effect of noise on the test results should be insignificant.
Handling, or contact between the laboratory personnel and the test
animals, can be a detrimental factor that reduces the standardization of
environmental factors (Hurni, 1970). Improper handling may produce
unnecessary stress or injury to the test animal or personnel (Short,
1967). Porter (1967) found that increased handling of mice affected
their reproduction and lactation and caused some reduction in weight
gain. On the other hand, proper handling produces tameness, which is
healthy for the animal and expedient for the researcher. However, even
with increased training of personnel and the use of the proper techniques,
such as those discussed by Short (1967), the chance for differential
treatment exists and should be considered, especially for comparative
studies (Hurni, 1970).
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2.3.11 Personnel
One of the basic factors in the design of a successful husbandry
program is the selection and training of staff personnel. The numbers
and types of personnel utilized and the chain of command developed depend
on the goals and role of each toxicity testing facility (U.S. Department
of Health, Education, and Welfare, 1974). Lane-Petter (1967) outlines
an example of one such staff setup. However, there are certain standards
that all staffs and personnel must meet. Basically the staff must
include animal technicians (usually at least junior and senior tech-
nicians; the number of levels depends on the size of facility) and
veterinary personnel (as supervisors or consultants) (National Academy
of Sciences, 1969). The personnel caring for and monitoring health of
the animals must be trained in the theoretical and practical aspects of
their jobs (Fox, 1977). Usually the theoretical or course work requires
certification by such organizations as the American Association of
Laboratory Animal Science (Fox, 1977) or the Canadian Association for
Laboratory Science Program (Arnold et al., 1977). Typically these
organizations certify personnel on three levels: assistant animal
technician, animal technician, and animal technologist. The individual
facility can then provide the practical onsite training, which ensures
the necessary training flexibility (Fox, 1977). To assist the veter-
inarian or toxicologist in the treatment, test procedures, and assess-
ment roles, veterinary technologists or toxicology technologists are
often employed (Fox,* 1977). By the use of such certified and special
personnel, many of the problems associated with husbandry requirements
can be minimized so that the test results are not affected.
2.3.12 Conclusions
Many husbandry factors contribute to a scientifically proper
toxicity evaluation. The selection of test animals should result in
subjects with standardized life cycle variables, uniform genetic compo-
sition, and, in some cases, controlled levels of health (e.g., SPF
strains). After selection, the animals are usually transported and
should be given time to acclimate before testing. They also should be
quarantined to prevent introduction of disease into the testing facility.
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23
The quarantine is part of a larger disease-control program based on proper
planning and thorough sanitary maintenance procedures. The design of
the testing facility is another variable that can contribute to the
success of a toxicity evaluation. A proper testing facility should
include separate human and animal areas, specialized laboratories for
maintenance and assessment of the animals, a supply-receiving area,
a quarantine area, and an incinerator. The design should also provide
for proper environmental controls. The cage design, especially solid
plastic models, affects the microclimate and thereby the toxicity
potential of the test chemical. Bedding material can be another source
of nontreatment toxicity and should not be composed of fresh softwoods
or hardwoods with high tannic acid content. The temperature and humidity
of the testing environment should be controlled within a range of 65° to
85°F and 30% to 70%, depending on the species, with only gradual changes.
The available light should be regulated to provide diffuse levels with a
standard photoperiod duration. Ventilation and filtration of the housing
atmosphere can control many other environmental factors and is often
designed to reduce contamination factors (e.g., "clean-dirty" corridor
system). Noise and handling variables can increase animal stress and
may invalidate the test results. The correct combination of well-
trained personnel likewise minimizes test interference. Only by control
of all these husbandry variables will the test results accurately reflect
the toxicity of the test chemical.
2.4 DIET
2.4.1 Introduction
The major issues necessary to the discussion of diets of laboratory
animals include general nutritional adequacy of diets, types of diets
available (commercial, open-formula, semisynthetic), and the presence in
the diet of toxic contaminants. Also important, in the context of this
document, are the effects of diet on the response of experimental animals
to toxic substances.
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24
2.4.2 Dietary Requirements for Laboratory Animals
Basic dietary requirements are similar for various common laboratory
animal species (Clarke et al., 1977). All require proteins, carbohydrates,
fats, minerals, micronutrients, and energy.
Dietary proteins are the source of the amino acids that an animal
requires to build its own proteins. The protein content of the diet
should ideally contain, in the correct proportions, all the "essential"
amino acids (those not formed at all in the animal's own tissues or not
formed at a rate fast enough to satisfy demand) (Clarke et al., 1977).
Table 2.3 is a summary of the attempts of various investigators to
determine the relative proportions in which the essential amino acids
are required by the growing rat (Coates et al., 1969). The composition
of two proteins, hen's egg and human milk, are also presented. These
are both known to be 100 percent utilizable by the rat and are assumed
to have practically ideal proportions of the essential amino acids.
Since the amino acid requirements for mice and rats seem to be similar
a single "target pattern," given in the last column of Table 2.3, is
proposed for both species. Clarke et al. (1977) suggested that the same
target pattern would be satisfactory for all common laboratory animals.
Carbohydrates are the major energy source in most diets for labora-
tory animals. It has been shown that energy requirements for maintenance
are closely related to basic metabolic rate; therefore, the requirement
for energy is higher during pregnancy, lactation, and periods of rapid
growth.
Lipids in the diets of laboratory animals are also a source of
energy and are necessary for utilization of fat-soluble vitamins.
Essential fatty acids are required for synthesis of tissue and cell
components.
Other general dietary requirements for laboratory animals include
vitamins A, D, E, K, B, and C and choline. Essential inorganic elements
or minerals include electrolytes (sodium, potassium, calcium, magnesium,
chloride, and phosphate), which contribute to ionic and osmotic balance
between cells, tissue fluids, and plasma. Minor and trace elements
include iron, copper, zinc, magnesium, iodine, cobalt, manganese, and
selenium, which take part in intracellular metabolic processes as
metalloprotein complexes and coenzymes.
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Table 2.3. Requirements for essential amino acids expressed as percentage of protein
Rat
Mouse
Human
milk
Whole
egg
Reference0
Arginlne
Histidine
Lysine
Tyrosine
Tryptophan
Phenylalanine
Methionine -f cystine
Threonine
Leucine
Isoleucine
Valine
1
4.0
10.1
1.0
4.6
7.5
5.0
8.0
5.0
7.0
2
5.0
2.5
9.0
3.0
1.1
4.2
5.0
5.0
7.0
5.5
5.5
3
9.0
2.9
11.8
1.0
7.7
5,4
3.5
7.3
4.8
5.4
4
1.8
5.2
1.0
4.9
4.7
4.1
7.8
4.3
5.0
567
4.6
2.0 2.0
3.7 6.5
4.7
1.5 1.3
6.3
4.4 4.6
2.6 4.2
9.5 8.2
5.1 5.0
6.2
1
2
7
1
7
5
4
6
4
5
8 9
.7
.5
.5
.3
.5
.0 3.0
.2
.7
.2
.8
10
1.9
3.4
9.0
4.8
1.9
14.0
8.0
6.8
12.5
8.7
9.7
11
4.0
2.2
6.4
5.7
1.7
4.8
4.4
4.6
9.2
6.6
6.7
11
6.6
2.4
6.4
4.2
1.6
5.8
5.5
5.1
8.8
6.6
7.3
"Target
pattern"
5.0
2.5
6.0
4.0
1.5
5.0
4.5
4.0
8.0
5.0
5.5
aHef. 1 — Rose, W. C., M. J. Oesterling, and M. Womach. 1948. Comparative Growth on Diets Containing
Ten and Nineteen Amino Acids with Further Observations upon the Role of Glutamlc and Aspartic Acids. J. Biol.
Chem. 176:753. Ref. 2 — Rama Rao, P. B., V. C. Metta, and B. C. Johnson. 1959. The Amino Acid Composition
and the Nutritive Value of Proteins. I. Essential Amino Acid Requirements of the Growing Rat. J. Nutr.
69:387. Ref. 3 — Rogers, Q. R., and A. E. Harper. 1964. Amino Acid Diet for Rats. Fed. Proc. Fed. Am. Soc.
Exp. Biol. 23:186. Ref. 4 - Bender, A. E. 1961. Determination of the Nutritive Value of Proteins by
Chemical Analysis. In: Progress in Meeting Protein Needs of Infants and Preschool Children. Publ. Natl.
Res. Coun., Wash. 843:407. Ref. 5 — Fisher, R. B. 1954. Protein Metabolism. London: Methuen. Ref. 6 —
HcLaughlin, J. M., and W. I. Illman. 1967. Use of Free Plasma Amino Acid Levels for Estimating Amino Acid
Requirements for the Growing Rats. J. Nutr. 93:21. Ref. 7 — Hartsook, W. E., and H. H. Mitchell. 1956. The
Effect of Age on the Protein and Methionine Requirement of the Rat. J. Nutr. 60:173. Ref. 8 — National
Research Council. 1962. Nutritional Requirements of Domestic Animals, Number 10: Nutritional Requirements
of Laboratory Animals. National Research Council, Washington. 990. Ref. 9 — Leveille, G. A.,
H. E. Sauberllch, and J. W. Shockley. 1961. Sulfur Amino Acid Requirements for Growth of Mice Fed Two Levels
of Nitrogen. J. Nutr. 75:455. Ref. 10 — Bauer, C. D., and C. P. Berg. 1943. The Amino Acids Required for
Growth in Mice and the Availability of the Optical Isomers. J. Nutr. 26:51. Ref. 11 — FAO. 1965. Protein
Requirements. FAO Nutr. Mtg. Rep. Ser. 37.
Source: Coates et al., 1969.
CO
Ui
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26
In selecting the proper diet the investigator must bear in mind
that basal dietary requirements, as well as specific nutritional require-
ments of animals, may vary with species or strain and with age, sex,
gestation, lactation, disease, season of the year, temperature, relative
humidity, ventilation, unknown internal physiological mechanisms, and
stress (of experimentation) (Hughes and Lang, 1978). Specific require-
ments for the various species of laboratory animals are discussed in
detail by Clarke et al. (1977) and in the National Academy of Sciences —
National Research Council (1972) document Nutrient Requirements of
Laboratory Animals.
2.4.3 Types of Diets for Laboratory Animals
In choosing a feed one should consider: (1) constancy of its major
ingredients and their sources; (2) timely delivery; (3) moisture content;
(4) freshness; (5) storage characteristics (Sontag, Page, and Saffiotti,
1976); and (6) purity.
Many commerical diets are available for laboratory animals. The
cost of a diet is a small part of the overall cost of bioassays, and the
investigator should buy the best diet to ensure long-term survival under
optimal nutritional conditions (Sontag, Page, and Saffiotti, 1976;
Weisburger, 1976).
The goal in testing must be to standardize the diet in and among
animal studies so that data can accurately be compared on a worldwide
level. Therefore, in the formulation of a standard diet international
availability of the ingredients should be considered.
The Food Safety Council (1978) recommends the use of standardized
diets in accordance with guidelines of the National Academy of Sciences'
1972 Subcommittee on Laboratory Animal Nutrition and 1978 Committee on
Laboratory Animal Diets.
Laboratory animal diets have been characterized by the American
Institute of Nutrition (1977) as:
Cereal-based diet Diet formulations composed predominantly of
Unrefined diet unrefined plant and animal materials that may
Nonpurified diet contain added vitamins or minerals — stock
diet, laboratory chow, etc.
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27
Open-formula Diet in which precise percentage composition
of each ingredient is available either in the
published scientific literature or in available
commercial information. No changes are per-
mitted in type or amount of ingredients.
Closed-formula Diet in which exact composition by type and
amount of each ingredient is not disclosed by
the manufacturer. Combination of ingredients
may change with market conditions.
Purified diet Diets composed primarily of refined ingredients
such as commercially refined proteins, carbo-
hydrates, and fats with vitamins and minerals
added. (Semisynthetic.)
Chemically defined Diets characterized by the nitrogen source being
diet provided by pure amino acids, carbohydrates from
refined mono- or disaccharides, and fats from
purified fatty acids or triglycerides. Minerals
are reagent grade, and vitamins are of high
purity.
Use of open-formula diets is encouraged by the Food Safety Council
(1978) and the American Institute of Nutrition (1977) and in Guidelines
for Carcinogen Bioassay in Small Rodents (Sontag, Page, and Saffiotti,
1976).
The open-formula, cereal-based diet (NIH-07) proposed by the
American Institute of Nutrition (1978) was developed by the National
Institute of Health in 1972 and is shown in Table 2.4. The NIH-07 open-
formula rat and mouse diet has been found to be satisfactory for repro-
duction, lactation, and maintenance of the animals. It is produced by
several feed manufacturers and is priced competitively with commercial
diets (American Institute of Nutrition, 1977). In spite of high cost,
the semisynthetic diet may be preferred for some studies (Page, 1977).
For example, practical experience has shown that semisynthetic diets
could be of use in subchronic studies, although they have not been
particularly satisfactory in chronic reproduction studies (Food Safety
Council, 1978).
The purified diet AIN-76 and the vitamin and mineral mixtures used
in this diet are shown in Tables 2.5, 2.6, and 2.7. This diet supports
growth, lactation, and reproduction in rats and mice comparable to the
NIH-07 diet (American Institute of Nutrition, 1977).
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28
Table 2.4. NIH-7 open-formula rat and
mouse ration0-
Ingredient %
Dried skim milk 5.00
Fish meal (60% protein) 10.00
Soybean meal (49% protein) 12.00
Alfalfa meal (dehydrated, 17% protein) 4.00
Corn gluten meal (60% protein) 3.00
Ground No. 2 yellow shelled corn 24.50
Ground hard winter wheat 23.00
Wheat middlings 10.00
Brewer's dried yeast 2.00
Dry molasses 1.50
Soybean oil 2.50
Sodium chloride 0.50
Dicalcium phosphate 1.25
Ground limestone 0.50
0.25
Total 100.00
Calculated proximate composition — crude
protein, 23.5%; crude fat, 5.0%; crude fiber, 4.5%;
ash, 7.0%.
Vitamin and mineral premixes shall provide per
kilogram of diet: vitamin A (stabilized), 6050 IU;
vitamin Da, 5060 IU; vitamin K, 3.1 mg; o-tocopheryl
acetate, 22 IU; choline 0.6 g; folic acid, 2.4 mg;
niacin, 33 mg; d-pantothenic acid, 20 mg; riboflavin,
3.7 mg; thiamin, 11 mg; vitamin B-12, 4.4 yg;
pyridoxine, 1.9 mg; biotin, 0.15 mg; cobalt, 0.44 mg;
copper, 4.4|mg; iron, 132 mg; manganese, 66 mg; zinc,
18 mg; iodine, 1.5 mg.
Source: American Institute of Nutrition, 1977.
Reprinted with permission of the publisher.
For special circumstances, controlled variation of experimental
diets to simulate the variety of human diets has been suggested by the
World Health Organization (1978) and the National Academy of Sciences
(1975). For example, animal diets could be shifted to a higher fat
content to allow for the expression of some cocarcinogenicity of fats or
to a lower protein content to simulate human deficiency states.
Regardless of the type of diet chosen, all test animals and their
corresponding controls should be maintained on identical diets, and when
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29
Table 2.5. AIN-76® purified d±eta»b (for rats and mice)
Ingredient %
Casein0 20.0
DL-Methionine 0.3
Cornstarch 15.0
Sucrose 50.0
Fiber** 5.0
Corn oile 5.0
AIM mineral mix/ 3.5
AIN vitamin mix 1.0
Choline bitartrate 0.2
Total 100.0
Trademark pending.
This diet is intended for growth and maintenance dur-
ing the first year of life. Investigators should be aware
that diets high in sucrose can be cariogenic and that some
strains of rats fed such diets may develop kidney lesions
after extended periods. The diet has been found to be sat-
isfactory for reproduction and lactation in both rats and
mice. If used for deficiency studies, modifications will be
necessary. If used in ultraclean environment, several trace
elements should be added (Federation Proc. 33, 1748-1773,
1974). The diet can be pelleted satisfactorily, if desired,
by addition of water (no binder).
Feed-grade casein having at least 85% protein.
Cellulose-type fiber.
a
Some commercial corn oils contain antioxidants (maximum
0.02%) and a surfactant (dimethyl silicone). These additives
should be innocuous for most nutritional studies, but inves-
tigators should be aware of their presence. It is recom-
mended that an oil with added antioxidant be used to prevent
rancidity. Diet should be stored at 4°C or colder, and it
is recommended that the diet not be kept longer than
4 months.
f
•"The total dietary content of some minerals, due to
their presence in casein, will be slightly higher and will
vary according to the casein used.
Source: American Institute of Nutrition, 1977.
Reprinted with permission of the publisher.
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30
Table 2.6. AIN-76 vitamin mixture0
„.„ . Per kilogram
Vitamin ,. . e
of mixture
Thiamin-HCl 600 mg
Riboflavin 600 mg
Pyridoxine-HCl 700 mg
Nicotinic acid* 3 g
D-Calcium pantothenate 1.6 g
Folic acid 200 mg
D-Biotin 20 mg
Cyanocobalamin (vitamin B-12) 1 mg
Retinyl palmitate or acetate *°
(vitamin A)
dl-a-Tocopheryl acetate 4**
(vitamin E)
Cholecalciferol (vitamin D3) 2.5 mge
Menaquinone (vitamin K)J 5.0 mg
Sucrose, finely powdered To make
1000.0 g
Based on the National Academy of Sciences-
National Research Council recommended levels for
rats (National Academy of Sciences-National
Research Council, 1972). To be used at 1% of diet.
Nicotinamide is equivalent.
CAs stabilized powder to provide 400 000 IU
vitamin A activity or 120 000 retinol equivalents.
As stabilized powder to provide 5000 IU
vitamin E activity.
e!00 000 IU. May be in powder form.
f
•'Menadione.
Source: American Institute of Nutrition, 1977.
Reprinted with permission of the publisher.
a change in diet manufacture occurs, all animals should be changed to
the new diet at the same time (Sontag, Page, and Saffiotti, 1976;
Weisburger, 1976). The diets should be well tried and readily available
to the animals (Magee, 1970).
In addition, feed must be protected from air, light, heat, chemical
fumigants, radiation, and contamination by microorganisms — all of which
may damage the nutrient quality of a diet (Clarke et al., 1977). Sontag,
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31
Table 2.7. AIN-76® mineral mixture*2
,. Grams per kilogram
Ingredient of mixture
Calcium phosphate, dibasic (CaHPO^) 500.0
Sodium chloride (NaCl) 74.0
Potassium citrate, monohydrate 220.0
(K3C6H507.H20)
Potassium sulfate ^SO^) 52.0
Magnesium oxide (MgO) 24.0
Manganous carbonate (43% to 48% Mn) 3.5
Ferric citrate (16% to 17% Fe) 6.0
Zinc carbonate (70% ZnO) 1.6
Cupric carbonate (53% to 55% Cu) 0.3
Potassium iodate (KI03) 0.01
Sodium selenite (Na2Se03-5H20) 0.01
Chromium potassium sulfate 0.55
Sucrose, finely powdered To make
1000.0
Based on the National Academy of Sciences-National
Research Council requirements for rats (National Academy of
Sciences-National Research Council, 1972) . To be used at
3.5% of the diet.
Source: American Institute of Nutrition, 1977.
Reprinted with permission of the publisher.
Page, and Saffiotti (1976) recommend sterilization of feed (without
degrading nutrients) when practical and consistent with the disease
control program of the laboratory.
2.4.4 Analysis for Nutrients and Contaminants
Contaminants in the diet and variations in the concentration of
essential nutrients can influence the response of animals in toxicity
tests and thus alter the interpretation of experimental data (Food
Safety Council, 1978; Fox, 1977; Newberne, 1975).
Variations in the concentration of essential nutrients can occur
for many reasons: batch or lot differences, regional and seasonal
differences, manufacturer variations, and processing. Most diets con-
tain nutrients in quantities sufficient for the growth, maintenance,
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32
and reproduction of a particular species. However, concentrations of
essential ingredients may vary from batch to batch of a formulation made
with different lots of natural ingredients, and at the same time the
guaranteed analysis shown on the label can remain correct (Newberne,
1975).
Differences in manufacturing procedures were pointed out by Porter,
Lane-Fetter, and Home (1963), who compared growth rates of groups of
young mice fed diets produced in the same region by three different
manufacturers and found considerable differences (cited by Hurni, 1970).
Also, nutritional quality may be seriously impaired during processing,
particularly by overheating, which can destroy amino acids or lead to
the formation of indigestible complexes (Clarke et al., 1977).
These examples help to illustrate the need for periodic analysis of
diets for nutrient content, as proposed by the National Research Council
(1977).
Because of the presence of a variety of contaminants in animal
diets it is also advisable to test periodically for pesticides, myco-
toxins, trace minerals, and industrial contaminants (such as polychlorin-
ated biphenyls, lead, and mercury) (Food Safety Council, 1978; National
Research Council, 1977; Newberne, 1975; Page, 1977). Low levels of
tf-nitrosamines have been detected in the diets of experimental animals
in Germany and in the United States (Edwards et al., 1979; Walker,
Castegnaro, and Griciute, 1979). The source of the nitrosamines is
thought to be fishmeal, and Knapka (1979) suggested that the ^-nitrosamines
are present only in diets purchased in meal form.
There is a growing interest in obtaining detailed open-formula infor-
mation for assurance that diets are free from pesticide residues and
other contaminants (Food Safety Council, 1978); if prospective analysis
for contaminants cannot be arranged with feed suppliers, retrospective
analysis could be conducted.
The ultimate solution to the problems of uniform constituents and
toxic contaminants in animal diets may be provided by the use of
rigorously purified semisynthetic diets or entirely synthetic diets
(Clarke et al., 1977; Food Safety Council, 1978).
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33
2.4.5 Effects of Diet on Toxicity Test Results
Nutritional imbalances may influence toxicity through physiological,
immunological, and/or biochemical mechanisms (Campbell and Hayes, 1974).
The main effects, however, can be attributed to the effects of nutritional
state on the drug-metabolizing enzymes located in the endoplasmic
reticulum of the hepatocyte. This enzyme complex, classified as a mixed-
function oxidase, is composed of cytochrome P-450, phosphatidycholine,
and a flavoprotein reductase. A wide variety of drugs and foreign
compounds are metabolized by the complex to products of greater or
lesser toxicity. The response of the mixed-function oxidase system to
toxic substances can be modified by deficiency or excess of dietary
constituents such as protein, carbohydrates, fats, lipotropes, vitamins,
and minerals. Examples of these effects in animals will be reviewed in
the following sections.
2.4.5.1 Protein — The dietary constituent most studied is protein.
Qualitative and quantitative changes in protein content can alter mixed-
function oxidase activities. McLean and McLean (1969) pointed out oppo-
site effects of protein deficiency on the toxicity of compounds that are
detoxified and the toxicity of those that are rendered toxic by biotrans-
formation. For example, protein-deficient diets protect against acute
poisoning from carbon tetrachloride and dimethylnitrosamine in rats
(McLean and McLean, 1966; McLean and Verschuuren, 1969), whereas the
toxicities of most pesticides are increased during protein deficiency
(Boyd, 1969). Protein-deficient rats have become more susceptible to
the toxicity of aflatoxin when administered alone and of chloroform
following enzyme induction with DDT; but chloroform toxicity in a single
oral dose is not altered by protein deficiency (Madhavan and Gopalan,
1965; McLean and McLean, 1969). Severe protein depletion has been
implicated in the production of increased resistance to mercuric chloride
poisoning in rats (Surthin and Yagi, 1958).
Metabolism of a number of carcinogens is affected by dietary protein
content. The carcinogenic action of dimethylnitrosamine has been enhanced
in rats by protein deficiency leading to suppression of microsomal
hydroxylation (Swan and McLean, 1968).
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34
Silverstone and Tannenbaum (1951) demonstrated that the formation
of spontaneous hepatomas was substantially retarded by a decrease (from
18% to 9%) in dietary protein, while increased protein intake (from 18%
to 45%) had no effect on tumor formation. The inhibition was found to
be due to a deficiency in sulfur-containing amino acids in the diet, not
to the difference in the proportion of total protein. Kawachi, Hirata,
and Sugimura (1968) demonstrated that addition of 1% tryptophan to the
diets of rats receiving a low level of #-nitrosodiethylamine increased
the liver cancer incidence almost fourfold. Bryan, Brown, and Price
(1964) reported that L-tryptophan enhanced the induction of bladder
tumors by 2-acetamidofluorene.
In other studies, deficiencies of protein and individual amino
acids have been related to congenital malformations in rats. Early
experiments in reproduction and embryogenesis were reviewed by Kalter
and Warkany (1959).
2.4.5.2 Lipids — Lipids provide energy and essential fatty acids
for synthesis of tissue and cell components. High fat diets sensitize
animals to the toxic effects of chloroform (Goldschmidt, Vars, and
Raudin, 1939).
Marshall and McLean (1969) showed that dietary addition of either
herring oil, linoleic acid, or 0.1% oxidized sitosterol was required for
maximum induction of cytochrome P-450 synthesis.
Rogers et al. (1974) and Rogers (1975) found that liver tumor
induction with various carcinogens was enhanced if the diet contained a
high fat content and was deficient in choline, methionine, and folic
acid. Carroll and Khor (1970) reported that Sprague-Dawley rats on a
high fat diet developed more mammary tumors after administration of
7,12-dimethylbenzanthracene than did rats on a low fat diet.
2.4.5.3 Lipotropes — Lipotropes are required for the synthesis of
phospholipids, which transport triglycerides. Lipotrope deficiency is
characterized by accumulation of triglycerides in the liver. Aflatoxin
has an increased hepatotoxic effect in rats on a low lipotrope diet
(Newberne, Rogers, and Wogan, 1968). Newberne and Rogers (1976) reported
a higher incidence of #-2-fluorenylacetamide- and dimethylbenzanthracene-
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35
induced mammary tumors in Sprague-Dawley rats on a low lipotrope, high
fat diet than in animals on a regular diet. Lipotrope-deficient Fischer
rats, on the other hand, which were resistant to mammary tumor induction
with tf-2-fluorenylacetamide, developed a significantly higher number of
hepatic carcinomas than did controls.
2.4.5.4 Vitamins — The role of vitamin deficiencies in the mixed-
function oxidase system has been reviewed comprehensively by Campbell
and Hayes (1974). The effects do not appear to be as serious as the
effects of protein deficiencies, although activities are affected to
some degree if the vitamin deficiencies are severe enough. The influence
of vitamins on the production of congenital malformations has been
reviewed by Kalter and Warkany (1959).
Reports of the effects of vitamin A on experimental tumor induction
are conflicting. Saffiotti et al. (1967) and Cone and Nettesheim (1973)
demonstrated inhibition by vitamin A of the induction of squamous
metaplasia and squamous cell tumors of the respiratory tract of rats and
hamsters with polycyclic hydrocarbons. However, Smith et al. (1975) and
Smith, Rogers, and Newberne (1975) reported enhancement by vitamin A of
the induction of respiratory tract tumors by benzo(a)pyrene.
2.4.5.5 Minerals — Deficiencies in dietary trace minerals, such as
iron, iodine, zinc, selenium, and copper, and their effects on the
mixed-function oxidase system have been reviewed by Campbell and Hayes
(1974). Moffitt and Murphy (1974) have shown that the trace mineral
concentration can influence the metabolism, distribution, and toxic
action of chemicals. The interactions between nutritional minerals and
toxic substances may be of a deleterious or protective nature (Brinkman
and Miller, 1961; Farkas, 1978). For example, Brinkman and Miller
observed that toxicity to molybdnenum was increased in rats whose diets
contained higher amounts of zinc than usual. Farkas, however, suggested
that selenium may play a role in decreasing the toxicity of cadmium and
mercury through its interactions with those metals.
2.4.6 Conclusions
Diets of experimental animals should contain proteins, carbohydrates,
fats, minerals, micronutrients, and energy in the proportions suitable
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36
for a particular species or strain. Various types of diets available
include nonpurified or cereal based, open-formula, closed-formula, puri-
fied or semisynthetic, and chemically defined.
There is growing concern regarding contaminants in the diet and
variations in the concentrations of essential nutrients and their con-
sequent effects on the response of test animals to toxic substances.
Periodic testing of diets for nutrient concentration and contaminants
has been proposed, but the ultimate solution to the problem may be
provided by the use of synthetic or semisynthetic diets.
2.5 PATHOLOGY
2.5.1 Introduction
The importance of proper pathology examination and reporting tech-
niques cannot be overemphasized (Prieur et al., 1973). They must be
planned before the start of the experiment because they influence the
test design and the selection of the animal models (Page, 1977). Quality
control must be emphasized at all times, because important lesions may
be lost at any of several steps: necropsy, trimming, fixation, paraffin
blocking, or slide preparation (Page, 1977). With the proper techniques
and quality control, a pathology workup gives information on the morphol-
ogy of the chemically induced lesions present at that time and some
indication of a dose-effect relationship (Zbinden, 1976). However, the
limitations of pathology must be recognized, including a lack of informa-
tion on early lesions that disappear before necropsy, the sequence of
lesion appearance, the reversibility of lesions, and the developmental
history (morphogenesis) of each lesion (Zbinden, 1976). The following
subsections will discuss the procedures that should be used to obtain
the desired pathology information.
2.5.2 Gross Examination
The gross examination or necropsy is the first step in a pathology
evaluation and the most influential factor in determining the toxic
effects of a chemical. Unless it is done properly, much valuable infor-
mation will be lost. Therefore the necropsy should either be performed
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37
directly by the pathologist (or the scientist who is to do the micro-
scopic examination) or by a trained technician with consultation from
the pathologist (Food Safety Council, 1978; Magee, 1970; Prieur et al.,
1973; Sontag, Page, and Saffiotti, 1976; U.S. Food and Drug Administra-
tion, 1971; World Health Organization, 1978). An extensive necropsy
(including blood counts, bone marrow smears, and organ weights) is a
desirable objective, but usually the design must be varied for each
experiment (Roe, 1965).
To ensure that the maximum information is obtained from the test
animals, a daily observation should be included to reduce loss from
postmortem degeneration (Magee, 1970; Roe, 1965). The necropsy should
be done as soon as possible after death and, if not, the animal should
be placed in a refrigerator for storage (Prieur et al., 1973). Even if
some tissue degeneration has occurred, a necropsy should be performed,
because it can indicate the general lesions to be expected (Magee, 1970;
World Health Organization, 1978). If an animal is moribund, sacrifice
is often required and should be determined based on a standard set of
criteria or on the experience of the observer (Magee, 1970). However,
the number of animals sacrificed should not exceed the short-term capac-
ity of the examiners (World Health Organization, 1978).
The organs examined should all be identified regardless of the
results (Roe, 1965). It is often easiest and most thorough to use a
checklist system (U.S. Food and Drug Administration, 1971; World Health
Organization, 1978). The examination of each organ should be performed
in a standard procedure, with the same slices and angles examined for
every test animal (Roe, 1965). During the examination, all gross
lesions observed should be recorded and described, including information
on the size, location, number, shape, color, and texture (Sontag, Page,
and Saffiotti, 1976; World Health Organization, 1978). The weight of
the organ can also be determined during a gross examination, but the
results are of doubtful significance for lifetime studies (Magee, 1970).
If weight is to be determined, it should be done as soon as possible to
reduce drying effects (World Health Organization, 1978).
The necropsy should start with an external examination including
orifices (Sontag, Page, and Saffiotti, 1976; World Health Organization,
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38
1978). Next, examine all the internal cavities and organs in situ and
do not separate the connective tissue until it also has been examined
(Prieur et al., 1973). Table 2.8 lists some tissues that have been sug-
gested for examination. Special procedures for individual organs have
also been recommended: (1) parenchymal and endocrine glands should be
thoroughly examined, including multiple cut slices, since these tissues
often contain deep-seated lesions (Sontag, Page, and Saffiotti, 1976;
Food Safety Council, 1978); (2) all hollow organs, including the urinary
bladder, gastrointestinal tract, and respiratory tract, should be cut
open and extensive examination of the mucosal surfaces performed (Food
Safety Council, 1978; Sontag, Page, and Saffiotti, 1976; World Health
Organization, 1978); (3) the skull and brain should be cut open and
examined, including examination of the pituitary (Magee, 1970); and (4)
Table 2.8. Tissues to be included in a
gross examination
Gross lesions Heart
Tissue masses or suspect tumors Thyroids
and regional lymph nodes Parathyroids
Skin Esophagus
Mandibular lymph node Stomach
Mammary gland Duodenum
Salivary gland Jejunum
Larynx Ileum
Trachea Spleen
Cecum Kidneys
Colon Adrenals
Rectum Bladder
Mesenteric lymph node Seminal vesicles
Liver Prostate
Thigh muscle Testes
Sciatic nerve Ovaries
Sternebrae, vertebrae, or femur Uterus
(plus marrow) Nasal cavity
Costochondral junction, rib Brain
Thymus Pituitary
Gallbladder Eyes
Pancreas Spinal cord
Lungs and bronchi
Source: Adapted from Sontag, Page, and Saffiotti,
1976.
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39
the spinal cord in rodents is best examined in situ (World Health
Organization, 1978) and often is not routinely examined (Magee, 1970).
In carcinogenicity studies, the gross examination is even more
important than usual because much of the lesion incidence data are gen-
erated during this procedure (Food Safety Council, 1978). To evaluate
the effectiveness of the necropsy to detect lesions, Frith et al. (1979)
and Kulwich et al. (1980) correlated observations of the gross and
microscopic examinations in carcinogenicity studies. Table 2.9 shows
how frequently the gross examination detected lesions also seen in
microscopic examinations in mice. Frith et al. (1979) concluded that
the size of the organ, the stage and nature of the lesion, and the
number of slices examined all affect the efficiency of the gross examin-
ation. Kulwich et al. (1980) found correlation rates between gross and
microscopic examinations of 70% and 76% for two chemicals in rats. They
concluded that gross examination is less successful for small organs,
because a single histological slice represents a larger portion of the
organ and can often find lesions not seen grossly. However, for tissues
like the skin and mammary gland, the gross examination is much more
successful. They also found that the size and nature of the lesions
affected the gross detection.
Table 2.9. Correlation between gross and microscopic lesions in
carcinogenic studies in mice
Organ
Liver
Thymus
Spleen
Mammary tissue
Uterus (adenocarcinomas)
Uterus (polyps)
Lung
Adrenal gland
Harderian gland
Testes
Pituitary gland
Lesions
seen in
gross exam
5 550
725
6 589
6 503
50
2 423
4 503
711
1 438
330
486
Lesions seen
microscopically
5 968
1 480
8 340
6 993
81
6 922
10 007
1 776
4 637
402
836
Percentage
found in
gross
93
49
79
93
62
35
45
40
31
82
58
Source: Adapted from Frith et al., 1979.
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40
2.5.3 Tissue Preservation and Storage
After the gross examination, tissues should be excised and preserved
to prevent further degeneration. This should be done as quickly as
possible, since degeneration can occur quite rapidly, especially with
mice (Magee, 1970). Several techniques for preservation are available,
with immersion being the most common (World Health Organization, 1978).
However, for some tissues (lung and central nervous system) and for
electron microscopy, perfusion is preferred. This involves draining the
blood, followed by injection of a proper fixative (World Health Organi-
zation, 1978). Perfusion cannot be used if organ weights are needed
(World Health Organization, 1978).
The fixative most recommended is neutral buffered formalin at
concentrations of 10% (Prieur et al., 1973; Sontag, Page, and Saffiotti,
1976; World Health Organization, 1978) or 4% (Zbinden, 1976). Special
preservatives are often required (e.g., Bouin's solution for paraffin-
embedded material) depending on the stain to be used and the tissue
involved (Prieur et al., 1973; Zbinden, 1976). The tissues should be
preserved at a maximum thickness of 0.5 cm (Prieur et al., 1973; Sontag,
Page, and Saffiotti, 1976; World Health Organization, 1978). Some tis-
sues, like the testicles, should first be immersed intact and then
sliced in 0.5-cm sections for further preservation (Zbinden, 1976). The
tissue/preservative ratio on a volume basis should be greater than 1:10
(World Health Organization, 1978) and preferably 1:15 to 1:20 (Prieur
et al., 1973). The tissues should be left in the preservative for at
least 24 h, and up to 72 h has been suggested (Prieur et al., 1973;
Sontag, Page, and Saffiotti, 1976; World Health Organization, 1978).
Often special procedures are suggested, including: covering tissues
that float with absorbant material (e.g., cheesecloth) for uniform
preservation (Prieur et al., 1973); skin, stomach mucosa, and nerves
need to be straightened out and often attached to a card or filter paper
to prevent crumpling (Prieur et al., 1973; World Health Organization,
1978; Zbinden, 1976); several thoracic-lumbar vertebrae should be fixed
in situ with the spinal cord (Sontag, Page, and Saffiotti, 1976); urinary-
bladder should be filled with fixative (Zbinden, 1976); and multiple
representative samples of variable tissue masses should be fixed (Sontag,
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41
Page, and Saffiotti, 1976). After preservation, the material should be
stored in plastic bags or jars and kept at least until the final con-
clusions are made for the study (Food Safety Council, 1978).
2.5.4 Trimming, Staining, and Embedding of Tissue
The preparation of tissues for microscopic evaluation includes
techniques for trimming, staining, and embedding. The tissues should be
sampled from the same specific sites for each organ (Prieur et al.,
1973), and the slices should be made so that the cut surfaces represent
the maximum possible area for examination (World Health Organization,
1978). Often a set trimming schedule should be used. The trimming
should be performed by or in the presence of the pathologist, with con-
sideration of the gross examination observations (Sontag, Page, and
Saffiotti, 1976). The tissues should be trimmed to a thickness of 2 to
3 mm for histologic processing (Sontag, Page, and Saffiotti, 1976; World
Health Organization, 1978). Specific recommendations for trimming of
certain organs have been made by the National Cancer Institute (Sontag,
Page, and Saffiotti, 1976) and the World Health Organization (1978).
These recommendations include: (1) multiple portions of large masses or
tumors should be submitted with some of the normal surrounding tissue;
(2) parenchymal organs (e.g., liver) should be sliced to give the
maximum observable area; (3) the kidneys should be sampled through the
cortex and medulla, one by a midlongitudinal section and the other by a
midtransverse section; (4) the lungs should be sectioned transversely
(parallel to the body axis) including the bronchi and carina; (5) at
least three brain cross sections are needed, with one through the
frontal cortex and basal ganglia, one through the parietal cortex and
thalamus, and one through the cerebellum with the pons; (6) the hollow
organs should be trimmed to include a cross-section slide from mucosa to
serosa; (7) if the larnyx is to be examined, the section should also
include the pharnyx; and (8) if the nasal cavity is to be examined,
three transverse sections are needed.
Routinely, the trimmed slices should be stained with hematoxylin
and eosin and embedded in paraffin (Zbinden, 1976). These are sliced to
4 to 6 ym for microscopic slides (Sontag, Page, and Saffiotti, 1976;
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42
World Health Organization, 1978). The paraffin embedding can be per-
formed using automation techniques. Often semithin slices (1 ym) are
needed for such organs as the bone marrow, kidney, or endocrine glands
(World Health Organization, 1978). Special stains are often employed,
for example in evaluations for the presence of fats, carbohydrates,
or special structures (World Health Organization, 1978; Zbinden, 1976).
These can in turn necessitate special preservation techniques. Obviously,
to obtain the maximum information from the ensuing microscopic evaluation,
proper planning prior to the testing is necessary, so that the special
techniques for tissue preservation, staining, and embedding can be
coordinated.
2.5.5 Microscopic Examination
The microscopic examination of properly prepared tissue slides is
usually the final step in a pathology evaluation. Although its importance
is overwhelmingly accepted, the details of the design are quite variable,
depending on the type of toxicity test employed. The main questions are
which tissues to examine, for how many animals and levels, and which
evaluation techniques to use.
Many recommendations have been made concerning the organs that
should be examined. Section 4.5 and Sect. 5.6.3 review some of the
specific literature discussions for individual toxicity tests. In
general, the choice depends on the scope of the test and is decided by
the pathologist (Magee, 1970). The primary problem is a conflict between
the desire to obtain the maximum amount of scientific information, and
the limits imposed by practical economic and time constraints. Therefore,
many discussions recommend only vague guidelines, although others are
extremely specific and encompassing (Page, 1977). As an attempt to
reach a compromise solution, Zbinden (1976) recommended a priority
system based on the frequency of morphological changes that are likely
to occur (Table 2.10). This system addresses both the specific organs
to be examined and the test animals from each dose level from which the
organs are to be taken. It is obvious that, with the number of chemicals
to be evaluated, the microscopic examination will have to be based on
some similar type of ranking system.
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43
Table 2.10. Organs and tissues to be examined in
routine toxicity tests
First Priority: All animals including controls
Liver (f)a
Kidney (f)
Adrenal glands (£)
Heart (left ventricle) (f)
Spleen (f)
Thymus
Testis
Epididymis
Lung
Bone marrow
Mesenterial lymph node
All organs showing gross changes
of shape, weight, color, or
structure
Second Priority: High dose animals and part of controls. Medium
and low dose animals only when significant changes observed in
high dose group
Thyroid
Parathyroid
Pituitary gland
Salivary glands
Stomach
Duodenum
Small intestine
Large intestine
Pancreas
Ovary
Uterus
Cervix uteri
Vagina
Seminal vesicles
Prostate
Coagulating gland
Tonsils
Brain
Spinal cord
Eye
Optic nerve
Peripheral nerve
Urinary bladder
Skin
Mammary gland
Bone-cartilage
Skeletal muscle
Gallbladder
Third Priority: Organs and tissues not examined in routine
experiments, unless indicated by clinical observations or motivated
by scientific interest
Heart valves
Purkinje fibers
Aorta and other blood vessels
Thoracic duct
Tongue
Teeth
Lips
Gingivae
Hard palate
Nasopharynx
Larynx
Trachea
Esophagus
Vermiform appendix
Rectum
Anus
Ureter
Urethra
Oviduct
Vulva
Penis
Paraurethral and preputial glands
Pineal gland
Spinal ganglions and roots
Sympathetic ganglions and trunk
Nerve fiber endings
Meninges
External ear
Middle ear
Inner ear
Olfactory organ
Subcutaneous lymph nodes
Tendons
Adipose tissue
Intervertebral disc
Synovial membrane
Lacrimal glands
(f) — frozen sections stained for fat obligatory.
Source: Adapted from Zbinden, 1976. Reprinted by permission
of the publisher.
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44
Another approach to reducing the amount of microscopic examination
has been proposed by Fears and Douglas (1977; 1978). On the basis of a
statistically valid sampling method, they suggest examination of all of
the treatment groups first and then limited examination of the controls.
The reduction occurs by limiting examination of the control groups to
tissues that were found to be affected in the treatment groups. They
also suggest several further modifications of this system, which result
in even less pathology. One involves random sampling of the treatment
groups based on gross observations to reduce pathology in those groups.
Another incorporates techniques to allow for "blind" evaluations where
the potential biases of the investigator are controlled. These approaches
can reduce the microscopic examination by 10% to 50%. However, Kulwich
et al. (1980) and Frith et al. (1979), in their evaluations of the
correlation between the gross and microscopic examinations, found that
one cannot rely totally on the gross examination to select affected
tissues for microscopic study. They stressed that a thorough microscopic
examination is still necessary. Therefore, the approach suggested by
Fears and Douglas still needs more refinement, but represents a potential
solution for reducing the amount of microscopic examination.
No matter what organs are examined microscopically, the information
will have to be evaluated and presented in a uniform manner. It is
often necessary to use a checklist to ensure that all intended organs
were examined (World Health Organization, 1978). Also, standard criteria
should be developed and reported for the descriptions and classifications
of the lesions, especially if these are done semiquantitatively (World
Health Organization, 1978). The formation of tumor tables incorporating
information on the group, organ, and sex variables for each lesion can
be useful for comparing chemical effects (World Health Organization,
1978).
The manner in which the slides are examined is also an issue of
debate. It has been suggested that the slides should be examined by the
pathologist without knowledge of the treatment or control group repre-
sented (U.S. Food and Drug Administration, 1971). This "blind" evaluation
would reduce possible biases introduced by the pathologist and produce
more objective conclusions (Fears and Schneiderman, 1974, as cited in
Page, 1977). However, many other reviewers stress that the danger of
-------
45
missing lesions or incorrectly interpreting the results far outweighs
bias problems, so that the pathologist should be aware of the sample
group (Roe, 1965; World Health Organization, 1978; Zbinden, 1976). The
Food Safety Council (1978) suggests a compromise situation where the
control groups are evaluated first and the treatment groups are done
"blindly." In any case, maximum use should be made of the results of
prior clinical studies and gross examinations (Food Safety Council,
1978; World Health Organization, 1978; Zbinden, 1976).
2.5.6 Conclusions
Pathology is extremely useful in toxicity testing and can provide
information on the chemically induced lesions present at the time of
sacrifice and any dose-effect relationship. To obtain a useful pathology
evaluation several requirements must be met: (1) the pathologist should
perform or supervise all steps in the protocol; (2) the gross necropsy
should be extremely thorough and include every animal in the study; (3)
the tissues should be properly preserved in an accepted fixative immedi-
ately after the necropsy; (4) the staining, embedding, and sectioning of
the tissues must be well planned and coordinated, particularly if
special stains are utilized; and (5) the microscopic examination should
be as thorough as practical limitations will allow. Only if these
requirements are met will the pathology contribute meaningful data for
evaluations of toxicity.
2.6 PHARMACOKINETICS
2.6.1 Introduction
Pharmacokinetics is the quantitative and qualitative description
of the time course of absorption, distribution, metabolism, and elimina-
tion of an agent in an intact animal (Young and Holson, 1978). The
following sections will examine some of the basic concepts of pharmaco-
kinetics and show how they can be used to supplement the routine
toxicological data and aid in the hazard evaluation of chemicals.
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46
2.6.2 Basic Concepts
Withey (1978) describes absorption, distribution, metabolism, and
elimination as the four major rate processes that are involved when a
substance is administered to a human or animal subject. He further
describes these as follows:
1. absorption — to the bloodstream from the site of administration;
2. distribution — to organs, tissues, storage depots, and receptor
sites;
3. metabolism — usually in the liver but in other organs also; and
4. elimination — mainly via the urine or feces but also via the
lung, from sweat and skin, and into hair.
Gehring, Watanabe, and Blau (1976) present a modification of a
graphical illustration published by Garrett in 1971 of a hydraulic
system used as an analogy of a three-compartment body model portraying
the general concepts of pharmacokinetics (Fig. 2.1). The amount of
chemical corresponding to the original dosage in the blood before any
distribution to other tissue compartments is represented by D , whereas
the amount of chemical at any given time in the vascular compartment is
represented by A . In both the a and b parts of Fig. 2.1, the amount of
chemical in the vascular compartment (A ) has equilibrated with the
amount of the chemical in the shallow or rapidly equilibrating tissue
compartment (T) but not with the amount of chemical in the deep-seated
or slowly equilibrating tissue compartment (2"); 2" is the maximum
in£i,x>
amount of chemical that will exist in the deep-seated tissue compartment.
The amount of chemical eliminated by the lungs and urine or metabolized
is represented by E and is equal to D - (A + T + 2"). As time
approaches infinity, E -+ E<» = D .
Gehring, Watanabe, and Blau (1976) define a compartment in the body
as those organs, tissues, and cells for which the rates of uptake and
clearance of a chemical are so similar as to not permit resolution. The
rapidly equilibrating compartment can be compared to those tissues with
a profuse compartment blood supply, whereas the slowly equilibrating
compartment can be compared to those tissues such as fat and bone that
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47
ORNL-DWG 80-18285
1
\
*
E°
r
^^
•-
>
i
|DO
1
1
I Av
1
T
? kF
— e fq
o
0
0
o
o
o
0
o
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T'
'MAX
T'
1
(a)
Av
J
T'
'MAX
T'
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TB-
O
O
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Fig. 2.1. Hydraulic analogy of three-compartment body model. Do
is the original dose in the vascular compartment prior to any equilibra-
tion at any time. AV is the amount in the vascular compartment at any
time. In both (a) and (b), Av has equilibrated with the shallow or
rapidly equilibrating compartment T. 3" represents the level in the
deep or slowly equilibrating compartment, and T'mayi is the maximum level
achieved in the deep compartment. E is the amount of chemical eliminated
or metabolized, and E<*> corresponds to the amount eliminated or metab-
olized at infinite time. ke represents the overall rate constant for
the elimination of the chemical from the body. Modified from Garrett.
(1971). Source: Gehring, Watanabe, and Blau, 1976. Reprinted with
permission of the publisher.
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48
have a more limited blood supply. Phannacokinetic rates may be
influenced by such factors as the physical characteristics of the sub-
stance (e.g., solubility, molecular weight, and surface area or solid
particles), pharmacologic properties (e.g., peristalsis and blood flow),
gastrointestinal functions (e.g., gastric emptying and intestinal
transit and motility), permeability of membrane barriers at the site of
absorption, local blood flow, metabolism, and specialized transport
mechanisms (Withey, 1978).
2.6.3 Utility of Pharmacokinetics
One example of the use of pharmacokinetics as an aid in the hazard
evaluation of chemicals is provided by a study of the absorption of
methylcellulose (Braun, Ramsey, and Gehring, 1974). Administration of
ll*C-labeled methylcellulose (viscosity of 3300 centipoise and molecular
weight of 77 000) in both single and repeated oral daily doses of
500 mg/kg to rats showed that virtually none of the methylcellulose was
absorbed, with essentially all being excreted in the feces. Therefore,
if it can be demonstrated that no absorption occurs there is no need
for further ingestion studies to ascertain systemic toxicity.
Another example of the utility of pharmacokinetics in the toxico-
logical evaluation of chemicals is the oral toxicity study of 6-chloro-
picolinic acid (6-CPA), a degradation product of 2-chloro-6-(trichloro-
methyl)pyridine (CTP) (Gehring, Watanabe, and Blau, 1976). Analysis of
the urine of rats given single doses of 10 mg/kg of llfC-labeled 6-CPA
revealed that 30% of the 1UC activity present was attributable to 6-CPA
and 62% to the glycine conjugate. The rapid (half-life, 1.1 ± 0.2. h) and
complete elimination of 6-CPA and its glycine conjugate from the rat
suggested that ingestion of trace amounts of 6-CPA does not constitute a
hazard. Gehring, Watanabe, and Blau state that, as expected from the
pharmacological evaluation, a 90-day toxicological study in which rats
were given daily doses of 6-CPA gave no indication of cumulative toxicity.
An example of the utility of pharmacokinetics to the pharmaceutical
industry is given by Nelson (1976). A hypothetical situation is described
of an antibiotic that is being developed for deep-seated tissue infection.
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49
Kinetic analysis of this antibiotic in animals or human volunteers shows
the drug to have a minimal distribution with a rapid elimination half-
life; furthermore, renal excretion kinetics indicate that it is elimin-
ated by both glomerular filtration and tubular secretion. It is
therefore doubtful that high serum and tissue levels of this antibiotic
could ever be realistically achieved, and thus the drug would not be
useful for deep-seated tissue infections. The renal excretion kinetics,
however, suggest that the antibiotic could be useful in treating urinary
infection. Nelson states that by performing such pharmacokinetic studies
many costly and needless clinical studies would be circumvented.
One other example of the value of pharmacokinetics in toxlcological
studies is shown by the work of Piper et al. (1973). The herbicide
2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was administered to rats and
dogs in single oral doses of 5 mg/kg with the resultant half-life
clearance values being 4.7 and 77.0 h, respectively. It was demonstrated
by Piper et al. that these rates of elimination can be correlated with
the toxicity of 2,4,5-T in rats and dogs. The slower rate of elimination
by dogs than by rats correlated with the higher toxicity in dogs for
which the single oral LDso is 10° mg/kg compared to 300 mg/kg for rats
(National Institute for Occupational Safety and Health, 1977). These
correlations support the statement of Gehring and Young (1978) that there
is an inverse relationship between the toxicity of an agent to an
individual or species and the ability of the individual or species to
eliminate the chemical and a direct relationship between toxicity and
the concentrations in the plasma.
In the testing of a chemical for teratogenicity, a knowledge of
its pharmacokinetics would be beneficial in explaining the teratogenic
effects or lack of such effects. Teratogenic drugs that are dissolved,
absorbed, metabolized, and excreted rather quickly and that are
administered during the critical period of organogenesis will cause
malformations because the action of the drug will occur shortly after
administration (Schardein, 1976). An example is 8-aminopropionitrile,
which is metabolized so quickly to its inactive metabolite, cyanoacetic
acid, that if day 15 (the critical period for induction of cleft palate)
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50
is not included in the dosing regime, very few malformations are
produced in the rat (King, Horigan, and Wilk, 1972). However, there are
some teratogenic drugs that are absorbed or metabolized so slowly or
incompletely that their teratogenicity would not be manifested if
administered during the critical period. An example of this type of
action is provided by the antihypercholesterolemic drug triparanol, now
withdrawn from use, which has been shown to be an active teratogen in
rats when administered as early as day 4 prior to implantation (Roux,
1964, as cited by Schardein, 1976).
2.6.4 Conclusions
The four rate processes of pharmacokinetics are absorption, distri-
bution, metabolism, and elimination. Each of these is important in
determining the fate of a chemical in the body and can be influenced by
such factors as the physical characteristics of the test substance,
pharmacologic properties, and gastrointestinal functions. Knowledge of
the pharmacokinetics of a chemical can aid in the hazard assessment
process by demonstrating in some cases the lack of potential toxicity
because of lack of absorption into the body or a fast elimination from
the body and in other cases by explaining why a chemical is more toxic
to one species of laboratory animal than to another.
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51
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Dymet, J. 1976. Air Filtration. In: Laboratory Animal Handbooks 7:
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3. ACUTE TOXIC ITY
3.1 INTRODUCTION
In toxicology the term acute is defined as a single dose or exposure,
or fractions of a dose, administered over a short period (National
Academy of Sciences, 1977). The toxic effects of such a dosing regime
may be observed within a few minutes or up to several days after dosing.
The effects may vary greatly with sex and age of the animal as well as
within and between different strains and species.
In acute oral LDso studies the test substance is commonly given by
gavage in a single dose or divided doses within 1 day, and the animals
are observed for 2 weeks; however, when possible, the route of exposure
should be that which is likely to be encountered in humans. The number
of deaths, time of death, clinical signs, and necropsy are parameters
used in assessing the acute toxicity of the chemical.
The Food Safety Council (1978) lists the following three purposes
for conducting acute toxicity tests of chemicals:
1. to give a quantitative measure of acute toxicity (LDso) for
comparison with other substances,
2. to identify the clinical manifestations of acute toxicity, and
3. to give dose-ranging guidance for other tests.
This section will discuss aspects of acute toxicity such as the
LDso, necropsy as a part of the protocol of an acute toxicity test, and
the length of the observation period following acute administration of
the test chemical.
3.2 LD50 DETERMINATIONS
3.2.1 Introduction
The most frequently determined index of toxicity is the LD50, the
dose that is lethal to one-half of a group of treated animals (National
Academy of Sciences, 1977). In addition, the LDso can be used as a
basis for determining doses to be used in subsequent subchronic and
59
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chronic toxicity studies. It can also aid the researcher in determining
which species and route of administration would be the most suitable for
his or her experiments. The following sections will show how the choice
of species, route of administration, and sex of the animal may affect
the LDso and acute toxicity response.
The information presented on acute LDs0s is available in the National
Institute for Occupational Safety and Health (NIOSH) Registry of Toxic
Substances (1977), which has entries in the following format:
BR09500 Ammonium, (2-hydroxyethyl)diisopropylmethyl-, bromide, xanthene-
9-carboxylate
TXDS: Oral-rat LD50: 370 mg/kg
Intraperitoneal-rat LDso: 25 mg/kg
Subcutaneous-rat LD5Q: 298 mg/kg
Intraduodenal-rat LDso: 125 mg/kg
Oral-mouse LDso: 445 mg/kg
Intraperitoneal-mouse LDso: 78 mg/kg
Oral-rabbit LDso: 750 mg/kg
Intravenous-guinea pig LDT : 51 mg/kg
LjO
This format provides information on the route of administration,
the species, and the toxic LDso dose or LDT dose (lowest published
LiO
lethal dose reported). Also included in the NIOSH document are synonyms,
Chemical Abstracts Service registry numbers, and the reference for each
item of toxicity data. Table 3.1 is a list of 96 compounds, arbitrarily
chosen, for which the LDso values were examined and compared for species
differences and for differences between routes of administration.
Although not discussed, it is recognized that the differences in reported
LDso values may have also been influenced by the age and strain of the
test animals, duration of the starvation period prior to dosing, ambient
temperature, concentration of the test substance, speed of injection,
and type of solvent or suspension medium (Zbinden, 1973), as well as by
interlaboratory variations in experimental procedures.
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Table 3.1. Compounds examined for LDso differences between
species and between routes of administration
Ammonium (2-hydroxyethyl)dlisopropylmethyl-, bromide, xanthene-9-carboxylate
Ammonium [(p-hydroxy-2-isobutyl)phenyl]trlmethyl-, iodide, methylcarbamate
Arsine, dichloro(2-chlorovinyl)-
Benzaldehyde, p-isopropyl-
Benzamide, 4-amino-ff-cyclopropyl-3,5-dichloro-
Benzimidazole, 2-(4-thiazolyl)-, hydrochloride
2-Benzimidazolinone, 5-chloro-l-[3-(diraethylamino)propyl]-3-phenyl-
Benzoic acid, benzyl ester
Benzole acid, 2,3,6-trlchloro-
2-H-Benzo(a)quinolizin-2-one, 1,3,4,6,7,llb-hexahydro-3-isobuty1-9,10-dimethoxy-
Benzoxazole, 2-amino-5-chloro-
Benzoxazoline, 3-methyl-2-(methylamino)-
2-Benzoxazollnone, 5-chloro-
Benzyl alcohol, alpha-(l-aminoethyl)-, hydrochloride, (+ -)-
Carbamic acid, methyl-, m-tolyl ester
Carbamic acid, methyl-, 3,4,5-trimethylphenyl ester
Carbanilic acid, m-chloro-, 4-chloro-2-butynyl ester
Carbon tetrachlorlde
2-Carboxymethylmercaptobenzenestrlbonic acid
Cerium chloride
Ethylamine, W-methy1-2-[(o-methyl-alpha-phenyIbenzyl)oxy]-,hydrochloride
Ethylene glycol
Guanidine, l-cyano-3-tert-pentyl-
3-Heptanone, 6-(dimethylamino)-4,4-diphenyl-, hydrochloride
1,6-Hexanediamine, N,N,Nl,N^-tetramethyl-, polymer with 1,3-dibromopropane
Hydrazine, methyl-, hydrochloride
Hydroquinone
Imidazole
3-3-Imidazoline, 4-amino-2,2,5,5-tetrakis(trifluoromethy1)-
1,3-Indandione, 2-[(p-chlorophenyl)phenylacetyl]-
IH-Indazole, l-benzyl-3-[3-(dimethylamino)propoxy]-, monohydrochloride
Indene-1-ethylamine, Af^ff-dimethyl-l-phenyl-, hydrochloride
Indole-3-acetic acid, l-(p-chlorobenzoyl)-5-methoxy-2-methyl-
Indole, 3-(2-aminoethyl)-5-methoxy-, hydrochloride
Isonicotinic acid, 2-[2-(benzylcarbamoyl)ethyl]hydrazide
Isonicotinic acid, 2-isopropylhydrazide
Isonipecotamide, 4-(p-chloropheny!)-!-[3-(p-fluorobenzoyl)propyl]-W,W-dimethyl-
Isonipecotic acid, l-(p-aminophenethyl)-4-phenyl-, ethyl ester
Meglumine hydroxamate
Mercury, [3-(alpha-carboxy-o-anisamide)-2-(2-hydroxyethoxy)propyl]hydroxy-, monosodium salt
Mercury, (3-cyanoguanidino)methyl-
Mercury (11) iodide
4-Metathiazanone, 2-(3,4-dichlorophenyl)-3-methyl-, 1, 1-dioxide
Methanesulfoanilide, 4'-[l-hydroxy-2-(methylamino)propyl]-, hydrochloride
Methanesulfonic acid, iodo-, sodium salt
2,6-Methano-3-benzazocln-8-ol, 1,2,3,4,5,6-hexahydro-3-ally1-6-ethyl-ll-methyl-
4,7-Methanoindene, l,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-
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62
Table 3.1 (continued)
Monomethylhydrazine nitrate
Morphinan-3, 14-diol, 17-(cyclopropylmethyl)-
Morphinan-3,6-alpha-diol, 7,8-didehydro-4,5-alpha-epoxy-17-methy1-
Morphinan, 17-(p-nitrophenethyl)-, hydrochloride (+ -)-
Morphinan-6-alpha-ol, 7,8-didehydro-4,5-alpha-epoxy-3-methoxy-17-methyl-
2-NaphChalenemethanol, alpha-[(isopropylamino)methyl]-
Naphthalimide, ff-hydroxy-, diethyl phosphate
2-Naphthol, 1,2,3,4-tetrahydro
Neomycin
Nickel (11) acetate(l:2)
Nicotinamide, tf,tf-diethyl-
2-Norbornanamine, ff-ethyl-3-phenyl-, hydrochloride
2-Norbornanamine, tf-3,3-trimethyl-
2-Norbornanamine, /Y,2,3,3-tetramethyl-, hydrochloride
l-Oxa-3,9-diazaspiro(5.5)undecan-2-one, 5-ethyl-9-[3-(p-fluorobenzoyl)propyl]-
1.2,4-Oxadiazole, 5-[2-(diethylamino)ethyl]-3-phenyl-
l,2,4-Oxadiaxolidine-3,5-dione, 2-(3,4-dichlorophenyl)-4-methyl-
2H-l,3-Oxazine-2,4(3H)-dione, 5,5-diethyldihydro-
Pactomycin
Penlcillanic acid, 6-phenoxyacetamido-
2,4-Pentanediol, 2-(p-chlorophenyl)-4-methyl-
4-Pentenoic acid, 2-[(2-diethylamino)ethyl]-2-phenyl-, ethyl ester
l-Penten-4-yn-3-ol, l-chloro-3-ethyl
Phenazine, 3-amino-7-(dimethylamino)-2-methyl-, hydrochloride
Phenethyl alcohol
Phenol, 2,4-dinitro-
Phenol, 2,4,5-trichloro-
Phenothiazine, 2-chloro-10-[3-(A'-cyclopentyl-W-methyl)amlnopropyl]-
Phenothiazine, 10-[2-(diethylamino)propyl]-
Phosphonothioic acid, phenyl-, 0-ethyl 0-(p-nitrophenyl) ester
Phosphoramidothioic acid, 0,5-dimethyl ester
Phosphoric acid, 2-chloro-l-(2,4-dichlorophenyl)vinyl diethyl ester
1,3-Propanediol, 2-(hydroxymethyl)-2-nitro-
12,-Propanediol, 3-(0-methoxyphenoxy)-, 1-carbaoate
Pyrrolidinium, 3-hydroxy-l,-dimethyl-, bromide,alpha-cyclopentylamandelate
3-Pyrrolidinol, l,2-dimethyl-3-phenyl-, propionate
Quinine, hexylbromide
Sodium pentafluorostannite
Streptomycin
Succlnic acid, cadmium salt (1:1)
Sulfanilamde
Sulfanilamide, JV1-2-thiazolyl-
5H-Tetrazoloazepine, 6,7,8,9-tetrahydro-
Theophy11ine, 8-benzy1-7-[2-(echy1(2-hydroxyethyl)amino)ethyl]-,hydrochloride
2H-l,3,5-Thiadiazine-2-thione, tetrahydro-3,5-dimethyl-
a-Triazine, 2-azido-4-(isopropylamino-6-(methylthio)-
8-Triazine,2,4-dichloro-6-(o-chloroanilino)-
Urea, 3-(hexahydro-4,7-methanoindan-5-yl)-l,l-dimethyl-
Urea, 1,1,3,3-tetramethyl-
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63
3.2.2 Difference Between Species
Table 3.2 uses the LD50 data for each compound listed in Table 3.1
to show the LD50 differences (expressed as percentage) between different
species categorized by route of administration. For example, the first
entry in Table 3.2, cat, is compared with the pigeon and rat for the
oral route of administration. The comparison of the cat with the rat
indicates that for one compound the cat had an LDso greater than the rat
LD50 by 1% to 25%, for three compounds the rat LD50 was greater than
the cat LD50 by 51% to 75%, and for one other compound the rat LD50
was greater by 76% to 100%. Therefore, for four of the five chemicals
compared, the rat showed a higher tolerance to the acute dose. Collec-
tive examination of data in Table 3.2 reveals that 5.8% of the species
compared showed no difference in LDso amounts, 23.3% of the species had
LD50 values differing by 1% to 25%, 29-4% had differing LD50 values of
26% to 50%, 27.7% of the species had LD50 values that differed by 51% to
75%, and 13.9% of the species had LD50 differences between 76% and 100%.
Therefore, less than one-half of the 374 species comparisons (41.7%)
showed LDso values that differed by more than 50%. A more detailed
examination of the data in Table 3.2 reveals the following:
1. The rat and mouse appear to be less sensitive indicators of toxicity
than the dog.
2. The rabbit is a less sensitive toxicity indicator than the rat but
slightly more sensitive than the mouse.
3. The mouse is a less sensitive toxicity indicator than the hamster.
4. The differences between LDso values for the mouse and rat were less
than 50% for 56% of the comparisons.
5. The mouse has a larger LD50 (less sensitivity) than the rat in the
majority of comparisons (60%).
The principal observations from these data are that the mouse and
the rat represent the most commonly used test species (84.3% of all
comparisons involved one or both) and that for slightly more than qne-
half (58%) of all the species comparisons the LDso values differed by
less than 50%.
-------
Table 3.2. Percent
differences between species
Species
Cat
Pigeon
Rat
Chicken
Guinea pig
Hamster
Mouse
Pigeon
Quail
Rabbit
Dog
Cat
Guinea pig
Hamster
Monkey
Pigeon
Rabbit
Rat
Duck
Chicken
Dog
Mouse
Pigeon
Quail
Route of
administration n . „,
0 1 to 25
Oral
Oral Cat
Oral Guinea pig
Oral
Subcutaneous Mouse
Oral Mouse (2)b
Oral
Intravenous
Oral
Oral
Oral Cat
Subcutaneous
Oral
Oral
Subcutaneous
Oral
Oral
Subcutaneous 1° Dog
Skin
Intravenous
Oral 1°
Intraperitoneal
Oral
Subcutaneous Rat
Intravenous Rat
Intraperitoneal 1°
Skin
Intravenous Chicken
Oral
Intravenous
Oral Pigeon
Intravenous
Oral
Percent
26 to 50
Guinea pig
Hamster
Quail
Dog(2)b
Dog
Monkey
Pigeon
Dog
Rabbit
Rat, dog(2)b
Dog
Rat
Rat(2)6
Rat
Quail
differences
51 to 75
Rat(3)b
Mouse
Mouse
Pigeon
Pigeon
Dog
Guinea pig
Dog
Dog
Rabbit ,
Rat, dog(2)C
Rat, dog
Chicken
Quail
76 to 100
Pigeon0
Rat
Rabbit
Dog
Monkey
Rat, dog
Dog
Dog
Duck
Pigeon
Pigeon
-------
Table 3.2 (continued)
Species
Guinea pig
Cat
Hamster
Monkey
Mouse
Pigeon
Rabbit
Rat
Mouse
Cat
Dog
Hamster
Monkey
Pigeon
Rabbit
Quail
Dog
Pigeon
Rat
Route of
administration
Oral
Intraperitoneal
Oral
Subcutaneous
Oral
Intraperitoneal
Subcutaneous
Intravenous
Oral
Subcutaneous
Skin
Oral
Oral
Subcutaneous
Skin
Intraperitoneal
Intravenous
Oral
Intraperitoneal
Oral
Subcutaneous
Intraperitoneal
Intravenous
Oral
Intraperitoneal
Subcutaneous
Oral
Intravenous
Oral
Subcutaneous
Intravenous
Oral
Oral
Oral
Percent differences
0 1 to 25
1°
Guinea pig
lc Mouse (2)*
Guinea pig
1°
Guinea pig
1° Guinea pig(2),fc
rat
1° Rat
Guinea pig
Dog, mouse
Mouse
1° Mouse(2)ft
Mouse
Mouse
Mouse ,
Rabbit (2)D
Pigeon
26 to 50
Mouse (A), guinea
Mouse, guinea pig
Guinea pig(2)"
Guinea pig
Rabbit
Guinea pig
Rabbit, guinea
Pig .
Guinea pig(2),fi
rat(2)Z)
Guinea pig
Cat
Mouse
Mouse(3)fc
Mouse
Mouse
Mouse
Rabbit (2),^
mouse ( 3) °
Mouse, rabbit (2)h
Rat
51 to 75
Cat, guinea pig
Monkey
Guinea pig
Guinea pig
t
Rabbit (2), fi
guinea pig
Guinea pig (4),
rat (3)"
Guinea pig, rat
Rat
1.
Mouse (3)°
Mouse
Dog
Mouse
Mouse
Hamster
Monkey
Mouse(2),b
rabbit
Mouse (2)°
Mouse (2)fc
Dog
Pigeon
Rat
76 to 100
Guinea pig
Mouse (2)fc
Guinea pig
Rat
Guinea pig
Mouse
Mouse (2)fc
Mouse
Rabbit (2)fc
Mouse(2)fc
Ui
-------
Table 3.2 (continued)
Species
Rabbit
Cat
Duck
Monkey
Rat
Duck
Hamster
Rat
Monkey
Mouse
Pigeon
Rabbit
Route of
administration _
Oral
Oral
Subcutaneous
Oral
Oral ; .•;•>'>
Intravenous
Oral
Intraperitoneal
Skin
Subcutaneous
Oral
Oral t*°
Intraperitoneal 2°
Intravenous 2°
Subcutaneous
Intramuscular
Oral
Intravenous
Oral 2°
Subcutaneous
Intraperitoneal 2°
Intravenous 2°
Skin
Intramuscular
Percent differences
1 to 25
Cat
Duck
Rat, hamster
Hanmter(3)k
Hamster
i
Mouse (9).°
rat(8)\
Mouse (5),
rat(4)6
Mouse (5), °
rat(2)£
Mouse(5)D
Rat(3)fc
Rabbit (2)
Rat, rabbit
Rabbit
26 to SO
Duck
Monkey
Hamster, rat
Monkey
Monkey
Mouse(7).fc
rat (6)\
Mouse (4), b
rat (3)*
Mouse(3),6
rat(4)6
Mouse(2),fe
rat(2)£
Rabbit
Rat, rabbit
Rat, rabbit
Rat(2),b rabbit
Rat
51 to 75 76 to 100
Monkey
Rat Rat, duck
Hamster (2)b
L t
Mouse (7), b Mouse (5), b
rat (10)* rat(3)6
Mouse (7),* Mouse
rat(2)6
Mouse(3),0 rat Mouse
Mouse (2). * Mouse
rat(2)0
Mouse
Pigeon
Rabbit (3),° rat Rabbit (3),° rat
Rat, rabbit Rat, rabbit (2)b
"indicates which animal of the comparison had the greatest LD50. Position of species indicates the per-
cent differences in the LD50s of the two species being compared.
Represents number of chemicals compared where LDso of species indicated was greater than species being
compared. If there is no number then only one chemical was compared.
°The number indicates how many times the
values were identical for the species compared.
-------
67
3.2.3 Difference Between Administration Routes
Table 3.3 shows the percentage difference between LDso values for
various pairs of routes of administration categorized by species.
Abbreviations in the appropriate columns indicate which route of the two
being compared is the greater. For example, the "inp" (intraperitoneal)
notation in the first entry for the mouse at 76% to 100% indicates that
the LDso for the intraperitoneal route of administration was 76% to 100%
greater than the LDso for the intracerebral route for the same compound.
Column by column examination of the data in Table 3.3 reveals that 2.4%
of the entries fall in the 0% (no difference in LD50) column, 20.4% in
the 1% to 25% column, 20.4% in the 26% to 50% column, 26.2% in the 51%
to 75% column, and 29.8% in the 76% to 100% column. These data indicate
that the LDso can vary significantly depending on the administration
route.
A more detailed examination of the data in Table 3.3 shows the
following:
1. For all compounds, the subcutaneous route of administration was
always associated with a higher LDso than the intravenous route.
2. Of the 328 entries, 116 (35%) compared the subcutaneous route with
other routes, 140 (42.6%) compared intravenous administration with
other routes, and 199 (60.6%) compared the oral route of administra-
tion with other routes. Thus the predominant use of the oral
administration route is shown.
3. When the intravenous route was compared to the oral and the intra-
peritoneal routes, respectively, the LDso values were greater for
the oral and intraperitoneal routes for most of the compounds. In
fact, in only four of 56 comparisons (two being equal) was the
intravenous LDso greater than the oral LDso [those four compounds
were methylhydrazine hydrochloride, 2-isopropylhydrazide isonico-
tinic acid, pactamycin, and 2-chloro-l-(2,4-dichlorophenyl)vinyl
phosphoric acid diethyl ester], and in only five of 35 comparisons
was the intravenous LDso greater than the intraperitoneal route
[those four compounds (two comparisons involved the same compound)
were methylhydrazine hydrochloride, 4-amino-2,2,5,5-tetrakis
-------
Table 3.3. Percent LD5o differences between routes of administration
Routes of
administration
Intracerebral and
intraperitoneal
Intracerebral and
Intravenous
Intracerebral and
subcutaneous
Intraperitoneal and
intramuscular
Intraperitoneal and
skin
Intraperitoneal and
subcutaneous
Intravenous and
intramuscular
Intravenous and
intraperitoneal
Intravenous and oral
Intravenous and skin
Intravenous and
subcutaneous
Species
0
Mouse
Mouse
Mouse -
Mouse
Rat
Rat
Hamster
Guinea pig
Rat 1°
Mouse
Rabbit
Rat
Mouse
Rat
Mouse
Rat
Mouse 1°
Dog
Guinea pig
Rabbit 1°
Rabbit
Rat
Rabbit
Mouse
Rat
Guinea pig
Percent differences
1 to 25
Inm, Inpa
Sub (5),* Inp(3)*
Inm
Inv, Inm
Inv , Inp
Inv(3),& Inp (3)*
Inv
0(4),* Inv
Sub (2)?
Sub (2)*
26 to 50
Inm
Inp, Sub
Sub (7)*
*
Inp (4)*
Inp (7)*
06 ft
0(2)*
0
Sub (6)*
Sub
51 to 75
Skin
Sub (3)*
Sub (3)*
Inp (4)*
Inp (8),° Inv
0(7),* Inv(2)*
Sub
Sub (8)°
Sub (3)
76 to 100
Inp
Inv
Sub
•L
Skin (2)*
Skin
Skin
Sub (2)*
Sub
Inm
Inm
Inp (2)
Inp
0(11)*
0(18)*
0
Skin
Skin
•L
Sub (2)?
Sub (7)*
Sub (7)*
Sub
00
-------
Table 3.3 (continued)
Routes of
administration
Oral and
intracerebral
Oral and
intramuscular
Oral and
intraperitoneal
Oral and
subcutaneous
Skin and oral
Subcutaneous and
intramuscular
Subcutaneous and
skin
0 1 to 25
Mouse
Rat
Mouse 1°
Rat 2° 0(5)i.fc Inp(2)&
Mouse 0(7)&
Hamster
Cat Inp
Rabbit Inp
Guinea pig 1°
Rat 0(9)\
Mouse 0(6), Sub (2)*>
Guinea pig lc
Rat
Mouse
Rabbit 0
Hamster
Guinea pig
Rabbit lira
Mouse Sub
Rat
Dog
Rabbit
Guinea pig
Percent differences
26 to 50 51 to 75
Inm
0
0(6) ,b Inp 0(10)fc
0(7), b Inp 0(11), b Inp(2)6
0 0(3)fc
t
0(3), £ Sub Sub .
0(7),° Sub 0(4),° Sub(3)°
i.
Skin 0, Skin(3)D
Skin
Sub
Skin
Skin
76 to 100
0
0
0(12),b Inp
0(9)&
0
t,
Skin(5)fi
0
Skin
Skin
Skin
Skin
Skin
alnm — intramuscular; Inp — intraperitoneal; Inv — intravenous; 0 — oral; Sub — subcutaneous.
Represents number of comparisons when route indicated was greater than the route being compared. If no
number is present then only one chemical was compared.
The number indicates how many times the
values were identical in a comparison.
-------
70
(trifluoromethyl)-3-imidazoline, 2-isopropylhydrazide isonicotinic
acid, and N1-2-thiazolylsulfanilamide].
4. The oral LD50 was greater than the subcutaneous LDso ^or 82.5% of
the comparisons; however, the difference was generally less than 50%.
5. A comparison between the oral LDso an<* intraperitoneal LDso showed
that for 89% of the entries the oral route had a greater LDso than
the intraperitoneal route [those compounds for which the intraperi-
toneal route was greater for at least one species were 2,3,6-tri-
chlorobenzoic acid, alpha-(1-aminoethyl)benzyl alcohol hydrochloride,
l-cyano-3-tert-pentylguanidine, l-(p-chlorobenzoyl)-5-methoxy-2-
methylindole-3-acetic acid, pactamycin, tetrahydro-3,5-dimethy1-
2H-l,3,5-thiadiazine-2-thione, 2,4-dichloro-6-(0-chloroanilino)-s-
triazine, 4-amino-#-cyclopropyl-3,5-dichlorobenzamide, and
2-(3,4-dichlorophenyl)-3-methyl-4-metathiazanone-l,l-dioxide].
The principal conclusion from these data is that the choice of
route of administration is an important part of the experimental design
because it can result in significant differences in toxicity response.
This emphasizes the importance of selecting the route of exposure that
will be the likely exposure route in man when conducting safety assess-
ments of chemicals.
3.2.4 Sex Differences in the Laboratory Rat
Male and female rats show differing susceptibilities to the acute
doses of some chemicals. Hayes (1975) states that the rat, which is
used more than any other species for studies in toxicology, apparently
shows more variation between the sexes in its response to chemicals than
does any other species. One example is provided by the research of Kast
et al. (1975i) with the amino-halogen-substituted benzylamine, fominoben-
HC1. The oral LDso f°r males was 6450 mg/kg and for females, 4500 rag/kg.
Other examples of sex differences in LDso response are seen in the
research of Gaines (1960, 1969). He reports that, in acute oral toxicity
tests with 98 pesticides, most compounds were more toxic to female than
to male rats. This is demonstrated with the organophosphorus pesticides
azodrin (male LD50, 126 mg/kg and female LD50, 112 mg/kg), Bayer 37289
-------
71
(male LD50, 180 mg/kg and female LD50, 64 mg/kg), and ethion (male LD50,
245 mg/kg and female LD50, 62 mg/kg). Hayes (1975) used the oral toxicity
LD50 data of Gaines (1960, 1969) on the rat for 69 pesticides and calcul-
ated the ratio of female to male oral LD5QS. His calculations showed
that the ratio ranged from 0.21 (indicating greater susceptibility of
the female) to 4.62 (indicating greater susceptibility of the male) and
averaged 0.94. Therefore, these studies indicate that both males and
females should be used as test animals when the rat is the animal of
choice for the acute toxicity testing of a chemical.
3.2.5 Test-Limiting Criteria
Some chemicals are relatively innocuous when given as single doses.
If this relative lack of acute toxicity can be determined at the begin-
ning of the acute toxicity test, there is no need for continuing the
acute testing. The National Academy of Sciences (1977) states that,
for most purposes, if animals survive single oral doses of 5 or 10 g/kg,
an adequate estimate of hazard is obtained. The Interagency Regulatory
Liaison Group's guidelines indicate that no further acute oral toxicity
testing of a chemical is necessary if no mortality is seen at a dose of
5 g/kg. Similarly, the guidelines for the Organization for Economic
Cooperation and Development state that, if 5 of 10 animals survive a
dose of 5 g/kg of a chemical for 14 days, no further acute toxicity
testing is necessary. If these criteria are met in one sex but not the
other acute toxicity testing must proceed with the sex that did not meet
the criteria for limiting the acute test.
3.2.6 Conclusions
The information in Sect. 3.2.2 and Sect. 3.2.3 clearly indicates
the importance of the selection of proper species and route of adminis-
tration. Specifically, 42% of all species comparisons (Table 3.2)
indicated differences in LD50 values of more than 50%, and 56% of all
comparisons involving administration routes (Table 3.3) showed differ-
ences between LD50 values of more than 50%. When the laboratory rat is
the test animal in acute toxicity studies, both male and female animals
-------
72
should be used because of the variation in response between the sexes.
If a chemical does not cause more than 50% mortality when administered
in acute doses of 5 g/kg, further acute toxicity testing is not required.
For safety assessment of chemicals with potential human exposure, it is
desirable to select a test animal that will reflect what is anticipated
to occur in man. In practice, however, the rat or mouse is usually the
species of first choice because they are easy to work with and relatively
inexpensive and there is a vast amount of information available on which
to base comparative assessments. The difference in LDsg values as a
result of using different exposure routes indicates the necessity of
selecting the exposure route that will be the likely exposure route in
man when chemicals are tested that have a potential for human exposure.
3.3 HUMAN VS ANIMAL RESPONSE
3.3.1 Introduction
This section will compare the responses of animals and humans to
acute exposures of chemical substances. For the most part, information
on dermal and ocular toxicity will not be presented, as this will be
covered in detail in a companion document.
3.3.2 Comparison of Lowest Published Lethal Doses (LD )
The source of the information in this section is the NIOSH Registry
of Toxic Substances (1977) in which the LDLo is defined as the lowest
published lethal dose. Twenty-seven compounds (Table 3.4) were chosen
at random and the human and animal LD data compared in Table 3.5. The
LiO
following observations are evident:
1. With the exception of two compounds, all comparisons involved the
oral route of administration.
2. In only one instance was the human and animal LIL equal, whereas
8.6% of the comparisons had LD differences between 1% and 25%,
17.3% between 26% and 50%, 15.2% between 51% and 75%, and 60.8%
between 76% and 100%.
-------
73
Table 3.4. Compounds selected for human vs animal
LD comparisons
.LiO
Oxalic acid 0.071
Quinine, monohydrochloride 0.230
Hydrocyanic acid 0.570
Phenol, 2,4-dinitro 4.3
Barbituric acid, 5-ethyl-5-hexyl- , sodium salt 5
Ouabain 5
Phosphoramidocyanidic acid, dimethyl-, ethyl ester 23
Mercury (11) chloride 29
Phenol, pentachloro- 29
Strychinine 30
Carbon tetrachloride 43
Chloral hydrate 50
Veriloid 50
Zinc, bis(dimethyldithiocarbamato)- 50
Quinine 50
Benzamide, p-amino-tf-(2-diethylamino)ethyl)0- 50
Ammonium, [4-(bis(p-(dimethylamino)phenyl)
methylene)-2,5-cyclohexadien-l-xylidene]
dimethyl-, chloride 50
Acetanilide, 4'-hydroxy 50
Methane , iodo- 50
2-Naphthol 50
Quinoline, 8[ (4-(diethylamino)-l-methylbutyl)
amino ] -6-methoxy- 50
Sodium fluoride 75
Benzoic acid 500
Ethyl alcohol 500
Sulfamic acid 500
2-Pentanone, 4-methyl- 500
1 , 4-Naphthoquinone , 2-methyl- 500
Compounds listed in order of decreasing toxicity.
-------
Table 3.5.
LD differences between humans and animals
Lo
Species
compared
Rat
Rabbit
Pigeon
Guinea pig
Chicken
Cat
Mouse
Dog
Route of
administration
Oral
Skin
Intravenous
Oral
Intravenous
Oral
Oral
Oral
Oral
Oral
Oral
Skin
Intravenous
Percent differences
0 1 to 25 26 to 50 51 to 75 76 to 100
Rata Human, rat (2r
Human
Rat
Rabbit Rabbit(2)Z? Human, rabbit(3)fc Rabbit(5),fc human
Rabbit
Pigeon Pigeon
Guinea pig(2)i> Guinea pig
Chicken
Cat
Mouse
1° Human Dog (7), human (2)
Dog
Dog
alndicates which animal of the comparison had the greatest LDLo. Position of species indicates the
percent differences in the LD^o of the human and animal species being compared.
Represents the number of chemicals compared where the LDLO of the species indicated was greater than
the species being compared. If there is no number then only one chemical was compared.
CFor one chemical, the human and the dog had identical LD^O values when administration was oral.
-------
75
3. Only 19.5% of the comparisons showed the human LDLQ value to be
greater than the animal LD value.
On the basis of these data, it can be concluded that there is a signifi-
cant difference between the human and animal acute LD, response and
that, in the majority of instances, the human showed less tolerance
(reflected by lower LDT values) to chemical insult than did the animal
LiO
model.
3.3.3 Comparison of Acute Toxicity Response
The studies of Goyer (1971) and Lock and Ishmael (1978) show the
effect of the acute administration of chemicals on the rat and human
kidney to be similar. In his review of the effects of lead on the
kidney, Goyer found that morphological and functional reactions of the
kidneys of rat and man to acute lead exposure have comparable features:
(1) the proximal tubular lining cells are affected, (2) intranuclear
inclusion bodies are formed, and (3) there is an associated aminoaciduria.
He also noted that although the impairment of mitochondrial function has
not been demonstrated in man as it has in rats, the impairment may be
inferred from morphological changes in human biopsy material. Lock and
Ishmael in studying the acute effects of paraquat and diquat found that
an oral dose of 680 micromoles/kg of either chemical would result in
marked diuresis, proteinuria, and glucosuria 6 to 24 h after dosing of
the male rat. In addition, histopathological kidney examination showed
mild hydropic change in the proximal convoluted tubules, and renal
clearance of insulin, p-aminohippuric acid (PAH), and /l/'-methylnicotamide
(NMN) was found to be markedly reduced 2 h after dosing. Lock and
Ishmael reported that these changes indicate that renal impairment is
similar in rat and man.
Hexachlorophene and ethchlorvynol have also been shown to produce
similar symptoms in humans and animals after acute exposure. Martinez,
Boehm, and Hadfield (1974) reported the accidental oral ingestion of
approximately 45 mL of hexachlorophene by a seven-year-old boy. Toxic
reactions included nausea, vomiting, anorexia, diarrhea, decrease in
visual acuity, blurred vision, blindness, somnolence, and disorientation,
-------
76
with respiratory and cardiac arrest occurring 61 h after hospital
admission. Further examination revealed the following: (1) interstitial
myocarditis, (2) pneumonitis and acute bronchiolitis, (3) edematous
brain tissue, (4) occasional neuronal degeneration, and (5) myelin
sheath disintegration and other neuronal changes. Martinez, Boehm, and
Hadfield noted that these changes have been produced in experimental
animals exposed to several chemicals including hexachlorophene.
In a case report of an intravenous injection of ethchlorvynol pre-
sented by Payne, Kerr, and Diaconis (1977), the individual developed
acute, severe pulmonary edema accompanied by hypoxemia and acidosis.
Similar toxic effects on the pulmonary vasculature were observed in rats
by Payne and coworkers after administering intravenous injections of
80 mg/kg ethchlorvynol, resulting in death from acute respiratory distress.
3.3.4 Conclusions
The information in Sect. 3.3.2 concerning lowest lethal doses
clearly indicates a difference between humans and animals with respect
to the size of the dose that will cause death; in most cases a smaller
dose was required to cause death in humans than in animals. Because of
this difference, an examination of the pathology and functional changes
induced by the acute exposure of humans and animals to chemicals (Sect.
3.3.3) might be expected to show corresponding differences. However,
the available literature showed that, for those chemicals compared, man
and animal acute toxicity was similar. This does not imply that there
are no chemicals for which the human and animal response would be dif-
ferent, but rather indicates the scarcity of information comparing human
and animal acute toxicity studies. Two possible reasons for the apparent
paucity of comparable data are that (1) many acute toxicity studies
performed on animals are done primarily to establish the LDsg and pro-
vide reference toxicity dose information for subchronic and chronic
testing and (2) most acute human exposures, with the possible exception
of dermal testing, occur only from accidental intake or suicides and,
unless the person is admitted to a hospital soon after intake, much
important data go unrecorded. Although it is not possible to satisfactorily
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77
appraise the comparative susceptibilities between man and animal with
only the few chemicals used as examples in Sect. 3.3.3, the examples do
support one of the major premises of toxicity research — that effects
observed in animals serve as an indicator of what a probable response
would be in man.
3.4 PATHOLOGY
3.4.1 Introduction
Evidence of tissue damage from the acute exposure of a test animal
to a chemical would be useful information for the hazard evaluation of
most chemicals, especially for those chemicals for which there is known
or potential acute human exposure. The following sections consider some
aspects of histopathology in acute toxicity testing of chemicals and
offer some concluding remarks concerning the degree of necropsy required
when evaluating the acute toxicity of a chemical.
3.4.2 Histopathology
Histopathological changes from acute exposures can be induced by a
variety of compounds (some examples are given in Table 3.6), a fact not
surprising since the experimental doses given are usually of such mag-
nitude that they kill at least one-half of the test animals. The stress
to the animal's system would in many cases produce lesions of various
organs, if the animal lives long enough for histologic damage to occur.
On the other hand, there are compounds that, when administered at doses
high enough to cause death, do not show any histopathology related to
the chemical. An example of this type of action was described by Robens
(1979) where several strains of rats were given high doses (2000 mg/kg)
of a short-chain chlorinated hydrocarbon. Several animals died as a
result of the exposure, but in no animals, living or dead, was there any
detectable histopathology related to the test chemical. Another example
is shown by the action of tf-2-fluorenylacetamide in mice where LDs0 doses
resulted in essentially normal tissues, as revealed by histopathological
examination (Haley, Dooley, and Harmon, 1973).
-------
Maytansine
Mirex
Ochratoxin A
Aflatoxin BI
Table 3.6. Examples of histopathological changes from acute exposures to chemicals
Test agent
3',4'-Dichloro-
proplonanilide
Cadmium chloride
Dichlorvos
Species
Rat
Rat
Dog
Route
Oral
Intravenous
Intravenous
and oral
Dose
50-700 mg/kg
Not given
2.2-22.0 mg/kg
Histopathology
Congestive hepatitis, gastroenteritis,
and patchy emphysematous lung changes
Lesions in endothelial clefts of small
vessels in target organs such as testis
and gasserian ganglion
Pulmonary changes with generalized con-
gestion and hyperemia and cardiovas-
Reference
Chand, 1973
Gabbiani et al. ,
1974
Snow, 1973
Rat
Subcutaneous
0.38-1.0 mg/kg
Rat and Intraperitoneal
mouse
Rat
Oral
Monkey Oral
330-700 ppm
15-50 mg/kg
1-3 mg/kg
cular changes
Lesions in gastrointestinal tract mucosa,
thymus, spleen, bone marrow, and tes-
tis; arrest of mitotic cell division;
hemorrhagic lesions in parenchymatous
organs and brain; and chromatolysis and
vacuolation of dorsal root ganglion
cells
Patchy lesions on surface of livers that
extended into the liver interior sev-
eral millimeters
Severe catarrhal or erosive enteritis in
the duodenum and jejunum
Liver damage characterized by hemorrhage,
necrosis, and massive accumulation of
lipid
Mugera and Ward,
1977
00
Kendall, 1974
Kanisawa et al.,
1977
Rao and Gehring,
1971
-------
79
What information do acute toxicity tests, in particular the
histopathological data, provide? Robens (1979) writes that in the
development of a drug, acute toxicity studies can: (1) establish some
measure of toxicity that can aid in choosing doses for studies of longer
duration, (2) help to assess whether the drug has efficacy in the non-
toxic range, (3) enable certain conclusions to be made regarding
mechanism of action, if the animals are carefully observed following
treatment and then necropsied, and (4) answer the question of how much
difference exists between dosages that seem to be safe and those that
are known to be highly toxic. Histopathological information derived
from acute toxicity tests can also aid physicians confronted with cases
of accidental poisoning or attempted suicides by providing information
on chemical effects parameters such as target organs.
Histologic evidence of damage to various organ systems as a result
of acute exposure often provides evidence that permits prediction as to
whether the organ system will likely be similarly affected after chronic
dosing, but this is not always the case. Three studies that illustrate
that the results from acute toxicity testing cannot always be used
to predict the results of chronic toxicity testing are reported by
Kanisawa et al. (1977), Diamond and Sleight (1972), and Pennarola,
Balletta, and DiPaolo (1969). Kanisawa et al. (1977) found that oral
administration of near-lethal single doses of ochratoxin A (a mycotoxin)
to rats did not result in renal damage, but several consecutive daily
doses produced massive acidophilic degeneration with necrosis and
desquamation of epithelium in the proximal tubules. Second, in a study
of the acute and subchronic effects of methylmercury dicyandiamide in
the rat, Diamond and Sleight (1972) reported that atrophy of the granular
cells of the cerebellum and hepatocytes was present only in those animals
subjected to repeated exposures. Third, in the research of Pennarola,
Balletta, and DiPaolo (1969), acute exposure of the rabbit to parathion
resulted primarily in hyperemia of the internal organs with pulmonary
emphysema and edema, whereas chronic exposure produced lesions of a
moderate nature; these lesions were more pronounced in the excretory
organs because of the longer contact of these organs with parathion and
its metabolites.
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80
3.4.3 Conclusions
Many acute toxicity studies are designed to give a quantitative
measure of acute toxicity (LDsg) for comparison with other substances
and/or dose-ranging guidance for subchronic or chronic tests; thus, the
necessity of having detailed histological data is minimal. Those toxicity
studies, however, that seek to completely characterize the acute toxicity
of a chemical would of course necessitate necropsy. Because histopatho-
logical changes that occur from acute exposures to chemicals may not be
the same as those changes that result from longer exposures and because
many acute exposures cause death relatively quickly (especially at LDsg
levels — see Table 3.7) and thus may not cause tissue damage, the
necessity of performing histopathological examinations for each chemical
tested for acute toxicity can be questioned. However, because some
chemicals do have the potential for acute exposure to humans and because
some knowledge of soft tissue changes would be useful (e.g., for com-
parison with other chemicals tested for acute toxicity) , gross necropsy
seems warranted for all animals when significant signs of toxicity are
observed, with consideration of histopathology for organs showing evidence
of gross pathology in animals surviving 24 h or more. This view is
supported by Zbinden (1973) , who states that histopathological examina-
tion of the organs is not recommended as a routine procedure because
(1) the organ changes of animals dying from acute overdosage are so
ambiguous that an intelligent assessment would be difficult and (2) the
information likely to be gained over that furnished by gross necropsy
would only occasionally justify the additional technical effort.
3.5 OBSERVATION PERIOD
3.5.1 Introduction
The LDso is the dose that will result in the death of one-half of
the test animals, to which it is administered. Inherent in this defini-
tion is an observation period of some finite length; however, after
reviewing several papers that describe the acute toxicities of various
chemicals, it is obvious that the observation periods are not always the
-------
Table 3.7. LD5o observation periods
Chemical
Methylmercury
dicyandiamlde
Ochratoxin A
Aflatoxin Bj
Maytansine
Proximp'ham
Guanethidine
0-Methyl-ff-(l-
napthyl) f luoro-
acetamide
Probocol
Phenoxarsine
oxide
Pentachlorophenol
Nefopam
Phenazine-5ff-oxide
Cyclopiazonic acid
Aroclor 1242
Budralazine
Species
Rat
Rat
Monkey
Rat
Mice
Rat
Mice
Rat
Guinea pig
Mice
Dog
Rat
Rat
Rat
Rat
Route
Intraperitoneal
Oral
Oral
Subcutaneous
Oral
Oral
Oral
Oral
Oral
Oral
Intravenous
Oral
Oral
Oral
Oral
(mg/kg)
13.35
28
2.2
0.48
1 300
1 050
250 to 370
5 280
24
74
20
6 600
36 (male)
63 (female)
4 250
620 (male)
730 (female)
Observation
period
(days)
7
3
14
14
21
14
14
7
21
7
14
7
10
14
7
Time deaths
occurred Reference
(days)
Diamond and Sleight,
1972a
Kanisawa et al. ,
1977*
4 Rao and Gehring,
1971°
4 Mugera and Ward,
1977a
Lewerenz, Lewerenz,
and Plass, 1970*
Hartnagel et al. ,
1976a
5 Hashimoto et al. ,
1968*
Molello, Gerbig, and
Robinson, 1973a
8 Ballantyne, 1978fc
Ahlborg and Lars son,
1978*
2 h Case, Smith, and
Nelson, 1975a
Koeda et al., 1976*
6 Purchase, 1971b
Bruckner, Khanna,
and Cornish, 197 3a
3 Onodera et al. ,
1978*
00
-------
Table 3.7 (continued)
Chemical
2,3-Dihydro-9H-
isoxasolo-(3,2-
b)quinazolin-
9-one
Oxepinac
Fhotomirex
Ethylidene
gyromitrin
Sulfotep
Benzene
Cadmium
Di-2-ethylhexyl
phthalate
Dichlorodiphenyl-
trichloroethane
Ethanol
Clindamycin
hydrochloride
Dipterex
Dimethyl
terephthalate
Peroxyacetyl
nitrate
Chloropyrifoa
Species
Dog
Rat
Rat
Rabbit
Mice
Mice
Rat
Rat
Mice
Mice
Rat
Mice
Mice
Rat
Rat
Rat
Rat
Route
Oral
Oral
Oral
Oral
Oral
Intraperitoneal
Intraperitoneal
Oral
Intraperitoneal
Oral
Oral
Intravenous
Oral
Oral
Intraperitoneal
Inhalation
Oral
. _ Observation
£«•> SJ3
700 ± 110
136
150 to 200 (male)
70
29.4
15.2
2 940
225
37 770
237
10 600
6.08
2 618
649
3 900
95 ppm (LC50)
118 to 245
14
7
28
3.5
14
14
14
7
10
1
7
7
7
14
14
Time deaths
occurred Reference
(days)
1 Banerjee et al. ,
1977a
6 Nomura et al., 1978fc
Hallett et al. ,
1978^
3.5 Makinen, Kreula. and
Krauppi, 19776
1 Kimmerle and
Klimmer, 19 7 V3
<1 Drew and Fouts, 1974a
Kotsonis and Klassen,
1977a
Lawrence et al. ,
1975a
Tomatis et al. , 1972b
Wiberg, Trenholm. and
Coldwell, 19706
Beliles, 1972°
Gray et al., 1972a
5 Edson and Noakes,
I9606
2 Krasavage, Yanno,
and Terhaar, 197 3a
1 Kruysse et al. ,
1977°
McCollister et al. ,
ts>
(various
strains)
1974°
-------
Table 3.7 (continued)
Chemical
trans- 2-Hexenal
Thiabendazole
hydrochloride
Cefazolin
SMA 1440-H resin
Alromine RU 100
Fenoterol HBr
Ethylene dibromide
Hexachlorophene
A204
Cadmium ions
1,1,3,3-Tetra-
ethoxypropane
2-Methylaniline
Species
Rat (male)
Rat
Mice
Rat (female)
Rat
Rat (male)
Rat (male)
Rat (male)
Rat
Rat
Rat
Rat
Route
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
._ Observation
(mg/kg) P«£J
780
3 100
>10 000 (no
deaths at 10 000)
>20 but <22
>3 200
2 300
146
69.2
7.62
130 to 180
1 610
900
14
10
7
14
10
7
14
5
7-14
14
14
14
Time deaths
occurred Reference
(days)
2 Gaunt et al., 1971a
Robinson, Stoerk,
and Graessle,
1965a
Birkhead, Briggs,
and Saunders, 1973a
Within Winek and Burgun,
3 days 1977a
Hunter and Stevenson,
1 Kast et al., 1975a*
Rowe et al., 1952a
3 Nakaue, Dost, and
Buhler, 197 3a
3 Worth et al. , 1970a
Lorke, 1978fc
4 Crawford et al.,
1965a
Jacobson, 1972a
Vs.
paper.
00
Foreign paper.
-------
84
same. This section primarily discusses the length of the observation
period of test animals following acute exposure to a chemical.
3.5.2 Purpose of Observation Period
The purpose of the observation period is to allow time for manifes-
tation of the toxic effect, which in determination of the LDso is either
survival or death. The observation period thus needs to be of sufficient
duration that the metabolism and elimination of the test substance from
the test animal is near completion when the LD5Q is determined. The
importance of this is revealed by the fact that many compounds have dif-
ferent rates of elimination. An example is that of three tetrachlorophenol
compounds: 2,3,5,6-, 2,3,4,6-, and 2,3,4,5-tetrachlorophenol. Ahlborg
and Larsson (1978) observed that following intraperitoneal injection to
rats, 2,3,5,6-tetrachlorophenol was eliminated within 24 h and 2,3,4,6-
tetrachlorophenol within 48 h, but only 60% of 2,3,4,5-tetrachlorophenol
was eliminated and subsequently recovered in the urine within 72 h.
The observation period following acute exposure must also be of
sufficient length to allow for the fact that different routes of admin-
istration can result in the toxic effect of a chemical being manifested
at different times. In a study by Nomura et al. (1978), mice that
received a lethal dose of oxepinac, an antiinflammatory drug, either
intraperitoneally or subcutaneously, exhibited convulsion and dyspnea,
with most deaths occurring within 24 h, whereas mice that received an
oral lethal dose showed anorexia, emaciation, and bloody feces with
deaths 2 to 6 days postadministration.
3.5.3 Length of Observation Period
Table 3.7 summarizes reported data from 43 studies. Examination of
this information shows that 12 studies had an observation period of
7 days and 18 studies had an observation period of 14 days, with the
remaining 13 studies having observation periods ranging from 3 to 28 days.
The Guidelines for CaroinoQen Bioassay in Small Rodents (Sontag,
Page, and Saffiotti, 1976) and guidelines proposed by the U.S. Environ-
mental Protection Agency (1978) for registering pesticides in the United
-------
85
States recommend at least a 14-day observation period. This 14-day
period is thus the choice of some U.S. regulatory and chemical testing
agencies. It is not surprising that many toxicological researchers in
the United States, many of whom contribute to the formulation of these
agencies' guidelines, also adhere to this 14-day period. Table 3.7
shows that 14 of 24 studies by U.S. researchers used the 14-day observa-
tion period.
In addition to showing variation in observation periods, the informa-
tion in Table 3.7 also raises the question of which length is the most
desirable. As previously mentioned (Sect. 3.5.2), the observation
period must be of sufficient duration to allow for manifestation of the
toxic effect; however, the time period cannot be so long that the
animal's death could be attributed to factors other than the primary
effect of the chemical being tested. Further examination of some of the
papers cited in Table 3.7 yields some interesting facts with regard to
time of death following acute administration. Rao and Gehring (1971)
subjected male cynomolgus monkeys (Macaco, ivus) to acute oral doses of
aflatoxin BI and found that all deaths occurred within the first 4 days
of the 14-day observation period. Similar results were seen when
2,3-dihydro-9H-isoxazolo(2,3-b-quinazolin-9-one was administered orally
to dogs (Banerjee et al., 1977). Within 24 h after dosing, 4 out of
4 dogs died at at dose of 1150 mg/kg and 2 out of 4 dogs died at a dose
of 750 mg/kg. The LDsgs of mice and rats were also determined in this
study, but no indication is provided as to when the animals died within
the 2-week observation period. Research with budralazine (Onodera et
al., 1978) and nefopam (Case, Smith, and Nelson, 1975) also indicated
that death from acute exposure may occur within a few hours or days.
The LDso studies with budralazine showed that most deaths occurred
within 48 h, with all survivors appearing normal 72 h after dosing,
whereas death of mice, rats, and dogs occurred even more quickly with
nefopam — 1 to 10 min after intravenous injection, 15 to 30 min after
intramuscular injection, and 30 to 120 min after oral dosage.
Still further evidence of short survival time following acute
administration is provided by the works of Kimmerle and Klimmer (1974)
-------
86
and Diamond and Sleight (1972). Kimmerle and Klimmer observed that
mice, rats, rabbits, cats, dogs, and hens exposed to sulfotep, either
orally or intraperitoneally, died within 24 h and that survivors
recovered within 1 to 4 days. Diamond and Sleight (1972) discovered
that, if rats acutely exposed to methylmercury dicyandiamide were going
to die, death would occur within 72 to 78 h.
3.5.4 Conclusions
Although only a relatively small number of papers were reviewed, it
seems evident that many U.S. researchers employ the 14-day observation
period following acute administration of a chemical, whereas many non—U.S.
researchers appear to use observation periods of various lengths. It is
also evident that in several instances test animals died well within the
specified observation period. Therefore, the 14-day observation period
suggested by most regulatory agencies seems sufficient for the determina-
tion of the LD for most chemicals.
-------
87
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ology. Aust. Vet. J. 49(3) .-120-125.
Sontag, J. M., N. P. Page, and U. Saffiotti. 1976. Guidelines for Carcin-
ogen Bioassay in Small Rodents. National Cancer Institute, NC1-CG-TR-1.
65 pp.
Tomatis, L., Y. Turusov, N. Day, and R. T. Charles. 1972. The Effect of
Long-Term Exposure to DDT on CF-1 Mice. Int. J. Cancer 10(3):489-506.
U.S. Environmental Protection Agency. 1978. CFR 40 No. 163.81.
Wiberg, G. S., H. L. Trenholm, and B. B. Coldwell. 1970. Increased
Ethanol Toxicity in Old Rats: Changes in LDso, In Vivo and In Vitro
Metabolism, and Liver Dehydrogenase Activity. Toxicol. Appl. Pharmacol.
16:718-727.
Winek, C. L. , and J. J. Burgun. 1977. Acute and Subacute Toxicology and
Safety Evaluation of SMA 1440-H Resin. Clin. Toxicol. 10(2):255-260.
Worth, H. M., D. B. Meyers, W. R. Gibson, and G. C. Todd. 1970. Acute
and Subacute Toxicity of A204. In: Antimicrobial Agents and Chemo-
therapy - 1970. pp. 357-360.
Zbinden, G. 1973. Acute Toxicity. In: Progress in Toxicology, Springer-
Verlag, Inc., New York. pp. 23-27.
-------
4. SUBCHRONIC TEST DESIGN
4.1 INTRODUCTION
The standard protocol for toxicity testing of chemicals generally
proceeds from single-dose tests of short duration (acute) to repeated-
dose tests of long duration (chronic). Part of this procedure is a
repeated-dose study of intermediate duration, referred to as a sub-
chronic or subacute test. The subchronic test provides information on
the toxic effects of chemicals that are likely to occur from repeated
exposures over a limited time period. Currently 14-, 28-, and 90-day
studies are commonly used; however, a review of the literature indicates
the duration of the study may vary widely.
The first recorded definition of a subchronic test was as follows:
a repeated exposure test with a duration of one-tenth the expected life
span of the experimental animal used (Food and Agriculture Organization/
World Health Organization Technical Report, 1958). Boyd (1961) thought
that for the rat this meant a duration of approximately 100 days. A 13-
week test with four dosage levels was the definition given for subacute
tests by Abrams, Zbinden, and Bagdon (1965). Loomis (1974) described
the repeated exposure test as a "prolonged test1' with daily exposure for
about 3 months. Guarino (1979) stated that traditionally three terms
(short-term chronic, subchronic3 or prolonged) are used interchangeably
to designate a 90-day test. Many similar definitions, based upon a
repeated dose given over a period of a few days to 3 months, have
appeared in the literature, including some by expert committees (National
Academy of Sciences, 1975; 1977). The cumulation of these efforts was
the definition and guidelines of the U.S. Environmental Protection
Agency (EPA) pesticide programs (Federal Register, 1978). This guide-
line set standards for subchronic tests covering dosage levels, routes
of exposure, number of species, number of test animals, type and degree
of pathology, hematology, biochemical and function evaluations, etc.
The choice of subchronic as the term of preference is followed in this
text and replaces all other terms. "However, the important thing is not
the choice of definition of a particular fraction of the life span but
93
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94
the selection of a testing interval that is as short as practicable and
yet will give meaningful information about the effect of absorbing the
toxicant during an entire lifetime" (Hayes, 1975). In addition to
summarizing the definition of a subchronic test, this quotation describes
the purpose of this chapter, which is to evaluate the currently employed
subchronic tests to determine whether they provide sufficient informa-
tion that makes longer-term tests unnecessary. The section does not
consider a comparison of time versus effect for dermal, ocular, or
inhalation toxicity and does not consider carcinogenic effect.
The two primary functions of subchronic tests, as they are currently
used, are (1) to assess toxic effects from repeated exposure over a
relatively short time so as to give maximum and minimum effect levels
with safety margins plus a prediction of possible effects from longer
exposures and (2) to determine the appropriate dose levels to be used if
longer exposures are planned (Benitz, 1970; Feck, 1968). The purpose of
this chapter is to evaluate the first of these two functions. To fulfill
this first function, a battery of biochemical, hematological, and patho-
logical tests is performed. These tests provide data upon which more
selective evaluations can be based. The target organ data are especially
useful. The value of such data has been recognized for several decades,
and, as stated by Barnes and Denz (1954), "It is in fact doubtful
whether it is worth proceeding beyond the subacute test." The second
function is an elaboration of the acute evaluation of effective doses
and should give maximum and minimum effect levels with safety margins.
The role of the subchronic test as a preliminary evaluation of appro-
priate dose levels for use in longer test designs (such as a carcino-
genicity bioassay) is evaluated in a separate review by Prejean (Appendix
A). However, as the time and cost requirements of toxicity testing
increase, so does the value of subchronic tests as reasonable evalua-
tions of chronic effects. If a more efficient subchronic test design
can be constructed, the need for longer, more costly chronic studies may
be reduced.
The standard or typical experimental design for a subchronic test
has often been discussed in the literature. Recent examples include a
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95
discussion of procedures for the preclinical toxicologic evaluation of
cancer chemotherapy agents (Prieur et al., 1973); a proposed system for
food safety assessments (Food Safety Council, 1978); a review of
principles and methods for evaluating chemical toxicities (World Health
Organization, 1978); and proposed designs being developed by the Inter-
agency Regulatory Liaison Group (IRGL) and Organization for Economic
Cooperation and Development (OECD). A representative sample of these
test designs is included in Table 4.1. Overall these systems are quite
similar. Selected areas of these standard designs will be reviewed in
this document to determine if modifications are possible. Thus, the
designs in Table 4.1 represent the baseline system for which changes
will be suggested.
4.2 SPECIES
4.2.1 Introduction
The debate over which and how many species to employ in subchronic
toxicity tests has been a part of toxicology for many years. Discussions
generally center on: (1) what species best represents human responses,
(2) what is the most sensitive species for a given test or effect, and
(3) what is the value of rodent and nonrodent combinations. To evaluate
these problems, this review will examine literature reviews and discus-
sions concerning species applicability and data from species comparison
studies.
4.2.2 Discussions in Literature Reviews Concerning Species Suitability
Barnes and Denz (1954) in their review of chronic tests discussed
the choice of test animals. They ranked the rat as the "first choice"
because of economic and scientific factors. The choice of a second,
nonrodent species should be based on the similarity to human responses.
Both the monkey and dog have disadvantages, particularly if the monkeys
are imported or the dogs are mongrel breeds. Further, Barnes and Denz
questioned how often these two species added to the information gained
by the rat. Only if inhalation studies (monkey) or large tissue/blood
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Table 4.1. Experimental design for recently proposed subchronic oral toxicity tests
Agency
Federal Insecticide,
Fungicide, and
Rodenticide Act
(Toxic Substances
Control Act) guide-
lines, 1978-1979a
Interagency Regulatory
Liaison Group, 1979a
Species
Two (rat and
nonrodent)
Two (rat and
dog)
Sex Age at start
Both Rats - 6 weeks
Dogs — 4 to
6 months
Both Rats - 6 weeks
Dogs — 4 to
6 months
Number of animals
per group
(sex per dose)
Rats - 20
Dogs — 6
Rats - 20 (30 days
to 6 months) , 10
(<30 days)
Number
Duration of
doses
Rodents - 90 days 3
Dogs — 6 months
Generally 90 days; 3r
shorter for spe-
cial situations
Dose levels
Highest - effect level,
but not more than
10Z mortality
Lowest — no evidence
of toxicity
Highest — toxic level,
but not excess
mortality
World Health Organi-
zation, 1979
Food Safety Council,
1978
Two (rodent and Both Rats - just after 10
nonrodent) weaning
Two (rodent and
nonrodent)
Both (Not indicated)
Rodents - 20
Nonrodents — 3 to
10% life span or 3
months
Rodents - 90 days
to 1 year
Nonrodents — 1 year
Lowest — no evidence
of toxicity
Highest — distinct
toxic level
Lowest — no detectable
toxic reaction
Highest — clearly
toxic, but not lethal
Lowest — no observable
adverse effect, but a
reasonably large mul-
tiple of estimated
human daily intake
vo
Proposed or unofficial guidelines.
Must have control groups also.
Source: Adapted from Page, 1979.
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97
samples (dog) are needed, do these species add significant data. They
concluded that the use of a second species should be flexible, with the
toxicologist choosing the best species, depending on the chemical or
test design requirements.
In later discussions more emphasis is placed on the relationship
between the test species and the human response rather than on economic
or logistic variables. A good example of this change is evident in a
discussion of inhalation tests (Roe, 1968), in which specific compari-
sons of the respiratory systems of several laboratory species are made.
By examining particle deposition patterns and mucous secretion mechanisms,
Roe dismissed the mouse, guinea pig, rabbit, and cat as suitable test
animals. On the basis of anatomical considerations, monkeys and dogs
resemble man more closely than do other species, and Roe recommended
these for "small-scale, short-term experiments." The value of the rat
apparently lies between these two groups, because it is more similar to
man in anatomy and response than the first group and of a more manageable
size and more certain genetic makeup than the dog or monkey. Thus, the
importance of similarity to the human response in species selection is
elevated in the present discussion to at least the same level as economic
and other practical considerations.
In a later review of chronic toxicity, Benitz (1970) documented the
frequency of use for various species and the reasons behind the choices.
He gave the following percentages for the use of each species based on
134 studies of 1 month or longer duration (these studies appeared in
Toxicology and Applied Pharmacology between 1959 and 1966): rat, 43.3%;
dog, 38.1%; monkey, 6.7%; mouse, 3.7%; and rabbit, 3.0%. The emphasis
on the rat and dog is supported in a report by Bushby, Lechat, and
Santarato (1966) in which 71.7% of all toxicity testing laboratories
used the dog and rat and only 16% used the rat, dog, and monkey. Benitz
attributed this to investigator familiarity with the rat and dog as test
species rather than to these species' superiority in sensitivity or
comparability to human response patterns.
Following the method of Pinkel (1956), several authors have evalu-
ated species suitability in anticancer drug studies by using doses based
on milligrams per square meter (surface area) instead of milligrams per
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98
kilogram (body weight). This technique is useful because surface area
correlates better with metabolic rate than body weight does. Therefore,
by giving doses on a surface area basis the researcher obtains tighter
control of the dosage variable, allowing a truer evaluation of species
differences. The results obtained when dosage sensitivity was compared
in this manner suggested that in most cases the monkey, the rat, and the
dog all give similar results (Freireich et al., 1966). However, in such
comparisons between the dog and monkey using the maximum tolerated dose,
Roman (1972) found that the dog was generally more sensitive and should
be preferred over the monkey.
In an evaluation of the predictive value of test species for the
metabolism of drugs, Smith (1979) compared the dog, monkey, and man. In
contrast to Roman (1972), Smith preferred the monkey. By discussing and
rating metabolic fate in 34 compounds for these species (Table 4.2),
Smith concluded that "(1) for a large group of relatively dissimilar
compounds the rhesus monkey is a significantly better predictor of
metabolic fate in man than either the dog or the rat and (2) that there
is little to choose between the latter two species, in both cases the
correspondence is poor for over half the compounds regardless of their
chemical classification."
Zbinden (1963) discussed species selection in evaluations of drug
toxicities. He favored the use of the dog and the rat, with additional
use of the monkey if confronted by unexplained toxic effects. However,
Table 4.2. Ranking of various animal models as predictors of
metabolic fate In man
Species
Rating
Good
Fair
Poor
No data
Total
Rat
Number
of compounds
5
7
19
3
34
Percent
14
21
53
12
100
Dog
Number
of compounds
6
8
20
34
Percent
18
23
59
100
Monkey
Number
of compounds
22
9
3
34
Percent
65
26
9
100
Source: Adapted from Smith, 1979. Reprinted with permission of the publisher.
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99
the key factor is metabolic similarity to man. Since absorption,
penetration, and distribution processes are similar in most species, the
primary differences between species are due to variations in biotrans-
formation pathways and in rates of inactivation. The final selection of
test species should reflect the species' similarity to man in metabolizing
the chemical under study.
In another review of drug safety evaluations, Peck (1968) stated
that the basis for experimental species selection should be a metabolic
similarity to man. However, as he pointed out, the human metabolic pat-
tern is generally unknown for new drugs. His final recommendation was
to include several species in drug tests in order to protect against
unknown metabolic variations.
However, Boyd (1968), in discussing species selection in his review
of drug testing, did not base the choice on metabolic similarity alone.
He felt another factor of equal importance is the ability to vomit (which
parallels the human protective reflex). Boyd suggested the use of a
species that has vomiting capabilities (recommending the dog) and one
nonvomiting species that is metabolically similar to man. Although Boyd
discussed only the vomiting ability of the test species, this factor is
representative of many such physiological traits. His conclusions can
be expanded to include the recommendation that the test species should
have a physiological functional pattern that is as similar as possible
to the human pattern for the specific test chemical. If such informa-
tion is not known, then "use as many species as may be possible."
This reliance on several species is recommended again by McNamara
(1976), although he did point out that many researchers feel comfortable
using only the rat. McNamara cited Hebold (1972) regarding the regula-
tions of six countries and three international policy-setting bodies re-
quiring the use of the rat, dog, and rabbit, or monkey as test species.
Hayes (1967a) also feels the need exists to use several species in
studies of pesticide toxicity. This is based on both the general
variability between animal and human responses and the importance of the
species selected in determining toxic results (second only to dose
levels employed). Guarino (1979), in his discussion of drug development
programs, saw a definite value in using many species. He recommended
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100
the use of "(1) mice and rats for experimental therapeutic data; (2)
dogs, monkeys, and mice for toxicologic studies; and (3) dogs, monkeys,
rats, and mice for pharmacologic information." This scheme is suggested
in contrast to the "self-perpetuating choice," based on economic or
convenience factors, of the rat and dog as test species.
Balazs (1976) discussed the rat and dog combination for testing
chemicals. He favored the rat because large numbers can be used, and it
has a relatively short life span. The choice of the dog allows for
"in-depth studies of organ systems" and provides greater test sensitivity.
The use of a third species is variable but "the omission of these species
[rat and dog] is seldom justified." This policy is also recommended by
the Food Safety Council (1978), and in the EPA Proposed Guidelines for
Pesticides (Federal Register, 1978). The EPA position is stated as:
"Testing shall be performed in at least two mammalian species. One
species shall be a generally recognized strain of laboratory rat. The
second species shall be a nonrodent. The nonrodent species should
usually be the dog. Selection of a nonrodent species other than the dog
will require full and adequate justification which should consider such
factors as the comparative metabolism of the chemical and species
sensitivity to the toxic effects of the test substance, as evidenced by
the results of other studies." Thus, it is assumed that for most
substances the combination of the dog and rat is preferred, although the
scientific basis of this conclusion is not stated.
However, in contrast to many discussions concerning the dog and rat
as test species, Aviado (1978) saw little advantage to including the
dog. In a two-part literature review, Aviado evaluated 110 studies in
which the dog and rat were used as test species during the period 1966
to 1978. In most studies the data obtained from the rat predicted
cardiovascular, bronchopulmonary, hepatorenal, digestive, nervous, and
somatic system toxicities as well as did data obtained from the dog.
Further, the rat studies were better than the dog studies for prediction
of pancreatic hypertrophy, hepatic porphyria, and nephropathy. He
concluded that the use of the dog or the rat in chronic studies is
sufficient and that using both species results in unnecessary duplica-
tion of scientific manpower and research effort.
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101
The review by the World Health Organization (1978) on toxicity
evaluations summarizes most of the primary factors discussed in litera-
ture reviews for the selection of test species. Of paramount importance
is the similarity of metabolism between the test species and man. If
sufficient data are available to allow a choice on this basis, the
species showing the greatest metabolic similarity to man should be used.
If such data are unavailable, then qualitative and quantitative response
patterns should be obtained for several test species to increase the
predictive value for the occurrence of similar toxic effects in man. In
most cases this necessitates the use of the dog and rat as a minimum in
subchronic studies. However, despite such reviews as the World Health
Organization document and Aviado's literature review, the conclusion of
Fancher (1978) in his review of animal models for toxicological studies
is in essence the current general practice in species selection: "In
spite of all that has been done, choice of species continues to be
based, in large part, on precedence, convenience, and economics."
4.2.3 Species Comparison Studies
Ansbacher, Corwin, and Thomas (1942) used the dog, cat, rabbit, and
monkey to test menadione and menadiol for toxicity. After subcutaneous
dosing for 4 to 11 days, all species except the monkey developed a weak
anemia, with the dog also showing hemoglobinuria. The dog and cat
showed some mortality at the higher doses. In the discussion of the
results, Ansbacher et al. felt that the hematologic changes could be
attributed to normal compensatory mechanisms. Thus, the mortality
exhibited by the cat and dog implies a greater sensitivity for these two
species than for the rabbit and monkey.
In an inhalation study of beryllium toxicity, Stokinger et al.
(1950) used 11 species, examining mortality, growth retardation, hema-
tology, and urinalysis factors. The animals were exposed 6 h per day
for 14 days at 100 mg/m3, 51 days at 47 mg/m3, 95 days at 10 mg/m3, and
100 days at 0.95 mg/m3. For mortality the rat and cat were the most
sensitive species at intermediate and high doses, with the dog showing
equal mortality rates at the high dose only. In growth response, the
monkey was the most affected species at the intermediate and high doses.
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102
The dog showed the most toxic effects in hematologic and urine analyses
In summation, the dog appeared to be the most sensitive species, but the
use of only five animals per test dose may limit the validity of this
study. Stokinger and Stroud (1951) tested the dog, rat, and rabbit once
again for hematological effects from beryllium inhalation. The exposures
were 6 h per day, 5 days a week for 6 weeks (continued for 23 weeks in
the rabbit) at 2.2 to 4.0 mg/m3. The result was a decrease in red blood
cells and an increase in mean corpuscle volume. Their comparison showed
that the degree of anemia varied among species, with the dog being the
most susceptible and the cat the least susceptible.
Rowe et al. (1952) compared the sensitivity of the rat, guinea pig,
rabbit, and monkey to ethylene dibromide using repeated 7-h per day
inhalation exposures with concentrations of 100, 50, and 25 ppm. Sub-
chronic exposures of less than 91 days showed toxic effects only at the
higher doses. All species showed some weight loss, but only changes in
the guinea pig were significant. Mortality levels were statistically
significant at the high dose in rats, as were increases in liver and
kidney weights. This increase in organ weight was also shown by the
guinea pig. Although Rowe et al. concluded that no species did partic-
ularly well at high dose, it is apparent that the rat and guinea pig
showed the greatest sensitivity to ethylene dibromide.
Litchfield (1961) designed a study to compare experimental animal
response and human response to toxic chemicals. By comparing the occur-
rence of toxic effects from tests performed on the rat and dog, and from
known human epidemiological studies, Litchfield was able to evaluate the
relationships for six chemicals. The tests included acute studies,
subchronic studies (1 to 6 months), and a 1-year rat study. The appear-
ance of toxic effects in each species and in each species combination
was the basis for his evaluation. The results are shown in Table 4.3.
The data support his conclusion that the dog better represented man's
response than did the rat. However, in summation, Litchfield noted that
the small number of chemicals examined reduced the statistical value of
this comparison, and he urged that more species be examined for use in
toxicity studies.
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103
Table 4.3. Occurrence of 39 physical signs from six drugs in three species
Physical sign
Weight loss
Weight gain
Muscle atrophy
Myositis
Lymphocytopenia
Neutropenia
Leukopenia
Anemia
Leukocytosis
Hyperglycemia
Liver damage
Jaundice
Fatty liver
Polydipsia
Polyuria
Oliguria
Hematuria
Crystalluria or renal
concretions
Renal damage
Gastroduodenal ulcer
Diarrhea
Salivation
Ataxia
Impaired reflexes
Decreased activity
Tremors
Ptosis
Catatonia
Priapism
Lacrimation
Urinary incontinence
Bacterial invasion
Parasitic invasion
Decreased thyroid function
Genital hypoplasia
Decreased adrenal function
(cortical)
Hypotension
Lung edema
Tachypnea
Total
Source: Adapted from
Absent Rat
3 1
4
5
4
5
3
1
3
5
2 1
5
5 1
5
2
4
3
5
3 1
4
1
5
2
3
3
3
5 1
5 1
5 1
5
4 1
5
4
4
4 2
5
2 1
5
5
146 11
Litchfield, 1961
Association. Reprinted with permission of
Rat
Dog and Man
dog
2
2
1
1
1
1 1
2 1
1 2
2
1
3
1
1
1
1
1 4
1
111
1 1
2
2
1
1
1
1
2
1
16 8 23
. Copyright 1961,
the publisher.
Rat,
Rat Dog d '
and and and'
"*" man man
4
1
1
1
1
2
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1 12 17
American Medical
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104
In a similar study of anticancer drug toxicities, Owens (1962) com-
pared the response of the rat, dog, and monkey to man in terms of bone
marrow, gastrointestinal, hepatic, renal, and neural toxicities. Table
4.4 summarizes the results of 4-week exposure tests. Owens concluded
that except for neurotoxicity all three species were suitable, and in
particular he saw no advantage in the use of the monkey rather than the
dog.
Weil and McCollister (1963) evaluated several factors in toxicity
tests, including species sensitivity. Although their study concentrated
on diet exposure studies in the rat, they also included some data on dog
Table 4.4. Organ system toxicities
Compound Rodent Dog Monkey Human
Bone marrow toxicity
Mechlorethamine + + + +
Cyclophosphamide + + + +
Myleran + + + +
1-Sarcolysin + + + +
2-Chloroethylmethanesulfonate + + ?
Methotrexate + + + +
6-Mercaptopurine + + +
4-Aminopyrazolo (3,4-D)pyrimidine + + +
Vincaleukoblastine + + +
Carzolamide - +
Actinomycin P2 + + +a
Mithramycin - +
Roseolic acid - + +
! Gastrointestinal toxicity
Mechlorethamine + + + +
Cyclophosphamide + + + +
Myleran + + + +
1-Sarcolysin + + + +
2-Chloroethylmethanesulfonate + + +
Methotrexate + + + +
6-Mercaptopurine + + +
4-Aminopyrazolo (3,4-D)pyrimidine + + +
Carzolamide + +
6-Azauracil i ~ "*"
Vincaleukoblastine + + +
Actinomycin P2 + + +
Mithramycin - ~ +
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105
Table 4.4 (continued)
Compound Rodent Dog Monkey Human
Nervous system toxicity
Mechlorethamine + + + +
Chloroquine mustard + + +
Carzolamide - - +
6-Azauracil + - - +,
Vincaleukoblastine - - +•,
Nitrofurazone - - 4-
NSC 38280 - - +°
Skin and appendages toxicity
(dermatitis and alopecia)
Cyclophosphamide +
Methotrexate - - +
Vincaleukoblastine - - +
8-Azaguanine - - +
Actinomycin P2 +
Methylglyoxal-bis-guanylhydrazone - - +
Hepatic toxicity
2-Chloroethylmethanesulfonate + + +
6-Mercaptopurine + + +
4-Aminopyrazolo (3,4-D)pyrimidine + + +
Carzolamide + + +
Mithramycin + + +
Roseolic acid - + +
Renal toxicity
2-Chloroethylmethanesulfonate + + +
Aminoiminomethanesulfinic acid + + +
Puromycin nucleoside + + +
Predominantly thrombocytopenia.
Peripheral neuropathy.
Extraocular palsies.
Source: Adapted from Owens, 1962. Reprinted with permission
of the publisher.
studies, for durations up to 2 years. They found 21 studies in which
the rat and dog were tested together. In none of these tests was the
dog more sensitive, and in seven tests it was less sensitive than the
rat. They concluded that the use of the dog should be limited to 90-day
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106
studies, and only when it was indicated as a more sensitive species
should it replace or augment the rat in long-term studies.
The use of the pig as a test species was discussed by Earl et al.
(1964). By injecting 1, 2, and 4 mg/kg of amphotericin B intravenously
for 3 days per week over 13 weeks, they were able to test the similarity
of the pig's response to that of man. Unlike previous rat and dog
studies on amphotericin B, the toxic effects appearing in the pig (renal
damage and anemia) were the same as human responses. They concluded
that the pig may be a useful test species because of these similarities
to man and because of the ease with which blood and organ samples are
taken.
In a study of the pesticide zenophos, Kohn, Kay, and Calandra
(1965) orally dosed the rat and dog for 90 days with diet levels of 0.5,
2, 8, and 25 ppm. At the high and intermediate doses both species
exhibited lowered food consumption, depressed growth rates, and depressed
cholinesterase activity. Additionally, the dog showed changes in its
hematologic pattern. The effects shown were attributed (except for
cholinesterase activity) to poor diet palatability and not to species
differences.
McNerney and MacEwen (1965) studied the inhalation toxicities of
ozone, carbon tetrachloride, and nitrous oxide in five test species
after continuous exposure for 2 weeks. The effects were generally the
same for all five species, except for the greater susceptibility to
nitrous oxide and a resistance to ozone exhibited by the monkey. In
contrast, the dog showed the opposite pattern of effects. Also, a
strain difference in the rats appeared significant, as Wistar rats were
extremely susceptible to carbon tetrachloride while Sprague-Dawley rats
were not.
A study using the rat and dog to test the oral toxicity of EX5004,
a sympatholytic drug, showed that after a 3-month exposure the dog was
more affected than the rat (Yeary, Brahm, and Miller, 1965). The
exposure levels were 10, 100, 500, or 1000 mg/kg in the diet for the rat
and for the dog 10, 100, or 500 mg/kg given by gelatin capsule. The dog
showed tachycardia and elevation of alkaline phosphatase activity at the
high and intermediate doses while the rat did not. This response was
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107
closer to the expected human response pattern, indicating that for these
variables the dog was the most sensitive test species.
Atkinson et al. (1966) studied the effects of cephaloridine, an
antibiotic, by using daily intramuscular or subcutaneous injections into
four species (cat, dog, rat, and rabbit) for 56 to 84 days. The major
significant effect was an increase in kidney weight in the cat (at 50
and 150 mg/kg), rabbit (at 30 and 45 mg/kg), and rat (at 50 and
150 mg/kg). However, the rat was the only species showing kidney
damage. There was also a slight, transient leukocytopenia in the rat
and dog and some enlargement of the liver (without histopathologic
damage) in the rabbit and cat. In this study it appeared that the rat
would have been sufficient as a test species.
Newberne, Gibson, and Newberne (1967) studied an analgesic drug in
the dog, rat, and monkey using oral dosing for 1 to 4 months. As shown
in Table 4.5, the dog was overwhelmingly more sensitive than the rat or
monkey, especially for histopathologic and clinical chemistry effects.
The only significant effect that appeared in the monkey and not in the
dog was the demyelination of the cerebral gyri. Thus, overall the dog
was the most sensitive species in this study.
Hagan et al. (1967) compared the toxicities of food flavorings
using oral exposure by diet or gavage for the dog (beagle) and the rat
(Osborne-Mendel). For 6-methylcoumarin, they found at the intermediate
dose (10 000 ppm for rat and 150 mg/kg for dog) that 20 rats showed no
effect after 14 weeks while the dog tested became moribund after 5 weeks,
exhibiting liver damage, skeletal muscle damage, and general weakness.
For methyl salicylate the rat showed growth retardation after 17 weeks
at the intermediate dose (10 000 ppm). The dog showed weight loss and
slight microscopic liver damage after no more than 29 days at 800 mg/kg.
One dog died after 4 days of dosing. With safrole ingested at inter-
mediate doses (5000 ppm), the rat exhibited growth retardation, liver
pathology, slight leucocytosis, and anemia after dosing for 2 years.
The dog, however, showed moderate kidney and liver damage and general
weakness after 96 to 116 days at 40 mg/kg. In these studies, the dog
generally appeared to be more sensitive to the additives than the rat,
even with shorter exposure periods.
-------
Table 4.5. Comparison of significant variations in the response of four species to an oral analgesic12
Parameter Dog
Clinical observations
Depressed weight gain or
weight loss +a
Depression or
unconsciousness +
Ernes is +
Icterus + —
Death +
Clinical chemistry
Elevated serum glutamic +
pyruvic transaminase
Elevated serum alkaline
phosphatase +
Bilirubinuria +
Necropsy observations
Discolored, yellow liver +
Histopathologic observations
Hepatic necrosis +
Myelin figures in
hepatocytes +
Excess bilirubin — liver
and kidney +
Hemosiderosis +
Depressed spermatogenesis +
Demyelination of cerebral
gyri
Dosage*
(mg/kg/day)
30
30
30
30
30
10
10
60
30
30
30
30
30
30
60
»» i Dosage
Monkey , /, / . »
(mg/kg/day)
+ 25
+ 30
+ 25
60
+ 30
60
60
60
60
60
+ 30
60
60
60
+ 30
Dosage*
(mg/kg/day)
+ 30
100
100
100
100
100
100
NTe NT
100
100
100
100
100
100
100
Rabbit
_
-
-
-
-
NT
NT
NT
—
NT
NT
NT
NT
NT
NT
Dosage*
(mg/kg/day)
80
80
80
80
80
NT
NT
NT
80
NT
NT
NT
NT
NT
NT
al-2-(l-Methyl-2-piperidyl)-l,l-diphenylethyl propionate hydrochloride.
Minimum dose at which an effect occurred or the highest dose given.
^Indicates a response in one or more animals at the indicated dose.
Indicates no abnormal response.
eNo test.
Source: Adapted from Newberne, Gibson, and Newberne, 1967. Reprinted with permission of the publisher.
o
oo
-------
109
In a toxicological study of aldrin and dieldrin, Hodge, Boyce,
Deichmanne, and Kraybill (1967) noted the subchronic response of several
species by comparing histopathologic data, body weight changes, mortality,
and organ weight changes. Using these criteria, they found wide vari-
ability in species response. They found the monkey was the most sensitive
for body weight and mortality criteria, and both the dog and rat were
sensitive to histopathology and liver weight criteria. In this study it
was the sensitivity of the species that was evaluated and not the
similarity to human responses. The significance of this study lies in
the selection of the rat, dog, and monkey as the best indicators of
toxicity from among a list of ten species.
Benitz, Roberts, and Yusa (1967) studied the morphological changes
in the rat, dog, monkey, and mouse after dosing for 27 to 38 days with
minocycline, a tetracycline antibiotic. In the rat, minocycline produced
a black discoloration of the thyroid (pigment deposition) with oral
doses of 8, 25, and 75 mg/kg per day. Hyperplastic changes were asso-
ciated with this discoloration. In the dog, the discoloration and
hyperplasia occurred with intravenous doses of 5, 10, 20, and 40 mg/kg
per day. Additionally, various degrees of hemolytic anemia occurred in
the dose range 10 to 40 mg/kg per day. In the monkey, the discoloration
occurred (pigmentation less pronounced) at an oral level of 30 mg/kg per
day, but no hyperplastic changes were observed. However, in the mouse,
no changes of any kind were noted with 250 mg/kg per day of oral dosing.
The results in this limited evaluation of toxicity indicated that the
rat and dog were the most sensitive species, followed by the monkey.
The mouse was the least sensitive species tested.
Schein et al. (1970) evaluated the monkey (four species) and dog
(mongrel and beagle) for their potential value in predicting qualitative
toxicities in man using 25 anticancer drugs. They used data from their
own tests (3 to 90 days of intravenous or peroral administration), data
found in the literature, and data from clinical studies in man. They
evaluated approximately 170 parameters to determine toxicities and, as
shown in Table 4.6, found good predictive value by both animals for most
organ systems. The dog was generally a better predictor for the occur-
rence of a toxic effect in man, while the monkey was better at predicting
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110
Table 4.6. Predictive abilities of the dog, monkey, and combination
Organ system
TP*2 FP
(%) (%) (%) CO TP + FN
Number of
compounds
Dog as a predictor for organ-specific toxicity in man
Injection site 16 36 40 8 33
Integument 12 32 40 16 57
Cardiovascular 28 24 36 12 30
Respiratory 16 64 16 4 20
Bone marrow 80 12 0 8 9
Lymphoid 4 72 24 0 0
Gastrointestinal 92 8 0 0 0
Liver 52 44 4 0 0
Renal 32 56 4 8 20
Neuromuscular 24 60 12 4 14
25
25
25
25
25
25
25
25
25
25
Monkey as a predictor for organ-specific toxicity in man
Injection site 13 26 52 9 40 23
Integument 13 17 57 13 50 23
Cardiovascular 22 26 30 22 50 23
Respiratory 13 48 30 9 40 23
Bone marrow 83 13 0 4 5 23
Lymphoid 0 31 65 4 100 23
Gastrointestinal 74 9 0 17 19 23
Liver 52 35 13 0 0 23
Renal 35 48 13 4 11 23
Neuromuscular 22 30 39 9 28 23
The combination of dog and monkey as a predictor for
organ-specific toxicity in man
Injection site
Integument
Cardiovascular
Respiratory
Bone marrow
Lymphoid
Gastrointestinal
Liver
Renal
Neuromuscular
16
24
36
16
88
4
92
52
36
24
36
36
32
76
12
76
8
48
56
60
40
36
28
4
0
20
0
0
4
12
8
4
4
4
0
0
0
0
4
4
33
14
10
20
0
0
0
0
10
14
25
25
25
25
25
25
25
25
25
25
True positive, toxicity was observed in both the animals and
in man.
False positive, toxicity was observed in the animals but not
in man.
True negative, no toxicity was observed in the animals and man.
HFalse negative, toxicity was not observed in the animals but
was recorded in man.
^Corrected false negatives — an index of false negative predic-
tion which analyzes for only those compounds which produced the
specific toxicity in man.
Source: Adapted from Schein et al., 1970. Reprinted with
permission of the publisher.
-------
Ill
the absence of a toxic effect in man. This complementary pattern is
demonstrated by the correct prediction of toxicity in nine out of the
ten organ systems by the combination of data from the dog and monkey
studies. However, this predictive ability was achieved only with a high
percentage of false positives.
Worth et al. (1970) tested the antibiotic A204 by oral exposure for
90 days in the rat (0.06, 0.025, 1.0 mg/kg per day) and dog (0.5, 0.8,
1.25 mg/kg per day). They found no significant species differences
measured by tests of hematology, clinical chemistry, or histopathology.
However, the mortality rate was slightly greater for the rat, particularly
at the intermediate dose, and so was the depression of the growth rate.
Therefore, in this study, the rat appeared to be more sensitive than the
dog.
Vogin et al. (1970) used the rat (T-DKL) and the dog (beagle) to
study azotrek, a tetracycline phosphate complex-sulfamethizole formula-
tion, in tests for oral toxicity with dosing for up to 33 weeks. The
rat showed no effects at any dose level (125, 250, 500 mg/kg per day).
However, the dog could not tolerate the high dose, exhibiting decreases
in hematocrit and hemoglobin levels, increased thyroid weight, and one
fatality. The dog appeared to be the most sensitive species in this
study.
Lyon et al. (1970) tested acrolein for inhalation toxicity in the
rat, dog, guinea pig, and monkey using repeated exposure studies (8 h
per day, 5 days per week, for 6 weeks, with concentration levels of
0.7 and 3.7 ppm) and continuous exposure studies (24 h per day for
90 days, with concentrations of 0.22, 1.0, and 1.8 ppm). In the repeated
exposure test, the only effect at the 0.7-ppm concentration was a chronic
inflammation in the lungs of the dog and monkey. At the 3.7-ppm level,
the effects included a significant decrease in weight gain for the rat,
mortality in the monkey, and nonspecific inflammatory changes in the
lung, liver, and kidney (most severe in the dog and monkey). The dog
also developed bronchopneumonia. In the continuous exposure test at
0.22 ppm, the dog and monkey showed specific inflammatory changes in the
lung, while the monkey, guinea pig, and dog had nonspecific inflammation
in the liver, kidney, and heart. At 1.0 ppm, the dog and monkey showed
-------
112
ocular and nasal irritation and histopathologic damage to the trachea.
Also, the rat suffered growth depression. At 1.8 ppm, the irritation
and tracheal damage were again noticed only in the dog and monkey. The
rat again showed a decrease in weight gain, and all species showed
nonspecific inflammation in the lung, liver, and kidney. The authors
concluded that the dog and monkey were the most sensitive species.
A comparison of the miniature pig and the dog (beagle) was made by
Earl et al. (1971) using subchronic tests of 1 to 148 days for the dog
and 1 to 69 days for the pig. The exposure levels were 5, 10, 25, 50,
100, 300, and 500 mg/kg per day for the pig and 5, 10, 25, 50, 100, 300,
400, and 500 mg/kg per day for the dog. Both species suffered mortality,
with the pig first affected at 5-mg/kg levels while the dog was not
affected until the 25-mg/kg level. Neither species showed a change in
hematological values, but the dog did exhibit erratic increases at the
high dose level in alkaline phosphatase, serum glutamic oxaloacetic
transaminase, lactate dehydrogenase, ornithine carbamyl transferase, and
amylase values. Histopathological examination revealed lesions of the
small intestine in the dog, and the pig had hemorrhaging of the heart
and fat deposits in the pancreas, in addition to the intestinal lesions.
These results indicated that the two species reacted comparably to the
pesticide diazon. The dog showed greater biochemical sensitivity;
the pig was more sensitive to lower doses and showed more lesion damage.
Knapp, Busey, and Kundzins (1971) compared the sensitivity of the
rat and dog to monochlorobenzene (MCB) for 93 or 99 days of oral dosing.
The dog was more sensitive to MCB, showing mortality, increases in the
activity of alkaline phosphatase and serum glutamic pyruvic transaminase,
increased numbers of immature leucocytes, and gross or microscopic
pathology for the liver, kidney, gastrointestinal mucosa, and hematopoietic
tissues. The majority of the effects were at the high dose (272.5 mg/kg
per day). The rat exhibited some growth depression and increased liver
and kidney weights at the high (250 mg/kg per day) and intermediate
(50.0 mg/kg per day) doses. The effects found in the rat were not as
severe as those found in the dog.
Jones, Strickland, and Siegel (1972) used the rat (Sprague-Dawley),
guinea pig (Hartley), monkey (squirrel), and dog (beagle) to test the
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113
inhalation toxicity of propylene glycol 1,2-dinitrate for 90 days with
daily exposure levels of 67, 108, and 236 mg/m3- The effects detected
were similar among all four species and included liver pathology and
changes in hematological-biochemical parameters. The appearance of
fatty deposits in the liver first occurred at the low dose for the
guinea pig and rat, but not until the intermediate-dose level for the
dog and monkey. Increased methemoglobin levels were found at the high
dose for all species but were more severe for the dog and monkey. The
dog also showed decreased hemoglobin and hematocrit levels at the high
dose. The monkey had decreased alkaline phosphatase and increased blood
urea nitrogen levels at the intermediate and high doses, and some
mortality at the high dose. The dog and monkey appeared to be the more
sensitive species for effects in hematological and biochemical parameters,
and the rat was more sensitive for liver and kidney changes.
Verschuuren, Kroes, and Tonkelaar (1973) compared variability in
species response to oral doses of an acaricide, tetrasul, over a 90-day
test period using diet levels of 50, 200, 1000, and 3000 ppm. Growth
retardation was evident for all species at the high dose, and for all
species except the rat and mouse at the higher intermediate dose. In-
creased liver weights were found at the lower intermediate dose for all
species except the rat, and for all species at higher doses. Histopatho-
logical damage of the liver was found in all species at and above the
lowest dose. In conclusion, Verschuuren and his associates ranked the
six species based on nine criteria with a rating of 1 to 6 for each
criteria. Based on this system, five species were in a range of ±2.5
points, with the mouse separated from this group by +10 points. In the
narrower context of the evaluation of chlorinated hydrocarbons, the
sensitivity ranks were as follows: the miniature pig and rat were the
most sensitive; the rabbit, guinea pig, and chicken were next; and the
mouse was the least sensitive.
King, Shefner, and Bates (1973) studied the effects of chlorinated
dibenzodioxins in the rat and mouse after oral dosing in the diet for
42 weeks. As shown in Table 4.7, the rat generally suffered higher
mortality rates and more liver or lung damage than the mouse. Using
these criteria, the rat was a more sensitive species than the mouse.
-------
Table 4.7. Summary of dioxin oral administration data
Rat (35 per group)
Compound
Controls
1% Dioxane
0.5% Dioxane
1% Unsubstituted
dibenzodloxin
0.5% Unsubstituted
dibenzodioxin
1% Dichloro-
dibenzodioxin
0.5% Dichloro-
dibenzodioxin
1% Octachloro-
dibenzodioxin
0.5% Octachloro-
dibenzodioxin
0.25% Octachloro-
dibenzodioxin
0.125% Octachloro-
dibenzodioxin
Sex
M
F —
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
Week
of
test
34
- 34
42
42
42
42
42
42
42
42
17
17
17
17
32
22
37
25
17
17
17
Number of
Survivors
35
35
24
20
26
32
33
32
31
35
35
35
35
35
0
0
0
0
28
15
30
Significant
pathology
Lung
8/11
3/5
6/6
3/3
2/2
1/2
3/4
1/5
°/5«-
1/5*
1/2
0/3
Liver
1/11
2/5
1/6
1/3
2/2
2/2
1/4
5/5
5/5
5/5
2/2
3/3
Mouse (50 per group)
Week
of
test
31
31
40
43
40
43
39
39
34
39
17
17
17
17
10
37
8
37
17
15
9
Number of
Survivors
50
50
50
49
49
49
48
29
50
49
49
48
50
49
0
5
0
45
1
0
0
Significant
pathology
Lung
3/14
0/1
1/2
0/5
0/5
0/5
2/2
Liver
7/14
0/1
1/2
5/5
6/6
5/5
1/2
Source: Adapted from King, Shefner, and Bates, 1973.
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115
Harris et al. (1973) compared the guinea pig (Hartley), rat (C-D),
and mouse (albino CD-I) for sensitivity to 2,3,7,8-tetrachlorodibenzo-
p-dioxin (TCDD). They exposed each species to TCDD in the diet for 30
consecutive days and/or once a week for 4 to 8 weeks. As shown in Table
4.8 the guinea pig appeared to be the most sensitive species, followed
by the rat.
Kast et al. (1975a) administered fentoterol-HBr to the mouse (ICR-
JCL) and rat (Sprague Dawley-JCL) by gavage daily for 30 days. At the
highest dose (1500 rag/kg), the rat showed growth depression; a high mor-
tality rate; increased weight of the salivary glands, heart, and liver;
and histopathological effects in the heart and salivary glands. At the
highest dose (150 mg/kg), the mouse showed all the above effects except
growth depression, liver weight increase, and heart histopathology.
However, the mice did exhibit histopathological damage to the liver.
Both species showed dose-dependent changes in hematology and biochemical
factors. For the rat, this included increased blood urea nitrogen
level, glutamic oxaloacetic transaminase activity, and glutamic pyruvic
transaminase activity and decreased platelet and glucose levels. In the
mouse, hemoglobin, red blood cells, and packed cell volume all increased
in a dose-dependent manner. The rat appeared to be slightly more sensi-
tive to fentoterol-HBr than the mouse.
In another study by Kast et al. (1975Z?) , the toxicity of fominoben-
HC1 was studied by dosing the mouse (ICR-JCL) and the rat (Sprague
Dawley-JCL) for one month by gavage. The daily exposure levels were
250, 500, 1000, and 2000 mg/kg for the rat and 500, 10DO, 2000, and
4000 mg/kg for the mouse. Mortality occurred only at the high dose for
both species. Pathological findings were limited to a significant dose-
dependent increase in liver weight. At the highest dose, this increase
occurred with a fatty degeneration in the mouse liver and with a focal
necrosis followed by a widening of interstitial connective tissue in the
rat liver. An increase of enzyme activity paralleled the lesion forma-
tions. In the rat, the enzymes and biochemical parameters affected
included bilirubin, cholesterol, alkaline phosphatase, glutamic oxalo-
acetic transaminase, and glutamic pyruvic transaminase. The parameters
affected in the mouse were not listed.
-------
Table 4.8. Summary of biological effects of TCDDa
Frequency of dose
and criteria
affected
Lethal dose
Single
Weekly
Daily
Body weight
Lowest dose effect
Single
Weekly
Daily
No effect
Single
Weekly
Daily
Thymus weight
Lowest dose effect
Single
Weekly
Daily
Rat
Dose MTD
(yg/kg) (days)
100 18
4 x 25 28
10 22
25
6x5
30 x 1
5
6x1
30 x o.l
5
6x5
30 x 0.1
Guinea pig Mouse
Mortality0 , D°*% ^ \ Mortality0 *»* ,
(yg/kg) (days) J (yg/kg)
6/14 3 18 9/10 >50
2/10 5xl 28 10/10 >4 x 25
15/16
1
8 x 0.2 4 x 25
50
8 x 0.04 4x5
10
8 x 0.04 4x5
a2,3,7,8-Tetrachlorodibenzo-p-dioxin.
Mean time to death after first exposure.
CNumber of animals dying per number of animals treated.
Source: Adapted from Harris et al., 1973.
-------
117
Villeneuve and Newsome (1975) tested hexachlorobenzene (HCB) in the
rat (Wistar) and guinea pig by administering a daily peroral dose of
500 mg/kg body weight for 16 days. Both species showed high mortality,
but the guinea pig was affected most. The rat showed high tissue con-
centrations indicating a greater resistance to HCB. Both species
experienced weight loss before death. No histopathology, hematology, or
biochemical tests were performed. On the basis of a greater mortality
rate and lower tissue concentrations, the guinea pig appeared to be the
more sensitive species to HCB.
The rat (Charles-River) and dog (beagle) were used to test TR2379,
an antihypertensive agent, for peroral toxicity over a 13-week period
(Hartnagel et al., 1975). For the rat the dose levels were 50, 200, and
820 mg/kg per day, and the dog was dosed at 30, 70, and 160 mg/kg per
day. Growth retardation occurred for male rats at the intermediate and
high doses and for female dogs at the high dose. Increased kidney,
adrenal, and thyroid weights were recorded for male rats at the high
dose, and while both sexes had increased liver and heart weights. In
the dog only males had organ weight increases, and these were limited to
the liver and kidney at all doses. Both species suffered mortality at
the high dose. The sensitivity of the two species was about equal,
with male dogs showing organ weight increases at low doses while male
rats showed more growth depression at lower doses.
Koeferl et al. (1976) reported the toxicity of cyclohexanone to the
rat, dog, and monkey after intravenous injection of 142 and 284 mg/kg
per day 5 days per week for 21 days. The rat exhibited the highest
mortality rate at an additional high dose (568 mg/kg). However, the dog
showed erythroid hyperplasia with increased hematocrit counts, a myeloid-
erythroid ratio below unity, and nucleated red blood cells in the
peripheral blood. This was termed the "most significant" finding,
indicating the authors' opinion concerning the value of the dog in this
toxicity evaluation.
In a study of the antibiotic sisomicin, Robbins and Tettenborn
(1976) tested intramuscular toxicity in the rat (Wistar and Sprague-
Dawley) and dog (beagle) for 3 to 5 weeks and 13 weeks. For the shorter
duration the daily dose levels were 2 to 100 mg/kg for the rat and 1 to
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118
60 mg/kg for the dog. During the longer duration, the daily levels were
2, 4, and 8 mg/kg (rat) and 2 to 16 mg/kg (dog). At the higher doses,
the rat experienced a 33% mortality rate, glycosuria, reduced body
weight, increased blood urea nitrogen and serum creatinine levels, and
renal histopathological effects in the shorter test. The 13-week exposure
showed a dose-dependent response for renal damage. In the 5-week dog
studies, renal damage and mortality were the primary effects, and this
trend continued for the 13-week exposure. The renal pathology was again
dose dependent and occurred to a greater degree at lower doses in the
dog than in the rat. The wider response spectrum shown by the rat
offsets the greater sensitivity at low doses shown by the dog, so neither
species showed a definite advantage in this study.
In a study of drug metabolism, Litterst et al. (1976) compared the
monkey (squirrel), rat (Sprague-Dawley), miniature pig (Hanford), and
common tree shrew (Tupaia glis) as replacements for the rhesus monkey.
Each species was evaluated for its enzymatic activity pattern by an
examination of 14 parameters of drug metabolism in hepatic microsomal
and soluble fractions, including liver weight to body weight ratio,
cytochrome P-450, glutathione s-aryltransferase activity, reduced
nicotinamide-adenine dinucleotide phosphate-cytochrome C reductase
activity, mixed-function oxidation, aminopyrine tf-demethylase activity,
ethylmorphine ff-demethylase activity, biphenyl hydroxylase activity,
aniline hydroxylase activity, aryl hydrocarbon hydroxylase activity,
UDP-glucuronyItransferase activity using both p-nitrophenol and
0-aminophenol acceptor substrates, and tf-acetyltransferase activity
using both p-aminobenzoic and sulfadiazine acceptor substrates. All
four species demonstrated activity in 13 of the 14 parameters, but no
one species was similar to the rhesus in all quantitative aspects.
Based on an arbitrary scale of favorable response, the similarity to the
rhesus is as follows: the miniature pig is closest, followed by the
rat, the squirrel monkey, and the tree shrew. In conclusion, the
authors felt that the miniature pig would be suitable if housing and
handling problems could be solved and that in any case a primate replace-
ment for the rhesus might not be the best choice.
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119
Sulfolane, a manufacturing solvent, was tested for inhalation
toxicity by repeated (8 h per day, 5 days per week, for 27 exposures)
and continuous (23 h per day for 85 to 110 days) exposures of the rat
(Sprague-Dawley), guinea pig (Hartley), monkey (squirrel), and dog
(beagle) (Anderson et al., 1977). In the repeated exposure test a dose
concentration of 495 mg/m3 was used, and in the continuous exposure test
the levels were 2.8, 4.0, 20, 159, and 200 mg/m3- All animals except
the monkey survived the repeated-dose test. Of nine monkeys exposed to
the high dose, three died and five were sacrificed in a moribund state
after 17 days. All had pale livers and hearts, and five showed fatty
metamorphosis of the liver. No significant hematological, body weight,
or biochemical changes were noted in the other species. All species
showed chronic lung inflammation, with the rat also showing liver
inflammation. In the continuous study, the monkey again exhibited high
mortality rates at the high dose level. The dog, which was exposed to
the high dose, exhibited highly aggressive behavior, seizures and
pneumonia. No body weight changes were noted in the rat or the guinea
pig although the histopathology examination revealed liver damage. The
guinea pig also showed some leukopenia and increased plasma transaminase
activity at the high dose. All species again showed chronically in-
flamed and hemorrhagic lung tissue at the two higher doses. In their
discussion of the results, the authors rated the susceptibility of the
species to sulfolane in a monkey-dog-rat progression.
Cavender et al. (1977) compared the sensitivity of the rat (male
Charles River) and guinea pig (female Hartley) to sulfuric acid mist
(5.0 and 10.0 mg/m3), ozone (1.0 and 2.0 ppm), and a combination of
both. Both species were exposed via inhalation for 2 and 7 days. The
guinea pig, which proved to be more sensitive to sulfuric acid mist than
the rat, exhibited lung damage at low doses and mortality at higher
doses. With ozone, both species showed lung damage and increased liver
weight to body weight ratios, but the rat adapted to this stress more
quickly than did the guinea pig. In conclusion, the authors pointed out
that the differences in sensitivity between the rat and the guinea pig
illustrate the importance of using "at least two species in inhalation
toxicity studies."
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120
In a test of the inhalation toxicity of acrolein, a cigarette smoke
component, Feron et al. (1978) exposed the rat (Wistar), rabbit (Dutch),
and hamster (Syrian Golden) to the chemical for 6 h per day, 5 days per
week, for 13 weeks. As shown in Table 4.9, all three species were
affected. The rat was the most sensitive. It suffered mortality and
showed toxic effects at the lowest dose, unlike the hamster and rabbit.
One aspect of species selection that has not been considered by
other reports is the opposition of the general public to the use of some
nonrodent species as test animals because of their status as domestic
pets. As a result of such a consideration, Cummings et al. (1979)
Table 4.9. Summary of treatment-related effects in hamsters, rats, and rabbits
repeatedly exposed to acrolein (ppm) for 13 weeks
Criteria affected
Symptomatology
Mortality
Growth
Food intake
Hematology
Urinary amorphous
material
Urinary crystals
Organ weights
Lungs
Heart
Kidneys
Adrenals
Gross pathology
Lungs
Histopathology
Nasal cavity
Larynx
Trachea
Bronchi and lungs
Hamster
0.4
Oa
0
0
NE*
0
0
0
0
0
0
0
0
0
0
0
0
1.4
X
0
0
NE
0
0
0
0
0
0
0
0
X
0
0
0
4.9
XXX
0
**
NE
X
+
*
-H-
+
+
0
0
XXX
X
XX
0
0.4
0
0
*
0
0
0
0
0
0
0
0
0
X
0
0
0
Rat
1.4 4.9
X XX
0 +f+
** ***
* **
0 0
0 +
0 *
0 4+
0 +
0 +
0 +++
0 x
XX XXX
0 xx
0 xxx
0 xxx
0.4
0
0
0
0
0
0
0
0
0
0
0
0
0
NE
0
0
Rabbit
1.4
X
0
*
*
0
0
0
0
0
0
0
0
0
NE
0
0
4.9
xxx
0
**
**
0
+
0
-H-
0
0
0
0
XX
NE
X
XX
fi u^t- •*ffe*r*+-t*A' v — C14»Vi#-1w **ffaf>t-aA' w — MnA&Trat-olv aff&rt'aA* vw —
\J HUk OL ± ^\- WCU 9 A *J ^ JL.*jlt ^ ±.J Bt^»-ww b w-w y r*n * •vHb*»tab^y V»~W*.WM j «»»•>
severely affected; * — Slightly decreased; ** - Moderately decreased; *** — Markedly
decreased; + — Slightly increased; -H- — Moderately increased; +++ — Markedly
increased.
Not examined.
Source: Adapted from Feron et al., 1978. Reprinted with permission of the
publisher.
-------
121
evaluated the rat as a replacement for "pet" species (the dog, cat,
etc.)- They specifically tested some physiological and behavioral
techniques, primarily used in larger animals, and evaluated their poten-
tial as toxicity indicators in the rat. Included in this evaluation
were techniques for detecting changes in blood pressure, heart rate,
electrocardiogram (EGG) patterns, respiratory measurements, treadmill
performance, various reflex and body regulatory mechanisms, body tem-
perature, and behavior (retaining learned behavior, learning new behaviors,
and maintaining unconditioned behaviors). Changes in these physiological
and behavioral traits can indicate toxic effects in the respiratory,
cardiovascular, neuromuscular, central nervous, and autonomic nervous
systems. The techniques were all performed using restrained but
unanesthesized rats to avoid interfering effects from the anesthetic.
The results indicated that the rat could be analyzed for toxic effects
using these techniques and would be a suitable replacement for larger
test species.
4.2.4 Conclusions
In evaluating the suitability of species for toxicity tests, one
constantly finds conflicting data. Depending upon the chemical used,
the rat, dog, monkey, and guinea pig have each been selected in various
studies as the most sensitive species. This trend indicates why there
is such reluctance to change from the standard dog-rat combination. In
papers discussing the value of rodents (mouse, guinea pig, rabbit, rat,
and hamster), the guinea pig and rat usually prove to be the most
sensitive. The studies by Kast et al. (1975a, 1975b), Benitz, Roberts,
and Yusa (1967), and King, Shefner, and Bates (1973) eliminate the mouse
as a potential test species for subchronic toxicity tests, while studies
by Stokinger et al. (1950) and Feron et al. (1978) eliminate the other
rodent species. In direct comparisons of the guinea pig and rat,
evidence can be found to support either species as the most sensitive.
In their studies, Jones, Strickland, and Siegel (1972) and Roe (1968)
ranked the rat as more sensitive than the guinea pig, but Harris et al.
(1973) and Villeneuve and Newsome (1975) found just the opposite.
-------
122
Additionally, Rowe et al. (1952) and Cavander et al. (1977) all con-
sidered the sensitivity of both species to be equal. The selection of a
preferred species in this case may have to be based on economic factors
or on individual species response to certain classes of chemicals.
The value of including a nonrodent species in the test protocols is
also a source of considerable contradiction and disagreement. Some
authors feel that the use of the dog-rat-monkey combination should be
encouraged (Anderson et al., 1977; Freireich et al., 1966; Guarino, 1979;
Hayes, 1967&; Hodge, Boyce, Diechmanne, and Kraybill, 1967; Jones,
Strickland, and Siegel, 1972; McNerney and MacEwen, 1965; Owens, 1962;
Zbinden, 1963) at least for some chemicals. Others feel that the rat
and dog are sufficient to detect most toxic effects (Balazs, 1976;
Hartnagel et al., 1975; Kohn, Kay, and Calandra, 1965; Robbins and
Tettenborn, 1976). If no information on the metabolism and pharmaco-
kinetics of the chemical in question is available, the literature cited
would support the recommendation that the studies be done using the dog
and the rat. However, as discussed in Sect. 4.2.2, if data on metabolism
in animals are available that can be related to man, this information
should be of paramount importance in choice of species. Before embarking
on lengthy, expensive animal studies, efforts should be made to ensure
that the animals chosen for the studies are not unique in the way they
metabolize the test chemical. The same effort should be made in regard
to the physiological functional pattern of the test species, since
differences in this area can be just as important as metabolic differences.
In direct comparisons, the dog is usually more sensitive than other
nonrodent species, such as the monkey or cat, although Smith (1979) did
find the monkey to be metabolically more similar to man than was the
dog. The dog has also been selected as the most sensitive species in
more studies (Ansbacher, Corwin, and Thomas, 1942; Hagan et al., 1967;
Knapp, Busey, and Kundzins, 1971; Koeferl et al., 1976; Litchfield,
1961; Newberne, Gibson, and Newberne, 1967; Stokinger et al., 1950;
Stokinger and Stroud, 1951; Vogin et al., 1970; Yeary, Braham, and Miller,
1965) than the rat (Atkinson et al., 1966; Verschuuren, Kroes, and
Tonkelaar, 1973; Weil and McCollister, 1963; Worth et al., 1970). The
review by Aviado (1978) suggested that the use of both the dog and rat
-------
123
is superfluous and that the rat alone may be sufficient. This trend is
also supported by the work of Cummings et al. (1979) in which physio-
logical and behavioral assessment techniques, usually restricted to use
with larger animals, were found to be applicable in rat studies. To
further complicate the issue, Earl et al. (1964, 1971) gave good evidence
supporting the use of the miniature pig in certain instances. When the
scientific data are conflicting, economic and species availability
factors should also be considered in the task of species selection.
It is obvious that, in the ideal situation of maximum detection of
toxic effects, a dog-rat combination should be used with selected incor-
poration of alternative species such as the guinea pig, monkey, or
miniature pig. The exact combination would depend on the chemical or
effect to be evaluated. The analysis by Gehring, Rowe, and McCollister
(1973) indicated that cost and time parameters could be very high for a
subchronic test using two species. Therefore one could infer that the
ideal situation would be to select a single test species to reduce these
costs. However, if by using two species, such as the dog and rat, a
more efficient determination of toxicity is achieved, then a savings in
both time and cost could be managed by reducing or eliminating the need
for costlier chronic or lifetime studies. This aspect of the problem
will be discussed further in the section on subchronic test durations.
4.3 DURATION
4.3.1 Introduction
The duration of a subchronic test has always been a poorly defined
parameter. As discussed in the introduction, even the definition of a
subchronic test, which is based in large part on duration, has not
specified a standard exposure duration. The closest to a standard
exposure duration is 90 days. Therefore, for this discussion the 90-day
subchronic test will be considered the standard duration. However,
tests of shorter durations, especially 28- or 30-day tests, in the liter-
ature are also discussed. Durations of 14, 28, and 90 days are commonly
used and guidelines indicate that these are acceptable. The 14- to 28-
day tests are generally used to provide information on the nature of the
-------
124
toxic effect, and likely "no effect" and "effect" doses. They do not
usually provide information on maturation or aging effects. The 90-day
duration can provide information on the effects of chemicals on the
maturation process, in addition to evaluating the nature of toxic effects
at lower prolonged doses. The purpose of this section is to evaluate
the effectiveness of 90-day and shorter duration tests by presenting
duration reviews from the literature and summarizing data from duration
comparison studies.
4.3.2 Duration Reviews
One of the first discussions of the length of exposure necessary
for a repeated-dose study was that of Smith (1950). As a result of the
need for antimalarial drugs during World War II, a short test of 11 to
14 days was designed (Wiselogle, 1946) to rapidly evaluate the toxicity
of these drugs. Smith later evaluated the effectiveness of this test,
including increasing the duration to 21 days, by comparing growth rate
curves and particularly the size of the standard deviations. He assigned
an arbitrary value of 100% for the sensitivity of the method at 21 days
and calculated what percent of this sensitivity was achieved at shorter
durations. The results were 15% to 30% sensitivity at 7 days, 68% to
85% sensitivity at 11 days, and 75% to 95% sensitivity in 14 days. He
concluded that for absolute toxicity evaluations the gain was significant
when the test was increased from 11 to 14 days but not when increased to
21 days. Thus, Smith recommended a subchronic test duration of 14 days.
In a discussion of the relationship between short- and long-term
oral toxicity studies, Weil and McCollister (1963) evaluated the effec-
tiveness of a 90-day diet test. Their evaluation was based on 33 chemicals
using the criteria of body weight gain, relative weight changes of the
liver and kidney, and liver and kidney pathology studies in the rat.
They compared both the minimum effect and maximum no-effect levels for
short-term (approximately 90 days) and long-term (2-year) studies,
arriving at a 90-day:2-year ratio for each level (Table 4.10). These
ratios indicated the degree to which these values changed with increased
test duration. In 95% of the studies the ratio was 6.0 or less, meaning
that the safe levels found for a chemical tested for 90 days could be
-------
125
Table 4.10. Relationship of dosage levels of short-term and 2-year
feeding of materials in the diet of rats
Percentage of materials in diet
Material
number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Duration
of short-
term test
105
90
90
120
90
90
97
90
130
30
30
90
90
90
130
50
98
90
29
210
90
130
90
90
30
90
90
90
93
91
90
90
142
Short-term
Minimum
effect
0.015
4.0
1.0
3.0
0.25
0.01
0.1
8.0
1.0
0.05
25.0
0.75
10.0
0.03
3.0
0.3
0.01
16.0
0.25
0.25
0.225
0.1
0.5
0.009
0.3
8.0
16.0
M°
M*
M*
M*
M*
M*
Maximum
no-effect
0.005
2.0
0.3
1.0
0.0625
0.003
0.03
4.0
0.3
0.012
10.0
0.375
3.0
0.01
1.0
0.1
0.003
8.0
0.06
0.05
0.15
0.03
0.25
0.003
0.1
4.0
8.0
3.0
5.0
0.18
1.0
2.5
25.0
2-years
Minimum
effect
0.03
8.0
2.0
5.0
0.256
0.01
0.1
8.0
1.0
0.04
20.0
0.40
5.0
0.0125
1.0
0.1
0.003
4.0
0.06
0.05
0.04
0.005
M°
M*
Vf1
M3
M3
3.0
5.0
0.06
MO
M*
M*
Maximum
no-effect
0.01
4.0
0.2
0.5
0.064
0.003
0.03
4.0
0.2
0.01
5.0
0.13
1.0
0.0062
0.2
0.03
0.001
2.0
0.02
0.01
0.02
0.0025
0.5
0.004
0.1
2.0
4.0
1.0
1.0
0.02
0.3
0.5
5.0
Ratio of short-term
to 2-year test
Minimum
effect
0.5
0.5
0.5
0.6
1.0
1.0
1.0
1.0
1.0
1.2
1.2
1.9
2.0
2.4
3.0
3.0
3.3
4.0
4.2
5.0
5.6
20.0
Maximum
no-effect
0.5
0.5
1.5
2.0
1.0
1.0
1.0
1.0
1.5
1.2
2.0
2.9
3.0
1.6
5.0
3.3
3.0
4.0
3.0
5.0
7.5
12.0
0.5*
0.8*
1.0*
2. Of
2.0fc
3.0C
5.0e
9.0*
3.3^
5.0^
5.0d
Indicates that the maximum no-effect level was the highest dosage level fed.
As the M level was on the 2-year test, the ratios are a maximum.
As the M level was on the short-term test, the ratios are a minimum.
As the M levels were on both the short-term and 2-year tests, the ratios are indicative
only of which levels were used.
Source: Adapted from Weil and McCollister, 1963. Reprinted with permission of the
Journal of Agricultural and Pood Chemistry. Copyright by the American Chemical Society.
used to predict results for a 2-year study, with a significant degree of
accuracy, by dividing the short-term value by 6.0. Since it is common
to apply a hundredfold decrease, as a safety factor, to the maximum no-
effect level determined with a 2-year study, the sixfold difference
requiting from the 90-day study would appear to be a reasonable estimate
of chronic effects. However, Weil and McCollister did not recommend
that the 2-year study be abandoned, stating only that in certain cases a
-------
126
90-day study would be sufficient. The determination of when the shorter
study would suffice is left up to the toxicologist based on his
experience with the chemical and its metabolic pattern.
In a later paper, Weil et al. (1969) examined the 90-day test in
the same manner to see if a shorter test could predict the results it
gives. Their evaluation included the prediction of 90-day repeated
exposure results from 7-day repeated exposure and acute LDso data. They
tested the oral toxicity of 20 chemicals in the diet of the rat using
acute LD50, 7-day durations, and 90-day durations and evaluating the same
criteria used by Weil and McCollister in 1963. By constructing minimum
effect level ratios between each duration group (acute, 7-, and 90-day)
as they had done with 90-day:2-year minimum effect levels, Weil et al.
(1969) were able to evaluate the predictive relationships (Table 4.11).
The relationship between the LDso data and the 90-day test was poor,
needing a ratio of 40 to achieve a predictive accuracy of 95%. The
relationship between LDso data and 7-day test data was not much better,
with a ratio of 20 for 95%. However, extrapolation from 7- to 90-day
tests was much better, with a ratio of only 6.2 for 95% of the results.
Table 4.11. Parameters of ratios of acute peroral LDsoS, 7- or 90-day
and 2-year minimum effect (MiE) dosage levels
Ratio
Percentile
25th
50th
75th
95th
Semiinterquartile
range
Coefficient of
rank correlation
Formula T „ _ _
. , LDcn to LDso to
or symbol -^ __D"
* 7-day 90-day
MiE MiE
Ql = P2s 1.2
P50 = median 2.3
$3 = P75 6.0
P95 20.0
(#3 - Qi)/2 2.4
P 0.831
2.
10.
25.
40.
11.
0.
3
0
0
0
4
782
7-day
MiE to
90-day
MiE
2
3
5
6
1
0
.2
.0
.2
.2
.5
.943
90-day
MiE to
2-year
MiE
1
1
3
5
1
0
.1
.8
.8
.7
.4
.946
Source: Adapted from Weil et al., 1969. Reprinted with permission
of the publisher.
-------
127
This compares favorably with the extrapolation results of the 2-year
studies done in 1963. As the authors stated: "Therefore, one can
predict 90-day results from a 7-day test with the same confidence as one
can the two-year results from a 90-day test." The formulas necessary to
predict the minimum effect dose for 50% to 95% of the studies are given
in Table 4.12. These two studies were among the first to present suf-
ficient significant data indicating that subchronic test durations could
be used to predict chronic effect levels without a substantial loss in
sensitivity.
Table 4.12. Prediction formulas
Predicted upper limit to achieve a
minimum effect level (MiE)
Value
For 90-day For 2-year
feeding study feeding study
Median 7-day MiE/3.0a 90-day MiE/1.8fl or
7-day MiE/5.4
95th percentile 7-day MiE/6.2 90-day MiE/5.7 or
7-day MiE/35.3
aThe denominators for the 7-day MiE/2-year MiE
relationships were obtained by multiplying those for 7
and 90 days [e.g.,. 5.4 = 3.0 (1.8)].
Source: Adapted from Weil et al., 1969. Reprinted
with permission of the publisher.
Peck (1968) discussed the value of shorter tests in drug evaluation
studies. He cited a World Health Organization Technical Report (1966)
and Davey (1964) as recommending that a 3- or 6-month test is adequate
to detect long-term toxicity. This 3- to 6-month duration is also
recommended by Boyd (1968), Bein (1963), Barnes and Denz (1954), and
Zbinden (1963). Peck gave data from studies done from the previous
15 years to support the use of a duration of 3 months. As shown in
Table 4.13, only one study showed additional toxicity after 3 months,
and only four showed added toxicity after 2 months. This again supports
-------
128
Table 4.13. Approximate duration of drug administration
required to define toxicitya in animals
Compound
Indomethacin
Hydrochlorothiazide
Amiloride HCl
Cyproheptadine
Amitriptyline
Methyl-DOPA
Penicillamine
Ethacrynic acid
Protriptyline HCl
Thiabendazole
Dexamethasone
Duration
(months)
1/2
J>
X
X
X
X
X
X
X
X
X
X
1
X
X
X
X
X
X
X
X
0
X
X
2
Oc
0
0
0
0
X
X
X
0
X
X
3
0
0
0
0
0
0
X
X
0
X
0
6
0
0
0
0
0
0
0
0
0
0
0
12 18 24
000
0 0
0
xd
0 0
0 0
000
By physical examination or hematologic, biochemical, and/or
anatomical studies.
x — Toxicity demonstrated.
^
0 — Continuing duration of study, no additional toxicity.
Additional finding of precipitate in kidneys. No earlier
sacrifice, so time of onset not known. Found in rats but not in
dogs or monkeys.
Source: Adapted from Peck, 1968 — An Appraisal of Drug
Safety Evaluation in Animals and the Extrapolation of Results to
Man. In: Importance of Fundamental Principles in Drug Evalua-
tion, D. H. Tedeschi and R. E. Tedeschi, eds. Raven Press, New
York. pp. 450-471. Reprinted with permission of the publisher.
the idea that shorter tests can provide reasonable indications of long-
term toxicity.
In contrast to the papers by Weil, Hayes (1972) stated that although
the 90-day or 70-exposure test is sufficient to determine most long-
term effects, reduction to lesser durations is not adequate. He recom-
mended this after consideration of some data on chemosterilants.
-------
129
Initially Hayes (19672?) felt that some drugs could be tested in 30 expo-
sures, but on reexamination he preferred the 90-day exposure test. Thus,
there is definitely some opposition to reduction of test duration.
McNamara (1976) compiled data similar to Weil and McCollister
(1963) from the literature using 82 studies evaluating 122 compounds
with a variety of test species. Despite the variability due to differ-
ences in species and experimental design, McNamara's data agree with
that of Weil and McCollister in recommending that 2-year studies need
not always be performed. The results are summarized in Table 4.14 in
which formulas are given designating the safe long-term dose levels
Table 4.14. Prediction of long-term
no-effect doses
Time in which
Dose repeated dosage
will produce
no effect
LDso/lOO 3 months
LD5o/1000 Lifetime
3-month no-effect
dose/10 Lifetime
Source: Adapted from McNamara,
1976. Reprinted with permission of
the publisher.
predicted by short-term tests. As with the other authors, McNamara
qualified this predictive value to exclude carcinogenetic and terato-
genetic effects. In conclusion he stated:
If any toxic effect occurs in 3 months, additional (and
perhaps more serious) effects may appear with continued dosing.
If no effect occurs in 3 months, there is a low likelihood that
any effect will occur on continued dosing for 1 year. Thus,
these dose relationships can be of great value in decreasing
the time involved in LT toxicological testing. Experimental
evidence strongly supports the view that LT no-effect doses
can be reliably predicted from ST studies.
-------
130
4.3.3 Comparison Summaries
Gaunt et al. (1965) studied selected aspects of the oral toxicity
of butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) in
the rat with dose levels of 0.1% for 16 weeks. Based on previous studies,
the authors concentrated on liver effects, although other toxicity
parameters were monitored. As shown in Table 4.15, the principal effects
were increases in ascorbic acid excretion, liver weight, and adrenal
weight. For BHT, the females showed significant changes in ascorbic
acid excretion and liver weight by the 4th week, but the adrenal weight
effects did not appear at a significant level until the 12th week. In
the male rat, a trend of increasing ascorbic acid excretion for BHT was
noted from the 4th week on, but not always at a significant level. The
same was true for increases in liver and adrenal weights and in general
for all parameters of the BHA studies in both sexes. In this study, the
12-week duration definitely predicted the longer-term effects of 16 weeks.
The 4-week duration did provide good indications of the toxicity trends
but not always at a significant level.
In a 90-day study of dibutyl(diethylene glycol bisphthalate) (DDGB),
a plasticizer, Hall, Austin, and Fairweather (1966) tested the oral
toxicity in rats. As shown in Table 4.16, the effect on body weight
gain was evident by the fourth week when a significant decrease was
noted for males at the high (2.5% of diet) and intermediate (1.0% of
diet) doses, and for females at the high dose. The only other toxic
effect noted was an increase in relative liver, heart, and brain weights
after 90 days. The authors felt that these changes were due to the body
weight loss since the absolute organ weights did not change. Therefore,
in this case, the only toxic effect attributable to DDGB would have been
detected by a 4- or 8-week test.
Misu et al. (1966) tested Sumithion, an insecticide, for 90 days
given in the diet of the rat at concentrations of 32, 63, 125, 250, and
500 ppm. Toxic effects began to appear at the high dose during the
first week of testing: the mean body weight was decreased (Fig. 4.1),
the testes and brain weights increased (Table 4.17), and the cholinestrase
activity decreased (Fig. 4.2). The growth rate decrease was most notice-
able after the first week, but began to parallel the control values as
-------
Table 4.15. Urinary ascorbic acid excretion and relative liver and adrenal weights of rats fed butylated hydroxyanisole (BHA)
or butylated hydroxytoluene (BHT) at 0.1% of the diet for 16 weeks0
Duration
Compound of feeding
Ascorbic acid*
(mg/kg/day)
Male
Arachis oil
BHT
BHA
Arachis oil
BHT
BHA
Arachis oil
BHT
BHA
Arachis oil
BHT
BHA
, control 4
, control 6
, control 12
, control 16
2.20 ±
4.74 ±
1.84 +
4.39 ±
6.90 ±
4.89 ±
2.10 ±
3.41 i
1.87 ±
2.50 ±
3.13 ±
2.29 ±
0.39
0.95
0.24
1.08
1.76
1.56
0.22
0.41
0.37
0.34
0.43
0.24
Female
3.25 ± 0.35
7.83 ± 1.27
4.16 ± 0.38
1.28 i 0.38
3.39 ± 0.51
1.67 ± 0.51
3.12 ± 0.57
4.83 ± 0.63
2.32 ± 0.36
0.98 * 0.14
2.37 ± 0.37
0.92 ± 0.17
aEach result represents the mean ± statistical error for six
Ascorbic acid is the mean of four days' excretion.
°P < 0.05.
dp < o.oi.
Organ
weight Co body weight ratio
Liver
Male
, 4.31
d 4.68
0 4.36
4.13
4.18
4.24
3.73
3.93
3.58
; 3.50
3.73
3.72
animals .
± Q.lf>d
± 0.15
± 0.05
± 0.06
± 0.12
i 0.07
± 0.07
t 0.14
+ 0.10
± 0.08
+ 0.09
Female
3.94
4.45
4.50
3.71
4.00
3.82
3.15
3.51
3.60
3.21
3.63
3.40
± 0.08
± 0.08*;
* 0.12d
+ 0.11,
± 0.09
± 0.12
t 0.10
i 0.08 ,
i 0.06
± 0.10
± o.ioe'
± 0.17
(2)
Adrenal
Male
0.010 i 0.0008
0.013 ± 0.0010°
0.012 ± 0.0007
0.010 ± 0.0005
0.010 ± 0.0004
0.010 ± 0.0011
0.009 i 0.0037
0.010 ± 0.0026
0.011 ± 0.0008
0.009 + 0.0081
0.010 ± 0.0061
0.011 ± 0.0005
Female
0.016
0.018
0.018
0.018
0.015
0.017
0.017
0.022
0.020
0.016
0.019
0.020
± 0.0005
± 0.0008
± 0.0011
± 0.0011
± 0.0009
± 0.0006
± 0.0008^
± 0.0006"
± 0.0009"'
* 0.0014
± 0.0009
± 0.0004G
P < 0.001.
Source: Adapted from Gaunt et al.
CO
1965. Reprinted with permission of the publisher.
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132
Table 4.16. Mean values of body weights, food consumption, and
dibutyl(diethylene glycol bisphthalate) (DDGB) intake of rats
fed DDGB at 0% to 2.5% of the diet for 11 weeksa
Dietarv Body wei8ht in grams
JeveF at end of week
(%:
0.0
) 0°
96
0.25 100
1.0 91
2.5 98
0.0 82
0.25 82
1.0
2.5
87
83
4
241
226,
2l4d
noe
169
156
160
U5e
8
317
301
280e
232e
205,
185d
196
me
11
352
335
315*
262e
220
199*
211"
190e
Food consumption in
grams per rat per
day at end of week
0
11
11
9
5
9
9
9
5
c
.9
.6
.5
.8
.5
.7
.5
.6
values are the means for groups of 10
DDGB intakes are calculated from data
Day one of
dP < 0.05.
feeding
•
4
Male
23.4
20.5
19.3
16.3
Female
15.7
14.5
14.6
14.2
animals
on body
8
18.7
20.2
17.1
15.5
13.9
13.1
13.5
13.6
weight
11
18.0
19.4
16.7
14.9
14.5
11.3
13.5
11.4
and
DDGB intake" in grams per
kg per day at end of week
0° 4 8
0.29 0.23 0.17
1.04 0.90 0.61
1.48 2.40 1.68
0.30 0.23 0.18
1.09 0.91 0.69
1.67 2.44 1.93
food consumption.
11
0.14
0.53
1.41
0.14
0.64
1.52
eP < 0.001.
Source: Adapted from Hall, Austin, and Fairweather, 1966. Reprinted with permission
of the publisher.
the exposure period progressed. However, the final body weight was
still significantly below control values. Testes weight was significantly
lower by the llth day and remained so until the 90th day. The same
pattern was found for the chclinestrase activity in the liver, kidney,
brain, and red blood cells. In each case the initial stress was the
greatest, producing the most effect. All toxic effects showed recovery
or stabilization at the 90th day. Some minor effects of the same type
as occurred at the high dose were noted with a dose of 250 ppm, and the
no-effect level was 125 ppm. Thus, it appears that all of the toxic
effects could have been detected by an 11- or 30-exposure test, and
only the stabilization trend would have been missed.
In a study of the oral toxicity of diuron, a herbicide, Hodge,
Downs, Fanner, Smith, Maynard, Clayton, and Rhodes (1967) utilized rat
tests with durations of 1 month, 3 months, or 2 years. The dose levels
employed were: 200, 400, 2000, 4000, and 8000 ppm (1 month); 50, 250,
500, 2500, and 5000 ppm (3 months); and 25, 125, 250, and 2500 ppm
-------
133
ORNL-DWG 79-43668R
400 -
350 ~
300 -
U)
o
o
CD
250 -
200 ~
450 -
400 -
I I I I I I I I I
—•—CONTROL
500 ppm
0 7
24 35 49 63
EXPERIMENT DAY
Fig. 4.1. Changes in the mean body weight of rats fed Sumithion
in the diet for 90 days. Source: Adapted from Misu et al., 1966.
Reprinted with permission of the publisher.
(2 years). Depression of the growth rate occurred at 1 month (2000,
4000, and 8000 ppm), at 3 months (2500 and 5000 ppm), and at 2 years
(2500 ppm). Mortality was evident at 1 month (6 of 10 rats at 8000 ppm)
and at 2 years (44 of 70 rats at 2500 ppm), but no mortality was seen at
3 months. However, the mortality was affected by disease vectors,
and the authors concluded that the death trend in the 2-year study was
not due to diuron levels. The red blood cell counts were decreased at
-------
Table A. 17. Mean testes and brain weights, as percentage of body weight, of control rats and rats fed a dietary level
of 500 ppm of Sumithion for various periods of time
Organ weight after days of feeding indicated
Organ
Left testis
Right testis
Brain stem
Brain cortex
11
Control
(%)
0.41 ± 0.02
0.42 ± 0.02
0.21 ± 0.00
0.58 ± 0.02
500 ppm
00
0.53a ± 0.02
0.52a ± 0.01
0.25* ± 0.01
0.79a ± 0.01
30
Control
(Z)
0.40 ± 0.02
0.40 ± 0.02
0.15 ± 0.04
0.46 i 0.01
500 ppm
(Z)
0.60a ± 0.40
0.59a ± 0.01
0.23a ± 0.01
0.54d ± 0.12
60
Control
(Z)
0.29 ± 0.02
0.29 ± 0.02
0.14 ± 0.01
0.32 ± 0.01
500 ppm
(Z)
0.38fc ± 0.03
0.39^ ± 0.03
0.14d ± 0.01
0.40^ i 0.02
90
Control
(Z)
0.27 ± 0.02
0.26 ± 0.02
0.13 ± 0.01
0.30 ± 0.01
500 ppm
(Z)
0.35° ± 0.02
0.34° ± 0.03
0.15° ± 0.01
0.35° ± 0.01
t-1
CO
4s-
differs from controls, P < 0.01.
Value differs from controls, 0.01 < P < 0.05.
°Value differs from controls, 0.05 < P < 0.5.
'Slot significant, P > 0.5.
Source: Adapted from Misu et al., 1966. Reprinted with permission of the publisher.
-------
ORNL-DWG 79-13665
UJ
CO
<
tr.
UJ
l-
co
UJ
z
-J
o
X
o
CO
UJ
X
o
^-
z
UJ
o
IT
UJ
CL
- ~ (OLIVER
100 -
50 -
0
n
R
i
^CONTROL
500ppm
250 ppm fl 125 ppm t 1 63 ppm
(rfRED BLOOD CELL
100 -ci
50 -
i
- (
-------
136
1 month (4000 and 8000 ppm), at 3 months (2500 and 5000 ppm), and at
2 years (2500 ppm). The hemoglobin concentrations were also depressed
at 1 month (4000 and 8000 ppm), at 3 months (5000 ppm), and at 2 years
(2500 ppm). The 1-month study indicated a decrease in hematocrit that
was not evident in the longer studies. Organ weights were also affected.
After 1 month at 8000 ppm, the liver and kidney weights were decreased,
and the spleen weight was increased. At 3 months the spleen weight was
increased (5000 ppm), but the liver and kidney weights were unaffected.
In the 2-year study the spleen weight was increased (2500 ppm) at 9 and
17 months, but not at 24 months. Histopathologic data were not signifi-
cantly different from the controls for all tests regardless of duration.
In conclusion, it appears that the major toxic effects were sufficiently
indicated by the 1-month duration. Extension to 3 months and 2 years
provided little additional information.
Ambrose et al. (1972) studied 3'-4'-dichloropropionanilide in rats
after 13 weeks and 2 years of oral exposure. In the 13-week study, dose
levels of 100, 330, 1000, 3300, 10 000, and 50 000 ppm were used, and
the 2-year study used levels of 100, 400, and 1600 ppm. The significant
effects recorded after 13 weeks of dosing included growth depression
(high and intermediate doses), a decrease in hemoglobin levels (high and
intermediate doses), and increases in weight for the heart (high dose),
spleen and liver (females at high and intermediate doses), and testes
(high dose). After the 2-year exposure period, the effects at the high
dose included growth rate depression, increased female liver and kidney
weights, and increased testes weight. These were all detected with the
13-week study. The hemoglobin level was also depressed at the high dose
in the 2-year study, but, in contrast to the 13-week study, results
occurred only in the female rats. Thus, in this study there was a good
prediction of chronic (2-year) effects from the 13-week data, suggesting
that the 13-week study would have been sufficient.
Lawrence et al. (1972) studied epichlorohydrin in the rat by intra-
peritoneal injection for duration's of 30 daily injections (with doses*of
0.00955 and 0.01910 mL/kg) or 12 weeks of three injections per week
(with doses of 0.00955, 0.01910, and 0.04774 mL/kg). In the cumulative
toxicity test (30 daily exposures), weight gain was significantly
-------
137
decreased at the high dose by day 15 and at the low dose by day 20
(Table 4.18). In the 12-week study, the high dose exhibited a signifi-
cant decrease in weight gain until the last week (Table 4.19). The
hematology and biochemical parameters showed no significant trends in
most tests. However, hemoglobin levels did show a significant decrease
at the high dose at the end of the cumulative toxicity test and a dose-
dependent decrease after the 12-week study. The measurement of organ
weights also showed some changes attributed to toxic effects. In the
30-day study, the kidney weight decreased significantly at both doses
(Table 4.20). After 12 weeks of dosing at the high dose, the kidney,
liver, and heart all showed significant weight increases, and the brain
showed a significant weight decrease (Table 4.21). In comparison of
these two experiments, the total number of injection exposures is
similar (30 vs 36), but the duration of exposure is not (4 weeks vs
12 weeks). The results from both studies are quite similar, which
suggests either that the shorter duration adequately predicts the results
of the longer duration or that the number of doses given is the key
factor in a repeated-dose study.
Table 4.18. Cumulative toxicity of epichlorohydrin: body
weight gain in grams (mean ± statistical error)
Day
5
10
15
20
25
30
Cottonseed
oil control
28.17 ± 1.96
66.00 ± 2.44
111.25 ± 3.39
145.67 ± 3.75
157.25 ± 4.38
183.33 ± 5.59
Epichlorohydrin
0.00955 mL/kg
36.25 ± 5.73
71.92 ± 6.15
105.25 ± 7.24
123.42 ± 4.83a
145.67 ± 6.44
167.67 ± 7.27
0.01910 mL/kg
26.00 ± 1.54
63.67 ± 2.68
91.33 ± 4.33a
131.58 ± 2.87a
153.92 ± 7.33
158.50 ± 6.82fc
Significantly different from controls at 99% level
(P = 0.01) by Student's t test.
Significantly different from controls at 95% level
(P = 0.05) by Student's t test.
Source: Adapted from Lawrence et al., 1972. Repro-
duced with permission of the copyright owner.
-------
138
Table 4.19. Subacute toxicity of epichlorohydrin: body
weight gain in grams (mean ± statistical error)
Week
1
2
3
4
5
6
7
8
9
10
11
12
Cottonseed
oil control
46.58 ± 2.88
76.50 ± 6.15
105.33 ± 5.86
186.17 ± 5.74
214.25 ± 8.14
254.83 ± 6.52
279.25 ± 7.16
296.17 ± 7.10
333.50 ± 9.72
345.50 ± 13.49
356.67 ± 14.75
358.92 ± 13.27
0.0095 mL/kg
38.58 ± 4.50
94.42 ± 3.05*
119.83 ± 3.72*
193.42 ± 4.74
228.08 ± 6.80
254.33 ± 11.07
267.08 ± 9.11
289.92 ± 5.98
330.75 ± 9.59
359.83 ± 9.64
370.42 ± 10.82
383.42 ± 11.22
Epichlorohydrin
0.0190 mL/kg
41.42 ± 2.40
86.50 ± 5.14
111.83 ± 6.79
175.58 ± 11.02
203.50 ± 10.73
242.75 ± 11.44
261.25 ± 12.27
276.00 ± 11.47
315.67 ± 21.92
334.00 ± 13.81
371.17 ± 12.08
380.92 ± 13.34
0.04774 mL/kg
24.00 ± 3.14a
55.09 ± 10.23
97.00 ± 9.27
128.30 ± 11.54a
172.00 ± 11.37*
195.60 ± 10.29a
218.50 ± 11.14a
246.80 ± 12.16a
266.60 ± 13.19a
293.70 ± 14.19*
286.70 ± 21.91*
328.33 ± 18.19
aSignificantly different from controls at 99% level (P = 0.01) by
Student's t test.
Significantly different from controls at 95% level (P = 0.05) by
Student's t test.
Source: Adapted from Lawrence et al., 1972. Reproduced with permission
of the copyright owner.
In two companion studies, Kociba et al. (1976; 1978) evaluated the
toxicity potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) admin-
istered in the diet of rats for 13 weeks (1.0 and 0.1 yg/kg) and 2 years
(0.1 and 0.01 yg/kg).| As shown in Table 4.22 the results were quite
similar for mortality, body weight gain, alkaline phosphatase, packed
cell volume, red blood cell count, hemoglobin concentration, relative
weights of the liver and thymus, and histopathologic changes in the
liver, lung, thymus, uteri, and lymphoid tissues. Differences between
the two studies consisted mainly of the more transient effects indicated
by the subchronic study, with only a few different effects indicated by
the chronic study. Although there were minor differences in serum
enzyme levels, hematology values, and relative organ weight changes, the
subchronic study did evaluate the major effects found in the chronic
study.
-------
139
Table 4.20. Cumulative toxicity of epichlorohydrin:
percent organ weight to body weight of rats
(mean ± statistical error)
Organ
Adrenals
Brain
Gonads
Heart
Kidneys
Liver
Lungs
Spleen
Cottonseed
oil control
0.014 ± 0.001
0.475 ± 0.017
0.975 ± 0.037
0.273 ± 0.006
0.654 ± 0.023
3.960 ± 0.140
0.381 ± 0.027
0.236 ± 0.010
Epichlorohydrin
0.00955 mL/kg
0.014 ± 0.001
0.567 ± 0.041
0.995 ± 0.051
0.309 ± 0.022
0.750 ± 0.025a
4.150 ± 0.230
0.409 ± 0.028
0.266 ± 0.017
0.01910 mL/kg
0.013 ± 0.002
0.543 ± 0.033
0.971 ± 0.037
0.378 ± 0.078
0.786 ± 0.035a
3.916 ± 0.056
0.407 ± 0.015
0.253 ± 0.012
(P = 0.05) by Student's t test.
Source: Adapted from Lawrence et al., 1972. Reproduced
with permission of the copyright owner.
Table 4.21. Subacute toxicity of epichlorohydrin: percent organ
weight to body weight of ratsa (mean ± statistical error)
Organ
Adrenals
Brain
Gonads
Heart
Kidneys
Liver
Spleen
Cottonseed
oil control
0.023 ± 0.013
0.444 ± 0.018
0.785 ± 0.028
0.291 ± 0.011
0.670 ± 0.034
2.983 ± 0.219
0.174 ± 0.012
0.0095 mL/kg
0.011 ± 0.001
0.426 ± 0.012
0.707 ± 0.034
0.302 ± 0.018
0.636 ± 0.032
3.232 ± 0.157
0.194 ± 0.010
Epichlorohydrin
0.0190 mL/kg
0.010 ± 0.001
0.439 ± 0.029
0.866 ± 0.054
0.306 ± 0.030
0.728 ± 0.049
3.276 ± 0.186
0.208 ± 0.031
0.04774 mL/kg
0.013 ± 0.002
0.324 ± 0.027fc
o
0.402 ± 0.029b
0.917 ± 0.054b
4.070 ± 0.276d
0.225 ± 0.021
a
cent organ weight to body weight.
Significantly different from controls at 99% level (P = 0.01) by
Student's t test.
Q
Gonadal weights were not determined in this group.
Significantly different from controls at 95% level (P = 0.05) by
Student's t test.
Source: Adapted from Lawrence et al., 1972. Reproduced with permission
of the copyright owner.
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140
Table 4.22. Comparison of 13-week and 2-year
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) studies
Test parameters with
significant changes
Mortality
Body weight
Food consumption
Packed cell volume
Red blood cell count
Hemoglobin concentration
Reticulocyte count
Thrombocyte count
Total leucocyte count
Total and direct bilirubin
Glutamic-pyruvic transaminase
Alkaline phosphatase
Relative thymus weight
Relative liver weight
Histopathologic changes6 in:
Thymus
Other lymphoid tissue
Uteri
Ovaries
Liver
Lung
Brain
Pituitary
13-week
1.0 ug/kg
*5
\d
\
4-M; tF
4-M; tF
4-M; tF
t
t
tF
t
t
4
t
M, F
M, F
F
F
M, F
M, F
study0
0.1 yg/kg
\
I
4-M
4-M
4-M
tF
tF
4-
t
M, F
M, F
M, F
2-year study
0.1 yg/kg 0.01 yg/kg
F
4- 4-F
4-F
4M
4-
tF
tF
4-F
tF tF
F
F
M, F M, F
M, F F
F
F
QAdapted from Kociba et al., 1976.
Adapted from Kociba et al., 1978.
Q
F — females only; M — males only.
4- — decrease in value vs control; t — increase in value vs control.
o
Histopathologic changes exclusive of carcinogenic tumors.
In another study of TCDD, Harris et al. (1973) tested the toxic
effects in female rats from gastric intubation for 31 consecutive days
with doses of 0.1, 1, and 10 mg/kg. At the high dose, significant
weight loss (Table 4.23) occurred by the 7th day, and 15 of 16 rats died
by a mean of 21.8 days after the start of dosing. For the intermediate
dose, weight gain was depressed significantly during the first 35 days,
but reversed to yield significant growth increases during a recovery
period on days 35 to 63. Organ weight changes occurred only in the
liver and the thymus (Table 4.24). Liver weight increased at all three
dose levels (although statistically erratic) by the 10th day of exposure.
Livers at the high dose showed a decrease in weight at the 17th and 24th
-------
Table 4.23. Weight gain of female rats receiving 31 daily doses of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
Daily Initial Weight gain
TCDD number Initial body weight (g ± standard deviation)
dose of (g ± standard deviation)
(yg/kg) animals Days 1 to 7 Days 1 to 35a Days 35 to 63a
0 16 183.3 ± 6.28 15.8 ± 6.6 65.8 ± 8.0 28.4 ± 9.7
1 12 185.6 ± 5.04 12.1 ± 6.4 34.8 ± 12.2b 42.0 ± 11.9°
10 16 184.0 ± 8.00 -21.4 ± 14.9fc
Values based on 5 and 8 rats at 0 and 1 yg/kg respectively; 15 of 16 rats died or were killed
when moribund at mean of 21.8 days.
bP < 0.01.
°P < 0.06.
Source: Adapted from Harris et al., 1973.
-------
Table 4.24. Liver and thymus weights of female rats receiving daily doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
Number
of
doses
3
6
10
13
17
24
31
Number
of
animals
4
4
4
4
4
4
4
Statistical
*A11
°P<
0 yg/kg
Liver
(g ± SE?)
7.80 ± 0.48
8.41 ± 0.36
9.14 ± 0.21
7.90 ± 0.31
9.26 ± 0.42
9.45 ± 0.31
9.70 ± 0.49
error.
dose response tests for
0.05.
Thymus ,
(mg ± SE)ft
530 ± 48
615 ± 59
525 ± 43
558 ± 87
0.1
Liver
(g ± SE)
8.62 ± 0.41
10.35 ± 0.45C
10.78 ± 0.41C
10.17 ± 0.94°
10.91 ± 0.37C
11.40 ± 1.05
11.78 ± 0.81
Mg/kg
Thymus
(mg ± SE)fc
492 ±
468 ±
412 ±
475 ±
81
22
28°
49
1.0 vg/kg
Liver Thymus
(g ± SE) (mg ± SE)fc
9.32 ± 0.45
9.00 ± 0.14,
11.53 + 0.40
9.76 ± 0.62,
11.33 ± 0.29d
11.39 ± 0.98
12.84 ± 0.84C
390 ± 58
270 ± 11"
372 ± 28^
260 ± 41a
10 yg/kg
Liver
(g ± SE)
9.58
10.84
10.53
9.80
7.52
4.67
± 0.43
± 0.97
± 0.36°
± 1.08
± 1.37,
± 0.21a
Thymus
(mg ± SE)b
142 ± 12°
110 ± 48°
32 ± lQd
thymus significant at P < 0.01.
P < 0.01.
Source: Adapted from Harris et al., 1973.
-------
143
days of exposure, caused primarily by reduced body weight. The thymus
weight was significantly decreased by day 17 at both the high and inter-
mediate doses. Results of this study indicated that the 30-day duration
would have been sufficient to detect body and organ weight changes and that
even 24 days would have indicated the majority of effects. The results of
this 30-day study also correlate relatively well with the results of the
longer 13-week and 2-year TCDD toxicity studies by Kociba et al. (1976;
1978).
Rosenkrantz et al. (1975) assessed the oral toxicity of A9-tetra-
hydrocannabinol, the major active ingredient in marijuana, in rats
exposed for 28, 90, and 180 days at concentrations of 2, 10, and 50 mg/kg
per day. Body growth depression was one of the primary effects, with an
8% to 12% rate decline at 28 and 90 days, increasing to 9% to 17% by
180 days. Hematological and biochemical parameters showed several
trends. At 28 days, although there were no statistically significant
changes, there were increases in lymphocyte levels of males, increases
in reticulocyte levels of females, and decreases in polymorphonuclear
cells of males. At 90 days, red blood cell numbers increased 11%,
hematocrit levels increased 8%, and for females the white blood cell
levels increased 19% to 74% in a dose-dependent manner. At 180 days,
hematocrit and red blood cells rose 11% and 7%, respectively, in the
females. Serum glutamic oxaloacetic transaminase (SCOT) and serum
glutamic pyruvic transaminase (SGPT) levels rose in the males at rates
of 44% and 65%. The relative adrenal weight increased 30% at 28 days,
and for males the pancreas weight increased 10% and the prostate
weight decreased 30%. After 90 days of treatment,.the brain weight in-
creased 3% to 25%, and the kidney weight increased 7% to 18%. Adrenal
weight remained elevated while the prostate weight continued to decrease,
but only at 6% to 21%. At 180 days, more organs revealed weight changes
including increases in the brain (16%), lung (10% to 23%), kidney (11%
to 20%), heart (12%), liver (10%), and adrenal glands (34% to 56%).
Changes in organ weight in the males were as follows: the thymus decreased
17%, the pancreas increased 96%, the prostate increased 13%, and the
testes increased 25%. In the females the weight of the uterus decreased
14%. Although many of these changes at 180 days were dose dependent,
-------
144
the significance was borderline. None of the histopathological changes
was considered significant, with lesions occurring only incidentally in
both control and treatment animals. In this study, the 28-day duration
revealed the same changes as the longer studies for growth rate depression
and weight changes in minor organs. However, the study failed to detect
weight changes in major organs and in hematology values and suggests
that a 28-day test does not adequately predict what happens at 90 days.
By the 90th day, toxic effects in these categories were detected.
Although the 180-day duration revealed more changes, the indication of
toxicity was sufficient at 90 days to suggest the potential danger of
the compound.
In a study of the oral toxicity of fominoben-HCl, Kast et al.
(1975&) dosed rats daily for 1 and 6 months. Even though the 6-month
test is not a subchronic duration, this study is useful for evaluating
the effectiveness of short-term tests (less than 90 days) to predict
long-term effects. In the 1-month study, dose levels of 250, 500, 1000,
and 2000 rag/kg were used, and the 6-month study used levels of 50, 100,
and 200 mg/kg. In the shorter test, mortality was evident by the
second week at the high dose, with 9 of 30 rats dying. Body weight gain
was depressed from the first week until the end of the 1-month test.
Other effects detected at the high dose were increases in SCOT, SGPT,
serum alkaline phosphatase, serum bilirubin, and serum cholesterol; a
decrease in total protein; liver pathology; atrophy of the gastric
mucosa; and decreases in thymus and testes weights in male rats. The
liver weight increased at a dose-dependent rate. In the 6-month experi-
ment, no mortality occurred, but body weight gain was decreased at the
high dose. The biochemical and hematological parameters were affected
less in the longer study than in the 1-month study, with increases in
SCOT and SGPT activities being the only significant changes. The
pathology was limited to liver damage at the high dose, and liver weight
increased at a dose-dependent rate. In comparing the results of the two
duration periods, it is apparent that the shorter test indicated the
major effects, particularly the liver damage, that were revealed in the
6-month study. Therefore, in this study, the shorter test would have
been a sufficient predictor of long-term effects.
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145
In another study by Kast et al. (1975a) the same experimental
design was used to test the toxicity of fenoterol-HBr. The doses used
were 15, 150, and 1500 mg/kg (1 month) and 3, 30, and 300 mg/kg (6 months).
In the shorter study, mortality at the high dose was high (64% for
males, 24% for females), although in the chronic study only one substance-
related death occurred. The body weight gain was significantly decreased
at the high dose in the 1-month test, but showed a significant increase
in the female after 6 months of testing. The hematological and bio-
chemical tests performed for the 1-month duration revealed, at the high
dose, decreases in platelet count (also at intermediate dose) and blood
glucose levels and increases in blood urea nitrogen, Ca+, SCOT, and SGPT
levels. In the 6-month study, an alkaline phosphatase increase at the
high dose was the only significant change, although blood glucose levels
showed a slight depression. Organ weights increased for the salivary
glands (high dose), heart (high and intermediate doses), and liver
(females at high and intermediate doses). However, the males showed a
decrease in liver and testes weights at the high dose. In the 6-month
study, only female heart weight increased at high and intermediate
doses. In the shorter test, histopathological examination revealed
salivary gland and heart (primarily ischemic lesions) damage at the high
dose. Testes were also atrophied in three males at the high dose. In
the 6-month test, only the heart damage (again with a predominance of
ischemic lesions) occurred at the high dose. Comparing the two experi-
ments, it appears the shorter test indicated more toxicity than the
longer evaluation. This may be caused by different dose magnitudes or
the effects of fenoterol-HBr being acute in nature, or it may indicate
an adaptation to the toxic effects which reduced the final effects in
the chronic study.
Kruysse et al. (1977) studied the inhalation toxicity of peroxyacetyl
nitrate (PAN) by exposing the rat for 4 and 13 weeks. The exposure
period was 6 h per day, 5 days per week, with dose levels of 0.9, 4.1,
and 11.8 ppm (4 weeks) and 0.2, 1.0, and 4.6 ppm (13 weeks). In a com-
parison of the effects of the two durations, both produced quite similar
results, especially at the high doses. In the 4-week test, mortality
was observed at the high dose, with 9 of 20 rats dying. No mortality
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146
was seen after the 13-week exposure. Growth depression occurred at the
high dose (4 and 13 weeks) and irregularly at the intermediate dose (4
weeks). This was accompanied in both tests by reduced food consumption.
In the 4-week test (high dose) hemoglobin, hematocrit, and red blood
cell counts were all significantly elevated; in the 13-week test (high
dose) hemoglobin and neutrophil levels were increased and lymphocyte
levels decreased. Relative organ weights were also affected, but the
authors stated that this may be due to severe growth depression, because
absolute organ values were below control values. After 4 weeks at the
high dose, heart, kidney, and lung weights increased, and the spleen
weight decreased. After 13 weeks at the high dose, testes, thyroid, and
lung weights all increased. Histopathology revealed lung and tracheal
damage after 4 and 13 weeks at the high and intermediate doses. The
degree and depth of damage increased with higher doses. The authors
concluded that "comparable PAN effects" were observed at the high dose
in 4- and 13-week tests. It appears that the 4-week test would be
sufficient in this case.
Gray et al. (1977) studied the toxicity of di-(2-ethylhexyl)phthalate,
a plasticizer, in the diet of the rat for 17 weeks at concentrations of
0.2%, 1.0%, and 2.0%. Toxic effects were evident early in the study,
particularly at the high dose. Loss of body fur was noticeable by the
1st week at the high dose and continued until the 17th week, with the
affected rats quite emaciated at that time. Decreases in the body
growth rate (Table 4.25) appeared by the 2nd day at the high dose and at
the intermediate dose by day 6 in the males. This decrease was caused
in part by lowered food consumption, which was especially prominent
during the first 48 h. As shown in Table 4.26, there were irregular
fluctuations in the packed cell volume, hemoglobin, and red blood cell
values throughout the study. The decrease in packed cell volume was the
only significant change occurring at both 6 and 17 weeks. As shown in
Table 4.27, changes in relative organ weights occurred by the 2nd week
of dosing. Increased weights were noted for the brain (females), liver,
stomach, small intestine (males), caecum (males), thyroid (males), and
adrenals. Decreases in weight were noted for the heart, spleen, and
testes. At 6 weeks, the only additional changes were an increase in
-------
Table 4.25. Mean body weights and water intake of rats fed diets containing 0% to 2.0%
di-(2-ethylhexyl)phthalate (DEHP) for up to 17 weeks
Dietary
level
(%)
0.0
0.2
1.0
2.0
0.0
0.2
1.0
2.0
aBody
Body weighta
0° 1 27
96 105 340
98 105 325,
98 100 297*
99 99 187
85 92 214
88 95 216
87 90 210
88 87 131*
in grams at day
55
478
455^
300*
273
277
259
164*
90
569
539
493*
413*
309
308-
2847
191*
120
628
588
546
447*
329
325d
201*
Water intake**
0°
18.3
17.6
18.0
18.4
15.9
17.9
17.4
18.3
1
Male
18.5
19.7
15.1
15.7
Female
15.7
18.9
15.4
14.8
in mL per rat per day at day
27
37.1
37.3
34.3,
24.9
21.5
24.9.
26. 6J
21.1
55
38.0
36.3,
27.9*
30.9
21.9
34.5
24.5
21.3
90
28.5
32.3
29.0,
34.4
18.1
26.5-
25. 97
18.9
Well.!
120 (mL per
26.3
24.7
27.7
26.9
19.4
22.1
22.6
16.7
Mean
>r intake
rat per day)
30.1
30.1
27.8-
25.7^
22.3-
25. 07
24. 37
19.7
weights are the means for 15 animals.
Values for water consumption
the 24-h period preceding the
*First
dp < o
day of treatment.
.01.
day
are the
shown.
means
for three cages
of five
animals
and were measured over
P < 0.001.
fp < 0.05.
Source: Adapted from Gray et al., 1977. Reprinted with permission of the publisher.
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148
Table 4.26. Mean hematological values2 for rats fed diets containing OX to 2.OX
di-(2-ethylhexyl)phthalate (DEHP) for 2, 6, or 17 weeks
Sex and
dietary
level
Male
0
1.0
2.0
Female
0
1.0
2.0
Male
0
1.0
2.0
Female
0
1.0
2.0
Male
0
0.2
1.0
2.0
Female
0
0.2
1.0
2.0
Number
of rats
5
5
5
5
5
5
5
5
5
5
5
5
15
15
15
15
15
15
15
15
Hb*
(g per 100 mL)
14.8
13.0^
14.8
15.1
14.4
14.8
15.1
15.0
14.9
15.8
15.4
14.4
16.0
15.4
14.5*
14. 5*
14.9
14.9
14.4
13.8
PCV0
(X)
Week
45
40
42
43
38
41
Week
48
45
46
49.
44*
431
Week
46
45
43?
43l
45
44
42*
42»
RBCd
(106 per mm3)
2
6.26
5.77^
6.48
6.50
6.24
6.82
6
7.08
6.96
6.86
7.81
7.90
7.49
17
7.57
7.44
6.97
7.60
7.14
7.05
7.26
6.78
Reticse
(X of RBC)
2.0
2.7
1.3
1.6
2.2.
0.8*
1.4
1.6
1.3
1.2
1.0
1.2
0.9
0.6
0.8
0.9
0.9
0.8
1.0
0.8
WB(/
(103 per mm3)
7.2
5.6
5.3
4.9
4.2
6.4
6.2
6.0
5.0
6.9
4.7
4.5
6.4
7.5
6.5
6.5
4.7
4.4
5.4
5.5
Figures are means for the numbers of rats shown.
Hb — Hemoglobin.
CPCV — packed cell volume.
TIBC — red blood cells.
a
Retics — reticulocytes.
f ' '
•'WBC — white blood cells.
9P < 0.01.
hP < 0.05.
1P < 0.001.
Source: Adapted from Gray et al., 1977. Reprinted with permission of the publisher.
heart weight, an increase in male brain weight, an increase in female
kidney weight, and an increase in ovary weight. However, by the 17th
week, no new organs were affected, although there were quantitative
changes. An examination of the data in this study reveals that the
majority of effects were indicated by the 2-week study, and certainly by
-------
Table 4.27. Relative organ weights of rats fed diets containing OX to 2.0% di-(2-ethylhexyl)phthalate (DEHP) for 2, 6, or 17 weeks
Sex and
dietary
level
tt)
Male
0
1.0
2.0
Female
0
1.0
2.0
Male
0
1.0
2.0
Female
0
1.0
2.0
Male
0
0.2
1.0
2.0
Female
0
0.2
1.2
2.0
Number
of rats
examined Brain
5 1.12
5 1.17
5 1.75
5 1.19
5 1.21,
5 2.041
5 0.56
5 0.62
5 1.03
5 0.80
5 0.89
5 1.63
15 0.37
15 0.39
15 0.42^
15 0.50°
15 0.64
15 0.64
15 0.69j
15 1.10
Relative organ weight (g per 100 g of body weight)
Heart Liver Spleen
0.46 3.15,
0.42 6.197,
0.42e 6.47a
0.45 3.61^
0.42* 5.28*
0.40 6.62°
0.35 2.99j
0.40* 4.72^
0.43d 6.76*
0.36 2.88,
0.38 4.40,
0.41* 5.83rf
0.27 2.31,
0.27 2.72,
0.28 3.450
0.30 4.12
0.30 2.25
0 . 30 2 . 60
0.31, 3.46^
0.39d 4.59d
""Relative weights of these organs are
Relative weights of the
°P <
dP<
0.01.
0.001.
female gonads
0.30
0.32
0.24
0.29
0.27
0.24e
0.21
0.19
0.28
0.26
0.24
0.25
0.14
0.15
0.14
0.16
0.16
0.17
0.17
0.19g
expressed
Kidneys
0.95
1.01
1.05
1.07
1.00
1.06
0.78
0.79
0.86
0.80
0.82
0.95"
0.56
0.59
0.64 ',
0.70 '
0.57
0.60,
0.65,
0.71"
in mg per
are expressed in mg
c. . Small
Stomach intestlne
Week
0.70
0.70
l.Ol''
0.69
0.65
0.98 '
Wet-k
0.45
0.47
0.78'
0.58
0.60 ,
0.98
Week
0.30
O.J1
0.33';
0.38
0.39
0.39
0.42^
0.72*
100 g of
per 100
2
3.39
J.76
4.41 '
3.62
3.79
4.39
6
2.42
3.15'^
4. 58*'
2.80
3.16 ,
3.80 '
17
1.45
1.55
1.66',
1 .91 '
2.01
2.09
3'.13'7
body weight.
Cecum
0.54
0.55
0.67 '
0.56
0.57
0.65
0.42
0.47
0.54
0.43
0.51
0.52
0.25
0.24
0.25
0.296
0.29
0.32^
0 . 34 ,
0 . 44"
Adrenals0
20.1
19.9
26.9"
36.0
25.8''
3 J.I
12.3
11.1
20.0''
22.5
23.0
27.7
8.9
9.5
10.1
10.9
17.2
19.0
21.9 '
20.4
Gonads
1.08
0.90
0.59^
57
50
41
0.88 ,
0. JO,
0.44
5r>
61
39''
0.60
0.61 y
0.41 ,
0.23'
30
29
37
25
Pituitary0
4.1
3.9
4.0
5.4
4.H
4.1
2.6
2.4
3.0
4.5
S.4
4.7
1.9
1.9
2.3 .
2 . 8
4.2
4.0
4. ft
4.0
Thyroid0
6.6
8.3
12.0 '
8.2
9. J
10.1
i.ft
4.7
H.I
6.2
7.1
10.1 '
J.8
i.4
5.1
5.1
7.5
6.5
7.8
9.1
g of body weight.
vo
eP < 0.05.
Source: Adapted from Gray et al., 1977. Reprinted with permission of the publisher.
-------
150
the 6th week. The only significant change found with 17 weeks of
testing was the quantitative degree of toxicity in some organs.
4.3.4 Conclusions
The literature contains many well-designed studies evaluating the
predictability of chronic (lifetime) effects from subchronic data. The
review papers by Weil and McCollister (1963) and McNamara (1976) presented
compilations of data demonstrating that the 90-day or 90-exposure test
is a significant predictor of chronic effects. Other authors generally
agreed with this conclusion, with many suggesting a duration of 3 to
6 months as sufficient (Barnes and Denz, 1954; Bein, 1963; Boyd, 1968;
Davey, 1964; Peck, 1968; World Health Organization Technical Report,
1966). In addition, several primary studies showed that chronic effects
are indicated at a statistically significant level by tests of 90 days
(Ambrose et al., 1972; Gaunt et al., 1965; Kociba et al., 1976, 1978;
Rosenkrantz et al., 1975). Thus, the evidence in the literature supports
the conclusion that a 90-day test duration does predict most chronic
effects.
There are many comparisons in the literature concerning test
durations shorter than 90 days. Generally, these conclude that the
shorter tests do indicate toxic effect patterns. However, one must
consider that many of the primary studies of short duration produced
effects only at the high dose, which reduced the reliability of their
conclusions. Papers by Smith (1950) and Weil et al. (1969) concluded
that short tests (7 to 14 days) could be sufficient to indicate toxic
effects and predict safe dose levels. Two papers by Kast et al. (1975a,
19753) indicated that a 1-month duration would adequately predict
6-month effects. Thirty-day durations have also been evaluated and
generally recommended as sufficient to indicate toxicity or dose levels
(Gray et al., 1977; Hall, Austin, and Fairweather, 1966; Harris et al.,
1973; Hodge, Downs, Panner, Smith, Maynard, Clayton, and Rhodes, 1967;
Krussye et al., 1977; Misu et al., 1966). However, Hayes (1972) con-
cluded that 30-day durations were insufficient, but in an early paper
(Hayes, 1967&) did agree that a 90-day duration was a good indicator of
-------
151
chronic effects. Thus, data from primary studies generally support the
use of 30-day duration, although there is little concurrence on what
toxic effects might be missed. Perhaps in the future, with more informa-
tion, specific recommendations can be made. In any case, the acceptance
of a 90-day test as a significant evaluation of chronic effects can
reduce the cost and time factors. This could reduce costs from approxi-
mately $255 800 for a complete chronic and subchronic study to $100 500
for a subchronic study design (Gehring, Rowe, and McCollister, 1973).
4.4 ROUTE OF EXPOSURE
4.4.1 Introduction
In subchronic test designs, various routes of administering the
chemical to the test animals have been utilized. The routes most often
employed are oral and inhalation. Alternate exposure methods such as
percutaneous application are used if advantageous for a specific chemical.
In 183 subchronic studies taken from the literature, the frequency for
employment of each route is as follows: oral, 51%; inhalation, 19%;
percutaneous, 2%; intravenous, 8%; intraperitoneal, 8%; subcutaneous,
4%; and others, 8%.
The choice of which route to use should be based on the expected
route of human exposure (Benitz, 1970; Federal Register, 1978; Food
Safety Council, 1978; Ministry of Health and Welfare Canada, 1975;
National Academy of Sciences, 1975, 1977; Peck, 1974; World Health
Organization, 1978). In addition to the metabolic reasons for using the
expected human exposure route, it is easier to extrapolate safe dose
levels from test data to the actual human conditions if the same routes
are used. However, in some cases it may not be advisable to use only
the expected human route. If there are several potential routes of
exposure, it may be necessary to test each one to determine the most
toxic route. This can be important when the major route of exposure is
not the most toxic (U.S. Environmental Protection Agency, 1979). The
effect at the site of exposure is another important consideration since
the concentration is controlled by the mode of exposure (U.S. Environmental
-------
152
Protection Agency, 1979; Weisburger, 1975; World Health Organization,
1978). Also, the availability of testing facilities may determine the
choice of exposure route, especially for inhalation studies. In most
cases, however, there is little or no conflict among these variables,
and the route chosen is the expected route of human exposure.
The following section will discuss aspects of individual routes
used most frequently in subchronic tests. An additional section will
briefly consider the primary literature base for comparison of routes
and the validity of extrapolation between routes.
4.4.2 Route Discussions
Oral administration of chemicals is the principal route used in
most toxicity experiments. This predominance results from both practical
considerations, including ease of application and low cost factors,
and experimental requirements (oral exposure represents the expected
primary route of human exposure for many substances). In general, oral
exposures produce quick responses of intermediate toxicity. Hayes
(1967a) rates oral exposure as more toxic than dermal applications,
while the World Health Organization (1978) rates oral (gavage) as more
toxic than dermal but less than inhalation exposures. On a more specific
level, oral administration of quickly absorbed chemicals produces more
liver toxicity than any other route (Loomis, 1974; World Health Organiza-
tion, 1978); slowly absorbed chemicals produce more gastrointestinal
damage (Ministry of Health and Welfare Canada, 1975).
Oral administration can be performed by various techniques, including
mixture in diet (food or drinking water), gavage (gastric intubation),
and gelatin capsules or pressed tablets. The relative frequency of use
for these techniques in 95 oral application studies surveyed was as
follows: gavage, 27%; capsule, 11%; and diet, 60% (89% in food; 11% in
water).
The most common technique for oral exposure of chemicals is by
mixing the test substance into the diet. One of the advantages of this
method is the reduced handling time due to the simplicity of administra-
tion. This saves valuable personnel time and disturbs the test animals
-------
153
less (Barnes and Denz, 1954). Diet mixtures also produce a low,
prolonged exposure with few concentration peaks (Benitz, 1970). This
exposure pattern is more representative of chronic human exposure than
other oral routes (Sontag, Page, and Saffiotti, 1976). However, one
must be aware of possible shortcomings or hazards involved in the use of
diet mixtures. These include: limited duration for stability of the
drug in the diet (Benitz, 1970; World Health Organization, 1978); adjust-
ment of the dose with animal maturation to maintain proper concentration
levels (Barnes and Denz, 1954; National Academy of Sciences, 1977; World
Health Organization, 1978); alteration of nutrient availability or
quality in the diet (Barnes and Denz, 1954; Food Safety Council, 1978);
unpalatability of the diet mixture to test animals affecting growth
rates (Barnes and Denz, 1954); nonhomogeneity of the diet-chemical
mixture (Sontag, Page, and Saffiotti, 1976); and measurement of daily
food consumption to calculate daily exposures (Barnes and Denz, 1954;
Benitz, 1970; Ministry of Health and Welfare Canada, 1975). The choice
between diet and gavage or capsule application is often based on such
considerations. Use of gavage or capsules eliminates problems of
palatability, drug stability, nutrient integrity, and consumption
calculations associated with diet administration. However, gavage or
capsules tend to produce peaks in substance concentrations, giving an
irregular cycle to blood and tissue levels (Benitz, 1970). Also, only
small amounts of a compound can be administered by gavage, thus limiting
its use to highly toxic substances (Food Safety Council, 1978). Other
disadvantages include higher mortality rates and more frequent need to
use a solvent to administer the chemical (Sontag, Page, and Saffiotti,
1976).
Often the best choice depends on the chemical being tested. Worden
and Harper (1963) and Bein (1963) gave examples of chemicals that are
more toxic by feed than by gavage and vice versa. If there is little
difference between the techniques, diet is to be preferred because of
its simplicity (World Health Organization, 1978). However, either
method is acceptable for exposure in tests involving subchronic durations
(Ministry of Health and Welfare Canada, 1975; National Academy of Sciences,
1977).
-------
154
Inhalation exposure is used in studies where the primary human
exposure is to be by inhalation or when information on the specific site
of entry (lung) damage is desired (Roe, 1968). The total effects of
inhalation can rarely be predicted (with a high degree of certainty)
from oral or parenteral studies, which necessitates the use of inhala-
tion exposure (Gage, 1970; World Health Organization, 1978). However,
unlike oral administration, use of inhalation exposure is neither simple
nor inexpensive.
For an adequate study of inhalation toxicity, specially designed
inhalation chambers are needed. The various chamber designs and advan-
tages are too numerous to discuss in this document, but Roe (1968), the
National Academy of Sciences (1977), and the World Health Organization
(1978) provide good reviews of this area. Additional important considera-
tions are the increased cost that this route of exposure entails and the
potential bottleneck represented by the limited number of quality
facilities available for testing.
One aspect of the test design that must be considered is the diffi-
culty associated with the determination of dose levels in an inhalation
study. The dose is usually measured by monitoring the chamber concen-
tration (a) of the toxicant and the time (£) that the test animal is in
the chamber (MacFarland, 1968; Roe, 1968). The dose is expressed as the
product (at) of these two variables (Ministry of Health and Welfare
Canada, 1975) and is used in lieu of actual dosage levels (National
Academy of Sciences, 1977). However, doses given in this manner are
difficult, if not impossible, to correlate with oral or injection studies
where the dose is expressed as milligrams per kilogram of body weight
(Barnes and Denz, 1954; Hayes 1967a). Additional problems associated
with inhalation dosage are the uncertainties regarding the actual amount
of the toxicant inhaled by the test animal, the amount of toxicant that
adsorbs onto the chamber walls, and possible variations in the flow rate
of the toxicant into the chamber (National Academy of Sciences, 1977;
World Health Organization, 1978). The amount of toxicant that enters
the test animal is influenced by its ventilation rate, the particle size
or vapor pressure of the toxicant, and the behavioral traits of the test
species (National Academy of Sciences, 1977; Roe, 1968).
-------
155
Another factor of importance in the chamber design is the expected
schedule of exposure durations. If continuous exposures (22 to 24 h/day,
7 days/week) are to be used, then the chamber must provide all food,
water, and other needs of the test animals. If intermittent exposures
(6 to 8 h/day, 5 days/week) are used, chambers of a simpler design can
be used. These two duration patterns produce different effects. The
nonexposure time in an intermittent test design allows the animal to
recover and tends to produce a pulsed chemical concentration pattern
(World Health Organization, 1978). In contrast, the continuous exposure
results in a steady-state pattern of toxin concentrations. The choice
of duration schedules should be related to the expected human exposure
pattern.
The generation and characterization of aerosols is another complexity
involved with inhalation studies. This is of little concern in vapor or
gas studies, but in aerosol mist or dust studies the aerosol particles
must be evaluated. Specialized equipment is required to generate the
aerosol particles, and some control is necessary to ensure uniform
particle size and density, at least within a size range that is suitable
for biological action (Ministry of Health and Welfare Canada, 1975;
National Academy of Sciences, 1977; Roe, 1968). Particle size must also
be monitored in the chamber itself to ensure that the exposure levels
remain consistent. The deposition sites (nose, trachea, bronchi, or
alveoli) and the degree of biological action of the inhaled particles
are determined by their solubility in tissue fluids, their reactivity
with lung tissue, and their size (Roe, 1968; World Health Organization,
1978). Generally the larger particles, 5 to 10 urn in diameter, are
trapped in the upper respiratory tract and nasal cavity (Ministry of
Health and Welfare Canada, 1975; World Health Organization, 1978).
Particles of less than 5 um disperse further into the lower respiratory
tract, with the depth of penetration increasing with decreasing particle
size (World Health Organization, 1978). It has been estimated that
approximately 25% of the inhaled particles are immediately expelled, 50%
are trapped in the upper respiratory tract, and only 25% reach the
alveoli (Morrow et al., 1966).
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156
After inhalation exposure, two types of toxic effects can occur.
Systemic effects, not related to the site of entry, are often observed
and in most studies represent the major toxic effects (Barnes and Denz,
1954; World Health Organization, 1978). These effects are quite similar
to parenteral or intravenous injection effects, at least in qualitative
data (Gage, 1970; World Health Organization, 1978). In contrast to oral
exposure, the circulatory transport of toxins entering through the lung
does not carry them through the liver first. Thus, other organs (e.g.,
brain, heart, and endocrine glands) are often sites of damage after
inhalation exposure (World Health Organization, 1978). Specific site of
entry effects (lung damage) can be studied only with inhalation exposure
(Roe, 1968). These effects result from both physical impairment of
respiratory function and slow metabolic transformation and transport by
lung epithelial tissue. Tests to evaluate this specific lung and
tracheal damage are reviewed by Roe (1968) and the World Health Organiza-
tion (1978). These include respiratory function tests, as well as
pathological damage evaluations.
A complication in evaluating toxic effects from inhalation is the
clearance mechanism. The mucociliary system often transports particles
out of the lung. These are either expelled or swallowed. If expelled,
the dose levels are not as potent as originally designed. If swallowed,
oral exposure results, which produces different types of toxic damage
(World Health Organization, 1978). These problems are most often
associated with the dust or aerosol studies.
The unique value of inhalation exposure in subchronic test designs
is primarily for the assessment of specific lung damage. Because of the
high cost, limited facilities, and additional personnel time involved,
inhalation exposure should be limited to such assessments and not used
for general assessment of systematic toxicity.
Other routes of exposure are infrequently used in subchronic studies,
including percutaneous and injection methods. Percutaneous or dermal
application is usually performed as a separate study. When percutaneous
exposure is used, several factors influence the test design and data
interpretation. These include slower activity and lower exposures than
-------
157
with oral exposure, difficulty of measuring absorbed dose, necessity of
using a vehicle or solvent in many cases, and ingestion by animals
during self-cleaning of doses applied to the skin (Benitz, 1970; Loomis,
1974; Ministry of Health and Welfare Canada, 1975; National Academy of
Sciences, 1977).
Parenteral injections are also used occasionally in subchronic
tests. These include intradermal, subcutaneous, intramuscular, intra-
venous, intraperitoneal, and intrathecal (Loomis, 1974). Intravenous
administration produces rapid distribution of toxins, with the degree of
metabolic transformation dependent upon the location of the used blood
vessel in the body. The other injection methods concentrate doses in
selected areas with slow diffusion to other tissues (Loomis, 1974). In
any injection method, rotation of injection sites is necessary (Benitz,
1970). Although this prevents excessive entry site damage, the use of
different target sites complicates the analysis of effects. In most
subchronic studies, the use of parenteral administration is not the
preferred route, because it is rarely an expected route of human exposure.
4.4.3 Route Comparisons
Few papers in the literature discuss in detail the comparative
value of exposure routes. The review articles used in Sect. 4.4.2
contain most of the available information. Primary studies designed to
evaluate this area are almost nonexistent. Even within individual
chemicals, comparisons are difficult. The use of different species,
doses, durations, and parameters of evaluation (pathology, hematology,
etc.) all combine to make route comparisons feasible only at a general
level. A few brief comparisons within chemicals are included as examples
of the difficulties. These are summarized in Table 4.28. Following the
chemical comparisons will be a brief discussion of route-to-route
extrapolation.
4.4.3.1 Arsenic — Bencko and Symon (1969) studied tissue levels of
arsenic in hairless mice following administration of white arsenic
(AS203) in drinking water. Continuous exposure to 50 mg or 250 mg of
-------
Table A.28. Summary of chemical examples tested for toxiclty by various routes of administration
Chemical
Arsenic
Beryllium salts
Insoluble salts
Soluble compounds
Beryllium chloride
Beryllium nitrate
Beryllium sulfate
Beryllium oxyfluorlde
Beryllium oxide
Beryllium phosphate
Beryllium
Beryllium sulfate
Beryllium fluoride
Beryllium sulfate
Beryllium oxide
Route
Oral
Inhalation
Oral
Inhalation " ~~
Inhalation
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Oral
Inhalation
Inhalation
Inhalation
Inhalation
Species
Mouse
Mouse
Guinea pig
Guinea pig
Guinea pig
Guinea pig
Guinea pig
Guinea pig
Guinea pig
Guinea pig
Guinea pig
Rat
Rat
Rabbit, dog
Dog, rat
Rabbit
Dose
10 to 15 mg/
kg/day
179. A UK As
per m*
0 to 30 mg/
kg/day
188.9 and 233
mg/ra3
27 and A3
mg/m3
0.1 g/day
0.1 g/day
0.1 g/day
0.1 g/day
0.1 g/day
0.1 g/day
6.6 and 66.6
Ug/day
34.25 pg
Be per m3
2.2 mg/m3
A mg/m3
80 mg/m3
Duration
32 days
6 h/day,
5 day /week.
6 weeks
11 to 29 weeks
30 to AO min/
day, 6 days/
week, 1 day to
15 weeks
30 to AO rain/day,
6 days/week,
1 and 10 days
A days
A days
A days
A days
4 months
A months
6, 12, 18, 2A
months
7 h/day, 5
days/week,
72 weeks
6 h/day, 5
days/week,
6 to 23 weeks
6 h/day, 5
days/week,
6 to 23 weeks
6 h/day, 5
days/week,
6 to 23 weeks
Effects noted by author
Transient accumulation in skin
and liver; weight loss
Transient accumulation in skin.
liver, and kidney
No adverse effects; low tissue
levels
No adverse effects; low tissue
levels
Severe symptoms; low tissue
levels
100Z mortality
100Z mortality
100Z mortality
100Z mortality
No mortality
No mortality
Decreased body weight; accumu-
lation in gastrointestinal
tract; skeleton, blood,
liver. No hepatic cell
destruction.
Inflammatory response; lung
tumors
Macrocytic anemia
Macrocytlc anemia
Macrocytic anemia
Reference
Bencko and Symon,
1969
Bencko and Symon,
1970
Hyslop, 19A3
Hyslop, 19A3
Hyslop, 19A3
Hyslop, 19A3
Hyslop, 19A3
Hyslop, 19A3
Hyslop, 19A3
Hyslop, 1943 M
Hyslop, 19A3 Ui
CO
Reeves, 1965
Reeves, Dei ten.
and Vorwald,
1967
Stokinger and
Stroud, 1951
Stokinger and
Stroud, 1951
Stokinger and
Stroud, 1951
Carbon tetrachlorlde Inhalation
Inhalation
Rat
Rat
Not given
10, SO, 100
ppm/day
3 to 5 min, 3
times per day
on alternate
days In 35 days
3 h/day, 6 to 8
weeks
Cirrhosis of liver
Elevated liver triglycerides,
SCOT,0 SGPT* unaffected
Reddy,
Krishnamurthy,
and Bhaskar,
1962
Shimizu, Nagase,
and Kawal, 1973
-------
Table 4.28 (continued)
Chemical
Dimethyl terephthalate
Fenoterol HBr
2-Methyl-A-
chlorophenoxy
Route
Oral
Oral
Oral
Subcutaneous
Subcutaneous
Subcutaneous
Subcutaneous
Subcutaneous
Intraperitoneal
Oral
Inhalation
Oral
Inhalation
Intravenous
Dermal
Species
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
House, rat
Rat
Rabbit
Rabbit
Dose
2 iiL per 100 g
150 to S20 ppm/
day
0.2 mL/kg/day
0.1 mL/100 g
1.3 mL/kg/day
1.3 mL/kg/day
1.3 mL/kg/day
1.3 mL/kg/day
0.06 mL per
100 kg
0.25, 0.5, IX
per day
16.5 and 86.4
mg/m3
15, 150, 1500
mg/kg/day
(rat)
1.5, 15, 150
mg/kg/day
(mouse)
0.01 to 1.0 rag/
kg/day
1.0 and 25 to
50 mg/kg/day
0.5, 1.0, 2.0
g/kg/day
Duration
1 day (acute)
6 weeks
3 days/week,
7 weeks
1 dose per
3 days in
90 days
2 days/week.
12 weeks
2 days/week,
12 weeks
2 days /week,
12 weeks
2 days /week.
12 weeks
I to 10 injec-
tions in
119 days
96 days
4 h/day, 5
days/week, 58
exposures
30 days
30 days
30 days
30 days
3 weeks
Effects noted by author
(Single dose) elevated SGOT,a
SGPT6
Trlglyceride accumulation in
liver
Increased liver fat
Cirrhosis
Cholanglofibrosis
Hepatic vein thrombosis
Neoplasia
Cirrhosis
Retardation of growth; elevated
SGPT; hepatic necrosis (tran-
sient); Inhibition of normal
mineralization of tooth
dentine
Decreased body weight
No adverse effects
Enlarged salivary glands;
enlarged hearts. Mortality:
rats — 642 male, 24X female;
mouse — 3X male and 3X female
No adverse effects; <1X
mortality
Enlarged salivary glands;
enlarged hearts; 6X mortality
Transient growth retardation;
erythema; decreased leuko-
Reference
Shimizu, Nagase,
and Kawai, 1973
Alumot et al. ,
1976
Friedman, Sage,
and Blendermann
1970
Reddy,
Krishnamur thy ,
and Bhaskar,
1962
Reuber and Glover,
1967a
Reuber and Glover,
1967Z>
Reuber and Glover,
1967e
Reuber and Glover,
1968
Hals, Bjorlln, and
Jacobsen, 1973
(-•
Ui
\0
Krasavage, Yanno,
and Terhaar, 1973
Krasavage, Yanno,
and Terhaar, 1973
Kast et al., 1975a
Kast et al., 1975a
Kast et al., 1975a
Verschuuren, Kroes,
and Tonkelaar,
i nt e
acetic acid
(MCPA)
cytes; hyperplaaia, hyper-
keratoais, and loss of
elasticity of skin
100X and 75X mortality in high
dose groups
1975
-------
Table 4.28 (continued)
Chemical
Route
Species
Dose
Duration
Effects noted by author
Reference
2-Methyl-A-
chlorophenoxy
propionic acid
(MCPP)
Oral
Dermal
Oral
Rat
Rabbit
Rat
50, 400, 3200 90 days
ppm
0.5, 1.0, 2.0 3 weeks
g/kg per day
50, 400, 3200 90 days
ppm
Growth retardation
Increased kidney weight
Increased erythrocyte size
Increased hemoglobin content
Transient growth retardation;
erythema, transient loss of
skin elasticity
Decreased red blood cell count;
decreased hemoglobin content;
increased alkaline phosphatase
activity, decreased hematocrit
and leucocyte values (male
only); Increased kidney weights
depression of ovary and
prostate weights
Verschuuren, Kroes,
and Tonkelaar,
1975
Verschuuren, Kroes,
and Tonkelaar,
1975
Verschuuren, Kroes,
and Tonkelaar,
1975
Nefopam hydrochloride Intraperitoneal
Intramuscular
Oral
Intravenous
Oral
Intramuscular
A9-Tetrahydrocannablnol Oral
Intravenous
Rat
Rat
Rat
Dog
Dog
Dog
Rhesus monkey
2 and 10 mg/
kg per day
1 and 2 mg/
kg per day
20, 40, 80
mg/kg per
day
1 and 5 mg/
kg per day
4, 10, 40
mg/kg
per day
1.5 and 3.0
mg/kg
per day
50, 250, 500
mg/kg
per day
5, 15, 45
mg/kg
per day
5 days/week.
4 weeks
7 days /week,
2 weeks
4 weeks
7 days/week.
4 weeks
7 days/week,
4 weeks
7 days/week,
3 weeks
28 days
28 days
Increased liver weights
No adverse effects
No adverse effects
Slight weight loss
Slight weight loss
No adverse effects
Summarized In Table 4.29
Case, Smith, and
Nelson, 1975
Case, Smith, and
Nelson, 1975
Case, Smith, and
Nelson, 1975
Case, Smith, and
Nelson, 1975
Case, Smith, and
Nelson, 1975
Case, Smith, and
Nelson. 1975
Thompson et al . ,
1974
SCOT — serum glutamlc oxaloacetic transaminase.
SGPT — serum glutamlc pyruvic transaminase.
-------
161
arsenic per liter (10 to 15 mg/kg per day) for 32 days produced levels
of the chemical in the skin and liver that peaked at 16 days, then
decreased significantly by 32 days.
In an inhalation experiment (Bencko and Symon, 1970), hairless mice
were exposed in a dust chamber to As203 dried onto fly ash at a mean
concentration of 179.4 yg arsenic per m3 for 5 days per week, 6 h daily,
for 6 weeks. Concentrations of arsenic in the liver, kidney, and skin
were measured after 1, 2, 4, and 6 weeks of exposure. Arsenic levels
increased rapidly in the liver and kidney up to 2 weeks, then decreased
sharply during the third and fourth weeks. Skin values peaked similarly
as a result of direct exposure in the dust chamber, but decreased more
gradually. The authors recognized that effects of inhaled particles
cannot be distinguished from effects of particles ingested as a result
of mucociliary clearance or licked from the skin during grooming, and
they concluded that the course of arsenic accumulation is similar fol-
lowing oral administration and inhalation. The drop in arsenic accumula-
tion in tissues after the initial peak was subsequently shown to be
caused by a stimulation of the excretory mechanism, which increased the
excretory rate of the animals (Bencko, Dvorak, and Symon, 1973).
4.4.3.2 Beryllium — In an extensive test for beryllium toxicity
(Hyslop, 1943), guinea pigs ingested up to 40 mg per kg of body weight
per day of various beryllium salts and exhibited no adverse effects
after 11 to 29 weeks of exposure. The total amounts of beryllium
retained in body tissues were small in comparison to the quantities
ingested. Inhalation of insoluble beryllium carbonate (188.9 mg/m3 or
233 mg/m3) 30 to 40 min/day, 6 days/week for 1 day to 15 weeks, failed
to produce toxic effects in guinea pigs. However, inhalation of soluble
compounds induced severe symptoms in a short time: 27 mg/m3 of beryllium
oxyfluoride and 43 mg/m3 beryllium sulfate induced 67% mortality after
10 days and 1 day of exposure, respectively. Distribution of the
chemical in tissues remained remarkably low following inhalation of both
soluble and insoluble beryllium salts. Intraperitoneal injections of
0.1 g of beryllium chloride, beryllium nitrate, beryllium sulfate, or
beryllium oxyfluoride produced 100% mortality in guinea pigs after 4
days; beryllium oxide and phosphate were not lethal after 4 months.
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162
Other studies of beryllium toxicity are summarized in Table 4.28.
These are of little value for route comparisons because of dose, duration,
and species differences.
4.4.3.3 Carbon Tetrachloride (CCl^) — Reddy , Kr ishnamurthy , and
Bhaskar (1962) exposed albino Wistar rats to carbon tetrachloride
vapors for 3 to 5 h, three times per day on alternate days for 35 days.
Inhalation of the vapors caused cirrhosis of the liver in 8 of 9 males
and 5 of 15 females tested. Eight of the 15 females developed patchy
fibrosis of the liver. Inhalation of 10, 50, or 100 ppm CClit per day,
3 h/day for 6 to 8 weeks resulted in temporarily elevated triglyceride
levels in the livers of male Sprague-Dawley rats, but no progressive or
cumulative changes were observed (Shimuzu, Nagase, and Kawai, 1973).
Transaminase activity was not affected by inhalation exposure, although
a single oral dose of CClt4 produced increased SCOT and SGPT values.
Elevated levels of liver fat content have been observed in rats
following oral administration of CCl^. After 6 weeks of feeding
fumigated mash containing a daily dose of 1% of the LD50 (5 g per kg of
body weight) , triglyceride accumulation in the liver was found to be the
most sensitive sign of chronic CCl^ poisoning (Alumot et al. , 1976).
Oral intubation of 0.2 mL CCl^ per kg of body weight three times a week
for 7 weeks also caused an increase in the liver fat content of rats
(Friedman, Sage, and Blendermann, 1970).
Subcutaneous injection of 0.1 mL per 100 g of body weight every
3 days for 90 days produced cirrhosis in 9 of 9 male rats and in 6 of
15 female rats (Reddy, Kr ishnamurthy, and Bhaskar, 1962). Five females
developed patchy fibrosis of the liver, and four females did not develop
liver lesions. The authors concluded that the frequency of CCl^-induced
cirrhosis was the same following inhalation (described previously) and
subcutaneous exposure. In a series of separate studies in which CClt*
was administered subcutaneously to Buffalo strain male and female rats,
effects of the chemical were manifested as a function of age and sex of
the animals (Reuber and Glover, 1967a, 19672>, 1967c, 1968).
Intraperitoneal injection of 0.06 mL of CCli, per 100 kg of body
weight (up to seven injections during 119 days) into albino rats induced
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163
an inhibition of weight gain, elevated SGPT values, and hepatic necrosis,
which then regenerated (Hals, Bjorling, and Jacobsen, 1973). Concom-
itantly, inhibition of normal mineralization of the dentine layer of
rat incisors was observed.
These ten studies on carbon tetrachloride, although using the same
species, contain wide variations in dose and duration. Also, the use of
different age groups in some studies further complicates the comparisons.
Thus, comparisons of routes using these data would be difficult.
4.4.3.4 Dimethyl Terephthalate (DMT) — DMT, shown previously to be
of a low order of toxicity when administered intragastrically, intraperi-
toneally, and subcutaneously to rats and mice (Slyusar and Cherkasov,
1964, and Prusakov, 1966, as cited in Krasavage, Yanno, and Terhaar,
1973), was further evaluated in various acute studies and in subchronic
feeding and inhalation studies by Krasavage, Yanno, and Terhaar (1973).
Neither feeding nor inhalation caused mortality. The toxicological
effect seen in either study was reduced weight gain in high dose animals
of the feeding study. Although this study was well designed for a
comparison of routes, the lack of toxic effects precludes comparisons.
4.4.3.5 Fenoterol HBr — Kast et al. (1975a) tested the beta-
sympathomimetic, fenoterol HBr, for toxic properties. Subacute exposures
(oral, intravenous, and inhalation) continued for 1 month. Inhalation
produced no symptoms in rats that could be related to fenoterol. However,
after oral administration enlarged salivary glands and enlarged hearts
(with some myocardial scarring) were evident in both rats and mice.
Less than 1% of the rats in the inhalation experiment died during the
exposure period, while oral administration of high doses produced mortality
rates of 64% for male rats, 24% for female rats, and 3% for male and
female mice. Rabbits receiving the drug intravenously developed enlarged
salivary glands and enlarged hearts. Six percent of the rabbits died
during the third week of the trial. In this study, the durations were
all comparable, but the use of various species and different doses
reduced the reliability of the comparisons.
-------
164
4.4.3.6 2-Methyl-4-Chlorophenoxy Acetic Acid (MCPA) — In a subacute
dermal study, doses of 0, 0.5, 1.0, and 2.0 g MCPA per kg of body weight
were applied to the skin of chinchilla rabbits in an aqueous paste
(Verschuuren, Kroes, and Tonkelaar, 1975). Four of four rabbits treated
with 2 g MCPA and three of four treated with 1 g MCPA died during the
treatment period, possibly as the result of dysbacteria (an illness
often seen in rabbits of that institute). Growth retardation was observed
in animals of the 0.5-g/kg group. However, growth rates improved during
the recovery period. Other major changes noted and attributed to dermal
application of MCPA were erythema; a decrease in the number of lympho-
cytes in the 0.5-g/kg group; and hyperplasia, hyperkeratosis, and loss
of elasticity of the skin.
In the subchronic oral study, male and female rats received 0, 50,
400, or 3200 ppm MCPA in their food for 90 days (Verschuuren, Kroes, and
Tonkelaar, 1975). During the first week's feeding of 3200 ppm the rats
exhibited unhealthy fur and cold extremities. There was no mortality
attributed to dosage. Decreased food consumption, decreased body weight
gain, and increased kidney weights were characteristic of the 3200-ppm
group. There was also an increase in erythrocyte size and a proportional
increase in hemoglobin content in rats of the high dose group.
4.4.3.7 2-Methyl-4-Chlorophenoxy Propionic Acid (MCPP) — In a sub-
acute dermal study, doses of 0, 0.5, 1.0, and 2.0 g MCPP per kg of body
weight were applied to the skin of chinchilla rabbits in an aqueous
paste (Verschuuren, Kroes, and Tonkelaar, 1975). One rabbit of the
1.0-g/kg group and one rabbit of the 2.0-g/kg group died. Growth retarda-
tion was seen in all groups, but some recovery was observed during the
2 weeks after termination of treatment. Erythema was observed and was
dose related. The skin lost its elasticity, but reverted to normal
condition during the recovery period. Organ weights of the treated
animals were no different from those of the controls.
In a subchronic oral study, male and female rats received 0, 50,
400, or 3200 ppm MCPP in their food for 90 days (Verschuuren, Kroes, and
Tonkelaar, 1975). Rats of both sexes treated with 3200 ppm had unhealthy
fur, decreased hemoglobin content, decreased erythrocyte counts, and
-------
165
increased alkaline phosphatase activity; hematocrit and leucocyte values
were decreased in males only. Some of the changes observed also occurred
with 400 ppm. Increased kidney weights were seen in rats at levels of
3200 and 400 ppm, and depression of ovary and prostate weights was seen
with 3200-ppm feedings.
These two studies of MCPA and MCPP are typical of many primary-
studies in the literature. Although two routes of exposure are used,
they differ in species, duration, and dose. This makes them of minimal
value for comparisons.
4.4.3.8 Nefopam Hydrochloride — Nefopam, a compound with nonnarcotic
analgesic activity, was tested for subacute toxic properties by Case,
Smith, and Nelson (1975). Carworth CFN rats were administered the drug
by three routes: intraperitoneally, in daily doses of 0, 2, and
10 mg/kg (5 days per week for 4 weeks); intramuscularly, in daily doses
of 0, 1, and 2 mg/kg (7 days per week for 2 weeks); and orally, in the
diet, in daily doses of 0, 20, 40, and 80 mg/kg for 4 weeks. Increased
liver weights were observed in female rats receiving 10 mg/kg of the
drug intraperitoneally (approximately 30% of the intravenous LDsg).
No indications of toxicity were seen in any of the other groups receiving
up to 2 mg/kg per day intramuscularly (approximately 3.5% of the intra-
muscular LDso) or up to 80 mg/kg per day orally (approximately 65% of
the oral U^g),
Mongrel dogs showed only slight weight loss after daily intravenous
administration of 5 mg/kg (approximately 25% of the intravenous LDso)
and after daily oral administration of 4 and 40 to 80 mg/kg (25% to 50%
of the oral LDsg) of nefopam. No toxic effects were observed following
daily intramuscular injections of up to 3 mg/kg of body weight (approxi-
mately 10% of the intramuscular LDso) °f t*ie drug.
Despite using three routes of exposure in both the rat and the dog,
the study by Case, Smith, and Nelson (1975) still differs in dose and
duration so that comparisons would be difficult.
4.4.3.9 A9-Tetrahydrocannabinol (A9-THC) — Thompson et al. (1974)
described the toxic effects of A^-tetrahydrocannabinol, the active
ingredient in marijuana, when adminstered intravenously or orally to
-------
166
primates for 28 days. In a preliminary acute study rhesus monkeys
survived up to 9000 mg/kg orally, while 128 mg/kg intravenously produced
100% mortality (two of two). The subacute study consisted of male and
female monkeys, either treated with oral doses of 0, 50, 250, or
500 mg/kg per day or injected intravenously with 0, 5, 15, or 45 mg/kg
per day. During oral administration, two male monkeys treated with
500 mg/kg per day and one monkey treated with 50 mg/kg became moribund
and were sacrificed on days 10, 14, and 16, respectively. Deaths in the
intravenous trial caused by acute hemorrhagic pneumonia occurred only in
the 45 mg/kg per day group at days 8 and 19. Table 4.29 summarizes the
main toxic effects observed in intravenously or orally treated monkeys.
Table 4.29. Main toxic effects produced by oral or
intravenous administration of
A9-tetrahydrocannabinola
Routes of administration
Oral Intravenous
Organ size
Thymus Atrophic Atrophic
Pancreas Atrophic
Testes Atrophic
Adrenal Enlarged
Liver Enlarged Enlarged
Body weight Decreased
Clinical effect
Leukocytosis
Myeloid hyperplasia
Anemia
Bromosulfophthalein
retention
Electrolyte balance
Proteinuria
Bradypnea
Anorexia
Ulcerative colitis
Other
Behavioral changes
Development of tolerance
Yes
Yes
No
No
Altered
Yes
Yes
Yes
effects
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
-------
167
This study has differences in dose levels that make the conclusions of
the authors questionable.
The preceding examples are typical of within-chemical comparisons.
Almost every comparison was complicated by test design variables. Any
conclusions that could be drawn from these would be tenuous at best.
However, this is unfortunately the current status of information on this
subject. A computer and manual search of over 30 000 titles and abstracts
produced no studies specifically designed to compare exposure routes for
subchronic toxicity tests. Without sufficient literature data, an
evaluation of the route-to-route extrapolations is limited to a general
discussion.
The value of extrapolating data from one route to another is limited.
The oral route of exposure has been sufficiently developed to the point
that it is easy to perform, requires a minimum of specialized equipment,
and produces good toxicity data. Thus, there is little need to extrapo-
late data from other routes to determine oral toxicity for most chemicals.
Injections are rarely primary routes of exposure and are relatively easy
to perform for durations of a subchronic nature. Extrapolation from
another route is not needed here.
Dermal and inhalation studies are the principal routes for which
extrapolation might prove useful. Difficulty in calculating exact dos-
ages taken in by test animals applies to both routes. Also the anatomy
and physiology of the entry sites for these routes differ between the
test species and man (especially for inhalation), thus affecting the
predictive value of data from these two routes. For inhalation, the
additional problem of special equipment requirements limits the number
of studies that can be performed. Extrapolation might be useful in
these routes, if the substituted methods solve these problems. For
example, an intravenous injection could be used to replace an inhalation
evaluation (at least for systemic toxicity) thus reducing the problem of
limited facilities. Dosages could also be more stringently controlled.
Another alternative route could be the oral route. This would require
previous testing to ascertain if any significant metabolic transforma-
tions by the gastrointestinal tract occur for that chemical. Of course,
these extrapolations would not provide information on toxicity at the
-------
168
site of entry. These data would have to be obtained from preliminary
studies (acute or subacute).
Before any of these route extrapolations can be employed, much lab-
oratory research needs to be performed. Specific comparisons over a
wide range of chemicals need to be carried out so that the background
data would be available. A basis for extrapolation should be sought in
pharmacokinetic and metabolism studies that could justify the quantita-
tive and qualitative validity from one route to the other. This research
would help to define the types of toxicants and the aspects of each
route, for which extrapolation would not be useful.
4.4.4 Conclusions
In selecting the route of exposure for subchronic toxicity tests,
the route by which human exposure is most probable is the preferred
choice. The routes used in most subchronic testing are oral and inhala-
tion exposures. Oral exposure can be by diet mixtures, gavage, or
capsule, all of which are relatively simple and effective. Inhalation
exposure requires special equipment, which results in higher costs per
test and acts as the limiting factor in determining the number of tests
that can be performed. Occasional use is made of percutaneous and
intraperitoneal exposures, depending on the chemical, its metabolic
pattern, and the expected human exposures.
In some cases, extrapolation of data from one route to determine
potential toxicity by another route may be feasible. Some substitution
for dermal or inhalation studies could theoretically occur by using data
from intravenous or oral studies. However, the literature is currently
insufficient to evaluate this area. The lack of data in the literature
stresses the need for research on route-to-route comparisons and
extrapolations.
4.5 PATHOLOGY
4.5.1 Introduction
Gross and microscopic pathology examinations are currently an
integral part of most toxicity tests. Particularly for the subchronic
-------
169
evaluations, extensive organ and tissue examination is required or
recommended. The EPA guideline for pesticide evaluations (Federal
Register, 1978) is a good example of this, recommending for oral admin-
istration the microscopic examination of approximately 30 organs or
tissues from all animals at the control and high doses. Additionally,
any lesions on other organs revealed by gross examination require a
histopathological follow-up. For intermediate and low doses, the tissues
to be examined depend on the toxic effect, target organ, and lesions
found in the gross examination or in the high-dose histology procedures.
Table 4.30 gives a typical list of tissues to be examined (Peck, 1968).
Table 4.30. Postmortem studies
Gross
External lesions Oral tissues
Tumors Voluntary muscle
Abdominal contents Bones
Pelvic contents Brain
Thoracic contents Spinal cord
Cervical tissues
Organ weights
Histomorphology
Thytnus Heart
Lymph node Large and small
Thyroid arteries
Parathyroid Lung
Adrenal Stomach
Pancreas Duodenum
Liver Jej unum
Gallbladder Ileum
Kidney Colon
Urinary bladder Bone (with marrow)
Ovary Marrow smears
Uterus Brain
Testes Pituitary
Prostate Voluntary muscle
Seminal vesicle Eyes
Mammary gland Other tissues as
indicated by
gross observations
and by drug activity
Source: Adapted from Peck, 1968 — An
Appraisal of Drug safety Evaluation in
Animals and the Extrapolation of Results to
Man. In: Importance of Fundamental
Principles in Drug Evaluation, D. H. Tedeschi
and R. E. Tedeschi, eds. Raven Press, New
York. pp. 450-471. Reprinted with permis-
sion of the publisher.
-------
170
Besides the general appearance and microscopic condition of the organs,
the organ weight change is also monitored during and at the end of the
exposure period. An extensive, detailed protocol for organ examination,
removal, fixation, and interpretation is given by Prieur et al. (1973).
The value of such extensive examinations is debatable, however, with
evidence and opinions supporting both the pro and con arguments. The
purpose of this section of the literature review is to give an overview
of opinions concerning the use of pathology and the relative predictive
efficiency for each organ.
4.5.2 Use of Pathology
In some earlier short-term tests, microscopic examination was not
performed because either the exposure periods were considered too short
to affect organ tissues (Bratton, 1945) or the ability of the histopatho-
logic examination to add quantitative information to the other observa-
tions was doubted (Smyth and Carpenter, 1948). The significance of
histopathology was considered to increase somewhat when the results of
the short test were to be a preliminary step for a 2-year study (Smyth
and Carpenter, 1948). Smith (1950) felt that histopathology could also
be useful if a series of compounds were being compared or ranked for
relative toxicities. Even in these cases, the tissues to be examined
were few, generally restricted to the liver, kidney, heart, and any
lesion-bearing organs. This trend toward reduced histopathology in
earlier short-term tests was continued by Smyth, Carpenter, and Weil
(1951) in their development of the range-finding test. As shown in
Table 4.31, these researchers found no material that caused microscopic
tissue damage as the first effect (at the lowest dose). They did find
that organ weight could be a significant early indicator of toxic
effects, particularly weight changes in the liver and kidney.
Barnes and Denz (1954), in a general review of chronic toxicity,
also expressed reservations about the value of pathology, particularly
histopathology. They identified some of the sources of variation in
pathological results as: the subjective nature of the examination; the
occurrence of natural disease; and the problems of age changes in the
test species. They saw little value in routine sectioning and histo-
-------
Table 4.31. Range-finding data on subacute oral toxicitya
Least dally dose causing symptom (g/kg)
Material studied
Acroleln<*
Aldol
Allyl alcohol"
l,3-Butanediol«
"Cellosolve" (2-ethoxyethanol)d'e
Dichloral urea
2,4-Dichlorophenoxy ethyl
sulfate, sodium
Diethanolaalne
(2,2'-lmlnodiethanol)e
Dl(2-ethylhexyl) adipate*
Di(2-ethylhexyl)
tetrahydrophthalate8
2-Dimethylaminoethanol
(dloethylethanolamlne)*
2 , 4-Dimethy 1-2-oethylene-
1,2,4-Chlodiazolidine thlone
Dimethyl methylene diphenol-p,p'e
Dimethyl methylene diphenol-p,p',
diglycidyl ether
Ethylene chlorhydrin
(2-chloroethanol)d
Ethylenediamlne"''
2-Ethyl-l , 3-hexanediole
2-Ethylhexanimido (diethyl-
2-ethyl hexanoate)
Glycidyl sorbate dimer
2-Hethyl-2,4-pentanedlol
Monoethanolamlne (2-amlnoethanol)e
Monolsopropanolamine8
Tetrabutyl thiodlsucclnate*
1,2,3, 4-Tet rathla-6 , 9-diazecane-
5,10-dithione
Triethanolamlne6
Maximum
dose
(g/kg)
0.0015
1.58
0.0097
5.60
1.89
5.33
0.66
0.68
4.74
2.67
0.89
0.10
0.52
0.35
0.042
0.31
0.70
1.53
0.27
0.31
2.67
2.22
10.3
0.55
2.61
Minimum
dose
(g/kg)
0.00017
0.026
0.0013
0.32
0.052
0.012
0.011
0.0051
0.16
0.053
0.045
0.0025
0.002
0.014
0.0024
0.036
0.20
0.10
0.012
0.043
0.16
0.14
0.26
0.04
0.0050
Reduced Reduced
growth appetite
0.0015
1.58
0.0097
0.74 0.74
0.18 5.33
0.66
0.68
2.92 2.92
2.67
0.10 0.10
0.31 0.12
0.70
0.27
10.3
0.55
1.27
Altered .
sf S
0.0015
0.0097
0.74 0.74
0.18 2.55
0.20
0.090 0.17
2.92 2.92
0.84
0.89
0.039
0.012
0.31 0.12
1.53
0.012 0.27
0.64 1.28
2.22
1.08 10.3
0.17
0.17 0.73
Death
0.0097
1.89
2.55
0.66
0.17
4.74
0.12
0.27
1.28
10.3
0.73
Maximum
dose Single-dose
having no oral U>so°
effect (g/kg)
(g/kg)
0.00017
0.43
0.0040
5.60J
0.21
0.048
0.047
0.020
0.61
0.19
0.18
0.010
0.52.T
t
0.35'
0.0024
0.036
0.48
0.44
0.0129
0.3V
0.32
0.60
0.26
0.04
0.08
0.046 (0.039-0.056)
2.18 (2.00-2.38)
0.064 (0.056-0.074)
22.8 (21.8-23.9)
3.00 (2.51-3.59)
32 RF
1.41 RF
1.82 (1.66-2.00)
9.11 (7.28-11.4)
114 RF
2.34 (2.26-2.42)
0.50 (0.48-0.52)
4.04 (3.73-4.38)
8.14 (7.25-9.14)
0.089 (0.067-0.117)
1.16 (0.98-1.37)
2.71 (2.52-2.93)
40 RF
5.75 (5.28-6.25)
4.76 (4.27-5.30)
2.74 (2.39-3.15)
4.26 (3.89-4.67)
100 RF
8.53 (6.12-11.9)
9.11 (8.45-9.82)
"Materials administered in the food to groups of ten rats for 30 days on each dose level unless noted otherwise.
fcThe dose producing microscopic lesions in liver, kidney, spleen or testis of any rat.
"When LDSO was derived from an advanced test, the range of 1.96 times the standard deviation is shown in parentheses. The letters "RF"
refer to data derived from a range-finding test.
^The dose was administered in the drinking water. In this case appetite is judged on the basis of mllllllters of water consumed.
*Doses administered for 90 days.
^The dose shown is the maximum that was administered, and It caused no symptoms.
sThe dose shown is the minimum administered, and it caused some symptoms.
Source: Adapted from Smyth, Carpenter, and Weil, 1951. Copyright 1951, American Medical Association. Reprinted with permission
cf the publisher.
-------
172
logical examination of 20 major organs and suggested that a thorough
gross examination would suffice. They also doubted the value of organ
weight changes as indicators of toxicity. Barnes and Denz cited Cameron
(1952) concerning the increase in organ weights caused by hypertrophy
without true damage to the tissues. They suggested that organ weights
be used as toxicity indicators only if histology also shows damage.
Zbinden (1963) also questioned the practice of examining all tissues
histologically. He felt that to reduce the extensive technical work
histology should be done only on high-dose animals and 25% to 50% of the
control animals. Further examination would be limited to organs showing
changes. In addition, Zbinden recommended a grading system that ranks
histopathological findings, thus increasing the accuracy for initial or
subtle effects. The role of organ weight changes is secondary when
autopsy and histology are done carefully.
In contrast to these earlier authors, more emphasis is placed on
organ weight changes than on histopathology as the predominant indicator
of pathological damage by Weil and McCollister (1963) in their eval-
uation of subchronic and chronic tests. As shown in Table 4.32, out of
33 short-term experiments only twice was histopathology the sole toxic
indicator at the lowest dose. However, organ weight change was the sole
low-dose toxicity indicator seven times. The same trend is shown for
2-year studies, indicating that histopathology is a poor short-term
indicator of chronic toxicity. Specifically, among the organs examined
only the liver, kidney, testes, and spleen showed weight changes indic-
ating histologic damage. The weight changes of the liver and the kidney
increased their value as toxicity predictors in the longer 2-year
studies. Based on these findings, Weil and McCollister recommended that
for determination of lowest dose level of effect, the liver and kidney
should be examined for weight changes and histological damage. Although
not as useful as organ weight, histology of the liver and kidney did
increase the predictive efficiency. These factors, plus body weight
gain, would serve to adequately indicate toxicity.
In a later paper, Weil et al. (1969) again commented on the value
of histopathology. In comparing 90-day, 1-week, and single exposure
tests, they used primarily the histology and weight changes of the
-------
Table 4.32. Summary of observations of effects detected in short-term and two-year oral studies
(At lowest dosage level in which any effect was detected)
Short-term
Criterion
of effect
Mortality
Food intake
Body weight
Organ weights
Liver
Kidney
Heart
Spleen
Testes
Lung
Brain
Thyroid
Stomach
Adrenal
Gross pathology
Micropathology
Liver
Kidney
Heart
Spleen
Testes
Lung
Adrenal
Pancreas
Bone marrow
Voluntary
muscle
Stomach
Intestine
Bladder
Brain
Sciatic nerve
Number of
studies in
which this
criterion
was
followed
33
30
33
27
27
17
17
16
15
3
0
1
0
33
30
30
22
21
21
20
16
16
1
1
3
3
2
3
2
Number of
pertinent
studies0
27
26
27
22
22
13
17
13
12
2
0
0
0
27
24
24
17
16
17
16
12
12
0
1
1
1
2
3
2
Number in
which this
criterion
was
affected
1
2
10
8
7
0
1
0
0
0
0
0
0
0
3
1
0
0
1
0
0
0
0
0
0
0
0
0
0
Number
in which
it was
the sole
effect
0
0
6
4
3
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
Number of
studies in
which this
criterion
was
followed
33
29
33
30
30
17
16
18
12
4
2
1
3
3
33
33
29
23
33
32
27
23
11
6
15
21
12
11
0
Two-year
Number of
pertinent
studies'2
25
21
25
22
22
13
10
12
10
3
1
0
0
25
25
25
21
16
25
25
20
17
6
3
10
14
9
8
0
Number in
which this
criterion
was
affected
2''
0
15
6
5
0
0
0
0
0
0
0
0
0
9
8
0
1
0
0
0
0
0
0
0
0
0
0
0
Number
in which
it was
the sole
effect
0
0
7
0
3
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Number this
criterion
affected at
next higher
dosage level*'
Short-
term
0
3
9
4
2
0
1
1
0
0
0
0
0
0
}
2
0
0
0
0
0
0
0
0
0
0
0
0
0
Two-
year
0
1
2
1
1
0
0
0
0
0
0
0
0
0
3
2
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
Table 4.32 (continued)
Short-term
Criterion
of effect
Hematology
Blood urea
nitrogen
Clinical urine
analysis
Central nervous
system
Neoplasm
Fertility
Total number
a-lcm-lf Icanf-lv nlf-m
Number of
studies in
which this
criterion
was
followed
19
10
1
1
33
0
of studies
"•oH at* anv t
Number of
pertinent
studiesa
16
7
1
1
27
0
in which this
1n*ana*j*> 1 o-iral
Number in
which this
criterion
was
affected
1
0
0
1
0
0
criterion was
Number
in which
it was
the sole
effect
0
0
0
1
0
0
followed
Number of
studies in N
criterion
was
followed
31
6
7
0
33
5
minus the studies
Two-year
lumber of
ertinent
studies0
24
3
5
0
25
3
in which
Number in
which this
criterion
was
affected
0
0
0
0
0
1
none of the
Number
in which
it was
the sole
effect
0
0
0
0
0
0
criteria were
Number this
criterion
affected at
next higher,
dosage level
Short-
term
0
0
0
0
0
0
Two-
year
0
0
0
0
0
0
•vj
-t-
Number of times that effects were demonstrated by the criteria when they were not affected at the next lower dosage level.
Highest level fed was the only one affected.
Source: Adapted from Weil and McCollister, 1963. Reprinted with permission of the Journal of Agricultural and Food Chemistry.
Copyright by the American Chemical Society.
-------
175
kidney and liver as indicators of toxic dosages. However, for the 90-day
studies, they examined additional organs for toxic changes. The results
from the additional organs were less sensitive than the other criteria
selected, and they again suggested that histopathology be limited to
certain organs such as the liver, kidney, or spleen.
Peck (1968), in his discussion of pathology in drug evaluations,
questioned the value of organ weight changes without a histopathological
examination as a follow-up. He stated that, when organ weight changes
are reflective of toxic effect, histopathology will also show results.
But if histopathology is negative, the organ weight change is most
likely not due to the toxic action of the drug. Therefore, to be sure
that the effect indicated by organ weight change is a result of the
tested substance, a histopathology examination is necessary. Peck also
stated that histopathology is useful in determining the significance of
spontaneous species-specific lesions. If the commonly occurring lesions
for a species are altered in time of appearance, number, character, or
severity, this could be due to the activity of the drug. However, as a
caution to the use of histopathology, Peck stressed the difficulty in
interpreting results (particularly for electron microscopy) and in
extrapolating results from the test species to man.
Benitz (1970), in his review of chronic toxicity, also stressed the
need for a complete histopathological examination. He emphasized that
to achieve the maximum results from pathology techniques, a thorough
gross necropsy is the critical starting procedure. If the organs are
excised improperly the extent of later microscopic examination can be
irrelevant. This includes, of course, all organs, because in his
opinion restricting histology to organs with gross lesions significantly
decreases the determination of complete toxicity. He also rejects the
procedures of examining only the liver and kidney since morphological
effects or target organs for new drugs are unknown; of examining only
the major organs because this is too imprecise and varies with the
pathologist's definition of major; and of examining organs with gross
lesions and all organs from a few representative test animals as this
may not yield the optimum amount of morphological data. Benitz does
allow for deviations from histopathological examination of all organs if
-------
176
previous studies for that chemical indicate certain toxicity patterns or
if results are needed only for certain organ systems. He stressed,
however, that all organ systems have demonstrated toxic effects from
chemicals, as is shown in Table 4.33. These effects can be characterized
as follows: 76.5% were degenerative changes, 14% were inflammatory
lesions, 7% were circulatory disorders, and 1.8% were neoplastic, precan-
cerous lesions. Benitz generally recommended weighing of the organs as
Table 4.33. Incidence (in percent) of drug-induced
pathological changes encountered over a ten-year
period in approximately 14 000 animals
System and organ
Cardiovascular system
Respiratory system
Alimentary system
Liver
Gastrointestinal tract
Pancreas
Urinary system
Kidney
Ureter and urinary bladder
Reproductive system
Males
Females
Ductless glands
Thyroid
Adrenal
Pituitary
Islets of Langerhans
Hematopoietic system
Lymphoid and reticuloendothelial
system
Central nervous system
Sensory system
Eye
Ear
Locomotor system
Skin
Incidence
By organ By system
15.8
3.8
1.0
12.0
1.0
4.8
2.9
11.5
4.8
5.3
0.5
1.4
0.5
4.3
5.3
20.6
13.0
7.7
22.1
3.3
7.2
3.3
1.9
10.5
1.0
Source: Adapted from Benitz, 1970. Reprinted with
permission of the publisher.
-------
177
an indicator of toxicity. However, he felt that use of relative organ
weights could be misleading, and if they are used the absolute organ and
body weights should also be given. This eliminates any uncertainty
regarding whether the change is caused by reduced body weight or actual
organ weight change.
Benitz, in his discussion of the significance of organ weight
changes, deals with the question raised by Peck (1968) concerning how
often organ weight changes correspond with positive histopathology
findings. Benitz cited Jackson and Cappiello (1964) and their conclu-
sion that in beagles 80% of the organ weight changes correlates with
histopathological damage. In assessing the extent of histological
damage, Benitz discussed both the role of electron microscopy and the
quantification of morphological changes. He felt electron microscopy is
useful for: (1) determining the earlier stages of morphological changes,
(2) better interpreting visible lesion damage, and (3) detecting ultra-
structural changes not visible through normal techniques. However,
because of the time and effort involved, the use of electron microscopy
must be adequately justified in each instance. Quantification of
morphological changes is of value because results are more precise and
comparable. Table 4.34 describes several generally used quantification
methods. As can be discerned from his lengthy treatment of histopathology,
Benitz felt it is highly important to the determination of toxicity.
Although some of this may be due to his consideration of chronic as well
as subchronic tests, the principles discussed apply equally to both
topics.
Changes in body weight can often give misleading results for relative
organ weight change. This can lead to errors in assessing toxic effects.
Stevens (1976, 1977) examined this problem and proposed a simple modifica-
tion of the standard relative weight determination. By obtaining initial
and terminal body and organ weights for untreated test animals, Stevens
constructed a simple linear regression of body weight and organ weight.
This regression is used to predict the expected mean organ weight for a
test group based on their mean body weight. Comparisons can be made
between the expected and the actual relative body weights. Differences
-------
178
Table 4.34. Summary of histological quantification methods and their
applications for chemically induced lesions
Procedure
Histological substrate
Comments
Counts
Linear
measurements
Area measurements
Nuclear volume
Composition of
organs
Nuclear density
Internal surface
measurements
Bile plugs, giant cells, mast
cells, calcification sites,
mitoses, etc.
Thinning of femoral epi-
physeal disc; hyperplasia of
zona glomerulosa in adrenal
cortex; reduction in height
of spermatogenic epithelium;
decrease of tubular diameter
of testes
Muscles: necrosis
Uterus: atrophy of mucosa
and myometrium
Brain: hydrocephalus
Hyperplasia of thyroid gland;
dystrophy of adrenal cortex;
atrophy of liver due to
starvation
Hyperplasia of thyroid gland:
epithelium colloid, open
capillaries, etc.
Atrophy of ovaries: ratio of
primary follicles to corpora
lutea
Cirrhosis of liver: density
of reticulum fibers
Composition of submaxillary
glands: ratio of mucous to
serous terminal portions
Dystrophy in zona fascicu-
lata of adrenal cortex
Alveolar surface in lungs
altered by inflammation,
fibrosis, or emphysema.
Trabecular surface in femoral
epiphysis altered by osteo-
dystrophy
Expressed as average number
per field; total number per
section, etc.
Calibrated micrometer,
results in micrometer units
or microns
Components expressed in
planimeter values obtained
from tracings or projections
Two perpendicular measure-
ments using computerized
formula of rotation ellip-
soid for calculating v3
Point sampling,
Components expressed in
percentage of organ volumes
Line sampling
Results expressed in
mm*/mm3 of organ volume
^Por methodology and examples see Hennig, 1959, and Benitz and Dambach, 1964.
Source: Adapted from Benitz, 1970. Reprinted with permission of the
publisher.
-------
179
indicate the actual toxic effect. By analyzing simulated results from
rat organ weights, Stevens was able to test this modification and found
it to be a significant improvement in relative organ weight data.
Several recent committee reviews of toxicity tests have discussed
procedures for pathology examinations. The National Academy of Sciences
review (1977) suggested that as a minimum all organs of animals at the
high dose and controls should be examined microscopically. Further
tissue examination at other dose levels should be based on gross obser-
vations and results from the minimal microscopic examinations. The Food
Safety Council (1978) also recommends this procedure, with additional
emphasis on gross examination.
The review by the World Health Organization (1978) is more exten-
sive in its discussions. It generally recommends the procedures endorsed
by the National Academy of Sciences and Food Safety Council, but points
out more hazards in data evaluation. In interpreting the data, the
pathologist should be aware of the rate of occurrence for spontaneous
lesions and allow for these in his examination. Also organ weight
changes should be evaluated carefully to determine if they resulted from
the toxin or as a consequence of stress phenomena or metabolic overloading.
Finally, they emphasize that microscopic examinations should be performed
only after all data from the gross examination, organ weight determina-
tions, and biochemical-hematological tests have been properly interpreted.
These data will help direct the microscopic examination and reduce
redundant efforts.
The U.S. Environmental Protection Agency (1979), in a support
document for chronic toxicity testing guidelines, follows the recommenda-
tions of the National Cancer Institute (Sontag, Page, and Saffiotti,
1976), which suggested microscopic examination of all test animals (both
control and treatment groups) in chronic studies. For subchronic studies,
Sontag, Page, and Saffiotti (1976) suggested that all animals from the
control, highest-dose, and next highest-dose levels be subjected to a
histopathologic examination covering 30 to 40 tissues plus tissues with
gross lesions. They feel the results obtained with this added pathology
are worth the increased time and costs, which can account for 40% of the
-------
180
overall study cost (Page, 1977). This represents the maximum recommended
microscopic examination recorded in the literature.
4.5.3 Basis for a Minimum Pathology Screen
The value of histopathology in subchronic tests is related to the
sensitivity of each organ to different chemicals. If certain organs
consistently show toxic effects in subchronic tests, they should be
routinely included in most histopathologic examinations. These organs
could comprise a minimum, basic pathology screen to which other organs
could be added depending on the suspected toxic effects from the test
chemical.
To aid in evaluating this concept, a rough tabulation of organ
testing frequency and frequency of positive findings was constructed
from 54 subchronic studies (Table 4.35). These subchronic studies
tested a wide range of chemicals (Appendix B), including various drugs,
herbicides, pesticides, fungicides, industrial chemicals and solvents,
food additives, resins, and natural substances. The cumulative data for
each organ are given in this table, combining three species (dog, rat,
and monkey) and three routes (oral, inhalation, and injection).
Appendixes B and C contain specific information on routes of exposure,
test species, chemicals used, and literature references for each organ
in Table 4.35. The table gives an indication of how often each organ is
examined and how informative it is in subchronic studies. The "total
studies" column refers to the number of studies in the literature that
were examined for these data. The "studies using organ" column refers
to the actual number of studies for which that specific organ was examined
for pathologic changes. The "positive results" column refers to the
number of studies in which pathologic changes were found in that organ.
The "percent used" column refers to the number of studies using that
organ as a toxicity indicator, out of the total number of studies reviewed.
The "percent positive" column refers to the percentage of studies finding
pathological effects in an organ out of the total number of studies
using change in that organ as a criteria of toxicity. For example, the
brain was evaluated for pathologic changes in 46 of 54 studies for a
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181
Table 4.35. Comparison of the usefulness of Individual organs to indicate
toxicity when pathologically examined in a subchronic study
Organa
Adrenal
Aorta
Bone
Bone marrow
Brain
Esophagus
Eye
Gallbladder
Heart
Kidney
Large intestine
Liver
Lung
Lymph node
Mammary gland
Muscle tissue
Nerve tissue
Ovaries
Pancreas
Pituitary
Prostrate
Salivary gland
Sciatic nerve
Skin
Small intestine
Spinal cord
Spleen
Stomach
Testes
Thymus
Thyroid
Trachea
Urinary bladder
Uterus
Total ,
studies
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
54
Studies
using
organ5
46
17
13
22
46
18
14
9
53
53
38
54
50
31
8
22
15
36
36
26
19
26
4
11
39
16
49
38
40
24
36
18
33
20
Positive
results1^
13
0
1
2
7
0
0
1
8
29
3
28
15
5
0
0
6
6
1
1
3
2
1
1
1
4
12
3
12
6
5
4
0
1
Percent
used6
85
31
24
41
85
33
26
17
98
98
70
100
93
57
15
41
28
67
67
48
35
48
7
20
72
30
91
70
74
44
67
33
61
37
Percent
positive-'
28
0
8
9
15
0
0
11
15
55
8
52
30
16
0
0
40
17
3
4
16
8
25
9
3
25
24
8
30
25
14
22
0
5
Appendices B and C contain specific information for each organ con-
cerning test species, routes of exposure, chemicals tested, and literature
references.
Total number of studies reviewed for the inclusion of an organ as an
indicator of pathologic changes.
Q
Total number of studies in which an organ was used to assess patho-
logic changes.
lumber of studies in which pathologic changes were detected in an
organ.
O
Frequency that an organ was used as a pathologic indicator
(b/a x 100).
f
•* Frequency that positive pathologic changes were found in an organ
(c/b x 100).
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182
frequency of use percentage of 85. However, out of the 46 studies
utilizing the brain in this manner only 7 showed positive pathological
data. Thus, the frequency of positive results for the brain was 15%.
With the relative criteria of a 20% use frequency and a 5% positive
result frequency, a minimum screen would consist of the following
organs: adrenal, bone, bone marrow, brain, heart, kidney, large
intestine, liver, lung, lymph node, nerve tissue, ovaries or testes,
prostate, salivary gland, skin, spinal cord, spleen, stomach, thymus,
thyroid, trachea, and uterus. This minimum screen does include repre-
sentative organs from all of the major systems, except for sensory (e.g.,
eye) and would seem to be a logical start for composing a pathology
evaluation.
4.5.4 Conclusions
In reviewing the literature on the use of pathology, the wide
variety of protocols is striking. In few cases"do the discussions agree
concerning the amount and type of pathology necessary. The opinions
regarding the value of gross examination are perhaps the closest to a
consensus. Most reviews recommend gross examination of all organs at
all dose levels (Barnes and Denz, 1954; National Academy of Sciences,
1977; Peck, 1968; World Health Organization, 1978), although the defini-
tion of all organs is often not specified. This procedure would seem to
be the best option, as time and cost factors are not excessive and the
information gained can be substantial (Food Safety Council, 1978).
The recommendations for microscopic examination are not as simple
or similar as for the gross examination. Initially the importance of
microscopic examination for subchronic tests was rated low, with few
tissues examined (Smyth and Carpenter, 1948; Smyth, Carpenter, and Weil,
1951; Weil and McCollister, 1963; Weil et al., 1969). As toxicity tests
became more extensive, the role of microscopic observations became
greater. Many reviewers recommend microscopic examination of selected
tissues (25 or less) at the high- and control-dose levels (Barnes and
Denz, 1954; Zbinden, 1963). These tissues are usually major organs and
lesion-bearing organs observed in gross examination. Other researchers
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believe that all organs (30 to 40) should be sampled at high and control
doses to adequately determine toxicity (Benitz, 1970; Food Safety
Council, 1978; National Academy of Sciences, 1977; World Health Organi-
zation, 1978). Yet other researchers believe that this is still insuf-
ficient and recommend examination of all tissues at all levels (Sontag,
Page, and Saffiotti, 1976; U.S. Environmental Protection Agency, 1979).
This last protocol is obviously the most sensitive in detecting toxicity,
but it is also the most costly. The data in Table 4.35 and the recom-
mendations of other researchers also indicate that the examination of
all tissues at two dose levels can be excessive, because some tissues
are poor indicators of toxicity.
The majority of researchers agree that a highly efficient protocol,
in terms of toxicity detection per time and personnel cost, is the
microscopic examination of specified organs and tissues at the high-
and control-dose levels with added examination of grossly visible lesion-
bearing organs. The exact number and type of tissues to be examined
would vary with the chemical and its suspected target organs, but,
at the least, every organ system should be represented.
The value of organ weight determinations is another controversial
aspect of pathology. Although on occasion changes in organ weights may
not indicate specific toxicity, since they may reflect problems such as
functional hypertrophy or metabolic overloading, they may still provide
information valuable in a toxicological assessment. Furthermore, many
researchers feel that it can be more signifcant than histopathology.
Since the procedures for determining organ weight (relative or absolute)
are relatively easy, the most efficient protocol would include them.
Even if considered insufficient alone as toxicity measures, changes in
organ weight can indicate the general trends and suggest specific organs
for microscopic examination.
Thus, the data presented in the literature surveyed indicate that
an efficient, yet relatively inexpensive, pathology protocol would
consist of a gross examination of all tissues and organs at all dose
levels, a microscopic examination of designated tissues at the high and
control levels, organ weights for major organs, and supplemental
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microscopic examination of tissues at other dose levels based on the
gross observations and the organ weight data.
4.6 CLINICAL LABORATORY TESTS
4.6.1 Introduction
Recent developments in automation, methodology refinement, and data
interpretation have greatly increased the predictive role of biochemical
tests on blood, urine, and enzyme systems. Almost all current testing
schemes include some biochemical tests requiring at least pre- and post-
treatment sample evaluation. If more thorough toxic effect data are
required, interim sacrifices or periodic sample removals are needed to
provide sufficient data for evaluation. Despite automation, this increases
time and cost factors in subchronic toxicity tests. The EPA guidelines
for pesticide evaluations (Federal Register^ 1978) recommend the following
31 biochemical tests: hematocrit, hemoglobin, erythrocyte count, total
and differential leukocyte counts, platelet count, reticulocyte count,
serum calcium, serum potassium, serum lactate dehydrogenase (LDH),
SGPT, SCOT, glucose, blood urea nitrogen, direct and total bilirubin,
serum alkaline phosphatase, total cholesterol, albumin, globulin, total
protein, serum chlorine, uric acid, blood creatinine, urine specific
gravity, urine pH, urine protein, urine glucose, urine ketones, urine
bilirubin, and urobilinogen. In addition to these, some toxicologists
also use the following determinations to assess toxic effects: ornithine
carbamyl transferase, creatinine phosphokinase, lipase, amylase, sodium,
prothrombin time, serum thyroxine, cholinesterase activities, packed
cell volume, mean corpuscle hemoglobin, mean corpuscle volume, glucose-
6-phosphatase, hexobarbital oxidase, methemoglobin, and many others.
All of these tests are used to enhance early detection of toxicity or to
help pinpoint toxic mechanisms. However, the actual value of this large
number of tests has been discussed infrequently in the literature. The
purpose of this section is to summarize the available literature on the%
use of hematological, biochemical, enzymatic, and urinalysis tests in
subchronic tests and to present some data on the predictive efficiencies
of these tests.
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4.6.2 General Clinical Testing
Weil and McCollister (1963), in their comparison of short- and
long-term feeding studies, evaluated the efficiency of clinical uri-
nalysis, blood urea nitrogen, and hematology as primary indicators of
toxic effects. As shown in Table 4.32, of 24 pertinent studies using
these criteria only one showed any effects. Furthermore, the effect
noticed was not at the lowest toxic dose, indicating that it contributed
only secondary toxicity information. They concluded that efficient
detection of toxicity did not need these types of tests for experiments
of a subchronic nature.
Peck (1968), in reviewing the state of drug safety evaluations,
commented on the value of biochemical, hematologic, and function tests.
He cited the Coggeshall Report of the Committee on Drug Safety (1964),
attributing the origin of biochemical toxicity tests as beginning with
disease evaluation in man. These tests, originally designed for human
systems, were incorporated in evaluations of animal toxicity effects,
with an assumption that the tests would give comparable results in both
man and test species. Both the Coggeshall report and Peck questioned
the original validity of this assumption, especially without verifying
studies. Peck also stated that the interpretation of data from these
tests needs improvement and refining.
The use of tests dealing with changes in organ function or enzymatic
activities for drug safety evaluations without supporting pathological
data is especially questionable. Here Peck's opinion is essentially the
same as his opinion on using organ weight changes without correlating
histopathology. In both situations, it is necessary to utilize data
from several methods of toxicity assessment if the evaluation is to be
conclusive. Peck felt that experience has shown that the tests are
generally valid for man and the test species, but any toxicity study
should consider the test development history in making evaluations or
recommendations.
The occurrence of abnormal results is also a problem in the interpre-
tation of biochemical or function tests. Average values within two
standard deviations and a knowledge of past aberrant biochemical test
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values for the species under study are essential to a proper evaluation
of data. This will help eliminate concern over spontaneous abnormal
values. If these factors are considered and compensated for, then the
use of biochemical, hematologic, and function tests can be valuable in
determining toxicity.
Benitz (1970), in a review of chronic toxicity, discussed the value
of clinical chemistry, hematology, and serum drug levels in evaluating
toxicity. In contrast to some authors, he stated that a large battery
of tests is not always better than a smaller set. If the small set is
constructed of tests better suited to evaluate the chemical's toxicity,
as indicated in short-term tests, the results will be more meaningful.
Use of interim function and clinical tests is helpful primarily in
aiding the postmortem evaluation, because these tests indicate the sites
of toxic damages. Benitz also suggested the use of tests similar to
those used in humans, with the stipulation that they be equally effective.
This agrees in principle with Peck's (1968) experiences in the use of
human tests in experimental species. As an aid in evaluating the test
results, Benitz suggested that baseline studies be done prior to testing
rather than using "normal" values from the literature or other laboratories.
Another important role of the pretest baseline examination is to determine
the health status of the test animal, which allows the researcher to
remove any potentially questionable subjects before treatment starts.
In assessing hematological data, one should be aware of the varia-
bility between samples taken from different sites in the vascular system
and of differences in reliability between methods. In addition to
numerical data, morphological or qualitative data can aid in assessing
toxicity. Serum levels of drugs are most useful for determining toxic
dose level and for comparison with levels to be used in man. By assess-
ing the serum level at which toxic effects occur, comparisons between
human clinical trials and animal toxicity studies are facilitated. In
addition, Benitz warned against a rigid predetermined schedule of
sampling. He stated that "these observations should be made when nec-
essary." "When necessary" is determined during the trial by toxic
effects from the high dose and from pretest short-term studies. To set
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up an inflexible schedule of sampling only invites unnecessary evaluations
and a waste of time and money. In conclusion, Benitz supported Peck's
(1968) association of pathology evaluations and clinical-hematologic
tests, stating that toxic effects are not always indicated only by
morphological damage or only by functional impairment.
McNamara (1976) briefly discussed hematology and biochemical tests,
indicating that he supported their inclusion in a test design. He
listed these tests as minimal requirements: blood urea nitrogen; sugar,
glutamic oxalacetic transaminase; alkaline phosphatase; glucose-6-
phosphatase; cholinesterase; lactic acid dehydrogenase; potassium
(albumin, globulin); sodium; calcium; chloride; carbon dioxide; hema-
tocrit; hemoglobin; total and differential white blood cells; and
erythrocytes. He also suggested that these tests be performed at expo-
sure days -14, -7, +3, +7, +30, +60, and +90. This schedule allows for
establishment of pretreatment baseline values and ensures a sufficient
number of determinations to detect most transient changes.
The National Academy of Sciences (1977) review on toxicity testing
briefly discussed biochemical and hematological examinations. These
investigators suggested selected use of clinical tests based on prior
test data or chemical class. They agreed with Benitz (1970) in recom-
mending that a rigid schedule of sampling is unnecessary; a flexible
schedule that can be modified by the health status of the test animals
is more practical. They suggested using large animals (nonrodent) for
most tests, especially organ function tests or special clinical examina-
tions (e.g., x rays or respiratory rate determinations). They did not
recommend any specific tests, suggesting that this be left to the
investigators.
The Food Safety Council (1978) stated that biochemical and hematolog-
ical examinations should be a part of every study, with the degree of
emphasis dependent on the substance. They also recommended a flexible
sampling schedule, with shorter intervals between samples when the
toxicity potential is unknown or unpredictable. They did not recommend
any specific set of tests but suggested a variety of tests including
some biochemical, hematological, urine, and fecal evaluations. However,
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in contrast to other reviews, they did not recommend baseline studies
but suggested that historic colony values are acceptable. They also
suggested that the final protocol be determined by the investigator.
In addition to these general comments on clinical tests, there are
comments in the literature concerning specific hematology, organ/enzyme
function, and urinalysis evaluations, which are discussed in the following
sections.
4.6.3 Hematology
Zbinden (1963) discussed in his review of drug toxicity the various
tests used for evaluations of hematological toxicity. For evaluation of
total hematological effect, he recommended starting with a group of
routine tests (hemoglobin, hematocrit, total red blood cells, total
leucocytes, and differential leucocytes). As the study progresses,
special tests (reticulocyte counts, Heinz-Ehrlich bodies, methemoglobin,
sulfhemoglobin, thrombocyte count, and sedimentation rate) are added
when indicated. The tests should be performed at monthly intervals,
evaluating some animals from all dose levels for nonrodents and from the
high-dose level for rodents.
Saslaw and Carlisle (1969) reviewed their toxicity data on various
chemicals and drugs in which they utilized nonhuman primates (primarily
rhesus monkeys) and monitored hematology parameters. They determined
that for hematology effects the monkey was a good animal model for
predicting effects in humans. Also, they discussed the value of
hematology tests in determining general toxicity. In their opinion,
hematology should be included in toxicity tests, as the laboratory work
is not too excessive and "this effort could possibly be rewarded with
observations that could be meaningful to groups or agencies faced with
the awesome task of predicting or assessing hematoxicity of the increas-
ingly complex preparations intended for eventual use in man." However,
few definitive studies in support of this statement are included in the
discussion. Saslaw and Carlisle also commented on the need for pre-
liminary baseline studies for each specimen to be used in the toxicity
tests. This would supply significant pretreatment "normal" values for
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the blood factors, which would allow easier assessment of changes
occurring during treatment. The use of "mean average count" data is not
as efficient as the individual baseline data, and its use should be
discouraged.
Bushby (1970) evaluated the role of hematological tests in toxicity
studies. In subchronic studies, the essential hematological tests
should detect anemia and alterations in leucocytes and platelets. If
these changes are observed, more testing should be performed to deter-
mine the causes. Bushby broke these two types of hematology tests
(detection of effects tests and site of action tests) into the three
groups listed in Table 4.36.
To detect anemia, Bushby recommended a hemoglobin count, a red
blood cell count, and a determination of the packed cell volume. All
three tests are interrelated, and a change in any of the three would
indicate a possible anemia. Determination of the packed cell volume
is easier and more reliable than the red blood cell count, so it is
preferred. Additional tests that can pinpoint the type of anemia or
site of action include mean corpuscular hemoglobin concentration, mean
corpuscular hemoglobin, circulating reticulocyte numbers, bilirubin, and
methemoglobin. For detecting changes caused by organic disease, Bushby
recommended use of the sedimentation rate of the red blood cell count,
an easily performed procedure.
For detection of alterations in the leucocytes and platelets,
Bushby suggested a direct count of their numbers. An examination of a
stained blood film will supply information on any gross morphological
changes in platelets and leucocytes. The type and number of leucocytes
present can also be determined with the film. This differential
leucocyte count should always be converted to absolute figures using the
total leucocyte numbers. Bushby did not recommend a routine test for
the determination of hemorrhagic disorders. If changes are indicated by
clinical evidence or platelet counts, the prothrombin and partial
thromboplastin times should be determined. Bushby concluded that in
most prolonged studies a few hematological parameter evaluations will
detect general blood damage but, if site of action information is desired,
more extensive and different tests are necessary.
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Table 4.36. Hematological studies during toxicity tests
Group
Primary tests and estimations
Hemoglobin estimation
Packed cell volume
Total and differential leucocyte count
Platelet count
Examination of stained film for polychromasia and abnormal
leucocytes and platelets
Sedimentation rate
Secondary tests
Red cell count
Calculation of mean corpuscular volume, mean corpuscular
hemoglobin, and mean corpuscular hemoglobin
concentration
Reticulocyte count
Detection of Heinz bodies
Examination for methemoglobin
Hemoglobinemia
Bilirubinemia
Bone marrow
Coagulation time
Bleeding time
Clot retraction
Prothrombin time
Partial thromboplastin (cephalin) test
Fragility test
Follow-up investigations
Schumm's test for methemoglobin
Coproporphyrins I and III in urine
Thromboplastin generation test
Platelet adhesion
Antiglobulin test for autoantibodies
Tourniquet or vacuum test
Osmotic fragility
FIGLU test
Folic acid and B12 deficiencies
Estimation of life span of red cell
Iron metabolism
Ferrioxamine test
Serum iron estimation
Serum iron-binding capacity
Siderocytes in bone marrow
Estimation of haptoglobin
Blood volume
Source: Adapted from Bushby, 1970. Reprinted with
permission of the publisher.
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191
Arnold et al. (1977) commented on hematological procedures in their
discussion of animal health monitoring in chronic studies. Generally,
they recommended testing on a preset schedule or in response to changes
in animal health. They also recommended using, but did not specify, a
basic test set with incorporation of additional tests when any of these
conditions are observed: obvious clinical anemia; preliminary conditions
of anemia (e.g., vaginal bleeding or hematuria); enlargement of the
spleen, liver, or lymph nodes; and signs of physical trauma.
The monitoring of hematological effects should begin with a gross
observation of the blood sample to detect changes such as altered color
or transparency. Next, standard erythrocyte and leucocyte evaluations
should be performed. Arnold et al. stressed the need for interrelated
and overlapping evaluations, especially the correlation of data from
cell counts and blood smear examinations. Similarly, differential
leucocyte counts must be translated into absolute numbers and given with
the total leucocyte counts. To screen for hemorrhagic effects, they
recommended the use of prothrombin time (one stage) and partial thrombo-
plastin time (activated). If additional information is needed, the
platelets should be counted and assessed for morphological changes, and
bleeding time should be determined. If these procedures are followed,
hematology can be a valuable tool in determining the type and degree of
toxicity.
Another review that provides some detailed comments on hematogical
data is a World Health Organization (1978) report on evaluations of
chemical toxicity. These investigators recommended sampling at 30-day
intervals with subgroups of rodent and nonrodent test species. They
stated that to adequately assess hematological parameters both quantifi-
cation and morphology of the blood cells are needed. They recommended a
standard protocol that included the determination of erythrocyte numbers,
reticulocyte numbers, total and differential leucocyte numbers, hema-
tocrit, and hemoglobin. The erythrocytes must also be examined for
morphological changes. For evaluating hemorrhagic effects, a screen
should consist of platelet counts, clot retraction, prothrombin time
(one stage), and partial thromboplastin time (activiated). A more
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detailed evaluation of hemorrhagic effects includes factor assays,
thrombin time, fibrinogin determination, platelet aggregation, pro-
thrombin consumption time, and euglobulin clot lysis time.
4.6.4 Biochemical and Organ Function
Wr6blewski and LaDue (1955) evaluated SCOT as an index of liver
cell injury. They primarily examined human patients with varying types
of liver diseases, but also included some rat studies. Among the aims
of their paper was to discover if the degree of SCOT activity was
related to liver cell destruction; whether SCOT activity in chronic
liver disorders indicated active liver cell destruction; what correla-
tion existed between SCOT activity and various tests of liver dysfunc-
tion; and whether changes in SCOT reflected the presence of liver
cancers. After evaluating their data, Wrdblewski and LaDue reached the
following conclusions: a rough relationship exists between the amount
of a toxin in the animal and the degree of elevation of SCOT; the SCOT
activity does not alter at the same rate or in the same direction as
other liver function tests; SCOT appears to be an index of liver cell
injury and not liver cell function changes; and an increase in SCOT
activity indicates the presence of liver metastases. Based on this
report, SCOT determinations appear to be a useful index of liver damage.
In addition to discussing hematology tests, Zbinden (1963) reviewed
the use of clinical chemistry and organ function tests. The tests he
listed as commonly utilized included SCOT; SGPT; serum lactate dehydro-
genase; blood urea nitrogen; blood glucose; serum cholesterol; alkaline
phosphatase; thymol turbidity; serum bilirubin; bromosulfalein excretion;
nonprotein nitrogen; and phenolsulfonphthalein. However, he had reserva-
tions about their usefulness, stating, "Despite the widespread use of
these tests, there is still not enough information about their signifi-
cance in toxicology." Liver function tests are slightly more reliable
than most of the tests and usually give results in the pathological
range that correlate well with the degree of organ damage. However, not
all degenerative changes that occur in the liver are detected. Thus,
the significance of these tests should not be overrated either. Their
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use should obviously be as a confirming or indicator test. Zbinden also
stressed the usefulness of drug blood level determinations. These allow
more reliable extrapolation of "safe" dose levels from test species to
man.
Hoe and O'Shea (1965) evaluated the predictive efficiency of bio-
chemistry tests for detection of kidney disease in the dog. They used
known kidney lesion-producing chemicals to assess the degree of correla-
tion between biochemical tests and histopathology. The biochemical
tests evaluated included blood urea; blood creatinine; inorganic phosphate;
serum protein; calcium; serum alkaline phosphatase; sodium; potassium;
chlorine; cholesterol; SGPT; and SCOT. Each was tested in dogs after
exposure to the chemicals. The results of the tests for calcium,
chlorine, and serum proteins did not correlate with kidney damage and
were of no predictive value. The evaluations of alkaline phosphatase,
SCOT, and SGPT were inconclusive regarding kidney damage due to exces-
sive interference resulting from liver damage. However, the other tests
did correlate with kidney damage. Blood urea increased in a parallel
fashion with kidney damage. However, the authors warned that other
researchers (Bloom, 1954) have found that urea values do not always cor-
relate in cases of nephritis. Creatinine also indicated kidney damage
but not as reliably as did urea changes. Inorganic phosphate showed a
correlation, as did sodium and potassium. But Hoe and O'Shea cautioned
that interpretation of these tests is complex, and careful evaluation is
necessary. In conclusion, the authors suggested that by combining the
biochemical tests and using urinalysis a forecast of 90% accuracy can
be achieved for kidney damage. They suggested that general protocols
cannot be developed and that every case or chemical should be assessed
on an individual basis.
Peck et al. (1967) discussed several aspects of the interpretation
of serum enzyme tests. They warn against assuming that a change in the
test value automatically indicates a toxic effect. The change could be
indicative only of reversible pharmacologic or physiologic actions of
the test chemical. To properly assess the significance of the changes,
one should consider the magnitude of the change, its incidence rate in
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194
the test groups, the presence or absence of a dose-response pattern,
the results after continued repeated exposure, and the results of
pharmacology and pathology evaluations. Another problem to be aware of
is the potential deleterious loss of blood with repeated sampling. To
prevent this, special studies or additional sample groups should be
used, rather than jeopardizing the main study by oversampling.
Street (1970) reviewed biochemical tests in toxicology and stated
that their primary functions are as early indicators of toxicity and for
identification of target organs and evaluation of reversibility of
changes. Most of the techniques employed are modified from human tests
and can be done on an automated basis. For proper evaluation of the
results, initial values and values from concurrent control groups are
needed for comparison. The initial values should be taken twice during
the acclimation period. Normal values published in the literature are
of limited value because of high variability. Every toxicologist should
establish the "normal" values at his own laboratory using his methods
and species. These can then be used to aid in evaluation.
The usefulness and shortcomings of individual tests are discussed,
and Street recommended analysis techniques for each one. For liver
damage, he suggested the use of SCOT, SGFT, and alkaline phosphatase
evaluations. Additional tests that can add information on other effects
besides liver damage include bilirubin and protein electrophoresis.
Street also recommended the use of isocitric dehydrogenase in place of
the dye tests, unless the dye tests can be designed to give full elimina-
tion curves. For the assessment of kidney damage, he recommended the
blood urea test correlated with urinalysis results. Other tests reviewed
include electrolyte determinations, blood lipids, and cholinesterase.
In conclusion, Street cautioned that biochemical test results usually
suggest the use of additional confirmatory tests and in any case their
findings should be checked against histopathology for correspondence of
effects.
Cornish (1971) reviewed the literature associated with the use of
serum enzyme changes as indicators of toxic effects. Cornish did not
limit his discussion to tests that indicate liver and kidney damage, but
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also reviewed tests that may indicate heart or lung damage. In general,
he refrained from recommending specific tests for each organ, preferring
to emphasize the advantages and disadvantages found by researchers for
each test. Several factors of test design are repeatedly stressed in
his discussion. A principal factor is the need for frequent sampling or
sampling during the periods when the changes in serum enzyme levels
would be most .probable. If sampling is done at inappropriate times,
many of the changes may be missed, since the toxicant stimulates a
short-term elevation of serum levels. Despite its often transient
nature, this immediate response can be a sensitive indicator of func-
tional and/or morphological damage. Although this might require a
predosing determination of enzyme level patterns or a more frequent
sampling schedule after dosing, the enzyme tests require only small
samples of serum and can eliminate the need for the more costly interim
sacrifices of test animals used to determine the onset of organ damage.
Also, the toxicologist should consider other factors, such as stress and
alterations in dietary intake, that can affect the serum enzyme levels
regardless of any organ damage.
In addition to the usual enzyme tests, Cornish also discussed
the potential value of serum isoenzyme determinations. Isoenzymes
usually have a distinctive distribution pattern for each organ, and the
pattern found in the blood after a chemical insult can indicate damage
to individual organs. Because most of the other enzyme tests are less
specific concerning the organ damaged, Cornish feels that the use of
isoenzyme determinations has a great potential in toxicology. In addi-
tion to pinpointing damage to specific organs, isoenzyme patterns can
often be more sensitive indicators of overall tissue damage than other
enzyme tests. However, as with serum enzyme tests, the schedule of
sampling for isoenzymes is important, because the pattern can change
with time and alter the resulting diagnosis.
Grice et al. (1971) compared the sensitivity of changes in SCOT,
lactate dehydrogenase (LDH), and LDH isozyme patterns with histopathology
to determine their usefulness in detecting liver and kidney damage.
After exposure of four toxic chemicals in rats, the test results showed
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that morphological damage occurs at lower doses and before any change in
the serum enzyme levels. The SCOT test was somewhat more sensitive than
LDH, but lacked the specificity of the isozyme pattern. Grice et al.
concluded that the serum enzyme levels and isozyme patterns could
contribute supplemental information, but they are not substitutes for
pathology evaluations.
Korsrud et al. (1973) also used histopathology to assess the sensi-
tivity of changes in several serum enzymes to indicate liver toxicity.
They used acute doses of three toxic chemicals and monitored the changes
in isocitrate dehydrogenase (ICD), fructose-1-P aldose (F-l-P ALD),
sorbitol dehydrogenase (SDH), glutamic-pyruvic transaminase (GPT),
glutamic-oxaloacetic transminase (GOT), malic dehydrogenase (MDH), LDH,
glutamic dehydrogenase (GDH), fructose-l,6-diphosphate aldolase (F-1,6-
diP ALD), and ornithine carbamyl transferase (OCT) in rats. As shown in
Table 4.37, histopathology was repeatedly the most sensitive index. Of
the enzyme tests, SDH was the most sensitive. The next most reliable
indicators were ICD, GPT, GOT, and F-l-P ALD. Korsrud et al. identified
several factors that could account for the differences in sensitivity,
such as different intracellular locations of the enzymes in the liver,
high control values for LDH, different release rates into the blood for
each enzyme, different turnover rates for the enzymes, and the enzyme
activity in the cell prior to release. The responses were also
dependent on the time of sampling. Korsrud et al. concluded that SDH
was the best choice for assessing minimal liver damage when using serum
enzyme levels.
Gray et al. (1972), in an evaluation of the toxicity of clindamycin
in dogs, discussed the value of serum transaminase tests. In particular,
they concentrated on the predictive value of changes in SGPT. They felt
that SGPT changes were not so much an indication of actual liver damage
as an indication of liver intolerance. They based this on the role of
SGPT in the transport efficiency of the membrane. They concluded that
no definite morphological effect could be related to SGPT changes;
therefore, SGPT could not be used as a predictor of liver damage.
Arnold et al. (1977) briefly commented on the usefulness of serum
enzyme determinations. They cautioned that the removal of blood and the
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Table 4.37. Relative sensitivity of liver
enzyme tests
Lowest dose at which significant
change occurred
Testa (mg/kg)
TA^ DMN*2 DBA
ICD
F-l-P ALD
SDH
GPT
GOT
MDH
LDH
GDH
OCT
F-l, 6-diP ALD
Histopathology
25.4
25.4
9.4
25.4
25.4
68.6
68.6
NCe
68.6
NC
3.5
13.7
13.7
5.1
13.7
13.7
37.0
37.0
NC
37.0
NC
1.9
800
800
800
800
800
800
1600
1600
1600
1600
400
ICD — isocitrate dehydrogenase; F-l-P ALD —
fructose-1-P-aldolase; SDH — sorbitol dehydrogenase;
GPT — glutamic-pyruvic transaminase; GOT — glutamic-
oxaloacetic transaminase; MDH — malic dehydrogenase;
LDH — lactic dehydrogenase; GDH — glutamic dehydro-
genase; OCT — ornithine carbamyl transferase; F-l,
6-diP ALD — fructose-l,-diphosphate aldolase.
TA — thioacetamide.
DMN — dimethylnitrosamine.
uEA — diethanolamine.
No change.
Source: Adapted from Korsrud et al., 1973.
trauma associated with it can significantly affect the test animal's
health. Therefore, serum enzyme tests should be performed only when
necessary. Also, many of the tests fail to detect minor damage or may
reflect only transient changes. As a result of these disadvantages,
the use of biochemical tests is limited to a confirmatory role.
These same disadvantages and cautions are also voiced by the World
Health Organization (1978). They suggested the use of large test spe-
cies, such as the dog, to reduce sample removal effects. They recom-
mended a set sampling schedule with serum withdrawal prior to the test,
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198
3 and 10 days after the start of dosing, and continuing at 30-day
intervals. They concluded that, in general, these tests cannot detect
minor or initial changes in organ function but can act as a guide for
further assessments.
Clampitt (1978) investigated the predictive value of various enzyme
tests for liver damage in the rat. He compared these results with
histopathological evaluations to determine their effectiveness. He
found that no single test gave an unquestionable indication of liver
damage. Many of the conventional liver damage tests were not the most
sensitive indicators of damage. These include alkaline phosphatase,
alanine transaminase, and aspartate transaminase. Clampitt did recom-
mend that to assess lipid metabolism changes, plasma cholesterol and
triglyceride levels should be monitored. Also, the time at which blood
samples are taken should be carefully monitored if transient effects are
desired. In conclusion, Clampitt suggested that minimal changes can be
detected if the "appropriate plasma constituents" are measured and used
with knowledge of the "intracellular location of the diagnostic enzymes."
4.6.5 Urinalysis
Balazs et al. (1963) discussed the value of urinalysis in assessing
renal damage. In general the authors believed that although kidney
function tests are not easily adapted to routine toxicity studies using
the rat, they are potentially very useful. Their data suggested that
the proximal convoluted tubules were the most vulnerable part of the
kidney. Their tests were therefore designed to evaluate damage in this
area. They found that the renal tests that provide the most reliable
information were the Addis count, urinary glutamic oxaloacetic trans-
aminase test (UGOT), and urinary concentration test. Blood urea nitrogen
was erratic and less reliable than the UGOT test because of extrarenal
interference. Also, the standard concentration tests were inappropriate,
and the authors suggested using a 6-h determination. This allowed minor
effects to be detected. Additional evidence of renal damage can be
obtained from observation of renal epithelial cells and casts in the
urine.
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199
Hoe and O'Shea (1965) evaluated biochemical analysis of renal dam-
age in the dog, including the value of urinalysis techniques. They rec-
ommended the use of urine specific gravity, proteinuria, and the presence
of casts in the urine to assess renal damage. As with their recommenda-
tions on enzyme tests, Hoe and O'Shea believed a combination of urinalysis
and function tests was necessary for a complete assessment of renal
damage. In this way, 90% of the cases could be diagnosed by biochemical
tests alone.
Urinalysis and its diagnostic value were discussed by Street (1970).
The use of the simple tablet or dipstick analyses techniques in any
capacity greater than a preliminary screen is questioned by Street. He
feels that the more complex tests add significant information, especially
for renal and hepatic damage. The recommended tests include observations
on the color and volume, pH (by meter, not paper), specific gravity,
protein electrophoresis, examination of deposits, and 16-h concentration
tests (in the dog). Simple screen tests can also be performed to check
for glucose, ketones, free hemoglobin, bile, and reducing substances.
Unlike Balazs et al. (1963), Street did not recommend the UGOT and Addis
counts as standard tests. The specific gravity and concentration tests
are sufficient to detect most renal damage. Urinary ascorbic acid can
be used to test for liver enlargement, but it requires a. series of
comparative assays. Overall, Street recommended the inclusion of urin-
alysis tests for a complete assessment of toxicity.
Grice (1972) reviewed some aspects of urinalysis, particularly
tests derived from human renal assessments. In general he feels the
tests to be used on small laboratory animals have not reached a similar
level of sophistication and are not as valuable as for humans. A major
problem, besides the interpretation of test results, is obtaining a
suitable sample. Collection is difficult, since the test animals "will
not urinate on command" and contamination with feces or other materials
is hard to prevent. He also discusses other techniques to evaluate
renal damage, such as renal cell excretion rates (useful for assessing
the degree of injury) and palpation of the kidney (often more sensitive
than either urinalysis or urinary enzyme tests). However, he concludes
that presently the tests are less sensitive and less reliable than
histopathology.
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200
Wright and Plummer (1974) evaluated the excretion rate of four
urinary enzymes (lactate dehydrogenase, alkaline phosphatase, acid
phosphatase, and glutamate dehydrogenase) as indicators of acute kidney
damage by administering several known nephrotoxic agents. The enzymes
were selected for their utility as "markers" for renal injury to specific
regions in the cell, and the changes were compared to baseline excretion
rates. They found that lactate dehydrogenase and alkaline phosphatase
were excreted at significantly higher rates after renal insult, and
these increases seemed to correlate with renal damage. The other two
enzymes were not appreciably affected. They concluded that excretion
rates of some enzymes could effectively detect acute renal damage.
Cottrell et al. (1976) conducted similar studies on the value of
excretion rates of creatinine, alkaline phosphatase, lactic dehydro-
genase, and leucine aminopeptidase after acute and repeated-dose expo-
sure to nephrotoxicants. They used both histology and histochemistry to
evaluate the renal damage. They found that for acute exposure, all the
enzymes but creatinine were affected, tactic dehydrogenase was the most
sensitive indicator. However, the excretion pattern after repeated
exposure to the nephrotoxicants did not continue to indicate the
increasing renal damage shown by histology and returned to normal rates
after three days. Therefore, they concluded that currently the value of
urinary excretion rates is confined to acute and very short-term expo-
sures. Their utility in subchronic tests would be quite limited.
Berndt (1976) reviewed the tests necessary for confirmation of the
kidney as a target organ. In addition to recommending the hematology
tests and blood urea nitrogen and plasma creatinine tests, Berndt recom-
mended a urinalysis determination. Specifically he stressed the use of
changes in urinary glucose levels as a good indicator of renal damage.
Also, the excretion of p-aminohipparate (PAH) and tetraethylammonia
(TEA) can be detected in the urine to indicate damage. These tests are
particularly good for evaluating the toxic effect on the nephrons.
Other recommended tests include the volume and concentration determina-
tions. Protein determinations will indicate the effects on the glomerulus.
Berndt concluded that, in comparison with urinalysis, histological and
anatomical techniques are the "least useful criterion for evaluation of
renal damage."
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201
Arnold et al. (1977) also discussed the use of urinalysis in tox-
icity diagnosis. They suggested collection of urine overnight or at
24-h interims for determinations of volume and the excretion patterns of
calcium, phosphate, creatinine, chloride, sodium, and potassium. Fresh
samples were needed, however, for pH, osmolarity, and the presence of
malignant cells. As a routine examination, the authors suggested the
use of strip tests covering pH, protein, glucose, ketones, blood, and
bilirubin. Depending on the results, more specific tests or more com-
plex analysis could be incorporated, including microscopic evaluations
of urine sediments. These urinalysis tests will elucidate renal
problems such as anuria, hematuria, or hypertrophic changes. Thus, the
authors recommended the inclusion of at least preliminary examination of
the urine for all toxicity assessments.
A recent review of renal function tests evaluated the effectiveness
of changes in glomerular filtration rate (GFR), renal plasma (RPF) and
red blood flow (RBF), total urinary protein excretion, urinary concentra-
tion, urinary acidification, urinary enzyme excretion rates, urinary
sodium excretion rate, and microscopic examination of sediments as
indicators of nephrotoxic effects (Digzi and Biollaz, 1979). The
literature they reviewed indicated that for GFR determinations urea and
creatinine excretion rates were poor tests, and clearance rates for
marker substances were preferred; RBF and RPF were extremely difficult
to interpret, especially for rodents; total protein and urinary concen-
tration can be useful screens but do not give site-specific information;
urinary enzyme excretion has been shown useful but because of a lack of
correlation with increases in renal damage, its utility is uncertain;
and sodium excretion and urinary sedimentation are useful only as
screening tools. They concluded the predictive value for human toxicity
of these elaborate tests is uncertain and their routine use is not
suggested. Also, the difficulty of sample collection and interpretation
of normal variations in values makes the general use of these tests less
practical. However, these tests can be used for more detailed informa-
tion when prior studies indicate renal damage or when the structure of
the chemical to be investigated suggests possible renal damage.
U.S. Environmental Protection Agency investigators, in a support
document for chronic tests (1979), stressed the value of urinalysis.
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202
They recommended a routine screening (semiquantitative) with a more
detailed follow-up for positive results. They suggested the use of
urinalysis to detect early renal damage, as most enzyme function tests
do not adequately assess this. Although the EPA document evaluated
chronic test applications, the principles should also apply to sub-
chronic applications.
4.6.6 Conclusions
Biochemical tests have become a large part of the modern toxicity
evaluation. In general, they provide early indications of the presence
of toxic effects and suggest specific target organs or contribute con-
firmatory evidence on the toxic effect. The wide variety of tests
available has presented the toxicologist with the problem of test selec-
tion. Most of the literature reviewed did not suggest a standard list
of required biochemical tests, but rather suggested the inclusion of
hematology, enzyme, and urinalysis evaluations. This is perhaps a good
policy because the effectiveness and applicability of the tests change
with the toxicologist (particularly his experience with these tests) and
the chemical to be examined.
For hematology evaluations there was general agreement that a set
of basic hematology tests should provide information on cellular damage
and hemorrhagic effects. To properly evaluate these parameters, several
lists of tests were suggested. Most schemes used a small routine set of
tests with more complex evaluations added as indicated by the results.
The routine tests included morphological and quantitative evaluations of
erythrocytes and leucocytes plus a temporal evaluation of hemorrhagic
effects. The scheme in Table 4.36 is representative of this.
The question of baseline studies or normal values for comparison
was discussed in terms of hematology tests, although it also applies to
enzyme function tests. Most reviews suggested that for nonrodent
species, baseline data compiled before treatment were more valuable. A
pretreatment examination also indicates the health status of the test
animals, confirming their suitability for test use. The use of normal
values was less acceptable because of variations between individual test
animals and between laboratories. If normal values are to be used, they
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203
should be developed at the individual laboratory. The same applies to
the use of historic colony values for rat studies. The use of baseline
or normal values for comparison would be in addition to the use of
concurrent properly designed control groups.
The sampling schedule was another area of discussion. Many reviews
recommended sampling at 30-day intervals with initial samples at 1 day
and 2 weeks. Others stress the value of flexible schedules adaptable to
changes observed in the animals' health. The best solution would be an
initial schedule of routine tests performed on a 30-day basis that is
flexible enough to incorporate additional tests as the study progresses.
The effect of sampling is greater for rodents than for nonrodents,
because of size and blood volume considerations. A recent paper by
Cardy and Warner (1979) discussed the effect of sequential bleeding on
rats with the removal of 1 mL of blood every 2 weeks over a 23-week
testing period. They found that the hematological values were not
significantly altered but that body weight gain was significantly
reduced on this sampling regime. Thus, if frequent sequential samples
are taken, consideration must be given to their effect on all parameters.
Enzyme and organ function tests are usually also performed when
hematology samples are taken with the emphasis on detecting liver and
kidney damage. The literature recommends several procedures for asses-
sing these kinds of damage. Liver tests usually recommended include
SCOT, SGPT, SDH, and alkaline phosphatase, with the emphasis varying
among species and chemicals. Kidney evaluations were primarily by blood
urea and creatinine tests associated with urinalysis results. As
indicated by several reviews, these results should be interpreted care-
fully and only used in connection with histopathological follow-up.
The value of urinalysis is not quite as accepted as hematology or
enzyme tests. The National Academy of Sciences (1977) saw little value
in routine urinalysis tests. The use of urinary enzyme excretion
changes is particularly limited for subchronic studies. However, other
reviews suggested the use of at least a dipstick check of the urine as a
preliminary screen. Based on evidence in several articles, it seems
that detailed assessments of kidney damage are best evaluated by using
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204
urinalysis tests and a few enzyme determinations. If used, the urin-
alysis examination should check physical (e.g., volume and specific
gravity), enzymatic (e.g., lactic dehydrogenase), and chemical (e.g.,
glucose and phosphate) parameters. However, because of problems with
sample collection and interpretation of test results, the routine use of
urinalysis is questionable and should be left to the discretion of the
investigator.
Thus, the value of hematology, enzyme function, and urinalysis
techniques is apparently sufficient to recommend that they be included
in standard subchronic toxicity protocols. The extent that each is
utilized should be left up to the investigator. Perhaps the best
utilization of the biochemical evaluations would be in conjunction with
the terminal pathology examination. With refinement, the biochemical
tests might suggest target organs and types of effects that could be
checked by pathology. The effectiveness would thus increase for both
procedures.
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205
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Appendix A
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SUBCHRONIC TOXICITY TESTS AS DOSE-LEVEL-SETTING STUDIES
FOR CHRONIC CARCINOGENICITY EVALUATIONS
David Prejean
INTRODUCTION
A subchronic toxicity test can be defined as an intermediate-length,
repeated-dose study designed to indicate the potential long-term toxic
effects of a chemical. If the study provides a sufficient basis for the
estimation of safe exposure conditions, it may function as an "end
result" study; however, if the data obtained are insufficient to esti-
mate safety and a longer chronic study is required for evaluation, the
subchronic toxicity test becomes a "dose-level—setting" study — the
topic of this discussion.
Subchronic toxicity tests as dose-level-setting studies have a
variety of applications, particularly as prechronic studies in terato-
genicity, reproduction, longevity, or carcinogenesis evaluations; however,
it is in the area of carcinogenicity that they have most recently been
applied. Therefore, the discussion that follows centers on the use of
subchronic toxicity tests to establish a maximum tolerated dose for
each sex and species used in a chronic carcinogenicity study.
TEST MATERIAL
It is essential that the chemical being assayed be characterized as
to purity and pertinent physical properties such as stability, solubility,
and vapor pressure. To ensure consistency, it is preferable, when pos-
sible, to use the same manufacturer's lot number for both the subchronic
and chronic studies. In addition, it is absolutely necessary to deter-
mine the accuracy and consistency with which the chemicals/vehicles
are prepared (Page, 1977).
Although not all of the above requirements have been specified in
performing the subchronic dose-level-setting studies in the past (National
Institutes of Health, 1978), current specifications for conducting
carcinogenesis bioassay studies, as set forth by both EPA (Federal
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Register, 1978) and the National Cancer Institute (Sontag, Page, and
Saffiotti, 1976), do require or recommend each of the above.
TEST ANIMALS
Species and Strain
Since the subchronic test is a dose-level-setting study, the spe-
cies, strains, and sexes selected for use should be the same as will be
used in the long-term study that follows. For chronic toxicity tests,
two species have usually been recommended — preferably a rodent and a
nonrodent (Page, 1977), with the rat and dog historically the most popu-
lar. Carcinogenicity studies, however, present a unique problem.
Whereas toxicity studies, even long-term ones, require relatively small
numbers of animals, carcinogenicity studies require a relatively large
number for statistical purposes. Thus, because of the cost and time
factors, small laboratory rodents, specifically the rat and mouse, have
emerged as the most expedient test species.
As with the selection of the test animals for toxicity studies, the
designation of a particular species to be used in a carcinogenesis study
is only the beginning. Other questions, such as the use of inbred or
random bred strains and the use of germ-free, specific pathogen-free,
or conventional animals, must be answered. Although a large number of
different rat and mouse strains have been utilized in the past (Boyland
and Sydnor, 1962; Dunning, Curtis, and Madsen, 1947; National Institutes
of Health, 1977; Pietra, Rappaport, and Shubik, 1961; Shimkin et al.,
1962), the Fischer-344 rat and B6C3F1 hybrid mouse have become the
standard for the National Cancer Institute Carcinogenesis Bioassay
Program (National Institutes of Health, 1977; National Institutes of
Health, 1978; Sontag, Page, and Saffiotti, 1976). Thus, although the
controversy over the use of inbred or outbred strains continues, for the
vast majority of long-term carcinogenicity studies performed between
1972 and 1979, the inbred and hybrid strains have been used.
The final consideration in the selection of a test animal has been
the health status. For the most part, germ-free animals have rarely
been used in chronic carcinogenesis testing because of the extensive
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labor requirements. Gnotobiotic, specific pathogen-free, and "extremely
clean" conventional animals have been used most frequently in long-
term carcinogenesis tests and thus have also been used most widely in
subchronic tests designed to establish chronic dose levels (National
Institutes of Health, 1978; Sontag, Page, and Saffiotti, 1976).
One final point should be made before leaving the area of species
and strain. With rare exceptions, the majority of long-term carcinogen-
esis bioassay studies have been performed utilizing cost-effective
systems rather than pursuing the question of carcinogenicity from the
standpoint of the response of the test system to the chemical in question
and the similarity to known responses in man. Future investigations
should attempt to tailor the subchronic study to delineate one or more
of a series of species and/or strains which would provide the most
suitable test system.
Number
The number of animals required for a subchronic dose-level—setting
study has varied considerably in the past. At one time the required
number of animals per dose level was five animals per species, and sex
was not taken into consideration (Prejean, 1979). Understandably, the
resulting dose levels selected for chronic studies were, at best,
guesses. In some cases the levels selected proved correct but, more
often than not, incorrect dosages were selected, producing invalid
chronic data.
The current trend in subchronic studies has been to use a minimum
of ten animals of each sex and each species in the long-term study
(Sontag, Page, and Saffiotti, 1976). This number has proved sufficient
as long as the parameters for evaluation were limited to mortality, body
weight gain, clinical signs, postmortem observation, and pathologic
diagnosis; however, the recent expansion of these parameters to include
hematology, clinical chemistry, and pharmacokinetic studies will, of
necessity, produce an increase in the number of animals required for the
subchronic study. There are indications that the role of the subchronic
study in the carcinogenesis bioassay program is likely to require more
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animals, probably a minimum of 20 animals per sex per species (National
Cancer Institute, 1979).
DOSES AND DOSE LEVELS
The doses selected for use in the subchronic study are usually
based on data obtained from other shorter-term studies (Sontag, Page,
and Saffiotti, 1976) but may, in some instances, be based on literature
information. In selecting doses to be used in the subchronic study, it
is important to remember that the ultimate objective of the study is to
establish the maximum tolerated dose(s) for use in the chronic study.
Thus, the more data available from the subchronic study, the more
accurately the chronic dose levels may be set. For this reason, it is
extremely important to select as the highest dose a level that will
produce an easily discernible, reproducible toxic effect and a series of
lower doses that provide a complete spectra of dose responses (Weisburger
and Weisburger, 1967) from toxic to "nontoxic." In this way, not only
can the dose levels for the chronic study be selected with more accuracy,
but representative toxic lesions and symptoms may be observed that could
be of value later in the chronic study.
Subchronic dose-level-setting studies should include, as a minimum,
a range of five equally spaced dose levels (Sontag, Page, and Saffiotti,
1976). The intervals used most frequently have been half-doses (100,
50, 25, etc.) (Sontag, Page, and Saffiotti, 1976) or half-log10 doses
(100, 31.6, 10, 3.16, etc.). Thus, assuming five dose levels, the
lowest level tested would be approximately 6/100 of the top dose if half-
doses are used or 1/100 of the top dose if half-log}Q doses are used;
either of these selections provides a reasonable span of doses from
which to gather toxicity data.
ROUTE
t
Administration of the chemical in the subchronic study should
closely simulate the route by which most human exposure occurs and
should be identical to that which will be used for the chronic study.
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With the exception of certain drugs, most chemicals enter the body by
one of three routes — orally, by inhalation, or by absorption through
the skin (Page, 1977). These then are the most common routes used to
test chemicals for carcinogenicity. When exposure occurs by more than
one route of administration, as in the case of pesticides, the route of
exposure chosen is usually based on pharmacokinetic data, which could
necessitate performing several subchronic studies before selecting the
route and dose levels for the chronic study.
In general, chemical vapors and particulate materials such as smoke
and asbestos are administered by inhalation, whereas food additives and
pesticides are examples of chemicals that would be administered orally
either by way of gastric intubation, the drinking water, or the feed.
Cutaneous application, though not so common as oral administration, is
the preferred route for administering cosmetics and certain drugs.
DURATION
Although the literature abounds with various specifications regard-
ing the appropriate length for an end result subchronic study (Peck,
1968; Smith, 1950; Weil and McCollister, 1963), the majority of the evi-
dence supports the conclusion that a 90-day test will predict most
chronic effects exclusive of carcinogenicity and teratogenicity (McNamara,
1976). Since the function of a dose-level-setting subchronic test is to
establish appropriate dose levels for a chronic study through the iden-
tification of subchronic toxic effects and their extrapolation to chronic
toxic effects, a 90-day test should be just as appropriate for a dose-
level-setting subchronic study as it is for an end result study. Of
course, if prior information suggests that the chemical or its metabolites
may be rapidly or slowly eliminated from the test species, then the
duration may be reduced or extended as necessary. This has, in fact,
proven to be the case. In the Guidelines for* Carcinogen Bioaseay in
Small Rodents the National Cancer Institute recommends that in a subchronic
study "The test agent should be administered to the animals for 90 days"
(Sontag, Page, and Saffiotti, 1976). Thus, as with the other parameters
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already discussed, the duration of end result and dose-level-setting
subchronic studies is compatible.
DATA COLLECTION
Of all the aspects of a subchronic test, regardless of whether it
is an end result study or a dose-level-setting study, the most important
is data collection. Without the accumulation of adequate and accurate
observations during the test period and pathological examinations, the
establishment of reliable chronic dose levels and/or the estimation of
the long-term toxic effects is very difficult. What then are the impor-
tant data collection points for a subchronic dose-level-setting study?
Clinical Observations, Body Weights, and Food and Water Consumption
During the course of a subchronic study much of the data must be
collected on a daily or, at most, a weekly basis. Clinical observations
as to the general health of each animal should be recorded twice daily,
7 days a week by a qualified technician (National Academy of Sciences,
1975). These observations should include an assessment of animal activ-
ity, posture, body temperature (extremes only), appearance of eyes and
hair or coat, excreta and bedding, and respiratory function.
In addition to the above observations, each animal should be weighed
weekly (Sontag, Page, and Saffiotti, 1976). Except in unusual instances,
palpation for subcutaneous or intraperitoneal masses would not be required.
Food and water consumption should be measured at least weekly and if
possible daily for water consumption. These data are especially useful
for those studies where the test chemical is administered by dosed-feed
or dosed-water. Not only are the data necessary for dose calculations,
they also provide an excellent indication of the effect of the chemical
on the palatability of the food and water and indicate the general
health status of the test animals.
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Pathology
The gross and microscopic examination of test animal tissues and
organs is an extremely important aspect of data collection on a subchronic
dose-level-setting study. Complete necropsies should be performed on
all animals (Sontag, Page, and Saffiotti, 1976). These necropsies
should be performed under the supervision of a qualified pathologist,
preferably board-certified. As a minimum, samples of the approximately
30 organs or tissues listed in the Guidelines for Carcinogen Bioaseay in
Small Rodents should be preserved (Sontag, Page, and Saffiotti, 1976).
In addition to a careful examination of all internal organs and tissues
prior to their removal from the animal, a gross necropsy should include
an external examination of all body surfaces and orifices.
A brief review of the literature indicates that there is general
agreement concerning the gross observations recommended above (Peck,
1968; World Health Organization, 1978). The recommendations from the
microscopic examination vary widely, however, ranging from none (Barnes
and Denz, 1954) to tissues from all control animals, the highest dose-
level animals without mortality, and the next higher dose-level group
(Sontag, Page, and Saffiotti, 1976). In practice, results of subchronic
toxicity studies performed with token microscopic examinations compared
to those with extensive examinations indicate that the selections of
dose levels for chronic studies were more accurate when a reasonable
amount of histopathology was available (National Institutes of Health,
1977; National Institutes of Health, 1978; Prejean, 1979).
Other Data
Recently, consideration has been given to the addition of biochemical
tests to subchronic dose-level-setting studies. In general, it is felt
that these tests could be extremely valuable in evaluating specific
toxic effects of selected chemicals; however, for the most part, the
literature does not specify a standard set of tests but recommends the
inclusion of hematology, clinical chemistry, and pharmacokinetic and
behavioral studies for each chemical.
-------
226
CONCLUSIONS
In reviewing the general parameters for a subchronic toxicity test
designed to establish dose levels for a chronic carcinogenicity study,
the degree of similarity between this type of study and one designed as
an end result study is remarkable. Both types require characterization
of the test chemical, an adequate number of test dose levels (usually
five), the selection of route based on exposure data in man, exposure
over a 90-day period, and the collection of pertinent data such as
clinical and gross observations and histopathologic evaluations. Although
the use of at least two species is recommended for both types of studies,
the end result test has usually relied on a rodent and a nonrodent,
whereas the dose-level-setting test has used two species of rodents
because of the economic considerations of the long-term study that will
follow. The only other major area of disagreement has been in the use
of biochemical tests. End result studies have relied on these tests for
several years, but it has been only recently that dose-level-setting
studies have also begun to take advantage of the data available in
selected hematologic or clinical chemistry tests. Thus, unless the
current trend changes, it appears that, except for the species difference,
which will probably always remain, end result and dose-level-setting sub-
chronic toxicity tests will be virtually identical in the next few
years.
-------
227
REFERENCES FOR APPENDIX A
Barnes, J. M., and F. A. Denz. 1954. Experimental Methods Used in
Determining Chronic Toxicity. Pharmcol. Rev. 6:191-242.
Boyland, E., and K. Sydnor. 1962. The Induction of Mammary Cancer in
Rats. Br. J. Cancer 16:731-739.
Dunning, W., M. Curtis, and M. Madsen. 1947. Induction of Neoplasms in
Five Strains of Rats with Acetylaminofluorene. Cancer Res. 7:134-140.
Federal Register. 1978. Nonclinical Laboratory Studies: Good Labora-
tory Practice Regulations. 43Fr(247):59986-60025.
McNamara, B. 1976. Concepts in Health Evaluation of Commercial and
Industrial Chemicals. In: Advances in Modern Toxicology, Vol. 1,
Part 1: New Concepts in Safety Evaluation, M. A. Mehlman, R. E.
Shapiro, and H. Blumenthal, eds. John Wiley and Sons, New York.
pp. 61-140.
National Academy of Sciences. 1975. Acute and Subchronic Toxicity.
In: Principles for Evaluating Chemicals in the Environment. National
Academy of Sciences, Washington, D.C. pp. 103-104, 327-330.
National Cancer Institute. 1979. Carcinogenicity Studies in Rodents.
National Institutes of Health. RFP No. N01 CP 95619-62. Bethesda,
Maryland.
National Institutes of Health. 1977. Bioassay of Tolbutamide for Pos-
sible Carcinogenicity. National Cancer Institute Carcinogenesis
Technical Report Series No. 47. Department of Health, Education, and
Welfare Publication No. (NIH) 77-831. Bethesda, Maryland. 83 pp.
National Institutes of Health. 1978. Bioassay of 4,4'-Thiodianiline
for Possible Carcinogenicity. National Cancer Institute Carcinogenesis
Technical Report Series No. 47. Department of Health, Education, and
Welfare Publication No. (NIH) 78-847. Bethesda, Maryland. 106 pp.
Page, N. 1977. Chronic Toxicity and Carcinogenicity Guidelines. J.
Environ. Pathol. Toxicol. 1:161-182.
Peck, H. 1968. An Appraisal of Drug Safety Evaluation in Animals and
the Extrapolation of Results to Man. In: Importance of Fundamental
Principles in Drug Evaluation, D. H. Tedeschi and R. E. Tedeschi, eds.
Raven Press, New York. pp. 450-471.
Pietra, G., H. Rappaport, and P. Shubik. 1961. The Effects of Carcino-
genic Chemicals in Newborn Mice. Cancer 14:308-317.
Prejean, J. 1979. Unpublished data. Southern Research Institute.
Birmingham, Alabama.
-------
228
Shimkin, M., S. Smith, P. Shimkin, and H. Andervont. 1962. J. Natl.
Cancer Inst. 28:1219-
Smith, C. 1950. A Short-Tern Chronic Toxicity Test. J. Pharmcol. Exp.
Ther. 100:408-420.
Sontag, J., M. Page, and U. Saffiotti. 1976. Guidelines for Carcinogen
Bioassay in Small Rodents. Department of Health, Education, and Welfare
Publication No. (NTH) 76-801. National Cancer Institute, Bethesda,
Maryland. 65 pp.
Weil, C., and D. McCollister. 1963. Relationship Between Short- and
Long-Tena Feeding Studies in Designing an Effective Toxicity Test. J.
Agric. Food Chem. 11(1):486-491.
Weisburger, J., and E. Weisburger. 1967. Tests for Chemical Carcinogens.
In: Methods in Cancer Research, Vol. I, H. Busch, ed. Academic Press,
New York. pp. 307-398.
World Health Organization. 1978. Acute, Subacute, and Chronic Toxicity
Tests. In: Principles and Methods for Evaluating the Toxicity of
Chemicals. Geneva, pp. 95-115.
-------
Appendix B
-------
Appendix B. Chemicals surveyed for Table 4.35
Namea
Primary
use
,b
Reference
Carbon tetrachloride
3',4'-Dichloropropion anilide
Tetrahydrothiophene-1,1-dioxide;
Sulfolane
Calcium carbimide
1,2,4-Trichlorobenzene
Acrolein; aerylaldehyde, 2-propenal
Ethylenebisisothiocyanate sulfide
Triphenyl tin hydroxide; fentin
hydroxide
Isooctyl isodecyl nylonate
Ponceau 4R; trisodium salt of l-(4-
sulpho-1-naphthylazo)-2-naphthol-
6,8-disulphonic acid
Orange G; disodium salt of 1-phenylazo-
2-naphthol-6,8-disulphonic acid
Hexachlorobenzene; HCB
Clindamycin hydrochloride; Cleocin
Trlethyl phosphate
Ponceau MX; disodium salt of l-(2,4-
xylylazo)-2-naphthol-3,6-disulphonic
acid
Dibutyl(diethylene glycol bisphtha-
late); DDGB
2,3,7,8-Tetrachlorodibenzo-p-dioxin;
TCDD
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidinium
bromide; MA 540
TR2379; trans isomer of N-(l,3,4,6,7-
hexahydro-llbH-benzo[a]quinolizin-
2-y1)propionanilide hydrochloride
ff-Methyl-ff-(1-naphthyl)fluoroaceta-
mlde; Nissol; MNFA
2-Methyl-4-chlorophenoxy acetic acid;
MCPA
2,5,4*-Trichlorobiphenyl
Fenterol-HBr; 1-(3,5-dihydroxy-phenyl)-
2-{[l-(4-hydroxybenzyl)-ethyl]-amino}
ethanol hydrobromide
Industrial chemical
Herbicide
Solvent
Antialcoholic drug
Solvent
Cigarette smoke
component
Fungicide
Insect reproduction
inhibitor
Plasticizer
Food color
Food color
Fungicide and indus-
trial by-product
Antibiotic drug
Whipping agent
Food color
Plasticizer
Impurity in pesticides
Antihypertensive drug
Antihypertensive drug
Antihypertensive drug
Pesticide
Herbicide
Transformer dielectric
fluid
Beta-sympathomimetic
drug
Adams et al., 1952
Ambrose et al., 1972
Anderson et al., 1977
Benitz, Kramer, and Dambach,
1965
Coate et al., 1977
Feron et al., 1978;
Lyon et al., 1970
Freudenthal et al., 1977
Gaines and Kimbrough, 1968
Gaunt et al., 1969
Gaunt et al., 1967
Gaunt et al., 1971
Gralla et al., 1977;
latropoulos et al., 1978;
latropoulos et al., 1976;
Kimbrough and Linder, 1974;
Kuiper-Goodman et al.,
1977
Gray et al., 1972
Gumbman, Gagne, and Williams,
1968
Hall, Lee, and Fairweather,
1966
Hall, Austin, and
Fairweather, 1966
Harris et al., 1973; Kociba
et al., 1976; Zinkl et al.,
1973
Hartnagel et al., 1976
Hartnagel et al., 1976
Hartnagel et al., 1975
Hashimoto et al., 1968
Hattula et al., 1977;
Verschuuren, Kroes, and
Den Tonkelaar, 1975
latropoulos et al., 1977
Kast et al., 1975a
231
-------
232
Appendix B (continued)
Namea
Primary
Reference
Fominoben-HCl; 3'-chloro-2'-[N-methyl-
[(morpholino-carbonyl)methyl]-amino-
methyl]benzanilide-hydrochlorlde
Mycotoxin produced by Aepergillus
fianigatua 121
Peroxyacetyl nitrate; PAN
Epichlorohydrin; l-chloro-2,3-
epoxypropane
Ferric dimethyl dithiocarbamate; Ferbam
Tetramethylthiuram disulfide; Thiram
AHR-2438B; sodium salt of a. lignosul-
phonate
Barthrin; 6-chloropiperonyl ester of
chrysanthemumic acid
Dimethrin; 2,4-dimethylbenzyl ester of
chrysanthemumic acid
Hexachlorophene; 2,2'-methylenebis
(3,A,6-trichlorophenol)
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol; A9-THC
2-Methyl-4-chlorophenoxy propionlc
acid; MCPP
Antitussive drug
Animal feed impurity
Photochemical product
in smog
Epoxy resin
Fungicide
Fungicide
Animal feed impurity
Insecticide
Insecticide
Antibacterial and anti-
fungicidal agent
Antischizophrenic drug
Natural ingredient in
marijuana plant
Herbicide
Kast et al., 1975Z>
Khor et al., 1976
Kruysse et al., 1977
Lawrence et al., 1972
Lee, Russell, and Minor,
1978
Lee, Russell, and Minor,
1978
Luscombe and Nicholls, 1973
Masri et al., 1964
Masri et al., 1964
Nakaue, Dost, and Buhler,
1973
Phillips et al., 1978
Thompson et al., 1974
Verschuuren, Kroes, and
Den Tonkelaar, 1975
rirst name listed is the one used in Appendix C.
Primary use as listed by the reference source.
-------
233
REFERENCES FOR APPENDIX B
Adams, E. M., H. C. Spencer, V. K. Rowe, D. D. McCollister, and
D. D. Irish. 1952. Vapor Toxicity of Carbon Tetrachloride Deter-
mined by Experiments on Laboratory Animals. Ind. Hyg. Occup. Med.
6:50-66.
Ambrose, A. M., P. S. Larson, J. F. Borzelleca, and R. G. Hennigar, Jr.
1972. Toxicologic Studies on 3',4'-Dichloropropionanilide. Toxicol.
Appl. Pharmacol. 23:650-659.
Anderson, M. E., R. A. Jones, R. G. Mehl, T. A. Hill, L. Kurlansik,
and L. J. Jenkins, Jr. 1977. The Inhalation Toxicity of Sulfolane
(Tetrahydrothiophene-l,l-Dioxide). Toxicol. Appl. Pharmacol.
40:463-472.
Benitz, K. F., A. W. Kramer, Jr., and G. Dambach. 1965. Comparative
Studies on the Morphologic Effects of Calcium Carbimide, Propylthiouracil,
and Disulfiram in Male Rats. Toxicol. Appl. Pharmacol. 7:128-162.
Coate, W. B., W. H. Schoenfisch, T. R. Lewis, and W. M. Busey. 1977.
Chronic Inhalation Exposure of Rats, Rabbits, and Monkeys to 1,2,4-
Trichlorobenzene. Arch. Environ. Health 32:249-255.
Cornish, H. H., and R. Hartung. 1969. The Subacute Toxicity of
1,1-Dimethylhydrazine. Toxicol. Appl. Pharmacol. 15:62-68.
Feron, V. J., A. Kruysse, H. P. Til, and H. R. Immel. 1978. Repeated
Exposure to Acrolein Vapor: Subacute Studies in Hamsters, Rats, and
Rabbits. Toxicology 9:47-57.
Freudenthal, R. I., G. A. Kerchner, R. L. Persing, I. Baumel, and
R. L. Baron. 1977. Subacute Toxicity of Ethylenebisisothiocyanate
Sulfide in the Laboratory Rat. J. Toxicol. Environ. Health
2:1067-1078.
Gaines, T. B., and R. D. Kimbrough. 1968. Toxicity of Fentin Hydroxide
to Rats. Toxicol. Appl. Pharmacol. 12:397-403.
Gaunt, I. F., J. Colley, P. Grasso, M. Creasey, and S. D. Gangolli. 1969.
Acute (Rat and Mouse) and Short-Term (Rat) Toxicity Studies on
Isooctyl Isodecyl Nylonate. Food Cosmet. Toxicol. 7:115-124.
Gaunt, I. F., M. Farmer, P. Grasso, and S. D. Gangolli. 1967. Acute
(Mouse and Rat) and Short-Term (Rat) Toxicity Studies on Ponceau 4R.
Food Cosmet. Toxicol. 5:187-194.
Gaunt, I. F., M. Wright, P. Grasso, and S. D. Gangolli. 1971. Short-
Term Toxicity of Orange G in Rats. Food Cosmet. Toxicol. 9:329-342.
-------
234
Gralla, E. J., R. W. Fleischman, Y. K. Luthra, M. Hagopian, J. R. Baker,
H. Esber, and W. Marcus. 1977. Toxic Effects of Hexachlorobenzene
After Daily Administration to Beagle Dogs for One Year. Toxicol. Appl.
Pharmacol. 40:227-239.
Gray, J. E., R. N. Weaver, J. A. Bollert, and E. S. Feenstra. 1972.
The Oral Toxicity of Clindamycin in Laboratory Animals. Toxicol. Appl.
Pharmacol. 21:516-531.
Gumbmann, M. R., W. E. Gagne, and S. N. Williams. 1968. Short-Term
Toxicity Studies of Rats Fed Triethyl Phosphate in the Diet. Toxicol.
Appl. Pharmacol. 12:360-371.
Hall, D. E., F. S. Lee, and F. A. Fairweather. 1966. Acute (Mouse and
Rat) and Short-Term (Rat) Toxicity Studies on Ponceau MX. Food Cosmet.
Toxicol. 4:375-382.
Hall, D. E., P. Austin, and F. A. Fairweather. 1966. Acute (Mouse and
Rat) and Short-Term (Rat) Toxicity Studies on Dibutyl (Diethylene
Glycol Bisphthalate). Food Cosmet. Toxicol. 4:383-388.
Harris, M. W., J. A. Moore, J. G. Vos, and B. N. Gupta. 1973. General
Biological Effects of TCDD in Laboratory Animals. Environ. Health
Perspect. 5:101-109.
Hartnagel, R. E., B. M. Phillips, E. H. Fonseca, and R. L. Kowalski.
1976. The Acute and Target Organ Toxicity of l-Methyl-3-Keto-4-
Phenylquinuclidinium Bromide (MA 540) and Guanethidine in the Rat
and Dog. Arzneim.-Forsch. 26:1671-1672.
Hartnagel, R. E., B. M. Phillips, P. J. Kraus, R. L. Kowalski, and
E. H. Fonseca. 1975. A Subchronic Study of the Toxicity of an
Orally Administered Benzoquinolizinyl Derivative in the Rat and
Dog. Toxicology 4:215-222.
Hashimoto, Y., T. Makita, H. Miyata, T. Noguchi, and G. Ohta. 1968.
Acute and Subchronic Toxicity of a New Fluorine Pesticide. #-Methyl-
tf-(l-Naphthyl)fluoroacetamide. Toxicol. Appl. Pharmacol. 12:536-547.
Hattula, M. L., H. Elo, H. Reunanen, A. U. Arstila, and T. E. Sorvari.
1977. Acute and Subchronic Toxicity of 2-Methyl-4-Chlorophenoxyacetic
Acid (MCPA) in Male Rat. I. Light Microscopy and Tissue Concentra-
tions of MCPA. Bull. Environ. Contain. Toxicol. 18:152-158.
Hunter, C. G., and D. E. Stevenson. 1967. Acute and Subacute Oral
Toxicity of Alromine RU 100 in Rats. Food Cosmet. Toxicol. 5:491-496.
latropoulos, M. J., J. Bailey, H. P. Adams, F. Coulston, and W. Hobson.
1978. Response of Nursing Infant Rhesus to Clophen A-30 or Hexachloro-
benzene Given to Their Lactating Mothers. Environ. Res. 16:38-47.
-------
235
latropoulos, M. J., G. R. Felt, H. P. Adams, F. Korte, and F. Coulston.
1977. Chronic Toxicity of 2,5,4'-Trichlorobiphenyl in Young Rhesus
Monkeys. II. Histopathology. Toxicol. Appl. Pharmacol. 41:629-638.
latropoulos, M. J., W. Hobson, V. Knauf, and H. P. Adams. 1976.
Morphological Effects of Hexachlorobenzine Toxicity in Female
Rhesus Monkeys. Toxicol. Appl. Pharmacol. 37:433-444.
Kast, A., Y. Tsunenari, M. Honma, J. Nishikawa, T. Shibata, and M. Torii.
1975a. Acute, Subacute, and Chronic Toxicity Studies of the Beta-
Sympathomimetic, Fenterol-HBr on Rats, Mice, and Rabbits. Oyo Yakuri.
Sendai 10(1):45-71.
Kast, A., Y. Tsunenari, M. Honma, J. Nishikawa, T. Shibata, and M. Torii.
19752?. Acute, Subacute, and Chronic Toxicity Studies of an Amino-
Halogen-Substituted Benzylamine (Fominoben) in Rats and Mice. Oyo
Yakuri. Sendai 10(1):31-43.
Khor, 6. L., J. C. Alexander, J. H. Lumsden, and G. J. Losos. 1976.
Safety Evaluation of AspergilZus fumigatus Grown on Cassava for Use
as an Animal Feed. Can. J. Comp. Med. 41:428-434.
Kimbrough, R. D., and R. E. Linder. 1974. The Toxicity of Technical
Hexachlorobenzene in the Sherman Strain Rat. A Preliminary Study.
Res. Commun. Chem. Pathol. Pharmacol. 8(4):653-664.
Kociba, R. J., P. A. Keeler, C. N. Park, and P. J. Gehrin. 1976.
2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD): Results of a 13-Week
Oral Toxicity Study in Rats. Toxicol. Appl. Pharmacol. 35:553-574.
Kruysse, A., V. J. Feron, H. R. Immel, B. J. Spit, and G. J. Van Esch.
1977. Short-Term Inhalation Toxicity Studies with Peroxyacetyl
Nitrate in Rats. Toxicology 8:231-249.
Kuiper-Goodman, T., D. L. Grant, C. A. Moodie, G. 0. Korsrud, and
I. C. Munro. 1977. Subacute Toxicity of Hexachlorobenzene in the
Rat. Toxicol. Appl. Pharmacol. 40:529-549.
Lawrence, W. H., M. Malik, J. E. Turner, and J. Autian. 1972. Toxicity
Profile of Epichlorohydrin. J. Pharmacol. Sci. 61(11):1712-1717.
Lee, C-C., J. Q. Russell, and J. L. Minor. 1978. Oral Toxicity of
Ferric Dimethyl Dithiocarbamate (Ferbam) and Tetramethylthiuram
Bisulfide (Thiram) in Rodents. J. Toxicol. Environ. Health 4:93-106.
Luscombe, D. K., and P. J. Nicholls. 1973. Acute and Subacute Oral
Toxicity of AHR-2438B, a Purified Lignosulphonate, in Rats. Food
Cosmet. Toxicol. 11:229-237.
Lyon, J. P., L. J. Jenkins, Jr., R. A. Jones, R. A. Coon, and J. Siegel.
1970. Repeated and Continuous Exposure of Laboratory Animals to
Acrolein. Toxicol. Appl. Pharmacol. 17:726-732.
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236
Masri, M. S., A. P. Hendrickson, A. J. Cox, Jr., and F. DeEds. 1964.
Subacute Toxicity of Two Chrysanthemumic Acid Esters: Barthrin and
Dimethrin. Toxicol. Appl. Pharmacol. 6:716-725.
Nakaue, H. S., F. N. Dost, and D. R. Buhler. 1973. Studies on the
Toxicity of Hexachlorophene in the Rat. Toxicol. Appl. Pharmacol.
24:239-249.
Phillips, W.E.J., J.H.L. Mills, S. M. Charbonneau, L. Tryphonas,
6. V. Hatina, Z. Zawidzka, F. R. Bryce, and I. C. Munro. 1978.
Subacute Toxicity of Pyridoxine Hydrochloride in the Beagle Dog.
Toxicol. Appl. Pharmacol. 44:323-333.
Thompson, 6. R., R. W. Fleischmann, H. Rosenkrantz, and M. C. Braude.
1974. Oral and Intravenous Toxicity of A9-Tetrahydrocannabinol in
Rhesus Monkeys. Toxicol. Appl. Pharmacol. 27:648-665.
Verschuuren, H. G., R. Kroes, and E. M. Den Tonkelaar. 1975. Short-
Term Oral and Dermal Toxicity of MCPA and MCPP. Toxicology 3:349-359.
Zinkl, J. G., J. G. Vos, J. A. Moore, and B. N. Gupta. 1973. Hema-
tologic and Clinical Chemistry Effects of 2,3,7,8-Tetrachlorodibenzo-
p-dioxin in Laboratory Animals. Environ. Health Perspect. 5:111-118.
-------
Appendix C
-------
Appendix C. Species, route of exposure, chemical, and reference data for Table 4.35
Chemical
Carbon tetrachloride
3, ' ,4'-Dichloropropionanilide
Calcium carbimide
Acrolein
Ethylenebislsothiocyanate sulfide
Triphenyl tin hydroxide
Ponceau 4R
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
2,3,7, 8-Tetrachlorodibenzo-p-dloxin
TR2379
Quanethidine
l-Methyl-3-keto-4-phenylquinuclldinium
bromide
ff-Methyl-ff- (1-naphthyl) f luoracetamide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Fominoben-HCl
Aspergillus fumlgatus
Peroxyacetyl nitrate
Bpichlorohydrin
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulf ide
Ponceau MX
DibutyKdiethylene glycol bisphthalate)
AHR-2438B
Acrolein
Barthrin
Dimethrin
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
Species
Adrenal
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Dog
Dog
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Rat
Rat
Dog
Monkey
Monkey
Route Result
Inhalation
Oral
Oral +
Inhalation +
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral +
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral
Oral +
Oral
Oral
Oral +
Oral
Oral
Inhalation -
Injection
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Injection
Oral +
Reference
1
2
4
6
7
8
10
9
11
12
22
24
28
31
13
13
14
17
29
19
19
18
18
18
18
20
21
40
40
25
26
27
30
32
33
33
15
16
34
35
36
36
38
39
39
239
-------
240
Appendix C (continued)
Chemical
Tetrahydrothiophene-l,l-dioxide
Ac role in
Ethylenebisisothiocyanate sulfide
leooctyl isodecyl nylonate
Ponceau 4R
Clindamycin hydrochloride
TR2379
Fominoben-HCl
Fenterol-HBr
2,3, 7 ,8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
A 9-Tet rahydrocannab ino 1
Calcium carbimide
Ethylenebisisothiocyanate sulfide
TR2379
Quanethidine
l-Methyl-3-keto-A-phenylquinuclidinium
bromide
Hexachlorobenzene
2,5,4' -Trichlorobiphenyl
Ferric dimethyl dithlocarbamate
Tetramethylthiuram disulfide
Carbon tetrachloride
3' ,4'-Dichloropropionanilide
Calcium carbimide
1 , 2 ,4-Trichlorobenzene
Hexachlorobenzene
Clindamycin hydrochloride
TR2379
ff-Methyl-ff- (1-naphthyl) f luoracetamide
Fenterol-HBr
Fominoben-HCl
Asperglllus fumigatus
Species
Aorta
Rat
Dog
Monkey
Rat
Rat
Rat
Rat
Dog
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Monkey
Monkey
Bone
Rat
Rat
Dog
Rat
Rat
Dog
Rat
Dog
Monkey
Monkey
Monkey
Rat
Rat
Bone Marrow
Rat
Rat
Rat
Rat
Monkey
Dog
Dog
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Route Result
Inhalation
Inhalation
Inhalation
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Injection
Oral
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral +
Inhalation
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral i
Oral
Oral
Reference
3
3
3
6
7
9
10
13
13
19
19
26
25
29
30
39
39
4
7
19
19
18
18
18
18
24
22
23
33
33
1
2
4
5
5
12
13
13
19
19
20
25
26
27
-------
241
Appendix C (continued)
Chemical
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
Ponceau MX
Dibutyl(diethylene glycol biaphthalate)
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2,5,4'-Trichlorobiphenyl
Carbon tetrachloride
3' ,4'-Dichloropropionanilide
Tetrahydrothiophene-1 , 1-dloxide
Calcium carbimide
1 , 2 ,4-Trichlorobenzene
Acrolein
Ethylenebisisothlocyanate sulfide
Triphenyl tin hydroxide
Ponceau 4R
Isooctyl iaodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
TR2379
tf-Methyl-tf- (1-naphthyl) f luoracetamide
2-Methyl-A-chlorophenoxy acetic acid
2-Methyl-A-chlorophenoxy propionic acid
Fenterol-HBr
Fominoben-HCl
Aapergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
Epichlorohydrln
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
Species
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Brain
Rat
Rat
Monkey
Dog
Rat
Rat
Rat
Monkey
Rat
Dog
Monkey
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Rat
Dog
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Route Result
Oral
Oral
Oral
Oral
Oral
Oral +
Injection
Oral
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Inhalation -
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Oral
Oral
Oral
Oral
Oral
Oral +
Inhalation
Injection
Oral
Oral
Reference
33
33
15
16
38
39
39
23
1
2
3
3
3
4
5
5
6
35
35
7
8
9
10
11
12
22
24
28
31
13
13
14
19
19
20
21
40
40
25
26
27
29
30
32
33
33
-------
242
Appendix C (continued)
Chemical
Ponceau MX
Dibutyl(diethylene glycol bisphthalate)
AHR-2438B
Hexach lorophene
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2,5,4' -Tr ichlorobiphenyl
Tetrahydrothiophene-1 , 1-dioxide
Ethylenebisisothiocyanate sulfide
Clindamycin hydrochloride
TR2379
Hexachlorobenzene
2,5,4' -Trlchlorobiphenyl
Fenterol-HBr
Fominob en-HC 1
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
AHR-2438B
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
1 , 2 ,4-Trichlorobenzene
Ethylenebisisothiocyanate sulfide
Hexachlorobenzene
2,5,4' -Trichlorophenyl
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
A 9-Tetrahydrocannabinol
Tetrahydrothiophene-1 , 1-dioxide
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
A9-Tetrahydracannabinol
Species
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Esophagus
Monkey
Dog
Rat
Rat
Dog
Rat
Rat
Dog
Monkey
Monkey
Monkey
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Monkey
Rat
Dog
Monkey
Monkey
Monkey
Rat
Monkey
Monkey
Gallbladder
Monkey
Dog
Rat
Dog
Dog
Rat
flat
Monkey
Monkey
Route Result
Oral
Oral
Oral
Oral +
Oral +
Oral
Injection
Oral +
Inhalation
Inhalation
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inj ection
Inhalation
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inj ection
Inhalation
Inhalation
Inhalation
Oral
Oral +
Oral
Oral
Oral
Injection
Reference
15
16
34
37
38
39
39
23
3
3
3
7
13
13
19
19
22
24
23
25
26
29
34
38
39
39
5
5
7
12
22
24
23
29
39
39
3
3
3
12
13
13
14
39
39
-------
243
Appendix C (continued)
Chemical
Carbon tetrachloride
3 ' , A ' -Dichloropropionanilide
Tetrahydrothiophene-1 ,1-dioxide
Calcium carbimide
1,2, 4-Tr ichlorobenzene
Acrolein
Ethylenebisisothiocyanate sulfide
Triphenyl tin hydroxide
Ponceau 4R
Isooctyl isodecyl nitrate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
TR2379
Quanethidine
l-Methyl-3-keto-A-phenylquinuclidinium
bromide
ff-Methyl-ff- (1-naphthyl) f luoracetamide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-A-chlorophenoxy propionic acid
Fenterol-HBr
Fomlnoben-HCl
Aspergillus fumigatus
Peroxyacetyl nitrate
Epichlorohydrin
Ferric dimethyl dithiocarbamate
Tetramethylthluram disulfide
Ponceau MX
Dibutyl(diethylene glycol blsphthalate)
AHR-2438B
Barthrin
Species
Heart
Rat
Rat
Rat
Dog
Monkey
Rat
Monkey
Rat
Rat
Rat
Dog
Monkey
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Dog
Dog
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Route Result
Inhalation
Oral +
Inhalation
Inhalation
Inhalation +
Oral
Inhalation
Inhalation
Inhalation +
Inhalation
Inhalation -
Inhalation -
Oral
Oral
Oral
Oral
Oral +
Oral
Oral
Oral
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Oral
Oral
Inhalation
Inj ection
Oral
Oral
Oral
Oral
Oral
Oral
Reference
1
2
3
3
3
4
5
5
6
35
35
35
7
8
10
9
11
12
22
24
28
31
13
13
14
17
29
19
19
18
18
18
18
20
21
40
40
25
26
27
30
32
33
34
15
16
34
36
-------
244
Appendix C (continued)
Chemical
Dimethrin
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2,5,4' -Trichlorobiphenyl
Carbon tetrachloride
3 ' ,4 ' -Dichloropropionanilide
Tetrahydrothiophene-1 , 1-dioxide
Calcium carbimide
1,2, 4-Tr ichlorobenzene
Acrolein
Ethylenebisisothiocyanate sulfide
Triphenyl tin hydroxide
Ponceau 4R
Isooctyl isodecyl nylonate
Orange G
Hexach lorob enz ene
Clindamycin hydrochloride
Trlethyl phosphate
2,3,7, 8-Tetrachlorodlbenzo-p-dioxin
TR2379
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
ff-Methyl-ff- (1-naphthyl) f luoractemide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Fominoben-HCl
Aspergillus fumlgatus
Peroxyacetyl nitrate
Epichlorohydrin
Ferric dimethyl dithiocarbamate
Species
Rat
Dog
Monkey
Monkey
Monkey
Kidney
Rat
Rat
Rat
Monkey
Dog
Rat
Rat
Monkey
Rat
Rat
Monkey
Dog
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Dog
Dog
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Route Result
Oral
Oral
Oral
InJ ection
Oral
Inhalation +
Oral +
Inhalation
Inhalation
Inhalation
Oral
Inhalation +
Inhalation
Inhalation +
Inhalation +
Inhalation +
Inhalation +
Oral +
Oral +
Oral
Oral +
Oral
Oral +
Oral +
Oral +
Oral +
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral +
Oral +
Oral +
Oral +
Oral
Oral
Oral +
Inhalation
Injection +
Oral
Reference
36
38
39
39
23
1
2
3
3
3
4
5
5
6
35
35
35
7
8
10
9
11
12
22
24
28
31
13
13
14
17
29
19
19
18
18
18
18
20
21
40
40
25
26
27
30
32
33
-------
245
Appendix C (continued)
Chemical
Tetramethylthiuram disulfide
Ponceau MX
Dibutyl(diethylene glycol bisphthalate)
AHR-2438B
Barthrin
Dimethrln
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2,5 ,4 '-Trichlorobiphenyl
Large
Carbon tetrachloride
3' ,4'-Dichloroproplonanilide
Tetrahydrothiophene-1 , 1 -dioxide
Acrolein
Ethylenebislsothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange G
Hexachlo rob enz ene
Clindamycin hydrochloride
Triethyl phosphate
TR2379
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
AT-Methyl-ff- (1-naphthyl) f luoracetamide
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Barthrin
Dimethrin
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
Species
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Intestine
Rat
Rat
Rat
Dog
Monkey
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Dog
Rat
Rat
Rat
Dog
Dog
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Route Result
Oral
Oral +
Oral
Oral +
Oral +
Oral +
Oral
Oral
Injection
Oral +
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Oral
Oral +
Oral
Oral
Injection
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Injection +
Reference
33
15
16
34
36
36
38
39
39
23
1
2
3
3
3
6
7
9
11
12
22
24
13
13
14
19
19
18
18
18
18
20
25
26
27
29
30
33
33
34
36
36
38
39
39
-------
246
Appendix C (continued)
Chemical
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
2,5,4' -Trichlorobipheny 1
Carbon tetrachlorlde
3 ' , 4 ' -Dichloropropionanllide
Tetrahydrothiophene-1 , 1-dioxlde
Calcium carbimlde
1 , 2 ,4-Tr ichlorobenzene
Acrolein
Ethylenebislsothiocyanate sulfide
Triphenyl tin hydroxide
Isooctyl isodecyl nylonate
Ponceau 4R
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
TR2379
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidinlum
bromide
ff-Methyl-ff- (1-naphthyl) fluoracetamide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy proplonic acid
Fenterol-HBr
Fomlnoben-HCl
Asperglllus fumigatus
Peroxyacetyl nitrate
Epichlorohydrin
Ferric dimethyl dithiocarbamate
Tetramethylthiuram dlsulfide
Species
Rat
Rat
Monkey
Liver
Rat
Rat
Rat
Dog
Monkey
Rat
Rat
Monkey
Rat
Rat
Dog
Monkey
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Dog
Rat
Dog
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Route Result
Oral
Oral
Oral
Inhalation +
Oral +
Inhalation
Inhalation
Inhalation +
Oral +
Inhalation +
Inhalation
Inhalation
Inhalation +
Inhalation +
Inhalation +
Oral
Oral
Oral +
Oral
Oral +
Oral
Oral
Oral +
Oral +
Oral +
Oral
Oral
Oral +
Oral +
Oral +
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral
Oral +
Oral
Oral
Oral +
Oral +
Oral
Inhalation
Injection
Oral
Oral
Reference
40
40
23
1
2
3
3
3
4
5
5
6
35
35
35
7
8
9
10
11
12
22
24
28
31
13
13
14
17
29
19
19
18
18
18
18
20
21
40
40
25
26
27
30
32
33
33
-------
247
Appendix C (continued)
Chemical
Ponceau MX
Dibutyl(dlethylene glycol bisphthalate)
AHR-2A38B
Barthrin
Dimethrin
Hexachlorophene
Pyrldoxine hydrochloride
A 9 -Tet rahydr o c annab inol
2,5,A'-Trichlorobiphenyl
Carbon tetrachlorlde
3' ,A'-Dichloropropionanilide
Tetrahydrothiophene-1 , 1-dioxide
Calcium carbimlde
1,2, A-Tr ichlorobenzene
Acrolein
Ethylenebisisothiocyanate sulfide
Triphenyl tin hydroxide
Isooctyl isodecyl nylonate
Orange G
Hexachl orob enz ene
Clindamycin hydrochloride
Triethyl phosphate
2,3,7, 8-Tet rachlorodibenzo-p-dioxin
TR2379
Quanethidine
l-Methyl-3-keto-A-phenylquinuclldinium
bromide
ff-Methyl-ff- (1-naphthyl) f luoracetamide
2-Methyl-A-chlorophenoxy acetic acid
2-Methyl-A-chlorophenoxy propionic acid
Fenterol-HBr
Fominoben-HCl
Species
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Lung
Rat
Rat
Dog
Rat
Monkey
Rat
Rat
Monkey
Rat
Rat
Dog
Monkey
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Dog
Rat
Dog
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Route Result
Oral +
Oral
Oral +
Oral +
Oral +
Oral
Oral
Oral +
Injection +
Oral +
Inhalation
Oral
Inhalation +
Inhalation +
Inhalation +
Oral
Inhalation
Inhalation -
Inhalation +
Inhalation +
Inhalation +
Inhalation +
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Reference
15
16
3A
36
36
37
38
39
39
23
1
2
3
3
3
A
5
5
6
35
35
36
7
8
9
10
12
2A
22
28
31
13
13
1A
17
29
19
19
18
18
18
18
20
21
AO
AO
25
26
-------
248
Appendix C (continued)
Chemical
Aspergillus fumigatus
Peroxyacetyl nitrate
Ep ichlorohydr in
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Barthrin
Dimethrin
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2,5,4'-Trichlorobiphenyl
Species
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Route Result
Oral
Inhalation +
Injection +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Injection +
Oral +
Reference
27
30
32
33
33
34
36
36
38
39
39
23
Lymph Node
Carbon tetrachloride
Tetrahydrothiophene-1 , 1 -dioxide
Calcium carbimide
Acrolein
Ethylenebisisothiocyanate sulfide
leooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
TR2379
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3, 7 ,8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Pyridoxine hydrochloride
A9-Tetrahydrocannablnol
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
2,5,4' -Tr ichlorobiphenyl
Rat
Rat
Dog
Monkey
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Dog
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Monkey
Inhalation
Inhalation
Inhalation
Inhalation
Oral +
Inhalation
Oral
Oral
Oral
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Inhalation
Oral +
Oral
Oral +
Oral
Oral
Injection
Oral
Oral
Oral
1
3
3
3
4
6
7
9
11
12
22
24
13
13
14
19
19
25
26
27
29
30
33
33
34
38
39
39 «
40
40
23
-------
249
Appendix C (continued)
Chemical
Species
Route Result
Reference
Mammary Gland
Ethylenebisisothiocyanate sulfide
Orange G
Hexachlorobenzene
2,5,4' -Trichlorobiphenyl
A 9-Tet rahy drocannabinol
Rat
Rat
Dog
Monkey
Monkey
Monkey
Monkey
Monkey
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Injection
7
11
12
22
24
23
39
39
Muscle Tissue
Carbon tetrachloride
3 ' ,4 '-Dichloropropionanilide
Acrolein
Triphenyl tin hydroxide
Orange G
Clindamycin hydrochloride
TR2379
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Fomlnoben-HCl
Aspergillus fumlgatus
2,3,7, 8-Tet rachlorodibenzo-p-d ioxin
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
Nerve
Carbon tetrachloride
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
Hexachlorobenzene
Aspergillus fumlgatus
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
2,5,4' -Trichlorobiphenyl
Rat
Rat
Rat
Rat
Rat
Dog
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Tissue
Rat
Dog
Rat
Dog
Rat
Monkey
Monkey
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Monkey
Inhalation
Oral
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral
Injection
Inhalation
Oral +
Oral +
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral +
Oral
Injection
Oral
Oral
Oral +
1
2
6
8
11
13
13
19
19
21
40
40
25
26
27
29
30
33
33
38
39
39
1
18
18
18
18
22
24
28
27
38
39
39
40
40
23
-------
250
Appendix C (continued)
Chemical
3' ,4'-Dichloropropionanilide
Calcium carbimide
Acrolein
Ethylenebisisothiocyanate sulfide
Triphenyl tin hydroxide
Ponceau MX
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
TR2379
JV-Methyl-ff- (1-naphthyl) f luoracetamide
Fenterol-HBr
Fominoben-HCl
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Epichlorohydrin
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
Ponceau MX
Dibutyl (diethylene glycol bisphthalate)
AHR-2438B
Barthrin
Dimethrln
Hexach lorophene
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
2,5,4' -Trichlorobiphenyl
Carbon tetrachloride
3* ,4'-Dichloropropionanilide
Tetrahydrothiophene-1 , 1-dioxide
Calcium carbimide
Acrolein
Ethvlenebisisothlocyanate sulfide
Species
Ovaries
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Dog
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Monkey
Pancreas
Rat
Rat
Rat
Dog
Monkey
Rat
Rat
Rat
Route Result
Oral
Oral +
Inhalation
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Injection
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inj ectlon
Oral
Oral +
Oral
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Oral
Inhalation
Oral
Reference
2
4
6
7
8
10
9
11
12
24
22
31
13
13
14
19
19
20
25
26
29
32
33
33
16
17
34
36
36
37
38
39
39
40
40
23
1
2
3
3
3
4
6
7
-------
251
Appendix C (continued)
Chemical
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
TR2379
tf-Methyl-ff- (1-naphthyl) f luoracetamide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Barthrin
Dimethrin
A9-Tetrahydrocannabinol
2,5,4' -Tr ichlorobiphenyl
Carbon tetrachloride
Calcium carbimide
Ethylenebisisothiocyanate sulfide
Isooctyl Isodecyl nylonate
Orange G
Hexachlorobenzene
2,5,4' -Trichlorobiphenyl
Clindamycin hydrochloride
Triethyl phosphate
TR2379
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7, 8-Tetrach lorodibenzo-p-dioxin
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Pyrldoxine hydrochloride
Species
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Dog
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Monkey
Monkey
Monkey
Pituitary
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Dog
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Route Result
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral +
Injection
Oral
Oral
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Reference
9
11
12
22
24
31
13
13
14
19
19
20
21
40
40
30
33
33
34
36
36
39
39
23
1
4
7
9
11
12
22
24
23
13
13
14
19
19
25
26
27
29
33
33
34
38
-------
252
Appendix C (continued)
Chemical
A9-Tetrahydrocannabinol
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-A-chlorophenoxy propionlc acid
Ethylenebislsothiocyanate sulfide
Hexachlorobenzene
Clindamycin hydrochloride
TR2379
2-Methyl-A-chlorophenoxy acetic acid
2-Methyl-A-chlorophenoxy propionic acid
Fenterol-HBr
Fominob en-HC 1
Aspergillus fumigatus
2,3,7, 8-Tet rachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulflde
A^-Tetrahydrocannabinol
2,5,A'-Trichlorobiphenyl
Species
Monkey
Monkey
Rat
Rat
Prostate
Rat
Rat
Rat
Dog
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Monkey
Monkey
Monkey
Route Result
Oral
Injection
Oral
Oral
Oral
Oral
Oral
Oral ff
Oral
Oral
Oral
Oral
Oral +
Oral +
Oral +
Oral
Oral
Inhalation
Oral
Oral
Oral
Injection
Oral
Reference
39
39
40
AO
7
12
13
13
19
19
21
AO
40
25
26
27
29
30
33
33
39
39
23
Salivary Gland
Calcium carbimide
Acrolein
Ethylenebisisothiocyanate sulfide
Triphenyl tin hydroxide
Isooctyl iaodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
TR2379
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2A38B
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Dog
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Oral
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral ,-
Oral *« •
Oral +
Oral
Oral
Oral
Oral +
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
A
6
7
8
9
11
12
22
2A
13
13
19
19
25
26
27
29
30
33
33
34
-------
253
Appendix C (continued)
Chemical
A9-Tetrahydrocannabinol
2,5,4' -Tr ichlorobiphenyl
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Species
Monkey
Monkey
Monkey
Rat
Rat
Route Result
Oral
Injection
Oral
Oral
Oral
Reference
39
39
23
40
40
Sciatic Nerve
Ethylenebisisothiocyanate sulfide
Hexachlorobenzene
2,3,7, 8-Tet rachlorodibenzo-p-dioxin
Pyridoxlne hydrochloride
3* ,4'-Dichloropropionanilide
1,2, 4-Trichlorobenzene
Acrolein
Hexachlorobenzene
Aspergillus fumigatus
2,3, 7 ,8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
AHR-2438B
A9-Tetrahydrocannabinol
Rat
Dog
Rat
Dog
Skin
Rat
Rat
Monkey
Rat
Dog
Rat
Rat
Rat
Rat
Monkey
Monkey
Oral
Oral
Oral
Oral +
Oral
Inhalation
Inhalation
Inhalation
Oral
Oral
Oral
Inhalation
Oral +
Oral
Injection
7
12
29
38
2
5
5
6
12
27
29
30
34
39
39
Small Intestine
Carbon tetrachloride
3' ,4'-Dlchloropropionanilide
Tetrahydrothiophene-1 , 1-dloxlde
Acrolein
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange G
Trlethyl phosphate
Hexachlorobenzene
Clindamycin hydrochloride
TR2379
Quanethldine
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
ff-Methyl-ff- (1-naphthyl) f luoracetamide
2-Methyl-4-chlorophenoxy acetic acid
Rat
Rat
Dog
Rat
Monkey
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Dog
Rat
Dog
Dog
Rat
Dog
Rat
Rat
Rat
Rat
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Inhalation
Oral
Oral
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
1
2
3
3
3
6
7
9
11
14
12
22
24
13
13
19
19
18
18
18
18
20
21
40
-------
254
Appendix C (continued)
Chemical
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Fominoben-HCl
Aapergillus fumigatus
2,3,7, 8-Tetr achlorodibenzo-p-d ioxin
Peroxyacetyl nitrate
Ferric dimethyl dithiocar hamate
Tetramethylthiuram disulfide
AHR-2438B
Barthrin
Dimethrin
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2,5,4' -Tr ichlorobiphenyl
1 , 2 ,4-Trichlorobenzene
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Hexachlorobenzene
Fenterol-HBr
Fominob en-HC 1
Aspergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Acrolein
Pyridoxine hydrochloride
2,5,4' -Trichlorobiphenyl
Carbon tetrachlorlde
3 ' , 4 ' -Dichloropropionanilide
Tetrahydrothiophene-1 , 1-dioxide
Calcium carbimide
1,2, 4-Tr ichlorobenzene
Acrolein
Ethylenebisisothiocyanate sulfide
Trinhenvl tin hydroxide
Species
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Spinal Cord
Rat
Monkey
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Dog
Monkey
Spleen
Rat
Rat
Rat
Dog
Monkey
Rat
Rat
Monkey
Rat
Rat
Dog
Monkey
Rat
Rat
Route Result
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Injection
Oral
Inhalation
Inhalation
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral
Inhalation
Inhalation
Oral +
Oral +
Inhalation +
Oral +
Inhalation
Inhalation
Inhalation
Oral +
Inhalation
Inhalation
Inhalation
Inhalation
Inhalation
Inhalation
Oral
Oral +
Reference
40
25
26
27
29
30
33
33
34
36
36
38
39
39
23
5
5
7
9
12
22
24
31
25
26
27
29
35
35
38
23
1
2
3
3
3
4
5
5
6
35
35
35
7
8
-------
255
Appendix C (continued)
Chemical
Isooctyl isodecyl nylonate
Ponceau 4R
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
TR2379
tf-Methyl-ff- (1-naphthyl) f luoracetamide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
Peroxyacetyl nitrate
Epichlorohydrin
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
Ponceau MX
Dibutyl(dlethylenc glycol bisphthalate)
AHR-2438B
Barthrln
Dimethrin
Hexachlorophene
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
Carbon tetrachloride
3' ,4'-Dichloropropionanilide
Tetrahydrothiophene-1 , 1-dioxide
Acrolein
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange 6
Hexachlorob enz ene
Species
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Stomach
Rat
Rat
Rat
Dog
Monkey
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Route Result
Oral
Oral
Oral +
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral +
Oral
Oral
Oral
Oral +
Oral
Oral
Oral
Oral
Oral
Inhalation
Injection
Oral +
Oral
Oral +
Oral
Oral +
Oral
Oral
Oral
Oral
Oral
Inj ection
Inhalation -
Oral
Inhalation
Inhalation
Inhalation
Inhalation
Oral
Oral
Oral
Oral +
Oral
Oral
Reference
9
10
11
12
22
24
28
31
13
13
14
17
29
19
19
20
21
40
40
25
26
27
30
32
33
33
15
16
34
36
36
37
38
39
39
1
2
3
3
3
6
7
9
11
12
22
24
-------
256
Appendix C (continued)
Chemical
Clindamycin hydrochloride
Triethyl phosphate
TR2379
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
#-Methyl-ff-(l-naphthyl)f luoracetamide
Fominoben-HCl
Fenterol-HBr
Aspergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Barthrin
Dimethrin
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-A-chlorophenoxy propionic acid
2,5,4' -Tr ichlorob ipheny 1
Carbon tetrachloride
3' ,4 '-Dichloropropionanilide
Calcium carbimide
Acrolein
Ethylenebislsothiocyanate sulfide
Triphenyl tin hydroxide
Isooctyl isodecyl nylonate
Ponceau 4R
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
TR2379
Species
Dog
Rat
Rat
Rat
Dog
Rat
Dog
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Monkey
Testes
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Dog
Route Result
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Injection
Oral
Oral
Oral
Inhalation +
Oral +
Oral +
Inhalation
Oral
Oral +
Oral
Oral
Oral
Oral +
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Oral
Oral
Reference
13
13
14
19
19
18
18
18
18
20
26
25
27
29
30
33
33
34
36
36
38
39
39
40
40
23
1
2
4
6
7
8
9
10
11
12
28
31
13
13
14
17
29
19
19
-------
257
Appendix C (continued)
Chemical
ff-Methyl-ff- (1-naphthyl) f luoracetamlde
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
Peroxyacetyl nitrate
Epichlorohydrin
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
Ponceau MX
DibutyKdiethylene glycol bisphthalate)
AHR-2438B
Barthrin
Dimethrin
Hexachlorophene
Fyridoxine hydrochloride
A 9-Tetrahydrocannab inol
2,5,4' -Trichlorobipheny 1
Calcium carbimide
Acrolein
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange G
2,3,7, 8-Tetrachlorodibenzo-p-d ioxin
Quanethidlne
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
AMtethyl-JV-(l-naphthyl)fluoracetamide
Hexachlorobenzene
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
Peroxyacetyl nitrate
TR2379
A9-Tetrahydrocannabinol
2-Methyl-4-chlorophenoxy acetic acid
2-Methvl-4-chlorophenoxy propionic acid
Species
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Thymus
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Rat
Dog
Rat
Rat
Monkey
Rat
Rat
Rat
Rat
Rat
Monkey
Monkey
Rat
Rat
Route Result
Oral +
Oral +
Oral
Oral
Oral +
Oral +
Oral
Inhalation +
Injection
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inj ection
Oral +
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral
Oral +
Oral
Oral +
Oral
Inhalation
Oral
Oral +
Injection +
Oral
Oral
Reference
20
21
40
40
25
26
27
30
32
33
33
15
16
34
36
36
37
38
39
39
23
4
6
7
9
11
17
29
18
18
18
18
20
24
25
26
27
30
34
39
39
40
40
-------
258
Appendix C (continued)
Chemical
3 , ' 4 ' -Dichloropropionanilide
Tetrahydrothiophene-1 ,1-dioxide
Calcium carbimide
Acrolein
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
TR2379
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
Ferric dimethyl dlthiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Barthrln
Dimethrin
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2,5,4' -Trichlorobiphenyl
Tetrahydrothiophene-1 ,1-dioxide
Acrolein
Ethylenebisisothiocyanate sulfide
Orange G
Hexachlo rob enz ene
Species
Thyroid
Rat
Rat
Dog
Monkey
Rat
Rat
Dog
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Dog
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Trachea
Rat
Dog
Monkey
Rat
Dog
Monkey
Rat
Rat
Monkey
Monkey
Route Result
Oral
Inhalation
Inhalation
Inhalation
Oral +
Inhalation
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral +
Oral
Oral
Inhalation +
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Injection
Oral
Inhalation
Inhalation
Inhalation
Inhalation +
Inhalation +
Inhalation +
Oral +
Oral
Oral
Oral
Reference
2
3
3
3
4
6
35
7
9
11
12
22
24
31
13
13
14
19
19
21
40
40
25
26
27
29
30
33
33
34
36
36
38
39
39
23
3
3
3
6
35
35
7
11
22
24
-------
259
Appendix C (continued)
Chemical
2,5,4' -Trichlorobiphenyl
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
A^-Tetrahydrocannabinol
Carbon tetrachloride
3' ,4'-Dichloropropionanilide
Tetrahydrothiophene-1 , 1-dioxide
Acrolein
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
TR2379
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Barthrin
Dimethrin
Pyrldoxine hydrochloride
A9-Tetrahydrocannabinol
2,5,4' -Trichlorobiphenyl
Species
Monkey
Rat
Rat
Rat
Rat
Rat
Monkey
Monkey
Urinary Bladder
Rat
Rat
Rat
Dog
Monkey
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Dog
Rat
Rat
Rat
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
2-Methyl-4-chlorophenoxy acetic acid Rat
2-Methyl-4-chlorophenoxy propionic
acid Rat
Route Result
Oral
Oral
Oral
Oral
Oral
Inhalation
Injection
Oral
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Injection
Oral
Oral
Oral
Reference
23
25
26
27
29
30
39
39
1
2
3
3
3
6
7
9
11
12
22
24
13
13
14
19
19
25
26
27
29
30
33
33
34
36
36
38
39
39
23
40
40
-------
260
Appendix C (continued)
Chemical
Acrolein
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
2,5,4' -Trichlorobiphenyl
Clindamycin hydrochloride
TR2379
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2-Methyl-A-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Species
Uterus
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Rat
Dog
Rat
Dog
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Rat
Route Result
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral +
Oral
Oral
Oral
Oral
Injection
Oral
Oral
Reference
6
7
9
11
12
22
24
23
13
13
19
19
29
33
33
38
39
39
40
40
-------
261
REFERENCES FOR APPENDIX C
1. Adams, E. M., H. C. Spencer, V. K. Rowe, D. D. McCollister, and
D. D. Irish. 1952. Vapor Toxicity of Carbon Tetrachloride Deter-
mined by Experiments on Laboratory Animals. Ind. Hyg. Occup. Med.
6:50-66.
2. Ambrose, A. M., P. S. Larson, J. F. Borzelleca, and R. G. Hennigar, Jr.
1972. Toxicologic Studies on 3',4'-Dichloropropionanilide. Toxicol.
Appl. Pharmacol. 23:650-659.
3. Anderson, M. E., R. A. Jones, R. G. Mehl, T. A. Hill, L. Kurlansik,
and L. J. Jenkins, Jr. 1977. The Inhalation Toxicity of Sulfolane
(Tetrahydrothiophene-l,l-Dioxide). Toxicol. Appl. Pharmacol.
40:463-472.
4. Benitz, K. F., A. W. Kramer, Jr., and G. Dambach. 1965. Comparative
Studies on the Morphologic Effects of Calcium Carbimide, Propylthio-
uracil, and Disulfiram in Male Rats. Toxicol. Appl. Pharmacol.
7:128-162.
5. Coate, W. B., W. H. Schoenfisch, T. R. Lewis, and W. M. Busey. 1977.
Chronic, Inhalation Exposure of Rats, Rabbits, and Monkeys to
1,2,4-Trichlorobenzene. Arch. Environ. Health 23:249-255.
6. Feron, V. J., A. Kruysse, H. P. Til, and H. R. Immel. 1978. Repeated
Exposure to Acrolein Vapour: Subacute Studies in Hamsters, Rats,
and Rabbits. Toxicology 9:47-57.
7. Freudenthal, R. I., G. A. Kerchner, R. L. Persing, I. Baumel, and
R. L. Baron. 1977. Subacute Toxicity of Ethylenebisisothiocyanate
Sulfide in the Laboratory Rat. J. Toxicol. Environ. Health
2:1067-1078.
8. Gaines, T. B., and R. D. Kimbrough. 1968. Toxicity of Fentin Hydroxide
to Rats. Toxicol. Appl. Pharmacol. 12:397-403.
9. Gaunt, I. F., J. Colley, P. Grasso, M. Creasey, and S. D. Gangolli.
1969. Acute (Rat and Mouse) and Short-Term (Rat) Toxicity Studies
on Isooctyl Isodecyl Nylonate. Food Cosmet. Toxicol. 7:115-124.
10. Gaunt, I. F., M. Farmer, P. Grasso, and S. D. Gangolli. 1967. Acute
(Mouse and Rat) and Short-Term (Rat) Toxicity Studies on Ponceau 4R.
Food Cosmet. Toxicol. 5:187-194.
11. Gaunt, I. F., M. Wright, P. Grasso, and S. D. Gangolli. 1971. Short-
Term Toxicity of drange G in Rats. Food Cosmet. Toxicol. 9:329-342.
-------
262
12. Gralla, E. J., R. W. Fleischman, Y. K. Luthra, M. Hagopian,
J. R. Baker, H. Esber, and W. Marcus. 1977. Toxic Effects of
Hexachlorobenzene After Daily Administration to Beagle Dogs for One
Year. Toxicol. Appl. Pharmacol. 40:227-239.
13. Gray, J. E., R. N. Weaver, J. A. Bollert, and E. S. Feenstra.
1972. The Oral Toxicity of Clindamycin in Laboratory Animals.
Toxicol. Appl. Pharmacol. 21:516-531.
14. Gumbmann, M. R., W. E. Gagne, and S. N. Williams. 1968. Short-Term
Toxicity Studies of Rats Fed Triethyl Phosphate in the Diet.
Toxicol. Appl. Pharmacol. 12:360-371.
15. Hall, D. E., F. S. Lee, and F. A. Fairweather. 1966. Acute (Mouse
and Rat) and Short-Term (Rat) Toxicity Studies on Ponceau MX. Food
Cosmet. Toxicol. 4:375-382.
16. Hall, D. E., P. Austin, and F. A. Fairweather. 1966. Acute (Mouse
and Rat) and Short-Term (Rat) Toxicity Studies on Dibutyl (Diethylene
Glycol Bisphthalate). Food Cosmet. Toxicol. 4:383-388.
17. Harris, M. W., J. A. Moore, J. G. Vos, and B. N. Gupta. 1973.
General Biological Effects of TCDD in Laboratory Animals. Environ.
Health Perspect. 5:101-109.
18. Hartnagel, R. E., B. M. Phillips, E. H. Fonseca, and R. L. Kowalski.
1976. The Acute and Target Organ Toxicity of l-Methyl-3-Keto-4-
Phenylquinuclidinium Bromide (MA 540) and Guanethidine in the Rat
and Dog. Arzneim.-Forsch. 26:1671-1672.
19. Hartnagel, R. E., B. M. Phillips, P. J. Kraus, R. L. Kowalski, and
E. H. Fonseca. 1975. A Subchronic Study of the Toxicity of an
Orally Administered Benzoquinolizinyl Derivative in the Rat and
Dog. Toxicology. 4:215-222.
20. Hashimoto, Y., T. Makita, H. Miyata, T. Noguchi, and G. Ohta. 1968.
Acute and Subchronic Toxicity of a New Fluorine Pesticide, #-Methyl-
tf-(l-Naphtyl)fluoracetamide. Toxicol. Appl. Pharmacol. 12:536-547.
21. Hattula, M. L., H. Elo, H. Reunanen, A. U. Arstila, and T. E. Sorvari.
1977. Acute and Subchronic Toxicity of 2-Methyl-4-Chlorophenoxy
Acetic Acid (MCPA) in Male Rat. I. Light Microscopy and Tissue
Concentrations of MCPA. Bull. Environ. Contain. Toxicol. 18:152-158.
22. latropoulos, M. J., J. Bailey, H. P. Adams, F. Coulston, and
W. Hobson. 1978. Response of Nursing Infant Rhesus to Clophen
A-30 or Hexachlorobenzene Given to Their Lactating Mothers. Environ.
Res. 16:38-47.
23. latropoulos, M. J., G. R. Felt, H. P. Adams, F. Korte, and F. Coulston.
1977. Chronic Toxicity of 2,5,4'-Trichlorobiphenyl in Young Rhesus
Monkeys. II. Histopathology. Toxicol. Appl. Pharmacol. 41:629-638.
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263
24. latropoulos, M. J., W. Hobson, V. Knauf, and H. P. Adams. 1976.
Morphological Effects of Hexachlorobenzene Toxicity in Female Rhesus
Monkeys. Toxicol. Appl. Pharmacol. 37:433-444.
25. Kast, A., Y. Tsunerari, M. Honma, J. Nishikawa, T. Shibata, and
M. Torii. 1975a. Acute, Subacute, and Chronic Toxicity Studies of
the Beta-Sympathomimetic, Fenterol-HBr on Rats, Mice, and Rabbits.
Oyo Yakuri. Sendai 10(1):45-71.
26. Kast, A., Y. Tsunenari, M. Honma, J. Nishikawa, T. Shibata, and
M. Torii. 1975&. Acute, Subacute, and Chronic Toxicity Studies
of an Amino-Halogen-Substituted Benzylamine (Fominoben) in Rats and
Mice. Oyo Yakuri. Sendai 10(1):31-43.
27. Khor, G. L., J. C. Alexander, J. H. Lumsden, and G. J. Losos.
1976. Safety Evaluation of Aspergillus fwrrlgatua Grown on Cassava
for Use as an Animal Feed. Can. J. Comp. Med. 41:428-434.
28. Kimbrough, R. D., and R. E. Linder. 1974. The Toxicity of Tech-
nical Hexachlorobenzene in the Sherman Strain Rat. A Preliminary
Study. Res. Commun. Chem. Pathol. Pharmacol. 8(4):653-664.
29. Kociba, R. J., P. A. Keeler, C. N. Park, and P. J. Gehring. 1976.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD): Results of a 13-Week
Oral Toxicity Study in Rats. Toxicol. Appl. Pharmacol. 35:553-574.
30. Kruysse, A., V. J. Feron, H. R. Immel, B. J. Spit, and G. J. Van Esch.
1977. Short-Term Inhalation Toxicity Studies with Peroxyacetyl
Nitrate in Rats. Toxicology 8:231-249.
31. Kuiper-Goodman, T., D. L. Grant, C. A. Moodie, G. 0. Korsrud, and
I. C. Munro. 1977. Subacute Toxicity of Hexachlorobenzene in the
Rat. Toxicol. Appl. Pharmacol. 40:529-549.
32. Lawrence, W. H., M. Malik, J. E. Turner, and J. Autian. 1972.
Toxicity Profile of Epichlorohydrin. J. Pharmacol. Sci. 61(11):
1712-1717.
33. Lee, C-C., J. Q. Russell, and J. L. Minor. 1978. Oral Toxicity of
Ferric Dimethyl Dithiocarbamate (Ferbam) and Tetramethylthiuram
Disulfide (Thiram) in Rodents. J. Toxicol. Environ. Health 4:93-106.
34. Luscombe, D. K., and P. J. Nicholls. 1973. Acute and Subacute Oral
Toxicity of AHR-2438B, a Purified Lignosulphonate, in Rats. Food
Cosmet. Toxicol. 11:229-237.
35. Lyon, J. P., L. J. Jenkins, Jr., R. A. Jones, R. A. Coon, and J. Siegel.
1970. Repeated and Continuous Exposure of Laboratory Animals to
Acrolein. Toxicol. Appl. Pharmacol. 17:726-732.
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264
36. Masri, M. S., A. P. Hendrickson, A. J. Cox, Jr., and F. DeEds. 1964.
Subacute Toxicity of Two Chrysanthemumic Acid Esters: Barthrin and
Dimethrin. Toxicol. Appl. Pharmacol. 6:716-725.
37. Nakaue, H. S., F. N. Dost, and D. R. Buhler. 1973. Studies on the
Toxicity of Hexachlorophene in the Rat. Toxicol. Appl. Pharmacol.
24:239-249.
38. Phillips, W.E.J., J.H.L. Mills, S. M. Charbonneau, L. Tryphonas,
G. V. Hatina, Z. Zawidzka, F. R. Bryce, and I. C. Munro. 1978.
Subacute Toxicity of Pyridoxine Hydrochloride in the Beagle Dog.
Toxicol. Appl. Pharmacol. 44:323-333.
39. Thompson, G. R., R. W. Fleischmann, H. Rosenkrantz, and M. C. Braude.
1974. Oral and Intravenous Toxicity of A9-Tetrahydrocannabinol in
Rhesus Monkeys. Toxicol. Appl. Pharmacol. 27:648-665.
40. Verschuuren, H. G., R. Kroes, and E. M. Den Tonkelaar. 1975. Short-
Term Oral and Dermal Toxicity of MCPA and MCPP. Toxicology
3:349-359.
-------
5. CHRONIC TOXICITY AND CARCINOGEN1CITY TESTING
5.1 INTRODUCTION
Tests designed to evaluate chronic toxicity and those designed to
evaluate carcinogenic effects have many similar considerations. Both
are subject to various design factors, including species, strain, sex,
age, and number of the test and control animals; duration of test; route
of exposures; dosage levels; and use of data evaluations. Both studies
are costly and involve extensive manpower and time requirements. However,
the two tests are also different in many ways. There is little uniformity
in the literature regarding the purposes, methods, and nomenclature of
tests for chronic toxicity. Some investigators seek only the demonstration
of a "safe" or "harmless" dose, while other workers attempt to observe
the nature and severity of toxic effects as well as to determine a maximum
"no-observed-adverse-effect" dose level (Friedman, 1973). In contrast,
for carcinogenicity tests, tumor1 formation and increased tumor incidence
are the major end points. Tumor incidence, tumor latency, and in some
cases tumor multiplicity are the parameters used to determine sensitivity
of the test animals to the carcinogenic challenge. Short-term in vivo
tests can also be useful, and several types are included as examples of
intermediates between the lifetime studies and the various short-term in
vitro tests. Therefore, in the following subsections, chronic toxicity
and carcinogenicity tests and the factors that affect them will be dis-
cussed together when possible, pointing out both the similarities and
dissimilarities. There will also be discussions of topics that pertain
only to each individual test. Additionally, some topics usually considered
part of chronic toxicity testing, such as reproductive and behavioral
effects, neurotoxicity, and teratogenicity, are discussed elsewhere in
this report or later publications and are not included in this section.
this report, the general term "tumor" will apply to either
benign or malignant neoplasms.
265
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266
5.2 TEST ANIMALS
Various aspects of the selection of species and strain of experimental
animal will be discussed in the context of long-term toxicity tests, which
include assays for chronic toxicity and carcinogenicity. Species- and
strain-specific responses and spontaneous tumor incidence in test animals
will be examined through a review of the literature.
5.2.1 Species
Because the usual purpose of chronic toxicity and carcinogenicity
testing is to predict adverse effects of chemicals in man, test animals
should be chosen that closely resemble man with respect to absorption,
distribution, metabolism, excretion, and target site effect of the toxic
substance (Weil, 1972). Unless information to the contrary is available
from previous metabolic, pharmacodynamic, or subchronic studies, the most
sensitive species and strain should be selected for chronic toxicity and
carcinogenicity testing (National Academy of Sciences, 1977).
No test animal has been found to be an ideal surrogate for man
under all test conditions (Krasovskii, 1976; Rail, 1969; Shubik, 1972);
furthermore, great variability of response exists among different species
(Hodge et al., 1967). Nevertheless, a variety of species can respond to
individual toxicants in a manner useful for chronic testing. Thus, dogs,
cats, and nonhuman primates, but not mice or rats, have been found suitable
for testing methylmercury compounds (National Academy of Sciences, 1977),
and the rhesus monkey is more similar to humans in the dermal absorption
of certain compounds such as benzoic acid, hydrocortisone, and testosterone
than is the pig, rat, or rabbit (Fancher, 1978). The chicken more closely
resembles man than other species tested for response to demyelinating effects
of triorthocresyl phosphate and other organophosphorus compounds (Barnes
and Denz, 1954). In a similar vein, monkeys are recommended for inhalation
experiments because of anatomical similarities to man, cats for compounds
likely to produce methemoglobinemia, pigtail monkeys for methanol toxicity,
and dogs or rats for cholinesterase inhibition studies (Fancher, 1978).
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267
5.2.1.1 Chronic Toxicity Testing - Despite the need for manlike
metabolic responses in test animals, in the ultimate analysis practical
considerations drastically limit the choice of animals for chronic
testing. Because the test period must encompass a major portion of the
life span of the test species, short-lived mammalian species usually
receive first consideration. Precedent, convenience, and economic
considerations dictate the use of rats or mice in the majority of all
chronic toxicity tests (Stevenson, 1979). For example, in more than
100 computer-selected publications on noncarcinogenic chronic toxicity
examined during the preparation of this chapter (listed in the Bibliog-
raphy) , the frequency of occurrence of test animals was: rat, 54%; dog,
24%; monkey, 9%; mouse, 6%; rabbit, 4%; guinea pig, 3%; and gerbil, 1%.
Only a few of these studies exceeded 2 years in duration. On the basis
of animals exposed to the test material over a major portion of their
life spans, the above data on animal frequency show a strong bias with
respect to short-lived rodents: rat, 88%; mouse, 8%; dog, 2%; and gerbil,
2%.
The dominating effect of practical considerations in the choice of
test animals for chronic toxicity testing is further reflected in animals
used in 134 long-term studies published in Toxicology and Applied Pharma-
cology between 1959 and 1966 (Benitz, 1970): rat, 43.3%; dog, 38.1%;
monkey, 6.7%; mouse, 3.7%; rabbit, 3.0%; chicken and guinea pig, 1.5%;
and gerbil, 0.7%. A survey of all studies published in the same journal
during 1975 revealed a continuing preference for use of short-lived
rodents: rat, 43.7%; mouse, 15.9%; rabbit, 7.6%; dog, 7.5%; primate,
7.3%; guinea pig, 3.7%; human, 3.4%; fish, 3.0%; cat, 2.2%; hamster,
1.1%; chicken, 0.9%; swine, 0.7%; duck, 0.6%; quail, 0.6%; cow, 0.4%;
sheep, 0.4%; frog, 0.2%; goat, 0.2%; sea lion, 0.2%; and snail, 0.2%
(Fancher, 1978).
When relatively long-lived nonrodent animals are the test species
of choice and it is not feasible to expose the animals to the test
material over a major fraction of their life span, some authorities
recommend conducting kinetic studies to determine when steady-state
tissue concentrations of the test chemical and its metabolites have been
achieved. Treatment of the animal for a substantial period after attaining
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268
steady-state kinetics would then partially substitute for a lifetime
study and provide added assurance of the validity of experimental conclu-
sions (National Academy of Sciences, 1977; World Health Organization,
1978).
When a rodent is chosen as the principal test animal in a chronic
toxicity test, most authorities recommend including a second species
that is not a rodent in order to reveal a broader range of toxic effects
(Barnes and Denz, 1954; Zbinden, 1973). Usually, the dog is chosen
(Federal Register, 1978; Goldenthal, 1968; Page, 1977Z?), but other
species may be selected if their metabolic processes are thought to
resemble those of man.
5.2.1.2 Carcinogenicity Testing — Rodents, mainly the rat, mouse,
and Syrian hamster, are generally selected for large-scale screening of
suspected carcinogens (Ministry of Health and Welfare Canada, 1975;
National Academy of Sciences, 1975; Page, 19772?). Utilization of these
species is based neither on the established similarities to man nor on
biochemical, physiological, or anatomical characteristics but on the
ability to test large numbers of compounds in these animals in a rela-
tively short time. Although the susceptibility of simians and the dog
to chemical carcinogenesis has been established with several groups of
chemicals, the long latency period in these species discourages their
use in carcinogenicity screening (Ministry of Health and Welfare Canada,
1975; National Academy of Sciences, 1975; U.S. Food and Drug Administration,
1971). Dogs and monkeys may be useful for extrapolation purposes, but
only in highly select situations (Page, 1977&).
The selection of the most appropriate animal system is an important
factor in tests for carcinogenicity, and species differences in response
to various chemical carcinogens must be a major consideration in that
selection. Responses of various mammalian species to chemical carcinogens
within selected chemical classes are reviewed in the following subsections.
5.2.1.2.1 Polynuclear aromatic carcinogens — Tumor induction with
polynuclear aromatic compounds has been demonstrated in a variety of
species using different routes of administration. The following section
includes comparisons of species' responses to a few of these compounds.
The tumorigenic response of animals to a particular carcinogen can vary
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269
according to route of administration of the test material, and this is
demonstrated in Sect. 5.3; thus, valid comparisons can be made between
species only when the same route of exposure is used. Therefore, the
studies are grouped according to mode, of administration of the carcinogen.
Tumors have been induced in many species by subcutaneous injection
of polycyclic aromatic carcinogens or by subcutaneous implantation of
special discs which have been impregnated with the carcinogens. For
instance, when injected subcutaneously into newborn mice, 7,12-dimethyl-
benz(a)anthracene has been found to be a potent leukemogen (Pietra,
Rappaport, and Shubik, 1961). Of 27 Swiss albino mice injected with 30
to 40 yg dimethylbenzanthracene in 1% gelatin, 29.6% developed malignant
lymphoma by 12 to 27 weeks after injection, and 85.4% developed pulmonary
tumors. However, when newborn Lewis rats were injected subcutaneously
with 10 to 1000 yg of dimethylbenzanthracene, the only tumors related to
the treatment were sarcomas induced at the site of injection (Toth and
Shubik, 1963). The number and latent periods of the tumors were dose
related. Thus, it seems that newborn Swiss mice respond systemically to
subcutaneously administered dimethylbenzanthracene, while newborn Lewis
rats respond locally.
Pott, Brockhaus, and Huth (1973) found the subcutaneous tissue of
the NMRI mouse to be more sensitive than that of the Wistar rat to
benzo(a)pyrene. Female NMRI mice developed malignant sarcomas at the
site of injection of benzo(a)pyrene in tricaprylin (Pott, Tomingas, and
Misfeld, 1977). Single doses of 3.3 to 270 yg of the carcinogen were
administered. A linear dose relationship was observed in the range of
3.3 to 90 yg for an induction time of 20 to 41 weeks. Survival time was
21 to 84 weeks, and the tumor incidence was 8% to 80%.
When sensitive strains of Syrian hamsters were injected subcutane-
ously with 500 yg of benzo(a)pyrene in tricaprylin, sarcomas were palpa-
ble at 20 weeks (Homburger et al., 1972). In the experiments of Pott et
al. and Homburger et al. the average induction time for the appearance
of subcutaneous tumors was the same for mice and hamsters. However, the
dose required to produce tumors in hamsters in 20 weeks was two times
the dose required to produce tumors in mice in the same time. If one
assumes that the dose response in hamsters is linear, the mice would be
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270
more sensitive. In the same hamster study, 500 yg of 3-methylcholanthrene
induced sarcomas at the injection site in 17 weeks; in an earlier
experiment (Homburger and Hsuch, 1970), sarcomas appeared in hamsters
only 3 weeks after subcutaneous injection of 7,12-dimethylbenzanthracene
in tricaprylin.
Hartley guinea pigs, on the other hand, exhibited relatively pro-
longed induction times of 7 to 17 months after methylcholanthrene was
injected into the abdominal wall (Dale et al., 1973). One or two doses
of 4 mg of the carcinogen in sesame oil produced sarcomas in 30% of the
animals surviving for 6 months. The dose of methylcholanthrene admin-
istered was 8 to 16 times higher than the dose that, in the study of
Homburger et al. (1972), induced subcutaneous tumors in hamsters in 17
weeks. Thus, the guinea pig seems relatively resistant to subcutaneous
tumor induction with 3-methylcholanthrene.
A few years earlier O'Gara and Kelly (1965) attempted to produce
tumors in rhesus and cynomalgus monkeys using conditions known to produce
malignant tumors in rodents. Dibenzo(a,i)pyrene and 3-methylcholanthrene
injected subcutaneously or intradermally induced local keratoacanthomas,
papillomas, and giant cell granulomas but no sarcomas or carcinomas.
The squirrel monkey (Saimiri soireus) is the second most widely
used subhuman primate in biomedical research, and by 1969 no reports had
been made of tumor induction in this animal. Steinmuller, Dillingham,
and Prehn (1969) attempted to induce tumors with subcutaneously implanted
discs of paraffin impregnated with 5% 3-methylcholanthrene (one disc
per 25 g of body weight). No tumors or neoplastic changes were found
4 years later in any of eight monkeys tested. In addition, autochthonous
skin fragments implanted subcutaneously with 3-methylcholanthrene crystals
failed to give rise to tumors in 4 years in the three monkeys treated.
No signs of toxicity were seen in monolayers of monkey kidney epithelial
cells grown in vitro with 5% 3-methylcholanthrene discs during 3 weeks
of exposure, although Alfred et al. (1964), using identical techniques,
had produced marked toxicity in mouse cells.
The most frequent explanation for the differences in tumor response
between rodents and primates is based on the assumption of a direct
relationship between the induction time of tumors and the life span of
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271
the animal. However, experiments of Sugiura, Smith, and Sunderland
(1956) do not support this but tend to emphasize differences In sensi-
tivity among the species to a particular carcinogen. Thus, skin painting
with MH101, a high boiling catalytically cracked oil, in repeated doses
produced papillomas in six rhesus monkeys and, later, malignant change
in three of them. This was the first demonstration of tumor induction
in Macaaus rhesus. In the same experiment, mice and rabbits developed
papillomas and cancers, but rats and guinea pigs did not. Table 5.1
demonstrates the lack of a relationship between life span and tumor
induction in various species (e.g., papilloma induction required 1/25 of
the life span of the mouse, 1/18 of the life span of the monkey, but
only 1/70 of the life span of the rabbit). Steinmuller, Dillingham, and
Prehn (1969) concluded from the experiment of Sugiura, Smith, and
Sunderland (1956) and from their own data that the species-specific
response to carcinogens was more evident than a relationship between
induction time and life span. They also suggested that primates are
more resistant than rodents to the polycyclic hydrocarbons.
In contrast to squirrel monkeys and rhesus monkeys, primates lower
in the phylogenetic scale seem to be responsive to the carcinogenic
effects of polycyclic hydrocarbons (Adamson, Cooper, and O'Gara, 1970)
with the difference in response occurring approximately at the evolu-
tionary level of the marmoset. Levy (1963) injected a marmoset subcu-
taneously with 2 mg of 3-methylcholarithrene, and a fibrosarcoma appeared
in 10 months; Noyes (1969) induced a fibrosarcoma and a rhabdomyosarcoma
in 16 months after injection of benzo(a)pyrene and dimethylbenzanthracene,
respectively, into opposite flanks of a cottontop marmoset. Also, Noyes
(1968) observed fibrosarcomas in three tree shrews (Tup-ia glis) 6 months
after injecting benzo(a)pyrene. In a similar study, Adamson, Cooper,
and O'Gara (1970) injected six tree shrews with single subcutaneous
doses of 10 mg methylcholanthrene dissolved in 1 mL olive oil. At the
same time, 12 galagos were injected intradermally or subcutaneously with
3 to 10 mg benzo(a)pyrene. The three tree shrews that survived developed
tumors at the injection site, the first appearing 14 months after injec-
tion. Of the surviving 12 galagos, one developed a tumor at the site of
injection in 26 months. The fibrosarcomatous nature of the tumors
-------
Table 5.1. Some factors in the experiments with MH 101 upon a variety of species
Species
Mouse
Rat
Guinea pig
Rabbit
Monkey
Time to first
appearance
(days)
Papilloma
20 (l/25)fc
0
0
26 (1/70)
322 (1/18)
Approximate
Dose weight of
(g) animal
tn\
Dose per kg
(g)
Area
treated*2
(cm2)
Dose per cm2
(g)
Approximate
life span
(years)
Cancer V6'
70
0
0
411
1373
(1/10)
(1/4)
(1/4)
0.015
0.1
0.1
0.5
0.2-1.0
25
200
400
3000
7000
0.6
0.5
0.25
0.17
0.03-0.14
1
4
4
16
4-35
0.
0.
0.
0.
0.
015
025
025
031
05-0.03
2
3
6
5
16
NJ
•vl
Ni
Refers to a single painted site.
Numbers in parenthesis represent portion of life span required for tumor induction.
Source: Adapted from Suguira, Smith, and Sunderland, 1956. Reprinted with permission of the publisher.
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273
induced in these species [and the failure to induce sarcomas in rhesus
and cynamolgus monkeys (O'Gara and Kelly, 1965) and in squirrel monkeys
(Steinmuller, Dillingham, and Prehn, 1969)] led Adamson et al. to suggest
that prosimians, which include tree shrews, lemurs, lorises, and tarsiers,
may resemble rodents more than the higher primates in their reactions to
polycyclic hydrocarbon carcinogens.
Although many skin carcinogenesis experiments have been conducted
using the two-stage, initiation-promotion technique, promotion studies
are omitted from this section to simplify discussion. Skin tumor induc-
tion times are compared for mice, rats, and hamsters in the following
examples.
Weekly cutaneous applications of 0.1% (or 0.025 mg per dose) 9,10-
dimethyl-l,2-benzanthracene to the skin of noninbred mice induced papil-
lomas in 5 to 6 weeks and carcinomas in 20 to 40 weeks (Neiman, 1968).
The induction times for both papillomas and carcinomas were reduced con-
siderably by presensitization with nontumorigenic doses of the carcin-
ogen. In a later study with C57 B1/6J mice, Wislocki et al. (1977)
noted the appearance of squamous cell carcinomas after 30 weeks of
biweekly applications (15 skin paintings) of benzo(a)pyrene.
Similar experiments have been conducted in rats and hamsters. Mul-
tiple weekly doses of 1% dimethylbenzanthracene applied to the skin of
hamsters gave rise to papillomas and squamous cell carcinomas. The
average latent period was 16 weeks (Delia Porta et al., 1956). Doses of
0.5% of the same carcinogen applied to the skin of Fischer rats produced
squamous cell carcinomas after 6 months of skin painting (Zackheim,
1964). Table 5.2 summarizes the results of the three experiments with
dimethylbenzanthracene. The differences in dose administered in the
three studies increase the difficulty of comparing responses of the
animals. Nonetheless, the limited data indicate that mice are more
sensitive than hamsters and rats to skin carcinogenesis with 9,10-
dimethyl-l,2-benzanthracene, supporting the view that mouse skin generally
exhibits greater sensitivity than rat skin to carcinogenesis with poly-
cyclic aromatic hydrocarbons (Weisburger, 1976).
Tumors of the respiratory tract have been induced by polycyclic
aromatic hydrocarbons in the various species by special techniques,
-------
274
Table 5.2. Induction of skin carcinomas in mice,
hamsters, and rats with weekly applications of
9,10-dimethyl-l,2-benzanthracene
Species
Dose
Induction time
Number of
applications
Inbred mice
Syrian
hamsters
Fischer rats
0.1% (0.25 mg)
1%
0.5%
5
4
6
to 10
months
months
, average
months , average
20
16
24
to 40
which include intratracheal injection. In the first report of tumor
induction by the intratracheal route of administration, weekly instil-
lations of 50 yg dimethylbenzanthracene in 1% gelatin for up to 45 weeks
produced tracheobronchial carcinomas in hamsters (Delia Porta, Kolb, and
Shubik, 1958). In a recent study Kektar et al. (1978) instilled benzo-
(a)pyrene, weekly, in Syrian golden hamsters at doses of 0.1, 0.33, or
1.0 mg (in bovine serum albumin). Respiratory tract neoplasms were
found in 19% to 22% of the males and 20% to 40% of the females treated.
Survival times of 10 to 40 weeks were dose related, but tumor incidence
was not.
Although hamsters have been the animal of choice for intratracheal
studies in the past, pneumonia-free specific pathogen-free (SPF) rats
are now used frequently because of their low incidence of infectious
diseases (Nettesheim and Griesemer, 1978). Davis et al. (1975) induced
squamous carcinomata in the lungs of Wistar rats with intratracheal
injections of benzo(a)pyrene. Using infusine as the vehicle, 0.5-, 1.0-,
and 2.0-mg doses were administered once every 2 weeks. Twenty-four of
48 rats developed squamous neoplasms of the lung. Unlike the results
with the hamster system (Ketkar et al., 1978), the tumor incidence in
rats was dose related. Tumors have also been induced by intratracheal
instillation of carcinogens in mice (Nettesheim and Hammons, 1971), in
prosimian galagos (Crocker et al., 1970), and in rabbits (Hirao et al.,
1968).
-------
275
Another mode of administration of chemical carcinogens is by direct
injection into the salivary gland. Glucksman and Cherry (1971) injected
0.1 mL of a 2% suspension of 9,10-dimethyl-l,2-benzanthracene in acetone
into the salivary glands of male and female Lister rats (18 males and 10
females) and observed carcinomas and/or sarcomas of the glands in 16 of
18 males and 10 out of 10 females. Carcinomas peaked at 100 days, and
sarcomas appeared as late as 770 days.
Wigley, Amos, and Brookes (1978) induced tumors in the salivary
glands of C57BL male mice by direct injection of 0.1, 0.5, or 2.0 mg
benzo(a)pyrene. Forty percent of the animals injected with 0.1 mg, 90%
injected with 0.5 mg, and 56% injected with 2 mg developed fibrosarcomas,
carcinomas, and/or rhabdomyosarcomas. (Two lymphosarcomas developed at
sites other than the injection site.) The first two tumors appeared ap-
proximately 110 days after injection of benzo(a)pyrene. The responses
of mice and rats to polynuclear aromatic carcinogens injected locally
into the salivary glands seem to be similar.
A broad spectrum of tumor types can be elicited in laboratory
animals with polycyclic aromatic hydrocarbons administered systemically.
For example, mice have been shown to develop gastric carcinomas, pulmonary
adenomas, and/or leukemias and lymphomas following oral administration
of benzo(a)pyrene; the organ specificity of the response is clearly
related to strain (Rigdon and Neal 1966; Rigdon et al., 1969). To be
specific, of 81 male Wistar rats given daily intragastric instillations
of 5 mg 3-methylcholanthrene, 28% developed 26 malignant neoplasms (10
mammary carcinomas, 4 sebaceous gland carcinomas, 7 cutaneous carcinomas,
and 5 leukemias). Of 122 female rats, 115 (98%) developed 117 malignant
neoplasms (106 mammary carcinomas, 10 leukemias, 1 sebaceous gland
carcinoma, and no skin carcinomas) (Gruenstein, Meranze, and Shimkin,
1966).
Huggins, Grand, and Brillantes (1961) observed preferential mammary
cancers in 100% of the 50-day-old Sprague-Dawley female rats 28 to 59
and 34 to 70 days after feeding them sesame oil solutions of 20 and 10
mg of 7,12-dimethyl-benzanthracene, respectively. Only ten animals per
group were used. Control groups were not included in the experiment,
but the investigators reported that only two mammary cancers had been
observed in 20 000 females less than 8 months old in their colony.
-------
276
Thus, female rats seem to be highly susceptible to mammary carcinogenesis
following oral administration of polycyclic aromatics.
The carcinogenic response of hamsters to 3-methylcholanthrene was
tested by Homburger et al. (1972), who administered, by gavage, 5 mg of
the chemical three times per week for 17 weeks and observed carcinomas
in males of the most susceptible strain tested 16 to 31 weeks later.
Tumors were found in the forestomach (20%), stomach (40%), small intestine
(45%), and large intestine (65%). Eighty-seven percent of the female
hamsters of that strain developed mammary tumors of various histological
types.
5.2.1.2.2 Aromatic amines — Bladder cancer in humans has been
reported in all countries with established chemical industries. A
correlation between aromatic amine exposure and human bladder cancer was
first reported by Rehn in 1895. Tumor induction in animals by aromatic
amines and subsequent- epidemiological studies have confirmed the corre-
lation reported by Rehn. Experimental details for the studies discussed
in this section are shown in Table 5.3.
Early attempts to produce bladder cancer in various species were
unsuccessful, but in 1937 Heuper and Wolfe reported bladder tumors in
dogs following oral and subcutaneous administration of 70 g of 2-naph-
thylamine. The tumors were first diagnosed by use of cystoscopy 21
months after the start of treatment.
Nelson and Woodard (1953) demonstrated the susceptibility of dogs
to o-aminoazotoluene (o-AAT). Administered orally for 30 to 62 months,
0-AAT produced urinary bladder tumors in two of four animals studied.
The other two dogs developed liver and gallbladder tumors.
Another aromatic amine, 4-aminodiphenyl, was used by Walpole,
Williams, and Roberts (1954) to produce bladder cancer in the dog.
Cumulative doses of 30 to 33 g, orally administered, induced tumors in
the two dogs tested within 2% years. Thirty control beagles developed
no bladder tumors during 3 to 9 years of observation.
Walpole's results were confirmed in a 3-year study by Deichmannvet
al. (1965), who were able to induce bladder tumors in 100% of six dogs
with smaller cumulative doses (5.4 to 7.3 g per dog) of 4-aminodiphenyl.
-------
Table 5.3. Tumor Induction in various species with aromatic amines
Chemical/species
2-Naphthylamlne
Dog
Syrian hamster
Mouse
IF mouse
CBA mouse
CBA mouse
IF mouse
Albino rat
Rabbit
2-Acetvlamino-
fluorene
Dog
Mouse
Guinea pig
Dose
70 g
l.OZ
1-2 mg per pellet
200 mg/kg body weight
in arachis oil
twice weekly
120 mg/kg body weight
in arachis oil
twice weekly
160 mg/kg body weight
once weekly
Saturated solution
in benzene
310 mg/kg body weight
per week
200 mg
twice per week
4-12 mg/kg body weight
per day
0.05Z
0.045Z or 0.032Z
1.5 and 1.6 mg per
Duration of
exposure
(weeks)
>36
22-39
30-72
30-89
40-89
40-99
20->90
140-272
272-364
56
112-128
9-22
Route of
exposure
Oral;
subcutaneous
Oral, diet
Bladder pellet
Oral, gavage
Oral , gavage
Oral, gavage
Skin
Oral, diet
(reduced
protein)
Oral, spoon
Oral, diet
Oral, diet
Oral, diet
Intraperitoneal
Tumor incidence
Bladder
50Z Bladder
carcinomas
0/8
10/25 Liver
cholangiomas
13/23 Hepatomas;
1 cholangloma
11/26 Hepatomas
0/25
3/15 Bladder
papillomas
1/6 Bladder
papilloma
4/4 Bladder,
liver
14/49 Bladder
carcinoma or
papilloma
0/16
0/26
Tumor
latency Reference
(weeks)
84 Heuper and Wolf,
1937
40 Saffiotti, 1966
Bonser et al. , 1952
50-72 Bonser et al., 1952
50-89 Bonser et al., 1952
60-89 Bonser et al., 1952
Bonser et al. , 1952
60-90 Bonser et al. , 1952
247 Bonser et al., 1952
272-364 Morris and Eyes tone,
1953
48 Miller, Miller, and
Enomota, 1964
Miller, Miller, and
Enomota, 1964
Miller, Miller, and
Monkey
100 g body weight
2-3 times per week
Dosage increased
with time from
10-200 rag/kg
body weight per
day, S days per
week
52-182
Oral, diet
0/16
Enomota, 1964
Dyer, Kelly, and
O'Cara, 1966
ro
-------
Table 5.3. (continued)
Chemical/species
2-Acetylamino-
fluorene
Hamster
o-Aminoazo-
toluene
Hamster
Rat
C3H mouse
Scraln C mouse
Strain A mouse
FI hybrids of
above
Dog
4-Aminodlphenyl
Dog
Duration of
Dose exposure
(weeks)
0.03X 30
1.5 mg per 100 g body 24-32
weight per day
3 times per week
0.1X 49
1.4-3.4 g total dose 21-49
10 mg once per 40
4 weeks in glycerol
20 rag/kg per day 8
5 mg/kg per day 128-248
Varied 140
5-20 mg/kg body weight
6 days per week
(Total - 2.9-3.3 g/kg)
1.0 mg/kg body weight 156
5 times per week
Route of Tumor incidence
exposure
Oral, diet 3/18 Liver
bile duct
carcinomas
Intraperitoneal 1/18 Small intestine
adenocarcinoma
Oral, diet 20/25 Bladder
19/24 Liver cell
3/15 Mammary
Oral 7/8 Hepatomas
Subcutaneous Varied with strain
Hepatic
Pulmonary
Hemangio-
endotheliomas
Oral 0/5 (all died
with liver damage)
Oral 2/5 Bladder
carcinomas
Oral, capsule 2/2 Bladder
Oral, capsule 6/6 Bladder
(4 malignant,
Tumor
latency Reference
(weeks)
52 Miller, Miller, and
Enomota, 1964
64 Miller, Miller, and
Enomota, 1964
45 Tomatis, Delia Porta,
and Shublk, 1961
21 Yoshida, 1932
Andervont, Crady, and
Edwards, 1942
Nelson and Woodard,
1953
Walpole, Williams,
and Roberts, 1954
Walpole, Williams,
and Roberts, 1954
Deichmann et al., 196:
to
«^l
00
Rat
3.6-5.8 g/kg body
weight total
36-53
Subcutaneous
17/23 (Variety
of sites. No
bladder tumor.)
Walpole, Williams,
and Roberts, 1952
-------
279
Similar doses of 2-naphthylamine failed to produce neoplasms in six
dogs, thus establishing 4-aminodiphenyl as the more potent carcinogen in
this study.
Morris and Eyestone (1953) induced liver and bladder tumors in each
of four dogs fed a total dose of 90 to 198 g of 2-acetylaminofluorene,
administered orally for 68 to 91 months. Experiments in which bladder
cancer has been induced in dogs by other aromatic amines are listed in
Table 5.4.
A distinct difference exists among the various species in their
responsiveness to carcinogenesis by aromatic amines. The following
demonstrate some of those differences: (1) 2-naphthylamine produced
bladder cancer in hamsters (Saffiotti et al., 1966) but not in rats,
mice, and rabbits (Bonser et al., 1952); (2) o-ATT produced bladder
cancer in hamsters (Tomatis, Delia Porta, and Shubik, 1961), but not in
rats (Yoshida, 1932) or mice (Andervont, Grady, and Edwards, 1942;
Andervont, 1950); (3) 4-aminodiphenyl failed to induce bladder tumors in
rats (Walpole, Williams, and Roberts, 1952); and (4) 2-fluorenylacetamide
produced bladder tumors in mice but not in hamsters, guinea pigs (Miller,
Miller, and Enomota, 1964), or monkeys (Dyer, Kelly, and O'Gara, 1966).
Because of the relative resistance encountered in other species, it
is felt that the dog should be used for testing carcinogenicity of
substances related to the aromatic amines (U.S. Food and Drug Adminis-
tration, 1971). However, the hamster can develop aromatic amine-induced
bladder tumors having short latent periods and may be a likely substitute
(Gak, Graillot, and Truhaut, 1976).
5.2.1.2.3 ff-Nitroso compounds — About 100 #-nitroso compounds have
been shown to be carcinogenic in experimental animals. The carcinogenic
properties of some of these compounds are compared in different species.
Diethylnitrosamine and dimethylnitrosamine — In 1967 Schmahl and
Osswald reviewed the effects of diethylnitrosamine in 11 different
animal species, paying particular attention to the organotropic activity
of the carcinogen. The data of Schmahl and Osswald (1967) and those of
other investigators are summarized in Table 5.5, which includes the
total dose (mg per kg of body weight) required to produce cancer in the
various species. Tumors were induced in all species and in almost 100%
-------
Table 5.4. Compounds, not previously listed, which have produced bladder cancer in the dog
Compound
Individual doses
Total dose per animal
that produced tumors
(g)
Incidence of tumors
Latent period
Benzidine
4-Dimethylamino-
azobenzene
4-Nitrobiphenyl
2-Naphthylamine plus
4-nitrobiphenyl
2-Naphthylamine plus
4-nitrobiphenyl plus
4-aminobiphenyl
200 mg per dog per day
(in capsules), 6 days/
week for 15 months,
followed by 300 mg per
dog, 6 days/week for
45 months; then treat-
ment discontinued
20 mg/kg per dog per
daya (dry or in corn
oil in capsules)
0.3 g per dog three
times a week (in
capsules)
0.1 g of each compound
per dog, three times
a week (in capsules)
0.1 g of each compound
per dog, three times
a week (in capsules)
325
1 of 6 mongrels
(1 male, 5 females)
7.5 years
98 to 129
Total: 17 to 20
Total: 22 to 25
2 (1 male and 1
female) of 10
mongrels
3 of 4 female
mongrels
5 of 5 female
beagles
3 of 3 female
beagles
3 years, 2 months
to 4 years
2 years, 1 month
to 2 years, 9
months
2 years, 5 months
to 2 years, 7
months
2 years, 1 month
to 2 years, 4
months
ait is not clear whether a certain dosage per day was given five or seven times per week.
Source: Adapted from Deichmann and Radomskl, 1963. Data collected from several sources.
oo
o
-------
Table 5.5. The carcinogenic action of diethylnitrosamine in different animal species
Species
Mouse
Rat
Hamster
Guinea pig
Rabbit
Dog
Pig
Monkey
Grass
parakeet
Brachydanio
rerio
Trout
Route of
administration
Oral
Oral
Oral
Oral
Oral
Oral and
subcutaneous
Oral
Oral
Intramuscular
Oral
Daily dose
(mg/kg)
3
3
40 (weekly)
3
3.4
3
4.4
2 to 50
70 (weekly)
10 to 100 ppm
Total dose (D50)
(mg/kg)
871 ± 124
700 ± 53
640
1200 ± 100
2500
560
1400
1400 to 25 700
2800 ± 400
10 to 13 weeks
Type of tumor
Hemangioendotheliomas
the liver
Hepatocarcinomas
Hepatocarcinomas
Hepatocarcinomas
Hepatocarcinomas
Leimyosarcoma of the
of
liver
Reticulosarcomas of the
liver
Hepatocarcinomas
Hepatocarcinomas
Hepatocarcinomas and
cholangiomas
Hepatocarcinomas
Source: Adapted from Schmahl and Osswald, 1967. Reprinted with permission of the
publisher.
00
-------
282
of the animals tested. Rabbits, dogs, and pigs treated with approxi-
mately 3 mg/kg diethylnitrosamine developed cirrhosis and liver cancer,
while mice, rats, and guinea pigs developed only liver cancer.
Although Schmahl and Osswald were unable to produce hepatomas in
monkeys, they referred to Kelly et al. (1966), who had done so in 6 of
15 monkeys (cynamolgus, rhesus, and capuchin) with oral doses of diethyl-
nitrosamine administered for more than 1 year. Experimental details of
this and the following studies with diethylnitrosamine and dimethylnitro-
samine are listed in Table 5.6.
Two cercopithecus monkeys given 1.6 and 2.2 g of diethylnitrosamine
intraperitoneally also developed hepatocarcinomas, with induction times
of 25 and 27 months (Kelly et al., 1966).
The monkeys in the preceding experiments did not develop tumors in
organs other than the liver. A strain of inbred guinea pigs, however,
developed tumors in both the lung and the liver when diethylnitrosamine
was administered in their drinking water (Argus and Hoch-Ligeti, 1963).
In these animals the first liver carcinoma and the first lung papilloma
were diagnosed at 16 weeks and 45 weeks, respectively. In a later study
with outbred Hartley guinea pigs, diethylnitrosamine in the water induced
only hepatomas, with a mean induction time of 15 months (Dale et al.,
1973).
Rats, on the other hand, developed tumors in a variety of organs
following oral administration of diethylnitrosamine (Lijinsky and Taylor,
1978). Diethylnitrosamine was administered in drinking water to groups
of 6 to 15 Sprague-Dawley rats for up to 29 weeks. The animals survived
for 33 weeks, and 100% of them developed tumors of the liver (carcinomas
and sarcomas), nasal turbinates, and/or esophagus. In a study by Reuber
(1976), even more extensive lesions were observed in Buffalo strain rats
of various ages fed diethylnitrosamine in their diet for 26 weeks.
Malignant tumors appeared in the esophagus, liver (carcinomas and sar-
comas), and prostate gland; the type and distribution of tumors depended
on the sex and age of the animals. The carcinogen was incorporated into
the diet in the amount of 0.0114%, and the first tumors were seen 24 to
36 weeks after the start of the experiment.
Similar to rats, BALB/c mice developed tumors of the liver and
esophagus as a result of ingesting diethylnitrosamine in drinking water
-------
Table 5.6. Tumor induction in various species with diethylnitrosamine and dimethyInitrosamine
Chemical /species
Diethylnitrosamine
Monkey
Monkey
Inbred guinea
Pig
Outbred Hartley
guinea pig
Sprague-Dawley
Dose
2-5 mg/kg body
weight/day
Total: 6-24 g
20-40 rag per 2 weeks
Total: 1.6-2 g
1.1-4.2 rag/day
Total: 200-300 mg
About 3 mg/day
3 days/week
Total: 203 mg
Duration
of
exposure
(weeks)
108
108
16 to 40
Up to 68
29
Route of
exposure
Oral
Intraperitoneal
Oral
Oral
Oral
Tumor incidence
6/18 hepatomas
2/2 hepatomas
14/15 hepatomas
7/15 lung tumors
2/15 bronchial
papillomas
35/35 hepatomas
10/12 liver tumors
Tumor
latency Reference
(weeks)
92 Kelly et al. , 1966
104 Kelly et al., 1966
16 to 46 Argus and Hoch-Ligeti
1963
60 Dale et al., 1973
About 20 Lijinsky and Taylor,
rat
Buffalo strain
rat (12 weeks
old)
Mice BALB/c
0.0114X in diet 26
Total: 515 mg/kg 41
body weight
Total: 1010 mg/kg 41
body weight
Oral
Oral
Oral
2/12 nasal
turbinate tumors
6/12 esophageal
papilloma
1/12 esophageal
carcinoma
1/7 prostate tumor
12/26 esophageal
carcinoma
Unclear liver
4/15 liver
heraangiosarcomas
6/15 esophageal
tumors
3/15 stomach tumors
0/15 lung tumors
14/60 liver
hemangiosarcomas
35/60 stomach tumors
2/60 lung tumors
1978
24 to 36 Reuber, 1976
Unclear Clapp, Tyndall, and
Otten, 1971
Unclear Clapp, Tyndall, and
Otten, 1971
ro
oo
-------
Table 5.6 (continued)
Chemical/species
Dose
Duration
of
exposure
(weeks)
Route of
exposure
Tumor incidence
Tumor
latency
(weeks)
Reference
Mice, Swiss
DimethyInitrosamine
Mice, BALB/c
Mice, Swiss
Chinese hamster
6 mg/kg body
weight/week
10
Intraperitoneal
Total: 300 mg/kg
body weight
6 mg/kg body
weight/week
Up to 41 Oral
10
Intraperitoneal
0.89-3.54 mg/kg
body weight/week
Total: 31-105 mg
Subcutaneous
30/38 lung tumors 66
4/38 vascular
tumors
3/38 forestomach
papillomas
0/38 kidney tumors
10/38 lymphomata
1/38 mammary
adenocarc inoma
3/15 hemangio- Unclear
sarcoma
7/15 lung tumors
37/39 lung tumors 59
12/39 vascular
tumors
0/39 forestomach
papilloma
4/39 kidney tumors
4/39 lymphomata
1/39 mammary
adenocarcinoma
100/108 liver 26
tumors
1/108 lung tumors
1/108 nasal tumor
Cardesa et al., 1974
Clapp, Tyndall, and
Otten, 1971
Cardesa et al., 1974
N>
oo
Reznik, 1975
-------
285
(Clapp, Tyndall, and Otten, 1971). Tumors were also found in the lung
and stomach. Dimethylnitrosamine, however, produced tumors only in the
lung and liver. Differences in susceptibility of the lung to diethyl-
nitrosamine (3% tumor incidence) and dimethylnitrosamine (47% tumor
incidence) indicate that different enzyme systems may be activated for
metabolism of each of the chemicals and/or that the system for metabo-
lizing diethylnitrosamine is not present in sufficient quantity in the
lung of the BALB/c mouse. Swiss mice, a strain having a 26% spontaneous
lung tumor incidence, also developed more lung tumors after intraperito-
neal injection of dimethylnitrosamine than after diethylnitrosamine, but
the difference in tumor incidence was less pronounced (Cardesa et al.,
1974). Liver tumors in both strains of mice were of vascular rather than
of hepatocellular origin. A similar response was reported in Chinese
hamsters injected subcutaneously with three doses of dimethylnitrosamine
(Reznik, 1975). Tumors were mainly in the liver and were all of vascular
origin (hemangioendotheliomas). Tumor incidence was nearly 100% and was
not dose dependent; the first liver tumor occurred in the 26th exper-
imental week.
Thus, in the above examples the most obvious species-related differ-
ence in the response of animals to diethylnitrosamine and dimethylnitro-
samine was in the type of liver tumors induced. Those in monkeys, rats,
guinea pigs, and hamsters were of vascular and/or liver cell origin,
while those in mice were of vascular origin only.
Other jy-nitroso compounds — In separate studies, species differences
were observed between rats and hamsters treated with diisopropanol-
nitrosamine (Mdhr, Reznik, and Pour, 1977; Pour, Kruger, and Althoff,
1974, 1975). Sprague-Dawley rats were given subcutaneous injections of
178 to 1425 mg per kg of body weight once weekly for 20 weeks, and
hamsters received 125 to 500 mg per kg of body weight per week for life.
The chief differences noted were: (1) pancreatic tumors were diagnosed
in all hamsters, but only one such tumor was observed among 150 rats;
(2) cholangiomas or cholangiocarcinomas were, found in the hamster but
not in the rat; and (3) adenomas and adenocarcinomas were found in the
thyroid glands of rats but not in hamsters.
-------
286
Lijinsky et al. (1967, 1970) tested the carcinogenicity of a cyclic
nitrosamine, nitroazetidine, in rats, mice, and hamsters. Five milli-
grams of the compound per day administered in drinking water for 46 days
(total dose 230 mg) induced tumors in 15 out of 20 Wistar rats, and 0.5
mg per day (total dose 23 mg) induced tumors in 15 out of 40 mice.
Hamsters were resistant to total doses of up to 500 mg. However, both
rats and hamsters were responsive to tumor induction by another cyclic
nitrosamine, nitrosoheptamethyleneimine (Lijinsky et al., 1967, 1970).
It may be concluded from the above examples of #-nitroso compound
carcinogenesis that the organ specificity of these chemicals is approxi-
mately the same in all species that respond to them, regardless of
route. However, differences do exist in general susceptibility of the
various species to specific compounds of this class of chemicals.
5.2.1.2.4 Miscellaneous compounds — Brief mention is made below of
species differences encountered in carcinogenesis studies with miscel-
laneous compounds of chemical classes other than those previously men-
tioned.
Minerals and Metals — Exposure in inhalation chambers to amosite,
crocidolite, and chrysotile was the basis for a comparison of the various
responses of rats (Charles River), guinea pigs (Camm-Hartley), rabbits
(Shankin Farms Dutch), mice (Swiss), and gerbils (Mongolian) of both
sexes (Reeves, Puro, and Smith, 1974). The animals were exposed 4 h per
day, 4 days per week for 2 years (1480 h cumulative total exposure).
Some animals were sacrificed after 3 to 6 months, but most were allowed
to survive until the exposure period ended. Table 5.7 contains results
of the study.
Reeves and associates were the first to obtain lung carcinoma and
pleural mesothelioma following exposure by inhalation to amosite. Rats
seemed to be the only species that displayed an increased incidence of
neoplasia of the respiratory tract (mice were excluded because of their
tendency to develop spontaneous bronchial cancers). It was also noted
that after equivalent asbestos exposure, pulmonary ferruginous bodies
were abundant in the guinea pig and rare in the rat. The authors sug-
gested the frequency of ferruginous bodies is inversely related to
carcinogenic effects attributable to asbestos inhalation.
-------
Table 5.7. Carcinogenicity of different forms of asbestos in various species
Species
Mouse*2
Gerbil
Rat
Rabbit
Guinea pig
Number
surviving
19
50
43
11
14
Chrysotile
Effect
None
None
One papillary carcinoma
of lung
One squamous carcinoma
of lung
One mesothelial fibro-
sarcoma of mediastinum
None
None
Crocidolite
Number
surviving
18
49
46
9
14
Effect
Two papillary
carcinomas of
bronchus
None
One adenocarci-
noma of lung
Three squamous
carcinomas of
lung
One papillary
adenocarc inoma
of lung
None
None
Number
surviving
17
51
46
9
13
Amosite
Effect
None
None
One metastatic osteo-
sarcoma of lung
One mesothelial fibro-
sarcoma of lung and
pleura
One fibrous mesothelial
of pleura
None
None
00
^1
aControl mouse had one papillary carcinoma of the bronchus. All other controls were free of neoplasma.
Source: Adapted from Reeves, Puro, and Smith, 1974. Reprinted with permission of the publisher.
-------
288
Sunderman (1971) reviewed the carcinogenic effects of beryllium,
cadmium, chromium, cobalt, iron, lead, nickel, selenium, zinc, and
titanium in various species by different routes of exposure.
jL>l-Trichloro-2,2-bis(p-chlgrophenyl) ethane (DDT) — DDT has been
shown to be primarily a hepatocarcinogen in mice, although tumors have
developed in other organs of more susceptible mouse strains (Innes et
al., 1969; Terracini et al., 1973). Tomatis and Turusov (1975) demon-
strated that the tumorigenicity of DDT in mice is dose related. In rats,
however, DDT has been noncarcinogenic (Cameron and Cheng, 1951) or mar-
ginally carcinogenic (Deichmann et al., 1967) and has had no effect on
hamsters and monkeys (Agthe et al., 1970; Durham, Ortega, and Hays,
1963).
Aflatoxin Bj — The hepatocarcinogenicity of aflatoxin Bj had been
demonstrated in rats (Newberne and Rogers, 1972; Wogan and Shank, 1971)
but not in mice until Vessilnovitch et al. (1972) induced heptatomas in
newborn mice with intraperitoneal injection of a total of 1.5 to 6 yg
per g of body weight (in one to five doses). Tumors were observed 52
weeks after exposure.
Adamson, Correa, and Dalgard (1973) reported the induction of liver
tumors in a rhesus monkey with aflatoxin BI} and carcinogenicity of
aflatoxin in the hamster has been described by Herrold (1969). Table
5.8 lists other species and target organs in which the tumorigenic
activity of aflatoxin has been demonstrated.
[Although this report is primarily concerned with mammalian tests
for carcinogenicity, Table 5.8 has been included because it concisely
relates the association between exposure to a chemical or a chemical
process and the occurrence of cancer in humans as reported by the Inter-
national Agency for Research on Cancer (Tomatis et al., 1978). These
data are correlated with the results of animal tests.]
5.2.2 Strain
The use of random-bred, rather than highly inbred, strains in
chronic toxicity and carcinogenicity testing remains controversial. In
broad-scale studies where little prior information exists, some workers
-------
Table 5.8. Chemicals or industrial processes associated with cancer induction in humans:
comparison of target organs and main routes of exposure in animals and humans
Chemical or
industrial process
Aflatoxins
4-Aminobiphenyl
Arsenic Compounds
Asbestos
Auramine
(manufacture of)
Benzene
Benzidine
Bis(chloromethyl)
ether
Humans
of^xposu're*
Environmental, Liver
occupational
Occupational Bladder
Occupational, Skin, lung, liver
medicinal, and
environmental
Occupational Lung, pleural cavity,
gastrointestinal
tract
Occupational Bladder
Occupational Hemopoietic system
Occupational Bladder
Occupational Lung
Main route. A_<_«I
of exposure**0 Animal
P.O., i.h. Rat
Fish, duck, mar-
moset, tree
shrew, monkey
Rat
Mouse, rat
Mouse
i.h., s. , Mouse, rabbit, dog
p.o. Newborn mouse
Rat
i.h., P.O., Mouse, rat, dog
s . Mouse
i.h., p.o. Mouse, rat, hamster,
rabbit
Rat, hamster
Rat
i.h., s., p.o. Mouse, rat
Rabbit, dog
Rat
i.h., s. Mouse
i.h., s., p.o. Mouse
Rat
Hamster
Dog
i.h. Mouse, rat
Mouse
Rat
Animals
Target organ
Liver, stomach,
colon, kidney
Liver
Liver, trachea
Liver
Local
Lung
Bladder
Liver
Mammary gland,
intestine
Inadequate, negative
Inadequate, negative
Lung, pleura
Local
Local ,
Various sites
Liver
Negative
Local, liver,
intestine
Inadequate
Liver
Liver
Zymbal gland, liver,
colon
Liver
Bladder
Lung, nasal cavity
Skin
Local, lung
Local
Route of
exposure"
p.o.
p.o.
i.t.
i.p.
s.c.
i.p.
p.o.
s.c.
s.c.
p.O. N3
t i v 00
t., l.V. ^
i.h. or i.t.
ipl.
i.p., s.c.
p.o.
p.o.
p.o.
s.c.
t., s.c.
s.c.
p.o.
s.c.
p.o.
p.o.
i.h.
t.
s.c.
s.c.
-------
Table 5.8 (continued)
Chemical or
industrial process
Cadmium-using indus-
tries (possibly
cadmium oxide)
Chloramphenicol
Chloromethyl methyl
ether (possibly
associated with
bis (chloromethyl) -
ether
Chromium (chromate-
producing
industries)
Cyclophosphamide
Die thy Is t ilbes t rol
Hematite mining
(? radon)
Isopropyl oils
Melphalan
Main type
of exposure*2
Occupational
Medicinal
Occupational
Occupational
Medicinal
Medicinal
Occupational
Occupational
Medicinal
Humans
^SS"
Prostate, lung** i.h., p.o. Rat
Hemopoietic system P.O., i.j.
Lung i.h. Mouse
Rat
Lung, nasal cavities^ i.h. Mouse, rat
Rat
Bladder p-c... i.j. Mouse
Rat
Uterus, vagina p.o. Mouse
Mouse
Rat
Hamster
Squirrel monkey
Lung i.h. Mouse, hamster,
guinea pig
Rat
Nasal cavity, larynx i.h.
Hemopoietic system P.O., i.j. Mouse
Rat
Animals
Target organ
Local, testis
No adequate tests
Initiator
Lungd
Local,, lung"
Local"
Local
Lung
Hemopoietic system,
lung
Various sites
Bladder^
Mammary gland
Various sites
Mammary
Mammary, lymph-
oreticular, testis
Vaeina
•«*o ,
Mammary, hypophysis"
bladder
Kidney
Uterine serosa
Negative
Negative
No adequate tests
Initiator
Lung, lymphosarcomas
Local
Route of
exposure0
s.c. or i.m.
s.
i.h.
s.c.
s.c.
s.c. , i.m.
i.b. impl.
i.p., s.c.
N)
vO
p.O. o
i.p.
i.p.
i.v.
p.o.
s.c. ,
s.c. impl .
Local
s.c. impl.
B.C.;
s.c. impl .
s.c. impl .
i.h., i.t.
s.c.
s.
i.p.
i.p.
-------
Table 5.8 (continued)
Chemical or
industrial process
Mustard gas
2-Naphthylamine
Nickel (nickel
refining)
/V,#-Bis(2-chlpro-
ethyl) 2-naphthy 1-
lamine
Oxymetholone
Phenacetin
Phenytoin
Soot, tars, and oils
t
Vinyl chloride
Main type
of exposure"
Occupational
Occupational
Occupational
Medicinal
Medicinal
Medicinal
Medicinal
Occupational,
environmental
Occupational
Humans
Target organ
Lung, larynx
Bladder
Nasal cavity, lung
Bladder
Liver
Kidney
Lymphoreticular
tissues
Lung, skin (scrotum)
Liver, braind, lungd
Main route A-.IIMI
of exposure* ^iaai
i.h. Mouse
i.h., s. , p.o. Hamster, dog,
monkey
Mouse
Rat, rabbit
I.h. Rat
Mouse, rat, hamster
Mouse, rat
p.o. Mouse
Rat
p.o.
p.o.
P.O., i.j. Mouse
i.h., s. Mouse, rabbit
i.h., s. Mouse, rat
Animals
Target organ
Lung
Local , mammary
Bladder
Liver, lung
Inadequate
Lung
Local
Local
Lung
Local
No adequate tests
No adequate tests6
Lymphoreticular
tissues
Skin
Lung, liver, blood
vessels, mammary,
Zymbal gland,
kidney
Route of
exposure0
i.h., i.v.
s.c.
p.o.
s.c.
p.o.
i.h.
s.c., i.m.
i.m. impl.
i.p.
s.c.
NS
\£>
P.O., i.p.
t.
i.h.
aThe main types of exposures mentioned are those by which the association has been demonstrated; exposures other than those mentioned
may also occur.
The main routes of exposure given may not be the only ones by which such effects could occur.
°p.o. - peroral; i.t. - intratracheal; i.p. - intraperitoneal; s.c. — subcutaneous; t. - topical; i.v. — intravenous; ipl. - intra-
pleural; i.h. - inhalation; i.m. - intramuscular; s. - skin; s.c. impl. - subcutaneous implantation; i.b. - intrabronchial implantation;
i.J. — injection.
d.
1977).
Indicative evidence.
The induction of tumors of the nasal cavities in rats given phenacetln has been reported recently (S. Odashima, personal communication,
-------
292
recommend using random-bred strains to ensure observing a species-
characteristic response (Benitz, 1970). Other investigators urge the
use of inbred animals whose genetic stability tends to reduce variations
in the incidence of spontaneous disease and contributes to greater
experimental precision (Food Safety Council, 1978; Womack, 1979).
However, Greenman, Delongchamp, and Highman (1979) warn that easy general-
ization cannot be made about the variability of inbred and hybrid mice.
Whether or not inbred or random-bred strains are used, it is important
that background clinical and pathological information be available for
test animals so that results in both control and treated animals can be
compared with known literature values (Fancher, 1978; World Health
Organization, 1978). Germ-free animals are rarely used in chronic
toxicity and carcinogenicity testing because maintenance is too laborious
and costly for long-term studies, but specific pathogen-free and gnoto-
biotic animals have been found useful by some investigators (Benitz,
1970).
Selection of the appropriate animal strain is an important part of
experimental design in bioassays for carcinogenicity. In terms of
numbers of strains available, selection is not easy. For example, there
are over 250 mouse strains, 100 rat strains, and 30 hamster strains
(Butler, 1979). Weisburger (1976) suggested several factors that deserve
consideration in choosing a strain. These include maintenance require-
ments, sensitivity to test chemicals, and occurrence of diseases, both
nonneoplastic and neoplastic. In addition, he reviewed information
about some of the strains of rats, mice, and hamsters used in bioassays
of carcinogenic agents. Because of variation in response to such agents
by animals of different strains, it is generally advisable to test the
agents in several strains of a species (Butler, 1979). Several studies
will expand upon some of these considerations.
5.2.2.1 Hamsters and Guinea Pigs — Inbred strains of Syrian golden
hamsters respond differently to subcutaneously injected methylcholanthrene
and benzopyrene (Homburger et al., 1972). The less sensitive strains
(BIO 82.73, BIO 1.5) have longer induction times than does the more sen-
sitive 87.20 line. This fact is important for determining the duration
of an experiment and, if not properly understood, could lead to misinter-
pretation of data. Another consideration in selection of an appropriate
-------
293
strain relates to tumor type. For example, some strains of hamsters
(BIO 54.7, BIO 82.73, BIO 86.93) are not susceptible to induction of
intestinal neoplasms and thus should not be used for bioassays concerned
with such tumors (Homburger et al., 1972).
A strain may be sensitive to one chemical and not another, as seen
in the Hartley and inbred ICRF guinea pigs. Diethylnitrosamine is an
effective carcinogen in both strains; methylcholanthrene is inconsistent
in the Hartley strain, but less so in the ICRF animals (Dale et al.,
1973).
5.2.2.2 Mice — The influence of genetic factors has been clearly
demonstrated in two strains of mice, BALB/c and C57BL/6, which after
treatment with diethylstilbestrol developed testicular teratomas and
pituitary neoplasms, respectively. The F^ hybrids of these strains
developed both tumors in response to the carcinogen (Greenman and
Delongchamp, 1979). In addition to variation in tumor types among
different strains, incidence, induction time, and susceptibility to an
agent can vary. In an extensive study of transplacentally injected 1-
ethyl-1-nitrosurea, Diwan and Meier (1974) demonstrated clear differences
in all these parameters for five mouse strains (AKR/J, SWR/J, DBA/2J,
C57L/6J, and C57L/J). They concluded that responses to the carcinogen
were dependent on both the strain involved and the fetal age at the time
of injection. The latent period for malignant lymphoma in "101" mice
treated with dimethylbenzanthracene (DMBA) was shorter than that of CBA
mice (Roe, Rowsen, and Salaman, 1961). Thiery and van Gijsegem (1965)
studied the induction of squamous cell carcinoma of the cervico-vaginal
epithelium by 3,4-benzo(a)pyrene in 12 mouse strains. On the basis of
tumor yield and induction time, strains C3H, A, 0.20, N, R, K, Q, and Mo
were highly sensitive, S and AKR were moderately sensitive, and H and W
were relatively insensitive to the carcinogen. Holland, Gosslee, and
Williams (1979) demonstrated that C57BL/6 mice were 2.4 times more
sensitive to the epidermal carcinogens bis(2,3-epoxycyclopentyl)ether,
2,2-bis(p-glycidyloxphenyl) propane, and m-phenylenediamine. Holland et
al. also noted that the sensitivity of an assay could be affected by the
choice of strain in the case of very weak carcinogens.
-------
294
5.2.2.3 Rats — Within a few weeks after one dose of dimetbylbenz-
anthracene, Sprague-Dawley rats developed high incidence of multiple
mammary gland tumors. In contrast, Long-Evans rats receiving the same
treatment had low incidence, few tumors, extended induction time, and,
in some cases, tumor regression. The sensitivity of the mammary glands
to the hydrocarbon can be correlated with inherited variation in pituitary
function. Fibroadenoma incidences after DMBA in Fj hybrids from recip-
rocal crosses of the two strains were nearly equal; hybrids from back-'
crosses in which the strain of origin was SD had high tumor incidence as
opposed to low ones from the LE strain. Foster nursing had no effect on
tumor incidence (Sydnor et al., 1962). Sydnor (1973) also tested nine
inbred strains of rats for susceptibility to benzo(a)pyrene. Sarcoma
incidence ranged from 38% in Long-Evans rats to 97% in Fischer rats. The
most sensitive strains in terms of incidence and induction time were
Sprague-Dawley, Fischer, and Werck-Stewart, and the least sensitive were
the Long-Evans rats. Sydnor concluded that susceptibility is genetically
determined by carcinogen dose.
In conclusion, careful selection of appropriate strains for experi-
mentation and substance evaluations is a prerequisite for meaningful
interpretation of data. Strain-related parameters that relate to carci-
nogenesis include spontaneous tumor incidence (discussed elsewhere),
age, induction time, and susceptibility. Superimposed on the genetic
framework are the many environmental factors that can modify the response
of a strain to a particular stimulus. These factors must be controlled
as extensively as possible. The great value of strain differences lies
in the potential for eventually determining the molecular mechanisms
that vary between strains. Ultimately, such knowledge may help to
determine those mechanisms that cause variation among individuals.
5.2.3 Spontaneous Tumors
Spontaneous tumors are relatively common among experimental animals,
both those bred for laboratory studies as well as wild animals maintained
in the laboratory (Andervont and Dunn, 1962). Such lesions occur in a
variety of organs and at different ages. Snell (1965) defined spontaneous
lesions as those for which a cause cannot be ascertained. Such neoplastic
-------
295
lesions appear in animals that have not been experimentally exposed to a
carcinogenic agent, Hoag (1963) pointed out that spontaneous tumors are
generally considered "naturally occurring"; however, when the etiology
of the particular tumor is understood, it can be induced experimentally.
Spontaneous tumor incidences in the various organs of animals used in
the national Cancer Institute Bioassay Program have been summarized by
Page (1977a) and are listed in Table 5.9. The B6C3Fj mouse, an PI
hybrid cross between the C57BL/6 female and the C3H male, has a rela-
tively high incidence of spontaneous tumors of the lung and liver. The
F/344 rat has a high incidence of testicular tumors in the male and of
pituitary tumors in the female. These strains have been used extensively.
They have the advantages of good survival, disease resistance, and a
relatively low spontaneous tumor incidence in organs other than those
mentioned.
In order to make valid interpretations and comparisons of results
in a variety of experimental situations — whether they are designed to
test carcinogenicity of specific agents, to screen substances for anti-
carcinogenic activity, or to look at mechanisms of neoplastic growth —
the investigators must be cognizant of the incidence of spontaneous
tumors in the animal tested. For example, the tumor response (i.e., the
type, frequency, latency, etc.) to carcinogenic agents may vary among
animals with different spontaneous tumor rates. For more information
relating to this point, see the discussion of strain differences.
Various factors can influence frequency, distribution, latency, and
morphology of spontaneously occurring tumors. Some variables known to
have effects are: breeding methods, genetics, age, diet, maintenance,
histologic screening, and statistical evaluation (Pollard and Kajima,
1970; Pour et al., 1976; Sass et al., 1975), as well as geophysical
parameters such as geography and climate (Gilbert et al., 1958). Sex
and age incidence of characteristic tumors for certain inbred strains of
mice have been summarized by Weisburger and Weisburger in Table 5,10.
Spontaneous tumor incidence can generally be viewed as the result of
both genetic and environmental influences whose relative contributions
must be defined for a particular animal strain. Genetic or strain
differences in spontaneous tumor frequency can be best evaluated when
environmental factors have been characterized (Gilbert et al., 1958).
-------
Table 5.9. Spontaneous tumor incidence in animals used in Che National Cancer Institute Bioassay Program
B6C3F1 Fischer
mouse0 344 rat*1
fl v rr in
Males
Brain <1
Skin/ subcutaneous 1.0
Mammary gland
Spleen <1
Lung/ trachea 9.2
Heart
Liver 15.7
Pancreas <1
Stomach/ intestines 1.3
Kidney <1
Urinary bladder
Testis <1
Ovary NA
Uterus NA
Pituitary <1
Adrenal <1
Thyroid 1.1
Parathyroid
Pancreatic islets <1
Thymus <1
Body cavities <1
Leukemia/lymphoma 1.6
Females Males
1176 846
1.3
<1 5.7
<1 1.0
<1 <1
3.5 2.4
1
2.5 1.2
<1 <1
<1 <1
<1 <1
<1 j <1
NAfl 76.2
<1 NA
1.9 NA
3.5 10.2
<1 8.7
<1 5.1
<1 3.2
<1 <1
6.8 6.5
Females
840
-------
297
Table 5.10.
Sex and age incidence of characteristic tumors
of inbred strains
Tumor type
and strain
Mammary gland tumor
C3H
DBA/ 2
A
DD
Lymphocytic leukemia
AKR
C58
Primary lung tumor
A
SWR
Hepatoma
C3HeB
Reticulum cell
neoplasm, type B SJL
Incidence
(%)
99
100
77
Lower
84
5
84
75
92
90
90
80
91
58
30
91
High
Type mouse
Breeding 99
Virgin 99
Breeding 99
Virgin 99
Breeding 99
Virgin 99
Breeding 99
Virgin 99
Breeding
-------
298
Table 5.10 (continued)
Tumor type
and strain
Incidence
Type mouse
Age
(months)
Testicular teratoma
129
Myoepithelioma
1
82
66
Male gonadal
ridges trans-
planted to adult
testes
Congenital
BALB/c
A
Skin papilloma
HR/De
Skin carcinoma
HR/De
Subcutaneous sarcoma
C3H/J
Harderian gland tumor
C3H
4
Similar
9 Hairless
3 Hairless
3 99
1
Av.
Av.
Av.
Av.
22
18
25
20.7
Source: Weisburger and Weisburger, 1967, as adapted from Murphy,
1966. Reprinted with permission of the publisher.
Genetic control of susceptibility to ileocecal immunocytomas in
certain strains of rats appears to reflect one or more dominant genes
for susceptibility in the LOD/c strain and one or more loci of resistance
in the OKA strain. Fj progeny from crosses of these strains have null
tumor incidence (Beckers and Bazin, 1978). High incidence of spontaneous
congenital testicular teratomas in an inbred subline of mice is probably
due to a single gene mutation (Stevens, 1973).
Evidence of genetic involvement at the molecular level is evident
in the inhibition of mammary tumors in mice by treatment with a bacterial
cell wall skeleton preparation which reduces the level of DNA synthesis
in normal mammary glands and lowers circulating prolactin levels, both
major factors in tumorigenesis (Nagasawa, Yanai, and Azuma, 1978).
Genetic influence on susceptibility has been demonstrated in certain
vy
strains of C3H mice prone to hepatomas and mammary tumors. The A gene
-------
299
increases the Incidence of both tumor types, perhaps by enhancing the
virulence or transmission of the mammary tumor virus by either parent or
by increasing the responsiveness of tissues. The gene's effects are
also correlated with increased body weight and are enhanced by hormonal
factors (Heston and Vlahakis, 1968; Vlahakis, Beston, and Smith, 1970).
Interesting results with this same CSH-A^ system have been reported
by Sabine, Horton, and Wicks (1973). Mice bred in Australia had 0%
incidence of mammary tumors compared with the incidence of nearly 100%
found in the united States. By using feed and bedding from the United
States, the high tumor incidence can be restored. The interactions of
genetic and environmental factors are clearly complex, and both deserve
careful attention.
Examples of the modification of spontaneous tumor incidence by
environmental factors have been reported in the C3H mouse hepatic tumor
system, which can be influenced by sex, population density, and diet
(level of protein and caloric intake) (Grahn and Hamilton, 1964; Heston,
Vlahakis, and Deringer, 1960; Peraino, Fry, and Staffeldt, 1973; Silverstone
and Tannenbaum, 1951; Tannenbaum and Silverstone, 1949). Heston (1958),
in studying four substrains of C3H mice, found that mammary tumor inci-
dence was influenced not only by genetic susceptibility, but also by
hormonal stimulation during breeding and by age. Riley (1975) described
a shortening of latent period.for mammary tumors in C3H/He mice carrying
the Bittner oncogenic virus and exposed to environmental stress factors.
Age of the animals and latency periods of the tumors can present
problems in design and interpretation of experiments. Tumor incidence
data from rats at 2 to 2.5 years of age could give a different picture
from that which might be seen if the animals lived to old age. If the
animals are more susceptible to tumors late in life, then testing only
in young animals could prevent detection of neoplasms. Studies over the
life span of the animal are necessary for the evaluation of the biologic
behavior of spontaneous tumors and, possibly, of some carcinogen-induced
tumors (Burek and Hollander, 1977). In addition to the value of life
span data, one must consider the latency period before the effect of a
carcinogen is detectable as well as the dose (which in some cases deter-
mines latency). Laboratory animals tend to be more susceptible to
-------
300
tumors as they age, but, as discussed earlier, additional factors are
involved (Gilbert et al., 1958). Table 5.11 summarizes some pertinent
literature on this subject.
5.2.4 Number
The number of animals used in chronic toxicity and carcinogenicity
tests represents a compromise between the need for a sufficiently large
number of animals to allow adequate statistical precision of the conclu-
sions and the need to place a reasonable limit on costs and the experi-
mental work load. For example, if the true frequency of a toxic effect
in test animals is 5%, a test group numbering at least 58 must be used
to discover the effect with a probability of P = 0.05, but 90 animals
are needed at the P = 0.01 level of significance, and 134 animals must
be used to achieve the P = 0.001 level of confidence (Barnes and Denz,
1954). Although high levels of statistical probability are obviously
desirable, large increases in group size may diminish the thoroughness
and care that are necessary for a successful completion of the study and
may be counterproductive. In general, more useful information can be
obtained by conducting thorough studies with a relatively small number
of animals than by performing incomplete experiments using an excessive
number of animals (Benitz, 1970).
Early investigators of chronic noncarcinogenic toxicity tended to
use 10 or fewer test animals (rodents) per group (Barnes and Denz,
1954); later, groups of 20 to 30 rodents per sex and dose level were
considered sufficient for practical purposes (Benitz, 1970). More
recently, a minimum of 50 rodents of each species and sex has been
considered necessary, especially in work subject to federal agency
regulation (Federal Register, 1978, 1979; Food Safety Council, 1978;
National Academy of Sciences, 1977). Fewer rodents per group may be
used if the number of dose levels is increased; however, little current
literature support exists for groups smaller than 20 animals.
Test groups are generally smaller than indicated in the previous
paragraph when the test species are nonrodents. If dogs, cats, or non-
human primates are used in chronic toxicity studies, a minimum of at
least four animals per dose and sex is usually considered necessary
-------
Table 5.11. Spontaneous tumors in laboratory animals
Animal
Tumor and organ
Incidence and age
nts
Reference
Monkey
Maaaca phillipieneie and
Macaaa mulatto
Lymphosarcoma — kidney
Hemangloendothelloma — vertebra
Meningiomatosls — lumbar cord
Lipoma — chorold plexus
Endotheliomatosls — lumbar cord
Overall - 0.08*
prepubertal
12 000 monkeys (1800 of
them cynomolgus) observed
over 3.5 years. Also looked
at granulomatous processes.
Jungherr, 1963
Nude mutant, most
with BALB/c, some
with C3H or C57/BL/6
backgrounds
Nude mutant from
BALB/c backgrounds
CD8F,
(BALB/CXDBA/8F1)'
SHN
129/terS
Strain A
CD^-lHaM/ICR
Mammary tumors
None
Lymphoreticular neoplasms
Adenocarclnoma mammary
(also metastasis to lung)
Mammary tumors
Teratomas, testicular
Lung tumors
Lymphoreticular mammary,
pulmonary, osteogenlc sarcoma,
hemanglosarcoma, renal and
hepatic tumors
Liver, mammary tumors
CSH-AfB
Reaches 87%
OX, generally die at
2 to 3 months of age
Estimated from graphic
material, tumor Incidence
for entire group was about
50X at 57 weeks (2Z at 35
weeks)
802, 10 months
41.32 at 7 months, 87.52
at 12 months
302, earliest seen were in
6-day fetuses
02 at 2 months, 402 at 12
months, 77.1Z at 18 months
Total incidence of tumors —
range of 0.77-50Z for age
groups ranging from 0+ to 20+
months
90-1002 or 02, ages from
7 to 22 months
Studied tumor regression
after 5-methyl cytidine.
11 000 mice studied from
birth to 3 months.
Cermfree conditions — can
maintain for over 20 months.
In germfree nu/+ females, no
mammary tumors seen.
Studied chemotherapeutic
activity.
Studied tumor suppression.
High incidence probably
due to a single gene
mutation.
Frequency of lung tumors has
been reproducible for more
than 30 years.
Percentages of a particular
tumor type varied with age.
Tumor Incidence apparently
related to type bedding used.
Strong and
Matsunaga, 1975
Rygaard and
Polvsen, 1974
Outzen et al.,
1975
Fugmann et al. ,
1977
Nagasawa, Yanai,
and Azuma, 1978
Stevens, 1973
Shimkin and
Stoner, 1975
Percy and
Jonas, 1971
Sabine, Horton,
and Wicks, 1973
U>
O
-------
Animal
C3Hf
C3Hff
C3Hf/2
C3Hf/An
C3H£B X YBR
C3H£B X YBR
C3Hfb X YBR
YBR X C3UfB
YBR X C3HfB
YBR X C3HfB
YBR X C3HfB
C3H/St
C3HX101
hybrids
A
GR
020
CBA
C3H
DBA
C57BL
Table 5.11
Tumor and organ
Mammary tumors: adeno- 22%,
carcinoma,
Adenoacanthoma, care in o- 29Z,
sarcoma
51Z,
27Z,
Hepatoma 100Z
94Z
63Z
52
95*
43*
41%
*+•*•:-,. QZ
Adenocarcinoma, mammary 41Z,
Os ceosar comas 20X
Lung tumors 71Z,
42Z,
58Z,
18%,
14Z,
9%,
7%,
(continued)
Incidence and age
20 months
19 months
20 months
20 months
4. 5 months
23 months
14 months
21 months
27 months
21 months
21 months
22 months
Comments
Deprived of mammary tumor
agent by foster nursing.
Genotypes: AYA males
Aa males
A^A females
AYA males
Aa males
AYA females
Aa females
Weight and growth factors
considered Important.
Study of effects of As and
Se on tumor incidence.
Authors cited data of
Bentvelzen and Szalay,
1966.
Reference
Heston, 1958
Heston and
Vlahakis, 1966
Schrauzer
et al. , 1978
Shlmkin and
Stoner, 1975
NMRl-Neuherberg
Wild house mice
(Hue mueculue)
Lymphoma
Lung tumors
Glomerulosclerosis
Ovarian neoplasms
Adenomatous hyperplasia of
the glandular stomach
Pulmonary, reticulem-cell
(type B), granuloaa-cell of the
ovary, hepatoma, hemangioendo-
thelioma, mammary, lymphocytic
neoplasm
41Z
16Z
13Z (50Z survival time
12Z is 600-700 days)
7Z
36Z, 2-24 months
64X, 25-33 months
U>
O
NJ
Luz, 1977
Tumors in mice received
in 1951 and their descendants
until 1961.
Andervont and
Dunn, 1962
-------
Table 5.11 (continued)
Animal
Tumor and organ
Incidence and age
Comments
Reference
C3Hf/Anl(Anl 70)
C3H/HeIH
GR/N
BALB/cfC3H
C57BL/6XC3HF.
(called i
B6C3F.)
AKR
BALB/c
C57BL/6_
C3H/HeN
C3H/HeJ
C3H/He
C3H/He
C3H/A/BOM
C3HeB/Fej
C3H
C3Hf
C3He
Liver tumors
Mammary tumors
C3H-A
vy
C3H-AvyfB
Mammary tumors
Mammary tumors
Hepatoma
Hepatoma
Hepatoma
Mammary tumors
Mammary tumors
Females 6-13Z, males 41-
682, before 12 months
99*, 7.2 months
70%, 10 months
<10Z, 8 months
OX, 8 months
32, 15 months
1Z, 15 months
5Z, 8 months
27%, duration of experiment,
2 years. (In another table,
incidence appears to be 100Z.)
80-100% within 8 to 18
months
66%, 22-25
received
starting
50%, 18-20
received
starting
25Z, 12-16
received
months, controls
rat albumin
at 9 months.
months, controls
adjuvant in PBS
at 2 months.
months, controls
adjuvant in PBS
85%
72%
78Z
All killed at 14 months
100Z, 12 months
100Z, 6-7 months
90Z, 15 months
20Z, 15 months
Studied enhancement of tumor
incidence.
Demonstration of mammary
tumor virus antibodies in
different strains. Highest
titers in tumor bearing
animals.
Peraino, Fry, and
Staffeldt, 1973
Ihle, Authur, and
Fine, 1976
Reduction of tumor incidence
by manipulation of estrogen
and prolactin interactions
with administration of 2-
bromo-d-ergocryptine, 17 B-
estradiol, or Enovid.
Strain carrying the Bittner
oncogenic virus incidence
greater under chronic stress.
Study of increased levels of
serum alpha-fetoprotein in
mice with spontaneous liver-
cell cancer.
In males.
C3Hf male fed Purina
chow instead of the NCI
pellets had a 57Z incidence.
Males (also nearly all
females at 16 months).
Females with MTV.
Female breeders free of MTV
but having NIV.
Female virgins free of MTV
but having NIV.
Helsch, 1976
Riley, 1975
Jalanko et al.,
1978
OJ
O
UJ
Heston, Vlahakis,
and Deringer, 1960
Heston and
Vlahakis, 1968
-------
Table 5.11 (continued)
Animal
Tumor and organ
Incidence and age
Comments
Reference
Hamster
Syrian, Eppley colony,
Hannover colony
BIO 4.24
BIO 45.5
Overall tumor Incidence
Primary tumors in respiratory,
digestive, urogenital, endocrine,
vascular, lymphatic systems
Adrenal tumors
32Z, average
41Z survival time for animals
with multiple tumors was 97
and 85 weeks, respectively
52Z, average age at autopsy,
80.3 weeks
17Z
Study of spontaneous tumors Pour et al.,
and diseases In Syrian hamster 1976
colonies.
Hotnburger and
Russfield, 1970
Rat
CD
CD-I
BN/BiRij
LOU/C
LOU/M
AXC
OKA
(LOU/CXAXC) F.
(LOU/CXAUG)F.
(LOU/CXOKA)F7
GG
Sarcoma, fibroma, adeno-
carcinoma, adenoma, hemangioma,
fibroadenoma, adenoma, leiomyoma,
neck, Jaw, flank, shoulder,
salivary gland, pituitary gland,
thyroid gland, spleen, mammary
gland, uterus
Papillary tumors - urinary
bladder
Squamous cell carcinomas -
ureter
Ileocecal immunocytomas
Ileocecal lymph node for most
NB
Phaeochromocytoma (adrenal
gland) the major tumor.
Numerous others also occurred
in breast, pituitary gland,
thyroid gland, testis, pancreas,
liver, kidney, ovary, salivary
glands, uterus, and meninges
Adrenal carcinoma
252, weeks of observation of
tumors ranged from 23 to 80
weeks
Males
28% (bladder), 6Z (ureter),
ages 7-48 months
Females
2% (bladder), 54* (ureter),
ages 7-54 months
8-12 months
242
1.7Z
2.6Z
OZ
16Z
122
OZ
74Z of all males,
50Z of all females
Controls in study of oral Lee, Russell, and
toxiclty of organic fungicides. Minor, 1978
Females about 52 at 12+
months
First report of high incidence
of such tumors in rats. These
spontaneous tumors are rare.
Tumor incidence not homo-
geneous - could get wide
variation among litters of
the same strain.
Tumor incidence influenced
by diet through effects on
endocrine glands, used other
strains also.
Spontaneous tumors seldom
found in males.
Boorman and
Hollander, 1974
Beckers and
Bazin, 1978
Gilbert et al.,
1958
Noble, Hochachka,
and King, 1975
Breast fibroadenoma
About 20Z at 12+ months
-------
Table 5.11 (continued)
Animal
Tumor and organ
Incidence and age
Comments
Reference
BDX
Wistar - Af/Han-EMD
BN/Bi
AC1/N
Sarcomas - connective tissue,
bone, neural system, skin
carcinomas, tumors of lung,
gastrointestinal tract, genito-
urinary tract, mammary glands,
testis, adrenal glands;
Malignancies of lymphoreticular
system
Tumors of the central nervous
system: oligodendroglioma,
estrocytoma, mixed glioma,
pleomorphic glioma, meningioma
Mammary gland — fibroadenoma
Pituitary tumors
Lynphoreticular sarcomas
Adrenal gland, cortical adenoma
Pancreas - islet adenoma
Cervix and vagina, sarcoma
Testes-lnterstitial cell tumors,
adrenal gland, pituitary gland,
urinary bladder, mammary gland,
thymus, lymph nodes, subcutaneous
tissue, heart, vagina, salivary
gland, others
66%, 13-30 months
Tumors were macroscopic,
malignant as proven by
histology and transplantation.
Zoller, Matzku,
and Goerttler,
1978
5.82, mean ages ranged from
796 to 963 days
11Z, 30 months (23-40)
Males, 14*, 28 months (15-42)
Females, 26Z, 31 months (17-39)
Males, 14X, 24 months (19-36)
Males, 12Z, 33 months (27-43)
Females, 19Z, 33 months (23-54)
Males, 152, 33 months (20-42)
Females, 11*, 33 months (23-40)
15Z, 27 months (18-38)
56Z in males
52Z in females
Developed in 169 weeks
Data for other tumor types
Included. Study of Inci-
dence of benign and malignant
neoplasms at different ages.
Sum! et al.,
1976
Burek and
Hollander, 1977
O
Ui
Maekawa and
Odashlma, 1975
-------
306
(National Academy of Sciences, 1977); however, currently proposed U.S.
Environmental Protection Agency regulations require six nonrodent animals
per exposure group (Federal Register, 1979). This number does not
include allowances for interim sacrifices and must be increased if that
practice is followed. Needless to say, toxic reactions with low incidence
rates are unlikely to be detected with such small groups (Barnes and
Denz, 1954). Consequently, nonrodent test animals are usually used only
as "second" species in conjunction with larger numbers of rodent test
animals.
For carcinogenicity testing, the number of animals (rodents) to be
used is influenced by two additional factors not pertinent to chronic
toxicity evaluations (Magee, 1970; Page, 1977b). First, a significant
number of animals must survive to tumor-bearing age. Second, the inci-
dence of spontaneous tumors in the control groups must be compensated
for in order to avoid jeopardizing the sensitivity of the test. Table
5.12 shows, in relation to animal numbers, how such an increase in
spontaneous tumors in the controls will affect the incidence of observed
tumors required for significance. Experience has shown that in most
general carcinogenicity tests, 50 animals per sex per dose level will be
sufficient to meet these two requirements (National Academy of Sciences,
1977; Page, 1977&; Sontag, Page, and Saffiotti, 1976). However, in any
case, the number surviving to tumor-bearing age should be at least 25
per sex per level (World Health Organization, 1978).
5.2.5 Controls
Each chronic toxicity or carcinogenicity test should be evaluated
with reference to a concurrent control group composed of animals of the
same species, age, sex, and weight as the treated groups (Fedeval Register,
1979; U.S. Food and Drug Administration, 1971; Weil, 1962; Weil and
Carpenter, 1969). These animals should be handled identically to the
test species except for treatment with the test material (World Health
Organization, 1978). Most investigators require that this negative
control group contain at least as many animals of each sex as the test
groups; other workers recommend that it contain twice as many (National
Academy of Sciences, 1977). In lieu of a negative control group, a
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307
Table 5.12. Incidence of tumors In treated
groups required for significance (P = 0.05)
depending on experimental group size and
spontaneous tumors in controls*2
Incidence of
tumors in controls
(%)
0
10
20
30
40
Number
10
50
70
80
90
100
of
25
20
40
52
64
72
animals
50
12
28
40
52
62
per group^
75 100
8 6
24 21
36 34
47 45
58 55
Calculations based on tabulations of Mainland and
Murray, 1952, as cited in Page, 19772).
Controls and treated groups of same size.
Source: Adapted from Page, 1977Z?. Reprinted with
permission of the publisher.
vehicle control group is often required when the test material is admin-
istered by gavage (World Health Organization, 1978). If the toxic
properties of the vehicle are not well documented, both negative and
vehicle control groups may be recommended (Federal Register, 1979; Page,
19772?).
The use of "positive controls," animals treated with a chemical of
known toxic or carcinogenic potential utilizing the same or a very
similar test design, is occasionally recommended for chronic toxicity or
carcinogenicity tests (Page, 1977Z?). In chronic tests, its usefulness
is not well established (Benitz, 1970), and it is only infrequently
suggested in order to detect borderline effects and minimal enhancement
of normal pathological or age-related conditions (Federal Register,
1979; National Academy of Sciences, 1977).
For carcinogenicity tests, positive controls can provide more
information, including: (1) establishing the test animals' ability to
respond to a carcinogenic insult; (2) helping detect genetic drift; (3)
providing baseline information on new test species or strains; (4)
revealing any accidental inclusion of extraneous factors that could
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308
affect the test; (5) providing an indirect evaluation of the laboratory
and its general bioassay program; and (6) assessing the relative carcino-
genic potential of the test chemical (Food Safety Council, 1978; Page,
1977Z?; World Health Organization, 1978). Despite all this potential
information, the applicability of positive controls is limited. Most
discussions suggest that it be applied only to series of analogous
chemicals and need not be done with every study (Food Safety Council,
1978; Page, 1977fc; Peck, 1974; U.S. Food and Drug Administration, 1971).
5.2.6 Age
The age at which animals are started on test is an important and
sometimes overlooked consideration in testing for chronic toxicity and
carcinogenicity. Because it is a basic principle of chronic toxicity
and carcinogenicity testing that test animals should be exposed to the
test material for a major portion of their life span, it is generally
agreed that animals should be put on test at the earliest practical age
(National Academy of Sciences, 1977). In most published screening
studies for carcinogenesis, weanling or immediately postweanling rats
and mice have been used (Loomis, 1974; Magee, 1970; Ministry of Health
and Welfare Canada, 1975; National Academy of Sciences, 1977; World
Health Organization, 1978). Effects of the age of the test animal on
its response to carcinogenic challenge are well documented, and the
remainder of this section will be devoted to a discussion of these
effects.
The use of weanlings or immediately postweanlings permits testing
under a variety of conditions not present in mature animals (e.g.,
during periods of active protein synthesis, cellular proliferation, and
sexual maturation). In all of these physiological stages the weanling's
response to carcinogens is expected to be more acute than that of adults
(Weisburger and Weisburger, 1967).
Numerous studies have demonstrated that infant, newborn, and even
in utero rodents may have greatly increased susceptibilities to certain
carcinogens compared with weanlings. For example, Pietra, Spencer, and
Shubik (1959) observed a 32% incidence of malignant lymphomas in 11- to
24-week-old Swiss mice following a single 30-iig subcutaneous dose of
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309
9,10-dimethyl-l,2-benzanthracene administered at age 12 h or less. Sim-
ilarly, Kelly and O'Gara (1961) induced pulmonary tumors in essentially
all 18-h-old albino mice treated subcutaneously with 0.02 mg dibenz(o,7z)-
anthracene or 0.011 mg 3-methylcholanthrene.
Many other workers have also demonstrated the extraordinary sensi-
tivity of newborn mice to certain carcinogens (Axelrad and van der Gaag,
1962; Berenblum, Boiato, and Trainin, 1966; Chieco-Bianchi et al., 1963;
De Benedictis et al., 1962; Doell and Games, 1962; Flaks, 1965, 1966;
Gargus, Paynter, and Reese, 1969; Kaye and Trainin, 1966; Klein, 1963;
Liebelt, Liebelt, and Lane, 1964; Liebelt, Yoshida, and Gray, 1961;
Nishizuka, Nakakuki, and Sakakura, 1964; Roe, Mitchley, and Walters,
1963; Toth, Magee, and Shubik, 1964; Toth, Rappaport, and Shubik, 1963;
Vesselinovitch and Mihailovich, 1966; Vesselinovitch, Milhailovitch, and
Itze, 1970; Vesselinovitch et al., 1972, 1975; Vesselinovitch, Rao, and
Milhailovitch, 1975). Similar and related studies have been performed
with hamsters (Lee, Toth, and Shubik, 1963) and rats (Baba and Takayama
1961; Howell, 1963; Toth and Shubik, 1963). Many of these studies were
critically reviewed by Toth (1968) or Delia Porta (1968).
In view of the apparently accentuated sensitivity of newborn animals
to selected carcinogens, some early workers believed that a relatively
quick and sensitive screening test for carcinogens might be achieved by
exposing newborn animals to single doses of the test material (Delia
Porta and Terracini, 1969; Epstein, Andrea, and Jaffe, 1967; Gorrod,
Carter, and Roe, 1968; Roe, Carter, and Adamthwaite, 1969; Roe, Rowsen,
and Salaman, 1961; Toth, 1968). However, subsequent research indicated
such exposures could be more complex than they first appeared. For
example, treatment with #-hydroxy-#-2-fluorenylacetamide produced no
detectable tumors in infant rats but did induce tumors in weanlings
under similar conditions (Weisburger et al., 1970). Also, dimethylnitro-
samine induced renal adenomas in mice exposed as adults, but not in
those exposed as newborns (Terracini et al., 1966). In retrospect, it
became apparent that since newborns differ from weanlings or adult
animals in metabolic capability, viral susceptibility, and hormonal
status, as well as other anatomical and physiological characteristics,
it is illogical to expect identical responses from each of these age
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310
groups (Ministry of Health and Welfare Canada, 1975; Rice, 1976). It is
reasonable to expect newborns to respond more sensitively than adults to
ultimate carcinogens because of reduced immunological and hormonal
competence. But because of the lack of well-developed metabolic capabil-
ities, responses of such animals to proximate carcinogens that require
metabolic activation will obviously be deficient or absent compared with
more mature animals (Page, 19772?). It is thus clear that negative
results from tests involving newborn animals cannot be the sole basis of
evaluating the carcinogenicity of a test material. Indeed, Roe (1975)
even questioned the wisdom of accepting positive results from such
experiments.
In principle, test sensitivity and age-related effects should be
maximized if animals are exposed to the test material during all prenatal
and postnatal phases of their lives (Munro, 1977; Page, 1977fc; U.S. Food
and Drug Administration, 1971). Such a procedure requires treatment of
the pregnant mother and continuing postnatal exposure of the offspring.
Such a bigenerational exposure was strongly recommended as a routine
approach to carcinogenesis testing by several national and international
authorities following the discovery of vaginal adenocarcinomas in daugh-
ters of women who had received diethylstilbestrol during pregnancy
(Greenwald et al., 1971; Herbst, Ulfelder, and Poscanzer, 1971; Munro,
1977). Furthermore, several subsequent studies showed that prenatal
exposures to certain known carcinogens in fact result in high incidences
of tumors in offspring (Andrianova, 1971; Goerttler and Lohrke, 1977;
Mohr, 1973; Spatz and Laqueur, 1967; Svenberg et al., 1972; Tomatis et
al., 1971; Turusov et al., 1973). Ivankovic (1973) reported transpla-
cental induction of tumors in rats with ethylnitrosourea. Five to
eighty mg per kg of body weight of the compound given as a single dose,
intravenously or orally, on day 13 to 23 of pregnancy produced a signif-
icant yield of tumors of the nervous system in the offspring. However
the 5-mg/kg dose was ineffective in adult rats observed throughout their
entire life span. In fact, a notable yield of tumors in the nervous
system of adults was obtained only when 140 to 200 mg/kg were given,
thus demonstrating increased sensitivity of the fetus to carcinogenesis.
-------
311
However, routine testing for carcinogenesis through prenatal and bigen-
erational exposures is complicated by several factors that have not yet
been adequately resolved. For example, dose determination and regulation
for animals in utero is uncertain, and tolerances are variable and age
dependent (Vesselinovitch, 1973). Also, many substances likely to be
tested as carcinogens are teratogenic and may jeopardize the outcome of
the cancer bioassay through reproductive deficiencies unless doses are
carefully selected and timely administered. Furthermore, certain classes
of compounds likely to be tested for carcinogenicity, such as chlorinated
aliphatic and aromatic hydrocarbons, tend to accumulate in body fat and
liver and may interfere with normal reproductive processes even though
they are not strongly teratogenic. In an effort to minimize such repro-
ductive interferences, some authorities recommend limiting in utero
exposures to the second half of pregnancy when organogenesis is complete
(Golberg, 1974); otherwise, teratogenicity studies would have to be done
and nonteratogenic doses would have to be used for in utero studies.
However, no generally accepted guidelines exist with respect to in utero
and bigenerational exposures for carcinogenesis testing (Munro, 1977).
Except for special studies, such as effects of certain food additives,
pesticides, and drugs on pregnant women (U.S. Food and Drug Administra-
tion, 1971), bigenerational and in utero exposures are not generally
made a part of routine carcinogenesis testing today.
5.2.7 Sex
Current practice calls for the use of equal numbers of male and
female test animals in assays for chronic toxicity (Federal Register,
1978; Food Safety Council, 1978; National Academy of Sciences, 1977;
World Health Organization, 1978), although early workers questioned the
need for equity (Barnes and Denz, 1954). Essentially all test guide-
lines require that both sexes of the test species be used in carcino-
genicity assays (Page, 1977i>). However, female mice are preferred by
some researchers because they are less aggressive and survive better
than males; in some instances, male animals may be preferred when females
are needed for breeding. In the following section, data will be presented
that illustrate sex-related differences in the response of animals to
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312
chemical carcinogens and the effects certain intrinsic and extrinsic
factors of the animal test system have on these differences. Comparisons
will be made of the responses of male and female animals to chemicals
within selected chemical classes: polycyclic hydrocarbons, nitroso
compounds and amines, and halogenated hydrocarbons.
Akamatsu and Barton (1974) compared the neoplastic response of both
sexes of five strains of mice that had received 1 mg of 3-methylcho-
lanthrene intragastrically. Tumor incidence varied with strain and, in
some cases, sex. Sixty-seven percent of the males of the BTO strain
developed gastric tumors while only 30% of the females did, and more
males (25%) of the C3H/HeOs strain had gastric tumors than did the
females (12%). The C57BL/60s females had an increased incidence of
lymphomas, 25%, while only 11% of the treated males responded. More
females of the BALB/cOs strain developed pulmonary tumors (62%) than did
the males (50%); methylcholanthrene-treated males in all five strains
developed amyloidosis more often than did the females. Females of two
strains of mice (BTOs and C3H/HeOs) had a high incidence of spontaneous
mammary tumors, which was not increased by methylcholanthrene; however,
strains with low mammary tumor incidence (BALB/cOs and C3H/HeOs), treated
with methylcholanthrene, developed statistically significantly more
mammary tumors than did their untreated controls. The C57BL/60s females,
and males of all strains, did not develop any mammary tumors. The data
(Table 5.13) in this experiment suggest that differences in tumor response
of mice to systemic administration of methylcholanthrene are sex related
and that sex-related differences are strain dependent.
Wistar rats given daily intragastric instillations of 3-methylcho-
lanthrene for 9 weeks (for a total of 270 mg) also displayed distinct,
sex-related differences in neoplastic responses (Gruenstein, Meranze,
and Shimkin, 1966). Results of the study are summarized in Table 5.14.
Of 122 treated females, 115 (94%) developed 120 total neoplasms. Of
these, 117 were malignant. Of 81 treated males, 23 (28%) developed 26
malignant neoplasms. The difference in the tumor incidences of male and
female rats can be accounted for mainly by the difference in the numbers
of mammary carcinomas (87% of the females, 12.4% of the males). However,
16% of the males developed benign and malignant cutaneous tumors, while
-------
Table 5.13. Systemic tumor induction in mice with 3-methylcholanthrene by the intragastric route
Strain
BTO
Treated0 male
Untreated*5 male
Treated female
Untreated female
C57B1/60
Treated male
Untreated male
Treated female
Untreated female
BALB/cO
Treated male
Untreated male
Treated female
Untreated female
C3HeB/0
Treated male
Untreated male
Treated female
Untreated female
C3H/HeO
Treated male
Untreated male
Treated female
Untreated female
Number
of
animals
51
93
20
174
85
90
20
40
36
18
42
104
70
142
32
193
24
253
43
166
Tumor incidence (X)
Gastric
67
0
30
0
15
0
20
0
11
6
14
1
34
0
38
0
25
0
12
0
Skin
4
2
5
1
3
0
5
0
3
0
3
4
0
0
6
0
9
0
0
0
Lymphomas
0
4
5
2
11
6
25
3
20
6
31
13
5
5
3
6
4
0
9
1
Hepatomas
4
11
0
1
1
1
0
0
3
0
3
2
3
18
0
0
4
34
0
0
Pulmonary
tumor
4
2
0
1
1
0
5
0
50
0
62
1
3
0
0
3
4
0
0
0
Bladder
tumor
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Mammary
tumor
85
70
0
0
24
12
6
2
93
96
Ovarian
tumor
0
2
0
0
5
4
11
O
0
0
Amyloidosis
28
19
25
2
13
2
10
3
6
0
0
0
11
0
0
0
4
0
0
0
Mice
with
tumors
67
19
95
76
35
7
45
3
50
11
81
36
29
22
56
13
46
34
96
96
aAll treated animals received 1 mg 3-methylcholanthrene in olive oil.
Controls received olive oil alone.
Source: Adapted from Akamatsu and Barton, 1974.
co
!-•
co
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Table 5.14. Influence of sex on response to carcinogens
Species
Wistar rat
Fischer rat
Lister rat
B6C3F} mouse
C3AF} mouse
Buffalo rat
C3H/HeN
mouse
C57BL/6JN
mouse
Albino mouse
Age at start
of experiment
6 weeks
3-6 months
2-3 months
1 day
15 days
42 days
1 day
15 days
42 days
1 day
15 days
42 days
1 day
15 days
42 days
4 weeks
12 weeks
24 weeks
52 weeks
8 weeks
8 weeks
8 weeks
Chemical
3-Methylcholan-
threne
3-Methylcholan-
threne
Dimethylbenz-
anthracene
Benzo(a)pyrene
Benzo(a)pyrene
Benzo(a)pyrene
Benzo (a) py rene
Diethylnitros-
amine
4-Hydroxyamino-
quinoline 1-
oxide (4HAQO)
4-HAQO
4HAQO
Dose
270 mg
(total)
1.6 mg/week
o os)0'5*
°-°T5Ji.o%
^2.0%
0 OS)0*5*
V • U Jl « ABl
. >1.0%
mL)2.0%
75 ug/g body
weight
150 yg/g body
weight
75 pg/g body
weight
150 ug/g body
weight
0.0114%
1 mg
1 mg
1 mg
Route
Intragastric
instillation
Skin painting
Salivary gland
Salivary gland
Intraperitoneal
Intraperitoneal
Intraperitoneal
Intraperitoneal
Diet for 26
weeks
Subcutaneous
Subcutaneous
Subcutaneous
Tumor
type
Sebaceous
gland
Cutaneous
Skin
Carcinoma
Sarcoma
Liver
Liver
Liver
Liver
Total
Sarcoma
Sarcoma
Sarcoma
Tumor
incidence „ ,
Reference
Male
18%
16%
Female
0.8% Gruenstein, Meranze,
and Shimkin, 1966
0.8%
No difference Zacheim, 1964
18%
75%
83%
91%
75%
73%
55%
60%
13%
81%
58%
9%
34%
27%
0%
46%
23%
3%
60%
54%
33%
0%
40%
10%
11%
0% Glucksmann and
33% Cherry, 1971
71%
49%
70%
100%
7% Vesselinovitch et
7% al., 1975
0%
18%
7%
0%
2%
2%
0%
2%
2%
0%
21% Reuber, 1976
38%
23%
0%
20% Shirasu, 1965
10%
9%
-------
Table 5.14 (continued)
Species Afe at s*a" Chemical
K of experiment
Holtzman
Fischer rat
Wistar rat
B6C3Fi mouse 6 weeks
Osborne- 3-4 months
Mendel rat
Osborne- 3-4 months
Mendel rat
Osborne- 3-4 months
Mendel rat
Wistar rat , 12 hours
ACX rat
Intact 2-4 months
Castrate
Castrate-
hormone
Ethionine
Ethionine
Ethionine
Benzidene
hydrochloride
p-Dimethylamino-
benzene-1-azo-
1-naphthalene
p-Dimethylamino-
azobenzene
p-Diraethylamino-
benzene-1-azo-
2-naphthalene
p-Dimethylamino-
azobenzene
2-Diacetylamino-
fluorene
Dose Route
Diet for 7.5
months
Diet for 7.5
months
Diet for 7.5
months
50 ppm Diet
150 ppm Diet
0.075% Diet
0.060% Diet
0.075% Diet
1.2 mg Subcutaneous
166-217 mg/kg Diet
body weight
Tumor
type
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Tumor
incidence
Male
60%
100%
100%
6%
44%
75%
85%
98%
Female
25%
90%
100%
26%
94%
17%
19%
100%
No difference
60%
33%
29%
12.5%
0%
86%
Reference
Farber, 1963
Vesselinovitch, Rao,
and Mihailovicch,
1975
Mulay and O'Gara,
1959
Baba and Takayama,
1961
Morris and
Ferminger, 1956
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316
the incidence of those tumors in females was less than 1%, and male rats
developed more sebaceous gland tumors (18% incidence) than females
(<0.8%). Other types of tumors were induced in this study, but no other
sex differences were obvious in the responses. The cutaneous tumor data
were at variance with the results of an earlier experiment in which
Zackheim (1964) studied the cutaneous response of several strains of
rats to skin painting with methylcholanthrene, anthramine, and 7,12-
dimethyl(a)benzanthracene. Although he reported differences in types of
skin tumor that were related to the chemicals administered, no significant
strain or sex-related differences were observed. Gruenstein et al.
suggested that the sex differences seen in their experiment were influenced
by route of administration. In Zackheim's experiment the route was
external (skin painting), whereas the route used in the Gruenstein et al.
study was internal (intragastric).
Glucksmann and Cherry (1971) reported that, when low concentrations
of 0.5% or 1.0% 9,10-dimethyl-l,2-benzanthracene were injected into rats
locally in the salivary gland, about twice as many sarcomas and carcinomas
were induced in males as in females (Table 5.14). These effects were
confirmed when the administration of female hormones, estrogens, to
males reduced the number of dimethylbenzanthracene-induced tumors by one-
half and the administration of a male hormone, testosterone, doubled the
number of tumors in carcinogen-exposed females. However, when 2% dimethyl-
benzanthracene was injected, the sex difference disappeared, suggesting
that the sex differences observed were related to carcinogen dose and
were therefore quantitative rather than qualitative.
In an experiment designed to test the effects of age, sex, and
strain on tumor induction with benzo(a)pyrene, two strains of mice of
various ages were injected intraperitoneally, with one dose of the carcin-
ogen per mouse (75 or 150 yg per g of body weight) (Vesselinovitch et
al., 1975). The life span study revealed specific effects associated
with each of the modifiers (Table 5.14). Sex-influenced differences
were most prominent in induction of liver and lymphoreticular tumors^.
Male rats of both strains developed significantly more liver tumors than
females, whereas females of both strains developed more lymphoreticular
tumors than the males. As the animals grew older, the response of the
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317
liver to the carcinogen decreased, the differential between the two
sexes decreased, and the sex response therefore appeared to be related
to age.
Reuber (1976) examined the effects of age and sex on the induction
of preneoplastic and neoplastic lesions in the esophagus of Buffalo
strain rats with diethylnitrosamine. Starting at 4, 12, 24, and 52
weeks of age, the animals were fed a diet containing 0.0114% diethyl-
nitrosamine for 26 weeks. At 26 weeks, male rats that had been 4 weeks
old when the treatment was started had a higher incidence of carcinomas
of the esophagus (60%) than females of the same age group (21%). Among
animals that began receiving treatment at 12 weeks of age, the differ-
ence between the sexes was slight by 28 weeks; in those that received
the first treatment at 24 weeks, tumor incidence between the two sexes
was practically the same by 36 weeks. The differences in the responses
of the males and females diminished with increasing age. This finding
supports the previously cited data of Vesselinovitch et al. (1975). See
Table 5.14.
As in the experiments of Akamatsu and Barton (1974) with methylcho-
lanthrene, the strain of test species was shown by Shirasu (1965) to
influence the difference in sex response of mice to 4-hydroxyaminoquin-
oline-1-oxide. Results of the experiment are summarized in Table 5.14.
Nine months after starting subcutaneous injection of 1 mg of the carcin-
ogen (divided into 10 weekly injections), 40% of the male mice of the
C3H/HeN strain had developed sarcomas at the injection site, while only
20% of the females developed the same type of tumor. Other strains
tested developed sarcomas with incidences of 9% to 40%, but no sex-
related differences were seen. Papilloma development appeared to be sex
related in the C3H/HeN strain (papillomas in 40% of the females and 0%
of the males), in the C57BL/6JN strain (papillomas in 0% of the females
and 20% of the males), and in a noninbred albino strain (papillomas in
9.1% of the females and 0% of the males). None of the females and only
10% of the males of the C57BL/6JN strain had leukemia. Subcutaneous
injection of 4-nitroquinoline-l-oxide produced sarcomas in 10% of the
C3H/HeN males but none in the females.
One other experiment that showed sex-related differences in response
to amino compounds was that of Farber (1963), who observed the response
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318
of Holtzman rats to hepatocarcinogenesis with ethionine. Feeding the
chemical in the diet for 7.5 months produced liver cancer in 60% of the
males and in 25% of the females. Almost 100% of both sexes of two other
more sensitive strains (Fischer and Wistar) tested in the same experiment
developed liver cancers (Table 5.14).
Benzidine dihydrochloride, administered to mice continuously in the
diet at doses of 50, 100, and 150 ppm from the 6th to the 90th week of
age, induced liver tumors at dose-related incidences of 6% to 44% in
males and 26% to 94% in females (Vesselinovitch, Rao, and Milhailovich,
1975). When the carcinogen was administered intermittently instead of
continuously, the sex difference was abolished (Table 5.15). Regardless
of the schedule of administration, males developed more tumors of the
Harderian gland than females. Lung adenomas were found in more males
(23%) than females (7%), but only when feeding was intermittent. When
the animals were fed the carcinogen twice (at 7 and then at 27 days of
age), 66% of the males and none of the females developed liver tumors,
whereas feeding at 42 and 62 days produced liver tumors in only 4% of
the males and, again, none in the females. These data demonstrate the
influence of age on tumor induction in males in particular.
Mulay and O'Gara (1959) demonstrated that Osborne-Mendel male rats
fed p-dimethylamino-benzene-1-azo-l-naphthalene (DAN) and p-dimethyl-
aminoazobenzene (DAB) were more sensitive to liver tumor induction than
females. (See Table 5.14.) Daily administration of DAN induced liver
tumors in 75% of the males and in 17% of the females, whereas DAB induced
liver tumors in 85% of the males and 19% of the females. Male and
female mice fed p-dimethylaminoazobenzene-l-azo-2-naphthalene (DA-2-N)
responded with the same incidence of liver tumors (98% and 100%), but
the tumors appeared more slowly in females, suggesting that they were
more resistant to DA-2-N than were males. Baba and Takayama (1961), on
the other hand, were unable to demonstrate sex differences in Wistar
rats injected subcutaneously with DAB. The investigators suggested that
if the experiment had been stopped on the 200th day, instead of on day
380, hepatoma incidence would have differed greatly between the two
sexes, as induction time was longer for females. They also speculated
that if the doses of carcinogen had been larger the difference in sex
might have been obscured. The authors concluded that in DAB carcino-
genesis sex of the animal does not affect the transformation of normal
-------
Table 5.15. Comparison of continuous versus intermittent administration of BZ-2HC1 on development of
liver, Harderian gland, and lung
Dose
(ppm)
50
100
Sex
M
F
M
F
Liver
Continuous
Ratio0
3/50
13/50
11/50
32/50
%
6
26
22
64
tumors
Harderian gland tumors
b
Intermittent
Ratio
3/75
4/75
12/75
17/75
%
4
5
16
23
Continuous
Ratio
9/50
3/50
18/50
3/50
%
18
6
36
6
Intermittent
Ratio
8/75
2/75
11/75
5/75
%
11
3
15
7
Lung adenomas
Continuous
Ratio
2/50
2/50
3/50
2/50
%
4
4
6
4
Intermittent
Ratio
17/75
5/75
19/75
4/75
%
23
7
25
5
Animals were given food containing specified amounts of BZ«2HC1.
Mice received BZ-2HC1 stomach intubation in amounts equivalent to specified dosages; mice were intubated twice weekly. The
amounts at each treatment were 0.5 mg (corresponding to 50 ppm series) or 1.0 mg (corresponding to 100 ppm series) per animal at
treatment.
^Number of mice bearing specified tumors per number of BZ*2HCl-treated animals.
Source: Adapted from Vesselinovitch, Rao, and Mihailovitch, 1975. Reprinted with permission of the publisher.
CO
M
vo
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320
cells to malignant ones, but that it does affect proliferation of the
cells and thus appearance of the cancer. This supports the opinion of
Glucksmann and Cherry (1971) that sex-related differences in tumor
induction are quantitative rather than qualitative.
Morris and Ferminger (1956), using 2-acetylaminofluorene, induced
tumors in male and female rats (castrated and normal) that, in some
cases, were treated with sex hormones. Most hepatomas were confined to
the livers of intact males and of castrated females treated with testos-
terone propionate. Intact females, castrated males, castrated females,
and castrated males treated with diethylstilbesterol were less suscept-
ible (Table 5.14). Therefore, it appeared that under those experimental
conditions the presence of androgen was important for the development of
hepatomas in the rat. Estrogen did not seem to have a protective effect
against tumor development.
Weisburger (1977) tested several halogen compounds for carcinoge-
nicity in B6C3Fj mice and in Osborne-Mendel rats. Some of the sex differ-
ences seen after 78 weeks of dosing by gavage were:
1. Chloroform-treated male rats had an increased incidence of kidney
tumors, and females developed more thyroid tumors.
2. l,2-Dibromo-3-chloropropane caused many mammary carcinomas in
female rats.
3. 1,2-Dichloroethane caused some increase in hepatocellular carcinomas
and stomach tumors in male mice and induced increased numbers of
mammary tumors in female rats.
4. lodoform increased thyroid tumors in male rats and hepatocellular
carcinomas in male mice.
Both sexes of mice have been found to be equally susceptible to the
hepatocellular effects of carbon tetrachloride (Andervont, 1958; Weisburger,
1977), whereas female Buffalo strain rats have appeared to be more
sensitive than males (Reuber and Glover, 1967).
5.2.8 Conclusions
The need for lifetime exposures in chronic toxicity and carcino-
genicity tests limits the choice of primary test animals to relatively
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321
short-lived rodents in all but a few exceptional cases. Rats are the
overwhelming choice of most investigators for chronic testing, with mice
and dogs distant second and third choices, respectively.
In addition to the primary test animal, most authorities recommend
the use of a second species in chronic toxicity and carcinogenicity
tests to reveal a broader range of toxic effects. Most, but not all,
authorities recommend a nonrodent species for chronic tests. Among
nonrodents, the dog is most frequently used, but other species may be
selected if their metabolic processes are thought to resemble those of
man more closely.
Rats, mice, and hamsters are generally selected for carcinogenicity
assays. No test animal has been found to be an ideal surrogate for man
in testing for long-term toxicity; however many species do respond to a
variety of toxic substances and are reliable subjects for testing.
Selection of the most appropriate species or strain for testing is
highly complex and can depend on sensitivity desired by the experimenter,
physiology, anatomy, and life span of the animal. Ideally, the test
animal that most closely resembles man would be chosen, but cost and
time requirements do not always allow this. The choice may be simplified
through such techniques as those involving the use of structure-activity
relationships, metabolism studies, and in vitro screening. These tech-
niques, in conjunction with standardized animal studies, could aid in
the efficient selection of the appropriate animal species. However,
literature searches revealed a distinct lack of experiments expressly
designed to demonstrate species differences in response to chemical
carcinogens. Thus, it was necessary in this document to make many
comparisons of nonstandardized data between experiments conducted in
different laboratories. There is a definite need for basic, standard-
ized animal experiments that allow for the comparison of the responses
of various species to chemical carcinogens or classes of carcinogens.
The number of animals used in chronic toxicity and carcinogenicity
tests represents a compromise between requirements for good statistical
precision and reasonable costs and work loads. Early investigators used
10 or fewer test animals per dose group, but most authorities now recommend
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322
using 50 rodents or 4 to 8 nonrodents per group. Equal numbers of male
and female animals are normally used.
Each long-term toxicity test should be evaluated with reference to
a concurrent control group composed of animals of the same species, age,
sex, and weight as the treated groups. These animals should be handled
identically to the test species except for treatment with the test mate-
rial. Each control group should contain at least as many animals as the
corresponding test group. Negative and vehicle control groups are com-
monly used in all long-term tests, but positive controls are more fre-
quently used in carcinogenicity testing.
General agreement exists among authorities on the need to start
animals on test at an early age to provide maximum exposure during
periods of active protein synthesis, cellular proliferation, and sexual
maturation, as well as adulthood. In most routine screening tests for
carcinogenesis, weanlings or immediately postweanling animals are used.
There is also general agreement that newborn animals are sometimes even
more sensitive to carcinogens than weanlings and adults; however, the
metabolic, hormonal, and immunological incompetence of prenatal animals
may induce responses uncharacteristic of more mature animals. Hence,
some responses from newborn animals, taken alone, may not be reliable
indicators of the carcinogenicity of the test material.
In principle, test sensitivity and age-related effects should be
maximized if animals are exposed to the test material during all prenatal
and postnatal phases of their lives. In practice, difficulties with
dose estimation or regulation and teratogenic or reproductive interfer-
ences of the test material so limit the effectiveness of this approach
that tests involving in utero and bigenerational exposures are performed
only infrequently. As a consequence, much more experience with these
methods will be required before their general application to carcino-
genesis testing can be considered. However, use of such tests may be
indicated whenever the chemistry or biology of the test compound, or its
pattern of use, suggests a high level of interaction with in utero,
infant, or preadolescent children.
The sex of the animal can influence the incidence, site, and latency
of tumors. The host environment, which is determined by factors such as
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323
sex, appears to regulate the growth of the induced neoplasm. The mecha-
nism of sex-influenced effects is not clear, and sex-related differences
cannot be explained on the basis of hormone effects alone or on the
basis of cell susceptibility. Homburger and Tregier (1960) described
studies in rodents in which tumors induced in males grew larger when
transplanted to males than when transplanted to females. These and
results of previous tests indicated to them that cells of the male and
female are equally susceptible to carcinogens but that the malignant
cells encounter a less favorable environment in the female than in the
male and that the tumors progress at a slower rate. Sex-related differ-
ences in the tumor response to chemical carcinogens can be altered by
factors such as strain, species, and age of the animal; route and schedule
of administration of the chemical; dose of the chemical; and duration of
the experiment. "Therefore, reliable testing of chemicals with unknown
characteristics would be virtually impossible using only one sex of
experimental animal.
5.3 ROUTES OF ADMINISTRATION
The mode of exposure of experimental animals to chemicals may
influence the results of chronic and carcinogenicity studies and should
closely simulate conditions under which human exposure would most likely
occur (Ministry of Health and Welfare Canada, 1975). Thus, the oral
route is preferred for testing compounds to which humans may be exposed
through ingestion (Ministry of Health and Welfare Canada, 1975; World
Health Organization, 1978). For gases, volatile industrial solvents,
and other air contaminants, inhalation studies are recommmended (Clark,
1977; Nettesheim and Griesemer, 1978). Section 5.3.1 provides an evalu-
ation of advantages and disadvantages of these exposure routes in tests
for chronic toxicity and carcinogenicity and includes a description of
other less common routes which have specific application in tests for
carcinogenicity. In Sect. 5.3.2 examples are presented to illustrate
differences or similarities observed in a given species following carcino-
gen exposure by different routes.
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324
5.3.1 Exposure Routes
5.3.1.1 Oral Route — Suitable for all species, the oral route of
exposure is used for testing compounds to which humans may be exposed
through ingestion. Oral administration may be effected by gavage, by
feeding in dietary mixtures, or by administration in drinking water.
All three methods are useful for long-term administration and permit
reliable quantitation if intake and body weight are measured routinely
(Ministry of Health and Welfare Canada, 1975). Sontag, Page, and
Saffiotti (1976) discussed the advantages and disadvantages of the three
oral routes.
The gavage method has the advantages of more effective hazard
control and better quantitation. The test agent can be freshly prepared
and, because less test agent is needed, less storage space is required.
Animal handling is increased, allowing frequent observation for clinical
symptoms. Gavage has certain disadvantages: (1) intake of the test
agent may be less than maximum, (2) a solvent is often required, (3)
mortality may be increased because of trauma, and (4) animals must be
closely matched by weight.
Feeding through diet mix and drinking water has other advantages
and disadvantages. Although these methods most closely simulate the
mode of human exposure and allow greater total intake, the quantity
ingested may vary, homogenicity and stability of the mix may be incon-
sistent, and palatability may be altered. Furthermore, the concentra-
tion of the test material in the diet must be altered weekly or biweekly
during the early part of the experiment to maintain a constant dose
level (in milligrams per kilogram of body weight), because toxicity is
related to dosage per unit of body weight and food consumption per unit
of body weight decreases with increasing animal age (Weil, 1973). Also,
potential exposure of workers who mix and dispense feed and water con-
taining toxic chemicals must not be overlooked.
Dietary feeding is generally recommended for adult animals (Weisburger
and Weisburger, 1967), and intubation techniques are reserved for new-
born and infant animals and for chemically unstable compounds (Weisburger
and Weisburger, 1967). Pills and capsules may be suitable for oral
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325
administration to large species. The Ministry of Health and Welfare
Canada (1975) suggested experimental use of the oral route of exposure,
in certain instances, even when it is not the primary mode of human
exposure. However, substitution of the oral route would be feasible
only if levels of the compound in the tissue are higher than those
produced following administration by the normal route for humans. In
any case, extrapolation from one route of exposure to another must be
approached with extreme caution.
5.3.1.2 Repiratory Routes — Experimental exposure of the respiratory
tract to chemical carcinogens is usually accomplished through inhalation
or by intratracheal instillation. These and two other specialized
techniques which are occasionally used for carcinogenicity studies
(pellet implantation and tracheal washing) will be discussed. The most
significant advantage of inhalation exposure is that it duplicates
conditions under which humans are exposed to air contaminants and it is
the most probable route of industrial exposure to toxic chemicals
(Nettesheim and Griesemer, 1978; Ministry of Health and Welfare Canada,
1975). The disadvantages of inhalation techniques, some of which add
difficulty to interpretation of results, were listed by Ministry of
Health and Welfare Canada (1975): (1) methods and equipment are complex
and expensive, (2) particle sizes must be exact, not to exceed 5 y, (3)
the respiratory anatomy of man and animals differs in several respects,
and (4) it is difficult to determine the actual dose inhaled.
Of the various species of experimental animals, the rat seems to be
most suited to inhalation studies. Size of the animal, economic consider-
ations, and similarities to man (demonstrated by the induction of squamous
cell carcinomas of bronchogenic origin) are desirable characteristics of
the rat model (Page, 19772?).
To circumvent the complexity of inhalation procedures, other useful
methods for direct exposure of the respiratory tract have been developed.
Intratracheal instillation of test material permits administration of a
controlled dose and can be used in cocarcinogenesis studies concomitantly
with inhalation exposure (National Academy of Sciences, 1975). In
addition, large particles and large doses can be administered, the nasal
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326
filter in rodents is bypassed, skin contamination is avoided, and there
is less risk to personnel from carcinogen exposure. Intratracheal
instillation is currently the method of choice for testing suspected
respiratory carcinogens (Nettesheim and Griesemer, 1978). Disadvantages
of the technique are site-to-site variability in deposition of the test
material and the formation of foci of chronic inflammation in the respi-
ratory tract. Hamsters appear to be the animal most used in experiments
requiring intratracheal injections. Size of the animals, their low
incidence of spontaneous respiratory infections, their low incidence of
spontaneous tumors, and their susceptibility to various carcinogens make
hamsters suitable for intratracheal instillation.
Implantation of carcinogen-containing pellets into respiratory
tissues of the rat produced squamous cell carcinomas in the bronchial
epithelium (Kuschner et al., 1957) and in the epithelium of heterotropic
tracheal transplants (Griesemer et al., 1977). In the pellet implant
system the carcinogen acts on a specific limited area determined by the
investigator, and dose-response relationships have been established.
The respective carcinogenicities of two occupational pollutants,
chromium compounds and polyurethane, have been detected using the bron-
chial implant. However, the acute and chronic traumas afforded by this
method are unavoidable and can be troublesome.
Schreiber, Schreiber, and Martin (1975) described the induction of
squamous cell carcinomas in the hamster using repeated localized tracheal
washings with a carcinogen. Tumors produced by this method are easily
detected (the animal shows respiratory distress as the tumor becomes
large) and may usually be diagnosed simply by dissecting the trachea
under the dissecting microscope (Nettesheim and Griesemer, 1978). This
relatively new exposure technique is complex and traumatic to the animal,
and little is known to date of its real potential.
5.3.1.3 Dermal Route — Of particular interest in carcinogenicity
studies, this route exposes the skin directly and may result in exposure
of the whole body following percutaneous absorption (Ministry of Health
and Welfare Canada, 1975).
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327
An obvious advantage of dermal application is the ease with which
the latency for cancerous or precancerous lesions can be established
(Page, 19772?). Also, the effective cutaneous dose can be smaller than
the effective oral dose, even when absorbed (however, the maximum dose
that can be given dermally is usually less than the maximum dose that
can be administered orally); tumors arise rapidly, and they are usually
multiple; the statistical analysis in skin carcinogenesis studies is
advanced; the method is well suited to the two-stage carcinogenesis
procedure; and the technique is useful for testing carcinogens that are
destroyed in the gut. As for disadvantages, the procedure is arduous
and expensive, the absorbed dose is not accurately known (whole body
exposure may occur when the test agent is licked off the skin), lesions
and infections caused by the vehicle may shorten the life span of the
animal, and it is unsuitable for chemicals requiring activation in the
gut (Ministry of Health and Welfare Canada, 1975).
Weisburger (1976) suggested that mice and rabbits are generally
more responsive to dermal tumorigenesis than are other species. Magee
(1970), on the other hand, concluded that there seemed to be no inherent
difference in the capacity of mouse and rat skin to undergo malignant
change. This conclusion was based partly on the results of Graffi,
Hoffman, and Schutt (1967), who induced skin tumors in mice, rats, and
hamsters by dermal application of tf-nitrosomethylurea. However, Fare
(1966) demonstrated that complete reliance on results of mouse skin
carcinogenesis studies could be misleading. An azo dye, 3-methoxy-4-
dimethylaminoazobenzene, when painted on the skin of ten male rats for
62 weeks, was a potent carcinogen, producing tumors in 100% of the
animals. But the carcinogen was found to be totally ineffectual when
painted on the skin of male and female mice for averages of 62 and 30
weeks, respectively.
5.3.1.4 Subcutaneous Injection — Subcutaneous injection is a
sensitive and frequently used mode of administration of chemical carcino-
gens (Page, 19772>). Doses can be accurately delivered, absorption is
slow, high concentrations in the blood are reached rapidly, and the
compound circulates without those changes that might occur in the gut
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328
following oral administration. Repeated injections can be time-consuming
and local reactions are likely to occur (Ministry of Health and Welfare
Canada, 1975), but the appearance of subcutaneous sarcomas at the site
of injection constitutes the most controversial characteristic of the
technique. The production of subcutaneous sarcomas has been an end
point in the testing of many compounds for carcinogenic potential. In
1941 Turner induced sarcomas in rats at the site of implantation of
bakelite. Oppenheimer, Oppenheimer, and Stout (1948) obtained similar
results using cellophane.
Grasso (1976), following extensive experiments performed in an
attempt to elucidate the mechanism of induction of subcutaneous tumors
at the site of injection or implantation, concluded that "a progressive
type of connective tissue reaction, which has been carefully characterized
histologically, is responsible for the malignant tumors that are produced
in the subcutaneous tissue of rats and mice by the following: (a)
solid implants, (2?) injection of hypertonic, acidic, or surface active
solutions, (0) injection of macromolecular substances or substances that
form local deposits." Thus, although the subcutaneous techniques are
still quite useful, caution is required in the interpretation of the
results of assays in which they are used.
Homburger and Tregier (1960) observed species differences in suscep-
tibility of hamsters, mice, rats, and monkeys to subcutaneously administered
3,4,9,10-dibenzopyrene. One monkey appeared to be resistant, but sarcomas
were induced in hamsters, mice, and rats. Also, strain differences were
observed among the mice tested.
5.3.1.5 Other Exposure Techniques — Intraperitoneal and intravenous
injections and bladder implantation of wax pellets are less frequently
used techniques.
The intraperitoneal route of administration is convenient for
single-dose exposure to a carcinogen, the liver being the first organ
encountered. With procarcinogens the tumor spectrum induced is the same
as by oral administration (Weisburger, 1976), and injection may be done
repeatedly if the carcinogen is soluble and if there is no vehicle
accumulation. Intraperitoneal injection does not resemble the usual
routes of human exposure.
-------
329
Intravenous injection is a rapid and efficient means of delivery of
test materials to various organs. However, it is an unlikely route for
human exposure and is not well suited for repeated injections (Ministry
of Health and Welfare Canada, 1975).
Jull (1951) introduced the technique of surgical implantation of
wax pellets containing carcinogens into the bladders of mice. Since
then other investigators, including Bonser et al. (1952) and Allen et
al. (1957), have used the technique successfully for testing aromatic
amines, polycyclic hydrocarbons, saccharin, and other potential carcino-
gens. However, the U.S. Food and Drug Administration (1971) felt that
the number of chemicals tested comparatively by both bladder implantation
and more conventional methods was insufficient to provide sound inter-
pretation of results obtained by the technique. Therefore, any chemical
clearly carcinogenic by bladder implantation would be considered suspi-
cious but should then be tested by more conventional assays.
5.3.2 Carcinogen Exposure by Different Routes
5.3.2.1 Diethylnitrosamine in the Hamster ~ The effects of route
of administration on the carcinogenic action of diethylnitrosamine have
been tested in the Syrian hamster (Herrold and Dunham, 1963; Herrold,
1964a, 1964&). The routes tested were intragastric, intratracheal,
subcutaneous, intradermal, topical, and intraperitoneal. Results of the
experiment are summarized in Table 5.16. Irrespective of the route of
administration of diethylnitrosamine, tumors were induced in the trachea,
bronchi, nasal cavity, and liver of the hamsters. The frequency of
tumors in the nasal cavity was considerably higher after subcutaneous
administration of diethylnitrosamine than after administration by intra-
gastric or intratracheal routes (Herrold, 1964a). Although liver tumors
did not develop in animals injected intratradically, atypism of the
hepatic cells was observed (Herrold and Dunham, 1963). There was no
local effect on the skin or in subcutaneous tissue (Herrold, 19642?).
Transplacental transfer of the carcinogen was not demonstrated by Herrold,
but Mohr (1973) described extensive experiments in which he and his
associates induced tumors transplacentally with diethylnitrosamine.
-------
Table 5.16. Induction of tumors in the Syrian hamster with diethylnitrosamine
Route Number of
animals
Buccal pouch 10
Intradermal 19
Intraperitoneal 18
Topical 8
Dose
0.1 mg in saline
(total - 7.5 mg)
3.5 mg in water
(total - 70-84 mg)
2 mg in saline
(total - 32-56 mg)
Undiluted/' then 1:1
in water
Exposure
schedule
Twice a. week for
about 9 months
Once a week for
5-6 months
Once .1 week for
4-7 months
Once a week for
1 month then
twice a month
for 3 months
Trachea
0/10
19/19
17/18
6/8
Tumor incidence
Nasal
Anterior
0/10 0/10 0/10
10/19 3/19 10/19
4/18 4/18 5/15
2/8 4/8 6/8
cavity
Posterior
0/10
13/19
11/15
4/8
Reterencu
Kidney0
Milievskaja and
Klseleva, 1976
Herrold, 1964i
Herrold, 1964&
Herrold, 1964;
CO
LO
Pregnant female 3
subcutaneous
5, 8, or 10 mg in
water
1-2 days preced-
ing delivery
(single dose)
3/3
1/3
0/3
1/3
2/3
Herrold, 19642)
Subcutaneous
Intratracheal
Intragastric
15
11 female
14 male
15 female
13 male
2 mg in water
(total - 32-48 mg)
0.05 raL of a 1:14 ,
solution in water
0.4 mL of a 1:250
solution in water
Twice 6 week for
4-6 months
Once a week for
6 months
Twice a week for
7 months
NSU
11/11
14/14
15/15
13/13
NS
4/11
10/14
6/15
4/13
NS
0/11
0/14
10/15
12/13
14/15
3/9
1/13
6/11
4/9
0/11
0/14
7/15
5/13
Herrold, 1964j
Herrold and
Dunham, 1963
Herrold and
Dunham, 1963
Microscopic lesions that may represent an early stage in tumorigenesis.
Dose and schedule were changed after animals died during the first month of treatment.
°NS — tumors observed but incidence not specified.
Htoses administered intratracheally and intragastrically were approximately the same.
-------
331
Milievskaja and Kiseleva (1976) attempted to induce tumors in the
buccal pouch of the Syrian hamster with injections of diethylnitrosamine.
Doses of 0.1 mg per animal twice a week for about 9 months failed to
produce local or remote tumors. The small number of animals (ten) or
the route of administration constitutes possible reasons for the lack of
carcinogenicity of diethylnitrosamine in the experiment. The total dose
administered to the buccal pouch, however, was only one-tenth of the
intradermal dose required to produce tumors in hamsters (Herrold, 1964&);
therefore, the lack of effect could be attributed to insufficient dose.
5.3.2.2 Methylcholanthrene in the Mouse — Skin carcinomas have
been induced in mice painted with single large doses (0.1 mL of 0.6%
solution four times in an hour) or with repeated smaller doses (0.15%
two times per week for 30 weeks) of 3-methylcholanthrene (Bielschowsky
and Bullough, 1949; Shubik, 1950). Table 5.17 contains experimental
details for local tumor induction with methylchloanthrene.
When methylcholanthrene is painted on the skin of mice in doses too
low to produce tumors, croton oil, applied repeatedly, enhances the
appearance of tumors (Klein, 1952). The promoting action of croton oil
in skin carcinogenesis appeared to be a factor in the induction of
sarcomas in albino mice following intramuscular injection of methylcho-
lanthrene (Klein, 1951). Single injections of 0.024 or 0.004 mg of
methylcholanthrene in croton oil produced tumors in a shorter time and
with a higher incidence than those produced by the same doses of carcino-
gen injected alone. However, croton oil did not influence tumor number
or latency when injected intramuscularly with 0.002, 0.0008, or 0.0004
mg methylcholanthrene doses, which produced few or no sarcomas at the
site of injection (Klein, 1953).
Subcutaneous administration of polycyclic hydrocarbons has been
used primarily for the induction of connective tissue tumors. Epidermal
changes that occur with this route of exposure are not often described.
Bhisey and Sirsat (1975) monitored histological changes in the skin of
Swiss albino mice at various times after subcutaneous injection of
0.5 mg of 20-methylcholanthrene in thiophene-free benzene. A mild focal
epidermal hyperplasia developed, which led to development of squamous
-------
Table 5.17. Local tumor induction in mice with 3-methylcholanthrene by various routes of administration
Number
Route Strain of
animals
Skin painting Kreyberg 30
CF-1 28
DBA 34
35
Intramuscular Albino A 40
40
32
32
35
36
36
35
33
37
Methylcholanthrene
dose
0.1 mL of 0.6Z in
acetone
0.15% in acetone
0.5* in olive~oil
0.5% in olive oil
0.024 mg in olive
oil
0.024 mg in olive
oil and croton
oil
0.004 mg in olive
oil
0.004 mg in olive
oil and croton
oil
0.002 mg in olive
oil
0.002 mg in olive
oil and croton
oil
0.0008 mg in olive
oil
0.0008 mg in olive
oil and croton
oil
0.0004 mg in olive
oil
0.0004 mg in olive
oil and croton
oil
Exposure schedule
4 applications at
15-min intervals
Twice & week for
30 weeks
Once, followed by
application of
5* croton oil in
olive oil 1-3
times per week
for 53 applica-
tions
Once, with no
additional
treatment
Single injection
Single injection
Single injection
Single injection
Single injection
Single injection
Single injection
Single injection
Single injection
Single injection
Average
induction
time
10.5 weeks
Not clear
59 days
160 days
95 days
168 days
123 days
183 days
185 days
254 days
272 days
Tumor
incidence
7/30 (skin)
23/28 (skin)
27/34 (skin)
0/35 (skin)
20/40 (muscle)
40/40 (muscle)
9/32 (muscle)
22/32 (muscle)
5/35 (muscle)
3/36 (muscle)
1/36 (muscle)
1/35 (muscle)
0/33 (muscle)
0/37 (muscle)
Reference
Comments
Bielschowsky 3/7 carcinomas (par-
and Bullough, tial autopsy)
1949
Shubik,
Klein,
Klein,
Klein,
Klein,
Klein,
Klein,
Klein,
Klein,
Klein,
Klein,
Klein,
Klein,
1950
1952
1952
1951
1951
1951
1951
1953
1953
1953
1953
1953
1953
2 malignancies in
69 total tumors
(partial autopsy)
Fibrosarcomas (par-
tial autopsy)
Fibrosarcomas (par-
tial autopsy)
Fibrosarcomas (par-
tial autopsy)
Fibrosarcomas (par-
tial autopsy)
Fibrosarcomas (par-
tial autopsy)
Fibrosarcomas (par-
tial autopsy)
Fibrosarcomas (par-
tial autopsy)
Fibrosarcomas (par-
tial autopsy)
w
-------
Table 5.17 (continued)
Number „«.!._, u
Route Strain of Methylcholanthrene schedule
animals dose
Subcutaneous Swiss 24 0.5 tag in thio- Single injection
phene-free
benzene
Average T,,n,«r
induction . T^or a
. . incidence
time
13/24 (skin)
Sirsat, 1975
2/24 (muscle)
19/24 (subcuta-
neous connec-
tive tissue)
3/24 (skin)
Reference
Bhisey and
Sirsat,
1975
Comments
Squamous cell carci'
noma (partial
autopsy)
Rhabdomyosarcomas
(partial autopsy)
Fibrosarcomas (par-
tial autopsy)
Papillomas (partial
6/24 (skin)
autopsy)
Sebaceous cysts (par-
tial autopsy)
Intratracheal BC3Fj
DBA/2
Bladder Albino
implant of '
pellets
36 0.5 mg in 0.2X
gelatin
50 0.5 mg in 0.2%
gelatin
37 12.5% suspension
38 20% suspension
Once a week for
6 weeks
Once a week for 4
weeks
Continuous up to
4 weeks
Continuous up to
4 weeks
First mouse 31/36 (lung)
died in
4 weeks,
last in
24 weeks
3/18 (lung)
18/37 (urinary
bladder)
22/38 (urinary
bladder)
Nettesheim and
Haramons, 1971
Nettesheim and
Hammons, 1971
Bonser et al. ,
1963
Bonser et al. ,
1963
Squamous cell
carcinomas
Squamous cell car-
cinomas (22
animals were still
alive)
Carcinomas (partial
autopsy) , 12% of
controls developed
tumors
Carcinomas (partial
autopsy) , 4.8% of
controls developed
tumors
CO
co
CO
aNumber of tumor-bearing mice per total number of mice at risk.
-------
334
cell carcinomas. Papillomas, sebaceous cysts, rhabdomyosarcomas, and
fibrosarcomas also developed. Bhisey compared the overall lack of
epidermal hyperplasia seen in these experiments to the massive epidermal
hyperplasia thought to be a required step during neoplastic transformation
of mouse skin topically painted with methylcholanthrene. He concluded
that "when methylcholanthrene is administered subcutaneously to Swiss
mice, epidermal hyperplasia is not a prerequisite for epidermal tumori-
genesis."
Examples of specialized techniques for local tumor induction in the
respiratory tract and urinary bladder include intratracheal injection
and pellet implantation, respectively. Repeated intratracheal injections
of 3-methylcholanthrene have induced squamous cell carcinomas in the
respiratory tracts of mice (Nettesheim and Hammons, 1971). When DBA/2
and BC3Fj mice were injected weekly with 0.5 mg of 3-methylcholanthrene
for 4 and 6 weeks, respectively, 31 of the 36 BC3Fi mice developed
squamous cell carcinomas, the first appearing 4 weeks after exposure.
Tumor incidence was much lower in the DBA/2 strain. Methylcholanthrene-
impregnated (12% or 20% methylcholanthrene) wax pellets implanted in the
urinary bladder of albino mice induced local squamous cell carcinomas in
approximately 55% of the animals during 25 weeks of observation (partial
autopsy) (Bonser et al., 1963).
Systemic effects of methylcholanthrene in mice have been reported
following oral, intravenous, subcutaneous, and vaginal exposure. Akamatsu
and Barton (1974) fed, by gavage, 1 mg of 3-methylcholanthrene in olive
oil to five inbred strains of mice. Tumors were induced at various
sites, including skin, forestomach, liver, lung, and lymphatics (Table
5.13). Treated animals also developed amyloidosis, which was signifi-
cantly correlated with gastric neoplasms.
Strain A mice, highly susceptible to chemical induction of pulmonary
adenomas, developed an increased number (as compared with controls) of
lung tumors 4 weeks after intravenous injection of 0.05 to 1.5 mg methyl-
cholanthrene in horse serum and cholesterol (Table 5.18) (Shimkin,
1940). The incidence of tumors was 100% in 3 months. Subcutaneous
injection of 0.25, 0.5, and 1.0 mg of methylcholanthrene in lard induced
lung tumors in strain A mice, with 70%, 80%, and 91% incidence developing
-------
Table 5.18.
Incidence of pulmonary tumors in strain A mice after intravenous
injection of methylcholanthrene
Dosage
1.5 mg
Time
(weeks)
3
4
5
6
13
20
Number
of
mice
6
7
10
4
5
Number
with
tumors
of the
lungs
1
7
10
4
5
Average
number of
tumors
of the
lungs
1
12
22
55
74
Number
of
mice
9
10
10
10
18
6
0.5 mg
Number
with
tumors
of the
lungs
1
8
10
10
18
6
Average
number of
tumors
of the
lungs
1
5
14
25
30
47
Number
of
mice
7
10
15
0.1 mg
Number
with
tumors
of the
lungs
3
8
15
Average
number of
tumors
of the
lungs
2
4
11
Ul
Source: Adapted from Shimkin, 1940. Copyright 1940, American Medical Association.
Reprinted with permission of the publisher.
-------
336
by 3 months. Differences seen in carcinogenic potency of 20-methylcho-
lanthrene (in horse serum) and 1,2,5,6-dibenzanthracene (in cholesterol),
when 0.5 mg of each compound was injected intravenously, disappeared
when 0.5 mg of each was injected subcutaneously (Tables 5.19 and 5.20).
These results suggest that the subcutaneous route is less sensitive than
the intravenous route for lung tumor induction in strain A mice.
Table 5.19. Incidence of pulmonary tumors in strain A mice after
intravenous injection of 0.5 mg of methylcholanthrene or 0.5 mg
of dibenzanthracene
Methylcholanthrene
Time
(weeks)
3
4
5
6
Number
_ c
or
mice
9
10
10
10
Number
with
tumors of
the lungs
1
8
10
10
Average
number of
tumors of
the lungs
1
5
14
25
Dibenzanthracene
Number
of
mice
10
10
11
10
Number
with
tumors of
the lungs
0
3
10
10
Average
number of
tumors of
the lungs
0
1
3
8
Source: Adapted from Shimkin, 1940. Copyright 1940, American
Medical Association. Reprinted with permission of the publisher.
Table 5.20. Incidence of pulmonary tumors in strain A mice given
subcutaneously 0.5 mg of methylcholanthrene or 0.5 mg of
dibenzanthracene dispersed in 0.5 cc of horse serum and cholesterol
Methylcholanthrene
Time
(weeks)
3
4
5
6
Number
mice
10
10
10
10
Number
with
tumors of
the lungs
0
2
5
7
Average
number of
pulmonary
tumors
0
1.0
1.6
2.2
Dibenzanthracene
Number
mice
10
7
6
7
Number
with
tumors of
the lungs
0
0
2
5
Average
number of
pulmonary
tumors
0
0
1.0
2.0
Source: Adapted from Shimkin, 1940. Copyright 1940, American
Medical Association. Reprinted with permission of the publisher.
-------
337
Intravaginal exposure of ICR Swiss mice to 20-methylcholanthrene
gave rise to tumors remote from the site of application (Campbell, Yang,
and Bolton, 1965). During a 6-week period, doses totaling 1.5 to 5.5 mg
of 20-methylcholanthrene were administered, and the mice were observed
for life. Of 106 female mice, 23% developed lung adenomas (two mice had
adenocarcinomas), 12% developed genital tract cancer, and genital tract
dysplasia occurred in 27%. Six of the 24 mice with lung adenomas also
had cancer and/or dysplasia of the vagina and/or cervix uteri.
5.3.2.3 Aromatic Amines in Mice, Rats, and Rabbits — Bonser et al.
(1952) compared carcinogenesis of 2-naphthylamine and 2-amino-l-naphthol
hydrochloride in several species by several routes of administration.
Implantation of 2-amino-l-naphthol pellets (10% to 15% carcinogen)
induced metaplasia, papilloma, and carcinoma in the bladders of mice,
and subcutaneous injection (5 mg per 100 g of body weight) induced
sarcomas in both mice and rats. Implantation of 2-naphthylamine pellets
in the bladder and skin painting with the carcinogen failed to produce
bladder tumors in mice, but oral administration (100 to 300 mg/kg weekly)
induced hepatomas in mice and benign bladder tumors in rats and rabbits
(Table 5.21).
Inhalation of benzidine causes bladder cancer in man (Goldwater,
Rosso, and Kleinfeld, 1965) and has been shown to be carcinogenic in
certain animal species. Rats, after inhaling benzidine for 13 months,
developed leukemia, fibroadenomas, carcinoma of the mammary glands,
carcinoma of the male mammary gland, and hepatoma, but no bladder tumors
(Zabezhinskii, 1970). Oral administration of benzidine to rats induced
hepatomas and rectal and acoustic duct carcinomas (Spitz, Maguigan, and
Dobriner, 1950), male mammary gland carcinomas, and leukemia (see
National Institutes of Health, 1968-1973). The absence of tumors of the
urinary bladder of rats receiving benzidine by inhalation and by subcu-
taneous and oral administration led Zabezhinskii to conclude that the
character of action of aromatic amines is dependent mainly on the species
of the experimental animal and not the mode of administration of the
substance.
-------
Table 5.21. Tumor Induction with aromatic amines by various routes of exposure
Chemical/species
2-Amino-l-naphthol
hydrochlorlde
Mouse
Mouse, "stock"
Mouse, R III
Rat, albino
2-Naphthylamine
Mouse
Mouse, IF
Mouse, CBA
Rat, albino
Mouse, IF
Rabbit
Route of
exposure
Bladder
pellet
Subcutaneous
Subcutaneous
Subcutaneous
Bladder
pellets
Gavage
Gavage
Diet
Diet (low
protein)
Skin painting
Spoon feeding
Dose
1-2 mg per pellet
5 mg per 100 g
body weight
every 2 weeks
5 mg per 100 g
body weight
every 2 weeks
5 mg per 100 g
body weight
every 2 weeks
1-2 mg per pellet
5 mg every 2 weeks
in arachis oil
120 mg/kg body
weight twice
per week
160 mg/kg body
weight per week
310 mg/kg body
weight per week
Saturated solution
100 mg/kg body
weight per week
Duration of
exposure
(weeks)
22 to 28
30 to 39
Up to 80
Up to 80
Up to 80
39
Up to 72
Up to 89
Continuous
>90
Up to 99
Up to about
275
Tumor incidence
0/6 bladder tumor
1/8 bladder
adenoma
5/8 bladder
carcinoma
2/15 subcutaneous
tumors
3/15 leukemias
A/15 hepatomas
1/8 subcutaneous
tumors
1/8 leukemia
(hepatoma and
leukemia in
controls)
5/14 subcutaneous
tumors
0/8 bladder tumor
Cholangioma of
bile duct
13/23 hepatomas
11/26 hepatomas
3/15 bladder
papillomas
0/25 skin tumor
0/25 hepatoma
1/6 bladder
papilloma
Tumor
latency Reference
(weeks)
Bonser et al.,
About 30 1952
Bonser et al. ,
1952
Bonser et al. ,
1952
OJ
00
Bonser et al. ,
1952
Bonser et al. ,
1952
Bonser et al. ,
1952
Bonser et al . ,
1952
About 60 Bonser et al. ,
1952
About 60 Bonser et al.,
1952
Bonser et al . ,
1952
Bonser et al. ,
1952
-------
Table 5.21 (continued)
Chemical/species
Route of
exposure
Duration of
Dose exposure Tumor incidence
(weeks)
Tumor
latency
(weeks)
Reference
Benzidine
Rat, noninbred
Benzidine,
technical
Rat
Inhalation 4 hours/day, 5 80
days/week
Subcutaneous 15 mg/week
Life
Benzidine, pure
Rat
Benzidine sulfate
Rat
Subcutaneous 15 mg/week
Life
Subcutaneous 15 mg/week
Oral
15 mg/week
Life
Life
5/28 leukemias
1/28 hepatoma
2/28 mammary
tumors
8/233 hepatomas
0/233 bladder
tumors
7/385 gastric
tumors
54/233 auditory
canal tumors
6/152 hepatomas
0/152 bladder
tumors
32/152 auditory
canal tumors
1/153 hepatomas
0/153 bladder
tumors
16/153 auditory
canal tumors
1/37 hepatomas
0/37 bladder
tumors
2/37 auditory
canal tumors
52
Zabizhinakii,
1970
Spitz, Maguigan,
and Dobriner,
1950
Spitz, Maguigan,
and Dobriner,
1950
Spitz, Maguigan,
and Dobriner,
1950
Spitz, Maguigan,
and Dobriner,
1950
so
-------
340
5.3.3 Conclusions
It is recommended that the route of exposure used in animal studies
closely resemble the means by which man would encounter the chemical in
the environment. Nevertheless, it is sometimes necessary to substitute
other routes. Because of the variables inherent in any animal test
system — metabolic, biochemical, species, and strain differences, for
example — route-to-route extrapolations should be made with extreme
caution. Experiments designed specifically to define the role of route
of administration of chemicals to animals in carcinogenesis studies are
not easily found in the literature. However, it is obvious that route
of administration is a major modifying factor that can determine site,
histological type, and incidence of tumors.
5.4 DOSE AND DURATION
Dose selection is a critical aspect of chronic toxicity and carcino-
genicity testing. If the dose level is set too low, no effects will be
observed; if it is set too high, excess mortality rates will reduce the
test population too low for adequate statistical analysis. Consequently,
dose rates for long-term testing should be set only after data from
acute and subchronic tests have been analyzed. When possible, pharmaco-
kinetic data should also be considered (Golberg, 1975; Munro, 1977).
Massive doses that result in nonlinear pharmacokinetics should be avoided
unless such data reflect actual conditions of human exposure (Watanabe,
Young, and Gehring, 1977).
In early chronic toxicity testing, the primary purpose was the
demonstration of no-observed-effect level; more recently, data needed
for dose-response analysis have also been sought (Barnes and Denz, 1954;
Federal Register, 1979; National Academy of Sciences, 1977; Winstead,
1978). For the establishment of a no-observed-effect level, at least
three dose rates are required. The highest level should produce some
signs of toxicity without seriously altering normal physiological func-
tion or causing excessive lethality during the course of the experiment.
The lowest level should be a fraction of the high dose that is not
expected to produce evidence of toxicity. The remaining dose should be
-------
341
set at a level intermediate between the high and low dose (World Health
Organization, 1978). Dose levels should also be influenced by the
anticipated level of human exposure as well as the margin of safety that
may be desired in setting subsequent exposure standards (National Academy
of Sciences, 1977). If the determination of a dose-response curve is an
objective of the chronic toxicity test, more than three dose levels may
be required (Federal Register, 1979; Food Safety Council, 1978).
Although a test for carcinogenic potential might occasionally
entail only a single well-selected dose, tests for risk estimation or
determination of a no-effect level usually require several dose levels
(Page, 1977a). If more than one dose is used, the highest level should
be within the toxic range but should be consistent with prolonged survival
of the majority of the animals. The lowest dose selected ideally should
produce no real increase in tumor incidence over controls (Ministry of
Health and Welfare Canada, 1975). The recommendations of several agencies
regarding the controversial subject of dose selection in carcinogenicity
studies are presented in Table 5.22.
Weisburger (1976) emphasized the selection of the maximum tolerated
dose (dose at which mortality is low in an 8-week test and which may
depress weight gain 5% to 20%, with an optimal depression of approxi-
mately 10%). The rationale given by Munro (1977) for use of maximum
tolerated dose (MTD) includes: (1) when weak carcinogens are tested,
high doses for long periods of time increase chances of seeing statisti-
cally increased numbers of tumors within the life span of the animals
(since cancer induction in animals is both time- and dose-related); and
(2) increased confidence can be placed in negative data because the
chemical has been tested at the maximum dose level compatible with
normal or near normal survival rates. Munro also points out several
problems associated with the use of MTD: (1) the fact that MTD may
result in a 10% weight loss is considered to be inappropriate because it
may affect biochemical or physiological processes that make the animal
more or less susceptible to carcinogenic challenge; (2) metabolic over-
loading may result in changes in normal handling of the test compound;
(3) simultaneously induced toxicity due to the test compound may enhance
or diminish its carcinogenic activity, and further use of MTD favors
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Table 5.22. Summary of recommendations on dose selection
Agency
Recommendation
Health Protection Branch,
Department of National
Health and Welfare
Canada, 1973
International Union Against
Cancer, 1969
National Cancer Institute,
1976
U.S. Food and Drug }
Administration, 1971
"The highest dose level within the toxic
range but consistent with prolonged
survival. The lower levels should
permit . . . good health . . . until
tumors develop."
"All substances submitted for carcino-
genicity testing should be examined
at 3 or more dose levels."
"The MTDa is defined as the highest dose
of the test agent given during the
chronic study that can be predicted
not to alter the animals' normal lon-
gevity from effects other than car-
cinogenicity. The MTD should be the
highest dose that causes no more than
10% weight decrement, as compared to
the appropriate control groups and
does not produce mortality, clinical
signs of toxicity, or pathologic le-
sions (other than those that may be
related to a neoplastic response) that
would be predicted to shorten the ani-
mal's natural life span. Although de-
pressed weight gain may be a clinical
sign of toxicity, it is acceptable
when estimating the MTD. Since the
data may not always be easily inter-
pretable, a degree of judgment is
often necessary in estimating the
MTD."
"At several dose levels — one likely to
yield maximum tumor incidence."
nfD — Maximum tolerated dose.
Source: Adapted from Munro, 1977. Reprinted with permission of the
publisher.
very toxic compounds that can be used only at lower dose levels, thus
reducing the chance of tumor formation; (4) there is difficulty in
handling data when the criteria for MTD are not met; and (5) MTD doses
usually far exceed human exposure levels.
In tests for carcinogenicity a dose-response relationship should be
established if at all possible, but, because of the number of animals
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required, this is highly impractical with doses that produce lower tumor
incidences (Terracini, Magee, and Barnes, 1967). Also, the limited life
span of the animals may frustrate attempts to establish a low dose
response. The simple concept of additive effects of repeated doses is
not applicable to every situation. Page (19772?) suggested that the
effectiveness of increasing exposure might decrease on a per dose basis
for some chemicals especially if the body's ability to retain the chemical
becomes saturated. This is illustrated by the study of Schepers (1971),
who compared the effects of beryllium oxide in rats after: (1) continuous
inhalation exposure up to 12 months, (2) continuous exposure for 6
months and (3) brief exposure for 1 month to high concentrations of the
chemical. The rate of tumor formation was enhanced in animals of the
interrupted (6-months) exposure group, whereas those exposed continuously
for a long period and those exposed continuously for a shorter period
(but to a higher concentration) developed tumors at about the same slow
rate. Schepers postulated that the effect might be attributed to saturation
of the lung with beryllium oxide.
Carcinogens are usually administered every day (to simulate human
exposure) over the life span of the animal; some experiments may be
terminated when mice are 18 months old and rats are 24 months old (Ministry
of Health and Welfare Canada, 1975). Termination of studies must be
carried out with caution since some tumors tend to develop late in the
life of the animal. For example, Nelson, Fitzhugh, and Calvery (1943)
reported that rats did not begin to develop liver tumors until 18 months
after they had been placed on a diet containing selenium. The tumors in
11 of 53 animals arose in cirrhotic livers, while control animals had a
spontaneous hepatic tumor incidence of less than 1%.
In carcinogenicity testing the route of administration often deter-
mines the frequency of exposure necessary for tumor development. Weisburger
and Weisburger (1967) pointed out that for cutaneous exposure to strong
carcinogens a weekly or even a single treatment is satisfactory. More
frequent administration of weak carcinogens is recommended. Intraperi-
toneal injections require a repeated schedule because of the higher
absorption rate of the internal organs, while intravenous administration
allows no more than approximately eight injections.
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344
In chronic toxicity evaluations the test compound should also be
administered 7 days per week (Benitz, 1970). Treatment 5 days a week
results in a 28.6% decrease of exposure and a 48-h weekly recovery
period. The administration of the equivalent of seven doses during 5
days as recommended by some workers (Boyd, 1968) does not constitute a
treatment equivalent to daily dosing and may not produce identical
results (Baker and Alcock, 1965, as cited in Benitz, 1970; Hayes, 1967).
Apart from dose rate, length of exposure is the principal character-
istic that distinguishes chronic tests from other forms of toxicity
testing. Most investigators agree that chronic testing should extend
over a large portion of the test animal's life span (Boyd, 1968; Food
Safety Council, 1978; National Academy of Sciences, 1975, 1977; World
Health Organization, 1978); however, there is no consensus concerning
the length of this period. Conceptually, the duration of a chronic
toxicity test should reflect the time for the test material, or its
active metabolites, to accumulate to toxic levels in the organism, plus
the time required for the target organ or system to respond once the
toxic concentration is achieved (Lawrence, 1976). Generally speaking,
most effects of chronic testing become apparent within 3 or 4 months.
For example, in one study of the chronic effects of 11 drugs on rats,
dogs, and monkeys, all compounds produced an observable effect within
the first 2 weeks, and only one compound caused an additional effect
after the first 3 months (Peck, 1968). Similarly, in a chronic study of
46 compounds, the Ciba Drug Company noted all indications of toxicity
within the first 8 weeks of the test (Bein, 1963, as cited in McNamara,
1976). In this connection, McNamara (1976) stated, "If no effect occurs
in 3 months, there is a low likelihood that any effect will occur on
continued dosing for 1 year." Barnes and Denz (1954) asserted that
little useful information can be extracted from chronic toxicity tests
after the first 3 to 6 months. Because of this characteristically early
detection of most chronic effects, many investigators feel that the
duration of chronic tests need not exceed 6 months (Barnes and Denz,
1954; McCollister, 1974, as cited in McNamara, 1976; Paget, 1963, as
cited in McNaraara, 1976; Weil and McCollister, 1963; Zbinden, 1973).
However, this conclusion appears to have gained wider acceptance among
investigators dealing with drugs, to which humans may be exposed for
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relatively brief intervals, than with investigators dealing with occupa-
tional and environmental pollutants, to which the population may be
exposed for intervals approximating a lifetime. For assessing the
effects to humans of lifetime exposures of toxic compounds, some scien-
tists insist that lifetime or near-lifetime exposures of laboratory test
animals are required (Food Safety Council, 1978; Loomis, 1974; National
Academy of Sciences, 1975; World Health Organization, 1978). Unlike the
shorter acute or subchronic tests, lifetime exposures are said to reveal
toxic effects associated with (1) repetitive exposures to a test material
and (2) changes occurring during the aging process, such as altered
tissue sensitivity, changing metabolic and physiological capability, and
spontaneous disease (World Health Organization, 1978). The logic of the
latter argument appears unassailable; however, examples illustrating its
validity do not abound in the literature.
Only occasionally are chronic effects reported that were not observed
during the first 90 days of treatment (Page, 1977Z?) . Among more than
100 computer-selected publications on chronic toxicity examined during
the preparation of this chapter (see Bibliography), only two such papers
were noted: (1) Rosenkrantz et al. (1975) reported observing hyperglycemia
in rats only after 180 days of treatment with A9-tetrahydrocannabinol,
and (2) Verschuuren, Kroes, and Van Esch (1973) detected stridor in rats
fed tetrasul only after 4 months of treatment. In a similar examination
of the literature, McNamara (1976) considered 82 long-term toxicity
studies involving 122 test materials and 566 dose levels. Only three
f
compounds and 15 dose levels produced toxic signs after, but not before,
3 months exposure. It thus appears that in most instances all significant
toxic effects are indeed observed within 3 to 4 months, but that excep-
tions occasionally occur. Because it is not possible to determine a
priori the relative significance of the occasional chronic effect missed
in short-term tests, confidence in the validity of toxicity test results
can be maximized only by performing life span exposures on test animals,
even though longer tests are much more expensive and will provide no
additional useful information most of the time. Justification of the
test duration thus becomes a discretionary decision that must be resolved
on the basis of risk-benefit analysis.
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When lifetime or near-lifetime exposures of test animals are required
in chronic toxicity tests, the actual duration of exposure will naturally
vary with test animal. Many strains of mice or rats will survive 30
months or more with good care; consequently, these animals are usually
exposed for 24 to 27 months, beginning at weaning (Food Safety Council,
1978; National Academy of Sciences, 1977). The normal life spans of
other common test animals are considerably greater than that indicated
for mice and rats. Table 5.23 shows that chronic tests of even 2 years
duration represent only a small fraction of the life span of most nonrodent
laboratory test animals. Needless to say, these animals are only rarely
chosen for life span exposures. Among the publications examined during
the preparation of this chapter, no life span exposures of nonrodents
were found, but one 7-year dog study and one 43-month monkey study were
noted.
When a long-lived animal is chosen as a second species to mice or
rats in chronic toxicity tests, exposures amounting to at least 10% of
the projected life span of the animal are generally recommended (National
Academy of Sciences, 1977; World Health Organization, 1978). On this
basis, dogs used as a second species in chronic toxicity tests should
usually be exposed for at least 1 year. However, several investigators
have reported previously unobserved toxic effects in dogs after periods
of exposure greater than 1 year (Braun et al., 1977; Kaplan and Sherman,
1977; Weil et al., 1971). Consequently, some authorities recommend
exposing dogs longer, for periods of 2 to 7 years (Goldenthal, 1968;
Loomis, 1974; National Academy of Sciences, 1977). In the chronic
toxicity papers analyzed in this study the typical duration of exposure
of dogs used as a second species was 2 years.
In conclusion, dose selection is a critical aspect of long-term
testing. Dose rates should be set only after acute and subchronic test
data have been analyzed and pharmacokinetic effects have been considered.
At least three dose rates are required to establish a no-observed-effect
level. The highest level should produce some signs of toxicity without
seriously altering normal physiological function or causing excessive
lethality during the course of the experiment. The lowest level should
be a fraction of the high dose that is not expected to produce evidence
of toxicity. The remaining dose should be set at an intermediate level
-------
Table 5.23. Time relationships among drug exposure, life span, and time equivalents in man
of study
(months)
1
2
3
6 •?.•
12 -
24
Life
span
(%)
4.1
8.2
12
25
49
99
Rat
Human
equivalent
(months)
34
67
101
202
404
808
Life
span
(%)
1.5
3.0
4.5
9.0
18
36
Rabbit
Human
equivalent
(months)
12
24
36
72
145
289
Life
span
(%)
0.82
1.6
2.5
4.9
9.8
20
Dog
Human
equivalent
(months)
6.5
14
20
40
81
162
Life
span
(%)
0.82
1.6
2.5
4.9
9.8
20
Pig
Human
equivalent
(months)
6.5
14
20
40
81
162
Monkey
Life
span
(%)
0.55
1.1
1.6
3.3
6.6
13
Human
equivalent
(months)
4.5
9
13
27
53
107
Source: Adapted from Benitz, 1970.
to
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348
between the high and low doses. Dose levels should also reflect antici-
pated human exposures as well as the margin of safety that may be desired
in setting subsequent exposure standards. If a dose-response relation-
ship is a goal of the test, then more than three dose levels may be
required.
The test compound should be administered 7 days per week; treatment
5 days a week or administration of the equivalent of seven doses during
5 days is not equivalent to daily dosing and may not produce identical
results. In carcinogenicity studies, the exposure pattern is often
modified by the route of exposure.
Most authorities agree that chronic toxicity and carcinogenicity
studies should extend over a large portion of the test animal's life
span. However, there is no consensus concerning the length of this
period. Conceptually, the duration of a chronic toxicity or carcino-
genicity test should reflect the time for the test material, or its
metabolites, to reach toxic concentrations in the test animal, plus the
time required for the target organ or system to respond once the toxic
concentration is achieved. Generally speaking, most effects of non-
carcinogenic chronic testing become apparent within 3 or 4 months, and
many authorities feel there is only a low likelihood that additional
effects will be observed after 6 months of treatment. Nevertheless, a
few new effects are occasionally seen during longer periods of treatment,
and in carcinogenicity studies the chance for late development of long-
term effects is even greater. Because it is not possible to determine a
priori the relative significance of the occasional chronic effect missed
in short-term tests, confidence in the validity of toxicity test results
can be maximized only by performing life span exposures on test animals,
even though the longer tests are much more expensive and will provide no
additional useful information most of the time.
When lifetime or near-lifetime exposures of test animals are required
in chronic toxic and carcinogenicity tests, the actual duration of
exposure naturally varies according to species. The life spans of mice
and rats are about 30 months, and they are usually exposed for 24 to 27
months, beginning at weaning. The nominal life spans of nonrodents are
much longer — for example, rabbit, 66 months; dog or pig, 120 months;
and monkey, 184 months. Consequently, these animals are rarely subjected
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349
to life span exposures. However, a few literature examples have been
noted of 7-year exposures for dogs.
When long-lived animals are chosen as second species to mice or
rats in noncarcinogenic chronic toxicity studies, exposures of at least
10% of the projected life span of the animal are usually recommended.
On this basis, dogs used as a second species in chronic toxicity tests
should be exposed for at least a year. In the noncarcinogenic chronic
toxicity publications analyzed in this study the typical exposure of
dogs used as a second species was 2 years.
5.5 INTERIM SACRIFICE
Interim sacrifices are generally considered useful in the study of
long-term toxic effects, pathogenesis, and reversible changes. Timely
sampling may also provide valuable insights into specific clinical chem-
istry or organ function tests that may be required (Food Safety Council,
1978; National Academy of Sciences, 1977). For these reasons, some
authorities recommend the inclusion of serial sacrifices in chronic
toxicity tests (Fitzhugh, 1955, as cited in National Academy of Sciences,
1975; National Academy of Sciences, 1975; World Health Organization,
1978). However, other workers consider interim sacrifices a needless
reduction in an otherwise limited number of test and control animals
and recommend that interim sacrifices be avoided in chronic toxicity
tests (Benitz, 1970). Obviously, extra animals could be taken initially
to compensate for losses in size of the test and control groups through
interim sacrifices. Frequently, however, similar information is available
from previously performed interim sacrifices during subchronic tests or
from earlier pharmacodynamic studies. The need for interim sacrifices
in long-term studies thus varies from study to study and should be
reassessed with each new study.
In actual practice, few investigators appear to make extensive use
of interim sacrifices during chronic toxicity studies. Among more than
100 chronic toxicity publications examined during the preparation of
this chapter, only 12 reported interim sacrifices, and only eight studies
involved the examination of several animals during several intervals of
the test period. Significant observations derived specifically from
interim sacrifices were cited in only four of these reports.
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350
In conclusion, the use of interim sacrifices in chronic toxicity
testing is controversial. Some authorities recommend the practice as a
valuable aid in the study of toxic effects, clinical chemistry, organ
function tests, and pathogenesis. Other experts acknowledge the value
of interim sacrifices for the purposes stated, but consider the practice
a needless waste of the relatively limited number of test and control
animals typically available in chronic toxicity tests. Much of the
information provided by interim sacrifices in chronic toxicity tests can
be provided by previously performed subchronic toxicity and pharma-
codynamic studies. In actual practice, few workers appear to make
extensive use of interim sacrifices during chronic toxicity studies. In
the literature sample represented by the bibliography for this section
interim sacrifices were reported in only 12 papers. Of these, only eight
studies involved the examination of several animals during several
intervals of the test period, and significant observations based on
interim sacrifices were reported in only four papers. It thus appears
that interim sacrifices are not a generally used, or needed, feature of
chronic toxicity tests.
5.6 DATA COLLECTION AND EVALUATION
Long-term tests are unique research experiments that are only
rarely amenable to standardized protocols (Food Safety Council, 1978).
Nevertheless, the success of a well-planned chronic toxicity or carcino-
genicity study strongly depends on adequate observations of the animal
during the test period and on the efficacy of the ensuing pathological
examinations. The following subsections address important aspects of
clinical and pathological procedures essential to the successful execution
of long-term tests.
5.6.1 Food Consumption and Body Weight
Growth and body weights are important indicators of adverse effects
of the test material, but these data are not uniformly collected by some
investigators. All animals should be weighed weekly during periods of
rapid growth and at least monthly thereafter (Benitz, 1970; Food Safety
Council 1978; Loomis, 1974; Magee, 1970; National Academy of Sciences,
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351
1977; Sontag, Page, and Saffiotti, 1976; World Health Organization,
1978). Food consumption should also be monitored weekly to observe the
effect of the test material on food intake and to provide an accurate
determination of dose when the test material is administered in the
diet. When the test compound is administered in drinking water, the
weekly consumption rate of this material is also required for dose
determinations.
5.6.2 Clinical and Laboratory Examinations
Careful clinical observation of test and control animals appears to
be the most neglected area in experimental toxicology (Food Safety
Council, 1978; World Health Organization, 1978). To remedy this situation
and minimize the effects of extraneous factors on the course of chronic
toxicity and carcinogenicity experiments, several authorities recommend
that qualified employees observe the general physical condition, viability,
and adverse behavioral changes of all test and control animals at least
twice daily, 7 days a week (Arnold et al., 1977; Food Safety Council,
1978; Fox, 1977; National Academy of Sciences, 1977; World Health Organi-
zation, 1978). Animals with obvious life-threatening conditions should
be isolated; those unlikely to survive an additional day should be
sacrificed and necropsied to preserve tissues for histological examination.
Each animal should be completely examined at least once a week by quali-
fied personnel for unusual behavioral patterns, respiratory signs,
bleeding, and palpable masses, as well as for abnormalities of the coat,
eyes, mouth, teeth, nose, and ears (Arnold et al., 1977; National
Academy of Sciences, 1977). In chronic toxicity studies, appropriate
biochemical tests are also essential for detecting and assessing toxic
effects at the clinical level, but no consensus exists regarding the
extent or frequency of their use. Loomis (1974) recommended clinical
blood chemistry tests, urinalysis, and blood cell counts for all animals
at 6- to 12-week intervals, but stated "Routine special types of bio-
chemical analyses of sample material, such as blood or urine, from
apparently healthy animals probably are indicated only when there is
reason to suspect that the chemical under investigation is capable of
producing specific toxic effects for which biochemical methods are
clinically of diagnostic value." A report of the National Academy of
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352
Sciences (1977) suggests clinical chemistry tests "should be judiciously
selected," based on preliminary or short-term toxicity tests, or on the
known chemical characteristics of the test compound. The Academy report
further states that a rigid sampling schedule is impractical and excess-
ively costly. Other workers recommend "periodic clinical examinations"
to identify and monitor the progression of specific abnormal findings
(Food Safety Council, 1978). There appears to be general agreement,
however, that clinical blood chemistry tests, blood cell counts, and
urinalysis tests should be performed on all animals that become ill or
show effects from exposure to the test material. Arnold et al. (1977),
Fox (1977), and Street (1970) provide lengthy discussions of these
tests, as well as extensive bibliographies of additional papers.
Less unanimity exists among investigators of chronic toxicity rela-
tive to prescheduled organ function tests. The minimum recommendation
of the Food Safety Council (1978) includes tests for liver, kidney, and
bone marrow function. The World Health Organization (1978) also recom-
mends the use of organ function tests but concedes that "In general,
these methods are not sufficiently standardized or reproducible to
detect minor alterations in organ function." Benitz (1970) and the
National Academy of Sciences (1977) questioned the benefits of presched-
uled organ function tests because of their low sensitivity to organ
damage. Instead of organ function tests, the latter workers recommended
exposing extra animals that can be later sacrificed for complete gross
and histopathological examinations. Other workers have also expressed
reservations about the use of organ function tests (Peck, 1968). When
organ function tests are included in chronic toxicity experiments, they
are most often limited to liver, kidney, and thyroid studies in larger
animals (National Academy of Sciences, 1977).
5.6.3 Pathological Examinations
Until recently, generally accepted procedures in long-term studies
required the necropsy of all nonrodent test animals, but only a fraction
of the total number of rodents (Benitz, 1970; Zbinden, 1973). Now, how-
ever, there appears to be general agreement among investigators that all
animals that die during the study, or are subsequently killed, should be
carefully necropsied (Arnold et al., 1977; Food Safety Council, 1978;
-------
353
National Academy of Sciences, 1977; Sontag, Page, and Saffiotti, 1976;
World Health Organization, 1978). Furthermore, current thinking stresses
the importance of adequate gross examination of tissues prior to histo-
logic sampling. In many instances, pertinent information missed during
the macroscopic examinations of the test animal cannot be recovered in
subsequent microscopic examinations of prepared tissues, even if the
utmost care is used (Benitz, 1970). Consequently, the necropsy procedure
should be considered one of the most important steps in the experimental
protocol (Food Safety Council, 1978).
There is general agreement in the current literature that samples
of all major organs and tissues should be taken from all test and control
animals for macroscopic examination and fixation (Benitz, 1970; Food
Safety Council, 1978; Loomis, 1974; National Academy of Sciences, 1977).
No consensus exists, however, concerning which tissues should be examined
microscopically. Early workers recommended microscopic examination of
all obviously altered tissues plus an additional quantity that generally
did not exceed 20 (Abrams, Zbinden, and Bagdon, 1965; Barnes and Denz,
1954). Other authorities left the microscopic observation of the sup-
plementary tissues to the discretion of the pathologist in charge (Magee,
1970) or specified "complete and accurate" pathological examination
without indicating more specific requirements (U.S. Food and Drug Adminis-
tration, 1971). More recent guidelines require microscopic examination
of all major tissues and gross lesions in all high dose and control
animals, as well as grossly altered tissues in all other dose groups
(Food Safety Council, 1978; National Academy of Sciences, 1977; Peck,
1974; World Health Organization, 1978). The National Cancer Institute
guidelines for chronic toxicity tests are even more stringent: they
require microscopic examination of approximately 40 tissues from all
test and control animals, except positive controls which may be exempted
from this requirement (Sontag, Page, and Saffiotti, 1976). Obviously,
careful microscopic examination of all tissues is highly desirable from
the experimental point of view; however, the Incremental increase in
information gained through such a practice must be balanced against the
added economic, temporal, and labor costs incurred by such a requirement.
According to recent estimates, the number of trained pathologists avail-
able to perform histologic examinations of tissues from tests required
-------
354
or expected under the Toxic Substances Control Act and other expanded
government programs is insufficient if microscopic examinations of 40 or
more tissues from all test and control animals are required (Page,
19772?).
In carcinogenicity testing, special emphasis is placed on the
criteria used to classify the lesions found in microscopic examination
(World Health Organization, 1978). The investigator should specify how
the classification was done and what is implied by the terms benign,
malignant, neoplastio, preneoplastic, and hyperneoplastio. Also, as
discussed before, the pathologist should be aware of the type and location
of the spontaneous tumors associated with the animal species/strain used
(Magee, 1970). The value of proper pathology is especially great in
carcinogenicity testing, since other evaluations are less frequently
incorporated in the test design (Page, 1977
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355
Each animal should be examined at least weekly for unusual behavioral
patterns, respiratory signs, excretory products, and palpable masses, as
well as other abnormalities. In noncarcinogenic chronic tests there is
general agreement that animals showing obvious signs of illness should
receive blood chemistry and urinalysis tests, but no consensus exists
concerning routine tests for healthy animals. Even less unanimity
exists relative to prescheduled organ function tests, which many authori-
ties regard as insufficiently standardized and reproducible to detect
minor alterations in organ function. When performed, these tests are
generally limited to the kidney and liver function of larger, nonrodent
test animals.
There is general agreement among experts that samples of all major
organs and tissues should be taken from all test and control animals for
macroscopic examination and fixation, but no consensus exists concerning
which tissues should be examined microscopically. Obviously, careful
microscopic examination of all sample tissues is highly desirable from
the experimental point of view, but, according to recent estimates,
there are too few trained pathologists to perform histologic examinations
of tissues from tests required or expected under the Toxic Substances
Control Act and other expanded government programs if microscopic exami-
nations of 40 or more tissues from all test and control animals are
required. It thus appears that, at least for the next few years, limita-
tions must be placed on (1) the number of long-term tests performed, if
total histologic tissue analysis of all animal samples is required, (2)
the number of animals to be examined histologically in each long-term
test, or (3) the number of tissues per animal to be examined histologically.
In the literature analyzed in this study, most workers performed histo-
logical examinations of "all major organs and tissues" of high dose and
control animals, plus any other tissues exhibiting gross lesions. In
most thorough studies, "all major organs and tissues" correspond to
approximately 30 histological samples. The analysis of this number of
samples from the high dose and control groups is recommended as the
minimum acceptable level for histological sampling at the present time.
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356
5.7 SHORT-TERM TESTS FOR CARCINOGENICITY
Because of time and cost requirements, application of the lifetime
rodent bioassay to the screening of all potential carcinogens is practi-
cally impossible. Short-term in vitro screening procedures have been
developed that provide limited information concerning the potential car-
cinogenicity of chemicals, under reasonable time and cost constraints.
Such tests, designed to detect genotoxic activity or morphological
transformation, include the following: the Ames Salmonella/microsome
assay; the polk+lpolk.- assay (differential toxicity in E. ooli); "Rec"
differential toxicity in Bacillus subtilis; mouse lymphoma assay (mutation
in cultured mouse cells); and assays for unscheduled DMA synthesis, in
vitro cell transformation, sex-linked recessive lethal mutation in
Drosophila, sister chromatid exchange, and in vitro chromosome aberra-
*-.
tions. These assays are being examined closely in the GENE-TOX workshop
series (sponsored by the U.S. Environmental Protection Agency) and
therefore will not be included in this discussion of short-term tests
for carcinogenicity. For evaluation of advantages and limitations of
the assays mentioned above, see Brusick (1978), Bridges (1976), and
Stoltz et al. (1974).
The discussion of short-term tests will be limited to brief descrip-
tions of in vivo (or in vivo-in vitro) protocols which allow for meta-
bolic activation of chemical carcinogens in the host animal. These
tests are designed to shorten tumor induction time or to detect cellular
alterations or markers that appear shortly after exposure to carcinogens.
5.7.1 Embryo Homograft
A promising technique for producing tumors with chemicals in a
relatively short time was introduced by Peacock in 1962 and was discussed
in more detail by Peacock and Dick (1963). Using BALB/c mice, tissues
from various organs of embryos were implanted surgically into the thigh
muscle of adults of the same strain. Skin, lung, stomach, and urinary
bladder yielded 100% growing implants. Kidney, adrenal, thymus, and
spleen implants were sometimes successful, but liver and brain tissues
failed to grow. When small quantities of each of 13 solid polycyclic
hydrocarbons were introduced along with the tissue, malignant tumors
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357
formed in the Implants. The quantity of carcinogen required to induce
tumors was estimated to be no more than 150 pg, and the tumors induced
were mainly squamous cell carcinomas (with some sarcomas and lung ade-
nomas) ; however, the incidence and types of embryo tissue affected
varied with each hydrocarbon. Only those hydrocarbons previously reported
to be carcinogens (by any method) produced tumors during the time of
observation. The results were obtained in 16 weeks. The surgical
technique used for implantation introduced the problem of possible
effect of the carcinogen on the healing process, but Davies, Major, and
Aberdeen (1971), using a nonsurgical implantation technique, were able
to induce adenomas in embryonic lung tissues exposed to 26 yg of 3,4-
benzo(a)pyrene (BAP) or 1,2,5,6-dibenzanthracene. Twenty percent of the
implants exposed to benzo(a)pyrene and 62% of the implants exposed to
dimethylbenzanthracene developed adenomas in 16 weeks. The technique
lacks quantitation, but that disadvantage can perhaps be eliminated by
in vitro exposure of organ cultures to accurately measured amounts of
carcinogen. For example, lung explants exposed to 20-methylcholanthrene,
3,4-benzo(a)pyrene, or 1,2,5,6-dibenzanthracene and implanted subcutane-
ously into a host animal developed adenomas (some as early as 3 months
later), as demonstrated by Laws and Flaks (1966) and Davies, Major, and
Aberdeen (1970).
5.7.2 jSite Transfer
Homburger and Baker (1967) described studies in which mice and
hamsters received subcutaneous injections of 25 y an^ 500 y> respectively,
of 3,4,9,10-dibenzopyrene. At various times after injection, the injec-
tion sites were removed. Four sites were pooled, minced, and injected
subcutaneously into recipients of the same age, sex, and strain. The
test animals were checked weekly for tumor formation. Control mice were
injected with a carcinogen, injection sites being left intact, and were
also checked for tumor development. One group of hamsters was also in-
jected with carcinogen and the injection sites were removed at various
times for histological study.
Homburger and Baker found that 10% of the secondary host mice
developed palpable tumors by 10 weeks, whereas 14 to 15 weeks were
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required for 10% of the primary hosts to develop palpable tumors. In
the hamster study, tumors palpable 4 weeks after transfer of the injec-
tion sites later turned out to be malignant fibrosarcomas. These partic-
ular injection sites had been transferred 2 days after carcinogen injec-
tion.
RNA-loaded atypical fibroblasts appeared at the site of administra-
tion soon after injection and increased in number until a fibrosarcoma
was formed. Homburger suggested that the fibroblasts represent premalig-
nant cells which, if left in the original host, slowly develop into a
fibrosarcoma but which, when transferred to a secondary host, express
their malignant behavior more readily. Homburger and Baker proposed
that (by using susceptible inbred strains of hamsters, by developing
harvesting methods for selective collection of fibroblasts, and by
transfer of such cells into fresh hamsters) a test is theoretically
possible requiring less than 1 month for the demonstration of carcino-
genic potency in vivo in adult mammalian cells that yield tumors as end
points.
5.7.3 Partial Hepatectomy
Using the 1938 study of Alexander Haddow as a starting point, Solt,
Medline, and Farber (1977) developed a new model for the sequential
analysis of liver carcinogenesis. In their description of this model
Solt et al. stated that cancer cells will emerge under conditions that
would inhibit or impair the growth of normal cells. Hyperplastic liver
lesions induced by the administration of hepatocarcinogens are resistant
to other hepatocarcinogens and hepatotoxins that impair the growth of
surrounding normal liver cells.
Single doses of diethylnitrosamine eventually produce liver cancer,
and during the first week after exposure diethylnitrosamine-induced
nodules arise on the surface of the liver. The model proposed by Solt,
Medline, and Farber (1977) contains the following features: initiation
by diethylnitrosamine, selective growth inhibition of normal cells by 2-
acetylaminofluorene and growth stimulation of carcinogen-altered hepato-
cytes by partial hepatectomy. The test can be completed in 4 weeks.
The procedure in male Fischer 344 rats was as follows: (1) the rats
were injected intraperitoneally with 200 mg/kg diethylnitrosamine, (2)
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after a 2-week recovery period the animals were placed on a basal diet
containing 0.02% acetylaminofluorene, and (3) after 1 week on the 2-
acetylaminofluorene diet the rats were subjected to two-thirds partial
hepatectomy. The animals were maintained on the carcinogen diet after
surgery until 24 h before sacrifice or for one more week. The liver
nodules continued to proliferate vigorously under these experimental
conditions, were characterized histologically by the investigators, and
were identified as precursor lesions for some hepatocellular carcinomas.
In experiments designed to study the cellular components involved
in hepatocarcinogenesis and the progression of such cells to malignancy,
Laishes and Farber (1978) isolated the carcinogen-altered hepatocyte
populations from the livers of treated rats. The cells, some bearing a
y-glutamyl transpeptidase (y-GT) marker, were transferred to the livers
of syngeneic host rats. Selective proliferation of the y-GT-positive
hepatocytes was then stimulated in the host rat liver by partial hepa-
tectomy, while proliferation of y-GT-negative host hepatocytes was
inhibited by dietary administration of 2-acetylaminofluorene. The
system is quantitative and rapid — macroscopic foci were observed within
10 days of cell transfer. In a recent report Laishes, Fink, and Carr
(1980) described in vitro purification of the carcinogen-altered cell
populations isolated from rats treated with the diethylnitrosamine/2-
acetylaminofluorene/partial hepatectomy regime. Primary hepatocyte
monolayer cultures from carcinogen-treated and untreated rats were
exposed for 24 h to acetylaminofluorene, to its derivatives, or to
certain chemicals known to be cytocidal but nonhepatocarcinogenic.
Acetylaminofluorene (AAF), its derivatives N-OH-AAF and N acetoxy-AAF,
and three chemotherapeutic drugs — methotrexate, cyclohexamide, and
adriamycin — were found to be selectively cytocidal to normal rat
hepatocytes. However, the hepatocyte phenotypes which developed during
hepato-carcinogenesis were highly resistant to the effects of the chemi-
cals. Studies are under way in which cells selected in vitro will be
transferred to syngeneic hosts for determination of colony-forming
ability.
Tatematsu et al. (1977) adapted the system of Solt, Medline, and
Farber (1977) as an in vivo screening test for hepatocarcinogens. Their
procedure was as follows: (1) rats were injected intraperitoneally with
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200 mg diethylnitrosamine per kg of body weight; (2) two weeks later the
animals were started for 2 weeks on the test chemical in the diet or
drinking water; (3) after 1 week of administration of the test chemical
partial hepatectomy was performed; and (4) one week later the rats were
killed. Various control groups were established. The end points of the
experiment were the number of liver hyperplastic nodules and the histo-
chemical observation of yGT, acid phosphatase (acid-Pase), glucose-6-
phosphatase (G-6-Pase), and adenosine triphosphatase (ATPase) activity.
The chemicals tested included N-2-fluorenylacetamide, 3'-methyl-4-
(dimethylamino)azobenzene, dimethylnitrosamine, diethylnitrosamine, DL-
ethionine, quinoline, 5,7-dibromo-8-hydroxy-quinoline, 8-hydroxyquinoline,
or 8-nitroquinoline. The first six chemicals, known carcinogens, in
combination with diethylnitrosamine and partial hepatectomy, induced
statistically significantly more hyperplastic liver nodules than did
diethylnitrosamine and partial hepatectomy alone. y-GT activity, not
demonstrated in normal hepatocytes or after partial hepatectomy, was
seen in hyperplastic nodules and in the epithelium of bile ducts and
ductules in the portal area. Acid-Pase and G-6-Pase were usually
decreased, and ATPase was usually absent in hyperplastic areas. Thus,
the presence of y-GT and absence of ATPase are good markers for detec-
tion of hyperplastic nodules.
Tatematsu et al. suggested further evaluation of the system, par-
ticularly to eliminate the possibility of a noncarcinogen acting as a
selective growth inhibitor. They suggested that it may be useful as an
in vivo short-term test in conjunction with in vitro assay systems and
together these tests would provide a secondary screening for chemicals
to be studied in long-term in vivo studies. Laishes, Fink, and Carr
(1980) have recently demonstrated that three noncarcinogenic chemothera-
peutic agents are selectively cytocidal to normal cells in vitro. (See
above.)
5.7.4 Alkaline Elution
Petzold and Swenberg (1978) described a procedure for detecting
single strand breaks in DNA of tissues taken from a wide range of organs
of rats following in vivo exposure to known carcinogens of several
classes.
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Newborn rats were injected intraperitoneally with (3H)dThd (triti-
ated deoxythymidine) for 3 weeks. The animals were exposed to chemicals
for various periods of time, and tissues were then removed and homoge-
nized. The (3H)DNA elution was carried out as described by Swenberg,
Petzold, and Harbach (1976). Increased alkaline elution was expressed
as the increase in elution of treated rats over that of vehicle control
rats.
Twenty-three chemicals were tested and the following organs were
evaluated for DNA damage: liver, lung, kidney, brain, thymus, duodenum
stomach, bone marrow, and mammary gland. The tissues tested were from
target and nontarget organs, and there was a high degree of correlation
between tissues with increased elution after in vivo exposure to carcino-
gens and target organs for tumor susceptibility. For example, the
hepatocarcinogens dimethylnitrosamine, diethylnitrosamine, 2-acetyl-
aminofluorene, and #-hydroxyacetylaminofluorene caused the greatest
elution in liver preparations. Less correlation was seen with chemicals
not requiring metabolic activation. The alkaline elution assay is rapid
(not as time-consuming as the alkaline sucrose gradient technique) and
is reproducible.
Petzold and Swenberg (1978) recommended the alkaline elution test
as a confirmation of in vitro tests when the carcinogenic potential of a
compound is unknown. It can help identify target organs for procarcino-
gens and can be utilized to assess DNA damage in organs that are subject
to toxic changes.
5.7.5 q-Fetoprotein in Serum
a-Fetoprotein has been found in animals bearing chemically induced
liver tumors. Watabe (1971) described the appearance of a-fetoprotein
in the serum of rats after the start of feeding of 4-dimethylaminoazo-
benzene. The protein was detected in the sera of approximately 20% of
41 rats by as early as 3 weeks and in 76% of the rats by 6 weeks. The
serum protein disappeared at 11 to 12 weeks and reappeared at 13 weeks
in 27 of 33 rats; 26 of these 27 rats developed hepatomas. Ninety-one
percent of the rats that developed "early" a-fetoprotein levels had
tumors after 19 weeks.
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362
Serum levels of the protein increased from 2 to 4 mg/dL in the
early stage up to 60 to 100 mg/dL in the later stage. Watabe suggested
that the early protein peak could be attributed to chemical toxicity not
related to carcinogenicity.
In a later study Kroes, Williams, and Weisburger (1973) tested the
effects of different dose levels of several hepatotoxins and hepatocar-
cinogens on the early appearance of a-fetoprotein. They found that high
levels of liver carcinogen were required to induce the "early" peak at 2
to 5 weeks. The protein was not detected in the sera of rats treated
with hepatotoxins. Kroes suggested possibly using the a-fetoprotein
assay as a short-term test to distinguish between hepatotoxins and
hepatocarcinogens.
5.7.6 Strain Susceptibility
Differences in the responses of various strains of animals to
chemical carcinogenesis are discussed in Sect. 5.2.2. Selection of
sensitive strains of test animals can shorten tumor induction time
considerably. The following are examples of such instances.
5.7.6.1 Hamster Fibrosarcoma System — Homburger and Hsuch (1970)
tested the susceptibility of inbred strains of hamsters to tumor induc-
tion with 7,12-dimethylbenz(a)anthracene. A dose of 500 pg of the
chemical, injected subcutaneously, gave rise to subcutaneous fibrosarcomas
in a relatively short time. The 15.16 line was the most sensitive, with
males and females developing tumors having average induction times of 10
and 9 weeks, respectively. The 82.73 line was the least sensitive, with
males and females developing tumors having average induction times of 18
and 15 weeks, respectively.
5.7.6.2 Rat Mammary Tumor System — Huggins, Grand, and Brillantes
(1961) reported preferential mammary tumor induction in female Sprague-
Dawley rats following single oral doses of polynuclear hydrocarbons.
All rats injected with 20 mg or 15 mg of 7,12-dimethylbenzanthracene
developed mammary tumors; the tumor induction time and the number of
active centers were dose related. Doses of 2 to 100 mg of 3-methylcho-
lanthrene induced tumors in 10% to 100% of the animals, and the effect
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was dose related; induction times and the numbers of active centers were
not. Doses of 100 mg and 500 mg of 2-acetylaminofluorene produced
tumors in 30% of the animals, and only the induction times were dose
related.
Hormonal treatments suppressed the development of mammary tumors
caused by 3-methylcholanthrene. 7,12-Dimethylbenzanthracene was the
most effective compound for the induction of mammary tumors (100% tumors
in 43 days).
In a later study, Huggins, Grand, and Fukunishi (1964) demonstrated
in the mammary tumor system that intravenous injection of a compound in
three equal doses was more effective in inducing tumors than the sum of
the doses given as a single injection. The latent period was shorter,
and more active centers per animal were produced, although in both cases
100% of the rats developed tumors.
Griswold et al. (1968) tested 35 compounds using the mammary tumor
induction system. Ten doses of the maximum tolerated dose were adminis-
tered to each animal, and the experiment was terminated 9 months later.
(The duration was increased to 9 months for added sensitivity.) Multiple
doses increased the number of tumor-bearing animals and the number of
tumors per rat; however, multiple doses were no more effective than the
single dose for determining general carcinogenicity of a compound. The
mammary tumor assay is a sensitive test for detection of polynuclear
aromatic hydrocarbons, polycyclic nitro and amino derivatives, and certain
heterocyclic compounds. The investigators recommend the test for rapid
screening of certain potential carcinogens.
5.7.6.3 Mouse Lung Adenoma System — The lung adenoma system was
first utilized as a bioassay of chemicals for carcinogenic potential by
Andervont and Shimkin (1940). In an exhaustive review of the lung
adenoma bioassay system, Shimkin and Stoner (1975) outlined the now
standardized procedure:
1. A susceptible inbred strain of mouse is selected, usually strain A.
Weanlings are generally used, and both sexes should be included in
initial bioassays.
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2. The maximum tolerated dose of the chemical must be determined,
and that dose is administered three times per week for 8 weeks.
The second dose level is one-half the first and that of the third
group is one-fifth the first. Chemicals are generally injected
intraperitoneally, but other routes can be used more effectively
with certain chemicals.
3. Animals can be sacrificed in 12 to 24 weeks. The nodules appearing
on the surface of the lung are counted and are usually statistically
evaluated; however, most tests are considered positive if the
number of lung tumors per mouse is increased by one or more, if
there is a dose-response relationship, and if the mean number of
lung tumors in the vehicle and untreated controls is that usually
found.
The advantages of the system are that it is a rapid, convenient,
and quantitative procedure. The criticisms are that the adenoma does
not have a counterpart in the human and that the tumors represent an
increase in incidences of spontaneous tumors instead of an inductive
process.
5.7.7 The Sebaceous Gland Test
The sebaceous gland suppression test has been suggested as a possible
screening test for skin carcinogens (Brune, 1977; National Academy of
Sciences, 1975). Exposure of the skin to polycyclic hydrocarbons leads
to thickened epidermis and atrophy of the sebaceous glands in a matter
of days. However, false positive results have been obtained with the
test (Weisburger, 1976), making it subject to criticism.
5.7.8 Host-Mediated In Vivo-In Vitro Assay
DiPaolo et al. (1973) described a combination in vivo-in vitro
bioassay for carcinogenicity utilizing transplacental exposure of Syrian
golden hamster embryos. Pregnant females were injected intraperitoneally
with test chemicals at 10 to 11 days gestation. The embryos were excised
at day 13 of gestation, and cells from the embryos were cultured in
Dulbeccos1 modified Eagle's or F-12 medium supplemented with 10% fetal
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365
bovine serum. Cells were passed every 4 to 6 days from secondary cultures.
Colonies, formed by cells transferred to feeder layers, were scored for
morphologic transformation and were further checked for tumorigenicity
by animal innoculation. The cells were harvested and were injected
subcutaneously into hamsters, which were watched closely for subsequent
tumor development. Sarcomas were produced in 3 to 16 weeks. Twelve
known chemical carcinogens, of which several were known to be inactive
in in vitro tests, were tested in this system, and all produced neoplas-
tic transformation; five noncarcinogens did not. The results were
reproducible, the assay seems to eliminate false negative results that
occur because of the requirement for metabolic activation, and spontaneous
transformation does not occur.
5.7.9 Conclusions
Short-term tests for carcinogenicity are not intended to replace
the conventional lifetime studies. However, these types of assays could
be valuable tests in the screening of chemicals for carcinogenicity or,
more precisely, for lack of carcinogenic potential. A battery of short-
term tests could be designed to identify safe compounds so that they can
be eliminated from further testing.
The procedures described above can be completed in from 3 weeks
(appearance of a-fetoprotein in the serum) to 3 to 4 months (the time
required for the detection of actual tumors, as in the lung adenoma
assay and the embryo homograft technique). Because exposure of cells to
chemicals occurs in vivo, these particular assays have the additional
advantage of allowing for metabolic activation. However, in order to
determine the best application of in vivo short-term assays to the
screening of chemicals for carcinogenicity, further evaluation of such
tests is necessary, and their limits of sensitivity must be established.
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TECHNICAL REPORT DATA
i-ase read fasfructions on the reverse before completing)
1. REPORT NO.
EPA-560/1-80-001
2.
4. TITLE AND SUBTITLE
Scientific Rationale for the Selection of Toxicity
Testing Methods: Human Health Assessment
5. REPORT DATE
Publication date-December 1980
6. PERFORMING ORGANIZATION CODE
3. RECIPIENT'S ACCESSIOWNO.
7. AUTHOR(S)
R. H. Ross, M. G. Ryon, M. W. Daugherty, J. S. Drury,
J. T. Ensminger, and M. V. Cone
8. PERFORMING ORGANIZATION REPORT NO.
ORNL/EIS-151
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Health and Environmental Studies Program
Oak Ridge National Laboratory
P.O. Box X
Oak Ridge, Tennessee 37830
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
80-D-X0856
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Pesticides and Toxic Substances
U.S. Environmental Protection Agency
401 M St. SW
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This document is the first of a two-part literature analysis of parameters
associated with the various toxicity testing methods (test animal selection, pathology
requirements, etc.) Acute, subchronic, chronic, and carcinogenic testing methods are
covered; a discussion of some basic experimental considerations is also included.
This report was prepared for the purpose of assisting and supporting the U.S. Environ-
mental Protection Agency in its efforts to develop guidelines for more efficient and
economical testing procedures.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
Reviews
Toxicity
Tests
Toxicity Testing
Methodology
06
13. DISTRIBUTION STATEMENT
Release unlimited
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
436
20. SECURITY CLASS {This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
OU.S. GOVERNMENT PRINTING OFFICE: 1980*40-245/405
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