ORNL/EIS-151
Contract No. W-7405-eng-26
Information Division
SCIENTIFIC RATIONALE FOR THE SELECTION OF TOXICITY
TESTING METHODS: HUMAN HEALTH ASSESSMENT
Robert H. Ross, John S. Drury, Michael G. Ryon,
Mary L. Williams, John T. Ensminger, and M. Virginia Cone
Health and Environmental Studies Program
Information Center Complex
Work sponsored by the Office of Toxic Substances, U.S. Environmental
Protection Agency, Washington, D.C., under fnteragency Agreement No.
78-D-X0453.
Project Officers
Norbert Page
Daljit Sawhney
June 1980
CAUTION
This document has not been given final patent clearance and the
dissemination of its information is only for official use. No
release to the public shall be made without the approval of the
Law Department of Union Carbide Corporation, Nuclear Di-
vision.
OAK RIDGE NATIONAL LABORATORY
Oak Ridge, Tennessee 37830
operated by
UNION CARBIDE CORPORATION
for the
DEPARTMENT OF ENERGY
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CONTENTS
Figures vii
Tables ix
Foreword xiii
Acknowledgments xiii
1. Summary 1-1
1.1 Basic Experimental Considerations 1-1
1.2 Acute Toxicity Testing 1-2
1.3 Subchronic Toxicity Testing 1-3
1.4 Chronic Toxicity and Carcinogenicity Testing 1-5
1.5 Teratogenicity „ 1-7
2. Basic Experimental Considerations 2-1
2.1 Introduction 2-1
2.2 Test Materials 2-1
2.2.1 Introduction 2-1
2.2.2 Chemical Stability 2-2
2.2.3 Chemical Impurities 2-2
2.2.4 Vehicles 2-3
2.2.5 Homogeneity in Vehicle 2-4
2.2.6 Conclusions 2-4
2.3 Husbandry '. 2-5
2.3.1 Introduction r 2-5
2.3.2 Animal Selection - 2-5
2.3.3 Transportation, Quarantine and Disease Control . . 2-6
2.3.4 Design of Testing Facility 2-8
2.3.5 . Caging 2-9
2.3.6 Bedding 2-10
2.3.7 Temperature and Humidity 2-13
2.3.8 Light 2-14
2.3.9 Ventilation 2-15
2.3.10 Noise and Handling 2-16
2.3.11 Personnel 2-18
2.3.12 Conclusions 2-19
2.4 Diet 2-20
2.4.1 Introduction 2-20
2.4.2 Dietary Requirements for Laboratory Animals .... 2-20
2.4.3 Types of Diets for Laboratory Animals 2-23
2.4.4 Analysis for Nutrients and Contaminants 2-29
2.4.5 Effects of Diet on Toxicity Test Results 2-31
2.4.6 Conclusions '.' 2-35
2.5 Pathology 2-35
2.5.1 Introduction 2-35
2.5.2 Gross Examination 2-36
2.5.3 Tissue Preservation and Storage 2-41
2.5.4 Trimming, Staining, and Embedding of Tissue .... 2-42
2.5.5 Microscopic Examination 2-44
2.5.6 Conclusions 2-47
iii
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iv
3. Acute Toxicity 3-1
3.1 Introduction 3-1
3.2 LDso Determinations 3-2
3.2.1 Introduction 3-2
3.2.2 Difference Between Species 3-7
3.2.3 Difference Between Administration Routes 3-12
3.2.4 Sex Differences in the Laboratory Rat 3-16
3.2.5 Test Limiting Criteria 3-17
3.2.6 Conclusions 3-17
3.3 Human vs Animal Response 3-18
3.3.1 Introduction 3-18
3.3.2 Comparison of Lowest Published Lethal Doses
(LDLo) „ 3-19
3.3.3 Comparison of Acute Toxicity Response 3-19
3.3.4 Conclusions 3-23
3.4 Pathology 3-24
3.4.1 Introduction 3-24
3.4.2 Histopathology 3-24
3.4.3 Conclusions 3-27
3.5 Observation Period 3-31
3.5.1 Introduction 3-31
3.5.2 Purpose of Observation Period 3-31
3.5.3 Length of Observation Period . . . .' 3-32
3.5.4 Conclusions ^ 3-34
4. Subchronic Test Design ;. 4-1
4.1 Introduction 4-1
4.2 Species 4-3
4.2.1 Introduction 4-3
4.2.2 Discussions in Literature Reviews Concerning
Species Suitability 4-5
4.2.3 Species Comparison Studies 4-11
4.2.4 Conclusions 4-35
4.3 duration .• v . 4-38
4.3.1 Introduction 4-38
4.3.2 Duration Reviews 4-39
4.3.3 Comparison Summaries 4-46
4.3.4 Conclusions 4-72
4.4 Route of Exposure 4-75
4.4.1 Introduction 4-75
4.4.2 Route Discussions 4-76
4.4.3 Route Comparisons . . 4-82
4.4.4 Conclusions ' 4-95
4.5 Pathology 4-96
4.5.1 Introduction 4-96
4.5.2 Use of Pathology 4-98
4.5.3 Basis for a Minimum Pathology Screen 4-109
4.5.4 Conclusions 4-112
4.6 Clinical Laboratory Tests 4-114
4.6.1 Introduction 4-114
4.6.2 General Clinical Testing 4-115
4.6.3 Hematology 4-119
4.6.4 Biochemical and Organ Function 4-123
4.6.5 Urinalysis 4-132
4.6.6 Conclusions 4-136
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Appendix A . ". 4 4-151
Appendix B 4-164
Appendix C 4-171
5. Teratogenicity 5-1
5.1 Introduction 5-1
5.2 General Considerations 5-2
5.2.1 Introduction 5-2
5.2.2 Dosage — Number and Levels 5-2
5.2.3 Dosage —Duration 5-5
5.2.4 Pharmacokinetics 5-7
5.2.5 Positive Controls 5-8
5.2.6 Number of Species 5-9
5.2.7 Number of Test Animals Per Dose Group 5-10
5.2.8 Administration Route 5-11
5.2.9 Fetal Examination 5-12
5.2.10 Conclusions 5-13
5.3 Teratogen, Mutagen, and Carcinogen Relationships 5-14
5.3.1 Introduction 5-14
5.3.2 Numerical Examination 5-14
5.3.3 Literature Overview 5-15
5.3.4 Conclusions 5-40
5.4 Teratogenesis and Time of Administration 5-41
5.4.1 Introduction '. 5-41
5.4.2 Organogenesis 5-41
5.4.3 Histogenesis and Fetal Period/Behavioral
Teratogenesis 5-44
5.4.4 Conclusions 5-46
5.5 Species Comparisons 5-47
5.5.1 Introduction 5-47
5.5.2 General Aspects 5-47
5.5.3 Rat . . . 5-65
5.5.4 Mouse 5-65
5.5.5 Rabbit 5-66
5.5.6 Hamster 5-67
5.5.7 Nonhuman Primates 5-68
5.5.8 Other Species 5-69
5.5.9 Human Teratogenic Chemicals Tested in Animal
Models 5-71
5.5.10 Conclusions 5-79
6. Chronic Toxicity and Carcinogenicity Testing . '. 6-1
6.1 Introduction 6-1
6.2 Test Animals i 6-2
6.2.1 Species 6-2
6.2.3 Strain 6-29
6.2.4 Spontaneous Tumors 6-36
6.2.5 Number 6-42
6.2.6 Controls 6-49
6.2.7 Age 6-52
6-.2.S Sex 6-56
6.2.9 Conclusions 6-68
6.3 Routes of Administration 6-71
6.3.1 Conclusions • 6-91
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vi
6.4 Dose and Duration 6-91
6.5 Interim Sacrifice 6-102
6.6 Data Collection and Evaluation 6-104
6.6.1 Food Consumption and Body Weight 6-104
6.6.2 Clinical and Laboratory Examinations 6-105
6.6.3 Pathological Examinations 6-107
6.6.4 Conclusions 6-109
6.7 Short-Term Tests for Carcinogenicity 6-111
6.7.1 Embryo Homograft 6-112
6.7.2 Site Transfer 6-113
6.7.3 Partial Hepatectomy 6-114
6.7.4 Alkaline Elution 6-117
6.7.5 a-Fetoprotein in Serum «• 6-118
6.7.6 Strain Susceptibility 6-119
6.7.7 The Sebaceous Gland Test 6-122
6.7.8 Host-Mediated In Vivo-In Vitro Assay ........ 6-122
6.7.9 Conclusions 6-123
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TABLES
2.1 Space recommendations for laboratory animals 2-11
2.2 Cage space requirements for rodents 2-12
2.3 Recommended levels for essential amino acids for
laboratory animals expressed as g/100 g protein ...... 2-21
2.4 NIH-7 open formula rat and mouse ration 2-25
2.5 AIN-76™ purified diet . 2-26
2.6 AIN-76™ vitamin mixture 2-27
2.7 AIN-76™ mineral mixture T 2-28
2.8 Tissue to be included in a gross examination 2-38
2.9 Correlation between gross and microscopic lesions in
carcinogenic studies in mice 2-40
2.10 Organs and tissues to be examined in routine toxicity tests. . 2-45
3.1 Compounds examined for LD30 differences between species and
between routes of administration 3-3
3.2 Percent LDSo differences between species . . . .' 3-8
3.3 Percent LD30 differences between routes'of administration. . . 3-13
3.4 Compounds-selected for human vs animal LDLo comparisons. . . . 3-20
3.5 LDLo differences between humans and animals 3-21
3.6 Examples of histopathological changes from acute exposures
to chemicals 3-25
3.7 LD30 observation periods 3-28
4.1 Experimental design for recently proposed subchronic oral
toxicity tests 4-4
4.2 Comparison of various animal models as predictors of
metabolic fate in man 4-8
4.3 Occurrence of 39 physical signs from 6 drugs in 3 species. . . 4-14
4.4 Organ system toxicities - 4-15
4.5 Comparison of significant variations in the response of four
species to an oral analgesic . .' 4-19
4.6 Predictive abilities of the dog, monkey, and combination . . . 4-22
4.7 Summary of dioxin oral administration data 4-27
4.8 Summary of biological effects of TCDD 4-28
4.9 Summary of treatment-related effects in hamsters, rats, and
rabbits repeatedly exposed to acrolein (ppm) for 13 weeks. . 4-34
4.10 Relationship of dosage levels of short-term and 2-year
feeding of materials in the diets of rats 4-40
4.11 Parameters of ratios of acute peroral LDsoS, 7- or 90-day
and 2-year minimum effect (MiE) dosage levels 4-42
ix
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4.12 Prediction formulas 4-44
4.13 Approximate duration of drug administration required to
define toxicity in animals 4-45
4.14 Prediction of long-term no-effect doses 4-47
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 " . 4-48
4.16 Mean values of body weights, food consumption, and dibutyl-
(diethylene glycol bisphthalate) (DDGB) intake of rats
fed DDGB at 0%-2.5% of the diet for 11 weeks 4-50
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 4-52
4.18 Cumulative toxicity of epichlorohydrin: body weight gain in
grams (mean ± SE) 4-56
4.19 Subacute toxicity of epichlorohydrin: body weight gain in
grams (mean ± SE) 4-57
4.20 Cumulative toxicity of epichlorohydrin: percenf organ to
body weight of rats (mean ± SE). . 4-59
4.21 Subacute toxicity of epichlorohydrin: percent organ to body
weight of rats (mean ± SE) 4-60
4.22 Comparison of 13 week and 2 year TCDD studies 4-61
4.23 Weight gain of female rats receiving 31 daily doses of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 4-63
4.24 Liver and thymus weights of female rats receiving daily doses
of 2,3,7,8-tBtrachlorodibenzo-r-p-dioxin (TCDD) 4-64
4.25 Mean body weights and water intake of rats fed diets contain-
ing 0%-2.0% di-(2-ethylhexyl)phthalate (DEHP) for up to 17
weeks 4-69
4.26 Mean haematological values for rats fed diets containing
0%-2.0% di-(2-ethylhexyl)phthalate (DEHP) for 2, 6, or 17
weeks 4-70
4.27 Relative organ weights of rats fed/diets containing 0%-2.0%
di-(2-ethylhexyl)phthalate (DEHP) for 2, 6, or 17 weeks. . . 4-73
4.28 Summary of chemical examples tested for toxicity by various
routes of administration 4-84
4.29 Main toxic effects produced by oral or intravenous
administration of A9-THC 4-93
4.30 Postmortem studies ". 4-97
4.31 Range-finding data on subacute oral toxicity 4-99
4.32 Summary of observations of effects detected in short-term
and-two-year oral studies 4-101
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xi
4.33 Incidence (in percent) of drug-induced pathological changes
encountered over a ten-year period in approximately
14,000 animals 4-105
4.34 Summary of histological quantification methods and their
applications for chemically induced lesions 4-107
4.35 Comparison of the usefulness of individual organs to indicate
toxicity when pathologically examined in a subchronlc study. 4-110
4.36 Haematological studies during toxicity tests 4-121
4.37 Relative sensitivity of liver enzyme tests 4-130
5.1 Literature survey of experimental parameters in testing
chemicals for teratogenicity *. 5-3
5.2 Compounds identified as both carcinogens and mutagens 5-16
5.3 Compounds identified as both mutagens and teratogens 5-17
5.4 Compounds identified as both carcinogens and teratogens. . . . 5-18
5.5 Compounds identified as carcinogens, mutagens, and teratogens. 5-23
5.6 Activation of carcinogens to mutagens 5-25
5.7 Presence or absence of mutagenicity of various nitrosamines
in the presence of liver fraction from PB-pretreated rats. . 5-34
5.8 Agents that are both carcinogenic and' teratogenic in man . . . 5-36
5.9 Effects of chemical agents which have been tested for both
oncogenicity and teratogenicity 5-39
5.10 Critical periods of organogenesis in animals 5-42
5.11 Species susceptibility to drugs 5-49
5.12 Thalidomide teratogenesis in primates 5-50
5.13 Chemicals tested for teratogenicity 5-52
5.14 Chemical agents known to be teratogenic in man 5-72
5.15 Aminopterin action in various species 5-73
5.16 Methotrexate action in various species 5-74
5.17 Thalidomide action in various species. ...» 5-75
6.1 Some factors in the experiments with MH 101 upon a variety
of species ' 6-9
6.2 Induction of skin carcinogens in mice, hamsters, and rats with
weekly applications of 9,10-dimethyl-l,2-benzanthracene. . . 6-12
6.3 Tumor induction in various species with aromatic amines. . . . 6-16
6.4 Compounds, not previously listed, which have produced bladder
cancer in the dog 6-19
6.5 The carcinogenic action of diethylnitrosamine in different
animal species 6-21
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xii
6.6 Tumor induction of various species with diethylnitrosamine
and dime thy Initrosamine 6-23
6.7 Carcinogenicity of different forms of asbestos in various
species. ..... 6-28
6.8 Chemicals or industrial processes associated with cancer
induction in humans: comparison of target organs and
main routes of exposure in animals and humans 6-30
6.9 Spontaneous tumor incidence in animals used in the
National Cancer Institute Bioassay Program 6-37
6.10 Sex and age incidence of characteristic tumors of inbred
strains , 6-39
6.11 Spontaneous tumors in laboratory animals 6-43
6.12 Incidence of tumors in treated groups required for
significance (p = 0.05) depending on experimental group
size and spontaneous tumors in controls 6-50
6.13 Systemic tumor induction in mice with 3-methylcholanthrene
by the intragastric route 6-58
6.14 Influence of sex on response to carcinogens 6-59
*
6.15 Comparison of continuosu versus intermittent administration
of BZ»2HC1 on development of liver Jrarderian gland, and
lung 6-65
6.16 Induction of tumors in the Syrian hamster with diethyl-
nitrosamine 6-79
6.17 Local tumor induction in mice with 3-methylcholanthrene by
various routes of administration 6-81
6.18 Incidence of pulmonary tumors in strain A mice after intra-
venous injection of methylcholanthrene • 6-85
6.19 Incidence of pulmonary tumors in strain A mice after intra-
venous injection of 0..5 g of methylcholanthrene or 0..5 rag
of dibenzanthracene 6-86
6.20 Incidence of pulmonary tumors in strain A mice given sub-
cutaneously 0.5 mg of methylcholanthrene or 0.5 mg of
dibenzanthracene dispersed in 0.5 cc of horse serum and
cholesterol 6-87
f
6.21 Tumor induction with aromatic amines by various routes of
exposure 6-89
6.22 Summary of recommendations on dose selection 6-93
6.23 Time relationships among drug exposure, life span, and time
equivalents in man 6-100
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FIGURES
4.1 Changes in the mean body weight of rats fed Sumithion in the
diet for 90 days 4-51
4.2 Changes in cholinesterase activity in the kidney, liver, red
blood cells, and brain cortex of rats fed Sumithion for
90 days 4-53
5.1 Numbers of mutagens and nonmutagens tested in our laboratory
and their correlation to carcinogenic activities 5-30
5.2 Chronology of overlapping of carcinogens and mutagens 5-32
5.3 Diagram of the successive stages in the pathogenesis of a
developmental defect, beginning with the initial types of
changes in developing cells or tissues (the mechanism) and
continuing to the final defect 5-38
5.4 Curve represents the susceptibility of the human embryo to
teratogenesis, beginning with fertilization and continuing
throughout intrauterine development 5-45
viz
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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: technical review and follow-up on chemical studies, arrang-
ing workshops, writing reviews on health effects of specific chemicals,
and conducting structure-activity oriented reviews of chemicals associ-
ated with mutagenic or teratogenic effects.
This document is a literature analysis of parameters associated
with the various toxicity testing methods. The information contained
herein has been compiled for the purpose of assisting EPA in its efforts
to develop guidelines for more efficient and more economical toxicity
testing procedures.
ACKNOWLEDGMENTS
The authors would like to thank Helga Gerstner, manager of the
Information Center Complex, for her support during the preparation of
this document. The advice and support of Norber Page, and Dal jit Sawhney,
EPA project officers, and the assistance of Helen Warren and other members
of the Toxicology Information Response Center of the Information Center
Complex are gratefully acknowledged. Special thanks are extended to
Peter Witschi of the Biology Division and to Guy Griffin of the Health
and Safety Research Division, Oak Ridge National Laboratory, and to
Harold Grice and Clifford Chappel, FDC Consultants, for their technical
review and helpful suggestions. The authors are also greatly indebted
to Pat Hartman and Donna Stokes, for typing the manuscript, and to
Carolyn Seaborn for her assistance in collection and organization
of data.
xiii
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1. SUMMARY
1.1 BASIC EXPERIMENTAL CONSIDERATIONS
To aid in obtaining reliable toxicity test results, careful atten-
tion should be given to certain aspects of test materials, husbandry,
diet, and pathological examination.
^
The chemical and physical properties of the test chemical should be
known prior to the initiation of a toxicity experiment. This is essential
because the chemical and physical properties will influence laboratory
storage practice and will permit the researcher to determine the stabil-
ity of the chemical under the conditions of the experiment (e.g., temper-
ature and pH). In addition, the chemical purity of the test material and
possible interactions with the vehicle must be investigated.
The husbandry conditions associated with toxicity testing can affect
the test results and should be controlled or standardized. Included among
the husbandry factors are the following: animal selection; transportation;
» -
quarantine; disease control; cage design; test facility design; regulation
of macro- and microclimate variables; and certification of personnel.
The 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 an essential part of the evaluation of a chemical's
toxicity, providing information on the morphology of the lesions and
indicating dose-effect relationships. A successful pathology evaluation
1-1
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1-2
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 tis-
sues 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.
ft
1.2 ACUTE TOXICITY TESTING
Acute toxicity studies are very useful because they provide base-
line information such as the LD3o (the most frequently determined index
of toxicity), the relative sensitivity of the various species, and the
comparative toxicity of various chemicals. They also permit identifica-
tion of the nature of the toxicity or the method of the toxic action and
provide guidance on doses to be used in subsequent experiments. Adminis-
tration may be single oral or single parenteral -injection or may be given
in food or drinking water over a 24 hr period.
The data from acutely dosed animals can provide some insight regard-
ing mechanism of chemical action and assist in establishing no effect/
effect levels. When the laboratory rat is used as the test7animal, both
f
sexes should be tested because of the possibility of sex related differ-
ences in LDso values.
The time following dosing, during which deaths are recorded for the
purpose of determining an LD50» is the observation period. This period
must be of sufficient duration for manifestation of lethal effects but
not so long -that there is doubt whether the animal died as a direct
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1-3
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 so that an assessment
of the nature and cause of death can be determined.
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 tar-
get 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 one area of
controversy 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. How-
ever whenever possible the choice of species should be governed by infor-
mation on pharmacokinetics and metabolic fate of the compound. Under
such circumstances other species might also be considered.
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1-4
The duration of exposure for a subchronic test is also debatable.
The literature suggests that a 90-day exposure duration can provide essen-
tially 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 additional
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 ques-
tion needs more data for analysis. Comparison studies or access to un-
published 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 shoirld 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. Extrapolation of data
obtained from one route to evaluate the hazard with exposure to another
route could provide a solution to the problems of inhalation tests. How-
ever, there is very 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 pathology and biochemical
tests as parameters to evaluate toxicity. Both pathology and biochemical
tests can provide acceptable data to assess toxicity when used individually.
However, much more significant data can be obtained when the two pro-
cesses are used in combination.
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1-5
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, due to 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 screen (evaluating cel-
lular damage and hemorrhaging) , a set of enzyme and or'gan function tests
(primarily evaluating liver and kidney damage), and a urinalysis screen
(primarily for kidney damage). Changes indicated by these tests can be
investigated further by more complex biochemical tests.
By combining pathology and biochemical testing a more thorough eval-
uation of*organ system toxicity can be achieved/ Without the comprehensive
evaluation provided by this combination, subchronic tests would be less
useful as precursors for chronic tests or as primary chronic evaluations.
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, diet synergists or antagonists, immunologic status,
duration of the experiment and other factors.
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1-6
The principle objective for conducting chronic toxicity tests is to
determine the toxic potential of a chemical when it is administered for
the greater part or all of the animals' lifetime. The major objective
in testing of chemicals for their carcinogenic potential may involve
studies that are designed simply to determine whether or not the chemical
is capable of inducing tumors in a particular strain or species of animals.
Since most chronic toxicity and carcinogenicity tests are conducted
over the major portion of the lifespan of the animal, short-lived rod-
ents are the overwhelming choice of most investigators. However, the
dog is occasionally used, particularly in tests for chronic toxicity.
Test duration is a controversial issue and attempts to standardize
this parameter have been unsuccessful. Generally, when mice and rats are
used, 24 to 27 months and approximately 24 rrfonths are recommended for
chronic toxicity and carcinogenicity tests respectively. Typically, dogs
are treated for one to two years in chronic toxicity studies.
The requirements for lifetime carcinogenicity studies may be reduced
by the introduction .of new strains of, animals which are especially suscep-
tible to tumor induction. Genetic factors which influence the spontaneous
tumor incidences in laboratory animals have been exploited in the develop-
ment of certain short-term assays such as the mouse lung adenoma system.
Other in vivo short-term bioassays have been developed which could be
f
considered as intermediate between in vitro short-term studies and the
conventional lifetime studies.
The number of test animals used is a compromise between requirements
for good statistical pr-ecision and reasonable costs and work loads. Most
authorities now recommend 50 rodents or 4 to 8 nonrodents per test group.
Concurrent control groups comprised of animals of the same species, age,
sex, weight, and number are also required.
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1-7
In tests for chronic toxicity at least three dose levels should be
used, and both treatment and observation of animals should be conducted
seven 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
pathogenesis of toxic events unless relevant data has been gathered in
previous subchronic or pharmacodynamic studies.
Adequate macroscopic examination of animals and microscopic examina-
tion of tissues should be made to justify the cost and labor of long
chronic toxicity tests. As a minimum, all -riiajor 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.
1.5 TERA*TOGENICITY
Some important considerations in the determination of the terato-
genicity of a chemical are the number, level, and duration of the dose,
pharaacokinetics of the chemical, number and choice of appropriate species,
number of test animals per dose group, and the extent of the fetal examina-
tion. The number and levels of the dose are important to insure that the
teratogenic potential of the chemical is fully investigated, whereas the
duration of administration is important because the teratogenicity of
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1-8
some chemicals has been shown to be dependent on whether administration
is acute or chronic. The pharmacokinetics of the chemical must be con-
sidered because the rate at which a chemical is absorbed, the time to
reach peak blood levels, the half-life, the placental transfer, the
nature of the metabolites, and the rates and extent of elimination are
all factors that can determine the teratogenic potential of the chemical.
The number and choice of appropriate species is an^important considera-
tion because there is species variation with respect to teratogenic
response. Although nonhuman primates have shown teratogenic responses
similar to humans to some chemicals (e.g., thalidomide), they have not
been susceptible to the teratogenic action of some drugs, such as aminop-
terin, which have a proven teratogenic potential for humans. Rats, mice,
and rabbits, even though they do not shown~a consistent correlation to
human teratogenic response, have been and will probably continue to be
used more frequently than the monkey primarily due to availability,
ease of handling, and economy. Finally, the extent of fetal examination
is important because the results of whether or not a chemical is terato-
genic is dependent on the fetal examination; therefore, a thorough exami-
nation is essential.
The relationships of chemical carcinogens, mutagens, and teratogens
has received considerable attention during the last decade, particularly
/•
the relationship of carcinogens and mutagens. Most chemical carcinogens
are believed to be mutagens, but the reverse relationship, that of muta-
gens being carcinogens, is not as well established.
The organogenesis period of prenatal development which usually varies
in length for different animal species, is considered the most sensitive to
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1-9
the action of teratogenic chemicals. In addition, a chemical administered
at one stage of organogenesis might induce cleft palate, whereas the same
chemical given at a different organogenesis stage might cause no effect or
possibly malformation to the limbs. Teratogenic insult during histogenesis,
which is the embryonic period following organogenesis, may cause minor
structural deviations; however, the abnormalities that are more likely to
occur are those that involve growth or functional aspects of development.
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2. ' BASIC EXPERIMENTAL CONSIDERATIONS
2.1 INTRODUCTION
This section will discuss such basic experimental parameters as the
purity and chemical stability of the test chemical, the importance of
selecting the appropriate vehicle, and the diet, husbandry, and patho-
logical examination of the test animal. 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
s
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 the result of the compound under investigation and not
due to 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 regulatory
purposes but can, in some instances, provide an erroneous impression 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 varia-
tion of the compound, chemical synthetic processes, packaging and handling
procedures, storage requirements, and interactions with other chemicals
must be considered. The following subsections will briefly describe
some aspects of the pretesting examination of the test material.
2-1
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2-2
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). This means that prior to the start
of the toxicity test the stability of the test chemical in the feed and/
or vehicle and at the temperature and/or pH to be used in the test must
be investigated. The stability of the test material under its conditions
*
of storage and/or administration will influence the frequency at which
^s
fresh treatment mixtures are prepared (Sontag, Page, and Saffiotti, 1976;
National Academy of Sciences, 1975).
When the test material is a chemical used in food processing, the
possibility exists that under conditions of use the chemical may be trans-
formed or degraded in foods and 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 it is possible that trace contaminants could be responsible for, or
at least modify the observed biological effects attributed to the test
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material under investigation, and as pointed out by the World Health Orga-
nization (1978), if the contaminants are unknown or their biological activ-
ity unsuspected, toxicity tests may lead to erroneous conclusions concerning
the test material.
A related issue which 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 puri-
fied, etc.) (World Health Organization, 1978). If the test material is
a commerical product which is known to contain impurities, then toxicity
testing of a highly purified sample could result in erroneous conclusions.
If the impurities are standardized, then testing of the commercial product
would be the solution; however, for those commerical products, chemicals
used in manufacturing, or chemicals incidentally released into the envi-
ronment 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 chem-
ical reacts chemically with the vehicle (also mentioned in section 2.1.2).
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 super heated 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). Two other
examples of vehicle interference in tests for carcinogenicity are described
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2-4
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-1-oxide was administered in olive oil and lecithin instead
of in a solvent mixture containing cholesterol.
^
2.2.5 Homogeneity in Vehicle
If a text mixture is prepared in sufficient quantity for the dosing
of several animals who are to receive the same concentration of the test
material, then the homogeneity in the vehicle is very essential. Sontag,
Page, and Saffiotti (1976) state tht in a long-term study where the test
material is incorporated into the feed, the'liomogeneity and concentration
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 procedures 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.
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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 which 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. ./^
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 tox-
icity studies. First the animals should all have standardized life cycle
variables, such as age, sexual maturity, and mating status, since these
factors can affect toxicity (Hurni, 1970). Next the genetic composition
should be uniform, since this insures consistency of response and repro-
ducibility of results (Zbinden, 1963; Food Safety Council, 1978). 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, which 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
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2-6
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). Since both these options have problems, the use of
hybrids from crosses of inbred strains is often utilized. These animals
are genetically uniform but are not homozygous (Hurni, 1970; Food Safety
Council, 1978). Festing (1979) reviews, in more derail, 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) which have accepted levels
of health and are known to be free of certain standard diseases and para-
sites (Meister, Hobik, and Metzger, 1967; Hurni, 1970). However, these
animals require special isolated colonies with "clean zone" barriers and
can be difficult to maintain (Hurni, 1970). Therefore as with other fac-
tors , the, nature of • the experiment and the risks involved will decide the
animal type and quality to be utilized (Page, 1977).
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 inhouse breeding colony to the testing facility) has affected
the blood status of mice for 3-4 days afterwards. More extensive trans-
portation can disrupt the normal state for up to six weeks (Hurni, 1970).
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2-7
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 two weeks (National
Academy of Sciences, 1969). As part of the quarantine process, it is a
good practice to include: (1) a physical examination on arrival; (2)
veterinary care to check for parasites, disease, and allow for immuniza-
tion; (3) general grooming; and (4) a pathological examination of a small
s
number of the test animals (U.S. Department of Health, Education, and
Welfare, 1974; Sontag, Page, and Saffiotti, 1976). 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:
f
(1) acquire and include (after quarantine) only healthy animals (2) pre-
vent introduction of pathogens into test facility; (3) maintain sanitary
conditions; and (4) utilize a well planned disease 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
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2-8
Academy of Sciences, 1969). The use of filters and filter caps is par-
ticularly helpful to control airborne diseases (dough, 1976). Sanitary
control should include facility and personnel cleanliness, proper waste
disposal practices, and vermin control (U.S. Department of Health, Edu-
cation, 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 fre-
quent 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) and 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
numbers 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; minimize disease factors; and minimize economic 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 con-
struction details are discussed in several rreviews (U.S. Department of
Health, Education, and Welfare, 1974; National Academy of Sciences,
1969; Grange, 1976).
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2-9
In general the rooms should be arranged to provide barriers between
individual rooms and the outside environment (National Academy of Sciences,
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 unidirectional corridor flow of
materials and personnel (Sontag, Page, and Saffiotti, 1976). Each room
has a separate entrance and exit door, with personnel'and materials being
s
decontaminated before entering, and leaving only through the exit door
(Page, 1977). This sytem 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 separaee 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.
f
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 micro- and macroenvironment of the testing facility (U.S.
Department of Health, Education, and Welfare, 1974; Clough, 1976). The
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certain cancers than groups of 1-5 mice. Hurni (1970) discussed several
studies in which isolated animals showed an increase in toxicity. How-
ever, 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
square inches necessary per rodent and the maximum number 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.
f
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) provides thermal insulation; (2)
absorbs fecal and urinary wastes, and water spillage; (3) used to build
nests; and (4) reduces stress by providing a protective, isolating cover
(National Academy of Sciences, 1969). The material selected should there-
fore be absorbant, nonedible, and innocuous but not dusty or highly resin-
our (National Academy of Sciences, 1969; Hurni, 1970; Clough, 1976). Some
f-
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,
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Table 2.1. Space recommendations for laboratory animals
Species
Mouse
Rat
Hamster
Guinea pig
Rabbit
Cat
Dog*
Primates"' d
Group 1
Group 2 .
Group 3
Group 4
Group 5
Weight
Up to 10 g
10-15 g
16-25 g
Over 25 g
Up to 100 g
100-200 g
201-300 g
Over 300 g
Up to 60 g
60-80 g
81-100 g
Over 100 g
Up to 250 g
250-350 g
Over 350 g
Up to 2 kg
2-4 kg
Over 4 kg •
Up to 4 kg
Over 4 kg
Up to 15 kg
15-30 kg
Over: 30 kg
Up to 15 kg
15-30 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/animal
(square)
39 cm (6 in)
52 cm (8 in)
77 cm (12 in)
97 cm (15 in)
110 cm (17 in)
148 cm (2*3 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)
b
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 six 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 six inches.
cThe 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.
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Table 2.2. Cage space requirements for rodents
Minimum square Maximum number
Inches per animal 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
(Soave, 1964).
Source: Adapted from National Academy of Sciences, 1969.
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2-13
can induce biological variations in the 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 carcinogenicity studies. In any case, the material must be
sterilized, preferably 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 magni-
tude, frequency, and duration of the changes; whether or not the test ani-
mals have behavioral modifications to adapt ,£0 these; and the current
physical and health status of the animals (Weihe, 1976a). The easiest
solution is to control them by air conditioning each animal room (U.S.
Department of Health, Education, and Welfare, 1974).
Weihe (1964 cited by Heinecke, 1967), by varying the environmental
* ' -
temperature, was able to affect the LDsos of chemicals. Heinecke (1967)
found that temperature changes also affected blood values. Hurni (1970)
reviewed several studies in which cold tempratures, or increases in ambi-
ent 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 it was found to be a prin-
cipal factor of ringtail in young rats (Flynn, 1967; Clough, 1976).
Thus it is very important to control these factors.
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2-14
The exact limits of temperature and humidity beyond which signifi-
cant metabolic adaptation will occur is not known, but if the changes are
moderate (± 5°K or 20-30% humidity) the animals should be able to adapt
behaviorally without damage (Weihe, 1976a). The National Academy of Sci-
ences (1969) recommends a fluctuation of no more than ±2°F in a range of
70°-80°F and 40-70% humidity for rats and mice. The U.S. Department of
Health, Education, and Welfare (1974) suggests levels of 65-85° and 30-70%
depending on the test species. Sontag, Page, and Saffiotti (1976) recom-
mend 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
s
Light is another environmental variable that should be controlled.
The damaging effects, of light are dependent on the intensity and duration
(Weihe, 1976i). 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,
1976i).
Strong light has proved to decrease the reproductive ability of mice
(Porter, 1967), damage the retina and indirectly the endocrine system (Weihe,
1976&; Fox, 1977). 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 300-500 lux for rodents. In computing the light
levels, the effect of opaque or translucent cage construction should be
considered, since this can greatly affect the level reaching the test
animal (Clough, 1976).
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2-15
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, 1976&). The daylight cycle should be designed to
suit the test species, for example, 10 hr for rats and mice, but 14 hr
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
contaminants (e.g., particulates, C0a, NH3, moisture,,and microorganisms),
reduce odors, regulate temperatures, and promote comfort (U.S. Department
of Health, Education, and Welfare, 1974; Clough, 1976). The ability to
limit odor depends on the species used, and the population densities as
well as a properly designed facility (U.S. Department of Health, Education,
and Welfare, 1974). The precise pattern of air movements in each room
i •
results from the interaction of convection currents arising from the test
animals; the air input by the ventilation system; and the deflections
caused by the cages, racks, and equipment (Clough, 1976). Nevins (1971)
discusses the design criteria that are needed to optimize the ventilation
patterns. The rooms used by humans and animals should each be ventilated
separately (U.S. Department of Health, Education, and Welfare, 1974;
National Academy of Sciences, 1969). The air should be changed frequently
without causing drafts, and incoming or recirculated air msut be filtered
(National Academy of Sciences, 1969; Hurni, 1970; Sontag, Page, and
Saffiotti, 1976). The recommended rates of air changes per hour include:
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2-16
6-15 (National Academy of Scineces, 1969); 15-23 (Hurni, 1970); and 10-15
(U.S. Department of Health, Education, and Welfare, 1974; Shaw, 1976;
Sontag, Page, and Saffiotti, 1976). The exact rate will depend on the
population density, refuse removal schedule, test design, and degree of
desired comfort (National Academy of Sciences, 1969; Hurni, 1970). Hurni
(1970) also suggests that the air should be negatively charged (about 2000
negative ions/cm3) to increase the oxidation of odojrs 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 back flow contamination. For a. lam-
inar 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, since turbulance 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).
2.3.10 Noise and Handling
Noise can be undesirable because of its effects, often underrated,
on test animals and personnel. Since there is always some unavoidable
background noise, it should be considered in designing test facilities
and toxicity studies (U.S. Department of Health, Education, and Welfare,
1974; Hurni, 1970). 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
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2-17
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 uncer-
tain since many of the critical factors such as species auditory levels,
frequency, intensity, and temporal patterns of common noises is unknown.
In general, the effects of noise should be minimized especially where the
exposure could be lengthy or the 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 per-
sonnel training, transferring cleaning chores outside the housing area,
and acclimating the animals to unavoidable background noises (U.S. Depart-
ment of Health, Education, and Welfare, 1974; Hurni, 1970; Fletcher, 1976).
By doing these, the effect of noise on the -test results should be
insignificant.
Contact between the laboratory personnel and the test animals,
handling, can be a detrimental factor which 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, plus 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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2-18
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, (number of levels depending on size of facility, but
usually at least junior and senior technicians), and veterinary personnel
(as supervisors, or consultants) (National Academy of Sciences, 1969).
The personnel caring for and monitoring health of the animals must be
s
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 they certify personnel on three levels:
Assistant Animal Technician, Animal Technician, and Animal Technologists.
The individual facility can then provide the practical on-site training,
which insures the necessary training flexibility (Fpx, 1977). To assist
the veterinarian or toxicologist in the .treatment, test procedures, and
assessment roles, veterinary technologists or toxicology technologists
are often employed (Fox, 1977). By using such certified and special
personnel, many of the problems associated with husbandry requirements
can be minimized so that the test results are not affected.
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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 composition, and in
some cases, controlled levels of health (e.g., SPF strains). After selec-
tion, the animals are usually transported and should be given time to
acclimate before testing. Also they should be quarantined to prevent
introduction of disease into the testing facility. The quarantine is
part of a larger disease control program utilizing 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. Also the design should provide for proper environ-
mental controls. The cage design, especially solid plastic models,
affects the microclimate and thereby the toxicity potential of the test
chemical. Bedding material can also be a 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 envi-
ronment should be controlled within a range of 65-85°F and 30-70%,
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 atmos-
phere 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
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the test results. It is also important to have the correct combination
of well trained personnel to minimize test interference. Only by con-
trolling all of these husbandry variables, will the test results accu-
rately 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 (commerical, 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
*s
to toxic substances. These topics will be considered in the following
section.
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 amino acids which 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).
The recommended levels of amino acids for laboratory animals were listed
in Table 2.3 by Coates, O'Donoghue, and Ward (1969) as cited by Clarke
et al. (1977).
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Table 2.3. Recommended levels for
essential amino acids for laboratory
animals expressed as g/100 g protein
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine+ cystine
Phenylalanine
Threonine
Tryptophan
Tyrosine
Valine
5.0
2.5
5.0
8.0
6.0
s 4.5
5.0
4.0
1.5
4.0
5.5
Source: Coates, O'Donoghue, and Ward,
1969,.cited by Clarke et al., 1977-
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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 during 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 linclude
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 elemnts include
iron, copper, zinc, magnesium, iodine, cobalt, manganese, and selenium
which take part in intracellular metabolic processes as metalloprotein
complexes, and co-enzymes.
In the selection of the proper diet the investigator must bear in
mind that basal dietary requirements as well as specific nutritional
requirements of animals may vary with species or strain and with age,
sex, gestation, lacatation, disease, season of the year, temperature,
relative humidity, ventilation, unknown internal physiological mechanism
and stress (of experimentation) (Hughes and Lang, 1979). Specific
requirements 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.
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2-23
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.
There are many commerical diets available for laboratory animals.
The cost of a diet is a small part of the overall cbst of bioassays and
the investigator should buy the best diet to insure long-term survival
under optimal nutritional conditions (Weisbarger, 1976; Sontag, Page, and
Saffiotti, 1976).
The goal in testing must be to standardize the diet in and among
^
animal studies so that data can accurately be compared.
x
The Food Safety Council (1978) recommends the use of standardized
diets as recommended by the National Academy of Sciences Committee's
guidelines (Subcommittee on Laboratory Animal Nutrition, 1972; Committee
on Laboratory Animal Diets, 1978).
Laboratory animal diets have been characterized by the American
Institute of Nutrition (1977):
Cereal Based Diet} Diet formulations composed predominantly of
Unrefined Diet >: unrefined plant and animal materials which may
Non-purified Diet) contain added vitamins or minerals. Stock
diet, laboratory chow, etc.
/'
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.
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2-24
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 Diet: Diets characterized by the nitrogen source being
provided by pure amino acids, carbohydrate from
refined mono- or disaccharides and fat from puri-
fied 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), the American Institute of Nutrition (1977) and in Guidelines for
Carcinogen Bioassay in Small Rodents (Sontag, Page, nd 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
f
and mouse diet has been found to be satisfactory for reproduction, lacta-
tion and maintenance of the animals. It is produced by several feed manu-
facturers 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 satisfac-
tory in chronic reproduction studies (Food Safety Council, 1978).
The purified diet, AIN-76TM, 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).
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
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2-25
Table 2.4. NIH-7 open formula rat and
mouse ration*2
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
Brewers dried yeast 2.00
Dry molasses f 1.50
Soybean oil 2.50
Sodium chloride , x" 0.50
Dicalcium phosphate 1.25
Ground limestone 0.50
Pre-mixesk 0.25
100.00
Calculated proximate composition = crude protein,
23.5%; crude fat, 5.0%; crude fiber, 4.5%; ash, 7.0%.
^Vitamin and mineral pre-mixes shall provide per
kg diet: vitamin A (stabilized), 6,050 IU; vitamin D3,
5,060 IU; vitamin K, 3.1 mg; a-tocopherylacetate, 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; bio-
tin, 0.15 mg; cobalt, 0.44 mg; copper, 4.4 ing; iron
132 mg; manganese, 66 mg; zinc, 18 mg; iodine, 1.5 mg.
Source: American Institute of Nutrition, 1977.
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2-26
Table 2.5. AIN-76™ purified dieta
(for rats and mice)
Ingredient
Casein
DL-Methionine
Cornstarch
Sucrose
Fiber0
Corn oil"
AIN mineral mix
AIN vitamin mix
Choline bitartrate
20.0
0.3
15.0
50.0
5.0
5.0
3.5
' 1.0
0.2
100.0
*Trade Mark pending.
This diet is intended for growth and
maintenance during 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 per-
iods. The diet has been found to be satis-
factory for reproduction and lactation in
both rats and mice. If used for deficiency
studies, modifications will be necessary.
If used in ultra-clean 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.
Some commercial corn oils contain
antioxidants (maximum 0.02%) and a surfac-
tant (dimethyl silicone). These additives
should be innocuous for most nutritional
studies, but investigators should be aware
of their presence. It is recommended 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.
eThe 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 for
Nutrition, 1977.
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2-27
Table 2.6. AIN-76™ vitamin mixturea
Vitamin
Per kg
mixture
Thiamin-HCl
Riboflavin
Pyridoxine«HCl
Nicotinic acid6
D-Calcium pantothenate
Folic acid
D-Biotin
Cyanocobalamin (vitamin B-12)
Retinyl palmitate or acetate
(vitamin A)
dl-o-Tocopheryl acetate
(vitamin E)
Cholecalciferol (vitamin D3)
Menaquinone (vitamin K)/
Sucrose, finely powdered
600 mg
600 mg
•700 mg
3 g
1.6 g
200 mg
20 mg
1 mg
2.5 m
5.0 mg
To make
1,000.0
Based on the NAS-NRC recommended levels for rats
(2). To be used at 1% of diet.
^Nicotinamide is equivalent.
CAs stabilized powder to provide 400,000 IU vita-
min A activity or 120,000 ret4.no! equivalents.
"As stabilized powder to provide 5,000 IU vitamin
E activity.
SJ.00,000 IU. May be in powder form.
^Menadione.
Source: American Institute of Nutrition, 1977.
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2-28
Table 2.7. AIN-76™ mineral mixturea
Ingredient ?/kg
mixture
Calcium phosphate, dibasic (CaHPO,,) 500.0
Sodium chloride (NaCl) 74.0
Potassium citrate, monohydrate
(K3C6Hs07«HaO) 220.0
Potassium sulfate (K2SO<,) 52.0
Magnesium oxide (MgO) 24.0
Manganous carbonate (43-48% Mn) 3.5
Ferric citrate (16-17% Fe) 6.0
Zinc carbonate (70% ZnO) 1.6
Cupric carbonate (53-55% Cu) 0'3
Potassium iodate (KI03) ^ 0.01 ,
Sodium selenite (NaaSe03»5HaO) ' 0.01 \
Chromium potassium sulfate
[CrK(SOja«12HaO] 0.55
Sucrose, finely powdered To make
1,000.0
Based on the NAS-NRC requirements for rats (2).
To be used at 3.5% of the diet.
» • •
Source: American Institute of Nutrition, 1977.
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2-29
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
a change in diet manufacture occurs all should be changed to the new diet
at the same time (Weisburger, 1976; Sontag, Page, and Saffiotti, 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, and radiation, and contamination by microorganisms-all of which
may damage the nutrient quality of a diet (Clarke et al., 1977). Sontag,
Page, and Saffiotti (1976) recommend sterilization of'feed (without
degrading nutrients) when practical, and whe'n 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 (Fox, 1977;
Newberne, 1975; Food Safety Council, 1978).
Variations in the concentration of essential nutrients can occur
for a variety of reasons: batch or lot'differences, regional and seasonal
differences, manufacturerer variations, and processing. Most diets con-
tain nutrients in quantities sufficient for growth, maintenance and repro-
duction of a particular species. However, concentrations of essential
ingredients may vary from batch to batch of a formulation made with dif-
ferent lots of natural ingredients, and at the same time the guaranteed
analysis shown on the label can remain correct (Newberne, 1975).
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2-30
Differences in manufacturing procedures were pointed out by Porter
et al. (1963) who compared growth rates of groups of young mice fed diets
produced in the same region by 3 different manufacturers and found con-
siderable differences (cited by Hurni, 1970). Also, nutritional quality
may be seriously impaired during processing, particularly by overheating
which can destroy amino acids or can lead to the formation of indigestable
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).
Becasue of the presence of a variety of contaminants in animal diets
it is also advisable to test periodically for pesticides, mycotoxins, trace
minerals, and industrial contaminants (such'as polychlorinated biphenyls,
lead and mercury) (Newberne, 1975; Page, 1977; Food Safety Council, 1978;
National Research Council, 1977). Low levels of N-nitrosamines have been
detected in the diets of experimental animals in Germany and in the United
States (Walker, Castegnaro, and Griciute, 1979;.Edwards et al., 1979). The
source of the nitrosamines is thought to be fishmeal, and Knapka (1979)
suggested that the N-nitrosamines are present only in diets purchased
in meal form.
There is a growing interest in obtaining detailed "open formula"
f
information for assurance that diets are free from pesticide residues
and other contaminants (Food Safety Council, 1978); and if prospective
analysis for contaminants cannot be arranged with feed suppliers then
retrospective analysis could be conducted.
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2-31
The ultimate solution to the problems of uniform constituents and
toxic contaminants in animal diets may be provided by the use of semi-
synthetic rigorously purified, or entirely synthetic diets (Food Safety
Council, 1978; Clarke et al., 1977).
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 comprised of cytochrome P-450, phosphatidycholine and a flavo-
protein 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 pro-
tein, 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 those that are rendered toxic by biotransformation.
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).
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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 hydrox-
ylation (Swan and McLean, 1968).
Silverstone and Tannenbaum (1951) demonstrated tht 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 dif-
ference ^n the proportion of total protein. Kawachi et al. (1968) demon-
strated that addition of 1% tryptophan to the diets of rats receiving a
low level of /V-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).
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2-33
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 et al., 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 N-2-fluorenylacetamide- and dimethylbenzanthracene-
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 N-2-fluorenylacetamide developed a significantly higher number of
hepatic carcinomas than controls.
2.4.5.4 Vitamins — The role of vitamin deficiencies in the mixed
function oxidase system have been reviewed comprehensively by Campbell
and Hayes (1974). The effects don't appear to be as serious as the
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2-34
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 mamlforaations 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 meta-
plasia and squamous cell tumors of the respiratory tract of rats and ham-
sters 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(
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2-35
2.4.6 Conclusions
Diets of experimental animals should contain proteins, carbohydrates,
fats, minerals, micronutrients, and energy in the proportions suitable
for a particular species or strain. Various types of diets available
include non-purified 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 the effects
they have 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.
s
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, participating in the test
design and the selection of the animal models (Page, 1977). Quality
control must be emphasized at all times, since 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
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2-36
limitations of pathology must be recognized including a lack of informa-
tion on: early lesions that disappear before necropsy; on the sequence
of lesion apperance; on the reversibility of lesions; and on the develop-
mental history (morphogenesis) of each lesion (Zbinden, 1976). The fol-
lowing 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 information will
be lost. Therefore the necropsy should either be directly performed by
the pathologist (or the scientist who is tpxdo the microscopic examina-
tion) or by a trained technician with consultation from the pathologist
(Magee, 1970; Food and Drug Administration, 1971; Prieur et al., 1973;
Sontag, Page, and Saffiotti, 1976; World Health Organization, 1978; Food
Safety Council, 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 insure that the maximum information is obtained from the test
animals, a daily observation should be included to reduce loss from post-
mortem degeneration (Roe, 1965; Magee, 1970). The necropsy should be
done as soon as possible after death and if not, then 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,
since it can indicate the general lesions to be expected (Magee, 1970;
World Health Organization, 1978). If an animal is moribund, sacrifice
-------
2- 37
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) and it is often easiest and most thorough to use a
check list system (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 (Sorftag, 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 life-time studies (Magee, 1970). If
weight is determined, it should be done as soon as possible to reduce
drying efrfects (World Health Organization, 1978).
The necropsy should start with an external examination including
orifices (Sontag, Page, and Saffiotti, 1976; World Health Organization,
1978). Next, examine all the internal cavities and .organs in situ and
do not separate the connective tissue until it has also 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 including: (1) parenchymal and endocrine glands
should be thoroughly examined including multiple cut slices, since these
tissue often contain deep seated lesions (Sontag, Page, and Saffiotti,
-------
2-38
Table 2.8. Tissues to be Included in a
gross examination
Gross lesions
Tissues masses or suspect tumors
and regional lymph nodes
Skin
Mandibular lymph node
Mammary gland
Salivary gland
Larynx
Trachea
Cecura
Colon
Rectum
Mesenteric lymph node
Liver
Thigh muscle
Sciatic nerve
Sternebrae, vertebrae, or femur
(plus marrow)
Costochondral junction, rib
Thymus
Qallbladder.
Pancreas
Lunga and bronchi
Heart
Thyroids
Parathyroids
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Spleen
Kidneys'
Adrenals
Bladder
Seminal vesicles
Prostate
Testes
Ovaries
Uterus
Nasal cavity
Brain
Pituitary
Eyes
Spinal cord
Source: Adapted from Sontag, Page, and Saffiotti,
1976.
-------
2-39
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
(Sontag, Page, and Saffiotti, 1976; Food Safety Council, 1978; World
Health Organization, 1978); (3) the skull and brian should be cut open
and examined including the examination of the pituitary (Magee, 1970);
and (4) 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 since much of the lesion incidence data is generated
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 the observations of the gross and
microscopic examinations in carcinogenicity studies. Table 2.9 shows
how frequently the gross examination detected lesions also seen in micro-
scopic examinations in mice. Frith et al. (1979) concluded that the size
of the osgan, the stage and nature of the lesion, and the number of
slices examined all affect the efficiency of the gross examination.
Kulwich et al. (1980) found correlation rates between gross and micro-
scopic examinations of 70% and 76% for two chemicals in rats. They con-
cluded that gross examination is less successful for small organs, since
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.
-------
2-40
Table 2.9. Correlation between gross and microscopic lesions in
carcinogenic studies in mice
Organ
Liver
Thyraus
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.
-------
2-41
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 possi-
ble, 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). How-
ever, 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 Organization,
1978). Perfusion cannot be used if organ weights are needed (World Health
Organization, 1978).
^
The fixative most recommended is neutral buffered formation at
/
concentrations of 10% (Sontag, Page, and Saffiotti, 1976; Prieur et al.,
1973; 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 in-
volved (Zbinden, 1976; Prieur et al., 1973). The tissues should be pre-
served at a maximum thickness of 0.5 on (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 1:15-20 (Prieur et al., 1973).
The tissues should be left in the preservative for at least 24 hr and
up to 72 hr has been suggested (Prieur et al., 1973; Sontag, Page, and
Saffiotti, 1976; World Health Organization, 1978). Often special proce-
dures are suggested including: covering tissues that float with absorbant
-------
2-42
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; Zbinden, 1976; World Health Organization, 1978); several
thoracio-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 tissues masses should be fixed (Sontag, Page, and Saffiotti,
1976). After preservation, the material should be stored in plastic
bags or jars and kept at least until the final conclusions are made for
that 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 max-
»
imum possible area for examination (World Health Organization, 1978).
Oftan a set trimming schedule should be used. The trimming should be
performed by or in the presence of the pathologists, with consultation
of the gross examination observations (Sontag, Page, and Saffiotti, 1976).
The tissues should be trimmed to a thickness of 2-3 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 Heatlh Organization (1978) including: (1) multiple portions
-------
2-43
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 mid-longitudinal section and
the other by a mid-transverse 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 neededy 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, then the section should
also include the pharnyx; and (8) if the nasal cavity'is to be examined,
s
then three transverse sections are needed.
Routeinely, the trimmed slices should be stained with hematoxylin
and eosin, then embedded in paraffin (Zbinden, 1976). These are sliced
to 4-6 ym for microscopic slides (Sontag, Page, and Saffiotti, 1976;
World Health Organization, 1978). The paraffin' embedding can be per-
formed using automation techniques. Often semi-thin slices (1 ym) are
needed for such organs as the bone marrow, kidney, or endocrine glands
(World Health Organization, 1978). Also special stains are often employed,
for example in evaluations for the presence of fats, carbohydrates or
special structures (Zbinden, 1976; World Health Organization, 1978).
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-.
-------
2-44
2.5.5 Microscopic Examination
The microscopic examination of properly prepared tissues slides is
usually the final step in a pathology evaluation. Although its Importance
is overwhelming accepted, the details of the design are quite variable and
change 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. Sections 4.5 and 6.6.3 review some of the specific litera-
ture 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
,s
maximum amount of scientific information, and the limits imposed by prac-
tical economic and time constraints. Therefore, many discussions recommend
only vague guidelines, while others are extremely specific and encompassing
(Page, 1977). As an attempt to reach a compromise solution, Zbinden (1976)
recommencfed a priority system based oh 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 with the number
of chemicals that will have to be evaluated, that the microscopic examina-
tion will have to be based on some similar type of ranking system.
Another approach to reducing the amount of microscopic examination
has been proposed by Fears and Douglas (1977; 1978). Based on a statis-
tically valid sampling mpthod, 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
-------
2-45
Table 2.10. Organs and tissues to be examined in
routine' toxicity tests
First Priority: All animals including controls
Liver (f)u
Kidney (f)
Adrenal glands (£)
Heart (left ventricle)
Spleen (f)
Thymus
Testis
Epldidymis
Lung
Bone marrow ,
(f) 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
Op*lc nerve
Peripheral nerve
Urinary bladder
Skin
Mammary gland
Bone-cartilage
Skeletal muscle
Gall bladder
Third Priority: Organs and tissues not examined in routine
experiments, unless indicated by clinical observations or motivated
by .scientific interest
Heart valves
Purkinje fibres
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 fibre endings
Meninges
External ear
Middle ear
Inner ear
Olfactory organ
Subcutaneous lymph nodes
Tendeons
Adipose tissue
Intervertebral disc
Synovial membrane
Lacrimal glands
(f) — frozen sections stained for fat obligatory.
Source: Adapted from Zbinden, 1976.
-------
2-46
that were found to be affected in the treatment groups. They also sug-
gest 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 poten-
tial 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 you cannot
rely totally on the gross examination to select affected tissues for micro-
scopic study. They stressed that a thorough microscopic examination is
still necessary. Therefore, the approach suggested by Fears and Douglas
still needs more refinement, but represents1^ 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 check list to insure 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 semi-quantitatively (World Health
Organization, 1978). The formation of tumor tables incorporating infor-
mation on the group, organ, and sex variables for each lesion can be use-
ful 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 represented
-------
2-47
(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 cited in Page, 1977). However,
many other reviewers stress that the danger of missing lesions or incor-
rectly interpreting the results far outweigh bias problems and the pathol-
ogist should be aware of the sample group (Roe, 1965; Zbinden, 1976; World
Health Organization, 1978). 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 i
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, especially if special
stains are utilized; and (5) the microscopic examination should be as
thorough as practical limitations will allow. Only by following these
requirements will the pathology contribute meaningful data for evaluations
of toxicity.
-------
2-48
SECTION 2
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Toxico^ogic Evaluation of Cancer Chemotherapeutic Agents: Protocols
of the Laboratory of Toxicology. Cancer Chemother. Rep. Part 3
Roe, F.J.C. 1965. Spontaneous Tumors in Rats and Mice. Food Cosmet.
Toxicol. 3:717-720.
Rogers, A. E. 1975. Variable Effects of a Lipotrope-Def icient High-
Fat Diet on Chemical Carcinogenesis in Rats. Cancer Res. 35:2469-2474.
/
Rogers, A. E., 0. Sanchez, F. M. Feinsod, and P. M. Newberne. 1974.
Dietary Enhancement of Nitrosamine Carcinogenesis. Cancer Res. 34:
96-99.
Saffiotti, U., F. Cefis, R. Montesano, and A. R. Sellakumar. 1966.
Induction of Bladder Cancer in Hamsters Fed Aromatic Amines. Indust.
Med. Surg. 35:564.
-------
2-53
Shaw, B. H. 1976. Air Movements within Animal Houses. In: Laboratory
Animal Handbooks 7: Control of the Animal House Environment. T. McSheehy,
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Short, D. J. 1967. Handling, Sexing and Palpating Laboratory Animals.
In: Husbandry of Laboratory Animals, Proceedings of the Third Inter-
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M. L. Conalty, ed. Academic Press, New York. pp. 3-15.
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*•
Smith, D. M., A. E. Rogers, B. J. Herndon, and P. M. Newberne. 1975.
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-------
2-54
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•'
s
-------
3. ACUTE TOXICITY
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 regimen may be
^
observed within a few minutes 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 LDSo studies the test substance is commonly given by gavage
in a single or divided doses within one day and the animals are observed
r
for 2 weeks; however, when possible the route of exposure should be the
exposure route that is likely to be encountered in humans. In acute
feeding studies several levels are tested in at least two species and
animals are fed for a week or more and the animals are observed as in the
LD5o studies. The number and 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.
3. To give dose-ranging guidance for other tests.
This section will discuss aspects of acute toxicity such as the LD30,
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
3.2 LD30 DETERMINATIONS
3.2.1 Introduction
The most frequently determined index of toxicity is the LDSO, the dose
that is lethal to half (50%) of a group of treated animals (National Academy
of Sciences, 1977). In addition, the LD50 can also be used as a basis for
determining doses to be used in subsequent subchronic and chronic toxicity
studies. It can also aid the researcher in determining which species and
route of administration would be the most suitable for his experiments. The
following sections will show how the choice of species, route of administra-
tion, and sex of the animal may affect the LD30 and acute toxicity response.
/•
The information presented on acute LD30,s is available in the NIOSH
Registry of Toxio Substances (1977) which has entries in the following
format:
BR09500 Ammonium, (2-hydroxyethyl)diisopropylmethyl-, bromide, xanthene-
9-carboxylate
TXDS: ^Oral-rat LDSO: 370 mg/kg
Intraperitoneal-rat LD30: 25 mg/kg
Subcutaneous-rat LD5o: 298 mg/kg
Intraduodenal-rat LD30: 125 mg/kg
Oral-mouse LD30: 445 mg/kg
Intraperitoneal-mouse LD50: 78 mg/kg
Oral-rabbit LD30: 750 mg/kg
Intravenous-guinea pig LDLo: 51 mg/kg
This format provides information on the route of administration, the
species, and the toxic LD30 dose or LDLo dose (lowest published lethal dose
reported). Also included in the NIOSH document are synonyms, Chemical
Abstracts Service registry numbers, and the reference for each item of tox-
icity data. Table 3.1 is a list of 96 compounds, arbitrarily chosen, for
-------
3-3
Table 3.1. Compounds examined for LDSO differences between
species and between routes of administration
Ammonium, (2-hydroxyethyl)diisopropylmethyl-, bromide, xanthene-9-
carboxylate N
Ammonium, ((p-hydroxy-2-isobutyl)phenyl)trimethyl-, iodide,
methylcarbamate
Arsine, dichloro(2-chlorovinyl)-
Benzaldehyde, p-isopropyl-
Benzamide, 4-amino-/P-cyclopropyl-3,5-dichloro-
Benzimidazole, 2-(4-thiazolyl)-, hydrochloride
2-Benzimidazolinone, 5-chloro-l-(3-(dimethylamino)propyl)-3-phenyl-
Benzoic acid, benzyl ester
Benzoic acid, 2,3,6-trichloro-
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-Benzoxazolinone, 5-chloro- ^
Benzyl alcohol, alpha-(1-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 tetrachloride
2-Carboxymethylmerc'aptobenzenestibonic acid
Cerium chloride
Ethylamine, ff-methyl-2-((o-methyl-alpha-phenylbenzyl)oxy)-,
hydrochloride
Ethylene glycol
Guanidine, l-cyano-3-tert-pentyl-
3-Heptanone, 6-(dimethylamino)-4,4-diph,enyl-, hydrochloride
1,6-Hexanediamine, W.W.^S^^tetramethyl-, polymer with 1,3-
dibromopropane
Hydrazine, methyl-, hydrochloride
Hydroquinone
Imidazole
-------
3-4
Table 3.1 (continued)
3-Imidazoline, 4-amino-2,2,5,5-tetrakis(trifluoromethyl)-
1,3-Indandione, 2-((p-chlorophenyl)phenylacetyl)-
X
1 H-Indazole, l-benzyl-3-(3-dimethylamino)propoxy)-, monohydrochloride
Indene-1-ethylamina, tf,tf-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-chlorophenyl)-l-(3-(p-fluorobenzoyl)propyl)-
//, //-dime thy 1-
Isonipecotic acid, l-(p-aminophenethyl)-4-phenyl-, ethyl ester
Meglumine hydroxamate
Mercury, (3-(alpha-carboxy-o-anisamido)-2-(2-hydroxyethoxy)propyl)
hydroxy-, monosodium salt
Mercury, (3-cyanoguanidino)methyl-
f
Mercury (11) iodide
s
4-Metathiazanone, 2-(3,4-dichlorophenyl)-3J-methyl-, 1, 1-dioxide
Methanesulfoanilide, 4'-(l-hydroxy-2-(methylamino)propyl)-,
hydrochloride
Methanesulfonic acid, iodo-, sodium salt
2,6-Methano-3-benzazocin-8-ol, 1,2,3,4,5,6-hexahydro-3-allyl-6-ethyl-
11-methyl-
4,7-Methanoindene, l,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-
Monoraethylhydrazine nitrate
Morphinan-3, 14-diol, 17-(cyclopropylmethyl)-
Morphinan-3,6-alpha-diol, 7,8-didehydro-4,5-alpha-epoxy-17-methyl-
Morphinan, 17-(p-nitrophenethyl)-, hydrochloride (+.-)-
Morphinan-6-alpha-ol, 7,8-didehydro-4,5-alpha-epoxy-3-methoxy-17-
methyl-
2-Naphthalenemethanol, alpha-((isopropylamino)methyl)-
Naphthalimide, N-hydroxy-, diethyl phosphate
2-Naphthol, 1,2,3,4-tetrahydro
Neoraycin
Nickel (11) acetate(l:2)
-------
3-5
Table 3.1 (continued)
Nicotinaraide, tf.tf-diethyl-
2-Norbornanamine, tf-ethyl-3-phenyl-, hydrochloride
2-Norbornanamine, /17,3,3-trimethyl-
2-Norbornanamine, tf,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-Oxadiazolidine-3,5-dione, 2-(3,4-dichlorophenyl)-4-methyl-
2H-l,3-Oxazine-2,4(3H)-dione, 5,5-diethyldihydro- '
Pactomycin
Penicillanic 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 s
Phenol, 2,4-dinitro-
Phenol, 2,4,5-trichloro-
Phenothiazine, 2-chloro-10-(3-(]7-cyclopentyl-^-methyl)aminopropyl)-
Phenothiazine, 10-(2-(diethylamino)propyl)-
Phosphonothioic acid, phenyl-, 0-ethyl O-(p-nitrophenyl) ester
Phosphotfamidothioic acid, 0,S-dimethyl ester
Phosphoric acid, 2-chloro-l-(2,4-dichlorophenyl)vinyl diethyl ester
1,'3-Propanediol, 2-(hydroxyraethyl)-2-nitro-
1,2-Propanediol, 3-(o-methoxyphenoxy)-, 1-carbamate
Pyrrolidinium, 3-hydroxy-l,l-dimethyl-, bromide,alpha-
cyclopentylmandelate
3-Pyrrolidinol, l,2-dimethyl-3-phenyl-,' propionate
Quinine, hexylbromide
Sodium pentafluorostannite
Streptomycin
Succinic acid, cadmium salt (1:1)
Sulfanilamide
-------
3-6
Table 3.1 (continued)
Sulfanilamide, N(sup l)-2-thiazolyl-
5H-Tetrazoloazepine, 6,7,8,9-tetrahydro-
s
Theophylline, 8-benzyl-7-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-,
hydrochloride
2H-l,3,5-Thiadiazine-2-thione, tetrahydro-3,5-dimethyl-
s-Triazine, 2-azido-4-(isopropylamino)-6-(methylthio)-
s-Triazine,2,4-dichloro-6-(o-chloroanilino)-
Urea, 3-(hexahydro-4,7-methanoindan-5-y1)-1,1-dimethy1-
Urea, 1,1,3,3-tetramethyl-
-------
which the LD30 values were examined and compared for species differences
and for differences between routes of administration. Although not dis-
cussed, it is recognized that the differences in reported LDSo values may
have also been influenced by the age and strain of the test animals, dura-
tion of the starvation period prior to dosing, ambient temperature, concen-
tration 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.
3.2.2 Difference Between Species
Table 3.2 uses the LDSo data for each compound listed in Table 3.1 to
f
show the LD30 differences (expressed as percent) between different species
s
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 LD30 by 1-25%, for
three compounds the rat LD30 was greater than the cat LD30 by 51-75%, and
for one other compound the rat LD30 was greater by 76-100%. Therefore,
for 4 of the 5 chemicals compared, the rat showed a higher tolerance to
the acute dose. Collective examination of data in Table 3.2 reveals that
5.8% of the species compared showed np'difference in LD30 amounts, 23.3%
of the species had LD30 values differing by 1% to 25%, 29.4% had differing
LD30 values of 26% to 50%, 27.7% of the species had LD30 values that dif-
fered by 51% to 75%, and 13.9% of the species had LD50 differences between
76% to 100%. Therefore, less than half of the 374 species comparisons
(41.7%) showed LD30 values that differed by more than 50%. A more detailed
examination of the data in Table 3.2 reveals the following:
-------
Table 3.2. Percent LDjo differences between species
Species
Cat
Pigeon
Rat
Chicken
Guinea pig
Hamster
Mouse
Pigeon
Quail
Rabbit
Dog
Cat
Guinea pig
Hamster
Monkey
Pigeon
Rabbit
Rat
Route of
administration Q i to 25
Oral
Oral Cac
Oral Guinea pig
Oral
Subcutaneous Mouse
Oral Mouse(2)fc
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
Percent
26 to SO
Guinea pig
Hamster
Quail
t,
Dog(2)\
Dog
Monkey
Pigeon
%
Dog
Rabbit
h
Rat, dog(2)
Dog
Rat .
Rat(2r
Rat
differences
51 to 75 76 to 100
. Pigeon0
Rat (3)° Rat
Mouse
Mouse
Pigeon
Pigeon
Rabbit
Dog
Guinea pig Dog
Monkey
1
Dog
Dog
Rabbit .
Rat, dog(2) Rat, dog
Rat , dog Dog
CO
CO
-------
Table' 3.2 (continued)
Species
Duck
Chicken
Dog
Mouse
Pigeon
Quail
Guinea pig
Cat
Hamster
Monkey
Mouse
Pigeon
Rabbit
Rat
Route of
administration
Intravenous
Oral
Intravenous
Oral
Intravenous
Oral
Oral
Intraperitoneal
Oral
Subcutaneous
Oral
Intraperitoneal
Subcutaneous
Intravenous
Oral
Subcutaneous
Skin
Oral
Oral
Subcutaneous
Skin
Intraperitoneal
Intravenous
Percent differences
0 1 to 25
Chicken
Pigeon
1°
Guinea pig
1° Mouse(2)fc
Guinea pig
1°
Guinea pig
1° Guinea pig(2)2),
rat
1 Rat
Guinea pig
26 to SO
Quail
Mouse (4) , guinea
pig(2)Z>
Mouse,' guinea pig
Guinea {Jig (2)fc
Guinea pig
Rabbit
Guinea pig
Rabbit, guinea
Pig » .
Guinea pig(2)°,
rat(2)*>
Guinea pig
51 to 75
Chicken
Quail
Cat, guinea pig
Monkey
Guinea pig
Guinea pig
j,
Rabbit (2) .
guinea pig
Guinea plg(4) ,
rat(3)fc
Guinea pig, rat
Rat
76 to 100
Dog
Duck
Pigeon
Pigeon
Guinea pig
Mouse (2)b
l Guinea pig
Rat
Guinea pig
-------
Table 3.2 (continued)
Species
Mouse
Cat
Dog
Hamster
Monkey
Pigeon
Rabbit
Quail
Dog
Pigeon
Rat
Rabbit
Cat
Duck
Monkey
Rat
Duck
Hamster
Route of
administration
Oral
' Intraperitoneal
Oral
Subcutaneous
Intraperitoneal
Intravenous
Oral
Intraperitoneal
Subcutaneous
Oral
Intravenous
Or.al
Subcutaneous
Intravenous
Oral
Oral
Oral
Oral
Oral
Subcutaneous
Oral
Oral
Intravenous
Oral
Percent differences
0 1 to 25
Dog, mouse
Mouse ,
1° Mouse (2)°
Mouse
Mouse
Mouse ,
Rabbit(2)
•
Pigeon
Cat
Duck
Rat, hamster
26 to 50
Cat
Mouse ,
Mouse (3)
Mouse
Mouse
Mouse
j.
Rabbit (2) ,
mouse (3)°
Mouse, rabbit (2)°
Rat
Duck
Monkey
Hamster, rat
51 to 75 76 to 100
Mouse (3)^ Mouse
.
Mouse Mouse (2)
Dog
Mouse
Mouse Mouse
Hamster
Monkey
b b
Mouse (2) , Rabblt(2)
rabbit
Mouse (2)0
Mouse(2)D Mou3e(2)°
Dog
Pigeon
Rat «
Monkey
Rat Rat, duck
U)
M
O
-------
Table 3.2 (continued)
Species
Rat
Monkey
Mouse
Pigeon
Rabbit
Route of
administration
Intraperitoneal
Skin
Subcutaneous
Oral
Oral
Intraperitoneal
Intravenous
Subcutaneous
Intramuscular
Oral
Intravenous
Oral
Subcutaneous
Intraperitoneal
Intravenous
Skin
Intramuscular
Percent differences
0 1 to 25
Hamster (3)
Hamster
a b
It Mouse(9)f,
rat (8)
2° Mouse (S)6,
rat (4)*>
t,at,\^f
2° Mouse(5)6,
rat (2)*1
Mpuse(5)t
,
Rat or
ft
2 Rabbit (2)
2° .
2° Rat, rabbit
Rabbit
26 to 50
Monkey
Monkey ,
Mouse (7)?,
rat (6r
Mouse (4)*,
rat (3}
Mouse (3) J,
rat (4)
Mouse (2)*,
rat(2)b
Rabbi t\
Rat , rabbit
Rat, rabbit
Rat(2)*>, rabbit
Rat
51 to 75
Hamster(2)
j,
Mouse (7)D,
rat (10)*
Mouse (7)6,
rat (2.}®
Mouse (3)fc, rat
Mouse(2)£.
rat(2)fc
Mouse
h
Rabbit (3) , rat
Rat, rabbit
1
76 to 100
,
Mouse (5) ,
rat (3)Z>
Mouse
Mouse
Mouse
Pigeon .
Rabbit (3) , rat
h
Rat, rabbit (2)
i
Indicates which animal of the comparison had the greatest LD30- Position of species indicates the per-
cent differences In the LD30s of the tuo species being compared.
^Represents number of chemicals compared where LD30 of species indicated was greater than species being
compared. If there is no number then only one chemical was compared.
cThe number indicates how many times the W,0 values were identical for the species compared.
CO
I
-------
3-12
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
but slightly more sensitive than the mouse.
3. The mouse is a less sensitive toxicity indicator than the hamster.
4. The data show that the differences between LDSO values for the mouse
*•
and rat were less than 50% for 56% of the comparisons.
5. The mouse is shown to have a larger LDSO (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 half (58%) of all
the species comparisons, the LD30 values differed by less than 50%.
3.2.3 Difference Between Administration Routes
Table 3.3 shows the percent difference between LD30 values for various
pairs o^E 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 mouse at 76% to 100% indicates that the /LD5<> for the intraperito-
neal route of administration was 76% to 100% greater than the LD50 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% or no
difference in LDSO 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 LD30 can vary significantly depending
on the administration route.
-------
Table 3.3. Percent LDjo differences between routes of administration
Routes of
administration
Intracerebral and
intraperitoneal
Intracerebral and
Intravenous
Intracerebral and
subcutaneous
Intraperitoneal and
intramuscular
Intraperitoneal and
skin
Intraperltoneal and
subcutaneous
Intravenous and
intramuscular
Intravenous and
intraperltoneal
Intravenous and oral
Intravenous and skin
Intravenous and
subcutaneous
Percent differences
0 1 to 25 26 to 50 51 to 75
Mouse
Mouse
Mouse
Mouse Inm, Inp
Rat Inm
Rat Skin
Hamster
Guinea pig
.
Rat 1° Inp, Sub Sub(3)^
Mouse Sub(5)*, Inp(3)* Sub(7)* Sub(3)
Rabbit '
Rat Inm ^
Mouse Inv, Inm
•
b b
Rat Inv, Inp Inp(4) Inp(4)
Mouse Inv(3)*, Inp(3)* Inp(7)* Inp(8)*, Inv ,
Rat Inv Oe 0(5)
Mouse lc 0(4)*, Inv 0(2)fc > 0(7)*, Inv(2)*
Dog 0
Guinea pig
Rabbit 1°
Rabbit
Rat
Rabbit . . Sub ,
Mouse Sub(2), Sub(6) Sub(8),
Rat Sub(2)D Sub Sub (3)
Guinea pig
76 to 100
Inp
Inv
Sub
•j,
Skin(2r
Skin
Skin
j,
Sub
,
Inm
Inm
Inp(2)b
Inp
0(11)*
0(18)*
0
Skin
Skin
V
Sub (2)?
Sub (7)?
Sub (7)
Sub
CO
M
U>
-------
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
Subcutanoues and
skin
0 1 to 25
Mouse
Rat
Mouse 1
Rat 2° 0(5)?. Inp(2)*
Mouse 0(7)
Hamster
Cat Inp
Rabbit Inp
Guinea pig 1
t
Rat 0(9)* b
Mouse o 0(6) , Sub(2)
Guinea pig 1
Rat
Mouse
Rabbit 0
Hamster
Guinea pig
Rabbit Inm
Mouse Sub
Rat
Dog
Rabbit
Guinea pig
Percent differences
26 to SO 51 to 75
Inm
0
0(6)* Inp 0(10)* .
0(7)°, Inp 0(11). Inp(2)°
0 0(3)0
1-
0(3)7, Sub Sub . .
0(7) , Sub 0(4)°, Sub(3)
Skin 0, Sklnpr
N Skin
Sub
,
Skin
Skin
76 to 100
0
0
0(12)fc. Inp
0(9)0
0
,
Skln(5r
0
Skin
Skin
Skin
Skin
Skin
,Inm — 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.
cThe number indicates how many times the LD30 values were idential In a comparison.
I
M
*-
-------
3-15
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 LD30 than the intravenous route.
2. From 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 adminis-
tration route is shown.
3. When the intravenous route was compared to the oral and intraperitoneal
routes, respectively, the LD30 values were greater for the oral and
*
intraperitoneal routes for most of the^compounds. In fact, in only 4
of 56 comparisons (2 being equal) was the intravenous LD30 greater
than the oral LD90 (those 4 compounds were methylhydrazine hydrochloride,
2-isopropylhydrazide isonicotinic acid, pactamycin, and 2-chloro-l-
[2,4-dichlorophenyl]vinyl phosphoric acid diethyl ester), and in only
» •
5 of 35 comparisons was the intravenous LDSO greater than the intra-
peritoneal route (those 4 compounds [2 comparisons involved the same
compound] were methylhydrazine hydrochloride, 4-amino-2,2,5,5-tetrakis
[trifluoromethyl]-3-imidazoline, 2-isopropylhy'drazide isonicotinic
acid, and N[sup l]-2-thiazolylsulfanilamide).
4. The oral LD30 was greater than the subcutaneous LD50 for 82.5% of the
comparisons; however, the difference was generally less than 50%.
5. A comparison between the oral LD30 and intraperitoneal LD50 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-(l-aminoethyl)benzyl alcohol hydrochloride,
-------
3-16
l-cyano-3-tert-pentylguanidine, l-(p-chlorobenzoyl)-5-methoxy-2-
methylindole-3-acetic acid, pactamycin, tetrahydro-3,5-dimethyl-
2H-1,3,5-thiadiazine-2-thione, 2 ,4-dichloro-6-(o-chloroanilino)-s-
triazine, 4-amino-N-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
emphazises the importance when conducting safety assessments of chemicals
of selecting the route of exposure that will be the likely exposure route
in man.
t
s
3.2.4 Sex Differences in the Laboratory ~Rat
Male and female rats do 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 any
other species. One example is provided by the research of Kast et al.
(19753) with the amino-halogen-substituted benzylamine, fominoben HC1.
The oral LDSO for males was 6450 mg/kg and for females, 4500 mg/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 LD30 112 mg/kg), Bayer 37289 (male LDSO 180
mg/kg and female LDso 64 mg/kg) and ethion .(male LDso 245 mg/kg and
female LDSO 62 mg/kg). Hayes (1975) used the oral toxicity LDSo data
-------
3-17
of Gaines (1960, 1969) on the rat for 69 pesticides and calculated the
ratio of female to male oral LDSo's. 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 then there is no further need for contin-
,/
uing 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 Regu-
latory 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 mg/kg. Similarly, the Organization for Economic Cooperation
and Development's guidelines state that if 5 of 10 animals survive a dose
of 5 rag/kg of a chemical for 14 days, then no further acute toxicity test-
ing is necessary. If these criteria are met in one sex but not the other
then acute toxicity testing must proceed with the sex that did not meet
the criteria for limting the acute test.
3.2.6 Conclusions
The information in Sections 3.2.2 and 3.2.3 clearly indicates the
importance of proper species and route of administration selection.
-------
3-18
Specifically, 42% of all species comparisons (Table 3.2) indicated dif-
ferences in LDso values of more than 50%, and 56% of all comparisons
involving administration routes (Table 3.3) showed differences between
LDso values of more than 50%. When using the laboratory rat as the test
animal in acute toxicity studies, both male and female animals should be
used because of the variation in response between the sexes. If a chemical
^
does not cause death when administered in acute doses of 5 g/kg then fur-
ther 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
f
they are easy to work with, relatively inexpensive, and there is a vast
amount of information available on which to base comparative assessments.
The difference seen in LDSO 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.
-------
3-19
3.3.2 Comparison of Lowest Published Lethal Doses (LDLo)
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 pub-
lished lethal dose. Twenty-seven compounds (Table 3.4) were chosen at
random and the human and animal LDLo data compared in Table 3.5. The fol-
lowing 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 LDLo equal, whereas
8.6% of the comparisons had LDLo differences between 1% and 25%,
17.3% between 26% and 50%, 15.2% between 51% and, 75%, and 60.8%
between 76% and 100%. '
3. Only 19.5% of the comparisons showed the human LDLo value to be
greater than the animal LDLo value.
On the basis of this data, it can be concluded is that there is a signifi-
cant difference between the human and animal acute LDLo response and that
in the majority of instances, the human showed less tolerance (reflected
by lower LDLo values) to chemical insult than the animal 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 kid-
ney to be similar. In his review of lead effects on the kidney, Goyer
found that morphological and functional reactions of the kidneys of rat
and man to acute lead exposure have features which are comparable: (1)
the proximal tubular lining cells are affected, (2) intranuclear inclusion
-------
3-20
Table 3.A. Compounds selected for human vs animal
LDL0 comparisons
(mg/kg)a
Chemical Dose
s
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-ff-(2-diethylamino)ethyl)0- 50
Ammonium, (4-(bis(p-(dimethylamino)phenyl)
methylene)-2,5-cyclohexadien-l-xylidene)
dimethyl-, chloride 50
Acet*anilide, 4'-hydroxy 50
Methane, iodo- 50
2-Naphthol 50
Quinoline, 8((4-(diethylamino)-1-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. LDLo differences between humans and animals
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 1
Skin
Intravenous
• Percent differences
1 to 25 26 to 50 51 to 75 76 to 100
Rata Human, Rat (2)
Human
Rat
b b b
Rabbit Rabbit (2) Human, Rabbit (3) Rabbit (5) , Human
Rabbit
Pigeon Pigeon
Guinea pig (2) Guinea pig
Chicken
Cat
Mouse
\ b b
x Human Dog (7) , Human (2)
Dog
Dog /
Indicates which animal of the comparison had the greatest' LDLO- Position of species indicates the
percent differences in the LDL0 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 LDLO values when administration was. oral.
U)
i
Si
-------
3-22
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 study-
ing the acute effects of paraquat and diquat found that an oral dose of
680 micromoles/kg of either chemical would result in marked diuresis, pro-
*•
teinuria, and glucosuria 6 to 24 hr 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 ff'-methylnicotamide (NMN) was found to be
markedly reduced 2 hr after dosing. Lock and Ishmael reported that these
changes indicate that renal impairment is similar in rat and man.
Hexachlorophene and etchlorvynol 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, somolence, and disorientation with res-
piratory and cardiac arrest occurring 61 hr after hospital admission.
Further examination revealed the following: (1) insterstitial 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 chem-
icals including hexachlorophene.
-------
3-23
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. Sim-
ilar toxic effects on the pulmonary vasculature were observed in rats by
Payne and co-workers after administering intravenous injections of 80 mg/
kg, resulting in death due to acute respiratory distress.
*
3.3.4 Conclusions
The information in Section 3.3.2 concerning lowest lethal doses
clearly indicates that there is a difference between humans and animals
with respect to the size of the dose that will cause death; in most cases
r
a smaller dose was required to cause death in humans than in animals. Based
s
on this difference, Section 3.3.3 which examines the pathology and func-
tional changes induced by the acute exposure of humans and animals to chem-
icals might be expected to show corresponding differences. However, the
available literature showed that for those chemicals compared, man and ani-
mal acute toxicity was similar. This does not imply that there are no
chemicals for which the human and animal response would be different, but
rather indicates the scarcity of information comparing human and animal
acute toxicity studies. Two possible reasons for Jthe apparent paucity of
comparable data are that (1) many acutg toxicity studies performed on ani-
mals are done primarily to establish the LD50 and provide 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 hos-
pital soon after intake, much important data goes unrecorded. It is
-------
3-24
realized chat it is not possible to satisfactorily appraise the comparative
susceptibilities between man and animal with only the few chemicals used
as examples in Section 3.3.3; however, 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 fallowing sections consider some
aspects of histopathology in acute toxicity testing of chemicals and offer
some concluding remakrs 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 magnitude
that they kill at least half of the test animals (LDSo). The stress to
the animal's system would in many cases produce lesions of various organs
providing the animal live long enough for histologic damage to occur.
On the other hand, there are compounds which when administered at doses
high enough to cause death do not show any histopathology related to the
-------
Table 3.6. Examples of histoputhological changes from acute exposures to chemicals
Test agent
3' ,V-Dichloro-
propionanilide
Cadmium chloride
Dichlorvos
Maytansine
M'irex
Ochratoxin A
Aflatoxin B,
Species
Rat
Rat
Dog
Rat
Rat and
mouse
Rat
Monkey
Route
Oral
Intravenous
Intravenous
and oral
Subcutaneous
Intraperitoneal
Oral
Oral
Dose Histopathology
50-700 mg/kg Congestive hepatitis, gastroenteritis,
and patchy emphysematous lung changes
Not given Lesions in endothelial clefts of small
vessels in target organs such as testis
and gasserian ganglion
2.2-22.0 mg/kg Pulmonary changes with generalized con-
gestion and hyperemia and cardiovas-
cular changes
0.38-1.0 mg/kg Lesions in gastrointestinal tract mucosa,
thymus, spleen, bone marrow, and tes-
tis; arrest of mitotlc cell division;
hemorrhagic lesions in parenchyma tous
organs and brain; and chroma tolysis and
vacuolation of dorsal root ganglion
330-700 ppm Patchy lesions on surface of livers which
extended into the liver interior sev-
eral millimeters
15-50 mg/kg Severe catarrhal or erosive enteritis in
the duodenum and jejunum
1-3 mg/kg Liver damage characterized by hemorrhage,
Reference
Chand, 1973
Gabbianl et al. ,
1974
Snow, 1973
Mugera and Ward,
1977
Kendall, 1974
/
Kanisawa et al. ,
1977
Rao and Gehring,
necrosis, and massive accumulation of
lipid
1971
U)
to
01
-------
3-26
chemical. An example of this type of action was described by Robens (1978)
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 his-
topathology related to the test chemical. Another example is shown by the
action of #-2-fluorenylacetamide in mice where LDSO doses resulted in essen-
*
tially normal tissues upon histopathological examination (Haley, Dooley,
and Harmon, 1973).
What information do acute toxicity tests, in particular the histopath-
ological data, provide? Robens (1978) writes that in the development of a
drug, acute toxicity studies can: (1) establish some measure of toxicity
f
which can aid in choosing doses for studie^ of longer duration, (2) help
to assess whether the drug has efficacy in the nontoxic range, (3) enable
certain conclusions regarding mechanism of action to be made, if the ani-
mals are carefully observed following treatment and then necropsied, and
(4) answer the question of how much difference does exist between dosages
» • . .
which seem to be safe and those that are known to be highly toxic. Histo-
pathological information derived from acute toxicity tests can also aid
physicians confronted with cases of accidental poisoning or attempted sui-
cides by providing information on chemical effects parameters such as
target organs.
Although histologic evidence of damage to various organ systems as a
result of acute exposure often provides evidence that permits prediction
that the organ system will likely be similarly affected after chronic dosing
this is not always the case. Three papers which illustrate this are Kanisawa
et al. (1977), Diamond and Sleight (1972), and Pennarola, Balletta, and
-------
3-27
Di Paolo (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. Secondly, in a study of the acute and subchronic
effects of methylmercury dicyandiamide in the rat, Diamond and Sleight
-------
Table 3.7. U)JO observation periods
Chemical
Mechylraercury
dicyandiamide
Ochratoxin A
Aflatoxin Bt
Maytansine
Proxlmpham
Cuanethidlne
JV-Methyl-ff-(l-
napthyl)fluoro-
acetamide
Probocol
Phenoxarsine
oxide
Pentachlorophenol
Nefopam
Phenazine-5N-oxide
Cycloplazonic acid
Aroclor 1242
Budralazine
Species
Rat
Rat
Monkey
Rat
Mice
Rat
Mice^ '
Rat
Guinea pig
t
Mice
Dog
Rat
Rat
Rat
Rat
Route
Intraperitoneal
*
Oral
Oral
Subcutaneous
Oral
Oral
Oral
Oral
Oral
Oral
Intravenous
Oral
Oral
Oral
Oral
Observation
LD»o period
(days)
1.335 rag/100 g
28 mg/kg
2.2 mg/kg
0.48 mg/kg
1.3 g/kg
1050 mg/kg
250-370 mg/kg
\
5280 mg/kg
24 mg/kg
74 mg/kg
20 mg/kg
6600 mg/kg
Male - 36 mg/kg
Female — 63 mg/kg
4.25 g/kg
Male - 0.62 g/kg
Female — 0.73 g/kg
7
3
14
14
21
14
14
7
21
„ 7
14
7
10
14
7
Tine deaths
occurred Reference
(days)
— Diamond and Sleight,
1972°
— Kanisawa et al.,
1977*
4 Rao and Centring,
1971a
4 Mugera and Ward,
1977a
— Lewerenz, Lewerenz,
and Plass, 1970"
— Hartnagel et al. ,
1976°
5 Hashimoto et al..
1968*
— Molello, Gerbig, and
Robinson, 1973"
8 Ballantyne, 1978*
«
— Ahlborg and Larsson,
1978&
2 hr Case, Smith, and
Nelson, 1974°
Koeda et al., 1976*
6 Purchase, 1971*
— Bruckner, Khanna,
and Cornish, 1973
3 Onodera et al.,
1977*
u>
Isl
00
-------
Table 3.7 (continued)
Chemical
2,3-Dihydro-9H-
isoxasolo-(3,2-
b)quinazolin-
9-one
Oxepinac
Photomirex
Ethylidene
gyromitrin
Sulfotep
Benzene
Cadmium
Di-2-ethylhexyl
phthalate
Dichlorodiphenyl-
trichloroethane
Ethanol
Clindamycin
hydrochloride
Dipterex
Dimethyl
terephthalate
Peroxyacetyl
nitrate
Chloropyrifos
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
LD,0
700 ± 110 mg/kg
136 mg/kg
Male - 150-200
mg/kg
70 mg/kg
29.4 mg/kg
15.2 mg/kg
2.94 g/kg
225 mg/kg
37.77 g/kg N
237 mg/kg
10.6 g/kg
6.08 mg/kg
2618 mg/kg
649 mg/kg
3900 mg/kg
95 ppra
118-245 mg/kg
Observation
period
(days)
14
7
28
3.5
14
14
14
7
10
1
, 7
7
7
14
14
-
Time deaths
occurred Reference
(days)
1 Danerjee et al. ,
1977" ;
i
.
6 Nomura et al., 1978 i
- Hallett et al.,
1978"
3.5 Maklnen, Kreula. and
Krauppi, 1977»
1 Kimmerle and , to
Klimmer, 19746 ^
£1 Drew and Fouts, 1974°
— Kotsonis and Klassen, '
1977a
— Lawrence et al., '
19753
- Tomatis et al., 1972*
- *Wiberg et al.. 1970
- Bellies, 1972
Gray et al., 1972°
5 Edson and Noakes,
19600
2 Krasavage, Yanno,
and Terhaar, 1973
1 Kruysse et al..
1977^
— McCol lister et al.,
(various
strains)
-------
Table 3.7 (continued)
Chemical
trans-2-Hexenal
Thiabendazole
hydrochlorlde
Cefazolln
SMA 1440-H resin
Alromine RU 100
Fenoterol HBr
Ethylene dlbromide
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
Observation
Route LDjo period
(days)
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
780 mg/kg
3100 mg/kg
>10,000 mg/kg (no
deaths at 10,000)
>20 but <22 mg/kg
>3.2 g/kg
2300 mg/kg
146 mg/kg
69.2 mg/kg
7.62 mg/kg N
130-180 mg/kg
1610 mg/kg
900 mg/kg
14
10
7
14
10
7
14
5
7-14
14
14
Time deaths
occurred Reference
(days)
2 Gaunt et al., 1971°
— Robinson, Stoerk,
and Graessle,
1965a
— Birkhead, Briggs,
and Saunders, 1973
Within Ulnek and Bur gun,
3 days 1977a
— Hunter and Stevenson,
1967h
1 Kast et al., 1975&
- Rowe et al., 1952a
3 Nakaue, Dost, and
Buhler, 1973a
3 Worth et al., 1970a
- Lorke, 1978*
4 Crawford et al.,
« 1965°
- Jacobson, 1972a
£u.S. paper.
Foreign paper.
OJ
UJ
o
-------
3-31
examinations for each chemical that is tested for acute toxicity can be ques-
tioned. However, because some chemicals do have the potential for acute ex-
posure to humans and because some knowledge of soft tissue changes would be
useful, e.g., comparison with other chemicals tested for acute toxicity, gross
necropsy but not histopathology of animals used in the acute testing of a
chemical seems warranted. This view is supported by Zbinden (1973) who states
^
that histopathological examination 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.
f
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 definition is
* • • -
an observation period of some finite length; however, after reviewing sev-
eral papers that describe the acute toxicities of various chemicals, it is
obvious that the observation periods are not always the same. This section
primarily discusses the length of the observation period of test animals
f-
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 such that the metabolism and elimination of the test substance
from the test animal is near completion when the LDSO 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 hr and 2,3,4,6-
tetrachlorophenol within 48 hr, but only 60% of 2,3,4,5-tetrachlorophenol
was eliminated and subsequently recovered in the urine within 72 hr.
The observation period following acute exposure must also be of suf-
ficient length to allow for the fact that different,routes of administra-
tion can result in the toxic effect of a oftemical being manifested at
different times. In a study by Nomura et al. (1978), mice that received
a lethal dose of oxepinac, a new anti-inflammatory drug, either intraperi-
toneally or subcutaneously, exhibited convulsion and dyspnea with most
deaths occurring within 24 hr, whereas mice that received an oral lethal
» • •
dose showed anorexia, emaciation, and bloody feces with deaths two to six
days postadministration.
3.5.3 Length of Observation Period
Table 3.7 summarizes data from 43- papers. Examination of this in-
formation shows that twelve papers had an observation period of 7 days and
eighteen papers had an observation period of 14 days, with the remaining
thirteen papers having observation periods ranging from 3 to 28 days.
The Guidelines for Carcinogen Bioassay in Small Rodents (Sontag, Page,
and Saffiottii, 1976) and the Environmental'Protection Agency's proposed
-------
3-33
guidelines for registering pesticides in the United States (40 CFR part
163.81-1) recommend at least a 14-day observation period. This 14-day
period is thus the choice of some American 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 fourteen
of twenty-four papers by U.S. researchers, used the 14-day observation period.
In addition to showing that there is variation in observation periods,
the data in Table 3.7 also raises the question of which length is the most
desirable. As previously mentioned (Section 3.5.2), the observation period
must be of sufficient duration to allow for manifestation of the toxic ef-
/•
feet; 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
» • i -
(Macaco, irus) 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 are seen when 2,3-dihydro-9H-isoxazolo(2,3-b)-quinazolin-9-one was
administered orally to dogs (Banerjee et al., 1977). They discovered that
within 24 hr after dosing, 4/4 dogs died at 1150 mg/kg and 2/4 dogs died
at 750 mg/kg. The LD30s of mice and rats were also determined in this
study, but no indication is provided as to when the animals died within
the two-week observation period. Research with budralazine (Onodera et
al., 1978) and nefopam (Case, Smith, and Nelson, 1975) also indicated that
-------
3-34
death from acute exposure may occur within a few hours or days. LD30
studies with budralazine showed that most deaths occurred within 48 hr
with all survivors appearing normal 72 hr after dosing, whereas death of
mice, rats, and dogs occurred even quicker 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 adminis-
tration is provided by the works of Kimmerle and Klimmer (1974) 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 hr, and that survivors recovered within one to four days.
Diamond and Sleight (1972) discovered that/'lf rats acutely exposed to methyl-
mercury dicyandiamide were going to die, death would occur within 72 to 78 hr.
3.5.4 Conclusions
Although only a relatively small number of papers were reviewed, it
» • .
seems evident that many American 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 animal's died well within the
specified observation period. Therefore, the fourteen day observation
period suggested by most regulatory agencies appears sufficient for the
determination of the LDSo for most chemicals.
-------
3-35
SECTION 3
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s
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Diamond, S. S., and S. D. Sleight. 1972. Acute and Subchronic Methyl-
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3-36
Food Safety Council. 1978. Proposed System for Food Safety Assessment.
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Gaines, T. B. 1969. Acute Toxicity of Pesticides. Toxicol. Appl.
Pharmacol. 14:515-534.
Gaunt, I. F., J. Colley, M. Wright, M. Creasey, P. Grasso, and S. D.
Gangolli. 1971. Acute and Short-Term Toxicity Studies on Trans-2-
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Goyer, R. A. 1971. Lead and the Kidney. Curr. Top. Pathol. 55:147-176.
Gray, J. E., R. N. Weaver, J. A. Bollert, and E. S. Feenstra. 1972. The
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s
Haley, T. J., K. L. Dooley, and J. R. Harmon. 1973. Acute Oral Toxicity
of iY-2-Fluorenylacetamide (2-FAA) in Several Strains of Mice. Proc. Soc.
Exp. Blol. Med. 143(4):1117-1119. (Abstract).
Hallett, D. J., K. S. Khera, D. R. Stoltz, I. Chu, D. C. Villeneuve, and
G. Trivett. 1978. Photomirex: Synthesis and Assessment of Acute Tox-
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26(2)-.388-391.
» • •
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-phenylquinucli-
dinium Bromide (MA 540) and Guanethidine in the Rat and Dog. Drug Res.
26(9):1671-1672.
Hasimoto, Y. , T. Makita, H. Miyata, T. Noguchi, and G. Ohta. 1968. Acute
and Subchronic Toxicity of a New Fluorine Pesticide, tf-Methy!-#-(!-
naphthyl) Fluoracetamide. Toxicol. Appl. Pharmacol. 12(3):536-547.
Hunter, C. G., and D. E. Stevenson. 1967. Acute and Subacute Oral Tox-
icity of Alromine RU 100 in Rats. Food Cosmet. Toxicol. 5:491-496.
Jacobson, K. H. 1972. Acute Oral Toxicity of Mono- and Di-Alkyl Ring-
Substituted Derivatives of Aniline. Toxicol. Appl. Pharmacol.
22:153-154.
Kanisawa, M., S. Suzuki, Y. Kozuka, and M. Yamazaki. 1977. Histopatholog-
ical Studies on the Toxicity of Ochratoxin A in Rats: I. Acute Oral
Toxicity. Toxicol. Appl. Pharmacol. 42(l):55-64.
-------
3-37
Kast, A., Y. Tsunenari, M. Honma, J. Nishikawa, T. Shibata, and M. Torii.
1975a. Acute, Subacute and Chronic Toxicity Studies of the Beta-
Sympathomimetic, Fenoterol HBr on Rats, Mice and Rabbits. Oyo Yakuri
Kast, A., Y. Tsunenari, M. Honma, J. Nishikawa, T. Shibata, and M. Torii.
19750. Acute, Subacute and Chronic Toxicity Studies of an Aminor-Halogen-
Substituted Benzylamine (Fominoben) in Rats and Mice. Oyo Yakuri
Kendall, M. W. 1974. Acute Histopathologic Alterations Induced in Livers
of Rat, Mouse, and Quail by the Fire-Ant Poison", Mirex. Anat. Rec. 178:
388.
Kimmerle, G. , and 0. R. Klimmer. 1974. Acute and Subchronic Toxicity of
Sulfotep. Arch. Toxicol. 33:1-16.
Koeda, T., M. Odaki, H. Sasaki, M. Yokota, T. Niizato, H. Watanabe, H.
Kawaoto, and T. Watanuki. 1976. Toxicological Studies on Phenazine-
5N-Oxide in Rats. Oyo Yakuri 12(3) : 483-499.
Kotsonis, F. N., and C. D. Klaassen. 1977. Toxicity and Distribution of
Cadmium Administered to Rats at Sublethai Doses. Toxicol. Appl. Pharmacol.
41:667-680.
Krasavage, W. J. , F. J. Yanno, and C. J. Terharr. 1973. Dimethyl Tere-
phthalate (DMT): Acute Toxicity, Subacute Feeding, and Inhalation
Studies in Male Rats. Amer. Ind. Hyg. Assoc. J. 34(1) :455-462.
Kruysse, A., V. J. Feron, H. R. Immel, B. J. Spit, and G, J. Von Esch.
1977. Short-Term Inhalation Toxicity Studies with Peroxyacetyl Nitrate
in Rats. Toxicology 8:231-249. '
Lawrence, W. H. , M. Malik, J. E. Turner, A. R. Singh, and J. Autian. 1975.
A Toxicological Investigation of Some Acute, Short-Term, and Chronic
Effects of Administering Di-2-Ethylhexyl Phthalate (DEHP) and other
Phthalate Esters. Environ. Res. 9:1-11.
Lewerenz, H. J. , G. Lewerenz, and R. Plass. 1970. Acute (Mouse and Rat)
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Toxicol. 8(5) -.517-526.
Lock, E. A., and J. Ishmael. 1978. The Effects of Paraquat and Diquat on
Rat Kidney (abstract). Toxicol. Appl. Pharmacol. 45(1) :227.
Lorke, D. 1978. New Studies on Cadmium Toxicology. Proc. 1st Int.
Cadmium Conf . pp. 175-180.
Makinen, S. M. , M. Kreula, and M. Kauppi. 1977. Acute Oral Toxicity of
Ethylene Gyromitrin in Rabbits, Rats and Chickens. Food Cosmet. Toxicol.
15(6) :575-578.
-------
3-38
Martinez, A. J., R. Boehm, and M. G. Hadfield. 1974. Acute Hexachloro-
phen Encephalopathy: Clinico-Neuropathological Correlation. Acta.
Neuropathol. 28(2):93-104.
McCollister, S. B., R. J. Kociba, C. G. Humiston, D. D. McCollister, and
P. J. Gehring. 1974. Studies of the Acute and Long-Term Oral Toxicity
of Chlorpyrifos (0,0-Diethy1-0-(3,5,6-trichloro-2-pyridyl)phosphorothi-
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Molello, J. A., C. G. Gerbig, and V. B. Robinson. 1973. Toxicity of
[4,4'-(Isopropylidenedithio)bis(2,6-di-t-butylphenol)], Probucol, in
Mice, Rats, Dogs and Monkeys: Demonstration of" a Species-Specific
Phenomenon. Toxicol. Appl. Pharmacol. 24:590-593.
Mugera, G. M., and J. M. Ward. 1977. Acute Toxicity of Maytansine in
F344 Rats. Cancer Treat. Rep. 61(7):1333-1338.
Nakaue, H. S., F. N. Dost, and D. R. Buhler. 1973. Studies on the Tox-
icity of Hexachlorophene in the Rat. Toxicol. Appl. Pharmacol. 24:
239-249.
^
National Academy of Sciences. 1977. Principles and Procedures for Evalu-
ating the Toxicity of Household Substance's. Printing and Publishing
Office, Washington, D.C. 130 pp.
NIOSH. 1977. Registry of Toxic Effects of Chemical Substances. E. J.
Fairchild, R. J. Lewis, and R. L. Tatken (eds.). Vol. II. 987 pp.
Nomura, M., T. Onodera, M. Kato, A. Yamada, H. Ogawa, and T. Akimoto.
1978. Acute, Subacute, and Chronic Toxicity of Oxepinac. Arzneim.-
Forsch. 28(3):445-451.
» • •
Onodera, T., S. Takayama, A. Yamada, Y. Ono, and T. Akimoto. 197 .
Toxicological Studies of l-[2-(l,3-Dimethyl-2-butenylidene)hydrazino]-
phthalazine, a New Anti-hypertensitive Drug, in Mice and Rats. Toxicol.
Appl. Pharmacol. 44(3):431-439.
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-------
3-39
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-------
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 subchronic or sub-
acute 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
f
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 test" with'daily exposure for about
three months. Guarino (1979) stated that traditionally three terms (short-
term chronic, subchronic, or prolonged tests) 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 three months, have appeared in
the literature including some by expert committees (National Academy of
Sciences, 1975, 1977). The cumulation of this was the definition and guide-
lines of the Environmental Protection Agency pesticide programs (Federal
-------
4-2
Register, 1978). This guideline set standards for subchronic tests cover-
ing dosage levels, routes of exposure, number of species, number of test
animals, type and degree of pathology, hematology, biochemical and func-
tion evaluations, etc. Their choice of subchronic as the term of prefer-
ence 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 lifespan but 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 quote
describes the purpose of this chapter, that is to evaluate the currently
employed subchronic tests to determine whether they provide sufficient
s
information that makes longer term tests unnecessary. The section does
not consider a comparison of time vs. 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, arft 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
to determine the appropriate dose levels to be used^if longer exposures
are planned (Peck, 1968; Benitz, 1970). .The purpose of this chapter is
/•
to evaluate the first of these two functions. To fulfill the first func-
tion, a battery of biochemical, hematological, and pathological tests are
performed. These tests provide data upon which more selective evaluations
can be based. The target organ data are especially useful for this. The
value of such data has been recognized for several decades, and as stated
by Barnes a-nd Denz (1954) , "It is in fact doubtful whether it is worth
. proceeding beyond the subacute test." The second function is an elaboration
-------
4-3
of the acute evaluation of effective doses and should give maximum/minimum
effect levels with safety margins. The role of the subchronic test as a
preliminary evaluation of appropriate dose levels for use in longer test
designs (such as a carcinogenicity bioassay) is evaluated in a separate
review by Prejean (Appendix A). However, as the time and cost requirements
of toxicity testing increases, so does the value of subchronic tests as
reasonable evaluations of chronic effects. If a more efficient subchronic
test design can be constructed then 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 dis-
cussion 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 Interagency 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. Over-
all these systems are quite similar. Selected areas of these standard de-
signs will be reviewed in this document to determine if modifications are
possible. Thus, the designs in Table 4.1 represent the baseline system for
f
which changes will be suggested.
4.2 SPECIES
4.2.1 Introduction
The debate over which and how many spe,cies to employ in subchronic
toxicity 'tests has been a part of toxicology for many years. Discussions
-------
Table 4.1. Experimental design for recently proposed subchronlc oral toxlcity tests
Agency
FIFRA (TSCA) guide-
lines, 1978-1979°
Interagency Regulatory
liaison group, 1979°
World Health Organi-
zation, 1979
Food Safety Council,
1978
Species
Two (rat and
nonrodent)
Two (rat and
dog)
Two (rodent and
nonrodent)
Two (rodent and
nonrodent)
.
Number of animals
Sex Age at start per group
(sex/dose)
Both Rats — 6 weeks Rats — 20
Dogs —.4-6 months Dogs — 6
Both Rats — 6 weeks Rats — 20 (30 days-
Dogs — 4-6 months 6 months) 10
(<30 days)
Dogs — 4
Both Rats — Just after 10
weaning
Both (Not indicated) Rodents — 20
Nonrodents — 3-4
\
Number
Duration of
doses
Rodents — 90 days 3
Dogs — 6 months
Generally 90 days; 3
shorter for spe-
cial situations
10% llfespan or 3 3
months
Rodents — 90 days- 5
1 year
Nonrodents — 1 year
«
Dose levels
Highest — effect level
but not more than
10% mortality
Lowest — no evidence
of toxicity
Highest — toxic level
but not excess
mortality
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
.Proposed or unofficial guidelines.
Must have control groups also.
Source: Adapted from Page, 1979.
-------
4-5
generally center on: (1) which 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 discussions con-
cerning 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"
due to 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 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 discus-
sion of inhalation tests (Roe, 1968). In this discussion 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.
Based on anatomical considerations, monkeys and dogs resemble man more
closely than do other species and Roe recommended these for "small-scale,
-------
4-6
short-term experiments." The value of the rat apparently lies between
these two groups, since 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 this discussion
to at least the same level as 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 one month or longer duration (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 backed up in a report by Bushby, Lechat, and Santarato (1966) in
which 71.7% of all toxicity 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 their super-
iority irt sensitivity or comparability to human' response patterns.
Several authors have evaluated species suitability in anticancer
drug studies by using doses based on mg/m2 (surface area) instead of mg/kg
(body weight) following the method of Pinkel (1956). 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 sen-
sitivity was compared in this manner suggested that in most cases the mon-
key, 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
-------
4-7
tolerated dose, Roman (1972) found that the dog was generally more sensi-
tive 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 classi-
fication."
s
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,
the key factor is metabolic similarity to man. Since absorption, penetra-
tion, and distribution processes are'similar in-most species, the primary
differences between species are due to variations in biotransformation
pathways and in rates of inactivation. The final selection of test spe-
cies should reflect their similarity to man in metabolizing that chemical.
In another review of drug safety evaluations, Peck (1968) also stated
f
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.
-------
4-8
Table 4.2. Comparison of various animal models as
predictors of metabolic fate in man
Rating
Good
Fair
Poor
No data
Total
Rat
Number
5
7
19
3
34
Percent
14
21
53
12
100
Species
Dog
Number Percent
6 18
8X 23
20 59
34 100
Monkey
Number
22
9
3
34
Percent
65
26
9
100
Source: Adapted from Smith, 1979.
-------
4-9
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 (par-
allels 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 only discussed
the vomiting ability of the test species, this factor is just representa-
tive of many such physiological traits. His conclusions can be expanded
to include the recommendation that the test species should have a physio-
logical functional pattern that is as similar as possible to the human
pattern for the specific test chemical. If such information is not
known, then "use as many species as may be possible."1
s
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 be-
tween 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 the use of "(1) mice and rats
for experimental therapeutic data; (2) dogs, monkeys, and mice for toxi-
cologic 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.
-------
4-10
Balazs (1976) discussed the rat and dog combination for testing chem-
icals. 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
X'
justification which should consider such factors as the comparative metab-
olism 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 papers 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, bron-
chopulmonary, hepatorenal, digestive, nervous, and somatic system toxicities
as well as data obtained from the dog. Further, the rat studies were better
than the dog studies for prediction of pancreatic hypertrophy, hepatic por-
phyria, 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 duplication of scientific manpower and research effort.
-------
4-11
The review by the World Health Organization (1978) on toxicity evalu-
ations summarizes most of the primary factors discussed in literature re-
views 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, then the species show-
ing 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 neces-
sitates Che use of the dog and rat as a minimum in subchronic studies. How-
ever, despite such reviews as the WHO document and Aviado's literature
review, the conclusion of Fancher (1978) in his review of animal models
s
for toxicological studies is in essence ttie 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.
-------
4-12
In an inhalation study of beryllium toxicity, Stokinger et al. (1950)
used 11 species, examining mortality, growth retardation, hematology, and
urinalysis factors. The animals were exposed 6 hr 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
rag/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. In hematologic and urine anal-
yses the dog showed the most toxic effects. 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
s
beryllium inhalation. The exposures were 6 hr 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 »ith 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 hr per day inha-
lation exposures with concentrations of .100, 50, and 25 ppm. Subchronic
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
-------
4-13
et al. concluded that at the high dose no species did particularly well;
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, subchronic
(one to six months), and a one-year rat study. The appearance 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
f
conclusion that the dog represented man's response better than the rat.
s
However, in summation, Litchfield noted that the small number of chemicals
examined reduced the statistical value of this comparison and urged that
more species be examined for use in toxicity studies.
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 four-week exposure tests. Owens concluded
that except for neurotoxicity all three species were suitable, and in par-
ticular 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
studies, for durations up to two years. They found 21 studies for which
the rat and dog were tested together. In none of these tests was the dog
more sensitive, and in 7 tests it was less sensitive than the rat. They
-------
4-14
Table 4.3. Occurrence of 39 physical signs from 6 drugs in 3 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 N
Total
Rat
Absent Rat Dog and
dog
2
3 1
4
5
4 1
5 1
3 1
1 2
3 1
5
212
5
5 I
5 I
2 s
4
3 1
5
311
4 1
1 1
5 1
2 11
3 1
3 2
3
5 1
5 1
5 1 • -•
5 1
4 ,.- 1
5
4
4 1
4 ' 2
5
2 1
5
5 1
146 11-16 8
Man
2
1
1
1
2
3
1
4
1
1
2
1
1
2
23
Rat Dog
and and
man man
1
1
1
1
2
1
1
1
1
1
1
1 12
Rat,
dog,
and
man
4
1
1
1
2
1
1
-
1
1
1
1
1
1
1
17
Source: Adapted from Litchfield, 1961.
-------
4-15
Table 4.4. Organ system toxicities
Compound Rodent Dog Monkey Human
Bone marrow toxicity
Mechlorethamine + + + +
Cyclophosphamide -f + + +
Myleran + + + +
1-Sarcolysin + + + +
2-Chloroethylmethanesulfonate •• + + ?
Methotrexate + + + +
6-Mercaptopurine + + +
4-Aminopyrazolo (3,4-D)pyrimidine + + +
Vincaleukoblastine + + 4-
Carzolamide - - +
Actinomycin P2 + + +
Mithramycin - +
Roseolic acid - + +
Gastrointestinal toxicity
Mechlorethamine + + 4 +
Cyclophosphamide + + + +
Myleran + + + +
1-Sarcolysin + . 4- + 4-
2-Chloroethylmethanesulfonate + + +
Methotrexate + + + +
6-Mercaptopurine + + +
4-Aminopyrazolo (3,4-D)pyrimidine + + +
jCarzolamide , - + +
6-Azauracil ' + - +
Vincaleukoblastine + + +
Actinomycin P2 + + +
Mithramycin - - +
Nervous system toxicity
Mechlorethamine . + + + +
Chloroquine mustard . + + +
Carzolamide - - +
6-Azauracil + - - +^
Vincaleukoblastine - - +,
Nitrofurazone - - +
NSC 38280 - - +
Skin and appendages toxicity
(dermatitis and alopecia)
Cyclophosphamide - - - +
Methotrexate • +
Vi,ncaleukoblastine - - +
8-Azaguanine - +
Actinomycin P2 - - +
Methylglyoxal-bis-guanylhydrazone - - +
-------
4-16
Table 4.4 (continued)
Compound Rodent Dog Monkey Human
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.
-------
4-17
concluded that the use of the dog should be limited to 90-day 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 three days per week over 13 weeks, they were able to test the similar-
ity of the pigs' 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 cholin-
esterase 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 two 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 just the opposite pattern of effects. Also, a strain dif-
ference in the rats appeared significant as Wistar rats were extremely
susceptible to carbon tetrachloride while Sprague-Dawley rats were not.
-------
4-18
A study using the rat and dog to test the oral toxicity of EX5004,
a sympatholytic drug, showed that after a three-month exposure the dog
was more affected than the rat (Yeary, Brahm, and Miller, 1965). The ex-
posure 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
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
s
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 en-
largemenS 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 one to four months. As
/•
shown in Table 4.5, the dog was overwhelmingly more sensitive than the
rat or monkey, especially for histopathologic and clinical chemistry ef-
fects. 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.
-------
Table 4.5. Comparison of significant variations in the response of four species to an oral analgesic
Parameter Dog
Clinical observations
Depressed weight gain or
weight loss + '
Depression or
unconsciousness +
Emesis 4-
Icterus +
Death +
Clinical chemistry
Elevated SGPT +
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
£l-2-(l-Methyl-2-piperidyl)-l,l-diphenylethyl
Minimum dose at which an effect occurred or
^Indicates a response in one or
Indicates no abnormal response.
No test.
Source: Adapted from Newberne,
more animals
Monkey , Do*a&e. , Rat
(mg/kg/day)
+
+
+
-
+
.
-
-
-
i
\
4-
-
-
1
+
propionate
the highest
25 +
30
25
60
30
60
60
60 NT
60
60
30
60
60
1 60
30
hydrochloride .
dose given.
Dosage
(mg/kg/day)
30
100
100
100
100
100
100
NT
100
100
100
100
' 100
100
100
D KKJ,- Dosage
Rabbit , /, /. \
(rag/kg/day)
J
- 80
80
80
80
80 j
NT NT
NT NT
NT NT
I
80 £
NT NT
NT NT
NT NT
NT NT
NT NT
NT NT
at the indicated dose.
Gibson, and Newberne, 1967.
-------
4-20
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 dam-
age after no more than 29 days at 800 mg/kg. One dog died after four days
of dosing. With safrole ingested at intermediate doses (5000 ppm), the
rat exhibited growth retardation, liver pathology, slight leucocytosis,
^
and anemia after dosing for two 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.
In a toxicological study of aldrin and dieldrin, Hodge, Boyce,
Deichmanhe, and Kraybill (1967) noted the subch'ronic response of several
species by comparing histopathologic data, body weight changes, mortality,
and organ weight changes. Using these criteria, they found wide variability
in species response. They found the monkey was the/most sensitive for
body weight and mortality criteria, while both the dog and rat were sensi-
tive 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
-------
4-21
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 associated with
this discoloration. In the dog, the discoloration and hyperplasia occur-
red with intravenous doses of 5, 10, 20, and 40 mg/kg per day. Addition-
ally, various degrees of hemolytic anemia occurred in the dose range 10
to 40 mg/kg per day. In the monkey, the discoloration occurred (pigmenta-
tion less pronounced) at an oral level of 30 mg/kg per day, but no hyper-
plastic 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
the absence of a toxic effect in man. .This complementary pattern is dem-
onstrated 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.
-------
4-22
Table 4.6. Predictive abilities of the dog, monkey, and combination
Organ system
Dog as a
Injection site
Integument
Cardiovascular
Respiratory
Bone marrow
Lymphoid
Gastrointestinal
Liver
Renal
Neuromuscular
Monkey as a
Injection site
Integument
Cardiovascular
Respiratory
Bone marrow
Lymphoid
Gastrointestinal
Liver
Renal
Neuromuscular
•The combination
Injection site
Integument
Cardiovascular
Respiratory
Bone marrow
Lymphoid
Gastrointestinal
Liver
Renal
Neuromuscular
"> .
predictor
16
12
28
16
80
4
92
52
32
24
predictor
13
13
22
13
83
0
74
52
35
22
of dog .and
16
24
36
16
88
4
92
52
36
24
£ X
for organ-specific
36 40
32 40
24 36
64 16
12 0
72 24
8 0
44 4
56 4
60 12
8
16
12
4
8
0
0
0
8
4
for organ-specific
26 52
17 57
26 30
48 30
13 0
31 65
9 0
35 13
48 13
30 39
monkey as -
toxicity in
36 40
36 36
32 28
76 4
12 0
76 20
8 0
48 0
56 4
60 12
9
13
22
9
4
4
17
0
4
9
FNSe
TP + FN
toxicity
33
57
30
20
9
0
0
0
20
14
toxicity
40
50
50
40
5
100
19
0
11
28
Number of
compounds
in man
25
25
25
25
25
25
25
25
25
25
in man
23
23
23
23
23
23
23
23
23
23
a predictor for organ-specific
man
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 map.
°False positive, toxicity was observed in the animals but not
in man.
*VTrue negative, no toxicity was observed in the animals and man.
False 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.
-------
4-23
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 meas-
ured by tests of hematology, clinical chemistry, or histopathology. How-
ever, 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 m»re sensitive than the
dog.
Vogin et al. (1970) used the rat (T-DRL) and the dog (beagle) to
test azotrek, a tetracycline phosphate complex-sulfamethizole formulation,
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
s
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, dogt guinea pig, and monkey using repeated exposure studies (8 hr
per day, 5 days per week, for 6 weeks, with concentration levels of 0.7
and 3.7 ppm) and continuous exposure studies (24 hr 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 inflamma-
/•
tion 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 bron-
chopneumonia. In the continuous exposure test at 0.22 ppm, the dog and
monkey showed specific inflammatory changes in the lung, while the monkey,
-------
4-24
guinea pig, and dog had nonspecific inflammation in the liver, kidney, and
heart. At 1.0 ppm, the dog and monkey showed 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,
f
300, and 500 mg/kg per day for the pig and 5, 10, 25, 50, 100, 300, 400,
s
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 hematolog-
ical 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 while the pig had hemorrhaging of the heart.and fat deposits in
the pancreas, in addition to the intestinal lesions. These results indi-
cated that the two species reacted comparably to the pesticide diazon. The
dog showed greater biochemical sensitivity while 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
-------
4-25
of alkaline phosphatase and serum glutamlc 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 in-
halation 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 sim-
/•
ilar among all four species and included liver pathology and changes in
s
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 nemo- .
globin and hematocrit levels at the high dose. The monkey had decreased
alkaline phosphatase and increased blood urea nitrogen levels at the inter-
mediate and high doses, and some mortality at the high dose. The dog and
monkey appeared to be the more sensitive/ species for effects in hematolog-
ical and biochemical parameters while 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
-------
4-26
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 one to six 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 sensi-
tivity ranks were as follows: the miniature pig and rat were the most
sensitive; the rabbit, guinea pig, and chicken were next; and the mouse
s
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.
Harris et al. (1973) compared the guinea pig (Hartley), rat (C-D),
and mouse (albino CD-I) for sensitivity to 2,3,7,8-fetrachlorodibenzo-p-
dioxin (TCDD). They exposed each species to TCDD in the diet for 30 con-
secutive days and/or once a week for four to eight weeks. As shown in
Table 4.8 the guinea pig appeared to be the most sensitive species, fol-
lowed 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
-------
Table 4.7. Summary of dioxin oral administration data
Rat (35 per group)
Compound
Sex
Week . Significant
of Survivors Pathology
test
Controls
1% Dloxane
0.5% Dioxane
1% Unsubstituted
dibenzodioxin
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
Source: Adapted
*
j,- ^ i "*!" , ' , ' i - -*.' . • , - - . )''..-, . ' -^ ?> — 'i * .. 'I "t.-'-1-
M
F
M
F
M
F
M
F
M
"-F
M
F
M
F
M
F
M
F
M
F
M
F
from
->
34
34
42
42
42
42
42
42
42
42
17
17
17
17
32
22
37
25
17
17
17
King,
..,.,. r ,,.,,,.,
35
'35
24
20
26
32
33
32
31
35
35
35
35
35
0
0
0
0
28
15
30
Shefner, and
Lung
8/11
3/5
6/6
3/3
2/2
1/2
3/4
\
\
1/5
0/5
1/5
1/2
0/3
Bates, 1973.
Liver
1/11
2/5
1/6
1/3
2/2
2/2
1/4
5A5
5/5
5/5
2/2
3/3
•'• •• . -
:'\
Mouse (50
Week
of
test
31
31
40
43
40
43
39
39
34
39
17
17
17
17
10
37
8
37
17
15
9
':
Survivors
50
50
50
49
49
49
48
29
50
49
49
48
50
4,9
0
5
0
45
1
0
0
per group)
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
NJ
«TVfi.ifc*..T»*
t£ftftrV£#
tfJ^RV';;^^^^
-------
Table 4.8. Summary of biological effects of TCDD
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 Guinea pig Mouse
Dose MTD „ -. O Dose MTD „ , . G Dose
(lig/kg) (days). Mortality (pg/kg) (days) Mortality ^^
100 18 6/14 3 18 9/10 >50
4 x 25 28 2/10 5x1 28 10/10 >4 x 25
10 22 15/16
25 - 1
6x5 8 x 0.2 4 x 25
~30 x 1
,
5 N 50
6x1 8 x 0.04 4x5
30 x 0.1
\
\
5 10
6x5 8 x 0.04 4x5
30 x o.l
a
,2,3,7,8-Tetrachlorodibenzo-p-dioxin.
Mean time to death after first exposure.
Number of animals dying per number of animals treated.
Source: Adapted from Harris et al., 1973.
to
oo
-------
4-29
highest dose (1500 mg/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. How-
ever, they did exhibit histopathological damage to the liver. Both spe-
cies 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 activ-
ity, 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 sensitive to fentoterol-HBr
s
than the mouse.
In another study by Kast et al. (1975&), the toxicity of forminoben-
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, 1000, 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 formations. In the rat, the
enzymes and biochemical parameters affected included bilirubin, cholesterol,
alkaline phosphatase, glutamic oxaloacetic transaminase, and glutamic
pyruvic transaminase. The parameters affected in the mouse were not
listed.
-------
4-30
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 concentra-
tions 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
s
820 mg/kg per day, while 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,
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
-------
4-31
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 (Wister and Sprague-Dawley) and
dog (beagle) for 3 to 5 weeks and 13 weeks. For tne shorter duration the
daily dose levels were 2 to 100 mg/kg for the rat and 1 to 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 exper-
ienced a 33% mortality rate, glycosuria, reduced body weight, increased
blood urea nitrogen and serum creatinine levels, and renal histopatholog-
s
ical 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 ^Zis) as replacements for the rhesus monkey.
By examining 14 parameters of drug metabolism in hepatic microsomal and
soluble fractions (including: liver/body weight ratio, cytochrome P-450,
glutathione s-aryltransferase activity, NADPH-cytochrome C reductase
-------
4-32
activity, mixed function oxidation, aminopyrine N-demethylase activity,
ethylmorphine N-demethylase activity, biphenyl hydroxylase activity, ani-
line hydroxylase activity, aryl hydrocarbon hydroxylase activity, UDP-
glucuronyltransferase activity using both PNP and OAF acceptor substrates,
and N-acetyltransferase activity using both p-aminobenzoic and sulfadia-
zine acceptor substrates), each species was evaluated for its enzymatic
activity pattern. All four species demonstrated activity in 13 of the
14 parameters, but no one species was similar to the rhesus in all quan-
titative aspects. Based on an arbitrary scale of favorable response, the
similarity to the rhesus is as follows: the miniature pig is closest,
then 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.
Sulfolane, a manufacturing solvent, was tested for inhalation toxic-
ity by repeated (8 hr/day, 5 days per week for 27 exposures) and continu-
ous (23 hr/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, while 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 re-
f
peated dose test. Of nine monkeys exposed to the high dose, three died
and five were sacrificed in a moribund state after 17 days. They 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
-------
4-33
monkey again exhibited high mortality rates at the high dose level. The
dog, exposed to the high dose, exhibited highly aggressive behavior, sei-
zures 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 transami-
nase 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
f
and 10.0 mg/ra3), ozone (1.0 and 2.0 ppm), and a combination of both. Both
s
species were exposed via inhalation for two and seven 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/body weight
ratios, tiut the rat adapted to this stress quicker than did the guinea
pig. In conclusion, the authors pointed out that the differences in sen-
sitivity between the rat and the guinea pig illustrate the importance of
using "at least two species in inhalation toxicity studies."
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 hr/day, five 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, while neither the, hamster nor the rabbit did.
-------
4-34
Table 4.9. Summary of treatment-related effects in hamsters, rats, and rabbits
repeatedly exposed to acrolein (ppm) for 13 weeks
Hamster
0.4 1.4
Symptomatology 0 x
Mortality 0 0
Growth 0 0
Food intake NE7* NE
Haematology 0 0
Urinary amorphous
material 0 0
Urinary crystals 0 0
Organ weights
Lungs 0 0
Heart 0 0
Kidneys 0 0
*•'• Adrenals 0 0
Gross pathology
Lungs 0 0
-Histopathology
Masai cavity 0 x
•'-- Larynx 0 0
Trachea 0 0
;" Bronchi and lungs 0 0
TO — N'ot affected.
'^x — Slightly affected.
^•xxx — Severely affected.
"""xx — Moderately affected.
^.+-H- — Markedly increased.
\"* — Moderately decreased.
?" — Slightly decreased.
„;«** — Markedly decreased.
^."E — N'ot examined.
V+ — Slightly increased.
"++ — Moderately increased.
Source: Adapted from Feron et
4.9 0.4
xxx 0
0 0
**f *9
NE 0
x 0
*i
+J 0
* o
fc
++* 0
+ 0
+ 0
0 0
0 0
xxx x
x 0
xx 0
0 0
/
al., 1978.
Rat
1.4 -4.9
X XX
O^^^^L
1 T 1
^
** ***"
^
0 0
0 +
0 *
0 -H-
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-
d
0
0
0
XX
NE
x
XX
-------
4-35
One aspect of species selection which has not been considered by other
reports, is the opposition of the general public to the use of some non-
rodent species as test animals because of their status as domestic pets.
As a result of such a considerstion, Cummings et al. (1979) evaluated the
rat as a replacement for "pet" species (the dog, cat, etc.). They specif-
ically tested some physiological and behavioral techniques, primarily used
in larger animals, and evaluated their potential "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 temperature, and behavior (retaining learned
/•
behavior, learning new behaviors, and maintaing unconditioned behaviors).
s
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. This avoided interfering effects from
the ane'sthetic. 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 stud-
ies 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 ham-
ster), the guinea pig and rat usually prove to be the most sensitive. The
-------
4-36
studies by Kast et al. (1975a, 1975&), 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 study, Jones,
Strickland, and Siegel (1972) and Roe (1968) ranked the rat as more sen-
sitive than the guinea pig, but Harris et al. (1973) and Villeneuve and
Newsome (1975) found just the opposite. Additionally, Rowe et al. (1952),
and Cavander et al. (1977) all considered the sensitivity of both species
to be equal. The selection of a preferred species in this case may have
f
to be based on economic factors or on their individual response to certain
s
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
(Hodge,* Boyce, Deichmanne, and KrayMll, 1967'; Owens, 1962; McNerney and
MacEwen, 1965; Jones, Strickland, and Siegel, 1972; Anderson et al., 1977;
Zbinden, 1963; Hayes, 1967&; Guarino, 1979; Freireich et al., 1966) at
least for some chemicals. Others feel that the rat and dog are sufficient
to detect most toxic effects (Kohn, Kay, and Calandra, 1965; Hartnagel et
al., 1975; Robbins and Tettenborn, 1976; Balazs, 1976). If no information
on the metabolism and pharmacokinetics of the chemical in question are
available then the literature cited would support the recommendation that
the studies be done using the dog and the rat. However, as discussed in
Section 4.2.2, if data on metabolism in animals are available which can
be related to man then this information should be of paramount importance
-------
4-37
in choice of species. Before embarking on lengthy expensive animals stud-
ies, 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 pat-
tern 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 the dog. Surprisingly, the dog has also been selected
as the most sensitive species in more studies (Litchfield, 1961; Ansbacher,
Corwin, and Thomas, 1942; Stokinger et al., 1950; Stokinger and Stroud,
1951; Yeary, Braham, and Miller, 1965; Newberne, Gibson, and Newberne,
1967; Hagan et al. , 1967; Vogin et al., 197(T; Knapp, Busey, and Kundzins,
1971; Koeferl et al., 1976) than the rat (Weil and McCollister, 1963;
Atkinson et al., 1966; Worth et al., 1970; Verschuuren, Kroes, and
Tonkelaar, 1973). The review by Aviado (1978) does suggest that the use
of both Jhe dog and. rat is superfluous and that, the rat alone may be suf-
ficient. This trend is also supported by Cummings et al. (1979) in which
physiological 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 (1964, 1971) gave good evidence sup-
*•
porting the use of the miniature pig in certain instances. When the
scientific data is conflicting, then 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 incorpora-
tion of alternative species such as the guinea pig, monkey, or miniature
-------
4-38
pig. The exact combination would depend on the chemical or effect to be
evaluated. The analysis by Gehring, Rowe, and McCollister (1973) indi-
cated that cost/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 arid rat, a more efficient deter-
mination 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 ,s
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 speci-
fied 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 literature are also dis-
cussed. Durations of 14, 28, and 90 days are commonly used and guide-
lines indicate that these are acceptable. The 14 to 28 day tests are
generally used to provide information on the nature of the 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 pro-
vide information on the effects of chemicals on the maturation process,
in addition to evaluating the nature of toxic effects at lower prolonged
-------
4-39
doses. The purpose of this section is to present duration reviews from
the literature and summarize data from duration comparison studies, to
evaluate the effectiveness of 90-day and shorter duration tests.
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). £s 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 test 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
s
100% for the sensitivity of the method at 21 days and then 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
toxiciOy evaluations the gain was significant-when the test was increased
from 11 to 14 days but not when increased to 21 days. Thus, Smith recom-
mended 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 effectiveness
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 (approx-
imately 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
-------
4-40
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-tern
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°
M2
M2
M3
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*
vP
M*
M°
M*2 ,
3.0
5.0
0.06
M8
M*
vF
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: short-term/
2-years
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. a
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. Ot
0.5?
0.8?
1.0*
2.0?
2.0i
3.0C
5.0C
9.0"%
3.3.5
5.0^
5.0^
?M = 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.
°Aa 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.
-------
4-41
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 used to predict results for a 2
year study, with a significant degree of accuracy, by dividing the short-
terra 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 resulting from the 90 day study would
appear to be a reasonable estimate of chronic effects. However, Weil and
McCollister did not recommend that the two-year study be abandoned, stating
only that in certain cases a 90-day study would be sufficient. The deter-
mination of when the shorter study would suffice is left up to the toxi-
cologist based on his experience with the chemical and its metabolic pattern.
f
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 re-
sults 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 LD30, 7-
and 9-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
LD30 data and the 90-day test was poor, needing a ratio of 40 to achieve
a predictive accuracy of 95%. The relationship between LD30 data and 7-
day test data is not much better with a ratio of 20 for 95%. However,
extrapolation from 7- to 90-day tests is much better, with a ratio of
only 6.2 for 95% of the results. This compares favorably with the extrap-
olation results of the two-year studies done in 1963. As the authors
-------
4-42
Table 4.11. Parameters of ratios of acute peroral ID50s, 7- or 90-day
and 2-year minimum effect (MiE) dosage levels
Ratio
Percentile
25th
50th
75th
95th
Semi-interquartile
range
Coefficient of
rank correlation
Formula _ n ,
or symbol ?_*°y
MiE
Qi = Pa5 1.2
Pso = median ,2.3
Qs = PTS 6.0
P,3 20.0
(S3-Si)/2 2.4
P 0.831
LD30/
90-day
MiE
,2.3
10.0
25.0
40.0
11.4
0.782
7-day
MiE/
90-day
MiE
2.2
3.0
5.2
6.2
1.5
0.943
90-day
MiE/
2-year
MiE
. 1.1
1.8
3.8
5.7
1.4
0.946
Source: Adapted from Weil et al., 1969.
-------
4-43
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 sufficient significant data indicating that
subchronic test durations could be used to predict chronic effect levels
without a substantial loss in sensitivity.
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 six- or three-month test is adequate
to detect long-term toxicity. This three- to six-month duration is also
recommended by Boyd (1968), Bein (1963), Barnes and Denz (1954), and Zbinden
s
(1963). Peck gave data from studies done from the previous 15 years to
support the use of a duration of three months. As shown in Table 4.13,
only one study showed additional toxicity after three months, and only
four showed added toxicity after two months. This again supports 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 i,s not adequate. He recommended
/•
this after consideration of some data on chemosterilants. Initially Hayes
(19672?) felt that some drugs could be tested in 30 exposures, but on re-
examination he prefered the 90-day exposure test. Thus, there is defi-
nitely 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 components with a
-------
4-44
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/a3.0 90-day MiE/a1.8 or
,7-day MiE/5.4
95th percentile 7-day MiE/fi.2 90-day MiE/5.7 or
7-day MiE/35.3
'zhe 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.
-------
4-45
Table 4.13. Approximate duration of drug administration
required to define toxicitya in animals
Compound
Indomethacin
Hydrochlorothiazide
Amiloride HC1
Cyproheptadine
Amitriptyline
Methyl-DOPA
Penicillamine
Ethacrynic acid
Protriptyline HC1
Thiabendazole
Dexamethasone
Duration
1/2
b
X
X
X
X
X
X
X
X
X
X
X
1
X
X
X
X
X
X
X
X
0
X
X
2
0°
0
0
0
0
X
X
s
X
0
X
X
3
0
0
0
0
0
0
*
X
X
0
X
0
(months)
6
0
0
0
0
0
0
0
0
0
0
0
12
0
0
0
d
X
0
0
0
18
0
0
0
0
0
24
0
0
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.
-------
4-46
variety of test species. Despite the variability due to differences in
species and experimental design, McNamara's data agree with that of Weil
and McCollister in recommending that two-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 predicted by short-
term tests. As with the other authors, McNamara qualified this predictive
value to exclude carcinogenetic and teratogenetio 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.
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 principle effects were in-
creases 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 stud-
ies in both sexes. In this study, the 12-week duration definitely predicted
-------
4-47
Table 4.14. Prediction of long-term
no-effect doses
Repeated dosage
Dose will produce
no effect in
LD3o/lOO , 3 months
LD5o/1000 Lifetime
3-month no-effect
dose/10 Lifetime
Source: Adapted from McNamara,
1976.
-------
Table A.IS. Urinary ascorbic acid excretion and relative liver and adrenal weights of rats fed butylated hydroxyanlsole (BI1A)
or butylated hydroxytoluene (BUT) at 0.1Z of the diet for 16 weeks'2
Compound
Duirdtion
of feeding
Ascorbic acid''
(mg/kg/day)
Male
Arachis oil, control
BUT
BHA
Arachis oil , control
BUT
BHA
Arachis' oil, control
BUT
BHA
Arachis oil, control
BUT
BHA
4 2
4
1
6 - - 4
6
4
12 2
3
1
16 2
3
2
.20
.74
.84
.39
.90
.89
.10
.41
.87
.50
.13
.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. ± 1.27a
4.16 ± 0.38°
1.28 ± 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
Organ weight to
body weight ratio (%)
Liver
Male
4.31 ± 0.11,
4.68 ± 0.16
4.36 ± 0.15
4.13 ± 0.05
4.18 ± 0.06
4.24 ± 0.12
3.73 ± 0.07
3.93 ± 0.07°
3.58 ± 0.14
3.50 ± 0.10
3.73 ± 0.08
3.72 ± 0.09
Female
3.94 t 0.08
4.45 ± 0.08;;
4.50 ± 0.12a
3.71 ± 0.11.
4.00 ± 0.09
3.82 ± 0.12
3.15 ± 0.10
3.51 ± 0.08,
3.60 ± 0.06
3.21 ± 0.10
3.63 ± 0.10°
»3.40 ± 0.17
Adrenal
Male
0.010
0.013
0.012
0.010
0.010
0.010
0.009
0.010
0.011
0.009
0.010
0.011
± 0.0008
± 0.0010°
± 0.0007
± 0.0005
± 0.0004
± 0.0011
± 0.0037
± 0.0026
± 0.0008
± 0,0081
± 0.0061
± 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
± 0.0004°
.Each result represents the mean ± SE for six animals.
Ascorbic acid is the mean of four days' excretion.
Cf < 0.05.
ap < o.oi.
P < 0.001.
•o
J>
oo
Source: Adapted from Gaunt et al., 1965.
-------
4-49
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) (DDBG),
a plasticizer, Hall, Austin, and Fairweather (1966) tested the oral toxic-
ity 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% pf 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 DDBG would have been detected "by a four- or eight-
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 noticeable after
the first week, but began to parallel the control values as the exposure
period progressed. However, the final body weight was still significantly
f-
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 cholinestrase 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
-------
Table 4.16. Mean values of body weights, food consumption, and
dibutyl(diethylene gfycol blsphthalate) (DDGB) intake of rats
fed DDGB at 0%-2.5% of the diet for 11 weeksa
Dietary
level
(%)
0.0
0.25
1.0
2.5
0.0
0.25
1.0
2.5
Body weight (g)
at end of week
0°
96
100
91
98
82
82
87
83
4 8
241 317
226 , 301
214^ 280e
17 Oe 2323
169 205 ,
156 185
160 196
11
352
335
315e
262e
220
199J
21la
1908
Food consumption
(g/rat/day)
at end of week
n°
0
11.9
11.6
- 9.5
5.8
9.5
9.7
9.5
5.6
4
Male
23.4
20.5
19.3
16.3
Female
15.7
14.5
14.6
14.2
8
18.7
20.2
17.1
15.5
\
13.9
13.1
13.5
13.6
i
11
18.
19.
16.
14.
14.
11.
13.
11.
DDGB intake (g/kg/day)^
at end of week
0°
0
4 0.29
7 1.04
9 1.48
5
3 0.30
5 1.09
4 1.67
4
0.23
0.90
2.40
0.23
0.91
2.44
8
0.17
0.61
1.68
0.18
0.69
1.93
11
0.14
0.53
1.41
0.14
0.64
1.52
,Values are the means for groups of 10 animals.
DDGB intakes are calculated from data on body weight and food consumption.
i)ay one of feeding.
P <0.05.
6P <0.001.
Source: Adapted from Hall, Austin, and Fairweather, 1966.
-------
4-51
ORNL-OWG 79-I3668R
400
I
o
Q
O
CD
UJ
5
350
300
250
200
150
100
I I I
I I I I
—•—CONTROL
500 ppm
07 21 35 49 63 77
EXPERIMENT DAY
91
Figure 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.
-------
Table 4.17. Mean cestes and brain weights, as percentage of body weight, of control rats and rats fed a dietary level
of 500 ppm of sumlthion for various periods of time
Organ
weight after days of feeding indicated
Organ " 30 60 90
Control
Left tescis 0.41 ± 0.02
Right testis 0.42 ± 0.02
Brain stem 0.21 ± 0.00
Brain cortex 0.58 ± 0.02
500 ppm Control
0.53? ± 0.02 0.40 ± 0.02
0.52a ± 0.01 0.40 ± 0.02
0.25^ ± 0.01 0.15 ± 0.04
0.79a ± 0.01 0.46 ± 0.01
500 ppm Control 500 ppm Control 500 ppm
(Z) «) (Z) GO (%)
0.60a t 0.40 .0.29 ± 0.02 0.38 ± 0.03 0.27 ± 0.02 0.35° ± 0.02
0.593 ± 0.01 0.2^ ± 0.02 0.39fc ± 0.03 0.26 ± 0.02 0.34° ± 0.03
0.23° ± 0.01 0.14 ± 0.01 0.14 ± 0.01 0.13 ± 0.01 0.15° ± 0.01
d b f /
0.54 ± 0.12 0.32 ± 0.01 0.40 ± 0.02 0.30 ± 0.01 0.35 ± 0.01
?Value differs from controls, P <0.01. %
Value differs from controls, 0.01
-------
4-53
- (a) KIDNEY
100
5O
o
X
u
i.
ORNL-OWG 79-13663
u (A)LIVER
K'l CONTROL
I 500 ppm
I 250 ppm (~1 125 ppm I I 63 ppm
too
50
II 30 60
EXPERIMENT DAY
90
30 60
EXPERIMENT DAY
32 ppm
- (dRED 8LOOD CELL
J
~ ,
'n
JT
i
i
' n m
• ini 1 1
1
ill
1!
-
r
J
x
- (
d
R
1
'
1
I*
*
E
|
5 P
^ 1
J
\
V
?
f
>
s
JT
1
|
|
90
Figure 4.2. Changes in cholinesterase activity in the kidney,
liver, red blood cells, and brain cortex of rats fed Sumithion for
90 days. Source: Adapted from Misu et al., 1966.
-------
4-54
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.
>J 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
(3 months); -and 25, 125, 250, and 2500 (2 years). Depression of the growth
rate occured 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
s
no mortality was seen at 3 months. Howevef, 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 1 month (4000 and 8000 ppm), at 3 months (2500 and 5000 ppm),
and at 2 iyears (2500 ppm). The hemoglobin concentrations were also de-
pressed at 1 month (4000 and 8000 ppm), at 3 months (5000 ppm), and at 2
years (2500 ppm). The 1-month study also indicated a decrease in hemato-
crit which was not evidentf 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 un-
affected. In the 2-year study the spleen weight was increased (2500 ppm)
at 9 and 17 months, but not at 24 months. Histopathologic data was not
significantly different from the controls for all tests regardless of dur-
ation. In conclusion it appears that the major toxic effects were suffi-
ciently indicated by the 1-raonth duration. Extension to 3 months and 2
years provided very little additional information.
-------
4-55
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 while 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 in-
termediate 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 nemo-
f
globin level was also depressed at the high dose in the two year study,
s
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 durations 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 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 significant 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
-------
4-56
Table 4.18. Cumulative toxicity of epichlorohydrin:
body weight gain in grams (mean ± SE)
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. 6f ± 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 ± 6.82^
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.
-------
Table 4.19. Subacute toxicity of epichlorohydrin:
body weight gain in grams (mean ± SE)
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
h
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
37Q.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
X34.00 ± 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
7j
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
^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.
•o
-------
4-58
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 signif-
icantly 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,
while the brain showed a significant weight decrease (Table 4.21). In com-
paring 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.
*•
In two companion studies, Kociba et al. (1976; 1978) evaluated the
s
toxicity potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administered
in the diet of rats for 13 weeks (1.0 and 0.1 yg/kg) and two years (0.1 and
0.01 ug/kg). As shown in Table 4.22 the results were quite similar for
mortality, body weight gain, alkaline phosphatase, packed cell volume, red
blood c*ell 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 were mainly due
to 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.
In another study of TCDD, Harris et al. (1973) tested the toxic
effects in female rats from gastric inturbation for 31 consecutive days
-------
4-59
Table 4.20. Cumulative toxicity of epichlorohydrin:
percent organ to body weight of rats (mean ± SE)
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 ± 0.05J.
0.3,09 ± 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.
-------
Table 4.21. Subacute toxicity of epichlorohydrin:
percent organ tx> body weight of rats'7 (mean ± SE)
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 ± 0.027^
a
0.402 ± 0.029h
0.917 ± 0.054*
4.070 ± 0.276d
0.225 ± 0.021
Calculated as (organ weight, g/body weight, g) x 100 =
body weight.
^Significantly different from controls at 99% level (P
Student's t test.
^Gonadal weights were not determined in this group.
"Significantly different from controls at 95% level (P
Student's t test.
Source: Adapted from Lawrence et al., 1972.
percent organ to
= 0.01) fcy
= 0.05) by
.e-
i
o\
o
-------
4-61
Table 4.22. Comparison of 13 week and 2 year 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 changes in:
Thymus
Other lymphoid tissue
Uteri t
Ovaries
Liver
Lung
Brain
Pituitary
13 week
1.0 ug/kg
Only FC
4-"
4-
4-M; tF
4-M; tF
4-M; tF
t
t
tF
t -x
—
t
4- .
t
M, F
M, F
F
F '
M, F
M, F
—
~~~
study
0.1 nag/kg
4-
4-
4-M
4-M
4-M
—
— ,
—
tF
—
tF
4-
t
M, F
M, F
—
—
M, F
— .
—
^™
2 year study
0.1 ug/kg 0.01 ug/kg
Only F
4- 4-F
— —
4-F -
4-M -
4- -
— —
— — _ .
— — ... •
— —
tF -
tF -
4-F -
tF tF
F -
— —
F -
_ . _
M, F M, F
M, F F
F -
F -
a
,Adapted from Kociba et al., 1976.
Adapted from Kociba et al., 1978..
J — females only; M — males only.''
4- — decrease in value vs control; t — increase in value vs control.
Histopathologic changes exclusive of carcinogenic tumors.
-------
4-62
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 days of exposure,
due primarily to reduced body weight. The thymus weight was significantly
decreased by day 17 at both the high and intermediate doses. Results of
this study indicated that the 30-day duration would have been sufficient
s
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).
Rosankrantz efal. (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/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. For
hematological and biochemical, parameters, there were several trends. At
28 days, although there were no statistically significant changes, there
were increases in lymphocyte levels of males and reticulocyte levels of
females, and a decrease in polymorphonuclear cells of males. At 90 days,
red blood cell numbers increased 11%, hematocrit increased 8%, and for
females the- white blood cell levels increased 19% to 74% in a dose-dependent
-------
Table 4.23. Weight gain of female rats receiving 31 daily doses of |
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
Daily Initial T . . , TI . , . . PT,
TCDD number K *nitialK Weight gain, g t SD
, ,. body weight
dose of , . f _ . _ , __a „_ ,^a
f /, «. . , (g ± SD) Days: 1-7 1-35 35-63
(vig/kg) animals & 7
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 ± 12.2 42.0 ± 11.9
10 16 184.0 ± 8.00 -21.4 ± 14.9
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.
°P <0.06.
Source: Adapted from Harris et al., 1973.
-------
Table A.24. Liver and thymus weights of female rats receiving daily doses of 2,3,7,8-tetrachlorodlbenzo-p-dioxin (TCDD)
Number
of
doses
3
6
10
13
17
24
31
Number
of
animals
4
4
4
4
4
4
4
0 yg/kg
Liver
(g ± SF.)
7.80 ± 0.48
8.41 ± 0.36
9.14 i 0.21
7.90 + 0.31
9.26 ± 0.42
9.45 ± 0.31
9.70 ± 0.49
Thymus
(rag ± SE)fl
530 ± 48
615 + 59
525 ± 43
558 ± 8.7
0.1 ug/kg
Liver -
(g ± SE)
8.62 ± 0.41,
10.35 ± 0.45?
10.78 ± 0.41?
10.17 + 0.94°
10.91 i 0.37
11.40 ± 1.05
11.78 ± 0.81
Thymus
(mg ± SE)a
492 ± 81
468 ± 22,
412 ± 28
475 ± 49
1.0 ug/kg
Liver
(g ± SE)
9.32 ± 0.45
9.'00 ± 0.14
11.53 ± 0.40
9.76 ± 0.62
11.33 ± 0.29°
11.39 ± 0.98.
12.84 ± 0.84
Thymus
(mg t SE)a
390 ± 58
270 ± 11?
372 ± 28°
260 ± 41
10 ug/kg
Liver
(g ± SE)
9.58
10.84
10.53
9.80
7.52
4.67
*
± 0.43
± 0.97,
i 0.36°
± 1.08
± 1.37
± 0.21°
Thymus
(mg ± SE)a
142 ±
110 ±
32 ±
12"
48J
10°'
a
.All dose response tests for thymus significant at P <0.01.
P <0.05.
CP <0.01.
Source: Adapted from Harris et al., 1973.
-------
4-65
manner. At 180 days, hematocrit and red blood cells rose 11% and 7%,
respectively, in the females. SCOT and 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 prostrate
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 prostrate 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 prostrate 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, the
significance was borderline. None of the histopathological changes were
considered significant, with lesions only occurring incidentally in both
control ^nd treatment animals. In th.is study, the 28-day duration revealed
the same changes as the longer studies for growth rate depression and weight
changes in minor organs. However, it 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
enough 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&) I /
dosed rats daily for one and six months. Even though the six-month test
is not a subchronic duration, this study is useful for evaluating the
-------
4-66
the effectiveness of short-term tests (<90 days) to predict long-term
effects. In the one-month study, dose levels of 250, 500, 1000, and
2000 mg/kg were used, while the six-month study utilized 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 one-month test.
Other effects detected at the high dose were increases in SCOT, SGPT, AP,
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 in a dose-dependent
rate. In the six-month experiment, no mortality occurred, but body weight
gain was decreased at the high dose. The biochemical'and hematological
s
parameters were affected less in the longer study than in the one 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 six-month study. Therefore, in this study, the shorter test would
have been a sufficient predictor of long-term effects.
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 (one month) and 3,'30, and 300 mg/kg (six months). In
the shorter study, mortality at the high dose was high (64% for males, 24%
for females) while in the chronic study only one substance-related death
occurred. The body weight gain was significantly decreased at the high
-------
4-67
dose in the one-month test, but showed a significant increase in the female
after six months of testing. The hematological and biochemical tests per-
formed for the one-month duration, revealed at the high dose, decreases in
platelet count (also at intermediate dose) and blood glucose levels, and
increases in BUN, Ca , SCOT, and SGPT levels. In the six month study, an
AP 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
six month study, only female heart weight increased at high and intermedi-
ate 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 six-month test, only the heart damage (again with a predominance of
ischemic lesions) occurred at the high dose. Comparing the two experi-
ments, i£ appears the shorter test indicated more toxicity than the longer
evaluation. This may be due to different dose magnitudes, the effects of
Fenoterol-HBr being acute in nature, or indicating 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 hr 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 comparing the ef-
fects 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 was seen after the 13 week
-------
4-68
exposure. Growth depression occurred at the high dose (4 and 13 weeks)
and irregularily 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, while 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 Ghat this may be due to
severe growth depression, since 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
s
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 t'oxicity of'di-(2-ethylhexyl)phthalate,
Xj
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 is due in part to lowered food consump-
tion, which was especially bad during the first 48 hours. As shown in
Table 4.26, there were irregular fluctuations in the packed cell volume,
hemoglobin and red blood cell values throughout the study with the decrease
-------
Table 4.25. Mean body weights and water intake of rats fed diets containing 0%-2.0%
di-(2-ethylhexyl)phthalate (DEHP) for up to 17 weeks
Dietary Body weight, ga at day
level
'
0.0 96 105
0.2 98 105
1.0 98 100
2.0 99 99
0.0 85 92
0.2 88 95
1.0 87 90
2.0" 88 87
, Body weights
27 55
340 478
325, 455
297J 417*
1878 300*
214 -. 273
216 277
210 259
13le 164
90
569
539
493e
413e
309
308f
284'
191*
1.20
628
588
546e
4476
329
325,
297d
20le
Water intake, ml/rat/day^ at day
0°
Male
18.3
17.6
18.0
18.4
Female
15.9
17.9
17.4
18.3
1
18.5
19.7
15.1
15.7
15.7
18.9
15. 4\
14.8
27
37.1
37.3
34.3,
24. 9d
21.5
24.9.
26. 6J
21.1
55
38.0
36.3,
27. 9d
30.9
21.9
34.5
24.5
21.3
90
28.5
32.3
29.0,
34. kd
18.1
26.5.
25. 9J
18.9
Mean
120 (ml/rat/day)
26
24
27
26
19
22
22
16
.3
.7
.7
.9
.4
.1
.6
.7
30.1
30.1
27,8.
25. 77
22.3.
25. 07
24. y
19.7
are the means for 15 animals. «
Values for water consumption
the 24-hr period preceeding the day
^First day of
P <0.01
IP
-------
Table 4.26. Mean haematological values for rats fed diets containing
di-(2-ethylhexyl)pttthalate (DEHP) for 2, 6, or 17 weeksa
'/—9 (
b £. « \
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
Number
of rats
examined
5
5
5
5
-• 5
5
5
5
5
5
5
5
15
15
15
15
Hb*
(g/100 ml)
14.8
13.00
14.8
15.1
14.4
14.8
15.1
15.0
14.9
15.8
15.4
14.4
16.0
15 '4fc
14 '5h
14.5
G d
PCV RBC
(%) (lOVmm3)
Week 2
45
40
42
43 .
38
41
Week 6
48
45
46
49.
44*
43*
Week 17
46
45
43?
43
6.26
5.770
6.48
6.50
6.24
6.82
\
7.08
6.96
6.86'
7.81
7.90
7.49
7.57
7.44
6.97
7.60
Re tics6 WBC-f
(% of RBC) (lOVmra3)
2.0
2.7
1.3
1.6
2'2h
0.8"
1.4
1.6
1.3
1.2
1.0
1.2
0.9
0.6
0.8
0.9
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
.p-
-------
Table 4.26 (continued)
Sex and
dietary
level
Female
0
0.2
1.0
2.0
Number
of rats
examined
15
15
15
15
(g/100 ml)
14.9
14.9
14.4
13.8
PCVC
45
44,
42ft
42^
(lOVmm3)
7.14
7.05
7.26
6.78
Retics6
(% of RBC)
0.9
0.8
1.0
0.8
WBC-'
(10s /mm3)
4.7
4.4
5.4
5.5
are means for the numbers of rats shown.
rFigures
Hb — haemoglobin.
,PCV — packed cell volume.
RBC — red blood cells.
n
/Jletics — reticulocytes.
•'WBC - white blood cells..
IP
-------
4-72
in packed cell volume the only significant change occurring at both 6 and
17 weeks. As shown in Table 4.27, changes in relative organ weights oc-
curred 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 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. In examining the data in this study, the majority of effects
were indicated in the 2-week study, and certainly by the 6th week. The
r
only significant change found with 17 weeks of testing was the quantita-
s
tive 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 three to six months
f
as sufficient (Peck, 1968; Boyd, 1968; Bein, 1963; Barnes and Denz, 1954;
World Health Organization Technical Report, 1966; Davey, 1964). In addi-
tion several primary studies showed that chronic effects are indicated at
a statisically significant level by tests of 90 days (Ambrose et al., 1972;
Kociba et al., 1976, 1978; Gaunt et al., 1965; Rosenkrantz et al. , 1975).
Thus, the evidence in the literature supports the conclusion that a 90-day
test duration does predict most chronic effects.
-------
Table 4.27. Relative organ weights of rats fed diets containing 02-2.OZ di-(2-ethylhexyl)phthalate (DE1IP) 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
1.2
2.0
Number
of rats
examined
5
5
5
5
5
5
5
5
5
5
5
5
15
15
15
15
15
15
15
15
Relative organ weight (g/100 g body weight)
Brain
1.12
1.17
1.75
1.19
1.21,
2.04d
0.56
0.62
1.03°
0.80
0.89
1.63
0.37
0.39
Q
0.50d
0.64
0.64
0.69 ,
i.iod
Heart
0.46
0.42°
0.426
0.45
0.426
0.40
0.35
0.40*
0.43d
0.36
0.38
O'.41e
0.27
0.27
0.28
0.30
0.30
0.30
0.31 ,
0.39d
Liver
3.15^
6.19j
6.47d
3.61,
5.28
6.62
2.99^
4.72*
6.76d
2.88^
4.40*
5,83d
2.31d
2.72*
4il2d
2.25
2.60
3.46^
4.59d
Spleen
0.30
0.32
0.24e
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
0.196
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^
0.70d
0.57
0.60 ,
0.65d
0.71d
Stomach . ma. .
Intestine
Week 2
0.70
0.70
1.01°
0.69
0.65
0.98°
Week 6
0.45
0.47
0.78°
0.58
0.60,
0.98d
Week 17
0.30
0.31
0.33^
0.38d
0.39
0.39
0.42,
0.72d
3.39
3.76
4.41°
3.62
3.79
4.39
2.42
~ 1 C
4!58e
2.80
3.16,
3.80d
.
1.45
1.55
1.66*1
1.91d
2.01
2.09
2.40 ,
3.13d
Caecum
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
0.29
0.20
0.32
0.34^
0.44d
Adrenals
20.1
19.9
26. 9e
36.0
25.8°
33.1
12.3
11.1
20. Oe
22.5
23.0
27.7
8.9
9.5
10.1
10.9
17.2
19.0
21.9°
20.4
b a
Gonads Pituitary
1.08
0.90
0.59°
57
50
41
0.88^
°'30d
0.44d
55
61
39e
0.60
0.61 ,
0.41d
0.23d ,
30
29
37
25
4.1
3.9
4.0
5.4
4.8
4.1
2.6
2.4
3.0
4.5
5.4
4.7
1.9
1.9
4.2
4.0
4.6
4.0
Thyroid0
6.6
8.3
12.0°
8.2
9.3
10.1
3.6
4.7
8.1
6.2
7.1
10. 1°
3.8
3.4
5.1
5.1
7.5
6.5
7.8
9.1
.Relative weights of these organs are expressed in mg/100 g body weight.
Relative weights of the female gonads are expressed in mg/100 g body weight.
~f <0.01
P <0.001
CO
P <0.05
Source: Adapted from Cray et al., 1977.
-------
4-74
There are also 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, 19752?) indicated that a one-
month duration would adequately predict six-month effects. Thirty day
durations have also been evaluated and generally recommended as sufficient
to indicate toxicity or dose levels (Misu et al., 1966; Hall, Austin, and
Fairweather, 1966; Hodge, Downs, Fanner, Smith, Maynard, Clayton, and
^
Rhodes, 1967; Harris et al., 1973; Krussye et al., 1977; Gray et al., 1977).
However, Hayes (1972) concluded that 30-day durations were insufficient,
but an early paper (Hayes, 19672?) did agree that a 90-day duration was a
good indicator of chronic effects. Thus, data from primary studies generally
support 1»he use of 30 day duration, but there is little concurrence on what
toxic effects might be missed. Perhaps in the future, with more information,
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 approximately $255,800
i
for a complete chronic and subchronic study to $100,500 for a subchronic
study design (Gehring, Rowe, and McCollister, 1973).
-------
4-75
4.4 ROUTE OF EXPOSURE
4.4.1 Introduction
In subchronlc test designs, various routes of administering the
chemical to the test animals have been utilized. The routes most often
employed are oral and inhalation. However, alternate exposure methods,
such as percutaneous application are used if advantagous for a specific
chemical. In 183 subchronic studies taken from the literature, the fre-
quency 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 basad on the expected
route of human exposure (Peck, 1974; World^Health Organization, 1978;
National Academy of Sciences, 1975, 1977; Ministry of Health and Welfare
Canada,. 1975; Benitz, 1970; Food Safety Council, 1978; Federal Register,
1978). In addition to the metabolic reasons for using the expected human
exposure route, it is also 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 (World Health Organization, 1978;
U.S. Environmental Protection Agency, 1979; Weisburger, 1975). Also, the
-------
4-76
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 principle route used in most
toxicity experiments. This predominance results from both practical con-
siderations (ease of application; low cost factors) and experimental re-
quirements (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 Organization, 1978), while 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).
-------
4-77
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 administration. This
saves valuable personnel time and disturbs the test animals 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 pos-
sible 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); adjustment of the dose
*
with animal maturation to maintain proper concentration levels (Barnes
s
and Denz, 1954; National Academy of Sciences, 1977; World Health Organi-
zation, 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);
nonhonuJgeneity of the diet-chemical mixture (Sontag, Page, and Saffiotti,
1976); and measurement of daily food consumption to calculate daily expo-
sures (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 elimin-
ates 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,
-------
4-78
1978). Other disadvantages include higher mortality rates and more fre-
quent 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 differ-
ence between the techniques, diet is to be prefer-ed due to 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).
Inhalation exposure is used in studies where the primary human expo-
f
sure is to be by inhalation, or information on the specific site of entry
s
(lung) damage is desired (Roe, 1968). The total effects of inhalation can
rarely be predicted (with a high degree of certainty) from oral or paren-
teral studies, which necessitates the use of inhalation exposure (Gage,
1970; World Health Organization, 1978). However, unlike oral administra-
tion, ttee of inhalation exposure is' neither simple nor inexpensive.
To adequately study inhalation toxicity, specially designed inhalation
chambers are needed. The various chamber designs and advantages 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 considerations are the increased
cost which this route of exposure entails and the potential bottleneck rep-
resented 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
-------
4-79
study. The dose is usually measured by monitoring the chamber concentra-
tion (c) of the toxicant and the time (t) that the test animal is in the
chamber (Roe, 1968; MacFarland, 1968). The dose is expressed as the prod-
uct (ct) 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 very difficult, if not
impossible to correlate with oral or injection sCudies where the dose is
expressed as rag/kg body weight (Barnes and Denz, 1954; Hayes 1967a).
Additional problems associated with inhalation dosage are the uncertain-
ties regarding the actual amount of the toxicant that is inhaled by the
test animal, the amount of toxicant that adsorbs onto the chamber walls,
*•
and possible variations in the flow rate of toxicant into the chamber
,s
(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 (Roe, 1968; National Academy
of Science, 1977)!
Another factor of importance in the chamber design is the expected
schedule of exposure durations. If continuous exposures (22-24 hr/day, 7
days/week) are to be used, then the chamber must provide all food, water,
and other needs of the test animals. If intermittant exposures (6 to 8
hr/day, 5 days/week) are used, then chambers of a simpler design can be
used. These two duration patterns produce different effects. The nonex-
posure time in an intermittant 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
-------
4-80
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 aer-
osol particles and some control is necessary to insure uniform particle
size and density, at least within a size range that is suitable for biolog-
ical action (Roe, 1968; Ministry of Health and Welfare Canada, 1975; National
Academy of Sciences, 1977). Particle size must also be monitored in the
chamber itself to insure that the exposure levels remain consistent. The
/•
deposition sites (nose, trachea, bronchi, or alveoli) and the degree of
s
biological action of the inhaled particles are determined by their solu-
bility in tissue fluids, their reactivity with lung tissue, and their size
(Roe, 1968; World Health Organization, 1978). Generally the larger par-
ticles, 5-10 ym in diameter, are trapped in the upper respiratory tract
and nas*al cavity (Minister Health and Welfare Canada, 1975; World Health
Organization, 1978). Particles less than 5 ym disperse further into the
lower respiratory tract with the depth of penetration increasing with de-
creasing 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 alevoli (Morrow et al., 1966).
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;
-------
4-81
World Health Organization, 1978). These effects are quite similar to par-
enteral or intravenous injection effects, at least in qualitative data
(Gage, 1970; World Health Organization, 1978). In contrast to oral expo-
sure, 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). SpeciJric site of entry effects
(lung damage) can only be studied 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 Organization (1978). 'These include respir-
s
atory 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
then the»dose levels are not as potent as originally designed. If swal-
lowed, then 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. Due to the high
cost, limited facilities, and additional personnel time involved, the use
of inhalation exposure should be limited to such assessments and not for
general assessment of systematic toxicity.
Other routes of exposure are infrequently used in subchronic studies,
including percutaneous and injection methods. Percutaneous or dermal appli-
cation is usually performed as a separate study. When using percutaneous
-------
4-82
exposure several factors influence the test design and data interpretation.
These include slower activity and lower exposures than with oral exposure,
difficulty of measuring absorbed dose, necessity of using a vehicle or sol-
vent in many cases, and ingestion by animals during self-cleaning of doses
applied to the skin (Loomis, 1974; Benitz, 1970; National Academy of Sci-
ences, 1977; Ministry of Health and Welfare Canada, 1975). A more detailed
review of dermal application techniques is given 4-n Section 3.
Parenteral injections are also used occasionally in subchronic tests.
These include intradermal, subcutaneous, intramuscular, intravenous, intra-
peritoneal, and intrathecal (Loomis, 1974). Intravenous administration
produces rapid distribution of toxins with the degree of metabolic trans-
formation, dependent upon the location of the used b'lood vessel in the
s
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 prefered route, since it
is rarely an expected route of human exposure.
4.4.3 Route Comparisons
f
There are very few papers in the literature that discuss in detail
the comparative value of exposure routes. The review articles used in
Section 4.4.2 contain most of the available information. Primary studies
designed to evaluate this area are almost nonexistent. Even within indi-
vidual chemicals, comparisons are difficult. The use of different species,
doses, durations, and parameters of evaluation (pathology, hematology, etc.)
-------
4-83
all combine to make route comparisons feasible only at a general level. A
few brief comparisons within chemicals are included as examples of the dif-
ficulties. 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 (Asa03)
in drinking water. Continuous exposure to 50 mg o* 250 mg arsenic/liter
(10 to 15 mg/kg per day) for 32 days produced levels of the chemical in
the skin and liver which 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 ug arsenic/m3 for 5"days per week, 6 hr 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 and then decreased sharply
during the third and fourth weeks. Skin values' peaked similarly as a re-
sult of direct exposure in the dust chamber, but decreased more gradually.
The authors recognized that effects of inhaled particles cannot be distin-
guished from effects of particles ingested as a result of mucociliary clear-
ance or licked from the skin during grooming, and concluded that the course
of arsenic acccmulation is similar following oral administration and inha-
lation. The drop in arsenic accumulation in tissues after the initial peak
was subsequently shown to be due to a stimulation of the excretory mecha-
nism 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/kg body weight per day of
-------
Table 4.28. Summary of clicmlc.il examples tested for toxlclty by various routes of .•ulniliilstr.ition
CliiMnlc.il
Arsunlc
Beryllium salts
Insoluble salts
Soluble compounds
Beryllium chloride
Beryllium nitrate
Beryllium sulfate
Beryllium oxyfluoride
Beryllium oxide
Beryllium phosphate
Beryllium
.
Beryllium sulfate
Beryllium fluoride
Beryllium sulfate
Beryllium oxide
Carbon tetrachloride
, Route
Oral
Inhalation
Oral
Inhalation
Inhalation
I. P.
I. P.
I. P.
I. P.
I. P. .
I. P.
Oral
Inhalation
Inhalation
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
Rat
Rat
Dose
10-15 in^/kg/
ituy
179.4 us As/ra'
0-30 mg/kg/day
188.9 and 233
mg/m3
27 and 43
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 ug Be/ms
2.2 mg/m3
r
4 mg/m3
80 mg/m3
Not given
10, 50, 100
ppni/day
Rural Ion
32 days
6 hr/day. 5
day /week, 6
weeks
11-29 weeks
30-40 tnin/day,
6 days /week,
1 day to 15
weeks
30-40 rain/day,
6 days/week,
1 and 10 days
4 days
4 days
4 days
4 days
4 months
4 months
6, 12, 18, 24
months ' x
\
7 hr/day, 5
days/week.
72 weeks
6 hr/day, 5
days /week,
6-23 weeks
6 hr/day, 5
days /week,
6-23 weeks
6 hr/day, 5
days/week,
6-23 weeks
3-5 rain, 3 times/
day on alter-
nate days in
35 days
3 hr/day, 6-8
weeks
Kffcrts n»tud by author
Transient accumulation In skin
and liver; wcic.ht 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
100% mortality
100% mortality
100% mortality
100% mortality
No mortality
No mortality
Decreased body weight; accumu-
lation in G.I. tract; skele-
ton, blood, liver. No hepatic
cell destruction.
Inflammatory response; lung
tumors
\
'Macrocytic anemia
Macrocytic anemia
Macrocytic anemia
Cirrhosis of liver
Elevated liver triglycerides
SCOT, SGPT unaffected
Reference
Bi-ncko and Syiium, 1969
Ui-ncko and Sycuon, 197O
llyslop, 1943
!
Hyslop, 1943
Hyslop, 1943
Hyslop, 1943
Hyslop, 1943
Hyslop, 1943 |
llyslop, 1943 00
llyslop, 1943 **
Hyslop, 1943
Reeves, 1965
—
Reeves, Deitch.^and
Vorwnld, 1967
Stoklnger and Stroud,
1951
Stokinger and Stroud,
1951
Stoklnger and Stroud,
1951
Reddy , Krishnamurthy ,
and Bhaskar, 1962
Shimizu, Nagase, and
Kawai, 1973
-------
Chemical
Dimethyl terephthalate
Fenoterol IIBr
2-Methyl-4-
chlorophenoxy
acetic acid
(MCPA)
Route Species
Oral Rat
Oral Rat
Oral Rat
S.C. Ra t
S.C. Rat
S.C. Rat
S.C. Rat
S.C. Rats
I. P. Rat
,
Oral Rat
Inhalation Rat
Oral Mouse, '.rat
Inhalation Rat
I.V. Rabbit
Dermal Rabbit
Table 4.
Dose
2 pi/100 g
150-520 ppm/day
0.2 ml/kg/day
0.1 ml/.lOO g
1.3 ml /kg/day
1.3 ml/kg/day
1.3 ml/kg/day
1.3 ml/kg/day
0.06 ml/ 100 kg
,
0.25, 0.5, !%/
day
16.5 and 86.4
mg/ra3-
15, 150, 1500
mg/kg/day
(rat)
1.5, 15, 150
mg/kg/day
(mouse)
0.01-1.0 rag/
kg/day
• 1.0 and 25-50
mg/kg/day
0.5, 1.0, 2.0
g/kg/day
28 (continued)
Duration
1 day (acute)
6 weeks
3 days/week, 7
weeks
1 dose/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
1-10 Injections
in 119 days
96 days
\
4 hr/day, 5
days/week, 58
exposures
30 days
30 days
30 days
30 days
3 weeks
Effects noted by author
(Single dose) elevated SCOT,
SCPT
Trlglycerlde accumulation In
liver
Increased liver fat
Cirrhosis
Cholangloflbrosis
Hepatic vein thrombosis
Neoplasla
Cirrhosis
Retardation of growth; elevated
SCPT; hepatic necrosis (tran-
sient); inhibition of normal
mineralization of tooth
dentine
Decreased body weight
No adverse effects
Enlarged salivary glands;
, enlarged hearts. Mortality:
rats — 64% male, 24% female;
mouse — 3% male and female
No adverse effects; <1%
mortality
Enlarged salivary glands;
enlarged hearts; 6% mortality
Transient growth retardation;
erythema; decreased leuko-
cytes; hyperplasia, hyper-
koratosis, and loss of
Reference
Shlmlzu, Nagase, and
Kawal, 1973
Alumot et al. , 1976
Friedman et al. , 1970
Reddy, Krishnamurthy ,
and Bhaskar, 1962
Reuber and Clover, 1967a
Reuber and Glover, 1967i>
Reuber and Clover, 1967c
Reuber and Glover, 1968
Hals, BJorlin, and
Jacobsen, 1973
Krasavage, Yanno, and
Terhaar, 1973
Krasavage, Yanno, and
Terhaar, 1973
Kast et al. , 1975
Kast et al. , 1975
Kast et al.. 1975
Verschuuren, Kroes , and
Tonkelaar, 1975
*>
Ul
elasticity of skin
100% and 75% mortality in high
dose groups
-------
Table 4.28 (continued)
Chemical Route
Oral
2-Methyl-4- Dermal
chlorophenoxy
propionlc acid
(MCPP) Oral
Nefopam hydrochloride I. P.
I.M. '
Oral
I.V.
Oral
I.M.
A9-Tetrahydrocannablnol Oral
I.V.
Species Dose
Rat 50, 400, 3200
ppra
Rabbit 0.5, 1.0, 2.0
. g /kg/day
Rat 50, 400, 3200
ppra
Rat 2 and 10 mg/
kg/day
Rat 1 and 2 mg/
kg/day
Rat 20, 40, 80
mg/kg/day
Dog 1 and 5 mg/
kg/day
Dog 4, 10, 40
mg/kg/day
Dog , 1.5 and 3.0
mg/kg/day
Rhesus monkey 50, 250, 500
mg/kg/day
5, 15, 45
rag/kg/day
Duration
90 days
3 weeks
90 days
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
Effects noted by author
Growth retardation
Increased kidney weight
Increased erythrocyte size
Increased hemoglobin content
Transient growth retardation;
erythema, transient loss of
skin elasticity
Decreased RBC count; decreased
hemoglobin content; increased
alkaline phosphatnse activity,
decreased hematocrit and leu-
cocyte values (male only);
increased kidney weights;
depression of ovary and
prostate weights
Increased liver weights
No adverse effects
No adverse effects
Slight weight loss
Slight weight loss
No adverse effects *
^
Summarized in Table 4.36
Reference
Verschuuren
Tonkelaar
Verschuuren
Tonkelaar
Verschuuren
Tonkelaar
Case, Smith
1975
Case, Smith
1975
Case, Smith
1975
Case, Smith
1975
Case, Smith
1975
Case, Smith
1975
Thompson et
, Kroes , and
, 1975
, Kroes , and
, 1975
, Kroes , and
, 1975
, and Nelson, oo
, and Nelson,
, and Nelson,
, and Nelson,
, and Nelson,
'
, and Nelson,
al.. 1974
-------
4-87
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 quite 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 per 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 and 1 days of exposure
respectively. Distribution of the chemical in tissues remained remark-
ably low following inhalation of both soluble and insoluble beryllium
salts. Intraperitoneal (I.P.) injections of 0.1 gm of beryllium chloride,
beryllium nitrate, beryllium sulfate, or beryllium oxyfluoride produced
100% mortality in guinea pigs after 4 days"while beryllium oxide and
phosphate were not lethal after 4 months.
Other studies of beryllium toxicity are summarized in Table 4.33.
These are of little value for route comparisons due to dose, duration,
and species differences.
4.4.3.3 Carbon Tetrachloride (CC1<.) — Reddy. Krishnamurthy, and
Bhaskar (1962) exposed albino Wistar rats to carbon tetrachloride (CC1A)
vapors for 3 to 5 hr, 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 the 15 females tested. Eight of the 15 females developed patchy
fibrosis of the liver. Inhalation of 10, 50, or 100 ppm CCl^/day, 3 hr/day
for six to eight 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 CC14 produced increased SCOT and SGPT values.
-------
4-88
Elevated levels of liver fat content have been observed in rats fol-
lowing oral administration of CC1<,. After six weeks of feeding fumigated
mash containing a daily dose of 1% of the LD90 (5 g/kg body weight), tri-
glyceride accumulation in the liver was found to be the most sensitive
sign of chronic CC1<, poisoning (Alumot et al., 1976). Oral intubation of
0.2 ml CCl<./kg body weight three times a week for seven weeks also caused
an increase in the liver fat content of rats (Friedman, Sage, and
Blendermann, 1970).
. Subcutaneous injection of 0.1 ml/100 gm body weight every 3 days for
90 days produced cirrhosis in 9 of 9 male rats and in 6 of 15 female rats
(Reddy, Krishnamurthy, 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 CC1<, induced cirrhosis was the same
following inhalation (described previously) and subcutaneous exposure. In
a series of separate studies in which CClu was administered subcutaneously
to Buffalo strain male and female rats, effects of the chemical were mani-
fested as a function of age and sex of the animals (Reuber and Glover,
1967a, 19672?, 1967e, 1968).
Intraperitoneal injection of 0.06 ml/100 kg body weight CC14 (up to
seven injections during 119 days) into albino rats induced an inhibition
of weight gain, elevated SGPT values, antf hepatic necrosis which then re-
/•
generated (Hals, Bjorling, and Jacobsen, 1973). Concomittantly, inhibi-
tion 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.
-------
4-89
A.A.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;
Prusakov, 1966 as cited in Krasavage, Yanno, and Terhaar, 1973), was fur-
ther evaluated in various acute studies and in subchronic feeding and in-
halation studies by Krasavage, Yanno, and Terhaar (1973). Neither feeding
nor inhalation caused mortality. The toxicologicel 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-sympatho-
mimetic, fenoterol HBr, for toxic properties. Subacute exposures (oral,
s
intravenous, and inhalation) continued for one month. Inhalation produced
no symptoms in rats which 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 rate in the inhalation experiment died during the exposure period,
while oral administration of high doses produced mortality rates of 64%
for male and 24% for female rats, and 3% for male and female mice. Rab-
bits receiving the drug intravenously developed enlarged salivary glands
and enlarged hearts. Six percent of the rabbits died during the third
r'
week of the trial. In this study, the durations were all comparible but
the use of various species and different doses reduces the reliability of
the comparisons.
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/kg body weight were
-------
4-90
applied to the skin of chinchilla rabbits in an aqueous paste (Verschuuren,
Kroes, and Tonkelaar, 1975). Four of four rabbits treated with 2 g and
three of four treated with 1 g MCPA died during the treatment period, pos-
sibly 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 lymphocytes 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,
f
400, or 3200 ppm MCPA in their food for 90 days (Verschuuren, Kroes, and
x
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 and body weight gain,
and increased kidney weights were characteristic of the 3200 ppm group.
There was also an increase in eryttirocyte 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/kg 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 retardation was
seen in all groups, but some recovery was observed during the two weeks
after termination of treatment. Erythema was observed and was dose re-
lated. The skin lost its elasticity, but reverted to normal condition
-------
4-91
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 in-
creased 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 stud-
ies 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 rag/kg (five
days per week for four weeks); intramuscularly, in daily doses of 0, 1,
and 2 mg/kg (seven days per week for two weeks); and orally, in the diet,
/
in daily doses of 0, 20, 40, and 80 mg/kg for four weeks. Increased liver
weights were observed in female rats'receiving 10 mg/kg of the drug intra-
peritoneally (approximately 30% of the intravenous LDSo)• 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 intramuscular LD30) or
up to 80 mg/kg per day orally (approximately 65% of the oral LD30).
-------
4-92
Mongrel dogs showed only slight weight loss after daily intravenous
administration of 5 mg/kg (approximately 25% of the intravenous LD30),
and after daily oral administration of 4 and 40 to 80 mg/kg (25% to 50%
of the oral LDSO) of nefopam. No toxic effects were observed following
daily intramuscular injections of up to 3 mg/kg body weight (approximately
10% of the intramuscular LD30) of the 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 dur-
ation so that comparisons would be difficult.
4.4.3.9 A9-Tetrahydrocannibinol (A9-THC) — Thompson et al. (1974)
described the toxic effects of A9-tetrahydrocannibinol, the active ingre-
r
dient in marihuana, when adminstered intravenously or orally to primates
s
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. This study has differences
in dose levels which make the conclusions of the authors questionable.
The preceeding examples are typical of within chemical comparisons.
Almost every comparison was complicated by test design variables. Any
-------
4-93
Table 4.29. Main toxic effects produced by oral or
intravenous administration of A9-THCa
Routes of administration
Oral Intravenous
Organ size
Thymus
Pancreas
Testes
Adrenal
Liver
Body weight
Clinical effect
Leukocytosis
Myeloid hyperplasia
Anemia
BSP retention
Electrolyte balance
Proteinuria
Bradypnea
Anorexia
Ulcerative colitis
Other effects
Behavioral changes
Development of tolerance
Atrophic
Atrophic
Atrophic
Enlarged
Enlarged
Decreased
'
/Yes
Yes
No
No
Altered
Yes
Yes
Yes
Yes
Yes
Atrophic
Enlarged
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
The authors concluded that pathologic changes
in deceased monkeys were associated with route of
administration.
/•
Source: Adapted from Thompson et al., 1974.
-------
4-94
conclusions that could be drawn from these would be tenuous at best. How-
ever, this is unfortunately the current status of information on this sub-
ject. 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 evalu-
ation of the route to route extrapolations is limited to a general level .
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, requiring a minimum of specialized equipment,
and producing good toxicity data. Thus, there is little need to extrapo-
late data from other routes to determine oral toxicity for most chemicals.
s
Injections are rarely primary routes of exposure and are relatively easy
to perform for durations of a subchronic nature. Thus 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
ability 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. Also
-------
4-95
dosages could be more stringently controlled. Another alternate route
could be the oral route. This would require previous testing to ascertain
if any significant metabolic transformations by the gastrointestinal tract
occur for that chemical. Of course, these extrapolations would not provide
information on toxicity at the site of entry. These data would have to
be obtained from preliminary studies (acute or subacute).
Before any of these route extrapolations can fee 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. Also a basis for extrapolation should be sought in
pharmacokinetic and metabolism studies that could justify the quantitative
and qualitative validity from one route to the other.' This research would
help to define the types of toxicants and tne 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 inhalation expo-
sures. 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 act as the
limiting factor in determining the number of tests that can be performed.
Occasional use is made of percutaneous and intraperitoneal exposures, de-
pending 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
-------
4-96
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 evaluations,
extensive organ/tissue examination is required or recommended. The EPA
r
guideline for pesticide evaluations (Federal Register, 1978) is a good
s
example of this, recommending for oral administration the microscopic ex-
amination 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 intermedi-
» • • -
ate 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). Besides the general appearance and microscopic
condition of the organs, the organ weigh't 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 debat-
able, however, with evidence and opinions supporting both the pro and con
arguments. The purpose of this section of the literature review is to give
-------
4-97
Table 4.30. Postmortem studies
Gross
External lesions Cervical tissues
Tumors Oral'tissues
Abdominal contents Voluntary muscle
Pelvic contents Bones
Thoracic contents Brain
Spinal cord
Organ weights
Histomorphology
Thymus s Heart
Lymph node Large and small
Thyroid arteries
Parathyroid Lung
Adrenal Stomach
Pancreas Duodenum
Liver Jejunum
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.
-------
4-98
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 the exposure periods were considered too short to affect
organ tissues (Bratton, 1945), or the ability of the histopathologic exam-
^
ination to add quantitative information to the other observations 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 two-year study (Smyth and Carpenter,
1948). Smith (1950) felt that histopathology could also be useful if a
series of compounds were being compared or i»anked for relative toxicities.
Even in these cases, the tissues to be examined were few, generally re-
stricted to the liver, kidney, heart, and any lesion-bearing organs. This
trend toward reduced histopathology in earlier short-term tests was con-
tinued by Smyth, Carpenter, and Weil (1951) in their development of the
» •
range finding test. As shown in Table 4.31, they found no material which
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 histo-
pathology. They identified some of the sources of variation in patholog-
ical results as: the subjective nature of the examination; the occurrence
of natural disease; and the problems of age changes in the test species.
-------
Table 4.31. Kun^c-l'inJIng Jala on subacute oral Coxlrlly
l.east dally Jose causing symptom (t;ni/kg)
M.iturlal studied
Acrolelir"
Aldol .
Ally I alcohol \
1 , 3-Butaned lol ,
"Cellosolvc" (2-ethoxyethanol) 'e
Dlchlor.il urea
2 ,4-Dichlorophenoxy ethyl
.sul Kile, sodium
Diethanolamine
(2,2'-im!nodiethanol)e
Dl(2-ethylliexyl) adipatefi
Dl(2-ethylhexyl)
tetrahydrophthalate
2-lHmetliylamlnoetlianol
(dlmetliyletlianolamine)
2,4-Dlmethyl-2-mcthylene-
1,2,4-thiodiazolidine Chione
Dimethyl methylene diphenol-p,p'
Dimethyl methylene diphenol-p,p ' ,
diglycidyl ether
Ethylene chlorhydrin
(2-chloroethanol)"
Ethylenediamine*'j s
2-Ethyl-l,3-hexanediole
2-Ethylhexanimido (diethyl-
2-ethyl hexanoate)
Glycidyl sorbate dimer
2-Methyl-2,4-pentanediol (4)
Monoethanolamlne (2-aminoethanol)
Mono isopropanol ami ne*
Tetrabutyl thiodisuccinate
l,2,3,4-Tetrathia-6,9-diazecane-
5,10-dithione
Tr iethanolaminee
Maxlimiro
dose
(Km/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
Ml lUr.uim
dose
(W/W
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
growth
_
1.58
—
_
0.74
0.18
0.66
_
2.92
2.67
-
0.10
—
—
—
0.31
0.70
—
0.27
—
-
—
10.3
0.55
1.27
Reduced
appetite
0.0015
-
0.0097
_
0.74
5.33
—
0.68
2.92
—
—
0.10
—
—
—
0.12
—
—
—
—
—
—
—
—
—
Altered
liver or
kidney
we luh I
0.0015
-
—
_
0.74
0.18
0.20
0.090
2.92
0.84
0.89
0.039
—
—
. 0.012 ,
0.31 \
—
1.53
0.012
—
0.64
2.22
1.08
0.17
0.17
Micro-
scopic
U-s Ions''
-
0.0097
_
0.74
2.55
—
0.17
2.92
-
—
—
—
-
—
0.12
—
—
0.27
—
1.28
—
10.3
—
0.73
!>.:.! Ill
-
0.0097
_
1.89
2.55
0.66
0.17
4.74
—
—
—
—
—
—
0.12'
—
—
0.27
—
1.28
—
10.3
—
0.73
Max I mini
dose
having no
effect
(f.n/kB)
0. (10(117
0.43
0.0040
5.60/
0.21
0.048
0.047
0.020
0.61
0.19
0.18
0.010
0.52/
0.35/
0.0024 '
0.036
0.48
0.44
0.0129
0.31/«
0.32
0.60
0.26
0.04
0.08
Single-dose
oral 1.1), »c'
/kK)
0.046 (0.039-0.056)
2.18 (2.00-2.3H)
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.
The dose producing microscopic lesions in liver, kidney, spleen or testis of any rat.
'Uhen l.D,o was derived from an advanced test, the range of 1.96 times the standard deviation is shown in parentheses.
refer .to data derived from a range-finding test.
The letters "RF"
'..The dose was administered in the drinking water. In this case appetite Is judged on the basis of mllllliters of water consumed.
^Boaes administered for 90 days.
•'The dose shown is the maximum which was administered, and it caused no symptoms.
The dose shown is the minimum administered, and it caused some symptoms.
Source: Adapted from Snyth, Carnenter, and Well, 1951.
I
VO
VO
-------
4-100
They saw little value in routine sectioning and histological examination
of 20 major organs and suggested that a thorough gross examination would
suffice. They also doubted the value of organ weight changes as indica-
tors of toxicity. Barnes and Denz cited Cameron (1952) concerning the
increase in organ weights due to hypertrophy without true damage to the
tissues. They suggested that organ weights be used as toxicity indica-
tors 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, his-
tology should only be done on high dose animals and 25% to 50% of the con-
trol animals. Further examination would be limited to organs showing
changes. In addition, Zbinden recommended a grading 'system which 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 rather than histopathology as the predominant indi-
cator 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
f
low dose toxicity indicator seven times. The same trend is shown for two-
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 indicating histo-
logic damage. The weight changes of the liver and the kidney increased
-------
Table 4.32. Summary of observations of effects detected in short-terra and two-year oral studies
At lowest dosage level in which any effect was detected
Short-term
Criterion Number of
" of effect studies in „ . ,
... . . Number of
which this ,
pertinent
criterion ,, a
studies"
Mortality
Food intake
Body weight
Organ weights
Liver
Kidney
Heart
Spleen
Testes
Lung
Brain
Thyroid
Stomach
Adrenal
Gross pathology
Mlcropathology
Liver
Kidney
Heart
Spleen
Testes
Lung
Adrenal
Pancreas
Bone marrow
Voluntary
muse] e
Stomach
Intestine
Bladder
Brain
Sciatic nerve
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
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 Number
where this where
criterion it was
was the sole
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
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
Two-year
Number of
studies in ... ,
which this Numb" off
. . pertinent
criterion ,, n
studies
was
followed
33
29
33
30
30
17
16
18
12
4
2
1
3
3
\
33 X
33
29
23
33
32
27
23
11
6
15
21
12
11
0
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
where 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
where
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
Ac next
higher dosage
level this
criterion
affected**
Short-
term
0
3
9
4
2
0
1
1
0
0
0
0
0
0
3
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
4>
M
O
-------
Table 4.32 (continued)
Ac lowest dosage
level in which any effect was detected
Short-terra
Criterion
of effect
llematology
Blood urea
nitrogen
Clinical urine
analysis
Central nervous
system
Neoplasm
Fertility
Number of
studies in
which this
criterion
was
followed
19
10
1
1
33
0
Number of
pertinent
studies'3
16
7
1
1
27
0
Number
where this
criterion
was
affected
1
0
0
1
0
0
»
Number
where
it was
the sole
effect
0
0
0
1
0
0
Number of
studies in
which this
criterion
was
followed
31
6
7
0
33
5
Two-year
Number of
pertinent
studies'1
24
3
5
0
25
3
Number
where this
criterion
was
affected
0
0
0
0
0
1
Number
where
it was
the sole
effect
0
0
0
0
0
0
higher dosage
criterion
affected"
Short-
term
0
0
0
0
0
0
Two-
year
0
0
0
0
0
0
Total number of studies in which this criterion was followed minus the studies in which none of the criteria were
significantly altered at any dosage level.
^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.
•c-
(-"
o
\
-------
4-103
their value as toxicity predictors in the longer two-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 a comparison of 90-day, one-week, and single exposure
tests, they used primarily the histology and weight changes of the 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
s
additional organs were less sensitive thati 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, then 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. He also stated that histopath-
ology is useful in determining the significance of spontaneous species-
specific lesions. If the commonly occurring lesions for that species are
altered in time of appearance, number, character, or severity, then this
-------
4-104
could be due to the activity of the drug. However, as a caution to the
use of histopathology, Peck stressed the difficulty in interpreting the
results (particularly for electron microscopy) and in extrapolating those
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, since in his opinion restricting
histology to organs with gross lesions significantly decreases the deter-
mination of complete toxicity. He also rejects the procedures of examin-
ing only the liver and kidney (since morphological effects or target organs
for new drugs are unknown), of examining just the major organs (too impre-
cise; varying with the pathologist's definition of major), and of examining
organs with gross lesions and all organs from a few representative test
animals (may not yield the optimum amount of morphological data). Benitz
does allow for deviations from histopathological examination of all organs
if previous studies for that chemical indicate certain toxicity patterns
or if results are only needed for certain organ systems. He stressed,
however, that all organ systems have demonstrated toxic effects from chem-
icals, 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—precancerous
lesions. Benitz also generally recommended weighing of the organs as an
indicator of toxicity. However, he felt that use of relative organ weight
-------
4-105
Table 4.33. Incidence (in percent) of drug-induced
pathological changes encountered over a ten-year
period in approximately 14,000 animals
Incidence
ay seem ana organ
Cardiovascular system
Respiratory system
Alimentary system
Liver
Gastrointestinal tract
Pancreas
Urinary system
Kidney
. Ureter and urinary bladder
Reproductive system
Hales
Females '
Ductless glands
Thyroid
Adrenal
Pituitary
Islets of Langerhans
Hematopoietic system
Lymphoid and reticuloen'dothelial •
system
Central nervous system
Sensory system
Eye
Ear
Locomotor system
Skin
By organ
4F
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
By system
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.
-------
4-106
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 due to 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 conclusion that in beagles
80% of the organ weight changes correlate 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
s. '
stages of morphological changes, (2) better interpretation of visible lesion
~. damage, and (3) detecting ultrastructural changes not visible through nor-
.;. mal techniques. However, due-to the time and effort involved, the use of
electron microscopy must be adequately justified in each instance. Quan-
tification of morphological changes ±s of value because results are more
precise and comparable. Table 4.34 describes several generally used quan-
tification 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
-------
4-107
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 fibres
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 num-
ber/field; total number/
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 y3
Point sampling
Components expressed in
percentage of organ volumes
Line sampling
Results expressed in
mm2/mm3 of organ volume
For methodology and examples see Hennig, 1958 and Benitz and Dambach, 1964.
Source: Adapted from Benitz, 1970.
-------
4-108
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 then be made
between the expected and the actual relative body weights. Differences
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 observations and
results from the minimal microscopic examinations. The Food Safety Council
(1978) also recommends this procedure, with additional emphasis on gross
examination.
TUe review by the World Health Organization (1978) is more extensive
in its discussions. It generally recommends the procedures endorsed by
the National Academy of Sciences and Food Safety Countil, but point 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 only be performed after all data from the
gross examination, organ weight determinations, and biochemical-hematological
tests have been properly interpreted. These data will help direct the
microscopic examination and reduce redundant efforts.
-------
4-109
However, in a support document for chronic toxicity testing guide-
lines, the U.S. Environmental Protection Agency (1979) follows the recommenda-
tions of the National Cancer Institute (Sontag, Page, and Saffiotti, 1976).
Their recommendation 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 riose levels be subjected
to a histopathologic examination covering 30-40 tissues plus tissues with
gross lesions. They feel the results obtained with this added pathology
is worth the increased time and costs, which can account for 40% of the
overall study cost (Page, 1977). This represents the maximum recommended
*
microscopic examination recorded in the literature.
s
4.5.3 Basis for a Mimimum Pathology Screen
The value of histopathology in subchronic tests is related to the
sensitivity of each organ to different chemicals. If certain organs con-
sistently show toxic effects in subchronic tests then 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 test-
ing 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, pes-
ticides, fungicides, industrial chemicals and solvents, food additives,
resins, and natural substances. The cumulative data for each organ are
-------
4-110
Table 4.35. Comparison of the usefulness of individual organs to indicate
toxicity when pathologically examined in a subchronic study
Organ
Adrenal
Aorta
Bone
Bone marrow
Brain
Esophagus
Eye
Gall bladder
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
organ^1
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
results'3
X
13
0
1
2
7
0
0
1
8
29
3
28
15
5
0
0
6
6
%/»l
1
3
2
1
1
1
4
12
3
12
6
5
4
0
1
Percent
used"
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
positive6
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
Total number of studies reviewed for the inclusion of an organ as an
indicator of pathologic changes.
''Total number of studies in which an organ was used to assess patho-
logic changes.
^Number of studies in which pathologic changes was detected in an organ.
frequency that an organ was used as a pathologic indicator (b/a x 100).
Frequency .that positive pathologic changes was found in an organ
(c/b x 100).
Appendices B and C contain specific information for each organ concern-
ing test species, routes of exposure, chemical? tested, and literature
references.
-------
4-111
given in this table, combining three species (dog, rat, and monkey) and
three routes (oral, inhalation, 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 iTor these data. The "stud-
ies 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 num-
s
ber of studies that were 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 £4 studies for a 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%. Using the relative criteria of a 20% use frequency and a 5% posi-
tive 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, prostrate, salivary
gland, skin, spinal cord, spleen, stomach, thytnus, thyroid, trachea, and
uterus. This minimum screen does include representative 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.
-------
4-112
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; Peck, 1968; National Academy of Sciences, 1977;
World Health Organization, 1978); although the definition 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
s
or similar as for the gross examination. Initially the importance of
microscopic examination for subchronic tests was rated low, with few tis-
sues examined (Smyth and Carpenter, 1948; Smyth, Carpenter, and Weil, 1951;
Weil and McCollister, 1963; Weil et al., 1969). Then as toxicity tests
became mere 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 believe that all organs
(30 to 40) should be sampled at high and control doses to adequately deter-
mine toxicity (Benitz, 1970; National Academy of Sciences, 1977; World
Health Organization, 1978; Food Safety Council, 1978). Yet other research-
ers believe that this is still insufficient and recommend examination of
all tissues at all levels (Sontag, Page, and Saffiotti, 1976; U.S. Environ-
mental Protection Agency, 1979). This last protocol is obviously the most
-------
4-113
sensitive in detecting toxicity, but is also the most costly. The data
in Table 4.35 and the recommendations of other researchers also indicate
N
that the examination of all tissues at two dose levels can be excessive,
since 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 micro-
scopic 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.
f
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 hyper-
trophy or metabolic overloading, they may still provide information which is
valuable in a toxicological assessment. Furthermore, many researchers feel
that it dan be more'signifcant than hlstopathology. Since the procedures
for determining organ weight (relative or absolute) are relatively easy,
the most efficient protocol would include them. Even if considered insuffi-
cient 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 micro-
scopic examination of designated tissues at the high and control levels, organ
weights for major organs, and supplemental microscopic examination of tis-
sues at other dose levels based on the gross observations and the organ
weight data.
-------
4-114
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 re-
quired, 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 Environmental Protec-
:S5 tion Agency guidelines for pesticide evaluations (Federal Register, 1978)
•* recommends the following 31 biochemical te'sts: hematocrit, hemoglobin,
'* erythrocyte count, total and differential leukocyte counts, platelet count,
•^ reticulocyte count, serum calcium, serum potassium, serum lactate dehydro-
*-•'•' genase (LDH) , serum glutamic pyruvic transaminase (SGPT) , serum glutamic
•'"•" oxaloacetic transaminase (SCOT), glucose, blo.od urea nitrogen (BUN), direct
and total bilirubin, serum alkaline phosphatase (AP), total cholesterol,
albumin, globulin, total protein, serum chlorine, uric acid, blood creati-
nine, 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 (OCT), creatinine phosphokinase (CPK), lipase,
amylase, sodium, prothrombin time, serum thyroxine, cholinesterase activi-
ties, packed cell volume, mean corpuscle hemoglobin, mean corpuscle volume,
glucose-6-phosphatase, hexobarbital oxidase, methemoglobin, and many others.
-------
4-115
All of these tests are used to inhance 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. „
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 urinalysis,
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, com-
mented on the value of biochemical, hematologic, and function tests. He
cited the Coggeshall Report of the Committee on Drug Safety (1964), attri-
buting the origin of biochemical toxicity tests as beginning with disease
/
evaluation in man. These tests, originally designed for human systems,
were then 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 improve-
ment and refining.
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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 histopath-
ology. 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 devel-
opment 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 ± two standard
deviations and a knowledge of past aberrant biochemical test values for
s
that species 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.
Be\iitz (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, then the results will be more meaning-
ful. Use of interim function and clinical tests is primarily helpful in
aiding the postmortem evaluation, since 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
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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 numer-
ical data, morphological or qualitative data can aid in assessing toxic-
f
ity. Serum levels of drugs are most useful for determining toxic dose
/
level and for comparison with levels to be used in man. By assessing
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 necessary." "When
necessary" is determined during the trial by toxic effects from the high
dose and from pretest short-term studies. To set up an inflexible sched-
ule of sampling only invites unnecessary evaluations and a waste of time
and money. In conclusion, he supported Peck's (1968) association of path-
ology evaluations and clinical-hematologic tests, stating that toxic ef-
fects 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
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oxalacetic transarainase; alkaline phosphatase; glucose-6-phosphatase;
cholinesterase; lactic acid dehydrogenase; potassium (albumin, globulin);
sodium; calcium; chloride; carbon dioxide; hematocrit; hemoglobin; total
and differential white blood cells; and erythrocytes. He also suggested
that these tests be performed at exposure days -14, —7, +3, +7, +30, +60,
and +90. This schedule allows for establishment of pretreatment baseline
values, and insures 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. They sug-
gested selected use of clinical tests based on prior test data or chemical
class. They agreed with Benitz (1970) in recommending that a rigid sched-
/
ule 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 examinations (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 empha-
sis 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, hemato-
logical, urine, and faecal evaluations. However, 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.
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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 (reticu-
locyte counts, Heinz-Ehrlich bodies, methemoglobin,'sulfhemoglobin, throm-
bocyte 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
chemicatls 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 the opinion of the authors, 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 increasingly complex prepa-
rations intended for eventual use in man." However, few definitive studies
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in support of this statement are included in the discussion. Saslaw and
Carlisle also commented on the need for preliminary baseline studies for
each specimen to be used in the toxicity tests. This would supply signif-
icant pretreatment "normal" values for 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 anaemia and alterations in leucocytes and platelets. If these
changes are observed, more testing should be performed to determine 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 anaemia, Bushby recommended a hemoglobin count, a red blood
cell count (RBC), and a determination of the packed cell volume (PCV). All
three tes,ts are interrelated and a change in any of the three would indicate
a possible anaemia. Determination of the PCV is easier and more reliable
than the RBC, so it is preferred. Additional tests that can pinpoint the
type of anaemia or site of action include mean corpuscular hemoglobin con-
centration (MCHC), mean corpuscular hemoglobin (MCH), circulating reticulo-
i
cyte numbers, bilirubin, and methemoglobin. To detect changes due to or-
ganic disease, Bushby recommended use of the sedimentation rate of RBC,
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
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Table 4.36. Haematological studies during
toxicity tests
Primary tests and estimations
Haemoglobin 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 MCV, MCH, and MCHC
Reticulocyte count
Detection of Heinz bodies
Examination for methaemoglobin
Haemoglobinaemia ^
Bilirub inaemia
Bone marrow
Coagulation time
Bleeding time
Clot retraction
Prothrombin time
Partial thromboplastin (cephalin) test
Eragility test
Follow-up investigations
Schumm's test for methaemoglobin
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 Bi2 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.
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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,
then the prothrombin and partial thromboplastin trimes should be determined.
Bushby concluded that in most prolonged studies, a few hematological param-
eter evaluations will detect general blood damage, but if site of action
information is desired, more extensive and different tests are necessary.
Arnold et al. (1977) commented on hematological procedures in their
s
discussion of animal health monitoring in chronic studies. Generally, they
s
recommended testing on a preset schedule or in response to changes in ani-
mal 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 anaemia; preliminary conditions of anaemia (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. They stressed the need for interrelated and over-
lapping 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 leuco-
cyte counts. To screen for hemorrhagic effects, they recommended the use
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of prothrombin time (one stage) and partial thromboplastin time (activated).
If additional information is needed, the platelets should be counted and
assessed for morphological changes, and bleeding time should be determined.
By following these procedures, hematology can be a valuable tool in deter-
mining 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 chem-
ical toxicity. They recommended sampling at 30-day intervals with subgroups
of rodent and nonrodent test species. They stated that to adequately assess
hematological parameters both quantification and morphology of the blood
cells are needed. They recommended a standard protocol which included the
determination of erythrocyte numbers, reticulocyte numbers, total and dif-
s
ferential leucocyte numbers, hematocrit, and hemoglobin. The erythrocytes
must also be examined for morphological changes. To evaluate hemorrhagic
effects, a screen should consist of platlet counts, clot retraction, pro-
thrombin time (one stage), and partial thromboplastin time (activiated).
For a more detailed evaluation of hemorrhagic effects, include factor
assays, thrombin time, fibrinogin determination, platelet aggregation,
prothrombin consumption time, and euglobulin clot lysis time.
4.6.4 Biochemical and Organ Function
f
Wr6blewski and LaDue (1955) evaluated serum glutamic oxaloacetic
transaminase (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 were to discover:
if the degree of SCOT activity was related to liver cell destruction;
whether SCOT activity in chronic liver disorders indicated active liver
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cell destruction; what correlation existed between SCOT activity and
various tests of liver dysfunction; and if changes in SCOT reflected the
presence of liver cancers. After evaluating their data, they reached
the following conclusions: there is a rough relationship 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 activ-
ity 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) also
reviewed the use of clinical chemistry and organ function tests. The
/
tests he listed as commonly utilized included: serum glutamic oxalo-
acetic transaminase (SCOT); serum glutamic pyruvic transaminase (SGPT);
serum lactate dehydrogenase (LDH); blood urea nitrogen; blood glucose;
serum cholesterol; alkaline phosphatase; thymol turbidity; serum bili-
rubin; bromosulf al'ein excretion (BSP) ; nonprotein nitrogen (NPN) ; and
phenolsulfonphthalein (PSP). However, he had reservations about their
usefulness stating, "Despite the widespread use of these tests, there
is still not enough information about their significance 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 use should obviously be as
a confirming or indicator test. Zbinden also stressed the usefulness of
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drug blood level determinations. These allow extrapolation of "safe"
dose levels from test species to man with more reliability.
Hoe and O'Shea (1965) tested the predictive efficiency of biochem-
istry tests for detection of kidney disease in the dog. They used known
kidney lesion-producing chemicals to assess the degree of correlation
between biochemical tests and histopathology. The biochemical tests
evaluated included: blood urea; blood creatininer; inorganic phosphate;
serum protein; calcium; serum alkaline phosphatase; sodium; potassium;
chlorine*; cholesterol; serum glutamic pyruvic transaminase (SGPT); and
serum glutamic oxaloacetic transaminase (SCOT). Each was tested in dogs
after exposure to the chemicals. Upon examining the results, the tests
^
for calcium, chlorine, and serum proteins did not correlate with kidney
/
damage and were of no predictive value. The evaluations of alkaline phos-
phatase, SCOT, and SGPT were inconclusive regarding kidney damage due to
excessive interference resulting from liver damage. However, the other
tests did correlate with kidney damage. Blood urea increased in a par-
allel 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 urea changes. Inorganic phosphate also 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 also using urinalysis, a forecast of 90% accuracy
can be achieved for kidney damage. They also suggested that general pro-
tocols cannot be developed and that every case or chemical should be
assessed on an individual basis.
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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 indica-
tive of only 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 the test
groups; the presence or absence of a dose responses-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 jeopardiz-
ing the main study by oversampling.
Street (1970) reviewed biochemical tests in toxicology and stated
that their primary functions are: early indicators of toxicity; identi-
fication 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. To properly evaluate the results, initial
values and values from concurrent control groups are needed for compari-
son. The initial values should be taken, twice during the acclimatization
period. Normal values published in the literature are of limited value
due to high variability. Every toxicolojgist 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, SGPT, and alkaline phosphatase evaluations.
Additional -tests that can add information on other effects besides liver
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damage include bilirubin and protein electrophoresis. Street also recom-
mended the use of isocitric dehydrogenase (ICID) in place of the dye tests,
unless the dye tests can be designed to give full elimination curves. For
the assessment of kidney damage, he recommended the blood urea test cor-
related 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 addi-
tional 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 does not
f
limit his discussion to tests that indicate liver and kidney damage, but
/"
also reviews tests that may indicate heart or lung damage. In general,
he refrains from recommending specific tests for each organ, prefering
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 sam-
pling during the periods when the changes in serum enzyme levels would
be most probable. If sampling is done at inappropriate times, then many
of the changes may be missed, since the toxicant stimulates a short-term
elevation of serum levels. Despite its/often transient nature, this im-
mediate response can be a sensitive indicator of functional and/or morpho-
logical 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
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determine the onset of organ damage. Also the toxicologist should con-
sider 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 discusses 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 indi-
vidual organs. Since 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 addition to pin-
pointing damage to specific organs, isoenzyme patterns can often be more
sensitive indicators of overall tissue damage than otner enzyme tests.
s
However, as with serum enzyme tests, the schedule of sampling for iso-
enzymes is very important, because the pattern can change with time and
alter the resulting diagnosis.
Grice et al. (1971) compared the sensitivity of changes in serum
glutamic «oxaloacetic transaminse (SCOT), lactate dehydrogenase (LDH),
and LDH isozyme patterns with histopathology to determine their useful-
ness in detecting liver and kidney damage. After exposure of four toxic
chemicals in rats, the test results showed that morphological damage occurs
at lower doses and before any change in the serum enzyme levels. SCOT
,•
was somewhat more sensitive than LDH, but lacked the specificity of the
isozyme pattern. They concluded that the serum enzyme levels and isozyme
patterns could contribute supplemental information but they are not sub-
stitutes' for pathology evaluations.
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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-1-P-ALD), sorbitol
dehydrogenase (SDH), glutamic-pyruvic transaminase (GPT), glutamic-
oxaloacetic transminase (GOT), malic dehydrogenase (MDH), lactic dehydro-
genase (LDH), glutamic dehydrogenase (GDH), fruct«se-l,6-diphosphate
aldolase (F-l,6-diPALD), 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. ICD,
GPT, GOT, and F-l-P ALD were the next most reliable indicators. Korsrud
^
et al. identified several factors that could account for the differences
s
in sensitivity, including: 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. Also the responses
were dependent on the time of sampling. They'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 serum glutamic
pyruvic transaminase (SGPT). They felt that SGPT changes did not indi-
cate actual liver damage as much 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
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Table 4.37. Relative sensitivity of liver
enzyme tests
Testa
ICD
F-l-P ALD
SDH
GPT
GOT
MDH
LDH
GDH
OCT
F-l, 6-diP ALD
His topathology
IA6
25. ke
25.4
9.4
25.4
25.4
68.6
68.6
NC/
68.6
NC
3.5
DMNC
13.7
13.7
5.1
13.7
13.7
37.0
37.0
NC
37.0
NC
1.9
DEAd
800
800
800
800
* 800
800
1600
1600
1600
1600
400
ICD — isocitrate dehydrogenase; F-l-
P ALD — fructose-1-P-aldolase; SDH — sorbi-
tol dehydrogenase; GPT — glutamic-pyruvic
transarainase; GOT — glutamic-oxaloacetic
transaminase; MDH — malic dehydrogenase;
LDH — lactic dehydrogenase; GDH — glutamic
dehydrogenase; OCT — ornithine carbamyl
transferase; F-l,6-diP ALD — fructose-1,6-
diphosphate aldolase.
^TA — thioacetamide.
Q
,DMN — dimethyInitrosamine.
T)EA — diethanolamine.
&
Lowest dose level at which signifi-
cant change occurred in mg/kg.
JNo change.
Source: Adapted from Korsrud et al.,
1973.
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4-131
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
trauma associated with it can significantly affect the test animal's
health. Therefore, serum enzyme tests should only be performed 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 do suggest the use of large test spe-
cies, such as the dog, to reduce sample removal effects. They recommended
a set sampling schedule with serum withdrawal prior to the test, 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 ini-
tial 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 histo-
pathological 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 indi-
cators of damage. These include alkaline phosphatase, alanine transami-
nase, and aspartate transaminase. Clampitt did recommend 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
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carefully monitored if transient effects are desired. In conclusion,
Clampitt suggested that minimal changes can be detected if the "appropri-
ate 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 func-
tion 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 gluteamic oxaloacetic transaminase test (UGOT),
and urinary concentration test. Blood urea nitrogen was erratic and less
reliable than the UGOT test due to extrarenal interference. Also, the
standard^ concentration tests were inappropriate and the authors suggested
using a 6-hr 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.
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 urinal-
ysis and function tests was necessary for a complete assessment of renal
damage. By doing this, 90% of the cases could be diagnosed by biochemical
tests alone.
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Urinalysis and its diagnostic value were discussed by Street (1970).
The use of the simple tablet or dipstick analyses techniques in any capac-
ity 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-hr 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 suf-
ficient to detect most renal damage. Urinary ascorbic acid can be used
s
to test for liver enlargement, but it requires a series of comparative
assays. Overall, Street recommended the inclusion of urinalysis 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 com-
/•
mande" 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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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 an 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 phosphataase, lactic dehydro-
genase, and leucine aminopeptidase after acute and repeated-dose exposure
to nephrotoxicants. They used both histology and histochemistry to evalu-
ate the renal damage. They found that for acute exposure, all the enzymes
but creatinine wer-e affected. Lactic 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. There-
fore, they concluded that currently the value of urinary excretion rates
/'
is confined to acute and very short-term exposures. 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 his recommendation of the
hematology tests, blood urea nitrogen and plasma creatinine, Berndt also
recommended a urinalysis determination. Specifically he stressed the
-------
4-135
use of changes in urinary glucose levels as a good indicator of renal
damage. Also, the excretion of p-aminohipparate (PAH) and tetraethyl-
ammonia (TEA) can be detected in the urine to indicate damage. These
tests are particularly good for evaluating the toxic effect on the neph-
rons. Other recommended tests include the volume and concentration de-
terminations. Protein determinations will indicate the effects on the
glomerulus. Berndt concluded that in comparison with urinalysis, histo-
logical and anatomical techniques are the "least useful criterion for
evaluation of renal damage."
Arnold et al. (1977) also discussed the use of urinalysis in tox-
icity diagnosis. They suggested collection of urine over night or at
f
24-hr interims for determinations of volume and the excretion patterns
s
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 complex
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
blood flow (RBF), total urinary protein excretion, urinary concentration,
urinary acidification, urinary enzyme excretion rates, urinary sodium
excretion fate, and microscopic examination of sediments as indicators of
-------
4-136
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, espe-
cially for rodents; total protein and urinary concentration can be useful
screens but do not give site specific information; urinary enzyme excre-
tion has been shown useful but due to a lack of oorrelation with increases
in renal damage, their 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 uncer-
tain and their routine use is not suggested. Also the difficulty of
sample collection, and interpretation of normal variations in values
s
makes the general use of these tests less practical. However, these
tests can be used for more detailed information when prior studies
indicate renal damage or when the structure of the chemical to be
investigated suggests possible renal damage.
The U.S. Environmental Protection Agency" in their support document for
chronic tests (1979), stressed the value of urinalysis. 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 is evaluating chronic test applications, the
principles should also apply to subchronic 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
-------
4-137
of toxic effects and suggest specific target organs, or contribute con-
firmatory evidence on the toxic effect. The wide variety of tests avail-
able has presented the toxicologist with the problem of test selection.
Most of the literature reviewed did not suggest a standard list of required
biochemical tests, but rather suggested the inclusion of hematology, en-
zyme, and urinalysis evaluations. This is perhaps a good policy since
the effectiveness and applicability of the tests ckange with the toxicol-
ogist (particularly his experiene 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
f
and hemorrhagic effects. To properly evaluate these parameters, several
s
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 a representative example 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 accept-
able due to variations between individual test animals and between labora-
tories. If normal values are to be used, they 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.
-------
4-138
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, due
to 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 two weeks over a 23 week testing per-
iod. 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, con-
sideration 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
f
by several reviews, these results should be interpreted carefully 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
-------
4-139
particularly limited for subchronic studies. However, other reviews sug-
gested 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 urinalysis tests
and a few enzyme determinations. If used the urinalysis examination should
check physical (e.g., volume and specific gravity), enzymatic (e.g., lactic
dehydrogenase), and chemical (e.g., glucose and phosphate) parameters. How-
ever, due to problems with sample collection and interpretation of test
results, the routine use of urinalysis is questionable, and should be
left to the investigator.
Thus, the value of hematology, enzyme function, and urinalysis tech-
niques is apparently sufficient to recommend that they be included in
s
standard subchronic toxicity protocols. TKe 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 sug-
gest target organs and types of effects that could be checked by pathology.
The effectiveness would thus increase for both procedures.
-------
4-140
SECTION 4
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*
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*•
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Page, N. P. 1979. Review of Current Activities in Test Standards Devel-
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f
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APPENDIX A
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SUBCHRONIC TOXICITY TESTS
AS DOSE LEVEL SETTING STUDIES
FOR CHRONIC CARCINOGENICITY EVAUATIONS
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 estimate safety
and a longer chronic study is required for evaluation, the subchronic tox-
f
icity 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 teratogenic-
ity, reproduction, longevity or carcinogenesis evaluations; however, it is
» •
in the area of carcinogenicity that they have most recently been applied.
Therefore, the discussion which follows centers on the use of subchronic
toxicity tests to establish a maximum tolerated dose (MTD) for each sex
and species used in a chronic carcinogenicity study.
TEST MATERIAL
It is essential that the chemical being assayed by 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
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and chronic studies. In addition, it is absolutely necessary to deter-
mine the accuracy and consistency with which the chemical/vehicle prepa-
rations 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 carcino-
genesis bioassay studies as set forth by both tfre Environmental Protection
Agency (Federal Register, 1978) and the National Cancer Institutes (Sontag,
Page, and Saffiotti, 1976) do require or recommend each of the above.
TEST ANIMALS
f
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 which follows. For chronic toxicity tests,
two species have usually been recommended — preferably a rodent and a
» • •
non-rodent (Page, 1977), with the rat and dog historically the most popu-
lar. Carcinogenicity studies, however, present a uniquely different prob-
lem. 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 particularly species to be used in a carcinogenesis study
is only the beginning. Other questions, such as the use of inbred or
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random bred strains and the use of germ-free, specific pathogen-free or
conventional animals, must be answered. Although a large number of dif-
ferent rat and mouse strains have been utilized in the past (Pietra,
Rappaport, and Shubik, 1961; Dunning, Curtis, and Madsen, 1947; Boyland,
and Sydnor, 1962; Shimkin et al., 1962; National Institutes of Health,
1977), the Fischer-344 rat and B6C3F1 hybrid mouse have become the stand-
ard for the NCI Carcinogenesis Bioassay Program«
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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 num-
ber 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, incor-
rect 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, 1977). This number has proved sufficient as long as
the parameters for evaluation were limited to mortality, body weight gain,
clinical signs, post-mortem observation and pathologic diagnosis; however,
the recent expansion of these parameters to include hematology, clinical
chemistry, and pharmacokinetic studies will, of necessity, produce an in-
crease 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 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,
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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 Max-
imum 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, repro-
ducible toxic effect and a series of lower doses which provide a complete
spectra of dose response (Weisburger and Weisburger, 1967) from toxic to
"non-toxic." 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 which could be of value later in the chronic
s
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, ett.) (Sontag, Page, and Saffibtti, 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-logio doses are,used; either of these
selections provides a reasonable span,of doses from which to gather tox-
icity data.
ROUTE
Administration of the chemical in the subchronic study should closely
simulate the route by which most human exp.osure occurs and should be iden-
tical to that which will be used for the chronic study. With the exception
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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 carcino-
genicity. 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 3ose 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 which would be administered orally
/•
either by way of gastric intubation, the drinking water, or the feed.
s
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 (Smith,
1950; Peck, 1968; 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 and1 teratogenicity (McNamara, 1976).
Since the function of a "dose level setting" subhcronic test is to estab-
lish appropriate dose levels for a chronic study through the identification
of subchronic toxic effects and their extrapolation to chronic toxic ef-
fects, a 90-day test should be just as appropriate for a "dose level set-
ting" subchronic study as it is for an "end result" study. Of course, if
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prior information suggests that the chemical and/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 fov Carcinogen Bioassay 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 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. Withouth 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 longrterra toxic effects are 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,
seven 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.
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In addition to the above observations, each animal should be weighed
weekly (Sontag, Page, and Saffiotti, 1976). Except in very unusual in-
stances, palpation for subcutaneous or intraperitoneal masses would not
be required.
Food and water consumption should be measured weekly and for water
daily if possible. This data is 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 excel-
lent 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.
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 Bioassay 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 ex-
amination 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
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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 (Prejean,
1979; National Institutes of Health, 1977; National Institutes of Health,
1978).
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.
CONCLUSIONS
In reviewing the general paramters 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 non-rodent; whereas the "dose level
-------
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setting" test has used two species of rodents due to the economic consid-
erations of the long-term study that will follow. The only other major
area of disagreement has been in the use of biochemical tests. "End re-
sult" 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 cfeanges, it appears that,
except for the species difference which will probably always remain, "end
result" and "dose level setting" subchronic toxicity tests will be virtu-
ally identical in the next few years.
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APPENDIX A
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Publication No. (NIH) 76-801. National Cancer Institute, Bethesda,
Maryland. 65 pp.
Weil, C., and D. McCollister. 1963. Relationship Between Short- and
Long-Term Feeding Studies in Designing an Effective Toxicity Test. J.
Agric. Food Chem. 11(1):486-491.
s
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.
-------
4-164
APPENDIX B
-------
4-165
Appendix B. Chemicals surveyed for Table 4.35
Name0
Carbon tctrachloride
3' ,4'-Dlchloropropion anilide
Tetrnhydrothiophene-l,l-dioxide;
Sulfolane
Calcium carbimlde
1,2,4-Trichlorobenzene
Acroleln; acrylaldehyde, 2-propenal
Ethyleneblslsothiocyanate sulfide
Trlphenyl tin hydroxide; fentin
hydroxide
Isooctyl Isodecyl nylonate
Ponceau 4R; trisodium salt of l-(4-
sulpho-l-naphthylazo)-2-naphthol-
6,8-disulphonic acid
Orange G; dlsodlum salt of 1-phenylazo-
2-naphthol-6,8-disulphonic acid
Hexachlorobenzene; HCB
Clindaaycin hydrochloride; Cleocin
Trlechyl phosphate
Ponceau MX; disodium salt of l-(2,4-
Primary
Industrial chemical
Herbicide
Solvent
Antialcholic 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
Reference
1
2
3
4
5
7, 37
a
9
10
11
12
13, 24, 26,
30, 33
14
15
16
xylylazo)-2-naphthol-3,6-disulphonic
acid
DlbucyKdiethylene glycol bisphtha-
lace); DDCB
2,3,7,8-Tetrachlorodibenzo-p-dioxin;
TCDD
Quanethidine
l-Mechyl-3-keco-4-phenylquinuclidinium
bromide; MA 540
TR2379; trans isomer of N-(l,3,4,6,7-
licxahydro-llbH-benzo[a]quinolizin-
2-yl)propionanilide hydrochloride
N-Methyl-S-(l-naphchyl)fluoroaceta-
r.tde; Nissol; UNFA
2-Methyl-4-chlorophenoxy acetic acid;
::CPA
2,5,4'-Trichlorobiphenyl
Fenterol-HBr; l-(3,5-dihydroxy-phenyl)-
2-{[l-(4-hydroxybenzyl)-ethyl]-amino}
echanol hydrobromide
Plasticizer 17
Impurity in pesticides IS, 31, 43
Antihypertensive drug 19
Antihypertensive drug 19
Antihypertensive drug 20
Pesticide 21
Herbicide : 22, 42
Transformer dielectric 25
fluid /
Beta-sympathomiaetic 27
drug
-------
4-166
Appendix B (continued)
Name
Primary
Reference
Fominoben-HCl; 3'-chloro-2'-[N-methyl-
[(morpholino-carbonyl)methyl]-amino-
methyl]benzanilide-hydrochloride
Mycocoxin produced by Aspergillue
fumigatua 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
Dimechrin; 2,4-dimethylbenzyl ester of
chrysanthemumic acid
Hexachlorophene; 2,2'-methylenebis
(3,4,6-trichlorophenol)
Pyridoxine hydrochlorlde
A9-Tetrahydrocannabinol; 49-THC
2-Methyl-4-chlorophenoxy propionic
acid; MCPP
Antitussive drug 28
Animal feed impurity 29
Photochemical product 32
in smog
Epoxy resin 34
^
Fungicide 35
Fungicide 35
Animal feed impurity 36
Insecticide 38
Insecticide 38
Antibacterial and anti- 39
fungicidal agent
f
Antischizophrenic drug 40
/Natural Ingredient in 41
marihauna plant
Herbicide 42
^First name listed is the one used in Appendix C.
Primary use as listed by the reference source.
-------
4-167
APPENDIX B
REFERENCES
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D. D. Irish. 1952. Vapor Toxicity of Carbon Tetrachloride Deter-
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7':128-162.
/
5. Coate, W. B., W. H. Schoenfisch, T/ R. Lewis, and W. M. Busey. 1977.
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9. Gaines, T. B., and R. D. Kimbrough. 1968. Toxicity of Fentin
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10. 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.
11. 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.
-------
4-168
12. 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.
13. Gralla, E. J., R. W. Fleischman, Y. K. Luthra, M. Hagopian, J. R.
Baker, H. Esber, and W. Marcus. 1977. Toxic Effects of Hexachlo-
robenzene after Daily Administration to Beagle Dogs for One Year.
Toxicol. Appl. Pharmacol. 40:227-239.
14. 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.
^
15. 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.
16. 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.
17. Hall, D. E., P. Austin, and F. A. Fairweatherv 1966. Acute
(Mouse and Rat) and Short-Term (Rat) Toxicity Studies on Dibutyl
(Diethylene Glycol Bisphthalate). JPtiod Cosmet. Toxicol. 4:383-388.
18. 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.
19. 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.
20. 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.
21. Hashimoto, Y., T. Makita, H. Miyata, T. Noguchi, and G. Ohta. 1968.
Acute and Subchronic Toxicity of/a New Fluorine Pesticide. N-Methyl-
N-(l-Naphthyl)Fluoroacetamide. Toxicol. Appl. Pharmacol. 12:536-547.
22. 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. Contam. Toxicol. 18:152-158.
23. 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.
-------
4-169
24. 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.
25. 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.
26. 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.
27. 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 Rab-
bits. Oyo Yakuri. Sendai 10(1):45-71.
28. 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 (F,ominoben) in Rats
and Mice. Oyo Yakuri. Sendai 10(l):31-43.
s
29. Khor, G. L., J. C. Alexander, J. H. Lumsden, and G. J. Losos. 1976.
Safety Evaluation of ^sperg'iiZws /Wffrigatus Grown on Cassava for Use
as an Animal Feed. Can. J. Comp. Med. 41:428-434.
30. 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.
31. 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.
32. Kruysse, A., V. J. Feron, H. R. Immel, B. J. Spit, and G. J. Van
Esch. 1977. Short-Term Inhalation Toxicity Studies with Peroxy-
acetyl Nitrate in Rats. Toxicology 8:231-249.
33. 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.
34. Lawrence, W. H. , M. Malik, J. E". Turner, and J. Autian. 1972.
Toxicity Profile of Epichlorohydrin. J. Pharmacol. Sci. 61(11):
1712-1717.
35. 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.
-------
4-170
36. 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.
37. 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.
38. 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.
39. 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.
40. 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.
41. 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.
42. 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.
43. 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.
-------
4-171
APPENDIX C
-------
4-172
Appendix C. Species, route of exposure, chemical,
Chemical
Carbon tecrachloride
3, ' ,4'-Dlchloroproptonanllide
Calcium carblmlde
Acrolein
Ethyleneblsisothlocyanate sulfide
Triphenyl tin hydroxide
Ponceau 4R
Isooctyl Isodecyl nylonate
Orange G
Hexachlorobenzene
Cllndamycln hydrochloride
Trlethyl phosphate
2,3,7, 8-Te C rachlorodibenzo-p-dioxin
TR2379
Quanethidlne
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
N-Methyl-N-(l-naphthyl)fluoracetamide
2-Methyl-i-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fonterol-HBr »
Fominoben-HCl
Asperglllus fuolgatus
Peroxyacetyl nitrate
Eplchlorohydrln
Ferric dlncchyl dithiocarbamate
Tctrnnechylthiuraa disulfide
Ponceau MX
Dlbutyl(dlechylene glycol bisphthalate)
AHR-2438B
Acrolein
Barthrln
Dlaethrln
Pyrtdoxine hydrochloride
i'-Tetrahydrocannablnol
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
and reference
Route
Inhalation
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral s
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
• Oral
Oral
Oral
Inhalation
Injection
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Injection
Oral
data for
Result
-
•
+
+
-
-
-
+
*
-
-
-
+
+
-
-
+
-
-
+
+
-
-
-
-
-
+
-
-
+
-
-
-
-
-
--'
-
-
-
-
-
-
-
-
+
Table 4.35
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
-------
4-173
Appendix C (continued)
Chemical
Tetrahydrothiophene-1 , 1-dioxide
Acrolein
Ethylenebisisochiocyanate sulfide
Isooctyl isodecyl nylonate
Ponceau 4R
Clindamycin hydrochloride
TR2379
Fominoben-HCl
Fenterol-HBr
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
49-Tetrahydrocannabinol
Calcium carbimide
Ethylenebisisothiocyanate sulfide
TR2379
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
Hexachlorobenzene
2,5,4' -Trichlorobipheny 1
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
Carbon tetrachloride
3' ,4'-Dichloropropionanilide
Calcium carbimide
1,2, 4-Trichlorobenzene
Hexachlorobenzene
Clindamycin hydrochloride
TR2379
N-Methyl-N-(l-naphthyl)fluoracet amide
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigacus
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
DOR
Rat
Rat
Rat
Rat
Route
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
Or.al
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Result
-
-
-
-
-
_
_
,-
-
-
-
-
-
+
-
_
-
-
-
-
_
-
-
-
+
-
-
_
_
-
-
-
-
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
-------
- 4-174
Appendix C (continued)
Chemical
Ferric dimethyl dithiocarbamate
Tetramethylthiurara disulfide
Ponceau MX
Dibutyl(diethylene glycol bisphthalate)
Pyridoxine hydrochlorlde
49-Tetrahydrocannabinol
2,5,4' -Tr ichlorob ipheny 1
Carbon tetrachloride
3" ,4'-Dichloropropionanilide
Tetrahydrothiophene-l,l-dioxide
Calcium carbimide
1 , 2 , 4-Trichlorobenzene
Acrolein
Ethylenebisisothiocyanate sulfide
Triphenyl tin hydroxide
Ponceau 4R
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindaraycin hydrochloride
%
Triethyl phosphate
TR2379
•
N-Methyl-N-(l-naphthyl)fluoracetamide
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
Epichlorohydrln
Ferric dimethyl dithiocarbamate
Tetramethylthiurara 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
Oral
Oral
Oral
Oral
Oral
Oral
Injection
Oral
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Inhalation
Inhalation
Oral
Oral
Oral S
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Injection
Oral
Oral
Result
-
-
-
-
-
+
-
-
-
*
-
-
-
-
-
-
-
-
-
i
-
-
-
-
-
+
+
-
-
-
-
-
-
-
-
+
-
--'
-
-
-
+
-
-
-
-
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
-------
4-175
Appendix C (continued)
Chemical
Ponceau MX
DibutyKJlethylene glycol bisphthalate)
AHR-2438B
Hexachlorophene
Pyridoxlne hydrochloride
i'-Tetrahydrocannablnol
2,5,4'-Trichloroblphenyl
Tetrahydrothlophene-l.l-dioxlde
F.thylenebislsothiocyanate sulfide
Cllndamycin hydrochloride
TR2379
Hexachlorobenzene
2.5,4'-Trlchloroblphenyl
Fenterol-HBr
Fomlnoben-HCl
2,3, 7 ,8-Tetrachlorodibenzo-p-dioxin
AHR-2438B
Pyridoxlne hydrochloride
i^-Tecrahydrocannabinol
1,2,4-Trlchlorobenzene
Ethyleneblslsocnlocyanate sulfide
Hexachlorobenzene
2,5,4 '-Trlchlorophenyl
2,3,7, 8-Tct rachlorodlbenzo-p-dioxin
u9-Tecrahydrocannabinol
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
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
Injection
Inhalation
Inhalation
' Oral
Oral
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
Gall Bladder
Tctrahydrothlophene-1 , 1-dioxide
Hexachlorobenzene
Cllndamycin hydrochloride
Trlcthyl phosphate
1^-Tccraliydracannabinol
Monkey
Dog
Rat
Dog
Dog
Rat
Rat
Monkey
Monkey
Inhalation
Inhalation
Inhalation
Oral
Oral +
Oral
Oral
Oral
Injection
3
3
3
12
13
13
14
39
39
-------
- — 4-176
Appendix C (continued)
Chemical
Carbon tetrachloride
3' ,4'-Dlchloroproplonanillde
Tetrahydrothlophene-l,l-dloxide
Calcium carbimlde
1,2,4-Trlchlorobenzene
Acrolein
Ethylenebislsothlocyanate sulflde
Triphenyl tin hydroxide
Ponceau 4R
Isooctyl Isodecyl nitrate
Orange C
Hexachlorobenzene
Clindamycln hydrochloride
Trlethyl phosphate
2,3,7, 8-Tetrachlorodibenzo-p-dioxln
TR2379
Quanethidlne
l-Methyl-3-keto-4-phenylquinuclidinium
bronide »
N-Methyl-N'-(l-naphthyl)fluoracet amide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl— »-chlorophenoxy propionic acid
Fenterol-HBr
Foninoben-HCl
Aspergillus fualgatus
Peroxyacetyl nitrate
Eplchlorohydrin
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
Ponceau MX
DlbutyKdlethylene glycol bisphthalate)
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
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Inhalation
Inhalation
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral , S
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
, Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Injection
Oral
Oral
Oral
Oral
Oral
Oral
Result
-
+
-
-
+
-
-
-
+
- •
-
^
-
-
-
-
+
-
-
-
+
- ,
-
-
-
-
-
+
+
-
-
-
-
-
-
-
-
+
-„•
-
-
-
-
-
-
-
-
-
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
-------
4-177
Appendix C (continued)
Chemical
Dlmethrln
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2,5,4' -Trichlorobiphenyl
Carbon tetrachloride
3 ' , 4 ' -Dichloropropionanilide
Tetrahydrothiophene-l,l-dioxide
Calcium carbimide
1 , 2 ,4-Trichlorobenzene
Acrolein
Ethylenebisisothlocyanate sulfide
Triphenyl tin hydroxide
Ponceau 4R
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Trlethyl phosphate
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
»
TR2379
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
N-Methyl-N-(l-naphthyl)fluoractemide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Fominoben-HCl
Aspergillus furaigatus
Peroxyacetyl nitrate
Epichlorohydrin
Ferric dimethyl dithiocarbamate
s
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
-------
4-178
Appendix C (continued)
Chemical
Tetraraethylthiuram disulfide
Ponceau MX
Dlbutyl(diethylene glycol bisphthalate)
AHR-2438B
Barthrin
Dimethrin
Pyridoxine hydrochloride
i9-Tetrahydrocannabinol
2,5.4' -Tr ichlorobiphenyl
Large
Carbon tetrachloride
3 ' ,4 ' -Dichloropropionanilide
Tetrahydrothiophene-l,l-dioxide
Acrolein
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
TR2379
Quanethidine
l-Methyl-3-keto*4-phenylquinuclidinium
bromide
N-Methyl-N-(l-naphthyl)fluoracetamide
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7,8-Tetrachlorodlbenzo-p-dioxin
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Barthrin
Dimethrin
Pyridoxine hydrochloride
A5-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
Inj ection
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
-------
4-179
Appendix C (continued)
Chemical
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
2.5,4'-Trichloroblphenyl
Carbon tctrachlorlde
3* ,4'-Dichloroproplonanllide
Tetrahydrothlophene-l,l-dloxide
Calcium carblralde
1 , 2 , 4-Tr ichio robenzene
Ac role In
Ethylcneblslsothlocyanate sulfide
Trlphenyl tin hydroxide
Isooctyl isodecyl nylonate
Ponceau 4R
Orange C
Hexachlo robenzene
Clindamycln hydrochloride
Trlethyl phosphate
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
TR2379
Quanethldlne
l-y.ethyl-3-keto-4-phenylquinuclidinium
bromide
N-Methyl-S-(l-naphthyl)fluoracetamide
2-Mechyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Fonlnoben-HCl
Asperglllus fumigatus
Pcroxyacetyl nitrate
Eplchlorohydrln
Ferric dimethyl dithiocarbamate
Tetranethylchluram disulfide
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 s
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
-------
- 4-180
Appendix C (continued)
Chemical
Ponceau MX
Dibutyl(diethylene glycol blsphthalate)
AHR-2438B
Barthrin
Dlmethrin
Hexachlorophene
Pyrldoxine hydrochloride
A9-Tetrahydrocannabinol
2,5,4' -Tr ichlorobipheny 1
Carbon tetrachlorlde
3' ,4'-Dichloropropionanilide
Tetrahydrothiophene-1 , 1-dioxide
Calcium carbimide
1,2, 4-Trichlorobenzene
Acrolein
Ethylenebisisothiocyanate sulfide
Triphenyl tin hydroxide
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
t
Clindamycin hydrochloride
Triethyl phosphate
2,3,7, 8-Tet rachlorodibenzo-p-dioxin
TR2379
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
N-Methyl-N-(l-naphthyl)fluoracetamide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Forainoben-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 ' r
Oral
Oral
Oral
Oral
Oral
"Oral
Oral
Oral
Oral
Oral
Reference
15
16
34
36
36
37
38
39
39
23
1
2
3
3
3
4
5
5
6
35
35
36
7
8
9
10
12
24
22
28
31
13
13
14
17
29
19
19
18
18
18
18
20
21
40
40
25
26
-------
4-181
Appendix C (continued)
Chemical
Asperglllu* funlgatus
Peroxyacetyl nltrace
Eplchlorohydrln
Ferric dimethyl dlthlocarbamate
Tetramethylthiurara dlsulfide
A11R-2438B
Barthrln
Olmethrln
Pyrldoxlnc hydrochlorlde
i9-Tecrahydrocannabinol
: . 5 ,4 ' -Trtchloroblphenyl
Carbon tetrachlorlde
Te t rahydroth lophene-1 , 1 -dioxide
Calcium carbinlde
Acroleln
Ethyleneblsisothlocyanate sulfide
Isooctyl Isodecyl nylonate
Orange C
Hexachlorobenzene
Cllndaoycln hydrochlorlde
Trlethyl phosphate
TK2379
Fenterol-HBr »
Foninoben-HCl
Asperglllus fumigatus
2,3, 7 ,8-Tetrachlorodibenzo-p-dioxin
Peroxyacecyl nitrate
Ferric dimethyl dithiocarbamate
Tetranethylthiuran disulfide
AHR-2438B
Pyrldoxine hydrochloride
;9-Tetrahydrocannablnol
2-Methyl-4-chlorophenoxy acetic acid
Species
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Lymph Node
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
2-Mechyl-4-chlorophenoxy propionic acid Rat
:,5.4'-Trlchlorobiphenyl
Monkey
Route
Oral
Inhalation
Injection
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Injection
Oral
Inhalation
Inhalation
Inhalation
Inhalation
Oral
Inhalation
Oral
Oral
Oral .S
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
. Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral
Oral
Injection
Oral
Oral
Oral
Result Reference
27
•f 30
+ 32
33
33
34
36
36
38
39
-t- 39
•«>
+ 23
1
3
3
3
+ 4
6
7
r
9
11
+ 12
22
24
13
13
14
19
- . 19
25
26
27
+ 29
30
+ 33
33
+ 34
38
39
39
40
40
23
-------
4-182
Appendix C (continued)
Cheaical
Species
Route
Result
Reference
Mammary Gland
Ethylenebisisothlocyanate sulfide
Orange C
Hexachlorobenzene
2.5,4'-Trichlorobiphenyl
i 9-Tet rahydrocannabinol
Rat
Rat
Dog
Monkey
Monkey
Monkey
Monkey
Monkey
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inj ection
-
-
_
-
-
-
-
-
7
11
12
22
24
23
39
39
Muscle Tissue
Carbon tetrachloride
3' ,4'-Dichloropropionanilide
Acrolein
Triphenyl tin hydroxide
Orange G
Clindaaycin hydrochloride
TS2379
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-H3r
Foainoben-HCl
Asperglllus fumigatus
2,3,7,8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
Ferric dinethyl dithiocarbamate
Tetranethylthiuran disulfide
Pyridoxine hydrochloride
i?-Tetrahydrocannabinol
Nerve
Carbon tetrachloride
Quanechidine
l-Methyl-3-keto-4-phenylquinuclidinium
broaide
Hexachlorobenzene
Aspergillus fumigatus
Pyridoxine hydrochloride
^'-Tetrahydrocannabinol
2-Methyl-4-chlorophenoxy acetic acid
2-Mothyl-H-chlorophenoxy propionic acid
2,5,4'-Trlchlorobiphenyl
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 - S
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral
Inj ection
Inhalation
Oral
Oral
Oral
Oral
Ora"!
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
-------
4-183
Appendix C (continued)
Chemical
3' ,4'-Dlchloropropionanilide
Calcium carbimide
Acrolein
Ethylenebislsothiocyanate sulfide
Triphenyl tin hydroxide
Ponceau MX
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Triethyl phosphate
TR2379
N-Methyl-H-(l-naphthyl)fluoracetamide
Fenterol-HBr
Fominoben-HCl
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Epichlorohydrin
Ferric dimethyl dithiocarbamate
Tetramethylthiuran disulfide
Ponceau MX
DibutyKdiethylene glycol bisphthalate)
AHR-2438B
Barthrin
Dimethrin
Hexachlorophene
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
2,5,4' -Trichlorobiphenyl
Carbon tetrachlorlde
3' , 4 '-Dichloropropionanilide
Tetrahydrothiophene-1 ,1-dioxide
Calcium carbimide
Acrolein
Ethylenebisisothiocyanate 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
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
Injection
Oral
Oral
Oral
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Oral
Inhalation
Oral
Result
-
+
-
-
-
-
+
+
^
_
-
-
-
-
- ,
*
-
-
-
-
-
-
•*
-
-
-
+
-'
.
-
:
-
-
-
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
-------
-4-184
Appendix C (continued)
Chemical
Isooctyl isodecyl nylonate
Orange G
Hexachlorobenzene
Clindamycin hydrochloride
Trlethyl phosphate
TR2379
N-Methyl-N-(l-naphthyl)fluoracetamide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiurao disulfide
AHR-2438B
Barthrin
Dimethrin
A9-Tetrahydrocannabinol
2,5,4' -Tr ichlorobiphenyl
Carbon tetrachloride
Calcium carbimide
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange G
HexachlorobenzeAe
2,5,4' -Trichlorobiphenyl
Clindamycin hydrochloride
Triethyl phosphate
TR2379
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Pyridoxine 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
Oral
Oral
Oral.
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Inj ection
Oral
Oral
Oral
Oral
Oral
Oral
' Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Result
-
-
-
-
-
-
-
-
-
-
-
-
^
-
-
-
-
-
-
-
-
+
-
-
-
+
-
-
-
'
-
-
-
-
-
-
-'
-
-
-
-
-
-
-
-
-
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
-------
4-185
Appendix C (continued)
Chemical
i'-Tetrahydrocannabinol
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Kthyleneblslsothlocyanate sulflde
Hexachlorobenzene
Cllndamycln hydrochloride
TR2379
2->!ethyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Foainoben-HCl
Aspergillus funigatus
2,3,7, 8-Tetrach lorodibenzo-p-dioxin
Peroxyacetyl nitrate
Ferric diaethyl dlthiocarbamate
Tetramethylthiuram disulflde
i'-Tetrahydrocannabinol
2.5,4' -Trlchlorobiphenyl
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
Oral
Injection '
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral S
Injection
Oral
Result Reference
39
39
40
40
7
12
13
13
19
19
- 21
40
+ 40
+ 25
+ 26
27
29
30
33
- ' 33
39
39
23
Salivary Gland
Calcium carbioide
Acroleln
Ethylenebisisothiocyanate sulfide
Trlphenyl tin hydroxide
Isooctyl Isodec^l nylonate
Orange C
Hexachlorobenzene
Cllndanyctn hydrochloride
TX2379
Fenterol-HBr
Foninoben-HCl
Aspergillus fuaigatus
2,3,7,8-Tetrachlorodlbenzo-p-dioxin
Peroxyacetyl nitrate
Ferric diaethyl dlthiocarbamate
Tctramochylthiuraa disulfide
AHR-243SB
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Dog
Dog
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rac
Oral
Inhalation
Oral
Oral
' Oral
Oral .
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
4
6
7
8
9
11
12
22
24
+ 13
13
19
19
+ 25
26
27
29
30
33
33
34
-------
._ 4-186
Appendix C (continued)
Chemical
i?-Tetrahydrocannablnol
: . 5,4 ' -Trlchloroblphenyl
2->!ethyl-4-chlorophenoxy acetic acid
;-Methyl-4-chlorophenoxy propionlc acid
Species
Monkey
Monkey
Monkey
Rat
Rat
Route Result
Oral
Injection
Oral
Oral
Oral
Reference
39
39
23
40
40
Sciatic Nerve
Ethyleneblsisothlocyanate sulflde
Hexachlorobenzene
2,3,7, 8-Tetrach lorodibenzo-p-dioxin
Pyrldoxine hydrochloride
3' ,4'-Dichloropropionanilide
1,2,4-Trichlorobenzene
Acroleln
Hexachlorobenzene
Aspergillus fuaigatus
2,3, 7 ,8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
AHR-2438B
J.?-Tetrahydrocannablnol
Small
Carbon tetrachloride
3* ,4'-Dlchloropropionanilide
Tetrahydrothiophene-l,l-dioxide
Acroleln
Ethyleneblsisothlocyanate sulfide
Isooccyl Isodecyl nylonate
Orange C
Triethyl phosphate
Hexachlorobenzene
Cl indarayc in hydrochloride
7S2379
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidiniuni
broalde
::-Methyl-::-(l-naphthyl)fluoracetamide
2->!ethyl-4-chlorophenoxy acetic acid
Rat
Dog
Rat
Dog
Skin
Rat
Rat
Monkey
Rat
Dog
Rat
Rat
Rat
Rat
Monkey
Monkey
Intestine
Rat
Rat
Dog
Rat
Monkey
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Rat
Dog
Rat
Dog
Dog
Rat
Dog
Rat
Rat
Rat
Rat
Oral
Oral
Oral
Oral +
Oral
Inhalation -
Inhalation
Inhalation
Oral
Oral
Oral
Inhalation
Oral + '
Oral
Inj ect-iwx
Inhalation
Oral
Inhalation
Inhalation
Inhalation
Inhalation
• Oral
Oral
Oral +
' Oral
Oral
Oral
Oral
Oral
Oral
Or'al
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
7
12
29
38
2
5
5
6
12
27
29
30
34
39
39
1
2
3
3
3
6
7
9
11
14
12
22
24
13
13
19
19
18
18
18
18
20
21
40
-------
4-187
Appendix C (continued)
Chemical
2-Methyl-4-chlorbphenoxy propionic acid
Fenterol-HBr
Fominoben-HCl
Asperglllus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dloxln
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiuram disulfide
AHR-2438B
Barthrin
Dimethrin
Pyridoxine hydrochloride
i'-Tetrahydrocannabinol
2,5,4' -Tr ichlorobiphenyl
1 , 2 ,4-Trichlorobenzene
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Hexachlorobenzene
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Acrolein
t
Pyridoxine hydrochloride
2,5,4'-Trichlorobiphenyl
Carbon tetrachloride
3' ,4'-Dichloropropionanilide
Tetrahydrothiophene-1 , 1-dioxide
Calcium carbimide
1 , 2 ,4-Trichlorobenzene
Acrolein
Ethylenebisisothiocyanate sulfide
Triphenyl tin hydroxide
Species
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
Monkey
Monkey
Spinal Cord
Rat
Monkey
Rat
Rac
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
Oral
Oral
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Injection
Oral '
Inhalation
Inhalation
Oral
Oral
Oral s
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
Result
-
-
-
-
-
-
-
-
-
-
«£_
-
-
-
-
-
-
-
— f
-
+
+
-
-
-
-
-
-
-
+
+
+
+
-'
-
-
+
-
-
-
-
-
-
-
+
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
-------
4-188
Appendix C (continued)
Chemical
Isooctyl isodecyl nylonate
Ponceau 4R
Or.inge G
Hexachlorobenzene
Cllndamycln hydrochloride
Trl ethyl phosphate
2,3,7 , 8-Tetrachlorodibenzo-p-dioxin
TR2379
N-Methyl-N-(l-naphthyl)fluoracetamide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy proplonic acid
Fenterol-HBr
Foainoben-HCl
Aspergillus fumigatus
Feroxyacecyl nitrate
Eplchlorohydrln
Ferric dimethyl dlthlocarbamate
Tetramethylthluram dlsulfide
Ponceau MX
Dibutyl(diethylene glycol bisphthalate)
AHR-2438B
Barchrin
Disethrin
Kexachlorophene
Pyridoxine hydrochloride
i'-Tetrahydrocannabinol
Carbon tetrachloride
3' ,4'-Dlchloropropionanilide
Tetrahydrothlophene-l.l-dioxide
Acrolein
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange C
Hexachlorobenzene
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
Injectidn
Oral +
Oral
Oral +
Oral
Oral +
Oral
Oral
' Oral
Oral
Oral
Injection
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
-------
4-189
Appendix C (continued)
Chemical
Clindamycin hydrochloride
Triethyl phosphate
TR2379
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidiniuni
bromide
N-Methyl-N- (1-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
Barthrln
Dimethrin
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy proplonic acid
2,5,4* -Tr ichlorobiphenyl
Carbon tetrachloride
3' ,4'-Dlchloropropionanilide
Calcium carbimide
Acrolein
Ethylenebisisothiocyanate 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
Tea tea
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
-------
4-190
Appendix C (continued)
Chemical
N-Methyl-N-(l-naphthyl)fluoracetamide
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionlc acid
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
Peroxyacetyl nitrate
Epichlorohydrin
Ferric dimethyl dichlocarbamate
Tetramethylthiuram disulfide
Ponceau MX
Dibutyl(diethylene glycol bisphthalate)
AHR-2438B
Barthrln
Dlmethrin
Hexachlorophene
Pyridoxine hydrochloride
i9-Tetrahydrocannabinol
2 , 5 , 4 ' -Tr ichlorobipheny 1
Calcium carbimide
Acrolein
Ethylenebisisothlocyanate sulfide
Isooctyl isodecyl nylonate
Orange G
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Quanethidine
l-Methyl-3-keto-4-phenylquinuclidinium
bromide
N-Methyl-N-(l-naphthyl)fluoracetamide
Hexachlorobenzene
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
Peroxyacetyl nitrate
TR2379
A9-Tetrahydrocannabinol
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-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
Injection
Oral +
Oral
S
Oral
Inhalation
Oral
Oral.
Oral
Oral +
Oral +
Oral
Oral
Oral
Oral
Oral
Oral +
Oral -
Oral +
Or,al
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
-------
4-191
Appendix C (continued)
Chemical
3. 'i'-Dlchloroproplonanilide
Tctrahydrothlophene-1 ,1-dioxide
Calcium carblmlde
Acroleln
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange C
Hexachlorobcnzene
Cllndaraycln hydrochlorlde
Triethyl phosphate
TR2379
2-Methyl-4-chlorophenoxy acetic acid
2-Methyl-4-chlorophenoxy propionic acid
Fenterol-HBr
Foninoben-HCl
Aspergillus fuolgatus
2,3. 7 ,8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
Ferric diaethyl dlthlocarbamate
Tctramethylthluram disulfide
AHR-2438B
Barthrin *
Dir.ethrin
Pyrldoxinc hydrochloride
I'-Tetrahydrocannabinol
2,5,4'-Trtchlorobiphenyl
Tecrahydrothlophene-1 ,1-dioxide
Acrolein
Ethylcnebisisochiocyanate sulfide
Orange C
Hexachlorobenzene
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
Oral
Inhalation
Inhalation
Inhalation
Oral
Inhalation
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral S
Oral
Oral
Oral
Inhalation
Oral
Oral
Oral
' Oral
Oral
Oral
Oral
Injection
Oral
Inhalation
Inhalation
Inhalation
Inhalation
Inhalation
Inhalation
Oral
Oral
Oral
Oral
Result 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
-------
4-192
Appendix C (continued)
Chemical
2,5,4' -Trichlorobipheny 1
Fenterol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7, 8-Tetrachlorodibenzo-p-dioxin
Peroxyacetyl nitrate
A9-Tetrahydrocannabinol
Carbon tetrachlorlde
3' ,4'-Dichloropropionanilide
Tetrahydrothiophene-l,l-dioxide
Acrolein
Ethylenebisisothiocyanate sulfide
Isooctyl isodecyl nylonate
Orange G
Hexachlorob enzene
Clindamycin hydrochloride
Triethyl phosphate
TR2379
Feneerol-HBr
Fominoben-HCl
Aspergillus fumigatus
2,3,7, 8-Tet rachlorodibenro-p-dloxin
Peroxyacetyl nitrate
Ferric dimethyl dithiocarbamate
Tetramethylthiurara disulfide
AHR-2438B
Barthrin
Dimethrin
Pyridoxine hydrochloride
A9-Tetrahydrocannabinol
2,5,4' -Tr ichlorobiphenyl
2-Methyl-4-chlorophenoxy acetic acid
Species
' Monkey
Rat
Rat
Rat
Rat
Rac
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
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
1Z
22
24
13
13
14
19
19
25
26
27
29
30
33
33
34
36
36
38
39
39
23
40
40
-------
4-193
Appendix C (continued)
Chemical
Acroleln
Echylcneblslsothiocyanate sulfide
Isooctyl Isodecyl nylonate
Orange G
Hexachlorobenzene
2.5.4' -Trlchlorobiphenyl
Cllndamycln hydrochloride
TR2379
2,3,7, 8-Tct rachlorodibenzo-p-dioxin
Ferric dimethyl dlthlocarbamate
Tetramechylthluram disulflde
Pyrldoxlne hydrochloride
A ' -Te t rahyd rocannab Inol
2-Methyl-4-chlorophenoxy acetic acid
2-Mechyl-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
Inhalation
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inj ection
Oral
Oral
Result Reference
6
7
9
11
12
22
24
23
13
13
- 19
19
+ 29
33
33
38
39
39
40
- , 40
-------
4-194
APPENDIX C
REFERENCES
1. Adams, E. M., H. C. Spencer, V. K. Rowe, D. D. McCollister, and
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s
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Term Toxicity Studies of Rats Fed Triethyl Phosphate in the Diet.
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v
15. Hall, D. E., F. S. Lee, and F. A. Fairweather. 1966. Acute (Mouse
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16. Hall, D. E., P. Austin, and F. A. Fairweather. 1966. Acute (Mouse
and Rat) and Short-Term (Rat) Toxicity Studies on Dibutyl (Diethyl-
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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
(3rally Administered Benzoquiriolizinyl Derivative in the Rat and
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20. Hashimoto, Y., T. Makita, H. Miyata, T. Noguchi, and G. Ohta. 1968.
Acute and Subchronic Toxicity of a New Fluorine Pesticide, N-Methyl-
N-(l-Naphtyl)fluoracetamide. Toxicol. Appl. Pharmacol. 12:536-547.
21. Hattula, M. L., H. Elo, H. Reunanen, A. U. Arstila, and T. E. Sorvari.
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Acetic Acid (MCPA) in Male Rat. I. Light Microscopy and Tissue Con-
centrations 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.
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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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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.
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Yakuri. Sendai 10(1):45-71.
26. Kast, A., Y. Tsunenari, M. Honma, J. Nishikawa, T. Shibata, and M.
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Oyo Yakuri. Sendai 10(l):31-43.
27. Khor, G. L., J. C. Alexander, J. H. Lumsden, and G. J. Losos. 1976.
Safety Evaluation of Aspergillus fumigatus 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 Technical
Hexachlorobenzene in the Sherman Strain Rat. A Preliminary Study.
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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 Peroxy-
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Efet. Toxicol. Appl. Pharmacol. 40:529-549.
32. Lawrence, W. H., M. Malik, J. E. Turner, and J. Autian. 1972. Tox-
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33. Lee, C-C., J. Q. Russell, and J. L. Minor. 1978. Oral Toxicity of
Ferric Dimethyl Dithiocarbamate (Ferbam) and Tetramethylthiuram
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34. Luscombe, D. K. , and P. J. Nicho'lls. 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 Ani-
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Subacute Toxicity of Two Chrysanthemumic Acid Esters: Barthrin and
Dimethrin. Toxicol. Appl. Pharmacol. 6:716-725.
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4-197
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.
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39. Thompson, G. R., R. W. Fleischmann, H. Rosenkrantz, and M. C. Braude.
1974. Oral and Intravenous Toxicity of A9-Tetrahydrocannabinol in
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f
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. TERATOGENICITY
5.1 INTRODUCTION
As Wilson (1979) states, "the notion that chemical and physical
agents in the environment need to be tested for their potential to cause
teratogenic effects when there is likelihood of exposure during pregnancy
is a relatively new concept." It was not until frhe thalidomide tragedy in
Che early 1960's that the world's attention was focused to the fact that
the human embryo is not isolated in an impervious maternal body where it
is shielded from all but genetic harm. Wilson further states that the year
1966 probably deserves special recognition because of the significant efforts
to minimize the risks to the unborn population from' radiations by the Inter-
s
national Committee for Radiation Protection Recommendations and from drugs
by the Food and Drug Administration Guidelines.
This section will not include a discussion of viruses and ionizing
radiation as teratogenic agents but will limit its coverage to congenital
malformations which are chemically'induced. The first subsection will
briefly discuss some basic test parameters, the second subsection will
examine the relationship between carcinogens, mutagens, and teratogens,
followed by a subsection on the influence of the Jtime of administration
with regard to inducing teratogenicity, and concluding with a discussion
concerning the choice of species for teratological testing. The term
embryolethality refers to the death of offspring and embryotoxicity refers
to both teratogenic and abortifacient responses to a chemical.
-------
5-2
5.2 GENERAL CONSIDERATIONS
5.2.1 Introduction
This section will briefly examine some of the basic parameters of
testing chemicals for teratogenicity. These include dosage (number,
levels, and duration), pharmacokinetics, positive controls, number of
species, number of test animals per dose group, administration route, and
fetal examination.
5.2.2 Dosage - Number and Levels
An examination of the data in Table 5.1 shows that the number of dose
levels used in the teratogenic testing of chemicals'is variable among dif-
ferent researchers; ranging in these cited instances from 1 to 8 with 4 of
20 papers indicating 4 doses and 5 of 20 papers indicating 3 doses. All
regulatory groups for which teratological test guidelines/standards have
been proposed recommend at least 3 dose levels. The Federal Insecticide,
Fungicide, and Rodenticide Act, the Interagency Regulatory Liason Group, and
the Organization for Economic Cooperation and Development each recommend at
least 3 doses, whereas the Food and Drug Administration recommends the use
of 4 doses.
Ideally, the high dose level should produce either maternal toxicity
f
but not lethality or embryotoxicity (either intrauterine death, malformation
or growth retardation) (Wilson, 1975; World Health Organization, 1967).
The low dose should, ideally, permit normal embryonic development and the
intermediate dose should produce malformed offspring if the test chemical
is teratogenic (Collins and Collins, 1976). If more than 3 doses are tested
-------
Table S.I. Literature survey of experimental parameters in testing uf chemicals for teralogenii:ity
Positive
controls used
Number of doses
and duration
Number animals
per group
Kxtt-nt of
fetal examination
Reference
No
No
No
No
No
No
No
No
No
No
3 dose levels; low dose on
gestation day 9, Intermediate
dose on day 7, 8, 9, 10 or
11, and high dose on day 5,
6, 6.5. 7, 8, 9, 10 or 11
4 dose levels; Intravenous In-
jection on day 8 of gestation
1 dose level; on gestation day
8-16, 2-9, or from 4 days be-
fore macing Co 3 days after
mating
5 dose levels; oral intubation
beginning on 6th or 7th day
of gestation and lasting 4 to
6 days
1 dose level
2 dose levels; single oral in-
jection on 10th, llth, 12th
or 13th gestation day
6 dose levels; 4 given on gesta-
tion days 12-15, 1 on days
11-14, and 1 on days 10-13
4 dose levels; given on gesta-
tion days, 1-20
5 dose levels; given on gesta-
tion days, 6-15
1 dose level; given on gestation
day, 14
6 dose levels; single injection
on gestation days 7, 8, 9, 10
or 11
Kats - Not given External, skeletal (1/3 Wilnun, 1966
* of earli lltli-r), soft
tissue (2/3 of each
litter)
Hamsters - 2 to 16 External, skeletal, and
soft tissue
Rabbits - 2 to 7
Mice - 8 to 57
Rats - Not given
Rats - Not given
Rats - 3 to 24
Rats - 18 to 29
Rats - 14 to 19
Rats - Hot given
Rats - 7 to 20
External, skeletal, and
gross necropsy
Form and Hanlon, 1974
Drobeck, Coulston, and
Cornelius, 1965
External, skeletal, and Dipaolo, Catzek, and Pickren,
soft tissue 1964
Not evident from paper
External and skeletal
\
External, skeletal, and
soft tissue
External, skeletal (1/2
of each litter), and
soft tissue (1/2 of
each litter)
External, skeletal (1/2
of each litter), and
soft tissue (1/2 of
each litter)
External, skeletal, and
soft tissue
External and skeletal
Delahunt, Lassen, and Riesser,
1966
Sadler and Kochhar, 1975
King, Weaver, and Narrod, 1965
Collins, Black, and Ruggles,
1975
Schwetz, Sparschu, and Gehring,
1971
Brummett and Johnson, 1979
Lu, Matsumoto, and Lijima,
1979
Ui
UJ
-------
Table 5.1 (continued)
Positive
controls used
No
No
No
No
No
No
No
Yes
Number of doses
and duration
Number animals
per group
Extent of
fetal examination
Reference
8 dose levels; given on day 10
of gestation
3 dose levels; given on days
6-15 of gestation
4 dose levels; given on gestation
days 6-15, 8-11, and 12-15
1 dose level; single subcutaneous
Injection on gestation day 7, 8,
9, 10, 11, 12, 13 or 14
1 dose level; in diet throughout
pregnancy
3 dose levels; in diet from day 5
through day 10 of .gestation
4 dose levels; given on gestation
days 7-15
3 dose levels; given on gestation
days 7-19
Rats»- 7 to 15
Rats - 3 to 23 .
Rats - 15 to 20
Mice - 10 to 11
Hamsters - 20
Mice - Not given
Mice - 24 to 34
External, skeletal, and Skalko and Cold, 1974
soft tissue
External, skeletal (1/2 Schwetz, I.eong, and Cehring,
of each litter), and 1974
soft tissue (1/2 of
each litter)
External, skeletal, (1/2 Schwetz, Keeler, and Gehring,
of each litter), and 1974
soft tissue (1/2 of
each litter)
External and skeletal Inouye and Murakami, 1977
External and skeletal
Homburger et al., 1965
External, skeletal, and Randall and Taylor, 1979
soft tissue
External, skeletal (all John et al. , 1979
of ebch litter), and
soft tissue (1/3 of
each litter)
Rabbits - 9 to 12 External, skeletal (all Sofia, Strasbaugh, and Banerjee,
fetuses), and soft
tissue (all fetuses)
1979
ui
I
No 3 dose levels; given on gestation
days 4 or 5-16
Rabbits - 2 to 13
External, skeletal, and Fratta, Sigg, and Maiorana, 1965
soft tissue
All fetuses examined for external malformations. Ratio of skeletal to soft tissue examination cited if given.
-------
5-5
then more than one intermediate dose would be administered. Extrapolation
between the low dose and the intermediate dose or doses would thus provide
an indication of the effect/no effect range of the test chemical.
5.2.3 Dosage - Duration
Schardein (1976) states that, in general, acute dosing of a chemical
results in a greater teratogenic insult than dose* chronic dosing. One
example is that of the anticancer antibiotic actinomycin D in rats. Mal-
formations were induced in 28% of the surviving offspring with a single dose
of 200 ug/kg given on day 9 of gestation, but when given as ten daily
injections of 25 pg/kg on each of days 0 to 9 of gestation, only 9% of the
*
surviving offspring were malformed (Wilson, 1966). Further experimentation
s
demonstrated that the dam was sensitized by chronic treatment with actinomycin
D at high doses so that for the dam or her offspring even moderately teratogeni
doses become lethal doses. The stimulation as a result of drug-metabolizing
enzymes in the liver microsomes by chronic dosing was suggested as the possible
mechanism of this phenomenon which "has been observed by Koppanyi and Avery
(1966) as cited by Schardein (1976) in experiments with more than 100 drugs.
Similar results were demonstrated with the administration of the
benzhydrylpiperazine antihistamine drug chlorcyclizine (King, Weaver, and
Narrod, 1965). Chlorcyclizine administered to pregnant rats in 25 mg/kg doses
over a 4-day period (days 12 to 15 of gestation) produced 16% malformed young,
whereas the same dosage given over a 16-day period (days 1 to 15) resulted in
malformations in only 2% of the offspring. Even more striking results were
seen when 50 mg/kg was administered. Treatment on days 10 to 15 of gestation
resulted in 82% malformed individuals, but .only 0.1% malformed offspring
-------
5-6
was produced when 50 mg/kg was given over days 1 to 15. A subsequent paper
(King, Horigan, and Wilk, 1972) showed that the explanation behind this lack
of teratogenic activity from chronic treatment of chlorcyclizine was self-
stimulation of its own metabolism.
Wilson (1975) and the World Health Organization (1967) also report
that the repetitive administration of certain chemicals during pregnancy
^
can alter or mask a teratogenic action because of the ability of these
chemicals to change their own metabolism. Wilson states that the induction
by catabolizing enzymes (microsomes) in the liver or other tissues which
increases metabolism (also mentioned above), the metabolism decreasing
inhibition of naturally present enzymes which degrades chemicals, and the
*
induction of impaired function or overt pafhology in important homeostatic
organs such as the liver or kidneys as three ways that some chemicals can
change the rate of their own metabolism. He further states that it is
assumed these changes can occur within three of four days after the beginning
of repeated treatments.
» .
What then is the optimum dosing period for testing chemicals for terato-
genicity? The foregoing paragraphs suggest that dosing for .only one day or
for short periods of 3 to 4 days during organogenesis would enable detection
of some chemical teratogens whose teratogenicity might go undetected if
dosing duration was increased. However, with single or short term dosing
there is no possibility of measuring the cumulative teratogenic effects
(Collins and Collins, 1976). As a possible solution, Wilson (1975) recommends
using both short term and repeated tests. His recommended procedure consists
of dividing the period of organogenesis into shorter dosage periods of 3 to 4
days but also dosing some animals throughout organogenesis. He states that
-------
5-7
fewer animals would be needed in each dose group than if dosage were given
throughout organogenesis because the range of likely developmental defects,
and hence overall variability is reduced by the shorter treatment span and
as mentioned, some animals would still be dosed throughout organogenesis to
detect any teratogenic effects that would result from the cumulative action
of the chemical. Collins and Collins (1976) list three reasons why the
*
single-dose method technique which they report as being recognized as
the most successful in producing abnormalities is not used in place of the
repeatative dosing method in screening studies. These are: (1) the necessity
of treating different animals for each day of organogenesis increases the cost
of the experiment, (2) without prior knowledge of the compound's effects, there
f
is no way of predicting whether a specific^organ or the entire developmental
sequence will be affected, and (3) compounds that are likely to be tested for
teratogenic action are usually encountered by humans as repeative doses, and
with single dosing cumulative effects cannot be measured.
5.2.4 iPharmacokinetics
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 would be
6-aminopropionitrile (See Sect. 5.4.2) which is metabolized so quickly to its
inactive metabolite, cyanoacetic acid, that if day 15 (the ctitical period for
induction of cleft palate) is not included in the dosing regime, very few
malformations are produced in the rat (King, Horigan, and Wilk, 1972). How-
ever, there are some teratogenic drugs that; are absorbed or metabolized so
-------
5-8
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 period to implantation (Roux, 1964, as
cited by Schardein, 1976).
^
5.2.5 Positive Controls
Positive controls have been recommended in some teratogenic test
protocols such as the 1966 Food and Drug Administration Guidelines in order
to demonstrate that the test animal will produce malformed offspring after
*
exposure to an established teratogenic chemical. Although this premise
seems sound, the usefulness of positive controls can be questioned. A sur-
vey of the past and current literature indicates that not many researchers
use positive controls when testing. Table 5.1, Chemicals for Teratogenicity,
lists 20 papers which were read and pertinent teratogenic test protocol data
» • - -
extracted. Only one paper, Sofia, Strasbaugh, and Banerjee (1979), documents
the-use of positive controls and these researchers were following the FDA
Guidelines and thus were required to do so.
The reason that the usefulness of the data generated from positive
controls can be questioned is that an/animal species can vary in its reaction
to teratogenic chemicals. Table 5.17 shows the teratogenic effects in several
animals including man from thalidomide. Man, monkeys, and rabbits are shown
to be susceptible to the teratogenic action of thalidomide but several strains
of rats, mice, and hamsters are not. The action of the human teratogen
aminopterin (Table 5.15) also shows apparent species variability. Teratogenic
-------
5-9
effects have been seen in man and mice but not in the species of rats and
monkeys listed in Table 5.14. The data in these tables clearly demonstrate
that an animal may be susceptible to the teratogenic action of one chemical
but not necessarily to another chemical. Therefore,, if a positive control
is established in the animal species being tested and the test chemical
does not produce teratogenic effects it cannot be assumed that the test
^
chemical would not induce teratogenic effects in another animal species or
even another strain of the same species. Conversely, if an established
teratogen such as thalidomide fails to induce teratogenicity in an animal
being used as a positive control, it cannot be assumed that the test chemical
will not produce teratogenic effects in the same species. Thus the only
r
definite conclusion that can be reached by using a positive control is that
the positive control chemical produced teratogenicity.
5.2.6 Number of Species
Tables 5.15, 5.16, and 5.17 (Sect. 5.5.9) indicate that the choice of
species'or strain can determine whether or not a chemical is identified as
a teratogen. Species variability to potential teratogenic agents would
thus dictate that at least two species should be used. The question that
arises based on the two species requirement is why. should tests in two
species be necessary if when the two species are tested separately instead
of concurrently, the first species indicates that the test chemical is
teratogenic. This is a logical question since in the interest of human
safety the test chemical would have to considered potentially teratogenic
to humans if one animal species produces malformed offspring. The real
-------
5-10
value of the second species when the first has indicated teratogenicity
would primarily be for species comparisons. Such information as the dif-
ference or similarity in dose or anatomical defects would become available
and would probably be beneficial in future teratogenic studies with these
same species. In addition, the use of two species would give better a under-
standing of the effect/no effect level of the test chemical.
^
The use of only two species (usually a rodent and rabbit combination)
appears to be justified in view of the fact stated by the World Health
Organization (1967) that all substances that have been shown to be teratogenic
in man have also shown teratogenic activity in the mouse, rat, and rabbit.
They also state, however, that negative results obtained by testing chemicals
/•
in these species provides no absolute assurance that the chemical will not
induce teratogenic effects in man. This situation would particularly be
of concern when the chemical tested is a drug intended for use during human
pregnancy. In these instances, a third species closer to man in physiological
function than the rodent or rabbit probably should also be used as a test
» • .
animal. This is similar to the testing scheme proposed by Wilson (1975)
except that he recommends the use of the rat, mouse, hamster, or rabbit as
species in which to establish the embryotoxic dose range and not as the
primary test animal for teratogenic studies.
<•
5.2.7 Number of Test Animals Per Dose Group
The number of pregnant animals per dose group is an important considera-
tion because as Weil (1970) points out, in performing a statistical analysis
-------
5-11
of Che results of an experiment designed to assess teratogenicity the number
of independent sampling units, N, is the number of dams or litters. Weil
states that some researchers have used the number of pups as N and conse-
quently have reached invalid conclusions.
Table 5.1 indicates that the number of animals used in the testing of
chemicals for teratogenicity is highly variable. The results of some of the
*
researchers cited in Table 5.1 might be questioned because too few animals
in some dose groups were used to permit reliable statistical analysis. The
World Health Organization (1967) although not specifying animal numbers,
states that the number of rodents used must be large enough to satisfy
statistical requirements. For species more closely related to man, the
•<»*• ^
... World Health Organization recommends that^he number of animals be as large
as practicable, in order to obtain reproducible results. The guidelines
of the Federal Insecticide, Fungicide, and Rodenticide Act and the Inter-
agency Regulatory Liaison Group recommend 20 pregnant rats, 20 pregnant mice,
20 pregnant hamsters, and 15 pregnant rabbits per dose group. The Organiza-
» -•
tion for Economic Cooperation and Development also recommends 20 pregnant
rats and 20 pregnant mice but only 12 pregnant rabbits per group.
5.2.8 Administration Route
The choice of administration route is usually dictated, by the present
(a chemical in use) or expected (a new chemical) human exposure to the test
chemical (Collins and Collins, 1976). In most instances, the chemical should
be administered orally, but administration by other methods such as inhalation,
may be necessary in order to simulate human exposure. Although inhalation
tests are being used to determine the possible teratogenic effects of some
-------
5-12
chemicals (Hardin and Hanson, 1980; Schwetz, loset, Leong, and Staples, 1979;
Murray, Nitschke, Rampy, and Schwetz, 1979; Schwetz, Smith, Leong, and
Staples, 1979; Murray, Schwetz, McBride, and Staples, 1979), Collins and
Collins (1976) indicate that the exact amount of a test chemical ingested
by test animals in inhalation tests is often difficult to determine. The
reason is that the animals would not only receive the chemical by inhala-
^
tion but also by mouth as the animals would attempt to lick their fur clean.
Therefore, oral administration (especially by gavage) of test chemicals should
be employed when possible, because of the ability to more precisely measure
the amount ingested. The Federal Insecticide, Fungicide, and Rodenticide Act,
the Interagency Regulatory Liaison Group, and the Organization for Economic
f
Cooperation and Development guidelines recommend oral administration by gav-
age unless the chemical and physical characteristics of the test chemical or
the use pattern indicate that another route is necessary.
5.2.9 Fetal Examination
The 1967 World Health Organization's report and the National Academy of
Sciences "Principles and Procedures for Evaluating the Toxicity of Household
Substances" (NAS, 1977) recommend that all fetuses should be examined. The
World Health Organization states that all fetuses'should be examined for
external malformations; a significant .number should also be examined for
defects in organ structures. The NAS publication recommends that % of the
fetuses be examined for skeletal malformations and h for neural and visceral
defects. Table 5.1 shows that of 20 papers collected from the general litera-
ture, 15 examined the fetuses for both skeletal and soft tissue defects. Of
these 15 papers only 7 indicated the ratio 'Of fetuses examined for skeletal
-------
5-13
defects to fetuses examined for soft tissue defects, and of the 7, 4
indicated that % of the fetuses were examined for skeletal defects and
% for soft tissue defects (1:1).
5.2.10 Conclusions
1. At least 3 dose levels of a chemical should be tested, the low dose
should allow normal development, the intermediate dose should show
teratogenicity if the chemical tested is a teratogen, and the high dose
should produce either maternal toxicity or embryotoxicity.
2. Careful consideration should be given to the duration of treatment since
some chemicals are more teratogenic when administered acutely or for
/•
short periods during organogenesis rather than chronically throughout
organogenesis.
3. Proper attention to the pharmacokinetics of the chemical (if known) is
important because this influences how quickly after administration that
the chemical will exert its teratogenicity.
» • .
4. Positive controls cannot provide certainty that the test chemical is
not teratogenic if tests indicate no teratogenicity.
5. For each chemical at least two species should be tested for teratogenicity.
6. The number of pregnant test animals per group must be sufficient to permit
reliable statistical analysis. Twe'nty pregnant animals of a rodent species
and fifteen pregnant rabbits has been recommended by several regulatory
groups.
7. The administration route should be oral intubation unless the physical
and chemical properties of the chemical or human use conditions dictate
otherwise.
-------
5-14
8. All fetuses should be examined for external malformations. Approximately
one-half should be examined for skeletal defects and the remaining half
for soft tissue malformations.
5.3 TERATOGEN, MUTAGEN, AND CARCINOGEN RELATIONSHIPS
5.3.1 Introduction
^
As the number of chemicals which require toxicity testing increases
each year, both researchers and regulatory agencies seek to find reliable
toxicity testing methods which can either shorten the time necessary to
evaluate the toxicity of a chemical or can provide information on different
types of toxic responses, e.g., carcinogenic and mutagenic, from a single
f
test. This section considers these possibilities by examining the rela-
tionships between carcinogens, mutagens, and teratogens.
5.3.2 Numerical Examination
The approximate number of potential carcinogens, mutagens, and ter-
atogens has been identified through a computer search of the NIOSH Regis-
try of Toxic Effects of Chemical Substances (RTEC) data base which is
part of the National Library of Medicine's data file system. The number
of suspected and positively identified carcinogens, mutagens, and teratogens
was 2500, 70, and 500 respectively. (.The compounds listed as mutagens
were identified only on the basis of animal tests.) With the identifica-
tion of these subsets, it is possible to correlate the number of compounds
chat are (1) mutagenic and carcinogenic, (2) mutagenic and teratogenic,
(3) carcinogenic and teratogenic, and (4) mutagenic, teratogenic, and
carcinogenic. These correlations were performed on the RTEC data system
with the following results:
-------
5-15
1. Seventeen compounds were identified as both mutagens and carcinogens
(Table 5.2).
2. Ten compounds were identified as both mutagens and teratogens
(Table 5.3).
3. One hundred twenty-three compounds were identified as both carcinogens
and teratogens (Table 5.4).
^
4. Seventeen compounds were identified as mutagenic, carcinogenic, and
teratogenic (Table 5.5).
The compounds in Table 5.5 were for identification purposes not in-
cluded in Tables 5.2, 5.3, and 5.4, but in reality this group of 17 com-
pounds would also belong to the subset of mutagens and carcinogens, mutagens
and teratogens, and carcinogens and teratQgens. It should be noted that the
listing of a compound as a carcinogenic, mutagenic, or teratogenic agent
means only that the compound was found to have one or more of these effects
in at least one published source. Thus, the effect may or may not have been
corroborated by other researchers.
» • •
5.3.3 Literature Overview
The efforts to correlate carcinogenicty and mutagenicity are based on
the assumption that both carcinogens and mutagens 'exert their toxic action
by modification of the genetic material. By definition a mutagen alters
the genetic structure of a cell and as Magee (1977) writes, "Much current
opinion favors somatic mutation or aberrant differentiation as the mech-
anism of carcinogenesis most consistent with the available evidence."
Teratogens can exert their efforts by direct action with genetic material,
-------
5-16.
Table 5.2. Compounds identified as both carcinogens and mutagens
Name CAS number
Acridiniura, 3,6-diamino-10-methyl-, chloride mixed
with 3,6-acridinediamine 8043-52-0
l,2-Benzisothiazolin-3-one, 1,1-dioxide, sodium salt 128-44-9
p-Benzoquinone, 2,3,5-tris(l-aziridinyl)- " 68-76f-8
1-Cyclohexene-l-carboxylic acid, 3,4,5-trihydroxy- 138-59-0
Dibenz(a,h)anthracene 53-70-3
Diethylamine, 2,2'-dichloro-tf-methyl-, tf-oxide 126-85-2
Diethylamine, 2,2'-dichloro-tf-methyl-, tf-oxide,
hydrochloride 302-70-5
Ethane, l,l,l-trichloro-2,2-bis(p-chlorophenyl)- 50-29-3
f
Guanidine, l-methyl-3-nitro-l-nitroso- 70-25-7
Hexamethylenetetramine " 100-97-0
Imidazolidinone, 1-nitroso- 3844-63-1
2-Imidazolidinone, l-(5-nitro-2-thiazolyl)- - 61-57-4
Methanesulfonic acid, methyl ester 66-27-3
Phosphine oxide, tris(l-aziridinyl)- 545-55-1
Phosphoric triamide, hexamethyl- 680-31-9
« • •
Sulfide, bis(2-chloroethyl)- 505-60-2
Sulfuric acid, diethyl ester 64-67-5
-------
',£ C-r T:
5-17
Table 5.3. Compounds identified as both mutagens and teratogens
Name CAS number
Butyric acid, 4-((5-bis(2-chloroethyl)amino-l-methyl)-(2H)-
benzimidazolyl)-, hydrochloride
Caffeine 58-08-2
4-Cyclohexene-l,2-dicarboximide, #-((1,1,2,2-
tetrachloroethyl)thio)- 2425-06-1
IH-cyclonona(1,2-c:5,6-c')difuran-1,3,6,8(4H)-tetrone-10-
((3,6-dihydro-6-oxo-2H-pyran-2-YL)hydroxymethyl)-
5,9,10,ll-tetrahydro-4-hydroxy-5-(l-hydroxyheptyl)- ' 21794-01-4
Ergoline-8-beta-carboxamide, 9,10-didehydro-#C#-diethyl-
6-methyl- 50-37-3
2-Imidazolidinethione mixed with sodium nitrite
Methanediol, dimethanesulfonate 156-72-9
Methanesulfonic acid, isopropyl ester 926-06-7
Purine-6-thiol . 50-44-2
9H-thioxan«then-9-one,' l-((2-(diethylamino)ethyl)amino)-4-
(hydroxymethyl)-, monomethane- sulfonate (salt) 23255-93-8
-------
5-18
Table 5.4. Compounds identified as both carcinogens and teratogens
Name
CAS number
Acetamide, tf.ff-diethyl-
Acetamide, tf-fluoren-2-YL-
Acetic acid, (2,4-dichlorophenoxy)-
Acetic acid, (2,4-dichlorophenoxy)-, isooctyl ester
Acetic acid, iodo-
Acetic acid, lead(2+) salt
Acetic acid, (2,4,5-trichlorophenoxy)-»
Acrylonitrile
Adriamycin
Ammonium, (2-chloroethyl)trimethyl-, chloride
Androst-4-ene-3,17-dione
Androst-4-en-3-one, 17-beta-hydroxy-17-methy1-
Aniline, tf,tf-dimethyl-p-((p-chlorophenyl)azo)-
Aniline, tf,tf-dimethyl-p-(3-fluorpphenylazo)-
Aniline, tf,tf-dimethyl-p-phenylazo-
Aniline, tf-methyl-tf-nitroso-
Aniline, tf-methyl-p-(phenylazo)-
Arsenic acid, sodium salt
Arsenious acid, monosodium salt
Barbituric acid, 5-ethyl-:5-phenyl-, sodium salt
Benz(a)anthracene-7-methanol, 12-methyl-
Benzene
Benzene, hexachloro-
Benzene, pentachloronitro-
2,2'-Bipyridine
Butyric acid, 2-amino-4-(ethylthio)-, DL-
Butyric acid, 4-(p-bis(2-chloroethyl)aminophenyl)-
Cadmium
Cadmium chloride
Cadmium sulfate (1:1)
Carbamic acid, ethyl ester
685-91-6
53-96-3
94-75-7
25168-26-7
64-69-7
301-04-2
93-76-5
107-13-1
23214-92-8
999-81-5
63-05-8
58-18-4
2491-76-1
332-54-7
60-11-7
614-00-6
621-90-9
7631-8^
7784-46-5
57-30-7
568-75-2
71-43-2
118-74-1
82-68-8
366-18-7
67-21-0
305-03-3
7440-43-9
10108-64-2
10124-36-4
51-79-6
-------
5-19
Table 5.4 (continued)
Name
CAS number
Carbamic acid, ethylnitroso-, ethyl ester
Carbamic acid, methyl-, ethyl ester
Carbamic acid, methyl-, 1-naphthyl ester
Carbamic acid, #-methyl-#-nitroso, ethyl ester
Carbamic acid, propyl ester
Carbonic acid, diethyl ester
*
Carbon tetrachloride
Chloroform
Cholanthrene, 3-methyl-
Cholesterol
Chromium(VI) oxide (1:3)
Colchicine, tf-deacetyl-tf-methyl-
Cyclopenta(c)f uro(3',2': 4,5)furo(2,3-7z) (l)benzopyran-l',
11-dione, 2,3,6a,9a-tetrahydro-4-methoxy-
Daunomycin
Dibenzo-p-dioxin, 2,3,7,8-tetrachloro-
Diethylamine, 2,2*-dichloro-tf-methyl-
Diethylamine, tf-nitroso-
1,4:5,8-Dimethanonaphthalene, 1,2,3,4,10,10-hexachloro-
6,7-epoxy-l,4,4a,5,6-7,8,8a-octahydro, endo.exo-
»
1,4:5,8-Dimethanonaphthalene, 1,2,3,4,10,10-hexachloro-
1,4,4a,5,8,8a-hexahydro-, endo,exo-
Estradiol
Estradiol, 3-benzoate
Estrone
Ethane, azo-
/•
Ethane, azoxy-
Ether, 2,4-dichlorophenyl-p-nitrophenyl
Ethyl alcohol
2-Furaldehyde, 5-nitro-, semicarbazo'ne
Glutamic acid, tf-(p-(((2,4-diamino-6-pteridinyl)methyl)
amino)benzoyl)-, L-
614-95-9
105-40-8
6.V25-2
615-53-2
627-12-3
105-58-8
56-23-5
67-66-3
56-49-5
57-88-5
1333-82-0
477-30-5
1162-65-8
20830-81-3
1746-01-6
51-75-2
55-18-5
60-57-1
309-00-2
50-28-2
50-50-0
53-16-7
821-14-7
16301-26-1
1836-75-5
64-17-5
59-87-0
54-62-6
-------
5-20
Table 5.4 (continued)
Name
CAS number
Heliotrine 303-33-3
Hydantoin, 5,5-diphenyl- 57-41-0
Hydantoin, 5,5-diphenyl-, monosodium salt. 63C-93-3
Hydrazine, 2-benzyl-l-methyl- 1030'i!-79-2
Hydrazine, 1,2-diethyl-, dihydrochloride
Hydrazine, methyl- 60-34-4
Imidazole-4-carboxamide, 5-(3,3-dimethyl-l-triazeno}-,
citrate 64038-56-8
2-Imidazolidinethione 96-45-7
lH-indole-3-acetic acid 87-51-4
Isonicotinamide, 2-ethylthio- 536-33-4
Isonicotinic acid hydrazide 54-85-3
Lactose 63-42-3
Manganese, (ethylenebis(dithiocarbamato))- 12427-38-2
Mannitol, l,6-dibromo-l,6-dideoxy-, D- - 488-41-5
Methanesulfonanilide, 2'-hydroxy-5'-(l-hydroxy-2-
(isopropylamino)ethyl)-, monohydrochloride 14816-67-2
Methanol, (methyl-onn-azoxy)- 590-96-5
Methanol, (methyl-onn-azoxy)-, acetate (ester) 592-62-1
1,2,4-Matheno-lH-cyclobuta(cd)pentalene,
l,la,2,«2a,3,3a,4,5,5a,5b,6-dodecachlorooctahydro- 2385-85-5
2,7-Naphthalenedisulfonic acid, 3,3'-((4,4'-
biphenylylene)bis(azo))-bis(5-amino-4-hydroxy-,
tetrasodium salt 2602-46-2
1,3-Naphthalenedisulfonic acid, 6,6'-((3,3I-dimethyl-
4,4'-biphenylylene)bis(azo))bis(4-amino-5-hydroxy-,
tetrasodium salt ' 314-13-6
2,7-Naphthalenedisulfonic acid, 3,3'-((3/,3'-dimethyl-
4,4"-biphenylylene)-bis(azo))bis(5-amino-4-hydroxy-,
tetrasodium salt 72-57-1
2,7-Naphthalenedisulfonic acid, 3-hydroxy-4-((4-sulfo-
l-naphthyl)azo)-, trisodium salt ^ 915-67-3
5-Norbornene-2,3-dimethanol, 1,4,5,6,7,7-hexachloro-,
cyclic sulfite 115-29-7
19-Nor-17-alpha-pregn-5(10)-en-20-yn-3-beta,l-7-beta-diol 297-76-7
-------
5-21
Table 5.4 (continued)
Name
CAS number
19-Nor-17-alpha-pregn-5(10)-en-20-yn-3-one, 17-hydroxy- 68-23-5
19-Nor-17-alpha-pregn-4-en-20-yn-3-one, 17-hydroxy- mixed
with 3-methoxy-19-nor-17-alpha-pregna-l,3,5(10)-trien-
20-yn-17-ol 801^-29-0
Phenol, 2-sec-butyl-4,6-dinitro-r 88-85-7
Phenol, pentachloro- 87-86-5
Pregna-4,6-diene-3,20-dione, 6-chloro-17-hydroxy-, acetate 302-22-7
(6-alpha)-pregn-4-ene-3,20-dione, 17-(acetyloxy)-6-methyl- 71-58-9
Progesterone 57-83-0
Purin-6-thiol, 3-tf-oxide 145-95-9
3,5-Pyrazolidinedione, 4-butyl-l,2-diphenyl- 50-33-9
2,4-Pyrimidinediamine, 5-(p-chlorophenyl)-6-ethyl- 58-14-0
Quinoline, 4-nitro-, 1-oxide 56-57-5
Rifomycin sv, 3-(ff-(4-methyl-l-piperazinyl)formidoyl)-' 13292-46-1
Sarkomycin . ^ 11031-48-4
Semicarbazide, monohydrochloride 563-41-7
Serine, diazoacetate (ester) 115-02-6
Spiro(benzofuran-2(3H),l-(2)cyclohexene)-3,4'-dione, 7-
chloro-2',4,6-trimethoxy-6'-beta-methyl 126-07-8
4,4'-Stilbenediol, alpha,alpha'-diethyl- 56-53-1
Testosterbne ' ' . 58-22-0
Testosterone, propionate 57-85-2
Theophylline 58-55-9
p-Toluidine, alpha,alpha,alpha-trifluoro-2,6-dinitro-
iV.tf-dipropyl- 1582-09-8
Triazene, diethyl- ' 63980-20-1
Triazene, 3,3-diethyl-l-(m-pyridyl)- / 21600-43-1
Triazene, 3,3-dimethyl-l-phenyl- 7227-91-0
Triazene, 3,3-dimethyl-l-(m-pyridyl)- . 19992-69-9
Triazene, 3-monomethyl-l-phenyl- . 16033-21-9
Uracil, 5-(bis(2-chloroethyl)amino)- 66-75-1
Uracil, 5-fluoro- 51-21-8
Uracil, 6-methyl-2-thio- 56-04-2
-------
5-22
Table 5.4 (continued)
Name CAS number
Urea, 1,3-dimethyl-l-nitroso- 13256-32-1
Urea, ethyl nitroso- 759-73-9
Urea, ethyl- and sodium nitrite (2:1) ;.
Urea, hydroxy- 127-07-1
Urea, methylnitroso- 684-93-5
Urea,
-------
5-23
Table 5.5. Compounds identified as carcinogens,
mutagens, and teratogens
Compound CAS number
Actinomycin D .50-76-0
Azirino(2',3',:3,4)pyrrolo(1,2-a)indole-4,7-dione,6-amino-
1,la,2,8,8a,8b-hexahydro-8-(hydroxymethyl)-8a-methoxy-
5-methyl-,carbamate (ester) 50-07-7
•" /
Benz(a)anthracene, 7,12-dimethyl- 57-97-6
Benzo(a)pyrene 50-32-8
-1,4-Butanediol dimethylsulfonate 55-98-1
Colchicine 64-86-8
4-Cyclohexene-l,2-dicarboximide,N-(trichloromethyl)thio- 133-06-2
Dimethylamine, 2,2'-dichloro-tf-methyl, hydrochloride 55-86-7
f
Glutamic acid,N-(p-(((2,4-diamino-6-pteridinyl)methyl)
methylamino)benzoyl)-, L- S 59-05-2
Methanesulfonic acid, ethyl ester 62-50-0
2H-1,3,2-oxazaphosphorine,2-(bis(2-chloroethyl)
tetrahydro)-,2-oxide 50-18-0
Phosphine oxide, tris(l-(2-methyl)aziridinyl)- 57-39-6
Phosphine sulfide, tris(l-aziridinyl)- 52-24-4
Phosphonie acid, (2,-2,2-trichloro-l-hydroxyethyl)-,
dimethyl ester 52-68-6
Phthalimide, ff-((trichloromethyl)thio)- 133-07-3
p-Toluamide, W-isopropyl-alpha-(2-methyldrazino)- 671-16-9
s-Triazine, 2,4,6-tris(l-aziridinyl)- 51-18-3
-------
5-24
but this is only one of several ways that teratogenesis can be induced
(Magee, 1977). The following sections will examine the relationship be-
tween teratogenic, carcinogenic, and mutagenic chemicals.
5.3.3.1 Carcinogens and Mutagens — Much of the work devoted to
establishing a direct relationship between carcinogenic and mutagenic
chemicals has been that of Bruce Ames and his colleagues of the University
^
of California, Berkeley (Ames et al., 1973; McCann et al., 1975; McCann and
Ames, 1976, 1978; Ames and Hooper, 1978). Their experimental research has
demonstrated that some carcinogens can be accurately predicted by short-term
mutagenicity tests using mutant strains of bacteria. The basic premise in
establishing that those compounds which are carcinogenic are also mutagenic
f
is (as mentioned in Sect. 5.3.3) that both.chemical carcinogens and chemical
mutagens exert their effects through the alteration of the genetic material.
Thus, there would be a common action (DNA alteration) which would enable
the detection of carcinogens and mutagens by the same test.
Ames et al. (1973) demonstrated that certain carcinogens have frame-
*
shift mutagenic action when tested on a mutant set of the bacteria Salmo-
nella titphimuriim. Since many carcinogens require metabolic activation
before their carcinogenic effects are expressed, Ames and co-workers sup-
plied rat or human liver homogenate to the Salmonella histidine mutants.
Incubation of 20 known carcinogens, including aflatoxin BI, benzo(a)pyrene,
acetylaminofluorene, benzidine, and dimethylamino-trons-stilbene, together
with a rat or human liver homogenate and the bacterial tester strain on a
petri plate shows that 18 of the compounds are mutagenic, but only when
the homogenate fraction (S-9) is added, thus showing the importance of
metabolic activation (Table 5.6). The two 'carcinogens tested which did
-------
5-25
Table 5.6. Activation of carcinogens to mutagens
Carcinogen
2-Arainoanthracene .
2-Arainofluorene
2-Acetylamino-
f luorene
Benzidine
4-Arainobiphenyl
4-Amino-trans-
stilbene
4-Dimethylamino-
trons-stilbene
p-(Phenylazo)-
aniline
»
4-(o-Tolylazo)-o-
toluidine
//,tf-Dimethyl-p-
(ra-tolylazo)-
aniline
2-Naphthylamine
1-Aminopyrene
6-Aminochrysene
Dose
(Wg)
20
20
0
10
10
0
50
50
0
50
50
0
100
100
0
10
10
0
10
10
0
100
100
0
10
10
0
100
100
0
100
100
0
10
10
0
1
1
0
Histidine revertants per
_y
TA1535
+ 318*
21
+ 16
+ 77
21
+ 77
+ 117
86
+ 118
+ 34
38
+ 38
+ 143
92
+ 118
+ x-
-
+
+ 57
68
+ 97
+ 31
15
+ '45
+ 71
94
+ 97
+
-
+
+ 330a
20
+ , 16
+ •' 41
66
+ " 42
+ 18
30
+ 14
TA1536
lla
0
3
2
0
0
2T
0
3
1
0
0
4
2
3 -
3
0
1
3
0
2
1
1
1
2
0
2
1
0
1
6
0
2
TA1537
180a
17
12
121a
14
12
92a
11
12
29
10
22
108a
8
12
23
7
10
25
6
12
8
12
18
20
8
12
34
8
18
136a
23
9
127a
3
18
plate
TA1538
ll,200a
,. 27
••; 46
ll,300a
39
29
13,600a
21
46
265a
16
36
980a
28
46
842a
17
42
896a
28
53
94a
13
31
305a
17
29
147a
13
31
85a
15
21
398a
59
29
638a
30
46
-------
5-26
Table 5.6 (continued)
Carcinogen
Benzo(a)pyrene
3-Methyl-
cholanthrene
7,12-Dimethyl-
benz (a) anthracene
Aflatoxin BI
Sterigmatocystih
Dose
(ug)
5
5
0
50
50
0
50
50
0
1
1
0
0.5
0.5
0
Histidine revertants per
" 0
TA1535
+b 46
77
+ 48
+ 22
13
+ 11
+ 25
35
+ 29
+° 35
23
+ 42
+C 13
30
+ 5
TA1536
4
1
1
5
0
0
1*
0
1
0
3
0
3
1
0 '
TA1537
148a
16
28
88a
2
9
225a
15
11
36a
5
6
32
18
13
plate
TA1538
505a
54
44
510a
21
27
< 88a
20
36
266a
26
26
121a
8
32
?Judged to be significantly different from the controls.
The S-9 preparation was from a 200-g rat that was injected intra-
peritoneally, 24 hr before it was killed, with 16 mg of 3-methylcholan-
threne dissolved in corn oil.
st
One-third the normal amount of S-9 fraction was used in the S-9 mix.
Source: Adapted from Ames et al., 1973.
-------
5-27
not give positive results, auramine 0 and /y,ff-dimethyl-p-(phenylazo)-aniline
(not shown in table), have dimethylamino side chains that, according to
Ames et al., require oxidation (not provided in this test system) for
activation.
In two more recent papers, Ames and co-workers show the results of
testing approximately 300 chemicals (both carcinogens and noncarcinogens)
^
for mutagenicity (McCann et al., 1975) and discuss their results (McCann
and Ames, 1976). They found that of 175 known carcinogens, 157 (90%) were
also shown to be mutagenic by the Salmonella/m±c-cosome tests, and that of
108 noncarcinogens, 94 (87%) were shown to be nonmutagenic, thus indicating
the sensitivity of the test for predicting both carcinogens and noncarcinogens,
*
In general, Ames and colleagues believe the false positives and negatives to
be explainable by either inadequate animal carcinogenicity tests or inade-
quacies in their in vitro metabolic activation system. An example of a
false positive (a noncarcinogen that showed mutagenic activity) is the
fungicide captan; an example of a false negative (a carcinogen that showed
»
no mutagenic activity) is the chlorinated hydrocarbon dieldrin, which is
presumed by McCann and Ames (1976) to require another type of metabolic
activation (possibly dehalogenation) other than that which their microsome
system provided. However, Burchfield and Storrs (1978) cited the work
of Fahrig (1974), McCann and Ames (1976), Shirasu et al. (1976), Bidwell
et al. (1975), and Baue and Frohberg (1972) as examples of researchers
who have not been able to establish the mutagenicity of dieldrin (in vivo
and in vitro), chlordane (in vivo), heptachlor (in vitro and in vivo), and
other chlorinated hydrocarbons. Burchfield and Storrs report that there
is a poor correlation between the carcinogenicity of these pesticides to
-------
5-28
rodents and their mutagenicity by a variety of bacterial and animal tests.
This evidence suggests that many chlorinated hydrocarbons may not be muta-
genic in vitro and in vivo and raises two interesting questions which need
answers before regulatory agencies can make decisions concerning chlorinated
hydrocarbons on a sound scientific basis: (1) Can mutagenicity be used as
an indicator of carcinogenicity for all classes of compounds? and (2) Are
^
the hepatocellular carcinomas and other tumors produced in hybrid rodents
during lifetime feeding studies an indication that these compounds are
carcinogenic in humans?
Corroboration of the research of Ames and his colleagues concerning
the relationship of carcinogenic and mutagenic chemicals comes from two
v
Japanese papers (Teranishi, Hamada, and Watanabe, 1975; Sugimura et al.,
1976). The work of Teranishi and co-workers showed that carcinogenic poly-
nuclear aromatic hydrocarbons (PAH) — 3-methylcholanthrene, dibenzo(a,i)
pyrene, benzo(a)pyrene, dibenzo(a,e)pyrene, dibenz(a,ft)anthracene,
benz(a)anthracene, and benzo(e)pyrene — which have been detected as air
t • ,
pollutants were shown to be mutagenic to mutant strains of Salmonella
typhirmrium. Four mutants, TA1535, TA1536, TA1537, and TA1538, were used
as test strains, but only TA1537 and TA1538 gave a significant number of
revertant colonies. One interesting aspect of this study was the use of
two different liver homogenate fractions. For metabolic activation both
fractions were prepared from rats, but with one group of rats phenobarbi-
tal was added to the drinking water one week prior to their sacrifice to
induce liver enzyme production; the other group of rats received injections
of 3-methylcholanthrene or dibenz(a,7z)anthracene dissolved in corn oil in
-------
5-29
addition to phenobarbital prior.to being killed. The three strongest car-
cinogens, 3-methylcholanthrene, dibenzo(a,£)pyrene, and benzo(a)pyrene,
could be shown to be mutagenic using the phenobarbital-induced liver prep-
arations, but dibenz(a,7z)anthracene, benz(a)anthracene, and benzo(e)pyrene
caused a significant number of revertant colonies only when PAH-induced
liver enzyme was added to the culture medium. In addition, a definite
^
quantitative correlation between carcinogenicity and mutagenicity of PAH
was shown with strain TA1538, but not with strain TA1537, when the rat
liver enzyme was induced with both dibenz(a)anthracene and phenobarbital.
These results show the importance in the testing of chemicals for muta-
genicity of the bacteria test strain and of achieving the proper metabolic
*
activation. /-
Using a metabolic activating system mix of phenobarbital- or polychlo-
rinated biphenyl-induced liver homogenate, NADPH, NADH, glucose-6-phosphate,
and glucose-6-phosphate dehydrogenase, Sugimura et al. (1976) tested 240
compounds for mutagenicity using Salmonella typhimurium strains TA100 and
»
TA98 which had been derived from strains TA1535 and TA1538 respectively.
Of these 240 compounds, only 146 had been tested for carcinogenicty and
therefore were the only compounds whose mutagenicity could be compared to
carcinogenicity. A graphic illustration of the carcinogenic/mutagenic
correlation is shown in Fig. 5.1.; most of the 146 compounds (86%)
were carcinogens as well as mutagens or were noncarcinogens that when
tested were also nonmutagens. Sugimura and his colleagues believe that
some of the compounds which they found to be mutagenic, but whose carci-
nogenicity has not been established, will in the future be shown to be
-------
5-30
CARCINOGEN
SO
40
20
NON-MUTAGEN •
ORNL-OWG 79-(3664
eo 40 ;:;:;&;:•:; 2 zo «o eo
•MUTAGEN
40
so
NON-CARCINOGEN
Figure 5.1. Numbers of mutagens and nonmutagens tested in our labora-
tory and their correlation to carcinogenic activities. Source: Sugimura
et al., 1976, Figure 4, p. 202.
-------
5-31
carcinogenic. In support of this belief, they presented an illustration
(Fig. 5.2) which shows the increased correlation between chemical carcino-
gens and chemical mutagens for 1960 to 1975. One of the more notable
examples is the food additive 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide
(AF-2) which was indicated as being mutagenic using bacterial test strains
(Yahagi et al., 1974) and carcinogenic one or two years later (Nomura, 1975)
-------
5-32
CARCINOGEN MUTAGEN
1960
1966
AAF HN
BA 4NQOlNaN°2
OMN lBud"
NaNOj
BudR
4NQO — 4.niuoqurnoline- 1-oxide: OMN — N, JV-dimethylnitroianiine: DAB — N. JV-dimethvl-4-aminoazobenzene;
AAF - 2-acttylaminofluorene: BA - benzlalamhracene: B8N - butvl-/V-(4-hydroxybutyl)nitr<»amine:
MNNG — /V-methylW-nitro-W-nitrosoguanidine; AF<2 — 2^2-furyt}-3-(S-nitro-2-furyl}acrvlamide; NaNOj ~ sodium nitrite;
BudR - bromodeoxyuridine: MMS - methyl methane sultanate
Figure 5.2. Chronology of overlapping of carcinogens and mutagens.
Source: Sugimura et al., 1976, Figure 1, p. 193.
-------
5-33
be noncarcinogenic in the golden hamster (IARC, 1973 and National Insti-
tute of Health, 1973, as cited by Huberman, 1978). However, the previously
mentioned work of Teranishi, Hamada, and Watanabe (1975) listed dibenz (a,7z)
anthracene as a carcinogen on the basis of the work of Arcos and Argus
(1968) and Iball (1939). In addition, the paper by McCann et al. (1975),
also previously discussed, listed both dibenz(a,o)anthracene and dibenz-
*•
(a,/z)anthracene as carcinogens (no source indicated). The compounds were
also shown to be mutagenic in these studies.
The work of Kuroki, Drevon, and Montesano (1977) also shows the
reliability of Chinese V79 hamster cells in correlating the carcinogenic
and mutagenic activity of chemicals. Similar to the work of Huberman
f
(1975), Kuroki and co-workers used 8-azagu>anine resistance as an indication
of mutagenic activity, but in contrast to Huberman (1975 and 1978), who used
lethally irradiated rodent cells, a microsome fraction prepared from rats
that had received phenobarbital sodium prior to death was used for metabolic
activation. Table 5.7 shows the results of the Chinese hamster cell muta-
• • •
genie test on nine carcinogenic and two noncarcinogenic ff-nitrosamines and
compares these with mutagenic tests using the bacteria Salmonella typhimurium.
The correlation of carcinogenic and mutagenic chemicals was established
except for /Y-nitrosomethylphenylamine, which showed no mutagenic effects
in either the bacteria or mammalian test system.
Although many investigators now believe that most carcinogens are
mutagens, it is known that not all mutagens are carcinogens, or at least
not potent ones (Magee, 1977). Magee cited the mutagenic base analogues
such as bromodeoxyuridine as examples of chemicals that have not been
reported as powerful carcinogens.
-------
5-34
Table 5.7. Presence or absence of mutagenicity of various
nitrosamines in the presence of liver fraction
from PB-pretreated rats
Mutagenic effect
#-Ni tr osamine
Salmonella
typTvLmuriwn
Chinese
hamster
cells
Carcinogenic
DMN
tf-Nitrosodiethylamine
#-Nitrosodi-n-propylamine
ff-Nitrosomethyl-n-propylamine
#-Nitrosodi-rt-butylamine
#-Nitrosodi-n-pentylamine
tf-Nitrosomethylphenylamine
tf-Nitrosomorpholine
#-Nitrosopyrrolidine
tf-Nitroso-tf1-methylpiperazine
Noncarcinogenic
^-Nitrosodi-phenylamine
/7-Nitrosomethyl-tert-butylamine
Source: Kuroki, Drevon, and Montesano, 1977, Table 4,
1050.
-------
5-35
With the increasing number of chemicals introduced each year, the
need for short-term tests for carcinogenicity becomes more and more essen-
tial. At present, these short-term methods serve mainly as screening
mechanisms for animal testing, but in the future, if the somatic mutation
theory of chemical carcinogens can be further established, and if more
mutagenic chemicals are shown to be carcinogenic by virtue of mammalian
^
tests, then in vitro tests for carcinogenicity could possibly become an
important part of the regulatory process.
5.3.3.2 Teratogens and Carcinogens — Miller (1977), DiPaolo and
Kotin (1966), and Bolande (1977) have shown varied relationships between
congenital malformations and neoplasms, the extent and complexity of which,
*
according to Bolande, is now only beginning to be appreciated. Bolande
further states that the kinship of teratogenesis and oncogenesis appears
to be of fundamental importance in human developmental pathobiology and
that the appreciation of this kinship can significantly add to the under-
standing of the neoplastic process in general. Miller (1977) states that
»
the study of congenital malformations among patients with specific forms
of neoplasia has resulted in a new understanding of the fundamental biology
of cancer. The initial insight generally comes from bedside observations
made by clinicians and involves disorders for which no animal models are
known. In these instances the malformations are genetically induced or are
of unknown cases. Miller states, however, there has been no corresponding
increment in understanding from the capacity of some environmental teratogens
to induce cancer in man. This is not surprising since that only five agents,
with one of these being ionizing radiation, are thought to be both teratogenic
and carcinogenic in man (Table 5.8).
-------
5-36
Table 5.8. Agents that are both carcinogenic and
and teratogenic in man
Agent
Cancer sites
Organs affected
Alcohol
Diphenylhydantoin
Ionizing radiation
Androgens
Mouth, larynx,
esophagus
Lymph glands
Various
Liver
Diethylstilbestrol Cervix or vagina
Brain, face
Brain, face, nails
(hypoplasia)
Brain, stature
Masculinization of the
external genitalia
of females
*
Cervix and vagina
Source: Adapted from Miller, 1977, Table 3, p. 473.
-------
5-37
The relationship of chemically induced teratogenesis and carcinogenesis
appears to be closer when research in laboratory animals is considered.
DiPaolo and Kotin (1966) list 26 chemical agents that have been tested for
both oncogenicity and teratogenicity (Table 5.9). Of these 26, 25 were
found to be teratogenic, 20 were indicated as carcinogenic, and 19 were
found to be both carcinogenic and teratogenic. DiPaolo and Kotin believe
^
many parallelisms exist between teratogenesis and carcinogenesis and suggest
that the proposed hypotheses concerning the mechanisms of carcinogenesis —
(1) somatic mutation, (2) alteration of immune response, (3) protein delet-
ion, and (4) altered regulatory circuits — may be applicable to studies in
teratogenesis.
*
Although DiPaolo and Kotin believe that several parallelisms exist be-
tween teratogenesis and carcinogenesis, Magee (1977) concludes, based on
articles by Kalter (1971, 1975), Sullivan (1970), Wilson (1972), and Poswillo
(1976), that even though some teratogenic effects may be due to agents that
are mutagenic and/or carcinogenic, this does not necessarily imply that the
•
mechanisms are the same, and that in many cases, the mechanisms of terato-
genesis (Fig. 5.3) are unrelated to those of carcinogenesis and mutagene-
sis. Therefore, it is apparent that more research is required before the
relationship of carcinogenesis and teratogenesis can be clearly understood.
5.3.3.3 Teratogens and Mutagens *- By definition, mutagenesis results
when changes take place in the DNA template and/or in DNA polymerases,
whereas teratogenesis occurs when congenital malformations are produced.
These congenital malformations may be the result of one or more reactions
within the embryo or germ cells of which one might be mutation. Figure
5.3 demonstrates this by outlining the vari'ous causes, mechanisms, and
-------
ORNL-DWG 79-13669
MECHANISMS
PATHOGENESIS
COMMON PATHWAYS
-*- FINAL-DEFECT
INITIAL TYPES OF CHANGES IN
DEVELOPING CELLS OR TISSUES
AFTER TERATOGENIC INSULT:
1. MUTATION (GENE)
2. CHROMOSOMAL BREAKS,
NONDISJUNCTION, ETC.
3. MITOTIC INTERFERENCE
4. ALTERED NUCLEIC ACID
INTEGRITY OR FUNCTION
5. LACK OF NORMAL PRE-
CURSORS, SUBSTRATES,
ETC.
6. ALTERED ENERGY
SOURCES
7. CHANGED MEMBRANE
CHARACTERISTICS
8. OSMOLAR IMBALANCE
9. ENZYME INHIBITION
ULTIMATELY MANIFESTED AS
ONE OR MORE TYPES OF ABNOR-
MAL EMBRYOGENESIS:
1. EXCESSIVE OR REDUCED
CELL DEATH
2. FAILED CELL INTER-
ACTIONS.
3. REDUCED BIOSYNTHESIS
4. IMPEDED MORPHOGENETIC
MOVEMENT
5. MECHANICAL DISRUPTION
OF TISSUES \
2.
TOO FEW CELLS OR
CELL PRODUCTS TO
EFFECT LOCALIZED
MORPHOGENESIS OR
FUNCTIONAL MATURA-
TION
OTHER IMBALANCES
IN GROWTH AND
DIFFERENTATION
Ui
I
to
00
Figure 5.3. Diagram of the successive stages in the pathogenesis of a
developmental defect, beginning with the initial types of changes in
developing cells or tissues (the mechanism) and continuing to the final
defect. Source: Adapted from Wilson, 1977, Figure 1, p. 55.
-------
5-39
Table 5.9. Effects of chemical agents which have been tested
for both oncogenicity and teratogenicity
_, . , Effects
Chemical
classification • . „
Oncogenic Teratogenic
Nitrogen mustard (mechlorethamine hydrochloride) + +
Triethylenemelamine + +
Chlorambucil + 4-
Busulfan (Myleran) + +
Dactinomycin + '+
Thalidomide 4- +
5-Bromodeoxyuridine - +
Oxygen •+• +
Aminopterin - +
Benzo(a)pyrene + +
3-Methylcholanthrene •*• +
Monomethylaminoazobenzene s +• +
4'-Methyl-4-dimethyl-aminoazobenzene + +
3'-Fluoro-4-dimethyl-aminoazobenzene + +
4'-Fluoro-4-dimethyl-aminoazobenzene + +
tf-2-fluprenylacetamide + +
Ethyl carbamate (urethan) + +
Ethionine * ' + -
Colchicine - +
Testosterone and derivatives - +
Estrogens + +
Trypan blue -• + +
Nicotine - +
Galactose + +
Lead + +
Sodium fluoride ' - +
Source: Adapted from DiPaolo and Kotin, 1966, p. 3.
-------
5-40
manifestations of teratogenesis. Although a few chemicals are known to
be both teratogenic and mutagenic, e.g., benzo(a)pyrene, aflatoxin, and
nitrogen mustard (Wilson, 1972), Kalter (1975) states, "Environmentally
induced malformations and mutations cannot be equated with each other and
used as mutual indicators in monitoring potential harmful effects in the
environment."
^
5.3.A Conclusions
The relationship of chemical carcinogens to chemical mutagens has
been extensively studied during the past decade, especially the last five
years, and consequently much is now known. As Magee (1977) states, "The
fact that most chemical carcinogens are mutagens is now generally accepted";
~s
however, more research is still needed, particularly with the reverse rela-
tionship, mutagens to carcinogens, before the results of such testing can
be used for regulatory decision making.
The relationship of chemical carcinogens to chemical teratogens and
of chemical teratogens to chemical tnutagens is apparently not as well de-
fined as the carcinogen/mutagen relationship. Further comparative testing
is necessary for a sound understanding of these relationships.
More research is, therefore, a prerequisite before such statements
as the following made by the World Health Organization (1972) can be
significantly altered: "In the present state of our knowledge, we can
say only that some chemicals have mutagenic, teratogenic, and carcinogenic
effects, but that no evidence exists that all chemicals exerting one of
these three effects are necessarily also able to exert either one or both
of the other two."
-------
5-41
5.4 TERATOGENESIS AND TIME OF ADMINISTRATION
5.4.1 Introduction
A chemical should be considered nonteratogenic only when proper
teratological experimental procedures have been observed. This section
discusses one of the most critical aspects of testing chemicals for terato-
genicity, the proper time during gestation for ch'emical administration.
5.4.2 Organogenesis
The period of embryological differentiation (organogenesis) is con-
sidered to be the most sensitive developmental stage to the action of
teratogenic chemicals (Wilson, 1975; Schardein, 1976). This critical
/
period for a number of animal species, including man, is shown in Table
5.10; the period of greatest susceptibility in man is similar to that of
the baboon and the rhesus monkey.
Although Table 5.10 indicates that there are several days that are
categorized as being in the critical period, the embryologic differenti-
ation of the various organs proceeds at varying rates; thus, the time of
teratogenic insult is important with regard to which tissues are affected.
Two examples are the research of Inouye and Murakami (1977) and Sadler
and Kochhar (1975). Inouye and Murakami studied the teratogenicity in
mice of the hair-dye constituent 2,5-diaminotoluene and found that sub-
cutaneous or intraperitoneal injections of 50 mg/kg body weight caused a
low incidence of exencephaly (cranial malformation) and prosoposchisis
(fissure of the face, e.g., hair lip) and a high incidence of skeletal
malformations in those animals treated on day 8 of gestation. No such
-------
5-42
Table 5.10. Critical periods of organogenesis
in animals
Species
Mean duration
of gestation
(days)
Critical perioda
(days)
Hamster, golden
Mouse
Rat
Rabbit
Ferret
Cat
16
19
21
31
43
63
4
7
9
8
8
5
to 14
to 16
to 17
to 21
to 28
to 58;
5 to 15
Dog
63
most favorable
1 to 48; 8 to 20
estimated
Guinea pig
Pig
Sheep
Monkey , rhesus
Monkey , baboon
Armadillo '
Human
Cow
Horse
68
114
150
168
175
225 -
278
284
336
'11 to 20
12 to 34
14 to 36
20 to 45; 22 to 30
most susceptible
22 to 47
1 to 30
20 to 55
8 to 25
?
.
Period of embryological organogenesis or period
of known susceptibility to teratogens.
Source: Adapted from Schardein, 1976, Table 2-5,
p. 17. Data collected from several sources.
-------
5-43
malformed fetuses, however, were found in those animals treated on days
10 to 14 of pregnancy, and only a very low incidence of vertebral and rib
anomalies was observed in the fetuses treated on gestation day 7 or 9.
In studying the teratogenic effects of a single oral injection (with
a blunted needle) of chlorambucil (14.2 or 20 mg/kg) on the 10th, llth,
12th, or 13th day, Sadler and Kochhar (1975) observed limb defects from
^
treatments on the llth or 12th days of gestation and tail defects from
treatment on all days. Treatment on the 13th day resulted in digital
defects but without the long-bone defects observed with day 11 and 12
treatments; defects were observed only at the highest dose level. Sadler
and Kochhar also found that in vitro limb bud response to chlorambucil was
similar whether the limb buds were taken f^om pregnant mice after chlorambucil
treatment or the chlorambucil applied to limb bud cultures after removal from
nontreated pregnant mice.
The significance of the critical period is also shown by the human
teratogen thalidomide. Thalidomide was taken by 113 pregnant women be-
»
tween August 1959 and December 1961, but only 7 took the drug during the
critical period (reported here to be between 34 and 50 days after the last
menstrual period) (Kajii, Kida, and Takahashi, 1973). Of these 7 women,
3 delivered malformed babies and 4 delivered babies without apparent mal-
formations. Possible explanations for the 4 women who delivered normal
babies include faulty pregnancy timing and thus thalidomide possibly was
not taken during the critical period or the fact that there was actually
some sort of resistance to thalidomide. Two additional women took the
drug before the critical period, on day 19 and day 23, respectively;
-------
5-44
both women aborted (induced and spontaneous abortions, respectively).
The remaining 104 women took thalidomide after the critical period with
all offspring apparently normal, except for one reported case of anal
stenosis. One other example of the importance of the critical period
is shown in rats administered B-aminopropionitrile, a chemical which in-
duces cleft palate (King, Horigan, and Wilk, 1972). In this instance,
^
there were a significant number of offspring with cleft palate only when
day 15 of the gestation period was included in the dosing schedule. All
of the viable fetuses had cleft palate when 875 mg/kg was given on day
15, or over days 13 to 15 or 14 to 15, but when a total dose of 4320 mg/kg
was administered over days 12 to 14, only 8% of the young were malformed.
f
5.4.3 Histogenesis and Fetal Period/Behavioral Teratogenesis
The period of prenatal development which slightly overlaps organo-
genesis but extends primarily into the fetal period is known as histogenesis
(Fig. 5.4). Teratogenic agents that come into contact with the developing
fetus during this time of tissue fdrmation and development can cause minor
structural deviations, but the abnormalities that are more likely to occur
during the fetal period are those that involve growth or functional aspects
of development (Wilson, 1975). Examples of what Coyle, Wayner, and Singer
(1976) refer to as "behavioral teratogenesis" are demonstrated with the
administration of vitamin A during the early- and mid-fetal period
(Hutchings and Gibbon, 1973; Hutchings and Gaston, 1974). Pregnant rats
administered a teratogenic dose of vitamin A during the early fetal
period (days 14 and 15 of gestation) produced offspring that showed a
generalized retardation in growth as evidenced by delayed onset of fur
-------
5-45
ORNL-DWG 79-136JO
TERATOGENIC SUSCEPTIBILITY IS GREATEST
DURING EARLY ORGANOGEMESIS
[•FUNCTIONAL MATURATION-*-
EMBRYONIC PERIOD FETAL PERIOD
ENTIRE DEVELOPMENTAL SPAN
Figure 5.4. Curve represents the susceptibility of the human embryo
to teratogenesis, beginning with fertilization and continuing throughout
intrauterine development. Source: Adapted from Wilson, 19750, Figure 4,
p. 662.
-------
5-46
growth, eye-opening, and reduced body weight as well as reduced brain
size with obvious microcephaly in one animal. In addition, a behavioral
deficit was induced which was characterized by a decreased ability to in-
hibit responding to an auditory signal which indicates nonreinforcement.
In contrast, pregnant rats treated with vitamin A during the mid-fetal
period (days 17 and 18 of gestation) produced offspring which had no re-
*
tardation in growth or in brain size, but which had a possible motor
deficit affecting the motor coordination that resulted in slower rates of
response than control animals to auditory stimuli.
As Coyle, Wayner, and Singer indicated (1976), however, behavioral
anomalies can also be induced in the offspring during organogenesis. They
*
cite Hoffeld and Webster (1965) and Mura±^1966), who found that when
chlorpromazine was administered during early pregnancy maze learning was
impaired, but it had no observable effects when applied late in pregnancy.
Therefore, as both Coyle, Wayner, and Singer (1976) and Hutchings and
Gaston (1974) indicate, further investigations are needed before the most
»
vulnerable prenatal developmental stage for the onset of behavioral dys-
functions can be determined. It should also be mentioned that methods
for assessing behavioral effects are not well established.
5.4.4 Conclusions
Although it is generally recognized that organogenesis is the most
sensitive prenatal developmental period to the action of teratogenic
chemicals, another factor must be considered when testing a chemical for
teratogenicity. That is the possibility of the test chemical inducing
behavioral teratogenic effects which with some chemicals occurs when
-------
5-47
administration is during the organogenesis period and with others when
administration is during the developmental period following organogenesis
known as histogenesis. It should be stated, however, that test methods
for assessing behavioral effects are not well established.
5.5 SPECIES COMPARISONS
^
5.5.1 Introduction
As will be shown, the selection of the proper species or strain for
the testing of a chemical for teratogenicity can determine whether or not
the chemical would be considered a teratogen. The large numbers of animals
that are necessary for toxicological research usually dictates that the test
species be easily available and economical^ The following sections will con-
sider some general aspects of species selection, briefly discuss the advantages
and disadvantages of the rat, mouse, hamster, rabbit, monkey, and other less
commonly used species as models for extrapolation to man, and compare the
response of three .known human teratpgens — aminopterin, methotrexate, and
thalidomide — with the response in animals.
5.5.2 General Aspects
No ideal species has been identified with regard to extrapolating
experimental results from teratogenic 'tests in animals to man (NAS, 1977;
Schardein, 1976; Palmer, 1978). That is, there is no one species which
fits all the criteria for an ideal test animal as listed by Wilson (1975):
(1) absorbs, metabolizes, and eliminates test substances as does man,
(2) transmits test substances and their metabolites across the placenta
as does man, (3) has embryos and fetuses with developmental and metabolic
-------
5-48
patterns similar to those of man, (4) is easily bred and has large lit-
ters and a short gestation period, (5) is inexpensively maintained under
laboratory conditions, and (6) does not bite, scratch, kick, howl, or
squeal. The problem of choosing the most appropriate species is further
complicated when the test chemical has no record of human administration
from which to draw a comparison. Thus, with little scientific data
^
available, the choice of species is often on the basis of availability,
economic feasibility, and ease of management (Brown, 1963, as cited by
Palmer, 1978).
Kalter (1968) writes that there are three different types of inter-
specific and intraspecific susceptibility: (1) an agent teratogenic in
/•
some species or groups of animals may be completely or almost completely
without teratogenic effect in others, (2) a teratogen may produce similar
defects in various species, stocks, or strains of animals but with vary-
ing susceptibility (frequency) between one strain and another or between
one species and another, and (3) a teratogen may induce one or more abnor-
»
malities in some species or groups but have entirely or somewhat different
effects in others. Kalter indicates that these, however, are not mutually
exclusive categories and certain situations may contain features of more
than one of the three. These differences are illustrated in Tables 5.11
and 5.12 and also in the following sections.
The rat followed by the mouse and the rabbit are the most commonly
used laboratory animals for the teratogenic screening of chemical agents
(Palmer, 1978; World Health Organization, 1967). Further substantiation
oE this fact comes by examination of the holdings of the Environmental
-------
Table 5.11. Species susceptibility to drugs
Drug
Aspirin
Azathioprine
Cortisone
Demecolcine
Ethyndiol
Meclizine
Methylprednisolone
Perphenazine
Dosage
* (mg/kg)
and route
500 P0a
10 PO
5 or 10 IM6
1.5 IPC or SCd
1 PO or SC
125 PO
1 IM or SC
25 PO
Malformed species (percent incidence)
Mouse
31
0
79
23
30
0
92
100
Rat
0
0
0
0
92
0
98
Rabbit Others
0 (monkey)
0 (guinea pig)
57
50
3 0 (monkey)
0
0
0
0
,PO — per os (oral).
IM — intramuscular.
,IP — intraperitoneal.
SC — subcutaneous.
Source: Adapted from Schardein, 1976, Table 2-8, p. 21.
from several sources.
Data collected
-------
Table 5.12. Thalidomide teratogenesis In primates
Species
Man
Cynomolgous monkey
Baboon
Rhesus monkey
Bushbaby
Japanese monkey
Stump-tailed monkey
Marmoset
Bonnet monkey
Teratogenic
dose
(mg/kg) ,
oral route
0.5 to 1.0
10
5
12 to 19
20
20
5 to 10
45
5 to 30
Gestation
days
treated
20 to 36
22 to 32
18 to 44
24 to 26,
27, or 30
16 to 30
24 to 26
24 to 30
25 to 35
24 to 29 or
41 to 44
Limbs
Limbs
Limbs
Limbs
Defect
(80%), ear (20%)
(67%), teratomas (33%)
and tail (40%)
(100%)
Limbs (100%), tail (17%),
central nervous system (17%)
Limbs
Limbs
Limbs
(100%), tail (20%)
, ear, and jaw (100%)
(45%), visceral (52%)
i
Source: Adapted from Schardein,
several sources.
1976, Table 2-11, p. 25. Data collected from
Ln
O
-------
5-51
Teratology Information Center of the Oak Ridge National Laboratory. There
are several thousand papers on various aspects of teratogenic testing of
which approximately 3800 used the rat as a test animal compared to approx-
imately 2350 for the mouse, 935 for the rabbit, 260 for the hamster, and
255 for the nonhuman primate. In addition, nearly 3200 papers discuss
teratogens and teratogenicity in relation to humans.
^
The holdings of the Environmental Teratology Information Center and
the NIOSH Registry of the Toxic Effects of Chemical Substances data book
were used to show a significant number of chemicals that have been tested
for teratogenicity in the rat, mouse, monkey, hamster, rabbit, and human
(Table 5.13). Chemicals for which teratogenicity has been established in
those species are indicated. The data provided by the Environmental Tera-
tology Information Center showed a species by species account of which
chemicals had been tested for teratogenicity. However, no information
was available on whether the tests were positive or negative without read-
ing thousands of papers. Therefore, the NIOSH data book was used because
»
it does identify, on a species by species basis, those chemicals which are
teratogenic. Since two distinct information sources which were not designed
to necessarily complement each other were used to create Table 5.13, the
absence of a positive identification does not imply that a chemical is
conclusively not a teratogen but only that the NIOSH document does not
record it as being teratogenic. Also, the information in the NIOSH docu-
ment sometimes represents only one published paper and thus the results may
not be corroborated.
-------
5-52
Table 5.13. Chemicals tested for teratogenicity
Species
Teratogen
Rabbit Rat Mouse Hamster Human Monkey
AC 3092
Acetazolamide
Acetazolamide sodium
Acetohydroxamic acid
Af-Acetyl-2-aminofluorene
Acetylsalicylic acid
Acth
Actinomycln D
Adenine
Adrenalin
Acrylic acid
Aflatoxin Bj
Afridol blue
Alcohol
Aldrin
Allylestrenol
Amaranth
Amethopterln
2-Amino-l,3,4-thiadlazole
hydrochloride
2-Amino-6-(l'methyl-4'-
nitro-5'-imidazolyl)-
mercaptopiirine
4-Aminopterolyglutamic
acid
Aminoacetonitrile
Aminoacetonitrile sulfate
6-Arainonicotinaraide
Aminophenurobutane
Aminophylline
B-Aminopropionitrile
S-Aminopropionitrile
fumarate
Aminopterin
Amphetamine sulfate, D
Androgen
Androstenedione
Androsterone
Angiotonin
X(+)
X X(+) X
XXX
X
X
X
X
X /•
x(-t-) x(+)
X<+) X
X(+)
X(-t-)
X
X
X(+)
-------
5-53
Table 5.13 (continued)
Species
Teratogen Rabbit Rat Mouse Hamster Human Monkey
Apholace
Aracytidine
Arsenic
Aspartlc acid X
Aspirin X(+) X(+) X ' X X X
8-Azaguanine X X(+) X
Azaserine X X(+) X
6-Azauridine X(+) X XX
Azoe thane
Azoxyethane
Bamifyllin
Basfungin
Benzene
Benzoctamine hydrochloride
3,4-Benzpyrene X(#) X(+) X
Betamethasone X X(+) X(+) X
Bradykinin
Bredinin
1-3-Bis(2-chlorethyl)-l-
nitrosoures X X(+)
Bis(beta-chlorethyl)
methylamine
5-Bromo-2'-deoxyuridine
5-Bromo-l-(2-deoxy-beta-D-
ribofuranosyl)-5-fluoro-
6-methoxy hydrouracil
5-Bromodeoxyuridine X(+) X(-f)
2-Bromo-D-lysergic acid
diethylamide
9-Butyl-6-mercaptopurine
Butyl carbobutoxymethyl
phthalate
ff-Butyl methacrylate
Busulfan X X(+)
BW 50-63 X
BW 57-323H X(+)
2-see-Bu tyl-4,6-dinitrophenol
BZ 55 X(+)
Cadmium chloride X X(+) X(+) X
Cadmium sulfate X X(-l-) X(+)
-------
5-54
Table 5.13 (continued)
Species
Teratogen
Caffeine
Calcium fluoride
Cannabis
Captan
Carbaryl
Carbomazepine
Carbon dioxide
Carbon tetrachloride
CarbuCamide
Rabbit Rat Mouse
x xc+> x(+)
x(+)
X(+) X(+)
X(+) X(+) X
X X(+) X -
X(+)
X(+) X(+) X
X X(+) X
X X(+) X
Hamster Human
X
X(+) X
X(+)
X X
X
X
X
X
Monkey
X
Celestone
Ce tyItrimechylammonium
bromide
Cephalorldine
Chlorambucil
Chlorocholine chloride
Chloramiphene
Chlorcycllzine
Chlorocyclizine hydrochloride
Chlordiazepoxide
Chlorldine
Chlormadinone
4-Chloro-2-methylphenoxy-
acctic acid
l-p-Chlorobenzensulfonyl)-
3-propylurea
Chlorocholine chloride
5-Chlorodeoxyuridine
l-(2-Chloroethyl)-3-
cyclohexyl-1-nitrosourea
Chloroform
5-(4'-chloropheny1)-2,4-
d iamino-6-e thyIpyrimid ine
Chlorophos
Chloroquine
Chloropropamide
Chromomycin A3
Chloropromazlne hydrochloride
Chondroitin sulfate
Clomiphene
s
X
X
X
X
X(-t-)
X
x(+)
X(+)
X
X(+)
X
X(+)
X(-t-)
X(H-)
X
X
X
-------
5-55
Teratogen
Clonazepam
Colcemid
Colchicine
Congo red
Comital
Compazine
Table 5.13 (continued) s
Species
Rabbit Rat Mouse Hamster Human
xw
X X X XX
XXX X(+) X
x x(+)
x(+) x(-t-) x
X(+) X
Monkey
X
Corpus luteum hormone
Corticosterone
Corticosterone acetate
Cortisol
Cortisol acetate
Cortisone acetate
C-quens
Cyclocytidine
Cyclopamine
Cyclophosphamide
Cyproterone acetate
Cytarabine
Cytoxyl alcohol
Cytoxyl amine
2,4-D
D 860
2,4-D butyl'ester
2,4-D diraethylamine salt
Daraprim
Demecolcin
iV-Deraethyldiazepam
Demeton
DEN
2-Deoxyglucose
ff-Desacetyl methylcolchicine
Deserpidine
Dexamethasone
Dexamphetamine sulfate
Di(2-ethylhexyl)phthalate
2,4-Diamino-5-p-chloro-
pheny1-6-ethyIpyr imidine
Dianabol
X
X
X
X
X
X
X
X
X
X
X
X(+)
X
X
-------
5-56
Table 5.13 (continued)
Species
Teratogen
Dlazepam
Diazlnon
Rabbit
X
X
Rat
X
X
Mouse Hamster
X(+) X
X(+) X
Human Monkey
X X
-------
5-57
Table 5.13 (continued)
Species
Teratogen
2-a, 3-a-Epithio-
5-o-androstan-17-B-ol
Ergotamine tartrate
17-B-Estradiol
Estradiol benzoate
Estrone
Ethamoxytripheto1
Ethanol
Ethionine, dl
Ethosuxlmide
Ethyl alcohol
Ethyl urethan
Ethoxzolamide
Ethyl ff-hydroxycarbamate
Ethyl ff-methylcarbamate
9-Ethyl-6-mercaptopurine
2-Ethylamine-1,3,4-
thiadiazole
Ethylene thiourea
Ethyl parathion
Ethyltrichlorophon
17-a-Ethynyl-estr-5(10)-
ene-3-o, 17-0 dlol
17-a-Ethynyl-estr-5(10)-
ene-3-B, 17-6 diol
Evans blue
FD&C red No. 2
Fenthion
Firemaster BP-6
5-Fluoro-2'-deoxycytidine
5-Fluoro-2-Deoxyuridine
5-Fluorocytosine
5-Fluorodeoxycytidine
Rabbit Rat Mouse ' Hamster Human Monkey
EDTA
EM 12
EMS
Endoxan
Endrin
Epinephrine
x(+)
X(+) X(+) X
X(+) X
X(+) X(+)
X X(-f) X
XX XX
X(+)
(+) x
(+) X X
X
X
X
X
X
X
X
X(+)
X
X
X(+)
X
X
X
X
X
X
X
X(+)
X(+)
X(+)
-------
5-58
Table 5.13 (continued)
Species
Teratogen
Rabbit Rat Mouse Hamster Human Monkey
5-Fluorodeoxyuridine
5-Fluor o-,V-(4) -methy 1-2' -
deoxycytidine
5-Fluoroorotic acid
5-Fluorouracil X
5-Fluorouridine
Folliculin X
Folpet X
Formamlde X
Galactose
Cradenal X(+)
Gestanon
Griseofulvin
Hadacidin
Haloperidol X
Halothane X
Heliotrine
Hercules 14503
Hexachlorobenzene X •
Hexachlorodlbenzo-p-dioxin
Hexachlorophene X
Hycanthone methanesulfonate X(+)
Hydantoin • X
Hydrazomethane X(+)
Hydrocortlsone X
Hydrocortisone-21-acetate
Hydroxylamine
B-Hydroxyethylcarbamate
7-Hydroxymethyl-12-methyl-
benz(a)anthracene
5-Hydroxytryptamine X <•
5-Hydroxytryptamine creatinine
sulfate
llydroxyurea X
//-Hydroxyurethan
Hydroxyzine
Hydroxyzine hydrochloride
Hydroxyzine pamoacc
X
X
X
X
X
X(-l-)
X
X
X(+)
X
X
X
X
X
X
x(-t-)
X(H-)
-------
5-59
Table 5.13 (continued)
Species
Teratogen
Hypoglycine-A
Hystep
ICRF-159
Idoxuridine
Imidan
Imipramine
Imipramine hydrochloride
Imipramine-ff-oxide hydro-
chloride
Indium nitrate
5-Iodo-2 ' -deoxyuridine
lodoacetic acid
5-Iododeoxyuridine
Isobutyl methacrylate
Isodecyl methacrylate
Isopropyl methanesulf onate
Jervine
Lactose
Lead acetate
Lead chloride
Leucine
Lithium
Lithium carbonate
Lithium carmine
Lithium chloride
LSD
Maneb
Marpharsen
Medroxyprogesterone acetate
Meprobamate
6-Mercaptopurine
Mercaptopurine-3-ff-oxide
6-Mercaptopurine riboslde
Mescaline
Methadone
Metepa
Methaqualone
Methedrine
Rabbit Rat Mouse ' Hamster
X X(+)
X(+) X(+)
X X(+)
X X(+)
X X(+)
X(+) X
X X(+)
X(+)
X(+)
X(+)
X(+)
x(£)
X(+)
X
X X(+)
X
X
X(+)
x(+)
X X(+)
X
X(+)
X(+)
X
X(+) X
X X
X / X(+)
X(+)
x(+)
X
X X(+)
X
X X(+)
x(+)
X(+)
x(+)
X(+)
*
X
x(+)
X(+)
X(+)
x(+)
f
X
X X(+)
X
X X(-t-)
X X(+)
X
x(+)
X(+)
X(+)
X X(+)
X
X
X -'
X(+)
X
X
x(+)
X X
x(+)
Human Monkey
X
X X
X
X
X X
X X
X
X
X
X
X
X
X
-------
5-60
Table 5.13 (continued)
Species
Teratogen
Rabbit Rat Mouse Hamster Human Monkey
Methotrexate
l-Methyl-2-benzylhydrazine
4-Methyl-2-thiouracil
2-Methyl-3-(0-tolyl)-4(3H)-
qu inazolinone
6-a-Methyl-17-acetoxy
progesterone
6-a-Methyl-17-a-
hydroxyprogesterone acetate
Methyl ethyl ketone
Methyl parathion
Methyl salicylate
Methylamphetamine
Methylazoxymethanol
Methylazoxymethanol acetate
Methylene dimethanesulfonate
4-Methylethylenethiourea
.V-Me t hy If ormamid e
Methylmercury chloride
Methylmercury dicyandlamide
Methyl-4-phthalimido-glutara-
mate, dl
Methylprednisolone
Methyltestosterone
Methylthiouracil
Metiaplne
Mlraosine
Mlrex
Misulban
Mitomycln C
Monomethylformamide
MSG
Mustlne hydrochloride
Myleran
Mysoline
l-Naphthyl-W-methyl carbamate
Hatulan
Niagara blue 2B
Niagara blue 4B
X
X
X
X
X(-t-)
X(-t-)
X
X(+)
X
X
X
X
X(+)
X(-t-)
X(+)
XOO
X
X
X(+)
-------
5-61
Table 5.13 (continued)
Species
Teratogen
Niagara sky blue
Nicotine
Nitrogen mustard
Rabbit
X
X
Rat
xw
X
X(+)
Mouse Hamster Human Monkey
X(+) X X
X X
Nitrosoethylanlline
Nitrosomethylaniline
Nitrosopropylurea
Nitrous oxide
Norepinephrine X
Norethynodrel X
Ochratoxin A
a-Olefin sulfonate
Oranil
Orphenadrine hydrochloride
Oxophenarsine hydrochloride
Paraquat
Parathion
Fentachlorobenzene
Pentachloronitrobenzene
Phaltan
Phenatine
Phenobarbital
Phenobarbital sodium X
l-Phenyl-3,3-dimethyltriazene
Phenylmercuric acetate
Phenytoin
Phosphoramide mustard
W-Phthaloyl-t-aspartic acid
Polybrominated biphenyls
Polychlorocamphene
Potassium oxonate
Prednisolone X
Primaclone
Primidone
Procarbazine
Prochlorperazine X
Progesterone X
ff-Propyl carbamate
X
X
X
X(+)
X
X
X
X
X
X
X
-------
5-62
Table 5.13 (continued)
Species
Teracogen
Prostaglandin E2
Provera
Pyrlmethamine
Quinine
Quinine sulfate
Rabbit
X
X(+)
X
X
Rat
X
X
X
X
X
Mouse Hamster Human
X(+) X X
X
X X(+) X
X
* X
Monkey
X
Red Mo. 2 X
Rcsperpine X
Retinoic acid
Retinol
Retinyl acetate
Rlfampicin X
Rubldomycin X
Rubratoxin 3
Salicylic Acid
Saline X
Sarkontycin
Serotonin X
Serotonin creatinine sulfate
Sevin
Sodium acetazolamide
Sodium arsenate
Sodium arsenite
Sodium barbital X
Sodium chloride X
Sodium fluoride X
Sodium naphthionate
Sodium pentobarbital
Sodium retinoate
Sodium salicylate X
Solanlne X
Solantyl
Solasodine
Stilbestrol
Strcpconigrin X
SU-13320
Sulpyrln
2,4.5-T X
,x
X
X
X
X
X
X
X(-l-)
X
X
X(-t-)
X X
X
X
-------
5-63
Table 5.13 (continued)
Species
Teratogen
Rabbit Rat Mouse Hamster Human Monkey
2,4,5-T butyl ester
2,4,5-T isooctyl ester
Tegretol
TEM X
Testosterone
Testosterone enanthate
Testosterone propionate X
2,3,7,8-Tetrachlorodibenzo-
p-dioxin X
Tetrahydrophthalimide X
1,1,3,3-Tetramethylurea X
Thalidomide
Thalidomide (-)
Thalidomide (+ and -)
Thalidomide (+)
Theophylline
Thioguanine
Thioguanine riboside
Thio-tepa X
Thiram X
Tofranil
Tolbutamide
TOK
Toxaphene
2'3'5'-Triacetyl-6-
azauridine
Triamcinolone X
Triamcinolone acetonide X
Trichlorfon
Trifluperidol
Trimethylene dimethane—
sulfonace
Triparanol
Triton W.R.1339 X
Trypan blue X
Uracil mustard
Urethan
Valium
s
x(-t-)
X(-l-)
X
X(-l-)
X(-t-) X
X
X
X
X
X
X
X
X
-------
5-64
Teratogen
Velban
Vinblastlne
Vincrlstine
Vitamin A
Vitamin A acetate
Table 5.13 (continued)
Species
Rabbit Rat Mouse Hamster
X X(+)
X(+) X(+) X X(+)
X X(+) X X(+)
X X(+) X(-l-) X
x x(-t-) xr-t-)
Human
X
X
X
X
Monkey
X
X
X
Vitamin A acid
Vitamin A palmitate X X X X(-l-)
WU 385 X(+)
Zarontin X(+)
Zearalenone X(-f-)
Zlneb
x represents substance tested for teratogenicity but does not imply posi-
tive or negative data; '
+ indicates positive results.
Source: Data compiled from various sources.
-------
5-65
5.5.3 Rat
Advantages of using the rat as a test animal for teratogenic testing
include (1) short duration of pregnancy (21 to 22 days), (2) high fertil-
ity rate, (3) large litters and a relatively good resistance to the toxic
effects of most drugs, (4) a fairly good developmental stability, and (5)
a low spontaneous rate of major malformations (1 per 1000 fetuses, 0.001%)
(Tuchmann-Duplessis, 1972; Palmer, 1978). The rat is also easily handled
and economically maintained, and test materials can be administered by a
wide variety of routes (Palmer, 1978). The main limitation of the rat is
its poor teratogenic susceptibility to drugs such as cortisone (Thompson
and Schweisthal, 1969; Hansson and Angervall, 1966), azathioprine
(Tuchmann-Duplessis and Mercier-Parot, 19-6r6) , and thalidomide (Somers,
1963). Thus, definite conclusions based only on tests with rats could be
misleading (Tuchmann-Duplessis, 1972).
5.5.4 Mouse
» .
The advantages listed for the rat (Sect. 5.5.3) also apply to the
mouse with the addition that the mouse is even more economical, has a
shorter gestation period (18 to 19 days), has a wide variety of defined
inbred strains for special studies, and is more susceptible than the rat
to some teratogens (Palmer, 1968; Tuchmann-Duplessis, 1972). Disadvantages
include (1) small size of the fetus and consequent difficulty in examina-
tion of malformations; (2) arrangement of the malformations in clusters,
which creates difficulty in assessment; (3) higher spontaneous malformation
rate than the rat (0.5% in the Swiss albino colony of Tuchmann-Duplessis);
and (4) resorption rates much higher than in the rat, thus necessitating
-------
5-66
a large control group (Palmer, 1978; Tuchmann-Duplessis, 1972). The mouse
is also known to respond differently than the rat to some drugs such as
cortisone.
With regard to the human teratogen thalidomide (see also Sect.
5.5.9.3), mice embryos are relatively insensitive. This is evident in
a study by Somers (1963) in which he found that oral administration of
^
doses up to 400 mg/kg throughout pregnancy did not reduce the number of
newborn mice or their ability to survive weaning.
5.5.5 Rabbit
The rabbit was the first laboratory animal shown to be susceptible
^
to the human teratogen thalidomide (Somers,, 1962) and has since been
considered by many biologists to be one of the most favorable animals for
teratogenic studies (Tuchmann-Duplessis, 1972). The susceptibility of the
rabbit to thalidomide was also reported by Somers (1963), who produced
limb defects in the offspring by administering 150 mg/kg to pregnant rab-
*
bits on day 8 to day 16 of gestation and by Shepherd (1976), who states
that limb defects in New Zealand rabbits were observed after administration
of 250 mg/kg on days 8 to 10 of pregnancy.
Advantages of using the rabbit include the larger size of the rabbit
fetus compared to the rat or mouse (Palmer, 1978), the ability of the rab-
bit to show a variety of spontaneous malformations which suggest that
this species would be more susceptible than some species to a variety of
teratogenic actions (Palmer, 1968, as cited by Palmer, 1978), and for com-
parison it is physiologically somewhat different from the rat and the
mouse (Tuchmann-Duplessis, 1972). The necessity of having large control
-------
5-67
groups, a higher spontaneous malformation rate (1.7% for a five-year pe-
riod) (Tuchmann-Duplessis, 1972), and the fact of the dependence on gut
flora for nutrition, which would prevent the testing of antibiotics and
would make incorporation into the diet a hazardous and inaccurate means
of administering test compounds (Palmer, 1978), are possible disadvantages
of using the rabbit as a model for testing compounds for teratogenicity.
^
5.5.6 Hamster
Palmer (1978) reports that the hamster is mainly a competitor for
the mouse in animal studies; it is almost as economical to maintain and
is reputed to be more stable genetically. From the available information,
*
it appears that the hamster has several advantages that make it worthy of
s
consideration. Among these are unique reproductive features such as the
ease of obtaining accurately timed matings, large litter size, and short
gestation period (16 days) which, according to Ferm (1967), makes the
hamster a potentially valuable animal for developmental and teratogenic
studies. Palmer (1978), however, lists several disadvantages of using
the hamster rather than the mouse: (1) aggressive behavior generally
necessitates individual caging, (2) achieving precisely timed mating al-
though not difficult is not so convenient as with1the mouse, (3) intra-
venous injection is more difficult than in the mouse (Ferm [1967] does
demonstrate a method requiring anesthesia whereby the injection is made
utilizing the lingual vein), and (4) as with the rabbit, the hamster's
gut flora renders this species susceptible to change in the diet and to
antibodies. Tuchmann-Duplessis (1972) concludes that from available data
the hamster does not have a definite advantage in the teratogenic screen-
ing of drugs.
-------
5-68
5.5.7 Xonhuman Primates
Much has been written concerning the use of nonhuman primates as
animal models in the teratological testing of chemicals (Wilson, 1968,
1971a, 1971&, 1978; Siddall, 1978; Tanimura, 1972; Hendrickx, 1972;
Poswillo, Hamilton, and Sopher, 1972). The'rhesus monkey (Maoaca mulatto)
has been the most popular nonhuman primate test species according to
Wilson (1978), but teratological studies have been performed using other
nonhuman primates such as the cotton-eared marmoset (Siddall, 1978;
Poswillo, Hamilton, and Sopher, 1972) and the baboon (Hendrickx, 1972).
One of the primary advantages of using nonhuman primates in the tera-
tological screening of chemicals is that they are similar in several res-
pects to man. Wilson (1978), in reviewing several papers including many
of his own, reports that: (1) in some instances the metabolism of drugs
in man approximates that of other higher primates; (2) the reproductive
physiology of Old World monkeys (rhesus monkeys and baboons) is known to
closely resemble that of man, particularly the menstrual cycle, spermato-
genesis, and parturition; (3) the placental structure of man and nonhuman
primates appears to be quite similar, although the similarities of function
during the critical periods of gestation have yet to be fully established;
(4) the anatomic and temporal aspects of embryonic development between the
f
higher nonhuman primates and man is strikingly similar; and (5) the well-
known similarity in response to thalidomide (the comparative response to
other agents, however, to be mentioned in the following section, has been
variable). Wilson (1978) cites the scarcity; the difficulties in handling,
especially the unpredictability of behavior and general susceptibility to
-------
5-69
infections; and the low fecundity as disadvantages for using nonhuman pri-
mates for teratological testing of drugs. Two other disadvantages of non-
human primates are the expense and the long gestation period.
It should be mentioned that prosimians, and to a certain extent New
World monkeys such as marmosets, have not been used extensively for tera-
tological evaluations of chemicals. The greater bush baby (.Galago eras-
^
siaaudata), a prosimian, was found by Hendrickx (1972) to give a negative
response to thalidomide and has since received little attention. The
marmoset has shown similar responses as man to thalidomide (Siddall, 1978;
Poswillo, Hamilton, and Sopher, 1972), but the fact that they do not men-
struate, which causes difficulty in following the reproductive cycle,
^
possibly accounts for their sparse use (Wilson, 1978). Because of the
advantages afforded by their smaller size, however, Wilson (1978) recom-
mends further testing of the suitability of New World monkeys and
prosimians as animal models in teratology.
5.5.8 (Other Species
Other animals such as dogs, cats, and swine have been used as test
species for teratological testing but not to the extent of the previously
mentioned species.
5.5.8.1 Dog — Palmer (1978) writes that the primary advantage of the
dog is its concurrent use as a nonrodent species for other toxicity tests
which may provide further information of the test material such as phar-
macokinetics. Earl, Miller, and Van Loon (1973) tested the beagle with the
known human teratogens thalidomide and aminopterin as well as with methyl
mercuric chloride, hydroxyzine, and hydroxy.urea. They found that (1) the
-------
5-70
dog does respond to known human teratogens, but not in a classical manner
to thalidomide, (2) the dog does not appear to be a sensitive indicator of
these compounds, and (3) the dog offers little advantage over other labora-
tory animals. In addition the frequency of estrus (twice a year) and high
cost of maintenance are also disadvantages.
5.5.8.2 Cat — The cat has been used infrequently in teratology stud-
-------
5-71
cited by Khera and Iverson, 1978). In a follow-up study, Iverson, Khera,
and Hierlihy (1980) suggested that the reason for the absence of teratogenic
effects in the cat was the ability of the cat to extensively metabolize
ethylenethiourea to its S-methyl derivative; examination of rat urine showed
no evidence of the S-methyl derivative.
5.5.8.3 Pig — Palmer (1968) and Tuchmann-Duplessis (1972) list several
^
advantages of using the pig: (1) easily available and highly prolific com-
pared to other nonrodent species, (2) embryology and genetics are well known,
(3) pregnancy can be obtained year round, and (4) incorporation of the test
material into the diet is easily and accurately accomplished because of the
pig's voracious eating habits. These same authors cite disadvantages of
f
using the pig as the fact that large amounts of the test material are required
due to the size of the pig and that floor space requirements are excessive.
Earl, Miller, and Van Loon (1973) observed malformed offspring in the minia-
ture swine which had been exposed to thalidomide, hydroxyurea, and aminopterin.
These researchers conclude that although the reaction to thalidomide was not
i
the classical response, miniature swine offer certain advantages such as low
incidence of spontaneous malformations, but they recognize that more data are
necessary before their impact on teratogenic studies can be determined.
5.5.9 Human Teratogenic Chemicals Tested in Animal Models
Table 5.14 shows 13 chemicals or chemical classes that are thought to be
taratogenic in humans. Three of these, aminopterin, methotrexate, and
thalidomide, were selected to compare the teratogenic response between humans
and several animal species; the data are summarized in Tables 5.15, 5.16, and
5.17.
-------
5-72
Table 5.14.: Chemical agents known to be
teratogenic in man
Aminopterin Thalidoraide
Antithyroid drugs Warfarin
Busulfan Alcohol
Organic mercury Androgens '
PCBs Uiethylstilbestrol
Diphenylhydantoin Methotrexate
Tetracycline
Source: Adapted from Miller, 1977, Tables 1
and 3, pp. 472 and 473.
-------
Table 5.15. Aminopterln action in various species
Animal
Man
Rat
Species/
strain
nose
Effects
Reference
Mouse ICg-DUB
Histar and
others
Histar
Monkey Rhesus
Rhesus and
cynomolgus
Rabbit Not given
29 mg over 10 days
20 rag over several
weeks
12 mg over 12 days
25 mg on day 11
or day 12 of
gestation
.0.2, 0.1, 0.075, and
0.05 mg/kg single
injections
Not given
0.12 mg/kg orally
on days 24, 25,
and. 26 of
gestation
0.1-1.0 kg/day
2 mg/kg
Excessively large head; nasal bridge
broad and flattened; eyes widely
separated; malformed ears; mandib-
ular hypoplasia; posterior cleft
palate; absence of parietal bones
in skull
Multiple skull anomalies; left talipes
equinovarus
Numerous head abnormalities — soft
skull, no ossification of parietal
bones, eyes wide apart, broad nasal
bridge, posterior cleft palate, and
others; large hands, other anomalies
also described
Short or small limbs; hemimelia; severe
ectrodactyly (absence of one or more
fingers or toes)
Injections on 7th and 10th day effec-
tive — embryo lethality
No teratogenicity — fetuses resorbed
with high closes
No injury to offspring
Emerson, 1962
Meltzer, 1956
Warkany, Beaudry,
and Harnsteln,
1959
Kochhar, 1975
Baranov, 1965
Murphy, 1962
Tanimura, 1972
Ul
I
Two aborted and one normal fetus, but Wilson, 1969
no teratogenicity
No injury to six-day-old blastocyst Hay, 1964
-------
Table 5.16. Methotrexate action In various species
Animal
Species/
strain
Dose
Effects
Reference
Man
Mouse
Rat
ICR
Wistar
Monkey
Rhesus
Rhesus
Rabbit
New Zealand
white
New Zealand
white
5 mg/day for
two months
2.5 mg/day for
five days
0.3 to 50.0
mg/kg on
day ten of
gestation
I to 50 mg/kg
0.2 mg/kg
0'.5 to 4.0
mg/kg
<3 to 12 mg/kg
9.6 mg/kg
19.2 mg/kg
Major abnormalities of skull of infant
Major anomalies — absence of frontal
bone, absent lambdoitl and coronal
sutures, multiple anomalous ribs,
unusual facitis, and absence of all
digits on right foot and all but one
on left foot
25 and 50 mg/kg produced congenital
defects, primarily cleft palate and
reduction of digits
Two fetuses — one had a bifid 13th rib
on the left side and the other showed
encephalocgle
103 embryos exposed — 64% resorptions
and 30% malformations
Five females delivered normal fetusfts
and one aborted
Most fetuses normal, three aborted and
one showed moderate gut rotation;
only 13 embryos exposed so trivial
teratogenicity might reflect small
sample size
50% fetal mortality and a 25% malforma-
tion rate in surviving fetuses
Multiple anomalies — facial clefts,
cleft palates, defects of fore and
hind limbs, etc.
Powell and Lkort,
1971
Milunsky, Graef, and
Gaynor, 1968
Skalko and Gold,
1974
Berry, 1971
Wilson, 1970, as
cited by Wilson,
1971*
Wilson, Fradkin, and
Hardman, 1968
Wilson, 1971b
Jordan, Terapane,
and Schumacher,
1970
Jordan, 1973
in
I
-------
Table 5.17. Thai Uomitle action In various
An In.11
Species/
strain
Dose
M.m
Mouse
Rat
Schofield
A, Call, Swiss
CF,. ICR, CS7,
CBA, SJL
Wistar
Sprague Dawley
Kffects
Reference
Charles River
Long Evans and
Dunning Fischer
Holtzmann
Not given
Mot exactly
known
400 rag/kg
throughout
pregnancy
31-250 rag/kg
per day
200 mg/kg
2 g/kg
0-400 mg/kg
20 and 50 mg
10 mg/kg per
day
150 rag/kg
25-500 mg per
day
Missing linbs; auricle or pinna of ear ttccreased
in st:•->.•; lesser deficiencies
Reports of several cases — hypuplasla ami Jul.isla
of the extremity or indlviilual bones, absence
and malformation of limbs; malformations of
other organs such as stenoses and malrotatlon
of the gastrointestinal tract and dysplaslas of
the external ears and eyes often associated
No malformed fetuses in albino mice
Skeletal malformation; open eye; enlarged skull;
curvature of back; kinky tail; phocomelia;
micromelia
No teratogenlc effecc but increased resorptions
in CF, strain
16 litters — two had abnormalities, complete
resorption in one, more than 502 resorption in
three, and normal offspring in ten; abnormali-
ties-consisted of stunting of extremities and
tails, absence of dorsal and lumbar vertebrae
and ribs, absence of digital bones, and curved
bones of the fore and rear extremities
Fetal resorptions but no abnormalities
520 young examined — 17 resorbed, 36 grossly mal-
formed (6.9%), 40 showed malformations after
clearing of fetuses, 76 total malformations
(14.6%); malformations included malrotation of
hind limbs, hamartoma of the palate with acces-
sory incisors, lack of a tall in one Instance,
and a subcutaneous cartilaginousvtype mass of
tissue from the roiddorsal region to the tail
Resorptions but not malformations
No apparent injury
More resorptions than controls; only one fetus
showed limb malformation: significant terato-
genic effect shown by missing sternebrae and
delayed ossification of the sternum; malfor-
mations different than humans
Smliliells. 1962
I'leilfer and Koseiiow,
19I>2; I.CM; and
Knapp, 1962
Somers, 1963
DiPaolo, Gatzek, and
Pickren, 1964
Fratta et al., 1965
Murphy, 1962
Wi
-vl
Ol
Somers, 1963
King and Kendrick,
1962
Delahunt, I.asscn,
and Rieser, 1966
Fratta, Sigg, and
.Maiorana, 1965
Dwornlk and Moore,
1965
-------
Table 5.17 (continued)
Animal
Hamster
Monkey
Species/
strain
Not given
Not given
Inbred and random
Rhesus and stump-
tailed Macaque
Bonnet (Hacaca
radiata)
Not given
Rose
8000 mg /kg
throughout
pregnancy
150 mg
350 rag/kg
per day
5 and 10
mg/kg per
day
10 and 30
mg/kg
Not given
Effects
Not ter«acogenlc
Not teratogenlc
Inbred lines showed 6.2% incidence of grossly
malformed fetuses — acranla or split cranium.
abnormal positioning of lugs, kinking of tail,
and cleft palate; random bred lines showed no
significant teratogenlcity
Severe deformities in most species — missing
digits, missing radii and ulnae, shortened
burner! , kinking and in some cases shortening
of tall
Temporal and mandibular bones malformed
External malformations similar to human thalido-
mide syndrome — enlarged head, missing limbs,
Reference
Somcrs, 1963
Fratta, Sigg, and
Maiorana, 1965
I'omhurger ct al.,
1965
Vondruska, Francher,
and Colandra, 1971
Hendrlckx and
Newman, 1972
Uelahunt, l.assen,
and Rieser, 1966
t_n
Baboon (Papio 5-300 mg/kg
cynocephalus),
bonnet (Hacaca
radiata), and
cynomolgus
(Ilaoaaa irus)
Rabbit New Zealand white Not given
and Dutch-Belted
New Zealand white 150 mg/kg
per day
New Zealand white 150 mg/kg
per day
phoconelia, etc.
Malformed fetuses — deformed limbs, splna bifida,
kinked tail, etc.
N
Low incidence of deformed appendages in New Zealand
whites; Increased incidence of limb defects in
Dutch-Belted — agenesis or hypogenesis of either
the ulna, radium, tibia, and/or fibula; absence
of some bones in limbs ,
Limb deformities in almost every litter
Sixty-seven percent of litters had deformed
fetuses — most predominant effects seen in limbs
(arthrogryposis, micromella, absence of digits),
but developmental failure of visceral organs
(kidneys, adrenals) and in bony structures also
observed
Hendrickx, 1970
Delahunt, 1965
Somers, 1963
Drobeck, Coulston,
and Cornelius,
1965
-------
5-77
5.5.9.1 Aminopterin — In addition to man, the folic acid antagonist
aminopterin (Table 5.15) is teratogenic in the mouse but not in the rat,
where embryolethality and resorptions were observed, or in the rhesus
(Macaaa. mulatto) and cynomolgus (Maoaaa ivus") monkeys. The one reference
concerning aminopterin toxicity in the rabbit reports no injury to a six-
day-old blastocyst, but the assumption from this data that no injury would
^
result to the fetus probably would not be very reliable.
5.5.9.2 Methotrexate — Methotrexate, also a folic acid antagonist
and a methyl derivative of aminopterin, is teratogenic to humans, mice,
rats, and rabbits but apparently not to monkeys, although abortions were
induced (Table 5.16). Examination of the data in Table 5.16 indicates
*
that (1) teratogenic defects in the man, njouse, rat, and rabbit are some-
what similar, especially with regard to rib and limb abnormalities; (2)
the rat showes a teratogenic response to methotrexate, but not to amino-
pterin (Table 5.15); and (3) the monkey seems to be resistant to the
teratogenic action of both methotrexate and aminopterin (Table 5.15).
i
Shalko and Gold (1974) point out, however, that no teratogenic effects
are produced in mice at doses that are teratogenic in humans, rats, and
rabbits and abortifacient in rhesus monkeys (0.3 to 10 mg/kg), thus sug-
gesting that the mouse embryo is more resistant to the embryotoxic effects
of methotrexate than any other mammalian species yet studied. The
minimum dose required to produce teratogenicity in the mouse was 25
mg/kg and is the same as that necessary for aminopterin to be terato-
genic (Table 5.15). In rabbits, methotrexate-induced abnormalities
increase as the dose increases, a fact that is related to metabolism
by the maternal system (Jordan, 1974). Jordan discovered that when
methotrexate is present in low concentrations, it is rapidly converted
-------
5-78
by the maternal enzyme aldehyde oxidase to a relatively inactive metabolite,
7-hydroxymethotrexate; at higher levels of methotrexate, the enzyme becomes
saturated and, consequently, more unmetabolized drug reaches the embryo.
5.5.9.3 Thalidomide — Table 5.17 indicates that human, monkey, and
rabbit embryos are highly susceptible to thalidomide but that mouse, rat,
and hamster embryos are not nearly as sensitive and show apparent strain
^
differences. Although both the monkey and the rabbit embryos exhibit
malformations which are similar to those observed in the human fetus,
especially with regard to limb defects, Delahunt (1965) concludes that the
thalidomide-induced abnormalities in man are much more similar to the monkey
than they are to the rabbit. Although Drobeck, Coulston, and Cornelius
(1965) do not disagree with Delahunt, they'do believe that the results
observed in the rabbit with thalidomide generally confirm what has been
reported in humans. They continue by stating that micromelia and phocomelia
in the human cases have been so popularized that the wide range of effects
that has actually.been observed has been overshadowed. This wide range
includes developmental failure of visceral organs such as the kidneys and
adrenals as well as developmental failure in bony structures which, according
to Drobeck and co-workers, is characterized quite well in the rabbit.
Detailed examination of the data on rats, hamsters, and mice in Table
5.17 illustrates the apparent strain differences in susceptibility to thalido-
mide. Fratta, Sigg, and Maiorana (1965) found no teratogenicity in mice
strains CFt, ICR, C,7, CBA, and SJL; DiPaolo, Gatzek, and Pickren (1964)
induced malformations in strain A, C3H, and Swiss. Similar results are seen
in hamsters where inbred lines are susceptible and random bred lines are not,
and in rats where the Sprague Dawley and Holtzmann (descended from Sprague
-------
5-79
Dawley) strains and one unidentified strain produced malformed young,
whereas the Wistar, Charles River, Long Evans, and Dunning Fischer strains
did not.
5.5.10 Conclusions
There is no ideal animal model for use in teratological research.
The monkey (primarily higher primates) has received considerable attention
in recent years, especially since the thalidomide syndrome appears to be
mimicked to a high degree. The monkey offers the advantage of having a
chorioallantoic placenta as does man in contrast to the inverted yolk sac
placenta of rodents and rabbits as well as having other anatomical and
f
physiological similarities to man (Sect. 5.5.7). Yet, studies using the
,x
folic acid antagonists methotrexate and aminopterin, known human teratogens,
have been unable to demonstrate any significant teratogenicity in the monkey,
although abortions have been induced. However, if a compound whose embryo-
toxicity is being tested was shown to have abortifacient action but not
necessaVily teratogenic potential, 'the use of the compound would probably
have similar restrictions for women of child-bearing age. Thus, the decision
on potential use of this compound by pregnant women would probably be the
same whether it induced malformations or abortions. Using the criteria of
embryotoxicity (embryolethality and teratogenicity), Wilson (1971), by citing
several authors, shows that the results between the effects induced by several
agents in man and the rhesus monkey (chemical and viral) are comparable.
Wilson acknowledges that before the rhesus monkey can be considered more reli-
able than other laboratory animals for anticipating embryotoxicity in man,
more detailed comparative information on drug metabolism and distribution
between laboratory animals is necessary.
-------
5-80
Rats, mice, and rabbits have and will probably continue to be used
more frequently than the monkey as laboratory test animals primarily due
to availability, ease of handling, and economy. Rats and mice have been
the most commonly used, but use of the rabbit has increased since the early
1960s because the rabbit was the first laboratory animal shown to be sus-
ceptible to thalidomide teratogenicity.
^
All chemicals would, of course, not require the same degree of
teratogenic testing. Providing that the potential human exposure can
be assessed, compounds for which human exposure, especially to pregnant
women, is considered a probability should receive more intensive terato-
logical testing than those chemicals for which human exposure is a remote
possibility. In the same vein, Wilson (19?8) believes that despite their
limited availability, testing in the higher primates such as macaques and
baboons is essential for those drugs which are needed for therapeutic
purposes during human pregnancy and, perhaps, for the environmental chem-
icals which are likely inadvertently to involve heavy exposure in women
t
prior to the diagnosis of pregnancy. Wilson continues by stating that
potential teratogenic effects to the central nervous system can be ade-
quately tested only in the higher primates which have at least a roughly
comparable range of mental and nervous activities as does man.
-------
5-81
SECTION 5
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/'
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-------
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»
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^
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^
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*
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»
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^
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^
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-------
6. CHRONIC TOXICITY AND CARCINOGENICITY TESTING
6.1 INTRODUCTION
Tests designed to evaluate chronic toxicity and/or 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; length of test duration,; 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 determine a maximum
"no-observed-adverse-effect" dose level (Friedman, 1973). In contrast,
for carcinogenicity tests, tumor1 formation and increased tumor incidence
are th^ major end.points. Tumor irjcidence, tumor latency, and in some
cases, tumor multiplicity are the parameters used to determine sensitivity
of the test animals to the carcinogenic challenge. Also, short-term in
vivo tests can 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 only
pertain to each individual test. Additionally, some topics usually
*In this report, the general term "tumor" will apply to either
benign or malignant neoplasms.
6-1
-------
6-2
considered part of chronic toxicity testing, such as reproductive and
behavioral effects, neurotoxicity, and teratogenicity are discussed
elsewhere in this report or later volumes and are not included in this
section.
6.2 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.
6.2.1 Species
Since the usual purpose of chronic toxicity and carcinogenicity test-
ing is to predict adverse effects of chemicals in man, test animals should
be chosen that closely resemble man with respect to absorption, distribu-
tion, metabolism, excretion, and target site effect of the toxic substance
»
(Weil, 1972). Unless information to the contrary is available from pre-
vious 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
-------
6-3
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 are 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 methaemoglobinaemia, pigtail monkeys for methanol toxicity,
and dogs or rats for cholinesterase inhibition studies (Fancher, 1978).
6.2.1.1 Chronic Toxicity Testing — Despite the need for man-like
metabolic responses in test animals, in the ultimate analysis practical
considerations drastically limit the choic'e 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 con-
sideration. 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 non-
carcinogenic chronic toxicity examined during the preparation of this chapter
(listed in bibliography), 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 two years in duration. On
the basis of animals exposed to the test material over a major portion of
their life span, 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%.
-------
6-4
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).
f
When relatively long-lived nonrodent animals are the test species of
s
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 concen-
trations of the test chemical and its metabolites have been achieved.
Treatment of the animal for a substantial period after attaining steady-
state kinetics would then partially substitute for a lifetime study and
provide added assurance of the validity of experimental conclusions
(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, 1977&) , but other species may be
selected if their metabolic processes are thought to resemble those of man.
-------
6-5
6.2.1.2 Carcinogenicity Testing — Rodents —mainly the rat, mouse,
and Syrian hamster, are generally selected for large-scale screening of
suspected carcinogens (National Academy of Sciences, 1975; Ministry of
Health and Welfare Canada, 1975; Page, 19776). 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 anim§ls in a relativey short
time. Although the susceptibility of simians and the dog to chemical car-
cinogenesis has been established with several groups of chemicals, the long
latency period in these species discourages their use in carcinogenicity
screening (National Academy of Sciences, 1975; Food and Drug Administration,
1971; Ministry of Health and Welfare Canada, 1975)., Dogs and monkeys may
be useful for extrapolation purposes, bufbnly in highly select situations
(Page, 19772?).
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.
6.2.1.2.1 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 accord-
ing to route of administration of the test material and this is demonstrated
in Section 6.3; thus, valid comparisons can be made between species only
-------
6-6
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 J.eukemogen (Pietra,
Rappaport, and Shubik, 1961). Of 27 Swiss albino mice injected with 30
to 40 .ug dimethylbenzanthracene in 1% gelatin, 29.6% developed malignant
lymphoraa 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
s
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 pg of the carcinogen were administered.
A linear dose relationship was observed in the range of 3.3 to 90 pg 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 palpable
-------
6-7
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. Assuming that the
dose response in hamsters is linear,.the mice would be more sensitive.
In the same hamster study, 500 pg 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 three 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 ef'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 which, 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,-£)pyrene and 3-methylcholanthrene
injected subcutaneously or intradermally induced local keratoacanthomas,
papillomas, and giant cell granulomas but no sarcomas or carcinomas.
The squirrel monkey (Saimivi scireus) 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
-------
6-8
Prehn (1969) attempted to induce tumors with subcutaneously implanted
discs of paraffin impregnated with 5% 3-methylcholanthrene (1 disc per
25 g body weight). No tumors or neoplastic changes were found four 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 four years in the three monkeys treated.
No signs of toxicity were seen in monolayers of ijonkey kidney epithelial
cells grown in vitro with 5% 3-methylcholanthrene discs during three 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
f
between rodents and primates is based on the assumption of a direct rela-
s
tionship between the induction time of tumors and the life span of the
animal. However, experiments of Sugiura, Smith, and Sunderland (1956) do
not support this but tend to emphasize differences in sensitivity among
the species to a particular carcinogen. Thus, skin painting with MH101,
a high'boiling cabalytically cracked oil, in repeated doses produced pap-
illomas in six rhesus monkeys and, later, malignant change in three of
them. This was the first demonstration of tumor induction in A/aca<7US
rhesus. In the same experiment, mice and rabbits,developed papillomas
and cancers, but rats and guinea pigs did not. Table 6.1 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
-------
Table 6.1. Some factors in the experiments with MH 101 upon a variety of species
Species
Mouse
Rat
Guinea pig
Rabbit
Monkey
^Refers
Numbers
Source:
Time to first
appearance of
Papilloma
(days)
20 (l/25)fc
0
0
26 (1/70)
322 (1/18)
Cancer
(days)
70 (1/10)
0
0
411 (1/4)
1373 (1/4)
to a single painted site.
in parenthesis represent
Adapted from Suguira et
Dose
(g)
0
0
0
0
0
.015
.1
.1
.5
.2-1.0
portion of
al.
. 1956.
Approximate
weight of Dose/kg
animal (g)
(8)
25 .
200
400
3000
7000
life span
0
0
0
0
0
.6
.5
.25
.17
.03-0.14
required for
Area
treated"
(cm1)
1
4
4
16 v
4-35
Dose/cm2
(8)
1
0
0
0
0
0
.015
.025
.025
.031
.05-0.03
Approximate
life span
(years)
2
3
6-
5
16
tumor induction. \
%
\
•vo
-------
6-10
than a relationship between induction time and life span. They also sug-
gested 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 evolutionary level
of the marmoset. Levy (1963) injected a marmoset subcutaneously with 2
mg of 3-methylcholanthrene and a fibrosarcoma appeared in 10 months, and
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
f
fibrosarcomas in three tree shrews (Tupia ^Zis) six months after injecting
benzo(a)pyrene. In a similar study, Adamson, Cooper, and O'Gara (1970) in-
jected six tree shrews with single subcutaneous doses of 10 mg methylchol-
anthrene 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 injection. Of the surviving 12 galagos,
one developed a tumor at the site of injection in, 26 months. The fibro-
sarcomatous nature of the tumors 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.
-------
6-11
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 among 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 noninb,red 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 carcinogen.
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 appli-
cations (15 skin paintings) of benzo(a)pytfene.
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 aver-
age 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 six months of skin painting (Zackheim, 1964). Table
6.2 summarizes the results of the three experiments with dimethylbenzan-
thracene. The differences in dose administered in the three studies in-
crease 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, support-
ing the view that mouse skin generally exhibits greater sensitivity than
rat skin to carcinogenesis with polycyclic aromatic hydrocarbons
(Weisburger, 1976).
-------
6-12
Table 6.2. Induction of skin carcinogens in mice, hamsters, and
rats with weekly applications of 9,10-dimethyl-l,2-benzanthracene
Species
Inbred mice
Syrian
hamsters
Induction time
Dose for carcinomas
0.1% (0.25 mg) 5-10 months
1% 4 months average
Number of
applications
20-40
16
Fisher rats 0.5% 6 months average 24
-------
6-13
Tumors of the respiratory tract have been induced by polycyclic
aromatic hydrocarbons in the various species by special techniques
which include intratracheal injection. In the first report of tumor
induction by the intratracheal route of administration, weekly instil-
lations of 50 ug 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 choi'ce 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
benzoGjOpyrene. Using infusine as,the vehicle, 0.5, 1.0, and 2.0 mg doses
were administered once every two weeks. Twenty-four of 48 rats developed
squamous neoplasms of the lung. Unlike the results with the hamster sys-
tem (Ketkar et al., 1978), the tumor incidence in rats was dose related.
Tumors have also been induced by intratracheal instillation of carcinogens
in mice (Nettesheira and Mammons, 1971), in prosimian galagos (Crocker et
al. , 1970) and in rabbits (Hirao et al., 1968).
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
-------
6-14
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
IS 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
s
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 exam-
ple, mice have been shown to develop gastric carcinomas, pulmonary adenomas,
and/or»leukemia 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-methylchol-
anthrene, 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 carci-
nomas) (Gruenstein, Meranze, and Shimkin, 1966).
Huggins, Grand, and Brillantes (1961) observed preferential mammary
cancers i'n 100% of the 50-day-old Sprague-Dawley female rats 28 to 59 and
-------
6-15
34 Co 70 days after feeding them sesame oil solutions of 20 and 10 mg 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 eight months old in their colony. 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-p'ercent of the female hamsters
of that strain developed mammary tumors of various histological types.
6.2.1.2.2 Aromatic amines — Bladder cancer in humans has been reported
in all countries with established chemical industries. A correlation be-
tween aromatic amine exposure and human bladder cancer was first reported
» " '
by Rehn in 1895. Tumor induction in animals by aromatic amines and sub-
sequent epidemiological studies have confirmed the correlated reported by
Rehn. Experimental details for the studies discussed in this section are
shown in Table 6.3.
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-naphthylamine.
The tumors were first diagnosed by use of cystoscopy 21 months after the
start of treatment.
-------
Table 6.3. Tumor Induction in various species with aromatic amines
Chemicu I/species
2-Naphthylumine
Dog
Syrian hamster
Mouse
IF mouse
CBA mouse
CBA mouse
IF mouse
Albino rat
Rabbit
2-Acetylamino-
f luorene
Dog
Mouse
Guinea pig
Dose
70 g
1.0%
1-2 rag/pellet
200 mg/kg body weight
in arachls oil
twice weekly
120 mg/kg body weight
in arachls 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 '•
0.05%
0.045% or 0.032%
1.5 and 1.6 mg/lOOg
Duration of
exposure
(weeks)
>36
22-39
30-72
30-89
40-89
40-99
2«->90
1
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
Tumor incidence latency
(weeks)
Bladder 84
50% Bladder 40
carcinomas
0/8
10/25 Liver 50-72
cholangiomas
13/23 Hepatomas; 50-89
1 cholangioma
11/26 Hepatomas 60-89
0/25
3/15 Bladder 60-90
papillomas
1/6 Bladder 247
papilloma
4/4 Bladder, ^ 272-364
liver
14/49 Bladder 48
carcinoma or
papilloma
0/16
0/26 1
Reference
Heuper and Wolf,
1937
Saffiottl, 1966
Bonser et al. , 1952
Bonser et al., 1952
Bonser et al. , 1952
Bonser et al. , 1952
Bonser et al. , 1952
Bonser et al. , 1952
Bonser et al. , 1952
Morris and Eyestone,
1953
Miller et al. , 1964
Miller et al. , 1964
Miller et al., 1964
Monkey
body weight
2-3 times per week
Dosage Increased
with time from
10-200 mg/kg
body weight per
day, 5 duys per
week
52-182
Oral, diet
0/16
Dyer et al.. 1966
-------
Table 6.3 (continued)
Chemical /species
2-Acetylamino-
f luorene
Hamster
o-Aminoa/.o-
toluene
Hamster
Rat
C3H mouse
Strain C mouse
Strain A mouse
F, hybrids of
above
Dog
4-Aminodiphenyl
Dog
Rat
Duration of
nose exposure
(weeks)
«
0.03% 30
1.5 mg/lOOg body 24-32
weight per day
3 times per week
0.1% 49
1.4-3.4 g total dose 21-49
10 rag once per 40
4 weeks in glycerol
"
20 mg/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
3.6-5.8 g/kg body During 36-53
weight total weeks
„ , Tumor
Route of m . . . ,
Tumor incidence latency
exposure , , »
v (weeks)
.
Oral, diet 3/18 Liver 52
bile duct
carcinomas
Intraperitoneal 1/18 Small intestine 64
adenocarcinoma
Oral, diet 20/25 Bladder 45
19/24 Liver cell
3/15 Mammary
Oral 7/8 Hepatomas 21
Subcutaneous Varied with strain
Hepatic
Pulmonary '
Hemangio-
endaothelioraas
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,
2 benign)
Subcutaneous 17/23 (Variety
. of sites. No
Reference
Miller et al. ,
Miller et al. ,
Tomatis et al. ,
Yoshida, 1932
Andervont, 1942
1964
1964
1961
Nelson and Woodard ,
1953
Ualpole et al. ,
1954
Ualpole et al. ,
1954
Delchmann, 1965
Walpole et al. ,
1952
bladder tumor.)
-------
6-18
Nelson and Woodard (1953) demonstrated the susceptibility of dogs to
-------
Table 6.4. Compounds, not previously listed, which h.we pmducud bladder cancer In the dog
Compound
Benzldlne
4-Dimethylaralno-
azobenzene
4-Nitrobiphenyl
2-Naphthylamine plus
4-nitrobiphenyl
2-Naphthylamine plus
4-nitrobiphenyl plus
4-aminobiphenyl
Individual doucs
200 mg/dog/day (In
capsules), 6 days/
week for IS months
followed by 300 mg/
dog, 6 days/week for
45 months, then treat-
ment discontinued
20 rag/kg dog/daya (dry
or in corn oil in
capsules)
0.3 g/dog three times a
week (in capsules)
0.1 g of each compound/
dog, three times a
week (in capsules)
0.1 g of each compound/
1 dog, three times a
week (in capsules)
\
Tot.nl dose th.it
produced tumors
325 g/dog
98 to 129 g/dog
Total: 17 to
20 g/dog
Total: 22 to
25 g/dog
Incidence of tuiaors
In 1 of 6 mongrels
(1 male, 5 females)
In 2 (1 male and 1
female) of 10
mongrels
In 3 of 4 female
mongrels
In 5 of 5 female
beagles>
In 3 of 3 female
beagles
Latent period
7.5 years
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
It is not clear whether a certain dosage per day was given five or seven times per week.
Source: Adapted from Deichmann and Radoraski, 1963. Data collected from several sources.
-------
6-20
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 (Food and Drug Administration,
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).
6.2.1.2.3 //-Nitroso compounds — About 100 tf-nitroso compounds have
*
been shown to be carcinogenic in experimental animals. The carcinogenic
s
properties of some of these compounds are compared in different species.
Diethylnitrosamine and dimethylnitrosamine — In 1967 SchmMhl and
Oswald reviewed the effects of diethylnitrosamine in 11 different animal
species, paying particular attention to the organotropic activity of the
carcinogen. The data of Schmahl arid Oswald (1967) and those of other
investigators are summarized in Table 6.5, which includes the total dose
(mg/kg body weight) required to produce cancer in the various species.
Tumors were induced in all species and in almost 100% of the animals
tested. Rabbits, dogs, and pigs treated with approximately 3 mg/kg
diethylnitrosamine developed cirrhosis as well as liver cancer while
mice, rats, and guinea pigs developed only liver cancer.
Although Schmahl and Oswald had been 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 diethylnitros-
araine administered for more than one year. Experimental details of this
-------
Tnb^c 6.5. The carcinogenic action of dlcChylnlcrosamlne in different animal species
Species
Mouse
Rat
Hamster
Guinea pig
Rabbit
Dog
Pig
Monkey -
Grass
parakeet
Brachydanio
rerio
Trout
Ruutc of
administration
Oral
Oral
Oral
Oral
Oral
Oral and
subcutaneous
Oral
Oral
Intramuscular
I
Oral
Dally dose
(rag/kg)
3
3
AO (weekly)
3
3.4
3
4.4
2-50
70 (weekly)
•10-100 ppm
Total dose (D,0)
(mg/kg)
871 ± 124
700 ± 53
640
1200 ± 100
2500
560
1400
1400-25,700
2800 ± 400
10-13 weeks
Type of tumor
Ilamanglocndothelloaas of
the liver
Hepatocarcinomas
Hepatocarcinomas
Hepatocarcinomas
Hepatocarcinomas
Leimyosarcoma of the liver
Reliculosarcomas of the
liver
Hepatocarcinomas
Hepatocarcinomas
Hepatocarcinomas and
cholangiomas
Hepatocarcinomas
i
a\
to
Source: Adapted from Schmahl and Osswald, 1967.
-------
6-22
and the following studies with diethylnitrosamine and dimethylnitrosamine
are listed in Table 6.6.
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
v
with outbred Hartley guinea pigs diethylnitrosamine in the water induced
S
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. Malig-
nant tumors appeared in the esophagus, liver (carcinomas and sarcomas),
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.
-------
Table 6.6. Tumor induction in various species with dlethylnitrosamine and dimethylnitrosamine
Chemical/species
Diethylnitrosamine
Monkey
Monkey
Inbred guinea
pig
Outbred Hartley
guinea pig
Sprague-Dawley
rat
Buffalo strain
rat (12 weeks
old)
Duration
Dose °£ R°Ute °f
exposure exposure
(weeks)
2-5 mg/kg body 108 Oral
weight/day
Total: 6-24 g
20-40 mg/2 weeks 108 Intraperitoneal
Total: 1.6-2 g
1.1-4.2 mg/day 16-40 Oral
Total: 200-300 rag
About 3 mg/day Up to 68 Oral
3 days /week
Total: 203 rag 29 Oral
0.0114% in diet 26 » Oral
\
Tumor
Tumor Incidence latency Reference
(weeks)
6/18 hepatomas 92 Kelly et al. , 1966
2/2 heptatomas 104 Kelly et al. , 1966
14/15 heptaomas 16-46 Argus and Hoch-Ligeti,
7/15 lung tumors 1963
2/15 bronchial
papillomas
35/35 heptaomas 60 Dale et al. , 1973
10/12 liver tumors About 20 Lijinsky and Taylor,
2/12 nasal ~ 1978
turbinate tumors Q^
6/12 esophageal 1
papilloma • ^
1/12 esophageal
carcinoma
1/7 prostate tumor 24-36 Reuber, 1976
12/26 esophageal
carcinoma
Mice BALB/c
Total: 515 mg/kg
body weight
41
Total: 1010 mg/kg 41
body weight
Oral
Oral
Unclear liver
4/15 liver Unclear
hemangiosarcomas
6/15 esophageal ^
tumors
3/15 stomach tumors
0/15 lung tumors
14/60 liver Unclear
hemangiosarcomas
35/60 stomach tumors
2/60 lung tumors
Clapp et al., 1971
Clapp et al., 1971
-------
Table 6.6 (continued)
Chcra 1 c.i 1 /spec Ivs Dose
Mice, Swiss 6 ing/kn body
welghlA.'Ui.'k
Dime thy Inltrosamine
Mice, BALB/c Total: 300 mg/kg
body weight
Mice, Swiss 6 mg/kg body
weight/week
lUir.u Inn • _
, , Timor
of Route of _ . . . . .. ,
Tumor incidence latvncv Reference
exposure exposure , , ;
... (weeks)
(weeks)
10 lntrapcritonc.nl 30/38 luni; tui^ors 66 Cardesa, 1974
• 4/38 vascular
tumors
3/38 forcstora.icli
papl llomas
0/38 kidney tumors
10/38 lymphomata
1/38 mammnry
adenocarcinoma
Up to 41 Oral 3/15 hemanglo- Unclear Clapp et al. , 1971
sarcoma
7/15 lung tumors
10 Intraperitoneal 37/39 lung tumors 59 Cardesa, 1974
12/39 vascular
Chinese hamster
0.89-3.54 rag/kg
body weight/week
Total: 31-105 mg
Subcutaneous^
tumors
0/39 forestomach
papilloma
4/39 kidney tumors
4/39 lymphomata
1/39 mammary
adenocarcinoma
100/108 liver
tumors
1/108 lung tumors
1/108 nasal tumor
to
26
Reznik, 1975
-------
6-25
Similar to rats, BALB/c mice developed tumors of the liver and
esophagus as a result of ingesting diethylnitrosamine in drinking water
(Clapp, Tyndall, and Otten, 1971). Tumors were also found in the lung
and stomach. DimethyInitrosamine, however, produced tumors only in the
lung and liver. Differences in susceptibility of the lung to diethyl-
nitrosamine (3% tumor incidence) and dimethyInitrosamine (47% tumor inci-
dence) indicate either that different enzyme systems may be activated for
metabolism of each of the chemicals and/or that the system for metaboliz-
ing 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 intraperitoneal
injection of dimethyInitrosamine 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 ham-
sters injected subcutaneously with three doses of dimethyInitrosamine
(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, and 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 dimethyInitros-
amine 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 iV-nitroso compounds — In separate studies, species differences
were observed between rats and hamsters treated with diisopropanol-nitrosamine
-------
6-26
(Mohr, Reznik, and Pour, 1977; Pour, Kruger, and Althoff, 1974, 1975).
Sprague-Dawley rats were given subcutaneous injections of 178 to 1425 mg/kg
body weight once weekly for 20 weeks and hamsters received 125 to 500 mg/kg
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 adenocarci-
nomas were found in the thyroid glands of rats but not in hamsters.
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
s
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 ff-nitroso compound
carcinogenesis that the organ specificity of these chemicals is approxi-
mately the same in all species which 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.
6.2.1.2.4 Miscellaneous compounds — Brief mention is made below of
species differences encountered in carcinogenesis studies with miscellaneous
compounds of chemical classes other than those previously mentioned.
Exposure in inhalation chambers to amosite, crocidolite, and chryso-
tile 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,
-------
6-27
1974). The animals were exposed 4 hr per day, four days per week for two
years (1480 hr cumulative total exposure). Some animals were sacrificed
after three to six months, but most were allowed to survive until the
exposure period ended. Table 6.7 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 which 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 inveirsely related to
carcinogenic effects attributable to asbestos inhalation.
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.
l<,l-TrichlorjQ-2,2-bis(p-chlorophenyl) 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 non-carcinogenic (Cameron and Chen, 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 Bx — The hepatocarcinogenicity of aflatoxin Bt had been
demonstrated in rats (Wogan and Shank, 1971; Newberne and Rogers, 1972)
but not in mice until Vessilnovitch et al. (1972) induced heptatomas in
-------
T.I!) 11- ft .7 . C.irr I no^t-it If 11 y of d I f I" IT rut forms of a:>h»'slos I it var 1 oils spec lea
Spcclns
(I
Mouse
Gurbil
Kat
Rabbit
Guinea pig
Kiiiiilii-r
surviving
19
50
43
11
14
Clirysui 1 le
Kf IVi:l
None
None
One papillary carcinoma
of lung
One squamous carcinoma
of lung
One mesothelial fibro-
sarcoma of mediastinum
None
None
Croclilol Itu
Nuiubfr Kfl.-ct t.iiml>«r
surviving tiurvlvlnt;
18 Two papillary 17
carcinomas of
bronchus
49 None 51
46 One adenocarci- 46
noma of lung
Three squamous
carcinomas of
lung
One papillary
adenocarcinoma
of lung
9 None 9
14 None 13
Amottlte
Effect
None
None
One metastatic osteo-
sarcoma of lung
One mesothelial fibro-
sarcoma of lung and
pleura
One fibrous mesothelial
of pleura
None •
None
ON
oo
Control mouse had one papillary carcinoma of the bronchus. All other controls were free of neoplasma.
Source: Adapted from Reeves, Puro, and Smith, 1974. \
-------
6-29
newborn mice with intraperitoneal injection of a total of 1.5 to 6 ug/g
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 BU and carcinogenicity of afla-
toxin in the hamster has been described by Herrold (1969). Table 6.8
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 6.8 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 International Agency
for Research on Cancer (Tomatis et al., 19^78). These data are correlated
with the results of animal tests.)
6.2.3 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 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 generalization 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
-------
Table 6.8. 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
Af latoxins
A-Aminoblphenyl
Arsenic compounds
.
Asbestos
Auramlne
(manufacture of)
Benzene
Benzidlne
Bls(chloromethyl)
ether
Humans
Main type _
, 'r a Target organ
or exposure
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. . , .
, b Animal
of exposure
p.o., I.h. Rat
Fish, duck, mar-
moset, tree
shrew, monkey
Rat
l 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
Animals
Target organ
Liver, stomach,
colon, kidney
Liver
Liver, trachea
Liver
Local
Lung
Bladder
Liver
Mnmmary gland.
intestine
Inadequate, negative
Inadequate, negative
Lung, pleura '
Local
1.0 C ill ,
Various sites
Liver
Negative
Local, liver,
Intestine
Inadequate
Liver
Liver
Zyrabal gland, liver,
colon
Liver
Bladder
Lung, nasal cavity
Skin
Local, lung
Route of
a
exposure
p.o.
p.o.
i.t.
l.p.
s.c. i.J.
CT>
P-°- oj
s.c. i.J. ,0
s.c. i.j.
p.o.
t., l.v.
I.h. or I.t.
ipl.
l.p. . s.c. 1.1 .
p.o.
p.o.
P.O.
B.C. i.j.
t., s.c. I.J.
s.c. l.j. .
p.o.
s.c. i.J.
p.o.
p.o.
I.h.
t.
s.c. i.J.
Rat
Local
s.c. i.J.
-------
Table 6.8 (continued)
Ciictnical or
industrial process
Cadmium using indus-
tries (possibly
cadmium oxide)
Chloramphenicol
Chlororaethyl methyl
ether (possibly
associated with
bls(chloromethyl)-
ether
Chromium (chromate—
producing
industries)
Cyclophosphamide
Diethylstilbestrol
Hematite mining
(? radon)
Isopropyl oils
Melphalan
Main type
of exposure
Occupational
Medicinal
Occupational
Occupational
Medicinal
Medicinal
x
Occupational
Occupational
Medicinal
Humans
„, Main route, . . ,
Target organ , b Animal
6 & of exposure
Prostate, lung I.h., p.o. Rat
Hemopoietic system P.O., I.j.
Lung i.h. Mouse
, Rat
Lung, nasal cavaties i.h. Mouse, rat
Rat
Bladder P.O., 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 IH
Hemopoietic system p.o. , I.J. Mouse
Rat
Animals
Target organ
Local, testis
No adequate tests
Initiator
Lungd d
Local, lung
Local"
Local
Lung
Hemopoietic system,
lung
Various sites
Bladder**
Mammary gland
Various sites
Mammary
M.'imllary , lymph-
oreticular, testis
Vagina ,
Mammary, hypophysis
bladder
Kidney
Uterine serosa
Negative
Negative
No adequate tests
Initiator
Lung, lymphosar comas
Local
Route of
exposure
s.c. or l.m.
I.J.
s.
i.h.
s.c. i.j.
s.c. I.j.
s.c. . i.m. 1. 1.
i.b. impl.
i.p. , s.c.
p.o.
i.p.
i.p.
i.v.
P.O.
s.c. i.J.,
s.c. impl.
Local
s.c. impl.
s.c. i.j.
s.c. impl.
s.c. imp 1 .
i.h., i.t. '
s.c. I.j.
8.
i.p.
i.p.
ON
-------
, Table 6.8 (continued)
Chemical or
Industrial process
Mustard gas
2-Naphchylamine
Nickel (nickel
refining)
/V,V-Bis(2-chloro-
ethy 1) 2-naphthyl-
lamlnc
Oxymetholone
Phenacetln
Phenytoln
Soot, tars, and oils
Vinyl chloride
Main type
of exposure
Occupational
Occupational
Occupational
Medicinal
Medicinal
Medicinal
Medicianl
Occupational,
environmental
Occupational
Humans
Target organ
Lung, larynx
Bladder
Nasal cavity, lung
Bladder
-
Liver
Kidney
Lymphoreticular
tissues
Lung, skin (scrotum)
Liver, brain , lung
Main routefc
of exposure
l.h. Mouse
l.h., s.,' p.o. Hamster, dog,
monkey
Mouse
Rat, rabbit
l.h. Rat
I Mouse, rat, hamster
Mouse, rat
p.o. Mouse
Rat
p.o.
p.o. '
P.O., i.j. Mouse
l.h., s. Mouse, rabbit
l.h., s. Mouse, rat
An 1 ma 1 s
Target organ
Lung
Loc a 1 , mammary
Bladder
Liver, lung
Inadequate
Lung
Local
Local
Lung
Local
No adequate tests
a
No adequate tests
Lymphoreticular
tissues
s.
4
Lung, liver, blood
vessels, mammary,
Zymbal gland,
kidney
Route of _
exposure"
l.h. , l.v. :
B.C. l.J. !
p.o.
s.c. i.j.
p.o.
l.h.
s.c. , i.m. i.j.
I.m. impl. CT>
1
i.p. W
s.c. I.j.
P.O., i.p.
t.
l.h.
The main types of exposures mentioned are those by which the association has been demonstrated; exposures other than Chose 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. -intravenous; i.p. - intraperitoneal; s.c. l.J. -subcutaneous injection; t. -topical; l.v. -Intravenous;
ipl. - Intrapleural; i.h. - inhalation; i.m. - intramuscular; s. - skin; s.c. impl. - subcutaneous Implantation; i.b. - intrabronclal
Implantation; i.j. — injection.
"Indicative evidence.
eThe Induction of tumors of the nasal cavities In rats given phenacetln has been reported recently (S. Odashlma, personal communcation,
1977).
-------
6-33
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
gnotobiotic 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 which deserve consideration
in choosing a strain. These include mainlfenance requirements, sensitivity
to test chemicals, and occurrence of diseases, both nonneoplastic and neo-
plastic. 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.
6.2.3.1 Hamsters and Guinea Pigs — Inbred strains of Syrian golden
f
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
-------
6-34
strain relates to tumor type. For example, there are strains of hamsters
(BIO 54.7, BIO 82.73, BIO 86.93) which 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. DEN is an effective carcino-
gen in both strains; MCA is inconsistent in the Hartley strain, but less
so in the ICRF animals (Dale et al., 1973).
6-2.3.2 Mice — The influence of genetic factors has been clearly
demonstrated in two strains of mice, BALB/c and C57BL/6, which after treat-
ment with diethylstilbestrol developed testicular teratomas and pituitary
*
neoplasms respectively. The F! hybrids of these strains developed both
s
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-l-nitrosurea, Diwan and Meier
(1974)'demonstrated clear differences in all these parameters for 5 mouse
strains (AKR/J, SWR/J, DBA/2J, C57BL/6J, and C57BL/J). From their results
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 DMBA was shorter
than that of CBA mice (Roe, Rowsen, and Salaman, 1961). Thiery and van
Gljsegem (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,
-------
6-35
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.
6.2.3.3 Rats — Within a few weeks after one dose of DMBA, Sprague-
Dawley rats developed high incidence of multiple^mammary gland tumors.
In contrast, Long-Evans rats receiving the same treatment had low inci-
dence, 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. Fibro-
adenoma incidences after DMBA in Fx hybrids from reciprocal crosses of
the two strains were nearly equal; hybrids^ from backcrosses 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 Fisher rats. The most sensitive strains
in terms of incidence and induction time were Sprague-Dawley, Fisher,
and Werck-Stewart, and least sensitive were the Long-Evans rats. Sydnor
concluded that susceptibility is genetically determined by, and is also
dependent on, carcinogen dose.
In conclusion, careful selection of appropriate strains for experimen-
tation and substance evaluations is a prerequisite for meaningful interpre-
tation of data. Strain-related parameters which relate to carcinogenesis
include spontaneous tumor incidence (discussed elsewhere), age, induction
time, and susceptibility. Superimposed on the genetic framework are the
-------
6-36
many environmental factors which 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 which
cause variation among individuals.
^
6.2.4 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 (1955) defined spontaneous
s
lesions as those for which a cause cannot be ascertained. Such neoplastic
lesions appear in animals which have not been experimentally exposed to
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 6.9. The B6C3F1 mouse, an Fl hybrid cross
between the C57B1/6 female and the C3H male, has a relatively 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.
-------
Table 6.9. Spontaneous tumor incidence in animals used in the National Cancer Institute Bioassay Program
Organ
Brain
Skin/subcutaneous
Mammary gland
Spleen
Lung/ trachea
Heart
Liver
Pancreas
Stomach/ intestines
Kidney
Urinary bladder
Testis
Ovary
Uterus
Pituitary
Adrenal
Thyroid
Parathyroid i
Pancreatic islets
Thymus
Body cavaties
Leukemia/ lymphoma
B6C3F1 Fisher
mousea 344 rat
Males Females Males
1,132° 1,176 '846
<1 - 1.3
1.0 <1 5.7
<1 1.0
<1 <1 ' <1
9.2 3.5 2.4
1
15.7 2.5 ' 1.2
<1 <1 <1
1.3 <1 <1
<1 <1 <1
/j
<1 NAa 76.2
MA <1 . NA
NA '• 1.9 NA
<1 3.5 10.2
<1 <1 8.7
1.1 <1 5.1
_
<1 <1 3.2
<1 - -
<1 <1 <1
1.6 6.8 6.5
Females
840
-------
6-38
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 anticarcino-
genic activity, or to look at mechanisms of neoplastic growth, the inves-
tigators 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 aaong animals with differ-
ent 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 occur-
ring tumors. Some variables known to have effects are: breeding methods,
f
genetics, age, diet, maintenance, histologic screening, and statistical
s
evaluation (Pollard and Kajima, 1970; Sass et al., 1975; Pour et al.,
1976), 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 6.10. 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).
Genetic control of susceptibility to ileocecal immunocytomas in
certain strains of rats appears to reflect one or more dominant genes
for susceptibility in the LOU/c strain and one or more loci of resistance
in the OKA strain. FI progeny from crosses of these strains have null
tumor incidence (Beckers and Bazin, 1978). High incidence of spontaneous
-------
6-39
Table 6.10. Sex and age incidence of characteristic tumors
of inbred strains
Tumor type
and strain
«v
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
Reticulum cell
neoplasm, type A
C57BL
Ovarian tumor
C3HeB/De
C3HeB/Fe
RIII
CE
Pituitary tumor
C57L
C57BR/cd
Hemangioendothelioma
HR/De
Adrenal cortical tumor
CE
Incidence
(%)
99
100
77
Lower
84
5
84
75
92
90
90
80
91
58
! 30
91
High
15
47
37
64
22
60
50
34
33
33
24
100
79
Type mouse
Breeding 99
Virgin 99 »
Breeding 99
Virgin 99
Breeding 99
Virgin 99
Breeding 99
Virgin 99
—
r
s
—
—
Breeding 66
Virgin 99
Breeding 99
Virgin 99
Breeding 99, 66
Breeding 99
Virgin 99
Breeding 99
Breeding 99
Virgin 99
Breeding 99
Virgin 99
99
Breeding 99
Breeding 99
66 and 99
Gonadectonized 99
Gonadectonized 66
Age
(months)
Av. 7.2
Av. 8.8
Av. 15
—
—
—
Av. 7.7
Av. 10.2
Av. 8.0
Av. 10.0
After 18
After 18
Av. 21.4
Av. 24.1
Av. 21.0
Av. 13.3
—
Av. 22.3
Av. 24.3
Av. 21.5
After 19
After 19
After 17
After 17
After 20
Old
Old
Av. 22
After 6
After 7
-------
6-40
Table 6.10
Tumor type Incidence
and strain (%)
Testicular teratoma
129 1
82
Myoepithelioraa
BALB/c 4
A Similar
Skin papilloma
HR/De 9
Skin carcinoma
HR/De 3
Subcutaneous sarcoma
C3H/J 3
Harderian gland tumor
C3H 1
(continued)
A go
Type mouse ., * .
(months)
dd Congenital
Male gonadaT —
ridges trans-
planted to adult
tests
_ _
— —
Hairless ' Av. 22
X
Hairless Av. 18
99 Av. 25
Av. 20.7
Source: Adapted from Weisburger and Weisburger, 1967.
-------
6-41
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
vv
strains of C3H mice prone to hepatomas and mammary tumors. The A gene
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, Heston, and Smith, 1970).
w
Interesting results with this same C3H-A * system have been reported
by Sabine, Horton, and Wicks, (1973). Mice bred in Australia had 0% inci-
dence qf 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 envi-
ronmental 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; Tannenbaum and
Silverstone, 1949; Silverstone and Tannenbaum, 1951; Heston, Vlahakis,
and Deringer, 1960; Peraino, Fry, and Staffeldt, 1973). Heston (1958) in
-------
6-42
studying four substrains of C3H mice found that mammary tumor incidence
was influenced not only by genetic susceptibility, but also by hormonal
stimulation during breeding, and by age. Riley (1975) described a short-
ening 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 of spon-
taneous tumor incidence over the life span of the animal are necessary
s
and may be so for certain carcinogens (Burek and Hollander, 1977). In
addition to the value of life span data, one must consider the latent
period before the effect of a carcinogen is detectable as well as the
dose (which in some cases determines latency). Laboratory animals tend
to be more susceptible to tumors as they age, but as discussed earlier,
there are additional factors involved (Gilbert et al., 1958). Table
6.11 summarizes some pertinent literature concerning this subject.
6.2.5 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 workload. 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
-------
Table 6.11. Spontaneous tumors in laboratory animals
An I ma1
Tumor and organ
Incidence and age
Comments
Reference
Hxaaaa phi'llipienaia and
friiuOLHZ mulatto
Lymphosarcoma — kidney
Hemangioendothelioma — vertebra
Meningioraatosis — lumbar cord
Lipoma — choroid plexus
Endotliellomatosls — lumbar cord
Overall - 0.08%
Prepubertal
12,000 monkeys (1,800 of
them cynomolgus) observed
over 3.5 years. Also looked
at granulomatous processes.
Jungherr, 1963
Mouse
C31I/ST
Nude mutant most-
with BALB/c, some
viith C3H, or C57/BL/6
backgrounds
Mammary tumors
None
Reaches 87%
0%, generally die at
two to three months of
age.
Studied tumor regression
after 5-methyl cytldlne
11,000 mice studied from
birth to three months
Strong and
Matsunaga, 1977
Rygaard and
Palvsen,' 1974
Nude mutant from
BALB/c backgrounds
CU8F,
(BALB/cXDBA/SFl)
SHN
129/terS
Strain A
CD^-lHaM/ICR
C31l-AVy,
C3H-AVyfB
Lymphoretlcular neoplasms
Adenocarcinoma mammary
(also metastasis to lung)
Mammary tumors I
Teratomas, testlcular
Lung tumors
Lymphoretlcular mammary,
pulmonary, osteogenic sarcoma,
hemangiosarcoma, renal and
hepatic tumors
Liver, mammary tumors
Estimated from graphic
material, tumor Incidence
for entire group was about
50% at 57 weeks (2% at 35
weeks)
80%, ten months
41.3% at 7 months, 87.5%
at 12 months
\
30%, earliest seen were In
six-day fetuses
0% at 2 months, 40% at 12
months, 77.1% at 18 months
Total Incidence of tumors —
range of 0.77%-50% for age
groups ranging from 0+ to 20+
months
902-100% or 0% ages from
7 to 22 months
Gennfree 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.
Outzen et al.,
1975
Fugmann et al.,
1977
Nagasawa et al.,
1978
Stevens, 1973
Shimkin and
Stoner, 1975
Percy and
Jonas, 1971
Sablnc et al.,
1973
-O
-------
T.'ililc 6.11 (ruiulnurtl)
Anlrj.il
C till
C Jill f
C'Jllf/2
C 311 f/ An
C3llfB X YBR
C3llfB X YBR
C3Hfb X YBR
YBR X C3HfB
YBR X C3IUB
YBR X C311fB
YBR X C3llfB
C3H/St
C311X101
hybrids
A
GR
020
CBA
C3H
- DBA
C57BL
HMRl-Neuherberg
Wild house mice
(l-his mitatmlux)
'1 umor .iiul org.iu
H.i;:.in.iry tumor*: iitli-nu-
t:;uv 1 1101:1.1 .
*jth-iu>.itMiiUioi[).j . care 1 no-
-
Ilupatoma
•
Adenocarcinoma, mammary
Outeosarcomas
Lung tumors
1
•
Lymphoma
Lut\g tumors
Glomerulosclerosis
Ovarian neoplasms
Adenomatous hypcrplasia of
Che glandular stomach
Pulmonary, retlculem-cell
(type B) , granulosa-cell of the
22Z,
2«,
511.
27Z,
100 %
94Z
63Z
5Z
95Z
43Z
41Z
OZ
41Z,
20Z
71Z,
42Z,
58Z,
18Z.
14Z,
9Z.
7Z.
41Z
16Z
13Z
12Z
7Z
36Z,
64Z,
1 nc IdriH'r ;uul ag<-
I'O montlis
H months
20 r.iunllii
20 months
4.5 months
23 months
14 months
21 months
2 7, months
21 months
21 months
22 months
50Z survival time
is 600-700 days
2-24 months
25-33 months
ovary, hepatoma, hemangioendo-
thelioma, mammary, lymphocytlc
neoplasm.
Cuxmcnlb
Gunotypi-s:
AYA males
Aa males
AYA females
AYA males
Aa males
AYA females
Aa females
Weight and growth factors
considered important
Study of effects of As, Se
on tumor Incidence
Data of Bentvelzen and
Szalay, 1966
Tumors In mice received
in 1951 and their descendants
until 1961.
Hi.-f
irlvcj of macmary lunur Huston,
•lit by futitcr musing.
llrstcm and
VlahaklB, 1966
Schrauzer
et al., 1978
Shimkin and
Stoner, 1975
Luz, 1977
Andervont and
Dunn, 1962
-------
Tahle 6.11 (<,.ill l,n..!)
Ait i tn.'i 1 Tumor ;nul 01 g;m
CllIf/AuKAiil 70) I.ivcr tunnr*
1' H'/IU-N*- H:ii:r.iai y tumors
I;K/::
rtAi.it/i-fc.'iii
I».C3K )
AK K
HAI.It/i:
CV/li|./d
C'tll/lloM
s Incidence and a^c Comments Reference
Ki-males dZ-132, males Al%- Stiull.-il i>nh.inccnicnt of lumor I'.-ralno tt nl.,
CU?. llefure 1^ raoiuliM. 1m Idi'nre. 1973
9'/x, 7.2 Motulis hic:un:it r.it Ion of K.:j-i::i.iry llili- it al.,
70Z, 10 montlus (uir.,ir virus untlbuJU-b In lV7b
different b I rains. Illyhi-st
•H>7.. a uunllis tll.-rs in tumor bearing
•l n 1 roa 1 a .
07, 11 cnuull,:!
3Z, 1'i Eiinilli.s
1Z, 15 months
'it, 8 month*
C3H/A/BOH
C.tll
C'IMe
C.III-AU
Hiimm.-iry tumors
Mammnry tnmovs
HepaCoina
2T/., duration of experiment ,
two years. (In another Uihlc,
incidence appears to be ]00%.)
80Z-100% within 8 to 18
months
months, controls
rat albumin
at 9 months.
months, controls
adjuvant i.n PBS
at 2 months.
months, controls
adjuvant in T11S
llepatoma
ilopntomn
Milimiiary lunior.'j
66%, 22-25
received
starting
50Z, 18-20
received
starting
25%, 12-16
received
85%
72% All killed at 14 months
78%
100%, 12 months
100%, 6-7 mouHiu
90%, 15 mant-liK
2UX, IS
Koiluct ion of tumor incidence
by manipulation of estrogen
and prolactln interactions
witli administration of 2-
brorao-d-ergocryptlne, 17 B-
estrndiol, 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
T n males.
C3llf male fed Purina
chow instead of the NCI
pcllctb- had a 57% incidence.
Mitii-s (also nearly all
females at 16 months)
1'cmales wUh MTV
Kinnalc liruudKrs free of MTV
Inn luivlny MIV
rViii.ilu vli'uliiu frt-'C of KI'V
lull luivlntj HIV
Welscli, 1976
Riley, 1975
Jalanko et al., ?*
1978 *«
Ol
Iloston et al.,
1960
lies ton and
Vluhakls, 1968
-------
Table 6.11 (continued)
Animal
Tumor and organ
Incidence and age
Comments
Reference
Hamster
Syrian, Kppley colony,
Hannover colony
BIO 4.24
BIO 45.5
Overall tumor incidence
Primary tumors in respiratory,
digestive, urogenital, endocrine,
vascular, lymphatic "systems
Adrenal tumors
32%, average
41% survival time for animals
with multiple tumors was 97
and 85 weeks respectively.
52%, average age at autopsy,
80.3 weeks
17Z
Study of spontaneous tumors Pour et al.,
and diseases in Syrian hamster 1976
colonies
Hamburger and
Russfield, 1970
Rat
CD
CD-I
BN/BiRij
LOU/C
LOU/M
AXC
OKA
(LOU/CXAXC) F
(l.OU/CXAUG)F.
(I.OU/CXOKA)F
GG
NB
Sarcoma, fibroma, adeno-
carclnoma, 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
lleocecal lymph node for most.
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.
Hales
28Z (bladder), 61 (ureter),
ages 7-48 months
Females
2Z (bladder), 542 (ureter),
ages 7-54 months.
8-12 months
24*
1.7Z
2.6%
0%
16Z
12%
0%
74% of all males,
50% of all females
Females about 5Z at 12+
months
Controls in study of oral Lee et al.,
toxlclty of organic fungicides. 1978
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, 1978
Beckers and
Bazin, 1978
Gilbert et al.,
1958
Noble et al. ,
1975
Breast fIbroadenoma
about 20% at 12+
months
-------
t!
Table 6.11 (continued)
An[mal
Tumor and organ
Incidence and age
Comments
Reference
UDX
Wlstar - Af/Han-EMD
/BN/Bi
ACI/N
Sarcomas - connective tissue,
bone, neural system, skin
carcinomas, tumors of lung,
gastrointestinal tract, genito-
urinary tract, mammary glands,
tcstis, adrenal glands.
Malignancies of lymphoreticular
system.
Tumors of the central nervous
system: oligodendroglloma,
estrocytoma, mixed glioma,
pleomorphic glioma,'menlngloma
Mammary gland — fibroadenoma
Pituitary tumors
Lymphoreticular sarcomas
Adrenal gland, cortical adenoma
Pancr'eas - islet adenoma
Cervix and vagina, sarcoma
Testes-interstltial 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 et al.,
1978
5.8% mean ages ranged from
796 to 963 days.
11%, 30 months (23-40)
Males, 14%, 28 months (15-42)
Females, 26%, 31 months (17-39)
Males, 14%, 24 months (19-36)
Males, 12%, 33 months (27-43)
Females, 19%, 33 months (23-54)
Males, 15%, 33 months (20-42)
Females, 11%, 33 months (23-40)
15%, 27 months (18-38)
56% in males
52% in females
Developed in 169 weeks
\
Data for other tumor types
included. Study of Inci-
dence of benign and malignant
neoplasms at different ages.
Sumi, 1976
Burek and
Hollander, 1977
Maekaua and
Odashima, 1975
-------
6-48
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 counter-
productive. 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 non-carcinogenic toxicity tended to
use 10 or less test animals (rodents) per group (Barnes and Denz, 1954);
s
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, espe-
cially 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 are usually considered necessary (National
Academy of Sciences, 1977); however, currently proposed Environmental Pro-
tection Agency regulations require six nonrodent animals per exposure group
-------
6-49
(Federal Register, 1979). This number does not include allowances for interim
sacrifices and must be increased if the latter practice is followed. Need-
less 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 con-
junction 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, 19772?). First, a significant
number of animals must survive to tumor-bearing age. Second, the incidence
of spontaneous tumors in the control groups must be compensated for in
order to avoid jeopardizing the sensitivity of the test. Table 6.12 shows,
in relation to animal numbers, how such aff 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 {Sontag, Page, and Saffiotti, 1976; Page, 19772?; National
Academy of Sciences, 1977). However, in any case, the number surviving to
tumor-bearing age should be at least 25 per sex per level (World Health
Organization, 1978).
6.2.6 Controls
Each chronic toxicity or carcinogenicity test should be evaluated with
reference to a concurrent control group comprised of animals of the same
species, age, sex, and weight as the treated groups (Federal Register,
1979; Weil and Carpenter, 1969; Weil, 1962; Food and Drug Administration,
1971). These animals should be handled identically to the test species
-------
6-50
Table 6.12. 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 contro]
0
10
20
30
40
Number of animals
i .-
10
50
70
80
90
100
25
20
40
52
64
72
50
12
28
40
s 52
62
per groups^
75
8
24
36
47
58
100
6
21
34
45
55
Calculations based on tabulations of Mainland and
Murray, 1952.
^Controls and treated groups of same size.
Source: Adapted from Page, 19772?.
-------
6-51
except for treatment with the test material (World Health Organization,
1978). Most investigators require that this negative control group con-
tain 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 vehicle control group is
often required when the test material is administered 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, 1977fo).
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 carcino-
genicity tests (Page, 1977Z?) . In chronic^tests, it's usefulness is not
well established (Benitz, 1970), and 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
Sciencfs, 1977). .
For carcinogenicity tests, positive controls can provide more infor-
mation, 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 affect the test;
(5) providing an indirect evaluation of the laboratory and its general
bioassay program; and (6) assessing the relative carcinogenic potential
of the test chemical (Page, 1977&; World Health Organization, 1978; Food
Safety Council, 1978). Despite all this potential information, the
applicability of positive controls is limited. Most discussions suggest
-------
6-52
that it only be applied to series of analogous chemicals and need not
be done with every study (Food and Drug Administration, 1971; Peck,
1974; Page, 19772?; Food Safety Council, 1978).
6.2.7 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 post-weanling rats and mice have
been used (Ministry of Health and Welfare Canada, 1975; Loomis, 197A; Magee,
1970; National Academy of Sciences, 19.77; World Health Organization, 1978).
Effects of the age of the test animal on its response to carcinogenic
challenge are well-documented and the ramainder of this section will be
devoted to a discussion of these effects.
The use of weanlings or immediately post-weanlings 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
-------
6-53
Shubik (1959) observed a 32% incidence of malignant lymphomas in 11- to
24-week-old Swiss mice following a single 30 yg subcutaneous dose of
9,10-dimethyl-l,2-benzanthracene administered at age 12 hr or less. Sim-
ilarly, Kelly and O'Gara (1961) induced pulmonary tumors in essentially
all 18-hr-old albino mice treated subcutaneously with 0.02 mg dibenz(a,h)-
anthracene or 0.011 mg 3-methylcholanthrene.
Many other workers have also demonstrated the extraordinary sensi-
tivity of newborn mice to certain carcinogens (Axelrod and 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,
Yoshida, and Gray, 1961; Liebelt, Liebelt, and Lane^ 1964; Nishizuka,
Nakakuki, and Sakakura, 1964; Roe, Mitchle^y, and Walters, 1963; Toth,
Rappaport, and Shubik, 1963; Toth, Magee, and Shubik, 1964; Vesselinovitch
and Mihailovich, 1966; Vesselinovitch, Milhailovitch, and Itze, 1970;
Vesselinovitch et al., 1972, 1975a, Vesselinovitch, Rowe, 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
f
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, Rowsen, and Salaman, 1961; Roe, Carter, and Adamthwaite,
1969; Toth, 1968). However, subsequent research indicated such exposures
-------
6-54
could be more complex than they first appeared. For example, treatment
with .'V-hydroxy-/V-2-fluorenylacetamide produced no detectable tumors in
infant rats but did induce tumors in weanlings under similar conditions
(Weisburger et al., 1970). Also, dimethylnitrosamine induced renal ade-
nomas in mice exposed as adults, but not in those exposed as newboms
(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 groups (Ministry of Health and Welfare
Canada, 1975; Rice, 1976). It is reasonable to expect newborns to respond
more sensitively than adults to ultimate carcinogen^ because of reduced
immunological and hormonal competence, bat because of the lack of well-
developed metabolic capabilities, responses of such animals to proximate
carcinogens that requir metabolic activation will obviously be deficient
or absent, compared with more mature animals (Page, 1977&). 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
f
maximized if animals are exposed to the test material during all prenatal
and postnatal phases of their lives (Food and Drug Administration, 1971;
Munro, 1977; Page, 1977&). 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
-------
6-55
following the discovery of vaginal adenocarcinomas in daughters 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). Ivancovic (1973) reported transplacental induction of tumors
in rats with ethylnitrosourea. Five to eighty mg/kg body weight .of the
compound given as a single dose, intravenously or orally on day 13-23 of
preganancy produced a significant 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 lifespan'T In fact, a notable yield of
tumors in the nervous system of adults was obtained only when 140-200
mg/kg were given, thus demonstrating increased sensitivity of the fetus
to carcinogenesis. However, routine testing for carcinogenesis through
prenatal and bigenerational exposures is complicated by several factors
that have not yet been adequately resolved. For example, dose determina-
tion 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, cer-
tain 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
-------
6-56
such reproductive 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 non-teratogenic doses would have to be used in 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 effeats of certain food addi-
tives, pesticides, and drugs on pregnant women (Food and Drug Administra-
tion, 1971), biogenerational and in utero exposures are not generally
made a part of routine carcinogenesis testing today.
6.2.8 Sex
s
Current practice cells for the use of equal numbers of male and
female test animals in assays for chronic toxicity (Federal Register, 1978;
National Academy of Sciences, 1977; Food Safety Council, 1978; World Heath
Organization, 1978), although early workers questioned the need for equity
(Barnes and Den2,' 1954). Essentially all test guidelines require that both
sexes of the test speices be used in carcinogenicity assays (Page, 1977&).
However, female mice are preferred by some researchers because they are less
aggressive and survive better than males; and in some instances, male animals
may be preferred when females are needed for breeding. In the following
section, data will be presented which will illustrate sex-related differences
in the response of animals to chemical carcinogens, and the effects certain
intrinsic and extrinsic factors of the animal test system have on these dif-
ferences. Comparisons will be made of the responses of male and female
animals to chemicals within selected chemical classes: polycyclic hydro-
carbons, -nitroso compounds and amines, and halogenated hydrocarbons.
-------
6-57
Akamatsu and Barton (1974) compared the neoplastic response of both
sexes of five strains of mice which had received 1 mg of 3-methylchol-
anthrene 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%).
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%) ; methyl-
cholanthrene-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 6.13) in this experiment
suggest that differences in tumor response of mice to systemic administra-
tion of methylcholanthrene are sex related and that sex-related differences
are strain dependent.
Wistar rats given daily intragastric instillations of 3-methylcholan-
f
threne for nine 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 6.14. Of
122 treated females, 115 (94%) developed 120 total neoplasms. Of these,
117 were malignant. Of 81 treated males, 23 (28%) developed 26 malignant
-------
Table 6.13. Systemic tumor Induction In mice with 3-methylcholanthrenc by the intragastrlc route
Strain
BTO
Treated male
Untreated 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
Jt
2
5
1
3
0
5
0
3
0
3
4
0
0
6
0
9
0
0
0
Lymph omas
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
ON
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
2
0
0
Amyloidosls
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
, All treated animals received 1 mg 3-mcthylcholanthrene in olive oil.
Controls received olive oil alone.
Source: Adapted from Akamatsu and Barton, 1974. ,
t_n
00
-------
Table 6.14. Influence of sex on response to carcinogens
Species
Wistar rat
Fisher rat
Lister rat
B6C3F! mouse
C3AFi mouse
Buffalo rat
C3H/HeN
mouse
C57BL/6JN
mouse
Albino mouse
Age at start chemical
of experiment
6 weeks
3-6 months
2-3 months
1 day
15 days
42 days
1 day
IS days
42 days
1 day
IS days
42 days
1 day
15 days
42 days
4 weeks
12 weeks
24 weeks
52 weeks
8 weeks
8 weeks
8 weeks
3-Methylcholan-
threne
3-Methylcholan-
threne
Dlmethylbenz-
anthracene
Benzo (a) py rene
,.
Benzo (a) py rene
Benzo (a) py rene
Benzo.(a)pyrene
Diethylnitros-
amine
4-Hydroxyamino-
quinoline 1-
oxide (4HAQO)
4-HAQO
4HAQO
Dose
270 mg
(total)
1.6 mg per
week
0 05 °'5Z
ml l-0%
1 2.0%
0.05 °'5%
"ml l-°*
1 2.0%
75 ug/g body
weight
150 yg/g body
weight
75 ug/g body
weight
150 iJg/g body
weight
0.0114%
1 mg
1 mg
1 mg
Route
Intragastric
instillation
Skin painting
Salivary gland
I
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% Cruensteln, 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% Glucksman and
33% Cherry, 1971
71%
49%
70%
100%
7% Vesselinovltch et
7% al., 1975a
0%
18%
7%
0%
2%
2%
0%
22
2%
0%
21% Reuber. 1976
38%
23%
0%
20% Shirasu, 1965
10%
9%
a\
Ul
vo
-------
Table 6.14 (continued)
<, , Age at start „. . ,
Species ^ . Chemical
of experiment
Holtzman —
Fischer rat —
Wistar rat —
B6C3F, 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-Dlmethylamino-
azobenzene
p-Dimethylaraino-
benzene-1-azo-
2-naphthalene
p-Dime thy lamino-
azobenzene
2-Diacetylamlno-
f luorlne
Dose Route
• — Diet for 7.5
months
— Diet for 7.5
months
— Diet for 7.5
months
50 ppm Diet
150 ppm Diet
I
0.075Z Diet
0.060Z Diet
0.075Z Diet
v
1.2 mg Subcutaneous
166-217 rag/kg Diet
body weight *
Tumor
type
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Tumor
incidence
Male
60Z
100Z
100Z
6Z
44Z
75Z
85Z
982
Female
25Z
90%
100Z
26Z
94Z
17Z
19Z
100Z
No difference
60Z
33Z
29Z
«
12. 5%
OZ
86Z
Reference
Farber, 1963
Vessellnovitch, Rao,
and Mlhallovltch,
1975b
Mulay and O'Gara,
1959
Baba and Takayama,
1961
Morris and
Ferminger, 1956
-------
Table 6.14 (continued)
Age at start
Species of experiment
Holtzman rat —
Fischer rat —
Wistar rat —
B6C3F, 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
AXC rat
Intact 2-4 months
Castrate
Castrate-
hormone
Chemical Dose
Kthloninc —
•
Ktliiimine —
Ethionine —
Bcnzidene 50 ppm
hydrochloride 150 ppm
'
p-Dimethylamino- 0.075Z
benzene-1-azo-
1-naphthalene
p-Dimethylamino- 0.060Z
azobenzene
p-Diraethylamino- 0.075Z
•; ; ..,'.:
p-Dimethylamino- 1.2 mg
azobenzene
/ *
2-Diethylamino- 166-217 mg/kg
fluorine body weight
Route
Diet for 7.5
months
Diet for 7.5
months
Diet for 7.5
months
Diet
Diet
Diet
Diet
Diet
Subcutaneous
Diet
/
1 /
Tumor
type
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Tuao r
incidence
Male
601
100Z
100Z
6Z
44Z
75Z
85Z
98Z
Female
25Z
90Z
100%
26Z
94Z
17Z
19Z
100Z
No difference
60Z
33Z
29Z
-
12. 5Z
OZ
86Z
Reference
Farber. 1963
Vesselinovitch, Rao,
and Mihallovltch,
1975i
Mulay and O'Cara,
1959
Baba and Takayama,
1961
f
-------
6-62
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
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 differ-
ences 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)benz-
f
anthracene. Although he reported differences in types of skin tumor which
s
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 admin-
istration. In Zackheim's experiment the route was external (skin painting),
whereas* the route "used in the othei? study was internal (intragastric).
Glucksman 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 6.14). These effects were con-
firmed 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% dimethylbenz-
anthracene was injected, the sex difference disappeared, suggesting that
the sex differences observed were related to carcinogen dose and were
therefore, quantitative rather than qualitative.
-------
6-63
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 per mouse (75 or 150
ug/g body weight) of the carcinogen (Vesselinovitch et al., 1975<2). The
life span study revealed specific effects associated with each of the mod-
ifiers (Table 6.14). 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 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 a"ge 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% diethylnitrosamine for 26
weeks. A^t 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 difference between the sexes was slight
by 28 weeks; in those that received the first treatment at 24 weeks tumor
f
incidence between the two sexes was practically the same by 36 weeks. The
differences in the responses of the males and females dimimisnhed with in-
creasing age and this finding supports the previously cited data of
Vesselinovitch et- al. (1975a). See Table 6.14.
As in the experiments of Akamatsu and Barton (1974), with methylchol-
anthrene, the strain of test species was shown by Shirasu (1965) to influence
-------
6-64
the difference in sex response of mice to 4-hydroxyaminoquinoline-l-oxide.
Results of the experiment are summarized in Table 6.14. Nine months after
starting subcutaneous injection of 1 mg of the carcinogen (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 C57B1/6JN
strain had leukemia. Subcutaneous injection of 4-nitroquinoline-l-oxide
oroduced sarcomas in 10% of the C3H/HeN males but none in the females.
One other experiment which showed sex-related differences in response
to amino coraoounds was that of Farber (1963) who observed the response of
Holtzman .rats to heoatocarcinoeenesis. with ethionine. Feeding the chem-
ical 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) which were tested in the same
experiment developed liver cancers (Table 6.14).
f
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, 19752?).
When the carcinogen was administered intermittently instead of continuously
the sex difference was abolished (Table 6.15). Regardless of the schedule
-------
Table 6.15. Comparison of continuous versus Intermittent administration of BZ-2HC1 on development of
liver, HarJerian gland, and lung
I.Ivor tumors
Dose
(ppm)
50
100
Sex
M
F
M
F
Cunt inuous
Ratio11
3/50
13/50
11/50
32/50
2
6
26
22
64
Intermittent
Ratio
3/75
4/75
12/75
17/75
1
4
5
16
23
llardurlan gland tumors
Cunt inuous
Ratio
9/50
3/50
18/50
3/50
t
18
6
36
6
Intermittent
Ratio
8/75
2/75
11/75
5/75
Z
11
3
15
7
Lung adenomas
Continuous
Ratio
2/50
2/50
3/50
2/50
X
4
4
6
4
Intermittent
Ratio
17/75
5/75
19/75
4/75
2
23
7
25
5
.Animals were given food containing specified amounts of BZ*2IIC1.
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/number of BZ*2HCl-treated animals.
Source: Adapted from Vesselinovitch, Rao, and Mihailovitch, 19752>.
-------
6-66
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 car-
cinogen 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 tumour induction in males in
particular.
Malay and O'Gara (1959) demonstrated that Osborn-Mendel male rats
fed p-dimethylamino-benzene-l-azo-l-naphthalene(DAN) and p-dimethylamino-
azobenzene(DAB) were more sensitive to liver tumor induction than females.
(See Table 6.14). 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 been
f
greatly different 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 carcinogenesis sex of the animal does not affect the
transformation of normal cells to malignant ones, but that it does affect
-------
6-67
proliferation of the cells, and thus appearance of the cancer. This sup-
ports the opinion of Glucksman and Cherry (1971) that sex-related differ-
ences 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) which, 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 diethyl-stilbesterol were less suscept-
ible (Table 6.14). 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 carcinogenicity
in B6C3F1 mice and in Osborn-Mendel rats. Some of the sex differences
seen after 78 weeks of dosing by gavage were:
1. ^Chloroform-rtreated male rats, had an increased incidence of
kidney tumors while 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 car-
cinomas 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.
-------
6-68
Both sexes of mice have been found to be equally susceptible to the
hepatocellular effects of carbon tetrachloride (Andervont, 1958; Weisburger,
1977) while female Buffalo strain rats have appeared to be more sensitive
than males (Reuber and Glover, 1967).
6.2.9 Conclusions
The need for lifetime exposures in chronic toxicity and carcinogenicity
^
tests limits the choice of primary test animals to relatively 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 dist-
ant second and third chioces respectively.
In addition to the primary test animal, most authorities recommend
the use of a second species in chronic tp^icity 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 a long-term toxicity; however many species do respond to a vari-
ety 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 which most
closely resembles man would be chosen, but cost and time requirements do
not always allow this. The choice may be simplified through use of
techniques such as those involving the use of structure-activity relation-
ships, metabolism studies, and in vitro screening. These techniques, in
-------
6-69
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 unstandardized
data between experiments conducted in different laboratories. There is
a definite need for basic, standardized animal experiments which allow
for the comparison of the responses of various species to chemical car-
cinogens or classes of carcinogens.
The number of animals used in chronic toxicitv and carcinoeenicitv
tests represents a compromise between requirements for good statistical
precision, and reasonable costs and work loads. Early investigators used
10 or less test animals per dose group, but'most authorities now recommend
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 arouo comorised 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 frequently
used in carcinogenicity testing.
General agreement exists among authorities on the need to start ani-
mals 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,
-------
6-70
weanlings or immediately post-weanling 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 new-
born animals, taken alone, may not be reliable indicators of the carcino-
genicity 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 interferences of
the test material so limit the effectiveness of thie approach that tests
involving in utero and bigenerational exposures are performed only infre-
quantly. As a consequence, much more experience with these methods will
be required before their general application to carcinogenesis 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 preado-
lescent 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
sex appears to regulate the growth of the induced neoplasm. The mechanism
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
-------
6-71
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 differences 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 chemical; and duration of the experiment. Therefore, reliable
testing of chemicals with unknown characteristics, using only one sex of
experimental animal, would be virtually impossible.
6.3 ROUTES OF ADMINISTRATION
f
The mode of exposure of expeirmentalxanimals 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 6.3.1 provides an evaluation of
advantages and disadvantages of these' exposure routes in tests for chronic
toxicity and carcinogenicity and will include a description of other less
common routes which have specific application in tests for carcinogenicity.
In section 6.3.2 examples are presented to illustrate differences or
similarities observed in a given species following carcinogen exposure
by different routes.
-------
6-72
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 mix-
tures, or by administration in drinking water. All three methods are useful
for long-term administration and permit reliable quantitation provided in-
take and body weight are measured routinely (Ministry of Health and Welfare
Canada, 1975). Sontage, Page, and Saffiotti (197.6) 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 since
less test agent is needed, less storage space is required. Animal handling
is increased, allowing frequent observation for clinical symptoms. Gav-
s
age has certain disadvantages: (1) intake of the test agent may be less
than maximum, (2) a solvent is often required, (3) mortality may be in-
creased because of trauma, and (4) animals must be closely matched by
weight.
Feeding through diet mix and drinking water have other advantages
and disadvantages. While 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 inconsistent and palat-
ability may be altered. Furthermore, .the concentration of the test mate-
rial in the diet must be altered weekly or biweekly during the early part
of the experiment to maintain a constant dose level (in mg/kg body weight)
since toxicity is related to dosage per unit of body weight, and food con-
sumption per unit body weight decreases with increasing animal age (Weil,
1973). Also, potential exposure of workers who mix and dispense feed
and water which contain toxic chemicals must not be overlooked.
-------
6-73
Dietary feeding is generally recommended for adult animals (Weisburger
and Weisburger, 1967), while 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 admin-
istration to large species. Ministry of Health and Welfare Canada (1975)
suggested experimental use of the oral route of exposure, in certain in-
stances, even when it is not the primary mode ofjiuman exposure. However,
substitution of the oral route would be feasible only if levels of the com-
pound 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.
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 occationally used for carcinogenicity studies (pellet implanta-
tion and tracheal washing) will be discussed. The most significant advan-
tage 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 inhala-
tion 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 u, (3) the respiratory anatomy of man and animals differ
in several respects, and (4) it is difficult to determine the actual dose
inhaled.
-------
6-74
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 concomittantly
with inhalation exposure (National Academy of Sciences, 1975). In addition,
large particles and large doses can be administered, the nasal filter in
rodents is bypassed, skin contamination is avoided, and there is less
s
risk to personnel from carcinogen exposure.' Intratracheal instillation
is currently the method of choice for testing suspected respiratory car-
cinogens (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 respiratory tract. Ham-
sters appear to be the most used animal in experiments requiring intra-
tracheal injections. Size of the animals, their low incidence of sponta-
neous 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
-------
6-75
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, 19J8). This relatively
new exposure technique is complex and traumatic to the animal, and little
is known to date of its real potential.
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).
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 which
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 which
-------
6-76
are destroyed in the gut. As for disadvantages, the procedure is arduous
and expensive, the absorbed dose is not accurately known (whole body expo
sure 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 (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 differ-
ence 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 der-
mal 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 car-
cinogen 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.
Subcutaneous injection — Subcutaneous injection is a sensitive and
frequently used mode of administration of chemical carcinogens (Page,
/•
19772>). Doses can be accurately delivered, absorption is slow, high
concentrations in the blood are reached rapidly, and the compound circu-
lates without those changes which might occur in the gut following oral
administration. Repeated injections can be time-consuming and local reac-
tions are likely to occur (Canada, 1975), but the appearance of subcutane-
ous sarcomas at the site of injection constitutes the most controversial
-------
6-77
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 implan-
tation 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 connec-
tive tissue reaction, which has been carefully characterized histologically,
is responsible for the malignant tumors that are produced in the subcutane-
ous tissue of rats and mice by the following: (a) solid implants, (ib) in-
jection of hypertonic, acidic, or surface active solutions, (a) 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.
Homb^urger 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, an rats. Also, strain differences were
observed among the mice tested.
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 admin-
istration (Weisburger, 1976), and injection may be done repeatedly if the
-------
6-78
carcinogen is soluble and if there is no vehicle accumulation. Intra-
peritoneal injection does not resemble the usual routes of human exposure.
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 carcinogens. How-
ever, the 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 interpretation of
results obtained by the technique. Therefore, any chemical clearly
carcinogenic by bladder implantation would be considered suspicious but
should*then be tested by more conventional assays.
Diethylnitrosamine in the Hamster — The effects of route of administra-
tion on the carcinogenic action of diethylnitrosamine have been tested in
the Syrian hamster (Herrold and Dunham, 1963; Herrold, 1964a, 1964Z?). The
routes tested were intragastric, intratracheal, subcutaneous, intradermal,
topical, and intraperitoneal. Results of the experiment are summarized in
Table 6.16. 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 intragastric or intratracheal routes (Herrold, 1964a).
-------
Table 6.16. liutiicclon of tumors In the Syrian hamster with illi-ihy lul troKaralne
Koulu
„„.....,, pl,lu.|,
Int radcrmal
Intraperitoneal
Topical
Pregnant female
subcutaneous
Subcutaneous
Tntratraclieal
Intragastric
titimlier of
Dose
animals
10 0. 1 ni£ in sjl I in-
(total » 7.5 my) *
19 3.5 mu in water
(total « 70-84 my)
18 2 mg in saline
(total = 32-56 mg)
8 Undiluted then 1:1
in water
3 5, 8, or 10 mg in
water
15 2 mg in water
(total = 32-48 mg)
11 female 0.05 ml of a 1:14 ,
solution in water
14 male
15 female 0.4 ml of a 1:250
solution in water
13 male
.schedule
Twice a week for
about 9 months
Once a week for
5-6 months
Oncu a week for
4-7 months
Once a week for
1 month then
twice a month
for 3 months
1-2 days preced-
ing delivery
(single dose)
Twice a week for
4-6 months
Once a week for
6 months
Twice a week for
} months
0/10
19/19
17/18
6/8
3/3
NS°
11/11
14/14
15/15
-13/13
\
0/10
10/19
4/18
2/8
1/3
NS
4/11
10/14
6/15
4/13
Tuintir incidence
Nasal cavity
Anterior Posterior
0/10 0/10 0/10
3/19 10/19 13/19
4/18 5/15 11/15
4/8 6/8 4/8
0/3 1/3 2/3
NS 14/15
0/11 3/9 0/11
•
0/14 1/13 0/14
10/15 6/11 7/15
12/13 4/9 5/13
Reference
Mlllevskaja and
Klseleva. 1976
Herrold, 19f>4b
Herrold, 19642>
Herrold, 1964b
i
!
Herrold, 1964i
Herrold, 1964a
<3\
Herrold and -^4
Dunham, 1963 ^>
Herrold and
Dunham, 1963
.Microscopic lesions which may represent an early stage In tumorigenesls.
_Dose and schedule were changed after animals died during the first month of treatment.
,NS — tumors observed but incidence not specified.
u
Doses administered intratracheally and intragastrlcally were approximately the same.
-------
6-80
Although liver tumors did not develop in animals injected intratracheally,
atypism of the hepatic cells was observed (Herrold and Dunham, 1963). There
was no local effect on the skin or in subcutaneous tissue (Herrold, 19643).
Transplacental transfer of the carcinogen was not demonstrated by Herrold,
but Mohr (1973) described extensive experiments in which he and his asso-
ciates induced tumors transplacentally with diethylnitrosamine.
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 nine months failed to
produce local or remote tumors. The small number of animals (ten) or the
route of administration are possible reasons for the lack of carcinogenic-
ity of diethylnitrosamine in the experiment. The total dose administered
s
to the buccal pouch, however, was only one-tenth of the intradermal dose
required to produce tumors in hamsters (Herrold, 19646); therefore, the
lack of effect could be attributed to insufficient dose.
Methylcholanthrene in the mouse — Skin carcinomas have been induced
in mice painted with single large doses (0.1 nil 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 6.17 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 appear-
ance 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 raethylcholanthrene (Klein,
-------
Table 6.17. Local tumor induction in mice with 3-methylcholantlirene by various routes of administration
Route Strain
Skin painting Kreyberg
CF-1
DBA
Number
of
animals
30
28
34
Mcthylcholanthrcne
dose
0.1 ml of 0.6% in
acetone
0.15% in acetone
0.5% in olive oil
Exposure schedule
4 applications at
15 min intervals
Twice a week for
30 weeks
Once , followed by
Average
induction
time
10.5 weeks
Not clear
59 days
Tumor
incidence
7/30 (skin)
23/28 (skin)
27/34 (skin)
Reference
Bielschowsky
and Bullough,
1949
Shubik, 1950
Klein, 1952.
Comments
3/7 carcinomas (par-
tial autopsy)
2 malignancies in
69 total tumors
(partial autopsy)
35
Intramuscular Albino A 40
4D
32
32
35
36
.. 36
35
33
37
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
application of
5% croton oil in
olive oil 1-3
times per week
for 53 applica-
tions
Once, with no
additional
treatment
Single injection
160 days
Single injection 95 days
Single injection 168 days
Single injection 123 days
Single infection 183 days
Single injection 185 days
Single injection 254 days
Single injection 272 days
Single Injection
Single injection
0/35 (skin)
Klein, 1952
20/40 (muscle) Klein, 1951
40/40 (muscle) Klein, 1951
9/32 (muscle) Klein, 1951
22/32 (muscle) Klein, 1951
5/35 (muscle)
3/36 (muscle)
1/36 (miscle)
1/35 (muscle)
Klein, 1953
Klein, 1953
Klein, 1953
Klein, 1953
0/33 (muscle) Klein, 1953
0/37 (muscle) Klein, 1953
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)
00
-------
Table 6.17 (cimtlmu-d)
.Icl liy] rliu l.nit liriMiL- .. , , , , , , liin..ir
Route strain ul , Lxposurv bchfiliilr Inilucllon .. a
ilu.s«- ' , inr Idunco
an I ma 1 H t im».'
Snliriit.iii.-ou:> Swiss J4 O.'i ny In llilo- Single Injection l'J/24 (skin)
|)lu'lli--f IV C
hi-nZel*C
2/24 (cuisclii)
19/24 (suhcuta-
lu-oiis connec-
tive tissue)
3/24 (skin)
6/24 (skin)
Intratraclieal BC3Fi 36 0.5 mg in 0.2X Once a week for First mouse 31/36 (lung)
gelatin 6 weeks died in
4 weeks,
last In
24 weeks
DBA/2 - 50 0.5 rag in 0.2% Once a week for 4 3/18 (lung)
gelatin weeks
.
Bladder Albino 37 12.5% suspension Continuous up to 18/37 (urinary
implant of 4 weeks bladder)
pellets
t
38 20% suspension Continuous* up to 22/38 (urinary
A weeks \ bladder)
Kttfvrviicc
Bhlsey .mil
Slrsat, 1975
Nettesheim and
Haramons. 1971
Nettescheim and
Mammons, 1971
Bonser ec al. ,
1963
Bonser et al. ,
1963
Coiitfii/nl s
S(|vi.n-.uub ci-11 corci-
nunia (part la 1
autopsy)
Klial>domvi>s.iri:o.~ias
(partial autopsy)
Klbrosarcoi:i.is (par-
tial autopsy)
1'api 1 lum.is (partial
autopsy)
Sebaceous cysts ([par-
tial autopsy) :
Squaraous cell
carcinomas
Squamous cell car-
cinomas (22 O\
animals were still .L
alive) N>
Carcinomas (partial
autopsy) 12Z of
controls developed
tumors
Carcinomas (partial
autopsy) 4.8Z of
controls developed
tumors
Number of tumor-bearing mice per total number of mice at risk.
-------
6-83
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 carcinogen injected alone. How-
ever, 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 hydsocarbons has been used
primarily for the induction of connective tissue tumors. Epidermal changes
which 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 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 topic-
ally painted with methylcholanthrene. He concluded that "when methylchol-
anthrene is administered subcutaneously to Swiss mice, epidermal hyperplasia
is not a prerequisite for epidermal tumorigenesis."
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 respira-
tory tracts of mice (Nettesheim and Hammons, 1971). DBA/2 and BC3Fj. mice
were injected weekly with 0.5 mg of 3-methylcholanthrene for four and six
-------
6-84
weeks respectively. Of the 36 BC3Ft mice, 31 developed squamous cell car-
cinomas, the first appearing four 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
f
including skin, forestomach, liver, lung, and lymphatics (Table 6.13).
s
Treated animals also developed amyloidosisi which was significantly
correlated with gastric neoplasms.
Strain A mice, highly susceptible to chemical induction of pulmonary
adenomas, developed an increased number — over controls — of lung tumors
four weefcs after intravenous injectidn of 0.05 to 1.5 mg methylcholanthrene
in horse serum and cholesterol (Table 6.18) (Shimkin, 1940). The incidence
of tumors was 100% in three 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 by three months. Dif-
ferences seen in carcinogenic potency of 20-methylcholanthrene (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 6.19 and 6.20). These results suggest
that the subcutaneous route is less sensitive than the intravenous route
for lung tumor induction in strain A mice.
-------
Table 6.18. Incidence of pulmonary tumors in strain A mice after intravenous
injection of methylcholanthrene I
1.5 mg
Time
(weeks)
3
4
5
6
13
20
Mouse
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
Mouse
9
10
10
10
18
6
Dosage
0.5 mg
Number
.with
tumors
of the
lungs
1
8
\ 10
10
18
6
Average
number of
tumors
of the
lungs Mouse
1
5
14
25 7
30 10
47 15
1
0.1 mg
Number
with
tumors
of the
lungs
3
8
15
Average
number of
tumors
of the
lungs
2
4
11
00
Source: Adapted from Shimkin, 1940.
-------
6-86
Table 6.19. Incidence of pulmonary tumors in strain A mice after
intravenous injection of 0.5 g of methylcholanthrene or 0.5 mg
of dibenzanthracene
Methylcholanthrene (0.5 mg)
Dibenzanthracene (0.5 mg)
Time
(weeks)
3
4
5
6
Mouse
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
Mouse
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,
-------
6-87
Table 6.20. Incidence of pulmonary tumors in strain A mice given
subcutaneously 0.5 mg of methylcholanthrene or 0.5 tng of
dibenzanthracene dispersed in 0.5 cc of horse serum and cholesterol
Methylcholanthrene
Dibenzanthracene
Time
(weeks)
3
A
5
6
Mouse
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
Mouse
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.
-------
6-88
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 six-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 (2 mice had
adenocarcinomas), 12% developed genital tract cancer, and genital tract
dysplasia occurred in 27%. Six of the 24 mice w^th lung adenomas also
had cancer and/or dysplasia of the vagina and/or cervix uteri.
Aromatic amines in mice, rats, and rabbits — Bonser et al. (1952)
compared carcinogenesis of 2-naphthylamine and 2-amino-l-naphthol hydro-
chloride in several species by several routes of administration. Implan-
tation of 2-amino-l-naphthol pellets (10% to 15% carcinogen) induced
metaplasia, papilloma, and carcinoma in the bladders of mice, and subcu-
taneous injection (5 mg per 100 g 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 6.21).
Inhalation of benzidine causes bladder cancer in men (Goldwater,
Rosso, and Kleinfeld, 1965) and has been shown to be carcinogenic in cer-
tain animal species. Rats, after inhaling benzidine for 13 months, devel-
/'
oped 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 PHS 149). The absence of
tumors of the urinary bladder of rats receiving benzidine by inhalation,
and by subcutaneous and oral administration led Zabezhenskii to conclude
-------
Table 6.21. Tumor Induction with aromatic amines by various routes of exposure
Chemical /species
2-Amino-l-naphthol
hydrochloride
Mouse
Mouse, "stock"
Mouse, R III
Rat, albino
2-Naph thy lamine
Mouse
Mouse, IF
Mouse, CBA
Rat, albino
Mouse, IF
Rabbit
Route of
exposure
Bladder
pellet
Subcutaneous
Subcutaneous
•
Subcutaneous
Bladder
pellets
Cavage
Gavage
, Diet
Diet (low
protein)
Skin painting
Spoon feeding
Dose
"l-2 mg/pellet
5 mg/100 g body
. weight/2 weeks
5 mg/100 g body
weight/2 weeks
5 mg/100 g body
weight/2 weeks
1-2 mg/pellet
5 mg/2 weeks in
arachis oil
120 mg/kg body
weight /twice/week
160 mg/kg body
weight/week
310 mg/kg body
weight /week
Saturated solution
100 mg/kg body
weight/week
Duration of
exposure
(weeks)
22-28
30-39
Up to 80
Up to 80
Up to 80
39
u*p to 72
Up to 89
Continuous
N
>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
4/15 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
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
CT>
OO
VO
-------
Table 6.21 (continued)
,.,,!! . K.IUI Ollf
(.licalcnl/spcclcs Dose
vxpusure
ItenziJlne
Rat, lion 1 lib red hih.il.it Ion 4«hmirs/day , 5
days /week
Benzidine,
technical
Rat Subcutaneous 15 mg/week
Benzidine, pure
Rat Subcutaneous 15 mg/week
.
,.
I
Benzidine sulfate
Rat Subcutaneous 15 rag/week
Oral 15 mg/week
Dur.it Ion of Tumor
exposure Tumor incidence latency
(weeks) (weeks)
80 5/2H Icukenl.ns 52
1/28 hep.itom.-i
2/28 B.-inun.iry
tumors
Life 8/233 hepatomas
0/233 bladder
tumors
7/385 gastric
tumors
54/233 auditory
canal tumors
Life 6/152 hepatomas
0/152 bladder
tumors
32/152 auditory
canal tumors
,
Lite 1/153 hepatomas
0/153 bladder
tumors
16/153 auditory
canal tumors 1
Life 1/37 hepatomas
" 0/37 bladder
tumors
2/37 auditory
canal tumors
Reference
Zablzlilnskii.
1970
Spitz et al. ,
1950
Spitz et al., 1
1950 o
Spitz et al. ,
1950
Spitz et al. ,
1950
-------
6-91
chat 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.
6.3.1. 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. Experi-
ments 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 which can determine site, histological type, and
incidence of tumors.
6.4 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 rate's 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 pharmacokinetic
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).
-------
6-92
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 set at a
level intermediate between the high and low dose (World Health Organiza-
tion, 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).
While a test for carcinogenic potential might occasionally entail
only a single well-selected dose, tests for risk estimation or determina-
tion 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 con-
troversial subject of dose selection in carcinogenicity studies are pre-
sented in Table 6.22.
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6-93
Table 6.22. Summary of recommendations on dose selection
Agency
Recommendation
Food and Drug Administration,
1971
Health Protection Branch,
Department of National
Health and_Welfare,
Canada, 1973
International Union Against
Cancer, 1969
National Cancer Institute,
1976
"At several dtfse levels — one likely to
yield maximum tumor incidence."
"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 MTD 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 les-
ions (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."
Source: Adapted from Munro, 1977.
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6-94
Weisburger (1976) emphasized the selection of the maximum tolerated
dose (dose at which mortality is low in an eight-week test and which may
depress weight gain 5% to 20% with an optimal depression of approximately
10%). The rationale given by Munro (1977) for use of maximum tolerated
dose (MTD) include: (1) when weak carcinogens are tested, high doses for
long periods of time increase chances of seeing statistically increased
numbers of tumors within the lifespan of the animals (since cancer induc-
tion in animals is both time and dose-related); and (2) increased confi-
dence 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) that MTD may result in a 10% weight loss 'is considered to be
inappropriate because it may affect biochemical or physiological processes
which make the animal more or less susceptible to carcinogenic challenge;
(2) metabolic overloading 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 very toxic compounds which can only be used at lower dose levels,
thus reducing the change of tumor formation; (4) there is difficulty in
handling data when the criteria for MTD is 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
required, this is highly impractical with doses which 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
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to every situation. Page (19776) 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: (a) continuous inhalation
exposure up to 12 months, (b) continuous exposure for six months, and
(c) brief exposure for 1 month to high concentrations of the chemical.
The rate of tumor formation was enhanced in animals of the interrupted
(six months) exposure group while 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
-------
6-96
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.
In chronic toxicity evaluations the test compound should also be
administered seven days per week (Benitz, 1970). Treatment five days a
week results in a 28.6% decrease of exposure and.results in a 48-hr weekly
recovery period. The administration of the equivalent of seven doses
during five 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; National Academy of
Sciencas, 1975, 1977; Food Safety Council, 1978; World Health Organization,
1978); however, there is no consensus concerning the length of this period.
Conceptionally, 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 three or four 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 two weeks and only one compound
caused an additional effect after the first three months (Peck, 1968).
Similarly, in a chronic study of 46 compounds, the Ciba Drug Company
-------
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noted all indications of toxicity within the first eight 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 three to six months. Because of
this characteristic early detection of most chronic, effects, many investi-
gators feel that the duration of chronic tests need not exceed six months
(Barnes and Denz, 1954; McCollister, 1974, as cited in McNamara, 1976;
Paget, 1963, as cited in McNamara, 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 relatively brief intervals, thanr^with investigators dealing
with occupational 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 scientists
insist that lifetime or near-lifetime exposures of laboratory test animals
are required (Loomis, 1974; National Academy of Sciences, 1975; Food Safety
Council, 1978; 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
f
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.
-------
6-98
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 four 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 compounds and
15 dose levels produced toxic signs after, but not before, three month's
exposure. It thus appears that in most instances all' significant toxic
effects are indeed observed within three to four months, but that excep-
tions occasionally occur. Since 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
maximize^ 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.
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
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6-99
test animals are considerably greater than that indicated for mice and
rats. Table 6.23 shows that chronic tests of even two year's duration
represent only a small fraction of the life span of most nonrodent labor-
atory test animals. Needless to say, these animals are only rarely chosen
for life span exposures.. Among the publications examined during the prep-
aration of this chapter, no life span exposures of nonrodents were found,
but one 7-year dog study and one 43-month monkey styady 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 one year. However, several investigators have reported
previously unobserved toxic effects in dogs after periods of exposure greater
than one year (Braun et al., 1977; Kaplan and Sherman, 1977; Weil et al.,
1971). Consequently, some authorities recommend exposing dogs longer, for
periods of two to seven years (Goldenthal, 1968; Loorais, 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 spe-
cies was two years.
In conclusion, dose selection is a critical astiect of lone-term
f
testine. 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
-------
Table 6.23. Time relationships among drug exposure, life span, and time equivalents in man
Duration
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
Huraan
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
(Z)
0.55
1.1
1.6
3.3
6.6
13
Human
equivalent
(months)
4.5
9
13
27
53
107
O
O
Source: Adapted from Benitz, 1970.
-------
6-101
of toxicity. The remaining dose should be set at an intermediate level
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 relationship
is a goal of the test, then more than three dose levels may be required.
The test compound should be administered seven days per week; treat-
ment five days a week or administration of the equivalent of seven doses
during five 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 conc'erning the length of this
period. Conceptionally, 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 three or four months,
and many authorities feel there is only a low likelihood that additional
effects will be observed after six months of treatment. Nevertheless,
a few new effects are occasionally seen during longer periods of treat-
ment and in carcinogenicity studies, .the chance for late development of
long-term effects is even greater. Since 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,
-------
6-102
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 subj ected to
life span exposures. However, a few literature examples have been noted
of seven-year exposures for dogs.
When long-lived animals are chosen as second species to mice or rats
in non-carcinogenic 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 two years.
6.5 INTERIM SACRIFICE
Interim sacrifices are generally considered useful in the study of
f
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 (National Academy of
Sciences, 1977; Food Safety Council, 1978). For these reasons, some
authorities recommend the inclusion of serial sacrifices in chronic
toxicity.tests (Fitzhugh, 1955, as cited in National Academy of Sciences,
-------
6-103
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. Fre-
quently, 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 8 studies involved
the examination of several animals during several intervals of the test
period. Significant observations derived specifically from interim sac-
rificaes ^were cited.in only 4 of these reports.
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
-------
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extensive use of interim sacrifices during chronic toxicity studies. In
the literature sample represented by the bibliography for this chapter,
interim sacrifices were reported in only 12 papers. Of these, only 8
studies involved the examination of several animals during several inter-
vals of the test period, and significant observations based on interim
sacrifices were reported in only 4 papers. It thus appears that interim
sacrifices are not a generally used, or needed, feature of chronic tox-
icitv tests.
6.6 DATA COLLECTION AND EVALUATION
»
Long-term tests are unique research experiments that are only rarely
amenable to standardized protocols (Food Safety Council, 1978). Neverthe-
less , the success of a well planned chronic'toxicity or carcinogenicity
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 patho-
logical procedures essential to the successful execution of long-term tests.
6.6.1 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; Magee, 1970;
Loomis, 1974; Sontag, Page, and Saffiotti, 1976; National Academy of Sci-
ences, 1977; Food Safety Council, 1978; 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
-------
6-105
of dose when the test material is administered in the diet. When the test
compound is administered in drinking water, the weekly consumption of this
material is also required for dose determinations.
6.6.2 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 mini-
mize the effects of extraneous factors on the course of chronic toxicity
and carcinogenicity experiments, several authorities recommend that quali-
fied employees observe the general physical condition, viability, and
adverse behavorial changes of all test and control^animals at least twice
daily, seven days a week (Arnold et al.,,i977; Fox, 1977; National Academy
of Sciences, 1977; Food Safety Council, 1978; World Health Organization,
1978). Animals with obvious life-threatening conditions should be isolated;
those unlikely to survive an additional day should be sacrificed and necrop-
sied to preserve tissues for histological examination. Each animal should
»
be completely examined at least once a week by qualified personnel for
unusual behavioral patterns, respiratory signs, bleeding, and palpatable
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 biochemical analyses of sample material, such as blood
-------
6-106
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 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 National Academy
of Sciences (1977) questioned the benefits of prescheduled 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.
-------
6-108
microscopically. Early workers recommended microscopic examination of
all obviously altered tissues plus an additional quantity that generally
did not exceed 20 (Barnes and Denz, 1954; Abrams, Zbinden, and Bagdon,
1965). 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 with-
out indicating more specific requirements (Food and> Drug Administration,
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 (Peck, 1974;
National Academy of Sciences, 1977; Food Safety Council, 1978; World
Health Organization, 1978). The National Cancer Institute guidelines
for chronic toxicity tests are even more stringent; they require micro-
scopic examination of approximately 40 tissues from all test and control
animals, except positive controls which may be exempted from this require-
ment (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 available to perform histologic examina-
/
tions of tissues from tests required or expected under the Toxic Substances
Control Act and other expanded government programs is insufficient if
microsconic examinations of 40 or more tissues from all test and control
animals are required (Page, 19772?).
-------
6-109
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, neoplastic, preneoplastic, and hyperneoplastic. Also as dis-
cussed 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 in-
corporated in the test design (Page, 1977a).
Microscopic analysis is usually performed on at least some tissues
in most currently performed long-term tests. Histoiogical examination of
tissues was reported in 92% of the primary' chronic toxicity studies refer-
enced at the end of this chapter. Sixty percent of these papers indicated
that "all major organs" or "all major organs and tissues" were so examined.
Ten percent of the references mentioned histological examination of six or
less specific organs, and 14% referred to microscopic examination of only
one or two specific organs. In 8% of the references, organs examined
microscopically were not identified. In a similar number of papers,
histological examination of tissues was apparently not performed at all.
6.6.4 Conclusions
The successful execution of chronic toxicity and carcinogenicity
tests requires careful collection and evaluation of clinical and patho-
logical data. All animals should be observed twice daily, seven days a
week, by qualified personnel for changes in physical condition, viability,
and behavior. Body weight should be measured weekly during periods of
-------
6-110
rapid growth; and at least monthly thereafter. Food consumption should
be similarly measured, except when the test material is administered in
the diet; then, weekly measurements are required for dose determinations.
Each animal should be examined at least weekly for unusual behavioral
patterns, respiratory signs, excretory products, and palpatable masses.
as well as other abnormalities. In noncarcinogenie chronic tests there
is general agreement that animals showing obvious s^gns 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 authorities
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 micro-
»
scopic examination of all sample tissues is highly desireable from the ex-
perimental 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 examinations of 40 or
more tissues from all test and control animals are required. It thus
appears that, at least for the next few years, limitations must be placed
on either the number of long-term tests performed, if total histologic
tissue analysis of all animal samples is required, or the number of
animals, or tissues per animal examined histologically must be substantially
-------
6-111
restricted. In the literature analyzed in this study, most workers per-
formed histological 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" cor-
respond 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.
6.7 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 practic-
+
ally impossible. Short-term in vitro screening procedures have been
developed which 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 trans-
formation include the following: the Ames Sa£fflone£Za/microsome assay;
»
the polA+/polA- assay (differential toxicity in E. aoH,); "Rec" differen-
tial toxicity in Bacillus subtilis; mouse lymphoma assay (mutation in
cultured mouse cells); and assays for unscheduled DNA synthesis, in vitro
cell transformation, sex-linked recessive lethal mutation in Drosophila,
sister chromatid exchange, and in vitro'Chromosome aberrations. These
assays are being examined closely in the gene-tox workshop series which
is sponsored by the 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).
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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 metabolic
activation of chemical carcinogens in the host animal. These tests are
designed to shorten tumor induction time or to detect cellular alterations
or markers which appear shortly after exposure to carcinogens.
6.7.1 Emb rvo Homo eraf t
^
A oromisine techniaue for oroducins tumors with chemicals in a rela-
tively 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 im-
plants 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 formed in the
implants. The quantity of carcinogen required to induce tumors was esti-
%
mated to be no more than 150 pg and the tumors induced were mainly squam-
ous cell carcinomas (with some sarcomas and lung adenomas); 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 intro-
duced the problem of possible effect of the carcinogen on the healing
process, but Davies, Major, and Aberdeen (1971) using a non-surgical im-
plantation technique, were able to induce adenomas in embryonic lung
tissues exposed to 26 yg of 3,4-benzo(a)pyren'e (BAP) or 1,2,5,6-dibenz-
anthracene. Twenty percent of the implants exposed to benzo(a)pyrene
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and 62% of the implants exposed to DMBA 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-methyl-
cholanthrene, 3,4-benzo(a)pyrene, or 1,2,5,6-dibenzanthracene and implanted
subcutaneously into a host animal developed adenomas (some as early as
three months later) as demonstrated by Laws and Flanks (1966) and Davies,
Major, and Aberdeen (1970).
6.8.2 Site Transfer
Homburger and Baker (1967) described studies in which mice and
hamsters received subcutaneous injections of 25y and 500y» respectively,
f
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 found that 10% of the secondary host mice developed pal-
pable tumors by 10 weeks while 14 to 15 'weeks were required for 10% of
the primary hosts to develop palpable tumors. In the hamster study,
tumors palpable four weeks after transfer of the injection sites later
turned out to be malignant fibrosarcomas. These particular injection
sites had been transferred two days after carcinogen injection.
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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 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 one month for the demonstration of carcinogenic potency in vivo in
adult mammalian cells which yield tumors as end points.
f
6.7.3 Partial Hepatectomy
s
Using earlier work of Alexander Haddow (1938) 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 which 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 which impair the growth of surrounding
normal liver cells.
Single doses of diethylnitrosamine' (DEN) 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 (2AAF), and growth stimulation of carcinogen-altered
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hepacocytes by partial hepatectomy (PH). The test can be completed in
four weeks. The procedure in male Fisher 344 rats was as follows: (a)
the rats were injected intraperitoneally with 200 mg/kg diethylnitros-
araine, (b) after a two-week recovery period the animals were placed on
a basal diet containing 0.02% acetylaminofluorene, (c) after one 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 hr 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-acetylaminofluorine. The
system is quantitative, and rapid — macroscopic foci were observed
within 10 days of cell transfer. In a recent reports Laishes, Fink,
and Carr (1980) described in vitro purification of the carcinogen —
altered cell populations isolated from rats treated with the DEN/AAF/PH
regimen. Primary hepatocyte monolayer cultures from carcinogen-treated
and untreated rats were exposed for 24 hr to acetylaminofluorine to
its derivatives, or to certain chemicals known to be cytocidal but
non-hepatocarcinogenic.
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AAF, its derivatives N-OH-AAF and N acetoxy-AAF, and three chemo-
therapeutic 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 chemicals. Studies are underway
in which cells selected in vitro will be transferred to syngenic 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: (a) rats were injected intraperitoneally with
200 mg/kg body weight diethylnitrosamine, (b) two weeks later the animals
were started for two weeks on the test chemical in the diet or drinking
water, (c) after one week of administration of the test chemical partial
hepatectomy was performed, (d) one week later the rats were killed. Var-
ious control groups were established. The end points of the experiment
were the number of liver hyperplastic nodules and the histochemical obser-
vation of, yGT, acid-pohsphatase (acid.-Pase), glucose-6-phosphatase (G-6-
Pase), and adenosine triphosphatase (ATPase) activity. The chemicals
tested included #-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 combina-
tion with diethylnitrosamine and partial hepatectomy, induced statistically
significantly more hyperplastic liver nodules than DEN and partial hepa-
tectomy alone. y~GT activity, not demonstrated in normal hepatocytes or
after partial hepatectomy, was seen in hyperplastic nodules and in the
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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 hyper-
plastic areas. Thus, the presence of y-GT and absence of ATPase are good
markers for detection 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 itr 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 non-carcinogsnic chemotherapeutic
agents are selectively cytocidal to normal cells in vitro. See above).
s
6.7.4 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.
Newborn rats were injected intraperitoneally with ( H)d Thd (tritiated
deoxythymidine) for three weeks. The animals were then exposed to chem-
icals for various periods of time, and tissues were then removed and
homogenized. The ( H)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, thyraus, 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 carcinogens
-------
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and target organs for tumor susceptibility. For example, the hepatocar-
cinogens dimethyInitrosamine, diethylnitrosamine, 2-acetylaminofluorene,
and /IMiydroxyacetylaminofluorene 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 Swenbere (1978) recommended the alkaline elution test
as a confirmation of in vitro tests when the carcinoeenic potential of a
compound is unknown. It can help identify tareet organs for procarcino-
gens and can be utilized to assess DNA damage in organs that are subiect
to toxic changes.
6.7.5 q-Fetoprotein in Serum
s
ot-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-dimethylaminoazoben-
zene. The protein was detected in the sera of approximately 20% of 41 rats
»
by as early as three weeks, and in 76% of the rats by six 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.
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.
-------
6-119
In a later study Kroes, Williams, and Weisburger (1973) tested the
effects of different dose levels of several hepatotoxins and hepatocarcin-
ogens on the early appearance of a-fetoprotein. They found that high levels
of liver carcinogen were required to induce the "early" peak at two to five
weeks. The protein was not detected in the sera of rats treated with hepato-
toxins. Kroes suggested possibly using the a-fetoprotein assay as a short-
terra test to distinguish between hepatotoxins and Kepatocarcinogens.
6.7.6 Strain Susceptibility
Differences in the responses of various strains of animals to chemical
carcinogenesis are discussed in Section 8.2.3. Selection of sensitive
strains of test animals can shorten tumor induction time considerably. The
following are examples of such instances.
6.7.6.1 Hamster Fibrosarcoma System — Homburger and Hsuch (1970)
tested the susceptibility of inbred strains of hamsters to tumor induction
with 7,12-dimethyIbenz(a)anthracene. 500 pg of the chemical, injected sub-
cutaneous^ly, gave rise to subcutaneous fibrosarcomas in a relatively short
tine.
The 15.16 line was the most sensitive, with males and females devel-
oping 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.
6.7.6.2 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.
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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-methylcholanthrene
induced tumors in 10% to 100% of the animals and the effect was dose re-
lated; 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 administered
to each animal and the experiment was terminated nine 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 of
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6-121
certain heterocyclic compounds, and the investigators recommend the test
for rapid screening of certain potential carcinogens.
6.7.6.3 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.
2. The maximum tolerated dose of the chemical must be determined and
that dose is administered three times per week for eight weeks. The
second dose level is half the first and''that of the third group is
one-fifth the first. Chemicals are generally injected intraperito-
neally, 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.
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6.7.7 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 Sci-
ences, 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."
6.7.8 Host-Mediated In Vivo-In Vitro Assay
Di Paolo 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 Dulbeccos'
modified Eagle's or F-12 medium supplemented with 10% fetal bovine serum.
Cells were passed every four to six 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 innoc-
ulation. 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 carcino-
f
gens, of which several were known to be inactive in in vitro tests, were
tested in this system and all produced neoplastic 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.
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6.7.9 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 three weeks
(appearance of a-fetoprotein in the serum) to three-four months (the time
required for the detection of actual tumors, as in the lung adenoma assay
and the embryo homograft technique). And since exposure of cells to
chemicals occurs in vivo these particular assays have' the additional
s
advantage of allowing for metabolic activation. However, in order to
determine the best application of in vivo short-term assays to the screen-
ing of chemicals for carcinogenicty further evaluation of such tests is
necessary and their limits of sensitivity must be established.
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SECTION 6
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