National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 59 

1978 

BIOASSAY OF 

ESTRADIOL MUSTARD 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 22966-79-6 

NCI-CG-TR-59 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 

National Institutes of Health 


BIOASSAY OF
 

ESTRADIOL MUSTARD
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
 

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-1309
 

BIOASSAY OF
 

ESTRADIOL MUSTARD
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

FOREWORDr This report presents the results of the bioassay of
 

estradiol mustard conducted for the Carcinogenesis Testing
 

Program, Division of Cancer Cause and Prevention, National Cancer
 

Institute (NCI), National Institutes of Health, Bethesda,
 

Maryland. This is one of a series of experiments designed to
 

determine whether selected chemicals have the capacity to produce
 

cancer in animals. Negative results, in which the test animals
 

do not have a greater incidence of cancer than control animals,
 

do not necessarily mean that the test chemical is not a
 

carcinogen, inasmuch as the experiments are conducted under a
 

limited set of circumstances. Positive results demonstrate that
 

the test chemical is carcinogenic for animals under the
 

conditions of the test and indicate that exposure to the chemical
 

is a potential risk to man. The actual determination of the risk
 

to man from animal carcinogens requires a wider analysis.
 

CONTRIBUTORS; This bioassay of estradiol mustard was conducted
 

by Southern Research Institute, Birmingham, Alabama, initially
 

under direct contract to NCI and currently under a subcontract to
 

Tracer Jitco, Inc., prime contractor for the NCI Carcinogenesis
 

Testing Program.
 

The experimental design and doses were determined by Drs. D. P.
 

Griswold*, J. D. Prejean*, E. K. Weisburger^, and J. H.
 

Weisburger^'-^. Mr. J. Belzer^ and Mr. I. Brown^ were responsible
 

for the care of the laboratory animals and the administration of
 

the test chemical. Data management and retrieval were performed
 

by Ms. C. A. Dominick-'-. Histopathologic examinations were
 

performed by Drs. S. D. Kosanke-*- and J. C. Peckham^-, and the
 

diagnoses included in this report represent their interpretation.
 

iii
 
Animal pathology tables and survival tables were compiled at EG&G
 

Mason Research Institute^. The statistical analyses were performed by Dr. J. R. Joiner^ and Ms. P. L. Yong^, using methods
 

selected for the bioassay program by Dr. J. J. Gart^. Chemicals
 

used in this bioassay were analyzed by Drs. P. Lim', E. long',
 

and J. Jee', and the results of the analyses were reviewed by Dr.
 

C. W. Jameson-'.
 

This report was prepared at Tracor Jitco-* under the direction of
 

NCI. Those responsible for the report at Tracor Jitco were Dr.
 

L. A. Campbell, Director of the Bioassay Program; Dr. S. S. Olin,
 

Deputy Director for Science; Dr. J. F. Robens, toxicologist; Dr.
 

R. L. Schueler, pathologist; Dr. G. L. Miller, Ms. L. A. Waitz,
 

and Mr. W. D. Reichardt, bioscience writers; and Dr. E. W.
 

Gunberg, technical editor, assisted by Ms. Y. E. Presley.
 

The following other scientists at NCI were responsible for
 

evaluating the bioassay experiment, interpreting the results, and
 

reporting the findings: Dr. Kenneth C. Chu, Dr. Cipriano Cueto,
 

Jr., Dr. J. Fielding Douglas, Dr. Dawn G. Goodman, Dr. Richard A.
 

Griesemer, Dr. Harry A. Milman, Dr. Thomas W. Orme, Dr. Robert A.
 

Squire", and Dr. Jerrold M. Ward.
 

^Southern Research Institute, 2000 Ninth Avenue South,
 

Birmingham, Alabama.
 

^Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York.
 

Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

^Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

iv
 
^Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute, National
 

Institutes of Health, Bethesda, Maryland.
 

^Stanford Research Institute, Menlo Park, California.
 

°Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

v
 

SUMMARY
 

A bioassay of the experimental anticancer drug estradiol mustard
 

for possible carcinogenicity was conducted by administering the
 

chemical by gavage to Sprague-Dawley rats and B6C3F1 mice.
 

Groups of 35 rats and 34-36 mice of each sex were administered
 

estradiol mustard at one of the following doses, either 0.62 or
 

1.25 mg/kg body weight for rats and either 15 or 30 mg/kg body
 

weight for mice. The vehicle used for the test chemical consisted of 0.05% polysorbate 80 in phosphate-buffered saline. The
 

rats and mice were dosed three times per week for 52 weeks, then
 

observed for an additional 30-34 weeks. Controls consisted of
 

groups of 10 rats and 15 mice of each sex that were not administered the chemical (untreated controls) and also of groups of 10
 

rats of each sex, 14 male mice, and 16 female mice administered
 

the vehicle alone (vehicle controls). Pooled controls were also
 

used. All surviving rats were killed at 84-86 weeks and all
 

surviving mice at 82-86 weeks.
 

Mean body weights of male rats and male and female mice administered estradiol mustard were lower throughout the greater part of
 

the study than those of corresponding vehicle or untreated controls; mean body weights of dosed female rats were unaffected.
 

Administration of the test chemical had no significant effect on
 

the survival of either male or female rats. A large number of
 

dosed mice died prior to the end of the study. The numbers of
 

dosed male mice which were at risk as long as 52 weeks were
 

sufficient, however, for development of tumors appearing up to
 

that time. Time-adjusted analysis and life-table analyses were
 

applied to data obtained with the mice.
 

In rats, no tumors were observed in a statistically significant
 

incidence in the animals administered estradiol mustard.
 

In mice, lymphoma or lymphocytic leukemia occurred at significant
 

incidences in low-dose (P = 0.018) and high-dose (P < 0.001)
 

Vii
 
groups of males compared with those in the pooled vehicle controls (controls 0/28, low-dose 6/32, high-dose 17/29) and at
 

significant incidences in low-dose (P = 0.020) and high-dose (P =
 

0.002) groups of females compared with those in the corresponding
 

vehicle controls (controls 0/14, low-dose 9/30, high-dose 11/23).
 

In addition, the incidences of lymphoma were statistically
 

significant for dose-related trend for both the males (P < 0.001)
 

and the females (P = 0.003). Since lymphoma was observed in male
 

mice as early as 25 weeks, life-table analyses of the incidence
 

in each sex were performed. The results indicated a dose association (P = 0.001) between the administration of estradiol mustard
 

and the time of observation of lymphoma in either sex of mice.
 

In mice, alveolar/bronchiolar adenoma or carcinoma occurred at a
 

significant incidence (P = 0.004) in the low-dose group of males
 

compared with the pooled vehicle controls (controls 2/28, low-


dose 12/30, high-dose 5/24) and at a significant incidence (P =
 

0.022) in the low-dose group of females compared with the pooled
 

vehicle controls (controls 1/28, low-dose 7/27, high-dose 1/18).
 

Sarcoma of the myocardium similarly occurred at a significant
 

incidence (P = 0.015) in the low-dose group of males compared
 

with the pooled vehicle controls (controls 0/28, low-dose 6/30,
 

high-dose 2/24) and at a significant incidence (P = 0.002) in the
 

low-dose group of females compared with the pooled vehicle controls (controls 0/28, low-dose 8/27, high-dose 1/12). The
 

survival of both high-dose males and high-dose females was
 

slightly lower than that of the respective low-dose groups and
 

may account for the higher numbers of pulmonary tumors and
 

myocardial sarcomas among low-dose mice of both sexes. The
 

association of myocardial sarcoma with administration of the
 

chemical in both dosed groups of each sex is strengthened by the
 

fact that these tumors of the myocardium have not occurred in the
 

more than 500 male and 500 female historical-control mice of this
 

strain at the laboratory.
 

Squamous-cell carcinoma of the stomach occurred in the dosed male
 

mice (high-dose 2/29) and in the dosed female mice (low-dose
 

2/26, high-dose 2/14) but was absent in all controls. Although
 

the incidences in this bioassay were too low to be statistically
 

significant, the fact that no squamous-cell carcinomas of the
 

stomach have occurred in the more than 500 male and 500 female
 

historical-control mice of this strain at this laboratory
 

viii
 
indicates that these gastric tumors were related to the
 

administration of the estradiol mustard.
 

It is concluded that under the conditions of this bioassay,
 

estradiol mustard administered in a buffered saline vehicle was
 

not carcinogenic in Sprague-Dawley rats. Estradiol mustard was
 

carcinogenic in both male and female B6C3F1 mice, inducing
 

lymphoma, sarcoma of the myocardium, alveolar adenoma or
 

carcinoma, and squamous-cell carcinoma of the stomach.
 

ix
 

TABLE OF CONTENTS
 

Page
 

I. Introduction	 1
 

II. Materials and Methods	 3
 

A. Chemical	 3
 

B. Dosage Preparation	 3
 

C. Animals	 4
 

D. Animal Maintenance	 4
 

E. Subchronic Studies	 7
 

F. Designs of Chronic Studies	 9
 

G. Clinical and Pathologic Examinations	 12
 

H. Data Recording and Statistical Analyses	 13
 

III. Results - Rats	 19
 

A. Body Weights and Clinical Signs (Rats)	 19
 

B. Survival (Rats)	 19
 

C. Pathology (Rats)	 22
 

D. Statistical Analyses of Results (Rats)	 23
 

IV. Results - Mice	 27
 

A. Body Weights and Clinical	 Signs (Mice) 27
 

B. Survival (Mice)	 27
 

C. Pathology (Mice)	 30
 

D. Statistical Analyses of Results (Mice)	 34
 

V. Discussion	 41
 

VI.	 Bibliography 45
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in Rats
 

Table Al Summary of the Incidence of Neoplasms in Male Rats
 

Administered Estradiol Mustard by Gavage 47
 

Administered Estradiol Mustard by Gavage 49
 

Table A2 Summary of the Incidence of Neoplasms in Female Rats
 

Administered Estradiol Mustard by Gavage 52
 

xi
 
Page 

Appendix B Summary of the Incidence of Neoplasms in Mice 

Administered Estradiol Mustard by Gavage 55 

Table Bl Summary of the Incidence of Neoplasms in Male Mice 

Administered Estradiol Mustard by Gavage 57 

Table B2 Summary of the Incidence of Neoplasms in Female Mice 

Administered Estradiol Mustard by Gavage 60 

Appendix C Summary of the Incidence of Nonneoplastic Lesions in 

Rats Administered Estradiol Mustard by Gavage 63 

Table Cl Summary of the Incidence of Nonneoplastic Lesions in 

Male Rats Administered Estradiol Mustard by Gavage.. 65 

Table C2 Summary of the Incidence of Nonneoplastic Lesions in 

Female Rats Administered Estradiol Mustard by Gavage. 69 

Appendix D Summary of the Incidence of Nonneoplastic Lesions in 

Mice Administered Estradiol Mustard by Gavage 73 

Table Dl Summary of the Incidence of Nonneoplastic Lesions in 

Male Mice Administered Estraidol Mustard 

by Gavage , 75 

Table D2 Summary of the Incidence of Nonneoplastic Lesions in 

Female Mice Administered Estradiol Mustard 

by Gavage 78 

Appendix E Analyses of the Incidence of Primary Tumors in Rats 

Administered Estradiol Mustard by Gavage 81 

Table El Analyses of the Incidence of Primary Tumors in Male 

Rats Administered Estradiol Mustard by Gavage 83 

Table E2 Analyses of the Incidence of Primary Tumors in Female 

Rats Administered Estradiol Mustard by Gavage 87 

Appendix F Analyses of the Incidence of Primary Tumors in Mice 

Administered Estradiol Mustard by Gavage 91 

Table Fl Analyses of the Incidence of Primary Tumors in Male 

Mice Administered Estradiol Mustard by Gavage 93 

Table F2 Analyses of the Incidence of Primary Tumors in Female 

Mice Administered Estradiol Mustard by Gavage 99 

xii
 
Page 

Table F3 Time-adjusted Analyses of the Incidence of Primary 

Tumors in Male Mice Administered Estradiol Mustard 

by Gavage 105 

Table F4 Time-adjusted Analyses of the Incidence of Primary 

Tumors in Female Mice Administered Estradiol Mustard 

by Gavage 110 

TABLES 

Table 1 Design of Chronic Studies of Estradiol Mustard 

in Rats 10 

Table 2 Design of Chronic Studies of Estradiol Mustard 

in Mice 11 

FIGURES 

Figure 1 Growth Curves for Rats Administered Estradiol 

Mustard by Gavage 20 

Figure 2 Survival Curves for Rats Administered Estradiol 

Mustard by Gavage 21 

Figure 3 Growth Curves for Mice Administered Estradiol 

Mustard by Gavage 28 

Figure 4 Survival Curves for Mice Administered Estradiol 

Mustard by Gavage 29 

Figure 5 Life Table for Male Mice Administered Estradiol 

Mustard by Gavage: Combined Incidence of Lymphoma 

and Lymphocytic Leukemia 37 

Figure 6 Life Table for Female Mice Administered Estradiol 

Mustard by Gavage: Lymphoma 38 

xiii
 

I. INTRODUCTION
 

Estradiol mustard (CAS 22966-79-6; NCI C01570) is an experimental
 

anticancer agent that has been proposed for use in patients with
 

cancer of the ovary, breast, and prostate (Vollmer et al.,
 

1973a). It is the ester of the estrogen, estradiol, and a
 

nitrogen mustard alkylating agent, chlorphenacyl.
 

The manipulation of hormones in the body has been used for over
 

75 years to treat cancer in hormone-responsive tissues. Surgical
 

removal of endocrine organs, as in ovariectomy, has been associated with the regression of certain breast tumors, and the
 

administration of estrogens has also produced some tumor regressions (Vollmer, 1973b). The effects are believed to reflect the
 

role of hormones in regulating growth and development of certain
 

tissues (NCI, 1976). Tissues that respond to estrogen contain
 

cellular components known as estrogen receptors. These receptors
 

bind estrogen and transport it from the cytoplasm to the nucleus
 

where it initiates the synthesis of proteins that will be used to
 

express hormonal effects (Wittliff et al. , 1976). The rationale
 

behind the synthesis of estrogen-nitrogen mustard compounds was
 

that the alkylating moiety, when coupled with an estrogen, could
 

be transported directly to the nucleus of cells such as those in
 

the mammary glands, ovaries, uterus, prostate, and testes, which
 

have estrogen receptors (Wall et al., 1969). Cytotoxic effects
 

1 
of the nitrogen mustards would then be expressed only in these
 

organs.
 

Estradiol mustard was selected for testing for carcinogenic
 

activity because of the possibility that this drug may be used on
 

a repeated basis in humans.
 

2 
II. MATERIALS AND METHODS
 

A. Chemical
 

Estradiol mustard (estradiol,bis((p-(bis(2-chloroethyl)amino)
 

phenyl)acetate)) was obtained in two batches for the chronic
 

study from the Upjohn Company, North Haven, Connecticut. The
 

purity of the batch from Lot No. 80-4 was determined to be 97 +
 

1% by ultraviolet spectroscopy and thin-layer chromatography
 

analyses at the Stanford Research Institute. Elemental analyses
 

(C, H, Cl, N) were correct for C42H50C14N204, the molecular
 

formula for estradiol mustard. The identity was confirmed by
 

nuclear magnetic resonance, infrared, and ultraviolet spectra,
 

which were in agreement with the structure and matched the
 

spectra for a reference sample of estradiol mustard.
 

The chemical was stored in the presence of a desiccant
 

(Drierite®) in a closed container at 5°C.
 

B. Dosage Preparation
 

The test solutions of the estradiol mustard were prepared fresh
 

for each chemical administration. The chemical was suspended in
 

a phosphate-buffered saline-polysorbate 80 vehicle by mixing in a
 

Potter-Elvehjem tissue grinder. The buffered saline vehicle
 

3 
(pH 6.9) contained 0.85% NaCl, 0.40% NaH2P04, 0.65% Na2HP04, and
 

0.05% polysorbate 80.
 

No concentration or stability analyses of the chemical in the
 

buffered-saline vehicle were performed.
 

C. Animals
 

For the chronic study, Sprague-Dawley rats and B6C3F1 mice were
 

obtained from Charles River Breeding Laboratories, Inc.,
 

Wilmington, Massachusetts, under a contract with the Division of
 

Cancer Treatment, National Cancer Institute. Male rats were
 

received at 29 days of age, female rats at 36 days of age, and
 

male and female mice at 37 days of age. On arrival at the
 

laboratory, all animals were quarantined (rats for 6 days, mice
 

for 12 days). Those animals with no visible signs of disease
 

were assigned to control or dosed groups and earmarked for
 

individual identification.
 

D. Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

rooms. The temperature range was 20-24°C, and the relative
 

humidity was maintained at 40-60%. The room air was changed 15
 

times per hour and passed through incoming and exhaust fiberglass
 

roughing filters. In addition to natural, light, illumination was
 

4 
provided by fluorescent light for 9 hours per day. Wayne Lab
 

Blox animal meal (Allied Mills, Inc., Chicago, 111.) and water
 

were supplied daily and were available ad libitum.
 

Rats were housed five per cage and mice seven per cage in solid-


bottom stainless steel cages (Hahn Roofing and Sheet Metal Co.,
 

Birmingham, Ala.). The rat cages were provided with Iso-Dri®
 

hardwood chip bedding (Carworth, Edison, N.J.), and cage tops
 

were covered with disposable filter bonnets beginning at week 16;
 

mouse cages were provided with Sterolit® clay bedding (Englehard
 

Mineral and Chemical Co., New York, N.Y.). Bedding was replaced
 

once per week; cages, water bottles, and feeders were sanitized
 

at 82°C once per week; and racks were cleaned once per week.
 

The rats and mice were housed in separate rooms. Control animals
 

were housed with respective dosed animals. Animals administered
 

estradiol mustard were maintained in the same rooms as animals of
 

the same species being administered the following chemicals:
 

RATS
 

Gavage Studies
 

cholesterol (p-(bis(2-chloroethyl)amino)phenyl)acetate
 

(phenesterin) (CAS 3546-10-9)
 

5 
Intraperitoneal Injection Studies
 

4'-(9-acridinylamino)methansulfon-m-aniside monohydrochloride
 

(MAAM) (NSC 141549)
 

acronycine (CAS 7008-42-6)
 

5-azacytidine (CAS 320-67-2)
 

beta-2'-deoxy-6-thioguanosine monohydrate (beta-TGdR)
 

(CAS 789-61-7)
 

1,4-butanediol dimethanesulfonate (busulfan) (CAS 55-98-1)
 

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

3,3'-iminobis-l-propanol dimethanesulfonate (ester)
 

hydrochloride [IPD] (CAS 3458-22-8)
 

(+)-4,4'-(1-methyl-l,2-ethanediyl)bis-2,6-piperazinedione
 

(ICRF-159) (CAS 21416-87-5)
 

N,3-bis(2-chloroethyl)tetrahydro-2H-l,3,2-oxazaphosphorin-2­

amine-2-oxide (isophosphamide) (CAS 3778-73-2)
 

N-(2-chloroethyl)-N-(l-methyl-2-phenoxyethyl)benzylamine
 

hydrochloride (phenoxybenzamine) (CAS 63-92-3)
 

N-(l-methylethyl)-4-((2-methylhydrazino)methyl)benzamide
 

monohydrochloride (procarbazine) (CAS 366-70-1)
 

tris(l-aziridinyl)phosphine sulfide (thio-TEPA) (CAS 52-24-4)
 

2,4,6-tris(dimethylamino)-s-triazine (CAS 645-05-6)
 

MICE
 

Feed Studies
 

4-acetyl-N-((cyclohexylamino)carbonyl)benzenesulfonamide
 

(acetohexamide) (CAS 968-81-0)
 

anthranilic acid (CAS 118-92-3)
 

l-butyl-3-(p-tolylsulfonyl)urea (tolbutamide) (CAS 64-77-7)
 

4-chloro-N-((propylamino)carbonyl)benzenesulfonamide
 

(chlorpropamide) (CAS 94-20-2)
 

5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine
 

(pyrimethamine) (CAS 58-14-0)
 

2,6-diamino-3-(phenylazo)pyridine hydrochloride
 

(phenazopyridine hydrochloride) (CAS 136-40-3)
 

L-tryptophan (CAS 73-22-3)
 

N-9H-fluoren-2-ylacetamide (CAS 53-96-3)
 

N-(p-toluenesulfonyl)-N'-hexamethyleniminourea
 

(tolazamide) (CAS 1156-19-0)
 

1-phenethylbiguanide hydrochloride (phenformin) (CAS 114-86-3)
 

pyrazinecarboxamide (pyrazinamide) (CAS 98-96-4)
 

4,4'-sulfonyldianiline (dapsone) (CAS 80-08-0)
 

4,4'-thiodianiline (CAS 139-65-1)
 

ethlonamide (CAS 536-33-4)
 

6 
Gavage Studies
 

cholesterol (p-(bis(2-chloroethyl)amino)phenyl)acetate
 

(phenesterin) (CAS 3546-10-9)
 

Intraperitoneal Injection Studies
 

4'-(9-acridinylamino)methansulfon-m-aniside monohydrochloride
 

(MAAM) (NSC 141549)
 

acronycine (CAS 7008-42-6)
 

5-azacytidine (CAS 320-67-2)
 

beta-2'-deoxy-6-thioguanosine monohydrate (beta-TGdR)
 

(CAS 789-61-7)
 

1,4-butanediol dimethanesulfonate (busulfan) (CAS 55-98-1)
 

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

3,3'-iminobis-l-propanol dimethanesulfonate (ester)
 

hydrochloride [IPD] (CAS 3458-22-8)
 

(+)-4,4'-(l-methyl-l,2-ethanediyl)bis-2,6-piperazinedione
 

(ICRF-159) (CAS 21416-87-5)
 

N,3-bis(2-chloroethyl)tetrahydro-2H-l,3,2-oxazaphosphorin-2­

amine-2-oxide (isophosphamide) (CAS 3778-73-2)
 

N-(2-chloroethyl)-N-(1-methyl-2-phenoxye thyl)benzylamine
 

hydrochloride (phenoxybenzamine) (CAS 63-92-3)
 

N-(l-methylethyl)-4-((2-methylhydrazino)methyl)benzamide
 

monohydrochloride (procarbazine) (CAS 366-70-1)
 

tris(l-aziridinyl)phosphine sulfide (thio-TEPA) (CAS 52-24-4)
 

2,4,6-tris(dimethylamino)-s-triazine (CAS 645-05-6)
 

E. Subchronic Studies
 

Subchronic studies were conducted to estimate the maximum
 

tolerated doses of estradiol mustard, on the basis of which two
 

concentrations (hereinafter referred to as "low doses" and "high
 

doses") were determined for administration in the chronic
 

studies. In the subchronic studies, estradiol mustard was
 

administered by gavage in a vehicle containing polysorbate 80 in
 

saline at doses of 0.25, 0.62, 1.25, 2.5, or 5.0 mg/kg body
 

weight to male Sprague-Dawley rats and at doses of 0.4, 1.0, 2.0,
 

7 
4.0, 8.0, 16.0, or 32.0 mg/kg body weight to male Swiss mice.
 

Animals were dosed three times per week for 45 days and observed
 

for an additional 45 days before termination of the study. Five
 

animals of each species were used at each dose, and 10 animals of
 

each species were used as untreated or vehicle controls.
 

In rats, there were no deaths at any of the doses tested. After
 

45 days of chemical administration, mean body weight gains were
 

depressed 13-18% at doses of 0.62, 1.25, and 2.5 mg/kg and 25% at
 

a dose of 5.0 mg/kg. The low and high doses for the chronic
 

studies using rats were set at 0.62 and 1.25 mg/kg.
 

In mice, the only death due to toxicity of the chemical occurred
 

in an animal at 0.4 mg/kg during week 3 of the study. Mean body
 

weight gains in groups of animals at doses of 8.0 mg/kg or lower
 

were similar to those of controls; mean body weight gains in
 

groups of animals at doses of 16 or 32 mg/kg were slightly lower
 

than those of controls. The low and high doses for the chronic
 

studies using mice were set at 15 and 30 mg/kg.
 

Doses for both rats and mice were purposely set low because of
 

the wide variation in the acute toxicity data previously reported
 

for this chemical (Vollmer et al., 1973).
 

8 
F. Designs of Chronic Studies
 

Designs of the chronic studies are shown in tables 1 and 2.
 

Since the numbers of animals in the untreated-control and
 

vehicle-control groups were small, pooled-vehicle control groups
 

also were used for statistical evaluation. For the rats, the 10
 

matched vehicle-control animals of each sex from the current
 

bioassay of estradiol mustard were combined with groups of 10
 

vehicle-control animals of each sex from the bioassay of
 

phenesterin (CAS 3546-10-9) to give pooled groups of 20 vehicle-


control animals of each sex. For the mice, the 14 male and 16
 

female matched vehicle-control animals from the current bioassay
 

of estradiol mustard were combined with groups of 15 vehicle-


control animals of each sex from the bioassay of phenesterin to
 

give pooled groups of 29-31 vehicle-control animals of each sex.
 

The vehicle-control groups of rats and mice for the different
 

test chemicals were of the same strains and from the same
 

supplier. The various control groups were started at times no
 

more than 3 months apart, all at Southern Research Institute, and
 

the diagnoses were made by the same pathologists. The vehicle
 

used for the controls in the bioassay of phenesterin was the same
 

as that used for the controls in the bioassay of estradiol
 

mustard.
 

9 
Table 1. Design of Chronic Studies of Estradiol Mustard in Rats
 

Estradiol 

Sex and Initial Mustard Time on Study 

Test No. of Dose*' Dosed Observed 

Group Animals3 (mg/kg) (weeks) (weeks) 

Male 

Untreated-Control 10 0 86 

Vehicle-Control 10 Oc 52 34 

Low-Dose 35 0.62 52 33 

High-Dose 35 1.25 52 33 

Female 

Untreated-Control 10 0 86 

Vehicle-Control 10 Oc 52 34 

Low-Dose 35 0.62 52 33-34 

High-Dose 35 1.25 52 32-33 

a

Male rats were 35 days of age and females were 42 days of age
 

when placed on study.
 

"Estradiol mustard was administered by gavage in a vehicle
 

consisting of 0.05% polysorbate 80 in phosphate-buffered saline
 

at a volume of 0.25 ml/100 g body weight three times per week;
 

doses were based on individual body weights.
 

c

Vehicle-control groups received only polysorbate 80 in phosphate-


buffered saline at the same volume as dosed rats.
 

10
 
Table 2. Design of Chronic Studies of Estradiol Mustard in Mice
 

Estradiol 

Sex and Initial Mustard Time on Study 

Test No. of Doseb Dosed Observed 

Group Animals3 (mg/kg) (weeks) (weeks) 

Male 

Untreated-Control 15 0 86 

Vehicle-Control 14 Oc 52 32 

Low-Dose 34 15 52 32 

High-Dose 35 30 52 30-31 

Female 

Untreated-Control 15 0 85-86 

Vehicle-Control 16 Oc 52 32 

Low-Dose 36 15 52 32 

High-Dose 35 30 52 24d 

a

Mice were 49 days of age when placed on study.
 

"Estradiol mustard was administered by gavage in a vehicle
 

consisting of 0.05% polysorbate 80 in phosphate-buffered saline
 

at a volume of 1 ml/100 g body weight three times per week; doses
 

were based on the mean weights of the animals in each cage.
 

c

Vehicle-control groups received only polysorbate 80 in
 

phosphate-buffered saline at the same volume as dosed mice.
 

"All high-dose females died or were killed by week 76.
 

11
 
G. Clinical and Pathologic Examinations
 

All animals were observed twice daily for signs of toxicity, and
 

animals that were moribund were killed and necropsied, except for
 

those dying prior to day 100, due, presumably, to toxicity of the
 

test chemical. Rats and mice were weighed individually each week
 

for 8 weeks and every 2 weeks thereafter. Palpation for masses
 

was carried out at each weighing.
 

The pathologic evaluation consisted of gross and microscopic
 

examination of major tissues, major organs, and all gross lesions
 

from killed animals and from animals found dead. The following
 

tissues were examined microscopically: skin, muscle, lungs and
 

bronchi, trachea, bone and bone marrow, spleen, lymph nodes,
 

thymus, heart, salivary gland, liver, gallbladder and bile duct
 

(mice), pancreas, esophagus, stomach, small intestine, large
 

intestine, kidney, urinary bladder, pituitary, adrenal, thyroid,
 

parathyroid, mammary gland, prosstate or uterus, testis or ovary,
 

brain, and sensory organs. Peripheral blood smears were prepared
 

from each animal whenever possible. Occasionally, additional
 

tissues were also examined microscopically. The different
 

tissues were preserved in 10% buffered formalin, embedded in
 

paraffin, sectioned, and stained with hematoxylin and eosin.
 

Special staining techniques were utilized when indicated for more
 

definitive diagnosis.
 

12
 
A few tissues from some animals were not examined, particularly
 

from those animals that died early. Also, some animals were
 

missing, cannibalized, or judged to be in such an advanced state
 

of autolysis as to preclude histopathologic evaluation. Thus,
 

the number of animals from which particular organs or tissues
 

were examined microscopically varies, and does not necessarily
 

represent the number of animals that were placed on study in each
 

group.
 

H. Data Recording and Statistical Analyses
 

Pertinent data on this experiment have been recorded in an automatic data processing system, the Carcinogenesis Bioassay Data
 

System (Linhart et al., 1974). The data elements include descriptive information on the chemicals, animals, experimental design,
 

clinical observations, survival, body weight, and individual
 

pathologic results, as recommended by the International Union
 

Against Cancer (Berenblum, 1969). Data tables were generated for
 

"erification of data transcription and for statistical review.
 

These data were analyzed using the statistical techniques
 

described in this section. Those analyses of the experimental
 

results that bear on the possibility of carcinogenicity are
 

discussed in the statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

13
 
procedure of Kaplan and Meier (1958) and are presented in this
 

report in the form of graphs. Animals were statistically
 

censored as of the time that they died of other than natural
 

causes or were found to be missing; animals dying from natural
 

causes were not statistically censored. Statistical analyses for
 

a possible dose-related effect on survival used the method of Cox
 

(1972) for testing two groups for equality and Tarone's (1975)
 

extensions of Cox's methods for testing for a dose-related trend.
 

One-tailed P values have been reported for all tests except the
 

departure from linearity test, which is only reported when its
 

two-tailed P value is less than 0.05.
 

The incidence of neoplastic or nonneoplastic lesions has been
 

given as the ratio of the number of animals bearing such lesions
 

at a specific anatomic site (numerator) to the number of animals
 

in which that site is examined (denominator). In most instances,
 

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic examination was required to detect lesions prior to histologic sampling
 

(e.g., skin or mammary tumors), or when lesions could have
 

appeared at multiple sites (e.g., lymphomas), the denominators
 

consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

14
 
significantly higher proportion of tumors than did the control
 

animals. As a part of these analyses, the one-tailed Fisher
 

exact test (Cox, 1970) was used to compare the tumor incidence of
 

a control group with that of a group of dosed animals at each
 

dose level. When results for a number of dosed groups (k) are
 

compared simultaneously with those for a control group, a correction to ensure an overall significance level of 0.05 may be made.
 

The Bonferroni inequality (Miller, 1966) requires that the P
 

value for any comparison be less than or equal to 0.05/k. In
 

cases where this correction was used, it is discussed in the
 

narrative section. It is not, however, presented in the tables,
 

where the Fisher exact P values are shown.
 

The Cochran-Armitage test for linear trend in proportions, with
 

continuity correction (Armitage, 1971), was also used. Under the
 

assumption of a linear trend, this test determines if the slope
 

of the dose-response curve is different from zero at the one-


tailed 0.05 level of significance. Unless otherwise noted, the
 

direction of the significant trend is a positive dose relationship. This method also provides a two-tailed test of departure
 

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

of tumors. In this analysis, deaths that occurred before the
 

15
 
first tumor was observed were excluded by basing the statistical
 

tests on animals that survived at least 52 weeks, unless a tumor
 

was found at the anatomic site of interest before week 52. When
 

such an early tumor was found, comparisons were based exclusively
 

on animals that survived at least as long as the animal in which
 

the first tumor was found. Once this reduced set of data was
 

obtained, the standard procedures for analyses of the incidence
 

of tumors (Fisher exact tests, Cochran-Armitage tests, etc.) were
 

followed.
 

When appropriate, life-table methods were used to analyze the
 

incidence of tumors. Curves of the proportions surviving without
 

an observed tumor were computed as in Saffiotti et al. (1972).
 

The week during which an animal died naturally or was sacrificed
 

was entered as the time point of tumor observation. Cox's
 

methods of comparing these curves were used for two groups;
 

Tarone's extension to testing for linear trend was used for three
 

groups. The statistical tests for the incidence of tumors which
 

used life-table methods were one-tailed and, unless otherwise
 

noted, in the direction of a positive dose relationship.
 

Significant departures from linearity (P < 0.05, two-tailed test)
 

were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared with its control was calculated
 

16
 
from the exact interval on the odds ratio (Gart, 1971). The relative risk is defined as Pt/pc where pt is the true binomial
 

probability of the incidence of a specific type of tumor in a
 

dosed group of animals and pc is the true probability of the
 

spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

a specific tumor in a dosed group and the proportion in a control
 

group corresponds to a relative risk of unity. Values in excess
 

of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in approximately 95% of a large number of identical experiments, the true
 

ratio of the risk in a dosed group of animals to that in a
 

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

significant result (P < 0.025 one-tailed test when the control
 

incidence is not zero, P < 0.050 when the control incidence is
 

zero) has occurred. When the lower limit is less than unity, but
 

the upper limit is greater than unity, the lower limit indicates
 

the absence of a significant result while the upper limit indi17
 
cates that there is a theoretical possibility of the induction of
 

tumors by the test chemical, which could not be detected under
 

the conditions of this test.
 

18
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of the dosed male rats were lower than those of
 

the corresponding controls, and the depressions in weight were
 

dose related (figure 1). Mean body weights of the dosed female
 

rats were approximately the same as those of either the untreated
 

or vehicle controls. Fluctuations in the growth curve may be due
 

to mortality; as the size of a group diminishes, the mean body
 

weight may be subject to wide variation. No other clinical signs
 

attributable to administration of estradiol mustard were
 

reported.
 

B. Survival (Rats)
 

Kaplan and Meier curves estimating the probabilities of survival
 

for male and female rats administered estradiol mustard by gavage
 

at the doses of this bioassay, together with those of the matched
 

vehicle and untreated controls, are shown in figure 2.
 

In each sex, the results of the Tarone test for positive dose-


related trend in mortality are not significant. In male rats,
 

all of the 35 high-dose animals, 34/35 (97%) of the low-dose
 

group, and all of the 10 animals in either the vehicle- or
 

untreated-control group were alive at week 52. In females, 34/35
 

19
 
Figure 1. Growth Curves For Rats Administered Estradiol Mustard by Gavage 

20 
Figure 2. Survival Curves For Rats Administered Estradiol Mustard by Gavage 

21 
(97%) of the high-dose group, 34/35 (97%) of the low-dose group,
 

8/10 (80%) of the vehicle controls, and all of the 10 untreated
 

controls were alive at week 52. A sufficient number of dosed
 

rats of each sex was at risk for the development of late-


appearing tumors.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

Appendix A, tables Al and A2; findings on nonneoplastic lesions
 

are summarized in Appendix C, tables Cl and C2.
 

Mammary gland tumors occurred in 3/35 (9%) low-dose and 5/33
 

(15%) high-dose male rats. None occurred in males of the
 

untreated (0/10) and vehicle (0/9) controls. In the females,
 

tumors of the mammary glands occurred in 5/10 (50%) untreated-


control, 4/8 (50%) vehicle-control, 20/34 (59%) low-dose, and
 

21/33 (63%) high-dose rats. The majority of the tumors in the
 

dosed groups were benign. Mammary tumors of the same type have
 

been observed previously as spontaneous lesions in Sprague-Dawley
 

rats (Prejean et al., 1973; Young and Hallowes, 1973), and the
 

incidences observed in this study do not support the conclusion
 

of a compound-related effect.
 

A variety of other neoplasms occurred both in the control groups
 

(untreated and vehicle) and in the dosed groups. Some of these
 

22
 
neoplasms occurred only, or with a greater frequency, in rats of
 

dosed groups as compared with controls. These lesions, however,
 

are not uncommon in this strain of rat independent of the
 

administration of any chemical.
 

In addition to the neoplastic lesions, a number of degenerative,
 

proliferative, and inflammatory changes were also encountered in
 

animals of the dosed and control groups (Appendix C). These
 

nonneoplastic lesions are commonly seen in aged Sprague-Dawley
 

rats.
 

Based on the histopathologic examination, estradiol mustard
 

administered by oral gavage to Sprague-Dawley rats may have had
 

an effect on the mammary gland under the conditions of this
 

bioassay.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

least two animals in one group and with an incidence of at least
 

5% in one or more than one group. Vehicle-control groups and
 

pooled vehicle-control groups are used in the statistical
 

analyses. The untreated-control groups are not included in the
 

statistical analyses, since the test conditions of the vehicle
 

controls are more similar to those of the dosed animals.
 

23
 
In male rats, the results of the Cochran-Armitage test for
 

positive dose-related trend in the incidence of fibroadenoma of
 

the mammary gland is significant (P = 0.043) when the pooled
 

vehicle controls are used, but the results of the Fisher exact
 

test are not significant. The results of the tests made on the
 

incidence of the combination of fibroadenoma or adenocarcinoma
 

are not significant. The results of the bioassay program at this
 

laboratory to date indicate a spontaneous tumor rate of fibroadenoma or adenocarcinoma in the mammary gland in male rats of
 

6/215 (2.8%).
 

In female rats, the results of the Cochran-Armitage test on the
 

incidence of adenocarcinoma of the mammary gland, the incidence
 

of fibroadenoma of the mammary gland, and the incidence of the
 

combination of adenoma, adenocarcinoma, or fibroadenoma of the
 

mammary gland are not significant. The results of the Fisher
 

exact test show levels of probability above 0.05 when the
 

incidences in either the low- or high-dose groups are compared
 

with those in the controls. The historical data for female rats
 

concerning the spontaneous occurrence of adenocarcinoma at this
 

laboratory to date show an incidence of 15/220 (6.8%).
 

In male rats, significant results in the negative direction are
 

indicated in the incidences of pituitary tumors, where the
 

24
 
incidence in the controls exceeds that observed in the dosed
 

groups.
 

In each of the 95% confidence intervals of relative risk shown in
 

the tables, except that for the incidence of pituitary tumors in
 

the high-dose males, one is included; this indicates the absence
 

of significant positive results. It should also be noted that
 

each of these intervals has an upper limit greater than one,
 

indicating the theoretical possibility of the induction of tumors
 

by estradiol mustard, which could not be detected under the
 

conditions of this test.
 

25
 

IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of both the low- and high-dose male and female
 

mice were markedly lower than those of either the untreated- or
 

vehicle-control mice throughout most of the bioassay (figure 3).
 

Fluctuations in the growth curve may be due to mortality; as the
 

size of a group diminishes, the mean body weight may be subject
 

to wide variation. No other clinical signs attributable to
 

administration of estradiol mustard were reported.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice administered estradiol mustard
 

by gavage at the doses of this bioassay, together with those of
 

the vehicle and untreated controls, are shown in figure 4.
 

In each sex, the results of the Tarone test for positive dose-


related trend in mortality are significant (P < 0.001). In the
 

male mice, 13/14 (93%) of the vehicle controls, all of the 15
 

untreaued controls, 30/34 (88%) of the low-dose group, and 24/35
 

(69%) of the high-dose group were alive at week 52. Thus, sufficient numbers of dosed male mice were at risk for 52 weeks for
 

development of tumors appearing up to that time. In females,
 

27
 
Figure 3. Growth Curves For Mice Administered Estradiol Mustard by Gavage 

28 
Figure 4. Survival Curves for Mice Administered Estradiof Mustard by Gavage 

29 
13/16 (81%) of the vehicle controls, 12/15 (80%) of the untreated
 

controls, 26/36 (72%) of the low-dose group, and 12/35 (34%) of
 

the high-dose group survived at least as long as 52 weeks on
 

study.
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

Appendix B, tables Bl and B2; findings on nonneoplastic lesions
 

are summarized in Appendix D, tables Dl and D2.
 

A variety of neoplasms occurred both in control groups (untreated
 

and vehicle) and in dosed groups. Some of the tumors appeared to
 

be compound related.
 

In the males, alveolar-cell (alveolar-bronchiolar) tumors were
 

observed in 2/15 (13%) untreated-control, 2/14 (14%) vehicle-


control, 12/32 (44%) low-dose, and 5/29 (17%) high-dose males.
 

In the females, 7/31 (23%) low-dose and 1/24 (4%) high-dose mice
 

had these lung tumors. No alveolar-cell tumors occurred in
 

untreated- (0/14) or vehicle-control (0/16) female mice. These
 

alveolar-cell tumors included alveolar-cell adenocarcinomas
 

(alveolar/bronchiolar carcinomas) which were present in 6/32
 

(19%) low-dose and 1/29 (3%) high-dose males and in 4/31 (13%)
 

low-dose females. Both the benign and malignant neoplasms were
 

comprised of cuboidal to columnar cell's aligned along the
 

30
 
alveolar septa. Often the cells projected into the alveolar
 

spaces, resulting in the formation of numerous papillary
 

structures. Adenomas were differentiated from carcinomas by size
 

and the degree of cellular pleomorphism, nuclear atypism, and
 

extension into adjacent tissues. The alveolar-cell carcinomas
 

were all well differentiated with few mitotic figures being
 

observed. These tumors did not metastasize.
 

The hematopoietic and lymphoreticular neoplasms were observed in
 

17/29 (58%) high-dose and 6/32 (18%) low-dose males and in 11/24
 

(45%) high-dose and 9/31 (29%) low-dose females. None occurred
 

in any control groups except for one lymphoma in the untreated-


control females. The malignant lymphomas were classified as
 

lymphocytic and histiocytic. The lymphocytic type was comprised
 

of cells having a small, darkly basophilic to large lightly
 

basophilic, vesicular nucleus and a rim of eosinophilic
 

cytoplasm. Malignant lymphomas composed of lymphoblastic
 

(undifferentiated) cells were included in the lymphocytic type.
 

The histiocytic type was comprised primarily of cells with a
 

large open-faced nucleus, distinct eosinophilic nucleolus, and
 

abundant eosinophilic cytoplasm. However, some histiocytic
 

tumors contained many cells having a smaller, pleomorphic, often
 

elliptical or indented nucleus. The malignant lymphomas, NOS
 

(not otherwise specified), could not be further classified. A
 

31
 
malignant lymphoma, lymphocytic type, was observed in 1/14 (7%)
 

untreated-control females.
 

The malignant lymphomas were observed to be either generalized,
 

involving several organs, or solitary, involving only one organ.
 

The lymphocytic type of lymphomas appeared first in the thorax,
 

with the thymus and lungs most frequently involved. Other
 

organs, such as the liver, spleen, kidneys, and lymph nodes, were
 

involved, but less frequently. In two mice, only the thymus was
 

involved. The histiocytic type involved primarily the spleen,
 

liver, lungs, and mesenteric lymph nodes. In one mouse, only the
 

spleen contained neoplastic cells.
 

Lymphocytic leukemia was differentiated from malignant lymphoma,
 

lymphocytic type, by the diffuse infiltration of the neoplastic
 

cells within the involved organs, especially the liver. In the
 

case of lymphoma, the neoplastic cells were more solid in
 

arrangement.
 

The myocardial sarcomas were observed in 6/32 (19%) low-dose and
 

2/29 (7%) high-dose males and in 8/31 (26%) low-dose and 1/24
 

(4%) high-dose females. The neoplastic cells were pleomorphic
 

and varied from those with a small round, basophilic nucleus to
 

those with a large, vesicular nucleus. The larger nuclei
 

contained one or more distinct: nucleoli. All cells had an
 

32
 
abundant eosinophilic cytoplasm. The origin of the neoplastic
 

cells was not determined, but they appeared to arise from the
 

perimysial connective tissue surrounding myocardial fibers. The
 

marked proliferation of neoplastic cells resulted in displacement
 

and distortion of normal parenchyma. In some of the mice, the
 

*
 

neoplastic cells had invaded through the endocardium and existed
 

as large tumor thrombi within the ventricles. The neoplastic
 

cells had metastasized to the lungs in 3/32 (9%) low-dose males
 

and in 5/31 (16%) low-dose females.
 

Carcinoma, NOS, or squamous-cell carcinomas arising from the
 

stomach were observed in 1/32 low-dose and 2/29 (7%) high-dose
 

males as well as in 2/31 (6%) low-dose and 2/24 (8%) high-dose
 

females. The carcinomas were characterized by large polyhedral
 

cells that extended deep into the muscular wall of the stomach.
 

The neoplastic cells had a large open-faced nucleus and prominent
 

nucleolus. Large keratin pearls were numerous throughout the
 

neoplasms. The primary tumor had invaded through the stomach
 

wall and transplanted to other abdominal organs in 1/31 (3%)
 

low-dose and 1/24 (4%) high-dose females. In the high-dose
 

female, the tumor also had metastasized to the lungs. In
 

addition, a carcinoma, NOS, of the stomach occurred in a low-dose
 

male.
 

In addition to the neoplastic lesions, a number of degenerative,
 

33
 
proliferative, and inflammatory changes were also encountered in
 

animals of the dosed and control groups (Appendix D). These
 

nonneoplastic lesions are commonly seen in aged B6C3F1 mice.
 

Administration of estradiol mustard by oral gavage to B6C3F1 mice
 

for 19 months resulted in an increased incidence of hematopoietic
 

tumors and myocardial sarcomas. It was also associated with a
 

slight increase in the incidence of primary lung neoplasms and of
 

squamous-cell carcinomas of the stomach. Because of the rare
 

occurrence of squamous-cell carcinomas as spontaneous tumors, the
 

few such tumors observed in this study must be considered as
 

related to the administration of estradiol mustard. Based on the
 

histopathologic examination, estradiol mustard administered by
 

oral gavage to B6C3F1 mice appeared to be carcinogenic under the
 

conditions of this bioassay.
 

D. Statistical Analyses of Results (Mice)
 

Tables F1-F4 in Appendix F contain the statistical analyses of
 

the incidences of those primary tumors that occurred in at least
 

two animals in one group and with an incidence of at least 5% in
 

one or more than one group. Vehicle-control and pooled vehicle-


control groups are used in the statistical analyses. The
 

untreated-control groups are not included in the statistical
 

34
 
analyses, since the test conditions of the vehicle controls are
 

more similar to those of the dosed animals.
 

Due to the high mortality in the high-dose female mice, time-


adjusted analyses are performed in female mice, eliminating
 

animals that died before week 52 on study, unless a tumor was
 

found at the anatomic site of interest before week 52. In such
 

instances, comparisons are based exclusively on the female mice
 

that survived at least as long as the animal in which the first
 

tumor was found. In male mice, time-adjusted analyses are also
 

performed on the incidences of tumors of the lung, liver, and
 

myocardium. Thus, the statistical narrative regarding these
 

incidences of tumors is based on time-adjusted data; however, the
 

non-adjusted statistical data are also included in tables Fl and
 

F2 of Appendix F.
 

In male mice, the results of the Cochran-Armitage test on the
 

combined incidence of lymphoma or lymphocytic leukemia are
 

significant (P < 0.001), and the results of the Fisher exact test
 

show that the incidence in the high-dose group is significantly
 

higher (P < 0.001) than the incidence in either group of
 

controls. The results of the Fisher exact test comparing the
 

incidence in the low-dose group with that in the pooled controls
 

is significant (P = 0.018). Since the earliest occurrence of
 

lymphoma was observed in male mice at week 25, the time35
 
adjustment process would not materially affect the results of
 

this analysis. Life tables affecting the time of observation of
 

lymphoma in both male and female mice are shown in figures 5 and
 

6. The results in the life-table analysis using vehicle-


control, low-dose, and high-dose groups indicate a dose
 

association (P < 0.001) between the administration of estradiol
 

mustard and the time of observation of lymphoma in either sex of
 

mice. In female mice, the results of the Cochran-Armitage test
 

indicate a probability level of 0.003, and the results of the
 

Fisher exact test show that the incidences in both the low- and
 

high-dose groups are significantly higher than that in the
 

vehicle-control group (P = 0.020 and P = 0.002, respectively).
 

Results using the pooled-control group are more significant. The
 

statistical conclusion suggests that the incidence of lymphoma in
 

mice is dose associated. The significance of the combined
 

incidence of lymphoma and lymphocytic leukemia in male mice is
 

contributed by the lymphoma and not by the leukemia, which was
 

reported only in one low-dose animal. No leukemia was observed
 

in the female mice.
 

In male mice, using the pooled-control group, the results of the
 

Cochran-Armitage test on the incidence of alveolar/bronchiolar
 

adenoma or carcinoma and on the incidence of sarcoma of the
 

myocardium are not significant, but departure from linear trend
 

36
 
Figure 5. Life Table for Male Mice Administered Estradiol Mustard by Gavage:

 

Combined Incidence of Lymphoma and Lymphocytic Leukemia 

37 
Figure 6. Life Table for Female Mice Administered Estradiol Mustard by Gavage: Lymphoma 

38 
is observed (P _< 0.020), because the incidences in the low-dose
 

groups are greater than those in the high-dose groups. The
 

results of the Fisher exact test indicate that the incidences of
 

these tumors in the low-dose groups are significantly higher than
 

those in the pooled vehicle-control groups (P _< 0.015), but no
 

positive results are indicated in the high-dose groups.
 

Similarly, in female mice, using the pooled controls, the results
 

of the Cochran-Armitage test on the incidence of alveolar/
 

bronchiolar adenoma or carcinoma and on the incidence of sarcoma
 

of the myocardium are not significant, but departure from linearity is observed (P _< 0.008), because the incidences in the
 

low-dose groups are greater than those in the high-dose groups.
 

The results of the Fisher exact test show a P value equal to or
 

less than 0.022 when the incidence of the combined lung tumors in
 

the low-dose groups and the incidence of the myocardium tumors in
 

the low-dose groups are compared with the incidences of their
 

corresponding pooled vehicle controls, but the probability levels
 

of comparisons made using the vehicle-control group are above the
 

0.025 level (P = 0.048 for the incidence in the lung, and P =
 

0.029 for the incidence in the myocardium) required by the
 

Bonferroni inequality criterion when multiple comparison is
 

considered. The results of the bioassay program at this
 

laboratory to date indicate no sarcomas of the myocardium in more
 

39
 
than 500 male and 500 female mice. The statistical conclusion is
 

that the occurrence of these sarcomas in male mice (20%) and in
 

female mice (30%) in this study is associated with the administration of estradiol mustard. The increased mortality in both
 

high-dose groups of mice after 40 weeks on study may have limited
 

the development of tumors in those groups.
 

The results of the Cochran-Armitage test on the combined
 

incidence of adenocarcinoma and carcinoma of the mammary gland in
 

the female mice indicate a probability level of 0.035 when the
 

vehicle-controls are used, but the results of the Fisher exact
 

test are not significant.
 

40
 
V. DISCUSSION
 

Mean body weights of male rats and male and female mice administered estradiol mustard by gavage were lower throughout the
 

greater part of the study than those of corresponding vehicle or
 

untreated controls. The mean body weights of dosed female rats
 

were unaffected when compared with controls, and administration
 

of the chemical had no significant effect on the survival of
 

either male or female rats. Thus, the female rats may have been
 

able to tolerate a higher dose. However, the rats were dosed for
 

only 52 weeks, a shorter period of time than is specified in the
 

carcinogensis bioassay (Sontag et al., 1975). A large number of
 

the dosed mice died prior to the end of the bioassay; however,
 

sixty-nine percent or more of the male mice in either dosed group
 

lived for at least 52 weeks on study. Thus, the number of dosed
 

male mice at risk was sufficient for development of tumors
 

appearing up to that time. Time-adjusted analysis and life-table
 

analysis were applied to data obtained with the mice in most
 

cases.
 

In rats, the incidence of fibroadenoma of the mammary gland in
 

the males was dose related (P = 0.043), using pooled vehicle
 

controls (controls 0/18, low-dose 2/35, high-dose 5/33), but the
 

incidence in neither dosed group was significantly higher than
 

that in the controls. Thus, the occurrence of mammary tumors in
 

41
 
the male rats cannot clearly be related to administration of the
 

test chemical. The incidence of mammary tumors in the dosed
 

females was not statistically significant.
 

In mice, lymphoma or lymphocytic leukemia occurred at significant
 

incidences in low-dose (P = 0.018) and high-dose (P < 0.001)
 

groups of males compared with those in the pooled vehicle
 

controls (controls 0/28, low-dose 6/32, high-dose 17/29) and at
 

significant incidences in low-dose (P = 0.020) and high-dose (P =
 

0.002) groups of females compared with those in the corresponding
 

vehicle controls (controls 0/14, low-dose 9/30, high-dose 11/23).
 

In addition, the incidences of lymphoma were statistically
 

significant for dose-related trend for both the males (P < 0.001)
 

and the females (P = 0.003). Since lymphoma was observed in male
 

mice as early as 25 weeks, life-table analyses of the incidences
 

in each sex were performed. The results indicated a dose association (P < 0.001) between the administration of estradiol mustard
 

and the time of observation of lymphoma in either sex of mice.
 

In mice, alveolar/bronchiolar adenoma or carcinoma occurred at a
 

significant incidence (P = 0.004) in the low-dose group of males
 

compared with the pooled vehicle controls (controls 2/28, low-


dose 12/30, high-dose 5/24) and at a significant incidence (P =
 

0.022) in the low-dose group of females compared with the pooled
 

vehicle controls (controls 1/28, low-dose 7/27, high-dose 1/18).
 

42
 
Sarcoma of the myocardium similarly occurred at a significant
 

incidence (P = 0.015) in the low-dose group of males compared
 

with the pooled vehicle controls (controls 0/28, low-dose 6/30,
 

high-dose 2/24) and at a significant incidence (P = 0.002) in the
 

low-dose group of females compared with the pooled vehicle
 

controls (controls 0/28, low-dose 8/27, high-dose 1/12).
 

Metastasis of the myocardial sarcoma to the lung occurred in
 

low-dose males and low-dose females. The survival of both
 

high-dose males and high-dose females was slightly lower than
 

that of the respective low-dose groups and may account for the
 

higher numbers of pulmonary tumors and myocardial sarcomas among
 

low-dose mice of both sexes. The association of myocardial
 

sarcoma with administration of the chemical in both dosed groups
 

of each sex is strengthened by the fact that these tumors of the
 

myocardium have not occurred in the more than 500 male and 500
 

female historical-control mice of this strain at the laboratory.
 

Squamous-cell carcinoma of the stomach occurred in the dosed male
 

mice (high-dose 2/29) and in the dosed female mice (low-dose
 

2/26, high-dose 2/14), but was absent in all controls. A
 

carcinoma, NOS, of the stomach also occurred in a low-dose male
 

mouse. Although the incidences in this bioassay were too low to
 

be statistically significant, the fact that no squamous-cell
 

carcinomas of the stomach have occurred in the more than 500 male
 

43
 
and 500 female historical-control mice of this strain at this
 

laboratory indicates that these gastric tumors were related to
 

the administration of the estradiol mustard.
 

Two investigations of the carcinogenic potential of the chemotherapeutic agent estradiol mustard have been reported. Stoner
 

et al. (1973), using a pulmonary tumor test system, found a high
 

incidence of lung tumors in male and female A/He mice intraperitoneally injected three times per week for 8 weeks with doses
 

totaling 1.6, 1.2, or 0.48 g/kg body weight, terminating the
 

experiment 24 weeks after the first injection. Schmahl and Habs
 

(1976) subcutaneously injected Sprague-Dawley rats once per week
 

with 10 mg/kg or 20 mg/kg body weight for their life spans. No
 

carcinogenic effects were observed, although the mean life span
 

was considerably reduced (low-dose rats 513 + 62 days, high-dose
 

rats 492 + 92 days, control rats 721 + 123 days.
 

It is concluded that under the conditions of this bioassay,
 

estradiol mustard administered in a buffered saline vehicle was
 

not carcinogenic in Sprague-Dawley rats. Estradiol mustard was
 

carcinogenic in both male and female B6C3F1 mice, causing
 

lymphoma, sarcoma of the myocardium, alveolar adenoma or
 

carcinoma, and squamous-cell carcinoma of the stomach.
 

44
 
VI. BIBLIOGRAPHY
 

Armitage, P. , Statistical Methods in Medical Research, John Wiley
 

& Sons, Inc., New York, 1971, pp. 362-365.
 

Berenblum, I., ed., Carcinogenicity Testing; _A Report of the
 

Panel on Carcinogenicity of the Cancer Research Commission
 

of the UICC, Vol. _2, International Union Against Cancer,
 

Geneva, 1969.
 

Cox, D. R., Regression models and life tables. J. R. Statist.
 

Soc. B 34(2);187-220, 1972.
 

Cox, D. R. , Analysis of Binary Data, Methuen & Co., Ltd., London,
 

1970, pp. 48-52.
 

Gart, J. J., The comparison of proportions: a review of
 

significance tests, confidence limits and adjustments for
 

stratification. Rev. Int. Statist. Inst. 39(2);148-169,
 

1971.
 

Kaplan, E. L. and Meier, P., Nonparametric estimation from
 

incomplete observations. J. Amer. Statist. Assoc.
 

51:457-481, 1958.
 

Linhart, M. S., Cooper, J. A., Martin, R. L. , Page, N. P., and
 

Peters, J. A., Carcinogenesis bioassay data system. Comp.
 

and Biomed. Res. 7:230-248, 1974.
 

Miller, R. G., Jr., Simultaneous Statistical Inference,
 

McGraw-Hill Book Co., New York, 1966, pp. 6-10.
 

National Cancer Institute, Drugs vs Cancer, U. S. Department of
 

Health, Education, and Welfare, National Institutes of
 

Health, DHEW Publication No. (NIH) 76-786, 1976, pp. 5 and
 

8.
 

Prejean, J. D., Peckham, J. C., Casey, A. E. , Griswold, D. P.,
 

Weisburger, E. K., and Weisburger, J. H., Spontaneous tumors
 

in Sprague-Dawley rats and Swiss mice. Cancer Res.
 

13:2768-2773, 1973.
 

45
 
Saffiotti, U., Montesano, R., Sellakumar, A. R. , Cefis, F., and
 

Kaufman, D. G., Respiratory tract carcinogenesis in hamsters
 

induced by different numbers of administrations of 

benzo(a)pyrene and ferric oxide. Cancer Res. 32;1073-1081, 

1972. 

Schmahl, D. and Habs, M., Absence of carcinogenic effects of
 

estradiol mustard (NSC-112259) in rats. Z. Krebsforsch.
 

8]_: 197-198, 1976.
 

Sontag, J. M. , Page, N. P., and Saffiotti, U., Guidelines for
 

Carcinogen Bioassay in Small Rodents, National Cancer
 

Institute, Bethesda, Maryland, 1975.
 

Stoner, G. D., Shimkin, M. B., Kniazeff, A. J., Weisburger, J.
 

H., Weisburger, E. K. , and Gori, G. B., Test for
 

carcinogenicity of food additives and chemotherapeutic
 

agents by the pulmonary tumor response in strain A mice.
 

Cancer Res. 33:3069-3085, 1973.
 

Tarone, R. E., Tests for trend in life table analysis.
 

Biometrika 62(3);679-682. 1975.
 

Vollmer, E. P., Screening of endocrine agents. In: Cancer
 

Medicine, Holland, J. F. and Frei, E., III, eds., Lea &
 

Febiger, Philadelphia, 1973a, pp. 891-892.
 

Vollmer, E. P., Taylor, D. J., Masnyk, I. J., Cooney, D., Levine,
 

B., and Piczak, C., Estradiol mustard (NSC-112259)
 

clinical brochure. Cancer Chemotherapy Reports, Part III
 

4^1):121-140, 1973b.
 

Wall, M. E., Abernethy, G. S., Jr., Carroll, F. I., and Taylor,
 

D. J., The effects of some steroidal alkylating agents on
 

experimental animal mammary tumor and leukemia systems. J.
 

Med. Chem. 12(5):810-818, 1969.
 

Wittliff, J. L., Beatty, B. W., Savlov, E. D., Patterson, W. B.,
 

and Cooper, R. A., Jr., Estrogen receptors and hormone
 

dependency in human breast cancer. Recent Results Cancer
 

Res. 57:59-77, 1976.
 

Young, S. and Hallowes, R. C., Tumors of the mammary gland. In:
 

Pathology of Tumors in Laboratory Animals, Vol. I., Tumors
 

of the Rat, Part _I^, Turusov, V. S. , ed., International
 

Agency for Research on Cancer, Geneva, 1973, pp. 31-74.
 

46
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN RATS
 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE
 

47
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE 

{JNTREATED VEHICLE
 

CONTROL CONTROL LOW DOSE HIGH DOSE
 

ANIHALS INITIALLY IN STODY 10 10 35 35
 * HI HALS HECBOPSIED 10 9 35 33
 ANIHALS EXAMINED HISTOPATHOLOGICALL1 r 10 9 35 33
 

INTEGUMENTARY SYSTEM
 

*SKIH (10) (9) (35) (33)
 

SQO/IBODS CELL PAPILLONA 1 (3X)
 FIBROMA 1 (3«)
 

*SOBCOT TISSDE (10) (9) (35) (33)
 

SEBACEOUS ADENOMA 1 (3X)
 SABCOBA, NOS 1 (3X)
 FIBROMA 1 <3X)
 

BESPIBATOBY SYSTEM
 

HONE
 

HEHATOPOIETIC SYSTEH
 

NONE
 

CIRCULATOBY SYSTEH
 

BONE
 

DIGESTIVE SYSTEH
 

NONE
 

DBINABY SYSTEH
 

NONE
 

* NDHBER OF AHIHALS HITH TISSUE EXAMINED MICROSCOPICALLY 

* NOHBEH OF ANIMALS 1TECBOPSIED 

49 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIC iH DOSE 

ENDOCRINE SYSTEM 

tPITUITAHY 

CHBOHOPHOBE ADENOMA 

CHROMOPHOBE CARCINOMA 

(8) 

2 (25%) 

2 (25%) 

(8) 

3 (38X) 

1 (13*) 

(35) 

u (11%) 1 (3%) 

(31) 

IADRENAL PHEOCHHOMOCYTOHA (10) (9) 

1 (11X) 

(35) (32) 

ITHYROID 

FOLLICULAH-CELL ADENOMA 

C-CELL CABCINOHA 

(10) (9) (3U) 

1 (3*) 2 (6X) 

(32) 

*PANCREATIC ISLETS 

ISLET-CELL ADENOMA 

(10) (9) (35) (32) 

1 (3X) 

REPBODOCTIVE SYSTEM 

*NAHHABY GLAND 

ADENOCAHCINOMA, HOS 

FIBBOADENOMA 

(10) (9) (35) 

1 (3%) 2 (6X) 

(33) 

5 (15X) 

ITESTIS 

INTEBSTITIAL-CELL TOHOR 

(10) 

1 (10*) 

(9) (35) (32) 

NERVOUS SYSTEM 

IBRAIN 

GLIOHA, NOS 

ASTBOCYTOHA 

(10) (9) (33) (32) 

1 

1 

(3X) 

(3X) 

SPECIAL SENSE ORGANS 

*EAB CANAL 

SQUAMOUS CELL CARCINOMA (10) (9) (35) 

1 (3«) 

(33) 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

•ABDOMINAL CAVITY 

SAECQHAi fiOS._ __ _ 

(10) (9) (35) 

__ . . , . 

(33) 

1 liSi_ 

t NOBBEH OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBBB OF ANIMALS NECROPSIED 

50
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

uINTREATED VEHICLE
 

CONTROL CONTROL LOW DOSE HIGH DOSE 

AIL OTHER SYSTEHS 

NONE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATDBAL DEATHS HOHIBOMD SACRIFICE SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED TERMINAL SACRIFICE ANIHAL HISSING 

10 

1 

9 

10 

1 

9 

35 

2 

1 

32 

35 

6 

3 

26 

1 INCLUDES AUTOLYZED ANIHALS 

TUMOR SUMMARY 

TOTAL ANIHALS WITH PRIHABY TUHORS* 

TOTAL PRIMARY TUBORS 

5 

5 

4 

5 

10 

1<1 

10 

12 

TOTAL ANIMALS WITH BENIGN TUMORS TOTAL BENIGN TUMORS 3 

3 

3 

a 

7 

8 

8 

9 

TOTAL ANIMALS WITH MALIGNANT TUMORS TOT1L MALIGNANT TUHORS 2 

2 

1 

1 6 6 

3 

3 

TOTAL ANIMALS WITH SECONDARY TUMORS! TOTAL SECONDARY TUMORS 

TOTAL ANIHALS WITH TDHORS UNCERTAIN­ HENIGI OR MALIGNANT TOTAL UNCERTAIN TUMORS 

TOTAL ANIHALS WITH TUMORS UNCERTAIN* PRIMARY OR MBTASTATIC TOTAL UNCERTAIN TUMORS 

* E'HIHAHT TOHOBS: ALL TUHORS EXCEPT SECONDARY TOHOBS • SECORDART TOHORS: HETASTATIC TUMORS OR TOHORS INVASIVE INTO AN ADJACENT ORGAN 

51 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE 

IINTREATED CONTROL VEHICLE 

CONTROL LOW DOSE 

ANIMALS INITIALLY IN STUDY ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTOPATH OLOGICALLY 

10 

10 

10 

10 

8 

8 

35 

3I» 

3« 

INTEGUMENTARY SYSTEM
 

*SDBCDT TISSUE (10) (8) (3U)
 

SARCOMA, DOS
 

FIBROMA 1 (3X)
 

RESPIRATORY SYSTEM
 

tLUNG (10) (8) (3K)
 

SQDAMOUS CELL CARCINOMA, METASTA
 

SARCOMA, SOS, METASTATIC
 

HEMATOPOIETIC SYSTEM
 

*MDLTIPLE ORGANS (10) (8) (34)
 

MiLIG.LYMPHOMA, HISTIOCYTIC TYPE
 

LYMPHOCYTIC LEUKEMIA 1 (3«)
 

CIRCULATORY SYSTEM
 

HONE
 

DIGESTIVE SYSTEM
 

NONE
 

URINARY SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

tPITUITARY (10) (6) (31)
 CHROMOPHOBE ADENOMA 5.150)11. " I"*). _ 9_J29X1
 

t NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

35
 

33
 

33
 

(33)
 

1 (3X)
 

(33)
 

1 (3X)
 

1 (3J)
 

(33)
 

1 (3%)
 

(33)
 

9 127X1
 

52
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CHROBOPHOBE CARCINOMA 

•THYROID 

POLLICOLAR-CBLL CARCINOMA 

C-CBLL ADENOM A 

C-CELL CARCINOMA 

tPANCREATIC ISLETS 

ISLET-CELL CARCINOMA 

REPRODUCTIV E SYSTEM 

*MAHHARY GLAND 

ADENOMA , NOS 

ADENOCARCINONA, NOS 

PIBROADENOM A 

tUTEROS 

SARCOMA , NOS 

ENDOMETRIA L STROMAL POLYP 

NERVOD S SYSTEM 

NON E 

SPECIAL SENSE ORGAN S 

•E AR CANA L 

SQUAHODS CELL CARCINOM A 

MOSCOLOSKELETAL SYSTEM 

NON E 

BODY CAVITIES 

NON E 

A LL OTHER SYSTEMS 

NON E 

t NUMBER OF ANIMAL S HITH TISSUE

* NOMBEfi OF iNIMALS NEC80PSIED 

UNTREATED VEHICLE

 

CONTROL CONTROL LOW DOSE 

2 (6X) 

(10) (7) (32) 

1 (10*) 

(10) (8) (3U) 

1 (3X) 

(10) (8) (3d) 

1 OX) 

3 (9X) 

5 (SOX) a (SOX) 16 (U7X) 

(10) (7) (34) 

1 (3X) 

1 (10X) 

(10) (8) (34) 

 EXAMINE D MICROSCOPICALLY 

HIGH DOSE 

2 (6X) 

(33) 

1 (3X) 

1 (3X) 

(33) 

(33) 

5 (15X) 

16 (48*) 

(33) 

(33) 

1 (3X) 

53
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

UN 

Cl 

TREATED 

ONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

ANIHAL DISPOSITION SUMMARY 

AHIHALS INITIALLY IH STUDY NATURAL DEATHS 

BOHIBOMD SACEIFICE 

SCHEDOLED SACBIFICE ACCIDENTALLY KILLED 

TEBHINiL SACBIFICE ANIMAL HISSING 

10 

10 

10 

a 

6 

35 

6 

a 

25 

35 

14 

10 

21 

» INCLUDES AOTOLYZED ANIHALS 

TUHOH SUMMARY 

TOTAL ANIHALS KITH PBIHAHY TUHOBS* TOTAL PBIHABY TUHOBS 7 

12 

5 

8 

27 

35 

26 

37 

TOTAL ANIHALS WITH BENIGN TUHOBS TOTAL BHNIGN TUHOBS 7 

12 

5 

8 

23 

27 

22 

25 

TOTAL ANIHALS WITH HALIGNANT TOHOBS TOTAL MALIGNANT TDMOBS 7 

8 

10 

12 

TOTAL ANIHiLS WITH SECONDARY TUMOBS* TOTAL SECONDARY TUHOBS 2 

2 

TOTAL ANIMALS WITH TUHOBS UNCEHTAIN­ BENIGN OB HALIGNANT 

TOTAL UNCEBTAIN TUHOHS 

TOTAL ANIHALS WITH TUMOBS ONCEHTAIN­ PRIHAHY OB HETASTATIC TOTAL UNCERTAIN TUHOBS 

* PRIHiBY TUHOBS: ALL TOHOBS EXCEPT SKCONDiBY TOHORS I SECOMDAHY TUHORS: METASTATIC TOMORS OH TOHORS INVASIVE IHTO AH ADJACENT ORGAN 

54
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MICE
 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE
 

55
 

TABLE B1. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE 

UNTREATED VEHICLE
 

CONTROL CONTROL
 LOW DOSE
 

ANIMALS INITIALLY IN STUDY 15 14 34
 

ANIMALS HECROPSIED 15 14 32
 

ANIMALS EXAMINED HISTOPATH OLOGIC ALLY 15 14 32
 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (15) (14) (32)
 

SARCOMA, NOS 1 (3X)
 

RESPIRATORY SYSTEM 

UUNG 

ALVBOLAR/BRONCHIOLAR ADENOMA 

ALTEOLAR/BRONCHIOLAR CARCINOMA 

SARCOMA, NOS, METASTATIC 

(15) 

2 (13X) 

(14) 

2 (14X) 

(32) 

8

6

3

 (25X) 

 (19X) 

 (9X) 

HEMATOPOIETIC SYSTEM
 

•MULTIPLE ORGANS (15) (14) (32)
 

MALIGNANT LYHPHOMA, NOS 1 (3X)
 

MALIG. LYHPHOMA, LYMPHOCYTIC TYPE 3 (9X)
 

HALIG. LYHPHOHA, HISTIOCYTIC TYPE 1 (3X)
 LYHPHOCYTIC LEUKEMIA 1 (3X)
 

ISPLEEN (15) (14) (32)
 

MALIG. LYHPHOHA. HISTIOCYTIC TYPE
 

*THYHUS (15) (14) (31)
 

HiLIG. LYHPHOHA, UNDIFFBR-TYPE
 

CIRCULATORY SYSTEM
 

tntOCARDIOH (15) (14) (32)
 

SARCOMA, NOS 6 (19X)
 

HEHANGIOHA
 

DIGESTIVE SYSIEB
 

ILIVBR (15) (1«) (32)
 

_ISi!AT_2C5iLJU,AB_Ap_5!OflA. 2 1H&1-.. _ _ 3 J9J1. _.
 

t ROBBKB OP ANIBALS BITH TISSUE EIAMIJED MICROSCOPICALLY
 

* NUHBBH OF ANIBALS NECHOPSIED
 

HIGH DOSE
 

35
 

29
 

29
 

(29)
 

(29)
 

4 (14X)
 

1 (3X)
 

(29)
 

7 (24X)
 

8 (28X)
 

(29)
 

1 (3X)
 

(29)
 

1 <3X)
 

(29)
 

2 (7X)
 

1 (3X)
 

(29)
 

1.1UI.
 

57
 
HEPATOCELLULAB CARCINOMA

 

SARCOHA, NOS, METASTATIC 

tSTOMACH 

CARCINOMA , NOS 

SQOABOUS CELL CARCINOM A 

URINAR Y SYSTEM 

NON E 

ENDOCRIN E SYSTEM 

NONE 

REPRODUCTIVE SYSTEM 

• MAMMAR Y GLAND 

ADENOCAHCINOMA , NOS 

*PREPUTIA L GLAN D 

ADNEXA L CARCINOMA 

NERVOU S SYSTEM 

NON E 

SPECIAL SENSE ORGANS 

NON E 

MUSCULOSKELETAL SYSTEM 

NON E 

BODY CAVITIES 

NON E 

ALL OTHER SYSTEMS 

NON E 

* NUHBER 0? ANIHALS WITH TISSUE

* NUFIBER OF ANIHALS NECBOPSIED 

58
 

TABLE B1. MALE MICE: NEOPLASMS (CONTINUED} 

UNTREATED VEHICLE 

CONTROL CONTROL 

1 <7X) 

(15) (1«) 

(15) (1«) 

(15) d«) 

 EXAHINE D MICROSCOPICALLY 

LOW DOSE 

2 (6«) 

1 (3%) 

(32) 

1 (3X) 

(32) 

1 (3X) 

(32) 

HIGH DOSE 

(29) 

2 (7%) 

(29) 

(29) 

1 (3X) 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

uINTREATED VEHICLE
 

CONTROL CONTROL LOW DOSE HIGH DOSE
 

ANIMAL DISPOSITIOM SDHHAHY
 

ANIMALS INITIALLY IN STUDY 15 1U 34 35
 NATURAL DEATHS 1 19 21
 HORIBDND SACRIFICE 1 3 4
 

SCHEDULED SACRIFICE
 ACCIDENTALLY KILLED 1
 

TERMINAL SACRIFICE 11 13 12 9
 ANIMAL HISSING
 

i INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS illTH PRIMARY TUMOBS* 6 4 24 23
 TOTAL PRIMARY TUMORS 7 4 34 29
 

TOTAL ANIMALS WITH BENIGN TUMORS 5 14 10 6
 TOTAL BENIGN TDBORS 6 4 11 6
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 1 18 20
 TOTAL MALIGNANT TUMORS 1 23 23
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 3
 TOTAL SECONDARY TDHOHS 4
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIHiBY TUMORS: ILL TOHOHS EXCEPT SECONDiRY TDHOBS
 t SECONDARY TUHOHS: HETASTATIC TUMOBS OR TOHOHS INVASIVE INTO AN ADJACENT ORGAN
 

59
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE

 

u
 

ANIMALS INITIALLY IN STUDY
 

ANIHALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGDHEHTARY SYSTEM
 

*SUBCOT TISSUE
 

SABCOMA, NOS
 

BESPIBATOHY SYSTEM
 

ILUNG
 

CABCINOMA, NOS, METASTATIC
 

ALVEOLAB/BSONCHIOLAB ADENOMA
 

ALVEOLAR/BRONCHIOLAR CARCINOMA
 

SABCOHA, NOS, HETASTATIC
 

HEMATOPOIETIC SYSTEM
 

•MULTIPLE ORGANS
 

MALIGNANT LYMPHOMA, NOS
 

MALIG.LYMPHOMA. LYMPHOCYTIC TYPE
 

MALIG.LYHPHOMA, HISTI3CYTIC TYPE
 

ITHYMOS
 

HALIG. LYMPHOMA, UNDIFFER-TYPE
 

CIRCULATORY SYSTEM
 

IMYOCARDIUM
 

SARCOMA, NOS
 

DIGESTIVE SYSTEM
 

tSTOHACH
 

SQUAMOUS CELL CARCINOMA
 

URINARY SYSTEM
 

--BONE. _ _ _
 

NTREATED
 

CONTROL
 

15
 

11
 

14
 

(14)
 

(14)
 

(14)
 

1 (7*)
 

(14)
 

(14)
 

(14)
 

VEHICLE
 

CONTROL
 

16
 

16
 

16
 

(16)
 

(16)
 

(16)
 

(16)
 

(16)
 

(16)
 

LOW DOSE
 

36
 

31
 

30
 

(31)
 

2 (6X)
 

(31)
 

3 (10X)
 

4 (13%)
 

5 (16X)
 

(31)
 

3 (10X)
 

2 (6X)
 

4 (13X)
 

(3D
 

(31)
 

8 (26X)
 

(31)
 

2 (6X)
 

HIGH DOSE
 

35
 

24
 

24
 

(24)
 

1 (4X)
 

(24)
 

1 (4*)
 

1 (4*)
 

(24)
 

1 (4X)
 

8 (33X
 

1 (4X)
 

(24)
 

1 {"»«>
 

(24)
 

1 (4X)
 

(24)
 

2 (8X)
 

» BOHBE8 OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* BOBBER OF ANIHALS NECROPSIED
 

60
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

ENDOCBIHE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEH
 

•MAMMARY	 GLAND
 

CARCINOMA, NOS
 

ADENOCARCINOMA, NOS
 

*VAGINA
 

SQUAMOUS CELL CARCINOMA
 

tDTEBUS
 

ADENOCARCINOMA, NOS
 

ENDOHETRIAL STROHAL POLYP
 

IOVABY
 

CYST ADENOMA, NOS
 

HILAH-CELL TOMOB
 

NEB70US SYSTEH
 

NONE
 

SPECIAL SENSE ORGANS
 

•HABDEBIAN GLAND
 

ADENOMA, NOS
 

•EAR	 CANAL
 

SQDAHOas CELL CARCINOMA
 

BOSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

•PERITONEUM
 

SO.OA90.aS. CBL.L._C»BCINOBAJ. MET1STA
 

UNTREATED
 

CONTROL
 

d«)
 

(U)
 

(1«)
 

1 (7%)
 

(1t)
 

(11)
 

(1U)
 

(It)
 

VEHICLE
 

CONTROL
 

(16)
 

(16)
 

(16)
 

(16)
 

(16)
 

(16)
 

(16)
 

LOW DOSE
 

(31)
 

(31)
 

(31)
 

1 (3X)
 

(31)
 

1 (3*)
 

(31)
 

(31)
 

1 <3X)
 

(31)
 

-_ _ I i38L _.
 

HIGH DOSE
 

(2t)
 

1 (t«)
 2 (8%)
 

(2<«)
 

1 ("*)
 

(2i»)
 

(2i»)
 

1 C*«)
 

(2t)
 

1 (<»X)
 

(2t)
 

(2«)
 

_
 

I ROBBER OP ANIMALS MITH TISSUE EXABINED MICROSCOPICALLY
 

* NOMBE8 OF ANIMALS NECHOPSIED
 

61
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

uNTREATED VEHICLE
 

CONTROL CONTROL LOW DOSE HIGH DOSE 

SARCOMA, NOS 1 OX) 1 (<•*) 

ALL OTHER SYSTEMS 

*MULTIPI.E ORGANS 

SQUAHOUS CELL CARCINOMA, HETASTA 

(14) (16) (31) (24) 

1 (<**) 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATHS 

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 

ANIMAL MISSING 

15 

1 

1 

2 

11 

16 

1 

2 

1 

12 

36 

24 

7 

1 

4 

35 

19 

14 

2 

i INCLUDES AUTOLYZED ANIMALS 

TUMOR SUMMARY 

TOTAL ANIMALS WITH PRIMARY TUMORS* 

TOTAL PRIMARY TUMORS 2 

2 

25 

32 

20 

23 

TOTAL ANIMALS WITH BENIGN TUMORS 

TOTAL BENIGN TUMORS 

1 

1 

U 

u 3 

3 

TOTAL ANIMALS WITH MALIGNANT TUMORS 

TOTAL MALIGNANT TUMORS 

1 

1 

25 

28 

19 

20 

TOTAL ANIMALS WITH SECONDARY TUMORS* 

TOTAL SECONDARY TOMORS 

6 

6 

2 

2 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS ilTH TUMORS UNCERTAINPRIMARY OR METASTATIC 

TOTAL UNCERTAIN TUMORS 

* PRIHABY TUMOHS: ALL TUMORS EXCEPT SECONDAHY TUMORS t SECONDAHY TOHOHS: HETASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN 

62
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN RATS
 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE
 

63
 

TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE

 

uNTREATED 

CONTROL VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 

ANIMALS HECROPSIED 

ANIMALS EXAHINBD HISTOPATHOLOGIC ALLY 

10 

10 

10 

10 

9 

9 

35 

35 

35 

35 

33 

33 

INTEGUHENTARY SYSTEM
 

*SKIN (10) (9) (35) (33)
 

DLCBH, CHRONIC 1 (3%)
 INFLAMMATION, CHRONIC NECBOTIZIN 1 (3%)
 

HYPE8KERATOSIS 1 (3%)
 

ACANTHOSIS 1 (3%)
 

RESPIRATORY SYSTEM
 

iTRACHEA (10) (9) (35) (33)
 

INFLAMMATION, SUPPURATIVE 1 (3%)
 INFLAMMATION, CHRONIC 1 (10%) 3 (33%) 5 (1U%) 5 (15%)
 INFLAMMATION, CHRONIC SUPPORATIV 2 (6%)
 

*L UNO/BRONCHIOLE (10) (9) (35) (33)
 

HYPEHPLASIA, LYHPHOID 1 (11«) 1 (3%)
 

*LUNG (10) (9) (35) (33)
 

EMPHYSEMA, NOS 1 (3%)
 

INFLAMMATION, INTERSTITIAL 1 (3%) 1 (3%)
 PNEUMONIA, LIPID 1 (3%)
 BRONCHOPNEUHONIA SUPPURATIVE 1 (10%) 1 (11%) 2 (6%)
 

BRONCHOPMEOHONIA CHR3NIC SOPPURA 2 (22%) 5 (14%) 1 (3%)
 

METAPLASIA, OSSEOUS 1 (3%)
 

HEBATOPOIETIC SYSTEM
 

•BONE NARROW (9) (8) (35) (32)
 

ATROPHY, NOS 6 (61%) 6 (75%) 17 (49%) 19 (59%)
 HYPERPLASIA, GHANOLOCYTIC 1 (3%)
 

tSPLEBN (10) (9) (35) (32)
 

INFLAMMATION* SUPPORATIVE. 1 J3%1_ _
 

t NHHBEH OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

65
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

HYPEBPLASIA, GBANDLOCYTIC 

HEIUTOPOIESIS 

1

1

 (3X) 

 (3X) 1 (3X) 

tSPLEHIC FOLLICLES 

ATBOPHY, SOS 

(10) (9) (35) 

1 (3X) 

(32) 

MANDIBOLA B L. MODE 

HYPEBPLASIA, LYMPHOID (D d)1 (100X) 

tHESENTEBIC L. NODE 

INFLAHHATIOH, CHBONIC SOPPUHATI 7 

IHPLAHaATION. CHBOMIC HECBOTIZIN 

(D 

1 (100X) 

(D 

1 (100X) 

CIRCULATOBY SYSIEH 

HOME 

DISESTIVE SYSTEH 

tLIVEB 

NECROSIS, COAGOLATIVE 

HYPEBPLASIA, GBAHOLOCYTIC 

(10) (9) (35) 

1 (3X) 

(32) 

1 (3X) 

tLIVEB/CENTBILOBDLAB 

LIPOIDOSIS 

(10) (9) (35) (32) 

1 (3X) 

IPANCBEAS 

PEHIABTEBITIS 

(10) (9) (35) (32) 

1 (3X) 

tPANCBEATIC

ATBOPHY.

 ACINDS 

 BOS 

(10) (9) (35) (32) 

2 (6X) 

• STOMACH 

NECROSIS, SOS 

(10) (9) (35) (32) 

1 (3X) 

•GASTRIC HOSCULARIS 

HINEB1LIZATION 

(10) (9) (35) (32) 

1 (3X) 

fDOODEHU H 

CORGEHITAL HILFOBHATIOH, NOS 

(10) (9) 

1 (1«) 

(35) (32) 

OBINAR Y SYSTEH 

tKIDNEI 

latliflSUiaHj. CHBOfilC _

(10) 

_ 5.J50S1 __ (9) 

8.J89S1. __

(35) 

 _23 J66S1. . 

(32) 

2,2-J69%J. 

» IDNBEB OF ABIWALS IITH TISSOE EXAHIMED BICBOSCOPICALLY 

* NDMBER OF ANIMILS HECBOPSIED 

66
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

LOW DOSE HIGH DOSE
 

(35)	 (32)
 

1 (3X)
 

(35)	 (31)
 

1 (3X>
 

(35)	 (33)
 

1 (3X)
 

(35)	 (32)
 

1 (3X)
 

(35)	 (33)
 

1 (3X)
 

(35)	 (33)
 

2 (6X)
 

(35)	 (33)
 

1 (3*)
 

*UBINART BLiDDEH
 

ULCER, NOS
 

EMDOCHIIIB SYSTEM
 

*PITUITARY
 

CYST. HOS
 

BEP80DUCTIVE SYSTEM
 

*HAHMARY GLAND
 

CYST. NOS
 

*PBOSTATE
 

INFLAMMATION, SDPPDRATIVE
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE OBGANS
 

*EYE/COHNEA
 

ULCER, CHRONIC
 

HDSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDOHINAL CAVITY
 

STEATITIS
 

*PLEDRA
 

METAPLASIA, OSSEOUS
 

ALL OTHER SYSTEMS
 

NOfiJ
 

t NOffBER OF ANIMAL S WITH TISSOE

* NUMBER OF ANIMALS NECROPSIED 

UNTREATED
 

CONTROL
 

(10)
 

(8)
 

(10)
 

(10)
 

(10)
 

(10)
 

1 (10*)
 

(10)
 

VEHICLE
 

CONTROL
 

(9)
 

(8)
 

(9)
 

(9)
 

(9)
 

(9)
 

(9;
 

 EXAMINE D MICROSCOPICALLY 

67
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOS£ 

SPECIAL MORPHOLOGY SUMMAR Y • 

NO LESION REPORTED 

AUTOLYSIS/NO NECROPSY 

2 

1 

1 2 

2 

I NUHBEB OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIHALS NBCROPSIED 

68
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

AHIHALS INITIALLY IN STUDY 

ANIMALS NECBOPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

10 

10 

10 

10 

8 

8 

35 

34 

34 

35 

33 

33 

INTEGUHENTAHY SYSTEM 

NONE 

BESPIBATORY SYSTEM 

tTRACHEA 

INFLAMMATION. CHRONIC SUPPORATIV 

(10) (8) (34) (33) 

2 (6X) 

ILUNG/BRONCHIOLE 

HYPERPLASIA, LYBPHOID 

(10) (8) (34) 

1 (3X) 

(33) 

tLONG 

BRONCHOPNEOMONIA SUPPORATITE 

BRONCHOPNEUMONIA CHRONIC SUPPUFA 

(10) 

1 (10X) 

(8) 

1 O3X) 

(34) (33) 

4 (12X) 

HEMATOPOIBTIC SYSTEM 

tBONE MARROW 

ATROPHY, NOS (9)

6 (67X) 

(8) 

4 (SOX) 

(32) 

16 (SOX) 

(32) 

14 (44X) 

*SPLEEN 

HEHATOPOIESIS (10) 

1 (10%) 

(8) 

1 (13X) 

(34) 

3 (9X) 

(33) 

3 (9X) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

ILIVER 

NECROSIS^ COAGOLATIVE 

(10) (8) 

. ­

(34) 

_ -1 J3X1 

(33) 

* NUMBER OF ANIMALS KITH TISSDE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECHOPSIED 

69 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

ILIYBR/CEMTHILOBULAH 

NECROSIS, DOS 

HECHOSIS, COAGOLATIVE 

(101 (8) 

1 (13%) 

(34) (33) 

1 (3*) 

1 (3X) 

IPANCHEAS 

INFLAMMATION, CHRONIC 

(10) (8) (31) 

1 (3X) 

(33) 

ORINARY SYSTEM 

tKIDNEI 

INFLAMMATION, CHRONIC (10) (8) (31) 

6 (18X) 

(33) 

8 (24%) 

ENDOCRINE SYSTEM 

(ADRENAL 

ANGIECTASIS (10) 

3 (30%) 

(8) 

1 (13X) 

(31) 

12 (35X) 

(33) 

7 (21%) 

HEPHODOCTIVE SYSTEH 

•MAMMARY GLAND 

CYST, KOS 

(10) 

3 (30%) 

(8) 

1 (13%) 

(3U) 

« (12X) 

(33) 

3 (9%) 

tUTERUS 

HEMORRHAGE 

(10) (7) (3U) (33) 

1 (3%) 

tUTERUS/ENDOMBTRIUN 

INFLAMMATION, SUPPOHATIVE 

INFLAMMATION. CHRONIC SOPPORATIV 

HYPERPLASIA, NOS 

HYPERPLASIA, CYSTIC 

(10) 

1 (10%) 

1 (10%) 

(7) 

1 (14%) 

(3U) 

6 (18X) U (12X) 

1 (3X) 

(33) 

5 (15%) 3 (9%) 

2 (6%) 

*0¥ARY 

CYST, NOS 

(10) (7) (34) 

1 (3X) 

(33) 

1 (3%) 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

*AURICDLAR CARTILAGE 

DYSPLASIAX NOS 

(10) (8) 

.__

(34) 

1 J3$> _. 

(33) 

I KDHBEH OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

70
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MUSCOLOSKELETAL SYSTEM
 

NONE
 

BOD! CAVITIES
 

*ABDOMINAL CAVITY
 

STEATITIS
 

ALL OTHEB SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUHHAHY
 

NO LESION REPORTED
 

ADTOLYSIS/NO NECROPSY
 

UNTREATED VEHICLE
 CONTROL
 CONTROL LOW DOSE HIGH DOSE
 

(10) (8) (34) (33) 

1 (3X) 

1 2 1 1 

2 1 2 

* NOBBED OF AMIHALS WITH TISSDE EX AHI NED HICROSCOPICALLY
 

* HUHBEE OF AHIHALS NECHOPSIED
 

71 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MICE
 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE
 

73
 

TABLE 01. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE 

UNTREATED VEHICLE
 CONTROL CONTROL LOW DOSE
 

ANIMALS INITIALLY IN STUDY 15 1t 34
 

ANIMALS NECROPSIED 15 11* 32
 ANIHALS EXAMINED HISTOPATHOLOGICALLY 15 14 32
 

INTEGOSENTABY SYSTEM
 

*SKIN (15) (14) (32)
 

OLCBR, FOCAL 1 (3X)
 

*SOBCOT TISSUE (15) (14) (32)
 

EDEMA. NOS 1 (3X)
 INFLAMMATION, CHRONIC 1 (7X)
 INFLAMMATION. CHRONIC FOCAL 1 OX)
 

RESPIRATORY SYSTEM
 

•LONG/BRONCHIOLE	 (15) (14) (32)
 

HYPEHPLASIA, PLASMA CELL 1 (3X)
 

tLUNG (15) (14) (32)
 

BRONCHOPNBUMONIA SUPPURATI7E 1 (7X)
 

HEMATOPOIETIC SYSTEM 

*BONE HARROW 

ATROPHY. NOS (1t) (13) (30) 

tSPLEEN ARGIICTASIS (15) d«) (32) 

HTPERPLASIA, LYNPHOID HBMATOPOIESIS 1 (7X) 2 (6«) 

fLYHPH NODE CYST, NOS (2) 

1 (50X) 

(3) 

HKNORRHAGE 1 (50*) 

tCERVICAL LYMPH NODE 

HIESBEIASJU llfliaaiB . (2) 

.

(3) 

 - 1 133SI . 

* NUHBBR OF ANIMALS WITH TISSUE EXAMINED HICBOSCOPICALLY * NUMBER OF ANIHALS NECBOPSIKD 

HIGH DOSE
 

35
 

29
 

29
 

(29)
 

(29)
 

(29)
 

(29)
 

1 (3«)
 

(26)
 

2 (8X)
 

(29)
 

1 (3X)
 

1 (3X)
 

3 (10X)
 

(3)
 

(3)
 ,_
 

75
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED VEHICLE
 CONTROL CONTROL
 

tMESENTEBIC L. NODE (2)
 INFLAMMATION BITH FIBBOSIS
 

HYPEBPLASIA. LYMPHOID
 

tPELVIC LYMPH NODE (2)
 

INFLAMMATION, SUPPURATIVE
 ABSCESS, NOS
 

#THYHOS (15) (It)
 HYPEBPLASIA, LYMPHOID
 

CIBCULAT08Y SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVER (15) (1U)
 

INFLAMMATION, SUPPURATIVE 1 (7*)
 NECBOSIS, FOCAL 1 (7X)
 NECROSIS, COAGOLATIVE
 LIPOIDOSIS 1 (7X)
 HYPEBPLASIA, NODULAR 1 (7X) 1 (7X)
 HYPERPLASIA, GRANDLOCYTIC 1 (7X)
 

*BILE DUCT (15) (11)
 HYPEBPLASIA, NOS 1 (7X)
 

tPANCBEAS (15) (1U)
 

INFLAMMATION WITH FIBROSIS
 

URINARY SYSTEM
 

IKIDNEY (15) (1U)
 

PYELONEPHRITIS SDPPDRATIVE 1 (7X)
 

ABYLOIDOSIS
 

tURINARY BLADDER (15) (11)
 

INFLAMMATION, CHRONIC
 ULCER, CHBONIC
 

ENDOCRINE SYSTEM
 

*ADBENAL (15) (11)
 AMYLOIDOSIS
 

* NOMBEB OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBEB OF ANIMALS NECBOPSIED
 

LOW DOSE
 

(3)
 

1 (33%)
 

(3)
 

1 (33»)
 

1 (33X)
 

(31)
 

(32)
 

(32)
 

(32)
 

1 (3X)
 

(32)
 

1 (3X)
 

(32)
 

1 (3X)
 

(32)
 

__ 1 13X1 .
 

HIGH DOSE
 

(3)
 

2 (67X)
 

(3)
 

(29)
 

1 (3X)
 

(29)
 

1 (3X)
 

1 (3X)
 

(29)
 

(29)
 

(29)
 

(29)
 

1 (3X)
 

(29)
 

76
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED VEHICLE 

CONTROL CONTROL 

REPRODUCTIV E SYSTEM 

IPROSTATE (15) (11) 

INFLAMMATION , SUPPURAIIV E 

NERVOU S SISTEH 

NON E 

SPECIAL SENSE OBGANS 

NON E 

HOSCULOSKELETAL SYSTEM 

*BONE (15) (1«) HEALED FRACTURE 

CALLUS 

BODY CAVITIES 

*PERITONEDM 

INFLAMMATION , CHBONI: FOCAL 

(15) (11) 

•MESENTERY 

NEC80SIS, PAT 

(15) 

1 (7X) 

(14) 

ALL OTHER SYSTEMS 

*nULTIPLE OBGANS (15) (14) 

HYPEBPLASIA. GRAHDLOCYTIC 1 (7X) 

SPECIAL MOBPHOLOGY SUMMARY 

NO LESION REPORTED 5 8 

NO NECROPSY PERFORMED 

ADTOLYSIS/NO NECROPSY 

f	 NUHBER OF ANIMALS IITH TISSUE EIAHIHBD HICHOSCOPICALLT
 

* NOHBEH OF AHIIULS NECROPSIED
 

LOW DOSE HIGH DOSE 

(32) (29)

 

1 OX) 

(32) 

(32) 

1

(32) 

 (3X) 

(29) 

1 (3X) 

1 (3X) 

(29) 

(29) 

(32) (29) 

6 

1 

1 

2 

1 

5 

77
 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE 

1JNTREATED VEHICLE 

CONTROL CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALLY IN STDDY 15 16 36 35 

ANIflAL S NECROPSIED 11 16 31 21 

AMIHAL S EXAMINE D HISTOPATHOLOGIC ALL5 r 11 16 30 2U 

INTEGOHENTAB Y SYSTEM 

• SOBCIIT TISSUE (11) (16) (31) (21) 

INFLAMMATION, NECRDTIZING 1 (6%) 

BESPIRATOHY SYSTEM 

ILUNG/BBONCHIOLE (11) (16) (31) (21) 

HYPERPLASIA, LYMPHOID 1 (3X) 

»LON G (it) (16) (31) (21) 

BRONCHOPHEOMONIA SUPP08ATIVE 2 (6X) 1 (1*) 

BRONCHOPNBOMONI A CHB3NIC SDPPUBJ t 1 (IX) 

HYPEBPLASIA. LYMPHOID 1 (6X) 

HEMATOPOIETIC SYSIEB 

IBONE MARRO W (12) (16) (30) (23) 

ATROPHY, HOS 3 (25%) 2 (13*) 1 (3X) 5 (22X) 

HYPERPLASIA, GRAHOLOCYTIC 1 (IX) 

tSPLEEN (1") (16) (31) (21) 

HYPERPLASIA. GHANULOCYTIC 3 (13X) 

HYPEBPLASIA, LYMPHOI D 1 (7*) 1 <6X) 

HBHATOPOIESIS 1 (7») 2 (13X) 1 (3*) 2 (8X) 

tHESEHTERIC L. NODE (D ID (D (3) 

CONGESTION, NOS 1 (100X) 

CIRCOLATOBY SYSTEM 

IBYOCABDIUM (1<» ) (16) (31) (21) 

I8Ei»aa*Siafii_snppnHA.Ti»E i-jisi I KDNBER Of ANIMALS WITH TISSDE EXAMINED MICROSCOPICALLY
 • HUMBEB OF ANIMALS NECROPSIED
 

78
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

DIGESTIVE SYSTEM
 

ILIVEB
 

INFLAMMATION, ACUTE/CHRONIC
 

ADHESION, NOS
 

HYPERPLASIA. NODULAR
 

ANGIECTASIS
 

HEMATOPOIESIS
 

ILIVER/CENTRILOBULAH
 

NECROSIS, NOS
 

URINARY SYSTEM
 

tKIDNBY
 

INFLAMMATION, SUPPURAIIVE
 

INFLAMMATION, CHROMIC
 

HYPEEPLASIA, LYKPHOID
 

tKIDNEY/GLOMERULHS
 ABYLOIDOSIS
 

tKIDNEY/PELVIS
 

HYPERPLASIA, LYMPHOID
 

ENDOCRINE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEM
 

»MAMMARY GLAND
 

CYST, NOS
 

*VAGINA
 

INFLAMMATION, SUPPUHATIVE
 

*UTEHOS/ENDOMETRIOM
 

INFLAMMATION, SBPPURATIVE
 

INFLAMMATION, HEMORRHAGIC
 HYPERPLASIA, CYSTIC
 

• OVARY
 

INFLAMMATION^. HEfiORHHAGIC
 

*	 NUMBER OF ANIMALS WITH TISSUE


*	 "UMBER OF ANIMALS NECBOPSIED
 

 (6%)
 

 (6X)
 

 (75X)
 

-__—_—— —
 

LOW DOSE HIGH DOSE
 

(30) (24)
 

1 (3X)
 

1 <3X)
 

1	 (3X)
 

1	 (4X)
 

(30)	 (24)
 

2 (7X)
 

(31) (24)
 

1 (4X)
 

2 (6X)
 

(31)	 (24)
 

1 (3X)
 

(31) (24)
 

(31)	 (24)
 

1 (3X) 2 (8X)
 

(31)	 (24)
 

1 (3X)
 

(31)	 (24)
 

1 (3X)
 

2 (6X)
 

6 (19X) 5 (21X)
 

(31)	 (24)
 

1 13*1- . .
 

UNTREATED
 

CONTROL
 

(14)
 

(11)
 

(1K)
 

(U)
 

(14)
 

1 (7X)
 

(14)
 

(14)
 

(14)
 

7 (SOX)
 

(14)
 

VEHICLE
 

CONTROL
 

(16)
 

1


(16)
 

(16)
 

1


(16)
 

(16)
 

(16)
 

(16)
 

(16)
 

12


(16)
 

 EXAMINED MICROSCOPICALLY
 

79
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

HTPEBPLASIA, CYSTIC
 

iEBVODS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

•EYE/LACRIMAL GLAND
 

HYPBRPLASIA, NOS
 

•EAR	 CANAL
 

INFLAMMATION. SOPPURATIVE
 

ABSCESS. NOS
 

SUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

•ABDOMINAL CAVITY
 

STEATITIS
 

•PERITONEUM
 

INFLAMMATION. CHRONIC
 ADHESION, NOS
 

ALL OTHEB SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED
 

ACCIDENTAL DEATH
 NO NECROPSY PERFORMED
 

AUTO/NECROPSY/NO HISTO
 

AUTOLYSIS/NO NECROPSY
 

Ij, NOBBBR OF ANIMALS HITH TISSUE


*' NUMBER OF ANIMALS NECROPSIED
 

UNTREATED VEHICLE
 

CONTROL. CONTROL
 

(14) (16) 

1 (6X) 

(11) (16) 

1 (6X) 1 (6X) 

(14)	 (16)
 

1 (7«)
 

(14) (16)
 

5 2
 

1
 

 EXAMINED MICROSCOPICALLY
 

LOW DOSE
 

1 (3*)
 

(31)
 

(31)
 

(31)
 

(31)
 

1 (3*)
 

1 (3X)
 

it
 

1
 

1
 

it
 

HIGH DOSE
 

(21)
 

(24)
 

(21)
 

(21)
 

1
 

1
 

10
 

80 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN RATS
 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE
 

81
 

Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Estradiol Mustard by Gavagea
 

Topography: Morphology
 

Pituitary: Chromophobe
 

Adenoma'3
 

P Valuesc'd
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe
 

Adenoma or Carcinoma'5
 

P Values0 »d
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative *Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Vehicle
 

Control
 

3/17 (18)
 

P = 0.025(N)
 

trol)f
 

5/17 (29)
 

P = 0.003(N)
 

trol)f
 

Vehicle
 

Control
 

3/8 (38)
 

P = 0.003(N)
 

86
 

4/8 (50)
 

P < O.OOl(N)
 

86
 

Low
 

Dose
 

4/35 (11)
 

N.S.
 

0.648
 

0.127
 

4.049
 

0.305
 

0.076
 

1.821
 

85
 

5/35 (14)
 

P = 0.046*(N)
 

0.486
 

0.135
 

1.867
 

0.286
 

0.099
 

1.209
 

79
 

High
 

Dose
 

0/31 (0)
 

P = 0.006*(N)
 

P = 0.039**(N)
 

0.000
 

0.000
 

0.888
 

0.000
 

0.000
 

0.408
 

—
 

0/31 (0)
 

P = 0.001*(N)
 

P = 0.004**(N)
 

0.000
 

0.000
 

0.421
 

0.000
 

0.000
 

0.261
 

—
 
__ 

__ 

__ 

__ 

Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 4
 

Pooled
 

Vehicle
 

Topography: Morphology Control
 

Thyroid: C-cell Carcinoma^ 0/18 (0)
 

P Values0>d N.S.
 

Relative Risk (Pooled Vehicle Con .trol)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma" 0/18 (0)
 

P Values0 »d P = 0.043
 

Relative Risk (Pooled Vehicle Con .trol)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Vehicle
 

Control
 

0/9 (0)
 

N.S.
 

0/9 (0)
 

N.S.
 

—
 

Low
 

Dose
 

2/34 (6)
 

N.S.
 

Infinite
 

0.164
 

Infinite
 

Infinite
 

0.088
 

Infinite
 

85
 

2/35 (6)
 

N.S.
 

Infinite
 

0.159
 

Infinite
 

Infinite
 

0.085
 

Infinite
 

85
 

High
 

Dose
 

0/32 (0)
 

N.S.
 

—
 

—
 

—
 

—
 

5/33 (15)
 

N.S.
 

Infinite
 

0.724
 

Infinite
 

Infinite
 

0.391
 

Infinite
 

85 
__ 

__ 

__ 

Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Estradiol Mustard by Gavagea
 

(continued) 

Pooled 

Vehicle 

Topography: Morphology Control 

Mammary Gland: Fibroadenoma
 

or Adenocarcinoma, NOS" 0/18 (0)
 

P Values0>d N.S.
 

Relative Risk (Pooled Vehicle Con trol)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Brain: Glioma, NOS, or
 

Astrocytomab 0/18 (0)
 

P Values0>d N.S.
 

Relative Risk (Pooled Vehicle Con :trol)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Vehicle
 

Control
 

0/9 (0)
 

N.S.
 

0/9 (0)
 

N.S.
 

—
 

Low
 

Dose
 

3/35 (9)
 

N.S.
 

Infinite
 

0.324
 

Infinite
 

Infinite
 

0.175
 

Infinite
 

85
 

0/33 (0)
 

N.S.
 

—
 

—
 

—
 

—
 

—
 

High
 

Dose
 

5/33 (15)
 

N.S.
 

Infinite
 

0.724
 

Infinite
 

Infinite
 

0.391
 

Infinite
 

85
 

2/32 (6)
 

N.S.
 

Infinite
 

0.174
 

Infinite
 

Infinite
 

0.093
 

Infinite
 

63
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Estradiol Mustard by Gavage3
 

(continued)
 

a

Treated groups received doses of 0.62 or 1.25 mg/kg body weight.
 

^Number of tumor-bearing animals/number of animals examined at site (percent).
 

c

Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the
 

incidence of tumors in a treated group is the probability level for the Fisher exact test for
 

the comparison of that treated group with the vehicle-control group (*) or with the pooled
 

vehicle-control group (**) when P < 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 

^A negative trend (N) indicates a lower incidence in a treated group than in a control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

^The 95% confidence interval of the relative risk between each treated group and the specified
 

control group.
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Estradiol Mustard by Gavagea
 

Pooled
 

Vehicle
 

Topography: Morphology Control
 

Pituitary : Chromophobe
 

Adenoma or Carcinoma'3 10/16 (63)
 

P Valuesc»d N.S.
 

Relative Risk (Pooled Vehicle Con trol)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland:
 

Adenocarcinoma, NOS^ 1/18 (6)
 

P Values0 »d N.S.
 

Relative Risk (Pooled Vehicle Con .trol)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Vehicle
 

Control
 

4/6 (67)
 

N.S.
 

73
 

0/8 (0)
 

N.S.
 

—
 

Low
 

Dose
 

11/31 (35)
 

N.S.
 

0.568
 

0.314
 

1.187
 

0.532
 

0.316
 

1.766
 

77
 

3/34 (9)
 

N.S.
 

1.588
 

0.142
 

80.808
 

Infinite
 

0.163
 

Infinite
 

77
 

High
 

Dose
 

11/33 (33)
 

N.S.
 

0.533
 

0.294
 

1.123
 

0.500
 

0.296
 

1.670
 

66
 

5/33 (15)
 

N.S.
 

2.727
 

0.346
 

124.585
 

Infinite
 

0.354
 

Infinite
 

53
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Estradiol Mustard by Gavagea
 

(continued) 

Pooled 

Topography: Morphology 

Vehicle 

Control 

Vehicle 

Control 

Low
 

Dose
 

High
 

Dose
 

Mammary Gland: Fibroadenoma^ 8/18 (44) 4/8 (50) 16/34 (47)
 16/33 (48)
 

c

'd P Value s N.S. N.S. N.S.
 N.S.
 

Relative Risk (Pooled Vehicle Con .trol)f 

Lower Limit 

1.059
 

0.556
 

1.091
 

0.574
 

Upper Limit 2.327
 2.384
 

Relative Risk (Vehicle Control)f 

Lower Limit 

0.941
 

0.480
 

0.970
 

0.495
 

Upper Limit 3. 105
 3,182
 

Weeks to First Observed Tumor 74 67
 63
 

Mammary Gland: Adenoma, NOS, 

Adenocarcinoma, NOS, or 

Fibroadenomab 9/18 (50) 4/8 (50) 20/34 (59)
 20/33 (61)
 

d P Values0 » N.S. N.S. N.S.
 N.S.
 

Relative Risk (Pooled Vehicle Con itrol)f 

Lower Limit 

Upper Limit 

1.176
 

0.682
 

2.301
 

1.212
 

0.703
 

2.350
 

Relative Risk (Vehicle Control)f 

Lower Limit 

1.176
 

0.621
 

1.212
 

0.640
 

Upper Limit 3.701
 3.780
 

Weeks to First Observed Tumor 74 67
 53
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 

a

Treated groups received doses of 0.62 or 1.25 rag/kg body weight.
 

^Number of tumor-bearing animals/number of animals examined at site (percent).
 

c

Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the
 

incidence of tumors in a treated group is the probability level for the Fisher exact test for
 

the comparison of that treated group with the vehicle-control group (*) or with the pooled
 

vehicle-control group (**) when P < 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 

"A negative trend (N) indicates a lower incidence in a treated group than in a control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

*The 95% confidence interval of the relative risk between each treated group and the specified
 

control group.
 

APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN MICE
 

ADMINISTERED ESTRADIOL MUSTARD BY GAVAGE
 

91
 

Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Estradiol Mustard by Gavagea
 

Topography: Morphology
 

Lung: Alveolar /Bronchiolar
 

Carcinoma"
 

P Valuesc'd
 

Departure from Linear Trend6
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control) f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Vehicle
 

Control
 

0/28 (0)
 

N.S.
 

P = 0.005
 

trol)f
 

Vehicle
 

Control
 

0/14 (0)
 

N.S.
 

P = 0.015
 

—
 

Low
 

Dose
 

6/32 (19)
 

P = 0.018**
 

Infinite
 

1.437
 

Infinite
 

Infinite
 

0.754
 

Infinite
 

72
 

High
 

Dose
 

1/29 (3)
 

N.S.
 

Infinite
 

0.053
 

Infinite
 

Infinite
 

0.027
 

Infinite
 

83 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 

Pooled
 

Vehicle Vehicle Low High
 

Topography: Morphology Control Control Dose Dose
 

Lung: Alveolar /Bronchiolar
 

Adenoma or Carcinoma*5 2/28 (7) 2/14 (14) 12/32 (38) 5/29 (17)
 

P Value sc»d N.S. N.S. P = 0.006** N.S.
 

Departure from Linear Trend6 P = 0.006 P = 0.037
 

Relative Risk (Pooled Vehicle Control)f 5.250 2.414
 

Lower Limit 1.321 0.436
 

Upper Limit 44.443 23.631
 

Relative Risk (Vehicle Control)f 2.625 1.207
 

Lower Limit 0.717 0.236
 

Upper Limit 21.934 11.666
 

Weeks to First Observed Tumor 84 59 68
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice 

Administered Estradiol Mustard by Gavagea 

(continued) 

Pooled 

Vehicle Vehicle Low High 

Topography: Morphology Control Control Dose Dose 

Hematopoietic System: Lymphoma 

or Lymphocytic Leukemia" 0/28 (0) 0/14 (0) 6/32 (19) 17/29 (59) 

P Values0»d P < 0.001 P < 0.001 P = 0.018** P < 0.001* 

P < 0.001** 

Relative Risk (Pooled Vehicle Con trol)f Infinite Infinite 

Lower Limit 1.437 5.435 

Upper Limit Infinite Infinite 

Relative Risk (Vehicle Control) ^ Infinite Infinite 

Lower Limit 0.754 2.848 

Upper Limit Infinite Infinite 

__ 

Weeks to First Observed Tumor 41 25 

Myocardium: Sarcoma, NOS^ 0/28 (0) 0/14 (0) 6/32 (19) 2/29 (7) 

P Values0»d N.S. N.S. P = 0.018** N.S. 

Departure from Linear Trend6 P = 0.016 P = 0.039 

Relative Risk (Pooled Vehicle Con trol)f Infinite Infinite 

Lower Limit 1.437 0.292 

Upper Limit Infinite Infinite 

Relative Risk (Vehicle Control)f Infinite Infinite 

Lower Limit 0.754 0.152 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor 54 81 — 
__ 

__ 

__ 

__ 

Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Estradiol Mustard by Gavage3
 

(continued)
 

Topography: Morphology
 

Stomach: Squamous-cell
 

Carcinoma"
 

P Values0 »d
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular
 

Carcinoma"
 

P Values0 »d
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Vehicle
 

Control
 

0/28 (0)
 

N.S.
 

trol)f
 

0/28 (0)
 

N.S.
 

trol)f
 

Vehicle
 

Control
 

0/14 (0)
 

N.S.
 

0/14 (0)
 

N.S.
 

—
 

Low
 

Dose
 

0/32 (0)
 

N.S.
 

—
 

—
 

—
 

—
 

—
 

2/32 (6)
 

N.S.
 

Infinite
 

0.264
 

Infinite
 

Infinite
 

0.138
 

Infinite
 

78
 

High
 

Dose
 

2/29 (7)
 

N.S.
 

Infinite
 

0.292
 

Infinite
 

Infinite
 

0.152
 

Infinite
 

0/29 (0)
 

N.S.
 

—
 

—
 

—
 

—
 

—
 

—
 

81 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued) 

Pooled 

Vehicle Vehicle Low High 

Topography: Morphology Control Control Dose Dose 

Liver: Hepatocellular
 

Adenoma or Carcinoma'5 6/28 (21) 2/14 (14) 5/32 (16) 1/29 (3)
 

P Valuesc»d P = 0.037(N) N.S. N.S. P = 0.046**(N)
 

Relative Risk (Pooled Vehicle Control)f 0.729 0.161
 

Lower Limit 0.198 0.004
 

Upper Limit 2.559 1.208
 

Relative Risk (Vehicle Control)f 1.094 0.241
 

Lower Limit 0.214 0.004
 

Upper Limit 10.641 4.322
 

Weeks to First Observed Tumor 84 78 83
 

a

Treated groups received doses of 15 or 30 mg/kg body weight.
 

"Number of tumor-bearing animals/number of animals examined at site (percent).
 

c

Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the
 

incidence of tumors in a treated group is the probability level for the Fisher exact test for
 

the comparison of that treated group with the vehicle-control group (*) or with the pooled
 

vehicle-control group (**) when P < 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 

"A negative trend (N) indicates a lower incidence in a treated group than in a control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

fxhe 95% confidence interval of the relative risk between each treated group and the specified
 

control group.
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Estradiol Mustard by Gavagea
 

Topography: Morphology
 

Lung: Alveolar/Bronchiolar
 

Carcinoma*5
 

P Values0>d
 

Departure from Linear Trend6
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled 

Vehicle Vehicle Low High 

Control Control Dose Dose 

0/31 (0) 0/16 (0) 4/31 (13) 0/24 (0) 

N.S. N.S. N.S. N.S. 

P = 0.007 P = 0.020 

trol)f Infinite 

0.946 — 

Infinite — 

— __ 

Infinite 

0.508 

Infinite — 

— 

64 — — 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 

Topography: Morphology
 

Lung: Alveolar /Bronchiolar
 

Adenoma or Carcinoma"
 

P Values0 »d
 

Departure from Linear Trend6
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled 

Vehicle Vehicle Low 

Control Control Dose 

1/31 (3) 0/16 (0) 7/31 (23) 

N.S. N.S. P = 0.042* 

P = 0.026** 

P = 0.007 P = 0.008 

trol)f 7.000 

0.984 

302.728 

Infinite 

1.068 

Infinite 

64 — 

High
 

Dose
 

1/24 (4)
 

N.S.
 

1.292
 

0.017
 

97.132
 

Infinite
 

0.037
 

Infinite
 

40 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued) 

Pooled 

Vehicle 

Topography: Morphology Control 

Hematopoietic System:
 

Lymphomab 0/31 (0)
 

P Values0 »d P < 0.001
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Vehicle 

Control 

Low 

Dose 

0/16 (0) 9/31 (29) 

P = 0.002 P = 0.015* 

P = 0.001** 

Infinite 

2.692 

Infinite 

Infinite 

1.448 

Infinite 

— 36 

High
 

Dose
 

11/24 (46)
 

P = 0.001*
 

P < 0.001**
 

Infinite
 

4.437
 

Infinite
 

Infinite
 

2.384
 

Infinite
 

27
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 

Topography: Morphology
 

Myocardium: Sarcoma, NOS^
 

P Valuesc»d
 

Departure from Linear Trend6
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Vehicle
 

Control
 

0/31 (0)
 

N.S.
 

P < 0.001
 

trol)f
 

Vehicle
 

Control
 

0/16 (0)
 

N.S.
 

P = 0.004
 

—
 

Low
 

Dose
 

8/31 (26)
 

P = 0.025*
 

P = 0.002**
 

Infinite
 

2.338
 

Infinite
 

Infinte
 

1.257
 

Infinite
 

52
 

High
 

Dose
 

1/24 (4)
 

N.S.
 

Infinite
 

0.070
 

Infinite
 

Infinite
 

0.037
 

Infinite
 

60 
__ 

__ 

__ 

Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 

Topography: Morphology
 

Stomach: Squamous-cell
 

Carcinoma^
 

P Values0 »d
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Adenocarcinoma
 

or Carcinoma, NOS^
 

P Values0 »d
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Vehicle
 

Control
 

0/31 (0)
 

N.S.
 

trol)f
 

0/31 (0)
 

P = 0.022
 

trol)f
 

Vehicle
 

Control
 

0/16 (0)
 

N.S.
 

0/16 (0)
 

P = 0.044
 

—
 

Low
 

Dose
 

2/31 (6)
 

N.S.
 

Infinite
 

0.301
 

Infinite
 

Infinite
 

0.161
 

Infinite
 

76
 

0/31 (0)
 

N.S.
 

—
 

—
 

—
 

—
 

—
 

High
 

Dose
 

2/24 (8)
 

N.S.
 

Infinite
 

0.389
 

Infinite
 

Infinite
 

0.208
 

Infinite
 

50
 

3/24 (13)
 

N.S.
 

Infinite
 

0.794
 

Infinite
 

Infinite
 

0.426
 

Infinite
 

40
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 

a

Treated groups received doses of 15 or 30 mg/kg body weight.
 

"Number of tumor-bearing animals/number of animals examined at site (percent).
 

c

Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the
 

incidence of tumors in a treated group is the probability level for the Fisher exact test for
 

the comparison of that treated group with the vehicle-control group (*) or with the pooled
 

vehicle-control group (**) when P < 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 

^A negative trend (N) indicates a lower incidence in a treated group than in a control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

*The 95% confidence interval of the relative risk between each treated group and the specified
 

control group.
 
Table F3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Estradiol Mustard by Gavagea
 

Topography: Morphology
 

Lung: Alveolar /Bronchiolar
 

Carcinoma (52)b
 

P Values0 »d
 

Departure from Linear Trend6
 

Pooled 

Vehicle Vehicle 

Control Control 

0/28 (0) 0/13 (0) 

N.S. N.S. 

P = 0.006 P = 0.020 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Low
 

Dose
 

6/30 (20)
 

P = 0.015**
 

Infinite
 

1.534
 

Infinite
 

Infinite
 

0.753
 

Infinite
 

72
 

High
 

Dose
 

1/24 (4)
 

N.S.
 

Infinite
 

0.063
 

Infinite
 

Infinite
 

0.031
 

Infinite
 

—
 83 
Table F3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Estradiol Mustard by Gavage3
 

(continued) 

Pooled 

Vehicle Vehicle Low High 

Topography: Morphology Control Control Dose Dose 

Lung: Alveolar /Bronchiolar
 

Adenoma or Carcinoma (52)b 2/28 (7) 2/13 (15) 12/30 (40) 5/24 (21)
 

P Values0>d N.S. N.S. P = 0.004** N.S.
 

Departure from Linear Trend6 P = 0.008
 

Relative Risk (Pooled Vehicle Control)f 5.600 2.917
 

Lower Limit 1.415 0.530
 

Upper Limit 47.024 28.113
 

Relative Risk (Vehicle Control)f 2.600 1.354
 

Lower Limit 0.721 0.270
 

Upper Limit 21.512 12.864
 

Weeks to First Observed Tumor 84 59 68
 
__ 

__ 

Table F3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Estradiol Mustard
 

(continued) 

Pooled 

Vehicle Vehicle 

Topography: Morphology Control Control 

Myocardium:
 

Sarcoma, NOS (52)b 0/28 (0) 0/13 (0)
 

P Values0 >d N.S. N.S.
 

Departure from Linear Trend6 P = 0.020
 

Relative Risk (Pooled Vehicle Con trol)£
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 

__
 

Liver: Hepatocellular
 

Carcinoma (52)b 0/28 (0) 0/13 (0)
 

P Values0 »d N.S. N.S.
 

Relative Risk (Pooled Vehicle Con trol)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Cotrol)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 —
 

by Gavage3
 

Low
 

Dose
 

6/30 (20)
 

P = 0.015**
 

Infinite
 

1.534
 

Infinite
 

Infinite
 

0.753
 

Infinite
 

54
 

2/30 (7)
 

N.S.
 

Infinite
 

0.282
 

Infinite
 

Infinite
 

0.138
 

Infinite
 

78
 

High
 

Dose
 

2/24 (8)
 

N.S.
 

Infinite
 

0.353
 

Infinite
 

Infinite
 

0.172
 

Infinite
 

0/24 (0)
 

N.S.
 

—
 

—
 

—
 

—
 

—
 

81 
Table F3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 

Pooled
 

Vehicle Vehicle Low High
 

Topography: Morphology Control Control Dose Dose
 

Liver: Hepatocellular Adenoma
 

or Carcinoma (52)b 6/28 (21) 2/13 (15) 5/30 (17) 1/24 (4)
 

P Value sc»d N.S. N.S. N.S. N.S.
 

Relative Risk (Pooled Vehicle Control)f 0.778 0.194
 

Lower Limit 0.212 0.004
 

Upper Limit 2.715 1.440
 

Relative Risk (Vehicle Control)f 1.083 0.271
 

Lower Limit 0.215 0.005
 

Upper Limit 10.475 4.791
 

Weeks to First Observed Tumor 84 78 83
 

a

Treated groups received doses of 15 or 30 mg/kg body weight.
 

"Number of tumor-bearing animals/number of animals examined at site (percent), based on animals
 

that lived at least as long as the number of weeks shown in the parenthesis, after the
 

description of morphology.
 

c

Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the
 

incidence of tumors in a treated group is the probability level for the Fisher exact test for
 

the comparison of that treated group with the vehicle-control group (*) or with the pooled-


control group (**) when P < 0.05 for either control group; otherwise, not significant (N.S.)
 

is indicated.
 
Table F3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Mice 

Administered Estradiol Mustard by Gavage3 

(continued) 

"A negative trend (N) indicates a lower incidence in a treated group than in a control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

•"-The	 95% confidence interval of the relative risk between each treated group and the specified
 

control group.
 
__ 

Table F4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Estradiol Mustard by Gavage3
 

Pooled 

Vehicle Vehicle Low High 

Topography: Morphology Control Control Dose Dose 

Lung: Alveolar/Bronchiolar
 

Carcinoma (52)b 0/28 (0) 0/13 (0) 4/26 (15) 0/13 (0)
 

P Values0 »d N.S. N.S. P = 0.047** N.S.
 

Departure from Linear Trend6 P = 0.012 P = 0.037
 

Relative Risk (Pooled Vehicle Control) f Infinite
 

Lower Limit 1.026
 —
 

—
 Upper Limit Infinite
 —
 

Relative Risk (Vehicle Control)f Infinite
 

Lower Limit 0.503
 

Upper Limit Infinite
 —
 

Weeks to First Observed Tumor 64
 —
 —
 
Table F4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 

Topography: Morphology
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma (40)b
 

P Values0 >d
 

Departure from Linear Trend6
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled 

Vehicle Vehicle Low 

Control Control Dose 

1/28 (4) 0/13 (0) 7/27 (26) 

N.S. N.S. P = 0.048* 

P = 0.022** 

P = 0.008 P = 0.012 

trol)f 7.259 

1.032 

311.223 

Infinite 

1.018 

Infinite 

_» 

64 

High
 

Dose
 

1/18 (6)
 

N.S.
 

1.556
 

0.021
 

115.144
 

Infinite
 

0.041
 

Infinite
 

40 
Table F4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Estradiol Mustard by	 Gavage3
 

[continued)
 

High
 

Dose
 

11/23 (48)
 

P = 0.002*
 

P < 0.001**
 

Infinite
 

4.353
 

Infinite
 

Infinite
 

2.206
 

Infinite
 

27
 

1/12 (8)
 

N.S.
 

Infinite
 

0.127
 

Infinite
 

Infinite
 

0.061
 

Infinite
 

60
 

Topography: Morphology
 

tiematopotetic System:
 

Lytnphoraa (27)b
 

P Values0 >d
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Myocardium: Sarcoma, NOS (52)b
 

P Values0 >d
 

Departure from Linear Trend6
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Vehicle Vehicle Low
 

Control Control Dose
 

0/29 (0) 0/14 (0)	 9/30 (30)
 

P < 0.001 P = 0.003	 P = 0.020*
 

P = 0.001**
 

trol)f	 Infinite
 

2.612
 

Infinite
 

Infinite
 

1.326
 

Infinite
 

36
 —
 

0/28 (0) 0/13 (0) 8/27 (30)
 

N.S.	 N.S. P = 0.029*
 

P = 0.002**
 

P = 0.004 P = 0.015
 

itrol)f	 Infinite
 

2.443
 

Infinite
 

Infinite
 

1.199
 

Infinite
 

52
 —
 
Table F4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Estradiol Mustard by Gavagea
 

(continued)
 

Topography: Morphology
 

Stomach: Squamous-cell
 

Carcinoma (50)b
 

P Values0 >d
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk. (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Adenocarcinoraa
 

or Carcinoma, NOS (40)b
 

P Values0>d
 

Relative Risk (Pooled Vehicle Con
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Vehicle
 

Control
 

0/28 (0)
 

N.S.
 

trol)f
 

0/28 (0)
 

P = 0.015
 

trol)f
 

Vehicle Low
 

Control Dose
 

0/13 (0) 2/26 (8)
 

N.S. N.S.
 

Infinite
 

0.326
 

Infinite
 

Infinite
 

0.159
 

Infinite
 

~"TM
 76
 

0/13 (0) 0/27 (0)
 

P = 0.035 N.S.
 

_—
 

—
 

—
 

—
 

—
 

—
 

—
 —
 

High
 

Dose
 

2/14 (14)
 

N.S.
 

Infinite
 

0.608
 

Infinite
 

Infinite
 

0.297
 

Infinite
 

50
 

3/18 (17)
 

N.S.
 

Infinite
 

0.964
 

Infinite
 

Infinite
 

0.472
 

Infinite
 

40
 
Table F4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Mice 

Administered Estradiol Mustard by Gavagea 

(continued) 

a

Treated groups received doses of 15 or 30 mg/kg body weight.
 

^Number of tumor-bearing animals/number of animals examined at site (percent), based on
 

number of animals that lived at least as long as the number of weeks shown in parentheses
 

after the description of morphology.
 

c

Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the
 

incidence of tumors in a treated group is the probability level for the Fisher exact test for
 

the comparison of that treated group with the vehicle-control group (*) or with the pooled-


control group (**) when P < 0.05 for either control group; otherwise, not significant (N.S.)
 

is indicated.
 

^A negative trend (N) indicates a lower incidence in a treated group than in a control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

^The 95% confidence interval of the relative risk between each treated group and the specified
 

control group.
 
Review of the Bioassay of Estradiol Mustard* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

April 26, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DHEW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be
 

exposed. The members of the Clearinghouse have been drawn
 

from academia, industry, organized labor, public interest
 

groups, State health officials, and quasi-public health and
 

research organizations. Members have been selected on the
 

basis of their experience in carcinogenesis or related
 

fields and, collectively, provide expertise in chemistry,
 

biochemistry, biostatistics, toxicology, pathology, and
 

epidemiology. Representatives of various Governmental
 

agencies participate as ad hoc members. The Data Evaluation/
 

Risk Assessment Subgroup of the Clearinghouse is charged
 

with the responsibility of providing a peer review of
 

reports prepared on NCI-sponsored bioassays of chemicals
 

studied for carcinogenicity. It is in this context that the
 

below critique is given on the bioassay of Estradiol
 

Mustard for carcinogenicity.
 

The primary reviewer noted that the compound is an
 

anti-neoplastic agent targeted against tumors thought to
 

have estrogen receptors. She agreed with the conclusion
 

in the report that Estradiol Mustard was carcinogenic in
 

both sexes of mice. Although the report concludes that
 

Estradiol Mustard was not carcinogenic in rats, the primary
 

reviewer noted that mammary tumors were observed in 8/68
 

treated males while only 6/215 mammary tumors have been
 

reported in historic male control rats. Given these findings,
 

she recommended that the report give greater emphasis to
 

the possible significance of the mammary tumors in treated
 

male rats. Although the small control group size and the
 

high mortality rate reduced the sensitivity of the study,
 

she said that the shortcomings did not invalidate the
 

conclusion on the carcinogenicity of Estradiol Mustard.
 

Based on the bioassay, the primary reviewer concluded that
 

Estradiol Mustard could pose a carcinogenic risk to humans.
 

115
 
A Program staff pathologist commented that 5 of the
 

mammary tumors in the treated male rats were fibroadenomas.
 A Subgroup member noted that a number of mammary tumors
 

also were found in the treated female rats. He pointed out
 

that the Sprague-Dawley strain is particularly sensitive
 

to the induction of mammary tumors. Another Subgroup member
 

opined that the mammary tumors may have been due to the
 

estrogen component of the Estradiol Mustard.
 

A motion was approved unanimously that the report on
 

the bioassay of Estradiol Mustard be accepted with the
 

proviso that greater emphasis be given to the possible
 

significance of the mammary tumors found in treated rats.
 

Members present were:
 

Michael Shimkin (Acting Chairman), University of California
 

at San Diego
 

Joseph Highland, Environmental Defense Fund
 

George Roush, Jr., Monsanto Company
 

Louise Strong, University of Texas Health Sciences Center
 

John Weisburger, American Health Foundation
 

*	 Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

6 U. S. GOVERNMENT PRINTING OFFICE : L978— 260-899/3157 

116 

DHEW Publication No. (NIH) 78-1309