National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 60 

1978 

BIOASSAY OF 

PHENESTERIN 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 3546-10-9 

NCI-CG-TR-60 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

PHENESTERIN
 

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) 78-1310
 

BIOASSAY OF
 

PHENESTERIN
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division οϊ Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

FOREWORD: This report presents the results of the bioassay of
 

phenesterin 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 chc.micals 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: The bioassay was conducted by Southern Research
 

Institute, Birmingham, Alabama, initially under direct contract
 

to NCI and currently under a subcontract to Tracor 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. Η.
 

Weisburger2>3. ^s. j # Belzer* and Mr. I. Brown·*· were responsible
 

for the care of the laboratory animals and administration of the
 

test chemical. Data management and retrieval were performed by
 

Ms. C. A. Dominickl. Histopathologic examination was performed
 

by Dr. J. C. Peckham··-, and the diagnoses included in this report
 

represent his interpretation.
 

Animal pathology tables and survival tables were compiled by EG&G
 

iii
 
Mason Research Institute^. The statistical analyses were performed by Dr. J. Β.. Joiner^, using methods selected for the
 

bioassay program by Dr. J· J. Gart · Chemicals used in this
 

bioassay were analyzed by Mr. E. Brunson^ and Ms. N. Gross^, and
 

the analytical results 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. Marshall Steinberg, Director of the Bioassay Program; Dr. L.
 

A. Campbell, 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. Υ. Ε.
 

Presley.
 

The statistical analysis was reviewed by members of the
 

Mathematical Statistics and Applied Mathematics Section of NCI^:
 

Dr. John J. Gart, Mr. Jun-mo Nam, Dr. Hugh M. Pettigrew, and Dr.
 

Robert E. Tarone.
 

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. Squire8
 

Dr. Jerrold M. Ward
 

•'•Southern Research Institute, 2000 Ninth Avenue South,
 

Birmingham, Alabama.
 

iv
 
^Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

^Now with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York.
 

^EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

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

Maryland.
 

"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.
 

'College of Pharmacy, University of Tennessee, Memphis,
 

Tennessee.
 

^Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay of phenesterin for possible carcinogenicity was
 

conducted by administering the chemical by gavage to Sprague-


Dawley rats and B6C3F1 mice.
 

Groups of 35 rats of each sex were administered phenesterin at
 

one of two doses, either 5 or 10 mg/kg body weight, three times
 

per week for 52 weeks, then observed for an additional 32 or 33
 

weeks. The vehicle used was 0.05% polysorbate 80 in buffered
 

saline. Controls consisted of groups of 10 rats of each sex
 

which received the vehicle (vehicle control) and 10 rats of each
 

sex which were untreated (untreated control). All surviving rats
 

were killed at 84 or 85 weeks.
 

Groups of 35 mice of each sex were administered the chemical at
 

one of two doses, either 15 or 30 mg/kg body weight, three times
 

per week for 52 weeks. The males receiving 15 mg/kg were
 

observed for an additional period of 29 weeks, and those
 

surviving to this time were then killed; the animals of the
 

remaining groups were observed for additional periods of only
 

10-22 weeks, due to early deaths. Seventy-seven weeks after the
 

foregoing groups were started, additional groups of 40 mice of
 

each sex were started and were administered the chemical at 7
 

mg/kg body weight three times per week; administration of the
 

chemical terminated at week 102 for the males and at week 88 for
 

the females, due to deaths of all females at this time. Controls
 

for the low-dose (7 mg/kg) groups of mice consisted of groups of
 

20 mice of each sex which received the vehicle (vehicle control)
 

and 20 mice of each sex which were untreated (untreated control);
 

controls for the mid-dose (15 mg/kg) and the high-dose (30 mg/kg)
 

controls consisted of groups of 15 mice of each sex similarly
 

receiving the vehicle or untreated. All surviving low-dose
 

controls were killed at 104 weeks, and all surviving mid- and
 

high-dose controls were killed at 81-84 weeks.
 

Phenesterin was toxic to rats and mice at the doses used, as
 

shown by reduced mean body weights and survival. Time-adjusted
 

analyses were used for evaluation of incidences of tumors in the
 

female mice.
 

vii
 
In female rats, a dose-related trend (P = 0·019) was present in
 

adenocarcinoma of the mammary gland, using the pooled controls,
 

and the incidences of the tumor in the individual dosed groups
 

were significant (P £ 0.009) when compared with those in the
 

pooled controls (controls 1/18, low-dose 12/29, high-dose 12/30).
 

In male mice, the incidence of alveolar/bronchiolar carcinomas or
 

combined alveolar/bronchiolar adenomas and carcinomas in the
 

low-dose group (18/40) was significantly higher (P £ 0.020) than
 

that in the low-dose vehicle-control group (0/16). In female
 

mice, seven low-dose animals had alveolar/bronchiolar adenomas 

and eight other low-dose animals had alveolar/bronchiolar 

carcinomas. When these tumors were combined, their time-adjusted 

incidence was significant (P = 0.004) when compared with that in
 

the low-dose vehicle controls (controls 1/18, low-dose 15/35).
 

The lower and nonsignificant incidences of these tumors- observed
 

in the mid- and high-dose groups may be due to the earlier
 

mortality in these groups compared with the low-dose groups.
 

In each sex of mid- and high-dose mice, incidences of lymphoma
 

and leukemia were dose related (P ^< 0.005), using vehicle
 

controls; they were also significant (P £ 0.018) in direct
 

comparisons of mid- and high-dose groups of both sexes with
 

respective vehicle controls (males: controls 0/14, mid-dose 9/29,
 

high-dose 11/25; females, time-adjusted: controls 0/15, mid-dose
 

14/18, high-dose 17/19). The significance of the incidence of
 

lymphoma and leukemia in the mid- and high-dose groups of males
 

was increased (P <_ 0.001) when the pooled-control group was used,
 

both in the test for dose-related trend and in tests for direct
 

comparisons of dosed groups with the controls.
 

In each sex of mice, sarcomas of the myocardium were found in all
 

groups of dosed animals, but in no control animals (males: low-


dose 5/40, mid-dose 7/29, high-dose 2/25; females: low-dose
 

8/34, mid-dose 2/7, high-dose 3/7). In males, the incidence in
 

the mid-dose group was significant when compared with that in the
 

pooled controls (P = 0.006); in females, the incidences in the
 

low- and high-dose groups were significant (P ^< 0.023).
 

viii
 
It is concluded that under the conditions of this bioassay,
 

phenesterin was carcinogenic in female Sprague-Dawley rats,
 

producing adenocarcinoiaas of the mammary gland, and in both sexes
 

of B6C3F1 mice, producing alveolar/bronchiolar carcinomas,
 

hematopoietic tumors, and myocardial sarcomas·
 

ix
 

TABLE OF CONTENTS
 

I· Introduction · 1
 

II· Materials and Methods 3
 

A· Chemical · 3
 

 · Dosage Preparation 3
 

C · Animals 4
 

D. Animal Maintenance · 4
 

· Subchronic Studies · 7
 

F. Designs of Chronic Studies 9
 

G. Clinical and Pathologic Examinations 13
 

H· Data Recording and Statistical Analyses 14
 

III. Results - Rats 21
 

A. Body Weights and Clinical Signs (Rats) 21
 

B. Survival (Rats) 21
 

C. Pathology (Rats) 24
 

D. Statistical Analyses of Results (Rats) 27
 

IV. Results - Mice 29
 

A. Body Weights and Clinical Signs (Mice) 29
 

B. Survival (Mice) 32
 

C. Pathology (Mice) 35
 

D. Statistical Analyses of Results (Mice) 40
 

V. Discussion · 45
 

VI. Bibliography 51
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms
 

in Rats Administered Phenesterin
 

by Gavage. ·· · 53
 

Table Al Summary of the Incidence of Neoplasms
 

in Male Rats Administered Phenesterin
 

by Gavage. ·· 55
 

xi
 
Table A2 Summary of the Incidence of Neoplasms 

in Female Rats Administered Phenesterin 

by Gavage 5 8 

Appendix Β Summary of the Incidence of Neoplasms 

in Mice Administered Phenesterin 

by Gavage 61 

Table Bl Summary of the Incidence of Neoplasms 

in Male Mice Administered Phenesterin 

by Gavage (Control Groups) 63 

Table B2 Summary of the Incidence of Neoplasms 

in Male Mice Administered Phenesterin 

by Gavage (Treated Groups)... 66 

Table B3 Summary of the Incidence of Neoplasms 

in Female Mice Administered Phenesterin 

by Gavage (Control Groups) 6 9 

Table B4 Summary of the Incidence of Neoplasms 

in Female Mice Administered Phenesterin 

by Gavage (Treated Groups) · · · · 72 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Administered 

Phenesterin by Gavage 7 7 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Administered 

Phenesterin by Gavage 79 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Administered 

Phenesterin by Gavage 83 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Administered 

Phenesterin by Gavage 87 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Administered 

Phenesterin by Gavage (Control Groups) 89 

xii
 
Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Administered 

Phenesterin by Gavage (Treated Groups) 9 2 

Table D3 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered 

Phenesterin by Gavage (Control Groups) 95 

Table D4 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered 

Phenesterin by Gavage (Treated Groups) 9 8 

Appendix Ε Analyses of the Incidence of Primary 

Tumors in Rats Administered 

Phenesterin by Gavage •....103 

Table El Analyses of the Incidence of Primary 

Tumors in Male Rats Administered 

Phenesterin by Gavage 10 5 

Table E2 Analyses of the Incidence of Primary 

Tumors in Female Rats Administered 

Phenesterin by Gavage · 110 

Appendix F Analyses of the Incidence of Primary 

Tumors in Mice Administered 

Phenesterin by Gavage · 115 

Table Fl Analyses of the Incidence of Primary 

Tumors in Low-Dose Male Mice 

Administered Phenesterin by Gavage 117 

Table F2 Analyses of the Incidence of Primary 

Tumors in Mid and High-Dose Male Mice 

Administered Phenesterin by Gavage 122 

Table F3 Analyses of the Incidence of Primary 

Tumors in Low-Dose Female Mice 

Administered Phenesterin by Gavage 127 

Table F4 Analyses of the Incidence of Primary 

Tumors in Mid and High-Dose Female Mice 

Administered Phenesterin by Gavage 132 

xiii 
Page
 

Table F5 Time-Adjusted Analyses of the Incidence
 

of Primary Tumors in Low-Dose Female
 

Mice Administered Phenesterin by Gavage....136
 

Table F6 Time-Adjusted Analyses of the Incidence 

of Primary Tumors in Mid- and High-Dose 

Female Mice Administered Phenesterin 

by Gavage 141 

TABLES 

Table 1 Design of Chronic Studies of 

Phenesterin in Rats 10 

Table 2 Design of Chronic Studies of 

Phenesterin in Mice 11 

FIGURES 

Figure 1

Figure 2

 Growth Curves for Rats Administered 

Phenesterin by Gavage ·

 Survival Curves for Rats Administered 

Phenesterin by Gavage

 2 2 

 23 

Figure 3 Growth Curves for Male Mice Administered 

Phenesterin by Gavage 30 

Figure 4 Growth Curves for Female Mice 

Administered Phenesterin by Gavage 31 

Figure 5 Survival Curves for Male Mice 

Administered Phenesterin by Gavage 33 

Figure 6 Survival Curves for Female Mice 

Administered Phenesterin by Gavage 34 

xiv
 
I. INTRODUCTION
 

Phenesterin (CAS 3546-10-9; NCI C01558), an experimental
 

anticancer agent, is a steroidal alkylating agent composed of the
 

carboxylic acid ester of cholesterol and an aryl nitrogen mustard
 

(Wall et al. , 1969). The linkage to cholesterol was selected to
 

improve the lipophilicity of the compound, and thereby, to
 

facilitate its transport across cell membranes. Phenesterin has
 

shown antitumor activity against a number of rat mammary tumors
 

and leukemias and has been tested in clinical trials (Ansfield et
 

al., 1971). Phenesterin was selected for testing for
 

carcinogenic activity because of the possibility that, as an
 

anticancer agent, it would be used on a chronic basis.
 

II . MATERIALS AND METHODS 

A. Chemical
 

The phenesterin (cholesteryl p-bis(2-chloroethyl)aminophenyiacetate) used in the chronic study was obtained from the Upjohn
 

Company, North Haven, Connecticut, as Lot Nos. 10328-BDA-8 and
 

10328-BDA-612. The identity and purity of Lot No. 10328-BDA-8
 

was confirmed in analyses at the University of Tennessee College
 

of Pharmacy. The melting point for this lot was 89-92°C, which
 

was consistent with the reported value of 90-90.5°C (Merck Index,
 

1976). Four trace impurities were detected by thin-layer
 

chromatography; no attempt was made to identify or quantitate
 

these impurities. Infrared and nuclear magnetic resonance
 

spectra were in agreement with the structure.
 

The bulk chemical was stored in the presence of a desiccant
 

(Drierite®) at 5°C.
 

B. Dosage Preparation
 

The dosage mixtures of the phenesterin were prepared fresh for
 

each administration. The chemical was suspended in a buffered
 

saline vehicle by mixing in a Potter-Elvehjem tissue grinder.
 

The buffered saline vehicle (pH 6.9) contained 0.85% NaCl, 0.40%
 

, 0.65% Na2HPO4, and 0.05% polysorbate 80.
 
No concentration or stability analyses of the chemical in the
 

buffered saline vehicle were performed.
 

C. Animals
 

Male Sprague-Dawley rats and male Swiss mice were obtained from
 

Charles River Breeding Laboratories, Inc., Wilmington,
 

Massachusetts, for use in the subchronic studies.
 

For the chronic studies, male and female Sprague-Dawley rats and
 

male and female B6C3F1 mice were obtained from Charles River
 

Breeding Laboratories under a contract with the Division of
 

Cancer Treatment, National Cancer Institute. Male rats were 30
 

days of age, female rats were 37 days of age, and male and female
 

mice were 31 days of age on arrival at the laboratory. Animals
 

were quarantined (rats for 5 days, mice for 12 days) prior to the
 

start of the chronic studies. B6C3F1 mice that were started
 

later in the study were received from Litton Bionetics, Inc., at
 

29 days of age and were quarantined for 12 days. At the end of
 

the quarantine period, all 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
 

4 
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 both intake and exhaust
 

fiberglass roughing filters· In addition to natural light,
 

illumination was 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 17;
 

mouse cages were provided with Sterolit® clay bedding (Englehard
 

Mineral and Chemical Co., New York, N.Y.). Low-dose mice and the
 

vehicle controls were housed similarly, except for the final 4
 

months of the study, during which time they were housed in cages
 

provided with a hardwood chip bedding (Betta-Chip, Northeastern
 

Products Corp., Warrenton, N.Y.). Bedding was replaced once or
 

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

at 82°C once per week; 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
 
phenesterin were maintained in the same rooms as animals of the
 

same species being dosed with the following chemicals:
 

RATS
 

Gavage Studies
 

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

(estradiol mustard) (CAS 22966-79-6)
 

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 [1PD] (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-(l-methyl-2-phenoxyethyl)benzylamine
 

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

N-(1-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-((propylamine)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)
 

ethionamide (CAS 536-33-4)
 

reserpine (CAS 50-55-5)
 

Gavage Studies
 

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

(estradiol mustard) (CAS 22966-79-6)
 

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-(l-methyl-2-phenoxyethyl)benzylamine
 

hydrochloride (phenoxybenzamine hydrochloride) (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)
 

adriamycin (CAS 23214-92-8)
 

E. Subchronic Studies
 

Subchronic studies were conducted with male Sprague-Dawley rats
 

7
 
and male Swiss mice to estimate the maximum tolerated doses of
 

phenesterin, on the basis of which lflowff doses and "high11 doses
 

were determined for the chronic studies. Phenesterin was
 

administered by gavage in a vehicle of 0.05% polysorbate 80 in
 

saline. Rats received doses of 1.0, 2.5, 5.0, 10.0, or 20.0
 

mg/kg and mice received doses of 1.7, 4.25, 8.5, 17.0, 34.0,
 

68.0, or 136.0 mg/kg. Animals were administered the chemical
 

three times per week for 45 days, and were observed for 45 days
 

following the administration of the chemical. Five animals of
 

each species were tested at each dose (except for 10 animals
 

tested at 34.0 mg/kg), 10 rats and 5 mice were maintained as
 

untreated controls, and 10 rats and 10 mice received the vehicle
 

only.
 

In rats, one animal administered 20 mg/kg died during week 10 of
 

the study; there were no deaths at lower doses. Mean body weight
 

gains were depressed 10% at 2.5 mg/kg, 11% at 5.0 mg/kg, 20% at
 

10 mg/kg, and 34% at 20 mg/kg by the end of the period of
 

administration of the chemical. Mean weight gains remained
 

depressed during the observation period. No gross abnormalities
 

were seen at necropsy. The low and high doses for the chronic
 

studies using rats were set at 5 and 10 mg/kg.
 

Mean weight gains in dosed mice did not show any trends and were
 

similar to those of controls in all except the highest dosed
 
group (136 mg/kg) where weight gain during the period of
 

administration of the chemical was depressed 45%· Except for
 

accidental deaths, there were no other deaths at any of the doses
 

tested. No gross abnormalities were observed at necropsy.
 

Because the toxic effects of this drug were known to be
 

cumulative, the high dose was set lower than would otherwise be
 

predicted from these data. The low and high doses for the chronic
 

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

F. Designs of Chronic Studies
 

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

Since the numbers of animals in the untreated- and vehicle-


control groups of rats and in the mid- and high-dose untreated-


and vehicle-control groups of mice were small, pooled vehicle-


control groups of rats and mice also were used for statistical
 

comparisons. The groups of 10 vehicle-control rats of each sex
 

from the bioassay of phenesterin were combined with corresponding
 

groups of 10 vehicle-control rats of each sex from a similar
 

bioassay of estradiol mustard. The pooled controls for statistical tests using rats thus consisted of 20 males and 20 females.
 

The groups of 15 mid- and high-dose vehicle-control mice of each
 

sex from the bioassay of phenesterin were combined with the
 

corresponding groups of 15 mid- and high-dose vehicle-control
 

9 
Table 1· Design of Chronic Studies of Phenesterin in Rats
 

Sex and 

Test 

Group 

Initial 

No. of 

Animalsa 

Phenesterin 

Doseb 

(m£ /k£) 

Time on 

Dosed 

(weeks) 

O

Study 

bserved 

(weeks) 

Male 

Untreated-Control 

Vehicle-Control 

Low-Dose 

High-Dose 

.

10 

 10 

35 

35 

0 

0c 

5 

10 

52 

52 

52 

85 

33 

32-33 

32 

Female 

Untreated-Control 

Vehicle-Control 

Low-Dose 

High-Dose 

10 

10 

35 

35 

0 

0C 

5 

10 

52 

52 

52 

85 

33 

33 

32 

aMale rats were 35 days of age and female rats were 42 days of age
 

when placed on study.
 

^Phenesterin was administered by gavage in a vehicle of 0.05%
 

polysorbate 80 in buffered saline three times per week at a
 

volume of 0.25 ml/100 g body weight. Doses were based on
 

individual weights.
 

c

Vehicle-control groups received only the vehicle of 0.05%
 

polysorbate 80 in buffered saline at the same volume and on the
 

same schedule as dosed rats.
 

10
 
Table 2. Design of Chronic Studies of Phenesterin in Mice
 

Sex and 

Test 

Group 

Initial 

No. of 

Animalsa 

Phenesterin 

Doseb 

(m£ /kg) 

Time on Study 

Dosed 

(weeks) 

Observed 

(weeks) 

Male 

Mid- and High-Dose 

Untreated-Control 

Mid- and High-Dose 

Vehicle-Control 

Low-Dose 

Untreated-Controld 

Low-Dose 

Vehicle-Controld 

Low-Dosed 

Mid-Dose 

High-Dose 

15 

15 

20 

20 

40 

35 

35 

0 

0c 

0 

0c 

7 

15 

30 

52 

103 

102e 

52 

52 

83-84 

29-30 

104 

1 

29 

22* 

Female 

Mid- and High-Dose 

Untreated-Control 

Mid- and High-Dose 

Vehicle-Control 

Low-Dose 

Untreated-Controld 

Low-Dose 

Vehicle-Controld 

Low-Dosed 

Mid-Dose 

High-Dose 

15 

15 

20 

20 

40 

35 

35 

0 

0c 

0 

0c 

7 

15 

30 

52 

103 

88e 

52 

52 

84 

30-31 

104 

1 

10* 

10* 

aHigh- and mid-dose mice and their controls were 43 days
 

of age when placed on study; low-dose mice and their controls
 

were 41 days of age.
 

^Phenesterin was administered by gavage in the vehicle of 0.05%
 

polysorbate 80 in buffered saline three times per week at a
 

volume of 0.1 ml/10 g body weight. Doses were based on the mean
 

weight of the animals in each cage.
 

c

Vehicle-control groups received only the vehicle of 0.05%
 

polysorbate 80 in buffered saline at the same volume and on the
 

same schedule as dosed mice.
 

11
 
Table 2. Design of Chronic Studies of Phenesterin in Mice
 

(continued)
 

^The low-dose groups and their controls were started 7 7 weeks
 

after the mid- and high-dose groups, because of deaths in these
 

groups·
 

Administration of the chemical terminated at the time indicated,
 

due to death of all animals.
 

^Observation terminated at the time indicated, due to death
 

of all animals·
 

12
 
mice of each sex from a similar bioassay of estradiol mustard to
 

give pooled-control groups of 30 mid- and high-dose vehicle-


control mice of each sex. The vehicle-control groups of rats and
 

the vehicle-control groups of mice that were used in the
 

respective pooled-control groups were each of the same strain,
 

obtained from the same supplier, and examined by the same
 

pathologists. Further, the different control groups were placed
 

on study at starting times differing by no more than 3 months.
 

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 were weighed once per week for 4 weeks and
 

once every 2 weeks thereafter; mid- and high-dose mice were
 

weighed every 2 weeks throughout the study; low-dose mice were
 

weighed every 2 weeks for 34 weeks and once per month 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,
 

13
 
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, prostate 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.
 

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
 

14
 
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
 

verification 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
 

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 Ρ values have been reported for all tests except the
 

departure from linearity test, which is only reported when its
 

two-tailed Ρ value is less than 0.05.
 

15
 
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
 

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
 

 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 narr16
 
ative section. It is not, however, presented in the tables,
 

where the Fisher exact Ρ 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
 

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.
 

17
 
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
 

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
 

18
 
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, Ρ < 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
 

indicates 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.
 

19
 

III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

The mean body weights of the dosed male and female rats were
 

lower than those of the untreated or vehicle controls throughout
 

most of the study, especially in the high-dose groups (figure 1).
 

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 variation. No other signs of toxicity related to the
 

administration of the chemical were recorded.
 

Because of respiratory disease in the colony, oxytetracycline was
 

administered in drinking water at 0.6 mg/ml during weeks 17-23
 

and at 0.3 mg/ml during weeks 23-27.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats administered phenesterin by
 

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

matched controls, are shown in figure 2.
 

In each sex, the Tarone test result for positive dose-related
 

trend in mortality is significant (P < 0.001), using the
 

high-dose, low-dose, and vehicle-control groups. In the males,
 

4/35 (11%) of the high-dose group, 8/35 (23%) of the low-dose
 

21
 
TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves For Rats Administered Phenesterin by Gavage 

22 
TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves for Rats Administered Phenesterin by Gavage 

23 
group, and 7/10 (70%) of the controls were alive at the end of
 

the bioassay. In females, 2/35 (6%) of the high-dose group, 9/35
 

(26%) of the low-dose group, and 8/10 (80%) of the controls were
 

alive at the end of the bioassay. Over 50% of each group of
 

males or females survived at least 5 2 weeks. The untreated-


control groups are not used in these comparisons, since the test
 

conditions of the vehicle controls more closely resembled those
 

of the dosed rats; however, the Kaplan and Meier curves of the
 

untreated-control groups are shown in figure 2.
 

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.
 

A variety of neoplasms occurred in both the control (untreated
 

and vehicle) and dosed groups. These lesions, however, are not
 

uncommon in this strain of rat independent of any treatment. The
 

following sarcomas occurred primarily in the dosed groups:
 

24
 
RATS 

MALE FEMALE 

Low High Low High 

Dose Dose Dose Dose 

Number of Animals Necropsied (32) (30) (29) (30) 

Subcutis 

-sarcoma, NOS* 2 1 0 1 

-fibrosarcoma 1 0 1 0 

Bone 

-osteosarcoma 1 1 0 0 

Abdominal cavity 

-sarcoma, NOS 1 2 1 0 

-hemangiosarcoma 1 0 0 0 

Multiple organs, other 

-sarcoma, NOS 1 0 0 0 

-sarcoma, NOS, metastatic 

( r ima ry unknown) 0 0 0 1 

Mammary gland 

-sarcoma, NOS 0 0 0 1 

Cranial Cavity 

-sarcoma, NOS 0 0 0 1 

Number of Animals 

With Tumors 7 4 2 4 

*Not otherwise specified
 

These sarcomas included fibrosarcomas, osteosarcomas, hemangiosarcomas, and undifferentiated sarcomas, and they occurred more
 

frequently in males than in females. Metastases to the lungs
 

occurred in two high-dose males. Only one control animal, an
 

untreated male, had a sarcoma (abdominal cavity).
 

Adenocarcinomas and fibroadenomas of the mammary gland were the
 

25
 
most frequent tumors in female rats· The incidence was as
 

follows:
 

RATS
 

Untreated Vehicle Low High
 

Control Control Dose Dose
 

FEMALE
 

Number of Animals Necropsied (10) (10) (29) (30)
 

Mammary Gland
 

-adenocarcinoma 0 1 12 12
 

«fibroadenoma 6 4 21 11
 

Number of Animals With Tumors 6 5 26 19
 

Metastases to the lung occurred in one high-dose animal·
 

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

proliferative, and inflammatory changes were encountered also in
 

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

nonneoplastic lesions are commonly seen in aged Sprague-Dawley
 

rats.
 

Based on this histopathologic examination, phenesterin
 

administered by gavage to Sprague-Dawley rats at doses of 5 or 10
 

mg/kg was associated with an increased frequency of tumors of the
 

mammary gland, especially adenocarcinomas, in dosed females·
 

26
 
D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix Ε 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
 

analysis. The untreated controls are not included in the tables
 

and analyses, since the test conditions of the vehicle controls
 

more closely resemble those of the dosed rats·
 

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

positive dose-related trend in the incidence of animals with
 

lymphoma or leukemia are significant (P - 0·03 7) using the pooled
 

controls, but the results of the Fisher exact test are not
 

significant. Statistical test results on this incidence of
 

tumors in female rats are not significant.
 

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

incidence of adenocarcinomas of the mammary gland are significant
 

(P = 0.019) when the pooled vehicle-control group is used. The
 

Fisher exact tests show that the incidences in both of the dosed
 

groups are significantly higher (P < 0.010) than the incidence in
 

the pooled controls. These tumors of the mammary gland were
 

first observed as early as 26 weeks in the low-dose group and 33
 

27
 
weeks in the high-dose group· The statistical conclusion is that
 

this incidence of tumors in female rats is dose associated·
 

The results of the Cochran-Armitage test and the Fisher exact
 

test on the incidence of fibroadenomas of the mammary gland in
 

female rats are not significant· Significant results in the
 

negative direction are observed in the incidence of chromophobe
 

adenomas of the pituitary in female rats, where the incidences in
 

the control groups exceed those in the dosed groups. Time-


adjusted analysis, eliminating animals that died before 52 weeks
 

on study, indicated similar levels of significance in the
 

negative direction. The time-adjusted data are 10/16 (63%) of
 

the pooled controls, 6/10 (60%) of the vehicle controls, 4/24
 

(17%) of the low-dose group, and 0/16 of the high-dose group·
 

Survival in the dosed groups was shorter than in the controls,
 

and this shortened survival may account for the low incidence of
 

the pituitary tumors in the dosed groups·
 

28
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

The mean body weights of the low-dose groups of dosed male and
 

female mice were unaffected during the first 20-30 weeks, but for
 

the males, were lower than those of the vehicle controls, and for
 

the females, were lower than those of both the untreated and
 

vehicle controls, for the remaining period of the bioassay
 

(figures 3 and 4). Progressive weight loss was recorded in
 

several individual animals of each sex. The mean body weights of
 

the mid- and high-dose groups of each sex were lower than those
 

of the controls throughout most of the study. 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 variation· No
 

other signs of toxicity related to the administration of the
 

chemical were recorded.
 

Some of the animals in the low-dose groups, together with corresponding controls, had signs of respiratory disease. These groups
 

were administered oxytetracycline in the drinking water at 0.6
 

mg/ml during weeks 14-15 and at 0.3 mg/ml during week 15. To
 

reduce the transmission of airborne microorganisms, propylene
 

glycol was vaporized in the mouse room during weeks 14-25.
 

29
 
TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves For Male Mice Administered Phenesterin by Gavage 

30 
10 20 30 40 50 60 70 80 90 100

TIME ON STUDY (WEEKS) 

Figure 4. Growth Curves For Female Mice Administered Phenesterin by Gavage 

31 

 110 
. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice administered phenesterin by
 

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

matched controls, are shown in figures 5 and 6.
 

In each sex, the Tarone test result for positive dose-related
 

trend in mortality is significant (P < 0.001), using the
 

high-dose, mid-dose, and corresponding vehicle-control groups.
 

Departures from linear trend are observed (P < 0.001 in males and
 

 = 0.021 in females), because of the sharp decrease in survival
 

in the dosed mice. The result of the Cox test comparing the
 

low-dose vehicle-control group and the low-dose group is not
 

significant in male mice, whereas in female mice, it is
 

significant (P < 0.001). The untreated-control groups are not
 

used for comparison, since the test conditions of the vehicle
 

controls more closely resembled those of the dosed mice; however,
 

the Kaplan and Meier curves of the untreated-control groups are
 

included in figures 5 and 6.
 

In male mice, none of the dosed animals were alive at the end of
 

the study, but over 55% of them lived at least as long as 52
 

weeks on study (20/35 [57%] in the high-dose group, 26/35 [74%]
 

in the mid-dose group, and 33/40 [83%] in the low-dose group).
 

32
 
TIME ON STUDY (WEEKS) 

Figure 5. Survival Curves For Male Mice Administered Phenesterin by Gavage 

33 
(-η 

Δ i "Η 

Δ
 

Δ
 I 

AΔ λ 

j 

Ο. 

< • * 

Δ cc Δ \ D
 o> ο 

>- ; 

ί < ! tn
 

Ο ι oc
 

Q. CCMAI c Mire κ
 MID AND HIGH DOSE ! 1 UNTREATED CONTROL • • -J 

ι
 

] VEHICLE CONTROL
 

MID AND HIGH DOSE Λ 

Δ . > MID DOSE ; c 

 HIGH DOSE 

• τ 

ft— 10 20 30 40 50 60 70 80 90 100 110
 

TIME ON STUDY (WEEKS) 

JlllBJJIW..
 j
 

Φ Λ i 5 

0 90 - Φ 1 

1 

1 ( 

\ 

0 80­

( i 

' 1 

4 

\ 

i 

0 40" 

•— Φ 

/Κ 0 30~ FEMALE MICE 

LOW DOSE 

• UNTREATED CONTROL 

0 20 ^ LOW DOSE 

 VEHICLE CONTROL C 

 LOW DOSE 

0 10­ —( k-·— 

0 00­

50
 

TIME ON STUDY (WEEKS) 

Figure 6. Survival Curves For Female Mice Administered Phenesterin by Gavage 

34
 
The proportions of animals alive at week 75 are 0/35 in the high-


dose group, 7/35 (20%) in the mid-dose group, and 24/40 (60%) in
 

the low-dose group·
 

In females, no dosed mice lived to termination of the study.
 

Although 3 7/40 (93%) of the low-dose group were alive at week 52,
 

only 5/35 (14%) of the high-dose group and 8/35 (23%) of the
 

mid-dose group were alive at least as long as week 52; therefore,
 

time-adjusted analyses were performed· At week 75 all of the
 

animals in the mid- and high-dose groups were dead and only 11/40
 

(28%) in the low-dose group were alive·
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

Appendix B, tables B1-B4; findings on nonneoplastic lesions are
 

summarized in Appendix D, tables D1-D4.
 

A variety of neoplasms occurred both in control groups (untreated
 

and vehicle) and in dosed groups. Some types of neoplasms
 

occurred only in mice of dosed groups, or with a greater
 

frequency in dosed groups when compared with controls· These
 

lesions are not uncommon in this strain of mouse independent of
 

any treatment. However, many of the tumors appeared to be
 

related to the administration of the chemical. These tumors were
 

all malignant, many had metastasized to one or more locations,
 

35
 
and, except for the malignant lymphomas and mammary gland
 

adenocarcinomas, were observed only in dosed mice.
 

Alveolar-cell adenocarcinomas were present in 14/40 (35%)
 

low-dose males and 8/38 (21%) low-dose females· Alveolar-cell
 

adenomas occurred in a few animals in all dosed groups as well as
 

in three control males and one control female. These neoplasms
 

consisted of cuboidal to columnar cells aligned along the
 

alveolar septa. Often the cells projected into the alveolar
 

spaces, resulting in the formation of numerous papillary
 

structures. Neoplastic cell nuclei ranged from small and darkly
 

basophilic to large and vesicular. The large open-faced nuclei
 

often contained a prominent nucleolus. Pulmonary tumors had
 

metastasized to the liver in 1/39 (3%) males and to the
 

mediastinal lymphatics in 1/36 (3%) females. In some of the
 

mice, the alveolar-cell adenocarcinomas were multiple in origin.
 

The hematopoietic neoplasms were observed in 11/25 (44%) high-


dose, 9/29 (31%) mid-dose, and 11/40 (28%) low-dose males; and in
 

17/32 (53%) high-dose, 14/27 (48%) mid-dose, and 12/38 (32%)
 

low-dose females. Among controls these neoplasms were observed
 

in 1/35 (3%) untreated-control and 1/30 (3%) vehicle-control
 

males, and in 5/35 (14%) vehicle-control females, but in no other
 

control groups. The malignant lymphomas were classified as
 

lymphocytic, histiocytic, and mixed type. The lymphocytic type
 

36
 
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 mixed-cell
 

type was a combination of the lymphocytic and histiocytic types.
 

The malignant lymphomas had cellular distortion which prevented
 

further classification. Lymphocytic leukemia was differentiated
 

from malignant lymphoma by the diffuse infiltration of the
 

neoplastic cells within the involved organs, especially the
 

liver. In lymphoma, the neoplastic cells were more solid in
 

arrangement.
 

The eosinophilic leukemia was characterized by marked infiltration of spleen and liver sinusoids with cells having a segmented
 

nucleus and numerous eosinophilic cytoplasmic granules.
 

Myocardial sarcomas were observed in 5/40 (13%) low-dose, 7/2 9
 

(24%) mid-dose, and 2/25 (8%) high-dose males; and in 8/36 (22%)
 

low-dose, 2/27 (7%) mid-dose, and 3/31 (10%) high-dose females.
 

The neoplastic cells were pleomorphic and varied from those with
 

37
 
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 abundant cytoplasm. The
 

marked interstitial proliferation of neoplastic cells resulted in
 

compression atrophy and necrosis of adjacent myocardial fibers.
 

The origin of the neoplastic cells was not determined, but they
 

appeared to arise from the perimysial connective tissue
 

surrounding myocardial fibers or from Antischkow cells. In some
 

of the mice, the neoplastic cells had invaded through the
 

endocardium and existed as large tumor thrombi within the
 

ventricles. The myocardial sarcomas had metastasized to the
 

lungs in 3/40 (8%) low-dose, 1/29 (3%) mid-dose, and 2/25 (8%)
 

high-dose males; and in 6/38 (16%) low-dose, 2/27 (7%) mid-dose,
 

and 2/31 (6%) high-dose females.
 

Adenocarcinomas of the mammary gland were present in 6/38 (16%)
 

low-dose and in 2/20 (10%) untreated-control females. The morphology of the mammary tumors varied considerably. The lobular
 

type consisted of multiple compact foci of cells having a large,
 

open-faced nucleus, prominent nucleolus, and a moderate amount of
 

cytoplasm. The foci of cells were separated by a fine fibrovascular stroma. The acinar type was comprised of cuboidal to
 

columnar cells aligned along a basement membrane. The lining of
 

the acini was often several cells thick, and the center of the
 

38
 
acinus often contained numerous concentric layers of keratin. In
 

1/38 (3%) low-dose mice, the mammary adenocarcinoma had
 

metastasized to the lungs.
 

Hemangiosarcomas involved the subcutaneous tissue, liver, and
 

abdominal cavity of dosed males and females; and the lungs,
 

spleen, and bone of dosed females only. The incidence of the
 

vascular tumor in any one organ in a group of dosed mice was low;
 

however, when the incidences in the various organs were added
 

together, the combined incidence was too significant to overlook.
 

Hemangiosarcomas involving Lhe liver were present in 4/39 (10%)
 

low-dose males and 1/37 (3%) low-dose females. The endothelial
 

lining of many hepatic sinusoids was markedly thickened, with
 

cells having a large, open-faced nucleus, one or more nucleoli,
 

and abundant cytoplasm. The neoplastic cells were locally
 

invasive, resulting in compression atrophy and necrosis of
 

hepatocytes in the centrolobular zone. The sinusoidal spaces
 

were observed to be greatly distended.
 

Hemangiosarcomas involving the other organs were similar in
 

morphology. The neoplastic cells were often large and contained
 

an open-faced nucleus, prominent nucleolus, and abundant
 

cytoplasm. The neoplastic cells were associated with various-


sized vascular spaces, and appealed to radiate out from these
 

39
 
spaces. In 1/2 7 (4%) mid-dose females, a peritoneal hemangiosarcoma had invaded the liver and one of the kidneys·
 

Based on this histopathologic examination, phenesterin
 

administered by gavage to B6C3F1 mice at doses of 7, 15, and 30
 

mg/kg was associated with an increase in both epithelial and
 

nonepithelial malignant tumors. These tumors included pulmonary
 

alveolar-cell adenocarcinomas, mammary gland adenocarcinomas,
 

hemangiosarcomas of several organs, malignant lymphomas and
 

leukemias, and sarcomas of the heart.
 

D. Statistical Analyses of Results (Mice)
 

Tables F1-F6 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. Mid- and high-dose vehicle-control
 

groups and mid- and high-dose pooled vehicle-control groups are
 

used in the statistical analyses. No pooled low-dose control
 

group is used, since there are no appropriate controls to be
 

combined. The untreated controls are not included in the tables
 

and analyses, since the test conditions of the vehicle controls
 

more closely resemble those of the dosed mice. Due to poor
 

survival of the dosed female mice, time-adjusted analyses are
 

performed, eliminating animals that died before 52 weeks on
 

40
 
study, unless a tumor was found at the anatomic site of interest
 

before week 52. When such an early tumor was found, comparisons
 

are based exclusively on animals that survived at least as long
 

as the animal in which the first tumor was found.
 

In the following paragraphs, the statistical narrative on female
 

mice is based on time-adjusted data only.
 

In male mice, the Fisher exact test shows that the incidence of
 

alveolar/bronchiolar carcinomas or combined alveolar/bronchiolar
 

adenomas and carcinomas in the low-dose group is significantly
 

(P <^ 0.020) higher than that in the low-dose vehicle controls,
 

but this positive result is not confirmed by the incidences in
 

the mid- or high-dose groups when compared with their controls.
 

In female mice, the Fisher exact comparison of the incidence of
 

alveolar/bronchiolar adenomas and carcinomas between the low-dose
 

group and the low-dose vehicle-control group shows a probability
 

level of 0.004. The incidences in the mid- and high-dose groups
 

are not significant, nor are the occurrences of carcinomas alone
 

in any group. The lower proportions of these tumors observed in
 

the mid- and high-dose groups of either sex might be attributed
 

to the earlier mortality in these groups compared with the low-


dose group. For example, the first tumor to be observed in the
 

low-dose groups of either sex was at week 68, and at that point
 

41
 
in the bioassay, 60/70 (83%) of the mid- and high-dosed males
 

were dead and all of the female mid- and high-dose group were
 

dead·
 

In each sex of mid- and high-dose mice, the results of the Fisher
 

exact test indicate that the incidences of lymphomas or leukemias
 

are significantly higher in each of the dosed groups than in
 

their respective controls. The results of the Cochran-Armitage
 

test for positive dose-related trend are significant (P j< 0.005)·
 

An indicated departure from linear trend is observed in the
 

females (P = 0.016), due to the steep increase of these tumors in
 

the dosed animals. In the low-dose groups of each sex, an
 

increased incidence of these tumors is observed when compared
 

with the respective control group, (males: controls 1/16 [6%] ,
 

low-dose 11/40 [28%]; females: controls 5/18 [28%], low-dose
 

12/36 [33%])· These tumors were observed as early as 32 weeks in
 

both the low-dose and the low-dose vehicle-control groups of male
 

mice and as early as 15 and 22 weeks in the dosed groups of
 

female mice. While the comparisons in the low-dose groups are
 

not statistically significant, the incidences are in the same
 

direction as those of the statistically significant mid- and
 

high-dose.
 

The analysis of the incidence of sarcomas of the myocardium is
 

shown in the tables. In male mice, this tumor is seen in signifi42
 
cant incidences only in the mid-dose group when compared with the
 

pooled controls (P = 0·006). In females, the high- and low-dose
 

groups were observed to have this tumor in significant incidences
 

(P <^ 0.023). These data suggest an association of this tumor
 

with administration of the chemical.
 

Although none of the results of the Fisher exact test of the
 

incidence of hemangiosarcomas were statistically significant, it
 

should be observed that each of the dosed groups of either sex
 

had a higher incidence than the respective control group. No
 

such tumor was reported in any control group, while dosed groups
 

had incidences ranging from 3% to 33%.
 

The Fisher exact test shows that the incidence of tubular
 

adenomas of the ovary in the low-dose females is significantly
 

higher (P = 0.024) than that in the low-dose vehicle controls,
 

but no such tumor was observed in the mid- and high-dose groups.
 

Significant results in the negative direction were observed in
 

the incidence of hepatocellular adenomas in male mice, where the
 

incidences in the mid- and high-dose groups are lower than those
 

in the controls, probably due to the early mortality of the dosed
 

animals.
 

In summary, while early mortality in the dosed groups may have
 

curtailed the number of animals at risk for the development of
 

43
 
late-appearing tumors, the data indicate an association between 

the administration of the chemical and tumors of the lung, 

hematopoietic system, and myocardium. 

44
 
V. DISCUSSION
 

Under the conditions of this bioassay, phenesterin was toxic to
 

rats and mice at the doses employed, as shown by reduced mean
 

body weights and survival· Survival was sufficient, however, for
 

the development of significant incidences of tumors in female
 

rats and in both sexes of mice. Time-adjusted analyses were used
 

for evaluation of the incidences of tumors in the female mice.
 

In male rats, a variety of sarcomas occurred in different organs
 

(pooled controls 1/18, vehicle controls 0/9, low-dose 7/32, high-


dose 4/30). The incidences in the individual dosed groups were
 

not statistically significant, however, when compared with either
 

the pooled or vehicle controls. Similar tumors occurred among
 

the female rats (pooled controls 0/18, vehicle controls 0/10,
 

low-dose 2/29, high-dose 4/30), but also at incidences that were
 

not statistically significant. A dose-related trend (P = 0.037)
 

was present in lymphoma and leukemia in male rats, using the
 

pooled controls; however, the incidences of these neoplasms in
 

the individual dosed groups were not significant when compared
 

with incidences in either the pooled or vehicle controls (pooled
 

controls 0/18, vehicle controls 0/9, low-dose 2/32, high-dose
 

5/30).
 

In female rats, a dose-related trend (P = 0.019) was present in
 

45
 
adenocarcinoma of the mammary gland, using the pooled controls,
 

and the incidences of the tumor in the individual dosed groups
 

were significant (P < 0.009) when compared with those in the
 

pooled controls (controls 1/18, low-dose 12/29, high-dose 12/30).
 

Metastases to the lung occurred in one high-dose animal.
 

In male mice, the incidence of alveolar/bronchiolar carcinomas or
 

combined alveolar/bronchiolar adenomas and carcinomas in the
 

low-dose group (14/40) was significantly higher (P < 0.020) than
 

that in the low-dose vehicle-control group (0/16). Alveolar/bronchiolar adenomas also occurred in the mid-dose (6/29) and high-


dose (2/25) groups, but these incidences were not significant
 

when compared with either the respective pooled or vehicle
 

controls. In female mice, seven low-dose animals had
 

alveolar/bronchiolar adenomas and eight other low-dose animals
 

had alveolar/bronchiolar carcinomas. When these tumors were
 

combined, their time-adjusted incidence was significant (P =
 

0.004) when compared with that in the low-dose vehicle controls
 

(controls 1/18, low-dose 15/35). Only two animals in the mid-


and high-dose females had alveolar/bronchiolar neoplasms. The
 

lower incidences of these tumors observed in the mid- and high-


dose groups may be due to the earlier mortality in these groups
 

compared with the low-dose groups.
 

In each sex of mid- and high-dose mice, incidences of lymphomas
 

46
 
and leukemias were dose related (P < 0.005) using vehicle
 

controls; they were also significant (P < 0.018) in direct
 

comparisons of mid- and high-dose groups of each sex with
 

respective vehicle controls (males: controls 0/14, mid-dose 9/29,
 

high-dose 11/25; females, time-adjusted: controls 0/15, mid-dose
 

14/18, high-dose 17/19). The significance of the incidence of
 

lymphomas and leukemias in the mid- and high-dose groups of males
 

was increased (P < 0.001) when a pooled-control group was used,
 

both in the test for dose-related trend and in tests for direct
 

comparisons of dosed groups with the controls. In the low-dose
 

groups of each sex, an increased incidence of these tumors was
 

observed when compared with the respective controls (males:
 

controls 1/16, low-dose 11/40; females: controls 5/18, low-dose
 

12/36). These incidences in the low-dose groups were not
 

statistically significant.
 

In each sex of mice, sarcomas of the myocardium were found in all
 

groups of dosed animals, but in no control animals (males:
 

low-dose 5/40, mid-dose 7/29, high-dose 2/25; females: low-dose
 

8/34, mid-dose 2/7, high-dose 3/7). In males, the incidence in
 

the mid-dose group was significant when compared with that in the
 

pooled controls (P = 0.006); in females, the incidences in the
 

low- and high-dose groups were significant (P < 0.023). In some
 

of the mice, the neoplastic cells had invaded the myocardium and
 

47
 
formed large thrombi within the ventricles· The myocardial
 

sarcomas had metastasized to the lungs in several animals in each
 

dosed group. No sarcomas of the myocardium have occurred in over
 

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

the laboratory.
 

Hemangiosarcomas were observed in mice at the following
 

incidences (males: low-dose vehicle controls 0/20, low-dose 6/40,
 

mid- and high-dose vehicle controls 0/14, mid-dose 1/29, high-


dose 3/25; females: low-dose vehicle controls 0/18, low-dose
 

5/35, mid- and high-dose vehicle controls 0/15, mid-dose 1/7,
 

high-dose 2/6). Although none of the direct comparisons of the
 

dosed groups with controls were statistically significant,
 

hemangiosarcomas occurred in each of the dosed groups of either
 

sex, but in no control animals. Thus, the occurrence of this
 

tumor may be related to administration of the test chemical.
 

In low-dose female mice, adenocarcinomas of the mammary gland
 

were observed at an incidence of 6/35 and tubular adenomas of the
 

ovary at an incidence of 8/34. Neither tumor was found in the
 

vehicle controls or in the mid- or high-dose groups of female
 

mice; however, there were two untreated-control mice with
 

adenocarcinomas of the mammary gland. The different response of
 

the low-dose mice may have resulted from their longer survival or
 

48
 
because they were started on study 75 weeks after the other
 

groups studied.
 

Previous work showed that the toxicity of phenesterin administered by subcutaneous injection is relatively low, with an LD50
 

for rats of 2.0 g/kg for a single injection (Larionov et al. ,
 

1962); this toxicity is less than that of some of the nitrogen
 

mustards in general use (Wall et al. , 1969). Chronic
 

administration to rats, dogs, and monkeys (Vollmer et al., 1973)
 

resulted, however, in cumulative toxicity, manifested by weight
 

loss and myelosuppression. Chronic treatment of cancer patients
 

(Ansfield et al. , 1971) also resulted in myelosuppression. The
 

carcinogenicity of phenesterin has been tested in a pulmonary
 

tumor test system in strain A mice administered the chemical
 

intraperitoneally three times per week for 8 weeks at total doses
 

of 2,400, 6,000, or 12,000 mg/kg (Stoner et al. , 1973). Under
 

these conditions, phenesterin induced significant incidences (P =
 

0.001) of tumors of the lung in the mice at all doses tested.
 

It is concluded that under the conditions of this bioassay,
 

phenesterin was carcinogenic in female Sprague-Dawley rats,
 

producing adenocarcinomas of the mammary gland, and in both sexes
 

of B6C3F1 mice, producing alveolar/bronchiolar carcinomas,
 

hematopoietic tumors, and myocardial sarcomas.
 

49
 

VI. BIBLIOGRAPHY
 

Ansfield, F· J., Carter, A· C·, Goldenberg, I. S·, and Segalof f,
 

Α., Phase I study of phenesterin (NSO104469). Cancer
 

Chemotherap. Reports Part I;55(3);259-263, 1971.
 

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 of Carcinogenicity of the Cancer Research Commission
 

of UICC, Vol. _2, International Union Against Cancer, Geneva,*
 

1969.
 

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

Soc. Β 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^ Am. Statist. Assoc. 53:457-481,
 

1958.
 

Larionov, L. F., Degteva, S. Α., and Lesnaya, Ν. Α., Experimental
 

data on the antitumourous preparation "phenesterine". Vopr.
 

Onkol. 8(4):12-14, 1962.
 

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

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

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

Merck Index, 9th Edition, Merck and Co., Rahway, N.J., 1976.
 

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

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

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. 3211073-1081, 1972.
 

51
 
Stoner, G. D. , Shimkin, M· B., Kniazeff, A. J., Weisburger, J.
 

Η ·, Weisburger, Ε. Κ., 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., Taylor, D. J., Masnyk, I. J., Cooney, D. , Levine,
 

B., Piczak, C., and Trench, L., Phenesterin (NSC-104469)­

clinical brochure. Cancer Chemotherap. Reports Part III
 

4(l):103-113, 1973.
 

Wall, M. E., Abernethy, G. S·, Jr., Carroll, F. 1., and Taylor,
 

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

experimental animal mammary tumor and leukemia systems. J. 

Med. Chem. 12:810-818, 1969. 

52
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS ADMINISTERED PHENESTERIN BY GAVAGE
 

53
 

TABLE ΑΙ.
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS
 

ADMINISTERED PHENESTERIN BY GAVAGE
 

———————— ——
 UNTREATED VEHICLE
 

CONTROL CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 10 10 35 35
 

ANIMAIS NECFCFSIED 10 9 32 30
 

ANIMALS EXAMINED HISTOFATHOLOGICALLY 10 9 32 30
 

INTEGUMENTARY SYSTEM
 

*SKIN (10) (9) (32) (30)
 

SQUAMOUS CELL PAPILLOMA 1 (3%)
 

•SUECUT TISSUE	 (10) (9) (32) (30)
 

SEEACECUS ADENOMA 1 (3%)
 

SARCCMA, NCS 2 (6%) 1 (3%)
 

EIEROMA 1 0%)
 

FIERCSARCCMA 1 0%)
 

IIFCBA 1 (10%)
 

CHCNDRCMA 1 (3%)
 

RESEIFATCRY SYSTEM
 

#LUNG (10) (9) (32) (30)
 

SAPCOMA, NOS, METASTATIC 1 (3%)
 

OSTEOSARCOMA, METASTATIC 1 (3%)
 

HEMATCPOIEIIC SYSTEM
 

•MULTIFLE CBGANS	 (10) (9) (32) (30)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (3%)
 

LYMPHOCYTIC LEUKEMIA 1 (3%) 3 (10%)
 

GRANULOCYTIC LEUKEMIA 1 (10%) 2 (7%)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

NONE
 

# KUHEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

• NUMEER OF AN.IMALS NECROPSIED
 

55
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED)
 

UNTREATED VEHICLE
 

CONTROL CONTROL
 

URINARY SYSTEM
 

#KIDNEY (10) (9)
 

LIPOMA
 

ENDOCRINE SYSTEM
 

#PITUITARY (10) (9)
 

CHROMOPHOBE ADENOMA
 

CHROMOPHOBE CARCINOMA 1 (11%)
 

#ADRENAL (10) (9)
 PHECCHROMCCYTOMA
 

#PANCREATIC ISLETS (10) (9)
 

ISLET-CELL ADENOMA
 

REEFCDUCTIVE SYSTEM
 

#TESTIS (10) (9)
 

INTEBSTITIAL-CELL TUMOR 1 (1C%)
 

NERVCUS SYSTEM
 

#BRAIN (10) (9)
 

ASTROCYTOMA
 

SPECIAL SFKSE ORGAKS
 

*EAR CANAL (10) (9)
 

SQUAMOUS CELL PAPILLOMA
 

MUSCULOSKELETAL SYSTEM
 

•BONE	 (10) (9)
 

CSTECSARCCMA
 

BODY CAVITIES
 

*ABDOMINAL CAVITY (10) (9)
 SARCCMA. NCS 1 (10%)
 

# NUKEEF OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

• NUMEER OF ANIMALS NECROESIED
 

LOW DOSE
 

(32)
 

1 (3%)
 

(29)
 

2 (7%)
 

1 (3%)
 

(32)
 

1 (3%)
 

(32)
 

1 (3%)
 

(32)
 

(30)
 

1 (3%)
 

(32)
 

2 (6%)
 

(32)
 

1 (3%)
 

(32)
 

1 (3%)
 

HIGH DOSE
 

(30)
 

(29)
 

(30)
 

(30)
 

<3C)
 

(30)
 

1 (3%)
 

(30)
 

(30)
 

1 (3%)
 

(30)
 

2 11%)
 

56
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED)

 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

HEHANGICSABCOMA 1 (3%) 

All CTHER SYSTEMS 

•FOITIFLE CEGANS 

SABCOMA, NCS 

(10) (9) (32) 

1 (3%) 

(30) 

ANIMAL EISEOSITICN SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL EEATHcD 

KORIEUNE SACRIFICE 

SCHEEULEE SACRIFICE 

ACCIEENTALLY KILLEE 

TERMINAL SACRIFICE 

ANIMAL MISSING 

10 10 35 

15 

12 

35 

10 

21 

d INCLUEES AUTOLYZED ANIMALS 

TLPCF SUMMARY 

TCTAL ANIMALS WITH EPIMARY

TCTAL ERIMARY TUMOFS 

 TUMCRS* 18 

21 

11 

11 

TOTAL ANIMALS WITH EENIGN

1CTAL EENIGN TUMORS 

 TUMORS 9 

10 

TCTAL ANIMALS WITH MALIGNANT

TCTAL MALIGNANT TUMORS 

 TUMCRS 11 

11 

10 

10 

TOTAL ANIMALS WITH SECONDARY

TCTAL SECONDARY TUMORS 

 TUMORSt 

TOTAL ANIMALS WITH TUMORS

BENIGN CR MALIGNANT 

TCTAL UNCERTAIN TUMORS 

 UNCERTAINTCTAL ANIMALS WITH TUMORS

EFIKARY CR METASTATIC 

TCTAL UNCERTAIN TUMORS 

 UNCERTAIN­

* ERIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

# SECONEARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVF INTO AN ADJACENT ORGAN 

57
 
TABLE A2. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED PHENESTERIN BY GAVAGE 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 

ANIMALS NECFOPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

10 

10 

10 

10 

10 

10 

35 

29 

29 

35 

30 

30 

INTEGUMENTARY SYSTEM 

•SUBCUT TISSUE 

SARCCMA, NOS 

FIBROSARCOMA 

(10) (10) (29) 

1 (3%) 

(30) 

1 (356) 

RESPIFATCRY SYSTEM 

#LUKG 

ADENOCARCINOMA, NOS, METASTATIC 

ALVECLAR/BRONCHIOLAR CARCINOMA 

(10) (10) (28) 

1 (<•%) 

(3C) 

1 (3%) 

1 (3*) 

HEMATOPOIETIC SYSTEM 

•MUITIPLE CBGANS

MAIIG.IYFPHCFA, HISTIOCYTIC

GRANULOCYTIC LEUKEMIA 

 TYPE 

 (10) (10) (29) 

2 (7%) 

(30) 

1 (3*) 

. 1 (3*) 

CIFCIIATCRY SYSTEK 

KCNE 

CIGES1IVE S YSTEK 

NONE 

UFIKAFY SYS ΊΕΜ 

NONE 

ENECCEINE SYSTEM 

«PITUITARY 

CHROMOPHOBEADENCM^ 

(10) (10) (27) (30) 

# NUMEER OF ANIMALS WITH TISSUE EXAMINED

* NUMEER OF ANIMALS NECROPSIED 

 MICROSCOPICALLY 

58
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)
 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH OOSE 

ifAUCBIATIC ISLETS 

ISLET-CEIL ADENOMA 

(10) (10) 

1 (10%) 

(29) (30) 

BliFCEOCTIVE SYSTEM 

•HAHHABY GLAND 

ADENOCARCINOMA, NOS 

SARCOMA, NOS 

FIBRCADEKCHA 

(10) 

6 (60%) 

(10) 

1 (10%) 

4 (40%) 

(29) 

12 (U1%) 

21 (72%) 

(30) 

12 (40%) 

1 (3%) 

11 (37%) 

•CTEEUS 

ADENOCARCINOMA, NOS 

ENDOMETRIAL STROMAL POLYP 

(10) (10) 

1 (10%) 

(29) 

1 (3%) 

5 (17%) 

(30) 

1 (3%) 

2 (7%) 

tCEFVIX UTEEI 

SQUAMOUS CELL CARCINOMA 

(10) (10) (29) 

1 (3%) 

(30) 

1 (3%) 

NEFVCUS SYSTEM 

hCNE 

SFECIAL SE&SE CBGANS 

•EYE/RETINA 

NEUROBLASTOMA 

(10) (10) (29) (30) 

1 (3%) 

HDSCULOSKEIETAL SYSTEM 

1>CNE 

ECCY CAVITIES 

•AEtCMINAL CAVITY 

ADENOCARCINOMA, NOS 

SARCCMA, NOS 

(10) 

1 (10%) 

(10) (29) 

1 (3%) 

(30) 

ALL CTREE SYSTEMS 

•KULTIFLE CEGANS , ADENOCARCINOMA. NCS. METASTATIC 

(10) 

 imL 

(10) 

# SUPEER OP ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUHEER OF ANIMALS NECROPSIED 

(29) 

1 (3%) 

(30) 

59
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)
 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

SARCOMA, NOS, METASTATIC 1 (3*) 

CRANIAL CAVITY 

SARCOMA, NOS 1 

ANIMAL EISECSITICN SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL EEATH3 

KORIEUND SACRIFICE 

SCHEEULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 

ANIMAL MISSING 

10 

2 

β 

10 

1 

1 

8 

35 

α 

12 

9 

35 

9 

24 

2 

d INCLUDES AUTCLYZED ANIMALS 

TUMOR S0MMARY 

TOTAL ANIMALS KITH PRIMARY TUMORS* 

TCTAI ERIMARY TUMORS 

6 

8 

7 

13 

28 

H9 

22 

34 

TOTAL ANIMALS WITH BENIGN TUMORS 

TOTAL BENIGN TUMORS 

6 

7 

7 

12 

23 

30 

13 

13 

TOTAL ANIMALS WITH MALIGNANT TUMORS 

TCTAL MALIGNANT TUMORS 

1 

1 

1 

1 

16 

19 

16 

21 

TOTAL ANIMALS WITH SECONDARY

TCTAL SECONDARY TUMORS 

 TUHORSt 1 

1 

1 

1 

2 

2 

TOTAL ANIMALS WITH TUMORS UNCERTAINEENIGN OR MALIGNANT 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC 

TOTAL UNCERTAIN TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TDHORS 

# SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN 

60
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE ADMINISTERED PUENESTERIN BY GAVAGE
 

61
 

TABLE B1. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE
 

ADMINISTERED PHENESTERIN BY GAVAGE (CONTROL GROUPS)
 

MID & HIGH DOSE
 

VEHICLE
 

CONTROL
 

15
 

14
 

14
 

(14)
 

(14)
 

(14)
 

4 (29%)
 

LOW DOSE
 

VEHICLE
 

CONTROL
 

20
 

16
 

16
 

(16)
 

2 (13%)
 

(16)
 

1 (6%)
 

(16)
 

3 (19%)
 

1 (6%)
 

ANIMALS INITIALLY IN STUDY
 

ANIHALS NECFCPSIED
 

ANIMALS EXAMINED HISTOFATHCLOGICALLY
 

ΙΝΤEG0ΜΕΝΤΑR SYSTEM
 

HCNE
 

RESFIFATCFY SYSTEM
 

#LUNG
 

HEPATOCELLULAR CARCINOMA, METAST
 

AIVECLAR/BFCNCHIOLAR ADENOMA
 

HEBATCECIETIC SYSTEM
 

•MULTIPLE	 ORGANS
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

CIBCOLATCRY SYSTEM
 

NCNE
 

DIGESTIVE SYSTEM
 

«LIVER
 

HEPATOCELLULAR ADENOMA
 

HEPATOCELLULAR CARCINOMA
 

HEMANGIOMA
 

ORINAFY SYSTEM
 

I.CNE
 

ENICCFIKE SYSTEM
 

tiCNE
 

MID & HIGH DOSE
 

UNTREATED
 

CONTROL
 

15
 

15
 

15
 

(15)
 

1 (7%)
 

1 (7%)
 

(15)
 

1 (7*)
 

(15)
 

1 (7%)
 

2 (13%)
 

LOW DOSE
 

UNTREATED
 

CONTROL
 

20
 

20
 

20
 

(20)
 

(20)
 

(19)
 

2 (11%)
 

2 (11%)
 

# NDMEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* KOMEER OF ANIMALS NECROPSIED
 

63
 
TABLE B1. MALE MICE (CONTROL GROUPS): NEOPLASMS (CONTINUED)

 

• " -" • "-" ' ­

MID & HIGH DOSE LOW DOSE UNTREATED UNTREATED CONTROL CONTROL 

BEPBCEtJCTIVE SYSIEB
 

NCNE
 

NEFVCOS SYSTEM
 

BCNE
 

SFECIAL SEKSE CBGAHS
 

•HAFEERIAN	 GLAND (15) (20)
 

ADENCHA, NOS
 

MUSCULOSKELETAL SYSTEM
 

HCNE
 

ECEY CAVITIES
 

•EEBITONEOH	 (15) (20)
 

SARCOHA, NOS 1 (7X)
 

All CTHER SYSTEMS
 

HONE
 

ANirAI DISECSITION SUMMARY
 

ANIBAiS INITIALLY IN STUDY 15 20
 

NATURAL DEATHS 1 7
 

HORIBOND SACRIFICE 11
 

SCHEDULED SACBIPICE
 ACCIDENTALLY KILLED
 TEBHINAL SACRIFICE 14 2
 

ANIBAL HISSING
 

I INCLUDES AUIOLYZED AHIHALS
 

• 10HBEB OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* IUHEER OF ANIMALS NECBOPSIED
 

MID & HIGH DOSE
 VEHICLE
 CONTROL
 

(14)
 

15
 

1
 

14
 

LOW DOSE
 VEHICLE
 CONTROL
 

(16)
 

1 (6%)
 

(16)
 

20
 

1
 

13
 

<l
 

2
 
TABLE B1. MALE MICE (CONTROL GROUPS): NEOPLASMS (CONTINUED) 

MID & HIGH DOSE UNTREATED CONTROL 

LOW DOSE UNTREATED CONTROL 

MID & HIGH DOSE 

VEHICLE CONTROL 

LOW DOSE VEHICLE CONTROL 

TOBCR SUMMARY 

TOTAL ANIMALS WITH EBIMARY TUMORS*

TOTAL EBIMARY TUMORS

 6

 6

 4

 4

 4 

4 

TOTAL ANIMALS WITH BENIGN TDMOBS

TOTAL BENIGN TUMORS

 2

 2

 2

 2

 4 

4 

TCTAL ANIMALS WITH MALIGNANT TUMORS

TOTAL MALIGNANT TDMOBS

 4

 4

 2 

2 

TOTAL ANIMALS WITH SECONDARY TUMORS#

TCTAL SECONDARY TOMORS

 1 

1 

TOTAL ANIMALS WITH TOMORS UNCERTAIN­ BENIGN OR MALIGNANT 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIHARY OR METASTATIC 

TOTAL UNCERTAIN TUMORS 

* PRIHARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

# SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN 

65
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED PHENESTERIN BY GAVAGE (TREATED GROUPS) 

HIGH DOSE
 

35
 

2
 

25
 

25
 

(25)
 

(25)
 

3 (12%)
 

(25)
 

2 (8%)
 

2 (8%)
 

(25)
 

1 (4%)
 

9 (36%)
 

1 (4%)
 

(25)
 

2 i8%)
 

ANIFALS INITIAILY IN STUDY
 

ANIMALS HISSING
 

ANIMALS NECECFSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

•SKIN
 

CARCINOMA,NOS
 

KERATCACANTHCMA
 

•SUECUT	 TISSUE
 

FIEBOSARCCMA
 

HEMANGIOSAECOMA
 

FESPIBATCEY SYSTEF
 

#LUNG
 

ALVECLAR/BFONCHIOLAR ADENOMA
 

ALVECLAR/EFONCHIOLAR CARCINOMA
 

TUBULAR-CELL ADENOCARCINOMA, MET
 

SARCCMA, NOSf METASTATIC
 

HEFATCECIETIC SYSTEM
 

•MULTIPLE	 CRGANS
 

MALIGNANT LYMPHOMA, NOS
 

MALIG.LYMPHOMA, UNDIFFER-TYPE
 

MALIG.LYrPHOMA, LYfPHCCYTIC TYEE
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

MALIGNANT LYMPHOMA, MIXED TYEE
 

LYMPHOCYTIC LEUKEMIA
 

EOSINOPHILIC LEUKEMIA
 

#I»ESENTERIC L. NODE
 

MALIC.LYMPHOMA, HISTIOCYTIC TYPE
 

CIFCULATCRY SYSTEM
 

#MYOCARCIUM
 

SARCCMA, NOS
 

LOW DOSE
 

40
 

40
 

40
 

(40)
 

1 (3%)
 

1 (3%)
 

(40)
 

2 (5%)
 

(40)
 

4 (10%)
 

14 (35%)
 

3 (8%)
 

(40)
 

2 (5%)
 

5 (13%)
 

1 (3%)
 

1 (3%)
 

1 (3%)
 

(35)
 

1 (3%)
 

(40)
 

5 (13%)
 

MID DOSE
 

35
 

29
 

29
 

(29)
 

(29)
 

1 (3%)
 

(29)
 

6 (21%)
 

1 (3%)
 

1 (3%)
 

(29)
 

7 (24%)
 

2 (7%)
 

(4)
 

(29)
 

7 (24%)
 

# MMEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

• NUMEER OF ANIMALS NECROPSIED
 

66
 
TABLE B2. MALE MICE (TREATED GROUPS): NEOPLASMS (CONTINUED) 

LOW DOSE MID DOSE HIGH DOSE 

DIGESTIVE SYSTEM
 

LIVER 

HEPATOCELLULAR ADENOMA 

HEPATOCEILULAF CARCINOMA 

ALVECLAR/ERONCHIOLAR CA,

KEMANGIOSAFCCMA 

#STCMACH 

SCUAMOUS CELL PAPILLOMA 

 METASTA 

(39) 

7 (18%) 

3 (8%) 

1 (3%) 

4 (10*) 

(<+0) 

1 (3%) 

(29) 

2

(29) 

 (7%) 

(25) 

(24) 

URIKARY SYSTEM 

#KIENEY 

TUEULAR-CELL AEENCCARCINOMA 

(UO) (29) 

1 (3%) 

(25) 

ENICCFIKI SYSTEM 

KCNE 

REEFCCUCTIVE SYSTEM 

KCKE 

KEFVCCS SYSTEM 

NCNE 

SFECIAL SENS! CRGANS 

•HAPCERIAN GLANE 

ADENOMA, NOS 

(40) 

2 (5%) 

(29) (25) 

1 (4%) 

MCSCUICSKEIETAI

NONE 

 SYSTEM 

BODY CAVITIE S 

•PERITONEOM SARCCMA. NOS 

(40) 

1 (3%) 

(29) (25) 

# MJfBER CF ANIMALS WITH TISSDE EXAMINED

* NDMEER OF ANIMALS NECROPSIED 

 MICROSCOPICALLY 

67
 
TABLE B2. MALE MICE (TREATED GROUPS): NEOPLASMS (CONTINUED)

 

HEMANGIOSARCOMA
 

ALL CTHEF SY£TEi3S
 

•MULTIPLE	 CRGANS
 

SARCCMA, NOS, METASTATIC
 

ANIMAL EISPOSITICN SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATUEAL DEATHS
 

MORIEUND SACRIFICE
 SCHEEULED SACRIFICE
 ACCIDENTALLY KILLED
 TERMINAL SACRIFICE
 ANIMAL MISSING
 

a) INCLUDES AUTCLYZED ANIMALS
 

TUMOR SUMMAFY
 

TOTAL ANIMALS WITH PRIMARY TOMCES*
 

TCTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TCTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TCTAL MALIGNANT TUMORS
 

TOTAL ANIMALS WITH SECONDARY TUMORSt
 

TCTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCEFTAIN­ BENIGN OR MALIGNANT
 TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ EFIMARY OR METASTATIC
 

TCTAL UNCERTAIN TUMORS
 

LOW DOSE MID DOSE HIGH DOSE 

2 (5%) 

(40) (29) (25) 

1 (3%) 

40 35 35 

9 19 18 

31 11 13 

2 

5 

2 

29 20 17 

58 26 19 

11 7 2 

15 8 3 

27 18 16 

43 18 16 

5 2 2 

5 2 2 

• ERIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

* SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

68
 
TABLE B3.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED PHENESTERIN BY GAVAGE (CONTROL GROUPS) 

MID & HIGH DOSE LOW DOSE MID & HIGH DOSE LOW DOSE
 UNTREATED UNTREATED VEHICLE VEHICLE
 CONTROL CONTROL CONTROL CONTROL
 

ANIMALS INITIALLY IN STODY 15 20 15 20
 

ANIMALS NECBCESIED	 15 20 15 20
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 15 20 15 20
 

INTEGUMENTARY SYSTEM
 

•SUECUT	 TISSUE (15) (20) (15) (20)
 

HEMANGIOMA 2 (10%)
 

RESPIBATCBY SYSTEM
 

«LONG (15) (20) (15) (20)
 

ALVEOLAR/ERONCHIOLAR ADENOMA 1 (7%)
 

ALVEOLAB/BRONCHIOLAR CARCINOMA 1 (5%)
 

HEMATOPOIETIC SYSTEM
 

•HUITIPLE	 CRGANS (15) (20) (15) (20)
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (5%)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 3 (15%)
 

MALIGNANT LYMPHOMA, MIXED TYPE 1 (5%)
 

«SPLEEN (15) (20) (15) (19)
 

HEMANGIOMA 1 (7%)
 

CIFCDIATCRY SYSTEM
 

KCSE
 

DIGESTIVE SYSTEM
 

«LIVER (15) (19) (15) (20)
 

HEPATOCELLULAR ADENOMA 2 (13%) 2 (10%)
 

HEPATOCELLULAR CARCINOMA 1 (5%)
 

URINARY SYSTEM
 

NONE
 

I «UMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMEER OF ANIMALS NECROPSIED
 

69
 
TABLE B3. FEMALE MICE (CONTROL GROUPS): NEOPLASMS (CONTINUED)
 

MID & HIGH DOSE LOW DOSE
 UNTREATED UNTREATED
 CONTROL CONTROL
 

ENICCEINE SYSTEM
 

#ΕΙΊϋΙΤΑΕΥ (14) (17)
 

CHROMOPHOBE ADENCBA
 

#THYBOID (HO (20)
 

FOLIICDLAR-CELL ADENOMA
 

REEFCEUCTIVE SYSTEM
 

*HAMt?ARY GLAND (15) (20)
 

ADENOCARCINOMA, NCS 2 (10%)
 

NEFVCUS SYSTEM
 

KCNE
 

SFHCIA1 SEKSE CRGANS
 

UCKE
 

MOSCOICSKEIETAI SYSTEM
 

• EONE	 (15) (20)
 

CSTECSAPCOMA 1 (5%)
 

ECCY CAVITIES
 

•EEBITONEDM	 (15) (20)
 

LIPOMA 1 (5%)
 

All CTHEF SYSTEMS
 

MCNE
 

* NOMEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

MID & HIGH DOSE
 VEHICLE
 CONTROL
 

(15)
 

1 (7%)
 

(15)
 

(15)
 

(15)
 

LOW DOSE
 VEHICLE
 CONTROL
 

(1U)
 

1 (7%)
 

(16)
 

(20)
 

(20)
 

(20)
 

70
 
TABLE B3. FEMALE MICE (CONTROL GROUPS): NEOPLASMS (CONTINUED)
 

MID & HIGH DOSE 

UNTREATED 

CONTROL 

LOW DOSE 

UNTREATED 

CONTROL 

MID & HIGH DOSE 

VEHICLE 

CONTROL 

LOW DOSE 

VEHICLE 

CONTROL 

ANIHAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATHS 

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 

ANIHAL HISSING 

15 

1 

14 

20 

3 

15 

2 

15 

15 

20 

5 

9 

6 

9 INCLUDES AUTCLYZED ANIMALS 

TUMOR SUMHAF* 

TOTAL ANIMALS WITH PRIMARY TUMORS* 

TCTAL PRIMARY TUMORS 

3 

3 

4 2 

2 

9 

12 

TOTAL ANIMALS WITH BENIGN TUMO$£ 

TCTAL BENIGN TUMORS 

3 

3 

1 

1 

2 

2 

α 

5 

TCTAL ANIMALS WITH MALIGNANT TUMORS 

1CTAL MALIGNANT TUMORS 

3 

3 

7 

7 

TOTAL ANIMALS WITH SECONDARY

TCTAL SECONDARY TUMORS

 TUHORSt 

f 

TOTAL ANIMALS WITH TUMORS UNCERTAINEEMGN OR MALIGNANT 

TCTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINFBIHARY CP METASTATIC ΤΓΤΛ ΠΝΓΕΒΤΛΤ ΤΠΜΩΡ5 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

# SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN 

71
 
TABLE B4.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED PHENESTERIN BY GAVAGE (TREATED GROUPS) 

HIGH DOSE
 

35
 

32
 

31
 

(32)
 

(32)
 

1 0%)
 

(3D
 

1 (3%)
 

2 (6%)
 

1 (3%)
 

(32)
 

2 (6%)
 

14 (44%)
 

(30)
 

(2)
 

(28)·
 

ANIMALS INITIALLY IN STDDY
 

ANIMAIS NECECFSIED
 

ANIMALS EXAMINED HISTOEATHOLOGICALLY
 

INTIGUMENTAFY SYSTEM
 

•SKIN
 

SQUAMOUS CELL PAFILLOMA
 

•SUECUT TISSUE
 

CARCINOMA,NCS
 

HEMANGICSAECOMA
 

FESEIFATCEY SYSTEM
 

«LUNG
 

ADENOCARCINOMA, NOS, METASTATIC
 

ALVECLAR/BRONCHIOIAR ADENOMA
 

AIVECLAR/BFCNCHIOLAF CARCINOMA
 

SARCCMA, NCS, METASTATIC
 

HEMANGIOSAFCCMA
 

HEKATCECIETIC SYSTEK
 

•BULTIFLE	 CFGANS
 

MALIGNANT LYKPHCFA, NOS
 

MALIG.LYMPHOMA, ONDIFFER-TYPE
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE
 

MALIG.IYPPHCMA, HISTIOCYTIC TYPE
 

LYMPHOCYTIC LEUKEMIA
 

#SPLEEN
 

HEMANGIOSARCCMA
 

#FEEIASTINAL L.NODE
 

AIVEOLAR/EEONCHIOLAR CA, MTTASTA
 

#THYKUS
 

MALIG.LYKPHOMA. DNCIFFEP-TYPE
 

LOW DOSE MID DOSE
 

UO 35
 38 27
 

38 27
 

(38)	 (27)
 

1 (3%)
 

(38) (27)
 

2	 (5%)
 

(38) (27)
 

1 (3%)
 7 (18%) 1 (4%)
 

8 (21%)
 

6 (16%) 2 (7%)
 

(38) (27)
 

U (11%)
 

2 (5%) 12 (44%)
 

U (11%)
 

1 (3%) 2 (7%)
 

(38)	 (27)
 

1 (3%)
 

(36)	 (4)
 1 (3%)
 

(35)	 (27)
 

1 (3%)
 

# KUMEER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMEER OF ANIMALS NECROPSIED
 

72
 
TABLE B4. FEMALE MICE (TREATED GROUPS): NEOPLASMS (CONTINUED)

 

-	 LOW DOSE MID DOSE
 

KALIG.LYMPHOMA, LYMPHOCYTIC TYPE
 

CIFCOLATOFY SYSTEM
 

#KYCCARDIUM (36) (27)
 

SARCOMA, NOS 8 (22%) 2 (7%)
 

DIGESTIVE SYSTEM
 

«LIVER (37) (27)
 

HEPATOCELLULAR CARCINOMA 1 (3%)
 

HEMANGIOSAFCCHA 1 (3%)
 

#STCMACH (36) (26)
 

SQUAMOUS CELL CARCINOMA 1 (3%) 1 (U%)
 

UFIN*FY SYSTEK
 

KCKE
 

ENECCFINE SYSTEM
 

NCNE
 

FEEFCEICTIVE SYSTEM
 

•MAMMARY	 GLAND (38) (27)
 

ADENOCARCINOMA, NOS 6 (16%)
 

#CTEFUS (38) (27)
 

ENDOMETRIAL STROMAL SARCCMA 1 (3%)
 

#CVAFY (37) (27)
 

CARCINCMA,NOS 1 (3%)
 

AEENCMA# NCS 1 (3%)
 

TUBULAR ADENCMA 8 (22%)
 

NEFVCUS SYSTEK
 

kCNE
 

# &UKEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* KUMEER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

1 («•%)
 

(31)
 

3 (10%)
 

(30)
 

(29)
 

(32)
 

(30)
 

(30)
 

73
 
TABLE B4. FEMALE MICE (TREATED GROUPS): NEOPLASMS (CONTINUED)

 

LOW DOSE MID DOSE HIGH DOSE
 

SEKIPL SENS! ORGANS
 

•HAFDEEIAN	 GLAND (38) (27) (32)
 

ADENOMA, NOS 1 (3%)
 

MUSCULOSKELETAL SYSTEM
 

*EONE (38) (27) (32)
 

FEMANGICSARCCMA 1 (3%)
 

EOEY CAVITIES
 

•EEFITONEUM	 (38) (27) (32)
 

HEMANGIOSAFCCMA 1 0**)
 

ALL CTfiEF SYSTEMS
 

•KULTIELE	 CEGANS (38) (27) (32)
 

SQUAMOUS CELL CARCINOMA, METASTA 1 <<»%)
 

FIEBOUS HISTIOCYTOMA, MALIGNANT 1 (3%)
 

HEMANGIOSARCOHA, METASTATIC 1 (*»%)
 

ANIMAL DISPOSITION SUMMARY
 

ANIKALS INITIALLY IN STUDY 40 35 35
 

NATURAL DEATHS 13 13 18
 

KCRIEUND SACRIFICE 27 20 16
 

SCHEDULED SACRIFICE
 ACCIDENTALLY KILLED 2 1
 

TERMINAL SACRIFICE
 ANIMAL MISSING
 

@ INCLUDES AOTCLYZED ANIMALS
 

# NDHEER OE ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

• KUMEER OF ANIMALS NECROPSIED
 

74
 
TABLE B4. FEMALE MICE (TREATED GROUPS): NEOPLASMS (CONTINUED) 

LOW DOSE MID DOSE HIGH DOSE 

TDKOB SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMCRS* 33 17 21
 TCTAL PRIMARY TUMORS 62 20 23
 

TOTAL ANIMALS WITH BENIGN TUMCES 15 1 1
 TCTAL BENIGN TUMORS 18 1 1
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 30 17 21
 TCTAL MALIGNANT TUMORS 44 19 22
 

TOTAL ANIMALS WITH SECONDARY TDMORS# 8 4 2
 TCTAL SECONDARY TDMORS 8 4 2
 

TOTAL ANIMALS WITH TUMORS UNCEFTAINeENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINEFIHAFY CR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

• CRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECCNDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

75
 

APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS ADMINISTERED PHENESTERIN BY GAVAGE
 

77
 

TABLE CI.
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS
 

ADMINISTERED PHENESTERIN BY GAVAGE
 

UNTREATED VEHICLE CONTROL CONTROL LOW DOSE 

ANIMAIS 1NITIAILY IN STODY 10 10 35 ANIMAIS NECRCESIED 10 9 32 ANIMALS EXAMINED HISTOFATHOLOGICALLY 10 9 32 

ΙΝΊΕGUΜΕΝΤΑRY SYS1EM
 

•SKIN	 (10) (9) (32)
 

EEIDERMAL INCLUSION CYST
 

INFLAMMATION, SUEEURATIVE 1 (3%)
 

INFLAMMATION, NECFCTIZING 1 (3%)
 

INFLAMMATICN, CHRONIC 2 (6%)
 

HYPERKERATOSIS 2 (6%)
 

•SUECUT	 TISSUE (10) (9) (32)
 

GRANULATION, TISSUE 1 (3%)
 

2 (6%)
 

RESPIRATORY SYSTEM
 

#TRACHEA (10) (9) (32)
 

INFLAMMATION, SUPPURATIVE 1 (10%) 1 (3%)
 

#LUNG/ERCNCHIOLE (10) (9) (32)
 

HYPERPLASIA, LYMPHOID 2 (6%)
 

#LUNG (10) (9) (32)
 

INFLAMMATION, INTERSTITIAL 4 (13%)
 

PNEUMONIA, LIPID 1 (3%)
 

ERCNCHCENEUMCNIA SUPPURATIVE 1 (3%)
 

AESCESS, NCS 1 (3%)
 

ERCNCHCEhEUMCNIA CHRONIC SUPEURA 2 (6%)
 

HEPATCECIFTIC SYSTEM
 

#BONE MARROW (9) (9) (32)
 

ATROPHY, NOS 8 (89%) 8 (89%) 18 (56%)
 

#SPLEEN (10) (9) (32)
 

HEMORRHAGE 1 (10%)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

35
 

30
 

30
 

(30)
 

1 (3%)
 

(30)
 

(30)
 

1 (3%)
 

(30)
 

(30)
 

2 (7%)
 

1 (3%)
 

3	 (10%)
 

(26)
 

14 (54%)
 

(29)
 

79
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)
 

INFLAMMATION WITH FIBROSIS
 

HEMATOPOIESIS
 

•MESENTERIC	 L. NODE
 CONGESTION, NOS
 

CIFCUIATCRY SYSTEM
 

«MYCCAREIUM
 INFLAMMATION, INTERSTITIAL
 INFLAMMATION, CHRCNIC
 

CALCIFICATION, METASTATIC
 

DIGESTIVE SYSTEM
 

#LIVER
 

LIFOIECSIS
 

tLIVEP/CENTBILOEULAR
 

NECRCSIS, NOS
 

«PANCREAS
 

ANEURYSM
 

#FANCBEATIC ACINUS
 

ATROPHY, NOS
 

UBINABY SYSTEM
 

tKIENEY
 

INFLAMMATION, INTERSTITIAL
 INFLAMMATION, CHRCNIC
 

INFLAMKATICN WITH FIBROSIS
 

ENECCBINE SYSTEM
 

«ADRENAL
 

ANGIECTASIS
 

«THYBCIE
 INFLAMMATION, INTERSTITIAL
 

UNTREATED
 CONTROL
 

1 (10%)
 

(10)
 

(10)
 

(10)
 

(10)
 

(10)
 

(10)
 

1 (10%)
 

 (40%)
 

(10)
 

1 (1C%)
 

(10)
 

VEHICLE
 CONTROL
 

(9)
 

(9)
 

(9)
 

(9)
 

(9)
 

1 (11%)
 

(9)
 

5 (56%)
 

(9)
 

(8)
 

LOW DOSE
 

3 (9%)
 

(1)
 

(32)
 

1 (3%)
 

1 (3%)
 

(32)
 

(32)
 

2 (6%)
 

(32)
 

1 (3%)
 

(32)
 

(32)
 

1 (3%)
 

18 (56%)
 

1 (3%)
 

(32)
 

(28)
 

HIGH DOSE
 

1 (3%)
 

1 (25%)
 

(30)
 

1 (3%)
 

(30)
 

1 (3%)
 

(30)
 

1 (3%)
 

(30)
 

(30)
 

(30)
 

16 (53%)
 

(30)
 

(27)
 

1 (4%)
 

# NDMEEP CF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

80
 
TABLE CI. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED VEHICLE 

CONTROL CONTROL 

REPECEDCTIVE SYSTEM
 

«PROSTATE (10) (9)
 

INFLAMMATION, SUPPURATIVE
 

NEBVCOS SYSTEM
 

#ERAIN/MENINGES (10) (9)
 

INFLAMMATION, NOS
 

iERAIN/EPENEYHA (10) (9)
 

INFLAMMATION, NOS
 

«ERAIN (10) (9)
 

PERIARTERITIS
 

SPECIAL SEKSE ORGANS
 

KCNE
 

MUSCOICSKEIEIAI	 SYSTEE
 

NONE
 

ΒΟΕ CAVITIES
 

•PERITONEUM	 (10) (9)
 

INFLAMMATION, FIBRINOUS
 

INFLAMMATION, CHFONIC
 

INFLAMMATION, CHRONIC NECROTIZIN 1 (10%)
 

•PLEURA	 (10) (9)
 

INFLAMMATION, CHRONIC
 

INFLAMMATION, CHRONIC SUPPURATIV 1 (11%)
 

ALL CTHEF SYSTEMS
 

ADIPOSE TISSUE
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

LOW DOSE	 HIGH DOSE 

(32)	 (29)
 

2 (7%)
 

(30)	 (30)
 

1 (3%)
 

(30)	 (30)
 

1 (3%)
 

(30)	 (30)
 

1 (3%)
 

(32)	 (30) 

1	 (3%) 

1 (3%) 

(32)	 (30) 

1 (3%) 

81
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

UNTREATED VEHICLE 

CONTROL CONTROL LOW DOSE HIGH DOSE 

SEICIAL KOREfcOLOGY SUFBARY 

NO LESION FEEORTED 

AOTOLYSIS/NC NECEOESY 

# NOFEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NDMEER OF ANIMALS NECROPSIED 

82 
TABLE C2.
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS
 

ADMINISTERED PHENESTERIN BY GAVAGE
 

UNTREATED VEHICLE 

CONTROL CONTROL LOW DOSE HIGH DOSE 

ANIHAIS IMTIAII Y IN STUDY 10 1 0 3 5 3 5 

ANIMALS NECFCFSIED 10 10 29 30 

ANIMALS EXAMINED HISTOFATHOLOGICALLY 10 10 2 9 30 

IKTEGUMENTAFY SYSTEM
 

•SUECUT TISSUE	 (10) (10) (29) (30)
 

INFLAMMATION, CHRONIC FOCAL 1 (3%)
 

RESFIFATOF* SYSTEM
 

•TRACHEA	 (10) (10) (28) (30)

INFLAMMATION, CHRONIC 1 (3%) 

#LUNG/ERONCHIOLE (10) (10) (28) (30) 

METAPLASIA, SQUAMOUS 1 (3%) 

(10) (10) (28) (30) 

INFLAMMATION, INTERSTITIAL 2 (20%) 2 (7%) 

EFCNCHCFNEUMONIA SUPPURATIVE 1 (10%) 2 (7%) 

AESCESS, NOS 1 (4%) 1 (3%) 

ERCNCHCFKEUMONIA, CHRONIC 1 (10%) 

ERCNCHCENEUMCNIA CHRONIC SUPPURA 1 (10%) 3 (11%) 3 (10? 

HEMATOPOIETIC SYSTEM
 

«ECNE NARROW ( 9) (10) (29) (28)

ATROPHY, NOS 3 (33%) 7 (70%) 7 (24%) 9 (32%) 

ISPLEEN (10) (10) (29) (3C) 

HEMATOPOIESIS α m% 11 (37%) ) 

#MANCIEULAR I. NODE ( 1) ( 2) 

HYPERPLASIA, LYMPHOID 1 (50%) 

fEESENTFFIC L. NODE ( 1) ( 2) 

CONGESTION, NOS 1 (50%) 

CIFCCIATCRY SY.STEM
 

# MKBEB CF ANIMALS fclTH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

83
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

DIGESTIVE SYSTEM
 

#LIVER
 

HEMORRHAGE
 EIBRCSIS
 

LIFCIDCSIS
 

HYPERPLASIA, GRANULOCYTIC
 

•LIVEB/CENTBILOEOLAR
 

NECROSIS, NOS
 

•PANCREATIC	 ACINOS
 

ATROPHY, NOS
 

OFINAFY SYSTEK
 

«KIDNEY
 INFLAMMATION, CHRONIC
 

ENDCCBINE SYSTEK
 

#ACRENAL
 

ANGIECTASIS
 

REFBCEOCTIVE SYSTEM
 

•HAMMABY	 GLAND
 CYST, NCS
 

«UTERUS
 

ABSCESS, NOS
 

METAPLASIA, SQUAMOUS
 

«DTEBUS/ENDCMETBIUH
 

INFLAMMATION, SUPPURATIVE
 INFLAMMATION, CHBONIC SUPPORATIV
 HYPERPLASIA, CYSTIC
 

fCVABY
 CYST, NOS
 

INFLAMMATION, CHBONIC SUPPURATIV
 

NEFVCUS SYSTEM
 

JiCNE
 

UNTREATED
 CONTROL
 

(10)
 

(10)
 

(10)
 

(10)
 

1 (10%)
 

(10)
 

(10)
 

3 (30%)
 

(10)
 

(10)
 

1 (10%)
 

1 (10%)
 

(10)
 

1 (10%)
 

VEHICLE
 CONTROL
 

(10)
 

2 (20%)
 

(10)
 

1 (10%)
 

(10)
 

(10)
 

3 (30%)
 

(10)
 

(10)
 

4 (40%)
 

(10)
 

(10)
 

1 (10%)
 

(10)
 

1 (10%)
 

LOW DOSE
 

(28)
 

1 (4%)
 

1 (U%)
 

1 (*•%)
 

(28)
 

(29)
 

(29)
 

5 (17%)
 

(29)
 

3 (10%)
 

(29)
 

2 (7%)
 

(29)
 

(29)
 

7 (2U%)
 

5 (17%)
 

1 (3%)
 

(29)
 

HIGH DOSE
 

(30)
 

(30)
 

1 (3%)
 

(30)
 

1 (3%)
 

(30)
 

3 (10%)
 

(30)
 

2 (7%)
 

(30)
 

<• (13%)
 

(30)
 

1 (3%)
 

1 (3%)
 

(30)
 

1 (3%)
 

2 (7%)
 

(30)
 

* KUHBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

• NOMEEB OF ANIMALS NECBOPSIED
 

84
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

SFECIAL SENSE ORGANS 

*EYE/CCFNEA 

INFLAMMATION, CHRONIC 

(10) (10) (29) (30) 

1 (3*) 

MUSCULCSKEIFTAL

NCNE 

 SYSTEM 

BCEY CAVITIES 

NCNE 

All CTHEF SYSTEMS 

ADIPOSE TISSUE INFLAMMATION, CHFONIC 

hECRCSIS, FAT 

SPECIAL fCRFFOLOGY SUMMARY 

NO LESION REPORTED AUTCLYSIS/NC NECFCFSY 

.i.NUMEER OF AMMALS WITH TISSUE EXAMINED * NUMEER OF ANIMALS NECROPSIED MICROSCOPICALLY 

85
 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE ADMINISTERED PHENESTERIN BY GAVAGE
 

87
 

TABLE D1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED PHENESTERIN BY GAVAGE (CONTROL GROUPS) 

MID & HIGH DOSE 

UNTREATED 

CONTROL 

LOW DOSE 

UNTREATED 

CONTROL 

MID & HIGH DOSE 

VEHICLE 

CONTROL 

LOW DOSE 

VEHICLE 

CONTROL 

ANIHALS INITIALLY IN STUDY 

ANIMAIS NECBCESIEC 

ANIHALS EXAMINED HISTOFATHOLOGICALLY 

15 

15 

15 

20 

20 

20 

15 

14 

14 

20 

16 

16 

INTEGUMENTARY SYSTEM 

NONE 

RESFIFATCRY SYSTEM 

#TRACHEA 

INFLAMMATION, SUPPURATIVE 

INFLAMMATION, CHRONIC 

INFLAMMATION, CHRONIC SUFPURATIV 

(15) (19) 

4 

1 

1 

(21%) 

(5%) 

(5%) 

(14) (16) 

3 (19%) 

#LUNG/EFCNCHICLF 

HYPERPLASIA, PLASMA CEIL 

(15) (20) 

1 (5%) 

(14) (16) 

2 (13%) 

#IUNG 

INFLAMMATION, INTERSTITIAL 

ERCNCHCFSEUMCNIA SUPPURATIVE 

ERCNCHCEKEUMONIA CHRONIC SUPPURA 

(15) (20) 

10 

5 

(50%) 

(25%) 

(14) 

2 (14%) 

(16) 

10 (63%) 

HFfATCFOIETIC SYSTEM 

#ECKE MARROW 

ATROFHY, NOS 

(14) (20) d<0 

1 (7%) 

(16) 

#SPIEIN 

HYPERPLASIA, LYMPHOID 

HEMATOPOIESIS 

(15) 

1 (7%) 

3 (20%) 

(19) (14) 

3 (21%) 

(16) 

#LYPFH NODE 

HYPERPLASIA, LYMPHOID 

(6) (18) (3) (16) 

1 (6%) 

«KESENTFEIC L. NODE 

CONGESTION, NCS 

INFLAMMATICN^ACUTE^CHRONIC 

(6) (18) 

„

(3) 

1 (33%) 

 1.J331L 

(16) 

# KUPEER OF ANIMALS WITH TISSUE EXAMINED

* NUF.EFR OF ANIMAIS NECROPSIED 

 MICROSCOPICALLY 

89
 
TABLE D1. MALE MICE (CONTROL GROUPS): NONNEOPLASTIC LESIONS (CONTINUED)
 

MID ft HIGH DOSE 

UNTREATED 

CONTROL 

LOW DOSE 

UNTREATED 

CONTROL 

MID ft HIGH DOSE 

VEHICLE 

CONTROL 

LOW DOSE 

VEHICLE 

CONTROL 

HYPERPLASIA, LYMPHOID 

HEMATOPOIESIS 

1 (17% ) 

1 (17% ) 

«INGUINAL LYMPH NODE 

INFLAMMATION, CHRONIC 

HYPERPLASIA, LYMFHOID 

(6) 

1 (17% ) 

1 (17%) 

(18) (3) (16) 

CIFCTJIATCRY SYSTEM 

«MYOCARDIUM 

MINERALIZATICN 

INFLAMMATION, SUPPURATIVE 

INFLAMBATICN, CHRONIC DIFFUSE 

INFLABMATICN, CHRONIC SUPPORATIV 

DEGENERATION, GRANULAR 

(15) (20) 

1 (5%) 

3 (15%) 

1 (5%) 

2 (10%) 

4 (20%) 

(14) (16) 

1 (6%) 

1 (6%) 

DIGESTIVE SYSTEM 

«IIVER 

CYTOLOGIC DEGENERATION 

HYFERELASIA, NODULAR 

HYPERPLASIA, LYMEHCID 

HEMATOEOIESIS 

(15) (19) 

6 (32%) 

3 (16%) 

(14) 

1 (7%) 

(16) 

2 (13%) 

1 (6%) 

«LIVER/CENTRILOEULAR 

NECROSIS, NOS 

(15) 

1 (7%) 

(19) 

(16) 

UE1NAEY SYSTEM 

«KIDNEY HYDRONEPHROSIS 

INFLAMBATICN, 

INFLAMMATION, 

CHRONIC 

CHFONIC FOCAL 

(15) 

2 (13%) 

(19) 

1 (5%) 

(16) 

1 (6%) 

•KIENEY/MEEULLA 

ATROPHY, NOS 

(15) 

1 (7%) 

(19) (14) (16) 

«URINARY ELADDER 

INFLAMMATION, CHECNIC 

(15) (19) 

1 (7%) 

(16) 

ENECCFINE SYSTEM 

«EANCEEATIC ISLETS 

U i a P^A S I ^ Ε F fus E. 

(15) (18) 

1 

(14) (16) 

# KOMEER OF ANIMALS WITH TISSUE EXAMINED

* NUHEER OF ANIMALS NECROPSIED 

 MICROSCOPICALLY 

90
 
TABLE D1. MALE MICE (CONTROL GROUPS): NONNEOPLASTIC LESIONS (CONTINUED)

 

MID ft HIGH DOSE 

UNTREATED 

CONTROL 

LOW DOSE 

UNTREATED 

CONTROL 

MID ft HIGH DOSE 

VEHICLE 

CONTROL 

LOW DOSE 

VEHICLE 

CONTROL 

REJFFCEOCTHE SYSTEB 

NONE 

NEFVCOS SYSTEB 

kCNE 

SPECIAL SE*SE CFGANS 

•FIEDIE EAR 

INFLABMATION, CHRONIC SOPPORATIV 

(15) (20) 

1 (5%) 

(16) 

2 (13%) 

BOSCOLCSKEIETAL SYSTIB 

•JCINT 

EXOSTOSIS 

(15) (20)

1 <5X) 

 (14) (16 ) 

ECEY CAVITIES 

I.CNE 

All C1HEF SYSTEMS 

•BOITIPIE CRGANS 

HYPERPLASIA, LYBEHCID 

(15) (20) ) 

1 (7%) 

(16) 

SPECIAL rOFE&OLOGY SOBBARY 

NO LESION REPORTED ACCIDENTAL DEATH AUTCLYSIS/NO NECROPSY 

* NOBEER OF ANIBALS WITH TISSUE EXAMINED

* RUBEEB OF ANIBALS NECROPSIED 

 BICEOSCOPICALLY 

91
 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED PHENESTERIN BY GAVAGE (CONTROL GROUPS) 

HIGH DOSE
 

35
 

2
 

25
 

25
 

(25)
 

(25)
 

(25)
 

(25)
 

(25)
 

1 {*%)
 

1 (4%)
 

(24)
 

2 (8%)
 

1^121%!
 

ANIFALS INITIAILY IN STUDY
 

ANIMALS MISSING
 

ANIMALS NECRCPSIEE
 ANIMALS EXAMINED HISTOFATHCLOGICALLY
 

INTEGUMENTARY. SYSTEM
 

•SKIN
 

INFLAMMATION, FIBRINOUS
 

ULCER, CHRONIC
 

BYFEFKEFATCSIS
 

ACANTHOSIS
 

•SUBCOT TISSUE
 

HEMORRHAGE
 INFLAMMATION, CHRONIC SUPPURATIV
 

RESFIFATCR* SYSTEM
 

«TRACHEA
 

INFLAMMATION, SUPPURATIVE
 INFLAMMATION, CHRONIC SUPPURATIV
 

#LUNG/EFCNCHIOLE
 

FIBROSIS
 

HYPERPLASIA, LYMPHOID
 

«LUNG
 

INFLAMMATION, INTERSTITIAL
 PNEUMONIA, LIPID
 ERCNCHCEKEUHONIA SUPPURATIVE
 ERCNCHOFNEUMONIA CHRONIC SUPEURA
 CHCLESTERCL DEEOSIT
 

HEMATOPOIETIC SYSTEM
 

#EC M MARROW
 

CONGESTION, NOS
 

MB9FHY, NCS
 

LOW DOSE
 

uo
 

ao
 

40
 

(40)
 

1 (3%)
 

1 (3%)
 

(40)
 

3 (8*)
 

(39)
 

3 (8%)
 

1 (3%)
 

(40)
 

(40)
 

10 (25%)
 

1 (3%)
 

(39)
 

MID DOSE
 

35
 

29
 

29
 

(29)
 

1 (3%)
 

1 (3%)
 

(29)
 

1 (3%)
 

(29)
 

(29)
 

1 (3%)
 

1 (3X)
 

(29)
 

2 (7X)
 

1 (3X)
 

(28)
 

« NOMEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMEER OF ANIMALS NECROPSIED
 

92
 
TABLE D2. MALE MICE (TREATED GROUPS): NONNEOPLASTIC LESIONS (CONTINUED)

 

LOW DOSE MID DOSE HIGH DOSE
 

HYPERPLASIA, NEUTROPHILIC 1 (<•%)
 

«SFLEEN (10) (29) (25)
 

HYPERPLASIA, NEUTROPHILIC 1 (4%)
 

HEMATOPOIESIS 5 (17%) 5 (20%)
 

#KESENTERIC L. NODE (35) (")
 

INFLAMMATION, SUPPURATIVE 1 (3%)
 

HYPERPLASIA, LYMPHOID 2 (50%)
 

CIBCD1ATCBY SYSTEM
 

#HYCCARDIUM (40) (29) (25)
 

INFLAMMATION, SUPPURATIVE 1 (3%)
 

INFLAMMATION, CHRONIC SUPPURATIV 1 (3%)
 

KECRCSIS, COAGULATIVE 1 (3%)
 

DIGESTIVE SYSTEM
 

«LIVER (39) (29) (25)
 

HEMORRHAGE 1 (3%)
 

KECRCSIS, NOS 2 (7%)
 

NECROSIS, COAGULATIVE 4 (10%)
 

ANGIECTASIS 1 (3%) 1 (3%)
 

HEMATOPOIESIS 1 (<»%)
 

#LIVER/CENTFILOBULAR (39) (29) (25)
 

NECROSIS, NOS 1 (3%)
 

KECRCSIS, COAGULATIVE 1 (3%)
 

URINABY SYSTEM
 

«KIDNEY (40) (29) (25)
 

INFLAMMATION, CHRONIC 1 (3%)
 

ENCCCBINE SYSTEM
 

hCKE
 

REEFCEUCTIVE SYSTEM
 

# 8UMEEP OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUHEER OF ANIMALS NECROPSIED
 

93
 
TABLE D2. MALE MICE (TREATED GROUPS): NONNEOPLASTIC LESIONS (CONTINUED) 

LOW DOSE MID DOSE HIGH DOSE 

NFFVCUS SYSTEM
 

NONE
 

SEECIA L SENS E CRGAN S 

NClvE 

MUSCLICSKELFTAL SYSTEM
 

NCNE
 

ECLY CAVITIES
 

•EEEITONEUM	 (40) (29) (25)
 

INFLAMPATICN, HEMORRHAGIC 1
 

INFLAMMATION, CHFCNIC SUFFURATIV 2 (5%)

^ECFCSIS, FAT 1 (3%)

 

*ELEURA	 (40 (40)) (29) (25)


1 (3%) INFLAMMATION, CHRONIC SUPPURATIV
 

ALL CTFEF SYSTEMS
 

bfhl
 

SPECIAL PCBEhCLCGY SUMMARY
 

NC LESION FEEORTED	 U 2
 

AMMAL MISSING/NC NECROPSY	 2
 

ACCIDENTAL DEATH	 2
 

NC NECROESY PERFORMED	 1 1
 

ADT0/NECF0ESY/HIS1C EERF	 1
 

AUTCLYSIS/NO NECROPSY	 5 5
 

# MJFEEK CF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* i^DFEER CF ANIMALS KECRCPSIED 

94 
TABLE D3.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED PHENESTERIN BY GAVAGE (CONTROL GROUPS) 

MID & HIGH DOSE 

UNTREATED 

CONTROL 

LOW D06E 

UNTREATED 

CONTROL 

MID & HIGH DOSE 

VEHICLE 

CONTROL 

LOW DOSE 

VEHICLE 

CONTROL 

ANIMALS INITIALLY IN STUDY 

ANIMAIS NECECESIED 

ANIMALS EXAMINED HISTO-PATHOLOGICALLY 

15 

15 

15 

20 

20 

20 

15 

15 

15 

20 

20 

20 

INTEGUMENTAEY SYSTEM 

*SKIN 

ULCER, CHRONIC 

(15) (20) 

1 (5%) 

(15) (20) 

RESFIEATCRY SYSTEM 

#TEACHEA 

INFLAMMATION, SUPPURATIVE 

HYPERPLASIA, PLASMA CELL 

(15) (20) 

3 (15%) 

1 (5%) 

(15) (2C) 

2 (ir%) 

1 (5T) 

#LUNG/ERCNCHUS 

INFLAMMATION, SUPPURATIVE 

(15) (20) 

1 (5%) 

(15) (20) 

#LUNG/EECNCHIOLE 

HYPERPLASIA, PLASMA CELL 

HYPERPLASIA, LYMPHOID 

(15) 

1 (7%) 

(20) 

9 (45%) 

(15) 

1 (7%) 

(21) 

3 (15%) 

2 (10%) 

#LUNG 

INFLAMMATICN, INTERSTITIAL 

FKEUMONIA, ASPIRATION 

ERCNCHCEi^EUMONIA SUPPURATIVE 

ERCNCHCENEUMCNIA CHRONIC SUPEURA 

HYPERPLASIA, PLASMA CELL 

(15) 

1 (7%) 

(20) 

1 (5%) 

15 (75%) 

(15) 

1 (7%) 

1 (7%) 

(20) 

1 (5%) 

9 (45%) 

1 (5%) 

1 (5%) 

HIPATCFCIETIC SYSTEM 

#EChE MARECW 

ATROPHY, NOS 

(13) 

3 (23%) 

(20) (14) 

3 (21%) 

(18) 

#SPLEEN 

HYPERPLASIA, LYMPHOID 

HEMATOPOIESIS 

(15) 

1 (7%) 

(20) (15) 

1 (7%) 

(19) 

ClfCEIAICEK SYSTIM 

# &UCEER CF ANIMALS WITH TISSUE EXAMINED

* NUMEER CF ANIMALS NECROPSIED 

 MICROSCOPICALLY 

95
 
TABLE D3. FEMALE MICE (CONTROL GROUPS): NONNEOPLASTIC LESIONS (CONTINUED)

 

DIGESTIVE SYSTEM
 

#LIVEE
 

HEMORRHAGE
 

hECRCSIS, CCAGULATIVE
 

HYPERPLASIA, LYMPHOID
 

OFINAFY SYSTEM
 

• #KIENFY
 

HYPERPLASIA, LYMPHOID
 

ENECCFINE SYSTEM
 

•EITUITARY
 

HE110PFHAGE 

«ADRENAL 

HEMORRHAGE
 

FEFFCEUCTIVE SYSTEM
 

«tTERUS/ENECMETRIUM
 

INFLAMMATION, SUPPURATIVE
 

HYPERPLASIA, CYSTIC
 

«CVAEY
 

CYST, NOS
 

NEFVCCS SYSTEM
 

#EFAIN
 

EPIDERMAL INCLUSION CYST
 

SPECIAL SENSE OFGANS
 

NCNE
 

MUSCULOSKELETA L SYSTEM
 

# NUHEER OF ANIMALS WITH TISSUE


• KUBEEB OF ANIMALS NECROPSIED
 

MID & HIGH DOSE LOW DOSE
 

UNTREATED UNTREATED
 

CONTROL CONTROL
 

(15) (19)
 

1 (5%)
 

(15) (20)
 

(14) (17)
 

(15) (20)
 

(15) (20)
 

1 (5%)
 

13 (87%) 1 (5%)
 

(15) (20)
 

(15)	 (20)
 

1 (5%)
 

 EXAMINED MICROSCOPICALLY
 

MID & HIGH DOSE LOW DOSE
 

VEHICLE VEHICLE
 

CONTROL CONTROL
 

(15)	 (20)
 

1 <5X)
 

1 (5S)
 

1 (5*)
 

(15)	 (19)
 

1 (7%)
 

(15)	 (14)
 

1 (7%)
 

(15)	 (20)
 

1 (5%)
 

(15) (20)
 

14 (93%)
 

(15)	 (20)
 

1 (7%)
 

(15) (19)
 

96
 
TABLE D3. FEMALE MICE (CONTROL GROUPS): NONNEOPLASTIC LESIONS (CONTINUED)
 

MID & HIGH DOSE 

UNTREATED 

CONTROL 

LOW DOSE 

UNTREATED 

CONTROL 

MID & HIGH DOSE 

VEHICLE 

CONTROL 

LOW DOSE*" 

VEHICLE 

CONTROL 

ΕΟΓ CAVITIES 

•FERITCNEUM 

INFLAMMATICN, 

INFLAMMATION, 

KECRCSIS, FAT 

CHRONIC 

CHEONIC DIFFUSE 

(15 ) 

1 (7%) 

(20) 

1 (5%) 

(15) (20) 

1 (5%) 

1 (5%) 

All CTHEF SYSTEMS 

•MULTIPLE CEGANS 

HYPERPLASIA, PLASMA CELL 

HYPERPLASIA, RETICULUM CELL 

HYPERPLASIA, LYMPHOID 

(15) (20) 

1 (5%) 

1 (5%) 

1 (5%) 

(15) (20) 

AEIPCSE TISSUE 

INFLAMMATICN, CHRONIC FOCAL 

SPECIAL FORFHOLOG* SUMMARY 

NC LESION REPORTED 

NUMEER OF ANIMALS SITH TISSUE EXAMINED

KUMEER OF ANIMALS NECROPSIED 

 MICFOSCOPICALLY 

97
 
TABLE 04.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED PHENESTERIN BY GAVAGE (TREATED GROUPS) 

LOW	 DOSE MID DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY ao 35 35
 

ANIMALS NECECPSIED 38 27 32
 

ANIMALS EXAMINED HISTOFATHOLOGICALLY 38 27 31
 

IH1EGUMENTAEY SYS1EM
 

•SUECUT TISSUE	 (38) (27) (32)
 

HEMATOMA, NOS 1 (3%)
 

RESEIEATCBY SYSTEM
 

#TB*CHEA (38) (27) (30)
 

IKFLAMMATICN, SUPPURATIVE 3 (8%)
 PLASMA-CELL INFILTFATE 1 (3%)
 

#IUKG/EBONCHIOLE (38) (27) (31)
 

HYPERPLASIA, PLASMA CELL 3 (8%)
 

#LUNG (38) (27) (31)
 

CONGESTION, NOS 1 (<*%)
 HEMORRHAGE 1 <<•%)
 INFLAMMATION, INTERSTITIAL 1 <<*%)
 FKEUMCNIA, LIFID 1 (3%)
 ERCNCHCFNEUMONIA SUPPURATIVE 5 (13%)
 

FKEUMONIA INTERSTITIAL CHRONIC 1 (3%)
 ERCNCHCENEUMONIA CHRONIC SUFEDRA 8 (30%)
 

CHOLESTEROL DEPOSIT 1 (3%)
 ALVEOLAR MACROPHAGES 1 (4X)
 

HEMATOPOIETIC SYSTEM 

#ECKE MAREOH (38) (27) (28) 

CONGFSTION, NOS 2 (7%) 

ATROPHY, NOS 6 (22X) 14 (50%) 

HYPERPLASIA, NEUTROPHILIC 2 (7*) 

#SFLEEN (38) (27) (30) 

„ CQJJ:TJA.£TURE ^ _ 

# KUMEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

98
 
TABLE D4. FEMALE MICE (TREATED GROUPS): NONNEOPLASTIC LESIONS (CONTINUED)

 

LOW DOSE MID DOSE HIGH DOSE
 

HYPERPLASIA, NEUTROPHILIC 2 (7%)
 

HEMATOPOIESIS 6 (22%) 3 (10%)
 

«SPLENIC FOLLICLES (38) (27) (30)
 

ATROPHY, NOS 1 (3%)
 

MESENTERIC L. NODE (36) (4) (2)
 

HYPERPLASIA, NEUTROPHILIC 1 (25%)
 

HYPERPLASIA, LYMPHOID 1 (3%)
 

CIFCCIATCEY SYSTEM
 

KCNE
 

DIGESTIVE SYSTEM
 

(37) (27) (30) 

FEMORRHAGE 1 (3%) 

INFLAMMATION, CHRONIC SUPPURATIV 1 (3%) 

ItECRCSIS, NOS 1 (3%) 

I^ECRCSIS, MIDZCNAL 1 (3%) 

CYTCLCGIC DEGENERATION 1 (3%) 

HYPERPLASIA, NODULAR 1 (3%) 

ANGIFCTASIS 1 (3%) 

tLIVER/CENTBILOEULAR (37) (27) (30)
 

NECROSIS, NOS 1 (3%)
 

1>ECRCSIS, COAGULATIVE 3 (8%)
 

URINARY SYSTEM
 

#KIENEY (38) (27) (30)
 

INFLAMMATION, CHRONIC 1 (3%)
 

AMYLOIDOSIS 1 (3%)
 

ENEOCRINE SYSTEM
 

REFFCEUCTIVE SYSTEM
 

iCTEBUS (38) (27) (30)
 

« MJMEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMEER OF ANIMALS NECROPSIED
 

99
 
TABLE D4. FEMALE MICE (TREATED GROUPS): NONNEOPLASTIC LESIONS (CONTINUED) 

LOW DOSE MID DOSE HIGH DOSE
 

#CVARY (37) (27) (30)
 

CYST, NOS 1 (3%)
 

CCNGESTICN, NCS 1 (3%)
 

HEMORRHAGE 9 (24%) 2 (7%)
 

IhFLAFFATICN, SUFFURATIVE 1 (4%)
 

NEFVCtS SYSTEM
 

SFiCIAI SlhSl CRGANS
 

•EYF/COFNEA	 (38) (27) (32)
 

INFLAMMATION, SUPPURATIVE 1 (3%)
 

MIECUICSKEIFTAL SYSTiM
 

ECCY CAVITIFS
 

• EEFITCNEUM	 (38) (27) (32)
 

PEMCFRHAGE 1 (3%)
 

INFLAMMATION, SUFEUFATIVE 1 (3%)
 

INFLAMFATICNr CHRONIC 1 (3%)
 

!>ECRCSIS, FAT 1 (3%)
 

ALL CTPiE SYSTEMS
 

ACIECSF TISSUE
 INFLAMMATION, CBRONIC 1
 

INFLAMMATION, CHRONIC SUPPURATIV 1
 

SPECIAL PCFEtOLOGY SUMMARY
 

HC LESION FEEORTED 3
 

ACCIDENTAL DEATH 2 1
 

^ECRCESY^EERF^NO.UISTO^PEJ^RUIC . J,
 

# ^UPBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* hUMEFR OF ANIMALS NECROFSIED
 

100
 
TABLE D4. FEMALE MICE (TREATED GROUPS): NONNEOPLASTIC LESIONS (CONTINUED) 

LOW DOSE MID DOSE HIGH DOSE
 

NC NECFOFSY FERFORMED 1
 

AUTOIYSIS/NC NECFCFSY 1
 

# NUMEER OF ANIMALS WITH TISSUE EXAMINED MICEOSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

101
 

APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

RATS ADMINISTERED PHENESTERIN BY GAVAGE
 

103
 

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

Administered Phenesterin by Gavagea 

Topography: Morphology 

Pooled 

Control 

Vehicle 

Control 

Low 

Dose 

High 

Dose 

Hematopoietic System: 

Granulocytic Leukemia*3 0/18 (0) 0/9 (0) 0/32 (0) 2/30 (7) 

 Value0 > d N.S. N.S. N.S. N.S. 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

Infinite 

0.186 

Infinite 

Ο 

Ui 

Relative Risk (Vehicle Control)f 

Lower Limit 

Upper Limit 

Infinite 

0.100 

Infinite 

Weeks to First Observed Tumor 37 

Hematopoietic System: Malignant 

Lymphoma or Lymphocytic 

Leukemia*5 0/18 (0) 0/9 (0) 2/32 (6) 3/30 (10) 

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

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

Infinite 

0.174 

Infinite 

Infinite 

0.379 

Infinite 

Relative Risk (Vehicle Control)f 

Lower Limit 

Upper Limit 

Infinite 

0.093 

Infinite 

Infinite 

0.204 

Infinite 

Weeks to First Observed Tumor — 75 50 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

(continued) 

Topography: Morphology 

Hematopoietic System: Lymphoma 

or Leukemia*5 

 Values0 > d 

Relative Risk (Pooled Control)

f 

Lower Limit 

Upper Limit 

H*

σ\

Relative Risk (Vehicle Control)* 

 Lower Limit 

 Upper Limit 

Weeks to First Observed Tumor 

Pituitary: Chromophobe Adenoma*5 

 Valuesc > d 

Relative Risk (Pooled Control)* 

Lower Limit 

Upper Limit 

Relative Risk (Vehicle Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Administered Phenesterin by Gavagea
 

Pooled Vehicle
 

Control Control
 

0/18 (0) 0/9 (0)
 

 = 0.037 N.S.
 

0/18 (5) 0/9 (0)
 

N.S. N.S.
 

——
 

Low
 

Dose
 

2/32 (6)
 

N.S.
 

Infinite
 

0.174
 

Infinite
 

Infinite
 

0.093
 

Infinite
 

75
 

2/29 (7)
 

N.S.
 

Infinite
 

0.192
 

Infinite
 

Infinite
 

0.103
 

Infinite
 

73
 

High
 

Dose
 

5/30 (17)
 

N.S.
 

Infinite
 

0.798
 

Infinite
 

Infinite
 

0.430
 

Infinite
 

0/29 (0)
 

N.S.
 

—
 

—
 

—
 

——
 

—
 

37 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats 

Administered Phenesterin by Gavagea 

(continued) 

Topography: Morphology
 

Pituitary: Chromophobe Adenoma
 

or Carcinoma*5
 

 Values0>d
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Ear Canal: Squamous-cell
 

Papillomab
 

 Values0>d
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Control
 

1/18 (6)
 

N.S.
 

0/18 (0)
 

N.S·
 

——
 

Low
 

Dose
 

3/29 (10)
 

N.S.
 

1.862
 

0.167
 

94.142
 

0.931
 

0.093
 

47.120
 

73
 

2/32 (6)
 

N.S.
 

Infinite
 

0.174
 

Infinite
 

Infinite
 

0.093
 

Infinite
 

49
 

High
 

Dose
 

0/29 (0)
 

N.S.
 

0.000
 

0.000
 

11.440
 

0.000
 

0.000
 

5.736
 

—
 

0/30 (0)
 

N.S.
 

——
 

Vehicle
 

Control
 

1/9 (11)
 

N.S.
 

85
 

0/9 (0)
 

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

Administered Phenesterin by Gavagea
 

(continued)
 

Pooled Vehicle Low High
 

Topography; Morphology Control Control Dose Dose
 

A ll Sites: Sarcomab 1/18 (6) 0/9 (0) 7/32 (22) 4/30 (13)
 

 Values c> d N.S. N.S. N.S . N.S.
 

Relative Risk (Pooled Control) f 3.938 2.400
 

Lower Limit 0.579 0.268
 

Upper Limit 170.622 113.825
 

Relativ e Risk (Vehicle Control) f Infinite Infinite
 

Lower Limit 0.621 0.315
 

Upper Limit Infinite Infinite
 ο
 

oo
 

Weeks to First Observed Tumor 50 41
 

a

Dosed groups received 5 or 10 mg/kg.
 

"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 Ρ < 0.05, otherwise, not significant (N.S.) is indicated. Beneath the
 

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

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


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

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

Administered Phenesterin by Gavagea
 

(continued)
 

^A negative trend (N) indicates a lower incidence in a dosed group than in a control group·
 

e

The probability level for departure from linear trend is given when Ρ < 0·05 for any comparison·
 

95 percent confidence interval of the relative risk between each dosed group and the
 

specified control group·
 

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

Administered

 opography: Morphology 

Pooled 

Control 

Hematopoietic System: 

Granulocytic Leukemia^ 0/18 (0) 

 Valuesc > d N.S. 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

Relative Risk (Vehicle Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Hematopoietic System: Lymphoma 

or Leukemia*5 0/18 (0) 

 Values0 > d N.S. 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

Relative Risk (Vehicle Control)^ 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

 Phenesterin by Gavagea 

Vehicle 

Control 

Low 

Dose 

High 

Dose 

0/10 (0) 2/29 (7) 1/30 (3) 

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

Infinite 

0.192 

Infinite 

Infinite 

0.033 

Infinite 

Infinite 

0.113 

Infinite 

Infinite 

0.019 

Infinite 

— 46 50 

0/10 (0) 2/29 (7) 2/30 (7) 

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

Infinite 

0.192 

Infinite 

Infinite 

0.186 

Infinite 

Infinite 

0.113 

Infinite 

Infinite 

0.109 

Infinite 

— 46 50 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Phenesterin by Gavagea
 

(continued) 

Topography: Morphology 

Pituitary: Chromophobe Adenomab 

 Values c , d 

Relative Risk (Pooled Control)^ 

Lower Limit 

Upper Limit 

Relative Risk (Vehicle Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Mammary Gland: Adenocarcinoma, NOSb 

 Valuesc,d 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

Relative Risk (Vehicle Control)f 

Lower Limit 

Upper Limit 

Pooled Vehicle 

Control Control 

10/16 (63) 6/10 (60) 

 < 0.00l(N) < 0.00l(N) 

64 

1/18 (6) 1/10 (10) 

 = 0.019 N.S. 

Low 

Dose 

High 

Dose 

4/27 (15) 0/30 (0) 

 = 0.012*(N) 

 = 0.002**(N) 

 < 0.001*(N) 

 < 0.001**(N) 

0.237 

0.076 

0.673 

0.000 

0.000 

0.169 

0.247 

0.083 

0.843 

0.000 

0.000 

0.194 

71 — 

12/29 (41) 12/30 (40) 

 = 0.007** = 0.009** 

7.448 7.200 

1.284 1.239 

300.235 291.162 

4.138 4.000 

0.780 0.753 

156.921 161.888 

Weeks to First Observed Tumor 84 26 33 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Phenesterin by Gavagea 

(continued) 

Pooled Vehicle Low High 

Topography: Morphology Control Control Dose Dose 

Mammary Gland: Fibroadenoma^ 8/18 (44) 4/10 (40) 21/29 (72) 11/30 (37) 

 Values0 » d N.S. N.S. N.S. N.S. 

Departure from Linear Trende 

 = 0.008 = 0.009 

Relative Risk (Pooled Control)f 1.629 0.825 

Lower Limit 0.923 0.392 

Upper Limit 3.090 1.942 

Relative Risk (Vehicle Control)f 1.810 0.917 

Lower Limit 0.882 0.387 

Upper Limit 5.317 3.277 

Weeks to First Observed Tumor 64 60 42 

Uterus: Endometrial Stromal 

Polypb 1/17 (6) 1/10 (10) 5/29 (17) 2/30 (7) 

 Valuesc > d N.S. N.S. N.S. N.S. 

Relative Risk (Pooled Control)f 2.931 1.133 

Lower Limit 0.375 0.065 

Upper Limit 133.079 64.588 

Relative Risk (Vehicle Control)f 1.724 0.667 

Lower Limit 0.240 0.041 

Upper Limit 78.324 38.024 

Weeks to First Observed Tumor 85 67 69 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Phenesterin by Gavagea
 

(continued) 

Pooled Vehicle Low High 

Topography: Morphology Control Control Dose Dose 

All Sites: Sarcomab 0/18 (0) 0/10 (0) 2/29 (7) 4/30 (13)
 

 Valuesc>d N.S. N.S. N.S. N.S.
 

Relative Risk (Pooled Control)f Infinite Infinite
 

Lower Limit 0.192 0.585
 

Upper Limit Infinite Infinite
 

Relative Risk (Vehicle Control)f Infinite Infinite
 

Lower Limit 0.113 0.345
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor — 60 34
 

a

Dosed groups received 5 or 10 mg/kg.
 

^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 Ρ < 0.05, otherwise, not significant (N.S.) is indicated. Beneath the
 

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

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


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

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

Administered Phenesterin by Gavagea
 

(continued)
 

^A negative trend (N) indicates a lower incidence in a dosed group than in a control group·
 

e

The probability level for departure from linear trend is given when Ρ < 0·05 for any comparison·
 

95 percent confidence interval of the relative risk between each dosed group and the
 

specified control group·
 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

MICE ADMINISTERED PHENESTERIN BY GAVAGE
 

115
 

Table Fl. Analyses of the Incidence of Primary Tumors in Low-Dose
 

Male Mice Administered Phenesterin by Gavagea
 

Low-Dose 

Vehicle Low 

Topography; Morphology Control Dose 

Lung: Alveolar/Bronchiolar Carcinoma 0/16 (0) 14/40 (35) 

 Values0>d  = 0·004 

Relative Risk (Low-Dose Control) e Infinite 

Lower Limit 1.857 

Upper Limit Infinite 

Weeks to First Observed Tumor 68 

Lung: Alveolar/Bronchiolar Adenoma 

or Carcinoma^ 2/16 (13) 18/40 (45) 

 Valuesc>d Ρ = 0. 020 

Relative Risk (Low-Dose Control)e 3. .600 

Lower Limit 1. .037 

Upper Limit 29. ,336 

Weeks to First Observed Tumor 77 68 
Table Fl. Analyses of the Incidence of Primary Tumors in Low-Dose 

Male Mice Administered Phenesterin by Gavagea 

(continued) 

Low-Dose 

Vehicle Low 

Topography: Morphology Control Dose 

Hematopoietic System: Malignant Lymphoma 

or Lymphocytic Leukemia^ 1/16 (6) 10/40 (25) 

 Valuesc>d N.S. 

Relative Risk (Low-Dose Control)e 4. .000 

Lower Limit 0, .662 

Upper Limit 168, .059 

Weeks to First Observed Tumor 32 49
 00 

Hematopoietic System: Lymphoma 

or Leukemia^ 1/16 (6) 11/40 (28) 

 Valuesc>d N.S. 

Relative Risk (Low-Dose Control)e 4. .400 

Lower Limit 0. ,745 

Upper Limit 183. ,149 

Weeks to First Observed Tumor 32 49
 
Table Fl· Analyses of the Incidence of Primary Tumors in Low-Dose 

Male Mice Administered Phenesterin by Gavagea 

(continued) 

Low-Dose
 

Vehicle Low
 

Topography: Morphology Control Dose
 

Liver: Hepatocellular Adenomab 3/16 (19) 7/39 (18)
 

 Valuesc , d N.S.
 

Relative Risk (Low-Dose Control)e 0.957
 

Lower Limit 0.261
 

Upper Limit 5.237
 

Weeks to First Observed Tumor 56 76
 

Liver: Hepatocellular Carcinomab 0/16 (0) 3/39 (8)
 

 Valuesc , d N.S.
 

Relative Risk (Low-Dose Control)e Infinite
 

Lower Limit 0.261
 

Upper Limit Infinite
 

Weeks to First Observed Tumor — 73
 
Table Fl. Analyses of the Incidence of Primary Tumors in Low-Dose 

Male Mice Administered Phenesterin by Gavagea 

(continued) 

Low-Dose 

Vehicle Low 

Topography: Morphology Control Dose 

Liver : Hepatocellular Adenoma 

or Carcinoma" 3/16 (19) 10/39 (26) 

 Valuesc>d N.S. 

Relative Risk (Low-Dose Control) e 1.368 

Lower Limit 0.425 

Upper Limit 7.013 

Weeks to First Observed Tumor 56 73 

Myocardium: Sarcoma, NOS^ 0/16 (0) 5/40 (13) 

 Valuesc>d N.S. 

Relative Risk (Low-Dose Control) e Infinite 

Lower Limit 0.535 

Upper Limit Infinite 

Weeks to First Observed Tumor —— 78 
Table Fl· Analyses of the Incidence of Primary Tumors in Low-Dose
 

Male Mice Administered Phenesterin by Gavagea
 

(continued) 

Low-Dose 

Vehicle Low 

Topography: Morphology Control Dose 

All Sites: Hemangiosarcoma^ 0/20 (0) 6/40 (15) 

 Values c, d N.S. 

Relative Risk (Low-Dose Control) e Infinite 

Lower Limit 0.835 

Upper Limit Infinite 

Weeks to First Observed Tumor — 78 

a

Dosed group received 7 mg/kg·
 

b

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

c

Beneath the incidence of tumors in the dosed group is the probability level for the Fisher
 

exact test for the comparison of the dosed group with the vehicle-control group when Ρ < 0·05;
 

otherwise, not significant (N.S·) is indicated.
 

d

A negative trend (N) indicates a lower incidence in the dosed group than in the control group.
 

e

The 9 5 percent confidence interval of the relative risk between the dosed group and the
 

control group.
 
Table F2. Analyses of the Incidence of Primary Tumors in Mid and High-Dose
 

Male Mice Administered Phenesterin by Gavagea 

Mid and Mid and 

High-Dose High-Dose 

Pooled Vehicle Mid High 

Topography: Morphology Control Control Dose Dose 

Lung: Alveolar/Bronchiolar 

Adenoma*5 2/28 (7) 0/14 (0) 6/29 (21) 2/25 (8) 

 Valuesc > d N.S. N.S. N.S. N.S. 

Departure from Linear Trende = 0.038 

Relative Risk (Pooled Control)f 2.897 1.120 

Lower Limit 0.575 0.087 

Upper Limit 27.279 14.392 

Relative Risk (Vehicle Control)f Infinite Infinite 

Lower Limit 0.833 0.177 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor — 71 73 
Table F2· Analyses of the Incidence of Primary Tumors in Mid and High-Dose
 

Male Mice Administered Phenesterin by Gavagea
 

(continued)
 

Topography: Morphology
 

Hematopoietic System:
 

Lymphoma or Leukemia^
 

 Values0>d
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mid and Mid and 

High-Dose High-Dose 

Pooled Vehicle Mid 

Control Control Dose 

0/28 (0) 0/14 (0) 9/29 (31) 

 < 0.001 - 0.005 ­ 0.018* 

 - 0.001** 

Infinite 

2.616 

Infinite 

Infinite 

1.372 

Infinite 

— 32 

High
 

Dose
 

11/25 (44)
 

 = 0.003*
 

 < 0.001**
 

Infinite
 

3.859
 

Infinite
 

Infinite
 

2.023
 

Infinite
 

49
 
Table F2. Analyses of the Incidence of Primary Tumors in Mid and High-Dose 

Male Mice Administered Phenesterin by

(continued) 

Mid and Mid and
 

High-Dose High-Dose
 

Pooled Vehicle
 

Topography: Morphology Control Control
 

Myocardium: Sarcoma, NOSb 0/28 (0) 0/14 (0)
 

 Valuec , d N.S. N.S.
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 ——
 

All Sites: Hemangiosarcomab 0/28 (0) 0/14 (0)
 

 Valuesc , d - 0.046 N.S.
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 —
 

Gavagea 

Mid
 

Dose
 

7/29 (24)
 

 - 0.048*
 

 = 0.006**
 

Infinite
 

1.928
 

Infinite
 

Infinite
 

1.012
 

Infinite
 

48
 

1/29 (3)
 

N.S.
 

Infinite
 

0.053
 

Infinite
 

Infinite
 

0.027
 

Infinite
 

74
 

High
 

Dose
 

2/25 (8)
 

N.S.
 

Infinite
 

0.339
 

Infinite
 

Infinite
 

0.177
 

Infinite
 

57
 

3/25 (12)
 

N.S.
 

Infinite
 

0.691
 

Infinite
 

Infinite
 

0.362
 

Infinite
 

66
 
Table F2. Analyses of the Incidence of Primary Tumors in Mid and High-Dose
 

Male Mice Administered Phenesterin by Gavagea
 

(continued)
 

Topography: Morphology
 

Liver: Hepatocellular Adenomab
 

 Valuesc , d
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

a
Dosed groups received 15 or 30 mg/kg.
 

Mid and
 

High-Dose
 

Pooled
 

Control
 

6/28 (21)
 

 » 0.008 (Ν)
 

Mid and 

High-Dose 

Vehicle Mid 

Control Dose 

4/14 (29) 2/29 (7) 

 = 0.005 (Ν) N.S. 

0.322 

0.034 

1.623 

0.241 

0.026 

1.498 

81 67 

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

High
 

Dose
 

0/25 (0)
 

 = 0.012* (Ν)
 

 = 0.016**(N)
 

0.000
 

0.000
 

0.677
 

0.000
 

0.000
 

0.577
 

—
 
Table F2· Analyses of the Incidence of Primary Tumors in Mid and High-Dose 

Male Mice Administered Phenesterin by Gavagea 

(continued) 

c

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


Armitage test when Ρ < 0·05, otherwise, not significant (N.S·) is indicated· Beneath the
 

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

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


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

is indicated·
 

^A negative trend (N) indicates a lower incidence in a dosed group than in a control group·
 

e

The probability level for departure from linear trend is given when Ρ < 0·05 for any comparison·
 

^The 95 percent confidence interval of the relative risk between each dosed group and the
 

specified control group·
 
Table F3· Analyses of the Incidence of Primary

Female Mice Administered Phenesterin

Low-Dose 

Vehicle 

Topography: Morphology Control 

Lung: Alveolar/Bronchiolar Carcinomab 1/20 (5) 

 Valuesc , d 

Relative Risk (Low-Dose Control)e
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 82
 

Lung: Alveolar/Bronchiolar Adenoma
 

or Carcinomab 1/20 (5)
 

 Valuesc , d
 

Relative Risk (Low-Dose Control)e
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 82
 

 Tumors in

 by Gavagea 

 Low-Dose 

Low
 

Dose
 

8/38 (21)
 

N.S.
 

4.211
 

0.638
 

180.874
 

71
 

15/38 (39)
 

 = 0.004
 

7.895
 

1.391
 

318.390
 

68
 
Table F3. Analyses of the Incidence of Primary Tumors in

Female Mice Administered Phenesterin by Gavagea 

(continued)
 

Low-Dose
 

Vehicle
 

Topography: Morphology Control 

Hematopoietic System: Lymphoma
 

or Leukemia*5 5/20 (25)
 

 Values0>d 

Relative Risk (Low-Dose Control) e
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 86 

oo 

Liver: Hepatocellular Carcinoma"3 1/20 (5) 

 Values0>d 

Relative Risk (Low-Dose Control) e
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 104 

 Low-Dose 

Low 

Dose 

12/38 (32; 

N.S. 

1.263 

0.499 

4.019 

51 

1/37 (3) 

N.S. 

0.541 

0.007 

41.317
 

54
 
__ 

Table F3. Analyses of the Incidence of Primary Tumors in Low-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

(continued) 

Low-Dose 

Vehicle Low 

Topography: Morphology Control Dose 

Liver: Hepatocellular Adenoma
 

or Carcinomab 3/20 (15) 1/37 (3)
 

 Valuesc , d N.S.
 

Relative Risk (Low-Dose Control)e 0.180
 

Lower Limit 0.004
 

Upper Limit 2.095
 

Weeks to First Observed Tumor 82 54
 

Myocardium: Sarcoma, NOSb 0/20 (0) 8/36 (22)
 

 Valuesc , d = 0.021
 

Relative Risk (Low-Dose Control)e Infinite
 

Lower Limit 1.327
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 62
 
Table F3· Analyses of the Incidence of Primary Tumors in

Female Mice Administered Phenesterin by Gavagea 

(continued) 

Low-Dose
 

Vehicle
 

Topography: Morphology Control
 

All Sites: Hemangiosarcomab 0/20 (0)
 

 Valuesc , d
 

Relative Risk (Low-Dose Control)e
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 —
 

Ο Mammary Gland: Adenocarcinoma, NOSb 0/20 (0)
 

 Valuesc , d
 

Relative Risk (Low-Dose Control)e
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

 Low-Dose 

Low
 

Dose
 

5/38 (13)
 

N.S.
 

Infinite
 

0.693
 

Infinite
 

62
 

6/38 (16)
 

N.S.
 

Infinite
 

0.879
 

Infinite
 

54
 
Table F3· Analyses of the Incidence of Primary Tumors in Low-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

(continued) 

Low-Dose 

Vehicle Low 

Topography: Morphology Control Dose 

Ovary: Tubular Adenoma*5 0/20 (0) 8/37 (22)
 

 Values0>d = 0.023
 

Relative Risk (Low-Dose Control)e Infinite
 

Lower Limit 1.291
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 67
 

a

Dosed group received 7 mg/kg.
 

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

c

Beneath the incidence of tumors in the dosed group is the probability level for the Fisher
 

exact test for the comparison of the dosed group with the vehicle-control group when Ρ < 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

d

A negative trend (N) indicates a lower incidence in the dosed group than in the control group.
 

e

The 95 percent confidence interval of the relative risk between the dosed group and the
 

control group.
 
Table F4. Analyses of the Incidence of Primary Tumors in Mid and High-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

Topography: Morphology 

Mid and 

High-Dose 

Pooled 

Control 

Lung: Alveolar/Bronchiolar 

Adenomab 1/31 (3) 

 Valuesc , d N.S. 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

Relative Risk (Vehicle Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Mid and 

High-Dose 

Vehicle 

Control 

Mid 

Dose 

High 

Dose 

1/15 (7) 1/27 (4) 1/31 (3) 

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

1.148 

0.015 

86.774 

1.000 

0.013 

75.984 

0.556 

0.008 

42.017 

0.484 

0.007 

36.791 

83 39 61 
Table F4· Analyses of the Incidence of Primary Tumors
 

Female Mice Administered Phenesterin by
 

(continued) 

Topography: Morphology 

Mid and 

High-Dose 

Pooled 

Control 

Hematopoietic System: Lymphoma 

or Leukemiab 0/31 (0) 

 Valuesc , d < 0.001 

Departure from Linear Trende = 0.018 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

Relative Risk (Vehicle Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Mid and
 

High-Dose
 

Vehicle
 

Control
 

0/15 (0)
 

 = 0.002
 

 = 0.036
 

—
 

in Mid and High-Dose
 

Gavagea
 

Mid
 

Dose
 

14/27 (52)
 

 < 0.001*
 

 < 0.001**
 

Infinite
 

5.181
 

Infinite
 

Infinite
 

2.624
 

Infinite
 

22
 

High
 

Dose
 

17/32 (53)
 

 < 0.001*
 

 < 0.001**
 

Infinite
 

5.399
 

Infinite
 

Infinite
 

2.735
 

Infinite
 

15
 
Table F4· Analyses of the Incidence of Primary Tumors in Mid and High-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

(continued)
 

Topography: Morphology 

All Sites: Hemangiosarcoma^ 

 Values0>d 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

Relative Risk (Vehicle Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

a


Mid and Mid and 

High-Dose High-Dose 

Pooled Vehicle Mid 

Control Control Dose 

0/31 (0) 0/15 (0) 1/27 (4) 

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

Infinite 

0.062 

Infinite 

Infinite 

0.031 

Infinite 

— 57 

Dosed groups received 15 or 30 mg/kg.
 

b

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

High 

Dose 

2/32 (6) 

N.S. 

Infinite 

0.291 

Infinite 

Infinite 

0.147 

Infinite 

51 
Table F4. Analyses of the Incidence of Primary Tumors in Mid and High-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

(continued)
 

c

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


Armitage test when Ρ < 0·05, otherwise, not significant (N.S.) is indicated· Beneath the
 

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

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


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

is indicated·
 

d

A negative trend (N) indicates a lower incidence in a dosed group than in a control group·
 

e

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

f

The 95 percent confidence interval of the relative risk between each dosed group and the
 

specified control group·
 
Table F5. Time-Adjusted Analyses of the Incidence of

Female Mice Administered Phenesterin

 Primary Tumors

 by Gavagea 

 in Low-Dose 

Topography: Morphology 

Low-Dose 

Vehicle 

Control 

Low 

Dose 

Lung: Alveolar/Bronchiolar Carcinomac 1/18 (6) 8/35 (23) 

 Valuesd , e N.S. 

Relative Risk (Low-Dose Control)f 

Lower Limit 

Upper Limit 

4.114 

0.631 

176.029 

Weeks to First Observed Tumor 82 71 

σ Lung: Alveolar/Bronchiolar Adenoma 

or Carcinoma0 1/18 (6) 15/35 (43) 

 Valuesd , e = 0.004 

Relative Risk (Low-Dose

Lower Limit 

Upper Limit 

Control)f 7.714 

1.380 

309.076 

Weeks to First Observed Tumor 82 68 
Table F5· Time-Adjusted Analyses of the Incidence of Primary Tumors in Low-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

(continued)
 

Low-Dose 

Vehicle Low 

Topography: Morphology Control Dose 

Hematopoietic System: Lymphoma 

or Leukemiab 5/18 (28) 12/36 (33) 

 Values d, e N.S. 

Relative Risk (Low-Dose Control)f 1.200 

Lower Limit 0.484 

Upper Limit 3.772 

Weeks to First Observed Tumor 86 51 

Liver: Hepatocellular Carcinoma0 1/18 (6) 1/34 (3) 

 Values d, e N.S. 

Relative Risk (Low-Dose Control)f 0.529 

Lower Limit 0.007 

Upper Limit 40.372 

Weeks to First Observed Tumor 104 54 
Table F5. Time-Adjusted Analyses of the Incidence of Primary Tumors in Low-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

(continued)
 

Topography: Morphology
 

Liver: Hepatocellular Adenoma
 

or Carcinoma0
 

e
 Valuesd

>
 

Relative Risk (Low-Dose Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Myocardium: Sarcoma, NOSC
 

e
 Valuesd

>
 

Relative Risk (Low-Dose Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Low-Dose 

Vehicle Low 

Control Dose 

3/18 (17) 1/34 (3) 

N.S. 

0.176 

0.004 

2.038 

82 54 

0/18 (0) 8/34 (24) 

 = 0.024 

Infinite 

1.276 

Infinite 

62 
Table F5. Time-Adjusted Analyses of the Incidence of Primary Tumors in Low-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

(continued)
 

Low-Dose
 

Vehicle Low
 

Topography: Morphology Control Dose
 

All Sites: Hemangiosarcomac 0/18 (0) 5/35 (14)
 

e
 Valuesd , N.S.
 

Relative Risk (Low-Dose Control)f Infinite
 

Lower Limit 0.682
 

Upper Limit Infinite
 

Weeks to First Observed Tumor —
 62
 

VO Mammary Gland: Adenocarcinoma, NOSC 0/18 (0) 6/35 (17)
 

e
 Valuesd

> N.S.
 

Relative Risk (Low-Dose Control)f Infinite
 

Lower Limit 0.866
 

Upper Limit Infinite
 

Weeks to First Observed Tumor — 54
 
Table F5· Time-Adjusted Analyses of the Incidence of Primary Tumors in Low-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

(continued) 

Low-Dose 

Vehicle Low 

Topography: Morphology Control Dose 

Ovary: Tubular Adenoma0 0/18 (0) 8/34 (24)
 

 Valuesd>e = 0.024
 

Relative Risk (Low-Dose Control)f Infinite
 

Lower Limit 1·276
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 67
 

a

Dosed group received 7 mg/kg·
 

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

at least 51 weeks of the study.
 

c

Number of tumor-bearing animals/number of animals examined at site (percent) which survived
 

at least 52 weeks of the study.
 

^Beneath the incidence of tumors in the dosed group is the probability level for the Fisher exact
 

test for the comparison of the dosed group with the vehicle-control group when Ρ < 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

e

A negative trend (N) indicates a lower incidence in the dosed group than in the control group.
 

95 percent confidence interval of the relative risk between the dosed group and
 

the control group.
 
Table F6· Time-Adjusted Analyses of the Incidence of Primary Tumors in Mid and High-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

Topography: Morphology 

Lung: Alveolar/Bronchiolar 

Adenoma0 

 Valuese>S 

Relative Risk (Vehicle Control)i 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Hematopoietic System: 

Lymphoma or Leukemia*3 

 Valuesf>S 

Departure from Linear Trend*1 

Relative Risk (Vehicle Control)* 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Mid and 

High-Dose 

Vehicle 

Control 

1/15 (7) 

N.S. 

83 

0/15 (0) 

 < 0.001 

 = 0.016 

— 

Mid
 

Dose
 

1/18 (6)
 

N.S. 

0.833 

0.011 

61.743 

39 

14/18 (78) 

 < 0.001 

Infinite 

4.089
 

Infinite
 

22 

High 

Dose 

1/5 (20) 

N.S. 

3.000 

0.041 

178.689 

61 

17/19 (89) 

 < 0.001 

Infinite 

4.966 

Infinite 

15 
__ 

Table F6. Time-Adjusted Analyses of the Incidence of Primary Tumors in Mid and High-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

(continued) 

Topography: Morphology
 

Myocardium: Sarcoma, NOSd
 

 Valuesf , g
 

Relative Risk (Vehicle Control)i•
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

All Sites: Hemangiosarcomae
 

 Valuese>f
 

Relative Risk (Vehicle Control)±
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mid and
 

High-Dose
 

Vehicle
 

Control
 

0/15 (0)
 

 = 0.012
 

0/15 (0)
 

 = 0.033
 

—
 

Mid
 

Dose
 

2/7 (29)
 

N.S.
 

Infinite
 

0.685
 

Infinite
 

62
 

1/7 (14)
 

N.S.
 

Infinite
 

0.119
 

Infinite
 

57
 

High
 

Dose
 

3/7 (43)
 

 = 0.023
 

Infinite
 

1.421
 

Infinite
 

42
 

2/6 (33)
 

N.S.
 

Infinite
 

0.802
 

Infinite
 

51
 

a

Dosed groups received 15 or 30 mg/kg·
 

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

at least 15 weeks of the study·
 

c

Number of tumor-bearing animals/number of animals examined at site (percent) which survived
 

at least 39 weeks of the study·
 
Table F6· Time-Adjusted Analyses of the Incidence of Primary Tumors in Mid and High-Dose
 

Female Mice Administered Phenesterin by Gavagea
 

(continued)
 

d

Number of tumor-bearing animals/number of animals examined at site (percent) which survived
 

at least 42 weeks of the study·
 

e

Number of tumor-bearing animals/number of animals examined at site (percent) which survived
 

at least 51 weeks of study.
 

f

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


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

incidence of tumors in a dosed group is the probability lev. for the Fisher exact test for
 

the comparison of that dosed group with the matched-control *;roup when Ρ < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

g

 negative trend (N) indicates a lower incide, ce in a doseo ^roup than in a control group.
 

h

The probability level for departure from linear trend is gi^an when Ρ < 0.05 for any comparison.
 

i

The 95 percent confidence interval of the rel ive risk between each dosed group and the
 

control group.
 

Review of the Bioassay of Phenesterin* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

March 6, 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 Phenestrin for carcinogenicity.
 

In a written review submitted by a Subgroup member,
 

the reviewer agreed with the conclusion in the report that,
 

under the conditions of test, Phenesterin was carcinogenic
 

in female rats and in both sexes of mice. He briefly
 

described the experimental design and the conditions under
 

which Phenesterin was tested. In his critique, he noted the
 

following points: the inadequate size of the treatment and
 

control groups; the use of excessively high dose levels;
 

and animals were housed in the same room in which other
 

chemicals were under test. The reviewer said that these
 

shortcomings detracted from the value of the study. He
 

added that extrapolation of the results was made difficult
 

by the use of excessive doses and other conditions under
 

which Phenesterin was tested.
 

The secondary reviewer noted the poor selection of
 

dose levels. He agreed that it would be difficult to
 

extrapolate to lower doses for purposes of assessing human
 

risk. He added that human risk is a secondary consideration
 

145
 
since Phenesterin is an anti-cancer agent. One Subgroup
 

member said he would agree if Phenesterin is an effective
 

anti-cancer agent. Another Subgroup member noted that
 

anti-cancer agents also may be used to treat other diseases.
 

Another reviewer commented on the excessive amount of
 

toxicity resulting from the high treatment doses administered,
 

He felt, however, that an adequate number of animals survived
 

to demonstrate the carcinogenicity of Phenesterin.
 

It was moved that the report on Phenesterin be accepted
 

as written. The motion was seconded and approved unanimously,
 

Members present were
 

Gerald N. Wogan (Chairman), Massachusetts Institute of
 

Technology
 

Arnold L. Brown, Mayo Clinic
 

Lawrence Garfinkel, American Cancer Society
 

E. Cuyler Hammond, American Cancer Society
 

Joseph Highland, Environmental Defense Fund
 

Henry Pitot, University of Wisconsin Medical Center
 

George Roush, Jr., Monsanto Company
 

Sheldon Samuels, Industrial Union Department, AFL-CIO
 

Michael Shimkin, University of California at San Diego
 

John Weisburger, American Health Foundation
 

Sidney Wolfe, Health Research Group
 

*	 Subsequent to this review9 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.
 

146
 

*U.S. GOVERNMENT PRINTING OFFiCE. 1978 260-899/3086 1-3 

DHEW Publication (NIH) No. 78-1310