BIDASSAY OF
 

3-SULFOLENE
 

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) 78-1352
 

REPORT ON THE BIOASSAY OF 3-SULFOLENE
 

FOR POSSIBLE CARCINOGENICITY
 

CARCINOGENESIS TESTING PROGRAM
 

DIVISION OF CANCER CAUSE AND PREVENTION
 

NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH
 

FOREWORD: This report presents the results of the bioassay of 3-sulfolene conducted for the Carcinogenesis Testing Program, Division of
 

Cancer Cause and Prevention, National Cancer Institute (NCI), National
 

Institutes of Health, Bethesda, Maryland. This is one of a series of
 

experiments designed to determine whether selected chemicals have the
 

capacity to produce cancer in animals. Negative results, in which the
 

test animals do not have a significantly greater incidence of cancer
 

than control animals, do not necessarily mean the test chemical is not
 

a carcinogen because 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 a potential risk to man. The actual determination of the risk to
 

man from animal carcinogens requires a wider analysis.
 

CONTRIBUTORS; This bioassay of 3-sulfolene was conducted by Hazleton
 

Laboratories America, Inc., Vienna, Virginia, initially under direct
 

contract to the NCI and currently under a subcontract to Tracer Jitco,
 

Inc., prime contractor for the NCI Carcinogenesis Testing Program.
 

The experimental design was determined by the NCI Project Officers, Dr. J. H. Weisburger (1,2) and Dr. E. K. Weisburger (1). The
 

principal investigators for the contract were Dr. M. B. Powers (3),
 

Dr. R. W. Voelker (3), Dr. W. A. Olson (3,4) and Dr. W. M. Weatherholtz (3). Chemical analysis was performed by Dr. C. L. Guyton
 

(3,5) and the analytical results were reviewed by Dr. N. Zimmerman
 

(6); the technical supervisor of animal treatment and observation
 

was Ms. K. J. Petrovics (3).
 

Histopathologic examinations were performed by Dr. R. H. Habermann (3) and reviewed by Dr. R. W. Voelker (3) at the Hazleton Laboratories America, Inc., and the diagnoses included in this report
 

represent the interpretation of these pathologists. Histopathology
 

findings and reports were reviewed by Dr. R. L. Schueler (7).
 

Compilation of individual animal survival, pathology, and summary tables was performed by EG&G Mason Research Institute (8); the
 

statistical analysis was performed by Mr. W. W. Belew (6), using
 

methods selected for the Bioassay Program by Dr. J. J. Gart (9).
 

111
 
This report was prepared at METREK, a Division of The MITRE Corporation (6) under the direction of the NCI. Those responsible for
 

this report at METREK are the project coordinator, Dr. L. W. Thomas
 

(6), the task leader, Dr. M. R. Kornreich (6), the senior biologist,
 

Ms. P. Walker (6), and the technical editor, Ms. P. A. Miller (6).
 

The final report was reviewed by members of the participating organizations.
 

The statistical analysis was reviewed by members of the Mathematical Statistics and Applied Mathematics Section of the NCI: Dr.
 

J. J. Gart (9), Mr. J. Nam (9), Dr. H. M. Pettigrew (9), and Dr. R.
 

E. Tarone (9).
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

the results, and reporting the findings: Dr. K. C. Chu (1), Dr. C.
 

Cueto, Jr. (1), Dr. J. F. Douglas (1), Dr. D. G. Goodman (1), Dr. R.
 

A. Griesemer (1), Dr. H. A. Milman (1), Dr. T. W. Orme (1), Dr. R. A.
 

Squire (1,10), Dr. J. M. Ward (1), and Dr. C. E. Whitmire (1).
 

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

2.	 Now with the Naylor Dana Institute for Disease Prevention, American Health Foundation, Hammon House Road, Valhalla, New York.
 

3.	 Hazleton Laboratories America, Inc.,- 9200 Leesburg Turnpike,
 

Vienna, Virginia.
 

4.	 No wit w h the Center for Regulatory Services, 2347 Paddock Lane,
 

Reston, Virginia.
 

5.	 No wit w h Rhodia, Inc., 23 Belmont Drive, Somerset, New Jersey.
 

6.	 Th MITR e E Corporation, METREK Division, 1820 Dolley Madison
 

Boulevard, McLean, Virginia.
 

7.	 Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

8.	 EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

IV
 
9.	 Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cance1

"
 

Cause and Prevention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
 

10.	 Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay of 3-sulfolene for possible carcinogenicity was conducted using Osborne-Mendel rats and B6C3F1 mice. 3-Sulfolene in
 

corn oil was administered by gavage, at either of two dosages, to
 

groups of 50 male and 50 female animals of each species. The 78-week
 

period of chemical administration was followed by an observation
 

period of 33 weeks for the high dose female rats and low dose rats
 

of both sexes. The last high dose male rat died in week 60. All
 

treated groups of mice were observed for an additional 13 weeks
 

following chemical administration.
 

Initial dosage levels for the chronic bioassay were selected on
 

the basis of a preliminary subchronic toxicity test. Subsequent
 

dosage adjustments were made during the course of the chronic bioassay. The time-weighted average high and low doses of 3-sulfolene in
 

the chronic study were, respectively, 372 and 197 mg/kg/day for male
 

rats, 240 and 120 mg/kg/day for female rats, 622 and 311 mg/kg/day
 

for male mice and 768 and 384 mg/kg/day for the female mice.
 

For each species, 20 animals of each sex were placed on test as
 

vehicle controls. These animals were gavaged with corn oil at the
 

same times that dosed animals were gavaged with the 3-sulfolene mixtures. Twenty animals of each sex were placed on test as untreated
 

controls for each species. These animals were not intubated.
 

There was a significant positive association between the administered dosages of 3-sulfolene and mortality in both sexes of rats
 

and mice. In all groups, except the high dose male rats and the high
 

dose male and female mice, adequate numbers of animals survived sufficiently long to be at risk from late-developing tumors.
 

There were no tumors in either sex of rats or mice for which a
 

significant positive association could be established between chemical administration and incidence.
 

Under the conditions of this bioassay, there was no evidence
 

for the carcinogenicity of 3-sulfolene to Osborne-Mendel rats or
 

B6C3F1 mice.
 

vn
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 2
 

A. Chemicals 2
 

B. Dosage Preparation 2
 

C. Animals 2
 

D. Animal Maintenance 3
 

E. Gastric Intubation 4
 

F. Selection of Initial Dose Levels 5
 

G. Experimental Design 6
 

H. Clinical and Histopathologic Examinations 10
 

I. Data Recording and Statistical Analyses 12
 

III. CHRONIC TESTING RESULTS: RATS 17
 

A. Body Weights and Clinical Observations 17
 

B. Survival 17
 

C. Pathology 20
 

D. Statistical Analyses of Results 22
 

IV. CHRONIC TESTING RESULTS: MICE 29
 

A. Body Weights and Clinical Observations 29
 

B. Survival 29
 

C. Pathology 33
 

D. Statistical Analyses of Results 34
 

V. DISCUSSION 40
 

VI. BIBLIOGRAPHY 42
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH 3-SULFOLENE A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH 3-SULFOLENE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 3-SULFOLENE C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 3-SULFOLENE D-l
 

IX
 
Figure Number
 

1
 

Table Number
 

Al
 

LIST OF ILLUSTRATIONS 

Page 

GROWTH CURVES FOR 3-SULFOLENE CHRONIC STUDY 

RATS 18 

SURVIVAL COMPARISONS OF 3-SULFOLENE CHRONIC 

STUDY RATS 19 

GROWTH CURVES FOR 3-SULFOLENE CHRONIC STUDY 

MICE 30 

SURVIVAL COMPARISONS OF 3-SULFOLENE CHRONIC 

STUDY MICE 31 

LIST OF TABLES 

Page 

DESIGN SUMMARY FOR OSBORNE-MENDEL RATS— 

3-SULFOLENE GAVAGE EXPERIMENT 

DESIGN SUMMARY FOR B6C3F1 MICE--3-SULFOLENE 

GAVAGE EXPERIMENT 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

3-SULFOLENE AND SURVIVING AT LEAST 52 WEEKS 23 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH 3-SULFOLENE AND SURVIVING AT LEAST 52 

WEEKS 24 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

3-SULFOLENE AND SURVIVING AT LEAST 52 WEEKS 35 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH 3-SULFOLENE AND SURVIVING AT LEAST 52 

WEEKS 37 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH 3-SULFOLENE A-3 
LIST OF TABLES (Concluded) 

Table Number Page 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH 3-SULFOLENE A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH 3-SULFOLENE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH 3-SULFOLENE B-6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH 3-SULFOLENE C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH 3-SULFOLENE C-10 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH 3-SULFOLENE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH 3-SULFOLENE D-6 

XI
 

I. INTRODUCTION
 

3-Sulfolene (NCI No. C04557), a small ring, heterocyclic sulfone,
 

was selected for bioassay by the National Cancer Institute because of
 

the structural similarity of this compound to 3,4-epoxysulfolane and
 

1-propanesulfonic acid-3-hydroxy- -sultone, both of which are carcinogenic in mice (Van Duuren et al., 1971).
 

The Chemical Abstracts Service (CAS) Ninth Collective Index (1977)
 

*
 

name for this compound is 2,5-dihydrothiophene 1,1-dioxide. It is
 

also known as l-thia-3-cyclopentene 1,1-dioxide; butadiene sulfone; or
 

simply sulfolene.
 

3-Sulfolene is an intermediate in the production of sulfolane,
 

which is used in the petroleum, plastics, and textile industries, and
 

in the synthesis of one or more fungicides or additional chemicals
 

(Rose and Rose, 1966). 3-Sulfolene is also used as a catalyst (Rose
 

and Rose, 1966).
 

Specific production data for 3-sulfolene are not available; however, the inclusion of this compound in the 1977 Directory of Chemical
 

Producers, U.S.A. (Stanford Research Institute, 1977) implies that it
 

is produced in commercial quantities (in excess of 1000 pounds or
 

$1000 in value annually).
 

The potential for exposure to 3-sulfolene is greatest for workers
 

in 3-sulfolene and sulfolane production facilities but may be considerable for other chemical manufacturing workers, as well.
 

The CAS registry number is 77-79-2.
 
II. MATERIALS AND METHODS
 

A. Chemicals
 

3-Sulfolene was purchased from Phillips Petroleum Company and
 

chemical analysis was performed by Hazleton Laboratories America,
 

Inc., Vienna, Virginia. Thin-layer chromatography utilizing seven
 

various concentration combinations of methanol:acetone and/or benzene
 

each revealed only one spot. Ultraviolet analysis showed one peak at
 

X = 274 nm. When compared with an ultraviolet standard assumed to
 

max
 

have a purity of 100 percent, the purity of the test chemical was indicated to be approximately 92 percent. Chemical analysis performed
 

twelve months later indicated decomposition.
 

Throughout this report the term 3-sulfolene is used to represent
 

this material.
 

B. Dosage Preparation
 

Fresh solutions of 3-sulfolene in Duke's corn oil (S. F. Sauer
 

Company, Richmond, Virginia) were prepared weekly, sealed and stored
 

in dark bottles at 1°C. These 3-sulfolene solutions were considered
 

generally stable for ten days under the indicated storage conditions.
 

The concentration of 3-sulfolene in corn oil ranged from 10 to 20
 

percent in rats, and from 2.3 to 8 percent in mice.
 

C.	 Animals
 

Two animal species, rats and mice, were used in the carcinogenicity bioassay. The Osborne-Mendel rat was selected on the basis of
 

a comparative study of the tumorigenic responsiveness to carbon
 
tetrachloride of five different strains of rats (Reuber and Glover,
 

1970). The B6C3F1 mouse was selected because it has been used by the
 

NCI for carcinogenesis bioassays and has proved satisfactory in this
 

capacity.
 

Rats and mice of both sexes were obtained through contracts of
 

the Division of Cancer Treatment, National Cancer Institute. The
 

Osborne-Mendel rats were procured from the Battelle Memorial Institute, Columbus, Ohio, and the B6C3F1 mice were obtained from the
 

Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts.
 

Upon receipt, animals were quarantined for at least 10 days, observed
 

for visible signs of disease or parasites, and assigned to the various
 

treated and control groups.
 

D. Animal Maintenance
 

All animals were housed by species in temperature- and humidity-


controlled rooms. The temperature range wa^ 20° to 24°C and the relative humidity was maintained between 45 and 55 percent. The air
 

conditioning system in the laboratory provided filtered air at a rate
 

of 12 to 15 complete changes of room air per hour. Fluorescent lighting was provided on a 12-hour-daily cycle.
 

The rats were individually housed in suspended galvanized-steel
 

wire-mesh cages with perforated floors, while mice were housed by sex
 

in groups of 10 in solid-bottom polypropylene cages equipped with
 

(R)
 filter tops. Sanitized cages with fresh bedding (Sanichips , Pine-


wood Sawdust Company, Moonachie, New Jersey) were provided once each
 
week for mice. Rats received sanitized cages with no bedding with
 

the same frequency. Food hoppers were changed and heat-sterilized
 

once a week for the first 10 weeks and once a month thereafter.
 

Fresh heat-sterilized glass water bottles and sipper tubes were provided three times a week. Food (Wayne Lab-Blox®, Allied Mills, Inc.,
 

Chicago, Illinois) and water were available ad libitum.
 

Rats dosed with 3-sulfolene and their vehicle controls were
 

*
 housed in the same room as other rats intubated with lodoform (75­

47-8) and hexachloroethane (67-72-1). Untreated control rats were
 

housed in a room with other rats intubated with 1,1,2-trichloroethane
 

(79-00-5) and tetrachloroethylene (127-18-4).
 

All mice used in the 3-sulfolene chronic bioassay were housed
 

with other mice being intubated with 1,1,2,2-tetrachloroethane (79-34­

5); chloroform (67-66-3); allyl chloride (107-05-1); methylchloroform
 

(71-55-6); chloropicrin (76-06-2); dlbromochloropropane (96-12-8);
 

1,2-dibromoethane (106-93-4); 1,2-dichloroethane (107-06-2); trichloroethylene (79-01-6); 1,1-dichloroethane (75-34-3); iodoform (75-47­

8); 1,1,2-trichloroethane (79-00-5); tetrachloroethylene (127-18-4);
 

carbon disulfide (75-15-0); hexachloroethane (67-72-1); trichlorofluoromethane (75-69-4); and carbon tetrachloride (56-23-5).
 

E. Gastric Intubation
 

Intubation was performed for five consecutive days per week on
 

a mg/kg body weight basis, utilizing the most recently observed group
 

*
 

CAS registry numbers are given in parentheses.
 
mean body weight as a guide for determining the dose. Mean body
 

weights for each group were recorded at weekly intervals for the
 

first 10 weeks and at monthly intervals thereafter. All animals of
 

one sex within a treated group received the same dose. Animals were
 

gavaged with test solutions under a hood to minimize extraneous exposure of other animals and laboratory personnel to the chemical.
 

F. Selection of Initial Dose Levels
 

In order to establish the maximum tolerated dosages of 3-sulfolene for administration to treated animals in the chronic studies,
 

subchronic toxicity tests were conducted with both rats and mice.
 

Animals of each species were distributed among six groups, each
 

consisting of five males and five females. 3-Sulfolene mixed with
 

corn oil was introduced by gavage to five of the six rat groups at
 

dosages of 56, 100, 178, 316, and 562 mg/kg/day and five of the six
 

mouse groups at dosages of 316, 562, 1000, 1780, and 3160 mg/kg/day.
 

The sixth group of each species served as a control, receiving only
 

the corn oil by gavage. Intubation was performed 5 consecutive days
 

per week for 6 weeks, followed by a 2-week observation period to
 

detect any delayed toxicity.
 

A dosage inducing no mortality and resulting in a depression
 

in mean group body weight of approximately 20 percent relative to
 

controls was selected as the initial high dose. When weight gain
 

criteria were not applicable, mortality data alone were utilized.
 
The only deaths observed among treated rats were two females,
 

one receiving 316 mg/kg/day and one receiving 562 mg/kg/day. Mean
 

body weight depression was 17 percent in males treated with 562 mg/
 

kg/day, 18 percent in females treated with 178 mg/kg/day, and 35 percent in females treated with 316 mg/kg/day. The high dosages of
 

3-sulfolene selected for use in the chronic bioassay were 560 mg/kg/
 

day for male rats and 200 mg/kg/day for female rats.
 

No mice treated with 562 mg/kg/day or less died during the sub-


chronic test. The only group exhibiting mean body weight depression
 

was the females receiving 316 mg/kg/day; in those groups for which
 

treated mice survived the study (i.e., those receiving 1000 mg/kg/day
 

or less) the mean body weight was greater than the mean body weight
 

of the control group. The high dosage of 3-sulfolene selected for
 

use in the chronic bioassay was 450 mg/kg/day for mice of both sexes.
 

G. Experimental Design
 

The experimental design parameters for the chronic bioassay (species, sex, group size, dosages administered, duration of treated and
 

untreated observation periods, and the time-weighted average dosages)
 

are summarized in Tables 1 and 2.
 

The treated and vehicle control rats shared the same median date
 

of birth and were all approximately 8 weeks old at the time they were
 

placed on test. The untreated controls were approximately 4 weeks
 

younger than the other rats and were started on test approximately
 

1 week after the other groups.
 
TABLE 1
 

DESIGN SUMMARY FOR OSBORNE-MENDEL RATS
 

3-SULFOLENE GAVAGE EXPERIMENT
 

INITIAL 

GROUP 

SIZE 

3-SULFOLENE 

DOSAGE3 

OBSERVATION PERIOD TIME-WEIGHTED 

TREATED 

(WEEKS) 

UNTREATED 

(WEEKS) 

AVERAGE 

DOSAGEb 

MALE 

UNTREATED CONTROL 

VEHICLE CONTROL 

LOW DOSE 

20 

20 

50 

0 

0 

280 

200 

200C 

0 

0 

0 

22 

10 

36 

110 

111 

10 

33 

0 

0 

197 

HIGH DOSEd 50 560 

400 

400C 

200C 

22 

10 

8 

14 

2 

4 

372d 

FEMALE
 

UNTREATED CONTROL 20 0 0 110 0
 

VEHICLE CONTROL 20 0 0 111 0
 

LOW DOSE 50 100 17 120
 

150 15
 

150C 36 10
 

0 33
 

HIGH DOSE 50	 200 17 240
 

300 15
 

300C 36 10
 

0	 33
 

Dosages, given in mg/kg body weight, were administered by gavage 5 consecutive days per week.
 

(dosage X weeks received)
 Time-weighted average dosage
 78 weeks
 

"These dosages were cyclically administered with a pattern of 1 dose-free
 

week followed by 4 weeks (5 days per week) of chemical administration at
 

the dosage level indicated.
 

Terminated in week 60; therefore, average dosage is calculated over a
 

60-week period rather than a 78-week period.
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

3-SULFOLENE GAVAGE EXPERIMENT
 

INITIAL OBSERVATION PERIOD TIME-WEIGHTED 

GROUP 3-SULFOLENE TREATED UNTREATED AVERAGE 

SIZE DOSAGE3 (WEEKS) (WEEKS) DOSAGEb 

MALE
 

UNTREATED CONTROL 20 0 0 90 . 0
 

VEHICLE CONTROL 20 0 0 90 0
 

LOW DOSE 50 225 2 311
 

300 9
 

400 10
 

300 57
 

0	 13
 

HIGH DOSE 50	 450 2 622
 

600 9
 

800 10
 

600 57
 

0	 13
 

FEMALE
 

UNTREATED CONTROL 20 0 0 90
 

VEHICLE CONTROL 20 0 0 90
 

LOW DOSE 50 225 2 384
 

300 9
 

400 67
 

0 13
 

HIGH DOSE 50	 450 2 768
 

600 9
 

800 67
 

0	 13
 

Dosages, given in mg/kg body weight, were administered by gavage 5 consecutive days per week.
 

b . . ,	 , £(dosage X weeks received)
 Time-weighted average dosage = -—-, , :—: : r,.
 S(weeks receiving chemical)
 
The dosages initially administered were 560 and 280 mg/kg/day
 

for male rats and 200 and 100 mg/kg/day for female rats. Throughout
 

this report the animals initially receiving the higher of the two
 

dosages are referred to as the high dose group and the animals initially receiving the lower of the two dosages are referred to as the
 

low dose group. In week 18 the dosages utilized for high and low
 

dose females were increased to 300 and 150 mg/kg/day, respectively,
 

while the dosages administered to high and low dose males were decreased to 400 and 200 mg/kg/day in week 23. In week 33 administration of 3-sulfolene to all rats ceased for 1 week followed by 4 weeks
 

of compound intubation. This cyclic pattern of dose administration
 

was maintained for the remainder of the bioassay. The dosage administered to the high dose males was decreased to 200 mg/kg/day in week
 

43. This dosage, too, was administered on a cyclic basis. All high
 

dose male rats surviving until week 60 were sacrificed at' that time.
 

All other treated rats were dosed for 78 weeks, followed by an untreated observation period of up to 33 weeks.
 

The untreated control and all treated mice shared the same median
 

date of birth and were approximately 6 weeks old at the time they were
 

placed on test. The vehicle control mice were approximately 4 weeks
 

older than the other mice and were started on test approximately 4
 

weeks before the other groups.
 

The dosages initially administered to both sexes of mice were
 

450 and 225 mg/kg/day. Throughout this report the animals initially
 
receiving the former dosage are referred to as the high dose group
 

and those receiving the latter dosage are referred to as the low dose
 

group. The dosages administered were increased twice; the first time,
 

in week 3, to 600 and 300 mg/kg/day and the second time, in week 12,
 

to 800 and 400 mg/kg/day. In week 22 the dosages administered to the
 

high and low dose males were reduced to 600 and 300 mg/kg/day, respectively. These dosages were maintained for the remainder of the 78­

week period of chemical administration, and treated mice were observed
 

for a subsequent period of up to 13 weeks.
 

H. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

experiment. Body weights, food consumption, and data concerning appearance, behavior, signs of toxic effects, and incidence, size, and
 

location of tissue masses were recorded at weekly intervals for the
 

first 10 weeks and at monthly intervals thereafter. From the first
 

day, all animals were inspected daily for mortality. The presence
 

of tissue masses was determined by observation and palpation of each
 

animal.
 

During the course of this bioassay several pathology protocols
 

were in effect, each for different periods of time. The minimum protocol required that, if possible, certain tissues were to be taken
 

and examined histopathologically from all control animals, from any
 

animal in which a tumor was observed during gross examination, and
 

from at least 10 grossly normal males and 10 grossly normal females
 

10
 
from each treated group. In addition, any tissues showing gross abnormalities were to be taken and examined histopathologically. Under
 

later protocols, some tissues were taken from additional dosed animals. The number of animals in each group from which a tissue was
 

examined is indicated in Appendices A through D.
 

A necropsy was performed on each animal regardless of whether it
 

died, was killed when moribund, or was sacrificed at the end of the
 

bioassay. The animals were euthanized by exsanguination under sodium
 

pentobarbital anesthesia, and were immediately necropsied. The histopathologic examination consisted of gross and microscopic examination
 

of major tissues, organs, or gross lesions taken from sacrificed animals and, whenever possible, from animals found dead.
 

Slides were prepared from the following tissues from selected
 

animals: skin, subcutaneous tissue, lungs and bronchi, trachea, bone
 

marrow, spleen, lymph nodes, thymus, heart, muscle, salivary gland,
 

liver, gallbladder (mice), pancreas, esophagus, stomach, small intestine, large intestine, kidney, urinary bladder, seminal vesicle,
 

pituitary, adrenal, thyroid, parathyroid, testis, prostate, brain,
 

uterus, ovary and mammary gland.
 

Tissues for which slides were prepared were preserved in 10 percent buffered formalin, embedded in paraffin, sectioned, and stained
 

with hematoxylin and eosin prior to microscopic examination. An
 

occasional section was subjected to special staining techniques for
 

more definitive diagnosis.
 

11
 
I. Data Recording and Statistical Analyses
 

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

(Linhart et al., 1974). The data elements include descriptive information on the chemicals, animals, experimental design, clinical observations, survival, body weight, and individual pathologic results,
 

as recommended by the International Union Against Cancer (Berenblum,
 

1969). Data tables were generated for 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) when testing two groups for
 

equality and used Tarone's (1975) extensions of Cox's methods when
 

testing a dose-related trend. One-tailed P-values have been reported
 

for all tests except the departure from linearity test, which is only
 

reported when its two-tailed P-value is less than 0.05.
 

12
 
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 was 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, pp. 48-52) was used to compare the tumor incidence of a control
 

group to that of a group of treated animals at each dose level. When
 

results for a number of treated 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, pp. 6-10) requires that the P-value for any comparison
 

be less than or equal to 0.05/k. In cases where this correction was
 

used, it is discussed in the narrative section. It is not, however,
 

presented in the tables, where the Fisher exact P-values are shown.
 

13
 
The Cochran-Armitage test for linear trend in proportions, with
 

continuity correction (Armitage, 1971, pp. 362-365), was also used
 

when appropriate. Under the assumption of a linear trend, this test
 

determined 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 was 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.
 

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 animals died naturally or were sacrificed was entered
 

as the time point of tumor observation. Cox's methods of comparing
 

14
 
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 to its control was calculated from
 

the exact interval on the odds ratio (Gart, 1971). The relative risk
 

is defined as p /p where p is the true binomial probability of the
 

incidence of a specific type of tumor in a treated group of animals
 

and p is the true probability of the spontaneous incidence of the
 

c
 

same type of tumor in a control group. The hypothesis of equality
 

between the true proportion of a specific tumor in a treated group
 

and the proportion in a control group corresponds to a relative risk
 

of unity. Values in excess of unity represent the condition of a
 

larger proportion in the treated 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 percent
 

of a large number of identical experiments, the true ratio of the
 

risk in a treated 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
 

15
 
be inferred that a statistically significant result (a P < 0.025 one-


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

the control incidence is zero) has occurred. When the lower limit is
 

less than unity but the upper limit is greater than unity, the lower
 

limit indicates the absence of a significant result while the upper
 

limit 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.
 

16
 
III. CHRONIC TESTING RESULTS: RATS
 

A.	 Body Weights and Clinical Observations
 

Mean group body weight depression was observed for high dose
 

male rats when compared to controls; this was not the case, however,
 

for the other treated rat groups (Figure 1). Fluctuations in the
 

growth curve may be due to mortality; as the size of the group diminishes, the mean body weight may be subject to wide variations.
 

No characteristic clinical signs were observed during the first
 

5 weeks of the study. Beginning in week 6 and until the end of compound administration in week 78, a hunched appearance was noted in an
 

increasing number of treated rats. Abdominal urine stains were also
 

observed, particularly among treated females.
 

Respiratory signs characterized by labored respiration, wheezing
 

or nasal discharge were observed at a low incidence during the study,
 

increasing in all groups as the animals aged. Other signs commonly
 

observed in aging rats and noted at a comparable frequency in treated
 

and control rats included body sores, alopecia, reddened or squinted
 

eyes, roughened or stained fur, tissue masses, and palpable nodules.
 

B.	 Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and 3-sulfolene-dosed groups are shown in Figure 2.
 

For male rats the Tarone test indicated a significant (P < 0.001)
 

positive association between dosage and mortality when dosed groups
 

17
 
MEAN BODY WEIGHT (GRAMS) MEAN BODY WEIG

I s

I

 I ° i I j

s 

I

 u 

m 

>

im 

33 

8­

o O z Z 

oo m m in en 

5 

m 

m 

c/> 

8- 8­

8 i o a- z 

H O 3 z O -I 

X 

o 

I 

i 
PROBABILIT

Y OF SURVIVAL PROBABILIT

Y O

F 

30 

O 

o -0 33 

I 

Vt 

O 2= I- 30 •n m 

m 

Z 

m 

O 

X 3D 

O 

n 

o ?: 

\ 

p

b 

m 

S 

> 

r 33 

> 

J 

8­

r 

^ 

/I 

g­
were compared to the vehicle controls. Due to the accelerated mortality in the high dose group the departure from linear trend was also
 

significant (P < 0.001). In the high dose group 50 percent (25/50)
 

were dead after only 27 weeks, with the six rats still surviving
 

sacrificed at week 60. Survival was somewhat better in the low dose
 

treated group where the median survival was 71 weeks. In the vehicle
 

control group seven males were sacrificed in week 60, with 55 percent
 

(11/20) of the rats surviving on test at least 90 weeks. In the
 

untreated control group 85 percent (17/20) survived on test at least
 

90 weeks. In the high dose group survival was not adequate.
 

For female rats the Tarone test also showed a significant (P =
 

0.002) positive association between dosage and mortality when dosed
 

groups were compared to the vehicle controls. Adequate numbers of
 

animals were at risk from late-developing tumors with 52 percent
 

(26/50) of the high dose, 82 percent (41/50) of the low dose, 85
 

percent (17/20) of the vehicle control, and 80 percent (16/20) of
 

the untreated control group surviving on test at least 90 weeks.
 

C. Pathology
 

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).
 

Pituitary tumors, which normally occur in elevated incidences
 

in this strain of rats, were observed in 22/50 (44 percent) of the
 

low dose females and in 6/50 (12 percent) of the high dose females.
 

20
 
Although the incidence of this tumor in the low dose females was not
 

appreciably different from the incidences observed in control females (i.e., 8/20 [40 percent] in the untreated control and 7/20 [35
 

percent] in the vehicle control), the incidence among the high dose
 

females was lower than expected. In addition, the incidences of
 

mammary fibroadenomas were 17/50 (34 percent) and 9/50 (18 percent)
 

in the low and high dose female rats, respectively. These neoplasms
 

occur frequently in this strain in incidences approximating 30 percent. As observed, the incidence in the low dose females approximated that expected while the incidence among the high dose females
 

was lower than expected. These instances of commonly observed tumors, occurring in unexpectedly low incidences are probably reflections of the poor survival among the high dose female rats.
 

The toxicity of 3-sulfolene leading to early mortality was
 

reflected morphologically primarily in the circulatory, urinary,
 

biliary, and genital systems. There was an increased incidence of
 

commonly occurring nonneoplastic lesions including metastatic calcification of the lung, testes and stomach; vascular calcification in
 

the aorta, pulmonary artery, mesenteric artery, and pancreatic artery; periarteristis of the pancreatic artery; hepatocellular fatty
 

change; bile duct hyperplasia; intrahepatic cholangiectasia; portal
 

bile stasis; chronic renal inflammation; cystitis; parathyroid hyperplasia; prostatitis; hypospermatogenesis; and testicular atrophy.
 

The severity and incidence of these lesions was greatly increased and
 

21
 
their occurrence at an earlier age indicated the chemical was involved
 

in this exacerbation of "spontaneous" lesions.
 

The early onset and increased incidence of chronic renal inflammation, with equivocal evidence of toxic tubular nephrosis apparently
 

resulted in secondary parathyroid hyperplasia with subsequent metastatic calcification of numerous tissues.
 

In conclusion, administration of 3-sulfolene via gastric intubation resulted in early mortality, which was associated with the occurrence of a variety of nonneoplastic lesions. Neoplasms that were
 

observed occurred in incidences that were within or below the range
 

of spontaneous incidence commonly observed in Osborne-Mendel rats.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

rats are summarized in Tables 3 and 4. The analysis is included for
 

every type of tumor in either sex where at least two such tumors were
 

observed in at least one of the control or 3-sulfolene-dosed groups
 

and where such tumors were observed in at least 5 percent of the
 

group. Because of the high early mortality noted in rats of both
 

sexes, these analyses were based on those rats surviving at least 52
 

weeks.
 

For female rats the Cochran-Armitage test indicated a significant (P = 0.029) negative association between dose and the incidence
 

of pituitary chromophobe adenomas. The Fisher exact tests, however,
 

were not significant.
 

22
 
TABLE 3 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT 

SPECIFIC SITES IN MALE RATS TREATED WITH 3-SULFOLENE AND 

SURVIVING AT LEAST 52 WEEKS8 

TOPOGRAPHY : MORPHOLOGY 

VEHICLE 

CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Subcutaneous Tissue: Flbrosarcoma 

P Values0 

2/18(0.11) 

N.S. 

2/41(0.05) 

N.S. 

0/9(0.00) 

N.S. 

Relative Risk (Vehicle Control)d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

—_ 

108 

0.439 

0.035 

5.743 

60 

0.000 

0.000 

6.011 

to 

co Pituitary: Chromophobe Adenoma 

P Values0 

Relative Risk (Vehicle Control)d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

2/18(0.11) 

N.S. ___ 

105 

3/41(0.07) 

N.S. 

0.659 

0.085 

7.463 

96 

1/9(0.11) 

N.S. 

1.000 

0.018 

15.823 

60 

Treated groups received time-weighted average doses of 197 or 372 rag/kg by gavage.
 

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

c

The probability level for the Cochran-Armitage test is given beneath the incidence of tumors in
 

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison of a treated group with the control group is
 

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not signifi­ cant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designa­ tion (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 
TABLE 4 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT 

SPECIFIC SITES IN FEMALE RATS TREATED WITH 3-SULFOLENE AND 

SURVIVING AT LEAST 52 WEEKSa 

VEHICLE LOW HIGH 

TOPOGRAPHY: MORPHOLOGY CONTROL DOSE DOSE 

Subcutaneous Tissue: Fibroma 0/20(0.00) 3/49(0.06) 0/39(0.00) 

P Values0 N.S. N.S. N.S. ___ 

Relative Risk (Vehicle Control) Infinite 

Lower Limit 0.255 

Upper Limit Infinite 

Weeks to First Observed Tumor 111 

Pituitary: Chromophobe Adenoma 7/20(0.35) 22/49(0.45) 6/39(0.15) 

P Values0 P = 0.029(N) N.S. N.S. 

Departure from Linear Trend P = 0.033 

Relative Risk (Vehicle Control) 1.283 0.440 

Lower Limit 0.657 0.147 

Upper Limit 3.044 1.342 

Weeks to First Observed Tumor 89 77 72 

Thyroid: Follicular-Cell Carcinoma 1/20(0.05) 3/49(0.06) 1/39(0.03) 

P Values0 N.S. N.S. N.S. ___ 

Relative Risk (Vehicle Control) 1.224 0.513 

Lower Limit 0.108 0.007 

Upper Limit 62.958 39.256 

Weeks to First Observed Tumor Ill 109 92 
___ 

TOPOGRAPHY: MORPHOLOGY
 

Thyroid: Fcllicular-Cel] Carcinoma or
 

Follicular-Cell Adenoraab
 

P Values0
 

Relative Risk (Vehicle Control)d
 

Lower Linit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pancreatic Islets: Islet-Cell Adenoma
 

or Islet-Cell Carcinoma'-'
 

ho 

P Values0
 

Relative Risk (Vehicle Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tutor
 

Mammary Gland : Fibre adenoma
 

P Values0
 

Relative Risk (Vehicle Control)0
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (CONTINUED) 

VEHICLE
 

CONTROL
 

2/20(0.10)
 

N.S.
 

Ill
 

0/20(0.00)
 

N.S.
 

6/20(0.30)
 

N.S.
 

106
 

LOW
 

DOSE
 

4/49(0.08)
 

N.S.
 

0.816
 

0.131
 

8.603
 

109
 

1/49(0.02)
 

N.S.
 

Infinite
 

0.023
 

Infinite
 

111
 

17/49(0.35)
 

.N.S.
 

1.156
 

0.530
 

3.142
 

75
 

HIGH
 

DOSE
 

2/39(0.05)
 

N.S.
 

0.513
 

0.040
 

6.709
 

92
 

3/39(0.08)
 

N.S.
 

Infinite
 

0.321
 

Infinite
 

102
 

9/39(0.23)
 

N.S.
 

0.769
 

0.295
 

2.306
 

102
 
___ 

___ 

TOPOGRAPHY:MORFHOLOGY
 

Mammary Gland: Adenoma NOS or
 

Adenocarcinorca NOS
 

P Values0
 

Relative Risk (Vehicle Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Adenocarcinoma NOS,
 Adenoma NOS, or Fibroadenoma''
 

to 

P Values0 

Relative Risk (Vehicle Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Ovary: Granulosa-Cell Tumor
 

P Values0
 

Relative Rick (Vehicle Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (CONTINUED) 

VEHICLE
 

CONTROL
 

1/20(0.05)
 

N.S.
 

Ill
 

7/20(0.35)
 

N.S.
 ___.
 

106
 

1/20(0.05)
 

N.S.
 

—_
 

Ill
 

LOW
 

DOSE
 

5/49(0.10)
 

N.S.
 

2.041
 

0.254
 

94.440
 

93
 

20/49(0.41)
 

N.S.
 

1.166
 

0.587
 

2. 810
 

75
 

1/48(0.02)
 

N.S.
 

0.417
 

0.006
 

32.058
 

111
 

HIGH
 

DOSE
 

5/39(0.13)
 

N.S.
 

2.564
 

0.320
 

117.857
 

102
 

11/39(0.28)
 

N.S.
 

0.806
 

0.351
 

2.117
 

102
 

4/39(0.10)
 

N.S.
 

2.051
 

0.225
 

98.244
 

71
 
TABLE 4 (CONCLUDED)
 

TOPOGRAPHY :MORPHCLOGY 

VEHICLE 

CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Uterus: Endometrial Sternal Polyp 

? Values0 

1/20(0.05) 

N.S. 

4/48(0.08) 

N.S. 

0/39(0.00) 

N.S. 

Relative Risk (Vehicle Control) 

Lower Limit 

Upper Limit 

1.667 

0.192 

80.315 

0.000 

0.000 

9.531 

Weeks to First Observed Tumor Ill 111 

treated groups received time-weighted average doses of 120 or 240 mg/kg by gavage.
 

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

The probability level for the Cochran-Armitage test is given beneath the incidence of tumors in
 

the control group when P < 0.05; otherwise, net significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison of a treated group with the control group is
 

giver beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 «
 

The probability level of the test for departure from linear trend is given beneath the control
 

group when P < 0.05.
 
None of the statistical tests for any site in rats of either sex
 

indicated a significant positive association between the administration of 3-sulfolene and tumor incidence. Thus, there was no evidence
 

that 3-sulfolene was a carcinogen in Osborne-Mendel rats.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

risk have been estimated and entered in the tables based upon the
 

observed tumor incidence rates. In all of the intervals shown in
 

Tables 3 and 4, the value one is included; this indicates the absence
 

of statistically significant results. It should also be noted that
 

all of the confidence intervals have an upper limit greater than one,
 

indicating the theoretical possibility of tumor induction in rats by
 

3-sulfolene that could not be established under the conditions of
 

this test.
 

28
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Mean body weight depression was not observed when comparing
 

treated mice with controls (Figure 3). Fluctuations in the growth
 

curve may be due to mortality; as the size of the group diminishes,
 

the mean body weight may be subject to wide variations.
 

There was no definitive evidence of compound effect on physical
 

appearance and behavior; however, a rapid decline in survival among
 

the high dose mice, beginning in week 18 of the study, was considered
 

to be a result of compound toxicity.
 

Signs commonly observed in group-housed laboratory mice were observed at a comparable rate in treated and control mice. These signs
 

included sores on the body, localized alopecia, a hunched appearance,
 

genital irritation, roughened or stained fur, bloated appearance, and
 

palpable nodules or tissue masses.
 

B.	 Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and 3-sulfolene-dosed groups are shown in Figure 4.
 

For male mice the Tarone test indicated a significant (P < 0.001)
 

positive association between dosage and mortality when comparing the
 

dosed group to the vehicle control. Due to the accelerated mortality
 

in the high dose group, the departure from linear trend was also
 

significant (P < 0.001). In the high dose treated group 52 percent
 

(26/50) had died after only 21 weeks, with 24 percent (12/50) surviving
 

29
 
-50 

1—40 

1-30 

MALE MICE 

30 45 60 75 

TIME ON TEST (WEEKS) 

90 

UNTREATED CONTROL 

VEHICLE CONTROL 

LOW DOSE 

HIGH DOSE 

105 

T 

1—20 

120 

50­ -50 

40—I 

< 

cc 

r 

45 

T 

60 75 

TIME ON TEST (WEEKS) 

90 

UNTREATED CONTROL 

VEHICLE CONTROL 

LOW DOSE 

HIGH DOSE 

105 

T 

\-20 

120 

FIGURES 

GROWTH CURVES FOR 3-SULFOLENE CHRONIC STUDY MICE 

30 
PROBABILITY OF SURVIVAL PROBABILITY 

o o P o o 0 o 

3 0 (0 * 

o Kj ^ b> bo i 

0 o

i 1 i 1 I i i 1 i 1 1 o — i 1 i 

o ~ 

1 \

1 s 

1 I 5 1 1 1 

ir~ 1

 i- (P —

1 I I 

i ° TM i 

g S I 5 

yj I i i| ^ 3 r 

- I 8 § SH / : 8 IK > rJ' 

m m "m j— _; S2 m 0 OS ff1

 f 0 CO r 3 r^ 

1§ ^— 8- 1 S j 

?l -f"^ ? i/ 

_ - ? 1 ?i 00 =! ft- z

2

! £­ s O m m O 

m

/ ' O 

r

f^ 

z 

H 

5q s- m I 

r sq sm r 

fTI "" m 

r m / 

^ i 

• 7^ 

o C/) C/) 

i

i 

3D Oi TM" ^l 

01 . 

t^»T 

• f 

o - - 1

r

j 

i 

o g- i 8­ r* 

2 ' 

o - Tl ­ m 2 S > > i- a- m S a- g _ s 

O o m m 

§o - i I i I i I i I i | fO 

o c 

1

 i ' 1 ' 

o o o o r* c c io * b> bo o c 0 o 

w '*• 
on test at least 65 weeks. Of the high dose mice that died before
 

week 66, only two were reported to have lesions of any type; thus
 

early mortality was not tumor-related. Survival was better in the
 

low dose and control groups, with 76 percent (38/50) of the low dose
 

treated group, 60 percent (12/20) of the vehicle controls, and 85
 

percent (17/20) of the untreated controls surviving on test at least
 

75 weeks. In the low dose, vehicle control and untreated control
 

groups, adequate numbers of mice were at risk from late-developing
 

tumors. In the high dose group, however, survival was not adequate.
 

For female mice the Tarone test also showed a significant (P <
 

0.001) positive association between dosage and mortality when comparing the dosed groups to the vehicle control. The accelerated mortality in the high dose group resulted in a significant (P < 0.001)
 

parture from linear trend. In the high dose treated group the median
 

survival was 33 weeks with only 22 percent (11/50) of the animals
 

surviving on test at least 52 weeks. Of the high dose females that
 

died before week 52 only two were reported to have lesions of any
 

type; thus early mortality was not tumor-related. Survival was good
 

in the other groups with 90 percent (45/50) of the low dose group,
 

85 percent (17/20) of the vehicle controls, and 95 percent (19/20) of
 

the untreated controls alive on test at the end of the study. In the
 

low dose group and in both control groups, adequate numbers of mice
 

were at risk from late-developing tumors. In the high dose group,
 

however, survival was not adequate.
 

32
 
C. Pathology
 

Histopathologic findings on neoplasms in mice are summarized in
 

Appendix B (Tables Bl and B2); findings on nonneoplastic lesions are
 

summarized in Appendix D (Tables Dl and D2).
 

Administration of 3-sulfolene resulted in high mortality in high
 

dose male and female mice. Only 10 percent of the high dose females
 

and 16 percent of the high dose males survived the experiment. Therefore, the low dose groups may be the only groups about which a conclusion of carcinogenicity can be made.
 

Hepatocellular carcinomas occurred in 2/20 (10 percent) untreated
 

control, 1/20 (5 percent) vehicle control, 11/50 (22 percent) low
 

dose, and 5/49 (10 percent) high dose males, and 1/49 (2 percent) low
 

dose females. However, early deaths occurred in the high dose male
 

and female groups from weeks 15 through 30 of the experiment, and
 

only eight high dose males and five high dose females remained alive
 

at the termination of the test (91 weeks). Survival in the low dose
 

male and female groups was 36/50 and 45/50, respectively. The incidence of hepatocellular carcinomas in the low dose male group was
 

considered to be within the normal range of such lesions seen in other
 

studies conducted at this laboratory.
 

The incidence of inflammatory, degenerative, and proliferative
 

lesions was also reduced in the high dose mice of both sexes due to
 

early deaths. There were no pathologic alterations attributed to
 

administration of the chemical.
 

33
 
In conclusion, the administration of the high doses of 3-sulfolene increased mortality in mice of both sexes, thus potential carcinogenic effect could not be evaluated in these groups. Survival of
 

animals receiving low doses was believed to be sufficient to conclude
 

that there was no tumorigenic effect at that concentration.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

mice are summarized in Tables 5 and 6. The analysis is included for
 

every type of tumor in either sex where at least two such tumors were
 

observed in at least one of the control or 3-sulfolene-dosed groups
 

and where such tumors were observed in at least 5 percent of the
 

group. Because of high early mortality noted in mice of both sexes,
 

these analyses were based on those mice surviving at least 52 weeks
 

or, in the event that the tumor of interest was observed earlier than
 

52 weeks, these surviving at least until the first tumor of interest
 

was observed.
 

In male mice an increased incidence of hepatocellular carcinomas
 

was observed in the treated groups compared to the controls. The
 

Cochran-Armitage test indicated a significant (P = 0.040) positive
 

association between dose and incidence. The Fisher exact tests, however, were not significant at these low sample sizes.
 

No other tests from sites in either male or female mice were
 

significant. Based upon these results there was no statistical
 

evidence of the carcinogenicity of 3-sulfolene in mice.
 

34
 
_ __ 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH 3-SULFOLENE AND
 

SURVIVING AT LEAST 52 WEEKS3
 

TOPOGRAPHY: MORPHOLOGY
 

Subcutaneous Tissue: Fibrosarcoma
 

P Values0
 

Relative Risk (Vehicle Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

OJ Lung: Alveolar /Bronchiolar Adenoma or
 

Alveolar /Bronchiolar Carcinoma 'e
 

? Values
 

Relative Risk (Vehicle Control)0
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Heraatopoietic System: Malignant
 

Lymphoma°
 

P Values0
 

Relative Risk (Vehicle Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First. Observed Tumor
 

VEHICLE
 

CONTROL
 

2/18(0.11)
 

N.S.
 

——
 

77
 

0/18(0.00)
 

N.S.
 

1/18(0.06)
 

N.S.
 

76
 

LOW
 

DOSE
 

0/45(0.00)
 

N.S.
 

0.000
 

0.000
 

1.341
 

4/50(0.08)
 

N.S.
 

Infinite
 

0.350
 

Infinite
 

17
 

6/45(0.13)
 

N.S.
 

2. 400
 

0.329
 

107.714
 

72
 

HIGH
 

DOSE
 

0/16(0.00)
 

N.S.
 

0.000
 

0.000
 

3.593
 

2/42(0.05)
 

N.S.
 

Infinite
 

0.132
 

Infinite
 

91
 

2/15(0.13)
 

N.S.
 

2.400
 

0.138
 

131.036
 

78
 
TABLE 5 (CONCLUDED)
 

VEHICLE LOW HIGH 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE DOSE 

Liver: Hepatocellular Carcinoma 1/18(0.06) 11/45(0.24) 5/15(0.33) 

P Values0 P = 0.040 N.S. N.S. ___ Relative Risk (Vehicle Control) 4.400 6.000 

Lower Limit 0.732 0.782 

Upper Limit 184.020 256.215 

Weeks to First Observed Tumor 72 73 81 

Treated groups received time-weighted average doses of 311 or 622 mg/kg by gavage.
 

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

°The probability level for the Cochran-Armitage test is given beneath the incidence of tumors in
 

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison cf a treated group with the control group is
 

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 

e

For sites where the first tumor of interest was observed earlier than 52 weeks, the analyses
 

were based upon all animals that survived until or past the date that the first tumor was observed.
 
TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH 3-SULFOLENE AND
 

SURVIVING AT LEAST 52 WEEKSa
 

TOPOGRAPHY :MCRPHOLOGY
 

Lung: Alveolar/Bronchiclar Adenoma or
 

Alveolar/Brcnchiolar Carcinoma*-1
 

P Values0
 

Relative Risk (Vehicle Control)0
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Malignant Lymphoma
 

P Values0
 

Relative Risk (Vehicle Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tuiror
 

Pituitary: Chromophobe Adenoma
 

P Values0
 

Relative Risk (Vehicle Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

VEHICLE	 LOW
 

CONTROL	 DOSE
 

1/18(0.06) 5/45(0.11)
 

N.S.	 N.S.
 

2.000
 

0.252
 

92.361
 

90 91
 

1/18(0.06) 3/45(0.07)
 

N.S.	 N.S.
 

1.200
 

0.107
 

61.573
 

90 91
 

0/18(0.00) 2/39(0.05)
 

N.S.	 N.S.
 

Infinite
 — —
 0.143
 

Infinite
 

91
 

HIGH
 

DOSE
 

1/11(0.09)
 

N.S.
 

1.636
 

0.022
 

116.035
 

91
 

0/11(0.00)
 

N.S.
 

c.ooo
 

0.000
 

28.506
 

0/6(0.00)
 

N.S.
 
00 

TABLE 6 (CONCLUDED)
 

treated groups received time-weighted average doses of 384 or 768 mg/kg by gavage.
 

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

The probability level for the Cochran-Armitage test is given beneath the incidence of tuiwjrs in
 

the control group When P < ti.05; otherwise, not significant (N.S.) is indicated. The probability
 

level for the Fisher exact test for the comparison of a treated group with the control grtup is
 

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not signifi­ cant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests s negative designa­ tion (N) indicates a lower incidence in the treated group(s) than in the control group.
 

Tlie 95% confidence interval on the relative risk of the treated group to the control group.
 
To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

risk have been estimated and entered in the tables based upon the
 

observed tumor incidence rates. In all of the intervals shown in
 

Tables 5 and 6, the value one is included; this indicates the absence
 

of statistically significant results. It should also be noted that
 

all of the confidence intervals have an upper limit greater than one,
 

indicating the theoretical possibility of tumor induction in mice by
 

3-sulfolene that could not be established under the conditions of
 

this test.
 

39
 
V. DISCUSSION
 

There was a significant positive association between the administered dosages of 3-sulfolene and mortality in both sexes of rats
 

and mice. In all groups, except the high dose male rats and the high
 

dose male and female mice, adequate numbers of animals survived sufficiently long to be at risk from late-developing tumors.
 

Among rats evidence of the toxicity of 3-sulfolene that led to
 

accelerated mortality was morphologically reflected primarily in the
 

circulatory, urinary, biliary, and reproductive systems. There were
 

no significant positive associations between administration of the
 

compound and the incidences of any tumor in either sex.
 

When hepatocellular carcinomas were statistically analyzed in
 

male mice surviving at least 52 weeks the incidences utilized were
 

1/18 (6 percent), 11/45 (24 percent), and 5/15 (33 percent) in the
 

vehicle control, low dose, and high dose groups. Although there was
 

a significant positive association between dosage and the incidences
 

of this neoplasm in males, this finding was not supported by significant results from any of the Fisher exact comparisons using these low
 

sample sizes. There were no other tumors in mice of either sex for
 

which a significant positive association between chemical administration and incidence could be established.
 

Under the conditions of this bioassay 3-sulfolene administered
 

by gavage was not carcinogenic to Osborne-Mendel rats or B6C3F1 mice.
 

40
 
These findings are supported by a previous study (Van Duuren -

et
 

al., 1971) in which 3-sulfolene was tested for carcinogenicity in
 

female ICR/Ha Swiss mice, along with a number of other small ring
 

compounds including several monoepoxides and one acetal. In contrast
 

to two structurally similar compounds, 3,4-epoxysulfolane and 1-propanesulfonic acid-3-hydroxy-Y-sultone, which induced a variety of
 

benign and malignant tumors under similar testing conditions, weekly
 

subcutaneous injections of 3-sulfolene in 30 mice for up to 93 weeks
 

produced no tumors of any nature.
 

41
 
VI. BIBLIOGRAPHY
 

Armitage, P., Statistical Methods in Medical Research, Chapter 14.
 

J. Wiley & Sons, New York, 1971.
 

Berenblum, I., editor, Carcinogenicity Testing. International Union
 

Against Cancer, Technical Report Series, Vol. 2. International
 

Union Against Cancer, Geneva, 1969.
 

Chemical Abstracts Service. The Chemical Abstracts Service (CAS)
 

Ninth Collective Index, Volumes 76-85, 1972-1976. American
 

Chemical Society, Washington, D.C., 1977.
 

Cox, D.R., Analysis of Binary Data, Chapters 4 and 5. Methuen and
 

Co., Ltd., London, 1970.
 

Cox, D.R., "Regression Models and Life-Tables." Journal of the Royal
 

Statistical Society, Series "B" 34:187-220, 1972.
 

Cart, J.J., "The Comparison of Proportions: A Review of Significance
 

Tests, Confidence Limits, and Adjustments for Stratification."
 

International Statistical Institute Review 39:148-169, 1971.
 

Kaplan, E.L., and P. Meier, "Nonparametric Estimation from Incomplete
 

Observations." Journal of the American Statistical Association
 

21:457-481, 1958.
 

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

"Carcinogenesis Bioassay Data System." Computers and Biomedical
 

Research 7:230-248, 1974.
 

Miller, R.G., Simultaneous Statistical Inference. McGraw-Hill Book
 

Co., New York, 1966.
 

Reuber, M.D., and E.L. Glover, "Cirrhosis and Carcinoma of the Liver
 

in Male Rats Given Subcutaneous Carbon Tetrachloride." Journal
 

of the National Cancer Institute 44:419-423, 1970.
 

Rose, A. and E. Rose, The Condensed Chemical Dictionary, Seventh
 

edition. Van Nostrand Reinhold Company, New York, 1966.
 

Saffiotti, U., R. Montesano, A.R. Sellakumar, F. Cefis, and D.G.
 

Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced
 

by Different Numbers of Administration of Benzo (a) Pyrene and
 

Ferric Oxide." Cancer Research 32:1073-1079, 1972.
 

42
 
Stanford Research Institute, 1977 Directory of Chemical Producers,
 

U.S.A. Menlo Park, California, 1977.
 

Tarone, R.E., "Tests for Trend in Life-Table Analysis." Biometrika
 

62_:679-682, 1975.
 

Van Duuren, B.L., S. Melchionne, R. Blair, B.M. Goldschmidt, and C.
 

Katz, "Carcinogenicity of Isoesters of Epoxides and Lactones:
 

Aziridine Ethanol, Propane Sultone, and Related Compounds."
 

Journal of the National Cancer Institute 46(1):143-149, 1971.
 

43
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH 3-SULFOLENE
 

TABLE Al

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH 3-SULFOLENE 

CONTBOL(UNTR)

01-1<MM

 CONTBOL(VEH) 

 01-101M 

LOU DOSE 

01-102M 

HIGH DOSE 

01-103M 

ANIMALS

ANIMALS

ANIMALS

 INITIALLY IN STUDY

 NECBOPSIED

 EXAMINED HISTOPATHOLOGICALLY**

 20

 20

 20

 20 

 20 

 20 

50 

50 

50 

50 

50 

50 

INTEGUHENTAB Y SYSTEM 

*SKIN 

BASAL-CELL CABCINONA 

(20) (20) (50) (50) 

1 <2X) 

*SOBCOT TISSUE 

FIBBOMA 

FIBROSABCONA 

(20)

1 <5X)

 (20) 

1 (5X) 

2 (10X) 

(50) 

1 (2X) 

2 (»X) 

(50) 

jiESPIBATORY SYSTEM 

ALVEOLAR/BRONCHIOLAR CABCINOHA 

(20) (20) (50) 

<2X) 

(50) 

HEMATOPOIE1IC SYSTEB 

'ABDOMINAL CAVITY

MALIG. LYMPHOMA, HISTIOCYTIC TYPE 

 (20) (20) (50) 

1 (2X) 

(50) 

ISPLEEN

HEMANGIOMA 

HEHANGIOSABCOMA

 (19) 

2 (11X) 

(20) 

1 <5X) 

(50) 

1 (2X) 

(50) 

IMESENTEBIC L. NODE

HEMANGICMA 

 (19) (18) 

1 (6X) 

(«5) (30) 

CIBCULATOBY SYSTEfl 

NONE 

DIGESTIVE SYSTEM 

•SALIVARY GLAND (10) (11) 

1.S3.U . 

(27) (1) 

* NUHBEB OF ANIMALS HITH TISSDE EXAMINED

* NUMBER OF ANIHALS NECEOPSIED 

**EXCLUDES PARTIALLY ABTOLYZED ANIMALS 

 MICROSCOPICALLY 

A-3 
TABLE Al (CONTINUED) 

CONTROL(UNTR)

01-111H

 CONTROL(VEH)

 01-101H

 LOU DOSE

 01-102M

 HIGH DOSE 

 01-103H 

•LIVER 

HEPATOCELLULAR CARCINOMA 

•PANCREAS 

FIBROUS HISTIOCYTOHA, METASTATIC 

• STOMACH 

LEIOMYOSA6COMA 

(20) 

(19) 

(20) 

(20) 

(20) 

1 (5X) 

(20) 

(50) 

1 (2X) 

(50) 

(50) 

(50) 

(50) 

(50) 

UBINARY SYSTEM 

•KIDNEY 

FIBROUS HISTIOCYTOHA, HETASTATIC 

HIKE D TDHOR, MALIGNAN T 

(19) 

1 (5*) 

(20) 

1 (5X) 

(50) (50) 

ENDOCRINE SYSTEM 

• PITUITARY 

CHROMOFHOBE

CHROMOPHOBE

 ADENOMA 

 CARCINOHA 

(19)

3 (16*) 

(19) 

2 (11*) 

(50) 

3 (6X) 

(18) 

1 (2X) 

• ADRENA L 

PHEOCHROHUCYTOMA 

•THYROID 

FOLLICULAR-CELL ADENOMA 

FOLLICULAR-CELL CARCINOHA 

C-CELL CAECINOHA 

(19) 

(19) 

1 (5*) 

(20) 

1 (-5*) 

(20) 

1 (5X) 

1 (5*) 

(50) 

(50) 

2 <4X) 

(50) 

(49) 

•PANCREATIC ISLETS 

ISLET-CELL ADENORA 

(19) 

2 (11*) 

(20) (50) (50) 

REPRODUCTIVE SYSTEM 

•MAHHAHY GLAND 

ADENOCARCINOHA, NOS 

PAPILLARY CYSTADENOCARCISONA.NOS 

MEDULLARY CARCINOMA 

FIBBOADENOHA 

(20) 

1 (5*) 

(20) (50) 

1 (2*) 

1 <2X) 

(50) 

NERVOUS SYSTEM 

• BRAIN (19) (20) (50) (50) 

• NUMBER

* NUMBER

 OF ANIMAL S

 OF ANIMAL S

 WITH TISSUE

 NECROPSIED 

 EXABINED MICROSCOPICALLY 

A-4 
TABLE Al (CONTINUED) 

CONTBOL(UNTH)

01-1U1H

 COMTBOL(VEH)

 01-101M

 LOW DOSE

 01-102M

 HIGH DOSE 

 01-103M 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELMAL SYSTEM 

•SKELETAL MUSCLE

FIBROUS HISTIOCYTOMA, METASTATIC

 (20) (20)

 1 (5*) 

 (50) (50) 

BODY CAVITIES 

•PERITONEU M

FIBROUS HISTIOCYTOMA, MALIGNANT

 (20) (20)

 1 (5«) 

 (SO) (50) 

ALL OTHER SYSTEMS 

•MULTIPLE ORGAN S

FIBROUS HISTIOCYTOMA, MALIGNAN T

 (20) (20) (50)

 1 (2%) 

 (SO) 

ANIMA L DISPOSITION SUMMAR Y 

i>

ANIMAL S INITIALLY IN STUD Y

NATURA L DEATHS

MORIBUN D SACRIFICE

SCHEDULED SACHIFICE

ACCIDENTALLY KILLED 

TERMINA L SACRIFICE

ANIMA L MISSING 

 INCLUDES tUIOLlfZED AHIMilS 

 20

 11t

 6 

 20

 S

 7 

8 

 50

 17

 1

2 

 50 

 43 

1 

6 

* NUMBER

* NUMBER

 OF

 OF

 ANIMALS WITH TISSUE

 ANIMAL S NECEOPSIED 

 EXAMINED MICROSCOPICALLY 

A-5 
TABLE Al (CONCLUDED) 

COHTBOL (UNTE) CONTROL (VEH) L OU DOSE HISH DOSE 

01-141M 01-101M 01-102M 01-103H 

TUMOR SUMMAR Y 

TOTAL ANIMALS KITH PRIHARY TUMORS' 9 9 13 

TOTAL PRIHARY TUMORS 12 13 17 

TOTAL ANIMALS KITH 6 BENIGN TUMORS 7 4 

TOTAL BENIGN TUMORS 7 7 5 

TOTAL ANIMALS VITR MALIGNANT TUMORS 5 4 10 

TOTAL MALIGNANT TUMORS 5 6 12 

TOTAL ANIMALS KITH SECONDARY TUMOBS* 1 1 

TOTAL SECONDARY TUMORS 1 4 

TOTAL ANIMALS HUH TUMOBS UNCERTAINBENIGN OR MALIGNAN T

 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TOHORS UNCERTAINPRIMARY OR METASTATIC

 

TOTAL UNCERTAI N TUMORS 

* PRIMAR Y TUMORS : ALL TUMORS EXCEPT SECONDARY TUMORS 

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

A-6 
TABLE A2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED WITH 3-SULFOLENE 

CONTROL (UNTR ) CONTROL (VEH) 

01-1U1F 01-101F 

ANIMALS INITIALLY IN STUDY 20 20 

ANIMALS NECROPSIED 20 20 

ANIMALS EXAMINE D HISTOPATHOLOGICALLY *» 20 20 

INTEGUMENTAR Y SYSTEM 

•SUBCUT TISSUE (20) (20) 

FIBROMA 

FIBKOSA8COHA 1 I5X)

 

FIBROUS HISTIOCYTOHA, MALIGNANT 

HEMANSIOSARCOMA 

RESPIRATORY SYSTEM 

• LUNG (20) (20)

 BILE DUCT CARCINOMA, METASTATIC

 

FIBROUS HISTIOCYTOMA, METASTATIC 

HEMATOPOIETIC SYSTEM 

tSPLEEN (20) (20)

 

BILE DUCT CARCINOMA, METASTATIC 

CIRCULATORY SYSTEM 

•MESENTERIC AiTEHY (20) (20)

FIBROUS HISTIOCYTOMA, METASTATIC 

DIGESTIVE SYSTEM 

• LIVER (20) (20)

NEOPLASTIC NODULE 1 (5X) 

FIBROUS HISTIOCYTOMA, METASTATIC 

•BILE DUCT (20) (20)

— £I,LE DUCT_ C.AECI.NP. JJA^ «« 

* NUMBE R OF ANIMALS HITK TISSUE EXAMINE D MICROSCOPICALLY 

> NUMBER OF ANIMALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

LOU DOSE 

01-1011? 

50 

50 

50 

(50) 

3 (6X) 

1 (2X) 

(50) 

1 (2») 

(50)

1 (2X) 

 (50) 

 (50) 

2 (<**) 

 (50) 

1.12SL 

HIGH DOSE 

01-105F 

50 

50 

50 

(50) 

1 (2«) 

1 (2*) 

(50) 

1 (2*) 

 (U9) 

(50) 

1 (2X) 

(50) 

1 (2X) 

(50) 

A-7 
TABLE A2 (CONTINUED) 

CONTROL (UNTR) CONTROL (VEH) LOB DOSE HIGH DOSE 

01-11I1P 01-101F 01-104F 01-105? 

•PANCREAS (20) (20) (50) (50) 

BILE DUCT

FIBROUS HI

 CARCINOMA , METASTATIC 

STIOCYTOMA, METASTATIC 

1 (2X) 

1 (2X) 

• STOMAC H (20) (20) (50) (50) 

BILE DUCT CARCINOMA, METASTATIC 1 <2X) 

•SMALL INTESTINE (20) (20) (50) (50) 

BILE DUCT CARCINOMA, METASTATIC 1 (2X ) 

URINAR Y SYSTEM 

•KIDNEY (20) (20) (50) (50) 

FIBBOUS HISTIOCYTOMA, METASTATIC 1 (2X ) 

LIPOHA 1 (2X) 1 (2X) 

HAHARTOM A + 1 (5X) 1 (2X) 1 <2X ) 

• URINAR Y BLADDER (20) (20) (50) (U9 ) 

FIBROUS HISTIOCYTOMA, HETASTATIC 1 (2X ) 

ENDOCRIN E SYSTEM 

•PITUITARY (20) (20) (50) (50) 

CHROMOPHOBE ADENOM A 8 (UOX) 7 (35X) 22 (UlIX) 6 (12X) 

• ADRENA L (20) (20) (50) (50) 

CORTICAL CARCINOM A 

PHEOCHROMOCYTOMA 1 <5X) 

•THYROID (19) (20) (50) (50) 

FOLLICULAR-CELL ADENOM A 1 (2X) 1 (2X) 

FOLLICULAB-CELL CABCINOBA 1 (5*) 3 <6X) 1 <2*> 

C-CELL ADENOMA 2 (11X) 2 (IX) 

•PANCREATIC ISLETS (20) (20) (50) (50) 

ISLET-CELi. ADENOM A 1 (5X) 1 <2X) 2 (IX) 

ISLET-CELL CARCINOMA 1 (2X) 

REPRODUCTIVE SYSTEM 

*MAMHAE Y GLAN D (20) (20) (50) (50) 

ADENOMA , NOS 1 <5X) 1 (2X) 

ADENOCARCINOMA , NOS 5 (10X) » <8X) 

6.H2SL. 2-IiaSL.-. 

* MUMBfiR OF ANIMALS HITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECBOPSIED 

+ THIS IS CONSIDERED TO BE A BENIGN FORM OF THE MALIGNANT MIXED TUMOR OF THE KIDNEY AND CONSISTS OF PROLIFERATIVE 

LIPOCYTES, TUBULAR STRUCTURES, FIBROBLASTS, AND VASCULAR SPACES IN VARYING PROPORTIONS. 

A-8 
TABLE A2 (CONTINUED) 

CONTROL(UNTR) 

D1-1U1 F 

CONTROL (VEH) 

01-101F 

L0» DOSE 

01-101F 

HIGH DOSE 

01-105F 

•VAGINA

ENDOMETBIA L STBOMAL SARCOMA , SET 

KUTEBU S

ADENOCARCINOMA, NOS 

ENDOMETBIA L STBOMA L POLYP

ENDOHETBIA L STBOHAL SARCOM A 

•OVAR Y

BILE DUCT CARCINOMA , METASTATIC

GSANULOSA-CELL TUMO R

SEBTOLI-CELL TUMO R

FIBROUS HISTIOCYTOMA, METASTATIC

 (20) 

 (20) 

1 (5X) 

 (20)

(20) 

1 (5X ) 

(20) 

1 (5%) 

1 (5X) 

1 (5X) 

 (20)

 1 (5»)

(50) 

a (8%) 

 (U9 )

 1 (2%) 

1 (2X)

 1 (2K) 

(50) 

(50) 

 (50) 

t (8X) 

1 (2*) 

NERVOU S SYSTEM 

HCEREBBU M

OLIGODENDSOGLIOM A

 (20) (20) (50)

 1 (2X) 

 (50) 

SPECIAL SENSE ORGAN S 

NON E 

MUSCULOSKELETA L

NON E 

 SYSTEM 

BODY CAVITIES 

NON E 

A LL OTHER SYSTEMS 

*MULTIPLE ORGAN S (20) (20)

£IBRO]JS_HISTIOCYTO«Ai_MALIGNAl!l­ ­

* NUMBE R OF AKIMAL S KITH TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S NECROPSIED 

 (50) (50) 

A-9 
TABLE A2 (CONCLUDED) 

COHTBOL(ONTR) 

01-141F 

CONTBOL(?£H) 

01-101P 

L OU DOSE 

01-104P 

HIGH DOSE 

01-105F 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURA L DEATHS 

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINA L SACRIFICE 

ANIMA L MISSING 

20 

8 

12 

20 

6 

14 

50 

16 

1 

33 

50 

28 

2 

20 

INCLUDES AUTOLYZED ANIMALS 

TUMOR SUMMARY 

TOTAL ANIMALS WITH PRIMARY TUMORS* 

TOTAL PRIMARY TUMORS 

13 

18 

14 

22 

39 

68 

21 

33 

TOTAL ANIMALS KITH BENIGN TUMORS 

TOTAL BENIGN TUMORS 

13 

16 

11 

17 

36 

53 

15 

21 

TOTAL ANIMALS KITH MALIGNANT TUMORS 

TOTAL MALIGNANT TUMORS 

1 

1 

4 

4 

11 

12 

8 

8 

TOTAL ANIMALS KITH SECONDARY TUMORS*

TOTAL SECONDARY TUMORS

 1

 1

 1 

6 

TOTAL ANIMALS KITH TUMORS UNCERTAINBENIGN OR MALIGNANT

TOTAL UNCERTAIN TUMORS

 1

 1

 1

 1

 3 

3 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OB HETASTATIC TOTAL UNCERTAIN TUMOBS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SEC3HDABY TUMOBS 

* SECONDARY TUMORS: HETASTATIC TONOBS 38 TUMORS INVASIVE INTO AN ADJACENT ORGAN 

A-IO
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH 3-SULFOLENE
 

TABLE BI

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH 3-SULFOLENE 

c:ONTROL (UNTR ) 

02-M101 

CONTROL (VEH )

02-M091

 LOW DOSE 

 02-M102 

HIGH DOSE 

02-M10 3 

ANIMAL S

ANIMALS

ANIMALS

 INITIALL Y I N STUDY 

 NECROPSIED 

 EXAMINE D HISTOPATHOLOGICALLY* * 

20 

20 

20 

20

20

20

 50 

50 

 50 

50 

50 

INTEGUMENTAR Y SYSTEM 

*SKIN 

SEBACEOUS ADENOM A 

FIBROSABCOMA 

(20) 

1 (5*) 

(20)

1 (5X) 

 (50) (50) 

1 (2X ) 

•SUBCUT TISSUE 

FIBBOSA8COMA 

(20) (20)

2 (10X) 

 (50) (50) 

BESPIBATORY SYSTEM 

• LUN G 

ALVEOLAB/BRONCHIOLA B

ALVEOLAB/BRONCHIOLA B

 ADENOM A 

 CARCINOM A 

(20) 

1 (5X) 

1 <5X) 

(20) (50) 

1 (8X) 

(18) 

HEMATOPOIETIC SYSTEM 

*MULTIPLE ORGANS 

MALIG.LYMPHOMA ,

MALIG.LYMPHOMA ,

 LYMPHOCYTI C

 HISTIOCYTIC

 TYPE 

 TYPE 

(20) 

2 (10X) 

(20) (50) . 3 <6X) 

1 (2X) 

(50) 

1 (2X) 

1 <2X ) 

•SPLEEN 

MALIG.LYMPHOMA ,

MALIG.LYMPHOMA ,

 LYMPHOCYTIC

 HISTIOCYIIC

 TYPE 

 TYPE 

(20) 

1 <5X) 

1 (5X) 

(20)

1 (5X)

 (50) 

2 (IX) 

(18) 

•MESENTERIC L. NOD E 

BALIG.LYMPHOMA , HISTIOCYTIC TYPE 

(19) (20) (50) (36) 

•LIVES 

MALIG.LYMPHOHA ,

MALIG.LYMPHOMA ,

 LYMPHOCYTIC

 HISTIOCYTIC

 TYPE 

 TYPE 

(20) 

1 (5X) 

(20) (50) («, 

•PROSTATE 

TYPE 

(20) (18) (43) 

» NUMBE R OF ANIMAL S KITH TISSUE EXAMINE D

* NUMBER OF ANIMAL S NECHOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 MICROSCOPICALLY 

B-3 
TABLE Bl (CONTINUED) 

CONTROL (DNTR) CONTROL(VEH) LOR DOSE HIGH DOSE 

02-H101 02-H091 02-M102 02-N103 

•SEMINAL VESICLE (20) (20) (50) (50) 

MAIIS.LYMPHOHA, HISTIOCKTIC TYPE 1 (5*) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEH 

*LIYER (20) (20) (50) (49) 

HEPATOCEL.1.ULAH CARCINOMA 2 (10*) 1 (5X) 11 (22X) 5 (10*) 

HSHANSIOSABCONA 1 (2X) 

U8INABY SYSTEM 

HONE 

ENDOCRINE SYSTEH 

tTHYHOID (19) (19) (17) (15) 

FOLLICULA.-1-CELL ADENOMA 1 (2X) 

HEPHODUCTIVE SYSTEM 

NONE 

NEHVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

NUSCULOSKELETAL SYSTEM 

NONE 

riODY CAVITIES 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIBD 

B-4 
TABLE Bl (CONCLUDED) 

CONTROL (UNTB) CONTBOL (VEH) LOU DOSE HIGH DOSE 

02-M101 02-M091 02-N102 02-N103 

ALL OIHEH SYSTEMS 

NONE 

ANIMA L DISPOSITION SUMMAR Y 

ANIHALS INITIALLY IN STUDY 20 20 50 50 

HATURA L DEATHS 9 11 12 11 

MORIBUN D SACRIFICE 1 2 

SCHEDULED SACRIFICE 1 

ACCIDENTALLY KILLED 

TERMINA L SACRIFICE 11 8 36 8 

ANIMA L MISSING 

9 INCLUDES AUIOLYZED ANIMALS 

TUMOR SUNMABY 

TOTAL ANIMAi-S KITH PRIMAR Y TUHORS« 7 5 20 8 

TOTAL PRIMARY TUMORS 11 9 23 10 

TOTAL ANIHALS WIT H BENIGN TUMORS 1 5 2 

TOTAL BENIGN TUMORS 1 5 3 

TOTAL ANIMAL S KITH MALIGNAN T TUMORS 6 5 15 7 

TOTAL MALIGNAN T TUMORS 10 9 18 7 

IOTAL ANIMALS WITH SECONDARY TUMORS* 

TOTAL SECONDAR Y TUMORS 

TOTAL ANIMALS MITH TUMORS UNCERTAINBENIGN OB MALIGNAN T 

TOTAL UNCERTAI N TUMORS 

TOTAL ANIMAL S HITH TUMORS UNCERTAIN ­

PRIMARY OS METASTATIC 

TOTAL UNCERTAI N TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

I SECONDAR Y TUMORS : METASTATIC TUMORS 3R TUMORS INVASIV E INTO AN ADJACEN T ORGA N 

B-5 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED WITH 3-SULFOLENE 

CONTROL(UNIR) 

02-F101 

CONTROL (VEH) 

02-F091 

LOW DOSE 

02-F104 

HIGH DOSS 

02-F105 

ANIMALS INITIALLY IN STUDY 

ANIMALS NECEOPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 

20 

20 

20 

20 

20 

20 

50 

19 

49 

50 

49 

48 

INTEGUMENTAR Y SYSTEM 

•SUBCUT TISSUE 

OSTEOSAECUMA 

(20) (20) 

1 (5*) 

(49) (49) 

RESPIRATOR Y SYSTE M 

(LUNG 

ALVEOLAR/riRONCHIOLA R

ALVEOLAR/BEONCHIOLA R

 ADENOM A 

 CARCINOM A 

(20) (20) 

1 (5«) 

(49) 

4 <8X) 

1 (2X) 

(47) 

<2X) 

HEMATOPOIETIC SYSTEM 

*MULTIPLE ORGAN S 

MALIG.LYMPHOMA , 

MALIG.LYMPHOMA , 

•SPLEEN 

MALIG.LYMPHOMA , 

HALIG.LYMPHONA , 

tMESENTERIC L. NODE 

MALIG.LYMPHOMA , 

LYMPHOCYTIC 

HISIIOCYTIC 

LYMPH OCYTIC 

HISTIOCYTIC 

HISTIOCYTIC 

TYPE 

TYPE 

TYPE 

TYPE 

TYPE 

(20) 

1

1

(20) 

2

(19) 

 (5X) 

 (5X) 

 (10*) 

(20) 

(19) 

(20) 

1 (5X) 

(49) 

2 (4X) 

(49) 

1 (2X) 

(47) 

(49) 

(48) 

(44) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

»LIVER 

HEPATOCELLULAR CARCINOMA 

(20) (19) (49) 

1 (2*) 

(48) 

URINARY SYSTEM 

NONE 

* NUMBE R OF AMIMAL S UITH TISSUE EXAMINE D

« NUMBER OF ANIMAL S NECROFSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 MICROSCOPICALLY 

B-6 
TABLE B2 (CONTINUED) 

CONTROL(UNTR ) CONTROL (VEH) LOU DOSE HIGH DOSE 

02-F101 02-FOS1 02-F10K 02-F105 

ENDOCRIN E SYSTEM 

IPITUITARY (18 ) (20) (42) (25) 

ChHOMOPHOBE ADENOM A 2 (5X) 

REPRODUCTIV E SYSTEM 

•MAMMAR Y GLAN D (20) (20) (49) (49) 

ADENOCARCINOKA , NOS 1 (2%) 

*UTERUS/ENDOMETRIU » (20) (20) (49) (45) 

ADENOCABCINOMA , NOS 1 (5«) 

*OVARY (20) (20) (49) (44) 

GRANULOSA-CEL L TUMO R 1 (2X) 

NERVOUS SYSTEM 

NON E 

SPECIAL SENSE ORGAN S 

NONE 

MUSCULOSKEIETA L SYSTEM 

NON E 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

t NUMBE R OF ANIMAL S KITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

B-7 
TABLE B2 (CONCLUDED) 

CONTROL(UNTH ) CONTROL(VEH) LOW DOSE HIGH DOSE 

02-F101 02-F091 02-F10H 02-F105 

ANIBAL DISPOSITION SUMBAR Y 

ANIMALS INITIALLY I N STUDY 20 50 50

 

NATURA L DEATHS 45

 

MORIBUND SACRIFICE 

SCHEDULE D SACBIFICE 1

 

ACCIDEHTA1LY KILLED 3

 

TERMINA L SACRIFICE 19 17 45

 

ANIMA L HISSING 

3 INCLUDES AUIOLYZ.I.D ANIMAL S 

TUBOR SOBBAB Y 

TOTAL ANIBALS WITH PRIMAR Y TUMORS * 4 <t 10 

TOTAL fBIMAB Y TUMORS 4 1 13 

TOTAL ANIBAL S WITH BENIG N TUBORS 1 6 

TOTAL BENIG N TUMORS 1 6 

TOTAL ANIMALS WITH MALIGNANT TUMORS <t 3 6 

TOTAL MALIGNAN T TUMORS 1 3 6 

TOTAL ANIBALS WITH SECONDARY TUBORSt 

TOTAL SECONDARY TUBORS 

TOTAL ANIBALS WITH TUMORS UNCERTAIN ­

BENIGN OK MALIGNAN T 1 

TOTAL UNCERTAI N TUMORS 1 

TOTAL ANIBALS KITH TUBORS UNCERTAINPKIMAR Y OB METASTATIC

 

TOTAL UNCERTAI N TUBORS 

* PRIBAR Y 1URORS: ALL TUBORS EXCEPT SECONDARY TUMORS 

t SECONDAR Y TUMORS: BETA3TATIC TUMOPS OH TUBORS INVASIVE INTO AN ADJACENT ORGAN 

B-8 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS TREATED WITH 3-SULFOLENE
 

TABLE Cl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

TREATED WITH 3-SULFOLENE 

" CONTROL (UNTR) 

01-1K1H 

ANIMALS INITIALLY IN STUDY 20 

ANIMALS NECROPSIED 20 

ANIMALS EXAMINE D HISTOPATHOLOGICALLY*' 20 

INTEGUMENTAR Y SYSTEM 

•SKIN 

EPIDERMAL INCLUSION

INFLAMMATION , NOS 

ULCER, FOCAL 

*SUBCUT TISSUE 

EPIDERMAL INCLUSION

RESPIRATOR Y SYSTEM 

• TRACHE A 

ULCER, NOS 

(20) 

 CYST 1 <5i) 

1 <5X) 

(20) 

 CYSI 1 <5X) 

(19) 

INFLAMMATION , CHRONIC 

*LUNG/BHONCHUS (20) 

INFLAMMATION , ACUTE SUPPURATIVE 

•LUNG/BRONCHIOLE 

INFLAMMATION, ACUTE 

• LUN G 

INFLAMMATION , SUPPURATIV E 

INFLAMMATION , ACUTE 

ABSCESS, NOS 

PNEUMONIA , CHRONIC MUEIN E 

INFLAMMATION , GRANULONATOU S 

PESIARTERITIS 

CALCIFICATION, DYSTBOPHIC 

CALCIFICATION, METASTATIC 

*LUNG/ALVEOLI 

CALCIFICATION, NOS 

* NUMBE R OF ANIMAL S WIT H TISSUE

* NUMBER OF ANIMAL S NECROPSIES 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

(20) 

(20) 

16 (SOX) 

(20) 

 EXAMINE D MICROSCOPICALLY 

C-3 

CONTROL (VEH) LOR DOSE HIGH DOSE 

01-101H 01-102H 01-103M 

20 50 50 

20 50 50 

20 50 50 

(20) (50) (50) 

1 (5X) 

1 <2X) 

(20) (50) (50) 

(20) (50) (49) 

1 (2X) 

2 (101) 1 (2X) 

(20) (50) (50) 

1 (2X) 

(20) (50) (50) 

1 (2X) 

(20) (50) (50) 

2 (IX) 

1 <2X) 

1 (2X) 2 (tX) 

15 (75X) 33 (66X) 22 (<t«X| 

1 <5X) 

1 (2X) 

1 (2X) 

1 (5X) 

(20) (50) 

5 (10X) 

(50) 

1 (2X) 
TABLE Cl (CONTINUED)
 

CONTROL (ONTR) CONTROL (VEH) 

01-1U1M 01-101M 

HEHATOPOIETIC SYSTEM 

•BONE MARFOH (20) (20)

 

HYPERPLASIA, HEMATOPOIETIC 1 (5*)

 

•SPLEEN (19) (20)

 

INFLAMMATION , GBANULOBATOOS 

HEMATOPOIESIS 1 (5*) 1 (5X)

 

• LYMPH NODE (19) (18)

 

HYPERPLASIA, NOS 1 (6X)

 

tTHYMUS (16) (10) 

ATEOPHY, NOS 1 (10X) 

CIRCULATOBY SYSTEM 

•HEART (20) (20)

 

THROMBUS, ORGANIZED 

CALCIDM DEPOSIT 2 (10*) 

CALCIFICATION, NOS 

CALCIFICATION, HEIASTATIC 1 (5%)

 

•MYOCARDIUM (20) (20)

 

INFLAMMATION , ACUTE 

FIBHOSIS 1 (SX)

 

FIB80SIS, FOCA1 

NECROSIS, NOS

 

CALCIFICATION, NOS 

CALCIFICATION, MEIASIATIC 

• ENDOCARDIU M (20) (20)

 

HYPERPLASIA, NOS 1 (5X) 

*ARTEBY (20) (20)

 

MINERALIZATION 1 (5X)

 

INFLAMMATION , NOS 1 (5*) 

MEDIAL CALCIFICATION 

NECROSIS, NOS 1 <5X)

 

HYPERPLASiA, HEMATOPOIETIC 1 (5X)

 

* AORTA (20) (20)

 

PERIAHTER1TIS 

MEDIAL CALCIFICATION 3 (15%) 1 (5X)

 c4tci£icAtiojit_!iEiS5;jiic 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-4
 

LOU DOSE 

01-102M 

(49)' 

1 (2X) 

(50) 

1 (2X) 

1 (2X) 

(US) 

(23) 

(50) 

1 (2X) 

3 (6X) 

3 (6X) 

(50) 

13 (26*) 

1 (2X) 

<• (8X) 

1 (2X) 

(50) 

(50) 

2 (UX) 

(50) 

1 (2X) 

10 (20X) 

1-J2SL 

HIGH DOSE 

01-103M 

(50) 

(50) 

(30) 

(21) 

(50) 

(50) 

1 (2X) 

1 (2X) 

1 (2X) 

(50) 

(50) 

(50) 

1 (2X) 

^g_^^^^««—A 
TABLE Cl (CONTINUED)
 

CONTROL (BUTE) CONTROL (TEH) 

01-1U1B 01-101H 

*CORONARY AHTEBY (20) (20)

 

MEDIAL CALCIFICATION 2 (10X) 

CALCIFICATION, METASTATIC 

*PULMONAR Y ASTERY (20) (20)

 

MEDIA L CALCIFICATION 

CALCIFICATION, ME1ASTATIC 

• MESENTERIC ARTER Y (20) (20)

 

MEDIA L CALCIFICATION 1 (5X) 1 (5X)

 

CALCIFICATION, NOS 

CALCIFICATION, METASTATIC 

*IESTICULA8 ARTEE Y (20) (20)

 

MEDIA L CALCIFICATION 

CALCIFICATION, NOS 

DIGESTIVE SYSTEM
 

• LIVER (20) (20)
 

BILE STASIS
 

INFLAMMATION, NOS 1 (5*)
 

INFLAMMATION, FOCAL
 

INFLAMMATION, ACUTE SUPPURATIVE
 

INFLAMMATION, ACUTE NECROTIZING
 

GBANULCMA, NOS
 

PERI ARTHRITIS
 

DEGENERATION, NOS
 

PELIOSIS KEPATIS 3 (15X)
 METAMORPHOSIS FATTY 1 (5X) 1 (5X)
 FOCAL CELLULAR CHANGE
 

ANGIECTASIS
 

*aEPATIC LOBULE (20) (20)
 INFLAMMATION, NECROTIZING
 

DEGENERATION, NOS
 

METAMOEEHOSIS FATTY
 

fLIVES/CENTEILOBULAR (20) (20)
 DEGENERATION, NOS
 

METAMORPHOilS FATTY
 

ILIVEE/PEEIPOETAL (20) (20)
 INFLAMMATION, ACUTE/CHRONIC 1 (5*)
 

* NUMBER OF ANIMALS UITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECEOPSIED
 

C-5
 

L OU DOSE 

01-102M 

(50) 

3 <6X ) 

1 <2X) 

(50) 

lit (28S) 

1 (2X) 

(50) 

9 (18S) 

1 (2X) 

3 (6X) 

(50) 

2 (UX ) 

1 (2X) 

(50)
 

18 (36*)
 

1 (2X)
 

1 (2X)
 

1 (2X)
 

1 (2X)
 <4 (8X)
 

1 (2X)
 

(50)
 

1 (2X)
 

1 (2X)
 

(50)
 

5 (10X)
 

(50)
 

1-12SL-.
 

HIGH DOSE 

01-103M 

(50) 

1 (2X) 

(50) 

1 <2X ) 

(50) 

(50) 

(50)
 

31 (62X)
 

1 (2X)
 

1 (2X)
 

1 (2X)
 

(50)
 

1 (2X)
 

(50)
 

1 (2X)
 

1 (2X)
 

(50)
 
TABLE Cl (CONTINUED) 

CONTROL (UNTR ) CONTROL (VEH) 

01-1U1M 01-101B 

'BILE DUCT (20) (20) 

DILATATION, NOS 

BILE STASIS 

INFLAMMATION , CHRONIC 

HYPEEPLASIA, NOS 5 (25X)

 

IPANCREA S (19) (20)

 

INFLAMMATION , ACUTE/CHRONIC 1 (5X)

 

FIBROSIS 

PERIAR1IRITIS 1 (5X)

 

ATROPHY , NOS 

*PANCREATIC DUCT (19) (20)

 

INFLAMMATION , CHRONIC 

FIBROSIS 

*STOMACH (20) (20)

 

CALCIUM DEPOSIT 3 (15X) 

CALCIFICATION, NOS 

CALCIFICATION, FOCAL 

CALCIFICATION, METASTATIC 1 (5X)

 

tLARGE INTESTINE (20) (18) 

PARASITISM 2 (11X) 

UBINAH Y SYSTEM 

fKIDNE Y (19) (20) 

HYDROHEEH80SI S 

HEMORRHAG E 

PYELONEPHRITIS, NOS 2 (11*) 

PYELONEPHRITIS , ACUTE 

ABSCESS, NOS 

INFLAMMATION , CHRONIC 13 (68X) It (70X) 

PYELONEPHRITIS, CHRONIC 

CALCIUM DEPOSIT 1 (5*) 

CALCIFICATION, NOS 3 (15X) 

CALCIFICATION, METASTATIC 1 (5X) 

tKIDNEY/CAESULE (19) (20)

 

HEMORRHAG E 

*UHETER (20) (20)

 

CALCIFICATION, NOS 

CALCIFICATION. .BETASTATIC _ 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER 0? ANIMALS NECROPSISD
 

C-6
 

HIGH DOSE 

01-103M 

1 <2X ) 

L OU DOSE 

01-102M 

(50) 

9 (18X) 

24 (H8X) 

(50) 

7 (UX) 

(50) 

(50) 

2 (UX ) 

1 (2X) 

10 (20X) 

<U9 ) 

2 (<tX) 

(50) 

1 (2X) 

1 (2*) 

13 (86X) 

1 <2X ) 

7 (14X) 

(50) 

(50) 

« (8X) 

1-I2SL 

(50) 

1

1

12

28

(50) 

1

1

(50) 

1

1

(50) 

1

1

(50) 

(50) 

1

1

»

23

3

1

(50) 

1

(50) 

 (2X) 

 (2X) 

(2 HI) 

 (56%) 

 (2X) 

 <2X ) 

 (2X) 

 (2X) 

 (2X) 

 (2X) 

 (2X) 

 (2X) 

 (8X) 

 (U6X) 

 (6X) 

 <2X) 

 (2X) 
TABLE Cl (CONTINUED)
 

CONTROL (UNTR) CONTROL (VEH) 

01-111K 01-101M 

tUHINAR Y BLALtDKR (19) (19) 

CALCULUS, NOS 

INFLAMMATION , NOS 1 (5X) 

ULCER, NOS 

INFLAMMATION , ACUTE 

ULCER, ACUTE 

INFLAMMATION , ACUTE/CHRONIC 

INFLAMMATION , CHRONIC 

CALCIFICATION, NOS 

HYPERPLASIA , EPITHELIA L 

ENDOCRIN E SYS1SM 

• PITUITARY 

HYPERPLASIA , CHEOMOPHOBE-CEL L 

(19) (19) 

1 (5X) 

• ADRENA L . 

CALCIFICATION, METASTATIC 

(19) (20) 

•ADRENA L CORTEX 

DEGENERATION,

ANGIECTASj. 3 

 NOS 

(19) (20) 

7 (35X) 

1 (5X) 

•THYROI D 

FOLLICULAE CYST, NOS 

(19)

1 <5X) 

 (20) 

• PARATHYROI D 

HYPERPLASIA , NOS 

(19)

1 (5*)

 (17) 

2 (12*) 

REPRODUCTIV E SYSTEM 

•PROSTATE (19) (16) 

INFLAMMATION , NOS 2 (11X) 

INFLAMMATION , SUPPURATIV E 

INFLAMMATION, ACUTE 

INFLAMMATION , ACUTE FOCAL 

INFLAMMATION , ACUTE/CHRONIC 

INFLAMMATION , CHRONIC 1 <6X ) 

• SEMINA L VESICLE (20 ) (20) 

INFLAMMATION , ACUTE 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSISD
 

LOW DOSE 

01-102M 

(50) 

1

1

2

(50) 

(50) 

2

(50) 

20

2

(50) 

(50) 

15

(39) 

1

5

2

1

(50) 

 (2X) 

 (2X) 

 (it*) 

 (It*) 

 (flOX) 

 (<t*) 

 (30*) 

 (3X) 

 (13*) 

 (5X) 

 (3X) 

HIGH DOSE 

01-103M 

(U9 ) 

2 (IX) 

1 (2X) 

3 (6X) 

» (8X) 

1 (2X) 

1 (2X ) 

(48) 

(50) 

(50) 

8 (16*) 

(19) 

(37) 

6 (16*) 

(27) 

1 (15*) 

2 (7X) 

2 <7X ) 

(50) 

1 (2X) 

C-7
 
tTESTIS 

PERIAR1EBITIS 

CALCIFICATION, NOS 

CALCIFICATION, METASTATIC 

ATROPHY , NOS 

KYPOSPEBHAT06ENESIS 

tTESTIS/TUBULE 

CALCIFICATION, NOS 

•S.PIDIDYMIS 

INFLAMMATION , ACUTE FOCAL 

PERIABTEBITIS 

NECROSIS, FAT 

NERVOU S SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

•EYE/COBNEA 

ULCEB, NOS 

NUSCULOSKELETA L SYSTEM 

• BONE 

FIBBOUS OSTEODYSTROPHY 

• STERNU M 

EXOSTOSIS 

•SKELETAL MUSCLE 

INFLAHHATION, NOS 

INFLAHHATION, ACUTE 

BODY CAVITIES 

• PERITONEU M 

INFLAMMATION, FOCAL 

INFLAJJH4T.IQ1I. ACyjE £QQ&L. 

f NUNBE B OF ANIMAL S KITH TISSUE

• NUMBE R OF ANIMAL S NECBOPSIED 

TABLE Cl (CONTINUED) 

CONTROL (ONTE) CONTROL (VEH)

01-1U1II 01-101B

(19) (20)

1 (5X) 

8 (U2X) 1 (5X)

1 (20S)

(19) (20)

(20) (20)

1 (5*) 

1 (5X) 

(20) (20)

(20) (20)

1 (5X)

(20) (20)

(20) (20)

1 (5X) 

(20) (20)

 EXAMINED MICROSCOPICALLY 

C-8 

 LOU DOSE 

 01-102M 

 (50) 

2 («X) 

1 (2X) 

9 (18X) 

 11 (22X) 

 (50) 

1 <2X) 

 (50) 

 (50) 

1 (2X) 

 (50) 

3 (6X) 

 (50) 

1 (2X) 

 (50) 

 (50) 

HIGH DOSE 

01-103M 

(50) 

1 (2X) 

2 (<tX) 

7 (1UX) 

(50) 

(50) 

1 (2X) 

(50) 

(50) 

(50) 

(50) 

1 (2X) 

(50) 

1 (2X) 

1-121L.... 
TABLE Cl (CONCLUDED)
 

CONTROL (UNIR) CONTROL (VEH) L OU DOSE HIGH DOSE 

01-1U1M 01-101H 01-102B 01-1P3M 

•PLEURA (20) (20) (50) (50) 

INFLAMMATION, SUPPUBATIVE 1 (2*) 

INFLAMMATION , ACOTE 1 (2*) 

•PERICARDIUM (20) (20) (50) (50) 

INFLAMMATION , ACUTE 1 (2X) 

INFLAMMATION , ACUTE SUPFURATIV E 1 <2X) 

•EPICA8DIUM (20) (20) (50) (50) 

INFLAMMATION , ACDIE 1 (2X) 

INFLAMMATION , CHRONIC FOCAL 1 (2*) 

•MESENTERY (20) (20) (50) (50)

 

PEBIARIERITIS 2 (10X) 3 (15*) 8 (16*) 

ALL OTHER SYSIEMS
 

ADIPOSE TISSUE
 INFLAMMATION, CHRONIC FOCAL
 

SPECIAL MORPHOLOGY SUMMARY
 

NO	 LESION REPORTED
 

t NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-9
 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

TREATED WITH 3-SULFOLENE 

c:ONTROL(UNTR) 

ANIMAL S INITIALLY IN STUDY 

ANIMALS NECROPSIED 

ANIMALS EXAMINE D HISTOPATHOLOGICALLY* * 

INTEGUMENTAR Y SYSTEM 

*SKIN 

CYST, NOS INFLAMMATION , NOS

 

ULCER, NOS 

INFLAMMATION , SUPPURATIV E 

*SUBCUT TISSUE 

HEMORRHAG E 

GRANULCMA , FOREIGN BODY CALCIFICATION, NOS

 

RESPIRATOR Y SYSTEM 

*NASAL CAVITY 

INFLAMMATION , ACUTE SUPPUKATIV E 

*TR'ACHEA 

INFLAMMATION, ACUTE SUPPURATIVE 

tLUNG 

PNEUMONIA , ASPIRATION 

INFLAMMATION, SUPPURATIVE 

INFLAMMATION , ACUTE INFLAMMATION , ACUTE SUPPURATIV E

 

ABSCESS, NOS 

PNEUMONIA , CHRONIC MURIN E 

INFLAMMATION , PYOGHANULOMATOU S 

CALCIFICATION, METASTATIC 

*LUNG/ALVEOLI 

CALCIFICATION, METASTATIC 

HEMATOPOIETIC SYSTEM 

tBONE NABKO K 

HIEElPiA5iAA_HJMA^O£OI£TJC 

01-1U1? 

20 

20 

20 

(20) 

1 (5X) 

(20) 

(20) 

(20) 

(20) 

19 (95X) 

(20) 

(20) 

H _— —_____.—— •• 

CONTROL (VEH) LOU DOSE HIGH DOSE 

01-101F 01-10UF 01-105F 

20 50 50 

20 50 50 

20 50 50 

(20) (50) 

2 («X) 

(50) 

1 (5X) 

1 (2X) 

(20) (50) (50) 

1 (2X) 

1 (2X) 

1 (2X( 

(20) (50) (50) 

1 (2X) 

(20) (50) (50) 

2 (UX) 

(20) (50) 

2

1

 (IX) 

 <2X) 

(50) 

1 (2X) 

2 (UX) 

1 (5X) 

1 (5X) 

18 (90X) 

3 (6X) 

3 (6X) 

39 (78X) 

1 (2X) 

1 (2X) 

23 (16X) 

1 <2X) 

1 (5X) 

(20) (50) (50) 

1 (2X) 

(20) (50) (50) 

a_i22S) 2-12SL. 2,_1!«SL_-, 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY « NUMBER OF ANIMALS NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-IO 
TABLE C2 (CONTINUED)
 

L OU DOSE 

01-10tF 

(50) 

4 (8X) 

(50) 

(50) 

(50)

1 (2*) 

(50) 

1 (2X) 

1 (2X) 

2 («X) 

(50) 

(50) 

(50) 

(50) 

(50) 

(50) 

1 (2%) 

(50) 

HIGH DOSE 

01-105P 

(49) 

2 (4X ) 

(50) 

(50) 

(50) 

(50) 

1 (2%) 

1 (2X) 

(50) 

1 (2X) 

(50) 

(50) 

1 (2X) 

(50) 

1 (2X) 

(50) 

1 <2X) 

(50) 

2 (IX) 

(50) 

1.I2SL. 

tSPLEEN 

HEMATOPOItlSIS 

IMANDIBULA B L. NODE 

INFLAMMATION , NOS

 

INFLAMMATION , ACUTE 

tCERVICAL LYMPH NODE 

HYPERPLASIA , LYBPHOID 

CI8CULATOHY SYSTEM 

IHEART 

INFLAMMATION , CHRONIC FOCAL 

• MYOCARDIU M 

INFLAMMATION , ACUTE 

INFLAMMATION , CHBONIC FOCAL 

FIBROSIS 

CALCIFICATION, METASIATIC 

(ENDOCARDIU M 

HYPERPLASIA , NOS 

•ARTERY 

MEDIA L CALCIFICATION 

CALCIFICATION, METASIATIC 

*AORTA 

MEDIAL CALCIFICATION 

•CORONARY ARTER Y 

MEDIAL CALCIFICATION 

• PULMONAR Y AETERY 

MEDIAL CALCIFICATION

 

•MESENTEHIC ARTERY 

MEDIA L CALCIFICATION

 

DIGESTHE SYSTEM 

tLIVER 

BIlB-SJiSIS 

CONTROL (UNTR) 

01-141F 

(20) 

3 (15X) 

(20) 

(20) 

(20) 

(20) 

(20) 

1 (5*) 

(20) 

(20) 

1 (5X) 

(20) 

(20) 

(20) 

(20 ) 

CONTROL (VEH ) 

01-101F 

(20) 

2 (10%) 

(20) 

1 (5X> 

1 <5X) 

(20) 

1 (5X) 

(20) 

(20) 

1 (5X) 

(20) 

(20) 

1 (5X) 

1 (5*) 

(20) 

(20) 

(20) 

(20) 

(20) 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

« NUMBER OF ANIMALS NECROPSIED
 

C-ll
 
iLIVEH (CON'T)

 

THROMBOSIS, NOS INFLAMMATION, NOS

 

PELIOSIS HEPATIS 

NECSOSIE, FOCAL 

METAMOBEHOSIS FATTY FOCAL CELLULAfi CHANG S

 

ANGIECTASIS 

*HEPATIC lOBULE HETAMOREHOSIS FATTY

 

tLIVER/CENTRILOBULAR 

INFLAMMATION , NECROTIZING 

METABOBPHOSIS FATTY 

(LI VEB/ HEP ATOCYTE S 

FOCAL CELLULAR CHANGE 

•BILE DUCT 

DILATATION, NOS 

INFLAMMATION , CHRONIC HYPERPLASIA , NOS

 

ANGIECTASIS 

(PANCREAS 

PEBIARTEBITIS 

ATROPHY , NOS 

(STOMACH 

ULCER, FOCAL 

CALCIFICATION, NOS CALCIFICATION, METASTATIC

 

HYPEBKERA10SIS 

(GASTRIC BUCOSA 

ULCER, NOS 

ULCEB, FOCAL 

(SHALL INTESTINE 

ULCEB, NOS 

(LARGE INTESTINE 

PARASITISM 

(COLON 

I NUMBER OF ANIMAL S HITH TISSUE

* NUMBE R OF ANIMALS NECROPSIED 

TABLE C2 (CONTINUED) 

CONTROL (UNTR) CONTROL (VEH) 

01-U1F 01-101F 

1 (5X) 

2 (10X) ' (5X) 

(20) (20) 

(20) (20) 

(20) (20) 

(20) (20) 

2 (10X) 6 (BOX) 

1 (SX) 

(20) (20)­

1 (5X) 

(20) (20) 

3 (15X) 

1 (5X) 

(20) (20) 

(20) (20) 

1 <5X) 

(20) (20) 

1 <5X) 

(20) (20) 

 EXAMINED MICROSCOPICALLY 

LOH DOSE 

01-IOItF 

1 (2X) 

1 (2X) 

(50) 

1 (2X) 

(50) 

1 (2X) 

(50) 

(50) 

2 (tX) 

1 (2X) 

11 (28X) 

(50)

2 ;*xi 

(50) 

3 (6X) 

(50) 

(50) 

(50) 

* (8X) 

(50) 

HIGH DOSE 

01-105F 

1 (2X) 

1 (2X) 

1 (2X) 

1 (2X) 

1 (2X) 

(50) 

1 (2X) 

(50) 

1 (2X) 

2 (IX) 

(50) 

1 (2X) 

(50) 

1 (2X) 

1 (2X) 

8 (16X) 

(50) 

2 (*X) 

1 (2X) 

(50) 

1 (2X) 

1 (2X) 

1 (2X) 

(50) 

2 (4X) 

1 (2X) 

(50) 

(50) 

(50) 

C-I2 
TABLE C2 (CONTINUED) 

CONTROL (UNTR ) CONTROL (VEH ) L OU DOSE 

01-111P 01-101F 01-104F 

•CECUM (20) (20) (50) 

PARASITISM 1 (2%) 

URINAR Y SYSTEfl 

• KIDNE Y (20) (20) (50) 

MINERALIZATIO N 2 (10%) 

HYD80NEPHEOSIS 1 (5%) 

PYELONEPHHITIS, NOS 1 (5%) 1 (2%) 

INFLAMMATION , CHRONIC 6 (30%) 4 (20%) 17 (34%) 

NEPHHOPATH Y 1 (5X) 

CALCIUM DEPOSIT 1 (5%) 

CALCIFICATION, NOS 7 (35%) 3 (6%) 

• URINAR Y BLADDER (20) (20) (50) 

ULCER, ACUTE 1 (2%) 

ULCER, CHRONIC 1 (2%) 

ENDOCRIN E SYSTEM 

• PITUITAR Y (20) (20) (50) 

HYPERPLASIA , CHROMOPHOBE-CELL 2 (10%) 2 (US) 

• ADRENA L (20) (20) (50) 

CALCIUM DEPOSIT 1 (5%) 

HYPOPLASIA, NOS 1 (2% ) 

• ADRENA L CORTEX (20) (20) (50) 

DEGENERATION , NOS 6 (30%) 11 (22% ) 

ANGIECTASIS 5 (25%) 3 (40%) 11 (224) 

•THYROID (19) (20) (50) 

FOLLICULA K CYST, NOS 2 (11*)

 

• PARATHYROI D (20) (18) (19)

 

HYPERPLASIA , NO S 

REPRODUCTIV E oYSTE M 

*MAMMAH Y GLAN D (20 ) (20) (50) 

GALACTOCELE 1 (2%) 

* NUMBE R OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S SECROPSIZD 

HIGH DOSE 

01-105F 

(50) 

(50) 

1 (2%) 

25 (50%) 

(19) 

(50) 

It (8%) 

(50) 

(50) 

11 (22%) 

11 (22% ) 

(50) 

2 (1%) 

(37) 

7 (19%) 

(50) 

C-I3 
TABLE C2 (CONTINUED) 

CONTROL (UNT8) CONTROL (TEH)

01-U1F 01-101P

*VAGINA (20) (20)

INFLAHMATION, NOS 1 (5X) 

INFLAMMATION , ACUTE SUPPURATIVE

tUTERU S (20) (20)

HYDROMETRA

tOTERUS/ENDOMETRIUM (20) (20)

INFLAMMATION , NOS 2 (10X) 1 (5X) 

INFLAHHATION, ACUTE

HYPERPLASIA , CYSTIC

*OTAHY/OVIDUCT (20) (20)

INFLAMMATION, ACUTE

tOVAHY (20) (20)

CYST, NOS 1 (5X) 

FOLLICULAH CYST, NOS 1 (5X) 

NEHVOUS SYSTEM 

«BaAIN/MENINGE S (20) (20)

INFLAMMATION , ACUTE

tBHAIN (20) (20)

HEMORRHAG E

SPECIAL SENSE ORGAN S 

• EYE/CRYSTALLINE LENS (20) (20)

DISLOCATION COMPLETE

HUSCULOSKELEIA L SYSTEM 

• SKELETAL MUSCLE (20) (20)

INFLAMMATION, PYOGRAN ULOMATO OS

PERIAR1£RITIS 1 (5X) 

• MUSCLE OF NSCK (20) (20)

INFLAMMATION , ACUTE/CHRONIC

BODY CAVITIES 

• ABDOMINA L CAVIT Y (20) (20)

• NUMBE R OF ANIMAL S HITH IISSUE EXAMINE D MICROSCOPICALL Y 

• NUMBE R OF ANIMAL S NECROPSIED 

C-14 

 LOB DOSE HIGH DOSE 

 01-104F 01-105F 

 (50) (50) 

1 (2X) 

 (09) (50) 

1 (2X) 

 (49) (50) 

5 (10X) 1 (2X) 

2 (4X) 

 (49) (50) 

2 (UX) 

 (t9) (50) 

 (50) (50) 

1 (2X) 

 (50) (50) 

1 (2X) 

 (50) (50) 

1 (2X) 

 (50) (50) 

1 (2X) 

 (50) (50) 

1 (2X) 

 (50) (50) 
TABLE C2 (CONCLUDED) 

CONTROL(UNTB) CONTROL(VEH) LOW DOSE HIGH DOSE 

01-1U1F 01-101F 01-1C4F 01-105F 

*PLEURA (20) (20) (50) (50) INFLAMMATION, NOS 

INFLAMMATION, ACUTE 5 (10*) INFLAMMATION, CHBONIC 1 (2*) FIBROSIS 1 (2%) 

*PESICAHDIUa (20) (20) (50) (50) INFLAMMATION, ACUTE 1 (2X) INFLAMMATION, CHRONIC 

'MESENTERY (20) (20) (50) (50) PEBIABTERIT1S 2 («) 

ALL OTHER SI SI EMS 

ADIPOSE TISSUE 

INFLANHATION, NOS 

INFLAMMATION , CHBONIC 

SPECIAL MOBPHOLOGY SUMMAR Y 

NO LESION REPORTED 

* NUMBER OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF AMtULS NECROPSIED 

C-I5 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE TREATED WITH 3-SULFOLENE
 

TABLE Dl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH 3-SULFOLENE 

c:ONTROL (ONTR ) CONTROL (VEH)

02-M101 02-M091

ANIMALS INITIALLY IN STUDY 20 20

ANIMALS NECBOPSIED 20 20

ANIMALS EXAMINE D HISTOPATH010GICALLY ** 20 20

INTEGUMENTAR Y SYSTEM 

•SKIN (20) (20)

INFLAMMATION, NOS 1 (5X) 

ACANTHOSIS .1 (5X) 

•SUBCUT TISSUE (20) (20)

ABSCESS, NOS 2 (10X) 1 (5X) 

BESPIBATORY SYSTEM 

tLONG (20) (20)

PREDHONIA , CHRONIC MBKIN E 

HEMATOPOIETIC SYSTEM 

tSPLEEN (20) (20)

AMYLOICOSIS 2 (10X) 5 (25X) 

•CERVICAL LYMPH NODE (19) (20)

HYPEHPLASIA, RETICULUM CELL 

tHESENTERIC L. NODE (19) (20)

INFLAMMATION, NOS 

ANGIECTASIS 1 (5*) 

CIRCULATOR! SYSTEM 

t ENDOCARDIUM (20) (20)

RYPERPLASIA, NOS 1 (5X) 

DIGESTIVE SYSTEM 

•LIVER (20) (20)

ISf.IiASMUO.Nt. SSS. 

* NUMBER OF ANIMALS KITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS HECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

 LOB DOSE HIGH DOSE 

 02-M102 02-M103 

 50 50 

 50 50 

 50 49 

 (50) (50) 

 (50) (50) 

 (50) (48) 

6 (12*) 1 (2X) 

 (50) (48) 

 (50) (36) 

1 (2X) 

 (50) (36) 

1 (2X) 

 (50) (49) 

 (50) (49) 

D-3
 
TABLE Dl (CONTINUED) 

CONTROL(UNTR )

02-H101

 CONTROL(VEH )

 02-H091

PELIOSIS nEP

INFABCT, DOS

HYPERPLASIA ,

ANGIECTASI S

ATIS

 NODULA R

 1 (5*)

 1 (5X) 

URINAK Y SYSTEM 

tKIDNE Y (20) (20) HYDRONEEHEOSI S 3 (15X) 7 (35%) 

CYSI, NOS

PYELONEPHRITIS , NOS 1 (5X) 

INFLAMMATION , CHRONIC 14 (70X) 9 (<*5X)

AMYLOIDOSIS t (20X) 5 (25X)

HSTAMOEPHOSIS FAT1Y 1 (5X) 

ENDOCRIN E SYSTE M 

NONE 

REPRODUCTIV E SYSTE M 

NONE 

NERVOU S SYSTEM 

IBRAIN/MENINGE S (20) (20)

INFLAMMATION, NOS

SPECIAL SENSE ORGAN S 

NONE 

MUSCULOSKEIETAL SYSTEM 

NON E 

BODY CAVITIES 

NONE 

A LL OTHER SYSTEMS 

_NONE 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

 LOW DOSE

 02-11102

 1 (2*) 

1 (2«) 

 HIGH DOSE 

 02-H103 

2 (IIX) 

 (50)

 3 (6X)

 (19) 

1 (2X) 

2 (IX) 

1 (2X) 

 (50)

 1 (2*| 

 (<!9) 

D-4
 
TABLEDl (CONCLUDED) 

CONTROL (UNTR) CONTROL (VEH) LOU DOSE HIGH DOSE 

02-N101 02-M091 02-M102 02-H103 

SPECIAL MORPHOLOGY SUMMAR Y 

DO LESION REPORTED 2 5 24 38 

AUTO/NHCROPSY/NO HISIO 1 

f NUMBER OF ANIMALS KITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER 0? ANIMALS NECROPSIED 

D-5 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH 3-SULFOLENE 

CONTBOL(UNTB) 

02-F101 

CONTROL (VEH) 

02-F091 

L OU DOSE 

02-F10U 

HIGH DOSE 

02-F105 

ANIMAL S

ANIMALS

ANIMALS

 INITIALLY IN STODY

 NECROPSIED

 EXAMINED HISTOPATHOLOGICALLY**

 20 

 20 

 20 

20 

20 

20 

50 

19 

«9 

50 

49 

18 

INTEGUMENIAH Y SYSTEM 

NON E 

RESPIRATOR Y SYSTEM 

HUNG 

PNEUMONIA , CHRONIC MUEIN E 

(20) (20) 

2 (10X) 7 (1UX) 

<M7) 

1 (2X) 

HEMATOPOIETIC SYSTEM 

•SPLEEN 

HYPEEPLASIA, LYMPHOID 

(20) 

1 (5S) 

(19) 

•BRONCHIAL LYMPH NODE 

INFLAMMATION, NOS (19) (20) (47) 

1 (2X) 

(UU ) 

*HESENTERIC L. NODE 

INFLAMMATION, NOS 

(19) (20) (t7) 

1 <2X) 

CIRCULATOR Y SYSTEM 

NONE 

DIGESTIVE SYSTEM 

•LIVER 

INFLAMMATION, NOS 

PELIOSIS HEPAI1S 

ANGIECTASIS 

(20) 

1 (5*) 

(19) (19) 

1 (2%) 

1 (2X) 

3 (6*) 

• STOMACH 

HYPEBKERAIOSIS 

(20) 

1 (5X) 

(20) (48 ) 

• NUMBER OF ANIMALS WITH TISSUE EXAMINED

* NUMBE R OF ANIMAL S NECEOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 MICROSCOPICALLY 

0-6 
TABLE D2 (CONTINUED) 

CONTROL(UNTR ) CONTROL (VEH) LOU DOSE HIGH DOSE 

02-F101 02-F091 02-F104 02-F105 

USINAR Y SYSTEM 

NOME 

ENDOCRINE SYSTEM 

«PITUITARY (18) (20) (42) (25) 

ANGIECTASIS 1 (6X) 

tTHYBOID (18) (20) (39) (39) 

FOLLICULAB CYST, NOS 1 (3%) 

REPRODUCTIVE iYSTEH 

tUTEBUS (20) (20) (49) (45) 

HYDROMETRA 4 (20%) 10 (20*) 3 (7%) 

INFLAMMATION, NOS 2 (10%) 1 (5X) 1 (2S) 1 (2X) 

*UTEBUS/ENDOMETRIUH (20) (20) (49) (45) 

HYPEBPLAS1A, CYSTIC 5 (25*) 10 (SOX) 9 (18*) 4 (9X) 

*0¥A8Y (20) (20) (49) (44) 

CYST, NOS 3 (15*) 6 (30X) 16 (33*) 2 (5X) 

INFLAHMATIOS, NOS 2 (10X) 1 (5X) 1 (2X) 1 (2X) 

NEBVOOS SYSTEM 

NONE 

SPECIAL SENSE ORGAN S 

*HABDERIA N Gi.AND (20) (20) (49) (49) 

HYPERPLASIA , NOS 1 (2X) 

MUSCULOSKELSTAL SYSTEM 

NONE 

BODY CAVITIES 

( NUMBE R OF ASIMALS MITH TISSUE EXAMIMSD MICROSCOPICALLY 

* NUMBER OF ANIMALS NECBOPSIED 

D-7 
TABLE D2 (CONCLUDED) 

CONTROL (UNTE) COHTBOL (VEU) LOU DOSE HIGH DOSE 

02-F101 02-F091 02-P101 02-F10S 

ALL OTHEB SYSTEMS 

HONE 

SPECIAL MORPHOLOGY SUMMAR Y 

HO LESION REPORTED tt 4 10 10 

AUTO/NECBOPSY/NO HISTO

AUTOLYSIS/NO NECROPSY 1

 1 

1 

* NUMBER OF AMIHALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

D-8 
Review of the Bioassay of 3-Sulfolene* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

March 7, 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 3-Sulfolene for carcinogenicity.
 

The primary reviewer noted that 3-Sulfolene is an intermediate of Sulfolene which is used in petroleums, plastics,
 

and textiles and in the synthesis of some fungicides. He
 

briefly described the experimental design and conditions
 

under which 3-Sulfolene was tested. The poor survival
 

among the high dose male rats precluded an evaluation of
 

the carcinogenicity of 3-Sulfolene in this group. Under
 

the conditions of test, no carcinogenic effect was observed
 

in the low dose males or either treatment group of female
 

rats. Excessive mortality also resulted among the high
 

dose male and female treated mice. An increased incidence
 

of hepatocellular carcinomas was observed among the male
 

mice, although it was not statistically significant when
 

compared to historical controls. The primary reviewer said
 

that the study was complicated by excessive mortality and
 

changes in dosages during the course of the chronic phase.
 

As a result, he concluded that the study was inadequate to
 

assess the carcinogenicity of 3-Sulfolene.
 

45
 
The secondary reviewer said that consideration should
 

be given to a retest of 3-Sulfolene based on the incidence
 

of hepatocellular carcinomas among treated male mice. He
 

concluded that the studies' shortcomings were so severe
 

that it was not possible to draw any conclusion on the
 

carcinogenicity of 3-Sulfolene.
 

A discussion ensued as to whether an assessment of
 

the carcinogenicity of 3-Sulfolene could be based on the
 

low dose treated animals. It was finally agreed that the
 

report sufficiently defined the limitations of the study.
 The primary reviewer therefore moved that the report be
 

accepted as written. The motion was seconded and approved
 

unanimously.
 

Members present were
 

Gerald N. Wogan (Chairman), Massachusetts Institute of
 

Technology
 

Arnold 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, APL-CIO
 

Michael Shimkin, University of California at San Diego
 

John Weisburger, American Health Foundation
 

Sidney Wolfe, Health Research Group
 

Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

46
 

«U.S. GOVERNMENT PRINTING OFFICE:1978 260-899/3126 1-3