1979 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 132 

BIOASSAY OF 

2,5-DITHIOBIUREA 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 142-46-1 

NCI-CG-TR-132 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

2,5-DITHIOBIUREA
 

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) 79-1387
 

REPORT ON THE BIOASSAY OF 2,5-DITHIOBIUREA
 

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
 

2,5-dithiobiurea 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 2,5-dithiobiurea was conducted by
 

Mason Research Institute, Worcester, Massachusetts, initially under
 

direct contract to the NCI and currently under a subcontract to
 

Tracor 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. E. Smith (3) and
 

Dr. A. Handler (3). Animal treatment and observation were supervised
 

by Mr. G. Wade (3) and Ms. E. Zepp (3). Chemical analysis was performed by Midwest Research Institute (4) and the analytical results
 

were reviewed by Dr. N. Zimmerman (5).
 

Histopathologic examinations were performed by Dr. R. W. Fleischman (3), Dr. A. S. Krishna Murthy (3), and Dr. D. S. Wyand (3) at the
 

Mason Research Institute, and the diagnoses included in this report
 

represent the interpretation of these pathologists. Histopathology
 

findings and reports were reviewed by Dr. R. L. Schueler (6).
 

Compilation of individual animal survival, pathology, and summary tables was performed by EG&G Mason Research Institute (7); the
 

statistical analysis was performed by Mr. W. W. Belew (5,8), using
 

methods selected for the Carcinogenesis Testing Program by Dr. J. J.
 

Gart (9).
 

iii
 
This report was prepared at METREK, a Division of The MITRE Corporation (5) under the direction of the NCI. Those responsible for
 

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

(5), task leader Dr. M. R. Kornreich (5,10), senior biologist Ms. P.
 

Walker (5), biochemist Mr. S. C. Drill (5), and technical editor Ms.
 

P. A. Miller (5). The final report was reviewed by members of the
 

participating organizations.
 

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 (1,10), Dr.
 

R. A. Griesemer (1), Dr. M. H. Levitt (1), Dr. H. A. Milman (1), Dr.
 

T. W. Orme (1), Dr. R. A. Squire (1,11), Dr. S. F. Stinson (1), 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.	 Mason Research Institute, 57 Union Street, Worcester, Massachusetts.
 

4.	 Midwest Research Institute, 425 Volker Boulevard, Kansas City,
 

Missouri.
 

5.	 The MITRE Corporation, METREK Division, 1820 Dolley Madison
 

Boulevard, McLean, Virginia.
 

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

Maryland.
 

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

Rockville, Maryland.
 

8.	 Now with the Solar Energy Research Institute, Cole Boulevard,
 

Golden, Colorado.
 

9.	 Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cancer
 

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

IV
 
10.	 Now with Clement Associates, Inc., 1010 Wisconsin Avenue, N.W.,
 

Washington, B.C.
 

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

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay of 2,5-dithiobiurea for possible carcinogenicity was
 

conducted using Fischer 344 rats and B6C3F1 mice. 2,5-Dithiobiurea
 

was administered in the feed, at either of two concentrations, to
 

groups of 50 male and 50 female animals of each species, with the
 

exception of high dose male rats, of which there were only 49. The
 

dietary concentrations used in the chronic bioassay were 0.6 percent
 

for the low dose rats and 1.2 percent for the high dose rats. The
 

dietary concentrations used for low and high dose mice were 1.0 and
 

2.0 percent, respectively. After a 78-week dosing period, observation of the rats continued for an additional 31 weeks and observation
 

of the mice continued for an additional 16 weeks. For each species,
 

50 animals of each sex were placed on test as controls.
 

In both species, adequate numbers of animals in all groups survived sufficiently long to be at risk from late-developing tumors.
 

Compound-related mean body weight depression was observed in mice but
 

not in rats. No consistent pattern of clinical signs was observed in
 

either species.
 

No tumors occurred at a significantly higher incidence in dosed
 

rats than in their controls.
 

Among female mice, the Cochran-Armitage test indicated a significant positive association between the incidence of hepatocellular
 

carcinoma and dietary concentration of 2,5-dithiobiurea. According
 

to results of the Fisher exact test, the incidence of hepatocellular
 

carcinoma was significantly higher in the high dose female mouse
 

group when compared to the corresponding control group but not when
 

compared to the laboratory historical control data. No neoplasms
 

occurred at a significantly higher incidence in dosed male mice than
 

in their controls.
 

Under the conditions of this bioassay, the evidence suggested,
 

but was insufficient to establish the carcinogenicity of 2,5-dithiobiurea for female B6C3F1 mice. The compound was not carcinogenic
 

to male B6C3F1 mice or to male or female Fischer 344 rats.
 

VI1
 

TABLE OF CONTENTS
 

Pago
 

I. INTRODUCTION	 1
 

II. MATERIALS AND METHODS	 3
 

A. Chemicals	 3
 

B. Dietary Preparation	 4
 

C. Animals	 5
 

D. Animal Maintenance	 5
 

E. Selection of Initial Concentrations	 8
 

F. Experimental Design	 9
 

G. Clinical and Histopathologic Examinations	 12
 

H. Data Recording and Statistical Analyses	 14
 

III. CHRONIC TESTING RESULTS: RATS	 19
 

A. Body Weights and Clinical Observations	 19
 

B. Survival	 19
 

C. Pathology	 22
 

D.	 Statistical Analyses of Results 22
 

IV. CHRONIC TESTING RESULTS: MICE
 32
 

A. Body Weights and Clinical Observations	 32
 

B. Survival	 32
 

C. Pathology	 35
 

D. Statistical Analyses of Results	 36
 

V. DISCUSSION	 42
 

VI. BIBLIOGRAPHY	 44
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH 2,5-DITHIOBIUREA A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH 2,5-DITHIOBIUREA B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 2,5-DITHIOBIUREA C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 2,5-DITHIOBIUREA D-l
 

IX
 
LIST OF ILLUSTRATIONS 

Figure Number Page 

1 CHEMICAL STRUCTURE OF 2,5-DITHIOBIUREA 2 

2 GROWTH CURVES FOR 2,5-DITHIOBIUREA CHRONIC 

STUDY RATS 20 

SURVIVAL COMPARISONS OF 2,5-DITHIOBIUREA 

CHRONIC STUDY RATS 21 

GROWTH CURVES FOR 2,5-DITHIOBIUREA CHRONIC 

STUDY MICE 33 

SURVIVAL COMPARISONS OF 2,5-DITHIOBIUREA 

CHRONIC STUDY MICE 34 

LIST OF TABLES 

Table Number Page 

DESIGN SUMMARY FOR FISCHER 344 RATS— 

2,5-DITHIOBIUREA FEEDING EXPERIMENT 10 

DESIGN SUMMARY FOR B6C3F1 MICE—2 ,5-DITHIOBIUREA FEEDING EXPERIMENT 11 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

2,5-DITHIOBIUREA 23 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH 2,5-DITHIOBIUREA 27 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

2,5-DITHIOBIUREA 37 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH 2,5-DITHIOBIUREA 39 
LIST OF TABLES (Concluded)
 

Table Number Page
 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MALE RATS TREATED WITH 2,5-DITHIOBIUREA A-3
 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

FEMALE RATS TREATED WITH 2,5-DITHIOBIUREA A-7
 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MALE MICE TREATED WITH 2,5-DITHIOBIUREA B-3
 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

FEMALE MICE TREATED WITH 2,5-DITHIOBIUREA B-6
 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MALE RATS TREATED WITH 2,5­

DITHIOBIUREA C-3
 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN FEMALE RATS TREATED WITH 2,5­

DITHIOBIUREA C-8
 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MALE MICE TREATED WITH 2,5­

DITHIOBIUREA D-3
 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN FEMALE MICE TREATED WITH 2,5­

DITHIOBIUREA D-7
 

XI
 

I. INTRODUCTION
 

2,5-Dithiobiurea (Figure 1) (NCI No. C03009), a component of
 

photographic chemicals, was selected for bioassay by the National
 

Cancer Institute because it is a dimer of thiourea, a liver, thyroid
 

and Zymbal's gland tumorigen in rats (International Agency for
 

Research on Cancer, 1974).
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is 1,2-hydrazinedicarbothioamide. ^
 

2,5-Dithiobiurea can be used in both photographic emulsions
 

(Kodak-Pathe, 1966; McBride, 1966) and bleach-fixing baths for color
 

films (Nimura et al., 1973) and papers (Nimura et al., 1974). It
 

can also be used as a fuel in pyrotechnic disseminating compositions
 

(Niles, 1975), and electroplating baths for copper (Fujino and Fueki,
 

1971) and tin-nickel plating (Fueki at al., 1974).
 

Specific production data for 2,5-dithiobiurea are not available;
 

however, this compound is produced in commercial quantities (in excess of 1000 pounds or $1000 in value, annually) by one U.S. company
 

(Stanford Research Institute, 1977).
 

The potential for exposure to 2,5-dithiobiurea is greatest for
 

persons using photographic chemicals, pyrotechnic devices, and electroplating baths which contain this compound.
 

*
 

The CAS registry number is 142-46-1,
 
H2N—C—NH—NH—C—NH2

 

FIGURE 1 

CHEMICAL STRUCTURE OF 2,5-DITHIOBIUREA 
II. MATERIALS AND METHODS
 

A. Chemicals
 

Two batches of 2,5-dithiobiurea were purchased from Eastman Kodak
 

Company, Rochester, New York by the NCI for Mason Research Institute,
 

Worcester, Massachusetts. Chemical analysis was performed by Midwest
 

Research Institute, Kansas City, Missouri.
 

For the batch used during the first five months of the bioassay,
 

the experimentally determined melting point range (205° to 208°C),
 

although narrow, suggested the presence of at least minor impurities
 

because of its deviation from the literature value of 214° to 215°C
 

(Boit, 1973). Slight deviation of the experimentally determined
 

elemental composition from C H N S , the molecular formula for
 264 2
 

2,5-dithiobiurea, also indicated the presence of impurities. Thin-


layer chromatography utilizing two solvent systems (ethyl acetate:
 

methanol and acetone), each visualized with ultraviolet light, potassium dichromate, and heat, indicated the presence of one nonmotile
 

impurity. High pressure liquid chromatography showed the presence
 

of two impurities. Titration of the thiocarbonyl function provided
 

a result that was approximately 94 percent of the theoretical value.
 

This indicates that purity cannot exceed 94 percent, but other compounds containing thiocarbonyl functional groups could be present.
 

Infrared analysis was consistent with the structure of the compound.
 

A second batch of the chemical, purchased five months later and
 

used for the duration of the bioassay, appeared to be of lesser
 

3
 
purity since the range of the experimentally determined melting point
 

for this batch (180° to 215°C) was wider. Results of elemental analysis approximated those expected for the molecular formula of the
 

compound. Thin-layer chromatography utilizing two solvent systems
 

(ethyl acetate:methanol and acetone), each visualized with 254 and
 

367 nm light, dichromate, and heat, indicated one nonmotile impurity.
 

High pressure liquid chromatography also showed the presence of one
 

impurity. Titration of the thiocarbonyl function provided a result
 

that was 108 percent of the theoretical. The possible presence of
 

impurities was supported by the results of infrared analysis and
 

nuclear magnetic resonance analysis.
 

Throughout this report the term 2,5-dithiobiurea is used to represent these two batches of the chemical.
 

B. Dietary Preparation
 

The basal laboratory diet for both dosed and control animals con­

®
 sisted of Wayne Lab-Blox meal (Allied Mills, Inc., Chicago, Illinois).
 

2,5-Dithiobiurea was administered to the dosed animals as a component
 

of the diet. The chemical was mixed with an aliquot of feed. Once
 

visual homogeneity was attained, the mixture was placed into a 6 kg
 

capacity Patterson-Kelley twin-shell stainless steel V-blender with
 

the remainder of the meal. After 20 minutes of blending, the mixtures were placed in double plastic bags and stored in the dark at
 

4°C. The mixtures were discarded 2 weeks after formulation.
 
C. Animals
 

Two animal species, rats and mice, were used in the carcinogenicity bioassay. Fischer 344 rats and B6C3F1 mice were obtained
 

through contracts of the Division of Cancer Treatment, National
 

Cancer Institute. All animals used in the chronic bioassay were
 

supplied by Charles River Breeding Laboratories, Inc., Wilmington,
 

Massachusetts, and all but the control mice were received in the same
 

shipment. Control mice were received approximately 5 weeks after the
 

other animals.
 

Upon arrival a sample of animals was examined for parasites and
 

other signs of disease. The remaining animals were quarantined by
 

species for 2 weeks prior to initiation of the test. Animals were
 

assigned to groups and distributed among cages so that the average
 

body weight per cage was approximately equal for a given sex and
 

species.
 

D. Animal Maintenance
 

All animals were housed by species in rooms having a temperature
 

®
 

range of 23° to 34°C. Incoming air was filtered through Tri-Dek
 

®
 15/40 denier Dacron filters (Tri-Dim Filter Corp., Hawthorne, New
 

Jersey) providing six changes of room air per hour. Fluorescent
 

lighting was provided on a 12-hour-daily cycle.
 

Rats were housed five per cage by sex. During quarantine and for
 

the first 16 months of the bioassay, rats were housed in galvanized-


steel wire-mesh cages suspended above newspapers. Newspapers under
 
cages were replaced daily and cages and racks were washed weekly. For
 

the remainder of the study, rats were maintained in suspended poly-


carbonate cages equipped with disposal nonwoven fiber filter sheets.
 

®
 Clean bedding and cages were provided twice weekly. SAN-I-CEL corncob bedding (Paxton Processing Company, Paxton, Illinois) was used
 

during the first 7 months that the rats were housed in polycarbonate
 

cages, while Aspen hardwood chip bedding (American Excelsior Company,
 

Baltimore, Maryland) was used for the remainder of the bioassay.
 

Stainless steel cage racks were cleaned once every 2 weeks, and
 

disposable filters were replaced at that time.
 

Mice were housed by sex in polycarbonate shoe box type cages.
 

During quarantine and periods of chemical administration, cages were
 

fitted with perforated stainless steel lids. Stainless steel wire
 

bar lids were used during the final observation period. Both types
 

of lids were from Lab Products, Inc., Garfield, New Jersey. Nonwoven
 

fiber filter bonnets were used over cage lids. Dosed mice were
 

housed ten per cage for the first 15 months of study and five per
 

cage thereafter. Control mice, initially housed ten per cage, were
 

changed to five per cage after 13 months. Clean cages, lids, and
 

bedding were provided three times per week when cage populations
 

.®


were reduced to five. Ab-sorb-dri hardwood chip bedding (Wilner
 

Wood Products Company, Norway, Maine) was used for the first 3 months
 

®
 

of the bioassay (only 2 months for controls). SAN-I-CEL was used as
 

bedding for the next 12 months, after which a second corncob bedding
 
(Bed-o-Cobs , The Andersons Cob Division, Maumee, Ohio) was provided
 

for the remainder of the study. Reusable filter bonnets and pipe
 

racks were sanitized every 2 weeks throughout the study.
 

Tap water was available ad libitum for both species from 250 ml
 

water bottles equipped with rubber stoppers and stainless steel sip-


per tubes. Bottles were replaced twice weekly and, for rats only,
 

refilled as needed between changes.
 

®
 

Wayne Lab-Blox was supplied ad libitum throughout the bioassay.
 

®
 Animals received Wayne Lab-Blox meal during the initial quarantine
 

®
 

and periods of compound administration. Alpine aluminum feed cups
 

(Curtin Matheson Scientific, Inc., Woburn, Massachusetts) containing
 

stainless steel baffles were used to distribute powdered feed for the
 

first 13 months of study for all rats, for the first 17 months for
 

dosed mice and for the first 16 months for control mice. For the
 

remainder of the period of compound administration, meal was available from stainless steel gangstyle feed hoppers (Scientific Cages,
 

Inc., Bryan, Texas). During the final observation period, mice were
 

fed pellets from a wire bar hopper incorporated into the cage lid and
 

rats were fed pellets on the cage floor. Food hoppers were changed
 

on the same schedule as were cages. Food was replenished daily in
 

®
 Alpine feed cups.
 

2,5-Dithiobiurea-dosed and control rats were housed in a room
 

it
 

with rats intubated with m-cresidine (102-50-1); and with other rats
 

*
 

CAS registry numbers are given in parentheses,
 
receiving diets containing fenaminosulf (140-56-7) and cupferron
 

(135-20-6).
 

All mice, including controls, in the 2,5-dithiobiurea study were
 

housed in a room with other mice receiving diets containing fenaminosulf (140-56-7); cupferron (135-20-6); 4-chloro-o-phenylenediamine
 

(95-83-0); o-anisidine hydrochloride (134-29-0); and p-anisidine
 

hydrochloride (20265-97-8).
 

E. Selection of Initial Concentrations
 

In order to establish the concentrations of 2,5-dithiobiurea for
 

administration to dosed animals in the chronic studies, subchronic
 

toxicity tests were conducted with both rats and mice. Animals of
 

each species were distributed among several groups (five for rats
 

and six for mice), each consisting of five males and five females.
 

2,5-Dithiobiurea was incorporated into the basal laboratory diet
 

and supplied ad libitum to four of the five rat groups in concentrations of 0.3, 0.15, 0.08, and 0.04 percent and five of the six
 

mouse groups in concentrations of 2.0, 1.0, 0.5, 0.25, and 0.125
 

percent. The fifth rat group and the sixth mouse group served as
 

control groups, receiving only the basal diet. The dosed dietary
 

preparations were administered for a period of 7 weeks, followed by
 

a 1-week observation period during which all animals were fed the
 

basal laboratory diet.
 

The highest concentration causing no deaths, no compound-related
 

gross abnormalities, and no mean group body weight depression in
 
excess of 20 percent relative to controls was selected as the high
 

concentration utilized for the chronic bioassay.
 

In rats, no deaths were observed and no gross pathology was
 

recorded at necropsy. Mean group body weight depression was 8.9 and
 

9.2 percent, respectively, in males and females receiving 0.3 percent
 

2,5-dithiobiurea, the highest concentration administered. The concentration selected for high dose male and female rats in the chronic
 

study was 1.2 percent.
 

For mice, no deaths were observed and no gross pathology was
 

recorded at necropsy. Mean group body weight depression was 24.0
 

and 11.7 percent, respectively, in males and females receiving 1.0
 

percent, while it was 16.0 and 15.5 percent, respectively, in males
 

and females receiving 2.0 percent 2,5-dithiobiurea. The concentration selected for high dose male and female mice in the chronic
 

study was 2.0 percent.
 

F. Experimental Design
 

The experimental design parameters for the chronic bioassay (species, sex, group size, actual concentrations administered and duration
 

of treated and untreated observation periods) are summarized in Tables
 

1 and 2.
 

At initiation of the bioassay all rats were approximately 6
 

weeks old and shared the same median date of birth. The doses of
 

2,5-dithiobiurea utilized for both male and female rats were 1.2 and
 

0.6 percent. Throughout this report those rats receiving the former
 
TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

2,5-DITHIOBIUREA FEEDING EXPERIMENT
 

INITIAL OBSERVATION PERIOD 

GROUP 2, 5-DITHIOBIUREA TREATED UNTREATED 

SIZE CONCENTRATION3 (WEEKS) (WEEKS) 

MALE 

CONTROL 50 0 0 110 

LOW DOSE 50 0.6 78 

0 31 

HIGH DOSE 49 1.2 78 

0 31 

FEMALE 

CONTROL 50 0 0 110 

LOW DOSE 50 0.6 78 

0 31 

HIGH DOSE 50 1.2 78 

0 31 

Concentrations given in percentages in feed, 

10
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

2,5-DITHIOBIUREA FEEDING EXPERIMENT
 

INITIAL OBSERVATION PERIOD 

GROUP 2,5-DITHIOBIUREA TREATED UNTREATED 

SIZE CONCENTRATION3 (WEEKS) (WEEKS) 

MALE 

CONTROL 50 0 0 98 

LOW DOSE 50 1.0 78 

0 16 

HIGH DOSE 50 2.0 78 

0 16 

FEMALE 

CONTROL 50 0 0 98 

LOW DOSE 50 1.0 78 

0 16 

HIGH DOSE 50 2.0 78 

0 16 

Concentrations given in percentages in feed. 

11
 
concentration are referred to as the high dose groups, while those
 

receiving the latter concentration are referred to as the low dose
 

groups. These concentrations were administered in the feed for a
 

period of 78 weeks, followed by an observation period of up to 31
 

weeks. Control rats were on test for 110 weeks.
 

At initiation of the bioassay all dosed mice were approximately
 

6 weeks old and shared the same median date of birth. Control mice
 

were approximately 7 weeks old when they were started on test approximately 5 weeks after the dosed mice. Control mice were observed for
 

98 weeks. The doses utilized for both male and female mice were 2.0
 

and 1.0 percent. Throughout this report those mice receiving the
 

former concentration are referred to as the high dose groups, while
 

those receiving the latter concentration are refered to as the low
 

dose groups. These concentrations were administered in the feed for
 

78 weeks, followed by an observation period of up to 16 weeks.
 

G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

experiment and body weights were recorded twice weekly for the first
 

12 weeks of the study and at monthly intervals thereafter. Food
 

consumption, for two cages from each group, was monitored for seven
 

consecutive days once a month for the first nine months of the bioasay and for three consecutive days each month thereafter. From the
 

first day, all animals were inspected twice daily. The presence of
 

12
 
tissue masses and lesions was determined by monthly observation and
 

palpation of each animal.
 

A necropsy was performed on each animal regardless of whether it
 

died, was killed when moribund, or was killed at the end of the bioassay. The animals were euthanized by carbon dioxide inhalation, and
 

were immediately necropsied. Gross and microscopic examinations were
 

performed on all major tissues, organs and gross lesions taken from
 

sacrificed animals and, whenever possible, from animals found dead.
 

Tissues 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.
 

Slides were prepared from the following tissues: skin, subcutaneous tissue, lungs and bronchi, trachea, bone marrow, spleen, lymph
 

nodes, thymus, heart, salivary gland, liver, gallbladder (mice), pancreas, esophagus, stomach, small intestine, large intestine, kidney,
 

urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis,
 

prostate, brain, uterus, mammary gland, and ovary.
 

A few tissues were not examined for some animals, particularly
 

for those that died early. Also, some animals were missing, cannibalized, or judged to be in such an advanced state of autolysis as
 

to preclude histopathologic interpretation. Thus, the number of animals for which particular organs, tissues, or lesions were examined
 

13
 
microscopically varies and does not necessarily represent the number
 

of animals that were placed on experiment in each group.
 

H. Data Recording and Statistical Analyses
 

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

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

as recommended by the International Union Against Cancer (Berenblum,
 

1969). Data tables were generated for 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
 

14
 
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.
 

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
 

15
 
used, it is discussed in the narrative section. It is not, however,
 

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

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

continuity correction (Armitage, 1971, 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
 

16
 
during which animals died naturally or were sacrificed was entered as
 

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

these curves were used for two groups; Tarone's extension to testing
 

for linear trend was used for three groups. The statistical tests for
 

the incidence of tumors which used life-table methods were one-tailed
 

and, unless otherwise noted, in the direction of a positive dose
 

relationship. Significant departures from linearity (P < 0.05, two-


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared to its control was calculated from
 

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

is defined as pt- /p where p is the true binomial probability of the
 t t-c t-t
 

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
 

17
 
would be within the interval calculated from the experiment. When
 

the lower limit of the confidence interval is greater than one, it
 

can be inferred that a statistically significant result (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.
 

18
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

No compound-related mean body weight depression was apparent in
 

dosed male or female rats when compared to controls (Figure 2).
 

Only isolated clinical signs were observed. Subcutaneous masses
 

developed on the hind leg in two high dose males and in the axillary
 

mammary region in one low dose male and one high dose female. One
 

low dose male developed a cutaneous lesion of the chin and one control male had a hard cutaneous lesion on the dorsal surface.
 

B. Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and 2,5-dithiobiurea-dosed groups are shown in Figure
 

3. The Tarone test for positive association between dosage and mortality was significant for both males and females.
 

For males five rats from the high dose and five from the control
 

group were sacrificed in week 78. Survival was relatively high in
 

all groups until about week 70, after which increased mortality was
 

seen—especially in the high dose group. Adequate numbers of male
 

rats were at risk from late-developing tumors, with 22/50 (44 percent) of the high dose, 38/50 (76 percent) of the low dose, and 32/50
 

(64 percent) of the control rats surviving on test until the termination of the study.
 

For females five rats from the high dose and five from the control group were sacrificed in week 78. However, survival was also
 

19
 
750­ •750 

600­ -600 

cc 

O 

450­ -450 

300­

1 

150­

— 300 

-150 

MALE RATS 

I 

15 30 45 60 75 

TIME ON TEST (WEEKS) 

90 105 120 

750 

h-600 

450 

-300 

-150 

15 30 

TIME ON TEST (WEEKS) 

FIGURE 2 

GROWTH CURVES FOR 2,5-DITHIOBIUREA CHRONIC STUDY RATS 

120 

20 
PROBABILIT

Y O

F SURVIVAL PROBABILIT

Y O

F SURVIV

p o P p o 

p

O NJ -^ O3 00 o b 

o -

1 1 1 1 1 1 1 1 1 

TI 

m 

<: C/3 "J> C 1

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< x 

> >

 

> H

 

en co r­Oi a­ T3 

> 

30 

§ 

Z 

CO _

i *.

° i

w

~ 

S , * V1

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i ­

0 0 i f 

INJ HC j ^ H m £ S ! 5q 8- cq 

Oco ' -

r

2 !

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S 

c

30 3 

i

i ii

> J2 

i

• 

m S o S

-

j

r 30 | O 

bl 0 j,,JP 23 c =>~ D | ^H• 

30 

> 

i ^ H-H 1 JJ-rrj

.T 3 i f**^ • H a- x r n j-f" j-f o - 0 0 j' " _H 0 i i J"* !jp 1

 ~ -1 

Cl 

i

§

i i 5 ISJ — ­

o 

1 1 1 1 1 1 1 1 1 1 o ~T o o o o ­ o ro '** oi bo c3 o 
adequate for females, with 24/50 (48 percent) of the high dose, 41/50
 

(82 percent) of the low dose, and 36/50 (72 percent) of the control
 

rats surviving on test until the termination of the study.
 

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).
 

A variety of neoplasms was observed with approximately equal
 

frequency in the dosed and control rats. There were instances in
 

this study, as noted in the summary tables, where neoplasms occurred
 

only in dosed animals, or with increased frequency when compared to
 

the control animals. The nature and incidence of these lesions were
 

similar to those known to occur spontaneously in aged Fischer 344
 

rats, and therefore, these neoplasms were not considered to be related to the administration of 2,5-dithiobiurea.
 

Nonneoplastic lesions which commonly occur in aging rats of this
 

strain were seen in dosed and control rats. None of these lesions
 

was considered to be compound-induced.
 

This pathology examination provided no evidence for the carcinogenicity of 2,5-dithiobiurea in Fischer 344 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 malignant tumor in either sex where at least two such
 

22
 
___ _ __ 

___ 

TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH 2,5-DITHIOBIUREA3
 

TOPOGRAPHY : MORPHOLOGY
 

Hematopoietic System: Leukemia
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or Malignant
 

Lymphomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Carcinoma NOS
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

10/50(0.20)
 

N.S.
 

78
 

10/50(0.20)
 

N.S.
 

78
 

0/45(0.00)
 

P = 0.011
 

LOW
 

DOSE
 

10/49(0.20)
 

N.S.
 

1.020
 

0.419
 

2.484
 

78
 

11/49(0.22)
 

N.S.
 

1.122
 

0.477
 

2.674
 

78
 

0/44(0.00)
 

N.S.
 

HIGH
 

DOSE
 

.14/48(0.29)
 

N.S.
 

1.458
 

0.670
 

3.298
 

73
 

14/48(0.29)
 

N.S.
 

1.458
 

0.670
 

3.298
 

73
 

4/39(0.10)
 

P = 0.043
 

Infinite
 

1.075
 

Infinite
 

109
 
Table 3 (Continued) 

TOPOGRAPHY : MORPHOLOGY
 

Pituitary: Adenoma NOS pr Carcinoma NOS
 

or Chromophobe Adenoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocy toma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

7/45(0.16)
 

N.S.
 

78
 

3/50(0.06)
 

N.S.
 

78
 

1/37(0.03)
 

N.S.
 

110
 

LOW
 

DOSE
 

6/44(0.14)
 

N.S.
 

0.877
 

0.264
 

2.801
 

104
 

4/49(0.08)
 

N.S.
 

1.361
 

0.243
 

8.854
 

109
 

3/41(0.07)
 

N.S.
 

2.707
 

0.230
 

138.498
 

109
 

HIGH
 

DOSE
 

6/39(0.15)
 

N.S.
 

0.989
 

0.299
 

3.134
 

7/46(0.15)
 

N.S.
 

2.536
 

0.619
 

14.390
 

106
 

2/37(0.05)
 

N.S.
 

2.000
 

0.109
 

114.740
 

109
 

95 
___ 

TOPOGRAPHY: MORPHOLOGY
 

Thyroid: C-Cell Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma*3
 

NJ P Values0 

Ol 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Testis: Interstitial-Cell Tumor
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (Continued) 

CONTROL
 

2/37(0.05)
 

N.S.
 

109
 

3/37(0.08)
 

N.S.
 

—_
 

109
 

42/50(0.84)
 

P = 0.049(N)
 

P = 0.002
 

78
 

LOW
 

DOSE
 

2/41(0.05)
 

N.S.
 

0.902
 

0.069
 

11.920
 

109
 

2/41(0.05)
 

N.S.
 

0.602
 

0.053
 

4.969
 

109
 

47/48(0.98)
 

P = 0.018
 

1.166
 

1.009
 

1.218
 

78
 

HIGH
 

DOSE
 

1/37(0.03)
 

N.S.
 

0.500
 

0.009
 

9.179
 

109
 

2/37(0.05)
 

N.S.
 

0.667
 

0.058
 

5.481
 

109
 

33/47(0.70)
 

N.S.
 

0.836
 

0.675
 

1.065
 

70 
TABLE 3 (Concluded)
 

Treated groups received doses of 0.6 or 1.2 percent in feed.
 

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 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 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.
 

NJ
 
___ 

TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH 2,5-DITHIOBIUREA3
 

LOW
 

TOPOGRAPHY: MORPHOLOGY CONTROL DOSE
 

Hematopoietic System: Leukemia 7/49(0.14) 7/50(0.14)
 

P Values0 N.S. N.S.
 

Relative Risk (Control) 0.980
 

Lower Limit 0.317
 

Upper Limit 3.032
 

Weeks to First Observed Tumor	 96 101
 

N3	 Pituitary: Carcinoma NOS 0/39(0.00) 3/41(0.07)
 

P Values0 N.S. N.S.
 

Relative Risk (Control) Infinite
 

Lower Limit 0.577
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 109
 

Pituitary: Adenoma NOS or Chromophobe
 

Adenoma or Carcinoma NOSb 17/39(0.44) 15/41(0.37)
 

P Values0	 N.S. N.S.
 

Relative Risk (Control) 0.839
 

Lower Limit 0.461
 

Upper Limit 1.524
 

Weeks to First Observed Tumor	 78 97
 

HIGH
 

DOSE
 

5/49(0.10)
 

N.S.
 

0.714
 

0.191
 

2.430
 

90
 

0/45(0.00)
 

N.S.
 

12/45(0.27)
 

N.S.
 

0.612
 

0.311
 

1.183
 

78
 
TOPOGRAPHY: MORPHOLOGY
 

Thyroid: C-Cell Carcinoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma or
 

C-Cell Carcinoma
 

P Values
 

N> Relative Risk (Control) oo Lower Limit 

Upper Limit 

Weeks to First Observed Tumor
 

Mammary: Fibroadenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (Continued) 

CONTROL
 

2/45(0.04)
 

N.S.
 

110
 

2/45(0.04)
 

N.S.
 

110
 

12/49(0.24)
 

N.S.
 

103
 

LOW
 

DOSE
 

5/46(0.11)
 

N.S.
 

2.446
 

0.425
 

24.643
 

109
 

6/46(0.13)
 

N.S.
 

2.935
 

0.560
 

28.500
 

109
 

6/50(0.12)
 

N.S.
 

0.490
 

0.164
 

1.291
 

80
 

HIGH
 

DOSE
 

1/32(0.03)
 

N.S.
 

0.703
 

0.012
 

12.848
 

109
 

1/32(0.03)
 

N.S.
 

0.703
 

0.012
 

12.848
 

109
 

9/49(0.18)
 

N.S.
 

0.750
 

0.308
 

1.757
 

75 
TABLE 4 (Concluded)
 

TOPOGRAPHY : MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Uterus: Endometrial Stromal Polyp 

P Values0 

5/46(0.11) 

N.S. 

9/49(0.18) 

N.S. 

4/46(0.09) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

1.690 

0.553 

5.969 

0.800 

0.169 

3.480 

Weeks to First Observed Tumor 110 109 75 

Adrenal: Pheochromocytoma 

P Values0 

3/49(0.06) 

N.S. 

0/48(0.00) 

N.S. 

1/48(0.02) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

0.000 

0.000 

1.695 

0.340 

0.007 

4.060 

Weeks to First Observed Tumor 110 109 

Treated groups received doses of 0.6 or 1.2 percent in feed.
 

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 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 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.
 
tumors were observed in at least one of the control or 2 ,5-dithiobiurea-dosed groups and where such tumors were observed in at least 5
 

percent of the group.
 

For male rats the Cochran-Armitage test for the incidence of
 

pituitary carcinoma NOS was significant (P = 0.011). The Fisher
 

exact test comparing the incidence of this tumor in the high dose
 

group to that in the control group yielded a probability level of
 

P = 0.043, a marginal result which was not significant under the
 

Bonferroni criterion. When the combined incidences of pituitary
 

carcinoma NOS, pituitary adenoma NOS, and pituitary chromophobe
 

adenoma was considered, however, no tests were significant.
 

In male rats the Cochran-Armitage test showed a significant
 

(P = 0.049) negative association between dose and the incidence
 

of interstitial-cell tumors of the testis. The Fisher exact test,
 

however, showed a significantly (P = 0.018) increased incidence of
 

interstitial-cell tumors in the low dose group compared to the control. The comparison of high dose to control was not significant.
 

Based upon these results there was insufficient evidence to
 

conclude that 2,5-dithiobiurea was a carcinogen in rats. No other
 

statistical tests for any site in rats of either sex indicated a
 

significant positive association between the administration of
 

2,5-dithiobiurea and tumor incidence.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

30
 
risk have been estimated and entered in the tables based upon the
 

observed tumor incidence rates. In many 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
 

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

indicating the theoretical possibility of tumor induction in rats by
 

2,5-dithiobiurea that could not be established under the conditions
 

of this test.
 

31
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Compound-related mean body weight depression was apparent in
 

both male and female mice from weeks 20 through 84 (Figure 4). There
 

was no difference in mean body weight gain of low and high dose mice.
 

No unusual signs were recorded for mice of either sex.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and 2,5-dithiobiurea-dosed groups are shown in Figure
 

5. For male mice the Tarone test for a positive association between
 

dosage and mortality was significant. For female mice the Tarone
 

test did not show a significant positive association between dosage
 

and mortality.
 

For males five mice were sacrificed from the high dose group in
 

week 78 and five from the control group in week 79. Adequate numbers
 

of male mice were at risk from late-developing tumors, with 35/50
 

(70 percent) of the high dose, 49/50 (98 percent) of the low dose,
 

and 42/50 (84 percent) of the control group surviving on test until
 

the termination of the study.
 

For females five mice were sacrificed from the high dose group
 

in week 78 and five from the control group in week 79. Survival
 

among females was also adequate, with 40/50 (80 percent) of the high
 

dose, 42/50 (84 percent) of the low dose, and 40/50 (80 percent) of
 

32
 
50- -50 

40 — -40 

QC 

2 so-l 

i- -30 

> 20­ Q 

O 

no 

10— 

MALE MICE 

I 

15 

I 

30 

I 

45 60 

I 

75 

TIME ON TEST (WEEKS) 

90 

CONTROL 

LOW DOSE 

HIGH DOSE 

I 

105 

-20 

-10 

120 

— 50 

— 40 

TIME ON TEST (WEEKS) 

120 

FIGURE 4 

GROWTH CURVES FOR 2,5-DITHIOBIUREA CHRONIC STUDY MICE 

33 
PROBABILITY OF SURVI

PROBABILITY OF SURVIVAL o 0 P O 

o P o o to ^ b> 

o 

r

9 b ro •** CD bo c b i 1 i 1 I 1 I 

i 1 i 1 1 1 i 1 i o — 0 

- Tl 2 

m > 

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S

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> O 

m r

o 

-

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S

2 o 

3D 

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00 ^ m Hc \ m 

2} S~ 

ES 3§- \ 

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2 5 i

m 

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rm

i A 

t/5 

S * 1 L.I 1 ^ — ~ s- 

o a — ^ X :\

30 r_n 

O /JP 

•» 1 i z 

o :] 1 _i 

' 

1 

CO 

f

j

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J

5 8-

i 

1 : IT" a 1 ,1

 

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m 5 S

s

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m 

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o 1 o o o p 

p o p p p o to A <n 

b b NJ iu CD bo 
the control group surviving on test until the termination of the
 

study.
 

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).
 

With the exception of liver neoplasms observed in the female
 

mice, the neoplasms observed in dosed mice were noted at incidences
 

similar to those which occur spontaneously in B6C3F1 mice.
 

The incidence of hepatocellular carcinomas in both low (8/47 [17
 

percent]) and high dose (9/48 [19 percent]) female mice was elevated
 

when compared with the control female mice (2/49 [4 percent]). In
 

addition, hyperplastic nodules were found in a few dosed female mice
 

(3/47 [6 percent] low dose and 2/48 [4 percent] high dose). Histologically, the hepatocellular carcinomas varied from well-differentiated
 

neoplasms with rather close resemblance to normal liver to neoplasms
 

with greater architectural and cytological deviation from normal liver.
 

In the less well-differentiated neoplasms, there were cytoplasmic
 

vacuolation, great variation in cell size, and cytoplasmic hyaline
 

bodies. Well-differentiated neoplasms were composed of nests and
 

cords of cells, and they lacked bile ducts. They compressed the
 

normal parenchyma. Some contained focal areas of more undifferentiated cells. Undifferentiated tumors commonly had cystic and
 

35
 
blood-filled spaces. Cords and nests of atypical cells were often
 

separated by dilated blood-filled sinusoids. A great variation in
 

the incidence of mitotic figures was observed. Metastases did not
 

occur in the dosed mice but did occur at a very low frequency in both
 

male and female controls. No compound-related effects on the livers
 

of male mice were observed.
 

A variety of inflammatory and degenerative lesions which commonly occur in mice of this strain was seen with approximately equal
 

frequency in the dosed and control mice. These nonneoplastic lesions
 

were not considered to be compound-induced.
 

Based upon this pathology examination, 2,5-dithiobiurea was carcinogenic to female mice. There was an increased incidence of hepatocellular carcinomas in dosed female mice when compared to control
 

female mice. Compound-related neoplasms were not observed in the
 

male mice in this study.
 

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 malignant tumor in either sex where at least two such
 

tumors were observed in at least one of the control or 2,5-dithiobiurea-dosed groups and where such tumors were observed in at least 5
 

percent of the group.
 

For female mice the Cochran-Armitage test indicated a significant (P = 0.023) positive association between dose and the incidence 

36 
TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH 2,5-DITHIOBIUREAa
 

CO
 

TOPOGRAPHY : MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Carcinoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar Adenoma or
 

Alveolar/Bronchiolar Carcinoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Malignant
 

Lymphoma"
 

P Valuesc
 

Relative Risk (Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

3/47(0.06)
 

N.S.
 

98
 

7/47(0.15)
 

N.S.
 

97
 

1/50(0.02)
 

N.S.
 

—_
 

97
 

LOW
 

DOSE
 

3/50(0.06)
 

N.S.
 

0.940
 

0.132
 

6.700
 

94
 

13/50(0.26)
 

N.S.
 

1.746
 

0.714
 

4.722
 

94
 

4/50(0.08)
 

N.S.
 

4.000
 

0.415
 

192.807
 

86
 

HIGH
 

DOSE
 

2/47(0.04)
 

N.S.
 

0.667
 

0.058
 

5.554
 

94
 

4/47(0.09)
 

N.S.
 

0.571
 

0.131
 

2.089
 

94
 

4/47(0.09)
 

N.S.
 

4.255
 

0.442
 

204.823
 

94
 
TABLE 5 (Concluded) 

LOW HIGH 

TOPOGRAPHY: MORPHOLOGY CONTROL DOSE DOSE 

Liver: Hepatocellular Carcinoma 15/49(0.31) 9/50(0.18) 7/47(0.15) 

P Values0 P = 0.039(N) N.S. N.S. 

Relative Risk (Control) 0.588 0.487 

Lower Limit 0.252 0.185 

Upper Limit 1.292 1.144 

Weeks to First Observed Tumor 94 94 92 

Treated groups received doses of 1.0 or 2.0 percent in feed.
 

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
 

u>
 in the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The proba­ oo
 bility 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 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.
 
TABLE 6 

ANALYSES OF THE INCIDENCE OF PRIMARY

SPECIFIC SITES IN FEMALE MICE TREATED WITH

TOPOGRAPHY : MORPHOLOGY CONTROL 

Lung: Alveolar /Bronchiolar Carcinoma 3/50(0.06) 

P Values0 N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 79

 

Lung: Alveolar /Bronchiolar Adenoma OJ

 

VD or Alveolar/Bronchiolar Carcinoma 4/50(0.08)

 

P Values0 N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 79 

Hematopoietic System: Malignant Lymphoma 6/50(0.12) 

P Values0 N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 98 

 TUMORS AT 

2,5-DITHIOBIUREA£ 

LOW 

DOSE 

1/47(0.02) 

N.S. 

0.355 

0.007 

4.229 

83 

5/47(0.11) 

N.S. 

1.330 

0.304 

6.316 

83 

12/48(0.25) 

N.S. 

2.083 

0.792 

6.219 

48 

HIGH 

DOSE 

1/48(0.02) 

N.S. 

0.347 

0.007 

4.143 

94 

6/48(0.13) 

N.S. 

1.563 

0.396 

7.090 

94 

10/48(0.21) 

N.S. 

1.736 

0.622 

5.365 

78 
TABLE 6 (Concluded)
 

TOPOGRAPHY: MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Liver: Hepatocellular

P Values0 

 Carcinoma 2/49(0.04) 

P = 0.023 

8/47(0.17) 

P = 0.039 

9/48(0.19) 

P = 0.023 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

4.170 

0.889 

38.627 

4.594 

1.017 

41.865 

Weeks to First Observed Tumor 98 94 78 

Pituitary:

P Values0 

 Adenoma NOS 0/42(0.00) 

N.S. 

1/44(0.02) 

N.S. 

2/37(0.05) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Infinite 

0.051 

Infinite 

94 

Infinite 

0.338 

Infinite 

94 

Treated groups received doses of 1.0 or 2.0 percent in feed.
 

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

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 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.
 
of hepatocellular carcinoma. This was supported by a significant
 

(P = 0.023) Fisher exact test comparing the incidence of this tumor
 

in the high dose group to that in the control group. The low dose to
 

control comparison had a probability level of P = 0.039, a marginal
 

result which was not significant under the Bonferroni criterion. In
 

historical data on untreated B6C3F1 mice at Mason Research Institute
 

in the NCI Carcinogenesis Testing Program, 19/275 (7 percent) control
 

female mice had this tumor—compared to the incidences in this bioassay of 2/49 (4 percent), 8/47 (17 percent), and 9/48 (19 percent) observed in the control, low dose, and high dose groups, respectively.
 

This, together with the fact that the control mice were not matched,
 

weakened the significance of the findings.
 

For male mice the Cochran-Armitage test for the incidence of
 

hepatocellular carcinoma showed a significant (P = 0.039) negative
 

association. The Fisher exact tests, however, were not significant.
 

The historical incidence of this tumor in male B6C3F1 untreated
 

control mice observed at Mason Research Institute was 88/275 (32
 

percent), compared to the incidence of 15/49 (31 percent) in the
 

controls for this bioassay.
 

Based on these statistical results, the administration of 2,5­

dithiobiurea was associated with an elevated incidence of hepatocellular carcinoma in female B6C3F1 mice under the conditions of this
 

experiment. No other statistical tests for mice of either sex were
 

significant.
 

41
 
V. DISCUSSION
 

In both species adequate numbers of animals in all groups survived sufficiently long to be at risk from late-developing tumors.
 

Compound-related mean body weight depression was observed in mice but
 

not in rats. No consistent pattern of clinical signs was observed in
 

either species.
 

In rats no tumors occurred at a significantly higher incidence
 

in groups of rats dosed with 2,5-dithiobiurea than in corresponding
 

control groups. Since no significant retardation of growth, or increased occurrence of clinical signs were associated with the feeding of 2,5-dithiobiurea, it is possible that the compound was not
 

administered to rats at the maximum tolerated concentration.
 

Among female mice, there was a significant positive association
 

between the incidence of hepatocellular carcinoma and the concentration of 2,5-dithiobiurea in the diet. The incidence of hepatocellular
 

carcinoma was significantly higher in the high dose group than in the
 

control group. The control group was not completely matched, however,
 

since it was started 5 weeks after the dosed animals, and the control
 

incidence of 4 percent hepatocellular carcinomas was lower than the
 

7 percent found in the laboratory's historical controls. Among male
 

mice, however, there was a significant negative association between
 

the incidence of hepatocellular carcinoma and dietary concentration.
 

No neoplasms occurred at a significantly higher incidence in dosed
 

male mice than in their controls.
 

42
 
Under the conditions of this bioassay, the evidence suggested
 

that 2,5-dithiobiurea was carcinogenic to female B6C3F1 mice, causing
 

an increased incidence of hepatocellular carcinomas, but was not carcinogenic to male B6C3F1 mice or to Fischer 344 rats of either sex.
 

43
 
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,
 

Boit, H.G., Beilsteins Handbuch der Organischen Chemie. Springer
 

Verlag, Berlin, 1973.
 

Chemical Abstracts Service, The Chemical Abstracts Service (CAS)
 

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

Chemical Society, Washington, B.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.
 

Fueki, S., K. Abe, and K. Ohsawa, "Electrolyte for Plating Bright
 

Tin-Nickel Coatings, Especially an Aqueous Pyrophosphate Electroplating Bath." Ger. Offen. 2,419,024 (Sony Corporation),
 

November 7, 1974; Chemical Abstracts 82, 104891a.
 

Fujino, T. and S. Fueki, "Cooper Electroplating From a Pyrophosphate
 

Bath." Japanese Patent 71 37,646 (Metal Treating Technology Research Laboratory, Ltd.), November 5, 1971; Chemical Abstracts
 

77, 147067a.
 

Gart, 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.
 

International Agency for Research on Cancer (IARC), IARC Monographs
 

on the Evaluation of Carcinogenic Risk of Chemicals to Man.
 

Volume 7, Some Anti-thyroid and Related Substances, Nitrofurans
 

and Industrial Chemicals. World Health Organization, IARC, Lyon,
 

France, 1974.
 

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

Observations." Journal of the American Statistical Association
 

53:457-481, 1958~
 

44
 
Kodak-Pathe, "Sensitized Photographic Products to Give Stable Images."
 

French Patent 1,455,581, October 28, 1966; Chemical Abstracts 74,
 

103936x.
 

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

"Carcinogenesis Bioassay Data System." Computers and Biomedical
 

Research 7:230-248, 1974.
 

McBride, C.E., "Light-Developable Silver Halide Emulsions." U.S.
 

Patent 3,287,135 (Eastman Kodak Co.), November 22, 1966; Chemical
 

Abstracts 66, 3,287,137.
 

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

Co., New York, 1966.
 

Niles, E.T., "Pyrotechnic Disseminating Formulation." U.S. Patent
 

3,929,530 (Dow Chemical Co.), December 30, 1975; Chemical Abstracts 84, 138048n.
 

Nimura, T., K. Jin, and H. Komatsu, "Bleach-Fixing Baths for Color
 

Photographic Films." Ger. Offen. 2,210,841 (Konishiroku Photo
 

Industry Co., Ltd.), September 20, 1973; Chemical Abstracts 79,
 

151608m.
 

Nimura, T., K. Jin, and H. Komatsu, "Processing Light-Sensitive Silver
 

Halide Color Photographic Materials." U.S. Patent 3,809,563
 

(Konishiroku Photo Industry, Co., Ltd.), May 7, 1974; Chemical
 

Abstracts 81, 44110n.
 

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 3^:1073-1079, 1972.
 

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.
 

45
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH 2,5-DITHIOBIUREA
 

TABLE Al

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH 2,5-DITHIOBIUREA 

CONTROL (UNTR) LOW DOSE 

01-0160 01-0100 

HIGH 

01-0 

DOSE 

1 10 

ANIMALS INITIALLY IN STUDY 

ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 

50

50

50

 50 19 

 49 

50 

1*8 

1(8 

INTEGUMENTARY SYSTEM
 

*SKIN (50) (1*9) (48)
 

FIBROMA 1 (2X)
 

FIBROSARCOMA 1 <2X)
 

*SU3CUT TISSUE (50) («9) (48)
 

SARCOMA, NOS 1 (2X)
 FIBROMA 1 (2X) 1 (2X)
 

FIBROSARCOMA 1 (2X)
 

RESPIRATORY SYSTEM
 

ILUNG (49) (49) (48)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 2 (US) 1 (2X)
 

ALV20LAR/BRONCHIOLAH CARCINOMA 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

*«ULTIPLS ORGANS (50) (U9) (48)
 

LEUKEMIA, NOS 1 <2X)
 MYELOMONOCYTIC LEUKEMIA 9 (18X) 10 (20X) 11 (23%)
 GBANULOCYTIC LEUKEMIA 1 (2X)
 

&SPLEEN (50) (49) (47)
 

MALIG.LYKPHOtlA, LYtlPHOCYTIC TYPE 1 (2X)
 

*LIVER (49) (U9) (48)
 

MYiLOMONOCYTIC LEUKEMIA 2 (»*)
 

CIRCULATORY SYSTEM
 

*HEART (48) (48) (47)
 

SARCOMA^. NOS^ METASTATIC 1 ±2*1
 

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

* NUMBE R OF AMIHAL S NflCROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-3 
DIGESTIVE SYSTEM 

*LIVER 

HiJPATOCELLULAR CARCINOM A 

*JEJUNU M 

LEIOMYOSARCOMA
 

URINARY SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

IPITOITARY
 

CARCINOMA, NOS
 

ADENOMA, NOS
 

CHHOMOPHOBE ADENOMA
 

*ADRENAL
 

PHEOCHROMOCYTOMA
 

PHEOCHROMOCYTCMA. MALIGNANT
 

tTHYROID
 

POLLICULAR-CELL CARCINOMA
 

C-CELL ADENOMA
 

C-CELL CARCINOMA
 

tPANCREATIC ISLETS
 

ISLET-CELL ADBNOHA
 

REPRODUCTIVE SYSTEM
 

*PR!!PUTIAL GLAND
 

CARCINOMA, NOS
 

ADENOMA, NOS
 

HESTIS
 

INTERSTITIAL-CELL TUMOR
 

NESVOL'S SYSTEM
 

fBHAIN
 

ASTROCYTOMA ._
 

TABLE A1 (CONTINUED) 

CONTROL (UNTR) 

01-0160 

(49)
 

(19)
 

(45)
 

5 (11*)
 

2 (4X)
 

(50)
 

3 (6X)
 

(37)
 

1 (3X)
 

1 (3X)
 

2 (5X)
 

(47)
 

1 (2X)
 

(50)
 

2 (4X)
 

(50)
 42 (84X)
 

(50)
 

LOW DOSE 

01-0100 

(19)
 

1 (2X)
 

(48)
 

(11)
 

6 (14X)
 

(49)
 

4 (8X)
 

(41)
 

3 (7X)
 

1 (2X)
 

2 (5X)
 

(48)
 

2 <4X)
 

(49)
 

(48)
 

47 (98X)
 

(48)
 

HIUH DOSE 

01-0110 

(48)
 

2 (4X)
 

(45)
 

1 (2X)
 

(39)
 

4 (10X)
 

2 (5X)
 

(46)
 

6 (13X)
 

1 (2X)
 

(37)
 

2 (5X)
 

1 (3X)
 

1 (3X)
 

(45)
 

(48)
 

1 (2X)
 

1 (2X)
 

(47)
 

33 (70X)
 

(46)
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

» NUMBER OF ANIMALS NECROPSIED
 

A-4
 
TABLE A1 (CONTINUED) 

COHTROL(UNTR )

01-0160

 LOW DOSE

 01-0100

 HIGH DOSE 

 01-0110 

iCERSBRAL CORTEX

GLIOMA, NOS

 (50)

 1 (2*) 

 (48) (l»6) 

SPECIAL SENSE ORGAN S 

*ZYi1BAL' S GLAN D

C2RUMINOUS CARCINOMA

 (50) (U9 ) (48) 

1 (2X) 

MUSCULOSKELETAL SYSTEM 

*VERTEBRA

OSTEOSARCOMA

 (50) (49)

 1 (2X) 

 (US) 

BODY CAVITIES 

*BODY CAVITIES

MESOTHELIOMA, NOS

MESOTHELIOMA. MALIGNANT

 (50) (49)

 1 (2X)

 (l»8) 

1 (2X) 

1 (2*) 

*ABDOMINAL CAVITY

LEIOMYOSAHCOMA

 (50) (49)

 1 (2%) 

 (48) 

ALL OTHER SYSTEMS 

THORACIC CAVITY 

HEPATOCELLULAR CARCINOMA, BETAS! 1 

ANIHAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY

NATURAL DEATHS

MORIBUND SACRIFICE

SCHEDULED SACRIFICE

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE

ANIMAL MISSING 

 50

 5

 8

 5

 32

 50

 5

 7

 38

 50 

 11 

 12 

5 

 22 

!_!HCLUD2S_AUTgLYZED_ANIMALS 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

A-5
 
TABLE Al (CONCLUDED)
 

crONTROL(UNTR) LOW DOSE HIGH DOSE
 

01-0160 01-0100 01-0110
 

TUMOR SUMMARY
 

TOTAL ANIMALS KITH PRIMARY TUMORS* 19 48 40
 TOTAL PRIMARY TUMOSS 76 82 75
 

TOTAL ANIMALS WITH BENIGN TUHORS 46 47 35
 TOTAL BENIGN TUBOBS 56 62 45
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 17 18 24
 

TOTAL MALIGNANT TUMORS 20 19 29
 

TOTAL ANIMALS WITH SECONDARY TUMORSt 1 1
 

TOTAL SECONDARY TUMORS 1 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT 1 1
 

TOTAL UNCERTAIN TUMORS 1 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY 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 2,5-DITHIOBIUREA 

CONTROL (UNTR) LOW DOSE

02-0160 02-0100

 HIGH DOSE 

 02-0110 

ANIMALS INITIALLY IN STUDY 50

ANIMALS MISSING 1 

ANIMALS NECROPSIED «9

ANIMALS EXAMINED HISTOPATHOLOGICALLY** <49

 50

 50

 50

 50 

 V9 

 U9 

INTEGUMENTARY SYSTEM
 

*SKIN (U9) (50) («9)
 

OSTSOSARCOMA 1 (2%)
 

*SUBCUT TISSUE (U9) (50) (t9)
 

FIBROMA 2 (US) 1 (2X)
 

FIBROSARCOMA 1 (2X)
 

LIPOMA 1 (2X) 1 (2X)
 

RESPIRATORY SYSTEM
 

ILUNG (1(9) (50) (U9)
 

ADENOCARCINOMA. NOS, METASTATIC 1 (2X)
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (2H)
 

OSTEOSARCOMA, METASTATIC 1 (2X)
 

HEMATOPOIETIC SYSTEM
 

•MULTIPLE OBGANS (49) (50)
 LEDKEMIA.NOS 1 (2X)
 

MYELOMONOCYTIC LEUKEMIA 6 (12X) 5 (10S) 5 (10X)
 

*M£DIASTINAL L.NODlS («t) (K6) (11)
 AD£NOCARCIMOMA, NOS, HETASTATIC 1 (2%)
 

fLIVER (18) (50)
 

MYELOMONOCYTIC LEUKEMIA 2 <«X)
 

CIRCULATORY SYSTEM
 

NONE
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-7 
TABLE A2 (CONTINUED)
 

HIGH DOSE
 

02-0110
 

(19)
 

1 (2%)
 

(19)
 

1 (2X>
 

(15)
 

12 (27X)
 

(18)
 

1 (2S)
 

(18)
 

1(2%)
 

(32)
 

1 OS)
 

1 (3X)
 

(18)
 

1 (6X)
 

(19)


1 (2X)
 9 (18*)
 

(19)
 

2 11X).
 

DIGESTIVE SYSTEM
 

ILIVSR
 

NEOPLASTIC NODULE
 

HEPATOCELLULAR CARCINOMA
 

URINAR Y SYSTEM
 

tKIBNEY
 

ALVEOLAR/BRUNCHIOLAR


TUBULAR-CEIL ADENOMA
 

ENDOCRINE SYSTEM
 

•PITUITARY
 

CARCINOMA, NOS
 

ADENOMA. NOS
 

CH80MOPHOBE ADENOMA
 

#ADRENAL
 

PIIEOCHROMOCYTOMA
 

 CA, METASTA
 

PHEOCHFOMOCYTOMA, MALIGNANT
 

tADSENAL CORTEX
 

ADENOCARCINOMA, NOS, METASTATIC
 

#THYROID
 

FOLLICULAR-CELL CARCINOMA
 

C-CELL ADENOMA
 C-CELL CARCINOMA
 

*PARATHYROID
 

ADENOMA, NOS
 

REPRODUCTIVE SYSTEM
 

»MA«BAR Y GLAND
 

ADENOCARCINOMA, NOS
 

FIBROADENOMA
 

*CLITORAL GLAND
 

CARCINOdAiUOS
 

CONTROL (UNTR)
 

02-0160
 

(18)
 

1 (2%)
 

(US)
 

(39)
 

15 (38X)
 

2 (5X)
 

(49)
 

3 (6X)
 

(19)
 

(15)
 

2 (UX)
 

(27)
 

(19)
 

12 (2UX)
 

(19)
 

LOH DOSE
 

02-0100
 

(50)
 

(50)
 

1 (2%)
 

(11)
 

3 (7%)
 12(29%)
 

(18)
 

(48)
 

(16)
 

1 (2%)
 

5 (11%)
 

(35)
 

(50)
 

6 (12X)
 

(50)
 

..2 mi _.
 * NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

» NUMBER OF ANIMALS NECROPSIED
 

A-8
 
ADENOMA, NOS

#UT£RUS


LEIOMYOSARCOMA


ENDOHETRIAL STROMAL POLYP


NERVOUS SYSTEM
 

#BRAIN


ASTROCYTOMA


SPECIAL SENSE ORGANS
 

*ZYMBAL'S GLAND


CERUMINOUS CARCINOMA


MUSCULOSKE1ETAL SYSTEM
 

•SKULL


OSTEOMA


BODY CAVITIES
 

*PER!TONZUM


ADSNOCARCINOHA, NOS, METASTATIC


ALL OTHER SYST2MS
 

DIAPHRAGM
 

ADENOCARCINOMA, NOS, METASTATIC


SIT2 UNKNOWN
 

ADENQCARCINOMA^.NOS


*	 NUMBER OF ANIMALS BITH TISSUE EXAMINED


*	 NUMBER OF ANIMALS' NECROPSIED
 

TABLE A2 (CONTINUED) 

CONTROL(UNTR)

02-0160

 1 ( 2 %) 

 («6)


 5 (11X)


 (149)


 1 (2%)
 

(49)


 1 (2%)
 

(149)


 (149)


 MICROSCOPICALLY
 

 LOB DOSE HIGH DOSE 

 02-0100 02-0110 

 (49)	 (146)
 

1 (2%)
 

9 (18X) U (9%)
 

 (49) (148)
 

 (50) (49)
 

 (50)	 (149)
 

1 (2%)
 

 (50)	 (<49)
 

1 (2%)
 

1
 

i
 

A-9
 
TABLE A2 (CONCLUDED)
 

CONTROL (UNTR) LOW DOSE
 

02-0160 02-0100
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 50 50
 NATURAL DEATHS 2
 

MORIBUND SACRIFICE 6
 

SCHEDUL3D SACRIFICE 5
 

ACCIDENTALLY KILLED
 TERMINAL SACRIFICE 36
 

ANIMAL MISSING 1
 

SI INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS HITH PRIMARY TUMORS* 31 36
 

TOTAL PRIMARY TUMORS 52 51
 

TOTAL ANIMALS WITH BENIGN TUMORS 27 25
 TOTAL BENIGN TUMOHS 10 32
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 10 16
 TOTAL MALIGNANT TUMORS 12 19
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS HITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN-


PaiilARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

« SECONDARY TUMORS: M2TASTATIC TUMORS OR TUMORS INVASIVE INTO
 

HIGH DOSE
 02-0110
 

50
 

13
 

8
 

5
 

21
 

32
 

U2
 

21
 

27
 

la
 

3
 

ADJACENT ORGAN
 

A-10
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH 2,5-DITHIOBIUREA
 

TABLEBl 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH

CONTROL (ONTR) LOK DOSE 

05-0160 05-0120 

ANIHALS INITIALLY IN STUDY 50 50 

ANIilALS MISSING 

ANIMALS NECROPSIBD 50 50 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** (,9 50 

INTEGUMENTARY SYSTEM
 

*SKIN (50) (50)
 

SQUAMOUS CELL PAPILLOMA
 

RESPIRATORY SYSTEM
 

*LUNG (17) (50)
 

HEPATOCELLULAR CARCINOMA, METAST 2 («X)
 

ALVEOLAP/SRONCHIOLAR ADENOMA H (9X) 10 (20X)
 

AlVEOLAR/BRONCHIOLAR CARCINOMA 3 (6X) 3 (6X)
 

HEMATOPOIETIC SYSTEM
 

•MULTIPLE ORGANS (50) (50)
 

MALIGNANT LYMPHOMA, NOS 1 (2X) 1 (2«)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (2*)
 

*SPLEEN (U9) (50)
 

HEMANGIOMA
 

HEKASGIQSARCOMA 1 (2%) 1 <2X)
 

MALIGNANT LYMPHOMA, NOS
 

4MEDIASTINAL L.NODji (HO) (tl)
 MALIGNANT LYMPHOMA, NOS 1 (2%)
 

CPANCREATIC L.NODfi («0) (11)
 MALIGNANT LYMPHOMA, NOS
 

•DUODENUM	 (19) (50)
 

MALIGNANT LYMPHOMA, NOS 1 (2%)
 

CIRCULATOR Y SYSTEM 

# NUMBER OF ANIHALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 2,5-DITHIOBIUREA 

HIGH DOSE
 

05-0130
 

50
 

1
 

47
 

17
 

(17)
 

1 (2X)
 

(17)
 

2 (IX)
 

2 (US)
 

(17)
 

1 (2X)
 

(17)
 

1 <2X)
 

1 (2%)
 

2 (IX)
 

(«0)
 

(10)
 

1 (3X)
 

(16)
 

B-3 
TABLE Bl (CONTINUED) 

CONTRO L (UNTR )

05-0160 LOW DOSE 05-0120

 HIGH DOSE 

 05-0130 

DIGESTIV E SYSTE M 

*LIVER

HEPATOCELLULAR CARCINOMA

*3TOMACH

ADENOMATOUS POLYP, NOS

 (149)

 15 (J1X)

 («9)

 1 (2%) 

 (50)

 9 (\S%)

 (50)

 (HI) 

7 (15X) 

 (U7) 

URINARY SYSTEM 

tKIDNE Y

TUB'JLAP-CELL ADJ5NOH A

 (<*9) (50) («7) 

1 (2%) 

SNDOCHINE SYSTEM 

*THY80ID

FOLLICULAR-CELL ADENOMA

FOLLICULAR-CELL CARCINOMA

 (1*2) (U7) (<tO) 

1 (3X) 

1 (3X) 

REPRODUCTIVE SYSTEM 

#TESTIS

INTERSTITIAL-CELL TUMOR

 (U9)

 1 (2X) 

 (50) (»6) 

NERVOUS SYSTEM 

#BRAIN

ASTROCYTOMA

 (1*8) (SO) (U6) 

1 (2S) 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NKCROPSIED 

B-4
 
TABLE Bl (CONCLUDED)
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 ANIMAL MISSING
 

8 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TOTAL MALIGNANT TUMORS
 

TOTAL ANIMALS WITH SECONDARY TUMORS*


TOTAL SECOKDARY TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 TOTAL UNCERTAIN TUMORS
 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

05-0160 05-0120 05-0130 

50 50 50 

3 6 

1 3 

5 5 

42 49 35 

1 

20 23 18 26 27 22 

6 10 6 

6 10 6 

18 15 13 

20 17 16 

2 

2 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 * SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-5
 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED WITH 2,5-DITHIOBIUREA 

HIGH DOSE
 

06-0130
 

50
 

1
 

48
 

48
 

(48)
 

(48)
 

5 (10X)
 

1 <2X)
 

C»8)
 

6 (13X)
 

1 (2X)
 

(47)
 

1 (2X)
 

(39)
 

 (39)
 

 (18)
 

1 (2X)
 

1 (2X)
 

 (17)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS MISSING
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY**
 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE
 

FIBROSARCOMA
 

HEMANGIOSABCOMA
 

RESPIRATORY SYSTEM
 

ILUNG
 

HEPATOCELLULAR CARCINOMA. METAST
 

ALVEOLAR/BRONCHIOLAR ADENOMA
 

ALVEOLAR/BHONCHIOLAR CARCINOMA
 

HEMATOPOIETIC SYSTEM
 

'MULTIPLE ORGANS
 

MALIGNANT LYMPHOMA, NOS
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

ISPLEEN
 

HEMANGIOSARCOMA
 

MALIGNANT LYMPHOMA, NOS
 

MALIG.LYMPHOBA, HISTIOCYTIC TYPE
 

*MANDIBULAR L. NODE
 

MALIGNANT LYBPHOMA, NOS
 

tMEDIASTINAL L.NODE


MALIGNANT LYMPHOMA. NOS


tLIVER


MALIGNANT LYMPHOMA, NOS


MALIG.LYMPHOMA, HISTIOCYTIC TYPE


tPEYERS PATCH


CONTROL (UNTR)
 

06-0160
 

50
 

50
 

50
 

(50)
 

1 <2X>
 

1 (2X)
 

(50)
 

1 (2X>
 1 (2X)
 

3 (6X)
 

(50)
 

3 (6X)
 

(49)
 

1 (2X)
 

1 (2X)
 

(40)
 

1 <3X)
 

(10)


 (U9)


 (49)


LOW DOSE
 

06-0120
 

50
 

48
 

48
 

(48)
 

1 (2X)
 

(47)
 

4 (9X)
 

1 (2X)
 

(18)
 

10 (21X)
 

(46)
 

1 (2X)
 

(33)
 

(33)


 1 (JX)
 

 (U7)


 (17)


* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIBD 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-6
 
CIRCULATORY SYSTEM
 

NCNE
 

DIGESTIVE SYSTEM
 

fLIVER


HEPATOCELLULAR CARCINOMA


U3INARY SYSTEM
 

NONE
 

ENDCCRINE SYSTEM
 

*PITUITARY


ADENOMA, NOS


*THYROID


FOLLICULAR-CfiLL ADENOMA


REPRODUCTIVE SYSTEM
 

tUTESUS


SNDOMETRIAL STROMAL SARCOMA


#OVARY


PAPILLARY ADclNOCARCINOMA


TUBULAR ADENOMA


NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

»HARDEHIAN GLAND


ADENCMA, NOS


MUSCULCSKELETAL SYSTEH
 

NONE _
 

TABLE B2 (CONTINUED) 

CONTROL (UNTR)


06-0160


 (49)


 2 (4X)


 (12)


 (11)


 («9)


 (48)


 (50)


 LOW DOSE HIGH DOSE
 

 06-0120 06-0130
 

 (47)	 (U8)
 

8 (17*) 9 (19J)
 

 (Kit) (37)
 

1 (2%) 2 (5X)
 

 (45)	 (45)
 

1 (2X)
 

 (U3) («7)
 

1 (2X)
 

 (<*0) (45)
 

1 (2X)
 

1 (3%)
 

 (48)	 (48)
 

1 (2%)
 

*	 NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS N3CROPSIED
 

B-7
 
TABLE B2 (CONCLUDED)
 

CONTROL (UNTR) LOS DOSE HIGH DOSE
 

06-0160 06-0120 06-0130
 

BODY CAVITIES
 

NONE
 

ALL OTHEB SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

NATURAL DEATHS 3 2
 

MORIBUND SACRIFICE 2 2
 

SCHEDULED SACRIFICE 5 5
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE UO U2 UO
 

ANIMAL MISSING 1
 

INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 12 2U 20
 

TOTAL PRIMARY TUMORS 15 30 30
 

TOTAL ANIMALS HITH BENIGN TUMORS 1 7 8
 

TOTAL BENIGN TUMORS 1 7 8
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 11 21 18
 

TOTAL MALIGNANT TUMORS 1* 23 22
 

TOTAL ANIMALS WITH SECONDARY TUMORSi 1
 

TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 2,5-DITHIOBIUREA
 

TABLE Cl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

TREATED WITH 2,5-DITHIOBIUREA

 

CONTROL (ONTR) 

01-0160 

LOW DOSS 

01-0100 

HIGH DOSE 

01-0110 

ANIMALS INITIALLY IN STUDY 

ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

50 

50 

**50 

50 

19 

49 

50 

48 

48 

INTEGUMENTARY SYSTEM
 

*SKIN (50) (19) (18)
 EPIDERMAL INCLUSION CYST 1 (2%)
 INFLAMMATION, SUPPURATIVE 1 <2X)
 

ALOPECIA 1 (2%)
 

HYPERKERATOSIS 1 (2X)
 ACANTHOSIS 1 (2X)
 

*SUBCUT TISSUE (50) (19) (18)
 ABSCESS, NOS 1 (21)
 

RESPIRATORY SYSTEM
 

#LUNG (19) (19) (48)
 

CONGESTION, CHRONIC PASSIVE 1 (2X)
 

INFLAMMATION. INTERSTITIAL 4 (8X)
 

FIBROSIS, DIFFUSE 1 (2X)
 

HYPE3PLASIA, NOS 1 (2X)
 

HYPEHPLASIA, ADENOMATOUS 1 (2X)
 

HYPERPLASIA, ALVEOLAR EPITHELIUM 1 (2%) 1 (2X)
 

<I.UN J/ALVEOLI (19) C*9) (48)
 

HEMORRHAGE 1 (2X)
 

HEMATOPOIETIC SYSTEM
 

»BONE MARRO W (48) (41) (47)
 

MYEI.OFIBROSIS 1 (2X) 1 (2%)
 

HYPERPLASIA, HEMATOPOIETIC 5 (IIS) 6 (13X)
 

*SPLEEN (50) (49) (47)
 

FIBROSIS 1 (2%)
 

SCAH
 

t NUMBE R OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NU.13ER OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-3 
TABLECl (CONTINUED)
 

CONTROL (UNTR) LOH DOSE HIUH DOSE 

01-0160 01-0100 01-0110 

NECROSIS, NOS
 

CALCIFICATION. NOS 1 (2X)
 

HEMOSIDEROSIS 2 (4)4)
 

EHYTHROPOIESIS 1 (2X)
 

*MANDIBULAR L. NODE (49) »6)
 

NECROSIS, NOS 1 (2%)
 

HYPEEPLASIA, PLASMA CELL 1 (2%) 1 (2%)
 

*MES£NTERIC L. NODE (49) (46)
 

HYPERPLASIA, NOS 1 (2%)
 HYPEHPLASIA, PLASMA CELL 1 (2%)
 

3RYTHROPOIESIS 1 (2*)
 

*RENA L LYMP H NODji (49) (46) (44) 

HYP.ERPLASIA, NOS 1 (2X) 1 (2X) 

CIRCULATORY SYSTEM
 

tHEABT/ATRIUM (U8) (48) (47)
 

THROMBOSIS, NOS 1 (2X)
 

tMYOCARDIUM (48) (48) (47)
 

INFLAMMATION, INTERSTITIAL 1 (2%)
 

FIBROSIS 2 (4%)
 

DEGENERATION. NOS 1 (2*)
 

*PULMONARY ARTERY (50) (48) 

CALCIFICATION, NOS 1 (2*) 

DIGESTIVE SYSTEM
 

#SALIVARY GLAND (50) (47)
 

HYPERPLASIA, INTRADUCTAL 1 (2X)
 

tLIVER (49) (48)
 

BILE STASIS 1 (2%)
 

INFLAMMATION, CHRONIC FOCAL 1 (2»)
 

INFLAMMATION. CHRONIC DIFFUSE 1 (2X>
 HEPATITIS, TOXIC 1 (2%)
 

NECROSIS, FOCAL 3 (6X) 1 (2X)
 

METAMORPHOSIS FATTY 2 (4X)
 

3ASOPHILIC CYTO CHANGE 1 (2X)
 

HYPSEPLAS!A«__FQCAL.
 

» NOilBEE OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-4
 
TABLECl (CONTINUED) 

CONTROL (UNTR) 

01-0160 

LOU DOSE 

01-0100 

ANGIECTASIS 

#LIVER/CENTRILOBULAB 

CONGESTION, PASSIVE 

NECROSIS, NOS 

<U9) 

1 (2X) 

(49) 

*BILE DUCT 

HYPERPLASIA, NOS 

HYPERPLASIA, DIFFUSE 

(50) 

2 («%) 

(49) 

1 «X) 

1PANCREAS 

INFLAMMATION, NOS 

INFLAMMATION, CHRONIC 

INFLAMMATION, CHRONIC FOCAL 

(47) 

2 <H%) 

1 (2X) 

(48) 

1

1

 (2X) 

 (2X) 

ISTOMACH 

HYPERKERATOSIS 

ACANTHOSIS 

(t9) 

1 (2X) 

1 (2X) 

(48) 

tGASTRIC MOCOSA 

ULCER, DOS 

ABSCESS, NOS 

NECROSIS, FOCAL 

(49) (48) 

1 (2X) 

IPEYERS PATCH 

HYPERPLASIA, NOS 

(49) 

1 (2%) 

(48) 

URINARY SYSTEM
 

KKIDNEY (50) (49)
 

CONGESTION, NOS 1 (2%)
 GLOMEEULONEPHKITIS, NOS U <8X) 1 (2*>
 

NEPHEOSIS, NOS 35 (70*) U7 (96%)
 

PIGMENTATION, NOS 1 (2X)
 

#KIDNEY/CORTEX (50) (49)
 

CYST, NOS 1 (2%)
 

*K1DNEY/?ELVIS (50) (49)
 

MINERALIZATION 1 <2X)
 

#URINARY BLADDER (50) (46)
 

INFLAMMATION, ACUTE HEMOREHAGIC
 

ENDOCRINE SYSTEM
 

•PITUITARY	 (45) (41)
 

CONGESTION^ NOS 1 12XL
 

* NUBBE K OF ANIMAL S KITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NlSCROPSIED 

HIGH DOSE
 

01-0110
 

1 (2X)
 

(48)
 

2 (MX)
 

(48)
 

1 (2X)
 

(45)
 

2 (»»)
 

(45)
 

(45)
 

1 (2X)
 

1 (2X)
 

(45)
 

(48)
 

35	 (73X)
 

(48)
 

(48)
 

(47)
 

1 (2X)
 

(39)
 

C-5 
#ADRENAL COBTEX
 

HYPERPLASTIC NODULE
 

*ABR2NAL MEDULLA
 

HYPERPLASTIC NODULE
 

HYPERPLASIA, FOCAL
 

#THYROID
 

CYSTIC FOLLICLES
 

HYPERPLASIA, C-C2LL
 

REPRODUCTIVE SYSTEM
 

*PRSPDTIAL GLAND


INFLAMMATION, NOS
 INFLAMMATION, SUPPUSATIVE
 

INFLAMMATION, ACUTE
 

INFLAMMATION, CHRONIC
 

• PROSTATE


INFLAMMATION, NOS


INFLAMMATION, ACUTE
 

INFLAMMATION, ACUTE FOCAL
 

tTESTIS


MINERALIZATION
 

PBBIVASCULITIS
 CALCIFICATION. NOS


CALCIFICATION, FOCAL
 ATROPHY, NOS
 HYPERPLASIA, INTERSTITIAL CELL


ITESTIS/TUBULE
 DEGENERATION, NOS.
 

CALCIFICATION, NOS
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EYE/RETINA


CATARACT


TABLE Cl (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

01-0160 01-0100 01-0110 

(50)	 (U9) (16)

 

1 (2%)
 

(50)	 (49) (46)
 

1 <2X)
 

(37)	 (41) (37)

 

1 (3X) 1 (2X)
 

2 <5X) 1 (2XJ
 

 (50)	 (<J9)
 

1 (2X)
 

1 (2X)
 

1 (2X)
 

1 (2*)
 

 (US) (H5) (Hit)
 

3 (6X)
 

3 (7X) 3 (7X)
 

1 (2X)
 

 (50) (48) (47)
 

1 (2X)
 3 (6X)
 

1 (2X)
 11 (22%) 18 (38X) 22 (17X)
 It (8X) 1 (2X) 1 (2X)
 

 (50) (U8) (47)
 1	 (2*) 1 (2X)
 

1 (2X)
 

 (50)	 (49) (48)
 

1_I2X).
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

C-6
 
TABLECl (CONCLUDED)
 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

01-0160 01-0100 01-0110 

ATROPHY, NOS 1 <2X) 

•EYE/CRYSTALLINE LENS (50) (49) (18)
 

CALCIFICATION, NOS <2X)
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

»PLEURA (50) (U9) (08)
 

FIBROSIS, DIFFUSE 1 (2*)
 

ALL OTHER SYSTEMS
 

ADIPOSE TISSUE
 

STEATITIS
 

INFLAMMATION, CHRONIC
 

INFLAMMATION, GRANULOMATOUS
 

NECROSIS, NOS
 

OMSNTUM
 

INFLAMMATION, CHRONIC
 

NECROSIS, FAT
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED
 

AUTOLYSIS/NO NECROPSY
 

* NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-7
 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

TREATED WITH 2,5-DITHIOBIUREA 

CONTROL(UNTR)

02-0160

ANIMALS INITIALLY IN STUDY

ANIMALS MISSING

ANIMALS NECHOPSIED

 50

 1 

 19

ANIMALS EXAMINED HISTOPATHOLOGICALLY** <t9


INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

*LUNG


CONGESTION, ACUT2 PASSIVE


INFLAMMATION, INTERSTITIAL


FIBROSI3, DIFFUSE


HYPE3PLASIA, NOS


HEHATOPOIETIC SYSTEM
 

#BCNE MARRO W

HYPERPLASIA, HEMATOPOIET 1C


#SPLEE N

INFLAMMATION , ACUTE

HEMOSIDEROSI S

SRYTHROPOIESI S

CIRCULATOR Y SYST2M 

*AP2 X OF HEART

SCAR

#MYOCAP.DIU M

FIBROSIS

DIGESTIV E SYSTIH 

tLIVER

INFLAMMATION i _ACUTE/CH£ONIC

* NUMBE R O F ANIMAL S WIT H TISSUE

* NUMBE R OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 (1(9)


 1 (2JS)
 

(15)

 (17)

 3 (6X) 

(18)

 (18)

 1 (2% ) 

 (US)

 i_I2%J 

 EXAMINE D MICROSCOPICALL Y 

 LOU DOSE

 02-0100

 50

 50

 50

 HIGH DOSE 

 02-0110 

 50 

 19 

 t9 

 (50)

 1 (2%) 

1 (2X) 

1 (2%) 

 (19) 

 (18)

 1

 (50)

 3

 (2%)

 (6X)

 (46) 

1 (2%) 

 (19) 

1 (2*) 

1 (2X) 

 (50)

 1

 (50)

 (2X) 

 (19) 

 (19) 

 (50) (19) 

C-8 
TABLE C2 (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

02-0160 02-0100 02-0110
 

INFLAMMATION, CHRONIC FOCAL 1 (2%)
 NECROSIS, FOCAL 1 (2%) 2 (»%)
 

METAMORPHOSIS FATTY 1 (2%) 2 (!»*)
 

3ASOPHILIC CYTO CHANGE 2 (4%)
 

HYPERPLASIA, NOS 1 (2XJ
 

HYPERPLASIA, FOCAL 1 <2X)
 

ANGIECTASIS 2 (i»X)
 

1LIVER/CENTRILOBULAR (48) (50) (49)
 

NECROSIS, NOS 2 (4%)
 

*BILE DUCT (49) (50) (49)
 

INFLAMMATION, CHRONIC DIFFUSE 1 (2X)
 

HYPERPLASIA, NOS 2 ("»*)
 

HYPERPLASIA, FOCAL 1 (2X)
 

•PANCREAS	 (48) (49) (46)
 

INFLAMMATION, CHRONIC FOCAL 1 (2X)
 

•STOMACH	 (49) (48) (47)
 

INFLAMMATION, NOS 1 (2X)
 

•GASTRIC SUBMUCOSA (49) (48) (47)
 

EDEMA, NOS 1 (2X) 1 (2%)
 

•PEYERS	 PATCH (49) (50) (46)
 

HYPERPLASIA. NOS 2 (4X)
 

•COLON	 (49) (47) (45)
 

PARASITISM 1 (2%)
 

URIJAP.Y SYSTEM
 

ttKIDNEY (48) (50) (49)
 

GLOMERULONEPtlRITIS, NOS 4 (8X)
 

NEPHEOSIS, NOS 29 (60X) 33 (66%) 21 (43X)
 

•KIDNEY/CORTEX	 (48) (50) (49)
 

METAMORPHOSIS FATTY 1 (2X)
 

ENDOCRINE SYSTEM
 

•ADRENAL CORTEX (49) (48) (48)
 

HYPERPLASIAt _NODULAR_
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-9
 
TABLE C2 (CONTINUED) 

CONTROL (UNTR) 

02-0160 

LOW DOSE 

02-0100 

HIGH DOSE 

02-0110 

HYPERPLASIA, FOCAL 1 (2S) 

fTHYROID 

HYPERPLASIA. C-CELL 

(45) 

2 (US) 

(46) 

2 (4%) 

(32) 

tPARATHYROID 

HYPERPLASIA, NOS 

(27) (35) 

1 (3%) 

(18) 

REPRODUCTIVE SYSTEM 

*MAMHAPY GLAND 

DILATATION/DUCTS 

(49) 

1 (2X) 

(50) (49) 

*MAMMAR Y DUCT 

HYPERPLASIA, CYSTIC 

(t9) 

1 (2%) 

(50) (49) 

tUTERUS 

HYDROMETPA 

CYST, NOS 

HEMATOHA. NOS 

POLYP, INFLAMMATORY 

(16) 

1 (2X) 

1 (2%) 

1 (2») 

(49) 

2 (4*) 

1 (2%) 

(46) 

tUTSRUS/ENDOMETRIUM 

HYPERPLASIA, CYSTIC 

(46) (49) 

3 (6X) 

(46) 

1 (2X) 

#OVARY 

INFLAMMATION, CHRONIC 

(t7) 

1 (2X) 

(49) (46) 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

«EYS/CRYSTALLINE LENS 

CALCIFICATION, NOS 

(49) (50) 

1 (2X) 

(49) 

*LENS CAPSULE 

CALCIFICATION, NOS 

(49) 

1 (2%) 

(50) (49) 

MUSCULOSKELETAL SYSTEM 

NONE 

t

»

 NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

 NUMBER OF ANIMALS NECROPSIED 

c-io
 
TABLE C2 (CONCLUDED) 

CONTROL(UNTR)

02-0160

 LOH DOSE

 02-0100

 HIGH DOSE 

 02-0110 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

ADIPOSE TISSUE 

INFLAMMATION. CHRONIC

NECROSIS, NOS

 1

 1

 1 

1 

SPECIAL MORPHOLOGY SUMMARY 

N O LESION REPORTED 2 2 

ANIMA L MISSING/NO NECROPSY 1 

AUTO/HECHOPSY/HISTO PEEF 1

AUTOLYSIS/NO NECROPSY

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NBCROPSIED 

8 

3 

1 

C-ll
 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 2,5-DITHIOBIUREA
 

TABLE Dl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH 2,5-DITHIOBIUREA

 

CONTROL (UNTR) LOW DOSE HIGH DOSS
 

05-0160 05-0120 05-0130
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

ANIMALS MISSING 1
 

ANIMALS NECEOPSIED 50 50 47
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY** 49 50 47
 

INTEGUMENTARY SYSTEM 

*SKIN

INFLAMMATION, CHRONIC 

HYPERPLASIA, NOS 

ACANTHOSIS 

 (50) (50) (47) 

1

1

1

 (2%) 

(2%) 

 (2X) 

*SUBCUT TISSUE

HSMATOMA, NOS

INFLAMMATION, ACUTE FOCAL

ABSCESS, NOS

 (50) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) (47) 

RESPIRATORY SYSTEM
 

ILUNG (47) (50) (47)
 

INFLAMMATION, INTERSTITIAL 1 (2*)
 

HEMATOPOIETIC SYSTEM
 

ISPLEEN (19) (50) (47)
 

THROMBOSIS, NOS 1 (2%)
 CONGESTION, NOS 1 <2X)
 

ATROPHY, NOS 1 (2%)
 ANGIECTASIS 1 (2X)
 HYPE8PLASIA, LYMPHOID 1 (2*)
 HEMATOPOIESIS 2 (4X) 1 (2X)
 

ERYTHBOPOIESIS 2 (4X) 1 (2%)
 

#PANCREATIC L.NODE (40) (41) (40)
 

HYPERPLASIA, LYMPHOID 1 (2S)
 

(LUMBAR LYMPH NODE (40) (41) (40)
 

HYPERPLASIA, PLASMA CELL 1 (2%)
 

*MES2NTERIC L. NODE (40) (41) (40)
 

INFLAMMATION^ ACUTE 1_13%)
 

« NUMBE R OF ANIMAL S WITH TISSUE E.XAHINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-3 
TABLE Dl (CONTINUED) 

CONTRO L (UNTR ) LOW DOSE HIG H DOSE 

05-0160 05-0120 05-013 0 

HYPERPLASIA, PLASMA CELL	 1 (2%)
 

•RENAL	 LYMPH NODE (40) (41) (40)
 

HYPSRPLASIA, NOS 2 (5%)
 

HYPERPLASIA, PLASMA CELL 1 (2%)
 

CIRCULATORY SYSTEM
 

•HEART	 (49) (50) (46)
 

PERIVASCULITIS 1 (2%)
 

•MYOCARDIUM	 (19) (50) (46)
 

INFLAMMATION, FOCAL 1 (2%)
 

INFLAMMATION, CHRONIC 1 (2S)
 

FIBHOSIS, FOCAL 1 (2%)
 

ANGIECTASIS 1 (2%)
 

•HEPATIC	 SINUSOID (U9) (50) (47)
 

LEUKOCYTOSIS, NOS 1 (2%)
 

DIGESTIVE SYSTEM
 

• LIVER (49) (50) (47)
 

NECROSIS, NOS 1 (235)
 

NECFOSIS, FOCAL 1 (2X)
 

METAMORPHOSIS FATTY 1 (2%) 1 (2%)
 

MEGALOCYT03IS 1 (2%) 1 (2%)
 

HYPERPLASTIC NODULE 2 (4X) 1 (2%)
 

ANGIECTASIS 1 (2%)
 

•LIVER/KUPFFER	 CELL (49) (50) (47)
 

HYPERPLASIA, NOS 1 (2%)
 

• PANCREAS (46) (44) (45)
 

CYSTIC DUCTS 1 (2%) 2 (5%)
 

PSHIVASCULITIS 1 (2%)
 

NECROSIS, FAT 1 (2%)
 

tSTCMACH (49) (50) (47)
 

CYST, NOS 1 (2X)
 

ABSCESS, NOS 1 (2%)
 

ACANTHOSIS 1 (2*)
 

• GASTRIC BUCOSA (49) (50) (47)
 

____ INFLAMMATIONi _ACUTE
 

NU.13ES OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

NUMBE R OF ANIMAL S NECROPSIED
 

D-4
 
TABLED! (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

05-0160 05-0120 05-0130
 

IFEYiiHS PATCH (49) (50) (46)
 

INFLAMMATION, ACUTE 1 (2X)
 HYPERPLASIA, LYMPHOID 1 (2X)
 

URINARY SYSTEM
 

#KIDNEY (49) (50) (47) 

HYDRONEPHROSIS 2 (UX) INFLAMMATION, CHRONIC 

3LOMEEULONEPHEITIS, CHRONIC 1 (2X) 

SLOWEEULOSCLEROSIS, NOS 3 (6X) HEBOSIDEROSIS 1 (2X) 

#KIDNEY/GLOMEEULU S (49) (50) (47) 

AtlYLOIDOSIS 1 (2X) 

#U. BLADD£R/MUCOSA (49) (50) (46) 

INFLAMMATION , ACUTE 1 (2X>

 

ENDOCRINE SYSTEM
 

tTHYROID (42) (U7) (HO)

 

HYPERPLASIA, FOCAL <2X) 

REPRODUCTIVE SYSTEM
 

*PREPUTIAL GLAND (50) (50)
 

DILATATION, NOS 1 (2X)
 

DILATATION/DUCTS 1 (2X)
 

*SEMINAL VESICLE (50) (50) (47)
 

INFLAMMATION, ACUTE SUPPURATIVE 1 <2X)
 

#TESTIS (49) (50) (46)
 

INFLAMMATION, ACUTE SUPPURATIVE 1 (2X)
 

#TESTIS/TUBHLE (49) (50) (46)
 

DEGENERATION, NOS <2X) 

NECROSIS, FOCAL 1 (2X)
 

NERVOUS SYSTEM
 

NONE
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-5
 
SPECIAL SENSE OKGANS
 

NCNE
 

MUSCULOSKELETAL SYSTEM
 

*SKELETAL MUSCLE


PARASITISM


BODY CAVITIES
 

'ABDOMINAL CAVITY


ADHESION, NOS


*MES£NTERY


SIEATITIS


ABSCESS, NOS


ALL OTHER SYSTEMS
 

ADIPOSE TISSUE
 

STEATITIS


NECROSIS, FAT


SPECIAL MOBPHOLOGY SUMMAi•
 

NO LESION REPORTED


iNIHAL HISSING/NO NECROPSY


AUTO/NECROPSY/HISTO PERF


AUTO/NECROPSY/NO HISTO


AUTOLYSIS/NO NECEOPSY


TABLE Dl (CONCLUDED)
 

CONTROL (UNTR)


05-0160


 (50)


 (50)


 1 (2X)
 

(50)


 1 (2X)
 

1 (2X)
 

1
 

2
 

 17


 1
 

1
 

 LO'J DOSE HIGH DOSE 05-0120 05-0130 

 (50) (U7) 

1 (2%) 

 (50) (47) 

 (50) (17) 

 18 21 

1 

2 

# NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-6
 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH 2,5-DITHIOBIUREA

 

CONTROL (ONTR) LOW DOSE

06-0160 06-0120

 HIGH DOSE 

 06-0130 

ANIMALS INITIALLY IN STUDY 50

ANIMALS MISSING

ANIMALS NECROPSIED 50

ANIHALS EXAMINED HISTOPATHOLOGICALLY** 50

 50

 <|8

 l»8

 50 

1 

 48 

 48 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (50) (48) (48)
 

HEMORRHAGE 1 (2%)
 

RESPIRATORY SYSTEM
 

• LUNG/BRONCHUS (50) (47) (1(8)
 

INFLAMMATION, NOS 1 (2%)
 

*LUNS (50) (17) (US)
 

PERIVASCULITIS 2 (4X)
 

HEMATOPOIETIC SYSTEM
 

*SPLEEN (<*9) (46) (47) 

ACCESSORY STRUCTURE 1 (2X) 

HYPERPLASIA, PLASMA CELL 1 (2X) 

HYPERPLASIA, LYMPHOID 1 (2%) 

ERYTHROPOIESIS 1 (2!!) 1 (2X) 

#MANBIEULAR L. NODS (10) (33) (39) 

HYPERPLASIA, PLASMA CELL 1 (3X) 

*«jiBIASTINAL L.NODE (40) (33) (39) HYPERPLASIA, NOS 1 (3%) 

HYPERPLASIA, PLASMA CSLL 1 (3X) 

iLUMBAR LYMPH NODE (40) (33) (39) HYPIRPLASIA, NOS 1 <3X) 

»MESENTERIC L. NODE (tO) (33) (39) 

HYPERPLASIA, LYMPHOID 1 (3X) 

#RENAL LYMPH NODji (40) (33) (39) HYPJRPLASIAi_NOS 

t NUMBE R OP ANIMAL S HITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED **EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-7 
TABLE D2 (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

06-0160 06-0120 06-0130
 

HYPERPLASIA, PLASMA CELL 1 (3X)
 

CIRCULATORY SYSTEM
 

#MYOCiRDIUM (50) (46) (48)
 

INFLAMMATION, NOS 1 (2X)
 

INFLAMMATION, ACUTE DIFFUSE 1 (2%)
 

DIGESTIVE SYSTEM
 

ILIVER (49) (47) (48)
 

NECROSIS, NOS 1 (2X)
 

NECROSIS, FOCAL 1 <2X)
 

INFARCT, NOS 1 (2S)
 

HYPEBPLASTIC NODULE 3 (6%) 2 (4X)
 

HYPERPLASIA, FOCAL 1 (2%)
 

*LIVER/C2NTRIL08ULAR (49) (47) (48)
 

NECROSIS, DIFFUSE 1 (2X)
 

*3ILE DUCT (50) (48) (48)
 

INFLAMMATION, CHRONIC 1 (2%)
 

INFLAMMATION, CHRONIC FOCAL 2 (4%)
 

((PANCREAS (47) (45) (46)
 

CYSTIC DUCTS 1 (2%)
 

ISTOMACH (49) (45) (47)
 

INFLAMMATION, ACUTE FOCAL 1 (2%)
 

INFLAMMATION, CHRONIC 1 (2%)
 FIBROSIS 1 <2X)
 

CALCIFICATION, NOS 1 (2%)
 

tGASTRIC MUCOSA (49) (45) (47)
 

INFLAMMATION, ACUTE 1 (2%)
 

tPEYERS PATCH (49) (47) (47)
 

HYPERPLASIA, LYMPHOID 1 <2X)
 

•COLON	 (50) (46) (45)
 

NEMATODIASI3 1 (2X)
 

URINAR Y SYSTEM
 

tKIDNEY (49) (47) (48)
 

LYMPHOCYTIC_INFLAMMATgRY_INFILTR 1_J2%)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS MECROPSIED
 

D-8
 
TABLE D2 (CONTINUED) 

CONTROL (UNTR) LOU DOSE HIGH DOSE
 

06-0160 06-0120 06-0130
 

INFLAMMATION, INTERSTITIAL 1 (2X)
 

INFLAMMATION, CHRONIC 2 (4X) 1 (2%)
 INFLAMMATION, CHRONIC FOCAL 1 (2X)
 PEBIVASCULITIS 1 (2X)
 SLOMERULOSCLEROSIS, SOS 1 (2%) 1 (2X)
 AHYLOIDOSIS 1 (2X)
 

*KIDN£Y/M-EDULLA (49) (47) (48)
 

AMYLOIDOSIS 1 (2X)
 

*URINARY BLADDER (50) (45) (47)
 

INFLAMMATION, CHRONIC FOCAL 1 (2X)
 

#U. ELADDER/MUCOSA (50) (45) (47)
 

INFLAMMATION, CHRONIC 1 (2X)
 INFLAMMATION, CHRONIC FOCAL 1 (2X)
 

INFLAMMATION, CHRONIC DIFFUSE 1 (2%)
 

HYPERPLASIA. LYMPHOID 1 (2X)
 

ID.BLADDER/SUBMUCOSA (50) (45) (47)
 

INFLAMMATION, CHRONIC 1 (2X)
 

INFLAMMATION, CHRONIC FOCAL 16 (32%)
 

PERIVASCULITIS 1 (2%}
 

*U.BLADDSR/MUSCULARIS (50) (45) (47)
 

CALCIUM DEPOSIT 1 (2X)
 

ENDOCRINE SYSTEM
 

#THYROID (41) (45) (45)
 

HYPERPLASIA. C-CELL 2 (5X)
 

HYPERPLASIA, FOLLICULAR-CELL 1 (2X)
 

RBPRODUCTIVE SYSTEM 

*UTEHUS (49) (43) (47) 

HYDROMETRA 5 (10X) 3 (7X) 7 (15X) 

NECROSIS, FAT 1 (2X) 

CALCIFICATION, NOS 1 (2X) 

IUTERUS/ENDOMETRIUM (49) (43) (47) 

INFLAMMATION, SUPPURATIV ATIV E 2 (4X) 4 (9X) 2 (4X) 

HYPERPLASIA, CYSTIC 32 (65X) 22 (SIX) 28 (60X) 

I NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-9
 
ttOVARY/OVIDUCT


HYPZRPLASIA. PAPILLARY


#OVARY


CYST, NOS


HEHOHBHAGIC CYST


INFLAMMATION. SUPPURATIVE


INFLAMMATION, CHRONIC


NEFVOUS SYSTEM
 

N3NE
 

SPECIAL SENSS ORGANS
 

NOSE
 

MUSCULOSKELETAL SYSTEM
 

N JNi
 

BODY CAVITI3S 

*MEDIASTINUM


INFLAMMATION, ACUTE/CHRONIC


'PE3ICARDIUM


INFLAMMATION, ACUTE/CHRONIC


*MESENTERY


ABSCESS, NOS


ALL OTHER SYSTEMS
 

*HULTIPLE ORGANS


AMYLOIDOSIS


OMSNTU H 

PERIVASCflLITIS

TABLE D2 (CONTINUED)
 

CONTROL (UMTR)


06-0160


 (19)


 (U8)


 6 (13X)


 1 (2%)
 

1 (2%)


 (50)


 (50)


 (50)


 (50)


 1 (2X)


 Jt 

 LOW DOSE HIGH DOSE
 

 06-0120 06-0130
 

 («3) (<«7)
 

1 (2%)
 

 («0) (U5)
 

2 (5%) « (9X)
 

1 (3X)
 

1	 (2X)
 

 (t8) (48)
 

1 (2X)
 

 (U8) («8)
 

1 (2%)
 

 (18)	 (U8)
 

1 (2X)
 

 (48)	 (U8)
 

1 (2X)
 

*	 NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NECROPSIED
 

D-10
 
TABLE D2 (CONCLUDED) 

CONTROL(UNTR) LOW DOSE HIGH DOSE 

06-0160 06-0120 06-0130 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED 2 5 U 

ANIMAL HISSING/NO NECROPSY 1 

AOTO/NECROPSY/HISTO PERF 2 1 

AUTOLYSIS/NO NECROPSY 2 1 

# NUSBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY * NUMBER OF ANIMALS NECROPSIED 

D-ll
 

Review of the Bioassay of 2,5-Dithiobiurea* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

October 25, 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, and State health officials.
 

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 2,5-Dithiobiurea for carcinogenicity.
 

The primary reviewer for the report on the bioassay of 2,5-Dithiobiurea
 

said that, under the conditions of test, the compound was not demonstrated
 

to be carcinogenic in treated rats or mice. He pointed out that different
 

batches of the compound with different purities were used. He said the
 

shortcoming was not significant since the study was negative. Although
 

the dosages administered to mice appeared to be adequate, the primary
 

reviewer indicated that those used for rats appeared to have been set
 

"arbitrarily." He said that the bioassay was probably still valid since
 

the doses used for the rats were sufficiently high, the study was conducted
 

for an adequate time, and the survival was satisfactory.
 

The secondary reviewer of the bioassay of 2,5-Dithiobiurea noted
 

the following experimental shortcomings: 1) the stability of the
 

compound in the diet was not determined, 2) the control group of mice
 

was initiated five weeks after the start of the treatment groups, and
 

3) the examination of the thyroids should have been given special
 

attention because of the relationship of the compound to thiourea.
 

Despite the shortcomings, he agreed with the conclusions in the report
 

and added that the results "give some assurance of safety" of 2,5-Dithiobiurea
 

for humans.
 

There was no objection to a recommendation that the report on the
 

bioassay of 2,5-Dithiobiurea be accepted as written.
 

47
 
Clearinghouse Members Present;
 

Arnold L. Brown (Chairman), University of Wisconsin Medical School
 

(David Clayson, Eppley Institute for Cancer Research, submitted a
 

written review)
 

Joseph Highland, Environmental Defense Fund
 

William Lijinsky, Frederick Cancer Research Center
 

Henry Pitot, University of Wisconsin Medical Center
 

Verne A. Ray, Pfizer Medical Research Laboratory
 

Kenneth Wilcox, Michigan State Health Department
 

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.
 

48
 

«J.S. GOVERNMENT PRINTING OFFICE: 1978 281-217/3302 1-3 

DHEW Publication No. (NIH) 79-1387