1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 126 

BIOASSAY OF 

2,5-TOLUENEDIAMINE SULFATE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 6369-59-1 

NCI-CG-TR-126 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

2,5-TOLUENEDIAMINE SULFATE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-1381
 

REPORT ON THE BIOASSAY OF 2,5-TOLUENEDIAMINE SULFATE
 

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-toluenediamine sulfate 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-toluenediamine sulfate was
 

conducted by Mason Research Institute, Worcester, Massachusetts,
 

initially under direct contract to the NCI and currently under a
 

subcontract to Tracer Jitco, Inc., prime contractor for the NCI
 

Carcinogenesis Testing Program.
 

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

principal investigators for the contract were Dr. E. Smith (3) and
 

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

by Mr. G. Wade (3) and Ms. E. Zepp (3).
 

Histopathologic examinations were performed by Dr. D. W. Hayden
 

(3), Dr. A. S. Krishna Murthy (3), Dr. A. Russfield (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 (4).
 

Compilation of individual animal survival, pathology, and summary tables was performed by EG&G Mason Research Institute (5); the
 

statistical analysis was performed by Mr. W. W. Belew (6,7), using
 

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

Gart (8).
 

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

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

(6), task leader Dr. M. R. Kornreich (6,9), senior biologist Ms. P.
 

Walker (6), biochemist Dr. B. Fuller (6), chemist Dr. N. Zimmerman
 

(6), and technical editor Ms. P. A. Miller (6). 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 (1,9), Dr. R.
 

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

W. Orme (1), Dr. R. A. Squire (1,10), 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.	 Tracer Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

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

Rockville, Maryland.
 

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

Boulevard, McLean, Virginia.
 

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

Golden, Colorado.
 

8.	 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.
 

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

Washington, D.C.
 

iv
 
10.	 No wit w h the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay for possible carcinogenicity of 2,5-toluenediamine
 

sulfate was conducted using Fischer 344 rats and B6C3F1 mice. 2,5­

Toluenediamine sulfate was administered in the feed, at either of two
 

concentrations, to groups of 50 males and 50 females of each species.
 

The high and low time-weighted average concentrations of the compound
 

were, respectively, 0.2 and 0.06 percent for rats and 0.1 and 0.06
 

percent for mice. Because compound administration to the high and
 

low dose groups of each species was not begun simultaneously, each
 

dosed group was assigned a control group. All control groups consisted of 50 animals, except for the high dose male and female rat
 

control groups which were composed of 25 animals. The dosing period
 

was for 78 weeks, followed by an additional 28 to 31 weeks of observation in rats and an additional 16 to 19 weeks in mice.
 

There was no significant association between compound administration and accelerated mortality in either sex of either species.
 

Adequate numbers of animals in all groups survived sufficiently long
 

to be at risk from late-developing tumors.
 

A statistically significant incidence of interstitial-cell neoplasms of the testis in dosed male rats was not considered attributable to administration of the compound since the spontaneous incidence
 

of these neoplasms in male Fischer 344 rats is both high and variable.
 

No neoplasms were observed in female rats at statistically significant
 

incidences.
 

A statistically significant increase in lung tumors in high
 

dose female mice was not considered convincing evidence of a
 

compound-related carcinogenic effect because high dose mice were
 

received in separate shipments from their controls and housed in
 

separate rooms from their controls.
 

Under the conditions of this bioassay, sufficient evidence was
 

not obtained to demonstrate the carcinogenicity of 2,5-toluenediamine
 

sulfate in either Fischer 344 rats or B6C3F1 mice.
 

vii
 

TABLE OF CONTENTS
 

I. INTRODUCTION
 

II. MATERIALS AND METHODS
 

A. Chemicals
 

B. Dietary Preparation
 

C. Animals
 

D. Animal Maintenance
 

E. Selection of Initial Concentrations
 

F. Experimental Design
 

G. Clinical and Histopathologic Examinations
 

H. Data Recording and Statistical Analyses
 

III. CHRONIC TESTING RESULTS: RATS
 21
 

A. Body Weights and Clinical Observations 21
 

B. Survival 21
 

C. Pathology 24
 

D. Statistical Analyses of Results 25
 

34
 IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations 34
 

B. Survival 34
 

C. Pathology 37
 

D. Statistical Analyses of Results 37
 

V. DISCUSSION 44
 

VI. BIBLIOGRAPHY ^6
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH 2,5-TOLUENEDIAMINE
 

SULFATE A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH 2,5-TOLUENEDIAMINE
 

SULFATE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 2,5-TOLUENEDIAMINE SULFATE C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 2,5-TOLUENEDIAMINE SULFATE D-l
 

ix
 
LIST OF ILLUSTRATIONS
 

Figure Number
 

1 CHEMICAL STRUCTURE OF 2,5-TOLUENEDIAMINE 

SULFATE 

GROWTH CURVES FOR 2,5-TOLUENEDIAMINE 

SULFATE CHRONIC STUDY RATS 22 

SURVIVAL COMPARISONS OF 2,5-TOLUENEDIAMINE SULFATE CHRONIC STUDY RATS 23 

GROWTH CURVES FOR 2,5-TOLUENEDIAMINE 

SULFATE CHRONIC STUDY MICE 35 

SURVIVAL COMPARISONS OF 2,5-TOLUENEDIAMINE SULFATE CHRONIC STUDY MICE 36 

LIST OF TABLES 

Table Number 

DESIGN SUMMARY FOR FISCHER 344 RATS— 

2,5-TOLUENEDIAMINE SULFATE FEEDING 

EXPERIMENT 12 

DESIGN SUMMARY FOR

2,5-TOLUENEDIAMINE

EXPERIMENT 

 B6C3F1 MICE— 

 SULFATE FEEDING 

13 

ANALYSES OF THE INCIDENCE OF PRIMARY 

TUMORS AT SPECIFIC SITES IN MALE RATS 

TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 26 

ANALYSES OF THE INCIDENCE OF PRIMARY 

TUMORS AT SPECIFIC SITES IN FEMALE RATS 

TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 30 

ANALYSES OF THE INCIDENCE OF PRIMARY 

TUMORS AT SPECIFIC SITES IN MALE MICE 

TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 38 

ANALYSES OF THE INCIDENCE OF PRIMARY 

TUMORS AT SPECIFIC SITES IN FEMALE MICE 

TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 40 
LIST OF TABLES (Concluded) 

Table Number Page 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH 2,5-TOLUENEDIAMINE 

SULFATE A-3 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH 2,5-TOLUENEDIAMINE 

SULFATE A-8 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH 2,5-TOLUENEDIAMINE 

SULFATE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH 2,5-TOLUENEDIAMINE 

SULFATE B-6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH 2,5­

TOLUENEDIAMINE SULFATE C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH 2,5­

TOLUENEDIAMINE SULFATE C-10 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH 2,5­

TOLUENEDIAMINE SULFATE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH 2,5­

TOLUENEDIAMINE SULFATE D~9 

xx
 

I. INTRODUCTION
 

2,5-Toluenediamine sulfate (Figure 1) (NCI No. C01832), a salt
 

of 2,5-toluenediamine and sulfuric acid, was selected for bioassay by
 

the National Cancer Institute in an attempt to determine those dye
 

intermediates which may be responsible for the increased incidence
 

of bladder cancer observed among workers in the dye manufacturing
 

industry (Wynder et al., 1963; Anthony and Thomas, 1970). Aromatic
 

amines are one of several classes of chemicals thought to contribute
 

to the increased cancer risk in this industry (Wynder et al., 1963).
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is 2-methyl-l,4-benzenediamine sulfate.
 

It is also called p-tolylenediamine sulfate; p-diaminotoluene sulfate;
 

fouramine standard; and Colour Index (C.I.) oxidation base 4 (C.I. No.
 

76043).
 

2,5-Toluenediamine is used in the synthesis of safranine, a family of dyes, some of which are useful as biological stains (Hawley,
 

1971), and as an oxidation base to dye furs a deep brown (Society of
 

Dyers and Colourists, 1971). In addition, 2,5-toluenediamine is a
 

common component of the "permanent" or oxidative-type hair dye formulations (Ames et al., 1975). It may also be contained in indelible
 

ink, antifreeze, and nail polish. Production statistics for 2,5­

toluenediamine or its sulfate salt are not available; however, U.S.
 

*
 

The CAS registry number is 6369-59-1,
 
CH 

H2N 

FIGURE 1 

CHEMICAL STRUCTURE OF 2,5-TOLUENEDIAMINE (SULFATE) 
imports of this chemical through principal U.S. customs districts
 

amounted to 64,680 pounds in 1974 (U.S. International Trade Commission, 1976).
 

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

among workers in the dye manufacturing industry, although fur dyers
 

and manufacturers of inks as well as printers, engravers and lithographers may also experience significant daily contact with the
 

chemical. Epidemiological studies suggest a relationship between
 

occupational exposure to printing ink and increased incidence of
 

cancer of both the bladder and the liver (Hoover and Fraumeni, 1975).
 

Exposure to 2,5-toluenediamine is widespread among the general
 

population due to the increasingly common practice of hair dyeing.
 

It has been estimated that approximately 33 million women in the
 

United States dye their hair, often monthly over a period of many
 

years (Staats, 1977).
 

2,5-Toluenediamine is toxic following both ingestion and inhalation (Sax, 1975). It can also be absorbed through the skin. Three
 

hours after application of 2,5-toluenediamine to the abdomen of dogs,
 

skin penetration was indicated by blood and urine measurements (Kiese
 

et al., 1968). When applied to the scalp of human volunteers in
 

conjunction with detergents, resorcinol and hydrogen peroxide, the
 

diacetyl derivative was excreted in the urine for two days following
 

application, indicating absorption and metabolism through this route
 

as well (Kiese and Rauscher, 1968). The compound has a toxic action
 
upon the liver and can cause fatty degeneration of that organ.
 

In addition, it is toxic to the central nervous system and produces
 

anemia by destruction of red blood cells. The compound is considered
 

to be highly irritating, producing irritation and blisters on the
 

fingers of sensitive individuals; permanent injury to an eye was
 

reported following use of 2,5-toluenediamine as an eyelash dye (Sax,
 

1975).
 

In studies by Ames et al. (1975), 2,5-toluenediamine was found
 

to cause frameshift mutations in a tester strain (TA 1538) of Salmonella typhimurium in the presence of rat liver microsomes. Oxidation
 

of 2,5-toluenediamine by hydrogen peroxide prior to testing resulted
 

in a fortyfold increase in mutagenic activity.
 
II. MATERIALS AND METHODS 

A. Chemicals
 

2,5-Toluenediamine sulfate was purchased from the Wayland Chemical Division of the Philip A. Hunt Chemical Corporation, Lincoln,
 

Rhode Island. Analysis by the manufacturer suggested a purity of 99
 

percent with 25 ppm iron, 0.6 percent volatiles, and a maximum of
 

0.1 percent moisture. Analysis was also performed by Mason Research
 

Institute, Worcester, Massachusetts. The melting point determination
 

showed decomposition above 275°C. No literature value was found for
 

comparison. Thin-layer chromatography utilizing two solvent systems,
 

hexane:benzene:methanol and hexane:ethyl acetate, did not indicate
 

the presence of any impurities. The evidence suggested a compound
 

of high purity.
 

Throughout this report the term 2,5-toluenediamine sulfate is
 

used to represent this material.
 

B.	 Dietary Preparation
 

The basal laboratory diet for both dosed and control animals
 

consisted of Wayne Lab-Blox (Allied Mills, Inc., Chicago, Illinois).
 

2,5-Toluenediamine sulfate was administered to the dosed animals as
 

a component of the diet. The chemical was mixed with the feed in a 6
 

kg capacity Patterson-Kelley standard model stainless steel twin-shell
 

V-blender. After 20 minutes of blending, the mixtures were placed in
 

double plastic bags and stored in the dark at 4°C. Mixtures were
 

prepared weekly and stored for not longer than 2 weeks.
 

5
 
C. Animals
 

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

through contracts of the Division of Cancer Treatment, National Cancer
 

Institute. All mice and the high dose rats and their controls were
 

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

Massachusetts. The low dose rats and their controls were supplied by
 

Laboratory Supply Co., Inc., Indianapolis, Indiana. Except for the
 

low dose rats and their controls, all the other dosed animals were
 

received in separate shipments from their respective controls.
 

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 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
 

(R)
 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 13 months of study, low dose rats and their controls
 
were kept in galvanized- or stainless-steel wire-mesh cages suspended
 

above newspapers. High dose rats and their controls were housed in
 

galvanized-steel wire-mesh cages during quarantine and for the first
 

11 months of study. Newspapers under cages were replaced daily and
 

cages and racks washed weekly. For the remainder of the study, all
 

rats were housed in suspended polycarbonate cages equipped with disposable nonwoven filter sheets. Clean bedding and cages were provided
 

twice weekly. Low dose rats and their controls received Bed-o-Cobs®
 

corncob bedding (The Andersons Cob Division, Maumee, Ohio) for the
 

first 8 months that they were housed in polycarbonate cages. There­

(R)
 after, SAN-I-CEL corncob bedding (Paxton Processing Company, Paxton,
 

Illinois) was used. High dose rats and their controls were provided
 

/S)
 

with SAN-I-CEL for the first 11 months that they were housed in
 

polycarbonate cages, and then Aspen hardwood chip bedding (American
 

Excelsior Company, Baltimore, Maryland) was provided for the remainder of the study. 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 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
 

supplied by Lab Products, Inc., Garfield, New Jersey. Nonwoven fiber
 

filter bonnets were used over cage lids. Low dose mice and their
 
controls were housed ten per cage for the first 18 months of study
 

and five per cage thereafter. The number of high dose mice per cage
 

was reduced from ten to five after 13 months. The number of high dose
 

controls per cage was reduced to five after 12 months. Clean cages,
 

lids, filters, and bedding were provided three times per week when
 

cage populations were ten and twice per week when cage populations
 

were reduced to five. Bedding was the same as that provided to rats.
 

Ab-sorb-dri ® (Wilner Wood Products Company, Norway, Maine) hardwood
 

chips were supplied for 2 months to the high dose mice and their controls and for 8 months to the low dose mice and their controls.
 

Both groups received SAN-I-CEL for the next 12 months. Bed-o-Cobs®
 

was used until the end of the study. Reusable filter bonnets and
 

pipe racks were sanitized every 2 weeks throughout the study.
 

Water was available to both species from 250 ml water bottles
 

equipped with rubber stoppers and stainless steel sipper tubes.
 

Bottles were replaced twice weekly and, for rats only, refilled as
 

needed between changes.
 

(R)
 Pelleted Wayne Lab-Bloxw

 was supplied to all animals during the
 

initial quarantine and final observation periods. During the dosing
 

®
 period, all animals were fed Wayne Lab-Blox meal containing the
 

appropriate concentration of 2,5-toluenediamine sulfate. Control
 

animals had untreated meal available ad libitum. Meal was supplied
 

throughout the study to all mice and to low dose rats and their con­

(R)
 trols in Alpine aluminum feed cups (Curtin Matheson Scientific, Inc.,
 
Woburn, Massachusetts) containing stainless steel baffles. High dose
 

rats and their controls were fed from Alpine® feed cups for the
 

first 14 months of study and from stainless steel gangstyle hoppers
 

(Scientific Cages, Inc., Bryan, Texas) for the remainder of the
 

study. 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
 

(R)
 

schedule as were cages. Food was replenished daily in Alpine^ feed
 

cups.
 

Dosed rats were housed in a room with other rats receiving diets
 

*
 containing acetylaminofluorene (53-96-3); a mixture of dulcin (150­

69-6) and L-arginine glutamate (4320-30-3); sodium nitrite (7632-00-0);
 

L-arginine glutamate (4320-30-3); N-butylurea (592-31-4); 2-chloro-pphenylenediamine sulfate (61702-44-1); N,N-dimethyl-p-nitrosoaniline
 

(138-89-6); 2,4-dinitrotoluene (121-14-2); 4-nitroanthranilic acid
 

(619-17-0); 1,5-naphthalenediamine (2243-62-1); N-( 1-naphthyDethylenediamine dihydrochloride (1465-25-4); and aniline hydrochloride (142­

04-1). Control rats were housed in a room with other rats receiving
 

diets containing 1-nitronaphthalene (86-57-7); 5-nitro-o-toluidine
 

(99-55-8); hydrazobenzene (530-50-7); 2-aminoanthraquinone (117-79-3);
 

6-nitrobenzimidazole (94-52-0); 3-amino-9-ethylcarbazole hydrochloride;
 

2,4-diaminoanisole sulfate (615-05-4); and APC (8003-03-0).
 

*
 

CAS registry numbers are given in parentheses.
 
High dose mice shared a room with other mice receiving diets
 

containing 5-nitro-o-toluidine (99-55-8); hydrazobenzene (530-50-7);
 

3-amino-9-ethylcarbazole hydrochloride; 1-nitronaphthalene (86-57-7);
 

6-nitrobenzimidazole (94-52-0); 5-nitro-o-anisidine (99-59-2); and
 

2,4-diaminoanisole sulfate (615-05-4). High dose control mice were
 

housed in a room with other mice receiving diets containing 2-methyl1-nitroanthraquinone (129-15-7); 4-chloro-m-phenylenediamine (5131­

60-2); acetylaminofluorene (53-96-3); p-cresidine (120-71-8); and
 

fenaminosulf (140-56-7). Low dose mice and their controls were in
 

a room with other mice receiving diets containing amitrole (61-82-5);
 

APC (8003-03-0); N,N-dimethyl-p-nitrosoaniline (138-89-6); 2,4-dinitrotoluene (121-14-2); 4-nitroanthranilic acid (619-17-0); 2-aminoanthraquinone (117-79-3); 3-amino-4-ethoxyacetanilide (17026-81-2);
 

3-amino-9-ethylcarbazole hydrochloride; l-amino-2-methylanthraquinone
 

(82-28-0); 5-nitro-o-anisidine (99-59-2); 1-nitronaphthalene (86-57­

7); 5-nitroacenaphthene (602-87-9); 3-nitro-p-acetophenetide (1777­

84-0); and 2,4-diaminoanisole sulfate (615-05-4).
 

E.	 Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

2,5-toluenediamine sulfate for administration to dosed animals in
 

the chronic studies, subchronic toxicity tests were conducted with
 

*
 both	 Fischer 344 rats and C57BL/6 mice. Rats and mice were distributed among five groups, each consisting of five males and five
 

*
 

This strain was used due to the unavailability at that time of
 

B6C3F1 mice.
 

10
 
females. 2,5-Toluenediamine sulfate was incorporated into the basal
 

laboratory diet and fed ad libitum to four of the five rat groups
 

and four of the five mouse groups in concentrations of 0.02, 0.05,
 

0.08, and 0.11 percent. The remaining group of each species served
 

as control groups, receiving only the basal laboratory diet. The
 

dosed dietary preparations were administered for a period of 4 weeks,
 

followed by 2 weeks of observation.
 

All animals survived until necropsy. Mean body weight depression relative to controls was observed in all dosed rat groups and
 

in dosed female mice; however, the depression was not dose-related.
 

No mean body weight depression was observed in male mice.
 

The high concentrations selected for administration in the
 

chronic study were 0.05 percent for rats and 0.06 percent for mice.
 

F. Experimental Design
 

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

of treated and untreated observation periods, and time-weighted
 

average concentrations) are summarized in Tables 1 and 2.
 

At initiation of the study, all animals were approximately 6
 

weeks old. High dose rats and their controls were placed on test
 

11 months after low dose rats and their controls. Each dosed rat
 

group was placed on test during the same week as its respective control group. Rats initially received 2,5-toluenediamine sulfate at
 

dietary concentrations of 0.05 and 0.03 percent. Because of a lack
 

11
 
TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

2,5-TOLUENEDIAMINE' SULFATE FEEDING EXPERIMENT
 

INITIAL


MALE
 

LOW DOSE CONTROL
 

HIGH DOSE CONTROL
 

LOW DOSE
 

HIGH DOSE
 

FEMALE
 

LOW DOSE CONTROL
 

HIGH DOSE CONTROL
 

LOW DOSE
 

HIGH DOSE
 

GROUP
 

SIZE
 

50
 

25
 

50
 

50
 

50
 

25
 

50
 

50
 

2,5-TOLUENEDIAMINE
 

 SULFATE


CONCENTRATION


(PERCENT)
 

0
 

0
 

0.05
 

0.06
 

0
 

0.2
 

0
 

0
 

0
 

0.05
 

0.06
 

0
 

0.2
 

0
 

 OBSERVATION PERIOD


 TREATED UNTREATED


(WEEKS) (WEEKS)


0 107


0 109


14
 

64
 

28
 

78
 

30
 

0 108


0 109


14
 

64
 

29
 

78
 

31
 

 TIME-WEIGHTED
 

 AVERAGE
 

CONCENTRATION3
 

0
 

0
 

0.06
 

0.2
 

0
 

0
 

0.06
 

0.2
 

s(concentration X weeks received)
 Ti mp— UP i crhfpH av<= >rape ron cent ration =
 

12
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

2,5-TOLUENEDIAMINE SULFATE FEEDING EXPERIMENT
 

MALE 

INITIAL

GROUP 

SIZE 

2,5-TOLUENEDIAMINE 

 SULFATE

CONCENTRATION

(PERCENT)

 OBSERVATION PERIOD 

 TREATED UNTREATED 

 (WEEKS) (WEEKS) 

LOW DOSE CONTROL 50 0 0 96 

HIGH DOSE CONTROL 50 0 0 98 

LOW DOSE 50 0.06

0

 78 

 16 

HIGH DOSE 50 0.1

0

 78 

 19 

FEMALE 

LOW DOSE CONTROL 50 0 0 97 

HIGH DOSE CONTROL 50 0 0 98 

LOW DOSE 50 0.06

0

 78 

 16 

HIGH DOSE 50 0.1

0

 78 

 19 

13
 
of observed mean body weight depression or other clinical signs at a
 

level of 0.05 percent, the groups receiving 0.03 percent were terminated 2 months after initiation, and a new group was initiated at a
 

concentration of 0.2 percent along with a new control group. Throughout this report those rats receiving a concentration of 0.2 percent
 

and their controls are referred to as the high dose and high dose control groups, respectively, while those rats initially receiving a
 

concentration of 0.05 percent and their controls are referred to as
 

the low dose and low dose control groups, respectively. From week 15,
 

the concentration received by the low dose group was increased from
 

0.05 to 0.06 percent.
 

Low dose mice were placed on test 6 months before high dose
 

mice. Low dose mice were placed on test 2 weeks before their controls. High dose mice were placed on test 2 months after their
 

controls. The initial dietary concentrations utilized for mice were
 

0.06 and 0.03 percent. Again, because of a lack of observed mean
 

body weight depression or other clinical signs, the group receiving
 

0.03 percent was discontinued 6 months after initiation and a new
 

group was initiated at a concentration of 0.1 percent, accompanied by
 

a new control group. Throughout this report those mice receiving a
 

concentration of 0.1 percent and their controls are referred to as
 

the high dose and high dose control groups, respectively, while those
 

mice receiving a concentration of 0.06 percent and their controls are
 

referred to as the low dose and low dose control groups, respectively.
 

14
 
G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

experiment. 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 bioassay and
 

for three consecutive days each month thereafter. The presence of
 

tissue masses and lesions was determined by monthly observation and
 

palpation of each animal. From the first day, all animals were
 

inspected twice daily for mortality.
 

A necropsy was performed on each animal regardless of whether
 

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

the bioassay. The animals were euthanized by carbon dioxide inhalation, and were immediately necropsied. The histopathologic examination consisted of gross and microscopic examination of 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
 

15
 
nodes, thymus, heart, salivary gland, liver, gallbladder (mice), pancreas, esophagus, stomach, small intestine, large intestine, kidney,
 

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

prostate, seminal vesicle, brain, tunica vaginalis, muscle, ear,
 

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 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.
 

16
 
Probabilities of survival were estimated by the product-limit
 

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

in the form of graphs. Animals were statistically censored as of the
 

time that they died of other than natural causes or were found to be
 

missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect
 

on survival used the method of Cox (1972) when testing two groups for
 

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

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

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

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

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.
 

17
 
As a part of these analyses, the one-tailed Fisher exact test (Cox,
 

1970, pp. 48-52) was used to compare the tumor incidence of a control
 

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

results for a number of treated groups, k, are compared simultaneously
 

with those for a control group, a correction to ensure an overall
 

significance level of 0.05 may be made. The Bonferroni inequality
 

(Miller, 1966, pp. 6-10) requires that the P-value for any comparison
 

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

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

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

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
 

18
 
tumor was found, comparisons were based exclusively on animals that
 

survived at least as long as the animal in which the first tumor was
 

found. Once this reduced set of data was obtained, the standard procedures for analyses of the incidence of tumors (Fisher exact tests,
 

Cochran-Armitage tests, etc.) were followed.
 

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

incidence of tumors. Curves of the proportions surviving without an
 

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

during which animals died naturally or were sacrificed was entered as
 

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

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

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

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

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

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


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

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

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

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

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

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

c
 

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

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

19
 
and the proportion in a control group corresponds to a relative risk
 

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

larger proportion in the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical analyses. The interpretation of the limits is that in approximately 95
 

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

of the risk in a treated group of animals to that in a control group
 

would be within the interval calculated from the experiment. When
 

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

can 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.
 

20
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

When compared to their respective controls, the high dose female
 

rats exhibited consistent mean body weight depression. This trend
 

was not as evident in the other groups of dosed rats (Figure 2).
 

Only isolated clinical observations were reported. These
 

included crusted lesions on the dorsolateral surface in one low dose
 

control male and one low dose male and on the side of the head in one
 

low dose female; tissue masses on the ear in two low dose males and
 

on the foreleg in one low dose male; a subcutaneous mass under the
 

base of the tail in one high dose male; and growths on the foreleg in
 

three low dose females and on the ear in another low dose female.
 

B. Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and 2,5-toluenediamine sulfate-dosed groups are shown
 

in Figure 3. For both male and female rats there was no significant
 

association between dosage and mortality.
 

There were adequate numbers of male rats at risk from late-


developing tumors despite the sacrifice in week 78 of five rats from
 

the low dose and each control group, and the sacrifice in week 29 of
 

ten additional rats from the low dose control group. Five males from
 

the high dose group died in week 78. Ninety percent (45/50) of the
 

high dose, 84 percent (42/50) of the low dose, 72 percent (18/25) of
 

21
 
750 750 

- 600 

450 

LOW DOSE CONTROL 

-300 

- HIGH DOSE CONTROL 

. LOW DOSE - 150 

HIGH DOSE 

~T 

75 

TIME ON TEST (WEEKS) 

~r 

90 105 120 

750­ •750 

EC 

<£ 

IO 

LU 

S 

Q 

O 

00 

600 — 

 450­

FEMALE RATS 

^—^ LOW DOSE CONTROL 

'—•'—•' HIGH DOSE CONTROL 

LOW DOSE 

___ HIGH DOSE 

-600 

-450 

— 300 

150­ -150 

15 

~l

30

 ' T 

 45 60 75 

TIME ON TEST (WEEKS) 

-I |

90

 1 1— 

 105 120 

FIGURE 2 

GROWTH CURVES FOR 2,5-TOLUENEDIAMINE SULFATE CHRONIC STUDY RATS 

22 
PROBABILITY OF SURVIVAL PROBABILITY OF SURV

c3 0 p o 

cD k: ^ U) 

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r 

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-

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the high dose control, and 58 percent (29/50) of the low dose control
 

group survived on test at least 85 weeks.
 

There were adequate numbers of female rats at risk from late-


developing tumors despite the sacrifice in week 78 of five rats from
 

the low dose and each control group and the sacrifice in week 29 of
 

ten additional rats from the low dose control group. Five of the
 

high dose females died in week 78. Eighty-six percent (43/50) of the
 

high dose, 84 percent (42/50) of the low dose, 68 percent (17/25) of
 

the high dose control, and 66 percent (33/50) of the low dose control
 

group survived on test at least 85 weeks.
 

C. Pathology
 

Histopathologic findings on neoplasms in rats are summarized in
 

Appendix A (Tables Al and A2); findings on nonneoplastic lesions are
 

summarized in Appendix C (Tables Cl and C2).
 

A variety of neoplasms which are routinely seen in this strain of
 

rat was recorded. The incidences of these tumors in dosed rats were
 

not judged to be attributable to the administration of 2,5-toluenediamine sulfate.
 

Nonneoplastic lesions which commonly occur in aging Fischer 344
 

rats and which were unrelated to the administration of the test compound were seen.
 

This pathologic examination provided no evidence for the carcinogenicity of 2,5-toluenediamine sulfate in Fischer 344 rats.
 

24
 
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 for every type
 

of tumor that was observed in more than 5 percent of any of the 2,5­

toluenediamine sulfate-dosed groups of either sex is included. In
 

addition to these analyses, time-adjusted analyses were performed
 

based upon animals surviving at least 52 weeks; the time-adjusted
 

analyses did not produce different conclusions from the analyses presented here.
 

In male rats, the Fisher exact test indicated a significant incidence of interstitial-cell tumors of the testis when the high dose
 

group was compared to the high dose control (P = 0.014). The comparison of low dose to low dose control had a probability level of
 

P = 0.039, which was not significant under the Bonferroni criterion.
 

These results were discounted, however, due to the well-known high
 

variation in the spontaneous incidence of this tumor (Cockrell and
 

Garner, 1976).
 

For females the Fisher exact test indicated a significantly
 

(P = 0.006) lower incidence of pituitary adenomas in the low dose
 

group than in the low dose control group. The high dose comparison
 

was not significant.
 

No other test for any site in either male or female rats was
 

significant under the Bonferroni criterion. Based upon these statistical results there was no convincing evidence of the carcinogenicity
 

of 2,5-toluenediamine sulfate in rats.
 

25
 
___ 

___ 

___ 

___ 

TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH 2,5-TOLUENEDIAMINE SULFATE*
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar /Bronchiolar Adenoma
 

or Alveolar/Bronchiolar Carcinoma*1
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

ON
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma°
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Adenoma NOS, Basophil
 

Adenoma or Chromophobe Adenoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

LOW DOSE
 

CONTROL
 

0/46(0.00)
 

2/46(0.04)
 

79
 

12/41(0.29)
 

HIGH DOSE
 

CONTROL
 

3/25(0.12)
 

78
 

4/25(0.16)
 

85
 

3/21(0.14)
 

LOW
 

DOSE
 

1/48(0.02)
 

N.S.
 

Infinite
 

0.051
 

Infinite
 

97
 

5/49(0.10)
 

N.S.
 

2.347
 

0.407
 

23.709
 

89
 

3/45(0.07)
 

P = 0.006(N)
 

0.228
 

0.044
 

0.772
 

HIGH
 

DOSE
 

0/49(0.00)
 

P - 0.035(N)
 

0.000
 

0.000
 

0.843
 

8/49(0.16)
 

N.S.
 

1.020
 

0.310
 

4.278
 

99
 

3/40(0.08)
 

N.S.
 

0.525
 

0.078
 

3.650
 

Weeks to First Observed Tumor 101 78 106
 108
 
_ __ ___ 

___ ___ 

__ _ 

_ __ ___ 

TOPOGRAPHY :MORPHOLOGY
 

Adrenal: Pheochromocytoma or
 

Pheochromocytoma Malignant'3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Papillary Adenocarcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

N3 

Thyroid: Adenocarcinoma NOS,
 Papillary Adenocarcinoma, Cystadenocarcinoma NOS, or Papillary
 

Cystadeno carcinoma NOS^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED) 

LOW DOSE HIGH DOSE
 

CONTROL CONTROL
 

6/43(0.14) 4/25(0.16)
 

107 68
 

0/45(0.00) 0/23(0.00)
 

2/45(0.04) 0/23(0.00)
 

107
 

LOW
 

DOSE
 

2/48(0.04)
 

N.S.
 

0.299
 

0.031
 

1.568
 

103
 

3/46(0.07)
 

N.S.
 

Infinite
 

0.590
 

Infinite
 

106
 

3/46(0.07)
 

N.S.
 

1.467
 

0.176
 

16.894
 

106
 

HIGH
 

DOSE
 

1/48(0.02)
 

P = 0.044(N)
 

0.130
 

0.003
 

1.237
 

108
 

0/47(0.00)
 

_——
 

3/47(0.06)
 

N.S.
 

Infinite
 

0.303
 

Infinite
 

105
 
TABLE 3 (CONTINUED) 

TOPOGRAPHY : MORPHOLOGY 

LOW DOSE 

CONTROL 

HIGH DOSE 

CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Pancreatic Islets: Islet-Cell 

Adenoma" 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

2/42(0.05) ___. 

___ 

2/25(0.08) ___ 

___ 

4/48(0.08) 

N.S. 

1.750 

0.266 

18.600 

1/48(0.02) 

N.S. 

0.260 

0.005 

4.803 

Weeks to First Observed Tumor 107 109 106 108 

to 

oo 

Preputial Gland:

Carcinoma NOSb 

P Values0 

 Adenoma NOS or 

0/46(0.00) ___ 2/25(0.08) _.__ 0/49(0.00) 

N.S. 

2/49(0.04) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

___ 0.510 

0.040 

6.750 

Weeks to First Observed Tumor 85 108 

Testis: Interstitial-Cell Tumor 

P Values0 

33/45(0.73) ___ 19/24(0.79) ___ 43/48(0.90) 

P = 0.039 

47/48(0.98) 

P = 0.014 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

._ 1.222 

0.981 

1.446 

1.237 

1.017 

1.331 

Weeks to First Observed Tumor 78 78 78 78 
TABLE 3 (CONCLUDED)
 

Treated groups received time-weighted average doses of 0.06 or 0.20 percent in feed.
 

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

C

The probability level for the Fisher exact test for the comparison of a treated group with its
 

control group is given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. A negative designation (N) indicates a lower incidence
 

in the treated group than in the control group.
 

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

S3
 

VO
 
___ 

_ _ _ 

___ 

__ _ 

TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH 2,5-TOLUENEDIAMINE SULFATE'
 

TOPOGRAPHY: MORPHOLOGY
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma"3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 UJ
 

o
 

Pituitary: Adenoma NOS or Chromophobe Adenoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocytoma or
 

Pheochromocytoma Malignant*3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

LOW DOSE
 

CONTROL
 

4/49(0.08)
 

101
 

18/43(0.42)
 

76
 

2/46(0.04)
 

HIGH DOSE
 

CONTROL
 

2/23(0.09)
 

106
 

8/21(0.38)
 

78
 

3/23(0.13)
 

LOW
 

DOSE
 

2/50(0.04)
 

N.S.
 

0.490
 

0.046
 

3.251
 

107
 

17/48(0.35)
 

N.S.
 

0.846
 

0.477
 

1.507
 

80
 

1/50(0.02)
 

N.S.
 

0.460
 

0.008
 

8.542
 

HIGH
 

DOSE
 

10/50(0.20)
 

N.S.
 

2.300
 

0.553
 

20.530
 

10/43(0.23)
 

N.S.
 

0.611
 

0.266
 

1.546
 

108
 

1/48(0.02)
 

N.S.
 

0.160
 

0.003
 

1.882
 

Weeks to First Observed Tumor 108 109 107
 109
 

81 
__ _ 

___ 

___ 

TABLE 4 (CONTINUED) 

TOPOGRAPHY: MORPHOLOGY
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma'3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Adenocarcinoma NOS,
 

u> Papillary Adenocarcinoma, Papillary 

Cystadenocarcinoma NOS or Infiltrating Duct Carcinoma'3 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

LOW DOSE
 

CONTROL
 

1/47(0.02)
 

107
 

2/49(0.04)
 

101
 

4/49(0.08)
 

HIGH DOSE
 

CONTROL
 

3/21(0.14)
 

109
 

3/23(0.13)
 

52
 

4/23(0.17)
 

LOW
 

DOSE
 

3/48(0.06)
 

N.S.
 

2.938
 

0.246
 

150.900
 

107
 

1/50(0.02)
 

N.S.
 

0.490
 

0.008
 

9.103
 

107
 

6/50(0.12)
 

N.S.
 

1.470
 

0.372
 

6.681
 

HIGH
 

DOSE
 

0/49(0.00)
 

P = 0.025(N)
 

0.000
 

0.000
 

0.707
 

1/50(0.02)
 

N.S.
 

0.153
 

0.003
 

1.810
 

108
 

9/50(0.18)
 

N.S.
 

1.035
 

0.332
 

4.234
 

Weeks to First Observed Tumor	 101 109 100
 108
 
TABLE 4 (CONCLUDED)
 

TOPOGRAPHY: MORPHOLOGY 

LOW DOSE 

CONTROL 

HIGH DOSE 

CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Uterus: Endometrial Stromal Polyp 10/48(0.21) 6/23(0.26) 7/49(0.14) 7/45(0.16) 

P Values0 N.S. N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

___ 

0.686 

0.241 

1.826 

0.596 

0.199 

1.932 

Weeks to First Observed Tumor 78 87 101 108 

Uterus: Adenocarcinoma NOS 

P Values0 

4/48(0.08) ___ 0/23(0.00) ___ 3/49(0.06) 

N.S. 

1/45(0.02) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

_—_ 0.735 

0.113 

4.114 

Infinite 

0.028 

Infinite 

Weeks to First Observed Tumor 95 92 109 

Treated groups received time-weighted average doses of 0.06 or 0.20 percent in feed.
 

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

The probability level for the Fisher exact test for the comparison of a treated group with its
 

control group is given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. A negative designation (N) indicates a lower incidence
 

in the treated group than in the control group.
 

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

of this compound, 95 percent confidence intervals on the relative
 

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

observed tumor incidence rates. In 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-toluenediamine sulfate that could not be established
 

under the conditions of this test.
 

33
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

The only group exhibiting distinct compound-related mean body
 

weight depression when compared to its control group was the high
 

dose female group (Figure 4). It should be noted, however, that the
 

growth pattern of the female high dose control group was unusual
 

(i.e., it did not level off as the animals approached maturity).
 

No clinical abnormalities were recorded for dosed or control
 

mice of either sex.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and 2,5-toluenediamine sulfate-dosed groups are shown
 

in Figure 5. There was no significant association between dosage and
 

mortality for male or female mice.
 

Five male and five female mice were sacrificed in week 78 from
 

each group except the low dose group. There were adequate numbers of
 

male mice at risk from late-developing tumors as 74 percent (37/50)
 

of the high dose, 94 percent (47/50) of the low dose, 74 percent
 

(37/50) of the high dose control, and 84 percent (42/50) of the low
 

dose control mice survived on test until the end of the study. There
 

were also adequate numbers of female mice at risk from late-developing
 

tumors as 66 percent (33/50) of the high dose, 78 percent (39/50) of
 

the low dose, 70 percent (35/50) of the high dose control, and 74
 

34
 
50 -50 

-40 

-30 

LOW DOSE CONTROL 

HIGH DOSE CONTROL 

LOW DOSE 

HIGH DOSE 

-20 

-10 

60 75 

TIME ON TEST (WEEKS) 

I

90

 ' I

 105 

' 

120 

50 -50 

— 40 

-30 

TIME ON TEST (WEEKS) 

LOW DOSE CONTROL 

HIGH DOSE CONTROL 

LOW DOSE 

HIGH DOSE 

I 

105 

— 20 

-10 

120 

FIGURE 4 

GROWTH CURVES FOR 2,5-TOLUENEDIAMINE SULFATE CHRONIC STUDY MICE 

35 
PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVAL 

c3 O P O 

c3 KJ "^ b) 

1 1 1 1 1 i 1 

CO ­ 30 i i
 | V
 Ol
 

I i ri

 r o 5 § 5 | 

1 

m

i I i I
 o
 2) 8- CO 

m m

l 1


? t
 i 

CO 

O 

-i ^~ en — 

01 

d o 

m 

O 

C-n 

m 

s- CO s­ O3D ^ 

A 

m CO 

CO 

01 —
 

-
 m 

O 

X CO
 3D o — 

O 

-n - 

m 

2 

I D o — Ol
 m
 

O 

m .0 

m
 

i o i | i | i | i 

P o o c o 3

 o P P 

b to bo (3 Kj *k C3) 
percent (37/50) of the low dose control mice survived on test until
 

the end 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).
 

Hepatocellular carcinoma occurred in 7/48 (15 percent) of the
 

low dose control males, 10/45 (22 percent) of the high dose control
 

males, 8/48 (17 percent) of the low dose males, and 16/49 (33 percent)
 

of the high dose males. None of the tumors was judged to be due to
 

the administration of the test chemical.
 

A variety of inflammatory and degenerative lesions which commonly
 

occur in aging mice of this strain was seen. These nonneoplastic
 

lesions were not considered to be compound-related.
 

This pathologic examination provided no evidence for the carcinogenicity of 2,5-toluenediamine sulfate in B6C3F1 mice.
 

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 for every type
 

of tumor that was observed in more than 5 percent of any of the
 

2,5-toluenediamine sulfate-dosed groups of either sex is included.
 

Elevated incidences of alveolar/bronchiolar adenomas and alveolar/
 

bronchiolar carcinomas were observed among dosed female mice. For
 

37
 
___ 

___ 

___ 

___ 

___ 

___ 

___ 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH 2,5-TOLUENEDIAMINE SULFATE*
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar/Bronchiolar
 

Carcinoma*5
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lo
 

00
 Lung: Alveolar/Bronchiolar Carcinoma or Alveolar/Bronchiolar
 

Adenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma*3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW DOSE
 

CONTROL
 

6/48(0.13)
 

96
 

6/48(0.13)
 

96
 

4/48(0.08)
 

__._
 

96
 

HIGH DOSE
 

CONTROL
 

4/45(0.09)
 

97
 

11/45(0.24)
 

78
 

2/46(0.04)
 

97
 

LOW
 

DOSE
 

1/47(0.02)
 

N.S.
 

0.170
 

0.004
 

1.326
 

94
 

6/47(0.13)
 

N.S.
 

1.021
 

0.294
 

3.548
 

94
 

5/49(0.10)
 

N.S.
 

1.224
 

0.281
 

5.823
 

94
 

HIGH
 

DOSE
 

5/49(0.10)
 

N.S.
 

1.148
 

0.264
 

5.453
 

96
 

10/49(0.20)
 

N.S.
 

0.835
 

0.353
 

1.955
 

96
 

4/49(0.08)
 

N.S.
 

1.878
 

0.284
 

19.990
 

72
 
_ __ 

___ 

___ 

TOPOGRAPHY: MORPHOLOGY
 

Liver: Hepatocellular Carcinoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma or
 

Neoplastic Nodule
 

u>
 VO P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 5 (CONCLUDED) 

LOW DOSE
 

CONTROL
 

7/48(0.15)
 

_—
 

78
 

7/48(0.15)
 

»__
 

78
 

HIGH DOSE
 

CONTROL
 

10/45(0.22)
 

93
 

10/45(0.22)
 

_._
 

93
 

LOW
 

DOSE
 

8/48(0.17)
 

N.S.
 

1.143
 

0.394
 

3.411
 

94
 

8/48(0.17)
 

N.S.
 

1.143
 

0.394
 

3.411
 

94
 

HIGH
 

DOSE
 

16/49(0.33)
 

N.S.
 

1.469
 

0.705
 

3.233
 

77
 

18/49(0.37)
 

N.S.
 

1.653
 

0.817
 

3.558
 

77
 

Treated groups received doses of 0.06 or 0.10 percent in feed.
 

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

The probability level for the Fisher exact test for the comparison of a treated group with its
 

control group is given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. A negative designation (N) indicates a lower incidence
 

in the treated group 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 TUMORS AT 

SPECIFIC SITES IN FEMALE MICE TREATED WITH 2,5-TOLUENEDIAMINE SULFATE* 

LOW DOSE HIGH DOSE LOW HIGH 

TOPOGRAPHY: MORPHOLOGY CONTROL CONTROL DOSE DOSE 

Lung: Alveolar/Bronchiolar Adenoma 

or Alveolar/Bronchiolar Carcinoma 4/46(0.09) 1/45(0.02) 6/42(0.14) 8/45(0.17) 

P Values0 N.S. P = 0.016 ___ 

Relative Risk (Control) 1.643 7.826 

Lower Limit 0.419 1.118 

Upper Limit 7.390 338.408 

Weeks to First Observed Tumor 96 98 94 78 

Hematopoietic System: Leukemia or 

Malignant Lymphoma 5/48(0.10) 12/46(0.26) 4/45(0.09) 8/47(0.17) 

P Values0 N.S. N.S. 

Relative Risk (Control) 0.853 0.653 

Lower Limit 0.180 0.256 

Upper Limit 3.711 1.568 

Weeks to First Observed Tumor 96 95 94 78 

Liver: Hepatocellular Carcinoma 1/47(0.02) 4/45(0.09) 2/42(0.05) 4/46(0.09) 

P Values0 N.S. N.S. 

Relative Risk (Control) 2.238 1.000 

Lower Limit 0.121 0.198 

Upper Limit 128.900 5.050 

Weeks to First Observed Tumor 96 78 93 94 
TABLE 6 (CONCLUDED)
 

LOW DOSE HIGH DOSE LOW HIGH 

TOPOGRAPHY : MORPHOLOGY CONTROL CONTROL DOSE DOSE 

Pituitary: Adenoma NOS or Carcinoma NOSb 3/42(0.07) 6/37(0.16) 1/38(0.03) 0/38(0.00) 

P Values N.S. P = 0.012(N) ___ Relative Risk (Control) _— 0.368 0.000 

Lower Limit 0.007 0.000 

Upper Limit 4.349 0.602 

Weeks to First Observed Tumor 96 98 94 

Ovary: Papillary Cystadenoma NOS 

or Tubular Adenoma*5 1/45(0.02) 0/41(0.00) 0/36(0.00) 2/39(0.05) ___ P Values0 N.S. N.S. 

Relative Risk (Control) —— 0.000 Infinite 

Lower Limit 0.000 0.313 

Upper Limit 23.152 Infinite 

Weeks to First Observed Tumor 78 97 

treated groups received doses of 0.06 or 0.10 percent in feed. 

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

""The probability level for the Fisher exact test for the comparison of a treated group with its
 

control group is given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not significant (N.S.) is indicated. A negative designation (N) indicates a lower incidence
 

in the treated group than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 
the combined incidence in females, the Fisher exact test comparing
 

high dose to high dose control was significant (P = 0.016). In
 

historical data collected by this laboratory for the NCI Carcinogenesis Testing Program 12/350 (3 percent) of the untreated B6C3F1
 

female mice had an alveolar/bronchiolar adenoma, alveolar/bronchiolar
 

carcinoma, alveolar-cell adenocarcinoma, or an adenoma NOS of the
 

lung/alveoli—compared to the 4/46 (9 percent), 1/45 (2 percent),
 

6/42 (14 percent), and 8/45 (17 percent) observed in the low dose
 

control, high dose control, low dose, and high dose groups, respectively, in this bioassay.
 

Based upon these results the statistical conclusion is that the
 

administration of 2,5-toluenediamine sulfate was associated with the
 

incidence of alveolar/bronchiolar neoplasms in female B6C3F1 mice.
 

For females the Fisher exact test indicated a significantly
 

(P = 0.012) lower combined incidence of pituitary adenomas NOS or
 

pituitary carcinomas NOS in the high dose group than in the high dose
 

control. The low dose comparison was not significant.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

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

of statistically significant results. It should also be noted that
 

many of the confidence intervals have an upper limit greater than
 

42
 
one, indicating the theoretical possibility of tumor induction in
 

mice by 2,5-toluenediamine sulfate that could not be established
 

under the conditions of this test.
 

43
 
V. DISCUSSION
 

There was no significant association between compound administration and accelerated mortality in either sex of either species.
 

Adequate numbers of animals in all groups survived sufficiently long
 

to be at risk from late-developing tumors.
 

Although the incidence of interstitial-cell neoplasms of the
 

testis was statistically significant in each dosed male rat group,
 

development of these tumors was not considered attributable to compound administration since spontaneous incidence of these neoplasms
 

in male Fischer 344 rats is both high and variable. It should also
 

be noted that control rats were housed in a separate room from dosed
 

rats. There were no other neoplasms occurring in male rats at
 

statistically significant incidences, and none of the incidences
 

of neoplasms observed in female rats were statistically significant.
 

The only site of significantly increased tumor incidence among
 

dosed female mice was the lungs. The combined incidence of alveolar/
 

bronchiolar adenomas and alveolar/bronchiolar carcinomas was statistically significant for the high dose group. The combined incidences
 

of these tumors in both high and low dose female mouse groups were
 

elevated relative to historical controls. However, it should be
 

noted that high dose control mice were housed in a separate room
 

from dosed mice and received in separate shipments from dosed mice.
 

Because of these factors, this increased incidence does not provide
 

44
 
sufficient evidence of a compound-related effect. No significant
 

increase in tumor incidence was observed among dosed male mice.
 

Under the conditions of this bioassay, sufficient evidence was
 

not provided to conclusively demonstrate the carcinogenicity of
 

2s5-toluenediamine sulfate in either Fischer 344 rats or B6C3F1
 

mice.
 

45
 
VI. BIBLIOGRAPHY
 

Ames, B.N., H.O. Kammen, and E. Yamasaki, "Hair Dyes are Mutagenic:
 

Identification of a Variety of Mutagenic Ingredients." Proceedings of the National Academy of Science U.S.A. 72:2423-2427,
 

1975.
 

Anthony, H.M., and G.M. Thomas, "Tumors of the Urinary Bladder: An
 

Analysis of the Occupations of 1,030 Patients in Leeds, England."
 

Journal of the National Cancer Institute 45:879-895, 1970.
 

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

J. Wiley & Sons, New York, 1971.
 

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

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

Union Against Cancer, Geneva, 1969.
 

Chemical Abstracts Service, The Chemical Abstracts Service (CAS)
 

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

Chemical Society, Washington, B.C., 1977.
 

Cockrell, B.Y. and F.M. Garner, "Interstitial-Cell Tumors of the
 

Testis in Rats." Comparative Pathology Bulletin 8:2-4, 1976.
 

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.
 

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.
 

Hawley, G.G., editor, The Condensed Chemical Dictionary, 8th edition.
 

Van Nostrand Reinhold Company, New York, 1971.
 

Hoover, R. and J. Fraumeni, Jr., "Cancer Mortality in U.S. Counties
 

with Chemical Industries." Environmental Research 9:196-207,
 

1975.
 

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

Observations." Journal of the American Statistical Association
 

53:457-481, 1958.
 

46
 
Kiese, M., and E. Rauscher, "The Absorption of p-toluenediamine
 

Through Human Skin in Hair Dyeing." Toxicology and Applied
 

Pharmacology 13:325-331, 1968.
 

Kiese, M., M. Rachor, and E. Rauscher, "The Absorption of Some Phenylenediamines Through the Skin of Dogs." Toxicology and Applied
 

Pharmacology 12:495-507, 1968.
 

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.
 

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

Co., New York, 1966.
 

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

Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced
 

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

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

Sax, N.I. Dangerous Properties of Industrial Materials. Van Nostrand
 

Reinhold Company, New York, 1975.
 

Society of Dyers and Colourists, Colour Index, 3rd edition, Volume 3.
 

Yorkshire, England, p. 3260, 1971.
 

Staats, E.B., Comptroller General of the United States. Cancer and
 

Coal Tar Hair Dyes: An Unregulated Hazard to Consumers. Report
 

to the Subcommittee on Oversight and Investigations, Committee
 

on Interstate and Foreign Commerce, House of Representatives,
 

Washington, D.C., December 6, 1977.
 

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

1975.
 

U.S.	 International Trade Commission, Import os f Benzenoid Chemicals
 

and Products, 1974. USITC Publication 762, U.S. Government
 

Printing Office, Washington, D.C., 1976.
 

Wynder, E.L., J. Onderdonk, and N. Mantel, "An Epidemiological Investigation of Cancer of the Bladder." Cancer 161 : 1388-1407, 1963.
 

47
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH 2,5-TOLUENEDIAMINE SULFATE
 

TABLE Al

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 

—, HIGH DOSE LOB DOSE CONTROL (UNTR) CONTROL (UNTR) LOU EOSE HIGH DOSE 

01-0084 01-0037 01-0039 01-3090 

ANIMAL S INITIALLY IN STUDY 25 50 50 50

 

ANIMAL S NECROPSIE D 25 16 49 49

 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY* * 25 1*6 1*8 1(9

 

INTEGUMENTAR Y SYSTEM 

*SKIN (25) (U6 ) (U9)

 

SQUAMOUS CELL CARCINOMA 1 (2%)

 

*SUBCUT TISSUE (25) C*6) (K9) (49)
 

FIBROMA 1 (2%) 1 (2X)
 LIPOMA 1 (2«)
 KMANGIOSARCOMA 1 (2J)
 NEUROFIBROMA 1 (2*)
 

BESPIRATCRY SYSTEM
 

#TRACHEA (11) (1*5) (46) (47)
 

ADENOCARCINOMA, NOS, METASTATIC 1 (2X)
 

tLUNG (25) (1*6) (48) (49)
 

UNEIFFERENTIATED CARCINOMA METAS 1 <2«)
 ADENOCAECINOMA, NOS, METASTATIC 1 (2X)
 

ALVEOLAR/BRONCHIOLAR AD2NOMA 2 (8<) 1 (2*)
 

ALVEOLAS/BPONCHIOLAR CARCINOMA 1 (4?)
 PAPIILARY ADENOCARCINOMA, METAST 1 (2%)
 PHEOCHEOMOCYTOMA, METASTATIC 1 (4X)
 

HEMATOEOIETIC SYSTEM
 

*MDLTIPIE ORGANS (25) (46) (49) (49)
 MALIGNANT LYMPHOMA, NOS 1 (256)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYP3 1 (2%)
 UNDIF"ERENTIATED LEUKEMIA 2 <8X) 1 (2%)
 

BYELCMONOCYTIC LEUKEMIA 2 («») 5 (10!S)
 

LYMPHOCYTIC LEUKEMIA 2 (g\) 1 (2*) 1 (2X)
 

# NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED ** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 
TABLE A1 (CONTINUED) 

—• —— HIGH DOSE CONTROL (UNTF) 

01-008U 

LOW DOSE CONTROL (DNTR) 

01-0037 

LOW DOSE 

01-0039 

HIGH DOSE 

01-0090 

•LYMPH NODE 

ADENOCAHCINOBA, NOS, METASTATIC 

tMEDIASTINAL L.NODE 

UNDIFFFRENTIATED CARCINOMA BETAS 

tM.BDOMINAL LYMPH NODE 

UNEIFFFRENTIATED CARCINOMA BETAS 

*LIVER 

BYELOBONOCYTIC LEUKEMIA 

GBANIIIOCYTIC LEUKEMIA 

*THYB:JS THYMOMA 

<2K) 

(2U) 

(21) 

(25) 

(22) 

(38) 

1 (3*) 

(38) 

(38) 

(46) 

(38) 

(39) 

(39) 

(39) 

(48) 

(39) 

1 <3X) 

(13) 

(43) 

1 (2*) 

(43) 

1 (2X) 

(49) 

1 (2X) 

1 (2X) 

(28) 

CIRCULATORY SYSTEM 

NOSF 

DIGESTIVE SYSTEM 

•LIVF.R 

UNDIFFERENTIATED CARCINOMA

MFOPLASTIC NODULE 

HEPATOCELLULAR CARCINOMA 

'PANCREAS 

UNDIFFEPENTIATED CARCINOMA 

ADS NOB*, NOS 

•STOMACH 

SC'JABOUS CELL PAPILLOflA 

BASAL-CELL CARCINOMA 

*JEJUNUB 

CYSTAD3NOCASCINOMA, NOS 

*COLON 

ADENOCABCINOMA, NOS 

 METAS 

(25) 

(25) 

(24) 

1 (UH) 

1 (»%) 

<2t) 

(24) 

(46) 

(42) 

(45) 

(43) 

(43) 

(48) 

1 (2*) 

1 (2X) 

(48) 

(47) 

(48) 

(47) 

(«9) 

1 (2%) 

(48) 

1 (2X) 

1 (2*) 

(49) 

(48) 

1 (2J) 

(48) 

1 (2X) 

URINAR Y SYSTEM 

*KIDNSY 

__ TUBULARz£2LL_iD5N03A 

* NIIM5E B O F ANIMAL S WIT H TISSUE

* NUBIE S O F ANIMAL S NSCROPSIE D 

(24) (46) 

 EXAMINE D MICROSCOPICALLY 

(48) .___. _«_____.._. (49) 

1 (2X1 

A-4 
TABLE A1 (CONTINUED) 

. , HIGH DOSE LOW DOSE CONTROL (UNTR) CONTROL (UNTS) LOW DOSS HIGH DOSE 

01-0084 01-0037 01-0039 01-0090 

IURINARY BLADDER (23) (42) (48) (45) PAPILLOMA, NOS 1 (2X) 1 (2X) TPANSITIONAL-CELL CARCINOMA 1 (2X) 

ENDOCRINE SYSTEM 

*PITNITABY 

ADENOMA, NOS 

(21) 

1 (5*) 

(41) 

2 (5X) 

(45) 

3 (7X) 

(40) 

3 <8X) 

CHROMOPHOBE ADENOMA 10 (24*) BASOPHIL ADENOMA 2 (MX) 

#ADRENAL 

ADENOCARCINOBA, NOS, NETASTATIC 

(25) (43) 

1 (2%) 

(48) (48) 

PHECCHROMOCYTOMA 2 (8X) 6 (14X) 2 (4X) 1 (2X) PHECCH80MOCYTOMA, MALIGNANT 2 (8X) 

tTHYROIC (23) (45) (46) (47) ADENOMA, NOS 1 (2X) ADENOCARCINOBA, NOS 

PAPILLARY ADENOCAHCINOMA 

2 (4X) 

3 (7X) 

1 (2X) 

C-CELL ADENOMA 1 (2X) C-CELL CARCINOMA 

CYSTADENOCARCIN08A, NOS 

1 (2X) 1 <2X) 

1 (2*) PAPILLARY CYSTADENOCASCINO.IA.NOS 1 (2J) 

#PANCREATIC ISLETS (25) (42) (48) (48) ISLET-CELL ADENOMA 2 (81) 2 <5X) 4 (8X) 1 (2X) 

REPRODUCTIVE SYSTEM 

*MA«MARY GLAND (25) (46) (49) (49) FIBROADENOMA 1 (4*) 1 (2X) 

*PREPUTIAL GLAND (25) (46) (49) (49) CARCINOMA, NOS 1 (4*) 2 («X) ADENCMA, NOS ' 1 (4*) 

fPROSTATE (23) (45) (47) (46) PARAGAN6LIOMA, NOS 1 <2X) 

UTESTIS 

_LHZE_BS.HT,IAL.-££ii_IIMP.fi 

(24)

19. J22S1.., (45) 

3.3_J7JSJ. . 

(48) 

43 (90SL _ (48) . _47 198%} 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

A-5 
TABLE Al (CONTINUED) 

HIGH DOSE LOW DOSE CONTROL (ONTH ) CONTROL (UHTB) L OU DOSE HIGH DOSE 

01-0084 01-0037 01-0039 01-0090 

*SCRCTU M (25) (46) (49) (49)

LEIOMYOM A 1 (2*) 

NERVOU S SYSTE M 

• BRAI N (25) (44) (48) (48)

 

ASTROCYTOM A 1 (2% ) 

SPECIAL SENSE ORGAN S 

*FAR (25) (46) (49) (49) 

FIBRCM A 1 (2*) 

*EA R CANA L (25) (46) (49) (49)

 

SQUANOO S CELL CARCINOM A 1 (4*)

 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES
 

*BODY CAVITIES (25) (46) (49) («9)
 

MFSOTHELIOHA, NOS 1 (2X)
 

*MEDIASTINUM (25) (46) (49) (49)
 

ALVEOLAH/BEONCHIOLAR CA, METASTA 1 (4%)
 

*PLEURA (25) (46) (49) (49)
 

ALVEOLAR/BRONCHIOLA E CA, NETASTA 1 (4% ) 

ALL OTHEE SYSTEBS
 

* NUMBE R OP ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

A-6 
ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUKOBS*
 

TOTAL PEIMARY TUMORS
 

TOTAL ANIMAL S HITH BENIGN TUNCES
 

TABLE Al (CONCLUDED) 

HIGH DOSE 

CONTROL (ONTR) 

01-0084 

25
 

3
 

4
 

5
 

13
 

22
 

41
 

20
 

TOTAL BENIGN TUMORS 31
 

TOTAL ANIMALS WITH MALIGNANT TUMOSS 9
 

TOTAL MALIGNANT TUMORS 10
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 2


TOTAL SECONDARY TUMORS 3


TOTAL ANIMALS WITH TUMORS UNCERTAINEFNIGN OR MALIGNANT


TOTAL UNCERTAIN TUMOES


TOTAL ANIMALS KITH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMOSS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

LOW DOSE CONTROL (UNTR) 

01-0037 

50
 

6
 

2
 

15
 

27
 

34
 

61
 

33
 

55
 

5
 

5
 

1


 4


 1


 1


LOW DOSE HIGH DOSE
 

01-0039 01-0090
 

50 50
 5 3
 5 11
 5
 

35 36
 

45 48
 72 77
 

44 47
 

58 59
 

9 17
 

12 18
 

1
 

1
 

2
 

2
 

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

A-7
 
TABLE A2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

TREATED WITH 2,5-TOLUENEDIAMINE SULFATE

 

HIGH DOSE LOW DOSE
 CONTROL (UNTR) CONTROL (UNTR) LOW COSE HIGH DOSE
 

02-0080 02-0037 02-0039 02-0090
 

ANIMAL S INITIALLY IN STUDY 25 50 50 50
 

ANIMAL S NECROPSIED 23 t9 50 50
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY** 23 49 50 50
 

INTEGUMENTARY SYSTEM
 

*SKIN (23) (1*9) (50) (50)
 

SQUAMOUS CELL CARCINOMA 1 <2X)
 

SEBACEOUS ADENOCARCINOMA 1 (1J)
 

FIBHOSARCOMA 1 <2X)
 

*SUBCOT TISSUE (23) (49) (50) (50)
 

SQDAMOUS CELL CARCINOMA 1 (2X)
 

RESPIRATORY SYSTEM
 

#LUNG (23) <<*9) (50) (50)
 

ADENOCARCINOMA, NOS, METASTATIC 1 (2%) 1 <2X)
 

HTPATOCFLLULAR CARCINOMA, METAST 1 (2*)
 

ALVEOLAR/BKONCHIOLAB ADENOMA i (<*%> 1 (2X)	 1 <2X)
 

HEMATOPOIFTIC SYSTEM
 

•MULTIPLE	 OFGANS (23) (49) (50) (50)
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 2 (<tX)
 

LEUKEMIA, NOS 1 (2X)
 

UNDIFFSBENTIATED LEUKEMIA 2 (9%)
 

NYELCMONOCYTIC LEUKEMIA 2 (U%) 8 (16X)
 

MONOCYTIC LEUKEMIA 2 (it«)
 

• PENA L LYMPH NODE (21) (41) (47) (49)
 

ADENOCARCINOMA, NOS, METASTATIC 1 (2X)
 

KTHYMUS (20) (42) (43) (4H)
 

MALIGNAN T LYMPHOMA, NOS 1 <2X)
 

CIFCULA103 Y SYSTE M 

HQND 

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

* NUMBE R O F ANIMAL S NECFOPSIE D 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-8 
TABLE A2 (CONTINUED) 

HIGH DOSE LOW DOSE
 CONTROL (ONTR) CONTROL (BNTR)
 

02-0081 02-0037
 

DIGESTIVE SYSTEM
 

#LIVER (23) (19)
 ADENOCAHCINOMA, NOS, METASTATIC 1 <2X)
 

NEOFLASTIC NODULE 2 (9X)
 HEPATOCELLULAR CARCINOMA 2 (IX)
 

*STOMACH (23) (48)
 

SQUAMOUS CELL PAPILLOMA
 

IILEUM (23) (47)
 

LEIOMYOSARCOMA
 

URINARY SYSTEM
 

tURINARY BLADDER (22) (41)
 

PAPILLOMA, NOS
 

ENDOCRINE SYSTEM
 

#PITUITARY (21) (13)
 

ADENOMA, NOS 1 <5X) 3 (7X)
 

ADENOCARCINOMA, NOS 2 <5X)
 

CHROMOPHOBE ADENOMA 7 (33*) 15 (35X)
 

#ADRENAL (23) (46)
 

PHEOCHROBOCYTOMA 2 (9X) 2 (4X)
 

PHEOCHROMOCYTOMA, MALIGNANT 1 (IX)
 

tTHYROID (21) (47)
 

ADENOMA, NOS 1 (2X)
 ADENOCARCINOMA, NOS 2 (IX)
 C-CELL ADENOMA 2 (10X) 1 <2X)
 C-CELL CARCINOMA 1 (5X)
 

*THYROIE FOLLICLE (21) (47)
 PAPILLARY CYSTADENOCARCINOMA.NOS 1 <5X)
 

REPRODUCTIV E SYSTE M 

*NAMt!AR Y GLAN D (23)

 

-1-12 Si 

t NDMBE R OF ANIMAL S KITH TISSOE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

A-9 

—— —~—
 LOK DOSE
 

02-0039
 

(50)
 

1 (2X)
 

(50)
 

(50)
 

(47)
 

(18)
 

17 (35X)
 

(50)
 

1 (2X)
 

(18)
 

3 (6X)
 

(18)
 

(50) 

HIGH DOSE
 

02-0090
 

(19)
 

1 (2»)
 

(19)
 

1 (2%)
 

(19)
 

1 (2X)
 

(50)
 

1 (2X)
 

(13)
 

10 (23%}
 

(48)
 

1 (2X)
 

(49)
 

1 (2X)
 

(49)
 

(50) 
TABLE A2 (CONTINUED) 

HIGH DOSE CONTROL (UNTR ) 

02-0084 

LOW DOSE CONTROL (UNTR) 

02-0037 

— ———— L OW DOSE 

02-0039 

., 

HIGH DOSE 

02-0090 

ADENCCARCINOMA , NO S 

PAPILLAR Y ADENOCARCINOMA 

PAPILLAR Y CYSTADENOBA, DOS 

PAPILLAR Y CYSTADENOCARCINOSA.NO S 

INFILTRATIN G DUCT CAHCINOHA 

FI3ROADENCM A 

*CLITORAL GLAND 

CARCINOMA , NOS 

#UTERUS 

ADENOCARCINOMA , NO S 

ENDOMETRIA L STROMA L POLYP 

HFMANGIOM A 

#UTERUS/ENDOMETRIU M 

ADENCCAHCINOMA, NOS 

#OVAR Y 

TUBULA R ADENOM A 

2 (9%) 

1 (4%) 

4 (17%) 

(23) 

(23) 

6 (26%) 

(23) 

(22) 

1 (2%) 

1 (2%) 

4 (8%) 

(49) 

(48) 

4 (8%) 

10 (21X) 

(48) 

(47) 

1 (2X) 

6 (12X) 

(50) 

(49) 

1 (2X) 

7 (14X) 

1 (2%) 

(49) 

2 (4%) 

(50) 

1 (2%) 

1 (2X) 

9 (18S) 

(50) 

1 (2%) 

(45) 

1 (2%) 

7 (16%) 

(45) 

(46) 

1 (2%) 

NERVOU S SYSTE M 

NON 3 

SPECIAL SENSE ORGAN S 

*EAR 

SQUAMOU S

*EAR CANA L 

FIBROM A 

 CELL CARCINOM A 

(23) 

(23) 

(49) 

(49) 

1 (2X) 

(50) 

2 (4X) 

(50) 

(50) 

(50) 

MUSCULOSKEL3TA L SYSTEM 

NONE 

BODY CAVITIF.S 

*BOD Y CAVITIES (23) (49) (50) (50) 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

A-10 
TABLE A2 (CONTINUED) 

HIGH DOSE LOW DOSE CONTROL (UNTR) CONTROL (UNTR) L OU DOSE HIGH DOSE 

02-008U 02-0037 02-0039 02-0090 

*ABDCMINAL CAVITY (23) (19) (50) (50) 

SAKCOMA, NOS 1 (2X) 1 12%) LEIOMYOSARCOHA 1 (2%) 

*PERITONEOM (23) (50) (50)
 

ADENOCARCINOMA, NOS, METASTATIC 1 (2X)
 LIPOMA 1 (2*)
 

ALL OTHER SYSTEMS
 

DIAPHRAGM
 

RHABDOMYOSARCOHA
 

ANIMAL DISPOSITION SUM8ARY
 

ANIMALS INITIALLY IN STUDY 25 50 50 50
 

NATURAL DEATHS 3 5 2.
 MORIBUND SACRIFICE 5 7 1 1 1 1
 

SCHICtJLED SACRIFICE 5 15 5
 ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 12 23 32 39
 

ANIMAL MISSING
 

• NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUHBEB OF ANIMALS NECROPSIED
 

A-ll
 
TABLB A2 (CONCLUDED) 

HIGH DOSE LOW DOSE 

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

02-0084 02-0037 02-0039 02-0090 

TUMOR SUMMARY
 

TOTAL ANIMALS KITH PRIMARY TOBORS* 19 32 3<t 33
 

TOTAL PRIMARY TUMORS 3« 56 «7 53
 

TOTAL ANIMALS WITH BENIGN TUHCRS 18 27 28 23
 

TOTAL BENIGN TOMORS 23 39 32 33
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 8 15 11 17
 TOTAL MALIGNANT TUMORS 9 17 15 20
 

TOTAL ANIMALS WITH SECONDARY TUHOBS* 2 1
 

TOTAL SECONDARY TUMORS K 2
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT 2
 

TOTAL UNCERTAIN TUMORS 2
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPHIMABY OR HFTASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TOHORS EXCEPT SECONDARY TUMORS
 

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

A-I2
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH 2,5-TOLUENEDIAMINE SULFATE
 

TABLE Bl

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 

11IGH DOSE LOW DOSE
 (rONTBOL (UNTR) CONTROL (UNTR) LOW DOSE HIGH DOSE
 

05-0077 05-0037 05-0039 05-0089
 

ANIMALS INITIALLY IN STUDY 50 50 50 50
 

ANIMALS NECROPSIED U6 "48 49 49
 

ANIMALS EXAMINED HISTOPATHOLOGICALL Y** 45 48 48 49
 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (46) (48) (49) (49)
 

OSTEOHA 1 (2X)
 

RESPIEATOBY SYSTEM
 

ILUNG (45) (48) (47) (49)
 

HEPATOCELLULAR CARCINOMA. BETAS! 1 (2X) 2 (4*)
 

ALVEOLAR/BRONCHIOLAH ADENOMA 7 (16*) 5 (11X) 5 (10X)
 

ALVEOIAR/BRONCHIOLAE CARCINOMA « (9X) 6 (13X) 1 (2X) 5 (10X)
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (46) (48) (49) (49)
 

MALIGNANT LYMPHOMA, NOS 2 (<•*) 4 <8X) 2 (4*)
 

MALIG. LYMPHOMA. HISTIOCYTIC TYPE 2 (4X)
 

#SPLEEN (45) (47) (48) (43)
 

HE'MANGIOMA 1 (2X)
 MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (2*) 1 (2«)
 

#MANDIBULAR L. NODE (35) (44) (44) (36)
 

MAIIG. LYMPHOMA, HISTIOCYTIC IYP3 1 (3X)
 

tMESSNTERIC L. NODE (35) (44) (44) (36)
 

MALIGNANT LYMPHOMA. NOS 1 <3X)
 

tLIVER (45) (48) (48) (49)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (2%)
 

CIRCULATORY SYSTEM
 

UQN.S _
 

* NUMBE R OF ANIMAL S HITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIEE 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-3 
TABLEBl (CONTINUED) 

HIGH DOSE LOW DOSE CONTROL(UNTR ) COMTROL (UNTR)

05-0077 05-0037

DIGESTIVE SYSTEM 

*LIVER (45) (48)


NEOPLASTIC NODULE


HEPATOCELLULAR CARCINOMA 10 (22%) 7 (15*)


HEMANGIOM A 1 (2X)
 

frSTOSACH (42 ) (47)

SQUAPIOU S CELL PAPILLON A 1 (2t) 

SQUAMO'J S CELL CARCINOM A 1 (2S) 

URINAR Y SYSTEM
 

NONE
 

ENDOCRINE SYSTEB
 

#ADHENAL (43) (45)


PHEOCHROMOCYTOMA


#THYROID (40) (47)


FOLLIC'JLAR-CELL ADENOMA 1 (2X)
 

REPRODOCTIVE SYSTEPI
 

NONE
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*HARDEEIAS GLAND (46) (48)


ADENOMA. NOS


PAPILLARY CYSTADEliOMA, NOS


*EAR CANAL (46) (48)


SQDAMOUS CELL CARCINOMA 1 (211)
 

HUSCULOSKELETAL SYSTEM
 

* NUMBER OP ANIMALS WITH TISSUE EXAMINED HICHOSCOPICALLY
 

* NUMBER OP ANIMALS NECROPSIED
 

B-4
 

 LOW DOSE HIGH DOSE 

 05-0039 05-0089 

 (48) (49)
 

2 (4*)
 

8 (17)1) 16 (33X)
 

 (46) (48) 

 (45)	 (44)
 

1(2%)
 

 (46) (46)
 

 (49) ' (49)
 

1 (2X)
 

1 (2X)
 

 (49) (49)
 
TABLE Bl (CONCLUDED) 

HIGH DOSE LOW DOSE CONTROL (DMTR) CONTROL (UNTR) LOW DOSE HIGH DOSE 

05-0077 05-0037 05-0039 05-0089 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMAR Y
 

ANIMALS INITIALLY IN STUDY 50 50 50 50
 NATURAL DEATHS) 7 3 3 7
 

MORIBUND SACRIFICE 1 1
 

SCHEEULED SACRIFICE 5 5 5
 ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 37 «2 U7 37
 

ANIMAL MISSING
 

a INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 21 17 19 27
 

TOTAL PRIMARY TUMORS 25 21 21 34
 

TOTAL ANIMALS WITH BENIGN TUMCRS 8 2 7 7
 

TOTAL BENIGN TUMORS 8 3 7 7
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 15 15 13 23
 TOTAL MALIGNANT TUMORS 17 18 11 25
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1
 

TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCESTAIN­ BSNIGN 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 CRGAN
 

B-5
 
___ 

TABLE B2 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 

HIGH DOSE LOW DOSE
 CONTROL (ONTfl)	 CONTROL (UNTR)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS MISSING
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY**
 

INTEGUMENTARY SYSTEM
 

*SKIS
 

FIBRCSARCOMA
 

*SUBCUT TISSUE
 

SAHCOMA, NOS
 

LEIOMYOSAHCOMA
 

RESPIRATORY SYSTEM
 

*LUNG
 

HEPATOCSLLULAR CARCINOMA, METAST
 

ALVEOLAR/BHONCHIOLAR ADENOMA
 

ALVEOHR/BRONCHIOLAR CARCINOMA
 

HEMATOPOIF'IC SYSTEM
 

*MULTIPL" ORGANS
 

MALIGNANT LYMPHOMA, NOS
 

MALIG. LYMPHOMA, UNDIFFER-TYEE
 

MALIG. LYMPHOMA, HISTIOCYTIC TYPE
 

LYMPHOCYTIC LEUKEMIA
 

•SPLEEN
 

HEMANGIOSARCOMA
 

MALIG. LYMPHOHA, HISTIOCYTIC TYPE
 

•LYMPH	 NODS
 

MALIGNANT LYEPHOMA, NOS
 

•PEYERS PATCH
 

_ M.ALIG^LY.KP.HOMA,. HISTIOCYTIC TIPS-­

06-0077	 06-0037
 

50	 50
 

16 18
 

1(6 1(7
 

(16)	 (18)
 

2 (««)
 

(16)	 (13)
 

1	 (2%)
 

(15)	 (16)
 

1 (2%)	 3 (7%)
 

1 (2S)
 

(16)	 (18)
 

3 (7*) 1 (2%)
 1 (2%)
 

6 (13*) 2 (IX)
 1	 (2*)
 

(13)	 (16)
 

1 (2%)
 

1 (2%)
 

(11)	 (39)
 

(13)	 (11)
 

J_12il
 

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

* NUMBE R OF ANIMAL S NECROPSIED 

** EXCLUDES PARTIALLY AUTOLYEED ANIMALS 

B-6 

Loa EOSE HIGH DOSE
 

06-0039 06-0089
 

50	 50
 

1
 

1(5 47
 

13
 16
 

(15)	 (17)
 

(15)	 (17)
 

1 (2X)
 

(12)	 (16)
 

1 (2%)
 

6 (1UX) 7 (15X)
 1 (2*)
 

(15)	 (17)
 

1 (2%) 3 (6%)
 

3 (7*) 1 (2%)
 

(12)	 (12)
 

1 (2%)
 

2 (5X)
 

(37)	 (32)


1 (3*)
 

(12)	 (12)
 
TABLE B2 (CONTINUED)
 

LOW DOSE
 CH

0INTR1OLS?UNTR) CONTROL (UNTE)
 

06-0077 06-0037
 

tDOODENUN (43) («4)
 MALIGNANT LYMPHOMA, NOS
 

tTHYMUS (27) (31)
 MALIGNANT LYMPHOMA, NOS 1 (3X)
 

CIRCULATORY SYSTEM
 

*POLBONAFtY ARTEKY (16) (48)
 

ADENOCARCINOHA, NOS, METASTATIC
 

DIGESTIVE SYSTEM
 

IIIVEB (15) (47)
 

HEPATOCELLULAR CARCINOMA 4 (9*) 1 (2X)
 

FIBEOSARCOMA 1 (2*)
 

*BILE DUCT (H6) (48)
 

BILE DUCT CARCINOMA
 

ISTOMACH (142) (44)
 

SCUANOUS CELL PAPILLOHA 3 (7J) 1 <2X)
 

tJEJUNUM (43) (44)
 

ADENOCARCINOMA, NOS
 

*COLON (41) (40)
 

LEIOMYOSARCOHA 1 (3X)
 

URINARY SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

IPITOITARY (37) (42)
 CARCINOMA, NOS 1 (2X)
 ADENOMA, NOS 6 (16*) 2(5%)
 

*ADRENAL (43) (45)
 

QQ3HCA.L .AP.EHOM- 1.1&L
 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OP ANIMALS NECROPSIED
 

B-7
 

LOH DOSE
 

06-0039
 

(42)
 

(29)
 

(45)
 

1 (2*)
 

(42)
 

2 (5X)
 

(45)
 

(42)
 

(42)
 

1 (2%)
 

(41)
 

(38)
 

1 <3X)
 

(38)
 

HIGH DOSE
 

06-0089
 

(42)
 

1 (2%)
 

(29)
 

(47)
 

(46)
 

4 (9%)
 

(47)
 

1 (2%)
 

(43)
 

(42)
 

(43)
 

(38)
 

(41)
 
TABLE B2 (CONTINUED) 

PHECCHROMOCYTOMA 

ITHYROIC 

PAPILLARY ADENOCARCINOMA 

#PANCREATIC ISLETS 

ISLET-CELL ADENOM A 

HIGH DOSE 

CONTROL(UNTR ) 

06-0077 

(30) 

1 (2*) 

LOW DOSE 

COHT80L (UNTR)

06-0037

1 (2*) 

(43) 

 LOU DOSE 

 06-0039 

(36) 

1 (3X) 

(42) 

HIGH DOSE 

06-0089 

(34) 

(12) 

REPRODUCTIVE SYSTEM 

*MAKMAR Y GLAND 

ADENOCARCINOMA, NOS 

•UTERUS 

LEIOHYOSARCOMA 

ENDOMETRIAL STKOMAL POLYP 

*OVARY

PAPILLARY CYSTADENOMA, NOS

LUTECMA

TUBULAR ADENOMA

(46) 

1 (2X) 

(43) 

 (41)

 1 (2X) 

<U8) 

(45) 

3 (7X) 

 (45)

 1 (2X)

(45) 

(41) 

 (36)

(47) 

(42) 

 (39) 

1 (3X) 

1 (3X) 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

*HARDSRIAN GLAND

PAPILLARY CYSTADENOMA, NOS

 (46) (48) (45) (47) 

1 (2X) 

MUSCULOSKELSTAL SYSTEM 

NONF 

BODY CAVITIES 

NONE 

ALL OTHEB SYSTEMS 

* NUMBER OP ANIMALS WITH TISSUE EXAHINED MICROSCOPICALLY 

* NUMBER OF ANIHALS NECROPSIED 

B-8 
TABLE B2 (CONCLUDED) 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 NATURAL DEATHS
 MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUKMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*


TOTAI PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TUMCRS
 TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORS*


HIGH DOSE 

CONTROL(UNTP ) 

06-0077 

50
 

35
 

 22


 32


 12
 13


 18


 19


TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OH METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

LOW DOSE 

CONTROL (ONT8) 

06-0037 

50
 

6
 

2
 

5
 

37
 

 20


 24


 11
 11


 11


 13


LOW EOSE HIGH DOSE 

06-0039 06-0089 

50 50 

10 

2 

5 

39 33 1 

 15 22 17 21 

7 10 7 10 

9 12 10 

2 

2 

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

B-9
 

APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 2,5-TOLUENEDIAMINE SULFATE
 

__________ __ 

TABLE Cl 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE RATS TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 

HIGH DOSE LOW DOSE CONTROL (ONTR) CONTROL (UNTR) L OU DOSE HIGH DOSE 

01-0084 01-0037 01-0039 01-0090 

ANIMAL S INITIALL Y I N STUD Y 25 50 50 50

 

ANIMAL S NEC80PSIED 25 46 49 49

 ANIMALS EXAMINE D HISTOPATHOLOGICALLY ** 25 46 48 49

 

INTEGUMENTARY SYSTEM
 

•SKIN	 (25) (46) (19) (49) 

EPIDERMAL INCLUSION CYST 1 (2%) ABSCESS, NOS 1 (2X) 

NECROSIS, NOS 1 (4%) 

•SUBCUT	 TISSUE (25) (16) (19) (49)
 

GRANULATION, TISSUE 1 (2X)
 

SCAR 2
 

NECROSIS, NOS 1 (2%)
 

RESPIRATORY SYSTEM
 

*LARYNX (25) (46) (49) (49)
 

INFLAMMHTION ACUTE AND CHRONIC 1 (4i)
 

INFLAMMATION, CHRONIC 7 (28X)
 

#TRACHEA (11) (45) (46) (47)
 

INFLAMMATION, NOS 1 (9*) 9 (20%) 1 (2%)
 

INFLAMMATION, CHRONIC 10 (22%)
 

fLUNG/BRONCHUS (25) (46) (48) (49)
 

BRONCHISCTASIS 2 (8%)
 

INFLAMMATION, FOCAL 1 (4%)
 

ABSCESS, NOS 1 (2%)
 

INFLAMMATION, CHRONIC 8 (17%)
 

tBRONCHIAL MUCOUS GLA (25) (46) (48) (49)
 

ABSCF.SS, NOS 1 (2%)
 

NECROSIS, NOS 1 (2X)
 

HYPERPLASIA, AD5NCMATOUS 1 (2%)
 

ILUNG/BRONCHIOLE (25) (46) (48) (49)
 

_NOS
 

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

* NUMBE R O F ANIMAL S NECROPSIE D 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-3
 
TABLECl (CONTINUED) 

COINTR)OLE(UNTR) 

01-0081 

LOW DOSE CONTROL (UNTR)

01-0037 LOU COSE 01-0039

 HIGH DOSE 

 01-0090 

INFLAMMATION, FOCAL 1 (2%) 

*LUNG 

EMBRYONAL DUCT CYST 

ATEL5CTASIS 

CONGESTION, NOS 

EDEMA, NOS 

INFLAMMATION, NOS 

INFLAMMATION, FOCAL 

INFLAMMATION, INTERSTITIAL 

INFLAMMATION, SUPPURATIVE 

BRONCHOPNEUMONIA. ACUTE 

ABSCESS, NOS 

PNEUMONIA, CHRONIC MURINE 

INFLAMMATION, CHRONIC 

GRANULCMA, NOS 

9ERIVASCULITIS 

(25) 

2 (8%) 

1 (1*) 

1 (U*) 

11 (11%) 

1 (1%) 

(16) 

1 

1 

1 

1 

3 

1 

1 

1 

1 

5 

(2%) 

(2%) 

(2%) 

(2*) 

(7*) 

(2«) 

(2%) 

(2«) 

(2%) 

(11%) 

(18)

1 (2%) 

 (19) 

HEMATOtOIETIC SYSTEM 

tBON E MARRO W 

HYPERPLASIA , HEMATOPOIETIC 

(25) 

2 (8%) 

(UK) (18) (16) 

tSPLEEN 

THROMBOSIS, NOS 

FIBROSIS 

INFARCT, HEALED 

HEMOSIDEROSIS 

HETICULOCYTOSIS 

HYPERPLASIA, HEMATOPOIETIC 

HYPERPLASIA, EEYTHROID 

HYPERPLASIA, RETICULUM CELL 

HEMATOPOIESIS 

ERYTHROPOIFSIS 

(25) 

1

1

1

 (4X) 

 (US) 

 (!»*) 

(U6) 

1 (2*) 1 (2%) 

1 (2%) 

1 (2%) 

12 (26X) 

8 (17S) 

2

1

 (IX) 

 (2*) 

(17) 

ILYMPH NODE 

INFLAMMATION, NOS 

HYPERPLASIA, NOS 

PLASMACYTOSIS 

HYPERPLASIA, RETICULUM CELL 

(2U) 

1 (U«) 

(38) 

1 (3*) 

1 (3*) 

3 (8%) 

(39) (13) 

tMEDIASTINAL L.NODE 

PLASMACYTOSIS 

(2t) (38) 

1 (3*) 

(39) (13) 

tRENAL LYMPH NODE (21) (38) (39) (13) 

» NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

C-4 
TABLE Cl (CONTINUED) 

HIGH DOSE LOW DOSE
 CONTROL (UNTE) CONTROL (UNTR)
 

01-0084 01-0037
 

CIRCULATORY SYSTEM
 

*LYMPHATIC VESSELS (25) (46)
 

INFLAMMATION, NOS 1 (2%)
 

•HEART	 (25) (46)
 

PERIARTEEITIS 1 («)
 

»HEART/VFNTHICLE (25) (46)
 

FIBROSIS
 

(MYOCARDIUM (25) (46)
 

INFLAMMATION, NOS 1 (2%)
 

INFLAMMATION, INTERSTITIAL 22 (48X)
 INFLAMMATION, CHRONIC FOCAL 3 (1%)
 FIBROSIS 7 (15X)
 FIBROSIS, FOCAL 1 (4%)
 DEGENERATION, NOS 10 (1(0%)
 

* AORTA (25) (46)
 INFLAMMATION, CHRONIC FOCAL 1 (2)5)
 CALCIFICATION, FOCAL 1 (1!!)
 

*PULMONARY ARTERY (25) (46)
 HYFEBTEOPHY, NOS 1 (2%)
 

DIGESTIVE SYSTEM
 

#SALIVARY SLAND (24) (38)
 CYST, NOS
 

ILIVER (25) (46)
 CONGESTION, CHRONIC PASSIVE 1 (14%)
 INFLAMMATION, CHBONIC
 

CHOLANGIOFIBROSIS 1 (4X)
 NECROSIS, FOCAL 1 (4*) 3 <7X)
 

NECROSIS, COAGULATIVE 1 (2*)
 METAMORPHOSIS FATTY it (16X) 1 (2*)
 HYPEEPLASIA, FOCAL 23 (50*)
 

#LIVER/EERIPORTAL (25) (46)
 

FIBROSIS 1 (2%)
 

tLIVER/HEPATOCYTES (25) (46)
 

DJGSflERATtQN, ,5Q5 _„ ,t _^
 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS N2CROPSIED
 

C-5
 

LOU DOSE
 

01-0039
 

(49)
 

(48)
 

(48)
 

1 (2%)
 

(48)
 

1 <2X)
 

(49)
 

(49)
 

(48)
 

1 (2%)
 

(48)
 

1 (2X)
 

1 (2*)
 

(48)
 

(48)
 

-LJ2SL_
 

—. —
 

HIGH DOSE
 

01-0090
 

(19)
 

(48)
 

(48)
 

(48)
 

1 (2%)
 

(49)
 

(49)
 

(48)
 

(49)
 

1 (2%)
 

1 <2X)
 

(49)
 

(49)
 

1 _(2X)
 
TABLE Cl (CONTINUED)
 

HIGH DOSE CONTROL (UNTE) 

01-0084 

LOW DOSE CONTROL (UNTR) 

01-0037 

HYPERPLASIA, FOCAL 

*BILE DUCT 

INFLARMATION, NOS 

INFLAMMATION, CHRONIC DIFFUSE 

HYPERPLASIA, SOS 

HYPERPLASIA, FOCAL 

(25) 

6 (24X) 

(46) 

6 (13X) 

32 (70X) 

1 (2*> 

tPANCREAS 

INFLAMMATION, NOS 

HYPERPLASIA, INTRADUCTAL 

(25) 

1 (IX) 

(42) 

10 (2<*X) 

1 (2X) 

iPANCREATIC DUCT 

HYPERPLASIA, NOS 

(25) 

1 (4X) 

(42) 

tPANCREATIC ACINUS 

INFLAMMATION, NOS 

DEGENERATION, NOS 

ATROPHY, NOS 

(25) (42) 

4 (10*) 

*STO«ACH 

EPIDERMAL INCLUSION

ULCER, NOS 

HYPERPLASIA, NOS 

HYPERKERATOSIS 

ACANTHOSIS 

CYST 

(24) 

1 (4X) 

(45) 

1 (2X) 

2 <4X) 

6 (13X) 

1 (2X) 

1 (2X) 

IGASTRIC NUCOSA 

ULCER, FOCAL 

(24) (15) 

*PEYERS PATCH 

HYPERPLASIA, NOS 

(24) 

2 (8X) 

(43) 

7 (16*) 

*COLON 

NEMATODIASIS 

(24) (43) 

3 (7X) 

UKINMfY SYSTEM
 

*KIDNEY (24) (46)
 

GLOMERULONEPHRITIS, NOS 5 (21*) 33 (72X)
 

INFLAMMATION, INTERSTITIAL 1 (2*)
 

NEPHROPATHY 1 (4*)
 

NEPHROSIS, NOS 16 (67*)
 

tKIDNEY/CORTEX (24, (46)
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-6
 

.. . ...,, 

LOU DOSE 

01-0039 

1 <2X) 

(49) 

1 (2*) 

6 (12*) 

— ——­-— HIGH DOSE 

01-0090 

1 (2X) 

(49) 

3 (6*) 

(48) (48) 

(48) (48) 

(48) 

1 (2X) 

1 (2*) 

(47) 

(48) 

1 (2X) 

(49) 

(17) 

(48) 

(49) 

1 (2X) 

(48) 

(47) (48) 

(48) 

1 (2X) 

42 (88X) 

(48) 

(49) 

46 (94X) 

(49) 
TABLE Cl (CONTINUED)
 

HIGH DOSE LOW DOSE CONTROL (UNTR) CONTROL (UNTR) 

01-0084 01-0037 

HEMORRHAGE 

METAMORPHOSIS FATTY 

#KIDNEY/TUBULE (24) (46) 

NECROSIS, NOS 

tURINARY BLADDER (23) (42) 

CALCULUS, NOS 3 (13X) INFLAMMATION, NOS 1 <2X) HYPFRPLASIA, EPITHELIAL 3 (7X) 

ENDOCRINE SYSTEM
 

•PITUITAKY	 (21) (41)
 

HYPEKPLASIA, NOS 3 (7X)
 HYPERPLASIA, CHROMOPHOBE-CELL 2 <5X)
 

tPITUITAM/BASOPHIL (21) (41)
 NODULE 1 (5*)
 

tADRBNAL (25) (43)
 LIPOIDOSIS
 

tADHENAL CORTEX (25) (43)
 HYFE6TROPHY, FOCAL 1 (4*) 1 (2X)
 HYPEBPLASIA, NOS 1 (2X)
 

*ADRENAL MEDULLA (25) (43)
 NECROSIS, NOS 1 (236)
 CALCIFICATION, NOS 1 (2X)
 HYPEKPLASIA, NODULAR 1 (2X)
 

HYPERPLASIA, NOS 6 (14X)
 

tTHYROIC (23) (45)
 HYPERPLASIA, ADENOMATOUS 1 <2X)
 HYPERPLASIA, C-CELL 1 (2*)
 

tPANCREATIC ISLETS (25) (42)
 

HYPERPLASIA, NOS 2 <5X)
 

REPRODUCTIVE SYSTEM
 

*MAMMAR Y 3LAND (25) (46)
 _ _H

I££RPiASIiA_NOS_ 1.1US.L 5 111SJ.
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECPOPSISD
 

C-7
 

LOU DOSE 

01-0039 

(48) 

1 (2X) 

(48) 

1 (2X) 

• 

HIGH DOSE 

01-0090 

1 (2X) 

1 (2X) 

(49) 

(45) 

2 (4X) 

(45) 

(45) 

(48) 

(48) 

(48) 

(40) 

(40) 

(48) 

1 (2X) 

(48) 

1 (2X> 

(48) 

(46) 

(48) 

(47) 

1

(48) 

 <2X) 

(49) 

Jj.lZSI , •

(49) 

 in ­ . n ­ ­
TABLE Cl (CONTINUED) 

,
 HIGH DOSE LOW DOSE
 CONTROL (UNTR) CONTROL (UNTR)
 

01-0081 01-0037
 

LACTATION 7 (28*)
 

*PRHPOTIAL GLAND (25) (U6)
 

DILATATION, DOS
 

ABSCESS, NOS 1 (2*)
 

HYPERPLASIA, NOS 1 (2*)
 

#PROSTATE (23) (45)
 

INFLAMMATION, NOS 1 (4X) 21 (47*)
 

INFLAMMATION, FOCAL 3 (7*)
 INFLAMMATION, SUPPURATI7S
 

INFLAMMATION, ACUTE
 

ATROPHY, NOS 4 (17X)
 

HYPERPLASIA, FOCAL 5 (11X)
 HYPERPLASIA, PAPILLARY 2 (4X)
 METAPLASIA, SQUAMOUS 5 (11*)
 

*SEMINAL VESICLE (25) (i»6)
 ATROPHY, NOS 1 (4X)
 

tTESTIS (24) (45)
 

CALCIFICATION, FOCAL 4 (17%)
 

ATROFHY, NOS 12 (SOX) 2 (<•*)
 ASPERMATOGENESIS 1 (2*)
 HYPERPLASIA, INTERSTITIAL CELL 2 (8*) 19 (42S)
 

tTESTIS/TUBULE (24) (U5)
 DFGENEHATION, NOS 6 (13*)
 

NERVOUS SYSTEM
 

tBRAIN (25) (U4)
 HEMORRHAGE 2 (8*)
 CALCIFICATION, FOCAL 1 («*)
 

SPECIAL SENSE ORGANS
 

*EYE (25) (16)
 

INFLAMMATION, NOS
 

*EYE/CORNEA (25) (16)
 

__ ai£Ei«. HSS _
 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C_0

o
 

—————«—
 LOM DOSE
 

01-0039
 

(19)
 

1 <2X)
 

1 (2»)
 

(47)
 

1 (2X)
 1 (2X)
 

(49)
 

(48)
 

(48)
 

(48)
 

(49)
 

1 (2X)
 

(49)
 

, 1 1?!H­

—HIG —
 H DOSE
 

01-0090
 

(49)
 

(46)
 

(49)
 

2 (4*)
 

(«8)
 

1 (2X)
 

(48)
 

1 (2X)
 

(48)
 

(49)
 

(49)
 
TABLE Cl (CONCLUDED) 

HIGH DOSE LOW DOSE CONTROL (ONTF) CONTROL (UNTE)


01-OOSit 01-0037


MUSCULOSKELETAL SYSTEM
 

*BONF (25) (46)


OSTEOSCLEROSIS


+ SK3LETAL MUSCLE (25) (46)


CALCIFICATION, FOCAL 1 (4)5)
 

*CARTILAGE,NOS (25) (46)


CYST, NOS 1 (2X)
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL COHFHOLOGY SUMMARY
 

NO LESION REPOETED


AUTO/NECEOPSY/HISTO PERF 1
 

AUTC/NECROPSY/NO HISTO


AOTOIYSIS/NO NECROPSY 4


* NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-9
 

 LOW DOSE HIGH DOSE 

 01-0039 01-0090 

 (49) (49) 

1 (2)1) 

 (49) (49) 

 (49) (49) 

1 

1 

1 1 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE RATS TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 

ANIMAI S INITIALLY IN STUDY 

ANIMAL S NKCROPSIE D 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY* * 

INTEGUMENTAR Y SYSTE M 

NON " 

RESPIRATOR Y SYSTE M 

*NASA L TURBINAT E 

INFLAMMATION , SUPPURATIV E 

*LAHYN X 

INFLAMMATION ACUTE AND CHRONIC
 

INFLAMMATION, CHRONIC
 

(TRACHEA
 

HENOERHUGE
 

INFLAMMATION, NOS
 

INFLAMMATION, CHRONIC
 

POLYP, INFLAMMATORY
 

#LUNG/BPONCHOS
 

BRONCHIECTASIS
 

INFLAMMATION, NOS
 

INFLAMMATION, CHRONIC
 

*LUNG/BRONCHIOLE
 

INFLAMMATION, NOS
 

tLUNG
 

CYST, NOS
 

INFLAMMATION, NOS
 

INFLAMMATION, FOCAL
 

INFLAMMATION, INTERSTITIAL
 

PNEUMONIA, CHRONIC MURINE
 

SfiAJLU L.2S&.«._ EQ REIGN _SODY
 

# NUMBE R O F ANIMAL S WIT H TISSUE

* NUMBE R O F ANIMAL S NECROPSIE D 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

HIGH DOSE CONTROL (ONTR)

02-008U 

25 

23 

23 

(23 ) 

(23 ) 

1 (U*) 

3 (13X) 

(5) 

(23) 

(23) 

(23) 

3 (13* ) 

8 (35% ) 

 LOW DOSE CONTROL (UNTR)

02-0037 

50 

«9 

U9 

9 (19X) 

10 (2H) 

1 (2«) 

(U9 ) 

1 (2*) 

1 (2X) 

9 (18X) 

1 (2X) 

(19) 

1 (2%) 

7 

2 

 EXAMINE D MICROSCOPICALLY 

C-IO 

 LOW DOSE HIGH DOSE 

02-0039 02-0090 

50 50

 

50 50 

50 50 

(50) (50) 

1 (2*) 

(50 ) (50) 

(19) (19)

 

1 (2*) 

1 (2X) 

(50 ) (50)

 

1 (2X) 

(50) (50) 

(50) (50)

 

1 (2X) 
TABLE C2 (CONTINUED) 

COIITHOMBNTB) 

LOW DOSE CONTROL (UNTE) 

02-0084 02-0037 

PEfaVASCULITIS 6 (12X) 

CALCIFICATION, FOCAL 1 (It*) 

HYPERPLASIA , EPITHELIAL 1 (US) 

HEMATOPOIETIC SYSTEM
 

*BONE MARROW (22) 

HYPEFPLASIA, HEMATOPOIETIC 1 (5*) 

tSPLIEN (23) 

HSMATOMA, NOS 1 (It*) 

HEMOSIDEROSIS 2 (9*) 

HYPERPLASIA, NOS 1 (2*) 

HYPERPLASIA, HEMATOEOIETIC 3 (13X) 3 (6S) 

HYPEHPLASIA, EEYTHROID « (17*) 17 <35X) 

HYPERPLASIA, PLASMA CELL 1 (2X) 

HYPERPLASIA, RFTICULUM CELL 11 (22X) 

HEMAIOPOIESIS 3 (13X) ERYTHEOPOIESIS 

*LYMPH NODE (21)
 INFLAMMATION, NOS 3 (7%)
 HYPERPLASIA, NOS
 

PLASMACYTOSIS 3 (7XJ
 HYPERPLASIA, PLASMA CELL 1 (2X)
 

tMFDIASTINAL L.NODE
 (21)
 HYPERPLASIA, PLASMA CELL
 

CIRCULATORY SYSTEM
 

((MYOCARDIUM (23) (49)
 INFLAMMATION, NOS 1 (2X)
 INFLAMMATION, INTERSTITIAL 21 (19X)
 INFLAMMATION, CHRONIC
 

FIBEOSIS 5 <10X)
 DEGENERATION, NOS 4 (17«)
 

*PORTAL VSIN (23) (19)
 THROMBUS, MURAL 1 (2X)
 

DIGESTIV E SYSTE M 

• LIVER (23) (19) 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-l
 

L OW DOSE HIGH DOSE 

02-0039 02-0090 

2 

(50) (19) 

1 (2X) 

3 (6X) 1 (2*) 

(17) (19) 

(17) (19) 

1 (2*) 

(50)	 (50) 

1 <2X)
 

1 (2X)
 

1 (2X)
 

(50) (50) 

(50) (19)
 
TABLE C2 (CONTINUED)
 

HIGH DOSE LOW DOSE CONTROL (UNTR) CONTROL (UNTR) 

02-0081 02-0037 

INFLAMMATION, CHRONIC 

INFLAMMATION, FOCAL GRANULOMATOU 

FIBROSIS 1 (2*) 

CHOLANGIOFIBROSIS 

PEHIVASCULITIS 

1 (1*) 

1 (2*) 

NECROSIS, SOS 

NECROSIS, FOCAL 1 (8X) 

NECROSIS, COAGULATIVE 2 (IX) 

METAMORPHOSIS FATTY 2 (9X) 1 (2X) 

BASOPHILIC CYTO CHANGE I* (17*) 

HYPEHPLASIA, NODULAR 1 (2*) HYPERPLASIA, FOCAL 3 (13%) 22 (15X) ANGIECTASIS 1 (2X) 

HEMATOPOIESIS 

•LIVER/CSNTPILOBULAR (23) (19) NECROSIS, DIFFUSE 

•LIVER/PERIPORTAL (23) (19) 

METAMORPHOSIS FATTY 

•BILE DUCT (23) (1*9) 

INFLAMMATION, NOS 5 (10%) 

INFLAMMATION, CHRONIC 

HYPERPLASIA, NOS 2 (9*) 27 (55X) 

•PANCREAS (22) (16) 

INFLAMMATION, NOS 7 (15X) INFLAMMATION, INTERSTITIAL 

PERIARTERITIS 

•PANCREATIC DUCT (22) (16) 

HYPERPLASIA, NOS 1 (2X) 

• PANCREATIC ACINUS. (22) (16) 

ATROEHY, NOS 2 (1%) 

•STOMACH (23) (18) 

INFLAMMATION, NOS 

INFLAMMATION, FOCAL 

2 (11) 

2 (IX) 

PERIARTERITIS 

HYPERPLASIA, EPITHELIAL 1 (2X) 

•GASTRIC MUCOSA (23) (18) 

H.XEJ.EEllSIi.i HO.S_ 1-12JX 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-12
 

._-— .— I.——­

LOU DOSE
 

02-0039
 

1 (2X)
 

1 (2X)
 

1 (2%)
 

1 (2*)
 

2 (IX)
 

1 (2*)
 

1 (2X)
 

1 (2X)
 

1 (2X)
 

(50)
 

1 (2X)
 

(50)
 

(50)
 

2 (IX)
 

(50)
 

1 (2X)
 

(50)
 

(50)
 

(50)
 

1 (2X)
 

(50)
 

,, ., ,_ .. ,_., ,r
 

HIGH DOSE
 

02-0090
 

1 (2*)
 

1 (2X)
 

1 (2X)
 

(19)
 

(19)
 

1 (2%)
 

(50)
 

1 (2X)
 

1 (2X)
 

(19)
 

1 (2*)
 

(19)
 

(19)
 

(19)
 

(19)
 
TABLE C2 (CONTINUED) 

— ____ 

HIGH DOSE CONTROL (UNTH) 

02-0084 

LOW DOSE CONTROL (UNTB) 

02-0037 

LOW DOSE 

32-0039 

HIGH DOSE 

02-0090 

•PEYPRS PATCH 

HYFEBPLASIA, NOS 

(23) 

4 (17*) <«7> 

6 (13*) 

(50) (49) 

•COLON 

NEflATODIASIS 

PARASITISM 

(22) 

2 (9J) 

(43) 

3 (7*) 

(48) (48) 

URINAR Y SYSTEM 

•KIDNEY 

HYDRONEPHROSIS 

GLONERULONEPHRITIS, NOS 

INFLAMMATION, INTERSTITIAL 

GLOMERULONEPHRITIS, MEMBRANOUS 

PYELONEPHRITIS, ACUTE 

INFLAMMATION, CHRONIC 

PYELONEPHRITIS, CHRONIC 

PERIARTERITIS 

NEPHROSIS, NOS 

GLOMERULOSCLEROSIS, NOS 

METAMORPHOSIS FATTY 

CALCIFICATION, FOCAL 

(23) 

4 (17X) 

1 (4X) 

1 (4X) 

10 (43*) 

1 (4*) 

(49) 

1 (2%) 

33 (67X) 

1 (2X) 

1 (2X) 

1 (2X) 

(50) 

2 (4*) 

1 (2X) 

32 (64*) 

1 (2*) 

1 (2*) 

(49) 

22 (45*) 

•KIDNEY/CORTEX 

CYST, NOS 

(23) (49) (50) 

1 (2X) 

(49) 

•KIDNEY/TUBULE 

NECROSIS, NOS 

PIGMENTATION, NOS 

(23) 

1 (4*) 

(49) (50) 

1 (2X) 

(49) 

•URINARY BLADDER 

INFLAMMATION, NOS 

HYPERPLASIA, EPITHELIAL 

(22) (41) 

1 (2X) 

(47) 

1 <2X) 

(50) 

ENDOCRINE SYSTEM 

fPITUITAFY 

HEMOERHAGIC CYST 

HYPERPLASIA, NOS 

HYPERPLASIA, FOCAL 

HYPERPLASIA, CHROMOPHOBE-CELL 

(21) 

1 (5*) 

1 (5*) 

(43) 

2 (5*) 

1 (2X) 

(48) (43) 

•ADRENAL (23) (46) (50) (48) 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

C-I3 
TABLE C2 (CONTINUED) 

. ­.—__«._ 

HIGH DOSE LOW DOSE CONTROL (UNTR) CONTROL (UNTR) 

02-008U 02-0037 

HYPERTROPHY, NOS 

IADRESAL CORTEX (23) (16) 

NODUIE 1 (2*) 

HYPERPLASIA, NOS 7 (15X) 

tADRENAt. MEDULLA (23) (16) 

HYPEKPLASIA, NOS U (9X) 

•THYROID (21) (17) 

HYPEKPLASIA, C-CELL 3 (11X) 

HYPERPLASIA, FOLLICULAR-CELL 1 (21) 

tPANCREATIC ISLETS (22) (16) 

HYPERPLASIA, NOS 1 (2X> 

REPRODUCTIVE SYSTEM
 

• MAMMAR Y QLAND (23) (19)
 

GALACTOCELE 1 (UX) 5 (10X)
 

CYSTIC DUCTS
 

HYPERPLASIA, NOS 1 (1*) 17 (35*)
 

HYPERPLASIA, FOCAL
 

HYPEHPLASIA, PAPILLARY 1 <2X)
 

HYPERPLASIA, CYSTIC
 

FIBROCYSTIC DISEASE
 

LACTATION 9 (39X)
 

*VAGINA (23) (19)
 

HYPERTROPHY, NOS
 

# UTERUS (23) (18)
 

DILATATION, NOS
 

HYDROMETRA 3 (6X)
 

HEMATOMA, NOS
 

INFLAMMATION, SUPPURATIVE 1 <2X)
 

PYOMETRA 3 (13%)
 

ABSCESS, NOS 2 (IX)
 

HYPERPLASIA, ADENOMATOUS 5 (10X)
 

tCERVI X UTERI (23) (18) 

INFLAMMATION , SHPPURATIV E 

tUTERUS/ESDOKETRIU M (23) (18) 

I NUMBER OF ANIMALS WITH 1I3SUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-14
 

LOW DOSE
 

02-0039
 

1 (2X)
 

(50)
 

(50)
 

(18)
 

(50)
 

(50)
 

1 (2X)
 

1 (2X)
 

(50)
 

1 (2X)
 

(19)
 

1 (2X)
 

1 (2X)
 

2 (IX)
 

(19)
 

(19)
 

HIGH DOSE
 

02-0090
 

(18)
 

(18)
 

(19)
 

(19)
 

(50)
 

1 (2X)
 

1 (2X)
 

(50)
 

(15)
 

2 (UX)
 

(15)
 

1 (2X)
 

(15) '
 
TABLE C2 (CONTINUED) 

HIGH DOSE LOW DOSE 

CONTROL (UNTR) COHTBOL (UNI8) LOW DOSE HIGH DOSE 

02-0084 02-0037 02-0039 02-0090 

INFLAMMATION. FOCAL 

INFLAMMATION, SOPPURATIVE 

INFLAMMATION, CHROMIC 

HYPERPLASIA, MOS 

HYPERPLASIA, CYSTIC HYPERPLASIA, ADENOHATOUS 

«0?ARY/OVIDUCT 

INFLAMMATION, NOS 

INFLAMMATION, ACUTE ABSCESS, NOS 

• OVARY CYST, NOS

ABSCESS, NOS . INFLABHATION, FOCAL GRANULOKATOO FIBEOSIS

HYPSRPLASIA, INTERSTITIAL CELL

1 (4*) 

1 <««> 

1 («) 

(23) 

1 

1 

(4X) 

(4X) 

 (22)

 3 (1Ut)

1 (2*) 

2 (4X) 

(2X) 

(»*) 

(2X) 

(*8) 

1 (2%) 

 (17) « (9%)

 1 (2%) 

1 (2X) 

8 (16«) 

1 (2*) U (8X) 

(H9) 

 (50)

 6 (12*) 

1 (2%) 

2 (1*1 

7 (16X) 

6 (13%) 

 («6) 

NERVOUS SYSTEM 

• BRAIN

HYEROC3PHALOS. NOS HEHORRHAGF

CALCIFICATION, FOCAL

 (23)

 1 (4X) 1 (4S) 

1 (4X) 

 (49) (50) (48) 

SPECIAL StNSE ORGANS 

NONE 

RUSCOLOSKELETAL SYSTEM 

NON3 

BODY CAVITIES 

*(IEDIASTIHD«I

PERIARTEHITIS

 (23) (49) (SO)

 1 (2X) 

 (SO) 

•MESENTERY PERIARTERITIS (23) (49) (SO) 1 (2X)  (SO) 

ALL OTHER SYSTEMS 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

C-I5 
TABLE C2 (CONCLUDED) 

.(UNTR) COBTBOC (OKTH) LOK DOSE HIGH DOS2 

02-0081 02-0037 02-0039 02-0090 

'SPECIAL MORPHOLOGY SUMMAR Y 

HO LESIO* BEPOP.TED 

AUTOLYSIS/NO NECBOPSY 

t NOHBEB OF ANIMAL S RITH TISSUE EIANINBD HICROSCOPICALLT 

* MOHBEB OF ANIMALS HECROPSIED 

C-16 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 2,5-TOLUENEDIAMINE SULFATE
 

TABLE Dl 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 

. (UNTR) 

05-0077 

(DNTR) 

05-0037 LOU DOSE 

05-0039 

HIGH DOSE 

05-0089 

IHALS INITIALLY IN STUDY

IHALS NECROPSIED

IHALS EXAMINE D HISTOPATHOLOGICALLY**

 50 

 46 

 45 

50 

48 

48 

50 

49 

48 

50 

49 

49 

TEGUMESTAR Y SYSTEM 

SKIN

EPIDERMAL INCLUSION CYST ULCER, FOCAL 

INFLAMMATION, ACUTE/CHRONIC INFLAMMATION, CHRONIC FOCAL 

?IBHOSIS 

ALOPECIA 

HYPSRKSRATOSIS 

ACANTHOSIS 

 (46) (48)

1 <2X) 

1 (2X) 

 (49) (4< » (2X) 

<2X) 

(2X) 

<2X) 

(2X) 

(2X) 

SBBCUT TISSUE NECROSIS, NOS  (46) (48)

1 (2X) 

 {49) (4< » 

SPIBATORY SYSTEM 

LOKG/BROICHUS INFLAMMATION, NOS 

INFLAMMATION, FOCAL 

(45) 

1 (2X) 

1 (2X) 

(47) (49) 

LUNG 

BF.ONCHOPNEDMONIA, NOS 

INFLAMMATION, ROS 

I NFL AH NATION, INTERSTITIAL 

INFLAMMATION, ACUTE FOCAL 

ARTERIOSCLEROSIS, IOS 

HYPERPLASIA, EPITHELIAL 

(45) 

1 (2f) 

(48) 

1 <2X) 14 (29X) 

(47) 

(2X) 

1 (2X) 

(49) 

1 (2X) 

LUNG/ALV30LI 

HENOBBHAGE INFLAMMATION, FOCAL 

(45) 

2 (4X) 

1 J2X1 

(47) (49) 

1 (2X) 

NUMBER OF ANIHAL S KITH TISSUE EXAMINED

NUMBER OF ANIHALS NECROPSIED 

aCLUDES PARTIALLY AUTOLTZED ANIMALS 

 MICROSCOPICALLY 

D-3 
TABLE Dl (CONTINUED)
 

HIGH ! 

CONTROL(UNTH) CONTROL (UHTR) 

05-0077 05-0037 

HEMATOPOIETIC SYSTEM
 

tSPLEEN
 

INFLAMMATION, HOS
 

FIBROSIS
 

HYPERPLASIA, NOS 2 (»*)
 

HYPERPLASIA, HEMATOPOIETIC 2 («*)
 

HYPERPLASIA, EHYTHHOID 2 («X)
 

HYPERPLASIA. RETICOLUH CELL 3 <7X)
 

HYPERPLASIA, LYMPHOID
 

HSMATOPOIESIS 1 (2X)
 

ERYTHROPOIESIS
 

•LYMPH	 NODE (35)
 

HENORRHAGIC CYST
 

INFLAMMATION, DOS 13 (30t)
 

DEGENERATION, CYSTIC
 

HYPERPLASIA, NOS 2 (5*)
 

HYPERPLASIA. HEMATOPOIETIC 1 (2*)
 

HYPERPLASIA, LYHPHOID 2 (5*)
 

HYELCID METAPLASIA
 

tSDBHANDIBOLAR L.NODE (35)
 

RYPERPLASIA. PLASMA CELL
 

tMEDIASTINAL L.NODE (35)
 

NECROSIS. NOS 1 (2X)
 

•PANCREATIC	 L.NODE (35) !•)
 

INFLAMMATION. NOS 1 (2U)
 

RYPERPLASIA. NOS
 

*NESENTERIC L. NODE (35)
 

CONGESTION, NOS
 

HEMORRHAGE 1 (2*)
 

INFLAMMATION, NOS 9 (20X)
 

HYPERPLASIA, NOS
 

ITHIHOS (19) »«)
 NECROSIS. NOS 1 (3*)
 

CIRCULATORY SYSTEM
 

fHEART/VENTRICLE (48)
 

2
 

* NUMBER OF AIIHALS HITH TISSOE EXAMINED MICROSCOPICALLY
 

* BOMBER OF ANIMALS NECHOPSIED
 

LOU DOSE HIGH DOSE 

05-0039 05-0089 

(U8)
 

2 (5X)
 

(36)
 

(36)
 

2 (6X)
 

(36)
 

(36)
 

(36)
 

5 (11X) 2 (6X)
 

(32) (33)
 

B-4 
TABLE Dl (CONTINUED) 

HIGH DOSE LOW DOSE CONTROL (UNTR) CONTROL (UHTR) LOU DOSE 

05-0077 05-0037 05-0039 

•MYOCARDIUM	 (11) (18) (17)
 INFLAMMATION. INTERSTITIAL 2 (IX)
 

FI3ROSIS 5 (10X)
 

*BLOOD VESSEL (16) (18) (19)
 INFLAMMATION, NOS 2 (IX)
 

*POLMONARY ARTERY (16) (18) (19)
 

MINERALIZATION 2 (IX)
 

DIGESTIVE SYSTEM
 

•SALIVARY GLAND (13) (17) (17)
 INFLAMMATION, NOS 2 (IX)
 PERIVASCULAR CUFFING 1 (2X)
 

•LIVER	 (15) (18) (18)
 HEMATOHA, NOS 1 (2X)
 INFLAMMATION, FOCAL 2 (IX)
 DEGENERATION, NOS 1 (2X)
 NECBCSIS, FOCAL 13 (27X) 1 (2X)
 METAMORPHOSIS FATTY 3 (7X) 3 (6X)
 HYPEBPLASIA, NODULAS 2 {««)
 HYPEBPLASTIC NODDLE
 

HYPEBPLASIA, FOCAL 1 (2X)
 ANGIECTASIS 1 (2*)
 HENATOPOIESIS
 

MYELOID METAPLASIA 1 <2X)
 

•LIVSR/fERI PORTAL (15) (18) (18)
 INFLAMMATION, NOS 1 (2X)
 

•LIVER/HEPATOCYTBS	 (15) (18) (18)
 DEGENERATION, NOS 1 (2X)
 

•GALLBLADDER	 (16) (18) (19)
 INFLAMMATION, FOCAL 1 (2X)
 

»BILE DUCT (16) (18) (19)
 INFLAMMATION, NOS 1 (2X)
 

•PANCREAS	 (11) (18) (15)
 

IqFLAqgATIQNt ,NO§. -- ,- -,,„- TM ,? JJSXl^ ,.,
 

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

HIGH DOSE
 05-0089
 

(19)
 

(19)
 

(19)
 

(15)
 

(19)
 

1 (2X)
 

1 (2X)
 

(19)
 

(19)
 

(19)
 

(19)
 

(13)
 

D-5
 
TABLE Dl (CONTINUED)
 

IHUgfioitR) 8WNTTo!(UNTR, LOH EOSE HIGH DOSE
 

05-0077 05-0037 05-0039 05-0089
 

INFLAMMATION, FOCAL 1 (2X)
 DEGENERATION, CYSTIC 1 (2X)
 METAMORPHOSIS FATTY 1 (2X)
 

•PANCREATIC	 DDCT (44) (48) (45) (43)
 

HYPEBPLASIA, IDS 1 (2X)
 

•PANCREATIC ACINOS (44) (48) (45) (43)
 

HYPERTROPHY, FOCAL 1 (2X)
 HYPERPLASIA, FOCAL 1 (2X)
 

•STOMACH	 (42) (47) (46) (U8)
 INFLAMMATION, NOS 13 (28X)
 ULCER, NOS 1 (2X)
 INFLAMMATION, FOCAL 1 (2X)
 INFLAMMATION, INTERSTITIAL 1 (2X)
 PEBIARTERITIS 1 (2X)
 HYPERPLASIA, NOS 1 (2X)
 HYPERPLASIA, FOCAL 1 (2X) 1 (2X)
 HYPERKERATOSIS 3 (6X)
 ACAITHOSIS 3 (6X)
 

•GASTRIC HOCOSA (42) (47) («6) (48)
 

HYPEBPLASIA, FOCAL 1 (2X)
 

tPEYERS PATCH (43) (48) (47) (48)
 

HYPERPLASIA, NOS 2 (4X)
 

•ILEUS	 (43) (48) (47) (48)
 

HEMORRHAGE 1 (2X)
 IIFLAHHATIOI, NOS 2 («X)
 

ICOLOI (38) (45) (43) (46)
 

PARASITISH 1 (2X)
 

OSINARY SYSTEM
 

•KIDNEY	 (45) (47) (48) (48)
 CALCDLDS, BOS 20 (44X)
 GLOMERDLOIEPHHITIS, BOS 6 (13X)
 IIFLAHHATIOI, BOS 1 (2%)
 INFLABHATIOI, INTERSTITIAL 5 (11X) 23 (49X)
 INFLAHHATIOB, CHRONIC 1 (2X) 1 (2X)
 

PEIIfASCOLITIS ...^. . .2^ JSSl.--...__— _..._._.-,
 

« KOHBBI OF AIIMALS WITH TISSUE EXAHIIED MICROSCOPICALLY
 

* WURBCR OF AIIHALS IECROPSIED
 

D-6
 
TABLE D-1 (CONTINUED)
 

HIGH DOSE LOW DOSE 

CONTROL (UNTR) CONTROL (UNTR) LOU DOS! BI9HMSI 

05-0077 05-0037 05-0039 «•*»*» 

ARTERIOSCLEROSIS, NOS 1 (2%) 

NEPHROSIS, NOS 1 (2X) 

GLOHERULOSCLSROSIS, NOS 1 (1SJ 

HYPERPLASIA, TUBULAR CELL 2 (««) 

•KIDNSY/T3BULF (45) (U7) (48) (••)

 

DEGENERATION , NOS 1 (2X)

 

NSCROSIS, POCAL 1 (2X)

 METAMORPHOSIS FATTY 9 (20X)

 

tlJHINAR Y BLADDER (44 ) (48) (47) <»TJ

 

INFLAM1ATION, NOS It (8X)

 

HYPERPLASIA , 3PITHELIAL 9 (19*) -*..'•' • •'•••-««, >* 

ENDOCRIN E SYSTEM 

• PITUITAR Y (36) (42) (41) (J0|

 

HYPERPLASIA , NO S 3 (7*)

 

HYPERPLASIA, FOCAL 3 (7«)

 

• ADRENA L CORTEX (43) (45) (45) |4«)

 

NODULE 1 (2*)

 

HYPERTROPHY , POCAL 1 (2*)

 

HYPSRPLASIA, NOS 1 (2*)

 

•ADRENAL 3EDULLA (43) (»5) <•*»
 DE3BNERATION, NOS 1 (2*)
 

•THYROID	 (40)
 

LYBPHOCYTIC INFLAMMATORY IHFILTR
 HYPSRPLASIA, PAPILLARY
 

HYPERPLASIA, POLLICOLAH-CELL 1 (2X)
 

•PANCREATIC ISLETS (48)
 HYPERPLASIA, NOS 2 («*)
 

REPRODUCTIVE SYSTEM
 

•PREPUTIAL GLAND <t6)
 

DILATATIOM/DUCTS («)
 INFLAMMATION, SUPPUHATIVE
 ABSCESS, NOS 2 («*)
 

•TESTIS/TOBOLE (45)	 (»«) !«*»
 9

*1
 

t NUMBER OP ANIMALS HITH TISSUE RXAHINED MICROSCOPICALLY
 

*. NUMBER OP ANIMALS NECROPSIED
 

D-7
 
TABLE Dl (CONCLUDED)
 

SE LOW DOSE -. (ONTR) CONTROL (UNTR) 05-0077 05-0037

CALCIFICATION, FOCAL

NERVOUS SYSTEM
 

ICEREBHAL CORTEX (45) (48)


MINERALIZATION 3 (6%)
 

SPECIAL SENSE ORGANS
 

HOHE
 

BOSCOLOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

•ABDOMINAL CAVITY (46) (48)


STEATITIS 1 (2«)
 

RECBOSIS, FAT


*HESEHTERY (46) (48)


PERIARTERITIS


AIL OTHER SYSTEMS
 

ADIPOSE TISSUE
 NECROSIS. HOS


SPECIAL RORPHOLOGY SOHHARY
 

RO LESIOK REPORTED 8


AUTO/mCROPST/NO HISTO 1
 AUTOLYSIS/NO NECROPSY 4 2
 

t BUHBEB OF AMIHALS HUH TISSUE EXAHINED HICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

 LOH DOSE HIGH DOSE
 05-0039 05-0089
 

1 (2X)
 

 (47) (44)
 

 (19) (19)
 

1 (2«)
 

 (49)	 («9)
 

2 (4X)
 

1
 

 20 17
 

1
 

1 1
 

0-8
 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH 2,5-TOLUENEDIAMINE SULFATE 

HIGH DOSE LOW DOSE
 CONTROL (UNTR) CONTROL (UNTR) LOi COSE HIGH DOSE
 

06-0077 06-0037 06-0039 06-0089
 

ANIMALS INITIALLY IN STUDY 50 50 50 50
 

ANIMALS MISSING 1
 

ANIMALS NECROPSIED 46 48 45 47
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY** 46 47 43 46
 

INTEGUMENTARY SYSTEM
 

*SKIN (46) (48) (45) (47)
 

FTBRCSIS 1 (2*)
 

FI3ROSIS, FOCAL 1 (2*)
 

»SUBCUT TISSUE (46) (48) (45) (47)
 

MINERALIZATION 1 (2X)
 FIBROSIS 1 (2X)
 

RESPIRATORY SYST3M
 

•LUNG/BRONCHUS	 (45) (46) (42) (46)
 INFLAMMATION, FOCAL 1 (2X)
 

• LUNG (45) (46) (42) (46)
 CONGESTION. NOS 1 (2X)
 INFLAMMATION, INTERSTITIAL 2 (4X) 10 (22«)
 PZHIAPTBRITIS 1 (2X)
 

HYPBRPLASIA, EPITHELIAL 3 (7X)
 

HEMATOPOIETIC SYSTEM
 

• BONE NAPROW (44) (45) (39) (41)
 MISLCFIBBOSIS 1 (2X)
 HYPIRPLASIA, HEMATOPOIETIC 1 <3X)
 

tSPLEEN (43) (46) (42) (42)
 PLASMA-CELL INFILTRATE 1 (2X)
 HYPIRPLASIA, HEMATOfOIETIC 16 (35X)
 

HYPERPLASIA. ERYTHBOID 6 (13X)
 HYPE8PLASIA, RrTICULUM CELL 2 (5X)
 

... 3T?EHP.l,A.§;A-. LXBEH2IC S.12SL lfl_U2Sl- -_ • 1 (?*'
 

t NUSBER OF ANIMAL S HITH TISSDE EXAMINED MICROSCOPICALLY 

* NUNSER OF ANIMAL S NECROPSIED 

•'EXCLUDES PARTIALLY AUTOLYZED ANIMALS 
TABLE D2 (CONTINUED)
 

 LOU DOSE
 06-0039
 

3 (7X)
 

1 (2X)
 

 (37)
 

 (42)
 

 (42)
 

 (45)
 

 (40)
 

 (42)
 

2 (5X)
 

 (42)
 

 (45)
 

• -————
 HIGH DOSE
 06-0089
 

(32)
 

(46)
 

(46)
 

(47)
 

(43)
 

1 <2X)
 

(46)
 

1 (2*)
 

(46)
 

(47)
 

HENATOPOI2SIS
 

SRYTHHOPOIESIS
 

MYELOPOIESIS
 

tLYHPH NODE
 

CYST. NOS
 

INFLAMMATION, «OS
 

HYPERPLASIA, NOS
 RETICULOCYTOSIS
 

HYPERPLASIA, HEHATOPOIETIC
 

MYELOID METAPLASIA
 

CIRCULATORY SYSTEM
 

*HEAHT/VENTRICLE
 MELANIN
 

(MYOCARDIUM
 CALCIFICATION, FOCAL
 

•PULMONARY ARTERY
 

HYPERPLASIA, NOS
 

DIGESTIVE SYSTEM
 

tSALIYARY GLAND
 INFLAMMATION. NOS
 

PEBIABTEBITIS
 PERIVASCOLAR COFFIN.G
 

» LIVER
 INFLAMMATION, ROS
 

IHFLAMMATIOV, FOCAL
 NECROSIS, FOCAL
 CYTOPLASHIC CHANGE, NOS
 

HYPERPLASTIC NODDLE
 HYPEBPLASIA, DIFFUSE
 ANGIECTASIS
 

HSHATOPOIF.SIS
 

tLIVER/PERIFORTAL
 INFLAMMATION, NOS
 

•GALLBLADDER
 

ISfLilBiUPIf NO,§IL _-,„.„,...
 

HIGH DOSE
 CONTROL (UNTR)


06-0077


1 (2X)


(41)


(45)


(45)


1 (2*)
 

(46)


1 <2X)
 

(43)


(45)


1 (2X)
 

1 <2X)
 

1 (2X)
 

(45)


1 (2X)
 

(46)


 LOW DOSE
 CONTROL (UNTR)


 06-0037


 1 (2X)


1 <2X)
 

(39)


1 (3X)
 

15 (38X)
 

1 (3X)
 

1 (3X)
 

2 (5X)
 

1 <3X)
 

(46)


4 (9X)
 

(46)


 (48)


 (45)


2 (4X)
 

4 (9X)
 

(47)


1 (2X)
 

22 (47X)
 

1 (2X)
 

1 (2X)
 

3 (6X)


 (47)


 (48)


2 IH1
 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NBCROPSIED
 

D-IO
 
___________ 

TABLE D-2 (CONTINUED) 

_________ 

HIGH DOSE LOW DOSE CONTROL (UNTR ) 

06-0077 

CONTROL (UNTR) 

06-0037 

BILE DUCT (16) (18) 

INFLAMMATION , NOS 1 (2<) 1 (2X) 

PANC3SAS (11) (11)

INFLAMMATION , NO S 5 (11X) 

INFLAMMATION , INTERSTITIAL 

PTRIAPT?RITIS 1 (2«) 

PANCREATIC PUCT (U1) 

LYMPHOCYTIC INFLAMMATORY INFILTR 1 (2*) 

STOMACH (12) (1 ()
 INFLAMMATION, NOS 1 (16X)
 ULCER, NOS 1 (?X)
 INFLAMMATION, FOCAL (2X)
 HYPERPLASIA, NOS <2X)
 HYP5HPLASIA, EPITHELIAL <2X)
 HYPSRPLASIA, APENOMATOUS (2«)
 HYPEHKEHATOSI S (2X)
 ACANTHOSIS (2X)
 

GASTRIC MOCOSA (12) (1 • )
 HYPEBPLASIA, FOCAL 1 (2X)
 

PAYERS PATCH (13) (1 »)


HYFEFPLASIA, NOS 1 <2X)
 

IHASY SYSTEM
 

KIDNEY
 

CONGESTION, NOS
 

GLOHIBOLOKSPHRITIS, NOS 11 (30X)
 INFLAMHATION, INTERSTITIAL 3 (7X) 16 (35X)
 GLOHEROLONEPHRITIS, CHRONIC
 

PEEIVASCOLITIS 1 (9X)
 

KIDNEY/GLOHERULUf »3) (16)
 AHYLCIDOSIS 1 (2X)
 

KIDNEY/PELVIS (16)
 INFLAMMATION, ACOTE/CHRONIC
 

URINARY BLADDEF (U1) (16) 

NUMBER OF ANIMAL S WITH TISSUE EXANIN.D MICROSCOPICALLY 

lOXBER OF AHIBALS NECROPSIED 

L OUCOS — E 

06-0039 

(15) 

(12) 

1 (2X) 

(12) 

 (^2)
 

 (U2)
 

 (12)
 

<2X) 

(13)
 

(13)
 

(31)
 

HIGH DOSE 

06-0089 

(17) 

(12) 

(12) 

(13)
 

(13)
 

(12)
 

1 <2») 

(15) 

D-ll 
TABLE D2 (CONTINUED)
 

HIGH DOSE
 06-0089
 

(41)
 

1 (2X)
 

(38)
 

(41)
 

(34)
 

1 <3X)
 

(47)
 

(42)
 

2 (SX)
 

1 (2X)
 

(42)
 

4 (10*)
 

28 (67X)
 

(42)
 

HYPEFPLASIA, EPITHELIAL
 

*0.3LAOEBR/SOBMOCOSA


INFLAMMATION, CHROMIC
 

ENDOCRINE SYSTEM
 

fPITUITARY
 HYPERPLASIA, FOCAL
 

iADRENAL CORTEX


MODULE
 

*THYROID
 FOLLICULAR CYST, NOS
 

INFLAMMATION, DOS
 INFLAMMATION, CHRONIC FOCAL
 

REPRODUCTIVE SYSTEM
 

*MAMHARY GLAND


GALACTOCELF
 HYPERPLASIA, NOS
 

tUTERDS
 HYERCMETRA


INFLAMMATION, ACUTE
 ABSCESS, NOS
 FIBRCSIS
 

tOTSRUS/ENDOMETRIOM
 DILATATION, MOS
 CYST, MOS
 INFLAMMATION, NOS
 INFLAMMATION, SUPPURATIVE
 INFLAMMATION, ACUTE
 INFLAMMATION, ACUTE SOPPURATIVE
 HYPERPLASIA, MOS


HYPERPLASIA, CYSTIC
 HYPERPLASIA, ADENOMATOOS
 

• OVARY/OVIDUCT


JNFLMM.ATION, SOS.,,..__.^ t


!WTioiE(UNTR,
 06-0077
 

 (41)
 

 (37)
 

 (43)
 

 (30)
 

 (46)
 

 (43)
 

4 (9X)
 

 (U3)
 

 -2 (SX)
 

1 (2X)
 

 35 (81X)
 

 (43)
 

.,.__ ._.„„„,
 

CONTROL (UNTR)
 

06-0037
 

10 (22X)
 

(46)
 

(42)
 

6 (14X)
 

(US)
 

3 <7X)
 

(43)
 

1 (2X)
 

1 (2X)
 

(48)
 

1 (2X)
 

<• <8X)
 

(45)
 

1 (2X)
 

3 <7X)
 

1 (2X)
 

(45)
 

10 (22X)
 

4 (9X)
 

4 (9X)
 

18 (40X)
 

1 (2X)
 

(45)
 

5 11 M
 

LOW DOSE
 06-0039
 

(34)
 

(38)
 

(38)
 

(36)
 

(45)
 

(41)
 

2 (5X)
 

1 (2X)
 

(41)
 

5 (12X)
 

1 (2X)
 

1 <2X)
 

4 (10X)
 

18 (44X)
 

(41)
 

•-•— M-*»Mi I I I
 

* NDHBSR OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

DH2
 
TABLE D2 (CONTINUED)
 

,(UNTR) 

06-0077 

.... -OSE CONTROL (UNTB) 

06-0037 

LOU COSE 

06-0039 HIGH DOSE 06-0089 

INFLAMMATION, SUPPURATIVE 2 <5X) 

tOVARY CYST. SOS INFLAMMATION, NOS 

LYMPHOCYTIC INFLAMMATORY INFILTB 

INFLAMMATION, SUPPUBATIVE INFLAMMATION, ACUTE 

INFLAMMATION, ACUTE SUPPURATIV5 ABSCESS, HOS 

INFLAMMATION, CHROMIC 

DEGENERATION, CYSTIC ATROPHY, NOS 

(41) 

1 <2X) 

(45) 

3 <7X) K (9X) 

1 (2X) 

10 (22*) 

» (9X) 

1 (2X) 

(36) 

2 (6X) 

1 (3%) 

1 (3X) 

u (11%) 

1 (3X) 

(39) 

4 (10X) 

6 (15%) 

(OVARY/FOLLICLE MULTIPLE CYSTS . (41) (45) (36) 1 (3X)  (39) 

iRVOUS SYSTEM 

NONE 

'ECIAL SENSE ORSANS 

NONE 

ISCOLOSKELETAL SYSTEM 

•BONE

RESORP7ION (46) (48) 3 (6%) 

 (45) (47) 

'VERTEBRA OSTEOSC15SOSIS (46) 1 (2X)  (43) (45) (47) 

)DY CAVITIES 

NONE 

,L OTHEH SYSTEMS 

OHBNTOM !(ECSCS^S. FAT 1 . 

NOHBER OF ANIHALS WITH TISSUE EXANIISO HICROSCOPICALLY 

0? ANIMALS XECROPSIED 

D-13
 
TABLE D-2 (CONCLUDED) 

:OL(UNT8 ) LOW DOSE 

CONTROL (DHTH) LOU COSE HIGH DOSE 

06-0077 06-0037 06-0039 06-0089 

SPECIA L fOPPHOLOG Y SUMMAR Y 

NO LESION REPORTED 1 1 II 5 ANIKAL HISSING/MO NECROPSY A'JTC/SFCPOPSY/HISTO PERF 2 2 

1 

1 

AUTO/NSCROPSY/NO HISTO 1 2 1 

AUTOLYSTS/NO NECROPSY u 2 1) 3 ­

* NUMEH R OF ANIfiALS WITH TISSUE EXAHINE D MICROSCOPICALLY * NUH3SB OF ANIMAL S NECROPSIED 

D-I4 
Review of the Bioassay of 2,5-Toluenediamine Sulfate*
 

for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

June 29, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DHEW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be exposed.
 The members of the Clearinghouse have been drawn from
 

academia, industry, organized labor, public interest groups,
 State health officials, and quasi-public health and research
 

organizations. Members have been selected on the basis of
 

their experience in carcinogenesis or related fields and,
 collectively, provide expertise in chemistry, biochemistry,
 biostatistics, toxicology, pathology, and epidemiology.
 Representatives of various Governmental agencies participate
 

as ad hoc members. The Data Evaluation/Risk Assessment
 

Subgroup of the Clearinghouse is charged with the responsi­ bility of providing a peer review of reports prepared on
 

NCI-sponsored bioassays of chemicals studied for carcinogenic­ ity. It is in this context that the below critique is given
 

on the bioassay of 2,5-Toluenediamine Sulfate for carcinogenic­ ity.
 

Although a carcinogenic response was not demonstrated,
 the reviewer said that the evidence was suggestive that the
 

compound may have a carcinogenic potential. He recommended
 

that it be considered for retest. In his critique, he noted
 

several experimental flaws, including the use of animals
 

from different shipments, the conduct of the subchronic
 

study in a different mouse strain than used in the chronic
 

phase, and the start of the high dose rats on test some
 

months after the initiation of the low dose animal group.
 The reviewer said the compound warranted further testing
 

because of the experimental design and study conduct deficien­ cies, as well as the fact that 2,5-Toluenediamine Sulfate
 

49
 
had been shown to be positive in the Ames assay. The reviewer
 

moved that the report on the bioassay of 2,5-Toluenediamine
 

Sulfate be accepted as written but that the compound be
 

considered for retest. The motion was approved without
 

objection.
 

Clearinghouse Members present:
 

Arnold L. Brown (Chairman), Mayo Clinic
 

Paul Nettesheim, National Institute of Environmental
 

Health Sciences
 

Verne Ray, Pfizer Medical Research Laboratory
 

Verald K. Rowe, Dow Chemical U.S.A.
 Michael B. Shimkin, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

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.
 

4U.S. GOVERNMEN T PRINTIN G OFFICE i 1978-260-899/3201
 

50
 

DHEW Publication No. (NIH) 78-1381