1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 145 

BIOASSAY OF 

3-CHLORO-p-TOLUIDINE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 95-74-9 

NCI-CG-TR-145 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

3-CHLORO-p-TOLUIDINE
 

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) 78-1701
 

REPORT ON THE BIOASSAY OF 3-CHLORO-p-TOLUIDINE
 

FOR POSSIBLE CARCINOGENICITY
 

CARCINOGENSSIS TESTING PROGRAM
 

DIVISION OF CANCER CAUSE AND PREVENTION
 

NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH
 

FOREWORD: This report presents the results of the bioassay of
 

3-chloro-p-toluidine conducted for the Carcinogenesis Testing Program,
 

Division of Cancer Cause and Prevention, National Cancer Institute
 

(NCI), National Institutes of Health, Bethesda, Maryland. This is
 

one of a series of experiments designed to determine whether selected
 

chemicals have the capacity to produce cancer in animals. Negative
 

results, in which the test animals do not have a significantly
 

greater incidence of cancer than control animals, do not necessarily
 

mean the test chemical is not a carcinogen because the experiments
 

are conducted under a limited set of circumstances. Positive results
 

demonstrate that the test chemical is carcinogenic for animals under
 

the conditions of the test and indicate a potential risk to man.
 

The actual determination of the risk to man from animal carcinogens
 

requires a wider analysis.
 

CONTRIBUTORS: This bioassay of 3-chloro-p-toluidine was conducted
 

by Litton Bionetics, Inc., Bethesda, Maryland, initially under direct
 

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

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

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

Weisburger (1,3). The principal investigators for the contract were
 

Dr. S. M. Garner (4,5) and Dr. B. M. Ulland (4,5). Mr. S. Johnson
 

(4) was the coprincipal investigator for the contract. Animal treatment and observation were supervised by Mr. R. Cypher (4), Mr. D. S.
 

Howard (4) and Mr. H. D. Thornett (4); Mr. H. Paulin (4) analyzed
 

dosed feed mixtures. Ms. J. Blalock (4) was responsible for data
 

collection and assembly.
 

Histopathologic examinations were performed by Dr. W. Busey (6),
 

at Experimental Pathology Laboratories, Inc., the pathology narratives were written by Dr. W. Busey (6), and the diagnoses included in
 

this report represent the interpretation of this pathologist. Histopathology findings and reports were reviewed by Dr. R. L. Schueler
 

(7).
 

111
 
Compilation of individual animal survival, pathology, and summary tables was performed by EG&G Mason Research Institute (8); the
 

statistical analysis was performed by Mr. W. W. Belew (9,10) and
 

Mr. R. M. Helfand (9), using methods selected for the Carcinogenesis
 

Testing Program by Dr. J. J. Gart (11).
 

This report was prepared at METREK, a Division of The MITRE Corporation (9) under the direction of the NCI. Those responsible for
 

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

(9), task leader Ms. P. Walker (9), senior biologist Mr. M. Morse
 

(9), biochemist Mr. S. C. Drill (9), chemist Dr. N. Zimmerman (9) and
 

technical editor Ms. P. A. Miller (9). 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 (1,12), Dr.
 

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

T. W. Orme (1), Dr. R. A. Squire (1,13), 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 U.S. Environmental Protection Agency, 401 M Street
 

S.W., Washington, D.C.
 

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

4.	 Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington, Maryland.
 

5.	 Now with Hazleton Laboratories America, Inc., 9200 Leesburg
 

Turnpike, Vienna, Virginia.
 

6.	 Experimental Pathology Laboratories, Inc., Route 636, Herndon,
 

Virginia.
 

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

Maryland.
 

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

Rockville, Maryland.
 

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

Boulevard, McLean, Virginia.
 

10.	 Now with The Solar Energy Research Institute, Cole Boulevard,
 

Golden, Colorado.
 

11.	 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.
 

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

Washington, D.C.
 

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

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay for the possible carcinogenicity of 3-chloro-ptoluidine was conducted using Fischer 344 rats and B6C3F1 mice.
 

3-Chloro-p-toluidine was administered in the feed, at either of two
 

concentrations, to groups of 50 male and 50 female animals of each
 

species. Twenty animals of each sex and species were placed on test
 

as controls. The time-weighted average dietary concentrations of
 

3-chloro-p-toluidine administered to rats of both sexes were 3269
 

and 1635 ppm for the high and low dose groups, respectively. The
 

high and low dietary concentrations of 3-chloro-p-toluidine administered to mice were, respectively, 1200 and 600 ppm for males and 600
 

and 300 ppm for females. The compound was administered in the diet
 

for 78 weeks, followed by an observation period of 24 weeks for high
 

dose male rats, 25 weeks for all other dosed rats, and 12 weeks for
 

mice.
 

There were no significant positive associations between the
 

concentrations of 3-chloro-p-toluidine administered and mortality
 

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

Mean body weight depression, relative to controls, was observed in
 

high dose rats and mice of both sexes, indicating that the concentrations administered to these animals may have approximated the
 

maximum tolerated dosages. The unusual incidences of nonneoplastic
 

spleen and liver lesions in high dose rats supports this assumption.
 

Under the conditions of this bioassay there was no convincing
 

evidence for the carcinogenicity of 3-chloro-p-toluidine in Fischer
 

344 rats or B6C3F1 mice.
 

Vll
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 4
 

A. Chemicals 4
 

B. Dietary Preparation 4
 

C. Animals 5
 

D. Animal Maintenance 6
 

E. Selection of Initial Concentrations 7
 

F. Experimental Design 9
 

G. Clinical and Histopathologic Examinations 12
 

H. Data Recording and Statistical Analyses 14
 

III. CHRONIC TESTING RESULTS: RATS 19
 

A. Body Weights and Clinical Observations 19
 

B. Survival 19
 

C. Pathology 19
 

D. Statistical Analyses of Results 22
 

IV. CHRONIC TESTING RESULTS: MICE 30
 

A. Body Weights and Clinical Observations 30
 

B. Survival 30
 

C. Pathology 30
 

D. Statistical Analyses of Results 33
 

V. DISCUSSION 39
 

VI. BIBLIOGRAPHY 41
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH 3-CHLORO-p-TOLUIDINE A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH 3-CHLORO-p-TOLUIDINE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 3-CHLORO-p-


TOLUIDINE C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 3-CHLORO-p-


TOLUIDINE D-l
 

IX
 
LIST OF ILLUSTRATIONS 

Figure Number Page 

1 CHEMICAL STRUCTURE OF 3-CHLORO-p-TOLUIDINE 2 

2 GROWTH CURVES FOR 3-CHLORO-p-TOLUIDINE 

CHRONIC STUDY RATS 20 

SURVIVAL COMPARISONS OF 3-CHLORO-p-TOLUIDINE 

CHRONIC STUDY RATS 21 

GROWTH CURVES FOR 3-CHLORO-p-TOLUIDINE 

CHRONIC STUDY MICE 31 

SURVIVAL COMPARISONS OF 3-CHLORO-p-TOLUIDINE 

CHRONIC STUDY MICE 32 

LIST OF TABLES 

Table Number Page 

DESIGN SUMMARY FOR FISCHER 344 RATS— 

3-CHLORO-p-TOLUIDINE FEEDING EXPERIMENT 10 

DESIGN SUMMARY FOR B6C3F1 MICE—3-CHLORO-pTOLUIDINE FEEDING EXPERIMENT 11 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

3-CHLORO-p-TOLUIDINE 23 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH 3-CHLORO-p-TOLUIDINE 26 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

3-CHLORO-p-TOLUIDINE 34 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH 3-CHLORO-p-TOLUIDINE 36 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH 3-CHLORO-p-TOLUIDINE A-3 
LIST OF TABLES (Concluded) 

Table Number Page 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH 3-CHLORO-p-TOLUIDINE A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH 3-CHLORO-p-TOLUIDINE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH 3-CHLORO-p-TOLUIDINE B-6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH 3-CHLOROp-TOLUIDINE C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH 3-CHLOROp-TOLUIDINE C-7 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH 3-CHLOROp-TOLUIDINE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH 3-CHLOROp-TOLUIDINE D-6 

XI
 

I. INTRODUCTION
 

3-Chloro-p-toluidine (Figure 1) (NCI No. C02040), a dye intermediate and avicide, was selected for bioassay by the National Cancer
 

Institute because of the increased incidence of bladder cancer
 

observed among workers in the dye manufacturing industry (Anthony
 

and Thomas, 1970; Wynder et al., 1963). Aromatic amines, of which
 

3-chloro-p-toluidine is one example, are among several classes of
 

chemicals believed to contribute to this increased cancer risk
 

(Clayson and Garner, 1976).
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

*
 (1977) name for this compound is 3-chloro-4-methylbenzenamine. It is
 

also called l-amino-3-chloro-4-methylbenzene; 4-amino-2-chlorotoluene
 

(CH =1); 2-chloro-4-aminotoluene (CH =1); 3-chloro-4-methylaniline
 

(NH =1); and CPT.
 

3-Chloro-p-toluidine is used as an intermediate in the production of at least one dye, Palatine Fast Yellow 6GN (Society of Dyers
 

and Colourists, 1956).
 

3-Chloro-p-toluidine is strongly nephrotoxic to birds of several
 

species, especially starlings, and is therefore used as a selective
 

avicide for starling control (Mull and Giri, 1972; Metcalf, 1967).
 

Specific production data for 3-chloro-p-toluidine are not available; however, this compound is produced in commercial quantities (in
 

*
 

The CAS registry number is 95-74-9.
 
FIGURE 1

 

CHEMICAL STRUCTURE OF 3-CHLORO-p-TOLUIDINE 
excess of 1000 pounds or $1000 in value annually) by one U.S. company
 

(Stanford Research Institute, 1977).
 

The potential for exposure to 3-chloro-p-toluidine is greatest
 

for workers in the chemical and dye manufacturing industries and pest
 

control workers.
 
II. MATERIALS AND METHODS
 

A. Chemicals
 

3-Chloro-p-toluidine was purchased from E.I. duPont de Nemours
 

& Company, Wilmington, Delaware. Chemical analysis was performed
 

by Litton Bionetics, Inc., Bethesda, Maryland. Thin-layer chromatographic (TLC) plates, developed utilizing two solvent systems (benzene:methanol and diethyl ether:ethyl acetate:acetic acid), each
 

revealed one spot. Visualization was by visible and ultraviolet
 

light, I vapor and ferric chloride-potassium ferricyanide spray.
 

Gas-liquid chromatography showed one peak and infrared (IR) analysis
 

was consistent with the structure of 3-chloro-p-toluidine.
 

TLC and IR analyses performed after a six-month interval showed
 

no significant changes from the original analyses. These results
 

suggested that this compound was of high purity with good stability.
 

Throughout this report, the term 3-chloro-p-toluidine is used
 

to refer to this material.
 

B.	 Dietary Preparation
 

The basal laboratory diet for both dosed and control animals
 

(S\
 

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

3-Chloro-p-toluidine was administered to the dosed animals as a component of the diet.
 

The chemical was removed from its container and a proper amount
 

was blended with an aliquot of the ground feed using a mortar and
 
pestle. Once visual homogeneity was attained, the mixture was placed
 

in a 6 kg capacity Patterson-Kelley standard model twin-shell stainless steel V-blender along with the remainder of the feed to be prepared. After 20 minutes of blending, the mixtures were placed in
 

double plastic bags and stored in the dark at 4°C. The mixture was
 

prepared once weekly.
 

The stability of 3-chloro-p-toluidine in feed was determined
 

spectrophotometrically. Ten days after preparation of diets containing 1500 and 3000 ppm concentrations of 3-chloro-p-toluidine,
 

61.6 _+ 0.6 percent of the initial concentrations were detected
 

in the feed, using the methods indicated. Analysis of the data
 

generated by the analytical methods used does not permit a distinction to be made between stability and the extent of extraction.
 

C. Animals
 

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

through contracts of the Division of Cancer Treatment, National Cancer Institute. Rats were obtained from Laboratory Supply Company,
 

Inc., Indianapolis, Indiana; A.R. Schmidt, Madison, Wisconsin; and
 

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

Mice were supplied by Charles River Breeding Laboratories, Inc.,
 

Wilmington, Massachusetts; and A.R. Schmidt, Madison, Wisconsin.
 

There was no indication that animals from a specific supplier were
 

assigned to a specific group.
 
Rats and mice were approximately 4 weeks old when received.
 

Upon receipt, animals were examined for visible signs of disease or
 

parasites. Obviously ill or runted animals were culled. The remaining animals were quarantined for 2 weeks prior to initiation of test.
 

Animals which did not manifest clinical signs of disease were placed
 

on test at this time. Animals were assigned to groups and distributed
 

among cages so that the average body weight per cage was approximately
 

equal for a given species and sex.
 

D. Animal Maintenance
 

All animals were housed by species in temperature- and humiditycontrolled rooms. The temperature range was 22° to 26°C and the relative humidity was maintained between 45 and 55 percent. Incoming air
 

was filtered through HEPA filters (Flanders Filters, McLean, Virginia)
 

at a rate of 12 to 15 complete changes of room air per hour. Fluorescent lighting was provided 8 hours per day (9:00 a.m. to 5:00 p.m.).
 

All rats were housed four per cage by sex and all mice five per
 

cage by sex. Throughout the study dosed and control animals of both
 

species were housed in polycarbonate cages (Lab Products, Inc., Garfield, New Jersey) suspended from aluminum racks. Racks were fitted
 

with a continuous stainless steel mesh lid over which a sheet of
 

filter paper was firmly secured. Filter paper was changed at 2-week
 

intervals, when the racks were sanitized. Clean cages and bedding
 

CR)
 

were provided twice weekly. Ab-sorb-dri hardwood chip bedding
 

(Wilner Wood Products Company, Norway, Maine) was used in polycarbonate cages for the entire bioassay.
 

6
 
Acidulated water (pH 2.5) was supplied to animals in water bottles filled by an automated metering device that was checked daily
 

for diluting accuracy. Water bottles were changed twice weekly and
 

sipper tubes were washed at weekly intervals. During the period of
 

chemical administration, dosed and control animals received treated
 

®
 

or untreated Wayne Lab-Blox meal as appropriate. The feed was supplied in hanging stainless steel hoppers which were refilled three
 

times per week and sanitized weekly. Food and water were available
 

ad libitum for both species.
 

All dosed and control rats were housed in a room with other rats
 

receiving diets containing^\ 2-nitro-p-phenylenediamine (5307-14-2);
 

5-chloro-o-toluidine (95-79-4); and nitrofen (1836-75-5).
 

All dosed and control mice were housed in a room with other mice
 

receiving diets containing 2-nitro-p-phenylenediamine (5307-14-2);
 

Michler's ketone (90-94-8); p-chloroaniline (106-47-8); 4,4'-methylenebis(N,N-dimethyl)-benzenamine (101-61-1); l-phenyl-2-thiourea
 

(103-85-5); trimethylthiourea (2489-77-2); dibutyltin diacetate
 

(1067-33-0); 5-chloro-o-toluidine (95-79-4); and N-phenyl-p-phenylenediamine hydrochloride (2198-59-6).
 

E. Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations
 

of 3-chloro-p-toluidine for administration to dosed animals in
 

the chronic studies, subchronic toxicity tests were conducted with
 

*
 

CAS registry numbers are given in parentheses,
 
both rats and mice. Rats were distributed among six groups, each
 

consisting of five males and five females. 3-Chloro-p-toluidine was
 

incorporated into the basal laboratory diet and supplied ad libitum
 

to five of the six rat groups in concentrations of 315, 680, 1465,
 

3155, and 6800 ppm. The remaining rat group served as a control
 

group, receiving only the basal laboratory diet.
 

Mice were distributed among six groups, each consisting of five
 

males and five females. 3-Chloro-p-toluidine was incorporated into
 

the basal laboratory diet and supplied ad libitum to five of the six
 

mouse groups in concentrations of 810, 1180, 1740, 2550, and 3750
 

ppm. The sixth mouse group served as a control group, receiving only
 

the basal laboratory diet.
 

The dosed dietary preparations were administered for a period
 

of 4 weeks, followed by a 2-week observation period during which all
 

animals were fed the basal laboratory diet. Individual body weights
 

and food consumption data were recorded twice weekly throughout the
 

study. Upon termination of the observation period, all survivors
 

were sacrificed and necropsied.
 

At the end of the subchronic test, mean body weight gain of male
 

and female rat groups receiving 6800 ppm was 9 percent less than the
 

mean body weight gain of their control groups. The mean body weight
 

gain of male rats receiving 3155 ppm was 17 percent greater than
 

that of their controls, while female rats receiving the same concentration displayed a mean body weight gain 6 percent less than that
 

8
 
of their controls. No deaths were reported for any rat group. The
 

high concentration selected for administration to dosed rats in the
 

chronic bioassay was 6000 ppm.
 

At the end of the subchronic test, mean body weight gain among
 

male mice receiving 1740 ppm was the same as that of their controls,
 

while female mice receiving the same concentration displayed a mean
 

weight gain 5 percent less than that of their controls. The mean
 

body weight gain among male mice receiving 1180 ppm was the same as
 

that of their controls, while female mice receiving the same concentration displayed a mean body weight gain 3 percent less than that
 

of their control. No deaths were reported in any mouse group. The
 

high concentrations selected for administration to dosed mice in the
 

chronic bioassay were 1200 and 600 ppm for males and females, respectively.
 

F. Experimental Design
 

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

treated and untreated observation periods, and time-weighted average
 

concentrations) are summarized in Tables 1 and 2.
 

All rats were approximately 6 weeks old at the time the test
 

was initiated and were placed on test simultaneously. The initial
 

dietary concentrations of 3-chloro-p-toluidine administered were
 

6000 and 3000 ppm. Throughout this report those rats initially
 

receiving the former concentration are referred to as the high
 
TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

3-CHLORO-p-TOLUIDINE FEEDING EXPERIMENT
 

INITIAL


GROUP


SIZE


MALE
 

CONTROL 20
 

LOW DOSE 50
 

HIGH DOSE 50
 

FEMALE
 

CONTROL 20
 

LOW DOSE 50
 

HIGH DOSE 50
 

a
 

Concentrations given
 

 3-CHLORO-p­ OBSERVATION PERIOD

 TOLUIDINE TREATED UNTREATED

 CONCENTRATION3 (WEEKS) (WEEKS)

0 0 103
 

3000 7
 

1500 71
 

0 25
 

6000 7
 

3000 71
 

0 24
 

0 0 103
 

3000 7
 

1500 71
 

0 25
 

6000 7
 

3000 71
 

0 25
 

in parts per million.
 

 TIME-WEIGHTED
 

 AVERAGE
 

 CONCENTRATION
 

0
 

1635
 

3269
 

0
 

1635
 

3269
 

b 2 (concentration X weeks received)
 

TT Imp— WP i1 ffh f pd avftrppf >rnnrpntratii in — -. :——
 

10
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

3-CHLORO-p-TOLUIDINE FEEDING EXPERIMENT
 

INITIAL OBSERVATION PERIOD 

GROUP 3-CHLORO-p-TOLUIDINE TREATED UNTREATED 

SIZE CONCENTRATION3 (WEEKS) (WEEKS) 

MALE
 

CONTROL 20 0 0 90
 

LOW DOSE 50 600 78
 

0 12
 

HIGH DOSE 50 1200 78
 

0 12
 

FEMALE
 

CONTROL 20 0 0 90
 

LOW DOSE 50 300 78
 

0 12
 

HIGH DOSE 50 600 78
 

0 12
 

Concentrations given in parts per million.
 

11
 
dose groups and those initially receiving the latter concentration
 

are referred to as the low dose groups. At the start of week 8 the
 

dietary concentrations of 3-chloro-p-toluidine were adjusted to 3000
 

and 1500 ppm. Dosed rats were supplied with feed containing 3-chlorop-toluidine for 78 weeks followed by a 25-week observation period;
 

except high dose males, which were observed for 24 weeks.
 

All mice were approximately 6 weeks old at the time the test was
 

initiated and were placed on test simultaneously. The concentrations
 

of 3-chloro-p-toluidine utilized for male mice were 1200 and 600 ppm.
 

Throughout this report, those male mice receiving the former concentration are referred to as the high dose group and those receiving
 

the latter concentration are referred to as the low dose group. The
 

dietary concentrations of 3-chloro-p-toluidine utilized for female
 

mice were 600 and 300 ppm. Throughout this report those female mice
 

receiving the former concentration are referred to as the high dose
 

group and those receiving the latter concentration are referred to as
 

the low dose group. Dosed mice were supplied with feed containing
 

3-chloro-p-toluidine for 78-weeks followed by a 12-week observation
 

period.
 

G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the experiment. From the first day, all animals were inspected twice daily
 

for mortality. Food consumption data were collected at monthly intervals from 20 percent of the animals in each group. Body weights
 

12
 
were recorded once a week for the first 6 weeks, every 2 weeks for
 

the next 12 weeks, and at monthly intervals thereafter.
 

All moribund animals or animals that developed large, palpable
 

masses that jeopardized their health were sacrificed. A necropsy
 

was performed on each animal regardless of whether it died, was sacrificed when moribund, or was sacrificed at the end of the bioassay.
 

The animals were euthanized by carbon dioxide asphyxiation, and were
 

immediately necropsied. The histopathologic examination consisted of
 

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

gross lesions taken from sacrificed animals and, whenever possible,
 

from animals found dead.
 

Tissues were preserved in a 10 percent neutral buffered formalin
 

solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin prior to microscopic examination.
 

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

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

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

prostate, brain, tunica vaginalis, 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
 

13
 
for which particular organs, tissues, or lesions were examined microscopically varies and does not necessarily represent the number of
 

animals that were recorded in each group at the time that the test
 

was initiated.
 

H. Data Recording and Statistical Analyses
 

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

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

as recommended by the International Union Against Cancer (Berenblum,
 

1969). Data tables were generated for verification of data transcription and for statistical review.
 

These data were analyzed using the statistical techniques described in this section. Those analyses of the experimental results
 

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

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

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

missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect
 

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

14
 
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.
 

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
 

15
 
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
 

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
 

16
 
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
 

t c t
 

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

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

c
 

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

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

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

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

larger proportion in the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

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

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

17
 
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.
 

18
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

Mean group body weight depression was apparent in high dose
 

male and in female rats during the period of compound administration (Figure 2).
 

No abnormal clinical signs were recorded.
 

B. Survival
 

The estimated probabilities of survival for male and female
 

rats in the control and 3-chloro-p-toluidine-dosed groups are shown
 

in Figure 3. For both males and females, the Tarone test did not
 

indicate a significant association between dosage and mortality.
 

For males adequate numbers of rats were at risk from latedeveloping tumors, as 46/50 (92 percent) of the high dose, 43/50
 

(86 percent) of the low dose, and 13/20 (65 percent) of the control
 

group survived on test until the end of the experiment. For females
 

survival was also adequate, as 48/50 (96 percent) of the high dose,
 

44/50 (88 percent) of the low dose, and 17/20 (85 percent) of the
 

control group survived on test until the end of the experiment.
 

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).
 

19
 
750- •750 

600 — -600 

H 450­ -450 X 

UJ 

g 

§300­ — 300 

CO 

2 

< 

UJ CONTROL 

S 

150 — LOW DOSE -150 

MALE RATS HIGH DOSE 

[ \ I I I ] 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

750- •750 

_

w 

S 

 600­ -600 

cc 

H 450­ -450 

g 300H 

CO 

< 

LU 

150 — 

-300 

-150 

FEMALE RATS 

~T 

15 

~T 

30 

~T 

45 

~T 

60 

~r 

75 

~T 

90 105 120 

TIME ON TEST (WEEKS) 

FIGURE 2 

GROWTH CURVES FOR 3-CHLORO-p-TOLUIDINE CHRONIC STUDY RATS 

20 
PROBABILITY OF SURVIVAL

 PROBABILITY OF SUR

c3 O P O O p p c3 io -^ cn bo c3 b CD 

1 1 ! 1 1 1 1 1 

- •n 

m 

2 

rn r~ i ~ m 3D 

3D 

s

CO r­^

o 

O

s

 CO 

 °~ 8­ -o 

1) 

§

Z 

M H *• O — 01 

11 — 

j w

 

r-

- n H

 ro

 O jr m 

3>C 2? S­

<? 

H CO m 

O m "" r" 7^ C to 

2 3­ z 1 m

J 

O ^ 1 3

1 O 1

 i- Z ^D » o — O 1

 S* 

V 

1 I rn

 f 

5 8 i ! D

1 

C 

8 § s rr 1

 s r

> s- s

10 1

 1 ' 1 ' 1 ' 1 1 o r 

m

O 

m

m 

1^ \ 

co 3 O O O O o o 

t3 Ko ^ 'CT> bo c3 
A variety of neoplasms was seen in both the male and female
 

rats. These neoplasms were generally equally distributed among
 

the control and dosed groups.
 

Endometrial stromal polyps were seen in the female rats from
 

both of the dosed groups. No endometrial stromal polyps were recognized in the control females; however, this lesion is common in aged
 

female Fischer 344 rats, and the variation in its incidence among
 

the control and dosed rats is probably due to the low number of control females. Nonneoplastic lesions were of the types commonly observed in aging Fischer 344 rats, except for those of the spleen and
 

liver. A high incidence of fibrosis of the splenic capsule (i.e.,
 

25/50 [50 percent] in high dose males and.37/50 [74 percent] in high
 

dose females) and hepatic fatty metamorphosis (i.e., 35/50 [70 percent] in high dose males and 34/50 [68 percent] in high dose females)
 

was observed, primarily in high dose rats.
 

Based on the results of this pathologic examination, 3-chloro-ptoluidine was not carcinogenic in male or female Fischer 344 rats
 

under the conditions of this bioassay; however, administration of
 

the compound was toxic to the spleen and liver.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

22
 
___ 

TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH 3-CHLORO-p-TOLUIDINE£
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Adenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 U) Malignant Lymphoma'3
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

1/20(0.05)
 

N.S.
 

—_
 

103
 

4/20(0.20)
 

P = O.OOl(N)
 

P = 0.004
 

66
 

2/18(0.11)
 

N.S.
 

98
 

LOW
 

DOSE
 

5/48(0.10)
 

N.S.
 

2.083
 

0.259
 

96.358
 

84
 

0/50(0.00)
 

P = 0.005(N)
 

0.000
 

0.000
 

0.427
 

2/42(0.05)
 

N.S.
 

0.429
 

0.034
 

5.612
 

103
 

HIGH
 

DOSE
 

3/50(0.06)
 

N.S.
 

1.200
 

0.106
 

61.724
 

102
 

0/50(0.00)
 

P - 0.005(N)
 

0.000
 

0.000
 

0.427
 

1/47(0.02)
 

N.S.
 

0.191
 

0.003
 

3.512
 

102
 
___ 

TOPOGRAPHY: MORPHOLOGY
 

Adrenal: Pheochromocytoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma^
 

P Values0
 

NJ Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pancreatic Islets: Islet-Cell Adenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED)
 

CONTROL
 

1/18(0.06)
 

N.S.
 

_—
 

103
 

1/16(0.06)
 

N.S.
 

98
 

1/19(0.05)
 

N.S.
 

_—
 

102
 

LOW
 

DOSE
 

1/47(0.02)
 

N.S.
 

0.383
 

0.005
 

29.452
 

103
 

4/39(0.10)
 

N.S.
 

1.641
 

0.185
 

78.628
 

103
 

5/46(0.11)
 

N.S.
 

2.065
 

0.259
 

95.429
 

103
 

HIGH
 

DOSE
 

5/48(0.10)
 

N.S.
 

1.875
 

0.236
 

86.718
 

0/43(0.00)
 

N.S.
 

0.000
 

0.000
 

6.936
 

0/50(0.00)
 

N.S.
 

0.000
 

0.000
 

7.102
 

57 
___ 

TABLE 3 (CONCLUDED)
 

LOW HIGH
 

TOPOGRAPHY :MORPHOLOGY CONTROL DOSE DOSE
 

Testis: Interstitial-Cell Tumor 18/20(0.90) 48/49(0.98) 49/50(0.98)
 

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

Relative Risk (Control) 1.088 1.089
 

Lower Limit 0.959 0.960
 

Upper Limit 1.174 1.174
 

Weeks to First Observed Tumor 86 76 81
 

Treated groups received time-weighted average doses of 1635 or 3269 ppm in feed.
 

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

Ul 

£» 

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

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

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

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

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

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

group when P < 0.05.
 
___ 

TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH 3-CHLORO-p-TOLUIDINE*
 

LOW
 

TOPOGRAPHY: MORPHOLOGY CONTROL DOSE
 

Lung: Alveolar/Bronchiolar Adenoma or
 

Alveolar/Bronchiolar Carcinoma*3 0/19(0.00) 2/48(0.04)
 

P Values0 N.S. N.S.
 

Relative Risk (Control) —_ Infinite
 

Lower Limit 0.122
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 103
 K>
 

Pituitary: Chromophobe Adenoma 4/17(0.24) 10/42(0.24)
 

P Values0 N.S. N.S.
 

Relative Risk (Control) 1.012
 

Lower Limit 0.354
 

Upper Limit 3.962
 

Weeks to First Observed Tumor 103 37
 

Mammary Gland: Fibroadenoma 1/20(0.05) 6/50(0.12)
 

P Values0 N.S. N.S.
 

Relative Risk (Control) -.__ 2.400
 

Lower Limit 0.325
 

Upper Limit 108.021
 

Weeks to First Observed Tumor 103 103
 

HIGH
 

DOSE
 

5/50(0.10)
 

N.S.
 

Infinite
 

0.501
 

Infinite
 

102
 

9/45(0.20)
 

N.S.
 

0.850
 

0.286
 

3.408
 

3/50(0.06)
 

N.S.
 

1.200
 

0.106
 

61.724
 

80 

102 
___ 

TABLE 4 (CONCLUDED)
 

LOW HIGH
 

TOPOGRAPHY: MORPHOLOGY CONTROL DOSE DOSE
 

Uterus: Endometrial Stromal Polyp 0/19(0.00) 4/50(0.08) 9/50(0.18)
 

P Values0	 P = 0.018 N.S. P - 0.044
 

Relative Risk (Control) Infinite Infinite
 

Lower Limit 0.368 1.045
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor	 103 102
 

a

Treated groups received time-weighted average doses of 1635 or 3269 ppm in feed.
 

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

C

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

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

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

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

d

The	 95% confidence interval on the relative risk of the treated group to the control group.
 
tumors were observed in at least one of the control or 3-chloro-ptoluidine-dosed groups and where such tumors were observed in at
 

least 5 percent of the group.
 

None of the statistical tests for male rats indicated a significant positive association between the administration of 3-chloro-ptoluidine and an increased tumor incidence.
 

For female rats the Cochran-Armitage test indicated a significant (P = 0.018) positive association between dosage and the incidence of endometrial stromal polyps of the uterus. The Fisher exact
 

tests, however, were not significant under the Bonferroni criterion.
 

It should also be noted that these tumors occurred in 28/284 (10 percent) of the untreated female Fischer 344 rats in the historical
 

control observed at this laboratory for the NCI Carcinogenesis
 

Testing Program as compared with the lower incidence of 0/19 observed
 

in the controls of this bioassay.
 

Thus, based upon these statistical results there was no convincing evidence that 3-chloro-p-toluidine was a carcinogen in male
 

or female Fischer 344 rats under the conditions of this experiment.
 

The possibility of a negative association between dose and the
 

combined incidence of leukemia or malignant lymphoma was noted in
 

male rats.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

28
 
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 3-chloro-p-toluidine that could not be established under the
 

conditions of this test.
 

29
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Distinct and consistent dose-related mean group body weight
 

depression was apparent in male mice. Mean body weight depression,
 

relative to the controls, was apparent throughout a major portion of
 

the bioassay for high dose females. This was not true for low dose
 

females (Figure 4).
 

No abnormal clinical signs were recorded.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and 3-chloro-p-toluidine-dosed groups are shown in
 

Figure 5. The Tarone test did not indicate a significant association
 

between dosage and mortality for either male or female mice.
 

For males adequate numbers of mice were at risk from latedeveloping tumors, as 48/50 (96 percent) of the high dose, 44/50 (88
 

percent) of the low dose, and 20/20 in the control group survived on
 

test until the end of the experiment. For females survival was also
 

adequate, as 44/50 (88 percent) of the high dose, 44/50 (88 percent)
 

of the low dose, and 20/20 in the control group survived on test
 

until the end of the experiment.
 

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).
 

30
 
-50 

-40 

x— 

-30 

-20 

CONTROL 

LOW DOSE 

HIGH DOSE 

-10 

T 

45 

I 

60 

T 

75 

TIME ON TEST (WEEKS) 

90 

I

105 

' 

120 

50­ -50 

40­ -40 

£30-1 ..•'.' -30 

£20H 

o 

m 

< 

LLJ 

10— 

FEMALE MICE 

CONTROL 

LOW DOSE 

HIGH DOSE 

-20 

-10 

I 

15 

I 

30 

I 

45 

I 

60 

I 

75 

TIME ON TEST (WEEKS) 

\ 

90 

i I 

105 120 

FIGURE 4 

GROWTH CURVES FOR 3-CHLORO-p-TOLUIDINE CHRONIC STUDY MICE 

31 
PROBABILITY OF SURVIVAL PROBABILITY OF S

c3 0 O 0 0 - C3 O O O 

c3 kj * bi co i: 3 C3 kj •f b> 

1 1 1 1 i 1 i 1 < 1 . 1 1 1 

o — o — 

-n - M m

 C ^

 

39 ^ ^ i—

 ^ < cr ^r- cr m

 

< m S

 

5 O

^ O

r~ _ m

 O m

 o

8­ 1 8­ 30 

_ 

i 

O _. ^ 

Tl Zj in — d fts 

6 m • m

 

I o It 0

 

i Z n ^ | § ^ H H INi 33

 C m m O X £/) 0) _ co g>

 

7 « i! ° 1

 

d" I 1

rTM rn - rn

 

c TM 

O w en

 

2 -J ^j

m 

i! m 

w 

o» m •4 0 

X 1 • • 

30 1

 

1 

n <e J

L 

o i i- n 

S- ' 0 ? o § z 0 - 1 0 § 

w * m" _ o -

o 

O m 2 

S g- §- 1 ° —

X

1 

o S O

1— m m 

8- 1

 1 ' 1 ' 1 I 1 ' §- i 1 1 1 1 

c3 O o o o -.1 (3 0 o o 

c3 Kj '*» 0> 03 <3 <3 to !c. CD 
A variety of neoplasms was present in both the dosed and control
 

groups. No meaningful differences were noted in the incidence of
 

neoplasms among the control and dosed mice. Nonneoplastic lesions
 

were of the types commonly observed in aging B6C3F1 mice.
 

Based on the results of this pathologic examination, 3-chlorop-toluidine was not carcinogenic in B6C3F1 mice under the conditions
 

of this bioassay.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

tumors were observed in at least one of the control or 3-chloro-ptoluidine-dosed groups and where such tumors were observed in at
 

least 5 percent of the group.
 

None of the statistical tests for any site in mice of either sex
 

indicated a significant positive association between the administration of 3-chloro-p-toluidine and an increased tumor incidence. Thus,
 

at the dose levels used in this experiment there was no evidence that
 

3-chloro-p-toluidine was a carcinogen in B6C3F1 mice.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

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

33
 
___ 

___ 

___ 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH 3-CHLORO-p-TOLUIDINE*
 

TOPOGRAPHY : MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Adenoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 U)
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma or
 

Hepatocellular Adenoma*3
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

0/20(0.00)
 

N.S.
 

3/20(0.15)
 

N.S.
 

90
 

4/20(0.20)
 

N.S.
 

90
 

LOW
 

DOSE
 

3/49(0.06)
 

N.S.
 

Infinite
 

0.255
 

Infinite
 

90
 

6/47(0.13)
 

N.S.
 

0.851
 

0.208
 

4.897
 

90
 

10/47(0.21)
 

N.S.
 

1.064
 

0.361
 

4.230
 

90
 

HIGH
 

DOSE
 

3/49(0.06)
 

N.S.
 

Infinite
 

0.255
 

Infinite
 

83
 

3/49(0.06)
 

N.S.
 

0.408
 

0.061
 

2.857
 

90
 

7/49(0.14)
 

N.S.
 

0.714
 

0.211
 

3.052
 

90
 
TABLE 5 (CONCLUDED)
 

Treated groups received doses of 600 or 1200 ppm in feed.
 

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

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

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

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

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

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

OJ
 

Ln
 
___ 

___ 

___ 

TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH 3-CHLORO-p-TOLUIDINE£
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar /Bronchiolar Adenoma or
 

Alveolar /Bronchiolar Carcinoma*3
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphomab
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

2/20(0.10)
 

N.S.
 

P - 0.030
 

90
 

2/20(0.10)
 

N.S.
 

90
 

0/20(0.00)
 

N.S.
 

LOW
 

DOSE
 

0/50(0.00)
 

N.S.
 

0.000
 

0.000
 

1.345
 

4/50(0.08)
 

N.S.
 

0.800
 

0.128
 

8.436
 

90
 

3/49(0.06)
 

N.S.
 

Infinite
 

0.255
 

Infinite
 

87
 

HIGH
 

DOSE
 

4/46(0.09)
 

N.S.
 

0.870
 

0.139
 

9.144
 

90
 

3/47(0.06)
 

N.S.
 

0.638
 

0.081
 

7.284
 

74
 

0/45(0.00)
 

N.S.
 
TABLE 6 (CONCLUDED)
 

TOPOGRAPHY rMORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Liver: Hepatocellular Carcinoma or 

Hepatocellular Adenoma" 0/20(0.00) 4/49(0.08) 2/45(0.04) 

P Values N.S. N.S. N.S. 

Relative Risk (Control)0 

Lower Limit 

Upper Limit 

Infinite 

0.394 

Infinite 

Infinite 

0.136 

Infinite 

Weeks to First Observed Tumor 87 90 

treated groups received doses of 300 or 600 ppm in feed.
 

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

C

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

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

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

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

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

e

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

group when P < 0.05;
 
of statistically significant results. It should also be noted that
 

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

indicating the theoretical possibility of tumor induction in mice by
 

3-chloro-p-toluidine that could not be established under the conditions of this test.
 

38
 
V. DISCUSSION
 

There were no significant positive associations between the
 

concentrations of 3-chloro-p-toluidine administered and mortality in
 

either species. Adequate numbers of animals in all groups survived
 

sufficiently long to be at risk from late-developing tumors. Mean
 

body weight depression, relative to controls, was observed in high
 

dose rats and mice of both sexes, indicating that the concentrations
 

administered to these animals may have approximated the maximum
 

tolerated dosages. The unusual incidences of nonneoplastic spleen
 

and liver lesions in high dose rats supports this assumption.
 

In female rats endometrial stromal polyps of the uterus were observed in dosed but not in control groups (i.e., 0/19, 4/50, and 9/50
 

in the control, low dose, and high dose groups, respectively). There
 

was a statistically significant positive association between dosage
 

and the incidence of these uterine tumors; however, the Fisher exact
 

tests were not significant. In addition, it should be noted that the
 

incidence of these tumors in the control female rats in this bioassay
 

(i.e., 0 percent) was considerably lower than the incidence seen in
 

historical untreated controls from this laboratory (i.e., 10 percent).
 

There were no other statistically significant positive associations
 

between the administration of 3-chloro-p-toluidine and tumor incidence
 

in rats of either sex.
 

39
 
No biologically unusual tumors were detected in mice of either
 

sex, and there were no statistically significant positive associations
 

between compound administration and tumor incidence.
 

Under the conditions of this bioassay, there was no convincing
 

evidence for the carcinogenicity of 3-chloro-p-toluidine in Fischer
 

344 rats or B6C3F1 mice.
 

40
 
VI. BIBLIOGRAPHY
 

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.
 

Artnitage, 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.
 

Clayson, D.B. and R.C. Garner, "Carcinogenic Aromatic Amines and
 

Related Compounds." Chapter 8 in Carcinogenic Aromatic Amines,
 

C.E. Searle, editor. American Chemical Society Monograph 173,
 

Washington, D.C., 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.
 

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

Observations." Journal of the American Statistical Association
 

_53_:457-481, 1958.
 

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.
 

Metcalf, R.L., "Poisons: Economic," in Kirk-Othmer Encyclopedia of
 

Chemical Technology, Vol. 15. Interscience, New York, 1967.
 

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

Co., New York, 1966.
 

41
 
Mull, R.L. and S.N. Giri, "Role of Renal Aromatic N-Deacetylase in
 

Selective Toxicity of Avicide 3-Chloro-p-toluidine in Birds."
 

Biochimica Biophysica Acta 273(1):222-228, 1972; Chemical
 

Abstracts 77, 97484.
 

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.
 

Society of Dyers and Colourists, Colour Index, 2nd edition, Volume
 

3. Yorkshire, England, 1956.
 

Stanford Research Institute, 1977 Directory of Chemical Producers,
 

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

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

62^:679-682, 1975.
 

Wynder, E.L., J. Onderdonk, and N. Mantel, "An Epidemiological Investigation of Cancer of the Bladder." Cancer 16:1388-1407, 1963.
 

42
 
Review of the Bioassay of 3-Chloro-p-Toluidine* 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, 19?6, in compliance with DREW 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 carcinogenicity. It is In this context that the below critique Is given
 

on the bioassay of 3-Chloro-p-Toluidine for carcinogenicity.
 

The reviewer agreed with the conclusion in the report
 

that 3-Chloro-p-Toluidine was not carcinogenic under the
 

conditions of test. The compound was obtained from the
 

commercial producer and was analyzed for purity and stability, both over time and in the dietary mixture. Although
 

she noted the small control group sizes and dosage changes
 

during the chronic phase, she still considered the study
 

valid. The reviewer moved that the report on the bioassay
 

of 3-Chloro-p-Toluidine be accepted as written. 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.
 

43
 »U.S. GOVERNMEN T PRINTIN G OFFICE - I 978-? 6 0-89 9 / M « f>
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH 3-CHLORO-p-TOLUIDINE
 

TABLE Al

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH 3-CHLORO-p-TOLUIDINE 

CONTROL (UNTF) LOU DOSF HIGH DOSE 

11-112"; 11-1123 11-1121 

ANIPALS INITIALLY IN S1TOY 2C 50 50 

MIHALS NFCFOPSIFD 20 50 50 

ANIMAL S FXAMINF D HI STOPATHOLO.GICALLY ** 20 19 50 

INTEG'IMFNTAF Y SYS1EH 

*SKI N (21) (50) (SO) 

UHDIFFFFFNTIATE D CAPCINON A 1 (2%) 

IDNFXP L ADFNOM A 1 (2*) 

NlTlpoFIEBOKA 1 (2H) 

*SBBCOT Tissil" (2") (50) (50) 

FIBR01A 2 (U*) 2 (US) 

IIPO» A 1 (2*) 

SFSPI^ATOKY SYSTEM
 

tLn« G (20) («8) (51) 

UNDIFPFRENTIATE D CJPCINOH A MFT?S 1 (2%) 

HVFOLAB/BPONCHIOLJR ADrNOI A 1 (5*) 5 (10X) 3 (6*) 

HF!"ATOrOI'rTIC SYSTFt" 

*MDITIPLF PBGAN S (20) (50) (50) 

HAIIGNAN T LYMPHorA, NOS 2 (K*)
 LEUK^dlA , HOS 1 (5») 

IJNDIFFERENaiATF D LEUKF|<IA 1 (5%) 

CIPCHLATOBY SYSTEM 

NON ' 

DIGFSTIVE SYSTF H 

• LIVE P (20 ) (U7) (50) 

* NU1BF P OF ANI1AL S WI1H TISSDF FXAKIkF T llfTSCOPICMLT 

* NIIC3" H OF ANIMAL S NFrROPSIB!) 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-3 
TABLE A1 (CONTINUED) 

CONTROL (ONTR) LOW DOSF 

11-1125 11-1123 

*ST1ACH 09) (49) 

SRRC01A, MOS 2 («) 

»rCfL L IN'tSTIHr (20) (47) 

A D T NOMA , NOS 1 (^%) 

OL^SE IM-'STIHF (19) (46) 

L'lOPYPI A 

UPINSP Y SYST'I 

tKIP'JF Y (2P) (49) 

TUP1ILJR-CFL L ADFN'OrAICIHOKl i 

FNTOrciN 1 SYST*"« 

«DITI, 1 ^ A P Y (18) (42) 

C HPOCOPHOD 1 fDFNOC A i (11*) 2 (5*) 

MDF-NA I (18) (47) 

P 1lOnnOinCY'rOHA 1 (b% ) 1 (2t) 

»lHY^rir (16) (39) 

FTLLIC J14R-CF1L CAFCI10C A 

r-rni AD^NO^ A 3 (8%) t-r"iL cfhciNo m 1 (6») 1 (3*) 

#PANrFF»"I C ISLETS (19) (46) ISL'l-C^LL ADENOM A 1 (e

>%) 5 (11*) 

*PF-otJTIP L 3LAN D (2C) (50) 

CUCI"0«A,NO S 1 (2X) 

»-"<:TI<- (20 ) (49) INT rS"-I m IAL-CFLL T"noc 13 (<)( %) US (981) 

N^BVCM S J-Y^T" ^ 

NON­

» NUH3~ P <'p A'JI^IAL1- HIH TISSDP "XMII1FD llnSCSCOPICALLY 

HIGH DOSE 

11-1121 

(48) 

(49) 

(49) 

1 (2%) 

(U9) 

1 (2*) 

(47) 

1 (2«) 

(48) 

5 (10*) 

(43) 

1 (2*) 

(50) 

(5") 

(5")

49 (98%) 

A-4 
TABLE A1 (CONTINUED) 

CONTPO L (UKTE) LOW DOSE HIGH DOSE 

11-1125 11-1123 11-1121 

SPECIA L S'N'S­ ORGAN' S 

NON " 

HUSCHLOSK^L^TA L SYFTF H 

NON " 

BODY CfVITIF S 

*APDOMINJL CAVITY (21) (50) (50) 

N'JSO'rHFLIOllA, NOS 2 (UK ) 

*TtTNIC A VASINALI S (20) (50) (50 ) 

«!?SOTH' 7LIO"IAr NOS 1 (2%) 

A LL OIHE P SYSTEM S 

NON E 

SNIPA L DISPOSITIO N SOH^AF V 

HSIHJL F INiriALL Y IN ST'JDY 20 5H 5Q 

NATURA L DTATH i 1 U 3 

MORIBUN D SACRIFICE 3 3 1 

SCHFDULF E SACRIFICE 

JCCIDTDTALL T KILIT 

TSRHINA I SACFIFICF 13 «3 «6 

ANIMA L (<ISSIN 3 

* KtlBBF P OF ANI1AL S KIT H TIFSIIE EXAMIMF D H ICBOSCOPICA LLY 

* MJ1SFF nF ANI1AL S NFCFOPSIF D 

A-5 
TABLEAl (CONCLUDED)
 

CONTRO L (OHTP) L0» DOS? HIGH DOSE 

11-1125 11-1123 11-1121 

T ^ T A L f'JIPAL. S HITd PPIHAS Y "UMORS* 20 «9 5"

 

TOTA I P'illAT Y TU(<^PS 29 • 7» 67 

rrTH L ^'IIl•^Lc

; KITH B"NIGN Tn«oES 19 U9 50 TV)T fl I 1EVIG N TIT»OI>S 21 68 62 

TPT» L f.NTHHL S WIT H MILIGNPN T TIIBOPS 5 5 3

 T-OT fl I 1ALId.1AK T TUfiOF S 5 5 3

 

TO"1?!. ANIHAL S WITH SECONDL Y TnflOSS* 1 i OT n I s^rnN^sn y TH»O»S 1 

T^T4 L fNIIAL S fclT H T010PS UNCFBHIN-

RT^ \ IG M OS "ALIGNJN T 1 2

 

Tm'A 1 HNCFBTM N TUMOP S 1 2

 

JIM«M.S KITH T(J»1OPS DHCEBTAINp fi^TPSTATIC

 

TT^II UNCFRTAI N THKOP S 

* P-I«.A:< Y TUIOftS : »LI. TUKOP.E EXCEPT SECONDA'tY TDB3PS 

« s-mnD«r Y •"'jnoFS: HFIAST^TIC THROBS O R TOIOFS INVASIY E INTO A N ADJACEN T ORGA N 

A-6 
TABLE A2

 

SUMMARY OF THE INCIDENCE OF NFOPLASMS IN FEMALE RATS TREATED WITH 3-CHLORO-p-TOLUIDINE 

»MF?L S INITIPLL Y IN STUDY

ANT*»L S KFCrOFSIE T

ANI"H S fXA^INt D HI STOP1THOLOGICALLY* *

mTTG'IfMfP T SYSTE M 

*'5KI M

FIBKjSaProH \

"USFTFATOF J SYST^ H 

Hit'*.";

JLVOLf/BPINCHIOLS' ' ADENON 8

nlViri AP/BFO»'ChIOLJ P fASCINOI A

C-r^LI Cft^CINONA , r"TA3TI>TI C

Hf1AT3POIF T I C StST^I 

*«nL' T 'IPIf OFGAN S

1^LI^^IA^ T LY«PHO?"» ,

*?rj,"NTr ^o S (jLf

M'SOTH^LIOKA , NOS

»OT"->n s

I«ALI3NP» T LY1PHOMA ,

CIPCMHTrP Y SYSTEM 

NON " 

DIG" < !TIV'r SYST'« 

»LIV- »

 <us

 N"5

CONTFOL (UNTP )

11-1126

 2"

 2?

 19

 (20 )

 (19)

 (2")

 1 (5*) 

(19)

 (19 )

 (18 )

 LOU DOSE HIGH DOSE 

 11-112U 11-1122 

 50 50 

 50 50 

 50 50 

 (50) (50) 

1 (2%) 

 (U8) (50) 

1 (2*) 1 (8*) 

1 (2%) 1 (2X) 

1 (2%) 

 (5") (50) 

 (ag) (50) 

1 (2X) 

 (50) (50) 

1 (2») 

 (51) (50) 

1.J2S1­

» NJIMB^ P UP ANIMAL S HITrt TISSUE FXABI15D MirEOSCOPICA LLY 

* tillxBT p OF ^NIHAL b N'FCPOPSIf P 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-7 
TABLE A2 (CONTINUED) 

CCINTBO L (UNTP ) LOB DOSE 

1 1-112f 11-1 1214 

«U"Ili»F Y BLAOD5 1 (18) (15)

 

PAPILLOM , NO S 1 (2X) 

FNDrCRIK r SYSTT 

IPITIITAF Y (17) (1*2)

 

CH^O^npH O JF fln^KOFA U (2U* ) 10 (214*) 

• ATP^NA L (19 ) (149) 

PH^or HSCrtOCYTOI A 1 (2*)

 

tTHYROJ C (15) (13)

 

C-CFLL UTRNOX J 1 (7» )

 

C-C-I L CARCINOM A 

«PA'iCtr»TI C ISLETS (19 ) (149) 

ISLrT- c -:LL ADiNOM J 1 (2X)

 

pi:pc^ L )UrTrv T SYSTE M 

*^A»i1»r Y GIAN D (20) (50)

 

FIBroAD t "(013 1 (5X ) 6 (12*) 

*P?T-prjTI«L ^LAN D (21) (51)

 

33UHCHS CfL L CATIMOIf 1 (2% )

 

• IITF'DS (19) (50)

 

A DF NH M ft, NO S 

fi n^NOCA^^INOH A , NC S 2 (US) 

' NDOWTTH L STHOKA.L POLY" H (8%)

 

• (-""VIX HT^R I (19) (5")

 

FlPPCiw f 

• OVJR Y (19) (19)

 PAPILLA . "-Y CYSTADENOC^PCTNOI S ,uos

 

(18) (1*9) 

JLIOfA ^ NOS 1_J2.SI_ . 

* NUMB"! - f^ ANIMAL S WlTd riSSU" FXAHINP i 1ICFCSr nPICA LLY 

* NU-^P OF ANIMAL ? NFCimrSII D 

HITH DOSF 

11-1122 

1

(15)

9

(50) 

(1*7) 

1

1

(1*8) 

(51) 

3

(50) 

(50) 

1

9

(51) 

1

(18) 

1

(149) 

 (2*) 

 (2f< ) 

 (2*) 

 (2*) 

 (6X) 

 (2%) 

 (18*) 

 (2*) 

(2%) 

A-8 
TABLE A2 (CONTINUED) 

CONTROL(ONTE )

11-1126 LOU DOSE 11-1121

 HIGH DOSE 

 11-112 2 

I«ENINGION A 1 (2*) 

SPECUL SENS" OPG4N S 

NON! 

BUSCULOSKPLF'TAL SYSTIf! 

•JONF 

BOOT CAVITIP S 

NONE 

A LL OTHEP SYSTEMS 

NON E 

ANINf L DISPOSITION SUMMAR Y 

»NI"1*LS INITIALL Y IN STUDY 2" SO ^ 

NATDRAL DEATHS 2 3 1 

dOEIBIINP SftCRIFIC'' 1 3 1 

5CHEDDLED SACRIPICF 

ACCIDENTALLY KILLED 

TER1INAL SACRIFICF 17 «l» HB 

ANIMA L BISSIN3 

* NITMBFR OF HN11ALS KITH TISSdF "XAflNE D MICROSCOPICB LLY 

» NnfBFP OF 8NI1ALS NECEOPSIED 

A-9 
TABLE A2 (CONCLUDED) 

CONTPOL (ONiE) LOW DOS" HIGH DOSE 

11-1126 11-1121 11-1122 

Tt'M^P SU11AF Y 

TOTH L »NIBAL S WIT H PFIRKR Y TUBOPS* 7 2 2 28 

TOTH PKI1AR Y T11NOPS 7 33 33

 

TC^L fNIIAL S WIT H BFNIG N TDMOF S 6 19 25 

T1T»L GllilGK TnilPF" 6 24 29

 

TOTU ANIMAL S WITH "'IISNAM TUMOR S 1 7 3 

TOTM "JIIGNAN T TU»nRS 1 8 3 

TOTAL JMJULS WITH SFCONBARY TIIMOPSI 1 

TOTAl STCONDAR Y TUMOR S 1 

T/1TA1 ANIMAL S WITH TfllOFS I'NTFBTAIN -

DFNIG X OF ''ALIGNAV T 1 1 

T1TAI MNCPt<TJI N TUMQPS 1 1 

TOTAI ANIMAL S hlT H 

PFI1AR Y Or 

TTTAI U N 

* P C I1A" Y TIJ10BS: Al l TtllOtlf "XCFPT STONDAIiY TtlMPFS 

* S rCOfT-fF I TUBOFS : (1F1SSTA1IC TUMOP S OB TU10FS INVASIV F INTO AN ADJACEN T OPGA N 

A-10 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH 3-CHLORO-p-TOLUIDINE
 

TABLEB1

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH 3-CHLORO-p-TOLUIDINE 

CONTROL (UNTR) LOU DOSE HIGH DOSE 

22-2125 22-2123 22-2121 

ANIMAL S INITIALLY IN STUDY 2« 50 SO ANIMAL S HISSIN G 1 1 ANIMAL S NFCROPSIFD 20 49 49 ANIMAL S EXAMIOE D HISTOPATHOT OGICALLY** 20 49 49 

INTEGOPENTAF Y SYSTEM 

*St!BCtIT TISS'IF (20) (49) (49)

 FIBPOSUPCOM A 1 (2X)

 

RESPIRATOR Y SYSTM 

• LUN G (20) (49) (49) NFOPLASM . NOS, NETFSTATIC 1 (2X) ALVFOLAP/BPONCHIOLAP . AD*NOB> 3 (6*) 3 (6«) 

HFMATOPOIFIIC SYSTEM 

*"ULTIPLF OR3AN S (20) (49) (49) HULIGNAN T LYMPHOMA . NO S 1 (2*) 

LE!JK*MIA,NO S 1 (2*) 

fSPLEF N (19) (40) (46)

 fNGinsurcoM A 1 (3X)

 

tSHALL INTESTINF (20) (47) (49) mLISmNT LYMPHOfA . NOS 1 (2*) 

CIRCHIATOPY SYSTE M 

NON F 

DIGFSTI7F SYSTE M 

ILIVP (20) (47) (49) 

NE2fiiSlli_NOSx_aiLJSiIifil 1 iZtl 

* NUUBF P OF ANIMAL S BITH TISSU? EXAMINE D MICROSCOPICALLY 

* NDM8F P OF SNIBAL S NECROPSIFD 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-3 
TABLE Bl (CONTINUED) 

KSPATOCPLLULAR SDEFONA

HJPATOCELL'JLAR CAPCINONA

H"Pi!'rOBI.ASTOMA

ANGIOS^RCOMA

CONTROL(UNTR)

22-2125

 1 (5X)

 3 (15X)

 LOS DOSE

 22-2123

 » (9%)

 6 (13X)

 1 (2*) 

1 (2X) 

 HISH DOSS 

 22-2121 

U (811) 

3 (6*) 

UPINAPY SYST'1 

FNDOCPIHF SYSTF1 

• THT^Oir

FOLLICHIAF-CELL ADFNOHA

 (20)

 1 (5%) 

 (36) (U3) 

RFPPODUCTIVF SYSTEM 

*T?STI5

INT^FSTITIAL-CFLL T'ldOR

 (20) (US) (48) 

1 (2X) 

NEPVOWS SYSTF.1 

NONE 

SFFCUL SENS' ORGANS 

NON* 

HUSC"tOSKFLFT»L SYSTEM 

NON? 

BODY CAVITIFS 

NON1! 

ALL OTHER SYSTFHS 

NONE 

I NUMBFE OF ANISALS VITH TISSUE EIAKINED HICROSCOPICALLT 

* NOPBEF OF ANIHALS NFCROPSIFC 

B-4
 
TABLE Bl (CONCLUDED)
 

CONTROL (OUTS) LOW DOSt HIGH DOST 

22-2125 22-2123 22-2121 

ANIHA L DISPOSITION SOBflAR Y 

AIIIHJLS INITIALL Y III STODT 20 50 50 

NATOPA L DEATH* 2 1 

NURIBUN D SACPIFICF. 3

 

SCHFDULED SACRIFICF 

ACCIDENTALLY KILLED 

T'PBINAL SACPIFICE 20 U K »8

 

ANIMA L MISSING 1 1

 

1 INCLUDFS ADTOLYZF D HKIHAL S 

TOHOF Sn(I«A8I 

TOTAL JNIHAL S »ITH PPIMAR T TDBOES* 5 15 1»

 TOTAL PEIHARI TDNORS 5 13 14

 

TOTAL ANINAL S VITH BENIG N TDBOK S 2 7 8

 

TOTAL BEHIG* THHOF5 2 7 8 

TOTAL AHIBAL S VIITH MALIGHAN T TOBOES 3 8 6 

TOTAI NALIGNAH T IUROBS 3 11 6 

TCTHL ANIHALS KITH SFCONDART TOflOSSI 1
 

TOTAL SfCOKDARI TUKOPS 1
 

TOTAL ANIBALS KITH THHORS DNCERTAIH-


BFNIGN OP HALIG*ANT
 

TOTAL UKCFFTAI* TUHOPS
 

TOTAL ANIHALS WITH TOMOBS BKCEFTAIN-


PPIHJ1RY OR M"TASTATIC
 

TOTAL ONCFETAIN TOBOPS
 

* PPI1APY THHORS: ALL TUMORS EXCEPT SECONDARY T010BS
 

* SECONDARY TOHORS: MFTASTATIC TDHOSS OR TUNORS IHVASITF INTO All ADJACENT ORGAN
 

B-5
 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED WITH 3-CHLORO-p-TOLUIDINE 

CONTROL (tJNTB) LOB DOSS HIGH DOSE 

22-2126 22-2121 22-2122 

ANIMAL S INITIALLY IN STUDY 20 50 50 

ANIMAL S MISSING 3 

ANIMAL S NSCPOPSIED 20 50 U7 

ANIMAL S FXAMISFD HISTOPATHOLOGICALLT** 2P 50 U7 

INTEG'IMFNTAF I SYSTFH 

NON E 

RFSPIRATPBY SYSTEM
 

«LUN G (20) (50) (16) 

ALVFOLAP./BP.ONCHIOLAR ADENOMA 2 (1PX) 3 (7%) 

ALVEO1 AP/BROHCHIOLAR CAPCINOM A 1 (2*) 

HEMATnPOI^TIC SYSTEM 

• HUI^IPl? ORGAN S (20) (50) (17) 

MALIGNAN T LYMPHOMA , NOS 1 (5X) 2 (»*) 

M,\LIG.LYHPHO«A , HISTinCITIC TYPE 1 (5X) 2 (4*) 

LEnK"MIA,NO S 1 (2J) 1 (2») 

ILUN G (20) (50) (*6) 

• ALIGNAN T LYMPHOH9 , NOS 1 (2*) 

CIPCriLBTOEY SYSTEM 

NON" 

DIGESTIVr SYSTEM
 

tLIVPP (20) (»9) («5) 

HTPATOCFLLULAR ADENOM A 1 (2») 2 (UX ) 

H"PfTOCFLLOLAR CPRCINOM A 3 (6*) 

UPINA" Y SYSTEM 

..NOS?. 

t NUMBF " OF ANIMALS WITH TISSOE EXAMINE D MICROSCOPICALLY 

* NUM815K OF ANIMAL S NECEOPSIED 

•EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-6 
TABLE B2 (CONTINUED) 

CONTROL(UNTR ) LOW DOSE HIGH DOSE 

22-2126 22-212* 22-2122 

EltDOCBIHF SYSTEH 

*PITniTART (9) (3tt) (32) 

CHPOBOPHOBE ADEHOIU 1 (11X) 

• THtROID (17) (12) («3) 

POLLICOHB-CELL ADFHOPf 1 (2») 

RFPfi"DOCTIVP SISTEH 

•OVAR Y (19) (3»| (30) 

P^PILLJRI CYST4DEHOH*. NOS 1 (3*) 

TNTOBA 1 (3*) 

NFRVOU S SISTEH 

NON " 

SPJCIAL SFNSJ ORGAH S 

XOSCnLOSKFLFIAL SISTFfl 

BODY CAVITIE S 

*HFSEHTFRT (20) (50) (U7) 

LIPOfIA 1 (2») 

ALL OTRfP STSTIIIS 

HOH" 

t NUHBE F OP AHIHALS »HH TISSUE EX»HINE D HIC'OSCOPICALLI * »Of"E» P 0? i»III»LS KECPOPSIPD 

B-7 
TABLE B2 (CONCLUDED)
 

CONTROL(ONTE) LOM DOSE HISH DOSE
 

22-2126 22-212U 22-2122
 

AKIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STDDY 20 50 SO NATURAL DFATHS
 

MOPIBUND SACRIFICF
 

SCHPCDLFD SACRIFICF
 

ACCIDENTALLY KILLED
 

TERMINA L SACRIFICF 20 U K UK 

AVIMA L !"ISSIN5 3 

3 INCLODyS MJTOLYZED ANIMAL S 

TUMO R SORHAfl Y 

TOTAL ANIMAL S SITH PFIHAR Y TDMOSS* "5 6 11 

TOTAL PRIMAR Y TUIIOFS *> 10 11 

TOTJL ANI1JLS KITH BFNIGN TDHORS 3 2 7 

TOTAL BHNIGN TtlMORE 3 3 7 

TOTAL ANIMAL S WITH 1ALIGHANT TOMOBS 2 6 U 

TOTAL 1AHGHANT TDMORS 2 7 * 

TOTAL ANI»ALS WITH SFCONDARY TDMORS*
 

TOTAL SECONDARY TOMOES
 

TOTAL ANIMULS KITH TUMORS OHCEHTAIN-


BENIGN OR HALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTJL ANIKMS WITH TU10F.S ONCEFTAIN-


PSIMAPY OP SFTASTATIC
 

TOTAL nUC^BTAIN TUMORS
 

* PRIMARY TU10RS: ALL TOMORS FICFPT SFCONDARY TUMORS
 

» SFCONDAFY T1JMORS: MFTH-JTATIC TOMOBS 0" TUMOPS INTASITH INTO AH ADJACENT ORGAN
 

B-8
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 3-CHLORO-p-TOLUIDINE
 

TABLE Cl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

TREATED WITH 3-CHLORO-p-TOLUIDINE 

CONTFOL(UNTH) 

11-1125 

LOW DOSE 

11-1123 

HIGH DOSF 

11-1121 

ANIMAL S INITIALLY IN STUDY 20 

AMBALS VFCPOPSIFB 20 

ANIMALS EXAMINED HI STOF'THOLOGICAILY ** 2? 

50 

SO 

U9 

50 

50 

50 

INT'G'JKFNTAP Y SYSTFM 

NCN 2 

FFSFI'tATOFY SYSTE M 

IIUNG/EVCNCHOS (20) (48) (SO) 

BRONCHIPCTASI S 1 (2X) 

• Il'NG (20) (U8) (50) 

AfFLFCTASI S 1 (5X) 1 (2X) pN"Hf«ONiA , ^HRONI C IURIN F t (20%) 20 (t2%) 11 (22X) 

HYPFPTLHSIA , ADENOPATOn S 1 (2«) 

HFMATOPOIFTI C SYSTFM 

•SPLEFN (20) («7) (51) 

CONST-STION, NOS 1 (2*) 

H^'ATOPOIESIS 1 (21) 1 (2%) 

HYPOPIASIA , IYMPHOI D 1 (2*) 

«Sfl"NIC CAPSUL F (2n

) («7) (51) 

FIBPOSI ^ 1 (2%) 

FIBFOSIS, F1CM 1 (2*) 2M (148%) 

HYPFFPLASIA , NU S 1 (2*) 

tCFRVICA L LYMP H NOD E (16) (38) (39) 

INFLAMMATION . NO S 1 (3*) 

»MFSFNTHPI C L. NODF (16) (38) (39) 

CYST, NOS 1 (6*) 

N^^POSJ^j ....» _..____..__..___—...__..____.«._­ . N0^_ _ , — _— 1-I2SL 

» NUMB^ P OF ANIMAL S MTU TISSUF F.XAMINF D MICROSCOPICALL Y 

* NUIB^ F 0" ANI1ALS NFOOPSIFD 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-3 
TABLE Cl (CONTINUED) 

HIGH DOS? 

11-1121 

(50) 

1 (2%) 

1 (2%) 

(5P) 

(50) 

35 (70%) 

(50) 

(51) 

1 U*l 

(50) 

6 (12%) 

(49) 

2 (4%) 

(49)

7 (14%) 

(49) 

33 (67%) 

(49) 

1 (2%) 

(48) 

— -—— -i— — — —— .-•»»»— _u__u_— Oil IUJCIPCULATCP Y SYSTFP 

««YOCAFDIU 1 

INFLAMMATION , NO S 

FIBROSIS, DlttVST 

DFGFFFRATIDN , VOS 

HYP^TOOPHY , NHS 

*P!ILMONAr Y AP.TFPY 

TIYPHTI-OPHY, NOS 

DIGFSTIVF SYSTF.H 

fLIVF P 

H'CP^SIS, FOCAL 

n'STfosmosis FJTTY 

tUViB/fPIPOpTAL 

FIBPOSIS 

#BIL* DtlC^ 

CYST, MOS 

HYfEFPLASIA , NOS 

»PANCPFf"lr *CINUS 

ATPOPHY , NOS 

• SMAL L INTFSTIN F 

HYPEPPHSIA , LYfPHOI D 

*IPRo F IFThSTIN" 

H^flA^ODIASI S 

IJSINPP Y SYSTF M 

*KIDVY 

INFLSflATIOtl , CH90KIC 

tKID^Y/COWX 

H^NOFPHATI C CYST 

FSDorsi»r SYSTf 

• ADTNA L COPTEX 

» NUI"»FR °F ^NII1ALS WITH TISSUE *

 NI!«"1T-'? OF ANIHHL S NFCPOPSIED 

COHTTOL(DNTR) L0» DOTF 

11-112* 11-1123 

(20) (47) 

1 (2») 1 (5») 

1 (2%) 

(20) (SO) 

1 (2%) 

(20) (47) 

1 (2%) 

2 (K«) 5 (11%) 

(20) (47) 

1 (5«) 

(20) (47) 

1 (5*) 2 (4%) 

(19) (46) 

1 (5%) 5 (11%) 

(20) (47) 

1 (5%) 3 («X) 

(19) (46) 

4 (21%) 11 (24%) 

(20) (49) 

13 (65%) 33 (73%) 

(2C) (49) 

(18) (47) 

FXXMIHI D (1ICROSCOPICH LLY 

C-4 
TABLE C-1 (CONTINUED) 

CONTROL (OHTR)

11-1125

 LOi DOSE

 11-1123

 HIGH DOS1 

 11-1121 

*J>DR*m> L HFDULL A

HYr*P.PL»SIA , NOS

• THY'PI D

HYTPBLASTA , NOS

»PA"CR"»Tl<~ ISLFTS

HYPEfiPL»SIA , NOS

 (13 )

 (16 )

 (19)

 (17)

 1 (2X) 

 (39)

 1 (3*) 

 («6)

 1 (2*) 

 («8) 

 (13) 

 (50) 

F^PFCCUCTIVF SJ<!TFI« 

»PFOST«T E

INFLAMMATION , NOS

NOS

HYP^FPLASIA, IN1FFSTITIAL CFLL

 (20)

 1 (5»)

(20)

 (37)

 1 (3*) 

 (H9)

 1 (2X) 

1 (2*) 

 («8) 

 (5") 

NTRVOHS EYSTEM 

NON"1 

SPIC1SL S^NSF ORGANS 

1OH' 

lUScniOSKLLETAL SYSTEM 

BODY 

tM'S'NTi'PY

N"C"OSIS, FAT (20)1 I6'*) (50)2 (14*) (50) 1(2%) 

ML PTHF.P SYSTEPS 

THORAX 

IF ANI1PL?

OF ANIMAL S

 WITH TISSU"

 NTC"OPSIPD 

 FXABIN^ D H ICECSCOPICALLY 

C-5 
TABLE Cl (CONCLUDED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE' 

11-1125 11-1123 11-1121 

'BSCESS, NOS 1 

SPFCIAL SOBFHOLOGY SUH><AJ> Y 

AIITO/NECSOPSY/SO HISTO 

OF ANIMALS WITH TISSHE EXAKIWD NIC8CSCOPICAILV 

» NURBF P OF ANIMAL S KFCROPSIED 

C-6 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

TREATED WITH 3-CHLORO-p-TOLUIDINE

 

CONTPOL (UKTF) tO» DOSE HIGH DOSF 

11-1126 11-1124 11-1122 

ANIMAL S INITIALLY IN STUDY 20 50 "H> 

ANIMAL S NFCPOPSIED 2P 50 "SO 

ANIMAL S FXSflNE D HISTOriTHOLOGICALLT** 19 50 50 

INTFGUMFNT1R Y SYSTFt! 

NON " 

RFSPIBATOP Y SYSTEM 

«LUN> 3 (19 ) (»8) (50) 

BTFl'CTASIS 1 (2*) 

FDFHA, NOS 1 (2*) 

PI'OIONH, CHPONIC HnilNI' 2 (11K ) 7 (15H) 8 (16*) 

ILUNG/ALVFOL I (19) (<»«) (50) 

HYPTPLBSIA , BDFKOMATOO S 1 (2») 

HFIBT^POIFTIC STSTT! 

• SPLEF N (19) J«9> (50) 

HSUOSIDFROSI":: 2 (««( 1 (2<) 

HffATOPoiFsis 5 C\n%) 

*SPIFHIC CAPSOLF (19) (<•») (50)
 

CYST. NOS t (111) 2 (»X)
 

"IBROSIS 3 (6*)
 

F1EPOSIS. FOCAL 3 (CS) 3* (68«)
 

CIPCUIATORY SYSTF>(
 

IfYOCABDIUr (19) (50) (50)
 

INFLAMMATION, FOCAL 1 (2%) 1 (2*)
 

FIBPPSIS 1 (5X)
 

DIGfSTIVF SYSTFM
 

ILIVFP (18) (50) (50)
 

t NnflB^ F OF ANIMAL S KITH TISSUE FIABINED SICROSCOPICMLI 

« NOHBF P OF ANIMAL S HFCROPSIFD 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-7
 
TABLE C2 (CONTINUED)
 

CONTPOL(IJNTE) LOW DOSE 

11-1126 11-112« 

INFLAMMATION , NO S 1 <2X) 

PIBPOSIS, FOCAL 1 (2X) 

DEGFNFRJTION , NOS 

N*CPOSIS, FOCAL 1 (2«) 

1FTAKORPHOSIS FATTY 

BASOPHILIC CITO CHfNG1 ? 

ANGITTASIS 

1 (6*) 

t (8*) 

5 (10%) 

OBII " DDCT (18) (50) 

HYPEFPLASIA , NOS 1 (6*) 1 (2%) 

IPANCPF.f'Ic ACINU S (19) (119) 

ATPOPHY , NOS 1 (5%) » (8)t) 

»SMAL L INTFSTINE (19) (50) 

FIBROSIS, FOOL 1 (2«) 

HYPFFPLASIA , IY1PH0ID 1 (5X) 5 (15%) 

»LAPG " INTESTIN" (19) (<(9) 

N"CATODIASI S 2 (11%) 12 (24%) 

*SFCTUH (20) (50) 

NECPOSIS, NOS 1 (2%) 

riFINAH Y SYSTEM 

*KIDNf;Y (19) (50) 

INFLANHATON , CHPONIC 2 (11%) 7 (U%) 

D'GENFBATIO N PIGBFNTAR Y 1 (2*) 

ENDCCRIN r SYSTEM 

*PITniTAP Y (17) (12) 

CYST, NOS 1 (2%) 

»ADR5NA L (19) (U9) 

H"BOPPHAGIC CYST 1 <5«) 1 (2%) 

LIPOIPOSIS 1 (5X) 2 (»%) 

IADHENA L CORTEX (19) (»9) 

HYPFPPLASIA , FOCAL 

HCP^NSL MEDULL A (19) (»9) 

CYST^ NOS 

*	 NUMBFF OF ANIMALS WITH TISSUE FIAMIHFD 1IC30SCOPICALLY
 OF ANI1ALS NECBOPSTED
 

HIGH DOSE 

11-1122 

1 (2%) 

3U (68%) 

1 (2%) 

1 (2%) 

(50) 

(«8) 

It (8%) 

(»9) 

(U9) 

6 (12%) 

(50) 

(50) 

16 (32%) 

(«5) 

(50) 

(50) 

1 (2%) 

(50) 

..... 1 (2S1 

C-8
 
TABLE C2 (CONTINUED) 

CONTROL (ONTP) L0» DOSE 

11-1126 11-112t 

tTHYHOID (15) (113) HYPERPLASIA, NOS 1 (2X) 

HFPPCCUCTIVF SYSTEM 

*MAH1AP Y GLAN D (20) (50) CYST, NOS 2 (UX) 

*PRrPUTIAL (3LAND (20) (50)

 ABSCESS, NOS

 

ftJTERU S (19) (50) HYDFPMFTR A 1 (2*) CYST, IDS 1 (2X) INFLAillATION , NOS 3 (16X) PYOflFTRA 

INFLAMMATION , CHFONIC 

• nT^HUS/ENDONFTRIU M (19) (50)

 HYPIPPLASIf , CYSTIC 1 (5X)

 

tOVAR Y (19) (19) CYST, NOS a (21*) 1 (2X) PAFCTARIA N CYST 2 (U*) NECROSIS, FAT 1 (2*) 

NE"VnS SYSTEM 

NONI ? 

SPECIAL SF.NST ORGAN S 

NON E 

HUSOJLOSKFLETA L SYSTFtl 

NONF 

BODY CAVITIE S 

NON E 

» NDNBE R OP ANIMAL S WIT H TISSOE EXAMINE D MICROSCOPICALLY 

* tiUMBFP OF UNIMAL S NFCP.OPSIF.D 

HIGH DOSF 

11-1122 

(U7) 

(50) 

(50) 

1 (2») 

(50) 

2 (»*) 

2 (U«) 

1 (2X) 

(50) 

(18) 

2 (IX) 

U (8X) 

C-9 
TABLE C2 (CONCLUDED) 

CONTROL ((TNTR) LOU DOSE HIGH DOSE 11-1126 11-112« 11-1122 

ALL OTHFP SYSTFHS 

NONE 

SP^CIAI PORBHOLOG Y STT1KARY 

NO LFSION REPORTE D 1 4 • 'JTO/M^CliOPSY/HISTO PEPF 1 

M!TP/N"CHOPSY/NO 

* NU13T OF ANIMAL S WITH TISSU? EXAHTHE D HICPOSCOPICSLLY 

OP ANIMAL S NECPOPSIED 

C-10 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 3-CHLORO-p-TOLUIDINE
 

TABLE Dl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH 3-CHLORO-p-TOLUIDINE 

CONTPOL (0NTP) LO» DOSE HIGH DOS'? 

22-2125 22-2123 22-2121 

ANIMAL S INITIALLY IN STUDY 20 50 50 

ANIMALS MISSIN G 1 1 

ANIPALS NECROPSIFD 20 19 09 

ANIRAL S FXAMINE D HI STOP* THOLOGIO LLY ** 20 49 19 

INTBGIJMFNTAR Y SYSTEI" 

*SKIN (20) (19) (49 

INFLJ1HATION , POCAI 1 (2«) 

SCAPIASIS 1 (5%) 

RESPIRATOR Y SYSTEB 

•LUN 5 (20) (49) (49) 

P1"U«ONIA, CHRONIC MORINT 2 (I"*) 5 (10J) 7 (14X) 

PNFOflONIA INTERSTITIAL CHRONIC 3 <6X) 

INFLAMMATION , CHRONIC SUPPORATIV 1 (2%) HYP'RPLASIB, EPITHELIAL 1 (5*) 2 {»%) 1 (2%) PPITHFLIALIZATION 1 (2X) HYP'RPLASIA, LYHPHOIP 2 (4X) 

HEHATOPOIETIC SYSTEM 

tSPLEEN (19) (40) (46) 

CONOFSTION, NOS 1 (3») 1 (2X) 

AMYLOID , NOS 1 (5X) 

HYP"PPLASIA, RET1COLUH CBLl 2 (4%) 

HYPPRPLASIA, LYHPHOID 1 (5X) 2 (5*) 4 (9«) 

• LYNP H NOD" (13) (39) (43) 

HYPP^PLASIA , RKTICIIWIM C'LL 1 (2%) 

HYPERPLASIA , LIMPHOID 1 (8«) 

*MESFNTPRIC L. NODE (13) (39) (43) 

INFLAMMATION . HfMOtBHAGIC 1 (8«) 1 (3X) 

HYPERPL»SIA , RETICI1LOM C?LL 3 (7%) 

HYPIBfLASIA. LYMPHOID 

* NUflBFR OF ANIMAL S KITH TISSUE EXAHINED MICROSCOPICALLY 

» NDMBFR OP ANIMAL S NFCROPSIFD 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

0-3 
TABLE Dl (CONTINUED)
 

CONTROL (UNTB) L0» DOSE HIGH DOSE 

22-2125 22-2123 22-2121 

CIRCULATOR Y SYSTEM 

• MYOCAPDIO B (20) (IB) (48)

 

INFLAMMATION , FOCAL 1 (2X)

 

DIGFSTIVF. SYSTEM 

*LIV!R (20) (47) (49) 

INFLAMMATION , ACUTE 1 121) 

NECROSIS, DIFFIISF 1 (2X) 

fTOATOCYTOHEGALY 1 (5X) 2 (4X) 

ANGIFCTASIS 1 (5%) 

• PANCREA S (20) (45) (48)

 

INFLAMMATION , NECROTIZING 1 (2%)

 

fS"ALL INTESTIN" (20) (47) (49) 

INFLAMMATION , NOS 1 (2X) 

HYP^BPLASIA , LYMPHOID 1 (5X) 1 (2X) 

tLAPG E INTESTINE (2f) (47) (49) 

NEPATODIASI S 3 (6*) 6 (12X) 

HYPFFPLASIA . LYMPHOI D 1 (5X) 

tlPINAR Y SYSTEM 

• KIDNE Y (20) (48) (49) 

GLONFRdLONEPHRITIS , BFNBPANOD S 1 (2X)

 

*RFNA L PAPILLA (20) (48) (49) 

I")FLAM«ATION , NF.CFOTIZING 1 (2«) 

FNDOCRIN F SYSTEM 

• PANCREATIC ISLFTS (20) (45) (48)

 

HYPL5PLASIA , NODULA R 1 (2X)

 

RFFPCmCTIV F SYSTEM 

*SEMINA 1 VTSICLF (21) (49) (49) 

* NUMBER OF ANIMALS SITH TISSUF EXAMINED "IICROSCOPICALIY
 

* N1"BEP OF ANIMALS NECPOPSIED
 

U-4
 
TABLE Dl (CONCLUDED) 

CONTROL(ONTR) LOI DOSE HIGH DOSF 

22-2125 22-2123 22-2121 

•TFSTIS (20) (H5) (U8) 

DEGENERATION, NOS 1 (5*) 3 (6») CTROPHY, NOS 1 (2*) 

ITESTIS/TUBOLE (20) (15) (»8) 

DEGEP^HTICN, NOS 1 (2%) 1 (2*) 

NFRVOUS SYST^l 

• BRAIB HYDFOCFPHALOS, INTFFMAL

CORPORA A1YHCFA

 (20)

1 (5%) 

8 (UPS)

 (118)

 11 (29*)

 (1*9) 

 1« (29%) 

SPFCIM EFNSF OPGANS 

NON*: 

HUSrHLOSKElETAL SYSTF1 

NONF 

BODY CAVITIES 

NOM" 

ALL OTH"E SYSTF^s 

SDIPOSF TISSO" 

HIN"PAIIZHTION 1 

SPECIM MORPHOLOG Y SnMMAR Y 

NO LFSI01 PEPOFTF D 3 11 10 

ANTllf L 1ISSIN3/NO NECROPSY 1 1 

» NUBBFR OF ANIMAL S KITH TISSUE "JUMINFD HICFOSCOPICALLY 

» NOKBF P OF ANI1ALS KFCBOPSI"D 

U-5 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH 3-CHLORO-p-TOLUIDINE 

CONTROL (UHTR) LOR DOSE 

22-2126 22-212t 

ANIMAL S INITIALL Y IN STCIDY 20 50 

ANIMAL ? KISSIN G 

HNI«AL S KFCPOPSIF D 20 50 

ANIMAL S FXA1INF D HISTOPATHOLOGICALLY** 20 50 

INTFGUMF f TAFST SYS1FM 

NON " 

RFSPI^ATCPY SYST^P
 

»IUNG/BFONCHUS (20) (50)
 

BBONCHIECTASIS
 

«LUN ; (20) (50) 

PNTHPONIA , CHRONIC MUPIN " 2 (10X) 10 (20X) 

FTP°OSIS, DIFFUS F 

HYPEFTLASIA , EPITHFLIA L 1 (2X) HYPFFPL P SIA, LYMPHOI D 

tLDNQ/ALVrOL I (2") (50) 

HYP"EPLASIA , AC"NOrATnu S 

HFfATOPOI' :"riC SYSTFH 

(19) (HI) 

HFnnsitiFaosis 1 (2*) 

•SPL^FN (18) (111) 

H'nosio- ROSIS 2 (11%) 3 (7*) 

HYP'FPLf SIA. SOS 1 (2X) 

HYP^FPLASIA , KFTICOLU P 1 (2X) 

IIYPFBPLASIA , LYHPHOID 1 (6%) 6 (15X) 

H>=:nATPPOIF.SIS 3 (7X) 

• LYMP H KOD F (12) (K 3) HYP r q PlASIA , NOS 1 (2X) 

OF ANI1ALS WITH TISSDE EXAMINE D MICROSCOPICALL Y 

NnngFt OF ANIMAL S NECROPSIPT) 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

HIGH DOSE 

22-2122 

50 

3 

87 

<I7 

1 (2X)
 

(«6) 

17 (37X) 

1 (2X) 

1 (2X) 

(«6) 

1 (2X) 

(13) 

(«5) 

3 (7*) 

2 (IX) 

8 (18X) 

2 («X) 

(38) 

1-.13S1 

0-6 
TABLE D2 (CONTINUED)
 

CONTPOL(HNTF ) 

22-2126 

LOI DOSE 

22-212U 

HIGH DOSE 

22-2122 

tSIJBIANDIBULA R

HYPFFPLJSIA ,

 L.NOD E 

 KETICULUfl CELL 

(12) («3) 

1 (2*) 

(38) 

•HESENTFBIC L. XODE 

HYPEFPLASIA , LTBPHOIP 

(12) (43) 

1 (2*) 

(38) 

CIRCULATOR Y SYSTFM 

•CSEDIAC VALV F 

SCLEROSIS 

(18) («7| (45) 

1 <2X ) 

DI3FSTIVE SYSTEM 

tSALIVAP Y GLAN D 

ISFLAMPIfTION , NOS 

(19) (46) 

1 (2%) 

(41) 

HLIV2P 

INFLAMMATION , FOCAL 

INFLA»HATION , HULTIFOCA L 

INFLAMMATION , CHRONIC 

INFLAMMATION , CHPONI C FOCAL 

CTTOPIASHIC VACIIOLIZATION 

BASOPHLLIC CYTO CHANG " 

H?PATOCYTOBEGAL Y 

ANGIF.CTSSIS 

HYPFRPLASIP , LYMPHOI D 

(20) 

1 (5%) 

1 (5*) 

1 C5<) 

(49) 

2 (1*) 

1 (2%) 

1 (2*) 

1 (2%) 

1 <2«) 

2 (4*) 

(45) 

1 (2%) 

1 (2*) 

2 (»«) 

1 (2X) 

*BIL? DUCT 

INFLJIftATION , MOS 

I>IFLMlnATION , GEHNUtOIATOtJ 1 ; 

I NFLJ FIXATION , FOCAL GBANULOBJTO U 

(21) 

1 (5*) 

(49) 

1

1

 (2*) 

 (2*) 

(45) 

• PANCEFA S 

CYSTIC PncTS 

I»IFLmMATION, ACUTF 

ATSOPHY, NOS 

HYPFFPLASIA , LYTHOID 

(19) 

1 (5*) 

1 (5%) 

(46) 

1 (2X) 

(38) 

1 (3«) 

1 (3%) 

*S«PLL INTSSTIN; 

AMYLOirnsiS 

HyprppLASIA , ADFNOrATOU S 

(20) 

1

1

 (5«) 

 (5*) 

(48) (45) 

IILFUM 

INJEiiflMAUONx «2£ 

(20) (48) (45) 

1 12*1 

t NUHBEP OF ANIMALS WITH TISSUE EXAMINED BICROSCOPICALLY
 * NHHBFE OF ANIHALS NICROPSIED
 

D-7
 
TABLE D2 (CONTINUED) 

CONTFOL (UKTB) LOI DOSE HIGH DOSE 

22-212( 22-212« 22-2122 

HYPFFPLfSIA , LY«PHOI D 1 (2X) 

tLABJ F INTfSTIH F (2") (119) (42) 

HYprppLASIA , RFTICtlLUM CELL 1 (2*) 

•COLON (20) (H9) (42) 

MLCFB, CHRONI C 1 (2%) 

HYP"PLASIA , LYNPHOI P 2 (It*) 1 (2%) 

• KIDNF Y (20) (50) (45) 

MYD^OlfPHBOSI S 1 (2%) 1 (2*) 

GLO"'"pnLOM' rPHRIIIS,

 M OS 1 (2*) 

INFLAflHATION , FOCAL 1 (2*) 

INFLAMMATION , FOCAL l5» AM1L01ATOO 1 (2*) 

SCt " 1 (2*) f!YL<"inr NOS 1 (2*) HYPTPLSSIA , PFTirriLUB C^L-L 1 (2*)

 

HYP'rpLASIJ, LIPPHOID 1 (2*) 

• KIDNFY/GIOI1FPULU S (2n

) (SO) (45) *"YLOinosiS 1 (2») 

"NDOCRIN F SY^TF M 

HPITIJIT&P Y (9) (34) (32) 

MJIICT&SI-, 1 (11%)

 

tADRFNS L rOB.T'X (18) (39) (42)

 1"TA"OBPHOSIS FATTY 1 (3*)

 

ITHY"OI P (17) (42) (43) 

rf5!N°FATION , NO S 1 (6%)

 

IPHPATHYROIC (5) (16) (20) • ^HYFnLOSSU L DHCT CT5" 1 (2C% ) 

RFPFOCUCTI V SYST^N 

tOTEBM S (19) (49) (44) 

I VIIIBT F 0" r,NI1AL S HIT!! TISSHF EXA1INF D HIC^OSCOPICA LLY 

* K'J"PFR OF ANIBUL S KECPOPSIrr 

D-8 
TABLE D2 (CONTINUED)
 

CONTROL (IINTK) LOB DOSE HIGH DOSS
 

22-212f 22-212« 22-2122
 

CYST, NOS 1 (5*) 1 (2X) 

PYOMFTS A 2 <«*) 

NF.CFOSIS, NOS 1 (2X) 

ANGIFCTASI S 1 (2X)

 

IIYPFSPLASIA, LIMPHOIt) 1 (2X) 

«t!TERD5/FNDOMFTPIOM (19) (U9) («")
 CYST, NOS 10 (53%) 13 (27X) 2" (U5%)
 

INFLAMMATION, NOS 1 (2*)
 

INFLAMMATION, SUPPOHATIVF 1 (2*)
 

HYPFPPLASIA, NOS
 

HYPfRPLPSIA, CYSTIC 1 (5*) 1 (2%)
 

tUTERns/MYOMFTRIUM (19)
 INFLAMMfTION, N"CPOTIZING 1 (2*)
 

INFLAMMATION, GRANULOMATOHS 1 (2*)
 

tOVAR Y (19) (3t) (30) 

CYST, NOS 1 (5%) 3 (9S) 2 (7*) 

FOLLICfJLAF CYST, NOS 1 (3*) 

P^POVABIA N CYST 2 (11*) 1 (3X) 2 (7*) 

HEMORFHAGI C CYST 1 (3X) 

INFLAMMATION , GR A NULOMATOO S 1 (3X) H Y n FFPLPSIA , LYMPHOI P 1 (3X) 

NFBVOUS SYSTEM
 

150) INFLAMMATION, NOS 1 (2%)
 INFLAMMATION, FOCAl 1 (5»)
 

IBEAIN/FPfNDYM" (2") (50) (46)
 

INFLf MIHTION, FOCAL 1 (2%)
 

tBEAIN (21) (50) 

COFPOPA AMYLPCFA (25%) 10 (20X) 8 (17%) 

SPFCIAL E ORGAN S 

NON ? 

BlISCULOSKELFTA L SYSTE N 

»SKELFTf l MBSCLF (20) (50) 

* NU«3FP OF ANIMALS WITH TISSOF EXAMINED NICPOSCOPICALIY
 

* NUMSPp OF ANIMALS NECBOPSIEP
 

D-9
 
TABLE D2 (CONCLUDED) 

COHTPOL(ONTP)

22-2126

 LOW DOS1

 22-212*

 HIGH DOSE 

 22-2122 

EOE Y CAVITIF S 

*PFPITON' rU tl

INFLAMMATION , ,JP ANULOPATOn S

 (20) (50)

 1 (2*) 

 (17) 

A L L OTHtr SYSTEH S 

SIT7 U"KNOil N 

HYp-»PLASIf , LYHPHOI D

^niecsi TISSUE INFH1 CATION , PYOaF'ANnLOXPTOU S

 1 

1 

SPrCHL rOPPHOLOG Y SIJNt«AP Y 

NO LFSIO N P»PORTFr

H»lj»f j "ISSING/NO NTCPOPSY

B'lTO/OrCPOPSY/HISTO P'BF

t "ilJKBI P OF »NI1ALS WITH TIPSOF

* KUN^F F OF ANIMJL S NFCPOPSIFD 

 EXAMINE D

 1

 HICPOSCOPICHLLT 

7 1 

3 

1 

0-10 




DHEW Publication No. (NIH) 78-1701