1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 85 

BIOASSAY OF 

4-CHLORO-m-PHENYLENEDIAMINE 

FOR POSSIBLE CARCINOGENICITY 

CAS NO. 5131-60-2 

NCI-CG-TR-85 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

4-CHLORO-m-PHENYLENEDIAMINE
 

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) 78-1335
 

REPORT ON THE BIOASSAY OF 4-CHLORO-m-PHENYLENEDIAMINE
 

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
 

4-chloro-m-phenylenediamine 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 4-chloro-m-phenylenediamine was
 

conducted by Mason Research Institute, Worcester, Massachusetts,
 

initially under direct contract to the NCI and currently under a subcontract to Tracor Jitco, Inc., prime contractor for the NCI Carcinogenesis Testing Program.
 

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

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

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

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

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

Histopathologic examinations were performed by Dr. A. S. Krishna
 

Murthy (3) and Dr. A. Russfield (3) at the Mason Research Institute,
 

the pathology narratives were written by Dr. A. S. Krishna Murthy
 

(3), and the diagnoses included in this report represent the interpretation of these pathologists. Histopathology findings and reports
 

were reviewed by Dr. R. L. Schueler (6).
 

Compilation of individual animal survival, pathology, and summary
 

tables was performed by EG&G Mason Research Institute (7); the statistical analysis was performed by Mr. W. W. Belew (5), using methods
 

selected for the Carcinogenesis Testing Program by Dr. J. J. Gart (8).
 

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

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

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

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

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

the participating organizations.
 

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

J. J. Gart (8), Mr. J. Nam (8), Dr. H. M. Pettigrew (8), and Dr. R.
 

E. Tarone (8).
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

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

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

Squire (1,9), Dr. J. M. Ward (1), and Dr. C. E. Whitmire (1).
 

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

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

3.	 Mason Research Institute, 57 Union Street, Worcester, Massachusetts.
 

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

Missouri.
 

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

Boulevard, McLean, Virginia.
 

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

Maryland.
 

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

Rockville, Maryland.
 

8.	 Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cancer
 

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

iv
 
9.	 Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay of 4-chloro-m-phenylenediamine for possible carcinogenicity was conducted using Fischer 344 rats and B6C3F1 mice.
 

4-Chloro-m-phenylenediamine was administered in the feed, at either
 

of two concentrations, to groups of 49 or 50 male and 50 female
 

animals of each species. The dietary concentrations used in the
 

chronic bioassay for low and high dose rats were 0.2 and 0.4 percent,
 

respectively. The time-weighted average dietary concentrations used
 

for low and high dose mice were 0.7 and 1.4 percent, respectively.
 

After a 78-week period of compound administration, observation of
 

rats continued for an additional 27 weeks and observation of mice
 

continued for an additional 17 weeks. For each species, 50 animals
 

of each sex were placed on test as untreated controls.
 

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

Among male rats, an increased incidence of adrenal pheochromocytomas was statistically associated with dosage of 4-chloro-m-phenylenediamine. The incidence of these tumors was significantly higher
 

in the high dose group than in the control group.
 

Among female mice, there was a significantly increased incidence
 

of hepatocellular carcinomas in the low dose group and a significantly
 

increased combined incidence of hepatocellular carcinomas and hepatocellular adenomas in both low and high dose groups as compared to controls.
 

No other neoplasms in either species were considered to be related to compound administration.
 

Under the conditions of this bioassay, dietary administration of
 

4-chloro-m-phenylenediamine was carcinogenic to the experimental
 

animals, causing an increased incidence of hepatocellular tumors in
 

female B6C3F1 mice and an increased incidence of adrenal pheochromocytomas in male Fischer 344 rats.
 

vii
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 3
 

A. Chemicals 3
 

B. Dietary Preparation 3
 

C. Animals 5
 

D. Animal Maintenance 5
 

E. Selection of Initial Concentrations 8
 

F. Experimental Design 9
 

G. Clinical and Histopathologic Examinations 12
 

H. Data Recording and Statistical Analyses 14
 

III. CHRONIC TESTING RESULTS: RATS 19
 

A. Body Weights and Clinical Observations 19
 

B. Survival 19
 

C. Pathology 22
 

D. Statistical Analyses of Results 23
 

IV. CHRONIC TESTING RESULTS: MICE 30
 

A. Body Weights and Clinical Observations 30
 

B. Survival 30
 

C. Pathology 33
 

D. Statistical Analyses of Results 34
 

V. DISCUSSION 41
 

VI. BIBLIOGRAPHY 43
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLARTIC
 

LESIONS IN RATS TREATED WITH 4-CHLORO-m-


PHENYLENEDIAMINE C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 4-CHLORO-m-


PHENYLENEDIAMINE D-l
 

Ix
 
Figure Number
 

1
 

Table Number
 

LIST OF ILLUSTRATIONS 

Page 

CHEMICAL STRUCTURE OF 4-CHLORO-m-PHENYLENEDIAMINE 

4 

GROWTH CURVES FOR

CHRONIC STUDY RATS 

 4-CHLORO-m-PHENYLENEDIAMINE 

20 

SURVIVAL COMPARISONS OF 4-CHLORO-m-PHENYLENEDIAMINE CHRONIC STUDY RATS 21 

GROWTH CURVES FOR

CHRONIC STUDY MICE 

 4-CHLORO-m-PHENYLENEDIAMINE 

31 

SURVIVAL COMPARISONS OF 4-CHLORO-m-PHENYLENEDIAMINE CHRONIC STUDY MICE 32 

LIST OF TABLES 

Page 

DESIGN SUMMARY FOR FISCHER 344 RATS— 

4-CHLORO-m-PHENYLENEDIAMINE FEEDING EXPERIMENT 10 

DESIGN SUMMARY FOR B6C3F1 MICE—4-CHLORO-mPHENYLENEDIAMINE FEEDING EXPERIMENT 11 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

4-CHLORO-m-PHENYLENEDIAMINE 24 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH 4-CHLORO-m-PHENYLENEDIAMINE 27 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

4-CHLORO-m-PHENYLENEDIAMINE 35 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH 4-CHLORO-m-PHENYLENEDIAMINE 37 
LIST OF TABLES (Concluded) 

Table Number 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE A ~ 3 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS 

IN FEMALE RATS TREATED WITH 4-CHLOROm-PHENYLENEDlAMINE A ~ 7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS 

IN MALE MICE TREATED WITH 4-CHLORO-mPHENYLENEDIAMINE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS 

IN FEMALE MICE TREATED WITH 4-CHLOROm-PHENYLENEDIAMINE B ~ 6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS TREATED WITH 

4-CHLORO-m-PHENYLENEDIAMINE C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS TREATED WITH 

4-CHLORO-m-PHENYLENEDIAMINE C-7 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE TREATED WITH 

4-CHLORO-m-PHENYLENEDIAMINE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE TREATED WITH 

4-CHLORO-m-PHENYLENEDIAMINE D ~ 7 

XI
 

I. INTRODUCTION
 

4-Chloro-m-phenylenediamine (NCI No. C03305), an intermediate in
 

the preparation of dyes, was selected for bioassay by the National
 

Cancer Institute because of the high incidence of bladder cancer reported among workers in the dye manufacturing industry (Anthony and
 

Thomas, 1970; Wynder et al., 1963). Occupational exposure to several
 

types of compounds, including aromatic amines, is 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 4-chloro-l,3-benzenediamine. It is
 

also called 4-chlorophene-l,3-diamine; 4-chloro-l,3-phenylenediamine;
 

and C.I. (Colour Index) 76027.
 

4-Chloro-m-phenylenediamine can be used as an intermediate in
 

the production of C.I. Mordant Brown 48, C.I. Acid Brown 145, Paranil
 

Brown G., C.I. Direct Black 11, and Diamine Fast Black XCD (Society
 

of Dyers and Colourists, 1956), and in the synthesis of disazo pigmemts (Mueller, 1975; Papenfuhs, 1975). The compound can also be
 

used as a coupler in hair dye preparations (Corbett, 1973).
 

Patents have been issued for several applications of 4-chlorom-phenylenediamine including use as a color forming agent in formulations applied to paper used for microfilm enlargements (Wiswell,
 

*
 

The CAS registry number is 5131-60-2.
 
1968), a color intensifier in photographic films (Matter and Price,
 

1970), and a cross-linking agent for polyurethane manufacture (Krisnan, 1975).
 

Specific production figures for 4-chloro-m-phenylenediamine are
 

not available; however, the inclusion of the compound in the 1977
 

Directory of Chemical Producers, U.S.A. (Stanford Research Institute,
 

1977) implies an annual production in excess of 1000 pounds or $1000
 

in value.
 

The potential for exposure to 4-chloro-m-phenylenediamine may be
 

considerable for workers in dye and chemical production facilities
 

and those engaged in research involving this compound.
 
II. MATERIALS AND METHODS 

A. Chemicals
 

4-Chloro-m-phenylenediamine (Figure 1) was purchased from Carroll
 

Products, Wood River Junction, Rhode Island and analysis was performed
 

by Midwest Research Institute, Kansas City, Missouri. The narrow
 

range of the experimentally determined melting point (87° to 90°C)
 

and its proximity to the literature value (91°C) suggested a compound
 

of high purity. Elemental analysis yielded results closely approximating the theoretical analyses. Thin-layer chromatography utilizing
 

two solvent systems (benzene:methanol and methanol:acetic acid) and
 

visualized with ultraviolet light and furfural revealed, respectively,
 

one and two impurities. Vapor-phase chromatography showed the
 

presence of one extraneous peak. Nonaqueous titration of the amine
 

function was approximately 97 percent of the theoretical amount.
 

This indicates maximum possible purity only, as other amine compounds
 

might also have been present.
 

Throughout this report the term 4-chloro-m-phenylenediamine is
 

used to represent this compound.
 

B. Dietary Preparation
 

The basal laboratory diet for both treated and control animals
 

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

4-Chloro-m-phenylenediamine was administered to the treated animals
 

as a component of the diet. The compound was ground in a Quaker City
 

crystal mill, added to an aliquot of ground feed, and hand mixed with
 
FIGURE 1

 

CHEMICAL STRUCTURE OF 4-CHLORO-m-PHENYLENEDIAMINE 
a mortar and pestle until visual uniformity was attained. This
 

premix was then placed into a 6 kg capacity Patterson-Kelley twinshell stainless steel V-blender along with the remainder of the meal
 

and blended for 20 minutes. Prepared diets 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.
 

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. The animals were received in several separate shipments from
 

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

Rats and mice to be treated were received 3 weeks later than rats and
 

mice to be used as 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 the test. Animals were
 

assigned to groups and distributed among cages so that the average
 

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

D.	 Animal Maintenance
 

All animals were housed by species in rooms having a temperature
 

(R)
 range of 23° to 34°C. Incoming air was filtered through Tri-Dekw
 

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, rats were kept in galvanized- or
 

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

weekly. For the remainder of the study, rats were held in suspended
 

polycarbonate cages equipped with disposable nonwoven fiber filter
 

sheets. Clean bedding and cages were provided twice weekly. Corn­

(R)
 cob bedding, SAN-I-CEL (Paxton Processing Company, Paxton, Illinois),
 

was used for the first 6 months that the rats were housed in polycarbonate cages. Thereafter, a hardwood chip bedding (Aspen bedding,
 

American Excelsior Company, Baltimore, Maryland) was used. Stainless
 

steel cage racks were cleaned every 2 weeks, and disposable filters
 

were replaced at that time.
 

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

Cages were fitted with perforated stainless steel lids (Lab Products,
 

Inc., Garfield, New Jersey) and nonwoven fiber filter bonnets. Mice
 

were housed ten per cage for the first 11 months of study and five
 

per cage thereafter. Clean cages, lids, and bedding were provided
 

three times per week when cage populations were ten and twice per
 

week when cage populations were reduced to five. SAN-I-CEL(S\ Wwas used
 

as bedding for the first 12 months of study. A second corncob
 

bedding (Bed-o-Cobs®, The Andersons Cob Division, Maumee, Ohio) was
 
used for the next 8 months. Aspen bedding was then used until the
 

end of study. Reusable filter bonnets and pipe racks were sanitized
 

every 2 weeks throughout the study.
 

Water was available ad libitum for 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)
 Wayne Lab-Blox^was used throughout the entire bioassay. Wayne
 

(R)
 Lab-Bloxw

meal containing the appropriate concentrations of 4-chlorom-phenylenediamine was supplied to treated animals during the period
 

of compound administration. Control animals had untreated meal
 

available. Meal was dispensed in Alpine^aluminum feed cups (Curtin
 

Matheson Scientific, Inc., Woburn, Massachusetts) with stainless
 

steel baffles for the first 13 months of study. After that time,
 

animals were fed from stainless steel gangstyle hoppers (Scientific
 

Cages, Inc., Bryan, Texas). Food hoppers were changed on the same
 

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

cups.
 

4-Chloro-m-phenylenediamine-treated and control rats were housed
 

*
 in a room with other rats receiving diets containing acetylaminofLuorene (53-96-3); p-cresidine (120-71-8); 2,3,5,6-tetrachloro-4­

nitroanisole (2438-88-2); 4-chloro-o-phenylenediamine (95-83-0); and
 

IH-benzotriazole (95-14-7).
 

*
 

CAS registry numbers are given in parentheses.
 
Treated and control mice shared a room with other mice receiving
 

diets containing 2-methyl-l-nitroanthraquinone (129-15-7); p-cresidine (120-71-8); fenaminosulf (140-56-7); and acetylaminofluorene
 

(53-96-3).
 

E.	 Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

4-chloro-m-phenylenediamine for administration to treated animals in
 

the chronic studies, subchronic toxicity tests were conducted with
 

both rats and mice. Animals of each species were distributed among
 

several groups, each consisting of five males and five females. The
 

chemical was incorporated into the basal laboratory diet and supplied
 

ad libitum to four of five rat groups in concentrations of 0.1, 0.3,
 

1.0, and 3.0 percent and five of six mouse groups in concentrations
 

of 0.03, 0.1, 0.3, 1.0 and 3.0 percent. The fifth rat group and the
 

sixth mouse group served as controls, receiving only the basal laboratory diet. The dosed dietary preparations were administered for a
 

period of 8 weeks. All survivors were sacrificed at the end of the
 

8-week period.
 

The highest concentration causing no deaths, no compound-related
 

abnormalities, and no mean group body weight depression in excess of
 

15 percent was selected as the high concentration utilized for the rat
 

and mouse chronic bioassays.
 

All rats receiving dietary concentrations of 3.0 percent 4-chlorom-phenylenediamine and three male rats receiving 1.0 percent died by
 
the end of the 8-week study. No other deaths were recorded among
 

rats in the subchronic test. At the end of the subchronic study,
 

males treated with 1.0 and 0.3 percent weighed approximately 80 and
 

97 percent, respectively, the weight of controls and females receiving
 

the same concentrations weighed approximately 84 and 104 percent the
 

weight of controls. A 4-chloro-m-phenylenediamine concentration of
 

0.4 percent was selected for use as the high dose in the rat chronic
 

bioassay for both males and females.
 

Two deaths were observed among mice treated with the compound;
 

one, a female fed a concentration of 3.0 percent and the other, a
 

female fed a concentration of 0.3 percent. Mean group body weight
 

depression was approximately 74 and 90 percent in males treated with
 

3.0 and 1.0 percent 4-chloro-m-phenylenediamine, respectively, and
 

85 and 88 percent in females receiving the same concentrations. The
 

concentration of the compound selected for use as the high dose for
 

males and females in the mouse chronic bioassay was 2.0 percent.
 

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.
 

At initiation of the study, the rats were approximately 6 weeks
 

old. Rats received dietary concentrations of 0.4 or 0.2 percent
 

throughout the 78-week period of compound administration. Throughout
 

this report the rat groups receiving the former concentration are
 
MALE
 

CONTROL
 

LOW DOSE
 

HIGH DOSE
 

FEMALE
 

CONTROL
 

LOW DOSE
 

HIGH DOSE
 

TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

4-CHLORO-m-PHENYLENEDIAMINE FEEDING EXPERIMENT
 

4-CHLORO-mINITIAL PHENYLENEDIAMINE OBSERVATION PERIOD 

GROUP CONCENTRATION TREATED UNTREATED 

SIZE (PERCENT) (WEEKS) (WEEKS) 

50	 0 0 106
 

50	 0.2 78
 

0 26
 

49	 0.4 78
 

0 27
 

50 0 0 106 

50 0.2 78 

0 26 

50 0.4 78 

0 27 

10
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

4-CHLORO-m-PHENYLENEDIAMINE FEEDING EXPERIMENT
 

MALE 

CONTROL

LOW DOSE

INITIAL

GROUP 

SIZE 

 50 

 50 

HIGH DOSE 50 

FEMALE 

CONTROL

LOW DOSE

 50 

 50 

HIGH DOSE 49 

a . 

4-CHLORO-m­

 PHENYLENEDIAMINE


CONCENTRATION
 

(PERCENT)
 

0
 

1.0
 

0.5
 

0
 

2.0
 

1.0
 

0
 

0
 

1.0
 

0.5
 

0
 

2.0
 

1.0
 

0
 

 TIME-WEIGHTED 

 OBSERVATION PERIOD AVERAGE 

TREATED UNTREATED CONCENTRATION*1 

(WEEKS) (WEEKS) (PERCENT) 

0 97 0
 

29 0.7
 

49
 

17
 

29 1.4
 

49
 

17
 

0 97 0
 

29 0.7
 

49
 

17
 

29 1.4
 

49
 

17
 

2 (concentration X weeks received)
 

2(weeks receiving chemical)
 

11
 
referred to as the high dose groups while those receiving the latter
 

are referred to as the low dose groups. Control animals had untreated
 

feed available. After the 78-week dosing period, rats were observed
 

for an additional 26 or 27 weeks.
 

At initiation of the study the mice were approximately 6 weeks
 

old. Mice initially received dietary concentrations of 2.0 or 1.0
 

percent. Throughout this report those mice initially receiving the
 

former concentration are referred to as the high dose groups while
 

those receiving the latter concentration are referred to as the low
 

dose groups. After 29 weeks, the dosages were lowered to 1.0 and 0.5
 

percent for high and low dose mice, respectively. These concentrations were maintained for the remainder of the 78-week dosing period.
 

After the period of chemical administration, mice were observed for
 

an additional 17 weeks.
 

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. From the
 

first day, all animals were inspected twice daily for mortality.
 

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.
 

12
 
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
 

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, uterus, mammary gland, and
 

ovary.
 

A few tissues were not examined for some animals, particularly
 

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

preclude histopathologic interpretation. Thus, the number of animals
 

for which particular organs, tissues, or lesions were examined
 

13
 
microscopically varies and does not necessarily represent the number
 

of animals that were placed on experiment in each group.
 

H. Data Recording and Statistical Analyses
 

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

(Linhart et al., 1974). The data elements include descriptive
 

information on the chemicals, animals, experimental design, clinical
 

observations, survival, body weight, and individual pathologic results, as recommended by the International Union Against Cancer
 

(Berenblum, 1969). Data tables were generated for verification of
 

data transcription and for statistical review.
 

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

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

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

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

missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect
 

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

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

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

14
 
for all tests except the departure from linearity test, which is only
 

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

The incidence of neoplastic or nonneoplastic lesions has been
 

given as the ratio of the number of animals bearing such lesions at a
 

specific anatomic site (numerator) to the number of animals in which
 

that site was examined (denominator). In most instances, the denominators included only those animals for which that site was examined
 

histologically. However, when macroscopic examination was required
 

to detect lesions prior to histologic sampling (e.g., skin or mammary
 

tumors), or when lesions could have appeared at multiple sites (e.g.,
 

lymphomas), the denominators consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a significantly higher proportion of tumors than did the control animals.
 

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

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

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

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

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

significance level of 0.05 may be made. The Bonferroni inequality
 

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

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

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

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

15
 
The Cochran-Armitage test for linear trend in proportions, with
 

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

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

determined if the slope of the dose-response curve is different from
 

zero at the one-tailed 0.05 level of significance. Unless otherwise
 

noted, the direction of the significant trend was a positive dose
 

relationship. This method also provides a two-tailed test of departure from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation of
 

tumors. In this analysis, deaths that occurred before the first
 

tumor was observed were excluded by basing the statistical tests on
 

animals that survived at least 52 weeks, unless a tumor was found at
 

the anatomic site of interest before week 52. When such an early
 

tumor was found, comparisons were based exclusively on animals that
 

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

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

Cochran-Armitage tests, etc.) were followed.
 

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

incidence of tumors. Curves of the proportions surviving without an
 

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

during which animals died naturally or were sacrificed was entered as
 

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

16
 
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, twotailed 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
 

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
 

17
 
can be inferred that a statistically significant result (a P < 0.025
 

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

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

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

the lower limit indicates the absence of a significant result while
 

the upper limit indicates that there is a theoretical possibility
 

of the induction of tumors by the test chemical which could not be
 

detected under the conditions of this test.
 

18
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

No consistent pattern of mean group body weight depression was
 

observed among rats of either sex (Figure 2).
 

Subcutaneous masses were detected in two control males, three
 

control females, three low dose males, one low dose female, five high
 

dose males, and four high dose females. Cutaneous lesions/masses
 

were observed in two control males, two low dose females, one high
 

dose male, and one high dose female. Abdominal distention and cyanosis were observed in one low dose male. Two control males exhibited
 

eye discoloration. No other clinical abnormalities were reported for
 

either males or females.
 

B. Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and 4-chloro-m-phenylenediamine-dosed groups are
 

shown in Figure 3.
 

In male rats the Tarone test did not indicate a significant
 

association between increased dosage and accelerated mortality. Five
 

rats from the high dose group and five rats from the control group
 

were sacrificed in week 78. There were adequate numbers of male rats
 

at risk from late-developing tumors with 73 percent (36/49) of the
 

high dose, 86 percent (43/50) of the low dose, and 64 percent (32/50)
 

of the control group surviving until the end of the study.
 

19
 
750 750 

r-600 

— 450 

— 300 

L-150 

TIME ON TEST (WEEKS) 

120 

750­ -750 

600­ -600 

CC 

I 

O 

LU 

450­ -450 

§300­

m 

I 

m 

150­

-300 

-150 

FEMALE RATS 

15 30 

~r 

45 

~T 

60 75 

TIME ON TEST (WEEKS) 

—I

90

 1 1

 105

 1 

 120 

FIGURE 2 

GROWTH CURVES FOR 4-CHLORO-m-PHENYLENEDIAMINE CHRONIC STUDY RATS 

20 
PROBABILITY OF SURVIVAL

 PROBABILITY OF S

p p c3 NJ ^ (D O3 b b CD 

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In female rats the Tarone test again did not indicate a significant association between increased dosage and accelerated mortality.
 

Five rats from the high dose group and five rats from the control
 

group were sacrificed in week 78. There were adequate numbers of
 

female rats at risk from late-developing tumors with 66 percent
 

(33/50) of the high dose, 78 percent (39/50) of the low dose, and 72
 

percent (36/50) of the control group surviving until the termination
 

of the study.
 

C. Pathology
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

summarized in Appendix C (Tables Cl and C2).
 

A variety of neoplasms were seen in both control and treated
 

rats. There were instances of neoplastic lesions occurring only in
 

treated animals, or with increased frequency in the treated groups
 

when compared to the control group. The nature and incidences of
 

the majority of the lesions were similar to those known to occur
 

spontaneously in aged Fischer 344 rats. However, the incidence of
 

adrenal pheochromocytoma was higher in the treated male rats (14/48
 

[29 percent] high dose, 7/48 [15 percent] low dose) than in the
 

control (4/46 [9 percent]) male rats in this study. Zymbals" gland
 

tumors occurred only in treated rats (2/49 [4 percent] high dose
 

males and 1/47 [2 percent] high dose females).
 

22
 
A variety of nonneoplastic lesions were observed in this study.
 

All of these lesions are commonly seen in aged Fischer 344 rats and
 

occurred with approximately equal frequency in the treated and control animals.
 

Based on the results of this histopathologic examination, the
 

administration of 4-chloro-m-phenylenediamine was associated with an
 

increased incidence of adrenal pheochromocytomas in male Fischer 344
 

rats.
 

D. Statistical Analyses of Results
 

The results of the statistical anlayses of tumor incidence in
 

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

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

observed in at least one of the control or 4-chloro-m-phenylenediamine-dosed groups and where such tumors were observed in at least 5
 

percent of the group.
 

In male rats the incidence of adrenal pheochromocytomas was increased in the treated groups. The Cochran-Armitage test indicated
 

a significant (P = 0.007) positive association between dosage and tumor incidence. The Fisher exact test comparing the high dose treated
 

males to the control supported these findings with significant (P =
 

0.011) results. The historical combined incidence rate for pheochromocytomas and malignant pheochromocytomas was 32/250 (13 percent)
 

in data collected on the untreated male Fischer 344 rats at Mason
 

Research Institute for the NCI Carcinogenesis Testing Program. Based
 

23
 
KS
 

TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH 4-CHLORO-m-pHENYLENEDIAMINE£
 

TOPOGRAPHY : MORPHOLOGY
 

Subcutaneous Tissue: Fibroma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

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"
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

3/48(0.06)
 

N.S.
 

105
 

3/48(0.06)
 

N.S.
 

105
 

7/48(0.15)
 

P = 0.007(N)
 

P = 0.036
 

80
 

LOW
 

DOSE
 

3/49(0.06)
 

N.S.
 

0.980
 

0.138
 

6.979
 

104
 

1/49(0.02)
 

N.S.
 

0.327
 

0.006
 

3.898
 

104
 

0/49(0.00)
 

P = 0.006(N)
 

0.000
 

0.000
 

0.504
 

HIGH
 

DOSE
 

4/49(0.08)
 

N.S.
 

1.306
 

0.233
 

8.495
 

104
 

0/49(0.00)
 

N.S.
 

0.000
 

0.000
 

1.628
 

1/49(0.02)
 

P - 0.028(N)
 

0.140
 

0.003
 

1.029
 

93
 
TOPOGRAPHY: MORPHOLOGY
 

Pituitary: Adenoma NOS
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocytoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma or
 

C-Cell Carcinomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (Continued)
 

CONTROL
 

10/45(0.22)
 

N.S.
 

103
 

4/46(0.09)
 

P = 0.007
 

78
 

5/43(0.12)
 

N.S.
 

95
 

LOW
 

DOSE
 

6/43(0.14)
 

N.S.
 

0.628
 

0.205
 

1.732
 

104
 

7/48(0.15)
 

N.S.
 

1.677
 

0.459
 

7.336
 

104
 

3/48(0.06)
 

N.S.
 

0.538
 

0.088
 

2.595
 

104
 

HIGH
 

DOSE
 

15/47(0.32)
 

N.S.
 

1.436
 

0.679
 

3.185
 

78
 

14/48(0.29)
 

P = 0.011
 

3.354
 

1.153
 

12.990
 

78
 

6/48(0.13)
 

N.S.
 

1.075
 

0.295
 

4.153
 

95
 
TABLE 3 (Concluded)
 

TOPOGRAPHY : MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Testis: Interstitial-Cell Tumor 37/48(0.77) 43/49(0.88) 45/49(0.92) 

P Values P = 0.027 N.S. P = 0.041 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

1.138 

0.926 

1.353 

1.191 

0.981 

1.366 

Weeks to First Observed Tumor 78 94 78 

a

Treated groups received time-weighted averaged doses of 0.2 or 0.4 percent in feed.
 

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

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

in the control group when P < 0.05; otherwise, 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.
 

e

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 4-CHLORO-m-PHENYLENEDIAMINE£
 

TOPOGRAPHY : MORPHOLOGY
 

Pituitary: Adenoma, NOS or
 

Chromophobe Adenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

t-o
 

Adrenal: Pheochromocytoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW HIGH 

CONTROL DOSE DOSE 

17/40(0.43) 15/46(0.33) 17/44(0.39) 

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

0.767 0.909 

0.417 0.514 

1.413 1.621 

101 68 78 

6/48(0.13) 2/49(0.04) 4/46(0.09) 

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

0.327 0.696 

0.034 0.154 

1.720 2.736 

106 104 78 

6/50(0.12) 4/50(0.08) 5/47(0.11) 

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

0.667 0.887 

0.147 0.229 

2.635 3.249 

106 85 68 
TABLE 4 (Concluded)
 

LOW HIGH
 

TOPOGRAPHY: MORPHOLOGY CONTROL DOSE DOSE
 

Uterus: Endometrial Stromal Polyp 2/48(0.04) 12/48(0.25) 5/46(0.11)
 

P Values0 N.S. P = 0.004 N.S.
 

Departure from Linear Trend P = 0.004
 

Relative Risk (Control)d 6.000 2.609
 

Lower Limit 1.438 0.453
 

Upper Limit 52.712 26.300
 

Weeks to First Observed Tumor 106 104 105
 

Treated groups received time-weighted average doses of .0.2 or 0.4 percent in feed.
 NJ
 

OO 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.
 o
 

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

group when P < 0.05.
 
on the results of these findings, the administration of 4-chloro-mphenylenediamine was associated with the incidence of adrenal pheochromocytoma in male rats.
 

For female rats an increase in the incidence of endometrial
 

stromal polyps was observed. The Fisher exact test comparing the
 

incidence of tumors in the low dose group to that in the control was
 

significant (P = 0.004). The historical incidence of this tumor in
 

data on untreated female Fischer 344 rats at Mason Research Institute
 

was 31/249 (12 percent), which was greater than that observed for
 

either the control or the high dose group. Based upon these results
 

the statistical conclusion was that there was inadequate evidence to
 

conclude that compound administration induced these uterine tumors.
 

For male rats the Cochran-Armitage test for interstitial-cell
 

tumors of the testis indicated a significant (P = 0.027) positive
 

association between dosage and incidence. The Fisher exact test
 

comparing the high dose treated males to the controls yielded a value
 

of P = 0.041, a marginal result which was not significant under the
 

Bonferroni criterion.
 

For male rats the possibility of a negative association between
 

chemical administration and the incidence of malignant lymphomas or
 

leukemia was noted.
 

29
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

A distinct pattern of dose-related mean group body weight depression was apparent in mice of both sexes (Figure 4).
 

No clinical abnormalities were reported for male or female mice.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and 4-chloro-m-phenylenediamine-dosed groups are
 

shown in Figure 5.
 

In male mice the Tarone test for association between increased
 

dosage and accelerated mortality was not significant. Five high dose
 

and five control males were sacrificed in week 78. Survival was relatively good with 80 percent (40/50) of the high dose, 82 percent
 

(41/50) of the low dose, and 84 percent (42/50) of the controls
 

living until the end of the study. Thus, there were adequate numbers
 

of male mice at risk from late-developing tumors.
 

In female mice the Tarone test did not indicate a significant
 

association between increased dosage and mortality. Five high dose
 

and five control females were sacrificed in week 78. There were adequate numbers of female mice at risk from late-developing tumors as
 

78 percent (38/49) of the high dose, 74 percent (37/50) of the low
 

dose, and 72 percent (36/50) of the controls survived until termination of the study.
 

30
 
50- -50 

40 — -40 

-30 

-20 

o 

00 

CONTROL 

10- LOW DOSE -10 

HIGH DOSE 

MALE MICE 

I \ \ I \ I i 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

5 Q- -50 

40 — -40 

OC 

2 30­ -30 

(3 

-20 

o 

CO 

CONTHOL 

—10 

LOW DOSE 

FEMALE MICE HIGH DOSE 

I I [ \ \ \ 1 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

FIGURE 4 

GROWTH CURVES FOR 4-CHLORO-m-PHENYLENEDIAMINE CHRONIC STUDY MICE 

31 
30 

PROBABILITY OF SURVIVAL PROBABILITY OF S

o P P p p p 

0> bo KJ ** 01 

_L _L 1 i I I I 

GO 

8 

S 

GO 

O 

o 

30 

92 

3 O 

rnm 

201 m 

m m A 

m 

O 

Z

30 

O 

0 

a- a m 

S O a m O 

p

k) 
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).
 

A variety of neoplasms occurred with approximately equal frequency in treated and control mice. In addition, there was an
 

increased incidence of hepatocellular neoplasms observed in the
 

treated female mice when compared with the control group. These are
 

summarized in the following table: 

Control Low Dose High Dose 

Number of Female Mice with Livers 

Examined Histopathologically 46 44 45 

Hepatocellular Adenomas 0 3 3 

Hepatocellular Carcinomas 0 8 5 

The incidence of hepatocellular neoplasms among male mice was similar
 

in the treated and control groups.
 

The hepatocellular adenomas consisted of small circumscribed
 

nodules lacking normal lobular architecture, comprised of large
 

hepatocytes with eosinophilic cytoplasm and vesicular nuclei.
 

Hepatocellular carcinomas replaced partial or entire lobes of the
 

liver. The hepatic architecture was distorted and the neoplastic
 

hepatocytes were large in size with eosinophilic cytoplasm and lipid
 

vacuoles in some cells. A pleomorphism in nuclear size was evident
 

and mitotic figures were numerous. Metastasis to the lung occurred
 

in one treated male but in none of the female mice.
 

33
 
The treated and control mice had a variety of nonneoplastic lesions which are commonly seen in aged B6C3F1 mice. The incidence and
 

severity of the lesions were similar in the control and treated mice.
 

The results of this histopathologic examination indicate that
 

4-chloro-m-phenylenediamine was carcinogenic in female B6C3F1 mice,
 

as it was associated with an increased incidence of hepatocellular
 

neoplasms under the conditions of this study. There did not appear
 

to be any compound-related neoplastic lesions in the male B6C3F1 mice
 

in this study.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

were observed in at least one of the control or 4-chloro-m-phenylenediamine-dosed groups and where such tumors were observed in at least
 

5 percent of the group.
 

For females an increase in the incidence of liver tumors was
 

observed in treated mice. The Fisher exact test indicated a significantly (P = 0.002) higher incidence of hepatocellular carcinomas in
 

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

the probability level was P = 0.026, a marginal result that was not
 

significant under the Bonferroni criterion. When incidences were
 

combined so that the numerator represented mice with either a hepatocellular carcinoma or a hepatocellular adenoma, the Cochran-Armitage
 

34
 
___ 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE£
 

HIGH
 

DOSE
 

3/48(0.06)
 

N.S.
 

0.446
 

0.078
 

1.828
 

95
 

1/50(0.02)
 

N.S.
 

0.250
 

0.005
 

2.508
 

95
 

15/48(0.31)
 

N.S.
 

1.563
 

0.732
 

3.486
 

95
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Adenoma
 

or Alveolar/Bronchiolar Carcinoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Malignant Lymphoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW
 

CONTROL DOSE
 

7/50(0.14) 4/47(0.09)
 

N.S. N.S.
 

__—
 0.608
 

0.139
 

2.226
 

97 92
 

4/50(0.08) 3/50(0.06)
 

N.S. N.S.
 

—_—
 0.750
 

0.115
 

4.206
 

97 92
 

10/50(0.20) 8/48(0.17)
 

N.S. N.S.
 

0.833
 

0.312
 

2.150
 

60 84
 
TABLE 5 (Concluded)
 

TOPOGRAPHY : MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Liver: Hepatocellular Adenoma or 

Hepatocellular Carcinoma"3 15/50(0.30) 10/48(0.21) 19/48(0.40) 

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

Relative Risk (Control) 

Lower Limit 

Upper Limit 

0.694 

0.311 

1.481 

1.319 

0.724 

2.437 

Weeks to First Observed Tumor 60 84 95 

Treated groups received time-weighted average doses of 0.7 or 1.4 percent in feed.
 

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

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

the control group when P < 0.05; otherwise, not significant (N.S.) is indicated. The probability level for the Fisher exact test for the comparison of a treated group with the control
 

group is given beneath the incidence of tumors in the treated group when P < 0.05; otherwise,
 

not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control
 

group.
 

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

TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE£
 

TOPOGRAPHY : MORPHOLOGY
 

Lung: Alveolar /Bronchiolar Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar Adenoma or
 

Alveolar/Bronchiolar Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

3/46(0.07)
 

N.S.
 

97
 

4/46(0.09)
 

N.S.
 

97
 

6/47(0.13)
 

N.S.
 

69
 

LOW
 

DOSE
 

1/42(0.02)
 

N.S.
 

0.365
 

0.007
 

4.332
 

95
 

1/42(0.02)
 

N.S.
 

0.274
 

0.006
 

2.620
 

95
 

7/46(0.15)
 

N.S.
 

1.192
 

0.371
 

3.971
 

86
 

HIGH
 

DOSE
 

0/45(0.00)
 

N.S.
 

0.000
 

0.000
 

1.694
 

1/45(0.02)
 

N.S.
 

0.256
 

0.005
 

2.452
 

95
 

3/46(0.07)
 

N.S.
 

0.511
 

0.089
 

2.284
 

77
 
___ 

TOPOGRAPHY: MORPHOLOGY
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Q.


Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Adenoma or 

OJ Hepatocellular Carcinoma^ oo 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Adenoma, NOS
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 6 (Continued) 

CONTROL
 

0/46(0.00)
 

N.S.
 

P = 0.020
 

0/46(0.00)
 

P = 0.011
 

P = 0.012
 

1/33(0.03)
 

N.S.
 

80
 

LOW
 

DOSE
 

8/44(0.18)
 

P = 0.002
 

Infinite
 

2.397
 

Infinite
 

92
 

11/44(0.25)
 

P < 0.001
 

Infinite
 

3.485
 

Infinite
 

92
 

2/32(0.06)
 

N.S.
 

2.063
 

0.113
 

117.749
 

61
 

HIGH
 

DOSE
 

5/45(0.11)
 

P = 0.026
 

—— —
 

Infinite
 

1.293
 

Infinite
 

8/45(0.18)
 

P = 0.003
 

Infinite
 

2.343
 

Infinite
 

95
 

0/27(0.00)
 

N.S.
 

0.000
 

0.000
 

22.441
 

95 
TABLE 6 (Concluded)
 

TOPOGRAPHY : MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Ovary: Papillary Adenoma or 

Papillary Cystadenoma,NOSb 1/40(0.03) 2/40(0.05) 0/41(0.00) 

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

Relative Risk (Control) 

Lower Limit 

Upper Limit 

2.000 

0.109 

114.994 

0.000 

0.000 

18.131 

Weeks to First Observed Tumor 97 95 

Treated groups received time-weighted average doses of 0.7 or 1.4 percent in feed.
 

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

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.
 p
 

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

group when P < 0.05.
 
test showed a significant positive (P = 0.011) association between
 

dosage and incidence. The Fisher exact tests also showed significant
 

comparisons between the incidence in the control and that in both the
 

high dose (P = 0.003) and low dose (P < 0.001) groups. The departure
 

from linear trend was also significant (P = 0.012) since the incidence
 

in the low dose group was greater than that in the high dose group.
 

The historical incidence rate in untreated female B6C3F1 control mice
 

at Mason Research Institute was 19/275 (7 percent).
 

Based upon these results the statistical conclusion is that the
 

administration of 4-chloro-m-phenylenediamine was associated with
 

the combined incidence of hepatocellular adenomas and hepatocellular
 

carcinomas in female B6C3F1 mice.
 

No statistical tests for tumors of other sites were significant
 

for either male or female mice.
 

40
 
V. DISCUSSION
 

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

An increased incidence of adrenal pheochromocytomas in male
 

rats was statistically associated with dosage of 4-chloro-m-phenylenediamine. The incidence in the high dose group (14/48 or 29 percent)
 

was significantly higher than the incidence in the control group.
 

These statistical findings indicate that the development of adrenal
 

pheochromocytomas was associated with administration of the chemical.
 

Six male control groups at this laboratory had a mean incidence of
 

adrenal pheochromocytomas of 33/241 (13 percent), with one group
 

having as high as a 26 percent incidence of this lesion. An increased
 

incidence of interstitial-cell tumors of the testis, although statistically associated with dosage, was not considered to be caused by
 

compound administration. The incidences in the high and low dose
 

groups were not significantly higher than that in controls and these
 

tumors commonly appear at high incidences in untreated Fischer 344
 

rats.
 

Among female rats, the incidence of endometrial stromal polyps
 

was significantly higher in the low dose group than in the control
 

group. The incidence was not, however, dose-related and the incidence among historical controls was higher than the incidence observed in either the control or high dose group. It was, therefore,
 

41
 
concluded that the incidence of uterine stromal polyps was not caused
 

by dietary administration of 4-chloro-m-phenylenediamine.
 

Among female mice, the incidence of hepatocellular carcinoma was
 

significant in the low dose group, but the incidence in the high dose
 

group was not significant under the Bonferroni criterion. When incidences of hepatocellular carcinoma and hepatocellular adenoma were
 

combined, the proportion of female mice in each dosed group having
 

either a carcinoma, an adenoma, or both was significant.
 

Under the conditions of this bioassay, dietary administration of
 

4-chloro-m-phenylenediamine was carcinogenic to the experimental
 

animals, causing an increased incidence of hepatocellular tumors
 

in female B6C3F1 mice, and an increased incidence of adrenal pheochromocytomas in male Fischer 344 rats.
 

42
 
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.
 

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

J. Wiley & Sons, New York, 1971.
 

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

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

Union Against Cancer, Geneva, 1969.
 

Chemical Abstracts Service. The Chemical Abstracts Service (CAS)
 

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

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

Corbett, J.F., "The Role of Meta Difunctional Benzene Derivatives in
 

Oxidative Hair Dyeing. I. Reaction with p-Diamines." Journal
 

of the Society of Cosmetic Chemists 24:103-134, 1973.
 

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
 

5.3:457-481, 1958.
 

Krisnan, R.M., "4-Chloro-l,3-benzenediamine." Ger. Offen. 2,428,141
 

(Goodyear Tire and Rubber Company), January 23, 1975; Chemical
 

Abstracts 82, 157140m.
 

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.
 

Matter, J.A., and L. Price, "Color Photographic Materials." Ger.
 

Offen. 1,937,230 (Scott Paper Co.), January 22, 1970; Chemical
 

Abstracts 73, 50722q.
 

43
 
Miller, R.G., Simultaneous Statistical Inference. McGraw-Hill Book
 

Co., New York, 1966.
 

Mueller, P., "Disazo Pigment." Ger. Offen. 2,518,560 (Ciba-Geigy
 

A.-G.), November 13, 1975; Chemical Abstracts 84, 61181r.
 

Papenfuhs, T., "Mixtures of Azomethine and Disazomethine Pigments."
 

Ger. Offen. 2,415,550 (Hoechst A.-G.), October 2, 1975; Chemical
 

Abstracts 84, 6481p.
 

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
 

j^:679-682, 1975.
 

Wiswell, C.M., "Translucent Impregnated Paper Photographic Plate."
 

U.S. Patent 3,394,391 (Scott Paper Co.), July 23, 1968; Chemical
 

Abstracts 69, 7285v.
 

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

44
 
Review of the Bioassay of 4-Chloro~m-phenylenediamine*
 

for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

April 26, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, 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 responsibility of providing a peer review of
 

reports prepared on NCI-sponsored bioassays of chemicals
 

studied for carcinogenicity. It is in this context that the
 

below critique is given on the~bioassay of 4-Chloro-m­ phenylenediamine for carcinogenicity.
 

The primary reviewer agreed with the conclusion that
 

4-Chloro-m-phenylenediamine was carcinogenic in mice and
 

rats. After a brief description of the experimental design,
 he noted a significant dose-related increase in the incidence
 

of adrenal pheochromocytomas in treated male rats and an
 

increased incidence of hepatocellular carcinomas and adenomas
 

in treated mice. The primary reviewer concluded that the
 

bioassay was adequate to assess the carcinogenicity of
 

4-Chloro-m-phenylenediamine in rats and mice. He said that
 

4-Chloro-m-phenylenediamine would appear to pose a carcinogenic
 

risk to humans.
 

The secondary reviewer also agreed with the conclusion
 

given in the report. A discussion followed on the adequacy
 

of the bioassay to provide useful dose-response data. A
 

Subgroup member pointed out that the studies were not
 

45
 
designed for this purpose. Traditionally, the lower dose
 

level was a backup in the event animals at the higher one
 

died from toxicity.
 

It was moved that the report on the bioassay of
 

4-Chloro-m-phenylenediamine be accepted. The motion was
 

seconded and approved unanimously.
 

Members present were:
 

Michael Shimkin (Acting Chairman), University of California
 

at San Diego
 

Joseph Highland, Environmental Defense Fund
 

George Roush, Jr., Monsanto Company
 

Louise Strong, University of Texas Health Sciences Center
 

John Weisburger, American Health Foundation
 

(Sidney Wolfe, Health Research Group, submitted a written review)
 

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.
 

«UA GOVERNMENT PRINTING OFFICE: 1978 260-899/3145 1-3 46 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE
 

TABLE A1

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE

 

CONTROL (DNTR) LOW DOSE HIGH DOSE
 

01-0220 01-0225 01-0230
 

ANIMALS INITIAILY IN STDDY 50 50 950
 ANIMALS HISSING 1
 

ANIMALS HECROPSIED t8 U9 49
 ANIMALS EXAMINED HI STOPATHOLOGICALLY ** 48 49 49
 

INTEGUMENTABY SYST*!"!
 

*SIJBCUT TISSUE («8) (49) (49)
 

FIBPOMA 3 (6%) 3 <6X) 4 (8X)
 

FIBROSARCOMA 2 {!»*) 1 (2*)
 

RESPIRATORY SYSTEM
 

fLDN3 (48) (49) (49)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 3 (6%)
 

ALVEOLAR/BPONCHI3LAR CARCINOMA 1 12%)
 

EIBROSARCOMA, METASTATIC 1 (2*)
 

HEMATOPOIBTIC SYSTEM
 

*MHLTIPL*I ORGANS (18) (49) (49)
 

MALIGNANT IYMPHOMA, NOS 1 (2%)
 

MALI3.LYNPHOMA, HTSTIOCYriC TYPE 1 <2X)
 ONDIFFERFNTIATED LEUKEMIA 2 (H%1
 

tSPLEEN (t8) (49) (49)
 MESOTHELIOMA, METASTATIC 1 (2%)
 MYELOKONOCYTIC LEUKEMIA « (8*)
 

tLYMPH NDDE (U3) (44) (47)
 C-CELL CARCINOMA, METASTATIC 1 (2*)
 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

OLIVER (48) (48) (49) 

NEOPLASTIC_NODnLE ^1-12*1. 1_ 

I NUMBE R 3F ANIMAL S HITH TISSUE EXAMINE D MICROSCOPICALLY 

» HOBBFR IF AHIMAL S NECROPSIED 

» 51 ANIMALS BFR" INITIALLY IH THE STODY, BUT ONE ANIMA L HA S FOOND 

TO BE A FEMAL R IN A MAL E GBOOP. 

**EXCLUDES PARTIALLY AUTOtYZED ANIMALS 

A-3 
TABLE A1 (CONTINUED)
 

CONTROL (UNTR) LOW DOSE 

01-0220 01-0225 

HEPATnCELLULA R CARCINOM A 

IJEJUNUd (46) (48) 

SARCOMA , NOS 

URINAR Y SYSTEM 

NON E 

FNDOC8INF SYSTEM 

tPITOITHR Y (45) (43) 

ADENOMA , NOS 10 (22X) 6 (14K) 

tADRENA L (46) (48) 

CORTICAL ADENOM A 

PHEOCHPOMOCYTOM A 4 (9X) 7 (15K) 

*THYRDID (43) (48) 

FOLLICOLAR-CELL ADENOM A 

FOLLICULAR-CFLL CARCINTM A 

C-CELL ADENOM A 3 (7X) 2 (4*) 

C-CELL CARCINOMA 2 (5%) 1 (2*) 

*PAPATHYROI D (25) (27) 

ADENOMA , NO S 1 (4X) 1 (4X) 

tPANCPEATI C ISLFTS (44) (48)

ISLET-CELL ADENOM A 2 (4X) 

REPRODUCTIV E SYSTEM 

*PRFP(JTI!1L GLAN D (48) (49) 

ADENOMA , NOS 1 (2X) 

#PBOSTATE (45) (47) 

ADENOMA , NOS 1 (2*) 

iTESTJS (48) (49) 

INTERSTITIAL-CELL TOCOS 37 (77X) 43 (88X) 

NESVODS SYSTE M 

&BRSI N (46) (49) 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NFCROPSIED
 

HIGH DOSE 

01-0230 

1 (2X> 

(49) 

1 (2X) 

(47) 

15 (32%) 

(48) 

1 (2X) 

14 (29X) 

(48) 

1 (2*) 

2 (4X) 

3 (5X) 

3 (6X) 

(25) 

(49) 

1 <2X ) 

(49) 

1 (2X) 

(44) 

(49) 

45 (92X) 

(49) 

A-4
 
TABLE A1 (CONTINUED) 

COHTROL (OHTB)

01-0220

 LOU DOSE

 01-0225

 HIGH DOSE 

 01-0230 

ASTROCYTOM A 1 (2%) 

SPECIAL SENSE ORGAN S 

*ZYMBAL'S GLAN D

StiBACEODS ADENOCARCIHOMA

 (t8) (19) (<49) 

2 (f%) 

MOSCULOSKKLETAL SYSTEM 

*SKULL

QSTEOSARCOMA

 (»8)

 1 (2%) 

 (19) (U9) 

BODY C6VITIES 

*BODY CAVITIES

MESOTHELIOBA, NOS

MESOTHELIOHA, MALIGNANT

 (t8)

 1 (2X)

 (»9)

 1 (2*)

 1 (2«) 

 (U9) 

2 (U%) 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SOHMABY 

ANIMALS INITIALLY IN STUDY 

NATDPftL HEATH? 

MORIBUND SACEIFIC11: 

SCHEDULED SACPIFICE 

ACCIDENTALIY KILLED 

TEBMISAL SACEIFICE 

ANIMJL MISSING 

ANIMAL DELETED (HEONG SEX) 

50 

8 

5 

5 

32 

50 

U3 

1 

50 

2 

6 

5 

36 

1 

I NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NOMB"R ->? ANIMALS NEC8OPSIED 

A-5
 
TABLE Al (CONCLUDED)
 

t:OHTROL(ONTR) LOW DOSE HIGH DOSE
 

01-0220 01-0225 01-0230
 

TDHOR SUHHAHY
 

TOTAL ANIHALS WITH PRIHARY TDHOBS* 42 16 U6
 TOTAL PBinSRY TOHORS 7I» 72 99
 

TOTAL ANIHALS WITH BENIGN TUHORS UO Ul| 46
 

TOTAL BENIGN TUHORS 61 66 85
 

TOTAL ANIHALS WITH HALIGNANT TUHORS 11 1) 10
 

TOTAL MALIGNANT TUHORS 12 it 11
 

TOTAL ANIHALS WITH SECONDARY TOHOBS* 2 2
 TOTAL SECONDARY TOHORS 2 2
 

TOTAL ANIHMS WITH TUHORS UNCERTAIN-


BENIGN OR HAIIGNANT 1 2 3
 

TOTAL UNCERTAIN TUMORS 1 2 3
 

TOT4L ANIHALS WITH TOHORS ONCERTAIN-


PRIMARY OR HFTASTATIC
 

TOTAL UNCERTAIN TUHORS
 

* PRIMARY TUHOPS: ALL TUMOHS EXCEPT SECONDARY TDHORS
 « SECONDARY TUHORS: METASTATIC TUMORS 3R TUHORS INVASIVE INTO AN ADJACENT ORGAN
 

A-6
 
TABLE A2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED 

WITH 4-CHLORO-m-PHENYLENEDIAMINE 

CONTROL (UNTR) LOB DOSE 

02-0220 02-0225 

ANIMALS INITIAILY IN STUDY 50 50 SNIHftLS HISSING 

ANIMALS NECROPSIED 50 50 ANIMALS EXAMINED HISTOPATHOLOGICALLY** 50 50 

INTEGUMENTARY SYSTEM
 

*SDBCtIT TISSUE (50) (50)
 SARCOMA, NOS 1 (2%)
 FIBROMA 1 (2«)
 FIBBOSARCOMA 1 (2«)
 

RESPIRATORY SYSTEM
 

#LUNJ (50) (50)
 ADENOCARCINOMA, NOS, METASTATIC 2 (4X)
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (2*)
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (50) (50)
 MALIGNANT LYMPHOBA, NOS 2 (4X)
 

tSPLEEN (50) (50)
 ADENOCARCINOMA, NOS, METASTATIC 1 (2X)
 HENAN3IOMA 1 (2X)
 MYELOMONOCYTIC LEUKEMIA 2 (US)
 

fLYMPH NODE (43) (50)
 ADENOCARCINOKA, NOS, METASTATIC 2 (4%)
 

tTHYMUS (31) (31)
 THY10MA
 

CIRCULATORY SYSTEM
 

*HEART (50) (50)
 

____ ADFNOCARCINOMA^.NOS^aETASTATIC
 

f NOHBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED **EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

HIGH DOSE
 

02-0230
 

50
 

1
 

47
 

47
 

(47)
 

(47)
 

(47)
 

(47)
 

\
 

1 (2X)
 

(45)
 

(35)
 

1 <3X>
 

(47) 

A-7 
TABLE A2 (CONTINUED)
 

CONTROL (IINTR) LOW DOSE
 

02-0220 02-0225
 

DIGESTIVE SYSTEM
 

*INTESTINAL TRACT (50) (50)
 

ADENOCARCINOMA, NOS 1 (2X)
 

iLIVER (50) (50)
 

NEOPLASTIC SODUIE
 

fPAFCREAS (46) (48)
 

ISLET-CELL ADENOMA
 

tSTONACH (49) (49)
 

ADENOCARCINOMA, NOS, METASTATIC 2 (!»*)
 

URINAR Y SYSTEM 

tltlDNEY (H9) (50)
 

ADENOCAFCINOMA , NOS, HETASTATIC 

ENDOCRINE SYSTEM
 

IPITUITAFY (40) (46)
 

CARCINOMA , NOS 1 (2%)
 

ADENOMA , NOS 16 (40X) 15 (33X)
 

CHROM3PHOBE ADENOM A 1 (3X)
 

•ADRENAL	 (48) (»9)
 

PHEOCHROMOCYTOMA 6 (13X) 2 (4X)
 

tTHYBOID (43) (45)
 

FOLLICOHR-CFLL ADBSOMA 1 (2X)
 

FOLLICDLAR-CELL CARCINOM A 1 (2X)
 

C-CELL ADENOMA 2 (4X)
 

C-CELL CARCINOMA
 

REPRODUCTIVE SYSTEM
 

*MAMHARY GLAND	 (50) (50)


ADENOMA, NOS	 1 (2X)
 

ADENOCARCINOMA, NOS	 1 (2X)


FIBKOADENOHA	 fi_J12SL l-ifiSl.


* NUMBER OF ANIMALS WITH TISSDE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECHOPSIED
 

HIGH DOSE
 

02-0230
 

(47)
 

(47)
 

2 (4X)
 

(46)
 

1 (2X)
 

(46)
 

(47)
 

(44)
 

17 (39X)
 

(46)
 

4 (9X)
 

(44)
 

1 (2X)
 

1 (2X)
 

 (47)
 

1 (2X)
 

5_J11S1_
 

A-8
 
TABLE A2 (CONTINUED) 

CONTROL (DNTR)

02-0220

 LOW DOSE

 02-0225

 HIGH DOSE 

 02-0230 

*CLITORAL GLAN D

CARCINOMA,NOS

SQOAMOU S CELL CARCINOM A

ADENOMA , NOS

*t)TERUS

ADENOCARCINOB8, NOS

FIBROMA

END08BTRIAL STROBAL POLYP

ESDOMETRIAL STROMAL SARCOMA

 (50)

 (48)

 1 (2X)

 2 (IX) 

2 (1%)

 2 (*X) 

 (50)

 1 (2%) 

1 (2*) 

 (48)

 2 (M) 

 12 (25X)

 (17) 

1 (2X) 

 (H6) 

5 (1 U) 

NERVOUS SYSTEM 

fBSAIN

ASJROCYTOMA

 (50) (50) (U7) 

1 (2%) 

*SPINAL CORD

ASTROCYTOMA

 (50) (50)

 1 (2%) 

 (t7) 

SPECIAL SENSE ORGANS 

*ZYMBAL' S GLAN D

SEBACEOUS ADENOCARCI1IOMA

 (50) (50) (H7) 

1 (2*) 

(lOSCULOSKELETAL

NONE 

 SYSTEM 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

__NONE 

i NUMBER OP ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OP ANIMALS NECBOPSIED 

A-9
 
1

 TABLE A2 (CONCLUDED)
 

COMTROL(UNTR) LOW DOSE HIGH DOSE
 02-0220 02-0225 02-0230
 

ANIHAL DISPOSITION SUHHARY
 

ANIMALS INITIALLY IN STUDY 50 50 50
 NATURAL DEATHS 7 If 6
 

MORIBUND SACRIFICE 2 7 5
 

SCHEDULED SACRI'ICE 5 5
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 36 39 33
 

ANIMAL MISSING 1
 

9 INCLUDES AOTOLYZED ANIHALS
 

TDMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 27 33 30
 TOTAL PRIMARY TUHORS »2 19 H2
 

TOTAL ANIMALS WITH BENIGN TUHOBS 23 31 27
 

TOTAL BENIGN TUMORS 33 40 3t
 

TOTAL ANIHALS WITH MALIGNAN 9 T TUMORS 9 6
 

TOTAL MALIGNANT TUMORS 9 9 6
 

TOTAL ANIMALS WITH SECONDARY TDBORSI 2
 

TOTAL SECONDARY TUMORS 9
 

TOTAL ANIMALS WI^H TUMORS UNCERTAIN-
 BENIGN OR MALIGNANT 2
 

TOTAL UNCERTAIN TUMORS 2
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN-
 PRIHARY OB METASTATIC
 TOTAL UNCERTAIN TUMORS
 

* PRISAPY TUMORS: ALL TUHOBS EXCEPT SECONDARY TUMORS
 * SECONDARY TUHORS: HETASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

A-10
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE
 

TABLE Bl 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE 

CONTROL(UNTR ) LOW DOSE HIGH DOSE 

05-0220 05-0235 05-02ttO 

ANIMAL S INITIALL Y IN STUD Y 50 50 50 

ANIMALS NECROPSIE D 50 50 50 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY* * 51 19 US 

INTEGUMENTARY SYSTEM 

NONE 

RESPIRATOR Y SYSTE" 

*LDNG (50) (17) (K8)
 HEPATOCELLULAR CARCINOMA, METAST 1 (2«)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 5 (10*) 2 («X) 2 («X)
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 2 <«*) 2 («*) 1 (2X)
 

IC SYSTEM 

*HIILTIPLE ORGAN S (50) (50) (50) 

MALIGNAN T LYfiPHOBA , NOS 1 (2X) 

HALIG.LYMPHOMA , HISTIOCYTIC TYPE 1 (2X) 

MALIGNAN T LYHPHOMA , MIXED TYPE 1 (2%) 

*SPLEEN (50) (<»8) (47)
 

HEMANGIOHA 1 (2X) 1 (2«)
 

HEMANGIOSARCONA 2 (15S)
 

*MESENTE"IC L. NODE («*) (H1) (43)
 

HEMANGIOSAECOMA 1 (2«)
 

MALIGNANT LYMPH1MA, NOS 1 (218) 1 (2%)
 

KALIS. LYHPHOMA, HISTIOCYTIC TYPE 1 (2X)
 

(LIVER (50) (US) (U8)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (2K)
 

(KIDNEY (50) (U8) (48)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (2X)
 

CIRCDLATORY SYSTFM
 

NONE
 

* NDMBER OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NOMB"S OF ANIMAL S NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-3 
DIGESTIVE SYSTFM
 

»LIVEP
 NEOPLASM, NOS
 

H2PATOCELLULAR ADENOMA
 

HEPATOCELLOLAR CARCINOMA
 

HEMAHGIOMA
 

HBBANSIOSAHCOMA
 

UBINABY SYSTEH
 

*KIDNEY
 

TDBOLAR-CEIL ADENOBA
 

ENDOCRINE SYSTEH
 

tPITUITABY
 

ADENOBA, NOS
 

•ADRENAL/CAPSULE
 

ADENOMA, NOS
 

fTHYEOID
 

FOLLICULAR-CE1L ADENOMA
 POLLICU1AR-CELI, CARCINOHA
 

REPBODBCTIVE SYSTEH
 

*TESTIS
 

ESBRYONAt CARCINOHA
 

NERVOUS SYSTEH
 

NONE
 

SPFCIAL SENSH ORGANS
 

*HARDERIAN SLAND
 

CYSTADENOBA, NOS
 

BOSCOLOSKELETAL SYSTEM
 

NOSE
 

* NOHBER OF ANIHALS 1IITH TISSUE

* NDHBER OF ANIMALS NECROPSIED 

TABLE Bl (CONTINUED) 

CONTROL (UNTB) LOW DOSE
 05-0220 05-0235
 

(50)	 (it 8)
 

1 (2*)
 

5 (10%) 2 («%)
 

10	 (20%) 8 (17X)
 

1 (2%)
 

1 (2%)
 

(50)	 (48)
 

1 (2*)
 

(3t) (25)
 

1 (UX)
 

(12) (47)
 

(39)	 (43)
 

1 (3%)
 

1 (3«)
 

(50)	 (48)
 

1 (2X)
 

(50)	 (50)
 

1 (2X)
 

 EXAMINED MICROSCOPICALLY 

HIGH DOSE
 

05-0240
 

(48)
 

4 (8X)
 

15 (31%)
 

(48)
 

(28)
 

(45)
 

1 (2%)
 

(«2)
 

1 (2%)
 

(48)
 

(50)


2 (4X)
 

B-4 
TABLE pi (CONCLUDED)
 

CONTROL (OHTE) LOW DOSE HIGH DOSF 

05-0220 05-0235 05-02UO 

BODY CAVITIE S 

NON E 

ALL OTH'R SYSTEMS 

SITE UNKNOW N 

HEMANGIOM A 1 

ANIMA L DISPOSITION SUMMAR Y 

ANIHAL S INITIALL Y IN STUDY 50 50 50 

- NATTJRA L DEATHS 3 6 5 

MORIBUN D SACRIFICE 3 

SCHEDULE D SACRIFICE 5 5 

ACCIDENTALL Y KILLFD 

TERMINA L SACRIFICE «2 1 1 <to

 ANIMA L MISSING

 

B INCLI!""S AUTOLYZE D ANIMAL S 

TUMOIi SUMMAR Y 

TOTAL ANIMAL S WITH PRIMAR Y TUHOHS* 22 19 25

 TOTAL P8IMAE Y TUMORS 33 23 29

 

TOTAL ANIMAL S WITH BENIGN TDMO8S 12 7 12

 TOTAL BENIG N TUMOBS 13 7 12

 

TOTAL ANIMAL S WITH MALIGNAN T TDMORS 16 1U 17 

TOTAL MALIGNAN T TUMOR S 20 15 17 

TOTAL ANIMALS WITH SFCONDAPY TUMORS* 1
 TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS WITH TOMOES UNCERTAIN-
 BENIGN OF MALIGNANT 1
 TOTAL UNCERTAIN TUMORS 1
 

TOTAL ANIMALS WITT TUMORS UNCERTAIN-


PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TOMOES
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

t SECONDARY TUMORS: METASTATIC TUMORS OB TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-5
 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED 

WITH 4^HLORO-m-PHENYLENEDIAMINE

 

CONTROL(UNTR) LOW DOSE HIGH DOSE 

06-0220 06-0235 06-0210 

ANIMALS INITIALLY IN STUDY 50 50 950 

ANIMALS HISSING 2 2 1 

AN III ft LS NECROPSIED 47 46 46 

ANIMALS SXAMTNE D HISTOPATHOLOGICHLLY** 47 44 t6 

INTEGUMENTARY SYSTEM 

*SKIN (07) (46) (U6) 

KBRATOACAHTHON A 1 (2X) 

*SUBCDT TISSUE (47) (46) (46) 

SQOANOU S CILL CARCINOMA 1 (2*) 

SARCOMA, NOS 1 (2X) 

HEMANGIONA 1 (2%) 

RESPIRATORY SYSTEM 

• LONG (46) (42) (45) 

ALVFOLAR/BRONCHIOLA R ADENOMA 1 (2«) 1 (2X) 

ALVEOLAR/BRONCHIOLAR CARCINOMA 3 (7() 1 (2X) 

HBMATOPOIETIC SYSTEM 

*MOLTIPLE ORGANS (47) (46) (46) 

MALIGNAN T IYMPHOMA. NOS 1 (2() 

MALIS.LYMPHONA , HISTIOCYTIC TYPE 3 (6») 2 (4*) 

UNDIPFERENTIATED LEUKEMIA 1 (2X) 

HALIG.LYMPHOMA , UNDIPPER-TYPE 1 (2%) 

tSPLEEN (45) (41) (45) 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 2 (4*) 

tMESENTESIC L. NODE (38) (39) (41) 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (2X) 

ILIVEP (46) (44) (K5) 

UNDIPFERENTIATED LEOKEHIA 1 <2X) 

tPEYEPS PATCH (44) (42) (45) 

_J_12SL 1.J2X1 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUBBFS OF ANIMALS NECROPSIED 

3 50 ANIMAL S HERE INITIALLY IH THE STUDY, BUT ONE ANIMA L WAS FOUND TO EE 

A HAL E IN A FEMALE GROUP. 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-6 
TABLE B2 (CONTINUED) 

CONTROL (DNTH) LOW DOSE HIGH DOSE 

06-0220 06-0235 06-0210 

IKIDNE Y (46) (44) (15) 

MALTG.LYHPHOMA , HISTIOCYTIC TYPE 1 (2%) 

HTHYMU S (30) (20) (29) 

MALIGNAN T LYMPHOMA . HOS 1 (5X) 

CIRCULATOR Y SYSTEM 

NON E 

DIGESTIVE SYSTEM 

*SAIIVAR Y GLAN D (42) (40) (44) 

TSANSITIONAL-CELL CABCINOMA ,

*LIVEE


HEPATOCE1LBLAE ADENOMA


HEPATOCELLHLAB CARCINOMA


UBINAEY SYSTEM
 

iDRINBRY BLADDER


TRANSITIONAL-CELL CARCINOBH


ENDOCRINE SYSTEM
 

fPITUITARY


ADENOMA, NOS


*ADPENAL/CAPSBLE


ADENOMA. NOS


»THYROID
 FOLLICULAR-CEH ADENOMA


REPRODOCTIVE SYSTEM
 

tOTEBDS

LSIOMYOSABCOMA

t NUMBE R 3F ANIMAL S WITH TISSDE

* SOMBER OF ANIMAL S NECROPSIED 

 THV 1 (2X) 

 (16) (41) («5)
 

3 (7X) 3 (7X)
 

8 (18X) S (1 IX)
 

 («5) (41) (45)
 

1 (2X)
 

 (33)	 (32) (27)
 

1 (3%) 2 (6X)
 

 (40)	 (41) (44)
 1 (3X)
 

 (29)	 (40) (32)
 1 (3%) 1 (3X) 1 (3X)
 

 (45) (41) (44) 

 1_12SL 

 EXAMINE D MICROSCOPICALLY 

B-7 
TABLE B2 (CONTINUED) 

CONTROL (UNTR)

06-0220 LOW DOSE 06-0235

 HIGH DOSE 

 06-02UO 

ENDOMETRIAL STROMAL POLTP 1 (2X) 

iOVABY

PAPILLAR Y ADENOMA

PAPILLARY CYSTADENOMA. SOS

HOCINOUS CYSTADENOCA, INVASIVE

TUBOLAR ADENOHA

 (KO)

 1 (3«) 

 (40)

 2 (5X) 

1 (3») 

 (11) 

1 (2%) 

NIRVODS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

HUSCOLOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

ANIBAL DISPOSITION SDMHABY 

ANIMALS INITIALLY IN STUDY

NATBEAL DEATH9

MORIBHND SACRIFICE

SCH1DDLED SACRIFICE

ACCIDENTALLY KILLED 

TEPMINA L SACRIFICE

ANIMAL MISSING

ANIMA L DELETED (»RONG SEX)

a_!ECLDDES_AOTOLYZED_ANIHJL.S 

 50

 U

 3 

5

 36

 2

 50

 9

2 

 37

 2

 50 

U 

1 

5 

 38 

1 

1 

* NDMBER

* NUMBER

 OF

 OF

 ANIMALS WITH TISSUE

 ANIMAL S NECBOPSIED 

 EXAMINED MICROSCOPICALLY 

B-8 
TABLE B2 (CONCLUDED)
 

COHTBOL(OHTR) LOU DOSE HIGH DOSE 

06-0220 06-0235 06-0210 

TUMOR SUMMAR Y 

TOTAL ANIBAL S WITH PRIMAR Y TUMOBS* 1<» 20 16

 

TOTAL PRIMAR Y TDNORS 15 27 17

 

TOTAL ANIMAL S WITH BEHIGH TUMORS 5 8 7

 

TOTAL BENIG N TDMORS 6 9 7

 

TOTAL ANIMAL S WITH MALIGNAN T TUMORS 9 16 10

 

TOTAL MALIGNAN T TUMORS 9 18 10 

TOTAL ANIMAL S WITH SECONDARY TUBORSt 1 1 

TOTAL SECONDARY TUMOR S 1 1 

TOTAL ANIMALS WITH TUMORS UNCERTAIN-


BENIGN OB MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN-


PRIMARY OR BETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

t SECONDARY TUMORS: METASTATIC TUMORS OR TUBORS INVASIVE INTO AN ADJACENT ORSAN
 

B-9
 

APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE
 

TABLE Cl 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE 

CONTROL (UNTR) 

01-0220 

LOR DOSE 

01-0225 

HIGH DOSE 

01-0230 

ANIMALS INITIALLY IN STUDY 50 

ANIMALS MISSING 

ANIMALS NECROPSIED 48 

ANIMALS EXAMINED HISTOPATHOLOGICALLY** 48 

50 

1 

49 

U9 

350 

49 

49 

INTEGUMENTAR Y SYSTEM 

*SKIN (48) (49) (49)
 

EPIDERMAL INCLUSION CYST 1 (2X)
 

INFLAMMATION, NOS 1 (2X)
 

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

ABSCESS, NOS 3 (6X)
 

RESPIRATORY SYSTEM 

*NASAL TIIRBINATF, (49) (49) INFLAMMATION, SUPPURATIVE <2X) 

fLUNG/BRONCHDS (48] I (49) (19)
 

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

fLUNG (48 ) C»9) (49)
 PNEUMONIA, CHRONIC MDRINE 1 (2X)
 HYPERPLASIA, EPITHELIAL 1 (2«) 1 (2X)
 

HEMATOPOIETIC SYSTEM
 

ISPLEFN (49)
 INFARCT. NOS
 

HEMAT3POIESIS 5 (10X) 4 (8X)
 

fMEDIASriNAL I.NODE (t3) (44) (47) 

PLASMACYTOSIS 1 (2X) 

tTHYMUS (32) (30) (32)
 HYPERPLASIAt_NOS 1-1381.
 

t NUMBER OF ANIMAL S WITH TISSDE EXAMINE D MICROSCOPICALLY 

* NHHBEP OF ANIMAL S NECROPSIED 

9 50 ANIMAL S WERE INITIALLY IN THE STUDY, BUT ONE ANIMA L WAS FOUND TO BE 

A FEMALE IN A MALE GROUP. 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-3 
CIRCULATORY SYSTEM
 

(MYOCARDIUM
 

INFLAMMATION, NOS
 

PIBROSIS
 

DEGENERATION, NOS
 

*AORTA
 

INFLAMMATION, NOS
 

DIGESTIVE SYSTEM 

tLIVER
 

NECROSIS, FOCAL
 

METAMORPHOSIS PATTY
 

BASOPHILTC CYTQ CHANGE
 

HYPERPLASIA, FOCAL
 

ANGISCTASIS
 

HEIUTOPOIESIS
 

*PANCREAS
 

INFLAMMATION, NOS
 

INFLAMMATION, FOCAL
 

*PANCREATIr ACINtIS
 

AIBOPHY, NOS
 

tCOLON
 

PARASITISM
 

DRINA8Y SYSTEM
 

(KIDNEY
 

NEPH»OPATH Y 

CALCIFICATION, NOS 

tRENA L PAPILL A 

MINERALIZATION 

(tKIDNEY/TUBtlLF 

I NUMBE R 3F ANIHAL S WITH TISSOE

* NnMB"S OF ANIMAL S NECROPSIED 

TABLE Cl (CONIINUED) 

CONTROL (UNTR) LOW DOSE 

01-0220 01-0225 

(48) (19)
 

9 (18X)
 

9 (18*)
 

(18)
 

2 (4X)
 

(18)	 (18)
 

7 (15*)
 

2 (IX) it (8X)
 

7 (15*)
 

3 (6X) 2 (IX)
 

1 (2X)
 

(It) (18)
 

2 (">«)
 

1 (2*)
 

(I4U) (18)
 

5 (10X)
 

(12)	 (11)


2 (5X)
 

35 (73X) 17 (96X) 

1 (2X) 

(18) (19) 

3 (6X) 

(18) (19) 

.__ 1.12XL-. 

 EXAMINE D MICROSCOPICALLY 

HIGH DOSE
 

01-0230
 

(19)
 

1 (2X)
 

6 (12X)
 

5 (10X)
 

(19)
 

(19)
 

1 (2X)
 U (8X)
 

9 (18*)
 

1 (2*)
 

1 (2X)
 

(19)
 

(19)
 

3 (6*)
 

(18)
 

(19)
 

U8 (98*)
 

(19)
 

(19)
 

C-4
 
TABLE Cl (CONTINUED) 

CONTROL(UNTR) LOW DOSE HIGH DOSE 

01-0220 01-0225 01-0230 

FNDOCRINE SYSTFM 

(•PITUITARY (45) (17)

 

INFLAMMATION , ACUTE 1 (2«)

 HYPERPLASIA, NOS	 1 (2%)
 HYPEPPLASIA, FOCAL 1 (2%)	 1 (2*)
 

•ADRENAL	 MEDULLA (16) (18) (48)
 HYPEBPLASIA, NODULAR 1 (2*)
 HYPERPLASIA, NOS 1 (2%) 1 (2H)
 HYPERPLASIA, FOCAL 3 (7X) 1 (2*)
 

*THYBOID (13) (18) (48)
 HJPERPLASIA, C-CELL 3 (6*) 1 (2X)
 

(PARATHYROID (25) (27) (25)
 HYPERPLASIA, NOS 1 (4*)
 

IPANCPF.ATIC ISLFTS (11) (48) (49)
 HYPERPLASIA, NOS 1 (2%) 2 (IS)
 

REPRODUCTIVE SYSTEM
 

*PROSTAT" (15) (17) (11)
 INFLAMMATION, N1S 1 (2*)
 

tTESTIS (48) (49) (19)
 MINERALIZATION 1 (2*) 3 (6*) 3 (fiX)
 ATROPHY, NOS 4 (8%) 7 (14X) 7 (145!)
 

HYPFPPI.ASIA, INTERSTITIAL CELL 3 (6*) 3 (6%) 3 (6«)
 

*EPIDIDYMTS (48) (49) (19)
 ABSCESS, NOS 1 (2%)
 GHANULOMR , SPERMATIC 2 (U%)
 

NERVOU S SYSTE M 

NON E 

SPFCTAL ^ ORGAN S 

*EYR (19) 

___ _ CATARAC T 

* NUMBER 3F ANIMALS WITH TISSUE EXAMINE D PI tCROSCOPICA LLY 

* NUMBE R OF ANIMAL S SFCROPSIFD 

C-5 
TABLE Cl (CONCLUDED) 

CONTROL (DHTR) LOW DOSE HIGH DOSE 

01-0220 01-0225 01-0230 

*EYE/RETINA (18) (»9) (H9) 

ATROPHY, KOS 2 (ItX) 

MOSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

OMTNTUM 

NECROSIS, NOS 1 

SPECIAL MORPHOLOGY SUMMAK Y 

NO LESION REPORTED 1 

ANIMA L HISSING/NO NECROPSY 1 

ADTO/NECROPSY/HISTO PEHF 1 

AUTOLYSIS/NO NECROPSY 2 

I NUMBER OP ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECHOPSIED 

C-6 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE

 

CONTROL (UNTR)
 

02-0220
 

ANIMALS INITIALLY IN STUDY 50
 ANIMALS MISSING
 

ANIMALS N3CROPSIED 50
 ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 50
 

INTEGUMENTARY SYSTEM
 

*SKIN
 

EPIDERMAL INCLUSION CYST
 

*SOBCOT TISSUE
 

MINERALIZATION
 

NECROSIS, NOS
 

RESPIRATORY SYSTEM
 

*NASAL TURBINATF
 

INFLAMMATION, NOS
 

*LUNG/BRONCHUS
 

INFLAMMATION, FOCAL
 

INFLAMMATION, NECROTIZING
 

fLUNG
 

INFLAMMATION, NOS
 

INFLAMMATION, INTERSTITIAL
 

ABSCESS, NOS
 

PNEUMONIA, CHRONIC BURINE
 

GRANULOMA, NOS
 

HEMATOPOIETIC SYSTEM
 

tSPLEFN
 

HEHATOPOIESIS
 

tLYMPH NODE
 

t NUMBE R 3F ANIMAL S WITH TISSUE

* NUMB"P OF ANIMALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

(50)
 

(50)
 

(50)
 

1 (2X)
 

(50)
 

(50)
 

1 (2X)
 t (2X)
 

2 (4X)
 

1 (2X)
 

1 (2X)
 

(50)
 

7 (14%)
 

(43)
 

 EXAMINE D MICROSCOPICALLY 

LOW DOSE
 

02-0225
 

50
 

50
 

50
 

(50)
 

1 (2X)
 

(50)
 

(50)
 

(50)
 

1 (2X)
 

(50)
 

3 (6X)
 

(50)
 

22 (44X)
 

(50)
 

HIGH DOSE
 

02-0230
 

50
 

1
 

47
 

47
 

(47)
 

(47)
 

1 (2X)
 

1 (2X)
 

(47)
 

(47)
 

1 <2X)
 

(47)
 

2 (4X)
 

1 (2X)
 

(47)
 

9 (19X)
 

(45)
 

C-7 
CIRCULATORY SYSTEM
 

INYOCARDIDN
 

FIBROSIS
 

DEGENERATION, NOS
 

*AORTA
 

INFLAMMATION, NOS
 

DIGESTIVE SYSTEM
 

*LIVER
 

NECROSIS, FOCAL
 METAMORPHOSIS FATTY
 

BASOPHILIC CYTO CHANGE
 

HYPERPLASIA, FOCAL
 

HEMATOPOIESIS
 

•PANCREAS
 

INFLAMMATION, NOS
 

*PANCREATIC ACINUS
 

ATROPHY, NOS
 

tSTOBACH
 

HYPERPLASIA, PAPILLARY
 

URINARY SYSTEM
 

tKIDNKY
 

MINERALIZATION
 HYDR3NEPHROSIS
 

RLOMERULONEPHRITIS, NOS
 NEPHROPATHY
 

IKIDNEY/TOBULE
 MINERALIZATION
 

ENDOCHINE SYSTEM
 

fPITUITARY
 

TABLE C2 (CONTINUED)
 

CONTROL (ONTR)
 

02-0220
 

(50)
 

(50)
 

(50)
 

4 (8X)
 

9 (18*)
 

(46)
 

(46)
 

(49)
 

1 (2X)
 

(49)
 

1 (2X)
 

18 (37X)
 

(49)
 

(40)
 

LOW DOSE
 02-0225
 

(50)
 

2 (4X)
 

4 (8X)
 

(50)
 

1 (2*)
 

(50)
 

4 (8()
 

27 (54X)
 

1 (2*)
 

(48)
 

1 (2*)
 

(48)
 

6 (13X)
 

(49)
 

(50)
 

1 (2X)
 

43 (86X)
 

(50)
 

5 (10*)
 

(46)
 

HIGH DOSE
 02-0230
 

(47)
 

2 (4X)
 

7 (15*)
 

(47)
 

(47)
 

1 (2*)
 

2 (4X)
 

18 (38X)
 

(46)
 

(46)
 

(46)
 

(47)
 

1 <2X)
 

39 (33«)
 

(47)
 

(44)
 

* NDBBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPIC*LLY
 * NUMBER OF ANIMALS NECROPSIED
 

C-8
 
tADREHA L

 

HYPERPLASIA , NOS 

*ADR£SAL COPTEX 

HYPCTPLASIA. NOS 

tTHYROID 

HYPERPLASIA , C-CEIL 

fPARATHYROI D 

HYPERPLASIA . NODOLA R 

REP8ODOCTIVE SYSTEM 

*MA1MAHY GLAN D 

GALACTOCE1E 

HYPBRPLASIA , NOS 

*CLITORAL GLAN D 

ABSCESS, NOS 

NECROSIS, NOS 

*DTERDS 

HYDROMETP.A 

INFLAMMATION, NOS 

PYOMETH A 

ABSCESS, NOS 

NECROSIS, NOS 

METAPLASIA, SQOAHOOS 

tUTEEUS/ENDOMETRIUM 

INFLAMMATION, NOS 

HYPERPLASIA , NOS 

*OVA8Y 

CYST, NOS 

FOLLICULAR CYST, NOS 

INFLAMMATION , NOS 

DEGENERATION, CYSTIC 

NERVOUS SYSTEM 

HONE_ . _ „ 

t NDHBER OF ANIHALS WITH TISSDE

* NUMBER OF ABTMALS NECROPSIED 

TABLE C2 (CONTINUED) 

CONTROL (UNTR) LOW DOSE 

02-0220 02-0225 

(»8) («9) 

1 <2X) 

(18) (U9) 

1 (2X) 

(U3) (45) 

1 (2X) 2 (14%) 

(31) (19) 

1 <3X) 

(50) (50) 2 (H%) 1 (2*) 

1 (2%) 

(50) (50) 

(<»8) (18) 

2 (M) 

6 (13X) 

1 (2X) 

1 (2X) 

1 (2X) 

1 (2*) 

(18) (t8) 

1 (2*) 

1 (2%) 

(t9) (46) 

1 (2X) 

1 (2%) 2 (4*) 

2 (»X) 

 EXAHINED MICROSCOPICALLY 

HIGH DOSE 

02-0230 

(46) 

(46) 

(44) 

1 <2«) 

(25) 

(47) 

3 (6*) 

(47) 

1 (2%) 

1 (2X) 

(46) 

4 (9X) 

4 (9X) 

(46) 

2 (4X) 

(46) 

2 <4X ) 

C-9 
TABLE C2 (CONCLUDED) 

CONTHOL(UNTR)

02-0220

 LOW DOSE

 02-0225

 HIGH DOSE 

 02-0230 

SPECIAL SENSE ORGANS 

*HARDERHN GLAND

INFLAMMATION, SOS

 (50) (50) (l»7) 

1 (2t) 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHi?R SYSTEMS 

OHENTOM NECROSIS, FAT 1 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION PEPOSTED 11

ANIMAL HISSING/NO NECROPSY

AUTO/NECROPSY/HISTO PERF 1 

AUTOLYSIS/NO NECROPSY

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

2 t 

1 

2 

C-IO
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE
 

TABLE Dl 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH 4-CHLORO-m-PHENYLENEDIAMINE 

COHTBOL(UNTP)

05-0220

ANIMALS INITIALL Y IN STDDY 50

ANIMAL S NEC80PSIF-D 50

•NIHM.S EXAMINE D HISTOPATHOLOGICALLY** 50

INTEGUMENTAR Y SYSTEM 

*SUBCDT TISSU!

ABSCESS, NOS

8ESPIHATORY SYSTEM 

*IONG/BRONCHUS

INFLAMMATION , NOS

INFLAMMATION , FOCAL

ILUNG

INFLAMMATION , INTERSTITIAL

HEBATOPOIETIC SYSTEM 

tSPLEFN


HYPERPLASIA, LYMPHOID


HEMATOPOTESIS


IBFSENTERIC L. HODE


HEMATOPOIFSIS


CIRCULATORY SYSTEM
 

*MYOCARDII»M


DEGENERATION, NOS


DIGESTIVE SYSTEM
 

*LIVER

NECEOSISJ l ._FOCAL 

* NUMBER OF ANIMALS KITH TISSUE

* NUMBE R OF ANIMAL S HECHOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

(50)

 1 (2%) 

(50)

 1 (2*) 

(50)

 (50)


 (44)


 1 (2X)


 (50)


 (50)

 EXAMINED MICROSCOPICALLY 

 LOH DOSE HIGH DOSE 

 05-0235 05-0210 

 50 50 

 50 50 

 49 48 

 (50) (50) 

 (47) (48) 

1 (2%) 

 (47) (48) 

1 (2%) 

 (48) (47)
 

1 (2«)
 

1 (2*) 2 (4X)
 

 (41)	 (43)
 

3 (7*)
 

 (48)	 (48)
 

1 (2«) 1 (2X)
 

 (48) (48) 

D-3 
NECROSIS, COAGULATIVE
 

HYPERPLASTIC NODULE
 HYPEHPLASIA, FOCAL
 

HYPERPLASIA, DIFFUSE
 HEMATOPOIESIS
 

•GALLBLADDER
 

INFLAMMATICN, NOS
 

#PRNCRE1S
 

DEGENERATION, CYSTIC
 

*PANCPEATIC ACINUS
 

ATROPHY, NOS
 

HYPERTROPHY, FOCAL
 

URINARY SYSTEM
 

tKIDNEY
 

HYDRONEPHHOSIS
 

CYST, NOS
 GLOMERULONEPHRITIS, NOS
 

INFLAMMATION, NOS
 

INFLAMMATION, INTERSTITIAL
 

*KIDNEY/TUBULE
 MINERALIZATION
 

ENDOCRINE SYSTEM
 

•PITUITARY
 

HYPERPLASIA, NOS
 

tADRBNAL/CAPSOLE
 

HYPERPLASIA, NOS
 

•ADRENRL CO8TEX
 

HYPFRTROPHY, FOCAL
 

tPANCREATIC ISLETS

INFLAMMATION , NOS

HYPERPLASIA^ADENOHATOnS.

TABLED! (CONTINUED)
 

CONTROL (UNTR)
 

05-0220
 

1 (2X)
 

(50)
 

(50)
 

(50)
 

1 (2X)
 

1 (2X)
 

(50)
 

1 <2X)
 

(50)
 

(34) 

(U2) 

(42) 

 (50) 

3 (6X) 

i_J2Sl_ 

LOW DOSE
 

05-0235
 

3 (6X)
 

1 (2X)
 

(50)
 

3 (6X)
 

(13)
 

1 <2X)
 

(03)
 

(18)
 

3 (6H)
 

1 (2X)
 

7 (15*)
 

(48)
 

(25)
 

(47) 

2 (4X) 

(47) 

1 (2X) 

HIGH DOSE
 05-0240
 

2 (4X)
 

1 (2%)
 

(50)
 

(46)
 

(46)
 

(48)
 

1 (2X)
 23 (1*811)
 

12 (25X)
 

(48)
 

1 (2X)
 

(28) 

1 (2X) 

(45) 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-4
 
TABLE Dl (CONTINUED) 

CONTROL(UNTR) LOW DOSE HIGH DOSE 

05-0220 05-0235 05-0240 

REPRODUCTIVE SYSTFM 

tTESIIS (50) (18) (48) 

ATROPHY , NOS 1 (2%) 

fTESTIS/TUBOLE (50) (48) (48) 

MINERALIZATION 2 (45S) 1 (2X) 

NERVOUS SYSTEM 

*BRAIN (50) (48) (47) 

CHOLESTEATOMA 1 (2%) 

SPECIAL SENSE ORGAN S 

*EYE/LACaiMA L GLAN D (50) (50) (50) 

HYP13RPLASIA, HOS 1 (2*) 

*HARDERIAN GLAND (50) (50) (50) 

INFLAMMATION , NOS 1 (2%) 

HYPERPLASIB , PAPILLAR Y 1 (2X) 

HUSCULOSKBLETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHFR SYSTBNS 

NONE 

SPECIAL MORPHOLOGY SUMMAR Y 

HO_LESIOS_REPORTED J2_ 12 j 

* NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

,D-5 
TABLE Dl (CONCLUDED) 

COHTROL(ONTR) LOW DOSE 

05-0220 05-0235 

NECROPSY PERF/NO HISTO PERFORHED 1 

AUTO/NECROPSY/HISTO PERF 1 

AUTO/NECROPSY/NO HISTO 

I NUMBER OF UNIMftL S HITH TISSUE EXAMINED HICHOSCOPICSLLT 

* NBBBFK OF ANIHAL S NECROPSIED 

HIGH DOSE 

05-02i»0 

1 

1 

1 

D-6 
TABLE D2 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH ^CHLORO-m-PHENYLENEDIAMINE

 

CONTROL (DNTB) 

06-0220 

LOW

06­

 DOSE 

0235 

HIGH DOSE 06-02»0 

ANIMALS INITIALLY IH STUD!

ANIHALS HISSING AHIHALS NECROPSIED

AHIMALS EXAMINED HTSTOPATHOLOGICALLI **

 50 

2 

 47 

 »7 

50 

2 

46 

U4 

350 

1 

46 

46 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSDE (»7) (46) (46)
 HEMORRHAGE 1 <2X)
 INFLAMMATION, NOS 1 (2%)
 

RESPIRATORY SYSTEM
 

*LUNG (15)
 INFLAMMATION, NOS 1 (2X)
 

HEMATOPOIETIC SYSTEM
 

tBONE MAPROR <39) (12) (U3) 

NYELOFIBROSIS 8 (19X) 1 (2X) 

tSPLEEN (15) 

HYPERPLASIA, LYMPHOID 6 (15X) 2 (U«) 

HEHAT3POIESIS 1 (2%) 1 (2X) 1 <2X> 

CIRCDLATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

ILIVER (45) 

NECROSIS, FOCAL 1 (2X) 3 (7X) 

METAMOBPHOSIS PATTY 1 (2X) 

HYPERPLASTIC NODULE 2 (5X) 1 (2%) 

l_i2SL_ 

» NOMBK R 0? ANIHALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

9 50 ANIHAL S HERE INITIALLY IN THE STUDY, BUT ONE ANIMA L WAS FOUND TO EE 

A MALE IN A FEMALE GROUP. 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-7 
*GALLBLADDER
 

HYPEBPLASIA, PAPILLARY
 

iPANCREATIC ACINUS
 

DEGENFRATION, NOS
 

HYPERTROPHY, FOCAL
 

URINARY SYSTEM
 

*KIDNFY
 

HYD8ONEPHROSIS
 

GLONBROLONEPHRITIS. NOS
 

INFLAMMATION, NOS
 

INFLAMMATION, CHRONIC
 

fKIDNEY/TDBOLE
 

MINERALIZATION
 

IDRINAPY BLADDER
 

INFLAMMATION, NOS
 

HYPERPLASIA, EPITHELIAL
 

ENDOCRINE SYSTEM
 

tPITOITARY
 

HYPERPLASIA, NOS
 

tADRENAL/CAPSHLF
 

HYPERPLASIA, NOS
 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND
 

HYPERPLASIA, NOS
 

tUTEBDS/ENDOHETBItlfl
 

INFLAMMATION, NOS
 

HYPERPLASIA, NOS
 

HYPERPLASIA, CYSTIC


•OVAHY/OVID1CT


DEGENERATKJN^NOS

TABLE D2 (CONTINUED) 

CONTROL (DNTR) LOW DOSE HIGH DOSE 

06-0220 06-0235 06-0210 

(17) (16) (16) 

2 (IX) 

(15) (12) (13) 

1 <2X) 

1 (2X) 

(16) (11) (15) 

1 <2X) 

1 (2%) 2 (5%) 1 (9X) 

2 (5%) 6 (13X) 

1 (2X) 

(16) (1U) (15) 

2 (5%) 

(15) (11) (15) 

1 (9X) 3 (7X) 

(33) (32) (27) 

2 (6X) 

(10) (11) 

3 (7X) 

(11) 

1 <9X) 

(17) (16) (16) 

1 (2%) 

(15) (11) (11) 

1 (2X) 

9 (20%) 

1 (2X) 

 (15)

3. [2X] 

 (11) (11) 

* NUMBER DF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUHBEH OF ANIMALS NECROPSIED
 

D-8
 
TABLE D2 (CONCLUDED)
 

CONTROL(ONTP) LOW DOSE HIGH DOSE
 06-0220 06-0235 06-02HO
 

*OVARY <UO) (ItO) (U1)
 

CYST, SOS 1 (3*) 2 (5X)
 

DEGENERATION, CYSTIC 1 (2X)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SF.NSI ORGANS
 

*HARDERIAN GLAND (U7) (l»6) (U6)
 HYPEPPLASIA, NOS 1 (2%)
 

BDSCDLOSKEIETAL SYSTEM
 

NODE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

HONE
 

SPECIAL BORPHOLOSY SUMMARY
 

NO LESION REPORTED 30 10 15
 ANIMAL MISSING/NO NECROPSY 2 2 1
 

NECROPSY PERF/NO HISTO PERFORMED 1
 AUTO/NECROPSY/HISTO PERF 1
 

AUTO/NECROPSY/NO HISTO 1
 AOTOLYSIS/NO NFCROPSY 1 2 2
 

* NOBBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUHBFR OF ANIMALS NECROPSIED
 

D-9
 


DHEW Publication No. (NIH) 78-1335