1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 63 

BIOASSAY OF 

4-CHLORO-O-PHENYLENEDIAMINE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 95-83-0 

NCI-CG-TR-63 

BIOASSAY OF
 

4-CHLORO-O-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) 78-1313
 

REPORT ON THE BIOASSAY OF 4-CHLORO-O-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-o-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 environmental chemicals have the capacity to produce cancer
 

in animals. Negative results, in which the test animals do not have
 

a 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-o-phenylenediamine was
 

conducted by Mason Research Institute, Worcester, Massachusetts,
 

initially under direct contract to the NCI and currently under a
 

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

Carcinogenesis Testing Program.
 

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

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

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

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

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) and Dr. A. Chu (7),
 

using methods selected for the Bioassay Program by Dr. J. J. Gart (8).
 

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

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

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

Ms. P. Walker (5) and the 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 (l), Dr. R. A.
 

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

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

2.	 No wit w h 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.
 

V 

SUMMARY
 

A bioassay for possible carcinogenicity of technical-grade
 

4-chloro-o-phenylenediamine was conducted using Fischer 344 rats and
 

B6C3F1 mice. 4-Chloro-o-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. For male and female rats, the high
 

and low time-weighted average dietary concentrations of 4-chloro-ophenylenediamine were 1.0 and 0.5 percent, respectively. For male and
 

female mice, the high and low time-weighted average dietary concentrations were 1.4 and 0.7 percent, respectively. After a 78-week period
 

of chemical administration, observation of the rats continued for up
 

to an additional 28 weeks and observation of the mice continued for up
 

to an additional 18 weeks. Fifty animals of each species and sex were
 

placed on test as controls for the chronic bioassay.
 

There was a statistically significant positive association
 

between increased dosage and accelerated mortality in female rats
 

and male mice; however, survival among all groups was adequate for
 

meaningful statistical analysis of late-developing tumors.
 

In male and female rats receiving the test chemical a significantly increased incidence of neoplasms of the urinary bladder
 

occurred. Neoplastic nodules in the liver and tumors of the fore-


stomach may also have been related to administration of the chemical.
 

A significantly increased incidence of hepatocellular carcinomas
 

occurred in chemically treated male and female mice.
 

It is concluded that under the conditions of this bioassay
 

4-chloro-o-phenylenediamine was carcinogenic in Fischer 344 rats and
 

B6C3F1 mice, inducing tumors of the urinary bladder and forestomach
 

in both sexes of rats and hepatocellular carcinomas in both sexes of
 

mice.
 

VII
 

TABLE OF CONTENTS

 

Page 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 3
 

A. Chemicals 3
 

B. Dietary Preparation 4
 

C. Animals 4
 

D. Animal Maintenance 5
 

E. Selection of Initial Concentrations 7
 

F. Experimental Design 8
 

G. Clinical and Histopathologic Examinations 9
 

H. Data Recording and Statistical Analyses 13
 

III. CHRONIC TESTING RESULTS: RATS 18
 

A. Body Weights and Clinical Observations 18
 

B. Survival 18
 

C. Pathology 21
 

D. Statistical Analyses of Results 23
 

IV. CHRONIC TESTING RESULTS: MICE 35
 

A. Body Weights and Clinical Observations 35
 

B. Survival 35
 

C. Pathology 38
 

D. Statistical Analyses of Results 39
 

V. DISCUSSION 46
 

VI. BIBLIOGRAPHY 49
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

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

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

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

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 4-CHLORO-OPHENYLENEDIAMINE C-l
 

ix
 
TABLE OF CONTENTS (Concluded)
 

Page 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MICE TREATED WITH 4-CHLORO-OPHENYLENEDIAMINE D-l 

X 
LIST OF ILLUSTRATIONS
 

Figure Number Page 

1 GROWTH CURVES FOR 4-CHLORO-O-PHENYLENEDIAMINE 

CHRONIC STUDY RATS 19 

2 SURVIVAL COMPARISONS OF 4-CHLORO-O-PHENYLENEDIAMINE CHRONIC STUDY RATS 20 

3 GROWTH CURVES FOR 4-CHLORO-O-PHENYLENEDIAMINE 

CHRONIC STUDY MICE 36 

4 SURVIVAL COMPARISONS OF 4-CHLORO-O-PHENYLENEDIAMINE CHRONIC STUDY MICE 37 

LIST OF TABLES 

Table Number Page 

1 DESIGN SUMMARY FOR FISCHER 344 RATS— 

4-CHLORO-O-PHENYLENEDIAMINE FEEDING EXPERIMENT 10 

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

3 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

4-CHLORO-O-PHENYLENEDIAMINE 24 

4 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH 4-CHLORO-O-PHENYLENEDIAMINE 29 

5 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

4-CHLORO-O-PHENYLENEDIAMINE 40 

6 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH 4-CHLORO-O-PHENYLENEDIAMINE 43 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH 4-CHLORO-O-PHENYLENEDIAMINE A-3 

XI
 
LIST OF TABLES (Concluded) 

Table Number Page 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH 4-CHLORO-O-PHENYLENEDIAMINE A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH 4-CHLORO-O-PHENYLENEDIAMINE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH 4-CHLORO-O-PHENYLENEDIAMINE B-6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH 4-CHLORO0-PHENYLENEDIAMINE C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH 4-CHLORO0-PHENYLENEDIAMINE C-8 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH 4-CHLORO-OPHENYLENEDIAMINE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH 4-CHLORO0-PHENYLENEDIAMINE D-8 

xii
 
I. INTRODUCTION
 

4-Chloro-o-phenylenediamine (NCI No. C03292), an aromatic amine
 

used as an intermediate in dye production, was selected for bioassay
 

by the National Cancer Institute because of the high incidence of
 

bladder cancer reported among dye manufacturing industry workers
 

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

one of several classes of chemicals thought to contribute to the
 

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

apparent lack of chronic toxicity data for 4-chloro-o-phenylenediamine
 

was an additional factor in its selection for testing.
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is 4-chloro-l,2-benzenediamine. It is
 

also known as 4-chloro-l,2-diaminobenzene and Ursol Olive 6G (C.I.
 

[Colour Index] 76015).
 

4-Chloro-o-phenylenediamine is used in the production of the dye
 

C.I. Vat Brown 22, as an oxidation base (^Society of Dyers and Colourists, 1956), and in the synthesis of substituted benzimidazothioxanthenoisoquinolinone dyes for polyester fibers (Kadhim and Peters,
 

1974). 4-Chloro-o-phenylenediamine has also been used to synthesize
 

experimental drugs tested for prolongation of barbiturate narcosis in
 

mice, such as 4-methyl-7-chloro-2,3-dihydro-lH-l,5-benzodiazepin-2-one
 

*
 

The CAS registry number is 95-83-0,
 

1 
(Stolyarchuk et al., 1975) and substituted carbomethoxyaminobenzimidazoles, which possess anthelmintic activity in mice, sheep, and dogs
 

(Actor and Pagano, 1975).
 

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

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

Organic Chemicals, United States Production and Sales, 1975 (U.S.
 

International Trade Commission, 1977) implies an annual commercial
 

production in excess of 1000 pounds or $1000 in value. C.I. Vat
 

Brown 22 is not produced in commercial quantities (U.S. International
 

Trade Commission, 1977).
 

Since 4-chloro-o-phenylenediamine is used solely as an intermediate in synthesis, serious risk of exposure is limited to workers
 

in the dye and chemical industries and those involved in pharmaceutical research.
 

2 
II. MATERIALS AND METHODS
 

A. Chemicals
 

Technical-grade 4-chloro-o-phenylenediamine was purchased from
 

Carroll Products, Wood River Junction, Rhode Island and chemical
 

analysis was performed by Midwest Research Institute, Kansas City,
 

Missouri. The experimentally determined melting point (69° to 72°C)
 

suggested a compound of fairly high purity due to its proximity to
 

the literature value (76°C) and narrow range. Elemental analysis was
 

also quite close to the theoretical. Thin-layer chromatography
 

utilizing two systems (ethyl acetate and benzenermethanol) and visualized with ultraviolet light and salicylaldehyde revealed the
 

presence of two impurities. Vapor phase chromatography also showed
 

the presence of two impurities. Nonaqueous titration of the amine
 

function was approximately 96 percent of the theoretical. It should
 

be emphasized that this test yields a definition of maximum purity
 

and may not be indicative of the actual purity of the compound due to
 

the presence of other amines. Infrared analysis was not inconsistent
 

with the structure of the compound. Ultraviolet analysis yielded
 

X at 247 and 304 nm with respective e values of 6860 and 3830.
 max
 

Values reporte v
 d in the literature were \ at 247 and 302.5 nm with
 

 max
 

respectiv v
 e e values of 6725 and 3220. Examination of the lower \
 max
 

indicated an approximate purity of 99 percent, assuming no other
 

impurities absorbed in this region. The value for the upper X was
 

max
 

higher than the literature value and this was probably due to the
 

3
 
presence of impurities absorbing in this region. However, the evidence does suggest a compound of relatively high purity.
 

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

used to represent this technical-grade material.
 

B. Dietary Preparation
 

The basal laboratory diet for both treated and control animals
 

was Wayne Lab-Blox (Allied Mills, Inc., Chicago, Illinois). 4-Chloroo-phenylenediamine was administered to the treated animals as a component of the diet. The compound was mixed in the diet using a 6 kg
 

capacity Patterson-Kelly stainless steel twin-shell V-blender. The
 

mixtures were prepared once weekly, placed in double plastic bags, and
 

stored in the dark at 4°C.
 

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. Animals of both species were supplied by Charles River Breeding
 

Laboratories, Inc., Wilmington, Massachusetts. Treated and control
 

rats arrived in separate shipments, and mice placed in treated groups
 

also arrived in separate shipments from their controls.
 

Upon arrival, a random sample of animals from each shipment was
 

examined for nematode infestation and other signs of disease. The
 

remaining animals were quarantined for 2 weeks prior to initiation of
 

test. Animals were assigned to groups and distributed among cages so
 

4 
that average body weight per cage was approximately equal for a given
 

sex and species.
 

D. Animal Maintenance
 

All animals were housed by species in rooms having a temperature
 

range of 23° to 34°C and a range in relative humidity of 10 to 85
 

percent. Incoming air was filtered through Tri-Dek 15/40 denier
 

Dacron filters (Tri-Dim Filter Corp., Hawthorne, New Jersey), providing six changes of room air per hour. Fluorescent lighting was
 

provided on a 12-hour-daily cycle.
 

Rats were housed five per cage by sex. During the quarantine
 

period and for the first 14 months of study, rats were housed in
 

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

the remainder of the study, rats were housed in suspended solid-bottom
 

polycarbonate cages equipped with disposable nonwoven fiber filter
 

(S)
 sheets. Corncob bedding (SAN-I-CEL , Paxton Processing Company,
 

Paxton, Illinois) or hardwood chip bedding (Aspen bedding, American
 

Excelsior Company, Baltimore, Maryland) and clean cages were provided
 

twice weekly. Stainless steel racks were cleaned once every two
 

weeks, and disposable filters were replaced at that time.
 

Mice were housed by sex in solid-bottom polycarbonate cages
 

fitted with stainless steel lids and nonwoven fiber filter bonnets.
 

Animals were housed ten per cage for the first 12 months of study
 

and five per cage thereafter. Cages, lids, and bedding were changed
 

5 
three times a week while mice populations were ten per cage and twice
 

a week when the cage populations were reduced to five. Hardwood chip
 

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

was used during quarantine and the first month of test. Corncob bed­

®
 ding (SAN-I-CEL ) was used for the next 11 months, then replaced by
 

(R)
 Bed-o-Cobs (The Andersons Cob Division, Maumee, Ohio) for the next 8
 

months. Aspen bedding was used for the remainder of the test. Once
 

every two weeks, filters for mouse cages were changed and pipe cage
 

racks were cleaned.
 

Water was available from 250 ml polycarbonate water bottles
 

fitted with rubber stoppers and stainless steel sipper tubes. Both
 

were replaced twice weekly when animals were housed five per cage,
 

and three times per week when cage populations were ten. Water for
 

rats was supplied as needed between changes. Tap water (chlorinated
 

to 1 ppm at Worcester City Water Department) was used for all animals.
 

Food and water were available ad libitum.
 

®
 Pelleted Wayne Lab-Blox was supplied on cage floors during the
 

quarantine period. During the chemical administration phase of study,
 

®
 Wayne Lab-Blox meal containing the appropriate concentration of the
 

chemical was available to all treated animals. Control animals had
 

untreated meal available. While rats were housed in wire-mesh cages,
 

meal was dispensed in Alpine aluminum feed cups (Curtin Matheson
 

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

steel baffles. While in polycarbonate cages, rats received meal from
 

6 
stainless steel gangstyle food hoppers (Scientific Cages, Inc., Bryan,
 

Texas). Mice were fed from Alpine feed cups for the first 14 months
 

of study and from gangstyle hoppers for the remainder of the study.
 

(R)
 Gangstyle hoppers were changed once per week and Alpine feed cups
 

twice per week.
 

Treated and control rats were housed in a room with other rats
 

receiving diets treated with p-cresidine (120-71-8); iH-benzotriazole
 

(95-14-7); 2,3,5,6-tetrachloro-4-nitroanisole (2438-88-2); 4-chloro-mphenylenediamine (5131-60-2); and acetylaminofluorene (53-96-3).
 

Treated mice were housed in a room with other mice receiving
 

diets treated with acetylaminofluorene (53-96-3); 4-chloro-m-phenylenediamine (5131-60-2); IH-benzotriazole (95-14-7); fenaminosulf
 

(140-56-7); cupferron (135-20-6); o-anisidine hydrochloride (134-29­

0); and p-anisidine hydrochloride (20265-97-8). Control mice were in
 

a room with other mice receiving diets treated with acetylaminofluorene
 

(53-96-3); fenaminosulf (140-56-7); and p-cresidine (120-71-8).
 

E. Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

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

the chronic bioassay, subchronic toxicity tests were conducted with
 

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

five groups, each consisting of five males and five females. 4­

Chloro-o-phenylenediamine was incorporated into the basal laboratory
 

*
 

CAS registry numbers are given in parentheses.
 

7 
diet and supplied ad libitum to four of the five rat groups and four
 

of the five mouse groups in concentrations of 0.03, 0.1, 1.0, and 3.0
 

percent. The fifth group of each species served as a control, receiving only the basal laboratory diet. The dosed dietary preparations
 

were administered for a period of 8 weeks. Individual body weights
 

were recorded at weekly intervals throughout the study. Food consumption was recorded during weeks 1, 4, and 7. All survivors were sacrificed and necropsied at the end of the subchronic test.
 

A dosage inducing no mortality, no gross abnormalities, and no
 

mean group body weight depression in excess of 30 percent relative to
 

controls in either sex was to be selected as the initial high concentration for the chronic bioassay.
 

No gross pathology was observed in any of the animals. Deaths
 

were recorded in all groups receiving 3.0 percent 4-chloro-o-phenylenediamine but not in any other group. Mean body weight depression
 

was approximately 71, 80, 85, and 88 percent in the male rats, female
 

rats, male mice, and female mice, respectively, receiving 1.0 percent
 

of the compound in their feed. The initial high concentration selected for administration to rats and mice in the chronic bioassay was
 

1.0 percent. A concentration of 2.0 percent, however, was utilized
 

for the mouse bioassay.
 

F. Experimental Design
 

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

8 
treated and untreated observation periods, and the time-weighted
 

average concentrations) are summarized in Tables 1 and 2.
 

The low dose, high dose, and control rats were all approximately
 

6 weeks old at the time they were placed on test, but control rats
 

were placed on test one week after dosed rats. The high and low concentrations of 4-chloro-o-phenylenediamine administered to both sexes
 

were 1.0 and 0.5 percent, respectively. At the end of the 78-week
 

dosing period, observation continued for up to an additional 28 weeks.
 

The low dose, high dose, and control mice were all approximately
 

6 weeks old at the time they were placed on test, but control mice
 

were placed on test one week after dosed mice. The high and low concentrations of 4-chloro-o-phenylenediamine initially administered to
 

both sexes were 2.0 and 1.0 percent, respectively. In week 34 the
 

concentrations administered to high and low dose mice were lowered to
 

1.0 and 0.5 percent, respectively, due to high mortality and excess
 

weight depression in high dose rats. At the end of the 78-week dosing
 

period, observation continued for up to an additional 18 weeks.
 

G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

experiment. From the first day, all animals were inspected twice
 

daily for mortality. Body weights were recorded twice weekly for
 

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

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

seven consecutive days once a month for the first nine months of
 

9 
TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

4-CHLORO-O-PHENYLENEDIAMINE FEEDING EXPERIMENT
 

4-CHLORO-OINITIAL PHENYLENEDIAMINE OBSERVATION PERIOD 

GROUP CONCENTRATION TREATED UNTREATED 

SIZE (PERCENT) (WEEKS) (WEEKS) 

MALE 

CONTROL 50 0 0 105 

LOW DOSE 49 0.5 78 

0 27 

HIGH DOSE 50 1.0 78 

0 28 

FEMALE 

CONTROL 50 0 0 106 

LOW DOSE 50 0.5 78 

0 28 

HIGH DOSE 50 1.0 78 

0 28 

10
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

4-CHLORO-O-PHENYLENEDIAMINE FEEDING EXPERIMENT
 

4-CHLORO-OINITIAL PHENYLENEDIAMINE OBSERVATION PERIOD TIME-WEIGHTED 

GROUP CONCENTRATION TREATED UNTREATED AVERAGE 

SIZE (PERCENT) (WEEKS) (WEEKS) CONCENTRATION3 

MALE 

CONTROL 50 0 0 97 0 

LOW DOSE 50 1.0 33 0.7 

0.5 45 

0 17 

HIGH DOSE 50 2.0 33 1.4 

1.0 45 

0 18 

FEMALE 

CONTROL 50 0 0 97 0 

LOW DOSE 50 1.0 33 0.7 

0.5 45 

0 18 

HIGH DOSE 50 2.0 33 1.4 

1.0 45 

0 18 

a . . , . S(concentration X weeks received)
 Time-weighted average concentration = -^~, : :—: : : rr
 2; (weeks receiving chemical)
 

11
 
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.
 

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 Lnd microscopic examination of major tissues,
 

organs, or gross lesions taken from sacrificed animals and, whenever
 

possible, from animals found dead.
 

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

nodes, thymus, heart, salivary gland, liver, gallbladder and bile
 

duct (mice), pancreas, esophagus, stomach, small intestine, large
 

intestine, kidney, urinary bladder, pituitary, adrenal, thyroid,
 

parathyroid, testis, prostate, seminal vesicle, brain, Zymbal's
 

gland, uterus, mammary gland, and ovary.
 

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

with hematoxylin and eosin prior to microscopic examination. An
 

occasional section was subjected to special staining techniques for
 

more definitive diagnosis.
 

A few tissues were not examined for some animals, particularly for
 

those that died early. Also, some animals were missing, cannibalized,
 

12
 
or judged to be in such an advanced state of autolysis as to preclude
 

histopathologic interpretation. Thus, the number of animals for which
 

particular organs, tissues, or lesions were examined microscopically
 

varies and does not necessarily represent the number of animals that
 

were placed on experiment in each group.
 

H. Data Recording and Statistical Analyses
 

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

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

recommended by the International Union Against Cancer (Berenblum,
 

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

These data were analyzed using the statistical techniques
 

described in this section. Those analyses of the experimental results
 

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

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
 

13
 
survival used the method of Cox (1972) for testing two groups for
 

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

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

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

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

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

histologically. However, when macroscopic examination was required
 

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

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

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

The purpose of the statistical analyses of tumor incidence is to
 

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

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

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

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

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

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

significance level of 0.05 may be made. The Bonferroni inequality
 

14
 
(Miller, 1966, pp. 6-10) requires that the P-value for any comparison
 

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

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

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

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

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

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.
 

15
 
When appropriate, life-table methods were used to analyze the
 

incidence of tumors. Curves of the proportions surviving without an
 

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

during which animals died naturally or were sacrificed was entered as
 

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

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

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

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

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

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


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

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

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

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

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
 

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.
 

16
 
The interpretation of the limits is that in approximately 95 percent
 

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

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

be within the interval calculated from the experiment. When the
 

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

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

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

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

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

the lower limit indicates the absence of a significant result while
 

the upper limit indicates that there is a theoretical possibility
 

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

detected under the conditions of this test.
 

17
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

Distinct dose-related mean body weight depression was apparent
 

among treated male and female rats from week 10 until the end of the
 

bioassay (Figure 1).
 

Two low dose males had palpable scrotal masses, one high dose
 

male had a firm abdominal mass, and four low dose females had subcutaneous masses. One low dose female displayed eye discoloration
 

and in another, an exudate from the left ear was observed. Among
 

controls, three females had firm subcutaneous masses, two males had
 

scrotal masses, one male had a subcutaneous nodular mass, one male
 

had a mass at the base of the tail, and several control males
 

exhibited discoloration of the eyes.
 

B. Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and 4-chloro-o-phenylenediamine-dosed groups are shown
 

in Figure 2.
 

For males the Tarone test did not indicate a statistically significant positive association between dose and mortality. The actual
 

survival was adequate for meaningful statistical analysis despite the
 

sacrifice of five high dose and five control rats in week 78, as 56
 

percent (28/50) of the high dose, 80 percent (39/49) of the low dose,
 

and 64 percent (32/50) of the control rats survived until the end of
 

the study.
 

18
 
FIGURE 1

 

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

19 
FIGURE 2

 

SURVIVAL COMPARISONS OF 4-CHLORO-O-PHENYLENEDIAMINE CHRONIC STUDY RATS 

20 
For females the Tarone test indicated a significant association
 

between increased dosage and accelerated mortality. The actual survival was adequate for meaningful statistical analysis despite the
 

sacrifice of five high dose and five control rats in week 78, as 54
 

percent (27/50) of the high dose, 84 percent (42/50) of the low dose,
 

and 72 percent (36/50) of the control rats survived until the end 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).
 

As shown in Tables Al and A2, the incidence of neoplasms in
 

treated rats was higher than in controls. Neoplastic nodules of the
 

liver and tumors of the forestomach and urinary bladder were the predominant neoplasms that occurred in treated rats.
 

The neoplastic nodules of the liver were found in 10 treated
 

rats (4/47 low dose males, 4/48 high dose males, and 2/46 high dose
 

females) but in none of the control rats.
 

Neoplasms of the forestomach developed in 9 high dose rats (4/48
 

males and 5/46 females). Five of these tumors were diagnosed as
 

squamous-cell papillomas and three as squamous-cell carcinomas. A
 

squamous-cell carcinoma of the stomach had metastasized to the liver
 

and lung in one rat.
 

21
 
A spectrum of changes occurred in the urinary bladders of treated
 

rats ranging from transitional-cell hyperplasia to transitional-cell
 

papilloma to transitional-cell carcinoma as summarized in the following table: 

MALES 

Low 

Control Dose 

High 

Dose Control

FEMALES 

Low 

 Dose 

High 

Dose 

Number of Animals with 

Tissues Examined Histopathologically 48 42 49 47 46 45 

Papilloma NOS 

Papillomatosis 

Transitional-Cell 

Papilloma 

Transitional-Cell 

Carcinoma 

Papillary Carcinoma 

Squamous-Cell Carcinoma 

Adenocarcinoma NOS 

0 

0 

0 

0 

0 

0 

0 

0 

0 

8 

7 

0 

0 

0 

2 

0 

5 

18 

0 

3 

1 

0 

0 

0 

0 

0 

0 

0 

1 

0 

9 

4 

1 

0 

0 

0 

2 

8 

22 

0 

0 

0 

A focal increase of cells in the urinary bladder epithelium was considered to be transitional-cell hyperplasia. A polypoid mass covered
 

with markedly cellular transitional-cell epithelium with a delicate
 

fibrovascular septa was diagnosed as transitional-cell papilloma.
 

This tumor was neither extensive nor infiltrating. Cells in this
 

growth were much like those in the normal bladder epithelium.
 

Transitional-cell carcinomas were characterized as solitary or multiple papillary tumors which grew into the lumen of the bladder. Tumor
 

cells were arranged in villi or as syncitia. There was a pleomorphism
 

in shape and size of cells. Mitotic figures were numerous. Squamous
 

metaplasia had occurred in a few tumors. Some of the tumors were
 

22
 
infiltrating. Emboli of tumor cells were occasionally recognized in
 

blood vessels or lymphatics. In two rats, this tumor had metastasized
 

to lymph nodes and the lung or spleen. Squamous-cell carcinoma of
 

the urinary bladder was diagnosed in 3/49 high dose male rats.
 

Except for hydronephrosis of the kidney in 1/47 low dose and
 

6/48 high dose males and 8/47 high dose females, there were no other
 

compound-related nonneoplastic lesions.
 

In conclusion, 4-chloro-o-phenylenediamine is considered to be
 

carcinogenic to Fischer 344 rats for the following reasons: tumors
 

of the urinary bladder occurred in 45 male and 47 female treated rats,
 

but in none of the controls, and neoplastic nodules of the liver and
 

tumors of the forestomach were observed in treated rats but not in
 

the control rats.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

observed in at least one of the control or 4-chloro-o-phenylenediaminedosed groups and where such tumors were observed in at least 5 percent
 

of the group.
 

Neoplasms were noted in large numbers of the urinary bladders of
 

treated rats of both sexes: 0/48 control, 15/42 low dose, and 30/49
 

high dose male rats had a urinary bladder tumor, as did 0/47 control,
 

15/46 low dose, and 32/45 high dose female rats. For male rats the
 

23
 
TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH 4-CHLORO-O-PHENYLENEDIAMINE2
 

TOPOGRAPHY : MORPHOLOGY
 

Subcutaneous Tissue: Fibroma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: 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
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW 

CONTROL DOSE 

3/48(0.06) 1/47(0.02) 

N.S. N.S. ___ 

0.340 

0.007 

4.058 

105 105 

3/48(0.06) 5/47(0.11) 

N.S. N.S. 

1.702 

0.354 

10.411 

105 105 

7/48(0.15) 4/47(0.09) 

N.S. N.S. 

0.584 

0.133 

2.135 

80 93 

HIGH
 

DOSE
 

1/50(0.02)
 

N.S.
 

0.320
 

0.006
 

3.822
 

91
 

4/48(0.08)
 

N.S.
 

1.333
 

0.237
 

8.665
 

91
 

4/50(0.08)
 

N.S.
 

0.549
 

0.125
 

2.011
 

95 
___ 

TOPOGRAPHY : MORPHOLOGY
 

Pituitary: Adenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocytoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinoma'3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (Continued) 

CONTROL
 

10/45(0.22)
 

N.S.
 

102
 

4/46(0.09)
 

N.S.
 

78
 

0/43(0.00)
 

P = 0.034
 

_«_
 

LOW
 

DOSE
 

5/39(0.13)
 

N.S.
 

0.577
 

0.168
 

1.678
 

101
 

5/46(0.11
 

N.S.
 

1.250
 

0.286
 

5.928
 

105
 

0/40(0.00)
 

N.S.
 

__.­

HIGH
 

DOSE
 

4/42(0.10)
 

N.S.
 

0.429
 

0.106
 

1.358
 

106
 

5/48(0.10)
 

N.S.
 

1.198
 

0.276
 

5.687
 

106
 

3/40(0.08)
 

N.S.
 

Infinite
 

0.647
 

Infinite
 

97
 
___ 

TABLE

TOPOGRAPHY : MORPHOLOGY
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma^3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Neoplastic Nodule
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Urinary Bladder: Squamous-Cell Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

3 (Continued) 

CONTROL
 

5/43(0.12)
 

P = 0.013(N)
 

94
 

0/48(0.00)
 

N.S.
 

0/48(0.00)
 

P = 0.042
 

LOW
 

DOSE
 

1/40(0.03)
 

N.S.
 

0.215
 

0.005
 

1.806
 

105
 

4/47(0.09)
 

N.S.
 

Infinite
 

0.948
 

Infinite
 

101
 

0/42(0.00)
 

N.S.
 

HIGH
 

DOSE
 

0/40(0.00)
 

P = 0.033(N)
 

0.000
 

0.000
 

0.846
 

4/48(0.08)
 

N.S.
 

Infinite
 

0.929
 

Infinite
 

78
 

3/49(0.06)
 

N.S.
 

Infinite
 

0.592
 

Infinite
 

89
 
___ 

___ 

TOPOGRAPHY : MORPHOLOGY
 

Urinary Bladder: Transitional-Cell
 

Carcinoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Urinary Bladder: Papilloma NOS,
 

Transitional-Cell Papilloma. or
 

Transitional-Cell Carcinoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Testis: Interstitial-Cell Tumor
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (Continued) 

CONTROL
 

0/48(0.00)
 

P < 0.001
 

0/48(0.00)
 

P < 0.001
 

37/48(0.77)
 

N.S.
 

78
 

LOW
 

DOSE
 

7/42(0.17)
 

P = 0.004
 

Infinite
 

2.229
 

Infinite
 

99
 

15/42(0.36)
 

P < 0.001
 

Infinite
 

5.407
 

Infinite
 

99
 

33/45(0.73)
 

N.S.
 

0.951
 

0.741
 

1.225
 

93
 

HIGH
 

DOSE
 

18/49(0.37)
 

P < 0.001
 

Infinite
 

5.642
 

Infinite
 

70
 

25/49(0.51)
 

P < 0.001
 

Infinite
 

8.070
 

Infinite
 

58
 

40/47(0.85)
 

N.S.
 

1.104
 

0.889
 

1.338
 

78 
TABLE 3 (Concluded)
 

Treated groups received time-weighted average doses of 0.5 or 1.0 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.
 
TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH 4-CHLORO-0-PHENYLENEDIAMINE£
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Adenoma
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or Malignant
 

Lymphoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Stomach: Squamous-Cell Papilloma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW HIGH 

CONTROL DOSE DOSE 

0/50(0.00) 5/49(0.10) 0/47(0.00) 

N.S. P = 0.027 N.S. 

P = 0.002 

Infinite 

1.282 

Infinite 

105 

4/50(0.08) 6/49(0.12) 5/49(0.10) 

N.S. N.S. N.S. ___ 

1.531 1.276 

0.386 0.292 

6.952 6.068 

101 92 89 

0/49(0.00) 0/49(0.00) 3/46(0.07) 

P = 0.033 N.S. N.S. ___ ___ 

Infinite 

0.638 

Infinite 

78 
_ __ 

TOPOGRAPHY : MORPHOLOGY
 

Thyroid: Follicular-Cell Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinomab
 

P Values0
 

U) 

o Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma
 

P Values0
 

Departure from Linear Trend6
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (Continued) 

CONTROL
 

0/43(0.00)
 

N.S.
 

_ —
 

0/43(0.00)
 

P = 0.038
 

6/50(0.12)
 

P = 0.041(N)
 

P = 0.008
 

105
 

LOW
 

DOSE
 

1/45(0.02)
 

N.S.
 

Infinite
 

0.052
 

Infinite
 

105
 

2/45(0.04)
 

N.S.
 

Infinite
 

0.285
 

Infinite
 

105
 

10/49(0.20)
 

N.S.
 

1.701
 

0.609
 

5.266
 

105
 

HIGH
 

DOSE
 

3/44(0.07)
 

N.S.
 

Infinite
 

0.593
 

Infinite
 

101
 

4/44(0.09)
 

N.S.
 

Infinite
 

0.910
 

Infinite
 

78
 

0/49(0.00)
 

P = 0.014(N)
 

0.000
 

0.000
 

0.637
 
___ 

___ 

TABLE 4 (Continued)
 

TOPOGRAPHY : MORPHOLOGY
 

Urinary Bladder: Papillary Carcinoma or
 

Transitional-Cell Carcinoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Urinary Bladder: Papillary Carcinoma,
 

Transitional-Cell Carcinoma, Papilloma
 

NOS, Papillomatosis, or Transitional-


Cell Papillomab
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Adenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

0/47(0.00)
 

P < 0.001
 

0/47(0.00)
 

P < 0.001
 

16/40(0.40)
 

P - 0.030(N)
 

101
 

LOW
 

DOSE
 

5/46(0.11)
 

P = 0.026
 

Infinite
 

1.292
 

Infinite
 

105
 

15/46(0.33)
 

P < 0.001
 

Infinite
 

4.828
 

Infinite
 

105
 

15/38(0.39)
 

N.S.
 

0.987
 

0.534
 

1.808
 

105
 

HIGH
 

DOSE
 

22/45(0.49)
 

P < 0.001
 

Infinite
 

7.517
 

Infinite
 

78
 

32/45(0.71)
 

P < 0.001
 

Infinite
 

11.322
 

Infinite
 

78
 

7/38(0.18)
 

P = 0.032(N)
 

0.461
 

0.182
 

1.038
 

101
 
TABLE 4 (Concluded) 

TOPOGRAPHY : MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Adrenal: Pheochromocytoma 

P Values0 

6/48(0.13) 

N.S. 

2/47(0.04) 

N.S. 

4/46(0.09) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

_ — 0.340 

0.035 

1.799 

0.696 

0.153 

2.741 

Weeks to First Observed Tumor 105 105 99 

Treated groups received time-weighted average doses of 0.5 or 1.0 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.
 

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

group when P < 0.05.
 
Cochran-Armitage test indicated a significant (P < 0.001) positive
 

association between dosage and the incidence of transitional-cell
 

carcinomas. The Fisher exact tests confirmed these findings with
 

significant comparisons of both high dose (P < 0.001) and low dose
 

(P = 0.004) to control. Additionally, when incidences were combined
 

so that the numerator represented male rats with either a transitional-


cell carcinoma, a transitional-cell papilloma, or a papilloma NOS,
 

the Cochran-Armitage test indicated a significant (P < 0.001) dose-


response association. Again, the Fisher exact test confirmed these
 

findings with significant comparisons for both high dose (P < 0.001)
 

and low dose (P < 0.001).
 

Results for the female rats were quite similar. Additionally,
 

when incidences were combined so that the numerator represented an
 

animal with either a papillary carcinoma, a transitional-cell carcinoma, a papilloma NOS, a papillomatosis, or a transitional-cell
 

papilloma of the urinary bladder, the Cochran-Armitage test indicated
 

a significant (P < 0.001) positive association between dosage and incidence. The Fisher exact tests confirmed this with significant (P <
 

0.001) comparisons of both high dose and low dose to control. When
 

incidences were combined so that the numerator represented female rats
 

with either a papillary carcinoma or a transitional-cell carcinoma of
 

the urinary bladder, the Cochran-Armitage test indicated a significant
 

(P < 0.001) association between dosage and incidence. The Fisher
 

exact test comparing high dose to control confirmed this with a
 

33
 
significant (P < 0.001) result; the Fisher exact test comparing low
 

dose to control had a probability level of P = 0.026, a marginal
 

result which was not significant under the Bonferroni criterion.
 

Based upon these statistical results the administration of
 

4-chloro-o-phenylenediamine was associated with an increased incidence of urinary bladder tumors in both male and female rats.
 

The Cochran-Armitage test indicated a significant positive association between dosage and the incidences of follicular-cell thyroid
 

tumors (P = 0.034) in males and of squamous-cell papillomas of the
 

stomach (P = 0.033) and follicular-cell thyroid tumors (P = 0.038) in
 

females. These results were not supported, however, by the findings
 

of the Fisher exact tests. For alveolar/bronchiolar adenomas in
 

females the Fisher exact test comparing low dose to control showed a
 

probability level of P = 0.027, a marginal result which was not significant under the Bonferroni criterion.
 

The possibility of a negative association between dosage and
 

incidence was observed for mammary fibroadenomas in females. In
 

historical data compiled by Mason Research Institute for the NCI
 

Bioassay Program 115/585 (20 percent) untreated female Fischer 344
 

rats had this neoplasm.
 

For C-cell thyroid neoplasms in the males and for pituitary adenomas in the females, the Cochran-Armitage test indicated a significant negative association between dosage and incidence. The Fisher
 

exact tests, however, were not significant.
 

34
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Compound-related mean body weight depression was observed in
 

male and female mice (Figure 3).
 

No adverse clinical signs were observed among treated or control
 

mice of either sex.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and 4-chloro-o-phenylenediamine-dosed groups are shown
 

in Figure 4.
 

For male mice the Tarone test indicated a significant positive
 

association between dosage and mortality. The actual survival,
 

however, was adequate for meaningful statistical analysis despite the
 

sacrifice of five high dose and five control mice in week 78, as 70
 

percent (35/50) of the high dose, 84 percent (42/50) of the low dose,
 

and 84 percent (42/50) of the control mice survived until the end of
 

the study.
 

For female mice no significant positive association between dose
 

and mortality was detected. The actual survival was adequate for
 

meaningful statistical analysis despite the sacrifice of five high
 

dose and five control mice in week 78, as 78 percent (39/50) of the
 

high dose, 88 percent (44/50) of the low dose, and 72 percent (36/50)
 

of the control mice survived until the end of the study.
 

35
 
FIGURES

 

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

35 
FIGURE 4

 

SURVIVAL COMPARISONS OF 4-CHLORO-O-PHENYLENEDIAMINE CHRONIC STUDY MICE 

37 
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).
 

The incidence of neoplasms in mice treated with 4-chloro-ophenylenediamine was higher than that in control mice. Hepatocellular
 

neoplasms accounted for most of the neoplasms in treated mice, and
 

their distribution is summarized in the following table:
 

MALES FE MALES
 

Low High Low High
 

Control Dose Dose Control Dose Dose
 

Number of Animals with
 

Tissues Examined Histopathologically 50 49 47 46 48 47
 

Hepatocellular
 *
 

Carcinoma 10 18 26 0 4 6
 

Hepatocellular Adenoma
 

or Carcinoma 15 28 34 0 11 10
 

*
 

Metastatic to lung (1).
 

The hepatocellular neoplasms occurred in more male than female mice
 

(control or treated). A small circumscribed area comprised of large
 

hepatocytes with eosinophilic cytoplasm and vesicular nuclei and loss
 

of lobular architecture was considered to be a hepatocellular adenoma.
 

Hepatocellular carcinomas had replaced a part of or a whole lobe of
 

the liver. The normal lobular architecture was distorted. Transformed
 

38
 
hepatocytes were large. Cytoplasm of the cells was eosinophilic; in
 

some cells it was vacuolated and suggested fatty metamorphosis.
 

Pleomorphism in nuclear size was evident. There were a few mitotic
 

figures. A hepatocellular carcinoma metastasized to the lung in one
 

high dose male mouse.
 

A variety of nonneoplastic lesions were present in treated and
 

control mice. The only compound-related nonneoplastic lesion was
 

hyperplasia of the gall bladder epithelium which occurred in eight
 

treated mice.
 

The results of this histopathologic examination indicate that
 

there was a compound-related increase in the incidence of hepatocellular neoplasms in male and female mice.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

mice are summarized in Tables 5 and 6. The analysis 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-o-phenylenediaminedosed groups and where such tumors were observed in at least 5 percent
 

of the group.
 

Significant numbers of liver neoplasms were observed in both male
 

and female mice. For males the Cochran-Armitage test indicated a significant (P < 0.001) positive association between dosage and the incidence of hepatocellular carcinomas. The significance (P < 0.001) of
 

the Fisher exact test comparing high dose to control confirmed this
 

39
 
___ 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH


TOPOGRAPHY : MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Carcinoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar Adenoma or
 

Alveolar/Bronchiolar Carcinoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or Malignant
 

Lymphoma
 

c
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

2/50(0.04)
 

N.S.
 

97
 

7/50(0.14)
 

N.S.
 

97
 

4/50(0.08)
 

N.S.
 

_ —
 

97
 

4-CHLORO-0-PHENYLENEDIAMINE£
 

LOW
 

DOSE
 

5/48(0.10)
 

N.S.
 

2.604
 

0.451
 

26.304
 

87
 

9/48(0.19)
 

N.S.
 

1.339
 

0.483
 

3.894
 

87
 

4/49(0.08)
 

N.S.
 

1.020
 

0.200
 

5.183
 

94
 

HIGH
 

DOSE
 

2/47(0.04)
 

N.S.
 

1.064
 

0.080
 

14.153
 

96
 

7/47(0.15)
 

N.S.
 

1.064
 

0.344
 

3.278
 

78
 

3/47(0.06)
 

N.S.
 

0.798
 

0.122
 

4.463
 

82
 
___ 

TOPOGRAPHY : MORPHOLOGY
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Adenoma or
 

Hepatocellular Carcinoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 5 (Continued) 

CONTROL
 

10/50(0.20)
 

P < 0.001
 

60
 

15/50(0.30)
 

P < 0.001
 

60
 

2/39(0.05)
 

N.S.
 

— _
 

97
 

LOW
 

DOSE
 

18/49(0.37)
 

N.S.
 

1.837
 

0.900
 

3.968
 

82
 

28/49(0.57)
 

P = 0.006
 

1.905
 

1.140
 

3.246
 

82
 

2/46(0.04)
 

N.S.
 

0.848
 

0.064
 

11.248
 

95
 

HIGH
 

DOSE
 

26/47(0.55)
 

P < 0.001
 

2.766
 

1.475
 

5.519
 

39
 

34/47(0.72)
 

P < 0.001
 

2.411
 

1.518
 

3.817
 

39
 

3/41(0.07)
 

N.S.
 

1.427
 

0.174
 

16.339
 

96
 
TABLE 5 (Concluded) 

a
 

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-O-PHENYLENEDIAMINE2
 

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 Values
 

Departure from Linear Trend
 

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.
 

P = 0.029
 

69
 

LOW
 

DOSE
 

1/48(0.02)
 

N.S.
 

0.319
 

0.006
 

3.809
 

96
 

2/48(0.04)
 

N.S.
 

0.479
 

0.045
 

3.171
 

96
 

11/48(0.23)
 

N.S.
 

1.795
 

0.669
 

5.438
 

87
 

HIGH
 

DOSE
 

0/45(0.00)
 

N.S.
 

0.000
 

0.000
 

1.694
 

3/45(0.07)
 

N.S.
 

0.767
 

0.118
 

4.275
 

96
 

3/48(0.06)
 

N.S.
 

0.490
 

0.083
 

2.148
 

88
 
___ 

TABLE 6 (Concluded)
 

TOPOGRAPHY : MORPHOLOGY
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Adenoma or
 

Hepatocellular Carcinoma
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

0/46(0.00)
 

P = 0.014
 

0/46(0.00)
 

P = 0.003
 

P = 0.048
 

LOW
 

DOSE
 

4/48(0.08)
 

N.S.
 

Infinite
 

0.891
 

Infinite
 

95
 

11/48(0.23)
 

P < 0.001
 

Infinite
 

3.325
 

Infinite
 

95
 

HIGH
 

DOSE
 

6/47(0.13)
 

P = 0.014
 

Infinite
 

1.570
 

Infinite
 

78
 

10/47(0.21)
 

P = 0.001
 

Infinite
 

3.043
 

Infinite
 

78
 

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).
 

G

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

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

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

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

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

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

group when P < 0.05.
 
finding. When incidences were combined so that the numerator represented a male with either a hepatocellular carcinoma or a hepatocellular adenoma, the results were stronger: The Fisher exact test
 

comparing low dose to control (P = 0.006), the high dose comparison
 

(P < 0.001), and the Cochran-Armitage test (P < 0.001) were all
 

significant.
 

For females the Cochrart-Armitage test also indicated a significant (P = 0.014) positive association between dosage and the incidence
 

of hepatocellular carcinomas. The comparison of high dose to control
 

(P = 0.014) confirmed these findings. When incidences were combined
 

so that the numerator represented females with either a hepatocellular
 

carcinoma or a hepatocellular adenoma, the Cochran-Armitage test was
 

again significant (P = 0.003). The departure from linear trend was
 

significant (P = 0.048), primarily because of the elevated incidences
 

observed in both dosed groups. The Fisher exact tests were significant (P _<_ 0.001) for both high and low dose females.
 

Based on these results the statistical conclusion is that there
 

was a positive association between the administration of 4-chloro-ophenylenediamine and the incidence of liver tumors in mice of both
 

sexes.
 

45
 
V. DISCUSSION
 

Survival among all groups in this bioassay was adequate for
 

meaningful statistical analysis of late-developing tumors.
 

In rats, a broad spectrum of neoplasms of the urinary bladder
 

was observed among treated males and females (i.e., the incidence of
 

total tumors of the bladder was 0/48 control males, 15/42 low dose
 

males, 30/49 high dose males, 0/47 control females, 15/46 low dose
 

females, and 32/45 high dose females). These tumors included primarily transitional-cell papillomas and transitional-cell carcinomas.
 

For males, when incidences were grouped so that the numerator represented rats with a papilloma NOS or transitional-cell papilloma of
 

the bladder, the Cochran-Armitage test indicated a significant positive association between dosage and incidence. This association was
 

substantiated by significant results of the low dose to control and
 

high dose to control Fisher exact comparisons. When the incidences
 

were grouped so that the numerator represented males with bladder
 

carcinomas (i.e., squamous-cell carcinoma, transitional-cell carcinoma, or adenocarcinoma NOS) the Cochran-Armitage test and both Fisher
 

exact comparisons were significantly positive. In the females, when
 

the numerator of the incidence represented those rats with papilloma
 

NOS, papillomatosis, or transitional-cell papilloma, the Cochran-


Armitage test indicated a significant positive association between
 

dosage and the incidence of these bladder tumors. In addition, the
 

high dose and low dose to control Fisher exact tests confirmed the
 

46
 
relationship. When the numerator of the incidence represented
 

females with papillary carcinomas or transitional-cell carcinomas,
 

the Cochran-Armitage test and the high dose to control Fisher exact
 

comparison were significantly positive.
 

Two types of unusual tumors were detected in the forestotnach of
 

high dose male and female rats (i.e., squamous-cell papillomas in
 

2/48 [4 percent] high dose males and 3/46 [7 percent] high dose
 

females and squamous-cell carcinomas in 2/48 [4 percent] high dose
 

males and 1/46 [2 percent] high dose females). Although these tumors were not present in statistically significant incidences they
 

do occur with sufficient rarity to be of concern and are considered
 

in this study to be related to administration of the test compound.
 

This is further supported by the finding of nonneoplastic proliferative lesions of the stomach in several of the treated animals.
 

In mice hepatocellular carcinomas occurred in 10/50 (20 percent),
 

18/49 (37 percent), and 26/47 (55 percent), of the control, low dose,
 

and high dose males, respectively, and in 0/46, 4/48 (8 percent), and
 

6/48 (13 percent) of the control, low dose, and high dose females,
 

respectively. In both sexes statistical analysis of these incidences
 

indicated a significant positive association between dosage and tumor
 

incidence and this association was confirmed by the high dose to control Fisher exact comparison. When incidences were combined so that
 

the numerator represented those animals having either hepatocellular
 

carcinomas or hepatocellular adenomas, for both males and females,
 

47
 
the low dose to control Fisher exact comparison was significant in
 

addition to the Cochran-Armitage test and the high dose to control
 

Fisher exact comparison. There were no other neoplasms occurring at
 

statistically significant incidences in male or female mice.
 

It is concluded that under the conditions of this bioassay
 

4-chloro-o-phenylenediamine was carcinogenic in Fischer 344 rats and
 

B6C3F1 mice, inducing tumors of the urinary bladder and forestomach
 

in both sexes of rats and hepatocellular carcinomas in both sexes of
 

mice.
 

48
 
VI. BIBLIOGRAPHY
 

Actor, P.P., and J.F. Pagano, "5(6)-Butyl- and propoxy-2-carbomethoxybenzimidazoles." U.S. Reissue 28:403 (Smithkline Corporation)
 

Chemical Abstracts 84, 4950s, April 1975.
 

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

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

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

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

J. Wiley & Sons, New York, 1971.
 

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

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

Union Against Cancer, Geneva, 1969.
 

Chemical Abstracts Service. The Chemical Abstracts Service (CAS)
 

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

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

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.
 

Kadhim, A.M., and A.T. Peters, "New Intermediates and Dyes for
 

Synthetic Polymer Fibers. Substituted Benzimidazothioxanthenoisoquinolinones for Polyester Fibers." Journal of the Society
 

of Dyers and Colourists 90(6):199-202; Chemical Abstracts 83,
 

61612m, 1974.
 

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

Observations." Journal of the American Statistical Association
 

j[3_:457-481, 1958~
 

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

"Carcinogenesis Bioassay Data System." Computers and Biomedical
 

Research 7:230-248, 1974.
 

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

Co., New York, 1966.
 

49
 
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.
 

Stolyarchuk, A.A., Y.N. Furman, V.L. Pikalov, Z.F. Solomko, and S.V.
 

Tkachenko, "Synthesis and Pharmacological Study of 2,3 Dihydro1H-1,5-benzodiazepin-2-ones." Khimiko-Farmatsevtichi cheskii
 

Zhurnal 9(8):19-21; Chemical Abstracts 84, 9925g, 1975.
 

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

i62:679-682, 1975.
 

U.S.	 International Trade Commission, Synthetic Organic Chemicals,
 

United States Production and Sales, 1975. USITC publication 804,
 

U.S.	 Government Printing Office, Washington, D.C., 1977.
 

Wynder, E.L., J. Onderdonk, and N. Mantel, 1963. "An Epidemiological
 

Investigation of Cancer of the Bladder." Cancer 16:1388-1407,
 

1963.
 

50
 
APPENDIX A 

SUMMARY OF THE INCIDENCE OF NEOPLASMS 

IN RATS TREATED WITH 4-CHLORO-O-PHENYLENEDIAMINE 

TABLE A1

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED 

WITH 4-CHLORO-O-PHENYLENEDIAMINE 

CONTBOL (Oilfi) LOW DOSE HIGH DOSE 

01-0220 01-0205 01-0210 

AHIKAL S INITIALLY I K STUDY 50 349 50 

AHIMAL S KECBOPSIED i«8 47 50 

ANIHALS EXAMINE D HISTOEATHOLOGICALLY ** 48 47 49 

INTEGUHENTAB Y SYSTEM 

*SUECUT TISSUE (U8) (47) (50) 

FIEBOBA 3 (6%) 1 (2%) 1 (2X) 

IIEEOSAECOHA 2 («%) 

FIEEOADEN'OMA 1 (2X) 

SESPIEA10BY SYSTEH 

• LUN G (48) (47) (48) 

SQUAHOUS CELL CARCIHCHA, (1E1ASTA 1 (2X) 

TBA1ISITIONAL-CELL CABCINOtlA, NET 1 (2X) 

ALVJEOLAB/BRONCHIOLAR ADHHOH A 3 (6%) 5 (11X) 4 (8X) 

FIEBOSAECOHA, MEIASTATIC 1 (2%) 

HEBA10POIETIC SYSTEM 

*MULIIEIE DEGA11S 

MALIGNAN T LYHFHOMA , KO S 

LEDKEHIA,NO S 

USDIFFERENTIATED LEUKEHI A 

MYELCHONOCSTIC LEUKEBIA 

(48) 

1 (2%) 

2 (4X) 

(47) 

2 («X) 

1 (2X) 

1 <2X) 

(50) 

1 (2X) 

3 (6X) 

tStLEE K 

TEAHSITIONAL-CELL CARCINOHA,

MYELOBONOCYTIC LEUKEHIA 

 BET 

(48) 

t (8X) 

(46) 

1 (2X) 

(48) 

•LYKP H MODE 

TRAHSITIONAL-CELL CAECISOBA, HET 

(43) (40) 

1 (3X) 

(39) 

1 (3X) 

CISCULATCEY SYSTEH 

NON E 

* NUMBE R OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY * NUM B IE 01 ANIMALS NEC60PSIEE 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

a NOTE: 50 ANIMALS HERE INITIALLY III STUDY BUT ONE HAS FOUND TO BE A FEMALE IN A BALE 

GBOUP AID IAS DELETED. 

A-3 
TABLE Al (CONTINUED) 

HIGH DOSE 

01-0210 

(48) 

1 (2X) 

« <8X) 

(48) 

2 (MX ) 

2 (4X) 

(35) 

1 (3X) 

(49) 

1 <2X) 

2 (IX) 

3 (6X) 

5 (10X) 

18 (37X) 

1 <2X) 

(42) 

4 (10X) 

(48) 

5 (10X) 

(48) 

1 <2X) 

(40) 

2 (5X) 

1 (3X) 

(22) 

(41) 

1 (2X) 

DIGESTIVE SVSTEM 

OLIVER 

SQUABOOS CELL CARCINOMA ,

NECFIASTIC NODDLE 

•STCHACfi 

SQUAHOUS CELL PAPILLCHA 

SQOAHOUS CELL CABCINOHA 

ICOLON 

ADEKCHATOOS POLYP, NOS 

UBINAB Y SYSTEM 

tUBINAB Y BIADDEB 

NECEIASM, NOS 

PAPILLOHA, NOS 

SCUAHODS CELL CABCINOHA 

TRANSITIONAL-CELL PAPIL10HA 

IEANSITIONAL-CELL CAECINOHA 

ADENOCABCINOHA,

ENDOCRIN E SYSTEM 

•PITUIIABY 

AEiKCHA , NOS 

•AEBEBAI 

EHICCHBOMOCYTOHA 

tALEENA l MEDULLA 

GAKGLIONEU&OHA 

tlHYBCIC 

fOIilCDLAE-CELL

F01LICULAB-CELL

C-CILl ADENOHA 

C-CELL CARCINOMA 

tPABATHYBOID 

ADIhCBA , NOS 

*PANCEIATIC ISLETS 

 NOS 

 ADENOM A 

 CABCINOHA 

ISIIT-CELi. ADENOMA 

* NUMBI 8 OF ANIMAL S HUH TISSUE

* MUBBI B OF ANIMALS NECBOPSIED 

CONTHOL (UNTB) 

01-0220 

(18) HETASTA 

<«8) 

(U2) 

(18) 

(<t5) 

10 (22*) 

(<(6) 

« (9X) 

(«6) 

(«3) 

3 (7X) 

2 (5*) 

(25) 

1 <4X) 

(««) 

 EXAMINED HICBOSCOPICALLY 

A-4 

LOH DOSE 

01-0205 

(47) 

» (9X) 

(46) 

(«3) 

(42) 

8 (19X) 

7 (17*) 

(39) 

5 (13X) 

(46) 

5 (1«) 

(46) 

(40) 

1 (3X) 

(28) 

(13) 

2 (5X) 
TABLE Al (CONTINUED) 

CONTROL (ONTB) LOW DOSE 

01-0220 01-0205 

BEPRCDtCTIVE SYSTEM 

*SEMINA L VESICLE (18) (17) 

ADENOMA , NOS 1 (2X) 

tTESTIS (18) (15) 

INTIBSTITIAL-CELL TOM08 37 (77X) 33 (73X) 

IIEBVOUS SYSTEM 

tBRAIN (16) (16) 

OSTICSARCOMA, METASTATIC 1 (2X) 

ASIBOCYTOMA 2 (<4X) 

SPECIAL SENSE ORGAN S 

»ZYMBAL« S GLAND (18) (17) 

SEBACEOUS ADENOCABCINONA 1 (2X) 

HUSCULOSKELFTAL SYSTEM 

*SKULL (18) (17)

 

CS1 JCSABCOMA 1 (2*)

 

BODY CAVITIES 

*BODY CAVITIES (18) (17) 

BESCIHELIOMA. NOS 1 (2X) 2 (IX) 

'ABDOMINAL CAVITY (18) (17) 

TBANSI1IONAL-CELL CABCINOHA, MET 1 <2X) 

ALL OTHEB SYSTEMS 

SCBZ 

f NUMBEB Of ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIMALS NECBOgSIED 

A-5 

HIGH DOSE 01-0210 

(50) 

(17)

40 (85X) 

(18) 

(50) 

(50) 

(50) 

(50) 
TABLE Al (CONCLUDED) 

(:CNTBOL(ONTB) LCH DOSE HIGH DOSE 

01-0220 01-0205 01-0210 

ANIBAL DISPOSITION SUMMARY 

AKIMAIS INITIALLY IN STODY 50 50 50

 

NATURA L DEATHS 8 5 10 

MORIBUN D SACEIPICE 5 5 7 

SCHEtULE D SACRIFICE 5 5 

ACCIDENTALLY KILLED 

IEKBINAL SACRIFICE 32 39 28 

ANICA L HISSING 

ANIHA l DELETED (MBON G SEt) 1

 

3 INCLGDES AUTOLYZE D ANIMALS 

TDeCE SUKMAEl f 

TOTAL ANIMALS KITH PRIMAB Y TOHOES* 42 42 46 

TOTAL EEIMAB Y TUMOES 74 81 103 

TOTAL ANIMALS KITH BENIGN TUHOBS 40 41 43 

TOTAI BENIG N TUMOE S 61 61 69 

TOTAL ANIMALS KITH MA1IGSANT TDHOBS 11 13 26 

T01AI MALIGNANT TUHOES 12 14 29 

TOTAI ANIMALS KITH SECONDASY 1UHOFS* 2 1 2 

TOTAL SECONDABY TDMOfiS 2 3 4 

10TAL ANIMALS WITH TOMOSS ONCIRTAINBINIGK OE MALIGNANT 1 5 5 

TOTAI UNCERTAIN TUMOES 1 6 5 

10TAL ANIMALS glTH TU80BS DNCIBTAINFIIMAEY CK METASTATIC

 

TOTAI ONCEETAIN TUMOES 

* PBIMAE I 10BORS: ALL TUHOBS EXCEPT SECCNDAB Y TOHOBS 

t SECOfcEABY TUHORS: MEIASTA1IC TUMOfiS OE TUMOBS INVASIVE INTO AN ADJACENT ORGAN 

A-6 
TABLE A2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED 

WITH 4-CHLORO-O-PHENYLENEDIAMINE 

CONTROL (UNTB) LOR DOSE HIGH DOSE 

02-0220 02-0205 02-0210 

ANIMAL S INITIALLY IN STUDY 50 50 50 

ANIMAL S HISSING 1 

ANIMALS KECBOPSIED 50 49 49 

ANIMALS EXAMINED HISTOEATHOLOGICALLY *1 < 50 <I9 48 

IN1EGUMENTAEY SYSTEM 

*SOBCll 1ISSUE (50) (19) (19)

 

SAECCMA, NO S 1 (2*)

 LEIOMYOSAECOHA 1 (2X)

 

RESPIBA10RY SYSTEM 

iLDN G (50) (19) <t7)

 ALVICLAE/6EONCHIOLAB ADENOMA 5 (10*)

 

HEHATOPCIETIC SYSTEM 

*HULTIEI E OBGANS (50) (149) (49) 

MALIGNAN T LYMPHOMA , NOS 2 (IX) 1 (2X) UNEIFFEEENTJATED LEUKEHIA 2 (H%) 

KYELOMONOCYTIC LEDKEBIA 3 (6«) 5 (10X) 

• SHEEN (50) (t8) (46) 

SQUAfiOU S CELL CARCINOMA , HE1ASTA 1 (2%) MYEIOMONOCYTIC LEUKEHIA 2 (<»X) 

CIECULA10EY SYSTEM 

NON E 

DIGESTIVE SYSTEM 

tLIVEE (50) (t9) (<»6) NECELASTIC NODULE 2 (4%) 

*S1CHACH (U9) (U9 ) (46) 

SQUABOUS CELL PAPILLOMA 3 (7X1 

t NUHBE B OF ANIMAL S KITH TISSUE EXAHINE D HICBOSCOPICALLY 

* NUMBIB OF ANIMALS NECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-7 
TABLE A2 (CONTINUED) 

CONTEOL(DNTB) LOU DOSE 

02-0220 02-0205 

SCCABOUS CELL CARCINOMA 

BASAL-CELL CABCINOBA 

DBINABJ SYSTEM 

*U£INABI BLADDEB (47) (46) 

PAFIILCBA, NOS 1 (2X) 

PAPILLAE! CARCINOMA 1 <2X) 

PAPILLOMATOSIS 

TRANSITIONAL-CELL PAPILLOBA 9 (20X) 

TRANSITIONAL-CELL CABCINOHA 4 (9X) 

ENDOCRINE SYSTEM 

tPIIUHABY (40) (38) 

ADIBOBA , NOS 16 (40X) 15 (39X) 

CHBOBOPHOBE ADENOM A 1 (3X> 

tADREKA L (48) (47) 

PHF.CCHBOMOCYTOBA 6 (13X) 2 (4X) 

tALEE(,A L COBTEX (48) (47) 

LIECBA 1 (2X) 

*THYBCir (43) (45) 

FOIIICULAH-CELL ADENOBA 1 (2X) 

FOLLICULAR-CELL CARCINOBA 1 (2X) 

C-CELL CABCINOBA 1 (2X) 

BEPBODUCIIVE SYSTEM 

• MAKMAB Y GLAND (50) (49) 

ADIKCBA , NOS 

ADENOCARCINOMA, NOS 1 (2X) 

FIEROADENOHA 6 (12X) 10 (20X) 

*CLITCEAl GLAN D (50) (49) 

ADIUCflA , NOS 1 (2X) 

*OTIBUS (48) (49) 

ADOCCAECINOMA, NOS 1 <2X ) 

FIEBOHA 2 (4X) 1 (2X) 

ENDOBETRIAL STBOMAL POLYP 2 (4X) 2 (4%) 

t SUBBEE OF ANIHALS KITH TISSUE EXAHINE D MICROSCOPICALLY 

* NUMBE E OF ANIMALS NECROPSIED 

A-8 

HIGH DOSE 

02-0210 

1 (2X) 

1 (2X) 

(45) 

2 («X) 

8 (18X) 

22 (49X) 

(38) 

7 (18X) 

(46) 

4 (9X) 

(46) 

(44) 

1 <2X) 

3 (7X) 

2 (5X) 

(49) 

2 (4X) 

(49) 

(46) 

1 (2X1 
TABLE A2 (CONTINUED) 

CONTROL (UNTR) LCD DOSE HIGH DOSE 

02-0220 02-0205 02-0210 

ENtCBITRIAL STROMA1 SARCOBA 2 («») 

NEBVOUS SYSTEM 

(BRAI N (50) (49) (46) 

ASTKCCYTOBA 1 (2X) 

SPECIAL SEKSE ORGANS 

*ZYMBAL' S GLAND (50) <U9) (49) 

SQUAHOUS CELL CARCINOMA 1 <2«) 

MUSCULOSKELETAL SYSTEM 

NOME 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

SITE CNKNOHN 

SQUABOU S CELL CARCINOHA 1

 

ANIMA L DISPOSITION SOMHAR Y 

AI.IHALS INITIALLY IN STUDY 50 50 50

 

NATURA L DEATHS 7 6 8

 

MORIBUND SACRIFICE 2 2 9 

SCHEDULED SACRIFICE 5 5 

ACCIDENTALLY KILLED 

lEBHINAL SACRIFICE 36 U2 27

 

ANIBA L MISSING 1

 

* INCLUDES AUTOLYZED ANIMAL S 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-9
 
TABLE A2 (CONCLUDED) 

CCNTBOL(UHTB) LCN DOSE HIGH DOSE 

02-0220 02-0205 02-0210 

10DCE SGBHAE J 

TOTAL ANIMALS WITH PBIHABl TUflOES* 27 35 37 

TOIAI FBIflABI TUMOBS 12 62 67 

TOIA1 ANIMALS KITH BENIGN TUMCBS 23 27 20 

TOIAI BENIGN TUMOES 33 18 28 

TOTAI ANIMALS KITH MAtlGNANI TUMORS 9 13 30

 

TOTAL MALIGNANT TUMOES 9 14 37 

TOTAI ASIMALS HITH SECONDABJ IOMOBS* 1 

TOTAI SECONDABK TtlMOBS 1 

TOTAL ANIMALS HITH TUHOBS UNCIBTAINBIHIGH OE MALIGNANT 2 

TOTAL UNCERTAIN TUMOBS 2 

TOTAL ANIMALS WITH TUMOBS UNCIBIAINFBIMAB Y CE METASTATIC

 

TOTAI UNCEBTAIN TUMOBS 

* PE1MAB1 TDHO£S: ALL TDHORS EXCEPT SICONDABY TUBOBS
 

* SECOHTABY TUBOBS: HETASTATIC 10HOBS OB TOBOES INVASIVE INTO AM ADJACENT OBGAH
 

A-10 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH 4-CHLORO-O-PHENYLENEDIAMINE
 

TABLE Bl

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED 

WITH 4-CHLORO-O-PHENYLENEDIAMINE 

HIGH DOSE 

05-0215 

50 

17 

17 

(17) 

1 (2X) 

(«7) 

1 <2X) 

5 (11X) 

2 (*X) 

(«7) 

1 (2X) 

2 (IX) 

(14) 

(35) 

(17) 

ANIMALS INITIALLY IN STUDY 

ANIMAL S HISSING 

ANIMAL S MCEOPSIED 

ANIMALS EXAMINE D HISTOEATHOIOGICALLY 

INTiGUBENTAE Y SYSTEM 

*SUBCCT 1ISSUE 

LEICMYCSAECOM A 

BESPIKATORY SYSTEM 

ILUN G 

HfEATOCELLULAB CAECINOHA, HETAST 

ALVEOLAB/BBONCHIOLAB ADENOMA 

ALVECLAB/BBONCHIOLAR CABCIMOHA 

HEBATOPOIEIIC SYSTEM 

*MULTIPLE OBGANS 

MALIGNAN T LYUPHOMA , NOS 

MALIG.LYMPHOHA , DNDIFFER-TYPE 

MALIG.LYMPHOHA , HISTIOCYTIC TYPE 

MALIGNAN T LYHPHOMA , MIXE D TYPE 

tSELEI N 

SABCCBA. NOS 

HEMANGIOM A 

HEMANGIOSABCOHA 

*(1IS£STIEIC L. NODE 

MALIGNAN T LYMPH OH A, NOS 

MAIIG.LYUPHOMA , HISTIOCYTIC TYPE 

tLIVEE 

KAIIG.LYMPHOMA , HISTIOCYTIC TYPE 

CIECULATCBY SYSTEM 

NONf 

CONTBOL(DNTB) LOW DOSE

 

05-0220 05-0210 

50 50 

1 

50 «9 

**50 19 

(50) (<t9) 

(50) (t8) 

5 (10%) t (8*) 

2 <UX) 5 (10X) 

(50) (19) 

1 (2X) 

1 (2X) 

1 <2X) 

2 («X) 

(50) (17) 

1 <2X) 

1 (2X) 

2 («X) 1 (2X) 

(It) (11)

 

1 (2«) 

1 (2%) 

(50) (19)

 

1 (2«) 

t NUM6E B OF ANIMALS HITH TISSUE EXAMINE D MICBOSCOPICALLY 

* NDMBE E OE ANIMALS NECBOPSIEE 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

B-3
 
TABLE 01 (CONTINUED) 

CCNTBOL(UNTB) LOU DOSE HIGH DOSE 

05-0220 05-0210 05-0215 

DIGESTIVE SYSTEM 

1LIVEE 

HEEATOCELLULAB ADENOM A 

(50) 

5 (10%) 

(19) 

10 (201) 

(U7) 

8 (17X) 

HEPATOCELLULAB CABCINOHA 10 (20X) 18 (37X) 26 (55X) 

UBINAR Y SYSTEM 

NON E 

ENDOCRIN E SYSTEM 

•AEBEKA I 

EHICCHBOMOCYTOHA 

(12) (1*5) 

1 (2X) 

(»D 

tTHYECIE (39) (16) (41) 

FOIIICDIAR-CELL ADENOMA 1 (3X) 2 (W) 3 (7X) 

FOLLICULAB-CELL CABCIHOHA 1 (3*) 

BEPEODUCTIV E SYSTEM 

tIESTIS (50) (49) (46) 

INTESSTITIAL-CELL TOMOB 1 (2X) 

EMEBYONA L CABCIHOMA 1 <2X) 

MEBVOOS SYSTEM 

DON E 

SPECIAL SENSE ORGAN S 

*BABE£B1AN GLAND (50) (19) (47) ADENCHA , NOS 1 (2X) FAFILLAEY ADENOMA 1 (2*) 1 (2X> CYS1ADENOMA, NOS 1 (2*) PAPILLARY CYSTADENOMA. SOS 1 (2X) 

HUSCOLOSKELETAL SYSTEM 

NON E 

* NUMbE E OF ANIMAL S WITH 1ISSDE EXAMINE D MICBOSCOPICALLY 

* NUffBE F CE ANIMALS NECBOPSIED 

B-4 
i TABLE Bl (CONCLUDED) 

CCNTBOL(ONTB) LCH DOSE HIGH DOSE 

05-0220 05-0210 05-0215 

BOD* CAVITIES 

NONE 

Ail CTHIE SYSTEMS 

MOK E 

ANIHA L IISECSITION SUMMAB Y 

ASIBAIS INITIALLY IN STUDY 50 50 50 NATOBA L DEATHS 3 6 7 

HOEIBUN D SACRIFICE 1 3 SCHEDULE D SACRIFICE 5 5 ACCIDENTALLY KILLED 

TEEHINAL SACBIFICE t2 142 35 ANKA L BISSING 1 

3 INCLCDE S AUTOLYZED ANIHALS 

TUBCB SOtHAB Y 

TOTAL ANIKALS HUH PBIHAEY TOHOBS* 22 37 39 TOTAL EEIHAEY TOMOES 33 US 51 

TOTAL ANIHALS WIT H BENIGN TDBOBS 12 17 18 TOTAL BENIG N TDHOES 13 19 19 

TOTAL *NIHALS KITH BALIGNAH T TOHOBS 16 25 31 I01AI BALIGNANT TUHOBS 20 29 32 

TOTAL AHIHALS HUH SECONDABY 10HOES* 1 TOTAL SECONDARY TOHOBS 1 

TOTAL ANIHA1S WITH TDHOBS OMCEBTAINBINIG N OB MALIGNAN T

 

TOTAL DNCEETAIN TOHOBS 

TOTAL ANIMALS WITH TUMORS ONCIBTAINESIMAE Y OE HEIASTATIC

 

TOTAL ONCEETAIN TUHOBS 

* PKIMAEl TUMORS: ALL TUMORS EXCEPT SECCNDABY TDHOBS
 

t SiCOSEABY TUMORS: ME1ASTATIC TOHOBS OB TUHOBS IMVASIVE INTO All ADJACENT OBGAM
 

B-5 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED 

WITH 4-CHLOROO-PHENYLENEDIAMINE 

CONTBOL (DNTB) LOU DOSE 

06-0220 06-0210 

ANIMAL S

A HI BAL E

 INITIALLY

 HISSING 

 IB STUDY 50 

2 

SO

1 

ANIMAL S NECEOPSIED 47 48 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** «7 48 

INTEGUMENTAR Y SYSTEM 

*SKIN (17) (48) 

KEEAIOACAHTHOH A 1 (2%) 

*SUBCBT TISSUE («7) (18) 

HEMANGIOSAECOMA 1 12%) 

EESPIBA10BY SYSTEM 

• LUN G (146) (48) 

ALVECLAE/BBONCHIOLAS ADENOMA 1 (2X) 1 (2%) 

ALVEOLAE/BEONCHIOLAE CABCINOBA 3 (7X) 1 (2X) 

HEMA10EOIETIC SYSTEM 

•MULTIPLE OEGAHS <i»7) (48) 

MALIGNAN T LYMPHOMA , NOS 1 (2%) 

SALIG. LYMPHOMA, UNDIFFER-TYEE 2 («X) 

BALIG. LYMPHOMA, HISTIOCYTIC TYPE 3 (6X) 5 (10%) 

USEIFFEEENTIATED LEUKEMIA 1 (2X) 

4SPLEIK (t5) (48) 

HEHAHGIOMA 1 (2X> HEHANGIOSABCOMA 1 (2X) 

HALIG. LYMPHOMA, HISTIOCYTIC TYPE 2 (««) 

• LIPP H 1.0 CE (38) (46) 

ALVECIAB/BBONCHIOLAB CA, ME1ASTA 1 (2«) 

MAIIG. LYMPHOMA, HISTIOCYTIC TYPE 1 (2X) 

»MESLNTEEIC L. NODE (38) (46) 

MALIGNANT LYMPHOMA, NOS 

*PIYEBS PATCH (UK) (46) 

MAIIGiLYMPHDMA , HISTIOCYTIC TYPE 2 (4X) 

* NUMBE R Of ANIMALS KITH TISSUE EXAMINE D MICBOSCOPICALLY 

* NUMBE R OF ANIMALS NECBOPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

HIGH DOSE 

06-0215 

50 

48 

48 

(48) 

(48) 

(45) 

3 (7X) 

(48) 

1 (2X) 

(18) 

1 (2») 

(38) 

(38) 

1 (3«) 

(46) 

1 <2M 

B-6 
TABLE B2 (CONTINUED) 

CONI801 (HNTE) LOU DOSE HIGH DOSE 

06-0220 06-0210 06-0215 

CIBCULATCBY SYSTEM 

NONE 

DIGISTIVB SYSTEM 

ILIVEF (i»6) (48) (17) HiEATOCELLULAR ADENOMA 7 (15X) <« (9X) HECA10CEL1ULAE CARCINOM A t (8X) 6 (13X) 

URINAR Y SYSTEM 

NONE 

ENDOCRI1.E SYSTEM 

iEIIUITAEY (33) (34) (35) ADIKCHA , NOS 1 (3*) 1 (3X)

 

fAERINAI/CAPSUL E <UO) (45) (47) ADEtCHA, NOS 1 (3X)

 

tALBEHA L BEDULLA (UO) (45) (47)

 

NEOBCBLASTOMA 1 (2X)

 

tTHYECIt (29) (46) (39) FOIIICDLAR-CELL ADENOHA 1 (3X) 1 (2X) 1 <3X) 

*PANCBIATIC ISLETS («5) («7) (42) ISIIT-CELL ADENOMA 1 (2X) 

BEPEODCCTIVE SYSTEM 

IUTEEUS (U5) (45) (4«) IEICBYCSABCOBA 1 (2X) HEMANGIOMA 1 (2X) 

tOVABY (1*0) (45) (38) PAFIIIARY ADENOMA 1 <3») 1 (3%) GRANULOEA-CELL TUMCE 1 (3X) 

NEBVOD E SYSTEM 

NCNE 

* NUMBER OF ANIMALS WITH TISSDE EIAMINED HICBOSCOFICALLI
 

* NUBBIR OF ANIMALS NECROPSIED
 

B-7 
SPECIAL SEKSE OfiGANS 

*HASD£BIA N GLAND 

PAPILLARY ADENOMA 

CYS1ADENOKA, DOS 

PAPILLABY CYSTADENOHA, HOS 

HOSCOLOSKELETAL SISTEB 

NCN E 

BODY CAVITIES 

NONE 

A LL C1HHE SYSTEMS 

NON E 

ANIMA L [ISEOSI1ION SOHHABY 

AKIHAI S INITIALLY IN STUDY 

NA1CBAL DEATHS 

HOEIBUND SACBIFICE 

SCBBCUIED SACBIPICE 

ACCIDENTALLY KILLED 

TLEtllNAL SACRIFICE 

ANIf.A L HISSING 

9 IKCLlipIS AUTOLY2ED ANIMALS 

» NUMBE R OF ANIMALS KITH TISSUE

* tlUMBEE Of ANIMALS NECBOPSIED 

TABLE B2 (CONTINUED) 

CONTfiOL(DNTB) LOH DOSE 

06-0220 06-0210 

(17) (48) 

1 (2X) 

50 50 4 5 

3 

5 

36 114 

2 1 

 EXAMINED MICROSCOPICALLY 

B-8 

HIGH DOSE 

06-0215 

C»8) 

2 («X) 

1 <2X) 

50 

5 

1 

5 

39 
TABLE B2 (CONCLUDED)
 

CCITBOL(OITB) LOH DOSE HIGH DOSE 

06-0220 06-0210 06-0215 

IUMCB £BMMAB Y 

TGTA1 ANIMAL S HUH PBIHABY 1DDOBS* 14 24 21 

TO1A1 FBIMABY TUMOK S 15 31 26 

TOTAL ANIMALS KITH BENIGN TDHCBS 5 10 12 

TOTAL BENIGN TUHORS 6 12 15 

TOTAL ASIMALS KITH MALIGNAN T TOMOBS 9 15 10

 

101AL HALIGVANT TUflOES 9 19 10 

TOTAL ANIMALS KITH SICOHDAEY TUHOBS* 1 

T01AI SECONDABY TUBOBS 1

 

TOTAL ANIMALS HITH TUMORS UliCBBTAINBENIGII OE UALIGNANT 1 

TOTAI ONCE8TAIN TUMORS 1 

TOTAL ANIMALS HUH TUHOBS DNCiBTAIN-

PBIHABl OF BETASTATIC 

TOTAI ONCEHTAIN TOMOBS 

* P8IHA8Y 1UMOBS: ALL TUMORS EXCEPT SECCMDABT TUHOBS
 

t SECOBEABY TUHOHS: METASTATIC TOHOBS OB TOHOBS INVASIVE IMTO AH ADJACENT OBGAH
 

B-9 

APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

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

TABLE Cl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

TREATED WITH 4-CHLORO-O-PHENYLENEDIAMINE 

CONTROL (ONTR) 

01-0220 

LOg DOSE 

01-0205 

HIGH DOSE 01-0210 

AHIBALS IKITIALLY IN S10DY 

AHIBALS NECROPSIED 

ABIBALS EXAMINED HISTOEATHOLOGICALLY 

50 

1*8 

¥v 48 

a 119 »7 

U7 

50 

50 

19 

INTEGUMENTARY SYSTEH
 

*SKIN («8) (»7) (50)
 EPIDEBHAL INCLUSION CYST 1 (2X) 2 («X)
 

*SUBCUT TISSUE <U8) (*7) (50)
 ABSCISS, SOS 3 16%)
 

BESPIBATOHY SYSTEH
 

*NASAL TOBBINATE (i»8) (i»7) (50)
 INFLABBATION, SUPPURATIVE 1(2%)
 

tTEACHIA (29) (16) (17)
 INFLAMMATION, SOS 1 (2X)
 INFIAHBATION. ACUTE/CHHONIC 2 (UX)
 

tLONG/EEONCimS (18) («7) (18)
 BEOKCHIECTASIS 3 (6X)
 INfLAHHATION, DOS 1 <2X) 2 (IX)
 INFLABHATION, FOCAL 1 (2X) 1 UX)
 

• LUNG (US) (17) (18)
 CONGESTION, NOS 2 (IX)
 INFLAMMATION. INTEESTITIAL 1 (2X) 2 («X)
 PNEUMONIA, CHBONIC HUBINE 1 (2X)
 HYPEHPLASIA, EPITHELIAL 1 <2X) 2 (IX)
 

HEBATOPOIETIC SYSTEM
 

tSPLEEN («8) (16) (18)
 COMGISTION, NOS 1 (2*)
 ABSCESS, NOS 1 <2X)
 INTABCT. NOS 1 -U!L_ 1 (2*1
 

* NUMBE R OF ANIMAL S HITH TISSUE EXAMINED MICROSCOPICALLY 

* SUBBI8 OF ANIBALS NECBOPSIED 

**EXCLODES PARTIALLY AUTOLYZED ANIMALS 

» NOTE: 50 ANIMALS HERE INITIALLY III STUDY BUT ONE DAS FOUND TO BE A FEMALE IN A HALE 

C-3 
TABLE Cl (CONTINUED) 

CONTBOL(DNTB) LOH DOSE HIGH DOSE 

01-0220 01-0205 01-0210 

HEHCSIDEBOSIS 9 (20X) 6 (13X) 

LYHPHOCYTOSIS 1 <2X) 

HYPEBPLASIA, HEM ATOPOIETIC 1 (2*) 

HYPEBPLASIA, EEYTHROID 1 <2X) flEMATOPOIESIS 16 (35X) 10 (21X) 

«iYKPH NODE (143} (40) (39)

 

DEGENERATION , CYSTIC 1 (3»)

 

HYEEBPLASIA, NOS 2 (5X)

 

tTHYMO S (32) (21) (24)

 

HYEEEPLASIA, NOS 1 OX)

 

CIBCOLATCBY SYSTEM 

tHEAET/TENTEICLE C»8) (47) (47)

 

THRCBBOSIS. NOS 1 (2X)

 

tHYOCABCIUH (48) (47) (47) 

INIIAMHATION. INTEESTITIAL 1 <2X) 3 (6X) 

FIEBOSIS 18 (38X) 14 (30X) 

tENDOCABDIOH (18) (47) (47) 

ENtCCABDITIS, BACIEEIAL 1 (2X)

 

*ABTEEY («8) (47) (50)

 

MINERALIZATION 1 (2X)

 

DIGESTIVE SYSTEH 

•LIVES (18) (47) (48) 

FIBBCSIS SEPTAL LIVEB 1 (2X) 

NECBOSIS, FOCAL 1 <2X) « (8X) 

NECBOSIS, COAGOLATI7E 1 (2X) 2 <4X) 

BE1AHOBPHOSIS FATT? 2 («») 8 (17X) 5 (10X) 

HYPEBFLASIA, FOCAL 3 (6X) 3 (6X) 10 (21X) 

HEMAI«POIESI5 1 (2X) 

tLIVES/EEEIPOBIAL (18) (47) (48)

 

EIBECSIS 1 (2*)

 

*BI1E IDCT (48) (47) (50)

 

INF1AHMATIOH, FOCAL 1 (2X)

 

UYPEBPLASIA. HOS 6 M3X) 3 (6X> 

t NUMBER OF ANIMALS KITH TISSUE EIAMINED BICBOSCOPICALLY
 

* NUMBEB OE ANIMALS NECBOPSIED
 

C-4i
 
tPANCBEAS

 

IHf LAMHATION, NOS 

FIEBOSIS 

DEGENEBATION. CYSTIC 

IS10MACH 

HINEBAIIZATION 

INF1AHHATION, NOS 

ABSCISS, NOS 

HYPEHPLASIA, BASAL CELL 

HYPEHKEBATOSIS 

ACANTHOSIS 

tPEYEBS PATCH 

HYEIBP1ASIA, NOS

 

tCOLOK 

PABASITISM 

UHINABJ SYSTEM 

tKIDNB Y 

HYIBCNEPHBOSIS 

GiCHEROLONEPHBITIS, NOS 

PYELONEPHBITIS. NOS 

ABSCESS, NOS 

EIEBOSIS, DIFFUSE 

NEPHBOPATHY 

HYEEBPLASIA, TDBOLAB CELL 

HYPEBPLASIA, EPITHELIAL 

IKIDNEY/MEDULLA 

HINEEALIZATION 

HEHOBBHAGE 

*KICNEY/GLOHEfiDLUS 

NECBCSIS, FOCAL 

tKICNEY/TUBUL E 

MlkEBALIZATIO N 

NECBOSIS, FOCAL 

tURINAB Y BLADDER 

HYflBPLASIA, EPITHELIAL 

TABLE Cl (CONTINUED)
 

COSTBOL (ONTB) 

01-0220 

(«t)

2 (5«) 

(*8) 

(i»6) 

(«2) 

(U8) 

35 (73*) 

(18) 

(U8) 

(t8) 

(08) 

LOi DOSE 

01-0205 

(«3) 

11 (26%) 

(16) 

3 (7*) 

1 <2X) 

7 (15*) 

2 (14») 

11 (24X) 

(15) 

6 (13X) 

(13) 

3 (7X) 

(«7) 

1 <2X) 

20 (K3J) 

24 (51X) 

1 <2X) 

5 (11X) 

1 (2X) 

(17) 

1 (2X) 

(U7) 

1 (2X) 

(17) 

1 (2%) 

(12) 

3 <7X> 

HIGH DOSE 

01-0210 

(*1)

9 (22*) 

1 (2*) 2 <5X) 

(t8) 

1 (2*) 

1 (2*) 

1 (8*) 

5 (10*) 

8 (17X) 

(16) 

3 (7*) 

(35) 

2 (6«) 

(18)

6 (13*) 

10 (21() 

3* (71 X) 

5 (10*) 

(18)

8 (17X) 

2 (IX) 

(18) 

(18) 

2 (1*) 

(19) 

6 J12X1 

* NUMB IB OF ANIHALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* MOHBEE OF ANIHALS NECBOPSIED
 

C-5
 
HYtllFLASIA, PA PI LI AS Y 

HE1APLASIA, SQUAHODS 

ENDOCRINE SYSTEM 

IPITUI1ABY 

CYST, DOS 

NECROSIS, HE MOB t HAG 1C 

HYPERPLASIA, FOCAL 

•ADEEKAI 

HYPHBPIASIA, DOS 

• ACREKA I COBIEX 

HYIEBTBOPHY, FOCAL 

•AtiEKAI MEDULLA 

HVEEBPLASIA , NOS 

HYPERPLASIA , FOCAL 

lIHYEOir 

FOLIICULAE CYST, NOS 

HYPEEPLASIA, C-CELL 

tlHYECII FOLLICLE 

CEGIHEBATIOll, NOS 

PIGMENTATION, NOS 

tPAEATBYEOID 

HYEIEPIASIA. NOS 

IPANCEIATIC ISLETS 

HYIEEPLASIA, HOS 

B£PEODUCTIVE SYSTEM 

*MAB«AB Y GLAN D 

GAIACTOCfiLB 

*PfiOSTATE 

INflABBATION, NOS 

*SEMI1JAL VESICLE 

ATBCEJJJ, NOS 

f NUMEE E OF ANIMALS 8ITH TISSOE

* NUMBE E OF ANIMALS NECEOPSIED 

TABLE Cl (CONTINUED) 

CONTSO1 (DNTB) LOU DOSE 01-0220 01-0205 

1 <2X) 

(45) (39) 

1 (3X) 

1 (3X) 

1 (2*) 8 (21X) 

(16) (16) 

1 (2X) 

(«6) («6) 

1 (2X) 

<«6> («6) 

1 (2%) 

3 (7«) 1 (2X) 

(<»3) (10) 

1 <3X) 

3 (8X) 

(«3) (10) 

2 <5X) 

(25) (28) 

2 (7X) 

(U«) (13) 

1 (2X) 2 (5X) 

(U8) (17) 

(«5) (11)

8 (18X) 

(«8) (17) 

1 (2X> 

 ElAMINED SICBOSCOPICALLY 

IC-6 

HIGH DOSE 01-0210 

1 <8X) 

1 (2X) 

(12) 

3 (7X) 

(18) 

(18) 

(18J 

1 (2X) 

1 (2X) 

(10) 

1 <3X) 

(40) 

1 (3X) 

4 (10X) 

(22) 

2 (9X) 

(41) 

(50) 

1 <2X) 

(11) 

1 <9X) 

(50J 
TABLE Cl (CONCLUDED) 

COIIIBOL(OMTB) LOU DOSE 

01-0220 01-0205 

tlESTIS (48) (45) 

MINIBALIZATIOS 1 (2«) « (««) 

ATBOPHY, HOS 4 (8*) 6 (13*) 

ATROPHY, FOCAL 2 (4X) 

HYPEBPLASIA, IHTEBSTITIAL CIIL 3 (6X) 4 (9») 

•TESTIS/IOBULE (<»8) (45) 

BIHHBALIZATIOH 2 (4%) 

*IPIDIEYMIS <i»8) (47)

 

ABSCESS, NOS 1 (2%)

 

HEBVOUE SYSTEM 

HOSE 

SPECIAL SEKSE OBGAHS 

*EYI (48) (47)

 

CA1ABACT 1 (2*)

 

*EYE/BHTI»A (48) (47)

 

ATBOEHY, HOS 2 (4X) 

MUSCULOSKELETAL SYSTEM 

hCN E 

BODY CAVITIES 

NOME 

A LL CTHEB SYSTEMS 

OREM1D M 

MINEBALIZATIOH 1

 

NECBOSIS, FAT 2 

SPECIAL eOBFHOLOGY SUMMAR Y 

Ayi5^(|55p9fg](^g|STo PEHF 4 1 

AUlC/BICiiOFSY/110 HISTO 

AUTC1YSIS/HO NECROPSY 2 2 

* NUKBIS CF ANIMALS HUH TISSUE EXABIHED HICHOSCOPICALLY
 * NUBBIE CB AM HALS NECEOFSIED
 

C-7 

HIGH DOSE 

01-0210 

(47) 

« (9») 

8 (17X) 

2 («%) 

(47) 

2 (4%) 

(50) 

(50) 

(50) 

2 

1 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

TREATED WITH 4-CHLORO-O-PHENYLENEDIAMINE 

HIGH DOSE
 

02-0210
 

50
 

1
 

19
 

18
 

(19)
 

(17)
 

1 (2X)
 

(17)
 

2 (IX)
 

1 (2X)
 

1 (2X)
 

1 <2X)
 

* OX)
 

(»6)
 

1 (2X)
 

9 (20 X)
 

<«7)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS MISSING
 

ANIMALS HCBOPSIED
 

ANIMALS EXAMINED HISTO PATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

NONE
 

BBSPIHATCBY SYSTEM
 

»NASAI TURBINATE
 

INFLAMMATION, NOS
 

tlONG/BBONCUUS
 

INFLAMMATION, NOS
 

INFLAMHATION. SUPPUBATIVB
 

tLONG
 

INJ IAMMATION, NOS
 

INFLAMMATION, INTERSTITIAL
 

ABSCESS, NOS
 

PNEUMONIA, CHBONIC HDBINE
 

INFLAMMATION, GEANULOBATOOS
 

GRANULOMA, NOS
 

HYFEBPLASIA, EPITHELIAL
 

HEMATOFOIETIC SYSTEM
 

tSPLEEN
 

HEMCSIDEROSIS
 

ATROPHY, NOS
 

HEMATOPOIESIS
 

CIBCULAIOBY SXSTEM
 

tMYOCABCIUH
 

INFLAMMATION. INTEBSTITJAL
 

CONTROL (UNTR)
 

02-0220
 

50
 

50
 «v 50
 

(50)
 

1 (2X)
 

(50)
 

(50)
 

1 (2X1
 

1 (2X)
 

2 («X)
 

1 (2X)
 

1 (2X)
 

(50)
 

7 (1»X)
 

(50)
 

LOS DOSE
 

02-0205
 

50
 

19
 

19
 

(19)
 

(19)
 

3 (6X)
 

(19)
 

6 (12X)
 

1 (2X)
 

(18)
 

22 (16X)
 

1 (2X)
 

20 (12X)
 

(19)
 

1 (2X)
 

* NUMBB.B OF AMIBALS WITH TISSUE EXAHIHED HICBOSCOPICALLY 

* NUMB IS OF ANIMAL S NECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-8!
 
TABLE C2 (CONTINUED) 

HIGH DOSE 

02-0210 

« <9X) 

(17) 

1 <2X) 

(16) 

1 (2») 

1 <2») 

1 (2*) 

1 (2X) 

1 UX) 

22 (18X) 

(16) 

(16) 

1 (2X) 

(19) 

1 (2*> 

(«5) 

8 (18X) 

(16) 

6 (13X) 

1 (9X) 

6 (13X) 

(15) 

(36) 

1 (3X) 

(17) 

8 (17*1 

FIBECSIS 

tENDOCAEDIDM 

INFLAMMATION, DOS 

DIGESTIVE SYSTEM 

tLIVEB 

FIBBCSIS SEPTAL LIVBB 

NECEOSIS, FOCAL 

NECROSIS. COAGOLATIVE 

METABOBPHOSIS PATTY 

BASOPHILIC CYTO CHANGE 

HYPIBPLASIA, FOCAL 

ANGIECTASIS 

*LIVEB/CENTBH.OBOLAB 

SICBCSIS, NOS 

•LIVEF/EEBI POSTAL 

FIBBCSIS 

*BILE EUCT 

INFLAMMATION , NOS 

HIFEEPLASIA, HOS 

tPANCBHAS 

INfLAMMATION , NOS 

tSTCMACH 

EEGINEBATION, NOS 

HYPEBPLASIA, PAPILLABI 

HYPEBPLASIA, BASAL CELL 

HXPEBKEBATOSIS 

ACAN1HOSIS 

tBEYEF S PATCH 

HYPIBPLASIA. NOS 

•COLON 

PABASITISM 

UfilNAEJ SYSTEM 

*KIDNE Y 

HYtECNEPHEOSIS 

COHTBOL(ONTB) 

02-0220 

(50) 

(50) 

» <ex) 

9 (18*) 

(50) 

(50) 

(50) 

(116) 

<t9) 

1 (2X) 

(17) 

(10) 

(19) 

LOU DOSE 

02-0205 

5 (10J) 

(19) 

(19) 

1 (2») 

3 (6») 

1 (2*) 

23 («7») 

1 (2%) 

(19) 

1 (2X) 

(19) 

1 (2*) 

(19) 

2 (IX) 

10 (20X) 

<«1) 

10 (23X) 

(19) 

1 (2X) 

7 (11J) 

1 (2X) 

1 (8X) 

(18) 

6 (13X) 

(10) 

1 (3*) 

(19) 

t NUMBE E OF ANIMALS WITH TISSUE EXAMINED HICBOSCOPICALLY 

* NUHBE E OF ANIMAL S NECBOPSIED 

C-9, 
CY£1, NOS 

GLOHEBULONEPHBITIS , NOS 

PYELCNEPHEITIS, NOS 

INFLAMMATION . ACUTE 

NEFHBOPATH Y 

DEGENERATION, NOS 

HYPESPLASIA, EPITHELIAL 

• KIDNEY/MEDULL A 

MINIBALIZATIOlt 

tUEINAS Y BLADDER

 

HYEIEPLASIA, EPITHELIAL 

HYFEBPIASIA, PAPILLAEY 

ENDOCEINE SYSTEfl 

tPHUIIAEY 

MlkEBALIZAIION 

hEMCBRHASE 

HYEEEPLASIA, FOCAL 

tADBENA l 

THRCBBCSIS, NOS 

iAEfiEKAl COE1EX 

HYEEBTROPHY , FOCAL 

tACRECA I MEDULLA 

HYEEEPLASIA , NOS 

*THYBCIE 

ULTIBOBEANCHIAL CYST

 

FOLLICULAK CYST. NOS 

HYPEfiPLASIA, C-CELL 

» PA EA THYROID 

BYEIBPLASIA, HODHLAB 

«PANCBBATIC ISLETS 

HYIIEPLASIA, NOS 

BEPBODUCTIVE SYSTEM 

*MAHMAE Y GLAN D 

GAIACTOCfiLE 

* NUMBE R OF ANIMALS illTH TISSDE

* NUMBE R OF ANIMALS NECEOPSIED 

TABLE C2 (CONTINUED) 

CONTROL (UNTE ) LOK DOSE 02-0220 02-0205 

1 <2X) 

1 (2%) 35 (71X) 

6 (12X) 

18 (31%) 

<t9) («9) 

(«7) (16) 

1 (9X) 

(tO) (38) 

1 (3*) 

2 (5X) 

2 (5X) 

(18) (17) 

1 <2X) 

<t8) (t7) 

1 (2X) 

<«8) (17) 

(13) (15) 

1 <2») 

1 <2X) 

1 (2X) 1 (2X) 

(31) (22) 

1 13%) 

(146) (11) 

(50) (19) 

2 («X> ...5 (10X) 

 EXAMINED MICEOSCOPICALL Y 

C-IO 

HIGH DOSE 02-0210 

1 <9X) 

38 (81X) 

1 <2X) 

1 (2X) 

1 (2X) 

(47) 

1 (2X) 

(15) 

2 (IX) 

2 (IX) 

(38) 

(16) 

(16) 

(16) 

1 <2X) 

(11) 

(30) 

(15) 

2 (IX) 

(19) 

5 (10XL 
____ — ­

HYPEBPLASIA, NOS 

•ClITCBAL GLAND 

HE1AFLASIA, SQUABOOS 

«OTEBUS 

HYIBCHETBA 

ABSCESS, NOS 

NECBOSIS, NOS 

• OTEBUS/INDOMETRIUM 

INflAHHATION, ABSCESS, NOS 

HYPEBPLASIA,

tOYARY/CVIDUCT 

INFIABBATION,

•OVARY 

CYST. NOS 

INFLAMMATION,

DEGENERATION,

HYCEBFLASIA,

NEBVOU£ SYSTEM 

tBRAIN/MENINGES 

 NOS 

 DOS 

 NOS 

 NOS 

 CYSTIC 

 NOS 

INFIABMATION. ACUTE 

SPECIAI SEKSE OKGANS 

»EYE 

CATARACT 

*EYE/CCBNIA 

INPIABHAIION, HE!10EEHAGIC 

*EYE/RETIIiA 

ATROPHY, NOS 

*HARDEEIA N GLAND 

INFLAHHATION, CHRONIC 

8DSCULOSKELETAL SYSTEM 

NON E 

TABLE C2 '(CONTINUED)
 

CCHTBOI(DIITB) 

02-0220 

1 (2X) 

(50) 

(«8) 

1 (2X) 

1 (2*) 

(U8) 

1 (2*) 

(48) 

(l»9) 

1 (2X) 

2 («X) 

(50) 

(50) 

(50) 

(50) 

(50) 

LOI DOSE HIGH DOSE 

02-0205 02-0210 

3 (6«) 

(49) (49)

 

1 (2%) 

(49) (46) 

1 (2*) 

1 <2X) 

(49) (46) 

1 (2*) 2 (4X)

 

1 (2%) 

1 (2X) 

(49) (46)

 

1 (2X) 

(49) (47)

 

1 (2X) 

2 (4X) 

(49) (46) 

1 <2X) 

(49) (49) 

1 (2X) 1 (2X) 

(49) (49) 

2 (4%) 

(49) (49) 

1 (2X) 

(49) (49) 

1 <2X) 1 <2X> 

* NDHEEB Of ANIMALS UITH TISSUE EXAMINED HICBOSCOPICALLY
 * NOMBEB CF ANIMALS NECEOESIED
 

C-ll 
BODY CAVITIES 

*PLEOSA 

HYPEBPLASIA. DOS

 

ALL OTHER SYSTEMS 

OMEN 1 OB 

NECBCSIS, PAT 

SPECIAL eOBPHOLOGY SUMMARY 

NO LESION REPORTED 

ANIRA L MISSIHG/NO NECROPSY 

AUTC/HECROPSY/H1STO PEBF 

AUIO/HECEOPSY/NO HISTO 

A01CLYSIS/NO iECROPSY 

* NUMEE F OF A8IHALS HUH TISSUE * HUBBI F OF ANIMALS NECBOPSIZC 

TABLE C2 (CONCLUDED) 

CONTROL (0 BIB) LON DOSE 

02-0220 02-0205 

(50) <«9) 

11 

1 

1 

 EXABIKE E MICROSCOPICALLY 

C-I2 

HIGH DOSE 

02-0210 

(U9) 

1 <2«) 

2 

1 

1 

1 

1 
APPENDIX D 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

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

TABLE Dl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH 4-CHLOROO-PHENYLENEDIAMINE 

ANIMALS INITIALLY IN STUDY 

ANIMALS KISSING 

ANIMALS NECEOPSIED 

ANIBAL S EXAMINED HI STOEATHOLOGICALL I 

INTEGUBEBTABY SYSTEM 

*SKIN 

INFIABHATION, NECBOTIZING 

*SUBCUT TISSUE 

ABSCESS. MOS 

INFLABHATION, CHRONIC 

HESFISAIOBY SJSTBH 

I1EACHEA 

HYEEBPLASIA , FOCAL 

tLUNG/BBONCHUS 

INE1AHHATION,

tLUHG/BBONCHIOLE 

INEIAHHATION,

*LUN G 

ItiflAHHATION,

 NCS 

 NOS 

 IHTEBSTITIAL 

PEEHAECOLITIS 

HYPEBPLASIA, EPITHELIAL 

HEMA10POIETIC SYSTEH 

«SPL£EN 

HYfEBPLASIA , MOS 

HYEESPLASIA, HE HATOEOIETIC 

HYPE8PLASIA, EEYTHEOID 

HYPE8PLASIA, LYMPHOID 

HEMATOP01ESIS 

tLYHP H KODE 

INFLABBAIIOli, MOS 

CONTEOL (0«TB) 

05-0220 

50 

50 

** 50 

(50) 

(50) 

1 (2*) 

(28) 

(50) 

1 (2X) 

(50) 

(50) 

(50) 

(«'») 

LOW DOSE HIGH DO SB 

05-0210 05-0215 

50 50 

1 

U9 47

 

49 47 

(49) (47) 

1 (2X) 

(49) (47) 

1 <2X) 

1 (2X) 

(4«) (40)

 

1 (2») 

(48) (47) 

7 (15X) 1 (21) 

(48) (47)

 

2 (4%) 

(48) (47) 

13 (27X) 3 (6X) 

1 (2X) 

1 (2X) 1 (2X) 

(47) (44) 

6 (13*) 2 (5X) 

2 (5X) 

2 (5X) 

2 (4X) 

3 (6X) 4 (9X) 

(44) (35)

 

1 (2X1 

* NUMBE E OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* HUMBE8 CF AMH/M.S NECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-3
 
HYlItELASIA, SOS 

RE1ICOLOCYIOSIS 

HYPEBPIA3IA, LYHFHOID 

HEflAlOPOIESIS 

4HESEBTIRIC L. MODE

 

HEHATOPOIESIS 

CIBCULATCBY SYSTEM 

"CARDICVASCULAB SYSTE 

PEBHASCULITIS 

fHEAET/VEHTBICLE 

BE1ANIK 

DIGESTIVE SYSTEH 

*IIVEB 

INFIAHMATION, INTEBSTITIAL 

NECBOSIS. FOCAL 

NECBOSIS, COAGULATIVE 

HETAHOBPHOSIS FAITY 

H1PEBPLASTIC NODDLE 

HYPEBPLASIA, HYEERP1ASIA,

HEBATOPOIESIS 

*GALLELADDER 

INFLABHATJON, HYPIEPLASIA,

f PA NCR HAS 

INFLAHHATION,

 FOCAL 

 HEHATOPOIETIC 

 ACUTE/CHHONIC PAPILLAE! 

 NOS 

*FANCBHATIC ACINUS 

DEGENERATION, CYSTIC 

ATBOPUY, NOS 

HYPEBTHOPHY, FOCAL 

*STOBACH 

INfLABBATION, NOS 

HYPEfiPLASIA, NOS 

HYflRlCEEATOSIS 

TABLE Dl (CONTINUED) 

CCNTROl (ONTB) 

05-0220 

<«!») 

1 (2») 

(50) 

(50) 

(50) 

1 (2X) 

(50) 

(50) 

(50) 

1 <2X) 

1 (2X) 

(50) 

LC8 DOSE 

05-0210 

2 <5X) 

2 <5X) 

(««) 

(19) 

1 <2X) 

(«5) 

* 19%) 

(49) 

1 (2X) 

2 («) 

3 (6X) 

5 (10X) 

3 (6X) 

(<»9) 

(««)

2 (5X) 

(4U) 

1 (2X) 

(t«) 

1 (2X) 

HIGH DOSE 

05-0215 

3 (9X) 

1 (3X) 

1 (3X) 

1 (3X) 

(35) 

(47) 

(«6) 

11 (211) 

(") 

1 <2X> 

2 (4X) 

7 (15X) 

1 (2X) 

1 (2X) 

(47) 

1 <2X) 

1 (2X) 

(43) 

1 (2X) 

(43) 

(42) 

2 (5X) 

4 I10X> 

t NUBBEB OF ANIHALS KITH TISSUE EIAHINED HICBOSCOPICALLY
 

* NUDBER OF AHISALS NECBOPSIED
 

D-4
 
TAfcLE D1 (CONTINUED) 

CONTEOL(OHTB) LCD DOSE 

05-0220 05-0210 

ACASIHOSIS 3 (7») 

I6ASTEJC HOCOSA (50) (Ml) 

DEGENERATION, NOS 

•PEKEBS PATCH (50) (15) 

HYPIBPLASIA, DOS 1 (9*) 

*COLO( (<»«) (13) 

PAEASITISH 1 (2J) 

UBIKAEI SYSTEM 

IKIDNEY (50) (18) 

HYIBCHIPHBOSIS 1 (2*) 1 (2*) 

GLCHEHULONEPH8ITIS. DOS 10 (21X) 

INILAHMATION, MOS 

INFLAHHATION, INTERSTITIAL 6 (13X) 

flEBOSIS, DIFFUSE 1 (2X) 

HEIAHOBPBOSIS FATTY 

HYPERPLASIA, TUBULA E CEIL 1 (2») 

tUBINABY BLADDER (50) (19) 

IHI1AHHAIIOH, ACUTE/CH80HIC 1 (2X) 

HlfERPLASIA, EPITHELIAL 2 (IX) 

HETAPLASIA, SQUANOOS 1 (2X) 

ENDOCRINE SYSTEM 

tPHDIlAEY (31) (31) 

CYST, NOS 1 <3X) 

HJEEBPLASIA, FOCAL 2 (6X) 

•ADRENAL/CAPSULE (12) (15) 

NOtOlE 1 (2X) 

*ACB£KAL COBTEX (U2) (15) 

HYEiKTBOPHY. FOCAL 

HYPEKPLASIA, NOS 3 (7X) 

HYFEfiPLASIA, FOCAL 1 (2X) 

•THYRCIC (39) (16) 

FOIIICULAR CYST, SOS 1 (2X) 

HYFEBPIASIA- FOCAL 1 12%) 

t NUtlBEB OF ANIHALS KIT H TISSUE EXAMINE D HICBOSCOPICALLY 

* NUHBE E OF ANIMALS MECROPSIED 

D-5 

HIGH DOSE 

05-0215 

2 (5X) 

(12) 

1 <2X) 

(11)

2 (5X> 

(36) 

(17) 

1 (2X) 

6 (13X) 

2 (UX) 

8 (17X) 

2 (IX) 

1 (2X) 

(11) 

1 (2X) 

(32) 

2 (6X) 

(11) 

(11) 

2 (5%) 

2 (5X) 

(11) 
HYEIEELASIA , FOLLICUL AR-CILL 

• PARATHYROI D 

HYEIRPLASIA , NOS 

tPANCEIATIC ISLETS 

INFLAHHATION , NOS 

HYEEEPLASIA, ADENCMATOOS 

fiEPEODUCUVE SYSTEM 

*PENIS 

CAICDLUS, NOS 

INFLAMMATION , ACUTE DIFFOSE 

•PREPUTIAL GLAHD 

INFLAMMATION , CHBONIC

 

HYEIEPLASIA, NOS 

4PEOSTATZ 

HYEIEPLASIA , FOCAL 

tlESHS 

ATECIHY , NOS 

ATBOPHY . FOCAL 

tTESTIS/TOBULE 

MINEBALIZA1ION 

NEBVOO S SYSTEM 

•BRAIN 

GBANULCMA , MOS 

SPECIAL SESSE ORGANS 

NONE 

MUSCUICSKELETAL SYSTEM 

NON E 

* NUDBE E OF ANIMALS «ITH TISSUE

* NUHBI E Of ANIMAL S NECEOPSIED 

TABLE Dl (CONTINUED) 

CONTROL (U NTH) 

05-0220 

(17) 

(50) 

3 (6X) 

1 (2%) 

(50) 

(50) 

(tl) 

(50) 

1 <2X) 

(50) 

2 (<t«) 

(50) 

 EXAMINE D MICROSCOPICALLY 

D-6
 

LOB DOSE 

05-0210 

(27) 

3 (11%) 

(4<l) 

(t9) 

(«9) 

1 <2X) 

(13) 

(19) 

1 (2X) 

(19) 

(18) 

1 <2X) 

HIGH DOSE 

05-0215 

1 <2X) 

(19) 

(13) 

(17) 

1 (2X) 

1 (2X) 

(17) 

1 (2X) 

(11) 

2 <5X) 

(16) 

(16) 

(16) 
BODY CAVITIES 

*PERITOKEOB 

INFLAMMATION, NOS 

*P1EUEA 

GRANDLOHA, NOS 

ALL OTHER SYSTEMS 

OHENlliM 

NECFCSIS, FAT 

SPECIAL MORPHOLOGY SUMHABY 

NO LESION REPORTED 

ANIHA L HISSING/NO NECROPSY 

AOIC/NECROPSY/HISTO PERF 

AH10LISIS/NO NECROPSY 

* NUMEE E OF AMIHALS IITH TISSUE

* NUBBIE OF ANIMALS NECEOPSIED 

TABLE Dl (CONCLUDED) 

CONTROL (UBTR) LOW DOSE 

05-0220 05-0210 

(50) <U9) 

(50) <"9) 

1 (2*) 

1 

22 1 

1 

1 

 EXA HI NED MICROSCOPICALLY 

D-7
 

HIGH DOSE 05-0215 

(47) 

1 (2X) 

(*7) 

2 

3 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH 4-GHlJORO-O-PHENYLENEDIAMINE 

HIGH DOSE 06-0215 

50 

48 

48 

(48) 

(45) 

(45) 

3 (7X) 

(48) 

1 (21)

5 (10X) 

1 (2X) 

1 (2X) 

4 (8X) 

(38) 

1 (3X) 

1 (3X) 

(28) 

(47) 

1 (2X) 

AHIBALE INITIALLY III STUDY 

AMIOAL5 HISSING 

AMIBALS SICfiOPSIED 

ANIHALS HXABINED HISTOEATHOLOGICALLY

IBTEGUMEHTABY SYSTEM 

•SUBCCI TISSUE 

INFLAMMATION , NOS 

BESPI6A10HY SYSTEM 

tLDNG 

INf LABBATION, I NTEBSTITIAL 

INfABCT, NOS 

HEBATOEOIETIC SYSTEH 

*BONE HABBOH 

CSTICSCIEKOSIS 

• SPLEE N 

ATBCEHY , NOS 

HYFERPIASIA, NOS 

BE1ICOLOCYTOSIS 

HYEEBPLASIA, LYHPHOID 

HEflATOPOIESIS 

tLYBCH (ODE 

INFlAflBAIION, MOS 

HYEEBPLASIA , DOS 

RETICOLOCYTOSIS 

HIPBRPLASIA, HEHATOPOIETIC 

HYPE8PLASIA, LYBPHOID 

•THYHDS 

HYEEBPIASIA, LYflPHCID 

CIBCU1ATCBY SYSTEH 

tHIAET 

INFiAHMATtON , CHBONIC 

i MDHBE B Of AMIHALS WITH TISSUE

* NUKBE E Of A HI HALS HECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

I:ONTBOL (ONTfi) 

06-0220 

50 

2 

*7 

 ** 17 

(1*7) 

1 (2X) 

(46) 

(39) 

<«5) 

1 (2X) 

(38) 

(30) 

(15) 

LOU DOSE 

06-0210 

50 

1 

U8 

<I8 

(48) 

(US) 

1 (2X) 

1 (2X) 

(»3) 

4 (9%) 

(48) 

4 <8X) 

1 (2X) 

1 (2X) 

9 (19*) 

(46) 

1 <2X) 

2 (4X) 

1 (2X) 

1 (2X) 

(36) 

2 (6X) 

(48) 

 EIAHIMED MICROSCOPICALLY 

D-8 
•HEAE1/VESTBICLE

 

fiEIABDt 

tBYCCABUOH 

FIBBOSIS, FOCAL 

*ABIEEY 

PEBIVASCULITIS 

DIGESTIVE SYSTEH 

•LIVER 

FIBBCSIS 

NECEOSIS, FOCAL 

NECBOSIS, COAGOLATIVE 

(1EIAHOBPHOSIS FATTY 

HYPEBPLASTIC MODULE 

HYPEBPLASIA, FOCAL 

*GA1LELADE£B 

HYFEBPLASIA, DOS 

HYPBBPLASIA, FOCAL 

HYSEBPLASIA, PAPILLABY 

IPANCSIATIC ACINUS 

EEGINEBATION. NOS 

tSTCHACH 

INFIAHBATION , NOS 

INfLAHHATION , FOCAL 

HYFEBPLASIA. EPITHELIAL 

HYPEBKERA10SIS 

ACAN1HOSIS 

tPEYEES PATCH 

SECRCSIS, NOS 

HYFEBPLASIA, DOS 

UBINAR1 SYSTEH 

•KIDNEI 

GLCBIHULONiPHEITIS , DOS 

INFLAHBAIION. INTERSTITIAL 

* NUMBER OF ANIMALS KITH TISSUE

* NUMBIfi OF ANIMALS NECBOPSIED 

TABLE D2 (CONTINUED) 

CCMTBOL(UIIB) LOB DOSE 

06-0220 06-0210 

(U5) (48) 

7 (15*) 

(»5) («B) 

(«) C»8) 

1 (2%) 

(H6) (18) 

6 (13*) 

1 (2X) 3 (6X) 

5 (10X) 

1 (2X) 

(17) (M8) 

2 <4X) 

3 (6X) 

(U5) (17) 

1 (2X) 

06) (1(6) 

1 (2X) 

1 (2X) 

2 (IX) 

7 (15X) 

(It) (46) 

2 («*) 

(U6) (18) 

1 (2X) 1 (2X> 

 EIAHINED HICBOSCOPICALLY 

D-9 

HIGH DOSE 

06-0215 

(47) 

3 (6X) 

(47) 

1 (2*) 

(48) 

1 (2X) 

(47) 

1 (2X) 

2 (4X) 

1 (2X) 

1 (2X) 

7 (15X) 

1 (9X) 

(48) 

1 (2X) 

1 (2X) 

(42) 

(46) 

1 (2X) 

2 (4X) 

5 (11X) 

7 (15X) 

(46) 

1 (2X) 3 <7«) 

(«8) 

3 (6*) 

3 (6X) 
TABLE D2 (CONTINUED)
 

CCKTBOL(OITB) LCD DOSE HIGH DOSE 06-0220 06-0210 06-0215 

INPIAMBAIION, CHBOHIC 1 (2») 

tUBINAB Y ELADDEB («5) <«7) C»2) 

HYEIBPLASIA, EPITHELIAL « (10*) 

METAPLASIA, SQUABODS 1 (2») 

FNDOCKIN E SYSIEH 

*PITUITABY (33) (3U) (35) 

HYFIBPLASIA, FOCAL 1 <3») 2 (61) 

tAEEZSAI COfiTtX (»0) («5) («7) 

NCtUlE 1 (2S) 

HJEEETEOPHY, FOCAL 1 (2*) 

HYPIBPLASIA, HOS 10 (22X) » (9*) 

MHYBCIt (29) (16) (39) 

FOI1ICDLAK CIST, DOS 1 (2») 

HYFEBPIASIA, FOCAL 1 (3*) 

HYEEBPLASIA, PAPI1LABI 1 (2») 

tPABATHYBOID (13) <2<t) (21)

HEIAHIH 2 (10*) 

UYPEBPLASIA, MOS 1 <5*> 

3BPBODCCTIVE SYSTEM 

• HAMMAE I GLAND C»7) («8) («8)

 

HYEEBP1ASIA, HOS 1 (2X)

 

fOlERUS («5) <«S) (««)

HYIBCMETEA 7 (16«) 3 (7«) 

HEIAPLASIA, SQUAHODS 1 (2«) 

tOTEBUS/IHDOHEIBIOD (15) (15) (»») INFLABHATIOB, MOS 1 <2») 1 (2X) 

HYPEBPLASIA, DOS 17 (38X) 18 (i»1») 

HYPEEPLASIA, CYSTIC 7 (16*) 3 (7*) 

iOVAKY/CVIDUCT («5) («5) (««) 

INF1AHBAIIOH, HOS 1 (2%) 

DEGENEEATION, NOS 1 (2*) 1 <2») 

HYPEBPLASIA, PAPIllABY 1 (2*) 

*OVABY (»0) (»5) (38)

 

fllPffMIiMIOH 1 (2S1

 

* BUBBIE OF ANIMALS WITH TISSUE IXAHI1ED HICBOSCOPICALLY
 

* NUMBEB Of ABIBALS BECBOPSIED
 

D-IO
 
CISI, DOS 

CIS IIC FOLLICLES 

HEHOBBHAGE 

IVflARHAXION, CHRONIC 

DEGEIEBATIOV. DOS 

BE6INEBATION, CYSTIC 

HtPEBPLASIA, GBANOLOSA-CBLL 

•OVABY/IOILICLE 

HERCBBHAGE 

NEBVOUS SYSTEM 

HO HE 

SPiCIAl SEIiSE ORGANS 

•HABDESIAB GLAMO 

HYFIBPLASIA, FAEILLABI 

BOSCULCSUIE1AL SISTEH 

HOME 

BOCI CAVITIES 

HOKE 

ALL OTHEE SYSTEMS 

HONE 

SPECIAl ROBPUOLOGY SUHHABY 

DO LESION BEPOBTED 

AKIBAL HISSING/MO NECBOFSY 

AOTC/NECBOPSY/HISTO PEEF 

AUIOLYSIS/NO HECBOPSY 

TABLE D2 (CONCLUDED)
 

CCBTROI.(ONTB) 

06-0220 

(<«0> 

(«7) 

30 

2 

1 

1 

LOI DOSE HIGH DOSE 

06-0210 06-0215 

2 («*) 1 (3*) 

2 (5X) 

2 <«*) 1 <3») 

1 (2X) 

1 (3*) 

3 (7») 1 <3X) 

1 (2S) 3 <8X) 

(15) (38) 

1 (3X) 

(»8) (US)

 

1 (2X) 

3 it 

1 

1 2 

* BOBBIE OF ANIMALS KITH TISSUE EXAMINED HICBOSCOPICALLY
 

* NUMB IB Of ANIMALS NECBOPSIED
 

D-ll 

Review of the Bioassay of 4-Chloro-o_-phenylenediamine 

*»
 

for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

March 6, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DHEW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be
 

exposed. The members of the Clearinghouse have been drawn
 

from academia, industry, organized labor, public interest
 

groups, State health officials, and quasi-public health and
 

research organizations. Members have been selected on the
 

basis of their experience in carcinogenesis or related fields
 

and, collectively, provide expertise in chemistry, biochemistry,
 biostatistics, toxicology, pathology, and epidemiology.
 Representatives of various Governmental agencies participate
 

as ad hoc members. The Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse is charged with the responsibility of
 

providing a peer review of reports prepared on NCI-sponsored
 

bioassays of chemicals studied for carcinogenicity. It is in
 

this context that the below critique is given on the bioassay
 

of 4-Chloro-o_-phenylenediamine for carcinogenicity.
 

The primary reviewer agreed with the conclusion in the
 

report that 4-Chloro-o-phenylenediamine was carcinogenic in
 

both sexes of treated rats and mice, under the conditions of
 

test. Tumors of the urinary bladder and forestomach were
 

induced in the rats and hepatocellular carcinomas in the mice.
 He opined that the increased incidence of liver neoplastic
 

nodules in treated male rats could be biologically significant,
 although a lesser number were found in females. The primary
 

reviewer said that the results were particularly significant
 

since the bladder tumors are similar to ones induced in humans
 

by certain aromatic amine carcinogens.
 

The secondary reviewer was critical of the selection
 

of the dose levels used in the chronic study. However, he
 

concurred with the conclusion that 4-Chloro-o_-phenylenediamine
 

was carcinogenic in the treated animals, under the conditions
 

of test. He added that the substance could pose a carcinogenic
 

risk to humans.
 

51
 
The primary reviewer moved that the report on the bioassay
 

of 4-Chloro-o-phenylenediamine be accepted as written. The
 

motion was seconded and approved unanimously.
 

Members present were
 

Gerald N. Wogan (Chairman), Massachusetts Institute of
 

Technology
 

Arnold Brown, Mayo Clinic
 

Lawrence Garfinkel, American Cancer Society
 

E. Cuyler Hammond, American Cancer Society
 

Joseph Highland, Environmental Defense Fund
 

Henry Pitot, University of Wisconsin Medical Center
 

George Roush, Jr., Monsanto Company
 

Sheldon Samuels, Industrial Union Department, AFL-CIO
 

Michael Shimkin, University of California at San Diego
 

John Weisburger, American Health Foundation
 

Sidney Wolfe, Health Research Group
 

*	 Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the
 

review or other reasons. Thus, certain comments and
 

criticisms reflected in the review may no longer be
 

appropriate.
 

«U.S. GOVERNMENT PRINTING OFFICE:1978 260-899/3101 1-3 52 

DHEW Publication No. (NIH) 78-1313 °°