1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 113 

BIOASSAY OF 

2-CHLORO-p-PHENYLENEDIAMINESULFATE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 61702-44-1 

NCI-CG-TR-113 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

2-CHLORO-p-PHENYLENEDIAMINE SULFATE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-1368
 

REPORT ON THE BIOASSAY OF 2-CHLORO-p-PHENYLENEDIAMINE SULFATE
 

FOR POSSIBLE CARCINOGENICITY
 

CARCINOGENESIS TESTING PROGRAM
 

DIVISION OF CANCER CAUSE AND PREVENTION
 

NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH
 

FOREWORD: This report presents the results of the bioassay of
 

2-chloro-p-phenylenediamine sulfate conducted for the Carcinogenesis
 

Testing Program, Division of Cancer Cause and Prevention, National
 

Cancer Institute (NCI), National Institutes of Health, Bethesda,
 

Maryland. This is one of a series of experiments designed to determine whether selected chemicals have the capacity to produce cancer
 

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

a significantly greater incidence of cancer than control animals,
 

do not necessarily mean the test chemical is not a carcinogen because
 

the experiments are conducted under a limited set of circumstances.
 

Positive results demonstrate that the test chemical is carcinogenic
 

for animals under the conditions of the test and indicate a potential
 

risk to man. The actual determination of the risk to man from animal
 

carcinogens requires a wider analysis.
 

CONTRIBUTORS: This bioassay of 2-chloro-p-phenylenediamine sulfate
 

was conducted by Mason Research Institute, Worcester, Massachusetts,
 

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

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

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

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

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

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

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

Murthy (3) at the Mason Research Institute, and the diagnoses included in this report represent the interpretation of this pathologist. Histopathology findings and reports were reviewed by Dr. R.
 

L. Schueler (6).
 

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

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

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

Gart (8).
 

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

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

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

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

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

participating organizations.
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

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

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

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

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

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

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

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

Missouri.
 

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

Boulevard, McLean, Virginia.
 

6.	 Tracer 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.
 

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

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

iv
 
SUMMARY
 

A biossay for possible carcinogenicity of 2-chloro-p-phenylenediamine sulfate was conducted using Fischer 344 rats and B6C3F1
 

mice. 2-chloro-p-phenylenediamine sulfate was administered in the
 

feed, at either of two concentrations, to groups of 50 male and 50
 

female animals of each species. The dietary concentrations used in
 

the chronic bioassay were 0.3 and 0.15 percent for the high and low
 

dose rats, respectively, and 0.6 and 0.3 percent for the high and
 

low dose mice, respectively. Compound administration was for 105 to
 

107 weeks in rats, 87 weeks in high dose mice, and 104 to 105 weeks
 

in low dose mice. The only groups observed during an untreated
 

period after dosing were the high dose mice, observed for 18 weeks
 

after compound administration ceased. For each species, 20 animals
 

of each sex were placed on test as controls.
 

There were no significant positive associations between the
 

administered dietary concentrations of 2-chloro-p-phenylenediamine
 

sulfate and mortality for rats of either sex or male mice. There
 

was a significant positive association between dosage and mortality
 

for female mice; however, adequate numbers of animals in all groups
 

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

There were no statistically significant po-sitive associations
 

between dietary exposure to the compound and the incidences of any
 

tumor in rats. There was an increased incidence of transitional-


cell hyperplasia of the renal pelvic epithelium in both male and female rats, and transitional-cell tumors of the urinary bladder were
 

present in three dosed rats. These lesions indicated a possible
 

carcinogenic effect, but are not considered as sufficient evidence
 

of carcinogenicity. In mice no tumors occurred in statistically
 

significantly higher incidences in the dosed mice than in controls.
 

Under the conditions of this bioassay there was insufficient
 

evidence that dietary administration of 2-chloro-p-phenylenediamine
 

sulfate was carcinogenic to Fischer 344 rats or B6C3F1 mice.
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 3
 

A. Chemicals 3
 

B. Dietary Preparation 5
 

C. Animals 5
 

D. Animal Maintenance 6
 

E. Selection of Initial Concentrations 8
 

F. Experimental Design 9
 

G. Clinical and Histopathologic Examinations 12
 

H. Data Recording and Statistical Analyses 1^
 

III. CHRONIC TESTING RESULTS: RATS 19
 

A. Body Weights and Clinical Observations 1°
 

B. Survival 19
 

C. Pathology 22
 

D. Statistical Analyses of Results 24
 

IV. CHRONIC TESTING RESULTS: MICE 32
 

A. Body Weights and Clinical Observations 32
 

B. Survival 32
 

C. Pathology 35
 

D. Statistical Analyses of Results 37
 

V. DISCUSSION 45
 

VI. BIBLIOGRAPHY 47
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 2-CHLORO-p-


PHENYLENEDIAMINE SULFATE C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 2-CHLORO-p-


PHENYLENEDIAMINE SULFATE D-l
 

vii
 
LIST OF ILLUSTRATIONS
 

Figure Number 

CHEMICAL STRUCTURE OF 2-CHLORO-p-PHENYLENEDIAMINE SULFATE 

GROWTH CURVES FOR 2-CHLORO-p-PHENYLENEDIAMINE 

SULFATE CHRONIC STUDY RATS 20 

SURVIVAL COMPARISONS OF 2-CHLORO-p-PHENYLENEDIAMINE SULFATE CHRONIC STUDY RATS 21 

GROWTH CURVES FOR 2-CHLORO-p-PHENYLENEDIAMINE 

SULFATE CHRONIC STUDY MICE 33 

SURVIVAL COMPARISONS OF 2-CHLORO-p-PHENYLENEDIAMINE SULFATE CHRONIC STUDY MICE 34 

LIST OF TABLES 

Table Number Page 

DESIGN SUMMARY FOR FISCHER 344 RATS—2-CHLOROp-PHENYLENEDIAMINE SULFATE FEEDING EXPERIMENT 10 

DESIGN SUMMARY FOR B6C3F1 MICE—2-CHLORO-pPHENYLENEDIAMINE SULFATE FEEDING EXPERIMENT 11 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

2-CHLORO-p-PHENYLENEDIAMINE SULFATE 25 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE 28 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

2-CHLORO-p-PHENYLENEDIAMINE SULFATE 38 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE 41 

viii
 
LIST OF TABLES (Concluded) 

Table Number 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE A ~ 3 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE A ~ 6 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE B ~ 3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE B ~ 6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH 2-CHLORO-pPHENYLENEDIAMINE SULFATE c~3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH 2-CHLOROp-PHENYLENEDIAMINE SULFATE c ~ 7 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH 2-CHLORO-pPHENYLENEDIAMINE SULFATE D~3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH 2-CHLOROp-PHENYLENEDIAMINE SULFATE D ~ 6 

ix
 

I. INTRODUCTION
 

2-Chloro-p-phenylenediamine sulfate (NCI No. C03316) is a salt
 

of 2-chloro-p-phenylenediamine and sulfuric acid. It is a component
 

of commercial hair dyes and was selected for bioassay by the National
 

Cancer Institute because of the increased bladder cancer incidence
 

noted among dye manufacturing industry workers (Anthony and Thomas,
 

1970; Wynder et al., 1963). This elevated cancer risk has been associated with occupational exposure to several classes of compounds,
 

including aromatic amines (Wynder et al., 1963). The widespread exposure to 2-chloro-p-phenylenediamine or its mono- or disulfate salts
 

among hair dye users and the suspected increase in the incidence of
 

bladder cancer among hairdressers (Anthony and Thomas, 1970) were
 

also important factors in its selection for testing.
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

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

It is also called 3-chloro-4-aminoaniline sulfate; o-chloro-p-phenylenediamine sulfate; and C.I. (Colour Index) Oxidation Base 13A (C.I.
 

76066). 2-Chloro-p-phenylenediamine is known as Ursol Brown 0 (C.I.
 

76065).
 

2-Chloro-p-phenylenediamine or the corresponding sulfates are
 

primary intermediates in at least 31 commercially available permanent
 

hair dyes (Richardson, 1977). These compounds react with other dye
 

components within the hair shaft to produce permanent hair colors
 

(Corbett and Menkart, 1973).
 

*The CAS registry number is 61702-44-1.
 

1
 
Filaments and fibers of aromatic polyamides, such as p-aminobenzoyl chloride-2-chloro-p-phenylenediamine-piperazine-terephthaloyl
 

dichloride copolymer (Fujiwara et al., 1974) and poly(2-chloro-pphenyleneterephthalamide) (Bair and Morgan, 1975) have been produced
 

experimentally using 2-chloro-p-phenylenediamine; however, these
 

polymers do not appear to be used commercially.
 

All persons whose hair is colored with dyes containing 2-chlorop-phenylenediamine or the mono- or disulfate salts experience dermal
 

contact and local absorption may occur. Hairdressers may also be
 

exposed through dermal contact. Approximately 40 percent of U.S.
 

women are regular hair dye users, and sales of the permanent type
 

of dyes account for about 75 percent of all hair dye expenditures
 

(Corbett and Menkart, 1973). Portions of 2-chloro-p-phenylenediamine
 

or its sulfate salts which fail to react with the other dye constituents may reach rivers and streams via domestic wastewater.
 

The potential for exposure to 2-chloro-p-phenylenediamine compounds may also be considerable for workers in the dye and chemical
 

manufacturing industries and for those engaged in research with the
 

various polyamide fibers.
 

Lowenstein Dyes and Cosmetics, Inc. (undated) reports the
 

following effects of 2-chloro-p-phenylenediamine sulfate exposure:
 

skin irritation, asthmatic symptoms, and eye damage.
 
II. MATERIALS AND METHODS
 

A. Chemicals
 

2-Chloro-p-phenylenediamine sulfate (Figure 1) was purchased
 

from Aldrich Chemical Company, Milwaukee, Wisconsin. Chemical
 

analysis was performed by Midwest Research Institute, Kansas City,
 

Missouri. The manufacturer's specified purity for this chemical was
 

98 percent. The experimentally determined melting point range was
 

250° to 262°C, compared to a reference value of 251° to 253°C (Aldrich
 

Chemical Company, Inc., 1977). The wide range suggested the presence
 

of impurities. Thin-layer chromatography utilizing two solvent systems (ethyl acetate: ammonium hydroxide and benzene:1,4-dioxane:
 

ammonium hydroxide), each visualized with ultraviolet light and furfural showed, respectively, one less motile and one nonmotile impurities and two less motile and one nonmotile impurities. Results of
 

elemental analysis were consistent with C1C.H0(NH ) • H SO., the
 

6 3 2 2 2 4
 

molecular formula for 2-chloro-p-phenylenediamine sulfate. High-


pressure liquid chromatography revealed one peak with a tail, again
 

suggesting the presence of impurities. Vapor-phase chromatography
 

showed one homogenous peak.
 

Throughout this report, the term 2-chloro-p-phenylenediamine
 

sulfate is used to refer to the commercially available product used
 

in this bioassay.
 
NH2 

FIGURE 1 

CHEMICAL STRUCTURE OF 2-CHLORO-p-PHENYLENEDIAMINE (SULFATE) 
B. Dietary Preparation
 

The basal laboratory diet for both treated and control animals
 

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

2-Chloro-p-phenylenediamine sulfate was administered to the treated
 

animals as a component of the diet.
 

The chemical was removed from its container and proper amounts
 

were weighed out under an exhaust hood. The compound was hand-blended
 

in an aluminum bowl with an aliquot of the ground feed. Once visual
 

homogeneity was attained, the mixture was placed in a 6 kg capacity
 

Patterson-Kelley standard model twin-sheel stainless steel V-blender
 

along with the remainder of the feed to be prepared. After 20 minutes
 

of blending, the mixture was placed in double plastic bags and stored
 

in the dark at 4°C. The mixture was prepared once weekly.
 

C. Animals
 

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

through contracts of the Division of Cancer Treatment, National Cancer
 

Institute. All rats and mice were supplied by Charles River Breeding
 

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

animals for each species were received in separate shipments.
 

Upon arrival, a sample of animals was examined for parasites
 

and other signs of disease. All animals were treated with 3.0 gm
 

piperazine adipate per litre of water given ad libitum for three days,
 

followed by three days of tap water and three more days of piperazine
 
adipate administration. All animals were quarantined by species for
 

2 weeks prior to initiation of the test. Animals were assigned to
 

groups and distributed among cages so that the average body weight
 

per cage was approximately equal for a given species and sex.
 

D.	 Animal Maintenance
 

All animals were housed by species in rooms having a temperature
 

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

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

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

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

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

for the first 15 months of study, all rats were housed in wire-mesh
 

cages (Fenco Cage Products, Boston, Massachusetts) suspended over
 

newspapers. Newspapers under cages were replaced daily and cages
 

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

were held in suspended polycarbonate cages (Lab Products, Inc.,
 

Garfield, New Jersey) equipped with disposable nonwoven fiber filter
 

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

hardwood chip bedding (American Excelsior Company, Baltimore, Maryland) was used in polycarbonate cages. Stainless steel cage racks
 

(Fenco Cage Products) were cleaned once every 2 weeks.
 

Mice were housed five per cage by sex in polycarbonate cages
 

fitted with perforated stainless steel lids (Lab Products, Inc.)
 

and nonwoven fiber filter bonnets. Clean cages, lids, and bedding
 
(R)
 were provided twice per week. SAN-I-CEL (Paxton Processing Company,
 

(R)
 Paxton, Illinois) and Bed-o-Cobs (The Andersons Cob Division,
 

Maumee, Ohio) corncob bedding were used for the initial 15 months
 

of study. Aspen bedding was used for the remainder of the study.
 

Reusable filter bonnets and pipe racks were sanitized every 2 weeks
 

throughout the study.
 

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

bottles equipped with rubber stoppers and stainless steel sipper
 

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

was supplied as needed between changes.
 

During the period of chemical administration, treated and con­

(R)
 trol animals received treated or untreated Wayne Lab-Bloxw

meal as
 

appropriate. The food, replenished daily, was supplied in Alpine®
 

aluminum feed cups (Curtin Matheson Scientific, Inc., Woburn, Massachusetts) for the first 15 and 9 months of study for rats and mice,
 

respectively. For the remainder of the bioassay, meal was provided
 

in stainless steel gangstyle food hoppers (Scientific Cages, Inc.,
 

Bryan, Texas), in which food was replenished daily.
 

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

containing N-butylurea (592-31-4); 1,5-naphthalenediamine (2243­

62-1); N,N-dimethyl-p-nitrosoaniline (138-89-6); 2,5-toluenediamine
 

sulfate (6369-59-1); 2,4-dinitrotoluene (121-14-2); 4-nitroanthranilic acid (619-17-0); N-(l-naphthyl)ethylenediamine dihydrochloride
 

*
 

CAS registry numbers are given in parentheses.
 
(1465-25-4); aniline hydrochloride (142-04-1); and p-anisidine
 

hydrochloride (20265-97-8).
 

All mice were housed in a room with other mice receiving diets
 

containing hydrazobenzene (530-50-7); 2,3,5,6-tetrachloro-4-nitroanisole (2438-88-2); tris(2,3-dibromopropyl)phosphate (126-72-7);
 

and aniline hydrochloride (142-04-1).
 

E. Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

2-chloro-p-phenylenediamine sulfate for administration to treated
 

animals in the chronic studies, subchronic toxicity tests were conducted with both rats and mice.
 

Animals of each species were distributed among six groups, each
 

consisting of five males and five females. 2-Chloro-p-phenylenediamine sulfate was incorporat.ed into the basal laboratory diet and
 

supplied ad libitum to five of the six groups of each species in
 

concentrations of 3.0, 1.0, 0.3, 0.1, and 0.03 percent. The sixth
 

group of each species served as a control group, receiving only the
 

basal laboratory diet. The dosed dietary preparations were administered for a period of 8 weeks. Individual body weights were recorded
 

weekly throughout the study. Daily food consumption per cage was
 

monitored during the test. At the end of the 8-week period, all
 

survivors were sacrificed and necropsied.
 

The highest concentration causing no deaths, no compound-related
 

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

8
 
15 percent relative to controls during the 8-week subchronic test was
 

selected as the high concentration utilized for the rat and mouse
 

chronic bioassays.
 

At a dietary concentration of 1.0 percent all the male and one
 

of the female rats died. No mortality occurred in rats receiving a
 

dietary concentration of 0.3 percent and mean body weight depressions
 

were 10.4 and 6.4 percent in male and female rats, respectively. A
 

dietary concentration of 0.1 percent 2-chloro-p-phenylenediamine sulfate induced no deaths or mean body weight depression in either males
 

or females. The high concentration selected for administration to
 

rats in the chronic bioassay was 0.3 percent.
 

At a dietary concentration of 1.0 percent, one male mouse died
 

and mean body weight depression was 3.8 and 14.4 percent for male and
 

female mice, respectively. A dietary concentration of 0.3 percent
 

induced no deaths in either sex, produced no mean body weight depression in males and produced mean body weight depression of 16.1 percent
 

in female mice.
 

The high concentration selected for administration to mice in
 

the chronic bioassay was 0.6 percent.
 

F. Experimental Design
 

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

treated and untreated observation periods) are summarized in Tables
 

1 and 2.
 
TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

2-CHLORO-p-PHENYLENEDIAMINE SULFATE FEEDING EXPERIMENT
 

2-CHLORO-p-


INITIAL PHENYLENEDIAMINE OBSERVATION PERIOD
 

GROUP CONCENTRATION TREATED UNTREATED
 

SIZE (PERCENT) (WEEKS) (WEEKS)
 

MALE
 

CONTROL 20 0 0 107
 

LOW DOSF 50 0.15 105 0
 

HIGH DOSE 50 0.30 106 0
 

FEMALE
 

CONTROL 20 0 0 108
 

LOW DOSE 50 0.15 106 0
 

HIGH DOSE 50 0.30 107
 

10
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

2-CHLORO-p-PHENYLENEDIAMINE SULFATE FEEDING EXPERIMENT
 

MALE
 

CONTROL
 

LOW DOSE
 

HIGH DOSE
 

FEMALE
 

CONTROL
 

LOW DOSE
 

HIGH DOSE
 

2-CHLORO-pINITIAL PHENYLENEDIAMINE OBSERVATION PERIOD 

GROUP CONCENTRATION TREATED UNTREATED 

SIZE (PERCENT) (WEEKS) (WEEKS) 

20 0 0 107 

50 0.3 104 0 

50 0.6 87 

0 18 

20 0 0 107 

50 0.3 105 0 

50 0.6 87 

0 18 

11
 
The treated and control rats were all approximately 7 weeks
 

old at the time the test was initiated. The initial dietary concentrations of 2-chloro-p-phenylenediamine sulfate were 0.3 and 0.15
 

percent. Throughout this report the rats receiving the former
 

concentration are referred to as the high dose groups and those
 

receiving the latter concentration are referred to as the low dose
 

groups. Treated rats were supplied with dosed feed for up to a total
 

of 107 weeks.
 

The treated and control mice were all approximately 7 weeks old
 

at the time the test was initiated. The initial dietary concentrations of 2-chloro-p-phenylenediamine sulfate were 0.6 and 0.3 percent.
 

Throughout this report mice receiving the former concentration are
 

referred to as the high dose groups and those receiving the latter
 

concentration are referred to as the low dose groups. High dose mice
 

were supplied with dosed feed for a total of 87 weeks, followed by an
 

18-week untreated observation period, due to excessive mortality.
 

Low dose mice were supplied with dosed feed for up to a total of 105
 

weeks.
 

G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

experiment. Body weights were recorded twice weekly for the first
 

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

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

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

12
 
bioassay and for three consecutive days each month thereafter. From
 

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

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 and microscopic examination of major tissues, organs,
 

and gross lesions taken from sacrificed animals and, whenever possible, from animals found dead.
 

Tissues were preserved in 10 percent buffered formalin, embedded
 

in paraffin, sectioned, and stained with hematoxylin and eosin prior
 

to microscopic examination. An occasional section was subjected to
 

special staining techniques for more definitive diagnosis.
 

Slides were prepared from the following tissues: subcutaneous
 

tissue, lungs and bronchi, trachea, bone marrow, spleen, lymph nodes,
 

thymus, heart, salivary gland, liver, gallbladder (mice), pancreas,
 

esophagus, stomach, small intestine, large intestine, kidney, urinary
 

bladder, pituitary, adrenal, thyroid, parathyroid, testis, prostate,
 

brain, uterus, mammary gland, and ovary.
 

A few tissues were not examined for some animals, particularly
 

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

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

14
 
equality and used Tarone's (1975) extensions of Cox's methods when
 

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

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

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

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

histologically. However, when macroscopic examination was required
 

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

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

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

The purpose of the statistical analyses of tumor incidence is to
 

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

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

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

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

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

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

significance level of 0.05 may be made. The Bonferroni inequality
 

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

15
 
be less than or equal to 0.05/k. In cases where this correction was
 

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

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

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

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

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

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

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

noted, the direction of the significant trend was a positive dose relationship. This method also provides a two-tailed test of departure
 

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation of
 

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

tumor was observed were excluded by basing the statistical tests on
 

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

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

tumor was found, comparisons were based exclusively on animals that
 

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

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

Cochran-Armitage tests, etc.) were followed.
 

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

incidence of tumors. Curves of the proportions surviving without an
 

16
 
observed tumor were computed as in Saffiotti et al. (1972). The week
 

during which animals died naturally or were sacrificed was entered as
 

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

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

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

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

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

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


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

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

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

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

t c t
 

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

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

c
 

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

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

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

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

larger proportion in the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

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

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

17
 
of the risk in a treated group of animals to that in a control group
 

would be within the interval calculated from the experiment. When
 

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

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

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

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

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

the lower limit indicates the absence of a significant result while
 

the upper limit indicates that there is a theoretical possibility
 

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

detected under the conditions of this test.
 

18
 
III. CHRONIC TESTING RESULTS: RATS
 

A.	 Body Weights and Clinical Observations
 

Mean body weight depression was only slight for both male
 

and female treated groups (Figure 2). Eye discoloration, swelling,
 

reddening, and crustation were observed in one high dose male, two
 

low dose males, two high dose females, and four low dose females.
 

Alopecia occurred in one high dose female and one low dose female.
 

Subcutaneous masses were recorded for three high dose males, two low
 

dose males, two high dose females, four low dose females, and two
 

control females. Abdominal distention was observed in one high dose
 

male and general pallor was observed in one low dose female. No
 

other clinical abnormalities were reported.
 

B.	 Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and 2-chloro-p-phenylenediamine sulfate-dosed groups
 

are shown in Figure 3. For male and female rats the Tarone test did
 

not indicate a significant association between dosage and mortality.
 

Adequate numbers of males were at risk from late-developing
 

tumors, as 80 percent (40/50) of the high dose, 94 percent (47/50) of
 

the low dose, and 90 percent (18/20) of the control group survived on
 

test until the end of the study.
 

Adequate numbers of females were at risk from late-developing
 

tumors as 94 percent (47/50) of the high dose, 86 percent (43/50) of
 

19
 
750 

-600 

-450 

300 

-150 

120 

TIME ON TEST (WEEKS) 

750- •750 

CONTROL 

LOW DOSE 

600 — HIGH DOSE - 600 

DC 

(D 

\- 450- -450 

X 

o 

LLJ 

3 

8 30°­ — 300 

CD 

150 — -150 

FEMALE RATS 

\ I \ I 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

FIGURE 2 

GROWTH CURVES FOR 2-CHLORO-p-PHENYLENEDIAMINE SULFATE CHRONIC STUDY RATS 

20 
30 

PROBABILITY OF SURVIVAL PROBABILITY OF SUR

o o o o o CO I 

m 

33 

33 

s 

IS] 

6 

x 

m 

TJ O O m 

2 

H m 

I- 30 3 8­ m m s m" m g m 

A > CO 

m 

co 

C 

<° _ m 

O 

X 

30 

O 

O 

CO s- o 

o c o o 

CO n—r I I o o o 

00 
the low dose, and 80 percent (16/20) of the control group survived on
 

test 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).
 

A variety of neoplasms were observed with approximately equal
 

frequency in the control and treated animals. There were instances
 

in this study, as noted in the summary tables, of neoplastic lesions
 

occurring only in treated animals or with increased frequency in
 

treated animals when compared with the controls. However, the nature
 

and incidences of these neoplasms are similar to those known to occur
 

spontaneously in aged Fischer 344 rats.
 

A variety of degenerative, inflammatory, and proliferative
 

lesions were observed in both the treated and control rats. Except
 

for transitional-cell hyperplasia of the renal pelvis, none of the
 

nonneoplastic lesions appeared to be compound-related.
 

The incidences of transitional-cell hyperplasia of the renal
 

pelvis and transitional-cell papilloma and carcinoma of the urinary
 

system occurring in these rats are summarized in the following
 

table.
 

22
 
MALES FEMALES
 

KIDNEY/RENAL PELVIS Control

Low

 Dose

 High

 Dose Control

 Low

 Dose

 High 

 Dose 

No. of Animals with 

Kidneys Examined 

Histopathologically (20) (49) (50) (20) (48) (49) 

Transitional-Cell 

Hyperplasia 0 17 30 0 14 8 

Transitional-Cell 

Carcinoma 0 1 0 0 0 0 

URINARY BLADDER
 

No. of Animals with
 

Urinary Bladders
 

Examined Histopathologically (18) (48) (47) (20) (48) (48)
 

Transitional-Cell
 

Papilloma 0 1 0 0 0 0 

Transitional-Cell 

Carcinoma 0 0 1 0 1 0 

Transitional-cell hyperplasia appeared to be dose-related in
 

male rats and was characterized by an increased cellularity and
 

papillary formation. Mitotic figures were found in areas of hyperplasia. In a few animals, Brunn1

s nests were cystic and contained
 

blood.
 

Transitional-cell tumors of the urinary bladder were found in
 

three treated rats (two low dose and one high dose). The bladder
 

epithelium was not hyperplastic in any of the animals examined.
 

23
 
Based upon the findings of this histopathologic examination,
 

2-chloro-p-phenylenediamine sulfate does not appear to be carcinogenic to Fischer 344 rats.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

tumors were observed in at least one of the control or 2-chloro-pphenylenediamine sulfate-dosed groups and where such tumors were
 

observed in at least 5 percent of the group.
 

There were no tests in either male or female rats indicating a
 

positive association between chemical administration and tumor incidence. Based upon these statistical results, there was no convincing
 

evidence of the carcinogenicity of this compound in rats.
 

For female rats the Cochran-Armitage test indicated a significant (P = 0.043) negative association between dosage and the combined
 

incidence of leukemia or malignant lymphomas. The Fisher exact tests,
 

however, were not significant. Similarly, the Cochran-Armitage test
 

indicated a significant (P = 0.026) negative association between dosage and the incidence of islet-cell adenomas of the pancreatic islets,
 

but the Fisher exact tests were not significant.
 

For female rats the possibility of a negative association
 

between dosage and incidence was noted for endometrial stromal polyps
 

and for pituitary adenomas NOS. It must be noted, however, that the
 

24
 
TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATEC
 

K5
 

tn
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Carcinoma
 

or Alveolar/Bronchiolar Adenoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma^3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

b
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

g
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

0/20(0.00)
 

N.S.
 

1/20(0.05)
 

N.S.
 

107
 

0/20(0.00)
 

N.S.
 

P - 0.044
 

LOW
 

DOSE
 

3/49(0.06)
 

N.S.
 

Infinite
 

0.255
 

Infinite
 

105
 

6/49(0.12)
 

N.S.
 

2.449
 

0.332
 

110.166
 

98
 

3/49(0.06)
 

N.S.
 

Infinite
 

0.255
 

Infinite
 

105
 

HIGH
 

DOSE
 

2/50(0.04)
 

N.S.
 

Infinite
 

0.123
 

Infinite
 

106
 

4/50(0.08)
 

N.S.
 

1.600
 

0.175
 

77.169
 

0/50(0.00)
 

N.S.
 

85 
___ 

___ 

TOPOGRAPHY: MORPHOLOGY
 

Liver: Hepatocellular Carcinoma or
 

Neoplastic Nodule
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Adenoma NOS N3 P Values 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Adrenal: Pheochromocytoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED) 

CONTROL
 

0/20(0.00)
 

N.S.
 

3/17(0.18)
 

N.S.
 

107
 

4/20(0.20)
 

N.S.
 

107
 

LOW
 

DOSE
 

5/49(0.10)
 

N.S.
 

Infinite
 

0.536
 

Infinite
 

98
 

9/45(0.20)
 

N.S.
 

1.133
 

0.336
 

5.984
 

105
 

4/48(0.08)
 

N.S.
 

0.417
 

0.088
 

2.063
 

105
 

HIGH
 

DOSE
 

3/50(0.06)
 

N.S.
 

Infinite
 

0.250
 

Infinite
 

7/45(0.16)
 

N.S.
 

0.881
 

0.237
 

4.872
 

106
 

6/50(0.12)
 

N.S.
 

0.600
 

0.147
 

2.385
 

56 

101 
TABLE 3 (CONCLUDED)
 

TOPOGRAPHY: MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Thyroid: C-Cell Adenoma or C-Cell 

Carcinoma" 0/18(0.00) 3/43(0.07) 1/49(0.02) 

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

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Infinite 

0.264 

Infinite 

Infinite 

0.020 

Infinite 

Weeks to First Observed Tumor 105 106 

Treated groups received doses of 0.15 or 0.30 percent in feed.
 

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

C

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

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

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

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

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

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

group when P < 0.05.
 
TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE*
 

TOPOGRAPHY : MORPHOLOGY
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

00	 Pituitary: Adenoma NOS
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocytoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

3/20(0.15)
 

P = 0.043(N)
 

101
 

14/20(0.70)
 

P = 0.004(N)
 

P = 0.034
 

94
 

2/20(0.10)
 

N.S.
 

_—_
 

107
 

LOW
 

DOSE
 

2/49(0.04)
 

N.S.
 

0.272
 

0.025
 

2.233
 

74
 

12/42(0.29)
 

P = 0.002(N)
 

0.408
 

0.246
 

0.770
 

82
 

1/47(0.02)
 

N.S.
 

0.213
 

0.004
 

3.909
 

74
 

HIGH
 

DOSE
 

1/49(0.02)
 

N.S.
 

0.136
 

0.003
 

1.599
 

105
 

13/46(0.28)
 

P = 0.002(N)
 

0.404
 

0.248
 

0.753
 

100
 

1/49(0.02)
 

N.S.
 

0.204
 

0.004
 

3.754
 

107
 
TOPOGRAPHY: MORPHOLOGY
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pancreatic Islets: Islet-Cell
 

Adenoma
 

to
 

P Values0
 

e
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (CONTINUED)
 

CONTROL
 

0/20(0.00)
 

N.S.
 

—_
 

2/20(0.10)
 

P = 0.026(N)
 

P = 0.048
 

»
 

107
 

3/20(0.15)
 

N.S.
 

106
 

LOW
 

DOSE
 

1/44(0.02)
 

N.S.
 

Infinite
 

0.025
 

Infinite
 

106
 

0/48(0.00)
 

N.S.
 

0.000
 

0.000
 

1.400
 

1/49(0.02)
 

N.S.
 

0.136
 

0.003
 

1.599
 

106
 

HIGH
 

DOSE
 

3/39(0.08)
 

N.S.
 

Infinite
 

0.321
 

Infinite
 

106
 

0/48(0.00)
 

N.S.
 

0.000
 

0.000
 

1.400
 

2/49(0.04)
 

N.S.
 

0.272
 

0.025
 

2.233
 

107
 
TABLE 4 (CONCLUDED)
 

LOW HIGH
 

TOPOGRAPHY: MORPHOLOGY CONTROL DOSE DOSE
 

Uterus: Endometrial Stromal Polyp 4/20(0.20) 6/48(0.13)	 0/49(0.00)
 

P Values0	 P = 0.004(N) N.S. P = 0.006(N)
 

Relative Risk (Control) 0.625 0.000
 

Lower Limit 0.171 0.000
 

Upper Limit 2.764 0.435
 

Weeks to First Observed Tumor 94	 105
 

Q
 

Treated groups received doses of 0.15 or 0.30 percent in feed.
 

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

o	 Th probabilit e y 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 .
 

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

group when P < 0.05.
 
incidence of pituitary adenomas in the control group (70 percent or
 

14/20) was somewhat higher than expected based upon the 36 percent
 

(90/249) observed in the historical data on untreated Fischer 344
 

female rats maintained by this laboratory for the NCI Carcinogenesis
 

Testing Program.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

Tables 3 and 4, the value one is included; this indicates the absence
 

of statistically significant results. It should also be noted that
 

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

indicating the theoretical possibility of tumor induction in rats by
 

2-chloro-p-phenylenediamine sulfate that could not be established
 

under the conditions of this test.
 

31
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Mean body weight depression was only slight for both male and
 

female treated mice (Figure 4). Fluctuations in the growth curve may
 

be due to mortality; as the size of the group diminishes, the mean
 

body weight may be subject to wide variations.
 

Alopecia was recorded for 24 high dose males, 48 low dose males,
 

15 control males, 8 high dose females, 6 low dose females, and 11
 

control females. Eye discoloration, swelling, and infection developed
 

in one high dose male, one low dose male, one high dose female, and
 

one control female. Posterior ataxia was reported in one high dose
 

female. Palpable abdominal masses were observed in three low dose
 

females, two control females, and one control male. Subcutaneous
 

masses were reported in one high dose male and two control females.
 

Lightening of fur color was observed in four low dose females. No
 

other clinical abnormalities were reported.
 

B. Survival
 

The estimated probabilities of survival for male and female
 

mice in the control and 2-chloro-p-phenylenediamine sulfate-dosed
 

groups are shown in Figure 5. For male mice the Tarone test did not
 

indicate a significant association between dosage and mortality. For
 

female mice the Tarone test indicated a significant (P < 0.001)
 

positive association between dosage and mortality.
 

32
 
50- -50 

40 — -40 

W 

-30 

I 

o 

HI 

£20 - -20 

O 

m 

CONTROL 

10 — -10 

LOW DOSE 

MALE MICE HIGH DOSE 

~~l 1 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

50- -50 

40 — -40 


 

cc 

30 — -30 

01 

g 

>­

Q

O 

m 

 20­ -20 

< 

01 

10— 

CONTROL 

LOW DOSE 

—10 

FEMALE MICE HIGH DOSE 

15 30 15 60 75 

TIME ON TEST (WEEKS) 

90 

\ 

105 

I 

120 

FIGURE 4 

GROWTH CURVES FOR 2-CHLORO-p-PHENYLENEDIAMINE SULFATE CHRONIC STUDY MICE 

33 
1.0­

> 

oc 

0.6­ u. 

O 

03 0.4­

m 

O 

cc 

o_ _ CONTROL 

0.2­

... LOW DOSE 

MALE MICE . _ HIGH DOSE 

0.0- ~ I ' I ' I 

15 30 45 60 75 90 105

TIME ON TEST (WEEKS) 

" "t'i' •»••»•; 4uus-| .. 

- «. 1 '"i 

<°'8~ 'X \ 

CE ^ L, "*"-'L 

C^O 0.6 — T 

O 

l

f_ -| 

m 0.4­ \. 00 

O 

cc 

0. 

_^_^__ CONTROL 

0.2­

• ••••••.•.• LOW DOSE 

- FEMALEMICE — HIGH DOSE 

i 1 i 1 i 1 i 1 i 1 i 1 i 1 i 

15 30 45 60 75 90 105

TIME ON TEST (WEEKS) 

FIGURES 

SURVIVAL COMPARISONS OF 2-CHLORO-p-PHENYLENEDIAMINE SULFATE CHRONIC STUDY MICE 

34 

-1.0 

-0.8 

\-0.6 

-0.4 

-0.2 

-0.0 

 120 

-1.0 

-

-0.8 

-

-0.6 

-

— 0.4 

~ 

-0.2 

-

00 

 120 
Adequate numbers of males were at risk from late-developing
 

tumors, as 74 percent (37/50) of the high dose, 90 percent (45/50) of
 

the low dose, and 75 percent (15/20) of the control group survived on
 

test until the end of the study.
 

Adequate numbers of female mice were at risk from late-developing
 

tumors as 50 percent (25/50) of the high dose, 80 percent (40/50) of
 

the low dose, and 80 percent (16/20) of the control group survived on
 

test at least 90 weeks.
 

C. Pathology
 

Histopathologic findings on neoplasms in mice are summarized in
 

Appendix B (Tables Bl and B2); findings on nonneoplastic lesions are
 

summarized in Appendix D (Tables Dl and D2).
 

A variety of neoplasms were observed in both treated and control
 

mice in this study. There was an. increased incidence of proliferative
 

hepatocellular lesions observed in the treated mice of both sexes when
 

compared with control mice. The proliferative hepatocellular lesions
 

observed in this study are summarized in the following table.
 

MALES

Low

Control Dose

 High

 Dose Control

 FEMALES 

 Low

 Dose

 High 

 Dose 

No. of Animals with 

Livers Examined 

Histopathologically (17) (49) (46) (19) (49) (44) 

Focal Hyperplasia 0 1 1 0 1 1 

Hepatocellular 

Adenoma 0 4 1 0 0 3 5 

Hepatocellular 

Carcinoma 4 8 10 2 2 4 

35
 
A localized increase in the number and size of hepatocytes was
 

considered to be focal hyperplasia. Hepatocellular adenoma was a
 

circumscribed mass involving a few lobules and compressing surrounding
 

parenchyma. Hepatocytes were large with eosinophilic cytoplasm; in
 

a few animals the cytoplasm was vacuolated. Nuclei were vesicular,
 

and there were a few mitotic figures. Hepatocellular carcinoma had
 

involved a part or an entire lobe of the liver. The lobular architecture was distorted and there was marked pleomorphism of hepatocytes.
 

Nuclei were vesicular, and mitotic figures were numerous. Areas of
 

necrosis and mineralization were seen in some of the carcinomas.
 

In two treated male mice, nodules of markedly basophilic cells
 

were found in conjunction with hepatocellular carcinoma. The nodules
 

were continuous with the carcinoma; no demarcation was apparent.
 

The nodules appeared to be well-vascularized. Many of the cells were
 

cuboidal, and a few were fusiform cells as well. The cytoplasm of
 

these cells was basophilic. Nuclei were vesicular and mitotic figures, numerous. These tumors were considered to be undifferentiated
 

hepatocellular carcinomas, some of which resembled hepatoblastoma.
 

A variety of inflammatory and degenerative lesions which commonly
 

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

lesions were not considered to be compound-induced.
 

Based upon the findings of this histopathologic examination, the
 

dietary administration of 2-chloro-p-phenylenediamine sulfate was
 

associated with an increased incidence of proliferative hepatocellular
 

36
 
lesions in the compound treated mice when compared with the untreated
 

control 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 malignant tumor in either sex where at least two such
 

tumors were observed in at least one of the control or 2-chloro-pphenylenediamine sulfate-dosed groups and where such tumors were
 

observed in at least 5 percent of the group.
 

For male mice the Cochran-Armitage test indicated a significant
 

(P = 0.038) positive association between dosage and the combined
 

incidence of hepatocellular carcinomas or hepatocellular adenomas.
 

The Fisher exact tests, however, were not significant.
 

For females a significant negative association between dosage
 

and the incidence of thyroid follicular-cell neoplasms was observed,
 

but the Fisher exact tests were not significant. For males the
 

combined incidence of hemangiosarcomas or hemangiomas was significantly (P = 0.003) lower in the low dose group than in the control;
 

however, the high dose comparison and the Cochran-Armitage test were
 

not significant.
 

No other statistical tests were significant for either males or
 

females under the Bonferroni criterion. Based on these statistical
 

results, there was no conclusive evidence of the carcinogenicity of
 

this compound in male or female mice.
 

37
 
00 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE*
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar /Bronchiolar Carcinoma
 

P Values0
 

Q
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar /Bronchiolar Carcinoma or
 

Alveolar/Bronchiolar Adenoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma^1
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

3/17(0.18)
 

N.S.
 

P = 0.030
 

107
 

3/17(0.18)
 

N.S.
 

107
 

3/17(0.18)
 

N.S.
 

_—
 

78
 

LOW
 

DOSE
 

1/50(0.02)
 

P = 0.047(N)
 

0.113
 

0.002
 

1.326
 

104
 

7/50(0.14)
 

N.S.
 

0.793
 

0.213
 

4.407
 

104
 

6/50(0.12)
 

N.S.
 

0.680
 

0.170
 

3.900
 

104
 

HIGH
 

DOSE
 

4/48(0.08)
 

N.S.
 

0.472
 

0.092
 

2.997
 

105
 

6/48(0.13)
 

N.S.
 

0.708
 

0.177
 

4.056
 

88
 

8/50(0.16)
 

N.S.
 

0.907
 

0.257
 

4.911
 

88
 
TOPOGRAPHY: MORPHOLOGY
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma or
 

Hepatocellular Adenoma
 

P Values0
 

UJ 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Circulatory System: Hemangioma or
 

Hemangiosarcoma"3
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 5 (CONTINUED) 

CONTROL
 

4/17(0.24)
 

N.S.
 

107
 

4/17(0.24)
 

P = 0.038
 

107
 

4/17(0.24)
 

N.S.
 

P = 0.002
 

72
 

LOW
 

DOSE
 

8/49(0.16)
 

N.S.
 

0.694
 

0.223
 

2.860
 

104
 

12/49(0.24)
 

N.S.
 

1.041
 

0.383
 

3.994
 

104
 

0/50(0.00)
 

P = 0.003(N)
 

0.000
 

0.000
 

0.361
 

HIGH
 

DOSE
 

10/46(0.22)
 

N.S.
 

0.924
 

0.322
 

3.639
 

105
 

20/46(0.43)
 

N.S.
 

1.848
 

0.761
 

6.559
 

105
 

4/46(0.09)
 

N.S.
 

0.370
 

0.080
 

1.812
 

96 
TABLE 5 (CONCLUDED)
 

Treated groups received doses of 0.30 or 0.60 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.
 

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

group when P < 0.05.
 
TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE*
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar /Bronchiolar Carcinoma or
 

Alveolar/Bronchiolar Adenoma*5
 

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
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

CONTROL
 

0/19(0.00)
 

N.S.
 

8/19(0.42)
 

N.S.
 

74
 

2/19(0.11)
 

N.S.
 

LOW
 

TX)SE
 

3/49(0.06)
 

N.S.
 

Infinite
 

0.243
 

Infinite
 

104
 

8/49(0.16)
 

P = 0.030(N)
 

0.388
 

0.158
 

1.034
 

78
 

2/49(0.04)
 

N.S.
 

0.388
 

0.031
 

5.108
 

HIGH
 

DOSE
 

2/44(0.05)
 

N.S.
 

Infinite
 

0.133
 

Infinite
 

105
 

8/44(0.18)
 

P = 0.048(N)
 

0.432
 

0.177
 

1.145
 

54
 

4/44(0.09)
 

N.S.
 

0.864
 

0.139
 

9.058
 

Weeks to First Observed Tumor 107 104
 105
 
___ 

_ __ 

TOPOGRAPHY: MORPHOLOGY
 

Liver: Hepatocellular Carcinoma or
 

Hepatocellular Adenoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Adenoma NOS
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 6 (CONTINUED) 

CONTROL
 

2/19(0.11)
 

N.S.
 

107
 

1/15(0.07)
 

N.S.
 

107
 

2/17(0.12)
 

P = 0.041(N)
 

107
 

LOW
 

DOSE
 

5/49(0.10)
 

N.S.
 

0.969
 

0.180
 

9.685
 

104
 

5/35(0.14)
 

N.S.
 

2.143
 

0.277
 

98.143
 

88
 

1/42(0.02)
 

N.S.
 

0.202
 

0.004
 

3.695
 

99
 

HIGH
 

DOSE
 

9/44(0.20)
 

N.S.
 

1.943
 

0.464
 

17.429
 

105
 

1/24(0.04)
 

N.S.
 

0.625
 

0.009
 

47.035
 

105
 

0/38(0.00)
 

N.S.
 

0.000
 

0.000
 

1.491
 
TABLE 6 (CONCLUDED)
 

"Treated groups received doses of 0.30 or 0.60 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.
 
To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

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

of statistically significant results. It should also be noted that
 

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

indicating the theoretical possibility of tumor induction in mice by
 

2-chloro-p-phenylenediamine sulfate that could not be established
 

under the conditions of this test.
 

44
 
V. DISCUSSION
 

There were no significant positive associations between the
 

administered dietary concentration of 2-chloro-p-phenylenediamine
 

sulfate and mortality for rats of either sex or for male mice. There
 

was a significant positive association between dosage and mortality
 

for female mice; however, adequate numbers of animals in all groups
 

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

Although slight mean group body weight depression was observed
 

in treated rats of both sexes when compared to controls, thereby indicating an observable effect of compound administration, there were
 

no statistically significant positive associations between dietary
 

exposure to 2-chloro-p-phenylenediamine sulfate and the incidences of
 

any tumor. The increased incidence of transitional-cell hyperplasia
 

of the renal pelvic epithelium in both male and female rats and the
 

presence of transitional-cell tumors of the urinary bladder in three
 

dosed rats is suggestive of, but not considered as sufficient evidence
 

of, carcinogenicity. The suggestion of carcinogenicity is supported
 

by the absence of these lesions in the concurrent controls and by the
 

historical record of the low incidence of bladder tumors (0/250 males
 

and 1/249 females) in control Fischer 344 rats at this laboratory.
 

A variety of proliferative hepatocellular lesions were observed
 

in the mice in this bioassay. When those male mice having either
 

hepatocellular carcinomas or hepatocellular adenomas were combined
 

and the resulting tumor incidences were statistically analyzed, there
 

45
 
was a significant positive association between the dietary concentration of the chemical and the incidences. The Fisher exact tests were
 

not, however, supportive. There were no other tumors in mice for
 

which a significant positive association could be established between
 

dosage and incidence. There were also no other tumors occurring in
 

significantly higher incidences in the treated mice than in controls.
 

Under the conditions of this bioassay there was no convincing
 

evidence that dietary administration of 2-chloro-p-phenylenediamine
 

sulfate was carcinogenic in Fischer 344 rats or B6C3F1 mice.
 

46
 
VI. BIBLIOGRAPHY
 

Aldrich Chemical Company, Inc., The 1977-1978 Aldrich Catalog/Handbook
 

of Organic and Biochemicals, Catalog No. 18. Milwaukee, Wisconsin, 1977.
 

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.
 

Bair, T.I. and P.W. Morgan, "Fibers and Threads of Aromatic Polyamides." Ger. Offen. 1,817,952 (duPont de Nemours, E.I. and
 

Co.), July 31, 1975; Chemical Abstracts 83, 195033g.
 

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.
 

Corbett, J.F. and J. Menkart, "Hair Colouring." CUTIS 12:190-197.
 

1973.
 

Cox, D.R., Analysis of Binary Data, Chapters 4 and 5. Methuen and
 

Co., Ltd., London, 1970.
 

Cox, D.R., "Regression Models and Life-Tables." Journal of the Royal
 

Statistical Society, Series "B" 34:187-220, 1972.
 

Fujiwara, T., K. Nagosawa, T. Tsumaki, S. Goto, and S. Fukuoka,
 

"Piperazine-Modified Aromatic Polyamide Fibers." Japan Kokai
 

74,116,322 (Asahi Chemical Industry Co., Ltd.), November 7,
 

1974; Chemival Abstracts 83, 12023m.
 

Gart, J.J., "The Comparison of Proportions: A Review of Significance
 

Tests, Confidence Limits, and Adjustments for Stratification."
 

International Statistical Institute Review 39:148-169, 1971.
 

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

Observations." Journal of the American Statistical Association
 

53:457-481, 1958.
 

47
 
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.
 

Lowenstein Dyes and Cosmetics, Inc., "Material Safety Data Sheet:
 

o-Chloro-p-phenylenediamine Sulfate." Brooklyn, New York,
 

undated.
 

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

Co., New York, 1966.
 

Richardson, E.L., Ph.D., Project Manager, Cosmetic Registries, Division
 

of Cosmetics Technology, Food and Drug Administration, Department
 

of Health, Education, and Welfare, Washington, D.C. Letter to
 

Dr. J. Donoso, The MITRE Corporation/METREK Division, McLean,
 

Virginia, June 23, 1977.
 

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.
 

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

62^679-682, 1975.
 

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

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

1963.
 

48
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH 2-CHLORO-p-


PHENYLENEDIAMINE SULFATE
 

TABLE Al

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATF,

 

ANIMALS INITIALLY IN STUDY


ANIHALS NEC80PSIED


ANIMALS EXAHINED HISTOPATHOLOGICALLY


INTEGDMENTABY SYSTEK
 

*SUBCUT TISSUE


FIBROMA


BFSPIEATOBY SYSTEM
 

tLDNG


ALVEOLAR/BBONCHIOLAR ADENOMA


AlVEOLAR/BRONCHIOLAR CARCINOMA


HEHATOPOIETIC SYSTEM
 

*KULTIPLE ORGANS


MALIGNANT LYHPHOMA. NOS


ONDIFFERENTIATED LEUKEMIA


MYELOMONOCYTIC LEUKEMIA


ISPLEEN


MYELOMONOCYTIC LEUKEMIA


CIRCULATOBY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

*LIVER


NEOPLASTIC NODULE


HEPATOCELLULAB CARCINOMA


CONTROL (UNTR)


01-0355


 20


 20


 20


 (20)


 1 (5%)


 (20)


 (20)


 1 (5X)


 (20)


 (20)


 LOW DOSE HIGH DOSE
 

 01-0340 01-0345
 

 50 50
 

 19 50
 

 U9 50
 

 (49)	 (50)
 

2 (4X)
 

 (49)	 (50)
 

1 (2X)
 

2 (1J) 2 (43S)
 

(U9) (50)
 

2 (4X)
 

1 (2X)
 

5 (10*) 1 (2X)
 

 (49)	 (50)
 

1 (2X)
 

 (49)	 (50)
 

2 (4X) 3 (6%)
 

 3_16$)
 

t NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-3
 
OEINABY SYSTEM
 

tKIDNEY/PELVIS
 

TRANSITIONAL-CELL CARCINOMA
 

*UBINABY BLADDER
 

TRANSITIONAL-CELL PAPILLOMA
 

TRANSITIONAL-CELL CARCINOMA
 

ENEOCBINE SYSTEM
 

•PITUITARY
 

ADENOMA, NOS
 

*ADRENAt
 PHEOCHEOMOCYTOMA
 

tTHYBOID
 

C-CELL ADENOMA
 C-CELL CARCINOMA
 

*PANCREATIC ISLETS
 

ISLET-CELL ADENOMA
 

ISLET-CELL CARCINOMA
 

REPRODUCTIVE SYSTEM
 

tTESTIS
 

INTEESTITIAL-CELL TUMOB
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*ZYMBAL'S GLAND
 

CARCINOMA, NOS
 

CABCINOSARCOMA
 

MUSCULOSKELETAL	 SYSTEM
 

NONE
 

TABLE Al (CONTINUED)
 

CONTROL (UNTR) LOW DOSE
 01-0355 01-0340
 

(20)	 (19)
 

1 (2%)
 

(18)	 (U8)
 

1 (2X)
 

(17)	 ((15)
 

3 (IBS) 9 (20X)
 

(20)	 (KB)
 

4 (20X) 4 (8)1)
 

(18)	 (13)
 

1 (2X)
 

2 (5*)
 

(18)	 (1*6)
 

1	 (6X) 1 (2*)
 

1 (2*)
 

(19)	 (U8)
 

17 (89%) 47 (98X)
 

(20)	 (49)
 

HIGH DOSE
 01-0345
 

(50)
 

(47)
 

1 (2%)
 

(45)
 

7 (16X)
 

(50)
 

6 (12X)
 

(49)
 

1 <2X)
 

(46)
 

1 (2X)
 

1 (2X)
 

(50)
 

48 (96X)
 

(50)
 

1 (218)
 

1 <2X)
 

#	 NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-4
 
TABLE Al (CONCLUDED) 

CONTROL (UNTB) LOW DOSE HIGH DOSE 

01-0355 01-0340 01-0345 

BODY CAVITIES 

*EODY CAVITIES (20) (49) (50) 

MESOTHELIOMA, NOS 1 (5X) 1 (2%) 1 (2*) 

NEUROFIBHOM A 1 (2%) 

All OTHEE SYSTEMS
 

NONE
 

ANIHAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 20 50 50
 NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 18 47
 ANIMAL MISSING
 

8 INCLUDES AUTOLYZED ANIMALS
 

TDMOH SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 18 49 50
 TOTAL PRIMARY TUMORS 28 82 80
 

TOTAL ANIMALS WITH BENIGN TUMORS 18 48 48
 

TOTAL BENIGN TUMOBS 26 64 65
 

TOTAL ANIMALS WITH MALIGNANT TUMOSS 1 13 11
 

TOTAL MALIGNANT TUMORS 1 15 11
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TOMORS UNCERTAINBENIGN OR MALIGNANT 1 3 4
 

TOTAL UNCERTAIN TUMORS 1 3 4
 

TOTAL ANIMALS WITH TUMORS UNCERTAINFHIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMOSS
 

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

A-5
 
TABLE A2
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS
 

TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE
 

CONTROL (UNTR) 

02-0355 

L01T DOSE 

02-0340 

HIGH DOSE 

02-0345 

ANIMALS INITIALLY IN STUDY 

ANIHA1S NECBOPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

20 

20 

20 

50 

49 

49 

50 

49 

49 

INIEGOHENTASY SYSTEM
 

*SUBCUT TISSUE (20) (19) (49)
 

FIBROMA 1 (5X) 1 (2X)
 

RESEIBATOKY SYSTEM
 

*LUNG (20) (19) (19)
 

SQUAMOUS CELL CARCINOMA, HETASTA 1 (5X)
 

ADENOCARCINOMA, NOS, HETASTATIC 1 (5»)
 

ALVEOLAR/BEONCHIOLAR ADENOMA 1 (555) 1 (2X) 1 (2%)
 

HEHATOPOIETIC SYSTEM
 

•MULTIPLE ORGANS (20) (U9) (1(9)
 

MALIGNANT LYMPHOMA. NOS 1 (2%)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (2X)
 

MYELOMONOCYTIC LEUKEMIA 3 (15X) 1 (2X)
 

CIBCUIATOBY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

ILIVER (20) (U9) («9)
 

NEOPLASTIC NODULE 1 (2X) 1 (2%)
 

ICOLON (20)
 

MUCINOUS_ADENOCARCI1!OMA
 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-6
 
TABLE A2 (CONTINUED) 

CONTROL (UNTR) LOW DOSE 

02-0355 02-0340 

URINARY SYSTEM
 

tKIDNEY (20) (48)
 

ADENOCAECINOMA, NOS, HETASTATIC 1 (5X)
 

IURINARY BLADDER (20) (48)
 

TRANSITIONAL-CELL CARCINOMA <2X)
 

ENDOCRINE SYSTEM
 

IPITUITARY (20) (42)
 

ADENOMA, NOS 14 (70X) 12 (29X)
 

•ADRENAL	 (20) (47)
 

PHEOCHROMOCYTOMA 2 (10X) 1 (2X)
 

tTHYROID (20) (44)
 

FOLLICULiR-CELL ADENOMA 1 (2X)
 FOLLICULAR-CELL CARCINOMA
 

C-CELL ADENOMA 1 (5X) 1 <2X)
 

C-CELL CARCINOMA 1 (2X)
 

tPANCREATIC ISLETS (20) (48)
 

ISLET-CELL ADENOMA 2 (10X)
 

REFROEUCTIVE SYSTEM
 

*HAMHARY GLAND (20) (49)
 

ADENOMA, NOS 1 <2X)
 

ADENOCARCINOMA, NOS 1 (5X)
 FIBSOADENOHA	 3 (15X) 1 (2X)
 

*CLITORAL GLAND (20) (49)
 

ADENOMA, NOS 1 (2X)
 

• UTERUS (20) (48)
 

ADENOCARCINOMA, NOS 1 (5X)
 

INDOMETRIAL STROHAL POLYP 4 (20X) 6 (13X)
 

•OVARY	 (20) (48)
 

GRANULOSA-CELL TUMOR
 

t NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

02-0345
 

(49)
 

(48)
 

(46)
 

13 (26X)
 

(49)
 

1 (2X)
 

(39)
 

3 (8X)
 

(48)
 

(49)
 

1 (2X)
 

2 (4X)
 

(49)
 

(49)
 

2 (4X)
 

(49)
 

A-7
 
TABLE A2 (CONTINUED) 

CONTROL(UNTH )

02-0355 LOW DOSE 02-0340

 HIGH DOSE 

 02-03U5 

NERVOUS SYSTEM 

*EFAIN

ASTEOCYTOMA

 (19) (48)

 1 (2X) 

 (49) 

SPICIAI SENSE ORGANS 

*HARDERIAN GLAND

CARCINOMA,NOS

 (20)

 1 (5X) 

 (49) (49) 

*ZYHBAL'S GLAND

SQUAMOUS CELL CARCINOMA

 (20)

 1 (5X) 

 (49) (49) 

HUSCULOSKELETAL SYSTEM 

•PEIVIC BONES

OSTEOSARCOMA

 (20)

 1 (5X) 

 (»9) (49) 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONI 

ANIMAI DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATHS 

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 

ABIBAL HISSING 

20 

16 

50 

43 

50 

47 

»_IHCiUOIS_AUTQLYZED_ANIMALS. 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

A-8
 
TUBOR SUHMARY
 

TOTAL ANIMALS WITH PRIHARY TUMORS*
 TOTAL PRIMARY TUHORS


TOTAL ANIMALS WITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIMALS KITH MALIGNANT TUMORS
 TOTAL MALIGNANT TUMORS


TABLE A2 (CONCLUDED)
 

CONTROL (UNTB)


02-0355


 17


 36


 17


 28


 5
 8


TOTAL ANIMALS KITH SECONDARY TUMORSt TOTAL SECONDARY TUHORS 2 3 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT TOTAL UNCERTAIN TUMORS

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUHORS EXCEPT SECONDARY TUMORS
 

 LOS DOSE HIGH DOSE
 02-0340 02-03U5
 

 26 19
 

 34 25
 

 21 16
 26 18
 

6 5
 

6 6
 

2 1
 2 1
 

f SECONDARY TUHORS: METASTATIC TUMORS OE TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

A-9
 

APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH 2-CHLORO-p-


PHENYLENEDIAMINE SULFATE
 

TABLE Bl

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE

 

CONTBOL(UNTH) LOW DOSE HIGH DOSE 

05-0355 05-031*5 05-0350 

ANIMALS INITIALLY IN STUDY 20 50 50 

ANIMAL S MISSING 2 

ANIMAL S NECEOPSIED 17 50 50 

ANIMALS EXAMINED HISTOPATHOLOGICALLY" 17 50 48 

INTEGUMENTARY SYSTEM
 

NONE
 

RESFIEATORY SYSTEM
 

ILUNG (17) (50) (48)
 

AIVEOLAR/BRONCHIOLAR ADENOMA 6 (12X) 2 (4JI)
 

ALVEOLAB/BSONCHIOLAR CARCINOMA 3 (18*) 1 (2X) 4 (8X)
 

PHEOCHROMOCYTOMA, METASTATIC 1 (2X)
 

HEMATCPOIETIC SYSTEM
 

*MULTIPLE ORGANS (17) (50) (50)
 

MALIGNANT LYMPHOMA, NOS 2 (12X) 1 (2X) 3 (6X)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (6X) 1 (2*) 2 (4X)
 

*SPLEEN (16) (50) (45)
 

HEMANGIOMA 1 (6X) 1 (2%)
 

HEMANGIOSA8COMA 1 (6X) 1 (2X)
 

MALIGNANT LYMPHOMA, NOS 1 (2X)
 

ILYMPH NODE (14) (46) (42)
 

HEMANGIOMA 1 (7X)
 

IMESENTERIC L. NODE (14) (46) (42)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 4 (9X) 2 (5X)
 

CIBCULATOEY SYSTEM
 

NONE
 

* NDMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NDMBE8 OF ANIMAL S NECROPSIED 

**EXCLUDES PARTIALLY AUTOHZED ANIMALS 

B-3 
TABLE Bl (CONTINUED) 

CONTROL (UNT8)

05-0355

 LOW DOSE

 05-0315

 HIGH DOSE 

 05-0350 

DIGESTIVE SYSTEM 

tLIVES

HEPATOCELLULAR ADENOMA

HEPATOCELLULAB CARCINOMA

HEMANGIOMA

HEMANGIOSARCOMA

*ESOPHAGUS

BASAL-CELL CARCINOMA

 (17)

 « (24S)

 1 (6X)

 1 ((,%)

 (16)

 (49)

 4 (8X)

 8 (16)1)

 (47)

 (16) 

 10 (22%) 

 10 (22%) 

1 (2X) 

2 (4X) 

 (42) 

1 (2%) 

OBINABY SYSTEM 

tKIDHEY

TDBOLAB-CELL ADENOMA

 (16) (50) (47) 

1 (2%) 

ENEOCEINE SYSTEM 

tADRENAL

PHEOCHROMOCYTOHA, MALIGNANT

 (16) (49)

 1 (2X) 

 (46) 

BIFBODDCTIVE SYSTEM 

#TESTIS

SEHINOMA/DYSGEBMINOMA

EHBBYONAL CABCINOMA

 (17)

 1 (6X) 

 (49)

 1 (2X) 

 (47) 

NEBVOOS SYSTEM 

• EKSIN

MENINGIOHA

 (17)

 1 (6X) 

 (50) (45) 

SPECIAI SEKSE OBGANS 

*HABDEEIAN GLAND

ACEMOMA, NOS

 (17) (50)

 2 <4X) 

 (50) 

MUSCUIOSKELETAL SYSTEM 

__NQNE 

* NOMEEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

B-4
 
TABLE Bl (CONCLUDED)
 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

05-0355 05-03U5 05-0350 

BODY CAVITIES 

NON E 

A LL CTHER SYSTEMS 

NONE 

ANIMA L DISPOSITION SUMMAR Y 

ANIMALS INITIALLY IN STUDY 20 50 50
 NATURAL DEATHS
 

HOBIBUHD SACRIFICE
 

SCHEDDLED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 13 1(5 37
 ANIMAL MISSING 2
 

3 INCLUDES AOTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 12 21 30
 TOTAL PRIMARY TUMOES 17 29 U1
 

TOTAL ANIMALS WITH BENIGN TUMORS 10 14
 TOTAL BENIGN TUMORS 12 15
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 10 1t 2<t
 TOTAL MALIGNANT TUMORS 1« 17 26
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1
 

TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS HITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

B-5
 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE

 

CONTROL, ("NTR) LOW DOSE HIGH DOSE 

06-0355 06-03H5 06-0350 

ANIHALS INITIALLY IN STUDY 20 50 50 

ANIMAL S HISSING 1 

ANIMAL S SECBOPSIED 19 49 44 

ANIMALS EXAMINE D HISTOPATHOLOGICALLY *v 19 49 44 

INTEGUMENTAR Y SYSTEM 

*SUECUT TISSUE

ADENOCARCINOMA , NOS

SARCOMA , NOS

FIBROSAHCOBA

 (19)

 .

 1 (51) 

 (49)

 2 (4X) 

 (44) 

1 (2X) 

RESPIRATOR Y SYSTEM 

tLUNG (19) (1(9) (1C()
 

ADENOCARCINOMA, NOS, METASTATIC 1 (2X)
 

ALVEOLAR/BBONCHIOLAR ADENOMA 1 (2X) 2 (5X)
 

ALVEOLA8/BRONCHIOLAR CARCINOMA 2 (4X)
 

HEMATCPOIETIC SYSTEM
 

• MULTIPLE ORGANS (19) (49) (44) 

MALIGNANT LYMPHOMA, NOS 3 <6X) 2 (5X) 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (5X) 2 (4X) 3 (7%) 

ONDIFFERENTIATED LEUKEMIA 2 <5X) 

ISPLEEN (18) (49) (43) HEMANGIOSA8COMA 2 (5X) MALIGNANT LYMPHOMA, NOS 3 (17*) 1 (2*) MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (6%) 

tLYMPH NODE (17) (US) (38) 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (6)1) 

#MESENTERIC L. NODE (17) (45) (38) 

MALIGNANT LYMPHOMA, NOS 1 (2X) 

ILIVER (19) (49) (44) 

MALIG.LYHPHOHA, HISTIOCYTIC TYPE J 

* NUMBE R OF ANIMAL S WITH TISSDE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

**EXCLDDES PARTIALLY ADTOLYZED ANIMALS 

B-6 
TABLE B2 (CONTINUED) 

CONTROL (ONTR) LOW DOSE HIGH DOSE
 

06-0355 06-03U5 06-0350
 

UNDIFFEHENTIATED LEDKEBIA 1 (SIS)
 

*PEYERS PATCH (18) (17) (39)
 

MALIGNANT LYMPHOMA, DOS 1 (2X)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

•LIVIR	 (19) (19) (Ut)
 

NEOPLASM, NOS 1 (2X)
 

HEPATOCELLULAR ADENOMA 3 (6%) 5 (11X)
 

HEPATOCELLULAH CARCINOMA 2 (11*) 2 (4X) H (9X)
 

HEMANGIOSAHCOBA 1 (2%)
 

URINARY SYSTEM
 

NONE
 

ENECCBINE SYSTEH
 

tEITUITARY (15) (35) (21)
 ADENOMA, NOS 1 (7X) 5 (1UX) 1 (IX)
 

•ADRENAL	 (18) (K6) (11)
 CORTICAL CABCINOMA 1 (2X)
 PHEOCHROMOCYTOMA 1 <2X)
 

*ADRENAL/CAPSULE (18) (16) (11)
 ADENOMA, NOS 1 (2*)
 

• THYROID (17) (12) (38) 

NEOPLASM, NOS 1 OX) 

FOLLICULAR-CELL ADENOM A 2 (12X) 

FOLLICULAR-CELL CARCINOM A 1 (2X) 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND (19) (19)
 

ADENOHAi_NOS
 

* NUMBER OF ANIMALS KITH TISSDE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NECROPSIED
 

B-7
 
TABLE B2 (CONTINUED) 

CONTROL (DNTR)

06-0355

 LOU DOSE

 06-0315

 HIGH DOSE 

 06-0350 

IUTEEOS

ENDOMETBIAL STROHAL POLYP

INDOMETRIAL STROHAL SARCOMA

tOVABY

CYSTADENOMA. NOS

PAPILLAR Y CYSTADENOMA , NOS

SBRTOLI-CELL TUHOR

 (17)

 1 <6X) 

 (17)

 2 (12)5) 

 (U7)

 1 (2X) 

 (39)

 1 (3X)

 1 (3X) 

1

 (38) 

 (38) 

 (3X) 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

*HARDERIAN GLAND

ADENOMA, NOS

 (19) (49)

 1 (2*) 

 (««) 

MUSCDIOSKELETAl SYSTEM 

NONE 

BCIY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY

NATURAL DEATHS

MORIBUND SACRIFICE

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE

ANIBAL MISSING

 20

 2

 1

 16

 1 

 50 

 15 

2 

 33 

50 

26 

5 

19 

» INCLUDES AUTOLYZED ANIMALS 

* NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

B-8
 
TABLE B2 (CONCLUDED)
 

TUHOB SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*
 TOTAL PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TUMORS
 TOTAL BENIGN TUMORS


TOTAL ANIMALS HITH MALIGNANT TUMOBS
 TOTAL MALIGNANT TUHORS


TOTAL ANIMALS KITH SECONDARY TUMORSI
 TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS HITH TUMORS UNCERTAINPBIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL (UNTR)


06-0355


 12
 18


 5
 7


 9
 11


 LOi DOSB HIGH DOSE
 

 06-03U5 06-0350
 

 27 22
 31 27
 

 11 9
 13 10
 

 16 13
 17 16
 

1
 

1
 

1 1
 

1 1
 

* PRIMARY TUMORS: ALL TUMORS EXC8PT SECONDARY TUMORS
 t SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-9
 

APPENDIX C
 

SUMMARY OF THE INCIDENCE OF 'NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 2-CHLORO-p-


PHENYLENEDIAMINE SULFATE
 

TABLE Cl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE 

ANIMAL S INITIALLY IS STUDY

ANIMAL S NECHOPSIED

ANIHAL S EXAMINE D HISTOPATHOLOGICALLY

INTEGUMENTAR Y SYSTEH 

*SUBCUT TISSUE

NECROSIS, NOS

RESPIBATORY SYSTEM 

*LUNG/BRONCHU S

BRONCHIECTASIS

INFLAMMATION, NOS

ILONG


INFLAMMATION, NOS


HYPERPLASIA, ALVEOLAR EPITHELIUM


HEMATOPOIETIC SYSTEM
 

*SPLEEN


INFABCT, HEALED


HEWATOPOIESIS


tSPLENIC FOLLICLES


NECROSIS, NOS


CIECDLATORY SYSTEM
 

tHEABT


INFLAMMATION, INTERSTITIAL


*MYOCAEDIUM


INFLAMMATION, INTERSTITIAL


FIBROSIS ,


CONTROL(ONTE) LOU DOSE HIGH DOSE 

01-0355 01-0310 01-0315 

 20 50 50 

 20 19 50 

 20 19 50 

 (20) (19) (50) 

1 (2X) 

 (20) (19) (50) 

2 (10X) 1 (2X) 

1 (5X) 

 (20) (19) (50) 

1 (5X) 1(2%) 

1 (5X) 2 (IX) 

 (20) (49) (50) 

1(2%) 

5 (10X) 5 (10*) 

 (20) (19) (50) 

1(2%) 

 (20) (19) (50) 

1 (2X) 

 (20) (19) (50) 

1 (2X) 

 3_115Sl 

I NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-3
 
DEGENERATION, NOS
 

DIGESTIVE SYSTEH
 

tLIVEF
 

NECROSIS, FOCAL
 

METAMORPHOSIS FATTY
 

BASOPHILIC CYTO CHANGE
 

EOSINOPHILIC CYTO CHANGE
 

IPANCREATIC ACINUS
 

ATROPHY, NOS
 

ISTOMACH
 

ACANTHOSIS
 

ICOLON
 

NEMATODIASIS
 

URINAB Y SYSTEM
 

*KIDNEY
 

MINERALIZATION
 

INFLAMMATION, NOS
 

INFLAMMATION, CHRONIC
 

FIBROSIS, DIFFUSE
 

PERIVASCULITIS
 

NEPHROPATHY
 

DEGENERATION, CYSTIC
 

NEPHROSIS, NOS
 

GLOMERDLOSCLEROSIS, NOS
 

NECROSIS, NOS
 

HYPERTBOPHY, NOS
 

HYPEEPLASIA. EPITHELIAL
 

tKIDNEY/PELVIS
 

HYPERPLASIA, EPITHELIAL
 

ENDOCRINE SYSTEM
 

•ADRENAL CORTEX
 

HYPERTROPHY, NOS
 

TABLE Cl (CONTINUED) 

CONTROL (II SIR)
 

01-0355
 

5 (25%)
 

(20)
 

1 (5X)
 

1 (5X)
 

(18)
 

1 (6X)
 

(18)
 

1 (6X)
 

(18)
 

1 (6X)
 

(20)
 

18 (90%)
 

(20)
 

(20)
 

L0» DOSE HIGH DOSE
 

01-0340 01-0345
 

4 (8*) 2 (4X)
 

(49) (50)
 

1 (2%) 2 (4X)
 

2 (4X) 1 12%)
 

1 (2%)
 

(46) (46)
 

(48)	 (50)
 

1 (2X)
 

(46) (49)
 

(49)	 (50)
 

1 (2%)
 

1 <2X)
 

1 (2*)
 

1 (2%) 33 (66X)
 

1 <2X)
 

47 (96X) 48 (96%)
 

1 (2*)
 

1 (2X)
 

3 (6X)
 

1 (2X)
 

1 <2X>
 14 (29X) 30 (60X)
 

(49)	 (50)
 3 (6%)
 

(48) (50)
 

i NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-4
 
TABLE Cl (CONTINUED) 

CONTROL (UNTB) LOW DOSE HIGH DOSE 

01-0355 01-0340 01-0315 

HYPERPLASIA, DOS <5X) 

tADRENAL MEDULLA (20) (48) (50) 

HYPEEPLASIA, NODULA R 1 (2%) 

HYPEEPLASIA , NOS 2 (UX) 

*THYBOID (18) (43) (49) 

FOLLICDLAR CYST, NOS 2 (UX) 

HYPERPLASIA, C-CELL 1 (6%) 1 (2X) 

#PARATHYROID (8) (15) (18)
 

HYPERPLASIA, NOS 1 (6%)
 

REPRODUCTIVE SYSTEM
 

*PROSTATE (19) (U6) (48)
 

INFLAMMATION, NOS 1 (2*)
 

#TESTIS (19) (48) (50)
 

MINEBALIZATION 1 (5X) 2 (4X)
 

INFLAMMATION, NOS 1 (2X)
 

ATROPHY, NOS 2 (11X) 5 (10X) 6 (12X)
 

HYPERPLASIA, INTERSTITIAL CELL 1 (2X)
 

NERVOUS SYSTEH
 

NONE
 

SEICIAL SENSE ORGANS
 

*EYE (20) (49) (50)
 

CATAEACT 1 (2X)
 

*EYE/CORNEA (20) (49) (50)
 

INFLAMMATION, NOS 1 (2X)
 

*HARDERIAN GLAND (20) (49) (50)
 

INFLAMMATION, NOS 1 (2%)
 

MOSCUIOSKEIETAL SYSTEM
 

NONE
 

* NUMBE R OF ANIMALS WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

C-5 
TABLECl (CONCLUDED) 

CONTROL (HNTB) LOU DOSE HIGH DOSE 

01-0355 01-0340 01-0345 

BODY CAVITIES 

NONE 

ALI OTHER SYSTEMS 

OMENTUM 

NECROSIS, PAT 

SPECIAL MORPHOLOGY SUMMARY 

iUTO/NECROPSY/HISTO PERF 

AUTOLYSIS/NO NECROPSY 

* NUMBER OF ANIMALS BITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

C-6 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

TREATED WITH 2-CHLORO p PHENYLENEDIAMINE SULhATL 

HIGH DOSE
 

02-0345
 

50
 

49
 

49
 

(47)
 

1 (2X)
 

(49)
 

1 (2X)
 

(49)
 

(46)
 

1 (2X)
 

(49)
 

15 (31X)
 

(49)
 

(49)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

NONE
 

BESEIBATOHY SYSTEM
 

»TRACHEA
 

INFLAMMATION, NOS
 

•LUNG/BRONCHUS
 

BRONCHIECTASIS
 

INPLAMMATION, NOS
 

*LONG
 

INFLAMMATION, NOS
 

PNEUMONIA, CHRONIC MDRINE
 

HYPERPLASIA, ALVEOLAR EPITHELIUM
 

HEMATOPOIETIC SYSTEM
 

tEONE HARROW
 

HISTIOCYTOSIS
 

ISPLEEN
 

HEMATOPOIESIS
 

CIECOLATOEY SYSTEM
 

*HEART
 

MINERALIZATION
 

IHYOCAEDIOM
 

FIBHOSIS
 

CONTROL (UNTR) LOW DOSE
 

02-0355 02-03HO
 

20 50
 

20 49
 

20 49
 

(5) (18)
 

(20) (49)
 

1 15*) 2 (IX)
 

(20)	 0»9)
 

1 (2*)
 

1 (2X)
 

1 (2*)
 

(19) (48)
 

(20)	 (48)
 

6 (30%) 16 (33%)
 

(20)	 (49)
 

1 <2X)
 

(20)	 (49)
 

1 1211
 

* NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

C-7 
TABLE C2 (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 02-0355 02-0310 02-0345
 

DEGENERATION, NOS	 2 (10X) 2 (tK) 2 (IX)
 

DIGESTIVE SYSTEM
 

ILIVEE (20) (19) (49)
 NECROSIS, FOCAL 1 (5X) 1 (2X) 1 (2X)
 METAMORPHOSIS FATTY 2 (10X) 1 12%)
 BASOPHILIC CYTO CHANGE 9 (45X) 10 (20X) 10 (20X)
 ANGIECTASIS 1 (2X)
 

*EILE DUCT (20) (19) (49)
 HYPEEPLASIA, NOS 1 (2X)
 

tSTOMACH (20) (18) (48)
 

HYPERKEEATOSIS 1 (2»)
 

ACANTHOSIS 2 (IX)
 

•COLON	 (20) <U7) (49)
 

PARASITISM 1 (5X)
 

UEINAEY SYSTEM
 

IKIDNEY (20) (48) (49)
 

MINERALIZATION 2 (IX)
 NEPHROPATHY 11 (55X) 37 (77X) 25 (51X)
 HYPERPLASIA, EPITHELIAL 11 (29X) 8 (16X)
 

ENDCCBINE SYSTEM
 

*ADHENAL CORTEX (20) (17) (49)
 HYPERTROPHY, FOCAL 1 (2X) 2 (IX)
 

#THYROID (20) (11) (39)
 FCLLICULAR CYST, NOS 1 (2X)
 

REFRODDCTIVE SYSTEM
 

*MAMMAEY GLAND (20) (19) (49)
 GALACTOCELE 4 (20X) 3 <6X) 1 <2X)
 HYPERPLASIA, NOS 1 (5%) 3 (6X)
 

JUTERUS (20) (48) (49)
 

HYDEOMETRA 1 I2SL 1 (2X>
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-8
 
TABLE C2 (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

02-03b5 02-0340 02-0345 

INFLAMMATION, NOS 9 (19X) 7 (14X)
 

HYPEKPLASIA, ADENOBATOOS 1 <2X)
 POLYP, INFLAMMATORY 1 (2X)
 

IUTEBUS/ENDOMETRIUM (20) (48) (49)
 

INFLAMMATION. NOS 5 (25*)
 HYPERPLASIA, NOS 1 (5X) 2 (4X) 3 (6X)
 

*OVAHY/OVIDUCT (20) (18) (49)
 

INFLAMMATION, NOS 1 (2X)
 

#OVARY (20) (48) (49)
 

CYST, NOS 1 (2X)
 

INFLAMMATION, NOS 1 (2X) 3 (6%)
 

INFLAMMATION, NECROTIZING 1 (2X)
 

DEGENERATION, CYSTIC 1 (2X)
 

NF.FVODS SYSTEM
 

NONE
 

SPECIAL SENS! ORGANS
 

*EYE (20) (49) (49)
 

CATARACT 1 (2X)
 

*EYE/RETINA (20) (49) (49)
 

ATROPHY, NOS 1 (2X)
 

*HARDERIAN GLAND (20) (49) (49)
 

INFLAMMATION, NOS 1 (2X)
 

MDSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

All OTHER SYSTEMS
 

OMENTUM
 MINERALIZATION 1
 

* NUMBER OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIHALS NECROPSIED
 

C-9
 
TABLE C2 (CONCLUDED)
 

CONTROL(UNIB) 

02-0355 

LOS DOSE 

02-0340 HIGH DOSE 02-03H5 

NECROSIS, FAT 

JPECIBL MORPHOLOGY SUMMARY
 

NO LESION REPORTED
 

AUTC/NECROPSY/HISTO PERF
 

AUTOLYSIS/NO NECROPSY
 

NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

NUMBER OF ANIMALS NECROPSIED
 

c-io
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 2-CHLORO-p-


PHENYLENEDIAMINE SULFATE
 

TABLE Dl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH 2-CHLORO-p-PHhNYLENEDIAMlNE SULFATE

 

CONTROL (UNTB) LOH DOSE HIGH DOSE 

05-0355 05-0345 05-0350 

ANIHAL S INITIALLY IN STUDY 20 50 50 

ANIMALS HISSING 2 

ANIHAL S NECBOPSIED 17 50 50 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY ** 17 50 08 

INTEGDMENTAB Y SYSTEM 

NONE 

RF.SPIFATOBY SYSTEM 

KLUNG (17) (50) (48) INFLAMMATION, NOS 1 (2X) 

PNEDMONIA, CHRONIC HORINE 1 (2%) 

HEMATCPOIETIC SYSTEH
 

•SPLEEN	 (16) (50) (45)
 

HYPEBPLASIA, NOS 1 (2X)
 

HEMATOPOIESIS 2 (13%) 3 (6X) 1 (2X)
 

*MESENTEBIC L. NODE (14) (46) (42)
 

CONGESTION, NOS 1 <7X)
 

HEMATOPOIESIS 1 (2X)
 

CIFCULATOBl SYSTEM
 

*CABDIOVASCDLAR SYSTE (17) (50) (50)
 

PEBIVASCOLITIS 1 (2X)
 

DIGESTIVE SYSTEM
 

#LIVIF (17 ) (49) (46) 

INFLAMMATION, NOS 1 <2X) 

INFLAMMATION, ACUTE 

* NUMBEB OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIHALS NECROPSIED 

**EXCLODES PARTIALLY AUTOLYZED ANIMALS 

D-3 
TABLE Dl (CONTINUED) 

CONTROL (UNTR) LOU DOSE HIGH DOSE 

05-0355 05-0345 05-0350 

NECROSIS, COAGULATIVE 1 (6X)
 

HYPERPLASIA, FOCAL <2X) 1 (2X)
 HEMATOPOIESIS 1 (2%)
 

*GALLELADDER (17) (50) (50)
 CALCULUS, NOS 1 (2X)
 

*BILE DUCT (17) (50) (50)
 HYPERPLASIA, NOS 2 (4X) 1 (2X)
 

•PANCREATIC	 ACINUS (16) (1*7) (143)
 ATROPHY, NOS 1 (6X)
 

ISTOMACH (15) (17) (UU)
 

ABSCESS, NOS 2 (IX)
 

URINAE Y SYSTEM 

IKIDNEY (16) (50) (17)
 

MINERALIZATION 1 (2X)
 

ENECCRINE SYSTEM
 

#THYROID (17) (U7) (12) 

FOLLICULA R CYST, NOS 1 (2X) 

HYPERPLASIA , FOLLICULAR-CELL 1 (6X) 

REEHOEUCTIVE SYSTEM
 

NONE
 

NIRVCUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MBSCUIOSKELETAL SYSTEM
 

NONE
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-4
 
TABLE ni (CONCLUDED)
 

CONTROL (UNTH) LOW DOSE HLGH DOSE
 05-0355 05-0315 05-0350
 

BOtY CAVITIES
 

NONE
 

ALL CTHIE SYSTEMS
 

*MD1TIPLE ORGANS (17) (50) (50)
 

H1MATOPOIESIS 1 (2%)
 

SPECIAL MORPHOLOGY SUMMARY
 

NC LESION REPORTED
 ANIMAL MISSING/NO NECROPSY
 AUTO/NECROPSY/HISTO PERF
 AUTO/NECROPSY/NO HISTO
 

AUTOLYSIS/tiO NECROPSY
 

NDMEER OF ANIMALS KITH TISSDE EXAMINED MICROSCOPICALLY
 

NUMBER OF ANIMALS NEC80PSIED
 

D-5
 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH 2-CHLORO-p-PHENYLENEDIAMINE SULFATE 

ANIMALS INITIALLY IN STUDY
 

ANIMALS MISSING
 

ANIMALS NECEOPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE
 

CHOLESTEATOBA
 

RESPIRATORY SYSTEM
 

ftLUNG
 

INFLAHMATION, NOS
 

INFLAMMATION, FOCAL
 

HEMATCPOIETIC SYSTEM
 

iSPLEEN
 

HYPEEPLASIA, LYMPHOID
 

HEMATOPOIESIS
 

•SPLENIC FOLLICLES
 

HYPEEPLASIA, NOS
 

IKESENTERIC L. NODE
 

HEMATOPOIESIS
 

CIPCUIATORY SYSTEM
 

tHEART
 

AMYLOIDOSIS
 

DIGESTIVE SYSTEM
 

#IIVEE
 

HYPEEPLASIAJL_FOCAL__
 

CONTROL (UNTR)
 

06-0355
 

20
 

1
 

19
 

** 19
 

(19)
 

(19)
 

(18)
 

2 (11%)
 

(18)
 

(17)
 

(19)
 

(19 ) 

LOU DOSE HIGH DOSE
 

06-0345 06-0350
 

50 50
 

49 44
 

49 44
 

(49)	 (44)
 

1 <2X)
 

(49)	 (44)
 

3 <6X)
 

1 (2X)
 

(49) (43)
 

1 (2X)
 4 (8*) 1 (2X)
 

(49)	 (43)
 

1 (2X)
 

(45)	 (38)
 

1 (2X) 1 (3X)
 

(49)	 (43)
 

1 (2X)
 

(44) 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-6 
TABLE D2 (CONTINUED)
 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 06-0355 06-0345 06-0350
 

HEMATOPOIESIS	 1 <2X) 1 <2X)
 

*GALLBLADDEB (19) (19) (44)
 INFLAMMATION, NOS 1 (5X)
 

•PANCREAS	 (18) (1(14) (40)
 

INFLAMMATION, CHRONIC 1 (2*)
 

*PANCBEATIC ACINUS (18) (H4) (40)
 

ATROPHY, NOS 1 (2X) 1 (3X)
 

JSTOMACH (17) (18) (40)
 

ABSCESS, NOS 2 (14%) 1 (3«)
 

#PEYERS PATCH (18) (17) (39)
 

HYPERPLASIA, DOS 1 <6X)
 

IDDODENDM (18) (17) (39)
 

AMYLOIDOSIS 1 (3X)
 

IJEJUNUM (18) (17) (39)
 

AHYLOIDOSIS 1 <3X)
 

#ILEUH (18) (47) (39)
 

AtlYLOIDOSIS 1 (3X)
 

OSINABY SYSTEM
 

#KIDNEY (18) (19) (43)
 

INFLAMMATION, NOS 1 (2*)
 

GLOMERULOSCLEROSIS, NOS 1 (2«)
 

ABYLOIDOSIS 1 (2*)
 

ENDOCBINE SYSTEM
 

tADRENAL/CAPSULE (18) (16) (11)
 HYPEEPLASIA, NOS 3 (17%) 1 (2«)
 

(ADRENAL CORTEX (18) (46) (41)
 

HYPERTROPHY, FOCAL 1 (6X)
 

ITHYROID (17) (42) (38)
 

FOLLICBLAS CYST. NOS 1 J6S1
 

» NUMBER OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NDMBER OF ANIMALS NECROPSIED
 

D-7
 
TABLE D2 (CONTINUED) 

CONTROL (UNTR) LOU DOSE HIGH DOSE 

06-0355 06-03K5 06-0350 

HYPERPLASIA, FO LLICULAR-CELL 3 (18X) 

REPRODUCTIVE SYSTEM
 

fUTEROS (17) (17) (38)
 

INFLAMMATION, NOS 1 (6X)
 

IDTERUS/ENDOMETRIUM (17) (17) (38)
 

HYPERPLASIA. NOS 5 (29X) 1 (2X) 1 (3X)
 

HYPERPLASIA, CYSTIC 1 (6S)
 

tOVARY/OVIDUCT (17) (17) (38)
 

DEGENERATION, NOS 1 (3X)
 

*OVARY (17) (39) (38)
 

MINERALIZATION
 

CYST, NOS U (10X)
 

DEGENERATION, CYSTIC 1 <3X)
 

AMYLOIDOSIS 1 (3X)
 

NEEVCUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NOSE
 

BUSCUIOSKEIETAL SYSTEM
 

NOSE
 

BCEY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

*MULTIPL E ORGAN S (19) (U9) (it)
 

AMYLOIDOSI S 1_. 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-8
 
TABU D2 (CONCLUDED)
 

CONTROL (UNTR) LOU DOSE HIGH DOSE
 06-0355 06-0315 06-0350
 

SPECUL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 1 11 15
 ANIMAL MISSING/NO NECROPSY 1
 AUTO/NECROPSY/HISTO PERF 1 3
 

AUTOLYSIS/NO NECROPSY 1 6
 

NOMBEF OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 NDMBEfi OF ANIMALS NECROPSIED
 

D-9
 

Review of the Bioassay of 2-Chloro-p-phenylenediamine Sulfate*
 

for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

April 26, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with 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 2-Chloro-p-phenylenediamine Sulfate for carcinogenicity.
 

The primary reviewer agreed that the study did not
 

provide statistically significant evidence of the carcinogenicity of 2-Chloro-p-phenylenediamine Sulfate in rats
 

or mice. She said that it was unclear as to how the tested
 

material related to the commercially-used dye. She questioned
 

whether the route of exposure was relevant to the human
 

situation. The primary reviewer noted that the treatment
 

of the high dose mice had to be terminated early due to
 

excessive mortality and also the unexplained toxicity,
 

manifested as alopecia, in both treated and control animals.
 She commented on the increased incidence of transitional-


cell tumors of the urinary bladder found in treated rats
 

but absent in the matched controls. The primary reviewer
 

suggested that a statement was needed in the summary with
 

respect to the Incidence of these lesions in historic
 

control animals. A Program staff pathologist commented
 
that hyperplasia of the renal pelvis Is a relatively rare
 

lesion and is usually associated with bladder carcinogens.
 

A motion was approved unanimously that the report on
 

the bioassay of 2-Chloro-p-phenylenediamine Sulfate be
 

accepted.
 

Members present were:
 

Michael Shimkin (Acting Chairman), University of California
 

at San Diego
 

Joseph Highland, Environmental Defense Fund
 

George Roush, Jr., Monsanto Company
 

Louise Strong, University of Texas Health Sciences Center
 

John Weisburger, American Health Foundation
 

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.
 

JJ. U.S. GOVERNMEN T PRINTIN G OFFICE : 1978-260-899/3162
 

DHEW Publication No. (NIH) 78-1368