1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

NO. 82 

BIOASSAY OF 

N-PHENYL-p-PHENYLENEDIAMINE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 101-54-2 

NCI-CG-TR-82 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

N-PHENYL-p-PHENYLENEDIAMINE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-1332 

BIOASSAY OF
 

N-PHENYL-p-PHENYLENEDIAMINE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

FOREWORD: This report presents the results of the bioassay of
 

N-phenyl-p-phenylenediamine conducted for the Carcinogenesis
 

Testing Program, Division of Cancer Cause and Prevention,
 

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

Bethesda, Maryland. This is one of a series of experiments
 

designed to determine whether selected chemicals have the
 

capacity to produce cancer in animals. Negative results, in
 

which the test animals do not have a greater incidence of cancer
 

than control animals, do not necessarily mean that the test
 

chemical is not a carcinogen, inasmuch as 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 that exposure to
 

the chemical- is a potential risk to man. The actual determination of the risk to man from animal carcinogens requires a wider
 

analysis.
 

CONTRIBUTORS: This bioassay of N-phenyl-p-phenylenediamine was
 

conducted by Litton Bionetics, Inc., Kensington, Maryland,
 

initially under direct contract to the NCI and currently under a
 

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

NCI Carcinogenesis Testing Program.
 

The experimental design and doses were chosen by Drs. E. K.
 

Weisburger1^ J. H. Weisburger1'2, N. P. Page1*3, F. M. Garner4
 

and B. M. Ulland4'5. Mr. R. Cypher4, Mr. H. D. Thornett4, and
 

Mr. D. J. Howard4 were responsible for administration of test
 

chemical and for care and observation of animals.
 

Histopathologic examinations were performed by Drs. B. Cockrell4

,
 

A. dePaoli , F. M. Garner4, E. Gorgas , C. Montgomery , and N.
 

iii
 
Wosu^ for the rat study, and by Drs. Cockrell, dePaoli, Garner,
 

Montgomery, and Wosu for the mouse study. Histologic sections of
 

all tumors and hyperplasia were reexamined by Dr. R. A. Montali^,
 

who also reviewed all diagnoses and prepared the initial
 

interpretive pathology summary.
 

Animal pathology tables and survival tables were compiled at EG&G
 

Mason Research Institute". The statistical analyses were performed by Dr. J. R. Joiner', using methods selected for the
 

bioassay program by Dr. J. J. Gart°. Chemicals used in this
 

bioassay were analyzed under the direction of Mr. H. Paulin , and
 

the results of these analyses were reviewed by Dr. S. S. Olin .
 

The structural formula was supplied by NCI .
 

This report was prepared at Tracer Jitco' under the direction of
 

NCI. Those responsible for the report at Tracor Jitco were Dr.
 

Marshall Steinberg, Director of the Bioassay Program; Dr. L. A.
 

Campbell, Deputy Director for Science; Drs. J. F. Robens and C.
 

H. Williams, toxicologists; Dr. R. L. Schueler, pathologist; Dr.
 

G. L. Miller, Ms. L. A. Waitz, and Mr. W. D. Reichardt,
 

bioscience writers; and Dr. E. W. Gunberg, technical editor,
 

assisted by Ms. Y. E. Presley and Ms. P. J. Graboske.
 

The statistical analysis was reviewed by members of the
 

Mathematical Statistics and Applied Mathematics Section of NCI°:
 

Dr. John J. Gart, Mr. Jun-mo Nam, Dr. Hugh M. Pettigrew, and Dr.
 

Robert E. Tarone.
 

The following other scientists at the NCI^ were responsible for
 

evaluating the bioassay, interpreting the results, and reporting
 

the findings: Dr. Kenneth C. Chu, Dr. Cipriano Cueto, Jr., Dr. J.
 

Fielding Douglas, Dr. Dawn G. Goodman, Dr. Richard A. Griesemer,
 

Dr. Harry Milman, Dr. Thomas W. Orme, Dr. Robert A. Squire^, and
 

Dr. Jerrold M. Ward.
 

iv
 
1-Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York.
 

%ow with the Environmental Protection Agency, 401 M Street,
 

S.W., Washington, D.C.
 

^Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington,
 

Maryland.
 

with Hazleton Laboratories America, Inc., 9200 Leesburg
 

Turnpike, Vienna, Virginia.
 

Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

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

Maryland.
 

"Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute, National
 

Institutes of Health, Bethesda, Maryland.
 

'Now with the Division of Comparative Medicine, Johns Hopkins
 

University School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

V 

SUMMARY
 

A bioassay of N-phenyl-p-phenylenediamine for possible carcinogenicity was conducted by administering the test chemical in the
 

diet to Fischer 344 rats and B6C3F1 mice.
 

Groups of 50 rats of each sex were administered N-phenyl-pphenylenediamine at one of two doses, either 600 or 1,200 ppm,
 

for 78 weeks and were then observed for 26 additional weeks.
 

Matched controls consisted of groups of 20 untreated rats of each
 

sex. All surviving rats were killed at 104 weeks.
 

Groups of 50 mice of each sex were initially administered
 

N-phenyl-p-phenylenediamine at one of the following doses, either
 

2,500 or 5,000 ppm for the males and either 5,000 or 10,000 ppm
 

for the females, for 31 weeks. Because of toxicity of the
 

chemical, the doses were lowered at that time and terminated at
 

48 weeks. The animals were then observed for 43 additional
 

weeks. Time-weighted average doses during the period of
 

administration were 2,057 or 4,114 ppm for the males and 3,672 or
 

8,170 ppm for the females. Matched controls consisted of groups
 

of 20 untreated mice of each sex. All surviving mice were killed
 

at 91 weeks.
 

Mean body weights of the dosed rats were only slightly lower than
 

those of the matched controls during the bioassay. Mean body
 

weights of the dosed mice were appreciably lower than those of
 

the matched controls, and mortality was high in the dosed groups
 

prior to reduction of the doses, particularly in the females.
 

Sufficient numbers of rats and mice of each sex were at risk for
 

the development of late-appearing tumors; however, the shortened
 

period used for administering N-phenyl-p-phenylenediamine to the
 

mice may not have been adequate for determining the carcinogenic
 

potential of the test chemical in this species.
 

In the male and female rats, the incidences of neoplasms in the
 

vii
 
groups receiving the test chemical were not significantly
 

different from those in the corresponding control groups.
 

In the male mice, the incidence of combined hepatocellular
 

adenomas and carcinomas was significantly higher (P = 0.022) in
 

the low-dose group than in the controls, but there was no
 

significant dose-related trend (controls 2/20, low-dose 18/49,
 

high-dose 10/50). Furthermore, since at this laboratory the
 

overall historical incidences of these combined lesions in male
 

mice have been 53/340 (15.6%) and have been as high as 7/20
 

(35%), these neoplasms could not be established as being compound
 

related. Unusually extensive hepatic inflammation occurred in
 

large numbers of the dosed males (controls 0/20, low-dose 23/49,
 

high-dose 24/50) and in lesser numbers of the dosed females
 

(controls 1/20, low-dose 8/49, high-dose 2/48).
 

It is concluded that under the conditions of this bioassay,
 

N-phenyl-p-phenylenediamine was not carcinogenic for Fischer 344
 

rats or for B6C3F1 mice.
 

viii
 
TABLE OF CONTENTS
 

I. Introduction
 

II. Materials and Methods
 

A. Chemical
 

B. Dietary Preparation
 

C. Animals
 

D. Animal Maintenance
 

E. Subchronic Studies
 

F. Designs of Chronic Studies
 

G. Clinical and Pathologic Examinations
 

H. Data Recording and Statistical Analyses
 

III. Results - Rats
 

A. Body Weights and Clinical Signs (Rats)
 

B. Survival (Rats)
 

C. Pathology (Rats)
 

D. Statistical Analyses of Results (Rats)
 

IV. Results - Mice
 

A. Body Weights and Clinical Signs (Mice)
 

B. Survival (Mice)
 

C. Pathology (Mice)
 

D. Statistical Analyses of Results (Mice)
 

V. Discussion
 

VI. Bibliography
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Fed N-Phenyl-p-Phenylenediamine in the Diet...
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Fed N-Phenyl-p-Phenylenediamine
 

in the Diet
 

ix
 

Page
 

1
 

3
 

3
 

4
 

5
 

5
 

7
 

8
 

11
 

12
 

17
 

17
 

17
 

20
 

21
 

23
 

23
 

23
 

26
 

29
 

31
 

35
 

37
 
Table A2
 

Appendix B
 

Table Bl
 

Table B2
 

Appendix C
 

Table Cl
 

Table C2
 

Appendix D
 

Table Dl
 

Table D2
 

Appendix E
 

Table El
 

Page
 

Summary of the Incidence of Neoplasms in
 

Female Rats Fed N-Phenyl-p-Phenylenediamine
 

in the Diet 43
 

Summary of the Incidence of Neoplasms in
 

Mice Fed N-Phenyl-p-phenylenediamine in the Diet... 47
 

Summary of the Incidence of Neoplasms in
 

Male Mice Fed N-Phenyl-p-Phenylenediamine
 

in the Diet 49
 

Summary of the Incidence of Neoplasms in
 

Female Mice Fed N-Phenyl-p-Phenylenediamine
 

in the Diet 52
 

Summary of the Incidence of Nonneoplastic
 

Lesions in Rats Fed N-Phenyl-p-Phenylenediamine
 

in the Diet 55
 

Summary of the Incidence of Nonneoplastic
 

Lesions in Male Rats Fed N-Phenyl-p-


Phenylenediamine in the Diet 57
 

Summary of the Incidence of Nonneoplastic
 

Lesions in Female Rats Fed N-Phenyl-p-


Phenylenediamine in the Diet 61
 

Summary of the Incidence of Nonneoplastic
 

Lesions in Mice Fed N-Phenyl-p-Pheriylenediamine 

in the Diet 67 

Summary of the Incidence of Nonneoplastic
 

Lesions in Male Mice Fed N-Phenyl-p-


Phenylenediamine in the Diet 69
 

Summary of the Incidence of Nonneoplastic
 

Lesions in Female Mice Fed N-Phenyl-p-


Phenylenediamine in the Diet 72
 

Analyses of the Incidence of Primary Tumors
 

in Rats Fed N-Phenyl-p-Phenylenediamine
 

in the Diet 75
 

Analyses of the Incidence of Primary Tumors
 

in Male Rats Fed N-Phenyl-p-Phenylenediamine
 

in the Diet 77
 

x
 
Table E2
 

Appendix F
 

Table Fl
 

Table F2
 

Table 1
 

Table 2
 

Figure 1
 

Figure 2
 

Figure 3
 

Figure 4
 

Page 

Analyses of the Incidence of Primary Tumors 

in Female Rats Fed N-Phenyl-p-Phenylenediamine 

in the Diet 83 

Analyses of the Incidence of Primary Tumors 

in Mice Fed N-Phenyl-p-Phenylenediamine 

in the Diet , 87 

Analyses of the Incidence of Primary Tumors 

in Male Mice Fed N-Phenyl-p-Phenylenediamine 

in the Diet 89 

Analyses of the Incidence of Primary Tumors 

in Female Mice Fed N-Phenyl-p-Phenylenediamine 

in the Diet , 92 

TABLES 

Design of N-Phenyl-p-Phenylenediamine Chronic 

Feeding Studies in Rats , 9 

Design of N-Phenyl-p-Phenylenediamine Chronic 

Feeding Studies in Mice 10 

FIGURES 

Growth Curves for Rats Fed N-Phenyl-pPhenylenediamine in the Diet 18 

Survival Curves for Rats Fed N-Phenyl-pPhenylenediamine in the Diet 19 

Growth Curves for Mice Fed N-Phenyl-pPhenylenediamine in the Diet 24 

Survival Curves for Mice Fed N-Phenyl-pPhenylenediamine in the Diet 25 

xi
 

I. INTRODUCTION
 

N-phenyl-p-phenylenediamine (CAS 101-54-2; NCI C02233) is an
 

industrial intermediate that is used in the production of several
 

different chemical products. It is an intermediate for photographic chemicals, pharmaceuticals, microbicides, and other
 

organics (Uniroyal Chemicals, 1976); it is used in the manufacture of dyes and dye reagents (Colour Index, 1956); and it reacts
 

with ketones to form derivatives of p-phenylenediamine which are
 

used as antiozonants in rubber (Shaver, 1968).
 

N-phenyl-p-phenylenediamine is listed as a suggested ingredient
 

of oxidation-type hair dyes (Wall, 1972) and is, or has been,
 

used in 39 diferent hair dye products, according to voluntary
 

reports from the cosmetics industry to the Food and Drug
 

Administration (Greif, 1977).
 

Because of the increasing concern for occupational safety and
 

health in the chemical industry and because N-phenyl-p-phenylenediamine is an aromatic amine, it was selected for testing in
 

the Carcinogenesis Testing Program.
 

1
 

II. MATERIALS AND METHODS
 

A. Chemical
 

N-PHENYL-p-PHENYLENEDIAMINE
 

The chemical used in the bioassay was technical-grade N-phenyl-pphenylenediamine, supplied in a single batch (Lot No. 0100700 GB)
 

by Uniroyal Chemical, Division of Uniroyal, Inc., Naugatuck,
 

Connecticut. Analyses at Litton Bionetics, Inc., confirmed its
 

identity. Infrared and nuclear magnetic resonance (nmr) spectra
 

were consistent with the structure. The purity was estimated to
 

be approximately 90%, as determined by thin-layer chromatography
 

(tic), column chromatography, and nmr. No attempt was made to
 

identify two impurities that were detected. The melting range
 

was 64.5-68.0°C, similar to the published value of 66°C (Weast,
 

1974) and the manufacturer's value of 69°C.
 

3
 
The chemical was reanalyzed by tic at 6-month intervals. No
 

change in the tic pattern was detected.
 

The chemical was stored at 4°C in the original container.
 

B. Dietary Preparation
 

A 6-kilogram batch of feed was prepared twice per week for mice
 

and three times per week for rats. To obtain each dietary concentration of N-phenyl-p-phenylenediamine, the appropriate weight of
 

the chemical was mixed with a small portion of Wayne® Lab Blox
 

animal meal (Allied Mills, Inc., Chicago, Ell.) using a mortar
 

and pestle. This premix was then added to the remaining weight
 

of feed and mixed in a twin-shell blender for at least 15
 

minutes. Feed preparations containing the test chemical were
 

stored at 1°C for no longer than 1 week.
 

The stability of the chemical mixed with feed was determined at
 

concentrations of 600 and 10,000 ppm. After 10 days at room
 

temperature, no change in concentration was detected at either
 

level.
 

Ground Wayne® Lab Blox animal meal not containing added test
 

chemical (basal diet) was used as the diet for the control groups
 

of animals.
 

4
 
C. Animals
 

Fischer 344 rats were obtained from A. R. Schmidt, Madison,
 

Wisconsin, and B6C3F1 mice from Charles River Breeding
 

Laboratories, Inc., Wilmington, Massachusetts, at 28 days of age.
 

These laboratories were under contract with the Division of
 

Cancer Treatment, National Cancer Institute, to provide the
 

animals used for testing. On arrival at the laboratory, the
 

animals were quarantined for 14 days. The animals were
 

considered acceptable for testing if they had no clinical signs
 

of disease and were within a weight range of 19-22 g for mice and
 

85-110 g for rats at the end of the period of quarantine. Cage
 

assignments were made by grouping the animals by weight and
 

choosing one animal from each group so that the total weight of
 

animals in each cage was approximately the same.
 

D. Animal Maintenance
 

Animals were housed in temperature- and humidity-controlled
 

rooms. The temperature was maintained at 21-25°C and the
 

relative humidity at 45-55%. Room air was changed 15 times per
 

hour and passed through both intake and exhaust HEPA (High
 

Efficiency Particulate Air) filters. The animal rooms were
 

positively pressurized with respect to the exit hall and negatively pressurized with respect to the entrance hall. Cool white
 

5
 
fluorescent lighting was provided 8 hours per day. Test and
 

control diets were available ad libitum; feed hoppers were
 

replenished three times per week. Tap water, acidified to pH
 

2.5, was also available ad libitum.
 

Rats were housed four per cage and mice five per cage in solid
 

polycarbonate cages. Each cage was covered with a wire mesh
 

screen and a sheet of filter paper. Heat-treated hardwood chip
 

bedding (Absorb-Dri®, Lab Products, Garfield, N.J.) was used in
 

the cages. Cages and water bottles were sanitized twice per week
 

and feed hoppers once per week at approximately 82°C; bedding was
 

replaced twice per week.
 

Rats and mice were housed in separate rooms. Control animals and
 

dosed animals were housed in the same room. Animals fed
 

N-phenyl-p-phenylenediamine were housed in the same room as
 

animals of the same species being fed the following chemicals:
 

e»
 

RATS
 

N-9H-fluoren-2-ylacetamide (CAS 53-96-3)
 

4,4'-diisocyanato-3,3'-dimethoxy-l,l'-biphenyl (CAS 91-93-0)
 

N,N'-bis(carboxymethyl)glycine (NTA) (CAS 139-13-9)
 

MICE
 

4,4'-methylenebis(N,N'-dimethyl)benzenamine (CAS 101-61-1)
 

4,4'-bis(dimethylamino)benzophenone (CAS 90-94-8)
 

phenylthiourea (CAS 103-85-5)
 

bis(acetyloxy)dibutylstannane (CAS 1067-33-0)
 

N,N,N'-trimethylthiourea (CAS 2489-77-2)
 

4-chlorobenzenamine (CAS 106-47-8)
 

6
 
3-chloro-4-methylbenzenamine (CAS 95-74-9)
 

5-chloro-o-toluidine (CAS 95-79-4)
 

2-nitro-l,4-benzenediamine (CAS 5307-14-2)
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted to estimate the maximum
 

tolerated doses of N-phenyl-p-phenylenediamine, on the basis of
 

which low and high concentrations (hereinafter referred to as
 

"low doses" and "high doses") were determined for use in the
 

chronic studies. In the Subchronic studies, N-phenyl-p-phenylenediamine was administered to rats at concentrations in the feed
 

of 2,200, 3,200, 4,600, 6,800, or 10,000 ppm, and to mice at
 

concentrations of 3,000, 4,400, 6,500, 9,500, 14,700, 21,600, or
 

31,500 ppm. Feed containing the test chemical was provided to
 

dosed groups of five male and five female animals of each
 

species, and feed not containing the test chemical was similarly
 

provided to control groups of five male and five female animals
 

of each species. Dosed animals received the test diets for 7
 

weeks and basal diets for the final week of the studies. All
 

animals were killed and necropsied at week 8.
 

At 2,200 ppm, the mean body weight gain in the male rats after 7
 

weeks was 72% of that of the controls, and in the females, 50% of
 

that of the controls. Mean body weight gains were progressively
 

lower at higher doses. Deaths occurred in three males and two
 

7 
females at 6,800 ppm and in four males and four females at 10,000
 

ppm. The low and high doses for the chronic studies using rats
 

were set at 600 and 1,200 ppm.
 

Mice administered the chemical at doses of 3,000 to 9,500 ppm did
 

not show any effects. When groups were restarted at doses of
 

14,700 21,600, or 31,500 ppm, the male mice still showed no
 

effects; however, mean body weight gains were depressed in the
 

female mice. The low and high doses for the chronic studies
 

using male mice were set at 2,500 and 5,000 ppm; using females,
 

they were set at 5,000 and 10,000 ppm.
 

F. Designs of Chronic Studies
 

The designs of the chronic studies are shown in tables 1 and 2.
 

By week 31 of the study, signs of central nervous system toxicity
 

occurred in the male and female mice administered either the low
 

or high initial dose. Consequently, the doses were reduced by
 

1/2 in the low- and high-dose male mice, discontinued in the
 

high-dose females, and reduced by 1/4 in the low-dose females.
 

At week 39, the high-dose females were given diets containing
 

2,500 ppm. At week 48, all mice were placed on basal diets until
 

termination of the period of observation at week 91.
 

8 
Table 1. Design of Chronic Studies of N-phenyl-p-phenylenediamine
 

in Rats
 

N-phenyl-pphenyleneSex and Initial diamine Time on Study 

Test No. of in Diet Dosed Observed 

Group Animalsa (ppm)b (weeks) (weeks) 

Male 

Control 20 0 104 

Low-Dose 50 600 78 26 

High -Dose 50 1,200 78 26 

Female 

Control 20 0 104 

Low-Dose 50 600 78 26 

High-Dose 50 1,200 78 26 

a

All animals were approximately 42 days of age when placed on
 

study.
 

b

Test and control diets were available ad libitum 7 days per week.
 

9 
Table 2. Design of Chronic Studies of N-phenyl-p-phenylenediamine
 

in Mice
 

Sex and
 

Test
 

Group
 

Male
 

Control
 

Low-Dose
 

High-Dose
 

Female
 

Control
 

Low-Dose
 

High-Dose
 

Initial


No. of


Animals3


20


50


50


20


50


50


N-phenyl-pphenylene­

 diamine
 

 in Diet
 

 (ppm)b
 

0
 

 2,500
 

1,250
 

0
 

 5,000
 

2,500
 

0
 

0
 

 5,000
 

1,250
 

0
 

 10,000
 

0
 

2,500
 

0
 

Time on Study
 

Dosed Observed
 

(weeks) (weeks )
 

91
 

31
 

17
 

43
 

31
 

17
 

43
 

91
 

31
 

17
 

43
 

31
 

7
 

10
 

43
 

Time-Weighted
 

Average Dosec
 

(ppm)
 

2,057
 

4,114
 

3,672
 

8,170
 

a

Animals were approximately 42 days of age when placed on study.
 

^Test and control diets were available ad libitum 7 days per week.
 

c

Time-weighted average dose - £(dose in ppm x no. of weeks at that dose)
 

Z(no. of weeks receiving each dose)
 

10
 
G. Clinical and Pathological Examinations
 

All animals were observed twice daily for signs of toxicity, and
 

animals that were moribund were killed and necropsied. Rats and
 

mice were weighed individually at regular intervals. Palpation
 

for masses was carried out at each weighing.
 

The pathologic evaluation consisted of gross and microscopic
 

examination of all major tissues, organs, or gross lesions from
 

killed animals and from animals found dead. The following
 

tissues and organs were routinely subjected to microscopic
 

examination: skin, lymph nodes, mammary gland, salivary gland,
 

bone marrow, trachea, lungs and bronchi, heart, thyroid, parathyroid, esophagus, stomach, small intestine, large intestine,
 

liver, gallbladder (mice), pancreas, spleen, kidney, adrenal,
 

urinary bladder, prostate or uterus, testis or ovary, brain, and
 

pituitary. The tissues were preserved in 10% buffered formalin,
 

embedded in paraffin, sectioned, and stained with hematoxylin and
 

eosin.
 

A few tissues from some animals were not examined, particularly
 

from those animals that died early. Also, some animals were
 

missing, cannibalized, or judged to be in such an advanced state
 

of autolysis as to preclude histopathologic evaluation. Thus,
 

the number of animals from which particular organs or tissues
 

11
 
were examined microscopically varies, and does not necessarily
 

represent the number of animals that were placed on study 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
 

12
 
a possible dose-related effect on survival used the method of Cox
 

(1972) for testing two groups for equality and Tarone's (1975)
 

extensions of Cox's methods for testing for 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 is 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) was used to compare the tumor incidence of
 

a control group with that of a group of dosed animals at each
 

dose level. When results for a number of dosed groups (k) are
 

13
 
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) requires that the
 

P value for any comparison be less than or equal to 0.05/k. In
 

cases where this correction was used, it is discussed in the
 

narrative section. It is not, however, presented in the tables,
 

where the Fisher exact P values are shown.
 

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

continuity correction (Armitage, 1971), was also used. Under the
 

assumption of a linear trend, this test determines 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 is 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
 

14
 
the first tumor was found. Once this reduced set of data was
 

obtained, the stcindard procedures for analyses of the incidence
 

of tumors (Fisher exact tests, Cochran-Armitage tests, etc.) were
 

followed.
 

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

incidence of tumors. Curves of the proportions surviving without
 

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

The week during which an animal died naturally or was 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 with its control was calculated
 

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

relative risk is defined as pt/pc where pt is the true binomial
 

probability of the incidence of a specific type of tumor in a
 

dosed group of animals and pc is the true probability of the
 

spontaneous incidence of the same type of tumor in a control
 

15
 
group. The hypothesis of equality between the true proportion of
 

a specific tumor in a dosed 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
 

dosed 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% of a large number of identical experiments, the true
 

ratio of the risk in a dosed 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 (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.
 

16
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

The mean body weights of the dosed male and female rats were
 

slighty lower than those of the corresponding matched controls
 

(figure 1). Fluctuation in the growth curve may be due to
 

mortality; as the size of a group diminishes, the mean body
 

weight may be subject to variation. No other clinical signs were
 

recorded.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats fed N-phenyl-p-phenylenediamine
 

in the diet at the doses of this bioassay, together with those of
 

the matched controls, are shown in figure 2.
 

The result of the Tarone test for positive dose-related trend in
 

mortality is not significant in either sex. At least 84% of the
 

males (42/50 of the high-dose group, 42/50 of the low-dose group,
 

and 18/20 of the. control group) and over 85% of the females
 

(46/50 of the high-dose group, 43/50 of the low-dose group, and
 

18/20 of the control group) were alive at week 104. Sufficient
 

numbers of rats of each sex were at risk for development of
 

late-appearing tumors.
 

17
 
TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves For Rats Fed N-Phenyl-p-Phenylenediamine in The Diet 

18 
TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves For Rats Fed N-Phenyl-p-Phenylenediamine in The Diet 

19 
C. Pathology (Rats)
 

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 tumors of the integumentary system were observed in
 

the dosed rats, but none were observed in the controls. These
 

and the remaining tumors that were observed in the dosed and
 

control rats were considered to be spontaneous; most of them
 

occurred at incidences that were to be expected for this age
 

group of the Fischer 344 rat. They included testicular
 

interstitial-cell tumors in the males and pituitary chromophobe
 

adenomas in the females. Although no chromophobe adenomas or
 

C-cell tumors of the thyroid glands occurred in the control male
 

rats, the numbers of these relatively common tumors for aging
 

rats that were in the dosed animals did not exceed expected
 

incidences. There was a relatively low incidence of leukemias in
 

both male and female rats. Most were compatible with leukemia
 

(undifferentiated) as usually observed in the Fischer 344 rat, or
 

were unclassifiable and occurred with approximately equal
 

frequency in dosed and control groups.
 

A variety of degenerative and inflammatory lesions of the type
 

usually encountered in aged Fischer 344 rats were observed, but
 

20
 
none was considered to be attributable to administration of the
 

test chemical.
 

Based on the histopathologic examination, there was no evidence
 

for the carcinogenicity of N-phenyl-p-phenylenediamine in rats
 

under the conditions of this bioassay.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

least two animals in one group and with an incidence of at least
 

5% in one or more than one group.
 

The results of the Cochran-Armitage test for positive dose-


related trend and of the Fisher exact test for direct comparison
 

of the incidences in dosed and control groups are not significant
 

in either sex. In the females, a significant trend in the
 

negative direction is observed in the incidence of fibroadenoma
 

of the mammary gland; however, this negative significance cannot
 

be accounted for by differences in survival in the dosed and
 

control groups.
 

In each of the 9.5% confidence intervals of relative risk, shown
 

in the tables, the value of one is included; this indicates the
 

absence of significant positive results. It should also be noted
 

21
 
that each of the intervals has an upper limit greater than one,
 

indicating the theoretical possibility of the induction of tumors
 

by N-phenyl-p-phenylenediamine, which could not be detected under
 

the conditions of this test.
 

22
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

The mean body weights of the dosed male mice were lower than
 

those of the matched controls throughout the bioassay, and those
 

of the high-dose animals were slightly lower than those of the
 

low-dose animals (figure 3). The mean body weights of the dosed
 

female mice also were lower than those of the matched controls,
 

but those of the low- and high-dose groups did not differ from
 

one another. Fluctuation in the growth curve may be due to
 

mortality; as the size of a group diminishes, the mean body
 

weight may be subject to variation. Clinical signs of effects on
 

the central nervous system were reported in the dosed mice; these
 

signs were not, however, further described.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice fed N-phenyl-p-phenylenediamine
 

in the diet at the doses of this bioassay, together with those of
 

the matched controls, are shown in figure 4.
 

In the male mice, the result of the Tarone test for positive
 

dose-related trend in mortality is not significant. At least 85%
 

of the males from each group (44/50 of the high-dose group, 46/49
 

23
 
TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves For Mice Fed N-Phenyl-p-Phenylenediamine in The Diet 

24 
TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves For Mice Fed N-Phenyl-p-Phenylenediamine in The Diet 

25 
of the low-dose group, and 17/20 of the control group) lived to
 

the end of the study. In the females, the result of the Tarone
 

test is significant (P = 0.021), with 29/50 (58%) of the high-


dose group, 34/50 (68%) of the low-dose group, and 17/20 (85%) of
 

the matched controls surviving to the termination of the study.
 

Approximately 25% of the low-dose and 40% of the high-dose female
 

mice died with signs indicative of a central nervous system (CNS)
 

disturbance. Deaths occurred from week 8 to 46, with the
 

majority occurring at about 30 weeks after the start of the
 

study. Sufficient numbers of mice of each sex were at risk for
 

development of late-appearing tumors.
 

C. Pathology (Mice)
 

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 significant morbidity and mortality occurred in the dosed
 

female mice; this was attributed to administration of the
 

N-phenyl-p-phenylenediamine. No pathological changes were
 

evident, however, in the CNS or elsewhere to account for the
 

toxic manifestations.
 

The incidence of hepatocellular neoplasms in the dosed groups of
 

26
 
male mice exceeded that in the controls, as shown in the
 

following table:
 

MICE 

Male Female 

Matched Low High Matched Low High 

Control Dose Dose Control Dose Dose 

Number of animals with
 

tissues examined
 

microscopically 20 49 50 20 49 48
 

Hepatocellular
 

carcinoma 2(10%) 6(12%) 5(10%) 0(0%) 0(0%) 0(0%)
 

Hepatocellular adenoma
 

or carcinoma 2(10%) 18(37%) 10(20%) 1(5%) 2(4%) 1(2%)
 

One unusual feature was the number of tumors interpreted as
 

adenomas. These usually occurred as single, discrete nodules of
 

enlarged cells that were either vacuolated or contained densely
 

eosinophilic cytoplasm. The cells that made up the nodules
 

formed sheets or fairly regular plates which compressed surrounding normal hepatic parenchyma. The hepatic tumors interpreted as hepatocellular carcinomas were mostly larger versions
 

of the adenomas and were adjudged carcinomas mainly on the basis
 

of their size, although several had irregular trabecular structures and glandular patterns.
 

Inflammatory and other lesions that were considered to be
 

significant and possibly related to administration of the test
 

27
 
chemical occurred in the livers of the dosed male mice, as shown
 

in the following table:
 

Male Mice 

Matched Low High 

Control Dose Dose 

Number of animals with tissues
 

examined microscopically 20 49 50
 

Inflammation, focal 0(0%) 23(47%) 24(48%)
 

Hepatocellular hyperplasia 0(0%) 0(0%) 2(4%)
 

Bile duct hyperplasia 0(0%) 2(4%) 3(6%)
 

Hepatocytomegaly 0(0%) 0(0%) 4(8%)
 

Although it is not unusual to see some mild chronic inflammation
 

in the livers of aged B6C3F1 mice, the inflammatory changes in
 

these animals were often extensive and consisted of mononuclear
 

cells occurring as foci within the parenchyma and infiltrates in
 

portal triads. These were accompanied by a proliferation of
 

Kupffer's cells and increased numbers of megalocytes. A few of
 

these livers had hyperplastic bile ducts. The changes occurred
 

in many of the livers that also had hepatocellular neoplasms.
 

The hepatocellular hyperplasia noted in the two high-dose male
 

mice were lobular proliferations of hepatocytes that did not form
 

true nodules. Hepatocellular giant cells were multinucleated and
 

often contained from 10 to 20 small nuclei.
 

Based on the histopathologic examination, there was no conclusive
 

28
 
evidence for the carcinogenicity of N-phenyl-p-phenylenediamine
 

in mice under the conditions of this bioassay.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

least two animals in one group and with an incidence of at least
 

5% in one or more than one group.
 

In male mice, the result of the Cochran-Armitage test for
 

positive dose-related trend in the combined incidence of hepatocellular adenomas or carcinomas is not significant; however, an
 

indicated departure from linear trend is observed (P = 0.010),
 

because the incidence in the low-dose group is higher than that
 

in the high-dose group* The results of the Fisher exact test
 

show that the incidence in the low-dose group is significantly
 

higher (P = 0.022) than that in the matched controls, but a
 

significant incidence is not indicated in the high-dose group.
 

In female mice, a significant incidence of lymphoma is observed
 

in the negative direction; this may be accounted for by the
 

higher mortality in the dosed groups of animals than in the
 

control group.
 

In each of the 95% confidence intervals of relative risk, sliown
 

29
 
in the tables, except that for hepatocellular adenoma or
 

carcinoma of the liver in the low-dose male mice, the value of
 

one or less than one is included; this indicates the absence of
 

significant results. It should also be noted that each of the
 

intervals (except that for the incidence of lymphoma in the
 

low-dose female mice) has an upper limit greater than one,
 

indicating the theoretical possibility of the induction of tumors
 

by N-phenyl-p-phenylenediamine, which could not be detected under
 

the conditions of this test.
 

30
 
V. DISCUSSION
 

Under the conditions of this bioassay, N-phenyl-p-phenylenediamine had no appreciable toxic effect on Fischer 344 rats, but
 

it was quite toxic to the mice at the doses administered. The
 

doses for the mice in the chronic studies were initially set
 

higher than those for the rats, because little toxicity was
 

observed in the mice in the subchronic studies. In the chronic
 

studies, however, the original doses of N-phenyl-p-phenylenediamine were toxic in the mice; therefore, the doses used in the
 

chronic studies were lowered during the course of the bioassay,
 

and administration of the test chemical was terminated at 48
 

weeks. Sufficient numbers of rats and mice of each sex were at
 

risk for the development of late-appearing tumors; however, the
 

shortened period used for administering N-phenyl-p-phenylenediamine to the mice may not have been adequate for determining
 

the carcinogenic potential of the test chemical in this species.
 

In the male and female rats, the incidences of neoplasms in the
 

dosed groups were not significantly different from those in the
 

corresponding control groups.
 

In the male mice, the incidence of combined hepatocellular
 

adenomas and carcinomas was significantly higher (P = 0.022) in
 

the low-dose group than in the controls, but there was no
 

31
 
significant dose-related trend, since the incidence in the
 

high-dose group was lower than that in the low-dose group
 

(controls 2/20, low-dose 18/49, high-dose ]0/50). Furthermore,
 

since at this laboratory the overall historical incidences of
 

these combined lesions in male mice have been 53/340 (15.6%) and
 

have been as high as 7/20 (35%), these neoplasms could not be
 

established as being compound related. No hepatocellular
 

carcinomas were observed in the dosed or control female mice, and
 

there were only two incidences of hepatocellular adenomas in the
 

low-dose females and one in the high-dose females, compared with
 

one in the controls. Unusually extensive hepatic inflammation
 

occurred in large numbers of the dosed males (controls 0/20,
 

low-dose 23/49, high-dose 24/50) and in lesser numbers of the
 

dosed females (controls 1/20, low-dose 8/49, high-dose 2/48).
 

The inflammation occurred in many of the livers in which hepatocellular neoplasms were also observed.
 

N-phenyl-p-phenylenediamine has been reported to have a low acute
 

oral LD5Q of 464 mg/kg body weight for rats (Naugatuck Chemicals,
 

1976). The chemical was not carcinogenic when administered to
 

strain A mice by subcutaneous injection for 14 or 16 months
 

(Shear and Stewart, 1941) or when administered orally to dogs for
 

6 years (Deichmann and Lampe, 1967). It was also negative for
 

mutagenicity in the Salmonella/microsome test (McCann et al.,
 

32
 
1975). Skin irritation and sensitization have been reported in
 

persons exposed to the chemical (Naugatuck Chemicals, 1976;
 

Greenberg and Lester, 1954).
 

It is concluded that under the conditions of this bioassay,
 

N-phenyl-p-phenylenediamine was not carcinogenic for Fischer 344
 

rats or for B6C3F1 mice.
 

33
 

VI. BIBLIOGRAPHY
 

Armitage, P., Statistical Methods in Medical Research, John Wiley
 

& Sons, Inc., New York, 1971, pp. 362-365.
 

Berenblum, I., ed., Carcinogenicity Testing; A Report of the
 

Panel on Carcinogenicity of the Cancer Research Commission
 

oj[ the UICC, Vol. 2^, International Union Against Cancer,
 

Geneva, 1969.
 

Colour Index, Vol 3^, The American Association of Textile Chemists
 

and Colorists, Lowell Technological Institute, Lowell,
 

Mass., 1956, pp. 3006, 3325, 3411, 3415, 3597, and 3762.
 

Cox, D. R., Regression models and life tables. J. R. Statist.
 

Soc. B 34(2);187-220, 1972.
 

Cox, D. R. , Analysis of Binary Data, Methuen & Co., Ltd., London,
 

1970, pp. 48-52.
 

Deichmann, W. B., Introduction, in Bladder Cancer - A Symposium,
 

Deichmann, W. B. and Lampe, K. F., eds. Aesculapius,
 

Birmingham, Ala. 1967, p. 3.
 

Gart, J. J., The comparison of proportions: a review of
 

significance tests, confidence limits and adjustments for
 

stratification. Rev. Int. Statist. 39(2):148-169, 1971.
 

Greenberg, L. A. and Lester, D., Handbook of Cosmetic Materials,
 

Interscience Publishers, Inc., New York, 1954, p. 44.
 

Greif, M. Division of Cosmetics Technology, Food & Drug
 

Administration. Personal Communication, 1977.
 

Kaplan, E. L. and Meier, P., Nonparametric estimation from
 

incomplete observations. J. Amer. Statist. Assoc.
 

53:457-481, 1958.
 

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

Peters, J. A., Carcinogenesis bioassay data system., Comp.
 

and Biomed. Res. 7:230-248, 1974.
 

McCann, J., Choi, E. , Yamasaki, E., and Ames, B. N., Detection of
 

carcinogens as mutagens in the Salmonella/microsome test:
 

Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA
 

12(12):5135-5139, 1975.
 

35
 
Miller, R. G., Jr., Simultaneous Statistical Inference,
 

McGraw-Hill Book Co., New York, 1966, pp. 6-10.
 

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

Kaufman, D. G., Respiratory tract carcinogenesis in hamsters
 

induced by different numbers of administrations of benzo(a)
 

pyrene and ferric oxide. Cancer Res. 32:1073-1081, 1972.
 

Shaver, F. W., Rubber chemicals. In: Kirk-Othmer Encyclopedia
 

of Chemical Technology, 2nd ed. Vol. 17, Standen, A., ed.,
 

Interscience Publishers, Inc., New York, 1968, pp. 529-530.
 

Shear and Stewart, 1941, In: Survey of Compounds Which Have Been
 

Tested for Carcinogenic Activity, Hartwell, J. L., ed.,
 

National Cancer Institute, National Institutes of Health,
 

Bethesda, Md., 1951.
 

Tarone, R. E., Tests for trend in life table analysis.
 

Biometrika 62(3);679-682. 1975.
 

Uniroyal Chemical, p-Aminodiphenylamine, Naugatack® Chemicals,
 

Division of Uniroyal, Inc., Naugatuck, Conn., 1976.
 

Wall, F. E. , Bleaches, Hair Colorings, and Dye Removers. In:
 

Cosmetics Science and Technology. 2nd ed. , Vol. 2, Balsam,
 

M. S., and Sagarin, E., eds., New York, Wiley-Interscience,
 

1972, pp. 306-308.
 

Weast, R. C., ed., CRC Handbook of Chemistry and Physics,
 

Cleveland, CRC Press, 1974, p. C-102.
 

36
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS FED N-PHENYL-p-PHENYLENEDIAMINE IN THE DIET
 

37
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMSIN MALE 

RATS FED N-PHENYL-p-PHENYLENEDIAMINE IN THE DIET 

MATCHED

CONTROL 

 LOW DOSE 

ANIMALS INITIALLY IN STUDY

ANIMAIS NECBCPSIED

ANIMALS EXAMINED HI STOP ATHOLOGICALLY

 20

 20

 20

 50 

 50 

 50 

INTEGUMENTARY SYSTEM
 

*SKIN (20) (50)
 

PAPILLOflA, NOS 1 (5%)
 

SCUAMOUS CELL CARCINOMA 1 (2%)
 

EASAL-CELL TUMOR 1 (2X)
 

FIBROMA
 

HEMANGIOSARCOMA 1 (2%)
 

*SOECDT TISSUE (20) (50)
 

SCUAMOUS CELL CA8CINOMA
 

EASAL-CELL TUMOR
 

FIBROMA
 

RESEIIATCEY SYSTEM
 

tLUNG (20) (50)
 

ALVEOLAR/ERONCHIOLAR ADENOMA 2 (10%)
 

AlVEOLAH/ERONCHIOLAR CARCINOMA
 

PIBROMA 1 (2X)
 

HEMATCEOIETIC SYSTEM
 

^MULTIPLE ORGANS (20) (50)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

IEUKEMIA.NOS 2 (10*) 1 (2%)
 

UNDIFFERENTIATED LEUKEMIA
 

LYMPHOCYTIC LEUKEMIA
 

GBANULOCYTIC LEUKEMIA 1 (5%) 1 (2X)
 

ClfCUIAICRY SYSTEM
 

NONE
 

t NUMBER CF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

50
 

50
 

50
 

(50)
 

1 (2%)
 

(50)
 

2 (U»)
 

1 (2%)
 

3 (6%)
 

(49)
 

3 (6%)
 

1 (2%)
 

(50)
 

1 (2%)
 

2 (H%)
 

1 (2%)
 

1 (2%)
 

39 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE
 

CONTROL
 

DIGESTIVE SYSTEM
 

#SALIVABY GLAND (17) (US)
 

ADENOMA, NOS
 

*SBALL INTESTINE (20) (50)
 

LEIOMYOMA 1 (2%)
 

UEINAEY SYSTEM
 

NONE
 

ENECCBINE SYSTEM
 

#EI1UITABY (20) (46)
 

CHBCMOEHCBE ADENOMA 3 (7X)
 

JAEBENAI (20) (50)
 

EHEOCHBOBOCYTOMA 1 (5%) 2 (H%)
 

4TBYBOID (20) (50)
 

C-CELL ADENOMA 1 (2*)
 

C-CELL CARCINOHA 1 (2X)
 

#PANCR£ATIC ISLETS (20) (48)
 

ISLET-CELL ADENOHA 3 (6«)
 

BEEFCEUCTIVE SYSTEB
 

*HAHBAEY GLAND (20) (50)
 

FIBBOADENOMA
 

*fEEEDTIAL GLAND (20) (50)
 

AEENOHA, NOS 1 (2X)
 

tTESTIS (20) (50)
 

INTEESTITIAL-CELL TUBOB 19 (95X) 46 (92X)
 

NEEVCDS SYSTEH
 

NONE
 

# SOBBEB CF ANIMALS BITH TISSUE EXAHINED HICHOSCOPICALLY
 

* NDMBEB OF ANIMALS NECBOPSIED
 

HIGH DOSE
 

(47)
 

1 (2X)
 

(50)
 

(48)
 

3 (6*)
 

(50)
 

3 (6%)
 

(50)
 

3 (6X)
 

(49)
 

(50)
 

1 (2%)
 

(50)
 

(49)
 

46 (94X)
 

40 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED

CONTROL 

 LOW DOSE HIGH DOSE 

SPECIAL SENSE C8GANS 

*EAE

SIBACEOOS ADENOCABCINOMA

 (20) (50)

 1 (2%) 

 (50) 

MUSCUIOSKELE1AL SYSTtM 

NONE 

BCEY CAVITIES 

NONE 

A ll C1HBE SYSTEMS 

NCNE 

ANIMA L DISECSI1ICN SUMMAR Y 

ANIMALS INITIALLY IN STUDY

NATURAL DEATHS

MORIBUND SACRIFICE

SCHEEULEE SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE

AMKAL MISSING 

 20

 2 

 18

 50

3 

6

 41

 50 

4 

4 

 42 

d) INCLUEES AUTOLYZKD ANIMALS 

# KUMEEE CF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NDBBEE OF ANIMALS NECBOPSIED 

41
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

TOMCR SUMMAR Y
 

TCTAL ANIMAL S WITH ERIMARY TUMORS*


TOTA1 PRIMARY TUMCRS


TCTAL ANIMALS WITH BENIGN TUMOES


TOTAL BENIGN TUMORS


TCTAL ANIMAL S WITH MALIGNANT TUMORS


TCTAL MALIGNANT TUMORS


TOTAL ANIMAL S WITH SECONDARY TUMORS*
 

TCTAL SECONDARY TUMORS
 

TCTAL ANIMALS WITH TUMORS UNCEFTAIN­ EENIGN OR MALIGNAN T
 

TOTAL UNCERTAIN TUMORS
 

TOTPL ANIMALS WITH TUMORS UNCERTAIN­ EEIMAEY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED LOW DOSE HIGH DOSE 

CONTROL 

 20 H8 49 

 26 65 73 

 20 46 U8 

 23 59 65 

3 6 8 

3' 6 8 

* EBIKAHY TUHOES: ALL TUMORS EXCEPT SECONDARY TUMORS
 * SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

42 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE 

RATS FED N-PHENYL-p-PHENYLENEDIAMINE IN THE DIET 

MATCHED

CONTROL 

 LOW DOSE 

ANIHALS INITIALLY IN STUDY

ANIMAIS NECBCFSIED

ANIKALS EXAMINED HISTOPATHOLOGICALLY

 20

 20

 20

 50 

 50 

 50 

INTEGUMENTARY SYSTEM
 

*SKIN (20) (50)
 

SQUAMOUS CELL CARCINOMA 1 (2%)
 

*SUECUT TISSUE (20) (50)
 

FIBBOADENCMA 1 (536) 1 (2%)
 

RESEIFATORY SYSTEM
 

#IUNG (19) (50)
 

ADENOCARCINOMA, NOS, METASTATIC 1 (2%)
 

ALVEOLAR/ERONCHIOLAR ADENOMA
 

AIVEOLAR/ERONCHIOLAR CARCINOMA
 

HEMATCECIETIC SYSTEM
 

*MUI1IPLE CRGANS (20) (50)
 

LEUKEMIA, NOS 2 (HI)
 

UNDIFFERENTIATED LEUKEMIA 3 (15%)
 

LYMFHOCYTIC LEUKEMIA
 

tSPLEEN (20) (50)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

NONE, . _ ...
 

# NUMEEE OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEEF OF ANIMALS NECROFSIED
 

HIGH DOSE
 

50
 

50
 

50
 

(50)
 

(50)
 

2 (U%)
 

(50)
 

2 (<*X)
 

1 (2%)
 

(50)
 

1 (2%)
 

2 <«X)
 

1 (2X)
 

(U9)
 

1 (2%)
 

43
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

-. — — — —. —i
 

MATCHED LOW DOSE HIGH DOSE
 

CONTROL
 

URINARY SYSTEM
 

NONE
 

ENECCBINE SYS1ZH
 

#EITUITAEY (20) («7) (46)
 

CHBOMCPHCBE ADENOMA 7 (35%) 14 (30%) 9 (20%)
 

CHEOHOPHOEE CARCINOMA 1 (2%)
 

tAERENSL (20) (48) (50)
 

PHEOCHEOMCCYTOMA 1 (5%) 2 (4%) 1 (2%)
 

•THYROID	 (19) (46) (49)
 

ADENOCABCINOMA, NOS 1 (2%)
 

C-CELL ADENOMA 2 (11%) 1 (2%) 1 (2%)
 

EEEECEUCIIVE SYSTEM
 

*HA«MAR Y GLAND (20) (50) (50)
 

LEIOMYOSA5COMA 1 (2%)
 

FIBROADENOMA 2 (10%)
 

*PB£PUTIAL GIANC (20) (50) (50)
 

ACENOMA, NOS 1 (2%) 2 (4%)
 

IUTEEUS (20) (49) (50)
 

ADENOCARCINOMA, NOS 1 (2%)
 

FIBEOMA 1 (5%)
 

ENDOHETRIAL STROHAL POLYP 2 (10«) 4 (8%) 4 (8%)
 

#CEPVIX UTERI (20) (49) (50)
 

LEICMYOSAHCOMA 1 (2%)
 

#CVARY (20) (49) (50)
 

IUTEOMA 1 (2%)
 

NEEVCUS SYSTEM
 

#EKAIN/MENINGES (20) (50) (48)
 

SQUABOUS CELL CARCINOMA, METASTA 1 (2X)
 

SPECIAL SENSE ORGANS
 

NONE
 

# NUMBE R CF AMHAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUKEE E CF ANIMAL S NECEOFSIE D 

44 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE 

CONTROL 

MUSCUICSKELETA L SYSTE M 

NONE 

BOD Y CAVITIE S 

NON E 

All OTHER SYSTEM S 

DON E 

ANIHA I DISPOSITION SUMHftR Y 

ANIMAL S INITIALLY IN STUDY 20 50 50 

NATURA L DEATH S 2 2 3 

MORIBUN D SACRIFICE 5 1 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINA L SACRIFICE 18 13 46 

AtilBA L HISSING 

3 INCLUDES AOTOLYZED ANIMAL S 

t JIOHBEF CF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY 

* NUHBE E OF ANIMAL S NECSOPSIED 

45 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE
 

CONTROL
 

TUBOE SUMMAR Y
 

TCTAL ANIHALS SI1H PRIMARY TUHORS* 13 26 2H
 

TCTAl PEIMAFY TUHCRS 19 31 28
 

TOTAL ANIMALS WITH BENIGN TUMORS 12 22 19
 

TOTAL BENIGN TUMORS 16 24 21
 

TCTAL ANIHALS WITH MALIGNANT TUMORS 3 6 7
 

TOTAL MALIGNANT TUMORS 3 7 7
 

TOTAL ANIMALS WITH SECONDARY TUMORS// 2
 

TOTAL SECONDARY TUMORS
 2
 

TOTAL ANIMALS HITH TUMOBS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUHORS
 

TOTAL ANIHALS HITH TUHORS UNCERTAIN­ EFIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* EBIMARY TUMORS: ALL TDHORS EXCEPT SECONDARY TUMORS
 * SECONDARY TUMORS: HETASTATIC TUMORS OR TUMORS INVASIVE IHTO AN ADJACENT ORGAN
 

46 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE FED M-PHENYL-p-PHENYLENEDIAMINE IN THE DIET
 

47
 

TABLE B1. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE 

MICE FED N-PHENYL-p-PHENYLENEDIAMINE IN THE DIET 

ANIBAIS INITIALLY IN STODY


ANIBAIS KECBCESIED


ANIBALS EXAMINED HISTOPATHOLOGICALLY


INTEGUMENTABY SYSTEM
 

NONH
 

EESEIEATCEY SYSTEM
 

#IUNG


HEFATOCELLULAR CABCINOMA, METAST


ALVEOLAR/BRONCHIOLAR ADENOMA


HEMATCECIETIC SYSTEM
 

*MU1TIELE ORGANS


MALIGNANT LYMPHOMA, NOS


MALIG.LYHPHOMA, LYMPHOCYTIC TYPE


MALIGNANT LYMPHOMA, MIXED TYPE
 

tLYMPH NODE


MALIGNANT LYMPHOMA, NOS


#MESENTERIC L. NODE


MALIG.LYHPHOMA, LYMPHOCYTIC TYPE
 

#S«AIL INTESTINE


MALIG. LYMPHOMA, LYMPHOCYTIC TYPE
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVIE


HEFATOCEILULAB ADENOMA
 

MATCHED


CONTROL
 

 20
 

 20
 

 20
 

 (20)
 

1 (5%)
 

4 (2055)
 

 (20)
 

1 (5%)
 

1 (5%)
 

 (19)
 

1 (5%)
 

 (19)
 

 (20)
 

 (20)
 

ILOW DOSE HIGH DOSE
 

aso	 so
 

U9 50
 

49 50
 

(49) (50)
 

4 (8«) 5 (10*)
 

(49)	 (50)
 

1 (2%)
 

1 (2»)
 

(48)	 (47)
 

1 (2%)
 

(48)	 (47)
 

1 (2%)
 

(48)	 (50)
 

1 (2%)
 

(49)	 (50)
 

12 <24%1 5 (10K)
 

t	 NUMEEE OF ANIMALS HITH TISSDE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMALS NECROPSIED
 

3) 50 ANIMALS HERE INITIALLY IN THE STODY, BUT ONE ANIBAL HAS DELETED WHEN FOUND
 

TO BE A FEMALE IN A MALE GROUP.
 

49
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED

CONTROL 

 LOW DOSE HIGH DOSE 

HEPATOCELLULAR CAKCINOMA

HEMANGIOSABCOMA

 2 (10X)

 1 (5*) 

6 (12X) 5 (10*) 

USIKAEY SYSTEM 

NONE 

ENICCFINE SYSTEM 

NONE 

PEEECCUCTIVE SYSTEM 

*TES1IS

INTEESTITIAL-CELL TOMOK

 (19) (48)

 1 (2%) 

 (50) 

NEFVCUS SYSTEM 

NONE 

SEKIAL SENSE ORGANS 

KCNE 

HUSCUICSKEIETAL SYSTEM 

NONE 

ECtY CAVITIES 

*MEEIASTIfJUM

EIBEOSAHCOMA

*AECCMINAL CAVITY

IIPOMA

 (20)

 (20)

 1 (5%) 

 (H9)

 1 (2%) 

 (49)

 (50) 

 (50) 

All CTHEB SYSTEMS 

NONE 

# NUMBER CF ANIMAIS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBSK CF ANIMALS NECROPSIED 

50 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE
 

CONTROL
 

ANIMAL EISPCSITICN SUMMARY
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

NATURAL DEATHS 3 3 5
 

MORIBUND SACRIFICE 1
 

SCHEEOLEC SACRIFICE
 

ACCIDENTALLY KILLED
 

TEPHINAL SACRIFICE 17 46 44
 

ANIMAL MISSING
 

ANIMAL DELETED (HRONG SEX) 1
 

3) INCIODES AUTOLYZED ANIHALS
 

TUMOE SUHMABY
 

TOTAL ANIMALS WITH PBIMAEY TUMORS* 10 25 15
 

TCTAL PRIMARY TUMORS 11 26 18
 

TOTAL ANIMALS WITH BENIGN TUMORS 5 17 9
 

TOTAL BENIGN TUMORS 5 17 10
 

TCTAL ANIHALS HITH MALIGNANT TUMORS 6 9 8
 

TCTAL MALIGNANT TDMORS 6 9 8
 

TOTAL ANIMALS WITH SECONDARY TUMORS! 1
 

TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TCTAL UNCERTAIN TUMORS
 

TOTAL ANIHALS WITH TUMORS UNCEHTAIN­ EEIMABY OB METASTATIC
 

TOTAL UNCERTAIN TUHORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TOMORS
 

t SECONDARY TUMORS: HETASTATIC TUMORS OB TDHORS INVASIVE IHTO AN ADJACENT ORGAN
 

51 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE 

MICE FED N-PHENYL-p-PHENYLENEDIAMINE IN THE DIET

 

HIGH DOSE
 

50
 

1
 

49
 

49
 

(49)
 

(49)
 

1 (2X)
 

(47)
 

(48)
 

(42)
 

1 (2X)
 

(48)
 

(t8)
 

1 (2%)
 

ANIMALS INITIALLY IN STUDY


ANIMAL S MISSING
 

ANIMALS NECBOPSIED


ANIMALS EXAMINED HISTOPATHOLOGICALLY


INTIGUBENTAEY SYSTEM
 

*SUBCUT TISSUE


SARCOMA, NOS
 

BESIIEATCEY SYSTEM
 

NONE
 

HEMATCFCIETIC SYSTEM
 

*BUITIELE CEGANS


MALIGNANT LYBPHCBA, NOS


BALIG.LYBPHOMA, LYBPHOCYTIC TYPE
 

#SPLEEN


MALIG.LYflPHOBA, LYBPHOCYTIC TYPE


#1IVER


MALIGNANT LYMPHOMA, NOS


#SBAIL INTESTINE


MALIG.LYMPHOBA, LYBPHOCYTIC TYPE
 

fKIDNEY


BALIGNANT LYBPHOBA, NOS


CIFCUIATCEY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVEE


HEPATOCEILULAB ADENOMA


MATCHED LOW DOSE
 

CONTROL
 

 20	 50
 

 20 50
 

 20 50
 

 (20)	 (50)
 

1 (2X)
 

 (20)	 (50)
 

1 (5X)
 

 (19)	 0»3)
 

1 (5S)
 

 (20)	 C»9)
 

1 (5%)
 

 (20) (50)
 

 (20)	 (50)
 

1 (5%)
 

 (20)	 (49)
 

1 15%) „ 2 J4X)
 

I	 NDMEEB OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALLY
 

*	 NDHBEB OF ANIMALS NECROPSIED
 

52
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE
 

CONTROL
 

EBMANGIOSARCOHA 1 (2%)
 

UEINAEY SYS1EH
 

NONE
 

ENICCEINI SYS1IM
 

NONE
 

REEECEUCTIVE SYSTEM
 

NONE
 

NIEVCUS SYSTEM
 

NONE
 

SPEC1JI SEKSI CBGAN S
 

NONE
 

MUSCULCSKELE1AL SYSTEM
 

NCNE
 

ECEY CSVITIES
 

NONE
 

ALL CTHEF SYSTEHS
 

NONE
 

t NOHBEB CF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUHEEB CF ANIMALS NECROPSIED
 

53
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE
 

CONTROL
 

ANIMA L DISPOSITION SUHHARY 

ANIMALS INITIALLY IN STUDY 20 50 50 

NATURA L DEATHS 2 1 H 20 

MORIBUND SACRIFICE 1 2 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 17 34 29 

ANIEAL MISSING 1

3 INCLUDES AUTOLYZED ANIMALS 

TUMCB SUMMAR Y 

TOTAL ANIMALS HITH PRIMAR 5 Y TOKOBS* 4 2 

TOTAL PRIMARY TUMCPS 5 4 3 

TOTAL ANIMALS WITH EENIGN TUMORS 1 2 1 

TOTAL BENIGN TUMORS 1 2 1 

TOTAL ANIMALS WITH MALIGNAN 4 T TUMORS 2 2 

TOTAL MALIGNAN T TUMORS 4 2 2 

TOTAL ANIMALS WITH SECONDARY TUMOBS* 

TOTAL SECONDARY TOMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OS MALIGNANT 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINEBIMARY OR METASTATIC 

TOTAL UNCERTAIN TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

f SICCNEARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

54
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED N-PHENYL-p-PHENYLENEDIAMINE IN THE DIET
 

55
 

TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

FED N-PHENYL-p-PHENYLENEDIAMINEIN THE DIET 

MATCHED LOW DOSE HIGH DOSE
 

CONTROL
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

ANIHAIS HECECPSIED 20 50 50
 

ANIMALS EXAMINED HISTOPATHCLOGICALLY 20 50 50
 

INTEGUMENTARY SYSTEM
 

*SKIN (20) (50) (50)
 

HYPERKERATOSIS 1 (2«)
 

*SUECUT TISSUE (.20) (50) (50)
 

ABSCESS, NOS 1 (2%)
 

BESEIEATCBY SYSTEM »
 

#I.UNG/BRONCHUS (20) (50) (49)
 

INFLAMMATION, CHRONIC 3 (6%)
 

#LUNG (20) (50) (49)
 

CONGESTION, NOS 2 (10%) 1 (2%) 1 (2*)
 

HEMORRHAGE 1 (5%) 1 (2%)
 

PNEUMONIA, CHRONIC MURINE 5 (25X) 12 (24X) 16 (33X)
 

PNEUMONIA INTERSTITIAL CHRONIC 1 (5«)
 

INFLAMMATION, FOCAL GRANULOMATOU 1 (2%)
 

FOAM-CELL 1 (5X)
 

HYFERPLASIA, LYMPHOID 1 (2%)
 

HEBATCEOIETIC SYSTEM
 

*EONE MARRO W (17) («5) (48)
 

HYEIOSCLEROSIS 1 (2%)
 

HYPERPLASIA, GRANOLOCYTIC 2 (12S) 1 (2«)
 

tSPLEEN (20) (50) (50)
 

CONGESTION, NOS 1 (2%)
 

FIEROSIS, FOCAL 1 (2X)
 

FIBEOSIS, MULTIFOCAL 1 (2X)
 

HEMOSIDEHOSIS 1 I5&I
 

* NUMEEB OF ANIMALS WITH TISSDE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

57 
TABLE C1. MALE RATS:NONNEOPLASTIC LESIONS (CONTINUED) 

HIGH DOSE
 

(49)
 

(49)
 

2 (455)
 

(«9)
 

1 (255)
 

1 (2»)
 

(49)
 

6 (1255)
 

1 (2X)
 

(47)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

(47)
 

(50)
 

2 (4*)
 

(49)
 

(50)
 

(50)
 

hEKATOPOIESIS
 

#LYMPH NODE
 

HYPEPFLASIA, LYMPHOID
 

#BANDIEULAB L. NODE
 

INFLAMMATION, CHRONIC
 

HYPEEPLASIA, PLASMA CELL
 

&MESENTERIC L. NODE
 

CONGESTION, CHBONIC
 

EEEMA, NOS
 

HYPEPPLASIA, BETICULOM CELL
 

HYPEEPLASIA, LYMPHOID
 

CIFCUIATCBY SYSTEH
 

#MYCCARDIOH
 

FIBBOSIS
 

EEGENEBATION, NOS
 

DIGESTIVE SYSTEM
 

#IIVER
 

INFLAMMATION, CHBONIC
 

NECROSIS, FOCAL
 

METAMOBEHOSIS FATTY
 

EASOPHILIC CYTO CHANGE
 

HYP2RPLASIA, NODDLAB
 

HYPEEPLASIA, FOCAL
 

#LIVEB/CENTBILOBULAR
 

EEGENEBATION, NOS
 

*BIIE DUCT
 HYPEEPLASIA, NOS
 

tPANCREAS
 

ATBOPHY, FOCAL
 

tSTOMACH
 

GEANULOMA, NOS
 

#SHALL INTESTINE
 

INFLAMMATION, SUPPUBATIVE
 

MATCHED
 

CONTROL
 

1 (5»)
 

(19)
 

(19)
 

1 (5%)
 

(19)
 

1 (5X)
 

(19)
 

2 (11%)
 

2 (1155)
 

(20)
 

2 (10X)
 

1 (5S)
 

(20)
 

(20)
 

2 (10X)
 

(20)
 

1 (5X)
 

(20)
 

(20)
 

LOW DOSE
 

(49)
 

1 (2%)
 

(49)
 

(49)
 

1 (2%)
 

1 (2%)
 

(48)
 

2 (U»)
 

2 (4X)
 

(50)
 

1 (2%)
 

1 (2X)
 

2 (4%)
 

1 (2%)
 

(f,0)
 

1 (2%)
 

(50)
 

2 (4X)
 

(48)
 

(49)
 

1 (2*)
 

(50)
 

2 14%)
 

# NDMEEB OF ANIHALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* SDHBEB OF ANIMALS NECBOPSIED
 

58 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

HIGH DOSE
 

(50)
 

(50)
 

15 (30%)
 

(50)
 

(50)
 

1 (2%)
 

32 (64%)
 

(50)
 

1 (2%)
 

(50)
 

(50)
 

(50)
 

1 (2%)
 

(*»9)
 

1 (2%)
 

(50)
 

(49)
 

1 (2%)
 

HYPERPLASIA, LYMPHOID
 

#ILEOM
 

HYPEEPLASIA, LYMPHOID
 

#IARGE INTESTINE
 

NEBATODIASIS
 

#CCICN
 

LYMEHOCYTIC INFLAMMATORY INFILTE
 

UEItiAEY SYSTEM
 

tKIDNEY
 

CYST, NOS
 

PYELONEPHRITIS, NOS
 

INFLAMMATION, CHRONIC
 

tKIENEY/CCRTEX
 

CYST, NOS
 

ENECCPINE SYSTEM
 

#AEFENAL
 

HEMORBHAGIC CYST
 

LIPOIEOSIS
 

#AERENA1 CORTEX
 

HYEERPLAEIA, NODDLAR
 

#THYROID
 

HYFEEFLASIA, C-CELL
 

#PANCREATIC ISLETS
 

HYPEBPLASIA, NOS
 

HYFERPLASIA, FOCAL
 

BEEBCEUCTIVE SYSTEM
 

*SEBINAL VESICLE
 

ABSCESS, NOS
 

tTESTIS
 

RYPEEPLASIA^ INTERSTITIAL CELL
 

MATCHED
 

CONTROL
 

(20)
 

1 (5«)
 

(19)
 

8 (42S)
 

(19)
 

1 (5%)
 

(20)
 

13 (65%)
 

(20)
 

(20)
 

1 (5%)
 

(20)
 

(20)
 

1 (5*)
 

(20)
 

(20)
 

(20)
 

LOW DOSE
 

1 (2%)
 

(50)
 

1 (2X)
 

(48)
 

19 (U0«)
 

(1*8)
 

(50)
 

1 (2«)
 

19 (38%)
 

(50)
 

(50)
 

2 (UX)
 

(50)
 

1 (2%)
 

(50)
 

1 (2X)
 

(48)
 

1 (2X)
 

(50)
 

1 (2%)
 

(50)
 

f NUMBER OF ANIMALS HITH TISSUE EXAMINED MICEOSCOPICALLY
 

* NUMBER CF ANIMALS NECROPSIED
 

59
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED

CONTROL 

 LOW DOSE HIGH DOSE 

NEBVCUS SYSTEM 

#EBAIN

HYDROCEPHALUS, INTERNAL

 (20)

 1 (5%) 

 (48) (50) 

SEECIAl SENSE ORGANS 

NCNE 

MUSCUIOSKEIETAL SYSTEK 

NONE 

BCEY CAVITIES 

*EEEITONEOH

INFLAMMATION, FOCAL

 (20) (50) (50) 

1 (2%) 

All CTHEE SYSTEMS 

NONE 

SPECIAL HCEEhOLOGY SUKBAPY 

KCKE 

# NUMEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMEEB OF ANIMALS NECROPSIED 

60
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

FED N-PHENYL-p-PHENYLENEDIAMINE IN THE DIET 

MATCHED

CONTROL 

I.OW DOSE

ANIMALS INITIALLY IN STUDY 

ANI8AIS KECFCESIED 

ANIMALS EXAMINED HISTGPATHOLOGICALLY 

20 

20 

20 

50

50

50

INTEGUMENTARY SYSTEM
 

*SKIN (20) (50)


CYST, NOS
 

EFIEEEMAL INCLUSION CYST 1 (2X)
 

INFLAMMATION, CHRONIC 1 (5X)
 

*SUECUT TISSUE (20) (50)


CYST, NOS 1 (2X)
 

RESFIEATOHY SYSTEM
 

#1UNG/EECNCHUS (19) (50)


INFLAMMATION, CHRONIC
 

#LUNG (19) (50)


HEMOEEHAGE 1 (5X) 1 (2X)
 

PNEUMONIA, CHRONIC MURINE 7 (31%) 13 (26X)


INFLAMMATION, CHRONIC SUPPURATIV 1 (2X)
 

GRANULOMA, NOS 1 (556)
 

HEKATCEOIETIC SYSTEM
 

tSPLEEN (20) (50)


HYPERPLASIA, LYMPHOID 1 (5X)
 

HEMATOPOIESIS
 

*MANDIEOLAR L. NODE (20) (47)


HYPEBPLASIA, RETICULUM CELL 1 (5X) 1 <2X)
 

HYPERPLASIA, LYMPHOID 1 (2X)
 

#MESENTERIC L. NODE (20) (47)


LYBPHANGIECTASIS 1 (2X)
 

* NUBEEE OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECHOPSIED
 

 HIGH DOSE
 

 30
 

 50
 

 50
 

 (50)
 

2 (t»)
 

 (50)
 

 (50)
 

1 (2X)
 

 (50)
 

 12 (24X)
 

 (49)
 

1 (2X)
 

 (48)
 

 (48)
 

61 
TABLE C2. FEMALE RATS : NONNEOPLASTIC LESIONS (CONTINUED) 

HIGH DOSE
 

(47)
 

1 (2X)
 

(47)
 

1 (2X)
 

1 (2X)
 

2 (<*X)
 

2 (4X)
 

1 (2X)
 

(50)
 

2 (4X)
 

(48)
 

(48)
 

(50)
 

(50)
 

(50)
 

(50)
 

1 (2X1
 

EDEMA, NCS
 

DEGENERATION, CYSTIC
 

HYPERPLASIA, RETICULOM CELL
 

HYPEBPLASIA, LYHPHOID
 

CIBCUIA1CBY SYSTEM
 

IMYCCARDIUH
 

FIBBOSIS, DIFFUSE
 

DEGENERATION, NOS
 

DIGESTIVE SYSTEM
 

#LIVER
 

INFLAMMATION, ACUTE/CHRONIC
 

GRANULOHA, NOS
 

METAMORPHOSIS FATTY
 

CYTOPLASMIC VACUOLIZATION
 

BASOPHILIC CYTO CHANGE
 

HEPATOCYTOBEGALY
 

HYPERPLASIA, NODULAR
 

HYPERPLASIA, FOCAL
 

HYPERPLASIA, DIFFUSE
 

*EILE DUCT
 

HYP£EPLASIA, NOS
 

#PANCREAS
 

INFLAMMATION, HOS
 

tPANCREATIC ACINUS
 

ATROPHY. NOS
 

tSHALL INTESTINE
 

HYPEBPLASIA, LYMPHOID
 

*PEYERS PATCH
 

HYPEEPLASIA, LYBPHOID
 

#IIEOH
 

HYPERPLASIA, LYHPHOID
 

•1ARGE	 INTESTINE
 

UlCEB, NOS
 

MATCHED
 

CONTROL
 

1 (5*)
 

(20)
 

1 (5X)
 

1 (5X)
 

(20)
 

3 (15X)
 

3 (15X)
 

2 (10X)
 

(20)
 

2 (10%)
 

(20)
 

1 (5%)
 

(20)
 

(20)
 

1 (5X)
 

(20)
 

1 (5X)
 

(20)
 

1 (5X)
 

(20)
 

LOW DOSE
 

1 (2X)
 

1 (2X)
 

1 (2X)
 

1(2%)
 

(t5)
 

2 (4X)
 

(50)
 

1 (2X)
 

2 (UX)
 

1 (2X)
 

8 (16X)
 

1 (2X)
 

6 (12%)
 

1 (2X)
 

(50)
 

2 («X)
 

(50)
 

(50)
 

1 (2*)
 

0*8)
 

(t8)
 

1 (2*)
 

(48)
 

2 (4X)
 

(<I7)
 

.
 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

62 
TABLE C2. FEMALE RATS : NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE
 

CONTROL
 

NIMATODIASIS 7 (35%) 13 (28X) 15 (30X)
 

#C01CN (20) (47) (50)
 

HYPEBFLASIA, LYBPHOID 1 (5%)
 

UBINAF Y SYS1EM
 

*KIDNEY (20) (50) (50)
 

CYST, NOS 1 (2%)
 

INFLAMMATION, INTERSTITIAL 1 (2%)
 

INFLAMMATION, CHRONIC 6 (30%) 18 (36%) 11 (22%)
 

ENECCEINE SYSTEM
 

#PITUITARY (20) (47) (46)
 

CYST, NOS 1 (5%) 2 (4%) 1 (2%)
 

hEBOPRHAGIC CYST 1 (5%) 1 (2%) 3 (7%)
 

#AERENA L (20) (48) (50)
 

HEHOERHAGIC CYST 2 (10%) 1 (2X)
 

KETAMORPHOSIS FATTY 2 (4%)
 

tAERENA L CORTEX (20) (48) (50)
 

NECROSIS, NOS 1 (2%)
 

#TfiYHOID (19) (46) (49)
 

HYPEPFLAEIA, C-CELi 1 (2%)
 

REIfCDUCTIVE SYSTEM
 

*MAMRAR Y GIAND (20) (50) (50)
 

EILATATION/DUCTS 1 (5%) 1 (2%) 1 (2%)
 

CYSTIC DUCTS 1 (5%)
 

frUTERUS (20) (49) (50)
 

HYEEOMETRA 1 (2%)
 

IIiFLAKMATION, SUPPURATIVE 1 (5%) 2 (4%)
 

fCERVIX UTERI (20) (49) (50)
 

INFLAMMATION, NOS 1 (5%)
 

AESCESS, NOS 1 (5%)
 

# UTERUS/EN DO MET RIUM (20) (49) (50)
 

INFLAMMATION,. NOS 1 12%)
 

f NUMEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMEE R CF ANIMALS NECBOPSIED
 

63
 
TABLE C2. FEMALE RATS : NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL 

LOW DOSE 

INFLAMMATION, SUPPURATIVB 

HYPEFPLASIA, CYSTIC 1 (5K) 

1 (2X) 

#OVARY/OVIDUCT

INFLAMHATION, NOS 

 (20) (49) 

#OVARY

CYST. NOS

INFLAMMATION, NOS 

INFLAMMATION, SUPPURATIVE 

INFLAMMATION, NECROTIZING 

INFLAMMATION, CHRONIC SUPPURATIV 

 (20) 

5 (25%) 

(49) 

10

1

1

1

 (20«) 

 (2%) 

 (2%) 

 (2%) 

NEEVCDS SYSTEM
 

*ERAIN (20) (50)
 

HYDROCEPHALUS, NOS
 

HYDRCCEPhALUS, INTERNAL 1 (5%) 1 (2%)
 

SPECIAL SENSE ORGANS
 

NONE
 

MDSCUIOSKEIETAL SYSTEM
 

NONE
 

BCEY CAVITIFS
 

*ELEUPA (20) (50)
 

INFLAMMATION, CHRONIC
 

*EPICABDIOM (20) (50)
 

INFLAMMATION, NOS
 

ALL C1HEE SYSTEMS
 

*PUITIPLE ORGANS (20) (50)
 

PERIVASCULITIS
 1 12SL
 

# NUBEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NDHBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

1 (2%)
 

1 (2%)
 

(50)
 

3 (6%)
 

(50)
 

i» (8*)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

(48)
 

1 (2%)
 

(50)
 

1 (2%)
 

(50)
 

1 (2%)
 

(50)
 

64
 
TABL • m »Ul_lE_ C2 WK>1. FEMAL « M>»* «•-E « RAT«. I S : NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED

CONTROL 

 LOW DOSE HIGH DOSE 

SPECIAL HCHEHOLOG Y SUMBAB Y 

NO LESIO N EEFCPTED 7 

# NUMBER OF ANIHALS WITH TLSSUE EXAMINED MICROSCOPICALLY 

* NDKBEE CF ANIMALS NECROPSIED 

65 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE FED N-PHENYL-p-PHENYLENEDIAMINE IN THE DIET
 

67
 

TABLE 01. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

FED N-PHENYL-p-PHENYLENEDIAMINE IN THE DIET 

ANIMALS INITIALLY IN STUDY


ANIMALS NECECPSIED


ANIMJLS EXAMINED HISTOPATHOLOGICALLY


INTEGUMENTAR Y SYSTEM
 

*SKIN


EPIDERMAL INCLUSION CYST
 

RESEIEATCBY SYSTEM
 

tLONG/EECNCHIOLE


HYPERPLASIA, FOCAL


*LUNG


PNEUMONIA. ASPIRATION
 

PNEUMONIA, CHRONIC BURINE


HYPZRPLASIA, FOCAL
 

HEKATCECIETIC SYSTEM
 

#SPIEEN


INFARCT, NOS


#IYBPH NODE


HYPEFPLASIA, LYHPHOID
 

CIBCULATORY SYSTEM
 

#HEABT


EERIVASCULITIS
 

#MYOCAfiDIUM


INFLASBATION, CHBCINIC
 

DIGESTIVE SYSTEM
 

#IIVEB


INFLAHHATIOU. FOCAL
 

MATCHED LOW DOSE HIGH DOSE 

CONTROL 

 20 850 50 

 20 49 50 

 20 49 50 

 (20) (49) (50) 

1 (2X) 

 (20) (49) (50) 

1 (5«) 

 (20) (49) (50) 

1 (2%) 

1 (5X) 2 (4*) 5 (10*) 

1 (2X) 

 (20) (43) (46) 

1 (5X) 

 (19) (48) (47) 

1 (2X) 

 (18) (44) (47) 

1 (2X) 

 (18) (44) (47) 

1 (2%) 

 (20) (49) (50) 

23 i47X) 24 J48X}^ 

* SUMEER OF ANIMALS BITH TISSUE EXAHINED HICROSCOPICALLY
 

* SOBEEE OF ANIMALS NECBOPSIED
 

3 50 ANIHALS HERE INITIALLY IH THE STUDY, BUT OHE ANIHAL HAS DELETED HHEH FOUND
 

TO BE A FEMALE IN A RALE GROUP.
 

69 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED

CONTROL 

 LOW DOSE 

GEANDLOHA, NOS 

INFABCT, NOS 

HEPATOCYTOMBGALY 

CYTOLOGIC DEGENERATION 

HYPERPLASIA, NOS 

HYPEEPLASIA, FOCAL 

LIPOMATOSIS 

1 (5%) 

1 (2X) 

1 (2%) 

#1IVEE/CENTRILOBULAE 

CYTOLOGIC DEGENERATION 

(20) (49) 

*BIIE DOCT 

HYPERPLASIA, NOS 

(20) (U9) 

2 (4S) 

#PANCBEAS 

EILATATICN/DDCTS 

HEMORBHAGIC CYST 

(19) (45) 

1 (2X) 

1 (2%) 

fPANCREATIC ACINOS 

ATRCPHY, NOS 

(19) (45) 

1 (2%) 

#IARGE INTESTINE 

NEMATODIASIS 

(18)

5 (28%)

 (49) 

6 (12S) 

URINARY SYSTEM
 

#KIENEY (20) (49)
 

INFLAMMATION, FOCAL
 

INFLAMMATION, INTERSTITIAL 1 (2X)
 

CALCINOSIS, NOS
 

ENtCCIIKE SYSTEM
 

NONE
 

REJFCIUCTIVE SYSTEM
 

NONE
 

NEEVCUS SYSTEM
 

#ERAIN (20) (49)
 

HYDROCEPHALUSX INTERNAL
 

# NUMBER OF ANIMALS W-ITH TISSUE EXAMINED MICROSCOPICALLY
 

* NOMEEE OF ANIMALS NECROPSIED
 

HIGH DOSE
 

1 (2X)
 

4 (8X)
 

1 (2X)
 

1 (2X)
 

(50)
 

1 (2X)
 

(50)
 

3 (6K)
 

(42)
 

(42)
 

(48)
 

1 (2X)
 

(49)
 

1 (2X)
 

1 (2«)
 

1 (2%)
 

(48)
 

1 (2%Y
 

70 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED

CONTROL 

 LOW DOSE HIGH DOSE 

CORPORA ANY1ACEA 6 (30%) 15 (31X) 17 (35*) 

SEICIAL SEtiSE CBGANS 

NONE 

MUSCUIOSKEIETAL SYSTEM 

NCNI 

BCCY CAVITIES 

NCNJ 

ALI CTHEP SYSTEMS 

AEIEOSE TISSUE 

INFLAMMATION, NECROTIZING

INFLAMMATION, GR ANULOHATOUS

 1 

1 

SEECIA1 RCEFHOLOGY SUMMAR Y 

NO LESION REPORTED 3 4 

AUTC/NECRCPSY/HISTO PERF

# NUMEF R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMEE E CF ANIMAL S NECHOPSIED 

6 

1 

71
 
TABLE 02. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

FEDN-PHENYL-p-PHENYLENEDIAMINE IN THE DIET 

MATCHED

CONTROL 

 LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY

ANIMALS HISSING .

ANIHALS NECBCPSIED

ANIMALS EXAMINED HISTOPATHOLOGICALLY

 20

 20

 20

 50

 50

 50

 50 

1 

 49 

 49 

INTEGUMENTARY SYSTEM 

NONE 

EESIIfATCfY SYS1IM 

#IUNG

CONGESTION. NOS

HEHORBHAGE

INFLAMMATION, FOCAL

INFLAHBATION, MOLTIFOCAL

INFLAMMATION, INTERSTITIAL

PNEUMONIA, ASPIRATION

PNEOMONIA, CHRONIC MURINE

HYPEFPLASIA, ADENOMATOOS

 (20)

 1 (5%) 

1 (5X)

 1 (5X)

 (49)

 1 (2%)

 3 (6%)

 1 (2») 

2 (<**)

 1 (2X) 

 (49) 

1 (2X) 

1 (2J5) 

1 (2)1) 

2 (4*) 

6 (12X) 

HEBATCECIETIC SYSTEM 

#SPIEEN

HYPEEPLASIA, NOS

HYPEBPLASIA, LYMPHOID

 (19)

 1 (5X) 

 (43)

 1 (2X) 

 (47) 

tTHYMUS

DEGENERATION, HYALINE

 (1) (2) (2) 

1 (SOS) 

CIECDLATOEY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

tSALIVSEY GLAND

IYMPHOCYTIC INFLAHHATORY INFILTF

 (17) (36)

 1 13%} 

 (38) 

f NUHEER OF ANIMALS WITH TISSDE EXAMINED HICROSCOPICALLT 

* NOMBEB OF ANIMALS NECROPSIED 

72 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

tllVIE
 

MINEEALIZATION
 

CYST, NOS
 

INFLAMMATION, FOCAL
 

ttHVER/PESIPORTAL
 

LYMPHOCYTIC INFLAMMATORY INFILTR
 

tIARGE INTESTINE
 

NEMATODIASIS
 

UEINAE Y SYSTEM
 

#KIENEY
 

NEPHROSIS, NOS
 

#EERIEENAL TISSUE
 

HEMATOHA, ORGANIZED
 

ENECCEINE SYSTEM
 

*T£YROID
 

HYPEBPLASIA, FOLLICUiAR-CELL
 

REEECEUCTIVE SYSTEM
 

#UTEEUS
 

CYST, NOS
 

HEMORRHAGE
 

INFLAMMATION, SUPPU8ATIVE
 

#UT EROS/ ENDO MET RIUM
 

CYST, NOS
 

INFLAMMATION, NOS
 

#OVARY
 

CYST, NOS
 

NEEVCUE SYSTEH
 

tERAIN
 

CCRFORA AMTLACEA
 

* NUMBEfi OP AHIHALS HITH TISSUE

* NUMBE R OF ANIMALS NECROPSIED 

MATCHED
 

CONTROL
 

(20)
 

1 (5%)
 

(20)
 

(19)
 

(20)
 

(20)
 

(16)
 

(20)
 

2 (10X)
 

(20)
 

7 (35%)
 

(18)
 

H (22%)
 

(19)
 

3 (16X1
 

LOW DOSE
 

(49)
 

8 (16%)
 

(U9)
 

1 (2%)
 

(U9)
 

1 (2*)
 

(50)
 

1 (2%)
 

(50)
 

(36)
 

1 (3X)
 

(U 8)
 

9 (19X)
 

(48)
 

5 (10%)
 

(38)
 

a (11X)
 

(19)
 

7 11«?]_
 

 EXAMINE D MICROSCOPICALLY 

HIGH DOSE
 

(48)
 

1 (2«)
 

1 (2X)
 

2 (4X)
 

(48)
 

(41)
 

1 (2%)
 

(48)
 

(48)
 

1 (2X)
 

(28)
 

(46)
 

1 (2X)
 

1 (2X)
 

2 (4X)
 

(46)
 

5 (11X)
 

1 (2%)
 

(38)
 

5 (13X)
 

(48)
 

4 f8X)
 

73 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED LOW DOSE HIGH DOSE
 

CONTROL
 

SPECIAL SENSE OBGANS
 

NONE
 

BUSCOLOSKELBTAL SYSTEM
 

*SKEIETAL BUSCLE (20) (50) (49)
 

LYHPHOCYTIC INFLABBATOBY IHFILTB 1 (2X)
 

BCDT CAVITIES
 

NONE
 

ALL C1H£B SYSTEMS
 

NONE
 

SPECIAL HOBPBOLOGY SOBHABY
 

NO LESION REPORTED 5 15 17
 

ANIBAL HISSIHG/HO NECROPSY 1
 

ADTO/NECBOPSY/HISTO PBBF 1 5
 

I HDBBEB OF iHIH»LS WITH TISSUE EXiHIHED HICBOSCOPICA1.LT 

* BUBBEB OF AHIHALS HBCBOPSIED 

74 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN RATS
 

FED N-PHENYL-p-PHENYLENEDIAMINE 1*1 THE DIET
 

75
 

77 

Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed N-Phenyl-p-Phenylenediamine in the Dieta
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Subcutaneous Tissue: Fibromab 0/20 (0) 0/50 (0) 3/50 (6)
 

P Values0 >d N.S. N.S. N.S.
 

Relative Risk — Infinite
 

Lower Limit 0.250
 

Upper Limit Infinite
 

Weeks to First Observed Tumor — 80
 

Subcutaneous Tissue:
 

Squamous-cell Carcinoma^ 0/20 (0) 0/50 (0) 2/50 (4)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Riskf Infinite
 

Lower Limit 0.123
 

Upper Limit Infinite
 

Weeks to First Observed Tumor — 70
 
__ 

Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed N-Phenyl-p-Phenylenediamine in the Dieta
 

78

 

(continued)
 

Topography: Morphology


Lung: Alveolar /Bronchiolar
 

Adenoma or Carcinomab


P Valuesc»d


Departure from Linear Trend6


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Hematopoietic System: Lymphoma,
 

Leukemia, NOS, Undiff erentiated
 

Leukemia, or Lymphocytic Leukemia**


P Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

 Control
 

 2/20 (10)
 

 N.S.
 

 P = 0.032
 

 104
 

 2/20 (10)
 

 N.S.
 

 77
 

Low
 

Dose
 

0/50 (0)
 

N.S.
 

0.000
 

0.000
 

1.345
 

1/50 (2)
 

N.S.
 

0.200
 

0.004
 

3.681
 

91
 

High
 

Dose
 

4/49 (8)
 

N.S.
 

0.816
 

0.131
 

8.603
 

104
 

5/50 (10)
 

N.S.
 

1.000
 

0.184
 

10.007
 

79
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed N-Phenyl-p-Phenylenediamine in the Diet3
 

79

 

(continued)
 

Tomography: Morphology


Hematopoietic System:
 

Lymphoma or Leukemia"


P Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Pituitary: Chromophobe Adenomab


P Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

 Control
 

 3/20 (15)
 

 N.S.
 

 77
 

 0/20 (0*)
 

 N.S.
 

—
 

Low
 

Dose
 

2/50 (4)
 

N.S.
 

0.267
 

0.024
 

2.190
 

91
 

3/46 (7)
 

N.S.
 

Infinite
 

0.272
 

Infinite
 

103
 

High
 

Dose
 

5/50 (10)
 

N.S.
 

0.667
 

0.147
 

4.014
 

79
 

3/48 (6)
 

N.S.
 

Infinite
 

0.261
 

Infinite
 

104
 
80 

Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed N-Phenyl-p-Phenylenediamine in the Dieta
 

(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

Adrenal: Pheochromocytoma'3 1/20 (5) 2/50 (4) 3/50 (6)
 

P Values0>d N.S. N.S. N.S.
 

Relative Risk^ 0.800 1.200
 

Lower Limit 0.045 0.106
 

Upper Limit 46.273 61.724
 

Weeks to First Observed Tumor 104 104 84
 

Thyroid: C-cell Adenoma
 

or Carcinoma^3 0/20 (0) 2/50 (4) 3/50 (6)
 

P Value sc»d N.S. N.S. N.S.
 

Relative Riskf Infinite Infinite
 

Lower Limit 0.123 0.250
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 104 104
 
__ 

Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed N-Phenyl-p-Phenylenediamine in the Diet3
 

(continued)
 

Topography^ Morphology


Pancreatic Islets: Islet-cell
 

Adenomab


P Valuesc»d


Departure from Linear Trend6


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Testis: Interstitial-cell Tumorb


P Valuesc>d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched Low 

 Control Dose 

 0/20 (0) 3/48 (6) 

 N.S. N.S. 

P = 0.043 

Infinite 

0.261 

Infinite 

— 104 

 19/20 (95) 46/50 (92) 

 N.S. N.S. 

0.968 

0.902 

1.174 

 77 82 

High
 

Dose
 

0/49 (0)
 

N.S.
 

—
 

—
 

—
 

46/49 (94)
 

N.S.
 

0.988
 

0.920
 

1.168
 

81

 

79 
82 

Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed N-Phenyl-p-Phenylenediamine in the Dieta
 

(continued)
 

a

Dosed groups received 600 or 1,200 ppm.
 

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

c

Beneath the incidence of tumors in the matched-control group is the probability level for the
 

Cochran-Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

the incidence of tumors in each dosed group is the probability level for the Fisher exact
 

test for the comparison of that dosed group with the matched-control group when P < 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

"A negative trend (N) indicates the lower incidence in a dosed group than in the control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

^The 95% confidence interval of the relative risk between each dosed group and the matched-


control group.
 
___ 

__ __ 

Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

83

 

Fed N-Phenyl-p-Phenylenediamine in the Dieta
 

Matched Low
 

Topography; Morphology Control Dose
 

Lung: Alveolar /Bronchiolar
 

Adenoma or Carcinoma^ 0/19 (0) 0/50 (0)
 

P Valuesc'd N.S. N.S.
 

Relative Riskf
 

Lower Limit
 

Upper Limit
 —
 

—
 

Weeks to First Observed Tumor
 

Hematopoietic System:
 

Lymphoma or Leukemia^5 3/20 (15) 2/50 (4)
 

P Values0 »d N.S. N.S.
 

Relative Riskf 0.267
 

Lower Limit 0.024
 

Upper Limit 2.190
 

Weeks to First Observed Tumor 61 16
 

High
 

Dose
 

3/50 (6)
 

N.S.
 

Infinite
 

0.238
 

Infinite
 

104
 

5/50 (10)
 

N.S.
 

0.667
 

0.147
 

4.014
 

87
 
84

 

Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed N-Phenyl-p-Phenylenediamine in the Diet3
 

(continued)
 

Topography: Morphology


Pituitary: Chromophobe Adenoma
 

or Carcinoma*5


P Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched Low High 

 Control Dose Dose 

 7/20 (35) 15/47 (32) 9/46 (20) 

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

0.912 0.559 

0.431 0.226 

2.295 1.553 

 104 81 104 

Thyroid: C-cell Adenomab 2/19 (11) 1/46 (2) 1/49 (2) 

P Values0'd N.S. N.S. N.S. 

Relative Riskf 

Lower Limit 

Upper Limit 

0.207 

0.004 

3.789 

0.194 

0.003 

3.563 

Weeks to First Observed Tumor 104 104 104 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed N-Phenyl-p-Phenylenediamine in the Dieta
 

(continued)
 

Topography: Morphology 

Mammary Gland: Fibroadenoma^ 

P Values0 »d 

Departure from Linear Trend6 

Relative Riskf 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Uterus: Endometrial Stromal 

Polypb 

P Values

c

'

d 

Relative Risk^ 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Matched

Control

2/20 (10)

P = 0.024(N)

P = 0.042 

104 

2/20 (10)

N.S.

104

 Low High 

 Dose Dose 

 0/50 (0) 0/50 (0) 

 N.S. N.S. 

0.000 0.000 

0.000 0.000 

1.345 1.345 

__ 

 4/49 (8) 4/50 (8) 

 N.S. N.S. 

0.816 0.800 

0.131 0.128 

8.603 8.436 

 104 104 

85 
86

 

Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed N-Phenyl-p-Phenylenediamine in the Dieta
 

(continued)
 

a

Dosed groups received 600 or 1,200 ppm.
 

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

c

Beneath the incidence of tumors in the matched-control group is the probability level for the
 

Cochran-Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

the incidence of tumors in each dosed group is the probability level for the Fisher exact
 

test for the comparison of that dosed group with the matched-control group when P < 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

^A negative trend (N) indicates the lower incidence in a dosed group than in the control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

^The 95% confidence interval of the relative risk between each dosed group and the matched-


control group.
 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN MICE
 

FED N-PHENYL-p-PHENYLENEDIAMINE IN THE DIET
 

87
 

Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Fed N-Phenyl-p-Phenylenediamine in the Diet3
 

89

 

Tomography: Morphology


Lung: Alveolar /Bronchiolar
 

Adenomab


P Valuesc»d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Hematopoietic System: Lymphomab


P Values0'd


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

 Control
 

 4/20 (20)
 

 N.S.
 

 91
 

 3/20 (15)
 

 N.S.
 

 87
 

Low
 

Dose
 

4/49 (8)
 

N.S.
 

0.408
 

0.086
 

2.022
 

91
 

2/49 (4)
 

N.S.
 

0.272
 

0.025
 

2.233
 

91
 

High
 

Dose
 

5/50 (10)
 

N.S.
 

0.500
 

0.124
 

2.322
 

91
 

3/50 (6)
 

N.S.
 

0.400
 

0.060
 

2.802
 

91
 
90

 

Table Fl. Analyses of the Incidence of Primary Tumors in

Fed N-Phenyl-p-Phenylenediamine in the Diet3 

(continued) 

Matched Low
 

Topography: Morphology Control Dose
 

Liver: Hepatocellular Carcinoma'3

 2/20 (10) 6/49 (12)
 

P Values0 »d N.S. N.S.
 

Relative Riskf 1.224
 

Lower Limit 0.248
 

Upper Limit 11.802
 

Weeks to First Observed Tumor 91 91
 

Liver: Hepatocellular
 

Adenoma or Carcinomab

 2/20 (10) 18/49 (37)
 

P Values0 >d N.S. P = 0.022
 

Departure from Linear Trend6 P = 0.010
 

Relative Riskf 3.673
 

Lower Limit 1.019
 

Upper Limit 30.643
 

Weeks to First Observed Tumor 91 85
 

 Male Mice 

High
 

Dose
 

5/50 (10)
 

N.S.
 

1.000
 

0.184
 

10.007
 

82
 

10/50 (20)
 

N.S.
 

2.000
 

0.488
 

17.808
 

82
 
91

 

Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Fed N-Phenyl-p-Phenylenediamie in the Dieta
 

(continued)
 

a

Dosed groups received time-weighted average doses of 2,057 or 4,114 ppm.
 

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

c

Beneath the incidence of tumors in the matched-control group is the probability level for the
 

Cochran-Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

the incidence of tumors in each dosed group is the probability level for the Fisher exact
 

test for the comparison of that dosed group with the matched-control group when P < 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

*^A negative trend (N) indicates the lower incidence in a dosed group than in the control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

*The 95% confidence interval of the relative risk between each dosed group and the matched-


control group.
 
__ 92 

Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed N-Phenyl-p-Phenylenediamine in the Dieta
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Hematopoietic System: Lymphomab 4/20 (20) 0/50 (0) 2/49 (4)
 

P valuesc»d N.S. P = 0.005(N) N.S.
 

Departure from Linear Trend6 P = 0.003
 

Relative Riskf 0.000 0.204
 

Lower Limit 0.000 0.020
 

Upper Limit 0.427 1.323
 

Weeks to First Observed Tumor 34 91
 

Liver: Hepatocellular Adenomab 1/20 (5) 2/49 (4) 1/48 (2)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Riskf 0.816 0.417
 

Lower Limit 0.046 0.006
 

Upper Limit 47.195 32.058
 

Weeks to First Observed Tumor 91 91 91
 
93

 

Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed N-Phenyl-p-Phenylenediamine in the Dieta
 

(continued)
 

a

Dosed groups received time-weighted average doses of 3,672 or 8,170 ppm.
 

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

G

Beneath the incidence of tumors in the matched-control group is the probability level for the
 

Cochran-Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

the incidence of tumors in each dosed group is the probability level for the Fisher exact
 

test for the comparison of that dosed group with the matched-control group when P < 0.05;
 

otherwise, not significant (N.S.) is indicated.
 

"A negative trend (N) indicates the lower incidence in a dosed group than in the control group.
 

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

^The 95% confidence interval of the relative risk between each dosed group and the matched-


control group.
 

Review of the Bioassay of N-Phenyl-p-phenylenediamine*
 

for Carcinogenicity by the
 

Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

March 6, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DHEW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay"program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be
 

exposed. The members of the Clearinghouse have been drawn
 

from academia, industry, organized labor, public interest
 

groups, State health officials, arid 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 N-Phenyl-p-phenylenediamine for carcinogenicity.
 

The primary reviewer briefly described the experimental
 

design under which N-Phenyl-p-phenylenediamine was tested
 

and the results of the study. Although some liver tumors
 

were found among the treated mice, they were not statistically
 

significant when compared to the historical control animals.
 

He agreed with the conclusion that N-Phenyl-p-phenylenediamine
 

was not carcinogenic in rats or mice, under the conditions
 

of test.
 

The secondary reviewer pointed out the higher incidence
 

of total tumors in treated mice as compared with matched
 

controls. He also noted the staff's conclusion that the
 

shortened treatment period may not have been long enough
 

to evaluate the carcinogenicity of N-Phenyl-p-phenylenediamine
 

in the mice. The secondary reviewer recommended that the
 

chemical be considered for retest.
 

95 
The primary reviewer commented, that the treated and
 

control animals should be compared on an organ site basis,
 

since total tumor incidence may be misleading. A discussion
 

ensued on the differences in carcinogenicity displayed by
 

N-Phenyl-p-phenylenediamine and its structural relatives,
 

such as 4-Chloro-o-phenylenediamine and N-Phenyl-o-phenylenediamine.
 

A subgroup member questioned the general practice of
 

considering a high incidence of liver tumors in the treated
 

animals as not statistically significant based on a single
 

case in which a similar increase was seen in a laboratory
 

control group. He cited the 37% incidence of liver tumors
 

in the treated low dose male mice as an example of such a
 

case. Another Subgroup member added that it was important
 

to evaluate an increased incidence with respect to the overall
 

experience in a given laboratory. A Program staff member
 

responded tha£ the high control incidence of liver tumors
 

was not unusual in this particular laboratory. He continued
 

that the liver tumor incidence in the treated mice was within
 

the usual control values. It was suggested that average
 

tumor incidences be given for comparative purposes in instances
 

of this nature.
 

A motion was made that the report on the bioassay of
 

N-Phenyl-p-phenylenediamine be accepted as written. The
 

motion was seconded and passed with Dr. Pitot, Dr. Weisburger,
 

Dr. Shimkin, Dr. Roush, Dr. Brown, Dr. Wogan, and Mr. Garfinkel
 

in favor and Dr. Highland, Dr. Wolfe, and Mr. Samuels opposed.
 

A minority opinion was voiced by Dr. Wolfe that there
 

appeared to be a statistically significant increase in liver
 

tumors (adenomas plus carcinomas) among the low dose male
 

mice. He noted that benign and malignant tumors have been
 

combined in the past for purposes of statistical analyses.
 

Mr. Samuels said he voted against the motion because the
 

study was too inadequate to make any judgment on the
 

carcinogenicity of N-Phenyl-p-phenylenediamine.
 

*	 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.
 

96
 

DREW Publication No. (NIH) 78-1332