1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 130 

BIOASSAY OF 

ANILINE HYDROCHLORIDE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 142-04-1 

NCI-CG-TR-130 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

ANILINE HYDROCHLORIDE
 

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) 78-1385
 

REPORT ON THE BIOASSAY OF ANILINE HYDROCHLORIDE
 

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 aniline hydrochloride conducted for the Carcinogenesis Testing Program,
 

Division of Cancer Cause and Prevention, National Cancer Institute
 

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

one of a series of experiments designed to determine whether selected
 

chemicals have the capacity to produce cancer in animals. Negative
 

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

greater incidence of cancer than control animals, do not necessarily
 

mean the test chemical is not a carcinogen because the experiments
 

are conducted under a limited set of circumstances. Positive results
 

demonstrate that the test chemical is carcinogenic for animals under
 

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

actual determination of the risk to man from animal carcinogens requires a wider analysis.
 

CONTRIBUTORS: This bioassay of aniline hydrochloride was conducted
 

by Mason Research Institute, Worcester, Massachusetts, initially
 

under direct contract to the NCI and currently under a subcontract
 

to Tracor Jitco, Inc., prime contractor for the NCI Carcinogenesis
 

Testing Program.
 

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

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

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

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

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

Histopathologic examinations were performed by Dr. D. W. Hayden
 

(3) and Dr. D. S. Wyand (3) at the Mason Research Institute, and the
 

diagnoses included in this report represent the interpretation of
 

these pathologists. Histopathology findings and reports were reviewed by Dr. R. L. Schueler (6).
 

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

statistical analysis was performed by Mr. W. W. Belew (5,8) and
 

111
 
Mr. R. M. Helfand (5), using methods selected for the Carcinogenesis
 

Testing Program by Dr. J. J. Gart (9).
 

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

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

(5), task leader Dr. M. R. Kornreich (5,10), senior biologist Ms. P.
 

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

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

participating organizations.
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

Cueto, Jr. (1), Dr. J. F. Douglas (1), Dr. D. G. Goodman (1,10), Dr.
 

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

T. W. Orme (1), Dr. R. A. Squire (1,11), Dr. S. F. Stinson (1), Dr.
 

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

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

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

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

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

Missouri.
 

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

Boulevard, McLean, Virginia.
 

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

Maryland.
 

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

Rockville, Maryland.
 

8.	 Now with the Solar Energy Research Institute, Cole Boulevard,
 

Golden, Colorado.
 

IV
 
9.	 Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cancer
 

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

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

Washington, B.C.
 

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

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay of aniline hydrochloride for possible carcinogenicity
 

was conducted using Fischer 344 rats and B6C3F1 mice. Aniline hydrochloride was administered in the feed, at either of two concentrations,
 

to groups of 50 male and 50 female animals of each species, with the
 

exception of 49 female mice in the high dose group. The high and low
 

dietary concentrations of aniline hydrochloride were, respectively,
 

0.6 and 0.3 percent for rats and 1.2 and 0.6 percent for mice. After
 

a 103-week period of compound administration, observation of the rats
 

and mice continued for up to an additional 5 weeks.
 

For rats and mice, respectively, 25 and 50 animals of each sex
 

were placed on test as controls and fed only the basal diet.
 

In male rats there were several types of mesenchymal tumors,
 

primarily of the spleen, associated with administration of the compound. Hemangiosarcomas of the spleen and the combined incidence of
 

fibrosarcomas and sarcomas NOS of the spleen were each statistically
 

significant in male rats. The combined incidence of fibrosarcomas
 

and sarcomas NOS of multiple body organs was also significant in male
 

rats. The number of female rats having fibrosarcomas or sarcomas NOS
 

of either the spleen alone or multiple organs of the body cavity was
 

significantly associated with increased dietary concentration of aniline hydrochloride. This result was not supported by Fisher exact
 

tests, but because of the rarity of these tumors, the observed incidences (0/24 in the control group, 1/50 [2 percent] in the low dose
 

group, 7/50 [14 percent] in the high dose group) were considered indicative of a compound-related carcinogenic effect.
 

In mice of both sexes no tumors occurred in statistically significant increased incidences among dosed groups when compared to
 

controls.
 

Under the conditions of this bioassay, dietary administration of
 

aniline hydrochloride was carcinogenic to male and female Fischer 344
 

rats, inducing hemangiosarcomas and a combination of fibrosarcomas and
 

sarcomas NOS of the spleen and a combination of fibrosarcomas and sarcomas NOS of multiple body organs. There was no evidence of compound-


induced carcinogenicity in B6C3F1 mice of either sex.
 

vii
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION !
 

II. MATERIALS AND METHODS 4
 

A. Chemicals 4
 

B. Dietary Preparation 4
 

C. Animals 5
 

D. Animal Maintenance "
 

E. Selection of Initial Concentrations °
 

F. Experimental Design 9
 

G. Clinical and Histopathologic Examinations ^
 

H. Data Recording and Statistical Analyses -^
 

III. CHRONIC TESTING RESULTS: RATS 19
 

A. Body Weights and Clinical Observations 19
 

B. Survival 19
 

C. Pathology 22
 

D. Statistical Analyses of Results 25
 

IV. CHRONIC TESTING RESULTS: MICE 39
 

A. Body Weights and Clinical Observations 39
 

B. Survival 39
 

C. Pathology 42
 

D. Statistical Analyses of Results 42
 

49
 V. DISCUSSION
 

VI. BIBLIOGRAPHY 52
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH ANILINE HYDROCHLORIDE A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH ANILINE HYDROCHLORIDE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH ANILINE
 

HYDROCHLORIDE C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH ANILINE
 

HYDROCHLORIDE D-l
 

IX
 
LIST OF ILLUSTRATIONS 

Figure Number Page 

1 CHEMICAL STRUCTURE OF ANILINE HYDROCHLORIDE 2 

2 GROWTH CURVES FOR ANILINE HYDROCHLORIDE 

CHRONIC STUDY RATS 20 

SURVIVAL COMPARISONS OF ANILINE HYDROCHLORIDE CHRONIC STUDY RATS 21 

GROWTH CURVES FOR ANILINE HYDROCHLORIDE 

CHRONIC STUDY MICE 40 

SURVIVAL COMPARISONS OF ANILINE HYDROCHLORIDE CHRONIC STUDY MICE 41 

LIST OF TABLES 

Table Number Page 

DESIGN SUMMARY FOR FISCHER 344 RATS-­

ANILINE HYDROCHLORIDE FEEDING EXPERIMENT 11 

DESIGN SUMMARY FOR B6C3F1 MICE—ANILINE 

HYDROCHLORIDE FEEDING EXPERIMENT 12 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

ANILINE HYDROCHLORIDE 26 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH ANILINE HYDROCHLORIDE 31 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

ANILINE HYDROCHLORIDE 43 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH ANILINE HYDROCHLORIDE 45 
LIST OF TABLES (Concluded) 

Table Number 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH ANILINE HYDROCHLORIDE A-3 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH ANILINE HYDROCHLORIDE A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH ANILINE HYDROCHLORIDE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH ANILINE HYDROCHLORIDE B-7 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH ANILINE 

HYDROCHLORIDE C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH ANILINE 

HYDROCHLORIDE C-8 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH ANILINE 

HYDROCHLORIDE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH ANILINE 

HYDROCHLORIDE D-7 

XI
 

I. INTRODUCTION
 

Aniline HC1 (Figure 1) (NCI No. C03736), a dye intermediate and
 

a commercially important salt of aniline, was selected for bioassay
 

by the National Cancer Institute because of the increased incidence
 

of bladder cancer among workers in the dye manufacturing industry and
 

the historical association of aromatic amines with this increased cancer
 

risk (Wynder et al., 1963; Hamblin, 1963).
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is hydrochloride benzenamide. It is
 

also called aniline salt and aniline chloride.
 

The major industrial use of aniline HC1 is as an intermediate in
 

the manufacture of dyes. The oxidation of aniline HC1 within textile
 

fibers to produce the dye Aniline Black (C.I. [Colour Index] Pigment
 

Black 1) (C.I. 50440) is one application (Kouris and Northcott, 1963).
 

Specific production statistics for aniline HC1 are not available;
 

however, the inclusion of aniline HC1 in Synthetic Organic Chemicals,
 

U.S. Production and Sales, 1975 (U.S. International Trade Commission,
 

1977) implies an annual commercial production in excess of 1000
 

o
 

pounds or $1000 in value. Approximately 4 x 10 pounds of aniline,
 

the immediate precursor of aniline HC1, were produced in 1975 (U.S.
 

International Trade Commission, 1977).
 

*
 

The CAS registry number is 142-04-1
 
FIGURE 1

 

CHEMICAL STRUCTURE OF ANILINE (HYDROCHLORIDE) 
The potential for exposure to aniline HC1 is greatest for workers
 

involved in the manufacture or use of the compound. The majority of
 

these workers are employed by the dye and textile industries.
 

The most prominent effect of acute exposure to aniline hydrochloride is methemeglobinemia and the accompanying cyanosis; headache,
 

vertigo and mental confusion may also occur (Hamblin, 1963; Windholz,
 

1976). In humans, chronic aniline HC1 exposure can result in anemia,
 

anorexia, weight loss, and cutaneous lesions (Windholz, 1976). Although aniline has been frequently associated with so-called "aniline
 

tumors" of the urinary bladder, the majority of evidence indicates
 

that other intermediates in the production of aniline dyes, especially
 

2-naphthylamine and benzidine, are responsible for these tumors
 

(international Agency for Research on Cancer, 1974; Windholz, 1976).
 
II. MATERIALS AND METHODS 

A.	 Chemicals
 

Aniline hydrochloride was purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. Chemical analysis was performed by Midwest Research Institute, Kansas City, Missouri. The narrow melting
 

point range (197° to 199°C) and closeness of the observed melting
 

point to the literature value (195°C) (Linstead and Braude, 1954)
 

suggested a compound of high purity. Thin-layer chormatography was
 

performed utilizing two solvent systems (acetone:benzene:ammonia and
 

ethyl acetate:chloroform), and plates were visualized with ultraviolet
 

light and furfural. Each plate showed one or more nonmotile impurities. Experimental elemental analysis approximated that expected on
 

a theoretical basis. Results of nonaqueous titration of the amine
 

functions were approximately 100 percent of the theoretical value.
 

Vapor-phase chromatography showed one homogeneous peak. Infrared
 

analysis was consistent with the structure of the compound. The data
 

suggest a compound of high purity.
 

Throughout this report, the term aniline HC1 is used to represent
 

this compound.
 

B. Dietary Preparation
 

The basal laboratory diet for both dosed and control animals
 

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

Aniline HC1 was administered to the dosed animals as a component of
 

the diet.
 
The chemical was removed from its container and proper amounts
 

were ground to a powder in a Quaker City crystal mill, sifted and
 

weighed out under an exhaust hood and ground with a mortar and pestle.
 

The compound was hand-blended in an aluminum bowl with an aliquot of
 

the ground feed. Once visual homogeneity was attained, the mixture
 

was placed in a 6 kg capacity Patterson-Kelley standard model twin-


shell stainless steel V-blender along with the remainder of the feed
 

to be prepared. After 20 minutes of blending, the mixtures were
 

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

mixture was prepared once weekly.
 

C. Animals
 

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

through contracts of the Division of Cancer Treatment, National
 

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

Breeding Laboratories, Inc., Wilmington, Massachusetts. Dosed and
 

control animals for both species were received in separate shipments.
 

Upon arrival, a sample of animals was examined for parasites
 

and other signs of disease. All animals were treated for parasites
 

with piperazine adipate at 3.0 gm/liter of water fed ad libitum foir
 

three days followed by three additional days of piperazine adipate
 

dosing. Animals were then quarantined by species for 2 weeks priox
 

to initiation of the test. Animals were assigned to groups and
 
distributed among cages so that the average body weight per cage was
 

approximately equal for a given species and sex.
 

D. Animal Maintenace
 

All animals were housed by species in rooms having a temperature
 

®
 

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

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

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

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

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

for the first 14 months of study, all rats were housed in wire-mesh
 

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

newspapers. Newspapers under cages were replaced daily and cages
 

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

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

Garfield, New Jersey) equipped with nonwoven fiber filter sheets.
 

®
 Clean cages and bedding were provided twice weekly. SAN-I-CEL
 

corncob bedding (Paxton Processing Company, Paxton, Illinois) was
 

used for the first 8 weeks in polycarbonate cages. Aspen hardwood
 

chip bedding (American Excelsior Company, Baltimore, Maryland) was
 

used for the remainder of the study. Stainless steel cage racks
 

(Fenco Cage Products) were cleaned once every 2 weeks, and disposable filters were replaced at that time.
 

Mice were housed by sex in polycarbonate cages. Cages were
 

fi.tted with perforated stainless steel lids (Lab Products, Inc.).
 
Nonwoven fiber filter bonnets were used over cage lids. Control mice
 

were housed ten per cage for the first month of study, and five per
 

cage thereafter. Dosed mice were housed five per cage throughout
 

the study. Clean cages, lids, and bedding were provided three times
 

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

(R)
 populations were reduced to five. SAN-I-CEL was supplied for the
 

first 9 months of study, followed by Bed-o-Cobs^ corncob bedding (The
 

Andersons Cob Division, Maumee, Ohio) for 8 months. Aspen bedding
 

was then used for the remainder of the study. Reusable filter bonnets and pipe racks were sanitized every 2 weeks throughout the study.
 

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

bottles equipped with rubber stoppers and stainless steel sipper
 

tubes. Bottles were replaced twice weekly. For rats, bottles were
 

refilled as needed between changes.
 

(R)
 Wayne Lab-Blox was supplied to all animals during the initial
 

quarantine and final observation periods. During the period of
 

compound administration, all dosed animals were fed Wayne Lab-Blox®
 

meal containing the appropriate concentration of aniline HC1. Control
 

animals had untreated meal available ad libitum. Meal was supplied
 

to rats and mice for the first 12 and 11 months, respectively, in
 

(R)
 Alpine aluminum feed cups (Cirtin Matheson Scientific, Inc.,
 

Woburn, Massachusetts). For the remainder of the study, meal was
 

supplied from stainless steel gangstyle food hoppers (Scientific
 

Cages, Inc., Bryan, Texas). Food hoppers were changed on the same
 
schedule as were cages. Food was replenished daily in Alpine feed
 

cups.
 

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

>v
 receiving diets containing N-butylurea (592-31-4); N,N-dimethylp-nitrosoaniline (138-89-6); 2,5-toluenediamine sulfate (6369-59-1);
 

2,4-dinitrotoluene (121-14-2); 4-nitroanthranilic acid (619-17-0);
 

1,5-naphthaienediamine (2243-62-1); N-(l-naphthyl)ethylenediamine
 

dihydrochloride (1465-25-4); 2-chloro-p-phenylenediamine sulfate
 

(61702-44-1); and p-anisidine hydrochloride (20265-97-8).
 

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

mice receiving diets containing 2,3,5,6-tetrachloro-4-nitroanisole
 

(2438-88-2); hydrazobenzene (530-50-7); tris(2,3-dibromopropyl)
 

phosphate (126-72-7); N-(l-naphthyl)ethylenediamine dihydrochloride
 

(1465-25-4); and 2-chloro-p-phenylenediamine sulfate (61702-44-1).
 

E. Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

aniline HC1 for administration to dosed animals in the chronic
 

studies, subchronic toxicity tests were conducted with both rats and
 

mice. Animals of each species were distributed among five groups,
 

each consisting of five males and five females. Aniline HC1 was
 

incorporated into the basal diet and fed ad libitum to four of the
 

five groups of each species in concentrations of 0.03, 0.01, 0.3,
 

and 1.0 percent. The remaining group of each species served as a
 

*
 

CAS registry numbers are given in parentheses.
 
control group, receiving only the basal laboratory diet. The dosed
 

dietary preparations were administered for a period of 8 weeks. At
 

the end of the observation period, all survivors were sacrificed and
 

necropsied.
 

The highest concentration causing no deaths, no compound-related
 

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

5 percent relative to controls during the 8 week subchronic test was
 

selected as the high concentration utilized for the rat and mouse
 

chronic bioassays.
 

At dietary concentrations of 0.3 percent or greater, black,
 

granular and enlarged spleens were observed in both species. No
 

deaths were observed at any dose tested. A dietary concentration of
 

0.3 percent produced no mean body weight depression, while a dietary
 

concentration of 1.0 percent produced mean body weight depressions
 

of 23 and 24 percent in male and female rats, respectively. The
 

high concentration selected for administration to rats in the chronic
 

bioassay was 0.6 percent.
 

A dietary concentration of 1.0 percent produced mean body weight
 

depressions of 3 and 2 percent in male and female mice, respectively.
 

The high concentration selected for administration to mice in the
 

chronic bioassay was 1.2 percent.
 

F. Experimental Design
 

The experimental design parameters for the chronic study (species, sex, group size, concentrations administered, and duration of
 
treated and untreated observation periods) are summarized in Tables
 

1 and 2.
 

The dosed and control rats were all approximately 6 weeks old
 

at the time the test was initiated. The initial concentrations of
 

aniline HC1 administered in the diet were 0.6 and 0.3 percent.
 

Throughout this report those rats receiving the former concentration
 

are referred to as the high dose groups and those receiving the latter concentration are referred to as the low dose groups. The dosed
 

rats were supplied with feed containing aniline HC1 for a total of
 

103 weeks followed by a 4- to 5-week observation period.
 

The dosed and control mice were all approximately 6 weeks old
 

at the time the test was initiated. The initial concentrations of
 

aniline HC1 administered in the diet were 1.2 and 0.6 percent.
 

Throughout this report those mice receiving the former concentration
 

are referred to as the high dose groups and those receiving the latter concentration are referred to as the low dose groups. The dosed
 

mice were supplied with feed containing aniline HC1 for a total of
 

103 weeks followed by a 4-week observation period.
 

G.	 Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

experiment. Body weights were recorded twice weekly for the first
 

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

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

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

10
 
TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

ANILINE HYDROCHLORIDE FEEDING EXPERIMENT
 

INITIAL ANILINE HC1 OBSERVATION PERIOD 

GROUP CONCENTRATION TREATED UNTREATED 

SIZE (PERCENT) (WEEKS) (WEEKS) 

MALE 

CONTROL 25 0 0 110 

LOW DOSE 50 0.3 103 

0 4 

HIGH DOSE 50 0.6 103 

0 5 

FEMALE 

CONTROL 25 0 0 110 

LOW DOSE 50 0.3 103 

0 4 

HIGH DOSE 50 0.6 103 

0 5 

11
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

ANILINE HYDROCHLORIDE FEEDING EXPERIMENT
 

INITIAL ANILINE HC1 OBSERVATION PERIOD 

GROUP CONCENTRATION TREATED UNTREATED 

SIZE (PERCENT) (WEEKS) (WEEKS) 

MALE 

CONTROL 50 0 0 109 

LOW DOSE 50 0.6 103 

0 4 

HIGH DOSE 50 1.2 103 

0 4 

FEMALE 

CONTROL 50 0 0 109 

LOW DOSE 50 0.6 103 

0 4 

HIGH DOSE 49 1.2 103 

0 4 

12
 
seven consecutive days once a month for the first nine months of the
 

bioassay and for three consecutive days each month thereafter. The
 

presence of tissue masses and lesions was determined by monthly observation and palpation of each animal.
 

A necropsy was performed on each animal regardless of whether it
 

died, was killed when moribund, or was sacrificed at the end of the
 

bioassay. The animals were euthanized by carbon dioxide inhalation,
 

and were immediately necropsied. The histopathologic examination
 

consisted of gross and microscopic examination of major tissues,
 

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

possible, from animals found dead.
 

Tissues were preserved in 10 percent buffered formalin, embedded
 

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

to microscopic examination. An occasional section was subjected to
 

special staining techniques for more definitive diagnosis.
 

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

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

urinary bladder, pituitary, adrenal, thyroid, parathyroid, ear, testis, prostate, brain, uterus, mammary gland, and ovary.
 

A few tissues were not examined for some animals, particularly
 

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

13
 
preclude histopathologic interpretation. Thus, the number of animals
 

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

animals that were placed on experiment in each group.
 

H. Data Recording and Statistical Analyses
 

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

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

as recommended by the International Union Against Cancer (Berenblum,
 

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

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

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

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

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

missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect
 

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

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

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

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

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

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

histologically. However, when macroscopic examination was required
 

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

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

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

The purpose of the statistical analyses of tumor incidence is to
 

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

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

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

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

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

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

significance level of 0.05 may be made. The Bonferroni inequality
 

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

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

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

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

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

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

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

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

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

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation of
 

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

tumor was observed were excluded by basing the statistical tests on
 

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

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

tumor was found, comparisons were based exclusively on animals that
 

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

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

Cochran-Armitage tests, etc.) were followed.
 

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

incidence of tumors. Curves of the proportions surviving without an
 

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

during which animals died naturally or were sacrificed was entered as
 

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

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

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

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

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

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


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

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

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

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

t c t
 

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

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

c
 

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

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

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

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

larger proportion in the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

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

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

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

would be within the interval calculated from the experiment. When
 

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

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

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

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

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

the lower limit indicates the absence of a significant result while
 

the upper limit indicates that there is a theoretical possibility
 

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

detected under the conditions of this test.
 

18
 
III. CHRONIC TESTING RESULTS: RATS
 

A.	 Body Weights and Clinical Observations
 

Slight mean body weight depression was apparent in dosed female
 

rats and high dose male rats (Figure 2).
 

Subcutaneous masses developed in one high dose male, one low
 

dose male, one control male, one high dose female, five low dose
 

females, and three control females. Cutaneous lesions were observed
 

in one low dose male, two control males, one high dose female, and
 

one control female. Abdominal distention was noted in two low dose
 

males and in one control female. Distention of the scrotal sac was
 

seen in three low dose males. Posterior ataxia was displayed by one
 

low dose male. Alopecia was seen in one high dose female. No other
 

clinical abnormalities were noted.
 

B. Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and aniline HCl-dosed groups are shown in Figure 3.
 

For both male and female rats the Tarone test did not indicate a significant positive association between dosage and mortality.
 

Adequate numbers of males were at risk from late-developing tumors, as 54 percent (27/50) of the high dose, 68 percent (34/50) of
 

the low dose, and 68 percent (17/25) of the control rats survived on
 

test until the end of the study. Adequate numbers of females were
 

also at risk from late-developing tumors, as 82 percent (41/50) of
 

19
 
MEAN BODY WEIGHT (GRAMS) MEAN BODY WEIGHT (GR

o

I 

3)

O 

T. 3) n > c 

30 

m 

V) 

-n 

O 

•33 

s 

m I f! O 

<5 

eg 8 ­

o 

m 

O

X 

30 

O 

O 

CO 

c 

o 

30 

I 

o 

T~ 

I 
c 

PROBABILITY OF SURVIVAL PROBABILITY OF SURV

p p b b 

CO 

3D 

m 33 

8 33 

C/3 

> 

33 8­

i 

CO 

m 

O m 

O 

E 5c 

o 

33 

D 

m 

m

m m

m 

O 

X 

30 

O 

B 

i 

o 

3D 

> a­

§ 

T^ I 

o p

b o 
the high dose, 88 percent (44/50) of the low dose, and 64 percent
 

(16/25) of the control rats survived on test until the end of the
 

study.
 

C. Pathology
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

summarized in Appendix C (Tables Cl and C2).
 

Several different mesenchymal tumors were associated with
 

compound administration. These tumors occurred in large numbers in
 

the spleens of dosed male rats; additionally, for dosed males and
 

for high dose females, these tumors were often observed in multiple
 

organs of the pleural and abdominal cavities in the same animal.
 

These tumors are summarized in the following table:
 

Spleens and Capsule
 

Number of animals with
 

tissues examined
 

h is topatho logically
 

Sarcoma NOS
 

Fibroma
 

Fibrosarcoma
 

Hemangiosarcoma
 

Lipoma
 

Hemangioma
 

Control
 

(25)
 

0
 

0
 

0
 

0
 

0
 

0
 

MALES
 

Low
 

Dose
 

(50)
 

4
 

7
 

3
 

19
 

0
 

0
 

High
 

Dose
 

(46)
 

2
 

6
 

7
 

20
 

0
 

0
 

Control
 

(23)
 

0
 

0
 

0
 

0
 

0
 

0
 

FEMALES
 

Low
 

Dose
 

(50)
 

0
 

0
 

0
 

1
 

0
 

0
 

High
 

Dose
 

(50)
 

3
 

0
 

0
 

2
 

1
 

1
 

22
 
MALES FEMALES 

Low High Low High 

Control Dose Dose Control Dose Dose 

Body Cavity,
 

Multiple Organs
 

Number of animals with
 

tissues examined
 

histopathologically (25) (50) (48) (24) (50) (50)
 

Fibrosarcoma 0 2 8 0 1 3
 

Leiomyosarcoma 0 0 2 1 0 0
 

Hemangiosarcoma 0 0 1 0 0 0
 

Sarcoma NOS 0 0 1 0 0 1
 

The incidence of hemangiosarcoma, the tumor most frequently
 

observed, was elevated in dosed males (19/50 [38 percent] low dose
 

and 21/48 [44 percent] high dose). Hemangiosarcomas were tumors of
 

vascular endothelium with blood-filled spaces varying from capillary
 

size to large hematomas enclosed by a rim of neoplastic cells which
 

projected villous-like structures into the blood spaces. Capillary-


like areas sometimes lacked blood in the spaces. Solid sarcomatous
 

areas of spindle cells were common. Cells were often pleomorphic
 

with numerous mitoses.
 

Fibrosarcomas consisted of pleomorphic fibroblasts and collagen.
 

Mitotic figures were usually common. With loss of differentiation,
 

there was increased hyperchromasia and pleomorphism, and the tumors
 

became more cellular with decreased collagen. Fibrosarcomas were
 

quite invasive with widespread extension seen in the abdominal
 

cavity. In some fibrosarcomas there were prominent areas of osseous
 

metaplasia. The diagnosis of sarcoma NOS may represent very poorly
 

differentiated fibrosarcomas.
 

23
 
Fibromas were more circumscribed than fibrosarcomas, less
 

cellular, and had mature fibroblasts and greater amounts of collagen.
 

Mitotic figures were rare.
 

In a few instances, organs contained two distinct tumors, fibrosarcomas and hemangiosarcomas combined with fibromas. While the
 

tumors were usually distinctly separate, in one spleen an area of
 

fibrosarcoma blended imperceptibly with an area of endothelial proliferation which might have been early hemangiosarcoma.
 

Adrenal pheochromocytomas occurred in 6/50 (12 percent) low dose
 

and 12/44 (27 percent) high dose males, and 5/48 (10 percent) high
 

dose females. There were 2/24 (8 percent) in control males and 1/24
 

(4 percent) in control females.
 

Several proliferative nonneoplastic lesions occurred only in
 

dosed rats. In the spleen these included fibrosis of the splenic
 

capsule and trabeculae and fatty metamorphosis, the occurrence of
 

scattered large fat cells in the splenic parenchyma. Many dosed
 

rats (18/50 [36 percent] low dose and 7/46 [15 percent] high dose
 

males; 23/50 [46 percent] low dose and 28/50 [56 percent] high dose
 

females) exhibited fine papillary projections from the surface of the
 

splenic capsule. These projections had dense fibrous cores and were
 

covered by reactive mesothelial cells, as was the remainder of the
 

splenic capsule. While these lesions had some resemblance to early
 

mesotheliomas, they were considered to be hyperplastic rather than
 

neoplastic lesions and were classifed as papillary hyperplasia.
 

24
 
Hemosiderosis of renal tubular epithelium was found in very high incidences in male (21/50 [42 percent] low dose, 34/48 [71 percent] high
 

dose) and female (46/49 [94 percent] low dose, 45/50 [90 percent]
 

high dose) rats. The tubular epithelial cells of the cortex contained
 

many yellow-brown cytoplastnic pigment granules. Hemosiderosis of the
 

liver characterized by Kupffer cells filled with the pigment occurred
 

in both high dose males (26/47 [55 percent]) and high dose females
 

(29/50 [58 percent]). Because of this marked degree of hemosiderosis
 

compared to trace amounts in controls, the finding is considered to
 

be compound-related. Hemosiderosis was occasionally found in other
 

tissues.
 

The incidence of endometrial stromal polyp was increased in low
 

dose females (15/48 [31 percent]) as compared with controls (2/24 [8
 

percent]). The increase was not dose-related as it occurred in only
 

7/50 (14 percent) high dose animals.
 

On the basis of this pathologic examination, it is concluded that
 

aniline HC1 was carcinogenic when fed to male and possibly to female
 

rats, inducing mesenchymal tumors. An increased incidence of adrenal
 

pheochromocytomas appeared related to compound administration. Renal
 

and Kupffer-cell hemosiderosis in male and female rats were also related to compound administration.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

25
 
TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH ANILINE HYDROCHLORIDE£
 

TOPOGRAPHY: MORPHOLOGY
 

Pituitary: Adenoma NOS
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocytoma or Pheochromocytoma, Malignant
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

2/22(0.09)
 

N.S.
 

98
 

2/24(0.08)
 

P = 0.022
 

110
 

2/21(0.10)
 

N.S.
 

97
 

LOW
 

DOSE
 

7/45(0.16)
 

N.S.
 

1.711
 

0.367
 

16.016
 

63
 

6/50(0.12)
 

N.S.
 

1.440
 

0.286
 

13.931
 

102
 

2/39(0.05)
 

N.S.
 

0.538
 

0.042
 

7.050
 

107
 

HIGH
 

DOSE
 

4/34(0.12)
 

N.S.
 

1.294
 

0.206
 

13.453
 

90
 

12/44(0.27)
 

N.S.
 

3.273
 

0.824
 

28.393
 

105
 

1/30(0.03)
 

N.S.
 

0.350
 

0.006
 

6.326
 

96
 
TOPOGRAPHY : MORPHOLOGY
 

Thyroid: C-Cell Carcinoma or C-Cell
 

Adenoma"3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Testis: Interstitial-Cell Tumor
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED) 

CONTROL
 

2/21(0.10)
 

N.S.
 

97
 

1/21(0.05)
 

N.S.
 

110
 

21/25(0.84)
 

N.S.
 

97
 

LOW
 

DOSE
 

3/39(0.08)
 

N.S.
 

0.808
 

0.102
 

9.161
 

107
 

2/39(0.05)
 

N.S.
 

1.077
 

0.060
 

61.897
 

104
 

34/50(0.68)
 

N.S.
 

0.810
 

0.658
 

1.122
 

90
 

HIGH
 

DOSE
 

1/30(0.03)
 

N.S.
 

0.350
 

0.006
 

6.326
 

96
 

0/30(0.00)
 

N.S.
 

0.000
 

0.000
 

12.911
 

29/46(0.63)
 

N.S.
 

0.751
 

0.601
 

1.061
 

93
 
___ 

___ 

00 

TOPOGRAPHY :MORPHOLOGY
 

Skin: Squamous-Cell Papilloma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Spleen: Fibroma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED) 

CONTROL
 

2/25(0.08)
 

P = 0.037(N)
 

110
 

1/25(0.04)
 

N.S.
 

110
 

0/25(0.00)
 

N.S.
 

LOW
 

DOSE
 

0/50(0.00)
 

N.S.
 

0.000
 

0.000
 

1.685
 

3/50(0.06)
 

N.S.
 

1.500
 

0.130
 

77.150
 

95
 

7/50(0.14)
 

N.S.
 

Infinite
 

0.997
 

Infinite
 

102
 

HIGH
 

DOSE
 

0/48(0.00)
 

N.S.
 

0.000
 

0.000
 

1.753
 

0/48(0.00)
 

N.S.
 

0.000
 

0.000
 

9.720
 

6/46(0.13)
 

N.S.
 

Infinite
 

0.894
 

Infinite
 

107
 
___ 

TOPOGRAPHY: MORPHOLOGY
 

Spleen: Fibrosarcoma or Sarcoma NOS
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Spleen: Hemangiosarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Body Cavities, Multiple Organs:
 

Fibrosarcoma or Sarcoma NOS^1
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED)
 

CONTROL
 

0/25(0.00)
 

P = 0.020
 

0/25(0.00)
 

P = 0.001
 

0/25(0.00)
 

P = 0.004
 

LOW
 

DOSE
 

7/50(0.14)
 

N.S.
 

Infinite
 

0.997
 

Infinite
 

102
 

19/50(0.38)
 

P < 0.001
 

Infinite
 

3.140
 

Infinite
 

94
 

2/50(0.04)
 

N.S.
 

Infinite
 

0.151
 

Infinite
 

99
 

HIGH
 

DOSE
 

9/46(0.20)
 

P = 0.015
 

Infinite
 

1.470
 

Infinite
 

79
 

20/46(0.43)
 

P < 0.001
 

Infinite
 

3.616
 

Infinite
 

93
 

9/48(0.19)
 

P = 0.017
 

Infinite
 

1.408
 

Infinite
 

83
 
TABLE 3 (CONCLUDED)
 

TOPOGRAPHY: MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Spleen or Body Cavities , Multiple Organs : 

Fibrosarcoma or Sarcoma NOS" 

P Values0 

0/25(0.00) 

P < 0.001 

5/50(0.10) 

N.S. 

18/48(0.38) 

P < 0.001 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Infinite 

0.648 

Infinite 

Infinite 

3.086 

Infinite 

Weeks to First Observed Tumor 99 79 

Spleen or Body Cavities, Multiple Organs:
 

Hemangiosarcoma" 0/25(0.00) 19/50(0.38) 21/48(0.44)
 

P Values0 P = 0.001 P < 0.001 P < 0.001
 

Relative Risk (Control) Infinite Infinite
 

Lower Limit 3.140 3.651
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 94 93
 

Treated groups received doses of 0.3 or 0.6 percent in feed.
 

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

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

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

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

given beneath the incidence of tumors in the treated group when P < 0 .05; otherwise, not signiflcant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 
TABLE 4 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT 

SPECIFIC SITES IN FEMALE RATS TREATED WITH ANILINE HYDROCHLORIDE* 

LOW HIGH 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE DOSE 

Hematopoietic System: Leukemia or 

Malignant Lymphoma" 3/24(0.13) 4/50(0.08) 0/50(0.00) 

P Values0 P = 0.019(N) N.S. P = 0.031(N) 

Relative Risk (Control) 0.640 0.000 

Lower Limit 0.120 0.000 

Upper Limit 4.113 0.793 

Weeks to First Observed Tumor 94 106 

Spleen: Sarcoma NOS 0/23(0.00) 0/50(0.00) 3/50(0.06) 

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

Relative Risk (Control) Infinite 

Lower Limit 0.285 

Upper Limit Infinite 

Weeks to First Observed Tumor • 103 

Pituitary: Adenoma NOS 6/21(0.29) 14/46(0.30) 8/40(0.20) 

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

Relative Risk (Control) 1.065 0.700 

Lower Limit 0.463 0.253 

Upper Limit 2.980 2.167 

Weeks to First Observed Tumor 91 87 85 
TABLE

TOPOGRAPHY: MORPHOLOGY
 

Pituitary: Adenoma NOS or Carcinoma NOS
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Cortical Adenoma or Cortical
 

Carcinoma^1
 

OJ P Values0 

l-o 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocytoma or Pheochromocytoma, Malignant"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

4 (CONTINUED) 

CONTROL
 

6/21(0.29)
 

N.S.
 

91
 

0/24(0.00)
 

N.S.
 

1/24(0.04)
 

N.S.
 

110
 

LOW
 

DOSE
 

15/46(0.33)
 

N.S.
 

1.141
 

0.505
 

3.156
 

87
 

2/50(0.04)
 

N.S.
 

Infinite
 

0.146
 

Infinite
 

107
 

0/50(0.00)
 

N.S.
 

0.000
 

0.000
 

8.966
 

HIGH
 

DOSE
 

9/40(0.22)
 

N.S.
 

0.787
 

0.299
 

2.373
 

4/48(0.08)
 

N.S.
 

Infinite
 

0.477
 

Infinite
 

107
 

5/48(0.10)
 

N.S.
 

2.500
 

0.306
 

115.634
 

85 

107 
TOPOGRAPHY: MORPHOLOGY
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinoma^
 u> 

OJ 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Manmary Gland: Fibroadenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (CONTINUED) 

CONTROL
 

1/21(0.05)
 

N.S.
 

109
 

1/21(0.05)
 

N.S.
 

—_ _
 

110
 

4/24(0.17)
 

P = 0.006(N)
 

109
 

LOW
 

DOSE
 

2/38(0.05)
 

N.S.
 

1.105
 

0.062
 

63.481
 

107
 

0/38(0.00)
 

N.S.
 

0.000
 

0.000
 

10.263
 

5/50(0.10)
 

N.S.
 

0.600
 

0.145
 

2.807
 

107
 

HIGH
 

DOSE
 

0/38(0.00)
 

N.S.
 

0.000
 

0.000
 

10.263
 

2/38(0.05)
 

N.S.
 

1.105
 

0.062
 

63.481
 

108
 

0/50(0.00)
 

P = 0.009(N)
 

0.000
 

0.000
 

0.514
 
TABLE 4 (CONTINUED)
 

TOPOGRAPHY : MORPHOLOGY CONTROL 

Uterus: Endometrial Stromal Polyp 

P Values0 

2/24(0.08) 

N.S. 

Departure from Linear Trend P = 0.008 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 102 

Body Cavities, Multiple Organs:

sarcoma or Sarcoma NOS^3 

P Values0 

 Fibro0/24(0.00) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Spleen or Body Cavities, Multiple Organs: 

Fibrosarcoma or Sarcoma NOS° 

P Values0 

0/24(0.00) 

P = 0.009 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

LOW
 

DOSE
 

15/48(0.31)
 

P = 0.027
 

3.750
 

0.988
 

31.911
 

107
 

1/50(0.02)
 

N.S.
 

Infinite
 

0.026
 

Infinite
 

88
 

1/50(0.02)
 

N.S.
 

Infinite
 

0.026
 

Infinite
 

88
 

HIGH
 

DOSE
 

7/50(0.14)
 

N.S.
 

1.680
 

0.356
 

15.815
 

107
 

4/50(0.08)
 

N.S.
 

Infinite
 

0.458
 

Infinite
 

83
 

7/50(0.14)
 

N.S.
 

Infinite
 

0.960
 

Infinite
 

83
 
TABLE 4 (CONCLUDED)
 

•a
 

Treated groups received doses of 0.3 or 0.6 percent in feed.
 

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

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

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

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

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, net significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact tests a negative designation (N) indicates a lower incidence in the treated group(s) than in the control group.
 

The 95% confidence interval on the relative risk of the treated group to the control group.
 £
 w The probability level of the test for departure from linear trend is given beneath the control
 

<-" group when P < 0.05.
 
every type of malignant tumor in either sex where at least two such
 

tumors were observed in at least one of the control or aniline HCI-


dosed groups and where such tumors were observed in at least 5 percent of the group.
 

In dosed male rats numerous sarcomas were observed both in the
 

*
 spleen and in multiple organs of the body cavities. The Cochran-


Armitage test indicated a significant (P = 0.001) positive association between dosage and the incidence of hemangiosarcomas of the
 

spleen. Both the Fisher exact tests showed a significantly (P <
 

0.001) greater incidence in comparing the respective dosed groups to
 

the control. The Cochran-Armitage test also indicated a significant
 

(P = 0.002) positive association between dosage and the combined
 

incidence of fibrosarcomas or sarcomas NOS of the spleen. Again,
 

the Fisher exact test comparing high dose to control was significant
 

(P = 0.015). In historical control data compiled by this laboratory
 

for the NCI Carcinogenesis Testing Program, none of the 250 male
 

untreated Fischer 344 rats had one of these tumors—compared to the
 

20/46 (43 percent) hemangiosarcomas and 9/46 (20 percent) fibrosarcomas or sarcomas NOS observed in the male high dose group.
 

When the combined incidence of fibrosarcomas or sarcomas NOS of
 

multiple organs of the body cavities was considered, for males the
 

*
 

In this report reference is made to neoplasms of "multiple organs
 

of the body cavities." Such a neoplasm was observed in more than
 

one of the organs located in the pleural or the abdominal cavity
 

(or both).
 

36
 
Cochran-Armitage test indicated a significant (P = 0.004) positive
 

association between dose and incidence. The high dose Fisher exact
 

comparison was also significant (P = 0.017).
 

When the combined incidence of fibrosarcomas or sarcomas NOS
 

either of the. spleen alone or of multiple organs of the body cavities
 

was considered, for males the Cochran-Armitage test indicated a significant (P < 0.001) positive association between dose and incidence.
 

For males the Fisher exact test comparing the high dose to the control was also significant (P < 0.001). For females the Cochran-


Armitage test was significant (P = 0.009), but the Fisher exact tests
 

were not. This tumor combination was also rare in untreate;d Fischer
 

344 historical control rats, as none of the 250 male or 249 females
 

had one of these tumors. Making the assumption of a binomial distribution with a probability of 1/250 of spontaneous incidence (the most
 

conservative estimate), the probability of observing 7 or more rats
 

with one of these tumors out of 50 females (as in the high dose females) was P < 0.00001, a significant result.
 

Based upon these statistical results the administration of aniline
 

HC1 was associated with the increased incidence of hemangiosarcomas of
 

the spleen and of fibrosarcomas or sarcomas NOS both of the spleen and
 

of multiple organs of the body cavity in male rats. There was also
 

the possibility of an association between administration and the
 

increased combined incidence of fibrosarcomas or sarcomas NOS either
 

37
 
of the spleen or of multiple organs of the body cavities in female
 

rats.
 

For males the Cochran-Armitage test indicated a significant
 

(P = 0.022) positive association between dosage and the incidence
 

of adren.al neoplasms. The Fisher exact tests, however, were not
 

significant. No other tests for either males or females indicated
 

a significant positive association under the Bonferroni criterion.
 

For males the Cochran-Armitage test indicated a significant
 

negative association both between dosage and the incidence of
 

squamous-cell papillomas of the skin and between dosage and the
 

incidence of adrenal neoplasms. In both cases, however, no Fisher
 

exact tests; were significant. Similarly, for females the Cochran-


Armitage test showed a significant negative association for the
 

incidence of leukemia or malignant lymphomas, but no Fisher exact
 

tests were significant under the Bonferroni criterion.
 

The possibility of a negative association between dosage and
 

the incidence of mammary fibroadenomas was observed in female rats.
 

38
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Mean body weight depression occurred in dosed male mice, but not
 

in dosed female mice (Figure 4).
 

Partial alopecia was observed in 35 high dose males, 33 low dose
 

males, 39 high dose females, 21 low dose females, 28 control males,
 

and 30 control females. Abdominal distent ion was reported in two low
 

dose females, one control male, and one control female. Swollen eyes
 

were observed in two high dose males. No other clinical abnormalities
 

were noted.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and aniline HCl-dosed groups are shown in Figure 5.
 

For both males and females the Tarone test did not indicate a significant association between dosage and mortality.
 

Adequate numbers of males were at risk from late-developing
 

tumors, as 82 percent (41/50) of the high dose, 86 percent (43/50)
 

of the low dose, and 66 percent (33/50) of the control mice survived
 

on test until the end of the study. Seven control males were autolyzed in weeks 11 to 13. Survival was also adequate among females
 

as 84 percent (41/49) of the high dose, 74 percent (37/50) of the
 

low dose, and 60 percent (30/50) of the control mice survived on
 

test until the end of the study.
 

39
 
120 

TIME ON TEST (WEEKS) 

r T 

45 60 90 105 120

 

TIME ON TEST (WEEKS) 

FIGURE 4 

GROWTH CURVES FOR ANILINE HYDROCHLORIDE CHRONIC STUDY MICE 

40 
1.0 

« 

0.8­

0.6­

no 0.4 — 

CD 

O 

DC 

O. 

0.2­

MALE MICE 

0.0­

15 30 45 60 75 

TIME ON TEST (WEEKS) 

1

^""^^^^"^ ~ 

^^"~1

< 0 8 

cc 

z> 

f> 0.6­

O 

| 

m o.4­

m 

O 

oc 

Q. 

0.2­

- FEMALE MICE

0 0 i | I | I 1 i 1 i I i

0 15 30 45 60 75

TIME ON TEST (WEEKS) 

FIGURE 5 

-0.8 

-0.6 

-0.4 

COMTROL 

- 0.2 

LOW DOSE 

HIGH DOSE 

-0.0 

90 105 120 

1.0 

 *""^«. ... •«. « ••••••• ^^_

 

 •-. LI 

h "'•• 

0.8 

'n

-0.6 

-

-0.4 

i ­

~ 

_^_^__ CONTROL 

- 0.2 

LOW DOSE 

- HIGH DOSE 

| i i 00 

 90 105 120 

SURVIVAL COMPARISONS OF ANILINE HYDROCHLORIDE CHRONIC STUDY MICE 

41 
C. Pathology
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

summarized in Appendix D (Tables Dl and D2).
 

None of the observed tumors were considered to be compound-


induced, as they were found in approximately equal numbers in control
 

and dosed groups.
 

There were also numerous degenerative and inflammatory lesions
 

which commonly occur in aging mice of this strain. A chronic inflammation of bile ducts was only seen in dosed male mice. Other non-


neoplastic lesions were not considered to be related to feeding of
 

aniline HC1.
 

The pathologic examination provided no evidence for the carcinogenicity of aniline HC1 in B6C3F1 mice under the conditions
 

of this test.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

tumors were observed in at least one of the control or aniline
 

HCl-dosed groups and where such tumors were observed in at least 5
 

percent of the group.
 

For males the Cochran-Armitage test indicated a significant
 

(P = 0.042) negative association between dosage and the incidence of
 

42
 
TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH ANILINE HYDROCHLORIDE*
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar Carcinoma or
 

Alveolar/Bronchiolar Adenoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

CONTROL
 

2/39(0.05)
 

N.S.
 

109
 

4/39(0.10)
 

N.S.
 

109
 

13/39(0.33)
 

N.S.
 

LOW
 

DOSE
 

3/49(0.06)
 

N.S.
 

1.194
 

0.144
 

13.758
 

107
 

8/49(0.16)
 

N.S.
 

1.592
 

0.465
 

6.757
 

107
 

7/49(0.14)
 

P = 0.031(N)
 

0.429
 

0.162
 

1.039
 

HIGH
 

DOSE
 

1/49(0.02)
 

N.S.
 

0.398
 

0.007
 

7.377
 

107
 

3/49(0.06)
 

N.S.
 

0.597
 

0.093
 

3.330
 

81
 

11/49(0.22)
 

N.S.
 

0.673
 

0.311
 

1.448
 

Weeks to First Observed Tumor 100 95
 73
 
TABLE 5 (CONCLUDED)
 

LOW	 HIGH
 

TOPOGRAPHY: MORPHOLOGY CONTROL DOSE
 DOSE
 

Liver: Hepatocellular Carcinoma 12/35(0.31) 9/49(0.18) 7/49(0.14)
 

P Values0	 P = 0.042(N) N.S. N.S.
 

Relative Risk (Control) 0.597 0.464
 

Lower Limit 0.250 0.173
 

Upper Limit 1.384 1.154
 

Weeks to First Observed Tumor	 86 81 107
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinoma"3 0/38(0.00) 3/43(0.07) 1/43(0.02)
 

P Values0	 N.S. N.S. N.S.
 •P­

•P-
 Relative Risk (Control) Infinite Infinite
 

Lower Limit 0.536 0.048
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor	 82 107
 

a

Treated groups received doses of 0.6 or 1.2 percent in feed.
 

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

C

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

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

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

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

The 95% confidence interval on the relative risk of the treated group to the control group.
 
TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH ANILINE HYDROCHLORIDE'
 

TOPOGRAPHY : MORPHOLOGY
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

-o
 

Ul Circulatory System: Hemangioma or
 

Hemangiosarcomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

13/49(0.27)
 

N.S.
 

58
 

1/49(0.02)
 

N.S.
 

109
 

1/46(0.02)
 

N.S.
 

109
 

LOW
 

DOSE
 

17/49(0.35)
 

N.S.
 

1.308
 

0.675
 

2.589
 

90
 

0/49(0.00)
 

N.S.
 

0.000
 

0.000
 

18.651
 

5/48(0.10)
 

N.S.
 

4.792
 

0.566
 

221.559
 

107
 

HIGH
 

DOSE
 

18/49(0.37)
 

N.S.
 

1.385
 

0.726
 

2.711
 

82
 

3/49(0.06)
 

N.S.
 

3.000
 

0.251
 

154.197
 

71
 

5/48(0.10)
 

N.S.
 

4.792
 

0.566
 

221.559
 

107
 
TABLE

TOPOGRAPHY : MORPHOLOGY
 

Pituitary: Adenoma NOS
 

P Values0
 

cl
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Pheochromocytoma or Pheochromocytona, Malignant
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinoma^3
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

6 (CONTINUED) 

CONTROL
 

3/34(0.09)
 

N.S.
 

109
 

4/46(0.09)
 

P = 0.027(N)
 

68
 

4/44(0.09)
 

P = 0.026(N)
 

80
 

LOW
 

DOSE
 

2/32(0.06)
 

N.S.
 

0.708
 

0.062
 

5.772
 

107
 

1/45(0.02)
 

N.S.
 

0.256
 

0.005
 

2.452
 

107
 

0/33(0.00)
 

N.S.
 

0.000
 

0.000
 

1.418
 

HIGH
 

DOSE
 

0/36(0.00)
 

N.S.
 

0.000
 

0.000
 

1.551
 

0/45(0.00)
 

N.S.
 

0.000
 

0.000
 

1.099
 

0/36(0.00)
 

N.S.
 

0.000
 

0.000
 

1.304
 
TABLE 6 (CONCLUDED)
 

o
 

Treated groups received doses of 0.6 or 1.2 percent in feed.
 

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

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

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

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

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

The 95% confidence interval on the relative risk of the treated group to the control group.
 
hepatocellular carcinomas. The Fisher exact tests, however, were not
 

significant. Similarly, for females Cochran-Armitage tests showed
 

significant negative associations between dosage and the incidence of
 

follicular-cell neoplasms of the thyroid and between dosage and the
 

combined incidence of pheochromocytomas or malignant pheochromocytomas
 

of the adrenal gland. In both cases, however, the Fisher exact tests
 

were not significant.
 

There were no other statistically significant test results for
 

either males or females under the Bonferroni criterion. Based upon
 

these results there was no statistical evidence that indicated aniline HC1 was a carcinogen in male or female mice.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

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

of statistically significant results. It should also be noted that
 

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

indicating the theoretical possibility of tumor induction in mice by
 

aniline HC1 that could not be established under the conditions of
 

this test.
 

48
 
V. DISCUSSION
 

There were no significant positive associations between the
 

administered dietary concentrations of aniline HC1 and mortality in
 

either sex of rats or mice. Adequate numbers of animals in all groups
 

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

Slight mean body weight depression, relative to controls, was observed
 

in dosed female and high dose male rats and in dosed male mice.
 

In male rats there were several types of mesenchymal tumors, primarily of the spleen, associated with compound administration. There
 

was a significant positive association between dietary concentration
 

of the chemical and the incidence of hemangiosarcomas of the spleen.
 

The high dose to control and low dose to control Fisher exact comparisons supported the finding. The combined incidence of fibrosarcomas
 

and sarcomas NOS of the spleen was also statistically significant in
 

male rats. Each of these types of sarcoma of the spleen is rare in
 

untreated male Fischer 344 historical control rats. When the numbers
 

of male rats having fibrosarcoma and/or sarcoma NOS of multiple body
 

organs were combined, there was a significant positive association
 

between aniline HC1 concentration and incidence. This finding was
 

supported by the high dose to control Fisher exact comparison.
 

Therefore, under the conditions of this bioassay, aniline HC1 was
 

carcinogenic to male Fischer 344 rats.
 

The number of female rats having fibrosarcomas or sarcomas NOS
 

of either the spleen alone or multiple organs of the body cavity was
 

49
 
significantly associated with increased dietary concentration of aniline HC1. Although this result was not supported by the Fisher exact
 

tests, these tumors are rare in female Fischer 344 rats. Historical
 

data indicate that none of the 249 female Fischer 344 control rats at
 

this laboratory had any of these tumors. Assuming a binomial distribution, the probability of the incidence observed in the high dose
 

group (7/50 [14 percent]) occurring by chance is less than 1 in
 

100,000. This result is, therefore, considered indicative of a carcinogenic effect of compound administration.
 

In mice no tumors occurred with significantly greater frequency
 

in dosed groups than in control groups.
 

Experimental data reviewed by the International Agency for
 

Research on Cancer was not considered adequate to indicate that aniline HC1 is carcinogenic to rats or mice (international Agency for
 

Research on Cancer, 1974). No tumors were observed in bladders,
 

livers, spleens or kidneys of randomly bred rats (sex unspecified)
 

surviving up to 750 days after initiation of oral intake of 22 mg/
 

day of aniline HC1 (Druckrey, 1950). Tumors were absent in 11 mice
 

(strain unspecified) 12 months after they received 13 subcutaneous
 

injections of aniline HC1 (4 mg in aqueous solution) (Hartwell and
 

Andervont, 1951; as cited in Hartwell, 1963).
 

Under the conditions of this bioassay, dietary administration
 

of aniline HC1 was carcinogenic to male and female Fischer 344 rats,
 

inducing hemangiosarcomas and a combination of fibrosarcomas and
 

50
 
sarcomas NOS of the spleen and a combination of fibrosarcomas and
 

sarcomas NOS of multiple body organs. There was no evidence of a
 

carcinogenic effect due to the administration of aniline HC1 to
 

B6C3F1 mice of either sex.
 

51
 
VI. BIBLIOGRAPHY
 

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

J. Wiley & Sons, New York, 1971.
 

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

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

Union Against Cancer, Geneva, 1969.
 

Chemical Abstracts Service. The Chemical Abstracts Service (CAS)
 

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

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

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

Co., Ltd., London, 1970.
 

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

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

Druckrey, H., "Beitrage zur Pharmakologie Cancerogener Substanzen.
 

Versuche mit Anilin." Arch, exp. Path. Pharmakol. 210:137, 1950.
 

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

Tests, Confidence Limits, and Adjustments for Stratification."
 

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

Hamblin, D.O., "Aromatic Nitro and Amino Compounds." Industrial
 

Hygiene and Toxicology, 2nd edition. Interscience Publishers,
 

New York, 1963.
 

Hartwell, J.L. and H.B. Andervont; as cited in Hartwell, J.L., Survey
 

of Compounds Which Have Been Tested for Carcinogenicity, 2nd
 

Edition. U.S. Department of Health, Education, and Welfare,
 

Public Health Service Publication 149, U.S. Government Printing
 

Office, Washington, D.C., 1963.
 

International Agency for Research on Cancer, IARC Monographs on the
 

Evaluation of Carcinogenic Risk of Chemicals to Man: Some Aromatic Amines, Hydrazine and Related Substances, N-Nitroso Compounds, and Miscellaneous Alkylating Agents, Volume 4. IARC,
 

Lyon, France, 1974.
 

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

Observations." Journal of the American Statistical Association
 

53:457-481, 1958.
 

52
 
Kouris, C.S., and H. Northcott, "Aniline and its Derivatives."
 

Kirk-Othmer Encyclopedia of Chemical Technology, 2nd edition,
 

Volume 2. Interscience Publishers, New York, 1963.
 

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

"Carcinogenesis Bioassay Data System." Computers and Biomedical
 

Research 7:230-248, 1974.
 

Linstead, R.P. and E.A. Braude, "Amines from Nitro Compounds." British Patent 705,919 (National Research Development Corp.), March
 

24, 1954; Chemical Abstracts 50, 1079g.
 

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

Co., New York, 1966.
 

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

Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced
 

by Different Numbers of Administration of Benzo (a) Pyrene and
 

Ferric Oxide." Cancer Research 32:1073-1079. 1972.
 

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

62^:679-682, 1975.
 

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

U.S. Production and Sales, 1975. USITC Publication 804, U.S.
 

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

Windholz, M., editor, The Merck Index: An Encyclopedia of Chemicals
 

and Drugs, Ninth edition. Merck and Co., Rahway, New Jersey,
 

1976.
 

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

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

1963.
 

53
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH ANILINE HYDROCHLORIDE
 

TABLE Al

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

TREATED WITH ANILINE HYDROCHLORIDE 

CONTROL (IINT8) 

01-0330 

LOW DOSE 

01-0315 

ANIMAL S INITIALLY IN STUDY 25 50 ANIMAL S NECROPSIED 25 50 ANIMALS EXAMINE D HI STOPATHOLOGICALLY ** 25 50 

INTEGOMENTAS Y SYSTEM 

*SKIN (25) (50) S3UAMOUS CELL PAPILLOMA 2 (8*) 

SQUASOUS CELL CARCINOMA 1 <2X) LIPOSARCOMA 1 (2X) 

*SUBCOT TISSUE (25) (50) FIBROM A 1 (4X) FIBSO SARCOMA 1 <2X ) 

RESPIRATORY SYSTEM 

*LUNG (25) (50) PHEOCHROMOCYTOMA. METASTATIC 1 (4*) 

HEMATOPOIETIC SYSTEM 

*MULTIPLE ORGANS (25) (50) NYELOMONOCYTIC LEUKEMIA 1 (4*) 2 («*) 

•SUBCUT TISSUE/HEAD (25) (50) MALIG.LYMPHOMA , LYMPHOCYTIC TYPE 1 (2X) 

IBONE MARRO W (23) («7) HEMANGIOM A 1 (2X) 

*SPLBEN (25) (50) SARCOMA , NOS 4 <3X) FIBROMA 7 (14X) FIBRO SARCOM A 3 (6%) HSMANGIOSARCOH A 19 (38X) 

tMANDIBBLAR L. NODE (24) («7) 

C-SELL CARCINOMA . MBTASTATIC J_J*S1 

i NUMBER OF ANIMAL S WITH TISS1E EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-3 

HIGH DOSE 

01-0320 

50 

48 

48 

(48) 

(48) 

1 (2X) 

(45) 

(48) 

(48) 

(45) 

(46) 

2 (4X) 6 (13X) 

7 (15*) 20 (1)3*) 

(35) 
TABLE Al (CONTINUED) 

CONTROL (UNTR) LOR DOSE HIGH D3SE 

01-0330 01-0315 01-0320 

CIRCULATOR Y SYSTEM 

NON E 

DIGESTIVE SYSTKtl 

*LIVER (25) (50) («) NEOPLASTIC NODDLE 1 (U%)

 

*STOHACH (24) (48) (45) 

SQUAHOUS CELL PAPILLOMA 1 (2»)

 

DRINAR Y SYSTEM 

»U. BLADDER/MDCOSA (25) (49) (4U) HEMANGIOMA 1 (2X)

 

ENDOCRINE SYSTEM 

«PIlaiTAEY (22) (»5) (34)

 ADENOMA , NOS 2 (9X) 7 (16*) 4 (12X)

 

IADRENA L (24) (50) (44)

 

CORTICAL ADENOMA 1 (2X) PHEOCHROMOCYTOMA 1 («*) 5 (10X) 11 (25X)

 PHEOCHROMOCYTOMA, MALIGNAN T 1 (<•*) 1 (2«) J (2X)

 

(•THYROID (21) (J9) (30) PAPILLAR Y ADENOCABCINOMA 1 (3X)

 FOLLICOLAR-CELL ADENOBA 1 (5X) 1 (3X)

 FOLLICULAR-CELL CARCINOM A 1 (3X)

 C-CELL ADENOM A 1 (3X)

 C-CELL CARCINOMA 2 (IDS) 2 (5X) 1 (3X)

 

tPANCREATIC ISLETS (25) («5) (37)

 

ISLET-CELL ADENOM A 1 (4X) 1 (2X)

 

REPBODUCriVE SYSTEM 

fTESIIS (25) (50) (46) INTERSTITIAL-CELL TOMOR 21 181*%). 3U (68%) 2s_jsasi. _ 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

A-4 
TABLE Al (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

01-0330 01-0315 01-0320 

NERVOU S SYSTEM 

tBRAIN (25) («9) (45) 

ASTROCYTOBA 2 (4*) 

SPECIAL SENSE ORGAN S 

*EAP CANA L (25) (50) (48) 

SQOAMOOS CELL CARCINOMA 1 (4*) 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

*BODY CAVITIES (25) (50) (48) 

MESOTHELIOMA, NOS 2 (4X) 2 (4*) 

ALL OTHER SYSTEMS 

•MULTIPLE ORGANS (25) (50) (48) 

SARCOMA, NOS 1 (2X) 

FIB80SARCOMA 2 (4X) 8 (17*) 

LEIOMYOSARCOHA 2 (4X) 

HEMANGIOSARCOMA 1 (2X) 

ANIMA L DISPOSITION SUMMAR Y 

ANIMALS INITIALLY IN STUDY 50 50 

N A T ORA L DEATHS 19 

HORIBDND SACRIFICE 4 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 17 34 27 

ANIMA L MISSING 

S_!NCLa_MS_iUTQLYZED_AN!MAI.S 

* NUMBER OF ANIMAL S WITH TISSUE EXAMINED HICKOSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

A-5 
TABLE Al (CONCLUDED) 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMOFS*


TOTAL PRIHARY IDMORS


TOTAL ANIMALS HITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TOMORSt


TOTAL SECONDARY TUMORS


TOTAL ANIMALS HITH TUMORS UNCERTAINBENIGN OR MALIGNANT


TOTAL UNCERTAIN TtTMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL (UNTR)

01-0330

 21


 35


 21


 29


 5


 5


 2
 

2
 

1


 1


 LOW DOSE HIGH DOSE 

 01-0315 01-0320 

 47 47
 

 102 97
 

 42 33
 

 60 50
 

 35 42
 

 40 45
 

2 2
 

2 2
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

* SECONDAPY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

A-6
 
TABLE A2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

TREATED WITH ANILINE HYDROCHLORIDE 

CONTROL (UNTR) L OU DOSE HIGH DOSE 02-0330 02-0315 02-0320 

ANIMAL S INITIALLY IN STUDY 25 50 50 

ANIMAL S NECROPSIED 21* 50 50 

ANIMAL S EXAMINE D HI STOPATHOLOGICALLY ** 2« 50 50 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (24) (50) (50) SARCOMA, NOS 1 (2%) FIBROSARCOMA 1 (1%) 

RESPIRATOR Y SYSTEM 

NONE 

HEMATOPOIETIC SYSTEM 

*MULIIPLE ORGAN S (2U) (50) (50)

 

MALIG.LYMPHOHA , HISTIOCYTIC TYPE 1 (U%) 

LEUKEMIA,NOS 1 (2%)

 

MYELOMONOCYTIC LEUKEMIA 2 (8X) 3 (6X)

 

ISPLEEN (23) (50) (50) SARCOMA, NOS 3 (6%) 

LIPOHA 1 <2X ) HEMANGIOMA 1 (2X) HEMANGIOSARCOHA 1 (2X) 2 (UX) 

CIRCULATOR Y SYSTFM 

NON E 

DIGESTIVE SYSTEM 

*LIVE3 (2H ) (50) (50) NEO_£L.ASTIC_NODULE_ J_J2$1_ 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-7 
URINAR Y SYSTEM 

iURINAR Y BLADDER 

PAPILLOMA , NOS 

TRANSITIONAL-CEL L PAPILLOM A 

ENDOCRIN E SYSTE M 

tPITUITAR Y 

CAPCINOMA , NOS 

ADENOMA , NOS 

*ADRENA L 

CORTICA L ADENOM A 

CORTICAL CARCINOM A 

PHEOCHROMOCYTOM A 

PHEOCHROMOCYIOMA , MALIGNAN T 

tTHYROID 

FOLLICDLAR-CEL L ADENOMA 

FOLLICIILAR-CEL L CAPCINOM A 

C-CELL ADENOM A 

C-CELL CAPCINOM A 

tPANCPEATIC ISLETS 

ISLET -CELL ADENOM A 

ISLET-CELL CARCINOM A 

EEPPODUCTIV E SYSTEM 

*MAM.1ARY GLAND 

FIBROADENOM A 

*CLIIORA L GLAN D 

CARCINOMA , NOS 

*VAGIN A 

SARCOMA , NOS 

*UTERUS 

ADENOCARCINOHA , NOS 

ENDOMETRIA L STROMA L POLYP 

* NUMBE R OF ANIMAL S H ITH TISSUE

* NUMBE R OF ANIMAL S NECPOPSIED 

TABLE A2 (CONTINUED) 

CONTROL (IIHIR) LOU DOSE 

02-0330 02-0315 

(24) (50) 

1 (4%) 

(21) (46) 

1 (2X) 

6 (29X) 14 (3 OX) 

(24) (50) 

2 (4X) 

1 (4X) 

(21) (38) 

1 (5*) 1 (3%) 

1 (5X) 1 (3%) 

(22) (49) 

1 (2X) 

1 (5%) 

(24) (50) 

4 (17X) 5 (10X) 

(24) (50) 

1 (it*) 

(24) (50) 

(24) (48) 1 (ill) 15 J31X1 _ 

 EXAMINE D MICROSCOPICALLY 

A-8 

HIGH DOSE 

02-0320 

(49) 

1 (2X) 

(40) 

1 <3X) 

8 (2 OX) 

(48) 

3 (6X) 

1 (2X) 

3 (6X) 

2 (4X) 

(38) 

1 (3X) 

1 (3X) 

(49) 

(50) 

(50) 

(50) 

1 (2X) 

(50) 

1 (2X) . 7..114X) 
TABLE A2 (CONTINUED) 

CONTROL(UNTH ) LOW DOSE HIG H DOSE 

02-0330 02-0315 02-0320 

ENDOHETRIA L STEOMAL SARCOMA 1 (<t% ) 

tUTERUS/ENDOMETBIU H (24) (US ) (50) 

LEIOMYOSARCOMA 1 ______- (2*) Y _ _____ _

 _ —__ _ _ „ — ____ _ 

NERVOU S SYSTEM 

IBRAI N (23) (U9 ) (50) ASTROCYTOIU 1 (2X)

 

SPECIAL SENSE ORGAN S 

*EAR CANA L (21*) (50) (50) 

SQUAHOOS CELL CARCINOM A 1 (U* ) 

MUSCOLOSKELETA L SYSTEM 

NON E 

BODY CAVITIES 

•ABDOMINAL CAVIT Y (2<4) (50) (50) 

LEIOSYOSAPCOMA 1 (UJ) 

A LL OTHER SYSTEMS 

*MULTIPLE ORGAN S (24) (50) (50) 

SARCOMA , NOS 1 (2*) 

FIBROSAHCOMA 1 (2*) 3 (6X ) 

ANIMA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALLY It) STUDY 25 50 50 

NATURA L DEATHS U U U 

MORIBUN D SACRIFICE 5 2 5 

SCHEDULED SACRIFICE 

ACCIDENTALL Y KILLED 

TERMINA L SACRIFICE 1 6 U U U1 

ANIMA L MISSING 

S_INCLUDES_AUTOLYZID_ANIMI!S ] 

* NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIBD 

A-9 
TABLE A2 (CONCLUDED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

02-0330 02-0315 02-0320 

TUMO R SUMMAR Y 

TOTA L ANIMAL S WITH PRIMAR Y TUMORS* 17 32 35 

TOTAL PRIMAR Y TUMORS 25 U8 44

 

TOTAL ANIMAL S WIT H BENIGN TUMORS 1 0 26 21 

TOTAL BENIG N TUMORS 14 38 25 

TOTAL ANIMALS WITH MALIGNANT TUMORS 10 9 17 

TOTAL MALIGNAN T TUMORS 11 10 13

 

TOTAL ANIMAL S WITH SECONDARY TUMORS* 

TOTAL SECONDARY TUMORS 

TOTAL ANIMAL S WITH TUMORS UNCERTAIN ­

BEBIG H OR MALIGNAN T 1 

TOTAL UNCERTAI N TUMORS 1 

TOTAL ANIMAL S WITH TUMORS UNCERTAIN ­

PRIMAR Y OR METASTATIC 

TOTAL UNCERTAI N TUMOSS 

* PRIMAR Y TUMORS : ALL TUMORS EXCEPT SECONDAR Y TUMORS 

t SKCONDAP Y TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN 

A-IO 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH ANILINE HYDROCHLORIDE
 

TABLE Bl

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

TREATED WITH ANILINE HYDROCHLORIDE 

HIGH DOSE
 

05-0325
 

50
 

1(9
 

1*9
 

(49)
 

2 (4%)
 

1 (2%)
 

(49)
 

5 (10X)
 

2 (4%)
 

1 (2%)
 

(49)
 

1 (2)5)
 

1 (2X)
 

(45)
 

(48)
 

1 (2%)
 

(26)
 

I I4%1
 

ANIMALS INITIALLY IN STUDY
 

ANIHAIS MISSING
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALL Y
 

INTEGUMENTAR Y SYSTE M 

NON E 

RESPIRATORY SYSTEM
 

tLONG
 

HEPATOCELLULAR CARCINOMA, METAST
 

ALVEOLAE/BHONCHIOLAR ADENOMA
 

ALVEOLAR/BBONCHIOLAB CARCINOMA
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS
 

MALIGNANT LYMPHOMA, NOS
 

MALIG.LYMPHOSA, HISTIOCYTIC TYPE
 

GRANULOCYTIC LEUKEMIA
 

ISPLEEN
 

HEMANGIOBA
 

HEMANGIOSARCOMA
 

MALIGNANT LYMPHOMA, NOS
 

#MESENTERIC L. NODJ.
 

MALIGNANT LYMPHOMA, NOS
 

MALIG. LYMPHOMA, HISTIOCYTIC TYPE
 

IPEYERS PATCH
 

MALIGNANT LYMPHOMA, NOS
 

tTHYMUS
 

MALIGNANT LYMPHOMA,. NOS
 

CCNTBOL (UNTR)
 

05-0330
 

50
 

2
 

39
 ** 39
 

(39)
 

2 (5«)
 

2 (5%)
 

2 (5*)
 

(39)
 

11 (28%)
 

(38)
 

1 (3%)
 

(36)
 

1 (3%)
 

(37)
 

(13)
 

LOU DOSE
 

05-0320
 

50
 

49
 

49
 

(49)
 

5 (10)5)
 

3 (6%)
 

(K9)
 

4 <8X)
 

(149)
 

1 (2%)
 

(45)
 

3 (7%)
 

(48)
 

(28)
 

* NUMBE R OF ANIMAL S KITH TISSUE EXAMINE D MICROSCOPICALLY » NUMBE R OF ANIMAL S NECROPSIE D 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-3 
TABLE Bl (CONTINUED) 

CONTROL (UNTH) LOW DOSE HIGH DOSE 

05-0330 05-0320 05-0325 

CIRCULATOR Y SYSTE M 

*CABDIOVASCULA E SYSTE (39) (49) (19) 

HBMANGIOSARCOM A 1 (2X ) 

DIGESTIV E SYSTEM 

ttLIVSR (39) (49) (19) 

HEPATOCELLULAR CARCINOMA 12 (31X) 9 (18X) 7 (14%)
 

•STOMACH (37) (49) (47)
 

SQUAMOUS CELL PAPILLOMA 1 (2%)
 

URINAR Y SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

tTHYBOID (38) (43) (43)
 

FOLIICULAR-CELL ADENOMA 2 (5S) 1 (2%)
 

FCLLICULA8-CELL CARCINOMA 1 (2%)
 

tPANCREATIC ISLETS (36) (45) (46)
 

ISLET-CELL ADENOMA 1 (2%)
 

8;!PEODUCTIVE SYSTEM
 

*TE3TIS (38) (48) (49)
 

INTERSTITIAL-CELL TUMOR 1 (2%)
 

NERVOUS SYSTEM
 

NCNE
 

SPECIAL SENSE OBGANS
 

*HARD£RIAN GLAND (39) (49) (49)
 

flQS JL-12S1
 

* NUMEE E OF ANIMALS WITu TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMALS NECROPSIED
 

B-4
 
MUSCOLOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NON E
 

ANIMA L DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURA I DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLBD
 

TERMINAL SACRIFICE
 

ANIMA L MISSING
 

S_INCLODES_AUTOLYZED_ANIM4LS_
 

TABLE Bl (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

05-0330 05-0320 05-0325 

50 50 50
 13
 

2
 

33
 

2
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

B-5
 
TABLEBl (CONCLUDED)
 

TUMOB SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUMORS*


TOTAL PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TUMORS


10TAL EENIGN TUMORS


TOTAL ANIMALS KITH MALIGNANT TUMORS
 TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORS*
 TOTAL SECONDARY TUMORS


TOTAL ANIMALS HITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL (UNTR)


05-0330


 21


 29


 2
 

2


 22
 27


 2
 

2
 

 LOW DOSE HIGH DOSE
 05-0320 05-0325
 

 27 22
 

 31 25
 

9 5
 

 10 5
 

 20 19
 21 20
 

* PEIMABY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 * SECCNCARY TUMORS: METASTATIC TUMORS OR TUMOBS INVASIVE INTO AN ADJACENT ORGAN
 

B-6
 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

TREATED WITH ANILINE HYDROCHLORIDE 

CCNTROL (UNTR) LOW DOSE HIGH DOSE
 

06-0330 06-0320 06-0325
 

ANIMALS INITIALLY IS STUDY 50 50 350
 

ANIMALS MISSING 1
 

ANIMALS BECROPSIED 19 19 19
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 19 1*8 19
 

INTIGUMENTAHY SYSTEM
 

NONE
 

BESPIRATCRY SYSTEM
 

NONE
 

HEMATCPOIETIC SYSTEM 

*MULTIPLE ORGANS (19) (19) (49) 

MALIGNANT LYMPHOMA. NOS 10 (20X) 7 (14%) 16 (333S) MALIG.LYMPHOMA. HISTIOCYTIC TYPE 1 (2%) 4 (8X) 2 (IX) 

GEANULOCYTIC LEUKEMIA 1 (2)5) 

#SPLEEN (45) (48) (19) 

HEMANGIOSARCOMA 1 <2«) 

MALIGNANT LYMPHOMA. NOS 3 (6*) 

tMEDIASTINAL L.NODE 

MALIGNANT LYMPHOMA, NOS (11) (37) 

1 (3%) 

(16) 

•PANCREATIC L.NODi (11) (37) (46) 

MALIGNANT LYMPHOMA, NOS 1 (2%) 

*LIVER (16) (18) (18) 

MALIGNANT LYMPHOMA, NOS 1 (2%) 

tKIDNEY (16) (48) (19) 

MALIGNANT LYMPHOMA, NOS 1 (2%) 

CIHCUIATOBY SYSTEM
 

NONE
 

» NUMBEE OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 a 50 ANIMALS WERE INITIALLY IN THE STUDY, BUT ONE ANIMAL HAS FOUND TO BE A MALE IN A FEMALE GROUP.
 

B-7
 
TABLE B2 (CONTINUED)
 

CONTROL (UNTE) LOW DOSE


06-0330 06-0320


DIGESTIVE SYSTEM
 

#LIVER (46) (48)
 HEPATOCELLULAR CARCINOMA 1 (2*) 5 (10%)


tSTOMACH (41) (46)


SQUAMOUS CELL PAPILLONA 1 (2%)
 

OEINSBY SYSTEM
 

NONE
 

<!NDCCHINE SYSTEM
 

#PITUITARY (34) (32)


ADENOMA, NOS 3 (9X) 2 (6S)
 

#ADRENAL (46) (45)


PHEOCHROHOCYTOMA 3 (1%) 1 (2%)
 PHEOCHEOMOCYTCMA, MALIGNANT 1 (2%)
 

ITHYROID (44) (33)
 FOLLICULAR-CELL ADENOMA 2 (5*)
 

FCLLICULAS-CELL CARCINOMA 2 (5X)
 

REPRODUCTIVE SYSTEM
 

tUTEBUS (44) (46)
 ENDOMETRIAL STROMAL POLYP 1 (2%)
 

#OVARY (44) (45)
 GRANULOSA-CELL TUMOR
 

TUBULAR ADENOMA 1 (2*)
 

HEflANSIOMA
 

NERVOU S SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

* NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

B-8
 

 HIGH DOSE
 

 06-0325
 

 (48)
 

5 (10X)
 

 (47)
 

 (36)
 

 (45)
 

 (36)
 

 (43)
 

 (47)
 

1 (2%)
 

2 (»%)
 

2 (Hit)
 
MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDCMINAL CAVITY
 

HEHANGIOSARCONA
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 TERMINAL SACRIFICE
 

ANIMAL MISSING
 ANIMAL DELETEDfWRONG SEX)
 

S_INCLUDES_AUTOLYZED_ANIMAtS_.
 

TABLE B2 (CONTINUED) 

CONTROL (UNTR)
 

06-0330
 

50
 

17
 

3
 

30
 

LOW DOSE HIGH DOSE 06-0320 06-0325 

(49) 

1 (2*) 

50 50 11 

1 

37 11 1 

1 

DUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

NUMBER OF ANIMALS NECROPSIED
 

B-9
 
TABLE B2 (CONCLUDED)
 

TUMCB SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUMORS*


TOTAL PHIMABY TUMORS


TOTAL .ANIMALS WITH BENIGN TUMOBS


TOTAL EENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMOBS UNCEHTAINEENIGN OF MALIGNANT


TOTAL UNCERTAIN TUMOBS


TOTAL ANIMALS WITH TUMOBS UNCERTAINPBIMABY OR METASTATIC
 

TOTAL UNCERTAIN TUMOBS
 

CONTROL (DNTR)


06-0330


 21


 28


 9


 10


 16


 18


 LOW DOSE HIGH DOSE 

 06-0320 06-0325 

 22 28 

 26 29 

4 <t 

U t 

 20 2U 

 22 2U 

1 

1 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

B-IO
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH
 

ANILINE HYDROCHLORIDE
 

TABLE Cl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

TREATED WITH ANILINE HYDROCHLORIDE

 

CONTROL (UNTR) LOW DOSE HIGH DOSE 01-0330 01-0315 01-0320 

ANIMAL S INITIALLY IN STUDY 25 50 50 

ANIMAL S NECROPSIED 25 50 US 

ANIMALS EXAMINED HI STOPATHOLOGICALLY ** 25 50 

INTEGUMENTAR Y SYSTEM 

*SKIN (25) (50) (t3)
 ULCER, ACUTE 1 (2X)
 ABSCESS, NOS 1 (2X)
 SCAR 1 («*>
 HYPERKERiTOSIS 1 (2X)
 ACANTHOSIS 1 (2X)
 

RESPIRATORY SYSTEM 

tLUNG/BRONCHU S (25) (50) (16) 

BRONCHIECTASIS 1 (2*) 

INFLAMMATION , SUPPURATIV E 1 (2X ) 

fLUNG/BEONCHIOLE (25) (50) (K6)

 

LYMPHOCYTIC INFLAMMATOR Y INFILTR 1 (2X) 

*LUN3 (25) (50) (U6 ) 

BRONCHOPNEUMONI A NECROTIZING 1 (IX) 1 (2X ) 

PNEUMONIA , CHRONIC MURIN E 2 (tX) 2 (IS) 

FIBR3SIS, DIFFUSE 1 (2X) 1 (2X) 

HYPEHPLASIA , ATYPICAL 1 (2X) 

HYPEPPLASIA, ALVEOLAR EPITHELIUM 1 (2X) 

HEMATOPOIETIC SYSTEM 

tSPLEEN (25) (50) 

THROMBOSIS, NOS 1 (2X) 

THROMBUS, ORGANIZED 1 (2X) 

CONGESTION, NOS 1 (US) 2 <4X) 

CONGESTION, PASSIVE 1 <2X) 

HEMORRHAGE 

» NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-3 
TABLE Cl (CONTINUED) 

CONTROL(UNTR) L OU DOSE 

01-0330 01-0315 

FIBROSIS 

NECROSIS, HEMORRHAGIC 

2 (IX) 

1 (2%) METAMORPHOSIS PATTY 

HEMOSIDEROSIS 

HYPERPLASIA. PAPILLARY 

2 <<tX ) 

3 <6X) 

18 (36%) METAPLASIA, OSSEOUS 

HBMATOPOIESIS 

E8YTHROPOIESIS 

1 (2%) 

6 (12X) 

#S PL EN 1C CAPSULE (25) (50) INFLAMMATION , CHRONIC 1 (2X) 

*MANDIBULA R L. NODE (21) (D7) 

HYPEBPLASIA, PLASMA CELL 2 (8%) 

IRENA L LYMP H NODE <2U ) (U7) HEMOSIDBROSIS 

HYPERPLASIA , NOS 

CIRCULATOR Y SYSTEM 

• MYOCARDIUM (25) (50) INFLAMMATION , NOS 

INFLAMMATION , CHRONIC 1 <«X) 

DEGENERATION , NOS 

"CORONAR Y ARTERY (25) (50) 

INFLAMMATION , NOS 1 (2X) 

•HEPATIC VEIN (25) (50) 

THROMBUS, ORGANIZED 

DIGESTIVE SYSTEM 

tLIVER (25) (50) 

THROMBOSIS, NOS 

HEMORRHAG E 

INFLAMMATION , FOCAL GHANDLOKATO U 1 (2X) 

DEGENERATION, NOS 1 (4X) 

NECROSIS, FOCAL 

INFARCT, NOS 

METAMORPHOSIS FATTY 1 (IX) 

BASOPHILIC_CY10_CHANGE J-iftSL 12_I20J}__ 

* NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY * NUMBER OF ANIMALS NECROPSIED 

C-4 

HIGH DOSE 

01-0320 

1 (2X) 

1 (2*) 

2 (tX) 

7 (15X) 

1 (2X) 

5 (11X) 

(«6) 

(35) 

(35) 

1 OX) 

1 OX) 

(46) 

1 (2X) 

1 (2X) 

(48) 

(U8) 

1 (2X) 2 (U») 

1 (2*) 

1 (2X) 
TABLE Cl (CONTINUED) 

CONTHOL(UNTR ) 

01-0330 

LOM DOSE 

01-0315 

HIGH DOSE 

01-0320 

HYPEPPLASIA , FOCAL 1 (4)i) 

ANGIECTASI S 1 (14*) 1 (21) 

•HEPATIC CAPSULE 

NECROSIS, COAGULATIVE 

(25) (50) (47) 

1 (2X) 

*LIVEP/C ENTRILOBULA R (25) (50) (47) NECROSIS, NOS 1 <2X > NECROSIS , DIFFDSE 1 (2%) 

BETABOHPHOSI S FATTY 1 (2S) 

*LIVER/KUPFFER CELL 

HEMOSIDEHOSI S 

(25) (50) 

2 (4X) 

(47) 

26 (55X) 

IPANCREATIC

ATROPHY ,

 ACINU S 

 NOS 

(25) (45) (37) 

2 <5X) HYPERPLASIA , EPITHELIAL 1 <3X ) 

ISTOMACH 

ULCER, NOS 

(24) (48) (45) 

1 (2X) 

tGASTRIC MUCOSA (21) (48) (45) NECROSIS, FOCAL 1 (2X) 

tGASTRIC MUSCULARI S 

INFLAMMATION , FOCAL GRANULOMATO U 

(24) (48) (45) 

1 (2X) 

URINAR Y SYSTEB 

KK1DNEY (25) (50) (48) CALCULUS , NOS 

HYDRONEPHROSI S 

NEPHROSIS, NOS 

HEMOSIDEROSI S 

21 (84X) 

1

7

21

 (2X) 

 (14X) 

 (42*) 

1

8

34

 (2X) 

 (17X) 

 (71X) 

tKIDNEY/COFTEX (25) (50) (48) MULTILOCBLA R CYST 1 (4X) 

IURINAR Y BLADDE R (25) (49) (44) 

INFLAMMATION , ACUTE 1 (2%) 

HYPERPLASIA , EPITHELIAL 1 (2X) 

ENDOCRIN E SYSTEB 

IADRENA L CORTEX 

HYPERPLASIA . FOCAL .

(21) _ 1 -14X1 

(50) 

1 i2XJL _ 

(44) 

* NUMBE R OF ANIMAL S WITH TISSOE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

C-5 
TABLE Cl (CONTINUED) 

CONTROL (UNTR) LOW DOSE 

01-0330 0 1-0315 

IADRENA L MEDULL A (24) (50)

 

HYPERPLASIA , FOCAL 4 (17*)

 

tTHYHOID (21) (39) 

COLLOID CYST 

HYPERPLASI& , C-CELL 1 (3*) 

HEPRODUC1IVE SYSTEM 

*MAMMAR Y GLAN D (25) (50)

 

HYPEPPLASIA, NOS 

*MAMMAR Y DUCT (25) (50)

 

HEMORRHAG E i (us) 

tPFOSTATE (25) (49) 

INFLAMMATION , SUPPDRAT1VE 2 (8%) 

INFLAMMATION , ACUTE 1 (2X > INFLAMMATION , ACUTE FOCAL 1 (2X) 

*SEMINA L VESICLE (25) (50) 

ATROPHY , NOS 1 (2*) 

*TESTIS (25) (50) 

DSGENERATION , NOS 1 (2S) 

ATROPHY , NOS 5 (20*) 5 (10*) 

HYPEEPLASIA , INTERSTITIAL CELL 5 (10X) 

ITESTIS/TUBULE (25) (50) 

DEGENERATION , NOS 1 <2X) 

NERVOU S SYSTE M 

NON E 

SPECIAL SENSE ORGANS 

NON E 

MUSCULOSKELETA L SYSTEM 

* NTIMBBR OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECBOPSIED 

C-6 

HIGH DOSE 

01-0320 

(«t)

2 (5«) 

(30) 

1 <3X) 

(18) 

1 (2X) 

(«8) 

(12) 

(48) 

(46) 

5 (11X) 

1 (2X) 

(16) 

1 (2X) 
TABLE Cl (CONCLUDED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

01-0330 01-0315 01-0320 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

ADIPOSE TISSUE
 

INFLAMMATION, CHRONIC
 

NECROSIS, NOS
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 

AUTOLYSIS/NO NECROPSY 

* NUMBER OF ANIMAL S KITH TISSOE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

C-7 
TABLE C2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

TREATED WITH ANILINE HYDROCHLORIDE 

CONTROL (ONTR) LOW DOSE HIGH DOSE 

02-0330 02-0315 02-0320 

ANIMAL S INITIALLY IN STUDY 25 50 50 

ANIMAL S NECPOPSIED 24 50 50 

ANIMAL S EXAMINE D HI STOPATHOLOGICALLY** 24 50 50 

INTEGUMENTARY SYSTEM 

NON E 

RESPIRATOR Y SYSTEM 

*LUNG (24 ) (50) (50) 

BRONCHOPNEUMONIA , ACUTE 1 (2X) 

INFLAMMATION , ACUTE DIFFUSE 1 (2X) 

PNEUMONIA , CHRONIC MURIN E 1 (2X) 

HYPERPLASIA, ADENOMATOUS 1 (2X) 

HEMATOPOIETIC SYSTEM 

*BONE MARRO W (21) (48) (50) 

HYPERPLASIA , HEMATOPOIETIC 1 (2X) 

*SPLEEN (23) (50) (50) 

DILATATION , NOS 1 (2*) 1 (2X) 

CYST, NOS 1 (2X ) CONGESTION, NOS 1 (2*) 11 (22 X) 

HEMORRHAG E 1 (2X ) 

FIBROSIS 1 <2X ) 

FIBROSIS, FOCAL 1 <2X ) 

METAMORPHOSIS FATTY 1 (2X) 1 (2X) 

PIGMENTATION , NOS 1 (2X) HEMOSIDEROSIS 6 (12%) 5 (10X) 

HYPERELASIA , PAPILLAR Y ' 23 (46X) 28 (56X) 

ERYTHROPOIESI S 1 (4%) 36 (12%) 30 (SOX) 

tSPLENIC CAPSULE (23) (50) (50) 

FIBPOSIS 1 (2X) 1 (2X) 

#SPLENIC SINUSOIDS (23) (50) (50) 

FIBROSIS 1 _[2 X.1 

t NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

» NUMBER OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 
TABLE C2 (CONTINUED) 

CONTROL (ONTR) LOW DOSE HIGH DOSE 

• MANDIBULA R L. NODE 

HYPERPLASIA, PLASMA CELL 

* MEDIA SII SAL L.NOD E 

INFLAMMATION, ACUTE
 

• MESENTERIC L. NODE
 

LYMPHANGIECTASIS
 

• RENAL LYMPH NODE
 

HYPERPLASIA, NOS
 

CIPCULATOBY SYSTEM
 

• MYOCARDIUM
 

CALCIFICATION, FOCAL
 

• ENDOCARDIUM
 

INFLAMMATION, NOS
 

*AORTA
 

MEDIAL CALCIFICATION
 

*CORONARY ARTERY
 

MEDIAL CALCIFICATION
 

DIGESTIVE SYSTEM
 

• LIVER
 

CYST, NOS
 

INFLAMMATION, ACOTE/CHRONIC
 

INFLAMMATION, FOCAL GRANULOMATOU
 

METAMORPHOSIS FATTY
 

BASOPHILIC CYTO CHANGE
 

HYPERPLASIA, FOCAL
 

ANGIECTASIS
 

HYPERPLASIA, BASOPHILIC
 

•HEPATIC	 CAPSDLE
 

HEMOEFHAGE
 

•LIVEH/CENTRILOBULAR
 

CONGESTION, NOS
 

# NOHBER OF ANIMALS WITH TISSOE

* NOHBER OF ANIMAL S NECSOPSIED 

02-0330 02-0315 02-0320 

(19) (HB) 

2 (11*) 

(19) (11) 

1 (2%) 

(19) (18) (41) 

1 (2X) 

(19) (18) (11) 

1 <2X) 

(21) (50) (50) 1 («X) 

(2«) (50) (50) 

1 <2X> 

(2U) (50) (50) 

1 (i*X) 

(21) (50) (50) 

1 {«%) 

(21) (50) (50) 

1 (2X) 

1 (2*) 

1 (2X) 

1 (2X) 1 (2X) 

10 (>12%) 38 (76X) 15 (30X) 

1 (2X) 

1 (it*) 1 (2X) 

1 (UX) 2 <»*) 

(24) (50) (50) 

1 (2X) 

(21) (50) (50) 

_.J-J2*1 

 EXAMINE D MICROSCOPICALLY 

C-l 
TABLE C2 (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

02-0330 02-0315 02-0320 

NECROSIS, DIFFUSE 1 (2X) 1 <2X) 

NECROSIS, COAGULATIVE 1 <2X ) 

#LIVEE/KaPFFER CELL (24) (50) (50) 

HEBOSIDEEOSIS 29 (58X) 

ILIVEE/HEPATOCYTES (2 it) (50) (50) 

NECROSIS , FOCAL 1 (2X) 

*BILE DUCT (24) (50) (50) 

INFLAMMATION, CHRONIC DIFFUSE 1 (2%) 1 (2X) 

IPANCREAS (22) (U9 ) (49) 

METAMORPHOSI S FATTY 1 (2X) 

ISTOMACH (21) (49) (48) 

I'LCEE, ACUT E 1 (2«) 

HYPEHKERATOSI S 1 <2X ) 

ACANTHOSI S 1 (2X) 

URINAR Y SYSTEM 

KKIDNE Y (21) (49) (50) 

HYDRONEPHROSI S 1 (2X) 

INFLAMMATION, CHRONIC FOCAL 1 (2X) 

NEPHROSIS , NOS 8 (33%) 1 (2%) 

METAMORPHOSI S FATT Y 1 (2X) 

HEMOSIDEROSI S 46 (94X) 45 (90%) 

HYPE8PLASIA, PAPILLAR Y 1 (2X) 

tKIDNEY/TUBUL E (21) (49) (50)

 

CALCIFICATION, NOS 1 (4*)

 

ENDOCRINE SYSTEM 

IPIT'JITARY (21) (46) (10)

 

HEHOSIDEROSIS 1 (5*) 

IADPEBA L MEDULL A (24) (50) (48) 

HYPEEPLASIA , FOCAL 1 (2*) 2 (4X) 

•THYROID (21) (38) (38) _ HYPERPLASIAt _C-CELL_ . __ J_15S)__ _ . 2 (5X) 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

C-IO 
TABLE C2 (CONCLUDED) 

CONTKOL(UNTR ) LOW DOSE HIGH DOSE 02-0330 02-0315 02-0320 

REPRODUCTIV E SYSTEM 

*MAMMAR Y GLAN D (50) (50) 

GALACTOCELE 1 (2*) 

HYPERPLASIA, NOS 1 (2*) 

tUTERUS/ENDOMETRIO M (24) (48) (50) 

INFLAMMATION , SUPPURATIV E 3 (13*) 1 (2X) 

HYPERPLASIA, CYSTIC 

METAPLASIA, SQUAMOUS 1 (14%) 

1 (2X) 

tOVARY/OVIDUCT (24) (50) 

INFLAMMATION , SDPPDRATIV E 5 (21*) 

tOVARY (24) (49) (50) 

CYST, NOS 2 (8X) 1 (2X) 4 (8X) 

INFLAMMATION, SUPPURATIVE 1 (45!) 1 (2*) 

INFLAMMATION, CHRONIC 1 (2X) 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

*EYE/CRYSTALLINE LENS (24) (50) (50) 

CALCIFICATION, NOS 1 (2X) 

CALCIFICATION, FOCAL 1 (2*) 

MUSCIILOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

_ NONE 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED 1 

AUTOLYSIS/NO NECROPSY 1 

C-ll 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH
 

ANILINE HYDROCHLORIDE
 

TABLE Dl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH ANILINE HYDROCHLORIDE 

CONTROL (UNTR) 

05-0330 

LOW DOSE 

05-0320 

HIGH DOSE 

05-0325 

ANIMALS IBITIALLY IN STUDY 50 

ANIMALS HISSING 2 

ANIMALS NECHOPSIED 39 

ANIBALS EXAMINED HISTOPATHOLOGICALLY ** 39 

50 

49 

1(9 

50 

49 

49 

INTEGUMENTARY SYSTEM
 

*SKIN (39) (49) (49)
 EPIDERMAL INCLUSION CYST 1 (3%)
 INFLAMMATION. CHHONIC 1 (3X)
 FIBROSIS 1 (3X)
 

RESFIBA10RY SYSTEM
 

tLUNG/ALVEOLI (39) (49) (49)
 

HISTIOCYTOSIS 1 (2X)
 

HEMATOPOIETIC SYSTEM
 

ISPLEEN (38) (49) (49)
 

HYPERPLASIA, LYMPHOID 1 <3X) 1 (2X)
 

HEMATOPOIESIS 1 (3*) 1 (2X) 1 (2X)
 2HYTHSOPOIESIS 3 (8X) 2 (4%) 4 (8%)
 

IMANDIBULAR L. NODE (36) (45) (45)
 

HYPERPLASIA, PLASMA CELL 1 (3%)
 

#PANCR£ATIC L.NODE (36) (45) (45)
 HYPERPLASIA, NOS 1 (2X)
 

iMESENTERIC L. NODE (36) (45) (45)
 

LYMPHANGIECTASIS 2 (4X) 3 (7X)
 INFLAMMATION, FOCAL GEANULOMATOU 1 (2%)
 HYPEEPLASIA, NOS 4 (11X) 2 (4X) 2 (4X)
 HYPEEPLASIA, PLASMA CELL 2 (4%)
 HYPEBPLASIA, LYMPHOID 4 (11X) 1 (2%)
 

CIRCULATORY SYSTEM
 

• MYOCARDIUM (39) (49) (49)
 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY * NUMBE R OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-3 
TABLEDl (CONTINUED)
 

CONTROL (UNTR) LOW DOSE
 

05-0330 05-0320
 

DIGESTIVE SYSTEM
 

tLIVER (39) (49)
 

DEGENERATION, NOS 1 (2%)
 

HEHOSIDEROSIS
 

HYPERPLASTIC NODULE 1 (2%)
 

HYPEHPUSIA. FOCAL 3 (8X)
 

HEMATOPOIESIS
 

•BILE	 EUCT (39) (49)
 

INFLAMMATION, CHRONIC 2 <UX)
 

INFLAMMATION, CHRONIC DIFFUSE 14 (29X)
 

HYPERPIASIA, DIFFUSE 1 (2X)
 

•PANCREAS	 (36) (45)
 

PERIARTERITIS
 

NECROSIS, DIFFUSE 1 (2X)
 

•GASTRIC	 NUCOSA (37) (49)
 

HYPEflPLASIA, FOCAL 1 (2%)
 

HYPEEE1ASIA, ADENOMATOUS
 

(GASTRIC SUBMUCOSA (37) (49)
 

INFLAMMATION, ACUTE FOCAL
 

•PEYERS	 PATCH (37) (48)
 

HYPERPLASIA, LYHPHOID
 

*JEJUNUM (37) (48)
 

AMYLOIDOSIS 1 (3X)
 

*ILEUM (37) (48)
 

AMYLOIBOSIS 2(5%)
 

OBISABY SYSTEM
 

*KIDNEY (39) (49)
 

HYDRONEPHROSIS 1 (2%)
 

INFLAMMATION, CHRONIC 1 (2%)
 

PEHIVASCULITIS 1 (2X)
 

GLOMERULOSCLEROSIS, NOS 3 (8*) 8 (16X)
 

IKIDNEY/CORTEX (39) (49)
 

SCiR _ .. __ 1 J3S1
 

* NUMBER OP ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-4
 

HIGH DOSE
 

05-0325
 

(49)
 

3 (6%)
 

1 (2%)
 

1 <2X)
 

(49)
 

13	 (27%)
 

1 (2*)
 

(46)
 

1 (2%)
 

(47)
 

1 (2X)
 

1 (2%)
 

(47)
 

1 (2%)
 

(48)
 

1 (2%)
 

(48)
 

(48)
 

(49)
 

2 (4%)
 

(49)
 
ItKIDNEY/GLOMERULUS


AMYLOIDOSIS


ENDOCBINE SYSTEM
 

*ADBENAL


AMYLOIDOSIS


ITBYROID


CYSTIC FOLLICLES


AMYLOICOSIS


HiPEBPLASIA, C-CSLL


HYPEBP1ASIA, FOLLICULAR-CELL


tPASATHYROID


HYPIRPLASIA, NOS


*PANCREAIIC ISLETS


HYPIRPLASIA, NOS


REPRODUCTIVE SYSTEM
 

• PROSTATE


PERIVASCULITIS


NERVOU S SYSTEM
 

NONE
 

SPECIAL SENSE OESANS
 

NONE
 

HOSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIE S 

TABLE Dl (CONTINUED)
 

CONTROL (BNTR)


05-0330


 (39)


 2 (5X)
 

(36)


 2 (651)
 

(38)


 1 (3X)


 1 (35!)
 

2 (55!)
 

(28)


 1 (l»5l)
 

(36)


 1 (35!)
 

(37)


 LOW DOSE HIGH DOSE
 

 05-0320 05-0325
 

 (H9) («9)
 

 («5) (16)
 

 (43)	 (13)
 

1 (2%)
 

1 (2%)
 

 (18) (26)
 

 (15) (46)
 

 (118)	 (19)
 

1 (2X)
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIBD
 

D-5 
TABLED! (CONCLUDED)
 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

05-0330 05-0320 05-0325
 

ALL OTHER SYSTEMS
 

*HULTIPLE ORGANS (39) (49) (U9)
 

AMYLOICOSIS 1 (3*)
 

SPECIAL MCBPHOLOGY SUMMARY
 

NO LESION REPORTED 5 10 7
 

ANIMA L HISSING/NO NECROPSY 2
 

AUTO/NECROPSY/HISTO PERF 1 1
 

AUTOLYSIS/NO NECROPSY 9 1 1
 

# NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-6
 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH ANILINE HYDROCHLORIDE

 

CONTROL (UNTR) 

06-0330 

LOW DOSE 

06-0320 

HIGH DOSE 

06-0325 

ANIMALS INITIALLY IN STUDY

ANIMALS MISSING 

ANIMALS NECROPSIED

ANIMALS EXAMINE D HISTOPATHOLOGICALLY

 50 

 49 

 ** 49 

50 

1 

49 

1)8 

350 

49 

49 

INTEGUMENTAR Y SYSTEM
 

*SKIN (49) (49) (49) 

INFLAMMATION, ACUT3 1 (2X) 

BESPIBATCBY SYSTEM
 

#LUNG (49) (48) (49)
 

PERIVASCULITIS 1 (2%)
 

HDNATOPOIETIC SYSTEM
 

tSPLEEN (15) (48) (49)
 

HYPERPLASIA, LYMPiWID 1 {2%) 3 (651)
 HEMATOPOIESIS 2 (4X)
 

EEYTHBOPOIESIS 4 (9X) 6 (13X) 13 (275i)
 

4LYMEH NODE OF THORAX (44) (37) (46)
 

HYPERPLASIA, NOS 1 (2«)
 

KPANCREATIC L.NODE (1)4) (37) (46)
 

HYPERPLASIA, DOS 1 (3%)
 

HEMATOPOIESIS 1 (251)
 

*LUMEAR LYMPH MODE (44) (37) (46)
 

HYPERPLASIA, NOS 1 (2*) 1 (2*)
 

*H£SENTERIC L. NODE (1)4) (37) (46)
 

HYPEHPLASIA, NOS 1 (2%) 2 (5%) 2 (4X)
 

HYPERPLjASIA^ LYMPHOID 1 J2J1 1 1231 __
 

* NUMBE R O F ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIE D 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

H> 50 ANIMAL S WER E INITIALL Y IN THE STUDY , BUT ONE ANIMA L HAS FOUND TO BE A HALE IN A FEMAL E GROUP . 

D-7
 
TABLE D2 (CONTINUED) 

CCNTKOL (UNTR) L0» DOSE HIGH DOSE
 

06-0330 06-0320 06-0325
 

HEMATOPOIESIS	 1 (2*)
 

tRENAL LYHPH BODE (44) (37) (46)
 

HEMATOPOIESIS 1 (2X)
 

CIRCULATORY SYSTEM
 

*AORTA (49) (19) (49)
 

BEDIAL CALCIFICATION 1 (2*)
 

*PULMONAPY ABTEHY (19) (U9) (49)
 

MEDIAL CALCIFICATION 1 <2X)
 

DIGESTIVE SYSTEM
 

*LIVER (46) (U8) (48)
 

NECROSIS, FOCAL 1 (2%)
 

HYPERPLASTIC NODULE 1 (2%)
 

HEMATOPOIESIS 1 (2X) 1 (2%)
 

'GALLBLADDER (U9) (19) (49)
 

INFLAMMATION, ACUTE/CHRONIC 1 (2X)
 

*BILB EUCT (19) <«9) (49)
 

INFLAMMATION, CHRONIC 1 (2%)
 

INFLAMMATION, CHRONIC DIFFUSE 1 (2%) 1 (2%)
 

•PANCREAS	 (38) (U5) (46)
 

CYSTIC DUCTS 1 (2*)
 

ISTOMACH (tl) (46) (47)
 

HYPEFFLASIA, CYSTIC 1 (2%)
 

iGASTRIC MUCOSA <t1) (46) (47)
 

CALCIFICATION. NOS 1 (2%)
 

HYPERPLASIA, ADENOMATOUS 1 (2%)
 

•GASTRIC FOVEOLAE (t1) (46) (47)
 

METAPLASIA, SQUAMOUS 1 (2X)
 

URINARY SYSTEM
 

(tKIDNEY (46) (48) (49)
 

GLOBERULOS£LSRQSISi_NOS 2_14X) 4__(821__
 

t NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

D-8
 
TABLE D2 (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE
 

06-0330 06-0320 06-0325
 

HEMOSIDEROSIS 1 (2X)
 

KKIDNEY/TUBULr) (16) (U8) (19)
 CALCIFICATION, NOS 1 (2%)
 

ENDCCBINE SYSTEM
 

#AD8ENAL/CAPSUL2 t»6) («5) (1*5)
 HYPERFLASIA. NOS 1 (2*)
 

tTHYROID (UK) (33) (36)
 

INFLAMMATION, ACUTE FOCAL 1 (2X)
 

INFLAMMATION, CHRONIC FOCAI 1 (3X)
 

HYPEBFLASIA, FOLLICULAB-CELL 2 (5X)
 

BEPBODUCTIVE SYSTEM
 

KUTERUS (U4) l«6) (U3)
 HYEROMETRA 1 (2X) 5 (12X)
 

tUTZRUS/ENDOMETRIUM (HU) (1*6) (13)
 

HEMORRHAGE 1 (2X)
 INFLAMMATION, SUPPURATIVE 1 (2X)
 HYPERPLASIA, NOS 1 (2X)
 HYPEBPLASIA, CYSTIC 30 (68X) 18 (39X) 13 (30X)
 

*OVARY ("»«) (1*5) (17)
 

CYST, NOS 7 (16*) 6 (13%) 2 (1%)
 

HEMOFRHAGIC CYST It (9X)
 

ABSCESS, NOS 1 (2*)
 INFLAMMATION. CHRONIC 2 (5*)
 

NEBVOOS SYSTEM
 

NONE
 

SPECIA L SENS E OBGAN S 

NON E 

.1USCULOSKE1ETA L SYSTE M 

*SKELETA L MUSCL E (19) (1*9) (1*9)
 

ABSCESS^NOS 

» NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMALS NECROPSIED
 

D-9
 
TABLE D2 (CONCLUDED)
 

CCNTROL (DNTR) LOW DOSE HIGH DOSE
 

06-0330 06-0320 06-0325
 

BODY CAVITIES
 

NONE
 

ALL QTHEB SYSTEMS
 

ADIPOSE TISSUE
 

STEATITIS 1
 

INFLAMMATION, CHHCNIC 1
 

FIBBOSIS 1
 

NECBOSIS, FAT 1
 

SPECIAL MOEPHOLOGY SUMMAR Y
 

NO LESION REPORTED 3 7
 

ANIMAL MISSING/NO N2CROPSY 1
 

KECROESY PERF/NO HISTO PERFORMED 1
 AUTO/NECROPSY/HISTO PERF 3 1
 

AUTOLYSIS/NO NECROPSY 1
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

OF ANIMALS NECROPSIED
 

D-IO
 
Review of the Bioassay of Aniline Hydrochloride* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

June 29, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DREW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer•Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be exposed.
 The members of the Clearinghouse have been drawn from
 

academia, industry, organized labor, public interest groups,
 State health officials, and quasi-public health and research
 

organizations. Members have been selected on the basis of
 

their experience in carcinogenesis or related fields and,
 

collectively, provide expertise in chemistry, biochemistry,
 biostatistics, toxicology, pathology, and epidemiology.
 

Representatives of various Governmental agencies participate
 

as ad hoc members. The Data Evaluation/Risk Assessment
 

Subgroup of the Clearinghouse is charged with the responsi­ bility of providing a peer review of reports prepared on
 

NCI-sponsored bioassays of chemicals studied for carcinogenic­ ity. It is In this context that the below critique is given
 

on the bioassay of Aniline Hydrochloride for carcinogenicity.
 

The reviewer agreed with the conclusion in the report
 

that Aniline Hydrochloride was carcinogenic in treated rats,
 

under the conditions of test. He pointed out that there was
 

marked hemosiderosis in the renal tubular epithelium and in
 

the liver of treated rats but none was reported in treated
 

mice. Based on the experimental findings, he said that the
 

compound may pose a possible carcinogenic risk to humans.
 The reviewer moved that the report on the bioassay of
 

Aniline Hydrochloride be accepted as written. The motion
 

was approved without objection.
 

Clearinghouse Members present:
 

Arnold L. Brown (Chairman), Mayo Clinic
 

Paul Nettesheim, National Institute of Environmental
 

Health Sciences
 

Verne Ray, Pfizer Medical Research Laboratory
 

Verald K. Rowe, Dow Chemical U.S.A.
 Michael B. Shimkin, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

55
 

J^U.S. GOVERNMEN T PRINTIN G OFFICE i 1978-260-899/3202
 

DHEW Publication No. (NIH) 78-1385