National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 154 

1979 

BIOASSAY OF 

AZOBENZENE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 103-33-3 

NCI-CG-TR-154 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

AZOBENZENE
 

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) 79-1710
 
ii

 
BIOASSAY OF
 

AZOBENZENE
 

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
 

azobenzene 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. A negative ^esult, in which the test animals
 

do not have a greater incidence of cancer than control animals,
 

does not: necessarily mean that a test chemical i^ not a
 

carcinogen, inasmuch as the experiments are conducted under a
 

limited set of circumstances. A positive results demonstrates
 

that a test chemical is carcinogenic for animals under the
 

conditions of the test and indicate that exposure to the chemical
 

is a potential risk to man. The actual determination of the risk
 

to man from chemicals found to be carcinogenic in animals
 

requires a wider analysis.
 

CONTRIBUTORS; This bioassay of azobenzene was conducted at the
 

NCI Frederick Cancer Research Center (FCRC) (1), Frederick,
 

Maryland, for NCI (2) by Litton Bionetics, Inc.
 

The manager of the bioassay at FCRC was Dr. B. Ulland, the
 

toxicologist was Dr. E. Gordon, and Drs. R. Cardy and D. Creasia
 

compiled the data. Ms. S. Toms was responsible for management of
 

data, Mr. D. Cameron for management of histopathology, Mr. L.
 

Callahan for management of the computer branch, and Mr. R. Cypher
 

for management of the facilities. Mr. A. Butler performed the
 

computer services. Histopathologic evaluations for rats and mice
 

were performed by Dr. B. Ulland (1). The diagnoses included in
 

this report represent his interpretations.
 

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

Mason Research Institute (3). Statistical analyses were
 

performed by Dr. J. R. Joiner (4) and Ms. P. L. Yong (4), using
 

methods selected for the bioassay program by Dr. J. J. Gart (5).
 

The chemicals used in this bioassay were analyzed at FCRC by Dr.
 

W. Zielinsky (1). The chemical narrative and analyses were
 

reviewed and approved by Dr. W. Lijinsky (1).
 

This report was prepared at Tracer Jitco (4) under the direction
 

of NCI. Those responsible for the report at Tracer Jitco were
 

Dr. C. R. Angel, Acting Director of the Bioassay Program; Dr. S.
 

S. Olin, Deputy Director for Science; Dr. J. F. Robens,
 

toxicologist; Dr. R. L. Schueler, pathologist; Dr. G. L. Miller,
 

Ms. L. A. Owen, Ms. M. S. King, and Mr. W. D. Reichardt, 

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

assisted by Ms. Y. E. Presley. 

The following scientists at NCI were responsible for evaluating
 

the bioassay experiment, interpreting the results, and reporting
 

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

J. Fielding Douglas, Dr. Richard A. Griesemer, Dr. Thomas E.
 

Hamm, Dr. William V. Hartwell, Dr. Morton H. Levitt, Dr. Harry A.
 

Milman, Dr. Thomas W. Orme, Dr. A. R. Patel, Dr. Sherman F.
 

Stinson, Dr. Jerrold M. Ward, and Dr. Carrie E. Whitmire.
 

(1) Frederick Cancer Research Center, P.O. Box B, Frederick,
 

Maryland.
 

(2) Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

(3) EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

(4) Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

(5) Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute,
 

National Institutes of Health, Bethesda, Maryland.
 

IV
 
SUMMARY
 

A bioassay of azobenzene for possible carcinogenicity was
 

conducted by administering the test chemical in feed to F344 rats
 

and B6C3F1 mice.
 

Groups of 50 rats of each sex were administered azobenzene at one
 

of two doses, either 200 or 400 ppm, for 105 or 106 weeks.
 

Matched controls consisted of 20 untreated rats of each sex. All
 

surviving rats were killed at the end of administration of the
 

test chemical.
 

Groups of 50 male mice were administered azobenzene at one of two
 

doses, either 200 or 400 ppm, for 105 weeks. Groups of 50 female
 

mice were administered the test chemical at one of two doses,
 

initially 400 or 800 ppm, for 38 weeks. Because of excessively
 

lowered body weights in the dosed groups of the females, doses
 

for the females were then reduced to 100 and 400 ppm,
 

respectively, and administration at the lowered doses was
 

continued for 67 or 68 weeks. The time-weighted average doses
 

for the female mice were either 208 or 545 ppm. Matched controls
 

consisted of 20 untreated mice of each sex. All surviving mice
 

were killed at the end of administration of the test chemical.
 

Mean body weights of dosed rats and mice of each sex were lower
 

than those of corresponding controls, and were generally dose
 

related throughout the bioassay. Mortality was dose related in
 

the male rats and the female mice, but was not significantly
 

affected in either the female rats or the male mice. Survival
 

was 70% or greater at week 90 on study in all dosed and control
 

groups of each species and sex; thus, sufficient numbers of 

animals were at risk in all groups for the development of 

late-appearing tumors. 

In rats, a large number of sarcomas, including fibrosarcomas,
 

hemangiosarcomas, and osteosarcomas in both males and females and
 

malignant hemangiopericytomas in females, occurred in the spleen
 

and other abdominal organs at incidences that were dose related
 

in each sex (P less than 0.001) and that in direct comparisons
 

were significantly higher (P less than 0.001) in the high-dose
 

groups of each sex than in the corresponding control groups
 

(males: controls 0/20, low-dose 6/49, high-dose 31/49; females:
 

controls 0/20, low-dose 5/50, high-dose 21/50).
 

In mice, no tumors occurred in either males or females at
 
incidences that were significantly higher in the dosed groups
 

than in the corresponding control groups.
 

It is concluded that under the conditions of this bioassay,
 

azobenzene was carcinogenic (sarcomagenic) for F344 rats,
 inducing various types of sarcomas in the spleen and other
 

abdominal organs of both males and females. The test chemical
 

was not carcinogenic for B6C3F1 mice of either sex.
 

vi
 
TABLE OF CONTENTS
 

I. Introduction. 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 3
 

C. Animals 4
 

D. Animal Maintenance 5
 

E. Subchronic Studies.... 7
 

F. Chronic Studies 11
 

G. Clinical and Pathologic Examinations 11
 

H. Data Recording and Statistical Analyses 14
 

III. Results - Rats 21
 

A. Body Weights and Clinical Signs (Rats) 21
 

B. Survival (Rats) 21
 

C. Pathology (Rats) 24
 

D. Statistical Analyses of Results (Rats) 27
 

IV. Results - Mice /. 31
 

A. Body Weights and Clinical Signs (Mice) 31
 

B. Survival (Mice) 31
 

C. Pathology (Mice) 34
 

D. Statistical Analyses of Results (Mice) 36
 

V. Discussion 37
 

VI. Bibliography 41
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Administered Azobenzene in the Diet 43
 

Table Al Summary of the Incidence of Neoplasms in Male
 

Rats Administered Azobenzene in the Diet 45
 

Table A2 Summary of the Incidence of Neoplasms in Female
 

Rats Administered Azobenzene in the Diet 49
 

vii
 
Page 

Appendix B Summary of the Incidence of Neoplasms in Mice 

Administered Azobenzene in the Diet 53 

Table Bl Summary of the Incidence of Neoplasms in Male 

Mice Administered Azobenzene in the Diet 55 

Table B2 Summary of the Incidence of Neoplasms in Female 

Mice Administered Azobenzene in the Diet 58 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Administered Azobenzene in the 

Diet 63 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Administered Azobenzene 

in the Diet 65 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Administered Azobenzene 

in the Diet 70 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Administered Azobenzene 

in the Diet 75 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Administered Azobenzene 

in the Diet 77 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered Azobenzene 

in the Diet 81 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Administered Azobenzene in the Diet.... 85 

Table El Analyses of the Incidence of Primary

in Male Rats Administered Azobenzene 

in the Diet

 Tumors 

 87 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Administered Azobenzene 

in the Diet 94 

viil 
Page 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Administered Azobenzene 

in the Diet 101 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Administered Azobenzene 

in the Diet 103 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Administered Azobenzene 

in the Diet 107 

TABLES 

Table 1 Azobenzene Subchronic Feeding Studies 

in Rats and Mice 8 

Table 2 Azobenzene Chronic Feeding Studies 

in Rats 12 

Table 3 Azobenzene Chronic Feeding Studies 

in Mice 13 

FIGURES 

Figure 1 Growth Curves for Rats Administered 

Azobenzene in the Diet 22 

Figure 2 Survival Curves for Rats Administered 

Azobenzene in the Diet 23 

Figure 3 Growth Curves for Mice Administered 

Azobenzene in the Diet 32 

Figure 4 Survival Curves for Mice Administered 

Azobenzene in the Diet 33 

ix
 

I. INTRODUCTION
 

Azobenzene (CAS 103-33-3; NCI
 

C02926) occurs as a by-product
 

during the manufacture of
 

benzidine (Noller, 1965; Lurie,
 

1964). Benzidine is a widely used
 

intermediate for the azo dyes and Azobenzene
 

other organic chemicals and is a
 

carcinogen (Department of Labor, 1974). Azobenzene itself has no
 

known uses as a dyestuff and is produced only in small quantities
 

for research purposes (International Agency for Research on
 

Cancer, 1975).
 

Since 1950, there has been documented evidence of an increased
 

risk of bladder cancer in persons employed in the dye industries
 

(International Agency for Research on Cancer, 1975). Although it
 

has not been possible to identify the causative dyes or
 

intermediates by these epidemiological studies, some compounds
 

have been shown to be carcinogenic in animal studies. Azobenzene
 

has been regarded in the literature as a noncarcinogen (Daoust
 

and Calamai, 1971; Eldredge and Luck, 1952), as a result of a
 
long-term study by Spitz et al. (1950) in which Sherman rats were
 

given subcutaneous injections of the compound for life. More
 

recently, azobenzene was tested by Innes et al. (1969) in a
 

large-scale screen of industrial compounds for carcinogenic
 

activity. Since the results of this preliminary bioassay in mice
 

did not clearly associate the incidence of any tumor with
 

administration of the test chemical, azobenzene was selected for
 

further testing in the Carcinogenesis Testing Program.
 
II. MATERIALS AND METHODS
 

A. Chemical
 

Azobenzene (diphenyldiimide; azobenzide) was obtained from
 

Eastman Chemical Company as a hard, dark-orange, crystalline
 

material. Its purity was determined at Frederick Cancer Research
 

Center using gas-liquid chromatography (GLC) to be 99.5%, with up
 

to six minor contaminants and a melting point of 66 C (literature: 68 C). Mass spectral analysis gave a molecular ion at
 

m/e 182 and a base peak at m/e 77. The infrared spectrum was
 

consistent with its structure, and was identical to that of a
 

standard.
 

B. Dietary Preparation
 

Test diets containing azobenzene were prepared in 6-kg batches at
 

the appropriate doses. A known weight of the chemical was first
 

mixed with an equal weight of autoclaved Wayne® Sterilizable
 

Lab Meal with 4% fat (Allied Mills, Inc., Chicago, 111.), using a
 

mortar and pestle. The mixing was continued with second and
 

third additions of feed, and final mixing was performed with the
 
remaining quantity of feed for a minimum of 15 minutes in a
 

Patterson-Kelly twin-shell blender with an intensifier bar.
 

Detailed GLC analyses of aliquots of azobenzene-feed mixtures
 

taken from various locations in the blender showed that the
 

mixture was homogeneous.
 

C. Animals
 

Male and female F344 (Fischer) rats and B6C3F1 mice were obtained
 

as 4-week-old weanlings, all within 3 days of the same age, from
 

the NCI Frederick Cancer Research Center animal farm (Frederick,
 

Md.). The animals were housed within the test facility for 2
 

weeks and were then assigned four rats to a cage and five mice to
 

a cage on a weight basis for each cage of animals of a given
 

species and sex. For use in the chronic study, the male rats
 

were required to weigh 90 to 105 g, averaging at least 100 g; the
 

female rats, 80 to 95 g, averaging at least 90 g; the male mice,
 

18 to 22 g, averaging at least 19.5 g; and the female mice, 17 to
 

21 g, averaging at least 18.5 g. Individual animals were
 

identified by ear punch.
 
D. Animal Maintenance
 

The animals were boused in polycarbonate cages (Lab Products,
 

Inc., Garfield, N.J.), 19 x 10-1/2 x 8 inches for the rats and
 

11-1/2 x 7-1/2 x 5 inches for the mice, which were suspended from
 

aluminum racks (Scientific Cages, Inc., Bryan, Tex.) and were
 

covered by nonwoven polyester-fiber 12-mil-thick filter paper
 

(Hoeltge, Inc., Cincinnati, Ohio). The bedding used was
 

®
 Absorb-dri hardwood chips (Northeastern Products, Inc.,
 

Warrenburg, N. Y.). The feed supplied was presterilized Wayne
 

Sterilizable Lab Meal, provided ad_ libitum in suspended stainless
 

steel hoppers and replenished at least three times per week.
 

Water, acidified to pH 2.5, was supplied ad_ libitum from glass
 

bottles. Sipper tubes (Lab Products, Inc.) were suspended
 

through the tops of the cages.
 

The contaminated bedding was disposed of through an enclosed
 

vacuum line that led to a holding tank from which the bedding was
 

fed periodically into an incinerator. The cages were sanitized
 

twice per week and the feed hoppers twice per month at 82 to
 

88 C in a tunnel-type cagewasher (Industrial Washing Corp.,
 

Mataway, N. J.), using the detergents, Clout® (Pharmacal
 

Research Laboratories, Greenwich, Conn.) or Oxford D'Chlor
 

(Oxford Chemicals, Atlanta, Ga.).
 
The glass bottles and sipper tubes were sanitized at 82 to 88 C
 

in a tunnel-type bottle washer (Consolidated Equipment Supply
 

Co., Mercersburg, Pa.) three times per week, using a Calgen
 

Commercial Division detergent (St. Louis, Mo.). The racks for
 

the cages were sanitized at or above 82 C in a rack washer
 

(Consolidated Equipment Supply Co.) once per month, using Calgen
 

Commercial Division detergent, and the filter paper was changed
 

at the same time.
 

The air in the aniiflal rooms was maintained at 22 to 24 C and 45
 

to 55% relative humidity. Fresh air was passed through a filter
 

of 65% efficiency and a bag filter of 95% efficiency at the
 

intake and through a "Z"-type roughing filter of 30% efficiency
 

and a bag system of 90 to 95% efficiency at the exhaust (American
 

Air Filters, Louisville, Ky.; Mine Safety Appliances, Pittsburgh,
 

Pa.); the air was not recirculated. Room air was changed 15
 

times per hour. The air pressure was maintained negative to a
 

clean hallway and positive to a return hallway. Fluorescent
 

lighting was provided automatically on a 12-hour-per-day cycle.
 

Rats administered azobenzene and their corresponding controls
 

were housed in the same room as rats on feeding studies of the
 

following chemicals:
 

(CAS 72-56-0) p,p'-ethyl-DDD
 

(CAS 120-62-7) piperonyl sulfoxide
 
Mice administered azobenzene and their corresponding controls
 

were housed in the same room as mice on feeding studies of the
 

following chemicals:
 

(CAS 128-66-5) C. I. vat yellow 4
 

(CAS 72-56-0) p,p'-ethyl-DDD
 

(CAS 20941-65-5) ethyl tellurac
 

(CAS 298-00-0) methyl parathion
 

(CAS 85-44-9) phthalic anhydride
 

(CAS 51-03-6) piperonyl butoxide
 

(CAS 86-06-2) 2,4,6-trichlorophenol
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted to estimate the maximum
 

tolerated doses (MTD's) of azobenzene, on the basis of which two
 

concentrations (referred to in this report as "low" and "high"
 

doses) were selected for administration in the chronic studies.
 

Groups of five rats of each sex and five mice of each sex were
 

fed diets containing azobenzene at one of several doses, and
 

groups of five control animals of each species and sex were
 

administered basal diet only. The test chemical was administered
 

for 7 weeks, followed by 1 week of additional observation. Each
 

animal was weighed twice per week. Table 1 shows the survival of
 

animals in each dose group at the end of the study and the week
 

on study when the last death occurred; the table also shows the
 

mean body weights of each dosed group at week 7, expressed as
 

percentages of mean body weights of controls. At the end of the
 

7
 
Table 1. Azobenzene Subchronic Feeding
 

Studies in Rats and Mice
 

Male Female 

Week on Week on 

Study Mean Weight Study Mean Weight 

When Last at Week 7 When Last at Week 7 

Dose Surviv­ Death as % of Surviv- Death as % of 

(ppm) al (a) Occurred Control al (a) Occurred Control 

Rats 

500 5/5 88 5/5 96 

700 5/5 75 5/5 83 

1,000 5/5 73 5/5 69 

2,200 5/5 33 0/5 6 

4,600 0/5 2 0/5 2 

Mice 

500 5/5 88 5/5 91 

700 5/5 86 5/5 91 

1,000 5/5 91 5/5 91 

2,200 4/5 5 89 5/5 91 

4,600 5/5 66 5/5 67 

(a) Number surviving/number in group. 
subchronic studies, all animals were killed using C0? and
 

necropsied.
 

Groups of male and female rats receiving doses of 2,200 or 4,600
 

ppm were observed during clinical examination to be emaciated.
 

At necropsy, the groups of rats receiving the four highest doses
 

had slightly enlarged livers. Histopathologic changes due to
 

administration of the azobenzene were noted in the kidneys and
 

livers of male and female rats dosed at 1,000 or 2,200 ppm. In
 

these animals the proximal convoluted tubules of the kidney
 

contained generally moderate amounts of granular,
 

yellowish-brown, intracytoplasmic pigment. Trace to moderate
 

amounts of centrilobular cytoplasmic vacuolation of hepatocytes,
 

suggestive of lipidosis, occurred in six male rats. Trace to
 

very small amounts of bile stasis were noted in the livers of
 

both males and females. The hepatic changes indicated mild
 

injury. Pigmentation of renal tubules was considered to
 

represent accumulation of lipofuscin in association with mild
 

degenerative changes in the tubular epithelium.
 

No clinical signs were observed during examination of male and
 

female mice in the groups dosed at 4,600 ppm. At necropsy the
 

groups of mice receiving the four highest doses had enlarged
 

spleens and mesenteric nodes. Trace to very slight stasis of the
 
bile, trace to very slight granular intracytoplasmic pigmentation
 

of the proximal convoluted tubular epithelium, and slight
 

pigmentation of the splenic red pulp were noted in all animals
 

examined.
 

Ten percent depression in body weight was the major criterion for
 

estimation of MTD's. The doses that were required to produce
 

this response were determined by the following procedure: first,
 

least square regressions of mean body weights versus days on
 

study were used to estimate mean body weights of each of the
 

dosed groups at day 49. Next, probits of the percent weights of
 

corresponding control groups were plotted against the logarithms
 

of the doses, and least squares regressions fitted to the data
 

were used to estimate the doses required to induce 10% depression
 

in weight.
 

Based on the data thus obtained, the low and high doses for
 

chronic studies using male and female rats were set at 200 and
 

400 ppm; using male mice, 200. and 400 ppm; and using female mice,
 

400 to 800 ppm.
 

10
 
F. Chronic Studies
 

The test groups, doses administered, and durations of the chronic
 

feeding studies are shown in tables 2 and 3. Due to excessive
 

weight depression in the dosed female mice, doses for the low-


and high-dose groups were reduced to 100 and 400 ppm,
 

respectively, after week 38.
 

G. Clinical and Pathologic Examinations
 

All animals were observed twice daily. Observations for sick,
 

tumor-bearing, and moribund animals were recorded daily.
 

Clinical examination and palpation for masses were performed each
 

month, and the animals were weighed at least once per month.
 

Moribund animals and animals that survived to the end of the
 

bioassay were killed using CCL and necropsied.
 

The pathologic evaluation consisted of gross and microsocopic
 

examination of major tissues, major organs, and all gross
 

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

embedded in paraffin, sectioned, and stained with hematoxylin and
 

eosin. The following tissues were examined microscopically:
 

skin, lungs and bronchi, trachea, bone marrow (femur), spleen,"
 

11
 
Table 2. Azobenzene Chronic Feeding Studies
 

in Rats
 

Sex and Initial Azobenzene Time on 

Test No. of in Diet (b) Study 

Group Animals (a) (ppm) (weeks) 

Male
 

Matched-Control 20 0 106
 

Low-Dose 50 200 106
 

High -Dose 50 400 105
 

Female
 

Mat ched-Contro 1 20 0 106
 

Low-Dose 50 200 106
 

High-Dose 50 400 105-106
 

(a)	 All animals were 6 weeks of age when placed on study.
 

(b)	 Test and control diets were provided ad libitum 7 days
 

per week.
 

12
 
Table 3. Azobenzene Chronic Feeding Studies
 

in Mice
 

Sex Initial Azobenzene Time on Time-Weighted
 

and Test No. of in Diet (b) Study Average Dose (c)
 

Group Animals (a) (ppm) (weeks) (ppm)
 

Male
 

Matched-


Control 20 0 106
 

Low-Dose 50 200 105
 

High -Dose 50 400 105
 

Female
 

Matched-


Control 20 0 106
 

Low-Dose 50	 400 38
 

LOO 68 208
 

High-Dose 50	 800 38
 

400 67 545
 

(a) All animals were 6 weeks of age when placed on study.
 

(b) Test and control diets were provided ad libitum 7 days
 

per week.
 

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

. of weeks receiving each dose) 

13 
lymph nodes (mesenteric and submandibular), thymus, heart,
 

salivary glands (parotid, sublingual, and submaxillary), liver,
 

pancreas, esophagus, stomach (glandular and nonglandular), small
 

and large intestines, kidney, urinary bladder, pituitary,
 

adrenal, thyroid, parathyroid, testis, prostate, mammary gland,
 

uterus, ovary, brain (cerebrum and cerebellum), and all tissue
 

masses. Peripheral blood smears also were made for all animals,
 

whenever possible.
 

Necropsies were also performed on all animals found dead, unless
 

precluded in whole or in part by autolysis or cannibalization.
 

Thus, the number of animals from which particular organs or
 

tissues were examined microscopically varies and does not
 

necessarily represent the number of animals that were placed on
 

study in each group.
 

H. Data Recording and Statistical Analyses
 

Pertinent data on this experiment have been recorded in an
 

automatic data processing system, the Carcinogenesis Bioassay
 

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

descriptive information on the chemicals, animals, experimental
 

design, clinical observations, survival, body weight, and
 

14
 
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 appropriate statistical
 

techniques described in this section. Those analyses of
 

experimental results that bear on the possibility of
 

carcinogenicity are discussed in the statistical narrative
 

section.
 

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

extensions of Cox's methods for testing for a dose-related
 

trend. One-tailed P values have been reported for all tests
 

except the departure from linearity test, which is only reported
 

when its two-tailed P values is less than 0.05.
 

15
 
The incidence of neoplastic or nonneoplastic lesions has been
 

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

at a specific anatomic site (numerator) to the number of animals
 

in which that site is examined (denominator). In most instances,
 

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic
 

examination was required to detect lesions prior to histologic
 

sampling (e.g., skin or mammary tumors), or when lesions could
 

have appeared at multiple sites (e.g., lymphomas), the
 

denominators consist of the number of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

animals. As a part of these analyses, the one-tailed Fisher
 

exact test (Cox, 1970) was used to compare the tumor incidence of
 

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

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

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

made. The Bonferroni inequality (Miller, 1966) requires that the
 

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

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

16
 
narrative section. It is not, however, presented in the tables,
 

where the Fisher exact P values are shown.
 

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

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

assumption of a linear trend, this test determines if the slope
 

of the dose-response curve is different from zero at the
 

one-tailed 0.05 level of significance. Unless otherwise noted,
 

the direction of the significant trend is a positive dose
 

relationship. This method also provides a two-tailed test of
 

departure from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

first tumor was observed were excluded by basing the statistical
 

tests on animals that survived at least 52 weeks, unless a tumor
 

was found at the anatomic site of interest before week 52. When
 

such an early tumor was found, comparisons were based exclusively
 

on animals that survived at least as long as the animal in which
 

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.
 

17
 
When appropriate, life-table methods were used to analyze the
 

incidence of tumors. Curves of the proportions surviving without
 

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

The week during which an animal died naturally or was sacrificed
 

was entered as the time point of tumor observation. Cox's
 

methods of comparing these curves were used for two groups;
 

Tarone's extension to testing for linear trend was used for three
 

groups. The statistical tests for the incidence of tumors which
 

used life-table methods were one-tailed and, unless otherwise
 

noted, in the direction of a positive dose relationship.
 

Significant departures from linearity (P less than 0.05,
 

two-tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared with its control was calculated
 

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

relative risk is defined as P../P where p is the true
 

binomial probability of the incidence of a specific type of tumor
 

in a dosed group of animals and p is the true probability of
 

the spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

a specific tumor in a dosed group and the proportion in a control
 

group corresponds to a relative risk of unity. Values in excess
 

18
 
of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in
 

approximately 95% of a large number of identical experiments, the
 

true ratio of the risk in a dosed group of animals to that in a
 

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

significant result (P less than 0.025 one-tailed test when the
 

control incidence is not zero, P less than 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.
 

19
 
20

 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of dosed male and female rats were lower than
 

those of corresponding controls and were dose related throughout
 

the bioassay (figure 1). Fluctuation in the growth curves may be
 

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

body weight may be subject to variation. Other clinical signs,
 

such as corneal opacity and tissue masses, occurred at low
 

incidences and were common to dosed and control groups.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats administered azobenzene in the
 

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

matched controls, are shown in figure 2. In male rats, the
 

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

mortality is significant (P less than 0.001). An indicated
 

departure from linear trend is observed (P less than 0.001)
 

because the low-dose animals survived longer than the control
 

21
 
TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves for Rats Administered Azobenzene in the Diet 

22 
TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves for Rats Administered Azobenzene in the Diet 

23 
animals. In females, the result of the Tarone test is not
 

significant.
 

In male rats, 35/50 (70%) of the high-dose group, 47/50 (94%) of
 

the low-dose group, and 17/20 (85%) of the control group lived at
 

least as long as week 90 on study. In females, 48/50 (96%) of
 

the high-dose group, 44/50 (88%) of the low-dose group, and 19/20
 

(95%) of the control group lived at least as long as week 90 on
 

study.
 

Sufficient numbers of rats of each sex were at risk for the
 

development of late-appearing tumors.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

Appendix A, tables Al and A2; findings on nonneoplastic lesions
 

are summarized in Appendix C, tables Cl and C2.
 

Long-term feeding of azobenzene to male and female rats was
 

associated with a marked increase in the frequency of malignant
 

mesenchymal tumors involving the abdominal viscera including
 

mesentery and omentum. None of these neoplasms were seen in
 

24
 
control animals. These tumors often spread throughout the
 

abdominal cavity involving multiple tissues and organs to such an
 

extent that determination of the primary site was precluded. The
 

spectrum of mesenchymal malignancies included a striking array of
 

forms, from bizarre undifferentiated sarcomas to relatively well-


differentiated fibrosarcomas, osteogenic sarcomas, and vascular
 

neoplasms.
 

The abdominal organ most constantly involved was the spleen, and
 

the most commonly observed tumors of the spleen were
 

fibrosarcomas. Some were characterized by proliferating
 

anaplastic spindle cells forming broad sheets, the occurrence of
 

tumor giant cells, the presence of numerous bizarre mitoses, and
 

large areas of ischemic necrosis. Others were more
 

differentiated, having varying amounts of collagen, and were
 

composed of proliferating spindle cells growing in sheets and
 

intersecting bundles. Occasionally, these tumors contained
 

mature adipose tissue. Osteogenic sarcomas observed were all
 

well-differentiated and contained large amounts of bone, often
 

trabecular, and osteoid tissue. Hemangiosarcomas observed in
 

dosed animals varied from solid tumors composed of proliferating
 

sheets of spindle cells containing a myriad of cleft-like
 

structures or vascular channels with varying numbers of
 

erythrocytes to massive cavernous blood-filled tumors with
 

25
 
thick fibrotic walls and intersected by traberculae lined by
 

neoplastic cells. A well-differentiated hemangiopericytoma, with
 

a typical "whorling" pattern of proliferating pericytes was also
 

observed in a dosed animal.
 

A large number of sarcomas were observed involving multiple
 

organs of the abdominal cavity. These probably represent
 

extensions of primary splenic neoplasms. Often, nearly every
 

abdominal organ and tissue, including scrotal fat, was affected,
 

testifying to the extreme invasiveness of these tumors.
 

A wide variety of other neoplasms were observed in all groups,
 

but there was no clear-cut relationship of these neoplasms to
 

azobenzene exposure.
 

Nonproliferative lesions associated with long-term dietary intake
 

of azobenzene were observed in several instances. Increased
 

amounts of hemosiderin were deposited in the spleen, liver, and
 

renal tubular epithelium of dosed female rats. Chronic
 

capsulitis of the spleen was observed in all dosed groups of
 

males and females, but particularly in the females. This was
 

characterized by a cystic papillary proliferation of serosal
 

cells, thickening of the capsule, and focal collections of
 

mononuclear cells and mineral deposits.
 

26
 
Several low-dose male and female rats had unusual accumulations
 

of mature-appearing adipose tissue within the spleen, and several
 

females had varying degrees of fibrosis of the splenic pulp.
 

These changes may be within the spectrum of proliferative lesions
 

already discussed.
 

A wide variety of lesions previously found in aged F344 rats
 

occurred in all groups without relationship to administration of
 

the test chemical.
 

Based on the histopathologic examination, azobenzene was
 

carcinogenic (sarcomagenic) to F344 rats, being associated with a
 

high incidence of malignant mesenchymal tumors that were not
 

observed in control animals, under the conditions of this
 

bioassay. The striking array of splenic proliferative lesions
 

suggests that azobenzene may have an effect on primitive
 

reticular cells that are the precursors of the various
 

differentiated components.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

27
 
least two animals of one group and at an incidence of at least 5%
 

in one or more than one group.
 

The result of the Cochran-Armitage test for positive dose-related
 

trend in the incidence of fibrosarcoma of the spleen is
 

significant in both male (P = 0.020) and female (P = 0.012) rats,
 

although the results of the Fisher exact test are not
 

significant. The historical records for the rats maintained as
 

controls at this laboratory show an incidence of tumors of 1/285
 

(0.4%) in males and 0/285 in females. Using the incidence of
 

1/285 as a parameter and assuming a binomial distribution, the
 

probability level of obtaining 7 or more such tumors out of 49 or
 

50 animals is less than 0.001.
 

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

incidence of fibrosarcoma of multiple organs is significant (P
 

less than 0.001). An indicated departure from linear trend is
 

observed (P = 0.030), due to the steep increase in the incidence
 

of tumors in the high-dose group. The Fisher exact test shows
 

that the incidence in the high-dose group is significantly higher
 

(P = 0.007) than that in the control group. The statistical
 

conclusion is that the incidence of fibrosarcoma of multiple
 

organs in male rats is associated with the administration of
 

28
 
azobenzene. No such tumor is observed at a significant incidence
 

in females.
 

In female rats, the result of the Cochran-Armitage test for the
 

incidence of osteosarcoma of the spleen is significant (P =
 

0.041), but the results of the Fisher exact test are not
 

significant. The historical records of this laboratory show no
 

such tumor among 285 control F344 female rats. Using 1/285 as a
 

parameter and assuming a binomial distribution, the probability
 

level of obtaining 5 such tumors out of 50 animals is less than
 

0.001.
 

When tests are performed using the incidences of animals with any
 

type of sarcoma in the abdominal cavity, the P values for
 

dose-related trend and for significance of direct comparisons of
 

high-dose and control groups are less than 0.001 for both male
 

and female rats.
 

Significant results in the negative direction are observed in the
 

incidences of adenomas of the pituitary and of interstitial-cell
 

tumors of the testis in male rats. The increased incidence in
 

the negative direction may be due to the earlier mortality of the
 

high-dose animals.
 

29
 
In summary, the incidences of male and female animals with 

sarcomas in the abdominal cavity are related to the 

administration of azobenzene. 

30
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of dosed male and female mice were lower than
 

those of corresponding controls, and for female mice were dose
 

related throughout the bioassay (figure 3). Fluctuation in the
 

growth curve may be due to mortality; as the size of a group
 

diminishes, the mean body weight may be subject to greater
 

variation. Other clinical signs such as alopecia, corneal
 

opacity, and tissue masses occurred at low incidences and were
 

common to both dosed and control groups.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice administered azobenzene in the
 

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

matched controls, are shown in figure 4. The result of the
 

Tarone test for positive dose-related trend in mortality is not
 

significant in male mice, but is significant (P less than 0.001)
 

in females.
 

31
 
TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves for Mice Administered Azobenzene in the Diet 

32 
TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves for Mice Administered Azobenzene in the Diet 

33 
In male mice, 46/50 (92%) of the high-dose group, 49/50 (98%) of
 

the low-dose group, and 18/20 (90%) of the control group lived at
 

least as 'long as week 90 on study. In females, 37/50 (74%) of
 

the high-dose group, 43/50 (86%) of the low-dose group, and 19/20
 

(95%) of the control group lived at least as long as week 90 on
 

study.
 

Sufficient numbers of mice of each sex were at risk for the
 

development of late-appearing tumors.
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

Appendix B, tables Bl and B2; findings on nonneoplastic lesions
 

are summarized in Appendix D, tables Dl and D2.
 

A variety of neoplasms occurred with approximately equal
 

frequency in dosed and control mice. The incidence,
 

distribution, and nature of these neoplasms are similar to those
 

of neoplasms commonly seen in aged B6C3F1 mice.
 

Several inflammatory, degenerative, and proliferative lesions
 

commonly seen in aged B6C3F1 mice occurred with approximately"1
 

34
 
equal frequency in dosed and control animals. The occurrence of
 

these lesions was unrelated to exposure to azobenzene.
 

Based on the histopathologic examination, changes related to
 

administration of azobenzene were not observed in B6C3F1 mice
 

receiving azobenzene under the conditions of this bioassay.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

least two animals of one group and at an incidence of at least 5%
 

in one or more than one group.
 

The results of the Cochran-Armitage test for positive
 

dose-related trend in the incidence of tumors and the results of
 

the Fisher exact test comparing the incidence of tumors in the
 

control group with that in each dosed group in the positive
 

direction are not significant in either sex. However,
 

significant results in the negative direction are observed in the
 

combined incidence of liver tumors in male mice.
 

35
 
In each of the 95% confidence intervals for relative risk, shown
 

in the tables, the value of one or less than one is included;
 

this indicates the absence of significant positive results. It
 

should also be noted that each of the intervals, except that for
 

the combined incidence of liver tumors in the high-dose male
 

mice, has an upper limit greater than one, indicating the
 

theoretical possibility of the induction of tumors by azobenzene,
 

which could not be detected under the conditions of this test.
 

36
 
V. DISCUSSION
 

Mean body weights of the dosed rats and mice of each sex were
 

lower than those of corresponding controls, and were generally
 

dose related throughout the bioassay. Mortality was dose related
 

in the male rats and the female mice, but was not significantly
 

affected in the female rats and male mice. Survival was 70% or
 

greater at week 90 on study in all dosed and control groups of
 

each species and sex; thus, sufficient numbers of animals were at
 

risk in all groups for the development of late-appearing tumors.
 

In rats, a large number of animals had various types of sarcomas
 

of the spleen and other abdominal organs. Fibrosarcomas of the
 

spleen occurred at incidences that were dose related in b-,th male
 

(P = 0.020) and female (P = 0.012) rats, although in direct
 

comparisons incidences of the tumors in individual dose'1 <?roups
 

were not significantly higher than those in corresponding
 

controls (males: controls 0/20, low-dose 2/49, high-dose 7/49;
 

females: controls 0/20, low-dose 1/50, high-dose 7/50). The
 

incidence of fibrosarcomas of the spleen in historical-control
 

male F344 rats at this laboratory is 1/285, and in female F344
 

rats it is 0/285; using 1/285 as a parameter and assuming a
 

binomial distribution, the probability that the occurrence of 7
 

37
 
such tumors in 49 male or 50 female high-dose rats in the present
 

bioassay was due to chance is less than 0.001.
 

Fibrosarcomas of multiple organs (organs other than the spleen)
 

occurred at incidences that were dose related (P less than 0.001)
 

in the male rats, and in a direct comparison the incidence in the
 

high-dose group was significantly higher (P = 0.007) than that in
 

the control group (controls 0/20, low-dose 0/49, high-dose
 

13/50). In females, fibrosarcomas of multiple organs occurred
 

only in one high-dose animal.
 

Osteosarcomas of the spleen occurred at incidences that were dose
 

related (P = 0.041) in the female rats, although in direct
 

comparisons the incidences in the individual dosed groups were
 

not significantly higher than that in the control group (controls
 

0/20, low-dose 1/50, high-dose 5/50). The incidence of
 

osteosarcomas of the spleen in historical-control female F344
 

rats at this laboratory is 0/285; using 1/285 as a parameter and
 

assuming a binomial distribution, the probability that the
 

occurrence of 5 Such tumors in the 50 high-dose female rats of
 

the present bioassay was due to chance is less than 0.001. In
 

males, osteosarcomas of the spleen occurred in one low-dose male
 

and one high-dose male, but in no control males. Osteosarcomas
 

also occurred in multiple organs in three additional high-dose
 

38
 
females and in three additional high-dose males, but in no
 

controls of either sex.
 

When all types of sarcomas of the abdominal cavity were combined,
 

the incidences of such tumors in both male and female rats were
 

dose related (P less than 0.001); and in direct comparisons the
 

incidences of these tumors in the high-dose groups of males and
 

females were significantly higher (P less than 0.001) than those
 

in the corresponding controls (males: controls 0/20, low-dose
 

5/49, high-dose 31/49; females: controls 0/20, low-dose 5/50,
 

high-dose 21/50).
 

No tumors occurred in the male or female mice at incidences that
 

were significantly higher in the dosed groups than in the
 

corresponding controls.
 

Essentially no evidence of carcinogenicity of azobenzene for rats
 

or mice was obtained in early work carried out from 1936 to 1952
 

(Hartwell, 1963; Eldredge and Luck, 1952; Spitz et al., 1950),
 

and the compound has generally been considered by cancer
 

investigators not to be carcinogenic. In the work of Innes et
 

al. (international Agency for Research on Cancer, 1975; Innes et
 

al., 1969; NTIS, 1968), however, it was reported that when
 

azobenzene was administered at 21.5 mg/kg body weight by stomach
 

39
 
tube for 3 weeks, then in the diet at 56 ppm for 18 months, to
 

hybrid mice (B6C3F1 and B6AKF1), an elevated incidence of
 

hepatomas (P = 0.01) was observed in the male B6C3F1 hybrids;
 

nevertheless, additional evaluation was proposed. The
 

observation of an increased incidence of tumors of the liver in
 

B6C3F1 mice in the study by Innes et al. was not confirmed by the
 

results of the present bioassay. Damage to the spleen of the
 

dosed F344 rats, characterized by hemosiderosis and capsulitis,
 

occurred in the present bioassay; similar damage was reported for
 

Wistar rats fed azobenzene in the diet in previous studies (Smith
 

et al., 1943).
 

It is concluded that under the conditions of this bioassay,
 

azobenzene was carcinogenic (sarcomagenic) for F344 rats,
 

inducing various types of sarcomas in the spleen and other
 

abdominal organs of both males and females. The test chemical
 

was not carcinogenic for B6C3F1 mice of either sex.
 

40
 
VI. BIBLIOGRAPHY
 

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

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

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

Panel o>f_ Careinogenicity o£ the Cancer Research Commission of the
 

UICC, Vol. 2^. International Union Against Cancer, Geneva, 1969.
 

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

London, 1970, pp. 48-52.
 

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

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

Daoust, R. and Calamai, R., Hyperbasophilic foci as sites of 

neoplastic transformation in hepatic parenchyma. Cancer Res. 

3J.: 1290-1296, 1971. 

Department of Labor, Carcinogens - Occupational Health and Safety
 

Standards. Federal Register 39 (20);3756-3757.
 

Eldredge, N. T. and Luck, J. M., Electrophoretic studies on the
 

water-soluble proteins of liver during azo dye carcinogenesis in
 

the rats. Cancer Res. 12^801-806, 1952.
 

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

significance tests, confidence limits and adjustments for
 

stratification. Rev. Int. Stat. Inst. 39_:148-169, 1971.
 

Hartwell, J. L., Survey £f Compounds Which Have Been Tested for
 

Carcinogenic Activity. Public Health Service Publication No.
 

149, National Cancer Institute, National Institutes of Health,
 

Bethesda, Md., 1963.
 

Innes, J. R. M., Ulland, B. M., Valeric, M. G., Petrucelli, L.,
 

Fishbein, L., Hart, E. R., Pallota, A. J., Bates, R. R., Falk, H.
 

L., Gart, J. J., Klein, M., Mitchell, I., and Peters, J.,
 

Bioassay of pesticides and industrial chemicals for
 

tumorigenicity in mice: a preliminary note. £. Natl Cancer Inst.
 

42:1101-1114, 1969.
 

41
 
International Agency for Research on Cancer, Azobenzene. In:
 

IARC Monographs on the Evaluation of_ the Carcinogenic Risk £f
 

Chemicals tx? Man - Some Aromatic Azo Compounds, Vol. 8^
 

International Agency for Research on Cancer, Lyon, France, 1975,
 

pp. 75-81.
 

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

incomplete observations. J_. Amer. Statist. Assoc. 53_:457-481, 

1958. 

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

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

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

Lurie, A. P., Benzidine. In: Kirk-othmer Encyclopedia £f
 

Chemical Technology, Vol. 3_, Interscience Publishers, New York,
 

1964, pp. 408-412 and 420.
 

Miller, R. G., Jr., Simultaneous Statistical Inference, McGraw-


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

National Technical Information Service (NTIS), Evaluation £f
 

Carcinogenic, Teratogenic, and Mutagenic Activities of Selected
 

Pesticides and Industrial Chemicals. Vol. _!_. Carcinogenic
 

Study. U.S. Department of Commerce, Washington, D.C., 1968.
 

Noller, C. R., Aromatic nitro compounds. In: Chemistry of Organic
 

Compounds, W. B. Saunders Co., Philadelphia, 1966, pp. 518-520.
 

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

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

induced by different numbers of administrations of benzo (a)pyrene
 

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

Smith, M. I., Lillie, R. D., and Stohlman, E. F., The toxicity
 

and histopathology of some azo compounds as influenced by dietary
 

protein. Publ. Hlth. Rep. 581:304-317, 1943.
 

Spitz, S., Maguigan, W. H., and Dobriner, K., The carcinogenic
 

action of benzidine. Cancer 3_:789-804, 1950.
 

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

62 (3):679-682, 1975.
 

42
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS ADMINISTERED AZOBENZENE IN THE DIET
 

43
 
44

 
TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED AZOBENZENE IN THE DIET 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUHbEH OF ANIMALS NECROPSIED
 

45
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

DIGESTIVE SYSTEM 

ILIVER 

NEOPLASTIC NODULE 

HEPATOCELLULAR CARCINOM A 

HS-MANGIOSABCOMA 

OSTBOSABCCEA, HETASTATIC 

IPANCREAS 

HiMANGIOSARCCM A 

#COLON 

BUCINOOS CYSTADENOCARCINCM A 

MUCINOOS ADENOCARCINOM A 

URINAR Y SYSTEH 

NONi, 

JNDOCBINE SYSTEM 

tPITUITARY 

ADENOMA , NOS 

CHROMOPHOBE ADENOM A 

#ADRENA L 

PHEOCHROMOCYTOMA 

fTHYROID 

C-CELL CARCINOM A 

REPRODUCTIVE SYSTEH 

*MAMMARY GLAND 

FIBROADENOM A 

*PREPUTIAL GIAND 

SQUAMOUS CELL CARCINCM A 

#TESTIS 

INTERSTITIAL-CELL TUMOR 

NERVOU S SYSTEM 

_ NONE 

MATCHED 

CONTROL 

(20) 

(20) 

(20) 

1 (5») 

(20) 

U (20X) 

(20) 

1 (536) 

(20) 

(20) 

(20) 

1 (5X) 

(20) 

17 (85%) 

LOW DOSE 

(1*9) 

1 (2%) 

1 (2X) 

(47) 

(47) 

1 (2«) 

C*9)

2 (US) 

2 (i*X) 

(49) 

1 (2X) 

(49) 

1 (28) 

(49) 

2 (4%) 

(49) 

(48) 

41 (85%) 

HIGH DOSE 

(50) 

2 (4S) 

1 (2X) 

1 (2%) 

1 (2S) 

(48) 

1 (2%) 

(50) 

(49) 

3 (68) 

(5C) 

1 (2») 

(48) 

1 (2%) 

(50) 

<5C) 

(49) 

31 (63%} 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

46 
TABLE A1. MALE RATS: NEOPLASMS

SPECIAL SENSE ORGANS
 

*EAR CANAL


KERATOACAKTHCHA


HUSCULOSKELE1AI SYSTEH
 

NONE
 

EODY CAVITIES
 

*ABDOflINAL CAVITY


HiMANGIOSABCOBA
 

*HES£NTERY


SAKCOHA, NCS
 

OSIEOSARCCl'.A, HETASTATIC


ALL OTHER SYSTEMS
 

*HULTIPLE ORGANS


SARCOMA, NCS
 

FIBBOSARCCBA
 

HiSOTHELICRA, HALIGNANT


OSTEOSARCOMA
 

ANIMAL DISPOSITION SUMHARY
 

ANIHALS INITIALLY IN STDDY


NATURAL DIATHS


HORIBOND SACRIFICE


SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE


ANIHAL HISSING


3 INCLUDES AUTCLYZED ANIHA1S
 

 (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 (20) (49) (50) 

1 (5«) 

 (20) (49) (50) 

1 (2X) 

 (20) (US) (50) 

1 (2X) 

1 (2%) 

 (20) (49) (50) 

2 (4S) 

13 (2f>%) 

1 (2%) 

3 (6X) 

 20 50 50 

5 4 2S 

2 4 5 

 13 41 16 

1 

# NUMBER OF ANIHALS HITH TISSUE EXAHINED MICROSCOPICALLY
 

* NUMBER OF AKIMALS NECROPSIED
 

47
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

TUMOR SUMMARY
 

TOTAL ANIMALS BITH PRIMARY TUMOBS*


TOTAL PBIBABY TOBOBS


TOTAL ANIHALS WITH BENIGN TDHOBS


TOTAL EEBIGN TUHOBS


TOTAL ANIMALS WITH MALIGNANT TUHORS


TOTAL MALIGNANT TUMOKS


TOTAL ANIMALS HITH SECONDARY TUMOBSf


TOTAL SECONDARY TUMORS


TOTAL ANIMALS BITH TUMORS UNCERTAIN­ BENIGN OR HAIIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS HITH TUMOBS UNCERTAIN­ PEMARY OR KETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 20 15 47 

 29 63 87 

 19 45 34 

 23 51 36 

6 11 42 

6 11 49 

1 2 

2 2 

1 2 

1 2 

* PRIMARY TOMCES: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

48
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED AZOBENZENE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUEY 20 5.) 50
 

ANIMALS NECBOE5IED 20 50 50
 

ANIMAi.S EXAHINIE HISTOPATHOLOGICALLY 20 50 50
 

INTEGUMENTARY SYSTEM
 

*SKIN (20) (50) (50)
 

SQUAMOUS CELL CARCINOMA 1 (2X)
 

*SUBCUT TISSUI (20) (5J) (50)
 

FIBROMA 2 (U%) 1 (2X)
 

FIBHOADENCMA 1 (2%)
 

RESPIBATORY SYSTEM
 

tLUN G (20) (50) (50) 

ALVEOLAR/BRONCHIOLAR ADiNOMA 1 (255) 

ALVEOLAR/E5CNCHIOLAR CARCINOM A 1 (2») 

MIXED TUBCE , METASTATIC 1 (2*) 

HEMATOPOIETIC SYSTEM 

*MULIIPLE ORGAN S (20) (50) (50) 

GBANULOCYTI C LEUKEMI A 2 (HI) 1 (2%) 

flONOCYTIC IEUKEHIA 1 (5X) 3 (6%) 

ISPL^EN (20) (50) (50) 

FiBHOSARCCBA 1 (2%) 1 (1435) 

lUflANGIOSABCOMA 1 (2%) H (8%) 

HEMANGIOIEflCYTOMA , MALIGNAN T 1 (2X) 

OSTEOSARCCMA 1 (2%) 5 (10*) 

#SPL£NIC CAPSCL£ (20) (50) (5CJ 

FIBROSARCCK A 1 (2X) 

ILYMPH NODE (20) (50) (50) 

MillG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2S) 

CIRCULATORY SYSTEM 

NQN£_ 

* NUHdEH OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 * NUHBEB OF AMIMALS NECROPSIED
 

49
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

—— — T ••-— ••- -r--r-— . —• J. - :-•-.-—. T-..- J "*—- ""• TM* -""•-" ^ ' ~ ' TM '• ' 

MATCHED 

CONTROL LOW DOSE 

DIGESTIVE SYSTEM 

4LIVER (20) (50) 

NEOPLASTIC NODDLE 

HEPATOCE1IDLAB CARCINOMA 1 (2X) 

t JEJUNUM (20) (50) 

SARCOMA, NOS 1 (2X) 

URINAB Y SYSTEM 

NONi 

ENDOCRINE SYS1EB 

4PITUITAHY (20) (49) 

ADENOMA , NOS 4 (20%) 7 (14X) 

CHBOMOPHCEE ADENOMA 1 <5X) 1 (2X) 

IADBENA L (20) (50)

 

PHEOCHROMOCYTOMA 

ITHYfiOID (20) (50) 

FOLLICOLAB-CELL ADENOMA 1 (2X) 

FOLLICULAB-CELL CARCINOMA 1 (2X) 

C-CELL CARCINOMA 1 (5X) 3 (6X) 

REPRODUCTIVE SYSTEM 

*MABHARY GLAhE (20) (50) 

UNDIFFERENTIATE D CARCINOMA 1 (5X) 

AuENOMA , NOS 1 (2X) 

ADENOCARCINOMA, NOS 1 (5X) 

MIXED TUMCR, MALIGNAN T 

FIBROADEKCK A 3 (15X) 5 (10X) 

*CLITOBAL GLAN D (20) (50) 

SQUAMOU S CELL CABCINOMA 1 (2%) 

f UTEBUS (20) (50) 

CARCINOMA^NOS 1, (2%1 

* NDMBER OF ASIHALS HITH TISSUE EXAMINED MICROSCOPICALLY
 * NDMBEfi OF AKIMAL S NECBOPSIED 

HIGH DOSE 

(50) 

2 (4X) 

(50 ) 

(50) 

1 (14X) 

(50) 

1 (2J8) 

(48 ) 

1 (2X) 

(50) 

1 (2%) 

1 (2X ) 

3 (6X) 

(50) 

1 (2%) 

(50) 

50 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ENDOMETEIAI STROMAL POLYP 2 (1051) 5 (1055) 1 (235)
 

liEHVOUS SYSTSK
 

tCERtBRUH (20) (50) (50)
 

ASTROCYTCKA 1 (555) 1 (255)
 

MENINGIOMA 1 (235)
 

SPECIAL SENSE ORGANS
 

NONE
 

BUSCOLOSKELETAI SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDOMINAL CAVITY (20) (50) (50)
 

OSTEOSAECCHA 1 (2*)
 

ALL OTHER SYSTEMS 

*BULXIPI.E OBGANS (20) (50) (50) 

FiBROSARCCEA 1 (2%) 

OSTEOSAECCMA 3 (6%) 

ANIBAi DISPOSI1ICN SUHBABY 

ANIBALS INITIALLY IN STUDY 20 50 50 

NATURAL DfATH3 2 9 9 

BOfilBUND SACRIFICE 3 3 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 18 38 38 

ANIMAL HISSING 

9 INCLUDES AH10LYZEP ANIflALS 

i NOHBER OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECBOPSIED 

51 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

10HOB SUMMARY
 

TOTAL ANIMALS BITH PRIMARY TUMORS*


TUTAL PBIHARY TUMOES


TOTAL ANIMAIS WITH BENIGN TUMOBS


TOTAL EENIGN TONOBS


TOTAL ANIMALS WITH MALIGNANT TUMOBS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS HITH SECONDARY TOMORSf


TOTAL SECCliDABY TOHOBS


TOTAL ANIMALS WITH TUBORS UNCERTAIN­ BEN1GN OB KAIIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMAIS WITH TUMOSS UNCEBTAIN-


PRIfiABY OB DITASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 10 32 33 

 15 43 45 

9 19 13 

 10 23 16 

5 18 24 

5 2J 27 

1 

1 

2 

2 

* PBIMABY TUMCBS: ALL TUHOHS £XCEPT SECONDABY TUMORS
 * SECONDARY TUf.ORS: METASTATIC TUMORS OR TUMOBS INVASIVE INTO AN ADJACENT ORGAN
 

52
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE ADMINISTERED AZOBENZENE IN THE DIET
 

53
 
54

 
TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED AZOBENZENE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

JNIHA^S INITIJILY IN STUE Y 20 50 50 

*N.IMAi.S MISSING 1 2 

ANIMALS NECROPSIED 23 49 48 

ANIBA.LS EXAMINE D HISTOPATHOLOGICAL!Y 20 49 48 

INTEGJflENIABY SYSTEM 

*SUBcUT TISSUE (20) (49) (48) 

SARCOMA, NCS 1 (2%) 

FIBROSARCOMA 1 (2%) 

BESPIdATORY SYSTEM 

*IUN G (20) (49) (47) 

HtPATOCEILULAS CARCINOMA , METAST 1 (5X) 

ALVEOLAR/EEONCHIOLA R ADENOM A 2 (105!) 3 (6S) 1 (255) 

ALVEOLAR/ERCNCHIOLA R CARCINOMA 1 (2X) 

HEMATOPOIETIC SYSTEM 

*MULTIP1E OFGAN S (20) (49 ) (48) 

MALIG.LYMEBCMA , UHDIFFER-TYPE 1 (5*) 2 (4X) 1 (2«) 

MALIG. LYMPHOMA , LYMPHOCYTIC TYPE 2 (10X) 3 (651) 

MALIG.LYMPHOMA , HISTIOCYTIC TYPE 2 (4X) 2 (45S) 

ISPL^EN (20) (49) (47)
 

HtMANGIOSARCCMA 1 (5%) 2 (4%) 2 (4«)
 

*M£ScNTEHIC L. NODE (20) (48) (47)
 

MALIG.LYBPfiCMA , UNDIFFEB-TYPE 1 (2%) MALIG.LYMPF.CMA , LYMPHOCYTIC TYPE 1 (2») 

#LJVi.E (20) (49) (48) 

MALIG . LYHP1-CMA, HISTIOCYTIC TYPE 1 (555) 

*MESi.NTEHY (20) (49) (48) 

MALI3. LYKFHCMA , LYMPHOCYTIC TYP E 1 (255) 

ITHYilUS (12) (40 ) (40 ) 

I NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NRCROPSTFn
 

55
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

CIRCOiATOHY SYSTEM 

NONil . 

ElfiESTIVE SJSTIM 

I1IVEH

NEOPLASTIC NODULE

HiPATOCEIIOLAR CARCINOMA

H£MANGIOSAICOH A

UEINA8 Y SYSTifl 

NONi 

iNDOCEINE SYS1IB 

lADRiiNAL

PHEOCHROHCCYTOB A

SAfiCOMA, NCS

*THYriOID

PAPILLARY 4D1JJOCARCINOMA

FOLLICULAR-CELL ADENOM A

REPRODUCTIVE SYSTE M 

NONfi 

KERVOUS SYSTIM
 

NON£
 

SPECIAL SENSE CHGANS
 

NONi
 

MUSCULOSKELETAL SYSTEM
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 (20) (49) (48) 

6 [30%) 8 (16%) 

3 (15X) 10 (20K) 2 (U») 

3 (6») 

 (20) (U8) (45) 

3 (7%) 

1 (2%) 

 (18) (49) (47) 

1 (2%) 

1 (2%) 

t NUMtiER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

56
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES 

1U OTHEB SYSTIMS 

HONE 

ANIHAi DISPOSITION SUMMAR Y 

ANIMALS INITIALLY IN STUDY 2) 5J 50 

NATURAL DEATHS 2 4 5 

MORIBUND SACRIFICE 2 2 

SCHEDULID SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 16 45 40 

ANIHAL MISSING 1 2 

9 INCLUDES AUTCLY2I D ANIMALS 

TDMO E SUHMAB Y 

TOTAL ANIMAL S WITH PRIMAR Y TOHOES* 13 25 2C 

TOTAL PRIMAR Y TOMORS 16 31 23 

TOTAL ANIMAL S WITH 2 BENIG N TUMORS 3 5 

TOTAL BENIG N TOMORS 2 3 5 

TOTAL ANIMAL S HITH MALIGNAN T TUMORS 8 19 17 

TOTAL MALIGNAN T TUMORS 8 20 18 

TOTAL ANIMAL S WIT H SECONDARY TUMORS* 1 

TOTAL SECCliDAKY TUMOES 1 

TOTAL ANIMALS WITH TUMORS UNCERTAINEENIGN OR MAIIGNANT 6 8
 

TOTAL UNCERTAIN TUMOES 6 8
 

TOTAL ANIMAIS HUH TUMORS UNCEfiTAIN-


PRIflABY OR KETASTATIC
 

TOTAL UNCEBTAIN TUMOES
 

* PRIdARY TUMCES: ALL TOMORS EXCEPT SECONDARY TUMORS
 

« SECONDARY TUBORS: METASTATIC TUMOES OR TUMORS INVASIVE INTC AN ADJACEM OR3AN
 

57
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED AZOBENZENE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAi-S INITIALL Y IN STUDY 20 53 50 

ANIMAL S MISSIN G 1 7 

ANIMALS NECROPSIEE 20 47 38 

ANIMAL S EXAMINE D HISTOPATHOLOGICAL!,! 20 17 38 

INTEGUMENTAR Y SYSTE M 

*SUBcUT TISSUE (20) (HI) (38) 

HiMANGIOSAECOM A 1 (2X) 

RESPIBATORY SYSTEM 

*LUNJ (20) (46) (36)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 2 (10K) 2 (4S) 2 (6*)
 

HEMATOPOIETIC SYSTEM
 

MULTIPLE OSGANS (20) (47) (38)
 

HALIG.iYBPKCHA, UNDIFFEB-TYPE 1 (5*) 1 (2X)
 

MALIG.LYMEEOHA, LYMPHOCYTIC TYPE 2 (10%) 1 (2X) 4 (11*)
 

BALIG.LYKEKCMA, HISTIOCYTIC TYPE 2 (10*) 2 (U%) 1 (3*)
 

UNDIFFERENTIATED LEUKEJSIA 1 (2X)
 

GttANULCCYTIC LEUKEMIA 1 (2X) 2 (5X)
 

ISPljiEN (20) (47) (38)
 

HiHANGIOSJECOBA 1 (2%)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYS1IM
 

ILIVtfR (20) (47) (36) 

NEOPLASM , NOS, METASTATIC 1 (2X) Hi£il2C!!iiLAR_CAR£!NOHA 2 (4X) 

* NUMBER OF ANIMALS HITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF AKIMAIS NECHOPSIID 

58 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

HEMANGIOSJFCCM A 

AiiGIOSABCCKA 

UHINAB Y SYSTE M 

NONA 

IHEOCBINE SYSTEM 

IPITJITARY 

ADENOMA , NCS 

JADRENA L 

PhEOCHROHCCYTOMA 

tTHYBOID 

FOLLICULAR-CELL ADENCH A 

REPRODUCTIVE SYSTEM 

*MAHHAHY GLAlit 

ADENOMA , NCS 

iUTEBUS 

HEBANGIOSSECCSA 

IOVAHY 

GBANULOSA-CELL CARCINOMA 

NERVOU S SYSTJB 

NONE 

SPECIAL SENSE CFGAK S 

*HABLlERIAN GLAN D 

ADENOMA, NCS 

MUSCULOSKELETAL SYSTEM 

*ABDOMINAL HCSCLE 

HBMANGIOSAFCOMA 

MATCHED 

CONTROL LOW DOSE 

(19) C*2) 

2 (11SS) 1 (2X) 

(20) (47)

2 (US) 

(20) (45) 

1 (5X) 

(20) (47) 

(20) (47) 

1 (2%) 

(19) (46) 

120) (47) 

3 (6X) 

(20) (47) 

1 12%) 

HIGH DOSE 

1 {3X) 

1 (3X) 

' (30) 

(37) 

1 (3S) 

(35) 

(38)

1 (3S) 

(37) 

(37)

1 (3X) 

(38) 

1 (3X) 

(38) 

I NDHQEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

59
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HI6H DOSE 

EOEY CAVITIES 

*MEDIASTINU M (20) (K7) (38) 

GRANULOSA-CELL CARCINOMA, METAST 1 (3X) 

*PERITONEUM (20) (47) P8)

 

SAECOHA, NCS 1 12X)

 

*MESENTER Y (20) (U7) (31)

 

HEMANGIOSARCOMA 1 (2X)

 

A LL OTHER SYSTEM S 

*MULTIPLE ORGAN S (20) (47) (38)

 

HEMANGIOSARCOMA 1 (2*)

 

ANIMAL DISPOSITION SUMMAR Y 

ANIMALS INITIALLY IN STUD Y 20 50 50

 

NATURA L DEATHS 1 9 16

 

MORIBUND SACRIFICE J 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 1 

TERMINA L SACRIFICE 19 40 23 

ANIMAL MISSING 1 7 

@ INCLUDES AUTOLYZED ANIMALS ^ , ^ 

I NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

60
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL
 

10MOR SOMMABX 

TOTAL ANIMALS WITH PRIMARY TUHORS* TOTAL PBIBABY TUMOBS 9 

 10 

TOTAL ANIBAIS WITH BENIGN TUMORS

TOTAL EENIGN TUHORS

 H 

5 

TOTAL ANIMALS HITH MALIGNANT

TOTAL MALIGNANT TUMORS

 TUMOHS 5 

5 

TOTAL ANIMALS WITH SECONDARY TUMOBS* 

TOTAL SECCKDARY TUMORS 

TOTAL ANIHALS WITH TUMORS UNCEBTAIN­ BEN1GN OR HALIGNANT 

TOTAL UNCERTAIN TOMOBS 

TOTAL ANIMALS UITH TUMORS

PRIMARY OE KBTASIATIC 

 UNCERTAINTOTAL UNCEFTAIN TUMORS
 

* PRIMARY TUMOBS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

LOW DOSE HIGH DOSE 

20 12 

23 15 

8 5 

8 5 

13 8 

15 10 

1 1 

1 1 

# SECONDARY TUBORS: METASTATIC TUMORS OE TUMORS INVASIVE INTC AN ADJACIKT ORGAN
 

61
 
62

 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN
 

RATS ADMINISTERED AZOBENZENE IN THE DIET
 

63
 
64

 
TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED AZOBENZENE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

SNIHilS INITIJLILY IN STUD Y 23 5J EC 

JNIBALS MISSING 1 

ANIBALS NECRCPSIEE 20 49 50 

ANIBAiS EXAMINE D HISIOPATHOlOGICAiLY 20 49 50 

INTEGUMENTAR Y SYSTE M 

*SKia (2')) (49) (50)

 

EPIDERMA L INCLUSION CYST 1 (2%) 

HYPERKERATOSI S 1 (58) 1 (2%) 

*SUBCUT TISSUE (20) (49) (53) 

HEMOREHAG E 1 (2X) 

HhMATOMA , 1>OS 1 (2X) 

BESPIEATORY SYSTEM 

»LUNG/BRONCHD S (20) (49) (50) 

BKONCHIECTflSIS 1 (2X) 

*LOS^ (20) (49) (50) 

EriONCHOPNEOMONIA, NOS 1 (2%) 

INFLAMBATICN , NOS 1 (5X) 1 (2%) 1 (2X) 

HYPERPLASIA , ALVEOLAR EPITHELIU M 1 (2X) 

HEMATOPOIETIC SYSTEM 

ISPLiiEN (23) (49) (49) 

CONGESTION, NOS 1 (2X) 

FIBROSIS 1 (2%) 

MiTAMORPHCSI S FATTY 3 (6») 

LYMPHOID EEPLETION 1 (2X) 

HYPERPLASIA, FOCAL 1 (2X) 

HYPERPLASIA , BESOTHELIAL 1 (2X) 

HYPERPLASIA , BETICULUfl CELL 3 (6X) 

*SPLtNIC CAPSULE (20) (49) (49) 

* NUMDER OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMfaER OF AMMAL S NECROPSIED 

65 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

INFLAMHATICN, FOCAL
 

INFLAMMATION, CHRONIC
 

SIDEEOSIS
 

•SPLENIC BEE EULP
 

HYPERPLASIA, NOS
 

•MANDIBULAR	 L. NODE
 

fl£MOBBHAGE
 

SIDEROSIS
 

CIBCULATORY SYSTEM
 

IHEAKT
 

THROMBOSIS, NOS
 

FIBROSIS
 

FIBROSIS, IIFFUSE
 

DEGENERATION, NOS
 

tMYOCABDIUM
 

INFLAMMATION, FOCAL
 

FIBROSIS
 

DEGENERATION, NOS
 

t ENDOCARDIUM
 

FIBROELASTCSIS, NOS
 

*PANCREATIC ABTERY,
 

INFLAMMATION, CHRONIC
 

3IGESTIVE SYSTIM
 

tPABGTID GLANI
 

INFLAMMATION, NOS
 

INFLAMMATION, NECROTIZING
 

INFLAMMATION, ACUTE
 

INFLAMMATION, CHRONIC
 

ISUBHAXILLABY GLAND
 

INFLAMMATION, NOS
 

INFLAMMATION, SECROTIZING
 

INFLAMMATION, ACUTE
 

INFLAMMATION, ACUTE/CHEONIC
 

t NUflbEE OF ANIMAL S WITH TISSUE

* NUMBER OF ANIMAL S NECROPSIED 

MATCHED
 

CONTROL LOW DOSE
 

11	 (22*)
 

(20)	 (49)
 

4 (8*)
 

(20)	 (49)
 

(20) (49) 

1 (2*) 

9 (458) 14 (29*) 

1 (2X) 

1 (2X) 

(20) (49) 

1 (5*) 1 (2X) 

2 (10*) 

(20) (49) 

1 (2X) 

(20) (49) 

1 (5X) 

(20)	 (49)
 

2 (4X)
 

4	 (20 X) 1 (2X)
 

1 (2*)
 

1 (2X)
 

(20)	 (49)
 

4 (8*)
 

3 (15*) 2 (4X)
 

... , .. ,
 2 J4£)
 

 EXAMINED BICBOSCOPICALLY 

HIGH DOSE
 

1 (2*)
 

10 (20X)
 

1 (2X)
 

(49)
 

1 (2%)
 

(50)
 

1 (2*)
 

1 (2X)
 

(50)
 

2	 (4X)
 

3	 (6X)
 

(SC)
 

1 (2*)
 

2 (4X)
 

2 (4X)
 

(50)
 

(50)
 

(50)
 

1 (2*)
 

1 (2*)
 

(5C)
 

1 (2X)
 . _ J (2*1
 

66 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

-1 -' ' "~ "~~lr"- — ————» — — —— ——.—._
 

MATCHED 

CONTROL LOW DOSE 

IMFLAHHATICN, CHEONIC 

A1ROPHY, NCS 

BiGENERAllCN, NOS 

ILIViR 

INFLAMMATION, NECROTIZING 

GRANULOMA, NOS 

FIBROSIS 

CHOLANGICf IBEOSIS 

CIRRHOSIS, NOS 

NECROSIS, KOS 

MiTABORPHCSIS FATTY 

FOCAL CELLULAR CHANGE 

(20)

11 (55)1)

 («9) 

11 (22X) 

 35 (71X) 

1 (2X) 

1 (2») 

3 (6X) 

ILIVIR/CENTRIIOEULAR 

CYTOPL&SHIC VACUOLIZATICH 

HLGALOCY1CSIS 

(20) (49) 

#BIL£ DUCT 

HYPERPLASIA, NOS 

HYPERPLASIA, FOCAL 

(20) (49) 

1 (2*) 

1 (2X) 

•PANCREAS 

PEBIARTEBITIS 

AT80PHY, NCS 

(20) (47) 

4 (9X) 

4 (9X) 

tSTOMACH 

INFLAMHATICN, CHRONIC 

(20) (49) 

1 [2%) 

URINAfcY SYSTIf
 

IKIDiJEY (20) (49)
 

NEPHROPATHY 15 (75*) 34 (69X)
 

INFARCT, NCS
 

PIGHENTA1ICN, NOS
 

HKIDNEY/CORTIX (20) (49)
 

CYST, NOS
 

#KIDNEY/TUEUII (20) (49) 

PIGHENTATICN , NOS 

fURINAR Y ELilEER (20) (47) 

TE 

f NUBtiER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

" —— — —•—
 

HIGH DOSE
 

1 12%)
 

1 (2%)
 

1 (2X)
 

(SC)
 

1 (2%)
 

3 (6X)
 

1 (2%)
 

13 (2655)
 

1 (2X)
 

3 (6X)
 

2 (4S)
 

4 (8X)
 

(5C)
 

1 (2X)
 

1 (2X)
 

(5C)
 

3 (6«)
 

(4£)
 

2 (4K)
 

2 (4X)
 

(5C)
 

2 (4X)
 

(50)
 

14	 (28%)
 

1 (2X)
 

1 (2X)
 

<5C)
 

1 (2X)
 

(50) 

(4X) 

(47) 

67 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

iNDOCalNE SYSTI M 

•PITUITARY (20) (49) 

HiMORHHAGIC CYST 1 (2%) 

HYPERPLASIA, NOS 1 (5X) 3 (6X) 

HYPERPIASIA, FOCAL 1 (5X) 3 (6X) 

4ADRENAL (20) (49) 

HETAMOBPHOSIS FATTY 1 (2X) 

#ADRENAL COR1IX (20) (49)

 

NECROSIS, NOS 

#ADRiNAL MEDUIL A (20) (49) 

HYPERPLASIA , NOS 7 (14«) 

tTHYHOID (20) (49) 

COLLOID CYST 1 (2X) 

HYPERPLASIA , NOS 

HYPERPLASIA, C-CELL 1 (5X) 2 (4») 

HYPERPLASIA, FOLLICULAR-CELL 1 (2X) 

REPRODUCTIVE SYSTEH 

*PREPOTIAL GIAN D (20) (49) 

HYPERPLASIA , CYSTIC 1 (2X) 

tPBOiTATE (20) (47) 

INFLAMMATION , ACUTE 8 (17X) 

*IESIJS (20) (48) 

GRANULOMA , SPERMATIC 1 (2%) 

Pt-RIABTEBIlIS 12 (25X) 

ATROPHY, NCS 1 (5X> 6 (13X) 

HYPERPLASIA , INTERSTITIAL CELL 3 (15X) 2 (4X) 

NERVOU S SYSTJB 

•CERriBBUM (20) (49) 

GLIOSIS 1 (2!!) 

SPECIAL SENSE ORGAN S 

NONE 

« NUHBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE 

(49) 

4 (8X) 

(50) 

1 (2%) 

(5J) 

1 (2X) 

(50) 

1 (2%) 

(48) 

1 <2X) 

(50) 

(47 ) 

l«9) 

1 (2X) 

2 (4X) 

(50) 

68
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

MUSCULOSKELETAL SYSTEM 

NON E 

£ODY CAVITIES
 

*MEDAASTINUM (20) (49) (50)
 

REMORRHAGIC CYST 1 (2X)
 

*RESENTERY (20) (U9) (50)
 

INFARCT, NCS 1 (235)
 

ALL OTHER SYSIIHS
 

ADIPOSE TISSUE
 

INFLAMMA3ICN, CHBONIC 1
 

SPECIAL BORPHCLOGY SUHHARY
 

ANIBAL MISSING/NO NECROPSY 1
 

i NUHbER OF ANIHALS WITH TISSUE EXAHINED BICBOSCOPICALIY
 

* NUMBER OF ANIMALS NECROPSIED
 

69
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED AZOBENZENE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITJAILY IN STOEY 20 50 SO 

ANIMALS NECBCESIED 20 50 50 

ANIMALS EXAMINE D HISTOPATHOLOGIC ALLY 20 50 50 

INTEGJBEHTAEY SYSTEM 

(20) (50) (50) 

EPIDEBBAL INCLUSION CYST 1 (2X) 

BESPIriATOHY SYSTEM 

SLUNu! (20) (50) (50) 

BRONCHOPKIUHONIA, NOS « <8X) 

INFLAMBA1ICN , NOS 3 (6X) 

INFLAMMATION , FOCAL 1 (2%) 

PNEUMONIA , LIPID 1 (2X) 

IHFLAMBA1ICN , CHBCNIC 1 (5X) 

GfiANULCBA , NOS 1 12%) 

HEHATOPOIETIC SYSTEM 

JtSPLjiEN (20) (50) (50) 

HEMOREHAG I 1 (2X) 

HEBATOMA, SOS 1 (2X) 

H^HATOMA, CHGANIZED 1 (2*) 

PIBBOSIS 1 (2X) 2 («X) 

PjiHIAHTEEITIS 1 (2X) 

DtGENERATICN , NOS 2 (4X) 

MaTAHORPHCSIS FATTY 5 (10») 5 (10X) 

PJ.GHENTA1ICN, NOS 4 (8%) 9 (18*) 

H£MOSIDEECSIS 2 (10X) 3 (6«) 

HfiBATOEOIESIS 1 (2X) 

ISPLtNIC CAPSDLE (20) (50) (SO) 

INPLAMBATICN, NOS 1 I2X) 2 [H%) 

INFLAMMATICN , CR80NIC 29 (58S) 31 (62X) 

iSPLiiNIC BED POLP (20) (50) (50)
 

FIBROSIS 

t NDHBEB OF ANIHALS HITH TISSUE EXABINED MICSOSCOPICA1LY
 

* NUBBER OF AKIBALS SECBOPSIED
 

70
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

PIGMENTATION, NOS 1 (2X) 1 (2X) 

HYPERPLASIA, NOS 2 (UX) 

ILYMPH NODE (20) (50) (5C) 

HEMORRHAGE 1 (2X) 

HYPERPLASIA, NOS 1 (2X) 

CIRCnLATORY SYSTEM 

IHEAB1 (20) (50) ' (50)
 

FIBROSIS 6 (30%) 3 (6X) 11 (22X)
 

IMY04-ABDIUM (20) (50) (50)
 

FIBBOSIS 1 (2X)
 

DEGENERATION, NOS 2 (<JX)
 

EIGES1IVE SYSTEM 

IPAROTID GLAN n (20) (50) (50) 

INFLAHMATICN , NOS 1 (2%) 1 (2%) 

INFLAMMATION , NSC ROTIZING 1 (5%) 1 (2%) 2 (4X ) 

INFLAMMATION , ACU TE/CHBONIC 2 (UX) 

INFLAMMATION , CHRONI ONC2 (4») 

tSUBflAXILLARY GLAND (20) (50) (SO) 

INFLAMMATION , NOS 7 (1UX ) 

INFLAMMATION , NEC ROTIZING 1 (5X) 7 (14X) a (8X) 

INFLAMMATION , ACUTTE 1 (5X) 1 (2X ) 

INFLAMMATION, ACUTEE/CHRCNI , C 2 (10X) 1 (2%) 1 (2X)

INFLAMMATION , CHRONI ONC2 (<*X) 3 (6X)

ILIVER (20) (50) «5C ) 

GRANULOMA , NOS 16 (32%) 15 (30%) 

INFLAMMATION , AL GRANULOMATO U 1 (2X ) 

CnOLANGICFIBROSIS 3 (15X) 17 (34%) 2 (4X) 

CIRRHOSIS, NOS 1 (5X) 

HEPATITIS, TOXIC 1 (2%) 

NiCHOSIS, KOS 1 (2%) 2 (4% ) 

NtCROSIS, IOCAL 1 (2X) 

NiCHOSIS, COAGULATIV TIVEE 1 (5X ) 

MiTABORPHCSIS FATTTY 1 (5X ) 2 (4») 1 (2%) 

PIGMENTATION , NOS 11 (22%) 

FOCAL_CEIIULAB CHANG ANGE 3 J15Jfl 7 J14XJ. 3_J5!i_

t NUHdE R OF AMMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF AMMAL S NEC80PSI1D 

71 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

#LIVt,R/CENTRIIOBULAR (20) (50)

 

NECROSIS, NOS 

*IIVi8/HEPATCCYTIS (20) (50)

 

H^GALOCYTCSIS 1 (5X)

 

1PANLREAS (20) (50) 

ATROPHY, NCS 1 (2!t) 

ISTOMACH (20) (50) 

U1CER, NCS 1 (5») 

ISPLAHHA1ICN, CHRONIC 1 (2%) 

URINAR Y SYSTEM 

IKIDNE Y (20) (50) 

PYELONEPHRITIS , NOS 

NEPHROPATH Y 2 (1055) 4 (6%) 

PIGHENTAIICN , NOS 27 (54%) 

#KIDNEY/TUBU1 I (20) (50)

 

PIGMENTATION , NOS 1 (5X)

 

*URINAR Y ELJEIE R (20) (50)

HYPERPLASIA, EPITHELIAL 1 (2*) 

ENDOCRIN E SYSTIM 

#PITUITABY (20) (49) 

CYST, NOS 1 (5X) 1 [2%} 

HjiMORRHAGIC CYST 1 (5X) 

HYPERPLASIA, NOS 2 (4X) 

HYPERPLASIA , FOCAL 2 (10!!) 2 (4X) 

IADRENAL 120) (50)

 

HuMORRHAG I 

NiCBOSIS, NOS 1 (5*)

 

HYPERPLASIA , FOCAL 

#ADR£NA L COBTI X (20) (50) 

HiiTAMORPBCSIS FATTY 

..HYPERPLASIA, NOS .. I i§*) 

t NUMBER OF ANIMALS WITH TISSUE EXAHIHED BICBOSCOPICALLY
 * NUMBER OF ANIMALS NECBOPSIED
 

HIGH DOSE 

(50) 

1 (2X ) 

(50) 

(50) 

1 (2X ) 

(50) 

(50) 

1 (2X) 

3 (655) 

2 (48) 

(50) 

5 (10X) 

(50) 

(50) 

1 (2X) 

S (1035) 

(50) 

1 (2%) 

1 (2%) 

(SC) 

1 (2X) 

72
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

TM ~TM ~~ "~ — '-"- "- — — — ~—~"~ -i-— _ — ———.—— 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

lADKJiNAL BEDUIL A (20) (50) (50) 

HYPERPLASIA , NOS 1 (2X) 6 (12XJ 

1THYROID [20) (50) l«8)

 

COLLOID CYST 1 (5«) 1 (2X) 

HYPERPLASIA, C-CELL 2 (105!) 4 (8X)

 

REPRODUCTIVE SYSTE M 

*HAHMAR Y GLAN I (20) (50) (50)

 

CYST, NOS 1 (5X) 

INFLAMMA1ICN , CHEONIC 1 (2X)

 

HYPERPLASIA , CYSTIC 1 (5X)

 

*PREP(JTIAL GLAN D (20) (50) (50) 

HYPERPLASIA, CYSTIC 1 (2X) 

«UTERU S (20) (50) (50) FIBROSIS 1 (5X) 1 (2X) i {«) 

*UTERUS/ENDCMETRIUB (20) (50) (50)

 

INFLAMHATICN , VESICULAR 2 (US)

 

IOVARY (20) (50) (50)

 

CYST, SOS 1 {5X)

 

«OVAiY/BEDULIA (20) (5J) (50) 

HYPERPLASIA, NOS 1 (5X) 1 (2X) 7 (UX) 

NERVOU S SYSTIB 

fCBBfiBELLUB (20) (50) (50)

 

HiBORRHAGE 1 (2*)

 

SPECIAL SENSE ORGANS 

*£YE/LACRIflAI GLAN D (20) (50) (50) 

INFLAMMATION , NOS 1 (2X) 

HOSCUiOSKELETAI SYSTEH 

NONfi 

» SOHBEE OF ANIHALS WITH TISSUE EXAMINED HICBOSCOPICALLY
 

* SUHBER OF ANIBALS NBCHOPSIED
 

73
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC

EOEY CAVITIIS 

*ABDOBINA1 CAVIT Y

STEATITIS

NiCBOSIS, FAT

*MESiNTEBY

PfcBIABTEBIlIS

A LL OTHER SYSTEMS 

NONE 

SPECIAL MOHEfcCIOGY SOHBAPY
 

NO LESIOK fEfOBTED


AUTO/NSCBCPSY/HISTO P£BF


 LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 {20) (50) (50) 

1 (2%) 

1 (2X) 

 (20) (50) (50) 

1 (2X) 

3
 

1
 

t NOHJEB OF AKIMALS WITH TISSUE EXAMINED BICBOSCOPICALLY
 

* NUMBEB OF AMMALS NECBOPSIED
 

74
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN
 

MICE ADMINISTERED AZOBENZENE IN THE DIET
 

75
 
76

 
TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED AZOBENZENE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 20 50 50 

ANIMAL S HISSING 1 2 

ANIflALS NECBOPSIED 20 49 18 

ANIMALS EXAHINE D HISTOPATHOLOGICALS 20 49 48 

INTEGUMENTAR Y SYSTEM 

*SKIil (20) (49) (48)
 

HYPERPIASIi. CYSTIC 1 (5X)
 

RESPIRATORY SYSTEM
 

ILUNG/BRONCHICLE (20) (49) (47)
 

HYPERPLASIA, LYHPHOID 3 (6«)
 

•LUNG	 (20) (49) |47)
 

INFLAMMATION, FOCAL 1 (5X)
 

HYPERPLASIA, ALVEOLAR EPITHZLIUM 1 (2«)
 

tEMATOPOIETIC SYSTEM
 

ISPLiEN (20) (49) (47) 

ATROPHY, NCS 2 (4X) 

HYPERPLASIA , LYMPHOID 2 (10*) 4 (8*) 1 (235) 

HLMATOPOIfSIS 1 (5%) 1 (2*) 

ILYMPH NOPE (20) (48) (47) 

HYPERPLASIA , LYMPHOI D 15 (328) 

ISUBflANDIBUIA R L.SODE (20) (48) (47)
 

HYPERPLASIA , IYMPHOID 1 (5«)
 

tCEKVICAL LYMP H NODE (20) (47)
 

MtTAHOHPfcCSIS FATTY 1 (5%)
 

HYPERPLASIA , LYMPHOI D 1
 

•PANCREATIC L.NOEE (20) (47)
 

HYPERPLASIA. LYHPHOID 

* NOHBER OF ANIMALS WITH TISSUE EXAMINED MICEOSCOPICALLY
 

* NUMriER OF ANIMALS NECROPSIED
 

77
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

~~ ——— — — ~°—•----­—J ——""• —­ „_ „. i , i . . ­- --r. "~ '~ "— ­ *~ "" — —• •—• 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

((MESENTERIC I. NODE 

ATROPHY, NCS 

HYPERPLASIA , LYMPHOID 

HiMATOEOIESIS 

(20) 

2

1

1

 (1JX) 

 (5X) 

 (5%) 

(48) 

3

3

 (6X) 

 (6%) 

((47) 

CIRCULATORY SYSTEM 

#BYOCARDIU M 

FIBROSIS 

FIBROSIS, JCCAL 

(20 ) (48) 

1

1

 (2X) 

 (2X) 

(46) 

DIGESTIVE SYS1IM 

4SALIVAR Y GLAN D 

INFLAMMATICN, NOS 

(19) (48) 

1 I2X) 

(47) 

*SUBM AXIL LAR K GLAN D 

INFLAMMATION , NOS 

(19) 

6 (32%) 

(48) 

16 (33%) 

(47) 

#LIV£R 

TUROMBOSIS, NOS 

HflMOBRHAGIC CYST 

INFLAMMATIC N ACUTE PUSTULA R 

INFLAMMATION , CHRONIC FOCAL 

INFARCT , NCS 

BASOPHILIC CY10 CHANGE 

FOCAL CELLULA R CHANGE 

MtGALOCYTCSIS 

HYPERPLAS1IC NODULE 

HYPERPLASIA , LYMPHOID 

(20) 

1 (5*) 

1 (5X) 

1 (5%) 

1 (5X) 

1 (5X) 

1 (5%) 

(49) 

1 (2X) 

1 (2X) 

5 (10%) 

2 (4S) 

{48) 

1 (2X) 

1 (2X) 

1 (2X) 

*LIVtR/CENTRIIOBULAR 

BILE STASIS 

CYTOPLASMIC VACUOLIZATION 

(20) (49) (4€) 

1 (2X) 

1 (2X) 

ILIV£R/KUEFFEE CELL 

HYPERPLASIA , NOS 

(20) 

1 (5X) 

(49) (48) 

ISTOBACH 

U^CER, NOS 

ABSCESS, NCS 

(20) (49) (HI) 

1 (2X) 

(2X) 

»PEYi,RS PATCB 

HYPERPLASIAJ_LYHPHQIE 

(20) (49) (47) 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ARIMALS NECBOPSIiD 

78 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

OBINAHY SYSTIM
 

IKIDNE Y (20) (49) (46) 

PYELONEPHRITIS, HOS 1 (2*) 

INFLAMMATION, CHRONIC 1 (2«) 

G LO HER ULCJiEP H BITIS, CHRONIC 1 (5!«) 

HYPERPLASIA , LYMPHOID 6 (30%) 17 (35%) 

#UHINAR Y ELiEEER (20) (49) (45) 

HYPERPLASIA , LYMPHOID 4 (88) 

EMDOCHINE SYSTEM 

IPITUITARY (17) (45) (38) 

CYST, NOS 1 (2%) 

REPRODUCTIVE SYSTEM 

IPROSTATfi (20) (48) (46) 

INFLAMMATION , CHRONIC 1 (5«) 1 (2%) 

*SCROTOM (20) (49) J48) 

NbCROSIS, IAT 1 (5X) 

NERVOUS SYSTie 

4CEREBRUM (20) (49) (46) 

CALCIFICATION, NOS 12 (60X) 17 (35%) 

SPECIAL SENSE CRGAN S 

HARDERIA N GLAN D (20) (49) (48) 

IHFLAMMATICN, NECROTIZING 1 (2X) 

ISFLAHMATICN, CHRONIC 1 (2S) 

JSUSCULOSKELE1A1 SYSTEfl 

*MUSCLE OF BACK (20) (49) (48) 

INFLAMMATION. CHBONIC
 1_J5JLL 

t NUMBER OF ANIMAL S HITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ASIMALS NECROPSIED 

79 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

EODY CAVITIES 

*ABDOHINAL CAVIT Y [20) (49) (148) 

NhCBOSIS, FAT 1 (5SS) 

INFARCT, HCS 1 (2%) 

*HES*NTERY (20) (49) (4£) 

NECROSIS, FAT 1 (5«) 

AIL OIHER SYSTIMS 

NON E 

SPECIAL HOREbCIOGY SDBHAEI
 

NO LESION BEPORTED 1 2 1C 

AMIMA L HISSING/NC NECROISY 1 2 

AUTO/NECBCJESY/HISTO PERF 1 

* NOMbEB OF AMHALS WITH TISSOE EXAMINED HICROSCCPICALLY 

* NUMBER OF AKIMAL S NECROPSIED 

80 
TABLE 02.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED AZOBENZENE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

MIBt-LS IIITIA1LI II STUDY 23 5J 50 

IBUALS HISSIBG 1 7 

tBIBALS BICBCrSIEC 20 47 38 

AHH1LS ZXABIIIA HISTOPATHOlOGICAilY 20 U7 38 

H7EGUBMT1BT SISIIfl 

MClit 

BESPI4ATOBI SYSTEH 

•IOB6/BBOBCKICLE (20) (46} (36) 

HYPIBPLAS1I. LTHPBOID 10 (22S) 

«LDHG (20) (46) (36) 

HEHORKHiG! 1 (2%) 

IMFLASB4TICH, HOS 1 (3X) 

HYPERPLASIA, FOCAL 1 (5%) 

HYPERPLASIA, LTHPHOID 6 (30X) 2 (4X) 

HEMATOPOIETIC SISTER 

IBOHfi HABflOB (20) (46) (37) 

SIDBROSIS 1 (3%) 

tSPLiEN (20) (47) (36) 

HiHOSIDERCSIS 6 (161) 

HYPEBPLASIA, LYMPHOID 7 (35S) 16 (34«) 4 (11X) 

HiHATOFOIISIS 7 (35*) 8 (178) 

•LYMPH NODE (20) (47) (35)
 

HYPERPLASIA, LYMPHOID 1 15*) 8 (23*)
 

*SUBi1ANDIBUIAR L.MODE (20) (47) (35) 

HYPERPLASIA, IYBPHOID 1 (2*) 

*CEBVICAL LYEP H NODE (20) (47) (35) 

f NUMBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NOMBEH OF ANIMALS NECBCPSIED
 

81
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

— - ~~ — 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

HYPERPLASIA , LYHPHOI D (J (20%) 

tMESENTERIC L. NODE [20) C*7) (35)

 

ATROPHY, NCS 1 (5X) 2 (H%)

 

HYPERPLASIA , NOS 1 (2X)

 

LYMPHOCYTOSIS 1 (2X) 

HYPERPLASIA, LYHPHOID 1 (5X) 2 («X) 

ITHYilUS (16) (41) (26)

 

A1ROPHY, tiCS 1 (6X)

 

CIRCULATOR Y SYSTEM 

NONE 

riGESIIVE SYSTIM 

• SALIVARY G1AK D (19) (45) (35)

 

INFLAMMATION, NOS 2 (11X) 1 (2X)

 

*SUBHAXILLAR Y GLAN D (19) (45) (35)

 

INFLAMMATION, NOS 7 (3755) 14 (31X)

 

*LIVEB (20) (47) (36) 

INFLAMMATION , NECROTIZING 1 (2X) 1 (3XJ 

NtCBOSIS, NOS 1 (3X) 

NiCBOSIS, fOCAI 1 (5X) 1 (2X) 

METAMORPHOSIS FATTY 4 (11X) 

FOCAL CEIIULAB CHANGI 1 (5X) 2 (4X) 

MtGALOCYTCSIS 1 (3X) 

HYPERPLASIA, LYMPHOID 1 (5X) 9 (19X) 

#LIVtB/CAODJT£ LOEE (20) (47) (36) 

INFARCT, NCS 1 (3X) 

IPANCBEAS (20) (47) . (3«)

 

INFLAHBATICN , NOS 1 (5X) 1 (2X)

 

INFLAHMAIICN , ACUTE 1 (2X)

 

1 STOMACH (20) (47) (37)

H&HOBRHAGf 1 (3X) 

DBINARY SYSTIM 

*KIDHEY (20) (<*7) (38) ___

 „«, INFLAMMATION. CHRONIC

 1 rsxi 

t NUMBEB OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUHBEB OF ANIMALS NECROPSIED
 

82
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

HYPERPLASIA. LYMPHOID 

IKIDNEY/CORTIX 

CYST, NOS 

IKIDHEY/TUBOII 

PXGHENTATICN, NOS 

fURIBARY BLAEEBH 

INFIAHHATICN, CHRONIC 

HYPERPLASIA, LYMPHOID 

10-Bi. ADDER/SO EMUCOS A 

HYPERPLASIA, LYMPHOID 

ENDOCRINE SYS1IM 

•PITUITARY 

HYPERPLASIA, FOCAL 

lADBJiNAi CORTIX 

DEGENERATION, NOS 

•ADRSNAi HIDDILA 

HYPERPLASIA, NOS 

REPRODUCTIVE SYSTEfl 

1 UTERUS 

HYCROHETBA 

H£HORRHAG£ 

HEHOHRHAGIC CYST 

HYPERPLASIA, LIHPHOID 

• UTEKUS/ENDCKITRIU M 

INFLAMMATION , NOS 

UJFLAHBA1ICN, VKSICOLAR 

WVASY/PAROVARIAN 

IHFAHCZ, NCS 

•OVARY 

CYST, NQS 

I NOHBEB OF ANIMAL S HITH TISSUE

* BO«bEF OF ANIMAL S NECROPSIED 

MATCHED 

CONTROL LOW DOSE 

10 (SOX) 13 (28X) 

(20) (47) 

(20) (47) 

(20) 

5 (25%) 

(47) 

1

10

 (2X) 

 (21X) 

(20) 

1 (5X) 

(47) 

(19) (42) 

1 (2X) 

(20) (47) 

1 (2*) 

120) (47) 

(20) (47) 

1 (2X) 

1 (2X) 

1 (2X) 

(20) (47) 

8 (40X) 27 (57X) 

(20) (47) 

2 (10X) 

(19) (46) 

1 J5XJ S-J11SI 

 EXAMINED MICROSCOPICALLY 

HIGH DOSE 

(38) 

1 (3X) 

(38) 

1 (3X) 

(36) 

(36) 

(30) 

(37) 

(37) 

2 (5X) 

(37) 

2 (5S) 

1 (3X) 

(37) 

11 (303S) 

4 (11X) 

(37) 

(37) 

2 (5»1_ 

83 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

THROMBOSIS, NOS 2 (4%) 1 (3») 

HJEMORRHAGIC CYST 1 (5%) 1 (3X) 

NERVOUS SYSTEM 

*CEBj,BRUH (20) (47) (36) 

CALCIFICATION, NOS 13 (65%) 12 (26%) 

SPECIAL SENSE CRGAN S 

NONE 

KDSCUiOSKELETAL STSTEH 

NONE 

EOEY CAVITIES
 

*ABDOMINAL CAVITY (20) (47) (38)
 

NiCROSIS, IA1 1 (2%)
 

INFARCT, KCS 1 (3«)
 

HYPERPLASIA, LYHPHOID 1 (2%)
 

*MESENTERY (20) (47) (36)
 

NECROSIS, JAT 1 (2%)
 

ALL OTHER SYSTEMS 

OHENTDH 

NiCBOSIS, FAT 1 

SPECIAL MORPHCIOGY SUMMAR Y 

NO LESION BEFOBTED 2 

ANIMAL MISSING/NO NECROPSY 1 7 

AUTOLYSIS/NO NECROPSY 2 5 

I NUflfiEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

84 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS ADMINISTERED AZOBENZENE IN THE DIET
 

85
 
86

 
Table El. Analyses of the Incidence of Primary Tumors

Administered Azobenzene in the Diet (a) 

 in Male Rats 

Topography: Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Hematopoietic System: 

Lymphoma (b) 1/20 (5) 0/49 (0) 3/50 (6) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.000 

0.000 

7.624 

1.200 

0.106 

61.724 

Weeks to First Observed Tumor 105 — 65 

CO 

-J 

Hematopoietic System: 

Monocytic Leukemia (b) 2/20 (10) 1/49 (2) 5/50 (10) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.204 

0.004 

3.754 

1.000 

0.184 

10.007 

Weeks to First Observed Tumor 80 94 78 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Azobenzene in the Diet (a)
 

(continued)
 

Topography: Morphology


Hematopoietic System:
 

Lymphoma or Leukemia (b)


P Values (c,d)


Departure From Linear Trend (e)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

oo
 

oo Weeks to First Observed Tumor


Liver: Hepatocellular Carcinoma
 

or Neoplastic Nodule (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

 Control
 

 3/20 (15)
 

 N.S.
 

P = 0.022
 

 80
 

 0/20 (0)
 

 N.S.
 

Low
 

Dose
 

1/49 (2)
 

N.S.
 

0.136
 

0.003
 

1.599
 

94
 

1/49 (2)
 

N.S.
 

Infinite
 

0.023
 

Infinite
 

106
 

High
 

Dose
 

8/50 (16)
 

N.S.
 

1.067
 

0.295
 

5.813
 

65
 

3/50 (6)
 

N.S.
 

Infinite
 

0.250
 

Infinite
 

91
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

(continued)
 

Topography; Morphology
 

Pituitary: Adenoma, NOS (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

co
 

VO Pituitary: Chromophobe
 

Adenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Administered Azobenzene in the Diet (a)
 

Matched Low 

Control Dose 

4/20 (20) 2/49(4) 

P = 0.002 (N) N.S. 

0.204 

0.020 

1.323 

105 106 

0/20 (0)	 2/49 (4)
 

N.S.	 N.S.
 

Infinite
 

0.125
 

Infinite
 

86
 

High
 

Dose
 

0/49 (0)
 

P - 0.006 (N)
 

0.000
 

0.000
 

0.435
 

—
 

3/49 (6)
 

N.S.
 

Infinite
 

0.255
 

Infinite
 

90
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats 

Administered Azobenzene in the Diet (a) 

(continued) 

Matched Low High 

Topography! Morphology Control Dose Dose 

Testis: Interstitial-cell 

Tumor (b) 17/20 (85) 41/48 (85) 31/49 (63) 

P Values (c,d) P = 0.012 (N) N.S. N.S. 

Relative Risk (f) 1.005 0.744 

Lower Limit 0.836 0.607 

Upper Limit 1.335 1.083 

weeks to First Observed Tumor 76 82 82 

Spleen: Fibrosarcoma (b) 0/20 (0) 2/49 (4) 7/49 (14) 

P Values (c,d) P = 0.020 N.S. N.S. 

Relative Risk (f) Infinite Infinite 

Lower Limit 0.125 0.826 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor 106 86 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Azobenzene in the Diet (a)
 

(continued)
 

Matched Low
 

Topography : Morphology Control Dose
 

All Sites: Hemangiosarcoma (b) 0/20 (0) 1/49 (2)
 

P Values (c,d) N.S. N.S.
 

Relative Risk (f) Infinite
 

Lower Limit 0.023
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 106
 —
 

VO
 

Multiple Organs: Fibrosarcoma (b) 0/20 (0) 0/49 (0)
 

P Values (c,d) P less
 " —
 than 0.001
 

Departure From Linear Trend (e) P = 0.030
 

Relative Risk (f)
 

Lower Limit
 —
 

Upper Limit
 —
 

—
 

Weeks to First Observed Tumor
 

High
 

Dose
 

4/50 (8)
 

N.S.
 

Infinite
 

0.386
 

Infinite
 

82
 

13/50 (26)
 

P = 0.007
 

Infinite
 

1.674
 

Infinite
 

76
 
__ 

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

Administered Azobenzene in the Diet (a)
 

(continued)
 

Matched Low
 

Topography; Morphology Control Dose
 

Multiple Organs: Osteosarcona (b) 0/20 (0) 0/49 (0)
 

P Values (c,d) N.S.
 

Relative Risk (f)
 

Lower Limit
 —
 Upper Limit
 —
 

Weeks to First Observed Tumor
 —
 

VO 

NJ
 Abdominal Cavity:
 

Sarcoma (b,g) 0/20 (0) 6/49 (12)
 

P Values (c,d) P less than 0.001 N.S.
 

Departure from Linear Trend (e) P « 0.027
 

Relative Risk (f) Infinite
 

Lower Limit 0.680
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 106
 

High
 

Dose
 

3/50 (6)
 

N.S.
 

Infinite
 

0.250
 

Infinite
 

97
 

31/49 (63)
 

P less than
 

0.001
 

Infinite
 

4.415
 

Infinite
 

76
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Azobenzene in the Diet (a)
 

(continued)
 

(a)	 Dosed groups received 200 or 400 ppm.
 

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

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P is less than 0.05; otherwise, not significant (N.S.) is indicated.
 

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

test for the comparison of that dosed group with the matched-control group when P is less than
 

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

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

(e) The probability level for departure from linear trend is given when P is less than 0.05 for any
 VO 

w comparison. 

(f) The 95 percent confidence interval of the relative risk between each dosed group and the control 

group. 

(g) Malignant neoplasm, NOS; sarcoma, NOS; fibrosarcoma; hemangiosarcoma; osteosarcoma; or 

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

Topography; Morphology


Hematopoietic System:
 

Monocytic Leukemia (b)


P Values, (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Hematopoietic System :
 

Lymphoma or Leukemia (b)


P Values, (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Administered Azobenzene in the Diet (a) 

Hatched Low High 

 Control Dose Dose 

 1/20 (5) 3/50 (6) 0/50 (0) 

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

1.200 0.000 

0.106 0.000 

61.724 7.475 

 106 89 — 

 1/20 (5) 6/50 (12) 1/50 (2) 

 M.S. N.S. N.S. 

2.400 0.400 

0.325 0.005 

108.021 30.802 

106 89 106 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

(continued)
 

Topography : Morphology


Pituitary: Adenoma, NOS (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


VO Thyroid: C-cell
 

Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Administered Azobenzene in the Diet (a)
 

Matched Low 

 Control Dose 

 4/20 (20) 7/49 (14) 

 N.S. N.S. 

0.714 

0.211 

3.052 

 106 106 

 1/20 (5) 3/50 (6)
 

 N.S. N.S.
 

1.200
 

0.106
 

61.724
 

106 104
 

High
 

Dose
 

7/50 (14)
 

N.S.
 

0.700
 

0.207
 

2.994
 

105
 

1/48 (2)
 

N.S.
 

0.417
 

0.006
 

32.058
 

100 
Table E2.


(continued)
 

Topography: Morphology


Manaary Gland:
 

Fibroadenoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

 Analyses of the Incidence of Primary Tutors in Female Rats
 

Administered Azobenzene in the Diet (a)
 

Matched Low
 

 Control Dose
 

 3/20 (15) 5/50 (10)
 

 N.S. N.S.
 

0.667
 

0.147
 

4.014
 

Weeks to First Observed Tumor 106 106 

VO 

** Uterus: Endometrial Stromal 

Polyp (b) 2/20 (10) 5/50 (10) 

P Values (c,d) N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

1.000 

0.184 

10.007 

Weeks to First Observed Tumor 106 106 

High
 

Dose
 

3/50 (6)
 

N.S.
 

0.400
 

0.060
 

2.802
 

105
 

1/50 (2)
 

N.S.
 

0.200
 

0.004
 

3.681
 

100
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Azobenzene in the Diet (a)
 

(continued) 

Topography: Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Spleen: Fibrosarcoma (b) 0/20 (0) 1/50 (2) 7/50 (14) 

P Values (c,d) P ­ 0.012 N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

0.022 

Infinite 

Infinite 

0.809 

Infinite 

Weeks to First Observed Tumor 106 91 

MD Spleen: Osteosarcoma (b) 0/20 (0) 1/50 (2) 5/50 (10) 

P Values (c,d) P ­ 0.041 N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

0.022 

Infinite 

Infinite 

0.525 

Infinite 

Weeks to First Observed Tumor 106 106 
__ 

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

Administered Azobenzene in the Diet (a)
 

(continued)
 

Matched Low
 

Topography; Morphology Control Dose
 

Spleen: Hemangiosarcoma (b) 0/20 (0) 1/50 (2)
 

P Values, (c,d) N.S. N.S.
 

Relative Risk (f) Infinite
 

Lower Limit 0.022
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 106
 

oo
 Multiple Organs: Osteosarcoma (b) 0/20 (0) 0/50 (0)
 

P Values, (c,d) N.S.
 —
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit —
 

—
 

Weeks to First Observed Tumor
 

High
 

Dose
 

4/50 (8)
 

N.S.
 

Infinite
 

0.386
 

Infinite
 

93
 

3/50 (6)
 

N.S.
 

Infinite
 

0.250
 

Infinite
 

102
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Azobenzene in the Diet (a)
 

(continued)
 

Topography ; Morphology 

Match

Contr

ed

ol

 Low 

 Dose 

High 

Dose 

Abdominal Cavity: 

Sarcoma (b,g) 0/20 (0) 5/50 (10) 21/50 (42) 

P Values (c,d) P less than 0.001 N.S. P less than 

0.001 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

0.525 

Infinite 

Infinite 

2.840 

Infinite 

Weeks to First Observed Tumor 106 91 

(a) Dosed groups received 200 or 400 ppm.
 

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

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P is less than 0.05; otherwise, not significant (N.S.) is indicated.
 

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

test for the comparison of that dosed group with the matched-control group when P is less than
 

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

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

(e) The probability level for departure from linear trend is given when P is less than 0.05 for any
 

comparison.
 

(f) The 95 percent confidence interval of the relative risk between each dosed group and the control
 

group.
 

(g) Fibrosarcoma, hemangiosarcoma, malignant hemagiopericytoma, osteosarcoma, or sarcoma, NOS.
 
100

 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN MICE ADMINISTERED AZOBENZENE IN THE DIET
 

101
 
102

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

Topography: Morphology
 

Lung: Alveolar/Bronchiolar
 

Carcinoma or Adenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System:
 

o
 

CO Lymphoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Administered Azobenzene in the Diet (a) 

Matched Low High 

Control Dose Dose 

2/20 (10) 4/49 (8) 1/47 (2) 

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

0.816 0.213 

0.131 0.004 

8.603 3.909 

106 92 85 

4/20 (20) 5/49 (10) 8/48 (17) 

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

0.510 0.833 

0.126 0.261 

2.367 3.459 

106 92 105 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Azobenzene in the Diet (a)
 

(continued)
 

Topography: Morphology


All Sites: Hemangiosarcoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Liver: Hepatocellular
 

Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low High 

 Control Dose Dose 

 1/20 (5) 2/49 (4) 5/48 (10) 

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

0.816 2.083 

0.046 0.259 

47.195 96.358 

 106 105 100 

 3/20 (15) 10/49 (20) 2/48 (4) 

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

1.361 0.278 

0.406 0.025 

7.138 2.278 

106 91 105 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Azobenzene in the Diet (a)
 

(continued)
 

Topography : Morphology
 

Liver: Hepatocellular
 

Carcinoma or Neoplastic
 

Nodule (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

o
 Weeks to First Observed Tumor
 Ln
 

Adrenal: Pheochromocytoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 

Matched
 

Control
 

8/20 (40)
 

P less than
 

0.001 (N)
 

106
 

0/20 (0)
 

N.S.
 

Low
 

Dose
 

16/49 (33)
 

N.S.
 

0.816
 

0.412
 

1.896
 

91
 

0/48 (0)
 

—
 

—
 

—
 

—
 

High
 

Dose
 

2/48 (4)
 

P = 0.001 (N)
 

0.104
 

0.012
 

0.469
 

105
 

3/45 (7)
 

N.S.
 

Infinite
 

0.278
 

Infinite
 

105 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Azobenzene in the Diet (a)
 

(continued)
 

(a) Dosed groups received 200 or 400 ppm.
 

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

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P is less than 0.05; otherwise, not significant (N.S.) is indicated.
 

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

test for the comparison of that dosed group with the matched-control group when P is less than
 

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

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

(e) The probability level for departure from linear trend is given when P is less than 0.05 for any
 

comparison.
 

(-• (f) The 95 percent confidence interval of the relative risk between each dosed group and the control
 

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

Administered Azobenzene in the Diet (a)
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Azobenzene in the Diet (a)
 

(continued)
 

Matched Low
 

Topography: Morphology Control Dose
 

All Sites: Hemangiosarcoma (b) 0/20 (0) 4/47 (9)
 

P Values (c,d) N.S. N.S.
 

Relative Risk (f) Infinite
 

Lower Limit 0.411
 

Upper Limit Infinite
 

Weeks to First Observed Tumor — 88
 

Pituitary: Adenoma, NOS (b) 2/19 (11) 1/42 (2)
 

P Values (c,d) N.S. N.S.
 o
 

CO
 

Relative Risk (f) 0.226
 

Lower Limit 0.004
 

Upper Limit 4.137
 

Weeks to First Observed Tumor 106 106
 

High
 

Dose
 

1/38 (3)
 

N.S.
 

Infinite
 

0.029
 

Infinite
 

0/30 (0)
 

N.S.
 

0.000
 

0.000
 

2.096
 

105 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Azobenzene in the Diet (a)
 

(continued)
 

Matched Low High
 

Topography ; Morphology Control Dose Dose
 

Harderin Gland: Adenoma,
 

NOS (b) 0/20 (0) 3/47 (6) 1/38 (3)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk (f) Infinite Infinite
 

Lower Limit 0.266 0.029
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 106 105
 

(a) Dosed groups received 208 or 545 ppm.
 

o
\o
 (b) Number of tumor-bearing animals/number of animals examined at site (percent).
 

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P is less than 0.05; otherwise, not significant (N.S.) is indicated.
 

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

test for the comparison of that dosed group with the matched-control group when P is less than
 

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

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

(e) The probability level for departure from linear trend is given when P is less than 0.05 for any
 

comparison.
 

( f) The 95 percent confidence interval of the relative risk between each dosed group and the control
 

group.
 
110

 
Review of the Bioassay of Azobenzene* for Carcinogenicity
 

by the Data EvaluationRisk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

December 13, 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 on the Institute's bioassay program to identify and 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, and State health officials.
 

Members have been selected on the basis of their experience in carcinogenesis or related fields and, collectively, provide expertise in
 

chemistry, biochemistry, biostatistics, toxicology, pathology, and
 

epidemiology. Representatives of various Governmental agencies participate as ad hoc members. The Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse is charged with the responsibility of providing
 

a peer review of reports prepared on NCI-sponsored bioassays of chemicals
 

studied for carcinogenicity. It is in this context that the below critique is given on the bioassay of Azobenzene.
 

The primary reviewer for the report on the bioassay of Azobenzene
 

agreed that the compound was carcinogenic in treated rats. After commenting on the conditions of test, he said that the bioassay was properly designed and conducted. Based on the results of the study, he
 

concluded that Azobenzene may pose a carcinogenic risk to humans.
 

The secondary reviewer said that the study was straightforward
 

and that she agreed with the conclusion in the report. It was moved
 

that the report on the bioassay of Azobenzene be accepted as written.
 

The motion was seconded and approved without objection.
 

Clearinghouse Members Present:
 

Arnold L. Brown (Chairman), University of Wisconsin Medical School
 

Joseph Highland, Environmental Defense Fund
 

William Lijinsky, Frederick Cancer Research Center
 

Henry Pitot, University of Wisconsin Medical Center
 

Verne A. Ray, Pfizer Medical Research Laboratory
 

Verald K. Rowe, Dow Chemical USA
 

Michael Shimkin, University of California at San Diego
 

111
 
Louise Strong, University of Texas Health Sciences Center
 

Kenneth Wilcox, Michigan State Health Department
 

*	 Subsequent to this review, changes may have been made in the
 

bioassay report either as a result of the review or other reasons.
 

Thus, certain comments and criticisms reflected in the review may
 

no longer be appropriate.
 

U.S. GOVERNMEN T PRINTIN G OFFICE : 1 979-281-217/3032
 

112
 

DHEW Publication (NIH) No. 79-1710