1979 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 128 

BIOASSAY OF 

3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 91-93-0 

NCI-CG-TR-128 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE
 

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) 79-1383
 

REPORT ON THE BIOASSAY OF 3,3'-DIMETHOXYBENZIDINE4,4 '-DIISOCYANATE 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 3,3'­

dimethoxybenzidine-4,4'-diisocyanate conducted for the Carcinogenesis
 

Testing Program, Division of Cancer Cause and Prevention, National
 

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

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

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

a significantly greater incidence of cancer than control animals, do
 

not necessarily mean the test chemical is not a carcinogen because
 

the experiments are conducted under a limited set of circumstances.
 

Positive results demonstrate that the test chemical is carcinogenic
 

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

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

carcinogens requires a wider analysis.
 

CONTRIBUTORS: This bioassay of 3,3'-dimethoxybenzidine-4,4'-diisocyanate was conducted by Litton Bionetics, Inc., Bethesda, Maryland,
 

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

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

Weisburger (1,3). The principal investigators for the contract were
 

Dr. F. M. Garner (4,5) and Dr. B. M. Ulland (4,5). Mr. S. Johnson
 

(4) was the coprincipal investigator for the contract. Animal treatment and observation were supervised by Mr. R. Cypher (4), Mr. D. S.
 

Howard (4) and Mr. H. D. Thornett (4); Mr. H. Paulin (4) analyzed
 

dosed feed mixtures. Ms. J. Blalock (4) was responsible for data
 

collection and assembly.
 

Histopathologic examinations were performed by Dr. B. C. Zook
 

(4) at Litton Bionetics, Inc., the pathology narratives were written
 

by Dr. B. C. Zook (4), and the diagnoses included in this report
 

represent the interpretation of this pathologist. Histopathology
 

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

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

statistical analysis was performed by Mr. W. W. Belew (8,9) and
 

Mr. R. M. Helfand (8), using methods selected for the Carcinogenesis
 

Testing Program by Dr. J. J. Gart (10).
 

This report was prepared at METREK, a Division of The MITRE Corporation (8) under the direction of the NCI. Those responsible for
 

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

(8), task leader Ms. P. Walker (8), senior biologist Mr. M. Morse
 

(8), biochemist Mr. S. C. Drill (8), chemist Dr. N. Zimmerman (8),
 

and technical editor Ms. P. A. Miller (8). The final report was
 

reviewad by members of the participating organizations.
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

Cueto, Jr. (1), Dr. J. F. Douglas (1), Dr. D. G. Goodman (1,11), Dr.
 

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

T. W. Orme (1), Dr. R. A. Squire (1,12), Dr. S. F. Stinson (1), Dr.
 

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

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

2.	 No wit w h the U.S. Environmental Protection Agency, 401 M Street
 

S.W., Washington, D.C.
 

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

4.	 Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington, Maryland.
 

5.	 No wit w h Hazleton Laboratories America, Inc., 9200 Leesburg
 

Turnpike, Vienna, Virginia.
 

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

Maryland.
 

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

Rockville, Maryland.
 

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

Boulevard, McLean, Virginia.
 

iv
 
9.	 No wit w h the Solar Energy Research Institute, Cole Boulevard,
 

Golden, Colorado.
 

10.	 Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cancer
 

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

11.	 No wit w h Clement Associates, Inc., 1010 Wisconsin Avenue, N.W.,
 

Washington, D.C.
 

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

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay for the possible carcinogenicity of 3,3"-dimethoxybenzidine-4,4'-diisocyanate was conducted using Fischer 344 rats and
 

B6C3F1 mice. 3,3"-Dimethoxybenzidine-4,4'-diisocyanate was administered, at either of two concentrations, to groups of 50 male and 50
 

female animals of each species, with the exception of 49 high dose
 

female rats. The compound was administered in the feed with the ex­ ception of the first 22 weeks of the rat bioassay, when it was administered by gavage. Twenty animals of each sex ami species were
 

placed on test as controls. During intubation the high and low dosages of 3,3'-dimethoxybenzidine-4,4'-diisocyanate administered to
 

rats were 3000 and 1500 mg/kg, respectively, while the high and low
 

concentrations administered in the feed to both rats and mice were
 

44,000 and 22,000 ppm, respectively. The compound was administered
 

for a period of 78 weeks, followed by an observation period of 26
 

weeks for rats and 25 weeks for mice.
 

There was a significant positive association between the admin­ istration of 3,3'-dimethoxybenzidine--4,4'-diisocyanate and mortality
 

in male and female rats, but not in male or female mice. Adequate
 

numbers of animals in all groups survived sufficiently long to be at
 

risk from late-developing tumors.
 

For both sexes of rats, there was a significant positive association between dosage and the incidence of leukemia and malignant
 

lymphoma. There was a significantly higher incidence of neoplasms
 

of the skin, excluding skin of the ear, when dosed ui'iie rats were
 

compared to controls. There was a significant positive association
 

between the dosages administered and the incidences of endometrial
 

stromal polyps in female rats. None of the statistical tests for
 

any site in male or female mice indicated a signifiesat positive
 

association between compound administration and tumor incidence.
 

Under the conditions of this bioassay, administration of 3,3'­ dimethoxybenzidine-4,4'-diisocyanate was carcinogenic to Fischer 344
 

rats, causing neoplasms of the skin (excluding skin of the ear) in
 

males, endometrial stromal polyps in females, and leukemia and malignant lymphoma in both sexes. The compound was also associated with
 

the development of a combination of squamous-cell carcinomas and
 

sebaceous adenocarcinomas of the Zymbal's gland and skin of the ear
 

in rats of both sexes. There was no evidence for the carcinogenicity
 

of the compound in B6C3F1 mice.
 

vli
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 4
 

A. Chemicals 4
 

B. Dosage Preparation 5
 

C. Dietary Preparation 5
 

D. Animals 6
 

E. Animal Maintenance 7
 

F. Selection of Initial Concentrations 9
 

G. Gastric Intubation 10
 

H. Experimental Design 10
 

I. Clinical and Histopathologic Examinations 14
 

J. Data Recording and Statistical Analyses 1^
 

21
 III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations 21
 2 B. Survival
 1


C. Pathology 24
 

D. Statistical Analyses of Results ^9
 

42
 IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations 42
 

B. Survival 42
 

C. Pathology 46
 

D. Statistical Analyses of Results 47
 

V. DISCUSSION 53
 

VI. BIBLIOGRAPHY 56
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH 3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATE A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH 3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 3,3'-DIMETHOXYBENZ'IDINE-4,4'-DIISOCYANATE C-l
 

ix
 
TABLE OF CONTENTS (Concluded) 

Page 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MICE TREATED WITH 3,3'-DIMETHOXYBENZIDINE-4,4' -DUSOCYAMATE D-l 

x
 
LIST OF ILLUSTRATIONS 

Figure Number Page 

1 CHEMICAL STRUCTURE OF 3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE 2 

GROWTH CURVES FOR 3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATE CHRONIC STUDY RATS 22 

SURVIVAL COMPARISONS OF 3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE CHRONIC STUDY RATS 23 

GROWTH CURVES FOR 3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATE CHRONIC STUDY MICE 43 

SURVIVAL PROBABLITY COMPARISONS OF 3,3'-

DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE CHRONIC 

STUDY MICE 44 

PERCENT SURVIVAL OF 3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATE CHRONIC STUDY MICE 45 

LIST OF TABLES 

Table Number 

DESIGN SUMMARY FOR FISCHER 344 RATS—3,31 -

DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE CHRONIC 

BIOASSAY 11 

DESIGN SUMMARY FOR B6C3F1 MICE—3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE FEEDING EXPERIMENT 12 

TIME-ADJUSTED ANALYSES OF THE INCIDENCE OF 

PRIMARY TUMORS AT SPECIFIC SITES IN MALE 

RATS TREATED WITH 3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATE 30 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH 3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE 36 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE 48 

XI
 
LIST OF TABLES (Concluded) 

Table Number Page 

6 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH 3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE 50 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH 3,3'-DIMETHOXYBENZIDINE-4, 4 '-DIISOCYANATE A-3 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH 3,3'-DIMETHOXYBENZIDINE-4, 4'-DIISOCYANATE A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH 3,3'-DIMETHOXYBENZIDINE-4, 4 '-DIISOCYANATE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH 3,3'-DIMETHOXYBENZIDINE-4, 4 '-DIISOCYANATE B-6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH 3,3'­

DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE 0-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH 3,3'-

DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE C-7 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH 3,3'-

DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH 3,3'­

DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE D-7 

XII
 
I. INTRODUCTION
 

3,3'-Dimethoxybenzidine-4,4'-diisocyanate (Figure 1) (NCI No.
 

C02175), a dimer of o-anisidine isocyanate, was selected for bioassay
 

by the National Cancer Institute because of the structural similarity
 

of this compound to 3,3'-dimethoxybenzidine, a carcinogen in Fischer
 

rats (Hadidian et al., 1968).
 

The Chemical Abstracts Service (CAS) Ninth Collective Index (1977)
 

name fcr this compound is 4,4'-diisocyanato-3,3'-dimethoxy-1,1'-bi­

*
 phenyl. It is also called dianisidine diisocyanate; isocyanic acid
 

3,3'-dimethoxy-4,4'-biphenylene ester; 4,4'-diisocyanato-3,3'-dimethoxybiphenyl; 3,3'-dimethoxybiphenylene-4,4'-diisocyanate; 3,3'-dimethoxy-4, 4'-biphenyl diisocyanate; and 3,3'-dimethoxy-4,4'-biphenylene
 

diisocyanate.
 

Several patents have been issued or applied for describing potential uses of 3,3'-dimethoxybenzidine-4,4'-diisocyanate. These include
 

synthesis of shock-absorbing polyurea-polyurethanes, which can be used
 

in railway car couplers (Chung, 1975); in situ manufacture of polyurethane gaskets and seals for metal pail and drum covers, aerosol mounting cups, and metal bottoms of fiber drums (Grace, 1974); synthesis of
 

heat stable thermoplastic urethane rubbers, useful as gaskets, seals,
 

0-rings, and wire coatings (Bonk and Shah, 1975); formulation of photocurable triazine-containing polyene-polythiol lacquers for steel
 

*
 

The CAS registry number is 91-93-0.
 
N=C=0 

CH3—0 0—CHs 

FIGURE 1 

CHEMICAL STRUCTURE OF 3,3'-DIMETHOXYBEIMZIDINE-4. 4'-DMSOCYANATE 
cans (Guthrie and Rendulic, 1975); and manufacture of abrasion-


resistant antireflection coatings for color photographs (Chiklis,
 

1975); however, these uses appear to be purely experimental.
 

Specific production data for 3,3'-dimethoxybenzidine-4,4'-dLisocyanate are not available; however, exclusion of this compound from
 

the 1977 Directory of Chemical Producers, U.S.A. (Stanford Research
 

Institute, 1977) implies that it is not currently produced in commercial quantities (in excess of 1000 pounds or $1000 in value annually)
 

in the United States.
 

The potential for exposure to 3J3l-dimethoxybenzidine-4,4'-diisocyanate is limited to researchers, particularly those involved in the
 

experimental synthesis of elastomers and polymeric coatings.
 
II. MATERIALS AND METHODS
 

A. Chemicals
 

3,3'-Dimethoxybenzidine-4,4'-diisocyanate was purchased from
 

Upjohn Company, Kalamazoo, Michigan. Chemical analysis was performed
 

by Litton Bionetics, Inc., Kensington, Maryland. The experimentally
 

determined range in melting point was 113° to 116°C. No literature
 

value was found for comparison. Thin-layer chromatography (TLC) was
 

performed utilizing two solvent systems (i.e., anhydrous acetone and
 

ethyl acetate). Each plate was visualized with visible and ultraviolet light, iodine vapor, and ferricyanide-nitroprusside spray.
 

The plate developed with acetone showed one contaminant that was less
 

motile than the major spot, while the plate developed with ethyl acetate revealed two contaminants, one of lesser and one of greater motility than the major spot. The results of infrared (IR) and nuclear
 

magnetic resonance (NMR) analyses were consistent with those expected
 

based on the structure of the compound. Ultraviolet/visible (UV/VIS)
 

spectrophotometry showed X at 310 and 285 nm with respective molar
 max
 

4 4
 extinction coefficients of 2.55 x 10 and 1.87 x 10 .
 

A second batch of the compound was purchased approximately 1.5
 

years later from the same manufacturer. The experimentally determined
 

range in melting point was 109.3° to 111.3°C. TLC was performed as
 

with the first batch. One impurity that was less motile than the
 

major spot was observed on each plate. The results of IR and NMR
 

analyses were consistent with those expected based on the structure
 

4
 
of the compound. UV/VIS analysis revealed \ at 307 and 280 nm
 max
 A
 

with respective molar extinction coefficients of 2.33 x 10 and
 

1.67 x 104.
 

Throughout this report the term 3,3'-dimethoxybenzidine-4,4'­

diisocyanate is used to represent this material.
 

B. Dosage Preparation
 

Fresh solutions of 3,3'-dimethoxybenzidine-4,4'-diisocyanate in
 

*
 steroid suspending vehicle (Litton Bionetics, Inc., Kensington, Maryland) were prepared on each day that intubation was performed. Excess
 

portions of the mixtures were disposed of rather than stored. The
 

concentration of 3,3l-dimethoxybenzidine-4,4'-diisocyanate in steroid
 

suspending vehicle ranged from 15 to 30 percent.
 

C. Dietary Preparation
 

The basal laboratory diet for both dosed and control animals
 

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

3,3'-Dimethoxybenzidine-4,4'-diisocyanate was administered to the
 

dosed mice as a component of the diet throughout the period of chemical administration, while it was administered to rats as a component
 

of the diet beginning with week 23 and continuing for the duration
 

of compound administration. (Rats were intubated with the chemical
 

for the first 22 weeks of the bioassay.)
 

*
 

Steroid suspending vehicle = 9 gm NaCl, 5 gm carboxymethylcellulose,
 

~4 ml polysorbate 80, and 9 ml benzyl alcohol q.s. to 1 liter with
 

distilled water.
 
The chemical was removed from its container and a proper amount
 

was blended with an aliquot of the ground feed using a mortar and
 

pestle. Once visual homogeneity was attained, the mixture was placed
 

in a 6 kg capacity Patterson-Kelley standard model twin-shell stainless steel V-blender along with the remainder of the feed to be prepared. After 20 minutes of blending, the mixtures were placed in
 

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

prepared once weekly.
 

Dosed feed preparations containing 23,000 and 46,000 ppm of 3,3'­

dimethoxybenzidine-4,4'-diisocyanate were analyzed spectrophototnetrically. The mean result immediately after preparation was 95.2 _+ 4.2
 

percent of theoretical, including correction for the analytical method
 

of recovery used. After ten days, at ambient room temperature, the
 

mean result was 100.3 +_ 4.4 percent of theoretical, including correction for the analytical method of recovery used.
 

D. Animals
 

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

contracts of the Division of Cancer Treatment, National Cancer Institute. Rats were supplied by A. R. Schmidt, Madison, Wisconsin, and
 

Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts.
 

All mice were supplied by Charles River Breeding Laboratories, Inc.
 
Rats and mice were approximately 4 weeks old when received. Upon
 

receipt, animals were examined for visible signs of disease or parasites. Obviously ill or runted animals were culled. The remaining
 

animals were quarantined for 2 weeks prior to initiation of test.
 

Animals which did not manifest clinical signs of disease were placed
 

on test at this time. Animals were assigned to groups and distributed
 

among cages so that the average body weight per cage was approximately
 

equal for a given species and sex.
 

E. Animal Maintenance
 

All animals were housed by species in temperature- and humidity-


controlled rooms. The temperature range was 22° to 26°C and the
 

relative humidity was maintained between 45 and 55 percent. Incoming
 

air was filtered through HEPA filters (Flanders Filters, McLean, Virginia) at a rate of 12 to 15 complete changes of room air per hour»
 

Fluorescent lighting was provided 8 hours per day (9:00 a.m. to 5:00
 

p.m.).
 

All rats were housed four per cage by sex and all mice were housed
 

five per cage by sex. Throughout the study dosed and control animals
 

of both species were housed in polycarbonate cages (Lab Products, Inc.,
 

Gartielc, New Jersey) suspended from aluminum racks. Racks were
 

fitted wjth a continuous piece of stainless steel mesh over which a
 

sheet of rilter paper was firmly secured. Filter paper was changed
 

at 2-week intervals, when the racks were sanitized* Clean cages and
 

®
 bedding were provided twice weekly. Ab-sorb-dri hardwood chip
 
bedding (Wilner Wood Products Company, Norway, Maine) was used in
 

polycarbonate cages for the entire bioassay.
 

Acidulated water (pH 2.5) was supplied to animals in water bottles filled by an automated metering device that was checked daily
 

for diluting accuracy. Water bottles were changed and washed twice
 

weekly, and sipper tubes were washed at weekly intervals. During the
 

period of chemical administration, dosed and control animals received
 

®
 treated or untreated Wayne Lab-Blox meal as appropriate. The feed
 

was supplied in hanging stainless steel hoppers which were refilled
 

three times per week and sanitized weekly. Food and water were
 

available ad libitum for both species.
 

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

*
 receiving diets containing N-phenyl-p-phenylenediamine hydrochloride
 

(2198-59-6); acetylaminofluorene (53-96-3); and nitrilotriacetic acid
 

(139-13-9).
 

All dosed and control mice were housed in a room with mice receiving diets containing EDTA trisodium salt. (150-38-9); diaminozide
 

(1596-84-5); N,N'-diethylthicurea (105-55-5); triphenyltin hydroxide
 

(76-87-9); carbromal (75-65-6); p-quinone dioxime (105-11-3); 4-amino2-nitrophenol (119-34-6); other mice intubated with lithocholic acid
 

(434-13-9); and other mice receiving I.P. injections of methiodol
 

sodium (126-31-8).
 

*
 

CAS registry numbers are given in parentheses.
 
F. Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

3,3'-dimethoxybenzidine-4,4'-diisocyanate for administration to dosed
 

animals in the chronic study, subchronic toxicity tests were conducted
 

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

among six groups, each consisting of five males and five females.
 

3,3'-Dimethoxybenzidine-4,4'-diisocyanate mixed with distilled water
 

was introduced by gavage to five of the six rat groups at dosages of
 

800, 1260, 2000, 3160, and 5000 mg/kg and to five of the six mouse
 

groups at dosages of 215, 464, 1000, 2150, and 4640 mg/kg. The sixth
 

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

suspending vehicle by gavage. Intubation was performed five times
 

per week for 4 weeks, followed by a 2-week observation period to
 

detect any delayed toxicity. Individual body weights were recorded
 

weekly. At the end of the observation period, all survivors were
 

sacrificed and necropsied.
 

At the end of the subchronic test, mean body weight gain among
 

male rats dosed with 2000 mg/kg was 1 percent greater than the mean
 

body weight gain of their controls, while female rats receiving the
 

same dosage displayed a mean body weight gain 4 percent less than
 

that of their controls. At a dosage of 3160 rag/kg, the mean body
 

weight gain among male rats was 9 percent less than that of their
 

controls, while female rats receiving the same dosage displayed a
 

mean body weight gain 14 percent less than that of their controls.
 
The high concentration selected for administration to dosed rats in
 

the chronic bioassay was 3000 mg/kg.
 

At the end of the subchronic test, mean body weight gain among
 

male mice dosed with 4640 mg/kg was 4 percent less than the mean
 

body weight gain of their controls, while female mice receiving the
 

same dosage displayed a mean body weight gain 11 percent less than
 

that of their controls. The high concentration selected for administration to dosed mice in the chronic bioassay was 44,000 ppm
 

(equivalent to 7040 mg/kg, assuming an average food consumption of
 

4 grams/day and an average body weight of 25 grams).
 

G. Gastric Intubation
 

Intubation was performed for 5 consecutive days per week on a
 

mg/kg body weight basis, utilizing the most recently observed group
 

mean body weight as a guide for determining the dose. All rats
 

were weighed and dosages adjusted once monthly, based on group mean
 

body weight. Thus, although the ratio of dose to weight remained
 

constant, the total dosage administered fluctuated with an increase
 

or decrease in group mean body weight. Rats of each sex within a
 

dosed group received the same dosage.
 

H. Experimental Design
 

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

treated and untreated observation periods, and the time-weighted
 

average concentrations) are summarized in Tables 1 and 2.
 

10
 
TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

3,3'-DIMETHOXYBENZIDINE-4,4l-DIISOCYANATE CHRONIC BIOASSAY
 

INITIAL OBSERVATION PERIOD 

GROUP 3,3'-DIMETHOXYBENZIDINE­ TREATED UNTREATED 

SIZE 4,4'-DIISOCYANATE3 » b (WEEKS) (WEEKS) 

MALE
 

VEHICLE CONTROL 20 104
 

LOW DOSE 50 1,500* 22
 

22,000 56
 

0 26
 

HIGH DOSE 50 3,000* 22
 

44,000 56
 

0 26
 

FEMALE
 

VEHICLE CONTROL 20 104
 

LOW DOSE 50 1,500* 22
 

22,000 56
 

0 26
 

HIGH DOSE 49 3 000* 22
 ' h
 

44,000 56
 

0 26
 

Dosages, given in rag/kg body weight, were administered by gavage 5
 

consecutive days per week. These dosages (i.e., 3000 and 1500 mg/kg)
 

are equivalent to respective concentrations of 44,000 and 22,000 ppm
 

(assuming average daily food consumption and body weight).
 

Concentrations given in parts per million in the feed,
 

Gavaged with steroid suspending vehicle for the first 22 weeks and
 

subsequently transferred to the basal laboratory diet.
 

11
 

c 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE FEEDING EXPERIMENT
 

MALE 

INITIAL 

GROUP 

SIZE 

3,3' -DIMETHOXYBENZIDINE4,4'-DIISOCYANATE 

CONCENTRATION3 

OBSERVATION PERIOD 

TREATED UNTREATED 

(WEEKS) (WEEKS) 

CONTROL 20 0 0 103 

LOW DOSE 50 22,000 

0 

78 

25 

HIGH DOSE 50 44,000 

0 

78 

25 

FEMALE 

CONTROL 20 0 0 103 

LOW DOSE 50 22,000 

0 

78 

25 

HIGH DOSE 50 44,000 

0 

78 

25 

Concentrations given in parts per million. 

12
 
The method of compound administration for the chronic bioassay
 

in rats, which was begun prior to the chronic bioassay in mice, was
 

initially intubation. Intubation was selected because it was believed that the compound was not stable in feed. After determination that the compound was stable in feed (as indicated on page 6)
 

the method of compound administration to rats was changed to dietary
 

and the mouse bioassay was initiated using dietary administration.
 

All rats were approximately 6 weeks old at the time the test was
 

initiated and were placed on test simultaneously. The dosages initially administered to males and females were 3000 and 1500 rag/kg,
 

by gavage (equivalent to dietary concentrations of 44,000 and 22,000
 

ppm). Throughout this report those rats initially receiving the former dosage are referred to as the high dose groups and those initially
 

receiving the latter dosage are referred to as the low dose groups.
 

All dosed rats were administered 3,3'-dimethoxybenzidine 4,4'-diisocyanate by gavage at the dosages indicated for 22 weeks. Beginning
 

in week 23, 3,3'-dimethoxybenzidine-4,4'-diisocyanate was mixed with
 

the feed and administered ad libitum in the diet. All dosed rats
 

were supplied feed containing 3,3'-dimethoxybenzidine-4,4'-dii8Ocyanate for the remaining 56 weeks of compound administration. During
 

this 56-week period, the high and low concentrations administered
 

were 44,000 and 22,000 ppm, respectively. The dosed rats were
 

observed for a period of 26 weeks after compound administration
 

ceased.
 

13
 
All mice were approximately 6 weeks old at the time the test was
 

initiated and were placed on test simultaneously. The dietary concentrations of 3,3'-dimethoxybenzidine-4,4'-diisocyanate administered
 

to both sexes were 44,000 and 22,000 ppm. Throughout this report
 

those mice receiving the former concentration are referred to as the
 

high dose groups and those receiving the latter concentration are
 

referred to as the low dose groups. Dosed mice were supplied with
 

feed containing 3,3!-dimethoxybenzidine-4,4'-diisocyanate for 78
 

weeks followed by a 25-week observation period.
 

I. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the experiment. From the first day, all animals were inspected twice daily
 

for mortality. Food consumption data were collected at monthly intervals from 20 percent of the animals in each group. Body weights of
 

rats were recorded once a week for the first 6 weeks, every 2 weeks
 

for the next 12 weeks, and once a month thereafter. Body weights of
 

mice were recorded once a week for the first 6 weeks, every 2 weeks
 

for the next 10 weeks and once a month for the remainder of the bioassay.
 

All moribund animals or animals that developed large, palpable
 

masses that jeopardized their health were sacrificed. A necropsy
 

was performed on each animal regardless of whether it died, was sacrificed when moribund, or was sacrificed at the end of the bioassay.
 

14
 
The animals were euthanized by carbon dioxide asphyxiation, and were
 

immediately necropsied. The histopathologic examination consisted of
 

gross and microscopic examination of all major tissues, organs, and
 

gross lesions taken from sacrificed animals and, whenever possible,
 

from animals found dead.
 

Tissues were preserved in a 10 percent neutral buffered formalin
 

solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin prior to microscopic examination.
 

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

lymph nodes, thymus, heart, salivary gland, liver, gallbladder (mice),
 

pancreas, esophagus, Zymbal's gland, stomach, small intestine, large
 

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

parathyroid, testis, prostate, brain, uterus, mammary gland, and
 

ovary.
 

A few tissues were not examined for some animals, particularly
 

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

preclude histopathologic interpretation. Thus, the number of animals
 

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

animals that were recorded in each group at the time that the test
 

was initiated.
 

15
 
J. Data Recording and Statistical Analyses
 

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

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

as recommended by the International Union Against Cancer (Berenblum,
 

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

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

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

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

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

missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect
 

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

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

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

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

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

16
 
The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

histologically. However, when macroscopic examination was required
 

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

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

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

The purpose of the statistical analyses of tumor incidence is to
 

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

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

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

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

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

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

significance level of 0.05 may be made. The Bonferroni inequality
 

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

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

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

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

17
 
The Cochran-Armitage test for linear trend in proportions, with
 

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

when appropriate0 Under the assumption of a linear trend, this test
 

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

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

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation of
 

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

tumor was observed were excluded by basing the statistical tests on
 

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

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

tumor was found, comparisons were based exclusively on animals that
 

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

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

Cocaran-Armitage tests, etc.) were followed.
 

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

incidence of tumors. Curves of the proportions surviving without an
 

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

during which animals died naturally or were sacrificed was entered
 

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

18
 
these curves were used for two groups; Tarone's extension to testing
 

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

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

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

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


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

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

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

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

t c t
 

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

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

c
 

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

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

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

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

larger proportion in the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

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

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

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

would be within the interval calculated from the experiment. When
 

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

19
 
can be inferred that a statistically significant result (a P < 0.025
 

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

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

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

the lower limit indicates the absence of a significant result while
 

the upper limit indicates that there is a theoretical possibility
 

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

detected under the conditions of this test.
 

20
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

Dose-related mean body weight depression was not apparent in
 

either male or female rats. There was, however, slight mean body
 

weight depression in dosed groups of both sexes when compared to
 

their respective controls (Figure 2).
 

No abnormal clinical signs were recorded.
 

B. Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and 3,3'-dimethoxybenzidine-4,4'-diisocyanate-dosed
 

groups are shown in Figure 3. For male rats the Tarone test for a
 

positive association between dosage and mortality was significant
 

(P = 0.002). While the Tarone test was not significant for females,
 

the Cox tests comparing high dose to control and low dose to control
 

indicated a significantly higher mortality among dosed rats when compared with the control group.
 

There were adequate numbers of male rats at risk from late-


developing tumors as 80 percent (40/50) of the high dose, 88 percent
 

(44/50) of the low dose, and 90 percent (18/20) of the control group
 

survived on test more than 80 weeks. Thirty-two percent (16/50) of
 

the high dose, 44 percent (22/50) of the low dose and 75 percent
 

(15/20) of the control group survived on test until the termination
 

of the study.
 

21
 
750-r •750 

600­ -600 

er 

o 

450­ h-450 

LU 

5 

gsoo­ -300 

CQ 

150­ -150 

MALE RATS 

I ~T 

15 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

750­

600 — -600 

CC 

O 

X 

UJ 

450 ­ -450 

D 

O 

m 

r-300 

150­ — 150 

FEMALE RATS 

15 

~T~ 

30 

~T 

45 CO 75 

TIME ON TEST (WEEKS) 

T 

90 105 120 

FIGURE 2 

GROWTH CURVES FOR 3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE CHRONIC STUDY RATS 

22 
PROBABILITY OF SURVIVAL PROBABILITY OF SU

o o p j> o> bo ca 

30 1 

Tl O m 

•o 

m 30

 33

 

§ 

u 

w 

H r! £­ O

 3 

DO Z} 

ro m OJ sm

 

m 

en 

8 

gO 

I O 

bo b 
Similarly, there were adequate numbers of female rats at risk
 

from late-developing tumors with 90 percent (45/50) of the high dose,
 

80 percent (40/50) of the low dose, and 95 percent (19/20) of the
 

control group surviving on test more than 80 weeks. At the termination of the study, 42 percent (21/50) of the high dose, 42 percent
 

(21/50) of the low dose and 75 percent (15/20) of the control group
 

were alive on test.
 

C. Pathology
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

summarized in Appendix C (Tables Cl and C2).
 

There were increased incidences of tumors of baso-squamous
 

epithelial origin in dosed male and female rats, when compared to
 

their respective controls. These tumors were commonly in the skin of
 

the head, inguinal area and back. The location of 28 tumors of the
 

head strongly suggested that these tumors arose from the Zymbal's
 

gland. The location and type of 17 tumors in the inguinal area suggested involvement of preputial glands. Grossly, they appeared as
 

irregular fungating or bulging ulcerated lesions measuring about 0.5
 

to 3.0 cm in greatest dimension.
 

Microscopically, the tumors thought to arise from the Zymbal's
 

gland were carcinomas of the sebaceous gland, squamous-cell carcinomas, and a carcinosarcoma as summarized in the following table:
 

24
 
Low High
 

Control Dose Dose
 

MALES
 

Number of Animals with '"ipsues
 

Examined Histopatholofe^>..-lly (20) (50) (50) 

ZYMBAL'S GLAND 

Squamous-Cell Carcinoma 0 2(4%) 5(10%) 

Sebaceous Adenocarcinoma 0 3(6%) 2(4%) 

Careinosarcoma 0 0 0 

FEMALES
 

Number of Animals with Tissues
 

Examined Histopathologically (20) (50) (48)
 

ZYMBAL'S GLAND
 

Squamous-Cell Carcinoma 0 1(2%) 4(8%)
 

Sebaceous Adenocarcinoma 0 7(14%) 2(4%)
 

Carcinosarcoma 1(5%) 0 0
 

These and other tumors of baso-squamous origin were classified
 

according to the apparent direction of maturation. The auditory tumors had both sebaceous and squamous differentiation as did nearly
 

all other skin neoplasms discussed later. The Zymbal's gland tumors
 

appeared to have the most malignant potential of any of the skin tumors. Two invaded the skull and two others metastisized to the lung.
 

Tumors arising from preputial and clitoral glands (i.e., 0/20, 1/50
 

[2 percent], and 3/50 [6 percent] in the control, low dose, and high
 

25
 
dose males, respectively, and 0/20, 8/50 [16 percent], and 4/48 [8 percent] in the control, low dose, and high dose females, respectively)
 

were the most consistent of the skin tumors in that they mostly resembled sebaceous glands and as a rule had little squamous differentiation.
 

These tumors appeared to have little malignant potential and were therefore regarded of low-grade malignancy. Additional skin tumors arose
 

from other locations as shown in the following table:
 

Low High
 

Control Dose Dose
 

MALES
 

SKIN
 

Number of Animals with Tissues
 

Examined Histopathologically (20) (50) (50) 

Neoplasms NOS 0 1(2%) 0 

Papilloma NOS 0 0 2(4%) 

Squamous-Cell Carcinoma 0 5(10%) 4(8%) 

Basal-Cell Tumor 0 4(8%) 5(10%) 

Trichoepithelioma 0 2(4%) 1(2%) 

Sebaceous Adenoma 0 2(4%) 0 

Sebaceous Adenocarcinoma 0 1(2%) 0 

Keratoacanthoma 1(5%) 4(8%) 5(10%) 

Carcinosarcoma 0 0 1(2%) 

FEMALES 

SKIN 

Number of Animals with Tissues 

Examined Histopathologically (20) (50) (48)
 

Neoplasms NOS 0 0 1(2%) 

Papilloma NOS 0 0 0 

Squamous-Cell Carcinoma 0 1(2%) 0 

Basal-Cell Tumor 0 0 1(2%) 

Trichoepithelioma 0 1(2%) 0 

Sebaceous Adenoma 0 0 1(2%) 

Sebaceous Adenocarcinoma 1(2%) 1(2%) 1(2%) 

Keratoacanthoma 0 0 0 

Carcinos arcoma 

26 

1(2%) 0 0 
The classification ot these tumors was seldom obvious. More often
 

than not, the tumors were mixtures of basal cells often with sebaceous
 

differentiation, squamous cells often forming keratin pearls and sometimes mimicked hair follicles. The direction of differentiation was
 

obscured by maturation in two or more directions simultaneously. The
 

impression was that all such skin tumors arose from similar pleuropotent cells and that the path of differentiation is variable, thus they
 

should all be considered to have a malignant potential, however, low-


grade.
 

Squamous-cell carcinomas were characterized by down growth of
 

clusters of basal cells with individual cell and group keratinization,
 

often with many keratin pearls. In some, invasion of the stroma led
 

to a desmoplastic response. Sebaceous carcinomas were composed of
 

lobules of basal type epithelium maturing toward sebaceous cells and
 

often filling cystic spaces with "ghosts" of necrotic cells. Basal-


cell tumors formed lobules of typical basophilic basal cells in a
 

dense fibrous connective tissue. There were always some keratin
 

pearls and sebaceous cells. Keratoacanthomas arose from greatly
 

thickened surface epithelium and tended to form inverted cysts filled
 

with laminated keratin. The cyst wall was thick, complex and contained keratin whorls and sebaceous glands, but was generally sharply
 

circumscribed.
 

Leiikemias were common in dosed male (i.e., 0/20, 18/50 [36
 

percent], and 16/50 [32 percent] in the control, low dose, and high
 

27
 
dose, respectively) and dosed female (i.e., 1/20 [5 percent], 8/50
 

[16 percent], and 12/48 [25 percent] in the control, low dose, and
 

high dose, respectively) rats and were nearly absent in controls.
 

The leukemias usually involved the spleen, causing splenomegaly, and
 

less commonly involved the liver, giving it a slightly enlarged and
 

mottled appearance.
 

Microscopically, in addition to the spleen and liver, the pulmonary capillaries were often filled with leukemic cells and sometimes
 

the bone marrow sections contained leukemic cells. In all cases in
 

which the tissues were not autolyzed, the leukemia was classified as
 

of the undifferentiated type. The affected cells contained large
 

nucleoli with evenly distributed chromatin. The nuclei were round
 

with various degrees of indentation.
 

There was a spontaneous occurrence of a variety of other tumors
 

in both the control and dosed groups. These lesions were of the type,
 

incidence, and distribution often observed in aged Fischer 344 rats,
 

and, therefore, were not attributed to compound administration.
 

The usual nonneoplastic lesions were seen in rats of all groups
 

and were not considered to be compound-related.
 

The results of this pathologic examination indicated that
 

3,3'-dimethoxybenzidine-4,4'-diisocyanate caused skin and adnexal
 

tumors in male and female rats, and may also be associated with
 

leukemia in male and female rats.
 

28
 
D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

tumors were observed in at least one of the control or 3,3'-dimethoxybenzidine-4,4'-diisocyanate-dosed groups and where such tumors
 

were observed in at least 5 percent of the group. Due to the early
 

mortality of a number of male rats the analyses for males have been
 

based solely upon those males surviving at least 52 weeks or, in the
 

event that the tumor of interest was observed earlier, at least as
 

long as the time at which the first tumor of interest was observed.
 

For male rats, the Cochran-Artnitage test indicated a significant
 

(P = 0.022) positive association between dose and the combined incidence of leukemia or malignant lymphomas. This result was supported
 

by significant Fisher exact test results for the low dose (P = 0.001)
 

and the high dose (P = 0.002) groups. For females, the Cochran-


Armitage test was also significant (P = 0.007) and was supported by
 

a significant (P = 0.016) Fisher exact test comparing high dose to
 

control. Based on these statistical results, the administration of
 

3,3'-dimethoxybenzidine-4,4"-diisocyanate was associated with the
 

increased combined incidence of leukemia or malignant lymphomas in
 

male and female Fischer 344 rats under the conditions of this bioassay.
 

Also in male rats, the Fisher exact tests for the incidence of
 

skin (excluding skin of the ear) neoplasms (i.e., papilloma NOS,
 

29
 
TABLE 3
 

TIME-ADJUSTED ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

a,f
 SPECIFIC SITES IN MALE RATS TREATED WITH 3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE


TOPOGRAPHY : MORPHOLOGY
 

Skin (Excluding Skin of Ear): Squamous-


Cell Carcinoma13
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 u>
 

o
 

Skin (Excluding Skin of Ear): Sebaceous
 

Adenoma or Sebaceous Adenocarcincma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Skin (Excluding Skin of Ear): Papilloma
 

NOS, Basal-Cell Tumor, Trichoepithelioma, Sebaceous Adenoma, or Keratoacanthomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

VEHICLE
 

CONTROL
 

0/18(0.00)
 

N.S.
 

,
 

0/13(0.00)
 

N.S.
 

0/18(0.00)
 

N.S.
 

LOW
 

DOSE
 

5/48(0.10)
 

N.S.
 

Infinite
 

0.496
 

Infinite
 

94
 

3/48(0.06)
 

N.S.
 

Infinite
 

0.236
 

Infinite
 

79
 

12/48(0.25)
 

P = 0.014
 

Infinite
 

1.445
 

Infinite
 

79
 

HIGH
 

DOSE
 

4/46(0.09)
 

N.S.
 

Infinite
 

0.380
 

Infinite
 

82
 

0/46(0.00)
 

N.S.
 

11/46(0.24)
 

? = 0.018
 

Infinite
 

1.366
 

Infinite
 

60
 
TOPOGRAPHY: MORPHOLOGY


Skin (Excluding Skin of Ear) : Squamous-


Cell Carcinoma, or Sebaceous Adenocarcinomab


P Values0


Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Skin (Excluding Skin of Ear) : Papilloma
 

NOS, Basal-Cell Tumor, Trichoepithelioma,
 

Sebaceous Adenoma, Keratoaeanthoma,
 

Squamous-Cell Carcinoma, or Sebaceous
 

Adenocarcinomab


P Values0


Departure from Linear Trend6


Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED) 

VEHICLE
 

 CONTROL
 

 0/18(0.00)
 

 N.S.
 

 0/18(0.00)
 

P = 0.045
 

P = 0.024
 

LOW
 

DOSE
 

6/48(0.13)
 

N.S.
 

Infinite
 

0.630
 

Infinite
 

94
 

17/48(0.35)
 

P = 0.002
 

Infinite
 

2.132
 

Infinite
 

79
 

HIGH
 

DOSE
 

4/46(0.09)
 

N.S.
 

Infinite
 

0.380
 

Infinite
 

82
 

14/46(0.30)
 

P = 0.005
 

Infinite
 

1.795
 

Infinite
 

60
 
___ 

TOPOGRAPHY : MORPHOLOGY 

Lung: Alveolar /Bronchiolar Adenoma 

P Values0 

Departure from Linear Trend 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Hematopoietic System:

•! 

 Leukemia or 

Malignant LymphomaD 

S3 

OJ 

P Values0 

Departure from Linear Trend 

Relative Risk (Control)d

 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Thyroid: C-Cell Adenoma or C-Cell 

Carcinoma 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

TABLE 3 (CONTINUED) 

VEHICLE 

CONTROL 

0/18(0.00) 

N.S. 

P = 0.002 

0/18(0.00) 

P = 0.022 

P = 0.028 

1/18(0.06) 

N.S. 

104 

LOW 

DOSE 

8/47(0.17) 

N.S. 

Infinite 

0.919 

Infinite 

79 

18/48(0.38) 

P = 0.001 

Infinite 

2.270 

Infinite 

72 

3/43(0.07) 

N.S. 

1.256 

0.112 

64.377 

98 

HIGH 

DOSE 

0/43(0.00) 

N.S. 

16/46(0.35) 

P = 0.002 

Infinite 

2.083 

Infinite 

80 

0/42(0.00) 

N.S. 

0.000 

0.000 

7.981 
TABLE 3 (CONTINUED) 

TOPOGRAPHY: MORPHOLOGY 

VEHICLE 

CONTROL 

Preputial Gland:

carcinoma'5 

P Values0 

 Sebaceous Adeno0/18(0.00) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

OJ 

OJ 

Testis: Interstitial -Cell Tumor 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

17/18(0.94) 

N.S. 

Weeks to First Observed Tumor 97 

Liver: Hepatocellular Adenoma or 

Hepatocellular Carcinoma*5 

P Values0 

1/18(0.06) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 104 

LOW
 

DOSE
 

1/48(0.02)
 

N.S.
 

Infinite
 

0.021
 

Infinite
 

97
 

39/48(0.81)
 

N.S.
 

0.860
 

0.793
 

1.139
 

77
 

8/48(0.17)
 

N.S.
 

3.000
 

0.457
 

129.949
 

104
 

HIGH
 

DOSE
 

3/46(0.07)
 

N.S.
 

Infinite
 

0.246
 

Infinite
 

94
 

36/45(0.80)
 

N.S.
 

0.847
 

0.779
 

1.128
 

79
 

3/46(0.07)
 

N.S.
 

1.174
 

0.104
 

60.276
 

86
 
___ 

TABLE 3 (CONTINUED) 

VEHICLE 

TOPOGRAPHY : MORPHOLOGY CONTROL 

Pituitary: Chromophobe Adenoma ' 3/16(0.19) 

P Values0 N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 39 

Adrenal: Pheochromocytoma 4/18(0.22) 

P Values0 P = 0.003(N) 

u> Relative Risk (Control) —_ 

-p- Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 104 

Zymbal's Gland, Ear, and Skin of Ear: 

Squamous-Cell Carcinoma or Sebaceous 

Adenocarcinoma'3 0/18(0.00) 

P Values0 P = 0.040 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

LOW 

DOSE 

7/44(0.16) 

N.S. 

0.848 

0.231 

4.673 

80 

3/48(0.06) 

N.S. 

0.281 

0.047 

1.530 

104 

5/48(0.10) 

N.S. 

Infinite 

0.496 

Infinite 

77 

HIGH 

DOSE 

2/34(0.06) 

N.S. 

0.314 

0.029 

2.516 

69 

0/45(0.00) 

P = 0.005(N) 

0.000 

0.000 

0.425 

8/46(0.17) 

N.S. 

Infinite 

0.939 

Infinite 

70 
TABLE 3 (CONCLUDED) 

VEHICLE LOW HIGH 

TOPOGRAPHY:MORPHOLOGY CONTROL DOSE DOSE 

Zymbal's Gland, Ear, and Skin of

Squamous-Cell Carcinoma, Sebac

Adenocarcinoma, Keratoacanthoma,

Neoplasm NOS, or Basal-Cell Tumor 

 Ear 

eous 

, 

1/18(0.06) 6/48(0.13) 9/46(0.20) 

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

Relative Risk (Control) 

Lower Limit 

Upper Limit 

2.250 

0.308 

101.140 

3.522 

0.556 

150.403 

Weeks to First Observed Tumor 104 77 70 

Treated groups received doses of 1500 or 3000 mg/kg by gavage for 22 weeks followed by concentrations of 22,000 or 44,000 ppm in feed for 54 weeks. 

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

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

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

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

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

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

£

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

group when P < 0.05. 

These analyses were based solely upon animals surviving at least 52 weeks, except for sites
 

where the first tumor of interest was observed earlier than 52 weeks in any group of this
 

sex and species, where the analyses were based upon all animals that survived until or past
 

the date that the first tumor was observed.
 
TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH 3,3'-DIMETHOXYBENZIDINETOPOGRAPHY : MORPHOLOGY
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma'3
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

4,4'-DIISOCYANATEa 

VEHICLE LOW HIGH 

CONTROL DOSE DOSE 

1/20(0.05) 8/50(0.16) 15/48(0.31) 

P = 0.007 N.S. P = 0.016 

3.200 6.250 

0.482 1.093 

138.771 255.482 

94 90 83 

4/18(0.22) 8/48(0.17) 8/45(0.18) 

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

0.750 0.800 

0.239 0.255 

3.097 3.292 

103 71 100 

0/18(0.00) 3/44(0.07) 0/41(0.00) 

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

P = 0.049 

Infinite 

0.258 

Infinite 

100 
TABLE 4 (CONTINUED)
 

TOPOGRAPHY: MORPHOLOGY 

VEHICLE 

CONTROL 

Mammary Gland: Fibroadenoma, Adenoma 

NOS, or Adenocarcinoma NOS*5 0/20(0.00) 

P Values0 N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

___ 

Weeks to First Observed Tumor 

Preputial Gland: Sebaceous Adenocarcinoma 

or Squamous-Cell Carcinoma*3 0/20(0.00) 

P Values0 N.S. 

Departure from Linear Trend 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

P = 0.043 ___ 

Weeks to First Observed Tumor 

Uterus: Endometrial Stromal Polyp 0/20(0.00) 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

P = 0.013 ___ 

Weeks to First Observed Tumor 

LOW
 

DOSE
 

5/50(0.10)
 

N.S.
 

Infinite
 

0.525
 

Infinite
 

103
 

8/50(0.16)
 

N.S.
 

Infinite
 

0.952
 

Infinite
 

71
 

5/48(0.10)
 

N.S.
 

Infinite
 

0.547
 

Infinite
 

74
 

HIGH
 

DOSE
 

6/48(0.13)
 

N.S.
 

Infinite
 

0.695
 

Infinite
 

100
 

4/48(0.08)
 

N.S.
 

Infinite
 

0.402
 

Infinite
 

93
 

10/48(0.21)
 

P = 0.022
 

Infinite
 

1.292
 

Infinite
 

86
 
TABLE 4 (CONCLUDED)
 

VEHICLE LOW HIGH
 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE DOSE
 

Zymbal's Gland: Squamous-Cell Carcinoma
 

or Sebaceous Adenocarcinoma^ 0/20(0.00) 8/50(0.16) 6/48(0.13)
 

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

Relative Risk (Control) Infinite Infinite
 

Lower Limit 0.952 0.695
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 44 72
 

Zymbal's Gland and Skin of Ear:
 

Squamous-Cell Carcinoma, Sebaceous
 

U) Adenocarcinoma or Trichoepitheliomab 0/20(0.00) 9/50(0.18) 6/48(0.13)
 oo
 

P Values0 N.S. P = 0.039 N.S.
 

Relative Risk (Control) Infinite Infinite
 

Lower Limit 1.096 0.695
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 44 72
 

Treated groups received doses of 1500 or 3000 mg/kg by gavage for 22 weeks followed by concentrations of 22,000 or 44,000 ppm in feed for 54 weeks.
 

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

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

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

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

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

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

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

group when P < 0.05.
 
basal-cell tumor, trichoepithelioma, sebaceous adenoma, keratoacanthoma, squamous-cell carcinoma, or sebaceous adenocarcinoma) was
 

significant when comparing both the high dose to control (P = 0.005)
 

and the low dose to control (P = 0.002). The Cochran-Artnitage test
 

was significant (P = 0.045) with a significant (P = 0.024) departure
 

from linear trend due to the higher incidence in the low dose group
 

relative to the high dose group. Based on these statistical results,
 

the administration of 3,3'-dimethoxybenzidine-4,4'-diisocyanate was
 

associated with the increased incidence of skin neoplasms in male
 

Fischer 344 rats under the conditions of this bioassay.
 

In male rats the Cochran-Armitage test indicated a significant
 

(P = 0.040) positive association between dose and the incidence of
 

squamous-cell carcinomas or sebaceous adenocarcinomas of the Zymbal's
 

gland and the skin of the ear. While the Fisher exact tests did not
 

support these results, the historical control data for untreated male
 

Fischer 344 rats at this laboratory indicated only a 0.3 percent (1/300)
 

incidence of these tumors in untreated males, indicating the rarity of
 

occur ence of these tumors. In female rats, none of the statistical
 

tests ror these tumors at this site were significant under the iionferroni criterion. However, as with male rats, historical control
 

data for untreated female Fischer 344 rats at this laboratory indicate squamous-cell carcinomas and sebaceous adenocarcinomas of the
 

Zymbal's gland and skin of the ear to be rarely occurring, with 0 percent (0/298) incidence. Based upon these results the data suggest
 

that the administration of 3,3"-dimethoxybenzidine-4,41-diisocyanate
 

39
 
was associated with the increased incidence of squamous-cell carcinomas or sebaceous adenocarcinomas of the Zymbal's gland and the
 

skin of the ear in male and female rats.
 

In female rats the Cochran-Armitage test indicated a significant
 

(P = 0.013) positive association between dose and the incidence of
 

endometrial stromal polyps. The high dose to control Fisher exact
 

test was also significant (P = 0.022). Based on these statistical
 

results the administration of 3,3'-dimethoxybenzidine-4,4'-diisocyanate was associated with the increased incidence of endometrial
 

stromal polyps.
 

The Cochran-Armitage test and the Fisher exact test comparing
 

high dose to control indicated a significant negative association
 

between dose and the incidence of adrenal pheochromocytomas in male
 

rats. However, the historical data for this laboratory indicated an
 

incidence of 7 percent (20/294) in male control Fischer 344 rats as
 

compared to the higher 22 percent (4/18) observed in control male
 

rats in this bioassay.
 

Tn summary, the statistical findings were that the administration of 3,3'-dimethoxybenzidine-4,41

-diisocyanate was associated
 

with the increased incidence of leukemia or malignant lymphomas in
 

male and female rats, the incidence of skin neoplasms (excluding
 

skin of the ear) in male rats, and the incidence of endometrial
 

stromal polyps in female rats. The data also suggest that the incidence of squamous-cell carcinomas or sebaceous adenocarcinomas of
 

40
 
the Zymbal's gland and the skin of the ear was associated with
 

chemical administration to both sexes.
 

41
 
IV. CHRONIC TESTING RESULTS: MICE
 

A.	 Body Weights and Clinical Observations
 

Slight dose-related mean body weight depression was apparent in
 

male mice. Female mice evidenced slight mean body weight depression
 

in comparison to the control group (Figure 4).
 

No abnormal clinical signs were recorded.
 

B.	 Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and 3,3'-dimethoxybenzidine-4,4'-diisocyanate-dosed
 

groups are shown in Figure 5. The Tarone test for association between dosage and mortality was not significant for either male or
 

female mice.
 

The percentage of mice surviving on test is shown in Figure 6.
 

In the male mice, despite the disappearance of 1 control in week 22,
 

1 low dose mouse in week 38, 3 low dose mice in week 41, and 1 low
 

dose mouse in week 92, and the accidental sacrifice of 1 high dose
 

mouse in week 44, there were adequate numbers at risk from late-


developing tumors. Ninety-two percent (46/50) of the high dose,
 

80 percent (40/50) of the low dose, and 90 percent (18/20) of the
 

control group survived on test until the termination of the study.
 

With 62 percent (31/50) of the high dose, 72 percent (36/50) of
 

the low dose and 90 percent (18/20) of the control group surviving
 

on test until termination of the study, there were adequate numbers
 

42
 
50- -50 

40- -40 

cc 

¥. 30H -30 

g 

£20­ -20 

O 

00 

CONTROL 

LOW DOSE 10— -10 

MALE MICE HIGH DOSE 

' I i \ r 

15 30 45 60 90 105 120 

TIME ON TEST (WEEKS) 

50- -50 

40- -40 

VI 

< 

-30 

X 

CD 

>- 20- -20 

O 

O 

CO 

CONTROL 

10 — —10 LOW DOSE 

FEMALE MICE ___ _ HIGH DOSE 

! \ I 

15 30 45 60 90 105 120 

TIME ON TEST (WEEKS) 

FIGURE 4 

GROWTH CURVES FOR 3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE CHRONIC STUDY MICE 

43 
- -

LO — 1.0 ­ _.........................*

 

^••- fr 

t-0.8 0.8­

-0.6 co 0.6­ u. 

O 

0.4­ -0.4 

m 

O 

cc _ CONTROL a. 

0.2 ,,., LOW DOSE -0.2 ­

MALE MICE . _ HIGH DOSE 

0.0- 1 -0.0 I I I I ' I T \ 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

— 1.0 

- "^l^ - , ' ^... 

_, 0.8- "~~U — 0.8 

t £ 

cc 

w -0.6 0.6­ u. 

O -

5 0.4- -0.4 

CD 

0 

CC ••

 

a. i^_^_i^ CONTROL 

0.2 -0.2 - LOW DOSE 

FEMALEMICE _ — HIGH DOSE 

00 0 0 i I i I i I i I i 1 i I i I i 

0 15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

FIGURE 5 

SURVIVAL PROBABILITY COMPARISONS OF 3,3'-DIMETHOXYBENZIDINE-*,4'-DIISOCYANATE CHRONIC STUDY MICE 

44 
1.0-

f—1.0 

-0.8 0.8­

-0.6 Sj 0.6­

W 

O 

£ 0.4 H -0.4 

a. 

CONTROL. 

-0.2 0.2-

LOW DOSE 

MALE MICE HIGH DOSE 

0.0-

0.0 

I \^ I ' I ' I \ ' \ ' 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

— 1.0 

0.8 — 0.8 ­

DC -0.6 0.6­

V) 

ILU O 

% 0.4 H -0.4 

CONTROI 

0.2­ -0.2 LOW DOSE 

FEMALE MICE HIGH OOSE 

0.0-

-0.0­

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

FIGURE 6 

PERCENT SURVIVAL OF 3,3'-DIMETHOXYBENZIOINE-4,4'-DIISOCYANATE CHRONIC STUDY MICE 

45 
of female mice at risk from late-developing tumors. Seven females
 

from the high dose group and 9 from the low dose group were missing
 

by week 28.
 

C. Pathology
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

summarized in Appendix D (Tables Dl and D2).
 

A variety of tumors occurred in both the control and dosed
 

groups. The type, incidence, and distribution of these lesions were
 

similar to those expected in aged B6C3F1 mice; therefore, these lesions were considered to be spontaneous and not related to compound
 

administration.
 

A few neoplasms occurred only, or with a greater frequency, in
 

dosed groups as compared with controls. This may be noted in male
 

mice in which benign and malignant hepatocellular neoplasms were
 

found in 21 percent of the high dose mice compared to 5 percent of
 

controls.
 

A variety of nonneoplastic, inflammatory, degenerative or fiforotic lesions occurred randomly in all groups and none appeared to be
 

compound-related.
 

The results of this pathologic examination indicated that
 

3 ,3"-dimethoxybenzidine-4,4"-diisocyanate was not carcinogenic in
 

male or female B6C3F1 mice under the conditions of this bioassay.
 

46
 
D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

tumors were observed in at least one of the control or 3,3'-dimethoxybenzidine-4,4'-diisocyanate-dosed groups and where such tumors
 

were observed in at least 5 percent of the group.
 

None of the statistical tests for any site of either male or female mice indicated a significant positive association between chemical administration and tumor incidence. Based upon these statistical
 

results there was no evidence that 3,3'-dimethoxybenzidine-4,4'-diisocyanate was a carcinogen in B6C3F1 mice under the conditions of this
 

bioassay.
 

In male mice the Cochran-Armitage test and the Fisher exact test
 

comparing high dose to control indicated a significant negative association between dose and the incidence of interstitial-cell tumors of
 

the testis. However, historical control data from the same laboratory
 

indicate an incidence of 2 percent (4/266) of these tumors in untreated
 

male B6C3F1 mice as compared with the 16 percent (3/19) in control male
 

mice in this bioassay.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

47
 
__ _ 

___ 

00 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH 3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATEa
 

LOW
 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE
 

Lung: Alveolar /Bronchiolar Carcinoma
 

or Alveolar /Bronchiolar Adenoma" 2/19(0.11) 8/43(0.19)
 

P Values0 N.S. N.S.
 

Relative Risk (Control) 1.767
 

Lower Limit 0.406
 

Upper Limit 16.114
 

Weeks to First Observed Tumor 103 103
 

Hematopoietic System: Leukemia or
 

Malignant Lymphomab 0/19(0.00) 4/44(0.09)
 

P Values0 N.S. N.S.
 

Relative Risk (Control) Infinite
 

Lower Limit 0.418
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 98
 

Liver: Hepatocellular Carcinoma 0/19(0.00) 5/43(0.12)
 

P Values0 N.S. N.S.
 

Relative Risk (Control) Infinite
 

Lower Limit 0.583
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 98
 

HIGH
 

DOSE
 

7/46(0.15)
 

N.S.
 

1.446
 

0.315
 

13.509
 

103
 

4/50(0.08)
 

N.S.
 

Infinite
 

0.368
 

Infinite
 

68
 

6/48(0.13)
 

N.S.
 

Infinite
 

0.662
 

Infinite
 

72
 
TABLE 5 (CONCLUDED)
 

TOPOGRAPHY : MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Liver: Hepatocellular Carcinoma cr 

Hepatocellular Adenoma" 1/19(0.05) 8/43(0.19) 10/48(0.21) 

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

Relative Risk (Control) 

Lower Limit 

Upper Limit 

3.535 

0.537 

152.566 

3.958 

0.639 

167.483 

Weeks to First Observed Tumor 103 98 72 

Testis: Interstitial-Cell Tumor 3/19(0.16) 1/42(0.02) 0/48(0.00) 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

P = 0.007(N) ___ N.S. 

0.151 

0.003 

1.760 

P = 0.020(N) 

0.000 

0.000 

0.651 

Weeks to First Observed Tumor 103 103 

Treated groups received doses of 22,000 or 44,000 ppm in feed.
 

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

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

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

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

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

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

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH 3,3'-DIMETHOXYBENZIDINETOPOGRAPHY : MORPHOLOGY
 

Lung: Alveolar /Bronchiolar Adenoma
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Ul Weeks to First Observed Tumor
 o
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

4,4'-DIISOCYANATEa 

LOW HIGH 

CONTROL DOSE DOSE 

3/20(0.15) 2/41(0.05) 1/40(0.03) 

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

0.325 0.167 

0.030 0.003 

2.651 1.945 

103 103 103 

3/20(0.15) 9/41(0.22) 10/43(0.23) 

N.S. N.S. N.S. ___ 1.463 1.550 

0.424 0.465 

7.728 8.072 

103 86 33 
TABLE 6 (CONCLUDED)
 

o
 

Treated groups received doses of 22,000 or 44,000 ppm in feed.
 

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

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

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

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

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

The 95% confidence interval on the relative risk of the treated group to the control group.
 
Tables 5 and 6, the value one is included; this indicates the absence
 

of statistically significant results. It should also be noted that
 

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

indicating the theoretical possibility of tumor induction in mice by
 

3,3'-dimethoxybenzidine-4,4'-diisocyanate that could not be established under the conditions of this test.
 

52
 
V. DISCUSSION
 

There was a significant positive association between the dosages
 

of 3,3'-dimethoxybenzidine-4,4'-diisocyanate administered and mortality in male rats. For female rats, mortality among the dosed groups
 

was significantly higher than that for the controls. There were no
 

significant positive associations between dosage and mortality among
 

male or female mice. Adequate numbers of animals in all groups survived sufficiently long to be at risk from late-developing tumors.
 

Mean body weight depression was slight, but detectable, when dosed
 

male and female mice were compared to their respective controls,
 

indicating that the dosages administered to these animals may have
 

approximated the maximum tolerated dosages.
 

For both sexes of rats, there was a significant positive association between dosage and the incidence of leukemia and malignant
 

lymphoma. The high dose to control and the low dose to control Fisher
 

exact comparisons for males and the high dose to control comparison
 

for females were also significant for the incidences of these tumors.
 

Excluding the skin of the ear in considering neoplasms of the skin
 

(i.e., a combination of papilloma NOS, basal-cell tumor, trichoepithelioma, sebaceous adenoma, keratoacanthoma, squamous-cell carcinoma,
 

and sebaceous adenocarcinoma) the Fisher exact comparisons were significant for both dose levels in male rats. There was a significant
 

positive association between the dosages of the chemical administered
 

and the incidences in female rats of endometrial stromal polyps.
 

53
 
The high dose to control Fisher exact comparison supported the finding. In male rats the Cochran-Armitage test provided a significant
 

positive association between dosage and the combined incidence of
 

squamous-cell carcinomas or sebaceous adenocarcinomas of the Zymbal's
 

gland, ear, or the skin of the ear. For females, the association was
 

not significant, and for both sexes the Fisher exact comparisons were
 

not significant. The historical incidences for these tumors in
 

Fischer 344 control rats maintained by this laboratory during the
 

Carcinogenesis Testing Program are 1/300 (0.3 percent) and 0/298 for
 

males and females, respectively. When compared with the incidences
 

of these neoplasms observed in the males (i.e., 5/50 [10 percent] in
 

the low dose and 8/50 [16 percent] in the high dose) and in the
 

females (i.e., 8/50 [16 percent] in the low dose and 6/48 [13 percent]
 

in the high dose) in this bioassay, it is probable that the incidences observed were related to compound administration.
 

None of the statistical tests for any site in male or female
 

mice indicated a significant positive association between compound
 

administration and tumor incidence.
 

Under the conditions of this bioassay, administration of 3,3'­

dimethoxybenzidine-4,4'-diisocyanate was carcinogenic to Fischer
 

344 rats, causing neoplasms of the skin (excluding skin of the ear)
 

in males, endometrial stromal polyps in females, and leukemia and
 

malignant lymphoma in both sexes. The compound was also associated
 

with the development of a combination of squamous-cell carcinomas
 

and sebaceous adenocarcinomas of the Zymbal's gland and skin of the
 

54
 
ear in rats of both sexes. There was no evidence for the carcinogenicity of the compound in B6C3F1 mice.
 

55
 
VI. BIBLIOGRAPHY
 

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

J. Wiley & Sons, New York, 1971.
 

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

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

Union Against Cancer, Geneva, 1969.
 

Bonk, H.W., and T.M. Shah, "Polyurethanes from 3,3'-dimethyldiphenyl4,4'-diisocyanate Polyester Diols and BisChydroxethyl ether) of
 

Hydroquinone." U.S. Patent 3,899,467 (Upjohn Co.), August 12,
 

1975; Chemical Abstracts 83. 194863r.
 

Chemical Abstracts Service, The Chemical Abstracts Service (CAS)
 

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

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

Chiklis, C.K., "Photographic Products Containing Antireflection
 

Layer." U.S. Public Patent Application B 354,008 (Polaroid
 

Corp.), January 28, 1975; Chemical Abstracts 83, 35653f.
 

Chung, D.A., "Polyurethane Shock Absorbers." Ger. Offen. 2,435,078
 

(Goodyear Tire and Rubber Co.), February 20, 1975; Chemical
 

Abstracts 83, 44171z.
 

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

Co., Ltd., London, 1970.
 

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

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

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

Tests, Confidence Limits, and Adjustments for Stratification."
 

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

Grace, W.R. and Co., "Polyurethane Gaskets." British Patent 1,374,745,
 

November 20, 1974; Chemical Abstracts 83, 116347m.
 

Guthrie, J.L. and F.J. Rendulic, "Photocurable Triazine-Containing
 

Polyene-Polythiol Lacquer Composition." U.S. Patent 3,900,594
 

(Grace, W.R., and Co.), August 19, 1975; Chemical Abstracts 83,
 

195438t.
 

56
 
Hadidian, Z., T.N. Frederickson, E.K. Weisburger, J.H. Weisburger,
 

R.M. Glass, and S. Msntel, "Tests for Chemical Carcinogens.
 

Report on the Activity of Derivatives of Aromatic Amines, Nitrosamines, Quinolines, Nitroalkanes, Amides, Epoxides, Aziridines,
 

and Purine Antimetabolites." Journal of the National Cancer
 

Institute 4_1_(4): 985-1025, 1968.
 

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

Observations." Journal of the American Statistical Association
 

53_:457-481, 19587"
 

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

"Carcinogenesis Bioassay Data System." Computers and Biomedical
 

Research 7_: 230-248, 1974.
 

Miller, R.G., ^imul.tanepus Statistical Inference. McGraw-Hill Book
 

Co. , New York," 1966.
 

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

Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced
 

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

Ferric Oxide." Can£er_Researcli 32:1073-1079, 1972.
 

Stanford Research Institute, 1977 Directory of Chemical Producers,
 

U.S_.A.. Menlo Park, California, 1977,,
 

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

62:679-682, 1975.
 

57
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH 3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANAT'r
 

TABLE A1 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED 

WITH3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE 

... ... ,._!. , 

CONTROL (VfH) LOS COSE 

11-1095 11-1093 

ANIMALS INITIALLY IN STUDY 20 50 

ANIMALS NECROPSIED 20 50 

ANIMALS EXJBINED HISTOPATHOLOGICALIY ** 20 50
 

INTEGUHENTAPY SYSTEM
 

*SKIN (20) (50)
 NEOPLASM, NOS 1 (2%)
 EAPILLOMA, NOS
 SQDAMOOS CELL CARCINOMA 5 (10X)
 

EASAL-CELL TUNOE t (8%)
 TEICHOEPITHELIOHA 2 (IX)
 

SEBACEOUS ADENOMA 2 (IX)
 SEBACEOUS ADENOCAECINOMA 1 (2*)
 

KFPATOACANTHOMA 1 (5%) <t (8X)
 CARCIHOSAFCOMA
 

•SUBCUT TISSUE (20) (50)
 FIBROMA 1 <2X)
 FIBHOSARCOMA 1 (2X)
 

RESPIRATORY SYSTEM
 

*LUNG (20) (19)


ALVEOLAB/B80NCHIOLAR ADENOMA a (16*)
 CAECINOSARCOMA, METASTATIC
 

HFMATOPOIETIC SYSTEM
 

*MtHTIPLE ORGANS (20) (50)
 

MALIGNANT LYHPHOHA, NOS 1 12%)


LEUKEMIA,DOS 12 (24*)
 

UNDIFFERENTIATED LEUKEMIA 6 (12X)
 

tCERTICAL LYMPH NODE (17) (19)
 CARCINOSARCOMA, METASTATIC
 

CIRCULATORY SYSTEM
 

SQSI
 

* NUMBE R OF ANIMAL S SITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIEP 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-3 

HIGH DOSE
 11-1091
 

50
 

50
 

50
 

(50)
 

2 (IX)
 

1 (8%)
 

5 (10X)
 

1 (2%)
 

5 (10*)
 

1 (2%)
 

(50)
 

(17)
 

1 (2*)
 

(50)
 

1 (2%)
 7 <1«X )
 

9 (18X)
 

(12)
 

<2X)
 
TABLE A1 (CONTINUED)
 

CONTROL (VEH) LOH DOSE HIGH EOSE
 11-1095 11-1093 11-1091
 

DIGESTIVF SYSTEM
 

OLIVEP (20) (50) (50)
 

HEPATOCELIULAR ADENOMA 1 (5%) 6 (12%) 2 <4X)
 

HEPATOCELLOLAR CARCINOMA 2 («») 1 <2X)
 

•PANCR2AS	 (20) (19) (50)
 

ACINAR-CELL ADENOMA 1 <2X)
 

*STCPIACH (20) (49) (48)
 PAPILLOMA, NOS 1 (2%)
 

ISflALI INTESTINE (20) (19) (49)
 ADENOMATODS POLYP, NOS 1 (2X)
 

tCOION (19) (47) (46)
 ADENOCARCINOHA, NOS 1 (2%)
 

DEINARY SYSTEM
 

IKIDNFY/PELVIS (20) (50) (49)
 

TRANSITIONAL-CELL CARCINOMA 1 (5X)
 

ENDOCRINE SYSTEM
 

tPITUITABY (16) (45) (36)
 CHROMOPHOB? ADENOMA 3 (19X) 7 (16%) 2 (6%)
 

*APRENAL (20) (50) (49)
 PHEOCHROMOCYTOMA 1 (20%) 3 (6%)
 

•THYROID	 (19) (45) (46)
 

FOLLICOLAR-CELL ADENOMA 1 (5%)
 

FOLLICOLAR-CELL CARCINOMA 1 (2%)
 C-CELL ADENOMA 1 (5%) 1 (2%)
 C-CELL CARCINOMA 2 <4X)
 

CYSTADENONS. NOS 1 (2%)
 

•PANCREATIC ISLETS (20) (49) (50)
 

ISLET-CELI ADENOMA 1 (5%) 1 (2%)
 

REPRODUCTIVE SYSTEM
 

*PREP(JTIAL GLAND (20) (50) <5<M
 SEBACEOBS ADENOCARCINOMA 1 {2%) 3_J6il
 

i NUMBER OP ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMAL S NECROPSIED
 

A-4
 
TABLE A1 (CONTINUED) 

CONTROL (VEH) LOW DOSE HIGH COSE 

11-1095 11-1093 11-1091 

tTESTIS (20) (50) (U9)
 

INTEBSTITIAL-CELL TUBOB 17 (85%) 39 (78X) 36 (73*)
 

*EPIDIDYMIS (20) (50) (50)
 

FIBFOSAECOMA, INVASIVE 1 (2X)
 

NFPVCUS SYSTEM
 

tBBAIN (19) (50) («7)
 

SQDABOUS CELL CARCINOMA, INVASIV 1 (2%)
 

GLIOBA, NOS 1 (2%)
 

ASTROCYTOBA 1 (5*)
 

CLIGODENDBOGLIOMA 1 (2*)
 

SPECIAL SENS! ORGANS
 

*EAF (20) (50) (50)
 

SQUAMOUS CELL CARCINOMA 1 (2%)
 

*ZYMBAL'S GLAND (20) (50) (50)
 

SQUAMOUS CELL CARCINOMA 2 (HX) 5 (10%)
 

SEBACEOUS ADENOCARCINOHA 3 (6X) 2 (<»%)
 

HUSCOIOSKELETAL SYSTEM
 

NONS
 

BODY CAVITIES
 

*MEDtASTINUM (20) (50) (50)
 

SAFCOMA, NOS 1 (2%)
 

*ABCO«INAL CAVITY (20) (50) (50)
 

LIPOBA 1 (2«)
 

ALL OTHER SYSTEMS
 

*MULTIPLE ORGANS (20) (50) (50)
 

CAFCINOSARCOMi
 

t NUBBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NOMBEF OF ANIMALS NECEOPSIFD
 

A-5
 
MESOTHELIOMA, NOS
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 NATURAL DEATHS
 

MORIBUND SACRIFICE
 SCHEDULED SACRIFICE
 

ACCIDENTALLY RULED
 TERMINAL SACRIFICE
 ANIMAL HISSING
 

INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMAR Y
 

TOTAL ANIHALS WITH PRINARl TOBORS*
 TOTAL PRIMARY TKCOST
 

TOTAL ANIMALS WITH BENIGN TUMORS
 TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMCRS
 

TOTAL MALIGNANT TUMORS
 

TOTAL ANIMALS WITH SECONDAPY TUMORS*
 TOTAL SECONDARY TDMORS
 

TOTAL ANIHALS WITH TUMORS USC?PTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMAL S HITH TUMOHE UNCERTAIN­ PBINAPY OF HETASTRTIC
 

TOTAL UNCERTAIN TUMORS
 

TABLE Al (CONCLUDED)
 

CONTROL (VEH)


11-1095


20


3


2


15
 

18
 

31
 

18
 

29
 

2
 

2
 

 LOW DOSE HIGH DOSE 11-1093 11-1091 

1 (2*) 

 50 50 

 16 12 

 12 22 

US 16 

119 95 

U5 13 

80 56 

30 31 38 38 

* PRIMARY TUMOSS: ALL TUMORS EXCEPT SECONDARY TUMORS
 * SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

A-6
 
TABLE A2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED 

WITH3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE 

CONTROL (VEH) 10* DOSE HIGH COSE
 

11-1096 11-109U 11-1092
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

ANIHALS NECPOFSIED 20 50 US
 ANIMALS EXAMINED HISTOPATHOLOGICALIY ** 2" SO U8
 

INTEGUBENTAB Y SYSTEM 

»SKI N (20) (5P) 

NEOPLASM, NOS 1 (2%) 

SQUAHOOS CELL CABCINOBA 1 (2X) 

BASAL-CELL TUMOB 1 (2«) 

TBICHOEPITHELIOHA 

SEBACEOUS ADENOMA 1 <2X) 

SEBACEOUS ADENOCABCINOMA 1 (5«) 1 (2«) 1 (2%) 

CARCINOSABCOMA 1 (5%) 

*SUBCDT TISSUE (20) (50) 

FTBBOSABCOWA 1 (2%) 

IIPOHA 1 (2X) 

PESPIBATORY SYSTEM
 

*HASAL CAVITY (20) (50) (48)
 

SQUAMOOS CELL CABCINOMA, INVASIV 1 (2X)
 

ILONG (20) («8) (US)
 

SQUAMOUS CELL CAECIMOMA, HETASTA 1 (2X)
 ADENOCAHCIHOMA, NOS, METASTATIC 1 (2X)
 

ALVEOLAR/EBONCHIOLAH ADENOMA 1 (5%) 2 (IX) 1 (2»)
 

SEBACEOUS ADENOCAECINOMA. METAST 1 (2%)
 

TIBROSABCCMA, HFTASTATIC 1 (2X)
 

CABCINOSABCOMA, HETASTATIC 1 (2X)
 

HEMATOPOIETIC SYSTEM
 

•MULTIPLE ORGANS	 (20) (50) (U8)
 MALIGNANT	 LYHPHONA, NOS 1 (2%)
 

6 (13V
 

* NUMBER OF ANIMAL S SITH TISSUE EXAMINE D MICBOSCOPICALlt 

* NUMBER OP ANIMALS NECROPSIED 

*«EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-7 
TABLE A2 (CONTINUED) 

UNDIFPEBENTIATED LEUKEMIA 

tBONE MARROW 

LEUKEMIA, NOS 

ISPLEEN 

UNDIFFERENTIATED LEUKEMIA 

CONTROL (VEH)

11-1096

1 (5*)

(19)

(20)

 LOW DOSE 11-1091 

3 <6X) 

 (15) 

 (18) 

HIGH COSE 11-1092 

6 (13*) 

(15) 

1 (2*) 

(18) 

1 (2%) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

iLIVER 

HEPATOCELLULAR ADENOMA 

fPANCREAS 

ADENOCARCINOMA, NOS 

tSTCHACS 

SQUAMOOS CELL CARCINOMA 

fSMALL INTESTINE 

CYSTADENOCARCINOMA, NOS 

tILEUM 

ADENOMATOUS POLYP, NOS 

tCOION ADENOCARCINOMA, NOS 

(20)

(20)

(20)

(20)

(20)

(18)

 (50) 

 (18) 

1 (2*) 

 (50) 

1 (2%) 

 (50) 

 (50) 

 (15) 

1 (2*) 

(18) 

2 (1*) 

(18) 

(18) 

1 (2%) 

(18) 

1 (2%) 

(18) 

1 (2*) 

(17) 

1 (2X) 

DRINARY SYSTBB 

tKIDNEY 

TUBULAR-CELL ADENOMA (20) (19) 

1 (2X) 

(18) 

ENDOCRINE SYSTEM 

iPITUITARY 

NSO PLASM, NOS 

(18) (18) 

f NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

("5) 

1 .12*1 . . . _, , 

A-8 
TABLE A2 (CONTINUED)
 

HIGH COSE
 11-1092
 

8 (18%)
 

(47)
 

1 (2%)
 

(41)
 

(48)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

4 (8%)
 

(48)
 

4 (8%)
 

(48)
 

1 (2%)
 

(48)
 

1 (2%)
 

10 (21%)
 

(48)
 

1 (2%)
 

(48)
 

1 (2%)
 

CHROMOPHOBE ADENOMA
 

*ADRENAL
 

CORTICAL ADENOMA
 FHFOCHROMOCYTOMA
 

•THYROID
 FOLLICOLAR-CELL ADENOMA
 C-CELL ADENOMA
 

C-CELL CARCINOMA
 

REPRODUCTIVE SYSTEM
 

*MAMHARY GLAND
 ADENOMA, NOS
 ADENOCARCINOMA, NOS
 

CYSTADENOHA, NOS
 FIBROADENOMA
 

*PHHPUTIAL GLAND
 

SQOAMOOS CELL CARCINOMA
 

SEBACEOOS ADENOCARCINOMA
 

*VAGINA
 PAPILLOMA, NOS
 

IUTF.ROS
 

SQOAMOOS CELL CARCINOMA
 

ADENOCARCINOMA, NOS
 

IEIOMYOMA
 

LEIOMYOSARCOMA
 

INDONETRIAL STPONAL POLYP
 

*UT!PUS/!NDOMETRIU«
 

CARCINOMA, NOS
 

ADENOCAPCINOMA, NOS
 

•OVARY
 

CYSTADENOCARCINOMA, NOS
 

NERVCOS SYSTEM
 

IBRAIN
 

SEBACEOOS ADENOCARCIHONA, INYASI
 

CONTROL (VHH)
 

11-1096
 

* (22%)
 

(20)
 

1 (5%)
 

(18)
 

(20)
 

(20)
 

(20)
 

(20)
 

(20)
 

1 (5%)
 

(20)
 

(20)
 

LOR DOSE
 11-109H
 

8 (17%)
 

(U9)
 

1 (2%)
 

1 (2%)
 

(UU)
 

1 (2%)
 

2 (5%)
 

1 (2%)
 

(50)
 

1 (2%)
 

5 (10%)
 

(50)
 

1 (2%)
 

^ (14%)
 

(50)
 

(48)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

5 (10%)
 

(48)
 

(U7)
 

(50)
 

* NDNBER OF ANIMALS »ITH TISSUE EXAMINED MICROSCOPICALLY
 

* SOMBER OF ANIMALS NECROPSIED
 

A-9
 
EPENDYMONA 

SPECIAL SENSE ORGANS
 

*ZYMBAL'S GLAND
 

SQOANODS CELL CARCINOMA
 

SEBACEOUS ADENOCARCINOMA
 

CARCINOSARCOHA
 

MOSCULOSKELFTAl SYSTEM
 

NONE
 

BODY CAVITIES
 

NONF
 

ALL OTHER SYSTEMS
 

•MULTIPLE ORGANS
 

ADEHOCAHCINONA, NOS, HETASTATIC
 

CARCINOSARCOMA
 

ANIBAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

ANIMAL DELETED (WRONG SEX)
 

9 INCLUDES_AUTQLTZEE ANIMALS
 

TABLE A2 (CONTINUED) 

COHTROL (VEH) 

11-1096 

(20)
 

1 (5*)
 

(20)
 

15
 

LOW DOSB HIGH COSE 

11-1094 11-1092 

1 (2«)
 

(50)
 

1 (2*) t (8*)
 

7 2 <«*)
 

(50)	 (18)
 

1 (2*)
 

1 (2*)
 

50 50
 

9 16
 

20 12
 

21 21
 

1
 

I NUMBER OF ANIMALS HITH TISSOE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-10
 
TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TDMOBS*
 TOTAL PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TUMORS
 TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TDMORS
 TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORSt
 TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS WITH THMOHS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

TABLE A2 (CONCLUDED)
 

CONTROL (VEH)


11-1096


 10
 11


 6


 6


 5
 5


 LOB DOSE HIGH EOSB 11-1091 11-1092 

 UO <t« 63 69 

 22 26 29 33 

 27 30 

 3M 3U 

5 2 

6 2 

2 

2 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

A-ll
 

APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH 3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATE
 

TABLE Bl 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH 

3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE 

~ CONTROL (ONTR) 

22-2095 LOB DOSE 22-2093 HIGH EOS? 22-2091 

ANIMALS INITIALLY IN STUDY 

ANIMALS HISSING, 

ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTOPATHOLOGIC ALLY 

20 

1 

19 ** 19 

50 

5 

I)U 

1IM 

50 

50 

50 

INTEGDMENTAR Y SYSTEM 

*SUECUT TISSUE (19) (UK) (50) 

LIPOMA 1 (2X) 

RESPIRATORY SYSTEM
 

tLUNG (19)
 

HEPATOCELLULAH CARCINOMA, METAST 2 (5X)
 

ALVEOLAB/EHONCHIOLAR ADENOMA 1 (5%) 8 (19X) 7 (15*)
 

ALVEOLAR/ERONCHIOLAR CARCINOMA 1 (5*)
 

HEBATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (19) (50) 

MALIGNANT LYMPHOHA, NOS 2 (5*) 1 (2*) 

MALIG.LYMPHCMA. HISTIOCYTIC TYPE 1 (2%) 

LEUKEMIA,NOS 1 (2«) 

tSPIEEN (18) (37) (15) 

HEMANGIOSARCOMA 1 (2*) 

*MESENTERIC L. NODE (18) (36) (38) 

MALIGNANT LYHPHOMA, NOS 1 (3*) 

MALIGNANT LYMPHOMA, BIXZB TYPE 1 (3K) 

tAXILLARY LYMPH NODE (18) (36) (38) 

MALIG.LYBPHOMA, LYMPHOCYTIC TYPE 

CIRCULATORY SYSTEM
 

•HEART	 (19) (U8)
 

HEMANGIOSARCOHi L.J2S1.
 

t NUMBE R OP ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OP ANIMAL S NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-3 
TABLE Bl (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH EOSE 

22-2095 22-2093 22-2091 

DIGESTIVE SYSTE M 

*LIVEE (19) (43) (18)
 

HEPATOCELLOLAR ADENOMA 1 (5%) 3 (7X) U (8*)
 

HEPATOCELL'JLAF CARCINOMA 5 (12S) 6 (13%)
 

• STOMACH (15) (41) (49)
 

JDENOPIATOnS POLYP, NOS 1 (7*)
 

URINARY SYSTEB
 

NOSE
 

ENDOCRINE SYSTEM
 

IAORENAL (13) (25) (36)
 

CORTICAL ADENONA 1 (31)
 

BEPHCEtlCTIVE SYSTEM
 

tTESTIS (19) (42) (48)
 

ISTEHSTITIAL-CELL TUHOR 3 (16«) 1 (2*)
 

NEEVCDS SYSTEH
 

NONE
 

SPECIAL SENS'? ORGANS
 

NON"
 

KDSCUIOSKELETAl SYSTEM
 

N3NE
 

BODY CAVITIES
 

NPJE. , ,
 

* NUMBER OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NOMBEP OF ANIMALS NECHOPSIED
 

B-4
 
TABLE Bl (CONCLUDED)
 

ALL CTHER SYST5MS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 NATURAL DEATHS


MORIBUND SACRIFICE
 SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED


TERMINAL SACRIFICE


ANIMAL MISSING


8 INCLUDES AUTOLYZED ANIMALS
 

TUMOE SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*


TOTAL PRIMARY TUMOBS


TOTAL ANIMALS WITH BENIGN TUMORS
 TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMC8S
 TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMCRS*
 TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL (UNTR)


22-2095


 20
 1


 18


 1


 7
 7


 6


 6


 1
 1


 LOW DOSE HIGH EOSE 22-2093 22-2091 

 50 50 2 2 

3 1 

1 

 »0 16 

5 

 17 21 22 2<1 

 11 12 13 12 

8 10 

9 12 

2 

2 

* PRIMARY TUMOHS: ALL TUMORS EXCEPT SECONDARY TUMORS
 # SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-5
 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED 

WITH 3,3'-DIMETHOXYBENZIDINE-4,4'-DlISOCYANATE

 

CONTROL(UNTR) LOW DOSE 

22-2096 22-2094 

ANIMAL S INITIALLY IN STUDY 2"

ANIMALS MISSING

ANIMALS NECROPSIED 20

ANIMALS EXAMINED HISTOPATHOLOGICALIY** 20

 50 

9 

 41 

 41 

INTEGUMENTARY SYSTEM
 

*SUBCDT TISSUE (20) (1*1)
 

SARCOMA, NOS
 

PFSPIBATORY SYSTEM
 

tLUNG (20) (1*1)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 3 (15X) 2 (5X)
 

HEMATOPOIETIC SYSTEM
 

'MULTIPLE ORGANS (20) (11)
 

MALIGNANT LYMPHOMA, NOS 2 (10%) 5 (12*)
 

MALIG.LYMPHOMA, LYHPHOCYTIC TYPE 1 (2X)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYEE
 

LEUKEMIA, NOS 3 (7»)
 

tSPLEEN (18) (41)
 

MALIGNANT LYMPHOMA, NOS
 

tMESENTERIC L. NODE (20) (40)
 

MALIGNANT LYMPHOMA, NOS 1 (5%)
 

*DUCDENOH (20) C41)
 

MALIG.LYHPHOHA, LYMPHOCYTIC TYPE
 

CIFCOLATOHY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tSAIIVARY GLAND (14) (30)
 

I NUMBE R OP ANIMAL S WITH TISSDE EXAMINE D MICROSCOPICALLY 

* NOMBEF OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

HIGH COSE
 22-2092
 

50
 

7
 

43
 

42
 

(13)
 

1 (2X)
 

(40)
 

1 (3*)
 

(43)
 

3 <7X)
 

1 (2X)
 

3 (7X)
 

1 (2X)
 

(U1)
 

1 <2X)
 

(37)
 

(38)
 

1 (3X)
 

(40)
 

B-6 
TABLE B2 (CONTINUED) 

CONTROL(UNTR)

22-2P96

 LOW DOSE

 22-2091

 HISH EOS1 

 22-2092 

tLIVER

ADENOCARCINOMA. NOS, METASTATIC

HEPATOCELLULAR ADENOMA

HBPATOCELLULAR CARCINOMA

tCECUM

LEIOSYOMA

 (20)

 1 (5*) 

 (19)

 («1)

 1 (2X) 

1 (2X) 

 C*0)

 1 (3X) 

 (12) 

 (37) 

URINARY SYSTEB 

NONE 

ENDOCRINE SYSTEB 

»PITOITARY

CHRONOPHOBE ADENORA

 (8) (13)

 1 (8X) 

 (25) 

REPRCCDCTIVH SYSTEB 

*NAH!IARY GLAND

ADENOCARCINOMA, NOS

tOTEROS

ADENOCARCINONA, NOS

 (20)

 (19)

 (41)

 1 (2X)

 (40)

 1 (3X) 

 (U3) 

1 (2%) 

 (37) 

NERVCDS SYSTEB 

NONE 

SPECIAL SENSE CRGANS 

NONE 

NDSCULOSKELETAL SYSTEB 

NONE 

BODY CAVITIES 

•MESENTERY (20) 

t NUMBER OF ANIMALS SITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

(U3) 

1 

B-7
 
TABLE B2 (CONCLUDED)
 

CONTROL (UNTR) LOU DOSE HIGH DOSE
 22-2096 22-2091 22-2092
 

ALL OTHER SYSTEMS
 

HONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 20 50 50
 NATURAL DEATHS 2 3 7
 MORIBUND SACRIFICE 2 5
 SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 TERMINAL SACRIFICE 18 36 31
 ANIMAL MISSING 9 7
 

a INCLUDES AtlTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS KITH PRIMARY TUMORS* 6 13 13
 TOTAL PRIMARY TUMORS 7 17 14
 

TOTAL ANIMALS WITH BENIGN TUHORS U 14 2
 TOTAL BENIGN TUHORS U 4 2
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 3 11 11
 TOTAL MALIGNANT TUMORS 3 13 12
 

TOTAL ANIMALS WITH SECONDARY TUHCRSt 1
 TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR HETASTATIC
 TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDAFY TDMORS
 * SECONDARY TDMOBS: METASTATIC TUMOBS OB TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-8
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTICJ
 

LESIONS IN RATS TREATED WITH 3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATE
 

TABLEC1

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

TREATED WITH 3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE

 

CONTROL (VEH) LOB DOSE HIGH DOSE
 

11-1095 11-1093 11-1091
 

ANIMALS INITIALLY IV STUDY 20 50 50
 

ANIMALS NECROPSIED 20 50 50
 ANIMALS EXAMINED HISTOPATHOLOGICSLLY** 20 50 50
 

INTEGUMENTAR Y SYSTE M 

NONE 

RESPIRATORY SYSTEM 

*TRACHEA (20) (149) (U6)
 INFLAMMATION, NOS 1 (5%)
 

ILUNG (20) (H9) («7)
 PNEUMONIA, ASPIRATION 2 (1*)
 INFLAMMATION, SOPPURATIVE 1 (2)1)
 ERONCHOPNEUMONIA, ACUTE 1 (2*)
 

PNEUMONIA, CHRONIC MURINE 3 (15%) 7 (1K») 6 (13*)
 INFLAMMATION, CHRONIC 1 (2*) 1 (2%)
 INFLAMMATION, CHRONIC SUPPURATIV 1 (2*)
 HYPERPLASIA, ALVEOLAR EPITHELIUM 1 (2*)
 

ILUNG/ALVEOLI (20) (149) («7)
 PNEUMONIA, ASPIRATION 1 (2*)
 

HFMATOPOIETIC SYSTEM 

ISP1EEN (19) (19) («9)
 

HEMOSIDEHOSIS 2 (UX) 2 (1%)
 

HYPERPLASIA, NODULAR 1 (2*)
 

HEMATOPOIESIS 1 (2K) 1 (2*)
 

tMANDIBULAP L. NODE (17) (H9) (U2)
 

HYPERPLASIA, PLASHA CELL 1 (2K)
 

tMESENTEHIC L. NODE (17) (49) (12)
 

____ HYPERPLASIA, LYHPHOID
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-3
 
TABLE CI (CONTINUED)
 

HIGH DOSE
 1 1-1091
 

(46)
 

(46)
 

4 (9*)
 

(50)
 

2 (4*)
 

1 (25)
 

1 ;2*j
 

4 (8*)
 

1 ( 2%)
 

1 (2*)
 

1 (2*)
 

(50)
 

1 (2*)
 

(50)
 

(50)
 

(48)
 

1 (2*)
 

(49)
 

1 (2%)
 

(49)
 

1 (2*)
 

(49j
 

CIRCULATORY SYSTEM
 

•HEART
 

THROMBOSIS, NOS
 

INFLAMMATION, CHRONIC
 

tflYCCASDIUM
 

INFLAMMATION, CHRONIC
 

FIB80SIS
 

FIBROSIS, DIFFUSE
 

DEGENERATION, NOS
 

DIGESTIVE SYSTiH
 

*LTVER
 

NECROSIS, I'OCAL
 

NECROSIS, DIFFUSE
 

NFCROSIS, COAGOLATIVE
 

KETAflORPHOSIS FATTY
 

GLYCOGENIC CELL
 

HYPERPLASIA, SODULAR
 

HYPERPLASTiC NODCI.E
 

HEMATOPOIESIS
 

*LIVER/HEPATOCYTES
 

HYPERPLASIA, FOCAL
 

*BILE DUCT
 

HYPEFPLftSIA, NOS
 

tPANCREAS
 

HBROSIS, FOCAL
 

tSTOMACH
 

HYPEBKERATOSI3
 

ISMALL INTESTINE
 

OLCER, NOS
 

tPEYERS PATCH
 

ABSCESS, NOS
 

IDUCDENUM
 

UIC.E.S* NOS _
 

CONTROL (VEH)
 1 1-1095
 

(19)
 

(19)
 

1 (31)
 

1 (5%)
 ? (11*)
 

(20)
 

(20)
 

(20)
 

1 (5!S)
 

(20)
 

(20)
 

(20)
 

(20)
 

(20)
 

LOB DOSE
 11-1093
 

(48)
 

1 12%)
 

2 (U%)
 

(US)
 

2 C* %)
 

2 14*)
 

1 (2*)
 

(50)
 

2 CiS)
 

4 (8X)
 

1 (2%)
 

1 (?X)
 

(50)
 

(50)
 

(49)
 

3 (6*)
 

(49)
 

(49)
 

(49)
 

(49)
 

1 J2X1 _
 

t NUMBER OF ANIHALS KITH TISSOE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-4
 
TABLE Cl (CONTINUED)
 

tLARGE INTESTINE
 

NEBATODIASIS
 

ICOIOH
 

HBBATODIASIS
 

URINARY SYSTEB
 

tKIDNEY
 

HYD80NEPHROSIS
 

INFLAHBATION, CHRONIC
 

GRANULOBA, NOS
 

EIGBENTATIOM, NCS
 

ENDOCRINE SYST5B
 

*PITOITARY
 

CYST, NOS
 

HYPEHPLASIA, FOCAL
 

HYPERPLASIA, CHEOBOPHOBE-CEII
 

tADRBNAL
 

IIPOIDOSIS
 

tADSENAL CORTEX
 

HYPERPLASIA, NOS
 

tADRENAL BEDULLA
 FIBROSIS
 

HYPERPLASIA, SOS
 

tTHYROID
 

COLLOID CYST
 

FOLLICDLAR CYST, NOS
 

HYPERPLASIA, C-CELL
 

tPANCREATIC ISLETS
 

HYPERPLASIA, HOS
 

HYPEBPLASIA, ADENOBATOUS
 

BEPRCDOCTIVF SYSTIB
 

*BABBABY GLAND
 

BULIILgCULAR CYST
 

CONTROL (VEH)
 11-1095
 

(19)
 

7 (37H)
 

(19)
 

(20)
 

1 (5X)
 

13 (65X)
 

(16)
 

2 (13%)
 

(20)
 

(20)
 

(20)
 

2 (10X)
 

(19)
 

(20)
 

(20)
 

LOW DOSE HIGH DOSE
 

11-1093 11-1091
 

(U7) ("6)
 

1 (2%)
 

(U7) (I46)
 

14 (30*) 6 (13»)
 

(50) (49)
 

23 (U6X) 19 (39X)
 

1 <2X)
 

1 <2X)
 

(15)	 (36)
 

1 (2X) 1 (3X)
 

3	 (7X)
 

(50)	 (<*9)
 

1 (2%)
 

(50)	 (U9)
 

1 <2X)
 

(50) (49)
 

1 (2%)
 

3 (6X)
 

(l»5) (1*6)
 

1 (2X)
 

1 (2X)
 

5 (11X) 1 (2%)
 

(19)	 (50)
 

1 <2X)
 

1 (2X)
 

(50)	 (50)
 

LJ2X1
 

» NOBBER OF AHIBALS SITH TISSUE EXABINED BICEOSCOPICALLY
 

» NDBBER OF ANIBALS NECROPSIED
 

C-5
 
1 ABLE Cl (CONCLUDED)
 

CONTROL (VEH) LOW DOSE HIGH COSE
 11-1095 11-1093 11-1091
 

•PROSTATE	 (16) (46)
 INFLAHMATION, NOS 1 (2*)
 HYPERPLASIA, NOS 1 (2*)
 HYPERPLASIA. CYSTIC 3 (7«)
 

*SEHINAL VESICLE (20) (50) (50)
 CAST, NOS 1 (5X)
 INFLAMMATION, SOPPDRATIVE 1 (2X)
 HYPERPLASIA. CYSTIC
 

*TESTIS (20) (50) (49)
 ATEOPHY, NOS 4 (20K) 2 (4X)
 

SEBVCOS SYSTEM
 

*SBAIN (19) (50) (47)
 ATROPHY, NOS 2 (4«)
 ATROPHY, PRESSURE 1 (5«)
 

SPECIAL SENSE ORGANS
 

NONT
 

MDSCOLOSKELETAI SYSTEH
 

NONE
 

BODY CAVITIES
 

*INGDINAL REGION (20) (50) (50)
 

K7CFOSIS, FAT 1 (2X)
 

ALL OTHER SYSTEHS
 

ADIPOSE TISSUE
 NECROSIS, FAT 1 2
 

SPECIAL MORPHOLOGY SDBHARY
 

AOTO/NECEOPSY/HISTO PERF	 1
 

I NOH8ER OF ANIMALS WITH TISSHE EIAHINED MICROSCOPICALLY
 * NDHEER OP ANIMALS NBCROPSIED
 HSAC: MORIBUND SACRIFICE OTHR: OTHER
 

C-6
 
TABLE C2 

SUMMARY OF THE INCIDENCE OF NONNEOPIASTIC LESIONS IN FEMALE RATS 

TREATED WITH 3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE 

CONTROL (VEH)

11-1096

 LOW DOSE

 11-1091

 HIGH EOSE 

 11-1092 

ANIMALS INITIALLY IN STUDY 20

ANIMALS NECHOPSIED 20

ANIMALS EXAMINED HISTOPATHOLOGICALIY** 20

 50

 50

 50

 50 

 18 

 18 

INTEGUMENTARY SYSTEM
 

•SKIN	 (20) (50) (18)
 EPIDERMAL INCLUSION CYST 1 (5X)
 

*SUECUT TISSUE (20) (50) (18)
 

HEMOBRHAGIC CYST 1 (2*)
 

RESPIRATOR Y SYSTE M 

*LARYNX (20) (50) (18)
 INFLAMMATION, CHRONIC 1 (2%)
 

•TRACHEA	 (20) (51) (17)
 

INFLAMMATION, CHRONIC SUPPURATIV 1 (2«)
 

• LONG (20) (18) (18)
 

PNEUMONIA, ASPIRATION 1 (2X)
 PNEUMONIA, CHRONIC HUBINE 7 (35X) 16 (33t) 11 (23%)
 

HYPERPLASIA, ADENOMATOUS 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

tBONE MARROW (19) (15) (15)
 

HYPEBPLASIA, NOS 1 (2*)
 

iSPLEEN	 (20) (18) (18)
 HEHOSIDEROSIS 2 («*) 2 (1«)
 

HYPEEPLASIA, HETICULOM CFLL 1 (211)
 

HEMATOPOIESIS 7 (15») 1 (2%)
 

*CERVICAL LYMPH NODE	 (20) (15) (18)
 

* NUMBER OF ANIMAL S KITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIFD 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-7
 
TABLE C2 (CONTINUED) 

—_ _ — —'———— ——
 

CONTROL (VEH) LOW DOSE HIGH DOSE
 11-1096 11-109U 11-1092
 

CIRCULATORY SYSTEM
 

tHYCCARDIOB (18) (49) (US)
 FIBROSIS 1 (6*) 1 (2X) 2 (1*)
 FIBROSIS, FOCAL 1 (2*)
 DEGENERATION, NOS 1 (2*)
 

DIGESTIVE SYSTIB
 •


ILIVER (20) (50) (48)
 CYST, SOS 1 (2*)
 HEBOSRHAGIC CYST 1 (2*)
 NECROSIS, NOS 1 (5*)
 NECROSIS, FOCAL t (8*)
 NECROSIS, BIDZONAL 1 (2*)
 METAMORPHOSIS FATTY t (8*) « (8*)
 HYPERPLASIA, NODULAR 1 (2*)
 HYPERPtASIA, FOCAL 1 (2*)
 

tLIVBR/CENTRILOBULAR (20) (50) («8)
 BETABORPHOSIS FATTY 1 (2K)
 

*9IIE DDCT (20) (50) (»8)
 HYPERPLASIA, NOS 1 (2X)
 

*PANCBEAS (20) (K8) (48)
 FIBROSIS, FOCAL 2 (10*) 1 (2*)
 ATROPHY, FOCAL 1 (2*)
 

tSBAIL INTESTINE (20) (50) (18)
 BYPERPLASIA. LYBPHOID 1 (2*)
 

tLARG? INTESTINE (18) (US) <K7)
 NEKATODIASIS 2 (11*) « (9X) 2 («*)
 

IC010N (18) (15) (17)
 NEBATODIASIS 2 (11*) "» (9*) 3 <6X)
 

URINARY SYSTF.B
 

•KIDNEY	 (20) («9) (t8)
 INFLAHBATION. CHRONIC 5_JL25*J 5 JJ511 1U (29*)
 

* NOBBER OF ANIBALS WITH TISSOE EXABINED BICROSCOPICALLY
 * NOMBEB OF ANIMALS NECROPSIED
 
TABLE C2 (CONTINUED)
 

CONTROL (VIH) LOS DOSE
 

11-1096 11-1191*
 

SORINARY BLADDER (12) (36)
 HYPERPLASIA, FPITBELIAL 1 (3*)
 

ENDOCRINE SYSTEM
 

tPITUITARY (18) («8)
 

CYST, NOS 1 (2*)
 HYPFBPLASIA, FOCAL 1 (6*)
 

HYPERPL1SIA, CHROMOPHOBE-CELI
 

*ADRENAL (20) <«9)
 

NECROSIS, FOCAL 1 (2*)
 

tADRENAL MEDULLA (20) (K9)
 HYPERPLASIA, SOS 1 (5*) 1 (2*)
 

*THYROID (18) (11)
 HYPERPLASIA, C-CELL 3 (7%)
 HYPERPLASIA, FOLIICULAR-CBLL 1 (2X)
 

REPRODUCTIVE SYSTEM
 

*MAM«AR Y GLAND (20) (50)
 HYPERPLASIS, NOS
 

»UTERUS (20) (IB)
 

HYDROHETRA
 

INFLAMMATION, SDPPURATIVE 1 (5*) 1 (2*)
 EYOMETRA 3 (6X)
 

INFLABMATIOS, ACDTE
 

INFLAMMATION ACUTE AND CHRONIC
 INFLAMMATION. CHRONIC
 

HYPERPLASIA, ADENONATODS 1 <5X)
 

ICERVIX UTERI (20) («8)
 

INFLAMMATION ACUTE AND CHRONIC
 

IUT5ROS/ENDOHETRIDB (20) (18)
 

INFLAMMATION, NOS 1 (2«)
 

INFLAMMATION, SDPPUEATIVE 2 (10*) 2 (it*)
 

INFLAMMATION, ACUTE 1 (5*) 3 <6X)
 

IJiFLiMJJA^ION ACOTE AND CHFOJJIC 1 J2*L
 

t NOMBEB OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANISALS NECHOPSIED
 

HIGH DOSE
 

11-1092
 

(37)
 

(15)
 

2 (HX)
 

3 (7X)
 

(17)
 

(1*7)
 

(11)
 

(«8)
 

1 (2%)
 

(18)
 

1 (2X)
 

1 (2%)
 2 («X)
 

1 (2%)
 

1 (2X)
 1 (2X)
 

1 (2%)
 

(18)
 

1 (2X)
 

(18)
 

1 (2X)
 2 (IS)
 

3 (6X)
 

..... . .
 

C-9
 
TABLE C2 (CONTINUED)
 

CONTROL (VEH) LOH DOSE HIGH COSE
 

11-1096 11-109U 11-1092
 

INFLAHHATICN, CH80NIC 1 (2*)
 HYPERPLASIA, NOS 2 («X) 1 <2X)
 HYPEBPLASIA, CYSTIC 1 (5X) « (3%) 6 (13*)
 

tOVARY/OVIDOCT (20) (US) (US)
 ABSCESS, NOS 1 (2*)
 

IOVAHY (20) (HI) (18)
 CYST, NOS 3 (15%) 2 (a*) 3 (6*)
 

FOLLICULAR CYST, NOS 1 (2%)
 fAROVABIAN CYST 1 <2X)
 INFLAHMATION, SUPPORATIVE 1 (2X)
 

NEBVOUS SYSTEM
 

•BRAIN	 (20) (50) (U8)
 ATROPHY, PRESSURE 2 (10X) 1 12%)
 

SPECIAL SENSE CRGANS
 

NONE
 

WUSCOLOSKELETAL SYSTEH
 

NONE
 

BODY CAVITIES
 

•ABDOMINAL	 CAVITY (20) (50) (48)
 NECROSIS, FAT 2 (4X)
 

*PEBITONEOH (20) (50) (18)
 NECROSIS, FAT 2 (4X)
 

•PLEURA	 (20) (50) (48)
 GRANULOMA, NOS 1 (5X)
 

•MESENTERY	 (20) (50) (48)
 CYST, NOS 1 (2X)
 

ALL OTHFB SYSTEMS
 

•MULTIPLE OBGANS (20) (50) (48)
 PIGMENTATION. NOS 1 J2X1 1 J2X)
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-10
 
TABLE C2 (CONCLUDED)
 

CONTROL (VEH) LOW DOSE HIGH EOSE
 11-1096 11-1091 11-1092
 

ADIPOSE TISSUE
 STEATITIS
 NECROSIS. FAT
 

SPECIAL MORPHOLOGY SUHHARY
 

NO LESIOH REPORTED 2 1
 

AOTO/NECHOPSY/HISTO PERF 1
 

AOTOLYSIS/NO NECROPSY
 

HISS: BISSIHG TSAC: THRHINAL SACRIFICE
 

HSAC: HOH1BDND SACRIFICE OTHR: OTHER
 

C-ll
 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH '3,3'-DIMETHOXYBENZIDINE4,4'-DIISOCYANATE
 

TABLE Dl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

TREATED WITH 3,3'-DIMETHOXYBENZIDINE-4,4'-DIISOCYANATE

 

CONTROL (UNTR)

22-2095 

ANIMALS INITIALLY IN STUDY 

ANIMALS MISSIN3 

ANIMALS NECROPSIES 

20 

1 

19 ANIMALS EXAMINED HISTOPATHOLOGICALIY ** 19
 

INTEGUMENTARY SYSTEM
 

*S(UN
 

INFLAMMATION, CHRONIC FOCAL
 

*SUBCUT TISSUE
 

ABSCESS, NOS
 

RESPIRATORY SYSTEM
 

ILUNG
 

ATELECTASIS
 

CONGESTION, NOS
 

HEMORRHAGE
 

PNEUMONIA, LIPIB
 

ABSCESS, NOS
 

PEBIVASCULAE CI3FFING
 

SLVEOLAP MACRCPHAGES
 

HISTIOCYTOSIS
 

HEMATOPOIETIC SYSTEM
 

tSPLEFN
 

CONGESTION, NOS
 

HYPE8PLASIA, RETICULUM CELL
 

HYPERPLASIA, LYMPHOID
 

•LYMPH	 NODE
 

HYPERPLASIA, NOS
 

HYPEBPLASIA, LYMPHOID
 

*MESENTERIC L. NODE
 

t NUMBE R OF ANIHAL S KITH TISSUE

* NUMBE R OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AOTOLYZED ANIMALS 

(19)
 

(19)
 

(19)
 

1 (5X)
 

1 (5V
 

1 (5V
 

1 (5%)
 

(18)
 

(IS)
 

1 (6X)
 

(18)
 

 EXAMINE D MICROSCOPICALLY 

D-3 

 LOU DOSE HIGH tOSE
 

22-2093 22-2091
 

5? 50
 5
 

u.-i 50
 itl 50
 

"41) (50)
 

1 (2X)
 

(11)	 (50)
 

1 (2*)
 

(13)	 (16)
 

1 (2*)
 

1 (2*)
 

1 (2*)
 

1 (2%) 2 (IX)
 

(37) <U5)
 

1 (3V
 1 (3X)
 

1 (3<J
 

(36) (38)
 

1 {3V
 

(36) (38)
 
TABLEDl (CONTINUED) 

CONTROL (UNTR) 

22-2095 LOW DOSE 22-2093 

HYPERPLASIi, LYNFHOID 

CIRCULATORY SYSTEM
 

IHEART (19) <«3)
 PERIARTERITIS 2 (5*)
 CALCIFICATION, FOCAL 1 (2*)
 

tMYOCARDIUM (19) (13)
 FIBROSIS 1 (2*)
 

DIGESTIVE SYSTEM
 

ILIVER (19) («3)
 IYBPHOCYTIC INFLAMMATORY INFILTR
 NECROSIS, FOCAL 2 (5*)
 METAMORPHOSIS FATTY 1 (5%)
 NUCLEAR ENLARGEMENT 1 (2X)
 HYPERPLASIA, NODULAR
 HYPERPLASIA, FOCAL
 

*BILE DUCT (19) (UK)
 

CYST, NOS 1 (2%)
 

fPANCREAS (18) («1)
 INFLAMMATION, NECROTIZING
 

ATROPHY, SOS 1 (2*)
 

tSTCMACH (15) (11)
 CYST, NOS 1 (2%)
 INFLAMMATION, NOS
 

ILARGE INTESTINE (18) (41)
 NEMATODIASIS 1 (6*) 1 (2%)
 

ICOION (18) (11)
 NEMATODIASIS 3 (17X) 3 (7X)
 

ORINBRY SYSTEM
 

tKIDNEY (19) (12)
 

HYDBONEPjfgOSlS .__ i_J51L__
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIEC
 

D-4
 

HIGH COSE
 22-2091
 

1 <3X)
 

(«8)
 

(18)
 

<«8)
 

1 (2X)
 

1 (2*)
 

2 <1*)
 

1 (2%)
 

(50)
 

(38)
 

1 (3*)
 

(149)
 

1 (2*)
 

(»8)
 

(18)


3 (6X)
 

<5P)
 ._. ._ , 2,, JUS) ____ _ ._ ___ t
 
TABLE Dl (CONTINUED)
 

CONTBOL(UNTB) LOS DOSE HIGH DOSE
 

22-2095 22-2093 22-2091
 

•KIDNEY/CORTEX	 (19) <t2) (50)
 

CYST, DOS 1 (2*)
 

ENDOCRINE SYSTEM
 

IADRENAL CORTEX 113) (25) (36)
 

HYPERPLASIA, NOS 1 (8%)
 

• THYROID (13) (36) (35)
 

COLLOID CYST 1 (8X) <3X) 2
 

FIBROSIS, FOCAL 1 (8*)
 

tPANCREATIC ISLETS (18) (41) (38)
 

HYPEPPLASIA, NOS 1 (6«) 1 (3*)
 

REPRODUCTIVE SYSTEM
 

• NAUMAR Y GLAN C (19) (HU) (50) 

INFLAPIBfTIOS , NOS 1 (5X) 

*SEMINAL VESICLE (19) (14) (50)
 

DILATATION/DUCTS 1 (2H)
 

NERVOUS SYSTEI"
 

•BRAIN	 (19) (t«) (50)
 

CORPORA AMYLACEA 1 (5X) 1lt (32«) 9 (18X)
 

SPECIAL SENSE ORGANS
 

NONE
 

HUSCOI.OSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NOSE
 

t NOMBEP OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NOMBEP OF ANIMALS NECBOPSIED
 

0-5
 
TABLE Dl (CONCLUDED)
 

CONTROL(DNTR) ICH COSE HIGH COSE
 22-2095 22-2093 22-2091
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESIOS DEPORTED

ANIMAL MISSING/NO NECROPSY

AUTO/NECROFSY/HISTO PERP

AOTOLYSIS/HO HECROPSY

 6

 1

HISS: MISSING

MSAC: MORIBUND SACRIFICE


 11 13 

5 

1 

1
 

 TSAC: TERMINAL SACRIFICE
 

 OTHR: OTHER
 

D-6
 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

TREATED WITH 3,3'-DIMETHOXYBENZIDINE^,4'-DHSOCYANATE

 

_— ___— I-"-'---­TM ­1 - —'——————1 • -.! -_• (••!•<• 

CONTROL (UNTR) LOU DOSE HIGH EOSE 

22-2096 22-209U 22-2092 

ANIMALS INITIALLY IV STUDY 20 50 50 

ANIMALS HISSING 9 7 

ANIHALS NECSOPSIED 20 41 143 ANIMALS EXAMINED HISTOPATHOLOGICAIIY** 20 U1 12 

INTEGUMENTARY SYSTEM
 

*SUECUT TISSUE (20) (41) (43)
 

STEATITIS 1 (2%)
 

RESPIRATORY SYSTEM
 

• LUNG (20) («1) (10)
 EMBOLISM, NOS 1 (3*)
 PNEUMONIA, LIFID 1 (3»)
 INFLAMMATION, SUPPURATIVE 1 (2*)
 PNEUMONIA, CHRONIC NURINE 2 (10*) 1 (2X) 4 (10*)
 GBANULONA, NOS 1 (2*)
 HYPESPLASIA, ADENOHATOUS 1 (2()
 HISTIOCYTOSIS 1 (2*)
 

HEMATOPOIETIC SYSTEM
 

«BOHE MARROW (1H) (38) (37)
 

HYPERPLASIA, HEMATOPOIETIC 1 (3*)
 

ISPLEEN (18) (41) (11)
 

HYPEHPLASIA, RETICOLDM CELL 1 (2*) 1 (2*)
 

HYPERPLASIA, LYMPHOID 1 (6X) 2 (5*) 4 (10*)
 

BEBATOPOIESIS 2 (11*) 1 (2*)
 

*LYMPH NODE (20) (40) (37)
 

HYPERPLASIA, NOS 1 (3*)
 

HYPERPLASIA, RETICOLOM CELL 1 (5*)
 

HYPERPLASIA, LYHPROID 2 (5*)
 

*MESENTERIC L. NODE (20) (40) (37)
 

NOS !_15JH
 

« NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIEP 

"EXCLUDES. PARTIALLY AUTOLYZED ANIMALS 

D-7 
TABLE D2 (CONTINUED)
 

HIGH DOSE
 

22-2092
 

(12)
 

1 (8%)
 

(12)
 

1 (2%)
 

1 (2%)
 

(28)
 

(38)
 

(37)
 

1 (3*)
 

(37)
 

2 (5*)
 

(«2)
 

2 (5*)
 

1 (2«)
 

1 (2t)
 

1 <2E)
 

1 (2%)
 

(12)
 

__ i 1251
 

tTHYKDS
 

HYPERPLRSIA, IYMPH01D
 

CIRCULATORY SYSTEM
 

NON*
 

DIGESTIVE SYSIEH
 

*LIVER
 

NECROSIS, FOCAL
 

"ETAHORPHOSIS FATTY
 

NUCLEAR ENLARGEMENT
 

EFKATOPOIESIS
 

tPANCREAS
 

LYMPHOCYTIC INFLAMMATORY INFILTR
 

FIBBOSIS, FOCAL
 

tPEYERS PATCH
 

HYPERPLASIA, LYMPHOID
 

tLARGE INTESTINE
 

NE«ATODIASIS
 

ICOION
 

NEHATODIASIS
 

URINARY SYSTEM
 

IKIDNF.Y
 HYDBONEPHROSIS
 

GLOBEROLONEPHPITIS, NOS
 

LYMPHOCYTIC INFLAMMATORY INFILTR
 

INFLAMMATION. CHRONIC
 

AMYLOID, NOS
 

CALCINOSIS, NOS
 

METAPLASIA. OSSEOUS
 

tKIENEY/NEDDLLA
 

LYHPHOCYTJC INFLAMMATORY IHFILTR
 

CONTROL (ONTR)
 

27-2096
 

(20)
 

(19)
 

(20)
 

(19)
 

(19)
 

(19)
 

(19)
 

T-- -,,_ *.L -.. -..
 

L0» DOSE
 22-209U
 

(»1)
 

1 (2«)
 

1 (2«)
 

1 (2%)
 

(39)
 

1 (3S)
 

1 (3X)
 

(11)


1 <2X)
 

(«0)
 

1 (3*)
 

(UO)
 

(11)
 

1 (2X)
 

1 (2X)
 

1 (2*)
 

(11_
 t
 )

t NDMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF AHIMALS NECROPSIED
 

0-8
 
TABLE D2 (CONTINUED) 

CONTROL (ONTR) LOW DOSS HIGH COSE 

22-2096 22-209U 22-2092 

ENDOCRINE SYSTEM 

•ADRENAL (1t) (37) (32) IIPOIDOSIS 1 <3X) 

•ADRENAL CORTEX 

HYPERPLASIA. NOS (11) (37) (32) 1 (3%) 

•THYROID (12) (36) (32) COLLOID CYST 2 (6X) HYPERPLASIA, FOCAL 2 (6X) HYPERPLASIA, FOLLICULAB-CELL 1 (3%) 

fPAllCREATIC ISLETS (19) (39) (28) HIPERPLASIA, NOS 1 (5*) 2 (5%) 2 (7%) 

EEPRCCDCTIVE SYSTEM 

*VAGINA INFLAMMATION, SUPPOHATIVE 

(20) 

2 (10X) 

(11) 

1 (2X) 

(13) 

1 (2%) 

•UTERUS (19) (tO) (37) 

CYST, NOS 1 (3%) HEMORRHAGIC CYST 1 (5*) INFLAMMATION, SUPPURATIVE 1 (5X) EYOHETRA 8 (K2%) 1 <3X) 1 (3%) 

•UTIRINE SEROSA (19) (UO) (37) 

INFLAMMATION, FOCAL 1 (3X) 

tUTERUS/ENDOMBTRIUH (19) (10) (37) 

CYST, NOS INFLAMMATION, SDPPtlSATIVE HYPERPLASIA, CYSTIC 2 (11*) U (21%) 6 (15%) 

1 (3%) 19 (U8%) 

1 (3%) 

2 (5%) 

21 (57%) 

IUTIRUS/MYOMETRIOM 

EEGENERATION, HYALINE 

(19) (1") (37) 

1 (3%) 

IOVARY/OVIDDCT (19) (10) (37) 

INFLAMMATION, NOS 1 (3%) 

tOVARY 

CYST,. NOS _ (17) _ _ 3_JJ8X1 _ 

(31) _ 7 121*1_ _

(22) 

_ 6_J27%J 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

D-9 
TABLE D2 (CONCLUDED)
 

CONTROL(DNTB)

22-2096

 LOU DOSE

 22-2091

 HIGH EOSE 

 22-2092 

FOLLICULAR CYST, NOS

INFLAMMATION, SUEPURATIVE

ABSCESS, NOS

INFLAMMATION , CHIiONIC

INFLAMMATION, GRANOLOMATOOS

 1 (6%) 

1 (3X) 

1 (3X) 

1 (3K) 

1 (5X) 

NERVOOS SYSTEM 

tBHAIN

COFFOPA AMYLACEA

 (19)

 2 (11%)

 (39)

 8 (21%)

 (UO) 

8 (20*) 

SPECIAL SENSE ORGANS 

NONF. 

MOSCU10SKELETAL SYSTEM 

*LIGAMENT

ABSCESS, NOS

 (20) («1)

 1 (2%) 

 (13) 

BODY CAVITIES 

*MESENTERY

NECROSIS, FAT

 ' (20) («1) (13) 

1 (21) 

ALL CTHER SYSTEMS 

"MULTIPLE ORGANS

LYMPHOCYTIC INFLAMMATORY INFILTR

 (20)

 1 (5%) 

 (11) (13) 

BROAD LIGAMENT 

ABSCESS, NOS 1 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED

ANIMAL MISSING/NO NECROPSY

AtJTO/N?CROPSY/NO HISTO

 9

 3 

7 

1 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS SECBOPSIED 

D-10
 
Review of the Bioassay of 3,3'-Dimethoxybenzidine-4,4'-Diisocyanate*
 

for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

August 31, 1978
 

The Clearinghouse on Environmental Carcinogens was established in
 

May, 1976, in compliance with DREW Committee Regulations and the Provisions
 

of the Federal Advisory Committee Act. The purpose of the Clearinghouse
 

is to advise the Director of the National Cancer Institute (NCI) on its
 

bioassay program to identify and to evaluate chemical carcinogens in the
 

environment to which humans may be exposed. The members of the Clearinghouse have been drawn from academia, industry, organized labor, public
 

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

research organizations. Members have been selected on the basis of
 

their experience in carcinogenesis or related fields and, collectively,
 

provide expertise in chemistry, biochemistry, biostatistics, toxicology,
 

pathology, and epidemiology. Representatives of various Governmental
 

agencies participate as ad hoc members. The Data Evaluation/Risk
 

Assessment Subgroup of the Clearinghouse is charged with the 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 3,3'-Dimethoxybenzidine-4,
 

4'-Diisocyanate for carcinogenicity.
 

The primary reviewer said that 3,3'-Dimethoxybenzidine-4,4'-


Diisocyanate induced a statistically significant increase in skin neoplasms
 

and in leukemia in both sexes of treated rats, as well as endometrial
 

stromal polyps in females. No evidence for the compound's carcinogenicity
 

was found in treated mice. He noted that the compound was first administered by gavage and subsequently in the feed. The primary reviewer
 

said that the study was acceptable and that the evidence was sufficient
 

to conclude that 3,3'-Dimethoxybenzidine-4,4'-Diisocyanate posed a
 

potential carcinogenic risk to man.
 

The secondary reviewer noted that the purity of 3,3'-Dimethoxybenzidine4,4'-Diisocyanate was not reported. Although he agreed that the skin
 

tumors in male rats were treatment-related, he thought that the incidence
 

was not sufficiently high in females to draw the same conclusion from a
 

statistical basis. He suggested that the skin tumors may have resulted
 

through direct skin contact with the compound. The secondary reviewer
 

agreed that the leukemia and endometrial polyps in treated rats appeared
 

to be treatment-related.
 

59
 
A motion was approved unanimously that the report on the bioassay
 

of 3,3'-Dimethoxybenzidine-4,4'-Diisocyanate be accepted as written.
 

Members present were:
 

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

Joseph Highland, Environmental Defense Fund
 

Michael Shimkan, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

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

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

Thus, certain comments and criticisms reflected in the review may
 

no longer be appropriate.
 

«r U.S. GOVERNMENT PRINTING OFFICE: 1978-281-217:3298 

60 

DHEW Publication No. (NIH) 79-1383