1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 114 

BIOASSAY OF 

2,3,5,6-TETRACHLORO-4-NITROANISOLE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 2438-88-2 

NCI-CG-TR-114 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

2,3,5,6-TETRACHLORO-4-NITROANISOLE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-1369
 

REPORT ON THE BIOASSAY OF 2,3,5,6-TETRACHLORO-4-NITROANISOLE
 

FOR POSSIBLE CARCINOGENICITY
 

CARCINOGENESIS TESTING PROGRAM
 

DIVISION OF CANCER CAUSE AND PREVENTION
 

NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH
 

FOREWORD: This report presents the results of the bioassay of
 

2,3,5,6-tetrachloro-4-nitroanisole conducted for the Carcinogenesis
 

Testing Program, Division of Cancer Cause and Prevention, National
 

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

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

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

a significantly greater incidence of cancer than control animals, do
 

not necessarily mean the test chemical is not a carcinogen because
 

the experiments are conducted under a limited set of circumstances.
 

Positive results demonstrate that the test chemical is carcinogenic
 

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

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

carcinogens requires a wider analysis.
 

CONTRIBUTORS: This bioassay of 2,3,5,6-tetrachloro-4-nitroanisole
 

was conducted by Mason Research Institute, Worcester, Massachusetts,
 

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. J. H. Weisburger (1,2) and Dr. E. K. Weisburger (1). The
 

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

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

by Mr. G. Wade (3) and Ms. E. Zepp (3). Chemical analysis was performed by Mason Research Institute (3) and Midwest Research Institute
 

(4) and the analytical results were reviewed by Dr. N. Zimmerman (5).
 

Histopathologic examinations were performed by Dr. R. L. Schueler
 

(6) as a consultant for Mason Research Institute, and the diagnoses
 

included in this report represent the interpretation of this pathologist.
 

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

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

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

Gart (9).
 

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

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

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

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

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

participating organizations.
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

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

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

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

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

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

2.	 No wit w h the Naylor Dana Institute for Disease Prevention, American Health Foundation, Hammon House Road, Valhalla, New York.
 

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

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

Missouri.
 

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

Boulevard, McLean, Virginia.
 

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

Maryland.
 

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

Rockville, Maryland.
 

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

Golden, Colorado.
 

9.	 Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cancer
 

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

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

Washington, D.C.
 

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

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay for possible carcinogenicity of 2,3,5,6-tetrachloro4-nitroanisole was conducted using Fischer 344 rats and B6C3F1 mice.
 

2,3,5,6-Tetrachloro-4-nitroanisole was administered in the feed, at
 

either of two concentrations, to groups of male and female animals
 

of each species. The high and low dietary concentrations used in the
 

chronic bioassay were 0.012 and 0.006 percent, respectively, for both
 

species. After a 104-week period of chemical administration, observation of rats continued for up to 3 weeks and observation of mice
 

continued for up to 1 week. For rats 50 animals of each sex were
 

placed on test as controls, while for mice 55 animals of each sex
 

were placed on test as controls.
 

There were no significant positive associations between the
 

dietary concentration of 2,3,5,6-tetrachloro-4-nitroanisole administered and mortality in rats or mice of either sex. Adequate numbers
 

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

from late-developing tumors.
 

No neoplasms, except for interstitial-cell testicular tumors
 

in males, occurred at statistically significant incidences in dosed
 

rats. Because of the high and variable spontaneous incidence of
 

these lesions in Fischer 344 rats, these tumors were not considered
 

to be associated with the administration of the test compound.
 

Among dosed male mice the combined incidence of leukemia and
 

malignant lymphoma was statistically significant. However, since
 

these lesions occur spontaneously and with high variation in B6C3F1
 

mice, the lesions were not considered to be associated with the
 

administration of the test compound. No neoplasms were of a statistically significant incidence in dosed female mice.
 

Under the conditions of this bioassay, dietary administration
 

of 2,3,5,6-tetrachloro-4-nitroanisole was not carcinogenic to male
 

or female Fischer 344 rats or B6C3F1 mice of either sex.
 

vii
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 4
 

A. Chemicals 4
 

B. Dietary Preparation 4
 

C. Animals 5
 

D. Animal Maintenance 6
 

E. Selection of Initial Concentrations 8
 

F. Experimental Design 10
 

G. Clinical and Histopathologic Examinations 13
 

H. Data Recording and Statistical Analyses 15
 

III. CHRONIC TESTING RESULTS: RATS 20
 

A. Body Weights and Clinical Observations 20
 

B. Survival 20
 

C. Pathology 23
 

D. Statistical Analyses of Results 24
 

IV. CHRONIC TESTING RESULTS: MICE 34
 

A. Body Weights and Clinical Observations 34
 

B. Survival 34
 

C. Pathology 37
 

D. Statistical Analyses of Results 39
 

V. DISCUSSION 48
 

VI. BIBLIOGRAPHY 50
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH 2,3,5,6-TETRACHLORO-4­

NITROANISOLE A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH 2,3,5,6-TETRACHLORO-4­

NITROANISOLE B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH 2,3,5,6­

TETRACHLORO-4-NITROANISOLE C-l
 

ix
 
 D-l 

TABLE OF CONTENTS (Concluded)
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH 2,3,5,6­

TETRACHLORO-4-NITROANISOLE


x
 
Figure Number
 

1
 

Table Number
 

LIST OF ILLUSTRATIONS 

Page 

CHEMICAL STRUCTURE OF 2 ,3 ,5 ,6-TETRACHLORO-4­

NITROANISOLE 

GROWTH CURVES FOR 2 ,3,5,6-TETRACHLORO-4­

NITROANISOLE CHRONIC STUDY RATS 21 

SURVIVAL COMPARISONS OF 2,3,5,6-TETRACHLORO-4-NITROANISOLE CHRONIC STUDY RATS 22 

GROWTH CURVES FOR 2,3,5,6-TETRACHLORO-4­

NITROANISOLE CHRONIC STUDY MICE 35 

SURVIVAL COMPARISONS OF 2,3,5,6-TETRACHLORO-4-NITROANISOLE CHRONIC STUDY MICE 36 

LIST OF TABLES 

DESIGN SUMMARY FOR FISCHER 344 RATS—2 ,3 ,5 ,6-

TETRACHLORO-4-NITROANISOLE FEEDING EXPERIMENT 11 

DESIGN SUMMARY FOR B6C3F1 MICE--2 ,3,5,6­

TETRACHLORO-4-NITROANISOLE FEEDING EXPERIMENT 12 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

2,3,5,6-TETRACHLORO-4-NITROANISOLE 25 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH 2,3,5,6-TETRACHLORO-4-NITROANISOLE 29 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

2,3,5,6-TETRACHLORO-4-NITROANISOLE 40 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH 2,3,5,6-TETRACHLORO-4-NITROANISOLE 44 

XI
 
LIST OF TABLES (Concluded) 

Table Number Page 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH 2,3,5,6-TETRACHLORO4-NITROANISOLE A-3 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH 2,3,5,6-TETRACHLORO-4-NITROANISOLE A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH 2,3,5,6-TETRACHLORO-4-NITROANISOLE B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH 2,3,5,6-TETRACHLORO-4-NITROANISOLE B-7 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH 2,3,5,6­

TETRACHLORO-4-NITROANISOLE C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH 2,3,5,6­

TETRACHLORO-4-NITROANISOLE C-9 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH 2,3,5,6­

TETRACHLORO-4-NITROANISOLE D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH 2,3,5,6­

TETRACHLORO-4-NITROANISOLE D-8 

xii
 
I. INTRODUCTION
 

2,3,5,6-Tetrachloro-4-nitroanisole (Figure 1) (NCI No. C03032),
 

an agricultural fungicide and acaricide, was selected for bioassay by
 

the National Cancer Institute because of its structural similarity to
 

pentachloronitrobenzene, a pesticide classified as tumorigenic by the
 

Secretary's Commission on Pesticides and their Relationship to Environmental Health (U.S. Department of Health, Education, and Welfare, 1969).
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is l-methoxy-4-nitro-2,3 ,5,6-tetrachlo­

*
 

robenzene. It is also known as tetrachloronitroanisole; ENT 22335;
 

and TCNA.
 

2,3,5,6-Tetrachloro-4-nitroanisole has been shown to control
 

fungi which cause flag smut of winter wheat (Purdy, 1963) and rust,
 

root and stem rot, and wilt in a variety of vegetables and grains
 

(Carey, 1963). The compound's singular effectiveness against flag
 

smut is a result of its ability to control infections arising from
 

both seed-borne and soil-borne flag smut spores (Purdy, 1963).
 

Nonetheless, 2,3,5,6-tetrachloro-4-nitroanisole is not currently
 

registered as a pesticide in the United States (Schaughnessy, 1977).
 

Although specific production figures for 2,3 ,5,6-tetrachloro-4­

nitroanisole are not available, its exclusion from Synthetic Organic
 

*
 

The CAS registry number is 2438-88-2.
 
OCHFIGURE 1

 

CHEMICAL STRUCTURE OF 2,3,5,6-TETRACHLORO-4-NITROANISOLE 
Chemicals: United States Production and Sales, 1975 (U.S. International Trade Commission, 1977) implies that it is not produced in
 

commercial quantities in the United States.
 

Since 2,3,5,6-tetrachloro-4-nitroanisole is not in present use
 

as a pesticide, the potential for exposure is greatest for those persons engaged in the synthesis of 2,3,5,6-tetrachloro-4-nitroanisole
 

or in agricultural research involving 2,3,5,6-tetrachloro-4-nitroanisole.
 
II. MATERIALS AND METHODS
 

A. Chemicals
 

2,3,5,6-Tetrachloro-4-nitroanisole was purchased from Carroll
 

Products, Wood River Junction, Rhode Island. Chemical analysis was
 

performed by Mason Research Institute, Worcester, Massachusetts and
 

Midwest Research Institute, Kansas City, Missouri. The experimentally
 

determined melting point range was 101° to 105°C. Two differing
 

literature values (i.e., 105° to 106°C [Berckmans and Halleman, 1925]
 

and 112° to 113°C [Peters et al., 1943]) were found with no adequate
 

explanation for the variation. Elemental analysis of the purchased
 

compound suggested the presence of at least minor impurities. Thin-


layer chromatography utilizing two solvent systems (i.e., ethyl
 

acetate:hexane and benzene:chloroform) revealed, respectively, two
 

spots and one spot. Vapor-phase chromatography indicated two impurities of motility similar to the major compound. Nuclear magnetic
 

resonance and infrared analyses were consistent with the structure of
 

the compound.
 

Throughout this report, the term 2,3,5,6-tetrachloro-4-nitroanisole is used to refer to this compound.
 

B. Dietary Preparation
 

The basal laboratory diet for both dosed and control animals consisted of Wayne Lab-Blox®(Allied Mills, Inc., Chicago, Illinois).
 

2,3,5,6-Tetrachloro-4-nitroanisole was administered to the dosed animals as a component of the diet.
 

4
 
The chemical was removed from its container and proper amounts
 

were sifted and weighed out under an exhaust hood. The compound was
 

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

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

capacity Patterson-Kelley twin-shell stainless steel V-blender along
 

with the remainder of the meal. The blender was sealed and operated
 

for 20 minutes. The mixtures were placed in double plastic bags and
 

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

unused portions were discarded 14 days after formulation.
 

C. An ima1s
 

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

contracts of the Division of Cancer Treatment, National Cancer Institute. All rats were supplied by Charles River Breeding Laboratories,
 

Wilmington, Massachusetts, and all mice were supplied by the Frederick
 

Cancer Research Center, Frederick, Maryland. Dosed and control animals for both species were received in separate shipments.
 

Upon arrival, a sample of anima'ls was examined for parasites and
 

other signs of disease. One group of high dose male rats (group 2 as
 

defined in Section II. F., Experimental Design) had parasite infestations and were treated with 3.0 gm piperazine adipate per liter in
 

drinking water for 3 days, followed by 3 days of tap water and 3 more
 

days of piperazine adipate administration. Animals to be used in the
 

chronic bioassay were quarantined by species for 2 weeks prior to
 
initiation of test. Animals were assigned to groups and distributed
 

among cages so that average body weight per cage was approximately
 

equal for a given sex and species.
 

D. Animal Maintenance
 

All animals were housed by species in rooms having a temperature
 

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

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

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

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

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

the first 12 months of study, dosed rats were held in galvanized- or
 

stainless-steel wire-mesh cages (Fenco Cage Products, Boston, Massachusetts) suspended over newspapers (except for the high dose group 2
 

males, which were housed in wire-mesh cages for the first 10 months).
 

Control rats were housed in wire-mesh cages for the first 14 months
 

of study. Newspapers under cages were replaced daily, and cages and
 

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

housed in suspended polycarbonate cages (Lab Products, Inc., Garfield,
 

New Jersey) equipped with disposable nonwoven fiber filter sheets.
 

®
 

Clean bedding and cages were provided twice weekly. SAN-I-CEL corncob bedding (Paxton Processing Company, Paxton, Illinois) was used
 

for the first 6 months of polycarbonate caging. Aspen hardwood chip
 

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

used for the remainder of the study. Stainless steel cage racks were
 
cleaned once every 2 weeks, and disposable filters were replaced at
 

that time.
 

Mice were housed five per cage by sex in polycarbonate shoe box
 

type cages. Cages were fitted with perforated stainless steel lids
 

(Lab Products, Inc.). Nonwoven fiber filter bonnets were used over
 

cage lids. Clean cages, lids, and bedding were provided twice per
 

(R)
 week. Bed-o-cobs corncob bedding (The Andersons Cob Division,
 

Maumee, Ohio) was used for the first 7 months of study for dosed
 

mice, and for the first 8 months of study for control mice. Aspen
 

bedding was used thereafter. Reusable filter bonnets and pipe racks
 

were sanitized every 2 weeks throughout the study.
 

Water was available ad libitum from 250 ml water bottles equipped
 

with rubber stoppers and stainless steel sipper tubes. Bottles were
 

replaced twice weekly and, for rats only, water was supplied as needed
 

between changes.
 

During the period of compound administration dosed animals were
 

(R)
 fed Wayne Lab-Blox meal containing the appropriate concentration of
 

2,3,5,6-tetrachloro-4-nitroanisole. Control animals received un­

®
 treated meal. Food was supplied to rats in Alpine aluminum feed
 

cups (Curtin Matheson Scientific, Inc., Woburn, Massachusetts) while
 

they were in wire-mesh cages. While in polycarbonate cages, food was
 

supplied to rats from stainless steel gangstyle feed hoppers (Scientific Cages, Inc., Bryan, Texas). Food was supplied to dosed and
 

(R)
 control mice from Alpine feed cups for the first 1 and 2 months of
 
study, respectively, and from gangstyle hoppers thereafter. Food
 

hoppers were changed on the same schedule as were cages. Food was
 

t'Si
 replenished daily in Alpine feed cups.
 

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

&
 

rats receiving diets containing 4-chloro-o-phenylenediamine (95-83­

0) ; acetylatninofluorene (53-96-3); p-cresidine (120-71-8); 4-chlorom-phenylenediamine (5131-60-2); and IH-benzotriazole (95-14-7).
 

Dosed mice were housed in a room with other mice receiving
 

diets containing hydrazobenzene (530-50-7); tris(2,3-dibromopropyl)
 

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

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

aniline hydrochloride (142-04-1). Control mice were housed in a room
 

with other mice receiving diets containing fenaminosulf (140-56-7);
 

2,5-dithiobiurea (142-46-1); 4-chloro-o-phenylenediamine (95-83-0);
 

o-anisidine hydrochloride (134-29-0); p-anisidine hydrochloride
 

(20265-97-8); and cupferron (135-20-6).
 

E. Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

2,3,5,6-tetrachloro-4-nitroanisole for administration to dosed animals in the chronic studies, subchronic toxicity tests were conducted
 

with both rats and mice. Rats were distributed among seven groups,
 

each consisting of five males and five females. 2,3,5,6-Tetrachloro4-nitroanisole was incorporated into the basal laboratory diet and
 

* .


CAS registry numbers are given in parentheses.
 
supplied ad libitum to seven of the eight rat groups in concentrations
 

of 0.003, 0.01, 0.025, 0.05, 0.1, 0.2, and 0.4 percent. Mice were
 

distributed among nine groups, each consisting of five males and five
 

females. The chemical was incorporated into the laboratory diet and
 

supplied ad libitum to eight of the nine mouse groups in concentrations of 0.003, 0.006, 0.0125, 0.025, 0.05, 0.1, 0.2, and 0.4 percent.
 

One rat group and one mouse group each served as a control group,
 

receiving only the basal laboratory diet. The dosed dietary preparations were administered for 8 weeks. Individual body weights were
 

recorded during the first, fourth, and seventh weeks of the subchronic
 

study. Survivors were sacrificed at the end of the test, and gross
 

necropsies were performed.
 

The highest concentration causing no deaths, no compound-related
 

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

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

selected as the high concentration utilized for the chronic bioassay.
 

All rats receiving concentrations of 0.025 percent or more died.
 

Each male rat receiving a concentration of 0.025 percent or higher
 

had a spotted or colored liver or thymus. No gross pathology was
 

observed in female rats. A dietary concentration of 0.01 percent
 

produced a mean body weight depression of 8.2 percent in male rats
 

and no mean weight depression in female rats. The high concentration
 

selected for administration to rats in the chronic study was 0.012
 

percent.
 
All mice receiving concentrations of 0.1 percent or higher died.
 

Two male and three female mice receiving 0.05 percent died, and one
 

male mouse receiving 0.025 percent died. Mesenteric lymph nodes were
 

moderately enlarged in female mice receiving 0.025 percent. A dietary concentration of 0.0125 produced mean body weight depressions
 

of 10.8 and 6.3 percent in male and female mice, respectively. A dietary concentration of 0.006 percent produced mean body weight depressions of 10.0 and 7.2 percent in male and female mice, respectively.
 

The high concentration selected for administration to mice in the
 

chronic study was 0.012 percent.
 

F. Experimental Design
 

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

treated and untreated observation periods) are summarized in Tables 1
 

and 2.
 

All rats were approximately 6 weeks old at the time they were
 

placed on test. The concentrations of 2 ,3 ,5,6-tetrachloro-4-nitroanisole in diets were 0.012 and 0.006 percent. Throughout this report
 

those rats receiving the former concentration are referred to as the
 

high dose groups and those receiving the latter concentration are
 

referred to as the low dose groups. The high dose male rat group was
 

improperly sexed, and all females that were included in the male group
 

were removed from the study. Therefore, approximately 6 weeks after
 

the start of the bioassay, a supplementary group of 25 male rats was
 

10
 
TABLE 1
 

DESIGN SUMMARY FOR FISCHER 344 RATS
 

2,3,5,6-TETRACHLORO-4-NITROANISOLE FEEDING EXPERIMENT
 

2,3,5,6-TETRACHLOROINITIAL 4-NITROANISOLE OBSERVATION PERIOD 

GROUP CONCENTRATION TREATED UNTREATED 

SIZE (PERCENT) (WEEKS) (WEEKS) 

MALE
 

CONTROL 50 0 0 105
 

LOW DOSE 49	 0.006 104
 

0 1
 

HIGH DOSE 1 24	 0.012 104
 

0 1
 

HIGH DOSE 2 25	 0.012 104
 

0 3
 

FEMALE
 

CONTROL 50 0 0 105
 

LOW DOSE 50	 0.006 104
 

0 1
 

HIGH DOSE 50 0.012 104
 

0 2
 

Initiated approximately 6 weeks after the other male rat groups.
 

11
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

2,3,5,6-TETRACHLORO-4-NITROANISOLE FEEDING


2,3,5,6-TETRACHLOROINITIAL 4-NITROANISOLE 

GROUP CONCENTRATION 

SIZE (PERCENT) 

MALE
 

CONTROL 55 0
 

LOW DOSE 55 0.006
 

0
 

HIGH DOSE 55 0.012
 

0
 

FEMALE
 

CONTROL 55 0
 

LOW DOSE 55 0.006
 

0
 

HIGH DOSE 55 0.012
 

0
 

12
 

 EXPERIMENT
 

OBSERVATION PERIOD
 

TREATED UNTREATED
 

(WEEKS) (WEEKS)
 

0 105
 

104
 

0
 

104
 

1
 

0 105
 

104
 

1
 

104
 

1
 
added. These rats are referred to as the high dose male group 2,
 

while the male members of the original high dose male rat group are
 

referred to as high dose male group 1. The dosed rats were supplied
 

with feed containing 2,3,5,6-tetrachloro-4-nitroanisole for a total
 

of 104 weeks, followed by an observation period of up to 3 weeks.
 

All mice were approximately 6 weeks old at the time they were
 

placed on test. The dietary concentrations of 2 ,3 ,5 ,6-tetrachloro-4­

nitroanisole administered were 0.012 and 0.006 percent. Throughout
 

this report those mice receiving the former concentration are referred
 

to as the high dose groups, while those receiving the latter concentration are referred to as the low dose groups. The dosed mice were
 

supplied with feed containing 2,3,5,6-tetrachloro-4-nitroanisole for
 

a total of 104 weeks, followed by an observation period of up to 1
 

week.
 

G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the experiment. Body weights were recorded twice weekly for the first
 

12 weeks of the study and at monthly intervals thereafter. Food consumption, for two cages from each group, was monitored for seven consecutive days once a month for the first nine months of the bioassay
 

and for three consecutive days each month thereafter. From the first
 

day, all animals were inspected twice daily for mortality. The presence of tissue masses and lesions was determined by monthly observation and palpation of each animal.
 

13
 
A necropsy was performed on each animal regardless of whether it
 

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

bioassay. The animals were euthanized by carbon dioxide inhalation,
 

and were immediately necropsied. The histopathologic examination consisted of gross and microscopic examination of major tissues, organs,
 

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

from animals found dead.
 

Tissues were preserved in 10 percent buffered formalin, embedded
 

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

to microscopic examination. An occasional section was subjected to
 

special staining techniques for more definitive diagnosis.
 

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

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

urinary bladder, pituitary, adrenal, thyroid, parathyroid, 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 placed on experiment in each group.
 

14
 
H. Data Recording and Statistical Analyses
 

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

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

as recommended by the International Union Against Cancer (Berenblum,
 

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

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

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

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

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

missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect
 

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

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.
 

15
 
The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

that site 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.
 

16
 
The Cochran-Armitage test for linear trend in proportions, with
 

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

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

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

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

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation of
 

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

tumor was observed were excluded by basing the statistical tests on
 

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

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

tumor was found, comparisons were based exclusively on animals that
 

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

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

Cochran-Armitage tests, etc.) were followed.
 

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

incidence of tumors. Curves of the proportions surviving without an
 

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

during which animals died naturally or were sacrificed was entered as
 

the time point of tumor observation. Cox's methods of comparing
 
these curves were used for two groups; Tarone's extension to testing
 

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

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

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

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


tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

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

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

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

t c t
 

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

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

c
 

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

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

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

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

larger proportion in the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

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

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

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
 

18
 
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.
 

19
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

Slight mean body weight depression was observed in dosed male
 

and female rats after 68 weeks (Figure 2).
 

Firm subcutaneous masses were observed in 1 control male, 7 high
 

dose females, and 3 control females. Cutaneous lesions were observed
 

in 2 low dose males, 3 control males, 2 high dose females, and 2 low
 

dose females. Pale discoloration of the eyes was observed in 1 high
 

dose male and 2 control males. Swollen, bloody eyes were observed
 

in a second high dose male. A dark crusted eye and exudate from the
 

nose were observed in a third high dose male. A swollen mouth was
 

displayed by 1 high dose male. A distended scrotal sac was observed
 

in 1 high dose male. Alopecia was observed in 1 high dose female.
 

No other clinical abnormalities were noted.
 

B. Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and 2,3,5,6-tetrachloro-4-nitroanisole-dosed groups
 

are shown in Figure 3. For both male and female rats no significant
 

positive association between dosage and mortality was observed.
 

For each sex five control rats were sacrificed in week 78. Adequate numbers of males were at risk from late-developing tumors, as
 

88 percent (21/24) of high dose group 1, 68 percent (17/25) of high
 

dose group 2, 92 percent (46/50) of the low dose group, and 72 percent
 

20
 
750 750 

— 600 

^-450 

— 300 

1-150 

60 75 

TIME ON TEST (WEEKS) 

120 

750­

600­

FEMALE RATS CONTROL 

LOW DOSE 

HIGH DOSE 

- 750 

-600 

IT 

a 

450­

UJ 

g 

g 300­

CD 

-450 

-300 

150­ — 150 

I 

15 

I 

30 

r 

45 60 

\ 

75 

TIME ON TEST (WEEKS) 

90 

\ 

105 120 

FIGURE 2 

GROWTH CURVES FOR 2,3,5,6-TETRACHLORO-4-NITROANISOLE CHRONIC STUDY RATS 

21 
— 1.0 

Z 

g 0.8­ O 

Z 

0.6 — 

^0.4H 

ct 

CO 

h- 0.2­ cc 

O 

Qo 

o: 

o. 

O.oO 

D 

Z 

5 0.8­ o 

Z 

D 

O 

X 0.6­

i 

Z 

^ 0.4­

oc 

CO 

z: 

o 

H 0.2­ cc 

o 

CL 

O 

tr 

a. 

00 

0

-0.8 

-0.6 

-0.4 

CONTROL 

MALE RATS 

15 30 45 60 75 

TIME ON TEST (WEEKS) 

90 

LOW DOSE 

HIGH DOSE 2 

HIGH DOSE 1 

105 

- 0.2 

-0.0 

120 

J_ ^L««BB

,

 • •••••••*•••••••• 

^^~I 1

1

 f

 j 

 L.t 

"^"1 

~>­

— 1.0 

-

— 0.8 

— 06 

— 0.4 

____^_ CONTROL 

LOW DOSE 

_ 

— 0.2 

FEMALE RATS

> 1 i

 15

 1

 30

 i 1 i 1 i 1

 45 60 75

TIME ON TEST (WEEKS) 

i 1

 90

 i

 HIGH DOSE 

1 i 

 105

~ 

0 0 

 120 

FIGURE 3 

SURVIVAL COMPARISONS OF 2,3,5,6-TETRACHLORO-4-NITROANISOLE CHRONIC STUDY RATS 

22 
(36/50) of the control group survived on test for at least 100 weeks.
 

Survival among females was also adequate as 78 percent (39/50) of the
 

high dose, 96 percent (48/50) of the low dose, and 78 percent (39/50)
 

of the control group survived on test at least 100 weeks.
 

C. Pathology
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

summarized in Appendix C (Tables Cl and C2).
 

A variety of neoplasms occurred with approximately equal frequency in dosed and control rats. These neoplasms are known to occur
 

t
 

spontaneously in Fischer 344 rats and their distribution indicated a
 

lack of association with chemical administration.
 

Hepatic neoplasms, not observed in control rats, were encountered
 

in limited numbers among dosed rats (i.e., neoplastic nodules—3/49
 

[6 percent] low dose males, 1/25 [4 percent] high dose group 2 males,
 

0/23 high dose group 1 males, 1/50 [2 percent] low dose females, 3/45
 

[7 percent] high dose females; hepatocellular carcinomas—2/49 [4 percent] low dose males, 0/25 high dose group 2 males, 3/23 [13 percent]
 

high dose group 1 males, 0/50 low dose females, 1/45 [2 percent] high
 

dose females).
 

Degenerative, inflammatory, and hyperplastic lesions, frequently
 

observed in aging Fischer 344 rats, were noted among dosed and control groups. The distribution of these nonneoplastic lesions did not
 

provide evidence for an association with chemical administration.
 

23
 
Occasional lesions appeared to be sex-related (e.g. chronic nephropathy in males), and these findings were compatible with the incidences observed in historical controls.
 

Based upon this histopathologic examination, convincing evidence
 

was not provided for the carcinogenicity of dietary administration of
 

2,3,5,6-tetrachloro-4-nitroanisole; however, there was an increased
 

incidence of hepatic lesions in both sexes.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

tumors were observed in at least one of the control or 2,3,5,6-tetrachloro-4-nitroanisole-dosed groups and where such tumors were observed in at least 5 percent of the group. In these analyses the
 

Cochran-Armitage test was not used for high dose group 2 since this
 

group was started on test approximately 6 weeks after all the other
 

groups.
 

For male rats the Cochran-Armitage test indicated a significant
 

(P = 0.020) positive association between dosage and the combined incidence of hepatocellular carcinomas and neoplastic nodules of the
 

liver when high dose group 1 was used. None of the Fischer exact test
 

results were significant, however, under the Bonferroni criterion.
 

Similarly, for liver neoplasms in the females the Cochran-Armitage
 

test was significant (P = 0.020), but the Fisher exact tests were not
 

24
 
___ 

TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC
 

SITES IN MALE RATS TREATED WITH 2,3,5,6-TETRACHLORO-4-NITROANISOLE£
 

TOPOGRAPHY: MORPHOLOGY
 

Subcutaneous Tissue: Fibroma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar
 

Adenoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

3/48(0.06)
 

N.S.
 

105
 

3/48(0.06)
 

N.S.
 

105
 

7/48(0.15)
 

N.S.
 

80
 

LOW
 

DOSE
 

2/49(0.04)
 

N.S.
 

0.653
 

0.055 .
 

5.345
 

105
 

0/49(0.00)
 

N.S.
 

0.000
 

0.000
 

1.628
 

7/49(0.14)
 

N.S.
 

0.980
 

0.311
 

2.969
 

92
 

HIGH DOSE
 

GROUP 2
 

0/25(0.00)
 

N.S.
 

0.000
 

0.000
 

3.111
 

1/25(0.04)
 

N.S.
 

0.640
 

0.012
 

7.396
 

106
 

3/25(0.12)
 

N.S.
 

0.823
 

0.147
 

3.217
 

36
 

HIGH DOSE
 

GROUP 1
 

0/23(0.00)
 

N.S.
 

0.000
 

0.000
 

3.366
 

0/23(0.00)
 

N.S.
 

0.000
 

0.000
 

3.366
 

6/23(0.26)
 

N.S.
 

1.789
 

0.550
 

5.356
 

89
 
TOPOGRAPHY : MORPHOLOGY
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma or
 

Neoplastic Noduleb
 

ho P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Adenoma NOS
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED) 

LOW HIGH DOSE HIGH DOSE 

CONTROL DOSE GROUP 2 GROUP 1 

0/48(0.00) 2/49(0.04) 0/25(0.00) 3/23(0.13) 

P = 0.015 N.S. N.S. P = 0.031 

Infinite Infinite 

0.290 1.265 

Infinite Infinite 

105 105 

0/48(0.00) 5/49(0.10) 1/25(0.04) 3/23(0.13) 

P = 0.020 P = 0.030 N.S. P ­ 0.031 

Infinite Infinite Infinite 

1.237 0.103 1.265 

Infinite Infinite Infinite 

105 106 105 

10/45(0.22) 9/43(0.21) 4/21(0.19) 4/20(0.20) 

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

0.942 0.857 0.900 

0.375 0.216 0.227 

2.320 2.543 2.651 

102 103 106 105 
___ 

TABLE 3 (CONTINUED) 

TOPOGRAPHY: MORPHOLOGY
 

Adrenal: Pheochromocytoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Carcinoma or
 

C-Cell Adenomab
 

K3 P Values0 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pancreatic Islets: Islet-Cell
 

Adenoma or Islet-Cell
 

Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

4/46(0.09)
 

N.S.
 __ _.
 

78
 

5/43(0.12)
 

N.S.
 

94
 

0/44(0.00)
 

N.S.
 

LOW
 

DOSE
 

4/49(0.08)
 

N.S.
 

0.939
 

0.185
 

4.761
 

103
 

2/45(0.04)
 

N.S.
 

0.382
 

0.038
 

2.194
 

103
 

2/46(0.04)
 

N.S.
 

Infinite
 

0.284
 

Infinite
 

105
 

HIGH DOSE
 

GROUP 2
 

3/25(0.12)
 

N.S.
 

1.380
 

0.215
 

7.405
 

106
 

0/22(0.00)
 

N.S.
 

0.000
 

0.000
 

1.496
 

1/25(0.04)
 

N.S.
 

Infinite
 

0.094
 

Infinite
 

106
 

HIGH DOSE
 

GROUP 1
 

4/22(0.18)
 

N.S.
 

2.091
 

0.421
 

9.986
 

105
 

2/22(0.09)
 

N.S.
 

0.782
 

0.078
 

4.277
 

105
 

2/21(0.10)
 

N.S.
 

Infinite
 

0.625
 

Infinite
 

105
 
___ 

TABLE 3 (CONCLUDED) 

LOW HIGH DOSE HIGH DOSE
 

TOPOGRAPHY :MORPHOLOGY CONTROL DOSE GROUP 2 GROUP 1
 

Testis: Interstitial-Cell Tumor 37/48(0.77) 46/49(0.94) 19/24(0.79) 22/23(0.96)
 

P Values0 P = 0.009 P = 0.018 N.S. P = 0.046
 

Relative Risk (Control) 1.218 1.027 1.241
 

Lower Limit 1.011 0.745 0.968
 

Upper Limit 1.364 1.302 1.331
 

Weeks to First Observed Tumor 78 92 36 103
 

Treated groups received doses of 0.006 or 0.012 percent in feed.
 

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

The probability level for the Cochran-Artnitage test for the comparison of the control group and
 

the low dose group with high dose Group 1 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 be­ neath the incidence of tumors in that 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.
 
___ 

N3
 

TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC
 

SITES IN FEMALE RATS TREATED WITH 2,3,5,6-TETRACHLORO-4-NITROANISOLE£
 

TOPOGRAPHY: MORPHOLOGY
 

Subcutaneous Tissue: Fibroadenoma
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma or
 

Neoplastic Nodule"
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

0/50(0.00)
 

N.S.
 

P = 0.006
 

4/50(0.08)
 

N.S.
 

101
 

0/50(0.00)
 

P = 0.020
 

_—_
 

LOW
 

DOSE
 

4/50(0.08)
 

N.S.
 

Infinite
 

0.927
 

Infinite
 

100
 

7/50(0.14)
 

N.S.
 

1.750
 

0.476
 

7.682
 

88
 

1/50(0.02)
 

N.S.
 

Infinite
 

0.054
 

Infinite
 

105
 

HIGH
 

DOSE
 

0/45(0.00)
 

N.S.
 

7/45(0.16)
 

N.S.
 

1.944
 

0.531
 

8.487
 

4/45(0.09)
 

P = 0.047
 

Infinite
 

1.031
 

Infinite
 

97 

105 
TABLE 4 (CONTINUED) 

LOW HIGH 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE DOSE 

Pituitary: Adenoma NOS 16/40(0.40) 15/48(0.31) 14/40(0.35) 

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

Relative Risk (Control) 0.781 0.875 

Lower Limit 0.418 0.462 

Upper Limit 1.470 1.641 

Weeks to First Observed Tumor 100 105 102 

Pituitary: Adenoma NOS or Chromophobe 

Adenoma^* 17/40(0.43) 15/48(0.31) 14/40(0.35) 

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

Relative Risk (Control) 0.735 0.824 

Lower Limit 0.399 0.441 

Upper Limit 1.360 1.518 

Weeks to First Observed Tumor 100 105 102 

Pituitary: Adenoma NOS or Carcinoma 

NOSb 16/40(0.40) 16/48(0.33) 15/40(0.38) 

P Values0 N.S. N.S. N.S. ___ Relative Risk (Control) 0.833 0.938 

Lower Limit 0.455 0.506 

Upper Limit 1.547 1.729 

Weeks to First Observed Tumor 100 105 102 
TOPOGRAPHY: MORPHOLOGY
 

Adrenal: Pheochrornocytoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma0
 

P Values0
 

g


Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (CONTINUED) 

CONTROL
 

6/48(0.13)
 

P = 0.006(N)
 

105
 

0/43(0.00)
 

N.S.
 

P = 0.022
 

—_
 

0/43(0.00)
 

N.S.
 

P = 0.045
 

LOW
 

DOSE
 

1/50(0.02)
 

N.S.
 

0.160
 

0.004
 

1.249
 

105
 

3/47(0.06)
 

N.S.
 

Infinite
 

0.553
 

Infinite
 

88
 

4/47(0.09)
 

N.S.
 

—_
 

Infinite
 

0.852
 

Infinite
 

88
 

HIGH
 

DOSE
 

0/44(0.00)
 

P = 0.017(N)
 

0.000
 

0.000
 

0.679
 

0/37(0.00)
 

N.S.
 

_.,—
 

1/37(0.03)
 

N.S.
 

Infinite
 

0.062
 

Infinite
 

106
 
TABLE 4 (CONCLUDED) 

LOW HIGH 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE DOSE 

Mammary Gland: Fibroadenoma 6/50(0.12) 0/50(0.00) 1/45(0.02) 

P Values0 P = 0.021(N) P = 0.013(N) N.S. 

Relative Risk (Control) 0.000 0.185 

Lower Limit 0.000 0.004 

Upper Limit 0.625 1.441 

Weeks to First Observed Tumor 105 106 

Uterus: Endometrial Stromal Polyp 2/48(0.04) 9/50(0.18) 8/45(0.18) 

P Values0 P = 0.036 P = 0.030 P = 0.036 

Relative Risk (Control) 4.320 4.267 

Lower Limit 0.957 0.911 

Upper Limit 39.430 39.438 

Weeks to First Observed Tumor 105 105 102 

Treated groups received doses of 0.006 or 0.012 percent in feed.
 

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

"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.
 a
 

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

group when P < 0.05.
 
significant under the Bonferroni criterion. In historical control
 

data collected by this laboratory for the NCI Carcinogenesis Testing
 

Program 6/250 (2 percent) of the male and 35/249 (14 percent) of the
 

female untreated Fischer 344 rats had one of these tumors. For endometrial stromal polyps in the females the Cochran-Armitage test was
 

significant, but the Fisher exact tests were not under the Bonferroni
 

criterion. In historical control data 31/249 (12 percent) of the untreated females had one of these tumors.
 

Significant positive associations were observed for interstitial-


cell tumors of the testis in males, but these results must be discounted due to the usually high spontaneous incidence of this tumor
 

(Cockrell and Garner, 1976).
 

For females the possibility of a negative association between
 

dosage and incidence was observed for adrenal pheochromocytomas and
 

for mammary fibroadenomas.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

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

of statistically significant results. It should also be noted that
 

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

indicating the theoretical possibility of tumor induction in rats by
 

2,3,5,6-tetrachloro-4-nitroanisole that could not be established under
 

the conditions of this test.
 

33
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Mean body weight depression, observed in both dosed male and
 

dosed female mice when compared to controls, was more apparent during
 

the first year of study for males and during the second year of study
 

for females (Figure 4).
 

Abdominal distention, with and without palpable masses, was
 

observed in 2 high dose males, 5 low dose females, and 1 control
 

female. Swelling of the urogenital or rectal area was observed in 1
 

high dose male, 2 low dose males, 1 control male, and 1 high dose female. Blood in the urogenital area was observed in 2 control males.
 

Subcutaneous masses were observed in 4 control males, 2 low dose females, and 2 control females. Swollen eyes were noted in 2 high dose
 

females, 1 low dose female, and 1 control male; in this male a mass
 

developed in the Harderian gland. Cutaneous lesions were observed in
 

2 high dose males, 3 low dose males, 1 control male, 1 high dose female, 1 low dose female, and 1 control female. Alopecia was observed
 

in 17 high dose males, 20 low dose males, 42 control males, 25 high
 

dose females, 23 low dose females, and 54 control females. No other
 

clinical abnormalities were noted.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and 2,3,5,6-tetrachloro-4-nitroanisole-dosed groups
 

are shown in Figure 5. For both male and female mice there was no
 

significant positive association between dosage and mortality.
 

34
 
50 -50 ­

40 — -40 

£30­ -30 

HI 

3 

> -20 

o 

OQ 

CONTROL 

10 — ... LOW DOSE -10 

MALE MICE . _ HIGH DOSE 

I I I \ I \ 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

50 — — 50 

40 -40 — 

-30 230­

X 

o 

HI 

-20 >- 20­ Q 

O 

CD 

CONTROL 

10 — -10 LOW DOSE 

HIGH DOSE FEMALE MICE 

T ~T 1 1 1 1 [— 1 T

0 15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

FIGURE 4 

GROWTH CURVES FOR 2,3,5,6-TETRACHLORO-4-NITROANISOLE CHRONIC STUDY MICE 

35 
oc 

0 ^___^ -1.0 

^_ ­

Z 

§ 0.8­ O 

•—iv 4. *^>J_k -0.8 

TMj ^L Z '•1. - l__ ~ 

o 

I -0.6 0.6­ 1­

3 -

>

50.4 -0.4 ­

DC 

p 

z

0 

1- 0.2­ cc 

0 , LOW DOSE 

o. 

_______ CONTROL 

- 0.2 

O M A 1 II l\/l IPC ­

DC ____ _ HIGH DOSE O00 0 0 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 

0 15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

— 1.0 <z '•"­ o 

s -

1- — i 1 ^".ll1

^:-.,*,,^.! ­ —f 

5 *"t^ **TX — 0.8 o.B­ o *•* 

z !••

'i -

^

o 

X0.6 — 0.6 ­

§ -

— 0.4 

cr 

^ 0.4­

13 

CO — 

O _______ CONTROL 

1- 0.2­ - 0.2 cc 

0 LOW DOSE Q.

O FEMALE MICE " 

EC ____ _ HIGH DOSE Q. 

0 0 0 0 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 

() 15 30 45 60 75 90 105 12 0 

TIME ON TEST (WEEKS) 

FIGURES 

SURVIVAL COMPARISONS OF 2,3,5,6 TETRACHLORO-4-NITROANISOLE CHRONIC STUDY MICE 

36 
Adequate numbers of males were at risk from late-developing
 

tumors as 91 percent (50/55) of the high dose, 89 percent (49/55) of
 

the low dose, and 89 percent (49/55) of the control group survived on
 

test at least 85 weeks. Survival among the females was also adequate
 

as 84 percent (46/55) of the high dose, 85 percent (47/55) of the low
 

dose, and 96 percent (53/55) of the control group survived on test
 

for at least 85 weeks.
 

C. Pathology
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

summarized in Appendix D (Tables Dl and D2).
 

A dose-related increase in the incidence of malignant lymphomas
 

was observed among dosed mice as shown in the following table:
 

Males Females 

Low High Low High 

Control Dose Dose Control Dose Dose 

No. of Animals 

Necropsied (55) (55) (53) (55) (54) (52) 

Malignant Lymphoma NOS 0 3 0 2 0 1 

Malignant Lymphoma, 

Undifferentiated 0 1 5 0 2 4 

Ma1ignant Lymphoma, 

Lymphocytic 0 4 2 2 15 13 

Malignant Lymphoma, 

Histiocytic 1 0 1 1 4 0 

Malignant Lymphoma, 

Mixed 3 0 4 9 4 7 

Lymphocytic Leukemia 0 0 0 4 0 0 

Granulocytic Leukemia 0 1 0 0 0 0 

Total number of animals 

with lymphomas or 

leukemias 4 9 12 18 25 25 

37
 
These data suggest that administration of 2,3,5,6-tetrachloro-4­

nitroanisole may have induced lymphomas, particularly in male mice.
 

The malignant lymphomas were classified as lymphocytic, histiocytic, mixed, or undifferentiated. The cell types were characterized
 

as follows:
 

Lymphocytic: Round, basophilic lymphocytes with little or no
 

cytoplasm, and often resembling normal lymphocytes. A moderate
 

degree of differentiation was usual.
 

Histiocytic: Round, ovoid, sometimes indented nuclei surrounded
 

by abundant, granular pink cytoplasm.
 

Mixed: A combination of lymphocytic and histiocytic cells.
 

The histiocytic tumor cells sometimes folded and occasionally
 

formed giant cells.
 

Undifferentiated: A uniform population of "blast" type cells
 

with large, pale nuclei and, commonly, a single nucleolus.
 

Cytoplasmic boundaries were indistinct.
 

Lymphomas with circulating malignant cells were termed "leukemic"
 

or lymphocytic leukemia.
 

A variety of other commonly occurring neoplasms was encountered
 

among dosed and control groups of both sexes. The incidence of these
 

neoplasms indicated that they were not associated with chemical administration.
 

Acanthosis, hyperkeratosis, or both were detected in the fore-


stomach in 4/52 (8 percent) low dose and 5/50 (10 percent) high dose
 

males; and in 4/51 (8 percent) low dose and 7/51 (14 percent) high
 

dose females. Although few in number, the distribution of these lesions, when compared to that in historical control mice, indicates
 

38
 
an association with chemical administration. There was no apparent
 

progression of these lesions to neoplasia.
 

In male mice the number of animals with hepatocellular lesions
 

was highest in the control group, less in the low dose, and least in
 

the high dose group.
 

Based upon this histopathologic examination, administration of
 

2,3,5,6-tetrachloro-4-nitroanisole to mice was associated with a
 

slightly increased incidence of malignant lymphomas, particularly in
 

males. A slight increase in the incidence of acanthosis and hyperkeratosis of the forestomach was noted in dosed mice of both sexes.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

tumors were observed in at least one of the control or 2,3,5,6-tetrachloro-4-nitroanisole-dosed groups and where such tumors were observed
 

in at least 5 percent of the group.
 

For male mice the Cochran-Armitage test indicated a significant
 

(P = 0.019) positive association between dosage and the combined
 

incidence of leukemia or malignant lymphomas. The Fisher exact tests
 

supported this result with a significant (P = 0.023) comparison of
 

high dose to control. For females no statistical tests were significant although leukemia or malignant lymphoma was observed in 25/52
 

(48 percent) of the high dose mice. In historical control data
 

39
 
TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC
 

SITES IN MALE MICE TREATED WITH 2,3,5, e-TETRACHLORO-A-NITRC-ANISOLE2
 

TOPOGRAPHY : MORPHOLOGY
 

Subcutaneous Tissue: Fibrosarcoma or
 

Leiomyo sarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 -e­ o
 

Lung: Alveolar/Bronchiolar Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar Carcinoma
 

or Alveolar/Bronchiolar Adenoma"
 

c
 

P Values
 

Relative Risk (Control) .
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

1/55(0.02)
 

N.S.
 

73
 

6/54(0.11)
 

N.S.
 

—__
 

105
 

12/54(0.22)
 

N.S.
 

79
 

LOW
 

DOSE
 

3/55(0.05)
 

N.S.
 

3.000
 

0.250
 

154.535
 

103
 

13/54(0.24)
 

N.S.
 

2.167
 

0.835
 

6.445
 

78
 

17/54(0.31)
 

N.S.
 

1.417
 

0.709
 

2.922
 

78
 

HIGH
 

DOSE
 

2/53(0.04)
 

N.S.
 

2.075
 

0.111
 

120.111
 

101
 

9/51(0.18)
 

N.S.
 

1.588
 

0.545
 

5.043
 

105
 

13/51(0.25)
 

N.S.
 

1.147
 

0.534
 

2.482
 

89 
___ 

TOPOGRAPHY : MORPHOLOGY
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma,
 

Hepatocellular Adenoma, or Mixed
 

Hepato/Cholangio Carcinoma^
 

P Values
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 5 (CONTINUED)
 

CONTROL
 

4/55(0.07)
 

P = 0.019
 

105
 

24/54(0.44)
 

P = 0.002(N)
 

53
 

28/54(0.52)
 

P = 0.002(N)
 

53
 

LOW
 

DOSE
 

9/55(0.16)
 

N.S.
 

2.250
 

0.672
 

9.455
 

73
 

13/52(0.25)
 

P = 0.029(N)
 

0.563
 

0.299
 

1.015
 

79
 

18/52(0.35)
 

N.S.
 

0.668
 

0.405
 

1.083
 

79
 

HIGH
 

DOSE
 

12/53(0.23)
 

P = 0.023
 

3.113
 

1.016
 

12.452
 

91
 

9/52(0.17)
 

P = 0.002(N)
 

0.389
 

0.179
 

0.775
 

92
 

12/52(0.23)
 

P = 0.002(N)
 

0.445
 

0.237
 

0.795
 

92
 
TOPOGRAPHY : MORPHOLOGY
 

Liver: Angiosarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Capsula r r Adenoma NOS or
 i
 

Cortical Adenoma0
 

P Values0
 

KJ
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinomab
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 5 (CONTINUED) 

CONTROL
 

0/54(0.00)
 

N.S.
 

6/50(0.12)
 

P = 0.003(N)
 

P = 0.070
 

105
 

0/48(0.00)
 

N.S.
 

P = 0.017
 

—_
 

LOW
 

DOSE
 

2/52(0.04)
 

N.S.
 

Infinite
 

0.307
 

Infinite
 

97
 

0/52(0.00)
 

P = 0.012(N)
 

0.000
 

0.000
 

0.602
 

3/49(0.06)
 

N.S.
 

Infinite
 

0.590
 

Infinite
 

104
 

HIGH
 

DOSE
 

3/52(0.06)
 

N.S.
 

Infinite
 

0.623
 

Infinite
 

105
 

0/49(0.00)
 

P = 0.014(N)
 

0.000
 

0.000
 

0.637
 

0/44(0.00)
 

N.S.
 
TABLE 5 (CONCLUDED)
 

a

Treated groups received doses of 0.006 or 0.012 percent in feed.
 

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

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

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

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

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

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

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

group when P < 0.05.
 
___ 

_ __ 

TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC
 

SITES IN FEMALE MICE TREATED WITH 2,3,5,6-TETRACHLORO-4-NITROANISOLE£
 

TOPOGRAPHY: MORPHOLOGY
 

Lung: Alveolar/Bronchiolar Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar Carcinoma
 

or Alveolar/Bronchiolar Adenoma0
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

1/55(0.02)
 

N.S.
 

105
 

4/55(0.07)
 

N.S.
 

105
 

18/55(0.33)
 

N.S.
 

86
 

LOW
 

DOSE
 

5/52(0.10)
 

N.S.
 

5.289
 

0.620
 

244.998
 

97
 

8/52(0.15)
 

N.S.
 

2.115
 

0.606
 

9.064
 

83
 

25/54(0.46)
 

N.S.
 

1.415
 

0.847
 

2.392
 

80
 

HIGH
 

DOSE
 

2/52(0.04)
 

N.S.
 

2.115
 

0.114
 

122.378
 

72
 

4/52(0.08)
 

N.S.
 

1.058
 

0.207
 

5.393
 

72
 

25/52(0.48)
 

N.S.
 

1.469
 

0.882
 

2.470
 

64
 
_ __ 

_ __ 

TOPOGRAPHY: MORPHOLOGY
 

Liver: Hcpatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma,
 

Hepatocellular Adenoma or Mixed
 

Hepato/Cholangio Carcinoma^
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Adenoma NOS
 

P Values0
 

Relative Eisk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 6 (CONTINUED) 

CONTROL
 

7/54(0.13)
 

N.S.
 

——
 

101
 

11/54(0.20)
 

N.S.
 

59
 

0/42(0.00)
 

N.S.
 

LOW
 

DOSE
 

1/53(0.02)
 

P = 0.032(N)
 

0.146
 

0.003
 

1.076
 

105
 

4/53(0.08)
 

N.S.
 

0.370
 

0.091
 

1.161
 

105
 

2/47(0.04)
 

N.S.
 

Infinite
 

0.266
 

Infinite
 

105
 

HIGH
 

DOSE
 

3/52(0.06)
 

N.S.
 

0.445
 

0.078
 

1.832
 

105
 

5/52(0.10)
 

N.S.
 

0.472
 

0.138
 

1.363
 

105
 

3/41(0.07)
 

N.S.
 

Infinite
 

0.620
 

Infinite
 

105
 
TABLE 6 (CONCLUDED)
 

TOPOGRAPHY : MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Pituitary: Adenoma NOS, Chromophobe 

Adenoma, or Basophil Adenoma'3 

P Values0 

3/42(0.07) 

N.S. 

2/47(0.04) 

N.S. 

3/41(0.07) 

M.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

0.596 

0.052 

4.956 

1.024 

0.145 

7.232 

Weeks to First Observed Tumor 105 105 105 

Treated groups received doses of 0.006 or 0.012 percent in feed. 

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

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

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

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

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

The 95% confidence interval on the relative risk of the treated group to the control group.
 
collected by this laboratory for the NCI Carcinogenesis Testing
 

Program, leukemia or malignant lymphoma was detected in 22/259 (8
 

percent) of the untreated control male and 44/270 (16 percent) of
 

the untreated control female B6C3F1 mice. Of the 6 groups of
 

untreated control male mice and the 6 groups of untreated control
 

female mice included in these historical control incidences, the
 

highest incidence of this combination of neoplasms was 11/39 (28
 

percent) for males and 11/50 (22 percent) for females.
 

For male mice the possibility of a negative association between
 

dosage and the incidences of hepatocellular carcinomas and of adenomas
 

NOS of the adrenal was noted. No other statistical tests for either
 

sex were significant under the Bonferroni.criterion.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

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

of statistically significant results. It should also be noted that
 

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

indicating the theoretical possibility of tumor induction in mice by
 

2,3,5,6-tetrachloro-4-nitroanisole that could not be established under
 

the conditions of this test.
 

47
 
V. DISCUSSION
 

There were no significant positive associations between the dietary concentration of 2,3 ,5,6-tetrachloro-4-nitroanisole administered
 

and mortality in rats or mice of either sex. Adequate numbers of
 

animals in all groups survived sufficiently long to be at risk from
 

late-developing tumors.
 

Although the incidences of interstitial-cell testicular tumors
 

in dosed male rats were statistically significantly higher than in
 

controls, this was discounted due to the high and variable spontaneous incidence of this lesion in Fischer 344 rats (Cockrell and
 

Garner, 1976). There was an association between dosage and the combined incidence of hepatocellular carcinomas and neoplastic nodules
 

in both male and female rats. However, neither these tumors nor any
 

other tumors occurred at significantly increased incidences when dosed
 

male or female rats were compared to their controls.
 

When those male mice having either leukemia or malignant lymphoma were combined and the resulting incidences analyzed, the Cochran-


Armitage test indicated a significant positive association between
 

the concentration of the compound administered and occurrence of these
 

neoplasms. The high dose to control Fisher exact comparison supported
 

the finding. These hematopoietic lesions occur spontaneously and with
 

great variation in B6C3F1 mice (i.e., historical control incidences
 

of 8 and 16 percent for untreated control males and females, respectively, with maximum incidences in one group of 28 and 22 percent for
 

48
 
males and females, respectively); therefore, the administration of
 

the compound was not considered to be associated with their development. No other tumors occurred in significant positive incidences
 

when dosed mice of either sex were compared to controls. There was
 

a negative trend for the incidences of hepatocellular carcinomas in
 

male mice, which was not attributable to poor survival among the
 

dosed groups.
 

Under the conditions of this bioassay, dietary administration of
 

2,3,5,6-tetrachloro-4-nitroanisole was not carcinogenic to either sex
 

of Fischer 344 rats or to B6C3F1 mice.
 

49
 
VI. BIBLIOGRAPHY
 

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

J. Wiley & Sons, New York, 1971.
 

Berckmans, V.S.F. and A.F. Bailsman, Recueil des travaux Chimiques
 

des Pays-has 44:851, 1925.
 

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

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

Union Against Cancer, Geneva, 1969. 

Carey, W.F., "Determination of Tetrachloronitroanisole (TCNA) Residues in Vegetables and Grains." Journal of the Association of
 

Official Analytical Chemists 46:876-878, 1963.
 

Chemical Abstracts Service. The Chemical Abstracts Service (CAS)
 

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

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

Cockrell, B.Y. and F.M. Garner, "Interstitial-Cell Tumors of the
 

Testis in Rats." Comparative Pathology Bulletin 8:2-4, 1976.
 

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.
 

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

Observations." Journal of the American Statistical Association
 

5_3_: 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., Simultaneous Statistical Inference. McGraw-Hill Book
 

Co., New York, 1966.
 

Peters, A.T., R.M. Rocoe, and D.M. Stead, Journal of the Chemical
 

Society, 233, 1943.
 

50
 
Purdy, L.H., "Comparative Effectiveness of Seed-Treatment Chemicals
 

Against Flag Smut of Wheat." Plant Disease Reporter 47(9):793­

796, 1963.
 

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

Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced
 

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

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

Schaughnessy, J., Registration Division, U.S. Environmental Protection
 

Agency, Washington, D.C. Personal communication, May 24, 1977.
 

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

62^679-682, 1975.
 

U.S.	 Department of Health, Education, and Welfare, Report of the
 

Secretary's Commission on Pesticides and their Relationship to
 

Environmental Health. U.S. Government Printing Office,
 

Washington, D.C. , 1969.
 

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

United States Production and Sales, 1975. USITC Publication
 

804, U.S. Government Printing Office, Washington, D.C., 1977.
 

51
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH 2,3,5,6-TETRACHLORO-4-NITROANISOLE
 

TABLE A1 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH 

2,3,5,6-TETRACHLORO4-NITROANISOLE 

1lONTROL (UNTR ) L OW DOSE HIGH DOSE 2 HIGH DOSE 1 

01-0220 01-0270 01-S275 01-0275 

ANIMAL S INITIALLY IN STUDY 50 49 25 »50

 

ANIMAL S NECFOPSIED 18 49 25 23

 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY* * 48 49 25 23 

INTEGUMENTAR Y SYSTEM 

*SKI N (48) (49) (25) (23)

 

SQUAMOU S CELL CARCINOM A 1 (2% ) 

*SUBCUT TISSU E (48) (49) (25) (23)

 

SQUAMOU S CELL CARCINOM A 1 <4<S )

 

FIBROM A 3 (6%) 2 (4%) 

FIBROSARCOM A 2 (4X ) 

RESPIRATOR Y SYSTEM 

*NASA L TURBINAT E (18) (49) (25) (23)

 

SQUAMOU S CELL CARCINOM A 1 (4X)

 

JLUN G (18) (49 ) (25) (23) 

NEOPLASM , NOS 1 (2*) 

ALVEOLAB/BRONCHIOLA R ADENOM A 3 (6%) 1 ((IX) 

FIBROSARCOMA , METASTATI C 1 (25S) 

HEMATOPOIETI C SYSTEM 

• MULTIPL E ORGAN S (48) (25) (23) 

MALIGNAN T LYMPHOMA , NO S 1 (2*) 

MALIG . LYMPHOMA , U NDIFFER-TYPE 1 (4X) 1 («X) 

LEUKEMIA , NOS 1 (2%) 

UNDIFFEPENTIATE D LEUKEMI A 2 (4*) 

MYELOMONOCYTI C LEUKEMI A 6 (12X ) 2 (3X) 4 (17X) 

(49) 

tSPLEEN (48) (49) (25) (23)

 

MYELOMONOCYTIC LEUKEMI A 4 (8%)

 

•LIVEP (48) (49) (25) (23) 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECEOPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

9 50 ANIMALS WERE INITIALLY IN THE STUDY, BUT 26 WERE FOUND TO BE FEMALES IN A MALE GSOOP. 

A-3 
TABLE Al (CONTINUED) 

CONTROL (UNTE)

01-0220 LOW DOSE 01-0270

 HIGH DOSE

 01-3275 2 HIGH DOSE 01-0275 1 

CIRCULATOR Y SYSTEM 

NONE 

DIGESTIV E SYSTE M 

• LIVER

NEOPLASTIC NODULE

HEPATOCELLULA R CARCINOM A

 (48) (19)

 3

 2

 (6X)

 (4%)

 (25)

 1 (IX) 

 (23) 

3 (13*) 

*STONAC H

FIBROSARCOM A

 (48) (48 ) (25) (23) 

1 (UX) 

tDUODENU M

ISLET-CELL CARCINOMA , METASTATIC

 (46) (49)

 1 (2X) 

 (24 ) (23) 

URINAR Y SYSTE M 

NON E 

ENDOCRIN E SYSTE M 

tEITUITAR Y 

NEOPLASM , NOS 

ADENOMA , NOS 10 (22%) 

(43) 

1 <2X) 

9 (21X) 

(21) 

4 (19X) 

(20) 

4 (20X) 

tADRENA L 

CORTICAL ADENOMA 

PHSOCHROMOCYTOM A 

(46) 

4 (9%) 

(19)

1 (2X) 

it (8X) 

(25) 

3 (12X) 

(22) 

4 (18X) 

tTKYROI D 

CARCINOMA,NO S 

FOLLICULAR-CELL CARCINOM A 

C-CF.LL ADENOM A 

C-CELL CARCINOMA 

(43) 

3 (7)5) 

2 (5)6) 

(45) 

1 (2X) 

1 (2X) 

1 (2X) 

(22) 

1 (5X) 

(22) 

1 <SX) 

1 (5X) 

1 (5X) 

IPASATHYROID 

ADENOMA , NOS 

• PANCHEATI C ISLETS 

I3LET^CELL_ADSNOMA

(25) 

1 («*) 

(44) ___________________________________(46) 

1 (2X1 

(7) 

(25) 

(3) 

(21) 

* NDMBE P

* NUMBER

 OF ANIMAL S

 OF ANIMAL S

 WITH TISSUE

 NECROPSIED 

 EXAMINE D MICROSCOPICALLY 

A-4 
TABLE Al (CONTINUED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE 2 HIGH DOSE 1 

01-0220 01-0270 01-S275 01-0275 

ISLET-CELL CAECINOM A 1 (2%) 

REPRODUCTIV E SYSTEM 

• MAMMAR Y GLAN D (48) <A9) (25) (23) 

FIBROADENOM A 1 <<**> 

ITESTIS (US) (U9 ) (24) (23) INTESSTITIAL-CELL TUMO R 37 (77X) 46 (91%) 19 (79*) 22 (96*) 

SERVOOS SYSTEM 

*BRAIN (46) (US) (24 ) (22) 

OSTEOSAECOMA, METASTATIC 1 (2*) 

SPECIAL SENSE ORGAN S 

NONE 

MUSCHLOSKELETA L SYSTEM 

*SKULL (48) (49) (25) (23) OSTEOSAFCONA 1 (2X )

 

BODY CAVITIES 

*BODY CAVITIES (48) (49) (25) (23) MESOTHELIOMA , NO S 1 (2%) 1 (2% ) 1 (4«)

 

ALL OTHER SYSTEMS 

__NONE 

* NUMBE R Of ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

A-5 
TABLE Al (CONCLUDED) 

CONTBOL(UNTR) 

01-0220 

L OD DOSE 

01-0270 

HIG H DOSE

01-3275 

2 HIGH DOSE

01-0275 

1 

ANIMAL DISPOSITION SUMMARY 

ANIMAL S INITIALL Y IS STUDY 

NATURA L DEATHS 

MORIBUN D SACRIFICE 

SCHEDULE D SACRIFICE 

ACCIDENTALL Y KILLED 

TKPMISA L SACRIFICE 

ANIMA L MISSING 

DELETED ANIMALfSRON G SEX) 

50 

32 U2 

25 

16 

50 

21 

26 

51 INCLUDES AUTOLYZE D ANIMAL S 

TUMOR SUMMAR Y 

TOTAL ANIMALS WITH PRIMARY TUMORS*

TOTAL PRIMARY TUMORS

 U2

 It

 48

 83

 20 

 37 

23 

«5 

TOTAL ANIMAL S WITH BENIGN TUKORS

TOTAL BENIGN TUMORS

 «0

 61

 1)7

 6U

 19 

 29 

22 

33 

TOTAL ANIMALS WITH MALIGNANT TUMORS

TOTAL MALIGNANT TUMORS

 11

 12

 13

 13

 6 

6 

10 

12 

TOTAL ANIMALS WITH SECONDARY TUMORS*

TOTAL SECONDARY TUMORS

 2

 2

 1 

1 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT

TOTAL UNCERTAIN TUMORS

 1

 1

 5

 6

 2 

2 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC 

TOTAL UNCERTAIN TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

* SECONDARY TUMOPS: METASTATIC TUMOFS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN 

A-6
 
TABLE A2 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED WITH 

2,3,5,6-TETRACHLORO^-NITROANISOLE 

CONTROL (ONTR) LOW EOSE HIGH DOSE 

02-0220 02-0270 02-0275 

MIHUS INITIALLY IN STODY 50 50 50 ANIMALS MISSING 1 

ANIMAL S NECROPSIBD 50 50 145 

ANIMAL S EXAMINED HISTOPATHOLOGICALLY** 50 50 45 

INTEGUMENTAR Y SYSTEM 

*SOBCnT TISSUE (50) (50) (45) BASAL-CELL TUMO R 1 (2*) SARCOMA. NOS 1 (2*) 

FIBEOSARCOMA 1 (2X) 1 (2X) FIBRO ADENOM A t (8X) 

RESPIRATORY SYSTEM
 

*N4SAL CAVITY (50) (50) (45)

 PAPILLOMA, NOS 1 (2X)

 

HEMATOPOIETIC SYSTEM 

•MULTIPLE ORGANS (50) (50) (45) 

MALIGNAN T LYMPHOMA , NOS 2 (4X) 

MALIG.LYMPHOMA , LYMPHOCYTIC TYPE 1 (2X) 

MYELOMONOCYTIC LEUKEMIA 6 (12X) 6 (13X) 

ISPLE3N (50) (50) (44)
 

MYELOMONOCYTIC LEUKEMIA 2 (UX) 1 (2X)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVSE (50) (45) 

NEQPLAST1C NODULE, .--3-12X1­

* NUMBER OF ANIMAL S KITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-7 
TABLE A2 (CONTINUED) 

CONTROL (UNTR) LOW DOSE 

02-0220 02-0270 

HEPATOCELLULAR CARCINOMA 

HEMANGIOM A 

flRINAP Y SYSTEM 

NONE 

ENDOCRINE SYSTEM 

IPITUITAPY (40) (48) 

CARCINOMA , NOS 1 (2X) ADENOMA , NOS 16 (40%) 15 (31X) 

CHROMOPHOBE ADENOM A 1 (3X) 

tADRENA L (48) (50) 

CORTICAL ADENOM A 1 (2X) P HE OC HRO MOC YT 0 MA 6 (13*) 1 (2X) 

CTHYROID (43) (47) 

NEOPLASM , NOS 

C-CELL ADENOMA 3 (6*) 

C-CELL CARCINOM A 1 (2*) 

tPANCREATIC ISLETS (46) (50) 

ISLET -CELL ADENOM A 1 (2*) 

REPRODUCTIV E SYSTEM 

*MAMMAP Y GLAN D (50) (50)

 

ADEtiOCARCINOMA , NOS 1 (2*) 

FIBROADENOM A 6 (12X)

 

*CLITORAL GLAN D (50) (50)

 

CARCINOMA , NOS 1 <2*>

 

KERATOACAHTHOM A 

tUTERUS (48) (50) 

ADENOCARCINOMA , NOS 1 (2X) 

FIBROM A 2 (4*) LEIOMYOM A 

LEIOMYOSARCOM A 

ENDOMETRIA L STROMAL POLYP 2 ("*) 9 (18*) 

# NUMBE R Of ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

HIGH DOSE 

02-0275 

1 (2X) 

1 <2X) 

(40) 

1 (3X) 

14 (35X) 

(44) 

1 <2X) 

(37) 

1 <3X) 

1 (3X) 

("2) 

(45) 

1 (2X) 

(45) 

1 (2X) 

(45) 

1 (2*) 

1 (2X)

a_jii8*j 

A-8 
TABLE A2 (CONTINUED) 

CONTROL (UNIR)

02-0220 LOM DOSE 02-0270

 HIGH DOSE 

 02-0275 

ENDOMETRIAL STROM AL SARCOMA 2 (it*) 

NERVOU S SYSTEM 

»BRAIN (50) (50) (45) CARCINOMA , NOS, INVASIVE 1 (2X) 1 (2«) 

ASTROCYTON A 1 (2*) 

SPECIAL SENSE ORGAN S 

NONE 

M'JSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

*ABDO«INAL CAVITY (50) (50) (45) LEIOMYOSARCOMA 1 (2%) 

ALL OTHER SYSTEMS 

NONE 

ANIMA L DISPOSITION SDMMAR Y 

ANIMAL S INITIALLY IN STUDY 50 50 50 

NATURA L DEATHS 7 4 11 

MORIBUN D SACRIFICE 2 3 2 

SCHEDULED SACRIFICE 5 

ACCIDENTALLY KILLED 

TERMINA L SACRIFICE 36 43 36 

ANIMA L MISSING 1 

t NDMBER OF ANIMAL S KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

A-9 
TABLE A2 (CONCLUDED)
 

CONTROL(UNTR) LOW DOSE HIGH DOSE 

02-0220 02-0270 02-0275 

TUMOR SUMMAR Y 

TOTAL ANIMAL S WITH PRIMARY TONORS* 27 3« 28 

TOTAL PRIMAR Y TDMOBS U2 US 45 

TOTAL ANIMALS WITH BENIGN TUHORS 23 30 20

 

TOTAL BENIG N T<JMOBS 33 35 28

 

TOTAL ANIMALS WITH MALIGNANT TOMOfiS 9 12 11

 

TOTAL MALIGNAN T TUMOES 9 12 13

 

TOTAL ANIMAL S HITH SECONDARY TUMORS* 1 1

 

TOTAL SECONDARY TUMORS 1 1

 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIG N O R MALIGNAN T 1 a

 TOTAL UNCERTAI N TUMORS 1 4

 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIBABY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

A-IO
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH 2,3,5,6-TETRACHLORO-4-NITROANISOLE
 

TABLE Bl 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH 

2,3,5,6-TETRACHLORO^t-NITROANISOLE 

CONTROL (UNTE ) LOW DOSE HIGH DOSE 

05-0360 05-0271 05-0276 

ANIMAL S INITIALLY IN STUDY 55 55 55 

ANIMALS MISSING 1 

ANIMAL S NECROPSIED 55 55 53 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY* * 5 5 514 52 

INTEG'JMENTAR Y SYSTEM 

*SKIN (55) (55) (53) SO.UAMOUS CELL PAPILLOMA 1 (2X)

 

*SUBCnl TISSUE (55) (55) (53) FIBROM A 2 (4%) 

FIBROSARCOM A 1 (2%) 1 (2X) 

LiSIOMYOSARCOMA 2 (MX) 2 (»*) HEMANGIOM A 1 (2X) 

RESPIRATOR Y SYSTEM 

#LUN G (51) (54) (51) HEPATOCELLULA R CARCINOMA , METAST 4 (7S) ALVEOLAR/BRONCHIOLA P ADENOM A 6 (11*) 4 (7X) i» (8X) 

ALVEOLAR/BRONCHIOLAR CARCINOMA 6 (11%) 13 (214%} 9 (18*) 

CORTICAL CARCINOMA , METASTATI C 1 (2*)' 

HEflATOPOIETIC SYSTEM 

*MULTIPLE ORGAN S (55) (55) (53 ) 

MALIGNAN T LYMPHOMA , NO S 3 (5%) 

MALIG.LYMPHOMA . UNDIFFER-TYP E 1 (2X) 3 (611) 

MALIG.LYMPHOMA , LYMFHOCYTIC TYP E 2 (UX) 1 <2X) MALIG.LYMPHOMA , HISTIOCYTIC TYPE 1 (2X) 1 (2X ) MALIGNAN T LYMPHOMA , MIXE D TYP E 2 (4R) 3 (6X) GRANULOCYTI C LEHKEHI A 1 (2X) 

*SPLEEN (51) (53) (50) 

NEOPLASM , NOS 1 (2X) 

HEMANGIOM A 1 (2X) 

HEMAN2IOSARCOM A 

* NUMBE R OF ANIMAL S WIT H IISSIIE EXAMINE D MICROSCOPICALL Y 

* NUMBER OF ANIMAL S NECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-3 
TABLE Bl (CONTINUED) 

CONTROL(UNIR ) L OW DOSE 

05-0360 05-027 1 

• MEDIASPINA L L. NODE (tB) (50) 

ALVEOLAR/BRONCHIOLA R CA, METAST A 1 <2X ) 

tBESENTEPIC L. NOD E (48) (50) HEPATOCELLULA R CARCINOBA, METAST 1 (2X ) 

HEBANGIOSARCOM A 1 (2t) MALIG.LYMPHOMA , UNDIFFER-TYP E 

MALIG.LYMPHOMA , LYMPHOCYTIC TYPE 1 (2X) MALIGNAN T LYMPHOMA , MIXE D TYP E 

tAXILLAP Y LYMP H NOD E (U8 ) (50)

 LEIOMYOSARCOMA , METASTATIC

 

• DUODENU M (50) (52) MALIG.LYBPHOBA , LYMPHOCYTIC TYPE 1 (2<S) 

•JEJUNU M (50) (52) MA L IS. LYMPHOMA , UNDIFFER-TYP E

 

MALIGNAN T LYMPHOMA , MIX2D TYPE 1 (2X)

 

CIPC'ILATOEY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

tLIVER (5«) (52) 

HEPAIOCELLULA R ADENOM A t (7K ) 3 (6X) 

HEPATOCELLDLA R CARCINOM A 2 U (4ltX) 13 (255!) 

MIXED HEPATO/CHOLANGIO CARCINOMA 2 C*X) ANGIOSARCOB A 2 (4X ) 

• STOMACH (51 ) (52) 

SQUAMOU S CELL CARCINOM A 1 (2% ) 

• DUODENU M (50) (52) ADENOBATOtl S POLYP , NOS 1 (2%) 

UBINAS Y SYSTE M 

• KIDNEY/PELVI S (5t) (53) 

* NUMBE R OF ANIMAL S KITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

B-4 

HIG H DOSE 

05-0276 

(K7) 

(17) 

1 (2*) 

1 (2*) 

1 (2X) 

(U7 ) 

1 (2%) 

(50) 

(50) 

1 (2X) 

(52) 

2 (tX) 

9 (17X) 

2 (UX) 

3 (6X ) 

(50) 

(50) 

(52 ) 
TABLE Bl (CONTINUED) 

CONTROL (UNIR) L OU DOSE HIG H DOSE 

05-0360 05-0271 05-0276 

ENDOCRINE SYSTEM 

tADPENA L (50) (52) (19)

 

CORTICAL ADENOM A 1 (2%)

 

CORTICA L CARCINOM A 1 <2X )

 

#ADRENAL/CAPSULE (50) (52) (149) ADENOMA , NOS 5 (10*)

 

ITHYROID (US ) (19) (11)

 FOLLICITLAR-CELL ADENOMA 1 (2X)

 

FOLi-lCULAR-CELL CARCINOM A 2 (IX) 

iPANCFEATIC ISLETS (49) (53) (19) 

ISLET-CELL ADENOM A 2 (4%) 

REPRODUCTIVE SYSTEM 

*PKEPUTIA L GLAN D (55) (55) (53) 

HEMANGIOSARCOM A 1 (2X ) 

NERVOU S SYSTEM 

NON E 

SPECIAL SENSE ORGANS 

*HAHDERIA N GLAN D (55) (55) (53) 

CYSTADENOMA , NOS 1 (2X ) 

*EXTERNA L EAR (55) (55) (53) PIBPOHS HISTIOCYTOMA 1 (2*) 

MUSC1LO SKELETAL SYSTEM 

NONE 

BODY CAVITIES 

__NON E 

* NUMBE R OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECP.OPSISD 

B-5 
TABLE Bl {CONCLUDED) 

COHIEOL(ONTH) LOW DOSE HIGH DOSE 

05-0360 05-0271 05-0276 

ALL 01 HEP SYSTEMS 

*MULIIPLE ORGAN S (55) (55) (53) 

NEOPLASM , NOS 2 (4X) 

SARCOMA, NOS 1 (2*) 

ANIMA L DISPOSITION SDMMAK Y 

ANIMAL S INITIALLY IN STUDY 55 55 55 NATURA L DEATHS 11 11

 

MORIBUN D SACRIFICE 14 1

 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 2 

TERMINA L SACRIFICE 44 to ANIMA L MISSING 1

 

3 INCLUDES AUTOLYZE D ANIMAL S 

TUMO R SUMMAR Y 

TOTAL ANIMAL S WITH PRIMAR Y TUMORS* 43 38 36 

TOTAL PRIMARY TUMORS 59 57 50 

TOTAL ANIMAL S WITH BENIGN TUMORS 22 8 6 

TOTAL BENIG N TUMOFS 24 10 7 

TOTAL ANIMAL S WIT H MALIGNAN T TUMORS 29 34 32 

TOTAL MALIGNAN T TDMOP.S 35 47 40 

TOTAL ANIMAL S WIT H SECONDAR 4 Y TUMORS* 2 1 

TOTA L SECONDARY TUMORS 5 2 1 

TOTAL ANIMAL S WIT H TUMORS UNCERTAINBENIG N OR MALIGNAN T 3 

TOTAL UNCERTAI N TUMORS 3 

TOTAL ANIMAL S WIT H TUMORS UNCERTAINPRIMAR Y OR METASTATIC

 

TOTAL UNCERTAI N TUMOSS 

* PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

* SECONDARY TUMORS : METASTATIC TUMOKS OR TUHORS INVASIVE INTO AN ADJACENT OBGAN 

B-6 
TABLE B2 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED WITH 

2,3,5,6-TETRACHLORO^-NITROANISOLE 

CONTROL (HNTR) L OW COSE HIGH DOSE 

06-0360 06-0271 06-0276 

ANIMAL S INITIALL Y IN STUDY 55 55 55 

ANIMAL S MISSIN G 1 1 

ANIMAL S NECROPSIE D 55 54 52 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY ** 55 54 52 

INTEGUMENTARY SYSTEM
 

*SKIN (55) (54) (52) 

SQUAMOU S CELL CARCINOM A 1 (2%) 

*SUBCUT TISSUE (55) (54) (52) FIBROSARCOM B 1 <2X ) 1 (21) HEMANGIOM A 1 (2X ) 

RESPIRATORY SYSTEM
 

ILUNG (55) (52) (52) HEPATOCELUILAR CARCINOMA , WETAS T 2 (4%) 1 (2X ) MIXE D HEPATO/CHOLANGIOCA, METAST 1 (21) ALVEOLAR/BRONCHIOLA R ADENOM A 3 (55) 3 (6X) 2 (4*) ALVEOLAR/BRONCHIOLA P CARCINOM A 1 (2*) 5 (IDS ) 2 (4X) 

HEMATOPOIETIC SYSTEM
 

*MULIIPLE ORGANS (55) (54) (52) MALIGNAN T LYMPHOMA , NO S 1 (2% ) 

MALIG . LYMPHOMA , UNDIFFER-TYPE 2 (4S) 3 (6X) MALIG . LYMPHOMA , LYMPHOCYTIC TYPE 2 (4%) 11 (20S) 10 (19X ) MALIG . LYMPHOMA , HISTIOCYTIC TYPE 1 (2X) 2 (4X) MALIGNAN T LYMPHOMA , MIXE D TYPE ' 6 (1U) 2 (4X) 5 (10X) LYMPHOCYTIC LEUKEMI A 1 <7X ) 

*SPLBEN (53) (52) (51) HEMANGIOSARCOM A 2 (4% ) 1 (2X) MALIG . LYMPHOMA , LYMPHOCYTIC TYPE 3 (6%) MALIGNAN T LYMPHOMA, MIXED TYPE 1 (2S) 1 (2«) 1 (2X) 

tMEDIASTINA L L.NOD E (47) (50) (46)

 

MIXED HEPATO/CHOLANGIOCA, METAST 1 (2%)

 

» NUMBE R OF ANIMAL S KITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECEOPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-7 
TABLE B2 (CONTINUED) 

CONTRO L (ONTR) L OU DOSE 

06-0360 06-0271 

tMESENTERI C L. NODE (47) (50) MA LIG. LY MPHOMA , LYMPHOCYTIC TYPE 1 <2X ) 

MALIGNAN T LYMPHOMA , MIXED TYPE 2 <<*% ) 1 (2%) 

#RENA L LYKP H NODE (47) (50)

 

SARCOMA , NO S 

*LIVER (54) (53) MALIGNAN T LYMPHOMA , NO S 1 (2*) MALIG. LYMPHOMA, HISTIOCYTIC TYPE 2 (4%) 

tILEU M (52) (53)

 MALI G .LYMPHOMA , LYMPHOCYTIC TYPE

 

tCOLON (48) (51)

 MALIG . LYMPHOMA , D NDIFFES-TY PE

 

tTHYTJS (35) (39) ALVSOLAR/B50NCHIOLA F CA , METAST A 1 (3X ) THYMOM A 1 (3%) MALIGNAN T LYMPHOMA, MIXED TYPE 

CIRCULATOR Y SYSTEM 

IHEAS T (55) (53) ALVEOLAR/BRONCHIOLA R CA , METAST A 1 (2*) HEMANGIOM A 1 (2«) 

DIGESTIV E SYSTEM 

KLIVE H (54) (53) HEPATOCELHILA R ADENOM A t (7%) 2 (4%) HEPATOCELLULA R CARCINOM A 7 (13*) 1 (2%) MIXE D HEPATO/CHOLANGI O CARCINOM A 1 (2X ) HEMANGIOM A 1 (2X) ANGIOSARCOMA 

• PANCREA S (49 ) (51) MIXE D HEPATO/CHOLANGIOCA , METAS T 1 (2X) 

fSTOMACH (53) (51) 

PAPILLOMA.^NOS,, 

* NIIMBE P OF ANIMAL S WITH TISSHE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

HIGH DOSE 

06-0276 

(46) 

2

(46 ) 

1

(52) 

1

(51) 

1

(49) 

1

(38) 

1

(52) 

(52) 

2

1

1

(50) 

(51) 

 (4*) 

 (2X) 

 (2X) 

 (2*) 

 (2X) 

 (3X) 

 (4X) 

 (6X) 

 (2*) 

B-8 
TABLE B2 (CONTINUED) 

SQUAMOU S CELL PAPILLOM A 

MIXE D HEPATO/CHOLANGIOCA ,

IDUODENU M 

ADENOMATOU S POLYP, NOS 

URINAR Y SYSTEM 

NONE 

ENDOCSIN E SYSTEM 

(t PITUITAR Y 

ADENOMA , NO S 

CHROMOPHOBE ADENOM A 

BASOPHIL ADENOM A 

SARCOMA , NOS , METASTATIC 

tADRENA L 

PHEOCHROMOCYTOM A 

#THYROID 

FOLLICULAR-CEL L ADENOM A 

FOLLICULAR-CELL CAPCINOM A 

REPPODUCTIV E SYSTEM 

*MAMMAB Y GLAN D 

ADENOCARCINOMA , NO S 

ACINAR-CELL CARCINOM A 

FIBROADENOM A 

*UTERU S 

NEOPLASM , NOS, MALIGNAN T 

I2IOMYOM A 

ENDONETRIA L STROMA L POLYP 

• OVAR Y

MIXE D HEPATO/CHOLANGIOCA,

CONTROL(UNIR) 

06-0360 

L OW DOSE 

06-027 1 

HIGH DOSE 

06-0276 

2 (1*) METAST 1 (2%) 

(52) (53) (51) 

1 U*) 

(12) (47 ) 

2 (1*) 

(11) 

3 (7X) 

2

1

 (51) 

 (2%) 

1 (2*) 

(50) (53) (19) 

1 (2%) 

(18) 

1 (2S) 

(39) (18) 

1

1

 (2*) 

 (2*) 

(55) (51) 

1 (2*) 

(52) 

1 (2%) 

1 (2S) 

1 (2X) 

(51) 

1 (2% ) 

(51) (52) 

1

2

 (2*) 

 (IX) 

 (50 ) (19) (19) 

 METAST 1 (2<) 

PAPILLAR Y CYSTADENOMA ,

CHOPIOCARCINOM A

HEHANG1 0 SARCOM A 

 NOS 1

 1

 (2*) 

 (2X) 

#

*

 NUMBE R

 NUMBE R

 OF

 OF

 ANIMAL S

 ANIMAL S

 WITH TISSUE

 NECEOPSIED 

 EXAMINE D MICROSCOPICALLY 

B-9 
TABLE B2 (CONTINUED) 

CONTROL ('J NTH) LOW DOSE HIGH DOSE 

06-0360 06-0271 06-0276 

NERVOU S SYSTEM 

IBRAI N (55) (52) (52) 

SARCOMA , NOS, METASTATIC 1 (2X) 

SPECIA L SENSE ORGAN S 

•HAEDERIA N GLAN D (55) (5U) (52) 

PAPILLAR Y ADENOM A 1 (2X) 

PAPILLAR Y CYSTADENOMA , NOS 1 (2X) 1 (2X) 

MIISCULOSKELETA L SYSTEM 

DONS 

BOD Y CAVITIE S 

*BODY CAVITIES (55) (5U) (52) 

MESOTHELTOMA , NOS 1 (2») 

A LL OTHER SYSTEM S 

*MULTIPL E ORGAN S (55) (5<t) (52) 

SARCOMA , NOS 1 (2%) 

HL'AD 

SARCOMA , NOS 1 

ANIMA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALL Y IN STUD Y 55 55 55 

NATURAL DEATHS 7 11 10 

MORIBUN D SACRIFICE H 5 1 

SCHEDULE D SACRIFICE 

ACCIDENTALL Y KILLE D 

TERMINA L SACRIFICE "4U 38 13 

ANIMA L MISSING 1 1 

S_INCLUDES_AUTOLYZED_ANIMAL S 

# NUMBE R OF ANIMAL S WITH TISSUE EXABIKED HICROSCOPICALL* 

* NUMBER OF ANIMAL S NECROPSIED 

B-IO 
TABLE B2 (CONCLUDED)
 

(:ONTEOL(UNTR ) L O W DOSE HIG H DOSE 

06-0360 06-027 1 06-0276 

TUMO R SUMMAR Y 

TOTAL ANIMAL S WITH PRIMAR Y TUMOPS* 3U J7 38 

TOTAL PHIMAP Y TUMORS 50 U 7 52 

TOTAL ANIMAL S KITH BENIG N TUHORS 15 9 12 TOTAL BENIG N TUMORS 19 1 1 13 

TOTAL ANIMAL S WITH MALIGNAN T TUMOR S 27 3t 32

 TOTAL MALIGNAN T TOHORS 30 36 39

 

TOTAL ANIMAL S WITH SECONDAR Y TUMOPS* 2 3 1 

TOTA L SECONDAR Y TUMORS 2 9 1 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT 1
 TOTAL UNCERTAIN TOMOHS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OH METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

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

APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH
 

2,3,5,6-TETRACHLORO-4-NITROANISOLE
 

TABLE Cl 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS TREATED WITH 

2,3,5,6-TETRACHLORO^t-NITROANISOLE 

CONTROL (UNTR ) LOW DOSE 

01-0220 01-0270 

ANIMAL S INITIALL Y IN STUDY 50 49 

ANIMAL S NECROPSIE D 48 49 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY** 48 49 

INTEGUMENTAR Y SYSTE M 

•SKIN (48) (49) 

EPIDERMA L INCLUSIO N CYST 1 (2*) 1 (2X) 

ULCER, ACUTE 1 (2X ) 

DSGENERATION , NOS 1 (2%) 

NECROSIS, NOS 1 (2)5) 

*SUBCUT TISSUE (46) (49) ABSCESS, NOS 3 (6«) 1 (2*) 

RESPIRATOR Y SYSTEM 

*NASAL TURBINAT E (48) (49) INFLAMMATION , SUPPURATIV E 1 (2X) 

tLUNS/BPONCHU S (49) 

BRONCHIECTASI S 1 (2X) 

tLUN G (48) (49) 

EDEMA , NOS 

INFLAMMATION , FOCAL 3HANULOM4TO U 2 (4X) 

PSPIVASCULA R CUFFING 1 (2*) 

HYPERPLASIA , ADENOMATOU S 2 (4X) 

HISTIOCYTOSIS 1 (2%) 

HYPERPLASIA , LYMPHOI D 

HEHATOPOIETIC SY5TEK
 

*BONE MARRO W (46) (47) 

MYELOFIBROSI S 

tSPLEEN («8) (49 ) 

NECRgSISi_FOCAL_. 

# NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

9 50 ANIHAL S WER E INITIALL Y IN THE STUDY, BUT ^6 WER E FOUN D TO

HIGH

01-S

 DOSE 2 

275 

HIGH DOSE 

01-0275 

1 

25 

25 

25 

150 

23 

23 

(25) (23) 

(25) (23) 

(25) (23) 

(25) (23) 

(25) (23) 

1 ("»«) 

1 (4X) 

1 ("*) 

1 <4X) 

(25) (22) 

(5X> 

(25) (23) 

 BE FEMALES IN A MALE GBODP. 

C-3 
TABLE Cl (CONTINUED) 

CONTROL (UNTR ) LOU DOSE 

01-0220 01-0270 

INFARCT , NOS

LEDKEHOI D REACTION 

1 (2% ) 

1 (2*) 

tMANDIB'JLA P L. NODE (U3 ) (U6 ) 

PLASMA-CEL L INFILTRAT E 2 (UX ) 

HISTIOCYTOSIS 1 (2% ) 

PLASNACYTOSIS 2 (US) 

*CEFVICA L LYMP K NODE (43) (16) 

PLASMA-CEL L INFILTRAT E 1 (2% ) 

HISTIOCYTOSIS 1 (2U ) 

• RENA L LYMP H NOD E («3) (46 ) 

PLASMACYTOSI S 

ITHYNU S (32) (314) 

CYST, NOS 

ATROPHY , NO S 

HYPERPLASIA , NOS 1 (3% ) 

CIPCULATOP.Y SYSTEM 

• HEAR T (U8 ) (1*9) 

THROMBOSIS , NOS 

FIBBOSIS 1 (2% ) 

FIBROSIS, FOCA L 19 (39%) 

DIGESTIV E SYSTEM 

• LIVER (U8 ) (09) 

THROMBOSIS, NOS 

HEMORRHAG E 

LYMPHOCYTI C INFLAMMATOR Y INFILTR 

NECROSIS , NOS 

NECROSIS , FOCA L 1 (2% ) 

METAMORPHOSIS FATTY 2 (U«) 

LIPOIDOSIS 

CYTOPLASMI C V A COOL IZAT ION 

BASOPHILIC CYTO CHANG E U (8% ) 

CLEAR-CEL L CHANG E 3 (6X ) 

HYPERPLASIA, FOCAL 3 (6%) 

ANGIECTASI S 2 («) 

*LIV£R/HEPATOCYTE S (U8 ) (19)

HYPERTROPHY , FOCAL 1-J2H_ 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIE D 

HIGH DOSE 2 

01-3275 

(23) 

(23) 

(23) 

(19) 

1 (5*) 

(25) 

5 (20*) 

(25) 

1 (U» ) 

1 (It*) 

1 (UX ) 

(25) 

HIGH DOSE 1 

01-0275 

(19) 

(19) 

(19) 

1 (5*) 

(16) 

1 (6X) 

(23) 

1 (UX ) 

11 (61X) 

(23) 

1 (UX) 

1 (UX ) 

1 (UX ) 

1 (UX ) 

1 («%) 

3 (13X) 

2 (9X) 

1 (4X) 

(23) 

C-4 
TABLE Cl (CONTINUED) 

CONTROL (UNTR) LOW DOSE 

01-0220 01-0270 

*BILE D'ICT (18) (49) 

HYPERPLASIA , NOS 6 (12% ) 

• PANCREA S (11) («6)

 

INFLAMMATION , NOS 2 <5X)

 

ATROPHY , FOCAL 

IPANCFEATIC ACINU S ft" ) (16) 

ATROPHY , NOS 1 <9X ) 

AT80PHY, FOCAL a (17%) 

•STOMACH (18) (18)

 

EDEMA , NOS 

INFLAMMATION , ACUTE/CHRONIC 

INFLAMMATION , CHRONIC 

HYPERPLASIA , PSEUDOEPITHELIOMATO

 

HYPERKERATOSI S 

ACANTHOSIS 

• GASTRIC flUCOSA (U8) (18)

 

HYPERPLASIA , DIFFUSE 

• JEJUNU M (16) (19)

 

"AHASITISM 

•ILEUM (16) (19)

 INFLAMMATION , FOCAL

 

•COLON (12) (19) 

LYMPHOCYTIC INFLAMMATOR Y INFILTP 

PARASITISM 6 (12%) 

URINAR Y SYSTEM 

•KIDNEY (18) (19) 

EMBOLUS, SEPTIC 

INFLAMMATION, FOCAL 

INFLAMMATION , SUPPURATIV 2 

NBPHROPATH Y 35 (73 X) 20 (11%) 

METAPLASIA, OSSEOUS 1 (2%) 

tKIDNEY/TUBULE (18) (19) 

PIGMENTATION. SOS 1_J2%1 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

* N'JMBEE OF ANIMAL S MECROPSIED 

HIGH DOSE 2 

01-S275 

(25) 

1 (1«) 

(25) 

1 (1%) 

(25) 

2 (8%) 

(25) 

1 (K% ) 

1 (1%) 

1 (IX) 

1 (IX) 

(25) 

1 («%) 

(21) 

(21) 

1 («X) 

(23) 

5 (22%) 

(25) 

11 (UU% ) 

(25) 

HIGH DOSE 1 

01-0275 

(23) 

9 (39%) 

(21) 

(21) 

1 (5%) 

3 (11%) 

(23) 

1 (1%) 

1 (IX) 

(23) 

(23) 

1 (1%) 

(23) 

(23) 

1 (U%) 

2 (9%) 

(23) 

1 (1%) 

1 (1*) 

1 (IX) 11

 (48%) 

(23) 

C-5 
TABLE Cl (CONTINUED) 

ENDOCRIN E SYSTE M 

*P1T'JITAP Y 

HYPEPPLASIA , FOCAL 

*ADPENA L 

NECROSIS , FOCA L 

ANGIECTASI S 

HEHAIOPOIESI S 

*ADPENA L CORTE X 

HEBOPRHAG E 

NECROSIS , NOS 

LIPOIDOSIS 

ANGIECTASI S 

• ADRENA L MBDTILL A 

HYPERPLASIA , NOS 

HYPEPPLASIA , FOCA L 

(THYROI D 

HYPEPPLASIA , C-CELL 

• PANCREATI C ISLSTS 

HYPERPLASIA , NOS 

REPRODUCTIV E SYSTE M 

*BAMMAR Y GLAN D 

DISPLACEMENT , NO S 

LACTATIO N 

• PROSTAT E 

INFLAMMATION , SU PPUR ATI VE 

INFLAMMATION , ACUTE 

INFLAMMATION , ACUTE/CHRONI C 

*SEMINA L VESICLE 

INFLAMMATION , SUPPUHATIV E 

#TESTIS 

MINERALIZATIO N 

ATROPHY^ . NOS 

* NUHBE1 ? OF ANIMAL S WIT H TISSUE

* NUMBE R O F ANIMAL S NECROPSIE D 

CONTPOL(UMTR ) LOW DOSE HIG H DOSE 2 

01-0220 0 1-0270 01-S275 

(45) (43 ) (21 ) 

1 (2%) 4 (9%) 

(46) (49 ) (25) 

1 (2% ) 

1 (2X ) 

1 (»X ) 

(46) (49 ) (25 )

1 (4X) 

1 (4*) 

1 (4%) 

1 (4X) 

(46) (49) (25) 1 (2% ) 

3 (7% ) 3 (6%) 1 (4« ) 

(43 ) (45) (22) 

1 (2% ) 

(44 ) (46 ) (25) 1 (2%) 1 (2%) 

(48) (49) (25) 

1 <2X ) 

1 (2% ) 1 (4*) 

(45) (49 ) (24) 

1 (4X ) 

1 (4X ) 

(48) (49) (25) 

(48) (49 ) (24) 1 (2K ) 

- S 18*1 2 J4*) 3 (13X) 

 EXAMINE D MICROSCOPICALLY 

HIGH DOSE ! 

01-C I275 

(20) 

(22) 

(22 ) 

(22) 

(22) 

(21) 

(23) 

(23)

1 (4X)

 2 (9X)

3 (13X) 

(23) 

1 ("X)

 

(23) 

C-6 
TABLE Cl (CONTINUED) 

HYPERELASIA , INTERSTITIAL

*EPIDIDYMIS

DILATftTION/DUCIS

ABSCESS, NOS

 CBLI

CONTROL (UNTH)

01-0220

 3 (6%)

 (U8)

 1 (2%) 

 LOW DOSE

 01-0270

 (49)

 1 (2%) 

 HIGH DOSE

 01-S275

 1 (tJ) 

 (25)

 2 HIGH DOSE

 01-0275 

 (23) 

1 

NERVOU S SYSTEM 

• BRAI N

EMBOLUS , SEPTIC

HEMORRHAG E

FIBROSIS

NECROSIS , HEMOPRHAGI C

 (U6 ) (US)

 1 (2% ) 

1 (2*) 

 (2U ) (22) 

1 (5*) 

1 (5X) 

SPECIAL SENSE ORGAN S 

*EY E

CATARAC T

*EYE/REriN A

ATfiOPHY , NOS

 (U8 )

 1 (2X)

 (48)

 2 (<t«) 

 (49)

 (49)

 (25)

 (25)

 (23) 

1 (IX) 

 (23) 

MUSCIILOSKELETA L

NONE 

 SYSTEM 

BODY CAVITIES 

• MESENTER Y

PERIAPTERITI S

 (US) (49)

 1 (2R) 

 (25) (23) 

A LL OTHER

NONE 

 SYSTEMS 

SPECIAL MORPHOLOGY SUMMAR Y 

NO_LESION_REPORTEB

* SUMBEB OF ANIMAL S WIT H TISSUE

* NUMBE R OF ANIMAL S NECROPSIED 

 EXAMINE D MICROSCOPICALLY 

2 

C-7 
TABLE Cl (CONCLUDED) 

AtnO/NECROPSY/HISTO PERF 

AUTO/NECROPSY/NO HISTO 

AOIOLYSIS/NO NECROPSY 

CONTROL (UNTH ) LOW DOSE 

01-0220 01-0270 

1 

1 

2 

t NUHBEP OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS WECROPSIED 

HIGH DOSE 2

01-S275

 HIGH DOSE 

 01-0275 

1 

1 
TABLE C2 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS TREATED WITH 

2,3,5,6-TETRACHLORO^-NITROANISOLE 

CONTROL (UNTE ) L OW DOSE HIGH DOSE 

02-0220 02-0270 02-0275 

ANIMAL S INITIALL Y I N STUD Y 50 50 50

 

ANIMALS MISSING 1

 

ANIMAL S NECROPSIED 50 50 U5

 

ANIMAL S EXAMINE D HISTOPATHOLOGICA LLY ** 50 50 1(5

 

INTEGUMENTAR Y SYSTEM 

*SKIN (50) (50) (15) 

EPIDiiRMAL INCLUSIO N CYST 1 (2*) 

FIBSOSIS, FOCAL 1 (2*) 

POLYPOID HYPERPLASI A 1 (2*) 

HISTIOCYTOSIS 1 <2X ) 

RESPIRATOR Y SYSTEM 

*NASA L TU'miNATE (50) (50) (US)

 

INFLAMMATION , NOS 1 (2«) 

*LUNG/BPONCHU S (50) (50)

 

BSONCHIECTASI S 1 (2%) 

#LBN G (50) (50) (tt) 

ATELECTASI S 1 (2%)

INFLAMMATION , NOS 1 (22!) 

LYMPHOCYTI C INFLAMMATOR Y INFI1TE (2X ) 

INFLAMMATION , INTERSTITIAL 1 (2t) 

BRONCHOPNEUMONI A SUPP'JRATIVE <2X ) 

ABSCESS, NOS 2 («*) 

PNEUMONIA , CHRONI C MURIN E 1 (2«) 

G3ANULOKA , NOS 1 (2*) 

HEMATOPOIETIC SYSTEM
 

#BONE MARROW (U8) (U7) (U2)
 MYELOFIBROSIS 1 (2X)
 

ISPLEEN (50) (50) (UU)
 

___ HEMAIOPOIESIS
 

# NUMBER OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

* EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

C-9 
TABLE C2 (CONTINUED) 

CONTROL(UNTR ) L OW DOSE HIGH DOSE 

02-0220 02-0270 . 02-0275 

*LUMBAR LYMPH NODE (43) (49) (43) HYPERPLASIA , NOS 1 (2X)

 

tPENA L LYMP H NODE (43) (49) (13)

 SIDEROSI S 1 (2X)

 

ITHYMU S (3D (40) (32) HEMORRHAG E 1 (3X) 1 (3X) ATROPHY , NOS 1 (3X) 

CIRCULATOR Y SYSTEM 

KHEART (50) (50) (44) FIBROSIS, FOCAL 1 (2X) 1 (2X) 

• ENDOCARDIU M (50) (50) (44)

 

INFLAMMATION , CHRONIC 1 (2X)

 

DIGESTIV E SYSTEM 

tLIVER (50) (50) (45) 

CONGESTION , NOS 1 (2X) 

HSMOPFHAG E 1 (2X) 

INFLAMMATION , CHRONIC (2X) 

INFLAMMATION , CHRONIC FOCAL 1 (2X) 

INFLAMMATION , FOCAL GRANULOBATO U (2X) 

CHOLANGIOFIBROSI S 1 UX) 

NECROSIS , NOS 1 (2X) 

NECROSIS , FOCA L 1 (2%) 

METAMORPHOSIS FATTY 4 (8X) 

BASOPHILIC CYTO CHANG E 25 (50%) 16 (36X) 

HYPERPLASIA , FOCAL 9 (18X) 

ANSIECTASI S 1 <2X ) 

*LIVER/CENTRILOBI'LA R (50) (50) 

NSCROSIS, NOS 1 (2X) 

•BILE DUCT (50) (50) (45) 

CYST, NOS 1 (2X) 

HYPERPLASIA , NOS 1 <2X) 

tPANCREATIC ACISU S (46) (50) (42)

 

ATRgPHYi _NOS 

« NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECPOPSIiD 

C-IO 
• • 

TABLE C2 (CONTINUED) 

CONTROL (UNTH) LOW DOSE HIGH DOSE 

02-0220 02-0270 02-0275 

ATHOPHY , FOCAL 4 (10%) 

#STOMAC H (49) (50) (44) 

FIBROSIS, FOCA L 1 (2X) 

HYP2RPLASIA , PAPILLAR Y 1 (2X ) 

• ILEU M (47) (50) (44) 

CALCIFICATION , METASTATIC 1 (2X) 

•COLON (40 ) (47) (42) PARASITISM 6 (13X) 4 (10%) 

URINAR Y SYSTE M 

• KIDNE Y (49) (50) (45) 

GLOMEFULONEPHRITIS , NOS 1 (2X ) NEPHROPATH Y 18 (37%) 3 (6%) 3 (7X) 

IKIDNEY/THBULE (49) (50) (45) 

PIGMENTATION , NOS 1 (2%) 

CYTOPLASMIC VACUOLIZAT I ON 1 (2X ) 

• URINAR Y BLADDER (47) (49 ) (45) 

HEMORRHAG E 1 <2X ) HYPERPLASIA , EPITHELIA L 1 (2X) 

ENDOCRIN E SYSTSM 

tPITUITAH Y (40 ) (48) (40) MINERALIZATIO N 1 (3*) 1 (3X) HE10RFHAGE 2 (5)5) SIDEfOSI S 1 (3X) HYPEFPLASIA , FOCAL 1 (2%) 

tADRENA L (48) (50) (44)
 

ANGIECTASIS 1 (2%)
 

•ADRENAL	 CORTEX (48) (50) (44)
 

CYTOPLASMIC VACUOL1ZATION 2 (4S)
 

ITHYROID (43) (47) (37) 

CYSTIC FOLLICLES 1 (2*) 1 (3X) 

HYPERPLASI A ^CYSTIC 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

» NUMBER OF ANIMALS NECPOPSIED
 

C-ll
 
TABLE C2 (CONTINUED) 

CONTROL (U NT R) 

02-0220 

L OU DOSE 

02-0270 

HYPEBPLASIA ,

HYPERPLASIA ,

 C-CELL 

 FOLLICULAR-CELL 

1 (2*) 1 (2*) 

HYPERPLASIA , LYMPHOID 

*PARATHYROI D (31) (14) 

HYPERPLASIA , NODULA R 1 <3X ) 

REPRODUCTIV E SYSTEM 

*MAHMAR Y GLAN D (50) (50) DISPLACEMENT, NOS 8 (16%) DILATATION/DUCTS 1 (2%) GALACTOCELE 2 (IK) FIBROSIS, DIFFUSE 

HYPERPLASIA , SOS 1 (2%) LACTATION 1 (2X) 

*ACINUS OF BREAST (50) (50) DILATATION , NOS 

*CLITORAL GLAN D (50) (50) CYST, NOS 

INFLAMMATION , ACUTE 

HYPERPLASIA , CYSTIC 1 (2X) 

IUTERU S (1*8) (50) DILATATION , NOS 6 (12%) HYDROMETR A 1 (2%) INFLAMMATION , SUPPURATIVE 1 (2%) INFLAMMATION , ACUTE 1 (2%) ABSCESS, NOS 1 (2*) ABSCESS, CHRONIC 1 (2%) NECROSIS, NOS 1 (2*) DECIDU A 

#CERVI X UTERI (48) (50) HEMATOMA , NOS 1 (2%) 

tUTERUS/ENDOMETRIU M (48) (50) CYST, NOS 

INFLAMMATION , NOS 1 (2%) HYPSRPLASIA , NOS 

HYPERPLASIA , CYSTIC 1 (2X) 

tOVAR Y (49) (50) CYST, NOS 4 (8X) 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICAL1Y 

* NUMBE R OF ANIMAL S NBCROPSIED 

HIGH DOSE 

02-0275 

3 (8%) 

1 (3*) 

1 (3X) 

(21) 

(45) 

6 (13X) 

2 (4X) 

1 (2X) 

(45) 

1 (2*) 

(45) 

1 (2X) 

1 (2X) 

1 (2X) 

(45) 

3 (7«) 

5 (11X) 

1 <2X ) 

(45) 

(45) 

2 (U% ) 

1 (2X) 

(44) 

2_J5X1 

C-I2 
TABLE C2 (CONCLUDED) 

CONTROL (UNTR) LOW DOSE HIGH DOSE 

02-0220 02-0270 02-0275 

INFLAMMATION , NO S 1 (2*) 

INFLAMMATION , SOPPURATIV E 1 (2*) 

INFLAMMATION , ACUTE 1 (2X) 

INFLAMMATION , CHBONIC 2 (5J) 

DEGENERATION , CYSTIC 2 (41!) 

NERVOU S SYSTEM 

tBRAI N (50) (50) («5) 

HEMORRHAG E 2 (»*) 

NECROSIS , NOS 2 (<*«) 

SPECIAL SENSE ORGAN S 

NONE 

MUSCULOSKELETA L SYSTEM 

NON E 

BODY CAVITIES 

NON E 

A LL OTHER SYSTEMS 

DIAPHRAG M 

HilRNIA, NOS 1 

SPECIAL MORPHOLOG Y SUMMAR Y 

NO LESION REPORTED 11 1 3 

ANIMA L MISSING/NO NECROPSY 1 

A'lTO/NECROPSY/HISTO PERF 1 1 

AUTOLYSIS/N O NECROPSY t 

I NUMBER OF ANIMAL S KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

C-13 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH
 

2,3,5,6-TETRACHLORO-4-NITROANISOLE
 

TABLE Dl 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE TREATED WITH 

2,3,5.6-TETRACHLORO-4-NITROANISOLE 

CONTROL (UNTE ) LOW DOSE HIG H DOSE 

05-0360 05-0271 05 -0276 

ANIMALS INITIALLY IN STUDY 55 55 55

 ANIMALS MISSIN G 1

 

ANIMAL S NEC30PSIED 55 55 53 ANIMAL S EXAMINE D H ISTOPATHOLOGICALLY** 55 54 52

 

INTEGUMENTAR Y SYSTEM 

*SK1N (55) (55) (53) EPIDERMAL INCLUSION CYST 1 (2%)

 

INFLAMMATION, GRANULOMATOUS 1 (2*)

 

ACANTHOSIS 1 (2%) 

POLYP, INFLAMMATORY 1 (2%)

 

RESPIHATOR Y SYSTEM 

tLUN S (54) (54) (51) EDEMA , NOS 1 (2%) HEMOBBHAG E 1 <2X ) 1 (2*) HYPERPLASIA , ADENOMATOU S 1 (2%) HYPBRPLASIA , ALVEOLA R EPITHELIUM 1 (2«) 

HEMATOPOIETIC SYSTEM 

*SPLEEN (51 ) (53) (50) INFLAMMATION , PYOGPANULO M ATOU S 1 (2%) 

ATPOPHY , NOS 1 (2%) 

PLASMACYTOSIS 1 (2X) 

HYPEEPLASIA , LYMPHOI D 1 (2X) 1 <2X ) HEMATOPOIESI S . 2 (UJS) 2 (4X) 6 (12X) 

tPANCREATIC L.NODE (18) (50) (47)

 

PLASMACYTOSI S 1 (2X) 

tAORTIC LYMP H NOD E (48) (50) (47 )

 

THROMBOSIS, NOS 1 (2X)

 

OLOMSAP LYMPH NODE (48) (50) (47) 

HEMOFEHAGE
 

* NUM3E B OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE E OF ANIMAL S NECROPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-3 
TABLED! (CONTINUED) 

COMTROL(UNTR ) LOU DOSE HIGH DOSE 

05-0360 05-0271 05-0276 

*MESENTERIC I. NODE (18) (50) (47) 

CONGESTION, HOS 6 (13X) 

HEMORRHAG E 1 (2«) 

HIPEBPLASIA, NOS 1 (2X) 

HISTIOCYTOSIS 1 (2X) 1 (2%) 1 (2X) 

PLASMACYTOSIS 1 (2X) 

HYPERPLASIA , LYMPHOI D 1 (2X) 2 (4X) 

HEMATOPOIESIS 15 (30%) 10 (21X) 

#SENA L LYMPH NODE (48) (50) (47)

 

HEMORBHAG E 1 (2X)

 

HYPEEPIASIA, LYMPHOID 1 (2X)

 

ITHYMUS (39) (36) (39; ATROPHY , NOS 1 <3X) 

CIRCULATOR Y SYSTEM 

tHEARr (55) (54) (52) 

THPOMBOSIS, NOS 1 (2X) 

fIBSOSIS 1 (2%) 

FIBROSIS, DIFFUSE 1 <2X) 

DIGESTIVE SYSTEH 

tLIVER (54) (52) (52) 

INFLAHHATION , GHA NULOMATOU S 1 (2X) 

INFARCT, NOS 2 (»X) 

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

BASOPHILIC CYTO CHANG E 1 (2X) 

EOSINOPHILIC CYIO CHANG E 1 (2X) 

CLEAR-CELL CHANGE 3 (6X) 1 (2X) 

HYPEHPLASIA , NOS 1 (2X) 

*LIVEB/CENTPILOBOLAR (54) (52) (52)

 

CYTOPLASMIC VACDOLIZATION 1 (2X)

 

*BILE DUCT (55) (55) (53) 

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

IPANCREAS (49) (53) (49)

 

DILATATION/DHCTS 1 <2X >

 

* NaMBER OF ANIMAL S WITJJ TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE R OF ANIMAL S BECROPSIED 

D-4 
TABLE Dl (CONTINUED) 

CONTROL (UNTR ) L OW DOSE HIGH DQSE 

05-0360 05-0271 05-0276 

ATROPHY , NOS 1 (2%) 

ATROPHY , FOCAL 1 (2X) 

• STOMACH (51) (52) (50) 

ULCEB , NOS 2 (««) 

LYWPHOCYTI C INFLAMMATOR Y INFILTR 1 (2*) 

INFLAMMATION , ACUTE 1 (2%) 1 <2X ) 

ATYPIA , NO S 1 (2X) 

HYPERKERATOSI S 3 (6X) 5 (10X) 

ACANTHOSI S 4 (8*) 5 (10X) 

• JEJUNU M (50) (52) (50) 

DIVER T ICULU K 1 (2*) 

HYPERPLASIA , LYMPHOI D 1 (2*) 

• ILEU M (50) (52) (50) 

HYPERPLASIA , LYMPHOI D 2 (4X) 2 (!»*) 

*COLON (45) (19) (U6 ) 

PARASITISM 1 (2X) 4 (9X) 

URINAR Y SYSTE M 

#KIDNE Y (53) (52)

 

HYDRONEPHROSI S 1 (2%)

 

PYELONEPHRITIS , ACBTE 1 (2%)

 

PYELONEPHRITIS , CHRONIC 1 (2%)

 

FIBROSIS, FOCA L 1 (2X) 

NEPHROPATH Y 1 (2X) 

NECROSIS , CORTICA L 1 (2*) 

IORINAR Y BLADD3R (48) (52) (51)

 

CALCULUS , NOS 1 (2X)

 

HYPERPLASIA , EPITHELIA L 1 (2X)

 

ENDOCRIN E SYSTE M 

tADPENA L (50) (52) (49)

NECROSIS , CORTICA L 1 (2X) 

tPANCREATIC ISLETS (49) (53) (49)

 

HYPESPLASIA . NOS 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE P OF ANIMAL S NECPOPSIED 

D-5 
TABLE Dl (CONTINUED) 

CONTROL(ONTR)

05-0360

 LOW DOSE

 05-0271

 HIGH DOSE 

 05-0276 

REPRODUCTIV E SYSTEM 

*PREP»TIAL GLAN D

CJLCttLIS, NOS

DILATATION/DUCTS

CYST, NOS

INFLAMMATION , SUPPUEATIVE

HYPEEPLASIA, CYSTIC

IPROSTATE

INFLAMMATION, ACUTE

HYPEBPLASIA , PAPILLAR Y

ITESTIS

ATROPHY , NOS

•EPIDIDYMIS

MULTINUCLEAT E GIANT-CELL

 (55)

 1 (2%) 

 (52)

 1 (2*) 

 (5U)

 (55)

 (55)

 1 (2% ) 

1 (2*)

 1 (2% ) 

1 (2% ) 

 (50)

 1 (2% ) 

 (52)

 3 (6X)

 (55)

 (53) 

1 (2X) 

 (<*9) 

 (50) 

1 (2X ) 

 (53) 

1 (2X) 

NEPVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

•EYE

INFLAMMATION ,

CATARAC T

 ACUTE

 (55)

 1 (2%) 

1 (2% ) 

 (55) (53) 

MtJSCnLOSKELETAL

NONE 

 SYSTEM 

BODY CAVITIES 

• ABDOMINA L CAVIT Y

NECROSIS, FAT

 (55)

 5 (9X) 

 (55) (53) 

ALL OTHER SYSTEMS 

OMENTU M 

HEMATOJAJ_NOS

* NUMBER OF ANIMAL S

* NUMBER OF ANIMAL S

 ilTH TISSDE

 NECROPSIED 

 EXAMINED

 1 

 MICROSCOPICALLY 

D-6 
TABLE Dl (CONCLUDED) 

CONTROL (ONTR) LOW DOSE HIG H DOSE 

05-0360 05-0271 05-0276 

SPECIAL MORPHOLOGY SUMMAR Y 

NO LESION REPORTED 8 5 4
 

ANIMAL MISSING/NO NECROPSY 1
 

AUTO/NECFOPSY/HISTO PERF 1
 

AUTO/NECFOPSY/NO HISTO 1 1
 

AUTOLYSIS/NO NECEOPSY 1
 

t NUMBER OF ANIHALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

D-7 
TABLE D2 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE TREATED WITH 

2,3,5,6-TETRACHLORO-4-NITROANISOLE 

CONTROL (UNTR ) LOW DOSE HIGH DOSE 

06-0360 06-0271 06-0276 

ANIMAL S INITIALLY IN STUDY 55 55 55

 ANIMAL S KISSIN G 1 1

 

ANIMAL S NECROPSIED 55 5U 52

 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY ** 55 5U 52

 

INTEGUMENTAR Y SYSTEM 

NONE 

RESPIRATOR Y SYSTEM 

*LUN G 

ATELECTASI S 

INFLAMMATION, SUPPUP.ATIVE 

(55) 

1 (2*) 

(52) (52) 

1 (2X) 

HSMATOPOIETIC SYSTEM 

*BONE MARRO W (52) <5t) (52) MYELOFIBROSI S 31 (60/i) 26 (U8S) 18 (35X) HYPERPLASIA , HEMATOPOIETIC 1 (2% ) 

• SPLEE N (53 ) (52 ) (51 ) HYPERPLASIA , LYMPHOI D 1 (2% ) 1 (2% ) 

HEMATOPOIESIS 1 (2%) 7 (13X) !» (S%) 

• LUMBA R LYMP H NODE (K7 ) (50) ("6) INFLAMMATION , SUPPUSATIV E 1 (2% ) 

HYPEHPLASIA, LYMPHOID 2 (4X) 1 (2X) 

tMESiNTEEIC L. NODE C*7) (50) (*6) THROMBOSIS , NOS 1 (2% ) CONGESTION , NOS 1 (2%) 

HYPKRPLASIA , NO S 2 (I»X ) 

HYPERPLASIA , LYMPHOI D 3 (6J) 1 (2X) HEMATOPOIESI S 2 (HX ) 

tPENA L LYMP H NODE (17) (50) (1*6) 

INFLAMMATION ^ ACUTE^CHRONIC 1 (25) 

* SUMBE P OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S NECBOPSIED 

** EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

D-8
 
TABLE D2 (CONTINUED)
 

CONTHOL(UNTH ) LOB DOSE HIGH DOSE 

06-0360 06-027 1 06-0276 

HYPEPPLASIA , LYMPHOI D 2 (1%) 

*AXILLAR Y LYMP H NODE (17) (50) (16) 

INFLAMMATION , SUPPURATIV E 1 (2X ) 

*THYMU S (35) (39) (38)

 

ATROPHY , NOS 1 <3X ) 

hYPERPLASIA , LYMPHOI D 1 '3%)

 

CIRCULATOR Y SYSTEM 

• HEAR T (55) (53) (52)

 

PEP.IASTERITIS 1 (2*,) 

DEGENERATION , MUCOID 1 (2«)

 

DIGESTIV E SYSTEM 

tLIVER (51) (53) (52) 

CYTOPLASMI C V ACUOLIZ ATION 2 (1%) 

CLEAR-CEL L CHANG E 1 (2*) 

IPANCREA S (19) (51) (50) 

DILAIATION/DUCT S 1 (2% ) 1 (2%) 3 (6X) 

INFLAMMATION , CHRONIC 2 (1%) 

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

ATROPHY , FOCAL 1 (2% ) 

ATROPHY , FATT Y 1 (2% ) 

*PANCHEATI C ACINU S (19) (51) (50)

 

ATROPHY , FATT Y 1 (2%)

 

• STOMAC H (53) (51 ) (51) 

ULCER, NOS 1 (2% ) 1 (2% ) 

ABSCESS, NOS 1 (2X ) 

INFLAMMATION , CHRONIC 1 (2% ) 

INFLAMMATION , CHRONIC FOCAL 1 (2X ) 

EROSION 1 (2*,) 

KYPERPLASIA , EPITHELIA L 2 (t%) 

HYPEFPLASIA , PSEUDOEPITHELIOMAT O 1 (255) 

HYPERKEP. ATOSIS 3 (6%) 7 (11%) 

ACANTHOSI S U (8%) 7 (11X) 

ORINARY SYSTEM
 

tKIDNE Y (55) (53 ) (52 ) 

___ _ EYELO NEPHRITI S ^CHRONIC 

# NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

D-9 
TABLE D2 (CONTINUED) 

CONTROL (UNT8) LOW DOSE HIGH DOSE 

06-0360 06-0271 06-0276 

INFLAMMATION , CHRONIC FOCAL 1 (2%)

 NEPHROPATH Y 1 <2X )

 

DEGENERATION , HYALIN E 1 (2X )

 

tUEINAR Y BLADDE R (50) (54) (49) LYMPHOCYTIC INFLAMHATORY INFILTE 1 (2X) ULCER, ACUTE 1 (2») INFLAMMATION , CHRONIC 1 (2X) 

SNDOCRIN2 SYSTEM 

iPITUITAR Y (12) (47) (41)

 HYPEP.PLASIA, FOCAL 1 (2%)

 

• ADRENA L (50) (53) (49) NECROSIS, NOS 1 (2X)

 ANGIECTASIS 1 (2%)

 

fTHYSOI D (48) (39) (48) FOLLICULA R CYST, NOS 1 (2*) HYPERPLASIA , CYSTIC 1 (3X) 1 (2K) 

UPANCEEATI C ISLETS (49) (51) (50)

 HYPERPLASIA, NOS 3 (6%)

 

REPRODUCTIV E SYSTEM 

tUTBRUS (54) (51) (52) HYDPOMETR A 3 <6T.) DEGENERATION . HYALIN E 2 <4X ) NECROSIS, FOCAL 1 (2«) 

*UTERUS/ENDOMETEIU M (54) (51) (52) HYPERPLASIA , CYSTIC 15 (28%) 35 (69X) 32 (62%) 

#OVAH Y (50) (49 ) (49) 

CYST, NOS 7 (14*) 1 (2X) FOLLICULAR CYST, NOS 5 (10X) 3 (&*) HEMORRHAGI C CYST 1 (2%) 4 <8X) 

ABSCESS, NOS 1 (2*) 1 <2X ) 

NERVOU S SYSTEM 

tBRAIN/MENINGE S (55) (52) (52)

 

INFLAMHATION t NOS _ -1-12*1. .

 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECSOPSIED 

D-IO 
TABLE D2 (CONCLUDED) 

CONTRO L (IINTR ) LOW DOSE HIG H DOSE 

06-0360 06-0271 06-0276 

*BRAIN (55) (52) (52) 

HYDROCEPHALUS , NOS 2 (Uit) 

DEGENERATION , NOS 1 (2% ) 

SPECIAL SENSE ORGAN S 

NON E 

MUSCHLOSKELETA L SYSTEM 

NONE 

BODY CAVITIES 

*ABDOHINA L CAVITY (55) (51) (52) 

NECROSIS, FAT 7 (13% ) 

*MESENTERY (55) (5U) (52) CYST, NOS 1 (2*) 

A LL OTHER SYSTEMS 

*MULTIPLE ORGANS (55) (5U ) (52) 

PERIARTEPITIS 1 (2*) 

SPECIAL MORPHOLOG Y SUMMAR Y 

NO LESION REPORTED 1 1 2 

ANIMA L MISSING/NO NECROPSY 1 1 

AOTO/NECROPSY/HISTO PEHF 2 1 

AUTOLYSIS/NO NECROPSY 2 

* NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBER OF ANIMAL S NECf.OPSIED 

D-ll 

Review of the Bioassay of 2,3,5,6-Tetrachloro-4-Nitroanisole*
 

for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

June 29, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DREW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be exposed,
 

The members of the Clearinghouse have been drawn from
 

academia, industry, organized labor, public interest groups,
 

State health officials, and quasi-public health and research
 

organizations. Members have been selected on the basis of
 

their experience in carcinogenesis or related fields and,
 collectively, provide expertise in chemistry, biochemistry,
 biostatistics, toxicology, pathology, and epidemiology.
 Representatives of various Governmental agencies participate
 

as ad hoc members. The Data Evaluation/Risk Assessment
 

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

NCI-sponsored bioassays of chemicals studied for carcinogenicity. It is in this context that the below critique is given
 

on the bioassay of 2,3,5,6-Tetrachloro-4-Nitroanisole for
 

carcinogenicity.
 

The reviewer noted an increased (though not statisti­ cally significant) incidence of hepatic neoplasms among
 

treated rats and lymphomas and leukemias among treated mice.
 Although the results indicated that the animals were
 

administered maximum tolerated doses of the compound, the
 

reviewer expressed surprise that the levels were so low
 

given the nature of the substance. He suggested that the
 

low dose levels imposed by the toxicity may have limited
 

the expression of a higher tumor rate at those sites
 

at which an increased incidence was observed. Despite the
 

apparent adequacy of the study, the reviewer felt that some
 

additional testing was appropriate. In this regard, he
 

suggested that short-term in vitro assays might provide
 

useful information. The reviewer concluded that the bioassay
 

53
 
did not clearly show 2,355,6-Tetrachloro-4-Nitroanisole to
 

be positive or negative under the conditions of test. His
 

conclusion was accepted without objection.
 

Clearinghouse Members present:
 

Arnold L. Brown (Chairman), Mayo Clinic
 

Paul Nettesheim, National Institute of Environmental
 

Health Sciences
 

Verne Ray, Pfizer Medical Research Laboratory
 

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

Louise Strong, University of Texas Health Sciences Center
 

Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

<H).S. GOVERNMENT PRINTIN G OFFICE t 1978-260-899/3182
 

54
 

DHEW Publication No. (NIH) 78-1369