1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 61 

BIOASSAY OF 

PENTACHLORONITROBENZENE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 82-68-8 

NCI-CG-TR-61 

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

Public Health Service 

National Institutes of Health 
BIOASSAY OF
 

PENTACHLORONITROBENZENE
 

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) 78-1311
 

REPORT ON THE BIOASSAY OF PENTACHLORONITROBENZENE
 

FOR POSSIBLE CARCINOGENICITY
 

CARCINOGENESIS TESTING PROGRAM
 

DIVISION OF CANCER CAUSE AND PREVENTION
 

NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH
 

CONTRIBUTORS: This report presents the results of the bioassay of
 

pentachloronitrobenzene conducted for the Carcinogenesis Testing
 

Program, Division of Cancer Cause and Prevention, National Cancer
 

Institute (NCI), National Institutes of Health, Bethesda, Maryland.
 

This bioassay was conducted by Hazleton Laboratories America, Inc.,
 

Vienna, Virginia, initially under direct contract to the NCI and
 

currently under a subcontract to Tracor Jitco, Inc., prime contractor
 

for the NCI Carcinogenesis Bioassay 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. M. B. Powers (3),
 

Dr. R. W. Voelker (3), Dr. W. A. Olson (3,4) and Dr. W. M. Weatherholtz (3). Chemical analysis was performed by Dr. C. L. Guyton (3,
 

5) and the analytical results were reviewed by Dr. N. Zimmerman (6);
 

the technical supervisor of animal treatment and observation was Ms.
 

K. J. Petrovics (3).
 

Histopathologic examinations were performed by Dr. R. H. Habermann (3) and reviewed by Dr. R. W. Voelker (3) at the Hazleton Laboratories America, Inc., and the diagnoses included in this report
 

represent the interpretation of these pathologists. Histopathology
 

findings and reports were reviewed by Dr. R. L. Schueler (7).
 

Compilation of individual animal survival, pathology, and summary tables was performed by EG&G Mason Research Institute (8); the
 

statistical analysis was performed by Mr. W. W. Belew (6) and Dr.
 

J. R. Joiner (7), using methods selected for the Bioassay Program
 

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

This report was prepared at METREK, a Division of The MITRE Corporation (6) under the direction of the NCI. Those responsible for
 

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

(6), the task leader, Dr. M. R. Kornreich (6), the senior biologist,
 

Ms. P. Walker (6), and the technical editor, Ms, P. A. Miller (6).
 

The final report was reviewed by members of the participating organizations .
 

111
 
The statistical analysis was reviewed by members of the Mathematical Statistics and Applied Mathematics Section of the NCI: Dr.
 

J. J. Gart (9), Mr. J. Nam (9), Dr. H. M. Pettigrew (9), and Dr. R.
 

E. Tarone (9).
 

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), Dr. R.
 

A. Griesemer (1), Dr. H. A. Milman (1), Dr. T. W. Orme (1), Dr. R. A.
 

Squire (1,10), and Dr. J. M. Ward (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.	 Hazleton Laboratories America, Inc., 9200 Leesburg Turnpike,
 

Vienna, Virginia.
 

4.	 Now with the Center for Regulatory Services, 2347 Paddock Lane,
 

Reston, Virginia.
 

5.	 Now with Rhodia, Inc., 23 Belmont Drive, Somerset, New Jersey.
 

6.	 Th MITR e E Corporation, METREK Division, 1820 Dolley Madison
 

Boulevard, McLean, Virginia.
 

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

Maryland.
 

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

Rockville, Maryland.
 

9.	 Mathematical Statistics and Applied Mathematics Section, Biometry
 

Branch, Field Studies and Statistics Program, Division of Cancer
 

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

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

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

IV
 
SUMMARY
 

A bioassay of technical-grade pentachloronitrobenzene (PCNB)
 

for possible carcinogenicity was conducted using Osborne-Mendel rats
 

and B6C3F1 mice. PCNB was administered in the feed, at either of two
 

concentrations, to groups of 50 male and 50 female animals of each
 

species. The time-weighted average dietary concentrations of PCNB
 

were, respectively, 10,064 and 5417 ppm for male rats, 14,635 and
 

7875 ppm for female rats, 5213 and 2606 ppm for male mice, and 8187
 

and 4093 ppm for female mice. After a 78-week period of compound
 

administration, observation of the rats continued for an additional
 

33 to 35 weeks and observation of the mice continued for 14 or 15
 

additional weeks.
 

For each species, 20 animals of each sex were placed on test as
 

controls and fed only the basal diet.
 

No rare or unusual tumors were observed during the histopathologic examinations and no statistically significant positive associations were demonstrated between chemical administration and the
 

incidence of neoplasms in either sex of either species.
 

It is concluded that under the conditions of this bioassay PCNB
 

was not carcinogenic in either Osborne-Mendel rats or B6C3F1 mice.
 

V 

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 5
 

E. Selection of Initial Concentrations 6
 

F. Experimental Design 8
 

G. Clinical and Histopathologic Examinations 12
 

H. Data Recording and Statistical Analyses 13
 

III. CHRONIC TESTING RESULTS: RATS 18
 

A. Body Weights and Clinical Observations 18
 

B. Survival 20
 

C. Pathology 20
 

D. Statistical Analyses of Results 22
 

IV. CHRONIC TESTING RESULTS: MICE 30
 

A. Body Weights and Clinical Observations 30
 

B. Survival 30
 

C. Pathology 33
 

D. Statistical Analyses of Results 33
 

V. DISCUSSION 38
 

VI. BIBLIOGRAPHY 42
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH PCNB A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH PCNB B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH PCNB C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH PCNB D-l
 

Vll
 
LIST OF ILLUSTRATIONS 

Figure Number Page 

1 GROWTH CURVES FOR PCNB CHRONIC STUDY RATS 19 

2 SURVIVAL COMPARISONS OF PCNB CHRONIC STUDY 

RATS 21 

3 GROWTH CURVES FOR PCNB CHRONIC STUDY MICE 31 

4 SURVIVAL COMPARISONS OF PCNB CHRONIC STUDY 

MICE 32 

LIST OF TABLES 

Table Number Page 

1 DESIGN SUMMARY FOR OSBORNE-MENDEL RATS—PCNB 

FEEDING EXPERIMENT 9 

2 DESIGN SUMMARY FOR B6C3F1 MICE--PCNB FEEDING 

EXPERIMENT 10 

3 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

PCNB 23 

4 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED 

WITH PCNB 26 

5 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

PCNB 34 

6 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH PCNB 36 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH PCNB A-3 

viii 
LIST OF TABLES (Concluded) 

Table Page 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH PCNB A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN

MALE MICE TREATED WITH PCNB 

 B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN

FEMALE MICE TREATED WITH PCNB 

 B-6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC

LESIONS IN MALE RATS TREATED WITH PCNB 

 C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC

LESIONS IN FEMALE RATS TREATED WITH PCNB 

 C-7 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC

LESIONS IN MALE MICE TREATED WITH PCNB 

 D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC

LESIONS IN FEMALE MICE TREATED WITH PCNB 

 D-6 

ix 

I. INTRODUCTION
 

Pentachloronitrobenzene (PCNB) (NCI No. C00419), a halogenated
 

benzene derivative and agricultural pesticide, was selected for
 

bioassay by the National Cancer Institute following its classification
 

as a tumorigenic agent 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 pentachloronitrobenzene. It is
 

also known as quintozene (the common name approved by The International Standards Organization [International Agency For Research on
 

Cancer, 1974]), terrachlor, and PCNB.
 

PCNB was introduced as a fungicide in Germany in the 1930s but
 

did not achieve commercial importance in the United States until the
 

early 1960s (international Agency for Research on Cancer, 1974).
 

PCNB is presently approved in the United States for use as a soil
 

fungicide on fruit (bananas) and on a wide variety of vegetables
 

(e.g., cabbages, potatoes, and tomatoes), field crops (e.g., cotton
 

and soybeans) and ornamentals (e.g., carnations, lilies, roses, and
 

grasses); it may also be applied as a seed protectant fungicide for
 

crops such as barley, corn, cotton, oats, rice and wheat (international Agency for Research on Cancer, 1974).
 

*
 

The CAS registry number is 82-68-8,
 

1 
Recent production statistics for PCNB are considered proprietary
 

and are therefore not available; however, the listing of PCNB in the
 

1976 Directory of Chemical Producers, U.S.A. (Stanford Research
 

Institute, 1976) implies that the compound is presently manufactured
 

in commercial quantities (greater than 1000 pounds or $1000 in value)
 

and 1971 production was estimated to be in excess of 3 million pounds
 

(Johnson, 1972;as cited in International Agency for Research on
 

Cancer, 1974).
 

Occupational exposure to PCNB may occur at pesticide production
 

and formulating facilities and among agricultural workers engaged in
 

the treatment of soil or seeds with the fungicide. Exposure of the
 

general population may occur either through ingestion of residues
 

accumulating in food crops grown in PCNB-treated soils, or through
 

ingestion of milk from cows fed contaminated feed. PCNB residues
 

were detected in endive leaves and roots (0.06 to 83 ppm), in a fruit
 

sample from a total diet residue study (0.003 ppm), and in potatoes
 

(as high as 0.1 ppm in the peel following treatment of the soil at a
 

rate of 25 pounds per acre, a representative application rate for
 

vegetables of this type) (International Agency for Research on Cancer,
 

1974). Trace levels of PCNB (0.001 to 0.01 ppm) were also detected
 

in cows' milk from animals fed milking chow found to be contaminated
 

with 0.002 to 0.006 ppm of the chemical (Borzelleca et al., 1971).
 

Although data concerning human exposure are limited, PCNB is
 

generally considered to be of a very low order of toxicity (Courtney
 

et al., 1976).
 

22 
PCNB was found to be mutagenic in a tryptophan-requiring strain
 

of E. coli, causing a tenfold increase in the number of revertant
 

colonies over that expected as a result of spontaneous reversion
 

(Clarke, 1971). The fungicide, however, did not exhibit mutagenic
 

activity in the sex-linked lethal test in Drosophila melanogaster
 

(Vogel and Chandler, 1974) or in the host-mediated bioassay in mice
 

using several test organisms (Buselmaier et al., 1973).
 

Purified PCNB exhibited some teratogenic activity in CD-I mice,
 

producing cleft palate in an average of 8 percent of each litter
 

following oral administration of 500 mg/kg/day on days 7 through 16
 

of gestation (Courtney et al., 1976). PCNB was not, however, teratogenic in either CD or Wistar rats (Courtney et al., 1976; Khera
 

and Villeneuve, 1975; Jordan et al., 1975).
 

3 
II. MATERIALS AND METHODS 

A. Chemicals
 

Two batches of technical-grade pentachloronltrobenzene (PCNB)
 

were purchased from the 01 in Mathieson Chemical Corporation. The
 

manufacturer's stated assay for PCNB is 98 percent. Analysis was
 

performed by Hazleton Laboratories America, Inc., Vienna, Virginia.
 

The wide range observed in the melting point (134° to 145°C) indicated the presence of impurities even though the values were close
 

to that reported in the literature (146°C).
 

Total area analysis by gas-liquid chromatography suggested a
 

purity of approximately 97 percent, with 12 impurities present. Similar results after 12 and 24 months suggested little or no change in
 

composition. The four impurities eluted from the column before PCNB
 

were identified by the manufacturer as pentachlorobenzene, chloranil,
 

tetrachloronitrobenzene, and hexachloronitrobenzene.
 

Throughout this report the term PCNB is used to represent this
 

technical-grade material.
 

B.	 Dietary Preparation
 

®
 The basal laboratory diet consisted of Wayne Lab-Blox (Allied
 

®
 Mills, Inc.) plus 2 percent Duke's corn oil (S.F. Sauer Company) by
 

weight. Fresh mixtures of PCNB in corn oil were prepared each week
 

and stored in the dark. The PCNB mixtures were incorporated into the
 

appropriate amount of laboratory diet in a twin-shell blender fitted
 

4 
with an accelerator bar so that the final concentrations of PCNB in
 

the diet varied from 1075 to 22,000 ppm.
 

C. Animals
 

Two animal species, rats and mice, were used in the carcinogenicity bioassay. The Osborne-Mendel rat was selected on the basis of a
 

comparative study of the tumorigenic responsiveness to carbon tetrachloride of five different strains of rats (Reuber and Glover, 1970).
 

The B6C3F1 mouse was selected because it has been used by the NCI for
 

carcinogenesis bioassays and has proved satisfactory in this capacity.
 

Rats and mice of both sexes were obtained through contracts of
 

the Division of Cancer Treatment, National Cancer Institute. The
 

Osborne-Mendel rats were procured from the Battelle Memorial Institute, Columbus, Ohio, and the B6C3F1 mice were obtained from the
 

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

Upon receipt, animals were quarantined for at least 10 days, observed
 

for visible signs of disease or parasites, and assigned to the various
 

dosed and control groups.
 

D. Animal Maintenance
 

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


controlled rooms. The temperature range was 20° to 24°C and the
 

relative humidity was maintained between 45 and 55 percent. The air
 

conditioning system in the laboratory provided filtered air at a rate
 

of 10 to 15 complete changes of room air per hour. Fluorescent lighting was provided on a 12-hour-daily cycle. Rats were individually
 

5 
housed in suspended galvanized-steel wire-mesh cages with perforated
 

floors, while mice were housed by sex in groups of ten in solid-


bottom polypropylene cages with filter tops. Sanitized cages with
 

fresh bedding (Sanichips , Shurfire) were provided once each week for
 

mice. Rats received sanitized cages with no bedding with the same
 

frequency. Food hoppers were changed and heat-sterilized once a week
 

for the first 10 weeks and once a month thereafter, while fresh
 

heat-sterilized glass water bottles were provided three times a week.
 

Food (Wayne Lab-Blox~0 and water were available ad libitum.
 

The rats treated with PCNB and their controls were housed in the
 

*
 

same room as other rats receiving diets treated with clonitralid
 

(1420-04-8) and amitrole (61-82-5). All mice used in this study were
 

housed in the same room as other mice receiving diets treated with
 

trifluralin (1582-09-8); dioxathion (78-34-2); sulfallate (95-06-7);
 

p,p'-DDT (50-29-3); methoxychlor (72-43-5); p,p'-DDE (72-55-9);
 

p,p'-TDE (72-54-8); dicofol (115-32-2); chlorobenzilate (510-15-6);
 

clonitralid (1420-04-8); nitrofen (1836-75-5); endosulfan (115-29-7);
 

mexacarbate (315-18-4); amitrole (61-82-5); acetylaminofluorene
 

(53-96-3); and safrole (94-59-7).
 

E. Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

PCNB for administration to treated animals in the chronic studies,
 

subchronic toxicity tests were conducted with both rats and mice.
 

*
 CAS registry numbers are given in parentheses.
 

6 
Animals of each species were distributed among six groups, each consisting of five males and five females. PCNB was premixed with a
 

small amount of corn oil. This mixture was then incorporated into
 

the laboratory diet and fed ad libitum to five of the six rat groups
 

and five of the six mouse groups in concentrations of 2150, 4640,
 

10,000, 21,500 and 46,400 ppm. The sixth group of each species served
 

as a control group, receiving only the mixture of corn oil and laboratory chow. The dosed dietary preparations were administered for a
 

period of 6 weeks, followed by a 2-week observation period during
 

which all animals were fed the basal diet.
 

A concentration inducing no mortality and resulting in a depression of mean group body weight of approximately 20 percent relative
 

to controls was to be selected as the initial high concentration for
 

the chronic study. When weight gain criteria were not applicable,
 

mortality data alone were utilized.
 

All of the male rats and one of the female rats receiving 46,400
 

ppm PCNB died before the 6-week period of compound administration was
 

over. In males, mean body weight depression was 7 and 42 percent at
 

10,000 and 21,500 ppm, respectively. In females, mean body weight
 

depression was 16 percent at 21,500 ppm. The initial high concentrations selected for male and female rats in the chronic bioassay were
 

15,000 and 22,000 ppm, respectively.
 

At a concentration of 2150 ppm, none of the mice died during the
 

8-week study. However, four of the five males receiving 4640 ppm
 

7 
died by week 4. Mean body weight depression was only 3 percent in
 

the males receiving 2150 ppm but, due to the severe mortality observed
 

at 4640 ppm, 2150 ppm was selected as the initial high concentration
 

for the male mice. Mean body weight depression was only 10 percent
 

in females treated with 4640 ppm. However, two of the five females
 

receiving 10,000 ppm died. The initial high concentration selected
 

for the chronic bioassay was 4640 ppm for the female mice.
 

F. Experimental Design
 

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

treated and untreated observation periods, and the time-weighted
 

average concentrations) are summarized in Tables 1 and 2.
 

The high dose, low dose, and control rats were all approximately
 

6 weeks old when the bioassay began. The high and low concentrations
 

of PCNB initially utilized for males were 15,000 and 7500 ppm, respectively. During week 14 of the experiment, when the male rats
 

were approximately 20 weeks old, the high and low concentrations were
 

decreased to 10,000 and 5000 ppm, respectively, in response to adverse
 

clinical reactions observed in the treated animals. In week 53 administration of PCNB to the high dose males ceased for 1 week, followed
 

by 4 weeks of feeding at the previous concentration of 10,000 ppm.
 

This cyclic pattern of dose administration, an effort to reduce total
 

chemical intake, was continued for the remainder of the 78-week
 

period.
 

8 
TABLE 1
 

DESIGN SUMMARY FOR OSBORNE-MENDEL RATS 

PCNB FEEDING EXPERIMENT 

TIME-WEIGHTED 

AVERAGE CONCENINITIAL OBSERVATION PERIOD TRATION 

GROUP PCNB TREATED UNTREATED OVER A 78-WEEK 

SIZE CONCENTRATION3 (WEEKS) (WEEKS) PERIODb 

MALE 

CONTROL 20 0 111 0 

LOW DOSE 50 7,500 13 5,417 

5,000 65 

0 33 

HIGH DOSE 50 15,000 13 10,064 

10,000 39 

10,000C 20 6 

0 34 

FEMALE 

CONTROL 20 0 113 0 

LOW DOSE 50 11,000 13 7,875 

7,250 65 

0 35 

HIGH DOSE 50 22,000 13 14,635 

14,500 35 

14,500° 24 6 

0 33 

a
 Concentrations given in parts per million
 

bm, , . , . S (concentration X weeks received)
 Time-weighted average concentration = =7: T
 

These concentrations were cyclically administered with a pattern of
 

1 treatment-free week followed by 4 weeks of treatment at the level
 

indicated.
 

9 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

PCNB FEEDING EXPERIMENT
 

INITIAL OBSERVATION PERIOD TIME-WEIGHTED
 

GROUP PCNB TREATED UNTREATED AVERAGE
 

SIZE CONCENTRATION3 (WEEKS) (WEEKS) CONCENTRATION
 

MALE
 

CONTROL 20 0 91 0
 

LOW DOSE 50 1075 4 2606
 

1500 4
 

2000 10
 

2500 14
 

3000 46
 

0 14
 

HIGH DOSE 50 2150 4 5213
 

3000 4
 

4000 10
 

5000 14
 

6000 46
 

0 14
 

FEMALE
 

CONTROL 20 0 92 0
 

LOW DOSE 50 2320 4 4093
 

3000 4
 

3500 10
 

4000 14
 

4500 46
 

0 15
 

HIGH DOSE 50 4640 4 8187
 

6000 4
 

7000 10
 

8000 14
 

9000 46
 

0 15
 

Concentrations given in parts per million
 

bm. . , . 2(concentration X weeks received)
 Time-weighted average concentration = -=-7 ; :—: : -. rr
 2(weeks receiving chemical;
 

10 
For female rats, the initial high and low concentrations of PCNB
 

used were 22,000 and 11,000 ppm, respectively. During week 14, when
 

the female rats were approximately 20 weeks old, the high and low
 

levels were decreased to 14,500 and 7250 ppm, respectively, due to
 

the observation of adverse clinical reactions in the treated animals.
 

In week 49, administration of PCNB to the high dose females ceased
 

for 1 week, followed by 4 weeks of feeding at the previous concentration of 14,500 ppm in an effort to reduce total chemical intake.
 

This cyclic pattern of dose administration was continued for the
 

remainder of the 78-week study period.
 

The high dose, low dose, and control mice were all approximately
 

5 weeks old when compound administration began. The high and low
 

concentrations initially administered to male mice were 2150 and 1075
 

ppm, respectively. Because the treated animals exhibited no adverse
 

reaction to the PCNB, the concentrations were increased four times
 

over the duration of the study. In week 5 the high and low concentrations were increased to 3000 and 1500 ppm, respectively. Concentrations were again raised in week 9 of the experiment, to 4000 and 2000
 

ppm, respectively, in week 19 to 5000 and 2500 ppm, and in week 33 to
 

6000 and 3000 ppm.
 

The high and low concentrations of PCNB initially administered
 

to female mice were 4640 and 2320 ppm, respectively. Because the
 

treated animals exhibited no adverse reaction to the chemical, the
 

concentrations were increased four times over the course of the
 

11
 
bioassay. In week 5 of the study the high and low concentrations
 

were raised to 6000 and 3000 ppm, respectively, in week 9 to 7000 and
 

3500 ppm, 10 weeks later to 8000 and 4000 ppm, and finally, in week
 

33, to 9000 and 4500 ppm, respectively.
 

G. Clinical and Histopathologi". Examinations
 

Animals were weighed immedi.ately prior to initiation of the
 

experiment. From the first day, all animals were inspected daily for
 

mortality. Body weights and food consumption and data concerning
 

appearance, behavior, signs of toxic effects, and incidence, size,
 

and location of tissue masses were recorded once weekly for the first
 

10 weeks and monthly thereafter. Incidence of tissue masses was
 

determined by observation and palpation of each animal.
 

During the course of this bioassay several pathology protocols
 

were in effect, each for different periods of time. The minimum protocol required that, if possible, certain tissues were to be taken
 

and examined histopathologically from all control animals, from any
 

animal in which a tumor was observed during gross examination, and
 

from at least 10 grossly normal males and 10 grossly normal females
 

from each treated group. In addition, any tissues showing gross
 

abnormalities were to be taken and examined histopathologically.
 

Under later protocols, some tissues were taken from additional dosed
 

animals. The number of animals in each group from which a tissue was
 

examined is indicated in Appendices A through D.
 

A necropsy was performed on each animal regardless of whether
 

it died, was killed when moribund, or was sacrificed at termination
 

12
 
of the bioassay. The animals were euthanized by exsanguination under
 

sodium pentobarbital anesthesia, and were immediately necropsied.
 

The histopathologic examination consisted of gross examination of all
 

major tissues, organs, or gross lesions taken from sacrificed animals
 

and, whenever possible, from animals found dead.
 

Slides were prepared from the following tissues from selected
 

animals: skin, subcutaneous tissue, lungs and bronchi, trachea, bone
 

marrow, spleen, lymph nodes, thymus, heart, salivary gland, liver,
 

gallbladder and bile duct (mice), pancreas, esophagus, stomach, small
 

intestine, large intestine, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, pancreatic islets, testis, prostate, brain,
 

muscle, uterus, mammary gland, and ovary.
 

Tissues for which slides were prepared 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.
 

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,
 

13
 
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) for testing two groups for
 

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

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

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

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

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

histologically. However, when macroscopic examination was required
 

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

14
 
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.
 

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

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

Under the assumption of a linear trend, this test determined if the
 

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


tailed 0.05 level of significance. Unless otherwise noted, the direction of the significant trend was a positive dose relationship.
 

This method also provides a two-tailed test of departure from linear
 

trend.
 

15
 
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
 

16
 
the exact interval on the odds ratio (Gart, 1971). The relative risk
 

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

incidence of a specific type of tumor in a 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
 

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.
 

17
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

Distinct, dose-related mean body weight depression was evident
 

in both male and female rats throughout the bioassay (Figure 1).
 

Clinical signs were observed in the PCNB-treated groups as early
 

as week 1 of the study. The predominant clinical signs were urine
 

staining in the abdominal area and a hunched or thin appearance indicative of body weight effects. Beginning in week 1, approximately 40
 

percent of the high dose males, 70 percent of the high dose females,
 

and a few low dose females showed abdominal urine stains. Concomitant
 

thin or hunched appearance was displayed by about 25 percent of the
 

animals in these groups and was noted in increasing numbers from week
 

1 until cessation of compound administration in week 78. Thereafter,
 

until termination of the study, a comparable number of treated and
 

control animals appeared hunched. Abdominal urine stains were persistently observed in most of the treated groups except in the low
 

dose males. Abdominal urine staining was infrequently observed in
 

this group and in the controls throughout the study.
 

Respiratory signs characterized by labored or difficult respiration, wheezing, and/or nasal discharge were observed at a low incidence in all groups including controls during the first part of the
 

second year. The incidence increased gradually in all groups during
 

the last six months of the study.
 

18
 
FIGURE 1

 

GROWTH CURVES FOR PCNB CHRONIC STUDY RATS 

19 
Other signs associated with aging were observed in comparable
 

numbers of control and treated animals during the second year. These
 

signs included rough or stained fur, alopecia, sores on parts of the
 

body, reddish discharge or crust around body orifices, palpable
 

masses, wart-like growths, and nodules. Isolated, apparently spontaneous signs noted in one to three animals in each group included
 

tremors, head tilt or circling, small-appearing testes, and partial
 

limb paralysis.
 

B.	 Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and PCNB-treated groups are shown in Figure 2.
 

For both male and female rats the Tarone test did not indicate
 

a significant positive association between dosage and mortality. For
 

males 64 percent (32/50) of the high dose, 42 percent (21/50) of the
 

low dose, and 50 percent (10/20) of the control group survived until
 

the end of the study. For females 74 percent (37/50) of the high
 

dose, 80 percent (40/50) of the low dose, and 80 percent (16/20) of
 

the control group survived at least 101 weeks. Thus in both sexes
 

survival was adequate for meaningful statistical analyses of tumor
 

incidence.
 

C.	 Pathology
 

Histopathologic findings on neoplasms in rats are tabulated in
 

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

tabulated in Appendix C (Tables Cl and C2).
 

20
 
FIGURE 2

 

SURVIVAL COMPARISONS OF PCNB CHRONIC STUDY RATS 

21 
A variety of neoplasms were observed among both treated and control rats. Each type of tumor observed has been encountered previously as a spontaneous lesion in Osborne-Mendel rats. No appreciable
 

difference in the incidence of neoplasia was noted between the control
 

and treated rats in this study.
 

Inflammatory, degenerative, and proliferative lesions seen in
 

treated and control animals were similar in number and kind to lesions
 

occurring naturally in aged Osborne-Mendel rats.
 

The results of this histopathologic examination did not indicate
 

evidence of carcinogenicity in Osborne-Mendel rats of either sex.
 

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 for every type
 

of tumor that was observed in more than 5 percent of any of the
 

PCNB-dosed groups of either sex is included.
 

For both male and female rats, no statistical tests showed a
 

significant positive association between dosage and tumor incidence.
 

The possibility of a negative association between compound administration and the incidence of mammary fibroadenomas was noted for
 

the male rats. The Fisher exact tests, however, were not significant.
 

To provide additional insight, 95 percent confidence intervals
 

on the relative risk have been estimated and entered in the tables
 

based upon the observed tumor incidence rates. In all of the
 

intervals shown in Tables 3 and 4, the value one is included; this
 

22
 
TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH PCNBa
 

TOPOGRAPHY: MORPHOLOGY
 

Subcutaneous Tissue: Fibroma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia or
 

Malignant Lymphoma'5
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Circulatory System: Hemangiosarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW 

CONTROL DOSE 

1/20(0.05) 3/48(0.06) 

N.S. N.S. 

0.250 

0.111 

64.251 

104 110 

1/20(0.05) 2/48(0.04) 

N.S. N.S. 

0.833 

0.047 

48.155 

106 85 

1/20(0.05) 1/48(0.02) 

N.S. N.S. 

0.417 

0.006 

32.057 

83 105 

HIGH
 

DOSE
 

2/49(0.04)
 

N.S.
 

0.816
 

0.046
 

47.195
 

112
 

4/49(0.08)
 

N.S.
 

1.633
 

0.179
 

78.704
 

3/49(0.06)
 

N.S.
 

1.224
 

0.107
 

62.958
 

95 

112 
TOPOGRAPHY : MORPHOLOGY
 

Kidney: Mixed Tumor Malignant
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma or
 

Follicular-Cell Carcinomab
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 (CONTINUED) 

CONTROL
 

0/20(0.00)
 

N.S.
 

2/20(0.10)
 

N.S.
 

_.—
 

-—
 

106
 

2/20(0.10)
 

N.S.
 

104
 

LOW
 

DOSE
 

3/43(0.07)
 

N.S.
 

Infinite
 

0.291
 

Infinite
 

68
 

3/42(0.07)
 

N.S.
 

0.714
 

0.091
 

8.119
 

111
 

2/43(0.05)
 

N.S.
 

0.465
 

0.037
 

6.107
 

68
 

HIGH
 

DOSE
 

1/37(0.03)
 

N.S.
 

Infinite
 

0.030
 

Infinite
 

112
 

7/37(0.19)
 

N.S.
 

1.892
 

0.411
 

17.507
 

86
 

2/37(0.05)
 

N.S.
 

0.541
 

0.043
 

7.057
 

103
 
TABLE 3 (CONCLUDED)
 

TOPOGRAPHY: MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Mammary Gland: Fibroadenoma 2/20(0.10) 4/48(0.08) 0/49(0.00) 

P Values0 P = 0.043(N) N.S. N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

0.833 

0.134 

8.776 

0.000 

0.000 

1.372 

Weeks to First Observed Tumor 104 104 

Treated groups received time-weighted average doses of 5417 or 10,064 ppm in feed.
 

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

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

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

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

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

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

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH PCNB*
 

TOPOGRAPHY : MORPHOLOGY
 

Thyroid: C-Cell Carcinoma
 

P Values0
 

Departure from Linear Trend 6
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma or C-Cell Carcinomab
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

0/20(0.00)
 

N.S.
 

P = 0.041
 

0/20(0.00)
 

N.S.
 

P = 0.018
 

7/20(0.35)
 

N.S.
 

86
 

LOW
 

DOSE
 

3/40(0.08)
 

N.S.
 

Infinite
 

0.313
 

Infinite
 

112
 

4/40(0.10)
 

N.S.
 

Infinite
 

0.484
 

Infinite
 

112
 

9/50(0.18)
 

N.S.
 

0.514
 

0.208
 

1.436
 

112
 

HIGH
 

DOSE
 

0/36(0.00)
 

N.S.
 

0/36(0.00)
 

N.S.
 

8/48(0.17)
 

N.S.
 

0.476
 

0.183
 

1.365
 

113
 
TOPOGRAPHY: MORPHOLOGY
 

Circulatory System: Hemangiosarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenoma
 

P Values0
 

Departure from Linear Trend
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Uterus: Endometrial Stromal Polyp
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 4 (CONTINUED)
 

CONTROL
 

1/20(0.05)
 

N.S.
 

_~
 

104
 

7/20(0.35)
 

N.S.
 

P = 0.038
 

90
 

1/19(0.05)
 

N.S.
 

109
 

LOW
 

DOSE
 

1/50(0.02)
 

N.S.
 

0.400
 

0.005
 

30.802
 

89
 

20/40(0.50)
 

N.S.
 

1.429
 

0.726
 

3.333
 

91
 

2/39(0.05)
 

N.S.
 

0.974
 

0.055
 

56.013
 

112
 

HIGH
 

DOSE
 

1/47(0.02)
 

N.S.
 

0.426
 

0.006
 

32.057
 

113
 

8/35(0.23)
 

N.S.
 

0.653
 

0.253
 

1.829
 

101
 

1/37(0.03)
 

N.S.
 

0.514
 

0.007
 

39.250
 

113
 
TABLE 4 (CONCLUDED) 

a
 

Treated groups received time-weighted average doses of 7875 or 14,635 ppm in feed.
 

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

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.
 

6
The probability level of the test for departure from linear trend is given beneath the control
 

group when P < 0.05.
 
indicates the absence of statistically significant results. It should
 

also be noted that all of the confidence intervals have an upper limit
 

greater than one, indicating the theoretical possibility of a significantly increased rate of tumor incidence induced in rats by PCNB that
 

could not be established under the conditions of this test.
 

29
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

No consistent dose-related mean body weight depression was evident among male mice. A slight dose-related depression of mean body
 

weight became apparent for female mice after week 35 (Figure 3).
 

During the first 6 months of the study, treated and control mice
 

showed comparable patterns of appearance and behavior. Clinical signs
 

often observed in group-housed laboratory mice, particularly in males,
 

were observed in a comparable number of treated and control mice.
 

Such signs included body sores (mostly from fighting), localized alopecia, abdominal urine stains, penile/anal/vulvar irritation, bloating,
 

eyes showing cloudiness, redness and/or discharge, swollen areas on
 

the body or extremities, and palpable nodules.
 

In week 34 a hunched appearance was evident in approximately 27
 

percent of the low dose males and 70 percent of the high dose males.
 

This sign persisted in the survivors to termination of the study in
 

week 90.
 

B.	 Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and PCNB-treated groups are shown in Figure 4.
 

For both male and female mice the Tarone test did not indicate
 

a significant positive association between dosage and mortality. For
 

males 64 percent (32/50) of the high dose, 70 percent (33/50) of the
 

low dose, and 75 percent (15/20) of the control group survived at
 

30
 
FIGURES

 

GROWTH CURVES FOR PCNB CHRONIC STUDY MICE 

31 
FIGURE 4

 

SURVIVAL COMPARISONS OF PCNB CHRONIC STUDY MICE 

32 
least 75 weeks. After 75 weeks, mortality of males was greatly accelerated so that by week 90, only 25 low dose, 17 high dose, and 4 control mice were available for terminal sacrifice. For females survival
 

was adequate to permit development of possible late-occurring tumors
 

as 78 percent (39/50) of the high dose, 86 percent (43/50) of the low
 

dose, and 95 percent (19/20) of the control group survived until the
 

end of the study,
 

C. Pathology
 

Histopathologic findings on neoplasms in mice are tabulated in
 

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

tabulated in Appendix D (Tables Dl and D2).
 

A variety of neoplasms were represented among both the treated
 

and control mice. Each type of tumor represented has been encountered previously as a naturally occurring lesion in B6C3F1 mice and
 

was without apparent relationship to administration of the chemical.
 

The relative low incidence of hepatocellular carcinomas in male
 

mice and the lack of a relationship of these lesions to dosage, suggest that the increased incidence in the low dose group was not significant. The results of this histopathologic examination did not
 

indicate evidence of carcinogenicity in B6C3F1 mice of either sex.
 

*-*• Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

mice are summarized in Tables 5 and 6. The analysis for every type
 

33
 
___ 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH PCNBa
 

HIGH
 

DOSE
 

5/48(0.10)
 

N.S.
 

1.042
 

0.191
 

10.410
 

72
 

2/48(0.04)
 

N.S.
 

0.417
 

0.033
 

5.490
 

77
 

4/42(0.10)
 

N.S.
 

0.952
 

0.154
 

9.980
 

65
 

TOPOGRAPHY: MORPHOLOGY
 

Subcutaneous Tissue: Flbrosarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: 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
 

CONTROL
 

2/20(0.10)
 

N.S.
 

—_
 

63
 

2/20(0.10)
 

N.S.
 

55
 

2/20(0.10)
 

N.S.
 

79
 

LOW
 

DOSE
 

3/45(0.07)
 

N.S.
 

0.667
 

0.085
 

7.596
 

87
 

1/45(0.02)
 

N.S.
 

0.222
 

0.004
 

4.070
 

76
 

8/35(0.23)
 

N.S.
 

2.286
 

0.525
 

20.614
 

86
 
TABLE 5 (CONCLUDED)
 

Treated groups received time-weighted average doses of 2606 or 5213 ppm in feed.
 

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

G

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

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

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

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

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

TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH PCNB£
 

LOW HIGH 

TOPOGRAPHY: MORPHOLOGY CONTROL DOSE DOSE 

Hematopoietic System: Malignant Lymphoma 1/20(0.05) 1/47(0.02) 5/46(0.11) 

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

Relative Risk (Control) 0.426 2.174 

Lower Limit 0.006 0.271 

Upper Limit 32.720 100.415 

Weeks to First Observed Tumor 91 75 80 

Liver: Hepatocellular Carcinoma 0/20(0.00) 0/14(0.00) 3/20(0.15)
 

P Values0 P = 0.043 N.S. N.S.
 

Relative Risk (Control) Infinite
 

Lower Limit 0.630
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 71
 

3.Treated groups received time-weighted average doses of 4093 or 8187 ppm in feed.
 

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

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

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

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

given beneath the incidence of tumors in the treated group when P < 0.05; otherwise, not signifi­ cant (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.
 
of tumor that was observed in more than 5 percent of any of the PCNB-


dosed groups of either sex is included.
 

For female mice the Cochran-Armitage test indicated a significant
 

(P = 0.043) positive association between dosage and the incidence of
 

hepatocellular carcinomas. The Fisher exact tests did not support
 

this finding, but a small number of tissues (20) were examined microscopically. In the historical data compiled by this laboratory for
 

the NCI Bioassay Program 3/380 (1 percent) of the untreated female
 

B6C3F1 mice had this tumor.
 

No other statistical tests for any site in mice of either sex
 

indicated a significant positive association between the administration of PCNB and tumor incidence. Thus, at the dose levels used in
 

this experiment there was not adequate evidence to conclude that PCNB
 

was a carcinogen in B6C3F1 mice.
 

To provide additional insight, 95 percent confidence intervals
 

on the relative risk have been estimated and entered in the tables
 

based upon the observed tumor incidence rates. In all of the intervals shown in Tables 5 and 6, the value one is included; this indicates the absence of statistically significant results. It should
 

also be noted that all of the confidence intervals have an upper
 

limit greater than one, indicating the theoretical possibility of a
 

significantly increased rate of tumor incidence induced in mice by
 

PCNB that could not be established under the conditions of this test.
 

37
 
V. DISCUSSION
 

Survival among male rats, female rats, and female mice was adequate for meaningful statistical analyses of possible late-developing
 

tumors. Although more than half of each male mouse group survived
 

the 78-week period of compound administration, mortality of male mice
 

was high during the observation period following compound administration with only 25 low dose mice, 17 high dose mice, and 4 control
 

mice remaining alive at termination of the mouse bioassay in week 90.
 

There is a possibility that if more male mice had survived the observation period, a higher incidence of tumors would have been observed
 

in treated or control groups at terminal sacrifice.
 

It appears that maximum tolerated doses were received by treated
 

groups. Dose-related depression of mean body weight was observed for
 

male and female rats. Abdominal urine staining in all treated rat
 

groups except low dose males was further indication that rats were
 

given a maximum tolerated dose. From about week 34 until termination
 

of the study, a slight dose-related mean body weight depression was
 

observed among female mice and a dose-related incidence of hunched
 

body posture was observed among male mice.
 

Among rats, chromophobe adenomas of the pituitary were observed
 

in 2/20 (10 percent), 3/42 (7 percent), and 7/37 (19 percent) of the
 

control, low dose, and high dose males, respectively, and in 7/20
 

(35 percent), 20/40 (50 percent), and 8/35 (23 percent) of the control, low dose, and high dose females, respectively. As there was
 

38
 
no consistent relationship between PCNB concentration received and
 

incidences of lesions observed, and as these tumors occur spontaneously with similar incidences in untreated Osborne-Mendel rats, no
 

significance was attributed to the incidences of these neoplasms in
 

treated animals. In addition, statistical analyses revealed no significant positive associations between compound administration and
 

the incidence of pituitary chromophobe adenoma or any of the other
 

neoplasms that were observed in rats.
 

In mice, hepatocellular carcinomas were found in 2/20 (10 percent), 8/35 (23 percent), and 4/42 (10 percent) of the control, low
 

dose, and high dose males, respectively, and in 0/20, 0/14, and 3/20
 

(15 percent) of the control, low dose, and high dose females, respectively. As the incidence was low and as there was no consistent
 

relationship between PCNB concentration received and incidences
 

observed, these neoplasms were not considered to be compound-related.
 

In addition, the increased incidences observed in the low dose male
 

mice and the high dose female mice were not statistically significant
 

when compared to controls.
 

Under the series of pathology protocols in effect during the
 

course of this bioassay, not all grossly normal tissues were examined
 

histopathologically. The incidences presented in this discussion and
 

the statistical analyses presented in Tables 3, 4, 5, and 6 are based
 

on the number of tissues histopathologically examined. According to
 

39
 
these statistical analyses, no significant increases in tumor incidence were observed in treated rats or mice, although sometimes the
 

sample sizes were quite low. If it is assumed that no tumors ocurred
 

in the grossly normal tissues which were not examined histopathologically, then the statistical analyses can be based on the total number
 

of tissues necropsied. This lowers the percentage of animals with
 

tumors in each group, but increases the sample size. Statistical
 

analyses performed on the basis of total number of animals necropsied
 

indicated no significant increase in tumor incidence related to PCNB
 

administration.
 

In an earlier study (Innes et al., 1969), orally administered
 

PCNB was found to be tumorigenic in male B6C3F1 mice, producing an
 

increased incidence of hepatomas. The dosage level giving positive
 

results in the Innes et al. study, 1206 ppm in the diet, was considerably less than the dosage levels used in this bioassay (time-weighted
 

average low and high concentrations of, respectively, 2606 and 5213
 

ppm for males and 4093 and 8187 ppm for females). The reasons that
 

results of the Innes et al. study differed from results of this bioassay are not readily apparent. A possible cause of the difference
 

is that PCNB treatment began at an earlier age in the Innes et. al.
 

study. Dosing by Innes et al. began at the age of 7 days and was
 

performed by gastric intubation (464 mg/kg body weight/day) until the
 

mice were weaned at the age of 4 weeks at which time PCNB was administered in the diet. In this bioassay PCNB was only administered in
 

40
 
the diet, beginning at 6 weeks of age. In the Innes et al. study,
 

the term "hepatoma" was used for all hepatic tumors except for those
 

rare cases where the hepatic tumor was accompanied by unmistakable
 

pulmonary metastases (U.S. Department of Health, Education and Welfare, 1969). The fact that these NCI analyses were based on hepatocellular carcinomas would not account for differences in the final
 

study, since no hepatocellular tumors except carcinomas were reported
 

in this study. Other carcinogenicity studies, including a feeding
 

study in rats (Finnegan et al., 1958) and a skin painting study in
 

mice (Searle, 1966) were either inadequately reported or inconclusive
 

(International Agency for Research on Cancer, 1974).
 

It is concluded that under the conditions of this bioassay PCNB
 

was not demonstrated to be carcinogenic in either Osborne-Mendel rats
 

or B6C3F1 mice.
 

41
 
VI. BIBLIOGRAPHY
 

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

J. Wiley & Sons, New York, 1971.
 

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

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

Union Against Cancer, Geneva, 1969.
 

Borzelleca, J.F., P.S. Larson, E.M. Crawford, G.R. Hennigar, Jr.,
 

E.J. Kuchar and H.H. Klein, "Toxicologic and Metabolic Studies
 

on Pentachloronitrobenzene." Toxicology and Applied Pharmacology 18:522-534, 1971.
 

Buselmaier, W., G. Roehborn and P. Propping, "Comparative Investigations on the Mutagenicity of Pesticides in Mammalian Test Systems." Mutation Research 21:25-26, 1973.
 

Chemical Abstracts Service, The Chemical Abstract'' Service (CAS)
 

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

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

Clarke, C.H., "The Mutagenic Specificities of Pentachloronitrobenzene
 

and Captan, Two Environmental Mutagens." Mutation Research 11:
 

247-248, 1971.
 

Courtney, K.D., M.F. Copeland, and A. Robbins, "The Effects of Pentachloronitrobenzene, Hexachlorobenzene and Related Compounds on
 

Fetal Development." Toxicology and Applied Pharmacology 35:239­ 256, 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.
 

Finnegan, J.K., P.S. Larson, R.B. Smith, Jr., H.B. Haag, and G.R.
 

Hennigar, "Acute and Chronic Toxicity Studies on Pentachloronitrobenzene." Arch. Int. Pharmacodyn 114:38, 1958.
 

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.
 

42
 
Innes, J.R.M., B.M. Ulland, M.G. Valerio, L. Petrucelli, L. Fishbein,
 E.R. Hart, A.J. Pallota, R.R. Bates, H.L. Falk, J.J. Gart, M.
 

Klein, I. Mitchell, and J. Peters, "Bioassay of Pesticides and
 

Industrial Chemicals for Tumorigenicity in Mice. A Preliminary
 

Note." Journal of the National Cancer Institute 42:1101, 1969.
 

International Agency for Research on Cancer, "Quintozene (Pentachlorophenol)." IARC Monographs on the Evaluation of Carcinogenic Risk
 

of Chemicals to Man—Some Organochlorine Pesticides JK 211-218 ,
 

1974.
 

Johnson, 0., "Pesticides '72." Chemical Week:30, July 26, 1972 as
 

cited in International Agency for Research on Cancer, 1974.
 

Jordan, R.L., F. Sperling, H.H. Klein, and J.F. Borzelleca, "A Study
 

of the Potential Teratogenic Effects of Pentachloronitrobenzene
 

in Rats." Toxicology and Applied Pharmacology 33:222-230, 1975.
 

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

Observations." Journal of the American Statistical Association
 

J53_:457-481, 1958.
 

Khera, K.S., and B.C. Villeneuve, Teratogenicity Studies on Halogenated Benzenes (Pentachloro- , Pentachloronitro- , and Hexabromo- )
 

in Rats." Toxicology 5:117-122, 1975.
 

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.
 

Reuber, M.D., and E.L. Glover, "Cirrhosis and Carcinoma of the Liver
 

in Male Rats Given Subcutaneous Carbon Tetrachloride." Journal
 

of the National Cancer Institute 44:419-423, 1970.
 

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.
 

Searle, C.E. Tumor Initiatory Activity of Some Chloromononitrobenzenes and Other Compounds." Cancer Research 26:12, 19-66.
 

Stanford Research Institute, 1976 Directory of Chemical Producers,
 

U.S.A. Menlo Park, California, 1976.
 

43
 
Tarone, R.E., "Tests for Trend in Life-Table Analysis." Biometrika
 

j>2.: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, Parts I and II. U.S. Government
 

Printing Office, Washington, D.C., 1969.
 

Vogel, E. and J.L.R. Chandler, "Mutagenicity Testing of Cyclamate
 

and Some Pesticides in Drosophila melanogaster." Experimentia
 

30:621-623, 1974.
 

44
 
Review of the Bioassay of Pentachloronitrobenzene*
 

for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

January 18, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976 under the authority of the National
 

Cancer Act of 1971 (P.L. 92-218). The purpose of the Clear­ inghouse is to advise on the National Cancer Institute's
 

bioassay program to identify and evaluate chemical carcino­ gens 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 organic chemistry, bio­ chemistry, biostatistics, toxicology, pathology, and epide­ miology. 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 NCI bioassay
 

reports on chemicals studied for carcinogenicity, In this
 

context, below is the edited excerpt from the minutes of the
 

Subgroup's meeting at which Pentachloronitrobenzene was
 

reviewed.
 

The primary reviewer commented that adjustments in
 

exposure levels had to be made during the chronic phase due
 

to overt toxicity. He concurred with the conclusion in
 

the report that Pentachloronitrobenzene (PCNB) was not
 

carcinogenic in rats or mice, under the conditions of test.
 There was no observation of rare or unusual tumors or
 

statistically significant associations between the incidence
 

of neoplasms and treatment.
 

The secondary reviewer also agreed with the conclusion
 

given in the report. He pointed out, however, that the
 

survival among the male mice was relatively poor and the
 

size of the control groups were too small.
 

It was moved that the bioassay report be accepted as
 

written. The motion was seconded and approved unanimously.
 

45
 
Members Present Were:
 

Arnold Brown (Acting Chairman), Mayo Clinic
 

Lawrence Garfinkel, American Cancer Society
 

Joseph Highland, Environmental Defense Fund
 

Charles Kensler, Arthur D. Little Company
 

Verald K. Rowe, Dow Chemical, U.S.A.
 Sheldon Samuels, Industrial Union Department, AFL-CIO
 

Louise Strong, University of Texas Health Sciences Center
 

Sidney Wolfe, Health Research Group
 

*	 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.
 

46
 MJ.S. GOVERNMENT PRINTING OFFICE: 1978 260-899/3056 1-3 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH PCNB
 

TABLE Al

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH PCNB 

riltiH DOSE
 01-BC43
 

50
 

49
 

37
 

(49)
 

2 (4X)
 

(49)
 

1 (2«)
 

1 U*>
 

(35)
 

2 (6S)
 

1 (3X)
 

(37)
 

1 (3X)
 

(36)
 

CONTROL (VEH)


01-MOU1
 

ANIMALS INITIALLY III STUDY 20
 

ANIMALS kECBOESIED 20
 

ANIMALS EXAMINED HISTOPATHOLOGICA LLY •« ' 20
 

INTEGUMEK1ABY SYS1EM
 

*SUBCUT TISSUE (20)
 FIBBOMA 1 <5S)
 FIBBCSABCOMA
 

LIPCKA
 HEMANGIOSAfCOMA 1 <5X)
 

BESPIBATCBY SYSTEM
 

NONE
 

HEMATOPCIETIC SYSTEM
 

'MULTIPLE ORGANS (20)
 

MALIG.LYMPhGMA, HISTIOCYTIC TYPE
 

GBAdULOCJTIC LEUKEMIA
 

•SPLEEN	 (20)
 HENANGIOSABCOMA
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (5X)
 

fKIDNEY (20)
 MALIG.LYMPHOMA. HISTIOCYTIC TYPE
 

CIBCULA1CBY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVEB (20)
 

ai£41QSBli!ii4R.C4S£I£2fl4
 

LOW CCSE
 01-M042
 

50
 

U8
 

U3
 

(48)
 

3 (6*)
 

1 (2*)
 

1 UX)
 

1 <2X)
 

(18)
 

2 (i*X)
 

(«2)
 

(43)
 

(43)
 

l_JiSl
 

t NUMBE B OF ANIMAL S UIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMEE B OF ANIMAL S NECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-3 
TABLE Al (CONTINUED)
 

CONTROL (VEH) LOM DOSE
 

01-MOU1 01-8042
 

•PANCREAS	 (20) (<*3)
 

ADENCCARCINOMA, NCS, (1E1AS1ATIC 1 (2'i)
 

URINARY SYSTEM
 

•KIDNEY	 (20) (43)
 

ADENOCA8CINOMA, NCS, MEIA5TAT1C 1 (2%)
 MIXED TUMOR, MALIGNANT 3 (7X)
 HrMANGIOSAFCOHA
 

BNEOCBIN E SYSTEM
 

•PITUITARY	 (20) (42)
 CHKOnOPHGBE ADENOMA 2 (10%) 3 (!%}
 

• ADRENA L	 (20) («3)
 PHEOCHROMCCYTOMA 1 (2%)
 

•THYROID	 (20) (U3)
 FOLIICULAR-CELL ADENOMA 1 (5X) 2 (:>*)
 FOLLICULAR-CELL CARCINOMA 1 (5%)
 C-CELL ADENOK A 1 (5X) 1 (2*)
 C-CELL CARCINOMA 1 (2%)
 

REPRODUCTIVE SYSTEM
 

*MAMMAR Y GLAND (20) (48)
 ADENOMA , NOS 1 (2%)
 ADENCCARCINOMA, NOS 1 (2%)
 FIBHOADENOMA 2 (10S) it (3X)
 

*EPIDIDIt!IS (20) C*8)
 MESOTHELIOMA, KETASTATIC 1 (5X)
 

NEBVCUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NCNE
 

» NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUBBEB OF ANIMALS NECROPSIED
 

HIGH DOSE
 

01-MC43
 

(36)
 

(37)
 

1 (3%)
 

1 (3S)
 

(37)
 

7 (19%)
 

(37)
 

(37)
 

2 (5%)
 

1 (3X)
 

(49)
 

2 (4%)
 

(49)
 

A-4
 
MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDOMINAL CAVITY
 

LlfCMA
 

*MESENTERY
 

MESOTnELIOMA, MALIGNANT
 

ALL OTHIB SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMABY
 

ANIHAIS INITIALLY IN STUDY
 

NATUfAI, DEATHS
 

MCRIEUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANI»AL MISSING
 

* Jj»Eiflfiifi_AfiISJ.irz££ amflits
 

TABLE Al (CONTINUED)
 

CONTROL (VEH)
 

01-MOU1
 

(20)
 

(20)
 

1 (5*)
 

20
 

10
 

10
 

LOW DOSE
 

C1-M04i!
 

(18)
 

1 (2*)
 

(48)
 

50
 

28
 

1
 

21
 

HIGH DOSE
 

01-HCU3
 

(49)
 

1 (2X)
 

(49)
 

50
 

18
 

32
 

* NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-5 
TABLE Al (CONCLUDED)
 

CONTROL (VEH) LOW DOSE HIGH DOSE
 

01-M041 01-MOU2 01-HOU3
 

TUMOR SUMMAR Y
 

TOTAL ANIMALS WITH ERIMARY TUMORS* 8 20 18
 

TOTAL PRIMARY TUMORS 11 27 23
 

TOTAL ANIMALS WITH BENIGN TUMORS 5 13 10
 TOTAL BENIGN TUMORS 7 17 12
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 4 10 11
 TOTAL MALIGNANT TUMORS u 10 1 1
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1 1
 TOTAL SECONDARY TUMORS i 2
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMOBS UNCERTAIN­ PRIMAEY OR BETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECONDARY TUMOBS: METASTATIC TUMORS OH TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

A-6
 
TABLE A2
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE


CONTROL (VEH)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS KISSING
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY


INTEGUMENTARY SYS1EM
 

*SKIN
 

SQUAMOUS CELL CARCINOMA
 

*SUBCU1 TISSUE
 

FIBBCMA
 

FIBROSARCOMA
 

LI EC HA
 

RESPISATCRY SYSTEM
 

#LUNG
 

ADENOCARCINOMA, NOS, METASTATIC
 

FIBBOSARCOMA, METASTATIC
 

HEMATOf CIETIC SYSTEM
 

•MULTIPLE	 ORGANS
 

BALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

#SPLEEN
 

HEMANGIOSABCOMA
 

»LUNG
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

CIRCULA1CBY SYSTEM
 

NONE
 

DIGES1IVE SYSTEM
 

NONE
 

 **
 

01-F041
 

20
 

20
 

20
 

(20)
 

1 (5*)
 

(20)
 

1 (5X)
 

(20)
 

(20)
 

(20)
 

1 <5X)
 

(20)
 

 RATS TREATED WITH PCNB
 

LCK DCSE HIGH DOSE
 

01-F044 01-FCU5
 

50	 50
 

1
 

50 48
 

HO 34
 

(50)	 (48)
 

(50) («8)
 

2 (4%)
 

1 (2%)
 

1 <2X)
 

(10)	 (37)
 

1	 (3%)
 

1 (3%)
 

(50)	 (48)
 

1 (2%)
 

(10)	 (36)


1 (3*) 1 (3%)
 

C*0) (37)
 

1 (3%)
 

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

* NUMBE R OF ANIMALS NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALi 

A-7 
TABLE A2 (CONTINUED)
 

CONTROL (VEH) LOW DOSE
 

01-F011 01-FCW4
 

URINAR Y SYSTEM
 

((KIDNEY (20) (39)
 

ADENCCARCINOMA. DCS, MEXASIATIC 1 (3%)
 MIXED TUMOR, MALIGNANT
 

«URINAR Y BLADDER (19)
 

PAPILLOMA, NOS
 

ENDOCRINE SYSTEM
 

4PITUITAEY (20) (10)
 CHRGMOPHOEE ADENOMA 7 (35%) 20 (50%)
 

•ADRENAL	 (20) (39)
 

CORTICAL ADENOMA 1 (5%)
 

ItTHYROID (20) (10)
 

FOllICULAK-CELL CARCINOMA 1 (3X)
 C-CELL ADENOMA 1 (3%)
 

C-CEI.L CARCINOMA 3 (8%)
 

•PANCREATIC	 ISLETS (20) (10)
 ISLJT-CELL ADENOMA 1 (3%)
 

REPRODUCTIVE SYSTEM
 

*MAMMAR Y GLAND (20) (50)
 

ADENOCARCINOMA, NCS 1 (2%)
 

FIBROADENOMA 7 (35%) 9 (18%)
 

tUTERUS (19) (39)
 

ADENOCARCINOMA, NOS 2 (5%)
 

ENDOMETRIAL STROMAl POLYP 1 (5%) 2 (5%)
 

tOVARY (20) (UO)
 

CYSIADENOMA, NOS 1 (3%)
 

SERTXLI-CELL TUMOR 1 (3X)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE CEGANS
 

NONE _
 

* NUMBE R OF ANIMALS WITH 1I5SUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMALS NECSOPSIED
 

HISH DOSE
 

01-FOUS
 

(37)
 

1 (3%)
 

(33)
 

1 (3%)
 

(35)
 

8 (23%)
 

(36)
 

(36)
 

1 <3X)
 

(36)
 

C*8)
 

8 (17%)
 

(37)
 

1 (3%)
 

(37)
 

A-8
 
MUSCULOSKELETAL SYSTEM
 

*MUSCLE HIP/THIGH
 

FIBECSARCGMA
 

BODY CAVITIES
 

*ABDOMINAL CAVITY
 

LIPCfA
 

ALL OTHEF SYSTEMS
 

NCNE
 

ANIMAL DISPOSITION SUMMAR Y
 

ANIMALS INITIALLY IN STUDY
 

NATUEA L DEATHS
 

MORIBUND SACBIFICE
 

SCHEEULED SACRIFICE
 

ACCIEENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

S	 INCLUDES AUTULYZED ANIMALS
 

TABLE A2 (CONTINUED) 

CONTROL (VEH)
 

01-F041
 

(20)
 

(20)
 

20
 

13
 

7
 

LOW DOSE HIGH DOSE
 01-FOUU O1-F0i*5
 

(50)	 (18)
 

1 (2%)
 

(50)	 (48)
 

1 (2%)
 

50 50
 

11 16
 

1
 

38 33
 

1
 

*	 NUMBE8 OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NECBOPSIED
 

A-9
 
TABLE A2 (CONCLUDED) 

CONTROL (VEH) LOW DOSE HIGH DOSE
 

01-F041 01-FOKU 01-F045
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH EBIHAEY IUMOBS* 1U 31 20
 

TOTAL PRIMARY TUMORS 19 51 23
 

TOTAL ANIMALS WITH BENIGN TUMORS 13 29 16
 TOTAL BENIGN TUMORS 17 UO 18
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 2 10 5
 TOTAL MALIGNANT TUMORS 2 11 5
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1 1
 

TOTAL SECONDARY TUMORS 2 1
 

TOTAL ANIMALS WITH TUMOES UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPSIMAEY 08 MITASTATIC
 TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

A-IO
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH PCNB
 

TABLE Bl

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH PCNB 

CONTROL (VEH) 

02-M037 

LOW DOSE 

02-M038 

HIGH DOSE 

02-M039 

ANIMAIS INITIALLY IN STUDY 

ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTCP ATHOLOGICALLY ** 

20 

20 

20 

50 

15 

33 

50 

48 

42 

INTfGUKIMABY SYSTEM
 

•SUBCUT	 TISSUE (20) (15) (18)
 FIBECMA 1 (2%)
 FIBECSAECCMA 2 (10%) 3 (6%) 5 (1C*)
 

UESPIKATCfiY SYSTEM
 

NONE
 

HEMATCECIETIC SYSTEM
 

*MULTIPLE ORGANS (20) (15) (18)
 MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (5%) 1 (2%)
 MALIG.LYMPHGMA, HISTIOCYTIC TYPE 1 (5*) 2 (UX)
 

*Hr.SENTERIC L. NODE (13) (20) (31)
 

HEMANGI03AKCOMA 1 (8X>
 

CIHCULA1CEY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

ILIVER (20) (35) (12)
 HEPATOCEILULAE CAfiCISOMA 2 (10*) 8 (23%) 14 (10JS)
 

UHINAH Y SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

NONE.
 

* NUMBE R O P ANIMAL S WIT H TISSU E EXAMINE D MICROSCOPICALL Y 

* NUI1BE H OF ANIMAL S NECBOPSIE D 

**EXCLUDESPARTIALLY AUTOLYZEDANIMALS 

B-3 
TABLE Bl (CONTINUED) 

CONTROL (VEH) LOU DOSE HIGH DOSE 

02-M037 02-M038 02-M039 

REPRODUCTIV E SYSTE M 

NON E 

NE8VCU S SYSTE M 

fBRAI N (20) (35) (11)

 EPENtYMOM A 1 <3X)

 

SPECIA L SENS E ORGAN S 

NON E 

MUSCULOSKELIIA L SYSTE M 

NON E 

BOCY CAVITIE S 

NCN E 

A LL OTHE R SYSTEMS 

NON E 

ANIHA 1 DISPOSITION SUKMAB Y 

ANIMAL S INITIALL Y I N STUDY 20 50 50

 NATURA L DEATH* 16 21 33

 MORIBUN D SACRIFICE

 

SCHEEULE D SACRIFICE 

ACCICENTALL Y KILLE D 1

 

TERMINA L SACRIFICE V 25 17

 

ANIKA L MISSING 

3_Il!£I,U£fSJUllQU2£D_£l!ttAIiS 

* NUHBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMAL S NECBOfSIED
 

B-4
 
TABLE Bl (CONCLUDED) 

TUMOR SUMMAR Y
 

TOTAL ANIMALS «ITH PRIMARY TUMORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS KITH BENIGN TUMORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMALS KITH flAIIGNANT TUMORS
 

TOTAL MALIGNANT TUMORS
 

TOTAL AKIKALS WITH SECONDARY TUMORS*
 TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS HITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR MITASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL (VEH)
 02-M037
 

6
 

7
 

6
 

7
 

LOW DOSE HIGH DOSE 

02-B038 02-M039 

11 12 

1U 12 

1 

1 

1 1 11 

11 11 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUHOBS
 

# SECONDARY TUMORS: ME1ASTATIC TUMOBS OH TUMORS INVASIVE INTO AN ADJACENT OHGAN
 

B-5
 
TABLE B2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED WITH PCNB 

CONTROL (VEH) LCH DCSE
 

02-F037 C2-F01»0
 

ANIMALS INITIALLY IN STUDY 20 50
 

ANIMALS KISSING 2
 ANIMALS NECROPSIED 20 47
 ANIMALS EXAMINED HISTOPATHOLOGICALLY** 20 23
 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (20) (47)
 

FIBfiCSARCCMA
 

BESPIBATOEY SYSTEM
 

»LUNG (20) (23)
 

ALVECLAR/BBONCHIOLAB ADENOMA
 

OSTEOSARCOMA, MEIASTATIC
 

HEMATOECIETIC SYSTEM
 

'MULTIPLE ORGANS (20) C*7)
 MAIIG.LYMPHOMA. LYMEHOCYTIC TYPE 1 (2*)
 MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (5X)
 

iSPLEEN (20) (23)
 HEMANGIOSARCOMA 1 (5») 1 (»*)
 

»MSSENTERIC L. NODE (20) (20)
 

FIriECSAHCCKA, METASTATIC
 

CIRCUIA1CBY SYSTEM
 

NOSE
 

DIGESTIVE SYS1EM
 

ffLIVEH (20) (1<t)
 HEPATOCELLULAR CARCINOMA
 

URINAR Y SYSTEM
 

NCJiE
 

* NUMBE R O P ANIMAL S WIT H TISSUE EXAMIN2 D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S NECEOPSIE D 

**EXCLUDESPARTIALLY AUTOLYZEDANIMALS 

HIGH DOSE
 02-EOU1
 

50
 

1
 

46
 

21
 

(46)
 

1 <2S)
 

(20)
 

1 (5%)
 

1 (5%)
 

(46)
 

5 (11X)
 

(20)
 

1 <5X)
 

(19)
 

1 (5X)
 

(20)
 3 n '•*!
 

B-6 
TABLE B2 (CONTINUED) 

ENDOCRINE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEM
 

*KAMMARY GLAND
 ADENCMA, NCS
 

ADENCCARCINOMA, NOS
 

NERVOUS SYSTEM
 

NCN E
 

SPECIAL SENSE CBGANS
 

NONE
 

MUSCULOSKEIETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NOSE
 

ANIMAL EISFCSITION SUMMARY
 

ANIMAI S INITIALLY I IS STUD!'
 HAlliSAL DEATHS
 

MOHtnUN U SACRIFICE
 SCHEDULED SACRIFICE
 ACCIEESTALLY KILLED
 TERMINAL SACRIFICE
 

ANIKA:. :«SSI«G
 

o. ;NCL!'Ot.S AUTOLYZED A8IHSLS
 

CONTROL (VEH)
 

02-F037
 

(20)
 

) ^
 

1
 

1 ^
 

LOB DOSE HIGH DOSE
 

02-F040 02-F011
 

(47)	 (1*6)
 

2 (H X)
 

1 <2X)
 

50 50
 

5 1C
 

t3 39
 2 1
 

* yUiMhE B Cf ANIMAL S KIT H TISSUE EXAMINE D MICROSCOPICALL Y * MBHEES OF ANIMAL S NECROPSIED 

B-7 
TABLE B2 (CONCLUDED)
 

CONTROL (VEH) LOW DOSE HIGH DOSE
 

02-F037 02-FOUO 02-FOU1
 

TUMOR SUMMARY
 

TOTAL ANIMALS KITH PRIMARY TUMORS* 2 5 10
 

TOTAL PRIMARY TUHOBS 2 5 11
 

TOTAL ANIMALS KITH BENIGN TUMORS 2 1
 

TOTAL BENIGN TUMORS 2 1
 

TOTAL ANIMALS KITH MALIGNANT TUMORS 2 3 9
 TOTAI MALIGNANT TUMORS 2 3 10
 

TOTAL ANIMALS KITH SECONDARY TUMORS* 2
 TOTAL SECONDARY TUMORS 2
 

TOTAL ANIMALS NITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL AKIMALS KITH TUMOBS UNCERTAINPEIMA6Y OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIHARX TUHORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 • SECONDARY TUMORS: ME1ASTAIIC TUMORS OB TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-8
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH PCNB
 

TABLE Cl

 

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

HIGH DOSE
 01-M043
 

50
 

49
 

37
 

(49)
 

(49)
 

(49)
 

(37)


9 (24X)
 

(37)
 

(35)
 

(36)
 

1 (3X)
 

(37)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS KECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY


INTEGUMENTARY SYSTEM
 

*SKIN
 

HYPERPLASIA, NOS
 

*SUBCUT TISSUE
 

ABSCESS. NOS
 

BESPIBATCRY SYSTEM
 

*ACCESSOfiY SINUS
 

INFLAMMATION, NOS
 

*LIJNG
 

PNEUMONIA, CHRONIC MURINE
 

HYPERPLASIA, NOS
 

HEMATOfOIETIC SYSTEM
 

IBONE HARROW
 

METAMORPHOSIS FATTY .
 

(SPLEEN
 HEHATOPOIESIS
 

tMESENTERIC L. NODE
 INFLAMMATION, NOS
 

CIRCUIATCRY SYSTEM
 

IHEABT
 

THBCBBOSIS, NOS
 

- _CAl£JJU_fiSfflSII_
 

CONTROL (VEH)
 01-M041
 

20
 

20
 

 ** 20
 

(20)
 

(20)


1 (5*)
 

(20)
 

(20)


8 (40X)
 

(20)
 

(20)


1 (5X)
 

(19)
 

(20)
 

1 (5*)
 

_2-Ji5Si


LOW DCSE
 G1-M042
 

50
 

UB
 

43
 

(48)
 

1 (2X)
 

(48)
 

(48)
 

1 (2*)
 

(43)
 

11	 (26*)
 

1 (2*)
 

(43)
 

1 (2%}
 

(42)
 

1 (2*)
 

(42)
 

(43)
 

 2.J521
 

* NUMBEfi OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMALS NECGOPSIED 

**EXCLUDESPARTIALLY AUTOLYZEDANIMALS 

C-3 
HYPERPLASIA, NOS
 

•MYOCARDIUM
 INFLAMMATION, NOS
 

DEGENERATION, NOS
 

•ENDOCARDIUM
 

INFLAMMATION, SOS
 

*AORTA
 

THRCMBOSIS, DOS
 

ARTERIOSCLEROSIS, NOS
 

CALCIUM DEPOSIT
 

*MESENTERIC ARTERY
 THROMBOSIS, NOS
 

CALCIUM DEPOSIT
 

DIGESTIVE SYSTEM
 

•LIVER
 THHOr.BUS, ORGANIZED
 

INFLAMMATION. NOS
 

PELICSIS HEEATIS
 

METAMORPHOSIS FATTY
 

*BILE DUCT
 HYPEHPLASIA, NOS
 

•STOMACH
 

ULCER, FOCAL
 CALCIUM DEPOSIT
 

•COLON
 PARASITISM
 

UHINAHY SYSTEM
 

•KIDNEY
 CYST, SOS
 PYELCNEPHBITIS, NOS
 INFLAMMATION, CHRONIC
 

T ABLE Cl (CONTINUED)
 

CONTROL (VEB)
 

01-B041
 

(20)
 

3 (15X)
 

(20)
 

(20)
 

1 (5X)
 

2 (10X)
 

1 (5*)
 

(20)
 

1 (5X)
 

(20)
 

(20)
 

(20)
 

2 (10X)
 

(20)
 

(20)
 

1 (5X)
 

10 (SOX)
 

LOU DOSE
 01-M042
 

1 (2X)
 

(43)
 

2 (5X)
 

(43)
 

(48)
 

5 (10X)
 

(48)
 

2 (4X)
 

(43)
 

2 (5X)
 

1 (2X)
 4 (9X)
 

(48)
 

1 (2X)
 

(43)
 

2 (5%)
 

5 (12X)
 

(42)
 

1 (2X)
 

(43)
 

2 (5X)
 

27 (63X)
 

HIGH DOSE
 01-M043
 

(37)
 

3 <8X)
 

3 (8X)
 

(37)
 

1 (3X)
 

(49)
 

(49)
 

(36)
 

1 (3?£)
 

1 (3%)
 

(49)
 

(37)
 

2 (5X>
 

(37)
 

1 (3X)
 

(37)
 

15 (41X)
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROESIED
 

C-4
 
ENDOCRINE SYSTEM
 

•PITUITARY
 

CYST, SOS
 

•THYROID
 

CYST, NOS
 

FOLLICULAH CYST, NOS
 

HYPE6ELASIA. C-CILL
 

HYPEBPLASIA, FOLLICULAR -CEll
 

•PARATHYROID
 

HYTESPLASIA, NOS
 

REPSOtUCTIVE SYSTEM
 

•PROSTATE
 

INFLAMMATION, (JOS
 

KTESIIS
 

CALCIUM DEPOSIT
 

ATRCfHY. NOS
 

HYPEBPLASIA. NOS
 

NERVOU S SY.STEK
 

• BHAIlVBENINGES
 

INFLAMMATION, NOS
 

SPECIAL SENSE C8GANS
 

*EYE
 

INEIABKATICN, NOS
 

CATARACT
 

MUSCUIOShELETAl SYSTEK
 

NCNE
 

BODY CAVITIES
 

*PERITCSEUM
 

IHEiififiAIISK* i!2S
 

TABLECl (CONTINUED) 

CONT30I. (»EH)
 

01-MO«1
 

(20)
 

1 (5X)
 

(20)
 

1 <5S)
 

1 (5«)
 

1 (5%)
 J (15*)
 

(1)
 

1 (10PJ)
 

(17)
 

(20)
 

7 (35«)
 

(20)
 

(20)
 

1 (5X)
 

(20)
 

LOti DOSE
 01-HOU2
 

(<42)
 

3 (7*)
 

(4J)
 

2 (vX)
 

1 (2*)
 

(6)
 

5 (100*)
 

(38)
 

3 (8*>
 

("3)
 

1 (2$)
 

15 (35)i)
 

1 (2X)
 

(«3>
 

(U8)
 

1 (2S)
 

(48)
 

rflJH DOSE
 01-MCa3
 

(37)
 

1 (3%)
 

(37)
 

(33)
 

2 (6X)
 

(37)
 

8 (22%)
 

(37)
 

1 (3S)
 

(«9)
 

(49)
 

1-i2*l
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICBOSCOPICALLI
 

* NUMBER OF ANIMALS NECBOPSIED
 

C-5
 
TABLE Cl (CONCLUDED)
 

CONTRO L (VEH ) L OW DOSE 

01-M04 1 C1-.1CU2 

*EERICAIiEIU n 120) (18)

 INFLAMMATION , NO S

 

*HESENTER Y (20 ) (US ) 

f EBIABTEE1TI S 5 (10S) 

A L L 01HEB SYSTEM S 

NCN E 

SPECIA L fCBEHCIOG Y StlrBAK Y 

N O LESIO N REPORTE D 1 3 

NECROPS Y PEhF/N O HISIO PERFORME D a

 AUTC/liECEOE-SY/N O HISTO 1

 AUTOLYSIS/N O NECROPS Y 2

 

« NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY
 

* NUMEE B OF ANIMALS NZCBOPSIED
 

HIG H DOSE 

01 -MOI4 3 

(U9 ) 

1 (2% ) 

(1*9) 

2 (IS) 

5 

1 1 

1 

1 

C-6
 
TABLE C2

 

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

HIGH DOSE
 

01-FC45
 

50
 

1
 

48
 

3U
 

(48)
 

(37)
 

3 (8X)
 

(37)
 

2 (5%)
 

(36)
 

(31)
 

(37)
 

2 (5%)
 

2 (58)
 

(37)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS KISSING
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICA LLYY*
 

INTEGUMENTABY SYSTEM
 

NCNE
 

RESPIRATORY SYSTEM
 

*ACCESSCBY SINUS
 

INFLAMMATION, NOS
 

*LUNG
 

PNEUMONIA, CHRONIC MU8IWE
 

CALCIUM DEPOSIT
 

HEtMATOECIETIC SYSTEM
 

tBONE MARRO W
 

METAMORPHOSIS FATTY
 

*SPLEEN
 

ANGIECIASJS
 

HEKATOEOIESIS
 

•CERVICAL	 LYMPH NODE
 

INFLAMMATION, NOS
 

CIRCULATORY SYSTEM
 

•HEART
 

THBCMBUS, ORGANIZED
 

CALCIUM DEPOSIT
 

MYOCARDIUM
 

eSSENEBATIOH^ NO.S
 

CONTROL (VEH)


01-F041
 

20
 

20
 

20
 

(20)
 

(20)
 

2 (10*)
 

1 (5%)
 

(20)
 

(20)
 

1 (5*)
 

(20)
 

(20)
 

1 (5«)
 

(20)
 

JLI521 .
 

LCH BCSE
 

01-F044
 

50
 

50
 

UC
 

(50)
 

2 (UX)
 

(10)
 

2 (5X)
 

(40)
 

3 (3%)
 

(U"))
 

1 (JX)
 

7 (13%)
 

(33)
 

1 <3X)
 

(40)
 

1 (3 X)
 

(40)
 

J J3%i
 

* NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

C-7
 
*AOHTA
 

ARTERIOSCLEROSIS, NOS
 

DIGESTIVE SYSTEM
 

*GUM
 

INFLAMMATION, NOS
 

•LIVER
 

INFLAMMATION, DOS
 

INFLAMMATION, FOCAL
 

METAMOKEHOSIS FATTY
 

FOCAL CELLULAR CHANGE
 

*STOMACH
 

ULCER, NOS
 

CALCIUM CEEOSIT
 

•COLON
 

PARASITISM
 

URINAR Y SYSTEM
 

•KIDNEY
 

INFLAMMATION, CHRONIC
 

CALCIUM DEPOSIT
 

•URINARY	 BLADDER
 INFLAMMATION, CHRONIC
 

ENEOCRINE SYSTEM
 

•PITUITARY
 CYST, NOS
 

•ADRENAL
 

CYST, NOS
 

ANGIECTASIS
 

•PARATHYROID
 

__ flYPJSPLASIi^ SOS
 

TABLE C2 (CONTINUED) 

CONTROL (VEH)
 

01-F011
 

(20)
 

2 (10X)
 

(20)
 

(20)
 

(20)
 

2 (10X)
 

(20)
 

(20)
 

4 (20S)
 

(19)
 

(20)
 

1 (5X)
 

(20)
 

1 (5X)
 

(1)


J ^ICOJi.
 

LOW DOSE
 C1-FC14U
 

(50)
 

(50)
 

1 US)
 

(40)
 

1 (3X)
 

1 (3%)
 

U (10%)
 

1 (3%)
 

(UO)
 

1 (3X)
 

1 (3%)
 

(HO)
 

2 (5X)
 

(39)
 

5 (13S)
 

1 (3%)
 

•	 C*0)
 

(39)
 

2 (5X)
 

3 (8*)
 

(1)


1 J100XJ.
 

HIGH DOSE
 01-F015
 

(48)
 

(U8)
 

(37)
 

1 (3%)
 

(37)
 

(37)
 

(37)
 

5 (1t)S)
 

1 (3%)
 

(33)


1 (3X)
 

(35)
 

(36)
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROFSIED
 

C-8
 
TABLE C2 (CONTINUED) 

CONTROL (VEH) LOW DOSE
 01-F011 C1-FOUII
 

REPRODUCTIVE SYSTEM
 

*MAMMAR Y GLAND (20) (50)
 GALACTOCELE 1 (2X)
 

*VAGINA (20) (50)
 INFLAMMATION, NOS 1 <2X)
 

*UTERUS (19) (39)
 HYtRCMETRA 2 (5X)
 INFLAMMATION, NOS 1 (5X) t (10S5)
 

tUTERUS/ENDOMETRIUM (19) (39)
 HYPERPLASIA, CYSTIC
 

•OVARY	 (20) («0)
 CYST, NOS
 

INFLAMMATION, NOS 1 (5X)
 ABSCESS, NOS 1 (3X)
 

NERVOUS SYSTEM
 

NCNE
 

SPECIAL SENSE ORGAN S
 

*EYE/CCRNEA	 (20) (50)
 INFLAMMATION, NOS	 1 <5X)
 

MUSCULOSKELETAL SYSTEM
 

*MUSCLE HIP/THIGH (20) (50)
 CALCIUM DEPOSIT 1 (2%)
 

BODY CAVITIES
 

'PERITONEUM (20) (50)
 INFLAMMATION. NOS 1 (2X)
 

*MESENTERY (20) (50)
 PEEIARTERI1IS 2 (10X)
 

ALL OTHER SYSTEMS
 

NCN£___	 .
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

HI3H DOSE
 01-FC1.5
 

(18)
 

(48)
 

(37)
 

r

j (lux)
 

2 (5S)
 

(37)
 

1 (3X)
 

(37)
 

1 <3X)
 

C*8)
 

(«8)
 

<«8)
 

C*8)


2 (4X)
 

C-9
 
TABLE C2 (CONCLUDED)
 

CONTROL (VEH) LOW DOSE HIGH DOSE
 

01-FOU1 C1-F04U 01-FOU5
 

SPECIAL fOREHOLOGY SUMMAR Y
 

NO LESION REPORTED 2 1 8
 

ANIMA L MISSING/NO NEC60FSY 1
 

NECROPSY PERF/NO HISTO PERFORMED 10 1 1
 

AUTC/NECROPSY/HISTO PERF 1
 

AUTC/NECRCESY/NO HISTO 3
 

AUTOIYSIS/NO NECROPSY 1
 

C-IO
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH PCNB
 

TABLE Dl

 

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

HIGH DOSE
 

02-M039
 

50
 

18
 

U2
 

(48)
 

(tl)
 

1 (2%)
 

(39)
 

1 (3%)
 

11 (28X)
 

(3t)
 

(41)
 

(41)
 

(<*2)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICA LLY


INTEGUMHKTAEY SYSTEM
 

*SUBCUT TISSUE
 

ABSCESS, NOS
 

RESPIRA1CRY SYSTEM
 

*LUNG
 

PNEUMONIA, CHRONIC MURINE
 

HEMATOECIETIC SYSTEM
 

tSPLEEN
 

INFIAMMATION, NOS
 

AMYICIDOSIS
 

HEMATOP01E3IS
 

*MESENTERIC L. NODE
 

INFLAMMATION, NOS
 

CIRCUIATCRY SYSTEM
 

*MYOCABDIUM
 

INFLAMMATION, NOS
 

IENDOCARDIUM
 

INFLAMMATION, NOS
 

DIGESTIVE SYSTEM
 

•LIVES
 

ClfSl, NGS _
 

* NUBBEf l O F ANIMAL S WIT H TISSU E

* NUMBE R OF ANIMAL S NECROESIE D 

**EXCLUDESPARTIALLY AUTOLYZEDANIMALS 

CONTROL (VEH)
 

02-M037
 

20
 

20
 

 ** ' 20
 

(20)
 

(20)
 

1 (5X)
 

(20)
 

9 («5%)
 

(13)
 

(20)
 

2 (10X)
 

(20)
 

2 (10X)
 

(20)
 

1_J5*1
 

LOW DOSE
 

02-M038
 

50
 

U5
 

33
 

(45)
 

1 (2%)
 

(35)
 

1 (3*)
 

(33)
 

3 (9*)
 5 (15X)
 

(20)
 

1 (5%)
 

(35)
 

1 (3X)
 

(35)
 

(35)
 

 EXAMINE D MICROSCOPICALL Y 

D-3 
INFLAMMATION, NOS
 

AMYLCIDCSIS
 

METAMORPHOSIS FATTY
 

HYPEEPLASIA, BETICULUM CELL
 

•PANCREATIC	 DUCT
 

CYST, NOS
 

ICOLON
 

PAEASITISM
 

UEINAEY SYSTEM
 

tKIDNEY
 

HYDBCNEPHBOSIS
 

CYST, NOS
 

PCLYCYSTIC KIDNEY
 

PYELONEPHRITIS. NOS
 

INFLAMMATION, CHECNIC
 

AMYLCIDCSIS
 

CALCIUM DEPOSIT
 

tURINAR Y BLADDER
 

CYST, NOS
 

INFLAMMATION, NOS
 

ENDOCBINE SYSTEM
 

NCNE
 

REPRODUCTIVE SYSTEM
 

*PEOSTATE
 

INFLAMMATION, NOS
 

*SEMINAL VESICLE
 

INFLAMMATION, NOS
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NOJiE
 

NUMBE R OF ANIMAL S HUH TISSUE

NUMBE R O F ANIMAL S NECROP3IE D 

TABLE Dl (CONTINUED) 

CONTROL (VEH) LOU DOSE
 

02-M037 02-M038
 

1 (3»)
 2 (10X) 1 <3X)
 

1 (5X)
 

(20) (30)
 

(18)	 (33)
 

1 ((,%)
 

(20)	 (35)
 

1 (5%)
 

1 (5%)
 

1 (5X)
 

2 (10X) 1 (3*)
 t (20%) 3 (9X)
 U (20%) 1 (3*)
 

1 (5S)
 

(20)	 (35)
 

1 (5*)
 

1 (5%)
 

(18)	 (31)
 

1 (6%)
 

(20)	 (45)
 

1 (5X)
 

 EXAP11NS U rtlCSOSCOr 1C A LLY 

HIGH DOSE
 

02-HC39
 

1 (2X)
 

(38)
 

1 <3X)
 

(36)
 

(42)
 

1 (2X)
 

2 (5X)
 

12 (29X)
 

7 (17X)
 

1 (2*)
 

(42)
 

(34)
 

(48)
 

D-4 
TABLE Dl (CONCLUDED) 

CONTROL (VEH) 

02-M03 7 

LOW DOSE 

02-MC3 8 

HI3h DOSE 

02 ­M 03 9 

MUSCULOSKELEIA L

NCN E 

 SYSTE M 

BOD Y CAVITIE S 

NCN E 

A L L OTKE S

NON E 

 SYSTEM S 

SPECIAL KCEFHCLOG Y SUMMAR Y 

N O LESIO N EEPORTE E 

ACCIDENTA L DEAT H 

NECROPS Y PESF/N O HISTO PERFORME D 

AUTC/NECRO f SY/HISIO PIRF 

AUTO/NECRO P 3Y/MO HISTO 

AUTCLYS1S/N O NECRCPS Y 

2 12 

1 

1 1 

$ 

1 

u 

13 

6 

2 

£. 

D-5
 
TABLE D2

 

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

CONTHOL(VEH) 

02-F037 

LOW DOSE 

C2-F040 

ANIMALS INITIALLY IN STUDY 

ANIMALS MISSING 

ANIMALS NECROfSIED 

ANIMALS EXAMINED HISTGPATHOLOGIt.AI.LY ** 

20 

20 

20 

50 

2 

«7 

23 

INTEGUMEKTAKY SYSTEM
 

NCNE
 

RESPIRATORY SYSTEM
 

• LUNG (20) (23)
 PNEUMONIA, CHRONIC MUBINE 1 (4%)
 

HEMATOECIETIC SYSTEM
 

•SPLEEN	 (20) (23)
 AMYL01DOSIS
 

HEMATOPOIISIS 2 (10%) 2 (9X)
 

»LYKPH NODE (20) (20)
 INFLAMMATION, NOS
 

•PANCRSAIIC	 L.NODE (20) (20)
 INF LABMATION, NOS 1 (5%)
 

fMESENTERIC L. NODE (20) (20)
 HYPEHPLASIA, NO5 1 15%)
 

CIRCUIAlCfiY SYSTEM
 

•MYOCARDIUM	 (20) (23)
 INFLAMMATION, NOS
 

DIGESTIVE SYSTEM
 

#LIVER (20) (14)
 

-IfilliBHAEIQl!*. NOS _ _ .
 

» NUMBE R O F ANIMAL S KIT H TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S NECROPSIE D 

**EXCLUDESPARTIALLY AUTOLYZEDANIMALS 

HIGH DOSE
 

Q2-F041
 

50
 

1
 

U6
 

21
 

(20)
 

2 (10JS)
 

(20)
 

1 (5X)
 

1 (5%)
 

(19)
 

1 (5%)
 

(19)
 

(19)
 

(20)
 

1 (5X)
 

(20)
 

1 j(5%l
 

D-6 
TABLE D2 (CONTINUED) 

CONTROL (VEH) LOB DOSE
 

02-F037 02-F040
 

*GALLBLADDER (20) C*7)
 

INFLAMMATION, SOS
 

»PANCREAS (19) (21)
 

INFLAMMATION, NOS
 

(tCOLON (20) (20)
 PARASITISM 1 (5*)
 

URINAB Y SYSTEM
 

KKIDNEY (20) (23)
 PYELONEPHRITIS, NOS
 

ENDOCRINE SYSTEM
 

NONE
 

REP80DUC1IVE SYSTEM
 

*UTEEUS (20) (21)
 HYDRONET8A 3 (15X) 1 (5*)
 INFLAMMATION, NOS b (30*) 7 (33X)
 

»U1ERU£/ENDOMETRIUM (20) l^D
 HYPEBPLASIA, CYSTIC 5 (25*) « (19%)
 

tOVARY/CVIDUCT (20) (21)
 INFLAMMATION, NOS 2 (10X)
 

tOVARY (20) (21)
 CYST. NOS 1 (20») 1 (5X)
 INFLAMMATION, NOS 3 (15X) tt (19X)
 

NERVOUS SYSTEM
 

NCNE
 

SPECIAL SENSE ORGANS
 

*EYE (20) (U7)
 __ iaFLAMM4H21!t_l!OS_ .
 

# NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

02-FCU1
 

(1*6)
 

1 (2X)
 

(20)
 

1 (5X)
 

(18)
 

(20)
 

1 (5X)
 

(19)
 

2 (11%)
 

4 (21X)
 

(19)
 

5 (26X)
 

(19)
 

(18)
 

5 (23%)
 

2 (11X)
 

(U6)
 

. 1 12%l
 

D-7
 
TABLE D2 (CONCLUDED)
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVI1IES
 

*PERITCNEUK
 

INFLAMMATION, NOS
 

ALL OTfiEf SYSTEMS
 

NONE
 

SPECIAI tCEPHCLCGY SUBMAB Y
 

NO LESION EEPOE1EE
 

ANIMA L MISSING/NO NECROPSY
 

NECHCPSY PERF/NO HISIO PERFORMED
 

AUTOLYSIS/NO NECROPSY
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED


* NUMBER OF ANIMALS NECROPSIED
 

CONIHOL (VEH)
 02-F037
 

(20)
 

2 (10%)
 

5
 

 MICROSCOPICALLY
 

LOW DOSE
 

02-FOHO
 

(47)
 

1 (2%)
 

7
 

2
 

21
 

1
 

HIGH EOSE
 02-FCU1
 

(46)
 

3 (7%)
 

2
 

1
 

25
 

3
 

D-8
 
DHEW Publication No. (NIH) 78-1311