o 

X 

5 

3 National Cancer Institute J 

CO CARCINOGENESIS 

Technical Report Series 

r- No. 41 Q) 1978 s

r* 

BIOASSAY OF 

CHLOROTHALONIL 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 1897-45-6 

NCI-CG-TR-41 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF 

CHLOROTHALONIL 

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) 78-841 

BIOASSAY OF
 

CHLOROTHALONIL
 

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
 

chlorothalonil 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 environmental chemicals have the
 

capacity to produce cancer in animals. Negative results, in
 

which the test animals do not have a greater incidence of cancer
 

than control animals, do not necessarily mean that the test
 

chemical is not a carcinogen, inasmuch as 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 that exposure to
 

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

of the risk to man from animal carcinogens requires a wider
 

analysis.
 

CONTRIBUTORS: This bioassay of chlorothalonil was conducted by
 

Gulf South Research Institute, New Iberia, Louisiana, initially
 

under direct contract to NCI and currently under a subcontract to
 

Tracor Jitco, Inc., Rockville, Maryland prime contractor for the
 

NCI Carcinogenesis Testing Program.
 

The experimental design was determined by Drs. J. H.
 

Weisburger^- >^ and R. R. Bates-*-'-^; the doses were selected by Drs.
 

T. E. Shellenberger^'^, J. H. Weisburger, and R. R. Bates.
 

Administration of the chemical and observation of the animals
 

were supervised by Drs. T. E. Shellenberger and H. P.
 

Burchfield , with the technical assistance of Ms. D. H.
 

Monceaux^, Mr. D. Broussard^, and Mr. R. J. Wheeler .
 

Histopathologic examination was performed by Drs. E. Bernal^ and
 

B. Buratto^ at Gulf South Research Institute, and the diagnoses
 

ill
 
included in this report represent the interpretation of these
 

pathologists.
 

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

Mason Research Institute". Statistical analyses were performed
 

by Dr. J. R. Joiner', using methods selected for the bioassay
 

program by Dr. J. J. Gart^. Chemicals used in this bioassay were
 

analyzed under the direction of Dr. H. P. Burchfield , and the
 

results of the analyses were reviewed by Dr. S. S. Olin'.
 

This report was prepared at Tracer Jitco^ under the direction of
 

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

L. A. Campbell, Director of the Bioassay Program; Dr. S. S. Olin,
 

Deputy Director for Science; Drs. J. F. Robens and C. H.
 

Williams, toxicologists; Dr. R. L. Schueler, pathologist; Dr. G.
 

L. Miller, Mr. W. D. Reichardt, and Ms. L. A. Waitz, bioscience
 

writers; and Dr. E. W. Gunberg, technical editor, assisted by Ms.
 

Y. E. Presley.
 

The following other scientists at NCI were responsible for
 

evaluating the bioassay experiment, interpreting the results, and
 

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

Jr., Dr. J. Fielding Douglas, Dr. Dawn G. Goodman , Dr. Richard
 

A. Griesemer, Dr. Harry A. Milman, Dr. Thomas Orme, Dr. Robert A.
 

Squire10, Dr. Sherman Stinson, Dr. Jerrold M. Ward, and Dr.
 

Carrie E. Whitmire.
 

^Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York.
 

with the Office of the Commissioner, Food and Drug
 

Administration, Rockville, Maryland.
 

iv
 
^Gulf South Research Institute, Atchafalaya Basin Laboratories,
 

P. 0. Box 1177, New Iberia, Louisiana.
 

^Now with the National Center for Toxicological Research,
 

Jefferson, Arkansas.
 

6

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

Rockville, Maryland.
 

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

Maryland.
 

^Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Studies and Statistics, Division of Cancer
 

Cause and Prevention, National Cancer Institute, National
 

Institutes of Health, Bethesda, Maryland.
 

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

Suite 660, Washington, D.C.
 

with the Division of Comparative Medicine, Johns
 

Hopkins University, School of Medicine, Traylor Building,
 

Baltimore, Maryland.
 

V 

SUMMARY
 

A bioassay of technical-grade chlorothalonil for possible
 

carcinogenicity was conducted by administering the test chemical
 

in the diet to Osborne-Mendel rats and B6C3F1 mice.
 

Groups of 50 rats of each sex were administered chlorothalonil at
 

one of two doses for 80 weeks, then observed for 30-31 weeks.
 

Time-weighted average doses for both males and females were 5,063
 

or 10,126 ppm. Matched controls consisted of groups of 10
 

untreated rats of each sex; pooled controls consisted of the
 

matched-control groups combined with 55 untreated male or female
 

rats from similar bioassays of five other test chemicals. All
 

surviving rats were killed at 110-111 weeks.
 

Groups of 50 mice of each sex were administered chlorothalonil at
 

one of two doses for 80 weeks, then observed for 11-12 weeks.
 

Time-weighted average doses for males were 2,688 or 5,375 ppm,
 

and for females, 3,000 or 6,000 ppm. Matched controls consisted
 

of groups of 10 untreated mice of each sex; pooled controls
 

consisted of the matched-control groups combined with 50
 

untreated male or female mice from similar bioassays of five
 

other test chemicals. All surviving mice were killed at 91-92
 

weeks.
 

Clinical signs that appeared with increasing frequency in dosed
 

rats included hematuria and, from week 72 until termination of
 

the study, bright-yellow urine. Since the dosed female mice did
 

not have depression in mean body weights or decreased survival
 

compared with the controls, they may have been able to tolerate a
 

higher dose.
 

In rats, adenomas and carcinomas of the renal tubular epithelium
 

occurred with a significant dose-related trend in both the males
 

(P = 0.030) and the females (P = 0.007). These neoplasms also
 

occurred at a higher incidence in the high-dose males (P = 0.035)
 

and the high-dose females (P = 0.016) than in the corresponding
 

controls (males: pooled controls 0/62, low-dose 3/46, high-dose
 

4/49; females: pooled controls 0/62, low-dose 1/48, high-dose
 

vii
 
5/50). These tumors included both adenomas and carcinomas which
 

are considered to be histogenically related. Thus these findings
 

are interpreted as sufficient evidence for the carcinogenicity of
 

chlorothalonil.
 

In mice, no tumors were found to occur at a greater incidence
 

among dosed animals than among controls.
 

It is concluded that under the conditions of this bioassay,
 

technical-grade chlorothalonil was carcinogenic to Osborne-Mendel
 

rats, producing tumors of the kidney. Chlorothalonil was not
 

carcinogenic for B6C3F1 mice.
 

viii
 
TABLE OF CONTENTS
 

Page
 

I. Introduction. 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 4
 

C. Animals 5
 

D. Animal Maintenance 6
 

E. Subchronic Studies 7
 

F. Chronic Studies 8
 

G. Clinical and Pathologic Examinations 11
 

H. Data Recording and Statistical Analyses 12
 

III. Results -Rats 19
 

A. Body Weights and Clinical Signs (Rats) 19
 

B. Survival (Rats) 21
 

C. Pathology (Rats) 23
 

D. Statistical Analyses of Results (Rats) 24
 

IV. Results - Mice 27
 

A. Body Weights and Clinical Signs (Mice) 27
 

B. Survival (Mice) 29
 

C. Pathology (Mice) 29
 

D. Statistical Analyses of Results (Mice) 31
 

V. Discussion 33
 

VI. Bibliography 35
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Fed Chlorothalonil in the Diet 37
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Fed Chlorothalonil in the Diet 39
 

Table A2 Summary of the Incidence of Neoplasms in
 

Female Rats Fed Chlorothalonil in the Diet 43
 

ix
 
Page 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Fed Chlorothalonil in the Diet 47 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Fed Chlorothalonil in the Diet 49 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Fed Chlorothalonil in the Diet 52 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Fed Chlorothalonil in the Diet 55 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Chlorothalonil 

in the Diet 57 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Chlorothalonil 

in the Diet 61 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Fed Chlorothalonil 

in the Diet 65 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed Chlorothalonil 

in the Diet 67 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed Chlorothalonil 

in the Diet 69 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Fed Chlorothalonil in the Diet 71 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Fed Chlorothalonil in the Diet 73 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Fed Chlorothalonil in the Diet 77 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Fed Chlorothalonil in the Diet 83 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Fed Chlorothalonil in the Diet 85 

x
 
Page 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Fed Chlorothalonil in the Diet 87 

Appendix G Analysis of Formulated Diets for 

Concentrations of Chlorothalonil 89 

TABLES 

Table 1 Chlorothalonil Chronic Feeding 

Studies in Rats 9 

Table 2 Chlorothalonil Chronic Feeding 

Studies in Mice 10 

FIGURES 

Figure 1 Growth Curves for Rats Fed

in the Diet

 Chlorothalonil 

 20 

Figure 2 Survival Curves for Rats Fed Chlorothalonil 

in the Diet 22 

Figure 3 Growth Curves for Mice Fed

in the Diet

 Chlorothalonil 

 28 

Figure 4 Survival Curves for Mice Fed Chlorothalonil 

in the Diet 30 

xi
 

I. INTRODUCTION
 

Chlorothalonil (CAS 1897-45-6;
 

NCI C00102) is a broad-spectrum
 

fungicide which has been in use
 

in the United States since 1963.
 

It is registered for foliar and
 

root applications on vegetables,
 

fruits, greenhouse plants, and
 

turf, and as a seed treatment for cotton (EPA Pesticide Data
 

Base, Compact Label File, 1976). Chlorothalonil is also used in
 

formulating paints and stains for mildew resistance.
 

Chlorothalonil was one of a series of pesticides selected for
 

study in the Carcinogenesis Testing Program because of the
 

potential for long-term human exposure to the chemical during
 

agricultural or industrial application or to residues in food
 

products.
 

1 

II. MATERIALS AND METHODS
 

A. Chemical
 

Chlorothalonil (2,4,5,6-tetrachloro-l,3-benzenedicarbonitrile)
 

was obtained from Diamond Shamrock Chemical Co., Agricultural
 

Chemicals Division, Cleveland, Ohio, in two batches of technical-


grade product for the chronic studies. These batches of chloro­

®
 thalonil (Daconil 2787') were used for all the chronic studies,
 

except for a 2-week period 6 months into the study when rats
 

received analytical-grade chlorothalonil (99.7%) rather than
 

technical grade.
 

Analyses at Gulf South Research Institute confirmed the identity
 

of the technical- and analytical-grade batches (melting range;
 

elemental analysis; and infrared, ultraviolet, and nuclear
 

magnetic resonance spectra). No attempt was made to identify or
 

quantitate impurities.
 

Samples of both batches were returned to Diamond Shamrock for
 

analysis after the bioassay was complete. It was found that
 

these batches were typical of the current product and that all
 

impurities were within Diamond Shamrock's current specifications
 

for technical chlorothalonil. According to the Diamond Shamrock
 

analyses, the first batch consisted of 98.50% chlorothalonil,
 

1.24% pentachlorobenzonitrile (CAS 20925-85-3), 0.06% other
 

3 
tetrachlorodicyanobenzene isomers, and smaller quantities of
 

other partially chlorinated dicyanobenzenes. The second batch
 

consisted of 98.0% chlorothalonil, 0.6% pentachlorobenzonitrile,
 

1.2% other tetrachlorodicyanobenzene isomers, and again smaller
 

quantities of other partially chlorinated dicyanobenzenes
 

(Diamond Shamrock, 1975, 1977).
 

The chemical was stored at 4°C in the original glass container.
 

B. Dietary Preparation
 

All diets were formulated using finely ground Wayne® Lab Blox
 

(Allied Mills, Inc., Chicago, 111.) to which was added the
 

required amount of chlorothalonil for each dietary concentration.
 

A given amount of the test chemical was first hand-mixed with an
 

approximately equal amount of feed. This mixture was then added
 

slowly with mechanical mixing to a larger quantity of feed to
 

give the desired concentration of the material. Acetone
 

(Mallinckrodt Inc., St. Louis, Mo.) and corn oil (Louana ,
 

Opelousas Refinery Co., Opelousas, La.) were then added to the
 

feed, each in an amount corresponding to 2% of the final weight
 

of feed. The diets were mixed mechanically for not less than 25
 

minutes to assure homogeneity of the mixture and evaporation of
 

the acetone. Formulated diets were stored at approximately 17°C
 

until used, but no longer than one week.
 

4 
The stability of chlorothalonil in feed was tested by determining
 

the concentration of the chemical in formulated diets at intervals over a 7-day period. Diets containing 5,000 or 10,000 ppm
 

chlorothalonil showed no change on standing at ambient temperature for this period. Extraction and analytical procedures are
 

outlined in Appendix G.
 

As a quality control test for the accuracy of preparation of the
 

diets, the concentration of chlorothalonil was determined in
 

different batches of formulated diets during the chronic study.
 

The results are summarized in Appendix G. At each dietary
 

concentration, the mean of the analytical concentrations for the
 

checked samples was within 1.5% of the theoretical concentration,
 

and the coefficient of variation was never more than 5.7%. Thus,
 

the evidence indicates that the formulated diets were prepared
 

accurately.
 

C. Animals
 

Rats and mice of each sex, obtained through contracts of the
 

Division of Cancer Treatment, National Cancer Institute, were
 

used in these bioassays. The rats were of the Osborne-Mendel
 

strain obtained from Battelle Memorial Institute, Columbus, Ohio,
 

and the mice were B6C3F1 hybrids obtained from A. R. Schmidt,
 

Madison, Wisconsin. On arrival at the laboratory, all animals
 

5 
were quarantined (rats for 7 days, mice for 15 days) and then
 

assigned to control or dosed groups.
 

D. Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

rooms. The temperature range was 22-24°C, and the relative
 

humidity was maintained at 40-70%. The air entering each room
 

was passed through fiberglass filters and was changed 10-12 times
 

per hour. Fluorescent lighting provided illumination 10 hours
 

per day. Food and water were available ad libitum.
 

The rats were housed individually in hanging galvanized steel
 

mesh cages, and the mice were housed in plastic cages with filter
 

bonnets (Lab Products, Inc., Garfield, N.J.), five per cage for
 

females, and two or three per cage for males. Initially, rats
 

were transferred every week to clean cages; later in the study,
 

cages were changed every 2 weeks. Mice were transferred every
 

week to clean cages with filter bonnets; bedding used for the
 

mice was Absorb-Dri® (Lab Products, Inc., Garfield, N. J.). For
 

rats, absorbent sheets under the cages were changed three times
 

per week. Feeder jars and water bottles were changed and
 

sterilized three times per week.
 

Cages for control and dosed mice were placed on separate racks in
 

the same room. Animal racks for both species were rotated later6

 
ally once per week; at the same time each cage was changed to a
 

different position in the row within the same column. Rats
 

administered chlorothalonil in feed, along with their matched
 

controls, were housed in a room by themselves. Control mice were
 

housed in the same room as the respective dosed mice. Mice
 

administered chlorothalonil were maintained in the same room as
 

mice being administered the following chemicals in the feed:
 

(CAS 133-90-4) chloramben
 

(CAS 1918-02-1) picloram
 

(CAS 72-20-8) endrin
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted with rats and mice to
 

determine the two concentrations (hereinafter referred to as "low
 

doses" and "high doses") to be administered in the chronic
 

studies. In these Subchronic studies chlorothalonil was added to
 

the animal feed in twofold increasing concentrations, ranging
 

from 1,250 to 20,000 ppm for rats and from 1,250 to 40,000 ppm
 

for mice. Dosed and control groups each consisted of five male
 

and five female animals. The chemical was provided in feed to
 

dosed groups for 6 weeks, followed by 2 weeks of observation.
 

During week 1, the mean weight gains in male and female rats
 

receiving 20,000 ppm chlorothalonil were 10 and 40%,
 

respectively, of those of the controls. By weeks 3 and 4, these
 

7 
groups had weight gains generally comparable to those of the
 

controls. Male and female rats receiving 10,000 ppm and male
 

rats receiving 5,000 ppm had lesser initial depressions in mean
 

weight gains than rats receiving 20,000 ppm. The low and high
 

doses for each sex of rats were set at 10,000 and 20,000 ppm for
 

the chronic studies.
 

Male and female mice receiving 40,000 or 20,000 ppm lost 2 to 6 g
 

of mean body weight during the first 2 to 3 weeks of the study;
 

and those animals receiving 10,000 ppm had lesser weight gains
 

than controls during this period. Three males receiving 40,000
 

ppm died during week 6; and one female receiving 40,000 ppm died
 

during week 3. The low and high doses for male and female mice
 

were set at 10,000 and 20,000 ppm for the chronic studies.
 

F. Chronic Studies
 

The test groups, doses administered, and times on study of the
 

chronic feeding studies are shown in tables 1 and 2.
 

Since the numbers of animals in the matched-control groups were
 

small, pooled-control groups also were used for statistical
 

comparisons. Matched controls from the current bioassay of
 

chlorothalonil were combined with matched controls from bioassays
 

of malathion (CAS 121-75-5), tetrachlorvinphos (CAS 961-11-5),
 

toxaphene (CAS 8001-35-2), lindane (CAS 58-89-9), and endrin (CAS
 

8 
Table 1. Chlorothalonil Chronic Feeding Studies in Rats
 

ChloroSex and

Test

Group

 Initial 

 No. of 

Animals3 

thalonil 

in Dietb 

(ppm) 

Time 

Dosedc 

(weeks ) 

on Study 

Observed01 

(weeks ) 

Time-Weighted 

Average Dose6 

(ppm) 

MALE 

Matched-Control 10 0 110 

Low-Dose 50 10,000 1 

5,000 79 5,063 

0 30 

High-Dose 50 20,000 1 

10,000 79 10,126 

0 31 

FEMALE 

Matched-Control 10 0 110 

Low-Dose 50 10,000 1 

5,000 79 5,063 

0 30-31 

High-Dose 50 20,000 1 

10,000 79 10,126 

0 30-31 

a

All animals were 35 days of age when placed on study.
 

^Doses of chlorothalonil were lowered after 1 week on study
 

since, based on the pattern of mortality, changes in body weight,
 

and the general condition of the animals in this and other
 

carcinogen bioassays, it was believed that excessive mortality
 

would occur before termination of the study.
 

C

A11 animals were placed on study on the same day.
 

^When diets containing chlorothalonil were discontinued, all
 

animals and their matched controls were fed control diets (2%
 

corn oil added).
 

e

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

£(no. of weeks receiving each dose)
 

9 
Table 2. Chlorothalonil Chronic Feeding Studies in Mice
 

Sex and

Test

Group

 Initial 

 No. of 

Animals3 

Chlorothalonil 

in Dietb 

(ppm) 

Time 

Dosedc 

(weeks ) 

on Study 

Observed*1 

(weeks ) 

Time-Weighted 

Average Dose6 

(ppm) 

MALE 

Matched-Control 10 0 91 

Low-Dose 50 10,000 

2,500 

0 

2 

78 

11-12 

2,688 

High-Dose 50 20,000 

5,000 

0 

2 

78 

12 

5,375 

FEMALE 

Matched-Control 10 0 91 

Low-Dose 50 10,000 

5,000 

2,500 

0 

2 

10 

68 

11 

3,000 

High-Dose 50 20,000 

10,000 

5,000 

0 

2 

10 

68 

12 

6,000 

a

All animals were 35 days of age when placed on study.
 

"Doses of chlorothalonil were lowered after 2 and 10 weeks on study
 

since, based upon the pattern of mortality, changes in body weight,
 

and the general condition of the animals in this and other
 

carcinogen bioassays, it was believed that excessive mortality
 

would occur before termination of the study.
 

C

A11 animals were placed on study on the same day.
 

10
 
72-20-8). All control animals received 2% corn oil in the feed.
 

The pooled controls for statistical tests using rats consisted of
 

65 males and 65 females; using mice, 60 males and 60 females. The
 

bioassays on chemicals other than chlorothalonil were also
 

conducted at Gulf South Research Institute and overlapped the
 

chlorothalonil study by at least 1 year. The matched-control
 

groups of rats for the different test chemicals which comprised
 

the pooled controls were of the same strain and from the same
 

supplier and were examined by the same pathologists. The other
 

control groups of mice which comprised the pooled controls were
 

of the same strain and were examined by the same pathologists,
 

but were received from a different supplier.
 

G. Clinical and Pathologic Examinations
 

All animals were observed twice daily for signs of toxicity,
 

weighed at regular intervals, and palpated for masses at each
 

weighing. Animals that were moribund at the time of clinical
 

examination were killed and necropsied.
 

The pathologic evaluation consisted of gross and microscopic
 

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

from killed animals and from animals found dead. The following
 

tissues were examined microscopically: skin, lungs and bronchi,
 

trachea, bone and bone marrow, spleen, lymph nodes, heart,
 

11
 
salivary gland, liver, gallbladder (mice), pancreas, stomach,
 

small intestine, large intestine, kidney, urinary bladder,
 

pituitary, adrenal, thyroid, parathyroid, mammary gland, prostate
 

or uterus, testis or ovary, and brain. Occasionally, additional
 

tissues were also examined microscopically. The different
 

tissues were preserved in 10% buffered formalin, embedded in
 

paraffin, sectioned, and stained with hematoxylin and eosin.
 

Special staining techniques were used when indicated for more
 

definitive diagnosis.
 

A few tissues from some animals were not examined, particularly
 

from those animals that died early. Also, some animals were
 

missing, cannibalized, or judged to be in such an advanced state
 

of autolysis as to preclude histopathologic evaluation. Thus,
 

the number of animals from which particular organs or tissues
 

were examined microscopically varies, and does not necessarily
 

represent the number of animals that were placed on study in each
 

group.
 

H. Data Recording and Statistical Analyses
 

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

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

descriptive information on the chemicals, animals, experimental
 

12
 
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) for testing two groups for equality and Tarone's (1975)
 

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

One-tailed P values have been reported for all tests except the
 

departure from linearity test, which is only reported when its
 

two-tailed P value is less than 0.05.
 

The incidence of neoplastic or nonneoplastic lesions has been
 

13
 
given as the ratio of the number of animals bearing such lesions
 

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

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

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic examination was required to detect lesions prior to histologic sampling
 

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

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

consist of the 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) was used to compare the tumor incidence of
 

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

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

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

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

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

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

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

where the Fisher exact P values are shown.
 

14
 
The Cochran-Armitage test for linear trend in proportions, with
 

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

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

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


tailed 0.05 level of significance. Unless otherwise noted, the
 

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

first tumor was observed were excluded by basing the statistical
 

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

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

such an early tumor was found, comparisons were based exclusively
 

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

the first tumor was found. Once this reduced set of data was
 

obtained, the standard procedures for analyses of the incidence
 

of tumors (Fisher exact tests, Cochran-Armitage tests, etc.) were
 

followed.
 

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).
 

15
 
The week during which an animal died naturally or was sacrificed
 

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

methods of comparing these curves were used for two groups;
 

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

groups. The statistical tests for the incidence of tumors which
 

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

noted, in the direction of a positive dose relationship-


Significant departures from linearity (P < 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 Pt/pc where pt is the true binomial
 

probability of the incidence of a specific type of tumor in a
 

dosed group of animals and pc is the true probability of the
 

spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

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

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

of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

16
 
analyses. The interpretation of the limits is that in
 

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

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

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

significant result (P < 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. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

The mean body weights of the rats fed chlorothalonil were consistently lower than those of the matched controls, and the data
 

indicate that the effects were dose related for both sexes
 

(figure 1). Fluctuation in the growth curve may be due to
 

mortality; as the size of a group diminishes, the mean body
 

weight may be subject to variation.
 

During the first year of the study, some animals had rough hair
 

coats; however, the dosed animals were generally comparable to
 

the controls in appearance and behavior. A few animals showed
 

evidence of an eye infection which was diagnosed as viral
 

conjunctivitis.
 

Clinical signs in all dosed groups were noted at a low or
 

moderate incidence during the first half of the second year, and
 

with gradually increasing frequency during the second half of
 

that year. These signs included loss of weight (figure 1),
 

decreased food consumption, rough hair coats, pale mucous
 

membranes, ataxia, tachypnea, epistaxis, dermatitis, hematuria,
 

hyperactivity, and vaginal bleeding. Several nodular masses
 

developed as abscesses that, in some cases, ruptured and exuded a
 

19
 
Figure 1. Growth Curves For Rats Fed Chlorothalonil In The Diet 

20 
purulent discharge. Beginning at week 72, all dosed animals had
 

discolored hair coats and bright-yellow urine.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats fed chlorothalonil in the diet
 

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

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

results of the Tarone test for positive dose-related trend in
 

mortality are not statistically significant (P > 0.05), but the
 

rates of survival of the dosed females decreased after week 60.
 

There is a significant (P = 0.002) decrease in survival of the
 

dosed males when compared with those in the pooled controls used
 

in this study. In female rats, the survival in the dosed groups
 

are comparable with that in the pooled controls (P = 0.061).
 

In male rats, all 49 of the high-dose animals, all 50 of the
 

low-dose animals, and all 10 of the matched controls survived
 

beyond week 52 on study. Neither the high-dose animals that died
 

before week 68 nor the low-dose animals that died before week 86
 

had tumors. While the rates of survival of the low- and
 

high-dose males showed little difference from one another, there
 

is a steep decline in survival in either dosed group after week
 

75 compared with the control group.
 

21
 
Figure 2. Survival Curves For Rats Fed Chlorothalonil In The Diet 

22 
Forty-nine out of fifty (98%) of the high-dose females, 48/49
 

(98%) of the low-dose females, and all 10 of the female matched
 

controls survived beyond week 52 on study.
 

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

development of late-appearing tumors.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

are summarized in Appendix C, tables Cl and C2.
 

The majority of neoplastic and nonneoplastic lesions were not
 

unusual findings, and their incidences in animals of the dosed
 

groups were comparable to those in the controls. Therefore,
 

these lesions are considered to have occurred spontaneously. In
 

some instances, the pathologic changes were found only in either
 

dosed or control rats; except for renal tumors the frequency of
 

these findings was, however, within normal range for the
 

Osborne-Mendel rats. Medial calcification of arteries and
 

parathyroid hyperplasia occurred with a relatively high incidence
 

in dosed rats. These lesions are often associated with chronic
 

inflammatory renal disease, however, and since they are commonly
 

observed in aging rats regardless of any treatment, it is
 

unlikely that chlorothalonil was the cause of induction.
 

23
 
Neoplasms of the tubular epithelium of the kidney were observed
 

in dosed but not in control rats; these tumors occurred as
 

follows:
 

Males Females
 

Matched Low High Matched Low High
 

Control Dose Dose Control Dose Dose
 

Carcinoma U/10 1/46 3/49 0/10 l/4b 2/50
 

Adenoma 0/1U 2/46 1/49 0/10 0/48 3/50
 

Total 0/10 3/46 4/49 0/10 l/4b 5/50
 

Renal tumors are rarely observed in other control Osborne-Mendel
 

rats at this laboratory. The relatively high incidence in this
 

bioassay of tubular-cell renal tumors in dosed rats indicates a
 

compound-related effect. In addition, a transitional-cell
 

carcinoma, a carcinosarcoma, a liposarcoma, and a hamartoma were
 

recorded in low-dose male rats.
 

Based on the histopathologic evaluation, the results indicate
 

that chlorothalonil induced renal neoplasms in the rats under the
 

conditions of this bioassay.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

24
 
least two animals of one group and at an incidence of at least 5%
 

in one or more than one group.
 

The results of the Cochran-Armitage test on the incidence of male
 

rats with carcinoma, tubular-cell adenocarcinoma, adenoma,
 

adenocarcinoma, or papillary adenoma of the kidney are
 

significant (P = 0.030) using the pooled controls. The Fisher
 

exact comparison of the incidence in the high-dose group with
 

that in the pooled-control group shows a P value of 0.035, which
 

is above the 0.025 level for significance when the Bonferroni
 

inequality criterion is used for multiple comparison. Historical
 

records of this bioassay program at this laboratory indicate an
 

incidence of tubular-cell adenomas in male rats of 3/240 (1.25%)
 

with no other renal tumors occurring.
 

In females, the results of the Cochran-Armitage test on the
 

incidence of animals with either adenomas or tubular-cell
 

adenomas of the kidney are significant (P = 0.028) using the
 

pooled controls, but the results of the Fisher exact test are not
 

significant. The results of the Cochran-Armitage test on the
 

incidence of female rats with adenomas, carcinomas, tubular-cell
 

adenomas, or tubular-cell adenocarcinomas are significant (P =
 

0.007), and the results of the Fisher exact test show that the
 

incidence in the high-dose group is significantly higher than
 

that in the pooled controls (P = 0.016). Historical records
 

25
 
indicate none of the above tumors were seen in 235 female control
 

rats at this laboratory. Overall, the statistical tests indicate
 

a dose association of this chemical with the kidney tumors in
 

both sexes of rats at the doses administered in this bioassay.
 

In male rats, the results of the Cochran-Armitage test for positive dose-related trend in the incidence of malignant fibrous
 

histiocytomas of the subcutaneous tissue in the integumentary
 

system are significant (P = 0.030) using the pooled controls;
 

however, the results of the Fisher exact test for the comparisons
 

of the incidences in the dosed groups with those in the control
 

groups are not significant (P > 0.05). Statistical tests on the
 

incidence of these tumors in female rats are not significant.
 

In female rats, a negative result is observed in the incidence of
 

chromophobe adenoma of the pituitary.
 

26
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of dosed male mice were lower than those of the
 

controls and were dose related throughout most of the bioassay
 

(figure 3). There was little difference among control and dosed
 

groups of female mice. Fluctuation in the growth curve may be
 

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

body weight may be subject to variation.
 

During the first year of the study, the dosed animals were
 

generally comparable to the controls in appearance and behavior.
 

A few animals had alopecia and were hyperexcitable.
 

Clinical signs were noted at a low incidence in all dosed groups
 

of both males and females during the second year of the study and
 

included alopecia, loss of weight, rough hair coats, abdominal
 

distention, and nodular masses, many of which were located in the
 

lower abdomen. On many of the low- and high-dose males, nodules
 

located in the inguinal area appeared intermittently during the
 

last year of the study. Several males also intermittently had
 

swollen testes. Beginning at week 62 and persisting until
 

termination of the study, a majority of the low- and high-dose
 

males were hyperexcitable.
 

27
 
a 

o

m 

111 

5 

Figure 3. Growth Curves For Mice Fed Chlorothalonil In The Diet 

28 
B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice fed chlorothalonil in the diet
 

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

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

for positive dose-related trend in mortality is not statistically
 

significant in either sex. In each sex the rates of survival of
 

the dosed groups are comparable to those of the respective pooled
 

controls.
 

In male mice, all 50 of the high-dose group, 42/50 (84%) of the
 

low-dose group, and 9/10 (90%) of the controls were still alive
 

at week 91. In female mice, 41/50 (82%) of the high-dose group,
 

47/50 (94%) of the low-dose group, and 7/10 (70%) of the controls
 

were still alive at week 91. Sufficient numbers of mice of each
 

sex were at risk for the development of tumors.
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

are summarized in Appendix D, tables Dl and D2.
 

The lesions observed in mice were not as numerous or of various
 

types as were those in rats. The distribution and frequency of
 

29
 
Figure 4. Survival Curves For Mice Fed Chlorothalonil In The Diet 

30 
these pathologic changes among the dosed and control mice
 

indicate no relationship with the exposure to chlorothalonil.
 

Based on the histopathologic examination, tumors were not related
 

to administration of chlorothalonil in the B6C3F1 hybrid mouse
 

under the conditions of this bioassay.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

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

in one or more than one group.
 

The results of the Cochran-Armitage test for positive dose-


related trend and of the Fisher exact test for direct comparisons
 

of the incidences in the control and dosed groups are not significant in either sex.
 

In male mice, a significant trend in the negative direction is
 

observed in the incidence of alveolar/bronchiolar adenomas of the
 

lung when the matched-control, low-dose, and high-dose groups are
 

used, because the incidence in the matched-control group exceeds
 

the incidences in the dosed groups. There is also a negative
 

association indicated by the incidences of hepatocellular
 

carcinoma when the incidence in pooled controls is used. These
 

31
 
negative associations cannot be explained by differential
 

survival.
 

In each of the 95% confidence intervals shown in the tables, the
 

value of one is included; this indicates the absence of significant positive results. It should also be noted that each of the
 

intervals has an upper limit greater than one, indicating the
 

theoretical possibility of the induction of tumors by
 

chlorothalonil which could not be detected under the conditions
 

of this bioassay.
 

32
 
V. DISCUSSION
 

Chlorothalonil is a chlorinated phthalonitrile of low acute oral
 

toxicity (oral LD5Q in rats > 10,000 rag/kg, Spencer, 1973; oral
 

LD5Q in female ICR mice 6,000 mg/kg, Yoshikawa and Kawai, 1966).
 

In this bioassay, Osborne-Mendel rats fed Chlorothalonil were
 

comparable to the controls in appearance and behavior during the
 

first year of the study. In the second year of the study,
 

hematuria, vaginal bleeding, abscesses, and rough hair coats were
 

observed with increasing frequency, particularly among the dosed
 

rats. From week 72 until termination of the study, discolored
 

hair and bright-yellow urine were noted only among dosed rats. A
 

majority of dosed male mice were hyperexcitable from week 62
 

until termination of the study. Mean body weights of rats of
 

each sex and of male mice were consistently lower than those of
 

the controls throughout the study. Since the dosed female mice
 

did not have a depression in mean weight or decreased survival
 

compared with controls, they may have been able to tolerate a
 

higher dose.
 

Survival rates of the dosed rats decreased sharply after week 60,
 

but sufficient numbers of rats were at risk for late-developing
 

tumors. Survival of the mice was higher than that of the rats.
 

33
 
In rats, neoplasms of the renal tubular epithelium occurred with
 

a significant dose-related trend in both the males (P = 0.030)
 

and the females (P = 0.007) and also occurred at a higher
 

incidence in the high-dose males (P = 0.035) and the high-dose
 

females (P = 0.016) than in the corresponding controls (males:
 

pooled controls 0/62, low-dose 3/46 [7%], high- dose 4/49 [8%];
 

females: pooled controls 0/62, low-dose 1/48 [2%], high-dose
 

5/50 [10%]). These tumors included both adenomas and carcinomas
 

which are considered to be histogenically related. Only adenomas
 

have been observed among historical-control Osborne-Mendel rats
 

at this laboratory (males 3/240 [1.3%], females 0/235). Thus the
 

tumors are rare and their incidence in the present study is
 

interpreted as sufficient evidence for the carcinogenicity of
 

chlorothalonil.
 

*
 

In mice, no tumors were found to occur at a significantly greater
 

incidence among dosed animals than among controls.
 

It is concluded that under the conditions of this bioassay,
 

technical-grade chlorothalonil was carcinogenic to Osborne-Mendel
 

rats, producing tumors of the kidney. Chlorothalonil was not
 

carcinogenic for B6C3F1 mice.
 

34
 
VI. BIBLIOGRAPHY
 

Armitage, P. , Statistical Methods in Medical Research, J. Wiley &
 

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

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

Panel of Carcinogenicity of the Cancer Research Commission
 

of the UICC, Vol. 2. International Union Against Cancer,
 

Geneva, 1969.
 

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

Soc. B_:187-220, 1972.
 

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

London, 1970, pp. 48-52.
 

Diamond Shamrock Chemical Co. , Product Label, Agricultural
 

Chemicals Division, Cleveland, Ohio, 1975 and 1977.
 

Environmental Protection Agency, EPA Pesticide Data Base, Compact
 

Label File, 1976.
 

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

significance tests, confidence limits and adjustments for
 

stratification. Rev. Int. Stat. Inst. 39;148-169. 1971.
 

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

incomplete observations. J. Amer. Statist. Assoc.
 

_53:457-481, 1958.
 

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

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

Biomed. Res. 7;230-248, 1974.
 

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


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

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

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

induced by different numbers of administrations of benzo(a)
 

pyrene and ferric oxide. Cancer Res J32:1073-1081, 1972.
 

35
 
Spencer, E. Y. , Guide to Chemicals Used in Crop Protection, 

Research Institute, University of Western Ontario, London, 

Ontario, 1973. 

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

62_ (3):679-682, 1975. 

Yoshikawa, H. and Kawai, K. , Toxicity of phthalodinitrile and 

tetrachlorophthalodinitrile. Ind. Health 4 (11): 11-15, 

1966. 

36 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS FED CHLOROTHALONIL IN THE DIET
 

37
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

FED CHLOROTHALONIL IN THE DIET 

CONTROL LOW DOSE
 

ANIMAL S INITIAIIY IK STUDY 10 50
 

ANIMALS NECBCPSIED 10 <i7
 

ANIMALS EXAMINED HISTOFATHCLOGIC ALLY 10 a?
 

INTEGUMENTAEY SYSIEM
 

*SUBCUT TISSUE (10) (47)
 

FIBRCMA 1 (2%)
 

FIBROUS HISTIOCYTOMA, MALIGNANT
 

RESEIEATGRY SYSTEH
 

#LUNG (10) (44)
 

FIBROUS HISTIOCYTOMA, METASTATIC
 

HEMATCP01ETIC SYSTEM
 

#SPLEEN (10) (46)
 

HEHANGIOSARCOMA
 

ANGIOMA 1 (10%)
 

HAMARTOM A
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE. SYSTEM
 

*CRAL MUCOUS MEMBRAN E (10) (47)
 

FIBROMA 1 (10%)
 

URINAR Y SYSTEH
 

#KIDNEY (10) (46)
 

CAECINOKA^NOS
 

# NUKEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

50
 

49
 

49
 

(49)
 

3 (6X)
 

(47)
 

1 (2%)
 

(49)
 

1 (2%)
 

1 (2%)
 

(49)
 

(49)
 

2 iitSl39
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE
 

TEANSITICNAL-CELL CARCINOHA 1 (2%)
 

ADENCCARCINOMA, NOS
 

PAPILLARY ADENOBA 1 (2%)
 

TUEULAB-CELL ADENOflA 1 (2%)
 

TUBULAR-CELL ADENOCARCINOH A 1 (2%)
 

LIPOSAfiCOMA 1 (2%)
 

CABCINOSAECOHA 1 (2%)
 

t HAHARTOMA 1 (2X)
 

ENDOCBINE SYSTEM
 

#FITUITARY (9) (46)
 

CARCINOMA, NOS 1 (11%)
 

ADENOCARCINOMA, NOS 1 12%)
 

CHBOMOPHOBE ADENOMA 3 (7%)
 

CHROHOFHOBE CARCINOMA 2 (4%)
 

tADRENAL (10) (45)
 

GANGLICNEOROBLASTCKA
 

tTHYfiOID (9) (1»U)
 

PAPILLARY ADENOflA 1 (11%)
 

FOLLICULAR-CELL ADENOMA 2 (5%)
 

C-CELL ADENOMA
 

tEANCEEATIC ISLETS (9) (45)
 

ISLET-CELL ADENOMA 2 (UX)
 

REPRODUCTIVE SYSTEM
 

*HA«MARY GLAND (10) (47)
 

AEENOHA, NOS 1 (2%)
 

tlESIIS (9) (<*5)
 

INTERSTITIAL-CELL TUMOR
 

NEEVCUS SYSTEM
 

#ERAI N/HENINGES (10) (45)
 

MENINGIGMA 1 (2%)
 

*ERAIN (10) (45)
 

GRANULAR-CELL TUMOR. MALIGNANT
 

SPECIAL SENSE ORGANS
 

NCNE _
 

# NUHEEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * KUCBER OF ANIMALS NICROPSIED
 

HIGH
 

1
 

1
 

(46)
 

1
 

5
 

(49)
 

1
 

(45)
 

3
 

1
 

(49)
 

(49)
 

(48)
 

1
 

(49)
 

(49)
 

1
 

DOSE
 

(2X)
 

<2X)
 

(2%)
 

(11«)
 

(2%)
 

(7%)
 

(2X)
 

(2%)
 

(2%)
 

t This is considered to be a benign form of the malignant mixed tumor
 

of the kidney and consists of lipocytes, tubular structures, and
 

fibroblasts in Vv.rying proportions.
 

40
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MUSCUIOSKELETAL SYSTEM
 

NONE
 

BOEY CAVITIES
 

*TUNICA VAGINALIS
 

MESOTHELIOMA. NOS
 

ALL C1HEB SYSTEBS
 

NCNE
 

ANIMAL E1SEOSITICN SUBMABY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

$ INCLUDES AUTCLY2EP ANigALS
 

* NUMBE R OF ANIMAL S WITH TISSUE

* NUMBE B OF ANIMALS NECROPSIED 

CONTROL LOW DOSE HIGH DOSE
 

(10)	 (47) (l»9)
 

1 (2X)
 

10 50 50
 

7 7
 

2 23 22
 

8 20	 20
 

 EXAMINED MICROSCOPICALLY 

41 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

(:ONTROL LOH DOSE HIGH DOSE
 

TUKOfi SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 4 19 16
 

TOTAL PRIMAR Y TUBOES 14 21 22
 

TOTAL ANIMALS BITH BENIGN TUHORS 3 10 9
 

TCTAL BENIGN TDMORS 3 12 12
 

TOTAL ANIMALS WITH MALIGNANT TUMOBS 1 8 10
 

TCTAL MALIGNANT TUHORS 1 8 10
 

TOTAL ANIMALS WITH SECONDARY TUMORSt 1
 

TOTAL SECONDARY TUHORS 1
 

TOTAL ANIHALS tllTH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT 1
 

TCTAL UNCERTAIN TUHORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAINEEIMARY OR METASTATIC
 

TCTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TOHORS
 

# SECONDARY TOHORS: HETASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACEHT ORGAS
 

42 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

FED CHLOROTHALONIL IN THE DIET 

CONTROL LOB DOSE
 

ANIMALS INITIALLY IN STUDY 10 50
 

ANIMALS NECSOPSIED 10 18
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 10 48
 

INTEGUMENTARY SYSTEM
 

*SKIN (10) (48)
 

FIBROUS HISTIOCYTOMA, MALIGNANT 1 (2%)
 

*SUBCUT TISSUE (10) (U8)
 

FIBROMA
 

RESFIEATORY SYSTEM
 

tLUNG (9) (47)
 

ALVEOLAR/BEONCHIOLAR ADENOMA
 

CORTICAL CARCINOMA, HETASTATIC 1 (2%}
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE CRGANS (10) ((48)
 

LYMPHOCYTIC LEUKEMIA
 

fTHYMUS d)


FIBROUS HISTIOCYTOMA, MALIGNANT 1 (100%)
 

CIRCULATORY SYSTEM
 

NCNE
 

DIGESTIVE SYSTEM
 

•SALIVARY GLAND (10) (48)
 

ADENOMA, NOS 1 (2%)
 

#PARGTIC GLAND __ (10) (48)
 

SfiJiAMQUS C£Lj. CA££IJJfiflA_ H2&1
 

# KUMEEP CF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * MJMBER CE ANIMALS NECROPSIED
 

HIGH DOSE
 

50
 

50
 

50
 

(50)
 

(50)
 

1 (2*)
 

(50)
 

1 (2%)
 

(50)
 

1 (2%)
 

(50)
 

(50)
 — _ ______
 

43
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

HIGH DOSE
 

(50)
 

2 (4X)
 

(50)
 

1 (2JS)
 

1 (2%)
 

2 (4%)
 

1 (2X)
 

1 (2%)
 

(47)
 

1 (2%)
 

(49)
 

6 (12X)
 

(50)
 

(41)
 

1 (2%)
 

1 (2%)
 

(50)
 

1 (2%)
 

(50)
 

1 (2%)
 

1 (2X)
 

1 (2%)
 

4 (8X)
 

(49)
 

i_I211_
 

#LIVER
 

NEOPLASTIC NODULE
 

UEINAfiY SYSTEM
 

tKIDNEY
 

CARCINOMA, NOS
 

ADENOMA, NOS
 

1UEU1AB-CELL ADENOMA
 

TUBULAB-CELL ADENOCARCINOH A
 

IIPOKA
 

tURINAR Y BLADDER
 

CARCINOMA, NOS, METASTATIC
 

ENDOCRINE SYSTEM
 

tPITUITARY
 

CHROMOFHCBE ADENCKA
 

#ADRENA L
 

CORTICAL ADENOMA
 

CORTICAL CARCINOMA
 

#IHYROID
 

FCLLICULAR-CELL ADENOMA
 

FOLLICULAR-CELL CAECINOMA
 

C-CELL ADENOMA
 

#PANCREATIC ISLETS
 

ISLET-CELL ADENOMA
 

REPRODUCTIVE SYSTEM
 

*MAMMAR Y GLAND
 

ADENOMA, NOS
 

PAPILLARY ADENOCABCINOMA
 

FIBROMA
 

FIEROADENOHA
 

#IJTERUS
 

CASCINCHA.tNOS
 

CONTROL
 

(10)
 

(10)
 

(9)
 

(10)
 

2 (20%)
 

(10)
 

1 (1C%)
 

(7)
 

(9)
 

(10)
 

1 (10X)
 

(9)
 

LOU DOSE
 

(1*8)
 

3 (6*)
 

(1*8)
 

1 (2%)
 

1 (2%)
 

(47)
 

(48)
 

1 (2%)
 

(48)
 

1 (2%)
 

(45)
 

2 (4«)
 

1 (2X)
 

(48)
 

(48)
 

2 (4S)
 

3 ((>%)
 

8 (17S)
 

(47)
 

# NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBE R OF ANIMALS NECROPSIED
 

44
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

HIGH DOSE
 

1 (2%)
 

1 (2%)
 

1 (2X)
 

2 (4*)
 

(50)
 

2 (4%)
 

50
 

2
 

12
 

36
 

AEENCCARCINOMA, NOS
 

SABCCMA, NOS
 

IEICMYCMA
 

INDCMEIEIAL STEOMAL POLYP
 

NEEVCUS SYSTEM
 

NCNE
 

SPECIAL SENSE ORGANS
 

NCNE
 

MUSCULOSKEIETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

KCNE
 

AIL OTHER SYSTEMS
 

"•MULTIPLE OBGANS
 

FIBROUS HISTIOCYTOBA, MALIGNANT
 

ANIHA L DISPOSITION SUMMARY
 

ANIMALS INITIA.LZY IN STUDY
 

NATUBAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULEC SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

? JHCWM§..AyTPIrIZSP ANiBAiS
 

CONTROL LOW DOSE
 

1 (11%) 2 (4X)
 

(10) (48)
 

10 50
 

7
 

5 11
 

5 31
 

* SDMEER OF ANIMALS WITH TISSUE EXAHIHED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

45
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

* PRIMARY TUMORS: ALL TUHORS EXCEPT SECONDARY TOHORS
 

# SECONDARY TUMORS: BETASTATIC TOHORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

46
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE FED CHLOROTHALONIL IN THE DIET
 

47
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

FED CHLOROTHALONIL IN THE DIET 

CONTROL LOW DOSE HIGH DOSE
 

ANIMAL S INITIALLY IN STUDY 10 50 

ANIKALS NECBOESIED 10 U9 

ANIMAL S EXAMINE D HI STCFATHOiOGICALLY 10 48 

INTEGUMENTARY SYSTEM
 

NCNE
 

RESPIEATORY SYSTEM
 

#LUNG (9) (46)
 

ALVEOLAR/360NCHIGLAR AEENOHA 1 (11*) a (9«)
 

HEKATOPOIETIC SYSTEM
 

#SPLEEN (9) <U4)
 

HEKANGIOM A 1 (2%)
 

*KICNEY (9) (45)
 

BALIG. LYKPHOMA, LYHPHOCYTIC TYPE 1 (2X)
 

CIBCULATCRY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVER (10) (46)
 

HEPATOCELLULAR CARCINOMA 2 (2055) 1 (2X)
 

•STOMACH	 (8) (46)
 

SQUAKOUS CELL PAPILLOMA 1 (2%)
 

SQUAKOUS CELL CARCINOMA 1 (2X)
 

URINAR Y SYSTEM
 

NCNS _ _
 

# NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R CF ANIMAL S NECROPSIED
 

50
 

50
 

50
 

(50)
 

(50)
 

(50)
 

(50)
 

1 (2%)
 

(50)
 

49
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

HIGH DOSE
 

(50)
 

50
 

50
 

SNDCCBINE SYSTEM
 

#AEEENAL
 

CCETICAL ADENOMA
 

REPRODUCTIVE SYSTEM
 

NONE
 

NERVOU S SYSTEM
 

NON E
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULCSKELETAL SYSTEM
 

NONE
 

BCDY CAVITIES
 

NCNE
 

A LL CTHEF SYSTEMS
 

NONE
 

ANIKA I CISECSITICN SUMMAR Y
 

ANIKALS INITIALLY IN STUDY
 

NATUBAL DEATHS
 

MCEIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINA L SACRIFICE
 

ANIMAL KISSING
 

a INCLUDES AUTCLYZED ANIMALS
 

CONTROL LOW DOSE
 »
 

(9)	 C*5)
 

1 (2X)
 

10 50
 

1 5
 

1
 

2
 

9 42
 

* NUMEER OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

50
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

1
CONTROL
 

TUMCE SUMMAR Y 

TOTAL ANIMALS WITH PEIMARY TUMORS* 

TOTAL ERIMARY TUMCKS 

3 

3 

TOTAL ANIMALS WITH BFNIGN TDHOBS 

TOTAL BENIGN TUMORS 

1 

1 

TOTAL ANIKALS WITH MALIGNANT TOMORS 

TOTAL MALIGNANT TUMORS 

2 

2 

TOTAL ANIMALS HITH SECONDARY TOHORSf 

TOTAL SECONDARY TUMORS 

TOTAL ANIMALS HITH TUMORS UNCERTAINBENIGN OE MALIGNANT 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIKALS WITH TUMOfiS UNCERTAIN­ EFIMARY OR METASTATIC 

TOTAL UNCERTAIN TUHOfiS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

LOH DOSE HIGH DOSE 

9 

10 

6 

7 

3 

3 

1 

1 

1 

1 

# SECONDAEY TUMOBS: METASTATIC TUHORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

51
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

FED CHLOROTHALONIL IN THE DIET 

CONTROL LOW DOSE HIGH DOSE
 

ANIKAL S INITIAILY IN STUDY 10 50 50
 

ANIHALS NECECPSIED 10 49 47
 

ANIMALS EXAMINED HISTOFATHOLOGICALLY 10 49 47
 

INTEGUMENTARY SYSTEM
 

*SUBCDT TISSUE (10) (49) (47)
 

FIBROMA 1(2%)
 

RESPIEATORY SYSTEM
 

tLUNG (10) (49) (46)
 

ALVEOLAfi/BBONCHIOLAR ADENOMA 1 (10X) 1 (2X) 1 (2X)
 

AIVEOLAR/BEONCHIOLAR CA8CINOHA 1 (2X) 2 (IX)
 

HEMATOPOIETIC SYSTEM
 

*KUITIPLE ORGANS (10) (49) (47)
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (2%)
 

1YMPHOCYTIC IEOKEMIA 1 (2*)
 

#SPIEEN (9) (48) (45)
 

HEMANGIOMA 1 (11X) 1 (2X)
 

MALIG.LYMPHOMA, LYHPHOCYTIC TYPE 1 (2X)
 

CIRCULATORY SYSTEM
 

SCNE
 

DIGESTIVE SYSTEH
 

*STCSACH (10) (49) (46)
 

FAPILLOMA, NOS 1 (2X)
 

SQUAMOUS CELL CARCINOMA 1 (2X)
 

URINABY SYSTEM
 

* KIDNEY (10) ___________ (49) _
 (46)
 

TUBULAR-CELL ADENOMA 1 <2K
 

* NUMBER OF ANIHALS WITH TISSUE EXAHINED HICROSCOPICALLY
 

* SOHBER OF ANIHALS MECROPSIED
 

52
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

HIGH DOSE
 

(44)
 

1 (2X)
 

(44)
 

(47)
 

(47)
 

(47)
 

1 (2%)
 

ENDOCRINE SYSTEM
 

•ADRENAL
 

SQUABOUS CELL CARCINOMA, METASTA
 

#PANCBEATIC ISLETS
 

ISLET-CELL ADENOMA
 

REPRODUCTIVE SYSTEM
 

*MAMHARY GLAND
 

ADENCMA. NOS
 

NERVCUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EYE/LACBIHAL GLAND
 

PAPILLARY ADENOMA
 

MUSCULOSKEIETAL SYSTEM
 

NONE
 

BOEY CAVITIES
 

*ABCCMINAL CAVITY
 

SQOAKCUS CELL CARCINOMA, METASTA
 

ALI CTHEE SYSTEMS
 

1ICN£
 

CONTROL LOW DOSE
 

(10) (49)
 

(9)	 (49)
 

1 (2X)
 

(10)	 (49)
 

1 (1035)
 

(10)	 (49)
 

1 (10X)
 

(10) (49)
 

#	 NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

53
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

ANIMAL EISECSITICN SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

KORIBUND SACRIFICE
 

SCHEDULES SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL HISSING
 

3 INCLUDES AUTOLYZED ANIHALS
 

TORCH SUMMAR Y
 

CONTROL
 

10
 

3
 

7
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 

TOTAL PRIMARY TUMORS 

3 

4 

TOTAL ANIMALS HITH BENIGN TUHOBS 

TOTAL BENIGN TUMORS 

3 

4 

TOTAL ANIMALS 8ITH MALIGNANT TOBOBS 

TOTAL MALIGNANT TUHORS 

TOTAL ANIMALS WITH SECONDARY TUHORSt 

TOTAL SECONDARY TUMORS 

TOTAL ANIMALS BITH TDBORS UNCEBTAIN­ EENIGN OB MALIGNANT 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS HITH TUHORS UNCEHTAIN­ EFIHARY OR METASTATIC 

TOTAL UNCERTAIN TUHORS
 

* PRIMARY IDHORS: ALL TUMORS EXCEPT SECONDARY TOHORS
 

LOW DOSE
 

50
 

1
 

2
 

47
 

6
 

6
 

3
 

3
 

3
 

3
 

HIGH DOSE
 

50
 

5
 

u :
 

41
 

8
 

a
 

4
 

4
 

4
 

4
 

1
 

2
 

t SECONDARY TUHORS: HETASTATIC TOHORS OR TOHORS INVASIVE INTO AN ADJACENT ORGAN
 

54
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED CHLOROTHALONIL IN THE DIET
 

55
 

TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

FED CHLOROTHALONIL IN THE DIET 

ANIMAL S IJHTIAUY IN S1UBY
 

ANIMAL S NECBCPSIED
 

ANIMALS EXAMINED HISIOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

*SKIN
 

ULCER, NOS
 

EESPIEATOBY SYSTEM
 

tLUNG
 

EMPHYSEMA, NOS
 

IDEMA, NOS
 

HEBOBRHAGE
 

INFLAMMATION, CHRONIC
 

HEMATCEOIETIC SYSTEM
 

tEONE MARROW
 

BYELOFIBBOSIS
 

*SPLEEN
 

CONGESTION, NOS
 

HEMORRHAGE
 

INFLAMMATION, CHRONIC FOCAL
 

ANGIEC1ASIS
 

HYPOPLASIA, LYMPHOID
 

•CERVICAL LYMPH NODE
 

INFLAMMATION, SOS
 

CIRCULATORY SYSTEM
 

#HEART
 

CALCIFICATION, FOCAL
 

*ARTERY
 

Ei£IA£l£RlTls
 

CONTROL LOU DOSE HIGH DOSE
 

10 50 50 

10 47 49 

10 1*7 49 

(10) (<*7) (49) 

1 (2%) 

(10) (14) (47) 

1 (2*) 

1 (10X) 

1 (10%) 

1 (2X) 

(10) (45) (49) 

1 (2X) 

(10) (46) (49) 

2 (M) 

1 (2X) 

1

1

 (2X) 

 (2X) 

1 <2X) 

(9)

1 (11X) 

(*2) (46) 

(8) (44) 

1 (2X) 

(45) 

(10) (47) (49) 

1.J2I1 2 (4X1 

t NUMBER OF ANIMALS KITH TISSUE EIABINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

57
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

HIGH DOSE
 

5 (10X)
 

(49)
 

2 (4X)
 

(49)
 

(49)
 

(48)
 

6 (13«)
 

(49)
 

(49)
 

4 <8X)
 

(49)
 

(47)
 

2 (4%)
 

(2)
 

1 (SOX)
 

(2)
 

1 (SOX)
 

(2)
 

(49)
 

1_J2S1_.
 

MEDIAL CALCIFICATION
 

*AOETA
 

MEDIAL CALCIFICATION
 

*KESENTERIC ARTERY
 

THROMBOSIS, NOS
 

*TESTICULAR ARTERY
 

INFLAMMATION, NOS
 

DIGESTIVE SYSTEM
 

tllVER
 

DEGENERATION, BALLOONING
 

DEGENERATION PARENCHYMATOUS
 

METAMORPHOSIS FATTY
 

ANGIECTASIS
 

*EILE DUCT
 

HYPERPLASIA, NOS
 

*EANCREAS
 

FERIARTEHITIS
 

tfANCREATIC ACINUS
 

ATROPHY, NOS
 

KSTCMACH
 

MINERALIZATION
 

CALCIFICATION, FOCAL
 

*LARGE INTESTINE
 

INFLAMMATION, HEMORRHAGIC
 

tCOLCN
 

INFLAMMATION, NOS
 

#CECUM
 

INFLAMMATION, CHRONIC
 

URINARY SYSTEM
 

#KIDNEY
 

!NZL£MMA.TICN,i_.N.OS_


CONTROL
 

(10)
 

(10)
 

(10)
 

(10)
 

6 (60%)
 

1 (10%)
 

(10)
 

(9)
 

(9)
 

(9)
 

(10)
 

.
 

LOW DOSE
 

6 (13X)
 

(47)
 

3 <6X)
 

(47)
 

1 (2%)
 

(47)
 

3 (6%)
 

(46)
 

1 (2%)
 

7 (15%)
 

2 (4X)
 

(47)
 

1 (2*)
 

(45)
 

4 (9X)
 

(45)
 

1 (2X)
 

(45)
 

1 (2X)
 

7 (16X)
 

(D
 

(D
 

d>
 

1 (100X)
 

(46)
 

3.-JZX1
 

# NUMBER OF ANIMALS WITH TISSOE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

58
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOff DOSE
 

INFLAMMATION, INTERSTITIAL 5 (50%)
 

INFLAMMATION, CHRONIC 2 (20%) 33 (72%)
 

ENEOCBINE SYSTEM 

•PITUITARY 

CYST, NOS 

HULTILOCULAR CYST 

(9) 

1 (11*) 

(46) 

2 (4X) 

6 (13X) 

ANGIECTASIS 1 (2X) 

#ADRENAL (10) (45) 

METAHOBPHOSIS FATTY 

ANGIECTASIS 

1

2

 (2%) 

(H%) 

#THYEOIE 

HYPEEPLASIA, C-CELL 

(9) (44) 

HYPEEPLASIA, FOLLICOLAB-CELL 1 (2X) 

f PARATHYROID (3) (32) 

HYPEEPLASIA, NOS 7 (22%) 

HYPEEPLASIA, EPITHELIAL 1 OX) 

REPRODUCTIVE SYSTEH
 

tPBCSTATE (9) (44)
 

INFLAMHATICN, NOS 2 (5S)
 

INFLAMMATION, SUPPURATIVE
 

INFLAMMATION, ACUTE 1 <2X)
 

INFLAMMATION, ACUTE SUPPUEATIVE 1 (11X)
 

#TESTIS (9) (45)
 

EDEMA, NOS 1 (11X) 2 (4%)
 

PERIARTERI1IS 1 (2X)
 

ATROPHY, NOS 3 (33X) 17 (38X)
 

NEBVCUS SYSTEM
 

NCNE
 

SPECIAL SENSE ORGANS
 

KCNE
 

MU5CUIOSKELETAL SYSTEM
 

__]<CNE_ __ __
 

# NUMBER OF ANIMALS WITH TISSUE EXAHINED MICROSCOPICALLY
 * NUMEER OF ANIMALS NECROPSIED
 

59
 

HIGH DOSE
 

36 (73X)
 

(46)
 

2 (4X)
 

(49)
 

1 (2X)
 

(45)
 

1 (2X)
 

1 (2%)
 

(35)
 

5 (14X)
 

1 (3X)
 

(47)
 

1 (2X)
 

(48)
 

10 (21X)
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

BODY CAVITIES
 

*HESENTERY (10) (U7) (49)
 

PERIAR1ERI1IS 8 (17%) 6 (12X)
 

ALL OTHER SYSTEMS
 

*MULTIPLE CBGANS (10) (47) (49)
 

PEBIARTERI1IS 1 (2%) 1 (2X)
 

SPECIAL HOfiPHOLOGY SUMMARY
 

NC LESICN REPORTED 1 2 4
 

AUTOLYSIS/NO NECROPSY 3
 

# KUREEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

60
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

FED CHLOROTHALONIL IN THE DIET 

CONTROL LOW DOSE HIGH DOSE
 

ANIMAL S INITIALLY IK STUDY 1C 50
 

ANIMALS NECKCPSIED 10 48
 

ANIMAL S EXAMINED HISTOFATHOLOGICALLY 10 48
 

INTEGUMEN1ABY SYSTEM
 

NONE
 

RESFIBATORY SYSTEM
 

#LUNG (9) (47)
 

CALCIFICATION, METASTATIC 1 (1U)
 

tLUNG/ALVEOLI (9) (47)
 

EMFHYSEHA, NCS 1 (11%)
 

HEMATOPOIETIC SYSTEM 

tSPLEEN 

INFLAMMATION, CHFONIC 

HEMOSIDE50SIS 

HYPERPLASIA, LYMPHOID 

(10) 

1 (10%) 

(47) 

1 (2%) 

CIRCULATORY SYSTEM
 

#MYOCARDIOM (9) (47)
 

INFLAMMATION, CHFONIC FOCAL 1 (2%)
 

*ARTERY (10) (48)
 

MEDIAL CALCIFICATION 1 (2«)
 

*AORTA (10) (48)
 

ARTERICSCLEROSIS, NOS 1 (10S)
 

*CORONABY ABTF.RY (10) (48)
 

MEDIAL CA|.CIFICA2IO£ 1_J2X1
 

# NUMBER OF ANIMALS HITH TISSOE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

50
 

50
 

50
 

(50)
 

(50)
 

(50)
 

1 (2%)
 

1 (2X)
 

(50)
 

(50)
 

(50)
 

(50)
 

61
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

DIGESTIVE SYSTEM
 

#LIVER (10) (48) (50)
 

FIBRCSIS, FOCAL 1 (2%)
 

EEGENERATION PARENCH YM ATOUS 2 (2CX)
 

CEGENEBATION, GRANULAR 1 (10*)
 

NECBOSIS, FOCAL 1 (10%)
 

KETAMCRfHOSIS FATTY 1 (10%) 1 (2%) 1 (2%)
 

ANGIECTASIS 5 (10%) 1 (2%)
 

#LIVER/CENTfiILOBULAR (10) (US) (50)
 

KETAMOBPHOSIS FATTY 2 (20%)
 

*BILE BUCT (10) (48) (50)
 

HYPERPLASIA, NOS 2 (4%)
 

#FANCREAS (9) (48) (50)
 PERIARTERITIS 2 (4%) 2 (4%)
 

#STCMACH (10) (48) (49)
 

CALCIFICATION, METASTATIC 1 (10%)
 

HYPERPLASIA, NOS 1 (2%)
 

•GASTRIC	 MUCOSA (10) (48) (49)
 

CALCIFICATION, NOS 1 (2%)
 

URINAR Y SYSTEM
 

((KIDNEY (10) (48) (50)
 

KYDRCNEPHRCSIS 1 (2%)
 

GLOflEBULCNEPHRITIS, NOS 1 (10%)
 

INFLAMMATION, INTEESTITIAL 2 (20%)
 

INFLAMMATION ACDTE AND CHRONIC 1 (2%)
 

INFLAMMATION, CHRONIC 1 (10%) 13 (27%) 6 (12%)
 

FIBBOSIS, FOCAL 1 (2%)
 

#URINARY BLADDER (9) (47) (47)
 

HYPEBPLASIA, EPITHELIAL 1 (2%)
 

ENDOCRINE SYSTEM
 

^PITUITARY (10) (48) (i»9)
 

HEMORRHAGE 1 MMi.
 

# NUMBER OF ANIMALS WITH TISSUE EXAHIHED MICROSCOPICALLY
 

* NOMBEB OF ANIMALS NECROPSIED
 

62
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE
 

ANGIEC1ASIS
 

SADRENA L (10) (48)
 

NECROSIS, HEMOERHAGIC
 

ANGIECTASIS 1 (2*)
 

#ADRENAL CORTEX (10) (48)
 

ANGIECTASIS 1 (10%)
 

#THYROID (7) (45)
 

HYPERPLASIA, C-CELL 1 (14*) 2 (4S)
 

HYPERPLASIA, FOLLICULAR-CELL
 

frPARATHYROIC (5) (26)
 

HYPERPLASIA, NOS 1 (20X) 2 (8X)
 

REPRODUCTIVE SYSTEM
 

*MAMBAFY GLAND (10) (48)
 

HYPERPLASIA, NOS 1 (10*)
 

ADENOSIS 1 (10*)
 

tUTERUS/ENDCHETRIUM (9) (47)
 

HYPERPLASIA, CYSTIC 1 (2X)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

HDSCOIOSKELETAL SYSTEB
 

NONE
 

BODY CAVITIES
 

*BESENTERY (10) (48)
 .___ «.______ __..
 ,PS8i&?T,E;RilJg ^ r_ 3, {§»!_,
 

# NUBEEH OF ANIHALS WITH TISSUE EXAHINED HICROSCOPICALLlf
 * NUBEER OF ANIBALS NECROPSIED
 

HIGH DOSE
 

2 (4S)
 

(50)
 

1 (2X)
 

6 (12*)
 

(50)
 

(41)
 

2 (5*)
 

1 <2X)
 

(26)
 

1 (4X)
 

(50)
 

(49)
 

(50)
 

? (6S)
 

63
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

ALL CTHER SYSTEMS 

*KULTIPLE OEGANS 

FERIARTERITIS 

HEDIAL CALCIFICATION 

(10) (48) 

2 (4X) 

1 (2X) 

(50) 

1 (2%) 

SPECIAL MORPHOLOGY SUHKARY 

NC LESION BEFORTFD 

AUTOLYSIS/NO NECfiCFSY 

1 

# NUF.EEB CF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

8 

1 

11 

64
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE FED CHLOROTHALONIL IN THE DIET
 

65
 

TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

FED CHLOROTHALONIL IN THE DIET 

CONTROL LOW DOSE HIGH DOSE
 

ANIMAIS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOFATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

NCNE
 

RESPIRATORY SYSTEM
 

*LUNG
 

ATELECTASIS
 

INFLAMMATION ACUTE AND CHEONIC
 

HYPERPLAEIA, ALVEOLAR EPITHELIUM
 

HEKATCFOIETIC SYSTEM
 

NCNE
 

CIRCULATORY SYSTEM
 

NCNE
 

DIGESTIVE SYSTEM
 

#LIVER
 

HYPERPLASIA, NODULAR
 

#STOMACH
 

EROSION
 

UBINAH Y SYSTEM
 

- NON E
 

10 50 50
 

10 49 50
 

10 U8 50
 

(9)	 (<*6) (50)
 

1 (11X)
 

1	 (2%)
 

1 (2%)
 

(10)	 (46) (50)
 

1 (10%) 2 (4%) 3 (6%)
 

(8)	 (46) (50)
 

1 (2*)
 

#	 NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUBEER OF ANIMALS NECROPSIED
 

67
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOH DOSE HIGH DOSE
 

ENDOCRINE SYSIEH
 

ttTHYEOID (9) (43) (U9)
 

HYPERPLASIA, FOLIICULAR-CELL 1 (2X)
 

REPRODUCTIVE SYSTEM
 

NONE
 

NERVCUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKEIETAL SYSTEM
 

NONE
 

BOEY CAVITIES
 

NONE
 

ALL OTHEB SYSTEMS
 

NONE
 

SPICIA1 MORPHOLOGY SUHHAHY
 

NO LESION BEEOETED 5 36 at
 

AUTO/MECROPSY/NO HISTO 1
 

AUTOLYSIS/NO NECBOPSY 1
 

# NOBEER OF ANIMALS Wllli TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

68
 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

FED CHLOROTHALONIL IN THE DIET 

ANIKAIS INITIALLY IK STUDY
 

ANIMALS NECRCPSIED
 

ANIMALS EXAMINED HISTOFATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

#LUNG
 

EDEMA, NOS
 

HEMATOEOIETIC SYSTEM
 

#SPLEEN
 

HEMATOPOIESIS
 

tSPLENIC FOLLICLES
 

HYPERPLASIA, NOS
 

#KESENTERIC L. NODE
 

INFLAMMATION. GRANULCMATOUS
 

CIECOIATCRY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVER
 

INFLAMBA1ION, ACUTE DIFFUSE
 

INFLAMMATION, ACUTE NECKOTIZING
 

INFARCT, FOCAL
 

HYPERPLASIA, NODULAR
 

URINARY SYSTIM
 

NCNf.
 

CONTROL LOW DOSE HIGH DOSE 

10 50 50 

10 49 47 

10 49 47 

(10) <U9) (46) 

1 12%) 

<9) (48) (45) 

1 (2%) 

(9)

1 (11%) 

(US) (45) 

(10) (HH) (43) 

1 (10S) 

(10) (49) (46) 

1 (2X) 

1 <2X) 

1 (10%) 

1

1

 (2X) 

 <2X) 

# NUKBER OF ANIMALS WITH TISSUE EXAHINED MICHOSCOPICALLY
 * KUKBFR OF ANIMALS NECROPSIED
 

69
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

ENEOCBINE SYSTEM
 

fTHYROID (9) (45) (44)
 

HYPERPLASIA, FOLLICULAR-CELL 1 (2X)
 

REPRODUCTIVE SYSTEM
 

*MAMMAR Y GLAND (10) C*9) (47)
 

HYPERPLASIA, NOS 1 (2X)
 

#LTERUS/ENDOMETRIUM (10) (48) (43)
 

HYPEEPLASIA, CYSTIC 2 (20%)
 

#OVARY (10) (49) (42)
 

HEMORRHAGIC CYST 1 (10S)
 

NEEVCUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

KCNE
 

MUSCULOSKELETAI SYSTEM
 

NCfiE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

KCNE
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 2 41 37
 __ 1
 AUTOLYSIS/NO_N£CiOPSY 3
 

# NUMEER OF ANIMALS WI1H TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEEK OF ANIHALS NECROPSIE0
 

70
 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS FED CHLOROTHALONIL IN THE DIET
 

71
 

__ 

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

Fed Chlorothalonil in the Dieta 

Topography: Morphology
 

Integumentary System:
 

Malignant Fibrous Histiocytoma
 

of the Subcutaneous Tissue"
 

P Valuesc»d
 

Relative Risk (Pooled Control)^
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe
 

Adenoma or Carcinoma"
 

P Values0 »d
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Control
 

0/59 (0)
 

P = 0.030
 

—
 

6/56 (11)
 

N.S.
 

—
 

Matched
 

Control
 

0/10 (0)
 

N.S.
 

—
 

0/9 (0)
 

N.S.
 

—
 

Low
 

Dose
 

0/47 (0)
 

N.S.
 

—
 

—
 

—
 

—
 

—
 

—
 

5/46 (11)
 

N.S.
 

1.014
 

0.260
 

3.722
 

Infinite
 

0.280
 

Infinite
 

103
 

High
 

Dose
 

3/49 (6)
 

N.S.
 

Infinite
 

0.721
 

Infinite
 

Infinite
 

0.136
 

Infinite
 

87
 

5/46 (11)
 

N.S.
 

1.014
 

0.260
 

3.722
 

Infinite
 

0.280
 

Infinite
 

87
 
__ 

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

Fed Chlorothalonil in the Dieta 

(continued) 

Topography: Morphology 

Thyroid: Follicular-cell 

Adenoma" 

P Values0 »d 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Kidney: Carcinoma, NOS, 

Adenocarcinoma or Tubular-cell 

Adenocarcinomab 

P Values0 >d 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor —

 

Low 

Dose 

2/44 (5) 

N.S. 

0.625 

0.059 

4.132 

Infinite 

0.068 

Infinite 

101 

1/46 (2) 

N.S. 

Infinite 

0.072 

Infinite 

Infinite 

0.013 

Infinite 

110 

High 

Dose 

3/45 (7) 

N.S. 

0.917 

0.140 

5.125 

Infinite 

0.136 

Infinite 

84 

3/49 (6) 

N.S. 

Infinite 

0.757 

Infinite 

Infinite 

0.136 

Infinite 

84 —

 

Pooled 

Control 

4/55 (7) 

N.S. 

0/62 (0) 

P = 0.044 

Matched 

Control 

0/9 (0) 

N.S. 

— 

0/10 (0) 

N.S. 
__ 

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

Fed Chlorothalonil in the Diet3 

(continued) 

Topography: Morphology
 

Kidney: Papillary Adenoma or
 

Tubular-cell Adenoma"
 

P Values0 >d
 

Relative Risk (Pooled Control) ^
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Kidney: Carcinoma, NOS,
 

Tubular-cell Adenoma,
 

Tubular-cell Adenocarcinoma,
 

Adenocarcinoma, or
 

Papillary Adenoma"
 

P Values0 »d
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 —
 

Low
 

Dose
 

2/46 (4)
 

N.S.
 

Infinite
 

0.397
 

Infinite
 

Inf intie
 

0.071
 

Infinite
 

103
 

3/46 (7)
 

N.S.
 

Infinite
 

0.807
 

Infinite
 

Infinite
 

0.145
 

Infinite
 

103
 

High
 

Dose
 

1/49 (2)
 

N.S.
 

Infinite
 

0.068
 

Infinite
 

Infinite
 

0.012
 

Infinite
 

95
 

4/49 (8)
 

P = 0.035**
 

Infinite
 

1.167
 

Infinite
 

Infinite
 

0.211
 

Infinite
 

84
 —
 

Pooled
 

Control
 

0/62 (0)
 

N.S.
 

0/62 (0)
 

P = 0.030
 

Matched
 

Control
 

0/10 (0)
 

N.S.
 

—
 

0/10 (0)
 

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

 

Fed Chlorothalonil in the Diet3

 

(continued) 

a

Dosed groups received time-weighted average doses of 5,063 or 10,126 ppm.
 

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

c

Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the
 

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

the comparison of that dosed group with the matched-control group (*) or with the pooled-


control group (**) when P < 0.05 for either control group; otherwise, not significant (N.S.)
 

is indicated.
 

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

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats

 

Fed Chlorothalonil in the Diet3

 

Topography: Morphology 

Pituitary: Chromophobe 

Adenoma^ 

P Values

c

»d 

Departure from Linear Trend6 

Relative Risk (Pooled Control)

f 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)

f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Thyroid: Follicular-cell 

Adenoma or Carcinoma*5 

P Values

c

»d 

Relative Risk (Pooled Control)

f 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Pooled 

Control 

9/56 (16) 

N.S. 

P = 0.024 

— 

0/58 (0) 

N.S. 

— 

Matched 

Control 

2/10 (20) 

N.S. 

P = 0.025 

100 

0/7 (0) 

N.S. 

— 

Low 

Dose 

1/48 (2) 

P - 0.015**(N) 

0.130 

0.003 

0.882 

0.104 

0.002 

1.883 

94 

2/45 (4) 

N.S. 

Infinite 

0.380 

Infinite 

Infinite 

0.054 

Infinite 

111 

High 

Dose 

6/49 (12) 

N.S. 

0.762 

0.239 

2.215 

0.612 

0.141 

5.791 

102 

2/41 (5) 

N.S. 

Infinite 

0.417 

Infinite 

Infinite 

0.059 

Infinite 

111 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats 

Fed Chlorothalonil in the Dieta 

(continued) 

Topography: Morphology 

Liver: Neoplastic Nodule*5 

P Values0 »d 

Relative Risk (Pooled Control)^ 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)^ 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Kidney: Carcinoma, NOS, or 

Tubular-Cell Adenocarcinoma^ 

P Values0 »d 

Relative Risk (Pooled Control)

f 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)^ 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Pooled 

Control 

1/59 (2) 

N.S. 

— 

0/62 (0) 

N.S. 

— 

Matched 

Control 

0/10 (0) 

N.S. 

— 

0/10 (0) 

N.S. 

— 

Low 

Dose 

3/48 (6) 

N.S. 

3.688 

0.307 

189.405 

Infinite 

0.139 

Infinite 

84 

1/48 (2) 

N.S. 

Infinite 

0.069 

Infinite 

Infinite 

0.012 

Infinite 

107 

High 

Dose 

2/50 (4) 

N.S. 

2.360 

0.126 

136.426 

Infinite 

0.065 

Infinite 

11 

2/50 (4) 

N.S. 

Infinite 

0.365 

Infinite 

Infinite 

0.065 

Infinite 

91 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats 

(continued) 

Topography: Morphology 

Kidney: Adenoma, NOS, or 

Tubular-Cell Adenomab 

P Values0 »d 

Relative Risk (Pooled Control)

f 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)

f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Kidney: Adenoma, Carcinoma, NOS, 

Tubular-cell Adenoma, or 

Tubular-cell Adenocarcinoma^ 

P Values0 »d 

Relative Risk (Pooled Control)

f 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Fed Chlorothalonil in the Dieta 

Pooled 

Control 

Matched 

Control 

Low 

Dose 

High 

Dose 

0/62 (0) 0/10 (0) 0/48 (0) 3/50 (6) 

P = 0.028 N.S. N.S. N.S. 

__ 

— 

— 

__ 

— 

— 

Infinite 

0.742 

Infinite 

Infinite 

0.134 

Infinite 

__ __ __ 97 

0/62 (0) 0/10 (0) 1/48 (2) 5/50 (10) 

P = 0.007 N.S. N.S. P = 0.016** 

Infinite 

0.069 

Infinite 

Infinite 

1.556 

Infinite 

Infinite 

0.012 

Infinite 

Infinite 

0.281 

Infinite 

— — 107 91 
Table E2. Analyses of the Incidence of Primary Tumors in Female
 

Fed Chlorothalonil in the Diet3
 

(continued)
 

Topography: Morphology
 

Mammary Gland: Fibroadenoma"
 

P Values0>d
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)f
 

Lower Limit
 

oo Upper Limit
 

o
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibromab
 

P Values0 »d
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Control
 

9/64 (14)
 

N.S.
 

—
 

1/64 (2)
 

N.S.
 

—
 

Matched
 

Control
 

1/10 (10)
 

N.S.
 

68
 

,0/10 (0)
 

N.S.
 

—
 

Low
 

Dose
 

8/48 (17)
 

N.S.
 

1.185
 

0.428
 

3.188
 

1.667
 

0.279
 

72.240
 

72
 

3/48 (6)
 

N.S.
 

4.000
 

0.333
 

205.455
 

Infinite
 

0.139
 

Infinite
 

101
 

Rats
 

High
 

Dose
 

4/50 (8)
 

N.S.
 

0.569
 

0.135
 

1.903
 

0.800
 

0.097
 

38.616
 

72
 

1/50 (2)
 

N.S.
 

1.280
 

0.017
 

98.511
 

Infinite
 

0.012
 

Infinite
 

111
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Chlorothalonil in the Dieta
 

(continued)
 

a

Dosed groups received time-weighted average doses of 5,063 or 10,126 pptn.
 

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

c

Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the
 

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

the comparison of that dosed group with the matched-control group (*) or with the pooled-


control group (**) when P < 0.05 for either control group; otherwise, not significant (N.S.)
 

is indicated.
 

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

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

^The 95% confidence interval of the relative risk between each dosed group and the specified
 

control group.
 

APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN MICE FED CHLOROTHALONIL IN THE DIET
 

83
 

__ __ 

Topography: Morphology
 

Lung: Alveolar /Bronchiolar
 

Adenoma"
 

P Values0 »d
 

Relative Risk (Pooled Control)^
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular
 

Carcinoma^
 

P Values0 »d
 

Departure from Linear Trend6
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

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

Fed Chlorothalonil in the Dieta 

Pooled Matched
 

Control Control
 

4/58 (7) 1/9 (11)
 

N.S. P = 0.040(N)
 

91
 

9/59 (15) 2/10 (20)
 

P = 0.005(N) N.S.
 

P = 0.041
 

85
 —
 

Low
 

Dose
 

4/46 (9)
 

N.S.
 

1.261
 

0.247
 

6.400
 

0.783
 

0.097
 

37.702
 

91
 

1/46 (2)
 

P = 0.022**(N)
 

0.143
 

0.003
 

0.969
 

0.109
 

0.002
 

1.963
 

92
 

High
 

Dose
 

0/50 (0)
 

N.S.
 

0.000
 

0.000
 

1.253
 

0.000
 

0.000
 

3.374
 

1/50 (2)
 

P - 0.016**(N)
 

0.131
 

0.003
 

0.895
 

0.100
 

0.002
 

1.810
 

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

 

Fed Chlorothalonil in the Dieta

 

(continued) 

a

Dosed groups received time-weighted averages doses of 2,688 or 5,375 ppm.
 

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

G

Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the
 

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

the comparison of that dosed group with the matched-control group (*) or with the pooled-


control group (**) when P < 0.05 for either control group; otherwise, not significant (N.S.)
 

is indicated.
 

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

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

^The 95% confidence interval of the relative risk between each dosed group and the specified
 

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

Fed Chlorothalonil in the Diet3
 

Pooled Matched Low High 

Topography: Morphology Control Control Dose Dose 

Lung: Alveolar/Bronchiolar 

Adenoma or Carcinoma" 3/58 (5) 1/10 (10) 2/49 (4) 3/46 (7) 

P Values0 >d N.S. N.S. N.S. N.S. 

Relative Risk (Pooled Control)f 0.789 1.261 

Lower Limit 0.068 0.176 

Upper Limit 6.598 8.980 

Relative Risk (Matched Control)f 0.408 0.652 

Lower Limit 0.025 0.063 

Upper Limit 23.619 33.512 

Weeks to First Observed Tumor 91 91 92 — 

a

Dosed groups received time-weighted average doses of 3,000 or 6,000 ppm.
 

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

c

Beneath the incidence of tumors in a control group is the probability level for the Cochran-


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the
 

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

the comparison of that dosed group with the matched-control group (*) or with the pooled-


control group (**) when P < 0.05 for either control group; otherwise, not significant (N.S.)
 

is indicated.
 

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

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.
 

^The 95% confidence interval of the relative risk between each dosed group and the specified
 

control group.
 

APPENDIX G
 

ANALYSIS OF FORMULATED DIETS FOR
 

CONCENTRATIONS OF CHLOROTHALONIL
 

89
 

APPENDIX G
 

Analysis of Formulated Diets for
 

Concentrations of Chlorothalonil
 

A 10-g sample of the dosage mixture to be analyzed was shaken
 

with 125 ml benzene at room temperature for 16 hours, then
 

filtered through Celite with benzene washes, and reduced in
 

volume to 10 ml. After appropriate dilutions, the solution was
 

quantitatively analyzed for chlorothalonil by gas-liquid
 

chromatography (electron capture detector, 10% DC-200 on Gas
 

Chrom Q column). Recoveries were checked with spiked samples,
 

and external standards were used for calibration.
 

Theoretical No. of Sample Coefficient of Range (ppm) 

Concentrations Samples Analytical Variation (%) 

in Diet (ppm) Mean (ppm) 

2,500 22 2,506 5.4% 2,271-2,835 

5,000 33 5,016 5.7% 4,375-5,648 

10,000 30 10,110 4.3% 9,500-10,960 

20,000 2 19,700 2.2% 19,400-20,000 

91
 

Review of the Bioassay of Chlorothalonil* 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 DHEW 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 Chlorothalonil for carcinogenicity.
 

The reviewer said that the compound was carcinogenic in
 

treated rats but "was not clearly carcinogenic" in treated
 

mice. The reviewer was particularly critical of the limited
 

number of matched control animals. She pointed out the
 

occurrence of certain tumors in the treated rats and mice
 

that may have taken on greater significance had the control
 

groups been larger. Other deficiencies noted by the reviewer
 

included the use of different batches of Chlorothalonil and
 

poor animal survival. Despite the shortcomings, she said
 

that the kidney tumors in both the treated male and female
 

rats appeared to be biologically significant.
 

In subsequent discussions, a staff pathologist described
 

the criteria for classifying kidney tumors, particularly with
 

respect to differentiating between benign and malignant
 

neoplasms. He noted greater incidences of renal toxicity
 

and hypoparathyroidism observed in treated animals than in
 

controls and he suggested that Chlorothalonil was probably a
 

renal toxin. A representative of the manufacturer of
 

93
 
Chlorothalonil said that the compound had been shown to
 

produce renal toxicity, which was probably exerted through
 

an indirect mechanism.
 

A motion was made that the report on the bioassay of
 

Chlorothalonil be accepted as written. The motion was
 

approved without objection.
 

Clearinghouse Members present:
 

Arnold L. Brown (Chairman), Mayo Clinic
 

Paul Nettesheim, National Institute of Environmental
 

Health Sciences
 

Verne Ray, Pfizer Medical Research Laboratory
 

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

Louise Strong, University of Texas Health Sciences Center
 

*	 Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

fcU.S . GOVERNMEN T PRINTIN G OFFICE ! 1978-260-899/318 5
 

94
 

'o

i 

£ 

CD 

DHEW Publication No. (NIH) 78-841