1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 90 

I 

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

Public Health Service 

National Institutes of Health 

BIDAS SAY OF
 

DICOFOL
 

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
 

DREW Publication No. (NIH) 78-1340
 

REPORT ON THE BIOASSAY OF DICOFOL
 

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 dicofol
 

conducted for the Carcinogenesis Testing Program, Division of Cancer
 

Cause and Prevention, National Cancer Institute (NCI), National Institutes of Health, Bethesda, Maryland. This is one of a series of experiments designed to determine whether selected chemicals have the
 

capacity to produce cancer in animals. Negative results, in which
 

the test animals do not have a significantly greater incidence of
 

cancer than control animals, do not necessarily mean the test chemical is not a carcinogen because the experiments are conducted under a
 

limited set of circumstances. Positive results demonstrate that the
 

test chemical is carcinogenic for animals under the conditions of the
 

test and indicate a potential risk to man. The actual determination
 

of the risk to man from animal carcinogens requires a wider analysis.
 

CONTRIBUTORS: This bioassay of dicofol was conducted by Hazleton
 

Laboratories America, Inc., Vienna, Virginia, initially under direct
 

contract to the NCI and currently under a subcontract to Tracer Jitco,
 

Inc., prime contractor for the NCI Carcinogenesis Testing Program.
 

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

principal investigators for the contract were Dr. 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 Carcinogenesis Bioassay
 

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

111
 
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), task leader Dr. M. R. Kornreich (6), senior biologist Ms. P.
 

Walker (6), biochemist Mr. S. C. Drill (6), and technical editor Ms.
 

P. A. Miller (6). The final report was reviewed by members of the
 

participating organizations.
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

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

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

Squire (1,10), Dr. J. M. Ward (1), and Dr. C. E. Whitmire (1).
 

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

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

3.	 Hazleton Laboratories America, Inc., 9200 Leesburg Turnpike,
 

Vienna, Virginia.
 

4.	 No wit w h the Center for Regulatory Services, 2347 Paddock Lane,
 

Reston, Virginia.
 

5.	 No wit w h 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 dicofol for possible carcinogenicity was conducted using Osborne-Mendel rats and B6C3F1 mice.
 

Dicofol was administered in the feed, at either of two concentrations,
 

to groups of 50 males and 50 females of each species. The high and
 

low time-weighted average concentrations of dicofol were, respectively,
 

942 and 471 ppm for male rats, 760 and 380 ppm for female rats, 528
 

and 264 ppm for male mice, and 243 and 122 ppm for female mice. For
 

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

The period of compound administration was 78 weeks, followed by 34
 

weeks of observation in rats and 14 or 15 weeks in mice.
 

There was no statistically significant positive association
 

between dietary concentration and mortality in either sex or species.
 

Hepatocellular carcinomas in dosed male mice were the only neoplasms that occurred in any dosed group of either species in statistically significant increased incidences when compared to controls.
 

The Cochran-Armitage test as well as the Fisher exact test for both
 

the high and low dose groups supported the association between compound administration and increased incidences of this tumor in the
 

male mice. No increase in hepatocellular carcinomas was observed
 

in dosed female mice.
 

Under the conditions of this bioassay, technical-grade dicofol
 

was carcinogenic in male B6C3F1 mice, causing hepatocellular carcinomas. No evidence for carcinogenicity was obtained for this compound in Osborne-Mendel rats of either sex or in female B6C3F1 mice.
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 4
 

A. Chemicals 4
 

B. Dietary Preparation 4
 

C. Animals 6
 

D. Animal Maintenance 6
 

E. Selection of Initial Concentrations 7
 

F. Experimental Design 9
 

G. Clinical and Histopathologic Examinations 13
 

H. Data Recording and Statistical Analyses 14
 

III. CHRONIC TESTING RESULTS: RATS 19
 

A. Body Weights and Clinical Observations 19
 

B. Survival 21
 

C. Pathology 21
 

D. Statistical Analyses of Results 23
 

IV. CHRONIC TESTING RESULTS: MICE 28
 

A. Body Weights and Clinical Observations 28
 

B. Survival 28
 

C. Pathology 32
 

D. Statistical Analyses of Results 32
 

V. DISCUSSION 39
 

VI. BIBLIOGRAPHY 41
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH DICOFOL A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH DICOFOL B-l
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH DICOFOL C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH DICOFOL D-l
 

vii
 
LIST OF ILLUSTRATIONS
 

Figure Number
 

1
 

2
 

Table Number
 

CHEMICAL STRUCTURE OF DICOFOL 

GROWTH CURVES FOR DICOFOL CHRONIC STUDY 

RATS 20 

SURVIVAL COMPARISONS OF DICOFOL CHRONIC 

STUDY RATS 22 

GROWTH CURVES FOR DICOFOL CHRONIC STUDY 

MICE 29 

SURVIVAL COMPARISONS OF DICOFOL CHRONIC 

STUDY MICE 30 

COMPARISONS OF DICOFOL CHRONIC STUDY MALE 

MICE SURVIVING WITHOUT OBSERVED HEPATOCELLULAR CARCINOMAS 38 

LIST OF TABLES 

Page 

DESIGN SUMMARY FOR OSBORNE-MENDEL RATS— 

DICOFOL FEEDING EXPERIMENT 10 

DESIGN SUMMARY FOR B6C3F1 MICE—DICOFOL 

FEEDING EXPERIMENT 11 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

DICOFOL 24 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED WITH 

DICOFOL 26 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

DICOFOL 33 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED WITH 

DICOFOL 36 

viii
 
LIST OF TABLES (Concluded)
 

Table Number 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE RATS TREATED WITH DICOFOL A-3 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH DICOFOL A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH DICOFOL B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH DICOFOL B-6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH DICOFOL C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH DICOFOL C-7 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH DICOFOL D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH DICOFOL D-6 

ix
 

I. INTRODUCTION
 

Dicofol (NCI No. C00486), a synthetic organochlorine acaricide,
 

was selected for bioassay by the National Cancer Institute because it
 

is an alcohol analog of the known tumorigen DDT (innes et al., 1969).
 

Its widespread use on edible crops was also an important factor in
 

its selection for testing.
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is 4-chloro-alpha-(4-chlorophenyl)­

*
 alpha-(trichloromethyl)benzenemethanol. It is also called 1,1-bis
 

(p-chlorophenyl)-2,2,2-trichloroethanol; 4,4'-dichloro-alpha-(trichloromethyl)benzhydrol; and 2 ,2,2-trichloro-l,l-di-(4-chlorophenyl)
 

ethanol.
 

Dicofol is a nonsystemic acaricide that is used to control mites
 

on cotton, corn, and other field crops; vegetables; citrus and non-


citrus fruits; and nursery and greenhouse crops (Martin and Worthing,
 

1977). In 1971, 447 thousand pounds of dicofol were used to treat
 

474 thousand acres of crops in the United States. Cotton was the
 

major single crop treated, accounting for 189 thousand pounds or 42
 

percent of total dicofol usage (Andrilenas, 1974). The vast majority
 

of dicofol usage (409 thousand pounds on 428,000 acres) took place in
 

the Pacific States (California, Oregon, and Washington).
 

*
 

The CAS registry number is 115-32-2,
 
Specific production statistics for dicofol are not available;
 

however, the inclusion of the compound in Synthetic Organic Chemicals,
 

U.S. Production and Sales, 1975 (U.S. International Trade Commission,
 

1977) implies an annual commercial production in excess of 1000 pounds
 

or $1000 in value. Approximately 4 million pounds of dicofol were
 

produced in 1971 (Ouellette and King, 1977). No imports of the pesticide were reported in the period 1970-1974 inclusive (U.S. Department
 

of Agriculture, 1975).
 

Agricultural workers have the greatest potential for exposure to
 

dicofol, although the production and storage of the compound may also
 

present a significant risk to workers in the pesticide manufacturing
 

industry. The general population may be exposed to dicofol in house
 

and garden pesticides for evergreens, shrubs, and flower and vegetable
 

gardens (Gosselin et al., 1975); to airborne dicofol after commercial
 

agricultural spraying; to residues in rivers and streams as a result
 

of industrial discharge; and to dicofol residues in crops and soils.
 

Dicofol has been found in concentrations of up to 0.066 ppm in "readyto-eat" fruits (Manske and Corneliussen, 1974). Residues in soil
 

decrease rapidly, but traces may persist for a year or longer (Martin
 

and Worthing, 1977). Industrial wastewater in the Soviet Union has
 

been found to contain as much as 0.397 mg/1 of dicofol (Diatlovitskaia
 

and Botvinova, 1971).
 

The effects of dicofol poisoning presumably resemble those of
 

DDT, although the latter is somewhat more toxic. The primary sites
 
of action appear to be the cerebellum and higher motor cortex (Gosselin et al., 1976).
 

Leukopenia, neutropenia, and a decrease in the hemoglobin level
 

were found in humans working with dicofol-chlorophos-copper oxychloride mixtures 8 to 10 hours daily for several months (Stuneeva, 1973).
 

Although it is stored to a certain extent throughout the body,
 

dicofol, like DDT, appears to be preferentially stored in fat; however, some dicofol is apparently converted into DDE, a DDT metabolite,
 

in rats (Brown, 1972).
 

Workers using a number of pesticides, including dicofol, were
 

found to have an abnormally large amount of lymphocyte chromosomal
 

damage (Yoder et al., 1973); however, the compound showed no mutagenic
 

activity in Escherichia coli, failing to induce reversions to prototype in a tryptophan-dependent mutant (WP2 Try-) (Ashwood-Smith et
 

al., 1972).
 

No indications of teratogenicity were found in mice fed dicofol
 

in their diet in amounts of up to 500 ppm over five generations
 

(Brown, 1972).
 
II. MATERIALS AND METHODS 

A. Chemicals
 

Technical-grade dicofol (Figure 1) [1,l-bis(p-chlorophenyl)­

2,2,2-trichloroethanol] was purchased from Rohm and Haas Chemical
 

Company and chemical analysis was performed by Hazleton Laboratories
 

America, Inc., Vienna, Virginia. The wide range observed for the
 

melting point (45° to 60°C) and the difference from the literature
 

value (77° to 78°C) suggested a compound of low purity. Although the
 

effectiveness of gas-liquid chromatography is limited due to thermal
 

decomposition of the compound, analysis using this technique suggested
 

a purity between 40 and 60 percent. Analyses performed twelve months
 

later revealed similar results. Within the next twelve-month period
 

a significant amount of the stored material liquified, suggestive of
 

substantial decomposition.
 

Throughout this report the term dicofol is used to represent this
 

technical-grade material.
 

B.	 Dietary Preparation
 

The basal laboratory diet for both treated and control animals
 

®
 consisted of 2 percent Duke's' corn oil (S. F. Sauer Company, Richmond,
 

®
 Virginia) by weight added to Wayne Lab-Blox meal (Allied Mills, Inc.,
 

Chicago, Illinois). Fresh mixtures of dicofol in corn oil were prepared each week and stored in the dark. The mixtures of dicofol in
 

corn	 oil were incorporated into the appropriate amount of basal laboratory diet in a twin-shell blender fitted with an accelerator bar.
 

4
 
FIGURE 1

 

CHEMICAL STRUCTURE OF DICOFOL 
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 12 to 15 complete changes of room air per hour. Fluorescent lighting was provided on a 12-hour-daily cycle.
 

The rats were individually housed in suspended galvanized-steel
 

wire-mesh cages with perforated floors. Mice were housed by sex in
 
groups of ten in solid-bottom, polypropylene cages equipped with filter tops. Sanitized cages with fresh bedding (Sanichips®, Pinewood
 

Sawdust Company, Moonachie, New Jersey) 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. Fresh
 

heat-sterilized glass water bottles and sipper tubes were provided
 

three times a week. Food and water were available ad libitum.
 

The dicofol-treated and control rats were housed in the same
 

room with rats receiving diets containing dioxathion (78-34-2);
 

mexacarbate (315-18-4); nitrofen (1836-75-5); endosulfan (115-29-7);
 

and trifluralin (1582-09-8).
 

All mice used in the dicofol study, including controls, were
 

housed in the same room as other mice receiving diets containing
 

trifluralin (1582-09-8); p,p'-DDE (72-55-9); dioxathion (78-34-2);
 

methoxychlor (72-43-5); p,p'-TDE (72-54-8); chlorobenzilate (510­

15-6); safrole (94-59-7); acetylaminofluorene (53-96-3); clonitralid
 

(1420-04-8); sulfallate (95-06-7); p,p'-DDT (50-29-3); amitrole (61­

82-5); pentachloronitrobenzene (82-68-8); nitrofen (1836-75-5); endosulfan (115-29-7); and mexacarbate (315-18-4).
 

E.	 Selection of Initial Concentration
 

In order to establish the maximum tolerated concentrations of
 

dicofol for administration to treated animals in the chronic studies,
 

*
 

CAS registry numbers are given in parentheses.
 
subchronic toxicity tests were conducted with both rats and mice.
 

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

small amount of laboratory diet. The mixture was then incorporated
 

into the basal laboratory diet and fed ad libitum to five of the six
 

rat groups in concentrations of 178, 316, 562, 1000, and 1780 ppm
 

and to five of the six mouse groups in concentrations of 100, 178,
 

316, 562, and 1000 ppm. The sixth group of each species served as a
 

control group, receiving only the basal laboratory diet. 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 laboratory diet.
 

A dosage inducing no mortality and resulting in a depression in
 

mean group body weight of approximately 20 percent relative to controls was selected as the initial high concentration. When weight
 

gain criteria were not applicable, mortality data alone were utilized.
 

Mean body weight depression was observed at all dosage levels in
 

both male and female rats. At a concentration of 562 ppm, the depression in mean group body weight was 3 percent in male rats and 2 percent in females. At 1000 pprn the depression in mean body weight was
 

20 percent in males and 11 percent in females. No deaths occurred
 

in groups receiving concentrations of 1000 ppm or less. The initial
 

concentration used in the chronic bioassay for high dose male and
 

female rats was 760 ppm. This was later increased for male rats to
 

1000 ppm.
 
Mean body weight depression in mice was observed in all treated
 

groups. For males the mean group body weight depressions were 20 percent in the group receiving 178 ppm and 28 percent in those receiving
 

316 ppm. One male receiving 178 ppm died. Among females mean body
 

weight depression was 19 percent at 100 ppm and 27 percent at 178 ppm.
 

No deaths were reported for any of the female groups. The initial
 

high concentrations used for male and female mice in the chronic
 

study were 300 and 110 ppm, respectively. This was later increased,
 

as shown in Table 2.
 

F. Experimental Design
 

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

of treated and untreated observation periods, and the time-weighted
 ;>
 

average concentrations) are summarized in Tables 1 and 2.
 

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

placed on test. The initial dietary concentrations of dicofol administered to rats were 760 and 380 ppm. Throughout this report
 

those rat groups initially receiving the former concentration are
 

referred to as the high dose groups and those initially receiving
 

the latter concentration are referred to as the low dose groups. In
 

week 20, the high and low concentrations for males were increased to
 

1000 and 500 ppm, respectively, as the treated males had apparently
 

tolerated the previous levels. These levels were maintained throughout the remainder of the dosing period. The high and low dose rats
 
TABLE 1
 

DESIGN SUMMARY FOR OSBORNE-MENDEL RATS
 

DICOFOL FEEDING EXPERIMENT
 

INITIAL OBSERVATION PERIOD TIME-WEIGHTED 

GROUP DICOFOL TREATED UNTREATED AVERAGE 

SIZE CONCENTRATION3

 (WEEKS) (WEEKS) CONCENTRATION 

MALE
 

CONTROL 20 0 110 0
 

LOW DOSE 50 380 19
 

500 59 471
 

0 34
 

HIGH DOSE 50 760 19
 

1000 59 942
 

0 34
 

FEMALE
 

CONTROL 20 0 111 0
 

LOW DOSE 50 380 78 380
 

0 34
 

HIGH DOSE 50 760 78 760
 

0
 

34
 

a
Concentrations given in parts per million.
 

b 2 (concentration X weeks received)
 Ti tnp— w<= i ffh fpH flvprafff ' rnrtrfnt-ra 11 nn = ; : : : :^
 

2(weeks receiving chemical)
 

10
 
TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE 

DICOFOL FEEDING EXPERIMENT 

INITIAL OBSERVATION PERIOD TIME-WEIGHTED 

GROUP DICOFOL TREATED UNTREATED AVERAGE 

SIZE CONCENTRATION3 (WEEKS) (WEEKS) CONCENTRATION 

MALE 

CONTROL 20 0 91 0 

LOW DOSE 50 150 4 264 

200 15 

250 14 

300 45 

0 14 

HIGH DOSE 50 300 4 528 

400 15 

500 14 

600 45 

0 14 

FEMALE 

CONTROL 20 0 91 0 

LOW DOSE 50 55 9 122 

85 10 

100 14 

150 45 15 

HIGH DOSE 50 110 9 243 

170 10 

200 14 

300 45 

0 15 

a
 

Concentrations in parts per million.
 

b ^(concentration X weeks received)
 Timp— WPI fhfpfl flvprfltrp reMirpnfrafinn =
 £(weeks receiving chemical)
 

11
 
were treated for 78 weeks followed by 34 weeks of observation during
 

which they received the basal laboratory diet.
 

All mice were approximately 6 weeks old at the time the experiment began. The initial dietary concentrations administered to male
 

mice were 300 and 150 ppm. Female mice received initial concentrations
 

of 110 and 55 ppm. Throughout this report males initially receiving
 

300 ppm and females initially receiving 110 ppm are referred t as the
 

high dose groups while males initially receiving 150 ppm and females
 

initially receiving 55 ppm are referred to as the low dose groups.
 

Dosage levels were increased on three separate occasions for both
 

male and female mice, as apparent tolerance of previous concentrations
 

was observed. The high and low concentrations administered to male
 

mice were increased to 400 and 200 ppm, respectively, during week 5.
 

In week 10, the high and low concentrations administered to the
 

female mice were increased to 170 and 85 ppm, respectively. During
 

week 20 the high and low concentrations administered to treated mice
 

were increased, respectively, to 500 and 250 ppm for the males and to
 

200 and 100 ppm for the females. Final increases in concentrations
 

were made during week 34, when high and low concentrations were increased to 600 and 300 ppm for males, and to 300 and 150 ppm for females. These dosage levels were maintained for the remainder of the
 

78-week period of chemical administration. A 14- to 15-week observation period followed, during which the animals received the basal
 

laboratory diet.
 

12
 
G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

experiment. Body weights, food consumption, and data concerning
 

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

and location of tissue masses were recorded at weekly intervals for
 

the first 10 weeks and at monthly intervals thereafter. From the
 

first day, all animals were inspected daily for mortality. The
 

presence 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 the end of
 

the bioassay. The animals were euthanized by exsanguination under
 

sodium pentobarbital anesthesia, and were immediately necropsied.
 

13
 
The histopathologic examination consisted of gross and microscopic
 

examination of major tissues, organs, and gross lesions taken from
 

sacrificed animals and, whenever possible, from animals found dead.
 

Tissues were preserved in 10 percent buffered formalin, embedded
 

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

to microscopic examination. An occasional section was subjected to
 

special staining techniques for more definitive diagnosis.
 

Slides were prepared from the following tissues from selected
 

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

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

gallbladder (mice), pancreas, esophagus, stomach, small intestine,
 

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

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

and ovary.
 

H. Data Recording and Statistical Analyses
 

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

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

as recommended by the International Union Against Cancer (Berenblum,
 

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

14
 
These data were analyzed using the statistical techniques described in this section. Those analyses of the experimental results
 

that bear on the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

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

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

missing; animals dying from natural causes were not statistically
 

censored. Statistical analyses for a possible dose-related effect on
 

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

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

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

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

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

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

histologically. However, when macroscopic examination was required
 

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

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

15
 
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
 

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

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

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

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

from linear trend.
 

16
 
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
 

17
 
the exact interval on the odds ratio (Gart, 1971). The relative risk
 

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

t c t
 

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

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

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.
 

18
 
III. CHRONIC TESTING RESULTS: RATS
 

A. Body Weights and Clinical Observations
 

Dose-related mean body weight depression was apparent in both
 

male and female rats throughout the bioassay (Figure 2).
 

During the first 30 weeks of the study, appearance and behavior
 

of the treated rats were generally comparable with those of the untreated controls. As the study progressed (from week 30 until cessation of chemical administration in week 78), a hunched appearance was
 

observed in a slightly greater number of treated rats than untreated
 

controls. During the subsequent observation period, this characteristic was noted in comparable numbers of treated and control animals.
 

Respiratory signs involving labored respiration, wheezing, and/or
 

nasal discharge were noted at a low incidence in all groups during the
 

study.
 

Clinical signs often associated with aging in laboratory rats
 

were observed at a comparable rate in control and treated animals
 

during the second year of the study. These included sores on the
 

body, abdominal urine stains, rough fur, localized alopecia, eyes
 

reddened or discharging, swollen areas of the body or bloating, and
 

tissue masses or palpable nodules. Isolated observations noted in
 

one to three rats during the study included circling, salivation,
 

undersized gonads, red vaginal discharge, and ataxia.
 

19
 
750­

600­

IT 

450­

150­

MALE RATS 

T~ 

15 30 45 60 

TIME ON TEST (WEEKS) 

90 

_ CONTROL 

... LOW DOSE 

. «. HIGH DOSE 

105 

TIME ON TEST (WEEKS) 

FIGURE 2 

GROWTH CURVES FOR DICOFOL CHRONIC STUDY RATS 

20 

•750 

-600 

— 450 

— 300 

-150 

120 

750 

- 600 

r-450 

-300 

-150 

120 
B.	 Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and dicofol-dosed groups are shown in Figure 3. For
 

both male and female rats there was no significant positive association between dosage and mortality.
 

Adequate numbers of male rats were at risk from late-developing
 

tumors as 72 percent (36/50) of the high dose, 64 percent (32/50) of
 

the low dose, and 55 percent (11/20) of the control group survived at
 

least 100 weeks. For female rats the survival was also adequate as
 

88 percent (44/50) of the high dose, 92 percent (46/50) of the low
 

dose, and 80 percent (16/20) of the control group survived at least
 

100 weeks.
 

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).
 

The types of tumors represented have been encountered previously
 

as naturally occurring lesions in the Osborne-Mendel rat and were
 

without apparent relationship to the administration of the chemical.
 

The incidences of inflammatory, degenerative, and proliferative
 

lesions were similar in treated and control animals and were consistent with spontaneous lesions found in untreated aged Osborne-Mendel
 

rats.
 

21
 
PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVAL 

p p p p b 01 bo b 

I 

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§ I v> 

? s 

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o 

o 

CO 

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CO 

\ ~ p p p p b o> bo b b 
This histopathlogic examination provided no evidence for the carcinogenicity of dicofol in Osborne-Mendel rats.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

tumors were observed in at least one of the control or dicofol-dosed
 

groups and where such tumors were observed in at least 5 percent of
 

the group.
 

For rats of both sexes none of the statistical tests indicated a
 

significant positive association between the administration of dicofol
 

and the incidence of any tumor. Thus, at the dose levels used in this
 

experiment there was no convincing statistical evidence that dicofol
 

was a carcinogen in Osborne-Mendel rats.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

Tables 3 and 4, the value one is included; this indicates the absence of statistically significant results. It should also be noted
 

that all of the confidence intervals have an upper limit greater than
 

one, indicating the theoretical possibility of tumor induction in
 

rats by dicofol that could not be established under the conditions of
 

this test.
 

23
 
TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH DICOFOL3
 

TOPOGRAPHY: MORPHOLOGY
 

Hematopoietic: Malignant Lymphoma
 

P Values0
 

Relative Risk (Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

(S3
 

-O	 Pituitary: Chromophobe Adenoma
 

P Values0
 

Relative Risk (Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Carcinoma
 

P Values0
 

Relative Risk (Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

0/20(0.00)
 

N.S.
 

6/18(0.33)
 

N.S.
 

95
 

1/19(0.05)
 

N.S.
 

110
 

LOW
 

DOSE
 

4/50(0.08)
 

N.S.
 

Infinite
 

0.386
 

Infinite
 

72
 

5/28(0.18)
 

N.S.
 

0.536
 

0.157
 

1.815
 

95
 

3/31(0.10)
 

N.S.
 

1.839
 

0.164
 

93.219
 

83
 

HIGH
 

DOSE
 

3/49(0.06)
 

N.S.
 

Infinite
 

0.255
 

Infinite
 

93
 

3/17(0.18)
 

N.S.
 

0.529
 

0.102
 

2.049
 

104
 

3/27(0.11)
 

N.S.
 

2.111
 

0.188
 

106.380
 

83 
TABLE 3 (CONCLUDED)
 

LOW HIGH 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE DOSE 

Thyroid: Follicular-Cell Adenoma 

or Follicular-Cell Carcinoma*3 1/19(0.05) 4/31(0.13) 3/27(0.11) 

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

Relative Risk (Control)d 2.452 2.111 

Lower Limit 0.272 0.188 

Upper Limit 116.423 106.380 

Weeks to First Observed Tumor 110 83 83 

Thyroid: C-Cell Adenoma or C-Cell 

Carcinoma 0/19(0.00) 3/31(0.10) 1/27(0.04) 

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

Relative Risk (Control)d Infinite Infinite 

Lower Limit 0.386 0.039 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor 105 112 

Treated groups received time-weighted average doses of 471 and 942 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 DICOFOL*
 

TOPOGRAPHY: MORPHOLOGY
 

Pituitary: Chromophobe Adenoma
 

P Values0
 

Relative. Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenoma or C-Cell
 

Carcinoma"
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

9/20(0.45)
 

N.S.
 

_—
 

84
 

1/19(0.05)
 

N.S.
 

Ill
 

3/19(0.16)
 

N.S.
 

Ill
 

LOW
 

DOSE
 

14/32(0.44)
 

N.S.
 

0.972
 

0.503
 

2.077
 

112
 

2/25(0.08)
 

N.S.
 

1.520
 

0.085
 

85.947
 

112
 

2/25(0.08)
 

N.S.
 

0.507
 

0.047
 

4.010
 

112
 

HIGH
 

DOSE
 

15/30(0.50)
 

N.S.
 

1.111
 

0.588
 

2.303
 

100
 

0/22(0.00)
 

N.S.
 

0.000
 

0.000
 

15.847
 

0/22(0.00)
 

N.S.
 

0.000
 

0.000
 

1.378
 
TABLE 4 (CONCLUDED) 

LOW HIGH 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE DOSE 

Mammary Gland: Fibroadenoma 5/20(0.25) 6/50(0.12) 5/50(0.10) 

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

Relative Risk (Control) 4.800 0.400 

Lower Limit 0.143 0.107 

Upper Limit 1.807 1.583 

Weeks to First Observed Tumor 86 102 86 

Uterus : Endometrial Stromal Polyp 2/20(0,10) 1/32(0.03) 4/31(0.13) 

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

Relative Risk (Control) 0.313 1.290 

Lower Limit 0.006 0.208 

Upper Limit 5.661 13.323 

Weeks to First Observed Tumor Ill 112 112 

Treated groups received doses of 380 and 76Q 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.
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Dose-related mean body weight depression was apparent in female
 

mice from approximately week 40 until the bioassay was terminated
 

(Figure 4). No dose-related mean body weight depression was apparent
 

in males.
 

Both the physical appearance and behavior of the treated and
 

control mice were comparable during the first 18 weeks of the study.
 

Following the dosage increases in weeks 20 and 34 of the study, a
 

hunched appearance was observed in approximately 75 percent of the
 

treated male mice. Only a few treated females and untreated control
 

males and females exhibited this sign during the first 78 weeks; however, during the last 14 to 15 weeks of the study, most of the surviving control and treated mice appeared hunched. Signs often observed
 

in laboratory mice, particularly in group-housed animals, were noted
 

at a comparable rate in control and treated animals with the incidences increasing as the animals aged. These included sores and/or
 

desquamation on parts of the body (more prevalent in males due to
 

fighting), localized alopecia, stains on fur, genital irritation,
 

palpable nodules or tissues masses, and bloated appearance.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and dicofol-dosed groups are shown in Figure 5. For
 

28
 
-40 

-30 

-20 

10 — ^____ CONTROL -10 

MALE MICE _ ^^_ HIGH DOSE 

1

 I ' I ' I 1 ' 1 ' 1 ' 1 ' 

) 15 30 45 60 75 90 105 15 '0 

TIME ON TEST (WEEKS) 

50­ -50 

40 — -40 

to 

-30 

LU 

g 

£20 - -20 

O 

CD 

CONTROL 

10 — -10 

LOW DOSE 

FEMALE MICE HIGH DOSE 

I I \ 1 I \ 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

FIGURE 4 

GROWTH CURVES FOR DICOFOL CHRONIC STUDY MICE 

29 
PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVAL 

p p p o w *> <7> 

_L 1 , 1 , 1 , 

I

o 

30 

8­

o 

o 

m 

P J o, 

0§ 

8S 

5 m m m m o ;* 

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3) 

o 

o 

CO 

o 

2

o 

8­

O — 

Ol 

m

2 

o 

m O 

m 

n~ o p o b> 
both male and female mice there was no significant positive association between dosage and mortality.
 

For male mice 76 percent (38/50) of the high dose and 76 percent
 

(38/50) of the low dose but only 35 percent (7/20) of the control
 

group survived until the end of the study. For the females the
 

survival was relatively good as 96 percent (48/50) of the high dose,
 

84 percent (42/50) of the low dose, and 95 percent (19/20) of the
 

control mice 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).
 

Hepatocellular carcinomas occurred in 3/18 (17 percent) control
 

males, 22/50 (44 percent) low dose males, 35/47 (74 percent) high
 

dose males, 1/20 (5 percent) control females, 0/44 low dose females,
 

and 0/50 high dose females. Microscopically, the hepatocellular
 

carcinomas varied greatly in appearance. Some lesions contained
 

well-differentiated hepatic cells that had a relatively uniform
 

arrangement of the cords, and others had very anaplastic liver cells
 

with large hyperchromatic nuclei, often with pseudo-inclusion bodies,
 

and with vacuolated, pale cytoplasm. Mitotic figures were often
 

present. Some of the tumors were characterized by discrete areas
 

of highly anaplastic cells. The hepatic neoplasms occurring in the
 

control mice were not different in appearance from those noted in
 

the treated mice.
 

31
 
Other neoplasms that occurred in this study were considered to
 

be lesions that occur naturally in untreated B6C3F1 mice. There were
 

no appreciable differences in frequency between the control and
 

treated groups.
 

Incidences of other inflammatory, degenerative, and proliferative lesions that occurred were without appreciable difference in
 

the control and treated mice.
 

Based upon this histopathologic examination dicofol was carcinogenic in male mice, as it was associated with an increased incidence
 

of hepatocellular carcinomas. There was no evidence of compound-


related neoplasia in the female mice.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

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

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

tumors were observed in at least one of the control or dicofol-dosed
 

groups and where such tumors were observed in at least 5 percent of
 

the group.
 

Significant numbers of liver tumors were observed in the treated
 

male mice. The Cochran-Armitage test indicated a significant (P <
 

0.001) positive association between dosage and the incidence of hepatocellular carcinomas. The Fisher exact test supported this result
 

with a significant (P < 0.001) comparison of the high dose group to
 

the control group; the comparison of the low dose group to the
 

32
 
TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH DICOFOL3
 

TOPOGRAPHY: MORPHOLOGY
 

Subcutaneous Tissue: Fibroma
 

P Values0
 

Departure from Linear Trend8
 

Relative Risk (Control)d
 

Lower Limit
 

Upper Limit
 

OJ Weeks to First Observed Tumor
 U)
 

Subcutaneous Tissue: Fibrosarcoma^
 

P Values0
 

Relative Risk (Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar Adenoma
 

P Values0
 

Relative Risk (Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

CONTROL
 

0/18(0.00)
 

N.S.
 

P = 0.006
 

2/18(0.11)
 

N.S.
 

91
 

1/18(0.06)
 

N.S.
 

83
 

LOW
 

DOSE
 

6/50(0.12)
 

N.S.
 

Infinite
 

0.605
 

Infinite
 

91
 

7/50(0.14)
 

N.S.
 

1.260
 

0.276
 

11.800
 

82
 

2/36(0.06)
 

N.S.
 

1.000
 

0.057
 

57.355
 

91
 

HIGH
 

DOSE
 

0/48(0.00)
 

N.S.
 

2/48(0.04)
 

N.S.
 

0.375
 

0.030
 

4.932
 

92
 

5/39(0.13)
 

N.S.
 

2.308
 

0.292
 

106.089
 

92
 
TOPOGRAPHY: MORPHOLOGY
 

Hematopoletic: Malignant Lymphoma
 

P Values0
 

Relative Risk (Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma or
 

Hepatocellular Adenoma^
 

P Values0
 

Relative Risk (Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 5 (CONTINUED) 

CONTROL
 

0/18(0.00)
 

N.S.
 

3/18(0.17)
 

P< 0.001
 

83
 

3/18(0.17)
 

P < 0.001
 

83
 

LOW
 

DOSE
 

1/50(0.02)
 

N.S.
 

Infinite
 

0.020
 

Infinite
 

91
 

22/50(0.44)
 

P = 0.035
 

2.640
 

0.950
 

12.504
 

61
 

23/50(0.46)
 

P = 0.025
 

2.760
 

1.000
 

13.003
 

61
 

HIGH
 

DOSE
 

3/48(0.06)
 

N.S.
 

Infinite
 

0.236
 

Infinite
 

90
 

35/47(0.74)
 

P< 0.001
 

4.468
 

1.737
 

18.987
 

64
 

36/47(0.77)
 

P< 0.001
 

4.596
 

1.797
 

19.249
 

64
 
TABLE 5 (CONCLUDED)
 

a

Treated groups received time-weighted average doses of 264 and 528 ppm in feed.
 

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

c

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

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

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

control group is given beneath the incidence of tumors in the treated group when P < 0.05;
 

otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact
 

tests a negative designation (N) indicates a lower incidence in the treated group(s) than
 

in the control group.
 

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

e

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

group when P < 0.05.
 

CO
 
TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH DICOFOL3
 

TOPOGRAPHY: MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Hematopoietic:

P Values0 

 Malignant Lymphoma 2/20(0.10) 

N.S. 

4/44(0.09) 

N.S. 

3/50(0.06) 

N.S. 

Relative Risk (Control)d 

Lower Limit 

Upper Limit 

0.909 

0.146 

9.544 

0.600 

0.076 

6.861 

Weeks to First Observed Tumor 91 62 93 

a

Treated groups received time-weighted average doses of 122 and 243 ppm in feed.
 

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

c

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

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

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

control group is given beneath the incidence of tumors in the treated group when P < 0.05;
 

otherwise, not significant (N.S.) is indicated. For both Cochran-Armitage and Fisher exact
 

tests a negative designation (N) indicates a lower incidence in the treated group(s) than
 

in the control group.
 

d

The 95% confidence interval on the relative risk of the treated group to the control group.
 
control group had a probability level of P = 0.035, a marginal result
 

which was not significant using the Bonferroni criterion. To further
 

examine these results an additional, life-table analysis was performed. Figure 6 shows the probability of survival without a known
 

hepatocellular carcinoma for male mice. The Tarone test indicated a
 

significant (P = 0.010) positive association between dosage and tumor
 

incidence.
 

Based upon these results the administration of dicofol was
 

associated with the elevated incidence of hepatocellular carcinomas
 

in male B6C3F1 mice.
 

No statistically significant positive association between dosage and tumor incidence was observed at any other site in either male
 

or female mice.
 

To provide additional insight into the possible carcinogenicity
 

of this compound, 95 percent confidence intervals on the relative
 

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

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

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

of statistically significant results. It should also be noted that
 

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

indicating the theoretical possibility of tumor induction in mice by
 

dicofol that could not be established under the conditions of this
 

test.
 

37
 
<r

 1'°­ — 1.0 

O 

Z 

3 0.8­ 0 z 

1-

o 

X 0.6­ H 

[_J 

ll

ll 

ii 

'*. 

— 0.8 

-

-0.6 

3 -

z 

-0.4 

DC 

D 

c/3 -

Z 

O i__—__^ CONTROL 

1- 0.2­ 1 — 0.2 

cc 

O LOW DOSE | 

Q. 

O 

cc MALE MICE HIGH DOSE Q. 

i i i i i i i I i i i I i | i 0 0 

15 30 45 60 75 90 105 120 

TIME ON TEST (WEEKS) 

FIGURES 

COMPARISONS OF DICOFOL CHRONIC STUDY MALE MICE SURVIVING WITHOUT 

OBSERVED HEPATOCELLULAR CARCINOMAS 
V. DISCUSSION
 

Dietary administration of dicofol was not associated with a
 

significant accelerated mortality in either sex of either species
 

although some mice died early from liver cancer. Adequate numbers of
 

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

late-developing tumors.
 

In rats the only apparent effect of dicofol administration was
 

distinct, dose-related mean body weight depression in males and females. No consistent unusual clinical observations were reported,
 

no unusual or rare neoplasms or nonneoplastic lesions were observed,
 

and none of the neoplasms that did occur were present in statistically
 

significant increased incidences when compared to controls.
 

In mice dose-related mean body weight depresion was evident in
 

treated females but not in males. There were no unusual clinical
 

observations recorded for either sex. Hepatocellular carcinomas were
 

observed in 3/18 (17 percent), 22/50 (44 percent), and 35/47 (74 percent) of the control, low dose, and high dose males, respectively,
 

and 1/20 (5 percent), 0/44, and 0/50 of the control, low dose, and
 

high dose females, respectively. Statistical analysis of the incidences of this neoplasm, using the Cochran-Armitage test and life-


table analysis, indicated a significant positive association between
 

dosage and incidence in the treated males. This finding was supported
 

by the Fisher exact comparison of high dose to control. No unusual
 

tumors were observed during the histopathologic examination, and
 

39
 
statistical significance was not attributed to the incidences of
 

other tumors.
 

Long-term ingestion of p,p'-DDE, p,p'-DDT or technical-grade
 

DDT (compounds with similar chemical structures to dicofol) has been
 

found to induce liver tumors in both sexes of several strains of
 

mice (International Agency for Research on Cancer, 1974). However,
 

in this bioassay of dicofol, these lesions were induced only in male
 

B6C3F1 mice.
 

Under the conditions of this bioassay, technical-grade dicofol
 

was carcinogenic in male B6C3F1 mice, causing hepatocellular carcinomas. No evidence for carcinogenicity was obtained for this compound
 

in Osborne-Mendel rats of either sex or in female B6C3F1 mice.
 

40
 
VI. BIBLIOGRAPHY
 

Andrilenas, P.A., Farmers' Use of Pesticides in 1971—Quantities.
 

Economic Research Service, U.S. Department of Agriculture,
 

Agricultural Economic Report No. 252, 1974.
 

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

J. Wiley & Sons, New York, 1971.
 

Ashwood-Smith, M.J., J. Trevino, and R. Ring, "Mutagenicity of
 

Dichlorvos." Nature 240:418-420, 1972.
 

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

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

Union Against Cancer, Geneva, 1969.
 

Brown, J.R., "The Effect of Dietary Kelthane on Mouse and Rat Reproduction." Fate of Pesticides in the Environment. A.S. Tahori,
 

editor. Gordon and Breach Science Publishers, New York, 1972.
 

Chemical Abstracts Service, The Chemical Abstracts Service (CAS)
 

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

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

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.
 

Diatlovitskaia, F.G., Jr., and L.E. Botvinova, Jr., "Separate Determination of DDT and Kelthane in Industrial Waste Waters."
 

Gigiena i Sanitariia 36(1):72-74, 1971.
 

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.
 

Gosselin, R.E., H.C. Hodge, R.P. Smith, and M.N. Gleason, Clinical
 

Toxicology of Commercial Products, 4th edition. The Williams
 

and Wilkins Company, Baltimore, Maryland, 1976.
 

Innes, J.R.M., B.M. Ulland, M.G. Valerio, L. Petrucelli, L. Fishbein,
 

E.R. Hart, A.J. Pallotta, 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(6);1101-1114,
 

1969.
 

41
 
International Agency for Research on Cancer (IARC), IARC Monographs
 

on the Evaluation of Carcinogenic Risk of Chemicals to Man.
 

Volume 5, Some Organochlorine Pesticides. World Health Organization, IARC, Lyon, France, 1974.
 

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

Observations." Journal of the American Statistical Association
 

53^:457-481, 1958.
 

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.
 

Manske, D.D., and P.E. Corneliussen, "Pesticide Residues in Total Diet
 

Samples (VII)." Feticides Monitoring Journal 8(2):110-124, 1974.
 

Martin, H., and C.R. Worthing, Pesticide Manual. British Crop Protection Council, 1977.
 

Miller, R.G., Simultaneous Statistical Inference. McGraw-Hill Book
 

Co., New York, 1966.
 

Ouellette, R.P., and J.A. King, Chemical Week Pesticides Register.
 

McGraw-Hill Book Company, New York, 1977.
 

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.
 

Stuneeva, G.I., "Sanitary-Hygienic Conditions of Use and Toxicological
 

Properties of a Mixture of Kelthane, Chlorophos, and Copper
 

Oxychloride." Gigiena i Sanitariia (8):101~103, 1973; Chemical
 

Abstracts 80, 598c.
 

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

6^:679-682, 1975.
 

U.S.	 Department of Agriculture, The Pesticide Revue. USDA, Washington,
 

D.C., 1975.
 

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

Production and Sales, 1975. USITC Publication No. 804, U.S.
 

Government Printing Office, Washington, D.C., 1977.
 

42
 
Yoder, J., M. Watson, and W.W. Benson, "Lymphocyte Chromosome Analysis
 

of Agricultural Workers During Extensive Occupational Exposure to
 

Pesticides." Mutation Research 21:335-340, 1973.
 

43
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH DICOFOL
 

TABLE Al

 

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

ANIMALS INITIALLY IN STUDY
 

ANIMALS KECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY**
 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE
 

FIBROMA
 

FIBBOSABCOMA
 

LIPOMA
 

BESPIEAICRY SYSTEM
 

*LUNG
 

CORTICAL CARCINOMA, METASTATIC
 

OSTEOSARCOMA, HETASTATIC
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS
 

BALIG.LYHPHOMA. LYMPHOCYTIC TYPE
 

MALIG.LYMPHOMA, HISIIOCYTIC TYPE
 

tSPLEEN
 

HEMANGIOSARCOMA
 

*LIVER
 

BALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

CIRCULATOR Y SYSTEM 

NON E 

DIGESTIVE SYSTEM 

tSALIVARI GLAN D 

CONTBOL(VEH)
 

01-M038
 

20
 

20
 

19
 

(20)
 

1 (5*)
 

1 (5X)
 

(19)
 

1 (5X>
 

(20)
 

(19)
 

(19)
 

(8) 

LOB DOSE HIGH DOSE
 

01-M039 01-M040
 

50 50
 

50 49
 

49 47
 

(50)	 (49)
 

1 (2X)
 

1 (2X)
 

1 (2*) 1 (2X)
 

(40)	 (38)
 

1 (3X)
 

(50) (49)
 

1 (2X)
 

1 (8*) 1 (2X)
 

(25)	 (19)
 

2 (8X)
 

(42)	 (40)
 

1 <3X)
 

(16) 

* NUMBER OF ANIMAL S KITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMALS NECBOPSIEO 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-3 
TABLE Al (CONTINUED)
 

CONTROL (VEH) LOU DOSE HIGH DOSE
 

01-M038 01-M039 01-MOUO
 

#LIVER (19) («2) (40)
 

HEPATOCELLULAB CABCINOMA 1 (2X) 1 (3X)
 COBTICAL CARCINOMA, METASTATIC 1 (3X)
 

UBINABY SYSTEM
 

tKIDNEY (19) (00) (18)
 MIXIC TUMOfi, MALIGNANT 1 (3X)
 

OURINARY BLADDER (18) (20) (11)
 PAPILLOMA, NOS 1 (7X)
 

ENDOCRINE SYSTEM
 

tPITUITARY (18) (28) (17)
 CHRCMOPHOBE ADENOMA 6 (33X) 5 (18X) 3 (18X)
 

IADRENAL (19) (25) (20)
 CORTICAL CABCINOMA 1 <5X)
 

*THYROID (19) (31) (27)
 

FOLLICULAR-CELL ADENOMA 1 <3X)
 FOLLICULAR-CELL CARCINOMA 1 (5X) 3 (10X) 3 (11X)
 

C-CILL ADENOMA 2 (6X>
 C-CELL CARCINOMA 1 <3X) 1 (IX)
 

#PANCREATIC ISLETS (19) (25) (11)
 ISLET-CELL ADENOMA 1 (5X) 1 (IX)
 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND (20) (50) (19)
 ADENCCARCINOHA, NOS 2 (<»X) 1 (2X)
 

tPROSTATE (11) (D
 FIBROMA 1 (7X)
 

NERVOUS SYSTEM
 

*BRAIN (19) (23) __ (16__
 ) E.PENDYMD8A 1-J4S1 .
 

» NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBEB OF ANIMALS NECBOPSIED
 

A-4
 
TABLE Al (CONTINUED) 

CONTROL (VEH) LOU DOSE HIGH DOSE 

01-H038 01-H039 01-HOUO 

ASTBOCYTOHA 1 (5X) 

SPECIAL SENSE OBGAMS 

NOME 

MOSCULOSKELETAL SYSTEM 

'VERTEBRAL COLUM N (20) (50) (49) 

OSTEOSARCOMA 1 (5X) 

*MUSCLE OF HEAD (20) (50) (U9) 

SQUAMOH S CELL CARCINOMA 1 (2%) 

*HUSCIE OF MECK (20) (50) (49) 

FIBBOSABCOHA 1 (2%) 

BODY CAVITIES 

*ABDOMINA L CAVITY (20) (50) (»9) 

LltOMA 1 (2%) 

NIXED TUMOR, MALIGNAN T 1 (2X) 

ALL OTHER SYSTEMS 

NON E 

ANIMAL DISPOSITION SUMMARY 

ANIMAL S INITIALLY IN STUDY 20 50 50
 

NATUBA L DEATHS 12 27 21
 

MORIBUN D SACRIFICE 

SCHEDULED SACRIFICE 

ACCICENTALLY KILLED 

TEBMINAL SACRIFICE 23 29
 

ANIMA L MISSING 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBEB OF ANIMALS NECBOPSIED
 

A-5
 
TABLE Al (CONCLUDED)
 

crONTROL (VEH) LOW DOSE HIGH DOSE 

01-M038 01-M039 01-MOtO 

TUMOR SUMMAR Y 

TOTAL ANIMALS HITH PRIMARY TUMORS* 10 23 16

 

TOTAL PRIHARY TUMORS 13 30 17

 

TOTAL ANIMALS KITH BENIGN TUMORS 8 9 6

 

TOTAL BENIGN TUMORS 9 11 6

 

TOTAL ANIMALS HITH MALIGNANT TUMORS u 16 10

 

TOTAL MALIGNAN T TUMORS a 19 11

 

TOTAL ANIMALS WITH SECONDARY TUMORS * 1 1 

TOTAL SECONDAR Y TUMORS 1 2 

TOTAL ANIMALS HITH TUMORS UNCE8TAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TOHORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

A-6
 
TABLE A2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED WITH DICOFOL 

c:ONTKOL (VEH ) LOU DOSE HIG H DOSE 

01-F038 01-F075 01-F076 

ANIMALS INITIALLY IN STUD Y 20 50 50 

ANIMALS NECROPSIED 20 50 50 

ANIMALS EXAMINE D HISTOPATHOLOGICALLY ** 20 <)9 49 

INTEGUMENTAR Y SYSTEM 

*SOBCUT TISSUE (20) (50) (50) 

CYSTADENOCA8CINOMA , NOS 1 (2%) FIBROMA 1 (5%) 

HEMANGIOSABCOM A 1 (5%) 1 (2%) 

BESPIBA1C8Y SYSTEM
 

NONE
 

HEBATOPCIETIC SYSTEM
 

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

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 2 (1%) 1 (2»)
 

IOVARY (20) (23) (23)
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (KX)
 

CIBCULATCHY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

fPANCHEAS	 (20) (23) (19) 

CYSTADENOCAHCINOMA , METASTATIC 1 ("»%) 

UBINABY SYSTEM
 

tKIDNEY (20) (23) (20)
 ____ HAMARTOMA^
 

# NUMBER OF ANIHALS WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

+ THIS IS CONSIDERED TO BE A BENIGN FORM OF THE MALIGNANT MIXED TUMOR OF THE KIDNEY AND CONSISTS OF PROLIFERATIVE LIPOCYTES, TUBULAR STRUCTURES, FIBROBLASTS, AND VASCULAR SPACES IN 

VARYING PROPORTIONS. 

A-7
 
tURINABY BLADDER
 

LEIGMYOSARCOMA
 

ENDOCRINE SYSTEM
 

IPITUITARY
 

CHROBOPHOBE ADENOMA
 

•ADRENAL
 

CORTICAL ADENOMA
 

CORTICAL CARCINOMA
 

•THYROID
 

FOLIICULAfi-CELL CARCINOMA
 

C-CELL ADENOMA
 

C-CEIL CARCINOMA
 

tPANCREATIC ISLETS
 

ISLIT-CELL ADENOMA
 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND
 

ADENCMA, NOS
 

ADENCCABCINOMA, NOS
 

FIBROADENOMA
 

•VAGINA
 

LEICMYOSARCOMA
 

#UTERUS
 

ADENCMA, NOS
 

ENDCMETBIAL STROMAL POLYP
 

tOVARY
 

CYS1ADENOCAECINOMA, NOS
 

GRANULOSA-CELL TUMOR
 

NERVOUS SYSTEM
 

tBRAIN
 

EPENDYMOMA
 

SPECIAL SENSE ORGANS
 

MONE
 

TABLE A2 (CONTINUED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

01-F038 01-F075 01-F076 

(20) (22) (2C) 

1 (5%) 

(20) (32) (30) 

9 (i»5X) 11 (HUX) 15 (SOX) 

(20) (23) (22) 

1 (i»») 

1 (4%) 1 (5X) 

(19) (25) (22) 

1 (5X) 

2 (11X) 

1 (S*) 2 (8*) 

(20) (23) (19) 

1 (4X) 

(20) (SO) (50) 

1 (2X) 1 (2%) 

1 (5*) 

5 (25%) 6 (12X) 5 (10X) 

(20) (SO) (50) 

1 (2%) 

(20) (32) (31) 

1 i-X) 

2 (10X) 1 (3X) 4 (13X) 

120) (23) (23) 1 (4%) 

1 (4%) 

(20) (23) (19) 

1 (5X) 

* NUMBER OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECEOPSIED
 

A-8
 
TABLE A2 (CONTINUED 

CONTBOL (VEH) tO» DOSE HIGH DOSE 

01-P038 01-F075 01-F076 

MUSCUIOSKELETAL SYSTEM 

BODY CAVITIES 

NON E 

ALL OTHER SYSTEMS 

*MULTIPIE ORGAN S (20) (50) (50) 

HEMANGIOSARCOMA 1 (2X) 

ANIMAL DISPOSITION SUMMAR Y 

ANIMAL S INITIALLY IN STDDY 20 50 50 

NATURA L DEATHS 8 7 9 

MORIBUN D SACRIFICE 

SCHEDULED SACRIFICE 

ACCIEEHTALLY KILLED 

TERMINAL SACRIFICE 12 13 41
 

ANIMAL HISSING
 

I NUMBER OF ANIMALS WITH TISSUE EXAMINED MICEOSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

A-9
 
TABLE A2 (CONCLUDED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

01-F038 01-F075 01-F076 

1UHOR SUMMARY
 

TOTAL ANIMALS KITH PRIMARY TUHORS* 16 28 28
 

TOTAL PRIMARY TUHORS 23 33 36
 

TOTAL ANIMALS HUH BENIGN TUHORS 15 22 22
 

TOTAL BENIGN TUMORS 20 25 27
 

TOTAL ANIMALS KITH MALIGNAN 3 T TUMORS 8 7
 

TOTAL MALIGNANT TUMORS 3 8 8
 

TOTAL ANIMALS HITH SECONDARY TUMORSt 1
 

TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS WITH TUMOBS UNCERTAINBENIGN OR MALIGNANT 1
 

TOTAL UNCERTAIN TUHOES 1
 

TOTAL ANIMALS WITH TUMOBS UNCERTAINPRIMARY OR HETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUHORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 * SECONDARY TUMORS: HETASTATIC TUMORS OR TUMOBS INVASIVE INTO AN ADJACENT ORGAN
 

A-IO
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH DICOFOL
 

TABLE Bl

 

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

CONTROL (VEH) LOH DOSE HIGH DOSE 

02-M032 02-M033 02-M034 

ANIMALS INITIALLY IN STUDY 20 50 50 

ANIMALS BISSING 2 1 

ANIMAL S SECROPSIED 18 50 48 

ANIMALS EXAMINE D HISTOPATHOLOGICALLY** 18 48 47 

INTEGUMENTAR Y SYSTEM 

*SKIN (18) (50) (48) 

SQUAMOU S CELL CAfiCINOMA 1 (2X) 

SEBACEOUS ADENOMA 1 (6X) 

*SUBCUT TISSUE (18) (50) (48) 

FIBECMA 6 (12X) 

FIBROSARCOMA 2 (11X) 7 (14X) 2 (IX) 

RESPIRATORY SYSTEM 

»LUNG (18) (36) (39)
 

ALVEOLAE/BRONCHIOLAB ADENOMA 1 (6X) 2 (6X) 5 (13X)
 

HEMATOECIETIC SYSTEM
 

*MULTIFLE ORGANS (18) (50) (48)
 

MALIG.LYMPHOMA, HISTIOCY1IC TYPE 1 (2X) 3 (6X)
 

CIRCULA1CEY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVER (18) (50) (47)
 

HEPATOCELLULAR ADENOMA 1 (2X) 1 (2X)
 

HEPATOCELLULAH CARCINOMA 3 (17X) 22 (44X) 35 (74X)
 

ISTOHACH (18) (31) (39)
 

SQUAMOUS CELL CARCINOMA 1 (3X)
 

URINARY SYSTEM
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMALS NECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-3 
ENDOCRINE SYSTEM
 

NONE
 

BEPRODUC1IVE SYSTEM
 

NONE
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MIJSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDOMINAL CAVITY


LIPCBA


ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY


NATURAL DEATHS


MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE


ANIKAL MISSING


a_ISCLU£IS_AUTOLYZED_ANIMALS
 

TABLE Bl (CONTINUED)
 

CONTROL(VEH)


02-H032


 (18)


 20


 11


 7


 2


 LOW DOSE HIGH DOSE
 

 02-M033 02-MC34
 

 (50)	 (U8)
 

1 (2X)
 

 50 50
 

 12 11
 

 38 38
 

1
 

I	 NUMBER OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

B-4
 
TABLE Bl (CONCLUDED)
 

CONTROL (VEH) LOB DOSE HIGH DOSE 

02-8032 02-M033 02-M03* 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 3<* 38
 

TOTAL PRIMARY TUMORS 11 1(7
 

TOTAL ANIMALS WITH BENIGN TUMORS 9
 TOTAL BENIGN TUMORS 10
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 28 37
 TOTAL MALIGNANT TUMORS 31
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMOBS
 

TOTAL ANIHALS WITH TUMORS UNCERTAIN ­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

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

B-5
 
TABLE B2

 

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

CONTROL (VEH)

02-F032

 LOW DOSE 

 02-P035 

ANIMALS INITIALLY IN STUD* 20 ANIMALS HISSING

ANIMALS NECROPSIED 20 ANIMALS EXAMINED HISTOPATHOLOGICALLY** 20

 50 

2 

U<i 

UH 

INTEGUBEKTABY SYSTEB
 

*SUBCUT TISSUE (20) (It)
 

FIBBCSAECOMA 1 (2%)
 LIPOSARCCBA 1 (2X)
 

RESPIRATORY SYSTEM
 

KLUNG (20) (19)
 

ALVEOLAH/BBONCHIOLAB CARCINOMA 1 (5X)
 

HEMATOECIETIC SYSTEM
 

*MULTIP1E ORGANS (20) (14)
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (5%)
 

(1ALIG.LYMPHOMA, HISTIOCYTIC TYPE 2 (5X)
 

(SPLEEN (20) (16)
 

HEMANGIOSASCOMA
 

BALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (5fc)
 

IPANCREATIC L.NODE (20) (15)
 

SQUABOUS CELL CARCINOMA, METASTA
 MALIG.LYMPHOMA. HISTIOCYTIC TYPE 1 (7)6)
 

IBESENTERIC L. NODE (20) (15)
 

SUUAMOUS CELL CARCINOMA, METASTA
 MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

• LIVER (20) (tl)
 

MALIG.LYMPHOBA, HISTIOCYTIC TYPE 1 (2X)
 

CIRCULATORY SYSTEM
 

NONE
 

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

» NUMBE R OP ANIMAL S NECBOPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

HIGH DOSE
 

02-F036
 

50
 

50
 

50
 

(50)
 

(19)
 

(50)
 

1 <2X)
 

1 (2X)
 

(18)
 

1 (6*1
 

(19)
 

1 (5«)
 

(19)
 

1 (5S)
 

1 (5*)
 

(50)
 

B-6 
TABLE B2 (CONTINUED)
 

CONTROL (VEH) LOB DOSE HIGH DOSE
 02-F032 02-F035 02-F036
 

DIGESTIVE SYSTEM
 

tLIVER (20) (It) (50)
 

HEPATOCELLULAB CABCINOMA 1 (5X)
 

UBINABY SYSTEM
 

NONE
 

ENDCCEIBE SYSTEM
 

tPITDITABY (20) (1<t) (15)
 

CHBOHOPHOBE ADENOHA 1 (7%)
 

REPRODUCTIVE SYSTEM
 

*t)ARMARI GLAND (20) («<*) (50)
 

ADENOCABCINOMA, NOS 1 (51)
 

IUTERUS (20) (26) (23)
 

SQUABOUS CELL CABCINOHA 1 (4X)
 

HEHANGICMA 1 (UX)
 

iOVARY (20) (17) (25)
 CYSIADENOHA, DOS 1 (U»)
 

HEBVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

HUSCULCSKBLETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

* NUHBEfi OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECBOFSIED
 

B-7
 
ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUKMARY
 

ANIMALS INITIALLY IN STUDS
 NATURAL DEATHS


MORIBUND SACRIFICE
 SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE


ANIMAL MISSING


a INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TABLE B2 (CONCLUDED)
 

CONTBOL (VEH)


02-F032


 20
 1


 19


TOTAL ANIMALS WITH ERIMABY TUMORS*

TOTAL PRIMARY TUMORS

 5 5 

TOTAL ANIMALS WITH BENIGN TUMORS

TOTAL BENIGN TUMORS

TOTAL ANIMALS WITH MALIGNANT TUMORS TOTAL MALIGNANT TUMORS 5 5 

TOTAL ANIMALS HITH SECONDARY TUMORS*

TOTAL SECONDARY TUMORS

TOTAL ANIMALS KITH TUMORS UNCERTAINBENIGN OR MALIGNANT TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

 LOW DOSE 02-F035 HIGH DOSE 02-F036 

 50

 6 50 2 

 42

 2 

 13 

6 

6 

6 

6 

8 

8 

3 

3 

5 

5 

1 

2 

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

B-8
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH DICOFOL
 

TABLE Cl

 

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

CONTROL (VEH)

01-M038

 LOU DOSE

 01-M039

 HIGH DOSE 

 01-MOU O 

ANIMALS INITIALLY IN STUDY

ANIMALS SECBOPSIED

ANIMALS EXAMINED HISTOPATHOLOGICALLY**

 20

 20

 19

 50

 50

 t9

 50 

 19 

 47 

INTEGUMENTAR Y SYSTEM 

*SKIN

HYPERKERATOSI S

ACANTHOSIS

 (20)

 1 (5X) 

1 (5X) 

 (50) (<»9) 

*SUBCUT TISSUE

ABSCISS, NOS

 (20)

 1 (5X) 

 (50) (49) 

BESPIBATCR Y SYSTEM 

#LUNG

INFLAMMATION, NOS

PNEUMONIA, CHRONIC MURINE

CALCIUM DEPOSIT

 (19)

 6 (32X)

 (HO)

 12 (30X)

 (38) 

1 (3X) 

 13 (3UX) 

1 (3X) 

HEMATOPCIETIC SYSTEM 

*BONE MARROW

METAMOBPHOSIS FATTY

 (19)

 1 (5X)

 (21) (1U) 

1 (75^ 

*SPLEEN

INFLAMMATION, NOS

HYPERTROPHY, NOS

HEMATOPOIESIS

 (19)

 3 (16S)

 (25)

 1 (tX) 

 (19) 

1 (5)1) 

1 (5X) 

CIRCUIAIORY SYSTEM 

tHEART

CALCIUM DEPOSIT

 (19) (23)

 1 (IX)

 (18) 

1 (6X) 

iMYOCARDIU H (19) (23) (18) 

* NUMBE 8 OF ANIMAL S WITH TISSUE EXAMINE D

* NUMBER OF ANIHALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

 MICROSCOPICALLY 

C-3 
TABLECl (CONTINUED) 

CONTROL (VEH) LOU DOSE
 

01-M038 01-M039
 

IENDOCARDIOM (19) (23)
 

HYPEHPLASIA, NOS 1 (4X)
 

*AORIA (20) (50)
 ARTERIOSCLEROSIS, NOS 3 <6X)
 

DIGESTIVE SYSTEM
 

*LIVER (19) (42)
 METAMORPHOSIS FATTY 3 (16*) 6 (11*)
 HYPEBPLASIA. NOS 1 (5X)
 

tPANCREAS (19) (25)
 PEBIABTEEITIS 1 (UX)
 

•STOMACH	 (19) (28)
 INFLAMMATION, NOS 1 (5X)
 ULCEB, FOCAL 1 (5X)
 CALCIUM DEPOSIT 3 (11X)
 

tCOLON (19) (21)
 PARASITISM 1 (5)1)
 

URINARY SYSTEM
 

tKIDNEY (19) (10)
 PYELONEPHRITIS, NOS 1 (3X)
 INFLAMMATION, CHRONIC 11 (58%) 18 (45«)
 CALCIUH DEPOSIT 3 (8X)
 

#URINARY BLADDER (18) (20)
 

CALCULUS, NOS
 

INFLAMMATION, NOS 1 (6X)
 

ENDOCRINE SYSTEM 

tPITUUARY (18) (28) 

CYST, NOS 2 (7X) HYPEBPLASIA, NOS 1 (UX) 

•ADRENAL 

ANGIECTASIS . . (13) (25) 

. 1 J«X1 

# NUMBER OF ANIMALS WITH TISSUE EXAMINSD MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECHOPSIED
 

HIGH DOSE
 

01-B040
 

(18)
 

3 J17X)
 

(49)
 

2 (UX)
 

(40)
 

8 (20X)
 

(14)
 

1 <7X)
 

(30)
 

3 (10X)
 

(15)
 

(18)
 

1 (6X)
 

8 (44X)
 

(14)
 

1 (7%)
 

1 (7X)
 

(17)
 

(20)
 

04
 
#THYROID


CIST, NOS


HYPERPLASIA, C-CELL


tPARATHYROID


HYPEBPLASIA. NOS


REPRODUCTIVE SYSTEM
 

ITESTIS


ATROPHY, NOS


*EPIDIDI11IS


ATROPHY, NOS


NERVOUS SYSTEM
 

*BRAIN


GLIOSIS


SPECIAL SENSE ORGANS
 

*EYE


CATABACT


MUSCULCSKELZTAL SYSTEM 

NON E 

BODY CAVITIES 

NON E 

ALL OTHER SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUMMAR Y 

fiO_£ESION_RE.PORTED 2

I NUMBER OF ANIMAL S WITH TISSUE

* NUMBE R OF ANIMALS NECROPSIED 

TABLECl (CONTINUED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

01-M038 01-M039 01-M040 

 (19)	 (31) (27)
 

7 (23X) 7 (26X)
 

1 (3X)
 

 (19)	 (24) (17)
 

4 (17X) 1* (24X)
 

 (19)	 (30) (17)
 

7 (37X) 14 (47S) U (24X)
 

 (20)	 (50) (49)
 

1 (5X)
 

 (19)	 (23) (16)
 

1 (6X)
 

 (20)	 (50) (49)
 

1 (2X) 2 (4X)
 

, 3 

 EXAMINE D MICROSCOPICALLY 

C-5 
TABLE Cl (CONCLUDED) 

CONTROL (VEH ) LOH DOSE HIG H DOSE 

01-8038 01-8039 01-MOU O 

NECROPSY PERF/NO HISTO PEBFOHHED 1 

AUTC/NECROPSY/N O HISTO 1 2 

AUTOLYSIS/NO NECROPSY 1 

C-6 
TABLE C2

 

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

CONTBOL(VEH)

01-P038

ANIMALS INITIALLY IN STUDY 20

ANIMALS NECROPSIED 20

AN I MA IS EXAMINED HISTOPATHOLOGICALLY** 20

INTEGUMEHTABY SYSTEH 

NONE 

RESPIRATORY SYSTEH 

*LUNG

INFLAMMATION , NOS

PNEUMONIA, CHBONIC HURINE

HBMATOICIETIC SYSTEM 

*BONE HARROW


METAMORPHOSIS FATTY


• SPLEEN


HEMATOPOIESIS


*»ESENTERIC L. HODE
 CYST. NOS


CIRCULATORY SYSTEH
 

tHYOCARDIUH
 DEGENERATION. SOS


• ENDOCARDIUM
 HYPERPLASIA, NOS


*CORONARY ARTERY
 INFLAMMATION, NOS


DIGESTIVE SYSTEH
 

fLIVEH


* NUHBER OF ANIMALS KITH TISSUE

* NUHBER OF ANIMAL S NECBOPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

(20)

 1 (5X) 

9 (45X)

 (20)


 2 (10*)


 (20)


 1 (5X)


 (19)


 (20)


 (20)


 (20)


 (20)


 EXAMINE D MICROSCOPICALLY 

 LOB DOSE HIGH DOSE 

 01-F075 01-F076 

 SO SO 

 50 SO 

 49 49 

 (34) (24) 

5 (15X) 10 (42X) 

 (23)	 (19)
 

1 (5X)
 

 (26)	 (20)
 

3 (12X) 1 (5X)
 

 (22)	 (19)
 

1 (5X)
 

 (24)	 (21)
 1 (4X) 2 (10X)
 

 (24)	 (21)
 

1 (5X)
 

 (50)	 (50)
 

1 (2X)
 

 (41) (40)
 

C-7
 
" "
 

METAMORPHOSIS FATTY
 

HYPEBPLASIA, NOS
 

tSTOMACH
 

ULCER, FOCAL
 

URINARY SYSTEM
 

*KIDNEY
 

PYELONEPHRITIS, NOS
 

INFLAMMATION, CHRONIC
 

tURINARY BLADDER
 

INFLAMMATION, NOS
 

ENDOCRINE SYSTEM
 

KPITUITARY
 

CYST, NOS
 

tADRENAL
 

ANGIECTASIS
 

tTHYROID
 

CYST, NOS
 

BEPROCUC1IVE SYSTEM
 

*VAGINA
 

INFLAMMATION, NOS
 

POLYP
 

« UTERUS
 

HYDBCMETfiA
 

INFLAMMATION, NOS
 

tUTERUS/ENDOMETRIUfl
 

INFLAMMATION, NOS
 

HYPEBPLASIA, CYSTIC
 

tOVARY
 

CYST, NOS
 

NEEVOBS SYSTEM
 

TABLE C2 (CONTINUED) 

CONTROL (VEH)
 

01-F038
 

2	 (10X)
 

(20)
 

1 (5X)
 

(20)
 

2 (10X)
 

1 (5X)
 

(20)
 

1 (5X)
 

(20)
 

1 (5X)
 

(20)
 

(19)
 

(20)
 

1 (5X)
 

(20)
 

1 (5*)
 

(20)
 

1 (5X)
 

3 (15X)
 

(20)
 

1 (5%)
 

LOH DOSE HIGH DOSE
 

01-F075 01-F076
 

2 (5X)
 1 (3X)
 

(24)	 (25)
 

1 (4X)
 

(23) (20)
 

1 (5X)
 5 (22X) 2 (10X)
 

(22) (20)
 

(32) (30)
 

(23)	 (22)
 

2 (9X) 2 (9X)
 

(25)	 (22)
 

2 (8X) 1 <5X)
 

(50) (50)
 

2 (4X)
 

(32)	 (3D
 6 (19X) 6 (19X)
 

2 (6X)
 

(32) (31)
 

1 <3X)
 

3 (9X)
 

(23)	 (23)
 

1 («X) 4 (17X)
 

 NUMBER OF ANIMALS MITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE B OF ANIMALS NECEOPSIED 

I
TABLE C2 (CONCLUDED) 

CONTROL (VEH) LO» DOSE HIGH DOSE 

01-F038 01-F075 01-F076 

SPECIAL SENSE ORGANS
 

NCNE
 

MUSCULCSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*ABDOHIBAL CAVITY (20) (50) (50)
 

ABSCESS, NOS 1 (5X)
 

ALL OTHI8 SYSTEHS
 

NONE
 

SPECIAL KCHPHOLOGY SUMMABY
 

NO LISION EEPOBTED 13 8
 

NECROPSY PERF/NO HISTO PERFORMED 1 1
 

« NUMBER OF ANIMALS 1IITH TISSOE EXA9INED MICROSCOPICALLY
 

* NUBBEB OF ANIMALS NECROESIED
 

C-9
 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH DICOFOL
 

TABLE Dl

 

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

CONTROL (VEH) LOW DOSE HIG H DOSE 

02-M032 02-H033 02-M034 

ANIMAL S INITIALL Y IN STUDY 20 50 50 

ANIMAL S BISSING 2 1 

ANIMAL S NECROPSIED 18 50 48 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY ** 18 1*8 U7 

INTEGUMENTAR Y SYS1EM 

*SKI» (18) (50) (48) 

EPIDERMAL INCLUSION CYST 2 (11%) 

INFLAMMATION , NOS 1 (6%) 

RESPIRATORY SYSTEM 

#LUNG (18) (36) (39)
 

PNEUMONIA, CHRONIC MUEINE 1 (3%)
 

HEMATOEOIETIC SYSTEM
 

tSPLEEN (16) (36) (40)
 

AMYLOIDOSIS 5 (31S) 3 (8%)
 

HEMATOPOIESIS 2 (5X)
 

CIRCUIA1CRY SYSTEM
 

• MYOCARDIUM (18) (31) (36)
 

INFLAMMATION, NOS 1 (6%)
 

• ENDOCARDIUM (18) (31) (36)
 

INFLAMMATION, NOS 1 (6%)
 

*AORTA (18) (50) (48)
 

PEBIARTERITIS 1 (6%)
 

DIGESTIVE SYSTEM
 

*1IVEB (18) (50) (47)
 

IHROMBOSJSX_NOS J_16S1 

t NUMBER OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROESIE D 

"EXCLUDES PARTIALLY MJTOLYZED ANIMALS 

D-3 
TABLEDl (CONTINUED) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

02-M032 02-B033 02-M03U 

INFLAMMATION , NOS 1 (2X) 2 (4X) 

HYPEBPLASIA, NODULA B 1 (2X) 2 (1%) 

*PANCREAS (18) (31) (36)
 

CYSTIC DUCTS 1 (3%)
 

AT8CEHY, NOS 1 (3X)
 

*REC1UM (18) (50) (48)
 

PBOLAPSE 2 (4X)
 

URINARY SYSTEM
 

tKIDNEY (18) (36) (141)
 

HYCHONEPHHOSIS 1 (6X) 1 (3X)
 

PYELCNEPHBITIS. NOS 1 (6X)
 

INFLAMMATION, CHRONIC 8 (UHJS) 6 (17X)
 

AMYLCIDOSIS 5 (28X) 1 (3X)
 

• URINARY BLADDER (17) (30) (38)
 

INFLAMMATION, BOS 1 (3X)
 

ENDOCRINE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEH
 

*MAMMABY GLAND (18) (50) (18)
 CYST, KOS 1 <6X)
 

»PREPU1IAL GLAND (18) (50) (48)
 ABSCESS, NOS 1 (2X)
 

tPROSTATE (18) (30) (36)
 INFLAMMATION, NOS 1 (6X)
 

*SEMINAL VESICLE (18) (50) (48)
 

INFLAMMATION, NOS 1 (6X)
 

NEEVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE.
 

* NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIHALS NECHOPSIED
 

D-4
 
TABLEDl (CONCLUDED) 

CONTROL(VEH) 

02-H032 

LOU DOSE 

02-8033 

HIGH DOSE 

02-H034 

HUSCULOSKEIETAL SYSTEH 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEHS 

NONE 

SPECIAL (OREHOLOGY SUMMARY 

NO LESION EEFOBTED 

ANIMAL HISSING/HO NECROPSY 

NECBCPSY PERF/NO HISIO PERFORMED AUTC/NECROPSY/HISIO PERF AUTO/NECBOPSY/liO HISTO 

AUTOLYSIS/NO NECROPSY 

10 

1 

1 

D-5
 
TABLE D2

 

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

CONTROL (VEH ) LOH DOSE HIGH DOSE 

02-F032 02-F035 02-F036 

ANIMAL S INITIALLY IN STUD Y 20 50 50 

ANIMAL S HISSING 2 

ANIMALS NECHOPSIED 20 44 50 

ANIMAL S EXAMINE D HISTOEATHO LOGICALLY ** 20 44 50 

INTEGUMENTARY SYSTEM
 

*SKIN (20) (44) (50)
 

VERRUCA 1 (2X)
 

*SUBCUT TISSUE (20) (44) (50)
 

NECROSIS, FAT 1 (2%)
 

RESPIBATCBY SYSTEM
 

*LUNG (20) (19) (19)
 

PNEUMONIA, CHBONIC HURINE 1 (5*)
 

HYPEBPLASIA. ADENCMATOUS 1 (5X)
 

METAPLASIA, SQUAMOUS 1 (5X)
 

HEMATOICIETIC SYSTEM
 

tSPLEEN (20) (16) (18)
 

HEMATOPOIESIS 2 (13X) 1 (6X)
 

CIBCUIAICEY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVER (20) (44) (50)
 

CYST, NOS 1 <2X)

 

THECMBOSIS, NOS 1 (2X) 

INFLAMMATION , NOS 1 (2)t)

 

tPANCREA S (20) (14) (17)
 

INFi*MJATI6Nt_NOS_ 

* NUMBE R OF ANIMAL S HITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-6
 
TABLE D2 (CONTINUED) 

CONTROL (VEH ) LOH DOSE HIGH DOSE 

02-F032 02-F035 02-F036 

URINARY SYSTEM
 

tKIDNEY (20) (18) (18)
 HYCBONEPHROSIS 1 (6X)
 INFLAMMATION. CHRONIC 1 (6X)
 

ENDOCRINE SYSTEM
 

NONE
 

EEPRCCUC1IVE SYSTEH
 

*UTERUS (20) (26) (23)
 HYDBCHETRA 2 (10*) 2 (8X) 6 (26%)
 

INFLAMMATION, NOS 2 (10*) 2 (8X) 3 (13X)
 

tBTERUS/ENDOMETRIUM (20) (26) (23)
 HYPHHPLASIA. CYSTIC 6 (30X) 13 (50%)
 

tOVARY (20) (17) (25)
 

CYST, NOS 2 (10*) H (2tX) 9 (36X)
 

INFLAMMATION. NOS 1 (UX)
 

NERVOUS SYSTEM
 

NCNE
 

SPECIAL SHNSE ORGANS
 

NONE
 

MOSCUIOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

t NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMA1S NECROfSlED
 

D-7
 
TABLE D2 (CONCLUDED) 

CONTBOL (VEH) LOU DOSE HIGH DOSE 02-r032 02-F035 02-F036 

SPECIAL HORPHOLOGi SUMMARY 

NO LESION REPORTED 5 20 26 AHIBAL HISSING/NO NECROPSY 2 

AUTOLYSIS/NO NECROPSY 4 

D-8
 
Review of the Bioassay of Dicofol* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

April 26, 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 ^he below critique is given on
 

the bioassay of Dicofol for carcinogenicity.
 

The primary reviewer thought that the conduct and the
 

design of the bioassay were adequate, although he disagreed
 

with the presentation of the conclusion on the carcinogenicity
 

of Dicofol. He said that the conclusion should focus on the
 

negative response found in three of the treatment groups
 

rather than on the positive in only a single sex of one
 

species. He concluded that: 1) Dicofol should not be
 

classified as a carcinogen without replication of the study
 

and 2) Dicofol would not appear to pose a carcinogenic risk
 

for man on the basis of this bioassay.
 

The secondary reviewer said that the inadequacies of
 

the bioassay made it difficult to interpret the significance
 

of the increased incidence of liver tumors found in treated
 

male mice, particularly in the absence of other positive
 

data. He was especially critical of: the lack of analytical
 

data on the stability of Dicofol during storage and in the
 

treatment diet (it was noted that a significant amount of
 

45
 
the stored Dicofol liquified); the inadequate subchronic
 

data for selecting chronic dose levels; the change in dose
 

levels during the chronic phase; and the poor survival
 

among control male mice. He concluded that the test was
 

poorly conducted and recommended that it be repeated.
 

A Program staff member commented that the survival
 

among control male mice was fairly high until about 78
 

weeks. He said that the incidence of hepatocellular
 

carcinomas among treated male mice was 74% in the high
 

dose group and 44% in the low dose one, as compared to
 

a high of 25% in historic controls at the testing laboratory.
 He opined that the induction of a statistically significant
 

incidence of hepatocellular carcinomas in one sex and species
 

was sufficient to base a conclusion of carcinogenicity.
 The primary reviewer repeated that such a limited positive
 

response was overemphasized in the report. He suggested
 

that Dicofol be termed no more than a hepatocarcinogen in
 

male mice. The secondary reviewer contended that the
 

bioassay was too inadequate to draw any conclusion.
 

A subgroup member offered an amendment to a motion
 

put forth earlier by the primary reviewer. The amended
 

motion read: "Under the conditions of this bioassay,
 technical grade Dicofol produced no evidence of carcinogenicity in Osborne-Mendel rats of either sex or in female
 

B6C3P1 mice; the failure to determine the stability of
 

Dicofol throughout the study prohibits drawing any
 

conclusion concerning its carcinogenicity." A vote on the
 

amended motion passed unanimously.
 

In further discussion, it was recommended that a
 

sample of the original Dicofol be analyzed to determine
 

its composition. Based on the results of the analysis,
 a decision could be made as to whether the compound should
 

be considered for retest.
 

Members present were:
 

Michael Shimkin (Acting Chairman), University of California
 

at San Diego
 

Joseph Highland, Environmental Defense Fund
 

George Roush, Jr., Monsanto Company
 

Louise Strong, University of Texas Health Sciences Center
 

John Weisburger, American Health Foundation
 

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.
 

ftU.S. GOVERNMENT PRINTING OFFICE: 1978 260-899/3170 1-3 46 

DHEW Publication No. (NIH) 78-1340