1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 35 

BIOASSAY OF 

METHOXYCHLOR 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 72-43-5 

NCI-CG-TR-35 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

METHOXYCHLOR
 

FOR POSSIBLE CARCINOGEN1CITY
 

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) 78-835
 

REPORT ON THE BIOASSAY OF METHOXYCHLOR
 

FOR POSSIBLE CARCINOGENICITY
 

CARCINOGENESIS TESTING PROGRAM
 

DIVISION OF CANCER CAUSE AND PREVENTION
 

NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH
 

CONTRIBUTORS: This report presents the results of the bioassay of
 

methoxychlor conducted for the Carcinogenesis Testing Program, Division of Cancer Cause and Prevention, National Cancer Institute (NCI),
 

National Institutes of Health, Bethesda, Maryland. This bioassay was
 

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

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

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

principal investigators for the contract were Dr. M. B. Powers (3),
 

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

(3,5); 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 (6).
 

Compilation of individual animal survival, pathology, and
 

summary tables was performed by EG&G Mason Research Institute (7);
 

the statistical analysis was performed by Dr. J. R. Joiner (6), and
 

Mr. W. W. Belew (8), using methods selected for the Bioassay Program
 

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

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

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

(8), the task leader, Dr. M. R. Kornreich (8), and the senior biologist, Ms. P. Walker (8). The final report was reviewed by members of
 

the participating organizations.
 

111
 
The statistical analysis was reviewed by one or more members of
 

the Mathematical Statistics and Applied Mathematics Section of the
 

NCI: Dr. J. J. Gart (9), Mr. J. Nam (9), Dr. H. M. Pettigrew (9),
 

and Dr. R. E. Tarone (9).
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment, interpreting
 

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

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

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

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

1.	 Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

2.	 Now with 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.	 Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

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

Rockville, Maryland.
 

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

Boulevard, McLean, Virginia.
 

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 Comp-<rative Medicine, Johns Hopkins
 

University, School of Medicin-', Traylor Building, Baltimore,
 

Maryland.
 

IV
 
SUMMARY
 

A bioassay for possible carcinogenicity of technical-grade
 

methoxychlor was conducted using Osborne-Mendel rats and B6C3F1 mice.
 

Methoxychlor was administered in the feed, at either of two concentrations, to groups of 50 male and 50 female animals of each species.
 

For each species, 20 animals of each sex were placed on test as controls. The time-weighted average high and low dietary concentrations
 

of methoxychlor were, respectively, 845 and 448 ppm for male rats,
 

1385 and 750 ppm for female rats, 3491 and 1746 ppm for male mice,
 

and 1994 and 997 ppm for female mice. After a treatment period of 78
 

weeks, the rat groups were observed for an additional 34 weeks and
 

the mouse groups for an additional 15 weeks. A dose-related mean
 

group body weight depression was observed in both rats and mice, but
 

no effect on survival was detected.
 

Under the conditions of this study, methoxychlor was not found
 

to be carcinogenic in Osborne-Mendel rats or B6C3F1 mice of either
 

sex.
 

V 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 5
 

A. Chemicals 5
 

B. Dietary Preparation 5
 

C. Animals 5
 

D. Animal Maintenance 6
 

E. Selection of Initial Concentrations 7
 

F. Experimental Design 8
 

G. Clinical and Histopathologic Examinations 11
 

H. Data Recording and Statistical Analyses 13
 

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 35
 

A. Body Weights and Clinical Observations 35
 

B. Survival 35
 

C. Pathology 38
 

D. Statistical Analyses of Results 38
 

V. DISCUSSION 43
 

VI. BIBLIOGRAPHY 45
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH METHOXYCHLOR A-l
 

APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH METHOXYCHLOR B-1
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH METHOXYCHLOR C-l
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH METHOXYCHLOR D"1
 

vii
 
LIST OF ILLUSTRATIONS
 

Figure Number Page 

1 GROWTH CURVES FOR METHOXYCHLOR CHRONIC STUDY 

RATS 20 

2 SURVIVAL COMPARISONS OF METHOXYCHLOR CHRONIC 

STUDY RATS 22 

3 GROWTH CURVES FOR METHOXYCHLOR CHRONIC STUDY 

MICE 36 

4 SURVIVAL COMPARISONS OF METHOXYCHLOR

STUDY MICE

 CHRONIC 

 37 

LIST OF TABLES 

Table Number Page 

1 DESIGN SUMMARY FOR OSBORNE-MENDEL RATS— 

METHOXYCHLOR FEEDING EXPERIMENT 9 

2 DESIGN SUMMARY FOR B6C3F1 MICE—METHOXYCHLOR 

FEEDING EXPERIMENT 10 

3 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

METHOXYCHLOR 24 

4 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE RATS TREATED WITH 

METHOXYCHLOR 30 

5 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

METHOXYCHLOR 39 

6 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS 

AT SPECIFIC SITES IN FEMALE MICE TREATED 

WITH METHOXYCHLOR 40 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE 

RATS TREATED WITH METHOXYCHLOR A ~ 3 

Vlll
 
LIST OF TABLES (Concluded) 

Table Number Page 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE RATS TREATED WITH METHOXYCHLOR A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

MALE MICE TREATED WITH METHOXYCHLOR B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN 

FEMALE MICE TREATED WITH METHOXYCHLOR B-6 

Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE RATS TREATED WITH 

METHOXYCHLOR C ~ 3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE RATS TREATED WITH 

METHOXYCHLOR C ~ 7 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN MALE MICE TREATED WITH 

METHOXYCHLOR D ~ 3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 

LESIONS IN FEMALE MICE TREATED WITH 

METHOXYCHLOR D ~ 6 

ix
 

I. INTRODUCTION
 

Methoxychlor (NCI No. C00497), a synthetic organochlorine insecticide and structural analog of DDT, was one of several widely used
 

agricultural pesticides selected for bioassay by the National Cancer
 

Institute because of a lack of adequate chronic toxicity data. The
 

suspect state of all DDT-related chemicals was an important additional
 

factor in its selection.
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is 1,1'-(2,2,2-trichloroethylidene)bis
 

(4-methoxy)-benzene. It is also known as 1,1,l-trichloro-2,2'-bis(pmethoxyphenyl)-ethane; dianisyl trichloroethane; dimethoxy DDT; and
 

DMDT.
 

Methoxychlor is effective against a wide range of insects that
 

attack fruits, vegetables, shade trees, home gardens, forage crops,
 

and livestock. It is also used for the spray treatment of barns,
 

grain storage bins, mushroom houses, dairies, and other agricultural
 

buildings (Spencer, 1973; Brooks, 1974; Farm Chemicals Handbook,
 

1976). In 1971, the most recent year for which data are available,
 

more than 3 million pounds of methoxychlor were used in the United
 

States. Of this amount, approximately 2 million pounds were used to
 

protect livestock and livestock buildings, with application to beef
 

and dairy cattle accounting for 95 percent of livestock-related usage.
 

The remaining 1 million pounds of methoxychlor were used to treat 725
 

*
 

The CAS registry number is 72-43-5.
 

1 
thousand acres of varied cropland. The major crop treated was alfalfa
 

which accounted for 49 percent of crop-related methoxychlor usage and
 

66 percent of total treated acreage (Andrilenas, 1974). Although its
 

activity range differs somewhat from that of DDT, methoxychlor is
 

considered to be an excellent replacement for DDT for certain applications (Spencer, 1973; Farm Chemicals Handbook, 1976). It may also
 

substitute in some cases for aldrin, dieldrin, chlordane and heptachlor, all of which have had their registrations cancelled (Ouellette
 

and King, 1976).
 

According to the U.S. International Trade Commission (1977)
 

6
 5.504 x 10 pounds of methoxychlor were produced in the United States
 

in 1975, an almost 70 percent increase over 1974 production figures
 

(3.248 x 10 pounds) (Fowler and Mahan, 1976).
 

The risk of exposure to methoxychlor is greatest for agricultural
 

or farm workers engaged in pesticide application, although workers at
 

pesticide formulating plants may also experience significant contact
 

with the chemical. Since methoxychlor is relatively ineffective
 

against soil organisms (Brooks, 1974), it is generally applied directly to crops via ground or aerial spraying. As a result, contamination of the atmosphere over treated lands and surrounding areas,
 

with possible inhalational exposure of persons residing in those
 

areas, may occur.
 

Methoxychlor possesses long residual insecticidal activity
 

against many species (Farm Chemicals Handbook, 1976) probably as a
 

2 
result of its low volatility and relative resistance to oxidation,
 

heat, moisture and ultraviolet irradiation (Brooks, 1974). Consequently, residues may persist on treated crops for a length of time
 

sufficient to permit low-level exposure among the general population.
 

As an example, 5.3 ppm of tnethoxychlor were detected on alfalfa 33
 

days following treatment of the crop with 1 pound of insecticide per
 

acre (Miles et al., 1964). A total diet study conducted in 1969-1970
 

revealed methoxychlor concentrations ranging from 0.008 to 0.05 ppm
 

(Corneliussen, 1972) while total U.S. dietary intake in 1970 amounted
 

to 0.02 p.g/kg body weight/day (Duggan and Corneliussen, 1972).
 

Exposure of the general population through ingestion of contaminated beef or milk probably contributes minimally to total dietary
 

intake since methoxychlor undergoes rapid biodegradation in mammals
 

and shows little tendency to bioaccumulate in tissues (International
 

Agency for Research on Cancer, 1974). An average level of 0.001 ppm
 

methoxychlor was detected in dairy products in the 1960s (Duggan,
 

1967) while no residues were detected in the body fat of cattle fed
 

25 ppm methoxychlor in the diet for 16 weeks (Claborn, 1956).
 

Methoxychlor is generally considered a relatively safe pesticide
 

with a low order of toxicity to humans and other warm blooded animals
 

(Farm Chemicals Handbook, 1976). However, available chronic toxicity
 

studies for assessing carcinogenic potential are either limited in
 

scope or inconclusive. Evaluation of carcinogenicity following skin
 

application or subcutaneous injection was performed only in the mouse.
 

3 
No tumors were observed in C3H/Anf mice following weekly skin application of 0.1 or 10 mg of methoxychlor in acetone for a total of up
 

to 980 mg or following a single subcutaneous injection of 10 mg of
 

the pesticide in trioctanoin (Hodge et al., 1966).
 

4 
II. MATERIALS AND METHODS 

A. Chemicals
 

Technical-grade methoxychlor [1,1'-(2,2,2-trichloroethylidene)­

bis(4-methoxy)-benzene] was purchased and analyzed by Hazleton
 

Laboratories America, Inc., Vienna, Virginia. The observed melting
 

point (86.5° to 87.5°C) suggested a compound of high purity. Gas-


liquid chromatography suggested a purity greater than 95 percent but
 

also indicated the presence of four minor contaminants. Analyses
 

performed 1 and 2 years later showed no substantial change in the
 

results and thus suggested that no decomposition had occurred.
 

Throughout this report, the term methoxychlor is used to represent this technical-grade material.
 

B. Dietary Preparation
 

The basal laboratory diet for both control and treated animals 

(R) consisted of 2 percent Duke's corn oil (S.F. Sauer Company) by 

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

mixtures of methoxychlor in corn oil were prepared weekly and stored 

in the dark. The mixtures of methoxychlor in corn oil were incorporated into the appropriate amount of basal laboratory diet in a twinshell blender fitted with an accelerator bar. 

C. Animals
 

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

5 
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 treatment and control groups. 

D. Animal Maintenance 

All animals were housed by species in temperature- and humiditycontrolled 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 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 10 in 

solid-bottom polypropylene cages equipped with filter tops. Sani­

(R) tized cages with fresh bedding (Sanichips , Shurfire) were provided 

once each week for mice. Rats received sanitized cages with no 

6 
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 were provided
 

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

The methoxychlor-treated and control rats were housed in the
 

same room with other rats receiving diets treated with p,p'-DDE (72­

55-9) and safrole (94-59-7). All mice used in the methoxychlor study,
 

including controls, were housed in the same room as other mice receiving diets treated with trifluralin (1582-09-8), dioxathion (78-34-2),
 

sulfallate (95-06-7), p,p'-DDT (50-29-3), chlorobenzilate (510-15-6),
 

p,p'-DDE (72-55-9), p,p'-TDE (72-54-8), dicofol (115-32-2), pentachloronitrobenzene (82-68-8), amitrole (61-82-5), acetylaminofluorene
 

(53-96-3), clonitralid (1420-04-8), nitrofen (1836-75-5), endosulfan
 

(115-29-7), mexacarbate (315-18-4), and safrole (94-59-7).
 

E. Selection of Initial Concentrations
 

In order to establish the maximum tolerated concentrations of
 

methoxychlor for administration to treated animals in the chronic
 

studies, 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. Methoxychlor was
 

premixed with a small amount of corn oil. This mixture was then
 

incorporated into the laboratory diet and fed ad libitum to five of
 

the six rat groups at concentrations of 316, 562, 1000, 1780, and
 

*
 

CAS registry numbers are given in parentheses.
 

7 
3160 ppm and to five of the six mouse groups at concentrations of
 

1000, 1780, 3160, 5620, and 10,000 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 diet of corn oil and laboratory
 

chow.
 

A dosage inducing no mortality and resulting in a depression in
 

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

weight gain criteria were not applicable, mortality data alone were
 

utilized.
 

The initial high concentrations selected for the chronic study
 

for male and female rats were 720 and 1500 ppm, respectively. The
 

initial high concentrations selected for the chronic study for mice
 

were 2800 ppm for males and 1500 ppm for females.
 

F. Experimental Design
 

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

treated and untreated observation periods, and the time-weighted
 

average concentrations) are summarized in Tables 1 and 2.
 

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

6 weeks old at the time they were placed on test. The high and low
 

concentrations of methoxychlor initially utilized for males were 720
 

8 
TABLE 1
 

DESIGN SUMMARY FOR OSBORNE-MENDEL RATS
 

METHOXYCHLOR FEEDING EXPERIMENT
 

INITIAL OBSERVAT. ION PERIOD TIME-WEIGHTED 

GROUP METHOXYCHLOR TREATED UNTREATED AVERAGE 

SIZE CONCENTRATION3 (WEEKS) (WEEKS) CONCENTRATION 

MALE 

CONTROL 20 0 111 0 

LOW DOSE 50 360 29 448 

500 49 

0 33 

HIGH DOSE 50 720 29 845 

1000 29 

1000° 16 4 

0 33 

FEMALE 

CONTROL 20 0 111 0 

LOW DOSE 50 750 78 750 

0 33 

HIGH DOSE 50 1500 55 1385 

1500C 17 6 

0 34 

a 

Concentrations given in parts per million. 

b_. . . , . 2(concentration X weeks received) Time-weighted average concentration - -^77 : :—: v 2(.weeks receiving treatment; 

c

These concentrations were cyclically administered with a pattern of 1 

dosage-free week followed by 4 weeks of treatment at the level indicated. 

9 
INITIAL
 

GROUP
 

SIZE
 

MALE
 

CONTROL 20
 

LOW DOSE 50
 

HIGH DOSE 50
 

FEMALE
 

CONTROL 20
 

LOW DOSE 50
 

HIGH DOSE 50
 

a

Concentrations given

b. . , j. line-weigh ted average

TABLE 2
 

DESIGN SUMMARY FOR B6C3F1 MICE
 

METHOXYCHLOR FEEDING EXPERIMENT
 

METHOXYCHLOR
 

CONCENTRATION 3


0
 

1400
 

1750
 

0
 

2800
 

3500
 

0
 

0
 

750
 

1000
 

0
 

1500
 

2000
 

0
 

OBSERVAT: [ON PERIOD TIME-WEIGHTED 

TREATED UNTREATED AVERAGE 

 (WEEKS) (WEEKS) CONCENTRATION 

92 

1 1746 

77 

14 

1 3491 

77 

15 

92 0 

1 997 

77 

15 

1 1994 

77 

15 

 in parts per million. 

. 2(concentration X weeks received) concentration = ; ; :—: r S( weeks receiving treatment) 

10 
and 360 ppm, respectively. At the end of week 30, the high concentration for males was increased to 1000 ppm and the low concentration
 

was increased to 500 ppm. For females, the initial high and low concentrations of methoxychlor were 1500 and 750 ppm, respectively. In
 

week 56 of the study, administration of methoxychlor to the high dose
 

female rats ceased for 1 week and was then followed by 4 weeks of
 

feeding at the previous concentration of 1500 ppm. This pattern of
 

cyclic administration continued for the remainder of the treatment
 

period. This same method of total intake reduction was employed for
 

the high dose male rats beginning with week 59. Final observations
 

on all rats were made 112 weeks after the experiment was initiated.
 

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

6 weeks old at the time the experiment began. The high and low concentrations initially administered to male mice were 2800 and 1400
 

ppm, respectively. Female mice received initial high and low concentrations of 1500 and 750 ppm, respectively. At the end of week 2,
 

the high and low concentrations of methoxychlor were increased, respectively, to 3500 and 1750 ppm for male mice and to 2000 and 1000
 

ppm for female mice. These dosages were maintained until the termination of treatment (week 78). Final observations on all mice were
 

made 93 weeks after the experiment was initiated.
 

G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

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

11
 
for mortality. 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. 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. The histopathologic examination consisted of gross and microscopic examination
 

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

animals and, whenever possible, from animals found dead.
 

12
 
Slides were prepared from the following tissues from selected
 

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

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

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

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

uterus, mammary gland, and ovary.
 

Tissues for which slides were prepared were preserved in 10 percent buffered formalin, embedded in paraffin, sectioned, and stained
 

with hematoxylin and eosin prior to microscopic examination. An
 

occasional section was subjected to special staining techniques for
 

more definitive diagnosis.
 

A few tissues were not examined for some animals, particularly
 

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

preclude histopathologic interpretation. Thus, the number of animals
 

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

animals that were placed on experiment in each group.
 

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
 

13
 
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 used Tarone's (1975) extensions of Cox's methods for
 

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

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

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

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

14
 
histologically. However, when macroscopic examination was required
 

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

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

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

The purpose of the statistical analyses of tumor incidence is to
 

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

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

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

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

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

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

significance level of 0.05 may be made. The Bonferroni inequality
 

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

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

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

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

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

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

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

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


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

15
 
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 cf 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.
 

16
 
The approximate 95 percent confidence interval for the relative
 

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

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

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

t c t
 

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

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

c
 

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

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

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

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

larger proportion in the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

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

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

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

would be within the interval calculated from the experiment. When
 

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

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
 

17
 
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
 

Distinct, dose-related mean body weight depression was apparent
 

throughout the treatment period in both males and females (Figure 1).
 

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

behavior of the treated rats were generally comparable with that of
 

the controls, except that occasional hunched appearance, reddened
 

eyes, and abdominal urine stains were noted in one to three treated
 

rats as early as week 4.
 

From week 34 to cessation of treatment (week 78), some clinical
 

signs were noted with slightly greater frequency in the treated
 

groups than in the controls. During the post-treatment observation
 

period these signs were observed at comparable rates in treated and
 

control animals. These signs included hunched appearance; urine
 

staining; body sores; rough fur; eyes that were cloudy, squinted, or
 

reddened with discharge or crust; and palpable nodules or tissue
 

masses. As the animals aged, the incidence of these signs in all
 

groups gradually increased. Respiratory signs characterized by
 

labored respiration, wheezing, and/or nasal discharge were observed
 

at a low incidence in all groups throughout the study. Isolated,
 

sporadic, and spontaneous observations in one to three rats included
 

circling, small testes, head tilt, "stiff" gait, hind-limb paralysis,
 

ataxia, and transient vaginal discharge.
 

19
 
FIGURE 1

 

GROWTH CURVES FOR METHOXYCHLOR CHRONIC STUDY RATS 

20 
B.	 Survival
 

The estimated probabilities of survival for male and female rats
 

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

For both male and female rats the Tarone test did not detect a significant positive association between increased dosage and elevated
 

mortality.
 

For males the actual survival was adequate as 86 percent of the
 

high dose, 74 percent of the low dose and 85 percent of the control
 

rats lived at least 100 weeks. For females the actual survival was
 

also adequate as 94 percent of the high dose, 94 percent of the low
 

dose, and 90 percent of the control rats lived 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).
 

Hemangiosarcomas were the only tumors observed at unusually high
 

incidences. The hemangiosarcomas occurred in the spleen of 1/20 control males, 6/44 low dose males, 2/42 high dose males, and 1/20 control females. Hemangiosarcomas also occurred as subcutaneous masses
 

in 2/50 low dose males and as an abdominal tissue mass in 1/50 low
 

dose male rats. Thus, there was an increased incidence of hemangiosarcomas at all sites in male rats receiving methoxychlor (9 low dose
 

and 2 high dose) as compared to a single control male and a single
 

control female with these tumors. The lack of a dose response in the
 

21
 
FIGURE 2

 

SURVIVAL COMPARISONS OF METHOXYCHLOR CHRONIC STUDY RATS 

22 
number of male rats with hemangiosarcomas makes this finding of questionable importance even though in our experience hemangiosarcomas
 

occur relatively infrequently in the Osborne-Mendel rat. With the
 

exception of a hemangiosarcoma in the spleen of one control female
 

there were no unusual tumors or any unusual incidences of spontaneous
 

tumors in the female rats.
 

Microscopically, the hemangiosarcomas consisted of the formation
 

of capillaries and cavernous vascular spaces that often contained
 

blood and were lined with hyperchromatic, plump anaplastic endothelial cells. There was piling up of these cells around the vascular
 

spaces. These cells invaded and replaced the normal architecture of
 

the spleen and involved tissues.
 

Other neoplasms observed during this examination were seen at
 

incidences often found to occur naturally in untreated aged Osborne-


Mendel rats.
 

Inflammatory, degenerative, and proliferative lesions as seen in
 

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

those spontaneous lesions occurring in aged Osborne-Mendel rats.
 

There was no definitive evidence of the carcinogenicity of
 

methoxychlor 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 for every type
 

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

methoxychlor-dosed groups of either sex is included.
 

23
 
TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH METHOXYCHLOR*
 

TOPOGRAPHY : MORPHOLOGY 

Subcutaneous Tissue: Fibroma or 

Fibrosarcomab 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Liver: Neoplastic Nodule or Hepato cellular 

Care inomab 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Pancreas: Liposarcoma" 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

LOW HIGH 

CONTROL DOSE DOSE 

1/20(0.05) 2/50(0.04) 3/50(0.06) 

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

0.800 1.200 

0.045 0.105 

46.273 61.724 

Ill 101 26 

0/19(0.00) 2/37(0.05) 0/37(0.00) 

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

Infinite 

0.157 

Infinite 

96 

0/19(0.00) 1/20(0.05) 0/20(0.00) 

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

Infinite 

0.052 

Infinite 

79 
TABLE 3

TOPOGRAPHY : MORPHOLOGY 

Kidney: Transitional-Cell Carcinoma 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Kidney: Fibrous Histiocytoma, Malignant 

P Values0 

Relati-e Risk (Control )d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Kidney: Mixed Tumor, Malignant 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

 (Continued) 

CONTROL 

0/19(0.00) 

N.S. 

0/19(0.00) 

N.S. 

1/19(0.05) 

N.S. 

Ill 

LOW 

DOSE 

1/21(0.05) 

N.S. 

Infinite 

0.050 

Infinite 

94 

1/21(0.05) 

N.S. 

Infinite 

0.050 

Infinite 

96 

2/21(0.10) 

N.S. 

1.810 

0.103 

100.601 

63 

HIGH 

DOSE 

0/28(0.00) 

N.S . 

0/28(0.00) 

N.S. 

1/28(0.04) 

N.S. 

0.679 

0.009 

51.519 

111 
TOPOGRAPHY : MORPHOLOGY 

*b Kidney: Hamartoma 

P Values0 

Relative Risk (Control)d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Pituitary: Chromophobe Adenoma 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Adrenal: Pheochromocytoma 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

TABLE 3 (Continued)
 

CONTROL 

0/19(0.00) 

N.S. 

3/18(0.17) 

N.S. 

105 

0/20(0.00) 

N.S. 

LOW HIGH 

DOSE DOSE 

1/21(0.05) 0/28(0.00) 

N.S. N.S. 

Infinite 

0.050 

Infinite 

111 

8/21(0.38) 4/22(0.18) 

N.S. N.S. 

2.286 1.091 

0.662 0.216 

11.364 6.582

 

69 111 

1/18(0.06) 1/19(0.05) 

N.S. N.S. 

Infinite Infinite 

0.061 0.058 

Infinite Infinite 

111 111 

*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.
 
TOPOGRAPHY : MORPHOLOGY 

Thyroid: Follicular-Cell Carcinoma 

P Values0 

Departure from Linear Trend 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Thyroid: Follicular-Cell Adenoma 

P Values0 

Relative Risk (Control)d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Thyroid: Follicular-Cell Adenoma or 

Follicular-Cell Carcinoma^ 

P Values0 

Relative Risk (Control)d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

TABLE 3 (Continued) 

CONTROL 

0/19(0.00) 

N.S. 

P = 0.044 

1/19(0.05) 

N.S. 

105 

1/19(0.05) 

N.S. 

105 

LOW 

DOSE 

4/27(0.15) 

N.S. 

Infinite 

0.685 

Infinite 

69 

1/27(0.04) 

N.S. 

0.704 

0.010 

53.206 

111 

5/27(0.19) 

N.S. 

3.519 

0.447 

159.091 

69 

HIGH 

DOSE 

1/24(0.04) 

N.S. 

Infinite 

0.044 

Infinite 

111 

2/24(0.08) 

N.S. 

1.583 

0.090 

89 . 314 

111 

3/24(0.13) 

N.S. 

2.375 

0.212 

118.982 

111 
TABLE 3 (Continued) 

TOPOGRAPHY: MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Thyroid: C-Cell Adenoma 

P Values0 

2/19(0.11) 

N.S. 

0/27(0.00) 

N.S. 

2/24(0.08) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

0.000 

0.000 

2.319 

0.792 

0.063 

10.089 

Weeks to First Observed Tumor 109 110 

Thyroid: C-Cell Adenoma

P Values0 

 or C-Cell Carcinoma 2/19(0.11) 

N.S. 

1/27(0.04) 

N.S. 

3/24(0.13) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

0.352 

0.006 

6.315 

1.188 

0.153 

13.079 

Weeks to First Observed Tumor 109 111 110 

Pancreatic Islets:

P Values0 

 Islet-Cell Adenoma 0/19(0.00) 

N.S. 

1/20(0.05) 

N.S. 

1/20(0.05) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Infinite 

0.052 

Infinite 

Infinite 

0.052 

Infinite 

Weeks to First Observed Tumor 101 111 
TABLE 3 (Concluded) 

TOPOGRAPHY: MORPHOLOGY CONTROL 

LOW 

DOSE 

HIGH 

DOSE 

Testis: Interstitial-Cell Tumor 

P Values0 

0/20(0.00) 

N.S. 

0/19(0.00) 

N.S. 

1/21(0.05) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Infinite 

0.052 

Infinite 

Weeks to First Observed Tumor 111 

Circulatory System: Hemangiosarcoma 

P Values0 

1/20(0.05) 

N.S. 

9/50(0.18) 

N.S. 

2/50(0.04) 

N.S. . 

Departure from Linear Trend P = 0.020 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

3.600 

0.562 

154.106 

0.800 

0.045 

46.273 

Weeks to First Observed Tumor 108 87 111 

Treated groups received time-weighted average doses of 448 or 845 ppm in feed. 

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

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

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

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

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

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

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

group when P < 0.05.
 
TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH METHOXYCHLOR£
 

HIGH
 

DOSE
 

0/50(0.00)
 

N.S.
 

0.000
 

0.000
 

7.475
 

1/50(0.02)
 

N.S.
 

0.400
 

0.005
 

30.802
 

10/36(0.28)
 

N.S.
 

0.694
 

0.308
 

1.722
 

TOPOGRAPHY : MORPHOLOGY
 

Subcutaneous Tissue: Fibroma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Subcutaneous Tissue: Fibroma or
 

Fibro sarcoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW 

CONTROL DOSE 

1/20(0.05) 4/50(0.08) 

N.S. N.S. 

1.600 

0.175 

77.169 

94 100 

1/20(0.05) 5/50(0.10) 

N.S. N.S. 

2.000 

0.249 

92.596 

94 100 

8/20(0.40) 7/32(0.22) 

N.S. N.S. 

0.547 

0.209 

1.474 

106 95 

72 

106 
TABLE 4

TOPOGRAPHY : MORPHOLOGY 

Thyroid: Follicular-Cell Adenoma 

P Values0 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Thyroid: C-Cell Adenoma 

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 

 (Continued) 

CONTROL 

0/20(0.00) 

N.S. 

0/20(0.00) 

N.S. 

0/20(0.00) 

N.S. 

LOW 

DOSE 

2/28(0.07) 

N.S. 

Infinite 

0.220 

Infinite 

111 

0/28(0.00) 

N.S. 

1/28(0.04) 

N.S. 

Infinite 

0.039 

Infinite 

111 

HIGH 

DOSE 

0/31(0.00) 

N.S. 

3/31(0.10) 

N.S. 

Infinite 

0.404 

Infinite 

112 

3/31(0.10) 

N.S. 

Infinite 

0.404 

Inf inite 

112 
TABLE 4 (Continued) 

TOPOGRAPHY : MORPHOLOGY CONTROL 

Mammary Gland: Ad eno carcinoma NOS 

P Values0 

1/20(0.05) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor Ill 

Mammary Gland: Adenoma or Adenocarcinoma 

NOSb 

P Values0 

1/20(0.05) 

N.S. 

Relative Risk (Control)d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor Ill 

Uterus: Endometrial Stromal Polyp 

P Values0 

- 0/19(0.00) 

N.S. 

Relative Risk (Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

LOW
 

DOSE
 

1/50(0.02)
 

N.S.
 

0.400
 

0.005
 

30.802
 

16
 

1/50(0.02)
 

N.S.
 

0.400
 

0.005
 

30.802
 

16
 

1/34(0.03)
 

N.S.
 

Infinite
 

0.031
 

Infinite
 

111
 

HIGH
 

DOSE
 

3/50(0.06)
 

N.S.
 

1.200
 

0.105
 

61.724
 

106
 

5/50(0.10)
 

N.S.
 

2.000
 

0.249
 

92.596
 

106
 

4/35(0.11)
 

N.S.
 

Infinite
 

0.526
 

Infinite
 

111
 
TABLE 4 (Concluded) 

Treated groups received time-weighted average doses of 750 or 1385 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. 
None of the statistical tests for rats of either sex indicated
 

a significant positive association between the administration of
 

methoxychlor and an elevated tumor incidence. Thus, at the dose
 

levels used in this experiment there was no statistical evidence that
 

methoxychlor was a carcinogen in Osborne-Mendel rats.
 

To provide additional insight, 95 percent confidence intervals
 

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

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

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

indicates the absence of statistically significant results. It
 

should also be noted that many of the confidence intervals have an
 

upper limit greater than one, indicating the theoretical possibility
 

of a significantly increased rate of tumor incidence induced in rats
 

by methoxychlor that could not be established under the conditions of
 

this test.
 

34
 
IV. CHRONIC TESTING RESULTS: MICE
 

A. Body Weights and Clinical Observations
 

Distinct, dose-related mean body weight depression was apparent
 

in treated females during the entire bioassay. There were no distinct
 

differences in weight gain patterns of low dose and control male mice;
 

high dose male mice gained slightly less weight than these groups
 

during the treatment period (Figure 3).
 

Throughout the study there was no definitive evidence of compound
 

effects with regard to physical appearance and behavior of the treated
 

mice. Signs often observed in group-housed laboratory mice, particularly in males, were observed at a comparable rate in treated and
 

control animals. These common signs included sores on the body and/
 

or extremities (more prevalent in the males); a hunched appearance;
 

localized alopecia; penile, vulvar, or anal irritation with apparent
 

sores and abscesses around the inguinal area (more prevalent in male
 

mice); rough or stained fur; bloating; and palpable nodules or tissue
 

masses. Isolated observations in one or two mice from all groups
 

included reddened or squinted eyes and labored respiration.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

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

For both male and female mice the Tarone test did not detect a significant positive association between increased dosage and elevated
 

mortality.
 

35
 
FIGURES

 

GROWTH CURVES FOR METHOXYCHLOR CHRONIC STUDY MICE 

36 
FIGURE 4

 

SURVIVAL COMPARISONS OF METHOXYCHLOR CHRONIC STUDY MICE 

37 
For males the actual survival was adequate as 69 percent of the
 

high dose, 58 percent of the low dose, and 45 percent of the control
 

mice survived over 81 weeks. For females the actual survival was
 

high as 98 percent of the high dose, 90 percent of the low dose, and
 

85 percent of the control mice survived until the end of the test.
 

C. Pathology
 

Histopathologic findings on neoplasms in mice are tabulated in
 

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

tabulated in Appendix D (Tables Dl and D2).
 

A variety of neoplasms were observed among both treated and
 

control mice. Each of the types of tumors represented has been
 

encountered previously as a naturally occurring lesion in B6C3F1
 

mice, and is without apparent relationship to the administration of
 

the chemical.
 

The inflammatory, degenerative, and proliferative lesions that
 

occurred in the control and treated animals were also without appreciable differences in number and kind from those spontaneous lesions
 

occurring in aged B6C3F1 mice.
 

No evidence for the carcinogenicity of methoxychlor in B6C3F1
 

mice was provided by this histopathologic examination.
 

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 for every type
 

38
 
TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH METHOXYCHLOR£
 

LOW HIGH 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE DOSE 

Subcutaneous Tissue: Fibrosarcoma 1/12(0.08) 2/44(0.05) 2/47(0.04) 

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

Relative Risk (Control) 0.545 0.511 

Lower Limit 0.032 0.031 

Upper Limit 31.472 29.570 

Weeks to First Observed Tumor 92 86 71 

Liver: Hepatocellular Carcinoma 3/13(0.23) 3/36(0.08) 6/32(0.19) 

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

Relative Risk (Control) 0.361 0.813 

Lower Limit 0.058 0.216 

Upper Limit 2.452 4.490 

Weeks to First Observed Tumor 91 85 92 

Treated groups received time-weighted average doses of 1746 or 3491 ppm in feed.
 

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

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

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

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

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

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

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH METHOXYCHLOR*
 

TOPOGRAPHY : MORPHOLOGY
 

Hematopoietic System: Malignant Lymphoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Uterus: Endometrial Stromal Polyp
 

P Values0
 

Relative Risk (Control)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW 

CONTROL DOSE 

1/20(0.05) 5/46(0.11) 

N.S. N.S. 

2.174 

0.271 

100.415 

92 79 

0/20(0.00) 1/17(0.06) 

N.S. N.S. 

Infinite 

0.064 

Infinite 

93 

1/20(0.05) 0/17(0.00) 

N.S. N.S. 

0.000 

0.000 

21.164 

92 

HIGH
 

DOSE
 

1/50(0.02)
 

N.S.
 

0.400
 

0.005
 

30.802
 

93
 

0/14(0.00)
 

N.S.
 

1/13(0.08)
 

N.S.
 

1.538
 

0.021
 

111.057
 

93 
TABLE 6 (Concluded) 

LOW HIGH 

TOPOGRAPHY : MORPHOLOGY CONTROL DOSE DOSE 

Ovary: Cystadenoma NOS 0/20(0.00) 0/17(0.00) 1/14(0.07) 

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

Relative Risk (Control) Infinite 

Lower Limit 0.079 

Upper Limit Infinite 

Weeks to First Observed Tumor 93 

Treated groups received time-weighted average doses of 997 or 1994 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.
 
of tumor that was observed in more than 5 percent of any of the
 

methoxychlor-dosed groups of either sex is included.
 

None of the statistical tests for mice of either sex indicated
 

a significant positive association between the administration of
 

methoxychlor and increased tumor incidence. Thus, at the dose levels
 

used in this experiment, there was no statistical evidence that
 

methoxychlor was a carcinogen in B6C3F1 mice.
 

To provide additional insight, 95 percent confidence intervals
 

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

based upon the observed tumor incidence rates. In 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 a
 

significantly increased rate of tumor incidence induced in mice by
 

methoxychlor that could not be established under the conditions of
 

this test.
 

42
 
V. DISCUSSION
 

Under the conditions of this bioassay, dietary administration of
 

methoxychlor was not associated with an increased incidence of any
 

type of neoplasm in rats or mice of either sex. No statistically
 

significant positive association was demonstrated between dosage and
 

mortality. In all treated groups there was dose-related mean body
 

weight depression when compared to controls.
 

Although hemangiosarcomas occur relatively infrequently in
 

Osborne-Mendel rats, the occurrence of these neoplasms in males (nine
 

low dose and two high dose) in this bioassay is not considered to be
 

compound-related, primarily due to the absence of a dose-related
 

response. No significant statistical association was established
 

between methoxychlor treatment and the incidence of any tumor in
 

either male or female rats.
 

The pathologist detected no tumors of histopathologic significance in mice of either sex, and no tumors in dosed mice were observed
 

at statistically significant incidences.
 

Methoxychlor has been previously assayed by the oral route in
 

the rat. In two studies (Radomski et al., 1965; Deichmann et al.,
 

1967), the incidence and distribution of both benign and malignant
 

tumors were similar in treated and control groups; while in a third
 

study (Hodge et al., 1952) the variety of tumors observed in the
 

treated animals, but not observed in the controls (with the exception
 

of one adenocarcinoma of the pancreas), was attributed to coincidence
 

43
 
since all these tumors commonly occurred in this particular colony of
 

rats. None of these three studies presented evidence of the carcinogenicity of methoxychlor in rats.
 

Under the conditions of the present bioassay, methoxychlor was
 

not found to be carcinogenic in Osborne-Mendel rats or B6C3F1 mice.
 

44
 
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, Washington, B.C., 1974.
 

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

J. Wiley & Sons, New York7~1971.~' ~ ~~
 

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

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

Union Against Cancer, Geneva, 1969.
 

Brooks, G.T., Chlorinated Insecticides. Volume I: Technology and
 

Application. CRC Press, Inc., Cleveland, Ohio, 1974.
 

Chemical Abstracts Service. The Chemical Abstracts Service (CAS)
 

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

Chemical Society, Washington, B.C., 1977.
 

Claborn, H.V., Insecticide Residues in Meat and Milk. U.S. Department
 

of Agriculture ARS-33-25, U.S. Government Printing Office,
 

Washington, D.C., 1956. (As cited in International Agency for
 

Research on Cancer, 1974.)
 

Corneliussen, P.E., "Pesticide Residues in Total Diet Samples (VI)."
 

Pesticide Monitoring Journal 5:513, 1972. (As cited in International Agency for Research on Cancer, 1974.)
 

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^~ ~
 

Deichmann, W.B., M. Keplinger, F. Salo, and E. Glass, "Synergism
 

Among Oral Carcinogens. IV: The Simultaneous Feeding of Four
 

Tumorigens to Rats." Toxicology and Applied Pharmacology 11:
 

88, 1967. (As cited in International Agency for Research on
 

Cancer, 1974.)
 

Duggan, R.E., "Chlorinated Pesticide Residues in Fluid Milk and Other
 

Dairy Products in the United States." Pesticide Monitoring
 

Journal 1:2, 1967. (As cited in International Agency for
 

Research on Cancer, 1974.)
 

45
 
Duggan, R.E., and P.E. Corneliussen, "Dietary Intake of Pesticide
 

Chemicals in the United States (ill), June 1968-April 1970."
 

Pesticide Monitoring Journal 5:331, 1972. (As cited in International Agency for Research on Cancer, 1974.)
 

Farm Chemicals Handbook. Meister Publishing Company, Willoughby,
 

Ohio, 1976.
 

Fowler, D.L., and J.N. Mahan, The Pesticide Review 1975. Agricultural Stabilization and Conservation Service, U.S. Department
 

of Agriculture, Washington, D.C., 1976.
 

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

Tests, Confidence Limits, and Adjustments for Stratification."
 

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

Hodge, H.C., E.A. Maynard, and H.J. Blanchet, Jr., "Chronic Oral
 

Toxicity Tests for Methoxychlor (2,2-di-[p-methoxyphenyl]-l,l,1­

trichloroethane) in Rats and Dogs." Journal of Pharmacology and
 

Experimental Therapeutics 104:60, 1952.(As cited in International Agency for Research on Cancer, 1974.)
 

Hodge, H.C., E.A. Maynard, W.L. Downs, J.K. Ashton, and L.L. Salerno,
 

"Tests on Mice for Evaluating Carcinogenicity." Toxicology and
 

Applied Pharmacology 9:583, 1966. (As cited in International
 

Agency for Research on Cancer, 1974.)
 

International Agency for Research on Cancer, "Methoxychlor." IARC
 

Monographs on the Evaluation of Carcinogenic Risk of Chemicals
 

to Man. Volume 5: Some Organochlorine Pesticides. 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.
 

Miles, J.R.W., W.W. Sans, H.B. Wressell, and G.F. Manson, "Growth-


Dilution as a Factor in the Decline of Pesticide Residues of
 

Alfalfa Grass Forage." Canadian Journal of Plant Science 44:
 

37, 1964. (As cited in International Agency for Research on
 

Cancer, 1974.)
 

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

Co., New York, 1966.
 

46
 
Ouellette, R.P., and J. King, "Pesticides '76." Chemical Week 118:
 

27-38, June 23, 1976.
 

Radomski, J.L., W.B. Deichmann, W.E. MacDonald, and E.M. Glass,
 

"Synergism Among Oral Carcinogens. I: Results of the Simultaneous Feeding of Four Tumorigens to Rats." Toxicology and
 

Applied Pharmacology 7:652, 1965. (As cited in International
 

Agency for Research on Cancer, 1974.)
 

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.
 

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

edition. Research Branch, Agriculture Canada, Publication 1093,
 

1973.
 

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

62_:679-682, 1975.
 

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

United States Production and Sales 1975. USITC Publication
 

804, Washington, B.C., 1977.
 

47
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH METHOXYCHLOR
 

TABLE A1

 

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

HIGH DOSE 

01-H02 5 

50 

50 

41 

(50) 

1 (2% ) 

(50) 

2 (4% ) 

1 (2%) 

(23) 

1 (4% ) 

1 (4%) 

(50) 

2 (4% ) 

(50) 

(42 ) 

2 (5%) 

ANIMALS INITIALL Y I N STUD Y 

ANIMALS NECROPSIED 

ANIMAL S EXAMINE D HI STOP AT HO LOGICALLY 

INTEGUMENTAR Y SYSTE M 

*SKIN 

FIBROSARCOH A 

*SUBCUT TISSUE 

FIBBOHA 

FIBBOSASCOBA 

LIPOSARCOM A 

HEMAtiGIOSA&COM A 

RESPIRATOR* SYSTEM 

#LUN G 

HEPATOCELLULA H CARCINOMA , METAST 

FISROSARCOMA , BETASTATI C 

MIXED TUMOR , METASTATIC 

OSTEOSARCCMA , METASTATIC 

HEMATOEOIETI C SYSTE M 

*MULTIPLE ORGAN S 

MALIG.LYMPHOMA , HISTIOCYTIC TYP E 

*SUBCUT TISSUE/BACK 

MALIG.LYKPHOMA , HISTIOCYTIC TYPE 

tSPLEE N 

HEMANGIOSABCOM A 

CIRCULATOR Y SYSTEM 

NON E 

CONTRO L (VEH ) 

01-M02 3 

20 

20 

** 20 

(20) 

(20) 

1 (5«) 

1 (5%) 

(20) 

1 (5*) 

(20 ) 

(20) 

(20 ) 

1 (5%) 

L OW DOSE 

01-M02 4 

50 

50 

44 

(50) 

(50) 

2 (4%) 

2 (4*) 

(20) 

1 (5X) 

1 (5*) 

(50) 

(50) 

1 (2%) 

(4H ) 

6 (14% ) 

* NUBBE R OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R OF ANIKAL S NECSOPSIED 

"EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-3 
TABLE Al (CONTINUED) 

DIGESTIVE SYSTE M 

fLIVER 

NEOPLASTIC NODULE 

HEPATOCELLULA R CARCINOM A 

tPANCREA S 

LIPOSARCOMA 

URINAR Y SYSTEM 

tKIDNE Y 

TRANSITIONAL-CELL CARCINOM A 

FIBROUS HISTIOCYTOMA, MALIGNAN T 

MIXED TUMOB, MALIGNANT 

OSTEOSARCCMA, BETASTATIC 

HAHARTCMA + 

ENDOCRIN E SYSTEM 

tPITUJCTAR Y 

CHKOMOPHOBE ADENOM A 

#ADRENA L 

CORTICAL CARCINOM A 

PHEOCHBOMOCYTOHA 

*THYROID 

FOLLICULAE-CELL

FOLLICULAR-CELL

C-CELL ADENOM A 

C-CELL CARCINOM A 

tPANCREATIC ISLETS 

 ADENOM A 

 CARCINOM A 

ISLET-CELL ADENOMA 

REPRODUCTIV E SYSTE M 

*MAMMAR Y GLAN D 

ADENOMA , NO S 

#TESTIS 

__IJ<UfiS!ITI4L-C£L,L_TUH.°H 

CONTROL (VEH ) 

01-M02 3 

(19) 

(19) 

(19) 

1 (5%) 

1 (5*) 

(18) 

3 (17%) 

(20) 

1 (5%) 

(19) 

1 (5*) 

2 (11*) 

(19) 

(20) 

(20) 

LOW DOSE 

01-M02 4 

(37) 

1 (3%) 

1 (3*) 

(20) 

1 (5X) 

(21) 

1 (5*) 

1 <5X) 

2 (10%) 

1 (5X) 

(21) 

8 (38%) 

(18) 

1 (6%) 

(27) 

1 (U%) 

"* (15%) 

1 (<*%) 

(20) 

1 (5%) 

(50) 

1 (2%) 

(19) 

HIGH DOSE 

01-H025 

(37) 

(20) 

(28) 

1 (4%) 

(22) 

4 (18%) 

(19) 

1 (5%) 

(2«) 

2 (8%) 

1 (U*) 

2 (8%) 

1 (t%) 

(20) 

1 (5%) 

(50) 

1 (2*) 

(21) 

._ _l_i5%J 

* NUMBER OF ANIMALS WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECHOPSIED 

+ 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-4
 
TABLE A 1 (CONTINUED) 

HIGH DOSE
 

01-M025
 

(50)
 

1 (2%)
 

(50)
 

(50)
 

1 (2X)
 

(50)
 

1 (2%)
 

50
 

13
 

37
 

*EPIDIDYMIS
 

FIBECSAECCMA
 

NERVOUS SYSTEM
 

NOME
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

*VEHTEBBAL COLUMN
 

OSIEOSARCOHA
 

*BONE/UPPER EXTREMITY
 

OSTEOSABCCMA
 

BODY CAVITIIS
 

*ABDOMINAL CAVITY
 

FXBBCSARCOBA
 

HEMANGIOSARCOMA
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL HISSING
 

a INCLUDES AUTOLY2£D ANIMALS
 

CONTROL (VEH)
 

01-M023
 

(20)
 

(20)
 

2 (10%)
 

(20)
 

(20)
 

20
 

11
 

1
 

8
 

LOW DOSE
 

01-M02«
 

(50)
 

(50)
 

(50)
 

(50)
 

1 (2%)
 

50
 

17
 

1
 

32
 

t NUMBE R OF ANIMALS WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMALS NECROPSIED 

A-5 
TABLE Al (CONCLUDED)
 

1CONTROL (VEH) LOU DOSE HIGH DOSE
 

01-M023 01-M02U 01-M025
 

TUMOR SUMMARY
 

TOTAL ANIMALS HUH PRIMARY TUMOfiS* 11 23 21
 TOTAL PRIMARY TUMOHS 13 37 26
 

TOTAL ANIMALS WITH BENIGN TUMOBS 7 13 11
 

TOTAL BENIGN TUMOBS 7 15 1U
 

TOTAL ANIMALS HITH MALIGNANT TUMORS 6 17 11
 

TOTAL MALIGNANT TUMORS 6 21 12
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1 2 2
 

TOTAL SECONDARY TUMORS 2 2 2
 

TOTAL ANIMALS WITH TDHORS UNCERTAINBENIGN OR MALIGNANT 1
 

TOTAL UNCERTAIN TUMORS 1
 

TOTAL ANIMALS HITH TUHORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TOHOBS
 

* SECONDARY TUMORS: ME1ASTATIC TUMORS OH TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

A-6
 
TABLE A2 

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

ANIBALS INITIALLY IN STUD Y 

ANIMALS NECEOPSIED 

ANIMAL S EXAMINE D HISTOPATHOLOGICA LLY ** 

INTEGUMENTAR Y SYSTE M 

*SUBCUT TISSUE 

SUUAMOU S CELL CARCINOM A 

FIBROM A 

FIBRCSARCCM A 

LIPCBA 

RESPIRATOR Y SYSTEM 

*LUN G 

SQUAMOU S CELL CARCINOMA , HEPATOCELi-ULA R CARCINOMA , METASTA 

 METAST 

HEMATOPOIETIC SYSTEM 

*MULTIPL E ORGAN S 

MALIG.LYMPHOMA ,

tSPLEE N 

HEMANGIOSAECOM A 

CIRCULATOR Y SYSTE M 

tENDOCABDIU M 

SARCOMA , NOS

 

DIGESTIV E SYSTE M 

*LIVER 

 LYMPHOCYTIC TYP E 

 1_J5S1 

» NUMBE R OF ANIMAL S HITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIE D 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

NEOPLA3TIC NODUL E 

HEPATOCELLULA R CARCIJJOMA_ _ __ _.

CONTRO L (VEH ) 

01-F023 

20 

20 

20 

(20) 

1 (5X) 

(20) 

1 (5X) 

(20 ) 

1 (5%) 

(20) 

1 (5%) 

(20) 

(20) 

L CW DOSE 

C1-F026 

50 

50 

1*7 

(50) 

1 (2%) 

4 (9%) 

1 (2%) 

1 <2X) 

(33) 

1 (3S) 

(50) 

C*6) 

(32) 

1 (3%) 

(467 

1 (2X) 

HIG H DOSE 

01-F027 

50 

50 

49 

(50) 

1 (2») 

(33) 

(50) 

(1*8) 

(30) 

(47) 

1 (2%) 

1_12£1_. 

A-7 
TABLE A2 (CONTINUED) 

*BILI DUCT 

BILE DUCT ADENOM A 

URINAR Y SYSTEM 

tKIDNE Y 

HAMARTOMA + 

ENDOCRIN E SYSTE M 

JPITUI1ARY 

CHRCMOPHOBE ADENOM A 

fTHYfiOID 

FOLLICULAH-CELL

C-CELL ADENOM A 

C-CELL CARCINOM A 

REPRODUCTIVE SYSTEM 

*MAMMAR Y GLAN D 

ADENOMA , NOS 

ADENOCARCINOMA,

FIBBCADENCM A 

*VAGIN A 

LEIOMYOSARCOMA 

*UTERUS 

 ADENOM A 

 NOS 

ENDOMETHIAL STROMA1 POLYP 

• OVAR Y 

CYSTADENOMA , NOS 

FIBROSARCOMA 

NERVOU S SYSTEM 

*CEREBRU M 

ASTROCYTOMA 

SPECIAL SENSE ORGAN S 

NONE _. 

CONTROL (VEH) 

01-F023 

(20) 

(20) 

1 (5X) 

(20) 

8 (14 OX) 

(20) 

(20) 

1 (5%) 

8 (4 0%) 

(20) 

(19) 

(19) 

(20) 

LOB DOSE HIGH DOSE 

01-F026 01-FC27 

(50) (50) 

1 <2X) 

(32) (30) 

(32) (36) 

7 (22%) 10 (28%) 

(28) (31)

 

2 (7X) 

3 (10X) 

1 (U%) 

(50) (50) 

2 (4X) 

1 (2%) 3 (6%) 

16 (32*) 9 (18%) 

(50) (50)

 

1 (2%) 

(34) (35) 

1 (354) a (11X) 

(30) (30) 

1 (3%) 

1 (JX) 

(32) (30) 

1 (3%) 

* NUMBER OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECBOPSIED 

+ 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-8 
TABLE A2 (CONTINUED)
 

CONTRO L (VEH ) 

01-F023 

LOW DOSE 

01-F026 

HIG H DOSE 

01-F027 

MUSCULOSKELETAL SYSTEM 

*VEE1EEBAL COLUM N (20) (50) (5C) 

OSTEOSAKCCMA 1 (2X) 

BODY CAVITIE S 

'ABDOMINA L CAVIT Y (20) (50) (50) 

FIBROMA 1 (2») 

OSTEOSARCOMA , METASTATIC 1 (2») 

A L L OTHE B SYSTEMS 

NON E 

ANIKA L DISPOSITION SUKMAK Y 

ANIMAL S INITIALL Y I N STUD Y 20 50 50 

NATURA L DEATH S 6 3 4 

MORIBUN D SACRIFICE 1 4 

SCHEEULE D SACRIFICE 

ACCIDENTALL Y KILLE D 

TERMINA L SACRIFICE 13 43 U6 

ANIHA L HISSING 

2_!fiCLU£ES_AUTOLXZED_ANJM£LS 

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

* NUMBE R OF ANIMAL S NECROESIED 

A-9 
TABLE A2 (CONCLUDED) 

;ONTROL (VEH) LOW DCSE HIGH DOSE
 

C1-F023 C1-F026 01-F027
 

TUMOR SUMMARY
 

TOTAL ANIMAL S KITH ERIMARY TUMORS* 12 30 30
 

TOTAL PRIMARY TUMORS 22 t1 37
 

TOTAL ANIMALS WITH BENIGN TUMCRS 12 2b 26
 TOTAL BENIGN TUMORS 18 32 30
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 4 8 6
 TOTAL MALIGNANT TUMORS 4 8 6
 

TOTAL ANIMALS BITH SECONDARY TUMOHS* 1 2
 

TOTAL SECONDARY TUMORS 1 2
 

TOTAL ANIMALS WITH IUMCBS UNCERTAINBENIGN OR MALIGNANT 1 1
 

TOTAL UNCERTAIN TUMORS 1 1
 

TOTAL ANIMALS WITH TUMOES UNCERTAINPRIMARY OH KFIASTATIC
 

TOTAL UNCERTAIN TUMOES
 

* PRIMARY lUtiORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

* SECONDARY TUMORS: NE1ASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT CRGAN
 

A-10 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH METHOXYCHLOR
 

TABLEBl

 

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

CONTROL (VEfl) LOW DOSt HIGH DOSE
 

02-H02<» 02-M025 02-M026
 

ANIMALS INITIALLY IN STUCK 20 50 50
 

ANIMALS MISSING 1
 

ANIMALS NECHOPSIED 12 44 U7
 

ANIMALS EXAMINED HISTCPATHOLOGICA LL Y** 12 35 31
 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (12) (UK) (47)
 

FIBROSARCOMA 1 (8%) 2 (5%) 2 (U%)
 

BESPIEATCBY SYSTEM
 

NCNE
 

HEHATOFCIETIC SYSTEM
 

*MULTIPLE OEGANS (12) (KK) (47)
 

GRANULOCYTIC LEUKEMIA 2 (US)
 

tCERVICAL LYMPH NODE (13) (36) (31)
 

FISBOSABCGHA, METASTATIC 1 (3X)
 

#MESENTERIC L. NODE (13) (36) (31)
 

FIBBOSAKCOMA, METASTATIC 1 (3%)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVER (13) (36) (32)
 

HEPATOCELLULAR CARCINOMA 3 (23X) 3 (3%) 6 (19X)
 

UBINARY SYSTEM
 

N2NE __
 

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

* NUMBE B OF ANIMAL S NECROPSIE D 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-3 
ENDOCRINE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEM
 

NONE
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NCNE
 

MUSCULOSKELEIAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHEB SYSTEMS
 

NONE
 

ANIMAL EISPCSITION SUMMAR Y
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHIEULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIf.AL MISSING
 

£_INCLUDES_AUTO.LYZED_ ANIMALS
 

TABLEBl (CONTINUED) 

CONTROL (VEH)
 

02-M02I*
 

20
 

12
 

8
 

LOU DOSE HIGH DOSE
 02-M025 02-M026
 

50 50
 

28 17
 

2
 

20 32
 

1
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECHOESIED
 

B-4
 
TABLE Bl (CONCLUDED) 

CONTROL (VEH ) L OW DOSE HIG H DOSE 

02-M02 U 02-M025 02-M026 

TUMO R SUMMAR Y 

TOTAL ANIMAL S WIT H PRIMAR Y TUMORS* 3 5 9 

TOTAL PRIMAR Y TUMORS 4 5 10 

TOTAL ANIMAL S WIT H BENIGN TUMORS 

TOTAL BENIG N TUHOBS 

TOTAL ANIMAL S WIT H MALIGNAN T TUMORS 3 5 9 

TOTAI MALIGNAN T TUMOR S 4 5 10

 

TOTAL ANIMAL S KITH SECONDAR Y TUMORS * 1 

TOTAL SECONDARY TUMORS 2 

TOTAL ANIMAL S WITH TUMOR S UNCERTAIN ­

BENIG N O R MALIGNAN T 

TOTAL UNCERTAI N TUMOBS 

TOTAL ANIMAL S WITH TUMORS UNCERTAIN ­

PRIMAR Y OB METASTATIC

 

TOTAL UNCERTAI N TUMOR S 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECONDARY TUMORS: ME1ASTATIC 1UMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-5 
TABLE B2

 

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

CONTROL (VEH) LOW DOSE HIGH DOSE 

02-F02H 02-F027 02-F028 

ANIMAL S JNITIAIL Y I N STUD Y 20 50 50 

ANIMAL S MISSING 1 

ANIMAL S KECROPSIED 20 46 50 

ANIMALS EXAMINE D HISTOEATHOLOGICALL Y* * 20 16 1U 

INTEGUMENTAR Y SYSTEM 

NON E 

RESPIRATOR Y SYSTEM 

NON E 

HEMATOECIETI C SYSTE M 

*MULTII>LE ORGAN S (20) (46) (50)

 

MALIG.LYMPHOMA , LYMPHOCYTI C TYP E 1 (5*)

 

MALIG.LYMPHOMA , HIS1IOCYTIC TYPE 1 (9%) 1 (2%)

 

tCOLON (20) (17) (14)

 

MALIG.LYMPHOMA , HISTIOCYTIC TYPE 1 (6»)

 

CIRCULATOR Y SYSTE M 

NON E 

DIGESTIVE SYSTEM 

ILIVER (20) (17) (1U)

 

HEPATOCEiLULAa CAfiCINOM A 1 (6%) 

URINAR Y SYSTE M 

NON E 

ENDCCRIS E SYSTE M 

#ADRENA L (19) (17) (13) 

COHTICAL_ABEHOH A J_I5»L — _

.__.__.._—.._—.— —.-.—..—, 

* NUMBE R OF ANIMAL S »ITH TISSUE EXAMINE D MICROiT.OPICALLK 

* NUMBE B OF ANIMAL S NECHOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-6 
TABLE B2 (CONTINUED) 

CONTROL (VEH) LOW DOSE HIGH DOSE
 

02-F02a 02-F027 02-F028
 

REPRODUCTIVE SYSTEM
 

tUTERUS (20) (17) (13)
 

ENDOMETRIAL SIHOWAL POLYP 1 <5K) 1 <8X)
 

tOVARY (20) (17) (14)
 

CYSTADENCMA, NOS 1 <7X)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHHB SYSTEMS
 

NOME
 

ANIMAL DISPOSITION SUMMABY
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

NATURAL DEATHS 3 5 1
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 17 44 49
 

ANIHAL MISSING 1
 

a INCLUDES AUTOLYZED ANIMAL S__ _.
 

# NUMBE B OF ANIMAL S WITH TISSUE EXAHINE D MICROSCOPICALLY 

* NUMBE B OP ANIMAL S NECfiOPSIED 

B-7
 
TABLE B2 (CONCLUDED)
 

CONTROL (VEH ) LOW DOSE HIGH DOSE 

02-F02U 02-F027 02-F028 

TUMO R SUMMAR Y 

TOTAL ANIMAL S WIT H PRIMAR Y lUliORS* 3 6 3 

TOTAL PRIMAR Y TUMORS 3 6 3 

TOTAL ANIMAL S WITH BENIG N TUMCRS 2 2 

TOTAL BENIG N TUMOBS 2 2 

TOTAL ANIMAL S WITH MALIGNAN T TUMOR S 1 6 1 

TOTAI MALIGNAN T TUMOBS 1 6 1 

TOTAL ANIMALS WITH SECONDARY TUMORS* 

TOTAL SECONDAR Y TUMORS 

TOTAL ANIHAL S WITH TUMOR S UNCEBTAINBENIG N O R MALIGNAN T

 

TOTAL UNCERTAI N TUMORS 

TOTAL ANIMALS KITH TUMOBS UNCERTAIN ­

PRIMAR Y 08 METASTATIC 

TOTAL UNCERTAI N TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECONDARY TUMORS: ME1ASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-8 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH METHOXYCHLOR
 

TABLE Cl 

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

HIGH DOSE
 

01-M025
 

50
 

50
 

41
 

(50)
 

1 (2X)
 

(50)
 

1 (2X>
 

(23)
 

1 (4X)
 

(23)
 

(20)
 

(42)
 

1 (2X)
 

2 (5X)
 

(20)
 

(20)
 

6_J30XI
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY **
 

INTEGUMIKTAEY SYSTEM
 

*SKIN
 

EPIDERMAL INCLUSION CYST
 

INFLAMMATION. NOS
 

ABSCESS, NOS
 

*SUBCUT TISSUE
 

ABSCESS, NOS
 

BESPIBATCRY SYSTEM
 

tLUNG
 

PNEUMONIA, CHBONIC MUBINE
 

#ALVEOLAR WALL
 

MIKEBALIZATION
 

HEMATOECIETIC SYSTEM
 

*BONE MABROW
 

METAMORPHOSIS FATTY
 

ISPLEEN
 

HYPERTROPHY, NOS
 

HEMATOPOIESIS
 

CIBCULAT08Y SYSTEM
 

*HEAKT
 

ABTEBIOSCLEBOSIS, NOS
 

CALCIUM DEPOSIT
 

tMYOCABDIUM
 

INFLAMMATION, NOS
 

DEGENERATION^. NOS
 

* NUBBEB OF ANIMAL S WITH TISSUE

* NUMBE R OF ANIHALS NECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

<;ONTROL (VEH)
 

01-M023
 

20
 

20
 

20
 

(20)
 

4 (20*)
 

1 (5»)
 

(20)
 

2 (10%)
 

(20)
 

3 (15X)
 

(20)
 

1 <5X)
 

(20)
 

1 (5*)
 

(20)
 

1 (5*)
 

(20)
 

1 (5%)
 

1 (5«)
 

(20)
 

._ 8_JU2«1
 

LOW DOSE
 

01-M024
 

50
 

50
 

44
 

(50)
 

2 <4X)
 

(50)
 

(20)
 

3 (15*)
 

<20)
 

(18)
 

(44)
 

3 (7*)
 

(21)
 

(21)
 

2 (W)
 

6_J29JL
 

 EXAMINE D MICROSCOPICALLY 

C-3 
____

•ENDOCARDIUM
 

INFLAMMATION, NOS
 

HYPEEPLASIA, NOS
 

*AORTA
 

ARTERIOSCLEROSIS, NOS
 

MEDIAL CALCIFICATION
 

CALCIUM DEPOSIT
 

*MESEN1ERIC ARTERY
 

ARTERIOSCLEROSIS, NOS
 

DIGESTIVE SYSTEM
 

*LIVER
 

CYST, NOS
 

THROMBOSIS, NOS
 

INFLAMMATION, NOS
 

INFLAMMATION, GBANULOMATOUS
 

METAHOREHOSIS FATTY
 

*BILE DUCT
 

HYPERPLASIA, NOS
 

tPANCREAS
 

PEBIARTERITIS
 

fSTOMACH
 

INFLAMMATION, NOS
 

ULCER, FOCAL
 

CALCIUM DEPOSIT
 

URINARY SYSTEM
 

tKIDNEY
 

INFLAMMATION, SUPPURATIVE
 

INFLAMMATION, CHRONIC
 

NEPHROPATHY, TOXIC
 

ENDOCRINE SYSTEM
 

tADRENAL
 

_ ANGIECTASIS.
 

TABLECl (CONTINUED)
 

CONTROL (VEH)
 

01-M023
 

(20)
 

1 (5X)
 

(20)
 

1 <5X)
 

2 <10X)
 

(20)
 

2 (10X)
 

(19)
 

1 (5X)
 

a (21*)
 

(20)
 

1 (5*)
 

(19)
 

1 (5«)
 

(20)
 

1 (5X)
 

3 (15X)
 

(19)
 

12 (63X)
 

(20)
 

__ _-l_15iL
 

LOR DOSE
 

01-M02U
 

(21)
 

2 (10X)
 

(50)
 

1 (2%)
 

1 (2X)
 

(50)
 

(37)
 

1 <3X>
 

3 <8X)
 

4 (11X)
 

(50)
 

(20)
 

(20)
 

1 (5X)
 

2 (10X)
 

(21)
 

2 (10X)
 

3 (1KX)
 

1 (5X)
 

(18 _ ) _
 

HIGH DOSE
 

01-M025
 

(20)
 

1 <5X)
 

(50)
 

(50)
 

(37)
 

1 (3X)
 

1 (3X)
 

3 (8X)
 

(50)
 

1 (2X)
 

(20)
 

(21)
 

1 (5X)
 

(28)
 

9 (32X)
 

1 (UX)
 

(19)
 _ ______———
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

C-4
 
tTHYROIE
 

CYSTIC FOLLICLES
 

HYPERPLASIA. C-CELL
 

tPARATHYROID
 HYPERPLASIA, NOS
 

REPRODUCTIVE SYSTEM
 

*MAKMARY GLAND
 

GALACTOCELE
 

#TESTIS
 

INFLAMMATION, NOS
 

CALCIUM DEPOSIT
 

ATROPHY, NOS
 

*EPIDIDYMIS
 

ATBOPHY, NCS
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE OfiGANS
 

*EYE
 

CATARACT
 

*EYE/CORNEA
 INFLAMMATION. NOS
 

*EYE/CILIARY BODY
 INFLAMMATION, NOS
 

*EYE/IRIS
 

INFLAMMATION, NOS
 

*EYE/RETINA
 

INFLAMMATION, NOS
 

*EYE/CONJUNCTIVA
 

TABLE C'l (CONTINUED)
 

CONTHOL(VEH)
 

01-M023
 

(19)
 

(19)
 

2 (11*)
 

(20)
 

1 (5%)
 

(20)
 

1 (58)
 

6 (30*)
 

(20)
 

3 (15*)
 

(20)
 

(20)
 

(20)
 

(20)
 

(20)
 

(20)
 

LOW DOSE HIGH DOSE
 

01-M02"* 01-M025
 

(27) (2«)
 

1 <4X)
 

1 (4*)
 

(27)	 (23)
 

3 (11X) 2 (9X)
 

(50)	 (50)
 

1 (2X)
 

(19)	 (21)
 

1 (5X)
 

2 (11X) 8 (38X)
 

(50) (50)
 

(50)	 (50)
 

1 (2*)
 

(50)	 (50)
 

1 (2X>
 

(50)	 (50)
 

1 (2X)
 

(50)	 (50)
 

1 (2*)
 

(50)	 (50)
 

1 (2X)
 

(50) (50)
 

*	 NUMBEfi OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 *	 NUMBER OF ANIMALS NECROPSIED
 

C-5
 
TABLECl (CONCLUDED) 

CONTROL (VEH ) LOW DOSE 

01-M023 01-M024 

HUSCULOSKELETA L SYSTE M 

*MDSCLE HIP/THIGH (20) (50) 

INFLAMMATION , NOS 1 (2X) 

DEGENERATION , NOS 

BODY CAVITIES 

*ABDOBINA L CAVIT Y (20) (50) 

NECROSIS, FAT 1 (5X) 3 (6X) 

*PERICARDIUM (20) (50) 

INFLAMMATION , NOS 1 (5%) 1 <2X) 

*MESENTER Y (20) (50) 

PERIAHTERITIS 1 (5X) 

NECROSIS, FAT 1 (2X) 

ALL OTHER SYSTEMS 

NON E 

SPECIAI HOBEHOLOG Y SUMMAR Y 

NO LESION REPORTED 1 12 

NECROPSY PERF/NO HISTO PERFORMED 6 

AOTC/NECROESY/HISTO PERF 1 

AUTO/NECROPSY/NO HISTO 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUHBEB OF ANIMALS NECHCPSIED
 

HIG H DOSE 

01-M025 

(50) 

1 (2%) 

(50) 

1 (2X) 

(50) 

(50) 

1 (2X) 

10 

6 

3 

C-6
 
TABLE C2 

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

CONTROL (VEH ) LOW DCSE 

01-F023 01-F026 

ANIMAL S INITIALLY IN STUDY 20 50 

ANIMAL S NECROPSIE D 20 50 

ANIMAL S EXAMINE D HISTOPATHOLOGICA LLY ** ' 20 HI 

INTEGUMENTAR Y SYSTEM 

NON E 

RESPIRA1CR Y SYSTEM 

tLUN G (20) (33) 

INFLAMMATION , SUPEURATIV E 

PNEUMONIA , CHRONIC MUEIN E 3 (15*) 1 (3S) 

HEHATOEOIETIC SYSTEM 

#SPLEE N (20) (16) 

HEMATOPOIESI S 3 (15*) 1 (2X) 

CIRCUIATOR Y SYSTE M 

tMYOCARDIU M 

DEGENERATION , NOS 

(20) 

2 (10%) 

(32) 

4 (13X) 

#ENDOCARDIU M 

HYPEBPLASIA, NOS 

(20) (32) 

1 (3*) 

DIGESTIV E SYSTE M 

*LIVER (20) C*6) 

INFLAMMATION , CHRONIC 1 (2«) 

METAMORPHOSIS FATTY 1 (2X) 

FOCAL CELLULA R CHANG E 

*BILE DUCT (20) (50) 

_ HYPEBPLASIA.I NOS J_i2Jl 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECHOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

HIGH DOSE 

01-F027 

50 

50 

U9 

(33) 

1 <3X ) 

2 (6%) 

C*8) 

2 (4%) 

(30) 

4 (13X) 

(30) 

(U7 ) 

1 (2X) 

(50) 

C-7 
TABLE C2 (CONTINUED) 

CONTROL (VEH ) LOK DOSE 

01-F023 01-F026 

#STOMACH (20) (31)

 

ULCER, FOCAL 2 (10%) 

UBINAfi Y SYSTE M 

tKIDNE Y (20) (32) 

HYERONEPHSOSI S 1 (3%) 

IHFLAKBATION , SUPPUEATIV E 1 (5%) 

INFLAMMATION , CHfiCNIC 3 (15%) 1 <3X) 

CALCIU M DEPOSIT 

ENCOCRIN E SYSTE M 

IADRENA L (20 ) (30) 

ANGIECTASI S 1 (3%) 

tPARAIHYROI D (19) (28)

 

HYPEEPLASiA , DOS 1 (5%)

 

REPRODUCTIV E SYSTE M 

*VAGIN A (20) (50)

 

INFLAMMATION . NOS 2 (10%)

 

POLY E 

IUIERU S (19) P<*) 

HYDEOMETR A 2 (11%) 3 (9X) 

CYST, NOS 

INFLAMMATION , NO S 1 (5%) 3 (9%) 

tCEFVI X OTERI (19) (31) 

EPIDERMA L INCLUSION CYST 

INFLAMMATION , NO S 1 (3S) 

tUTERUS/ENDOMETRIU H i19) (3U) 

HYPE8PLAS1A, CYSTIC 5 (15X) 

NERVOU S SYSTEM 

HON E 

* N'JMBER OF ANIMAL S WIT H TISSUE EXAMINE D SICHOSCOPICALLY 

* NUMBE R OF ANIMALS NECBOESIED 

HIG H DOSE 

01-F027 

(31) 

(30) 

5 (17*) 

1 (3%) 

(31) 

1 (3X) 

(31) 

(50) 

1 (2%) 

(35) 

H (11*) 

1 (3%) 

(35) 

1 (3») 

(35) 

2 (6X) 

r_c L o 
TABLE C2 (CONCLUDED) 

SPECIAL SENSE ORGAN S 

*EYE 

SYNECHIA , POSTERIOR 

CATARACT 

CALCIU M DEPOSIT

 

*EYE/RETIN A 

INFLAMMATION , NOS 

MUSCULOSKELETA L SYSTE M 

NON E 

BODY CAVITIES 

*ABDOHINA L CAVIT Y 

NECROSIS, FAT 

*MESENTER Y 

NECROSIS, FAT 

A LL OTHEfi SYSTEM S 

NON E 

SPECIAL KORPHOLOG Y SUMMAR Y 

NO LESION REPORTE D 

NECROPS Y PERF/NO HISTO PERFORME D 

CONTROL (VEH ) LOW DOSE HIG H DOSE 

Q1-P023 01-F026 01-F027 

(20) (50) (50) 

1 (2*) 

2 (14%) 2 (4%) 

1 (2%) 

(20) (50) (50) 

1 (2%) 

(20) (50) (50) 

1 <2X ) 

(20) (50) (50) 

1 (2% ) 

5 12 12 

3 1 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

C-9
 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH METHOXYCHLOR
 

TABLE Dl 

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

(:ONTROL (VEH) 

02-M024 

LOU DOSE 

02-M025 

HIGH DOSE 

02-M026 

ANIMALS INITIALLY IN STUDY 

ANIMALS MISSING 

ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY** 

20 

12 

12 

50 

44 

35 

50 

1 

47 

31 

INTEGUMENTARY SYSTEM
 

• SKIN (12) (44) (47)
 

INFLAMMATION, NOS 2 (5X)
 

*SUBCUT TISSUE (12) (44) (47)
 

ABSCESS, NOS 3 (6%)
 

RESPIRATORY SYSTEM
 

ILUNG (13) (36) (33)
 

PNEUMONIA, CHRONIC MURINE 2 (6X)
 

HEMATOPCIETIC SYSTEM
 

* SPLEEN (12) (36) (33)
 AMYIOIDOSIS 4 (33X) 9 (25X) 2 (6X)
 

HEMATOPOIESIS 1 (3X) 1 <3X)
 

tHESENTERIC L. NODE (13) (36) (31)
 CONGESTION, NOS 1 (8X)
 

EDEMA, NOS 1 (8X)
 

CIRCUIATORY SYSTEM
 

IHEARI (13) (36) (33)
 THROMBOSIS, NOS 2 (6X)
 

•MYOCARDIUM	 (13) (36) (33)
 

INFLAMMATION, NOS 1 (3*)
 DEGENERATION, NOS 1 (3X)
 

•AORTA	 (12) (44) (47)
 

* NUMBER OF ANIMALS WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMALS NECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-3 
TABLEDl (CONTINUED) 

CONTROL (VEH) LOW DOSE HIGH DOSE
 

02-M02U 02-M025 02-M026
 

DIGESTIVE SYSTEM
 

•LIVER	 (13) (36) (32)
 

INFLAMMATION, NOS 1 (3X)
 METAHOBPHOSIS FATTY 1 (8%)
 

UBINARY SYSTEM
 

IKIDNEY (12) (36) (33)
 

HYCBCNEPHBOSIS 1 (8X) 1 (3X)
 CYST, NOS 1 (3X)
 

PYELONEPHRITIS. NCS 1 (8S) 2 <6S) 1 (3%)
 

INFLAMMATION, CHRONIC 3 (25%) 8 (22%) 1 (3*)
 

AdYLCIDOSIS 1 (8X) U (11S)
 

CALCIUM DEPOSIT 2 (6X)
 

iURINARY BLADDER (11) (35) (33)
 

INFLAMMATION, NOS 1 (3S)
 

ENDOCRIKI SYSTEM
 

NONE
 

REPRODUC1IVE SYSTEM
 

*PREPU1IAL GLAND (12) (UH) (47)
 

INFLAMMATION, NOS 1 (8*)
 

*SEMINAL VESICLE (12) (44) (47)
 

INFLAMMATION, NOS 1 (2%)
 

ATROPHY, NOS 1 (2%)
 

*TES1IS (11) (35) (32)
 

CALCIUM DEPOSIT 1 (3X)
 

ATRCEHY, NOS 3 (9%)
 

*EPIDIDYHIS (12) (44) (47)
 

INFLAMMATION, NOS 1 (2X)
 

GRANULOMA, SPERMATIC 5 (11»)
 

*SCROTUM (12) (44) (47)
 

INFLAMMATION, NOS 2 <5X)
 

NERVOUS SYSTEM
 

NONE
 

f NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

D-4
 
TABLE Dl (CONCLUDED) 

CONTROL (VEH ) 

02-S02H 

LOW DOSE 

02-M025 

HIGH DOSE 

02-M026 

SPECIAL SENSE OfiGAN S 

NON E 

MUSCULOSKELETA I SYSTEM 

NON E 

BODY CAVITIE S 

NON E 

A LL OTHE R SYSTEMS 

NCN E 

SPECIAL HORFHOLOG Y SUMMAR Y 

NO LESION REPORTED 

ANIMA L MISSING/NO NECROPSY 

NECROPSY PERF/NO HISTO PEEFORMED 

ADTO/NECfiOPSY/HISTO PEHF 

AUT01YSIS/NO NECBOPSY 

14 

a 

11 

9 

u 

6 

13 

1 

16 

2 

D-5 
TABLE D2 

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

CONTROL (VEH) LOW DOSE HIGH DOSE
 02-F024 02-F027 02-J028
 

ANIMAIS INITIALLY IN STUDY 20 50 50
 ANIHALS MISSING 1
 

ANIKALS NECROPSIED 20 46 50
 ANIMALS EXAMINED HISTOEATHQLOGICALL Y** 20 16 1U
 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

NONE
 

HEMATOEOIETIC SYSTEM
 

KCNE
 

CIRCUIATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#STOMACH (20) (17) (It)
 INFLAMMATION, NOS 1 (7»)
 

URINARY SYSTEM
 

NONE
 

ENDCCRItiE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEM
 

*UTERUS (20) (17) (13)
 HYCRCMETRA 4 (20%) 1 (6»)
 

INFLAMMATION.. NOS l_-li*l
 

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

* NUMBER OF ANIMALS NECBOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

D-6 
TABLE D2 (CONCLUDED) 

hlbH DOLE
 

Ci-f 026
 

(13)
 

(11)
 

(50)
 

10
 

36
 

#UTERU£/ENDCMETRIUM
 

HYPIRPLASIA, CYSTIC
 

#OVARY
 

CYST. DOS
 

INFLAMMATION, NOS
 

NERVOUS SYSTEM
 

NCNE
 

SPECIAL SENSE ORGANS
 

*EYE
 

INFLAMMATION, NOS
 

CATAEAC1
 

MUSCULOSKELETAL SYSTEM
 

NCNE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL BOREhOLOGY SUMMAR Y
 

NO LESION REPORTED
 

ANIMAL MISSING/NO NECROPSY
 

NECROPSY PEHF/NO HISTO PERFORMED
 

AUTOLYSIS/NO NECROPSY
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED


* NUMBER OF ANIMALS NECHOPSIED
 

LO'STfcOL (VT-h)
 

02-1 CiU
 

(20)
 

3 (15*)
 

(20)
 

5 (25X)
 

(20)
 

1 (5%)
 

1 (5%)
 

6
 

 MICROSCOPICALLY
 

LO» LOSE
 

Gl'-F 0^; 7
 

(17)
 

'4 (21%)
 

(17)
 

2 (12S)
 

1 (6%)
 

(U6)
 

6
 

1
 

JO
 

j
 

D-7
 

Review of the Bioassay of Methoxychlor* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

November 28, 1977
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976 under the authority of the National
 

Cancer Act of 1971 (P.L. 92-218). The purpose of the
 

Clearinghouse is to advise on the National Cancer Institute's
 

bioassay program to identify and 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 organic chemistry, bio­ chemistry, 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 NCI
 

bioassay reports on chemicals studied for carcinogenicity.
 

In this context, below is the edited excerpt from the
 

minutes of the Subgroup's meeting at which Methoxychlor was
 

reviewed.
 

The primary reviewer agreed with the conclusion stated
 

in the report, i.e., that Methoxychlor was not carcinogenic
 

in either the rats or mice under the conditions of test. An
 

experimental shortcoming, mentioned by the reviewer, was
 

that other chemicals were tested at the same time and in the
 

same room as Methoxychlor. He also noted that the treated
 

animals were stressed as a result of the high dosages tested
 

and that the number of matched control animals were inadequate
 

In conclusion, he opined that Methoxychlor is a safe pesticide
 

with respect to human risk.
 

A motion was made that the report on the bioassay of
 

Methoxychlor be accepted. The motion was seconded and
 

approved unanimously.
 

49
 
Members present were:
 

Gerald N. Wogan (Chairman), Massachusetts Institute of
 

Technology
 

Lawrence Garfinkel, American Cancer Society
 

Henry C. Pitot, University of Wisconsin Medical Center
 

George Roush, Jr., Monsanto Company
 

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
 

John H. 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.
 

50
 

MIS GOVERNMENT PRINTING OFFICE: 1978 J60-899/3014 1-3 

DHEW Publication No. (NIH) 78-835