1979 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 135 

BIOASSAY OF 

MALAOXON 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 1634-78-2 

NCI-CG-TR-135 

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

Public Health Service 

National Institutes of Health 

BIO AS SAY OF
 

MALAOXON
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20205
 

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

Public Health Service
 

National Institutes of Health
 

NIH Publication No. 79-1390
 

BIOASSAY OF
 

MALAOXON
 

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
 

malaoxon conducted for the Carcinogenesis Testing Program, Division
 

of Cancer Cause and Prevention, National Cancer Institute (NCI),
 

National Institutes of Health, Bethesda, Maryland. This is one of a
 

series of experiments designed to determine whether selected environmental chemicals have the capacity to produce cancer in animals. A
 

negative result, in which the test animals do not have a greater
 

incidence of cancer than control animals, does not necessarily mean
 

that a test chemical is not a carcinogen, inasmuch as the experiments
 

are conducted under a limited set of circumstances. A positive
 

result demonstrates that a test chemical is carcinogenic for animals
 

under the conditions of the test and indicates that exposure to the
 

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

risk to man from chemicals found to be carcinogenic in animals
 

requires a wider analysis.
 

CONTRIBUTORS; This bioassay of malaoxon was conducted by Gulf South
 

Research Institute (GSRI), New Iberia, Louisiana, initially under
 

direct contract to NCI and currently under a subcontract to Tracer
 

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

The experimental design for this bioassay is based on guidelines for
 

carcinogen bioassays in small animals that have been established by
 

NCI (Sontag et al., 1976). The doses for the chronic studies were
 

selected by Drs. E. E. Storrs (1) and 0. G. Fitzhugh (2,3). The
 

principal investigator was Mr. R. J. Wheeler (1). Histologic examination of rats was performed, by Dr. E. Bernal (1), and histologic
 

examination of mice was performed by Dr. B. Buratto (2). The diagnoses included in this report represent the interpretation of these
 

pathologists.
 

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

Mason Research Institute (4). Statistical analyses were performed
 

by Dr. J. R. Joiner (2) and Ms. P. L. Yong (2), using methods
 

selected for the bioassay program by Dr. J. J. Gart (5). Chemicals
 

ill
 
were synthesized and analyzed at GSRI by Dr. E. Green (1) and dosed
 

feed mixtures by Mr. S. M. Billedeau (1). The results of these
 

analyses were reviewed by Dr. C. W. Jameson (2,7).
 

This report was prepared at Tracer Jitco (2) under the direction of
 

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

R. Angel, Acting Director of the Bioassay Program; Dr. S. S. Olin,
 

Deputy Director for Science; Dr. J. F. Robens (8), toxicologist; Dr.
 

R. L. Schueler, pathologist; Ms. L. A. Owen and Mr. W. D. Reichardt,
 

bioscience writers; and Dr. E. W. Gunberg, technical editor,
 

assisted by Ms. Y. E. Presley.
 

The following scientists at NCI (6) were responsible for evaluating
 

the bioassay experiment, interpreting the results, and reporting the
 

findings: Dr. Kenneth C. Chu, Dr. Cipriano Cueto, Jr., Dr. J.
 

Fielding Douglas, Dr. Richard A. Griesemer, Dr. Charles K.
 

Grieshaber, Dr. Thomas E. Haran, Dr. William V. Hartwell, Dr. Morton
 

H. Levitt, Dr. Harry Mahar, Dr. Harry A. Milman, Dr. Thomas W. Orme,
 

Dr. A. R. Patel, Dr. Marcelina B. Powers, Dr. Sherman F. Stinson,
 

Dr. Jerrold M. Ward, and Dr. Carrie E. Whitmire.
 

(1) Gulf South Research Institute, Atchafalaya Basin
 

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

(2) Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

(3) 4208 Dresden Street, Kensington, Maryland.
 

(4) EG&G Mason Research Institute, 1530 East Jefferson
 

Street, Rockville, Maryland.
 

(5) Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute,
 

National Institutes of Health, Bethesda, Maryland.
 

(6) Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes
 

of Health, Bethesda, Maryland.
 

(7) Now with Carcinogenesis Testing Program.
 

(8) Now with the Bureau of Veterinary Medicine, Food and Drug
 

Administration, 5600 Fishers Lane, Rockville, Maryland.
 

iv
 
SUMMARY
 

A bioassay of malaoxon, the oxygen analogue of malathion (an
 

organophosphate insecticide), for possible carcinogenicity was
 

conducted by administering the test chemical in feed to F344 rats
 

and B6C3F1 mice.
 

Groups of 50 rats and 50 mice of each sex were fed diets containing
 

500 or 1,000-ppm malaoxon for 103 weeks and were then observed for
 

up to an additional 2 weeks. Matched controls consisted of groups
 

of 50 untreated rats and 50 untreated mice of each sex. All
 

surviving animals were killed at 103 to 105 weeks.
 

The only effects that could be related to administration of malaoxon
 

at the doses used were increased mortality among male mice, decreased
 

mean body weights of female mice, gastric ulcers in male and female
 

rats, and possibly C-cell adenomas or carcinomas of the thyroid
 

among treated female rats. The incidence of C-cell adenomas or
 

carcinomas among historical controls, however, precluded relating
 

the incidence of these tumors to administration of the chemical.
 

It was concluded that under the conditions of this bioassay malaoxon
 

was not carcinogenic for F344 rats or B6C3F1 mice.
 

TABLE OF CONTENTS
 

Page
 

I. Introduc tion	 1
 

II. Materials and Methods	 3
 

A. Chemical	 3
 

B. Dietary Preparation	 3
 

C. Animals	 5
 

D. Animal Maintenance	 5
 

E. Subchronic Studies	 6
 

F. Chronic Studies	 10
 

G. Clinical Examinations and Pathology	 10
 

H. Data Recording and Statistical Analyses	 13
 

III. Results - Rats	 17
 

A. Body Weights and Clinical Signs (Rats)	 17
 

B. Survival (Rats)	 19
 

C. Pathology (Rats)	 21
 

D. Statistical Analyses of Results (Rats)	 22
 

IV. Results - Mice	 35
 

A. Body Weights and Clinical Signs (Mice)	 35
 

B. Survival (Mice)	 37
 

C. Pathology (Mice)	 39
 

D. Statistical Analyses of Results (Mice)	 39
 

V. Discussion	 47
 

VI.	 Bibliography 51
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Table Al Summary of the Incidence of Neoplasms in
 

Rats Administered Malaoxon in the Diet 53
 

Male Rats Administered Malaoxon in the Diet.... 55
 

Table A2 Summary of the Incidence of Neoplasms in
 

Female Rats Administered Malaoxon in the Diet.. 59
 

vii
 
Appendix B Summary of the Incidence of Neoplasms in 

Mice Administered Malaoxon in the Diet 63 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Administered Malaoxon in the Diet.... 65 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Administered Malaoxon in the Diet.. 69 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Administered Malaoxon 

in the Diet 73 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Administered Malaoxon in 

the Diet 75 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Administered Malaoxon in 

the Diet 79 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Administered Malaoxon in 

the Diet 83 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Administered Malaoxon in 

the Diet 85 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered Malaoxon in 

the Diet 88 

Appendix E Analysis of Malaoxon 91 

Appendix F Analyses of Formulated Diets 

for Concentrations of Malaoxon 97 

TABLES 

Table 1 Experimental Design for Subchronic Malaoxon 

Feeding Studies in Rats 8 

Table 2 Experimental Design for Subchronic Malaoxon 

Feed ing Stud ies in Mice 9 

viii
 
Table 3
 

Table 4
 

Table 5
 

Table 6
 

Table 7
 

Table 8
 

Figure 1
 

Figure 2
 

Figure 3
 

Figure 4
 

Figure 5
 

Figure 6
 

Page 

Experimental Design for Chronic Malaxon 

Feed ing Stud ies in Rats 11 

Experimental Design for Chronic Malaoxon 

Feeding Studies in Mice 12 

Analyses of the Incidence of Primary Tumors 

in Male Rats Administered Malaoxon 

in the Diet 24 

Analyses of the Incidence of Primary Tumors 

in Female Rats Administered Malaoxon 

in the Diet 30 

Analyses of the Incidence of Primary Tumors 

in Male Mice Administered Malaoxon 

in the Diet 41 

Analyses of the Incidence of Primary Tumors 

in Female Mice Administered Malaoxon 

in the Diet 44 

FIGURES 

Growth Curves for Rats Administered 

Malaoxon in the Diet.., 18 

Survival Curves for Rats Administered 

Malaoxon in the Diet 20 

Growth Curves for Mice Administered 

Malaoxon in the Diet 36 

Survival Curves for Mice Administered 

Malaoxon in the Diet 38 

Infrared Absorption Spectrum of Malaoxon 95 

Nuclear Magnetic Resonance Spectrum of 

Malaoxon 96 

ix
 

I. INTRODUCTION
 

Malaoxon, 0,0-dimethyl S-l,2-bis
 

(ethoxycarbony1)ethyl phosphoroCH30
 

thioate (CAS 1634-78-2; NCI CO ^P—SCHCOOC H
 

S* . 2 Ei
 

8628), is an oxygen analogue of CH^U Q^J COOC H
 

malathion, a widely used organophosphate insecticide. Malathion
 

Malaoxon
 

is converted to malaoxon by the
 

substitution of oxygen for sulfur;
 

this conversion is catalyzed rn
 

vivo by mixed function oxidases in mammals and insects, by peroxidases in plants, and by sunlight (Eto, 1974). This step appears to
 

4
 be an activation process, since malaoxon is 10 times more potent
 

as a cholinesterase inhibitor than malathion (Eto, 1974) and is more
 

toxic in acute oral doses than malathion. Malaoxon has an intraperitoneal LDr-n in male Sprague-Dawley rats of 25 mg/kg, compared with
 

900 mg/kg for malathion (Brodeur and Dubois, 1967). Malaoxon is
 

relatively nonpersistent in the environment, having a half-life of
 

approximately 7 days in slightly acidic Illinois loam soil (Paschal
 

and Neville, 1976).
 

Malaoxon was selected by the Carcinogenesis Bioassay Program because
 
it is the active metabolite of malathion, a chemical also tested in
 

the program (NCI, 1978; NCI, 1979).
 
II. MATERIALS AND METHODS
 

A. Chemical
 

Malaoxon is not available commercially. The material used for the
 

chronic phase of/ the study was synthesized in one batch by Gulf
 

South Research Institute by the method of Ailman (1965) and purified
 

by large-scale column chromatography and/or distillation. Analysis
 

of the malaoxon at Gulf South Research Institute included elemental
 

analysis, thin-layer and vapor-phase chromatography, and infrared
 

and nuclear magnetic resonance spectrometry (Appendix E). The
 

results identified the material as malaoxon of high purity (i.e.,
 

greater than 95%). The bulk material was stored at 5 C until
 

mixed with the feed.
 

®* Dietary Preparation
 

®
 All diets were formulated using Wayne Lab-Blox Meal (Allied
 

Mills, Chicago, Illinois) to which was added the required amount of
 

malaoxon for each dietary concentration. The test compound was
 

first dissolved in a small amount of acetone (Mallinckrodt.
 

Chemicals, St. Louis, Mo.) which was then added to the feed. Corn
 
oil (Louana , Opelousas Refinery, Opelousas, Louisiana) was also
 

added to the feed, primarily as a dust suppressant, and the diets
 

were mixed mechanically for not less than 25 minutes to assure
 

homogenity and to allow for evaporation of the acetone. Final
 

diets, including those for control groups of animals, contained corn
 

oil equal to 2% of the final weight of feed. Formulated diets were
 

stored at room temperature until used, but not longer than 1 week.
 

The stability of malaoxon in feed was tested by determining the
 

concentration of the compound in formulated diets at intervals over
 

a 7-day period. Analysis of the diets containing 500 and 1,000 ppm
 

malaoxon showed no significant change in concentration on standing
 

at ambient temperature for this period.
 

As a quality control analysis for accuracy of preparation of the
 

diets, the concentration of malaoxon was measured in randomly
 

selected batches of formulated diets at 8-week intervals during the
 

chronic study. At each dietary concentration, the mean of the
 

analytical concentration was within 2.1% of the theoretical
 

concentration, and the coefficient of variation did not exceed 3.4%
 

(Appendix F).
 
C. Animals
 

Male and female F344 rats and B6C3F1 mice, 4 and 5 weeks of age
 

respectively, were obtained from the Frederick Cancer Research
 

Center, (Frederick, Maryland). The animals were acclimated for 14
 

days and then assigned to control or dosed groups.
 

D. Animal Maintenance
 

Rats were housed individually in hanging galvanized steel mesh cages
 

(Hoeltge, Inc., Cincinnati, Ohio), and mice were housed in polypropylene cages (Lab Products, Inc., Garfield, N.J.), five females
 

or two or three males per cage. Mouse cages were covered with
 

polyester filter bonnets (Lab Products, Inc.). Rat racks and cages
 

were washed every 2 weeks and mouse cages each week. Cages and
 

racks were washed in an industrial washer (Industrial Washing
 

Machine Corp., Matawan, N.J.) at 82 C with Acclaim detergent
 

(Economics Laboratory, Inc., St. Paul, Minn.) and then rinsed.
 

/a
 

Absorbent Kimpak cage liners (Kimberly Clark Corp., Neenah, Wis.)
 

were placed under the rat cages and changed three times per week.
 

Absorb-dri hardwood chip bedding (Lab Products, Inc.), used in
 

the mouse cages, was provided two times per week for males and three
 

times per week for females. Filter bonnets were washed each week.
 
Feed jars and water bottles were changed and washed three times per
 

week; sipper tubes and stoppers were washed two times per week; the
 

filter bonnets, feed jars, water bottles, sipper tubes, and stoppers
 

were washed in a Vulcan Autosan washer (Vulcan Autosan, Louisville,
 

Ky.). Cage racks for each species were rotated to a new position in
 

the room once each week; at the same time, each cage was moved to a
 

different row within the same column of a rack. Control and dosed
 

rats were housed on the same rack, whereas cages for control and
 

dosed mice were placed on separate racks in the same room. The rats
 

and mice receiving malaoxon were housed in separate rooms with their
 

respective controls. Malaoxon was the only compound on study in
 

each room.
 

Air was maintained at 22 to 24 C, and relative humidity was 40 to
 

70%. Fresh air, filtered through permanent air maze filters (Air
 

Maze Incom International, Cleveland, Ohio), was changed 10 to 12
 

times per hour. Fluorescent lighting provided illumination 10 hours
 

per day. Food and tap water were provided ad libitum. Twice a week
 

excess remaining feed was discarded and fresh feed was provided.
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted to determine the two
 
concentrations used in the chronic studies (referred to in this
 

report as "low" and "high" doses). Groups of 10 rats and 10 mice of
 

each sex were fed diets containing malaoxon at one of several doses
 

for 13 weeks, and groups of 10 control animals of each species and
 

sex were fed basal diet only. Animals were weighed each week.
 

Tables 1 and 2 show the doses fed, the survival of animals in each
 

dosed group at the end of the study, and the mean body weights of
 

dosed animals at week 13, expressed as percentages of mean body
 

weights of the corresponding controls. At the end of the 13-week
 

period, all surviving animals were killed and necropsied. Tissues
 

from all rats on feed containing 1,000 and 2,000 ppm and from all
 

mice on feed containing 2,000 and 4,000 ppm were examined
 

microscopically for pathologic change.
 

All rats fed 4,000 and 8,000 ppm died, and 16 of 20 mice fed 8,000
 

ppm died. There was no depression in weight gain compared with
 

controls in either rats or mice in groups having no deaths. No
 

gross or microscopic pathologic changes were observed that could be
 

related to administration of the test chemical in the rats or the
 

mice.
 

Based on the mortality data and on previous experience with the
 

estimation of doses for chronic studies with organophosphates at
 
Table 1. Doses, Survival, and Mean Body Weights
 

of Rats Fed Malaoxon for 13 Weeks
 

Male Female 

Dose(a) 

(ppm) Survival (b) 

Mean Weight 

at Week 13 

as % of 

Control Survival (a) 

Mean Weight 

at Week 13 

as % of 

Control 

0(c) 10/10 100 10/10 100
 

125 10/10 102 10/10 103
 

250 10/10 100 10/10 105
 

500 10/10 102 10/10 104
 

l,000(d) 10/10 104 10/10 100
 

2,000(d) 10/10 99 10/10 96
 

4,000 0/10 0/10
 

8,000 0/10 0/10
 

(a) No gross pathologic changes were found at necropsy in any of the
 

test groups.
 

(b) Number surviving/number in group.
 

(c) One male control showed mild diffuse parenchymatous hepatic
 

degeneration.
 

(d) Tissues from males and females administered these doses were
 

examined microscopically. One male in the 1,000 ppm group showed
 

focal fatty degeneration of the liver.
 
Table 2. Doses, Survival, and Mean Body Weights 

of Mice Fed Malaoxon for 13 Weeks 

Male Female 

Mean Weight Mean Weight 

at Week 13 at Week 13 

Dose(a) as % of as % of 

(ppm) Survival (b) Control Survival (a) Control 

0 10/10 100 10/10 100 

125 10/10 101 10/10 93 

250 10/10 102 10/10 96 

500 10/10 95 10/10 101 

1,000 10/10 99 10/10 100 

2,000(c) 10/10 105 10/10 105 

4,000(c) 10/10 101 10/10 99 

8,000 3/10 98 1/10 88 

(a) No gross pathologic changes were found at necropsy in any of the
 

test groups.
 

(b) Number surviving/number in group.
 

(c) Tissues from males and females administered these doses were
 

examined microscopically and were found to be essentially normal.
 
this laboratory, the low and high doses for the chronic studies were
 

set at 500 and 1,000 ppm for both the rats and the mice.
 

F. Chronic Studies
 

The test groups, doses administered, and durations of the chronic
 

feeding studies are shown in tables 3 and 4.
 

G. Clinical Examinations and Pathology
 

All animals were observed twice per day for signs of toxicity,
 

weighed at 2-week intervals, and palpated for masses at each
 

weighing. Observations of sick, tumor-bearing, and moribund animals
 

were recorded daily. Moribund animals and animals that survived to
 

the end of the bioassay were killed using pentobarbitol and
 

necropsied.
 

Pathology consisted of gross and microscopic examination of major
 

tissues, major organs, and all gross lesions from killed animals and
 

from animals found dead. The following tissues were examined
 

microscopically: skin, lungs and bronchi, trachea, bone and bone
 

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

10
 
Table 3. Experimental Design for Chronic Malaoxon
 

Feeding Studies in Rats
 

Sex and Initial Malaoxon Time on Study
 

Test No. of Doses (b) Dosed Observed
 

Group Animals (a) (ppm) (weeks) (weeks)
 

Male
 

Matched-Control 50 0 0 103-105
 

Low-Dose 50 500 103 0-1
 

High-Dose 50 1,000 103 0-1
 

Female
 

Matched-Control 50 0 0 103-105
 

Low-Dose 50 500 103 0-1
 

High-Dose 50 1,000 103 0-2
 

(a) All animals were 6 weeks of age when placed on study.
 

(b) Test and control diets were made available ad libitum.
 

11
 
Table 4. Experimental Design for Chronic Malaoxon
 

Feeding Studies in Mice
 

Sex and Initial Malaoxon Time on Study
 

Test No. of Doses (b) Dosed Observed
 

Group Animals (a) (ppm) (weeks) (weeks)
 

Male
 

Matched-Control 50 0 0 103-105
 

Low-Dose 50 500 103 0-2
 

High-Dose 50 1,000 103 0-2
 

Female 

Matched-Control 50 0 0 103-105

 

Low-Dose 50 500 103 0-1

 

High-Dose 50 1,000 103 0-1

 

(a) All animals were 7 weeks of age when placed on study. 

(b) Test and control diets were made available ad libitum. 

12 
gallbladder (mice), pancreas, stomach, small intestine, large
 

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

parathyroid, mammary gland, prostate or uterus, testis or ovary, and
 

brain. Occasionally, additional tissues were also examined microscopically. The tissues were preserved in 10% neutral buffered
 

formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Special staining techniques were utilized as
 

necessary for more definitive diagnosis.
 

Necropsies were also performed on all animals found dead, unless
 

precluded in whole or in part by autolysis or cannibalization. Thus,
 

the number of animals from which particular organs or tissues were
 

examined microscopically varies and does not necessarily represent
 

the number of animals that were placed on study in each group.
 

H. Data Recording and Statistical Analyses
 

Data on this experiment were 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 (Berenblura, 1969).
 

13
 
Probabilities of survival were estimated by the product-limit
 

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

report in the form of graphs. Animals were statistically censored
 

as of the time that they died of other than natural causes or were
 

found to be missing; animals dying from natural causes were not
 

statistically censored. Statistical analyses for a possible
 

dose-related effect on survival used the method of Cox (1972) for
 

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

Cox's methods for testing for a dose-related trend. One-tailed P
 

values have been reported for all tests except the departure from
 

linearity test, which is only reported when its two-tailed P value
 

is less than 0.05.
 

The incidence of neoplastic or nonneoplastic lesions has been given
 

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

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

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

denominators included only those animals for which that site was
 

examined histologically. However, when macroscopic examination was
 

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

or mammary tumors), or when lesions could have appeared at multiple
 

sites (e.g., lymphomas), the denominators consist of the numbers of
 

animals necropsied.
 

14
 
The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

animals. As a part of these analyses, the one-tailed Fisher exact
 

test (Cox, 1970) was used to compare the tumor incidence of a control
 

group with that of a group of dosed animals at each dose level.
 

When results for two dosed groups are compared simultaneously with
 

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

significance level of 0.05 may be made.
 

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

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

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

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

0.05 level of significance. Unless otherwise noted, the direction
 

of the significant trend is a positive dose relationship. This
 

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

trend.
 

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 lower and upper limits of the confidence interval of the
 

15
 
relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in approximately
 

95% of a large number of identical experiments, the true ratio of
 

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

would be within the interval calculated from the experiment. When
 

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

can be inferred that a statistically significant result (P less than
 

0.025 one-tailed test when the control incidence is not zero, P less
 

than 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.
 

16
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

There was no appreciable effect of administration of malaoxon on
 

mean body weights of male or female rats (figure 1).
 

During the first 4 months of the study, the dosed and control rats
 

were generally comparable in appearance. One tissue mass was noted
 

during week 2 in the high-dose male group. At week 4 the low- and
 

high-dose males were noted as being hyperexcitable, but this
 

condition did not persist.
 

During the remainder of the test, alopecia, rough discolored hair
 

coats, disfcolored urine, tachypnea, loss of weight, poor food
 

consumption, higher incidence of vaginal bleeding in the low-dose
 

females, hyperexcitability (not general, but a few individual cases
 

in all groups), lethargy, pale mucous membranes, abdominal distension, cyst-like growths, and palpable nodules and tissue masses were
 

noted in the controls and dosed groups with increasing frequency.
 

At week 78 the majority of control and dosed animals rejected their
 

feed. This almost total rejection continued for 4 days, at which
 

time all animals were given freshly mixed control feed containing 2%
 

17
 
a

o 

o 

\­

m 

0 

MALE RATS 

a D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKS) 

X 

o 

o 

o 

m 

LU 

s 

FEMALE RATS 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves for Rats Administered Malaoxon in the Diet 

18 
corn oil. After the animals were given the control diet for 4 days,
 

they were returned to their assigned test diets. As a result of the
 

food rejection, mean body weight decreased in all groups, but the
 

decrease was much greater in the males.
 

B. Survival (Rats)
 

Estimates of the probabilities of survival for male and female rats
 

administered malaoxon in the diet at the doses of this bioassay,
 

together with those of the matched controls, are shown by the Kaplan
 

and Meier curves in figure 2. The result of the Tarone test for
 

dose-related trend in the proportions surviving is not significant
 

in either sex. In female rats, an indicated departure from linear
 

trend (P = 0.041) is observed because the control animals did not
 

survive as long as the dosed animals. The result of the Cox test
 

comparing the survival of the control and low-dose groups is
 

significant (P = 0.025) in the negative direction.
 

In male rats, 37/50 (64%) of the high-dose group, 41/50 (82%) of the
 

low-dose group, and 40/50 (80%) of the control group were alive at
 

week 90 on study. In females, 40/50 (80%) of the high-dose group,
 

45/50 (90%) of the low-dose group, and 41/50 (82%) of the control
 

group were alive at week 90 on study.
 

19
 
PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVAL 
Sufficient numbers of rats of each sex were at risk for the development of late-appearing tumors.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

summarized in Appendix C tables Cl and C2.
 

A wide variety of degenerative inflammatory, proliferative, and
 

neoplastic lesions were observed in rats of the dosed and control
 

groups. The incidences of these lesions, with few exceptions, were
 

approximately equal in control and dosed animals. Occasionally, a
 

lesion occurred principally in rats of either dosed or control
 

groups; however, the incidences of these changes were generally low
 

(less than 5%) and were not considered to be related to the
 

administration of the chemical.
 

Gastric ulcers were seen* in increased incidences in dosed rats —
 

males: control 2/48 (4%), low-dose 6/50 (12%), high-dose 7/48
 

(15%); females: control 0/49 (0%), low-dose 1/49 (2%), high-dose
 

3/49 (6%); it was most commonly seen in the forestomach. The lesion
 

21
 
was usually solitary and sometimes accompanied by epithelial
 

hyperplasia and submucosal inflammation.
 

The histopathologic examination provided no evidence that malaoxon
 

was carcinogenic under the conditions of this bioassay in F344 rats.
 

D. Statistical Analyses of Results (Rats)
 

Tables 5 and 6 contain the statistical analyses of the incidences of
 

those primary tumors that occurred in at least two animals of one
 

group and at an incidence of at least 5% in one or more than one
 

group.
 

In male rats, the results of the Cochran-Armitage test for dose-


related trend in incidences of tumors and those of the Fisher exact
 

test comparing the incidences of tumors in the control group with
 

those in each dosed group, are not significant.
 

In female rats, the result of the Cochran-Armitage test for the
 

combined incidence of C-cell carcinomas and adenomas of the thyroid
 

is significant (P = 0.009). The Fisher exact test shows that the
 

incidence in the high-dose group is significantly higher (P = 0.024)
 

than that in the control group. The historical record of this
 

22
 
laboratory shows an incidence of female F344 rats with C-cell
 

adenomas or carcinomas of 16/223 (7%), compared with 0/50 in the
 

control group, 1/49 (2%) in the low-dose group, and 5/47 (11%) in
 

the high-dose group of this study. This indicates that the
 

incidence of C-cell tumors of the thyroid in female rats of the
 

present study is comparable to that usually seen in control animals.
 

In female rats, the Fisher exact comparison of the incidences of
 

fibroadenomas of the mammary gland in the low-dose and control
 

groups shows a P value of 0.026, which is above the 0.025 level
 

required for significance when the Bonferroni inequality criterion
 

is used for multiple comparison. The result of the Cochran-Armitage
 

test on the incidence of this tumor is not significant, and no
 

significant incidence is observed in the high-dose group.
 

23
 
Table 5. Analyses of the Incidence

Administered Malaoxon

 of

 in

 Primary Tumors in

 the Diet (a) 

 Male Rats 

Topography: Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Integumentary System: Fibroma 

of the Subcutaneous Tissue (b) 3/50 (6) 1/50 (2) 0/50 (0) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.333 

0.007 

3.983 

0.000 

0.000 

1.662 

Weeks to First Observed Tumor 98 104 — 

Hematopoietic System:

or Leukemia (b) 

 Lymphoma 

19/50 (38) 13/50 (26) 16/50 (32) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.684 

0.352 

1.292 

0.842 

0.463 

1.517 

Weeks to First Observed Tumor 73 81 
Table 5. Analyses of the Incidence of Primary Tumors in Male Rats 

Administered Malaoxon in the Diet (a) 

(continued) 

Topography: Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Pituitary: Carcinoma, NOS (b) 3/45 (7) 1/45 (2) 2/45 (4) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk ( f) 

Lower Limit 

Upper Limit 

0.333 

0.007 

3.964 

0.667 

0.058 

5.542 

Weeks to First Observed Tumor 72 98 86 

NJ 

Ln 

Pituitary: Adenoma, NOS (b) 17/45 (38) 20/45 (44) 18/45 (40) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

1.176 

0.682 

2.044 

1.059 

0.597 

1.882 

Weeks to First Observed Tumor 79 62 75 
Table 5. Analyses

Adm

 of

inis

 the

tered

 Incidence of

 Malaoxon in

 Primary

 the Diet

 Tumors

 (a) 

 in Male Rats 

(continued) 

Topography: Morphology 

Matched

Control

 Low 

 Dose 

High 

Dose 

Pituitary:

Adenoma,

 Carcinoma,

 NOS (b) 

 NOS or 

20/45 (44) 21/45 (47) 20/45 (44) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk ( f ) 

Lower Limit 

Upper Limit 

1.050 

0.639 

1.728 

1.000 

0.601 

1.664 

Weeks to First Observed Tumor 72 62 75 

Adrenal: Pheochromocy toma (b) 3/47 (6) 4/49 (8) 6/49 (12) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk ( f ) 1.279 1.918 

Lower Limit 0.229 0.437 

Upper Limit 8.316 11.263 

Weeks to First Observed Tumor 105 80 68
 
Table 5. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Malaoxon in the Diet (a)
 

(continued)
 

Topography: Morphology


Thyroid: Follicular-cell Adenoma
 

or Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Thyroid: C-cell Adenoma or
 

Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low High 

 Control Dose Dose 

 3/49 (6) 0/45 (0) 2/49 (4) 

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

0.000 0.667 

0.000 0.058 

1.805 5.565 

 100 102 — 

 2/49 (4) 0/45 (0) 4/49 (8) 

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

0.000 2.000 

0.000 0.302 

3.671 21.298 

100 72 
00 

Table 5. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Malaoxon in the Diet (a)
 

(continued)
 

Matched Low
 

Topography: Morphology Control Dose
 

Pancreatic Islet: Islet-cell Carcinoma
 

or Adenoma (b) 5/48 (10) 6/49 (12)
 

P Values (c,d) N.S. N.S.
 

Relative Risk (f) 1.176
 

Lower Limit 0.321
 

Upper Limit 4.557
 

Weeks to First Observed Tumor 77 96
 

Test is: Interstitial-cell Tumor (b) 41/50 (82) 44/49 (90)
 

P Values (c,d) N.S. N.S.
 

Relative Risk (f) 1.095
 

Lower Limit 0.914
 

Upper Limit 1 . 268
 

Weeks to First Observed Tumor 73 80
 

High
 

Dose
 

2/50 (4)
 

N.S.
 

0.348
 

0.038
 

2.219
 

104
 

41/49 (84)
 

N.S.
 

1.020
 

0.840
 

1.231
 

68 
Table 5. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Malaoxon in the Diet (a)
 

(continued)
 

(a) Dosed groups received 500 or 1,000 ppm.
 

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

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P is less than 0.05, otherwise, not significant (N.S.) is indicated. Beneath
 

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

for the comparison of that dosed group with the matched-control group when P is less than 0.05;
 

otherwise not significant (N.S.) is indicated.
 

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

(e) The probability level for departure from linear trend is given when P is less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
 
Table 6. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Malaoxon in the Diet (a)
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Hematopoietic System: Lymphoma
 

or Leukemia (b) 13/50 (26) 5/50 (10) 10/50 (20)
 

P Values (c,d) N.S. P = 0.034 (N) N.S.
 

Relative Risk (f) 0.385 0.769
 

Lower Limit 0.116 0.334
 

Upper Limit 1.054 1.715
 

Weeks to First Observed Tumor 22 104 86
 

Liver: Neoplastic Nodule or
 

Hepatocellular Carcinoma (b) 0/46 (0) 3/50 (6) 0/49 (0)
 

P Values (c,d) N.S. N.S.
 —
 

Departure from Linear Trend (e) P = 0.016
 

Relative Risk (f) Infinite
 —
 Lower Limit 0.555
 —
 Upper Limit Infinite
 —
 

Weeks to First Observed Tumor 104
 
Table 6. Analyses of the Incidence of Primary Tumors in Female Rats 

Administered Malaoxon in the Diet (a) 

(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

Pituitary: Carcinoma, NOS (b) 4/49 (8) 1/49 (2) 6/49 (12) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk ( f ) 0.250 1.500 

Lower Limit 0.005 0.380 

Upper Limit 2.409 6.811 

Weeks to First Observed Tumor 69 104 63 

Pituitary: Carcinoma, NOS or 

Adenoma, NOS (b) 35/49 (71) 34/49 (69) 32/49 (65) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 0.971 0.914 

Lower Limit 0.737 0.686 

Upper Limit 1.284 1.228 

Weeks to First Observed Tumor 69 83 63 
Table 6. Analyses of the Incidence of

Administered Malaoxon in

(continued) 

Matched
 

Topography: Morphology Control
 

Thyroid: C-cell Carcinoma or
 

Adenoma (b) 0/50 (0)
 

P Values (c,d) P = 0.009
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

NJ 

Mammary Gland: Fibroadenoma (b) 2/50 (4)
 

P Values (c,d) N.S.
 

Departure from Linear Trend (e) P = 0.002
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 94
 

 Primary Tumors in Female Rats 

 the Diet (a) 

Low
 

Dose
 

1/49 (2)
 

N.S.
 

Infinite
 

0.055
 

Infinite
 

94
 

9/50 (18)
 

P = 0.026
 

4.50"0
 

0.995
 

41.081
 

83
 

High
 

Dose
 

5/47 (11)
 

P = 0.024
 

Infinite
 

1.342
 

Infinite
 

96
 

1/50 (2)
 

N.S.
 

0.500
 

0.009
 

9.290
 

105
 
Table 6. Analyses of the Incidence of Primary Tumors in Female Rats 

Administered Malaoxon in the Diet (a) 

(continued) 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Uterus: Endometrial Stromal
 

Polyp (b) 11/46 (24) 8/47 (17) 11/48 (23)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk (f) 0.712 0.958
 

Lower Limit 0.274 0.419
 

Upper Limit 1.760 2.194
 

Weeks to First Observed Tumor 93 104 86
 

u> 

OJ 

(a) Dosed groups received 500 or 1,000 ppm.
 

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

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P is less than 0.05, otherwise, not significant (N.S.) is indicated. Beneath
 

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

for the comparison of that dosed group with the matched-control group when P is less than 0.05;
 

otherwise not significant (N.S.) is indicated.
 

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

(e) The probability level for departure from linear trend is given when P is less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
 
34

 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

There was no appreciable effect of administration of malaoxon on
 

mean body weights of male mice (figure 3). Mean body weights of
 

high-dose female mice were lower than those of the controls
 

throughout most of the bioassay, and mean body weights of the
 

low-dose females were lower than those of the controls after about
 

week 34.
 

During the first year of the study, the dosed and control mice were
 

generally comparable in appearance and behavior. At week 12 the
 

high-dose males and females appeared hyperexcitable, but this
 

condition did not persist. Although all male mice were observed
 

fighting, the resulting wounds and rough or missing hair were more
 

severe among the dosed males.
 

Clinical signs that were noted with increasing frequency during the
 

second ffir of the study included alopecia, pale mucous membranes,
 

abdominal distension, and palpable nodules and tissue masses.
 

Several aairoa's exhibited a hunched, slab-sided appearance. These
 

signs were observed in all groups but were predominant in the dosed
 

35
 
DO DDDQ pgnAo ofiAoinnn _A 

IH 30­

D 

O 

CO 

Z 20-

< 

MALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

40 BO 60 70 

TIME ON STUDY (WEEKS) 

D 

DDDD D 

H 3()_ DD Q°°.OOOOAAAAAQQOOOOO Q/ 

S OOoSA

AAA

 A

 O 

A 

tn OA 

Z 20 

UJ 

FEMALE MICE 

a MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

10 20 30 40 50 60 70 80 90 100 

TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves for Mice Administered Malaoxon in the Diet 

36 
males. At week 72, 10% of the high-dose females and at week 73, 30%
 

of the low-dose females appeared hyperexcitable, but this condition
 

did not persist.
 

B. Survival (Mice)
 

Estimates of the probabilities of survival for male and female mice
 

administered malaoxon in the diet at the doses of this bioassay,
 

together with those of the matched controls, are shown by the Kaplan
 

and Meier curves in figure 4. The result of the Tarone test for
 

positive dose-related trend in the proportions surviving is
 

significant (P = 0.028) in male mice but not in the females.
 

In male mice, 37/50 (74%) of the high-dose group, 42/50 (84%) of the
 

low-dose group, and 45/50 (90%) of the control group were alive at
 

week 103. In females, 45/50 (90%) of the high-dose group, 38/50
 

(76%) of the low-dose group, and 39/50 (78%) of the control group
 

were alive at week 103.
 

Sufficient numbers of mice of each sex were at risk for the development of late-appearing tumors.
 

37
 
PROBABILITY OF SURVIVAL PROBABILITY OF SURVIV

o p o o o 

-> O O 

MA 

oo 

tf 
C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

summarized in Appendix D, tables Dl and D2.
 

A variety of tumors occurred in both the control and dosed groups.
 

Some types of neoplasms occurred only, or with a slightly greater
 

frequency, in the dosed groups; however, the incidence of tumors did
 

not appear to be related to the administration of malaoxon.
 

In addition to the neoplastic lesions, a large number of degenerative, proliferative, and inflammatory changes which showed no
 

relationship to administration of the test chemical were encountered
 

in animals of the dosed and control groups.
 

The histopathologic examination provided no evidence that malaoxon
 

was carcinogenic in B6C3F1 mice under the conditions of this
 

bioassay.
 

D. Statistical Analyses of Results (Mice)
 

Tables 7 and 8 contain the statistical analyses of the incidences of
 

39
 
those primary tumors that occurred in at least two animals of one
 

group and at an incidence of at least 5% in one or more than one
 

group.
 

In each sex, the results of the Cochran-Armitage test for positive
 

dose-related trend in the incidence of tumors and the results of the
 

Fisher exact test comparing the incidence of tumors in the control
 

group with that in each dosed group in the positive direction are
 

not significant. Although the first observation of a hepatocellular
 

tumor in male mice occurred at week 65 in the high-dose group,
 

compared with week 97 in the control group and week 95 in the
 

low-dose group, the overall comparison of the time of observation of
 

hepatocellular tumors in the three groups by life table methods was
 

not found to be significant. Significant results in the negative
 

direction are observed in the incidences of liver tumors in each sex.
 

In each of the 95% confidence intervals for relative risk shown in
 

the tables, the value of one or less than one is included: this
 

indicates the absence of significant positive results. It should
 

also be noted that each of the intervals, except that for the
 

incidence of hepatocellular carcinomas in low-dose male mice, has an
 

upper limit greater than one, indicating the theoretical possibility
 

of tumor induction by malaoxon, which could not be detected under
 

the conditions of this test.
 

40
 
Table 7. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Malaoxon in the Diet (a)
 

Topography: Morphology


Integumentary System: Fibrosarcoma
 

of the Subcutaneous Tissue (b)


P Values (c,d)


Departure from Linear Trend (e)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Lung: Alveo lar /Bronch io lar
 

Adenoma or Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

 Control
 

 0/50 (0)
 

 N.S.
 

 P=0.013
 

—
 

 6/50 (12)
 

 N.S.
 

103
 

Low High 

Dose Dose 

3/49 (6) 0/50 (0) 

N.S. — 

Infinite 

0.614 

Infinite 

94 — 

5/48 (10) 6/50 (12) 

N.S. N.S. 

0.868 1.000 

0.224 0.287 

3.185 3.489 

96 89 
Table 7. Analyses of the Incidence of Primary Tumors in Male Mice 

Administered Malaoxon in the Diet (a) 

(continued) 

Topography: Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Hematopoietic System:

or Leukemia (b) 

 Lymphoma 

5/50 (10) 2/49 (4) 4/50 (8) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.408 

0.040 

2.358 

0.800 

0.168 

3.499 

Weeks to First Observed Tumor 88 95 82 

-e­ r-o 

Liver: Hepatocellular Carcinoma (b) 12/50 (24) 2/49 (4) 13/50 (26) 

P Values (c,d) N.S. P = 0.004 (N) N.S. 

Departure from Linear Trend (e) P = 0.002 

Relative Risk (f) 

Lower Limit 

Upper Limit 

1.701 

0.019 

0.711 

1.083 

0.507 

2.334 

Weeks to First Observed Tumor 97 95 65 
Table 7. Analyses of the Incidence of

Administered Malaoxon in

 Primary Tumors

 the Diet (a) 

 in Male Mice 

(continued) 

Topography: Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Liver: Hepatocellular Carcinoma or 

Adenoma (b) 12/50 (24) 5/49 (10) 17/50 (34) 

P Values (c,d) N.S. N.S. N.S. 

Departure from Linear Trend (e) P = 0.010 

P­

.0 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 97 

0.425 

0.127 

1.190 

95 

1.417 

0.716 

2.892 

65 

(a) Dosed groups received 500 or 1,000 ppm.
 

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

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P is less than 0.05, otherwise, not significant (N.S.) is indicated. Beneath
 

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

for the comparison of that dosed group with the matched-control group when P is less than 0.05;
 

otherwise not significant (N.S.) is indicated.
 

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

(e) The probability level for departure from linear trend is given when P is less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed gr^up and the control group.
 
Table 8. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Malaoxon in the Diet (a)
 

Topography: Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Hematopoietic System:

or Leukemia (b)

 Lymphoma 

 7/47 (15) 8/47 (17) 7/48 (15) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

1.143 

0.395 

3.404 

0.979 

0.318 

3.019 

Weeks to First Observed Tumor 81 76 93 

Liver: Hepatocellular Carcinoma (b) 3/47 (6) 1/47 (2) 0/48 (0)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk ( f) 0.333 0.000
 

Lower Limit 0.007 0.000
 

Upper Limit 3.972- 1.626
 

Weeks to First Observed Tumor 101 104
 
Table 8. Analyses of the Incidence of Primary Tumors in Female Mice 

Administered Malaoxon in the Diet (a) 

(continued) 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Liver: Hepatocellular Carcinoma or
 

Adenoma (b) 6/47 (13) 3/47 (6) 1/48 (2)
 

P Values (c,d) P = 0.034 (N) N.S. N.S.
 

Relative Risk (f) 0.500 0.163
 

Lower Limit 0.085 0.004
 

Upper Limit 2.191 1.272
 

Weeks to First Observed Tumor 101 103 104
 

Ul
 

Pituitary: Carcinoma, NOS
 

or Adenoma, NOS 1/33 (3) 1/33 (3) 3/33 (9)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk (f) 1.000 3.000
 

Lower Limit 0.013 0.257
 

Upper Limit 76.152 152.391
 

Weeks to First Observed Tumor 105 101 103
 
Table 8. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Malaoxon in the Diet (a)
 

(continued)
 

(a) Dosed groups received 500 or 1,000 ppm.
 

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

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P is less than 0.05, otherwise, not significant (N.S.) is indicated. Beneath
 

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

for the comparison of that dosed group with the matched-control group when P is less than 0.05;
 

otherwise not significant (N.S.) is indicated.
 

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

(e) The probability level for departure from linear trend is given when P is less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
 
V. DISCUSSION
 

Feeding of malaoxon had no appreciable effect on the mean body
 

weights of male or female rats or male mice. Mean body weights of
 

the high-dose female mice were lower than those of the controls
 

throughout most of the bioassay, and mean body weights of the
 

low-dose female mice were lower than those of the controls after
 

about week 34. No clinical signs related to administration of the
 

test chemical were recorded for male or female rats other than a
 

brief period of hyperexcitability noted early in the study in dosed
 

males. Dosed groups of male and female mice appeared hyperexcitable
 

at times during the course of the study, but this condition did not
 

persist. Mortality was not increased in any of the dosed groups of
 

male or female rats or female mice. Mortality was increased in a
 

dose-related manner in the male mice after week 65. However,
 

survival was 74% or greater at week 103 in all groups of male and
 

female mice. In rats, survival was 64% or greater at week 90 in
 

both dosed and control groups. Thus, there were few clinical signs
 

and only minimal effects on weight and survival that could be
 

related to administration of the test chemical. Sufficient numbers
 

of rats and mice were at risk for the development of late-appearing
 

tumors.
 

47
 
Gastric ulcers were seen in increased incidences in do-sed male and
 

female rats. The lesions were most commonly seen in the forestomach
 

and were usually solitary and sometimes accompanied by epithelial
 

hyperplasia and submucosal inflammation. This lesion was similar to
 

those found in male and female F344 rats administered malathion
 

(NCI, 1979).
 

In female rats, C-cell adenomas or carcinomas of the thyroid occurred in the high-dose group at an incidence that was significantly
 

higher (P = 0.024) than that in the control group, and the overall
 

incidences were dose related (P = 0.009). However, the historical
 

records at this laboratory show an incidence of 16/223 (7%) C-cell
 

adenomas or carcinomas among female control rats, which suggests
 

that the 0/50 incidence observed in the control female rats in the
 

present study was unusually low. In a previous bioassay of
 

malathion (NCI, 1979) using F344 rats, the incidences of C-cell
 

adenomas of the thyroid were not significantly higher than those
 

observed in control rats. No tumors in the present study occurred
 

at significant incidences in the positive direction by any test in
 

male rats and in male and female mice.
 

In two separate carcinogenesis bioassays of malathion, the parent
 

compound of malaoxon, conducted previously at the same laboratory as
 

the present study (NCI, 1978; NCI, 1979), there was no clear
 

48
 
evidence of the association of any tumor incidence with the dietary
 

administration of the compound to Osborne-Mendel rats, F344 rats, or
 

B6C3F1 mice. In the malathion study using F344 rats, it was noted
 

that the females may have been able to tolerate higher doses.
 

Although in the present malaoxon studies clinical signs and effects
 

on weight and survival were limited, it is considered that the
 

animals received approximately a maximum tolerated dose of malaoxon.
 

In the rats, this consideration is based mainly on the development
 

of gastric ulcers; in the mice, it is based on a decreased mean body
 

weight gain of females and on decreased survival of males.
 

Under the conditions of this bioassay, malaoxon was not carcinogenic
 

in F344 or B6C3F1 mice of either sex.
 

49
 

VI. BIBLIOGRAPHY
 

Ailman, D. E., Synthesis of 0,0-dimethyl S-(l,2-dicarbethoxy)­

ethyl phosphorothioate (maloxon) and related compounds from 

trialkyl phosphites and organic disulfieds. J_. Org. Chem. 

30:1074-1077, 1965. 

Armitage, P., Statistical Methods in Medical Research, John Wiley
 

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

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

Panel o_f Carcinogenicity p_f the Cancer Research Commission p_f
 

UICC, Vol. 2, International Union Against Cancer, Geneva, 1969.
 

Brodeur, J. and DuBois, K. P., Studies on factors influencing the
 

acute toxicity of malathion and malaoxon in rats. Can. J_.
 

Physiol. Pharmacol. 4_5:621-630, 1967.
 

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

1970, pp. 48-52.
 

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

Soc. B34;187-220, 1972.
 

Eto, M., Metabolism. In: Organophosphorus Pesticides; Organic
 

and Biological Chemistry, CRC Press, Inc., Cleveland, Ohio, 1974,
 

pp. 162-163.
 

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

significance tests, confidence limits and adjustments for
 

stratification., Rev. Int. Stat. Inst. 39:148-169, 1971.
 

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

incomplete observations. J_^ Amer. Statist. Assoc. 53:457-481,
 

1958.
 

Keith, L., Garrison, A., and Alford, A., The high resolution NMR
 

spectra of pesticides. I. Organophsophorus pesticides. J_.
 

Assoc. Of fie. Anal. Chem. _5_1:1063-1094, 1968.
 

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

Peters, J. A., Carcinogenesis bioassay data system. Cpmp. and
 

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

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

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

51
 
NCI, Technical Report No. 24, Bioassay £f_ Malathion for Possible
 

Carcinogenicity, DREW Publication No. (NIH) 78-824, U. S.
 

Department of Health, Education, and Welfare, Public Health
 

Service, National Institutes of Health, 1978.
 

NCI, Technical Report No. 192, Bioassay £f_ Malathion for Possible
 

Carcinogenicity, DHEW Publication No. (NIH)79r1748, U. S.
 

Department of Health, Education, and Welfare, Public Health
 

Service, National Institutes of Health, 1979.
 

Paschal, D. C. and Neville, M. E., Chemical and microbical
 

degradation of malaoxon in an Illinois soil. J. Environ. Qual.
 

_5(4):441-443, 1976.
 

Sontag, J. M., Page, N. P., and Saffiotti, U., Guidelines for
 

Carcinogen Bioassay in Small Rodents, NCI Technical Report 

Series, No. 1, DHEW Publication No. (NIH) 76-801, U. S. 

Department of Health, Education, and Welfare, Public Health 

Service, National Institutes of Health, 1976.
 

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

Biometrika 62:679-682, 1975.
 

52
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS ADMINISTERED MALAOXON IN THE DIET
 

53
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED MALAOXON IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 50 50 50
 

ANIMALS NECROPSIED 50 50 50
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 50 50 50
 

INTEGUMENTARY SYSTEM
 

XSKIN (50) (50) (50)
 

SQUAMOUS CELL CARCINOMA 2 (4%)
 

BASAL-CELL CARCINOMA 1 (2%) 1 (2%)
 

XSUBCUT TISSUE (so) (50) (so)
 

FIBROMA 3 (6%) 1 (2%)
 

FIBROUS HISTIOCYTOMA, MALIGNANT 1 (2%)
 

LIPOMA 1 (2%)
 

RESPIRATORY SYSTEM
 

SLUNG (49) (49) (49)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (2%)
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (2X)
 

FIBROUS HISTIOCYTOMA, METASTATIC 1 (2%)
 

OSTEOSARCOMA 1 (2%) 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

^MULTIPLE ORGANS (50) (50) (50)


MALIG.LYMPHOMA, HISTIOCYTIC TYPE 2 (4%)
 

LEUKEMIA,NOS 13 (26%) 9 (18%) 9 (18%)
 

LYMPHOCYTIC LEUKEMIA 2 (4%)
 

MONOCYTIC LEUKEMIA 5 (10%) 4 (8%) 2 (4%)
 

tSPLEEN (49) (49) (50)


MALIGNANT LYMPHOMA, NOS 1 (2%)
 

#LIVER (49) (50) (49)
 

LEUKEMIA,NOS 1 (2%)
 

CIRCULATORY SYSTEM
 

NONE
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

55
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

HIGH DOSE 

(50)
 

(49)


1 (2%)
 

(50)


2 (4%)
 

(48)
 

(49)


1 (2X)
 

(45)


2 (4X)
 

18	 (40%)
 

(49)


1 (2%)
 

4 (8%)
 

2 (4%)
 

(49)


1 (2*)
 

1 (2%)
 

2 (4%)
 

2 (4%)
 

(50)


2 (4%)
 

DIGESTIVE SYSTEM
 

#PAROTID GLAND
 

SQUAMOUS CELL CARCINOMA
 

SLIVER
 

NEOPLASTIC NODULE
 

•PANCREAS
 

ACINAR-CELL ADENOMA
 

•GASTRIC	 MUCOSA
 

SQUAMOUS CELL PAPILLOMA
 

URINARY SYSTEM
 

•KIDNEY
 

TUBULAR-CELL ADENOMA
 

ENDOCRINE SYSTEM
 

•PITUITARY
 

CARCINOMA, NOS
 

ADENOMA, NOS
 

CRANIOPHARYNGIOMA
 

•ADRENAL
 

CORTICAL ADENOMA
 

CORTICAL CARCINOMA
 

PHEOCHROMOCYTOMA
 

PHEOCHROMOCYTOMA, MALIGNANT
 

•THYROID
 

FOLLICULAR-CELL ADENOMA
 

FOLLICULAR-CELL CARCINOMA
 

C-CELL ADENOMA
 

C-CELL CARCINOMA
 

•PANCREATIC	 ISLETS
 

ISLET-CELL ADENOMA
 

ISLET-CELL CARCINOMA
 

CONTROL 

(49)
 

(49)
 

(48)
 

(48)
 

(50)
 

(45)
 

3 (7%)
 

17 (38%)
 

(47)
 

2 (4%)
 

1 (2%)
 

(49)


2 (4%)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

(48)


4 (8%)
 

1 (2%)
 

LOW DOSE 

(50)


1 (2%)
 

(50)
 

(49)


1 (2X)
 

(50)


1 (2X)
 

(50)


1 (2M)
 

(45)


1 (2X)
 

20 (44%)
 

1 (2X)
 

(49)
 

1 (2X)
 

4 (8X)
 

(45)
 

(49)


6 (12%)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

56
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

HIGH DOSE
 

(50)
 

1 (250
 

(50)


1 (25O
 

(49)


41 (845O
 

(49)
 

(50)
 

1 (25O
 

(50)
 

(50)
 

(50)
 

REPRODUCTIVE SYSTEM
 

XMAMMARY GLAND
 

CARCINOMA, NOS
 

FIBROADENOMA
 

XPREPUTIAL GLAND
 

CARCINOMA, NOS
 

»TESTIS
 

INTERSTITIAL-CELL


NERVOUS SYSTEM
 

ttBRAIN
 

EPENDYMOMA
 

SPECIAL SENSE ORGANS
 

XZYMBAL'S GLAND
 

CARCINOMA, NOS
 

MUSCULOSKELETAL SYSTEM
 

XSKULL
 

OSTEOSARCOMA
 

BODY CAVITIES
 

XABDOMINAL CAVITY
 

LIPOMA
 

XPERITONEUM
 

MESOTHELIOMA, NOS
 

ALL OTHER SYSTEMS
 

NONE
 

 TUMOR
 

CONTROL


(SO)


1 (25O
 

(50)


(50)


41 (825O


(49)


1 (25O
 

(50)


1 (250
 

(50)


1 (2%)
 

(50)


(50)


 LOW DOSE
 

 (50)
 

 (50)
 

 (49)
 

 44 (90%)
 

 (50)
 

 (50)
 

 (50)
 

(50)


1 (25O
 

(50)


2 (45O
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

57
 
TABLE A1. MALE RATS: NEOPLASMS

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE **
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TOTAL BENIGN TUMORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TOTAL MALIGNANT TUMORS
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

 (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

50 50 50 

2 1 7 

23 22 18 

2 

23 27 25 

50 49 48 

104 102 1 01 

47 47 44 

71 79 74 

28 19 26 

32 20 26 

1 

1 

1 3 1 

1 3 1 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

**
 

Animals are in fact early terminal sacrifices, but appear
 

as scheduled sacrifices due to system interpretation.
 

58
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED MALAOXON IN THE DIET 

CONTROL LOW DOSE
 

ANIMALS INITIALLY IN STUDY 50 50
 

ANIMALS NECROPSIED 50 50
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 50 50
 

INTEGUMENTARY SYSTEM 

*SKIN

SQUAMOUS CELL CARCINOMA 

 (50) (50) 

1 (2%) 

XSUBCUT TISSUE

SARCOMA, NOS 

FIBROSARCOMA 

 (50) (50)

1

1

 (2%) 

 (2X) 

XMAMMARY GLAND

FIBROUS HISTIOCYTOMA, MALIGNANT

 (50) 

1 (25i) 

(50) 

RESPIRATORY SYSTEM 

SLUNG

SQUAMOUS CELL CARCINOMA 

ALVEOLAR/BRONCHIOLAR ADENOMA

ALVEOLAR/BRONCHIOLAR CARCINOMA 

 (49) 

1 (2%) 

(50) 

1 (2%) 

HEMATOPOIETIC SYSTEM 

^MULTIPLE ORGANS

LEUKEMIA, NOS

MONOCYTIC LEUKEMIA

 (50) 

9 ( 18%) 

3 (6%) 

(50) 

3 (6%) 

2 (4%) 

*SPLEEN

MALIGNANT LYMPHOMA, NOS

 (50) 

1 (2%) 

(50) 

CIRCULATORY SYSTEM 

NONE 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

50
 

50
 

50
 

(50)
 

(50)
 

1 (2%)
 

(50)
 

(50)
 

1 (2%)
 

(50)
 

9 (18%)
 

1 (2%)
 

(46)
 

59
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE 

DIGESTIVE SYSTEM
 

ULIVER (46) (50)
 

CARCINOMA, NOS, METASTATIC 1 (25O
 

NEOPLASTIC NODULE 1 (2*)
 

HEPATOCELLULAR CARCINOMA 2 (4X)
 

#JEJUNUM (47) (48)
 

LEIOMYOMA
 

URINARY SYSTEM
 

8KIDNEY (50) (50)
 

LIPOMA 1 (25O
 

MIXED TUMOR, MALIGNANT 1 (250
 

ENDOCRINE SYSTEM 

»PITUITARY 

CARCINOMA, NOS 

ADENOMA, NOS 

CHROMOPHOBE ADENOMA 

(49) 

4 (850 

31 (6350 

1 (250 

(49) 

1 (250 

33 (67X) 

SADRENAL 

CORTICAL ADENOMA 

PHEOCHROMOCYTOMA 

(49) 

1 (25O 

(49)

2 (4X) 

2 (450 

KTHYROID 

FOLLICULAR-CELL ADENOMA 

(50) (49) 

C-CELL ADENOMA 

C-CELL CARCINOMA 

1 (2X) 

REPRODUCTIVE SYSTEM
 

*MAMMARY GLAND (50) (50)


PAPILLARY ADENOMA 2 (43O
 

FIBROADENOMA 2 (450 9 (18X)
 

XCLITORAL GLAND (50) (50)
 

CARCINOMA, NOS 1 (25O
 

#UTERUS (46) (47)
 

CARCINOMA, NOS 1 (25O
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY x NUMBER OF ANIMALS NECROPSIED 

HIGH DOSE 

(49)
 

(49)
 

1 (25O
 

(50)
 

(49)


6 (125O
 

26 (535O
 

(48)
 

2 (4%)
 

2 (4%)
 

(47)
 

1 (25O
 

4 (95O
 

1 (2X)
 

(50)


1 (250
 

1 (25i)
 

(50)


1 (2%)
 

(48)
 

60 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)
 

CONTROL LOW DOSE HIGH DOSE
 

LEIOMYOSARCOMA 1 (2X)
 

ENDOMETRIAL STROMAL POLYP 11 (24%) 8 (17%) 11 (23%)
 

SUTERUS/ENDOMETRIUM (46) (47) (48)
 

ADENOMA, NOS 1 (2%)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

^ABDOMINAL CAVITY (50) (50) (50)
 

LIPOMA 1 (2%) 2 (4%) 1 (2%)
 

ALL OTHER SYSTEMS
 

LOWER LEG
 

OSTEOSARCOMA 1
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

NATURAL DEATHS! 3
 

MORIBUND SACRIFICE 20 17
 

SCHEDULED SACRIFICE ** 2 2
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 25 38 31
 

ANIMAL MISSING
 

a INCLUDES AUTOLYZED ANIMALS
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

**
 

Animals are in fact early terminal sacrifices, but appear
 

as scheduled sacrifices due to system interpretation.
 

61
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 45 44 44
 

TOTAL PRIMARY TUMORS 70 75 70
 

TOTAL ANIMALS WITH BENIGN TUMORS 38 41 36
 

TOTAL BENIGN TUMORS 49 60 50
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 20 12 18
 

TOTAL MALIGNANT TUMORS 21 14 20
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1
 

TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT 1
 

TOTAL UNCERTAIN TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 * SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE ADMINISTERED MALAOXON IN THE DIET
 

63
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED MALAOXON IN THE DIET 

HIGH
 

50
 

50
 

50
 

(50)
 

(50)
 

(50)
 

5
 

1
 

(50)


1
 

1
 

1
 

1
 

(50)
 

(48)
 

(47)
 

DOSE
 

(10%)


(2X)
 

(2X)
 

(250


(2X)
 

(25O
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

*EAR
 

FIBROUS HISTIOCYTOMA
 

*SUBCUT TISSUE
 

FIBROSARCOMA
 

RESPIRATORY SYSTEM
 

ttLUNG
 

HEPATOCELLULAR CARCINOMA, METAST
 

ALVEOLAR/BRONCHIOLAR ADENOMA
 

ALVEOLAR/BRONCHIOLAR CARCINOMA
 

HEKATOPOIETIC SYSTEM
 

^MULTIPLE ORGANS
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

MALIGNANT LYMPHOMA, MIXED TYPE
 

LEUKEMIA, NOS
 

UNDIFFERENTIATED LEUKEMIA
 

GRANULOCYTIC LEUKEMIA
 

MONOCYTIC LEUKEMIA
 

XSKIN
 

MAST-CELL TUMOR
 

8LYMPH NODE
 

MALIGNANT LYMPHOMA, NOS
 

#JEJUNUM
 

MALIGNANT LYMPHOMA, MIXED TYPE
 

CONTROL
 

50
 

50
 

50
 

(50)


1 (2%)
 

(50)
 

(50)


1 (2X)
 

6 (12X)
 

(50)
 

2 (4X)
 

2 (45O
 

(50)


1 (2X)
 

(48)
 

(45)


1 (2X)
 

LOW DOSE
 

50
 

49
 

49
 

(49)
 

(49)


3 (6X)
 

(48)
 

4 (8%)
 

1 (2X)
 

(49)
 

1 (2%)
 

(49)
 

(41)
 

1 (2%)
 

(42)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

65
 
TABLE B1. MALE MICE: NEOPLASMS

CIRCULATORY SYSTEM
 

#SPLEEN


HEMANGIOSARCOMA


#LIVER


HEMANGIOMA


DIGESTIVE SYSTEM
 

#LIVER


HEPATOCELLULAR ADENOMA


HEPATOCELLULAR CARCINOMA


#JEJUNUM


ADENOCARCINOMA, NOS


URINARY SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

#PITUITARY


CARCINOMA,NOS


8THYROID


FOUICULAR-CELL ADENOMA


REPRODUCTIVE SYSTEM
 

#TESTIS


INTERSTITIAL-CELL TUMOR


NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

XEYE/LACRIMAL GLAND
 

PAPILLARY CYSTADENOMA, NOS
 

 (CONTINUED) 

CONTROL

 (49)


 (50)


 (50)


 12 (24%)


 (45)


 (35)


 (45)


 1 (2X)
 

(49)


(50)


2
 

 LOW DOSE HIGH DOSE 

 (45)	 (50)
 

1 (2%)
 

 (49)	 (50)
 

1 (25O
 

 (49)	 (50)
 

3 (6%) 4 (8%)
 

2 (4%) 13 (26X)
 

 (42)	 (47)
 

1 (2%)
 

 (24)	 (30)
 

1 (3X)
 

 (44)	 (44)
 

 (49)	 (48)
 

1
 

(49) (50)
 

#	 NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NECROPSIED
 

66
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

^MULTIPLE ORGANS (50) (49) (50)
 

OSTEOSARCOMA 1 C2X)
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

NATURAL DEATHS 2 4 3
 

MORIBUND SACRIFICE 3 4 10
 

SCHEDULED SACRIFICE ** 5 5 5
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 40 37 32
 

ANIMAL MISSING
 

a INCLUDES AUTOLYZED ANIMALS
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

**
 

Animals are in fact early terminal sacrifices, but appear
 

as scheduled sacrifices due to system interpretation.
 

67
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED)
 

CONTROL LOW DOSE HIGH DOSE
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 23 18 26
 

TOTAL PRIMARY TUMORS 28 18 30
 

TOTAL ANIMALS WITH BENIGN TUMORS 10 8 10
 

TOTAL BENIGN TUMORS 10 8 10
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 16 10 19
 

TOTAL MALIGNANT TUMORS 17 10 20
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1
 

TOTAL SECONDARY TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT 1
 

TOTAL UNCERTAIN TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

X PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

68
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED MALAOXON IN THE DIET 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

^MULTIPLE ORGANS
 

FIBROUS HISTIOCYTOMA, MALIGNANT
 

RESPIRATORY SYSTEM
 

#LUNG
 

ADENOCARCINOMA, NOS, METASTATIC
 

ALVEOLAR/BRONCHIOLAR ADENOMA
 

OSTEOSARCOMA, METASTATIC
 

HEMATOPOIETIC SYSTEM
 

^MULTIPLE ORGANS
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

MALIGNANT LYMPHOMA, MIXED TYPE
 

UNDIFFERENTIATED LEUKEMIA
 

MONOCYTIC LEUKEMIA
 

ttSPLEEN
 

MALIGNANT LYMPHOMA, MIXED TYPE
 

SMESENTERIC L. NODE
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

SLIVER
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

#KIDNEY
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE
 

50
 

47
 

47
 

(47)
 

(47)
 

1 (2%)
 

(47)
 

1 (2%)
 

3 (6%)
 

1 (250
 

(47)
 

(43)
 

(47)
 

1 (25O
 

(45)
 

50
 

47
 

47
 

(47)
 

1 (25O
 

(47)
 

1 (250
 

(47)
 

1 (2X)
 

3 (6X)
 

2 (4%)
 

(47)
 

(42)
 

(47)
 

1 (2%)
 

(47)
 

S NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

69
 

50
 

48
 

48
 

(48)
 

(48)


1 (2%)
 

1 (2%)
 

(48)
 

1 (2%)
 

2 (4%)
 

1 (2K)
 

(48)
 

1 (2%)
 

(41)
 

1 (2X)
 

(48)
 

(48)
 

1 (2%)
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (25O 

*UTERUS

MALIG.LYMPHOMA, HISTIOCYTIC TYPE

 (45) (46) 

1 (2%) 

CIRCULATORY SYSTEM 

SSPLEEN

HEMANGIOSARCOMA

 (47) (47) 

1 (2X) 

DIGESTIVE SYSTEM 

SLIVER

HEPATOCELLULAR ADENOMA

 (47)

 3 (6X)

 (47) 

2 (4X) 

HEPATOCELLULAR CARCINOMA 3 (6X) 1 (2X) 

URINARY SYSTEM 

NONE 

ENDOCRINE SYSTEM
 

8PITUITARY (33) (33)
 

CARCINOMA, NOS 1 (3X)
 

ADENOMA, NOS 1 (3X)
 

#ADRENAL (45) (45)
 

CORTICAL ADENOMA
 

REPRODUCTIVE SYSTEM
 

XMAMMARY GLAND (47) (47)
 

ADENOCARCINOMA, NOS
 

SUTERUS (45) (46)
 •ADENOCARCINOMA, NOS	 1 (2%)
 

#OVARY	 (45) (43)
 

CYSTADENOMA, NOS 1 (2%)
 

NERVOUS SYSTEM
 

NONE
 

• NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

X NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

(46)
 

(48)
 

(48)


1 (2X)
 

(33)
 

3 (9X)
 

(46)


2 (4%)
 

(48)


1 (2X)
 

(46)
 

(44)
 

70
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL SENSE ORGANS
 

*EYE/LACRIMAL GLAND (47) (47) (48)
 

PAPILLARY CYSTADENOMA, NOS 1 (2%)
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 50 50 50
 

NATURA L DEATHS 3
 

MORIBUND SACRIFICE 8
 

SCHEDULED SACRIFICE ** 5
 

ACCIDENTALLY KILLED 1
 

TERMINAL SACRIFICE 33 38 44
 

ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

« NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

**
 

Animals are in fact early terminal sacrifices, but appear
 

as scheduled sacrifices due to system interpretation.
 

71
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOWOOSE HIGH OOSE 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 14 15 15
 

TOTAL PRIMARY TUMORS 16 16 15
 

TOTAL ANIMALS WITH BENIGN TUMORS 5 3 7
 

TOTAL BENIGN TUMORS 6 3 7
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 9 12 8
 

TOTAL MALIGNANT TUMORS 10 13 8
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1 1
 

TOTAL SECONDARY TUMORS 1 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

72
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS ADMINISTERED MALAOXON IN THE DIET
 

73
 

TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE 

RATS ADMINISTERED MALAOXON IN THE DIET 

HIGH DOSE
 

50
 

50
 

50
 

(50)
 

1 (2%)
 

1 (2X)
 

(49)


2 (45O
 

1 (250
 

(50)
 

1 (25O
 

(42)
 

(50)


2 (45O
 

(50)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

XSUBCUT TISSUE
 

INFLAMMATION, NOS
 

INFLAMMATION, GRANULOMATOUS
 

CALCIFICATION, DYSTROPHIC
 

RESPIRATORY SYSTEM
 

SLUNG
 

INFLAMMATION, ACUTE
 

INFLAMMATION, FOCAL GRANULOMATOU
 

HYPERPLASIA, ALVEOLAR EPITHELIUM
 

HEMATOPOIETIC SYSTEM
 

#SPLEEN
 

CONGESTION, NOS
 

SCLEROSIS
 

FIBROSIS, FOCAL
 

HYPERPLASIA, LYMPHOID
 

KMANDIBULAR L. NODE
 

CYST, NOS
 

HYPERPLASIA, LYMPHOID
 

CIRCULATORY SYSTEM
 

^AURICULAR APPENDAGE
 

THROMBOSIS, NOS
 

THROMBUS, ORGANIZED
 

MYOCARDIUM
 

INFLAMMATION, FOCAL
 

CONTROL


50


50


50


(50)


1 (2%)
 

(49)


2 (450
 

(49)


1 (250
 

1 (25O
 

1 (25O


2 (455)
 

(43)


(49)


2 (45O
 

(49)


 LOW DOSE
 

 50
 

 50
 

 50
 

 (50)
 

 (49)
 

 (49)
 

2 (45O
 

(45)


1 (250
 

1 (25O
 

 (50)
 

(50)


1 (250
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

75
 
TABLE C1. MALE RATS: NONNEOPLASTIC

INFLAMMATION, INTERSTITIAL
 

SHEPATIC SINUSOID
 

CONGESTION, NOS
 

DIGESTIVE SYSTEM
 

SLIVER
 

INFLAMMATION, NOS
 

INFLAMMATION, MULTIFOCAL
 

NECROSIS, FOCAL
 

METAMORPHOSIS FATTY
 

CYTOPLASMIC CHANGE, NOS
 

CYTOPLASMIC VACUOLIZATION
 

ANGIECTASIS
 

SBILE DUCT
 

HYPERPLASIA, NOS
 

SPANCREATIC ACINUS
 

ATROPHY, NOS
 

SSTOMACH
 

ULCER, NOS
 

HYPERPLASIA, EPITHELIAL
 

URINARY SYSTEM
 

UKIDNEY
 

HYDRONEPHROSIS
 

INFLAMMATION, CHRONIC
 

8URINARY BLADDER
 

HEMORRHAGE
 

INFLAMMATION, HEMORRHAGIC
 

ENDOCRINE SYSTEM
 

((PITUITARY


CYST, NOS
 

HEMORRHAGE
 

HYPERPLASIA, FOCAL
 

 LESIONS (CONTINUED) 

HIGH DOSE
 

(49)


1 (2%)
 

(49)
 

1 (2%)
 

1 (2%)
 

2 (4%)
 

1 (2%)
 

(49)


1 (2%)
 

(50)


3 (650
 

(48)


7 (15%)
 

1 (2%)
 

(49)
 

34 (69%)
 

(48)
 

1 (2%)
 

(45)
 

2 (4%)
 

CONTROL
 

1 (2%)
 

(49)
 

(49)
 

1 (2%)
 

1 (2%)
 

(49)


5 (10%)
 

(48)


2 (4%)
 

(48)


2 (4%)
 

(50)
 

1 (2%)
 

41 (82%)
 

(48)
 

(45)
 

1 (2%)
 

4 (9%)
 

LOW DOSE
 

(50)
 

(50)


1 (2%)
 

3 (6%)
 

1 (2%)
 

1 (2%)
 

(50)
 

(49)
 

5 (10%)
 

(50)
 

6 (12%)
 

1 (2%)
 

(50)
 

35 (70%)
 

(45)


1 (2%)
 

(45)


1 (2%)
 

3 (7%)
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

76
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

ANGIECTASIS
 

•ADRENAL
 

METAMORPHOSIS FATTY
 

HYPERPLASIA, FOCAL
 

•ADRENAL	 CORTEX
 

CYTOPLASMIC VACUOLIZATION
 

HYPERPLASIA, NOS
 

SADRENAL MEDULLA
 

HYPERPLASIA, NOS
 

HYPERPLASIA, FOCAL
 

•THYROID
 

CYSTIC FOLLICLES
 

HYPERPLASIA, C-CELL
 

•PANCREATIC	 ISLETS
 

HYPERPLASIA, FOCAL
 

REPRODUCTIVE SYSTEM
 

XMAMMARY GLAND
 

ABSCESS, NOS
 

ttPROSTATE
 

INFLAMMATION, NOS
 

XSEMINAL VESICLE


HEMORRHAGE
 

•TESTIS
 

CALCIFICATION, DYSTROPHIC
 

ATROPHY, NOS
 

NERVOUS SYSTEM
 

•BRAIN
 

HEMATOMA, NOS
 

GLIOSIS
 

•CEREBELLUM
 

NECROSIS, HEMORRHAGIC
 

* NUMBER OF ANIMALS WITH TISSUE x NUMBER OF ANIMALS NECROPSIED 

CONTROL LOW DOSE 

1	 (2X)
 

(47)	 (49) 

1 (2X) 

(47) (49)


1 (25O
 

1 (25O
 

(47) (49)
 

1 (2%)
 

1 (2%) 1 (2%)
 

(49)	 (45)
 

1 (2%)
 

6 (13%)
 

(48)	 (49)
 

1 (2%)
 

(50)	 (50)


1 (2%)
 

(43)	 (41)
 

1 (2%)
 

(50)	 (50)
 

1 (2%)
 

(50) (49)


1 (250
 

4 (850 1 (2%)
 

(49) (50)
 

1	 (2X)
 

(49) (50)
 

2	 (4%)
 

 EXAMINED MICROSCOPICALLY 

HIGH DOSE 

1 (2%)
 

(49)
 

(49)
 

(49)
 

3 (6X)
 

(49)
 

10 (20%)
 

(50)
 

(50)
 

(41)
 

(50)
 

(49)
 

(49)


1 (2X)
 

(49)
 

77
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

XABDOMINAL CAVITY (50) (50) (50)
 

LIPOGRANULOMA 4 (8X) 3 (65O
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 1
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

78
 
TABLE C2. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE 

RATS ADMINISTERED MALAOXON IN THE DIET 

ANIMALS INITIALLY IN STUDY


ANIMALS NECROPSIED


ANIMALS EXAMINED HISTOPATHOLOGICALLY


INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

• LUNG


EMPHYSEMA, NOS


ATELECTASIS


HEMATOPOIETIC SYSTEM
 

•SPLEEN


CONGESTION, NOS


HYPERPLASIA, LYMPHOID


•LYMPH NODE


CONGESTION, NOS


HYPERPLASIA, LYMPHOID


CIRCULATORY SYSTEM
 

•AURICULAR	 APPENDAGE


THROMBOSIS, NOS


•MYOCARDIUM


INFLAMMATION, INTERSTITIAL


DIGESTIVE SYSTEM
 

•SALIVARY GLAND
 

INFLAMMATION. NOS
 

CONTROL LOW DOSE HIGH DOSE 

 50 50 50 

 50 50 50 

 50 50 50 

 (49) (50) (50) 

2 (<t%) 

1 (2X) 

 (50)

 1 (2%) 

 (50) (46) 

4 (9%) 

 (41) (40)

 1 (3X) 

1 (3X) 

 (31) 

 (50) (50) (49) 

1 (2X) 1 (2X) 

 (50) (50)

 1 (2%) 

 (49) 

(50)

1 (2%) 

(50) (47) 

« NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

79
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE
 

*LIVER	 (46) (50)


INFLAMMATION, NOS 1 (2X)
 

INFLAMMATION, MULTIFOCAL 3 (7X) 3 (6X)
 

NECROSIS, FOCAL 1 (2X)
 

METAMORPHOSIS FATTY 3 (7X) 7 (14%)
 

PIGMENTATION, NOS
 

FOCAL CELLULAR CHANGE	 1 (2X)
 

•PANCREATIC ACINUS	 (49) (50)


ATROPHY, NOS	 1 (230 3 (6%)
 

•STOMACH	 (49) (49)


ULCER, NOS	 1 (2X)
 

ftDUODENUM (47) (48)


FIBROSIS, FOCAL 1 (2X)
 

URINARY SYSTEM
 

•KIDNEY	 (50) (50)
 

HYDRONEPHROSIS t (2%)
 

INFLAMMATION, CHRONIC	 27 (545O 28 (56%)
 

ENDOCRINE SYSTEM
 

•PITUITARY	 (49) (49)
 

CYST, NOS
 

CONGESTION, NOS 1 (23O
 

HEMORRHAGE	 1 (25O
 

HYPERPLASIA, NOS	 1 (23O
 

HYPERPLASIA, FOCAL	 1 (2X) 2 (4X)
 

ANGIECTASIS	 6 (12SO 11 (22X)
 

•ADRENAL	 (49) (49)
 

HEMORRHAGE t (250
 

METAMORPHOSIS FATTY	 4 (83O
 

ANGIECTASIS	 1 (2X)
 

•ADRENAL CORTEX	 (49) (49)
 

DEGENERATION, NOS	 3 (6X)
 

FOCAL CELLULAR CHANGE
 

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

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

(49)
 

2 (4X)
 

5 (10X)
 

1 (2>O
 

(48)
 

3 (6X)
 

(49)
 

3 (6*)
 

(49)
 

(50)
 

18	 (36X)
 

(49)
 

3 (65C)
 

1 (2X)
 

2 (4X)
 

7 (14X)
 

(48)
 

1 (2X)
 

(48)
 

1 C2X)
 

80
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

HIGH DOSE
 

(47)
 

1 (2%)
 

6 (13%)
 

(50)
 

(48)
 

(48)
 

1 (2%)
 

(48)
 

(50)
 

1 (2%)
 

(50)
 

(50)
 

2 (4%)
 

ItTHYROID
 

CYST, NOS
 

HYPERPLASIA, C-CELL
 

HYPERPLASIA, FOLLICULAR-CELL
 

REPRODUCTIVE SYSTEM
 

KMAMMARY GLAND
 

INFLAMMATION, NOS
 

SUTERUS
 

HYDROMETRA
 

8UTERUS/ENDOMETRIUM
 

DEGENERATION, CYSTIC
 

HYPERPLASIA, CYSTIC
 

#OVARY
 

FOLLICULAR CYST, NOS
 

NERVOUS SYSTEM
 

#BRAIN
 

HYDROCEPHALUS, INTERNAL
 

NECROSIS, HEMORRHAGIC
 

CALCIFICATION, DYSTROPHIC
 

UCEREBELLUM
 

HEMORRHAGE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

^ABDOMINAL CAVITY
 

LIPOGRANULOMA
 

CONTROL
 

(50)
 

6 (12%)
 

2 (4%)
 

(50)
 

1 (2%)
 

(46)
 

3 (7%)
 

(46)
 

1 (2%)
 

(49)
 

(50)
 

3 (6%)
 

(50)
 

(50)
 

1 (2%)
 

LOW DOSE
 

(49)
 

8 (16%)
 

(50)
 

(47)
 

2 (4%)
 

(47)
 

(46)
 

2 (4%)
 

(50)
 

1 (2%)
 

(50)
 

1 (2%)
 

(50)
 

» NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

81
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

XMESENTERY

STEATITIS

 (50) (50) (50) 

1 (.2%) 

ALL OTHER SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY * NUMBER OF ANIMALS NECROPSIED 

1 2 

82
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE ADMINISTERED MALAOXON IN THE DIET
 

83
 

TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE 

MICE ADMINISTERED MALAOXON IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 50 50 50
 

ANIMALS NECROPSIED 50 49 50
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 50 49 50
 

INTEGUMENTARY SYSTEM
 

*SKIN (50) (49) (50)
 

EPIDERMAL INCLUSION CYST 3 (6X)
 

*SUBCUT TISSUE (50) (49) (50)
 

EPIDERMAL INCLUSION CYST 1 (2%)
 

ABSCESS, NOS 1 (2X)
 

RESPIRATORY SYSTEM
 

*LUNG (50) (48) (50)
 

BRONCHOPNEUMONIA, FOCAL 1 (2X) 1 (2X)
 

INFLAMMATION, FOCAL 8 ( 16X) 6 (13%) 2 (4X)
 

INFLAMMATION, CHRONIC FOCAL 1 (2%)
 

HYPERPLASIA, ALVEOLAR EPITHELIUM 8 (16%) 6 (13%) 2 (4%)
 

HEMATOPOIETIC SYSTEM
 

ULYMPH NODE (48) (41) (48)
 

HYPERPLASIA, NOS 1 (2%)
 

SMESENTERIC L. NODE (48) (41) (48)
 

HEMORRHAGE 1 (2%)
 

INFLAMMATION, NOS 1 (2X)
 

INFLAMMATION, ACUTE 1 (2%)
 

HYPERPLASIA, NOS 1 (2%) 1 (2%)
 

8INGUINAL LYMPH NODE (48) (41) (48)


HYPERPLASIA, NOS 1 (2%)
 

CIRCULATORY SYSTEM
 

NONE
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

85
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE
 

DIGESTIVE SYSTEM
 

#LIVER (50) (49)
 

ABSCESS, NOS 1 (2%)
 

NECROSIS, FOCAL 1 (2%)
 

INFARCT, NOS
 

SPANCREAS (48) (48)
 

DILATATION/DUCTS
 

ATROPHY, NOS
 

SPEYERS PATCH (45) (42)
 

HYPERPLASIA, NOS 1 (2%)
 

URINARY SYSTEM
 

SKIDNEY (49) (49)
 

PYELONEPHRITIS, ACUTE
 

#URINARY BLADDER (50) (43)
 

INFLAMMATION, ACUTE/CHRONIC 1 (2X)
 

ENDOCRINE SYSTEM
 

#PITUITARY (35) (24)
 

CYST, NOS 1 (4X)
 

&ADRENAL CORTEX (50) (48)


INFARCT, NOS 1 (2%)
 

SADRENAL MEDULLA (50) (48)
 

HYPERPLASIA, FOCAL 1 (2%)
 

OTHYROID (45) (44)
 

HYPERPLASIA, FOLLICULAR-CELL 2 (4X)
 

REPRODUCTIVE SYSTEM
 

SPROSTATE (47) (45)
 

INFLAMMATION, ACUTE/CHRONIC 1 (2X)
 

« NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 x NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE
 

(50)
 

(50)
 

1 (2X)
 

(47)
 

(48)
 

(49)
 

(30)
 

(48)
 

(48)
 

(44)
 

1 (2%)
 

(46)
 

1 (250
 

86
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

NERVOUS SYSTEM
 

ftLEPTOMENINGES (49) (49) (50)
 

CHOLESTEATOMA 1 (2%)
 ___________ _ — __,_______..___ — -»_—.._ — — _ — .— _ — _ — —._ — _ — .— — .— -. — — — — ».— — —— — -. — — *— — — — — — —. — — — — — — — — — — —­

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

*SKELETAL MUSCLE (50) (49) (50)
 

PARASITISM 1 (25O
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

^MULTIPLE ORGANS (50) (49) (50)
 

INFLAMMATION, FOCAL GRANULOMATOU 1 (2%)
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 20 21 17
 

AUTOLYSIS/NO NECROPSY 1
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

87
 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE 

MICE ADMINISTERED MALAOXON IN THE DIET

 

CONTROL LOW DOSE 

ANIMALS INITIALLY IN STUDY 50 50 

ANIMALS NECROPSIED 47 47 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 47 47 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

SLUNG (47) (47)
 

EDEMA, NOS
 

HEMORRHAGE
 

BRONCHOPNEUMONIA, FOCAL 3 (6X)
 

INFLAMMATION, FOCAL 7 (155i) 6 (13*)
 

BRONCHOPNEUMONIA, ACUTE 2 (4X)
 

HYPERPLASIA, ALVEOLAR EPITHELIUM 7 (15X) 6 (13X)
 

HEMATOPOIETIC SYSTEM
 

*MAMMARY GLAND (47) (47)
 

DYSPLASIA, NOS
 

ADENOSIS 1 (2X)
 

#SPLEEN (47) (47)
 

HYPERPLASIA, LYMPHOID 1 (2%)
 

SLYMPH NODE (43) (42)
 

HYPERPLASIA, NOS
 

SMESENTERIC L. NODE (43) (42)


INFLAMMATION, CHRONIC 1 (2X)
 

HYPERPLASIA, LYMPHOID 1 (2*)
 

*JEJUNUM (45) (45)


HYPERPLASIA, LYMPHOID 1 (2X)
 

CIRCULATORY SYSTEM
 

NONE
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE 

50
 

48
 

48
 

(48)
 

1 (2X)
 

1 (2%)
 

1 (2%)
 

1 (2%)
 

(48)
 

1 (2*)
 

1 (2K)
 

(48)
 

(41)
 

(41)
 

(47)
 

88
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE 

DIGESTIVE SYSTEM
 

ftLIVER (47) (47) (48)
 

FOCAL CELLULAR CHANGE 1 (2X)
 

ftBILE DUCT (47) (47) (48)
 

HAMARTOMA 1 (25O
 

8PANCREAS (47) (46) (48)
 

DILATATION/DUCTS 1 (2%)
 

ATROPHY, NOS 2 (45O
 

URINARY SYSTEM
 

#KIDNEY (45) (47) (48)
 

INFARCT, NOS 1 (2%)
 

ftURINARY BLADDER (41) (43) (44)
 

INFLAMMATION, CHRONIC 1 (2X)
 

ENDOCRINE SYSTEM
 

ftPITUITARY (33) (33) (33)
 

HEMORRHAGE 1 (3K)
 

HYPERPLASIA, FOCAL 4 (12X) 2 (65i) 1 (35O
 

ttTHYROID (43) (41) (43)
 

HYPERPLASIA, FOLLICULAR-CELL 2 (5*/.)
 

REPRODUCTIVE SYSTEM
 

*MAMMARY LOBULE (47) (47) (48)
 

HYPERPLASIA, NOS 1 (25O 2 (4%)
 

ttOVARY (45) (43) (44)
 

CYST, NOS 2 (4X)
 

HEMORRHAGE 1 (2%)
 

ABSCESS, NOS 1 (2%)
 

NERVOUS SYSTEM
 

NONE
 

ft NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

89
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAl SYSTEM
 

^SKELETAL MUSCLE (47) (47) (48)
 

PARASITISM 1 (2X)
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 19 18 26
 

AUTOLYSIS/NO NECROPSY 3 3 2
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

90
 
APPENDIX E 

ANALYSIS OF MALAOXON 

91 

APPENDIX E
 

Analysis of Malaoxon
 

A. Elemental Analysis
 

Element: C H P S 

Theory: 38.21 6.09 9.86 10.20 

Found: 37.95 6.09 9.60 10.40 

B. Boiling Point
 

Literature: 132°C at 0.1 mm Hg (Ailman, 1965).
 

Found: Not determined.
 

C. Thin-Layer Chromatography
 

Plate used: Silufol (100 n silica gel on Al foil).
 

Visualization: 12 vapor.
 

System I System II
 

Hexane, ether, acetone (10:3:1) Hexane, ether, acetone (4:4:1)
 

Results: Rf 0.07, trace Results: Rf 0.57, trace
 

at 0.51 at 0.82
 

D. Vapor-Phase Chromatography
 

Instrument: H-P 7610
 

Detector: EC-pulsed 150 at 280°C
 

Column: 10% DCZOO on GCQ, 4' x 1/4", glass at
 

165°C
 

Inlet Temp: 200°C
 

Results: Major peak at 6.5 minutes, with trace
 

impurities ( < 1%) at 2.0 and 5.0 minutes
 

93
 
E. Spectral Data
 

1. Infrared: The infrared absorption spectrum (figure 5)
 

was consistant with the spectrum given in the literature
 

(Ailman, 1965).
 

2. Nuclear Magnetic Resonance: The nuclear magnetic
 

resonance spectrum (figure 6) was consistent with the
 

spectrum given in the literature (Keith et al., 1968).
 

94
 
Oi
 

SOLVEN1 REMARKS Jj . »cU ,sr N SPEED Tlwt 

CONCENTRATION ICL ? r,

C£ L PATH «»

PART HO 457 WOt REFERENCE P 

Figure 5. Infrared Absorption Spectrum of Malaoxon
 
MOHMNO Italaowa 

Figure 6. Nuclear Magnetic Resonance Spectrum of Malaoxon
 
APPENDIX F
 

ANALYSES OF FORMULATED DIETS FOR
 

CONCENTRATIONS OF MALAOXON
 

97
 

Appendix F
 

Analyses of Formulated Diets for Concentrations
 

of Malaoxon
 

A 10-g sample of the diet mixture containing malaoxon was
 

extracted with 250 ml of benzene by shaking on a wrist-action
 

shaker for 3 to 4 hours. Appropriate aliquots of the extract
 

were taken and after appropriate dilutions, the solution was
 

quantitatively analyzed for malaoxon by gas-liquid chromatography
 

(flame photometric detector in phosphorus mode, 10% DC 200
 

column). Recoveries were checked with spiked samples, and
 

external standards were used for calibration.
 

Theoretical No. Samp le Coef ficienct 

Dietary Level of Analytical of Variation Range 

(ppm) Samples Mean (ppm) (%) ppm 

500 12 507.5 3.38 487-529 

1000 12 979.8 3.15 923-1032 

2000 11 2026.3 2.57 1934-2096 

99
 

Review of the Bioassay of Malaoxon* for Carcinogenic!ty
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

May 1, 1979
 

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 of the 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, and State health officials.
 

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 carcinogenic!ty. It is in this context that the below critique is given on the bioassay of Malaoxon.
 

The primary reviewer for the report on the bioassay of Malaoxon
 

said that the compound was the oxygen analogue of Malathion. The report
 

indicated that Malaoxon was not carcinogenic in rats or mice, under the
 

conditions of test. After briefly commenting on the experimental
 

design, he said that the study was straightforward. He did note,
 

however, that results from the subchronic study indicated that both
 

rats and mice may have been able to tolerate higher chronic dose
 

levels. The primary reviewer also pointed out the unusually high
 

incidence of lymphomas and leukemias in control rats. A staff member
 

commented that the incidence of these tumors are quite variable in
 

different groups of Fischer rats.
 

The secondary reviewer also noted that the chronic dose levels
 

could have been higher, as indicated from the subchronic findings.
 

Overall, however, he thought the study was adequate.
 

It was moved that the report on the bioassay of Malaoxon be
 

accepted as written. The motion was seconded and approved unanimously.
 

Clearinghouse Members Present:
 

Arnold L. Brown (Chairman), University of Wisconsin Medical School
 

David B. Clayson, University of Nebraska Medical Center
 

Joseph Highland, Environmental Defense Fund
 

101
 
Willia m Lijinsky, Frederick Cancer Research Center 

Sheldon Samuels, AFL-CIO 

Michael Shimkin, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

Kenneth Wilcox, Michigan State Health Department
 

*	 Subsequent to this review, changes may have been made in the bioassay report either as a result of the review or for other reasons,
 

Thus, certain comments and criticisms reflected in the review may
 

no longer be applicable.
 

*US GOVERNMENT PRINTING OFFICE 1979-2812173219 

102 

NIH Publication No. 79-1390