National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 16 

1979 

BIOASSAY OF 

PHOSPHAMIDON 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 13171-21-6 

NCI-CG-TR-16 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

PHOSPHAMIDON
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 79-816
 
11

 
BIOASSAY OF
 

PHOSPHAMIDON
 

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
 

phosphamidon conducted for the Carcinogenesis Testing Program,
 

Division of Cancer Cause and Prevention, National Cancer
 

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

Maryland. This is one of a series of experiments designed to
 

determine whether selected environmental chemicals have the
 

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

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

control animals, do not necessarily mean that the test chemical
 

is not a carcinogen, inasmuch as the experiments are conducted
 

under a limited set of circumstances. Positive results
 

demonstrate that the test chemical is carcinogenic for animals
 

under the conditions of the test and indicate that exposure to
 

the chemical is a potential risk to man. The actual
 

determination of the risk to man from chemicals found to be
 

carcinogenic in animals requires a wider analysis.
 

CONTRIBUTORS; This bioassay of phosphamidon was conducted by
 

Gulf South Research Institute, New Iberia, Louisiana, initially
 

under direct contract to NCI and currently under a subcontract to
 

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

NCI Carcinogenesis Testing Program.
 

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

(1,2) and R. R. Bates (1,3); the doses were selected by Drs. T. E.
 

Shellenberger (4,5,), J. H. Weisburger, and R. R. Bates.
 

Chemical administration and observation of animals were
 

supervised by Drs. T. E. Shellenberger and H. P. Burchfield (4),
 

iii
 
with the technical assistance of Ms. D. H. Monceaux (4) and Mr.
 

D. Broussard (4). Histopathology was performed by Drs. E. Bernal
 

(4) and B. Buratto (4) at Gulf South Research Institute, and the
 

diagnoses included in this report represent the interpretations
 

of these pathologists.
 

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

Mason Research Institute (6). Statistical analyses were
 

performed by Dr. J. R. Joiner (7) and Ms. P. L. Yong (7), using
 

methods selected for the bioassay program by Dr. J. J. Gart (8).
 

Chemicals used in this bioassay were analyzed under the direction
 

of Dr. H. P. Burchfield, and the results of the analyses were
 

reviewed by Dr. S. S. Olin (7).
 

This report was prepared at Tracor Jitco (7) under the direction
 

of NCI. Those responsible for the report at Tracor Jitco were
 

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

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

Williams, toxicologists; Ms. Y. E. Presley, technical writer; and
 

Dr. E. W. Gunberg, technical editor, assisted by Ms. P. J.
 

Graboske.
 

The following scientists at NCI were responsible for evaluating
 

the bioassay experiment, interpreting the results, and reporting
 

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

J. Fielding Douglas, Dr. Dawn G. Goodman (9), Dr. Richard A.
 

Griesemer, Dr. Thomas E. Hamm, Dr. William V. Hartwell, Dr. Harry
 

A. Miltnan, Dr. Thomas W. Orme, Dr. Robert A. Squire (10), Dr. 

Sherman F. Stinson, Dr. Jerrold M. Ward, and Dr. Carrie E. 

Whitmire. 

(1) Carcinogenesis Testing Program, Divison 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, Hammond House Road, Valhalla,
 

New York.
 

(3) Now with the National Institute of Environmental Health
 

Sciences, P.O. Box 12233, Research Triangle Park, North
 

Carolina.
 

(4) Gulf South Research Institute, Atchafalaya Basin
 

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

iv
 
(5)	 Now with the National Center for Toxicological Research,
 

Jefferson, Arkansas.
 

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

Rockville, Maryland.
 

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

Maryland.
 

(8)	 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.
 

(9)	 Now with Clement Associates, Inc., 1010 Wisconsin Avenue,
 

N.W., Suite 660, Washington, D. C.
 

(10) Now with the Division of Camparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 
v i

 
SUMMARY
 

A bioassay of technical-grade phosphamidon for possible
 

carcinogenicity was conducted using Osborne-Mendel rats and
 

B6C3F1 mice. The test material was administered in feed to 50
 

rats and 50 mice of each sex at one of two doses, either 80 or
 

160 ppm. The rats were fed the test chemical for 80 weeks, then
 

observed without compound administration for 30 or 31 weeks; the
 

low-dose male mice were fed for 71 weeks, then observed for 19
 

weeks; the high-dose male mice were fed for 62 weeks, then
 

observed for 28 weeks; and the low- and high-dose female mice
 

were fed for 80 weeks, then observed for 10 or 11 weeks. Matched
 

controls consisted of groups of 10 untreated rats or 10 untreated
 

mice of each sex; pooled controls consisted of the matched
 

controls combined with 85 male and 85 female untreated rats or 80
 

male and 80 female untreated mice from similar bioassays of eight
 

other test chemicals. All surviving rats were killed at 110 or
 

111 weeks; all surviving mice were killed at 90 or 91 weeks.
 

Hyperexcitability and tremors, both indications of phosphamidon
 

toxicity, were observed in dosed rats and mice. However,
 

sufficient numbers of all groups of both species were at risk for
 

the development of late-appearing tumors.
 

In male rats, the combined incidence of hemangiomas and
 

hemangiosarcomas in the spleen showed a statistically significant
 

(P • 0.012) dose-related trend. However, the comparison with
 

matched controls was not significant, and the historical records
 

of this laboratory on untreated males of this strain show a tumor
 

incidence of 6/240 (3%) with incidences in individual control
 

groups as high as 3/9 (33%) and 2/9(22%), compared with 5/49
 

(10%) seen in the high-dose group in this study. No hemangiomas
 

or hemangiosarcomas were found in the females.
 

In female rats, the Cochran-Armitage test for dose-related trend
 

was significant (P = 0.003) for C-cell adenomas and carcinomas of
 

the thyroid when pooled controls were compared with the dosed
 

groups. The incidences of these tumors were also significant
 

when low-dose females (P = 0.003) and high-dose females (P *
 

0.004) were compared directly with pooled controls. However, the
 

historical records of this laboratory show a tumor incidence of
 

16/235 (7%) in untreated female rats of this strain of female
 

rats, with incidences in individual control groups as high as 3/9
 

(33%) and 3/10 (30%); these data are therefore considered
 

marginal and insufficient to establish an association between the
 

tumors and administration of the chemical. In males, the
 

incidence of these tumors was not statistically significant.
 

vii
 
In mice, no tumor occurred at a higher incidence in dosed animals
 

than in controls.
 

It is concluded that under the conditions of this bioassay,
 technical- grade phosphamidon was not carcinogenic for B6C3F1
 

mice. The data obtained in this bioassay with Osborne -Mendel
 

rats are insufficient to allow the interpretation that
 

technical-grade phosphamidon is carcinogenic in this species.
 

viii
 
TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 3
 

C. Animals 5
 

D. Animal Maintenance 5
 

E. Sub chronic Studies 7
 

F. Chronic Studies 8
 

G. Clinical and Pathologic Examinations 11
 

H. Data Recording and Statistical Analyses 12
 

III. Results - Rats 19
 

A. Body Weights and Clinical Signs (Rats) 19
 

B. Survival (Rats) 21
 

C. Pathology (Rats) 23
 

D. Statistical Analyses of Results (Rats) 25
 

IV. Results - Mice 29
 

A. Body Weights and Clinical Signs (Mice) 29
 

B. Survival (Mice) 31
 

C. Pathology (Mice) 33
 

D. Statistical Analyses of Results (Mice) 33
 

V. Discussion 35
 

VI. Bibliography 39 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Fed Phosphamidon in the Diet 41
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Fed Phosphamidon in the Diet 43
 

Table A2 Summary of the Incidence of Neoplasms in
 

Female Rats Fed Phosphamidon in the Diet 47
 

ix
 
Page 

Appendix B Analyses of the Incidence of Primary Tumors 

in Mice Fed Phosphamidon in the Diet 51 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Fed Phosphamidon in the Diet 53 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Fed Phosphamidon in the Diet 56 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Fed Phosphamidon in the Diet... 59 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Phosphamidon in 

the Diet 61 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Phosphamidon 

in the Diet 64 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Fed Phosphamidon in the Diet... 67 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed Phosphamidon 

in the Diet 69 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed Phosphamidon 

in the Diet 72 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats fed Phosphamidon in the Diet 75 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Fed Phosphamidon in the Diet 77 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Fed Phosphamidon in the Diet.... 80 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Fed Phosphamidon in the Diet 85 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Fed Phosphamidon in the Diet 87 
Page 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Fed Phosphamidon in the Diet.... 89 

Appendix G Analysis of Formulated Diets for 

Concentrations of Phosphamidon 93 

TABLES 

Table 1

Table 2

 Phosphamidon Chronic Feeding Studies 

in Rats

 Phosphamidon Chronic Feeding Studies 

in Mice

 9 

 10 

FIGURES 

Figure 1 Growth Curves for Rats Fed Phosphamidon 

in the Diet 20 

Figure 2 Survival Curves for Rats Fed Phosphamidon 

in the Diet 22 

Figure 3 Growth Curves for Mice Fed Phosphamidon in 

the Diet 30 

Figure 4 Survival Curves for Mice Fed Phosphamidon 

in the Diet 32 

xi
 
x i i

 
I. INTRODUCTION
 

Phosphamidon (CAS 13171-21-6; NCI C00588) is an organophosphorus
 

compound used as a broad-spectrum insecticide in agriculture
 

since 1956 (Spencer, 1973). It is toxic both systemically and by
 

contact, and acts through the inhibition of cholinesterase
 

(Hazleton and Weir, 1958). Phosphamidon is currently registered
 

for use by both ground and aerial applications on vegetables,
 

fruits, and field crops with tolerances for residues from 0.1 to
 

1.0 ppm (EPA Compendium, 1974).
 

Phosphamidon was one of a series of pesticides selected for
 

screening for carcinogenic activity because of the potential for
 

long-term human exposure to the chemical during agricultural
 

application or to residues in food products.
 

II. MATERIALS AND METHODS
 

A. Chemical
 

Technical-grade phosphamidon (dimethyl 2*chloro-2-diethylcarbamoyl-1-methylvinyl phosphate) was obtained from Chevron
 

Chemical Company, Oreho Division (San Francisco, Calif.), in one
 

batch for the chronic study. The identity of the chemical was
 

confirmed by analyses at Gulf South Research Institute. Elemental
 

analysis (C, H, N, Cl, P) was correct for the molecular formula
 

of phosphamidon, CJQH^CINOJP . Infrared, nuclear magnetic
 

resonance, and mass spectra were as expected for phosphamidon.
 

No attempt was made to identify or quantitate any impurities that
 

may have been present. The chemical was stored at 5 C in the
 

original amber glass container until used.
 

B

* Dietary Preparation
 

All diets containing phosphamidon were formulated once per week
 

using Wayne® Lab Blox animal meal (Allied Mills, Inc., Chicago,
 

111.) to which was added the required amount Of phosphamidon for
 

each dietary concentration. The test chemical was first
 
dissolved in a small amount of acetone (Mallinckrodt, Inc., St.
 

Louis, Mo.), which was then added to the feed. Corn oil
 

(LouAna , Opelousas Refinery Co., Opelousas, La.) was also
 

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

were mixed mechanically to assure homogeneity of the mixtures and
 

evaporation of the acetone. Final diets, including those for the
 

control groups of animals, contained corn oil equal to 2Z of the
 

final weight of feed. The diets were stored at approximately
 

17 C until used, but no longer than 1 week.
 

The stability of phosphamidon in feed was tested by determining
 

the concentration of the chemical in formulated diets at
 

intervals over a 7-day period. Diets containing either 80 or 160
 

ppm phosphamidon showed no change in concentration on standing at
 

ambient temperature for this period.
 

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

diets, the concentration of phosphamidon was determined in
 

different batches of formulated diets during the chronic study.
 

The results are summarized in Appendix G. The means of the
 

analytical concentrations for the checked samples were 81.7 *_ 3.8
 

and 161.5 +_ 6.3 ppm for target concentrations of 80 and 160 ppm,
 

respectively.
 
C. Animals
 

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

Division of Cancer Treatment, National Cancer Institute, were
 

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

strain obtained from Battelle Memorial Institute, Columbus, Ohio,
 

and the mice were B6C3F1 hybrids obtained from Charles River
 

Breeding Laboratories, Inc., Wilmington, Massachusetts. On
 

arrival at the laboratory, all animals were quarantined (rats for
 

6 days, mice for 13 days) and then were assigned to either
 

control or dosed groups.
 

D. Animal Maintenance
 

All animals were housed by species in rooms maintained at 22 to
 

24 C, and 40 to 70% relative humidity. The air in each room
 

was filtered (Air Maze Incom International, Cleveland, Ohio), and
 

room air was changed 10 to 12 times per hour. Fluorescent
 

lighting provided illumination 10 hours per day. Food and water
 

were provided ad_ libitum. Fresh feed was provided daily, and
 

excess remaining feed was discarded.
 

The rats were housed individually in hanging galvanized steel
 
mesh cages (Hoeltge, Inc., Cincinnati, Ohio), and the mice were
 

housed in polypropylene cages (Lab Products, Inc., Garfield,
 

N.J.), five females per cage or two or three males per cage.
 

Mouse cages were covered with polyester filter bonnets (Lab
 

Products, Inc.). The rat racks and cages were sanitized every 2
 

weeks. The mouse cages were sanitized each week. These cages
 

and racks were washed in an industrial washer at 82 C with
 

Acclaim detergent (Economics Laboratory, Inc., St. Paul,
 

Minn.) and then rinsed. Absorbent Kimpak cage liners (Kimberly
 

Clark Corp., Neenah, Wis.) were placed under the rat cages and
 

were 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 sanitized each week. Peed jars
 

and water bottles were changed and sanitized three times per
 

week. Sipper tubes and stoppers were sanitized two times per
 

week.
 

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

stoppers were washed in a Vulcan Autosan Washer (Louisville, Ky.).
 

Cage racks for each species were rotated to a new position in the
 

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

different row within the same column of a rack. Rats receiving
 
phosphamidon, along with their matched controls, were housed in a
 

room by themselves. Mice were maintained in a room housing mice
 

from the following studies:
 

Feed Studies
 

(CAS 56-38-2) parathion
 

(CAS 60-51-5) dimethoate
 

E. Subchronic Studies
 

Subchrom'.c feeding studies were conducted with rats and mice to
 

estimate the maximum tolerated doses of phosphamidon, on the
 

basis of which two concentrations (referred to in this report as
 

"low" and "high" doses) were selected for administration in the
 

chronic studies. In the subchronic studies, phosphamidon was
 

added to the animal feed in twofold increasing concentrations,
 

ranging from 10 to 1,280 ppm for both rats and mice. The
 

chemical was provided in feed to dosed groups of five male and
 

five female animals of each species for 6 weeks, followed by
 

observation for 2 weeks. Untreated-control groups consisted of
 

five animals of each species and sex.
 

At 320 ppm and higher, all rats and all male mice died by week
 

2. No animals died at 160 ppm, although weight gains in dosed
 
rats were lower than those of their controls during the second
 

week of the study; for the remainder of the study, the behavior
 

of these animals was generally similar to that of the controls.
 

Male mice fed 160 ppm lost weight during the first week, but
 

generally gained weight throughout the remainder of the study.
 

The weight gains in the female mice at 160 ppm were similar to
 

those of their controls throughout the 8-week study. The low and
 

high doses for the chronic studies were set at 80 and 160 ppm for
 

both rats and mice.
 

F. Chronic Studies
 

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

feeding studies are shown in tables 1 and 2.
 

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

small, pooled-control groups also were used for statistical
 

comparisons. For rats, matched controls from the current
 

bioassay of phosphamidon were combined with matched controls from
 

the bioassays of tetrachlorvinphos (CAS 961-11-5), toxaphene (CAS
 

8001-35-2), endrin (CAS 72-20-8), lindane (CAS 58-89-9),
 

chlorothalonil (CAS 1897-45-6), chloramben (CAS 133-90-4),
 

picloram (CAS 1918-02-1), and malathion (CAS 121-75-5). The
 

8
 
Table 1. Phosphamidon Chronic Feeding Studies in Rats
 

Sex and 

Test 

Group 

Male 

Mat ched-Cont ro1 

Low-Dose 

High-Dose 

Female 

Matched-Control 

Low-Dose 

High-Dose 

Initia l Phosphamidon 

No. of Doses 

Animals (a) (ppm) 

10 0 

50 80 

0 

50 160 

0 

10 0 

50 80 

0 

50 160 

0 

Time 

Dosed 

(weeks) 

80 

80 

80 

80 

on Study 

Observed (b) 

(weeks) 

110 

30 

30 

111 

31 

31 

(a)	 All animals were 5 weeks of age when placed on study.
 

(b) When diets containing phosphamidon were discontinued,	 all
 

rats received control diets (2% corn oil added) until
 

termination.
 
Table 2. Phosphamidon Chronic Feeding Studies in Mice
 

Sex and Initial Phosphamidon Time on Study 

Test No. of Doses Dosed Observed (b) 

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

Male 

Matched-Control 10 0 90-91 

Low-Dose 50 80 71 

0 19 

High-Dose 50 160 62 

0 28 

Female
 

Matched-Control 10 0 90-91
 

Low-Dose 50 80 80
 

0 11
 

High-Dose 50 160 80
 

0 11
 

(a) All animals were 5 weeks of age when placed on study.
 

(b) When diets containing phosphamidon were discontinued,
 

high-dose males received control diets without corn oil added
 

for 8.5 weeks, then control diets (2% corn oil added) for
 

19.5 weeks. Low-dose male and all female mice were fed
 

control diets until termination.
 

10
 
pooled controls for statistical tests consisted of 95 rats and 90
 

mice of each sex. These studies overlapped the bioassay of
 

phosphamidon by at least 1 year. The pooled-control animals were
 

of the same strain as the test animals (Osborne-Mendel rats or
 

B6C3P1 mice) and from the same supplier, and their tissues were
 

diagnosed by the same pathologists.
 

G. Clinical and Pathologic Examinations
 

All animals were observed twice daily for deaths. Observations
 

to identify sick, tumor-bearing, and moribund animals were made
 

daily. Animals were weighed every 2 weeks for the first 12
 

weeks and monthly thereafter, and palpated for masses at each
 

weighing. Moribund animals and animals that survived to the end
 

of the bioassay were killed using ether and necropsied.
 

The pathologic evaluation consisted of gross and microscopic
 

examination of major tissues, major organs, and all gross
 

lesions. The tissues were preserved in 10% neutral buffered
 

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

indicated. The following tissues were examined microscopically:
 

skin, lungs and bronchi, trachea, bone and bone marrow, spleen,
 

11
 
lymph nodes, heart, salivary gland, liver, gallbladder (mice),
 

pancreas, stomach, small intestine, large intestine, kidney,
 

urinary bladder, pituitary, adrenal, thyroid, parathyroid, mammary
 

gland, prostate or uterus, test is or ovary, and brain. Occasionally, additional tissues were also examined microscopically.
 

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
 

Pertinent data on this experiment have been recorded in an
 

automatic data processing system, the Carcinogenesis Bioassay
 

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

descriptive information on the chemicals, animals, experimental
 

design, clinical observations, survival, animal weight, and
 

individual pathologic results, as recommended by the
 

International Union Against Cancer (Berenblum, 1969). Data
 

12
 
tables were generated for verification of data transcription and
 

for statistical review.
 

These data were analyzed using the statistical techniques
 

described in this section. Those analyses of the experimental
 

results that bear on the possibility of carcinogenicity are
 

discussed in the statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

report in the form of graphs. Animals were statistically
 

censored as of the time that they died of other than natural
 

causes or were found to be missing; animals dying from natural
 

causes were not statistically censored. Statistical analyses for
 

a possible dose-related effect on survival used the method of Cox
 

(1972) for testing two groups for equality and Tarone's (1975)
 

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

trend. One-tailed P values have been reported for all tests
 

except the departure from linearity test, which is only reported
 

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

The incidence of neoplastic or nonneoplastic lesions has been
 

fci.ven as the ratio of the number of animals bearing such lesions
 

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

13
 
in which that site is examined (denominator). In most instances,
 

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic
 

examination was required to detect lesions prior to histologic
 

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

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

denominators consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

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

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

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

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

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

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

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

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

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

where the Fisher exact P values are shown.
 

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

14
 
continuity correction (Armitage, 1971), is also used. Assuming a
 

linear trend, this test determines if the slope of the dose-


response curve is different from zero at the one-tailed 0.05
 

level of significance. Unless otherwise noted, the direction of
 

the significant trend is a positive dose relationship. This
 

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

trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

first tumor was observed were excluded by basing the statistical
 

tests on a,m'mals that survived at least 52 weeks, unless a tumor
 

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

such an early tumor was found, comparisons were based exclusively
 

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

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

obtained, the standard procedures for analyses of the incidence
 

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

followed.
 

When appropriate, life-table methods were used to analyze the
 

incidence of tumors. Curves of the proportions surviving without
 

an observed tumor were computed as in Saffiottt et al. (1972).
 

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

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

methods of comparing these curves were used for two groups, and
 

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

groups. All 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 less than 0.05, two-tailed test)
 

were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared to its control was calculated
 

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

relative risk is defined as p /p where p is the true
 

binomial probability of the incidence of a specific type of tumor
 

in a dosed group of animals and p is the true probability of
 

the spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

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

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

of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

The lower and upper limits of the confidence interval of the
 

16
 
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 left endpoint of the confidence interval is
 

greater than zero, the occurrence of 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.
 

17
 
18

 
III. RESULTS - RATS
 

A, Body Weights and Clinical Signs (Rats)
 

The mean body weights of the high-dose male and high-dose female
 

rats were lower than those of the matched controls for most of
 

the study (figure 1). The mean body weights of the low-dose
 

males and low-dose females were essentially unaffected by the
 

administration of phosphamidon. Fluctuation in the growth curve
 

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

mean body weight may be subject to wide variation.
 

During the first year of the study, the high-dose males and the
 

low-dose females were generally comparable to the controls in
 

appearance and behavior. The high-dose females appeared to be
 

hyperexcitable from weeks 6 to 10. At week 1.6, one low-dose male
 

and one high-dose male showed generalized body tremors.
 

Convulsions were observed in one low-dose male at week 19.
 

Beginning at week 28, a majority of the low-dose males had
 

generalized alopecia.
 

During the second year of the study, clinical signs were noted
 

with increasing frequency in both matched controls and dosed
 

19
 
Figure 1. Growth Curves for Rats Fed Phosphamidon in the Diet 

20 
animals. These signs included loss of weight, pale mucous
 

membranes, diarrhea, dermatitis, vaginal bleeding, rough hair
 

coats, epistaxis, and tachypnea. A majority of the dosed animals
 

had wet and urine-stained hair coats during the second year.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats administered phosphamidon in
 

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

the matched controls, are shown in figure 2. In neither sex is
 

the Tarone test result significant (P greater than 0.05) for
 

positive dose-related trend in mortality over the bioassay.
 

In male rats, 36/50 (72%) of the high-dose group, 34/50 (68%) of
 

the low-dose group, and 5/10 (50%) of the matched-control group
 

survived to the end of the bioassay. In females, 39/50 (78%) of
 

the high-dose group, 41/50 (82%) of the low-dose group, and 9/10
 

(90%) of the matched-control group lived to termination of the
 

study. Thus, sufficient numbers of rats of each sex were at risk
 

for the development of late-appearing tumors.
 

21
 
Figure 2. Survival Curves for Rats Fed Phosphamidon in the Diet 

22 
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 variety of neoplasms are represented among both the dosed and
 

the control rats. In general, these lesions are common in this
 

strain of rat, independent of any treatment. However,
 

hemangiosarcomas of the spleen occurred in one control male,
 

three low-dose males, and five high-dose males; a hemangioma
 

occurred in one other low-dose male. No hemangiomas or
 

hemangiosarcomas of the spleen occurred in any of the
 

matched-control or dosed females. The hemangiosarcomas in the
 

spleen of the male rats varied from very cellular areas in the
 

red pulp with minimal vascularity, to lesions with prominent
 

vascular spaces, hemorrhage, and necrosis. The tumor cells in
 

the stroma and lining vesicular spaces were large, hyperchromatic
 

oval to spindle cells, with prominent nuclei and nucleoli, and
 

several mitotic figures. The boundaries of the tumors were
 

irregular and indistinct, and some lesions involved the splenic
 

capsule. Although these tumors have been encountered in control
 

animals in the past, their occurrence at the incidences noted in
 

this study is infrequent.
 

23
 
C-cell adenomas of the thyroid also occurred with relatively high
 

incidences in low- and high-dose rats of each sex but not in any
 

of the matched controls. C-cell carcinomas were found in one
 

matched-control male, one low-dose female, and one high-dose
 

female. C-cell hyperplasias of the thyroid were also observed.
 

Proliferative lesions of the C-cells occurred as follows:
 

Males Females
 

Matched Low High Matched Low High 

Control Dose Dose Control Dose Dose 

Number of Animals with 

Tissue Examined 

Microscopically (8) (48) (47) (10) (50) (46) 

Thyroid 

C-cell Hyperplasia 0 7 3 0 4 0 

C-cell Adenoma
 0 7
 5
 0
 8

1
 

7
 

C-cell Carcinoma
 1 0 0 0
 1


Although these thyroid lesions have occurred in control rats of
 

this strain used in other similar studies, the incidence with
 

which they occurred in this experiment may indicate an
 

association with the administration of phosphamidon.
 

In addition to the neoplastic lesions, a number of degenerative,
 

proliferative, and inflammatory changes were encountered in
 

animals of the control and dosed groups. For the most part,
 

these nonneoplastic lesions were similar to those commonly
 

encountered in aged rats.
 

24
 
D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

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 Cochran-Armitage test for positive dose-related
 

trend in proportions for combined heraangioma and hemangiosarcoma
 

is significant (P = 0.012) using the pooled controls. The result
 

of the Fisher exact test shows a significantly higher incidence
 

of this tumor in the high-dose group (P = 0.020) than in the
 

pooled controls. The historical records of this laboratory show
 

an incidence of these tumors of 6/240 (3%) in untreated male rats
 

of this strain of male rats, with incidences in individual
 

control groups as high as 3/9 (33%) and 2/9 (22%), compared with
 

5/49 (10%) seen in the high-dose groups in this study. This
 

tumor was not observed in female rats.
 

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


related trend in the incidence of C-cell adenoma of the thyroid
 

in male rats are not significant. The Fisher exact comparison of
 

incidence between the low-dose and pooled-control groups shows a
 

P value of 0.031, which is above the 0.025 level required for
 

25
 
significance when the Bonferroni inequality criterion is used for
 

multiple comparison.
 

In females, the results of the Cochran-Armitage test for positive
 

dose-related trend in the incidence of animals with either C-cell
 

adenoma or carcinoma of the thyroid is significant (P * 0.003)
 

using the pooled controls. The result of the Fisher exact test
 

shows significantly higher incidences of this tumor both in the
 

low-dose group (P = 0.003) and the high-dose group (P • 0.004)
 

than in the pooled controls. The historical records of this
 

laboratory show an incidence of these tumors of 16/235 (7%) in
 

untreated female rats of this strain, with incidences in
 

individual control groups as high as 3/9 (33%) and 3/10 (30%),
 

compared with 8/46 (17%) seen in the high-dose group in this
 

study.
 

In male rats, the incidence of cortical adenoma of the adrenal in
 

the low-dose group is significantly higher (P * 0.023) than that
 

in the pooled-control group, but not higher than that in the
 

matched-control group. The incidence in the high-dose group is
 

not significant, nor are the results of the Cochran-Armitage test
 

using either control group.
 

26
 
Significant trends in the negative direction are observed in the
 

incidence of carcinoma of the pituitary in female rats27
 
28

 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

The mean body weights of the dosed male mice were lower than
 

those of the matched controls, and there was a dose-related
 

effect (figure 3). Fluctuation in the growth curve may be due to
 

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

weight may be subject to wide variation. The mean body weights
 

of the dosed females were essentially the same as the control
 

values.
 

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

generally comparable to the controls in appearance and behavior.
 

At week. 29, a majority of the dosed and control males were
 

observed fighting, which resulted in alopecia and in wounds.
 

During the second half of the study, clinical signs including
 

alopecia, rough hair coats, and abdominal distention were noted
 

with increasing frequency in both control and dosed animals. A
 

few low-dose males and a majority of high-dose males showed
 

generalized tremors at week 60. Tremors persisted in the
 

high-dose males until week 80. At week 69, a majority of
 

29
 
Figure 3. Growth Curves for Mice Fed Phosphamidon in the Diet 

30 
low-dose and high-dose males had rough hair coats and generally
 

appeared to be in poor physical condition.
 

B

« Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice administered phosphamidon in
 

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

the matched controls, are shown in figure 4. In neither sex is
 

the result of the Tarone test significant (P greater than 0.05)
 

for positive dose-related trend in mortality.
 

In male mice, 42/50 (84%) of the high-dose group, 45/50 (90%) of
 

the low-dose group, and 7/10 (70%) of the control animals lived
 

to the end of the study. In females, 45/50 (90%) of the
 

high-dose group, 46/50 (92%) of the low-dose group, and all 10 of
 

the control animals survived to the end of the study. Thus,
 

sufficient numbers of mice of each sex were at risk for the
 

development of late-appearing tumors.
 

31
 
Figure 4. Survival Curves for Mice Fed Phosphamidon in the Diet 

32 
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.
 

Neoplasms occurred at approximately equal incidences in control
 

and dosed mice. Hepatocellular adenomas and carcinomas, which
 

occurred in both control and dosed mice, were of the type usually
 

seen in aging mice of this strain. Under the conditions of this
 

study, there was no evidence that phosphamidon was carcinogenic
 

to B6C3F1 mice.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

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

in one or more than one group.
 

The results of the Cochran-Armitage test are not significant (P
 

greater than 0.05) in either sex for positive dose-related trends
 

in the proportions of tumors at any site, and none of the results
 

33
 
of the Fisher exact test are significant. However, in each of
 

the 95% confidence intervals for relative risk, shown in the
 

tables, the value of one is included; this indicates the absence
 

•jf significant positive results. It should also be noted that
 

each of the intervals has an upper limit greater than one,
 

indicating the theoretical possibility of the induction of tumors
 

by phosphamidon, which could not be detected under the conditions
 

of this test.
 

34
 
V. DISCUSSION
 

Phosphamidon is a member of the organophosphorus class of
 

pesticides whose predominant mode of toxicity is inhibition of
 

cholinesterase. Toxic effects characteristic of these chemicals
 

were evident in the animals in this bioassay. During the first
 

year of the study, both male and female rats were
 

hyperexcitable. Several males were observed to have convulsions.
 

In mice, the hyperexcitability and generalized tremors were more
 

pronounced in males than in females and developed at a later time
 

than in rats. Other clinical signs, such as rough hair coats,
 

dermatitis, epistaxis, and vaginal bleeding, increased in
 

frequency with age and could not definitely be correlated with
 

the administration of phosphamidon. Administration of
 

phosphamidon resulted in low mean body weights in high-dose male
 

and female rats and in high-dose male mice, but had no adverse
 

effect on the survival of either rats or mice. Sufficient
 

numbers of all groups of both species were at risk for the
 

development of late-appearing tumors.
 

The incidence of the combination of hemangioma and
 

hemangiosarcoma in both high- and low-dose male rats was
 

statistically significant (P = 0.012) when compared with the
 

35
 
pooled controls (pooled controls 1/91, matched controls 1/7, low-


dose 3/50, high-dose 5/49). The comparison with matched controls
 

was not significant. All of these tumors occurred in the spleen
 

in the animals in the present study. However, the historical
 

records of this laboratory on untreated males of this strain show
 

a tumor incidence of 6/240 (3%) with incidences in individual
 

control groups as high as 3/9 (33%) and 2/9 (22%). The data from
 

this bioassay with male rats are considered marginal and
 

insufficient to establish an association between the tumors and
 

administration of the chemical. These lesions did not occur in
 

female rats.
 

In male rats, the incidence of cortical adenoma of the adrenal in
 

the low-dose group was significantly higher (P • 0.023) than that
 

in the pooled control group (pooled controls 0/8, matched
 

controls 2/90, low-dose 6/49, high-dose 2/49). However, the
 

incidence in the high-dose group was not significant, and the
 

occurrence of the tumor was not considered to be related to
 

administration of the test chemical.
 

In female rats, the Cochran-Armitage test for dose-related trend
 

was significant (P « 0.003) for C-cell adenomas and carcinomas of
 

the thyroid when pooled controls were compared with the dosed
 

groups (pooled controls 2/82, matched controls 0/10, low-dose
 

36
 
9/50, high-dose 8/46). The incidence of these tumors was also
 

significant when low-dose females (P = 0.003) and high-dose
 

females (P = 0.004) were compared with corresponding pooled
 

controls. However, the historical records of this laboratory
 

show a tumor incidence of 16/235 (7%) in untreated female rats of
 

this strain, with incidences in individual control groups as high
 

as 3/9 (33%) and 3/10 (30%). The data from this bioassay with
 

female rats are considered marginal and insufficient to establish
 

an association between these tumors and administration of the
 

chemical. In males, the incidence of these tumors was not
 

statistically significant.
 

In mice, no tumor occurred at a statistically significant
 

incidence.
 

Sachsse and Voss (1971) reported that phosphamidon was not
 

carcinogenic in rats. In their study, rats were fed diets
 

containing 0.1 to 5 mg/kg of phosphamidon daily for 2 years, and
 

various organs, including the thyroid glands and spleens of the
 

high-dose animals, were examined. A different strain of rat from
 

the one used in the present bioassay (Carworth CFN vs.
 

Osborne-Mendel) and a lower maximum dose (5 mg/kg/day vs.
 

approximately 10 mg/kg/day) were used.
 

37
 
It is concluded that under the conditions of this bioassay,
 

technical-grade phosphamidon was not carcinogenic for B6C3F1
 

mice- The data obtained in this bioassay with Osborne-Mendel
 

rats are insufficient to allow the interpretation that
 

technical-grade phosphamidon is carcinogenic in this species.
 

38
 
VI. BIBLIOGRAPHY
 

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

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

Berenblum, I., ed., Carcinogenicity Testing, UICC Technical
 

Report Series, Vol. 2_. International Union Against Cancer,
 

Geneva, 1969.
 

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

Soc. B 34:187-220, 1972.
 

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

London, 1970, pp. 48-52.
 

Environmental Protection Agency, EPA Compendium of Registered
 

Pesticides, U. S. Government Printing Office, Washington, D. C ,
 

1974, III-P-12.1-12.5.
 

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

significance tests, confidence limits and adjustments for
 

stratifiation. Rev. Int. Statist. Inst. 39(2):148-169, 1971.
 

Hazleton, L. W. and Weir, R. J., Organic Phosphate. In:
 

Industrial Hygiene and Toxicology, Patty, F. A., ed.,
 

Interscience Publishers, New York, 1958, pp. 1935-1952.
 

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

incomplete observation. J_^ Amer. Statist. Assn. 53[:457-48l, 1958.
 

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

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

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

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

McGraw-Hill, New York, 1966.
 

Sachsse, K. R. and Voss, G., Toxicology of phosphamidon.
 

Residue Reviews 37:61-88,
 

1971.
 

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

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

induced by different numbers of administration of benzo(a) pyrene
 

and ferric oxide. Cancer Res. 32:1073-1081, 1972.
 

39
 
Spencer, E. Y., Guide £o Chemicals Used _in Crop Protection, Research Institute, University of Western Ontario, London, Ontario, 1973, pp. 410-411. 

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

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

40
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS FED PHOSPHAMIDON IN THE DIET
 

41
 
42

 
TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

FED PHOSPHAMIDON IN THE DIET

 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 10 50 50
 

ANIMALS NECROPSIED 9 50 49
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 9 50 49
 

INTEGUMENTAFY SYSTEM
 

*SKIN (9) (50) (49)

 

FIBROUS HISTIOCYTOMA 1 (2%) 

HEMANGIOPERICYTCMA, MALIGNANT 1 (11%)

 

RESPIRATORY SYSTEM 

#LUNG (8) (49) (49)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

•MULTIPLE ORGANS	 (9) (50 ) (49)

 

UNDIFFERENTIATED LEUKEMIA
 

i (2i) 

#SPLEEN (7) (50) 

HEMANGIOMA 1 (2%) (49) 

HEMANGIOSARCOMA 1 (14X) 2 (4%) 

HAKARTOMA 1 (2%) 5 (10%) 

CIRCULATORY SY3T ]M 

N0N2 

DIGESTIVE SYSTEM 

CSALIVARY GLAND (9) (50) (47) 

ADENOMA, NOS 1 (27.) 

#LIV2B (9) (49) (45) 

NE2£LiSTI_C_NODULE 1-1'iAl. 

# NUK3EP. OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* IIUMBER OF ANIMALS NECROPSIED 

43 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

#STOMACH (7) (50) (45) 

SQ'JAMOUS CELL CABCIKOMA 1 (2%) 

#COLON (38) (40) 

ADENOCARCINOMA, NOS 1 (3S) 

URINARY SYSTEM 

#KIDNEY (9) (49) (49)
 

f HAMARTOMA 1 (2%)
 

#URINARY BLADDER (6) (46) (47) 

TRANSITIONAL-CELL CARCINOMA 1 (2 4) 

ENDOCRINE SYSTEM
 

#PITUITARY (8) (49) (48)
 

CARCINOMA,NOS 1 (2%) 1 (255)
 

ADENOMA, NOS 1 (2%)
 

CHROMOPHOBE ADENOMA 2 (25%) 8 (16%) 3 (65)
 

•ADRENAL (8) (49) (49) 

CORTICAL ADENOMA 6 (12%) 2 (4%) 

PHEOCHROMOCYTOMA 1 (2X) 

•THYROID (8) (48 ) (47) 

FOLLICULAR-CELL CARCINOMA 1 (2«) 

C-CELL ADENOMA 7 (1555) 5 (11») 

C-CELL CARCINOMA 1 (13%) 

tPANCSEATIC ISLETS (7) (47) (49) 

ISLET-CELL ADENOMA 2 (4%) 2 (4J) 

REPRODUCTIVE SYSTEM 

•MAMMARY GLAND (9 ) (50 ) (49 ) 

FIBKOMA 1 (11%) 

NERVOUS SYSTEM 

#BRAIN (9) (50) (47) 

GRANULAR-CELL TUMOR, BENIGN 1_[2XJ 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALLY 

* NUMBER OF ANIMALS NECBOPSIED 

+ NONNEOPLASTIC PROLIFERATIVE LESION 

COMPOSED OF LIPOCYTES, TUBULAR STRUCTURES,
 AND FIBROBLASTS IN VARYING PROPORTIONS.
 

44
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL SENSE OR ANS 

NONE 

MOSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALI OTHER SYSTEMS 

•MULTIPLE ORGAN (9) (50) (49)

 

FIBROOS HISTIOCYTOMA 1 (2%)

 

FIBROUS HISTIOCYTOHA, MALIGNANT 1 (2%)

 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 10 50 50 

NATURAL DEATHS 4 

MORIBUND SACRIFICE 12 

SCHEDULED SA? RIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 34 36 

ANIMAL MISSING 

S _ IN C L U D ES_ A UT0 L_YZ E D_AN IM A L S_ 

# NUMBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

45
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

T'JMOB SUMMARY
 

TOTAL ANIMALS KITH PRIMARY TUMORS* 4 26 20
 

TOTAL PPIMARY TUMORS 6 34 27
 

TOTAL ANIMALS WITH BENIGN TUMORS 2 24 10
 

TOTAL BENIGN TUMORS 3 30 15
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 3 4 10
 

TOTAL MALIGN NT TUMORS 3 4 11
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECOND RY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALT NANT 1
 

TOTAL UNCERTAIN TUMORS 1
 

TOTAL ANIMALS WITH TUMOBS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECONDARY TUMORS: METASTATIC TUHO6S OR TUKORS INVASIVE INTO AN ADJACENT ORGAN
 

46
 
TABLE A2. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATFED PHOSPHAMIDON IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS

ANIMALS

ANIMALS

 INITIALLY IN STUDY

 NECROPSIED

 EXAMINED HISTOPATHOLOGICALLY

 10 

 10 

 10 

50 

50 

50 

50 

49 

49 

INTEGUMENTARY SYSTEM 

*SUBCUT TISSUE 

FI3P0US HISTIOCYTOMA 

(10) (50) 

1 (28) 

(49) 

EESPIRATOBY SYST IH 

#LUNG 

ALVEOLAR/BRONCHIOLAR ADENOMA 

C-CELL CARCINOMA, METASTATIC 

(10) (50) 

1 (2X) 

1 (2%) 

(49) 

HEMATOPOIETIC SYSTEM 

NONE 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

•LIVER 

NEOPLASTIC NODULE 

(10 ) (50) 

1 (2%) 

(49 ) 

URINARY SYSTEM 

#KIDNEY 

•f HAHARTOHA 

(10 ) (50 ) 

1 (2%) 

(49 ) 

ENDOCRINE SYSTEM 

#PITUITAPY 

CARCINOMA,NOS 

(10 ) 

._iLi20Xl_ 

(50 ) 

# NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

t NONNEOPLASTIC PROLIFERATIVE LESION 

COMPOSED OF LIPOCYTES, TUBULAR STRUCTURES, 

AND FIBROBLASTS IN VARYING PROPORTIONS. 

(45) 

47
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

CHROflOPHOBE ADENOMA 3 (305!) 7 (14*) 7 (16X) 

•ADRENAL (10) (50) (48) 

CARCINOMA,NOS 1 (10S) 

CORTICAL ADENOMA 2 (20%) 6 (12%) 6 (13«) 

CORTICAL CAR INOMA 1 (2%) 

#THYROID (10) (50) (46) 

FOI.IICULAR-CELL CARCINOMA 1 (2%) 

C-C2LL ADENOMA 8 (16X) 7 (15X) 

C-CELL CARCINOMA 1 (2%) 1 (2X) 

•PANCREATIC ISLETS (10) (49) (4 8) 

ISLET-CELL ADENOMA 1 (255) 

REPRODUCTIVE SYSTEM 

•KAMMARY GLAND (10) (50) (49) 

ADENOMA, NOS 1 (255) 

FIBROMA 1 (2%) 

FIBROADENOMA 2 (2 OX) 6 (12%) 3 (6X) 

•UTERUS (10) (48) (48) 

PAPILLARY ADENOMA 1 (10X) 

L2I0BY0HA 1 (2%) 

LEIOMYOSARCOMA 1 (2%) 

F.NDOMETRIAL STROMAL POLYP 1 (2X) 3 (6X) 

#OVARY 

GRANULOSA-CELL TUMOR 

(10) («9) 

1 (255) 

(49) 

1 (2%) 

SARCOMA, NOS 1 (2X) 

NERVOUS SYSTEM 

#BRAIN (9) (50) (49) 

GRANULAB-CELL TUMOR, BENIGN 1 (2X) 

OLIGODENDPOGLIOMA 1 (1IX) 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

NONE 

# NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIHALS NECROPSIED 

48 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 10 50 50
 

NATURAL DEATHS 

MORIBUND SACRIFICE 

SCHEDULED SA; RIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE «1 39
 

ANIMAL MISSIK3 

5 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 7 29 26
 

TOTAL PRIMARY TUMORS 12 40 31
 

TOTAL ANIMALS KITH BENIGN TUMORS 26 23
 

TOTAL BENIGN TUMORS 34 28
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 4 4 2
 

TOTAL MALIGN NT TUMORS 4 4 2
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1
 

TOTAL SECOND RY TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ BENIGN OR MALIC NANT 2 1
 

TOTAL UNCERTAIN TUMORS 2 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMOHS
 

# SECONDARY TUMORS: METASTATIC TUMORS OR TUMOHS INVASIVE INTO AN ADJACENT OK3AN
 

49
 
50

 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE FED PHOSPHAMIDON IN THE DIET
 

51
 
52

 
TABLE B1. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

FED PHOSPHAMIDON IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS

ANIMALS

ANIMALS

 INITIALLY IN STUDY

 N2CR0PSIED

 EXAMINED HISTOPATHOLOGICALLY

 10 

9 

9 

50 

49 

49 

50 

49 

48 

INTEGUMENTARY SYSTEM 

•SKIN

FIBROSARCOMA 

FIBROUS HISTIOCYTOMA 

 (9) (49) 

1 (2%) 

(49 ) 

1 (2%) 

•SUBCUT TISSUE

FIBROMA 

FIBROUS HISTIOCYTOMA, MALIGNANT 

 (9) (49) 

1 (2«) 

1 (2%) 

(49) 

1 (2«) 

RESPIRATORY SYST 1M 

#LUNG

ALVEOLAR/BRONCHIOLAR

ALVEOLAR/BRONCHIOLAE

 ADENOMA

 CARCINOMA

 (9)

 1 (11J)

 (49)

 2 (4«)

 1 (2»)

 (45) 

 1 (2%) 

1 (2*) 

HEMATOPOIETIC SYSTEM 

•MULTIPLE ORGANS

MALIGNANT LYMPHOMA, NOS

 (9) (49)

 1 (2S) 

 (49) 

CIRCULATORY SYST IB 

NONE 

DIGESTIVE SYSTEM 

#LIVER

HEPATOCELLULAR ADENOMA

NEOPLASTIC NODULE

HEPATOCELLULAR CARCINOMA

 (9)

 Z-llZ%\.

 (49)

 2 (4%) 

 2_JHiI

 (46) 

2 (4X) 

1-111H. 

# NUMBER

* NUMBER

 OF ANIMALS

 OP ANIMALS

 WITH TISSUE

 NECROPSIED 

 EXAMINED MICROSCOPICALLY 

53 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE 

HEMANGIOMA 1 (2%) 

URINARY SYSTEM 

NONE 

ENDOCRINE SYSTEM 

NONE 

REPRODUCTIVE SYSTEM 

NONE 

NSRVOOS SYSTEM 

NONE 

SPECIAL SENSE OR ANS 

NOME 

HUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES 

NONE _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ • _ _ . . _ _ _ _ _ _ _ — _ — _ , _ _ _ _ _ _ - — . — — _ — __—_-»__— ­ — . — — _ _ _ — _ — _——..- . — . — — — — — — — — — • — — • 

ALL OTHER SYSTEMS 

NONE. 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUK3EP OF ANIMALS NECROPSIED 

54 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED)

 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY


NATURAL DEATHS


MORIBUND SACRIFICE


SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TSPMINAL SACRIFICE


ANIMAL MISSI NG
 

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 MALIGN NT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECOND RY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OS MALIi NANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL


 10


 1


 2


 7


 3


 3


 1


 1


 2


 2


 LOW DOSE HIGH DOSE
 

 50 50
 

1 5
 

4 3
 

 45 42
 

 14 11
 

 17 11
 

6 3
 

6 3
 

 10 6
 

 11 6
 

2
 

2
 

* PRIMARY TUMOHS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECONDARY TUMORS: METASTATIC TUMOHS OB TUMORS INVASIVE INTO AN ADJACENT 0R5AN
 

55
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

FED PHOSPHAMIDON IN THE DIET

 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 10 50 50
 

ANIMALS NECROPSIED 10 50 49
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 10 49 49
 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (10) (50) (49)
 

FIBROUS HISTIOCYTOMA, MALIGNANT 1 {/%)
 

BESPIRATORY SYSTEM
 

• LUNG	 (10) (49) (49) 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (1OX) 2 (4%) 1 (2») 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (2%) 

HEMATOPOIETIC SYSTEM
 

•MULTIPLE ORGANS	 (10) (50) (49)
 

MALIGNANT LYMPHOMA, NOS 2 (4%)
 

LEUKEMIA,NOS 1 (2*)
 

LYMPHOCYTIC LEUKEMIA 1 (2S)
 

#SPLEEN (10) (49) (49) 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 [2%) 

#LYM?H NODE (9) (44) (45) 

MALI3NANT LYMPHOMA, NOS 1 [2Z) 

CIRCULATORY SYST M 

NONE 

DIGESTIVE SYSTEM 

#LIVER (10) (49) (49) 

HEPATOCELL_DLA.R_AP-I£OM.A I_i2%l 

# NUM3ER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECPOPSIED 

56 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

NEOPLASTIC NODULE 1 (2X) 

HEPATGCBLLULAR CARCINOMA 5 (10%) 

URINARY SYSTEM 

NONE 

ENDOCRINE SYSTEM 

•PITUITARY (10) (48) (42) 

CHROMOPHOBE ADENOMA 1 12%) 

REPRODUCTIVE SYSTEM 

#OVARY (10) (47) (47) 

CYSTADENOCARCINOJ1A, NOS 1 (2S) 

JERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

*EYE/LACRIHAL GLAND (10) (50) (49) 

PAPILLARY ADENOMA 1 (2X) 

1USCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHEE SYSTEMS 

NONE 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

57 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED)

 

ANIMAL t>IS POSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY


NATURAL DEAT!S)


MORIBUND SACRIFICE


SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE


ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOB SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMOES*


TOTAL PHIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGN NT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECOND RY TUMORS
 

TOTAL ANIMALS KITH TUMORS UNCSRTAIN­ 3ENIGN OR MALI- NANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL


 10


 10


 1


 1


 1


 1


 LOW DOSE HIGH DOSE
 

 50 50
 

2 1
 

2 U
 

 1*6 U5
 

 11 9
 

 11 10
 

3 3
 

3 3
 

8 6
 

8 6 .
 

1
 

1
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMOES
 

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

58
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED PHOSPHAMIDON IN THE DIET
 

59
 
60

 
TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE RATS FED PHOSPHAMIDON IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 10 50 50 

ANIMALS NECROPSIED 9 50 49 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 9 50 i*9 

INTEGUMENTAR Y 5YSTF.M
 

*SUBCUT TISSUE (9) (50) (49) 

GRANULOMA, NOS 2 (4%) 1 (2X) 

RESPIRATORY SYST IK 

#LUNG/ALVEOLI (8) (49) (49)
 

CALCIFICATION, NOS 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

#SPLSEN (7) (50) (49) 

FI3ROSIS, FOCAL 3 (6%) 

INFARCT, NOS 1 (2X) 

CIRCULATORY SYSTEM
 

fHEART (8) (50) (49) 

ARTERIOSCLEROSIS, NOS 1 (2*) 

CALCIFICATION, FOCAL 1 (2%) 

MYOCARDIUM (8) (50) (49) 

FI3R0SIS 3 (6S) FIBROSIS, FOCAL 1 (13*) 1 (2X) 

#ENDOCARDIUM (8) (50) (49) 

FIBROSIS, DIFFUSE 1 (2X) 

•AORTA (9) (50) (49)

 

MEDIAL CALCIFICATION 1 .12%) 2

 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

61
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE HIGH DOSE 

DIGESTIVE SYSTEM 

#SALIV&RY GLAND (9) (50) (47) 

FI3R0SI S 2 (4%) 

#LIVER (9) (49) (46) 

DEGENERATION, NOS 1 (11%) 

DEGENERATION PARENCHYMATOOS 1 (1H | 

METAMOBPHOSIS FATTY 1 (11%) 2 (H%) 4 (9S) 

FOCAL CELLUL iR CHANGE 1 (2%) 6 (13X) 

ANGIECTASIS 1 (11%) 

#BILE DUCT (9) (49) (46) 

HYPERPLASIA, DIFFUSE 1 (2%) 

•PANCREAS (7) (47) (49) 

PERIARTERITIS 1 (2XJ 

ATROPHY, NOS 1 (2X) 

#GASTRIC MUCOSA (7) (50) (45) 

CALCIFICATION , FOCAL 1 (2%) 

URINARY SYSTEM 

#KIDNEY (9) (49) (49) 

GLOMERULONEPHRITIS, NOS 

PYELONEPHRITIS, ACUTE 

2 (22%) 

1 (2%) 

INFLAMMATION CHHONIC 3 (33%) 12 (24%) 18 (37%) 

METAPLASIA, NOS 1 (2%) 1 (2 5) 

ENDOCRINE SYSTEM 

#PITUITARY (8) (49) (48) 

CYST, NOS 

HYPERPLASIA, NOS 

2 (4%) 5

2

 (10%) 

 (4S) 

HYPERPLASIA, FOCAL 2 (4%) 4 (8%) 

#ADRENAL (3) (49) (49) 

CYST, NOS 2 (4%) 

CONGESTION, NOS 1 (2%) 

HEMORRHAGE 2 (4%) 

#ADRENAL CORTEX (8) (49) (49) 

HYPERPLASIA­ FOCAL .J-ilJSl. 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECEOPSIED 

62 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

#THYROID 

FOLLICULAR CYST, NOS 

HYPERPLASIA, C-CELL 

HYPERPLASIA, FOLLICULAB-CELL 

#PAPATHYROID 

HYPERPLASIA, NOS

 

REPRODUCTIVE SYSTEM
 

•PROSTATE
 

INFLAMMATION, SUPPURATIVE
 

INFLAMMATION ACUTE 4ND CHRONIC
 

•TESTIS
 

ATROPHY, NOS
 

ATROPHY, FOCAL
 

NEPVOUS SYSTEM
 

NONE
 

SPECIAL SENSE OR. ANS
 

NONE
 

M0SCULOSKE1ETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

•MESENTERY
 

PERIARTERITIS
 

ALL OTHER SYSTEMS 

ADIPOSE TISSUE 

FIBROS IS

 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPOHTED 

AUTOLYSIS/NO NECROPSY 

CONTROL LOW DOSE HIGH DOSE 

(8)
 

(2)
 

(?)
 

(9)
 

(<»8) (47)
 

1 (2%)
 

7 (15%) 3 (6»)
 

1 (2%) 6 (13%)
 

(27)	 (39)
 

2 (5JS)
 

(45) (47)
 

1 (2*)
 

1 (2%)
 

(49) (48)
 

1 (11*)	 7 (14%)
 15 (31%)
 

1 (11%)
 

(9) (50) (49) 

2 (4X) 1 (2%) 

• NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMBER OF ANIMALS NECROPSIED 

63 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN FEMALE RATS FED PHOSPHAMIDON IN THE DIET 

ANIMALS INITIAL!. IN STUDY

ANIMALS NECROPSIED

ANIMALS EXAMINED HISTOPATHOLOGICALLY

INTEGUMENTARY SYSTEM
 

* 3 KI N
 

INFLAMMATION ACUTE AND CHRONIC
 

RESPIRATORY SYSTEM
 

#LUNj
 

BRONCHOPNEUMONIA, NOS
 

PNEUMONIA, ASPIRATION
 

GRANULOMA, FOREIGN BODY
 

HEMATOPOIETIC SYSTEM 

#SPI,EEH 

CONGESTION, NOS

 

HEMORRHAGE 

ATROPHY, FOCAL 

HYPEHPI.ASI A, NOS

 

CIRCULATORY SYSTEM 

MYOCARDIUM

INFLAMMATION, CHRONIC FOCAL

DIGESTIVE SYSTEM 

#LIVER

INFARCT, FOCAL

MSTAMORPHOSI' FATTY

ZO£AL_CEL,LULAR_CJiANGE

CONTROL LOW DOSE HIGH DOSE 

 10 50 50 

 10 50 49 

 10 50 49 

(10) (50) (49) 

1 (2%) 

(10) (50) (49) 

1 (2*) 

1 (28) 

1 (2%) 

(9) (50) (48) 

1 |11() 

1 (11*) 

1 (2X) 

1 <2X) 

 (10) (50) (4 9) 

1 (2*) 

 (10) (50) (49) 

1 (2%) 

1 (2%) 

. l^ik&l Z-1±%L­

(t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICRO SC3 PIC AIL Y 

* NUMBER OF ANIMALS NECPOPSIED 

64 
TABLE C2. FEMALE RATS: NONNEOPLASTtC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

ANGIECTASIS 1 (10%) 

#BILE DUCT (10) (50) (49) 

CYST, NOS 2 (4£) 

HYPERPLASIA, NOS 2 (4X) 

URINARY SYSTEM 

tKIDNEY (10) (50) (49) 

GLOHERULONEPHRITIS, NOS 1 (10%) 

PYELONEPHRITIS, ACUTE 1 (2%) 

INFLAMMATION CHRONIC 2 (2 0%) 2 (4%) 2 (454) 

ENDOCRINE SYSTEM
 

•PITUITARY	 (10) (50) (45) 

EYPERPLASIA, NOS 2 (4%) 

HYPERPLASIA, FOCAL 6 (12%) 4 (9%) 

#ADRENAL (10) (50 ) (4 8) 

CYST, NOS 4 (8*) 

HEMORRHAGE 6 (12%) 4 (8* ) 

DEGENERATION, CYSTIC 5 (10%) 1 (2%) 

#ADEENAL CORTEX (10) (50 ) (48) 

DEGENERATION, CYSTIC 3 (6%) 1 (2%) 

HYPERPLASIA, NOS 1 (2%) 

HYPERPLASIA, FOCAL 1 1.2%) 4 (8%) 

ANGIECTASIS 1 £10%) 

#THYROID (10) (50) (46)

 

HYPERPLASIA, C-CELL 4 (8%)

 

REPRODUCTIVE SYSTEM 

•MAMMARY GLAND (10) (50 ) (49) 

DYSPLASIA, NOS 1 (2S) 

•MAMMARY LO3ULE	 (10) (50) (49 ) 

HYPERPLASIA, NOS 4 1 [2%) 

NERVOUS SYSTEM 

NONE. 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

• NUM3ER OF ANIMALS NECR0PSI2D
 

65
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL SENSE OR ANS 

NONE 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHE8 SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED 1 15

AUTOLYSIS/NO NECROPSY

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

 13 

1 

66
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE FED PHOSPHAMIDON IN THE DIET
 

67
 
68

 
TABLE D1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS 

IN MALE MICE FED PHOSPHAMIDON IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 10 50 50 

ANIMALS NECBOPSI D 9 49 49 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 9 49 48 

INTEGUMENTARY SYSTEM
 

•SKIN	 (9) (49) (49)

 

INFLAMMATION, CHRONIC 1 (2%)

 

•SUBCUT TISSUE (9) (49) (49)

 

FIBROSIS, FOCAL 1 (2X)

 

RESPIRATORY SYSTEM 

#LUN3 (9) (49) (45) 

EMPHYSEMA, NOS 1 (21) 

HYPERPLASIA, ALVEOLAR EPITHELIUM 2 (»X) 

HEMATOPOIETIC SYSTEM
 

#LYMPH NODE (9) (44) (39)
 

INFLAMMATION, HEMORFHAGIC 1 (296)
 

CIPCULATORY SYSTEM
 

NONS
 

DIGESTIVE SYST3M
 

#LIVSR (9) (49) (46)

 

GRANULOMA, NOS 1 (117.)

 

•PANCREATIC	 ACINUS (3) (49) (44) 

ATROPHY, NOS 1 (2%) 

•PEYERS PATCH (9) (47) (44)

 

INFLAMMATION. NOS J1_J2%1_ 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

69 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

URINARY SYSTEM 

NONE 

ENDOCRINE SYSTEM 

NONE 

REPRODUCTIVE SYSTEM 

•COAGULATING GLAND (9) (49) (49) 

DILATATION, NOS 1 (2X) 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

M'JSCULOSKELETA-L SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUBMABY 

NO LESION REPORTED 5_ 32 33 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMBER OF ANIMALS NECROPSIED 

70 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

AUTO/NECROPSY/HISTO PERF

AUTO/NECPOPSY/NO HISTO

AUTOLYSIS/NO NECROPSY 1 1

 1 

1 

1 

# NUMBER

• NUMBER

 OF

 OF

 ANIMALS

 ANIMALS

 WITH TISSUE

 NECROP3IED 

 EXAMINED MICROSCOPICALLY 

71 
TABLE 02. 

SUMMARY OF THE INCID

IN FEMALE MICE FE

ENCE OF NONNE

D PHOSPHAMIDO

OPLASTIC LESIONS 

N IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS

ANIMALS

ANIMALS

 INITIAL ! IN STDDY

 NECROPSIED

 EXAMINED HISTOPATHOLOGICALLY

 10 

 10 

 10 

50 

50 

49 

50 

49 

49 

INTEGUMENTARY SYSTEM 

NONE 

RESPIRATORY SYSTEM 

#LUNG 

HYPBRP.LASIA, ALVEOLAE EPITHELIUM 

(10) (49 ) (49) 

1 (2X) 

HEMATOPOIETIC SYSTEM 

•SPLEEN 

HYPERPLASIA, LYMPHOID 

#MESENTERIC L . NODE 

INFLAMMATION, NOS 

(10) 

(9) 

(49) 

1

1

 (2%) 

 (2%) 

(49) 

3 (6%) 

(45) 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM 

fLIVER 

INFLAMMATION, NOS 

(10) (49) 

1 (255) 

(49) 

#PANCREAS 

DILATATION/DUCTS 

(10) (4 9) 

1 (2«) 

(49) 

1 (2%) 

•STOMACH

HYPERPLASIA. EPITHELIAL

 (10) (49) 

1_12XJ 

(48) 

# NUMBER OF ANIMALS WITH TISSUE EXA

* NUMBER OF ANIMALS NECROPSIED 

MINED MICROSCOPICALLY 

72
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

CONTROL LOW DOSE 

URINARY SYSTEM 

•KIDNEY (10 ) (48 ) 

CYST, NOS 1 (2%) 

A1YLOIDOSIS 

#URINARY BLADDER (10 ) (48 ) 

INFLAMMATION, CHRONIC 1 (2%) 

ENDOCRINE SYSTEM 

•PITUITARY (10 ) (48 ) 

CYST, NOS

 

HYPERPLASIA, CHROMOPHOBE-CELL 

REPRODUCTIVE SYSTEM 

#UTERUS/ENDO«ET! JUH (10 ) (48 )

 

HYPERPLASIA, NOS 

HYPERPLASIA, CYSTIC 

#OVARY/OVIDUCT (10 ) (48 ) 

RETENTION FLUID 1 [2%) 

•OVARY (10 ) (47 ) 

FOLLICULAR CYST, NOS 2 (2035) 1 (2%) 

INFLAMMATION, NOS 5 (50%) 3 (6%) 

INFLAMMATION, SUPPURATIVE 2 (4 35) 

INFLAMMATION CHRONIC 1 (10%) 

NE3VOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELETAL SYSTEM
 

NONE
 

# NUM3ER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

• NUMBER OF ANIMALS NECP.OPSIED
 

HIGH DOSE 

(49) 

1 (2X) 

(42) 

(42) 

1 (235) 

1 (235) 

(46) 

1 (2X) 

1 [2%) 

(46) 

(47) 

1 (2%) 

1 (2S) 

73
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES 

NONE 

ALL OTHER

NONE 

 SYSTEMS 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED

AUTO/NECROPSY/NO HISTO

AUTOLYSIS/NC NECROPSY

# NUMBER OF ANIMALS WITH TISSUE

* N0H32R OF ANIMALS NECROPSIED 

 EXAMINED

 4 28

 1 

 MICROSCOPICALLY 

 32 

1 

74 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS FED PHOSPHAMIDON IN THE DIET
 

75
 
76

 
Table El. Analyses of the Incidence of Primary Tumors
 

in Male Rats Fed Phosphamidon in the Diet (a)
 

Topography: Morphology
 

Spleen: Hemangioma
 

or Hemangiosarcoma (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

^ Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe
 

Adenoma (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/7 (14)
 

N.S.
 

110
 

2/8 (25)
 

N.S.
 

95
 

Pooled
 

Control
 

1/91 Cl)
 

P * 0.012
 

13/81 (16)
 

N.S.
 

Low
 

Dose
 

3/50 (6)
 

N.S.
 

0.420
 

0.045
 

21.638
 

5.460
 

0.451
 

280.710
 

97
 

8/49 (16)
 

N.S.
 

0.653
 

0.182
 

5.823
 

1.017
 

0.391
 

2.434
 

97
 

High
 

Dose
 

5/49 (10)
 

P = 0.020**
 

0.714
 

0.110
 

33.104
 

9.286
 

1.076
 

429.368
 

111
 

3/48 (6)
 

N.S.
 

0.250
 

0.038
 

2.777
 

0.389
 

0.074
 

1.324
 

101
 
Table El. Analyses of the Incidence of Primary Tumors
 

in Male Rats Fed Phosphamidon in the Diet (a)
 

(continued)
 

Matched Pooled
 

Topography: Morphology Control Control
 

Adrenal:
 

Cortical Adenoma (b) 0/8 (0) 2/90 (2)
 

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

Departure from Linear Trend (e) P - 0.016
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

JjJ Upper Limit
 

Weeks to First Observed Tumor —
 

Thyroid: C-cell Adenoma or
 

Carcinoma (b) 1/8 (13) 3/81 (4)
 

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

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 110
 

Low
 

Dose
 

6/49 (12)
 

P = 0.023**
 

Infinite
 

0.302
 

Infinite
 

5.510
 

1.027
 

53.857
 

103
 

7/48 (15)
 

P = 0.031**
 

1.167
 

0.198
 

51.419
 

3.937
 

0.945
 

22.494
 

110
 

High
 

Dose
 

2/49 (4)
 

N.S.
 

Infinite
 

0.055
 

Infinite
 

1.837
 

0.136
 

24.532
 

101
 

5/47 (11)
 

N.S.
 

0.851
 

0.124
 

39.383
 

2.872
 

0.584
 

17.657
 

107
 
Table El. Analyses of the Incidence of Primary Tumors
 

in Male Rats Fed Phosphamidon in the Diet (a)
 

(continued)
 

(a) Dosed groups received 80 or 160 ppm.
 

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

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


Armitage test when P 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 (*) or with the pooled-


control group (**) when P is less than 0.05 for either control group; 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 specified control group.
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Phosphamidon in the Diet (a)
 

Topography: Morphology
 

Pituitary: Carcinoma, NOS (b)
 

P Values (c,d)
 

Departure from Linear Trend (e)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

CD
 

o
 Weeks to First Observed Tumor
 

Pituitary: Chromophobe
 

Adenoma (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

2/10 (20)
 

P = 0.008 (N)
 

P = 0.001
 

111
 

3/10 (30) 

N.S. 

110
 

Pooled
 

Control
 

6/88 (7)
 

P = 0.019 (N)
 

14/88 (16) 

N.S. 

Low
 

Dose
 

0/50 (0)
 

P = 0.025
 

(N)*
 

0.000
 

0.000
 

0.667
 

0.000
 

0.000
 

1.104
 

7/50 (14) 

N.S. 

0.467 

0.143 

2.518 

0.880 

0.319 

2.152 

83
 

High
 

Dose
 

0/45 (0)
 

P = 0.030
 

(N)*
 

0.000
 

0.000
 

0.739
 

0.000
 

0.000
 

1.223
 

4/45 (9) 

N.S. 

0.296 

0.066 

1.824 

0.559 

0.140 

1.650 

111
 
00 

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

Fed Phosphamidon in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Adrenal:
 

Cortical Adenoma or Carcinoma
 

P Values (c,d)
 

Relative Risk (Matched Control)
 

Lower Lin^t
 

Upper Limit
 

Relative Risk (Pooled Control) (
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-cell Adenoma
 

or Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (Matched Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

(b)
 

(f)
 

f)
 

(f)
 

f)
 

Matched
 

Control
 

2/10 (20)
 

N.S.
 

110
 

0/10 (0)
 

N.S.
 

—
 

Pooled
 

Control
 

6/89 (7)
 

N.S.
 

2/82 (2)
 

P = 0.003
 

Low
 

Dose
 

7/50 (14)
 

N.S.
 

0.700
 

0.172
 

6.445
 

2.077
 

0.629
 

7.030
 

94
 

9/50 (18)
 

P = 0.003**
 

Infinite
 

0.587
 

Infinite
 

7.380
 

1.605
 

67.559
 

106
 

High
 

Dose
 

6/48 (13)
 

N.S.
 

0.625
 

0.144
 

5.907
 

1.854
 

0.521
 

6.517
 

111
 

8/46 (17)
 

P = 0.004**
 

Infinite
 

0.555
 

Infinite
 

7.130
 

1.495
 

66.171
 

73
 
(continued) 

Topography:

Table E2.

 Morphology 

 Analyses of the Incidence of Primary Tumors in Female Rats 

Fed Phosphamidon in the Diet (a) 

Matched 

Control 

Pooled 

Control 

Low 

Dose 

High 

Dose 

Mammary Gland: Fibroadenoma (b) 2/10 (20) 13/94 (14) 6/50 (12) 3/49 (6) 

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

Relative Risk (Matched Control) (f) 

Lower Limit 

Upper Limit 

0.600 

0.138 

5.679 

0.306 

0.044 

3.435 

Relative Risk (Pooled Control) (f) 

Lower Limit 

Upper Limit 

0.868 

0.285 

2.272 

0.443 

0.084 

1.510 

00 Weeks to First Observed Tumor Ill 77 64 

Uterus: Endometrial Stromal 

Polyp (b) 0/10 (0) 7/90 (8) 1/48 (2) 3/48 (6) 

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

Relative Risk (Matched Control) (f) 

Lower Limit 

Upper Limit 

Infinite 

0.012 

Infinite 

Infinite 

0.139 

Infinite 

Relative Risk (Pooled Control) (f) 

Lower Limit 

Upper Limit 

0.268 

0.006 

1.985 

0.804 

0.139 

3.323 

Weeks to First Observed Tumor — 110 64 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Phosphamidon in the Diet (a)
 

(continued)
 

(a) Dosed groups received 80 or 160 ppm.
 

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

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


Armitage test when P 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 (*) or with the pooled-


control group (**) when P is less than 0.05 for either control group; 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.
 oo
 

(f) The 95% confidence interval of the relative risk between each dosed group and the specified control group.
 
84

 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

MICE FED PHOSPHAMIDON IN THE DIET
 

85
 
86

 
00 

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

Fed
 

Topography: Morphology
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

--J Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

or Adenoma, or Neoplastic
 

Nodule (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Phosphamidon in
 

Matched
 

Control
 

1/9 (11)
 

N.S.
 

90
 

2/9 (22)
 

N.S.
 

89
 

the Diet (a)
 

Pooled
 

Control
 

6/85 (7)
 

N.S.
 

19/88 (22)
 

N.S.
 

Low
 

Dose
 

3/49 (6)
 

N.S.
 

0.551
 

0.055
 

28.360
 

0.867
 

0.145
 

3.845
 

85
 

9/49 (18)
 

N.S.
 

0.827
 

0.229
 

7.284
 

0.851
 

0.364
 

1.799
 

85
 

High
 

Dose
 

2/45 (4)
 

N.S.
 

0.400
 

0.025
 

23.103
 

0.630
 

0.064
 

3.332
 

91
 

7/46 (15)
 

N.S.
 

0.685
 

0.174
 

6.258
 

0.705
 

0.267
 

1.599
 

91
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Fed Phosphamidon in the Diet (a)
 

(continued)
 

(a) Dosed groups received 80 or 160 ppm.
 

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

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


Armitage test when P 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 (*) or with the pooled-


control group (**) when P is less than 0.05 for either control group; 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.
 

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

00

 comparison.
 

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

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

Fed Phosphamidon in the Diet (a)
 

Topography; Morphology
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

oo
 Weeks to First Observed Tumor
 

Liver: Hepatocellular Adenoma
 

or Carcinoma, or Neoplastic
 

Nodule (b)
 

P Values (c,d)
 

Departure from Linear Trend (e)
 

Relative Risk (Matched Control) (f)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Control) (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/10 (10)
 

N.S.
 

90
 

0/10 (0)
 

N.S.
 

P = G. t'+2 (N)
 

Pooled
 

Control
 

5/88 (6)
 

N.S.
 

3/87 (3)
 

N.S.
 

P = 0.013 (N)
 

Low
 

Dose
 

3/49 (6)
 

N.S.
 

0.612
 

0.060
 

31.507
 

1.078
 

0.172
 

5.262
 

90
 

6/49 (12)
 

N.S.
 

Infinite
 

0.365
 

Infinite
 

3.551
 

0.793
 

20.973
 

86
 

High
 

Dose
 

1/49 (2)
 

N.S.
 

0.204
 

0.003
 

15.723
 

0.359
 

0.008
 

3.070
 

91
 

1/49 (2)
 

N.S.
 

Infinite
 

0.012
 

Infinite
 

0.592
 

0.011
 

7.098
 

91
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed Phosphamidon in the Diet (a)
 

(continued)
 

Matched Pooled Low High
 

Topography; Morphology Control Control Dose Dose
 

Hematopoietic System: Lymphoma or
 

Leukemia (b) 0/10 (0) 2/89 (2) 2/50 (4) 4/50 (8)
 

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

Relative Risk (Matched Control) (f) Infinite Infinite
 

Lower Limit 0.065 0.206
 

Upper Limit Infinite Infinite
 

Relative Risk (Pooled Control) (f) 1.780 3.560
 

Lower Limit 0.132 0.525
 

Upper Limit 23.790 38.057
 

o Weeks to First Observed Tumor 90 79
 

(a) Dosed groups received 80 or 160 ppm.
 

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

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


Armitage test when P 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 (*) or with the pooled-


control group (**) when P is less than 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed Phosphamidon in the Diet (a)
 

(continued)
 

(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
 

compar ison.
 

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

vo
 
92

 
APPENDIX G
 

ANALYSES OF FORMULATED DIETS FOR
 

CONCENTRATIONS OF PHOSPHAMIDON
 

93
 
94

 
APPENDIX G
 

Analysis of Formulated Diets for
 

Concentrations of Phosphamidon
 

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

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

filtered through Celite with benzene washes and reduced in volume
 

to 10 ml. After appropriate dilutions, the solution was
 

quantitatively analyzed for phosphamidon by gas-liquid
 

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

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

and external standards were used for calibration.
 

Theoretical No. of Sample Coefficient of Range (ppm) 

Concentrations Samples Analytical Variation (%) 

in Diet (ppm) Mean (ppm) 

80 42 81.7 4.7% 74-89
 

160 40 161.5 3.9%. 150-174
 

95
 
96

 
Review of the Bioassay of Phosphamidon* for Oarcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

July 25, 1977
 

The Clearinghouse on Environmental Carcinogens was established
 

in May, 1976, in compliance with DHEW Committee Regulations and the
 

Provisions of the Federal Advisory Committee Act. The purpose of the
 

Clearinghouse is to advise the Director of the National Cancer Institute
 

(NCI) on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be exposed. The
 

members of the Clearinghouse have been drawn from academia, industry,
 

organized labor, public interest groups, State health officials, and
 

quasi-public health and research organizations. Members have been
 

selected on the basis of their experience in carcinogenesis or related
 

fields and, collectively, provide expertise in chemistry, biochemistry,
 

biostatistics, toxicology, pathology, and epidemiology. Representatives
 

of various Governmental agencies participate as ad hoc members. The
 

Data Evaluation/Risk Assessment Subgroup of the Clearinghouse is
 

charged with the responsibility of providing a peer review of reports
 

prepared on NCI-sponsored bioassays of chemicals studied for carcinogenicity. It is in this context that the below critique is given on
 

the bioassay of Phosphamidon for carcinogenicity.
 

The primary reviewer for the report on the bioassay of Phosphamidon
 

said that the survival of the rats and mice was adequate. No significant
 

difference in tumor incidence was found between treated mice and control
 

animals. In treated rats, however, a statistically significant increase
 

was reported in the incidence of hemangiosarcomas of the spleen and in
 

C-cell adenomas of the thyroid. The primary reviewer pointed out that
 

the incidence of C-cell adenomas was at least as high in control rats
 

from the Dichlorvos bioassay (Technical Report No. 10), as well as in
 

controls from another study reported in the literature. The Dichlorvos
 

control rats also had an incidence of hemangiosarcomas of the spleen
 

approaching the incidence found in the treated Phosphamidon animals.
 

The primary reviewer said that the data were inadequate to classify
 

Phosphamidon as a carcinogen. He suggested, however, that Phosphamidon
 

might promote the incidence of spontaneously occurring tumors.
 

A Program staff member commented that the finding of two different
 

tumor types of borderling significance, as opposed to only one, took
 

on greater meaning and was the basis for the conclusion that Phosphamidon
 

97
 
was carcinogenic in rats. Another Program staff member said that the
 

difference between the Dichlorvos and Phosphamidon control rats could
 

be related to variations between diagnoses made by the pathologists.
 

It also was suggested that a difference in tumor incidence could have
 

resulted from random variations in animals or from missed tumors when
 

tissues were selected or processed. Although pathologists may use
 

varying terminology for the same lesion, a staff pathologist said that
 

a difference in incidence probably was not related to variations in
 

the ability of diagnostic pathologists to detect tumors.
 

A Subgroup member questioned the significance of the dose-related
 

trend reported for tumors found in treated rats. He emphasized the need
 

to compare the treated animals with a program pool of control data. He
 

noted that it is only in this way that low incidence tumors can be
 

properly evaluated. Another Subgroup member suggested that questionable
 

studies could be reexamined after the control data are accumulated and
 

analyzed.
 

It was moved that the results from the bioassay should be considered
 

to be equivocal and, therefore, no conclusion drawn on the carcinogenicity
 

of Phosphamidon. The motion was seconded and approved unanimously.
 

Menibj?_r_s___p_resent were:
 

Gerald Wogan (Chairman), Massachusetts Institute of Technology
 

Arnold Brown, Mayo Clinic
 

E. Cuyler Hammond, American Cancer Society
 

Henry C. Pitot, McArdle Laboratory for Cancer Research
 

Verald K. Rowe, Dow Chemical USA
 

Michael Shimkin, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

.John Weisburger, American Health Foundation
 

Sidney Wolfe, Health Research Group
 

Subsequent to this review, changes may have been made in the
 

bioassay report either as a result of the review or other
 

reasons. Thus, certain comments and criticisms reflected in
 

the review may no longer be appropriate.
 

^U.S . GOVERNMEN T PRINTIN G OFFICE : ,9 7 8 - 28 I " 2 1 7 / 32 7 2
 

98