1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 77 

BIOASSAY OF 

PYRIMETHAMINE 

FOR POSSIBLE CARCINOGEN ICITY 

CAS No. 58-14-0 

NCI-CG-TR-77 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

PYRIMETHAMINE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 2001A
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-1327
 

BIOASSAY OF
 

PYRIMETHAMINE
 

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
 

pyrimethamine 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 no necessarily mean that the test
 

chemical is not a carcinogen, inasmuch as the experiments are
 

conducted under a limited set of circumstances. Positive results
 

demonstrate that the test chemical is carcinogenic for animals
 

under the conditions of the test and indicate that exposure to
 

the chemical is a potential risk to man. The actual determination of the risk to man from animal carcinogens requires a wider
 

analysis.
 

CONTRIBUTORS; This bioassay of pyrimethamine was conducted by
 

Southern Research Institute, Birmingham, Alabama, 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 and doses were determined by Drs. D. P.
 

Griswold1, J. D. Prejean*, E. K. Weisburger2, and J. H.
 

Weisburger2'^. Ms. J. Belzer^ and Mr. I. Brown^ were responsible
 

for the care of the laboratory animals and administration of the
 

test chemical. Data management and retrieval were performed by
 

Ms. C. A. Dominick . Histopathologic examinations were performed
 

by Drs. S. D. Kosanke , J. C. Peckham^-, and R. B. Thompson , and
 

the diagnoses included in this report represent their
 

interpretation.
 

ill
 
Animal pathology tables and survival tables were compiled by EG&G
 

Mason Research Institute'*. The statistical analyses were performed by Dr. J. R. Joiner , using methods selected for the
 

bioassay program by Dr. J. J. Gart". Chemicals used in this
 

bioassay were analyzed under the direction of Dr. E. Murrill ,
 

and the results of the analyses were reviewed by Dr. C. W.
 

Jameson-1.
 

This report was prepared at Tracor Jitco-5 under the direction of
 

NCI. Those responsible for the report at Tracor Jitco were
 

Dr. Marshall Steinberg, Director of the Bioassay Program; Dr. L.
 

A. Campbell, Deputy Director for Science; Drs. J. F. Robens and
 

C. H. Williams, toxicologists; Dr. R. L. Schueler, pathologist;
 

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

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

assisted by Ms. Y. E. Presley. 

The statistical analysis was reviewed by members of the
 

Mathematical Statistics and Applied Mathematics Section of NCI°:
 

Dr. John J. Gart, Mr. Jun-mo Nam, Dr. Hugh M. Pettigrew, and Dr.
 

Robert E. Tarone.
 

The following other scientists at NCI were responsible for
 

evaluating the bioassay experiment, interpreting the results, and
 

reporting the findings:
 

Dr. Kenneth C. Chu
 

Dr. Cipriano Cueto, Jr.
 

Dr. J. Fielding Douglas
 

Dr. Dawn G. Goodman
 

Dr. Richard A. Griesemer
 

Dr. Harry A. Milman
 

Dr. Thomas W. Orme
 

Dr. Robert A. Squire*5
 

Dr. Jerrold M. Ward
 

^•Southern Research Institute, 2000 Ninth Avenue South,
 

Birmingham, Alabama.
 

iv
 
^Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

%ow with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York.
 

Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

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

Maryland.
 

"Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute, National
 

Institutes of Health, Bethesda, Maryland.
 

'Midwest Research Institute, 425 Volker Boulevard, Kansas City,
 

Missouri.
 

"Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

SUMMARY
 

A bioassay of pyrimethamine, a prophylactic antimalarial, for
 

possible carcinogenicity was conducted by administering the test
 

chemical in feed to Fischer 344 rats and B6C3F1 mice.
 

Groups of 35 rats and 35 mice of each sex were administered
 

pyrimethamine 5 days per week at one of two doses, either 200 or
 

400 ppm for the rats and either 500 or 1,000 ppm for the mice.
 

The animals were administered the chemical for 78 weeks, then
 

observed for 26 or 27 additional weeks. Matched controls
 

consisted of 15 untreated rats and 15 untreated mice of each sex;
 

pooled controls consisted of the matched controls combined with
 

30 untreated rats and 30 untreated mice from similar bioassays of
 

two other test compounds. All surviving rats and mice were
 

killed at 102-105 weeks.
 

Mean body weights of the rats and mice fed diets containing
 

pyrimethamine were slightly lower than those of the matched
 

controls. Survival of the rats was not affected adversely by the
 

chemical. In mice, survival rates of both dosed and matched-


control males were low, with nearly two-thirds of the dosed and
 

one-half of the control mice dying by week 52. Some of the
 

deaths were associated with respiratory infections and may not
 

have been related to administration of the chemical. Numbers of
 

animals at risk in the dosed and control groups of female mice
 

were adequate, however, for the development of late-appearing
 

tumors.
 

In rats of each sex, no neoplastic lesions were found at a
 

statistically significant incidence in the groups fed the
 

pyrimethamine as compared with control groups. An increased
 

frequency of bone-marrow atrophy occurred in both male and female
 

dosed groups.
 

In male mice, the markedly decreased life spans may have
 

prevented the observation of late-appearing tumors, since only
 

vii
 
two tumors were observed, one in a high-dose mouse and one in a
 

low-dose mouse. In female mice, no neoplastic lesions were found
 

at a statistically significant incidence in the groups fed the
 

pyrimethamine as compared with control groups.
 

It is concluded that under the conditions of this bioassay,
 

pyrimethamine was not carcinogenic for male or female Fischer 344
 

rats or for female B6C3F1 mice. The carcinogenic potential of
 

pyrimethamine for male B6C3F1 mice cannot be assessed by this
 

bioassay, because of the markedly reduced life span.
 

viii
 
TABLE OF CONTENTS
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 3
 

C. Animals 4
 

D. Animal Maintenance 5
 

E. Subchronic Studies.. 7
 

F. Designs of Chronic Studies 9
 

G. Clinical and Pathologic Examinations 12
 

H. Data Recording and Statistical Analyses 13
 

III. Results -Rats 19
 

A. Body Weights and Clinical Signs (Rats) 19
 

B. Survival (Rats) , 19
 

C. Pathology (Rats) 22
 

D. Satistical Analyses of Results (Rats) 23
 

IV. Results - Mice 25
 

A. Body Weights and Clinical Signs (Mice) 25
 

B. Survival (Mice) 25
 

C. Pathology (Mice) 27
 

D. Statistical Analyses of Results (Mice) 30
 

V. Discussion 33
 

VI. Bibliography 35
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Fed Pyrimethamine in the Diet 37
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Fed Pyrimethamine in the Diet 39
 

Table A2 Summary of the Incidence of Neoplasms in
 

Female Rats Fed Pyrimethamine in the Diet 42
 

ix
 
Page 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Fed Pyrimethamine in the Diet 45 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Fed Pyrimethamine in the Diet 47 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Fed Pyrimethamine in the Diet 50 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Fed Pyrimethamine in the Diet 53 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Pyrimethamine in the Diet. 55 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Pyrimethamine in the Diet 58 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Fed Pyrimethamine in the Diet 61 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed Pyrimethamine in the Diet. 63 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed Pyrimethamine in the Diet 66 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Fed Pyrimethamine in the Diet 71 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Fed Pyrimethamine in the Diet 73 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Fed Pyrimethamine in the Diet 77 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Fed Pyrimethamine in the Diet 81 

Table Fl Analyses of the Incidence of Primary Tumors 

in Female Mice Fed Pyrimethamine in the Diet 83 
TABLES 

Table 1 Design of Pyrimethamine Chronic Feeding Studies 

in Rats 10 

Table 2 Design of Pyrimethamine Chronic Feeding Studies 

in Mice 11 

FIGURES 

Figure 1 Growth Curves for Rats Fed Pyrimethamine 

in the Diet 20 

Figure 2 Survival Curves for Rats Fed Pyrimethamine 

in the Diet 21 

Figure 3 Growth Curves for Mice Fed Pyrimethamine 

in the Diet 26 

Figure A Survival Curves for Mice Fed Pyrimethamine 

in the Diet 28 

xi
 

I. INTRODUCTION
 

Pyrimethamine (CAS 58-14-0; NCI C01683) was developed in the
 

early 1950's as an antimalarial drug with both prophylactic and
 

suppressive properties. Currently, it is used to prevent
 

falciparum malarial infection ana to suppress both vivax and
 

falciparum infections in areas where these species of Plasmodium
 

are not resistent to it. Repeated administration of the drug
 

suppresses the erythrocytic stage of the infection and controls
 

the periodic episodes of chills and fever, although it does not
 

cure the infection. It has been used with either dapsone or a
 

sulfonamide in areas where multiresistent strains are endemic.
 

Pyrimethamine binds to and interferes with plasmodial dihydrofolate reductase, an enzyme converting folic acid to folinic
 

acid; thus, it impairs the synthesis of the purine and pyrimidine
 

bases needed for nucleic acid synthesis (Rollo, 1975; AMA
 

Department of Drugs, 1973). Pyrimethamine has antitumor activity
 

in cancer chemotherapeutic screens In vivo, and its mechanism is
 

identical to that of the important antileukemic drug methotrexate
 

(Johns and Bertino, 1973; Calabresi and Parks, 1975).
 

Pyrimethamine was selected for testing for carcinogenic activity
 

because of its long-term administration to humans to prevent or
 

control malaria.
 

II. MATERIALS AND METHODS
 

A. Chemical
 

Pyrimethamine (5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine)
 

was obtained in two batches from a single lot (Lot No. 51416)
 

from the Burroughs Wellcome Company, Research Triangle Park,
 

North Carolina. The purity of this lot, according to the
 

manufacturer, met United States Pharmacopeia specifications. The
 

identity and purity of this batch was confirmed by analysis at
 

Midwest Research Institute. The melting point was 241-244°C, in
 

agreement with the reported value of 238-242°C (Stecker, 1968).
 

Nonaqueous titration of the amine function with perchloric acid
 

gave 100.3 i 0.4% of the theoretical value. High-pressure liquid
 

chromatography indicated a 0.2% impurity which was not
 

identified. Elemental analyses (C, H, Cl, N) were correct for
 

^•12^13^-'-^4> tne molecular formula of pyrimethamine. Nuclear
 

magnetic resonance and infrared spectra were consistent with the
 

structure.
 

The chemical used for the chronic study was stored in the
 

original container at 5°C.
 

B. Dietary Preparation
 

Feed mixtures containing the test chemical were prepared every 2
 
weeks by mixing a known amount of sifted pyrimethamine with a
 

small amount of Wayne® Lab Blox animal meal (Allied Mills, Inc.,
 

Chicago, 111.) in a portable mixer. This mixture was then added
 

to the required amount of animal meal and mixed in a twin-shell
 

blender for 10 minutes.
 

No analysis of concentration or determination of stability of the
 

chemical in feed were performed. The prepared diets were stored
 

at room temperature in sealed plastic containers.
 

C. Animals
 

For the subchronic studies, female Sprague-Dawley rats and male
 

and female Swiss mice were obtained from Charles River Breeding
 

Laboratories, Inc., Wilmington, Massachusetts. Female Fischer
 

344 rats were obtained from Laboratory Supply Co., Indianapolis,
 

Indiana. Rats were approximately 5 weeks of age, and mice 4
 

weeks of age when received from the supplier. All animals were
 

quarantined for 1 week prior to testing.
 

For the chronic studies, male and female Fischer 344 rats and
 

male and female B6C3F1 mice were obtained from Charles River
 

Breeding Laboratories under a contract with the Division of
 

Cancer Treatment, National Cancer Institute. Rats were received
 

at 30 days of age and mice at 37 days of age. On arrival at the
 

laboratory, all animals were quarantined (rats for 11 days, mice
 
for 18 days). Animals with no visible signs of disease were
 

assigned to control or dosed groups, and earmarked for individual
 

identification.
 

D. Animal Maintenance
 

Animals were housed in temperature- and humidity-controlled
 

rooms. The temperature range was 20-24°C, and the relative
 

humidity was maintained at 40-60%. The air was changed 15 times
 

per hour, and passed through both intake and exhaust fiberglass
 

roughing filters. In addition to natural light, illumination was
 

provided by fluorescent light for 9 hours per day. Food and
 

water were supplied daily and were available ad libitum.
 

Rats were housed five per cage and mice seven per cage in solid-


bottom stainless steel cages (Hahn Roofing and Sheet Metal Co.,
 

Birmingham, Ala.). The rat cages were provided with Iso-Dri®
 

hardwood chip bedding (Carworth, Edison, N.J.), and cage tops
 

were covered with disposable filter bonnets; mouse cages were
 

provided with Sterolit® clay bedding (Englehard Mineral and
 

Chemical Co., New York, N.Y.) and covered with filter bonnets
 

during the latter part of the study. Bedding was replaced once
 

per week; cages, water bottles, and feeders were sanitized at
 

82°C once per week; and racks were cleaned once per week.
 

The rats and mice were housed in separate rooms. Control animals
 
were housed in the same room as the respective dosed animals.
 

Animals fed pyrimethamine were maintained in the same rooms as
 

animals of the same species being administered the following
 

chemicals:
 

RATS
 

Feed Studies
 

4-acetyl-N-((cyclohexylamino)carbonyl)benzenesulfonamide
 

(acetohexamide) (CAS 968-81-0)
 

anthranilic acid (CAS 118-92-3)
 

l-butyl-3-(p-tolylsulfonyl)urea (tolbutamide) (CAS 64-77-7)
 

4-chloro-N-((propylamino)carbonyl)benzenesulfonamide
 

(chlorpropamide) (CAS 94-20-2)
 

2,6-diamino-3-(phenylazo)pyridine hydrochloride
 

(phenazopyridine hydrochloride) (CAS 136-40-3)
 

L-tryptophan (CAS 73-22-3)
 

N-9H-fluoren-2-ylacetamide (CAS 53-96-3)
 

N-(p-toluenesulfonyl)-N'-hexamethyleniminourea
 

(tolazamide) (CAS 1156-19-0)
 

1-phenethylbiguanide hydrochloride (phenformin) (CAS 114-86-3)
 

pyrazinecarboxamide (pyrazinamide) (CAS 98-96-4)
 

4,4'-sulfonyldianiline (dapsone) (CAS 80-08-0)
 

4,4'-thiodianiline (CAS 139-65-1)
 

ethionamide (CAS 536-33-4)
 

MICE
 

Feed Studies
 

4-acetyl-N-((eyelohexylamino)carbonyl)benzenesulfonamide
 

(acetohexamide) (CAS 968-81-0)
 

anthranilic acid (CAS 118-92-3)
 

l-butyl-3-(p-tolylsulfonyl)urea (tolbutamide) (CAS 64-77-7)
 

4-chloro-N-((propylamino)carbonyl)benzenesulfonamide
 

(chlorpropamide) (CAS 94-20-2)
 

2,6-diamino-3-(phenylazo)pyridine hydrochloride
 

(phenazopyridine hydrochloride) (CAS 136-40-3)
 

L-tryptophan (CAS 73-22-3)
 

N-9H-fluoren-2-ylacetamide (CAS 53-96-3)
 

N-(p-toluenesulfonyl)-N'-hexamethyleniminourea
 

(tolazamide) (CAS 1156-19-0)
 
1-phenethylbiguanide hydrochloride (phenformin) (CAS 114-86-3)
 

pyrazinecarboxamide (pyrazinamide) (CAS 98-96-4)
 

4,4'-sulfonyldianiline (dapsone) (CAS 80-08-0)
 

4,4'-thiodianiline (CAS 139-65-1)
 

ethionamide (CAS 536-33-4)
 

Gavage Studies
 

cholesterol (p-(bis(2-chloroethyl)amino)phenyl)acetate
 

(phenesterin) (CAS 3546-10-9)
 

estradiol bis((p-(bis(2-chloroethyl)amino)phenyl)acetate)
 

(estradiol mustard) (CAS 22966-79-6)
 

Intraperitoneal Injection Studies
 

4'-(9-acridinylamino)methansulfon-m-aniside monohydrochloride
 

(MAAM) (NSC 141549)
 

acronycine (CAS 7008-42-6)
 

5-azacytidine (CAS 320-67-2)
 

beta-2'-deoxy-6-thioguanosine monohydrate (beta-TGdR)
 

(CAS 789-61-7)
 

1,4-butanediol dimethanesulfonate (busulfan) (CAS 55-98-1)
 

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

3,3'-iminobis-l-propanol dimethanesulfonate (ester)
 

hydrochloride [IPD] (CAS 3458-22-8)
 

(+)-4,4'-(1-methyl-l,2-ethanediyl)bis-2,6-piperazinedione
 

(ICRF-159) (CAS 21416-87-5)
 

N,3-bis(2-chloroethyl)tetrahydro-2H-l,3,2-oxazaphosphorin-2­ amine-2-oxide (isophosphamide) (CAS 3778-73-2)
 

N-(2-chloroethyl)-N-(l-methyl-2-phenoxyethyl)benzylamine
 

hydrochloride (phenoxybenzamine) (CAS 63-92-3)
 

N-(l-methylethyl)-4-((2-methylhydrazino)methyl)benzamide
 

monohydrochloride (procarbazine) (CAS 366-70-1)
 

tris(l-aziridinyl)phosphine sulfide (thio-TEPA) (CAS 52-24-4)
 

2,4,6-tris(dimethylamino)-s-triazine (CAS 645-05-6)
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted to estimate the maximum
 

tolerated doses of pyrimethamine, on the basis of which low and
 

high concentrations (hereinafter referred to as "low doses" and
 

"high doses") were determined for administration in the chronic
 
studies. Dosed animals were administered the chemical in the
 

diet 7 days a week for 45 days and were then observed for an
 

additional 45 days. Five animals were tested at each dose, and
 

groups ranging in size from 5 to 20 animals were maintained as
 

untreated controls.
 

Pyrimethamine was administered initially at doses of 1,200,
 

3,000, 6,000, 15,000 or 30,000 ppm to female Sprague-Dawley rats.
 

Death occurred in all rats at doses of 3,000 ppm and above, and
 

in 2/5 animals fed the test chemical at 1,200 ppm. The chemical
 

was then retested at doses of 100, 300, 600, or 1,200 ppm in
 

female Fischer 344 rats. At these doses, death occurred only in
 

the group at 1,200 ppm, where there was one death at the end of
 

the feeding period. Mean weight gain at the end of 45 days in
 

animals fed pyrimethamine at 100 or 300 ppm was comparable to
 

that of controls, and at 600 ppm was 74% of controls. The low
 

and high doses for the chronic studies using rats were set at 200
 

and 400 ppm.
 

Pyrimethamine was initially tested in male Swiss mice at doses of
 

2,000, 5,000, 10,000, 25,000, or 50,000 ppm. Death occurred in
 

all animals administered doses of 5,000 ppm and above, and in 3/5
 

animals administered a dose of 2,000 ppm. A second test was then
 

performed using female Swiss mice and doses of 100, 250, 500,
 

1,000, or 2,000 ppm. There were no deaths in any of these dosed
 

8
 
groups. Body weight gains in animals fed the chemical at doses
 

of up to 500 ppm were comparable to those of controls, and weight
 

gains in animals given doses of 1,000 or 2,000 ppm were only
 

slightly lower than those of controls after 45 days of
 

administration of the chemical. The low and high doses for the
 

chronic studies using mice were set at 500 and 1,000 ppm.
 

F. Designs of the Chronic Studies
 

The designs of the chronic studies are shown in tables 1 and 2.
 

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

small, pooled-control groups also were used for statistical
 

comparisons. Matched controls of each species and sex from the
 

current bioassay of pyrimethamine were combined with matched
 

controls of each species and sex from bioassays of pyrazinamide
 

and L-tryptophan. The pooled controls for statistical tests
 

using either rats or mice consisted of 45 males and of 45
 

females. The bioassays of pyrimethamine, pyrazinamide, and
 

L-tryptophan were all conducted at Southern Research Institute at
 

the same time. The matched-control groups for the pyrazinamide
 

and L-tryptophan bioassays were of the same strains (Fischer 344
 

rats and B6C3F1 mice) and from the same supplier as those for the
 

pyrimethamine bioassay, and they were examined by the same
 

pathologists.
 
Table 1. Design of Pyrlmethamine Chronic Feeding Studies in Rats
 

Sex and 

Test 

Group 

Initial

No. of

Animals3

 Pyrimethamine 

 Dose Dosed 

(ppm)" (weeks) 

Time on Study 

Observed 

(weeks) 

Male 

Matched-Control 15 0 105 

Low-Dose 35 200 78 26 

High-Dose 35 400 78 26 

Female 

Matched-Control 15 0 105 

Low—Dose 35 200 78 27 

High-Dose 35 400 78 26 

a

Rats were 41 days of age when placed on study.
 

''Dosed animals were given test diets 5 days per week and
 

control diets 2 days per week.
 

10
 
Table 2. Design of Pyrimethamine Chronic Feeding Studies in Mice
 

Sex and Initial Pyrimethamine Time on Study
 

Test No. of Dose Dosed Observed
 

Group Animalsa (ppm)k (weeks) (weeks)
 

Male
 

Matched-Control 15 0 102
 

Low-Dose 35 500 78 26
 

High-Dose 35 1,000 78 26
 

Female
 

Matched-Control 15 0 104
 

Low-Dose 35 500 78 26
 

High-Dose 35 1,000 78 26
 

a

Mice were 55 days of age when placed on study.
 

"Dosed animals were given test diets 5 days per week and
 

control diets 2 days per week.
 

11
 
G. Clinical and Pathologic Examinations
 

All animals were observed twice daily for signs of toxicity, and
 

animals that were moribund were killed and necropsied, except for
 

those dying prior to day 100, due, presumably, to toxicity of the
 

test chemical. Rats and mice were weighed individually once
 

every 2 weeks for 20 months and once per month thereafter.
 

Palpation for masses was carried out at each weighing.
 

The pathologic evaluation consisted of gross and microscopic
 

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

from killed animals and from animals found dead. The following
 

tissues were examined microscopically: skin, muscle, lungs and
 

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

thymus, heart, salivary gland, liver, gallbladder and bile duct
 

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

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

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

brain, and sensory organs. Peripheral blood smears were prepared
 

from each animal whenever possible. Occasionally, additional
 

tissues were also examined microscopically. The different
 

tissues were preserved in 10% buffered formalin, embedded in
 

paraffin, sectioned, and stained with hematoxylin and oosin.
 

Special staining techniques were utilized when indicated for more
 

definitive diagnosis.
 

12
 
Tissues from some animals were not examined, particularly from
 

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

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

autolysis as to preclude histopathologic evaluation. Thus, the
 

number of animals from which particular organs or tissues were
 

examined microscopically varies, and does not necessarily
 

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

group.
 

H. Data Recording and Statistical Analyses
 

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

System (Linhart et al., 1974). The data elements include descriptive information on the chemicals, animals, experimental design,
 

clinical observations, survival, body weight, and individual
 

pathologic results, as recommended by the International Union
 

Against Cancer (Berenblum, 1969). Data tables were generated for
 

verification of data transcription and for statistical review.
 

These data were analyzed using the statistical techniques
 

described in this section. Those analyses of the experimental
 

results that bear on the possibility of carcinogenicity are
 

discussed in the statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

13
 
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.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

14
 
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
 

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

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

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


tailed 0.05 level of significance. Unless otherwise noted, the
 

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

15
 
first tumor was observed were excluded by basing the statistical
 

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

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

such an early tumor was found, comparisons were based exclusively
 

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

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

obtained, the standard procedures for analyses of the incidence
 

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

followed.
 

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

incidence of tumors. Curves of the proportions surviving without
 

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

The week during which an animal died naturally or was sacrificed
 

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

methods of comparing these curves were used for two groups;
 

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

groups. The statistical tests for the incidence of tumors which
 

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

noted, in the direction of a positive dose relationship.
 

Significant departures from linearity (P < 0.05, two-tailed test)
 

were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared with its control was calculated
 

16
 
from the exact interval on the odds ratio (Gart, 1971). The
 

relative risk is defined as p^/pc where pt is the true binomial
 

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

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

spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

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

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

of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in approximately 95% of a large number of identical experiments, the true
 

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

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

significant result (P < 0.025 one-tailed test when the control
 

incidence is not zero, P < 0.050 when the control incidence is
 

zero) has occurred. When the lower limit is less than unity, but
 

the upper limit is greater than unity, the lower limit indicates
 

the absence of a significant result while the upper limit
 

17
 
indicates that there is a theoretical possibility of the
 

induction of tumors by the test chemical, which could not be
 

detected under the conditions of this test.
 

18
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

During the period of administration of pyrimethamine in the diet,
 

mean body weights of the low- and high-dose male rats were
 

slightly lower than those of the matched controls. Mean body
 

weights of the low- and high-dose female rats were comparable to
 

those of controls for the first 30 weeks of administration, but
 

were lower thereafter (figure 1). Fluctuations in the growth
 

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

the mean body weight may be subject to variation. No clinical
 

signs related to administration of the test chemical were
 

recorded.
 

B. Survival (Rats)
 

Kaplan and Meier curves estimating the probabilities of survival
 

for male and female rats fed pyrimethamine in the diet at the
 

doses of this bioassay, together with those of the matched
 

controls, are shown in figure 2.
 

In male rats, the results of the Tarone test for positive
 

dose-related trend in mortality are not significant, with 30/35
 

(86%) of the high-dose group, 28/35 (80%) of the low-dose group,
 

and 10/15 (67%) of the matched controls living to the end of the
 

19
 
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 LOW DOSE 

 HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKS) 

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FEMALE RATS 

D MATCHEDCONTROL 

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TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves For Rats Fed Pyrimethamine In The Diet 

20 
IRVIVAL

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Figure 2. Survival Curves For Rats Fed Pyrimethamine In The Diet 

21 
study. In females, the results of the Tarone test are significant in the negative direction (P = 0.015), with 33/35 (94%) of
 

the high-dose group, 30/35 (86%) of the low-dose group, and 10/15
 

(67%) of the matched controls living to end of the study.
 

Sufficient numbers of dosed and control 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 variety of neoplasms occurred with approximately equal
 

frequency in the matched-control and dosed groups. Some types of
 

neoplasms occurred only in the dosed groups or with greater
 

frequency in the dosed than in the control groups. These
 

lesions, however, are not uncommon in this strain of rat
 

independent of administration of the test chemical.
 

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

proliferative, and inflammatory changes also were encountered in
 

animals of the dosed and control groups (Appendix C). These
 

nonneoplastic lesions are commonly seen in aged Fischer 344 rats.
 

An increased frequency of bone-marrow atrophy occurred in both
 

male and female dosed groups (males: controls 1/15 [77,], low22
 
dose 15/34 [44%], high-dose 15/32 [47%]; females: controls 2/14
 

[14%], low-dose 29/35 [83%], high-dose 23/35 [66%]).
 

In the judgment of the pathologists, pyrimethamine was not
 

carcinogenic when fed to Fischer 344 rats at doses of 200 or 400
 

ppm for 78 weeks.
 

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 in one group and with 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 and those of the Fisher exact
 

test for higher incidences in either dosed group than in either
 

control group are not significant for any of the tumors that were
 

observed. Significant results in the negative direction are
 

observed in the combined incidence of lymphoma and leukemia and
 

in the incidence of follicular-cell carcinoma of the thyroid,
 

where the incidences in the control groups exceed those in the
 

dosed groups; however, the dosed animals lived longer than the
 

controls.
 

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

23
 
dose-related trend in the incidence of C-cell adenoma of the
 

thyroid are significant (P = 0.038) using the matched controls,
 

but the results of the Fisher exact tests for direct comparisons
 

of incidences of this tumor in dosed and control groups are not
 

significant.
 

In each of the 95% confidence intervals of relative risk, shown
 

in the tables, a 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 for the
 

incidence of hematopoietic tumors in high-dose male rats compared
 

with the corresponding pooled-control group) has an upper limit
 

greater than one, indicating the theoretical possibility of the
 

induction of tumors by pyrimethamine, which could not be detected
 

under the conditions of this test.
 

24
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of both male and female mice were slightly
 

lower than those of the matched controls during the period of
 

administration of pyrimethamine in the diet, but were roughly
 

comparable during the observation period following administration
 

of the chemical (figure 3). Fluctuations in the growth curve may
 

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

body weight may be subject to variation.
 

No signs of toxicity related to administration of the test
 

chemical were recorded in the mice. Some animals showed signs of
 

respiratory disease, and all animals received oxytetracycline in
 

the drinking water for 5 days at a dose of 0.6 rag/ml during week
 

69 and for 5 days at 0.3 mg/ml during week 70. To arrest the
 

transmission of airborne microorganisms, propylene glycol was
 

vaporized in the mouse room during weeks 68-79.
 

B. Survival (Mice)
 

Kaplan and Meier curves estimating the probabilities of survival
 

for male and female mice fed pyrimethamine in the diet at the
 

doses of this bioassay, together with those of the matched
 

25
 
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FEMALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

TIME ON STUDY (WEEKS) 

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

26 
controls, are shown in figure 4. This figure indicates that few
 

animals of either sex survived to the end of the study.
 

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

mortality are not significant in either sex. In male mice, the
 

survival is low. Only 10/35 (29%) of the high-dose group, 12/35
 

(34%) of the low-dose group, and 8/15 (53%) of the matched
 

controls lived beyond 1 year. Three of the matched-control
 

animals were reported as missing at week 35. No tumor was
 

observed in the control group and only two tumors in the dosed
 

animals, one in the low-dose group at week 104 and one in the
 

high-dose group at week 87. These early deaths may have
 

prevented the observation of late-appearing tumors in the males.
 

In females, however, over 50% of the animals in the three groups
 

(22/35 [63%] of the high-dose group, 19/35 [54%] of the low-dose
 

group, and 10/15 [67%] of the matched controls) were at risk for
 

at least as long as 1 year; however, only 15/35 (45%) of the
 

high-dose females survived to the end of the study.
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

are summarized in Appendix D, tables Dl and D2.
 

The small number of tumors observed in mice may have been
 

27
 
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influenced by the decreased life span in both the matched-


control and dosed groups of male and female mice. Respiratory
 

infections and inflammatory lesions in the lungs were a factor in
 

the shortened life spans of all groups of mice. The incidences
 

of animals with respiratory lesions were: 3/8 (38%) control
 

males, 7/14 (50%) low-dose males, 2/13 (15%) high-dose males,
 

5/12 (42%) control females, 11/25 (44%) low-dose females, and
 

1/21 (5%) high-dose females.
 

Mortality was high, especially in the male mice. Some of these
 

deaths were associated with respiratory infections, while in
 

other animals the cause of death was undetermined. Many of the
 

latter animals had advanced autolysis which precluded histologic
 

evaluation. In addition, animals that died prior to 100 days on
 

study were not evaluated grossly or histologically. The
 

disposition of male and female mice was as follows:
 

MICE
 

MALE FEMALE
 

Matched Low High Matched Low High 

Control Dose Dose Control Dose Dose 

Animals starting study 15 35 35 15 35 35 

Autolysis/no necropsy 2 0 13 1 4 2 

No necropsy performed 1 21 8 2 4 11 

Animals missing 3 0 0 0 0 0 

Number of animals at 

terminal sacrifice 0 2 4 2 6 15 

Total animals necropsie.ed a

(percent) (60%) (40%) (40%) (80%) (77%) (63%) 

29
 
In addition to the neoplastic lesions, a number of degenerative,
 

proliferative, and inflammatory changes also were encountered in
 

animals of the dosed and control groups (Appendix D). For the
 

most part the nonneoplastic lesions are those commonly seen in
 

aged mice.
 

Administration of the pyrimethamine to the mice resulted in few
 

tumors. Most of the neoplastic lesions appeared unrelated to
 

administration of the chemical. The effectiveness of the
 

carcinogenesis bioassay was reduced by a decrease in life span.
 

In the judgment of the pathologists, the results of this bioassay
 

failed to define the carcinogenic activity of pyrimethamine in
 

B6C3F1 mice when fed at doses of 500 or 1,000 ppm for 78 weeks.
 

This may have been due to the high early mortality, especially in
 

the males, and to the small number of animals examined
 

histologically.
 

D. Statistical Analyses of Results (Mice)
 

Table Fl in Appendix F contains the statistical analyses of the
 

incidences of those primary tumors that occurred in at least two
 

animals in one group and with an incidence of at least 5% in one
 

or more than one group of the female mice. The incidences of
 

tumors in the male mice are not. included in the tables and
 

analyses, because only two tumorc were observed among the dosed
 

30
 
and control groups of animals. A sebaceous adenoma of the skin
 

was found in the low-dose group and a histiocytic type of
 

malignant lymphoma of the hematopoietic system was found in the
 

high-dose group. The early deaths of the male mice may have
 

prevented the observation of late-appearing tumors.
 

In female mice, the results of the Cochran-Armitage test for
 

positive dose-related trend and those of the Fisher exact test
 

for direct comparisons of control and dosed groups are not
 

significant. In each of the 95% confidence intervals of relative
 

risk, shown in table Fl, one is included; this indicates the
 

absence of significant positive results. It should also be noted
 

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

indicating the theoretical possibility of the induction of tumors
 

by pyrimethamine, which could not be detected under the
 

conditions of this test.
 

31
 

V. DISCUSSION
 

In this bioassay of pyrimethamine administered in feed, mean body
 

weights of rats were only slightly lower than those of controls,
 

and mortality was not related to administration of the chemical.
 

In the mice, mean body weights of the dosed animals were lower
 

than those of the controls throughout the administration period,
 

and mortality was high, particularly in the males. The early
 

deaths may not have been related to administration of the
 

pyrimethamine, since there was a high incidence of respiratory
 

infections among all groups of mice.
 

In rats of each sex, no neoplastic lesions were found in dosed
 

groups at incidences significantly different from those of either
 

matched or pooled controls. An increased frequency of bone-


marrow atrophy occurred in both male and female dosed groups.
 

In male mice, the markedly decreased life spans may have
 

prevented the observation of late-appearing tumors, since only
 

one tumor was found in a high-dose male and one in a low-dose
 

male. No tumor occurred in the dosed female mice at an incidence
 

significantly above those of the control animals.
 

No long-term studies on the carcinogenicity of pyrimethamine have
 

been reported.
 

33
 
It is concluded that under the conditions of this bioassay,
 

pyrimethamine was not carcinogenic for male or female Fischer 344
 

rats or for female B6C3F1 mice. The carcinogenic potential of
 

pyrimethamine for male B6C3F1 mice cannot be assessed by this
 

bioassay, because of the markedly reduced life span.
 

34
 
VI. BIBLIOGRAPHY
 

AMA Department of Drugs, Antimalarial agents. In: AMA Drug 

Evaluations, Publishing Sciences Group, Inc., Acton, 

Massachusetts, 1973, pp. 607-613. 

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

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

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

Panel on Carcinogenicity of the Cancer Research Commission
 

of the UICC, Vol. _2. International Union Against Cancer,
 

Geneva, 1969.
 

Calabresi, P. and Parks, R. E., Jr., Alkylating agents,
 

antimetabolites, hormones, and other antiproliferative
 

agents. In: The Pharmacological Basis of Therapeutics,
 

Goodman, L. S. and Gilman, A., eds., Macmillan Publishing
 

Co., Inc., New York, 1975, p. 1057, 1269.
 

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

Soc. B 34(2):187-220, 1972.
 

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

1970, pp. 48-52.
 

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

significance tests, confidence limits and adjustments for
 

stratification. Rev. Int. Statist. Inst. _39; 148-169, 1971.
 

Johns, D. G. and Bertino, J. R., The chemotherapeutic agents. In:
 

Cancer Medicine, Holland, J. F. and Frei, E. , III, eds., Lea
 

& Febiger, Philadelphia, 1973, pp. 742-743.
 

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

incomplete observations. J. Am. Statist. Assoc. 53;457-481,
 

1958.
 

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

J., Carcinogenesis bioassay data system. Comp. and Biomed.
 

Res. 7:230-248, 1974.
 

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

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

35
 
Rollo, I. M., Drugs used in the chemotherapy of malaria. In:
 

The Pharamcological Basis of Therapeutics, Goodman, L. S.
 

and Gilman, A., eds., MacMillan Publishing Co., Inc., New
 

York, 1975, pp. 1045-1048 and 1055-1056.
 

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

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

induced by different numbers of administrations of benzo (a)
 

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

Stecker, P. G. , Editor, Merck Index, 8th Edition, Merck and
 

Company, Inc., Rakway, N. J., 1968, pp. 892-893.
 

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

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

36
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS FED PYRIMETHAMINE IN THE DIET
 

37
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

FED PYRIMETHAMINE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

AMIMALS INITIALLY IN STUDY

ANIMALS N£CBOPSI£D

ANIHALS EXAMINED HISTOPATHOLOGICALLI

 15

 15

 15

 35

 35

 35

 35 

 33 

 33 

INTEGUMEHTABY SYS1EM
 

*SUBCUT TISSUE (15) (35) (33)
 

FIBHOMA 1 (3X)
 

FIBROSARCOMA 1 (3X) 1 (3X)
 

RESPIfiATOBY SYSTEM
 

HONE
 

HEHATOPOIETIC SYSTEM
 

*HULTIPLE OF.GANS (15) (35) (33)
 

BALIG.LYMPHOMA, UNDIFPEB-TYPE 1 (7X)
 

LEUKEMIA,NOS 1 (3X)
 

UHDIFFEBENTIATID LEUKEMIA 2 (13X) 1 (3X) 1 (3X)
 

CIBCULATOfiY SYSTEM
 

HONE
 

DIGESTIVE SYSTEM
 

fCOLOB (14) (35) (32)
 

ADEHOHATOUS POLYP, NOS 1 (3»)
 

ORIBABY SYSTEM
 

•UBIHAHY BLADDEB (15) (30) (32)
 

IHAMSITIONAL-CELL CABCIMOMA 1_l31f\,
 

• HUHBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIHALS NECBOPSIED
 

39
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

ENDOCRINE SYSTEM
 

•PITUITARY
 

CHROMOPHOBE ADENOMA
 

•ADBENAL
 

PHEOCHROMOCYTOMA
 

PHEOCHROHOCYTOHA. MALIGNANT
 

•THYROID
 

FOLLICULAR-CELL CARCINOMA
 

C-CELL CARCINOMA
 

•PANCBEATIC ISLETS
 

ISLET-CELL ADENOMA
 

ISLIT-CELL CARCINOMA
 

BEPBODOCTIVE SYSTEM
 

•TESTIS
 

INTERSTITIAL-CELL TUMOR
 

NEBVODS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*BAB CANAL
 

SQOAMOUS CELL CAECINOMA
 

HDSCULOSKELETAL SYSTEM
 

HONE
 

BODY CAVITIES
 

*PEBITONEOM
 

HESOTHELIOJM, NOS
 

MATCHED
 

CONTROL


(11)


2 (18X)


(15)


1 (7X)


(14)


2 (14X)


(14)


1 (7X)


(14)


13 (93X)


(15)


(15)


HIGH DOSE
 

(31)
 

1 (3X)
 

(33)
 

1 (3X)
 

(32)
 

3 (9X)
 

(33)
 

1 (3X)
 

2 (6X)
 

(33)
 

27 (82X)
 

(33)
 

1 (3X)
 

(33)
 

 LOW DOSE
 

 (33)
 

8 (24X)
 

 (35)
 

1 (3X)
 

 (34)
 

1 (3X)
 

3 (9X)
 

 (35)
 

2 (6X)
 

 (35)
 

 30 (86X)
 

 (35)
 

1 (3X)
 

 (35)
 

1 13X1
 

< NUflBER OF ANIMALS WITH TISSUE EJCABINED MICROSCOPICALLY 

* NDHBEH OF ANIMALS NECBOPSIED 

40 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

*PLEURA (15) (35) (33)
 

MESOTHEL1CMA BENIGN 1 (7X)
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 15 35 35
 

NATURAL DEATHS 3
 

MORIBUND SACRIFICE 4
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 10 28 30
 

ANIHAL MISSING
 

9 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 14 33 30
 

TOTAL PRIMABY TUMORS 23 51 40
 

TOTAL ANIMALS WITH BENIGN TUMORS 14 32 29
 

TOTAL BENIGN TUMORS 17 41 31
 

TOTAL ANIMALS WITH MALIGNAN 6 T TUMORS 8 9
 

TOTAL MALIGNANT TUMORS 6 9 9
 

TOTAL ANIMALS WITH SECONDARY TUMOBS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT 1
 

TOTAL UNCERTAIN TUMORS 1
 

TOTAL ANIMALS WITH TUMORS UNCEBTAIN­ PHIMABI OR METASTATIC
 

TOTAL UNCERTAIN TUMOBS
 

* PBIMARY 1UMOES: ALL TUHOBS EXCEPT SECONDABT TOMOBS
 

i SECONDARY TUMORS: METASTATIC TUMOBS OB TUHOBS INVASIVE INTO AN ADJACENT OBGAN
 

41
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

FED PYRIMETHAMINE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

AHIMALS INITIALLY IN STUDY 15

ANIMALS KECROPSIED 14

ANIMALS EXAMINED HISTOPATHOLOGICALLY 14

 35

 35

 35

 35 

 35 

 35 

INTEGUMENTARY SYSTEM
 

*SOBCUT TISSUE (14) (35) (35)
 

SARCOMA, NOS 1 (3X)
 

BESPIBATORY SYSTEM
 

tLUNG (13) (34) (35)
 

ADENOCARCINOMA, NOS, METASTATIC 1 (8X)
 

ALVEOLAR/BFONCHIOLAE ADENOMA 1 (3X)
 

ALVEOLAR/BfiCNCHIOLAR CARCINOMA 1 (3X)
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (14) (35) (35)
 

UNDIFFERENTIATED LEUKEMIA 1 (7X) 1 (3X)
 

CI8CULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

NONE
 

URINARY SYSTEM
 

NONE
 

BNDOCRIBE SYSTEM
 

•PITUITARY	 (11) (32)
 

CHROHOPHOBE ADENOMA 4 (36X1 8 <24%) 14
 

* NU8BEE OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NDMBER OF ANIMALS NECBOPSIED
 

42
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

tTHYBOID (13) (35) (3<4)
 

FOLI.ICULAB-CELL CARCINOHA 1 (3*)
 

C-CELL ADENOMA 4 (12X)
 

•PANCREATIC ISLETS (14) (34) (35)
 

ISLET-CELL CARCINOMA 1 (3X) 1 (3%)
 

REPRODUCTIVE SYSTEM
 

*BABBARY GLAND (14) (35) (35)
 

IHPILTfiATING DUCT CABCINOHA 1 (3X)
 

FIBHOAEEHOfA 2 (14X) 4 (11%) 4 (11X)
 

tUTEBOS (14) (35) (35)
 

ADENOCARCINOBA, NOS I (7%)
 

BNDOBETRIAL STKCKAL POLYP 3 (9X) 3 (9X)
 

BERVOUS SYSTFM
 

NONE
 

SPECIAL SENSF OFGANS
 

NONE
 

HUSCDLOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*PERITONEUM (14) (35) (35)
 

ADENOCARCINOHA, NOS, BETASTATIC 1 (7Jt)
 

*PLEURA (14) (35) (35)
 

ADENOCAECINOMA, NOS, HETASTATIC 1 (7X)
 

ALL OTHER SYSTEMS
 

* HOHBEB OF ANIBALS WITH TISSUE EXAHINED HICfiOSCOPICALLY
 * HHBBER OF ANIBALS NECROPSIED
 

43
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIHAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 15 35
 

NATURAL DEATHd)
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 10 30 33
 

ANIMAL MISSING
 

d INCLUDES AUTOLYZED ANIMALS
 

TOMOfi SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TOMORS* 7 16 26
 

TOTAL PRiMAkY TUMORS 8 19 29
 

TOTAL ANIMALS WITH BENIGN TUMORS 6 14 24
 

TOTAL ES1IIGN TUMORS 6 16 25
 

TOTAL ANIMALS WITH MALIGNAN 2 T TUMOKS 3 4
 

TOTAL MALIGNANT TUMORS 2 3 4
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1
 

TOTAL SECONDARY TUMORS 3
 

TOTAL ANIMALS HITH TUMORS UNCERTAIN­ BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUROBS
 

TOTAL ANIMALS WITH TUMORS UNCEBTAINPBIHABY OR METASTATIC
 

TOTAL UNCEETAIN TUMORS
 

* PBIUARY TUMORS: ALL TUHOHS EXCEPT SECONDABY TOHOBS
 * SECONDABY TUMORS: METASTATIC TUMORS OB TUMOBS INVASIVE INTO AN ADJACENT OBGAN
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE FED PYRIMETHAMINE IN THE DIET
 

45
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

FED PYRIMETHAMINE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIHALS INITIALLY IN STUDY 15 35 35
 

AHIHALS HISSING 3
 

ANIMALS NECROPSIED 9 14 14
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 9 14 14
 

INTEGUMENTARY SYSTEM
 

*SKIH (9) (14) (14)
 

SEBACEOUS ADENOMA 1 (7X)
 

HESPIBATOBY SYSTEM
 

NONE
 

HEHATOPOIETIC SYSTEM
 

*MULTIPLE OP.GANS (9) (14) (14)
 

MALIG.LYMPHOHA, HISTIOCYTIC TYPE 1 (7X)
 

CIBCDLATORY SYSTEM
 

HONE
 

DIGESTIVE SYSTEM
 

NONE
 

OBINAHY SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

NONE
 

BEPBODUCTIVE SYSTEM
 

BONE .
 

t NUMBER OF ANIHALS WITH TISSUE EXAMINED MICHOSCOPICALLY
 * NUHBEB OF ANIMALS NECBOPSIED
 

47
 
TABLE 81. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

HEBVOOS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

BOSCULOSKEIE7A1 SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

ANIHAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY

NATUEAL DEATHS

HOHIBUND SACRIFICE

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TEBMINAL SACRIFICE

ANIMA L HISSING

 15

 8

 4 

3 

 35

 31

2 

2

 35 

 29 

2 

4 

a INCLUDES AUTQL1ZED AMIMALS 

« NUMBEfi OF ANIMALS WITH TISSUE EXABINED HICHOSCOPICALLI 

* HUHBEH OF ANIHALS NECROPSIED 

48
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED
 

pONTROL LOW DOSE HIGH DOSE
 

TOHOB SUMMARY
 

TOTAL AHIMALS WITH PRIMARY TUMOBS* 1 1
 

TOTAL PRIMARY TUBOES 1 1
 

TOTAL AHIBALS WITH BENIGN TUMOHS 1
 

TOTAL BENIGN TUBORS 1
 

TOTAL AHIBALS WITH BALIGNANT TUBORS 1
 

TOTAL MALIGNANT TUMORS 1
 

TOTAL ANIBALS WITH SECONDARY TOHOHS*
 

TOTAL SECOHDABY TUMORS
 

TOTAL ANIBALS WITH TUMORS UNCERTAIN­ BEHIGN OB MALIGNANT
 

TOTAL UNCERTAIN TUB08S
 

TOTAL ANIBALS HITH TUMORS UNCERTAINPEIHABY OR MSTASTATIC
 

TOTAL UNCERTAIN TDKORS
 

* PRIMARY TUBOES: ALL TUBORS EXCEPT SECONDARY TUMORS
 * SECONDARY 7UKOP.S: METASTATIC TUBORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

49
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

FED PYRIMETHAMINE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 15 35 35 

ANIHALS NECBOPSIED 12 27 22 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 12 27 22 

INTEGUMENTAR Y SYSIEM 

NON E 

RESPIRATORY SYSTEM 

•LONG	 (12) (25) (21)
 

ALVEOLAE/EHONCHIOLAR ADENOMA	 2 (10X)
 

HEMATOPOIE1IC SYSTEM
 

•MULTIPLE OiiGANS (12) (27) (22)
 

MALIG.LY.flPHOMA, UNDIFFEH-TYPE 1 (4X)
 

HALIG.LYMPHOMA, HISTIOCYTIC TYPE 1 (H%) 4 (18%)
 

•KIDNEY	 (10) (27) (22)
 

BALIG.LYHPHOMA, HISTIOCYTIC TYPE 1 (4X)
 

CIHCULATOfiY SYSTEK
 

NONE
 

DIGESTIVE SYSTEM
 

•PANCBEAS	 (10) (24) (22)
 

ADBNOCARCIHOMA, NOS, METASTATIC 1 (5X)
 

OBINARY SYSTSW
 

•KIDNEY	 (10) (27) (22)
 

^DENQCARCIMpHA. NOS.. MET»STAT^C 1 <SX1
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

50
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

tDBINAEY BLADDER

ADEHOCARCINOMA, NOS, METASTATIC

 (8) (23) (21) 

1 (5%) 

ENDOCRINE SYSTEM 

•THYBOID

FOLLICOLAR-CELL ADENOMA

 (10) (25) (22) 

1 (5%) 

BEPBODOCTIVE SYSTEH 

*HABHA8Y GLAND

FIBBOADENOHA

• UTERUS

ADENONA, NOS

ADENOCARCINOHA. NOS

EHDOMBTBIAL STROHAL POLYP

•OVARY

ADENOCARCINOBA, NOS, BETASTATIC

 (12)

 (10)

 1 (10X) 

 (10)

 (27)

 1 (4X) 

 (25)

 1 (4X) 

 (24)

 (22) 

 (22) 

2 (9X) 

 (21) 

1 (5X) 

NBHVOOS SYSTEM 

HONE 

SPECIAL SENSE ORGANS 

HONE 

HUSCULOSKE1ETAL SYSTEM 

HOHE 

BODY CAVITIES 

NONE 

ALL OTHEB SYSTEMS 

HOME _ _ l L _ _ 

• NUHBEB OF AHIHALS HITH TISSUE EXAMINED MICROSCOPICALLY * NDHBEB OF AHIMALS NECROPSIED 

51
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

ANIHAL DISPOSITION SUMMARY
 

ANIHALS INITIALLY IN STUDY
 

NATUBAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TEHHIHAL SACRIFICE
 

ANIMAL MISSING
 

a INCLUDES AUTOLYZE0 ANIMALS
 

TUMOB SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*


TOTAL PRIMARY TUMORS


TOTAL ANIMALS KITH BENIGN TUMORS


TOTAL BENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMOBS*


TOTAL SECONDARY TUMORS


TOTAL ANIMALS WITH TUMOBS UNCERTAINBENIGN OB MALIGNANT
 

TOTAL UNCERTAIN TUMOBS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPBIHARY OR MSTASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

15 35 35 

8 15 14 

4 12 6 

1 2 

2 6 15 

1 5 9 

1 5 9 

1 2 3 

1 2 3 

3 6 

3 6 

1 

4 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECCNDARX TUMORS
 

t SECONDARY TUMORS: METASTATIC TUMORS OB TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

52
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED PYRIMETHAMINE IN THE DIET
 

53
 

TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE 

RATS FED PYRIMETHAMINE IN THE DIET 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

AHIHALS EXAMINED HISTO PATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

*SKIH
 

INFLAMMATION, CHRONIC
 

*SOBCOT TISSUE
 

ULTIMOBRANCHIAL CYST
 

EPIDERMAL INCLUSION CYST
 

HEHOBBHAGE
 

HESPIHATOBY SYSTEM
 

•TRACHEA
 

INFLAMMATION, SUPPUBATIVE
 

INFLAMMATION, CHRONIC SUPPUBATIV
 

•LONG
 

PNEUMONIA, CHRONIC MURINE
 

HBBATOPOIETIC SYSTEM
 

•BONE MARROW
 

ATROPHY, NOS
 

•SPLEEN
 

HEHATOPOIESIS
 

•MASDIBULAH L. FODE
 

HIPEBPLASIA, PLASMA CELL
 

•CEBVICAL LYMPH NODE
 

LYMPHANGIECTASIS
 

CIHCOLATOBY SYSTEM
 

HONE
 

MATCHED
 

CONTROL
 

15
 

15
 

15
 

(15)
 

(15)
 

(15)
 

(15J


3 (20X)
 

(15)
 

1 (7X)
 

(15)
 

(11)


1 (9X)
 

(11)
 

LOW DOSE
 

35
 

35
 

35
 

(35)
 

(35)
 

(35)
 

1 (3X)
 

(35)
 

19 (54*)
 

(34)
 

15 (44X)
 

(35)
 

2 (6X)
 

(33)
 

1 (3X)
 

(33)
 

HIGH DOSE
 

35
 

33
 

33
 

(33)
 

1 (3X)
 

(33)
 

1 <3X)
 

1 (3X)
 

1 (3X)
 

(33)
 

7 (21X)
 

5 (15X)
 

(33)
 

4 (12X)
 

(32)
 

15 (47X)
 

(33)
 

1 (3X)
 

(27)
 

(27)
 

1 (4X)
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED NICBOSCOPICALIY
 

* HDMBEB OF ANIMALS NECBOPSIED
 

55
 
TABLE C1. MALE RATS: NONNEOPLASTIC

DIGESTIVE SYSTEM
 

•LIVER
 

NECROSIS, COJGULATIVE
 

CYTOPLASKIC VACUOLIZATICB
 

HYPERPLASIA, NODULAR
 

ILIVEfi/CENTFILOBULAR
 

NECROSIS, COAGULATIVE
 

CYTOLOGIC DEGENERATION
 

tPANCREAS
 

FIBBOSIS, DIFFUSE
 

tSTCMACH
 

ULCER, FOCAL
 

UBINABY SYSTEM
 

tKIDNEY
 

INFLAMMATION, CHRONIC
 

INFARCT, NOS
 

ENDOCRINE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEM
 

*BAMMABY GLAND
 

CYST, NOS
 

fPBOSTATE
 

IHFLAMMATIOK, SUPPURATIVE
 

MATCHED
 

CONTROL
 

(15)
 

1 (7X)
 

(15)
 

1 (7X)
 

(14)
 

1 (7X)
 

(15)
 

(15)
 

12 (SOX)
 

(15)
 

1 (7X)
 

(15)
 

1 (7X)
 

(13)
 

LOW DOSE
 

(35)
 

1 (3X)
 

(35)
 

1 (3X)
 

(35)
 

(35)
 

(35)
 

30 (86X)
 

(35)
 

(33)
 

1 (3X)
 

(34)
 

HIGH DOSE
 

(32)
 

1 (3X)
 

2 (6X)
 

(32)
 

(33)
 

(33)
 

1 (3X)
 

(33)
 

23	 (70X)
 

1 (3X)
 

(33)
 

2 (6X)
 

(33)
 

(33)
 

1 (3X)
 

INFLAMMATION, CHRONIC SUPPUHATI?
 

NERVOUS SYSTEM
 

tBBAIN
 

HEMOBHHAGE
 

 LESIONS (CONTINUED) 

t NUMBER OF ANIMALS WITH TISSUE EXAHINED MICBOSCOPICALLI
 

* NUMBER OF ANIMALS NECHOPSIED
 

56
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

MALACIA 1 (3X) 

SPECIAL SENSE ORGAN S 

*HIDDLE EAR (15) (35) (33)
 

INFLAMMATION. CHRONIC SOPPORATIV 1 (3X)
 

HUSCULOSKELETAL SYSTEM
 

*SKELETAL MUSCLE (15) (35) (33)
 

INFLAMMATION, CHRONIC FOCAL 1 (3X)
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEHS
 

ADIPOSE TISSUE
 

INFLAMMATION, CHBONIC 1
 

INFLAMMATION, CHRONIC FOCAL 1
 

INFLAMMATION, CHRONIC DIFFUSE 1
 

SPECIAL MORPHOLOGY SUMMARY
 

NO NECROPSY PERFOBMED 2
 

t NUHBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIBD
 

57
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

FED PYRIMETHAMINE IN THE DIET 

J

 -1— n-TTT 1— TT— JIL ««MB_
 

ANIMALS INITIALLY IN STUDY


ANIMALS NECROPSIED


ANIMALS EXAMINED H1STOPATHOLOGICALLY


INTEGUMENTARY SYSTEM
 

*SKIN


ULCEB, CHRONIC
 

INFLAMMATION, CHRONIC FOCAL
 

RESPIRATORY SYSTEM
 

•TRACHEA


INFLAMMATION, SUPPURATIVE
 

PLASMA-CSLL INFILTRATE
 

HYPEHPLASIA, PLASMA CELL
 

• LUNG


PNEUMONIA, CHRONIC HURINE


HYPEBPIASIA, ALVEOLAR EPITHELIUM
 

HEMATOPOIETIC SYSTEM
 

•BONE HABROH


ATROPHY, NOS


•SPLEEN


HEHATOPOIESIS
 

•HEDIASTINAL L. NODE


HYPEBPLASIA, PLASMA CELL
 

CIBCULATOBY SYSTEM
 

• NUMBER OF ANIMALS WITH TISSUE EXAMINED


* NUMBER OF ANIMALS NECROPSIED
 

MATCHED 

CONTROL LOW DOSE 

 15 35 

 14 35 

 14 35 

 (14)	 (35)
 

1 (3X)
 

1 (3%)
 

 (14)	 (35)
 

1	 (3X)
 

 (13) (3ft)
 

4	 (31X) 16 (47X)
 

1 (3X)
 

 (14)	 (35)
 

2 (1<*X) 29 (83X)
 

 (14)	 (33)
 

1 (3X)
 

 (14)	 (34)
 

 MICROSCOPICALLY
 

HIGH DOSE
 

35
 

35
 

35
 

(35)
 

(35)
 

1 (3X)
 

1 (3X)
 

(35)
 

3 (9X)
 

(35)
 

23 (66X)
 

(35)
 

(30)
 

1 (3X)
 

58
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

DIGESTIVE SYSTEM
 

tLIVEB (14) (34) (35)
 

INFLAMMATION, CHRONIC FOCAL 1 (3X)
 

•LIVEB/CENTEILOBULAR	 (14) (34) (35)
 

CYTOLOGIC DEGENERATION 1 (3X)
 

UHINABY SYSTEM
 

•KIDNEY	 (14) (35) (35)
 

INFLAMMATION, CHRONIC 8 (57X) 7 (20X) 4 (11X)
 

BNDOCBINE SYSTEM
 

• THYHOID (13) (35) (34)
 

CYSTIC FOLLICLES 1 (3X)
 

HYPEBPLASIA, C-CELL 1 (8X) 1 (3X)
 

BEPBODOCTIVE SYSTEM
 

*MAMMABY GLAND (14) (35) (35)
 

CYST, NOS 1 (3X)
 

•OTBBUS/ENDCMETRIUM	 (14) (35) (35)
 

INFLAMMATION, SUPPURATIVE 7 (50%) 8 (23X) 7 (20X)
 

INFLAMMATION, CHRONIC SOPPDBATIV 1 (7X) 2 (6X) 4 (11X)
 

INFLAMMATION, FOCAL GBANOLOMATOO 1 (3X)
 

HYPEBPLASIA. CYSTIC 1 (3X)
 

#OVABY/OVIDUCT (14) (35) (35)
 

INFLAMMATION, SUPPURATIVE 1 (3X) 3 (9X)
 

•OVABY	 (14) (35) (35)
 

CYST, NOS 5 (14X) 12 (34X)
 

INFLAHHATION, SUPPUBATIVE 1 (7X) 2 (6X) 4 (11X)
 

INFLAMMATION, CHBONIC SUPPUBATIV 1 (7X) 1 (3X) 2 (6X)
 

MEBVODS SYSTEM
 

HONE
 

i HDBBEB OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECBOPSIED
 

59
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC

SPECIAL SENSE ORGANS
 

NONE
 

HDSCULOSKELETAL SYSTEB
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

ADIPOSE TISSUE 

INFLAMMATION, CHRONIC FOCAL


SPECIAL MORPHOLOGY SUHMARY
 

NO LESION REPORTED


AUTOLYSIS/NO NECROPSY


 LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

1
 

1
 

1
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

60
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE FED PYRIMETHAMINE IN THE DIET
 

61
 

TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

FED PYRIMETHAMINE IN THE DIET 

'~TM~1-"'J " •- ——. ......	 .


MATCHED
 

CONTROL LOW DOSE
 

ANIHALS INITIALLY IN STUDY 15 35
 

ANIMALS HISSING 3
 

ANIHALS NECBOPSIED 9 14
 

ANIHALS EXAMINED HISTOPATHOLOGICALLY 9 14
 

INTEGUMENTARY SYSTEM
 

NONE
 

BESPIBATOBY SYSTEM
 

tTBACHEA (7) (14)
 

INFLAMMATION, SUPPURATIVE 1 (7*)
 

fLUNG (8) (14)
 

CONGESTION, NOS 1 (7X)
 

EDEHA, NOS 1 (7X)
 

HEMORRHAGE 2 (14X)
 

PNEUBONIA, ASPIRATION
 

INFLAMMATION, SUPPUPATIVE 1 (13X)
 

BHONCHOPNEUMONIA SUPPUBATIVE 2 (25X) 3 (21X)
 

INFLAMMATION, CHRONIC FOCAL 1 (7X)
 

INFLAMMATION, CHRONIC SUPPURATIV 2 (14X)
 

BBONCHOPHEUMONIA CHRONIC SUPPURA 1 (7*)
 

HYPEEPLASIA, LYMPHOID
 

HEMATOPOIETIC SYSTEM
 

tBONE MAKROK (8) (14)

 

ATROPHY, NOS 2 (25X)

 

DEPLETION 1 (13X) 

•SPLEEN	 (8) (13) 

LIHPHOID DEPLETION 1 (13X) 

HYPERPLASIA, LYMPHOID 4 (31X) 

HEBATOEOIESIS 3 (38X) 6 (46X) 

•THTHOS	 (6) (9)

AIBOPHY. NOS	 1 mxi 

» NUBBEH OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUHBEB OF ANIMALS NECROPSIED
 

 -— i-r iii--r--i-.-r IL -•• TT jrr u ::• • nui •- • mr r.n
 

HIGH DOSE
 

35
 

14
 

14
 

(13)
 

1 (8%)
 

(13)
 

1 (8X)
 

1 (8S)
 

1 (8X)
 

4 (31X)
 

(13)
 

(14)
 

5	 (36X)
 

(12)
 

63
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE
 

CIBCU1ATOBY SYSTEM
 

IHYOCABDIUM (8) (14)
 

HEMORRHAGE 1 (7X)
 

INFLAMMATION, CHRONIC 2 (14X)
 

INFLAMMATION, CHRONIC FOCAL 2 (14X)
 

INFLAMMATION, CHRONIC SUPPURATIV 2 (14X)
 

DIGESTIVE SYSTEM
 

#LIVER (8) (13)
 

INFLAMMATION, FOCAL 1 (8%)
 

DEGENERATION, GRANULA R
 

NECBOSIS, COAGULATIVE 2 (15X)
 

CYTOPLASKIC V ACUOLIZ ATICN 1 (13X)
 

HYPERPLASIA, NODULAR 1 (13X) 1 (8%)
 

•LIVEB/HEPA^OCYTES	 (8) (13)
 

DEGENERATION, GRANULAR
 

*RECTUM (9) (14)
 

ULCER, NOS
 

*PERIANAL TISSUE (9) (14)
 

INFLAMMATION, CHPONIC FOCAL
 

UBINARY SYSTEM
 

tKIDNEY (8) (14)
 

LYMPHOCYTIC INFLAMMATORY INFILTR 2 (14X)
 

INFLAMMATION, CHROMIC
 

INFABCT, FOCAL 1 (7X)
 

HYPEBPLASIA, LYKPHOID 1 (7X)
 

ENDOCRINE SYSTEM
 

NONE
 

BEPBODUCTIVE SYSTEM
 

*HAMMJVB Y GLAN D (9) (14) 

DUCTS 

* NUHBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 » NUMBEB OF ANIMALS NECROPSIBD
 

PT

 *"" "- --L ~v _r «.- —
 

HIGH DOSE
 

(13)
 

3 (23X)
 

2 (15X)
 

(14)
 

1 (7X)
 

1 (7X)
 

(14)
 

1 (7X)
 

(14)
 

1 (7X)
 

(14)
 

1 (7X)
 

(1«)
 

2 (1UX)
 

(14)
 

64
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

tTESTIS (8) (1U) (14)
 

HEMORRHAGE 1 (7X)
 

INFLAMMATION, NOS 1 (7X)
 

DEGENERATION, NOS 2 (14X) t (29%)
 

ATROPHY, NOS 1 (7»)
 

NERVOUS SYST3M
 

•CEREBELLUM	 (7) (14) (13)
 

HEMORRHAGE 1 (7X)
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL HORPHOLOGY SUMMARY
 

NO LESION REPORTED 3 2
 

ANIHAL MISSING/NO NECROPSY 3
 

NO NECROPSY PERFORMED 1 21 8
 

AUTO/NECROPSY/HISTO PERF 1 2
 

AOTOLYSIS/NO NECROPSY 2 13
 

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

65
 
TABLE 02.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE 

MICE FED PYRIMETHAMINE IN THE DIET 

MATCHED
 

CONTROL
 

15
 

12
 

12
 

 (10)
 

 (12)
 

5 (<*2X)
 

 (10)
 

1 (10*)
 

 (8)
 

 (12)
 

1 (8.XJ
 

LOW DOSE
 

35
 

27
 

27
 

(26)
 

5 (19«)
 

2 (8X)
 

(25)
 

1 (4X)
 

1 (4X)
 

11	 (44X)
 

2 (8X)
 

(25)
 

6 (24X)
 

(23)
 

4 (17%)
 

(26)
 

HIGH DOSE
 

35
 

22
 

22
 

(22)
 

(21)
 

1 (5X)
 

7 (33X)
 

(22)
 

1 (5X)
 

1 (5X)
 

3 (14X)
 

4 (18X)
 

(18)
 

(22)
 

1 (5X)
 

ANIHALS
 

ANIMALS
 

ANIMALS
 

INITIALLY
 

NECROPSIED
 

EXAMINED H
 

IN STUDY
 

ISTO PATHOLOGICALLY
 

INTEGOHENTAHY SYS1EH
 

NONE
 

RESPIRATORY SYSTEM
 

•TRACHEA


INFLAMMATION, SUPPURATIVE
 

INFLAMMATION, CHRONIC SUPPOHATIV
 

•LUNG


PNEUMONIA, ASPIRATION
 

INFLAMMATION, SUPPURATIVE
 

BRONCHOPNEUMONIA SUPPURATIVE


HYPERPLASIA, LYMPHOID
 

HEHATOPOIETIC SYSTEM
 

tSPLEEN


LYHPHOID DEPLETION
 

HIPERPLASIA, RETICULUB CELL
 

HYPERPLASIA, LYMPHOID
 

HEHATOPOIESIS


tTHYHUS


ATROPHY, NOS
 

CIRCULATORY SYSTEM
 

MYOCARDIUM


INFLAMMATION, NOS
 

IHF1AMAIIOB, INTERSTITIAL


t NUMBER OF ANIHALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIHALS NECEOPSIED
 

66
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

INFLAMMATION, SUPPUBATIVE 

INFLAMMATION, CHRONIC 

INFLAMMATION, CHRONIC FOCAL <8X) 

1 (<*X) 

DIGESTIVE SYSTEM
 

•LIVEB	 (9) (25) 

NECROSIS, FOCAL 2 (22X) 

NECBOSIS, COAGULATIVE 1 (11X) 

CYTOPLASMIC VACUOLIZATION 

HYPEBPLASIA, NODULAB 

iLIVEB/HEPATOCYTES (9) (25) 

DEGENERATION, GRANULAR 

•PANCBEAS	 (10) (24) 

INFLAMMATION, CHRONIC
 

•PEYERS PATCH	 (10) (24) 

HYPERPLASIA, LYMPHOID 

UBINABY SYST3M
 

•KIDNEY	 (10) (27)
 

PEBIABTEEITIS
 

NECBOSIS, ISCHSMIC 1 (4X)
 

HYPE8PLASIA, LYMPHOID
 

•KIDNEY/PELVIS	 (10) (27)
 

LYHPHOCYTIC INFLAMMATOBY INFILTR
 

•UBINABY BLADDER (8) (23) 

HYPEHPLA3IA, LYHPHOID 

ENDOCRINE SYSTEM
 

•THYBOID	 (10) (25) 

CYSTIC FOLLICLES 2 (8X) 

REPRODUCTIVE SYSTEM
 

•HAMHARY	 GLAND (12) (27) 

DILATATION/DUCIS 1 (4%i 

* NOHBER OF ANIMALS WITH TISSUE EIAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE 

4 (18X) 

(22) 

1 (5X)
 

1 (5X)
 

(22)
 

1 (5X)
 

(22)
 

1 (5X)
 

(22) 

(5X)
 

(22) 

2 (9X)
 

(22)
 

1 (5X)
 

(21)
 

1 (5X)
 

(22) 

(22) 

67 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

tOTERUS/ENDOflilTRlTJM

CYST, NO.'!

INFLAMMATION, SUPPUHATIVE

HYPEHPLA3IA, FOCAL

HYPEBPIASIA, CYSTIC

 (10)

 2 (20%)

 1 (10%)

 5 (50%)

 (25)

 1

 7

 (4%) 

 (28%)

 (22) 

1

 1

 14

 (5%) 

 (5%) 

 (64%) 

*OVABY

FOLLICULAK CYST, NOS

INFLAMMATION, SUPPURATIVE

 (10)

 1 (10%) 

 (24)

 3 (13%)

 (21) 

5 (24%) 

MEBVOUS SYSTEM 

*BBAIN

LYMPHOCYTOSIS

 (11) (25)

 1 (4%) 

 (22) 

SPECIAL SENS2 ORGANS 

NONE 

MOSCOLOSKEIETAL SYST-SM 

NONE 

BODY CAVITIES 

*PEBITONEUM

INFLAMMATION, FIBBINOUS

 (12) (27) (22) 

1 (5%) 

ALL OTHER SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION EEP08T£D 

NO NECROPSY PERFORMED 

AUTQ/NECEQPSX/HISTO PEJF 

1 

11 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED HICHOSCOPICALLI 

* NUHBER OF ANIMALS NECBOPSIED 

68 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

AOTOLYSIS/NO NECROPSY 1 4

t NUMBEfl OF ANIMALS WITH TISSUE EXAMINED HICBOSCOPICALLI * NUHEEfi OF ANIMALS NECBOPSIED 

2 

69
 

APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS FED PYRIMETHAMINE IN THE DIET
 

71
 

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

Fed Pyrimethamine in the Diet3
 

Pooled Matched
 

Topography: Morphology Control Control
 

Heraatopoietic System:
 

Lymphoma or Leukemiab

 9/44 (20) 3/15 (20)
 

P Valuesc»d P - O.OIO(N) N.S.
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

to
 

Weeks to First Observed Tumor — 103
 

Pituitary: Chromophobe
 

Adenoma1* 4/32 (13) 2/11 (18)
 

P Valuesc'd N.S. N.S.
 

Departure from Linear Trend6 P - 0.026
 

Relative Risk (Pooled Control) ^
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor — 85
 

Low
 

Dose
 

2/35 (6)
 

N.S.
 

0.279
 

0.031
 

1.237
 

0.286
 

0.027
 

2.289
 

63
 

8/33 (24)
 

N.S.
 

1.939
 

0.582
 

7.964
 

1.333
 

0.339
 

11.794
 

97
 

High
 

Dose
 

1/33 (3)
 

P - 0.024**(N)
 

0.148
 

0.003
 

0.985
 

0.152
 

0.003
 

1.737
 

92
 

1/31 (3)
 

N.S.
 

0.258
 

0.005
 

2.419
 

0.177
 

0.003
 

3.167
 

104
 
__ 

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

Fed Pyrimethamine in the Dieta
 

(continued)
 

Pooled Matched Low High
 

Topography: Morphology Control Control Dose Dose
 

Thyroid: C-cell Carcinomab

 1/42 (2) 0/14 (0) 3/34 (9) 3/32 (9)
 

P Valuesc»d N.S. N.S. N.S. N.S.
 

Relative Risk (Pooled Control)f 3.706 3.938
 

Lower Limit 0.314 0.333
 

Upper Limit 188.485 199.811
 

Relative Risk (Matched Control)f Infinite Infinite
 

Lower Limit 0.265 0.282
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 104
 

Thyroid: Follicular-cell
 

Carcinomab 3/42 (7) 2/14 (14) 1/34 (3) 0/32 (0)
 

P Values0 »d N.S. P - 0.039(N) N.S. N.S.
 

Relative Risk (Pooled Control)f 0.412 0.000
 

Lower Limit 0.008 0.000
 

Upper Limit 4.836 2.149
 

Relative Risk (Matched Control)f 0.206 0.000
 

Lower Limit 0.004 0.000
 

Upper Limit 3.714 1.447
 

Weeks to First Observed Tumor 105 104
 

104 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed Pyrimethamine in the Diet3
 

(continued)
 

Pooled Matched Low High
 

Topography: Morphology Control Control Dose Dose
 

Pancreatic Islets: Islet-cell
 

Carcinomab 0/43 (0) 0/14 (0) 0/35 (0) 2/33 (6)
 

P Valuesc»d N.S. N.S. M.S. N.S.
 

M_
 

Relative Risk (Pooled Control)* Infinite
 

Lower Limit 0.388
 

Upper Limit — Infinite
 

._
 —
 

Relative Risk (Matched Control)* Infinite
 

Lower Limit 0.134
 

Upper Limit — Infinite
 

__ „
 —
 

Weeks to First Observed Tumor 104
 

Pancreatic Islets: Islet-cell
 

Adenoma or Carcinoma^5 2/43 (5) 1/14 (7) 2/35 (6) 3/33 (9)
 

P Valuesc»d N.S. N.S. N.S. N.S.
 

Relative Risk (Pooled Control)* 1.229 1.955
 

Lower Limit 0.093 0.237
 

Upper Limit 16.144 22.183
 

Relative Risk (Matched Control)* 0.800 1.273
 

Lower Limit 0.047 0.117
 

Upper Limit 45.853 64.698
 

Weeks to First Observed Tumor 75 104 92
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed Pyrimethamine in the Dieta
 

(cont inued) 

Pooled Matched Low High 

Topography: Morphology Control Control Dose Dose 

Testis: Interstitial-cell
 

Tumorb 39/43 (91) 13/14 (93) 30/35 (86) 27/33 (82)
 

P Valuesc»d N.S. N.S. N.S. N.S.
 

Relative Risk (Pooled Control)f 0.945 0.902
 

Lower Limit 0.805 0.760
 

Upper Limit 1.125 1.100
 

Relative Risk (Matched Control)f 0.923 0.881
 

Lower Limit 0.831 0.790
 

Upper Limit 1.259 1.235
 

Weeks to First Observed Tumor — 85 87 104
 

a

Groups fed pyrimethamine received doses of 200 or 400 ppm.
 

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

G

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


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

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

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


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

is indicated.
 

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

e

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

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 Pyrlmetharaine in the Diet3
 

Topography: Morphology

Pooled 

 Control 

Matched

Control

 Low 

 Dose 

Pituitary:

Adenoma^

 Chromophobe 

 9/37 (24) 4/11 (36) 8/34 (24) 

P Values0 » d N.S. N * S • N*S • 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

0.967 

0.367 

2.487 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

0.647 

0.236 

2.524 

Weeks to First Observed Tumor — 98 101 

Thyroid: C-cell Adenomab 2/42 (5) 0/13 (0) 0/35 (0) 

P Valuesc » d N.S. P ­ 0.038 N.S. 

Relative Risk (Pooled Control)* 

Lower Limit 

Upper Limit 

0.000 

0.000 

4.013 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

__ 

— 

— 

Weeks to First Observed Tumor — __ — 

High
 

Dose
 

14/32 (44)
 

N.S.
 

1.799
 

0.843
 

3.978
 

1.203
 

0.520
 

4.177
 

4/34 (12)
 

N.S.
 

2.471
 

0.378
 

25.875
 

Infinite
 

0.383
 

Infinite
 

89 

104 
00 

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

Fed Pyrimethamine in the Diet3
 

(continued)
 

Pooled Matched Low
 

Topography; Morphology Control Control Dose
 

Mammary Gland: Fibroadenomab 10/43 (23) 2/14 (14) 4/35 (11)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Pooled Control)f 0.491
 

Lower Limit 0.122
 

Upper Limit 1.536
 

Relative Risk (Matched Control)f 0.800
 

Lower Limit 0.135
 

Upper Limit 8.252
 

Weeks to First Observed Tumor — 103 102
 

Uterus: Endometrial Stromal
 

Polypb 6/42 (14) 0/14 (0) 3/35 (9)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Risk (Pooled Control)f 0.600
 

Lower Limit 0.103
 

Upper Limit 2.581
 

Relative Risk (Matched Control )f Infinite
 

Lower Limit 0.258
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 105
 

High
 

Dose
 

4/35 (11)
 

N.S.
 

0.491
 

0.122
 

1.536
 

0.800
 

0.135
 

8.252
 

98
 

3/35 (9)
 

N.S.
 

0.600
 

0.103
 

2.581
 

Infinite
 

0.258
 

Infinite
 

104
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Pyrimethamine in the Diet3
 

(continued)
 

a

Groups fed pyrimethamine received doses of 200 or 400 ppm.
 

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

c

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


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

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

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


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

is indicated.
 

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

VO
 e

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

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

control group.
 

APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN FEMALE MICE FED PYRIMETHAMINE IN THE DIET
 

81
 

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

Fed Pyrimethamine in the Diet3
 

Topography Morphology

Pooled 

 Control 

Matched

Control

 Low 

 Dose 

Lung: Alveolar /Bronchiolar 

Adenoma*5 2/38 (5) 0/12 (0) 0/25 (0) 

P Valuesc » d N.S. N.S. N.S. 

Relative Risk (Pooled Control)^ 

Lower Limit 

Upper Limit 

0.000 

0.000 

5.012 

00 

Relative Risk (Matched Control)* 

Lower Limit 

Upper Limit 

— 

— 

— 

Weeks to First Observed Tumor — —— — 

Hematopoietic System: 

Lymphomab 2/38 (5) 0/12 (0) 3/27 (11) 

P Valuesc'd N.S. N.S. N.S. 

Relative Risk (Pooled Control)f 

Lower Limit 

Upper Limit 

2.111 

0.258 

23.658 

Relative Risk (Matched Control)* 

Lower Limit 

Upper Limit 

Infinite 

0.291 

Infinite 

Weeks to First Observed Tumor 80 

High
 

Dose
 

2/21 (10)
 

N.S.
 

1.810
 

0.138
 

23.025
 

Infinite
 

0.183
 

Infinite
 

104
 

4/22 (18)
 

N.S.
 

3.455
 

0.537
 

35.023
 

Infinite
 

0.554
 

Infinite
 

71
 
__ 

--__ 

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

Fed Pyrimethamine in the Diet3
 

(continued)
 

Pooled Matched Low
 

Topography: Morphology Control Control Dose
 

Hematopoietic System:
 

Lymphoma, Hist iocy tic Typeb 2/38 (5) 0/12 (0) 2/27 (7)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Risk (Pooled Control)f 1.407
 

Lower Limit 0.107
 

Upper Limit 18.229
 

Relative Risk (Matched Control )f Infinite
 

oo Lower Limit 0.142
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 80
 

Uterus: Adenocarcinoma, NOSb 0/36 (0) 0/10 (0) 0/25 (0)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Pooled Control)f
 

Lower Limit
 

Upper Limit —
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 —


—
 

Weeks to First Observed Tumor
 — —
 

High
 

Dose
 

4/22 (18)
 

N.S.
 

3.455
 

0.537
 

35.023
 

Infinite
 

0.554
 

Infinite
 

71
 

2/22 (9)
 

N.S.
 

Infinite
 

0.491
 

Infinite
 

Infinite
 

0.149
 

Infinite
 

96
 
Table Fl. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed Pyrimethamine in the Dieta
 

(continued)
 

a

Groups fed pyrimethamine received doses of 500 or 1000 ppm.
 

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

c

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


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

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

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


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

is indicated.
 

°A negative trend (N) indicates a lower incidence in a dosed group than in a control group,
 oo
 

Ul
 e

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

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

control group.
 

Review of the Bioassay of Pyrimethamine* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

January 18, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976 under the authority of the National
 

Cancer Act of 1971 (P.L. 92-218). The purpose of the Clear­ inghouse is to advise on the National Cancer Institute's
 

bioassay program to identify and evaluate chemical carcino­ gens in the environment to which humans may be exposed. The
 

members of the Clearinghouse have been drawn from academia,
 industry, organized labor, public interest groups, State
 

health officials, and quasi-public health and research
 

organizations. Members have been selected on the basis of
 

their experience in carcinogenesis or related fields and,
 collectively, provide expertise in organic chemistry, bio­ statistics, biochemistry, toxicology, pathology, and epide­ miology. Representatives of various Governmental agencies
 

participate as ad hoc members. The Data Evaluation/Risk
 

Assessment Subgroup of the Clearinghouse is charged with
 

the responsibility of providing a peer review of NCI bioassay
 

reports on chemicals studied for carcinogenicity. In this
 

context, below is the edited excerpt from the minutes of
 

the Subgroup's meeting at which Pyrimethamine was reviewed.
 

The primary reviewer noted that the mode of action of
 

Pyrimethamine is similar to cancer chemotherapeutic agents
 

such as methotrexate. Dr. Kensler briefly described the
 

conditions of test and pointed out that the subchronic study
 

was conducted in different strains of rats and mice then
 

used in the chronic phase. He agreed with the staff's con­ clusion that, under the conditions of test, Pyrimethamine
 

was not carcinogenic in rats and that the mouse bioassay
 

was inadequate for evaluation. Based on these results,
 he concluded that P., 'imethamine would not appear to pose
 

a carcinogenic risk to humans.
 

The secondary reviewer opined that survival was
 

sufficient in the female mice to conclude that a carcinogenic
 

effect was not produced by Pyrimethamine. A subgroup member
 

commented that there was an inadequate number of animals in
 

the treated and control groups of both species to draw any
 

conclusion regarding the carcinogenicity of Pyrimethamine.
 

87
 
It was moved that the report be accepted as written.
 

The motion was seconded and approved by all the Subgroup
 

members except Mr. Garfinkel, who opposed it.
 

Members Present Were:
 

Arnold Brown (Acting Chairman), Mayo Clinic
 

Lawrence Garfinkel, American Cancer Society
 

Joseph Highland, Environmental Defense Fund
 

Charles Kensler, Arthur D. Little Company
 

Verald K. Rowe, Dow Chemical, U.S.A.
 Sheldon Samuels, Industrial Union Department, AFL-CIO
 

Louise Strong, University of Texas Health Sciences Center
 

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.
 

<HI.S. GOVERNMENT PRINTING OFFICE: 1978 260-899/3062 1-3 88 

DHEW Publication No. (NIH) 78-1327