1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 51 

BIOASSAY OF 

TOLAZAMIDE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 1156-19-0 

NCI-CG-TR-51 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

TOLAZAMIDE
 

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) 78-1301 

BIOASSAY OF
 

TOLAZAMIDE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

CONTRIBUTORS; This report presents the results of the bioassay
 

of tolazamide conducted for the Carcinogenesis Testing Program,
 

Division of Cancer Cause and Prevention, National Cancer
 

Institute (NCI), Bethesda, Maryland. The bioassay 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.
 

Weisburger^'-^. Ms. J. Belzer-*- and Mr. I. Brown-'- were responsible
 

for the care and feeding of the laboratory animals. Data
 

management and retrieval were performed by Ms. C. A. Dominick-*-.
 

Histopathologic examinations were performed by Drs. J. C.
 

Peckham1, and R. B. Thompson-'-, and the diagnoses included in this
 

report represent their interpretation.
 

Animal pathology tables and survival tables were compiled at 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 analytical results were reviewed by Dr. C. W. Jameson^.
 

This report was prepared at Tracer Jitco^ under the direction of
 

NCI. Those responsible for the report at Tracer 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;
 

iii
 
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 the National Cancer Institute
 

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. Squire8
 

Dr. Jerrold M. Ward
 

^•Southern Research Institute, 2000 Ninth Avenue South,
 

Birmingham, Alabama.
 

^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.
 

4

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

Rockville, Maryland.
 

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

Maryland.
 

iv
 
^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.
 

%ow with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

V 

SUMMARY
 

A bioassay of the hypoglycemic drug tolazamide 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
 

tolazamide at one of two doses, either 5,000 or 10,000 ppm, for
 

103 weeks. Matched controls consisted of 15 rats and 15 mice of
 

each sex. All surviving rats and mice were killed at 104 or 105
 

weeks.
 

Survival rates for the dosed rats of each sex were higher than
 

those for the matched controls, and were adequate for the 

development of late-appearing tumors. Survival rates for the 

mice were lower than those for the rats, particularly for the 

dosed females (matched controls 67%, low-dose 34%, high-dose
 

32%). However, a large number of these deaths in the dosed
 

females occurred after 90 weeks on study, and survival of both
 

males and females was adequate for the development of late-


appearing tumors.
 

All observed tumors were of types commonly found in the strains
 

of animals used, and there were no statistically significant
 

increases in the incidence of tumors in the dosed animals as
 

compared with controls.
 

It is concluded that under the conditions of this bioassay,
 

tolazamide was not carcinogenic for Fischer 344 rats or B6C3F1
 

mice.
 

vii
 

TABLE OF CONTENTS
 

Page
 

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	 9
 

H. Data Recording and Statistical Analyses	 12
 

III. Results - Rats	 19
 

A. Body Weights and Clinical Signs (Rats)	 19
 

B. Survival (Rats)	 19
 

C. Pathology (Rats)	 22
 

D. Statistical Analyses of Results (Rats)	 23
 

IV. Results - Mice	 25
 

A. Body Weights and Clinical Signs (Mice)	 25
 

B. Survival (Mice)	 25
 

C. Pathology (Mice)	 28
 

D. Statistical Analyses of Results (Mice)	 29
 

V. Discussion	 31
 

VI.	 Bibliography 33
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Table Al Summary of the Incidence of Neoplasms in
 

Rats Fed Tolazamide in the Diet 35
 

Male Rats Fed Tolazamide in the Diet 37
 

Table A2 Summary of the Incidence of Neoplasms in
 

Female Rats Fed Tolazamide in the Diet 41
 

ix
 
Page 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Fed Tolazamide in the Diet 45 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Fed Tolazamide in the Diet 47 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Fed Tolazamide in the Diet 50 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Fed Tolazamide in the Diet 53 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Tolazamide 

in the Diet 55 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Tolazamide 

in the Diet 58 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Fed Tolazamide 

in the Diet 61 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed Tolazamide 

in the Diet 63 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed Tolazamide 

in the Diet 66 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Fed Tolazamide in the Diet 69 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Fed Tolazamide in the Diet 71 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Fed Tolazamide 

in the Diet 77 

X 
Page 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Fed Tolazamide in the Diet 85 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Fed Tolazamide in the Diet 87 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Fed Tolazamide in the Diet 90 

TABLES 

Table 1 Design of Tolazamide Chronic Feeding 

Studies in Rats 10 

Table 2 Design of Tolazamide Chronic Feeding 

Studies in Mice 11 

FIGURES 

Figure 1 Growth Curves for Rats Fed Tolazamide 

in the Diet 20 

Figure 2 Survival Curves for Rats Fed Tolazamide 

in the Diet 21 

Figure 3 Growth Curves for Mice Fed Tolazamide 

in the Diet 26 

Figure 4 Survival Curves for Mice Fed Tolazamide 

in the Diet 27 

xi
 

I. INTRODUCTION
 

Tolazamide (CAS 1156-19-0; NCI C03327) is an oral hypoglycemic
 

agent of the arylsulfonylurea type, similar to tolbutamide,
 

chlorpropamide, and acetohexamide. It is approximately five
 

times more potent than tolbutamide in the human diabetic and was
 

developed in an effort to achieve secondary responses in patients
 

who relapsed following initial control by tolbutamide (The Upjohn
 

Co., 1977). The hypoglycemic effects of the arylsulfonylureas
 

are due to their ability to stimulate pancreatic secretion of
 

insulin and are used, therefore, only in patients with at least
 

minimal pancreatic function, as in maturity-onset diabetics
 

(Larner and Haynes, 1975). Controlled studies have shown that
 

the oral hypoglycemics may be no more effective than dietary
 

modifications in controlling the symptoms of maturity-onset
 

diabetes on a long-term basis and may be associated with an
 

increase in cardiovascular mortality (Shen and Bressler, 1977).
 

Tolazamide was selected for testing in the carcinogenesis program
 

in an attempt to evaluate the carcinogenicity of certain drugs
 

that are used extensively and for prolonged periods in humans.
 

1 

II. MATERIALS AND METHODS
 

A. Chemical
 

Tolazajnide (N-(p-toluenesulfonyl)-N'-hexamethylenirainourea) was
 

obtained in two batches (Lot Nos. 555BD and 824BK) from the
 

Upjohn Company, North Haven, Connecticut. Lot No. 555BD was used
 

in the prechronic study; Lot No. 824BK was used in the chronic
 

study. The purity and identity of Lot No. 824BK were established
 

by analyses at Midwest Research Institute. The melting point was
 

171-172°C (literature: 170-173°C). Elemental analyses (C, H, N,
 

S) were correct for C^^j^C^S, the molecular formula of
 

tolazamide. Nuclear magnetic resonance, infrared, and ultraviolet spectra were in agreement with the structure.
 

Lot No. 824BK. was stored in the original cardboard container at
 

5°C; Lot No. 555BK. was stored in the original container at 22°C.
 

B• Dietary Preparation
 

Test diets containing tolazamide were prepared every 2 weeks by
 

mixing a known amount of sifted tolazamide with a small amount of
 

®
 Wayne Lab Blox animal meal (Allied Hills, Inc., Chicago, 111.)
 

in a portable mixer, then adding this mixture to the required
 

amount of animal meal and mixing in a twin-shell blender for 10
 

minutes. The prepared diets were stored at room temperature in
 

sealed plastic containers.
 

3
 
The stability of tolazamide in feed was tested at Midwest
 

Research Institute by determining the concentrations of
 

tolazamide in formulated diets stored at room temperature (25°C)
 

for 2 weeks. The results of these analyses indicated that
 

tolazamide mixed with animal meal is stable for 2 weeks at room
 

temperature.
 

C. Animals
 

For the subchronic study, female Fischer 344 rats were obtained
 

from Harlan Industries, Cumberland, Indiana, and female B6C3F1
 

mice from Charles River Breeding Laboratories, Inc., Wilmington,
 

Massachusetts.
 

In the chronic study, male and female Fischer 344 rats were
 

obtained from Charles River Breeding Laboratories and male and
 

female B6C3F1 mice from A. R. Schmidt, Madison, Wisconsin,
 

through a contract with the Division of Cancer Treatment,
 

National Cancer Institute. On arrival at the laboratory, the
 

animals were 30 days of age. All animals were quarantined (rats
 

for 24 days, mice for 26 days). Animals with no visible signs of
 

disease were assigned to control or treated groups, and then
 

earmarked for individual identification.
 

4 
D. Animal Maintenance
 

All 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 room 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 and mice were housed five per cage in solid-bottom stainless
 

steel cages (Hahn Roofing and Sheet Metal Co., Birmingham, Ala.).
 

(R)
 The bottoms of the rat cages were lined with Iso-Dri hardwood
 

chips (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 second
 

year of the study. Bedding was replaced once per week; cages,
 

water bottles, and feeders were sanitized at 80°C once per week
 

for the first year of the study and twice per week for the second
 

year; and racks were cleaned once per week.
 

The rats and mice were housed in separate rooms. Control animals
 

5 
were housed with respective treated animals. Animals treated
 

with tolazamide were maintained in the same rooms as animals of
 

the same species being treated with 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)
 

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

(pyrimethamine) (CAS 58-14-0)
 

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)
 

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)
 

reserpine (CAS 50-55-66)
 

MICE
 

Feed Studies
 

4-acetyl-N-((cyclohexyland, no)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)
 

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

(pyrimethamine) (CAS 58-14-0)
 

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)
 

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

6 
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)
 

reserpine (CAS 50-55-6)
 

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-1,3,2-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)
 

adriamycin (CAS 23214-92-8)
 

E. Subchronic Studies
 

Subchronic studies were conducted to estimate the maximum
 

tolerated doses of tolazamide, on the basis of which low and high
 

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

7 
doses") were determined for use in the chronic study. Tolazamide
 

was administered in the diet at doses of 2,500, 5,000, 10,000,
 

20,000, or 40,000 ppm to female Fischer 344 rats and at doses of
 

2,000, 6,000, 12,000, 25,000, or 50,000 ppm to female B6C3F1
 

mice. Five animals were tested at each concentration, and 10
 

animals were maintained as untreated controls. Treated animals
 

received the test diets 7 days per week for 45 days and were then
 

observed for an additional 45 days.
 

In rats, 2/5 animals treated at 40,000 ppm died by week 3. No
 

deaths occurred in any of the other groups. By the end of the
 

period of treatment, the mean body weight of animals treated at
 

2,500 ppm was 81% of that of the controls, while weights of
 

animals treated at 5,000 and 10,000 ppm were comparable to those
 

of the controls. In animals treated at 20,000 ppm, the mean body
 

weight was 91% of that of the controls, and in animals treated at
 

40,000 ppm, 67% of that of the controls. By the end of the
 

study, weights of treated groups of animals were generally
 

comparable to those of the controls, except at 2,500 ppm, where
 

the final weight was low (81% of that of controls), and at 40,000
 

ppm, where the final weight was 90% of that of the controls. No
 

gross abnormalities were noted at necropsy. The low and high
 

doses were set at 5,000 and 10,000 ppm for the chronic study in
 

both male and female rats.
 

8 
In mice, death occurred during the final week of the study in one
 

animal at 50,000 ppm and in one animal at 25,000 ppm. Mean body
 

weights of the mice were essentially unaffected by the treatment
 

with tolazamide. No gross abnormalities were detected at
 

necropsy. The low and high doses were set at 5,000 and 10,000
 

ppm for the chronic study in both male and female mice.
 

F. Designs of Chronic Studies
 

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

G. Clinical and Pathologic Examinations
 

All animals were observed twice daily for signs of toxicity and
 

animals that were moribund were killed and necropsied. Rats and
 

mice were weighed individually every 2 weeks for about 47 weeks
 

and once monthly for the remainder of the study. 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
 

9 
Table 1. Design of Tolazamide Chronic Feeding Studies in Rats
 

Sex and 

Test 

Initial 

No. of 

Tolazamide 

in Dietb 

Time 

Treated0 

on Study 

Untreated*1 

Group Animals3 (ppm) (weeks) (weeks ) 

Male 

Matched-Control 15 0 105 

Low-Dose 35 5,000 103 2 

High-Dose 35 10,000 103 1 

Female 

Matched-Control 15 0 105 

Low-Dose 35 5,000 103 2 

High-Dose 35 10,000 103 1 

a

All animals were 54 days of age when placed on study.
 

"The treated animals were fed test diets 5 days per week and
 

control diets 2 days per week.
 

c

Test diets were withdrawn for 3 days during week 25 and
 

discontinued for 5 days during week 58.
 

"Observation period following the administration of the chemical.
 

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

Sex and 

Test 

Initial 

No. of 

Tolazamide 

in Dietb 

Time 

Treated0 

on Study 

Untreated11 

Group Animals3 (ppm) (weeks ) (weeks ) 

Male 

Matched-Control 15 0 104-105 

Low-Dose 35 5,000 103 2 

High -Dose 35 10,000 103 1 

Female 

Matched-Control 15 0 105 

Low-Dose 35 5,000 103 2 

High-Dose 35 10,000 103 1 

a

All animals were 56 days of age when placed on study.
 

"The treated animals were fed test diets 5 days per week and
 

control diets 2 days per week.
 

c

Test diets were withdrawn 3 days during week 27 and discontinued
 

entirely during weeks 28 and 29, while animals were being treated
 

with oxytetracycline for respiratory disease. They were also
 

discontinued for 5 days during week 60 following a decrease in
 

mean weights of both treated and control animals.
 

^Observation period following the administration of the chemical.
 

11
 
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. 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 eosin. Special staining techniques
 

were utilized when indicated for more definitive diagnosis.
 

A few 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
 

12
 
pathologic results, as recommended by the International Union
 

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

verification of data transcription and for statistical review.
 

These data were analyzed using the statistical techniques
 

described in this section. Those analyses of the experimental
 

results that bear on the possibility of carcinogenicity are
 

discussed in the statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

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

report in the form of graphs. Animals were statistically
 

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

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

causes were not statistically censored. Statistical analyses for
 

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

(1972) for testing two groups for equality and 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
 

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 treated animals at each
 

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

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

made. The Bonferroni inequality (Miller, 1966) 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
 

14
 
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
 

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
 

15
 
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 treated group compared to its control was calculated
 

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

relative risk is defined as Pt/pc where pj- is the true binomial
 

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

treated 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 treated group and the proportion in a
 

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

excess of unity represent the condition of a larger proportion in
 

the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

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

16
 
ratio of the risk in a treated group of animals to that in a
 

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

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

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

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

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

the absence of a significant result while the upper limit
 

indicates that there is a theoretical possibility of the
 

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

detected under the conditions of this test.
 

17
 

III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of the tolazamide-treated rats were lower than
 

those of the controls (figure 1). Body weights of the females
 

were depressed more than those of the males during the second
 

year on study. Fluctuations in the growth curve may have been
 

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

body weight may be subject to variation. No other signs of
 

drug-related toxicity were recorded in the rats. Rales were
 

noted in several animals of both treated and control groups
 

particularly during the second year of the study.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats fed tolazamide in the diet at
 

the doses of this experiment, together with those of the matched
 

controls, are shown in figure 2.
 

In each sex, the Tarone test result for positive dose-related
 

trend in mortality is not significat. In male rats, 31/35 (89%)
 

of the high-dose group, 34/35 (97%) of the low-dose group, and
 

9/15 (60%) of the matched-control group lived to the end of the
 

study. In females, the respective percentages of survival were
 

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

20 
Figure 2. Survival Curves for Rats Fed Tolazamide in the Diet 

21 
28/35 (80%), 28/35 (80%), and 9/15 (60%). A sufficient number
 

of rats of each sex was at risk for 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 both in the matched-control
 

groups and in the treated groups. The neoplasms listed in
 

Appendix A appeared with approximately equal frequency in treated
 

and control rats, or appeared in insignificant numbers. These
 

lesions are not uncommon in this strain of rat independent of any
 

treatment. There was a slight increase in the number of thyroid
 

tumors in the treated male and female rats. Although they
 

occurred mainly in treated animals, the incidence of these
 

neoplasms is within the range of that commonly observed among
 

untreated Fischer 344 rats. The pancreas was examined, and no
 

chemical-related lesions were observed.
 

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

proliferative, and inflammatory changes were also encountered in
 

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

nonneoplastic lesions are commonly seen in aged rats.
 

22
 
In the judgment of the pathologists, tolazamide fed in the diet
 

did not appear to be carcinogenic in Fischer 344 rats under the
 

conditions of this bioassay.
 

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 were observed in
 

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

or more of the groups.
 

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


related trend and of the Fisher exact test for direct comparison
 

of incidences between the matched-control group and each of the
 

treated groups in the positive direction are not significant. In
 

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

the tables, a value of one or of less than one is included,
 

indicating the absence of positive significant results. It
 

should also be noted that each of the intervals (except that for
 

the incidence of fibroadenoma of the mammary gland in high-dose
 

female rats) has an upper limit greater than one, indicating the
 

theoretical possibility of the induction of tumors by tolazamide,
 

which could not be detected under the conditions of this test.
 

Significant results in the negative direction were observed in
 

23
 
the incidence of f ibroadenoma of the mammary gland and in the
 

incidence of lymphoma or leukemia in the hematopoietic system in
 

female rats.
 

24
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of both male and female treated mice, when
 

compared with those of the controls, were markedly depressed
 

starting at approximately week 40 (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
 

other signs of drug-related toxicity were noted in the mice.
 

Since there was evidence of respiratory disease in some animals,
 

oxytetracycline was administered in the drinking water at a dose
 

of 0.6 mg/ml during week 28 and at 0.3 mg/ml during the following
 

week. In an effort to decrease the transmission of bacteria,
 

propylene glycol was vaporized in the animal rooms for about 2
 

months beginning at week 28.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice fed tolazamide in the diet at
 

the doses of this experiment, together with those of the matched
 

controls, are shown in figure 4.
 

In each sex, the Tarone test result for positive dose-related
 

trend in mortality is not significant. In male mice, 25/35 (71%)
 

25
 
Figure 3. Growth Curves for Mice Fed Tolazamide in the Diet 

26 
Figure 4. Survival Curves for Mice Fed Tolazamide in the Diet 
of the high-dose group, 22/35 (63%) of the low-dose group, and
 

9/15 (60%) of the matched-control group lived to the end of the
 

study, providing an adequate number of male mice at risk for
 

development of late-appearing tumors. In females, although only
 

11/34 (32%) of the high-dose group and 12/35 (34%) of the
 

low-dose group survived to termination of the study, all treated
 

females lived beyond week 52 on study, and no tumor was found
 

before this time. The percentage of survival in the matched-


control group was 10/15 (67%).
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

are summarized in Appendix D, tables Dl and D2.
 

A variety of neoplasms occurred both in matched-control groups
 

and in treated groups. As shown in Appendix B, some types of
 

neoplasms occurred only, or with a greater frequency, in mice of
 

treated groups compared with controls. These lesions, however,
 

are not uncommon in this strain of mouse independent of any
 

treatment. The pancreas was examined, and no chemical-related
 

lesions were observed.
 

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

proliferative, and inflammatory changes were also encountered in
 

28
 
animals of the treated and control groups (Appendix D). Most of
 

these nonneoplastic lesions are commonly seen in aged mice. The
 

suppurative lesions involving the middle ear (otitis media) were
 

associated with increased deaths or decreased life spans in the
 

treated groups of female mice (matched controls 3/15 [20%],
 

low-dose 11/33 [33%], high-dose 19/34 [56%]).
 

In the judgment of the pathologists, tolazamide fed in the diet
 

did not appear to induce tumors in B6C3F1 mice under the
 

conditions of this bioassay.
 

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 were observed in
 

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

or more of the groups.
 

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


related trend and of the Fisher exact test for direct comparison
 

of incidences between the matched-control group and each of the
 

treated groups in the positive direction are not significant. In
 

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

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

indicating the absence of positive significant results. It
 

should also be noted that each of the intervals (except those for
 

29
 
the incidence of lymphoma in low-dose female mice and in high-


dose male mice) has an upper limit greater than one, indicating
 

the theoretical possibility of the induction of tumors by
 

tolazamide, which could not be detected under the conditions of
 

this test. In mice, a significant result in the negative
 

direction occurs in the incidence of lymphoma, which was observed
 

in 4/14 (29%) of the male control animals and 6/15 (40%) of the
 

female control animals.
 

30
 
V. DISCUSSION
 

The dietary administration of tolazamide resulted in depression
 

of the mean body weights of both rats and mice. No other
 

clinical signs related to administration of the chemical were
 

observed.
 

Survival rates for the dosed rats of both sexes were higher than
 

those for the matched controls, and were adequate for the
 

development of late-appearing tumors. Survival rates for the
 

mice were lower than those for the rats, particularly for the
 

dosed females (matched controls 67%, low-dose 34%, high-dose
 

32%). However, a large number of these deaths in the dosed
 

females occurred after 90 weeks on study, and survival of both
 

males and females was adequate for the development of late-


appearing tumors.
 

In rats, the numbers of C-cell adenomas and carcinomas of the
 

thyroid were slightly higher in the dosed male rats than in the
 

matched controls (matched controls 1/15 [7%], low-dose 5/34
 

[15%], high-dose 6/35 [17%]). This incidence of tumors, however,
 

was not statistically significant.
 

All observed tumors were of types commonly found in the strains
 

of animals used and there were no statistically significant
 

31
 
increases in the incidence of tumors in the dosed animals as
 

compared with controls.
 

Tolazamide is an oral hypoglycemic agent of the sulfonylurea type
 

in common use in the United States. Long-term feeding studies
 

conducted at the Upjohn Company in rats and dogs have been
 

reported by Beckett and Donovan (1965). Rats administered
 

tolazamide orally at 25 to 100 mg/kg for 1 year developed
 

pancreatic islet cell hyperplasia, and dogs similarly
 

administered with 12.5 to 50 mg/kg body weight for 6 months
 

developed hypertrophy of the pancreatic islets.
 

It is concluded that under the conditions of this bioassay,
 

tolazamide was not carcinogenic for Fischer 344 rats or B6C3F1
 

mice.
 

32
 
VI. BIBLIOGRAPHY
 

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

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

Beckett, A. G. and Donovan, R. J., Tolazamide (Tolanase) - A new
 

sulphonylurea. Brit. J. Clin. Pract. 19(5):275-279, 1965.
 

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

Panel on Carcinogenicity of the Cancer Research Commission
 

of the UICC, Vol. 2_, International Union Against Cancer,
 

Geneva, 1969.
 

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

Soc. B 34(2):187-220, 1972, pp. 48-52.
 

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

1970.
 

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.
 

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

incomplete observations. J. Amer. Statist. Assoc.
 

.53:457-481, 1958.
 

Larner, J. and Haynes, R. C., Jr., Insulin and oral hypoglycemic
 

drugs; glucagon. In: The Pharmacological Basis of
 

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

Publishing Co., Inc., New York, 1975, pp. 1507 and
 

1520-1523.
 

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

Peters, J. A., 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.
 

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.
 

33
 
Shen, S. W. and Bressler, R., Medical Intelligence: Clinical
 

pharmacology of oral antidiabetic agents (second of two
 

parts). N. Engl. J. Med. 296(14);787-793.
 

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

j>2:679-682, 1975.
 

l*R)
 The Upjohn Company, Tolanase^ tablets. Physicians' Desk
 

Reference, Medical Economics Co., Oradell, N. J. , 1977, pp.
 

1652-1654.
 

34
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS FED TOLAZAMIDE IN THE DIET
 

35
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE 

RATS FED TOLAZAMIDE IN THE DIET 

CONTROL LOW DOSE
 

ANIMALS INITIALLY IN STUDY 15 35
 

ANIMALS NECBOPSIED 15 35
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 15 35
 

"
 

INTEGUMENTARY SYS1EM
 

*SKIN (15) (35)
 

SQUAMOUS CELL PAPILLOHA
 

*SUECOT TISSUE (15) (35)
 

EASAL-CELL CARCINOMA
 

FIBBOHA
 

LIPCHA 1 (3X)
 

RESPIRATORY SYSTEM
 

*TfiACHEA (15) (35)
 

PAPILLOBA, NOS 1 (3X)
 

tLUNG [15) (35)
 

ALVEOLAR/BRONCHIOLAR ADENOMA
 

ALVEOLAB/BBONCHIOLAB CARCINOMA
 

HEMATOPOIETIC SYSTEM
 

*NERVOUS SYSTEM (15) (35)
 

MALIGNANT LYMPHOMA, MIXED TYPE
 

*HULTIPLE ORGANS (15) (35)
 

BALIG.LYHPHOHA, HISTIOCYTIC TYPE
 

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

*HANDIBULAR L. NODE (15) (35)
 

SQUAMOUS CELL CARCINOMA, METASTA 1 <7X)
 

tCERTICAL LYMPH NODE (15) (35)
 

C-CELL CARCINOMA, BET ASTATIC 1 (3X)
 

CIRCULATORY SYSTEM
 

NONE
 

* N0HBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* BOMBER OF ANIMALS NECROPSIED
 

37
 

HIGH DOSE
 

35
 

35
 

35
 

(35)
 

1 (3X)
 

(35)
 

1 (3X)
 

1 (3%)
 

(35)
 

(35)
 

1 (3X)
 

1 (3X)
 

(35)
 

1 (3X)
 

(35)
 

1 (3X)
 

3 (9X>
 

(35)
 

(35)
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

EIGES1IVE SYSTEM
 

#PAflCREAS
 

SARCOMA, NOS
 

#STOMACH
 

SQUAMOOS CELL PAPILLOMA
 

#SMALL INTESTINE
 

SARCOMA, NOS
 

*ANUS
 

FIBROSAfiCOMA
 

URINARY SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

#PITUITARY
 

CHROMOPHOBE ADENOMA
 

#ADRENAL
 

PHEOCHROHOCYTOHA
 

PHEOCHROMOCYTOMA, MALIGNANT
 

#THYEOID
 

FOLLICULAR-CELL ADENOMA
 

FCLLICULAR-CELL CARCINOMA
 

C-CEtL ADENOMA
 

C-CELL CARCINOMA
 

•PANCREATIC	 ISLETS
 

ISLET-CELL ADENOMA
 

ISLET-CELL CARCINOHA
 

REPRODUCTIVE SYSTEM
 

*HAHMARY GLAND
 

FIBBQADENOM.A
 

CONTROL LOW DOSE
 

(15)	 (35)
 

1 (3%)
 

(15)	 (34)
 

(15)	 (35)
 

(15)	 (35)
 

1 (755)
 

(15)	 (33)
 

1 (7*) 6 (18X)
 

(15)	 (35)
 

1 (7X) 2 (6X)
 

(15)	 (34)
 

2 (6*)
 

2 (6X)
 

3 (9X)
 

1 (1%) 2 (6X)
 

(15)	 (35)
 

1	 (7X) 1 OX)
 

1 (3X)
 

(15)	 (35)
 

l_im__.
 

HIGH DOSE
 

(35)
 

(35)
 

1 (3%)
 

(35)
 

1 (3%)
 

(35)
 

(30)
 

4 (13X)
 

(35)
 

2 (6X)
 

1 (3X)
 

(35)
 

1 (3X)
 

3 (9X)
 

3 (9X)
 

(35)
 

1 (3X)
 

(35)
 

*	 NUMBER OF ANIHALS »ITH TISSUE EXAMINED MICBOSCOPICAILI
 

*	 NUMBER OF ANIMALS NECROPSIED
 

38
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE
 

flESHS (15) (35)
 

INTERSTITIAL-CELL TUHOR 13 (87X) 32 (91JS)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EAR CANAL (15) (35)
 

SQUAHOUS CELL CARCINOMA 1 (7%)
 

MUSCULOSKEIETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

*PERITONEUM (15) (35)
 

WESOTHELIOMA, NOS 1 (7%)
 

*MhSENTERY (15) (35)
 

SARCCMA, NOS 1 (7%)
 

LIPOMA 1 (3%)
 

ALL OTH£R SYSTEMS
 

•MULTIPLE	 ORGANS (15) (35)
 

SARCOMA, NOS 1(1%)
 

MESOTHELIOMA. NOS 1 (7%)
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 15 35
 

NATURAL DEATHS 1
 

MORIBUND SACRIFICE 6
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 9 34
 

ANIMAL MISSING
 

3 INCLUDES AUTOLYZED ANIMALS
 

f NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

39
 

HIGH DOSE
 

(35)
 

31 (89X)
 

(35)
 

1 (3«)
 

(35)
 

(35)
 

(35)
 

35
 

1
 

3
 

31
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

TUMOR SOHMABY
 

TOTAL ANIMALS WITH PRIMARY TDMORS* 14 35 35
 

TOTAL PRIMARY TUMORS 26 56 59
 

TOTAL ANIMALS WITH BENIGN TUMORS 13 35 33
 

TOTAL BENIGN TUMORS 17 49 45
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 5 6 10
 

TCTAL MALIGNANT TUMORS 7 7 14
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1 1
 

TOTAL SECONDARY TUMORS 1 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OF MALIGNANT 2
 

TOTAL UNCERTAIN TUMORS 2
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN­ PEIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

40
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE 

RATS FED TOLAZAMIDE IN THE DIET 

HIGH DOSE
 

35
 

35
 

35
 

(35)
 

(35)
 

(35)
 

2 (6»)
 

(34)
 

(29)
 

12 J41X1
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INT1GUMENTARY SYSTEM
 

*SKIN
 

SQUAMOUS CELL PAPILLOMA
 

RESEIEATORY SYSTEM
 

#LUNG
 

ALVEOLAR/BRONCHIOLAR ADJSNO.MA
 

ALVEOLAE/BRONCHIOLAR CARCINOMA
 

HEMATOPOIEllC SYSTEM
 

*MUITIPLE ORGANS
 

UNEIFFERENTIATED LEUKEMIA
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVER
 

HEPATOC£LLULAR ADENOMA
 

URINAR Y SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

#PITUITARY
 

CHROHOPHOBE ADtNOMA
 

CONTROL LOW DOSE
 

15 35
 

15 33
 

15 33
 

(15)	 (33)
 

1 (7*)
 

(15) (33)
 

1 (1%) 1(3%)
 

2 (6%)
 

(15)	 (33)
 

4 (21%) 3 (98)
 

(15)	 (33)
 

1 (3»)
 

(11)	 (31)
 

8 157J1. 13 1U2%1_
 

#	 NUMBER OP ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMEER OF ANIMALS NECROPSIED
 

41
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

HIGH DOSE
 

(35)
 

1 (3X)
 

(35)
 

1 (3%)
 

2 (6«)
 

2 (6%)
 

(35)
 

1 (3%)
 

(35)
 

1 (3S)
 

(35)
 

(3<*)
 

2 (6%)
 

2 (6«)
 

(34)
 

(34)
 

(35)
 

1 (3%)
 

(35)
 

1 J3S1
 

*AER£NAL
 

PHEOCHROMOCYTOMA
 

ITHYROID
 

FOLIICULAR-CELL ADENOMA
 

FOLLICULAR-CELL CARCINOMA
 

C-CELL ADENOMA
 

C-CELL CARCINOMA
 

tPANCREATIC ISLETS
 

ISL£T-CELL ADENOMA
 

REPRODUCTIVE SYSTEM
 

*MAKHAR Y GLAND
 

FIBROADSNCMA
 

*PBEFUTIAL GLAND
 

ACNEXAL ADENOMA
 

(tUTERUS
 

ENDOMETRIAL STROMAL POLYP
 

ENDOMETRIAL STROMAL SARCOMA
 

f U1ERUS/ENDOMETRIUH
 

ADENOCARCINOMA, NOS
 

PAPILLARY ADENOMA
 

#OVARY
 

ADENOCARCINOMA, NOS
 

IIPOMA
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SE.NS2 ORGANS
 

*HARDERIAN GLAND
 

ACENCMA, NOS
 

*2AR CANAL
 

S.QUAMOOS CELL CARCINOMA
 

CONTROL
 

(15)
 

(15)
 

1 (7%)
 

1 <7«)
 

(15)
 

(15)
 

4 (21%)
 

(15)
 

(15)
 

2 (13%)
 

(15)
 

1 (7%)
 

(15)
 

(15)
 

(15)
 

LOW DOSE
 

(33)
 

1 (3X)
 

(33)
 

3 (9X)
 

2 (6X)
 

(33)
 

(33)
 

U (12X)
 

(33)
 

1 (3X)
 

(33)
 

1 (3«)
 

2 {(>%)
 

(33)
 

1 (3»)
 

(33)
 

1 (3»)
 

1 (3%)
 

(33)
 

(33)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUME.ER OF ANIMALS NECROPSI3D
 

42
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NCNE 

ALL CTHEE SYSTEMS 

ADIPOSE TISSUE 

LIPOHA 1 

ANIMAL DISPOSITION SUHMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATHS 

MOBIEUND SACBIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TEBHINAL SACRIFICE 

ANIflAL HISSING 

15 

2 

4 

9 

35 

2 

5 

28 

35 

1 

6 

28 

3 INCLUDES AUTQLYZED ANIHALS __ 

* NUHBEB OF ANIMALS WITH TISSUE EXAMINED

* NUMBER OF ANIMALS NECROPSIED 

 MICROSCOPICALLY 

43
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

TOMOS SUMMAR Y
 

TOTAL ANiaALS HITH PRIMARY TUMORS* 13 23 20
 

TOTAL PRIMARY TUMORS 23 37 29
 

TOTAL ANIMALS WITH BENIGN TUMORS 10 17 17
 

TOTAL EENIGN TUMORS 16 25 22
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 7 10 7
 

TOTAL MALIGNANT TUMORS 7 12 7
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS HITH TUMOfiS UNCERTAINEENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS HITH TUflORS UNCEHTAINPEIMARY CR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PEIMABY TUBOES: ALL TUMORS EXCEPT SECONDARY TUMORS
 

# SECONDARY TUMORS: HETASTATIC TUMORS OR TUMORS INVASIVE IHTO AH ADJACENT ORGAN
 

44
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE FED TOLAZAMIDE IN THE DIET
 

45
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

FED TOLAZAMIDE IN THE DIET 

HIGH DOSE
 

35
 

34
 

34
 

(34)
 

1 (3%)
 

(34)
 

(34)
 

1 (38)
 

(34)
 

(34)
 

3 (9%)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS KECHOPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYS1EM
 

NONE
 

RESPIRATORY SYSTEM
 

#LUNG
 

ALVEOLAR/ERONCHIOLAR ADENOMA
 

AIVEOLAH/BRONCHIOLAR CARCINOMA
 

HEMATOPOIETIC SYSTEM
 

*NERVOUS SYSTEM
 

MALIG.LYMPHOHA, HISTIOCYTIC TYPE
 

^MULTIPLE ORGANS
 

MAIIG.LYMPHOMA, HISTIOCYTIC TYPE
 

MALIGNANT LYMPHOMA, MIXED TYPE
 

#PEYERS PATCH
 

MALIGNANT LYMPHOMA, MIXED TYPE
 

CIRCULATORY SYSTEM
 

NO'NE
 

DIGESTIVE SYSTEM
 

#LIVER
 

HEPATOCELLULAR ADENOMA
 

HLPATCCELLULAS CARCINOMA
 

URINAR Y SYSTEM
 

NONE
 

CONTROL LOW DOSE
 

15 35
 

14 35
 

1« 35
 

(14)	 (35)
 

1 (3S)
 

1 [3%]
 

(14)	 (35)
 

2 (14%) 3 (9X)
 

(14) (35)
 

1 (7%) 1 (3%)
 

1 (7*)
 

(14)	 (35)
 

1 (3«
 

(14) (35)
 

3 (21X) 1 (33t)
 

1 (3%)
 

# DUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

47
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

ENDCCBINE SYSTEM
 

#PITUITARY
 

CARCINOMA, NOS
 

CHBOHOEHOBE ADENOMA
 

#THYBOID
 

FOLLICULAR-CELL ADENOMA
 

EEPBODUCTIVE SYSTEM
 

NONE
 

NERVOUS SYSTEM
 

*TRIGEBINAL GANGLION
 

SARCCMA, NOS
 

SPECIAL SEKSE ORGANS
 

*HARDERIAN GLAND
 

ACENCBA, NOS
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NJN E
 

ALL OTHER SYSTEMS
 

NCNE
 

CONTROL
 

(13)
 

d")
 

1 (7%)
 

(14)
 

(14)
 

1 (1%)
 

LOW DOSE
 

(28)
 

1 (4X)
 

(34)
 

(35)
 

1 (3«)
 

(35)
 

2 ((>%)
 

HIGH DOSE
 

(26)
 

1 (4S)
 

(31)
 

1 (3%)
 

(34)
 

(34)
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBE R OF ANIMALS NjSCROPSISD
 

48
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

ANIMAL DISPOSITION SUMMARY
 

ANIHALS INITIALLY. IN STUDY 15 35 35
 

NATUKAL D2ATHO) 3 4 2
 

MORIBUND SACRIFICE 3 9 8
 

SCHBDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 9 22 25
 

ANIMAL MISSING
 

o> INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 8 11 5
 

TOTAL PRIMARY TUMORS 9 13 7
 

TOTAL ANIMALS WITH BENIGN TUMORS 5 5 4
 

TOTAL BENIGN TUMORS 5 5 5
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 4 8 2
 

TOTAL MALIGNANT TOMORS t 8 2
 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUHORS
 

TOTAL ANIMALS HITH TUMORS UNCERTAINEENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIHALS WITH TDMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUHORS
 

* PBINABY 10BOBS: ALL TUMORS EXCEPT SECONDARY TOMOBS
 

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

49
 
TABLE B2. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE 

MICE FED TOLAZAMIDE IN THE DIET 

ANIMALS INITIALLY IK STUDY
 

ANIMALS NECROPSI2D
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGDMENTARY SYSTEM
 

NCNI
 

RESPIRATORY SYSTEM
 

*LUNG
 

ALVEOLAR/BRONCHIOLAR ADENOMA
 

HEMAIOPOIETIC SYSTEM
 

#ERAIN
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

*MULTIPLE ORGANS
 

BALIG.LYMPHOMA, LYHPHOCYTIC TYPE
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

MALIGNANT LYMPHOHA, MIXfD TYPE
 

•SPLEEN
 

HfMANGIOMA
 

#OVAEY
 

MALIG.LYMPHOflA, HISTIOCYTIC TYPE
 

CIRCUIATOBY SYSTEH
 

NON£
 

DIGESTIVE SYSTEM
 

#1.1 VER
 

HEPA20CEUOLAR ABEHOBA
 

CONTROL LOW DOSE HIGH DOSE
 

15 35 335
 

15 33 34
 

15 33 3U
 

(15)	 (32) (34)
 

1 (7») 1 (3X)
 

(14)	 (32) (33)
 

1 (3X)
 

(15) (33) (34)
 

2 (6%}
 

4 (21%) 1 (3S)
 

1 (7X) 2 (6%)
 

(15)	 (3D (34)
 

1 (3X)
 

(15) (33) (34)
 

1 n»)
 

(15) (33) (34)
 

1 nsi 1	 (3*J
 

# NUMBER OP ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIHALS NECROPSIED
 

a 35 ANIMALS HERE INITIALLY IN THE STUDY, BUT ONE ANIMAL WAS POUND TO BE A MALE
 

IN A FEMALE GROUP.
 

50
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

HIGH DOSE
 

(26)
 

(32)
 

(34)
 

(34)
 

(34)
 

(34)
 

2 (6%)
 

HiPiTOCELLULAR CARCINOMA
 

DRINABY SYSTEM
 

NONE
 

ENDCCHINZ SYSTEM
 

#PITUITARY
 

CHROMOPHOBS ADENOMA
 

4THYROID
 

FOLLICULAR-CELL CARCINOMA
 

tPANCREATIC ISLETS
 

ISLfT-CELL CARCINOMA
 

REPRODUCTIVE SYSTEM
 

*VSGINA
 

SQUAMOUS CELL CARCINOMA
 

#UTERUS
 

£NDOMETRIAL STROHAL POLYP
 

NERVOUS SYSTEM
 

NCNE
 

SPECIAL SENSE ORGANS
 

*HARDERIAN GLAND
 

ACENCHA, NOS
 

HUSCULOSKBIETAL SYSTEM
 

H3NE
 

BODY CAVITIES
 

NONE
 

CONTROL
 

(13)
 

1 (8%)
 

(14)
 

1 (1%)
 

(15)
 

(15)
 

(15)
 

(15)
 

1 (7%)
 

LOW DOSE
 

1 OX)
 

(27)
 

(31)
 

(33)
 

1 (3*>
 

(33)
 

1 (3X)
 

(33)
 

1 (3X)
 

(33)
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

51
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

Ail CTHEB SYSTEMS
 

NCNE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

1EBMINAL SACRIFICE
 

ANIMAL HISSING
 

ANIMAL DELETED (WRONG SEX)
 

3 INCLUDES AUTOLYZED ANIMALS
 

TUMOB SUMMARY
 

TOTAL ANIMALS WITH PBIMAflY TUMORS*
 

TOTAL EHIBARY TUMORS
 

TOTAL ANIMALS HITE BENIGN TUMORS
 

TOTAL EENIGN TUEORS
 

TCTAL ANIMALS WITH MALIGNANT TUMOKS
 

TOTAL MALIGNANT TUMOES
 

TOTAL ANIMALS WITH SECONDARY TUMO&St*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMALS WITH TUMOHS USCERTAINE£NIGN OP MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTJL ANIMALS WITH TUMORS UNCERTAIN­ PBIKABY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

CONTROL
 

15
 

1
 

U
 

10
 

8
 

11
 

4
 

it
 

7
 

7
 

LOW DOSE
 

35
 

8
 

15
 

12
 

6
 

6
 

1
 

1
 

5
 

5
 

HIGH DOSE
 

35
 

10
 

13
 

11
 

1
 

9
 

q
 

5
 

5
 

4
 

U
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

52
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED TOLAZAMIDE IN THE DIET
 

53
 

TABLE C1. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN 

MALE RATS FED TOLAZAMIDE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 15 35 35
 

ANIMALS NECROPSIED 15 35 35
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 15 35 35
 

INTEGUMENTARY SYSTEM
 

*SKIN (15) (35) (35)
 

EPIDEBMAL INCLUSION CYST 1 (3»)
 

RESPIBATORY SYSTEM
 

fLUNG (15) (35) (35)
 

PNiUMONIA, CHRONIC MURINE 3 (9«) 2 (6X)
 

HYPEEPLASIA, ALVEOLAR EPITHELIUM 1 (7%)
 

HEMATOPOIE1IC SYSTEM
 

NONE
 

CIRCOLATORY SYSTEM
 

•MYOCARDIUM	 (15) (35) (35)
 

INFLAMMATION, CHRONIC 4 (21%) 4 (11X) 1 (3X)
 

INFLAMMATION, CHRONIC FOCAL \ (1%)
 

DIGESTIVE SYSTEM
 

tLIVER/CENTBILOBULAR (14) (34) (35)
 

CYTOPLASHIC VACOOLIZATIOM 1 (3X)
 

*BILE DUCT (15) (35) (35)
 

HYPEHPIASIA, NOS 1 (7X)
 

tPANCREAS (15) (35) (35)
 

INILAHMATION. CHRONIC « J11JX 5^i14Sl
 

# NUMBED OF ANIMALS BITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

55
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE 

URINARY SYSTEM
 

•KIDNEY	 (15) (35) (35)
 

INFLAMMATION, DIFFUSE 1 (3X)
 

INFLAMMATION. SUPPURATIV£ 1 (7X)
 

INFLAMMATION, CHRONIC 4 (11%) 3 (9X)
 

INFLAMHATIOS, CHBONIC FOCAL 2 (6X)
 

INFLAMHATIGN, CHRONIC DIFFUSE 11 (73X) 11 (3 IX) 5 (14X)
 

•UKINARY	 BLADDER (14) (33) (32)
 

ULCER. NOS 1 (7X)
 

ENDOCRINE SYSTEM
 

•THYROID	 (15) (34) (35)
 

HYPERPLASIA, C-CELL 2 (6X) 4 (11X)
 

•PARATHYROID	 (14) (29) (29)
 

HYPERPLASIA, NOS 1 (7X)
 

REPRODUCTIVE SYSTEM
 

•PROSTATE	 (13) (34) (33j
 

INFLAHHATION, SOPPORATIVE 1 (8X)
 

NERVOOS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EYE/RETINA (15) (35) (35)
 

ATROPHY, NOS 1 (3X)
 

*MIDDLE EAR (15) (35) (35)
 

IBFLIBHATION. SUPPOHATIVE 1 (3X)
 

MUSCDLOSKEIET1L SYSTEM
 

NONE
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

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

CONTROL LOW DOSE HIGH DOSE 

# NUBEEP OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

57
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE 

RATS FED TOLAZAMIDE IN THE DIET 

HIGH DOSE
 

35
 

35
 

35
 

(35)
 

1 (3%)
 

(35)
 

(30)
 

2 (6%)
 

(35)
 

3 (9%)
 

(35)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTEM
 

NCSE
 

RESPIRATORY SYSTEM
 

*LUNG/ERONCHOS
 

HYPERPIASIA, LYMPHOID
 

HEHATOPOIETIC SYSTHM
 

#SPLEEN
 

HYPERPLA5IA, RETICULUM CELL
 

HEMATOPOIESIS
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVER
 

NECROSIS, COAGULATIVfc
 CYTOFLASMIC V ACUOLIZ ATION
 

f PANCREAS
 

INFLAMMATION, CHRONIC
 

OEINARY SYSTEM
 

#KIDNEY
 

INFLAMMAliOIi_CHRQNIC_2IHUS£
 

CONTROL LOW DOSE
 

15 35
 

15 33
 

15 33
 

(15) (33)
 

(15)	 (33)
 

1 (7%)
 

2 (13%)
 

(15)	 (33)
 

1 (7%)
 

(15)	 (33)
 

1 <7S) 2 (6%)
 

(15)	 (33)
 

1_1Z«L__„
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICBOSCOPICALLY
 

* NUME2R OF ANIMALS NECROPSIF.D
 

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

CONTROL LOW DOSE
 

#UFINARY BLADDER (15) (33)
 

HYPERPLASIA, EPITHELIAL 1 <7X)
 

ENDOCRINE SYSTEM
 

#PITUITARY (14) (31)
 

HYPERPLASIA, CHROMOPHOEE-CE1L
 

ITHYROID (15) (33)
 

HYPERPLASIA, C-CELL 1 (3%)
 

REPRODUCTIVE SYSTEM
 

#UlERUS/ENDOMETflIUM (15) (33)


INFLAMMATION, SUPPURATIVE 2 (13%) 2 (6%;
 

HYPBRPLASIA, EPITHELIAL 1 (3*)
 

HYPERPLASIA, CYSTIC 2 (13X) 5 (15%)
 

#OVARY/OVIDUCT (15) (33)
 

INFLAMMATION, SUPPURATIVE 1 (3%)
 

JOVARY (15) (33)
 

INFLAKHATION, SUPPURATIVE 1 (3%)
 

INFLAKMATION, CHRONIC SOPPURATIV
 

KERVOUS SYSTEM
 

NONE
 

SPECIAL SINSE ORGANS
 

*EYE/RETINA (15) (33)
 

AlfiOPHY, NOS
 

*HIDDLE EAR (15) (33)
 

INFIAHMATION, SUPPORATIVE
 

BUSCULOSKEIETAL SYSTEM
 

NONE
 

* NOMBEE OF ANIMALS BITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUflEER OF ANIMALS NECROPSIED
 

59
 

HIGH DOSE
 

(35)
 

(29)
 

1 (3%)
 

(35)
 

1 (3%)
 

(34)
 

2 (6%)
 

3 (9%)
 

(34)
 

(34)
 

1 (3%)
 

1 (3%)
 

(35)
 

1 (3%)
 

(35)
 

1 (3%)
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

BODY CAVITIES
 

NONE
 

ALL C1HEE SYSTEMS
 

*NUITIPLE OBGANS (15) (33) (35)
 

HYPERPLASIA, RETICULUM CELL 1 (3«
 

SPECIAL HOBPHOLOGY SUHHARY
 

NC LESION BEEOBTID 6 12
 

ADTOLYSIS/SO NECROPSY 2
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINEE HICBOSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

60
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE FED TOLAZAMIDE IN THE DIET
 

61
 

TAILE 01. 

SUMMARY OF THE INCIDENCE OF NONNEOfLASTIC LESIONS IN MALE MICE 

FED TOLAZAIMDE IN THE DIET 

HHGflDOSE
 

35
 

34
 

34
 

(34)
 

(34)
 

2 (6%)
 

(34)
 

4 (12%)
 

2 (6X3
 

(34)
 

1 (3%)
 

(34)
 

(34)
 

ANIHALS INITIALLY	 IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HISTO PATHOLOGICALLY
 

INTEGUHENTARY SYSTEM
 

*SKIN
 

INFLAMMATION, CHRONIC NECHOTIZIN
 

RESEIFATORY SYSTEM
 

tTRACHEA
 

INFLABMATION, CHRONIC SUPPORATIV
 

#LUNG
 

EfiONCHOPNEUaONIA 3UPPURATIVE
 

ERCNCHOfNEOaONIA CHBONIC SUPPURA
 

HYPERPLASIA. ALVEOLAR 2PITHELIUR
 

HEBATOPOIETIC SYSTEM
 

fMESENTERIC L. NODE
 

HYPERPLASIA, LYRPHOID
 

CIRCOIATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

tLIVER
 

NECROSIS, COAGULATIViJ
 

HYPEBPLASIA, FOCAL
 

URINARY SYSTEM
 

#KIDNEY
 

INfLABBATION^ CHRONIC
 

CONTROL LOW DOSE
 

15 35
 

ia 35
 

14 35
 

(14)	 (35)
 

1 (7%)
 

(14)	 (35)
 

(14)	 (35)
 

12 (34*; )


1 (1%)
 

1 (7%) 1 (3X)
 

(14)	 (34)
 

(14)	 (35)
 

1	 (7X) 1 (3XJ
 

2 (6%)
 

(14)	 (35)
 

1 .P3L
 

f	 NUMBEB OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUHEER OF ANIMALS NECROPSIED
 

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

INFLAMMATION, CHBONIC FOCAL
 

ENDOCRINE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEM
 

tPROSTATE
 

INFLAMMATION, CHRONIC SUPPORATIV
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EYE/CORNEA
 

INFLAHMATION, CHRONIC SUPPUPATIV
 

*MIEDLE EAR
 

INFLAMMATION, CHRONIC SUPPURATIV
 

HUSCULOSKEIiTAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

NC LISION REPORTED
 

CONTROL LOW DOSE HIGH DOSE
 

1 (3X)
 

(14)	 (35) (34)
 

1 (7*)
 

(14)	 (35) (34)
 

1 (3%)
 

(14)	 (35) (34)
 

4 (29%) 11 (31%) 11 (32%)
 

4 1 1	 17
 

* NUMBER OF ANIHALS HITH TISSUE EXAHIUEE MICROSCOPICALLY
 

* NUMEER OF ANIMALS NECROPSIED
 

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

CONTROL LOW DOSE HIGH DOSE 

SUTOIYSIS/NO NECEOPSY 1 1 

# NUHEER OF ANIMALS KITH TISSUE 2XAMINEE MICROSCOPICALLY * NOMfcER OF ANIHALS NECROPSIED 

65 
TABLE 02. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

FED TOLAZAMIDE IN THE DIET 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 15 35 5)35
 

ANIHAIS NECROPSIED 15 33 3H
 

ANIMALS EXAMINED HISTO PATHOLOGICALLY 15 33 34
 

INTEGUMENTARY SYSI2M
 

*SUECUT TISSUE (15) (33) (34)
 

HiHOEHHAGE 1 (3%)
 

HESEIBATOHY SYSTEM
 

f LUNG (15) (32) (3U) 

INFLAMMATION, INTERSTITIAL 

ERCNCHOPNEUMONIA SUPPUBATIVE 

1

3

 (3X) 

 (9X) 1 (3%) 

BBCNCHCPNEOMCNIA CHRONIC SOPPURA 1 (3X) 

HYPERPLASIA, ALVEOLAR EPITHELIUM 1 (3X) 

HEMATOPOIETIC SYSTEM
 

tPANCREATIC i.NODE (15) (32) (33J
 

NJ-CROSIS, COAGULATIVE 1 (7X)
 

CIRCDIATOBY SYSTEM
 

NONE
 „
 

DIGESTIVE SYSTEH
 

fLIVER (15) (33) (34)
 

NECROSIS, COAGULATIVE 1 (3X)
 

HYPEBP1ASIA, FOCAL 1 (7X)
 

tPANCREAS (15) (33) (34)
 

CYSTIC DUCTS 1 (7X)
 

•PAHCHEATIC ACINUS (15) (33) (34)
 

ATROPHY. MOS 1 J3X1 _^.
 

* JiUHBEB OF AHIBALS HITH TISSUE EXAMINED MICBOSCOPICALLI
 

* NOHEEB OF AHIHALS NECROPSIED
 

8 35 ANIMALS JIERE INITIALLY IN THE STUDY, BUT ONE ANIHAL BAS POUND TO BE A HALE
 

IN A FBHALE GROUP.
 

66
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE
 

ATBCPHY, DIFFUSE 1 (3X)
 

#PEYERS PATCH (15) (33)
 

HYPERPLASIA, LYtlPHOID 1 (3»)
 

URINARY SYSTEM
 

tKIDNEY (15) (33)
 

HYDRONEPHKOSIS 1 (3%)
 

tUfclNARY ELADDiiR (15) (33)
 

INFLAMMATION, CHRONIC SUPPURATIV
 

ENDOCRINE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEM
 

NONE
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*MID£LE EAR (15) (33)
 

INFLAMMATION, CHRONIC SUPPURATIV 3 (20%) 11 (33X)
 

MDSCULOSKE1ETAL SYSTEM
 

NON£
 

BODY CAVITIES
 

*MESENTERY (15) (33)
 

STEATITIS _ , i_im__ _ 1 jm__
 * NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUHEER OF ANIJJALS NECROPSIED
 

HIGH DOSE
 

(3<0
 

(34)
 

(34)
 

1 (3%)
 

(34)
 

19 (56%)
 

(34)
 

67
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL LOW DOSE HIGH DOSE
 

IECRCSIS, FAT 1 (7*)
 

ALL OTHER SYSTEMS
 

"•MULTIPLE ORGANS (15) (33) (34)
 

HYPEEPLASIA, LYMPHOID 1 (3X) 1 (3%)
 

SPECIAL MORPHOLOGY SUMMARY
 

NC LESION REPORTED 5 1« 12
 

AUTOLYSIS/NO NECROPSY 2
 

# NUMBER OF ANIMALS HITH TISSUE 3XAIJINEC MICROSCOPICALLY
 * NUME£R OF ANIMALS NECROPSIED
 

68
 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN RATS
 

FED TOLAZAMIDE IN THE DIET
 

69
 

__ 

__ 

__ 

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

Topography: Morphology
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma'1
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Leukemia**
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Tolazamide in the Diet3
 

Matched
 

Control
 

0/15 (0)
 

N.S.
 

2/15 (13)
 

N.S.
 

104
 

Low
 

Dose
 

0/35 (0)
 

N.S.
 

—
 

—
 

1/35 (3)
 

N.S.
 

0.214
 

0.004
 

3.876
 

105
 

High
 

Dose
 

2/35 (6)
 

N.S.
 

Infinite
 

0.135
 

Infinite
 

104
 

3/35 (9)
 

N.S.
 

0.643
 

0.085
 

7.208
 

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

(continued)
 

Toposraphy: Morphology
 

Hematopoietic System: Lymphoma or
 

Leukemia*3
 

P Valuesc«d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenoma*'
 

P Values0>d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Tolazaraide in the Diet3
 

Matched Low
 

Control Dose
 

2/15 (13) 1/35 (3)
 

N.S. N.S.
 

0.214
 

0.004
 

3.876
 

104 105
 

1/15 (7) 6/33 (18)
 

N.S. N.S.
 

2.727
 

0.385
 

121.009
 

105 105
 

High
 

Dose
 

5/35 (14)
 

N.S.
 

1.071
 

0.207
 

10.495
 

104
 

4/30 (13)
 

N.S.
 

2.000
 

0.228
 

94.875
 

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

(continued)
 

Topography: Morphology
 

Adrenal: Pheochromocytoma"
 

P Valuesc»d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-cell
 

Carcinoma"
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Tolazamide in the Diet3
 

Matched Low 

Control Dose 

1/15 (7) 2/35 (6) 

N.S. N.S. 

0.857 

0.050 

49.128 

105 85 

0/15 (0)	 2/34 (6)
 

N.S.	 N.S.
 

Infinite
 

0.138
 

Infinite
 

105
 —
 

High
 

Dose
 

3/35 (9)
 

N.S.
 

1.286
 

0.117
 

65.497
 

104
 

1/35 (3)
 

N.S.
 

Infinite
 

0.024
 

Infinite
 

104
 
__ 

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

Fed Tolazamide in the Diet3
 

(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

Thyroid: Folltcular-cell
 

Adenoma or Carcinoma** 0/15 (0) 4/34 (12) 1/35 (3)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f Infinite Infinite
 

Lower Limit 0.437 0.024
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 105 104
 

Thyroid: C-cell Carcinomab 1/15 (7) 2/34 (6) 3/35 (9)
 

d
 P Values0

 » N.S. N.S. N.S.
 

Relative Risk (Matched Control)f 0.882 1.286
 

Lower Limit 0.051 0.118
 

Upper Limit 50.522 64.499
 

Weeks to First Observed Tumor 105 105 104
 
Table Ei. Analyses of the Incidence of Primary Tumors in Hale Rats
 

(continued)
 

Topography: Morphology
 

Thyroid: C-cell Adenoma
 

or Carcinoma"
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pancreatic Islets: Islet-cell
 

Adenoma or Carcinoma**
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Tolazamide in the Dieta
 

Matched Low
 

Control Dose
 

1/15 (7) 5/34 (15)
 

N.S. N.S.
 

2.206
 

0.286
 

100.914
 

105 105
 

1/15 (7) 2/35 (6)
 

N.S. N.S.
 

0.857
 

0.050
 

49.128
 

104 105
 

High
 

Dose
 

6/35 (17)
 

N.S.
 

2.571
 

0.363
 

114.375
 

104
 

1/35 (3)
 

N.S.
 

0.429
 

0.006
 

32.715
 

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

Fed Tolazamide in the Diet3
 

(continued) 

Topography; Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Testis:

Tumorb 

 Interstitial-cell 

13/15 (87) 32/35 (91) 31/35 (89) 

P Values0 » d N.S. N.S. N.S. 

Relative Risk (Matched Control)f 1.055 1.022
 

Lower Limit 0.881 0.852
 

Upper Limit 1.352 1.366
 

Weeks to First Observed Tumor 80 105 92
 

a

Treated groups received doses of 5,000 or 10,000 ppm in feed.
 

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

c

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


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

incidence of tumors in a treated group is the probability level for the Fischer exact test for
 

the comparison of that treated group with the matched-control group when P < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

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 treated group and the
 

control group.
 
__ 

--

_ _ 

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

Fed Tolazamide in the Diet3
 

Topography: Morphology
 

Lung: Alveolar/Bronchiolar
 

Carcinoma
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma
 

P Valuesc'd
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

0/15 (0)
 

N.S.
 

1/15 (7)
 

N.S.
 

105
 

Low
 

Dose
 

2/33 (6)
 

N.S.
 

Infinite
 

0.142
 

Infinite
 

105
 

3/33 (9)
 

N.S.
 

1.364
 

0.124
 

69.321
 

105
 

High
 

Dose
 

0/35 (0)
 

N.S.
 

_.­

.._
 

0/35 (0)
 

N.S.
 

0.000
 

0.000
 

7.949
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Tolazamide in the Diet3
 

(continued)
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Heraatopoietic System: Lymphoma
 

or Leukemia*3 4/15 (27) 3/33 (9) 2/35 (6)
 

P Values0 »d P - 0.040 (N) N.S. N.S.
 

Relative Risk (Matched Control)f 0.341 0.214
 

Lower Limit 0.059 0.022
 

Upper Limit 1.805 1.347
 

Weeks to First Observed Tumor 89 81 73
 

Pituitary: Chroraophobe Adenoma" 8/14 (57) 13/31 (42) 12/29 (41)
 

P Values0 >d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f 0.734 0.724
 

Lower Limit 0.403 0.391
 

Upper Limit 1.615 1.613
 

Weeks to First Observed Tumor 95 78 72
 
__ 

__ 

(continued)
 

Topography: Morphology
 

Thyroid: Follicular-cell
 

Carcinoma"
 

P Values0 »d
 

Departure from Linear Trend6
 

Relative Risk (Matched Control)*
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-cell
 

Adenoma or Carcinoma^
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

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

Fed Tolazamide in the Diet3
 

Matched
 

Control
 

0/15 (0)
 

N.S.
 

P - 0.036
 

0/15 (0)
 

N.S.
 

—
 

Low
 

Dose
 

3/33 (9)
 

N.S.
 

Infinite
 

0.290
 

Infinite
 

105
 

3/33 (9)
 

N.S.
 

Infinite
 

0.290
 

Infinite
 

105
 

High
 

Dose
 

0/35 (0)
 

N.S.
 

—
 

—
 

w«
 

1/35 (3)
 

N.S.
 

Infinite
 

0.024
 

Infinite
 

105 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Tolazamide in the Diet3
 

(continued)
 

Topography: Morphology
 

Thyroid: C-cell Carcinoma'3
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-cell Adenoma
 

or Carcinoma
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

1/15 (7)
 

N.S.
 

105
 

1/15 (7)
 

N.S.
 

105
 

Low High 

Dose Dose 

0/33 (0) 2/35 (6) 

N.S. N.S. 

0.000 0.857 

0.000 0.050 

8.417 49.128 

__ 

104 

2/33 (6) 4/35 (11) 

N.S. N.S. 

0.909 1.714 

0.053 0.196 

51.999 81.832 

105 104 
(continued)
 

Topography: Morphology
 

Mammary Gland: Fibroadenoma'5
 

P Values0 «d
 

Relative Risk (Matched Control)*
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Uterus: Endometrial Stromal
 

Polypb
 

P Values0 >d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

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

Fed Tolazamide in the Diet3
 

Matched
 

Control
 

4/15 (27)
 

P - 0.013 (N)
 

98
 

0/15 (0)
 

N.S.
 

—
 

Low
 

Dose
 

4/33 (12)
 

N.S.
 

0.455
 

0.102
 

2.174
 

95
 

1/33 (3)
 

N.S.
 

Infinite
 

0.025
 

Infinite
 

105
 

High
 

Dose
 

1/35 (3)
 

P - 0.024 (N)
 

0.107
 

0.002
 

0.986
 

105
 

2/34 (6)
 

N.S.
 

Infinite
 

0.138
 

Infinite
 

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

(continued)
 

Topography: Morphology
 

Uterus: End ome trial Stromal
 

Sarcoma0
 

P Value sc»d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Uterus: Endome trial Stromal
 

Polyp or Sarcoma*1
 

P Values0 »d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

a

Treated groups received doses of

Fed Tolazamide in the Dieta 

Matched Low High 

Control Dose Dose 

2/15 (13) 2/33 (6) 2/34 (6) 

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

0.455 0.441 

0.037 0.036 

5.871 5.706 

105 105 105 

2/15 (13) 3/33 (9) 3/34 (9) 

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

0.682 0.662 

0.090 0.087 

7.623 7.410 

105 105 104 

 5,000 or 10,000 ppm in feed. 

^Number of tumor-bearing animals/number of animals examined at site (percent), 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Fed Tolazamide in the Diet3
 

(continued)
 

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


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

incidence of tumors in a treated group is the probability level for the Fischer exact test for
 

the comparison of that treated group with the matched-control group when P < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

e

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

f

The 95% confidence interval of the relative risk between each treated group and the
 

control group.
 

APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN MICE
 

FED TOLAZAMIDE IN THE DIET
 

85
 

__ 

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

Fed Tolazamide in the Diet3
 

Topography: Morphology
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma'3
 

P Values0 »d
 

Relative Risk (Matched Control)*
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Lymphoma"
 

P Valuesc»d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

0/14 (0)
 

N.S.
 

4/14 (29)
 

P - 0.011 (N)
 

81
 

Low
 

Dose
 

2/35 (6)
 

N.S.
 

Infinite
 

0.125
 

Infinite
 

105
 

5/35 (14)
 

N.S.
 

0.500
 

0.134
 

2.235
 

98
 

High
 

Dose
 

1/34 (3)
 

N.S.
 

Infinite
 

0.023
 

Infinite
 

104
 

1/34 (3)
 

P - 0.021 (N)
 

0.103
 

0.002
 

0.942
 

104
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Fed Tolazamide in the Diet3
 

(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

Liver: Hepatocellular
 

Adenoma or Carcinoma"3 3/14 (21) 2/35 (6) 3/34 (9)
 

P Values0 >d N.S. N.S. N.S.
 

Relative Risk (Matched Control)f 0.267 0.412'
 

Lower Limit 0.026 0.065
 

Upper Limit 2.131 2.796
 

Weeks to First Observed Tumor 81 105 71
 

Harderian Gland: Adenoma, NOS
 

(not otherwise specified)" 1/14 (7) 2/35 (6) 0/34 (0)
 

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

Relative Risk (Matched Control)f 0.800 0.000
 

Lower Limit 0.047 0.000
 

Upper Limit 45.853 7.633
 

Weeks to First Observed Tumor 105 92
 —
 

a

Treated groups received doses of 5,000 or 10,000 ppm in feed.
 

"Number of tumor-bearing animals/number of animals examined at site (percent)
 
Table Fl. Analyses of the Incidence of Primary Tumors In Male Mice
 

Fed Tolazamide in the Dieta
 

(continued)
 

c

Beneath the incidence of tumors in the 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 treated group is the probability level for the Fischer exact test for
 

the comparison of that treated group with the matched-control group when P < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

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 treated group and the
 

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

Topography: Morphology
 

Hematopoietic System: Lymphomab
 

P Values0>d
 

Departure from Linear Trend6
 

Relative Risk (Matched Control)

f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Harderian Gland: Adenoma, NOSb
 

P Valuesc»d
 

Relative Risk (Matched Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Fed Tolazamide in the Dieta 

Matched Low High 

Control Dose Dose 

6/15 (40) 2/33 (6) 4/34 (12) 

P = 0.036 (N) P = 0.008 (N) P = 0.033 (N) 

P = 0.016 

0.152 0.294 

0.018 0.077 

0.744 1.076 

72 96 78 

1/15 (7) 0/33 (0) 2/34 (6) 

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

0.000 0.882 

0.000 0.051 

8.417 50.522 

105 95 — a

Treated groups received doses of 5,000 or 10,000 ppm in feed.
 

^Number of tumor-bearing animals/number of animals examined at site (percent)
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed Tolazamide in the Dieta
 

(continued)
 

c

Beneath the incidence of tumors in the 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 treated group is the probability level for the Fischer exact test for
 

the comparison of that treated group with the matched-control group when P < 0.05; otherwise,
 

not significant (N.S.) is indicated.
 

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

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 treated group and the
 

control group.
 

Review of the Bioassay of Tolazamide* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

March 6, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DHEW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be
 

exposed. The members of the Clearinghouse have been drawn
 

from academia, industry, organized labor, public interest
 

groups, State health officials, and quasi-public health and
 

research organizations. Members have been selected on the
 

basis of their experience in carcinogenesis or related fields
 

and, collectively, provide expertise in chemistry, biochemistry,
 biostatistics, toxicology, pathology, and epidemiology.
 Representatives of various Governmental agencies participate
 

as ad hoc members. The Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse is charged with the responsibility of
 

providing a peer review of reports prepared on NCI-sponsored
 

bioassays of chemicals studied for carcinogenicity. It is in
 

this context that the below critique is given on the bioassay
 

of Tolazamide for carcinogenicity.
 

The primary reviewer agreed with the conclusion that,
 

under the conditions of test, Tolazamide was not carcinogenic.
 He said that the pure grade compound was tested and that the
 

study was well-conducted.
 

The secondary reviewer said that the high dose administered in both species achieved the maximum tolerated dose,
 based on an examination of weight loss. He noted that the
 

treated animals survived longer than the associated controls.
 A staff member commented that an analysis of five oral hypo­ glycemic agents tested in bioassays, showed negative trends
 

in mammary adenocarcinomas or endometrial stromal polyps in
 

female rats. He suggested that the negative trends may be
 

due to the weight loss rather than specifically to the action
 

of the agents. Similarly, he suggested that increased
 

longevity may be associated with the weight loss.
 

A Subgroup member said that an older manufacturing
 

process of Tolazamide resulted in its contamination with
 

93
 
nitrosamines. He suggested that it would be worthwhile to
 

determine if the supply used in the bioassay was manufactured
 

under this old process.
 

One Subgroup member commented that the number of surviving
 

animals at the end of the study was too few to allow any
 

conclusion on the bioassay. Another Subgroup member disagreed,
 

stating that there was still a sufficient number on which to
 

evaluate the carcinogenicity of Tolazamide.
 

The primary reviewer moved that the report on the bioassay
 

of Tolazamide be accepted as written. The motion was seconded
 

and approved by all except Mr. Garfinkel, who opposed it.
 

Members present were:
 

Gerald N. Wogan (Chairman), Massachusetts Institute of
 

Technology
 

Arnold Brown, Mayo Clinic
 

Lawrence Garfinkel, American Cancer Society
 

E. Cuyler Hammond, American Cancer Society
 

Joseph Highland, Environmental Defense Fund
 

Henry Pitot, University of Wisconsin Medical Center
 

George Roush, Jr., Monsanto Company
 

Sheldon Samuels, Industrial Union Department, APL-CIO
 

Michael Shimkin, University of California at San Diego
 

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.
 

ft U.S. GOVERNMENT PRINTING OFFICE: 1978 — 260-899/3122 

94 



DHEW Publication No. (NIH) 78-1301