1978 

'- Od~0?? 

National Cancer Institute 

CARCINOGENESIS OFFICIAL NTP
 

Technical Report Series FILE COPY
 

No. 99
 

DO NOT REMOVE FROM THIS OFFICE
 

BIOASSAY OF 

PHENAZOPYRIDINE HYDROCHLORIDE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 136-40-3 

NCI-CG-TR-99 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

PHENAZOPYRIDINE HYDROCHLORIDE
 

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) 78-1349
 

BIOASSAY OF
 

PHENAZOPYRIDINE HYDROCHLORIDE
 

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
 

phenazopyridine hydrochloride 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 chemicals have the
 

capacity to produce cancer in animals. Negative results, in
 

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

than control animals, do not necessarily mean that the test
 

chemical is not a carcinogen, inasmuch as the experiments are
 

conducted under a limited set of circumstances. Positive results
 

demonstrate that the test chemical is carcinogenic for animals
 

under the conditions of the test and indicate that exposure to
 

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

analysis.
 

CONTRIBUTORS; This bioassay of phenazopyridine hydrochloride 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.
 

Griswold*, J. D. Prejean*, E. K. Weisburger^, and J. H.
 

Weisburger^»3. Ms. J. Belzer^ and Mr. I. Brown^ were responsible
 

for the care of the laboratory animals and the administration of
 

the test chemical. Data management and retrieval were performed
 

by Ms. C. A. Dominick*. Histopathologic examinations were
 

performed by Drs. R. B. Thompson^ and J. C. Peckham*, and the
 

diagnoses included in this report represent their interpretation.
 

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

Jameson-*. The chemical structure was supplied by NCI.
 

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

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

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

Deputy Director for Science; Dr. J. F. Robens toxicologist; Dr.
 

R. L. Schueler, pathologist; Dr. G. L. Miller, Ms. L. A. Waltz,
 

and Mr. W. D. Reichardt, bioscience writers; and Dr. E. W.
 

Gunberg, technical editor, assisted by Ms. Y. E. Presley and Ms.
 

P. J. Graboske.
 

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

^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.
 

Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

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

Maryland.
 

iv
 
^Mathematical Statistics and Applied Mathematics Section,
 

Bioiaetry 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.
 

SUMMARY
 

A bioassay of phenazopyridine hydrochloride 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
 

phenazopyridine hydrochloride at one of the following doses,
 

either 3,700 or 7,500 ppm for rats and either 600 ppm or 1,200
 

ppm for mice. The rats were administered the chemical for 78
 

weeks, then observed for 26 or 27 additional weeks; the mice were
 

administered the chemical for 80 weeks, then observed for 25-27
 

additional weeks. Matched controls consisted of 15 untreated
 

rats and 15 untreated mice of each sex. All surviving rats were
 

killed at 104 or 105 weeks, all surviving mice at 105-107 weeks.
 

Mean body weights of the dosed rats and mice of each sex were
 

consistently lower than those of corresponding control animals,
 

and the depressions in mean body weight were dose related.
 

Mortality in the groups of rats and mice did not, however, show
 

dose-related trends, and sufficient numbers of animals of both
 

dosed and control groups were at risk for the development of
 

late-appearing tumors.
 

In male rats, adenomas or adenocarcinomas of the large intestine
 

(colon or rectum) occurred at incidences having a significant
 

dose-related trend (P = 0.015). The direct comparison of the
 

incidences in each of the dosed groups with that in the control
 

group was not significant (controls 0/14, low-dose 4/34,
 

high-dose 8/35). In the females, 3/33 low-dose and 5/32
 

high-dose animals, but no control animals, had this tumor. In
 

addition, sarcomas were observed in the colon of one low-dose
 

male and one high-dose female. The laboratory historical records
 

showed no incidence of adenomas or adenocarcinomas of the large
 

intestine in 260 females and only one adenomatous polyp in 260
 

males. Assuming a spontaneous incidence of 1/261 (0.038%) and a
 

binomial distribution of such tumors, the occurrence seen in the
 

male and female high-dose groups are both significantly (P <
 

0.001) different from the expected value. Thus, these tumors are
 

considered to be related to administration of the test chemical.
 

vii
 
In female mice, the combined incidence of hepatocellular adenomas
 

and carcinomas showed a significant dose-related trend (P =
 

0.002), and the incidence in the high-dose group was significant
 

(P = 0.003) when compared with that in the control group
 

(controls 2/15, low-dose 11/34, high-dose 19/32). The incidence
 

of hepatocellular carcinomas, considered alone, also was
 

significant in the female mice, showing a dose-related trend (P =
 

0.010) and a P value of 0.039 for the comparison of the high-dose
 

group with the control group. In the males, the combined 

incidence of hepatocellular adenomas and carcinomas was not 

significant. 

It is concluded that under the conditions of this bioassay,
 

phenazopyridine hydrochloride was carcinogenic in Fischer 344
 

rats, inducing adenocarcinomas of the colon in both males and
 

females. Although phenazopyridine hydrochloride was not
 

carcinogenic in male B6C3F1 mice, the chemical was carcinogenic
 

in females, inducing hepatocellular adenomas and carcinomas.
 

viil
 
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. 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) 25
 

IV. Results - Mice 27
 

A. Body Weights and Clinical Signs (Mice) 27
 

B. Survival (Mice) 27
 

C. Pathology (Mice) 30
 

D. Statistical Analyses of Results (Mice) 33
 

V. Discussion. 35
 

VI. Bibliography 39
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Fed Phenazopyridine Hydrochloride
 

in the Diet 41
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Fed Phenazopyridine Hydrochloride
 

in the Diet 43
 

ix
 
Table A2 Summary of the Incidence of Neoplasms in 

Female Rats Fed Phenazopyridine Hydrochloride 

in the Diet , 47 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Fed Phenazopyridine Hydrochloride 

in the Diet 51 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Fed Phenazopyridine Hydrochloride 

in the Diet 53 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Fed Phenazopyridine Hydrochloride 

in the Diet 56 

Appendix C Summary of the Incidence of Nonneoplastic Lesions 

in Rats Fed Phenazopyridine Hydrochloride 

in the Diet 59 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Fed Phenazopyridine 

Hydrochloride in the Diet 61 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Phenazopyridine 

Hydrochloride in the Diet 66 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Fed Phenazopyridine 

Hydrochloride in the Diet 71 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Fed Phenazopyridine 

Hydrochloride in the Diet 73 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Fed Phenazopyridine 

Hydrochloride in the Diet 76 

Appendix E Analyses of the Incidence of Primary Tumors 

in Rats Fed Phenazopyride Hydrochloride 

in the Diet 79 
Page 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Fed Phenazopyridine Hydrochloride 

in the Diets 81 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Fed Phenazopyride Hydrochloride 

in the Diet 85 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Fed Phenazopyridine Hydrochloride 

in the Diet 87 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Fed Phenazopyridine Hydrochloride 

in the Diet 89 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Fed Phenazopyridine Hydrochloride 

in the Diet 93 

TABLES 

Table 1 Phenazopyridine Chronic Feeding Studies 

in Rats 10 

Table 2 Phenazopyridine Chronic Feeding Studies 

In Mice 11 

FIGURES 

Figure 1 Growth Curves for Rats Fed Phenazopyridine 

in the Diet 20 

Figure 2 Survival Curves for Rats Fed Phenazopyridine 

in the Diet 21 

Figure 3 Growth Curves for Mice Fed Phenazopyridine 

in the Diet 28 

Figure 4 Survival Curves for Mice Fed Phenazopyridine 

in the Diet 29 

xi
 

I. INTRODUCTION 

= « V
 

PHEIMAZOPYRIDINE (HYDROCHLORIDE)
 

Phenazopyridine hydrochloride (CAS 136-40-3; NCI C01672) is the
 

generic name for an azo dye which has been used for 40 years as
 

an analgesic drug to reduce pain associated with urinary tract
 

infections (American Medical Association, 1973; Billups, 1977;
 

Harvey, 1975). It is marketed both alone and in combination with
 

the sulfonamide urinary tract antiseptics.
 

This chemical was selected for testing because it is in common
 

use and could be used on a prolonged basis in the treatment of
 

persistent urinary tract infections.
 

II. MATERIALS AND METHODS 

A. Chemical
 

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

monohydrochloride, hereinafter referred to as phenazopyridine)
 

was obtained for the chronic studies in a single batch (Lot No.
 

26A-2023-DR4) from the Warner-Lambert Pharmaceutical Company,
 

Morris Plains, New Jersey. The identity and purity of this batch
 

was confirmed in analyses at Midwest Research Institute. The
 

melting point was 244-245°C with decomposition, (literature:
 

235°C with decomposition, National Formulary, 1970). Nonaqueous
 

titration of the amine function indicated 96.5 + 1.4% purity.
 

High-pressure liquid chromatography showed two impurities which
 

totaled ~1%; the impurities were not identified. Elemental
 

C1N5 were
 analyses (C, H, Cl, N) for C^H^2 slightly low for
 

carbon, chlorine, and nitrogen. The infrared and nuclear
 

magnetic resonance spectra were consistent with the structure.
 

The chemical used for the chronic study was stored at 5°C.
 

B. Dietary Preparation
 

Test diets were prepared every 2 weeks by mixing a known amount
 

of the sifted phenazopyridine 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 not
 

less than 10 minutes, to assure homogeneity of the mixture.
 

No analysis of concentration or determination of stability of the
 

chemical in feed was performed. The prepared diets were stored
 

at room temperature in sealed plastic containers.
 

C. Animals
 

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

and male Swiss mice were obtained from Charles River Breeding
 

Laboratories, Inc., Wilmington, Massachusetts. All animals were
 

quarantined for approximately 1 week prior to the start of the
 

studies.
 

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

obtained from Laboratory Supply Company, Indianapolis, Indiana,
 

and male and female B6C3F1 mice from Charles River Breeding
 

Laboratories. These animals were 30 days of age when received.
 

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

for 12 days, mice for 19 days). Animals with no visible signs of
 

disease were then assigned to control or dosed groups and
 

earmarked for individual identification.
 
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%. There were 15 changes of room
 

air per hour. Air was 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 consumed 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.). 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 phenazopyridine were maintained in the same rooms as
 
animals of the same species being administered the following
 

chemicals:
 

RATS
 

Feed Studies
 

(CAS 968-81-0) 4-acetyl-N-((cyclohexylamino)carbonyl)benzenesulfonamide (acetohexamide)
 

(CAS 118-92-3) anthranilic acid
 

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

(CAS 94-20-2) 4-chloro-N-((propylamino)carbonyl)benzenesulfonamide (chlorpropamide)
 

(CAS 58-14-0) 5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine
 

(pyrimethamine)
 

(CAS 73-22-3) L-tryptophan
 

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

(CAS 1156-19-0) N-(p-toluenesulfonyl)-N'-hexamethyleniminourea
 

(tolazamide)
 

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

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

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

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

(CAS 536-33-4) ethionamide
 

MICE
 

Feed Studies
 

(CAS 968-81-0) 4-acetyl-N-((cyclohexylamino)carbonyl)benzenesulfonamide (acetohexamide)
 

(CAS 118-92-3) anthranilic acid
 

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

(CAS 94-20-2) 4-chloro-N-((propylamino)carbonyl)benzenesulfonamide (chlorpropamide)
 

(CAS 58-14-0) 5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine
 

(pyrimethamine)
 

(CAS 73-22-3) L-tryptophan
 

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

(CAS 1156-19-0) N-(p-toluenesulfonyl)-N'-hexamethyleniminourea
 

(tolazamide)
 

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

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

(CAS 80-08-0) 4,4'-sulfonyldianiline (dapsone)
 
(CAS 139-65-1) 4,4'-thiodianiline
 

(CAS 536-33-4) ethionamide
 

Gavage Studies
 

(CAS 3546-10-9) cholesterol (p-(bis(2-chloroethyl)amino)phenyl)acetate (phenesterin)
 

(CAS 22966-79-6) estradiol bis((p-(bis(2-chloroethyl)amino)phenyl)acetate) (estradiol mustard)
 

Intraperitoneal Injection Studies
 

(NSC 141549) 4'-(9-acridinylamino)methansulfon-m-aniside
 

monohydrochloride (MAAM)
 

(CAS 7008-42-6) acronycine
 

(CAS 320-67-2) 5-azacytidine
 

(CAS 789-61-7) beta-2'-deoxy-6-thioguanosine monohydrate
 

(beta-TGdR)
 

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

(CAS 316-42-7) emetine dihydrochloride tetrahydrate
 

(CAS 3458-22-8) 3,3'-iminobis-l-propanol dimethanesulfonate
 

(ester) hydrochloride [IPD]
 

(CAS 21416-87-5) (+)-4,4'-(1-methyl-l,2-ethanediyl)bis-2,6­

piperazinedione (ICRF-159)
 

(CAS 3778-73-2) N,3-bis(2-chloroethyl)tetrahydro-2H-l,3,2­

oxazaphosphorin-2-amine-2-oxide (isophosphamide)
 

(CAS 63-92-3) N-(2-chloroethyl)-N-(l-methyl-2-phenoxyethyl)benzylamine hydrochloride (phenoxybenzamine hydrochloride)
 

CAS 366-70-1) N-(l-methylethyl)-4-((2-methylhydrazino)methyl)benzamide monohydrochloride (procarbazine)
 

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

E. Subchronic Studies
 

Subchronic feeding studies were conducted to estimate the maximum
 

tolerated doses of phenazopyridine, on the basis of which two
 

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

doses") were determined for administration in the chronic
 

studies. In these Subchronic studies, phenazopyridine was added
 

to the animal feed at concentrations of 300, 800, 1,500, 3,000,
 

or 6,000 ppm for the male Sprague-Dawley rats; concentrations of
 
7,500 or 15,000 ppm for the female Sprague-Dawley rats; and
 

concentrations of 500, 1,200, 2,500, 5,000, or 10,000 ppm for the
 

male Swiss mice. Dosed animals received the diets containing the
 

test chemical 7 days per week for 45 days and were then observed
 

for an additional 45 days. Five animals of each species were
 

tested at each dose, and 10 animals of each species were
 

maintained as untreated controls.
 

In rats, the only deaths occurred during weeks 4-6, when 3/5
 

females administered 15,000 ppm died. After 45 days of administration of the chemical, mean body weight gain of male rats was
 

depressed 11% at 300 ppm, 18% at 800 ppm, 33% at 1,500 ppm, 41%
 

at 3,000 ppm, and 57% at 6,000 ppm. In the female rats, mean
 

body weight gain was depressed 54% at 7,500 ppm, and the mean
 

body weight of females administered 15,000 ppm at day 45 fell
 

below the initial value. All groups showed signs of recovery
 

during the observation period and had final weight gains
 

comparable to those of the controls. No gross abnormalities were
 

noted at necropsy. The low and high doses for the chronic
 

studies using rats were set at 3,700 and 7,500 ppm.
 

In mice, deaths occurred in 0/5 animals at doses of 500 ppm, 1/5
 

at 1,200 ppm, 2/5 at 2,500 ppm, 1/5 at 5,000 ppm, and 5/5 at
 

10,000 ppm. Most deaths occurred after week 4. After 45 days of
 

administration of the chemical, mean body weight gains in animals
 

8
 
administered 500 and 1,200 ppm were comparable with those of the
 

controls. Weight gain was depressed 10% at 2,500 ppm and 70% at
 

5,000 ppm. No gross abnormalities were seen at necropsy. The
 

low and high doses for the chronic studies using mice were set at
 

600 and 1,200 ppm.
 

F. Chronic Studies
 

The test groups, doses administered, and times on study of the
 

chronic feeding studies are shown in tables 1 and 2.
 

G. Clinical and Pathologic Examinations
 

All animals were observed twice daily for signs of toxicity.
 

Rats and mice were individually weighed every 2 weeks for 21-23
 

months, then once per month thereafter. Palpation for masses was
 

carried out at each weighing. Moribund animals and those animals
 

that survived to the end of the bioassay were killed using C02
 

anesthesia and necropsied.
 

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 (mice),
 
Table 1. Phenazopyridine Chronic Feeding Studies in Rats
 

Sex and
 

Test
 

Group
 

Male
 

Ma tched-Control
 

Low-Dose
 

High-Dose
 

Female
 

Matched-Control
 

Low-Dose
 

High-Dose
 

Initial
 

No. of
 

Animals3
 

15
 

35
 

35
 

15
 

35
 

35
 

Phenazopyridine
 

in Dietb
 

(ppm)
 

0
 

3,700
 

7,500
 

0
 

3,700
 

7,500
 

Time on Study
 

Dosed Observed
 

(weeks) (weeks)
 

105
 

78 27
 

78 26
 

105
 

78 27
 

78 27
 

a

Rats were 42 days of age when placed on study.
 

^Dosed animals were fed test diets 5 days per week and
 

control diets 2 days per week.
 

10
 
Table 2. Phenazopyridine Chronic Feeding Studies in Mice
 

Sex and
 

Test
 

Group
 

Male
 

Matched-Control
 

Low -Dose
 

High-Dose
 

Female
 

Matched-Control
 

Low-Dose
 

High-Dose
 

Initial
 

No. of
 

Animals3
 

15
 

35
 

35
 

15
 

35
 

35
 

Phenazopyridine
 

in Dietb
 

(ppm)
 

0
 

600
 

1,200
 

0
 

600
 

1,200
 

Time on Study
 

Dosed Observed
 

(weeks) (weeks)
 

106
 

80 25
 

80 25
 

106
 

80 27
 

80 26
 

a

Mice were 49 days of age when placed on study.
 

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

control diets 2 days per week.
 

11
 
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 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 may have
 

been 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 descrip12
 
tive 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
 

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
 

cwo-tailed P value is less than 0.05.
 

The incidence of neoplastic or nonneoplastic lesions has been
 

13
 
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
 

significantly higher proportion of tumors than did the control
 

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

exact test (Cox, 1970) war 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.
 

14
 
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
 

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).
 

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

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

methods of comparing these curves were used for two groups;
 

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
 

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

relative risk is defined as Pt/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
 

16
 
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
 

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 dosed rats were consistently lower than
 

those of corresponding control rats, and the depressions in mean
 

body weight were dose related (figure 1). Fluctuation in the
 

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

diminishes, the mean body weight may be subject to variation.
 

Clinical signs, such as noisy breathing and presence of palpable
 

masses, were noted in both dosed and control animals.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats fed phenazopyridine in the diet
 

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

controls, are shown in figure 2. The result of the Tarone test
 

for positive dose-related trend in mortality is not significant
 

in either sex.
 

In the males, over 70% of the animals in each of the groups
 

(28/35 [80%] in the high-dose group, 25/35 [71%] in the low-dose
 

group, and 12/15 [80%] in the control group) were still alive at
 

week 104. In the females, 28/35 (80%) of the high-dose group,
 

30/34 (88%) of the low-dose group, and 8/15 (53%) of the controls
 

19
 
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 LOW DOSE 

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10 20 30 40 50 60 70 

TIME ON STUDY (WEEKS) 

90 100 110 

Figure 1. Growth Curves for Rats Fed Phenazopyridine in the Diet 

20 
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were alive at week 104. Sufficient numbers of animals 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.
 

The incidence and distribution of tumors in the large intestine
 

(colon and rectum) were as follows:
 

MALES FEMALES
 

Matched Low High Matched Low High
 

Control Dose Dose Control Dose Dose
 

Number of Animals with
 

Tissues Examined
 

Microscopically (14) (34) (33) (14) (33) (32)
 

Adenoma, NOS* 0 0 1 0 0 0
 

Adenocarcinoma, NO S 0 4 9 Q 3 5
 

Sarcoma, NO S 0 1 0 0 0 1
 

*Not otherwise specified
 

The most common tumors observed in the colon were adenocarcinomas. These tumors were polypoid masses that projected into the
 

lumen of the colon and were supported by a stalk composed of
 

fibrous connnective tissue. In some cases the muscularis mucosa
 

was pulled upward into the stalk. The epithelial cells of these
 

tumors usually formed glands and crypts. Neoplastic epithelial
 

22
 
cells were usually tall columnar cells with elongated or oval
 

basilar nuclei and acidophilic cytoplasm. In many instances, the
 

epithelial cells stained darkly basophilic. The deep portions of
 

the crypts had extensive hyperplasia and piling up of cells.
 

Dark-staining carcinomatous cells were usually present on the
 

surface of the tumors, and mitotic figures were commonly seen.
 

Many of the neoplastic cells appeared to have lost their mucous-


secreting ability and were distinguishable from normal epithelial
 

cells adjacent to the polypoid structures. Some ducts and glands
 

in the tumors were cystic and filled with necrotic cellular
 

debris.
 

The stalks of the polypoid colonic adenocarcinomas were composed
 

of loose or dense connective tissue and contained numerous
 

vessels in addition to an inflammatory cell infiltrate composed
 

of lymphocytes, plasma cells, histiocytes, and neutrophils.
 

There was carcinomatous infiltration into the stalk of some
 

tumors; however, the incidence of this type of change was
 

difficult to evaluate because the stalk was not always sectioned
 

with the tumors.
 

Metastatic lesions were not observed in other organs, which may
 

indicate that the tumors were low-grade, polypoid adenocarcinomas
 

and were less agressive than highly invasive flat carcinomas.
 

23
 
A colonic adenoma was observed in one high-dose male rat. The
 

adenoma was an elevated spherical mass on the mucosal surface,
 

consisting of a fibrous connective tissue stroma and a moderate
 

number of intestinal crypts. The cells covering the adenoma and
 

lining the crypts were well-differentiated colonic epithelial
 

cells with mucous-secreting ability. The adenoma lacked areas of
 

epithelial proliferation, cellular basophilia, and numerous
 

mitotic figures seen in the polypoid colonic adenocarcinomas.
 

A poorly differentiated sarcoma was detected in the colon of 1/34
 

(3%) of the low-dose males and 1/32 (3%) of the high-dose
 

females. An additional polypoid adenocarcinoma occurred in the
 

rectum of 1/35 (3%) of the high-dose male rats. No lesions of
 

the colon or rectum occurred in the control groups. Furthermore,
 

tumors of the intestine are rare in this strain of rat.
 

There was a decreased incidence of interstitial-cell adenomas of
 

the testis in the high-dose male rats. The incidence of inter-


stitial-cell tumors were: control males 14/14 (100%), low-dose
 

males 22/34 (65%), high-dose males 7/33 (21%). The decreased
 

incidence of these tumors in the high-dose male rats was not
 

associated with increased mortality or decreased life spans.
 

A variety of other neoplasms occurred in both the matched-control
 

and dosed groups. Some types of these neoplasms occurred only in
 

24
 
rats of dosed groups, or with greater frequency in dosed groups
 

when compared with controls. These lesions, however, are not
 

uncommon in this strain of rat independent of any treatment.
 

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

proliferative, and inflammatory changes were also encountered in
 

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

nonneoplastic lesions are commonly seen in aged rats.
 

Based on the histopathologic examination, it was concluded that
 

phenazopyridine fed in the diet increased the incidence of
 

polypoid adenocarcinomas in the colon and rectum of Fischer 344
 

rats under the conditions of this bioassay. A reduction of
 

testicular adenomas in high-dose male rats appeared to be
 

compound and dose related. However, verification of this
 

relationship would require further evaluation.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

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

in one or more than one group.
 

Adenomas or adenocarcinomas of the large intestine (colon or
 

rectum) were found in both male and female dosed groups of rats.
 

25
 
The result of the Cochran-Armitage test for positive dose-related
 

trend in the incidence of the tumors is significant (P = 0.015)
 

in the male rats, but not in the females. The result of the
 

Fisher exact comparison of the incidence of the tumor in the
 

high-dose group with that in the control group in the males shows
 

a P value of 0.034, which is above the 0.025 level required for
 

significance when the Bonferroni inequality criterion is used
 

for multiple comparison. The historical records of the bioassays
 

conducted for the program at this laboratory show no incidence of
 

adenoma or adenocarcinoma of the large intestine in 260 females
 

and only one adenomatous polyp in 260 males. Assuming a
 

spontaneous incidence of 1/261 (0.38%) and a binomial
 

distribution of such tumors, the occurrences of 8/35 (23%) and
 

5/32 (16%) seen in the male and female high-dose groups,
 

respectively, are both significantly (P < 0.001) different from
 

the expected value (Fears et al., 1977).
 

Significant results in the negative direction are observed in the
 

incidence of interstitial-cell tumors of the testis in the male
 

rats and in the incidence of fibroadenomas of the mammary gland
 

in the females, due to higher incidences in the control groups
 

than in the dosed groups.
 

In summary, the tumors of the large intestine appear to be
 

related to the administration of the chemical.
 

26
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of the dosed mice were consistently lower than
 

those of corresponding control mice, and the depressions in mean
 

body weight were dosed related (figure 3). Fluctuation in the
 

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

diminishes, the mean body weight may be subject to variation.
 

Other clinical signs were common to both control and dosed
 

animals.
 

To control respiratory disease, oxytetracycline was added to the
 

drinking water at 0.6 mg/ml during weeks 81-82 and at 0.3 mg/ml
 

during week 82; also, propylene glycol was vaporized in the mouse
 

room for approximately 2 months, beginning at week 81.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice fed phenazopyridine in the diet
 

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

controls, are shown in figure 4. The result of the Tarone test
 

for positive dose-related trend in mortality is not significant
 

in either sex.
 

At least 60% of the animals in each group lived to the end of the
 

27
 
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Figure 3. Growth Curves for Mice Fed Phenazopyridine in the Diet 

28 
I

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PROBABILITY OF SURVIVAL PROBABILITY OF SU

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bioassay (25/35 [71%] in the high-dose males, 26/35 [74%] in the
 

low-dose males, and 9/15 [60%] in the male controls; 21/35 [60%]
 

in the high-dose females, 26/35 [74%] in the low-dose females,
 

and 10/15 [67%] in the female controls). Sufficient numbers of
 

mice were at risk for the development of late-appearing tumors.
 

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.
 

With the exception of hepatocellular neoplasms in male and female
 

mice, the neoplasms listed in Appendix B appeared with
 

approximately equal frequency in dosed and control mice or
 

appeared in insignificant numbers.
 

There was an increased incidence of hepatocellular adenomas and
 

carcinomas in dosed mice when compared with corresponding
 

controls, as follows:
 

30
 
MALES FEMALES 

Matched Low High Matched Low High 

Control Dose Dose Control Dose Dose 

Number of Animals with
 

Tissues Examined
 

Microscopically


Liver
 

Hepatocellular
 

Carcinoma


Hepatocellular Adenoma
 

or Carcinoma


 (15) (35) (33) (15) (34) (32)
 

2 5 9 2 6 1 4
 

5 15 15 2 11 19
 

Hepatocellular adenomas were variable in size, ranging from small
 

spherical lesions to large irregular lesions equivalent in size
 

to several liver lobules. These lesions were usually well
 

demarcated from the surrounding parenchyma. Compression of the
 

surrounding parenchyma was occasionally seen but was not severe.
 

Cells were arranged in solid sheets or irregular plates that were
 

one or more cells thick. Sinusoids were dilated or compressed.
 

The adenomas frequently contained acidophilic, basophilic, or
 

clear cell foci. Cytologic alterations included nuclear atypia
 

which was characterized by enlargement, hyperchromasia, and
 

sometimes enlarged nucleoli. Marked cytomegaly was present in
 

some adenomas. Mitotic figures were occasionally seen but were
 

not abundant.
 

In contrast to adenomas, hepatocellular carcinomas were
 

extensive, irregularly shaped lesions involving numerous lobules
 

31
 
or entire lobes of the liver. Extensive peripheral compression
 

was frequently seen. The carcinomas were characterized by the
 

formation of trabeculae, sheets, or plates of irregularly
 

arranged cells in linar, papillar, or pseudoacinar patterns.
 

Plates and trabeculae of tumor cells were often 2 to 4 cells
 

thick. Cytologic alterations included nuclear atypia,
 

cytomegaly, and variable numbers of mitotic figures. Hepatic
 

sinusoids were extensively dilated in some tumors and compressed
 

in others. Metastasis of a hepatocellular carcinoma to an other
 

organ was observed in the female mice. Pulmonary metastases were
 

observed in 1/15 (7%) control females and 2/32 (6%) high-dose
 

females. Renal metastasis was detected in 1/5 (7%) control
 

females. Metastatic hepatocellular carcinomas were not observed
 

in the male mice.
 

In addition to the neoplastic lesions, a large number of
 

degenerative, proliferative, and inflammatory changes were
 

encountered in animals of the dosed and control groups (Appendix
 

D). For the most part, these nonneoplastic lesions are commonly
 

seen in aged mice and were not associated with increased
 

mortality.
 

Based on the histopathologic examination, it was concluded that
 

phenazopyridine fed in the diet increased the incidence of hepato32
 
cellular adenomas and carcinomas 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 occurred in at
 

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

in one or more than one group.
 

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

positive dose-related trend in the incidence of hepatocellular
 

carcinomas is significant (P = 0.010); however, the result of the
 

Fisher exact comparison of the incidence of the tumors in the
 

high-dose group with that in the control group shows a P value of
 

0.039, which is above the 0.025 level for significance when the
 

Bonferroni inequality criterion is used for multiple comparison.
 

When hepatocellular adenomas and carcinomas are combined for
 

analyses in female mice, the significance of the Cochran-Armitage
 

test increases (P = 0.002), and the Fisher exact test shows that
 

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

0.003) than that in the controls. The incidence seen in the
 

control group of female mice, 2/15 (13%), is the highest
 

percentage of any control group reported from this laboratory and
 

may reflect the small sample size. Despite this high incidence
 

33
 
in the control group, a significant difference is indicated by
 

the Fisher exact test when the incidence in the high-dose group,
 

19/32 (59%), is compared with this control group. The
 

statistical conclusion is that the combined incidence of
 

hepatocellular adenomas and carcinomas in female mice is
 

associated with administration of the phenazopyridine. Results
 

of the statistical tests on the incidence of the combination of
 

hepatocellular carcinomas and adenomas in male mice are not
 

significant.
 

A significant linear trend in the negative direction is observed
 

in the incidence of lymphoma of the hematopoietic system in
 

female mice, due to a higher incidence in the control group than
 

in the dosed group.
 
V. DISCUSSION
 

Both rats and mice administered phenazopyridine had a dose-


related depression in mean body weights. Mortality in the groups
 

of rats and mice did not, however, show dose-related trends, and
 

sufficient numbers of animals in both dosed and control groups
 

were at risk for the development of late-appearing tumors.
 

In male rats, aidenomas or adenocarcinomas of the large intestine
 

(colon or rectum) occurred at incidences having a significant
 

dose-related trend (P = 0.015). Direct comparisons of the
 

incidence in each dosed group with that in the control group were
 

not significant (controls 0/14, low-dose 4/34, high-dose 8/35).
 

In the females, 3/33 low-dose and 5/32 high-dose animals, but no
 

control animals, had this tumor. In addition, sarcomas were
 

observed in the colon of one low-dose male and one high-dose
 

female. The laboratory historical records showed no incidence of
 

adenomas or adenocarcinomas of the large intestine in 260 females
 

and only one adenomatous polyp in 260 males. Assuming a
 

spontaneous incidence of 1/261 (0.038%) and a binomial
 

distribution of such tumors, the occurrence seen in the male and
 

female high-dose groups are both significantly (P < 0.001)
 

different from the expected value. Thus, these tumors are
 

considered to be related to administration of the test chemical.
 

35
 
In female mice, the combined incidence of hepatocellular adenomas
 

and carcinomas showed a significant dose-related trend (P =
 

0.002), and the incidence in the high-dose group was significant
 

(P = 0.003) when compared with that in the control group
 

(controls 2/15, low-dose 11/34, high-dose 19/32). The incidence
 

of hepatocellular carcinomas, considered alone, also was significant in the female mice, showing a dose-related trend (P = 0.010)
 

and a P value of 0.039 for the comparison of the high-dose group
 

with the control group. In the males, the combined incidence of
 

hepatocellular adenomas and carcinomas was not significant.
 

When previously tested by intraperitoneal injection of total
 

doses of 0.31, 0.78, and 1.55 mg/kg body weight over a 24-week
 

period in an "A" m'ouse pulmonary tumor system (Stoner et al.,
 

1973), phenazopyridine induced no significant increases in
 

incidences of pulmonary tumors. When pellets of phenazopyridine
 

in cholesterol were implanted into the urinary bladders of 20
 

"stock" mice in a 52-week study (Allen et al., 1957), two dosed
 

mice out of 14 that survived longer than 30 weeks developed
 

carcinoma of the bladder and two others developed papilloma or
 

adenoma of the bladder; of 28 controls implanted with cholesterol
 

alone, 24 survived 30 weeks, and one of these developed carcinoma
 

of the bladder.
 

It is concluded that under the conditions of this bioassay,
 

36
 
phenazopyridine hydrochloride was carcinogenic in Fischer 344
 

rats, inducing adenocarcinomas of the colon in both males and
 

females. Although administration of phenazopyridine
 

hydrochloride was not carcinogenic in male B6C3F1 mice, the
 

chemical was carcinogenic in females, inducing hepatocellular
 

adenomas and carcinomas.
 

37
 

VI. BIBLIOGRAPHY
 

Allen, M. J., Boyland, E., Dukes, C. E., Horning, E. S., and
 

Watson, J. G., Cancer of the urinary bladder induced in mice
 

with metabolites of aromatic amines and tryptophan. Brit. J.
 

Cancer 11(2);212-228. 1957.
 

American Medical Association, AMA Drug Evaluations, 1973, 

Publishing Sciences Group, Inc., Acton, Mass., 1973, pp. 

903-904. 

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

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

Berenblum, I., ed., Carcinogenicity Testing; ^ Report of the
 

Panel of Carcinogenicity of the Cancer Research Commission
 

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

1969.
 

Billups, N. F., American Drug Index 1977, J. B. Lippincott Co.,
 

Philadelphia, 1977, pp. 495-496.
 

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

Soc. B34;187-220, 1972.
 

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

1970, pp. 48-52.
 

Fears, T. R., Tarone, R. E., and Chu, K. C., False-positive and
 

false-negative rates for Carcinogenicity screens. Cancer
 

_3_7:1941-1945, 1977.
 

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

significance tests, confidence limits and adjustments for
 

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

Harvey, J. C., Antiseptics and disinfectants; fungicides;
 

ectoparasitides. In: The Pharmacological Basic of
 

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

Publishing Co., Inc., New York, 1975, p. 1009.
 

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

incomplete observations. J. Amer. Statist. Assoc.
 

Jx3457-481, 1958.
 

39
 
Linhart, M. S., Cooper, J. A., Martin, R. L. , Page, N. P. and
 

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

and Bipmed. Res. 7:230-248, 1974.
 

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


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

National Formulary, XIII ed., American Pharmeceutical
 

Association, Washington, D.C., 1970, p. 538.
 

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.
 

Stoner, G. D., Shimkin, M. B., Kniazeff, A. J., Weisburger, J.
 

M., Weisburger, E. K., and Gori, G. B., Test for
 

carcinogenicity of food additives and chemotherapeutic
 

agents by the pulmonary tumor response in strain A mice.
 

Cancer Res. 33:3069-3085, 1973.
 

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

Biometrika 62:679-682, 1975.
 

40
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS FED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET
 

41
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET

 

MATCHED
 

CONTROL LOW DOSE


ANIMALS INITIALLY IN STUDY 15 35


ANIMALS NECROPSIED 14 34


ANIMALS EXAMINED HISTOPATHOLOGICALLY 14 34


INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (14) (34)


SQOAMOUS CELL PAPILLOMA 1 (7X)
 

FIBROMA 1 (7X) 1 (3X)
 

RESPIRATORY SYSTEM
 

fLUNG (12) (33)

SQUAMOOS CELL CARCINOMA , METASTA

ALVEOLAR/dRONCHIOLAR ADENOMA 3 (9X) 

HEMATOPOIETIC SYSTEM 

fBRAIN (13) (33)

MALIGNAN T RETICULOSIS 2 (6X) 

*MULTIPLE ORGAN S (14) (34)

MALIG.LYMPHOMA , LYMPHOCYTIC TYPE 1 (7X)

#5PLEEN (14) (34)

MALIG.LYHPHOMA , HISTIOCYTIC TYPE 1 (3X) 

CIRCULATOR Y SYSTEM 

NON E 

DIGESTIVE SYSTEM 

#LIVEF (14) (34)

HEPATQCELLULAR ADENOM A I__[3£l 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIBD 

 HIGH DOSE
 

 35
 

 35
 

 35
 

 (35)
 

 (35) 

1 (3X) 

 (35) 

 (35) 

1 (3X) 

 (35) 

 (34) 

43 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) _ 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

HEPATOCELLULAR CARCINOMA 1 (3X) 

•ESOPHAGUS

PAPILLOMA, NOS 

 (14) (33) (35) 

1 (3X) 

•COLON

ADENOMA, NOS 

ADENOCARCINOHA,

SARCOMA, NOS 

 NOS 

 (14) (34) 

4 (12X) 

1 (3X) 

(33) 

1 (3X) 

8 (24 X) 

*RECTUM

ADENOCARCINOHA, NOS 

 (14) (34) (35) 

1 (3X) 

UBINABY SYSTEM 

•KIDNEY (14) (34) (35) 

TUBULAR-CELL ADENOCARCI NOM A 1 (3X) 

•URINARY BLADDER (11) (30) (32) 

UNDIFFERENTIATED CARCINOMA 1 (9X) 

ENDOCRINE SYSTEM 

•PITUITARY (5) (25) (23) 

CHROMOPHOBE ADENOMA 1 (20X) 

•ADRENAL (13) (34) (34) 

CORTICAL ADENOH A 1 (3X) 

PHEOCHROMOCYTOMA 2 (15X) 1 (3X) 1 (3X) 

•THYROID (14) (32) (32) 

FOLLICULAfl-CELL ADENOMA 1 (3X) 

C-CELL ADtfNOBA 2 (14X) 1 (3X) 

•PANCREATIC ISLETS (14) (34) (35) 

ISLET-CEH, ADENOM A 1 (7X) 1 (3X) 1 (3X) 

REPRODUCTIVE SYSTEM 

tTESTIS (14) (34) (33) 

iSTJRSTITJli-£ELL. T.UJJQg 14 J100X.1 . _ 22_J65X1_ . 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECSOPSIED
 

44
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

NERVOU S SYSTEM 

#BRAIN

GLIOHA, DOS

SPECIAL SENSE ORGANS 

*EYE

SARCOMA, NOS

*EAR CANA L

SQUABOUS CELL CARCINOMA

BUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

*PERITONEUM

MESOTHELIOMA BENIGN

*PELVIS

LIPOMA

ALL OTHEB SYSTEMS 

NONE 

ANIMA L DISPOSITION SOHMARY 

ANIHALS INITIALLY IS STUDY

NATURAL DEATHS

MORIBUN D SACRIFICE

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE

ANIMAL HISSING 

a INCLUDES ADIOLY2ED ANIMALS 

* NUMBER OF ANIHALS BITH TISSUE

* NUMBER OF ANIMALS NECROPSIED 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 (13) (33) (35) 

1 (3*) 

 (14) (34) (35) 

1 (3X) 1 (3X) 

 (14) (34) (35) 

1 (3%) 1 (3%) 

 (14) (34) (35) 

1 (3X) 

 (14) (34) (35) 

1 (3X) 

 15 35 35 

2 3 2 

1 7 5 

 12 25 28 

 EXAMINED MICROSCOPICALLY 

45 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*


TOTAL PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TUMORS


TOTAL BENIGN TUHORS


TOTAL ANIMAi.5 WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TOMORSt


TOTAL SECONDARY TUMORS


MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 14 29 17
 

 24 44 . 26
 

 14 24 10
 

 22 32 13
 

2 12 11
 

2 12 13
 

1
 

1
 

TOTAL ANIMALS WITH TUMORS UNCESTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS ONCEHTAINPRIMARY OR HETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUHORS EXCEPT SECONDARY TUMORS
 

* SECONDARY TUMORS: METASTATIC TUMORS OR TUHORS INVASIVE INTO AN ADJACE8T ORGABT
 

46
 
TABLE A2. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY

ANIMALS NECROPSIED

ANIMALS EXAMINED HISTOPATHOLOGICALLY

 15

 15

 15

 35

 33

 33

 35 

 32 

 32 

INTEGUMENTARY SYSTEM
 

NONE
 

RESPIKATORY SYSTEM
 

fLUNG (1U) (33) (32)
 

ADENOCARCiNOMA, NOS, BETASTATIC 1 (3%)
 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (3S)
 

HEMATOPOIETIC SYSTEM
 

*MOLTIPLE ORGANS (15) (33) (32)
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (7X)
 

UNDIFFERENTIATED LEUKEMIA 1 (3X)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYS1EM
 

tCOLON (14) (33) (32)
 

ADENOCARCINOMA, NOS 3 (9%) 5 (16S)
 

SARCOMA, NOS 1 (3X)
 

URINARY SYSTEM
 

#KIDNEY (15) (33) (32)
 

AD£NOCARCiNOMA. NOS. METASTATIC
 

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

47
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

•URINARY	 BLADDER (13) (28) (27)
 

PAPILLOMA, NOS 1 (8X)
 

ENDOCBINE SYSTEM
 

tPITOITARY (9) (30) (25) 

CHROMOPHOdE ADENOHA 2 (22%) 1 (3X) 1 (4X) 

fADRENAL (14) (33) (31)

PHEOCHROMOCYTOBA 1 (3X) 

ITHYROID (11) (32) (31) 

FOLLICULAR-CELL ADENOH A 1 (3X) 1 (3X) 

C-CELL ADENOM A 1 (3X) 1 (3X) 

•PANCREATIC ISLETS (14) (33) (32) 

ISLET-CELL ADENOH A 1 (3X) 

REPRODUCTIVE SYSTEM 

*MAMMAR Y GLAN D (15) (33) (32) 

FIBROADENOMA 2 (13X) 

#UTERUS (15) (33) (32) 

ADENOCARCINOJ1A, NOS 1 (3X) 

ENDOHETRIA L STROMAL POLYP 1 (7X) 1 (3X) 1 (3X) 

NERVOU S SYSTEM 

NON E 

SPECIAL SENSE ORGAN S 

*EAB CANAL (15) (33) (32)
 

SQUAMOUS CELL CARCINOMA 1 (3X)
 

MUSCULOSKELETAL SYSTEM
 

_NQNE
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

48
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES 

*PLEURA (15) (33) (32) 

BESOTHELIOHA, MALIGNANT 1 (3X) 

ALL OTHER SYSTEMS 

DIAPHRAG M 

ADENOCARCINOMA , tfOS, HETASTATIC 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 15 35 35
 

NATURAL DEATHS 3 2 5
 

MORIBUND SACRIFICE 4 2 2
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 8 30 28
 

ANIMAL HISSING
 

ANIMAL DELETED (WRONG SEX) 1
 

a INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMAR Y 

TOTAL ANIMALS WITH PRIMAR Y TUMORS* 5 10 13 

TOTAL PRIMAR Y TUMORS 7 10 14 

TOTAL ANIMALS WITH 4 BENIGN TUMORS 5 5 

TOTAL BENIGN TUMORS 6 5 6 

TOTAL ANIMALS WITH MALIGNAN 1 T TUMORS 5 8 

TOTAL MALIGNANT TUMORS 1 5 8 

TOTAL ANIMALS WITH SECONDARY TUBORSt 1 

TOTAL SECONDARY TUMORS 3 

TOTAL ASIMALS HITH TUMOBS UNCERTAINBENIGN OR MALIGNANT

 

TOTAL UNCERTAIN TUMOBS 

TOTAL ANIMALS HITH TUMORS UNCEBTAINPRIHARY OR METASTATIC

 

TOTAL UNCERTAIN TUMOBS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

* SECONDARY TUMORS: METASTATIC TUMOBS OB TOHORS INVASIfE INTO AH ADJACENT ORGAN 

49 

APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE FED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET
 

51
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS 

ANIMALS 

ANIMALS 

INITIALLY IN STUDY 

NECROPSIED 

EXAMINED HISTOPATHOLOGICALLY 

15 

15 

15 

35 

35 

35 

35 

34 

34 

INTEGUMENTARY SYSTEM
 

NONE
 

BESPIRATORY SYSTEM
 

tLUNG (15) (35) (34) 

ALVEOLAR/BRONCHIOLAR ADENOMA 1 (7X) 3 (9X) 4 (12XJ 

ALVEOLAR/bRONCHIOLAR CARCINOMA 1 (3X) 

HEHATOPOIETIC SYSTEM 

'MULTIPLE ORGANS (15) (35) (34) 

UNDIFFERENTIATED LEUKEMIA 1 (3X) 

•MESENTERIC L. NODE (13) (31) (31) 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (3X) 

MALIGNAN T LYMPHOMA, MIXED TYPE 1 (3X) 

fPEYEHS PATCH (15) (35) (34) 

MALIGNANT LYMPHOHA, MIXED TYPE 1 (7X) 

CIRCDLATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

fLIVEH (15) (35) (33)
 

HEPATOCELLULAB ADENOMA 3 (20X) 10 (29X) 6 (18X
 

HEPATOCELLULAH CARCINOMA 2 (13X) 5 9 (27X
 

DBINARY SYSTEM
 

_»SNE
 

t NUMBER OF ANIMALS BITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIBD
 

53
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ENDOCRINE SYSTEM 

tTHYROID

FOLLICULAR-CELL ADENOM A

 (1<*)

 1 (7X) 

 (30) (31) 

HEPBODOCTIVE

HONE 

 SYSTEM 

NERVOUS

NONE 

 SYSTEM 

SPECIAL SENSE ORGANS 

*HARDEBIAN GLAN D

ADENOMA, NOS

 (15) (35) (34) 

1 (3X) 

.MUSCULOSKELETAL

NON E 

 SYSTEM 

BODY CAVITIES 

NON E 

ALL OTHER

NON E 

 SYSTEMS 

ANIMA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALLY IN STUD Y

NATURA L DEATHS

MORIBUND SACRIFICE

SCHEDULE D SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE

ANIMA L MISSING 

 15

 2

 4 

9

 35

 7

2 

 26

 35 

U 

6 

 25 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

54 
TABLE 81. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

TUMOB SUMMARY 

TOTAL ANIMALS WITH PRIMARY TUMORS*

TOTAL PRIMARY TUMORS

 7

 8

 17

 19

 20 

 23 

TOTAL ANIHALS WITH BENIGN TUMORS

TOTAL BENIGN TUMORS

 5

 5

 13

 13

 10 

 11 

TOTAL ANIMALS WITH MALIGNANT TUMORS

TOTAL MALIGNANT TUMORS

 3

 3

 5

 6

 12 

 12 

TOTAL ANIMALS WITH SECONDARY TOMORSI 

TOTAL SECONDARY TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUBURS: ALL TUMOfiS EXCEPT SECONDARY TUMORS
 

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

55
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET 

1MATCHED 

1CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 15 35 35 

iNIHALS NECBOPSIED 15 34 32 

ANIMALS EXAMINED HI STOPATHOLOGICALLY 15 34 32 

INTEGUMENTAR Y SYSTEH 

*SUBCOT TISSUE (15) (34) (32) 

MALIGNAN T MELANOMA 1 (1%) 

SARCOMA, MOS 2 <6X) 

HEMANGIOM A 1 (3X) 

RESPIRATORY SYSTEM 

tLUN G (15) (34) (32) 

HEPATOCELLULAR CARCINOMA, HETAST 1 (7X) 2 (6X) 

ALVEOLAR/riBONCHIOLAR ADENOM A 1 (7X) 1 (3X) 2 (6X) 

HEMATOPOIETIC SYSTEM 

*MULTIPLE ORGAN S (15) (34) (32) 

MALIGNANT LYHPHOMA, NOS 1 (3X) 

MALIG.LYMPHOBA, LYHPHOCYTIC TYPE 1 (7X) 1 <3X) 

MALIG.LYMPHOHA, HISTIO'CYTIC TYPE 1 (3X) 1 OX) 

MALIGNAN T LYMPHOMA , MIXED TYPE 3 (20%) 2 (6X) 

•PEYERS PATCH (15) (34) (32) 

MALIGNANT LYMPHOMA, MIKED TYPE 1 (3X) 

* UTERUS (15) (34) (32) 

MALIG.LYMPHOBA, HISTIOCYTIC TYPE 1 (3X) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

tLIVER (15) (34) (32) 

HEPATOCBLLULAR ADENOBi 1 (7XL 5 (15X) 7 122%) 

t NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

56
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

HEPATOCEILULAR CARCINOMA 2 (13X) 6 (18S) 14 (44X) 

URINAR Y SYSTEM 

•KIDNE Y

HEPATOCELLULAR CARCINOMA, METAST

 (15)

 1 (7X) 

 (34) (32) 

ENDOCRIN E SYSTE M 

tPITUITARY

CHROHOPHOBE ADENOM A

 (10)

 1 (10X)

 (30)

 2 (7X) 

 (23) 

#THYROI D

FOLLICULAH-CELL ADENOH A

 (14) (30) (29) 

1 (3X) 

REPRODOCTIVE SYSTEM 

*MAMMAB Y GLAND

FIBBOADENOMA

 (15) (34) (32) 

1 (3X) 

#UTEBOS

ENDOBETRIAL STRONAL POLYP

 (15) (34)

 1 (3X) 

 (32) 

#OVARY

PAPILLAR Y CYSTADENOHA, BOS

 (15) (34)

 1 (3X) 

 (32) 

NERVOU S SYSTEM 

NONE 

SPECIAL SENSE OBGANS 

*HABDEBIAN GLAND

ADENOMA, NOS

*EAR CANA L

SQUAHOUS CELL CARCINOHA

 (15)

 (15)

 (34)

 2

 (34)

 (6X)

 (32) 

3 (9%) 

 (32) 

1 (3X) 

MUSCDLOSKELETAL SYSTEM 

HONE 

t NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

57
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIES 

*ABDOMINAL CAVITY (15) (34) (32)
 

FIBROUS HISTIOCYTOMA, MALIGNAN T 1 (3S)
 

*PLEURA (15) (34) (32) 

SARCOMA, BIOS 1 (3%) 

ALL OTHEB SISTEMS
 

NONE
 

INIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 15 35 35 

NATURAL D£ATH3 2 5 7 

MORIBUND SACRIFICE 3 4 7 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 10 26 21 

ANIMAL MISSING 

INCLUDES AUXOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 8 20 27
 

TOTAL PRIMARY TUMORS 10 26 34
 

TOTAL ANIMALS WITH BENIGN TUMORS 2 11 14
 

TOTAL BENIGN TUMORS 3 13 14
 

TOTAL ANIMALS WITH MALIGSANT TUMORS 7 12 18
 

TOTAL MALIGNANT TOMOBS 7 13 20
 

TOTAL ANIMALS WITH SECONDARY TUMORS* 1 2
 

TOTAL SECONDARY TUMORS 2 2
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIG N OR MALIGNAN T

 

TOTAL UNCERTAI N TUMORS 

TOTAL ANIMALS WITH TUMORS UNCERTAINPBIHAR Y OR flETASTATIC

 

TOTAL UNCERTAI N TUMORS 

* PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

I SECONDAR Y TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGA N 

58 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS FED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET
 

59
 

TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE 

ANIMALS INITIALLY IN STUDY 15 35 

ANIMALS NBCHOPSIED 1<t 34 

ANIMALS EXAMINED HIS1 rOPATHOLOGICALLY 14 34 

INTEGUMENTARY SYSTEM
 

*SKIN (14) (34)
 

EPIDERMAL INCLUSION CYST 1 (7X)
 

*SUBCUT TISSUE (14) (34)
 

HYPE8PLASIA, BASAL CELL 1 (7X)
 

RESPIRATORY SYSTEM
 

•TRACHEA (U) (33) 

LYMPHOCYTIC INFLAMMATORY ISFILTR 1 (7X) 5 (15*) 

INFLAMMATION , SUPPURATIVE 12 (86X) 20 (61*) 

INFLAMMATION , ACUTE/CHRONIC 

INFLAMMATION, CHRONIC SUPPURATIV 

•LUNG/BRONCHUS (12) (33) 

BRONCHIECTASIS 2 (17*) 1 (3X) 

INFLAMMATION, SUPPURATIVE 1 (8%) 

POLYP, INFLAMMATORY 1 (3*) 

HYPERPLASIA, LYMPHOID 1 (8%) 1 (3X) 

tLUNG (12) (33) 

HEMORRHAGii 1 (3X) 

INFLAMMATION, INTERSTITIAL 5 (42X) 1 (3X) 

BRONCHOPNiiUMONI A SUPPURATIV E 3 (9X) 

ABSCESS, NOS 1 (3*' 

PNEUMONIA INTERSTITIAL CHRONIC 1 (8X) 

BRONCHOPNiiUMONIA CHRONIC SUPPURA 

INFLAMMATION , FOCAL GRANULOMATO U 

FIBROSIS 

fLUNG/ALVEOLI (12) (33) 

HJMOHRHMfi 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE 

35 

35 

35 

(35)
 

(35)
 

(35)
 

10 (29X)
 

17 (49X)
 

2 (6X)
 

1 (3X)
 

(35)
 

(35)
 

9	 (26X)
 

1 (3X)
 

1 (3X)
 

1 (3X)
 

(35)
 

1(3X1.
 

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

HEMATOPOIETIC SYSTEM
 

• BONE MARROW
 

ATROPHY, NOS
 

•SPLEEN
 

FIBROSIS
 

ATROPHY, NOS
 

HEMATOPOIiSIS
 

•LYMPH	 NODE
 

CYST, NOS
 

iMESENTERIC L. NODE
 

LYMPHANGIECTASIS
 

#THYMUS
 

ABSCESS, NOS
 

ATROPHY, NOS
 

CIRCULATORY SYSTEM
 

#HEART
 

INFLAMMATION, INTERSTITIAL
 

•MYOCARDIUM
 

INFLAMMATION, INTERSTITIAL
 

FIBROSIS
 

FIBROSIS, DIFFUSE
 

ATROPHY, NOS
 

•ENDOCARDIUM
 

INFLAMMATION, FOCAL
 

*AORTA
 

MEDIAL CALCIFICATION
 

*PULMONABY ARTERY
 

MEDIAL CALCIFICATION
 

DIGESTIVE SYSTEM
 

•SALIVARY GLAND
 

INFLAMMATION. CHRONIC DIFFUSE
 

MATCHED
 

CONTROL
 

(14)
 

(14)
 

(14)
 

(14)
 

(13)
 

(14)
 

(14)
 

6 (43X)
 

2 (14X)
 

1 (7%)
 

2	 (14X)
 

(14)
 

(14)
 

1 (7X)
 

(14)
 

(14)
 

LOW DOSE
 

(3.3)
 

(34)
 

19 (56%)
 

(10)
 

(10)
 

(26)
 

1 (4%)
 

(33)
 

(33)
 

30 (91X)
 

(33)
 

(34)
 

(34)
 

2 (6%)
 

(30)
 

HIGH DOSE
 

(35)
 

3 (9X)
 

(35)
 

1 (3X)
 

1 (3X)
 

5	 (14S)
 

(14)
 

1 (7X)
 

(14)
 

2 (14X)
 

(26)
 

4	 (15X)
 

(35)
 

1 (3X)
 

(35)
 

32	 (91X)
 

3 (9)1)
 

(35)
 

1 (3X)
 

(35)
 

(35)
 

(34)
 

1 13%)
 

• NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

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

MATCHED
 

CONTROL LOWE >OSE HIGH DOSE
 

fLIVEH (14) (34) (34)
 

NECROSIS, FOCAL 2 (6X) 1 (3X)
 

METAMORPHOSIS FATTY 1 (3S)
 

CYTOPLASMiC VACUOLIZATION 2 (6X) 1 (3X)
 

CYTOLOGIC DEGENERATION 1 (3X)
 

HYPERPLASIA, NODULAR 1 (3X) 1 (3X)
 

ANGIECTASIS 1 (3X) 1 (3X)
 

tLIVER/CENTRILOBULAR (14) (34) (34)
 

CYTOPLASMIC VACUOLIZATION 2 (6X) 1 (3X)
 

*BILE DUCT (14) (34) (35)
 

HYPERPLASIA, NOS 1 (7X) 3 (9X) 1 (3X)
 

•PANCREAS	 (34) (35)
 

FIBROSIS, DIFFUSE 1 (7X)
 

PERIARTERITIS 1 (75S)
 

•PANCREATIC ACINUS (34) (35)
 

ATROPHY, NOS 1 (7X) 2 (6X)

 

ATROPHY, FOCAL 8 (24X)

 

•ESOPHAGUS	 (33) (35) 

DIVERTICULUM 1 (3X) 

#STOMACH (34) (35) 

ULCER, FOCAL 1 (3X) 

INFLAMHATION, SUPPURATIVE 1 (3X) 

HYPERKERA10SIS 1 OX) 

tILEUH (1*0 (33) (34) 

ULCER, FOCAL 1 (3X) 

•CECUM	 (34) (33)

 

ULCER, NOS 1 (3X)

 

METAPLASIA, OSSEOUS 1 (3X)

 

URINARY SYSTEM 

tKIDNEY (14) (34) (35) 

INFLAMHATION, CHRONIC 13 (93X) 22 (65X) 5 (14X) 

NECROSIS. MEDULLARY LJZSL 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NBCROPSIBD
 

63
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

ENDOCRIN E SYSTEM 

tADRENAL CORTEX (13) (34) 

HYPERPLASIA , FOCAL 1 (8X)

 

ITHYROID (14) (32) 

HYPERPLASIA , C-CELL 2 (6*) 

*PARATHYROI D (12) (22) 

HYPERPLASIA , NOS 1 (5*) 

BEPRODUCTIVE SYSTEM 

' *MAHHAR Y GLAN D (14) (34) 

DILATATION/DUCTS 

CYST, NOS 1 (7X) 1 (3%) 

•PROSTATE (11) (34) 

INFLAMMATION , SOPPURATIV E 6 (188) 

*SEBINAL VESICLE < U > (34) 

CYST, NOS 

#TESTIS (14) (34) 

ATROPHY , NOS 

NERVOU S SYSTEM 

#BRAIN/MENINliES (13) (33)

 

INFLABMATION , ACUTE/CHRONIC 

SPECIAL SENSE ORGAN S 

*EYE (14) (34)

 

SYNECHIA , POSTERIOR 

*EYE/RETINA (14) (34)

 

ATROPHY, NOS 

*EAR (14) (34)

 

INFLAMMATION , CHRONIC SUPPURATIV 1 <7X ) 

t NUMBER OF AHIMALS WITH TISSUE EXAMINE D HICROSCOPICALLY
 

* NUMBER OF ANIHALS NECBOPSIED
 

HIGH DOSE 

(34) 

(32) 

5 (16*) 

(24) 

(35) 

1 (3*) 

(34) 

1 (3X) 

(35) 

1 (3X) 

(33) 

5 (15S) 

(35) 

1 (3X) 

(35) 

1 (351) 

(35) 

1 (3*) 

(35) 

64
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

*EAB CANA L

INFLAMMATION, SOPPOBATIVE

 (14) (34) (35) 

1 (3X) 

MUSCULOSKELETAL

NONE 

 SYSTEM 

BODY CAVITIES 

*PLEORA

INFLAMMATION, CHRONIC SUPPOBATIV

FIBROSIS

 (14) . (34) (35) 

1 (3%) 

1 (3X) 

ALL OTHER SYSTEMS 

ADIPOSE TISSUE 

INFLAMMATION, CHRONIC FOCAL 1 

SPECIAL MORPHOLOGY SUMMARY 

NO NECROPSY PERFORMED 1 

AUTOLYSIS/NO NECROPSY 1 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECHOPSIED 

65
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET 

ANIMAL S INITIALL Y IN STUDY

4NIMAL S NECHOPSIED

ANIMAL S EXAMINE D HI STOPATHOLOGIC ALLY

INTEGUMENTAR Y SYSTEM 

*SUBCUT TISSUE

EDEflA, NOS 

INFLAMMATION, CHRONIC SUPPURATIV

RESPIRATORY SYSTEM 

#TRACHE A

LYMPHOCYTIC INFLAMMATOR Y INFILTR

INFLAMMATION , SUPPURATIVE

METAPLASIA, SQUAMOUS

tLUNG/BRONCHUS

BRONCHIECIASIS 

HYPERPLASIA, LYMPHOID 

#LUNG/BRONCHIOLE

ABSCESS, NOS 

tLUNG

INFLAMMATION , INTERSTITIAL

BRONCHOPNEUMONI A SUPPURATIVE 

PNEUMONI A INTEBSTITIAL CHRONIC

BRONCHOPNfiUBONIA CHRONIC SHPPURA 

FIBROSIS, FOCAL 

HEHATOPOIETIC SYSTEH 

#BONE MARRO W

ATROPHY, NOS 

HYPERPLASIA, RETICULUH CELL 

tSPLEEN

HEHOSIDEROSIS 

MATCHED 

CONTROL 

 15 

 15 

 15 

 (15) 

1 (7X) 

 (15) 

3 (20X) 

7 (47%) 

1 (7X) 

 (14) 

 (14) 

 (14) 

5 (36X) 

1 (7X) 

 (14) 

 (15) 

LOW DOSE HIGH DOSE 

35 35 

33 32 

33 32 

(33) (32) 

1 (3X) 

(33) (32) 

6 (18X) 6 (19X) 

18 (55X) 18 (56X) 

(33) (32) 

1 (3X) 

3 (9X) 

(33) (32) 

1 (3X) 

(33) (32) 

1 (3X) 2 (6X) 

1 (3X) 

1 (3X) 

(33) (30) 

17 (57X) 

1 (3X) 

(33) (32) 

1 (3X1 

* NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

66
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

HEMATOPOIESIS 1 (7X) 27 (82X) 21 (66S) 

tMESENTERIC L. NODE (13) (16) (12) 

LYMPHANGIfiCTASIS 1 (6X) 5 (42X) 

•IHYMUS (11) (27) (26) 

HEMORRHAG E 1 (UX) 

ATBOPHY, NOS 

CIRCULATORY SYSTEM 

•MYOCARDIUM (15) (32) (32) 

INFLAMMATION, INTERSTITIAL 2 (13X) 30 (9UX) 25 (78X) 

INFLAMMATION , SUPPURATIV E 1 (3X) 

FIBROSIS, DIFFUSE 1 (7X) 

DIGESTIVE SYSTEM 

ILIVER (15) (33) (31) 

NECROSIS, FOCAL 2 (6X) 

FOCAL CELLULAR CHANGE 1 (3X) 

HEMATOPOIcSIS 1 (7X) 

•LIVEH/CENTR1LOBULAR (15) (33) (31) 

CONGESTION, NOS 1 (3X) 

•PANCREAS (14) (33) (32)

 

FIBHOSIS, DIFFUSE 3 (21X) 

•PANCREATIC ACINUS (33) (32) 

ATROPHY, NOS 2 (14X) 3 (9X) 

ATROPHY, FOCAL 6 (18X) 

DBINARY SYSTEM 

•KIDNEY (15) (33) (32) 

PYELONEPHRITIS, NOS 

INFLAHHATION, CHRONIC 5 (33X) 3 (9X) 1 (3X) 

HECROSIS, ISCHEMIC 

INFARCT, HEALED 1 (3X) 

•URINARY BLADDER (13) (28) (27)

 

HYPBBPLASIA. EPITHELIAL 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF AMIHALS NECHOPSIED
 

67
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

BETAPIASIA, SQUAMOUS 1 (8%) 

ENDOCBINE SYSTEM 

IADEENA L (14) (33)

 

HYPERPLASIA , NOS 

#THYROID (11) (32) 

HYPEBPLASIA, C-CELL 2 (6%) 

BEPRODUCTIVE SYSTEM 

*MAMMAR Y GLAN D (15) (33)

 

DILATATION/DUCTS 1 (7%) 

IUTERUS (15) (33)

 

CYST, NOS 1 (7%)

 

lUTEBUS/ENDOi-JETRIUM (15) (33) 

INFLAMMATION , SUPPURATIV E 2 (1395) 3 (9X) 

INFLAMMATION , CHRONIC SUPPURATI V 1 (7X) 

HYPERPLAS1A, NOS 2 (13*) 

HYPERPLAS1A, CYSTIC 1 (7X) 3 (9«) 

DECIDOAL ALTERATION, NOS 1 (7%) 1 (3X) 

#OVARY/OVIDUCT (15) (33)

 

INFLAMMATION , SUPPURATIVE H (21%) 

INFLAMMATION , CHRONIC SUPPURATI V 1 (7X)

 

#OVAR Y (13) (33) 

CYSTIC FOLLICLES 1 (3X) 

FOLLICULAtt CYST, NOS 1 (3%) 

MULTIPLE CYSTS 

INFLAMMATION , SUPPURATIV E 1 (8X) 4 (12X) 

NERVOU S SYSTEM 

tflRAIN/flENIHGE S (14) (33)

 

INFLAMMATION , NOS 

SPECIAL SENSE ORGAN S 

NON E 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

HIGH DOSE 

(31) 

1 (3X) 

(31) 

(32) 

(32) 

(32) 

6 (19X) 

(32) 

1 (3X) 

(32) 

2 (6*) 

1 (3XJ 

2 (6X) 

(32) 

1 (3%) 

68
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BUSCULOSKELETAL SYSTEM 

NONE 

EODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUMMABY 

NO LESION EEPOBTED

NO NECROPSY PERFORMED

AUTO/NECRJPSY/HISTO PERF 1 

AUTOLYSIS/NO NECROPSY

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

1

 1 

2 

1 

69
 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE FED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET
 

71
 

TABLE 01. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET 

ANIMALS INITIALLY IN STUDY


ANIMALS NECBOPSIED


ANIMALS EXAMINED HI STOPATHOLOGICALLY


INTEGUMENTARY SYSTEM
 

NONE
 

BESPIRATORY SYSTEM
 

«LUNG


PNEUMONIA, LIPID


PNEUMONIA INTERSTITIAL CHBOhIC
 

BRONCHOPNEUHONIA CHRONIC SDPPUBA
 

HEMATOPOIETIC SYSTEM
 

•SPLEEN


HEJJATOPOIESIS


IMES ENTERIC L. NODE


CONGESTION, 80S


INFLAMMATION, SUPPURATIVB
 

ATBOPHY, SOS
 

HYPEBPLASiA, LYMPHOID
 

CIRCULATORY SYSTEM
 

•MYOCARDIUM


INFLAMMATION, SUPPURATIVE
 

DIGESTIVE SYSIEB
 

ILIVER


INFLAMBATION, HECBpTIZING
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 15 35 35 

 15 35 34 

 15 35 34 

 (15) (35) (34) 

1 (7X) 

1 (3X) 1 (3X) 

1 (3X) 1 (3X) 

 (15) (35) (34) 

1 (7X) 2 (6X) 

 (13) (31) (31) 

1 (8X) 7 (23X) 

3 (10X) 

1 (3X) 

1 (3X) 1 (3X) 

 (15) (35) (34) 

1 (3X) 

 (15) (35) (33) 

1 (3X) 

I NUMBER OF ANIMALS BITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECBOPSIED
 

73
 
TABLE D1. MALE MICE: NONNEOPLASTIC

——— I _ .. . ir — .. in i 

NECECSIS, COAGULATIV E 

CYTOPLASMiC VACUOL7ZATIO N 

FOCAL CELLULA R CHANG E 

ANGIECTASI S 

HYPERPLASIA , LYMPHOID 

HEMATOPOIiSIS 

tLIVER/HEPATOCYTES 

CYTOPLASHIC VACtJOLIZATION 

• PANCREAS 

INFLAMMATION, INTERSTITIAL 

#PANCREATIC ACINU S 

ATROPHY , ilOS 

*COLON 

NEMATODIASIS 

URINAR Y SYSTErf 

#KIDNE Y 

HYDRONEPHHOSI S 

ENDOCRIN E SYSTEM 

NON E 

REPRODUCTIVE SYSTEM
 

NONE
 

NERVOUS SYSTEM
 

*TRIGEMINAL GANGLION
 

INFLAMMATION , CHRONIC 

SPECIAL SENSE ORGANS
 

*HIDDLE EAR
 

INFL&aHATION,. SUPPUSATIVE
 

 LESIONS (CONTINUED) 

MATCHED 

CONTROL

1 (7%) 

1 (7%) 

(15)

(15)

(15)

(15)

(15)

(15)
 

2 (13X)
 

(15)
 

 LOW DOSE HIGH DOSE 

1 (3%) 1 (3X) 

1 (3%) 1 OX) 

2 ((>%) 

1 (3X) 

1 OX) 

 (35) (33) 

2 (6%) 

 (35) (33)

 

1 (3X) 

 (35) (33)

 

1 (3X) 

 (35) (3U)

 

1 (3S) 

 (35) (34)

 

1 (3X) 

(35) (3U)
 

(35)	 (34)
 

-1-13J
 

*	 NUMBER OF ANIBALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NECROPSIID
 

74
 
TABLE 01. MALE MICE NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

INFLAMMATION , CHRONIC SDPPURATI V 1 (3X) 

flUSCULOSKELETAL

NON E 

 SYSTEM 

BODY CAVITIES 

*MESENTER Y

NECROSIS, FAT

 (15) (35) (34) 

1 (3%) 

ALL OTHER SYSTEMS 

ADIPOSE TISSUE 

INFLAMMATION , CHRONIC 1 

SPECIAL aOfiPHOLOGY SUMMAR Y 

NO LESION REPORTED

AOTOLYSIS/NO NECROPSY

# NUMBE R OF ANIMAL S WITH TISSUE

* NUMBE R OF AiilMALS NECROPSIED 

 I* 13

 EXAMINE D MICROSCOPICALLY 

8 

1 

75 
TABLE 02.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET 

ANIMALS INITIALLY IN STUDY
 

ANIMALS NECROPSIED
 

ANIMALS EXAMINED HI STOPATHOLOGICALL Y
 

INTEGUMENTARY SYSTEM
 

NONE
 

HESPIRATORY SYSTEM
 

tTRACHEA
 

INFLAMMATION, CHRONIC SUPPURATIV
 

tLUNG/BRONCHUS
 

INFLAMMATION, CHRONIC SUPPURATIV
 

*LUNG/BRONCHIOLE
 

HYPERPLASIA, LYMPHOID
 

#LUNG
 

PNEUMONIA INTERSTITIAL CHRONIC
 

BRONCHOPNfiUMONIA CHRONIC SUPPURA
 

HEMATOPOIETIC SYSTEM
 

*SPLEEN
 

ANGIECTASJ.S
 

HEMATOPOIriSIS
 

tMANDIBULAR n. NODE
 

HYPERPLASIA, PLASMA CELL
 

#PANCREATIC L.NODE
 

CONGESTION, NOS
 

HYPERPLASiA, LYMPHOID
 

#MESENTERIC j,. NODE
 

CONGESTION. NOS
 

MATCHED
 

CONTROL LOW DOSE
 

15 35
 

15 34
 

15 34
 

(15)	 (34)
 

1 (3X)
 

(15)	 (34)
 

1 (3X)
 

(15)	 (34)
 

1 (7X)
 

(15) (34)
 

1 (3X)
 

1 (7X) 3 (9X)
 

(15) (34)
 

3	 (9«)
 

(13) (33)
 

(13) (33)
 

(13) (33)
 

HIGH DOSE
 

35
 

32
 

32
 

(32)
 

(32)
 

(32)
 

(32)
 

1	 (3X)
 

(32)
 

1 (38)
 

1 (3X)
 

(29)
 

1 <3X)
 

(29)
 

1	 (3%)
 

(29)
 

2 J7XJ.
 

t	 NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

*	 NUMBER OF ANIMALS NBCROPSIED
 

76
 
TABLE 02. FEMALE MICE: NONNEOPLASTIC

ATROPHY , HOS 

KYPERPLASIA , LYMPHOI D 

tINGUINA L LYilPH NOD? 

HYPERPLASiA , PLASMA CELL 

CIRCULATOR Y SYSTEM 

NON E 

DIGESTIV E SYSTEM 

tLIVEfi 

CYTOPLASMIC V ACUOLIZ ATION 

HYPERPLASIA , NODULA R 

ANGIECTASI S 

HEMATOPOIiSSIS 

fPANCREA S 

HEMORRHAG E 

NECROSIS, FAT 

#PANCREATIC ACINU S 

ATROPHY , NOS 

URINAR Y SYSTEM 

IKIDNE Y 

HYPEfiPLASIA,

ENDOCRIN E SYSTEM 

tTHYROID 

HYPERPLASIA,

 LYHPHoiD 

 FOLLI COL AR-CELL 

REPRODUCTIV E SYSTEM 

tOTERUS 

HEMORRHAG E 

tUTEHUS/ENDOaETRIUM 

INFL^HHATION. SUPPOH1TIVE_ 

 LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

2 (6X) 1 (3X) 

1 (3X) 

(13) (33) (29) 

1 (3X) 

(15) (34) (32)

 

2 (13*) 1 (3X)

 

1 (3*) 

1 (3%) 1 (3X) 

1 (3X) 

(15) (34) (32) 

1 (3X) 

1 (3X) 

(15) (34) (32)

 

1 (3X) 

(15) (34) (32) 

1 (3X) 1 (3X) 

(14) (30) (29) 

1 (3X) 

(15) (34) (32) 

1 (3X) 1 (3X) 

(15) (34) (32)

 

1 (3X) 

* NUMBER OF AfllMALS WITH TISSUE EXAHINED HICBOSCOPIC4LLY 

* NUMBE R OF ANIMALS NECBOPSIED 

77 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

HYPEBPLASIA, CYSTIC 10 (67X) 31 (91S) 23 (12%)
 

tOVARY (15) (34) (32)
 

CYST, NOS 2 (13») 12 (35X) 5 (16X)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

*EYE/CORNEA (15) (34) (32)
 

INFLAMMATION, CHRONIC SOPPORATIV 1 (355)
 

HUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED 1
 

AUTO/NECHOPSY/HISTO PEHF 1
 

AOTOLYSIS/NO NECROPSY 1 3
 

I NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

78
 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS FED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET
 

79
 

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

Fed Phenazopyridine Hydrochloride in the Dieta
 

Topography; Morphology 

Lung: Alveolar/Bronchiolar 

Adenoma^ 

P Values0 >d 

Departure from Linear Trend6 

Relative Riskf 

Lower Limit 

Upper Limit CD
 

Weeks to First Observed Tumor 

Hematopoietic System: 

Malignant Reticulosis^ 

P Values

c

'

d 

Relative Riskf 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Matched Low High 

Control Dose Dose 

0/12 (0) 3/33 (9) 0/35 (0) 

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

P = 0.048 

Infinite — 0.238 

Infinite — 

— 

81 — 

0/13 (0) 2/33 (6) 0/35 (0) 

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

__ Infinite 

0.125 — Infinite — 

73 — 
Table El. Analyses of the Incidence of Primary Tumors in Male
 

Fed Phenazopyridine Hydrochloride in the Dieta
 

(continued)
 

Topography: Morphology


Large Intestine (Colon or
 

Rectum): Adenocarcinoma, NOSb


P Valuesc>d


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 oo
 

ho
 

Large Intestine (Colon or
 

Rectum): Adenocarcinoma, NOS
 

or Adenoma, NOSb


P Valuesc'd


Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Matched
 

 Control
 

 0/14 (0)
 

P = 0.028
 

—
 

 0/14 (0)
 

P = 0.015
 

—
 

Low
 

Dose
 

4/34 (12)
 

N.S.
 

Infinite
 

0.410
 

Infinite
 

105
 

4/34 (12)
 

N.S.
 

Infinite
 

0.410
 

Infinite
 

105
 

Rats
 

High
 

Dose
 

8/35 (23)
 

N.S.
 

Infinite
 

0.985
 

Infinite
 

98
 

9/35 (26)
 

P = 0.034
 

Infinite
 

1.134
 

Infinite
 

98
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed Phenazopyridine Hydrochloride in the Dieta
 

(continued)
 

Matched Low
 

Topography: Morphology Control Dose
 

Adrenal: Pheochromocytoma^ 2/13 (15) 1/34 (3)
 

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

Relative Riskf 0.191
 

Lower Limit 0.004
 

Upper Limit 3.442
 

Weeks to First Observed Tumor 105 105
 

oo Thyroid: C-cell Adenomab 2/14 (14) 0/32 (0)
 

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

Relative Riskf 0.000
 

Lower Limit 0.000
 

Upper Limit 1.447
 

Weeks to First Observed Tumor 93
 —
 

High
 

Dose
 

1/34 (3)
 

N.S.
 

0.191
 

0.004
 

3.442
 

104
 

1/32 (3)
 

N.S.
 

0.219
 

0.004
 

3.935
 

101
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Fed Phenazopyridine Hydrochloride in the Diet3
 

(continued)
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Testis: Interstitial-cell Tumorb 14/14 (100) 22/34 (65) 7/33 (21)
 

P Value sc>d P < O.OOl(N) P = 0.008(N) P < O.OOl(N)
 

Relative Riskf 0.647 0.212
 

Lower Limit 0.000 0.000
 

Upper Limit 0.868 0.349
 

Weeks to First Observed Tumor 92 81 101
 

oo aDosed groups received 3,700 or 7,500 ppm.
 -P»
 

"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 dosed group is the probability level for the Fisher exact test for
 

the comparison of that dosed group with the matched-control group when P < 0.05; 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 control
 

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

Fed	 Phenazopyridine Hydrochloride in the Diet3
 

Matched Low
 

Topography: Morphology Control Dose
 

Colon: Adenocarcinoma, NOSb

 0/14 (0) 3/33 (9)
 

P Valuesc»d N.S. N.S.
 

Relative Riskf Infinite
 

Lower Limit 0.273
 

Upper Limit Infinite
 

Weeks to First Observed Tumor	 105
 

CO	 Pituitary: Chromophobe Adenoma** 2/9 (22) 1/30 (3)
 

P Valuesc»d N.S. N.S.
 

Relative Riskf 0.150
 

Lower Limit 0.003
 

Upper Limit 2.655
 

Weeks to First Observed Tumor 97 105
 

High
 

Dose
 

5/32	 (16)
 

N.S.
 

Infinite
 

0.594
 

Infinite
 

105
 

1/25 (4)
 

N.S.
 

0.180
 

0.004
 

3.156
 

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

Fed Phenazopyridine Hydrochloride in the Dieta
 

(continued) 

Matched Low High 

Topography; Morphology Control Dose Dose 

Mammary Gland: Fibroadenoma^ 2/15 (13) 0/33 (0) 0/32 (0) 

P Values0 »d P = 0.032(N) N.S. N.S. 

Relative Riskf 0.000 0.000 

Lower Limit 0.000 0.000 

Upper Limit 1.506 1.551 

Weeks to First Observed Tumor 97 — 

°£ aDosed groups received 3,700 or 7,500 ppm.
 

^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 dosed group is the probability level for the Fisher exact test for
 

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

not significant (N.S.) is indicated.
 

d

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

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

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

group.
 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN MICE FED PHENAZOPYRIDINE HYDROCHLORIDE IN THE DIET
 

87
 

Table Fl. Analyses of the Incidence of Primary Tumors in

Fed Phenazopyridine Hydrochloride in the Dieta 

 Male Mice 

Topography; Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Lung: Alveolar/Bronchiolar Adenoma^1 1/15 (7) 3/35 (9) 4/34 (12) 

P Valuesc»d N.S. N.S. N.S. 

Relative Riskf 

Lower Limit 

Upper Limit 

1.286 

0.117 

65.499 

1.765 

0.200 

84.137 

Weeks to First Observed Tumor 106 100 91 

oo Lung: Alveolar/Bronchiolar

or Carcinomab

 Adenoma 

 1/15 (7) 4/35 (11) 4/34 (12) 

P Valuesc»d N.S. N.S. N.S. 

Relative Riskf 

Lower Limit 

Upper Limit 

1.714 

0.195 

81.831 

1.765 

0.200 

84.137 

Weeks to First Observed Tumor 106 100 91 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Fed Phenazopyridine Hydrochloride in the Dieta
 

(continued)
 

Matched Low High 

Topography: Morphology Control Dose Dose 

Hematopoietic System: Lymphoma"-1 1/15 (7) 0/35 (0) 2/34 (6) 

P Valuesc»d N.S. N.S. N.S. 

Relative Riskf 0.000 0.882 

Lower Limit 0.000 0.051 

Upper Limit 7.949 50.523 

Weeks to First Observed Tumor 106 105 — 

vo Hematopoietic System: Lymphoma or 

° Leukemiab 1/15 (7) 0/35 (0) 3/34 (9) 

P Valuesc»d N.S. N.S. N.S. 

Relative Riskf 0.000 1.324 

Lower Limit 0.000 0.121 

Upper Limit 7.949 67.356 

Weeks to First Observed Tumor 106 — 105 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Fed Phenazopyridine Hydrochloride in the Diet3
 

(continued) 

Matched Low 

Topography: Morphology Control Dose 

Liver: Hepatocellular Carcinomab

 2/15 (13) 5/35 (14) 

P Valuesc.d N.s. N.S.
 

Relative Riskf 1.071
 

Lower Limit 0.206
 

Upper Limit 10.495
 

Weeks to First Observed Tumor 90 100
 

Liver: Hepatocellular Adenoma
 

or Carcinomab 5/15 (33) 15/35 (43)
 

P Valuesc»d N.S. N.S.
 

Relative Riskf 1.286
 

Lower Limit 0.573
 

Upper Limit 3.811
 

Weeks to First Observed Tumor 90 64
 

a

Dosed groups received 600 or 1,200 ppm.
 

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

High
 

Dose
 

9/33 (27)
 

N.S.
 

2.045
 

0.509
 

17.921
 

82
 

15/33 (45)
 

N.S.
 

1.364
 

0.609
 

4.002
 

82
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice 

Fed Phenazopyridine Hydrochloride in the Diet3 

(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 dosed group is the probability level for the Fisher exact test for
 

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

not significant (N.S.) is indicated.
 

<*A negative trend (N) indicates a lower incidence in a dosed group than in the 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 control
 

group.
 

VO
 

Ni
 
__ 

Table F2. Analyses of the Incidence of Primary

Fed Phenazopyridine Hydrochloride in

Matched
 

Topography: Morphology Control
 

Integumentary System: Sarcoma, NOS,
 

of the Subcutaneous Tissueb 0/15 (0)
 

P Valuesc»d N.S.
 

Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor —
 

vo 

Lung: Alveolar/Bronchiolar Adenoma** 1/15 (7)
 

P Valuesc»d N.S.
 

Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 106
 

 Tumors in

 the Dieta 

Low
 

Dose
 

0/34 (0)
 

N.S.
 

—
 

—
 

—
 

1/34 (3)
 

N.S.
 

0.441
 

0.006
 

33.649
 

106
 

 Female Mice 

High
 

Dose
 

2/32 (6)
 

N.S.
 

Infinite
 

0.147
 

Infinite
 

88
 

2/32 (6)
 

N.S.
 

0.938
 

0.054
 

53.563
 

90
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed Phenazopyridine Hydrochloride in the Diet3
 

(continued)
 

Topography; Morphology
 

Hematopoietic System: Lymphomab
 

P Valuesc»d
 

Relative Riskf
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma'3
 

P Valuesc»d
 

Relative Risk^
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low 

Control Dose 

4/15 (27) 6/34 (18) 

P = 0.044(N) N.S. 

0.662 

0.193 

2.825 

90 89 

2/15 (13) 6/34 (18) 

P = 0.010 N.S. 

1.324 

0.281 

12.462 

90 90 

High
 

Dose
 

2/32 (6)
 

N.S.
 

0.234
 

0.024
 

1.469
 

90
 

14/32 (44)
 

P = 0.039
 

3.281
 

0.918
 

26.847
 

94
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Fed Phenazopyridine Hydrochloride in the Dieta
 

(continued) 

Matched Low 

Topography: Morphology Control Dose 

Liver: Hepatocellular Adenoma
 

or Carcinomab 2/15 (13) 11/34 (32)
 

P Valuesc»d P = 0.002 N.S.
 

Relative Riskf 2.426
 

Lower Limit 0.639
 

Upper Limit 20.695
 

Weeks to First Observed Tumor 90 90
 

Pituitary: Chromophobe Adenomab 1/10 (10) 2/30 (7)
 

p Valuesc»d N.S. N.S.
 

Relative Riskf 0.667
 

Lower Limit 0.041
 

Upper Limit 38.024
 

Weeks to First Observed Tumor 106 105
 

High
 

Dose
 

19/32 (59)
 

P = 0.003
 

4.453
 

1.327
 

34.479
 

94
 

0/23 (0)
 

N.S.
 

0.000
 

0.000
 

7.962
 

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

Fed Phenazopyridine Hydrochloride in the Diet3 

(continued)
 

Matched Low High
 

Topography; Morphology Control Dose Dose
 

Harderian Gland: Adenoma, NOS° 0/15 (0) 2/34 (6) 3/32 (9)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Riskf Infinite Infinite
 

Lower Limit 0.138 0.300
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 105 105
 —
 

a

Dosed groups received 600 or 1,200 ppm.
 •-o 

CTi 

^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 dosed group is the probability level for the Fisher exact test for
 

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

not significant (N.S.) is indicated.
 

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

group.
 
Review of the Bioassay of Phenazopyridine Hydrochloride*
 

for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

April 26, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DREW 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 Phenazopyridine
 

Hydrochloride for carcinogenicity.
 

The primary reviewer noted that the compound induced
 

colonic polyps in treated rats; the report did not indicate
 

whether they were multiple or whether metastases were
 

detected. An elevated but non-statistically significant
 

incidence of hepatomas was observed in treated male mice.
 He suggested that the report could be strengthened if it
 

indicated "the low nature of the response" and the questionab
 

malignant character of the colonic polyps. Given Phenazo­ pyridine Hydrochloride!

s use as a therapeutic agent, the
 

primary reviewer said that the carcinogenic risk to man is
 

minimal.
 

A Program staff pathologist commented that the colon
 

tumors were identical to those induced by known colon
 

carcinogens. He noted that some were polypoid and had a
 

degree of Invasion in the stalk when it was present. He
 
added that most colon carcinogens also induce liver tumors,
 as was the case in this bioassay.
 

A Subgroup member said that the data were adequate to
 

classify Phenazopyridine Hydrochloride as a weak colon
 

carcinogen. A Program staff member argued that tumor
 

incidence alone should not be the determinant in classifying
 

a carcinogen as weak or strong. A discussion ensued regarding
 

the assessment of strong versus weak carcinogens.
 

A motion was approved unanimously that the report on
 

the bioassay of Phenazopyridine Hydrochloride be accepted.
 

Members present were:
 

Michael Shimkin (Acting Chairman), University of California
 

at San Diego
 

Joseph Highland, Environmental Defense Fund
 

George Roush, Jr., Monsanto Company
 

Louise Strong, University of Texas Health Sciences Center
 

John Weisburger, American Health Foundation
 

Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

•ftU.S. GOVERNMEN T PRINTIN G OFFICE l 1978-260-899/3165
 

DHEW Publication No. (NIH) 78-1349