National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 18 

1978 

BIOASSAY OF 

3,3'-IMINOBIS-1-PROPANOL 

DIMETHANESULFONATE (ESTER)

HYDROCHLORIDE (IPD) 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 3458-22-8 

NCI-CG-TR-18 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

3,3'-IMINOBIS-l-PROPANOL
 

DIMETHANESULFONATE (ESTER) HYDROCHLORIDE [IPD]
 

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) 78-818
 

BIOASSAY OF
 

3,3'-IMINOBIS-l-PROPANOL
 

DIMETHANESULFONATE (ESTER) HYDROCHLORIDE [IPD]
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

CONTRIBUTORS; This report presents the results of the bioassay
 

of 3,3'-iminobis-l-propanol dimethanesulfonate (ester) hydrochloride [IPD] for possible carcinogenicity, conducted for the
 

Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute (NCI), Bethesda, Maryland·
 

The bioassay was conducted by Southern Research Institute,
 

Birmingham, Alabama, initially under direct contract to NCI and
 

currently under a subcontract to Tracor Jitco, Inc., prime
 

contractor for the NCI Carcinogenesis Testing Program.
 

The experimental design and doses were determined by Drs· D· P·
 1 1 2
 Griswold , J. D· Prejean , E. K. Weisburger , and J. Η.
 

Weisburger2

* 3# Ms· J. Belzer·* and Mr· I. Brown* were
 

responsible for the administration of the chemical and the care
 

of the laboratory animals· Data management and retrieval were
 

performed by Ms· C· A· Dominick*· Histopathologic examinations
 

were performed by Drs· S. D· Kosanke* and J· C· Peckham*, and the
 

diagnoses included in this report represent their interpretation·
 

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

Mason Research Institute^· The statistical analyses were
 

performed by Dr· J· R· Joiner", using methods selected for the
 

bioassay program by Dr· J. J. Gart'· Chemicals used in this
 

bioassay were analyzed under the direction of Dr· W· J· Haggerty,
 

Jr·^, and the results of the analyses were reviewed by Dr· S· S.
 

Olin6

·
 

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

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

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

iii
 
Campbell, Deputy Director of Science; Dr· J. F. Robens,
 

toxicologist; Dr. R· L. Schueler, pathologist; Dr. G. L· Miller
 

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

Gunberg, technical editor, assisted by Ms· Y· E· Presley·
 

The statistical analysis was reviewed by members of the
 

Mathematical Statistics and Applied Mathematics Section of NCI?:
 

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

Robert E. Tarone.
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment,
 

interpreting the results, and reporting the findings:
 

Dr. Kenneth C. Chu
 

Dr. Cipriano Cueto, Jr·
 

Dr. J. Fielding Douglas
 

Dr. Dawn G. Goodman
 

Dr. Richard A. Griesemer
 

Mr· Harry A· Milman
 

Dr. Thomas^ W· Or me
 

Dr· Robert A· Squire^
 

Dr. Jerrold M. Ward
 

^Southern Research Institute, 2000 Ninth Avenue South,
 

Birmingham, Alabama·
 

^Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland·
 

% o w with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York·
 

^Now with Battelle Pacific Northwest Laboratories, Battelle
 

Boulevard, Richland, Washington·
 

Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland·
 

iv
 
6Tracor Jitco, Inc· , 1776 East Jefferson Street, Rockville,
 

Maryland·
 

'Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute, National
 

Institutes of Health, Bethesda, Maryland·
 

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

Missouri·
 

^Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland·
 

SUMMARY
 

A bioassay of 3,3'-iminobis-l-propanol dimethanesulfonate (ester)
 

hydrochloride [IPD] for possible carcinogenicity was conducted by
 

administering the test chemical intraperitoneally to Sprague-


Dawley rats and B6C3F1 mice.
 

The IPD was injected three times per week to groups of 35
 

animals, using doses of 12, 24, or 48 mg/kg for the rats, and 20
 

or 40 mg/kg for the mice. Rats at 12 mg/kg were treated for 52
 

weeks. Because of the toxicity of the chemical, administration
 

of IPD for the group receiving 24 mg/kg was discontinued at week
 

34. Rats receiving 48 mg/kg were treated until all had died at
 

week 23 (males) and week 27 (females). Both groups of mice were
 

treated for 52 weeks. All survivors were killed after post-


administration periods that varied among groups.
 

With rats, untreated and vehicle-control groups, each consisting
 

of 10 males and 10 females, were started with the high- and mid-


dose groups and additional untreated and vehicle-control groups
 

of the same size were started with the low-dose groups. With
 

mice, untreated and vehicle-control groups each consisted of 15
 

males and 15 females.
 

The toxicity of IPD was associated with lower mean body weights
 

and lower rates of survival of both the rats and mice. The 

shortened life spans, particularly in the rats, reduced the 

likelihood of the development of tumors. 

In rats, peritonitis and fibrous adhesions, possibly, from direct
 

irritation by the test chemical were observed in most treated
 

rats at necropsy. Sarcoma, fibroma, or fibrosarcoma of the
 

peritoneum occurred in two low-dose male, one mid^dose male, and
 

three mid-dose female rats, but not in any control animals.
 

Because of this low incidence, and because irritation by the test
 

chemical may have been involved in the pathogenesis, these tumors
 

may have been due to local effects of the chemical.
 

vii
 
In mice, lymphomas were observed at the following incidences
 

(males: controls 0/14, low-dose 0/26, high-dose 3/21; females:
 

controls 1/15, low-dose 2/29, high-dose 6/27). The Tarone test
 

for life-table analysis of the probability of survival without
 

lymphoma indicated a significant positive dose-related increase
 

of lymphomas with a probability level of 0.011 for male mice and
 

0.003 for female mice.
 

Squamous-cell carcinoma was noted in the mice (low-dose males
 

6/26, high-dose females 2/27). Seven of these tumors were
 

observed in subcutaneous tissue in the inguinal region near the
 

sites of injection. Although not statistically significant, this
 

tumor may be associated with administration of IPD.
 

Tumors of the peritoneum in rats and tumors in the subcutaneous
 

tissue in mice may have been due to local effects related to
 

administration of the test chemical. The lymphomas in mice,
 

although marginally significant, were too few in number to
 

clearly be related to dosing.
 

Conclusions from this study are limited by early deaths and
 

toxicity, but the appearance of tumors in the peritoneum near the
 

injection sites in both rats and mice indicate the carcinogenic
 

potential of IPD.
 

viii
 
TABLE OF CONTENTS
 

Page 

I· Introduction · 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

. Dosage Preparation 3
 

C. Animals 4
 

D. Animal Maintenance 4
 

E. Subchronic Studies 7
 

F. Designs of Chronic Studies 8
 

G. Clinical and Pathologic Examinations 12
 

H. Data Recording and Statistical Analyses 13
 

III. Results - Rats 19
 

A. Body Weights and Clinical Signs (Rats) 19
 

B. Survival (Rats) 19
 

C. Pathology (Rats) 22
 

D· Statistical Analyses of Results (Rats) 26
 

IV. Results - Mice 29
 

A. Body Weights and Clinical Signs (Mice) 29
 

B. Survival (Mice) 29
 

C. Pathology (Mice) 32
 

D. Statistical Analyses of Results (Mice) 35
 

V. Discussion. · · · · 39
 

VI. Bibliography 43
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms
 

in Rats Given Intraperitoneal Injections
 

of IPD 47
 

Table Al Summary of the Incidence of Neoplasms
 

in Male Rats Given Intraperitoneal
 

Injections of IPD (Control Groups) 49
 

ix
 
Page 

Table A2 Summary of the Incidence of Neoplasms 

in Male Rats Given Intraperitoneal 

Injections of IPD (Treated Groups) 52 

Table A3 Summary of the Incidence of Neoplasms 

in Female Rats Given Intraperitoneal 

Injections of IPD (Control Groups) 55 

Table A4 Summary of the Incidence of Neoplasms 

in Female Rats Given Intraperitoneal 

Injections of IPD (Treated Groups) 58 

Appendix Β Summary of the Incidence of Neoplasms 

in Mice Given Intraperitoneal Injections 

of IPD 61 

Table Bl Summary of the Incidence of Neoplasms 

in Male Mice Given Intraperitoneal 

Injections of IPD 63 

Table B2 Summary of the Incidence of Neoplasms 

in Female Mice Given Intraperitoneal 

Injections of IPD 66 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Given Intraperitoneal 

Injections of IPD 69 

Taole CI Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Given Intraperitoneal 

Injections of IPD (Control Groups) 71 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Given Intraperitoneal 

Injections of IPD (Treated Groups) 74 

Table C3 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Given Intraperitoneal 

Injections of IPD (Control Groups) 77 

Table C4 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Given 

Intraperitoneal Injections of IPD 

(Treated Groups) 80 

χ 
Page 

Appendix D Summary of the Incidence ,of Nonneoplastic 

Lesions in Mice Given Intraperitoneal 

Injections of IPD 83 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Given Intraperitoneal Injections of IPD 85 

Table Ό2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Given Intraperitoneal Injections of IPD 88 

Appendix Ε Analyses of the Incidence of Primary 

Tumors in Rats Given Intraperitoneal 

Injections of IPD 91 

Table El Analyses of the Incidence of Primary 

Tumors in Male Rats Given Intraperitoneal Injections of IPD 93 

Table E2 Analyses of the Incidence of Primary 

Tumors in Female Rats Given Intraperitoneal Injections of IPD 94 

Table E3 Analyses of the Incidence of Primary 

Tumors in Female Rats Given Intraperitoneal 

Injections of IPD 95 

Table E4 Time-adjusted Analyses of Peritoneal 

Tumors in Female Rats Given Intraperitoneal 

Injections of IPD 99 

Appendix F Analyses of the Incidence of Primary 

Tumors in Mice Given Intraperitoneal 

Injections of IPD 101 

Table Fl Analyses of the Incidence of Primary 

Tumors in Male Mice Given Intraperitoneal Injections of IPD 103 

Table F2 Analyses of the Incidence of Primary 

Tumors in Female Mice Given Intraperi

Injections of IPD 105 

xi
 
Page 

Table F3 Analyses of the Incidence of Primary 

Tumors in Female Mice Given 

Intraperitoneal Injections of IPD 106 

Table F4 Time-adjusted Analyses of Hematopoietic 

Tumors in Female Mice Given 

Intraperitoneal Injections of IPD 109 

Appendix G Histologic Features of Tumors Observed 

in Sprague-Dawley Rats Given IPD in 

Buffered Saline Intraperitoneally Three 

Times Weekly for One Year Ill 

TABLES 

Table 1 Design of Chronic Studies of IPD 

in Rats 9 

Table 2 Design of Chronic Studies of IPD 

in Mice 11 

FIGURES 

Figure 1 Growth Curves for Rats Treated with IPD.... 20 

Figure 2 Survival Curves for Rats Treated with 

IPD 21 

Figure 3 Growth Curves for Mice Treated with IPD.... 30 

Figure 4

Figure 5

 Survival Curves for Mice Treated with 

IPD

 Life Table for Mice Treated with IPD: 

Malignant Lymphoma

 31 

 37 

xii
 
I. INTRODUCTION
 

3,3'-Iminobis-l-propanol dimethanesulfonate (ester) hydrochloride
 

(CAS 3458-22-8; NCI C01547), hereinafter called IPD, was
 

synthesized from bis(3-hydroxypropyl)amine and methanesulfonic
 

acid anhydride (El-Merzabani and Sakurai, 1965)· It was found to
 

have antitumor activity against a number of experimental tumors
 

that were naturally resistant to nitrogen mustard and has been
 

used in Japan for the treatment of myelogenous leukemia
 

(El-Merzabani and Sakurai, 1965; Hirano et al. , 1972). IPD was
 

selected for carcinogen bioassay as one agent in a series of
 

anticancer drugs that are administered chronically in the
 

treatment of human cancer·
 

II. MATERIALS AND METHODS
 

A. Chemical
 

The IPD was supplied by the Drug Development Branch, Division of
 

Cancer Treatment (DCT), National Cancer Institute (NCI). It was
 

purchased from Yoshitomi Pharmaceutical Industries, Limited, 35
 

Hiranomachi 3-chome, Higashi-ku, Osaka, Japan.
 

Analyses of each of two batches were provided through contracts
 

of the Division of Cancer Treatment, NCI, and showed that the
 

material consisted of > 99% of the designated chemical. The
 

analytical methods included melting point, infrared and nuclear
 

magnetic resonance spectroscopy, thin-layer chromatography (three
 

solvent systems), and elemental analysis. No impurities were
 

detected.
 

The IPD was stored at -20°C in the original glass container until
 

used in this study.
 

B. Dosage Preparation
 

The IPD was prepared in phosphate-buffered saline as a fresh
 

solution immediately prior to use. The actual mixing of the drug
 

and the vehicle was performed in the animal laboratory, in a
 

10-ml Potter-Elvehjem tissue grinder with a Teflon pestle. The
 

concentrations administered were 0.48%, 0.96%, or 1.92% (w/v) for
 

3 
rats, and 0.2% or 0.4% (w/v) for mice. The test chemical
 

solution or the vehicle was administered to the treated animals
 

or vehicle controls intraperitoneally, using one needle for each
 

injection group at a constant volume of 0.25 ml/100 g body weight
 

for rats, or 1.0 ml/100 g for mice. Unused solutions of IPD were
 

discarded each day of administration.
 

C. Animals
 

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

mice of each sex, obtained from the Charles River Breeding
 

Laboratories, Inc., Wilmington, Massachusetts, through contracts
 

of the Division of Cancer Treatment, NCI, were used. For the
 

chronic studies, Sprague-Dawley rats anjd B6C3F1 mice, obtained
 

from Charles River Breeding Laboratories, Inc., were used. On
 

arrival at the laboratory, all animals were quarantined for an
 

acclimation period (rats for 5 days, mice for 8 days), assigned
 

to control or treated groups, and then 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%. In addition to natural light,
 

illumination was provided by fluorescent light for 9 hours per
 

4 
day. Wayne® Lab Blox animal meal (Allied Mills, Inc., Chicago,
 

111.) and water were supplied daily and were made available ad
 

libitum.
 

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

solid-bottom stainless steel cages (Hahn Roofing and Sheet Metal
 

Co., Birmingham, Ala.). The bottoms of the rat cages were lined
 

with Iso-Dri® hardwood chips (Carworth, Edison, N. J.), and cage
 

tops were covered with disposable filter bonnets beginning at
 

week 22; 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; racks were cleaned once per
 

week.
 

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

were housed with respective treated animals. Animals treated
 

with IPD were maintained in the same rooms as animals of the same
 

species being treated with the following chemicals:
 

RATS
 

Gavage Studies
 

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

(phenesterin) (CAS 3546-10-9)
 

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

(estradiol mustard) (CAS 22966-79-6)
 
Intraperitoneal Injection Studies
 

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

(MAAM) (NSC 141549)
 

acronycine (CAS 7008-42-6)
 

5-azacytidine (CAS 320-67-2)
 

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

(CAS 789-61-7)
 

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

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

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

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

N,3-bis(2-chloroethyl)tetrahydro-2H-l,3,2-oxazaphosphorin-2­

amine-2-oxide (isophosphamide) (CAS 3778-73-2) 

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

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

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

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

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

MICE
 

Feed Studies
 

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

(acetohexamide) (CAS 968-81-0)
 

anthranilic acid (CAS 118-92-3)
 

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

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

(chlorpropamide) (CAS 94-20-2)
 

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

(pyrimethamine) (CAS 58-14-0)
 

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

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

L-tryptophan (CAS 73-22-3)
 

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

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

(tolazamide) (CAS 1156-19-0)
 

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

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

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

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

ethionamide (CAS 536-33-4)
 

6 
Gavage Studies
 

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

(phenesterin) (CAS 3546-10-9)
 

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

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

Intraperitoneal Injection Studies
 

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

(MAAM) (NSC 141549)
 

acronycine (CAS 7008-42-6)
 

5-azacytidine (CAS 320-67-2)
 

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

(CAS 789-61-7)
 

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

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

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

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

N,3-bis(2-chloroethyl)tetrahydro-2H-l,3,2-oxazaphosphorin-2­

amine-2-oxide (isophosphamide) (CAS 3778-73-2)
 

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

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

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

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

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

E. Subchronic Studies
 

Subchronic studies were conducted with female Sprague-Dawley rats
 

and male and female Swiss mice to estimate the maximum tolerated
 

doses of IPD, on the basis of which low and high concentrations
 

(hereinafter referred to as "low doses11 and "high doses") were
 

determined for administration in the chronic studies. In these
 

subchronic studies, IPD was administered intraperitoneally three
 

times per week for 45 days to female rats ana mice of each sex in
 

twofold increasing concentrations, followed by 45 days of
 

7 
observation. The treated groups consisted of five animals each;
 

the vehicle controls consisted of 10 animals each. The rats
 

received 24, 48, 96, or 192 mg/kg/dose; the mice received 40, 80,
 

160, or 320 mg/kg/dose.
 

All rats receiving doses of 96 or 192 mg/kg died. The mortality
 

rates for rats receiving 24 or 48 mg/kg were 20% and 40%, respectively; at these doses there were no differences in mean body
 

weight between the 90-day survivors and the controls.
 

All mice receiving doses of 160 or 320 mg/kg died. The mortality
 

rate for mice receiving 80 mg/kg was 90%. At 40 mg/kg, all mice
 

survived, but there was a 30% depression in mean body weight,
 

compared with the controls.
 

Low and high doses for the chronic study were set at 24 and 48
 

mg/kg, respectively, for rats and 20 and 40 mg/kg for mice.
 

F. Designs of Chronic Studies
 

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

Originally, doses of 24 or 48 mg/kg were administered to groups
 

of rats of each sex; however, toxicity resulted at the high dose,
 

and low-dose groups at 12 mg/kg were started on study at week 56.
 
Table 1. Design of Chronic Studies of IPD in Rats
 

Sex and Initial
 

Test No,. of
 

Group Animalsa
 

Male
 

Low-Dose
 

Untreated-Controls0 10
 

Mid- and High-Dose
 

Untreated-Control 10
 

Low-Dose
 

Vehicle-Controls0 10
 

Mid- and High-Dose
 

Vehicle-Control 10
 

Low-Dose 35
 

Mid-Dose 35
 

High-Dose 35
 

Female
 

Low-Dose
 

Untreated-Controls0 10
 

Mid- and High-Dose
 

Untreated-Control 10
 

Low-Dose
 

Vehicle-Controls0 10
 

Mid- and High-Dose
 

Vehicle-Control 10
 

Low-Dose 35
 

Mid-Dose 35
 

High-Dose 35
 

IPD
 

Doseb
 

(mg/kg)
 

0
 

0
 

0d
 

0d
 

12
 

24
 

48
 

0
 

0
 

0d
 

0d
 

12
 

24
 

48
 

Time on
 

Treated
 

(weeks)
 

52
 

52
 

34e
 

238
 

52
 

52
 

52
 

34e
 

278
 

Study
 

Untreated
 

(weeks)
 

89
 

89
 

37
 

37
 

3
 

38f
 

89
 

89
 

37
 

37
 

28
 

37*
 

aHigh- and mid-dose males, with controls, were 35 days of age when
 

placed on study; females with controls were 42 days of age. Low-


dose males and females, with controls, were 55 days of age when
 

placed on study·
 

bIPD was administered in buffered saline by intraperitoneal
 

injection three times per week at a volume of 0.25 ml/100 g body
 

weight. Doses were based on individual weights.
 

9
 
Table 1. Design of Chronic Studies of IPD in Rats
 

(continued)
 

cTen controls were started initially with the mid- and high-dose
 

groups, the other 10 were started concurrently with the low-


dose group.
 

^Vehicle-control groups received only buffered saline ^solution,
 

at the same volume as treated rats.
 

eMid-dose male and female animals were treated only 34 weeks due
 

to the toxicity of the chemical.
 

fMid-dose male and female animals were observed only 38 and 37
 

weeks, respectively, due to the death of all animals.
 

^High-dose male and female animals were treated only 23 and 2 7
 

weeks, respectively, due to the death of all animals.
 

10
 
Table 2. Design of Chronic Studies of IPD in Mice
 

Sex and Initial IPD Time on Study 

Test No. of Doseb Treated Untreated 

Group Animalsa (mR/kg) (weeks) (weeks) 

Male 

Untreated-Control 15 0 86 

Vehicle-Control 15 0c 52 34 

Low-Dose^ 34 20 52 25 

High-Dose 35 40 52 11 

Female 

Untreated-Control 15 0 86 

Vehicle-Control 15 0c 52 34 

Low-Dose" 36 20 52 34 

High-Dose 35 40 52 25 

aAll animals were 38 days of age when placed on study.
 

°IPD was administered in buffered saline by intraperitoneal
 

injection three times per week at a volume of 1 ml/100 g body weight.
 

Doses were based on the mean weights of the animals in each cage.
 

c

Vehicle-control groups received only buffered saline solution,
 

at the same volume as treated mice.
 

d
The low-dose group consisted of 34 males and 36 females instead
 

of 35 animals of each sex, because of missexing during the
 

initiation of the study·
 

11
 
G. Clinical and Pathologic Examinations
 

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

and animals that were moribund were killed and necropsied. Rats
 

(mid- and high-dose) and mice were weighed individually each week
 

for the first 8 weeks, once every 2 weeks for the next 72 weeks,
 

and once per month for the remainder of the study. Low-dose rats
 

were weighed once every 2 weeks for 66 weeks and once per month
 

thereafter. Palpation for masses was carried out at each
 

weighing.
 

The pathologic evaluation consisted of gross and microscopic
 

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

from killed animals and from animals found dead. The following
 

tissues were examined microscopically: skin, muscle, lungs and
 

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

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

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

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

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

brain, and sensory organs. Peripheral blood smears were taken
 

from each animal. Occasionally, additional tissues were also
 

examined microscopically. The different tissues were preserved
 

in 10% buffered formalin, embedded in paraffin, sectioned, and
 

12
 
stained with hematoxylin and eosin· Special staining techniques
 

were utilized when indicated for more definitive diagnosis.
 

A few tissues from some animals were not examined, particularly
 

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

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

of autolysis as to preclude histopathologic evaluation. Thus,
 

the number of animals from which particular organs or tissues
 

were examined microscopically varies, and does not necessarily
 

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

group.
 

H. Data Recording and Statistical Analyses
 

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

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

clinical observations, survival, body weight, and individual
 

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
 

13
 
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 Ρ values have been reported for all tests except the
 

departure from linearity test, which is only reported when its
 

two-tailed Ρ value is less than 0.05.
 

The incidence of neoplastic or nonneoplastic lesions has been
 

given as the ratio of the number of animals bearing such lesions
 

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

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

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic examination was required to detect lesions prior to histologic sampling
 

(e.g., skin or mammary tumors), or when lesions could have appear14
 
ed at multiple sites (e.g., lymphomas), the denominators consist
 

of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

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

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

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

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

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

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

 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 Ρ values are shown.
 

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

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

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

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


tailed 0.05 level of significance. Unless otherwise noted, the
 

direction of the significant trend is a positive dose relation15
 
ship· This method also provides a two-tailed test of departure
 

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

first tumor was observed were excluded by basing the statistical
 

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

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

such an early tumor was found, comparisons were based exclusively
 

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

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

obtained, the standard procedures for analyses of the incidence
 

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

followed.
 

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

incidence of tumors. Curves of the proportions surviving without
 

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

The week during which an animal died naturally or was sacrificed
 

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

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
 

16
 
noted, in the direction of a positive dose relationship·
 

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

were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each treated group compared to its control was calculated
 

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

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

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

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

spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

a specific tumor in a treated group and the proportion in a
 

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

excess of unity represent the condition of a larger proportion in
 

the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in
 

approximately 95% of a large number of identical experiments, the
 

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

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

17
 
significant result ( < 0.025 one-tailed test when the control
 

incidence is not zero, Ρ < 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.
 

18
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

The mean body weights of rats of each sex treated with IPD were
 

consistently lower than those of the vehicle controls (figure 1)·
 

Although the suppression of mean body weights was more marked in
 

the males than in the females, the data indicate dose-related
 

effects for both sexes. The growth rates of the untreated
 

controls, not shown, were similar to those of the vehicle
 

controls. Fluctuations in the growth curve may be due to
 

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

weight may be subject to wide variation. As the study
 

progressed, all treated animals developed a poor physical
 

condition; however, no other clinical signs were recorded.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats receiving IPD at the doses used
 

in this study, together with those of the controls, are shown in
 

figure 2. The following table shows the numbers of weeks on
 

study at which 50% and 100% mortality occurred in the treated and
 

control rats.
 

19
 
TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves for Rats Treated with IPD 

20 
Figure 2. Survival Curves for Rats Treated with IPD
 

21 
Time to Death (Weeks on Study)
 

Treated Group 50% Mortality 100% Mortality
 

Male Female Male Female
 

High-Dose 11 11 23 27
 

Mid-Dose 37 37 72 71
 

Low-Dose 38 47 55 80
 

Vehicle-Control 89+ 89+ 89+ 89+
 

The data show that as the dose increased, the time to death
 

decreased. The survival curves show highly significant
 

(P < 0.001) dose-related positive trends in mortality, which are
 

not linear (P < 0.001). The nonlinearity is due to the steep
 

declines in survival of the treated groups compared with the
 

vehicle-control groups.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

Appendix A, tables A1-A4; findings on nonneoplastic lesions are
 

summarized in Appendix C, tables C1-C4.
 

A variety of tumors occurred in both the control (untreated and
 

vehicle) and chemical-treated groups. Some types of neoplasms
 

occurred only, or with greater frequency, in rats of treated
 

groups compared with those of control groups. These lesions,
 

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

the administration of any test chemical.
 

22
 
A small number of spindle-cell tumors occurred in the subcutaneous and peritoneal tissues with metastasis to the lungs and
 

mediastinal lymph nodes. The subcutaneous tumors included one
 

fibroma (1/18 [6%] in the combined groups of vehicle-control
 

females) and two fibrosarcomas (1/20 [5%] in the cpmbined
 

vehicle-control males and 1/33 [3%] in the low-dose females).
 

The peritoneal tumors occurred in six rats: one fibroma (1/28
 

[4%] in the mid-dose males); two fibrosarcomas (1/28 [4%] in the
 

mid-dose males and 1/31 [3%] in the mid-dose females); and four
 

undifferentiated or pleomorphic spindle-cell sarcomas coded as
 

sarcoma, NOS (not otherwise specified), (2/32 [6%] in the low-


dose males and 2/31 [6%] in the mid-dose females). In one mid-


dose female rat, a peritoneal fibrosarcoma had metastasized to
 

the mediastinal lymph nodes. In another mid-dose female rat, a
 

peritoneal sarcoma of unspecified type had metastasized to the
 

lungs. No sarcomas were found in untreated control animals.
 

Both the subcutaneous and the peritoneal spindle-cell tumors
 

varied in the degree of differentiation and had various degrees
 

of collagenous formation. The fibromas had well-differentiated
 

fibroblasts with ample collagen, whereas the fibrosarcomas were
 

more pleomorphic, more anaplastic, and more variable in the
 

amount of collagen deposited. The poorly differentiated spindle-


cell tumors with little or no production of collagen were classi23
 
fied as sarcoma, NOS. One of the subcutaneous fibrosarcomas and
 

two of the sarcoma, NOS, lesions were pleomorphic with formation
 

of bizarre multinucleated giant cells. Two of the nonspecified
 

sarcomas had extensively infiltrated the smooth muscle of the
 

digestive tract. The confusing blend of neoplastic and nonneoplastic tissue made further classification of the sarcomas
 

virtually impossible. The possibility of a leiomyosarcoma was
 

not ruled out. Adenocarcinomas were observed in the large
 

intestine in 2/28 (7%) of the mid-dose males. Both lesions were
 

well differentiated, and one had large glandular spaces lined by
 

columnar, cuboidal, and squamous epithelial cells. These spaces
 

were filled by large amounts of mucin. Glands of this tumor had
 

invaded the muscle layers.
 

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

proliferative, and inflammatory changes were also encountered in
 

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

most part, these nonneoplastic lesions are commonly seen in aged
 

rats; however, the proliferative nonneoplastic lesions of the
 

connective tissues lining the peritoneum were associated with
 

treated groups. The incidences of these nonneoplastic lesions
 

together with incidences of neoplastic peritoneal lesions, were
 

as follows:
 

24
 
MALES
 

Number of animals
 

necropsied
 

Peritoneum
 

Chronic Inflammation
 

Fibrous Adhesion, NOS
 

Fibroma
 

Fibrosarcoma
 

Sarcoma, NOS
 

(spindle-cell)
 

FEMALES
 

Number of animals
 

necropsied
 

Peritoneum
 

Chronic Inflammation
 

Fibrous Adhesion, NOS
 

Fibrosarcoma
 

Sarcoma, NOS
 

(spindle-cell)
 

Untreated
 

Control
 

(20)
 

0
 

0
 

0
 

0
 

0
 

(20)
 

0
 

0
 

0
 

0
 

Vehicle Low Mid High 

Control Dose Dose Dose 

(20) (32) (28) (30) 

0 30 23 7 

0 21 5 0 

0 0 1 0 

0 0 1 0 

0 2 0 0 

(18) (33) (31) (31) 

0 32 28 7 

0 30 8 0 

0 0 1 0 

0 0 2 0 

Chronic peritonitis and needle trauma may have had an important
 

role in the pathogenesis of these peritoneal neoplasms. Needle
 

injuries may also have been a factor in the induction of the
 

subcutaneous fibromas and fibrosarcomas.
 

The small number of tumors observed may have been influenced by
 

complications of severe chronic peritonitis and bone-marrow
 

atrophy, with resulting decreased life span. With the reduced
 

25
 
period at risk, however, the tumors that were observed may have
 

greater importance.
 

Injection of rats with IPD resulted in few tumors· The majority
 

of the neoplastic lesions appeared unrelated to administration of
 

the chemical. Adenocarcinomas of the large intestine and
 

peritoneal sarcomas may have significance. The effectiveness of
 

the carcinogenesis bioassay was reduced by an associated decrease
 

in life span resulting from bone-marrow atrophy and severe
 

chronic peritonitis. In the judgment of the pathologist, the
 

results of this study failed to define the carcinogenic activity
 

of IPD in Sprague-Dawley rats.
 

Histologic features of the tumors referred to above are presented
 

in Appendix G.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix Ε contain the statistical analyses
 

of the incidences of those primary tumors that were observed in
 

at least 5% of one or more than one treated group of either sex.
 

No pooled-control groups are used in the statistical analyses,
 

since there are no controls from other studies that are suitable
 

for pooling. The untreated-control groups are not used in the
 

analyses, since the conditions of the vehicle-control groups are
 

more nearly comparable to the conditions of the treated groups.
 

26
 
Two separate analyses using the Cochran-Armitage test for linear
 

trend are included in the tables. The first line in the tables
 

shows the results of the analysis of the incidences of tumors in
 

the four groups of animals (vehicle-control, low-dose, mid-dose,
 

and high-dose groups), while the second line shows the results of
 

the analysis of the incidences seen in the three groups of
 

animals (vehicle-control, low-dose, and mid-dose groups)·
 

In each sex, neither the Cochran-Armitage tests for positive
 

dose-related trend in proportions for the incidence of tumors at
 

any site (using either three doses or two doses) nor any of the
 

Fisher exact tests for the comparison of the incidences of tumors
 

in a treated group with that in the controls in the positive
 

direction is significant at the 0·05 level. The survival was so
 

poor in the treated groups that little reliance can be placed on
 

the negative results of this test. Significant results in the
 

negative direction are observed in the incidences of mammary and
 

pituitary tumors, which may be accounted for by the early
 

mortalities of the treated animals.
 

In all of the 95% confidence intervals shown in the tables,
 

values of one or less than one are included, indicating the
 

absence of positive statistically significant results. It should
 

also be noted that in each of the intervals with an upper limit
 

greater than one, there is the theoretical possibility of the
 

27
 
induction of that particular tumor by IPD, which could not be
 

detected under the conditions of this test.
 

Time-adjusted analysis on the proportions of sarcoma, NOS,
 

fibroma, or fibrosarcoma of the peritoneum in male rats is shown
 

in table E2. The results of the Cochran-Armitage test are
 

significant (P = 0·031 when control, low-, mid and high-dose
 

groups are used, and Ρ = 0.045 when only control, low and mid-


dose groups are used), but the Fisher exact tests are not
 

significant.
 

The time-adjusted analysis on the incidence of sarcoma, NOS, or
 

fibrosarcoma of the peritoneum in female rats is shown in table
 

E4. The results of the Cochran Armitage test are significant
 

(P < 0.001), but departures from linear trend are observed (P <
 

0.001). These departures from a linear effect result from the
 

higher proportion observed in the mid-dose group when compared
 

with either the low-dosed or high-dosed groups. The Fisher exact
 

test shows that the incidence in the mid-dose rats is significantly higher (P = 0.003) than that in the matched vehicle controls.
 

These statistical test results suggest the possibility of dose
 

association; however, it should be noted that the sample size
 

used is very small, especially that in the mid-dose group, which
 

is only 4. The zero incidence observed in the high-dose rats is
 

probably due to the severe early mortality of this group of rats.
 

28
 
IV· RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

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

groups of mice were consistently lower in a generally dose-


related manner than those of the vehicle controls (figure 3) .
 

The growth rates of the untreated controls, not shown, were
 

similar to those of the vehicle controls. Fluctuations in the
 

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

diminishes, the mean body weight may be subject to wide
 

variation. No other clinical signs were recorded.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats receiving IPD at the doses used
 

in this study, together with those of the controls, are shown in
 

figure 4. The following table shows the numbers of weeks that
 

elapsed before 50% and 100% mortality occurred in the treated and
 

control mice.
 

Time to Death (Weeks on Study)
 

Treated Group 50% Mortality 100% Mortality 

Male Female Male Female 

High-Dose 53 63 63 77 

Low-Dose 63 65 77 86 

Vehicle-Control 86+ 86+ 86+ 86+ 

29
 
TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves for Mice Treated with IPD 

30 
Figure 4. Survival Curves for Mice Treated with IPD
 

31 
The data show that as the dose increased, the time to death
 

decreased·
 

As in rats, the results of the Tarone test for mice showed a
 

highly significant dose-related trend in mortality (P < 0·001 for
 

each sex). Neither males nor females showed a significant
 

departure from linear trend in relation to dose.
 

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 squamous-cell carcinomas and malignant
 

lymphomas, the neoplasms listed in Appendix Β appeared with
 

approximately equal frequency in the treated and control mice, or
 

appeared in insignificant numbers. Most of the squamous-cell
 

carcinomas were located in the inguinal region and were believed
 

to have originated from the preputial glands. Since the region
 

where these arose was also near the sites of injection, local
 

factors may have been involved in the pathogenesis.
 

Squamous-cell carcinomas in eight mice were recorded. The
 

incidences were 6/26 (23%) in the low-dose males and 2/27 (7%) in
 

the high-dose females. Seven of the squamous-cell carcinomas,
 

32
 
(in the six low-dose males and in one of the high-dose females)
 

occurred in subcutaneous tissue in the inguinal area near the
 

sites of injections. The presence of cyst walls in organization
 

of the tissues suggests that these tumors arose from the
 

epithelium of preputial glands. The lesions were similar in
 

appearance, with abundant well-differentiated keratin-forming
 

pearls in the center and poorly organized pleomorphic squamous
 

cells in the walls. Some of the lesions were atypical with
 

bizarre giant tumor cells. Several lesions had numerous mitotic
 

figures. One lesion had invaded a perineural lymphatic. The
 

eighth tumor was a squamous-cell carcinoma of the sebaceous
 

glands in the ear canal of a female mouse. Neoplastic squamous
 

cells had replaced the sebaceous glands and resulted in
 

epithelial atypism and hypercellularity. (A tissue mass from
 

another male mouse consisted cnly of keratin and probably
 

represented a ninth squamous-cell carcinoma; however, this lesion
 

was not included in the summary tables.)
 

Malignant lymphomas involved one or more organs in 13 mice. The
 

majority of the affected mice were female (males: high-dose 3/21
 

[14%]; females: untreated controls 1/13 [8%], vehicle controls
 

1/15 [7%], low-dose 2/29 [7%], high-dose 6/27 [22%]). The organs
 

with lymphoid tumors were kidney, liver, spleen, thymus,
 

mesenteric and mediastinal lymph nodes, heart, lungs, stomach,
 

33
 
adrenal, ovary, uterus, urinary bladder, and bone marrow. One
 

malignant lymphoma, grossly identified as an abdominal mass,
 

probably originated in the mesenteric lymph node· Seven mice had
 

disseminated malignant lymphomas involving multiple organs. Of
 

the 13 malignant lymphomas, nine were of the well-differentiated
 

lymphocytic type; the remaining four included two undifferentiated lymphomas and two types unspecified. Lesions classified
 

as a lymphocytic type had a uniform population of small cells
 

with little cytoplasm and small round to oval nuclei having small
 

inconspicuous nucleoli and coarse, dark chromatin. Cells of the
 

lymphoblastic type were similar to cells of the lymphocytic type,
 

with an increased amount of basophilic cytoplasm and larger, more
 

varied nuclei having finer reticulated chromatin and more
 

distinct nucleoli. The unspecified lymphomas had cellular
 

distortion which prevented further classification.
 

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

proliferative, and inflammatory changes were also encountered in
 

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

most part these nonneoplastic lesions were similar to those
 

commonly observed in aged mice. The chronic fibrous peritonitis
 

that was observed in rats given IPD was also present to a lesser
 

extent in the mice. Two of these mice had peritoneal osseous
 

metaplasia. Both the peritonitis and osseous metaplasia appeared
 

34
 
to be related to intraperitoneal injection, since these lesions
 

also occurred in the vehicle-control group. In addition to the
 

peritonitis, respiratory infections and bone-marrow injury may
 

also have had a role in reducing the life spans of mice during
 

this study. The extent to which reduced life spans influenced
 

the number of tumors observed could not be determined.
 

In the judgment of the pathologist, the results of this study
 

indicate that IPD given intraperitoneally to B6C3F1 mice was
 

responsible for squamous-cell carcinomas of the inguinal region
 

and an increased frequency of malignant lymphomas.
 

D· Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that were observed in
 

5% of one or more than one treated group of either sex. No
 

pooled-control groups are used in the statistical analyses, since
 

there are no controls from other studies that are suitable for
 

pooling. The untreated-control groups are not used in the
 

analyses, since the conditions of the vehicle controls are more
 

nearly comparable to the conditions of the treated groups.
 

In male mice, the Cochran-Armitage test for positive dose-related
 

trend in proportions for malignant lymphomas has a probability
 

level of 0.045, with an incidence of 3/21 (X4%) in the high-dose
 

35
 
group and none in the low-dose or vehicle-control groups. The
 

female mice also show a larger proportion of this tumor in the
 

high-dose group; however, due to the relatively small numbers in
 

the groups, the results of the Fisher exact test do not show a
 

significant difference between the high-dose and control groups
 

in either sex. The results of the Cochran-Armitage test on the
 

incidence in female mice are not significant. The time-adjusted
 

analyses on the incidence of malignant lymphoma in both male and
 

female mice are shown in tables F2 and F4, respectively. After
 

adjustment, the male mice show an incidence of 3/20 (15%), 0/24,
 

and 0/12 in the high-dose, low-dose, and vehicle-control groups,
 

respectively. In female mice, the time-adjusted incidence
 

becomes 1/15 (7%) in the vehicle-control group, 2/29 (7%) in the
 

low-dose group, and 6/26 (23%) in the high-dose group. The
 

results of the statistical tests using time-adjusted data are not
 

significant. The life-table method was performed, using as an
 

adjustment the week on study at which each malignant lymphoma was
 

observed. Based on these data, figure 5 shows the Kaplan and
 

Meier estimate of the probability of survival without the observation of a tumor. The Tarone test results indicated a significant
 

positive dose-related trend for both males and females with a
 

probability level of 0.011 for male mice and 0.003 for female
 

mice. In neither sex is departure from linear trend indicated.
 

36
 
TIME ON STUDY (WEEKS) 

Figure 5. Life Table for Mice Treated with IPD: Malignant Lymphoma 

37 
In each of the 95% confidence intervals shown in the statistical
 

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

of positive statistically significant results. It should also be
 

noted that each of the intervals has an upper limit greater than
 

one, indicating the theoretical possibility of the induction of
 

tumors by IPD, which could not be detected under the conditions
 

of this test.
 

38
 
V. DISCUSSION
 

The doses of IPD used in the bioassay were toxic, as shown by the
 

lowered mean body weights and rates of survival of the treated
 

rats and mice. The shortened life spans, particularly in the
 

rats, reduced the likelihood of the development of tumors.
 

High-dose rats of both sexes had all died by week 27. It should
 

also be noted that animals were treated for a maximum of only 52
 

weeks, which is a shorter period of time than used in other
 

current bioassays.
 

In rats, peritonitis and fibrous adhesions of the peritoneum were
 

observed in most of the treated animals at necropsy. Sarcoma,
 

fibroma, or fibrosarcoma of the peritoneum occurred only in
 

treated animals. Metastases to the lung or the lymph nodes occurred in two of the mid-dose females. Using time-adjusted analyses
 

of the incidences of these tumors in mid and low-dose animals
 

surviving at least 44 weeks (male) and 52 weeks (female), there
 

were significant dose-related trends (P - 0.045 in males, and Ρ •
 

0.001 in females), and the incidence in the mid-dose females was
 

significantly higher (P - 0.003) than that in the vehicle
 

controls. However, these significant results were based on
 

tumors in only six animals (two low-dose males, one mid-dose
 

male, and three mid-dose females). Because of this low incidence
 

and because irritation by the test chemical and chronic
 

39
 
peritonitis may have been involved in the pathogenesis, these
 

tumors may have been due to local effects of the injection of
 

IPD. Therefore, these tumors are not considered as evidence of
 

carcinogenicity of the test chemical.
 

Atrophy of the bone marrow was observed in all of the high-dose
 

animals of both sexes that were necropsied and examined
 

histopathologically.
 

In mice, lymphomas were observed at the following incidences
 

(males: controls 0/14, low-dose 0/26, high-dose 3/21; females:
 

controls 1/15, low-dose 2/29, high-dose 6/27). The results of
 

the unadjusted and time adjusted analysis are not significant;
 

however, the Tarone test for life-table analysis of the probability of survival without lymphoma indicated a significant positive
 

dose-related increase of lymphomas with a probability level of
 

0.011 for male mice and 0.003 for female mice. These significant
 

results are based on tumors in only three male and eight treated
 

female mice.
 

Squamous-cell carcinomas occurred in 6/26 low-dose male and 2/27
 

high-dose female mice, but in no other group. Seven of these
 

carcinomas occurred in subcutaneous tissue in the inguinal area
 

near the sites of injection and probably arose from the epithelium of the preputial glands; the eighth, in a female, was in the
 

40
 
sebaceous glands of the ear canal. None of the statistical tests
 

for these tumors was significant; however, since most tumors
 

arose near the sites of injection, they may have been related to
 

the repeated intraperitoneal injections of the test chemical,
 

irritation by the test chemical, or both.
 

IPD is an antitumor agent that has immunosuppressive properties,
 

as shown by reduction of leukocytes in Donryu rats (Tsukagoshi
 

and Sakurai, 1970), reduction of spleen and bone-marrow cells in
 

CDF1 mice (Vadlamudi et al., 1971a), and suppression of hemagglutinin synthesis in CDFl mice (Vadlamudi et al., 1971b). These
 

immunosuppressive properties may, in turn, be responsible for the
 

apparent increase in tumors of the lymphoid system in mice, which
 

was observed in the present bioassay. Results from a pulmonary
 

tumor test system have shown that intraperitoneal injections of
 

IPD into strain A mice at doses of 46 mg/kg three times per week
 

for 8 weeks induced statistically significant numbers of
 

pulmonary tumors (Stoner et al., 1973).
 

Tumors of the peritoneum in rats and tumors in the subcutaneous
 

tissue in mice may have been due to local effects related to
 

administration of the test chemical. The lymphomas in mice,
 

although statistically significant, were too few in number to be
 

clearly related to dosing.
 

41
 
Conclusions from this study are limited by early deaths and
 

toxicity, but the appearance of tumors in the peritoneum near the
 

injection sites in both rats and mice indicate the carcinogenic
 

potential of IPD.
 

42
 
VI. BIBLIOGRAPHY
 

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

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

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

Panel on Carcinogenicity of the Cancer Research Commission
 

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

1969.
 

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

Soc. Β 34(2):187-220, 1972.
 

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

1970, pp. 48-52.
 

Davey, F. R. and Moloney, W. C , Postmortem observations on
 

Fischer rats with leukemia and other disorders. Lab Invest.
 

23(3):327-334, 1970.
 

Davis, R. K. , Stevenson, G. T. , and Busch, Κ. Α., Tumor
 

incidence in normal Sprague-Dawley female rats. Cancer Res.
 

16:194-197, 1956.
 

Dunning, W. F. and Curtis, M. R., Multiple peritoneal sarcoma in
 

rats from intraperitoneal injection of washed, ground Taenia
 

larvae. Cancer Res, j>: 668-670, 1946.
 

El-Merzabani, M. M. and Sakurai, Υ., A new alkylating antitumor
 

agent effective on experimental tumors resistant to nitrogen
 

mustard. Gann 56:589-598, 1965.
 

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

significance tests, confidence limits and adjustments for
 

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

Hirano, M., Miura, M., Kakizawa, H., Morita, A·, Uetani, Τ. ,
 

Ohno, R., Kawashima, Κ. , Nishiwaki, Η. , and Yamada, Κ. ,
 

Effect of two new sulfonic acid esters of aminoglycols on
 

chronic myelogenous leukemia. Cancer Chemother. Rep.
 

^6:47-52, 1972.
 

Jacobs, B. B. and Huseby, R. Α., Neoplasms occurring in aged
 

Fischer rats, with special reference to testicular, uterine,
 

and thyroid tumors. J. Natl. Cancer Inst. 39:303-307, 1967.
 

43
 
Kaplan, Ε. L. and Meier, P., Nonparametric estimation from
 

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

1958.
 

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

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

and Biomed. Res. V. 230-248, 1974.
 

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

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

Pradham, S. N., Chung, E. B., Ghosh, B., Paul, B. D., and
 

Kapadia, G. J., Potential carcinogens, I: Carcinogenicity of
 

some plant extracts and their tannin-containing fractions in
 

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Weisburger, E. K. , and Weisburger, J. Η. , Spontaneous tumors
 

in Sprague-Dawley rats and Swiss mice. Cancer Res.
 

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Robbins, S. L. , Pathology, 3rd ed. , W. B. Saunders Co.,
 

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J. Η., Weisburger, Ε. Κ., and Gori, G. B., Test for
 

carcinogenicity of food additives and chemotherapeutic
 

agents by the pulmonary tumor response in strain A mice.
 

Cancer Res. J33:3069-3085, 1973.
 

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

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

Thompson, S. W., Huseby, R. Α., Fox, Μ. Α., Davis, C. L. , and
 

Hunt, R. D., Spontaneous tumors in the Sprague-Dawley rat.
 

J. Natl. Cancer Inst. 27(5):1037-1057, 1961.
 

44
 
Tsukagoshi, S. and Sakurai, Υ. , Cancer chemotherapy screening
 

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Cancer Chemother. Rep. _54:311-318, 1970.
 

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A., Effect of treatment with two related dimethanesulfonates
 

on the life span and spleen and bone marrow cells of 

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1971b. 

45
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS GIVEN INTRAPERITONEAL INJECTIONS
 

OF IPD
 

47 

TABLE A1

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF IPO (CONTROL GROUPS)

 

MID-AND HIGH-DOSE 

UNTREATED 

CONTROL 

ANIMALS INITIAILY IN STUDY 10 

ANIMALS NECPOPilED 10 

ANIMALS EXAMINEE HISTOPATHOLOGICALLY 10 

INTEGUMENTARY SYSTEM 

• SKIN (10 )

 

KERATOACANTHCMA 

• SUBCUT TISSUE (10 )

 

FIBROSARCOMA 

RESPIRATORY SYSTEM 

NONE 

HEMATOPOIETIC SYSTEM 

NONE 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

NONE 

URINARY SYSTEF 

NONE 

INEOCRINE SYSTEK 

IPITUITAFY (10 ) 

CHROHOPHOEE CARCINOMA 1 MO* ) 

LOW-DOSE 

UNTREATED 

CONTROL 

10 

10 

10 

(10 ) 

(10 ) 

( 8 ) 

MID-AND HIGH-DOSE 

VEHICLE 

CONTROL 

1C 

10 

10 

(10) 

1 (10%) 

(10) 

(10) 

1 f10%) 

LOW-DOSE 

VEHICLE 

CONTROL 

10 

10 

10 

(10) 

(10) 

1 (1 

(8) 

# NUMBFR OF ANIMALS WITH TISSUE EXAMINED MICROSCCPICALLY 

* NUMBER OF ANIMALS NECROPSIEE 

49 
TABLE A1 CONTROL MALE RATS: NEOPLASMS (CONTINUED)

 

REPRODUCTIVE SYSTEM 

•MAMMARY GLAND 

ADENOCARCINOMA, NOS 

FIBROADENOMA 

«TESTIS 

INTERSTITIJL-CELL TUMOR 

NERVOUS SYSTEM 

NONE 

SPECIAL SENSE CEGANS 

•FAR CANAL 

SQUAMOUS CELL CARCINOMA 

MUSCULOSKELETAL SYSTEM 

NONE 

EOEY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATHS 

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTAIIY KILLED 

TERMINAL SACRIFICE 

ANIMAL MISSING 

9 INCLUDES AU1CLY2ED ANIMALS 

# NUMBER OF ANIMALS WITH TISSUE

• NUMBER OF ANIMAIS NECROPSIED 

MID-AND HIGH-DOSE 

UNTREATED 

CONTROL 

(10) 

(9) 

(10) 

10 

1 

9 

LOW-DOSE 

UNTREATED 

CONTROL 

(10) 

(10) 

1 (10%) 

(10) 

1 (1C*) 

10 

10 

 EXAMINED MICROSCCPICALLY 

MID­ AND HIGH-DOSE 

VEHICLE 

CONTROL 

(10) 

(10) 

(10) 

10 

10 

LOW-DOSE 

VEHICLE 

CONTROL 

(10) 

1 (10*) 

1 (10*) 

(10) 

(10) 

10 

1 

2 

7 

50 
TABLE A1 CONTROL MALE RATS: NEOPLASMS (CONTINUED)

 

MID· AND HIGH-DOSE LOW-DOSE 

UNTREATED UNTREATED 

CONTROL. CONTROL 

TUMOR SOBBAEY 

TOTAL ANIMALS WITH PRIHARY TUBORS* 1 1

 

TOTAL PBIMARY TOBORS 1 2

 

TOTAL ANIBAIS WITH BENIGN TOBORS 1 

TOTAL BINIGN TUflORS 1

 

TOTAL ANIBALS WITH BALIGNANT TUBORS 1 1

 

TOTAL BALIGNANT TUMORS 1 1 

TOTAL ANIBALS WITH SECONDARY TUBORS* 

TOTAL SICONEARY TUBORS 

TOTAL ANIBALS WITH TUBORS UNCERTAINBENIGN OR BAIIGNANT

 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIBAIS WITH TUBORS UNCERTAINPRIBARY OR METASTATIC

 

TOTAL UNCERTAIN TUBORS 

• PRIBARY TUBORS: ALL TUBORS EXCEPT SECONDARY TUBORS 

t SECONDARY TUTORS: METASTATIC TUflORS OR TUflORS INVASIVE INTO

MID-AND HIGH-DOSE 

VEHICLE 

CONTROL 

2 

2 

1 

1 

1 

1 

 AN ADJACEI? ORGAN 

LOW-DOSE 

VEHICLE 

CONTROL 

3 

3 

1 

1 

2 

2 

51 
TABLE A2

 

SUMMARY OF THE INCIOENCE OF NEOPLASMS IN MALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF IPO (TREATED GROUPS) 

LOW DOSE MID DOSE HIGH DOSE 

JNIMALS INITIAIIY IN STUDY 35 35 

ANIMALS NECB0E5IED 32 28 30 

ANIMALS EXAMINEE HISTOPATHOLOGICALLY 32 28 30 

INTEGUMENTARY SYSTFM
 

NONE
 

FESPIRATORY SYSTEM
 

NONE
 

HEMATOPOIETIC SYSTEM
 

NONE
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

•COLON	 (32) (2 8) (24)
 

ADENOCARCINOMA, NOS 1 (<•*)
 

«CECUM	 (32) (28) (2U)
 

MUCINOUS JEENCCARCINOMA 1 (*»%)
 

ORINARY SYSTEP
 

NONE
 

ENDOCRINE SYSTEM 

• NUMBER OP ANIMALS WITH TISSUE EXAMIHED MICROSCOPICALLY 

• NUMBER OF AKIMALS NECROPSIED 

52 
TABLE A2 TREATED MALE RATS: NEOPLASMS (CONTINUED)

 

LOW DOSE MID DOSE 

REPRODUCTIVE SYSTEM 

NONE 

NfRVOUS SYSTEM 

«ERAIN (29) (28) ASTROCYTOMA 1 <3S)

 

SPECIAL SENSE ORGANS 

•EAR CANAL (32) (28) KERATOACANTHCMA 1 (*%) 

PUSCULOSKELETAI SYSTEM 

NONE 

BODY CAVITIES 

•PERITONEUM (32) (28) SARCOMA, NCS 2 (651) 

FIBROMA 1 (4*) FIBROSARCOMA 1 [H%) 

AIL OTHER SYSTEMS 

NONE 

JNIMAL CISPOSITICN SUMMARY 

ANIMALS INITIALLY IN STUDY 35 35

 

NATURAL DEATHS 17 16

 

MORIBUND SACRIFICT 18 19

 

SCHETULIE SACRIFICF 

ACCIDENTAIIY KILLED 

TERMINAL S/CRIFIC E 

ANIMAL MISSING 

3 INC^iJDlS^A U2CLUED.ANIMALS 

# NUMBFR OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMBER OF AMMAIS NECROPSIED 

HIGH DOSE 

(22) 

1 (5%) 

(30) 

(30) 

35 

22 

13 

53 
TABLE A2 TREATED MALE RATS: NEOPLASMS (CONTINUED)

 

LOW DOSE MID DOSE HIGH DOSE
 

TCMOR SDMMAEY • 

TO^AL ANIMA1S WITH PRIMARY TUMORS*

TOTAL PRIMARY TUMOPS

 3 

3 5 

1 

1 

TOTAL ANIHAIS WITH BENIGN

TOTAt EENICN TUMORS 

 TUMORS 2 

2 

TOTAL ANIMALS WITH MALIGNANT TUMORS

TOTAL MALICNANT TUMORS

 3 

3 

3 

3 

1 

1 

TOTAL ANIMALS WITH SECONDARY TUMORS* 

TOTAL SECONDARY TUMORS 

TOTAL ANIMALS WITH TUM3RS

BENIGN OR MAIIGNANT 

TOTAL UNCERTAIN TUMORS 

 UNCERTAINTOTAL ANIMAIS WITH TUMORS UNCERTAINFRIMARY OR MFTASTATIC 

TOTAL UNCERTAIN TUMORS 

* FRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

f SECONDARY TUfCES: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACEM ORGAN
 

54
 
TABLE A3 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS
 

GIVEN INTRAPERITONEAL INJECTIONS OF IPD (CONTROL GROUPS)
 

MID-AND HIGH-DOSE 

VEHICLE 

CONTROL 

10 

8 

θ 

(8) 

1 (13%) 

48) 

3 (38%) 

<€)

1 113%) 

LOW-DOSE 

VEHICLE 

CONTROL 

10 

10 

10 

(10) 

(10) 

5 (50%) 

(10) 

MID-AND HIGH-DOSE
 

UNTREATED
 

CONTROL
 

ANIMALS INITIALLY IN STUDY 10 

JKIMALS KECFCESIEE 10 

ANIMALS EXAMINED HISTOPATHOIOGICALLY 10 

INTEGUMENTARY SYSTEM 

• SUBCUT TISSUE (10 )
 

FIBROMA
 

RESPIRATORY SYSTEM 

NONE 

HEMATOPOIETIC SYSTEM 

NONE 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

NONE 

URINARY SYSTEM 

NONE 

ENDOCRINE	 SYSTEC 

«PITUITARY (10) 

CHROHOPHOEE ADENOMA 

«ADRENAL (10) 

CORTICAL iCENCMA 

LOW-DOSE
 

UNTREATED
 

CONTROL
 

10 

10 

10 

(10) 

(6) 

1 (17%) 

(10) 

# NUMBER OP ANIMALS βΙΤ TISSUE EXAMINED BICHOSCCPICA1LY
 

• NUMBER OF ANIMALS NECROPSIEC
 

55
 
TABLE A3 CONTROL FEMALE RATS: NEOPLASMS (CONTINUED)

 

MID-AND HIGH-DOSE LOW-DOSE 

UNTREATED UNTREATED 

CONTROL CONTROL 

«THYROIE (5) (7) 

C-CELL CARCINCMA 1 (2051) 

REPRODUCTIVE SYSTEM 

•MAMMARY GLANE (10 ) (10) ADENCMA, NOS 

ADENCCARCIKCMA, NOS 1 (10*) FIBROADENOMA 2 (20*) 3 (30%) 

# UTERUS (10) (10) 

ENDOMETRIAL STROMAL POLYP 1 (10*) 

KERVOUS SYSTEP 

«ERAIN (10) (9) ASTROCYTOMA 

SPECIAL SENSF CFGANS 

NONE 

fUSCULOSKELETAI SYSTEM 

NONE 

ECEY CAVITIES 

• MESENTERY (10) (10) 

LIPOMA 1 (10*) 

AIL OTHER SYSTEMS 

NONE 

# NUMBER OF ANIMALS WITH TISSUE EXAHIMED MICROSCOPICALLY 

• NUMBER OF AKIMAIS NECROPSIED 

MID-AND HIGH-DOSE 

VEHICLE 

CONTROL 

<8) 

(8) 

3 (38%) 

(8) 

1 (13%) 

(8) 

1 (13%) 

(8) 

LOW-DOSE 

VEHICLE 

CONTROL 

(10) 

(10) 

1 (10*) 1 (10*) 2 (20*) 

(10) 

(10) 

(10) 

56 
TABLE A3 CONTROL FEMALE RATS: NEOPLASMS (CONTINUED)
 

MID­ AND HIGH-DOSE LOW-DOSE MID· AND HIGH-DOSE LOW-DOSE 

UNTREATED UNTREATED VEHICLE VEHICLE 

CONTROL CONTROL CONTROL CONTROL 

ANIMAL DISPOSITION SOMHARY 

ANIMALS INITIAIt Y I N STUDY 

NATDEAL DEATHS 

MORIBUND SACRIFICE 

SCHEEOLIE SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 

ANIMAL MISSING 

10 

1 

1 

8 

10 

1 

9 

10 

3 

1 

6 

10 

10 

 INCLUDES AUTCLYZFD ANIMALS 

TUMOR SUMMARY 

TOTAL ANIffALS WITH PRIMARY

TOTAL PRIMARY TUMORS 

 TUMORS* 5 

6 

U 

4 

6 

10 

6 

9 

TOTAL ANIMALS WITH BENIGN

TOTAL BENIGN TUMORS 

 TUMORS 

4 

4 

4 

6 

9 

6 

8 

TOTAL ANIMALS WITH MALIGNANT

TOTAL MALIGNANT TUMORS 

 TUMORS 2 

2 

1 

1 

1 

1 

TOTAL ANIMALS WITH SECONDARY

TOTAL SECCl^EAFY TUMORS 

 TUMORS» 

TOTAL ANIMALS WITH TUMORS

EENIGN OR MAIIGNANT 

TOTAL UNCERTAIN TUMORS 

 UNCERTAIN _ 

TOTAL ANIMAIS WITH TUMORS

FRIMAFY OB METASTATIC 

TOTAL UNCEFTAIN TUMORS 

 UNCERTAIN _ 

* PPIMARY TUMORS: ALL TUMORS EXCEFT SECONDARY TUMORS 

# SECONDARY TUMORS: MFTASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACEKT ORGAN 

57
 
TABLE A4
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS
 

GIVEN INTRAPERITONEAL INJECTIONS OF IPD (TREATED GROUPS)
 

ANIMALS INITIAII Y IN STUDY 

ANIMALS NECFOFSIED 

ANIMALS EXAMTNIE HISTOPATHOLOGICALLY 

INTEGUMENTARY SYSTEM 

•SUBCUT	 TISSUE
 

FIBROSARCCfA
 

INSPIRATORY Μ STEM 

«LONG
 

SARCCNA, NCS, METASTATIC
 

HF.MATOPOIFTIC 5YSTEM 

fMEDIASTINAL I.NOD S
 

FIBROSARCCfA, METASTATIC
 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

«LIVER
 

HEPATOCELLULAR ADENOMA
 

URINARY SYSTEF 

NONE 

EXOCRINE SYSTEM 

«PITUITARY
 

CHROMOPHOEB ACENOMA
 

# NUMBFR OF ANIMALS WITH TISSUE

• NUMBER OF ANIMAIS NECROPSIEE 

LOW DOSE MID DOSE HIGH DOSE 

35 35 c 

33 31 31 

33 31 i 1 

(33) (31) (31) 

1 (3*) 

(33) (31) (30) 

1 (3%) 

(2) (9) (12) 

1 (11%) 

(33) (31 ) (31) 

1 (3%) 

(29) (27) (22) 

2 17%) 

 EXAMINED MICROSCCPICALLY 

58
 
TABLE A4 TREATED FEMALE RATS: NEOPLASMS (CONTINUED)
 

LOW DOSE MID DOSE HIGH DOSE 

REPRODUCTIVE SYSTEM 

•MAMMARY GLANE (33) (31 ) (31) 

ADENCMA, KCS 2 (651) 

ADENOCARCINOMA, NOS 1 O*) 

FIBROADENOMA 4 (12*) 

HEMANGIOMA 1 (3%) 

FERVOUS SYSTFr 

NONE 

SPECIAL SENS? CFGANS 

NONE 

PUSCULOSFELETAI SYSTEM 

NONE 

ICCY CAVITIES 

•PERITONEUM (33 ) (31) (3 1) 

SARCOMA, NCS 2 (6X) 

FIBROSARCOMA 1 (3*) 

Al l OTHER SYSTEMS 

NONE 

ANIMAL EISPCSITIC N SUMMARY 

ANIMALS INITIALLY IN STUDY 35 3 5 35 

NATURAL ΠΕΑΤΗ3 8 15 21 

MORIBUNE SACRIFICE 26 20 

SCHEEULEC SACFIFICF 

ACCIDENTALIY KILLED 

TERMINAL SACRIFICE 1 

ANIMAL MISSING 

a INCLUDES AC1CLYZED ANIMALS 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCCPICALIT 

• NUMBER OF AMMAIS NECROPSIED 

59
 
TABLE A4 TREATED FEMALE RATS: NEOPLASMS (CONTINUED)

 

LOW DOSE MID DOSE HIGH DOSE
 

[CHOP SDHMAFY
 

TOTAL ANIMAIS RITH PRIMARY TUMORS* 8 3
 TOTAL PFIMAFY TOMORS 10 5
 

TOTAL ANIMAIS WITH BENIGN TDMOBS 7 2
 TOTAI EEN1GN TUMORS 8 2
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 2 3
 TOTAL MAIIGNANT TUMORS 2 3
 

TOTAL ANIMALS WITH SECONDARY TUMORSt 2
 TOTAL SECONDARY TUMORS 2
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MAIIGNANT
 

TOTAL UNCEFTAIN TUMORS
 

TOTAL ANIMAIS WITH TUMORS UNCERTAIN-


FRIflARY OF METASTATIC
 

TOTAL UNCEFTAIN TUMORS
 

* PRIMARY TUMCRS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

60
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE GIVEN INTRAPERITONEAL INJECTIONS
 

OF IPD
 

61
 

TABLE B1 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

GIVEN INTRAPERITONEAL INJECTIONS OF IPD 

UNTREATED VEHICLE 

CONTROL CONTROL LOW DOSE 

ANIMALS INITIALLY IN STUDY 15 15 34 

ANIMALS MISSING 1 

niMAL S NECFOFSIEE m 14 26 

JNIMALS EXAMINED HISTOPATHOLOGICALL Υ 1U 14 26 

IKTEGUMENTAFTf EYSTFM 

•SUBLUT	 TISSUE (14 ) (14 ) (26) 

SQUAMOUS CFIL CARCINOMA 6 (23X) 

FFSPIRATCRY SYSTFW 

«LUNG (14 ) (26) 

CARCINOMA,NOS 1 (*»X) 

FEMATOPCIETIC SYSTEM 

•MULTIPLE	 CBGANS (14 ) (14 ) (26)
 

MALIG.LYMPFOMA, LYMPHOCYTIC TYPE
 

«MEDIASTINAL I.NODE (2) (1) CARCINOMA, NOS, METASTATIC 1 (1OCX) 

«THYMUS (11 ) (14 ) (22)
 

MALIC.LYMPHOMA, LYMPHOCYTIC TYPE
 

CIRCULATORY SYSTEM 

NONE 

riCESTIVE SYSTEM 

«STOMACH <14) (14 ) 423)
 

SQUAMOUS CELL PAPILLOMA
 

URINASY SYSTEM 

# NUMBER OF ANIMALS WITH TISSU E EXAMINED MICFOSCCPICALLY 

* NUMBER OF AMMAtS NECROPSIEC 

HIGH DOSE 

35 

21
 

21
 

(21 ) 

(22) 

(21) 

1 <5X) 

(3) 

(20) 

2 (10X) 

(22) 

1 (5*) 

63
 
TABLE B1 MALE MICE: NEOPLASMS (CONTINUED)
 

UNTREATED VEHICLE 

CONTROL CONTROL LOW DOSE HIGH DOSE 

INDOCRINE SYSTEM 

#PANCREATIC ISLETS (13 ) <2U) (21 ) 

ISLET-CEI I JEENOMA 1 (7%) 

FEPRODUCTIVE SYSTEM 

NONE 

NBRVCUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

KUSCULOSFEIETAI SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

JNIMAL DISPOSITION SUMMAPY 

ANIMALS INITIALLY IN STUDY 15 15 34 35 

NATURAL DEATHS 3 5 20 26 

MORIBUNE SACRIFICT 13 9 

SCHETOLEE SACRIFICF 

ACCIDENTAIIY KILLED 

TERMINAL S/CRIFICE 12 10 

ANIMAL MISSING 1 

3 TNCLIinF? ΑΓΤΓΤΥ7^ RNTMRT ^ 

# NUMBER OF ANIMALS WITH TISSUE FXAMINFD MICROSCCPICALLY 

• NUMBER OF AMMAIS NECROPSIEC 

64
 
TABLE B1 MALE MICE: NEOPLASMS (CONTINUED)
 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

TUMOR SUMMARY 

TOTAL ANIMALS WITH PRIMARY

TOTAL PBIBARY TUMORS 

 TUMORS* 1 

1 

6 

7 

u 

α 

TOTAL ANIMALS WITH BENIGN

TOTAL BEMGN TUMORS 

 TUMORS 1 

1 

1 

1 

TOTAL ANIMALS WITH MALIGNANT

TOTAI MALIGNANT TUMORS 

 TUMORS 6 

7 

3 

3 

TOTAL ANIMAIS WITH SECONDARY

TOTAL SECONDARY TUMORS 

 TUMORS# 1 

1 

TOTAL ANIMALS WITH TUMORS

EENIGN OB MAIIGNANT 

TOTAL UNCERTAIN TUMORS 

UNCFRTAIN

TOTAL ANIMAIS WITH TUMORS

PRIMARY OB METASTATIC 

TOTAI UNCEFTAIN TUMORS 

UNCERTAIN

• PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

i SECONDARY TUMOFS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACEM ORGAN 

65
 
TABLE B2
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE
 

GIVEN INTRAPERITONEAL INJECTIONS OF IPD
 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS

ANIMALS

ANIMALS

JMMALS

 INITIALLY IN STUDY 

 MISSING 

 BECECfSIFC 

 EXAMINED HISTOPATHOLOGICAILY 

15 

1 

13 

13 

15 

15 

15 

36 

29 

35 

27 

26 

IKTEGUMENTAFY SYSTEM 

• SUBCUT TISSU E 

SQUAMOUS CELL

FIBRCSAFCCfA 

 CAPCTNOMA 

(13 ) (15 ) 429) (27) 

1

1

 (<•*) 

 ("%) 

PESPIRATCBY SYSTEr 

ILUNG 

ALVEOLAR/ERCNCHIOLAR ADENOMA 

ALVEOLAP/EFONCHIOLAR CARCINOMA 

ADENCSQUAPCUS CARCINOMA, METASTA 

(13 ) (15) 

1 (7%) 

(29) 

2 (7%) 

(24) 

2

1

 (8* ) 

 (*»%) 

HEMATOPOIETIC SYSTEM 

•MULTIPIE CKGANS 

MALIGNANT IYMPHOMA, NOS 

MALIG. LYBFHCKA, UNDIFFER-TYPE 

MALIG. LYPEFCMA, LYMPHOCYTIC TYPE 

(13 ) 

1 (8%) 

(15) <29) (27) 

2

2

1

 (7%) 

 (7%) 

{*%) 

• AEDOMINAL CANITY 

MALIG.LYMEHOMA, LYMPHOCYTIC TYPE 

(13 ) (15) <29) 

1 (3*) 

(27) 

ILIVER 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 

(13 ) (15) 

1 (7%) 

(29) (2a) 

IKIDNEY 

MALIG.LYHPHCMA, LYMPHOCYTIC TYPB 

(13 ) (15) 428) 

1 l*%) 

(24) 

«THYMUS 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE 

(13 ) (15) (25) (19) 

1 (5%) 

CIRCULATORY SYSTEP 

NONE 

# NUMBER

* NUMBER

 OF

 OF

 ANIMALS

 AMMAIS

 WITH TISSUE

 NECROPSIED 

 EXAMINED ΗICROSCCPICALLY 

66
 
TABLE B2 FEMALE MICE: NEOPLASMS (CONTINUED)

 

UNTREATED VEHICLE 

CONTROL CONTROL 

EIGESTIVE SYSTEM 

NCNE 

URINARY SISTER 

NCNE 

ENDOCRINE SYSTfM 

NCNE 

REPFODOCTIVE SYSTEM 

• MAMMARY 6LANC (13) (15)

 

ADENCCARCIKCHA, NOS 

IOVARY (8) (1H) 

ADENOMA, NCS 

PAPILLARY JDENONA 

CYSTADENCUA, NOS 1 (7*) 

TOBOLAR AEENCMA 

UERVOUS SYSTEM 

NONE 

SPECIAL SENSI CFGANS 

• EAR CANAL (13) (15)

 

SQUAMOUS CELL CARCINOMA 

MUSCULOSKELETAL SYSTEM 

NONE 

ECEY CAVITIES 

•ABDOMINAL CAVITY (13) (15)

 

CARCINOMA.NOS 

# NUMBER OP ANIMALS WITH TISSUE EXAMINED HICROSCCPICALLY
 

• NUMBER OP AKIHAIS NPCROPSIEE
 

LOW DOSE 

(29) 

(25) 

9 (36X) 

1 (<*%) 

(29) 

(29) 

HIGH DOSE 

(27) 

1 i**%) 

(18) 

1 (6%) 

1 (6%) 

(27) 

1 (***) 

(27) 

1 (4%) 

67
 
TABLE B2 FEMALE MICE: NEOPLASMS (CONTINUED) 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

MUCINOUS ADENOCARCINOMA 1 (3%) 

•PELVI S 

MESOTHELIOMA, MALIGNANT 

(13 ) (15 ) <2S) 

1 0%) 

(27) 

M L OTHER SYSTEMS 

NONE 

JNIMAL DISPOSITION SDHMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATHS 

MORIBUND SACRIFICE 

SCHEDULE! SACRIFICE 

ACCIDENTAIIY KILLED 

TERHINAL S/CRIFIC E 

ANIKAL MISSING 

15 

1 

1 

12 

1 

15 

15 

36 

14 

14 

1 

1 

35 

24 

11 

3 INCLUDES AUTCLY2ED ANIMALS 

TUMOR SUMMARY 

TOTAL ANIMALS WITH PRIMARY

TOTAL PRIFARY TUMORS 

 TUMORS* 1 

1 

3 

3 

13 

16 

14 

15 

TOTAL ANIMALS WITH BENIGN

TOTAL BESIGN TUMORS 

 TUMORS 1 

1 

1C 

10 

U 

TOTAL ANIMALS WITH MALIGNANT

TOTAI MAIIGNANT TUMORS 

 TUMORS 1 

1 

2 

2 

c 

~6 

10 

11 

TOTAL ANIMALS WITH SECONDARY

TOTAL SECONDARY TUHOFS 

 TOMORSt 1 

1 

TOTAL ANIMALS WITH TUMORS

BENIGN OR MALIGNANT 

TOTAL UNCEETAIN TUMORS 

 UNCERTAINTOTAL ANIMAIS WITH TUMORS

PRIMARY OR METASTATIC 

TOTAL UHCEFTAIN TUMORS 

 UNCERTAIN­

• PRIMARY TDMCRS: ALL TUHORS EXCEPT SECONDARY TUHOBS 

# SECONDARY TUFORS: METASTATIC TUMORS OR TUMORS INVASIVE IHTO AN ADJACEM OBGAN 

68
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS GIVEN INTRAPERITONEAL
 

INJECTIONS OF IPD
 

69
 

TABLE C1

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF IPD (CONTROL GROUPS) 

MID -AND HIGH-DOSE LOW-DOSE MID-AND HIGH-DOSE 

UNTREATED UNTREATED VEHICLE 

CONTROL CONTROL CONTROL 

ANIMALS INITIALLY I N STUDY 10 10 1C 

ANIflALS NECBOPSIED 10 10 1C 

ANIMALS EXAMINEE HISTOPATHOLOGICALLY 10 10 10 

INTEGUMENTARY SYSTEM 

• SKIN (10 ) (10) (10) 

INFLAMMATION, CHRONIC SUPPURATIV 

INFLAMMATION, FOCAL GRANOLOMATOO 1 (10%) 

INFLAMMATION WITH FIBROSI S 1 (10%) HYPERKERATOSIS 1 (10%) PARAKERATOSIS 1 (10%) 

•SUBCUT TISSU E (10 ) (10) (10) 

EPIDERMAL INCLUSION CYST 

BISPIfiATCRY SYSTEM 

«TRACHEA (9) (10) (9) INFLAMMATION, CHRONIC 

INFLAMMATION, CHRONIC SUPPURATIV 1 (10%) 

«LUNG (10 ) (10) (10) 

PNEUMONIA, CHRONIC MfJRINE 1 (10%) 1 (10%) 

HEMATOPOIETIC SYSTEM 

tBONE MARROW (10 ) (10) (9) ATROPHY, NCS 5 (50%) 

«SPLEEN (10 ) (10) (10) 

HEMATOPOIESIS 

CIRCULATORY SYSTFM 

NONE 

DIGESTIVE SYSTEM 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMBER OF ANIMALS NFTROPSIEE 

LOW-DOSE 

VEHICLE 

CONTROL 

10 

10 

10 

(10 ) 

1 (10%) 

(10 ) 

1 (10%) 

(10) 

2 (20%) 

(10) 

1 (10%) 

(10) 

3 (30%) 

(10) 

1 (10%) 

71 
TABLE CI CONTROL MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

URINARY SYSTEM 

fKIDNEY 

HYDRONEPPFCSIS 

INFLAMMATION, INTFPSTITIAL 

INFLAMMATION, CHRONIC 

ENDOCRINE SYSTEM 

«THYROID 

CYSTIC FOIIICIES 

tPARATHYROIt 

HYPERPLASIA, NOS 

REPRODUCTIVE SYSTEM 

NONE 

NERVOUS SYSTIf 

NONE 

SPECIAL SENSE CBGANS 

NONE 

PUSCULOSKELETAI SYSTEM 

NONE 

ECEY CAVITIES 

NONE 

AIL OTHER SYSTEMS 

NONE 

t NUMBER OF ANIMALS WITH TISSUE

• NUMBER OF AMBAIS N^CROPSIJEJ) 

MID-AND HIGH-DOSE 

VEHICLE 

CONTROL 

(10) 

2 (20*) 

(10) 

(6) 

LOW DOSE 

VEHICLE 

CONTROL 

(10) 

3 (30*) 

(7) 

1 d<»X) 

(2) 

MID-AND HIGH-DOSE 

UNTREATED 

CONTROL 

(10) 

1 (10*) 

3 (30*) 

(2) 

 EXAMINED MICRCSCCPICALLY 

LOW-DOSE 

UNTREATED 

CONTROL 

(10) 

1 (10%) 

6 (6C%) 

(8) 

(6) 

1 (17%) 

72
 
TABLE CI CONTROL MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MID- AND HIGH-DOSE LOW-DOSE MID· AND HIGH-DOSE LOW DOSE 

UNTREATED UNTREATED VEHICLE VEHICLE 

CONTROL CONTROL CONTROL CONTROL 

SPECIAL MORPHOLOGY SUMMARY 

NO LiSIC N BEFCPTED 2 6 2 

# NUMBER OF AMMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMBER OF ANIMALS NECROPSIED 

73 
TABLE C2
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF IPD (TREATED GROUPS) 

LOW DOSE MID DOSE HIGH DOSE 

35 

ANIMALS NECFOP5IED 32 28 30 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 32 28 30 

ANIMALS INITIALLY IN STUDY 35 35 

INTEGUMENTARY SYSTEM 

•SOBCUT	 TISSUE (32) (28) (30) 

EDEMA, NOS 1 (3%) 

HEMORRHAGE 4 (13%) 

INFLAMMATION, HEMORRHAGIC 1 (3%) 

INFLAMMATION, CHRONIC 1 (3%) 

BESPIBATORY SYSTEM 

«TRACHEA 

INFLAMMATION, ACUTE/CHRCNIC 

(30) (27) 122) 

1 (5%) 

«LUNG/BRONCEUS 

OLCER, NOS 

(31) (28) (25) 

1 (4% ) 

«LUNG 

PNEUMONIA, CHRONIC MURINE 

(31) 

1 (3%) 

(28) 

7 (25%) 

(25) 

9 (36%) 

HEMATOPOIETIC SYSTEM 

#BONE HARROW (30) (27) <29) 

ATROPHY, NOS 1 (3*) 8 (30%) 28 (97%) 

«SPLEEN (32) (28) (23) HEM ΑΤ POI IS IS 2 (6%) 1 (4% ) 

«LYMPH NODE (8) (8) 

HYPERPLASIA, PLASMA CELL 1 (13%) 

«MEDIASTINAL L.NCDF (8) ( 6 ) 

HYPERPLASIA, PLASMA CELL 2 (25%) 

«MESENTERIC I . NCDE (8) (β ) 

INFLAMMATION. HEMORRHAGIC 1 (13%) 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMBER OF AMMAIS NECROPSIED 

74
 
TABLE C2 TREATED MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

CIRCULATORY SYSTFP 

«MYOCARDIUM 

INFLAMHATICN, INTERSTITIAL 

fCARDIAC VALVi 

FIBROSIS 

DEGENERATION, NOS 

•PULMONARY AFTERY 

THROMBUS, CFGANIZED 

DIGESTIVE SYSTEfi 

fLIVER 

HEMORRHAGE 

HEMATOMA, NOS 

NECROSIS, FCCAL 

NECRCSIS, COAGULATIVE 

tLIVER/CENTRIlOEULAR 

DEGENERATION, NOS 

NECROSIS, NOS 

•PANCREAS 

INFLAMMATION, INTERSTITIAL 

fCOLON 

HEMORRHAGE 

ULCER, NOS 

1CECUH 

HEMORRHAGE 

UBINAfiY SYSTEP 

•KIDNEY 

HEMORRHAGE 

PYELCNEEHBITIS, NOS 

INFLAHMATICN, CHRONIC 

FKDOCRINE SYSTEM 

fADRENAI COFTEX 

HYPERPLASIi^NQDOLAR 

#

 NUMBER OF ANIMALS WITH TISSUE

• NUMBER OF AHIMALS NECROPSIID 

LOW DOSE MID DOSE 

(31 ) (28) 

1 (4%) 

(31 ) (28) 

1 (358) 

1 (3X) 

(32) (28) 

1 (<**) 

(32) (27) 

1 (**) 

(32) (27) 

(32) (27) 

(32) (28) 

1 (3*) 

1 (3*) 

(32) (28) 

1 <3*) 

(32) (28) 

1 <**) 

1 (3*) 

(32) (28) 

2 17%) 

 EXAMINED MICROSCOPICALLY 

75 

HIGH DOSE 

423) 

{23) 

<30) 

(27) 

2 (7%) 

3 (11%) 

2 (7%) 

1 (<•%) 

(27) 

1 (4X) 

1 (**) 

(23) 

(2<l) 

1 (4%) 

1 («%) 

<2<O 

(2<0 

1 («*) 

<23) 

1 
TABLE C2 TREATED MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)
 

LOW DOSE MID DOSE
 

REPRODUCTIVE SYSTEM
 

«PROSTATE (32) (27)
 

INFLAMMATION, ACUTE SUPPURATIVE 1 (4%)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAI SYSTEM
 

NONE
 

BODY CAVITIES
 

•PERITONEUM	 (32) (28)
 

INFLAMMATION, SUPPURATIVE 3 (9%) (14%)
 

INFLAMMATION, HEMORRHAGIC
 

INFLAMMATION, CHRONIC 30 (94*) 23 {82%)
 

ADHESION, NOS 21 (66*) 5 (18%)
 

HYPERPLASIA, MESOTHELIAL 1 (»X)
 

•PLEURA	 (32) (28)
 

INFLAMMATION, SUPPURATIVE 1 (3*)
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORFHCIOGY SUMMARY
 

NO LESION FEFORTED 2 3
 NO NECROPSY PERFORMED 1
 

AUTOLYSI5/NO NECROPSY 2 7
 

I NUMBER OF ANIMALS HITH TISSUE EXAMINED MICRCSCOPICALLΥ
 

• NUMBER OF ANIMALS NECROPSIED
 

76
 

HIGH DOSE
 

(23)
 

(30)
 

1 (3%)
 

7 (23%)
 

<3C)
 

5
 
TABLE C3 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF IPD (CONTROL GROUPS)
 

MID­ AND HIGH-DOSE LOW-DOSE MID-AND HIGH-DOSE 

UNTREATED UNTREATEO VEHICLE 

CONTROL CONTROL CONTROL 

ANIMALS INITIALLY IN STUDY 10 10 10 

AKIMALS NECFCESIEE 10 10 8 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 10 10 8 

INTEGUMENT ΑΡ SYS1FM 

NONE 

RESPIRATORY SYSTEM 

«TRACHEA (10) (10) ( 8 ) 

INFLAMMATICN, ACUTF/CHRCNIC 1 (10*) 1 (13* ) 

INFLAHMATICN, CHRONIC 

INFLAMMATICN, CHRONIC SUFPURATIV 

«LUNG (10) (10) Cβ) 

PNEUMONIA, CHRONIC MURINE 1 (10*) 

HEMATOPOIETIC SYSTEM 

#BONE MARROW (9) (10) 18) 

ATROPHY, NCS 4 (αο*) 

CIBCULATCRY SYSTEE 

NONE 

CIGESTIVE SYSTEM 

IPANCREAS (10> (10) (8) 

NECROSIS, NOS 1 (10X) 

UBINARY SYSTEF 

«KIDNEY ( 9 ) (10 ) < 8 ) 

HYDRONEPHROSIS 1 (11*) 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICBOSCOPICALLY
 

• NUMBER OF ANIMALS NECROPSIED
 

LOW-DOSE
 

VEHICLE
 

CONTROL
 

10
 

10
 

10
 

(10) 

1 (10*) 

1 (10*) 

(10) 

( 9 ) 

4 (44*) 

(10) 

(10) 

77
 
TABLE C3 CONTROL FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)
 

MID AND HIGH-DOSE
 

VEHICLE
 

CONTROL
 

<e> 

(8) 

(8) 

Cβ) 

2 (25%) 

Cβ) 

1 (13%) 

LOW DOSE
 

VEHICLE
 

CONTROL
 

2 (20f) 

(10) 

(10) 

2 (2 0%) 

(10) 

(10) 

1 (10%) 

(10) 

MID­ AND HIGH-DOSE 

UNTREATED 

CONTROL 

INFLAMMATION, CHRONIC 

«URINARY BLADDER (10 ) 

HYPERPLASIA, EPITHELIAL 1 (10* ) 

ENDOCRINE SYSTFM 

«PITUITARY (10 ) 

HYPERPLASIA, CHROMOPHOBE-CELL 

«ADRENAL (10 ) 

ANGIECTAS1S 

REPRODUCTIVE SYSTEM 

«UTERUS/ENECFITFTUM (10 ) 

INFLAMMATION, SUPPURATIVE 

INFLAMMATION, CHRONIC SUPPURATIV 

«OVARY ( 8 ) 

INFLAMMATION, SUPPURATIVE 

NBRVCUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

Al l OTHER SYSTEMS 

KONE 

LOW-DOSE
 

UNTREATED
 

CONTROL
 

(10) 

( 6 ) 

2 (33%) 

(10) 

2 (20X) 

(10 ) 

3 (30%) 

1 (10%) 

(10 ) 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICBCSCCPICALLY 

• NUMBER OF AMMAIS NFCROPSIED 

78
 
TABLE C3 CONTROL FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

MID-AND HIGH-DOSE LOW-DOSE MID-AND HIGH-DOSE LOW-DOSE 

UNTREATED UNTREATED VEHICLE VEHICLE 

CONTROL CONTROL CONTROL CONTROL 

SPECIAL MORPHCIOGY SUWHARY 

NO LESICN FEECRTED 2 1 

AUTOLYSIS/NO NECROPSY 2 

# NUMBER OP AN1MAIS WITH TISSUE EXAMINED BICFOSCCPICALLT 

* NUMBER OF ANIMALS NECROPSIED 

79
 
TABLE C4
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF IPD (TREATED GROUPS) 

LOW DOSE MID DOSE HIGH DOSE
 

JNIMALS INITIAIIY IN STUDY 35 35 c
 

ANIMALS NECFOESIED 33 31 31
 ANIMALS EXAMINEE HISTOPATHOLOGICALLΥ 33 31
 

INTEGUMENTARY SYSTEM 

•SUBCOT TISSO F (33) (31) (31) EDEMA, NCS 1 (3%)
 

HEMOERHAGF 1 (3%)
 

RESPIHATCRY SYSTEM 

«TRACHEA (32) (31) <25) 

INFLAMMATION, ACUTE/CHRCNIC 2 (6X) 

INFLAMMATION, CHRONIC SOPPURATIV 1 (3*) 

#LUNG (33) (31) <3C) 

EDEMA, NOS 2 (7%) 

HEMOPRHAGE <* (13X) 

PNEUPCNIA, CHBONIC MURINE 4 <13%) 15 (50%) 

HEMATOPOIETIC SYSTEM 

IBONE MARECW (32) (31) (31) 

ATROPHY, NCS 2 (6%) 30 (97%) 

ISPLEEN (33) (31) 13 1) PERIARTERITIS 1 (3%) 

ATROFHY, KCS 2 (6%) 

HEMATOPOIESIS 5 (15??) 1 (3%) 

«MEDIASTINAL I.NCDE (2) (9) <1ί) 

HYPERPLASIA, PIASMA CELL 2 (17% ' 

IMESENTERIC I . NODE (2) (9) (12 ) 

HEMORRHAGE 1 (8%) 

CIRCULATCRY SYSTEM 

NONE 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

• NUMBER OF ANIMALS NECROPSIED
 

80
 
TABLE C4 TREATED FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

 

HIGH DOSE
 

(3D
 

3 (10%)
 

1 (3%)
 

1 (3%)
 

2 (6%)
 

(3D
 

1 (3%)
 

3 (10%)
 

(31)
 

1 (3%)
 

1 (3%)
 

(31)
 

1 (3%)
 

(3 1)
 

(1C)
 

(30)
 

430)
 

(25)
 

tICESTIVB SYSTEM
 

«LIVER
 

HEMATOMA, NOS
 

NECROSIS, NOS
 

NECRCSIS, ICCAL
 

NECRCSIS, COAGULATIVE
 

HEMATOPOIESIS
 

«LIVER/CENTRIIOEULAR
 

DEGENERATION, NOS
 

NECROSIS, NOS
 

«ILEUM
 

INFLAMMATION, ACUTE/CHRONIC
 

INFLAMMATION, CHRONIC
 

URINARY SYSTFP
 

«KIDNEY/TUBUII
 

NEPHROPATHY
 

ENDOCRINE SYSTiM
 

«ADRENAL
 

ANGIECTASIS
 

«THYROID
 

INFLAMMATION, CHRONIC
 

REPRODUCTIVE SYSTEM
 

«UTERUS
 

CYST, NOS
 

«UTERUS/ENtOFETRIUM
 

INFLAMMATION, SUPPURATIVE
 

INFLAMMATION, ACUTE SUPPURATIVE
 

«OVARY
 

CYST, NOS
 

INFLAMMATION, SUPPURATIVE
 

&ERVOUS SYSTEM
 

NONE
 

LOW DOSE
 

(33)
 

1 O%)
 

(33)
 

(33)
 

(33)
 

(33)
 

3 (9%)
 

(27)
 

(33)
 

(33)
 

1 (3S)
 

(33)
 

2 (6*)
 

1 (3%)
 

MID DOSE
 

(31)
 

2 16%)
 

(31)
 

1 (3%)
 

(28)
 

(31)
 

(31)
 

(27)
 

1 (<•*)
 

(28)
 

1 <«»%)
 

(28)
 

1 (*»%)
 

(22)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMBER OF ANIMALS NECROPSIID 

81 
TABLE C4 TREATED FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)
 

SPECIAL SENSF CRGiNS 

•MIDDLE	 *AF 

INFLAMMATION, CHRONIC SOFFUFATIV 

FUSCULOSKELETAI SYSTEM 

•JCINT 

INFLAMMATION,

INFLAMMATION,

ECCY CAVITIES 

•PERITCNEUH 

INFLAHMATICN#

INFLAMMATION,

ADHESICN, NOS 

•MESENTERY
 

HEMORRHAGE
 

il l OTH^R SYSTEMS 

NONE 

SPECIAL EOREHCICGY

 ACUTE/CHRCNIC 

 CHPCNIC 

 ^HPPURATIVE 

 CHRONIC 

 SUMMARY 

NO LESICN FEFORTED 

AUTOIYSIS/NO NECROPSY 

LOW DOSE MID DOSE HIGH DOSE 

(33) (31) (3 1) 

1 <3X) 

(33) <31) (3 1) 

1 (3%) 

1 (3%) 

(33) (31) (31) 

1 (33) 5 (16%) 

32 (97*) 2 8 (9C%) 7 (23%) 

30 (91S) 8 (26%) 

(33) (31) (31) 

1 (3S) 

1 

2 α 

# NUMBER OF AMMAIS WITH TISSUE EXAMINED MICROSCOPICALLY
 

• NUMBER OF ANIMAIS NECROPSIED
 

82
 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE GIVEN INTRAPERITONEAL
 

INJECTIONS OF IPD
 

83
 

TABLE D1
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN
 

MALE MICE GIVEN INTRAPERITONEAL INJECTIONS OF IPD
 

UNTREATED VEHICLE 

CONTROL CONTROL LOW DOSE 

INIMALS INITIAII Y IN STUDY 15 15 34 

ANIMALS MISSING 1 

ANIMALS NECROFSIEE 14 14 26 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 14 14 26 

INTEGUMENTARY SYSTEM 

NCNE 

EESPIRATORY SYSTEP 

ILUNG (14) <26) INFLAMMATION, INTERSTITIAL 5 (36*) 2 (14%) 2 (8%) 

BRONCHOPNEUMONIA SUPPURATIVE 2 (14%) 

fcEMATOPOIETIC SYSTEM 

«ECNE HARROW (9) (13 ) (21)
 ATROPHY, NCS
 

ISPLEEN (14) (14 ) <24) 

HEMATOPOIISIS 2 (8%) 

«MESENTERIC I . NODE (2) ID
 HYPERPLASIA, IYMPHOID 1 (50%)
 

CIRCULATCRY SYSTEM 

ΜΟΝΕ 

tIGESTIV E SYSTEM 

«LIVER (14) (14 ) 

NECROSIS, COAGULATIVE 1 («X) HYPEFPLA£IA, NODULAR 1 (7X) 1 (4%) 

ANGIECTASIS 1 P%) 1 (7%) 

OBINAHY SYSTEM 

NONE 

• NUMBER OF ANIMALS WITH TISSUE EXAMINED MICBOSCCPICALLY
 

• NUMBER OF ANIMALS NECROPSIEE
 

HIGH DOSE 

35 

21
 

21
 

(22) 

1 (5X) 

(19) 

2 (11% 

(20) 

(3) 

(20) 

85
 
TABLE D1 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)
 

UNTREATED VEHICLE 

CONTROL CONTROL LOW DOSE HIGH DOSE 

INDOCRINf SYSTEM 

ΜΟΝΕ 

BIPBODOCTIVE SYSTEM 

• SEBINAL VESICLE 4 (14 ) (14) 126) (21) 

HYPEEPLASIA, LYHPHOID 1 (7%) 

ITESTIS 

ATROPHY, NOS 

(13 ) (14) (22) (20) 

1 (5%) 

IEBYOUS SYSTEM 

NONE 

SPECIAL SENSE CFGANS 

NONE 

HOSCOLOSFEtlTAI SYSTEM 

NONE 

EODY CAVITIES 

•PERITONEUM (14) (14) (26) (21) 

INFLAMMATION, SUPPURATIVE 1 (4%) 

INFLAHMATICN, FIBRINOUS 1 (5*) 

INFLAMMATICN, CHRONIC 1 (7*) 2 (8%) 5 (24*) 

METAPLASIA, OSSEOUS 1 (7*) 

Ml OTHER SYSTEMS 

NONE 

SPECIAL rOBEHCICGY SUMMARY 

NO LESION FEFORTED g 7 13 q 

f NUMBER OF AMMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• NUMBER OF AHIMAIS NECROPSIED 

86
 
TABLE D1 MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)
 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

AMIHAL BISSIKG/HO NECFOEST 

MO MECBOBSI PERFORMED 

AUTOLYSIS/KC BECROPST 1 1 

1 

7 

1 

13 

i NUMBER OF ASIBAIS WITH TISSUE EXAMINED ΗICBOSCOPICALIT 

• NUMBER OP AHIHAIS NECROPSIBD 

87
 
TABLE D2
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN
 

FEMALE MICE GIVEN INTRAPERITONEAL INJECTIONS OF IPD
 

HIGH DOSE
 

35
 

27
 

26
 

(24)
 

3 (13*)
 

2 (8*)
 

(24)
 

(2)
 

(19)
 

1 (5*)
 

(24)
 

(25)
 

1 (4*)
 

ANIMALS INITIJILY IN STUDY
 

JNIMALS HISSING
 

JKIMALS HECFOFSIED
 

ANIMALS EXABINED HISTOPATHOLOGICALLΥ
 

IKTEGUHENTABY £Υ5ΊΕΜ
 

NOME
 

fESPIRATOBY SYSTEM
 

•LONG
 

INFLAMMATION, INTERSTITIAL
 

BRONCHOPNEUMONIA SUPPURATIVE
 

BEBATOPOIETIC SYSTEM
 

ISPLEEN
 

HEMATOPOIESIS
 

IMESENTEHIC I. HODE
 

INFLAMMATION, GRANULOMATOUS
 

tTHYMDS
 

ATROPHY, NOS
 

CIRCULATCBY SYSTEM
 

NONE
 

EIGESTIYE SYSTEM
 

ILIVER
 

HYPBRPLASIJ, NODDLAR
 

•STOMACH
 

ULCER, HOS
 

URINARY SYSTEB
 

# NDHBEB OF ANIHALS WITH TISSUE

• NUMBER OF ANIHAIS NECROPSIID 

UNTREATED
 

CONTROL
 

15
 

1
 

13
 

13
 

(13)
 

2 (15*)
 

(13)
 

(13)
 

(13)
 

(13)
 

VEHICLE
 

CONTROL
 

15
 

15
 

15
 

(15)
 

6 (40%)
 

(15)
 

(3)
 

1 (3 3%)
 

(15)
 

(15)
 

(15)
 

 EXAMINED MICROSCOPICALLY 

LOW DOSE
 

36
 

29
 

29
 

(29)
 

3 (10%)
 

1 (3%)
 

«26)
 

1 (<•%)
 

(25)
 

(29)
 

2 (7%)
 

(29)
 

88
 
TABLE D2 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

INEOCRINE SYSTEM 

«THYROID POIIJCLE 

HYPERPLASI A PAPILLARY 

REPRODUCTIVE SYSTEM 

«UTERUS/ENECPETFIUM 

INFLAMMATION, SUPPURATIVE 

HYPERPLASIA, CYSTIC 

«OVARY 

ATROPHY, NCS 

NERVOUS SYSTEP 

NOME 

SPECIAL SENSE CPGANS 

NONE 

MUSCULOSKELETAL SYSTEM 

•FEMUR 

OSTECPOROSIS 

FIBRCDS OSTEODYSTROPHY 

BODY CAVITIES 

•PERITONEUM 

INFLAMMATION, CHRONIC 

HETAFLASIA, CSSEOUS 

ALL OTHER SYSTEMS 

ANIMAL MISSING/NO NECROPSY 

UNTREATED 

CONTROL 

(12 ) 

(13 ) 

9 (69%) 

(8) 

(13) 

(13) 

1 

VEHICLE 

CONTROL 

(13 ) 

1 (8%) 

(15 ) 

1 (7%) 

8 (53%) 

(14 ) 

(15) 

(15) 

LOW DOSE 

<23) 

129) 

10 (34%) 

<25) 

(29) 

1 (3%) 

1 (3%) 

(29) 

2 (7%) 

1 (3%) 

HIGH DOSE 

(18 ) 

(23) 

(18) 

1 (6%) 

(27) 

(27) 

6 (22%) 

« NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

89
 
TABLE D2 FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)
 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL LOW DOSE HIGH DOSE 

5FECIAL HORPHOIOGY SOHHARY 

NO LESIOB FIFCRTED 

NBCBOPST FERF/NO HISTO

MO HECBOESY PERFORMED 

AUTOLTSIS/NO H1CROPSY 

 PERFORMED 

3 

1 

3 12 

1 

6 

8 

1 

θ 

• NUMBER OF

* NDHBER OF

 AKIHALS WITH TISSUE EXAMINED

 ANIMALS NECROPSIED 

 MICBCSCCPICALLΥ 

90
 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS GIVEN INTRAPERITONEAL INJECTIONS
 

OF IPD
 

91
 

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

Given Intraperitoneal Injections of IPDa
 

Vehicle Low Mid High
 

Topography: Morphology Control Dose Dose Dose
 

Peritoneum: Sarcoma, NOS,
 

Fibroma, or Fibrosarcoma^ 0/20 (0) 2/32 (6) 1/28 (4) 0/30 (0)
 

 Values (Control, Low,
 

Mid, High Dose)c

>d N.S. N.S. N.S. N.S.
 

 Values (Control, Low,
 

Mid Dose)c

>d N.S. N.S. N.S. N.S.
 

Relative Risk (Vehicle Control)f Infinite Infinite
 

Lower Limit 0.192 0.039
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 44 72 —mmm
 

a

Treated groups received doses of 12, 24, or 48 mg/kg three times per week.
 

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

c

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

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

incidence of tumors in each treated group is the probability level for the Fisher exact tests for the
 

comparison of that treated group with the vehicle-control group when Ρ < 0.05; otherwise not
 

significant (N.S.) is indicated.
 

d

A negative trend (N) indicates a lower incidence in a treated group than in the vehicle-control
 

group.
 

e

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

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


control group.
 
Table E2. Time-adjusted Analyses of the Peritoneal Tumors in Male Rats 

Given Intraperitoneal Injections of IPDa 

Vehicle Low Mid High
 

Topography: Morphology Control Dose Dose Dose
 

Peritoneum: Sarcoma, NOS,
 

Fibroma, or Fibrosarcoma*5 0/19 (0) 2/14 (14) 1/3 (33) 0/0 (0)
 

 Values (Control, Low,
 

Mid, High Dose)c>d 

 = 0.031 N.S. N.S. —
 

 Values (Control, Low,
 

Mid Dose)c>d  = 0.045 N.S. N.S.
 

Relative Risk (Vehicle Control)f Infinite Infinite
 

Lower Limit 0.421 0.345
 

Upper Limit Infinite Infinite
 

vo a

Treated groups received doses of 12, 24, or 48 mg/kg, three times per week.
 

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

at least 44 weeks of study.
 

c

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

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

incidence of tumors in each treated group is the probability level for the Fisher exact tests for
 

the comparison of that treated group with the vehicle-control group when Ρ < 0.05; otherwise not
 

significant (N.S.) is indicated.
 

d

A negative trend (N) indicates a lower incidence ina treated group than in the vehicle-control
 

group.
 

e

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

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


control group.
 
__ 

Table E3. Analyses of the Incidence of Primary Tumors in Female Rats
 

Given Intraperitoneal Injections of IPDa
 

Vehicle Low
 

Topography: Morphology Control Dose
 

Peritoneum: Sarcoma, NOS
 

or Fibrosarcomab 0/18 (0) 0/33 (0)
 

 Values (Control, Low,
 

Mid, High Dose) c , d N.S. N.S.
 

 Values (Control, Low,
 

Mid Dose) c , d N.S. N.S.
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma^ 5/18 (28) 4/33 (12)
 

 Values (Control, Low,
 

Mid, High Dose) c , d 

 = O.OOl(N) N.S.
 

 Values (Control, Low,
 

Mid Dose) c , d 

 - 0.003(N) N.S.
 

Relative Risk (Vehicle Control)f 0.436
 

Lower Limit 0.102
 

Upper Limit 1.800
 

Weeks to First Observed Tumor 79 47
 

Mid
 

Dose
 

3/31 (10)
 

N.S.
 

N.S.
 

Infinite
 

0.367
 

Infinite
 

56
 

0/31 (0)
 

 = 0.004(N)
 

 - 0.004(N)
 

0.000
 

0.000
 

0.446
 

High
 

Dose
 

0/31 (0)
 

N.S.
 

0/31 (0)
 

 = 0.004(N)
 

0.000
 

0.000
 

0.446
 
__ __ 

Table E3. Analyses of the Incidence of Primary Tumors in Female Rats
 

Given Intraperitoneal Injections of IPDa
 

(continued)
 

Vehicle Low Mid High
 

Topography: Morphology Control Dose Dose Dose
 

Mammary Gland: Adenoma, NOS^ 1/18 (6) 2/33 (6) 0/31 (0) 0/31 (0)
 

 Values (Control, Low,
 

Mid, High Dose) c , d N.S. N.S. N.S. N.S.
 

 Values (Control, Low,
 

Mid Dose) c , d N.S. N.S. N.S.
 

Relative Risk (Vehicle Control)f 1.091 0.000 0.000
 

Lower Limit 0.062 0.000 0.000
 

Upper Limit 62.383 10.726 10.726
 

Weeks to First Observed Tumor 80 29
 

Mammary Gland: Adenoma, NOS
 

or Fibroadenoma^ 6/18 (33) 6/33 (18) 0/31 (0) 0/31 (0)
 

 Values (Control, Low,
 

Mid, High Dose) c , d < 0.00l(N) N.S. = 0.00l(N) p = 0.001(N)
 

 Values (Control, Low,
 

Mid Dose) c , d 

 = 0.00l(N) N.S. = 0.00l(N)
 

Relative Risk (Vehicle Control)f 0.545 0.000 0.000
 

Lower Limit 0.178 0.000 0.000
 

Upper Limit 1.774 0.351 0.351
 

Weeks to First Observed Tumor 79 29
 
Table E3. Analyses of the Incidence of Primary Tumors in Female Rats
 

Given Intraperitoneal Injections of IPDa
 

(continued)
 

Vehicle Low Mid
 

Topography: Morphology Control Dose Dose
 

Pituitary: Chromophobe
 

Adenoma*5 8/18 (44) 2/29 (7) 0/27 (0)
 

 Values (Control, Low,
 

Mid, High Dose)c

>d 

 < 0.00l(N) = 0.004(N) < O.OOl(N)
 

 Values (Control, Low,
 

Mid Dose)c

>d 

 < 0.00l(N) = 0.004(N) < O.OOl(N)
 

Departure from Linear Trend
 

(Control, Mid, High Dose)e 

 = 0.001
 

Relative Risk (Vehicle Control)f 0.155 0.000
 

Lower Limit 0.019 0.000
 

Upper Limit 0.675 0.279
 

Weeks to First Observed Tumor 80 58
 

a

Treated groups received doses of 12, 24, or 48 mg/kg three times per week.
 

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

High
 

Dose
 

0/22 (0)
 

 = O.OOl(N)
 

0.000
 

0.000
 

0.338
 
Table E3. Analyses of the Incidence of Primary Tumors in Female Rats
 

Given Intraperitoneal Injections of IPDa
 

(continued)
 

c

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

Cochran-Armitage test when Ρ < 0·05; otherwise* not significant (N.S.) is indicated. Beneath the
 

incidence of tumors in each treated group is the probability level for the Fisher exact tests for
 

the comparison of that treated group with the vehicle-control group when Ρ < 0.05; otherwise not
 

significant (N.S.) is indicated.
 

d

A negative trend (N) indicates a lower incidence in a treated group than in the vehicle-control
 

group.
 

e

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

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


control group.
 
Table E4. Time-adjusted Analyses of Peritoneal Tumors in Female Rats
 

Given Intraperitoneal Injections of IPDa
 

Vehicle Low Mid High
 

Topography: Morphology Control Dose Dose Dose
 

Peritoneum: Sarcoma NOS or
 

Fibrosarcomab 0/18 (0) 0/16 (0) 3/4 (75) 0/0 (0)
 

 Values (Control, Low,
 

Mid, High Dose) c , d 

 < 0.001 N.S. - 0.003 N.S.
 

Departure from Linear Trende < 0.001
 

 Values (Control, Low,
 

Mid Dose) c , d 

 - 0.001 N.S. - 0.003
 

Departure from Linear Trende < 0.001
 

Relative Risk (Vehicle Control)f Infinite
 

Lower Limit 3.119
 

Upper Limit Infinite
 

a

Treated groups received doses of 12, 24, 48 mg/kg three times per week.
 

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

at least 52 weeks of study.
 
Table E4. Time-adjusted Analyses of Peritoneal Tumors in Female Rats
 

Given Intraperitoneal Injections of IPDa
 

(continued)
 

c

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

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

incidence of tumors in each treated group is the probability level for the Fisher exact tests for
 

the comparison of that treated group with the vehicle-control group when Ρ < 0.05; otherwise not
 

significant (N.S.) is indicated.
 

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

group.
 

e

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

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


control group.
 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN MICE GIVEN INTRAPERITONEAL INJECTIONS
 

OF IPD
 

101
 

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

Given Intraperitoneal Injections of IPDa
 

Topography: Morphology
 

Subcutaneous Tissue:
 

Squamous-cell Carcinoma^
 

 Values0

>d
 

Departure from Linear Trende
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System:
 

Malignant Lymphoma^
 

 Values0

>d
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Vehicle
 

Control
 

0/14 (0)
 

N.S.
 

 = 0.003
 

0/14 (0)
 

 - 0.045
 

——
 

Low
 

Dose
 

6/26 (24)
 

N.S.
 

Infinite
 

0.931
 

Infinite
 

64
 

0/26 (0)
 

N.S.
 

—
 

—
 

—
 

High
 

Dose
 

0/21 (0)
 

N.S.
 

....
 

—
 

—
 

3/21 (14)
 

N.S.
 

Infinite
 

0.431
 

Infinite
 

29 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Given Intraperitoneal Injections of IPDa
 

(continued)
 

a

Treated groups received doses of 20 or 40 mg/kg three times per week·
 

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

c

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

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

incidence of tumors in each treated group is the probability level for the Fisher exact tests for
 

the comparison of that treated group with the vehicle-control group when Ρ < 0.05; otherwise not
 

significant (N.S.) is indicated.
 

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

group.
 

e

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

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


control group.
 
Table F2. Time-adjusted Analyses of Hematopoietic Tumors in Male Mice
 

Given Intraperitoneal Injections of IPDa
 

Vehicle Low High 

Topography: Morphology Control Dose Dose 

Hematopoietic System: Malignant 

Lymphoma" 0/12 (0) 0/24 (0) 3/20 (15) 

 Values 0>d N.S. N.S. N.S. 

Relative Risk (Vehicle Control)f Infinite 

Lower Limit 0.395 

Upper Limit Infinite 

a

Treated groups received doses of 20 or 40 mg/kg three times per week.
 

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

at least 29 weeks of study.
 

c

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

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

incidence of tumors in each treated group is the probability level for the Fisher exact tests for
 

the comparison of that treated group with the vehicle-control group when Ρ < 0.05; otherwise not
 

significant (N.S.) is indicated.
 

d

A negative trend (N) indicates a lower incidence in a treated group than in the vehicle-control
 

group.
 

e

T he probability level for departure from linear trend is given when Ρ < 0.05 for any comparison.
 

f

T he 95% confidence interval of the relative risk between each treated group and the vehicle-


control group.
 
Table F3.


Topography: Morphology
 

Subcutaneous Tissue:
 

Squamous-cell Carcinoma*5
 

 Values0»d
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System:
 

Malignant Lymphoma*5
 

 Values0>d
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

 Analyses of the Incidence of Primary Tumors in Female Mice
 

Given Intraperitoneal Injections of IPDa
 

Vehicle Low 

Control Dose 

0/15 (0) 0/29 (0) 

N.S. N.S. 

— 

— 

1/15 (7) 2/29 (7) 

N.S. N.S. 

1.034 

0.060 

58.874 

86 65 

High
 

Dose
 

1/27 (4)
 

N.S.
 

Infinite
 

0.031
 

Infinite
 

63
 

6/27 (22)
 

N.S.
 

3.333
 

0.473
 

146.288
 

25
 
Table F3. Analyses of the Incidence of Primary Tumors in Female Mice
 

Given Intraperitoneal Injections of IPDa
 

(continued)
 

Topography: Morphology
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma^
 

 Valuesc , d
 

Relaitve Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Ovary: Cystadenomab
 

 Valuesc , d
 

Departure from Linear Trende
 

Relative Risk (Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Vehicle Low High 

Control Dose Dose 

1/15 (7) 2/29 (7) 2/24 (8) 

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

1.034 1.250 

0.060 0.073 

58.874 70.551 

86 84 64 

1/14 (7) 9/25 (36) 0/18 (0) 

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

 - 0.002 

5.040 0.000 

0.838 0.000 

207.589 14.053 

86 65 
Table F3. Analyses of the Incidence of Primary Tumors in Female Mice
 

Given Intraperitoneal Injections of IPDa
 

(continued)
 

a

Treated groups received doses of 20 or 40 mg/kg three times per week·
 

b

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

c

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

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

incidence of tumors in each treated group is the probability level for the Fisher exact tests for
 

the comparison of that treated group with the vehicle-control group when Ρ < 0.05; otherwise not
 

significant (N.S.) is indicated.
 

d

A negative trend (N) indicates a lower incidence in a treated group than in the vehicle-control
 

group.
 

e

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

f

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


control group.
 
Table F4. Time-adjusted Analyses of Hematopoietic Tumors in Female Mice
 

Given Intraperitoneal Injections of IPDa
 

Vehicle Low High
 

Topography: Morphology Control Dose Dose
 

Hematopoietic System: Malignant
 

Lymphoma^ 1/15 (7) 2/29 (7) 6/26 (23)
 

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

Relative Risk (Vehicle Control)f 1.034 3.462
 

Lower Limit 0.060 0.429
 

Upper Limit 58.874 151.557
 

a

Treated groups received doses of 20 or 40 mg/kg three times per week.
 

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

at least 25 weeks of study.
 

c

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

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

incidence of tumors in each treated group is the probability level for the Fisher exact tests for
 

the comparison of that treated group with the vehicle-control group when Ρ < 0.05; otherwise not
 

significant (N.S.) is indicated.
 

d

A negative trend (N) indicates a lower incidence in a treated group than in the vehicle-control
 

group.
 

e

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

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


control group.
 

APPENDIX G
 

HISTOLOGIC FEATURES OF TUMORS OBSERVED IN
 

SPRAGUE-DAWLEY RATS GIVEN INTRAPERITONEAL
 

INJECTIONS OF IPD IN BUFFERED SALINE
 

THREE TIMES PER WEEK FOR ONE YEAR
 

111
 

Appendix G
 

Histologic Features of Tumors Observed in Sprague-Dawley
 

Rats Given Intraperitoneal Injections of IPD
 

in Buffered Saline Three Times per Week for One Year
 

Skin and Ear Canal - Three epithelial neoplasms were observed in
 

male rats. Two lesions had diagnoses of keratoacanthomas; one
 

was on the back and the other involved the external ear canal·
 

Another related lesion was a papillomatous squamous-cell carcinoma with extensive keratinization and focal secondary suppurative
 

inflammation. Each tumor occurred in a rat from a different
 

group: the untreated-controls, the vehicle-controls, and the mid-


dose group.
 

Subcutis - Three pelvic mesenchymal tumors occurred in the pelvic
 

tissues of three rats, two females and one male. All were spin-


dle-cell tumors of various degrees of differentiation. One was
 

classified as a well-differentiated fibroma and a second as well-


differentiated fibrosarcoma. The latter had an extensive ulcerative epidermitis over the neoplastic tissue. The third lesion
 

was a pleomorphic fibrosarcoma with bizarre multinucleated giant
 

cells.
 

113
 
Mammary Glands - One mid-dose female had a hemangioma composed of
 

large cavernous blood spaces filled with erythrocytes and lined
 

by low cuboidal or squamoid cells. No glandular tissues were
 

involved· The adenomas, adenocarcinomas, and fibroadenomas of
 

the mammary gland were typical of those previously described by
 

Davis et al. (1956) and Thompson et al. (1961).
 

Liver - A hepatocellular lesioii was observed in one mid-dose
 

female rat. This lesion was termed a hepatocellular adenoma and
 

consisted of a focal area of pleomorphic hepatocytes with great
 

variation in their size and shape, as well as macronucleosis.
 

The hepatocytic plates were disrupted and thickened. Marked
 

fatty changes were observed in the hepatocytes of the affected
 

area. Following the classification of Squire and Levitt (1975),
 

the lesion would be classified as a neoplastic nodule.
 

Large Intestines - Two adenocarcinomas were observed in the
 

gastrointestinal tracts of two mid-dose male rats. One lesion
 

had large glandular spaces lined by columnar, cuboidal, and
 

squamoid epithelial cells and was filled by large amounts of
 

mucin. Glands of this tumor had invaded the muscular layers.
 

Peritoneum - Six rats, three males and three females, had eight
 

spindle-cell mesenchymal tumors which were classified as one
 

fibroma, three fibrosarcomas, and sarcomas, type unspecified.
 

114
 
Similar or related lesions have been termed also as peritoneal
 

sarcomas (Dunning and Curtis, 1946), malignant fibrous histiocytomas (Pradham et al. , 1974) and malignant mesotheliomas
 

(Dunning and Curtis, 1946; and Robbins, 1967). They occurred
 

only in low- and mid-dose rats. These rats had chronic
 

peritonitis with extensive adhesions involving the abdominal
 

viscera. Two rats had active suppurative inflammation. One rat
 

had three peritoneal masses: one mass classified as a fibroma
 

with well-differentiated fibroblasts and collagen, and two masses
 

as fibrosarcomas. In another rat, multiple well-differentiated
 

spindle-cell tumors (fibrosarcomas) were observed. These lesions
 

involved the liver, stomach, intestines, and pancreas with
 

metastasis to the mediastinal lymph nodes.
 

The four lesions given the diagnosis of sarcoma, NOS were similar
 

in many respects to the above fibrosarcomas. One rat had multiple abdominal masses. One mass consisted of mature spindle-cells
 

which had infiltrated the muscular wall from the serosa and
 

blended into the small muscle, making identification of the
 

neoplastic cells difficult. Much of this tumor resembled a
 

fibrosarcoma, but a leiomyosarcoma could not be ruled out.
 

Another rat had multifocal areas of spindle-cell proliferation
 

involving the spleen, liver, adrenals, kidneys and pancreas. The
 

muscle layers of the stomach wall were bisected by neoplastic
 

115
 
tissues with a confusing mixture of neoplastic and nonneoplastic
 

cells. Unusual cells, probably regenerative and reactive leiomyocytes, were seen with fibroblasts and collagen. A pleomorphic
 

spindle-cell sarcoma occurred in two animals. In the former,
 

attached to the serosal of the speen, was a mass composed of
 

spindle-cells and bizarre giant cells. The latter had a poorly
 

differentiated sarcoma, with some areas resembling histiocytes;
 

other areas had stromal spindle-cells. The lesions had a mixed
 

population of small and large cells having various amounts of
 

cytoplasm and round to oval nuclei with small nucleoli and delicate chromatin. A few areas had epithelioid cells. The liver,
 

pancreas, large intestine, uterus, and mesenteric lymph nodes
 

were involved. A vascular embolus of large vacuolated histiocytes was present in the lungs. No invasion of the pulmonary
 

parenchyma was seen.
 

A lipoma was also seen in the mesentery of one rat. This lesion
 

consisted of normal-appearing, well-differentiated lipocytes.
 

Endocrine glands - Tumors of the pituitary, adrenals, and thyroid
 

were similar to those previously observed in Sprague-Dawley rats
 

(Davis et al. , 1956; Thompson et al., 1961; and Prejean et al.,
 

1973).
 

Reproductive tract - Multiple interstitial-cell adenomas were
 

116
 
seen in the testes of one untreated male. These were typical of
 

those described by Jacobs and Huseby (1967) and by Davey and
 

Moloney (1970). Stromal polyps in the uteri of two rats were
 

similar to those described by Jacobs and Huseby (1967), and Davey
 

and Moloney, 1970.
 

Brain - Three astrocytomas of the cerebral cortex were observed;
 

one in a vehicle-control female, one in a low-dose male, and one
 

in a high-dose male. Two were classified as astrocytoma, NOS,
 

and one as a gemistocytic astrocytoma· In the latter lesion, a
 

large number of large neuron-like cells were localized around a
 

blood-filled space. The lesion resembled a hyperplastic basal
 

ganglion; however, the number of cells, their size, and
 

orientation around a vascular space supports a diagnosis of gemistocytic astrocytoma. The other lesions were well-differentiated
 

highly cellular neoplasms composed of a homogenous population of
 

small astrocytes having round to oval nuclei with delicate chromatin and small nucleoli. These cells tended to blend into the
 

adjacent neural tissues.
 

* U.S. GOVERNMENT PRINTING OFFICE 1978-260-899/320 7 

117 

Review of the Bioassay of 3,3T

-Iminobis-1-Propanol Dimethane
 

Sulfonate (Ester) Hydrochloride*[IPD] by the
 

Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

November 28, 1977
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976 under the authority of the National
 

Cancer Act of 1971 (P.L. 92-218). The purpose of the Clearinghouse
 

is to advise on the National Cancer Institute's bioassay
 

program to identify and evaluate chemical carcinogens in the
 

environment to which humans may be exposed. The members of
 

the Clearinghouse have been drawn from academia, industry,
 

organized labor, public interest groups, State health officials,
 

and quasi-public health and research organizations. Members
 

have been selected on the basis of their experience in
 

carcinogenesis or related fields and, collectively, provide
 

expertise in organic chemistry, 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 NCI bioassay reports on chemicals studied for
 

carcinogenicity. In this context, below is the edited
 

excerpt from the minutes of the Subgroup's meeting at which
 

IPD was reviewed.
 

The bioassay of IPD was part of a program to study the
 

carcinogenicity of a number of cancer chemotherapeutic
 

drugs. The primary reviewer briefly described the experimental
 

design. As shortcomings, the reviewer said the control
 

groups were too small and the dose levels tested were too
 

high. Many of the treated animals died early in the study
 

due to the severe toxicity of IPD. .Pointed out was a number
 

of tumor types seen in the treated animals, including squamouscell carcinomas, but absent in the controls. The significance
 

of the tumors, however, were confounded by the poor survival
 

of animals and relatively short duration of the study.
 

Despite the problem of determining the significance of the
 

tumors, the reviewer opined that the conclusion in the
 

report that "no clear carcinogenic effects of IPD were
 

demonstrated in either species" was not consistent with the
 

findings. The reviewer suggested that a more appropriate
 

conclusion would include a statement noting the increased
 

tumor incidence in treated animals, experimental shortcomings,


and the need to retest IPD to clearly define its carcinogenic
 

potential.
 
The secondary reviewer said that no conclusion could be
 

drawn from the study· He suggested that the study be
 

repeated at more appropriate dosages. Another Subgroup
 

member proposed that IPD could be effectively tested in the
 

Strain- pulmonary adenoma induction system, since the drug
 

is an alkylating agent.
 

A motion was made that, because of the inadequate
 

experimental design and conditions of test, only limited
 

conclusions can be drawn from the bioassay of IPD. However,
 

the high incidence of tumors at the injection site in both
 

species and sexes and the dose-related incidence of lymphomas
 

in teated mice suggest that IPD may be carcinogenic. The
 

motion was seconded and accepted by all present except Mr.
 

Garfinkel, who opposed it based on the small animal groups
 

and high early mortality.
 

Members present were:
 

Gerald N. Wogan (Chairman), Massachusetts Institute of Technology
 

Lawrence Garfinkel, American Cancer Society
 

Henry C. Pitot, University of Wisconsin Medical Center
 

George Roush, Jr., Monsanto Company
 

Verald K. Rowe, Dow Chemical U.S.A.
 Michael B. Shimkin, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

John H. 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.
 

DHEW Publication No. (NIH) 78-818