1977 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 32 

BIOASSAY OF 

ISOPHOSPHAMIDE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 3778-73-2 

NCI-CG-TR-32 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

ISOPHOSPHAMIDE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogen Bioassay and Program Resources Branch
 

Carcinogenesis Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

DREW Publication No. (NIH) 77-832
 

BIOASSAY OF
 

ISOPHOSPHAMIDE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

CONTRIBUTORS; This report presents the results of the bioassay
 

of isophosphamide for possible carcinogenicity, conducted for the
 

Carcinogen Bioassay and Program Resources Branch, Carcinogenesis
 

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
 

bioassay program.
 

The experimental design and doses were determined by Drs. D. P.
 

Griswold1, J. D. Prejean1, E. K. Weisburger2, and J. H.
 

Weisburger2* . Mr. I. Brown^ was responsible for the care of
 

laboratory animals and Ms. J. Belzer performed the treatments.
 

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. Pathologists at NCI
 

and at Tracor Jitco have reviewed selected slides and concur with
 

the overall pathologic evaluation of the study.
 

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 Drs. E. Tong , P.
 

Lim^, and Mr. C. Hewitt**, and the analytical results were
 

reviewed by Dr. S. S. Olin^.
 

This report was prepared at Tracor Jitco under the direction of
 

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

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

J. F. Robens and C. H. Williams , toxicologists; Ms. L. A.
 

Waitz , bioscience writer; and Dr. E. W. Gunberg , technical
 

editor, assisted by Ms. Y. E. Presley .
 

The following scientists at National Cancer Institute were
 

responsible for evaluating the bioassay experiment, interpreting
 

the results, and reporting the findings:
 

Dr. Kenneth C. Chu
 

Dr. Cipriano Cueto, Jr.
 

Dr. J. Fielding Douglas
 

Dr. Dawn G. Goodman
 

Dr. Richard A. Griesemer
 

Dr. Thomas W. Orme
 

Dr. Robert A. Squire9
 

Dr. Jerrold M. Ward
 

Southern Research Institute, 2000 Ninth Avenue South,
 

Birmingham, Alabama.
 

^Carcinogenesis Program, Division of Cancer Cause and Prevention,
 

National Cancer Institute, National Institutes of Health,
 

Bethesda, Maryland.
 

%ow with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York.
 

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

Rockville, Maryland.
 

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

Maryland.
 

"Mathematical Statistics and Applied Mathematics Section,
 

Field Studies and Statistics Branch, Division of Cancer
 

Cause and Prevention, National Cancer Institute, National
 

Institutes of Health, Bethesda, Maryland.
 

^Stanford Research Institute, Menlo Park, California.
 

iv
 
°Drug Development Branch, Division of Cancer Treatment,
 

National Cancer Institute, National Institutes of Health,
 

Bethesda, Maryland.
 

'Now with the Division of Comparative Medicine, John Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

v
 

SUMMARY

 

A bioassay of the anticancer drug isophosphamide for possible
 

carcinogenicity was conducted by injecting the test chemical
 

intraperitoneally into Sprague-Dawley rats and B6C3F1 mice.
 

Groups of 35 rats and 35 mice of each sex were given the
 

injections at one of two doses three times per week for 52 weeks.
 

Doses for rats were either 6 or 12 mg/kg, and for mice either 10
 

or 20 mg/kg. After the period of administration of the
 

isophosphamide, the surviving rats were observed for 31 weeks and
 

the mice for 28 weeks. Untreated controls as well as vehicle
 

controls (buffered saline) were used. The matched vehicle-


control groups each consisted of 10 rats or 15 mice of each sex;
 

pooled vehicle-control groups, used for statistical evaluation,
 

consisted of the matched vehicle controls of each species
 

combined with 20 male and 20 female matched vehicle-control rats
 

or 15 male and 15 female matched vehicle-control mice from a
 

similar bioassay of another test chemical. All surviving rats
 

were killed at 79-84 weeks, all surviving mice at 79-81 weeks.
 

Mean body weights of the high-dose rats of either sex were lower
 

than those of the matched vehicle controls after approximately 25
 

weeks on study. Survival was low among the high-dose male and
 

female rats, but in the low-dose groups it was adequate for
 

meaningful statistical analyses of the incidences of tumors.
 

Mean body weights of the mice did not show any consistent effect
 

from the isophosphamide treatment. Survival was adequate for
 

meaningful statistical analyses in both groups of female mice,
 

while survival of the males was 31% for both treated groups at
 

the end of the study.
 

In male rats, tumors of the hematopoietic system included six
 

malignant lymphomas and two granulocytic leukemias. With the
 

unadjusted analysis, these tumors showed a dose-related trend in
 

male rats using pooled vehicle controls (controls 0/29, low-dose
 

3/32, high-dose 5/34, P = 0.032) and a higher incidence in the
 

high—dose males than in the pdoled vehicle controls (P = 0.040).
 

These tumors were not significant when compared with matched
 

vii 
vehicle controls using adjusted analyses, and they cannot clearly
 

be associated with treatment. However, it should be noted that
 

five rats with these tumors were observed in the high-dose group
 

whose median survival was only 35 weeks.
 

In female rats, the incidence of uterine leiomyosarcoma was
 

significant in the low-dose group using pooled vehicle controls
 

(controls 0/27, low-dose 15/32, P < 0.001). There was also a
 

significant incidence of mammary fibroadenoma among low-dose
 

females using pooled vehicle controls (controls 8/28, low-dose
 

28/33, P < 0.001). The incidence of each tumor was also significant when compared with matched vehicle controls using time-


adjusted analyses. The low survival of the high-dose group
 

(median time on study, 35 weeks) may explain the lower incidences
 

of the uterine leiomyosarcoma and the mammary fibroadenoma in
 

this group. In some rats, the leiomyosarcomas metastasized to
 

the lungs, urinary bladder, spleen, and other abdominal sites.
 

In female mice, the incidence of malignant lymphoma of the
 

hematopoietic system showed a significant dose-related trend
 

using either matched vehicle controls (controls 0/14, low-dose
 

3/32, high-dose 13/34, P = 0.001) or pooled vehicle controls
 

(controls 1/29, P < 0.001). Further, incidences of this tumor in
 

the high-dose females were significantly higher than incidences
 

in the matched vehicle (P - 0.005) or pooled vehicle (P - 0.001)
 

controls.
 

It is concluded that under the conditions of this bioassay,
 isophosphamide was not carcinogenic in male Sprague-Dawley rats
 

or in male B6C3F1 mice. However, the incidence of
 

leiomyosarcomas of the uterus indicates that isophosphamide was
 

carcinogenic in female Sprague-Dawley rats, and the incidence of
 

fibroadenoma of the mammary gland in female rats was associated
 

with treatment with isophosphamide. Isophosphamide was
 

carcinogenic in female B6C3F1 mice, producing malignant lymphomas
 

of the hematopoietic system.
 

viii
 
TABLE OF CONTENTS
 

]
 Page
 

I. Introduction
 

II. Materials and Methods
 

A. Chemical
 

B. Dosage Preparation
 

C. Animals
 

D. Animal Maintenance
 

E. Subchronic Studies
 

F. Designs of Chronic Studies
 

G. Clinical and Pathologic Examinations
 

H. Data Recording and Statistical Analyses
 

III. Results - Rats
 

A. Body Weights and Clinical Signs (Rats)
 

B. Survival (Rats)
 

C. Pathology (Rats)
 

D. Statistical Analyses of Results (Rats)
 

IV. Results - Mice
 

A. Body Weights and Clinical Signs (Mice)
 

B. Survival (Mice)...
 

C. Pathology (Mice)
 

D. Statistical Analyses of Results (Mice)
 

V. Discussion
 

VI. Bibliography
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms
 

in Rats Treated With Isophosphamide
 

Table Al Summary of the Incidence of Neoplasms
 

in Male Rats Treated with
 

Isophosphamide
 

1
 

3
 

3
 

3
 

4
 

4
 

7
 

8
 

8
 

12
 

17
 

17
 

17
 

21
 

23
 

27
 

27
 

27
 

30
 

31
 

35
 

39
 

41
 

43
 

ix
 
Table A2
 

Appendix B
 

Table Bl
 

Table B2
 

Appendix C
 

Table Cl
 

Table C2
 

Appendix D
 

Table Dl
 

Table D2
 

Appendix E
 

Page 

Summary of the Incidence of Neoplasms 

in Female Rats Treated with 

Isophosphamide 47 

Summary of the Incidence of Neoplasms 

in Mice Treated with Isophosphamide 51 

Summary of the Incidence of Neoplasms 

in Male Mice Treated with 

Isophosphamide 53 

Summary of the Incidence of Neoplasms 

in Female Mice Treated with 

Isophosphamide 56 

Summary of the Incidence of Nonneoplastic 

Lesions in Rats Treated with 

Isophosphamide 59 

Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Treated with 

Isophosphamide 61 

Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Treated with 

Isophosphamide 64 

Summary of the Incidence of Nonneoplastic 

Lesions in Mice Treated with 

Isophosphamide 69 

Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Treated with 

Isophosphamide 71 

Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Treated with 

Isophosphamide. 74 

Analyses of the Incidence of Primary 

Tumors in Rats Treated with 

Isophosphamide. 77 

X 
Table El
 

Table E2
 

Table E3
 

Table E4
 

Appendix F
 

Table F1
 

Table F2
 

Table F3
 

Table 1
 

Table 2
 

Figure 1
 

Page 

Analyses of the Incidence of Primary 

Tumors in Male Rats Treated with 

Isophosphamide 79 

Analyses of the Incidence of Primary 

Tumors in Female Rats Treated with 

Isophosphamide 86 

Time-adjusted Analyses of the 

Incidence of Primary Tumors in 

Male Rats Treated with Isophosphamide 96 

Time-adjusted Analyses of the 

Incidence of Primary Tumors in 

Female Rats Treated with Isophosphamide.... 102
 

Analyses of the Incidence of Primary
 

Tumors in Mice Treated with
 

Isophosphamide 109
 

Analyses of the Incidence of Primary
 

Tumors in Male Mice Treated with
 

Isophosphamide 1ll
 

Analyses of the Incidence of Primary
 

Tumors in Female Mice Treated with
 

Isophosphamide 113
 

Time-adjusted Analyses of the
 

Incidence of Primary Tumors in
 

Male Mice Treated with Isophosphamide 115
 

TABLES
 

Design of Chronic Studies of
 

Isophosphamide in Rats 9
 

Design of Chronic Studies of
 

Isophosphamide in Mice 10
 

FIGURES
 

Growth Curves for Rats Treated with
 

Isophosphamide 18
 

xi
 
Page 

Figure 2 Survival Curves for Rats Treated with 

Isophosphamide 19 

Figure 3 Growth Curves for Mice Treated with 

Isophosphamide 28 

Figure 4 Survival Curves for Mice Treated with 

Isophosphamide 29 

xii
 
I. INTRODUCTION
 

Isophosphamide (Ifosfamide; CAS 3778-73-2, NSC 109724, NCI
 

C016380) is a cyclophosphamide analog differing from the parent
 

chemical by transposition of a chloroethyl group from the side
 

chain to the ring nitrogen. Isophosphamide requires microsomal
 

activation for cytotoxicity, which proceeds by alkylation and
 

chain scission of DNA (Allen and Creaven, 1972). Developed and
 

tested in Germany by the Asta-Werke Chemical Company during the
 

late 1960's, the drug was active in leukemia L1210, Lewis lung,
 

Ehrlich ascites, and Yoshida sarcoma test systems (Carter, 1972)
 

and produced positive clinical results with oat-cell tumors of
 

the lung, ovarian cancer, breast cancer, and lymphomas (Slavik
 

and Carter, 1973). Isophosphamide has undergone phase II
 

clinical evaluations in the United States with breast cancer and
 

colorectal cancer (Ahmann et al., 1974; Kovach et al., 1974).
 

Isophosphamide was one of a series of anticancer agents
 

administered chronically in clinical practice that was selected
 

for screening for carcinogenicity in the bioassay program.
 

1
 

II. MATERIALS AND METHODS
 

A. Chemical
 

Isophosphamide is the generic name for 3-(2-chloroethyl)-2-[(2­

chloroethyl)amino]tetrahydro-1,3,2-oxazaphosphorine-2-oxide. The
 

material used in this study was manufactured by Asta-Werke AG
 

(Brackwede/Westphalia, Germany) and was obtained through the Drug
 

Development Branch, Division of Cancer Treatment, National Cancer
 

Institute (NCI). The purity of the batch used was tested at
 

Stanford Research Institute, Menlo Park, California, and found to
 

be 99 — 1% pure. Elemental analyses (C, H, N, Cl, P) were
 

correct for C7H]^502N2Cl2P, the molecular formula for 

isophosphamide. Water content was — 0.18% by Karl Fischer 

determination. Infrared and nuclear magnetic resonance spectra 

were compatible with the structural formula for isophosphamide.
 

Gas-liquid chromatography showed one main component and two minor
 

contaminants (— 1%). No attempt was made to identify or
 

quantitate the impurities. On receipt at the test laboratory,
 

isophosphamide was stored in glass bottles at -20°C.
 

B. Dosage Preparation
 

Test solutions were prepared by dissolving the drug in a buffered
 

saline vehicle. Four concentrations (0.48, 0.24, 0.20, and
 

3 
0.10%) were prepared. Solutions were made daily and administered
 

immediately to avoid decomposition.
 

C. Animals
 

Male Sprague-Dawley rats and male Swiss mice were used in the
 

subchronic studies. Sprague-Dawley rats and B6C3F1 mice of both
 

sexes were used in the chronic studies. All animals were
 

obtained from Charles River Breeding Laboratories, Inc.,
 

Wilmington, Massachusetts, through contracts of the Division of
 

Cancer Treatment, NCI. Upon arrival at the laboratory, the
 

animals were quarantined (rats for 5 days, mice for 12 days) as
 

an acclimation period and were then earmarked and assigned to
 

control and test groups.
 

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 40-60%. There were 15 changes of room air per hour.
 

Air was passed through both incoming and exhaust fiberglass
 

roughing filters. In addition to natural light, illumination was
 

provided by fluorescent light for 9 hours per day. Wayne® Lab
 

Blox (Allied Mills, Inc., Chicago, 111.) and water were supplied
 

daily and were available ad libitum.
 

4 
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 34;
 

mouse cages were provided with Sterolit® clay bedding (Englehard
 

Mineral and Chemical Co., New York, N.Y.). Bedding was replaced
 

weekly, and cages, water bottles, feeders, and racks were
 

sanitized weekly.
 

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

were housed with respective treated animals. Animals treated
 

with isophosphamide were maintained in the same rooms as animals
 

of the same species being treated with the following chemicals:
 

RATS
 

5-azacytidine (CAS 118-92-3)
 

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

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

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

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

acronine (CAS 7008-42-6)
 

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

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

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

(phenesterin) (CAS 3546-10-9)
 

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

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

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

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

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

monohydrochloride (procarbazine hydrochloride) (CAS 366-70-1)
 
4'-(9-acridinylamino)methansulfon-m-aniside monohydrochloride
 

(MAAM) (No CAS or NSC number available)
 

3,3'-iminobis-1-propanol dimethanesulfonate (ester)
 

hydrochloride (CAS 3458-22-8)
 

MICE
 

anthranillc acid (CAS 118-92-3)
 

pyrazinamide (CAS 98-96-4)
 

L-tryptophan (CAS 73-22-3)
 

3,3'-iminobis-1-propanol dimethanesulfonate (ester)
 

hydrochloride (CAS 3458-22-8)
 

5-azacytidine (CAS 320-67-2)
 

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

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

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

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

acronine (CAS 7008-42-6)
 

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

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

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

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

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

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)
 

2,6-diamino-3-(phenylazo)pyridine hydrochloride (CAS 136-40-3)
 

2-ethyl-4-pyridinecarbothioamide (ethionamlde) (CAS 536-33-4)
 

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

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

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

(pyrimethamine) (CAS 58-14-0)
 

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

(chlorpropamide) (CAS 94-20-2)
 

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

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

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

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

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

(acetohexamide) (CAS 968-81-0)
 

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

(MAAM) (No CAS or NSC number available)
 

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

(tolazamide) (CAS 1156-19-0)
 

6 
E. Subchronic Studies
 

Subchronic studies were conducted to estimate the maximum
 

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

high doses were determined for administration in the chronic
 

studies. Sprague-Dawley male rats and Swiss male mice were
 

administered isophosphamide by intraperitoneal injection three
 

times per week for 45 days. Following treatment, all animals
 

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

species were used at each dose, 10 animals were used as untreated
 

controls, and 10 animals as vehicle (buffered saline) controls.
 

In rats 3/5 animals died at each of the highest doses (24 and 48
 

mg/kg). At lower doses (2.4, 6.0, and 12 mg/kg) there were no
 

deaths, and body weight gain in these groups was no more than 15%
 

lower than that of the controls. No gross abnormalities were
 

seen in any animals at necropsy. The low and high doses for rats
 

in the chronic studies were set at 6 and 12 mg/kg.
 

In mice 3/5 animals died at 80 mg/kg, 2/5 at 40 mg/kg, 1/5 at 20
 

mg/kg, and 0/5 at 10 or 4 mg/kg. Depression in weight gain
 

occurred during the treatment period in the group receiving 80
 

mg/kg; however, weights were similar among surviving mice in all
 

groups by the end of the study. The low and high doses for mice
 

in the chronic studies were set at 10 and 20 mg/kg.
 

7 
F. Designs of Chronic Studies
 

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

Since the numbers of animals in the matched vehicle-control
 

groups (hereinafter referred to as "vehicle controls") were
 

small, pooled vehicle-control groups (hereinafter referred to as
 

"pooled controls") also were used for statistical comparisons.
 

Vehicle controls from the current tests on isophosphamide were
 

combined with vehicle controls from tests performed on
 

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

(IPD). The pooled controls for statistical tests consisted of 30
 

male and 30 female rats and 30 male and 30 female mice. The
 

tests on IPD were also conducted at Southern Research Institute
 

and overlapped the isophosphamide study by at least 16 months for
 

rats and 14 months for mice. The pooled controls were of the
 

same strain and from the same supplier, they were administered
 

the same vehicle by the same route of administration, and they
 

were examined by the same pathologists.
 

G. Clinical and Pathologic Examinations
 

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

palpated for masses at each weighing. Rats and mice were weighed
 

individually each week (rats for 20 weeks, mice for 13 weeks) and
 

every 2 weeks for the remainder of the study. Those animals that
 

8
 
Table 1. Design of Chronic Studies of Isophosphamide in Rats
 

Sex and 

Treatment 

Group 

Initial 

No. of 

Animals3 

Isophosphamide 

Dos age" 

(mg/kg) 

Time 

Treated0 

(weeks ) 

on Study 

Untreated 

(weeks ) 

Male 

Untreated-Control 10 0 83 

Vehicle-Control 10 0^ 52 30-31 

Low-Dose 35 6 52 31 

High-Dose 35 12 52 27 

Female 

Untreated-Control 10 0 84 

Vehicle-Control 10 0^ 52 31 

Low-Dose 35 6 52 30-31 

High-Dose 35 12 52 27 

a

Male rats were 35 days of age when placed on study; female rats
 

were 42 days of age.
 

"Isophosphamide was administered intraperitoneally in buffered
 

saline three times per week at a volume of 0.25 ml/lOOg body
 

weight. Doses were based on individual weights.
 

C

A11 animals were placed on study on the same day.
 

^Vehicle-control groups received only buffered saline solution
 

at the same volume as the treated animals.
 

9 
Table 2. Design of Chronic Studies of Isophosphamide in Mice
 

Sex and 

Treatment 

Group 

Initial 

No. of 

Animals3 

Isophosphamide 

Dosage" 

(rag/kg) 

Time 

Treated0 

(weeks ) 

on Study 

Untreated 

(weeks ) 

Male 

Untreated-Control 15 0 80 

Vehicle-Control 15 Od 52 28 

Low-Dose 35 10 52 28 

High-Dose 35 20 52 27 

Female 

Untreated-Control 15 0 81 

Vehicle-Control 15 Od 52 28 

Low-Dose 35 10 52 28 

High-Dose 35 20 52 27 

a

All animals were 42 days of age when placed on study.
 

^Isophosphamide was administered intraperitoneally in buffered
 

saline three times per week at a volume of 1.0 ml/lOOg body
 

weight. Doses were based on the mean weight of animals in a
 

cage.
 

C

A11 animals were placed on study on the same day.
 

^Vehicle-control groups received only buffered saline solution
 

at the same volume as the treated animals.
 

10
 
were moribund at the time of clinical examination were killed and
 

necropsied.
 

The pathologic evaluation consisted of gross and microscopic
 

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

from killed animals and from animals found dead. The following
 

tissues were examined microscopically: skin, muscle, lungs and
 

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

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

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

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

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

brain, and sensory organs. Peripheral blood smears were prepared
 

from each animal. Occasionally, additional tissues were also
 

examined microscopically. The different tissues were preserved
 

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

stained with hematoxylin and eosin. Special staining techniques
 

were utilized when indicated for more definitive diagnosis.
 

A few tissues from some animals were not examined, particularly
 

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

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

of autolysis as to preclude histopathologic evaluation. Thus, the
 

number of animals for which particular organs or tissues were
 

examined microscopically varies, and does not necessarily
 

11
 
represent the number of animals that were placed on experiment in
 

each group.
 

H. Data Recording and Statistical Analyses
 

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

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

clinical observations, survival, animal weight, and individual
 

pathologic results, as recommended by the International Union
 

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

verification of data transcription and for statistical review.
 

The data of the experiments in this bioassay program are subjected to the statistical analyses described in the subsequent
 

paragraphs of this section. The analyses of the experimental
 

results that bear on the possibility of carcinogenicity are
 

discussed in the statistical narrative sections.
 

Probabilities of survival are estimated by the product-limit
 

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

report in the form of graphs. Animals are statistically censored
 

as of the time they are missing or are dead due to other than
 

natural causes; animals dying from natural causes are
 

statistically uncensored. Statistical analyses for a possible
 

12
 
dose-related effect on survival employ the method of Cox (1972)
 

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

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

One-tailed P values are reported for all tests except the
 

departure from linearity test, which is noted when its two-tailed
 

P value is less than 0.05.
 

The incidence of neoplastic or nonneoplastic lesions is 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 include only those animals for which such sites
 

are examined histologically. However, when macroscopic examination is required to detect lesions and when this examination is
 

followed by histologic sampling (e.g., skin or mammary tumors),
 

or when lesions could appear at multiple sites (e.g., lymphomas),
 

the denominators consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of the incidences of
 

tumors is to determine whether animals receiving the test
 

chemical develop a significantly higher proportion of tumors than
 

do control animals. Statistical analyses of the incidences of
 

specific types of tumors are made using the one-tailed Fisher
 

exact test (Cox, 1970) to compare a control group with groups of
 

treated animals at each dose. When results for a number of
 

13
 
treated groups (k) are compared simultaneously with those for a
 

control group, a correction which ensures an overall significance
 

level of 0.05 may be made. The Bonferroni inequality (Miller,
 

1966) requires that the P value for any comparison be less than
 

or equal to 0.05/k. When appropriate the correction is discussed
 

in the narrative section, but it is not used in the tables, where
 

the Fisher exact P values are shown.
 

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

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

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

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


tailed 0.05 level of significance. Unless otherwise noted, the
 

direction of the significant trend is a positive dose
 

relationship. This method also provides a two-tailed test of
 

departure from linear trend.
 

An alternative analysis is applied when early deaths result from
 

causes that are not associated with the formation of tumors. In
 

this analysis, deaths that occur before the first tumor is
 

observed are excluded by basing the statistical tests on animals
 

that survive at least as long as 52 weeks, unless a tumor is
 

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

an early tumor is found, comparisons are based exclusively on
 

animals that survive at least as long as the animal in which the
 

14
 
first tumor is found. Once this reduced set of data is obtained,
 

the standard procedures for analyses of the incidence of tumors
 

(Fisher exact test, Cochran-Armitage test, etc.) are followed.
 

When appropriate, life-table methods are applied to the incidence
 

of tumors. Curves of the proportions surviving without a tumor
 

being observed are computed according to Saffiotti et al. (1972).
 

The. times at which animals die naturally or are killed are
 

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

comparing these curves are used for two groups, and Tarone's
 

extension to testing for linear trend is used for three groups.
 

The tests for the incidence of tumors using life-table methods
 

are one-tailed and, unless otherwise noted, in the direction of a
 

positive dose relationship. Significant departures from
 

linearity (< 0.05, two-tailed test) are also noted.
 

The approximate 95% confidence interval for the relative risk
 

between each of the treated groups and its control is calculated
 

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

 wnere relative risk is Pt/Pc Pt
 is tne

 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 that in a control group is expressed
 

15
 
by 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 are included in the tables of statistical analyses.
 

The interpretation of the limits is that in approximately 95% of
 

a large number of similar 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, the
 

occurrence of a statistically significant result (P < 0.025
 

one-tailed test when the control incidence is not zero, P < 0.050
 

when the control incidence is zero) will also obtain. When the
 

lower limit is less than unity and the upper limit is greater
 

than unity, the former indicates the absence of a significant
 

result while the latter 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.
 

16
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights both of male and female rats administered
 

isophosphamide were comparable with those of the vehicle controls
 

for the first 25 weeks; thereafter, the mean body weights of the
 

high-dose groups were slightly lower (figure 1). Weights of
 

vehicle-control and treated male rats were less than those of
 

untreated controls. A sudden loss of weight during weeks 32 to
 

34 in both control and treated groups, particularly in the males,
 

coincided with an onset of rales. The animals were treated with
 

oxytetracycline in the drinking water at 0.6 mg/ml in weeks
 

35-41, and at 0.3 mg/ml in weeks 41-45. The rales were observed
 

in higher incidences in the treated rats than in the controls.
 

However, no clinical signs clearly associated with treatment with
 

isophosphamide were recorded.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of survival for the treated male and female rats, together with those
 

for the untreated and vehicle controls, are shown in figure 2.
 

In male rats, the Tarone test result for positive dose-related
 

trend in mortality of treated animals compared with vehicle
 

17
 
TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves For Rats Treated With Isophosphamide 

18 
TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves For Rats Treated With Isophosphamide 

19 
controls over the period is significant (P < 0.001), with a
 

similar significant level of departure from linear trend (P <
 

0.001), due to the steep increase in the rate of mortality in the
 

high-dose animals. The median time on study of the high-dose
 

group was 35 weeks, and that of the low-dose group was greater
 

than 83 weeks. Seventy percent of the untreated controls, 80% of
 

the vehicle controls, 55% of the low-dose animals, and 6% of the
 

high-dose animals lived to the end of the study. By week 52 of
 

the study, only 9/35 high-dose male rats had survived; of those
 

that died by week 52, five had developed tumors.
 

In females, the Tarone test result is also significant (P <
 

0.001), with a similar significant level of departure from linear
 

trend (P < 0.001), due to the sharp increase in the rate of
 

mortality in the treated groups, especially in the high-dose
 

group. One hundred percent of the untreated controls, 70% of the
 

vehicle controls, 42% of the low-dose animals, and 3% of the
 

high-dose animals lived to termination of the study. The median
 

time on study of the high-dose group was only 34 weeks, and that
 

of the low-dose group was 74 weeks. Early deaths of the treated
 

animals were not tumor associated, since few tumors were seen in
 

the animals that died early. By week 52 of the study, only 8/35
 

high-dose female rats had survived; of those that died by week
 

52, two had developed tumors, one as early as week 33. Due to
 

20
 
the numerous deaths in the high-dose groups early in the study,
 

the statistical analysis was made using the data concerning those
 

animals that lived longer than week 52 on study or lived beyond
 

the week in which the first tumor appeared, with separate
 

consideration given to each tumor site.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

Appendix A, tables Al and A2; findings on nonneoplastic lesions
 

are summarized in Appendix C, tables Cl and C2.
 

There was a spontaneous and random occurrence of a variety of
 

tumors both in the matched-control groups (untreated and vehicle,
 

phosphate-buffered saline) and in the treated groups. Some types
 

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

treated groups as compared with controls. These neoplasms,
 

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

any treatment.
 

Mesenchymal stromal tumors, which included fibromas, fibrosarcomas, leiomyosarcomas, and undifferentiated spindle-cell
 

sarcomas coded as sarcomas, NOS (not otherwise specified),
 

occurred in 5/32 low-dose males, 3/34 high-dose males, 17/33
 

low-dose females, and 2/34 high-dose females. None of these
 

lesions occurred in the control groups, 0/17 male and 0/20 female
 

21
 
rats. The majority of the stromal tumors in the females were
 

poorly differentiated leiomyosarcomas which were characterized by
 

spindle cells without collagen formation arising from the smooth
 

muscle of the uterus. One leiomyosarcoma was observed in the
 

pelvic subcutaneous tissues without definitive involvement of the
 

uterus. Metastatic leiomyosarcomas occurred in the lungs,
 

urinary bladder, spleen, and other abdominal sites.
 

The other stromal tumors, which were composed of spindle cells
 

having various degrees of collagen formation and differentiation,
 

occurred in a variety of sites in both males and females. One
 

osteosarcoma occurred in the subcutis of a vehicle-control female
 

rat.
 

The incidence of mammary fibroadenoma in female rats was highest
 

in the low-dose group (vehicle controls 3/10, low-dose 28/33,
 

high-dose 6/34).
 

A few rats had malignant lymphomas and granulocytlc leukemias.
 

These hematopoietic tumors occurred only in treated rats, but
 

have been observed previously at a low frequency in rats of this
 

strain.
 

In addition to the neoplastic lesions, a large number of degenerative, proliferative, and inflammatory changes were also
 

encountered in animals of the treated and control groups
 

22
 
(Appendix C). For the most part, these nonneoplastic lesions are
 

commonly seen in aged rats. A high incidence of respiratory
 

infections, especially bronchopneumonias, in the high-dose male
 

and female rats is correlated with decreased life spans and is
 

believed to have been a factor in the small number of neoplastic
 

lesions which occurred in these groups. The incidence of
 

peritonitis and peritoneal adhesions was low, indicating that
 

irritation and infection were not major problems despite the
 

frequent intraperitoneal injections.
 

In the judgment of the pathologists, the results of the study
 

indicate that isophosphamide in phosphate-buffered saline
 

administered intraperitoneally was responsible for a high
 

frequency of sarcomas in male and female Sprague-Dawley rats,
 

particularly uterine leiomyosarcomas. In addition, mammary
 

fibroadenomas were induced in low-dose female rats.
 

D. Statistical Analyses of Results (Rats)
 

Tables E1-E4 in Appendix E contain the statistical analyses of
 

the incidences of those specific primary tumors that were
 

observed in at least 5% of one or more treated groups of either
 

sex. The untreated controls are not included in these tables,
 

since the test conditions of the vehicle controls are closer to
 

those of the treated animals. Due to the severe mortality rates
 

23
 
in the treated rats of both sexes, time-adjusted analyses were
 

performed, eliminating animals that died before week 52 on study;
 

however, when a tumor was found at the specific site before week
 

52 of the study, the incidence was then based on animals that
 

survived at least as long as the animal in which the first tumor
 

appeared. These time-adjusted analyses are shown in tables E3
 

and E4. The statistical narrative below is based on the adjusted
 

analyses.
 

In male rats, the results of the Cochran-Armitage test for
 

positive dose-related trend on the time-adjusted proportions and
 

the Fisher exact test for direct positive comparisons of
 

time-adjusted incidences between the vehicle controls and each of
 

the treated groups are not significant. The incidences of
 

pituitary tumors in control male and female rats were significantly greater than in the treated animals.
 

In female rats, the result of the Cochran-Armitage test on the
 

time-adjusted incidence of fibroadenoma of the mammary gland is
 

significant (P = 0.009), and an indicated departure from linear
 

trend is observed (P = 0.028), due to the steep increase in
 

incidence in the treated groups. The Fisher exact test shows
 

that the incidence in the low-dose group is significantly higher
 

(P = 0.003) than that in the vehicle controls, and the lower
 

limit of the 95% confidence interval of this relative risk has a
 

24
 
value greater than one. The statistical conclusion indicates
 

that this incidence in the low-dose group is dose associated.
 

Although the high-dose percentage is also high (86%), it is based
 

on an incidence of only 6/7 animals. This small sample size
 

reduces the power of the test, and as result, the incidence in
 

the high-dose group is not statistically significant.
 

Leiomyosarcomas of the uterus occurred exclusively in the treated
 

groups. The Fisher exact test shows that the incidence in the
 

low-dose group is significantly higher (P = 0.012) than that in
 

the vehicle controls. The incidence in the high-dose group is
 

not significant, but the power of the test in this instance is
 

weak because of the small sample size of the vehicle-control and
 

high-dose groups (eight animals and seven animals, respectively).
 

In rats, there is no other tumor at any specific site for which
 

the incidence is significant in the positive direction. When the
 

incidences of types of tumors are combined for statistical
 

analysis (as, for example, granulocytic leukemia and lymphoma of
 

the hematopoietic system), the incidences of the individual
 

components of the grouping are not included in the statistical
 

analyses in the tables unless at least one group was observed to
 

have 5% or more of such tumor; however, a list of the incidences
 

of each type of tumor is provided in tables Al and A2 of Appendix
 

A.
 

25
 

IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of the mice showed no consistent effect of the
 

isophosphamide in either sex or at either dose (figure 3), and no
 

other clinical signs of toxicity were observed. No evidence of
 

outbreak of clinical disease was recorded.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of survival for the treated male and female mice, together with those
 

for the untreated and vehicle controls, are shown in figure 4.
 

In male mice, the Tarone test result for positive dose-related
 

trend in mortality of treated animals compared with vehicle
 

controls over the period is not significant at the 0.05 level.
 

Over 90% of the untreated controls, 7% of the vehicle controls,
 

and 31% of the treated animals lived to the end of the study.
 

The median times on study of the high-dose, low-dose, and
 

vehicle-control groups were 53 weeks, 44 weeks, and 29 weeks,
 

respectively. One hepatocellular adenoma of the liver occurred
 

as early as week 44 on study. Only three male mice in the
 

vehicle-control group lived beyond week 44 on study, and no tumor
 

was observed in this group. In the low—dose group, 18/35 male
 

27
 
TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves For Mice Treated With Isophosphamide 

28 
U 1U *W JU tV 3V OU /U OU 

TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves For Mice Treated With Isophosphamide 

29 
mice survived until week 44 of the study, and in the high-dose
 

group, 22/35 male mice survived until week 44. The early deaths
 

of the treated male mice may have suppressed the incidences of
 

late-developing tumors, and analysis of the male mice is made
 

using only those animals surviving 44 weeks or more. A
 

pooled-control group is used for comparison with the treated
 

groups in the analyses without time-adjustment.
 

In females, the Tarone test result of the mortality over the
 

period is significant (P = 0.004), with over 90% of the controls,
 

77% of the low-dose group, and 66% of the high-dose group living
 

to the end of the study, providing sufficient animals for
 

meaningful statistical analyses of the incidence of
 

late-developing tumors.
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

are summarized in Appendix D, tables Dl and D2.
 

With the exception of malignant lymphomas in female mice, the
 

neoplasms listed in Appendix B appeared with approximately equal
 

frequency in treated and matched-control mice, or appeared in
 

insignificant numbers.
 

30
 
Malignant lymphomas, histiocytic type, occurred in 3/32 low-dose
 

and 13/34 high-dose females, but in none of the untreated or
 

vehicle controls. In addition to the neoplastic lesions, a large
 

number of degenerative, proliferative, and inflammatory changes
 

were encountered in animals of the treated and control groups
 

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

commonly seen in aged mice and were not associated with increased
 

deaths or decreased life spans.
 

In the judgment of the pathologists, the results of the study
 

indicate that isophosphamide .in phosphate-buffered saline given
 

intraperitoneally is responsible for an increased incidence of
 

malignant lymphomas in female B6C3F1 mice.
 

D. Statistical Analyses of Results (Mice)
 

Tables F1-F3 in Appendix F contain the statistical analyses of
 

the incidences of those specific primary tumors that were
 

observed in at least 5% of one or more treated groups of either
 

sex. The untreated controls are not included in tables Fl and
 

F2, since the test conditions of the vehicle controls more
 

closely resemble those of the treated groups. Due to the severe
 

mortality rates in the treated male mice, time-adjusted analyses
 

on the incidences of liver tumors were performed. These time-


adjusted analyses are based on animals that lived at least as
 

31
 
long as week 44 of the study, when the first liver tumor
 

occurred, and are shown in table F3. The statistical narrative
 

below on male mice is based on the time-adjusted analyses.
 

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

positive dose-related trend on the time-adjusted proportions and
 

the Fisher exact test on the direct comparisons of time-adjusted
 

incidences of hepatocellular adenoma or carcinoma between the
 

vehicle controls and each of the treated groups are not significant; however, it should be noted that the power of the tests is
 

weak due to the small size of the control-group sample, only
 

three animals. When the time-adjusted incidence in the pooled
 

vehicle controls is used in the Fisher exact test, a probability
 

level of 0.043 results, but this value is above the 0.025
 

required by the multiple-comparison criterion. The incidence of
 

hepatocellular carcinoma is not included in the statistical
 

tables, because the incidences in the treated groups are less
 

than 5%; however, a list of the incidences of each type of tumor
 

is provided in tables Bl and B2 of Appendix B.
 

In the analyses of the incidences of malignant lymphoma of the
 

hematopoietic system in female mice, the Cochran-Armitage test
 

shows highly significant positive dose-related trends of P =
 

0.001 and P < 0.001 using the vehicle controls and the pooled
 

controls, respectively. The Fisher exact test shows that the
 

32
 
incidence in the high-dose group is significantly higher than
 

that in either the vehicle controls (P - 0.005) or the pooled
 

controls (P - 0.001). The statistical conclusion is that the
 

occurrence of malignant lymphoma in female mice is associated
 

with isophosphamide at the doses of this experiment. There is no
 

incidence of granulocytic leukemia in female mice, and there is
 

no other tumor at any specific site for which the statistical
 

tests are significant.
 

33
 

V. DISCUSSION
 

In this bioassay, mean body weights of both male and female rats
 

administered isophosphamide were comparable with those of the
 

controls for the first 25 weeks; thereafter, the mean body
 

weights of the high-dose groups were slightly lower. There was a
 

positive dose-related trend in survival in both sexes of rats.
 

In the high-dose rats, the median survival among males was 35
 

weeks, and only two animals survived until termination of the
 

study at week 79. Among females, the median survival was 35
 

weeks, and only one animal survived until termination of the
 

study. Inflammatory lesions of the lungs, including
 

bronchopneumonia, were observed in higher incidences in the
 

treated rats than in the controls, and may have been responsible,
 

at least in part, for the lower body weights and survival rates
 

observed in the high-dose groups.
 

Mean body weights of the mice did not show any consistent effect
 

from the isophosphamide treatment, and no other clinical signs
 

attributable to the chemical were recorded for the mice.
 

Survival of all groups of females was sufficient (vehicle
 

controls 93%, low-dose 77%, high-dose 66%) for meaningful
 

statistical analyses of the incidence of tumors, while survival
 

of the males was low (vehicle controls 7%, low-dose 31%,
 

high-dose 31%). The median times on study of the vehicle
 

35
 
controls, low-dose group, and high-dose group of male mice were
 

44 weeks, 53 weeks, and 29 weeks, respectively.
 

In male rats, tumors of the hematopoietic system included six
 

malignant lymphomas and two granulocytic leukemias. With the
 

unadjusted analyses, these tumors showed a dose-related trend in
 

male rats using pooled controls (controls 0/29, low-dose 3/32,
 

high-dose 5/34, P = 0.032) and a higher incidence in the
 

high-dose males than in the pooled controls (P = 0.040). These
 

tumors were not significant when compared with those in the
 

vehicle controls using time-adjusted analyses, and they cannot
 

clearly be associated with treatment. However, it should be
 

noted that the five rats with these tumors were observed among
 

the high-dose animals whose median survival was only 35 weeks.
 

The incidence of uterine leiomyosarcoma in female rats was
 

statistically significant in the low-dose group using pooled
 

controls (15/32, P < 0.001). The incidence was also significant
 

when compared with vehicle controls using time-adjusted analyses.
 

The poor survival rate of the high-dose females (median time on
 

study, 35 weeks) may account for the low incidence of this lesion
 

at this dose. Metastatic leiomyosarcomas occurred in the lungs,
 

urinary bladder, spleen, and other abdominal sites. These
 

results indicate that the incidence of uterine leiomyosarcomas in
 

female rats is related to treatment.
 

36
 
There was a highly significant incidence of fibroadenoma of the
 

mammary gland in the low-dose female rats (28/33) when compared
 

with the incidence in the vehicle controls (3/10, P = 0.001) and
 

with that in the pooled controls (8/28, P < 0.001). The
 

incidence was also significant when compared with matched vehicle
 

controls using time-adjusted analyses. This tumor was found in
 

only six high-dose animals and the low survival rate of this
 

group must be considered when assessing the low incidence.
 

Although fibroadenoma is a type of mammary tumor commonly found
 

in control female Sprague-Dawley rats in the bioassay program at
 

this laboratory (untreated controls 66/220, vehicle controls
 

75/245), the higher incidence in the low-dose group indicates
 

that this tumor is associated with treatment. Other epithelial
 

tumors of the mammary gland including adenoma, adenocarcinoma,
 

cystadenoma, and cystadenocarcinoma were found among the animals
 

having fibroadenoma and in one additional low-dose and vehicle-


control female, respectively.
 

In mice, five low-dose males had hepatocellular adenoma or
 

carcinoma of the liver. In comparison with the pooled vehicle
 

controls, a probability level of 0.043 resulted using time-


adjusted analysis, but this value is above the 0.025 required by
 

the multiple-comparison criterion and these tumors are not
 

clearly related to treatment. Malignant lymphomas of the
 

37
 
hematopoietic system in female mice showed a significant dose-


related incidence (vehicle controls 0/14, P = 0.001; pooled
 

controls 1/29, P < 0.001; low-dose 3/32, high-dose 13/34). The
 

incidence of malignant lymphomas in the high-dose females was
 

significantly higher than that in the vehicle controls (P =
 

0.005) or pooled controls (P = 0.001) using unadjusted analyses.
 

These tumors were not found in males.
 

Since isophosphamide is an anticancer drug, most of the testing
 

has been in clinical studies with humans (Carter 1972; Ahmann et
 

al., 1974; Kovach et al., 1974). Animal studies have been
 

focused upon chemotherapeutic effects rather than carcinogenic
 

effects (Finklestein, 1975).
 

It is concluded that under the conditions of this bioassay,
 

isophosphamide was not carcinogenic in male Sprague-Dawley rats
 

or in male B6C3F1 mice. However, the incidence of
 

leiomyosarcomas of the uterus indicates that isophosphamide was
 

carcinogenic in female Sprague-Dawley rats, and the incidence of
 

fibroadenoma of the mammary gland in female rats was associated
 

with treatment with isophosphamide. Isophosphamide was
 

carcinogenic in female B6C3F1 mice, producing malignant lymphomas
 

of the hematopoietic system.
 

38
 
VI. BIBLIOGRAPHY
 

Allen, L. M. and Creaven, P. J., Effect of microsomal activation
 

of interaction between isophosphamide and DNA.
 

Communications 61(12);2009-2011. 1972.
 

Ahmann, D. L., Bisel, H. F., and Hahn, R. G., Phase II clinical
 

trial of isophosphamide (NSC-109724) in patients with
 

advanced breast cancer. Cancer Chematherapy Reports Part _1
 

18(6):861-865, 1974.
 

Armitage, P., Statistical Methods .in Medical Research. J. Wiley &
 

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

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

Panel on Carcinogenicity of the Cancer Research Commission
 

of UICC. Vol. 2^, International Union Against Cancer, Geneva,
 1969.
 

Carter, S. K., New drugs on the horizon

carcinoma. Cancer 30;1402-1409. 1972. 

 in bronchogenic 

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

Soc. B 34;187-220. 1972. 

 J. R. Statist. 

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

1970, pp. 48-52.
 

Finklestein, J. Z., Tittle, K. , Meshnik, R., and Weiner, J. ,
 

Murine neuroblastoma: Further evaluation of the C1300 model
 

with single antitumor agents. Cancer Chemotherapy Reports
 

Part J_.59(5):975-983, 1975.
 

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

significance tests, confidence limits and adjustments for
 

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

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

incomplete observations. J. Am. Statist. Assoc. 53;457-481.
 1958.
 

Kovach, J. S., Schutt, A. J., Uahn, R. G., Reitemeier, R. J., and
 

Moertel, C. G., A phase 2 study of intermittent high dose
 

isophosphamide therapy of advanced colorectal cancer.
 

Oncology 29:34-39. 1974.
 

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

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

and Biomed. Res. _7:230-248, 1974.
 

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

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

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

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

induced by different numbers of administrations of benzo (a)
 

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

Slavik, M. and Carter, S. K., Bronchogenic carcinoma: New drugs
 

available for study. Cancer Chemotherapy Reports Part ^
 

4.(2): 265-269, 1973.
 

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

Biometrika 62:679-682. 197.5.
 

40
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS TREATED WITH ISOPHOSPHAMIDE
 

41
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF ISOPHOSPHAMIDE

 

CONTROL (OUTS) CONTROL (VEH) 

AHINALS INITIALLY IN STUDY 10 10 

iHIHALS NBCHOPSIED 8 9 

ANIMAL S EXAMINED HISTOPATHOLOGICALLY 8 9 

IHTEGUHENTAP.Y SYSTEM 

• SKIH (8) (9)

 SEBACEOUS ADENOM A

 

*SOBCOT TISSUE (8) (9)

 

SARCOMA, NOS 

FIBROMA 

RESPIRATORY SYSTEM 

ILONG (7) (9) ADENOCARCINOMA , NOS, HETASTATIC 1 (1«) 

ALVEOLAR/BPONCHIOLAR ADENOMA 

PIBPOSARCOMA, METASTATIC 

HEMATOPOIETIC SYSTEM 

*MOITIFLE ORGAN S (8) (9)

 

MALIG.LYMPHOMA , UND I PPEH-TYPE 

NALIG.LYMPHONA , LYMPHOCYTIC TYPE 

MALIG.LYBPHOMA , HISTIOCYTIC TYPE 

GRANDLOCYTIC LEUKEMI A 

*BCNE MAFRO W (8) (8)

 

IULIG.LYHPHOMA, HISTIOCYTIC TYPE 

CIRCULATOBY SYSTEM 

tHEART (7) (8)

 

PIBRCSARCCMA 

DIGESTIVE SYSTEM 

_,,BP8 5 , ., , , 

» NUMBER OP ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OP ANIMALS NECROPSIED 

LOU DOSE 

35 

32 

32 

(32) 

1 (3«) 

(32) 

1 (3%) 

2 (6*) 

(32) 

2 (6*) 

(32) 

2 <6«) 

1 (3%) 

(31) 

(29) 

1 (3%) 

HIGH DOSE 

35 

34 

3U 

(3U) 

(3i») 

(3«) 

1 (3«) 

(3») 

2 (6X) 

1 (3%) 

1 (3*) 

(3<t) 

1 (3«) 

(3») 

43 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED)

 

URINAR Y SYSTEM 

tKIDNEY 

ADENCCARCINOMA , NOS 

• URINAR Y BLADDER 

TRANSITIONAL-CELL CARCINOM A 

ENDOCRIN E SYSTEM 

tPITOITAFY 

CHRONOPHOBE ADENOM A 

CHROHOPHOBE CARCINOMA 

tADRENA L 

CORTICAL ADENCN A 

PHEOCHPOMOCYTOMA 

• PANCREATIC ISLETS 

ISLIT-CELL ADENOM A 

REPRODUCTIVE SYSTEM 

• MAMMAR Y GLAND 

FIBPO ADENOM A 

tTESTIS 

INTERSTITIAL-CILL TUMOR 

NEPVCUS SYSTEM 

tBRAIN/HEHINGES 

SARCOMA , NOS 

.SPECIAL SENSE ORGANS 

NON E 

HUSCULOSKELETAL SYSTEM 

fififii 

t NUMBER 0? ANIMAL S KITH TISSUE

» NUMBE R OF ANIMAL S NECFOPSIED 

CONTROL (UNTR) CONTROL (VEH) 

(7) (9) 

1 <11X) 

(8) (9) 

1 (13*) 

(8) (7) 

1 (13*) 3 (tt 3*) 

1 (13*) 1 (1««) 

(8) (9) 

1 (13X) 

1 (13*) 

(8) CM 

1 (13X) 

(8) (9) 

1 (11X) 

(8) (9) 

1 (13*) 

(7) (8) 

 EXAMINE D MICROSCOPICALLY 

44 

LOW DOSE 

(32) 

(28) 

(29) 

3 (10*) 

1 (3*) 

(31) 

2 (6*) 

2 (6*) 

(32) 

(32) 

(30) 

(30) 

1 (3*) 

HIGH DOSE 

(3*) 

(30) 

(32) 

(31) 

2 <SX ) 

2 <6X) 

(33) 

(3*1 

(33) 

(33) 

1 (3*) 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL (UNTR) CONTROL (VEH) LOU DOSE HIGH DOSE 

BODY CAVITIES 

• MEDIASTINU M 

PIBRCSARCOM A 

<8> (9) (32) (3U) 

1 (3*) 

•PERITONEU M (8) (9) (32) (3«) 

PIBSOSARCOMA 1 <3«) 

•MESENTERY (8) (9) (32) (3K) 

LIPCNA 1 (11«) 

ALL OTHER SYSTEMS 

• MULTIPLE ORGAN S (8) (9) (32) (3U) 

"IBROSARCOMA 

MESOTHELIOMA, MALIGNAN T 

1

1

 (3*) 

 (3«) 

ANIKA L DISPOSITION SUMMARY 

ANIMAL S INITIALLY IN STUDY 10 10 35 35 

NATURA L DEATHS 3 2 8 11 

MORIBUN D SACRIFICE 7 22 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINA L SACRIFICE 7 8 20 2 

ANIRA L HISSING 

4_I!!CiUD£s_A.UTgUZE2_iNI3AL.S 

* NDNBEG OF ANIMAL S KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

45 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL (UNTR) CONTROL (VEH) LOW DOSE HIGH DOSE 

TUMOR SUMMAR Y 

TOTAL ANIMALS 1IITH PRIMAR Y

TOTAL PRIMAR Y TUMORS

 TDMORS* 4

 7

 5

 7

 17

 21

 12 

 12 

TOTAL ANIMAL S WITH BENIG N

TOTAL BENIGN TUBORS

 TUMORS u

 5

 n

 5

 1 1

 1 2

 l| 

t 

TOTAL ANIMALS KITH MALIGNAN T

TOTAL MALIGNAN T TUMORS

 2 TUMORS

2 

2 

2 

9 

9 

3 

8 

TOTAL ANIMALS ilTH SECONDARY

TOTAL SECONDARY TUMORS

 TUMORS! 1

 1

 1 

1 

TOTAL ANIMAL S 8ITH TUMORS

BENIGN OR MALIGNAN T 

TOTAL UNCERTAI N TUMORS 

 UNCERTAINTOTAL ANIMALS KITH TUMORS

PRIMAR Y OR METASTATIC 

TCTAL UNCERTAIN TUMORS 

 UNCERTAIN­

* PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDARY TDMORS 

* SECONDAR Y TUMORS : METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN 

46 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF ISOPHOSPHAMIDE 

CONTROL (UNTR) CONTROL (VEH) LOW DOSE HIGH D3 SB 

ANIMALS INITIALLY IN STUDY 10 10 

ANIMAL S NECROPSIED 10 10 

ANIMAL S EXAMINE D HISTOPATHOLnGICALLY 10 10 

INTEGUMENTARY SYSTEM 

*SUBCUT TISSUE (10) (10) 

SQUAMOU S CELL CARCINOM A 

"IBROMA 

LEIOMYOSARCOM A 

OSTEOSARCOMA 1

RESPIRATOR Y SYSTEM 

tLUN G (9) (10) 

LEIOMYOSABCCMA, METASTATIC 

OSTEOSAPCOMA , METASTATIC 1

HEMATOPOIZTIC SYSTEM 

•MULTIPLE ORGAN S (10) (10) 

MALIG.LYMPHOMA , LYMPHOCYTIC TYPE 

*BCN E MARRO W (10) (9) 

G"ANULCCYTIC SARCOMA 

• SPLEE N (10) (10) 

LEIOMYCSARCCMA , METASTATIC 

HEMANGICSARCOM A 

CIPCULATOPY SYSTEM 

• ENDOCARDIU M (9) (9) 

FIBHCSARC01A 

DIGESTIVE SYSTEM 

• LIVER (10) (10) 

HEPATOCELLULA R ADENOM A 

* NDMBE1! OF ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

 (10%) 

 (10X) 

35

33

33

 35 

 3<» 

3H 

(33)

1

1

1

 (3X) 

 (3X) 

 (3%) 

 (3U) 

(33)

1 (3X) 

 (33) 

(33)

1

(32)

2

(33)

1

1

 (3%) 

 (6%) 

 (3») 

 (3%)

 (3K) 

 (32) 

 <3U ) 

1 (3%) 

(33)

1 <3X ) 

 (33) 

(33)

_ 1 13SL _ 

 (33) 

47 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

URINAR Y SYSTEM 

• URINAR Y BLADDER 

SQUA100S CELL CARCINOMA 

TRANSITIONAL-CELL PAPILLOMA 

TRANSITIONAL-CELL CARCINOM A 

LEIOMYOSAPCOflA , METASTATIC 

ENDOCRINE SYSTEM 

IPITUITASY 

CHROMOPHOBE ADENOM A 

CHROMOPHOBE CAPCIHOMA 

tADRENA L 

CORTICAL ADENOM A 

REPRODUCTIV E SYSTE M 

'MAMMAR Y GLAN D 

ADENCMA, NOS 

ADENOCARCINOMA , NOS 

CYSTADENOMA , NOS 

CYSTADENOCABCINOMA , HOS 

FIBROSARCOBA 

PIBPO ADENOM A 

'VAGINA 

SQUAMOU S CELL CARCINOM A 

*UTERUS 

SQUAMOU S CELL CARCINOM A 

LEIOMYOSARCOM A 

ENDOMETRIAL STROMAL POLYP 

tUTERUS/ENDCMETRIO N 

LEICMYCSARCOM A 

NERVOU S SYSTEM 

IBRAI N 

_ -§iB£CBi..i £OSa_INVA.SIVE _ 

I NUMBE P OF ANIMAL S KITH TISSUE

* NUMBE R OF ANI1ALS NSCPOPSIED 

CONTROL (ONTR) CONTROL (VEH) 

(10) (10) 

1 (10X) 

(10) (10) 

5 (SOX) 3 (30X) 

1 (10%) 4 (HOX ) 

(10) (10) 

3 (30X) 3 (30X) 

(10) (10) 

1 (10«) 1 (10X) 

1 (10X) 

3 (30X) 3

 (3 OX) 

(10) (10) 

(") (9) 

1 (11X) 

(9) (9) 

(10) (10) 

 EXAMINE D MICROSCOPICALLY 

48 

LOW DOSE HIGH DOSE 

(26) (32) 

1 (<•*) 

1 («*) 

1 (<*«) 

(31) (3U) 

7 (23X) 

1 (3X) 1 (3X) 

(33) (31) 

8 (21X) 1 <3X) 

(33) (3U) 

2 (6X) 

2 (6X) 2 (6X) 

3 (9«) 1 (3X) 

2 (6X) 

1

28

 (31) 

 (85X) 6 (18X) 

(33) (3U) 

1 (3X ) 

(32) (3«) 

1 <3X) 

iu (uux ) 

1 (3X) 

1 <3X ) 

(32) (3d ) 

1 (3X) 

(31) (31) 

1 1JXL _ 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL (UHTH) CONTROL (VEH) LOW DOSE HIGH DOSE 

ASTSOCYTOM A 1 (10S) 

tCEREBELLU N

HABARTC1 A

 (10) (10) (31)

 1 (3X) 

( 3U) 

SPECIAL SENS E ORGANS 

NON E 

9USCOLOSKELETA L SYSTE1 

*MUSCLE OF HEAD

SARCOMA , NOS

 (10) (10) (33)

 1 (3%) 

 (3U) 

BODY CAVITIES 

*ABDCHINA L CAVITY

SARCCHA , NOS

LEIOBYOSARCOMA, METASTATIC

 (10) (10) (33)

 1 (3«) 

 (31) 

1 (3%) 

ALL OTHER SYSTEMS 

NON E 

ANIBA I DISPOSITION SOHHAR Y 

ANIHAL S INITIALLY IN STODY

NATURA L DEATHS

MORIBUND SACRIFICE

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINA L SACRIFICE

ANIHA L HISSING 

 10

 10

 10

 3

 7

 35

 9

 11

 15

 35 

 15 

 19 

1 

» IB<;LBDES AOTOUZE.P. AgiH&L§ . _ _ T 1 . ,_ . . _,_ . _ . _ . _ _ 

t NUHBER OF ANIMALS BITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIHALS NECEOPSIED 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

C( DNTRO L (UNTR )

TUMOR SUMMAR Y 

TOTAL ANIMAL S WIT H PRIMAR Y

TOTAL PRIMAR Y TUMORS 

 TUMOPS* 7 

15 

TOTAL ANIMAL S KITH BENIGN

TOTAL BENIG N TUMORS 

 TUMORS 7 

13 

TOTAL ANIMALS WITH MALIGNANT

TOTAL MALIGNAN T TUMORS 

 TO 10RS 2 

2 

TOTAL ANIMALS WIT H SECONDAR Y TUMORS* 

TOTAL SECONDAR Y TUMORS 

TOTAL ANIMAL S WITH TUMORS

BENIG N OR MALIGNAN T 

TOTAL UNCERTAIN TUMORS 

 UNCS3TAINTOTAL ANIMAL S WITH TUMORS

PRIMAR Y OR METASTATIC 

TOTAL UNCERTAIN TUMORS 

 UNCERTAIN ­

 CONTROL (VEH ) LOW DOSE HIGH DOSE 

9 

17 

32 

84 

9 

15 

7 

10 

31 

52 

7 

8 

6 

7 

23 

32 

6 

7 

1 

1 

4 

5 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

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

50 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE TREATED WITH ISOPHOSPHAMIDE
 

51
 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

GIVEN INTRAPERITONEAL INJECTIONS OF ISOPHOSPHAMIDE

 

CONTROL (UNTH) CONTROL (VEH) LOW DOSE HIGH DOSE 

ANIMALS

ANINAI S

ANIMAL S

 INITIALLY IN STUDY

 NECHOPSIED

 EXAMINE D HISTOPATHOLOGICALL Y

 15

 14

 14

 15

 14

 14

 35

 30

 29

 35 

 27 

 11 

INTEGUMENTARY SYSTEM 

NON E 

RESPIRATORY SYSTEM 

HUNG

ADENOCAHCINONA , NOS,

ALVEOLAR/BRONCHIOLAR

 UNC PRIM OR

 CARCINOMA

 (14) (14) (29)

 1 ( 3X) 

•

 (27) 

1 (4X) 

HEMATOPOIETIC SYSTEM 

NONE 

CIRCOLATORY SYSTEM 

NO>IE 

DIGESTIVE SYSTEM 

*LIVEH

HIPATOCELLULAR

HEPATOCELLULAR

 ADENOMA

 CARCINOMA

 (14)

 2 (14X)

 (14) (29)

 4

 1

 (14%)

 (3() 

 (27) 

2 (7%) 

URINAR Y SYSTEM 

NON E 

INDOCFINE SYSTEM 

ftADRENAL

P.fl£CCflj|O.J!S£Xig.lJA.

t NUMBER OF ANIMALS WITH TISSUE

* NUMBER OF ANIMAL S NECROPSIED 

 (14) (14)

 EXAMINED MICROSCOPICALLY 

 (29)

 J ^JJl. 

 (27) 

53 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

CONTROL (UHTR) CONTROL (VE H) LOB DOSK HIGH DOSE 

REPRODUCTIVE SYSTEM 

NONE 

NERVCOS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

*HARDERIAN GLAND

ADENOMA, NOS

 (14) (14) (30) (27) 

1 («*) 

MUSCULOSKEIETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

• MULTIPLE ORGANS

IDBNOCARCINOMA, NOS, HETASTATIC

 (10) (11) (30)

 1 (3») 

 (27) 

AHIHAL DISPOSITION SUMMAR Y 

ANIMALS INITIALLY IN STUDY

NATURAL DEATHS

MORIBUND SACRIFICE

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE

ANIMA L HISSING 

 15

 1

 1«

 15

 10

 «

 1

 35

 24

 11

 35 

 16 

R 

 11 

9 WCL.BPBS ABIPLIZED ANIMALS 

t NUMBER

* NUMBER

 OF ANIMALS

 OF ANIMALS

 ilTH TISSUE

 NBCHOPSIED 

 EXAMINED MICROSCOPICALLY 

54 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

CONTROL (UNTR) CONTROL (TEH) LOR DOSE HIGH DOSE 

TUMOR SUMMAR Y 

TOTAL ANIMALS WITH PRIMAR Y

TOTAL PRIMAR Y TUMORS

 THHORS* 2

 2

 6

 7

 » 

» 

TOTAL ANIMAL S KITH BENIGN

TOTAL BENIGH TUMORS

 TOMOPS 2

 2

 5 

S 

3 

3 

TOTAL ANIMALS WITH MALIGNAN T

TOTAL MALIGNANT TUMORS

 TUMORS 1

 1

 1 

1 

TOTAL ANIMALS WITH SECONOAPY

TOTAL SECONDARY TUMORS

 TUMORSt 1 

1 

TOTAL ANIMAL S WITH TUMORS

BENIG N OR MALIGNAN T 

TOTAL UNCERTAIN TUMORS 

 UNCERTAIN ­

TOTAL ANIMALS WITH TUMORS

PRIMAR Y OR HETASTATIC

TCTAL UNCERTAI N TUMORS

 UNCERTAIN ­

1 

1 

«

t

 PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

 SECONDARY TUMORS: ME1ASTATIC TUNORS OP TUMORS INVASIVE INTO AN ADJACENT ORGAN 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

GIVEN INTRAPERITONEAL INJECTIONS OF ISOPHOSPHAMIDE 

CONTROL (UNTR) CONTROL (VEH) LOR DOSE HIGH DOSE 

ANIMAL S INITIALLY IB STUDY 15 15 35 35 

ANIMAL S HISSING 1 

AMI MAIS NECROPSIBD 15 1» 32 3U 

ANIMAL S EXAMINED HISTOPATHOLOGICALLY 15 1» 32 3« 

INTEGDMENTAR Y SYSTEM 

*SKIN (15) (11) (32) (3«)

 

ADENOSQOAMOUS CARCINOMA 1 (3%)

 

KERATOACANTHOMA 1 (3*)

 

*SUBCOT TISSUE (15) (1U) (32) (3«) 

SARCOMA, NOS 1 (3*)

 

RESPIRATORY SYSTEM 

NON E 

HEBATOPOIITIC SYSTEM 

*MULTIPLE ORGANS (15) (11) (32) (3"») 

MALIG.LYMPHOBA, HISTIOCYTIC TYPE 3 (9«) 12 (35*) 

fSPLEE N (15) (11) (32) <3») 

MALIG.LYMPHOMA , HISTIOCYTIC TYPE 1 (3») 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

ILIVHH (15) (1«) (32) (33)

 

HIPATOCELLDLAR CARCINOMA 1 (3*)

 

fSTCMACH (15) (11) (32) (3«) 

LEICMYOSA8CCNA 1 (3*) 

URINAR Y SYSTEM 

BOHJ 

* NUMBER Of ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* HDNBEP OF ANIMALS NECROPSIED 

56 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

CONTROL (ONTR) CONTROL (VEH) LOW DOSE HIGH DOSE 

ENDOCRIN E SYSTEM 

NON E 

REPRODUCTIV E SYSTEM 

•UTERUS (11) Ot) (32) (33) 

LEIOMYOSARCOM A 1 (3*) 1 <3X) 

tOVARY (1U) (IK ) (32) (31) 

HEHANGIQH A 1 (3») 

Nipvnus SYSTEM 

NONE 

SPECIAL SENSE OPGANS 

NONE 

MUSCULO SKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NON E 

A LL CTHER SYSTEMS 

NCK E 

* NUHBE R OF ANIMAL S HITH TISSUE EXABIHE D MICROSCOPICALLY 

* NUNBE P OF ANIMAL S NECROPSIED 

57 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

CONTROL (ONTR) CONTROL (VEH) LOB DOSE HIGH DOSE 

ANIMA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALL Y IN STUD Y

NATURA L DEATHS

MORIBUN D SACRIFICE

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED

TERMINA L SACRIFICE

ANIMA L MISSING

 15

 15

 15

 1

 1 

 13

 35

 5

 3

 26

 1 

 35 

6 

6 

 23 

3 INCLUDES AUTOLYZED ANIMAL S 

TU10S SUPNAR Y 

TOTAL ANIMAL S WITH PRIMAR Y TUMORS* TOTAL PRIMAR Y TUMOPS 7

 8

 15 

 16 

TOTAL ANIMAL S MIT H BENIG N

TOTAL BENIGN TUMOP.S

 TUMC'S 1

 1

 1 

1 

TOTAL ANIMAL S WIT H MALIGNAN T TUMOP S

TCTAL MALIGNAN T TUMORS

 7

 7

 1» 

 15 

TOTAL ANIMAL S WITH SECONDA3Y

TOTAL SECONDAR Y TUMORS 

 TU10PS* 

TOTAL ANIMAL S WITH TUMOPS

BENIG N OR lALIGNAN T 

TOTAL UNCERTAIN TUMORS 

 UNCERTAIN ­

TOTAL ANIMAL S WITH TUMORS

PSIMAB Y CR META STATIC 

TOTAL UNCERTAI N TUMORS 

 UNCEBTAIN ­

* PRIMAR Y TUMOES: ALL TUMORS EXCEPT SECONDARY TUMORS 

I SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIV E INTO AN ADJACENT ORGA N 

58 
SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS TREATED WITH ISOPHOSPHAMIDE
 

59
 

TABLE C1. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF ISOPHOSPHAMIDE 

CONTROL (UNTR) CONTROL (VEH) LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 10 10 35 35
 

ANIMALS NECROESIED 8 9 32 31
 

ANIMALS EXAMINED HISTOPATHOIOGICALLY 8 9 32 31
 

INTEGUMENTARY SYSTEM
 

*SKIN (8) (9) (32) (31)
 

INFLAMMATION, CHRONIC 1 (3X>
 

INFLAMMATION, GR ANULOM ATOUS 1 <3X)
 

*SUBCUT TISSUE (8) (9) (32) (31)
 

ABSCESS, NOS 1 (3X)
 PEHIARTERITIS 1 (3*)
 

RESPIRATORY SYSTEM 

tTRACHEA

INFLAMMATION, NOS

INFLAMMATION, SUPPURATIVE 

INFLAMMATION, ACUTE/CHRONIC

INFLAMMATION, CHRONIC 

 (8) 

1

 3

 (13%) 

 (38X) 

(8)

5 (63X)

 (30) 

1

 20

1

 (3X) 

 (67X) 

 (3%) 

(31) 

17

3

3

 (55%) 

 (10%) 

 (10%) 

ILUNG/BRCNCHUS BROUCHIECTASIS

INFLAMMATION, NOS INFLAMMATION, CHRONIC 

 (7) 

1 (11%) 

(9)

1 (11%)

 (32) 

5

1

3

 (16X) 

 (3X) 

 (9X) 

(31) 

7

1

1

 (21%) 

 (12%) 

 (3%) 

*LUNG

BRONCHOPNEUNONIA, NOS

INFLAMMATION, NOS 

INFLAMMATION, INTERSTITIAL 

ABSCESS, NOS 

 (7) 

1 (11X) 

(9) (32) 

5

1

1

 (16%) 

 (3X) 

 (3%) 

(31) 

21

1

2

 (62%) 

 (3%) 

 (6%) 

HEHATOPOIETIC SYSTEM
 

tBONE HARROW (8) (8) (31) (31)
 

ATROPHY, NOS 2 (6%)
 

tLYHPH NODE (2) (5) (9) (20)
 

t NUMBER OF ANIMAL S IITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

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

CONTROL (ONTH) CONTROL (VEH ) LOW DOSE 

ITHYMUS (7) (6) (23)

 

ATRCPHY , 1103 

CIRCULATORY SYSTEM 

*HEART (7) (8) (29) 

PEHIARTERITIS 1 (3%) 

DIGESTIVE SYSTEM 

#LIVER (8) (9) (32)

 

HEHORRHAGIC CYST 

NECROSIS, NOS 1 (13%) 

ISTOHACH (7) (9) (31)

 

DLCER, NOS 1 (11%)

 

ULCER, CHRONIC 

*COLON (8) (9) (31)

 

HEMORRHAGE 

INFLAMMATION , ACUTE 

URINAR Y SYSTEM 

•KIDNEY (7) (9) (32) 

INFLAMMATION , CHRONIC 3 (43%) 1 (11S) 5 (16%) 

ENDOCRINE SYSTEM 

NON E 

BEPRODOCTIVE SYSTEM 

*PROSTATE (8) (9) (31)

 

INFLAMMATION, SUPPUHATIVE 1 (13%)

 

IHFLAMHATION, CHRONIC 1 (13%) 

*SEMINAL VESICLE (8) (9) (32)

 

INFLAMMATION , ACUTE/CHRONIC 1 (13%)

 

NERVCO S SYSTEM 

NONJ 

f NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

HIGH DOSE 

(22) 

1 (5%) 

<3U) 

(3«) 

1 (3%) 

(33) 

1 (3%) 

1 (3%) 

(33) 

1 (3%) 

1 (3%) 

(31) 

3 (9%) 

(33) 

(31) 

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

I NUMBE R OF ANIMALS WITH TISSUE EXAMINE E MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

63 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF ISOPHOSPHAMIDE

 

CONTROL (UNTR) CONTROL (VEH) LOU DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY 10 10 35 35
 

ANIMALS NECROPSIED 10 10 33 34
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 10 10 33 34
 

INTEGUMENTARY SYSTEM
 

•SKIN	 (10) (10) (33) (34)
 

INFLAMMATION, GR ANULOM ATOOS 1 (10X)
 

RESPIRATORY SYSTEM
 

•LARYNX	 (10) (10) (33) (34)
 

INFLAMMATION, SUPPURATIVE 1 <3X)
 INFLAMMATION, ACUTE/CHRONIC 1 (10*)
 

•TRACHEA	 (9) (10) (29) (29)
 

INFLAMMATION, NOS 6 (21X)
 INFLAMMATION, SUPPUPATIVE 1 (3X)
 INFLAMMATION, ACUTE 2 <7X)
 

INFLAMMATION, ACUTE/CHRONIC 6 (67X) 4 (40X) 12 (41X) 8 (28X)
 

INFLAMMATION, CHRONIC 2 (7X)
 

ILUHG/BBCNCHUS (9) (10) (33) (33)
 BRONCHIBCTASIS 1 (11%) 2 (20X) 4 (12X) 6 (18X)
 

INFLAMMATION, NOS 4 (12X)
 

tLUNG (9) (10) (33) (33)
 

EDEPA, NOS 1 (3X)
 BRONCHOPNEOMONIA, NOS 1 (10X) 6 (18X) 13 (39X)
 INFLAMMATION, INTERSTITIAL 4 (12X)
 

INFLAMMATION, NECROTIZING 1 (3X)
 

ABSCESS, NOS 1 <3X)
 

INFLAMMATION, CHPONIC 1 ( 1 1X) 1 (3X)
 PLASBACYTOSIS 1 <3X)
 

HEMATOPOIETIC SYSTEM
 

tBONE NARROW (10) (9) (32) (32)
 

JIBSOJIS J_13.SL__.
 

t NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECBOPSIED 

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

CONTROL (ONTR) CONTROL(VEH) 

ATROPHY, NOS 1 (11*) 

tSPLEEN (10) (10)

 

NECROSIS, NOS 

tLYHP H NODE (6)

 

INFLAMMATION , NZCROTIZING 

PLASMACYTOSIS 

ITHYMUS (6) (10)

 

PLASMACYTOSIS 

CIRCULATORY SYSTEM 

•HEART (9) (9)

PERIAHTEHITIS 1 (11X) 

PLASMACYTOSIS 

t ENDOCARDIUM (9) (9)

 

FIBRCSIS 

DIGESTIVE SYSTEM 

•LIVER (10) (10)

 

HEMORRHAG E 

NECROSIS, COAGULATIVE 

tPANCREAS (10) (10)

 

HEMORRHAG E 

INFLAMMATION, INTERSTITIAL 

ATROPHY, NOS 

tSTCMACH (10) (10)

 

ULCER, NOS 

• GASTRIC SUBNUCOSA (10) (10) 

HEMORRHAG E 

tCECUH (9) (10) 

HEMORRHAGIC CYST 

URINAR Y SYSTEM 

•KIDNEY (10) (10) 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

LOO DOSE 

(33) 

1 (3*) 

(2U) 

(27) 

(33) 

(33) 

1 (3%) 

(33) 

(31) 

(33) 

2 (6*) 

(33) 

(33) 

(33) 

HIGH DOSE 

6 (19%) 

(3«) 

(30) 

1 (3*) 

1 (3*) 

(28) 

1 ("»%) 

(3J» 

1 <3») 

1 (3J) 

(33) 

(33) 

1 (3*) 

1 (3%) 

(33) 

1 (3%) 

1 (3%) 1 (3*) 

(34) 

(31) 

1 13%) 

(3U) 

1 (3*) 

(31) 

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

CONTROL (ONTR) CONTSOL(VEH) LOU DOSE HIGH DOSE 

tKIDNEY/PELVI S 

INPLAMBATICN , CHRONIC 

(10) (10) (33) (34) 

1 (3*) 

ENDOCRIN E SYSTEH 

NONE 

REPRODUCTIVE SYSTEM 

*NAHNAR Y GLAN D 

HYPERPLASIA, CYSTIC 

(10)

1 (10X)

 (10) (33) (3U) 

2 (6%) 

•UTERUS 

HEMORRHAG E 

HEMORRHAGE ,

PYOMETRA 

ANGIECTASIS 

 CHRONIC 

(9)

3 (33X)

 (9) (32)

1

 3

1

 (3«) 

 (9«) 

 (3X) 

 (3I») 

1 (3*) 

tUTERUS/BNDOMETRIUH 

INFLANHATICN, NOS 

(9) (9) (32)

1 (3*) 

 (3<l) 

tOVARY/OVIDOCT 

INFLAMMATION, NOS 

(9) (9) (32) (3«) 

1 (3*) 

» OVARY 

CYST, NOS 

HEMORRHAGE 

IN FL ABB ATION , SDPP DR AT IV E 

(7) (10) (21)

1 (5«) 

 (26) 

1

1

 (»X) 

 (»X) 

NERVOOS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

HONE 

MOSCOLOSKELETAL SYSTEM 

* JOINT 

IHFLJBHATION. CHgQNIC SUPPU?A.! 

(10) (10)

CU__ —

» NDMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

— _ —

 (33) (3«) 

2-J651-

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

CONTROL (ONT R) CONTROL (VEH ) LOW DOSE HIGH D3SE 

BOOT CAVITIES 

*PERITOHEOH 

HEMORRHAG E 

INFLAMMATION , 

INFLAMMATION , 

FIBRINOUS 

CHRONIC 

(10) (10) (33) (34) 

2

2

2

 (6*) 

 (««) 

 (6X) 

•PLEURA 

INFLAMMATION , 

INFLAMMATION , 

NOS 

CHKONIC 

(10) (1C) (33) (31) 

2 (6%) 

2 (6X) 

•PERICARDIUM 

INFLAMMATION , CHRONIC 

(10) (10) (33) (3U) 

1 (3*) 

A LL CTHEH SYSTEMS 

•FULTIPLE ORGANS 

PLASMACYTOSIS 

HYPERPLASIA, iLYHPHOID 

(10) (10) (33) (31) 

1

1

 (IX) 

 (3X) 

SPECIAL MOHPHOLOG't SUMMAR Y 

NO LESION REP 

AOTOLYS IS/NO 

ORTED 

NSCHOPSY 

1 

2 1 

I NUMBE R

* NUMBER

 OP ANIMALS

 OF AMIHALS

 WITH TISSUE

 NECROPSIED 

 EXAHINE E MICROSCOPICALLY 

67 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE TREATED WITH ISOPHOSPHAMIDE
 

69
 

TABLE D1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

GIVEN INTRAPERITONEAL INJECTIONS OF ISOPHOSPHAMIDE 

ANIMAL S INITIALLY IN STUDY

ANIMAL S KECROPSIED

ANIMAL S EXAMINE D HI STOPATHOLOGICALL Y

INTEGUMENTAR Y SYSTEM 

*SKIN

INFLAMMATION, SUPPUBATIVE 

ABSCESS, NOS 

RESPIRATOR Y SYSTEM 

tLUN G

INFLAMMATION , INTERSTITIAL

BPONCHOPNEOBONIA SUPPURATIV E

INFI AMMATION , ACUTE SUPPURATIVE 

HEMATOPOIF.TIC SYSTEM 

tSPLEEN

ATROPHY, NOS 

HEMATOPOIESIS

IMEDIASTINAL L. NODE 

ATROPHY , NOS 

*PANCREATIC L.NODE 

HYPERPLASIA , LYMPHOID 

IMESENTERIC L. NODE 

HYPERPLASIA, LYMPHOID 

tTHYMU S

ATROPHY , HOS 

CIRCULATORY SYSTEM 

NONE 

CONTROL (UNTR) 

 15 

1 4 

 14 

 (11) 

 (14) 

1 (7%) 

1 (7%) 

 (14) 

1 (7%) 

 (14) 

CONTROL (VEH) LOW DOSE HIGH DOSE 

15 35 35 

14 30 27 

14 29 21 

(14) (30) (27) 

1

1

 ("»%) 

 (4%) 

(14) (29) (27) 

1 (3%) 2 (7%) 

1 (3%) 

(1«) (28) (24) 

1 (7%) 2 (7*)

1

 4 (»*) (17X) 

(1) (1) (5) 

1 (20%) 

(1) (D (5) 

1 (20») 

(1) (D (5)

1 (20%) 

(14) (29) (26) 

1 («%) 

I NDMEE R OF ANIMALS WITH TISSUE EXAMINED 1ICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

71 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL (ONTR) CONTROL (VEH) LOU DOSE 

DIGESTIVE SYSTEM 

•LIVER (14) (14) (29) 

HYPERPLASIA, NODULA R 1 ( 3X) 

tLIVEP/CENTRILOBOLAR (1t) (14) (29) 

DEGENERATION , NOS 

ORINAR Y SYSTEM 

iKIDNE Y (14) (14) (29) 

INFLAMMATION , NOS 

INFLAHMATION, SUPPOBATIVE 1 (7X) 

PYELONEPHRITIS SUPPURATIV E 2 (14%) 

INFLAMMATION , CHHOHIC SOPPORATIV 

HYPERPLASIA , LYMPHOID 

•KIDNEY/PELVIS (14) (14) (29) 

INFLAFBATION, NOS 

tORINAR Y BLADDER (14) (14) (29) 

INFLAMMATION , SOPPORATIVE 1 (7%) 

HYPERPLASIA, EPITHELIAL 

INDOCSINE SYSTEM 

NONE 

HBPRODDCTIVE SYSTEM 

IPROSTATE (14) (14) (29) 

INFLAMMATION , SOPPORATIVE 1 (7X) 1 (3%) 

INFLAHHATION, CHRONIC 

INFLAHHATION, CHRONIC SDPPORATIV 

•SEHINAL VESICLE (14) (14) (30) 

HEMORRHAG E 

LYMPHOCYTIC INFILTRATE 

INFLAMHATION, SOPPORATIVE 

GRANOLOHA , NOS 

NECROSIS, NOS 

HYPERPLASIA, LYHPHOID 

NERVOOS SYSTEM 

HONE _ _ 

t NONEER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBE D OF ANIMALS NECROPSIED 

HIGH D3SE 

(27) 

1 (4«) 

(27) 

1 (4%) 

(27) 

1 (**) 

1 (4%) 

1 (4X) 

2 <7X) 

(27) 

1 (4X) 

(27) 

1 (4X) 

(27) 

2 (7%) 

1 (4*) 

1 (4%) 

(27) 

1 ("•«) 

1 (4X) 

2 (7%) 

1 (4*) 

1 (4%) 

1 (»«) 

72 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL (UNTP) CONTROL(VEH) LOB DOSE HIGH DOSE 

SPECIAL SENS E ORGAN S 

NON E 

NDSCULCSKELETA L SYSTEM 

NON E 

BODY CAVITIE S 

*PERITONEOM

INFLAMMATION , NO S

INFLAMMATION , SUPPURATIV E

•MESENTER Y

NECRCSIS, FAT

 (1U)

 (11)

 ( 1 It)

 1 (7X)

 (1U )

 1 (7*) 

 (30)

 1

 (30)

 (3%) 

 (27) 

1 (U%) 

 (27) 

A LL CTHER SYSTEMS 

NON E 

SPECIAL BCRPHOLOGY SUMMAR Y 

NO LESIO N REPORTE D

AUTC/NECROPSY/NO HISTO

AUTOLYS IS/NO NECSOPSY

I NUMBE R OF ANIMAL S WITH TISSUE

* NUMBE R OF ANIMAL S NEC30PSIED 

 10

 1 

 EXAMINE D MICROSCOPICALLY 

9

1 

 17

 1 

5 

 13 

8 

73 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

GIVEN INTRAPERITONEAL INJECTIONS OF ISOPHOSPHAMIDE

 

CONTROL (UNTR) CONTROL (VEH) LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY

ANIMAL S HISSING 

ANIMALS NECROPSIED

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY

 15

 15

 15

 15 

1 <t 

 1U 

35 

1 

32 

32 

35 

3U 

3U 

INTEGUMENTAR Y SYSTEM 

(ONE 

RESPIRATOR Y SYSTEM 

ILUN6/BHCNCHIOLE

HYPESPLASIA , PLASMA CELL 

HYPEHPLASIA. LYMPHOID

 (15)

 1 (7%) 

 (1«) (32) 

1

1

 (3«) 

 (3«) 

(3U) 

1 (3%) 

tLONG

INFLAMMATION , INTERSTITIAL

BRONCHOPNEUMONIA SOPPURATIV E

HYPERPLASIA , LYHPHOID

 (15)

 2 (13%)

 (11) 

1 (7%) 

1 (7%) 

1 (7%) 

(32) 

1 (3%) 

3 (9%) 

2 (6«) 

<3<t) 

1 (3%) 

1 (3%) 

1 (3%) 

HEMATOPOIBTIC SYSTEM 

IBONE MARRO W

ATROPHY , NOS

 (1U)

 1 (7%) 

( 1 «) (29) (3«) 

ISPLEEN

HYPERPLASIA , RETICOL'JM

HYPERPLASIA , LYMPHOI D 

HEMATOPOIESIS

 CELL 

 (15)

 1 (7%) 

 (1«) (32) 

2 (6%) 

11 (3H*( 

(31) 

1 (3%) 

5 (15%) 

*MEDIASTINAL L. NODE 

ATROPHY , NOS 

(2) (7) 

1 (1«%) 

tHESENTERIC L. NODE 

HYPE8PLASIA , LYMPHOI D 

(2) 

1 (50%) 

(7) 

ITHYHU S

A.l£SPH.X._Nfis (15) (Itt) (31) 

. _

(31) 

2 i«L 

» NUMBER

* NUMBER

 OF ANIMAL S WITH TISSUE

 OF ANIMAL S NECROPSIED 

 EXAMINE D MICROSCOPICALLY 

74 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CIRCULATORY SYSTEM 

•MYOCARDIUM 

INFLAMMATION , SUPPDR1TIVE 

DIGESTIVE SYSTEM 

HONE 

URINAR Y SYSTEM 

fKIDSEY 

HYPEHPLASIA , LYBPHOID 

ENDOCRINE SYSTEM 

NONE 

REPRODUCTIV E SYSTEM 

*BAMHAR Y GLAN D 

HYPERPLASIA , CYSTIC 

*UTERUS 

METAPLASIA , SQUAMOU S 

tUTERUS/ENDCMETRIU M 

INFLAKMATION , SOPPORATIVE 

HYPERPLASIA , NOS 

HYPERPLASIA , CYSTIC 

»OVAH Y 

CYST, NOS 

THROMBOSIS, NOS 

INFLAMMATION , SUPPURATIV E 

NERVOU S SYSTEM 

NON E 

SPECIAL SENS E ORGAN S 

NQNE _ 

* NUMBER OF ANIMAL S MITH TISSUE

* NUMBE R OF ANIMAL S NECROPSIED 

CONTROL (UHTR) CONTROL (VEH)

(15) (1«)

1 <7X) 

(11) (11)

2 (11X)

(15) (1t)

(1t) (1<M

(1U) (U)

10 (71X) 11 (79X)

(11) (11)

1 (7%) 

 EXAMINE D MICROSCOPICALLY 

 LOU DOSE HIGH DOSE 

 (32) (31) 

 (32) (3U) 

1 (3X) 

 (32) (3U) 

1 (3*) 

 (32) (33) 

1 (3X) 

 (32) (33) 

1 (3%) 

It (12X) 

 15 (17%) 25 (76%) 

 (32) (31) 

2 (615) 

1 (3«) 

75 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

CONTROL (UNTR) CONTROL (VEH) LOiDOSE HIGH DOSE 

HUSCDLOSKELETAL SYSTEM 

NON E 

BODY CAVITIES 

•PERITONEUM (15) (14) (32) (31) 

INFLAMMATION, CHRONIC 1 (3*) 

ALL OTHER SYSTEMS 

*MOITIPLE ORGANS (15) (1«) (32) (31) 

INFLAMMATION, GRANULOMATOOS 1 <3» 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED 3 1 5 2 

ANIMA L MISSING/NO NECROPSY 1 

ACCIDENTAL DEATH 1 

AOTOLYSIS/NO NECROPSY 2 1 

* NUMBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

76 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

RATS TREATED WITH ISOPHOSPHAMIDE
 

77
 

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

Treated with Isophosphamide3
 

Topography: Morphology


Subcutaneous Tissue: Fibromab


P Valuesc»d


Departure from Linear Trend6
 

Matched
 

Vehicle
 

 Control
 

 0/9 (0.00)
 

 N.S.
 

79

 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled 

Vehicle Low High 

Control Dose Dose 

0/29 (0.00) 2/32 (0.06) 0/34 (0.00) 

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

P = 0.045 

Infinite 

0.093 

Infinite 

Infinite 

0.272 

Infinite 

72 
80

 

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

Treated with Isophosphamide3

 

(continued) 

Matched Pooled
 

Vehicle Vehicle Low High
 

Topography; Morphology Control Control Dose Dose
 

Lung: Alveolar /Bronchiolar
 

Adenoma" 0/9 (0.00) 0/29 (0.00) 2/32 (0.06) 0/34 (0.00)
 

d
 P Values0

 » N.S. N.S. N.S. N.S.
 

Departure from Linear Trend6 P = 0.041
 

Relative Risk (Matched Vehicle Control)f Infinite
 

Lower Limit 0.093
 

Upper Limit Infinite
 

Relative Risk (Pooled Control)^ Infinite
 

Lower Limit 0.272
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 79
 —
 
Table El.


(continued)
 

Topography: Morphology


Hematopoietic System:
 

Malignant Lymphomab


P Valuesc»d


 Analyses of the Incidence of Primary Tumors in Male Rats
 

Treated with Isophosphamide3
 

Matched
 

Vehicle
 

 Control
 

 0/9 (0.00)
 

 N.S.
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor —
 

Hematopoietic System:
 

Leukemia or Lymphomab 0/9 (0.00)
 

P Values0 »d N.S.
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pooled
 

Vehicle


Control


0/29 (0.00)


N.S.


0/29 (0.00)


P = 0.032


 Low
 

 Dose
 

 2/32 (0.06)
 

 N.S.
 

Infinite
 

0.071
 

Infinite
 

Infinite
 

0.233
 

Infinite
 

79
 

 3/32 (0.09)
 

 N.S.
 

Infinite
 

0.140
 

Infinite
 

Infinite
 

0.417
 

Infinite
 

45
 

High
 

Dose
 

4/34 (0.12)
 

N.S.
 

Infinite
 

0.200
 

Infinite
 

Infinite
 

0.056
 

Infinite
 

34
 

5/34 (0.15)
 

P = 0.040**
 

Infinite
 

0.270
 

Infinite
 

Infinite
 

1.099
 

Infinite
 

34
 

81

 
__ 

__ 

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

Treated with Isophosphamide3
 

(continued)
 

Topography;


Pituitary:


Adenoma^
 

P Values0 »d
 

 Morphology
 

 Chromophobe
 

Relative Risk (Matched Vehicle
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe
 

Carcinoma
 

P Values0 »d
 

Matched
 

Vehicle
 

Control
 

3/7 (0.43)
 

P = 0.002 (N)
 

Control)f
 

Control)f
 

83
 

1/7 (0.14)
 

N.S.
 

Pooled
 

Vehicle


Control


4/25 (0.16)


P = 0.024 (N)


1/25 (0.04)


N.S.


 Low
 

 Dose
 

 3/29 (0.10)
 

 N.S.
 

0.241
 

0.049
 

1.361
 

0.647
 

0.120
 

3.215
 

79
 

 1/29 (0.03)
 

 N.S.
 

0.241
 

0.007
 

8.740
 

0.862
 

0.024
 

31.358
 

83
 

High
 

Dose
 

0/32 (0.00)
 

P = 0.003* (N)
 

P = 0.032** (N
 

0.000
 

0.000
 

0.344
 

0.000
 

0.000
 

0.825
 

0/32 (0.00)
 

N.S.
 

0.000
 

0.000
 

4.069
 

0.000
 

0.000
 

13.482
 

82

 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 80
 
__ 

Fable -&t. Analyses of the Incidence of Primary Tumors in Male Rats
 

Treated with Isophosphamide3
 

83

 

(continued) 

Matched 

Vehicle 

Topography: Morphology Control 

Pituitary: Chromophobe
 

Adenoma or Carcinoma" 4/7 (0.57)
 

P Values0 »d P < 0.001 (N)
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Cortical Adenoma 0/9 (0.00)
 

P Values0 »d N.S.
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control )f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 —
 

Pooled
 

Vehicle


Control


5/25 (0.20)


P = 0.012 (N)


0/27 (0.00)


N.S.


 Low
 

 Dose
 

 4/29 (0.14)
 

P = 0.030* (N)
 

0.241
 

0.080
 

1.059
 

0.690
 

0.166
 

2.738
 

79
 

 2/31 (0.06)
 

 N.S.
 

Infinite
 

0.077
 

Infinite
 

Infinite
 

0.217
 

Infinite
 

79
 

High
 

Dose
 

0/32 (0.00)
 

P < 0.001* (N)
 

P = 0.013** (N
 

0.000
 

0.000
 

0.219
 

0.000
 

0.000
 

0.605
 

„
 

2/34 (0.06)
 

N.S.
 

Infinite
 

0.071
 

Infinite
 

Infinite
 

0.200
 

Infinite
 

33
 
__ 

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

Treated with Isophosphamidea
 

(continued)
 

Topography; Morphology
 

Adrenal: Phenochromocytoma
 

P Values0 »d
 

Relative Risk (Matched Vehicle
 

Lower Limit
 

Upper Limit
 

Matched
 

Vehicle
 

Control
 

0/9 (0.00)
 

N.S.
 

Control)f
 

84

 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Fibroadenoma" 1/9 (0.11)
 

P Values0 »d N.S.
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 83
 

Pooled 

Vehicle Low 

Control Dose 

0/27 (0.00) 2/31 (0.06) 

N.S. N.S. 

Infinite 

0.077 

Infinite 

Infinite 

0.217 

Infinite 

74 

2/29 (0.07) 0/32 (0.00) 

N.S. N.S. 

0.000 

0.000 

5.218 

0.000 

0.000 

3.015 

— 

High
 

Dose
 

2/34 (0.06)
 

N.S.
 

Infinite
 

0.071
 

Infinite
 

Infinite
 

0.200
 

Infinite
 

45
 

0/34 (0.00)
 

N.S.
 

0.000
 

0.000
 

4.919
 

0.000
 

0.000
 

2.847
 

—
 
85 

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

Treated with Isophosphamide3 

(continued) 

a

Treated groups received doses of 6 or 12 mg/kg.
 

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

c

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


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the incidence of tumors in a treated group is the probability level for the Fisher exact test for the
 

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

vehicle-control group (**) when P < 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 

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

e

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

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

control group.
 
86

 

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

Treated with Isophosphamidea
 

Matched Pooled 

Vehicle Vehicle Low High 

Topography; Morphology Control Control Dose Dose 

Subcutaneous Tissue: Fibroma 0/10 (0.00) 1/28 (0.04) 1/33 (0.03) 0/34 (0.00)
 

P Values0 »d N.S. N.S. N.S. N.S.
 

Relative Risk (Matched Vehicle Control)f Infinite
 

Lower Limit 0.018
 

Upper Limit Infinite
 

Relative Risk (Pooled Vehicle Control)f 0.849 0.000
 

Lower Limit 0.023 0.000
 

Upper Limit 30.876 15.225
 

Weeks to First Observed Tumor 64
 —
 

Hematopoietic System:
 

Malignant Lymphoma 0/10 (0.00) 0/30 (0.00) 1/33 (0.03) 0/34 (0.00)
 

P Values0 »d N.S. N.S. N.S. N.S.
 

Relative Risk (Matched Vehicle Control)f Infinite
 

Lower Limit 0.018
 

Upper Limit Infinite
 

Relative Risk (Pooled Vehicle Control )f Infinite
 

Lower Limit 0.053
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 33
 —
 
__ 

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

Treated with Isophosphamidea
 

(continued)
 

87

 

Matched


Vehicle


Topography; Morphology Control


Pituitary: Chromophobe
 

Adenoma 3/10 (0.30)


P Valuesc»d P = 0.003 (N)


Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor 57
 

 Pooled
 

 Vehicle
 

 Control
 

 11/28 (0.39)
 

 P < 0.001 (N)
 

Low
 

Dose
 

7/31 (0.23)
 

N.S.
 

0.753
 

0.228
 

3.431
 

0.575
 

0.226
 

1.390
 

60
 

High
 

Dose
 

0/34 (0.00)
 

P = 0.009* (N)
 

P < 0.001** (N
 

0.000
 

0.000
 

0.472
 

0.000
 

0.000
 

0.242
 
88

 

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

Treated with Isophosphamidea
 

(continued) 

Matched Pooled 

Vehicle Vehicle Low 

Topography: Morphology Control Control Dose 

Pituitary: Chromophobe
 

Carcinoma 4/10 (0.40) 4/28 (0.14) 1/31 (0.03)
 

P Values0 »d P = 0.004 (N) N.S. P = 0.009* (N)
 

Departure from Linear Trend6 P = 0.008
 

Relative Risk (Matched Vehicle Control)f 0.081
 

Lower Limit 0.003
 

Upper Limit 0.681
 

Relative Risk (Pooled Vehicle Control)f 0.226
 

Lower Limit 0.010
 

Upper Limit 2.013
 

Weeks to First Observed Tumor 74 80
 

High
 

Dose
 

1/34 (0.03)
 

P = 0.007* (!
 

0.074
 

0.003
 

0.622
 

0.206
 

0.009
 

1.839
 

70
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Treated with Isophosphamide3
 

(continued)
 

68

 

Topography ; Morphology
 

Pituitary: Chromophobe
 

Adenoma or Carcinoma
 

P Values0 »d
 

Relative Risk (Matched Vehicle
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Adrenal: Cortical Adenoma*5
 

P Values0 »d
 

Departure from Linear Trend6
 

Relative Risk (Matched Vehicle
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched


Vehicle


Control


7/10 (0.70)


P < 0,001 (N)


Control)f
 

Control)f
 

57
 

3/10 (0.30)


P = 0.009 (N)


Control)*
 

Control)f
 

57
 

 Pooled
 

 Vehicle
 

 Control
 

 15/28 (0.54)
 

 P < 0.001 (N)
 

 4/28 (0.14)
 

 N.S. (N)
 

P - 0.024
 

Low
 

Dose
 

8/31 (0.26)
 

P = 0.017* (N)
 

P = 0.027** (N)
 

0.369
 

0.208
 

0.923
 

0.482
 

0.218
 

1.014
 

60
 

8/33 (0.24)
 

N.S.
 

0.808
 

0.264
 

4.147
 

1.697
 

0.515
 

6.917
 

60
 

High
 

Dose
 

1/34 (0.03)
 

P < 0.001* (N)
 

P < 0.001** (N
 

0.042
 

0.002
 

0.271
 

0.055
 

0.003
 

0.333
 

70
 

1/34 (0.03)
 

P = 0.032* (N)
 

0.098
 

0.004
 

0.978
 

0.206
 

0.009
 

1.839
 

35
 
Table E2.


(continued)
 

Topography: Morphology
 

Mammary Gland: Adenoma or
 

Adenocarcinoma, NOS"
 

P Values0 »d
 

 Analyses of the Incidence of Primary Tumors in Female Rats
 

Treated with Isophosphamide3
 

Matched
 

Vehicle
 

Control
 

1/10 (0.10)
 

N.S.
 

90

 

Relative Risk (Matched Vehicle Control)*
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland:
 

Cyst adenoma, NOSb
 

P Values0 »d
 

Relative Risk (Matched Vehicle
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

83
 

0/10 (0.00)
 

N.S.
 

Control)f
 

Control)f
 

—
 

Pooled 

Vehicle Low High 

Control Dose Dose 

3/28 (0.11) 3/33 (0.09) 2/34 (0.06) 

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

0.606 0.588 

0.045 0.044 

16.771 16.279 

0.566 0.549 

0.068 0.066 

3.998 3.882 

72 69 

0/28 (0.00) 3/33 (0.09) 1/34 (0.03) 

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

Infinite Infinite 

0.147 0.017 

Infinite Infinite 

Infinite Infinite 

0.400 0.048 

Infinite Infinite 

80 68 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

(continued)
 

Topography; Morphology
 

Mammary Gland:
 

Cystadenocarcinoma, NOS"
 

P Values0>d
 

Departure from Linear Trend6
 

Relative Risk (Matched Vehicle
 

Lower Limit
 

Upper Limit
 

Treated with Isophosphamidea 

Matched 

Vehicle 

Control 

Pooled 

Vehicle

Control

 Low

 Dose

 High 

 Dose 

0/10 (0.00) 0/28 (0.00) 2/33 (0.06) 0/34 (0.00) 

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

P = 0.050 

Control)f Infinite 

0.077 

Infinite 91

 

Relative Risk (Pooled Vehicle Control)f Infinite 

Lower Limit 0.215 

Upper Limit Infinite 

Weeks to First Observed Tumor 60 — 
92

 

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

Treated with Isophosphamide3
 

(continued) 

Matched Pooled 

Vehicle Vehicle Low High 

Topography: Morphology Control Control Dose Dose 

Mammary Gland: Cystadenoma or
 

Cystadenocarcinoma, NOS 0/10 (0.00) 0/28 (0.00) 5/33 (0.15) 1/34 (0.03)
 

P Values0 »d N.S. N.S. P = 0.040** N.S.
 

Departure from Linear Trend6 P = 0.041 P = 0.009
 

Relative Risk (Matched Vehicle Control)f Infinite Infinite
 

Lower Limit 0.303 0.017
 

Upper Limit Infinite Infinite
 

Relative Risk (Pooled Vehicle Control) f Infinite Infinite
 

Lower Limit 1.099 0.048
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 60 68
 —
 
93

 

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

Treated with Isophosphamide3
 

(continued) 

Matched Pooled 

Vehicle Vehicle Low High 

Topography: Morphology Control Control Dose Dose 

Mammary Gland:
 

Fibroadenoma 3/10 (0.30) 8/28 (0.29) 28/33 (0.85) 6/34 (0.18)
 

P Values0 »d P = 0.004 (N) N.S.	 P = 0.002* N.S.
 

P < 0.001**
 

Departure from Linear Trend6 P < 0.001 P < 0.001
 

Relative Risk (Matched Vehicle Control) f 2.828 0.588
 

Lower Limit 1.245 0.165
 

Upper Limit 9.795 2.778
 

Relative Risk (Pooled Vehicle Control)f 2.970 0.618
 

Lower Limit 1.677 0.208
 

Upper Limit 5.030 1.768
 

Weeks to First Observed Tumor 57	 63 68
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Treated with Isophosphamide3
 

(continued)
 

Topography; Morphology
 

Uterus: Leiomyosarcoma"
 

P Values0>d
 

Departure from Linear Trend6
 

Matched
 

Vehicle
 

Control
 

0/9 (0.00)
 

N.S.
 

P < 0.001
 

94

 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 —
 

a

Treated groups received doses of 6 or 12 mg/kg.
 

Pooled
 

Vehicle


Control


0/27 (0.00)


N.S.


P < 0.001
 

 Low
 

 Dose
 

 15/32 (0.47)
 

P = 0.009*
 

P < 0.001**
 

Infinite
 

1.513
 

Infinite
 

Infinite
 

4.113
 

Infinite
 

64
 

High
 

Dose
 

1/34 (0.03)
 

N.S.
 

Infinite
 

0.016
 

Infinite
 

Infinite
 

0.043
 

Infinite
 

69
 

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

c

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


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the incidence of tumors in a treated group is the probability level for the Fisher exact test for the
 

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

vehicle-control group (**) when P < 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Treated with Isophosphamide3
 

(continued)
 

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

e

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

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

control group.
 

95 
__ 

__ 

96

 

Table E3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Rats
 

Treated with Isophosphamide3
 

Matched
 

Vehicle Low High
 

Topography; Morphology Control Dose Dose
 

Subcutaneous Tissue: Fibroma'5 (52) 0/8 (0) 2/31 (6) 0/9 (0)
 

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

Relative Risk (Matched Vehicle Control)f Infinite
 

Lower Limit 0.087
 —
 Upper Limit Infinite
 —
 

Lung: Alveolar /Bronchiolar
 

Adenomab (52) 0/8 (0) 2/31 (6) 0/9 (0)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Matched Vehicle Control)f Infinite
 

Lower Limit 0.087
 

Upper Limit Infinite
 —
 

—
 
97

 

Table E3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Rats
 

Treated with Isophosphamide3
 

(continued)
 

Matched
 

Vehicle Low High
 

Topography; Morphology Control Dose Dose
 

Hematopoietic System: Malignant
 

Lymphoma" (34) 0/8 (0) 2/32 (6) 4/26 (15)
 

P Valuesc»d N.S. N.S. N.S.
 

Relative Risk (Matched Vehicle Control)f Infinite Infinite
 

Lower Limit 0.084 0.329
 

Upper Limit Infinite Infinite
 

Hematopoietic System: Leukemia or
 

Lymphoma'3 (34) 0/8 (0) 3/32 (9) 5/26 (19)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Matched Vehicle Control)f Infinite Infinite
 

Lower Limit 0.173 0.450
 

Upper Limit Infinite Infinite
 
Table E3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Rats
 

Treated with Isophosphamide3
 

(continued)
 

Topography: Morphology


Pituitary: Chromophobe Adenoma
 

or Carcinomab (52)


P Valuesc»d


Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Adrenal: Cortical Adenomab (33)


P Values0 »d


Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Matched
 

Vehicle
 

 Control
 

 4/6 (67)
 

P = 0.005(N)
 

 0/8 (0)
 

 N.S.
 

Low
 

Dose
 

4/27 (15)
 

P = 0.020(N)
 

0.222
 

0.094
 

0.941
 

2/31 (6)
 

N.S.
 

Infinite
 

0.087
 

Infinite
 

High
 

Dose
 

0/8 (0)
 

P = 0.015(N)
 

0.000
 

0.000
 

0.661
 

2/34 (6)
 

N.S.
 

Infinite
 

0.079
 

Infinite
 

98

 
Table E3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Rats
 

Treated with Isophosphamide3
 

(continued)
 

Topography; Morphology
 

Pituitary: Chromophobe Adenoma" (52)
 

P Values0>d
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Pituitary: Chromophobe
 

Carcinomab (52)
 

P Values0>d
 

Departure from Linear Trend6
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Matched
 

Vehicle
 

Control
 

3/6 (50)
 

P = 0.018(N)
 

3/6 (50)
 

P = 0.008(N)
 

P = 0.027
 

Low
 

Dose
 

3/27 (11)
 

N.S.
 

0.222
 

0.055
 

1.388
 

1/27 (4)
 

P = 0.014(N)
 

0.074
 

0.002
 

0.789
 

High
 

Dose
 

0/8 (0)
 

N.S.
 

0.000
 

0.000
 

1.044
 

0/8 (0)
 

N.S.
 

0.000
 

0.000
 

1.044
 

99

 
100

 

Table E3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Rats
 

Treated with Isophosphamidea
 

(continued) 

Matched 

Vehicle Low High 

Topography ; Morphology Control Dose Dose 

Adrenal: Pheochromocytoma (45) 0/8 (0) 2/31 (6) 2/34 (6)
 

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

Relative Risk (Matched Vehicle Control)f Infinite Infinite
 

Lower Limit 0.087 0.079
 

Upper Limit Infinite Infinite
 

Mammary Gland: Fibroadenoma^3 (52) 1/8 (13) 0/31 (0) 0/9 (0)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Matched Vehicle Control)f 0.000 0.000
 

Lower Limit 0.000 0.000
 

Upper Limit 4.780 15.254
 
101

 

Table E3. Time-adjusted Analyses of the Incidence of Primary Tumors in Male Rats 

Treated with Isophosphamide3 

(continued) 

a

Treated groups received doses of 6 or 12 mg/kg.
 

^Number of tumor-bearing animals/number of animals examined at site (percent), based upon
 

animals that survived at least as long as 52 weeks, unless a tumor was found at the specific
 

site before 52 weeks. In such an instance, it is based upon animals that survived at least as
 

long as the animal in which the first tumor was found. The week to first observed tumor is
 

indicated in the parentheses after footnote b.
 

c

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


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

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

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

otherwise, not significant (N.S.) is indicated.
 

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

e

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

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

control group.
 
__ 

__ 

Table E4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Rats
 

Topography ; Morphology
 

Subcutaneous Tissue: Fibroma'3 (52)
 

P Valuesc'd
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Hematopoietic System: Malignant
 

Lymphomab (33)
 

P Valuesc»d
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Treated with Isophosphamide3
 

Matched
 

Vehicle
 

Control
 

0/9 (0)
 

N.S.
 

0/9 (0)
 

N.S.
 

Low
 

Dose
 

1/32 (3)
 

N.S.
 

Infinite
 

0.016
 

Infinite
 

1/32 (3)
 

N.S.
 

Infinite
 

0.016
 

Infinite
 

High
 

Dose
 

0/7 (0)
 

N.S.
 

—
 

—
 

0/7 (0)
 

N.S.
 

—
 

—
 

102

 
Table E4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Rats
 

Treated with Isophosphamidea
 

(continued)
 

Topography: Morphology
 

Pituitary: Chromophobe Adenoma*' (52)
 

P Valuesc»d
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Pituitary: Chromophobe
 

Carcinomab (52)
 

P Values0 »d
 

Departure from Linear Trend6
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Matched
 

Vehicle
 

Control
 

3/9 (33)
 

N.S.
 

4/9 (44)
 

P = 0.049(N)
 

P = 0.013
 

Low
 

Dose
 

7/30 (23)
 

N.S.
 

0.700
 

0.225
 

3.650
 

1/30 (3)
 

P = 0.007(N)
 

0.075
 

0.002
 

0.658
 

High
 

Dose
 

0/7 (0)
 

N.S.
 

0.000
 

0.000
 

1.781
 

1/7 (14)
 

N.S.
 

0.321
 

0.008
 

2.307
 

103

 
104

 

Table E4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Rats
 

Treated with Isophosphamide3
 

(continued)
 

Matched
 

Vehicle Low High
 

Topography: Morphology Control Dose Dose
 

Pituitary: Chromophobe Adenoma
 

or Carcinomab (52) 7/9 (78) 8/30 (27) 1/7 (14)
 

P Values0 »d P = 0.006(N) P = 0.009(N) P = 0.020(N)
 

Relative Risk (Matched Vehicle Control)f 0.343 0.184 

Lower Limit 0.218 0.008 

Upper Limit 0.837 0.958 

Adrenal: Cortical Adenomab (35) 3/10 (30) 8/32 (25) 1/18 (6)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Matched Vehicle Control)f 0.833 0.185
 

Lower Limit 0.272 0.004
 

Upper Limit 4.253 1.997
 
Table E4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Rats
 

Treated with Isophosphamide3
 

(continued)
 

Topography; Morphology
 

Mammary Gland: Adenoma or
 

Adenocarcinoma, NOB** (52)
 

P Valuesc»d
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Mammary Gland:
 

Cystadenoma, NOSb (52)
 

P Values0 »d
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Matched 

Vehicle Low High 

Control Dose Dose 

1/9 (11) 3/32 (9) 2/7 (29) 

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

0.844 2.571 

0.084 0.166 

42.831 124.556 

0/9 (0) 3/32 (9) 1/7 (14) 

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

Infinite Infinite 

0.191 0.076 

Infinite Infinite 

105

 
__ 

106

 

Table E4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Rats
 

Treated with Isophosphamide3
 

(continued) 

Matched 

Vehicle Low High 

Topography: Morphology Control Dose Dose 

Mammary Gland:
 

Cystadenocarcinoma, NOSb (52) 0/9 (0) 2/32 (6) 0/7 (0)
 

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

Relative Risk (Matched Vehicle Control)f Infinite
 

Lower Limit 0.093
 

Upper Limit Infinite
 —
 

—
 

Mammary Gland: Cyst adenoma or
 

Cystadenocarcinoma, NOSb (52) 0/9 (0) 5/32 (16) 1/7 (14)
 

P Values0 »d N.S. N.S. N.S.
 

Relative Risk (Matched Vehicle Control)f Infinite Infinite
 

Lower Limit 0.402 0.076
 

Upper Limit Infinite Infinite
 
Table E4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Rats
 

107

 

Treated with Isophosphamidea 

(continued) 

Matched 

Vehicle Low High 

Topography ; Morphology Control Dose Dose 

Mammary Gland: Fibroadenoma^ (52) 3/9 (33) 28/32 (88) 6/7 (86) 

P Valuesc » d P = 0.009 P = 0.003 N.S. 

Departure from Linear Trend6 P = 0.028 

Relative Risk (Matched Vehicle Control)f 2.625 2.571 

Lower Limit 1.188 0.860 

Upper Limit 8.163 4.393 

Uterus: Leiomyosarcoma" (52) 0/8 (0) 15/31 (48) 1/7 (14) 

P Valuesc » d N.S. P = 0.012 N.S. 

Departure from Linear Trend6 P = 0.006 

Relative Risk (Matched Vehicle Control)f Infinite Infinite 

Lower Limit 1.417 0.068 

Upper Limit Infinite Infinite 
108

 

Table E4. Time-adjusted Analyses of the Incidence of Primary Tumors in Female Rats 

Treated with Isophosphamide3 

(continued) 

a

Treated groups received doses of 6 or 12 mg/kg.
 

"Number of tumor-bearing animals/number of animals examined at site (percent), based upon
 

animals that survived at least as long as 52 weeks, unless a tumor was found at the specific
 

site before 52 weeks. In such an instance, it is based upon animals that survived at least as
 

long as the animal in which the first tumor was found. The week to first observed tumor is
 

indicated in the parentheses after footnote b.
 

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


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

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

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

otherwise, not significant (N.S.) is indicated.
 

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

e

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

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

control group.
 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

MICE TREATED WITH ISOPHOSPHAMIDE
 

109
 

__ 

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

Treated with Isophosphamide3
 

Matched


Vehicle


Topography ; Morphology Control


Liver: Hepatocellular
 

Adenoma 0/14 (0.00)


P Values0 »d N.S.


Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Ill

 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular
 

Adenoma or Carcinoma
 

P Valuesc»d
 

Departure from Linear Trend6
 

Relative Risk (Matched Vehicle
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

0/14 (0.00)


N.S.


Control)f
 

Control)f
 

 Pooled 

 Vehicle Low 

 Control Dose 

 0/28 (0.00) 4/29 (0.14) 

 N.S. N.S. 

Infinite 

0.481 

Infinite 

Infinite 

0.916 

Infinite 

44 

 0/28 (0.00) 5/29 (0.17) 

 N.S. P = 0.028** 

P ­ 0.032 

Infinite 

0.656 

Infinite 

Infinite 

1.247 

Infinite 

44 

High
 

Dose
 

2/27 (0.07)
 

N.S.
 

Infinite
 

0.164
 

Infinite
 

Infinite
 

0.313
 

Infinite
 

79
 

2/27 (0.07)
 

N.S.
 

Infinite
 

0.164
 

Infinite
 

Infinite
 

0.313
 

Infinite
 

79
 —
 
112

 

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

Treated with Isophosphamide3
 

(continued)
 

a

Treated groups received doses of 10 or 20 mg/kg.
 

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

c

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


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the incidence of tumors in a treated group is the probability level for the Fisher exact test for the
 

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

vehicle-control group (**) when P < 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 

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

e

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

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

control group.
 
__ __ 

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

Treated with Isophosphamide3
 

113

 

Matched
 

Vehicle
 

Topography ; Morphology Control
 

*
 L


Liver: Hepatocellular
 

Carcinoma 0/14 (0.00)
 

P Values0 »d N.S.
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System:
 

Malignant Lymphoma 0/14 (0.00)
 

P Values0 »d P = 0.001
 

Relative Risk (Matched Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Relative Risk (Pooled Vehicle Control)f
 

Lower Limit
 

Upper Limit
 

Pooled
 

Vehicle


Control


0/29 (0.00)


N.S.


1/29 (0.03)


P < 0.001


 Low
 

 Dose
 

 1/32 (0.03)
 

 N.S.
 

Infinite
 

0.025
 

Infinite
 

Infinite
 

0.049
 

Infinite
 

77
 

 3/32 (0.09)
 

 N.S.
 

Infinite
 

0.282
 

Infinite
 

2.719
 

0.234
 

139.098
 

High
 

Dose
 

0/33 (0.00)
 

N.S.
 

13/34 (0.38)
 

P = 0.005*
 

P = 0.001**
 

Infinite
 

1.786
 

Infinite
 

11.088
 

1.853
 

449.268
 

Weeks to First Observed Tumor 73
 63
 —
 
114

 

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

Treated with Isophosphamide3
 

(continued)
 

a

Treated groups received doses of 10 or 20 mg/kg.
 

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

c

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


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the incidence of tumors in a treated group is the probability level for the Fisher exact test for the
 

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

vehicle-control group (**) when P < 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 

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

e

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

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

control group.
 
Table F3. Time-Adjusted Analyses of the Incidence of Primary Tumors in Male Mice
 

115

 

Treated with Isophosphamide3 

Matched Pooled 

Vehicle Vehicle Low High 

Topography: Morphology Control Control Dose Dose 

Liver: Hepatocellular Adenoma** 0/3 (0) 0/14 (0) 4/18 (22) 2/22 (9) 

P Valuesc » d N.S. N.S. N.S. N.S. 

Relative Risk (Matched Vehicle Control)f Infinite Infinite 

Lower Limit 0.240 0.062 

Upper Limit Infinite Infinite 

Relative Risk (Pooled Vehicle Control)f Infinite Infinite 

Lower Limit 0.780 0.201 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor — 44 79 

Liver: Hepatocellular Adenoma 

or Carcinomab 0/3 (0) 0/14 (0) 5/18 (28) 2/22 (9) 

P Valuesc » d N.S. N.S. P = 0.043** N.S. 

Departure from Linear Trend6 P = 0.018 

Relative Risk (Matched Vehicle Control)f Infinite Infinite 

Lower Limit 0.326 0.062 

Upper Limit' Infinite Infinite 

Relative Risk (Pooled Vehicle Control)^ Infinite Infinite 

Lower Limit 1.065 0.201 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor — 44 79 
116

 

Table F3. Time-Adjusted Analyses of the Incidence of Primary Tumors in Male Mice 

Treated with Isophosphamidea 

(continued) 

a

Treated groups received doses of 10 or 20 mg/kg.
 

^Number of tumor-bearing animals/number of animals examined at site (percent), based on
 

number of animals that lived at least as long as 44 weeks on study.
 

c

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


Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the incidence of tumors in a treated group is the probability level for the Fisher exact test for the
 

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

vehicle-control group (**) when P < 0.05 for either control group; otherwise, not significant
 

(N.S.) is indicated.
 

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

e

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

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

control group.
 

DHEW Publication No. (NIH) 77-832