National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 42 

1978 

BIOASSAY OF 

5-AZACYTIDINE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 320-67-2 

NCI-CG-TR-42 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

5-AZACYTIDINE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-842
 

BIOASSAY OF
 

5-AZACYTIDINE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

CONTRIBUTORS: This report presents the results ol. the bioassay
 

of 5-azacytidine for possible carcinogenicity conducted for the
 

Carcinogenesis Testing Program, Divison of Cancer Cause and
 

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

The bioassay was conducted by Southern Research Institute,
 

Birmingham, Alabama, initially under direct contract to NCI and
 

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

contractor for the NCI Carcinogenesis Testing Program.
 

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

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

Weisburger2>3. Ms. J. Belzer1 and Mr. I. Brown* were
 

responsible for the care and treatment of the laboratory animals.
 

Data management and retrieval were performed by Ms. C. A.
 

Dominick.1. Histopathologic examinations were performed by Drs.
 

S. D. Kosanke* and J. C. Peckham^-, and the diagnoses included in
 

this report represent their interpretation. The neoplasms and
 

chemical-related hyperplastic lesions were reviewed by Dr. J. F.
 

Hardisty^, who also prepared the interpretive pathology narrative
 

included in this report.
 

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

Mason Research Institute-*. 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 by Drs. P. Lim®, A. Cheung8

, and R. Yee8, and the
 

analytical results were reviewed by Dr. S. S. Olin^.
 

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

NCI. Those responsible for the report at Tracor Jitco were
 

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

F. Robens and R. W. Fogleman, toxicologists; Dr. R. L. Schueler,
 

pathologist; Ms. M. S. King and Mr. W. D. Reichardt, technical
 

writers; and Dr. E. W. Gunberg, technical editor, assisted by
 

Ms. Y. E. Presley.
 

The statistical analysis was reviewed by members of the
 

Mathematical Statistics and Applied Mathematics Section of NCI^:
 

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

Robert E. Tarone.
 

The following other scientists at the National Cancer Institute
 

were responsible for evaluating the bioassay experiment,
 

interpreting the results, and reporting the findings:
 

Dr. Kenneth C. Chu
 

Dr. Cipriano Cueto, Jr.
 

Dr. J. Fielding Douglas
 

Dr. Dawn G. Goodman
 

Dr. Richard A. Griesemer
 

Mr. Harry A. Milman
 

Dr. Thomas W. Orme
 

Dr. Robert A. Squire9
 

Dr. Jerrold M. Ward
 

^Southern Research Institute, 2000 Ninth Avenue South,
 

Birmingham, Alabama.
 

^Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

-^Now with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammond House Road, Valhalla,
 

New York.
 

^Experimental Pathology Laboratories Inc., P.O. Box 474, Herndon,
 

Virginia.
 

iv
 
5

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,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute, National
 

Institutes of Health, Bethesda, Maryland.
 

^Stanford Research Institute, Menlo Park, California.
 

^Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 

V 

SUMMARY

 

A bioassay of 5-azacytidine for possible carcinogenicity was
 

conducted by administering the test chemical by intraperitoneal
 

injection to Sprague-Dawley rats and B6C3F1 mice.
 

Groups of 35 rats of each sex were administered 5-azacytidine at
 

one of two doses, either 2.6 or 5.2 mg/kg body weight, in
 

buffered saline three times per week for 34 weeks, and were then
 

observed for 46 or 47 weeks. Controls consisted of groups of 15
 

rats of each sex that received injections of buffered saline
 

(vehicle controls) and 15 rats of each sex that were untreated
 

(untreated controls). All surviving rats were killed at 80 or 81
 

weeks.
 

Groups of 35 mice of each sex were administered the chemical at
 

one of two doses, either 2.2 or 4.4 mg/kg body weight, in
 

buffered saline three times per week for 52 weeks, and were then
 

observed for 29 or 30 weeks. Controls consisted of groups of 15
 

mice of each sex that received injections of buffered saline
 

(vehicle controls) and 15 mice of each sex that were untreated
 

(untreated controls). All surviving mice were killed at 81 or 82
 

weeks.
 

5-Azacytidine was toxic to the animals in this bioassay, since
 

mean body weights of both treated rats and treated mice were
 

lower than those of the corresponding vehicle controls, and since
 

none of the high-dose male and female rats and high-dose female
 

mice lived to the end of the bioassay. In treated male and
 

female rats and male mice, survival was inadequate for meaningful
 

statistical analyses of the incidences of tumors.
 

Only one male and three female high-dose rats had tumors, and
 

none of the tumors in the low-dose group of either sex were
 

present at a significantly increased incidence using any of the
 

statistical tests. Bone-marrow atrophy was present in both
 

treated groups of both sexes of rats.
 

vii
 
Only five high-dose male mice and one high-dose female mouse had
 

neoplasms. In low-dose female mice, however, lymphocytic and
 

granulocytic neoplasms of the hematopoietic system occurred in 17
 

animals, even though only 54% survived until week 81. Granulocytic neoplasms were observed in 10/29 low-dose female mice, but
 

in no other group, and were significant (P = 0.010) compared with
 

the vehicle controls. The incidence of combined lymphoma and
 

granulocytic neoplasms' was highly significant in the low-dose
 

females (vehicle controls 0/14, low-dose 17/29, P < 0.001). No
 

tumors were observed at a significant incidence in male mice.
 

Bone-marrow atrophy was present in high-dose female mice.
 

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

short life span and short duration of treatment of Sprague-Dawley
 

rats of either sex and of male B6C3F1 mice precluded evaluation
 

of the carcinogenicity of 5-azacytidine in these groups; however,
 

the induction of tumors of the hematopoietic system in female
 

B6C3F1 mice was associated with the administration of
 

5-azacytidine.
 

viii
 
TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dosage Preparation 3
 

C. Animals 4
 

D. Animal Maintenance 4
 

E. Subchronic Studies 7
 

F. Designs of Chronic Studies 9
 

G. Clinical and Pathologic Examinations 9
 

H. Data Recording and Statistical Analyses 12
 

III. Results - Rats 19
 

A. Body Weights and Clinical Signs (Rats) 19
 

B. Survival (Rats) 19
 

C. Pathology (Rats) 22
 

D. Statistical Analyses of Results (Rats) 24
 

IV. Results - Mice 27
 

A. Body Weights and Clinical Signs (Mice) 27
 

B. Survival (Mice) 27
 

C. Pathology (Mice) 30
 

D. Statistical Analyses of Results (Mice). 32
 

V. Discussion 35
 

VI. Bibliography 39
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms
 

in Rats Given Intraperitoneal Injections
 

of 5-Azacytidine 41
 

Table Al Summary of the Incidence of Neoplasms
 

in Male Rats Given Intraperitoneal
 

Injections of 5-Azacytidine 43
 

ix
 
Page 

Table A2 Summary of the Incidence of Neoplasms 

in Female Rats Given Intraperitoneal 

Injections of 5-Azacytidine 46 

Appendix B Summary of the Incidence of Neoplasms 

in Mice Given Intraperitoneal Injections 

of 5-Azacytidine 49 

Table Bl Summary of the Incidence of Neoplasms 

in Male Mice Given Intraperitoneal 

Injections of 5-Azacytidine 51 

Table B2 Summary of the Incidence of Neoplasms 

in Female Mice Given Intraperitoneal 

Injections of 5-Azacytidine 54 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Given Intraperitoneal 

Injections of 5-Azacytidine 57 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Given Intraperitoneal 

Injections of 5-Azacytidine 59 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Given Intraperitoneal Injections of 5-Azacytidine 62 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Given Intraperitoneal 

Injections of 5-Azacytidine 65 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Given Intraperitoneal Injections of 5-Azacytidine 67 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Given Intraperitoneal Injections of 5-Azacytidine 70 

Appendix E Analyses of the Incidence of Primary 

Tumors in Rats Treated with 

5-Azacytidine 73 

x
 
Page 

Table El Analyses of the Incidence of Primary 

Tumors in Male Rats Treated with 

5-Azacytidine 75 

Table E2 Analyses of the Incidence of Primary 

Tumors in Female Rats Treated with 

5-Azacytidine 77 

Appendix F Analyses of the Incidence of Primary 

Tumors in Mice Treated with 

5-Azacytidine 79 

Table Fl Analyses of the Incidence of Primary 

Tumors in Male Mice Treated with 

3~Azacytidine 81 

Table F2 Analyses of the Incidence of Primary 

Tumors in Female Mice Treated with 

5-Azacytidine 82 

TABLES 

Table 1 Design of Chronic Studies of 

5-Azacytidine in Rats 10 

Table 2 Design of Chronic Studies of 

5-Azacytidine in Mice 11 

FIGURES 

Figure 1 Growth Curves for Rats Treated with 

5-Azacytidine 20 

Figure 2 Survival Curves for Rats Treated with 

5-Azacytidine. 21 

Figure 3 Growth Curves for Mice Treated with 

5-Azacytidine 28 

Figure 4 Survival Curves for Mice Treated with 

5-Azacytidine 29 

xi
 

I. INTRODUCTION
 

5-Azacytidine (CAS 320-67-2; NCI C01569), a synthetic analogue of
 

cytidine, has been used as an investigational anticancer drug in
 

the United States since 1970 (Von Hoff et al. , 1976). Chemical
 

synthesis was first reported in 1964 by a Czech group, and later
 

the chemical was isolated as an antibiotic from a culture
 

filtrate of Streptoverticillium ladakanus (Heidelberger, 1973).
 

The exact mechanism of action of 5-azacytidine is not known. It
 

is rapidly phosphorylated in mammalian tissues and incorporated
 

into both RNA and DNA. By disrupting the processes of translation of nucleic acid sequences into protein, it inhibits the
 

synthesis of protein. Moreover, by inhibiting orotodylic acid
 

decarboxylase, the chemical also affects de novo pyrimidine
 

synthesis. 5-Azacytidine is rapidly cleared from plasma, is
 

concentrated in lymphatic tissues, and is rapidly excreted in the
 

urine as both unchanged drug and metabolites (Carter and Slavik,
 

1976).
 

5-Azacytidine was selected for screening in the carcinogenesis
 

program in an attempt to evaluate the carcinogenicity of certain
 

drugs that may be used in humans for prolonged periods.
 

1
 

II. MATERIALS AND METHODS
 

A. Chemical
 

5-Azacytidine, which is a common name for 4-amino-l-beta-Dribofuranosyl-1,3,5-triazine-2(lH)-one, was obtained as a single
 

batch (Lot No. AP-V-128) from Ash-Stevens, Inc., Detroit,
 

Michigan, by the Drug Development Branch, Division of Cancer
 

Treatment, National Cancer Institute (NCI).
 

The identity and purity of the chemical were confirmed in
 

analyses at Stanford Research Institute. Elemental analyses (C,
 

H, N, 0) were correct for CgHj^N^C^, the molecular formula of
 

5-azacytidine. Infrared, ultraviolet, and nuclear magnetic
 

resonance spectra were as expected for this chemical and were
 

identical to spectra of a reference standard. No free 5-azacytosine or D-ribose was detected by paper chromatography. On the
 

basis of these results, the purity was estimated to be > 99%.
 

The powdered 5-azacytidine was stored at 5°C in small bottles
 

®
 enclosed in sealed plastic bags containing Drierite .
 

B. Dosage Preparation
 

Concentrations of 5-azacytidine of 0.1 or 0.2% for rats and 0.02
 

or 0.04% for mice were prepared in buffered saline (pH 6.9) for
 

intraperitoneal injection of the chemical. Aqueous solutions
 

3 
were not stored, because they are unstable at roon temperature.
 

The drug and the vehicle were .nixed in a 10-ml glass Potter-


Llvehjem tissue grinder with a Teflon pestle. Fresh solutions in
 

exact amounts for each administration were prepared preceding
 

injection.
 

C. Animals
 

Female Sprague-Dawley rats and male and female Swiss mice were
 

used in subchronic studies.
 

Sprague-Dawley rats and H6C3F1 mice of both sexes, obtained
 

through contracts of the Division of Cancer Treatment, NCI, were
 

used in chronic studies. The Sprague-Dawley rats were obtained
 

from Charles River Breeding Laboratories, Inc., Wilmington,
 

ilassachusetts, and the B6C3F1 mice were obtained fron Charles
 

River Laboratories and from A. R. Schmidt, cladison, Wisconsin.
 

On arrival at the laboratory, the male rats were 30 days old, the
 

female rats were 37 days old, and the mice were all 30 days old.
 

The animals were quarantined for an acclimation period (rats for
 

5 days, mice for 4-5 days), assigned to control and treated
 

groups, and earmarked for individual identification.
 

D, Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

4 
rooms. The temperature range was 20-24°C, and the relative
 

humidity was maintained at 40-60%. There were 15 changes of room
 

air per hour. Air was passed through both intake and exhaust
 

fiberglass roughing filters. In addition to natural light,
 

illumination was provided by fluorescent light for 9 hours per
 

day. Wayne® Lab Blox (Allied Mills, Inc., Chicago, 111.) and
 

water were supplied daily and were available ad libitum.
 

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

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

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

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

tops were covered with disposable filter bonnets beginning at
 

®
 week 26; mouse cages were provided with Sterolit clay bedding
 

(Englehard Mineral and Chemical Co., New York, N.Y.). Bedding
 

was replaced once per week; cages, water bottles, and feeders
 

were sanitized at 82°C once per week; and racks were cleaned once
 

per week.
 

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

were housed with respective treated animals. Animals treated
 

with 5-azacytidine were maintained in the same rooms as animals
 

of the same species being treated with the following chemicals:
 

5 
RATS
 

Gavage Studies
 

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

(phenesterin) (CAS 3546-10-9)
 

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

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

Intraperitoneal Injection Studies
 

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

(MAAM) (NSC 141549)
 

acronycine (CAS 7008-42-6)
 

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

(CAS 789-61-7)
 

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

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

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

hydrochloride (CAS 3458-22-8)
 

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

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

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

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

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

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

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

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

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

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

adriamycin (CAS 23214-92-8)
 

MICE
 

Feed Studies
 

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

(acetohexamide) (CAS 968-81-0)
 

anthranilic acid (CAS ]18-92-3)
 

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

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

(chlorpropamide) (CAS 94-20-2)
 

5-(4-chlorophenyl)—6-ethy1-2,4-pyr imidinediamine
 

(pyrimethamine) (CAS 58-14-0)
 

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

hydrochlo.ide) (CAS 136-40-3)
 

ethionamide (CAS 536-33-4)
 

L-tryptophan (CAS 73-22-3)
 

6 
N-9H-fluoren-2-ylacetamide (CAS 53-96-3) 

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

(tolazamide) (CAS 1156-19-0)
 

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

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

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

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

ethionamide (CAS 536-33-4)
 

Gavage Studies
 

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

(phenesterin) (CAS 3546-10-9)
 

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

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

Intraperitoneal Injection Studies
 

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

(MAAM) (NSC 141549)
 

acronycine (CAS 7008-42-6)
 

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

(CAS 789-61-7)
 

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

emetine dihydrochloride tetrahydrate (CAS 316-42-7)
 

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

hydrochloride (CAS 3458-22-8)
 

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

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

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

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

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

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

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

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

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

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

adriamycin (CAS 23214-92-8)
 

E. Subchronic Studies
 

Subchronic studies were conducted in female Sprague-Dawley rats
 

and male and female Swiss mice to estimate the maximum tolerated
 

7
 
doses of 5-azacytidine, on the basis of which low and high doses
 

were determined for administration in the chronic studies. The
 

animals were administered 5-azacytidine by intraperitoneal
 

injection three times per week for 45 days, then 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 were used as vehicle (saline) controls.
 

In rats, administration of the five doses originally selected
 

(0.13, 0.33, 0.65, 1.3, and 2.6 mg/kg body weight) resulted in no
 

deaths and in no weight depression exceeding the 15% guideline,
 

when treated animals were compared with untreated controls. A
 

second study was performed using doses of 2.6, 5.2, 10.4, and
 

20.8 mg/kg. At 20.8 mg/kg, three animals died in week 4 and all
 

died by week 6. Two animals receiving 10.4 mg/kg died, one in
 

week 6 and one in week 8. At doses of 2.6 or 5.2 mg/kg, no
 

deaths occurred, no lesions were observed, and weight depression
 

did not exceed the 15% guideline. The low and high doses for
 

rats were set at 2.6 and 5.2 mg/kg for the chronic studies.
 

In mice, the subchronic study was initially conducted in males at
 

doses of 0.22, 0.6, 1.1, 2.2, and 4.4 mg/kg. No deaths attributable to drug toxicity and no weight depression exceeding the 15%
 

guideline resulted. A second study was performed in females,
 

using doses of 4.4, 8.8, 17.6, and 35.2 mg/kg. All animals
 

8
 
receiving 17.6 or 35.2 ing/kg died by week 6, and 3/5 at 8.8 rag/kg
 

died by week 8; however, there were no deaths at 4.4 mg/kg.
 

Weights of animals surviving to the end of the study were similar
 

to those of controls, and no lesions were seen at necropsy. The
 

low and high doses for mice were set at 2.2 and 4.4 mg/kg for the
 

chronic studies.
 

F. Designs of Chronic Studies
 

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

Treatment of the rats was terminated at week 34, due to excessive
 

mortality.
 

G. Clinical and Pathologic Examinations
 

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

those that were moribund were killed and necropsied. Rats and
 

mice were weighed individually each week for 2 months and every 2
 

weeks for the remainder of the study. Palpation for masses was
 

carried out at each weighing.
 

The pathologic evaluation consisted of gross and microscopic
 

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

frou killed animals and from animals found dead. The following
 

tissues were routinely examined microscopically: skin, muscle,
 

lungs and bronchi, trachea, bone and bone narrow, spleen, lymph
 

9 
Table 1. Design of Chronic Studies of 5-Azacytidine in Rats
 

Sex and 

Treatment 

Initial 

No. of 

5-Azacytidine 

Doseb 

Time 

Treated0 

on Study 

Untreated 

Group Animals3 (ing /kg) (weeks ) (weeks ) 

Male 

Untreated-Control 15 0 81 

Vehicle-Control 15 Od 34 46-47 

Low-Dose 35 2.6 34 46 

High-Dose 35 5.2 34 10e 

Female 

Untreated-Control 15 0 81 

Vehicle-Control 15 Od 34 47 

Low-Dose 35 2.6 34 46 

High-Dose 35 5.2 34 lie 

a

Male rats and their controls were 35 days of age when placed on
 

study; females were 42 days of age.
 

°5-Azacytidine was administered in buffered saline by
 

intraperitoneal injection three times per week at a volume of
 

0.25 ml/100 g body weight. Doses were based on individual
 

weights.
 

c

Treatment terminated at 34 weeks rather than at 52 weeks
 

because of high mortality in the high-dose groups.
 

^Vehicle controls received only buffered saline solution,
 

at the same volume as treated rats.
 

e

The remaining high-dose males were killed at week 44 and
 

high-dose females at week 45.
 

10
 
Table 2. Design of Chronic Studies of 5-Azacytidine in Mire
 

Sex and
 

Treatment
 

Group
 

Male
 

Untreated-Control
 

Vehicle-Control
 

Low-Dose
 

High-Dose
 

Female
 

Untreated-Control
 

Vehicle-Control
 

Low-Dose
 

High-Dose
 

Initial
 

No. of
 

Animals3
 

15
 

15
 

35
 

35
 

15
 

15
 

35
 

35
 

5-Azacytidine
 

Doseb
 

iia/ka.!
 

0
 

c
 o
 

2.2
 

4.4
 

0
 

Oc
 

2.2
 

4.4
 

Time on Study
 

Treated Untreated
 

(weeks ) (weeks)
 

81-82
 

52 29-30
 

52 29
 

52 29
 

82
 

52 29-30
 

52 29
 

52 9d
 

a

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

^5-AzacytLdine was administered in buffered saline by
 

intraperitoneal injection three times per week at a volume of
 

1 ml/100 g body weight. Doses were based on the mean weight of
 

the animals in each cage.
 

c

Vehicle controls received only buffered saline solution,
 

at the same volume as treated mice.
 

^The remaining high-dose female mice were killed at week 61.
 

11
 
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 may have
 

been judged to be in such an advanced state of autolysis as to
 

preclude histopathologic evaluation. Thus, the number of animals
 

from which particular organs or tissues were examined microscopically varies, and does not necessarily represent the number
 

of animals that were placed on study in each group.
 

H. Data Recording and Statistical Analyses
 

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

System (Linhart et al., 1974). ""he data elements include descriptive information on the chemica;

s, animals, experimental design,
 

12
 
clinical observations, survival, body weight, and individual
 

pathologic results, as recommended by the International Union
 

Against Cancer (Berenbluni, ly6y). Data tables were generated for
 

verification of data transcription and for statistical review.
 

These data were analyzed using the statistical techniques
 

described in this section. Those analyses of the experimental
 

results that bear on the possibility of carcinogenicity are
 

discussed in the statistical narrative sections.
 

Probabilities of survival were estimated by the product-limit
 

procedure of Kaplan and tieier (lySo) and are presented in this
 

report in the form of graphs. Aniraals were statistically
 

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

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

causes were not statistically censored. Statistical analyses for
 

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

(1972) for testing two groups for equality and Tarone's (iy?5)
 

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

One-tailed P values have been reported for all tests except the
 

departure fron linearity test, which is only reported when its
 

two-tailed P value is less than 0.05.
 

The incidence of neoplastic or nonneoplastic lesions has been
 

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

13
 
at a specific anatomic site (numerator) to the number of animals
 

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

the denominators included only those animals for which that site
 

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

(e.g., skin or mammary tumors), or when lesions could have
 

appeared at multiple sites (e.g., lymphomas), the denominators
 

consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

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

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

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

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

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

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

P value for any comparison be less than or equal to 0.05/k. In
 

cases where this correction was used, it is discussed in the
 

narrative section. It is not, however, presented in the tables,
 

where the Fisher exact P values are shown.
 

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

14
 
continuity correction (Armitage, 1971), was also used. Under the
 

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

of the dose-response curve is different from zero at the onetailed 0.05 level of significance. Unless otherwise noted, the
 

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

from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

first tumor was observed were excluded by basing the statistical
 

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

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

such an early tumor was found, comparisons were based exclusively
 

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

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

obtained, the standard procedures for analyses of the incidence
 

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

followed.
 

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

incidence of tumors. Curves of the proportions surviving without
 

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

The week during which an animal died naturally or was sacrificed
 

15
 
was entered as the time point of tumor observation. Cox's
 

methods of comparing these curves were used for two groups;
 

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

groups. The statistical tests for the incidence of tumors which
 

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

noted, in the direction of a positive dose relationship.
 

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

were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each treated group compared to its control was calculated
 

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

relative risk is defined as pt/Pc where pt is the true binomial
 

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

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

spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

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

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

excess of unity represent the condition of a larger proportion in
 

the treated group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in
 

16
 
approximately 95/o of a large number of identical experiments, the
 

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

control ^roup would be within the interval calculated frora the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically significant result (P < 0.025 one-tailed test when the control
 

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

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

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

the absence of a significant result while the upper limit
 

indicates that there is a theoretical possibility of the
 

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

detected under the conditions of this test.
 

17
 

III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Among males, the mean body weights were lower in the treated
 

groups than in the controls starting at about week 4 of the
 

study, with progressively greater differences occurring as the
 

study progressed, up to approximately week 34 (figure 1). When
 

treatment was discontined at week 34, the mean body weights of
 

the low-dose group markedly increased, as did those of the four
 

remaining high-dose animals. Among females, only the high-dose
 

group had slightly lower mean body weights compared with the
 

controls. Fluctuations in the growth curve may be due to
 

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

weight may be subject to wide variations. No other signs of
 

toxicity were recorded for the rats during the bioassay.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats administered 5-azacytidine by
 

intraperitoneal injection at the doses of this experiment,
 

together with those of the controls, are shown in figure 2.
 

In each sex, the result of the Tarone test for positive doserelated trend in mortality over the period is significant (P <
 

19
 
TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves For Rats Treated With 5-Azacytidine 

20 
TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves For Rats Treated With 5- Azacytidine 

21 
0.001), and a departure from linear trend is present (P < 0.001),
 

due to the steep increase in mortality in the high-dose rats, of
 

which all died before the end of the study. The median time on
 

study was only 13 weeks for these rats, and the high mortality at
 

the high dose may have reduced the occurrence of late-developing
 

tumors. In the low-dose rats, 21/35 (60%) of the males and
 

17/35 (49%) of the females lived to the end of the study at week
 

81. Time-adjusted analysis was performed on female rats that
 

lived at least 26 weeks, which is the earliest time of an
 

observed tumor. There were 15 such animals in the untreated
 

controls, !'• in the vehicle controls, 31 in the low-dose group,
 

and 10 in the high-dose group.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

are summarized in Appendix C, tables Cl and C2.
 

A variety of neoplasms were seen with approximately equal
 

frequency in the control and treated rats. Neoplasms were seen
 

more frequently in the female rats than in the males. The most
 

frequently observed neoplasms in the females involved the
 

pituitary gland and the mammary gland. Chromophobe adenomas of
 

the pituitary were observed frequently in the control females
 

22
 
only. Benign and malignant mammary gland neoplasms were seen
 

frequently in both control and low-dose female rats. The low
 

incidence of neoplasia in the male and female high-dose groups
 

was probably due to the unusually high number of early deaths
 

that occurred in both high-dose groups.
 

Several inflammatory and degenerative lesions occurred with
 

approximately equal frequency in treated and control animals.
 

Hepatocellular .degeneration and necrosis, usually centrilobular,
 

were present in several of the treated animals and were the only
 

nonneoplastic lesions that appeared to be directly related to
 

exposure to the chemical. None of the control rats had similar
 

hepatic lesions. These lesions were present in 1/31 low-dose and
 

5/31 high-dose females. Necrotizing liver lesions were also
 

present in 5/31 low-dose and 15/33 high-dose male rats. All of
 

the animals with liver necrosis died prior to the end of this
 

study. Bone-marrow atrophy was present in 5/30 low-dose and
 

26/33 high-dose males and in 2/30 low-dose and 15/29 high-dose
 

females, but its significance in relation to the early deaths
 

could not be evaluated, due to the lack of clinical pathology
 

data.
 

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

indicate that the administration of 5-azacytidine at the doses
 

used in this study caused hepatotoxicity and bone-marrow atrophy
 

23
 
in Sprague-Dawley rats. No carcinogenic effect was observed
 

during the shortened life spans of the animals.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that were observed in
 

at least two animals with an incidence of at least 5% in either
 

the vehicle-control or low-dose groups. Due to the shortened
 

survival in the high-dose groups, results for these groups are
 

not analyzed in the tables.
 

Although there was no incidence of chromophobe adenoma of the
 

pituitary in the low-dose female rats (0/30), this tumor is
 

listed in table E2 because of the incidence observed in the
 

vehicle-control group (4/15 [27%]); this incidence gave a
 

significant result in the negative direction. The absence of
 

tumors in the treated group may be accounted for by the high
 

mortality observed in this group. A time-adjusted analysis,
 

eliminating animals that died before week 52 on study, indicated
 

that the difference between the incidences in the treated (0/25)
 

and control (4/14) groups was still significant in the negative
 

direction.
 

There is no incidence of tumors at any specific site in either
 

sex that is statistically significant in the positive direction.
 

24
 
The absence of such tumors, however, may be due to the abnormally
 

short treatment periods and life spans in the treated animals,
 

rather than to absence of carcinogenicity of the test chemical in
 

the rats.
 

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

in the tables, except for the comparison involving the pituitary
 

tumors in female rats, the value of one is included; this
 

indicates the absence of positive significant results. It should
 

also be noted that these intervals have upper limits greater than
 

one, indicating the theoretical possibility of the induction of
 

tumors by 5-azacytidine, which could not be detected under the
 

conditions of this test.
 

25
 

IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

The mean body weights of the treated mice were dose related
 

throughout the period of the study, and were lower than those of
 

the vehicle controls (figure 3). When treatment was stopped at
 

week 52, some increases in the mean body weights of the remaining
 

animals occurred. A greater difference occurred between body
 

weights of treated and control females than between those of
 

treated and control males. Fluctuations in the growth curve may
 

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

mean body weight may be subject to wide variations. No
 

significant signs of toxicity were recorded for the mice during
 

the bioassay.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice administered 5-azacytidine by
 

intraperitoneal injection at the doses of this experiment,
 

together with those of the controls, are shown in figure 4.
 

In male mice, the result of the Tarone test for positive doserelated trend in mortality over the period is significant (P <
 

0.001), with 20% of the high-d ,se group, 37% of the low-dose
 

27
 
TIME DIM ST OY (WEEKS) 

Figure 3. Growth Curves For Mice Treated With 5-Azacytidine 

28 
TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves For Mice Treated With 5-Azacytidine 

29 
group, 63% of the vehicle controls, and 93% of the untreated
 

controls living to the end of the study. The early deaths of the
 

treated mice may have reduced the occurrence of late-developing
 

tumors.
 

In female mice, the result of the Tarone test is also significant
 

(P < 0.001), and a departure from linear trend is observed (P <
 

0.001), because of the steep increase in mortality among the
 

high-dose mice, of which all died before the end of the study.
 

These early deaths of the high-dose female mice may have
 

suppressed the occurrence of late-developing tumors. Of the
 

low-dose female mice, 49% survived until termination of the study
 

at week 82. In male mice, a time-adjusted analysis was performed
 

on those animals living at least 52 weeks. There were 15 such
 

animals in the untreated controls, 11 in the vehicle controls,
 

and 18 in both the low- and high-dose groups.
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

are summarized in Appendix D, tables Dl and D2.
 

A variety of neoplasms were present in both treated and control
 

groups. With the exception of the hematopoietic system,
 

neoplasms occurred infrequently and were distributed equally
 

30
 
among the organ systems and groups studied. Several neoplasms of
 

the hematopoietic system Involving multiple organs and tissues of
 

the spleen, lymph node, liver, and lung were present in both the
 

male and female mice. Both lymphocytic and granulocytic
 

neoplasms were present in the tissues examined. With the
 

exception of one untreated-control female mouse with malignant
 

lymphooa, all of the heiaatopoietic neoplasms occurred in treated
 

animals. liematopoietic neoplasms were diagnosed most frequently
 

in the low-dose groups (male 4/31, female 17/29), and the lower
 

incidence of hematopoietic neoplasms in the high-dose groups
 

(male 3/32, female 0/30) may be the result of the high number of
 

early deaths in these groups. In general, the hematopoietic
 

neoplasms present in the low-dose groups were poorly
 

differentiated, and precise classification of cell type was
 

extremely difficult. The neoplasms observed histologically were
 

classified into the following cell types: malignant lyraphoma,
 

lymphocytic type; malignant lyinphoraa, histiocytic type; malignant
 

lymphoma, undifferentiated type; granulocytic sarcoma; and
 

granulocytic leukemia.
 

Several inflammatory, degenerative, and proliferative lesions
 

commonly seen in B6C3F1 hybrid mice occurred with approximately
 

equal frequency in treated and control animals. The most common
 

of these lesions were interstitial pneumonia with perivascular
 

31
 
and peribronchiolar lymphocytio hyperplasia and extraraedullary
 

hematopoiesis in the spleen. Necrosis of the liver was
 

occasionally observed. Mice that died prior to termination of
 

the study had no consistent lesions, except bone-marrow atrophy
 

in high-dose females, that would account for the early deaths.
 

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

indicate that the administration of 5-azacytidine at the doses
 

used had a carcinogenic effect upon the hematopoietic system in
 

the B6C3F1 hybrid female mouse. This carcinogenic effect was
 

manifested by the apparent induction of lymphocytic and granulocytic neoplasms in the low-dose female mice. There appeared to
 

be only a slight increase in the number of hematopoietic
 

neoplasms in the high-dose groups; however, the high number of
 

early deaths in this group may have accounted for this
 

observation.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

of the incidences of those primary tumors that were observed in
 

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

or more than one group.
 

In male mice, there is no incidence of tumors at any specific
 

site that is statistically significant. When time-adjusted
 

32
 
analyses, eliminating animals that died before week 52 of the
 

study, were performed on the incidences of lymphoma of the
 

hematopoietic system, the incidences (vehicle controls 0/11,
 

low-dose 4/18 [22%], high-dose 3/18 [17%]) still remain not
 

significant.
 

In female mice, the analyses of the incidences of lymphoma show
 

that the Fisher exact comparison of incidences in the low-dose
 

(7/27 [24%]) and vehicle-control (0/14) groups has a probability
 

level of 0.048; however, this probability is above the 0.025
 

level required by the Bonferroni inequality criterion when the
 

multiple comparison test is applied. Granulocytic leukemia or
 

sarcoma of the hematopoietic system was found exclusively in the
 

low-dose female mice. The Fisher exact test shows that the
 

incidence in the low-dose group (10/29 [34%]) is significantly
 

higher (P =» 0.010) than that in the vehicle controls (0/14).
 

The analyses of the combination of lymphoma, granulocytic
 

leukemia, and sarcoma in female mice indicates that the incidence
 

of these tumors in the low-dose group (17/29) differs significantly (P < 0.001) from that in the vehicle-control group (0/14),
 

in which no such tumors were seen. The untreated-control group,
 

not shown in the table, was observed to have a 1/15 (7%) incidence of lymphoma. The early mortality in the high-dose group
 

may account for the absence of these tumors in this group. The
 

33
 
incidence of these types of tumors compiled to date in the
 

bioassay studies at this laboratory is 49/265 (19%) in female
 

mice, compared with 17/29 (59%) observed in the low-dose female
 

mice in this study; however, the laboratory results to date show
 

only 1/265 granulocytic sarcoma in vehicle controls compared with
 

9/29 (31%) in the study. The statistical conclusion is that
 

there is an association between the incidence of hematopoietic
 

tumors and the administration of the low dose of the chemical to
 

female mice.
 

34
 
V. DISCUSSION
 

The test chemical, 5-azacytidine, was toxic to both the rats and
 

the mice in this bioassay, as demonstrated by (1) the lower mean
 

body weights of the treated animals compared with the controls
 

and (2) the high mortality which occurred during the study. The
 

mean body weights of the treated male rats were much lower than
 

those of the controls during the treatment period of 34 weeks.
 

Because of the high mortality, treatment was discontinued; the
 

low-dose males then gained weight rapidly. Nearly all high-dose
 

animals had died by this time; the median time on study for all
 

high-dose rats was only 13 weeks. Among the low-dose rats, 60%
 

of the male and 49% of the females survived until termination of
 

the study at week 81. Both the low survival and the short
 

duration of treatment prevented evaluation of carcinogenlcity of
 

this compound in rats.
 

Treated mice had lower mean body weights than controls throughout
 

the study, and survival was dose related in both male and female
 

mice. All high-dose females died prior to termination of the
 

study, and only 7 high-dose males, 13 low-dose males, and 17
 

low-dose females survived to the end of the study at week 81.
 

Only one male and three female high-dose rats had tumors, and
 

none of the incidences of the tumors in the low-dose group of
 

35
 
either sex were significantly increased using any of the
 

statistical tests. Bone-marrow atrophy , was observed in 17% of
 

the low-dose and 79% of the high-dose male rats, in 7% of the
 

low-dose and 52% of the high-dose female rats, and in only 2% of
 

the combined controls. Centrilobular degeneration and necrosis
 

of hepatocytes were observed in 16% of the low-dose and 45% of
 

the high-dose male rats, in 3% of the low-dose and 16% of the
 

high-dose female rats, and in none of the combined controls.
 

Only five high-dose male mice and one high-dose female mouse had
 

neoplasms. In low-dose female mice, neoplasms of the hematopoietic system including malignant lymphoma, granulocytic sarcoma,
 

and granulocytic leukemia occurred in 17 aniamls, even though
 

only 54% survived until week 81. Malignant lymphoma occurred in
 

greater numbers in the low-dose female mice (7/29) than in the
 

vehicle controls (0/14). The probability level for direct
 

comparison with the vehicle controls was P = 0.048; however, this
 

is above the 0.025 level required by the Bonferroni inequality
 

criterion for significance in multiple comparisons. Granulocytic
 

leukemia or sarcoma was observed in 10/29 low-dose female mice,
 

but in no other group, and 7/29 of the low-dose females died
 

prior to the first observation of this tumor at week 53. The
 

incidence was significant when compared with that of the vehiclecontrol group (P = 0.010). The incidence of combined lymphoma
 

36
 
and granulocytic neoplasms was highly significant in the low-dose
 

females (vehicle controls 0/14, low-dose 17/29, P < 0.001).
 

Early deaths in the high-dose animals may have precluded the
 

observation of neoplasms, and thus, the low incidence of tumors
 

in the high-dose group does not contradict the association with
 

treatment of the tumors observed in the low-dose females. No
 

tumors were observed at a significant incidence in male mice.
 

Hepatocellular necrosis similar to that seen in the rats was
 

occasionally observed, but the occurrence of this lesion does not
 

explain the early deaths. Bone-marrow atrophy was present in
 

10/29 (34%) of the high-dose female mice.
 

No other long-term studies of 5-azacytidine have been reported;
 

however, humans treated with the drug have developed leucopenia
 

(lowered total white cell count) and, occasionally, hepatic
 

toxicity (Von Hoff et al., 1976).
 

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

short life span and short duration of treatment of Sprague-Dawley
 

rats of either sex and of male B6C3F1 mice precluded evaluation
 

of the carcinogenicity of 5-azacytidine in these groups; however,
 

the induction of tumors of the hematopoietic system in female
 

B6C3F1 mice was associated with the administration of
 

5-azacytidine.
 

37
 

VI. BIBLIOGRAPHY 

Armitage, P., Statistical Methods in Medical Research, John 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_ the UICC, Vol. J2, International Union Against Cancer,
 

Geneva, 1969.
 

Carter, S. K. and Slavik, S., Investigational drugs under study
 

by the United States National Cancer Institute. Cancer
 

Treatment Reviews, 1976, pp. 451-468.
 

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

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

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

1970, pp. 48-52.
 

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

significance tests, confidence limits and adjustments for
 

stratification. Rev. Int. Statist. Inst. 39(2):148-169,
 

1971.
 

Heildelberger, C., Pyrimidine and pyrimidine nucleoside
 

antimetabolites, In: Cancer Medicine, Holland, J. E. and
 

Frei, E., III, eds. , Lea and Febiger, Philadelphia, 1973,
 

pp. 784-786.
 

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

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

1958.
 

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

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

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

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

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

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

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

induced by different numbers of administrations of benzo (a)
 

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

39
 
Tarone, R. E., Tests for trend in life table analysis.
 

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

Von Hoff, D. D. , Slavik, M., and Muggia, F. M., 5-Azacytidine -


A new anticancer drug with effectiveness in acute
 

myelogenous leukemia. In: Advances in Pharmacology and 

Chemotherapy. Report of the Division of Cancer Treatment, 

NCI, August 1976. 

40
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS GIVEN INTRAPERITONEAL INJECTIONS
 

OF 5-AZACYTIDINE
 

41
 

TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF 5-AZACYTIDINE

 

UNTREATED VEHICLE LOW DOSE HIGH DOSE 

CONTROL CONTROL 

ANIMAL S INITIALL Y IN STUD Y 15

ANIMAL S NFCROPSIE D

ANIMAL ^ EXAMINE D HI STOP ATHOLOGICALLY

 1U

 1 H

 15 

 15 

 15 

35 

32 

32 

35 

33 

33 

IN'EGUHENTAR Y SYSTE M 

*SKIN

PAPTLLOMA , NOS 

SQOAMOU S CELL CARCINOM A 

 (14) (15) (32) 

1

1

 (3%) 

 (3H) 

(33) 

*S(JBCUT TISSUE

[^DIFFERENTIATED CARCINOMA 

BASAL-CEL L CARCINOM A 

SARCOMA, NOS 

"TBROSARCON A

 (11)

 1 (7X) 

 (15) (32) 

1 (3«) 

1 (3*> 1 (3X) 

(33) 

RESPIRATORY SYSTEM 

NON E 

HEMATOPOIETIC SYSTEM 

*MULTIPL E ORGAN S

HALIG.LYMPHOMA , UNDIFFER-TYPE 

GRAKOLOCYTI C LEOKEMI A 

GRANULOCYTI C SARCOM A 

 (1t) (15) (32) 

1

2

1

 (3*) 

 (6t) 

 (3*) 

(33) 

CIRCULATOR Y SYSTEM 

NON E 

DIGESTIVE SYSTEM 

NON E 

URINAR Y SYSTEM 

HQJ1E 

* NOHBER

* NUMBE R

 OF ANIMALS

 OF AHIMALS

 KITH TISSUE

 HECROPSIED 

 EXAMINED MICROSCOPICALLY 

43 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

UNTREATED

CONTROL

 VEHICLE

 CONTROL 

 LOW DOSE HIGH DOSE 

ENDOCRINE SYSTEM 

(PITUITAR Y

CHRONOPHOBE CARCINOM A

»A.DRENAL

PHEOCHROHOCYTOM A

 (13)

 (1U)

 (15)

 1

 (15)

 (7X)

 (29)

 1

 (31)

 1

 (3«) 

 (3«) 

 (31) 

 (33) 

REPRODUCTIV E SYSTEM 

*NABMAR Y GLAN D

ADENOCAPCINOMA , NOS

FIBROADENOM A

tTSTIs

INTERSTITIAL-CELL TUMO R

 (1t)

 (114)

 (15)

 (15)

 (32)

 1

 (28)

 2

 (3*) 

 (7«) 

 (33) 

1 (3%) 

 (32) 

NER700S SYSTEM 

IBRAI N

ASTPOCYTOM A

 (1l»)

 1 (1%) 

 (15) (30) (32) 

SPECIA L SENSE ORGAN S 

MON E 

NDSCULOSKELETA L SYSTEM 

NON E 

BOD Y CAVITIES 

NON E 

A L L OTHER SYSTEHS 

NON E __ _ _ 

* NUMBER OF ANIMALS

* NUMBER OF ANIMALS

 KITH TISSUE EXAHINED MICROSCOPICALLT 

 NECROPSIED 

44 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

UNTREATED

CONTROL

 VEHICLE

 CONTROL 

 LOW DOSE HIGH DOSE 

ANIHA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALL Y IN STUD Y

NATURA L DEATH *

MORIBUN D SACRIFICE

SCHEDULED SACRIFICE 

ACCIDENTALL Y KILLE D 

TERMINA L SACPTFICE

ANIMA L MISSIN G 

 15

 14

 3

 8

 15

 15

 35

 11

 3

 21 

 35 

 20 

 15 

i INCLUDE S AUIOLYZE D ANIMAL S 

TtJICP SUMPIAP Y 

lOTH L ANIMAL S WIT H PRIMAR Y

TOTA L PRIMAR Y TUMOR S

 2 TUMORS *

2 

1 

1 

1 

1 

1 

1 

1 

1 

TOTA L PNIMAL S WIT H BENIG N

TOTA L BENIG N TUMOR S

 TUMOR S 14

 1

 1 

1 

TOTA L ANIMAL S WIT H MALIGNAN T

TOTA L MALIGNAN T TUMOR S

 TOMOR S 2

 2

 1

 1

 9 

 10 

TOTAL ANIMAL S WIT H SECONDAR Y

I^TAL SECONDAF Y TUMOP S 

 TUMOPS * 

TOTA L ANIMAL S WIT H TUMOP S

BENIG N O R MALIGNAN T 

TOTA L UNCERTAI N TUMOR S 

 UKCEPTAIN -

TOTA L ANIMAL S WIT H TUMOR S

PRIMAP Y O P METASTATT C 

TOTA L UNCERTAI N TUMOR S 

 UNCERTAIN ­

* PFIMAP Y TUMORS : UL L TUMOR S EXCEP T SECONDAR Y TUMOP S 

* SECOND»R Y TUMORS : METASTATI C TUMOR S O R TUMOR S INVASIV E INT O A N ADJACEN T OR3S.N 

45 
TABLE A2. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF 5-AZACYTIDINE 

UNTREATED

CONTROL

 VEHICLE

 CONTROL 

 LOW DOSE HIGH DOSE 

AN1HHI.S INITIALLY IN STUDY

ANIMALS HECROPSIED

ANIHALS EXAMINE D HISTOPATH OLOGIC ALLY

 15

 15

 15

 15

 15

 15

 35

 31

 31

 35 

 31 

 31 

INTEGUMENTARY SYSTEM 

•SUBCOT TISSUE

FIBROMA

 (15) (15)

 1 (7*) 

 (31) (31) 

RESPIRATOR Y SYSTEM 

ILUN G

ADENOCARCINONA , NOS , NETASTATIC

 (15)

 1 (7%)

 (1U) (30)

 2 (7*) 

 (31) 

HEMATOPOIETIC SYSTEM 

•MULTIPLE ORGAN S

LYHPHOCYTIC LEUKEMIA

 (15) (15) (31)

 1 (3*) 

 (31) 

CIRCULATOR Y SYSTEM 

NON E 

DIGESTIVE SYSTEM 

HON E 

URINAR Y SYSTEM 

MIDNEY

TUBULA R -CELL ADENOCARCINOH A

 (15)

 1 (7X) 

 (13) (31) (31) 

ENDOCEINE SYSTEM 

tPITUITftRY

CHSOMOPHOBE

CHHOHOPHOBE.

 ADENOM A

 CARCINOM A

 (1»)

 5 (36X)

 (15)

 H (27*) 

 1_12*L 

 (30) (27) 

I NUMBER OF ANIHALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

46 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

UNTREATED VEHICLE 

CONTROL CONTROL 

REPR000CTIVE SYSTEM 

*MAHHAR Y GLAN D

ADENOCARCINOSA, HOS

PAPILLAR Y ADENOCARCINOH A 

FIBROADENOHA

 (15)

 »

 2

 (27*) 

 (13%)

 (15) 

3 (20%) 

tUTEROS

LEIOMYOSARCONA 

ENDOHETRIAL STROHAL POLYP 

 (15| (15) 

NERVOUS SYSTEH 

IBRAIN (1«) (1<t) 

CHPOMOPHOBE CARCIHOHA, HETASTATI 1 (7*) 

SPECIAL SENSE ORGAN S 

*EAR CANA L (15) (15) 

KERATOACANTHOH A 

BUSCULOSKELETA1 SYSTEH 

HON E 

BOD Y CAVITIES 

*MESBNTERY (15) (15) 

LTPOBA 1 (7«) 

AIL OTHER SYSTEMS 

NON E 

t NOHBER OF ANIMALS HITH TISSUE EXAHINED BICBOSCOPICALLI
 

* NUBBEP O» ANIMALS NECROPSIED
 

LOW DOSE 

(31) 

9 (29*) 

1 (3*) 

6 (19*) 

(30) 

1 (3*) 

1 (3X) 

(30) 

(31) 

1 (31) 

(3D 

HIGH DOSE 

(31) 

1 (3*) 

1 (3*) 

1 (3«) 

(29) 

(30) 

(31) 

(311 

47 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

1 

UNTREATED

CONTROL

 VEHICLE

 CONTROL 

 LOW DOSE HIGH DOSE 

U N I HAL DISPOSITION SUMMAR Y 

ANIMALS INITIALLY IN STUDY

NATURA L DEATH S

MORIBUND SACRIFICE

SCHEDULE D SACRIFICE 

ACCIDENTALLY KILLED 

TERMINA L SACRIFICE

ANIMA L HISSING 

 15

 1

 3

 11

 15

 1

 5 

9

 35

 10

8

 17 

 15 

 22 

 13 

a> INCLUDES AUTOLYZED ANIMALS 

TU10P SUMPIAP Y 

TOTA L ANIMAL S rflTH PRIMAR Y

TO^L PPISAP Y TUHOR S

 TUMORS * 11

 13

 7

 9

 15

 20

 3 

3 

TOTA L ANIMAL S WIT H BENIGN

•^OTAL BFNIGN TUMORS

 8 TUMORS

8 

6 

8 

7 

8 

1 

1 

TOTAL ANIMAL S WITH MALIGNAN T

TOTA L MALIGNAN T TUMORS

 TUHORS 5

 5

 1

 1

 1 1

 12

 2 

2 

"OTAL ANIMAL S WITH SECONDAR Y

TOTAL SECONDARY TUMORS

 TDMOPS* 1

 1

 1

 1

 2 

2 

TOTA L ANIMAL S WIT H TUMOR S

BENIG N OP MALIGNAN T 

TOTAL UNCERTAI N TUMORS 

 UNCEPTAIN -

TOTA L ANIMAL S HITH TUMOR S

PPIMAP Y O R MSTASTATIC 

TOTAL UNCERTAI N TUMORS 

 UNCERTHIN ­

* PRIMAR Y TUMORS : ALL TUMOR S EXCEPT SECONDAR Y TUMORS 

» SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AH ADJACENT ORGAN 

48
 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE GIVEN INTRAPERITONEAL INJECTIONS
 

OF 5-AZACYTIDINE
 

49 

TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

GIVEN INTRAPERITONEAL INJECTIONS OF 5-AZACYTIDINE 

LOW DOSE HIGH DOSE 

CONTROL CONTROL 

UNTREATED VEHICLE 

ANIMALS INITIALLY IN STUDY 15 16 35 35 

ANIMAL S NECPOPSIED 15 13 31 32 

ANIHALS EXAMINE D HISTOPATH OLOGICALLY 15 13 30 30 

INTEGUMENTAR Y SYSTEB 

NON E 

RESPIRATOR Y SYSTEM 

• LON G (15) (13) (29) (30) 

DWDIPFERENTIATED CARCINOMA BETAS 1 (7X) 

ALVEOLAR/BHONCHIOLA R ADENOM A 1 (8J) 1 (3% ) 

HEMATOPOIETIC SYSTEM 

• MULTIPL E ORGAN S (15) (13) (31) (32) 

HALIG.LYMPHOHA , LYMPHOCYTIC TYPE U (13X) 2 (6*) 

MALIG. LYMPHOBA , HISTIOCYTIC TYPE 1 (3*) 

tHEDIASTINAL L.NODE (15) (3) (23) (20)

 

UNDIFPERENTIATE D CARCINOM A BETAS 1 (7*) 

CIRCULATORY SYSTEB 

NONE 

DIGESTIVE SYSTEB 

niVBR (15) (13) (30) (30) 

(^DIFFERENTIATED CARCINOM A 1 (7%) 

HEPATOCELLULAR ADENOBA 1 (7«) 1 (8*) 1 O«) 1 (3*) 

ORIHAR I SYSTEH 

__NON E __ 

I NUMBE R OF ANIBALS WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OF ANIBALS NECROPSIED 

51 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

UNTREATED VEHICLE

CONTROL CONTROL 

ENDOCPIN E SYSTE M 

ITHYFOID (11) (7)

ADENOMA , NOS

REPRODUCTIV E SYSTE M 

NON E 

NERVOU S SYSTE M 

«JON E 

SPECIAL SENSE ORGAN S 

*EAR CANA L (15) (13)

KERATOACANTHOH A

NUSCULOSKELETA L SYSTEM 

NON E 

BODY CAVITIE S 

NON E 

A LL OTHER SYSTEMS 

NON E 

ANIMA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALLY IN STUDY 15 16

NATURA L DEATHS 5

MORIBUN D SACRIFICE 1 1

SCHEDULE D SACRIFICE 

ACCIDENTALL Y KILLED 

TERMINA L SACRIFICE 1U 10

ANIMA L HISSING 

* INCLUDES AUTOLYZED ANIHALS _ _ 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NOMBE P OF AHIMAL S NECROPSIED 

 LOW DOSE HIGH DOSE 

 (2U) (20) 

1 (!»*) 

 (31) (32) 

1 (3%) 

 35 35 

 18 15 

 II 1 3 

 13 7 

52 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

UNTREATED

CONTROL

 VEHICLE 

 CONTROL 

LOW DOSE HIGH DOSE 

TUMO R SUHMiH Y 

TOTA L ANIHAL S WITH PRIMAR Y

TOTAL PRIMAR Y TUMORS

 TUMORS * 2

 2

 2 

2 

6

6

 5 

6 

TOTAL ANIHAL S (IITH BENIG N

TOTAL BENIGN TOMORS

 TtJHOPS 1

 1

 2 

2 

2

2

 2 

3 

TOTAL ANIMAL S WITH MALIGNAN T

TOTAL MALIGNAN T TUHOPS

 TUMORS 1 

1 

H

U

 3 

3 

TOTAL ANIMAL S WITH SECONDAR Y

TOTAL SECONDAR Y TUMORS

 TUMORS* 1 

2 

TOTAL ANIMALS WITH TUMORS

BENIG N O B MALIGNAN T 

TOTAL UNCERTAI N TUMORS 

 UNCERTAINTOTA L ANIHAL S KITH TUMOR S UNCERTAIN -

PRIMfP Y O R METASTATIC 

TOTA L UNCERTAI N TUMOtiS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS t SECONDARY TUHORS: METASTATIC TUMORS OH TUMORS INVASIVE INTO AN ADJACENT ORGAN 

53 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

GIVEN INTRAPERITONEAL INJECTIONS OF 5-AZACYTIDINE

 

UNTREATED VEHICLE LOW DOSE HIGH DOSE 

CONTROL CONTROL 

ANIMAL S INITIALL Y IN STUDY 15 11 35 35 

ANIMAL S NECPOPSIE D 15 1U 29 30 

ANTHAL S EXAMINE D HI STOPATH OLOGIC ALL Y 15 13 29 30 

INTEGUMENTAR Y SYSTEM 

*SUBCnT TISSUE (15) ( 1 U) (29 ) (30) 

EA'AL-CELL CARCIN01A 1 (3») 

RESPIRATOR Y SYSTEM 

*LUNG (15) (13) (28) (30) 

ONDIFFEPENTIATE D CAPCINOH A META S 1 (7«) 

BASfL-CELL CARCINOMA, HETASTATIC 1 («*)

 

AT.VSOLAE/BEONCHIOLA 8 ADENOM A 1 (S%) 

ALVEOLAR/BROtiCHIOLA R CARCINOM A 1 (B%) 

SARCOHA , NOS, METASTATI C 1 (UK )

 

HEMATOPOIETTC SYSTEM 

• MULTIPL E ORGAN S (15) (11) (29) (3C)

 

MALIS.LYMPHOMA , UNO IFFE8-TYPE 1 (3*)

 

MALIG . LYMPHOMA , LYBPHOCYTTC TYPE 1 (7%) 1 (11%)

 

GFANULOCYTI C LEUKEMI A 1 (3*)

 

GFANBLOCYTIC SARCOMA 9 (31*)

 

*LIVFR (15) (13) (29) (3") 

XALIG.LYMPHOMA , LYMPHOCYTI C TYPE 1 (3*)

 

IKIDNE Y (15) (13) (29 ) (29 )

 

MALIG . LYHPHOMA , LYMPHOCYTIC TYPE 1 (3?)

 

CIRCULATORY SYSTEM 

tHEAR T (11) (12) (28) (30)

 

SAPCOMA X NOS _ _ _. 1.J141

 

I NUMBE P 0? ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S NECROPSIE D 

54 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

UNTREATED VEHICLE 

CONTROL CONTROL 

DIGESTIVE SYSTEM 

tLIVEE (15) (13) 

HEPATOCELLULA R ADENOMA 1 (7X) 

LBIOHYOSARCOMA, HETASTATIC 

URINAR Y SYSTEM 

NONE 

ENDOCRINE SYSTEM 

tADRENA L (1U ) (13) 

LEIOMYOSARCOHA, HETASTATIC 

REPRODUCTIV E SYSTEM 

*MAHBAR Y GLAN D (15) (11) 

ADENOCARCINOBA , NO S 

tOTEROS (15) (13) 

SARCOMA, NOS 

LEIOMYOSABCOHA 

NERVOU S SYSTEM 

NON E 

SPECIAL SENSE ORGAN S 

*EAR CANA L (15) (14) 

BASAL-CELL CARCINOMA 

HUSCOLOSfELETAL SYSTEM 

NON E 

BODY CAVITIES 

•PERITONEUM (15) (11) 

LE!QMIOS1££OHA_._SEI.AST4IIC__ 

t NOHBER OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NOHBER OF ANIMALS NECROPSIED 

LOW DOSE HIGH DOSE 

(29) (30) 

1 (3«) 

(27) (29) 

1 («%) 

(29) (30) 

2 (7X) 

(28) (27) 

1 («) 

2 (7X) 

(29) ( 30) 

1 (3«) 

(29) (30) 

1-11SL 

55 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

UNTREATED VEHICLE LOW DOSE HIGH DOSE
 CONTROL CONTROL
 

*PLEURA (15) (14) (29) (30)
 

LETOMYOSARCOMA, METASTATIC 1 (3%)
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STODY 15 1<» 35 35
 

NAT DEAL DEATH9 1 1 14 11
 

MORIBUND SACRIFICE 3 3 H 24
 

SCHEDULED SACRIFICE
 ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 11 10 17
 

ANIMAL MISSING
 

9 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS KITH PRIBAPY TUMORS* 2 2 22 1
 

TOTAL PRIMARY TUMORS 2 2 2U 1
 

TOTAL ANIMALS WITH BENIGN TUMORS 1 1
 

TOTAL BENIGN TUMORS 1 1
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 1 1 22 1
 

TOTAL MALIGNANT TUMORS 1 1 2U 1
 

TOTAL ANIMALS WITH SECONDARY TUHORSt 1 3
 

TOTAL SECONDARY TUMORS 1 6
 

TOTAL ANIMALS WITH TOMORS UNCERTAIN-


BENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUHORS
 

TOTAL ANIMALS HITH TUMORS UNCERTAIN-


PRIMARY OP HETASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUHORS: ALL TUMORS EXCEPT SECONDARY TUMOHS
 * SECONDARY TUMOBS: METASTATIC TUMOHS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

56
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS GIVEN INTRAPERITONEAL INJECTIONS
 

OF 5-AZACYTIDINE
 

57
 

TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF 5-AZACYTIDINE

 

UNTREATED VEHICLE 

CONTROL CONTROL 

ANIMAL S INITIALL Y IN STUDY 15 15 

ANIMALS NECROPSIED 14 15 

ANIHAL S EXAHINB D HISTOPATHOLOGICALL Y 14 15 

INTEGUMENTAR Y SYSTEM 

NONE 

RESPIRATOR Y SYSTEM 

ITRACHEA (12) (15) 

INFLAMMATION , ACOTE/CHRONIC 1 (7») 

tLONG/BRONCHIOLE (14) (13) 

HYPERPLASIA , LYMPHOI D 

ILUNG (14) (13) 

INFLAMMATION , INTERSTITIAL 1 (8«) 

BRONCHOPNEOHONI A SOPPORATIV E 3 (21*) 

BRONCHOPNBDHONIA, CHRONIC 

BROHCHOPNEOHONI A CHRONIC SUPPUR A 

HYPERPLASIA , LYMPHOI D 1 (8*) 

HEMATOPOIETIC SYSTEM 

*BONE MARRO W (13) (15) 

ATSOPHY , NOS 2 (13X) 

tSPLEEM (14) (15) 

INFLAMMATION , NECROTIZIN G 

NECROSIS, NOS 

tLYMP H NODE (7) (13 ) 

INFLAMMATION, NECROTIZIWG 

CIRCDLATOR Y SYSTEM 

tHEART/ATRIU M (U) (13) 

THPOHBOSJS.i_NOS _ 

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

LOW DOSE 

35 

32 

32 

(28) 

« (•"•*) 

(32) 

1 (3*) 

(32) 

4 (13*) 

5 (16X) 

2 (6*) 

U (13*) 

2 (6*) 

(30) 

5 (17X) 

(31) 

(22 ) 

(32) 

HIGH DOSE 

35 

33 

33 

(33) 

(32) 

(32)

1

 (3*) 

1 (3*) 

(33) 

26 (79*) 

(32) 

1 (3*) 

3 (9*) 

(28) 

1 (4*) 

(32|

i.iasi_ 

59
 
TABLE CI. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED VEHICLE LOW DOSE HIGH DOSE CONTROL CONTROL 

• MYOCARDIUM (14) (13)
 

INFLAMMATION, NECROTIZING
 

DEGENERATION, NOS
 

DIGESTIVE SYSTEM 

•LIVER	 (1*0 (14)
 

CONGESTION, PASSIVE
 

CONGESTION, CHRONIC PASSIVE
 

INFLAMMATION, NECROTIZING
 

NECROSIS, COAGULATIVE
 

NECROSIS, CENTRAL
 

•LIVER/CENTPILOBULAR	 d1

») (14)
 

NECROSIS, **OS
 

NECROSIS, COAGULATIVF
 

•BILE	 DUCT (14 ) (15)
 

INFLAMMATION, CHRONIC
 

•GASTRIC	 SUBMUCOSA (14 ) (15)
 

ABSCESS, CHRONIC
 

URINARY SYSTEM 

tKIDNEY (14 ) (15) 

HYDRONEPHROSIS 1 (7%) 

INFLAMMATION, INTERSTITIAL 

INFLAMMATION, SUPPURATIVE 

GLOMERULONEPHRITIS, MEMBRANOUS 1 (7%) 

PYELONGPHRITIS, ACUTE/CHRONIC 

IN^IAMMATION, CHRONIC 

ENDOCRTNE SYSTEM 

ΜΟΝΕ 3 (21% ) 

REPRODUCTIVE SYSTEM 

#PROS7A*E (15)
 

INFLAMMATION, SUPPURATIVE
 

NERVOUS SYSTEM 

NONE (1**) 

# NUMRFR 0 * ANIMALS KI^H TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER n* ANIMALS NECRO^SIED 

(32) 

(31) 

5 (16%) 

(31) 

(32) 

1 (3%) 

(31 ) 

(31 ) 

2 (6*) 

1 (3%) 

1 (3%) 

(28) 

(32) 

1 (3%) 

1 (3* ) 

(33) 

1 (3%) 

1 (3* ) 

1 (3%) 

2 (6%) 

3 (9%) 

(33) 

1 (3%) 

8 (24%) 

(33) 

(33) 

1 (3%) 

(32) 

1 (3%) 

1 (3%) 

(30) 

1 (3%) 

60
 
TABLE C1. MALE RATS: IMONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED VEHICLE LOW DOSE HIGH DOSE 

CONTROL CONTROL 

SPECIAL SENSE ORGANS 

NONB 

HUSCOLO SKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SUMMARY 

NO LESION REPORTED 7 12 K 1 

AUTOLYSIS/NO NECBOPSY 1 3 2 

t NUMBE R OF ANIHALS WITH TISSDE EXAMINE D MICROSCOPICALLY 

* NOMBE P OF ANIMALS NECROPSIED 

61
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

GIVEN INTRAPERITONEAL INJECTIONS OF 5-AZACYTIDINE

 

UNTREATED CONTROL VEHICLE CONTROL LOW DOSE HIGH DOSE 

ANIMAIS INITIALLY IN STUDY ANIMALS NECROPSIED

ANIMALS EXAMINED HI STOPATHOLOGICALLY

 15

 15

 15

 15 

 15 

 15 

35

31

31

 35 

 31 

 31 

INTEGUMENTARY SYSTEM 

NONE 

RESPIRATORY SYSTEM 

ITRACHEA

INFLAMMATION, ACUTE/CHRONIC 

 (1U) (15) (29)

1 <3X) 

 (28) 

*LONG

INFLAMMATION, INTERSTITIAL

BHONCHOPNEUHOMIA SUPPUR ATIVE

BRONCHOPNEOHONIA NECHOTIZING 

BHONCHOPNEUMONIA CHRONIC SUPPORA HYPERPLASIA. LYMPHOID

 (15)

 1 (7X) 

1 (7*) 

 (1«) 

1 (7%) 

1 (7%) 

(30)

1 (3«) « (13X) 

 (30) 

2 (7*) 

1 (3X) 

1 <3X) 

HEMATOPOIETIC SYSTEM 

*BONE MARROW ATPOPHY, NOS  (15) (15) (30)

2 (7%)

 (29) 

 15 (52%) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

tLIVER/CENTRILOBULAR NECROSIS, NOS 

NECROSIS, COAGOLATIVE CYTOLOGIC DEGENERATION 

 (14) (1t) (31)

1 (3X) 

 (31) 

H (13«) 

1 (3%) 

URINARY SYSTEM 

NO^E _ 

* NUHBEB OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

_ — — — 

62
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED

CONTROL

 VEHICLE

 CONTROL 

 LOW DOSE HIGH DOSE 

EiDOCBINE STSTBH 

NONE 

REPRODUCTIVE SYSTEH 

'MAMMAR Y GLAN D

HYPEPPLASIA , CYSTIC

IOTERUS/ENDOHETRIO M

INFLAMMATION , SOPPORATIVE

INFLAMMATION , CHRONIC SOPPORATIV

tOVAR Y

INFLAMMATION , SOPPORATIVE

INFLAMMATION , CHRONIC SOPPORATIV

 (15)

 1 (7X) 

 (15)

 1 (7%)

 (10)

 (15)

 (15)

 2

 (13)

 (13*)

 (31)

 (30)

 3

 3

 (23)

 1

 3

 (10X) 

 (10%) 

 (41) 

 (13X)

 (31) 

 (29) 

 (27) 

1 (U« ) 

NERVOU S SYSTEM 

HON E 

SPECIA L

NON E 

 SENSE ORGAN S 

MOSCOLOSKELETA L SYSTEM 

NON E 

BODY CAVITIES 

NON E 

ALL OTHER SYSTEMS 

..NONE

» NUMBER Of ANIMAL S

* NDMBER OF ANIMALS

 ilTH TISSDE

 NECBOPSIED 

 EXAMINE D

 . 

 MICROSCOPICALLY 

63 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED

CONTROL

 VEHICLE

 CONTROL 

 LOW DOSE HIGH DOSE 

SPECIA L NOPPHOLOG Y SUMMAR Y 

NO LESION REPORTED 

AUTO/NECROPSY/HISTO PERF 

AUTOLYSIS/N O NECROPSY 

« 7 9

4

 10 

2 

4 

t SOMBE R

* NUMBEP

 0"

 OF

 ANIMAL S

 ANIMAL S

 HITH TISSUE

 NECROPSIED 

 EXAMINE D MICROSCOPICALLY 

64 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE GIVEN INTRAPERITONEAL INJECTIONS
 

OF 5-AZACYTIDINE
 

65
 

TABLE 01. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

GIVEN INTRAPERITONEAL INJECTIONS OF 5-AZACYTIDINE 

UNTREATED 

CONTROL 

VEHICLE

CONTROL 

 LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY

ANIMALS NECBOPSIED

ANIMALS EXAMINED HI STOPATHOLOGICALLY

 15 

 15 

 15 

16

13

13

 35

 31

 30

 35 

 32 

 30 

INTEGUMENTARY SYSTEM
 

*SK!N (15) (13) (31) (32)
 EPIDERMAL INCLUSION CYST 1 (7*)
 

RESPIRATORY SYSTEM
 

tLUMG (15) (13) (29) (30)
 INFLAMMATION, INTERSTITIAL 1 (8X) 4 (14X)
 

BRONCHOPNEUMONIA SUPPORATIVE 1 (3H)
 

PERIVASCULITIS 1 (3«)
 

HYPERPLASIA, ALVEOLAR EPITHELIUM 1 (3*)
 

HEMATOPOIETIC SYSTEM
 

tBONE MARROH (15) (13) (29) (25)
 

ATROPHY, NOS 1 (8X) 3 (10%)
 

*SPLEEN (15) (13) (26) (28)
 

HYPERPLASIA, HEMATOPOIETIC 2 (8X)
 

HYPERPLASIA, RETICOLUM CELL 1 (<•*) 1 (IX)
 HEHATOPOIESIS 1 (8X)
 

#MESENTERIC L. NODE (15) (3) (23) (20)
 

INFLAMMATION, SOPPORATIVE 1 (UK)
 INFLAMMATION, HEMORRHAGIC 1 (t«)
 

CIRCULATOHY SYSTEM
 

NONE
 

I NUMBE R OF ANIMAL S WITH TISSUE EXAMINED HICBOS COPICALtY 

* NUMBER OF AHIH4LS NECROPSIED 

67 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED VEHICLE 

CONTROL CONTROL 

DIGESTIV E SYSTE M 

*LIVER (15) (13) 

PEPTVASCULITI S 

NECROSIS, NOS 

NECROSIS , COAGULATIV E 

NECROSIS, CENTRA L 

HYPERPLPSIA , NODULA R 2 (13*) 

HYPERPLASIA , HEM ATOPOI ET 1C 

'JRINAH Y SYSTE M 

tKIDNE Y (15) (13) 

PYELONGPHRITIS , ACUTE/CHRONIC 

INFLAMMATION , CHRONI C 1 (8*) 

*0. ELADDER/SUBMUCOSA (1H) (12) 

HEMORRHAG E 1 (8X) 

ENDOCRIN E SYSTEM 

NON E 

REPRODUCTIV E SYSTEM 

*SEHINAL VESICLE (15) (13) 

INFLAMMATION , SDPPDRATIV E 1 (8X) 

NERVOO S SYSTEM 

NON E 

SPECIAL SENS E ORGAN S 

NON E 

MUSCULOSKELETA L SYSTEM 

NON E _ _ 

t	 NDHBER OF ANIHALS KITH TISSOE EXAMINED MICHOSCOPICA LLI
 

*	 NUMBER OF ANIMALS NECEOPSIED
 

LOW DOSE HIGH DOSE 

(30) (30) 

1 (3*) 

1 (3X) 

1 (3*) 

1 (3*) 

1 (3X ) 

(28) (29) 

1 (l»«) 

(26) (21) 

(31) (32) 

68
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED VEHICLE LOW DOSE HIGH DOSE 

CONTROL CONTROL 

BODY CAVITIES 

*PERITONEOM (15) (13) (31) (32) 

INFLAMHATION, CHRONIC 1 (8*) 1 (3*) 

"-HESBNTERY (15) (13) (31) (32) 

NECROSIS, FAT 1 (3%) 

ALL OTHER SYSTEMS 

NON E 

SPECIAL MORPHOLOGY SUMMAR Y 

NO LESION REPORTED 10 8 13 22 

AUTO/NECROPSY/NO HISTO 1 2 

AUTOLYSIS/NO NECROPSY 3 K 3 

t NUMBE R 0' ANIMAL S WITH TISSUE FX'MINE D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S NECROPSIED 

69 
TABLE D2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

GIVEN INTRAPERITONEAL INJECTIONS OF 5-AZACYTIDINE

 

UNTREATED 

CONTROL 

VEHICLE 

CONTROL 

LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 

ANIMALS NECROPSIED 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 

15 

15 

15 

14 

14 

13 

35 

29 

2 9 

35 

30 

3 0 

INTEGUMENTARY SYSTEM 

NONE 

RESPIRATORY SYSTEM 

• LUNG 

INFLAMMATION, INTERSTITIAL 

HYPERPLASIA, LYMPHOID 

(15) 

2 (13%) 

3 (20%) 

(13) 

1 (8%) 

(28) 

1

2

 (4%) 

 (7%) 

(30) 

HEMATOPOIETIC SYSTEM 

•BONE MARROW 

ATROPHY, NOS 

(13) (13) (28) (29) 

10 (34%) 

•SPLEEN 

HYPERPLASIA,

HYPERPLASIA,

HYPERPLASIA,

 HEMATOPOIETIC 

 RETICULUM CELL 

 LYMPHOID 

(12) (13) 

1 (8%) 

(29) 

1

1

2

 (3%) 

 (3%) 

 (7%) 

(29) 

1 (3%) 

•MESENTERIC L . NODE 

HYPERPLASIA, RETICULUM CELL 

(10) (9) (24) 

1 (4%) 

(23) 

•THYMUS 

HYPERPLASIA, HEMATOPOIETIC 

(7) (8) (10) 

1 (10%) 

CIRCULATORY SYSTEM 

•HEART 

PERIARTERITIS 

(14) 

1 (7%) 

(12) (28) (30) 

DIGESTIVE SYSTEM 

•LIVER 

NECROSIS, FOCAL 

NECROSIS, COAGULATIVE 

HYPERPLASIA­ FOCAL 

(15 ) (13) (29) 

1 (3%) 

(30) 

1

1

 (3%) 

 (3%) 

• NUMBER

* NUMBER

 OF ANIMALS

 O? ANIMALS

 WITH TISSUE

 NFCROPSIED 

 EXAMINED MICROSCOPICALLY 

70 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED VEHICLE
 

CONTROL CONTROL
 

*BILE DUCT (15) (1<t)
 INFLAMMATION, ACOTE/CHBONIC 1 <7X)
 

•PANCREAS	 (13) (13)
 INFLAMMATION, CHEOHIC
 

NECROSIS, FOCAL
 

ORIHARY SYSTEM
 

«KIDNEY (15) (13)
 

AMYLOIDOSIS
 

tKIDNEY/GLOHERULUS (15) (13)
 

NECROSIS, FOCAL
 

ENDOCRINE SYSTEM
 

iTHYROID (13) (9)
 

NECPOSIS, FOCAL
 

IPARATHYROID (<») (2)
 PEHIARTERITIS 1 (25%)
 

REPRODUCTIVE SYSTEM
 

#UTERUS (15) (13)
 HYPEBPLASIA, RETICULUH CELL
 

tOTEROS/EHDOHETRIDM (15) (13)
 INFLAMMATION, HECFOTIZIN6 1 (8X)
 HYPEHPLASIA, CYSTIC 1 (7X)
 

tUTEHDS/HYOSETRIUN (15) (13)
 

INFLAMMATION, NECROTIZING 1 (8*)
 

NERVOUS SYSTEM
 

NONE
 

SPECIAL SEKSE ORGANS
 

_.S2S5
 

t NOHBE H OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

LOW DOSE HIGH DOSE
 

(29) (30)
 

(25) (27)
 

1 (1*)
 

1 <t«)
 

(29)	 (29)
 

1 (3%)
 

(29)	 (29)
 

1 (3«)
 

(13)	 (21)
 

1 (5*)
 

(5) (7)
 

(28)	 (27)
 

1 (»*)
 

(28) (27)
 

(28) (27)
 

71 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

UNTREATED

CONTROL

 VEHICLE

 CONTROL 

 LOW DOSE HIGH DOSE 

HUSCUI.OSKEI.ETiL SYSTEM 

NON E 

BODY CAVITIES 

*PERITONEUM

INFLAMMATION , CHRONIC

 (15) (1U ) (29) (30) 

1 (3%) 

A LL OTHER

NON E 

 SYSTEMS 

SPECIA L MORPHOLOGY SUMMAR Y 

MO LESION 3EPORTED 7

AUTO/NECHOPSY/NO HISTO

AOTOLYSIS/NO NECROPSY

t NUMBER OF ANIMALS WITH TISSOE EXAMINED MICROSCOPICALLY 

* NUMBER OF fNINALS NECROPSIED 

9

 1 

5

 6

 15 

5 

72 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

RATS TREATED WITH 5-AZACYTIDINE
 

73
 

75 

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

Treated with 5-Azacytidinea
 

Vehicle Low
 

Topography: Morphology Control Dose
 

Hematopoietic System: Lymphoma,
 

Granulocytic Leukemia, or Sarcoma" 0/15 (0) 4/32 (13)
 

P Values0 »d N.S.
 

Relative Risk (Vehicle Control)6 Infinite
 

Lower Limit 0.463
 

Upper Limit Infinite
 

Weeks to First Observed Tumor
 73
 

Testis: Interstitial-cell
 

Tumor

b 0/15 (0) 2/28 (7)
 

P Values0 'd N.S.
 

Relative Risk (Vehicle Control)6 Infinite
 

Lower Limit 0.168
 

Upper Limit Infinite
 

Weeks to First observed Tumor 80
 

a

Treated groups received doses of 2.6 or 5.2 rag/kg by injection three times per week for 34 weeks.
 

"Number of tumor-bearing animals/number of animals examined at site (percent).
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Treated with 5-Azacytidinee
 

(continued)
 

G

Beneath the incidence of tumors in the treated group is the probability level for the Fisher
 

exact test for the comparison of that group with the vehicle—control group when P < 0.05;
 

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

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

e

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

76 
77 

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

Treated with 5-Azacytidinea
 

Vehicle Low
 

Topography: Morphology Control Dose
 

Pituitary: Chroraophobe
 

Adenoma 4/15 (27) 0/30 (0)
 

P Values0>d P = 0.009(N)
 

Relative Risk (Vehicle Control)6 0.000
 

Lower Limit 0.000
 

Upper Limit 0.521
 

Weeks to First Observed Tumor 65
 

Mammary Gland: Papillary 

Adenocarcinoma, Fibroadenoma, 

or Adenocarcinoma. NOS * L. 

(not otherwise specified) 3/15 (20) 13/31 (42) 

P Valuesc » d N.S. 

Relative Risk (Vehicle Control)6 2.097
 

Lower Limit 0.716
 

Upper Limit 10.013
 

Weeks to First Observed Tumor 76 26
 

a

Treated groups received doses of 2.6 or 5.2 rag/kg by injection three times per week for 34 weeks.
 

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

Treated with 5-Azacytidinea
 

(continued)
 

c

Beneath the incidence of turaors in the treated group is the probability level for the Fisher
 

exact test for the comparison of that group with the vehicle-control group when P < 0.05;
 

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

G

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

e

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

control group.
 

78 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

MICE TREATED WITH 5-AZACYTIDINE
 

79
 

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

Treated with 5-Azacytidinea
 

Vehicle Low High
 

Topography: Morphology Control Dose Dose
 

Hematopoietic System: Lymphoraa 0/13 (0) 4/31 (13) 3/32 (9)
 

P Valuesc»d N.S. N.S.
 

Relative Risk (Vehicle Control)6 Infinite Infinite
 

Lower Limit 0.421 0.265
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 73 74
 

00 a

Treated groups received doses of 2.2 or 4.4 mg/kg by injection three times per week for 52 weeks.
 M
 

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

c

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 vehicle-control group when P < 0.05;
 

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

°Since survival in the high-dose group was short, the tests for dose-related trend are
 

not reported.
 

e

The 95% confidence interval of the relative risk between each treated group and the control group.
 
82 

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

Treated with 5-Azacytidinea
 

Topography; Morphology 

Vehicle

Control

 Low

 Dose

 High 

 Dose 

Multiple Sites: 

Lymphoma 0/14 (0) 7/29 (24) 0/30 (0) 

P Valuesc'd P = 0.048 N.S. 

Relative Risk (Vehicle Control)6 

Lower Limit 

Upper Limit 

Infinite 

1.010 

Infinite 

Weeks to First Observed Tumor 43 

Hematopoietic System: 

Granulocytic Leukemia or 

Sarcoma 0/14 (0) 10/29 (34) 0/30 (0) 

P Valuesc » d P = 0.010 N.S. 

Relative Risk (Vehicle Control)6 

Lower Limit 

Upper Limit 

Infinite 

1.554 

Infinite 

Weeks to First Observed Tumor 53 
__ 

--

__ 83

 

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

Treated with 5-Azacytidinea
 

(continued)
 

Vehicle Low High
 

Topography: Morphology Control Dose Dose
 

Hematopoietic System: Lymphoma,
 

Granulocytic Leukemia, or
 

Sarcoma 0/H (0) 17/29 (59) 0/30 (0)
 

P Valuesc»d P < 0.001 N.S.
 

Relative Risk (Vehicle Control)6 Infinite
 

Lower Limit 2.848
 

Upper Limit Infinite
 —


Weeks to First Observed Tumor 43
 

Mammary Gland: Adenocarcinoma'' 0/14 (0) 2/29 (7) 0/30 (0)
 

P Valuesc>d N.S. N.S.
 

Relative Risk (Vehicle Control)6 Infinite _.*
 

Lower Limit 0.153
 

Upper Limit Infinite —
 

—
 

Weeks to First Observed Tumor 61
 ——
 
__ 

84

 

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

Treated with 5-Azacytidinea
 

(continued) 

Vehicle Low High 

Topography; Morphology Control Dose Dose 

Uterus: Sarcoma, NOS, or
 n
 Leiomyosarcoma 0/13 (0) 3/28 (11) 0/27 (0)
 

P Valuesc»d N.S. N.S.
 

Relative Risk (Vehicle Control)6 Infinite
 

Lower Limit 0.300
 

Upper Limit Infinite —
 

—
 

Weeks to First Observed Tumor 81
 ——
 a

Treated groups received doses of 2.2 or 4.4 mg/kg by injection three times per week for 52 weeks.
 

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

c

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 vehicle-control group when P < 0.05;
 

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

d

Since survival in the high-dose group was short, the tests for dose-related trend are
 

not reported.
 

e

The 95% confidence interval of the relative risk between each treated group and the control group.
 
Review of the Bioassay of 5-Azacytidine*for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

November 28, 1977
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976 under the authority of the National
 

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

Clearinghouse is to advise on the National Cancer Institute's
 

bioassay program to identify and evaluate chemical carcinogens in the environment to which humans may be exposed.
 The members of the Clearinghouse have been drawn from
 

academia, industry, organized labor, public interest groups,
 

State health officials, and quasi-public health and research
 

organizations. Members have been selected on the basis of
 

their experience in carcinogenesis or related fields and,
 

collectively, provide expertise in organic chemistry, bio­ chemistry, biostatistics, toxicology, pathology, and
 

epidemiology. Representatives of various Governmental
 

agencies participate as ad hoc members. The Data Evaluation/
 

Risk Assessment Subgroup of the Clearinghouse is charged
 

with the responsibility of providing a peer review of NCI
 

bioassay reports on chemicals studied for carcinogenicity.
 In this context, below is the edited excerpt from the
 

minutes of the Subgroup's meeting at which 5-Azacytidine was
 

reviewed.
 

The primary reviewer commented that the severe toxicity
 

of the drug resulted in so many deaths that meaningful
 

statistical analyses could not be done on the treated rats
 

or male mice. In female mice, tumors of the hematopoietic
 

system were associated with treatment. Bone marrow atrophy
 

was a significant finding in the treated rats. The reviewer
 

agreed with the staff's conclusion that 5-Azacytidine induced
 

lymphomas and granulocytic leukemias in the treated female
 

mice. The secondary reviewer also agreed with the conclusion
 

given in the report. A discussion ensued as to the possible
 

mechanism by which 5-Azacytidine exerts its effect.
 

Despite the inadequacies of the study, a motion was
 

made that there was sufficient evidence to conclude that 5-
 Azacytidine induced hematopoietic neoplasms. The motion was
 

seconded and approved by all present except Mr. Garfinkel,
 who opposed it based on the small animal groups and high
 

early mortality.
 

85
 
Members present were:
 

Gerald N. Wogan (Chairman), Massachusetts Institute of
 

Technology
 

Lawrence Garfinkel, American Cancer Society
 

Henry C. Pitot, University of Wisconsin Medical Center
 

George Roush, Jr., Monsanto Company
 

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

Louise Strong, University of Texas Health Sciences Center
 

John H. Weisburger, American Health Foundation
 

*	 Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

<HJ S GOVERNMENT PRINTING OFFICE 1978 260-899/3017 1-3 86 

DHEW Publication No. (NIH) 78-842