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~4LAJ, MSC; LilIi ic 1). Witcher, SGT, USA; Don W. Korte, Jr.,

TIME COiVERED

ATE OF REPORT (Year, Month, Day)

1

-12, -!an81:n8J'uly

15. PAGE COUNT

1988

15

16, SJPPLrMENTARY NOTATION

17

ro~n'I CODES I

FIELD

I

fl~

PUo

~(Continue

SUB-GROUP

18. SUBJECT TERMS (Continue on reverse it necessary and identify by block number)

/;DNA damage,, Genet 'ic toxicology, Sister chromatid exchange, Nitroguanidine

on reverse of necessary and identy by block number)

potential of nitroguanidine-M.A-R--Code Ntiiber TP3i'to induce Sister Chromatid Ex(SCI:SC-,) was assessed using Chinese Hamister Ovary (010O) cells both with and without C~w'~nOIisetiinolic activation providcd by rat liver S-9). Cells were exposvd to test coma. t~±',cn:~atinsranging from 4 m%./ml to 10.01 mg/mI in cultures without exogenous metahah 2:ix'it nanid 3.9 mqg/mi to 0.01 Mg/ml inl Cultures with exogenous metabolic activai . NitlcOw'lan iiinc did not induce a statist ica lly sign ificant increase in SCI's in either the pCee or abs.ence of exoL'enoiis retabol ic ;'Ct ;v.-tion These results illtqt that fl1'J~inid~c as not an inducer of SCEs under the contditions of this su7

S20

CISTRISBL7ON AVAIL.ABILITY OF ABSTRACT UN CLA S.S1, FC,1N L I ri'T ED 1_- SAME AS RPT 22a. NAME 0; RESP-"' '% -)'~V'J

EU. Ii.

r t dIce, 1

DD Form 1473, JUN 86

21

ABSTRACT SECUR'TY CLAS~iICAIIN

DTIC USERS

-

122?b TELEPHONE (Include Area Code)

C_(.C415

50 1 - 3 60(1 Previous editions are obsolete

22c OFFICE SYMBOL

SGRD-111, SECUQ'TY CLASSIFICATION OF THIS PAGEN

ABSTRACT The potential of nitroguanidine (LAIR Code Number TP036A) to induce Sister Chromatid Exchanges (SCEs) was assessed using Chinese Hamster Ovary (CHO) cells both with

and without exogenous metabolic activation provided by rat liver S-9. Cells were exposed to test compound concentrations ranging from 4 mg/ml to 0.01 mg/ml in cultures

without exogenous metabolic activation and 3.9 mg/ml to 0.01 mg/ml in cultures with exogenous metabolic activation. Nitroquanidine did not induce a statistically significant increase in SCEs in either the presence or absence of exogenous metabolic activation. These results indicate that nitroguanidine was not an inducer of SCEs under the conditions of this study. Key Words:

DNA Damage, Genetic Toxicology, Sister Chromatid Exchange, Nitroguanidine

For 1NTTS GRA&I DTIC TAB

IAccession

jUna

nn

ouneed

E3 El

Distribution/

IAvailability

Ccdes Avail and/ or Dit Spocial

,-,

.

S

PREFACE

TYPE REIPORT:

Sist-er Chromatid Exchanqe

As:>'y

IP

Study Report

TESTING FACILITY:

US Army Medical Research and Development Command Letterman Army Institute of Research Presidio of San Francisco, CA 94129-6800 SPONSOR: US Army Medical Research and Development Command US Army Biomedical Research and Development Laboratory Frederick, MD 21701-5010 Gunda Reddy, PhD Project Officer:

PROJECT/WORK UNIT/APC:

85036

GLP STUDY NUMBER: STUDY DIRECTOR:

#3E162720A835/180/TLB0

MAJ Don W. Korte, Jr.,

PRINCIPAL INVESTIGATOR:

PhD, MSC

MAJ John W. Harbeil,

PhD, MSC

A copy of the final report, REPORT AND DATA MANAGEMENT: retired SOFs, study protocol, retired stability and purity data on the test compound, and an aliquot of the test compound will be retained in the LAIR Archives. TEST SUBSTANCE:

CAS # 556-88-7

Nitroguanidine

INCLUSIVE STUDY DATES:

15 Jul 85

- 14 Jar

86

OBJECTIVE: The objective of this study was to determine the potential of nitroguanidine (TP036A) to induce sli ter chromatid exchanges by usinq CHO cells in the presence and absence of exogenous metabolic act ivcatLI u

S ""

.

iii

-

*

~-

-

- - -

- -

-

-

ACKNOWLEDGMENTS

Joannue

Woncl

pc.ovidod research

assistance

dnrj-r.q thi-, Stu-d.

ivS

SIGNATURES

OF

PRINCIPAL

SCIENTISTS

AND

MANAGERS

We, the undersigned, declare that GLP study number 85036 was performed under our supervision, accordinq to the procedures described herein, and that this report is an accurate record of the results obtained.

DON W. KORTE, JR, MAJ, MS

-

Date

hD

Study Director

6

HN W. HARBELL, PhD / Date 1~,MS

Principal Investigator

/_ Dat-

LIILIf- D SGT, U-A

WITCHER,

i

CC.iKBA2 R. DAC

WHEELER,

PhD /

Date

Analytical Chemist

v

•N

DEPARTMENT OF THE ARMY LETTERMIAN ARMY INSTITUTE OF RESEARCH PRESIDIO OF SAN4 RANCISCO, CALIFORNIA 94129-6800

SGRD-ULZ-QA

17 June 1988

(70-in)

MEMORANDUM FOR RECORD SUBJECT:

GLP Compliance for GLP Study 85036

This is to certify that in relation to GLP Study I. 85036, the following inspections were made: -

29 March 1985 27 August 1985

0

Protocol Review Isolation and Fixation of Metaphase Cells

The institute report entitled "Sister Chromatid Exchange 2. Assay of Nitrogualidine in Chinese Hamster Ovary Cells," Toxicology Series 191, was audited on 25 May 1988.

CAROLYN M. LEWIS Chief, Quality Assurance

a

I

V,

TABLE

OF

CONTENTS

S

Abstract ........................................... Preface ................................................. iii

Acknowledgments ..........................................

iv

Signatures of Principal Scientists ........................ Report of the Quality Assurance Unit....................... vi Table of Contents ....................................... vii BODY .F

TIHE REPORT

INTRODUCTION ......................................... Objec:ive of the Study..............................2 MAT ER At,S AND>

4i'I'HODS ................................

..

Test Compound ................................... 2 Chemical Preparation ............................ 3 Positive Contros ................................................. 3 Cells

...........................................

3

Medium ........................................... 3 Metabolic Activation System ..................... 4 Assay Format ........................................ 4 Data Evaluation ................................. 7 Changes/Deviations .................................7 Stcrage of Raw Data anid Final Report ............. 8 RE SUL TS ..............................................

8

D)i S)C:U3SiON................................................

8

CONCLUS I ON.............................................

1"

REFER..........................................-

,

APPENDIX ............................................ 12 OFFICIAL DISTRIBUTION LIST ...............................

vii

15

7

Sister Chromatid Fxchange Assay of Chinese Hamster Ovary Cells--Harbell

Nitroguanidine et 31.

in

INTRODUCTION roguanidne,-a primary component of US Army triplebase rrpeilant cow Srocuced in a Govcr:rnent-owned ccntractor-operateu ammunziion pian. The US Army Biomedical Researcn and Development Laboratory 'R:A}, as part of its mission to evaluatethe environmental and heai:h hazards of military-uniqu- propellants genrated by US Army munitions manufacTuring facilities, nducted a ioview o- the nitracuanidine da abQs, nd identified siunificant oaps in zhe tox:icity data -he DivisiQn .ol___oi a IR, was tasked r-o-evelopia genetic and mammalian toxicity profile for nitroguanidine, related interimedaates/by-products of its manufacture, and its environmental deqradation p~r_~-.~. 'This study evaluated the clerio o>:iacc: .'rt], of nitroguanidine by using the Sist-er Chrqrdtid '-:chacge (SCE) As-:i% ..

1 xchanges between sister chromatids are detected by growi_ cells in the presence of bromodeoxyuridine (BrdU), Pecause DNA-repr-ication is semiconservativo, cfells grown-with BrdU would contain chromosomes with substituted chromatids s:-_ :.ne ro,nd of replication. The presence of BrdU allows for d rferential staining of chromatids with the fluorescence-plus-Giemsa (FPG) tech .ique. If DNA damage occurred, then the damage corrected by post-reolicative DNA repair processes would involve recombinational events (exchanges) between sister chromatids. The recombinational events are detected as a varied pattern of dark and light stainirg in the chromat id segment-s. The f reu('!,encies oC these SCEs aie analyzed since a direcl. correlation between the number of SCEs and the amount of DNA damage is assumed. Many compounds can be converted into mutagenic agents by enzymes associated with normal metabolism. To detect these promutagens, the SCE assay is performed both wzth and without metabolic activation.

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