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UNCLASSIFIEDSECURITY CLASSIFICATION OF THIS PAGE

Form ApprovedREPORT DOCUMENTATION PAGE IOMB No. 0704-0188

la REPORT SECURITY CLASSIFICATION 10 RESTRICTIVE MARK(INGS

2a SECURITY CLASSIFICATION AUTHORITY 3 DISTRIBUTION /AVALABILITY OF REPORTApproved for public release; distribution

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4 PERFORMING ORGANZATION REPORT NUMSER(S) S. -MONITORING ORGANIZATION REPORT NUMBER(S)

Institute Report No. 2736a. NAME OF -ERFQRMING ORGANIZATION 6b OFP!CE SYMBOL 7a. NAME OF MONITORING ORGANIZATION

Genctic Toxicology Branch I (if applicable) US5 Army Biomedical Research and DevelopmentPh L--i0l' Of TOXiCology SCRD-UL-TO-G Laboratory

6c. ADOR' ( City, Ste. and ZIP Code) 7b. ADDRESS(City. State, and ZIP Code)

Lcttorraan Arm), Institute of Research Ft. Detrickl'recs;d:,o of San Francisco, CA 94129-b800 Frederick, 1\11 21701-5010

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F rode 1i1ck , \l 1 50 ELEMENT NO NO INO. ACCESSION NO02720A 835 AB DA 303913,

11,. ITLF (i-c'Iuae S~.t lsiiainSister Chrom.Tatid 'Exchange As--ay of Nitro uannidine in Chinese Hamster Ovary CellsI 1

'PERSONAL AUTHOR(3) .'ohnj W. HarbellI , >LAJ, MSC; LilIi ic 1). Witcher, SGT, USA; Don W. Korte, Jr.,1- -NE1;7 PEPORT '13b TIME COiVERED 1 ATE OF REPORT (Year, Month, Day) 15. PAGE COUNT

nst t I;te oROMja -12, -!an 81:n8J'uly 1988 1516, SJPPLrMENTARY NOTATION

17 ro~n'I CODES 18. SUBJECT TERMS (Continue on reverse it necessary and identify by block number)FIELD I PUo SUB-GROUP /;DNA damage,, Genet 'ic toxicology, Sister chromatid exchange,

Nitroguanidine

I fl~ ~(Continue 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 withoutC~w'~nOIisetiinolic activation providcd by rat liver S-9). Cells were exposvd to test com-

a. t~±',cn:~atinsranging from 4 m%./ml to 10.01 mg/mI in cultures without exogenous meta-hah 2:ix'it nanid 3.9 mqg/mi to 0.01 Mg/ml inl Cultures with exogenous metabolic activa-i . 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 T hese results illtqt thatfl1'J~inid~ c as not an inducer of SCEs under the contditions of this su7

S20 CISTRISBL7ON AVAIL.ABILITY OF ABSTRACT 21 ABSTRACT SECUR'TY CLAS~iICAIIN

UN CLA S.S1, FC,1N L I ri'T E D 1_- SAME AS RPT DTIC USERS -

22a. NAME 0; RESP-"' '% -)'~V'J 122?b TELEPHONE (Include Area Code) 22c OFFICE SYMBOL

EU. t Ii. r 1 dIce, C_(.C415 50 1 - 3 60(1 SGRD-111,

DD Form 1473, JUN 86 Previous editions are obsolete SECUQ'TY CLASSIFICATION OF THIS PAGEN

ABSTRACT

The potential of nitroguanidine (LAIR Code NumberTP036A) to induce Sister Chromatid Exchanges (SCEs) wasassessed using Chinese Hamster Ovary (CHO) cells both withand without exogenous metabolic activation provided by ratliver S-9. Cells were exposed to test compoundconcentrations ranging from 4 mg/ml to 0.01 mg/ml in cultureswithout exogenous metabolic activation and 3.9 mg/ml to 0.01mg/ml in cultures with exogenous metabolic activation.Nitroquanidine did not induce a statistically significantincrease in SCEs in either the presence or absence ofexogenous metabolic activation. These results indicate thatnitroguanidine was not an inducer of SCEs under theconditions of this study.

Key Words: DNA Damage, Genetic Toxicology, SisterChromatid Exchange, Nitroguanidine

IAccession For

1NTTS GRA&IDTIC TAB E3jUnan n ouneed El

Distribution/ S

IAvailability CcdesAvail and/ or

Dit Spocial

,-, .

PREFACE

TYPE REIPORT: Sist-er Chromatid Exchanqe As:>'y IP Study Report

TESTING FACILITY:US Army Medical Research and Development CommandLetterman Army Institute of ResearchPresidio of San Francisco, CA 94129-6800

SPONSOR:US Army Medical Research and Development CommandUS Army Biomedical Research and Development LaboratoryFrederick, MD 21701-5010Project Officer: Gunda Reddy, PhD

PROJECT/WORK UNIT/APC: #3E162720A835/180/TLB0

GLP STUDY NUMBER: 85036

STUDY DIRECTOR: MAJ Don W. Korte, Jr., PhD, MSC

PRINCIPAL INVESTIGATOR: MAJ John W. Harbeil, PhD, MSC

REPORT AND DATA MANAGEMENT: A copy of the final report,retired SOFs, study protocol,retired stability and purity dataon the test compound, and analiquot of the test compound willbe retained in the LAIR Archives.

TEST SUBSTANCE: Nitroguanidine CAS # 556-88-7

INCLUSIVE STUDY DATES: 15 Jul 85 - 14 Jar 86

OBJECTIVE: The objective of this study was to determine thepotential of nitroguanidine (TP036A) to inducesli ter chromatid exchanges by usinq CHO cells inthe presence and absence of exogenous metabolicact 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 theprocedures described herein, and that this report is anaccurate record of the results obtained.

DON W. KORTE, JR, hD DateMAJ, MSStudy Director 6

HN W. HARBELL, PhD / Date1~,MS

Principal Investigator

LIILIf- D WITCHER, i /_ Dat-SGT, U-A

CC.iKBA2 R. WHEELER, PhD / DateDACAnalytical Chemist

v •N

DEPARTMENT OF THE ARMYLETTERMIAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN4 RANCISCO, CALIFORNIA 94129-6800

SGRD-ULZ-QA (70-in) 17 June 1988

MEMORANDUM FOR RECORD

SUBJECT: GLP Compliance for GLP Study 85036

I. This is to certify that in relation to GLP Study85036, the following inspections were made:

29 March 1985 - Protocol Review 027 August 1985 - Isolation and Fixation of

Metaphase Cells

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

CAROLYN M. LEWISChief, 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 ................................... 2Chemical Preparation ............................ 3Positive Contros ................................................. 3Cells ........................................... 3Medium ........................................... 3Metabolic Activation System ..................... 4Assay Format ........................................ 4Data Evaluation ................................. 7Changes/Deviations ................................. 7Stcrage 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 ............................... 15

7vii

Sister Chromatid Fxchange Assay of Nitroguanidine inChinese Hamster Ovary Cells--Harbell et 31.

INTRODUCTION

- roguanidne,-a primary component of US Army triple-base r rpeilant cow Srocuced in a Govcr:rnent-ownedccntractor-operateu ammunziion pian. The US Army BiomedicalResearcn and Development Laboratory 'R:A}, as part of itsmission to evaluatethe environmental and heai:h hazards ofmilitary-uniqu- propellants genrated by US Army munitionsmanufacTuring facilities, nducted a ioview o- thenitracuanidine da abQs, nd identified siunificant oaps inzhe tox:icity data -he DivisiQn .ol___oi a IR, wastasked r-o-evelopia genetic and mammalian toxicityprofile for nitroguanidine, related interimedaates/by-productsof its manufacture, and its environmental deqradationp~r_~-.~. 'This study evaluated the clerio o>:iacc: .'rt], ofnitroguanidine by using the Sist-er Chrqrdtid '-:chacge (SCE)As-:i%

.. 1 xchanges between sister chromatids are detected bygrowi_ cells in the presence of bromodeoxyuridine (BrdU),Pecause DNA-repr-ication is semiconservativo, cfells grown-withBrdU would contain chromosomes with substituted chromatidss:-_ :.ne ro,nd of replication. The presence of BrdU allowsfor d rferential staining of chromatids with thefluorescence-plus-Giemsa (FPG) tech .ique. If DNA damageoccurred, then the damage corrected by post-reolicative DNArepair processes would involve recombinational events(exchanges) between sister chromatids. The recombinationalevents are detected as a varied pattern of dark and lightstainirg in the chromat id segment-s. The f reu('!,encies oC theseSCEs aie analyzed since a direcl. correlation between thenumber of SCEs and the amount of DNA damage is assumed.

Many compounds can be converted into mutagenic agents byenzymes associated with normal metabolism. To detect thesepromutagens, the SCE assay is performed both wzth and withoutmetabolic activation.

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MATERIALS ND METHOD

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Nitroguanidine was dissolved direct ly in the cellculture medium (GEM 1717), withour fetal bovine se urn (FBS),at a concentration of 4.4 mg/ml and was ilter-sterilized.This concentration is clcse to saturation for ni'troguanidinein an aqueous medium (1). All test article dosing solutionswere prepared from this stock solution. For the nonactivated%cultures, FBS was added to the culture meiu. to achieve afinal concentration of 10%. Dilutions were prepared usingGEM 1717 4ith 10% FBS. For cultures with metabolicao....vation, two batches of cofactor mixture (see below) werepre aed. One coor.. ined nitroganidine while the otner didnot , 2nd nit r uani dine dilu._ ii cns were nreparo:iby mixina thetwos OQ< Aliions.

The p.:oi!ive for cultures without metabolicactuovati-on vas eth s fonate I dilit insterile distill-, water and added to the culture to achieve afinal concentration of 0.320 ml/.mi. The positive control foractivated cultures was cycioposnhamide which was alsodissolved in sterile distilled water and added to the cultureto achieve a final concentration of 1.33 pg/mi.

Cells

Chinse Ham,-,ter Ovary (CHO0 Cells were obtained fromDr. Sheldon Wolff, University o California, San Francisco.Tht CHO cells were qrown in an atmospher.e ot 5% C02 at 37±1Cin GEM 1717 with 10% FBS. The cell cyclc time ,jas 10-12hours Twenty-four hours before treatment, 6 x 105 cells wereseeded into each 75 cm 3 flask, which contained 15 ml of freshmedium, to ensure that the cells were in the logarithmic phaseof growth when the test corrpou:,. was added. Cells were testedand shown to be negative for mycoplasma (3).

yedium

EEPES buffered (10 mM3) .- .'!7, which was usedthrouqhout, was prepared frcm 64PME 160 (Gibc r ® Laboratories,Grand Island, NY) according to OP-STX-l2 (2). Except when Cthe medium was used to prepare the act ivatio; cofactormixture (see below), it contained 10% (v/v) Bb-S when incontact with the cells.

Y~atbe" : Cr: -fl--....

Z~~A ~2 ~~:va* io Syst em

mr-Tr c;c activation system was composed of Aroclor-9)00 x a supernatant fraction (S-9) (Litton

s MD) and in NADPH-rgenetatinq sysec-mh" -olaictor mixtiure. The cofactor mixtureNA'P (2 mo/mi) and a reduced carbon sourc,

1 ', 4.- ' s/inl). The cofactors were dis:c .

'7! , w ut serum, and filter-sterilized. Thisw, -.r fsuop]emented to make the complete metabol ic

" , item which contaired 2% FBS and 10% S-9. The

i-.-i ,in system waQ prepared immediat<.ly befoL-e

-'" - O -¢ _

" o.i re used was a modification of the proceduresb.; Ferry and Evans (4) and Stetka and Wolff (5). -Gu . a. - ] t as the positive and neqativew. ...,lu:e, both with and without metabolic

-.t.nrd e posiie periods (2). Ln testf:- Tr t. :)] . t . ation, the test compound ,*as

.. rh J.]7i with i@', FBS (15 ml per f!3sk)L. wI R - d. After aniition of the fresh medium,

', LrdK,, rhe 1eK s were .rown in th - d irk a, "•'ours. Frasks were wrapoed in foil to assure

S . 7-en coichicine was 'ded to each flask

.e cultures weto incubated for an additional. -re the cells were harvested.

Tn tests performed with metabolic activation, ther:-- ' .'riJ_u: ,ris removed, and 15 ml of activation mixture,

test artic"., or positive control, wascultiures were exposed to the test compound- t:-:ture for 2 hours. This shorter excosur-"

- the activation mixture was used to. . -n' t cyt.c--. c effects induced by the

"' Aft er tne 2--hour exposure period, the

. : . .test compound and the metabolic:: a,;pi. ated, the cells were washed three.a;d fresh, complete GEM 171'7 containing

t rIUl was..; added. The cultures wore "ncuot ed

in 1. hours. Colchicine was then added at a

.. )f 0.-1 kiq/m, as described above an'i the

'pL

I~ U -. V U - S - S - - - - - - - - - - - - ,

Harbe 1 1 a 1--'

cultures were incubated for _:i ,itional 7. hours b~forethe cells were harvested.

Harvest:

After 2.5 hr in colchicice, all of the cells in eachculture were harvested. At harvest, the medium, whichcontained dividing cells, was removed from the culture flasks

and saved. Each flask was rinsed once with 10 ml of Ca t+Mg+ - Free Hank's Balanced Salt Solution O1B52), and this washwas combined with the culture medium in a 50-ml tube. Ten mlof Cat+ Mg + + Free HBSS (with 0.02% EDTA) were added to therinsed flasks, which were then shaken for approximately 30min, until almost all the remaining cells were detached.While the flasks were shaking, the contents of each 50-mltube were spun down (1000 r 'm for 5 mmn), and all but 3 ml ofthe supernatant above each cell pellet was discarded. Eachcell pellet was resuspended ir. is 3 ml of supernatant, andthese cell suspensions iere transferre tc preiabelied 15-mlcentrfuge tubes. Each tube was labelled with the protocolnumber, date of the experiment, and the cu Loure (dose)number. The cell suspension from each flask was added to theappropriate tube (thus combining all the ce] s from a givenflask), and the cells were centrifuged (1000 rpm for 5 min).Most of the supernatant was aspirated, and each cell pelletwas gently resuspended in th e remaininq supernatant(approximately 0.2 t - 0.3 ml,, tube). Ten m! of hypotonic KCI(0.075 M) were added slowly (eve: about 2 min) to each tube,and the tubes were held at room temperature for approximately2 min. The tubes were again centrifu.ged (1000 rpm for 5min), the supernatant aspirated, and the cell pellet gentlyresuspended in the approximately 0.3 ml of remainingsupernatant. Freshly prepared Carnoy's fixative(methanol:acetic acid 3:1) wa- th-n added slowly to eachtube. The first 2 ml of fic:ative were added while gentlymixing the resuspended cells. After a total of 8 ml had beenadded, the cells were allowed to fix for at least 10 min.The cells were again cent:ifuged into a pellet and thefixation procedure was repeated. The cells were allowed tofix overnight. In the morning, the cells were centrifugedand the fixation procedure was; repeated. Pefore preparingthe slides, the cells were centrifuged, all but about 0.5 ml(depending on the cell pellet size) ot supernatant wasremoved, and the pellet was resuspended. Prelabelled cleanslides were wetted in cold distilled water. Up to 4 drops ofcell suspension were dropped onto each slide.

* wt, wro sA i ied by, a urc t-J1fi oU.' 1PG t r chnpii 5ol H1ec! t_ M3 ( Lg/ml in wfst r) were

!C(" ide, ind -vers]. ip wa:;, app i c'J . After-2 ~ ~ P io -f''1%r'n, the covers Iis v~ere removed

1'-' 4 .ed several 'Limes in *:.ao water andul JT "I o r everl tl d Qp. of MeIlvaine's burter (pv; S/

W ~ ~ no h s ie, -ind a new covers Ii n wa-~a~id

SI h P'P A bT (5) .Tlh cvr~ p workS-~ ~ilI Ii..wert rjnised !-everai t-imes in ip

Wa'Otii h teywcerc stii nod in dilute Gi-emsa stainI'' 1 1i~'' a 1)1 .i) , irli f'd, I-nci air dried1; then It

I n q roajz', t.r- JplIcI ate sl1iders we re prepared arndho iinimvor otf i s - econd-, anid UVid--di vision

!,i o S W r( :-co rod (whP re p)ss ibl e, 10 0 mot apha ses wo Let xi) N' :n! 11 y, 10 f second-division met aphases, conta i ni iq

/ ~ c'~ f ~ wore suor f for SCEs in each preparat i .i- ~ ~ 1:ere wer e ls;than 40 second-divi sionf

-(.I >' or- !lie i,,nmher of SC~s per cell wa1si-o1v7 cIi2 th~it ti wetc- I Is needed t o 1be cun nt ed t oV

U t t al flr iranc. I n t he f irst case, a I1>~~~ .'T -ii;;;(i h('romrosomn-e.,,) woi o sco-r-eu on ,;i I

A I i 1d t i t co CI I cut ed f r :m coded slIi de s The da-t I21 '' nV s Ve umor(codJed) , (quaI i t-y -)I I hi :; 1 d

(is ' 1 ) t hoe nuimiber &f f i t- seccid' -, andit I iV-.':: Iii r.; r- ech nti-t apia se ;co red, the,

- ii .2 *i ri -ai. jcn t the number ot cliror'osoiii'-'s,1.0 ni i-tn Ii 1.;, thli !--ier s501 t * iqs, art]id i 0p(to1np

I c 11 :.t ,I ph ias I I- s c I i ! i V r was pholtCqraplied t oid. I *'': it 1( it i f i iit i I-nT o f tI II-it moit Ap'h.i du r i nq

* 1 01 *I IO J T(lftit 2 iT I Af t e r ;('or i T2i o) f the,tl-i I I. ' lt i, tIlk i ides wore 1c/'( andi LillL

.1 ~ ~ ~ ~ ~ ~ -f f~rnt:,h -uc-u T ' I r IA_ ind socj-di.vi S- itT)mWr 21 i' ' ild 2t41.:: per (-,I w(-rr' mtched Ia t) ra ei

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liarbell et al--7

Stati stical Ariys s:

Stati st ical evaLuat: ion of Ih!t :;(F dat a w .1; a two-;'. r!

process involving the use of a one-way ailysis o: varianl,_to compare variance within treatments (du, to .<coringdifferences and sample difference) with variance amongtreatments (the effect of test compound) followed by aStucaent-Newman-Keuls test to indicate which groups(treatments) were different. Thius, the ANOVA was used toindica-e a difference among grou-ps aoid the Student-Newman-

Keuls test was used to indicate which specific dose or groupof doses was different from each other or controls. Thepositive and negative controls were compared by using the t-test. The p _ 0.05 confidence limit, was used for the t-test,ANOVA, and Student-Newman-Keuls test.

Data Evaluation

Cilteria for a Valid Test:

A valid SCE assay is one in which (]) tesrtinci wasperformed to the maximum concentration expectod to yieldsufficient second-division metaphases for analysis, thelimits of solubility, or the limits of compound availability(a maximum of 5000 .g/ml or 0.5% for liquids), and (2) thepositive control induced a statistically significant increasein the SCE frequency.

Interpretation of Results:

Positive: A test compound will be considered to haveelicited a positive response in the SCE assay if it induces astatistically significant (p < 0.05) increase in the SCEfrequencies compared to the negat. ve control rate (by theStudent-Newman-Keuls test) and the SCE frequency exhibited acorrelated dose response.

Negative: A test compound will be considered to haveelicited a negative response in the SCE assay if the criteria(statistical significance or dose response) for a positiveresponse are not met.

Changes/Deviations

The actual conduct of the study differed slightly fromthe SOP during the harvesting phase. Instead of shaking eachrinsed flask for 10 min with HBSS, the flasks were shaken for

h. - WV ," VXTYV y'1V V.' .- VW

Harbell ec al-8

310 min. This Provided sufficient ztime for the cells stiliacohering to th-e flask to detac-

Storaar o-f aw Data and Final Leport_

A copy of rthe final report, study protocolr, za-v data,retired SOPs, ar-i an dliquot cit the teFst compuund will bere tained in the: LAIR Archives.

RESULTS

Nic~rogruanidine was tested over a concentration ranoe of4.0 to 0.01 mg/mil without exogenous metabolic activation and3.q to C.01 mg/mi with activation. Spocific doses are aiyerin. TIable I. Nitroguanidine dic induce some toxicity. At theh-ichest dose without metabolic activation (4.0 mg/mI), 91', ofthe retaphase7s isolated were found to be from first-di-lvi .i-:cells, and the total yield of metaphases was too small forSCE sccring. The 2 mg/ml dose f.nduced some toxicity duringthe 24-hour exposure but failed to induce a significant rise

in C~ Nine of the low:c-r doseis of nitroguanidine .nducedr n i. yor rroa!.-~s in 51:.The assay with mnetabol iciv it ion 11,;(d 2-hi u teXP0s;'~ Period. In this Case,

Nrthle 1itgtw(:;t_ dose (3. 9 ru/iml ) nor the lower onesinduceo any apeibetoxicity or increase in SCEs.

Botn positive controls induced statistically significantIncrasesinSOs

DI SCU SS 10

Nitroguanidine was tested for its ability to induce SCEsin CR0 cells both with and without exogenous metabolicactivation. Even at the hiqhest doses tested, nitroguanidl-ledid not induue a statirti call)y s:ie nificant increase in SCEsE

wasLneic n apa ~rt. ce~ ~~~ndnLincrease,( in SCF-fsTv~ : i t. dsel t e'st~ed wasv- d; c'ct ed by the limits of

,o1utr> li (Y. Pot)i posit-ive con!.s induced siqnificantincre-ases ir tnhe S Fhs. Thus-, all the conditions for a valid,ne-sat- ye assay were Met..

Ni tr1om7iani~dino h~i i'u e-01 relpo-rted to cause significantLchoooe1damage in r, ,,4nese h imster fibroblasts (6)

Is.hi.date and Oda:chima use(d a do.;e of 4 mg/ml for 24 hours andf osnd 2,,% of th1-e metaphases t~o niave chromosomal aberrations,pr ircipa-lly citps, brea'Ks, anid t.:ans1locatio_-ns. Our data from

It~~~~i V.0 r

Harbell et al--9

Table I: NITROGUANIDINE (NG) S [STER CIIROMAT [D EXCH1,,NGE-

DOSES %FDa nb SCE ±ISD COMMENT

WITHOUT S-9 (24-hour exposure)

Neg Control 2 40 6.58 ±2.86

EMS 0.32 mg/ml 12 6 61.00 ±9.74 p<0.0001

NG 4.0 mq/ml 91 40 metdphases not scorableNG 2.0 mg/ml 14 40 7.28 ±2.53 NSNG 1.0 mg/m] 3 40 7.30 ±2.66 NsNG 0.5 mg/ml 2 40 7.20 ±3.03 NSNG 0.1 mg/ml 1. 40 6.78 ±2.83 NSNG 0.05 mq/ml 7 40 6.25 ±2.26 NSNC 0.01 mg/ml 4 40 7.25 +2.65 NS

WITH S-9 (2-hour exposure)

Neg Control 1 39 9.31 ±2.57

Cyciophosphamide1.33 gg/ml 9 ?0 34.5 ±11.75 p<0.0001

NG 3.9 mg/ml 1 39 9.03 +3.27 NSNG 2.0 mg/ml 1 19 6.53 ±2.57 NSNG 1.0 mg/ml 2 28 8.00 ±3.10 NSNG 0.5 mg/ml 1 40 7.85 ±3.31 NSNG 0.1 mg/ml 4 20 8.60 ±3.22 NSNG 0.05 mg/ml 0 40 8.65 ±3.32 NSNG 0.01 mg/ml 4 40 7.00 ±2.66 NS

a %FD = percentage of metaphases in first divisionb n = number of metaphases scored for SCEs

Negative controls and nitroguanidine groups were comparedusing ANOVA.Negative controls and positive controls were compared usingthe t-test.Abbreviations: EMS = ethyl met hanesulfonate, NG :nitroguanidine, SCE = sister chromatid exchange, SD =

standard deviation, NS = not significant. (P<0.05).

. , - . .. -, - -, . 'I

Harbell et al--10

the SCE assay shcwed NG to be very toxic at that cose over a24-hour ex' osure. Most of the mnetaphases detected wi, fromfirst-division cells, and even those that showed somedifferential staining had not gone through two completereplication cycles in the BrdU-containinq medium. Rath..,these cells appeared to have been in S phase when thenitrocuanidine and BrdU were added and went on to completethis cycle. After the second S phase, these cells contaireda mixture of thymidine, thymidine and BrdU, and Br-iC-containing strands so that they were not scorable for SCEs.Fxamination of the metaphases from this dose group did notreveal the chromosomal aberrations reported previously. Thusour data are at odds with those of Ishidate and Odashira (6)though the test systems were by no means identical (e.g.different cell lines, presence of BrdU, and differentchromosome preparation). In addition, no specific chemicfalpurity data were provided (6).

Nitroguanidine was also found to be negative in the AmesSalmonella/Microsome assay and Mouse Lymphoma TK -/- ForwardMutation assay (7). In these studies, nitroguanidine ,astested to the limits of solubility for each assay system bothwith and without exogenous metabolic activation.

CONCLUSION

Nitroguanidine was not a significant inducer of SCEsunder the coodit ions of this assay either with or withoute ogenous metabolic activation.

v

REFERENCES

1. Kenyon KF. A data base assessment cf environrental fateaspects of nitroguanidine. TechnicaI Report 8214(ADA125591) . Ft. Detrick, Freoerick, Maryland: US Arm-Medical Bioengineering Research and DevelopmentLaboratory, December 1982.

2. The Sister Chromatid Exchange Assay. Letterman ArmyInstitute of Research Standard OCerating Procedure 0-STX-72. Presidio of San Francisco, California:Letterman Army institute of ?esfarch, 1985.

3. Detection of mycoplasma in cell cultures. Letterman2'.rmy Institute of Research F, an¢4ard Oper t.rq Procedurt,OP-STX-92. Presidio of San Fran.:sc', California:Letterman Army Institute of Research, 1985.

4. Perry P, Evans HJ. Cytological detection of mutagen-carcinogen exposure. Sister Chromatid Exchange.Nature 1975; 258: 121-125.

5. Stetka DG, Wolff S. Sister chromatid exchanges as anassay for genetic damage induced. Mutacens/Carcinogens.Part II. In vitro test for compounds requiringmetabolic activation. Mutat Res 1976; 41: 343-350.

6. Ishidate M, Odashima S. Cl.romosome tests with 134compounds on Chinese hamster cells in vitro--a screeningfor chemical carcinogens. M.tat Res 1977; 48: 337-354.

7. Harbell JW, Witcher LD, Sebastian SE, Korte DW. Studieson the mutagenic potential of nitrocuanidine andnitrosoguanidine. 1987 3A.NAF Safety and EnvironmentalProtection Subcommittee Meeting. Laurel, Maryland:Chemical Propulsion Information Acency, 1987; CPIAPublication No. 466; pp. 357-3167.

Vc 0

[i, t t i I Ot I l-- I

Appendix: CHEMICAL DATA

Chemical name: 'i tIoqnIII dine (NC)

Other listed naries: Guanidine, Nitro; alpha-Nitrogi:or'djne;beta-Nitroguanidine

Chemical Abstracts Service Registry No.: 556-89- "

LAIR Co-de: PC36A

Structural forrmula:

H2N'C=N NO 2H2 N -

Molecular formula: C 14N402

Molecular weight : 104. 1

pH range of dosing suspensions: 6.7 - 7.4(l)

Physical state: White Powder

Melting point: 2320(2)

S -,Crce: Hercules ,erospace DivisionSunflower Ammlnition PlantDeSoto, Kansas

Lot No. SOW84KlOA001

Purity: 99.2% (data sheet attached)

1 . Wheeler CR. ;i rocel lofosc'-Nit rog]anid- ne ProtectsLaboratory Notebook p-12- p . P sidio ofFanoco, CA : rt t erm in A' my, I u7t i t itn ( -f Res,earchc

2. Feedorotf BT, 0hffield CE. Encyclopedia of e>.plosivosand related items. Vol V. D)over, New Jersey: PicatinnyArsenal 1975: G154.

larbel] et j - - 1

Appendix (cont.) : CHEMICAL DATA

Analytical data: The majo otpes 1n the infrafr!cspectrum ot trie crmpound weroobserved a! 3450, 2 {96, 342, 3278,3201, 16f, I634, 1525, ], 1U,, 1314,

1151, 1045, 782 cm- t( 3 ) The spec:lmobtained fc. the test compound in ourlaboratory was 'cint-ical to theSadtler standard soectrum for

nitroguanida n,( 4 ) HPLC showed on.lyone peak (reenti.on time 4.9 min)The condit.ons employed were as follows:column, Brownlee RP-18 (4.6 x 250 7-m);solvent 10% methanol/90% water, f!cw rate- ,0.7 mi/min; ov,en tempe.,at:re, 5COC;monitoring wavelength, 265c o nm.

1

Wheeler CR. Nitrocellulose-Nitroguranidirne Projects.

Laboratory Notebook #85-12-022, p. 22-23. Presidio cfSan Francisco, CA: Letterman Army Instiitute ofResearch.

4. Sadtler Research Laboratory, Inc. Sadtier standardspectra. Philadelphia: The Sadtler ResearchLaboratory, Inc., 1962: Infra-red spectrogram #21421.

5. Wheeler CR. Nitrocellulcse-NitrcQuanidi'ne Projects.Laboratory Notebook #85-12-022, pp. 24-25. Presidio ofSan Francisco, CA: Letterman A:-my institute ofResearch.

I

I

HarbelI et al--14

Appendix (cont.) CHEMICAL DATA

DESC'.'PTION SHEET FOR EXPLOSIVES, CHEMICALS, ETC IEXE.4PI.IPI- 7.2o

£ssaPp-:0~O9IAR 335 - 15

US m mnamentMni Sunf lower Ar-m.' Anr.unj t ic, Flant CA

-,d Ch i Coinand DeSoto. Kansas 5b018 -T RAttn: DSSMC-OAD ;taundrRo k .1 d ILL. 61299 Ni oguar r

MANUFCCTU5ER 2.. AHiercules Aerospace Division, Hercules Inc. ,AA3~~7-C-) I C1. 4l5

)N84K010IOA001 "I I"R .'"111 paul;

RequireTment Aayi

Pronertv n . M'

Purity, Z Q9.0 99.20Ash Content, 0.30 0.i2pir4 Value 4.5 7.0 6.0Acidity (as 112S04), Z 0.06 0.0Total Volatiles. Z 0.-5 0.16ISulfates (as 143504), Z 0.20 0.17Impurities, 1120 Insoluble, 0-20 0.02Particle Size. microns 3.4 6.0 3.5Color Wh i it e

ursistency Crystalline. Free FlA.,ang

*Combined averages of sampling taken in iaccordaince with I--JA.9 ia. 4.-.3-.

I)Packa~zing: Level C Fiber druoss per qpecffi, iton POT 2lCt'02) This lot was manufactur-rd 5 1) 'obpr II134 ari Is submitted as iirst Article In coIU)! lance

w.i th Parngrn-ph 4.3 of l.Nhih.3) Cuanidine Nitrate supplied by ,K,;-Amer icain 'csch was used In man,;,.turo if this Int .

$[_____________________ 1_____ 14C -Cp.FC IO

_ _ _ _ _ _00.1_ _ _ .. t 1S. -- ' ,I,

Herc les Aer spa e Di irin Ro~jE [ T - ISCEAIF~ D TUC 'I)ccpf T

$T, (E 8

Harbell et al--15

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V W -bd m I" ItAl.