Document dVLDED4q5695vq0Oa8Z6QrLQ

REPORT EVALUATION OF THE ABILITY OF T5869 TO INDUCE CHROMOSOME ABERRATIONS IN CULTURED PERIPHERAL HUNAN LYWPHOCYTES (WITH INDEPENDENT REPEAT) HNOOTTOOXX SPurbosjteacntce11358914936 - page 10f 26 - RECEIVED HOV a-9ng1994 TOACOLOLY 005605 T5869 - NOTOX Project 11593 STATEMENT OF GLP COMPLIANCE -- cr ----------------------imme NOTOX B.Y.., *s-Hertogenbosch, The Netherlands Trheecesnttudeyditdieosncriofb:ed in this report was conducted fn compliance with the most The OECD Principles of Good Laboratory Practice Which are essentially in conforaity with: The United States Food and Drug Adafnistration. Title 21 Code of Federal Regulations Part 58. TFehdeerUanlitReedguSltaattieosnsEnvPiarrotns1e60n.tal Protection Agency (FIFRA). Title 40 Cade of TFehdeerUanliteRedguSltaattieosnsEnvPiarrotna7e52n.tal Protection Agency (TSCA). Title 40 Code of With the following exception: Stability of the test substance in the vehicle vas unknown. Study Director Ing. E.J. van de Vaart /---- vate! osforfizg ~ page 2 - 005606 T5869 - QUALITY. _-- ASSURRNCE STATEMENT NOTOK 8.Y.., *s-Hertogenbosch, The Netherlands. NOTOX Project 115543 Sstpuedcyifipcropcreodcueressseswerweeresubaljseoctitnosppeecrteidodiatc pienrsipoedcitcionisntearnvdalgse.neral non study Tahnidsrreespuolrtts eacscuraautdeiltyedrbeyfletchte NthOeTOXrawQuadaltiat.y Assurance Unit and the methods DATES OFAuQDAUITSINSPECTIONS! JFaenburaurayry241,6, 1199949 MFaerbcrhuar23y, 231,8941994 Apri 19) 1594 REPORTING DATES JFaenburauraryy 241,4, 19199944 MFaerbcrhua2r4y, 231,9941994 Apri 19, 1994 Quality Assurance Manager .3. Mitchel B.Sc. Date: 1-5-Qu. - page 3 - 005607 7-589 - NOTOX Project 115943: -- REPO-- RT A-- PPRO-- VAL ------------------------------ STUDY DIRECTOR: Ing. .3. van de Vaart J vate: og/slogs MANAGEMENT: Tore.chnIiCc.al EDniirnegcator aq -- | nate: oblos hgglu - page 4 - 05608 T-5869 - NOTOX Project 11594-3 PREFACE _-- spansor Study Monitor Testing Facility Study Director Technical Coordinator Study Plan 3CanaBdealsgtirunaat- 1C1hemical EBC 8Be-l2g0i7u0mZWLINDRECHT Mr. RH. Cox NHOaTnObXakeBn.Yw.etering 3 T5h2e31NeDtDhe*rsl-aHnedrstogenbosch Ing. E.0. van de Waart A.M.C. Bertens SCotmpalertetd:: MJaarncuhar~y 2360,, 11999944 TEST SUBSTANCE Identification Description Batch Purity Specific Gravity Itnessttruscutbisotnasnceforstorage coSntdabiitliiotnys under storage Expiry date 4Sthaobulres fionr at least vehicle T-5869 Crean solid 2408 95% 15 At room temperature in the dark stable January 01, 1996 Dimethylsulphoxide: not indicated VEHICLE Tqhuealtietsyt(sMeurbcskt)a.nceTewsatssudbisstsaonlcveedcoinncednitmreatthiyolnssulwpehroexidperepofaresdpecdtirreocstcloypicprior to use. Twhiev)f.inal concentration of the solvent in the culture medium amounted to 0.9 % - page 5 - 05609 T-5869 - NOTOX Project 115943 GUIDELINES _-- ee Tguhiedesltiundeys:procedures described in this report were based on the following - OGurigdaenliisnaetsionforforTesEtcionngomiocf CChoe-moipcearlast,ionGuiadnedliDneevelnoo.pme47n3t: ("OEGCeDn)e,ticOECD T19o8x3i)c.ology: In Vitro Mammalian Cytogenetic Test*, (adopted May 26, - EEEuCropDeiarnectEicvoenom6i7c/54C8o/mEmEuCn,ityPar(tEECB:),MeDtihroedcstivfeor92t/h6e9/DEeEtCe.rmAinnnaetixonV ooff the TCoyxtiocgietnye;tic8.1T0e:st*.OthEeErC EPfufbelcitcsa-tMiuotnagneon.icLi3t8y3: (IandoVpitterodDWeacenmmbaelri,an1992). ARCHIVING NprOoTtOoXcoBl.,. rweiplolrt,arctehsitvearttheiclfeollroewfienrgencdeatasamfpolre,atallleasstpec1i0meyneasrsa:nd raw data. OBJECTIVE Purpose of the study Ttoheionbdujceectistvreucotfurtahlischsrtoumdoysowmaes atobeerrvaatliuoantse itnhecutletsutresdubhsutsaanncelyfaorphoictysteasb,ility either in the presence or absence of a metabolic system (S9-mix). - page 6 - 605610 T-5869 NOTOX Project 11594-3 Justification and rationale of the test system Sitnidmiucaltaotresd ocfulctluarsetdogheunmiacn lacytmipvhiotcyytoefs waerberoaudsedrabngeecaousfechtehmeiycaalreclsaesnsseist.iveIn cchoemnbiicnaaltionnutawgietnhs,a mia.em.maltihoasnemerteaqbuoilriinzgingmetsaybsotleimc(ts9r-amnisxf)ormaaltsiooninidnitreoctreactive Fionltleorwniendgiatterse,atmceonutl,d bceelltedsitveidsifoorn wcalsastaorgreenstiecdefinfectthse minetavipthraos.e stage of the ccehlalngecsyclseuchbyaasdbdrietaikosn,ofgaptsh,e smipniuntdelse,podiiscoenntrcioclschiacnidnee.xchStarnugcetufriaglurecshrwoenroesone erexsaunlitnsedvmeriecrcoosmcpoapriecdallwyithintchuosleturoefsthtereactonetdrowlith(vtehheictlees-ttresautbesdt)ancceultaunrdest.he mCihtroosmioss.omeTheabearprpaetairoannscewoerfethgeenefriarsltlypoesvta-lturateeadtmeinntthmeitofsiirsstcpooulsdt-tbreeatnent chaornvseisdteerdablayt d24elahyeadn,d 4d8uehtaoftteorxicbegiinnsnulitngofofthtereacetlmlesn.t tToherceofvoerre,thecelilnstervvearle Aintwehstichartmiacxliemunwhiacbherriantdiuocnedfarepquoesnictyivewasreesxppoencsteedf.n this assay was presuned to be a potential mammalian cell clastogenic agent. - page 7 - 005611 T-5869 " NOTOX Project 115943 MATERIALS AND METHODS TEST SYSTEM Test Systen Rationale Source CELL CULTURE Cultured peripheral husan lymphocytes Rreeccoognnniezneddedbytestthesyisntteenrna(tei.og.nalEPAg,uidOeEClDi,nesEECa)s. the pHielaoltthyStuaddyult: maaglee28volAuGTnte=er1s5:.4 h (Mar.'S4) eexxppeerriinmeenntt 21:: aaggee 3314 AAGGTT == 1156..66 hh ((vDeecc..''9933)) AGT = Average Generation Tine 8100d samples F10 complete culture medius Cell culture conditions Environmental conditions Bmalloeodvsoalmupntleeesrvebryevetnaakpeunncftruorneaushienaglthtyheadult Vsyesntoejnectwitmhultaipslueitasbalmeplesizbelosotderciollelecvtesisnegl wceornetafstnoirnegdsoadtaiumtehneppaerriant.ureThebetbwleoeond 4samanpdles 2Wi5th,in 4 h ater withdrawal lysphocyte cultures were started. FF1100 ncoemdpiluentewictuhlotuutrethymeadiiduinnecaonndsihsytpeodxaonfthHianme's i(nGaibcctoi)v,atseudpp(l5e6mCe;nt3e0d awiint)h f2o0eKtahleatc.alf serun p(e6nitbccto1)1,inL/-sgtlruetpatmoinnyeci(n2 a(k5)0,U/a) and 50 g/ml 3r0espUreacltivheelpya)r,in.sodium bicarbonate (2 9/1) and WcuhlotluereblomoeddiuwnaswictuhltuPrheydtohianesFa1g0glcuotmipnliente P(eHrurecxu)l.ture (5 ml F10 complete culture medium aPnhdyt0o.h4ae1maggwlhoulteiniblnovoads) 0ad.d1edm.1 (9 ng/ml) AaTt)nosipnhceurbeati(o8n0s-95w%e)recocnatrariineidngout5%inCO,a hiunmiadir ainndthCeOpd-aprekrceantta3g7eC.verTehemotneiapteorraetdurdeu,rinhgusitdhiety experifent. - page 8 - 005612 7-5869 : NOTOX Project 115943 REFERENCE SUBSTANCES _-- TVNheegeatinve iconoTtFroel: the test article. Positive controls: FWriothmouet metabo(lRiEc CacCtRiSvatmoi.on50(--0597-7mix): Sigma, acting mutagen at a final concentration of 0U.2.S.uAg./)ml wa(ssoulvseendt:asHBaSSd)irefocrt a 2p4erihodt.reatment period and 0.1 ug/ml (solvent: HBSS) for a 48h treatment EWityhcmeTtaoboplihc oac(stCiPvp:athiCoaen an(:4t+59d-a1sxS)0:8-10. Endosan-sta, Asta-Werke, F.R.G.) waasfinuasledcoansceanntriantdiiornectofa1c5tinugg/mmluta(gseonl,venrte:quHiErSiSn)g mfeotrabaol3hic tacrteiavtamteinotn, at period (24 h fixation period). B SHBoSlSva en=t l fHaonrk'a srefernencecsSuabe lsttanSd coelsution without calcium and megnesius. METABOLIC ACTIVATION SYSTEM ---------------------------- Preparation of $9-homogenate: ROFatSplriavgeuremiDcarwolseoynarlatse,nzwyhneischwewreereroobuttaiinneeldy pfrroemparBReLd, fSrwoimtzaedrullatndm.ale Wistar TThaeboarnaitmoarlyscownedrietihoonuss,edasatdeNsOcTOrXibeidn ainsptehceiaSlOP'rso.omThuenderratsstwaenrdeardinjected wienitgrhatp)eriintonceoranllyof]w.ithFivaesdoalyustiolnate(r2,0%twh/evy)weorfeArkoicllloerd b1y254dec(a5p0i0tantgi/okng; body s(atchreiyfivceer)e. dTehnetedTiavcecrsessoftothefoordatsforveraet rleemaosvted12ahsoeputriscaplrleyc,edianngd washed fn cNaogl-dED(T0A.C)Ssutbesreiqlueent0l.y1 thesoldiivuenrsphowesrpehatmeincbeudffefrn a(pbHle7n.4d)ercaonndtaihnoimnogge0n.i1zedaM wians'3cveonlturmiefusgeodf pfhoorsp15hamtienbuatff9e0r00wigt.h TahePotstueprernhaotmaognetni(z5e9r). wTashethroamnosgfeenrarteed into sterile ampules, which were stored fn 11quid nitrogen (-196C). *) Acnaersc,inBo.gNe.n,s MacnCdannnu,tagJ.ensandwitYhamatshaekiS,almE.o,nel1l97a5/,ManMmeatlhioadns fmoircrodseotmeecting mutagenicity test, Mutation Res., 31, 347-364. - page 9 - 0605613 T-5869 NOTOX Project 115543 Preparation of s9-atx: S$ph99o--smmpiihxxatwceao;sntap3i.rn4eepdmagrpeedNrADfP1n;a:c4d1l.ay0ato2e]lyngHEbPHeEofSColrpea.n6duHsge00.;5and2ml.4k6$e9p.tngoTnKhCeIf;acbeo1v.de7urimsngoglugttlihuoencstoesste.. WWvehetiraecbhomlviiaxcsed aadcadtndeidvaFatifilttoeenrrvaf(si0i.2at2cehriu-ensv)te-edsrtielbryiizlaaidtdziieondngo(f0a.p2tahretR1 Sf91r-iovmmeirxtheCSoom-Spm9oi-nxfernattcost)is.oinc:h cell suspension. EXPERIMENTAL PROCEDURE Cytogenetic test (1T9h8e4)t%e.st was carried out with ninor modifications as descr: ibed by Evans tTwhoe tiensdtepseunbdsetnatnceexpewraisnetnetsst.ed both with and without S3-atx in duplicate in wLdeoyrsaeepshcocuoylftteutrheecdulttefusotrres4s8ub(hs0t.a4anncda1etbhfleororoead2f4toefhraanedhxepao4l3stehdhywiimntahldoeuuptldiocnSoa9rt-eaiixntoo5rsme1floermcetde3idhus) wtthiheethctSea9sn-tcneicnxot.mrpaAotnufnodnapspvarwsohpirdciihaftfceiacuusrleatdngeitonhofdiibsidsotsoielovneloefvIenltshaeqwuamesiotuoscthioScsoelnuitnidteooxn.sdeIttnehreaciansee thBeridegiahutenns.etntCc,oonntccheeennttrcreaalttliisoonnsevxapesoxscededeedttieonrgait5nheedgt/ebasylttwsheuerbesstoanlnouctbei1teiistntyetdhI.en Atfhteercu3lthure presence of S9- mfpoeirrxiovaden)ro.ethTeFhrien2se0cde-l2l2osnchewh(iwFciithrhstw5erfeailxtartoeifaotnHeBdSpSerfioaorndd)24fonrhcuafbnoadrted4484i-hn45inhATt(hseegcraoobnvsdtehnfcimexeadtoiifuonn Sh9-anmdix48verh.e arrested by nDauodrtdiintrgiionnstheedofaltfahtseetr3sptihrnedaolftemethnietnhicabunildttowurreerecpoelfrciihxoieddc,iniecnesle(ld0i.a5dtievulgi/ysailoanftevras24 nPeadiu(n1)5.0 gT)heraendafttehre pellet were swollen sthuepercenlaltanctulwtausresrewmoevreed.ceCnetlrlisfugiendthfeorr5emaaiinninagt c1e3l0l0 by a 5 min treatnent wiih hypotonic 0.56 potassium 3chlcohranigdeessooflutmieotnhanaotl:37aCc.etiAcftearcidhypfoitxoantiivcetr(e3a:t1nevn/tv,). cells were fixed vith For the independent. repeat the 24 h fixation period was needed only. *) oEvfanCsh,romHo.3s.o,me19A8b4e,rraHtuimoannsPeirnipMhuetraaglenBTleosotds.LyIrnp:hoHcayntdebsoofkoroftheMuAtnaagleynsiicisty T40e5s-t42P7r,oceEdlusreevsi,erB.SJc.ienKcielbePyU,SIMT.sheLresgatBo.rV,.,.AssNtiecrhdoalns. and C. Rane] eds, page 10 005614 7-5869 NOTOX Project 115943 Preparation of slides F1:i1xedmixcteulrles woefre96%droeptpheandolo/nettoheprrevanidouscllyeanceldeanweitdh (a24thiosusures) iamnmdersmeadrkeidn a s(lwiidtehs thweereNOTpOrXepasrteuddypeirdecnutlitfuircea.tioSnlidneusmbweerreandallgorwoeudp ntuombderry) ansdlidtehse.reaTfoter sThtearienaefdtefror s1l0i-d3e0s wmeirnewirtihns5e%d i(nv/vt)apG-iweantsear asnodluatliloonweidn ttoapdrwya.terT.he dry sDleiPdeeXsawnedremoucnlteeadredwitbhy daicpopvienrgsltihpe.n in xylene before they were embedded in Mitotic index/dose selection Tmheetapmhiatsoetsicpeirnde1x000ofceelalcsh. cFuolrturtehewafsirsdtetefrimxianteidonbytimceoun(t2i4ngh hthaervensutm)ber of cihnrhoimboistoimoensoofftmheetampihtaosteicspirnedaedxsowferabeouatna5ly0s-e2d0%of(itfhopsreesecnutl)turwehserewaisthtahen msiotlovteinct cionndterxolo.f Atlhesolcouwletsutredsosetrelaetveeld wwaisthapapnroixnitmeartmeeldyiattehedossaemewearse the ehaxravmeisnte)d ofnoer caphprroomporsioamteeabdeorsreatlieovnesl. waFsorsetlheectseedconfdorfsicxaartiinogn otfimechr(o4m8oshome Ianberorradteirontso. obtain the appropriate concentration range for the chromosome waebreerraitdieontnictaelsttoa pthiolsoet einxptehreimecnhtromwaossompeerfaobremrerda.tioEnxpetersitm,enteaxlcecpotndtihtaitonosne cfuilxtautrioenpeprericoodnceinntrtahteiopnresweansceuseodf aSn9d-aiwxi.th the onission of the 48 h Analysis of slides for chromosome aberrations cFhorromcoosntormoel aboefrrbiaatsi,onsallandslisdcoersedw.ereAn raadnhdeosmliyvecoldaebedl bweifotrheNOeTxOaXminsatutdiyon of 1i0d0enmteitfaipchaatsieonchnruormboesromaendspcroedeadswasperstcuuckltuorveerwetrhee meaxrakmeidnedslibdye.1igAhttleast mcihcrroomsocsoopmyesfworerechranoamloyssoemde. aTbheerrantuimobnesr. ofOnlceyllmsetwaipthahseasbercroanttiaoinnsingand46the nusber of aberrations were calculated. ACCEPTABILITY OF ASSAY Afolclhorowmionsgomceritaebreirar:ation test was considered acceptable 1f it met the a) cThueltunruessbersshouolfd chrreoamsoosnaobmleyabfaelrlratwiiothnisnfotuhendlaibnortahteorsyolvheisnttorciocnatlrolcontrol b) Tdhaetaporsanigtei.ve control substances should produce a statistically wsiitghnifcihcraonntoso(nCehia-bseqruraarteiotness.t, P < 0.05) increase in the number of cells - page 11 005615 T-5869 - NOTOX Project 11594-3 OATA EVALUATION AND STATISTICAL PROCEDURES. Aabetresrtatisounbsttaenscte17w:as considered positive (clastogenic) in the chromosose 8) b) PAIt<stia0n.td0iu5sc)teidciaanlcldryoeassese-irgenilinaftitechdaentnsutsaibtneicrsrteioacsfaelcleiylnlssthiegintfihrfiecqchaurneotnmcoy(sCoohmfei.-asbaqeburearrrraeattitioeonsntss,.vas orbesseurlvtesdweirnethreeparbosdeuncciebleofna Canleairnddeopseen.dreenstploynsreeperaetleadtioenxpsehrip,kenbtu.t the cAhrtoemsotsosmuebstaabnecreratwiaosncotnesstideifr:ed negative (not clastogenic) in the 2) Nc(ohCnrheoin-oossfqouantreheeabtteeersstrt,aetdiPocn<osn.0c.e0n5t)ratiinocnrseasienduincedthea nsutmabteirstoifcalcleyllssiwgintihficant Tenhteerpreincteodintghecrfiintaelriaevavlerueatinoont daebcsiosliuotne. and other modifying factors might Tacbhoeemrpraiarntecidiodnteosn',cethIaontfclouafsbievtrehreanotrsolcevexelcnlltsusic(vocneetlrlgosalpsw)uifsthionrgoneeCahciohr-sqmtourraeeraetmceshntrtaotmiogssrtooimuceps:was X2 = (a(sNh-)1) xera) _(a(da-vob)e)2 (Bed WhereNgbd=== tthhee the ttoottaall total nnuuaabbeerr nuaber ooff of cnaeoblnelarsbrearsnrctaorncetedllcseilnlistnheitnhecotnchteornotclroonltcruolcltuulrtceuusrl.etsu.res. @ G = = tcthhoeempattrootteaadll winntuuhsmbbeetrrhe oocffonantobrneoarlbr.sarnrtanctelclesllisn treated cultures to in treated cultures be to be nWy Z= cstuohmmepaotrfoetadnlgwiantnudhsbetnryheofconcterlolls: scored in the treated cultures. I~, I we |12x> E em N-E 1) x (adS -Ebc)e2 || (two-tailed) gcciroshonrtusorpmnoaollsilsognreC(ooPuna<psbie0d.rfe0rs5ra)etrdieotjnhteseoctbefhsedy.posatnthidhegensitsifahsimeceantnuhftmaolbtryertbdhoieotffhfieatnrhbceeeinrdtrtearnncefteartoencodefltclhaseenldlcisontnhtetwrihoteslhotlevgsertonutp at the 95% confidence level. - page 12 - 05616 T-5869 - NOTOX Project 115943 RESULTS 00SE SELECTION _-- c4IgnouTla-d5p8rn6eo9ltimpbeiernatrmelsytecsdtu,uldtyubreecbalumoseoeddioucnfultwtuhiertehsoavwndersewoiltutbhrioeluaittteydS9wo-ifitxht.he1,Hitge3,hsetr10s,uCbosn3tc3aennactnerdati1in0o0ns the culture mediua. TABLE 1 MoIFTOTT-I5C869INOEX OF OONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS PILOT STUDY ------ ee Tcoensctesnutbrsattiaonnce (ug/al) . a Nusber of metaphases per 10r 00 ca ells of control 28 1 fixstion period Without veabelic setivation (SVEN 31 Controld. 6776 w110r0 133 1000) 532 87 0 & 281 rhxstion pertod 13 Control. 3338 129070 133 1000) 593 112400 27 n 24 h fiexateion period Con1tro1) 103 13003%) CTT en metabotic activation (s59-mix) a7s" 110008 &2 10a8s 87 1008 ba)) SDTMiSgOnt precipitation of the test substance in culture medius. - page 13 05617 T-5869 NOTOX Project 115943 Bsaesleedcteodn tfohre trheesuclthsroomfosotmhiesabpeirlroattisotnudytestth:e following dose levels were Experisent 1 + 2 WiitthhoSu3t-mSi9x-mix : 33,, 1100,, 3333 aanndd 110000 uugg//mmll ccuullttuurree mmeeddiiuumm Traalbelesdon2ora)ndt3resahtoewd twihtehmivtaortiiocusitnedsetx osfubsctualntcuerescon(cfernomtrabltoioodnsoofrawihtehaltthhey positive of negative control substances. Bcahsreodnosononethaebseerroabtsieornvsa:tions the following doses were selected for scoring of Experiment 1 Without S9-nix With S9-mix : 1(02,4 3h3fiaxndati1o0n0 upge/rmilod)culture medium 1100,0 u3g3/malnd(14080hugF/iaxlatciuonltuprereiomde)dium 1(0204 hug/fmilxat(i4o8nhpefriixoadt)ion period). Experinent 2 Without S9-nix With S9-mix : 1(02,4 h33fiaxndati1o0n0 upge/rmilod)culture medium (102,4 h33fainxdati1o0n0 puegr/imlod)culture medium CYTOGENETIC TEST TIhyempahboiclyitteys woafsT-i5n8v6e9stitgoatiendd.uceThechrtoesmtosovmaes caabrerrireadtioonust iinn dhuumpalnicapteeripihnertawlo ianndde8p.enTdheentesxcpoerresinfeonrts.theThneuabreerssulotfs oabfedrurapnlticacteellscul(tiunrcelsusiavree ainnddiceaxtceldusibvyeA vgaaprsi)ousandconthceennturaabteirosnsooffthtehevatreisotussubtsytpeasnceofarcehrpormeosseomnetedabeinrraTtaibolness 4a-t9.theThe criteria Appendix 1a.ccording to which the aberrations vere classified are outlined in sBtoatthisitnictahlelypreasnedncbeioalnodgicaablsleyncesigonfifSi9c-amnitx with chrososone aberrations. tihnecretaesste siunbsttheancneusbdeird nooftceilnldsuce a - page 14 - 05818 T-5869 - NOTOX Project 115943. Tc1h.ue1ltun(rueesabsnerw+eroefstcwaeinltdlhasirnd wditethhveiaclthairbooonmr)oastooamrbeyerrahaibnsettrorracitecilalolsns cpeofrnoturn1od0l0 indmaetttahaephraassnoeglseven((twi.ietc.hoontu1rt.o0lS5sRnpiirxxo;;ducggeaadppsssteeaxxtcclilusudtdeiedcd)a)l.lanydThse0i.g6npiofs+iict0ai.nv7teacbioennrctrrraeoanlstesccehleinlmsictphaeelrsfr1(e0M0qCu-meCenctyaanpdhofasCePa)sberb(rowtaihntth $9. ctehlalts.theItmevtasabotlhiecreafcotrievactoinocnludseydstetmhat(St9h-enixt)estfucnocntdiiotniedonsprwoepreerloyptimal and FTi-n5a8l6l9y,isitnotisclcaosntcolguedneidcthuandterthitsheteexsptersihmoeunltdalbecocnodnistiidoenrsedofvaltihdisantdestt.hat - page 15 - 005619 T-5869 - NOTOX Project 115943 - TABLE 2 MpITeOTTrrICreeoTNOE1X OF DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS TLeosntcenstubrsattainocne (ug/ml) Number of setaphases per 1000 ceellls by of control 201 riation period 53} Contro13) W10E0S:) 0.2 vat 48h fixation period 225 Contro13) 100) MMC-C; 0.1 ug/ml bb2F9All- ii50 h1iii00 Fa41-31 %91 3aFo5all- a332% 1iAi00 45 - 38 124 23 - 24 70 241 rr igeion pee riod 55 Contro18) 18 100) gsm 48h ration period 25 Contro12) 33 100) ith aetabotic activation (+69a16) w75[6l3-8]857 1i3%00 un 76 - 75 293 8Ga7 -np64 31i0n0 8g - Od 18 87 - 101 125 03o)) SoSvhoiocnatperecciuplittuartesion of the test substance in culture medium, d) Culture infected with fungi - page 26 - 005620 T-5869 - NOTOX Project 115943 TABLE 3 OMIFTOTT-I5C869INDEX OF DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS Exper nent 2 coTnecsetnsturbasttiaonnce (vam) Ca2n6trhof1iax)ation period 103 10303) HHC-C; 0.2 ug/ml RNuasbreer sof metaphases per 1000eceells b) of control FTERGUE ReCabeTic sctivation (-S3-875) 2a -- 3a6 1m0w 0 023--2466 18056 3a9-a43 110008 24 hCfrixamtion pertod Cont3ro1d) 1303 1c0P;01)5 g/m With metabolic activation (+59-mix) 077--6664 10080 67-35566 8Ed 754.-7324 n&m ab)) oduwpslicate cultures ) Slight precipitation of the test substance in culture medius. = page 17 - 005621 T-5869 : NOTOX Project 115943 Fn foe fo] TABLE 4 2CCHO4ANhDCHEDNFSTiORxMAaETtiIoOAnNSSERpReOArFTtIo1Od-N5S669IN(DOitNhOoRutCULST5-UaR1ExS)8T)REATED WITH VARIOUS Experiment 1 o oa core ooaoftowas 0 wel Te lie Spur]nn Bi oe Seiaued Eel. C odesfon es To Tos1The | nT w TaeTs TanTosTow| - Te = 1TT = 1TT wo [T TvT ee w [ 1T 1 T fawer|TTT[T ee] Cay 4) ) ASSSEbohibipErrpeieinrvoiiiinacetiyi1n2otnoysTndsusieafdeemvreeeferroensritcMmvaaefatrsoeiesSosueaiscauoitttyionepoeeesssevssoeafhrndatoAsbaeerrbrnsrapagttLeiioLootnnsi1e:soantrheee(lotoinsnytesedaoneidnntionss above. SEERA Tne page 18 - 605622 T-5869 : NOTOX Project 115943 - Et TF Bi i Le es pee wo Je [ne Te] e |TT. vw [oa [TeJs ew| wT 1s. rr] - Trrr wo | Ts marl r [oer [oe [or e foe | EE 0605623 5869 - NOTOX Project 11594-3 CONCENTRATIONS OF T-5869 (Without $9-a1x)3) Bh [onfe [rom| BE Lee] BT eTn L Te T TeTTTeee]] T FR EE T] =] 1] rr " Significantly ustrerentfroncontrol gros (Chi-square test), 605624 re CONCENTRATIONS OF T-5869 (With S9-mix)a) ales ped Sfo ian ae d T TT fT] -- TT fersrppe ) Abbreviations used for various types of aberrations are listed in 005625 T-5869 = NOTOX Project 11594-3 TABLE CHRONOS JOCRAATIONS Th DONOR CULTURES TGEATED WITH VARIOUS 2ECxO6pNheCrEiNfmTieRxnAatTtIiO2oNnSpeOrFioTd-5869 (Without S9-mix)a. BrBi e foee on] Sm [WS fem om core foowal 0 malowel 0aahew fi loco nooden os i SShoanin EE noah Crist itn sme te IE es [o TasTea Te = TTTT ee TTT Tee w [TT Tee = TT T --7T = 1TTTT wao[ [T TT TT TT e TT me |TTTT p mar T ar L eee fae] Ca ) + SFpAbhobimirnieieverikiaecttnyi1tolneyosottruysteensdowneefrsnoiertckvnsaFTatrooiveeoCaruraoscittaoittaoniysnoptsneasassnossotafnhndebaEebebsleedorCsunOrepgaSabittAnieicgooanentst:ioroanrtheenblannitseytssedssheientionad above. SRL ELL page 22 - 605626 T-5869 - NOTOX Project 115943 TABLE 9 CC2AO4RNhGCHEONFSTiORxNAa:TtIiOAoNSnSERpROeAFrTiIoTOd-N5S865IN(DWOiNtOhR5C9U-L8TiUxR)E3S. TREATED WITH VARIOUS Experiment 2 [em arm ours [ovwaso wel onal o ash ow] B Cma es Bl [of bes Seiad sen o ee w ia aT es wee| wee Cr TT wo |TT Te] ~T we | ee Ean 3 e strertah tion eresdefnosivarsieoEuisattiyopness osfrdasbtesrsrmaitiicoantsioanre (sontie)d sInnd oy Binhaetrriyervrseree twTtuereceactbpooennsteoaetgar7Seosloon(egrEianhdtsitaconatnhetcoosehantiond shove. - page 23 - 605627 : 7-586 - NOTOX Project 115943 DAEPFIPNIETNIODNSIXOF C1HROMOSOME ABERRATION SCORED IN METAPHASE PORTRAITS aberration Chromatid gap Abbreviation oe Chromosome gap 0 Chronatid break bv Chrososoe break bo Chromatid deletion ' Minute Double minutes aw Dicentric chrososone dic cThrriocneonstorniec tric Description Aann uacnhsrtoamianteidcrelgeisoinoniwnhitchhe caphpreoamrastidas sarmna,lletrhetshainzetohef wwihditchh oifs teqhuealchrtoomaotrid tahned cthheromaaptpiadreantrlmyar*ebroiknena*ligsnemgemnetn.ts of aann uancshtraoimnateidcrleegsiioonniwnhibcothhapcpheraormsatiasds aist etqhuealsamteo oporsistmiaolnl,ertthehansizteheofwidwthhich o"fbrotkheenchrsoemgaateindtsanodf tthhee acphproamraetnitdlsy are in alignment. Aanrn,acthhreomastiizce olfeswiohnichinias clharrogemratitdhan stheeg'newnitdsthofofthtehecchhrroommaattiidd.arTmhearberoken aligned or unaligned. aAnt tahcehrosmaameticposlietsiioonn, inthebostihzechroofmawthiidcsh is larger chromatid. tThhaen btrhoekewnidtshegonefnttsheof the chromatids are aligned or unaligned. Dcehlreotmeadtimdataerrei.al at the end of a Achrsionrgaltei,d luascukailnlgy acircceunltarro,merpea.rt of a cTwhor,omuastuiadlllyackciinrgculaarc,entpraormtesreo.f a cAencthrroonmeorseosm.e containing two cAencthrroomneorseosn.e containing three - page 24 - (05628 T-5869 : NOTOX Project 115943 APPENDIX 1 continued _-- A-v_e--rration Ring chrososone Exchange figure Cinhtrroamcohsaonmgee pchurlovmeorsiazseeds Abbreviation r exch. intra Description A ring structure with a distinct Tusen. Acohfnroeanxoctsrhioa-nnegseo(rsr)meosbrueel-ttaiwrnengeenditncwootnhfeoirgfusororarmteaitoino.n Aacfhtrceohrmroosmrooessjeoo.mieninigntorfachaanlgeesioins swictohriend one A fraguented or pulverized chronosone abteirrpalteions na ABormeetoafphatshee sabporveeadmecnotnitoaniendingabeternratorions i(ncchlruodmeadt)i.d and chronosone gaps not Polyplotay poly oAfch`trhoesonsorosnaelnduispblerotdthanutsb1esr.a aultiple Endoreduplication endo Acenftorroameorfepcoolnynpelcotisdytwion worhicfhoureacphairs r-- ere ------opafirc-- .hromatids -- instead of -- the noraal one - page 25 - 0605629 T-5869 : NOTOX Project 115943" APPENDIX 2 STATISTICAL EVALUATION OF TEST RESULTS chi-square Test TOTAL NUMBER OF CELLS WITH ABERRATIONS; TREATHENT/CONTROL COMPARISON, (INCLUSIVE/EXCLUSIVE GAPS). e Experinent 1 ee T00RSEEATH(EuNgT/an) SIMIX OPS tPwVoA-LsUiEded CDOENCFIISDIEONNCEATL9E5VE%L ---- 24h fixation period mec (0.2) - .: <00..00000088 Ssiiggnniiffiiccaanntt @ (15) . .: 00..00000046 ssiiggnniiffiiccaanntt 48h tixation period wee (0.1) . .: 00..00000046 ssiiggnniiffiiccaanntt e--e ------------------------------re--e--. --Expe--rtn--ent--2 ------------------------------------ T0R0ESAETH(EvNgTa) SIMIX GPS Pt-vVoA-LsUfEded CDOENCFIISDIEONNCEATLE5V5E%L ----t---------------------------- 24h tixation period wee (0.2) - .: 00..00000068 ssiiggnniiffiiccaanntt (15) . .: 000.0000044 ssiiggnniiffiiccaanntt e-- r --------------------rar-- ersr-- ee page 26 - 605630