Document vB5xoo7Lv4pV76o59b0R12ar6

(ven AR)E-0248. p= NOTOX er rm nse REPORT EVALUATION OF THE ABILITY OF Tsans TO INDUCE CHROMOSOME ABERRATIONS IN CULTURED PERIPHERAL HUMAN LYMPHOCYTES (WITH TNDEPENDENT REPEAT) NNDoTTOOXX Spurbosjteacntce11358911697 page 1 of 26 - yi log oo Mi eer e e ce | 3, 496 TT Fl-UILT (eroATg T tec HECEIVED T= NOV ~9 1994 7.0. Box 3476, 52H0a5mbbra"knanogenoe3. me NESRIMEmOLOGY r----e---------------------------------------------- ; ' T-594 2 REPORT CYALUATION oF THE ABILITY oF -- 0 INDUCE CHROKISOHE ABERRATIONS IV CULTURED PERIPHERAL HUNAN LYHPOCYTES (METH THDEPENOENT REPEAT) WWOOTTOX fpraojdecte11J59o1m9 RT 004497 RECEIVED NOV -9 1994 3 T-5874 - i STATEMENT OF GLP COMPLIANCE NOTOX Project 115919 NOTOX B.V., 's-Hertogenbosch, The Netherlands i Trheecesnttudeyditdieosncriofb:ed fn this report was conducted in compliance with the most 3 The OECD Principles of Good Laboratory Practice which are essentially in conformity with: TRheeguUlnaittieodnsStPaatrtes58F.ood and Drug Adainistration. Title 21 Code of Federal TFheederUanliteRdegSutlaatteisonEsnvPiarrotnm1e6n0t.al Protection Agency (FIFRA). Title 40 Code of TFheederUanliteRdeguSltaatteisonsEnvPiarrotnm7e5n2t.al Protection Agency (TSCA). Title 40 Code of With the following exception: Stability of the test substance in the vehicle was unknown. Study Director ] i 4 A Ing. E.J. van de Waart Ye vate: 05/bs/lsg% a 004498 3 1.5674 ] ompemeeeee NOTOX project 115919 Study procedures were subject to periodic inspections and general non study This report was audited by the NOTOX Quality Assurance Unit and the methods April 19, 1994 April 19, 1994 Quality Assurance Manager C.J. Mitchell B.Sc. Date: 1) - 5-9 page 3 - 004499 3 T-5874 : REPORT APPROVAL STUDY DIRECTOR: NOTOX Project 115919 Ing. E.3. van de Waart y//Yet Ee MANAGEMENT : Tore.chnIi.Cc.al EDninriencgator /N J - page 4 - 004500 T-5874 ~ NOTOX Project 115919 PREFACE sponsor Study Monitor Testing Facility 5BSM2a2n0as7de0alsgtivurEnaNaOt-RCE1h1CeHmTical ac Belgium We. RH Cox NOTOX B.V. Hambakenwetering 3 5231 DD 's-Hertogenbosch The Netherlands Study Director Ing. E.J. van de Waart Technical Coordinator Study Plan Test susstance A.M.C. Bertens start January 26, 1994 Completed : March 30, 1994 Identification description Tossa Crean solid Batch 2334 Purity specific Gravity Instructions for test substance storage Stability under storage conditions Expiry date 17100% At roon temperature in the dark Stable January o1, 1996 Stahboluers foTrh vatenileeraest Dvaitmeerthyl sulphoxide + nnaot indicated venteLe The test substance was dissolved fn dinethylsulphoride of spectroscopic Squaarlity (Merck). Test substance concentrations were prepared directly prior to The final concentration of the solvent in the culture medium amounted to 0.9 % ( page 5 - 004501 viv). ] } 1-5874 NOTOX Project 115919 GUIDELINES gThueidesltiundeys:procedures described in this report were based on the following - OGrugiadneilsianteisonforforTesEtcionngomiocf CChoe-moipcearlast,ionGuiadnedliDneevelnoo.pme4n73t: (O"EGCeDn)e,tiOcECD T1o9x8i3)c.ology: In Vitro Mammalian Cytogenetic Test, (adopted May 26, - EEEuCropDeiarnectEicvoenom6i7c/54C8o/mEmEuCn,ityPar(tEECB):,MeDtihroedcstivfeor92t/he69/DEeEtCe.rmiAnnnaetxionV ooff the TCoyxtiocgietnye;ticB.1T0e:st".OthEeErC EPfufbelcitcsa-tMiuotnagneon.icLi3t8y3: (IandoVpitterodDeMcaemmmbaelri,an1992). ARCHIVING pNrOoTtOoXcoBl.,V. rweiplolrt,arctheisvtearthteiclfeollroewfienrgencdeatasamfpolre,atallleasstpec10imeyneasrsa:nd raw data. OBJECTIVE Purpose of the study Tthoeionbdjuecectisvterucotfurtahlischstruodmyosowmaes atoberervaatliuoantse tinhecutletsutresdubshtuamnancelyfonrphoictysteasb,ility either fn the presence or absence of a metabolic system (59-mix). - page 6 - 004502 T-5874 NOTOX Project 115919 3 - Justification and rationale of the test system Sitnidniuclaattoerds coufltculraesdtogheunmiacn lacytnipvhiotcyyteosf waerberoaudsedranbgeecauosfechtehmeiycaalreclsaesnsseist.iveIn ccohmembiicnaaltiomnutwagietnhs,a mia.em.maltihaonsemerteaqbuoilriiznigngmetsaybsotelmic(5t9r-amnisxf)oramlastoionindiinrteoctreactive Fionltleorwmeidnigatterse,atmceonutl,d bceelltesdtievdisifoonr cwlaasstaorgreensitcedefifnectthse minetvaipthraos.e stage of the ccehlalngecsyclseuchbyasaddbrietaikosn, ofgaptsh,e smipniuntdelse, podiiscoenntrcioclschiacnidnee.xcShtarnugcetufriaglurecshrowmeorseome erxeasnuilntesdwemriecrocsocmoppairceadllwyithin tchuosleturoefs thtereactoendtrowlith(vtehheicltee-sttresautbesdt)ancceultaunrdest.he Cmhirtoosmioss.omeTheabearprpaetairoannsceweorfe tgheenefriarlslty peovsatl-utarteeadtmeinntthmeitofsiirsst copuolsdt-tbereatment chaornvseisdteerdablayt d24elahyeadn,d d4u8ehtoaftteorxicbegiinnsnuilntgofofthterecaetlmlesn.t Ttoherceofvoerre,thecelilnsterwvearle Aintewshtichartmiacxlienunwhaibcehrriantdiuocnedfraeqpuoesnictyivweasreesxppoencsteedi.n this assay was presumed to be a potential mammalian cell clastogenic agent. - page 7 - 004503 i T5874 } MATERIALS AND METHODS NOTOX Project 115919 TEST SYSTEM Test systen Rationale Source Cultured peripheral huran Tynphocytes Rreeccoognnniezneddedbytestthesyisntteemrna(tei.og.nalEPAg,uidOeEClDi,nesEECa)s. the pHielaoltthystuaddyult: magaele2v8olAuGnTte=er1s5:.4 h (Mar.'94) eexxppeerriimmeenntt 12:: aaggee 2381 AAGGTT == 1155..46 hh(D(Meacr..''9934)) AGT = Average Generation Tine CELL CULTURE Blood samples F10 complete culture medium Cell culture conditions Environmental conditions 8ma1l0e0 voslaumnplteeserwebrye vteankaepnuncftruornea uhseianglthtyhe adult Vseynsotjeemctwitmhultaipslueitasbalmeplesizbeloosdterciollelecvtesisnegl wceornetaisntionrgedsoatdiuantehemppaerriant.ureThebetbwleoeond 4samapnldes 2W5itCh.in 4 h after withdrawal lyaphocyte cultures were started. FF1100 mceodmipulmetewitchuolutturethymmeiddiiunnecoanndsihstyepdoxaonfthHianne's i(Gniabcctoi)v,atesdupp(l56emCe;nte3d0 mwiint)h 2f0oe%tahleatc-alf serun p(eGinbiccoi)i,inL/-sgtlruetpatnoimnyeci(n2 n(H5)0,'U/ml and 50 ug/ml 3r0esUp/emcltivheelpya)ri,n.sodium bicarbonate (2 g/1) and cWhuollteureblmoeoddiuwamswictuhltuPrheydtohianemFa10gglcuotmipnliente P(eMrurecxu)l.ture (5 ml F10 complete culture medium aPnhdyt0o.h4aenm1agwghloulteinibnloowda)s 0a.d1dedm.l (9 mg/ml) aAlt)nosipnhceurbeati(o8n0s-95w%e)reccoanrtraiiendingout5%inCO,a hiunmiadir iannd tCheO,-dpaerrkceantta3g7eC.werTehe motneimtpoerreatdurdeu,rinhgumitdhiety experineenntt. Soysoy T-5874 BN NOTOX Project 115919 REFERENCE SUBSTANCES _--_-- TNeRgaetSivveecnoTnotaFrotlTe:he test article. Positive controls: HWaicittohiRonyugetTnnmuteCatgaeb(noHlEiaLctE;aactCfAiiSnvaaltToi.ocnon3c0e(-n-05t79r7-am.tiixo)S:nlamoaf,0U..25.u4g./)mlva(ssoulvseendt:asHBaSSd)irefocrt a 2p4erihodt.reatment perfod and 0.1 ug/ml (solvent: HBSS) for a 48 h treatment WEityh cmeTtaobolpichao(c0tsi7v:aptiRoRneans(:+5T9S-0maaixbs)-:0. Endoxan-Asta, Asta-Werke, F.R.G.) waasfinuasledcaosnceanntriantdiiornectofac15tinugg/mmluta(gseonl,venrte:quHiBrSiSn)g mfeortabaol3ich tacrteiavtamteinotn, at period (24 h fixation period). HSB BoSlSvea n=tHl afnork'sa refen rencc e Ssuae blsttand Scoelsution without calcium and magnesium. METABOLIC ACTIVATION SYSTEM _--mm Preparation of $9-honogenate*: RoratSplriavgeuremiDcarwolseoymarlatse,nzywmheischwewreereroobuttaiinneeldy pfrreopmarBeRLd, frSowmitzaedrullatndm.ale Wistar Tlhaeboarnaitmoarlyscownedrietihoonuss,edasatdeNsOTcOrXibeidn ainsptehceiaSlOP'rso.omThuenderratsstawnedraerdinjected wienitgrhatp)eriitnonceoarlnly011w.ithFivaesodlayustiolnate(r2,0%thw/evy)weofreArkoicllloerd b1y254dec(a5p0i0tamtgi/okng; body s(atchreiyfiwceer)e. dTenhieedliavcecrsessoftothefoordatsforweraet lreeamsotved12asheoputriscaplrleyc,edianndg washed in cNional'gd3-EDv(To0Al.Cu)meSssutbesorefiqlupeehnots0lp.y1hatMtehesobdulifiufvneerrsphwiowtsehrpehaatmeiPnoctbetudefrfeirnhoma(opgHbelne7in.zd4e)err.coannTdhteaihnohimonomggoege0n.ni1azteedmi wianstocsetnetrriilfeugeamdpufloers,15whmiinchavter9e000stog.redTheinsulpieqruindatnainttro(g9e)n w(a-s196trCa)n.sferred *) Acmaersc,inoBg.Ne.n,s MacnCdannnu,tagJ.ensandwitYhamatshaekiS,al.no,ne119l7a5/,MaMnmeatlhioadns mfiocrrodseotmeec.ting mutagenicity test, Mutation Res., 31, 347-364. C----- 004505 9) T-5874 - NOTOX Project 115919 Preparation of $9-mix: $S99-.nniixx vcaosntapirneepdarepderimm1m:ed1i.a0t2elyngbeMfooCrley.6uHsye0;an2d.k4e6ptngonKCIi;ce1.d7urinnggglthuecotsees6t-. phosphate; 3.4 ng NADP; 4 umol HEPES and 0.5 mi 59. The above solutions were mixed wMheitcahbowlaisc and filter aadcdteidvataifotner (0.22 um)-sterilized wafsilatcehri-esvteedrilbyizaatdidoinng (apart o0f.2thenl from t S9T-imveirx h e S9-fraction, cSo3m-pmoinxen0ts)e.ach cell suspension. EXPERIMENTAL PROCEOURE Cytogenetic test T(h1es84t)e+s.t was carried out with minor modifications as described by Evans Tthneo tiensdtepesnudbestnatnceexpwearsimetnetsst.ed both with and without S9-mix in duplicate in wLdeoyrsmeepshocucolyfttuetrheecdu:lttefuosrrtes4s8ub(sh0t.4aanncdmle tbhfloeroroeda2f4toefhr aanehdxepao4ls8tehdhy imniatldheuopudltoincSoa9rt-emiixtno 5orsme1lfeormcetde3iduhm) With S9-mix. An appropriate range of dose levels was chosen to deteraine tthhee tceosntcenctornaptoiuonndsvawshicdhifcfaiucsueldt tionhdiibsistoilonveofin tahequemoiutsotiscoluitndieoxn.s tInhecase hmeidgihuems.t cCoonncceennttrraattiioonnswaesxcdeeetdeirnmgin5edmg/bmyl thweeresolnoutbilteisttyedi.n Atfhteercul3tuhre treatment, the cells exposed to the test substance in the presence of $9- mfpoeirrxiowadne)or.teheTrrhien2s0ec-de2l2losnchewh(wificithrhstwe5rfemilxattroifeoantHeBdSpSerfioarondd)24ionrhcubfaaontrded4448-i4nh65ihnm1t(hsegercoaowbnstdehnfcmieexdaiotufimon S9-mix vere not rinsed after treatnent and vere fixed insediately after 26 h and 48 arrested h. by During the last addition of the 3 h of the culture period, cell division spindle inhibitor colchicine (0.5 ug/ml was mpendiu(m1)5.0 Tg)heraenadfttehre tshuepercenlaltanctultwuasresrewmoevreed.ceCnetlrlisfugiend thfeor r5emmaiinninagt c1e3l0l0 pellet were swollen by a 5 min treatment with hypotonic 0.56% potassium 3Fcohrlcohrtaihndegeesisnoodfleuptmeienotdhneanntaotlr:e37peaCac.tetiAtchfetear2c4idhyhpfofitixoxanatiticivoentr(pe3e:a1rtmieovnd/tv,)w.ascelnlesedewdereonlfyi.xed with *) Evans, H.J., 1984, Human Peripheral Blood Lymphocytes for the Analysis oT4f0e5s-tC4h2rP7ro,omcoesEdolumsreeevsiA,ebrer8.rS3ac.tiieonKnciselbePiyun,bMlui.tsahgeLerensgatTBoe.rsV,t.s,.W.AnIsnN:tiecrHhdoaalnnsd.boaonkd oC.f MRuatnealgenedisc,ity pesos - 004506 T-5874 . NOTOX Project 115919 3 4 Preparation of slides F1:i1xedmixcteulrles woefre96%dreotphpaendolo/nettoheprrevainoduscllyeanceldeanweidth(2a4tihsosuures) iamnmdermsaerdkeidn a s(lwiidtehsthweereNOTpOrXepasrteuddypeirdencutlitfuircea.tioSnlidneusmbevrereandalglroowuepd ntoumbderry) asndlidtehse.reaTfwtoer Tshtearienaefdtefrorsl1i0d-e3s0 wmeirnewirtihns5e%d i(nv/vt)ap-Gwiaetmesra asnodlutailolnoweidn ttoapdrwya.terT.he dry sDeTPiedXesawnedremoculnetaerdedwitbhy daipcpovienrgsltihpe.m in xylene before they were embedded 1n Mitotic index/dose selection Tmheetapmhiatsoetsicpeirnde1x00o0fceelalcsh.cuFlotrurtehewafsirdsettefrimxianteidonbyticmoeun(t2i4ngh htahervensuts)ber of cihnrhoimboistoimoens ofof thmeetampihtaosteicspirnedaedxs ofweraebouatnal5y0s-e2d0%of(ifthopsreesecnutl)turwehserweiatsh thaen msiotlovteinct iconndterxolo.f tAhlesolcouwletsturedsosetrelaetveeld wwaisthapanproixnitmeartmeeldyiattehedossaemewearsethe ehaxravneisnte)d ofnoer cahprpornoopsroinaeteabdeorsreatlieovnesl. waFsorsethleectseedconfdorfisxcaotriionng tofinechr(o48mohsome aInberorradteirontso. obtain the appropriate concentration range for the chromosome awebreerraitdieonntictaelst tao ptihlosoet ienxpethreimecnhtrowmaossompeerfaobremrerda.tioEnxpetresitm,enteaxlcepctondtihtaitonosne cfuilxtautrieonpeprericoodnceinntrtahetiopnrewsaesnceuseodf aS9n-dmiwxi.th the omission of the 48 h Analysis of sides for chromosome aberrations Fcohrrocnoonstornoel aobferrbaiatsi,onsallandslisdceosredw.ereAnraadnhdeosmilyvecoldaebedl bewfiothreNOeTxOaXminsattuidoyn of 1i0d0enmteitfaipchaatsieonchnrusobmeorsomaendspcrodeeadswasperstuccukltuorveerwetrhee meaxrakmeidnedslibdye.1iAgthtleast mchircormoosscoompeysfworerechranoamloyssoemde. abTehrerantuimobnesr. ofOnlcyellmsetawipthhaseasbercroanttiaoinnsingand46the number of aberrations were calculated. ACCEPTABILITY OF ASSAY Afoclhlroowimnogsomceritaebreirar:ation test was considered acceptable if it met the a) Tchueltunruemsbersshouolfdchrreoamsoosnoambelyabfaelrlratwiiotnhsinftohuendlainbotrhaetorsyolvheinsttorciocnatlrolcontrol b) dThaetaporsaingtei.ve control substances should produce a statistically swiitghnifcihcraonmtoso(nCehi-asbqeurarraetiotness.t, P < 0.05) increase in the nuaber of cells 004507 i T-5874 - NOTOX Project 115919 DATA EVALUATION AND STATISTICAL PROCEDURES A test substance was cansidered positive (clastogenic) in the chromosome aa)berItratiinodnucetdesta idfo:se-related statistically significant (Chi-square test, b) AP<st0a.t0i5s)ticianlclryeasseigniinfitchaenntumibnecrreaofseceilnlsthweitfhrecqhureonmcoysoomfeaabbeerrrraattiioonnss.was orbesseurlvtesdweirnethreeparbosdeuncciebleofian calneairndedpoesned-ernetslpyonsreeperaetleadtioenxspheirpi,menbtu.t the Achrtoemstososmuebstaabnecreratwaisoncotnessitdeirf:ed negative (not clastogenic) in the a) N(oCnhei-soqfuatrhee tteesstt,edPco<n0c.e0n5t)ratiinocnrseasienduicnedthea snutmabteirstiocfalcleyllssigwniitfhicant chronosone aberrations. Tehneterpreicnetodintghecrfiitnaelriaevawlerueatinoont adebcsiosliuotne. and other modifying factors might Tahbeerriantciiodnesn,ceionfcluasbievreranort ceexlcllsusi(vceellgsapsw)ithforoneeacohr mtorreeatmchernotmogsroomuep was compared to' that of the solvent control using Chi-square statistics: X2 = ((aN+-b1)) (xesd)(ad(-abvce))2 (B90) wherebd == tthhee ttoottaall nnuusmbbeerr ooff anboenrarbaenrtranctellcsellisn tihnethcoentcroonltroclultcuurletsu.res. ng3 == tthhee ttoottaall nnuusmbbeerr ooff caeblelrsranstcorceedllsin itnhetrecoantterdolculctuultruersest.o be = cthoempatroteadl wintuhabetrheocfonntornoalb.errant cells in treated cultures to be ny = ctohenpatroteadl wintuhabetrheocfoncterlolls. scored in the treated cultures. W = sun of ng and ny I i we |IX2> TEE (N(-1)) x @(aod-bcG)2a | (two-tailed) 1cshrosmraolslone(P<ab0e.r0r5a)tiotnhes hiyspotthheessiasmethfaotr tbhoethinthceidetnrceeateodf caenldlsthweitsholvent cgornoturpol1sgrcoounpsidiesrerdejteoctebde asnidgnitfheicannutmlbyerdoifffaebreernrtanftrocmeltlhse icnonttrhoeltegsrtoup at the 95% confidence level. - pege=z2 - 004508 T-5874 HOTOX Project 115919 RESULTS DOSE SELECTION Iung aT-p5r8e7l4impienrarnly csutlutduyreblomoeddiucmultwuirtehsanwderewitthroeuattedS9-wmiitxh. 3,Hig10h,er33c,onc1e00ntraantdio3n3s3 tchoeuldculntoutrebemetdeisutne.d, because of the low solubility of the test substance in TABLE 1 MOIFTOTT-I5C674INDEX OF DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS PILOT STUDY, Tceosntcensturbasttiaonnce (ug/m) Co24nthrof1i3x)ation period 103 10303 3330) 48 h fixation period Cont3ro13) 1303 100 3330) NumberRosoTuoEef metaphases per 1000acteells of control Without metabolic activation (CSTR) 868 1800 7724 8824 a a7 27 31 3365 110003 3a5 1u070 33 9 12 34 C2o4nthrof1idx)ation period 103 10303 333%) With metabolic activation (s59-mix) 19057 11030 8937 10827 108 109 12 118 ab)) OTHesStO substance precipitated slightly in culture medium. - Taped - 004509 T-5674 ; TTT worox project 11s919 Bsaesleedctoend ttohederteesrunlitnseofa of the mitotic index: tchoinscenptirlaottiosntudwyhicthhe cafuosleldowitnhge adopsperoplreivaetles wienrheibition Experiment 1 Without S9-mix : 1(02,4 3h3,fi5x6a,tio1n00,per1i7o8d)and 333 ug/ml culture medium With S9-nix 3130,, 31030,,101078anadnd333333ygu/gn/lml cu(l48turhefimxeadituinon period) (24 hand 48 h fixation period) Experinent 2 Without 9-mix With S9-mix : 1(02,4 h33,Fi5x6a,tio1n00,per1i7o8d)and 333 ug/ml culture medium 1(02,4 3h3,fix1a00tioanndpe3r3i3odu)g/ml culture medium pmTaaolbselietsidvoen2oroa)rndnter3geaastthieovdwewtichotenhtmrivotalortiioscuusbsittnaednsectxesso.fubsctualtnucreescon(cfernotmrabltoioodnsoforawihtehaltthhye cBahsreodnosoonnethaesbeerroabtsieornvsa:tions the following doses were selected for scoring of Experiment 1 Without S9-mix With S9-mix Experiment 2 5(62,4 h100fixaandtio1n78peurgi/mold)culture mediun : 3137,8 u1g0/0mland(48333h uFgi/xmaltiocnultpuerrieodm)edium 1(0204 ahndfi3x3a3tiopn/pmeri(o4d8)h Fixation period). Without S9-mix 3(32,4 h100fixaandtio1n78peurgi/mold) culture mediun With S9-mix : 3(32,4 h100fixaandti3o3n3peurgi/mold)culture mediun CYTOGENETIC TEST ------------------------------ Tlhyempahboiclyitteys independent ewoxafpseTr-ii5nm8ve7en4sttsit.goatTiehnded.ucreTehseculhtrtsoemstoosfowmadesuplcaiabcrearrtireeadtciuoolunttsuriienns dhuuamrpealniciapntederiicpaihnteerdtawlboy A vgaaanprdsi)o8.us`aTnhcdeontchseecnontruremasbteifroosnrsotofhefthtnehuenbveatrressitouossfubtasybtpeearsnrcaenotfarcceehlrlposrmeoss(eoinmnteceldausbieivnrerTaatabinlodenssex4ac-tl9u.sthievTehe Acprpietnedriixa a1.ccording to which the aberrations were classified are outlined in sBiotttahhticsihtnrioctsaholesloypnreeasndeanbceberiroalatonigdoincasab.lsleyncesigonfif$i9c-aanitx ithnecretaessst isnubsttheancnuembdeir.d6nbot4i5nduoce a T-5674 NOTOX Project 115919 1cT.hu1eltun(rumemesbaenrwe+roefstwaceinltdlhasirndwitdtheheviaclthairbooonmr)oastoaombreeyrrhaaibnsettrorrcaietclialolsnspceofrnoturn1od0l0 imdnaetttahaephrasasoneglseven((tiw.iect.ohnotu1rt.o0lS53pmmiirxxo;;ducggeaadppsssetexxacctlliuusddteeiddc))a)l.layndThsei0.g6npiofs+iict0ai.nv7teacbioennrctrrraeonaltsesccehleilmnsictaphleesrfr1(0eM0qMuCe-mnCectyaapndhoafsCePa)sberb(rowatinhtth s9tcehlalts.theItmewtaasbotlhiecrefaocrteivactoinocnludseydstetmhat(5t9h-emixt)estfucnocntdiiotneidonsprowpeerrelyo.ptimal and TFi-n5a8l7l4y,is intotis clcaosntcolguedneidcthuandterthitsheteesxtpersihmoeunltdalbecocnodnistiidoenrsedofvaltihdis antdestt.hat 3 - Pages.- i 004511 T-5874 NOTOX Project 115919 TABLE 2 OMFITO1T-I5C674INDEX OF DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS -_-- Experiment 1 coTnesctensturbasttiaonnce (ug/ml) NumberRosaosf metae phasees pPeerrc1e0n0t0eagceeelles b) of control 24 h fixation period C1on0tro13) 5363 17080 MH3C3-3C;) 0.2 ug/ml C4o8nthro1tai.tion period 10303 178 MI3C33-C); 0.1 ug/ml Without metabolic 2278 -- 3337 283--3082 388--217 295.. 211 activation 10908 113s9 16071 383 (CSR 3366- -234 1u0w 0 23--2211 E0d 221-. 283 ia8 C2o4nthrof1i3x)ation 1330 100 3CP3;361)5 ug/ml 48 h fixation Con1t0ro13) 10303 3336) period period -_-- ab)) odmuspolicate cultures ) Test substance precipitated With metabolic activation 690 -- 8935 10902 8886-- 10852 19180 5388-- 7370 7389 6588 -- 7651 18030 67-- 662 8988 29-19 34 slightly in culture medium. (s59-mix) - pegs - | 004512 g 2 H T-5874 S- ro HOTOX Project 115919 TABLE 3 OMFITOTT-I5C874INDEX OF DONOR CULTURES TREATED WITH VARIOUS CONCENTRATIONS _--Exm perinent 2 coTnecstensturbasttiaonnce (ug/m) E NumRhbseorTuesof mee taphases pere 1000 cells b) of control 24 h fixation period Cont10ro1d) 536 117080 H3C3C3;) 0.2 ug/ml Without retanoTic activation (Sang 2169--322 10704 228-- 231 1072% 210--1251 570n 57--195 5228 Co2n4throf1i3x)ation period 3103 130303) cP; 15 ug/ml -_-- ba)) douspolicate cultures ) Test substance precipitated With metabolic activation 566 -- 5610 1800 550.-5571 8854 535--3499 8671 slightly in culture mediun. (+59-mix) - prgodin 004513 i != I -- NOTOX Project 115919 El fnfs nfu] TTABLTE 4 CCHORNOCMEONSTORMAETIOANBSERROAFTITO-N5S874IN(DWOiNtOhRoutCULST9U-rRiExS)3T)REATED WITH VARIOUS 24 h fixation period Experiment 1 som oem am foe [He sire foo aol 0 wofsonal 0 male 0 sal SSoiinttntaainn Eoieoarin Le ems e [T T e a] Tes = le |vm Te, ~ TTT mr lo o f] vo| w [TT rr we [| ee pny *) ) ASSSbooiEbipvrneReinivraoiiiAacsetiyin1otnlGsyTooedufsitemtduemmweeeefEronirete-snvLesafetrreiovronsauorsacciratoittineyinrspoesenlssarooestfwhneodcasosebrrteesorSeepnrbegaLiaitinticsoeianntasbeioeoanrtehetolaeoisnse}etssedemneianntioned abeve. SEL I T Poses 004514 T-se74 NOTOX Profect 115919 TASLE 5 CHROMOSOME ABERRATION IN DONOR CULTURES TREATED WITH VARIOUS TT E2C4xOpNehCrEiNfnTieRxnAatTtIiOo1NnSpeOrFioTd-5874 (With $9-mix)a) Ean BE HENNE Eehen Le s w [o TeT T TT ] T ] > [TT ee ew 1 = rT = rT TT ea Lo [l oe[on, faa Chm. ) Abbreviations used for various types of aberrations are listed in | pages - 004515 Tos74 NOTOX Project 115919 TABLE 6 CHROMOSOME AGERRATIONS IN DONOR CULTURES TREATED WITH VARIOUS E4CxO8pNheCrEiNfnTieRxnAatTtIiOo1NnSpeOrFioTd-5874 (Without S9-mix)8) soon En Cam -: | ve | 2Te [ew| : 1T - Ey Con ) abbreviations used for various types of aberrations are listed in Baio 004516 CONCENTRATIONS OF T-5874(With$9-mix)a) store eT FCE ERS I EN-- PEE S TTT] ) Abbreviations used for various types of aberrations are listed in ozs 0045717; H ~ 3 T-5874 en co - NOTOX Project 115919 TABLE - 8 24h fixation period CCHORNOCMEONSTORMAETIOANBSERROAFTITO-N5S674IN (DWOiNtOhRoutCULST9U-mRiExS)3T)REATED WITH VARIOUS Experiment 2 Sore [teres vin [rms care Tro woh 0 sols + woh 5seh a aa Bel [LL ihaardreet)inon YSJoe.setorri noa.h ww " Fol Lo | e [se e| ew 1 TT ea w TT = TT = 1TT eo |Te a 1TT 1 TT me | El os fo ) ) ACSHSeboiilbpgnvrneepeinivrcoiiikacnetiiyin3sot.nlt(syTreoabeudTysiasermndreaevrorefueirossnrc)taavlSuTaotereeiacsossrusiassuatontinitioeeyoneepneselssassooopkhfnmoedbnaeeymbrreesrSmenreepambintiniisoeeenns4t3iaSortheeCl?oiinsetsedseinntioned avove. mL ne a - - Meee. oo 004518 i . -- on TT CONCENTRATIONS OF T-5874 (With So-mix)) Eh Le Mn -- TC e r T TseT eT ] Te FT ES T NO T IT W I T Fra TTT] esl Lo [en| ) Abbreviations used for various types of aberrations are listed in -- 004519 3 " i T5674 aroun ne - NOTOX Project 115919 DAEPFIPNIETNIODNSIXOF C1HROMOSOME ABERRATIONS SCORED IN METAPHASE PORTRAITS e-- Aberrr-- ation--eeAbbe reviat tion eDes-- cript-- ion ---------- Chromatid gap 9" An achromatic lesion which appears as an unstained region in the chromatid asarnnmda,iltehtrehetashpipazaneretnhoteflywwhiid*ctbhhrokioesfn*etqhuesaelgcrhertnootmsaotriodf the chromatid arm are in alignment. Chromosome gap 9" chromatid break br An achromatic lesion which appears as an unstained region in both chromatids aiotsf tetqhhueealcsharmteoomaportosiidstmiaaolnndl,erthtehtehaanpspiaztreheenotwfliydwthihch i"n"braolkiegn*nmesnetg.ments of the chromatids are An achromatic lesion in a chromatid arm, the size of which is larger than the width of the chromatid. The broken saelgigmneendtsorofuntahleigcnherdo.matid arm are Chromosome break b* Chronatid deletion Aaitns atThcaehrrgeosramnaettihcapnoslietthsieioonwn,iditnthhebootfshiztehcehrofomwahtiicdhs cchhrroommaattiidd.s aThree barloikgennedseorgmeunntalsigonfedt.he Deleted material at the end ofa chromatid arm. Minute mn Double minutes Dicentric chromosone dic Tcrhircoemnotsroimce tric A single, usually circular, part of a chromatid lacking a centromere. Tcvhor,omuastuiadlllyackciinrcgulaarc,entpraormtesreo.f a Acencthrroommeorseosm.e containing two Acencthrroommeorseosm.e containing three - Page 24.- 004529 3 T-s674 - APPENDIX 1 continued NOTOX Project 115919 Aberration Ring chromosome Exchange figure Chromosome intrachange Puelhrvaeorsiozneeds Multiple aberrations Polyploidy Endoreduplication Abbreviation . exch. intra P na poly endo Description A ring structure with a distinct lumen. An exchange(s) between two or more chromosomes resulting in the formation of a tri- or more-armed configuration. A chromosome intrachange is scored after rejoining of a lesion within one chromosome. A fragmented or pulverized chromosome A metaphase spread containing ten or m(ocrheroomfatitdheanadbovcehrommeonstoimoenedgapasbenrroattions included). A chromosome number that is a multiple of the normal diploid number. Acenftorrammeorfepocloynpnleocitdsytwion worhicfhoureapcahirs par. of chromatids instead of the normal one Tees 004521 T-5874 NOTOX Project 115919 APPENDIX 2 STATISTICAL EVALUATION OF TEST RESULTS chi-square Test T(OITNACLLUNSUIMVBE/EERXCOLFUSCIEVLELSGAWPIST)H. ABERRATIONS; TREATMENT/CONTROL COMPARISON, Experiment 1 DTORSEEATM(EuNgT/ml) SI-MIX GAPS Pt-wVoA-LsUiEded CDOENCFIISDIEONNCEATLE9V5E%L 24 h fixation period MiC-C (0.2) - .- 00..00000044 ssiiggnniiffiiccaanntt cP (15) . .- 00..00000044 ssiiggnniiffiiccaanntt 48h fixation period MHC-C (0.1) - .- <00..00000048 ssiiggnniiffiiccaanntt Experiment 2 DTORSEEATM(EuNgT/a) S9-MIX GAPS 24 h fixation period MiC-C (0.2) - .- cp (15) + .- P-twVoA-LsUiEded 00..00000044 00..00000044 CDOENCFIISDIEONNCEATL9E5VE%L ssiiggnniiffiiccaanntt ssiiggnniiffiiccaanntt -cpage--se - 004522