Document ByV7aME30ojedYzgdwY69Qwo8

o AR22e-0431 `CORNING Hazleton MUTAGENICITY TEST ON T6564 ; MEACSHUIRNEISNEGHCAHMRSOTMEORSOOVMAARLYA(BCEHROR)ACTEILOLSN:S IN WITH A CONFIRMATORY ASSAY WITH MULTIPLE HARVESTS EINALREPORT : AUTHOR : Hemalatha Murl, Ph.D. . PERFORMINGLABORATORY Comi9n2g0H0azLleeetsobunrIgncP.ik(e.CHV) ~~ I Vienna, Virginia 22182 LABOPRR OJEA CTIT DENTOIFIRCATY ION CHV Study No.: 17750-0437CO SUBMITTETDO 3M Corporation Building 2202E-02 3M Center St Paul, Minnesota 55144-1000 SCTUODYMPLETIDOATNE September 16, 1996 CHV Study No.: 17750.0437C0 Tof30 i C00202 `CORNING Hazleton i QUALITY ASSURANCE STATEMENT Project Title: Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells: With a Confirmatory Assay With Multiple Harvest Project No.: 20990 Assay No.: 17750 Protocol No.: 437CO Edition No.: 4, Modified for 3M Corporation `pUQrnuoiajtleicattnydwAeasrcsecuorcraodnnicdneugcittnoesdptehacectgcieoonrnedsorifanltghrteeoqtushiterueSdmtyeaannntddsarordfetvOhipeeewraaoptfpitrnhogperPifraiotncaeleGdrouerpoeodrsotLofaftbtohhreaetQaourbayloivPteryarcAetsfisecrueernacnecde raengdultaotitohnes.stuFdiynddiinregcstofrroomn tthhee ifnoslpleocwtiinognsdaatensd: final report review were reported to management i InspectionDate EndingsReported Auditor Dosing/0620/1996 0621/1996 C. Orantes Draft Report Review/0821/19% 0822199 C.Orantes Final Report Review/09/16/1996 09/16/1996 C. Orantes i Quality Assurance i CHV Study No.: 17750-0-437CO : Date 2/Fe0l6 fe 2 00203 `CORNING Hazleton STUDY COMPLIANCE AND CERTIFICATION study design and evaluation criteria. `The described study was conducted in compliance with the Good Laboratory Practice regulations RaesgsueltaftoirotnhsinPartthe58F,oiosdsuaenddDDercuegmAbdemrin2i2s,t1r9a7t8i,on(e(fFfeDcAt)ivTeitJluen2e120o,ft1h9e79U).Sw.itChoadneyofapFpeldiecraballe amendments. There were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrityofthe study or the interpretationofthe test results. `The raw data have been reviewed by the Study Director, who certifies that the evaluationofthe test article as presented herein represents an appropriate conclusion within the contextofthe Al test and control results in tis report are supported by an experimental dat record an this record has been reviewed by the Study Director. All raw data, documentation, records, protocol . afancdilaitceospoyfCofotmhienfginaHlazrelpeotrotngIencn.efroaattaesdlaastreosnuletoyfeatrhifsosltluodwyinwgilslubbemiasrscihoinvoefdthinetfhienasltorreapgoer.t 0 :: ! tt`mihameteeS,rpiooarnlssseornrte.ttaAinfaetdsetriontrhtaehgeeonsfetaocyirelaiagtrey pfdaecriieloidts,ietsoihefbgSCypoontmnhisaeonSrgptomHnaeasyzoldreel.teocnt tIonch.afvoer tahneaadfdoirtieomneanltipoernieodd of Submitted By: `Study Director: Hsin Moi : MDHaeemmpmaaalralmtiehanatnMouCfryltGioe,gneePnthe.itDci.casnd Cellular Toxicology elo Date Study Completion i CHV Study No.: 17750-0-437C0 ; 3 00204 to TAOFBCOL NTEE NTS `CORNINGHazleton Page No. 10 SPONSOR ....oooiiiiiiiiiiiiennieiiiiie eee] 20 MATERIAL(TESTARTICLE) .....ooviiiiiiiiia. 21 Client' Identification 7 22 Date Received 23 Physical Description : 24 Genetics Assay No. 30 TYPEOFASSAY .......ooooiiiiiiiiiiiiiiiiiiiiiiiininaaanaaee 1 : 40 PROTOCOLNO............oooiiiiiiiiiiiiiniiiiiiiiiiiiiiiiininin nl] 50 STUDYDATES .........occoiiiiiiiiiiiiiiiiiiiiiiiiiininnnaaenen] 5.1 InitiationDate 52 `ExperimStearnttDaatle 53 Experimental Termination Date > 60 SUPERVISORY PERSONNEL .......0oviiiiiieuuiiiiiiiiiininananenT 6.1 Study Director 62 Laboratory Supervisor 70 OBJECTIVE .....iiiiiiiiiiiiiiiiiiiiie eee 7 80 RATIONALE............ocoiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiniii 8 90 EXPERIMENTALDESIGN ..........ccooviiiiiiiiiiiiiiiiiiiiiiiino 8 100 MATERANIDMAETLHODSS ......oooviiniiiiiiiiiiiiiieeeeieee 101 TestCells 9 . 102 Cell Culture Medium 103 Negative and Solvent Controls 104 Positive Control Agents 105 Rangefinding Assays 106 Aberrations Assay Without Metabolic Activation \ CHVStudy No.: 17750-0-437CO 4 ! C00205 i CORNINGHazleton 10.7 Abemations Assay With Metabolic Activation 108 HarvestProcedure . 109 Slide Preparation and Staining 10.00 Aberrations Analysis and Assay Evaluation TO RESULTS ooo ILI Solubility and Dose Determination 13 112 113 RRaannggeeffiinnddiinngg AAssssaayy WWiitthhoMuettMaebtoalbioclAicctAicvtaitviaotnion : 114 ILS CChhrroommoossoommaall AAbbeerrrraattiioonnss AAssssaayy WWiitthhoMuettMaebtoalbioclAicctAicvtaitviaotnion ! 120 CONCLUSIOoN.oo 18 130 REFERENCES ..... srtasrssssasenseipeacsncasaenrened : 140 DEV FROI MTHA ESIT GNEI DPROO TOCN OL ............oeeeene... 18 150 EXPERIMENTALDATATABLES ................c.ccoeourrinnnenin 19 : 160 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CHVStudy No.: 17750.0437C0 i i 5 GCOZ06 | `CORNING Hazleton ABSTRACT "Theobjectiveofthisinvitoassaywas to evaluate the ability ofT-6564 to induce chromosomal `aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation. a`Tnhdehsipesciwfaisc gursaevdittyoofcatlhcuelanteeatthteesdtoasritnicglestwoacsk peevaplaucaatteodna.s D1i.l2u2tgi/omnlsoifn tthhee etessttianrgicllaebowreartoery `prepared in sterile deionized water. For the dose rangefinding assays with and without metabolic cw`ayectrtoietvoatxteiisoctnie,tdy.cowAnalcsednotobrssaietnirgovnwesadosifn0atc.hh1ei6e9cvu,eld0tu.ur5se0is8n,dgo1sa.e6d9do,sw5ii.nt0gh8,vSo01l68.u09m,ue5go0/f.m8l,1.w10i6%t9,h(a510n08d,uwm1il6t)9h0o.,utaCnmodemt5ps0lb8ea0tlecpg/ml uacgt/imvaltiwoint.hoAutrmeedtuacbtoiolniocfac9t2iv%atwioansaosbcsoermvpeadreidn twhiethmitthoetiscolivnednetxocofnttrhoelccuullttuurree.doRseedducwtiithon1s6o9f0 `4a4n%d ,161950%,uga/nmdl 3w8it%hwemreteabooblsiecravcetdiviantitohne, mrietsopteicctiivnedliyc,eassof tcohmepacurletdurweistdhotsheedswoiltvhent16c9o,nt5r0o8l, culture. analyzed for chromosomal sbemations. No significant increas in cells with chromosomal Based on the data from the dose rangefinding assay, replicate cultures of CHO cells were 1 incubated with 62.5, 125, 250, 500, 1000, 1500, and 2000 pg/ml without metabolic activation * `and with 250, 500, 1000, 2000, 3000, and 4000 pg/ml with metabolic activation in 20.0 hour assays. Cultures treated with 125, 250, 500, and 1000 ug/ml from the assay without metabolic `activation assay and with 250, 500, 1000, and 2000 ug/ml with metabolic activation were aberrations was observed at the concentrations analyzed. Inthe confirmatory trial, replicate culturesofCHO cells were incubated with 100, 200, 400, 600, . i 8`w0i0t,ho1u0t0m0e,taanbdoli1c20a0ctuigv/amtilonanwditwhit20h.510.a0n,d 14040.,22h0o0u,r4h0a0r,ve6s0t0s,, 8r0e0s,pec1t0i0v0e,lya.ndIn12t0he0 apsgs/amylwith . `metabolic activation, cultures were incubated with 500, 1000, 1500, 2000, 2250, 2500, 2750,and : 3000 pg/ml with 20.1 and 44.2 hour harvests. Cultures treated with 200, 400, 600, and 800 pg/ml from the 20.1 hour nonactivation assay, with 100, 200, 400, and 600 pg/ml from the 44.2 hour nonactivation assay, with 1500, 2000, 2250, and 2500 pg/ml from the 20.1 hour . w`aabecertreirvaaattniiaoolnnysawzseasdasyo,orbasncedhrrwvoiemtdohas2to0mt0ha0el,c2ao2bn5ec0er,ntti2ro5an0ts0i.o,nasNnaodnas2li7gy5nzi0efdipcgea/xncmtelipnftcrrfoeomarsttehheeinh4ei4ge.hl2ehssotwuirdtoahscectihavrnaoatmlioyonszeoadmsaslay ; (2500 ug/ml from the 20.1 hour activation assay and 2750 g/ml from the 44.2 hour activation assay) from the activation. T6564 was considered negative for inducing chromosomal aberations in CHO eels with and s`iwgintihfoiutcamnettoabxoiliicyactivation, except at a single dose level with metabolic activation that induced : CHV Study No.: 17750-0-437CO : 6 00207 CORNINGHazleton Chromos`WoimtahlaACboenrfriartmiaotnosriynACshsianyesWeiHtahmMsutletripOlveaHrayrv(eCsHtOs) Cells: : With T6564 10 SPONSOR: 3M Corporation 20 MATERIAL (TEST ARTICLE): 21 Clients Identification: T-6564 L-13167 FC-1015-X 22 Date Received: May 30, 19% : 23 Physical Description: Clear, colorless liquid i 24 Genetics Assay No: 17750 : 30 TYPE OF ASSAY: C`hWirtohmaosCoonmfailrmAabteorrryatAisonssayinWCihtihneMsuletHipalmestHearrvOevsatrsy (CHO) Cells: 40 PROTOCOL NO.: 437CO, Edition 4, Modified for 3M Corporation 50 STUDY DATES: 51 Initiation Date: June 4, 1996 : 52 Experimental Start Date: June 13, 1996 ! 53 Experimental Termination Date: July 26, 1996 60 SUPERVISORY PERSONNEL: 6.1 Study Director: Hemalatha Murti, Ph.D. i 62 Laboratory Supervisor: Carol. Spicer, B.S. 70 OBJECTIVE: c`ThhreoombojsecotmiavleoafbterhriastiinonistirnoCahsisnaeysweahsamtsoteevralouvaatreyt(heCaHbOi)litcyeollfs,Tw-i6t5h6a4n1d0 wiintdhuocuet `metabolic activation. CHV Study No.: 17750-0437C0 7 00208 `CORNING Hazleton 80 RATIONALE: `wTihtehacseslalsy tios diensdiucgenecdhtrooemsotasbolmieshbwrehaektsh.erCthheemitecsatlalrytiiclneduocreidtslmeestiaobnoslmitaeys rceasnulitntienract obrreraeksusltinicnhvriosmibalteindtahmaatgaer.ecAibtehrerratriepoanisraerdbe yactohnesceeqllueinncseuocfhfaaiwlauryeaosrtmoisbteauknedsetinecrteapbalier processes 1962). such that breaks do not rejoin of rejoin in abnormal configurations (Evans, 90 EXPERIMENTAL DESIGN: Results from the rangefinding assay the chromosomal aberrations assay. were used to determine the dose range In the rangefinding assay, the cultures to be were used in thhaervceusltteudre2s0w.0erheouervsalaufaiteerdinfiotriaetviiondoefncteroefattmoexincti.tyM.itAotsicumimndaerxyaofndthveisutarleaotbmseenrtvation of schedule for the rangefinding assay is given below. i D. find I 5: Test Test Anicle -s9 0 +59 0 Wash Colcemid Fixation 178 180 200 30 180 200 `Ianndthneegcahtriovmeoasnodmasollvaebnetrrcaotnitornoslsa.ssaSyisn,glreepcluilctautreecsuwleturreesusweedrefoursceadcahot featcwhododsoeselseovfelt,he positive control. The aberrations the initial trial and with 20.1 and assays were conducted 44.2 hour harvest times with a in the 20.0 hour harvest time confirmatory trials. in i fCrhormomoonseoofmtahleabpeorsriattiivoencsownterroel adnosaelsy.zedAfsruommmtahreycouflttuhreesttrreeaattmeednattsfcohuerdduolseefolrevtehles and chromosomal aberrations assays is given on the following page. : CHV Study No.: 17750-0437CO 1 : 8 G00209 po i i CORNINGHazleton i . Test Test Aticle Wash _Coleemid Fixation InitialTrial -59 0 178 180 200 +59 0 30 180 200 ConfimatoryTrial -59 0 178 181 201 +59 0 30 1 201 -s9 0 as a2 442 } +59 0 30 a2 42 100 MATERIALSAND METHODS: 10.1 Test Cels: p`TehremCahniennetscelhlalmisnteearnodvwareyreceolrlisg(inCaHllOy-oWbtBaLi)nedusferdoimntthheislaasbosraaytworeyreoffDrro.mSa. i s`eWcollofn,eUdntiovemrasiinttyaionfCkaalriyfootrynpiiac,SsatanbilFirtayn.ciTshcoi.s ceTlhlelicneelhlsashaavneasvienrceagbeeceyncle time 0f 1210 14 hours with a modal chromosome numberof21. 102 Cell Culture Medium: "sTuhpepCleHmeOntceeldlswiwtehre1g0r%owfnetailn bMocvCionye'sserSuamcu(lFtBuSr)e,m1e%diLu-mglwuhtiamcihnew,asand 1% { p5e%nicCiOll,ininanird.streptomycin, at approximately 37C, in an atmosphereofabout 103 Negative and Solvent Controls: cIenltlhseannodncauclttiuvraetimoendiasusma.ys,Sonlevgeantitvceonctornotlrsolwsewreerceulctuulrteusrecsonwthaiicnhincgontthaeinsoolnvleynt : CHV Study No.: 17750-0437C0 9 00210 1. CORNING Hazleton : i cfuolrttuhreest,es1t0.ar0tiuclmel, .sterIinlethdeeiaocntiivzaetdiownataesrs,ayast,tthheenhiegghaetsitvceonacnedntsroaltvieonnt cuosnetdroilnstest. mweirxeintchleusdeadm.e as described in the nonactvation assays but with the S9 activation 104 Positive Control Agents: (ThMeMpCo)siftoirvethceonntornoalctaigveanttsiownhsiecrhiews earned ucsyecdloipn htohsepahsasmaiydsewe(rCeP)miintotmheycmeitnaCbolic cacltaisvtaotgieonntsheartiedso.csMintotomryecquiinreCme(tCaAbSoHli5c0a-c0t7i-va7t,ioSni.gmCay,cLlootph#o2s5pHh0a6m1i9d)e i(sCeAS # a6c0t5i5v-e19in-t2e,rmSeidgimaat,esLboyt#mi6c7r0o1so5m5a)ldeonezsynmoetsa.ctIndtihreectclhyrboumtomsuosmtalbeabceornrvaetritoends to a1s0s.a0ysg,/tmwo)cwoenrceenutsreadtitoonsinodfuMceMcChro(0m.o0s8o2mnadl0a.1beugr/mrla)taiinndotnhCsePC(5H.O0 acneldls, One ! oCfPthweerdeosdeislseovlevlsedwianswaantaelr.yzed in eachofthe aberration assays. Both MMC and 105 Rangefinding Assays: tIrnetahtemseentasbsyaysse,etdhiengcealplsprwoexriemactueltluyre0d.3fxor1a0ppcreolxlismpaetrel2y5 2c4mhofulrasskpriinotro toml of complete McCoy's Sa culture medium. 105.1 Assay Without Metabolic Activation: : `TThheenc,utlhteurteesstwaerrteicliencwuabsatweadswhietdhftrheomtetshtencielclls wfiotrh1p7h.o8shpohuartseabtuf=f3e7reCd. : scaolnicneentarnadtifornes0h.1coumgp/lmelt)ewmaesdaidudmedc.onTthaienicunlgtuCroelscweemriedth(efinntalrypsinized and harvesied 2.0 hours late. (See Sections on Harvest and Slide : Preparation and Staining). : 10.52 Assay With Metabolic Activation: aItn =t3hi7asCsainy,tthheeprCeHseOncceelolsfawerareievxeprose9drteoactthieontemstixatrtuircele(f9or 1t5hrleelho,urs ; N(MAolDePcu1l.a5rmTgox/imc,olaongdy,isIoncci.t,riLcoatc#i0d528.37 umsge/dm)f.or dTohseeSra9nfgreafcitnidoinng assay and initial rialof chromosomal aberrations assay; Lot# 0667, usedforthe CHV Study No.: 17750-04370 10 i 0211 : CORNING Hazleton : confirmatory trialof chromosomal aberrations assay ) was derived from wtihethliAvteorcolfomral12e5S4p1r0aignudeu-cDeatwhleeymirxatesdwfhuincchtihonadoxbiedeansepreenvzioyumselsy wtrheiacthed are capableof metabolizing chemicals to more active forms. The three `mhioxutruirnecumbiagthitonbetitmoexiwcatso uthseedceblelscaaunsdetphreoelnonzgyemdeeaxctpiovistuy rtoofeSt9heisS9lost rapidly at about 37C. The medium did not have FBS during the exposure `period to avoid possible inactivationofshort lived and highly reactive intermediates produced by the S9 enzymes by binding to serum proteins. sAafltienre.thCeoemxpploestuereMpceCroiyo'dsth5eacmelelds iwuemrewwaassahdeddetdwtiocethweictuhlbtuurfefserwehdich : w0c.ue1lrtegur/temhselwn)eiranedcdutbehadetnfeodtrrftyohprsi1ln7ai.zs8e2dh,.o0uhrhasrovuwerissttheodC,cooflliclxeeecdmt,imdaentd(afspiinhadalesscewonceecrlelesn,tprraTethpieaorned : and stained as was described for th nonactivation rangefinding assay. i 1053 Assay Evaluation: i Mitotic index was analyzed from the highest four (nonactivation assay) and three (activation assay) surviving dose levels by analyzing the number `of metaphases present in 1000 consecutive cells. 10.6 Aberrations Assay Without Metabolic Activation: . Caunldtu0.r8esXwe1r0eceilniltsia(t4e4d.b2yhosueredaisnsgaya)pppreorx7i5mactme?lyfl1a.s2kxin1t0o 1c0elmlslo(f=2c0ohmopulretaessay) i McCoy's Sa medium. One day after culture initiation, the cells were incubated at c=u3l7turCewsiwtehrtehtehetnestwaarsthieclde waittphrebdueffteerremdinseadlidnoesaensdfcorom1p7l.e8oterM4c1.C8oyho'usrs5.a. The : . `medium containing 0.1 pg/ml Colcemid was placed back onto the cells. Two hours later, the cells were harvested and air dried slides were made. The slides 4 were then stained in 5% Giemsa solutionfor the analysisofchromosomal iE aberrations. ; 107 Aberrations Assay With Metabolic Activation: Cultures were initiated by seeding approximately 1.2 x 10 cells (=20 hourassay) aMncdCo0y.8'sX 51a0mecedlilusm(.44.O2nheoudrayasasfatyr) cpuelrtu7r5e cimni'tifaltaisonk,itnhteo c1u0lmtulroesftchoatmpwleertee 3 CHV Study No: 17750-0437C0 n 00212 CORNINGHazleton ttrheraeteehdouunrdseirntthheecpornedsietnicoenosoffthmeettaesbtolaritcicalcetiavnadtitohnewSe9rereiancctuibonatmeidxtatur=e3i7nC for `MwceCreoyw'asshSeadmtewdicieuwmiwtihtbhuofufterFeBdS.salAifnieeartnhdetthherceeellhsowuerreexrpeofseudrweiptehricoodmptlheetceells MupctCooyt'hestSiamemoefdihuamr.vesTthweitchel0l.s1weurge/milncCuoblacteemdifdor tphreesreensttodfutriengctuhletulraestp2e.r0iod chyotuorgseonfeitniccuabnaatliysoins.. The metaphase cells were then harvested and prepared for 108 Harvest Procedure: : `PTrhieosretoobtsheervhaatrivoensstoifntchleudceudltaunreass,sevisssumaelnotobsfetrhveatpieorncsoenfttocxoincfiltuyenwceeroeftmhadeec.ell t `pmroensoelnacyeeorfmwiittohitnicth(elacrugleturroeufnldaesdks.cTehleorcludlsetau)rdescewlelsreflaolastoinegvailnutahteedmefdoirutmh.e i `mTihteotciucltaunrdesinftreormphtahseedcoesllesraanndgewfeirneditnrgeaatsesdaywiwtehr0e.t0r7y5psMinKizCeId fhiyrpstottoonciocllect csohlruotmioons.omTehsi.s tTrheaetcmuelnttuhreeslpwsetroestwheelnlftihxeedcewllisthanadntahbussolduitsepemrestehtahneol: glacial ! acetic slides acid were (p3r:e1p,avrevd).fixative and were washed several times before air-dried 109 Side Preparation and Staining: `STlhiedessliwdeesreweprreepsatraeidnebdywdirtohpp$i%ngGitheemhsaarvseoslutteidocnuflotrurtehseoannacllyesains solfides. ! maintdotciocveirndselxipapnedd cushirnogmoDseopmeaxl amboeurrnattiionngs.medAlilumsl.ides were then air-dried 10.10. Aberrations Analysis and Assay Evaluation: : Cceelnltsrowmeerreesseelqeucatledtofotrhegomooddamlonrupmhobleorg2y1an2d1on(rlaynegeel2s0-w2i2t)h twheerenuamnableyrzeodf. Othneetehsutnadrrtiecdlecealnlds,iffrpoomsstihbelnee,gratoimveeaancdhsroelpvleincattceocnutlrtoulrecualttufroeusrwdeorsee alnevaellyszeodf f1o6r.0t)h.e dAitflfeearsetnt2t5cypeelslosfwecrheroamnoaslyozmeadlfoarbecrrhartoimonosso(mEvaalnsa,ber1r9a6t2i;onSseferSoemctoinoen of ctehlelspowsiitthivoenceonotrrmolorcueltaubreersraatniodnsf.roFmotrhcoosnetrcoulltoufrebsiatsh,atalhlasdligdreesateexrcetphtanfo5r0t%heof positive controls were coded prior to analysis. Cells with aberrations were : CHV Study No.: 17750-0437CO 12 : 06213 CORNING Hazleton . : recorded on the data sheets by the microscope stage `eaxspsersesesdsbesydsaanapleyrzcienngtatgheeonfummibteortoicfmcieltlost.ic cells in location. M 1000 cells itotic index and the ratio was was `cThhreofmoolsloomwainlgafbaecrtroartsiownesredattaa:ken into account in the evaluationofthe Lhe percentageof cll with any aberrations. 23.. "dATonhsyee,peeLve, rpicdoeensntciaegteofiofrvcieenlcdlrosesaewsiirnteghspamomonorsueen.tthsanofdonaemaabgererawtiitonh.increasing Chromatid and isochromatid gaps, ifobserved, were noted in the raw data and i : ra`bweieplrrieetsyteanobtfutlrahuteeedtc.estThahreriyewoleormeibnonordteus,achkeooscwahemnrvdeoamrm,oalscyoomnpasilodaerbseedrsbaieniiiotnnhbedsulescviaeylndubcayttihtoeonyxoimfctaihytyenot : Percent polwyasapnallyzeodfriomtdhe4y4.2hourassayandresulwetrse tabulated. Historical control data are presented in Table 8. that itis minimum number. A cell classified as "GT" is considered to contain 10 aberrations for statistical purposes but a ">" is also included in the tables for this classification to indicate Statistical analysis employed the Fisher's Exact Test (Sokal and Rohlf, 1981) to recsoumlptasrfertohme tpheercseonltveangteocfocnetrlollss.wiAthlaibneearrrattrieonndstiensteoafcihnctrreeaatsmienngtngurmobueprowiftchetlhles a`wwneitcrheatbasekiregrnnaitifinioconasnawcciectowhuanistnecasrntedaabstlihinesghfdeiodnsawehe(evArarlemuipat<ta0igo.ne0,1o.ft19hA7el1)efwsaasaracilcasslooesparwetaresfsodbrampsereeddv.iuoTpuesoslnty scientific judgement. 110 RESULTS: 111 Solubility and Dose Determination ihe same pHa that of MeCoy's $ culture medium) was determined. The `The test article was soluble in water. A clear, colorless solution was obtained ata concentrationof500 mg/ml in water. Thissolution was dosed in the absence of - cells using a 1.0% (10 ul/ml) dosing volume in McCoy's Sa culture medium. Ata dosed concentrationof5000 ug/ml, a clear solution was obtained and a pHof 8.0 CHV Study No.: 17750-0-437CO 13 : 00214 1 CORNING Hazleton i s1p.e2c2ifgi/cmlg.ravTihtiysosfptehceifitcesgtraarvtiitcylwe awassuscealdcutloatperdepianrethdeotseisntginsgtolcabkosrfaotrortyheasassay. tShtiesrialsesadye.ionAilzleddowsaitnegrw(aBsatacchhi#ev1e9d, wprietpahare1d.a0t%C(H10.V0 ) uwlamsiu)seddosaisngtoheftvehheicdloesiinng dsetiocoknsizaenddwtahteers.olCvoenntcecnotnrtaotliocnusoltfur0e.1w6a9s,d0o.s5e0d8,wi1t.6h9,105..008i,m16i.9o,f5s0t.e3r,il1e69, 508, of16t9h0e, atensdt a5r0t8ic0lepugn/dmelrwtehreeptreesptaerdatiinotnhaenddosdeosriannggecfoinnddiitnigonasssuasyesd. iTnhtehisstaabsislaiytyis the responsibiltyofthe sponsor. 112 Rangefinding Assay Without Metabolic Activation A<5d%eacdelclemllonmoolnoalyaeyrerc,onnfoluveinscieblweemrietootbiscecrevlesd,ifnlotahteicnugldteuareddcoelslesdawnidthdebris, and ; 5vi0s8i0blpegm/imtlo.ticAcnelulsn,hefallotathiyngcedlleamdocneolllasyaenrd, adesbervies,reanreddu=c4t5io%nriendtuhcetinounmibnetrheofcell : : monolayer confluence slightly unhealthy cell were observed in the culture dosed monolayer, floating dead cells and with 1690 pg/ml. debris, and =30% A ; wreidtuhct5i0o8ngi/nmtlh.e ceFllloamtoinnogldaeyberriscoannfdlu=e1n5ce%wreerdeucotbisoenrivnetdhienctehlel mcuolntoulraeydeorsed confluence were was observed in observed in the culture dosed with the culture dosed with 50.8 pg/ml. 167 ug/ml. Floating Mitotic indices were debris analyzed frerdoumcttihoenocufl9tu2re%s dwoassedobwsietrhve5d0.i8n, t1h6e9,mi5t0o8t,icainndde1x6o90ftphge/mciul(tuTraebldeos1)e.dAwith i t1h6e9i0niptgia/lmalbearsrcatoimopnasraesdsawyitwhitthhoeustomlveetnatbocloinctraoclticvualttiuroen.waBsasceodndouncttheedsewirtehsualts, i 2200.000hpogu/rmlh.arvest testing concentrationsof 62.5, 125, 250, 500, 1000, 1500, and 113 Rangefinding Assay With Metabolic Activation { wNiothce5ll08m0onuogl/amly.erNaondotnhoervissiigbnlse omfitvoitsiucalcetlolxsiwcietryewoebrseerovbesderivnetdheincaulntyuroefdtohesed 1 other cultures. 508, and 1690 gM/itmolti(cTianbdliece1s).weRredeuacntailoynzsoedff4r4o%m,th1e5c%u,ltaunreds3d8o%sewdewrieth 169, . o1b6s9e0rvuge/dmiln,trheespmeicttoitvieclyi,ndaiscecsoofmptahreedcuwlitturhetshdeossoeldvewnittchon1t6r9o,l5c0u8l,tuaren.d Based on t`whietsheare2s0u.l0tsh,othur hinairtvieasltabeesrtriantgiocnosncaesnstaryatwiiotnhsomeft2a5b0ol,i5c0a0c,ti1v0a0t0i,on20w0a0s,c3o0n0d0u,ctaendd : 4000 pg/ml. { CHV Study No.: 17750-0437C0 1 i 00215 CORNINGHazleton 11.4 Chromosomal Aberrations Assay Without Metabolic Activation Initial Tia Dead cell monolayers, floating dead cells and debris, no visible mitotic cells, and =95% reduction in the cell monolayer confluence were observed in the cultures - dosed with 2000 pg/ml. Unhealthy cell monolayers, severe reductionsinthe `numberofvisible mitotic cells, floating dead cells and debris, and =45% reduction in the cell monolayer confluence were observed inthe cultures dosed `with 1500 pg/ml. Unhealthy cell monolayers, severe reductions in the number of visible mitotic cells, floating dead cells and debris, and =30% reduction in the cell monolayer confluence were bserved in the cultures dosed with 1000 p/m. Slightly unhealthy cell monolayers, floating debris, and =15% reduction in the cell monolayer confluencewere observed in the cultures dosed with 500 pg/ml. Chromosomal aberrations were analyzed from the cultures dosed with 125, 250, i 500, and 1000 pg/ml (Table 2). Reductionsof 25%, 30%, 87%, and 96% were concentrations analyzed. 3 o1b5s0e0rvpegd/miln,trheespmeicttoitvieclyi,ndaiscceosomfptahreedcuwlitutrhetshdeossoeldvewnittchon1t2r5o,l2c5u0l,tu1re0s0.0,Naond a significant increases in cells with chromosomal aberrations were observed at the i Basedonthe results from thistrial,the confirmatory trialofthe nonactivation aberrations assay was conducted testing concentrations of 100, 200, 400, 600, - 800, 1000, and 1200 g/ml and 50.0, 100, 200, 400, 600, 800, 1000, and 1200 pg/ml with 20.1 and 44.2 hour harvests, respectively. Confirmatory Trial In the 20.1 hour assay, very unbelthy cll monolayers, severe reductions inthe numberofvisible mitotic cells, floating dead cells and debris, and =15% reduction in the cell monolayer confluence were observed in the cultures dosed ; s`weivtehre12r0e0dupcgt/imoln.s iUtnhheeanltuhmybecerllofmvoinsoilbalyeemrist,otfilcoacteilngsdeweardeceolblssearvnedd in the debris,and i crueldtuucrteisondsosiendthweinthu1m00b0opfgv/imsli.blUenmhietaolttichyceclellsl wmeorneoloabyseerrsv,edflioattihneg cduelbtruirse,sand cdluiolgstheutdrerwseiddtuohcst8ei0do0nwsiptginh/mt6lh0.e0nSuulgim/gmbhlet.lryoCfuhnvhriesoaimlbotlsehyommictealolltimacobneeoerllaasiyowenressr,weoflrboseaetarinvnaeglddyiezbnerdtishf,eraonmdthe c8u4l9t%u,raensddo9s5ed% wwietrhe2o0b0s,e4r0v0e,d 6t0he0,maitnodti8c00inudgi/cmelof(Ttahbelecu3l)t.urReesdduocsteidonwsitohf 87030%,, CHV Study No: 17750.0437C0 15 ! 00216 CORNING Hazleton } c1u0l0tu0r,esa.ndN1o20s0igpnigf/imcla,ntreisnpcercetaisveeslyi,nacselclsomwpiathrecdhrwoimthostohemaslolavbeenrtractoinotnrsolwere ob- served at the concentrations analyzed. dIenatdhece4l4l.s2ahnodudrebarsissa,y,anddea=d4c5el%l mroendoucltaiyoenrsi,n tnhoevcieslilbmleonmoitloatyiecrcceloln,flfuleonactienwgere moobnsoelravyeedrisn,thfeloactuilntgurdeesaddocseeldlsawintdh d1e0br0i0s,ansdev1e2r0e0riegd/umclt.ionVseirnytuhnehneuamlbtheyrcoefll ovbissiebrlveemditionttihceccelullst,uraensdd=os3e0d%wrietdhuc8t0i0onpgi/nmtlh.e cUenlhlemaolntohlyacyeelrl mcoonnofllauyeencres,were aflnodat=i1ng5d%eardedcuecltlisoannidn dtehbericse,llrmedouncotliaoynesrincotnhfelnuuemncbeewreorfveiosbisbelrevmeidtoitnitchceells, cdeublrtiusr,esadnodsseldigwhtitrhed6u0c0tigo/nmsli.n thSelinguhtmlbyeurnohfevailstihbylceelmlitmootnioclcaeylelrssw,efrleoaotbisnegrved ifnrotmhetchuelctuulrteusrdeossdeodsweidtwhi4th0010p0g,/m2l0.0,C4h0r0,omaonsdo6m0a0liagb/emrrla(tiToanbslewe4r).e aRneadluyczteidons : odfos1e2d%w,i6th1%5,0.60,3%4,00a,n6d009,2a%ndwe8r0e0oubgs/emrlv,edretshpeemcittiotvieclayis,ndcicoemsopfartehdewicutlhtutrhees t: `saoblevrernatticoonnstorrolincuplotulryepsl.oiNdyowseirgneiofibcsaenrtviendcraetatshees cionncceelnltsrwaititohncsharnoamlyozseodm.al s"Thhoewsnenbsiytitvhityinocfrtehaesecdelflrceuqluteunrceyofofraibnedrurcattiioonnosfinchtrheomcoelslosmeaxlpoasbeerdratotions is `fomritionmdyucciinngC,chtrheompoossiotmiavel caobnetrrroaltiaognesna.nTdhpeoltyepsltoairdtyiculendies rconnosniadcetrievdatnieognative conditions. 115 Chromosomal Aberrations Assay With Metabolic Activation Initial Trial ; Ndoosecedllwimtohno3l0a0y0earnsdan4d00n0ouvgi/smilb.le mSiltiogthitclycuelnlhsewaletrheyocbeslelrmvoendolinayteherscualntdur=es30% rweidtuhc2ti0o0n0iungt/hmel.celAl mroenduocltaiyoenrocfon=f1l5ue%ncinetwheerceelolbsmeornvoeldayienrthceoncfulltuuernecsedwoasesd. observed analyzed finrotmhetchuelctuulrteusrdeossdeodsewditwhit1h002050g,/5m0l0., Chromosomal 1000, and 2000 aberrations were g/m (Table 5). R2e5d0uuctiognsaos/fco2m%mpawrleerde owbistehrtvheedisnoltvheentmciotnottrioclicnudlitcuerseosf.thNeocsuilgtnuirfeiscadnotsiendcrweiatshes i liynzceedl.ls with chromosomal aberrations were observed at the concentrations ana- ; CHV Study No.: 17750-0437CO 16 3 00217 CORNING Hazleton : Basassaeydvoinththmeetraesbuolltiscfarcotmivtathioinniwtiaasl rcaoln,dtuhcetecdontefsitrimnagtcoornycernitarlaotfitohnesoafber5r0a0t,ions, h1a0r0v0e,st1s.500, 2000, 2250, 2500, 2750, and 3000 g/m with 20.1and 44.2 hour Confirmatory Trial vIinstihbele2m0i.t1othiocurcealslss,aya,ndde<a5d %celclelmlomnoonloalyearyse,r fcloonaftliunegndceeawdecreellosbasnedrvdeedbriins,thneo crueldtuucrteisondsosiendthweitnhum2b7e5r0oafnvdis3i0b0l0e mpigt/omlt.ic cSellisg,htfllyoautnihnegadlethaydcceellllsmoannodldaeyberriss,, `caunldtu=re4s5d%osreedduwcittihon2i5n0t0heugc/emlll.moFnloolaatyiengr dceobnrfilsu,esnlcieghwterreeduocbtsieornvseidnitnhethneumber owfevriesiobblseermviteodtiicn ctehlelsc,ulatnudre=s 3d0os%edrewdiutchti2o2n5i0n ptghe/mcle.ll Rmeodnuocltaiyoenrscoofnf=l1u5en%cein the : ! `ceClhlrmoomnoosloamyaelracboenrrfaltuieonncsewweerreeaonbasleyrzveeddfirnotmhethceulctuulrteusredsodsoesdewdiwtiht2h010300p0,g2m0.00, 2250, and 2500 g/m (Table 6). Reductionsof 13%, 38%, 15%, 11%, and 30% : `2w2e5r0e,oabnsder2v5e0d0ign/thmel,mitreostpieccitnidveilcye,soafstchoempcualrteudrewsidtohstehdewsiotlhve1n0t0c0o,nt1r5o0l0, 2000, csuelrtvuereds.at tNhoe csoingcneifnitcraanttiionncsraenaasleysziend,ceelxlscewpitthincthhreocmuolstuormeasldoabseerdrawtiitohns were ob- 2th5e0c0ulutgu/rmels. doIsneadddwiittihon2,2a5n0ianncdre2a5se00inge/ndmo.reduplicated cells were observed in Ivinstihbele4m4i.t2ohtiocurcealsls,aya,ndde=a9d5c%ellremdouncotliaonyeirnst,hfelcoealtlinmgodneoaldayceerllcsoannfdludeenbcreisw,enroe osebvseerreverdediuncttihoencsulintutrhees nduosmebderwoiftvhi3si0b0l0epmgi/tmolt.icUcnehlela,lftlhoyatcienlgl dmeoandolcaeyllesrsa,nd cduelbtriusr,esandods=ed55wi%thre2d7uc5t0ioung/imnlt.hScleilglhmloynuonlhaeyaelrthcyoncfellluemnocneolwaeyreerso,bsfelroavteidngin the : dobesaedrcveeldlinantdhedecburlitsu,reasnddo=se4d5w%itrhed2u5ct0i0onpgi/nmtlh.e cReeldlumcotnioolnsayoefr=c1on5f%luienntcheevceelrle mCohnroolmaoyseormcaolnfalbueerrnacteiownesrweeorbesearnvaeldyziendtfherocmultthuerecsuldtousreeds dwoistehd22wi5t0hp2g0/0ml0,, 2250, 2ob5s0e0rvaendd i2n7t5h0e gmi/tomtic(Tianbdliece7s)o.ftRhedecuuclttiuornesosfdo1se4d%,wi1t7h%,105060%,,22a5n0d,52350%0,wearned 2si7g5n0ifiucga/nmtl,inrcersepaescetsivienlcye,llasswciotmhpacrherdomwoistohmtahle asboelrvreantticoonnstwreolrecuolbtusreers.vedNaot the i `adcdointcieonnt,raatisoingsniafniaclaynzteidn,cerxeacseeptininpotlhyepcluolitduyrewsadsosoebdsewrivtehd2i7n5t0hepgc/umllt.ureIsndosed CHV Study No: 17750-0-437C0 17 . 000218 : CORNINGHazleton : with 2250, 2500, and 2750 g/m. The dose levels with increased ! h`eonudroraesdsuayp.lication was in the cultures dosed with 2250 and 2500 ug/ml in the 20.1 i"Tnhceidseunccceeossffuclelasctwiivtathiconhrboymtohseommeatlabsobleirrcastyiosntseminstihleucsutlatutreeds biyndtuhceedinwcirtehased nceygcaltoipvheosfoprhaimndiudcei,ntgh chprosoimtoisveomcaonltarboelrargaetnito.nsTehxecetpetstaatraicilnegilsechoingshiddeorseedthat iinndduucciendgseingndiofriecdaunptltiocxaitciitoyn.anIdn apdodliytpiloonidty.h test articlei considered positive for 120 CONCLUSION: ; aTn-d65w6i4thwoaust mcoentsaibdoelriecdancetigvaattiivoenfeoxrcienpdtaucinsgincghlreomdoossoemlaelvealbweirrtahtmieontsabionlCicHaOctcievlaltsiownith that induced significant toxicity: | 150 REFERENCES: AYromrikt,agNeY,,P.19S7t1a.tistical Methods in Medical Research, John Wile&y Sons, Inc. New REveavnise,woHfJC.ytCohlroogmyo,s1o1m2a2l1a3be2r1r,at1i9on6s2.produced by ionizing radiation. International i : S1o9k8a1l, RR, and Robi, .J: Biometry, Ed. 2, W.H. Freeman and Company, New York, 140 DEVIATION FROM THE SIGNED PROTOCOL: : lEenvdeolrsedwuiptlhiicnactrieoanseadndenpdoolryepdluopildiycawteiroen aanndalsyozemdesweiptahraptoellyyplsoiindcye.thTeheisweardedesdogmreeadtoesre i scientific information t the study. CHV Study No. 17750-0437C0 1s 00219 1 `CORNING Hazleton ! 150 EXPERIMENTAL DATA TABLES + CHV Study No.: 177500437CO i 19 00220 CORNINGHazleton TABLE1 RANGEFINDING ASSAY FOR ASSESSING TOXICITY AssayNo:17750 Compound: T-6564 Metabolic Acivarion: 59 Trial Nou:1 Date: 06/13/96 Lab No.: CY6156 -- Treament [%CoSnotluvcennct+e Control NEGATIVECONTROL Maceoyn 3 ow SovENTCONTROL war owe 20 ww . TESTARLE arm 0 i wn 49 as i spon 7 on wom 02 5 : Metabolic Activation: +59 ! Tresment [%CoSnotluveenncte Conol NEGATIVE CONTROL _ corion wi mw SOLVENTCONTROL war oom 105 10 TEST ARTICLE own 60 + swe 53 ww oom 65 # Thhahrivseysetnodfpetihenemiteshetbraasaepdcuhuhpaeeodsolncseve.lilspAsuaccluahaltsclytbleoccoonaunltwfshiic.nhAoaLekhmeeandmenepdorafontyfhoe he : MoCvonilnogcacietoobserevlatsiaonndhdeefaldacskls.we atevansfor espaceof McSCaocuyrem'edisom. CHV Study No.: 17750-0437CO 20 : 0021 2 CHROMOSOME ABERRATIONS IN CHINESE HAMSTEROVARY CELLS `Cel Fined20.0 thoesAf Treament 5 Taser igoEt SnllEalel] ]rers e t] [Foawe ohe Aos ne Immo FE, -- 22 EEE: g a se te RnY ll i yo. f f: fn cormi Ee mer omonss rinss ww oem rene As i BR wm salle] EEed mi, EE raliisaly -- EL [re aasie 2 BEER gies ADS we , RE : pion: . Pr of on oa 82 5 g rs "CellsFixed442 Hows Aer Treatment 2 cour. counex [hee Ss wv. x "am ! wowouonon tr :I om & + Chvomosome hemaions otsyed debn igsdes vibefo ysis. 2 # wonononon +4Chramossme heraionssoksydduis xcs vic, !Zz g 2 pp 8 cv ns owC esins o av s mnnovs os eteei 59 safer EE Ee Pot EEN Bonu on ta oon 2p Cmmm mms ----, -- Tg g8 : a F fi cnomosous gman Eeaasteroes vay css | 7 wns wns oe ans waren eect 9 | 2 dor x ww 8 sel lr eee EE ER RE rs EE ' moss erumas oe 20 21 [a i IEEE - Bann wow om omon RT i 8 i i2 EJwenn i. TR we CHROMOSOMEAk DERATNsCHe sES asTEROVARY LS ocr wo. Wet alr gm ART aie EEE J23 2u esl Po moaponmou Q Comm mm--Amww eL eWiT34l-- 3aia -- i2L. -- b bwla,e4-- as w-- omonowm_ur nane ioew nu 2 g EEE 8 i s o . CORNINGHazleton TABLES `CONTROL DATA OF CHROMOSOME9A5BTEHRRROAUTIGOHNS12I5N5CHINESE HAMSTER OVARY CELLS Acivaion AoPcrFmCOirdolr_sAveWmOWiIioCnnHsS_AbeHWrOaaIieCoHlnSs so"gCmaese rpCooysn e eG woos wwxn oowm T aooo uas Sao osso ANS EeBon am a%n as a"m Ste Con woos wn ew a 0 0 a wWxG eoosn x% osmo ooso %% a0m awex : FoNnotemCaont waow Mn ou mo a woo 3% we ao i Nxo anm wnm wns : re ieCont wo w wn oeow o1sn i0o o5 o0ns Wxe w2n iae onw es aEn] :: SevenCosel win wMxx oono w7o a"o i0o uae FoieCant AxG em um ax ouw aEBm) Copan Wen L MN a0Emo so . Nxe oa mana aam CHV StudyNo: 17750-0437C0 2 C0028 3 CORNINGHazleton i 160 DCEEFLILNSITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED ? NOT COMPUTED TG Chromatid gap: : on("eticdhgraopm)at.idA,nthaecshirzoemoatfiwch(iucnshtiasienqeuda)lrtegoioorn in `snmoatleldebrutthanonttuhseuwailldythinocflaudcehdrionmaftiinda.l tTothaelsseofare aberrationsastheymaynotalbetruebreaks. SG Chromosome gap: b(u`tiasotcthhreosmaatmiedglaopc,us1Gi"n).boStahsmisetaesr cchhrroommataitdisd.gap i: UC Uncoiled chromosome: Fabaeirlruarteioofnc.hromatin packing. Probably nota true i PP Polyploid cell: Anucmebllerco(nnt)aoifnicnhgrmomuolsiopmleesc.opiNeostocftouhnetehdapilnoitdhe cells scored for aberrations. i E_ Endoreduplication: 4hanscfeallileidn.whNiocthcsoeupnatreadtiinotnohfeccehlrlsomsocsoroemdefpoarirs aberrations. SIMPLE . TB Chromatic break: Atnhe awcihdrtohmoatfaiccrhreogmiaotnidin. onTehecharsosmoactiiadt,eldafrrgaegrtmehnatn may be partially or completely displaced. SB Chromosome break: Cabhnroormmoaslo(mdeelehtaesd)a cclheraormborseaokm,efwoirtmhinagn aancentric ! ifnrcalgumdeensttthheataciesndtirsilcocfartaegd.menTthi(sAFc)l.assTihfiecaAtFionwansow : different from the SB only in that it was not apparenily related to any specific chromosome. : DM "Double Minute* fragment: `tTehremsienaarledeslmeatlilondsoaunbldesdoomtse,isntoemrsetoitfiawlhdieclhetiaornes : i d`ainsdtipnrgoubiashbalbyles.mall ings. Their origins are not : CHV Study No.: 17750-04370 i 2 00229 : CORNING Hazleton COMPLEX ID Interstitial deletion: Lengthofchromatid "cut out" from midregion ofa cychhirrnoogmmaabtteiisdid.dreesauslthionrgteinnead scmharlolmaftriadgmoefnatogfarpiningthe TR Triradial: : QR Quadri radial: `An exchange between two chromosomes, or one crehsrulotmsoisnoamteharnede-aanmaecdenctornifcigfurraagtmieonnt., which As triradial, but resultiinnag four-armed configuration. 3 D Dicentric: i Arensuletxschinanagcehbreotmwoeseonmtewwoicthhrtowmoocseonmtersomwehreisc.h `Thisi often associated wianatcenthric fragment in `whichcase it is classified as DF. DF TC Tricensic: : Dicentric with fragment. An exchange between three chromosomes which `rOeefsxtucelhntasnagisenssoaccicaahntreiodnmwvoiosltvhoemtemwaownittyohtcthhhrrreeoeemAocFes.notmrSoeumscehraensd. are named as follows: : QPCC PQeumadarciecnetnrtirci:c: HC Hexacentic: i R Ring i sffiioxvuerceccneetnnrttorrmooemmererereses,s,,uupupp0ttoosiffxiovuAerFAAFF cAencthrroomemroes.omTehiwshiiscohftfeonramsssocciiractleed cwiotnhaianning a acentric fragment in which case it is classed as RF. . RC Ring Chromatid: Single chromatid ring (acentric), RF C1 Chromosome Intrachange: Ring with associated acentric fragment. dEoxecshannotgeinwciltuhdientahechenrtoimreoscohmreo;moes.og,mae.ring that i CHV Study No.: 17750-0437C0 | | ! 2 00230 i: T Translocation: AB . . Other { GT/> i 1} `CORNINGHazleton chromosomesresulting in two aboormal Obvious transfer ofmaterial between two chromosomes. When identifiable, scoredas "T" not "2Ab." Absormalmonocenticchromosome.Thisisa chromosome whose morphology is abnormal for the pkaerrycoetnytpiec, iannvderosfitoenn,tehte.rCelsauslstiofifactatriaonnswloecadtiiofn, `abnormalitycannotbeascribedto;.g., areciprocal translocation. assowcitihaatritceemtidc. Ahceaevlillywdhiacmhagceondtcaeilnlssmhoorueldtbhaenan1a0laybzeerdrattoioindse.ntiAfy thetypesof aberraatnidomnaysnotactually have >10, e.g., multiple fragsm uce h anstt hosse found i CHV Study No. 17750:0437C0 0 00231