Document NGypEJdgdxdpGR69ke2xn54bw

CORNINGHazleton MUTAGENICITY TEST ON T-6342 MEASURING CHROMOSOMAL ABERRATIONS IN CHINESE HAMSTER OVARY (CHO) CELLS: WITH A CONFIRMATORY ASSAY WITH MULTIPLE HARVESTS FINAL REPORT AUTHO Hemalatha Murli,Ph.D. PERFORMING LABORATORY Coming HazletonInc.(CHV) 9200 LeesburgPike Vienna,Virginia22182 LABORATORY PROJECT IDENTIFICATION CHV StudyNo.: 17073-0-437CO SUBMITTED TO 3M Corporation Building220-2E-02 3M Center St.Paul,Minnesota 55144-1000 STUDY COMPLETION DATE September 16,1996 CHV StudyNo.: 17073-0-437CO I of 35 CORNINGHazleton QUALITY ASSURANCE STATEMENT ProjeTcittleC:hromosomaAlberratioinCshinesHeamsteOrvary(CHO)CellsW:itha ConfirmatoryAssay With MultipleHarvests ProjectNo.: 20990 Assay No.: 17073 ProtocolNo.: 437CO EditionNo.:4,Modifiedfor3M Corporation QualityAssuranceinspectionosf thestudyand reviewof thefinalreportof theabove referenced projectwere conductedaccordingtotheStandardOperatingProceduresof theQualityAssurance Unit and accordingtothegeneralrequirementsoftheappropriatGeood LaboratoryPractice regulationsF.indingsfrom theinspectionasnd finalreportreviewwere reportedtomanagement and tothestudydirectoorn thefollowingdates: Inspection/Date Findini!Rseported Audito Dosing-09/06/1995 09/06/1995 M. Murphy DraftReportReview-1 1/06,07/1995 11/07/1995 C. Orantes,K. Newland RevisedDraftReportReview-0 1/04/1996 01/04/1996 C. Orantes FinalReportReview-09/16/1996 09/16/1996 C. Orantes QualityAssuranceUnit CHV StudyNo.: 17073-0-437CO ]JateIteleased 2 CORNINGHazleton STUDY COMPLIANCE AND CERTIFICATION The describedstudy was conducted incompliance with theGood LaboratoryPracticeregulations as setforthintheFood and Drug Administration(FDA) Title21 of the U.S. Code of Federal RegulationsPart58, issuedDecember 22, 1978,(effectivJeune 20, 1979) with any applicable amendments. There were no significandteviationsfrom theaforementionedregulationosr the signedprotocolthatwould affectthe integritoyf thestudyor theinterpretationfthe testresults. The raw datahave been reviewed by the Study Director,who certifietshatthe evaluationof the testarticlaes presentedhereinrepresentsan appropriateconclusionwithinthe contextof the study design and evaluationcriteria. All testand controlresultsinthisreportaresupportedby an experimentaldatarecordand this recordhas been reviewed by the Study Director.All raw data,documentation,records,protocol and a copy of the finalreportgeneratedasa resultof thisstudywillbe archivedinthe storage facilitioefsComing Hazleton Inc.foratleastone year followingsubmissionof the finalreportto theSponsor. Afterthe one yearperiod,the Sponsor may electtohave theaforementioned materialsretainedinthestoragefacilitioefsComing HazletonInc.foran additionapleriodof time,or senttoa storagefacilitdyesignatedby the Sponsor. Submitted By: Study Director: id Hemalatha Murli,Ph.D. Mammalian Cytogenetics Department of Geneticand CellularToxicology Studyto MP'letion Date CHV Study No.: 17073-0-437CO 3 CORNINGHazleton TABLE OF CONTENTS PageNo. ABSTRACT ..............................................6.......... 1.0 SPONSOR .........................................8.......... 2.0 MATERIAL (TEST ARTICLE) ..............................8 .......... 2.1 Client'Isdentification 2.2 Date Received 2.3 PhysicalDescription 2.4 GeneticsAssay No. 3.0 TYPE OF ASSAY .....................................8.......... 4.0 PROTOCOL NO ......................................8........... 5.0 STUDY DATES ......................................8 .......... 5.1 InitiationDate 5.2 ExperimentalStartDate 5.3 ExperimentalTerminationDate 6.0 SUPERVISORY PERSONNEL .............................8........... 6.1 StudyDirector 6.2 LaboratorySupervisor 7.0 OBJECTIVE ........................................8.......... 8.0 RATIONALE ........................................9.......... 9.0 EXPERIMENTAL DESIGN ................................9 .......... 10.0 MATERIALS AND METHODS .............................1.0 .......... 10.1 TestCells 10.2 CellCultureMedium 10.3 Negativeand SolventControls 10.4 PositivCeontrolAgents 10.5 RangefindingAssays 10.6 AberrationAsssay WithoutMetabolicActivation CHV StudyNo.: 17073-0-437CO 4 11.0 12.0 13.0 14.0 15.0 CORNINGHazieton 10.7 10.8 10.9 10.10 AberrationAsssay With MetabolicActivation HarvestProcedure SlidePreparatioannd Staining AberrationsAnalysisand Assay Evaluation RESULTS 11.1 11.2 11.3 11.4 11.5 ...........................................1.5......... Solubilitaynd Dose Determination RangefindingAssay WithoutMetabolicActivation RangefindingAssay With MetabolicActivation Chromosomal AberrationAsssay WithoutMetabolicActivation Chromosomal AberrationAsssay With MetabolicActivation CONCLUSION .......................................2.0.......... REFERENCES .......................................2.1........... EXPERIMENTAL DATA TABLES ............................2.2 ......... DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS .............................................33........... CHV StudyNo.: 17073-0-437CO 5 CORNINGHazleton ABSTP,ACT TheobjectiovfethiisnYitmassawyastoevaluatheeabiliotfyT-6342toinduccehromosomal aberrationisn Chinesehamsterovary(CHO) cellswithand withoutmetabolicactivation. For thedose rangefindinagssaywithand withoutmetabolicactivationt,hetestarticlweas dissolvedinsterildeeionizedwaterata concentratioonf 498 mg/ml. Concentrationsof0.166, 0.498,1.66,4.98,16.6,49.8,166,498, 1660,and 4980 gg/ml were testedinthedose rangefindinagssays.Alldosingwas achievedusinga dosingvolume of I% (10 pi/ml).Inthe nonactivatioanssay,completecytotoxicitwyas observedintheculturedosed with4980 Ag/ml, and reductionsof 100% inthemitoticindexas compared withthesolventcontrolculturewsere observedintheculturedosed with 1660 gg/ml. Intheactivatioanssay,reductionsof 19%, 33%, 27%, and 63% as compared with thesolventcontrolcultureswere observedintheculturesdosed with 166,498, 1660,and 4980 pg/ml,respectively. Based on thedatafrom thedose rangefindinagssay,replicatceulturesofCHO cellswere incubatedwith 125,250,500,750,1000, 1500,and 2000 pg/ml withoutmetabolicactivatioannd with250,500,1250,2500,3750, and 5000 gg/ml withmetabolicactivatioinn 20.0hour aberrationasssays.Culturestreatedwith750,1000,1500,and 2000 gg/ml withoutmetabolic activatioannd with 1250,2500, 3750,and 5000 gg/ml withmetabolicactivatiownere analyzed forchromosomal aberrationsN.o significanitncreaseincellswithchromosomal aberrationwsas observedinthenonactivatioanssay.A significanitncreaseincellswithchromosomal aberrationwsas observedintheculturedsosed with3750 and 5000 gg/mi withmetabolic activation. Intheconfirmatorytrialr,eplicatceultureosf CHO cellswere incubatedwith 125,249,498,746, 995, 1490,and 1990 gg/ml withoutmetabolicactivatioannd with249,498, 1250,2490, 3730, and 4970 gg/ml withmetabolicactivatioinn20.0and 44.1hourassays.Culturestreatedwith 746,995, 1490,and 1990 gg/ml from the20.0hourassayand with249,498, 746 and 995 pg/ml from the44.1hour assaywithoutmetabolicactivationa,nd with 1250,2490, 3730, and 4970 gg/ml from the20.0and 44.1hourassayswithmetabolicactivatiownere analyzedfor chromosomal aberrationsN.o significanitncreaseincellswithchromosomal aberrationwsas observedinthenonactivatioanssay.A significanitncreaseincellswithchromosomal aberrationwsas observedintheculturesdosed with 4970 Ag/ml inthe20.0 hour assaywith metabolicactivationA. significanitncreaseinpercentpolyploidywas observedinthecultures dosed with4970 gg/ml inthe44.1hourassaywithmetabolicactivationT.hispositiveresponse was investigateudnder nonactivatiocnonditionsby treatincgellsfor3 hoursunder thesame conditionsastheassaywith metabolicactivation. CHV StudyNo.: 17073-0-437CO 6 CORNINGHazlcton Inthirsepeattriarle,plicactuelturoefsCHO cellwsereincubatweidth250,500,1250,2490, 3740,and 4980 pg/ml withoutmetabolicactivatiofnor3 hoursand harvested20.0and 44.0 hoursafterinitiatiofntreatment.Culturestreatedwith250,500, 1250 and 2490 gg/ml from the 20.0hour assayand with 500, 1250,2490,and 3740 pg/ml from the44.0 hour assaywere analyzedforchromosomal aberrationsN.o significanitncreaseincellswithchromosomal aberrationwsas observedinthe20.0hourassay.A weakly significanitncreaseincellswith chromosomal aberrationwsas observedintheculturedsosed with3740 gg/ml inthe44.0hour assay.A significanitncreaseinpercentpolyploidywas observedintheculturesdosed with 3740 @ig/mlinthe 44.0 hour assay. The testarticleT,-6342,was consideredweakly positivfeorinducingchromosomal aberrations inCHO cellswithoutmetabolicactivatiownitha threehourtreatment.T-6342 was considered positivfeorinducingchromosomal aberrationisnCHO cellswithmetabolicactivationT.-6')42 was consideredpositiveforinducingpolyploidywithand withoutmetabolicactivation. CHV StudyNo.: 17073-0-437CO 7 CORNINGHazieton ChromosomaAlberratioinCshinesHeamsteOrvary(CHO)CellsW:itha ConfirmatoryAssay With MultipleHarvestsWith T-6342 1.0 SPONSOR: 3M Corporation 2.0 MATERIAL (TEST ARTICLE): 2.1 Client'IsdentificatioTn-:6342 2.2 Date Received:July21, 1995 2.3 PhysicalDescriptionC:lear,colorleslsiquid 2.4 GeneticsAssay No.: 17073 3.0 TYPE OF ASSAY: Chromosomal AberrationisnChineseHamster Ovary (CHO) Cells 4.0 PROTOCOL NO.: 437CO, Edition4,Modifiedfor3M Corporation 5.0 STUDY DATES: 5.1 InitiatiDoante: August 15,1995 5.2 ExperimentalStartDate: August 24, 1995 5.3 ExperimentalTerminationDate:December 04, 1995 6.0 SUPERVISORY PERSONNEL: 6.1 StudyDirector:HemalathaMurli,Ph.D. 6.2 LaboratorySupervisor:Carol S.Spicer,B.S. 7.0 OBJECTIVE: The objectiveofthisinyi= assaywas toevaluatetheabilitoyf thetestarticleT,-6342, toinducechromosomal aberrationisnChinesehamsterovary(CHO) cellsw,ithand withoutmetabolicactivation. CHV StudyNo.: 17073-0437CO 8 CORNINGHazleton 8.0 RATIONALE: Theassayisdesignetdoestabliwshhethetrhetesatrticolreitmsetabolictaensinteract withcellstoinducechromosome breaks.Chemicallyinducedlesionsmay resultin breaksinchromatinthatareeitherepairedby thecellinsuch a way as tobe undetectable orresultinvisibledamage. Aberrationsarea consequenceof failurienrepairprocesses suchthatbreaksdo notrejoinor rejoininabnormalconfiguration(sEvans,1962). 9.0 EXPERIMENTAL DESIGN: Resultsfrom therangefindingassaywere used todeterminethedose rangetobe used in thechromosomal aberrationasssay.Intherangefindingassay,thecultureswere harvested20.0hoursafterinitiatiofntreatment.Mitoticindexand visualobservations of theculturewsere evaluatedforevidenceof toxicityA. summary of thetreatment scheduleforthe rangefindinagssay isgivenbelow. Summary of RangefindingAssay TreatmentScheduleinHours Test - S9 +s9 TestArticle 0 0 Wash 17.8 3 Colcemid' Fixation 17.9 20 17.9 20 In thechromosomal aberrationasssays,replicatceultureswere used ateach dose level, and negativeand solventcontrols.Singlecultureswere used foreach of two doses ofthe positivceontrol.The aberrationasssayswere conductedwitha 20.0hour harvesttimein theinitiatlriaalnd with20.0and 44.1hourharvesttimesintheconfirmatorytrials. Chromosomal aberrationwsere analyzedfrom theculturestreatedatfourdose levelsand from onlyone of thepositivceontroldoses.A summary of thetreatmentscheduleforthe chromosomal aberrationasssaysison thefollowingpage. CHV Study No.: 17073-0-437CO 9 CORNINGHazleton SUMMMZ@ofChromosomAablerratiAosnssayTreatmeSnctheduilneHours Test TestArticle Wash InitiaTlrial - S9 0 17.8 +s9 0 3 ConfirmatoryTrial - S9 0 17.6 +s9 0 3 - S9 0 41.8 +s9 0 3 Rel2eatTrial - S9 0 3 -s9 0 3 Coicemid" Fixation 18 20 18 20 18 20 17.9 20 42.1 44.1 42.1 44.1 18 20 42 44 10.0 MATERIALS AND METHODS: 10.1 TestCells: The Chinesehamsterovarycells(CHO-WBL) usedinthisassaywere froma permanentcelllineandwere originalloybtainedfromthelaboratoroyfDr.S. Wolff,UniversitoyfCaliforniSaa,n FranciscoT.he cellhsavesincebeen reclonedtomaintainkaryotypisctabilitTyh.iscelllinehasan averagecycletime of 12 to 14 hourswitha modal chromosome number of21. 10.2 CellCultureMedium: The CHO cellsweregrown inMcCoy's 5aculturmeedium which was supplementedwith10 % fetablovineserum(FBS),1% L-glutaminea,nd 1% penicillaind streptomyciant,approximatel3y7'C, inan atmosphereofabout 5%CO2 inair. CHV StudyNo.: 17073-0437CO 10 CORNINGHazleton 10.3 Negativeand SolventControls: In thenonactivatioanssays,negativecontrolwsere culturewshich containedonly cellsand culturemedium. Solventcontrolwsere culturecsontainingthesolvent forthetestarticlaetthehighestconcentratiounsed intestculturesI.n the activatioanssays,thenegativeand solventcontrolswere thesame asdescribedin thenonactivatioanssaysbutwiththeS9 activatiomnix included. 10.4 PositiveControlAgents: The positivecontrolagentswhich were used intheassayswere mitomycin C (MMC) forthenonactivatiosneriesand cyclophospharnid(eCP) inthemetabolic activatiosneriesM.itomycin C (CAS# 50-07-7,Sigma,Lot # 25HO619) isa clastogenthatdoes notrequiremetabolicactivationC.yclophosphamide (CAS 6055-19-2,Sigma, Lot # 67FO 155)does notactdirectlbyutmust be convertedto activeintermediatebsy microsomal enzymes. In thechromosomal aberrations assays,two concentrationosf MMC (0.08and 0.10 gg/ml,initiaalnd confirmatorytrials0;.50and 1.0gg/ml,thirdtriala)nd CP (5.0and 10.0gg/ml) were used toinducechromosomal aberrationisntheCHO cells.One of thedose levelswas analyzedineach oftheaberratioanssays.Both MMC and CP were dissolvedinwater. 10.5 RangefindingAssays: In theseassays,thecelIswere culturedforapproximately24 hourspriorto treatmentby seedingapproximately0.3x 10'cellsper25 CM2 flaskinto5 ml of completeMcCoy's 5a culturemedium. 10-5.1Assay WithoutMetabolicActivation: The culturewsere incubatedwiththetestarticlfeor17.8hoursat=37*C. Then, thetestarticlweas washed from thecellswith phosphatebuffered salineand freshcompletemedium containinCgolcemid' (final concentratio0n.1 gg/ml)was added. The culturewsere thentrypsinized and harvested2.0hourslater.(See Sectionson Harvestand Slide Preparatioannd Staining). 10.5.2Assay With MetabolicActivation: Inthisassay,theCHO cellswere exposedtothetestarticlfeorthreehours CHV Study No.: 17073-0437CO II CORNINGHazleton at=37*CinthepresencoefaratliveSr9reactimoinxtur(eS915pl/ml, NADP 1.5mg/ml, and isocitraiccid2.7mg/ml). The S9 fraction (MolecularToxicology,Inc.,Lot #0583)was derivedfrom theliverof male Sprague-Dawleyratswhich had been previouslytreatedwithAroclor 1254 toinducethemixed:ftmctionoxidaseenzymes which arecapableof metabolizingchemicalsto more activeforms. The threehour incubation time was used becauseprolongedexposuretotheS9 mixturemight be toxictothecellsand theenzyme activitoyf S9 islostrapidlyatabout 37*C. The medium didnothave FBS duringtheexposureperiodtoavoid possibleinactivationfshortlivedand highlyreactiveintermediates produced by theS9 enzymes by bindingto serum proteins. Aftertheexposureperiodthecellswere washed twicewithbuffered saline.Complete McCoy's 5a medium was added tothecultureswhich were thenincubatedfor16.8hourswithCoicemid' (finacloncentration 0.1 gg/ml)added forthelast2.0hourstocollectmetaphasecells.The cultureswere then trypsinizedh,arvestedf,ixed,and slideswere prepared and stainedaswas describedforthenonactivatiornangefindingassay. 10.5.3Assay Evaluation: Mitoticindexwas analyzedfrom thehighestfivesurvivingdose levelsby analyzingthenumber of metaphasespresentin 1000 consecutivecells. 10.6 AberrationsAssay WithoutMetabolicActivation: Cultureswere initiatebdy seedingapproximately1.2x 106 cells(20.0hourassay) and 0.8X 10'cells(=44.0hour assay)per75 cml fiaskinto10 in]ofcomplete McCoy's 5a medium. One day aftercultureinitiatiofno,rtheinitialnd confirmatorytrialst,hecellswere incubatedat=370C withthetestarticlaet predetermineddoses forabout 17.7(20.0hourassay)and 41.8(44.1hour assay) hours.The culturewsere thenwashed withbufferedsalineand complete McCoy's 5a medium containing0.1 gg/ml Colcemid' was placedback ontothe cells.Approximatelytwo hourslatert,hecellswere harvestedand airdriedslides were made. The slideswere thenstainedin5 % Giemsa solutionfortheanalysis of chromosomal aberrationsF.or thethirdtrialo,ne day aftercultureinitiation, thecultureswere incubatedat=37 *C forthreehours inthepresenceof thetest articlienMcCoy's 5a medium withoutFBS. Afterthethreehour exposureperiod thecellswere washed twicewithbufferedsalineand thecellswere refedwith completeMcCoy's 5a medium. The cellswere incubatedfortherestofthe CHV StudyNo.: 17073-0-437CO 12 CORNINGHazieton cultuprerioudptothetimeofharveswtith0.1gg/mlColcemidp"resendturing thelast=2.0 hoursof incubation.The metaphasecellswere thenharvestedand preparedforcytogeneticanalysis. 10.7 AberrationsAssay With MetabolicActivation: Cultureswere initiatebdy seedingapproximately1.2x 10'cells(20.0hour assay) and 0.8 X 10'cells(44.1hourassay)per75 CM2 flaskinto10 ml of complete McCoy's 5a medium. One day aftercultureinitiatiotnh,eculturesthatwere treatedundertheconditionosf metabolicactivatiownere incubatedatz3 7*C for threehoursinthepresenceof thetestarticlaend theS9 reactionmixturein McCoy's 5a medium withoutFBS. Afterthethreehourexposureperiodthecells were washed twicewithbufferedsalineand thecellswere refedwithcomplete McCoy's 5a medium. The cellswere incubatedfortherestof thecultureperiod up tothetime ofharvestwith 0.1 gg/ml Colcemid' presentduringthelast=2.0 hoursof incubation.The metaphasecellswere thenharvestedand preparedfor cytogeneticanalysis. 10.8 HarvestProcedure: Priortotheharvestoftheculturesv,isualobservationosftoxicitwyere made. These observationsincludedan assessmentof thepercentconfluenceof thecell monolayer withinthecultureflasks.The culturewsere alsoevaluatedforthe presenceof mitotic(largeroundedcellso)r dead cellsfloatinignthemedium. The culturesfrom thedoserangefindinagssaywere trypsinizefdirstocollect mitoticand interphasceellsand were treatedwith0.075M KCL hypotonic solution.Thistreatmenthelpsto swellthecellsand thusdispersethe chromosomes. The culturewsere thenfixedwithan absolutemethanol:glacial aceticacid(3:1,v:v)fixativaend were washed severaltimesbeforeair-dried slideswere prepared. 10.9 SlidePreparationand Staining: Slideswere preparedby droppingtheharvestedcultureson cleanslides.The slideswere stainedwith5% Giemsa solutiofnortheanalysisof mitoticindexand chromosomal aberrationsA.ll slideswere thenair-drieadnd coverslippedusing Depex" mounting medium. CHV StudyNo.: 17073-0437CO 13 CORNINGHazieton 10.10 AberratioAnnsalysiasndAssayEvaluation: Cellswere selectedforgood morphology and onlycellswiththenumber of centromeresequaltothemodal number 21 I (range20-22)were analyzed. One hundredcellsi,fpossiblef,rom eachreplicatceultureatfourdoselevelsof thetestarticlaend from thenegativeand solventcontrolcultureswere analyzed forthedifferenttypesofchromosomal aberration(sEvans,1962;See Section 15.0).At least25 cellswere analyzedforchromosomal aberrationfsrom one of thepositivecontrolculturesF.or controlof bias,allslidesexceptforthepositive controlswere coded priortoanalysis.Cellswithaberrationwsere recordedon the datasheetsby themicroscopestagelocation.Mitoticindexwas assessedby analyzingthenumber of mitoticcellsin 1000 cellsand theratiowas expressedas a percentageof mitoticcells. The followingfactorwsere takenintoaccountintheevaluationof the chromosomal aberrationdsata: I . The percentageof cellswith any aberrations. 2. The percentageof cellswithmore thanone aberration. 3. Any evidenceforincreasinagmounts of damage withincreasing dose,i.e.a,positivedose response. Chromatid and isochromatidgaps,ifobserved,were noted intheraw dataand were tabulated.They were not,however,consideredintheevaluationof the abilitoyf thetestarticlteoinducechromosomal aberrationssincetheymay not representruechromosomal breaksand may possiblybe inducedby toxicity. Percentpolyploidywas analyzedfrom the=44.0 hour assayand resultwsere tabulated.HistoricaclontroldataarepresentedinTable 10. A cellclassifieads "GT" isconsideredtocontain10 aberrationfsorstatistical purposesbuta ">" isalsoincludedinthetablesforthisclassificatitoonindicate thatitisa minimum number. Statisticaanlalysiesmployed theFisher'EsxactTestwithan adjustmentfor multiplecomparisons(Sokaland Rohlf,1981)tocompare thepercentageof cells withaberrationisneachtreatmentgroup withtheresultfsrom thesolvent controls.A lineatrrendtestof increasinngumber of cellswithaberrationwsith CHV Study No.: 17073-0-437CO 14 CORNINGHazleton increasidnogse(Armitag1e9,71)wasalspoerformedT.estarticsliegnificance was establishewdhere p<0.01. Allfactorass statedpreviouslywere takeninto accountand thefinalevaluatioonf thetestarticlweas basedupon scientific judgement. 11.0 RESULTS: 11.1 Solubilitaynd Dose Determination T-6-)'42 was describedas beingfreelysolublein water,so itssolubilitfyorthis studywas evaluatedinsterildeeionizedwater(Lot # 17,prepared06/16/95at CHV). A clearc,olorlesssolutionwas obtainedata concentratioonf498 mg/ml. Thissolutiownas dilutetdoI% (10 gl/ml)inMcCoy's 5a culturmeedium inthe absenceof any cells.At a concentratioonf 4980 pg/ml inculturemedium, the testarticlreemainedcompletelysolubleand a pH of 7.5was determined.Sterile deionizedwaterwas thereforcehosenasan appropriatveehicleforthisassay.A stockconcentratioonf 498 mg/ml and dilutiontshereofwere preparedfordosing therangefindinagssays.Alldosingwas achievedwitha I% (10 PI/ml)dosingof each stocksolutiona,nd thesolventcontrolculturewas dosed with 10 41/mlof sterildeeionizedwater.Concentrationosf 0.166,0.498,1.66,4.98,16.6,49.8, 166.498, 1660,and 4980 gg/mi were testedintherangefindingassayswith and withouttheS9 metabolicactivatiosnystem. The stabilitoyf thetestarticluender thepreparatioannd dosingconditionsused in thisassay istheresponsibiliotfythe Sponsor. 11.2 RangefindingAssay WithoutMetabolicActivation Intheculturetreatewdith4980 gg/ml, no visiblmeitoticellswere observed;the cellmonolayer was <5% confluenta,nd thecultureconsistedonlyofdebrisand dead cellsf,loatinogr attached.A severereductioninthenumber ofvisible mitoticcellswas observedintheculturedosed with 1660 gg/mi. The cell monolayer lookedunhealthyand thedegreeof confluencywas reducedabout 15%; floatindgead cellsand debriswere noted. Floatingdebrisalsowas observedintheculturedosed with498 gg/mi,butthecultureotherwisewas similartothenegativecontrol. Mitoticindiceswere analyzedfrom theculturedsosed with 16.6,49.8,166,498, and 1660 pLg/ml(Table1)and compared tothesolventcontrol.At 1660 gg/ml, themitoticindexwas essentialzleyro.However, littloer no reductioninmitotic CHV StudyNo.: 17073-0-437CO 15 CORNINGHazieton indexwasobserveadtthenextlowerdoosfe498gg/ml.Basedonthesreesults, thefirstriaolf thechromosomal aberrationasssaywithoutmetabolicactivation was conductedwitha 20 hour harvestattestarticlceoncentrationosf 125,250, 500, 750, 1000,1500,and 2000 gg/ml inordertoensurea wide toxicitryange. 11.3 RangefindingAssay With MetabolicActivation With S9 metabolicactivationa,n unhealthycellmonolayer was observedinthe culturedosed with4980 gg/ml. The cellmonolayerconfluencewas reducedby approximately85%, and a severereductioninthenumber of visiblmeitoticcells occurred(about63%, Table 1). At thenextlowerdose of 1660 gg/ml,the monolayer confluencewas thesame as thesolventcontrola,nd themitoticindex was onlysomewhat reduced(about27%). Based on theseresultst,heinitial aberrationasssaywithmetabolicactivatiownas conductedwith a 20 hour harvest attestarticlceoncentrationosf 250,500, 1250,2500,3750 and 5000 pg/ml. 11.4 Chromosomal AberrationAsssay WithoutMetabolicActivation InitiaTlrial Chromosomal aberrationwsere analyzedfrom theculturesdosed with 750, 1000, 1500,and 2000 gg/mi(Table2). No significanitncreaseisncellswithchromosomal aberrationwsere observedfortheculturesanalyzed. Thisassaycovereda wide rangeoftoxicityU.nhealthycellmonolayers,floating dead cellsand debriss,everelossofvisiblmeitoticcellsa,nd about25% reduction incellmonolayer confluencewere observedintheculturesdosed with 1500 and 2000 gg/ml. Slightluynhealthycellmonolayers,floatindgeadcellsand debris,a reductioninthenumber of visiblmeitoticcellsa,nd about 15% reductionincell monolayer confluencewere observedintheculturesdosed with 1000 pLg/ml.At thelowestanalyzeddose of750 pg/ml,thecellmonolayersappearedtobe almost normal,withonlya slighrteductioninthenumber of visiblemitoticellsand abouta 15% reductioninthedegreeof confluence.Severereductionsinmitotic indexrelativteothesolventcontrolculturesoccurredatalldose levelse,g.,95%. 86%, 67%, and 57% at2000, 1500,1000.and 750 pg/ml,respectively. Based on thetoxicitioebstainedinthistrialt,heconfirmatorytriaolf the nonactivatioanberrationasssaywas conductedattestarticlceoncentrationosf 125,249,498,746,995,1490,and 1990 gg/ml,usingtwo harvesttimesof 20 and 44.1 hours. CHV StudyNo.: 17073-0437CO 16 CORNINGHazleton ConfirmatoTrryial Forthe20hourharvescth,romosomaalberratiwoenrseanalyzefdromthecultures dosed with 746,995,1490,and 1990 gg/ml (Table3). No significainntcreases incellswith chromosomal aberrationwsere observedforany of thecultures analyzed.As obtainedinthefirsttrialt,hetoxicitryangewas wide, rangingfrom a severe95% reductioninmitoticindexat 1990 pg/ml to 25% reductionat 746 ;ig/ml.The cellmonolayerconfluencewas reducedby about25% at 1990 gg/ml,and only 83 metaphasescouldbe scoredfrom one of thereplicate cultures.To compensate,117 metaphaseswere scoredfrom the replicatceulture, inordertoscorea totalof200 metaphasesforthisdose. For the44.1 hour harvestc,hromosomal aberrationwsere analyzedfrom theculturesdosed with249,498,746,and 995 jig/ml(Table4). Again,no significant increasesincellswith chromosomal aberrationwsere observedforany of the cultureasnalyzed.The percentpolyploidyisscoredatthisharvesttime,and no significanitncreasewsere noted.The concentrationosf testarticlweere lower relativteothe20 hour harvestbecausethelongerexposuretime caused greater toxicityT.he two highestapplieddosesof 1990 gg/mi and 1490 gg/ml were lethal.Treatmentwith995 gg/mi was severelytoxic,resultinignabout40% reductionisnmonolayerconfluenceand 79% reductioninmitoticindex.At 746 gg/ml,however,thetoxicitwyas much less(about15% reductionin confluencyand 18% reductioninmitoticindexcompared tothesolventcontrol cultures). The resultosbtainedfrom thetwo assaytrialisntheabsenceof an S9 metabolic activatiosnystemappearedtoshow thelackofany clastogeniacctivitbyy thetest articleH.owever, inanotherassayhavingan exposuretimeof only3 hours,the inductionof chromosomal aberrationwsas demonstrated(seeSection11.6). 11.5 Chromosomal AberrationsAssay With MetabolicActivation InitiaTlri I Chromosomal aberrationwsere analyzedfrom theculturesdosed with 1250,2500. 3750, and 5000 gg/ml inthepresenceof theratliverS9 metabolicactivation system (Table5).Significanitncreaseisncellswithchromosomal aberrations were observedintheculturedsosed with-'175a0nd 5000 gg/ml.The treatment with 3750 gg/mi was onlyslightltyoxic,butthe5000 gg/ml treatmentwas highly toxic,causingabout95% reductioninmonolayerconfluencyforone cultureand CHV StudyNo.: 17073-0437CO 17 CORNINGHazleton about75% reductifoonrtherepliccautletureF.loatidnegadcellasnddebris were abundant,and only 94 metaphaseswere analyzablefrom theculturewith greatertoxicityH.owever, themitoticindexwas reducedby only 13% compared to thesolventcontrolcultures. Based on theseresultst,heconfirmatorytriawlas conductedwith essentialltyhe same testarticlceoncentrationsu,singtwo harvesttimesof 20 and 44.1 hours. Confirmatoa Trial For the20 hour harvest,a significanitncreaseincellswith chromosomal aberrationswas observedonlyfortheculturedsosedwith4970 Ag/ml (Table6). This toxictreatmentreducedtheconfluencyby about70% and causeda 43% reduction inthemitoticindex.The treatmentwith3730 pg/ml was much lesstoxic,causing about30% reductioninconfluencyand 18% reductioninmitoticindex.Even though thetreatmentwith 3730 pg/ml appearedtobe somewhat more toxicthan thesimilardose intheinitiatlrialt,herewas no hintof any clastogeniacctivity. Therefore,onlythehighlytoxictreatmentatthedose limitof theassay (5000 gg/mi)induceda repeatableb,ut largei,ncreaseinpercentcellswith chromosomal aberrationsI.n bothtrialsb,othsimpleand complex aberrations were observed. For the44.1 hour harvestc,hromosomal aberrationwsere analyzedfrom theculturesdosed with498,1250,2490,and 3730 gg/ml (Table7).No significant increasesincellswith chromosomal aberrationwsere observedforany of the cultureasnalyzed. Althoughthe4970 gg/mitreatmentwas tootoxicto yield sufficienmtetaphasesforanalysisi,twas neverthelesnsotedthatthepercent polyploidcellsincreasesdignificantlAyt.3730 pg/ml,therewas littloer no toxiciteyvident. The successfualctivatiobny themetabolicsystemwas illustratbeyd thelarge increaseinpercentcellswithchromosomal aberration(sat20 hoursharvest)in thecultureesxposed tocyclophosphamide,thepositivecontrolarticlew,hich requiresmetabolicactivatiotnobecome clastogenic. The initiailnterpretatioofntheseresultwsas thatthetestarticlweas convertedto clastogeniscubstance(si)nthepresenceof theS9 metabolicactivatiosnystem. However, accordingto informationprovidedby theSponsor,thetestarticliesnot metabolized.The appearanceof clastogeniacctivitiyntheactivatiopnortionof theassaywas notindoubt,butthisactivitsyhouldalsohave been observedunder CHV StudyNo.: 17073-0-437CO 18 CORNINGHazieton nonactivatcioonnditioinftsheS9systehmadnoeffecotnthetesatrticlTeh.is situatiornaisedthepossibilityhatan unrecognizedtoxicitiynthenonactivation assaywas actuallyeliminatintghecellswithinducedaberrationbseforethe cultureswere analyzed.The treatmentperiodsunder nonactivatiocnonditions were onlyabout2 hourslessthantheharvesttimes,whereas thetestarticlweas removed after3 hoursexposurefortheactivatioanssays.Ifthecontinued presenceof thetestarticlweas causingthedemise of cellswith induced aberrationst,hentheaberrationsshouldbe observableifthetreatmenttime was made equivalentotheactivatioanssay,ie,reducedto3 hours.Accordingly,the nonactivatioanssaywas repeated,usinga 3 hour exposureperiod. 11.6 Chromosomal AberrationAsssay WithoutMetabolicActivation3;-Hour Treatment The positiveresponseobservedin theassaywithmetabolicactivatiownas investigatebdy treatincgellsunder thesame conditionwsithoutmetabolic activationC.ellswere treatedfor3 hoursattestarticlceoncentrationosf 250, 500. 1250,2490, 3740,and 4980 pg/ml,usingharvesttimesof 20 and 44 hours aftertheinitiatiofntreatment. For the20 hour harvest,chromosomal aberrationwsere analyzedfrom thecultures dosed with250,500, 1250,and 2490 gg/ml (Table8).No significanitncreaseisn cellswithchromosomal aberrationwsere observed.Unfortunatelyt,hese treatmentswere notverytoxic(about30% reductioninconfluencyat 2490 gg/ml),and thenexthigherdoseof 3740 gg/ml was so toxicthatonlya smallnumber ofmetaphasescouldbe examined.As footnotedinthetable,7 of 38 metaphasesobtainedfrom thereplicatceulturehsad chromosomal aberrations. Thus, approximately18% of thecellswere indicateads havingaberrations.Due totheseveredepressionof mitoticindex,thepercentcellswithaberrationlsikely would have been higherata longerharvesttime. Obviously,much closerdose stepswould have been necessaryto achievetoxicitiecsomparabletotheactivation assaysand to compare theobservableclastogenicresponses. Itisinterestintgonotethatthetestarticlweas clearlymore toxicforthe3 hour treatmentperiodthanwhen theS9 activatiosnystemwas present.Genotoxic events(mutationor chromosomal changes)arenecessarilayssociatedwith toxicitya,nd themetabolismoftestarticletsoclastogeniscubstancesistherefore expectedto be accompaniedby increasedtoxicityn,otlesstoxicityT.hus,the observationof more toxicitiyntheabsenceof S9 was very suggestivethatthe CHV Study No.: 17073-0-437CO 19 CORNINGHazleton controllfiancgtionrobservicnlgastogeancitcivfiotrtyhitsesatrticwlaesthe attainmenotfa certaihnightoxicitfyora shorttimeperiod,and notthemetabolic conversionof thetestarticle. For the44 hour harvestc,hromosomal aberrationwsere analyzedfrom thecultures dosedwith500,1250,2490,and 3740 4g/ml(Table9).A small,butsignificant 7.5% of cellswithchromosomal aberrationwsas observedintheculturedsosed with3740 gg/ml.A significainntcreasienpercentpolyploidyalsowas observed intheseculturesT.he 3740 4g/mi treatmenrtesulteidnabout55% reductionin monolayerconfluencyrelativteothesolventcontrolsa,nd the 2490 ;IglmI* treatmentgaveabout30% reductionT.he mitoticindexwas alsosignificantly reduced(57%) forthehighdose. These resultwsereconsistenwtiththeinterpretatitohnatahighlytoxictreatment withthistestarticlaep,pliedfora shortperiodoftime,willgiverisetocells containincghromosomalaberrationtshatcan beobservedinthe 20 or44 hour timeperiodafteirnitiatioofntreatmentT.he harvestimefortheobservatioonf maximum numbers ofaberranctellswas notdetermined.The additioonfS9 to theculturesimplyreducedthetestarticlteoxicitsyo, thatfineradjustmentsinthe dose levelsthanachievedinthisstudy arenecessarytomake comparisons betweenequi-toxitcreatmentusnderactivatioannd nonactivatioanssay conditionsT.he initioablservationosfclastogeniacctivitoynlyinthepresenceof theS9 activatiosnystemcan be interpretetdobe a consequenceofa more limited toxicactionover3 hoursexposure,compared tothelongexposuresforthe standardnonactivatioanssays,ratherthanbeingduetoa metaboliceffect.The testarticliesthereforeevaluateads clastogeniocnlyforhighlytoxictreatments forshortenough exposureperiods(eg,3 hours)suchthatthecellscontaining chromosome aberrationdso notdisintegrabteforetheycanbe observedat20 to 44 hour harvesttimes. 12.0 CONCLUSION: The testarticleT,-6342,was evaluateadsbeingclastogenitcoculturedCHO cellsfor highlytoxictreatmentosfshortduratio(neg.,3 hours)bothinthepresenceand absence ofan S9 metabolicactivatisoynstem.Polyploidwyas alsoinducedforthehighlytoxic treatments. CHV Study No.: 17073-0-437CO 20 CORNINGHazleton 13.0 REFERENCES: Armitage, P. StatisticMaelthods inMedical Research,John Wiley & Sons, Inc.,New York, NY, 197 1. Evans, H.J.:Chromosomal aberrationpsroduced by ionizingradiation.International Review of Cytology,U:221-321, 1962. Sokal,R.R.,and Rohlf,F.J.:Biometry,Ed. 2,W.H. Freeman and Company, New York, 1981. CHV Study No.: 17073-0437CO 21 CORNINGHazleton 14.0 EXPERIMENTAL DATA TABLES CHV StudyNo.: 17073-0-437CO 22 CORNINGHazleton TABLE I RANGEFINDING ASSAY FOR ASSESSING TOXICITY Compound: T-6342 Assay No.: 17073 TrialNo.:I Date:08/24/95 MetabolicActivation-:S9 Lab No.:CY8225 Treatment NEGATIVE CONTROL McCoy's 5a SOLVENT CONTROL Water 10.0 TEST ARTICLE 16.6 49.8 166 498 1660** Metabolic Activation: +S9 Lab No.: CY8225 Ill/ml gg/mi pg/ml pg/mi Ag/m, pgtml % Mitotic Index 9.8 2.0 5.4 9.7 9.1 9.2 0.0 Confluence % Solvent Control 100 100 100 100 too 100 85 Trial No.:l NEGATIVE CONTROL SOLVENT CONTROL TEST ARTICLE Treatment mccov's Sa Water 10.0 pilml 49.8 gg/ml 166 pgtmi % Mitotic Index 14.8 17.1 17.3 13.9 Confluence % Solvent Control 100 too too 100 498 Ag/m, 11.5 100 1660 pg/ml 12.5 100 4980 gg/ml 6.3 14 This endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. Actual cell counts are not taken and any hypertrophy of the attached cells cannot be evaluated. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells. ** Toxic dose level. CHV Study No.: 17073-0-437CO 23 C@IROMOSOME TABLE2 ABERRATIONS IN CIIINESE IIAMSTER CellsFixed 20.0 tioursAfterTreatment OVARY CELLS Assay No.:17073 Trial I Lab #: CY9055 MetabolicActivation:-S9 Compound:T-6342 Date:09/06/95 NOMBI:R AN[)'I'Ylll0:1:A[31-.RRA'I'ION CONTROLS NEGATIVE: McCoy's 5a SOLVENT: Waler POSITIVE: mmc NO'I' N 01'. % cohiplirF[) SINIPI,E COMPLEX ()]'IIEPABERRA-CELL ....................................................................'..I..'.I.O.N.S...W.I.T.[.I......... CFT,I,S TG SG uc 'I'B SB ID I'R QR: CR D R Cl t)F GT PER ADERR SCORED CELL TIONS A 100 2 1 B 100 3 A+13 200 5 1 10,0111/nil A 11 100 2 100 2 1 A+B 200 4 1 0.08ligInil A 25 9 27 5 1 1 1 1 3 0.01 1.0 0.00 0.0 0.01 0.5 0.01 1.0 0.00 0.0 0.01 0.5 0,60 28.0* TEST ARTICLE 750lig/mi A B 100 2 1 100 4 A+B 200 6 1 1000lig/nil A B 100 8 1 100 4 A+B 200 12 1 1500fig/nil A B 100 4 100 A+B 200 4 2000lig/mi A 11 100 100 4 A+B 200 4 Significantlgyreaterthan thesolventcontrols,p<0.01 Significantlgyreaterthan thesolventcontrolsp,<0.05 1 0,01 1.0 0.00 0.0 1 0.01 0.5 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 2 0.02 2.0 2 0.01 1.0 0.00 0.0 0.00 0.0 0.00 0.0 CHROMOSOME TABLE3 ABERRATIONS IN CHINESE HAMSTER OVARY CellsI:ixe2d0.01lotirAsfter'l're;itiiieiii CELLS Assay No.:17073 Compound:T-6342 4 Trial#:2 Lab #: CY9155 MetabolicActivation:-S9 Date:09/13/95 NUMB[-'R ANI)'I'YPE 01:A131-"RRA'I'ION -4 CONTRUI.S CEI.LS SCORE[) NOT OF % COMPU-FEI) SIMPLE ....................... COMPLEX OTIIEP ABERRA- CEI-L ............................................................................ TG SG tic rB SB ID TR QR **-CR D ]'IONS WITI R ci DF G'r PER ABE" CELL TION NEGATIVE: McCoy's 5a A 100 8 1 B 100 8 1 1 0.01 1.0 1 0.02 2.0 SOLVENT: Water A+B 200 16 1 1 IO.Opl/nil A 100 7 1 1 B 100 1 2 1 1 0.02 1.5 0.01 1.0 0.00 0.0 Post IIVE: MNIC A+B 200 8 3 1 0.1001ig/iiii A 25 3 6 1 74 2 1 1 0.01 0.88 0.5 52.0* TEST ARTICLE 746pg/ml A 100 3 1 B 100 4 995pg/mi A+B 200 7 A 100 9 1] 100 11 1 1 1 A+B 200 10 1 2 1490lig/iiii A 11 100 3 1 100 4 1 1 A+B 200 7 1 1 1 199olig/mi A 83 41 1 1 li 117 12 1 1 A+B 200 16 2 2 1 Significantglryeaterthanthesolventcontrolsp,<0.01 1 0.02 2.0 1 0.01 1.0 2 0.02 1.5 1 1 0.03 3.0 0.01 1.0 1 1 0.02 2.0 1 0.02 2.0 1 0.01 1.0 2 0.02 1.5 0.01 1.2 0.00 0.0 0.01 0.5 CL 1-01 z Assay No.:17073 p Compound:T-6342 CONTROLS NEGATIVE: McCoy's 5a SOLVENT: Watcr TEST ARTICLE CHROMOSOME TABLE4 ABERRATIONS IN CHINESE HAMSTER CellsFixed 44.1 Hours AfterTreatment OVARY CELLS Trial#:2 Lab #:CY9155 Metabolic Activation-.-S9 Date:09/13/95 NUMBER ANI)']'YII0E1:ABERRA'I'ION CEI.TS SCORED NOT OF % COMPU'FEI) SIMPLE COMPLEX Ol'il[:HAIIERRAC-ELI, ................................................................................T..I...O.N.S....W.I.T.I........... I'G SG [IC 'VB: SB 11) TR QR CR D R C I D F C,I' PER ABER CELL I'ION A 100 3 2 B too I A+B 200 3 3 [O.Opi/nil A 100 3 B 100 2 1 1 A+B 200 5 1 1 0.00 0.0 1 0.01 1.0 1 0.01 0.5 0.00 0.0 1 0.02 2.0 1 0.01 1.0 249pgtmi A B 100 4 2 100 2 2 Afil 200 6 4 498lig/nil A B 100 4 2 100 2 1 A+B 200 6 3 746pg/nil A B 100 3 1 too 5 A4[3 200 8 1 995pg/ful A B 100 2 1 100 3 At[) 20o 5 1 2 2 11 11 3 1 21 511 1 1 12 11 23 1 1 2 0.04 3.0 0.03 3.0 0.04 3.0 0.01 1.0 0.00 0.0 0.01 0.5 0.05 4.0 0.02 2.0 0.04 3.o 0.03 3.0 0.01 1.0 0.02 2.0 C1+4 z Assay No.: 17073 p Compound: T-6342 CHROMOSOME TrialM: 3 TABLE5 ABERRATIONS IN CHINESE HAMSTER OVARY CellsFixed 20.0 Hours AfterTreatiiient CELLS [.abli:CY 11275 MetabolicActivation-:S9 Date: 11/28/95 NLIMIII-'ARNI)'I'YllOlf.-,ABII:RRA'I'ION -4 0 tONIR01,S NEGATIVE: McCoy's 58 SOLVENT: Water POSITIVE: mmc N OF 0/1 comptil'il)SIMPI.[: COMPI.I:X OTIIERABERRA- CEI, .........................................................................I..'.IONS Wi CELLS I'G::SG (IC TB. SB ID TR SCOPE[) CR: 1) R Cl 1:I)F GT PER ABEV CFI,I, TIO A 100 2 1 B 100 2 A+B 200 4 1 IO.Olil/mi A B 100 2 too I A+B 200 2 1 0.500@ig/mi A 25 5 3 1 1 I 1 6 1 26 4 1 1 3 11 41 1 0.01 1 0.01 1. 0.01 1- 0.03 3. 0.03 3. 0.03 3. 0.80 64, TEST ARTICLE 250lig/nil A B 100 100 2 A+B 200 2 500ilglilif A 100 2 1 1 B 100 1 A+B 200 2 1 1 1 1250pg/nil A 11 100 4 100 1 1 A+B 200 5 1 241)(Ilig/iiAil 100 2 1 0 100 10 1 2 A+B 200 12 1 21 3740lig/nil** A 0 11 0 3 1 0.03 3, 0.01 1 . 31 0.02 2. 3 0,04 4. 3 0.04 4. 6 0.04 4. 1 0.01 1. 1 0.01 1. 2 0.01 1. 1 0.02 2. 2 1 0.05 5. 3 1 0.04 3. All] 0 4 Significantlgyreaterthanthe solventcoiiirolsp,<0.01. (Itl%leevel.Very few tiletilillit,w,iistslcistie5s/.20;111112/1i8iielitpli,f.rits)cicsitiilttirAe,s.iii1d1,i-espectivellyi,-,cildiroiiii)soiilibieirirati CHROMOSOME TABLE6 ABERRATIONS IN CHINESE HAMSTER CellsFixed 44.0 floursAfterTreatment OVARY CELLS 2@ Assay No.: 17073 5) Compound: T-6342 TrialH: 3 l.ab#: CY 11275 Date: 11/28/95 MetabolicActivation:-S9 6 CONTR LS NEGATIVE: McCoy's Sa SOLVENT: Watcr 10.0111/mi NUMBER AN[) TYPE 01-ABERRAI'ION NOT COMPLI rED SIMPLE ........................ COMPI-EX OF % Ol'IlEFABERRA- CE[, .......................................................................... cl:[.[-ST(i.SG I SC'Okl:l) i 'I'illsit 11) I'R QR CR TIONS WIT 1) R (,1 1)1: G'I' PER ABER CEI.L TIOP A 100 2 1 B 100 2 A+B 200 2 3 A 100 3 1 B too I A+B 200 4 1 1 1 11 1 1 2 1 1 12 1 321 21 21 0.04 4.0 0.05 4.0 0.05 4.0 0.03 3.0 0.01 1.0 0.02 2.0 TEST ARTICLE 5OOpg/mi A B 100 2 3 100 3 A+B 200 5 3 1250pg/nil A 11 100 5 1 100 2 A+B 200 7 1 2490lig/iiiiA B 100 3 1 100 2 A+B 200 5 1 3740lig/nil A 11 100 6 100 6 A+li 200 12 Significantlgyreaterthanthe solventcontrolsp,<0.0 1. Significantlgyreaterthanthe solventcontrols,p<0.05. 1 11 21 1 12 22 21 21 1 1 1 1 2 2 51 71 0.00 0.0 0.03 3.0 0.02 1.5 0.00 0.0 0.01 1.0 0.01 0.5 0.02 2.0 0.03 3.0 0.03 2.5 1 >0.13 2 >0.29 3 >0.21 4.0 li.0 7.5* t-i 00 CHROMOSOME TABLE7 ABERRATIONS IN CHINESE HAMSTER CellsFixed 20.0 Hours AfterTreatment OVARY CELLS 2@ Assay No.:17073 p Compound:T-6342 C) 4 Trial I Lab #: CY9055 MetabolicActivation:+S9 Date:09/06/95 NUMBI@R'AND'I'Ylli.A'l(l)lI..'RRA'I'ION CON-1-ROLS NEGATIVE: McCoy's Sa SOLVENT: Watcr POSITIVE: CP CELLS SCORED A 100 B 100 A+B 200 IO.Opi/mi A 100 B 100 A+B 200 5.00ng/nil A 25 TEST ARTICLE 1250pg/mi A too B 100 A+B 200 2500lig/mi A 100 B 100 A+B 200 3750lig/nil A 100 B 100 A+B 200 5000lig/nil A 94 B 106 A+B 200 Signiflcantlgyreaterthan thesolventcontrolsp,<0.01 NO I' ol: COMP(J'FED SIMPLE COMPLEX OTIIEP ABERRA- ................................................................................................... TIONS TG:* SG UC 'rB SB 11) TR QR CR D R cl DF GT PER CELL CEI Wi ADET ]'I 14 21 35 21 21 42 9 1 1 1 1 1 1 2 1 11 1 21 1 12 4 1 2 7 1 0.01 1. 0.03 2. 0.02 1. 1 0.11 2. 0.03 3. 1 0.07 2.5 2 1.88 56.0 1 2 11 31 1 1 32 2 11 1 1 2 4 1 4 1 2 1 12 1 1 24 1 6 1 1 1 2 1 1 13 4 6 13 31 1 15 5 1 5 2 2 6 11 1 2 2 19 4 1 11 2 3 9 14 2 3 17 29 4 2 17 4 2 4 6 4 1 2 4 23 2 14 3 4 10 21 4 4 2 11 52 6 2 31 7 6 14 27 8 5 4--15 0.04 1.0 0.02 2.0 0.03 1.5 0.06 4.0 0.07 6.0 0.07 5.0 0.21 0.31 11.0 18.0 0.26 14.5 1 0.57 25.5 0.69 36.8 1 0.64 31.5 CL k.< z Assay No.:17073 Compound:T-6342 CHROMOSOME Trial#:2 TABLE8 ABERRATIONS IN CHINESE []AMSTER CellsFixed 20.0 Hours AfterTreatment OVARY CELLS Lab #:CY9155 Date:09/13/95 MetabolicActivation:+S9 CONTROLS NEGATIVE: McCoy's 5a SOLVENT: Water POSITIVE: CP CELLS SCORED A 100 B 100 A+B 200 IO.Opi/mi A 100 B 100 A+B 200 5.0ong/mi A 25 TEST ARTICI.E 1250pg/mi A 100 B 100 A+B 200 2490pg/mi A 100 11 100 3730pg/ml A+B 200 A 100 B 100 4970pg/mi A+li 200 A 75 13 75 A+B 150 Significantlgyreaterthan thesolventcontrolsp,<0.01 Significantlgyreaterthanthe solventcontrolsp,<0.05 NI)MBI:RANI)'I'Ylll.'AO[lI:LRRA'I'ION NOT COMPUTED SIMPLE COMPLEX OF % .........................................................................0..1..'..I..I..E..FA..B..L..R..R.A.- CELL TG SG tJC 'rB:SB ID TR QR:: CR 1) R Cl [)F GT TIONS WITII PER ABERP, CELL TIONI 2 52 72 4 11 6 1 10 11 1 11 4 3 4 3 3 3 1 1 11 1 2 0.03 2.0 0.00 0.0 0.02 1.0 0.03 3.0 0.00 0.0 0.02 1.5 0.49 36.0* 61 2 2 2 21 81 2 2 21 5 71 9 11 91 1 21 11 1 12 1 18 1 1 1 1 1 29 1 2 17 9 2 9 10 1 2 32 3 17 2 3 10 19 4 2 61 4 2 34 11 5 19 29 5 4 18 5 1 13 0.03 2.0 0.02 2.0 0.03 2.0 0.02 2.0 0.01 1.0 0.02 1.5 0.02 2.0 ().Ol 1.0 0.02 1.5 0.79 41.3 0.83 44.0 0.81 42.7* CHROMOSOME TABLE9 ABERRATIONS IN CHINESE HAMSTER CellsFixed44.1 "ours AfterTreatment OVARY CELLS Assay No.:17073 Trial#:2 Lab #: CY9155 MetabolicActivation:+S9 Compound:T-6342 CONTROLS NEGATIVE: McCoy's 5a SOLVENT: Water Date:09/13/95 NUMBER ANI)'I'YIIOLl-Atil-RRA'I'ION CELLS SCORED NOI' 9 OF % compu,rED SIMI'LE COMPI.EX 01'IIEHABFRRA- CELLS .............................................................................]...'..I.O.N.S...W.I.T.I.I......... TG SG TBI SR 10 TR QR:: CR D R Cl DF GT PER ABERR CELL 'I'IONS A 100 3 1 1 B too 2 1 A+B 200 5 1 1 1 IO.Opilnil A 100 2 2 1 B 100 2 2 11 3 A+B 200 4 4 12 3 0.01 1.0 0.01 1.0 0.01 1.0 0.01 1.0 0.05 5.0 0.03 3.0 TEST ARTICLE 498pg/mi A B 100 6 12 1 100 4 1 A+B 200 10 1 1 2 1 1250pg/mi A B 100 3 100 5 2 A+B 200 8 2 2490pg/nil A B 100 1 2 100 6 2 1 1 A+B 200 7 4 11 3730pg/iiii A 100 2 2 2 B 100 3 1 1 Atli 200 5 3 3 4970pgtml** A 0 B0 A+B 0 Significantlgyreaterttiaitihe solventcoiilrt)lps<,0.0I Toxic dose level. 1 1 2 1 1 1 1 1 1 O@04 3.0 0.01 1.0 0.03 2.0 0.00 0.0 0.01 1.0 0.01 0.5 0.02 2.0 0.01 1.0 0.02 1.5 0.03 2.0 0.01 1.0 0.02 1.5 CORNINGHazieton CONTROL DATA OF CHROMOSOME TABLE 10 ABERRATIONS IN CHINESE HAMSTER 6/95 THROUGH 8/95 OVARY CELLS Negative Control SolventControl PositiveControl Mitomycin C Negative Control SolventControl PositiveControl Cyclophosphamide Activation Without Without Without With With With MIN MAX AVG N MIN MAX AVG N MIN MAX AVG N MIN MAX AVG N MIN MAX AVG N MIN MAX AVG N # Of AberTations Per Cell 0.00 0.06 0.015 35 0.00 0.03 0.010 35 0.14 2.88 0.553 26 0.00 0.03 0.014 34 0.00 0.12 0.020 34 0.36 4.28 1.299 25 % Of Cells with Aberrations 0.0 4.0 1.19 35 0-0 2.0 0.74 35 14.0 68.0 31.38 26 0.0 3.0 1.10 34 0.0 7.0 1.44 34 24.0 88.0 51.20 25 % Of Cells With >I Aberrations 0.0 0.5 0.07 35 0.0 0.5 0.04 35 0.0 60@O 11.38 26 0.0 1.0 0.09 34 0.0 4.0 0.29 34 8.0 72.0 30.40 25 CONTROL DATA OF POLYPLOIDY IN CHINESE HAMSTER OVARY CELLS 4/95 THROUGH 9/95 Negative Control Solvent Control MIN MAX AVG N MIN MAX AVG N Without Activation 0.00 4.00 0.540 46 0.00 2.00 0.430 59 With Activation 0.0 4.0 0.63 46 0.0 3.0 0.50 58 CHV Study No.: 17073-0437CO 32 CORNINGHazleton 15.0 DEFINITIONS OF CHROMOSOME CELLS NOT COMPUTED ABERRATIONS FOR GIEMSA STAINED TG Chromatid gap: SG Chromosome gap: UC Uncoiledchromosome: PP Polyploidcell: E Endoreduplication: SIMPLE ("tigdap").An achromatic(unstainedr)egionin one chromatid,the sizeofwhich isequaltoor smallerthanthe width of a chromatid. These are notedbut not usuallyincludedinfinaltotalsof aberrationsas they may not allbe truebreaks. ("isochromatigdap,IG"). Same as chromatidgap butatthe same locusinboth sisterchromatids. Failureof chromatinpacking. Probably not a true aberration. A cellcontainingmultiplecopiesof thehaploid number (n)of chromosomes. Not countedinthe cellsscored foraberrations. 4n cellinwhich separationof chromosome pairs has failed.Not counted inthecellsscoredfor aberrations. TB Chromatic break: SB Chromosome break: DM "DoubleMinute " fragment: An achromaticregioninone chromatid,larger thanthe width of a chromatid. The associated fragmentmay be partialloyr completely displaced. Chromosome has a clearbreak,forming an abnormal (deleted)chromosome withan acentnc fragment thatisdislocated. These aresmall double dots,some of which are terminaldeletionsand some interstitidaelletions and probablysmallrings.Theiroriginsare not distinguishable. CHV Study No.: 17073-0437CO 33 COMPLEX ID Interstitdiealetion: TR Triradial: QR Quadriradial: D Dicentric: DF TC Tricentric: QC Quadricentric: PC Pentacentric: HC Hexacentric: R Ring RC Ring Chromatid: RF CHV StudyNo.: 17073-0-437CO CORNINGHazleton Lengthofchromatid"cutout"from midregionof a chromatidresultinigna smallfragmentof ring lyingbesidea shortenedchromatidof a gap inthe chromatid. An exchange between two chromosomes, orone chromosome and an acentrifcragmenl which resultisna three-armedconfiguration. As triradiablu,tresultinigna four-armed configuration. An exchange between two chromosomes which resultisna chromosome with two centromeres. This isoftenassociatewdith an acentricfragment inwhich caseitisclassifieads DF. Dicentricwithfragment. An exchange between threechromosomes which resultisna chromosome with threecentromeres. Oftenassociatewdithtwo tothreeAF. Such exchangescan involvemany chromosomes and arenamed as follows: fourcentromeresu,p tofourAF fivecentromeres,up tofiveAF sixcentromeres,up tosixAF A chromosome which forms a circlecontaininga centromere.Thisisoftenassociatedwith an acentrifcragmentinwhich caseitisclassedas RF. Singlechromatidring(acentric). Ring with associateadcentricfragment. 34 Cl Chromosome Intrachange: T Translocation: AB Other GT/> CORNINGHazieton Exchange withina chromosome; e.g.a, ringthat doesnotincludetheentircehromosome. Obvioustransfeorfmaterialbetweentwo chromosomes resultinigntwo abnormal chromosomes. When identifiablsec,oredas "T" not"2Ab." Abnormal monocentricchromosome. Thisisa chromosome whose morphology isabnormalfor thekaryotypea,nd oftentheresultofa translocatiopne,ricentrincversione,tc. Classificatiuosnedifabnormalitycannotbe ascribedto;e.g.a, reciprocatlranslocation. A cellwhich containsmore than10 aberrations. A heavilydamaged cellshouldbe analyzedto identiftyhetypesofaberrationasnd may not actuallhyave>I 0,e.g.m,ultiplefragmentssuch asthosefoundassociatewditha tricentric. CHV StudyNo.: 17073-0-437CO 35