Document 85a2DQYqBYeYyMLbRjgnDG3Mo

CORNINGHazleton MUTAGENICITY TEST ON T-6564 MEASURING CHROMOSOMAL ABERRATIONS IN CHINESE HAMSTER OVARY (CHO) CELLS: WITH A CONFIRMATORY ASSAY WITH MULTIPLE HARVESTS FINAL REPORT AUTHOR HemalathaMurli,Ph.D. PERFORMING LABORATORY Coming HazletonInc.(CHV) 9200 LeesburgPike Vienna,Virginia22182 ---------- LABORATORY PROJECT IDENTIFICATION CHV StudyNo.: 17750-0-437CO SUBMITTED TO 3M Corporation Building220-2E-02 3M Center St.Paul,Minnesota55144-1000 STUDY COMPLETION DATE September16,1996 CHV StudyNo.: 17750-0-437CO I of30 CORNINGHazleton QUALITY ASSURANCE STATEMENT ProjecTtitle:ChromosomalAberrationisnChineseHamsterOvary(CHO) CellsW:itha ConfirmatoryAssay With MultipleHarvests ProjectNo.: 20990 ProtocolNo.:437CO Assay No.: 17750 EditionNo.: 4,Modified for3M Corporation QualityAssurance inspectionsofthestudyand review of thefinalreportof theabove referenced projectwere conducted accordingtotheStandardOperatingProceduresof theQualityAssurance Unit and accordingtothegeneralrequirementsoftheappropriateGood LaboratoryPractice regulationsF.indingsfrom theinspectionsand finalreportreview were reportedto management and tothe studydirectoron thefollowingdates: Insl2ection/Date FindingsReported Auditor Dosing/06/20/1996 06/21/1996 C. Orantes DraftReport Review/08/21/1996 08/22/1996 C. Orantes FinalReport Review/09/16/1996 09/16/1996 C. Orantes In A <---@OuQaulai@lAisAtssyusr@uarnacne@c@e@ L DDaa@ttee ele ed CHV StudyNo.: 17750-0437CO 2 CORNINGHazleton STUDY COMPLIANCEAND CERTIFICATION The describedstudywas conductedincompliancewiththeGood LaboratoryPracticeregulations assetforthin theFood and Drug Administratio(nFDA) Title21 oftheU.S.Code ofFederal RegulationsPart58,issuedDecember 22, 1978,(effectiJvuene20,1979)withany applicable amendments. Therewereno significadnetviationfsromtheaforementionerdegulationosrthe signedprotocotlhatwould affectheintegritoyfthestudyortheinterpretatioofnthetestresults. The raw datahave beenreviewedby theStudyDirectorw,ho certifitehsattheevaluatioonf the testarticlaespresentedhereinrepresentasn appropriatceonclusionwithinthecontextof the studydesignand evaluatiocnriteria. Alltestand controlresultisnthisreportaresupportedby an experimentadlatarecordand this recordhasbeenreviewedby theStudyDirector.Allraw data,documentationr,ecordsp,rotocol and a copy ofthefinalreportgeneratedas a resulotf thisstudywillbe archivedinthestorage facilitioefsComing HazletonInc.foratleastone yearfollowingsubmissionofthefinalreporto theSponsor.Aftertheone yearperiod,theSponsormay electto have theaforementioned materialrsetaineidnthestoragefacilitioefsComing HazletonInc.foran additionapleriodof time,orsenttoa storagefacilitdyesignatebdy theSponsor. SubmittedBy: StudyDirector: HemalathaMurli,Ph.D. Mammalian Cytogenetics DepartmentofGeneticand CellulaTroxicology Study Cor@pletion Date CHV StudyNo.: 17750-0437CO 3 CORNINGHazleton TABI,IFOF CONTEM PageNo. ABSTRACT ...................................................6.............. 1.0 SPONSOR ..............................................7............. 2.0 MATERIAL (TEST ARTICLE) .................................7......... 2.1 Clienfs Identification 2.2 Date Received 2.3 PhysicalDescription 2.4 GeneticsAssay No. 3.0 TYPE OF ASSAY .........................................7............ 4.0 PROTOCOL NO ...........................................7............ 5.0 STUDY DATES ..........................................7............ 5.1 InitiationDate 5.2 ExperimentalStartDate 5.3 ExperimentalTerminationDate 6.0 SUPERVISORY PERSONNEL .................................7......... 6.1 Study Director 6.2 LaboratorySupervisor 7.0 OBJECTIVE .............................................7 ............. 8.0 RATIONALE ............................................8............. 9.0 EXPERIMENTAL DESIGN ...................................8.......... 10.0 MATERIALS AND METHODS .................................9......... 10.1 Test Cells 10.2 CellCultureMedium 10.3 Negative and SolventControls 10.4 PositiveControlAgents 10.5 RangefindingAssays 10.6 AberrationsAssay WithoutMetabolicActivation CHV Study No.: 17750-0-437CO 4 CORNINGHazleton 10.7 10.8 10.9 10.10 AberrationAsssay With MetabolicActivation HarvestProcedure SlidePreparatioannd Staining AberrationsAnalysisand Assay Evaluation 11.0 RESULTS ...........................................I .............. 11.1 Solubilitaynd Dose Determination 11.2 RangefindingAssay WithoutMetabolicActivation 11.3 RangefindingAssay With MetabolicActivation 11.4 Chromosomal AberrationAsssay Without MetabolicActivation 11.5 Chromosomal AberrationAsssay With MetabolicActivation 12.0 CONCLUSION ........................................18.............. 13.0 REFERENCES ........................................18............. 14.0 DEVIATION FROM THE SIGNED PROTOCOL .....................18...... 15.0 EXPERIMENTAL DATA TABLES ............................1.9......... 16.0 DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS ........................................................... CHV StudyNo.: 17750-0-437CO 5 CORNINGHazleton ABSTRACT The objectivoef thisinvitroassaywas toevaluatetheabilitoyfT-6564 toinducechromosomal aberrationisn Chinesehamsterovary(CHO) cellswith and withoutmetabolicactivation. The specifigcravityoftheneattestarticlweas evaluatedas 1.22glml inthetestinglaboratory and thiswas usedto calculattehedosingstockpreparationD.ilutionsofthetestarticlweere preparedinsterildeeionizedwater.For thedoserangefmdingassayswithand withoutmetabolic activationc,oncentrationosf0.169,0.508,1.69,5.08,16.9,50.8,169,508, 1690,and 5080 gg/ml were testedA.lldosingwas achievedusinga dosingvolume of 1.0% (10gl/ml).Complete cytotoxicitwyas observedintheculturesdosed with5080 gg/ml with and withoutmetabolic activationA. reductionof92% was observedinthemitoticindexof theculturedosed with 1690 gg/ml withoutmetabolicactivatioans compared withthesolventcontrolculture.Reductionsof 44%, 15%, and 38% were observedinthemitoticindicesof theculturesdosed with 169,508, and 1690 pg/ml withmetabolicactivatiorne,spectivelays, compared with thesolventcontrol culture. Based on thedatafrom thedose rangefindingassay,replicatceulturesof CHO cellswere incubatedwith 62.5,125,250, 500, 1000,1500,and 2000 pg/ml withoutmetabolicactivation and with250,500,1000,2000,3000,and 4000 gg/ml withmetabolicactivatioinn20.0hour assays.Culturestreatedwith 125,250,500,and 1000 gg/ml from theassaywithoutmetabolic activatioanssayand with250,500,1000,and 2000 pg/n-dwithmetabolicactivatiownere analyzedforchromosomal aberrationsN.o significanitncreaseincellswithchromosomal aberrationwsas observedattheconcentrationasnalyzed. In theconfirmatorytrialr,eplicatceulturesof CHO cellswere incubatedwith 100,200,400,600, 800,1000,and 1200 gg/ml and with 50.0,100,200,400,600,800,1000,and 1200 gg/ml withoutmetabolicactivatiownith20.1and 44.2hour harvestsr,espectivelyI.n theassaywith metabolicactivationc,ultureswere incubatedwith 500, 1000,1500,2000,2250,2500,2750, and 3000 gg/ml with20.1and 44.2hour harvests.Culturestreatedwith200,400, 600,and 800 gg/ml from the20.1hournonactivatioanssay,with 100,200,400,and 600 gg/ml from the 44.2hournonactivatioanssay,with 1500,2000,2250, and 2500 pg/ml from the20.1hour activatioanssay,and with2000,2250,2500, and 2750 gglml from the44.2hour activatioanssay were analyzedforchromosomal aberrationsN.o significanitncreaseincellswithchromosomal aberrationwsas observedattheconcentrationasnalyzedexceptforthehighestdose analyzed (2500 pg/ml from the20.1houractivatioanssayand 2750 pg/ml from the44.2hour activation assay)from theactivation. T-6564 was considerednegativeforinducingchromosomal aberrationisnCHO cellswithand withoutmetabolicactivatione,xceptata singledose levelwithmetabolicactivatiotnhatinduced significatnotxicity. CHV Study No.: 17750-0-437CO 6 CORNINGHazleton ChromosomAablerratiionnCshinesHeamsteOrvary(CHO)Cells: With a ConfirmatoryAssay With MultipleHarvests With T-6564 1.0 SPONSOR: 3M Corporation 2.0 MATERIAL (TEST ARTICLE): 2.1 Clienfs IdentificatioTn-:6564 L- 13167 FC- 1015-X 2.2 DateReceived:May 30,1996 2.3 PhysicalDescriptionC:lear,colorleslsiquid 2.4 GeneticsAssayNo.: 17750 3.0 TYPE OF ASSAY: Chromosomal AberrationisnChineseHamsterOvary (CHO) Cells: With a Confu-matoryAssay With MultipleHarvests 4.0 PROTOCOL NO.: 437CO, Edition4,Modifiedfor3M Corporation 5.0 STUDY DATES: 5.1 InitiatiDoante:June4,1996 5.2 ExperimentalStartDate:June 13,1996 5.3 ExperimentaTlerminationDate:July26,1996 6.0 SUPERVISORY PERSONNEL: 6.1 StudyDirectorH:emalathaMurli,Ph.D. 6.2 LaboratorySupervisor:CarolS.Spicer,B.S. 7.0 OBJECTIVE: The objectivoefthisinyi= assaywas toevaluatetheabilitoyfT-6564 toinduce chromosomalaberrationisnChinesehamsterovary(CHO) cellsw,ithand without metabolicactivation. CHV StudyNo.: 17750-0-437CO 7 CORNINGHazleton 8.0 RATIONALE: 'Me assayisdesignedtoestabliswhhetherthetestarticloeritsmetabolitecsan interact withcellstoinducechromosome breaks.Chemicallyinducedlesionsmay resulitn breaksinchromatinthatareeitherepairedby thecellinsucha way astobe undetectable orresulitnvisibldeamage. Aberrationasrea consequenceoffailuroermistakesinrepair processessuchthatbreaksdo notrejoinorrejoininabnormalconfiguratio(nEsvans, 1962). 9.0 EXPEFJMENTAL DESIGN: Resultsfrom therangefindinagssaywere usedto determinethedoserangetobe usedin thechromosomal aberrationasssay.Intherangefindinagssay,theculturewsere harvested20.0hoursafteirnitiatioofntreatment.Mitoticindexand visualobservatioonf theculturewsere evaluatedforevidenceoftoxicityA. sununaryofthetreatment schedulefortherangefindinagssayisgivenbelow. Summary ofDose RangefindingAssav_TreatmentSchedulein Hours Test TestArticle Wash Colcemid' Fixation -s9 0 17.8 18.0 20.0 +s9 0 3.0 18.0 20.0 Inthechromosomal aberrationasssays,replicatceulturewsere used ateach doselevel, and negativeand solventcontrols.Singleculturewsere usedforeach of two dosesof the positivceontrol.The aberrationasssayswere conductedwitha 20.0hourharvestimein theinitiatlriaalnd with20.1and 44.2hourharvestimesintheconfirmatortyrials. Chromosomal aberrationwsere analyzedfrom theculturetsreateadtfourdose levelsand from one ofthepositivceontroldoses.A summary ofthetreaunentscheduleforthe chromosomal aberrationasssaysisgivenon thefollowingpage. CHV StudyNo.: 17750-0-437CO 8 CORNINGHazieton Summ= ofChromosomAablerratiAosnssavTreatmeSnctheduilneHours Test InitiTarlial S9 +s9 ConfirmatoEvT_rial -s9 +s9 -s9 +s9 TestArticle Wash 0 17.8 0 3.0 0 17.8 0 3.0 0 41.8 0 3.0 ColcemidO Fixation 18.0 20.0 18.0 20.0 18.1 20.1 18.1 20.1 42.2 44.2 42.2 44.2 10.0 MATERIALS AND METHODS: 10.1 TestCells: The Chinesehamsterovarycells(CHO-WBL) usedinthisassaywere from a permanentcelllineand were originalloybtainedfromthelaboratoroyfDr.S. Wolff,UniversitoyfCaliforniSaa,n Francisco.The cellhsave sincebeen reclonedtomaintainkaryotypisctabilitTyh.iscelllinehasan averagecycletime of 12to14 hourswitha modal chromosome number of21. 10.2 CellCultureMedium: The CHO cellwsere grown inMcCoy's 5a culturmeedium which was supplementedwith10% fetablovineserum (FBS),1% L-glutaminea,nd 1% penicillaind streptomycina,tapproximately37*C, inan atmosphereofabout 5%Co2in air. 10.3 Negativeand SolventControls: Inthenonactivatioanssays,negativecontrolwsere culturewshich containonly cellsand culturemedium. Solventcontrolwsere culturecsontainintghesolvent CHV StudyNo.: 17750-0437CO 9 CORNINGHazleton forthetesatrticsltee,ridleeionizweadtera,tthehighecsotncentrautsieodinntest cultures1,0.0gl/ml.Intheactivatioanssays,thenegativeand solventcontrols were thesame asdescribedinthenonactivatioanssaysbutwiththeS9 activation mix included. 10.4 PositivCeontrolAgents: The positivceontrolagentswhich were used intheassayswere mitomycin C (MMC) forthenonactivatisoenrieasndcyclophosphamid(eCP) inthemetabolic activatiosneriesM.itomycinC (CASN 50-07-7,Sigma,Lot # 25HO619) isa clastogetnhatdoesnotrequiremetabolicactivationC.yclophosphamide(CAS 6055-19-2,Sigma,Lot# 67FO 155)does notactdirectlbyutmust be convertedto activeintermediatebsy microsomalenzymes. Inthechromosomal aberrations assayst,wo concentratioonfsMMC (0.08and 0.1jig/mla)nd CP (5.0and 10.0gg/ml)were usedtoinducechromosomal aberrationisntheCHO cellsO.ne ofthedose levelwsas analyzedineach of theaberratioanssays.Both MMC and CP were dissolveidnwater. 10.5 Rangefmding Assays: Intheseassays,thecellswere culturedforapproximately24 hourspriorto treatmentby seedingapproximatel0y.3x 10"cellsper25 cm' flaskinto5 ml of completeMcCoy's 5a culturemedium. IO.S.I Assay WithoutMetabolicActivation: The culturewsereincubatedwiththetestarticlfeor17.8hoursat 7 C. Then,thetestarticlweas washed from thecellswithphosphatebuffered salinaend freshcompletemedium containinCgolcemid'(final concentratio0n.1;ig/mlw)as added.The culturewserethentrypsinized and harveste2d.0hourslater.(SeeSectionson Harvestand Slide Preparatioannd Staining). 10.5.2Assay WithMetabolicActivation: Inthisassay,theCHO cellswere exposedtothetestarticlfeorthreehours at-37*C inthepresenceofa ratliverS9 reactiomnixture(S9 15 PI/ml, NADP 1.5mg/ml,andisocitraiccid2.7mg/ml). The S9 fraction (MolecularToxicologyI,nc.L,ot #0583 usedfordoserangefindinagssay and inititarliaolfchromosomal aberrationasssay;Lot 0667,used forthe CHV StudyNo.: 17750-0-437CO 10 CORNINGHazleton confirmattorrioyaflchromosomaablerratiaosnssa)ywasderivferdom theliverofmale Sprague-Dawleyratswhich had beenpreviousltyreated withAroclor1254toinducethemixed functionoxidaseenzymes which arecapableofmetabolizincghemicalstomore activeforms.The three hourincubationtimewas usedbecauseprolongedexposuretotheS9 mixturemightbe toxictothecellsand theenzyme activitoyfS9 islost rapidlyatabout37*C. The medium didnothaveFBS duringtheexposure periodtoavoidpossiblienactivatioofnshortlivedand highlyreactive intermediatepsroducedby theS9 enzymes by bindingtoserum proteins. Aftertheexposureperiodthecellswerewashed twicewithbuffered saline.CompleteMcCoy's 5a medium was added totheculturewshich were thenincubatedfor17.8hourswithColcemid* (finacloncentration 0.1gg/ml)added forthe last2.0hoursto collecmtetaphasecells.The culturewsere then trypsinizehda,rvestedf,ixed,and slideswere prepared and stainedaswas describedforthenonactivatiornangefmdingassay. 10.5.3Assay Evaluation: Mitoticindexwas analyzedfromthehighestfour(nonactivatiaosnsay) and three(activatiaosnsay)survivindgoselevelsby analyzingthenumber of metaphasespresentin 1000 consecutivecells. 10.6 AberrationAsssay WithoutMetabolicActivation: Cultureswere initiatbeydseedingapproximatel1y.2x 10"cells(=20 hourassay) and 0.8X 10"cells(44.2hourassay)per75 cm2 flaskinto10ml ofcomplete McCoy's 5amedium. One day aftecrulturienitiatitohne,cellswere incubatedat =37*C withthetestarticlaetpredeterminedosesfor17.8or41.8hours.The culturewsere thenwashed withbufferedsalineand completeMcCoy's 5a medium containin0g.1 gg/ml Colcemid*was placedbackontothecells.Two hourslatert,hecellswere harvestedand airdriedslideswere made. The slides were thenstainedin5 % Giemsa solutiofnortheanalysiosfchromosomal aberrations. 10.7 AberrationAsssay With MetabolicActivation: Cultureswereinitiatbeyd seedingapproximately1.2x 101cells(=20hourassay) and 0.8X 10'cell(s44.2hourassay)per75 cm' flaskinto10ml ofcomplete McCoy's Sa medium. One day aftecrulturienitiatiotnh,eculturetshatwere CHV StudyNo.: 17750-0437CO CORNINGHazieton treateudndetrheconditioonfmsetabolaictivatwieorneincubataetd=37*Cfor threehoursinthepresenceof thetestarticlaend the S9 reactionmixturein McCoy's 5a medium withoutFBS. Afterthethreehour exposureperiodthecells were washed twicewithbufferedsalineand thecellswere refedwithcomplete McCoy's 5a medium. T*hecellswere incubatedfortherestof thecultureperiod up to thetimeof harvestwith0.1 gg/ralColcemid" presentduri.-tihgelast2.0 hoursof incubation.The metaphase cellswere thenharvestedand preparedfor cytogenetiacnalysis. 10.8 HarvestProcedure: Priortotheharvestof theculturesv,isualobservationosf toxicitwyere made. These observationisncludedan assessmentofthepercentconfluenceofthecell monolayerwithinthe cultureflasks.The cultureswere alsoevaluatedforthe presenceofmitotic(largeroundedcellso)rdead cellsfloatinignthemedium. The culturesfrom thedose rangefmdingassaywere trypsinizefdirsttocollect mitoticand interphasceellsand were treatedwith 0.075M KCI hypotonic solutionT.histreatmenthelpstoswellthecellsand thusdispersethe chromosomes. The cultureswere thenfixedwith an absolutemethanol:glacial aceticacid(3:1,v:v)fixativaend were washed severaltimesbeforeair-dried slideswere prepared. 10.9 SlidePreparatioannd Staining: Slideswere preparedby droppingtheharvestedcultureson cleanslides. The slideswere stainedwith5% Giemsa solutionfortheanalysisof mitoticindexand chromosomal aberrationsA.llslideswere thenair-dried and coverslippedusingDepexe mounting medium. 10.10 AberrationsAnalysisand Assay Evaluation: Cellswere selectedforgood morphology and only cellswiththenumber of centromeresequaltothemodal number 21 I (range20-22)were analyzed. One hundredcellsi,fpossiblef,rom eachreplicatceultureatfourdose levelsof thetestarticlaend from thenegativeand solventcontrolcultureswere analyzed forthedifferenttypesofchromosomal aberration(sEvans,1962;See Section 16.0).At least25 cellswere analyzedforchromosomal aberrationfsrom one of thepositivecontrolculturesand from thoseculturesthathad greaterthan50% of cellswithone ormore aberrationsF.or controlof bias,allslidesexceptforthe positivceontrolwsere coded priortoanalysis.Cellswithaberrationwsere CHV StudyNo.: 17750-0-437CO 12 CORNINGHazleton recordoendthedatsaheetbsythemicroscospteagleocatiMoint.otincdewxas assessedby analyzingthenumber of mitoticcellsin1000 cellsand theratiowas expressedas a percentageofmitoticcells. The followingfactorwsere takenintoaccountintheevaluatioonfthe chromosomal aberrationdsata: I. The percentageof r-ellwsithany aberrations. 2. The percentageof cellswithmore thanone aberration. 3. Any evidenceforincreasinagmountsofdamage withincreasing dose,i.e.a,positivedoseresponse. Chromatidand isochromatigdaps,ifobserved,were notedintheraw dataand were tabulatedT.hey were not,however,consideredintheevaluatioonfthe abilitoyfthetestarticlteoinducechromosomal aberrationssincetheymay not representruechromosomal breaksand may possiblybe inducedby toxicity. Percentpolyploidywas analyzedfrom the44.2hour assayand resultwsere tabulatedH.istoricaclontroldataarepresentedinTable8. A cellclassifieads"GT" isconsideredtocontain10aberrationfsorstatistical purposesbuta ">"isalsoincludedinthetablesforthisclassificattioinndicate thatitisa minimum number. Statisticaanlalysiesmployed theFisheesExactTest(Sokaland Rohlf,1981)to comparethepercentageof cellswithaberrationisneach treatmengtroupwith the resultfsromthesolventcontrolsA. lineatrrendtestofincreasinngumber ofcells withaberrationwsithincreasindgose(Armitage,1971)was alsoperformed.Test articlseignificancweas establishewdhere p<0.01.Allfactorassstatedpreviously were takenintoaccountand thefinalevaluatioonfthetestarticlweas basedupon scientifjiucdgement. 11.0 RESULTS: 11.1 Solubilitaynd Dose Determination The testarticlweas solubleinwater.A clearc,olorlesssolutiownas obtainedata concentrationf500 mg/ml inwater.Thissolutiownas dosedintheabsenceof cellussinga 1.00/(a10gl/ml)dosingvolume inMcCoy's 5a culturmeedium. At a dosedconcentratioonf 5000 ;Lg/mla, clearsolutiownas obtainedand a PH of 8.0 (thesame pH asthatofMcCoy's 5a culturmeedium) was determined.The CHV StudyNo.: 17750-0-437CO 13 CORNINGHazleton specifgircaviotfythetesatrticwlaescalculaitnetdhetestilnagborataosry 1.22g/ml.Thisspecifigcravitywas usedto preparedosingstocksfortheassay. Sterildeeionizedwater(Batch# 19,preparedatCHV) was usedasthevehiclein thisassay.Alldosingwas achievedwitha 1.00/(o10.0pl/ml)dosingofthedosing stocksand thesolventcontrolculturweas dosedwith10.0PI/mlofsterile deionizedwater.Concentrationosf0.169,0.508,1.69,5.08,16.9,50.8,169,508, 1690,and 5080 pg/ml were testedinthedoserangefindinagssays.The stability ofthetestarticluenderthepreparatioannd dosingconditionussed inthisassayis theresponsibiliotfythesponsor. 11.2 RangefindingAssay WithoutMetabolicActivation A deadcellmonolayer,no visiblmeitoticellsf,loatindgeadcellsand debrisa,nd <5% cellmonolayerconfiuencweere observedintheculturedosedwith 5080 gg/ml.An unhealthycellmonolayer,a severereductioninthenumber of visiblmeitoticellsf,loatindgeadcellsand debrisa,nd =45% reductioninthecell monolayerconfluencewere observedintheculturdeosedwith 1690 @Lg/ml.A slightluynhealthycellmonolayer,floatindgeadcellsand debrisa,nd =30% reductioninthecellmonolayetconfluencewere observedintheculturedosed with508 gg/ml.Floatingdebrisand = 15% reductioninthecellmonolayer confluencewereobservedintheculturedosedwith167 gg/ml. Floatingdebris was observedintheculturedosedwith50.8pg/ml.Mitoticindiceswere analyzed from theculturedsosedwith50.8,169,508,and 1690 gg/ml(Table1).A reductioonf92% was observedinthemitotiicndexofthecult= dosedwith 1690 4g/mlascompared withthesolventcontrolcultureB.ased on theseresults, theinitialberrationasssaywithoutmetabolicactivatiownas conductedwitha 20.0hourharvesttestincgoncentrationosf 62.5,125,250,500,1000,1500,and 2000 pg/ml. 11.3 RangefindingAssay With MetabolicActivation No cellmonolayerand no visiblmeitoticellswere observedintheculturedosed with5080 gg/ml.No othersignsofvisualtoxicitwyereobservedinany ofthe otherculturesM.itoticindiceswere analyzedfromtheculturedsosedwith169, 508,and 1690 gg/ml(Table1).Reductionsof44%,15%, and38% were observedinthemitoticindiceosf theculturedsosed with 169,508,and 1690 ILg/mlr,espectivelays,compared withthesolventcontrolcultureB.ased on theseresultst,heinitialberrationasssaywithmetabolicactivatiownas conducted witha 20.0hourharvesttestincgoncentrationosf 250,500, 1000,2000,3000,and 4000 gg/ml. CHV StudyNo.: 17750-0437CO 14 CORNINGHazleton 11.4 ChromosomalAberratioAnsssayWithoutMetaboliAcctivation InitiaTlrial Dead cellmonolayers,floatingdead cellsand debris,no visiblemitoticcellsa,nd =95% reductioninthe cellmonolayer confluencewere observed inthecultures dosed with 2000 jig/ml.Unhealthy cellmonolayers,severereductionsinthe number of visiblemitoticcells,floatingdead cellsand debris,and =45% reductioninthecellmonolayer confluencewere observed intheculturesdosed with 1500 gg/ml. Unhealthy cellmonolayers,severereductionsinthe number of visiblemitoticcellsf,loatingdead cellsand debris,and =30% reductioninthecell monolayer confluencewere observed intheculturesdosed with 1000 @ie/ml. Slightlyunhealthycellmonolayers,floatingdebris,and =15% reductioninthe cellmonolayer confluencewere observed intheculturesdosed with 500 gg/mi. Chromosomal aberrationswere analyzedfrom the culturesdosed with 125,250, 500,and 1000 gg/ml (Table2). Reductionsof 25%, 30%, 87%, and 96% were observed inthemitoticindicesof theculturesdosed with 125,250, 1000, and 1500 gg/ml,respectivelya,s compared with thesolventcontrolcultures.No significanitncreasesincellswith chromosomal aberrationswere observed atthe concentrationsanalyzed. Based on theresultsfrom thistrialt,heconfumatory triaolf thenonactivation aberrationasssaywas conducted testingconcentrationsof 100,200, 400, 600, 800, 1000,and 1200 gg/ml and 50.0,100,200, 400, 600, 800, 1000,and 1200 jig/mlwith 20.1 and 44.2 hour harvests,respectively. ConfirmatoryTrial Inthe20.1 hour assay,veryunhealthycellmonolayers, severereductionsinthe number of visiblemitoticcells,floatingdead cellsand debris,and = 15% reductioninthecellmonolayer confluencewere observed intheculturesdosed with 1200 gg/ml. Unhealthy cellmonolayers,floatingdead cellsand debris,and severereductionsinthenumber of visiblemitoticcellswere observed inthe culturesdosed with 1000 gg/ml. Unhealthy cellmonolayers,floatingdebris,and reductionsinthenumber ofvisiblemitoticcellswere observed inthecultures dosed with 800 pg/ml. Slightlyunhealthycellmonolayers,floatingdebris,and slightreductionsinthenumber of visiblemitoticcellswere observed inthe culturesdosed with 600 gg/ml. Chromosomal aberrationwsere analyzedfrom the culturesdosed with200, 400, 600, and 800 gg/ml (Table3). Reductionsof 73%, 84%, and 95% were observed themitoticindicesof theculturesdosed .Vith800, CHV Study No.: 17750-0-437CO 15 CORNINGHazleton 1000a,nd1200pg/mlr,espectivaescloym,parewdiththesolvecnotntrol culturesN.o significainntcreaseisncellswithchromosomal aberrationwsere observedattheconcentrationasnalyzed. Inthe44.2hourassay,deadcellmonolayers,no visiblmeitoticellsf,loating dead cellsand debrisa,nd =45% reductioninthecellmonolayerconfluencewere observedintheculturedsosedwith1000and 1200Ag/ml.Very unhealthycell monolayers,floatindgead cellsand debriss,everereductionsinthenumber of visiblmeitoticellsa,nd =30% reductioninthecellmonolayerconfluencewere observedintheculturedsosedwith 800 tLg/ml.Unhealthyc.ellmonolayers, floatindgeadcellsand debrisr,eductionisnthenumber ofvisiblmeitoticells, and =15% reductioinn thecellmonolayerconfluencewere observedinthe culturedsosedwith600 pg/ml.Slightluynhealthycellmonolayers,floating debrisa,nd slighrteductionisnthenumber ofvisiblmeitoticcellswere observed intheculturedsosedwith400 gg/ml.Chromosomal aberrationwsere analyzed from theculturedsosedwith100,200,400,and 600 pg/ml (Table4).Reductions of 12%, 61%, 63%, and 92% were observedthemitoticindicesofthecultures dosedwith50.0,400,600,and 800 gg/ml,respectivelays,compared withthe solventcontroclulturesN.o significainntcreaseisncellswithchromosomal aberrationosrinpolyploidywere observedattheconcentrationasnalyzed. The sensitiviotfythecellcultureforinductioonfchromosomal aberrationiss shown by theincreasefdrequencyof aberrationisnthecellsexposedto mitomycinC,thepositivceontrolagent.The testarticliesconsiderendegative forinducingchromosomal aberrationasnd polyploiduyndernonactivation conditions. 11.5 Chromosomal AberrationAsssay With MetabolicActivation InitiTarlial No cellmonolayersand no visiblmeitoticellswereobservedinthecultures dosedwith3000 and 4000 gg/ml.Slightluynhealthycellmonolayersand =30% reductioinnthecellmonolayerconfluencewere observedintheculturedsosed with2000 pg/ml.A reductioonf= 15% inthecellmonolayerconfluencewas observedintheculturedsosedwith1000gg/ml. Chromosomal aberrationwsere analyzedfrom theculturedsosedwith250,500,1000,and 2000 Ag/ml (Table5). Reductionsof2% were observedinthemitoticindicesoftheculturedsosedwith 250 pg/ml ascompared withthesolventcontrolculturesN.o significainntcreases incellswithchromosomal aberrationwsere observedattheconcentrationasnalyzed. CHV StudyNo.: 17750-0437CO 16 CORNINGHazleton Basedon theresulftrsomtheinititarliatlh,econfirmattoriyaolftheaberrations assaywith metabolicactivatiownas conductedtestingconcentrationosf 500, 1000,1500,2000,2250,2500, 2750,and 3000 gg/ml with 20.1 and 44.2hour harvests. ConfirmatoryTrial In the20.1hour assay,dead cellmonolayers,floatindgead cellsand debris,no visiblmeitoticcellsa,nd <5% cellmonolayer confluencewere observedinthe culturedsosed with2750 and 3000 gg/ml. Slightluynhealthycellmonolaycrs, reductionsinthenumber ofvisiblemitoticcellsf,loatingdead cellsand debris, and =45% reductioninthecellmonolayer confluencewere observedinthe culturedsosed with2500 gg/ml.Floatingdebriss,lightreductionisnthenumber of visiblmeitoticellsa,nd =30% reductioninthecellmonolayerconfluence were observedintheculturedsosed with2250 gg/rnl.Reductionsof = 15% inthe cellmonolayer confluencewere observedintheculturesdosed with2000 gg/ml. Chromosomal aberrationwsere analyzedfrom theculturesdosed with 1500,2000, 2250,and 2500 gg/ml(Table6).Reductionsof 13%,38%,15%, 11%, and 30% were observedinthemitoticindicesof theculturesdosed with 1000,1500,2000, 2250,and 2500 gg/mi,respectivelays,compared withthesolventcontrol culturesN.o significanitncreaseisncellswithchromosomal aberrationwsere ob servedattheconcentrationasnalyzed,exceptintheculturesdosed with 2500 gg/mi. In additiona,n increaseinendoreduplicatecdellswere observedin theculturedsosed with2250 and 2500 gg/ml. Inthe44.2 hour assay,dead cellmonolayers,floatingdead cellsand debrisn,o visiblmeitoticcellsa,nd =95% reductioninthe cellmonolayer confluencewere observedintheculturesdosed with3000 gg/ml. Unhealthycellmonolavers, severereductionisnthenumber of visiblmeitoticcellsf,loatingdead cellsand debris,and =55% reductioninthecellmonolayer confluencewere observedinthe culturedsosed with2750 gg/ml. Slightluynhealthycellmonolayers,floating dead cellsand debris,and =45% reductioninthecellmonolaver confluencewere observedintheculturesdosedwith 2500 @Lg/ml.Reductionsof = 15% inthecell monolayer confluencewere observedintheculturesdosed with2250 gg/ml. Chromosomal aberrationwsere analyzedfrom the culturesdosed with 2000,2250, 2500 and 2750 gg/ml (Table7).Reductionsof 14%, 17%, 56%, and 53% were observedinthemitoticindicesoftheculturesdosed with 1000,2250,2500,and 2750 gg/ml,respectivelays,compared withthesolventcontrolculturesN.o significanitncreasesincellswith chromosomal aberrationwsere observedatthe concentrationasnalyzed,exceptin theculturesdosed with2750 gg/ml. In additiona,significanitncreaseinpolyploidywas observedintheculturesdosed CHV StudyNo.: 17750-0-437CO 17 CORNINGHazleton with22502,500a,nd2750gg/mlT.hedoselevelwsithincreased endoreduplicatiwoans intheculturedsosedwith 2250 and 2500 pg/mlinthe20.1 hour assay. The successfualctivatiobny themetabolicsystemisillustratbeydtheincreased incidenceofcellswithchromosomal abertationisnthecultureisnducedwith cyclophosphamidet,hepositivceontrolagent.The testarticliesconsidered negativeforinducingchromosomal aberrationesxceptata singlehighdosethat inducedsignificatnotxicityI.n additiotnhetestarticliesconsiderepdositivfeor inducingendoreduplicatiaond polyploidy. 12.0. CONCLUSION: T-6564was considerendegativeforinducingchromosomal aberrationisnCHO cellswith and withoutmetabolicactivatioenxceptata singledoselevelwithmetabolicactivation thatinducedsignificatnotxicity. 13.0 REFEREN rc!. Armitage,P.StatisticMaelthods inMedicalResearch,John Wiley & Sons,Inc.,New York,NY, 1971. Evans,H.J.:Chromosomal aberrationpsroducedby ionizinrgadiationI.nternational Review ofCytology,.U:221-3211,962. Sokal,R.R.,and Rohlf,F.J.:Biometry,Ed. 2,W.H. Freeman and Company, New York, 1981. 14.0 DEVIATION FROM THE SIGNED PROTOCOL: Endoreduplicatioand polyploidywere analyzedseparatelsyincetherewere some dose levelwsithincreaseedndoreduplicatiaond some withpolyploidy.Thisadded greater scientifiincformatiotnothestudy. CHV StudyNo.: 17750-0-437CO 18 CORNINGHazleton 15.0 EXPERIMEENTAL DATA TABLES CHV StudyNo.: 17750-0-437CO 19 CORNING Hazleton TABLE I RANGEFINDTNG ASSAY FOR ASSESSING TOXICITY Assay No.: 17750 Compound: T-6564 TrialNo.:I Date:06/13/96 Lab No.:CY6156 MetabolicActivation-:S9 Treatment NEGATIVE CONTROL SOLVENT CONTROL TEST ARNCLE McCoy's5a Water % Confluence Mitotic % Solvent Index Control 2.5 100 10.0PUMI 2.4 too 50.9 pgfml 4.7 100 169 pg/mi 4.9 as 508 pgfml 5.7 71 1690 jig/mi 0.2 57 MetabolicActivation+:S9 Treatment NEGATIVE CONTROL SOLVENT CONTROL TEST ARTICLE McCoy's Sa Water % Confluence Mitotic% Solvent Index Control 10.2 100 10.0 pi/mi 10.9 100 169 pg/mi 6.1 100 509pgtml 93 100 1690 pgtml 6.8 100 *Thisendpointisbasedupon visualobservationwshich aremade priortothe harvestofthemetaphasecells.Actualcellcountsarenottakenand any hypertrophyoftheattachedcellcsannotbe evaluated.At thetimeofthe confluenceobsmation theflasksarealsoevaluatedfortheappearanceof floatinmgitoticellsand deadccl[s. McCoy's 5a- culturemedium CHV StudyNo.: 17750-0-437CO 20 r) TABLE2 CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS E! CellsFixed20.0Hours AflerTreatment Z Assay No.: 17750 Trial I Date:06/20/96 Lab H:CY6]96 Metal Compound: T-6564 NUMBER AND TYPE OF ABERRATION CON I'ROLS NEGATIVE: SOLVENT: POSITIVE: TEST ARTICLE NOT COMPI CELI.S ........ ............C....O..M.P..I.,..E...X.......... SCORED TG : SO UC Tn en irtvn McCoy's 53 A 100 2 1 B 100 5 1 2 A+B 200 7 2 2 Water IO.Opl/ml A 100 B 100 4 1 A+R 200 4 1 mmc 0.0801ig/mi so I 1 812 2 1 125jig/mi A 100 1 1 D 100 1 A+B 200 1 2 250lig/mi A 100 2 1 B 100 2 A+B 200 4 1 500 pg/ml A 100 1 B 100 3 1 A+n 200 3 1 1 1000)igtml A 100 2 1 B 100 2 A+D 200 4 1 N.o"rI. ABERRATIONS PER CELI, %-to CEI.LS wi,rii ADERP, TION 0.00 0.0 0.02 1.0 001 0.5 0.00 0.0 0.01 1.0 0.01 0.5 0.29 22,0* 0.02 2.0 0.01 1.0 0.02 1.5 0.01 1.0 0.00 0.0 0.01 0.5 0.00 0.0 0.01 1.0 0.01 0.5 0,01 1.0 0.00 0.0 0.01 0.5 15001tgtml**A+B 0 Signiricentglryeatetrhanthesolventcontrolsp,<0.01. McCoy's5a- cieltumreeditim MMC = Milomycin C Chromosome aberrationnsotanaly7.cddue toexcessivetoxicity. CIIROMOSOME TABLE3 ABERRATIONS IN CtliNESE IIAMSTER OVARY CellsFixed20.1"ours AfterTreatment CEI.LS Assay No.: 17750 TrialN:2 Date:07/10/96 t,ab9:CY7116 Metabol Compound: T-6564 NUM13ER AND TYPE Ol'AIIERRA'I'ION CD 0 CONTROLS NEGATIVE: McCoy's52 CULI.S SCORED NOT N OF % ADERRA- CR-I.LS COMPUTED .S.I.M.P.L.E..............C.O.M.P.L.E.X..................O..T..[..T..E..R....T..I..O..N.S.....W.I.T.I]........... ....:......:.......... PER ABERPA- TO :SO UC TD! sn ID TR !QR 'CR D R ci Inr GT CEI@L. TIONS A 100 1 1 B 100 2 1 1 0.01 1.0 0.01 1.0 A+D 200 3 1 2 SOLVENT: Water 10.0111/ml A 100 2 1 11 1 1 n too 1 0.01 1.0 O@04 2.0 0.00 0.0 A+D 200 2 2 11 1 1 0.02 1.0 POSITIVE: mmc O.Ogopgtml so 2 44 5 71 1 0.44 28.0$ TEST ARTICLE 100ligtml$* A+13 0 200pgtml A 100 5 n 100 6 A+B 200 11 400 jig/mi A 100 4 B 100 4 1 A+D 200 8 1 600ligtmi A 100 3 11 100 A+B 200 3 800 pgtml A 100 3 0 100 4 1 A+D 200 7 1 2 1 3 2 2 1 1 0.02 2.0 0.01 1.0 0.02 1.5 0.02 2.0 0.00 0.0 0.01 1.0. 0.00 0.0 0.00 0.0 0.00 0.0 0.01 1.0 0.00 0.0 0.01 0.5 1000iigfttil*A0+4D 0 1200pgtml**O A4n 0 0 Signiricantglryeaterthanthesolventcontrolsp,<0.01. 00 Chromosome aberrationnsotanalyzeddue tohigherdosesavailablreoranalysis. McCoy's5a -culturmeedium MMC=Mil(iitiycinC Chromosome aberrationnso(analyzeddue toexcessiv CHROMOSOME TABLE4 ABERRATIONS IN CHINESE HAMSTER CellsFixed 44.2floursAfterTreatment OVARY CELLS Assay No.: 17750 Compound: T-6564 -L4it lp 0 CONTROLS NEGATIVE: SOI.VENT: McCoy's 5& Water TrialH:2 Date:07/10/96 Lab H:CY7116 Me CELLS SCORED NUMBER AND TYPE OF ABERRATION - NOT 0 or % ADERRA- CELLS CC COMP.U.TE.D.........S..I..M..P..L.E..............................C.O.M.P.L.E.X................O...'..n..r..E..R.....T..I.O.N.S.... WITII WIT TG .SO !UC TB SB ID TR QR CR D R :1Cl DF GT PER ADERRA-ADE CE'LL TIONS Ti A 100 n 100 2 A4D 200 2 10.0111/mi A 100 5 1 1)100 3 1 A+B 200 9 2 0,()0 0.0 0 0.00 0.0 0 0.00 0.0 0 1 0.01 1.0 0 0.00 0.0 0 1 0.01 0.5 0 TEST ARTICLE SO.Opgtml* A+B 0 100fig/ml A 100 1 1 n 100 1 A+B 200 2 1 200lig/mi A 100 1 n 100 1 A+B 200 1 1 400pgtml A 100 1 1 n 1()0 A+D 200 .I 1 600lig/iiil A 100 4 B 100 2 1 3 1 1 Afl)200 6 13 1 1 ROOligl"ilA*-*tn0 Chromosome aberrntionnsotanalyzeddue tohigherdosesavailablfeoranalysis. McCoy's 5a - culturieiictlitoiii a Includes polyploidy and endoreduplicalion 1 1 1 1 0.01 1.0 0 0.00 0.0 0 0.01 0.5 0 0.00 0.0 0 0.01 1.0 0. 0.01 0.5 0 0.00 0.0 0 0.00 0.0 0 0.00 0.0 0. ().Ol on 0. 0.02 2.0 0. 0.02 1.0 0 Chromosome aberrationsotanalyzeddue toexcessiveinxi CHROMOSOME TABLE5 ABERRATIONS IN CHINESE IIAMSTER CellsFixed20.0Hours AfterTrea(ment OVARY CELLS Assay No.: 17750 Compound: T-6564 Trial I Date:06/20/96 Lab #: CY6196 NUMBRR AND TYPF..OF ABERRATION C) CONTROLS NEOATIVE: SOLVENT: POSITIVE: McCoy's 58 Water CP [O.Opl/mi 5.00 pgtml CELLS SCORED NOT COMPUTED SIMPLE COMPLEX .......................................................................................... TO :SO UC TB: SO ID I TR QR: CR I 1) R I-T Cl DF CIT A 100 1 1 0 100 5 1 A+B 200 6 2 A 100 2 0 100 6 1 A+B 200 6 2 1 25 7 6 1 6 11 6 5 3 TEST ARTICLE 250pg/ml A 100 3 D 100 3 1 A+D 200 61 1 1 1 1 500 pgtml A 100 6 1 1 n 100 1 1 A+B 200 6 1 1 1 1 1000lig/ini A 100 3 1 n 100 A+B 200 3 1 2000JIg/ml A 100 ' 2 1 n 1()0 2 1 1 3 A+B 200 4 2 1 3 Signiricantglryeaterthanthesolventcontrolsp,<0.01. McCoy's5a - ctilitmier(elitim CP = Cycloplinspli-,tmi(fc Metaboli 0 OF ANERPATIONS PER CELL % CELLS WITII AF)RRKA. TIONS 0.00 0.00 0.00 0.00 0.01 0.01 1.52 0.0 0.0 0.0 00 1.0 0.5 64.00 0.00 0.0 0.02 2.0 0.01 1.0 0.02 2.0 o@01 1.0 0.02 1.5 0.01 10 0.00 0.0 0,01 0.5 0.04 3.0 0,00 0.0 0.02 1.5 TABLE6 CIIROMOSOME ABERRATIONS IN CIIINESE 14AMSTER OVARY CELLS L2 CellsFixed20.11foursA (terTrea(ment 1< Z Assay No.: 17750 p Trial9:2 Date:07/10/96 Lab CY7]16 Met - Compound: T-6564 4 A tA NUMBER AND TYPE OF ABI-RRATION lp C) CONTROLS NEGATIVE: McCoy's 58 CF-:LLS SCORED NOT N OF ADEPRA- COMPUTED .......S..I.M.P.I...E...................C.OM.P.L.E.X.......................O...N...[..E..R....T.I.O.N.S ...................... PER TO !:SO UC TO SD ID 1:TR itQR CR D iiR Cl DF O'TT@ CELL A 100 3 tllOO 3 1 1 0.01 0.01 % CELLS W[Tit ABERRATIONS CE WIT AIIE Ti 1.0 0 1.0 0. SOLVENT: A+D 200 6 1 Water IO.Opi/ml A 100 2 B 100 2 1 1 0.01 1.0 0. 1 0.01 1.0 0 1 0.01 1.0 0 A+B 200 4 1 2 0.01 1.0 0 POSITIVE: CP 5.001it/mi 25 4 42 1 14 1 0.52 29.0* 12 TEST ARTICLE 1000ligtml** A+13 0 1500 pglml A 100 1 n 100 2 2 A+B 200 3 2 2000 jig/ml A 100 5 I)100 1 1 A+B 2DO 6 1 2250lig/ml A 100 4 B 100 2 A+B 200 6 2500lig/nil A 100 B 100 7 54 A+B 200 12 4 12 1 12 1 4 44 1 13 3 2 4 9 17 7 6 4 10 1 1 11 1 21 1 2 1 2 2 3 1 3 3 6 0.00 0.0 0 0.01 1.0 0 0.01 0.5 0 O@02 2.0 0 0.01 1.0 0. 0.02 1.5 0. 0.07 4.0 2. 0.00 0.0 0. 0.04 2.0 1. 0.19 too 5. 0.35 15.0 10@ 0.27 - 12.5* 7. .Jignificiiigirteliytietrliiiilicst)lvciciti)iilr(pi<l0s.,01. McCoy's5a- cultumree(litini Cl' Cyclopliosplilitii(le *0 Clir()tiio%oiottli)ccrrniinioitiiisiiiiily(;i,iciiiel(iliigli(elroseosvailabicrotniialysis. Fn(lorcdtiplicatisocnorc(dlue to noticibliencreiseinincidence. CHROMOSOME TABLE7 ABERRATIONS IN CHINESE HAMSTER OVARY CellsFixed 44.2Hours AflerTreatment CELLS Assay No.: 17750 TrialN:2 Date:07/10/96 Lab CY7116 MetabolicA Compound: T-6564 LA 0 CONTROLS NEGATIVE: SOLVENT: McCoy's 5a Water IO.Opl/mi NUMBER AND TYPE OF ABERPAI'ION CEI.IS SCORED #OF NOT ABI RKA- COMPUTED SIMPLE COMPLEX OniER TI'ONS .....................................r..................I ...........................I.......................................P.ER TG i SG UC TB SD ID TR QR 1!CR D R 3 Cl DF GT CELL % CELLS WITII ADERILATIONS CE WIT ABE Ti A 100 3 1 0 100 1 A+D 200 4 1 A 100 1 1 0 100 1 A+D 200 1 1 1 0.00 0.0 0 0.00 0.0 0 0-00 0.0 0 0.01 1.0 0 0,00 0.0 0 0.01 0.5 0 TEST ARTICLE 10OOpg/ml*$ A+B 0 15DOligfml$* A+B 0 2000 pg/mi A 100 1 B 100 1 3 1 A+B 200 2 3 1 2250pgtml A 100 3 n 100 1 1 1 A+D 200 4 1 1 25OOpg/mi A 100 3 1 n 100 .1 1 A+B 200 4 1 1 2750pgtml A 81 B 94 6 1 126 4 13 2 A+B 165 10 2 4 2 8 1 2 21 1 1 2 1 2 4 1 4 42 62 10 2 4 2 0.00 0.0 0 o@ol 1.0 0 0.01 0.5 0 0.01 1.0 0 0.01 1.0 0 0.01 1.0 0 0.03 3.0 0 0.03 3.0 0 O@03 3.0 0 0.23 13.6 4 0.14 11.9 2 0.19 12.71 3 Signiricantlgyreaterthan thesolventcontrols,p<0.01. McCoy's Sa = culturemedium liicitedeIimilyploiilyanticntlt)rc(iiiplicniiiiii Chromosome aberrationsnot onaly7,cddtictohigher doses availablefor analysis. (3N CORNINGHazleton TABLES CONTROL DATA OF CHROMOSOME ABERRATIONS IN CHINESE HAMSTER OVARY CELLS 8/95THROUGH 12/95 Negative Control SolventControl Positive Control Mitomycin C NegativeControl SolventControl PositiveControl Cyclophosphamide Activation Without Without Without With With With MIN MAX AVG N MIN MAX AVG N MFN MAX AVG N MIN MAX AVG N MIN MAX AVG N MIN MAX AVG N Of %Ofcelis % Of Celis % Endort- Aberrations With With >1 duplicated Per Cell Aberrations Aberrations Cells 0.00 0.0 0.0 0.0 0.04 4.0 0.5 5.0 0.012 1.04 0.01 0.19 36 36 36 39 0.00 0.05 0.012 36 0.0 5.0 0.93 36 0.0 0.5 0.01 36 0.0 2.0 0.14 39 0.24 2.96 0.770 27 20.0 94.0 39.70 27 0.0 60.0 16.52 27 0.00 0.0 0.0 0.0 0.03 2.5 1.0 3.0 0.017 1.40 0.10 0.45 31 31 31 28 0.00 0.0 0.0 0.0 0.12 7.0 4.0 4.0 0.023 1.78 0.26 0.64 29 29 29 28 0.40 8.88 1.728 24 32.0 100.0 57.17 24 8.0 100.0 32.93 24 % POINploid-, Celts 0.0 9.5 1.93 45 0.0 10.0 2.20 45 0.0 12.5 2-33 30 0.0 13.0 2.29 30 CHV StudyNo.: 17750-0437CO 27 CORNINGHazleton 16.0 DEFINITIONOSF CHROMOSOME ABERRATIONSFOR GIEMSA STAINED CELLS NOT COMPUTED TG Chromatidgap: SG Chromosome gap: UC Uncoiledchromosome: PP Polyploidcell: E Endoreduplication: SIMPLE ("tigdap").An achromati(cunstainerde)gionin one Chromatid,thesizeof which isequalto or smallerdm thewidthofa chromatid.Theseare notedbutnotusuallyincludedinfinatlotalosf aberrationasstheymay not-allbe truebreaks. ("isochromatigdap,IG").Same aschromatidgap butatthesame locusinbothsistecrhromatids. Failureofchromatinpacking.Probablynota true aberration. A cellcontaininmgultiplecopiesofthehaploid number (n)ofchromosomes. Not countedinthe cellsscoredforaberrations. 4n cellinwhich separatioonfchromosome pairs hasfailedN.ot countedinthecellscoredfor aberrations. TB Chromaticbreak: SB Chromosome break: DM "DoubleMinute fragment: An achromaticregioninone chromatid,largetrhan thewidthofa chromatid.The associatefdragment may be partialloyrcompletelydisplaced. Chromosome hasa clearbreak,formingan abnormal(deletedc)hromosome withan acentric fi-agmentthatisdislocatedT.hisclassificatniown includetsheacentrifcragment(AF).The AF was differenftrom theSB onlyinthatitwas not apparentlryelatedtoany specificchromosome. Thesearesmalldoubledots,some ofwhich are terminaldeletionasnd some interstitdiealletions and probablysnudirings.Theiroriginasrenot distinguishable. CHV StudyNo.: 17750-0-437CO 28 CONTLEX ID Interstitdiealletion: TR Triradial: QR Quadriradial: D Dicentric: DF TC Tricentric: QC Quadricentric: PC Pentacentric: HC Hexacentric: R Ring RC Ring Chromatid: RF Cl Chromosome Intrachange: CORNINGHazleton Lengthofchromatid"cutout"from midregionofa chromatidresultinigna smallfragmentofring lyingbesidea shortenedchromatidof a gap inthe chromatid. An exchangebetween two chromosomes, orone chromosome and an acentrifcragment,which resultisna three-arrnecdonfiguration. As triradiablu,tresultinigna four-armed configuration. An exchangebetween two chromosomes which resultisna chromosome withtwo centromeres. Thisisoftenassociatewdithan acentrifcragmentin which caseitisclassifieads DF. Dicentriwcithfragment. An exchangebetweenthreechromosomes which resultisna chromosome withthreecentromeres. Oftenassociatewdithtwo tothreeAF. Such exchangescan involvemany chromosomes and are named as follows: fourcentromeresu,p tofourAF fivecentromeresu,p tofiveAF sixcentromeresu,p tosixAF A chromosome which formsa circlceontaininag centromere.Thisisoftenassociatewdithan acentrifcragmentinwhich caseitisclassedas RF. Singlechromatidring(acentric). Ring withassociateadcentrifcragment. Exchange withina chromosome; e.g.a,ringthat does notincludetheentirechromosome. CHV StudyNo.: 17750-0437CO 29 T Translocation: AB Other GT/> CORNINGHazieton ObviousU=fer ofmateriabletweentwo chromosomes resultinigntwo abnormal chromosomes. When identifiabslceo,redas"T" not 112Ab.m Abnormal monocentricchromosome. Thisisa chromosome whose morphology isabnormalforthe karyotypea,nd oftentheresultof a u-dnslocation, pericentrincversione,tc.Classificatiuosnedif abnormalitycannotbe ascribedto;e.g.a,reciprocal w,tnslocation. A cellwhichcontainmsore d= 10 aberrationAs. heavilydamaged cellshouldbe analyzedtoidentify thetypesofaberrationasnd may notactuallyhave >I 0,e.g.m,ultiplefragmentssuch asthosefound associatewditha tricentric. CHV StudyNo.: 17750-0-437CO 30