Document XRMEyMd4g5ozboajp2dRbpyyy

CORNINGHazieton MUTAGENICITY TEST ON 11c T-6342 10 MEASURING CHROMOSOME ABERRATIONS HUMAN WHOLE BLOOD LYMPHOCYTES WITH A CONFIRMATORY ASSAY WITH MULTIPLE IN HARVESTS FINAL REPORT lr",g AUTHOR Hemalatha Murli,Ph.D. PERFORMING LABORATORY Coming HazletonInc.(CHV) 9200 LeesburgPike Vienna,Virginia22182 LABORATORY PROJECT IDENTIFICATION CHV StudyNo.: 17073-0-449CO SUBMITTED TO 3M Corporation Building220-2E-02 3M Center St.Paul,Minnesota 55144-1000 STUDY COMPLETION DATE November 1,1996 CHV StudyNo.: 17073-0-449CO I of 29 CORNINGHazleton QUALITY ASSURANCE STATEMENT ProjeTcittleC:hromosomeAberratiionHsuman WholeBloodLymphocyteWsitha ConfirmatoryAssay With MultipleHarvests ProjectNo.: 20990 Assay No.: 17073 ProtocolNo.: 449CO EditionNo.:2,Modifiedfor3M Corporation QualityAssuranceinspectionosf thestudyand reviewofthefinalreportoftheabove referenced projectwere conductedaccordingtotheStandardOperatingProceduresoftheQualityAssurance Unitand accordingtothegeneralrequirementosf theappropriatGeood LaboratoryPractice regulationsF.indingsfrom theinspectionasnd finalreportreviewwere reportedtomanagement and tothestudydirectoron thefollowingdates: Insl2edtion/Date Additionof Colcemid/08/15/1996 FindingsReported 08/15/1996 Audito C. Orantes DraftReportReview/09/18-20/1996 09/20/1996 C. Orantes FinalReportReview/I 1/01/1996 11/01/1996 C. Smith l(o@:)(inlAistsyuranceUnit Date Released CHV StudyNo.: 17073-0-449CO 2 CORNINGHazleton STUDY COMPLIANCE AND CERTIFICATION The describedstudywas conductedincompliancewiththeOrganizationforEconomic Cooperationand Development PrincipleosfGood LaboratoryPracticCe(81)30(Final)Annex 2, issued1979-1980(effectiv1e981)withany applicablaemendments. There were no significant deviationsfrom theaforementionedregulationosr thesignedprotocolthatwould affecthe integritoyf thestudyortheinterpretatioofnthetestresultsT.he raw datahave been reviewedby theStudy Directorw,ho certifietshattheevaluatioonfthetestarticlaes presentedherein representasn appropriatceonclusionwithinthecontextofthestudydesignand evaluation criteria. Alltestand controlresultisnthisreportaresupportedby an experimentaldatarecordand this recordhas been reviewedby theStudy Director.Allraw data,documentation,records,protocol and a copy of thefinalreportgeneratedas a resulotf thisstudywillbe archivedinthestorage facilitioefsComing HazletonInc.foratleasotne yearfollowingsubmissionof thefinalreportto theSponsor. Aftertheone yearperiod,theSponsormay electtohave theaforementioned materialrsetainedinthestoragefacilitioefsComing HazletonInc.foran additionapleriodof time,or senttoa storagefacilitdyesignatedby theSponsor. SubmittedBy: Study Director: Hemalatha Murli,Ph.D. Mammalian Cytogenetics Department of Geneticand CellulaTroxicology qt Study'C'Ompletion Date CHV Study No.: 17073-0-449CO 3 CORNINGHazleton TABLE OF CONTENTS Page No. ABSTRACT 6 1.0 SPONSOR 7 2.0 MATERIAL (TEST ARTICLE) ...............................7.......... 2.1 Client'Isdentification/ 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 CellsUsed 10.2 CellCultureMedium 10.3 Negativeand SolventControls 10.4 PositiveControlAgents 10.5 Dose RangefindingAssay forMitoticSuppressionand Dose Determination 10.6 AberrationAsssay WithoutMetabolicActivation CHV Study No.: 17073-0-449CO 4 CORNINGHazieton 11.0 12.0 13.0 14.0 15.0 10.7 10.8 10.9 10.10 AberrationsAssay With MetabolicActivation HarvestProcedure SlidePreparatioannd Staining AberrationsAnalysisand Assay Evaluation RESULTS 11.1 11.2 11.3 11.4 11.5 ..........................................I............ Solubilitaynd Dose Determination Dose RangefindingAssay WithoutMetabolicActivation Dose RangefindingAssay With MetabolicActivation Chromosomal AberrationAsssay WithoutMetabolicActivation Chromosomal AberrationAsssay With MetabolicActivation CONCLUSION ........................................16........... REFERENCES ........................................17........... EXPERIMENTAL DATA TABLES ............................1.8 ......... DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS .............................................27........... CHV Study No.: 17073-0-449CO 5 CORNING Hazleton ABSTRACT The objectiveof thisi-nyiim assaywas toevaluatetheabilitoyf T-6"342toinducechromosomal aberrationisnculturedwhole bloodhuman lymphocyteswithand withoutmetabolicactivation. The testarticlweas dissolvedindeionizedwaterata concentratioonf500 mg/ml. The test articlseolutionasnd thevehiclecontrold,eionizedwater,were dosed witha dosingvolume of I% (10pl/ml)forthisassay. For thedose rangefindingassaywith and withoutmetabolicactivationc,oncentrationosf0.167, 0.500,1.67,5.00,16.7,50.0,167,500, 1670,and 5000 Ag/mi were tested.Reductionsof 35%, 6%, 38%, 15%, 18%, 57%, and 100% were observedinthemitoticindicesof theculturedsosed with0.500,1.67,5.00,16.7,500, 1670,and 5000 pg/ml withoutmetabolicactivation, respectivelays,compared with thesolventcontrolculture.Reductionsof 8%, 5%, 4%, 1%, and 100% were observedinthemitoticindicesoftheculturedsosed with 1.67,50.0,500, 1670,and 5000 @ig/mlwith metabolicactivatiorne,spectivelays,compared with thesolventcontrolculture. Based.on thesedata,theinitiatlriaolf thechromosomal aberrationasssaywas conductedtesting concentrationosf 127,253, 505, 1010,1510,2010,and 2510 pg/ml withoutmetabolicactivation and of 253, 505, 1010, 1510,2010,2510,3010,and 4010 gg/ml withmetabolicactivatioinn 22.0 hour assays.Culturesdosed with 253,505, 1010,and 1510 Ag/mi withoutmetabolic activatioannd with 505, 1010, 1510 and 2010 gg/mi withmetabolicactivatiownere evaluated forchromosomal aberrationsN.o significainntcreaseincellswithchromosomal aberrations, polyploidy,or endoreduplicatiownas observedattheconcentrationasnalyzed. In theconfirmatoryassay,replicatceultureswere incubatedwith 125,250,500, 900,1200, 1600, and 2000 gg/ml ina 22.1 hour assayand with 62.5,125,250,500,900, 1200,1600,and 2000 gg/ml ina 46.0hourassayundernonactivatiocnonditionsR.eplicateculturewsere incubatedwith250,500, 1000,1500,2000,2500,and 3000 @Lg/miin22.1and 46.0hourassays withmetabolicactivationC.ulturesdosed with125,250,500,and 900 gg/ml from the22.1and 46.0 hour nonactivatioanssays,with250, 500.1000,and 1500 Ag/ml from the22.1hour activatioanssay,and with 500, 1000,1500,and 2000 gg/ml from the46.0hour activatioanssay were evaluatedforchromosomal aberrationsN.o significanitncreaseincellswithchromosomal aberrationsp,olyploidy,orendoreduplicatiownas observedattheconcentrationasnalyzed, exceptfora weak increaseinendoreduplicatioant2000 pg/ml,an extremelytoxicdose level, from the45.9 hour activatioanssay. The testarticleT,-6342,was considerednegativeforinducingchromosomal aberrationisn culturedwhole blood human lymphocyteswithand withoutmetabolicactivationT.hese results were verifieidnindependentlyconductedconfirmatortyrials. CHV Study No.: 17073-0-449CO 6 CORNINGHazleton ChromosomaAlberratioinHsuman WholeBloodLymphocytes With a ConfirmatoryAssay With MultipleHarvests With 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 AberrationisnHuman Whole Blood Lymphocytes With a ConfirmatoryAssay With MultipleHarvests 4.0 PROTOCOL NO.: 449CO, Edition2,Modifiedfor3M Corporation 5.0 STUDY DATES: 5.1 InitiatiDoante:July9,1996 5.2 ExperimentalStartDate: July31,1996 5.3 ExperimentalTerminationDate:August 26,1996 6.0 SUPERVISORY PERSONNEL: 6.1 Study Director:HemalathaMurli,Ph.D. 6.2 LaboratorySupervisor:CarolS. Spicer,B.S. 7.0 OBJECTIVE: The objectiveofthisinMilM assaywas to evaluatetheabilitoyf thetestarticle, T-6342,toinducechromosomal aberrationisnculturedhuman whole bloodlymphocytes, withand withoutmetabolicactivation. CHV StudyNo.: 1707')-0-449CO 7 CORNINGHazieton 8.0 RATIONALE: The assayisdesignedtoestabliswhhetherthetestarticloer itsmetabolitescan interact withcellsto inducechromosome breaks.Chemicallyinducedlesionsmay resultin breaksinchromatinthatareeitherepairedby thecellinsuch a way as tobe undetectable or resultinvisibldeamage. Aberrationsarea consequenceoffailuroer mistakesinrepair processessuch thatbreaksdo notrejoinorrejoininabnormalconfiguration(sEvans, 1962). Inordertodetectchromosomal aberrationsc,ellsmust be inmetaphase so that chromosomes arevisibleT.he lymphocytesinblooddo notusuallydivideand are stimulatetdodivideincultureby phytohemagglutini(nPHA). The cellsarelaterarrested attheappropriatsetage(metaphase)by Colcemid'. 9.0 EXPERIMENTAL DESIGN: Resultsfrom thedose rangefindingassay were used todeterminethedose rangetobe used in thechromosomal aberrationasssay.In thedose rangefindingassay,thecultures were harvested20.1hoursafterinitiatioofntreatment.Mitoticindicesof thecultures were evaluatedforevidenceoftoxicityA. summary of thetreatmentscheduleforthe preliminarytestisgivenbelow. Summaiy of Dose RanizefindinAgssay TreatmentScheduleinHours Test - S9 +s9 TestArticle 0 0 Wash Colcemid" Fixation 19.5 20.1 22.1 3 20.1 22.1 Inthechromosomal aberrationasssays,replicatceultureswere used ateach dose level, and negatives,olventcontrolsa,nd foreachdose of thepositivceontrol.The aberrations assayswere conductedwitha 22.0 hour harvestime intheinitiatlriaalnd with 22.1and 46.0hour harvesttimesintheconfirmatortyrialsC.hromosomal aberrationwsere analyzedfrom theculturetsreatedatfourdose levelsand from one of thepositivecontrol doses.A summary ofthetreatmentscheduleforthechromosomal aberrationasssaysis givenbelow. CHV StudyNo.: 17073-0-449CO 8 CORNINGHazleton Summamo-fC-h hromosomAablerratiAosnsayTreatmenStchedlueinHours Test Culture Chemical Initiation InitiaTlrial -s9 -48 0 +s9 -48 0 ConfirmatoryTrial -s9 -48 0 +s9 -48 0 - S9 -48 0 +s9 -48 0 Wash 19.2 3.0 19.3 3.0 43.3 3.0 Colcemid' Fixation 20.0 22.0 20.0 22.0 20.1 22.1 20.1 22.1 44.0 46.0 44.0 46.0 10.0 MATERIALS AND METHODS: 10.1 CellsUsed: Human venous bloodfrom a singlen,ormal,healthymale donor was drawn into sterileh,eparinizeVdacutainersC.ultureswere initiatewdith0.3ml of bloodper 5 ml cultureforthedose rangefindinagssayand 0.6ml of blood per 10.0ml cultureforthechromosomal aberrationasssaysin 15 ml centrifugteubes. 10.2 CellCultureMedium: The cellswere incubatedatabout37'C on a slope,with loosecaps,inan atmosphereof about5% C02 inair.The culturemedium used was RPMI 1640 (JRH Biosciencess)upplementedwith 15% fetablovineserum (FBS; Biochemed, Lot#E5')')1,dose rangefindinagssay;LotNo.:T06024,chromosomal aberrations assay),1% phytohemagglutini(nPHA-M; Gibco),penicill(i1n00units/ml; QualityBiologicalasn)d streptomyci(n100pg/mi-Q,ualityBiologicalsa)n,d 2mM L-glutamine(QualitByiologicals). 10.3 Negativeand SolventControls: Inthenonactivatioanssays,negativecontrolswere cultureswhich containonly cellsand culturemedium. Solventcontrolwsere culturescontainingdeionized wateratthehighestconcentratiounsedintestculture(sI% or 10.0@Ll/ml)I.nthe CHV StudyNo.: 1707'3-0-449CO 9 CORNINGHazleton activataisosnaytsh,enegatiavnedsolvenctontrowlesrethesameasdescribiend thenonactivatioanssaysbutwiththeS9 activatiomnix included. 10.4 PositiveControlAgents: The positivceontrolagentswhich were used intheassayswere mitomycin C (MMC) forthenonactivatiosnerieasnd cyclophosphamide(CP) inthemetabolic activatiosneries.Mitomycin C (CAS# 50-07-7,Sigma,Lot # 40H2508) isa clastogenthatdoesnotrequiremetabolicactivationC.yclophosphamide (CAS 6055-19-2,Sigma,Lot # 43HO269) does notactdirectlbyutmust be convertedto activeintermediatebsy microsomalenzymes. Inthechromosomal aberrations assays,threeconcentrationosfMMC (0.1,0.2,and 0.3gg/mi)and CP (20,30, and 50 gg/ml)were used toinducechromosomal aberrationsO.ne of thedose levelswas analyzedineach of theaberratioanssays.Both MMC and CP were dissolvedinwater. 10.5 Dose RangefindingAssay: In thesetestsc,ulturewsere initiatewdith 0.3ml of bloodper 5 ml cultureand were incubatedfortwo days priorto treatment. 10.5.1TestWithoutMetabolicActivation: The lymphocyteswere incubatedwiththetestarticlfeor19.5hoursat =37'C. Then,thetestarticlweas washed from thecellswith phosphate bufferedsalineand freshcompletemedium containingColcemid' (final concentratio0n.1 gg/ml)was added. The cultureswere then harvested2.0 hourslater.(SeeSectionson Harvestand SlidePreparatioannd Staining). 10.5.2 TestWith MetabolicActivation: Inthistestt,helymphocyteswere incubatedtothetestarticlfeorthree hoursat=37'C inthepresenceofa ratliverS9 reactionmixture(S9 15 lil/miN,ADP 1.5mg/ml,and isocitraicid2.7mg/ml). The S9 fractio(nMolecularToxicology,Inc.,Lot #0667)was derivedfrom the liverof male Sprague-Dawleyratswhich had been previouslytreatewdith Aroclor1254 toinducethemixed functionoxidaseenzymes which are capableofmetabolizincghemicalstomore activeforms. The threehour incubationtimewas used becauseprolongedexposuretotheS9 mixture might be toxictothecellsand theenzyme activitoyf S9 islostrapidlyat CHV StudyNo.: 17073-0-449CO 10 CORNINGHazieton about37*C.ThemediumdidnothaveFBS durintgheexposurpeeriotdo avoidpossibleinactivatiofnshortlivedand highlyreactivientermediates produced by theS9 enzymes by bindingtoserum proteins. Aftertheexposureperiodthecellswere washed twicewith buffered saline.Complete RPMI culturemedium was added totheculturewshich were thenincubatedfor18.5hourswithColcemid' (finacloncentration 0.1 gg/ml)added forthelast2.0hourstocollecmtetaphase cells.The cultureswere thenharvestedf,ixed,and slideswere preparedand stained as was describedforthenonactivatiodnose rangefindingassay. 10.5.3Assay Evaluation: Mitoticindexwas analyzedfrom thesurvivingdoselevelsby analyzing thenumber of metaphasespresentin 1000 consecutivecells. 10.6 AberrationsAssay WithoutMetabolicActivation: Cultureswere initiatetdwo dayspriortotreatmentwith0.6ml of whole bloodper 10.0mi culturein 15 ml centrifugteubes.Two daysaftercultureinitiatiotnh,e cellswere treatedwiththetestarticlaetpredeterminedconcentrationfsorabout 19.-a'nld 43.")hours.The culturewsere thenwashed with bufferedsalineand completeRPMI 1640 medium containing0.1 gg/mi Colcemid" was placedback onto thecells.Two hourslatert,hecellswere harvestedand airdriedslideswere made. The slideswere thenstainedin5 % Giemsa solutionfortheanalysiosf chromosomal aberrations. 10.7 AberrationsAssay With MetabolicActivation: Cultureswere initiatetdwo dayspriortotreatmenwtith0.6ml of whole bloodper 10.0mi culturein 15 ml centrifugteubes.Two daysaftercultureinitiatiotnh,e cultureswere incubatedat=37*C forthreehoursinthepresenceof thetestarticle and theS9 reactionmixture.Afterthethreehour exposureperiod,thecellswere' washed twicewith bufferedsalineand thecellswere refedwith completeRPMI 1640 medium. The cellswere incubatedfortherestofthecultureperiodup tothe timeofharvestwith0.1 gg/ml Coicemid'presentduringthelast2.0hoursof incubation.The metaphasecellswere thenharvestedand preparedforcytogenetic analysis. CHV StudyNo.: 17073-0-449CO CORNINGHazleton 10.8 HarvestProcedure: The cellsuspensionwas centrifugedt.hesupernatantwas discardedand cellswere treatewdithhypotonicKCI (0.075M) forapproximately10minutes.This treatmenthelpstoswellthecellsand thusdispersethechromosomes. After centrifugatioand removaloftheKCI, thecellswere washed threetimeswith freshlypreparedfixativ(eabsolutmeethanol:glaciaacleticacid,')1:,v:v).Airdriedslideswere preparedfrom theharvestedcells. 10.9 SlidePreparatioannd Staining: Slideswere preparedby droppingtheharvestedcultureosn cleanslides. The slideswere stainedwith 5% Giemsa solutionfortheanalysisof mitoticindexand chromosomal aberrationsA.llslideswere thenair-dried and coverslipped. 10.10 AberrationsAnalysisand Assay Evaluation: Cellswere selectedforgood morphology and onlycellswiththenumber of centromeresequaltothemodal number 46 were analyzed. One hundredcellsi,favailablef,rom each replicatceultureatfourdose levelsof thetestarticlet,henegatives,olventa,nd positivceontrolcultureswere analyzed forthedifferenttypesofchromosomal aberration(sEvans,1962;See Section 15.0).At least25 cellswere analyzedforchromosomal aberrationfsrom those cultureswith>25% cellswithchromosomal aberrationsF.or controlof bias,all slideswere coded priortoanalysis.Cellswithaberrationwsere recordedon the datasheetsby themicroscopestagelocation.Mitoticindexwas assessedby analyzingthenumber of mitoticellsin 1000 cellsand theratiowas expressedas a percentageofmitoticcells. The followingfactorswere takenintoaccountintheevaluationofthe chromosomal aberrationdsata: I. The percentageofcellswith any aberrations. 2. The percentageofcellswith more thanone aberration. 3. Any evidenceforincreasinagmounts of damage with increasing dose,i.e.a,positivedose response. CHV Study No.: 17073-0-449CO 12 CORNINGHazleton Chromatiadndisochromagtaipdsi,fobservewde.renotedintherawdatand were tabulated.They were not,however,consideredintheevaluatioonf the abilitoyf thetestarticlteoinducechromosomal aberrationssincetheymay not representtruechromosomal breaksand may possiblybe inducedby toxicity. Percentpolyploidyand endoreduplicatiownere analyzedand resultwsere tabulated.HistoricaclontroldataarepresentedinTable 8. A cellclassifieads "GT" isconsideredtocontain10 aberrationfsorstatistical purposesbuta ">" isalsoincludedinthetablesforthisclassificatitoonindicate thatitisa minimum number. Statisticanlalysisemployed a Cochran-Armitagetestforlineartrendand Fisher's Exact Test(Thakuretal.,1985)tocompare thepercentageofcellswith aberration(sand,ifapplicablet,hepercentageof cellswithmore thanone aberration)p,olyploidya,nd endoreduplicatiiontreatedcellswithresultfsrom vehiclecontrolsT.estarticlseignificancweas establishewdhere p<0.01.All factorsas statedpreviouslywere takenintoaccountand thefinalevaluatioonf the testarticlweas basedupon scientifjiucdgement. 11.0 RESULTS: 11.1 Solubilitaynd Dose Determination Deionizedwater(PreparedatCHV, Lot # 20)was thesolventofchoiceforthis assay.T-6342 was dissolvedindeionizedwaterata concentratioonf 500 mg/ml forthedose rangefindinagssay. The testarticlseolutionasnd thevehiclecontrol, deionizedwater,were dosedwitha dosingvolume of I% (10 gi/ml)forthis assay. Concentrationosf0.167,0.500,1.67,5.00,16.7,50.0,167,500, 1670,and 5000 gg/ml were testedinthedose rangefindinagssaywithand withoutmetabolic activationT.he stabiliotfythetestarticluenderthepreparatioannd dosing conditionsused inthisassayistheresponsibiliotfythesponsor. 11.2 Dose RangefindingAssay WithoutMetabolicActivation Hemolysis was observedpriortowash intheculturesdosed with5000 jig/mi. Mitoticindiceswere analyzedfrom theculturesdosed with0.167,0.500,1.67, 5.00,16.7,50.0,167,500,1670,and 5000 gg/ml (Table1).Reductionsof '35%, 6%, 38%, 15%, 18%, 57%, and 100% were observedinthemitoticindicesof the culturesdosed with0.500,1.67,5.00,16.7,500, 1670,and 5000 gg/ml. Based on theseresultst,heinitiatlriaolf theaberrationasssaywithoutmetabolicactivation CHV StudyNo.: 17073-0-449CO CORNINGHazleton wasconductewditha 22.0hourharvesttesticnogncentratoifo1n2s7,253,505. 1010,15102,010a,nd2510gg/m1. 11.3 Dose RangefindingAssay With MetabolicActivation Hemolysis was observedpriortowash intheculturesdosed with 5000 gg/mi. Mitoticindiceswere analyzedfrom theculturesdosed with 0.167,0.500,1.67, 5.00,16.7,50.0,167,500, 1670,and 5000 gg/ml (Table1).Reductionsof 8%, 5%, 4%, 1%, and 100% were observedinthemitoticindicesof theculturedsosed with 1.67,50.0,500, 1670,and 5000 gg/ml,as compared with thesolventcontrol culture.Based on theseresultst,heinitiatlriaolftheaberrationasssaywithmetabolicactivatiownas conductedwith a 22.0hour harvesttestincgoncentrationsof253, 505, 1010,1510,2010,2510,30 10,and 40 10 gg/ml. 11.4 Chromosomal AberrationsAssay WithoutMetabolicActivation INITIAL TRIAL Hemolysis was observedpriortowash intheculturedsosed with25 10 gg/ml. Reductionsof 38%, 18%, 18%, 50%, 55%, and 95% inthemitoticindicesas compared with thesolventcontrolcultureswere observedintheculturetsreated with 127,253,505,1010,1510,and 2010 pg/ml,respectivelyC.hromosomal aberrationwsere analyzedfrom theculturestreatewdith 25-')5,05, 1010,and 15 10 gg/mi (Table2).No significanitncreaseincellswith chromosomal aberrationsp,olyploidyo,r endoreduplicatiownas observedattheconcentrations analyzed. Based on theseresultst,heconfirmatorytriaulndernonactivatiocnonditionwsere conductedtestindgose levelsof 125,250, 500,900, 1200,1600,and 2000 gg/ml with a 22.1harvestand 62.5,125,250, 500,900, 1200,1600,and 2000 pg/ml with a 46.0 harvest. CONFIRMATORY TRIAL In the22.1-hourconfirmatorytrialh,emolysiswas observedpriortowash ofthe culturesdosed with 1600 and 2000 gg/ml,and a slightevidenceofhemolysiswas evidentatharvestof theculturedsosed with 1600 gg/ml. Reductionsof 2%, 14%, 64%, 74%, 93%, and 93% inthemitoticindicesas compared withthe solventcontrolcultureswere observedintheculturesdosed with 125,500,900, 1200,1600,and 2000 gg/ml,respectivelyC.hromosomal aberrationwsere ana- CHV StudyNo.: 17073-0-449CO 14 CORNINGHazleton llvzferdomtheculturterseatweidth1252,50.500,and900@Lg/m(iTabl3e).No significanitncreaseincellswithchromosomal aberrationsp,olyploidyo,r endoreduplicatiownas observedattheconcentrationasnalyzed. Inthe46.0-hourconfirmatortyrialh,emolysiswas observedpriortowash of the culturesdosed with 1600 and 2000 @Lg/ml.Reductionsof 59%, 93%, 98%, and 98% inthemitoticindicesas compared withthesolventcontrolcultureswere observedintheculturedsosed with 900, 1200,1600,and 2000 gg/ml, respectivelyC.hromosomal aberrationwsere analyzedfrom theculturestreated with 125,250,500,and 900 gg/ml (Table4). No significanitncreasiencellswith chromosomal aberrationsp,olyploidyo,rendoreduplicatiownas observedatthe concentrationasnalyzed. The sensitivitoyfthecellcultureforinductionofchromosomal aberrationiss shown by theincreasedfrequencyof aberrationisnthecellsexposedtoMMC, the positivecontrolagent.The testarticliesconsiderednegativeforinducing chromosomal aberrationsp,olyploidya,nd endoreduplicatiounndernonactivation conditions. 11.5 Chromosomal AberrationAsssay With MetabolicActivation INITIAL TRIAL Hemolysis was observedpriortowash intheculturesdosed with 2010,25 10, 3010,and4OlOpg/ml. No cellswere visiblperiortotheadditioonfColcemid"to theculturesdosed with4010 Ag/ml,and hemolysiswas observedpriortothe additionof Colcemid"intheculturedsosedwith3010 gg/ml. Reductionsof 15%, 20%, 15%, 4-)%,77%, and 95% inthemitoticindicesascompared withthe solventcontrolculturewsere observedintheculturestreatewdith 253,505, 1010, 1510,2010,and 25 10 pg/mi,respectivelyC.hromosomal aberrationwsere analyzedfrom theculturetsreatedwith505,1010,1510,and 2010 pg/ml (Table5). No significanitncreaseincellswithchromosomal aberrationsp,ol.yp loidy,or endoreduplicatiownas observedattheconcentrationasnalyzed. Based on theseresultst,heconfirmatorytriaulnderactivatiocnonditionwsere conductedtestindgose levelsof250,500, 1000,1500,2000,2500,and 3000 gg/rrywlith 22.1and 46.0hour harvests. CHV Study No.: 17073-0-449CO 15 CORNING Hazleton CONFIRMALORY TRIAL In the22.1-hourconfirmatorytrialh,emolysiswas observedpriortowash and priortoharvestof theculturesdosed with2000,2500,and 3000 @Lg/ml. Reductionsof 15%, 5%, 69%, 82%, 97%, and 100% inthemitoticindicesas compared withthesolventcontrolculturewsere observedintheculturestreated with 500, 1000,1500,2000,2500,and 3000 pg/ml,respectivelyC.hromosomal aberrationwsere analyzedfrom theculturetsreatewdith 250, 500, 1000,and 1500 gg/ml (Table6).No significainntcreasiencellswithchromosomal aberrationsp,olyploidy,orendoreduplicatiownas observedattheconcentrations analyzed. Inthe46.0-hourconfirmatorytrialh,emolysiswas observedpriortowash and priorto harvestof theculturedsosed with2000,2500, and 3000 Ag/ml. Reductionsof 15%, 80%, 100%, and 100% inthemitoticindicesas compared with thesolventcontrolcultureswere observedintheculturestreatewdith 1500, 2000,2500, and 3000 gg/ml,respectivelyC.hromosomal aberrationwsere analyzedfrom theculturetsreatedwith 500, 1000,1500 and 2000 pg/ml (Table7). Due totoxicityo,nly 52 metaphaseswere availablfeoranalysisinone of the culturesdosedwith2000 pg/ml. No significainntcreaseincellswith chromosomal aberrationsp,olyploidyo,r endoreduplicatiownas observedatthe concentrationasnalyzed,exceptfora weak increaseinendoreduplicatioant 2000 gg/ml,an extremely.toxidcoselevel. The successfualctivatioonf themetabolicsystemisillustratbeyd theincreased incidenceof cellswith chromosomal aberrationisntheculturesinducedwith cyclophosphamide,thepositivceontrolagent.The testarticliesconsidered negativeforinducingchromosomal aberrationpso,lyploidya,nd endoreduplicatiounnderconditionsofmetabolicactivatione,xceptfora weak increaseinendoreduplicatioantan extremeint,oxicdose levelinthe46.0hour assay. 12.0 CONCLUSION: The testarticleT,-6342,was considerednegativeforinducingchromosomal aberrations inculturedwhole blood human lymphocytescellswithand withoutmetabolicactivation. These resultwsere verifieidnindependentlyconductedconfirmatorytrials. CHV StudyNo.: 17073-0-449CO 16 CORNINGHazieton 13.0 REFERENCES: Evans,H.J.(1962) Chromosomal aberrationpsroducedby ionizingradiation, InternationaRleview of Cytology,13,221-321. Thakur,A.J.,Ben-y,K.J.,and Mielke,P.W. Jr(1985) A FORTRAN program fortesting trendand homogeneityinproportionsC,omputer Programs inBiomedicine,19,229-23"). CHV StudyNo.: 17073-0-449CO 17 CORNING Hazleton 14.0 EXPERIMENTAL DATA TABLES CHV StudyNo.: 17073-0-449CO 18 CORNINGHazleton TABLE I DOSE RANGEFINDING ASSAY Assay No.: 17073 TrialNo.:I Date:07/31/96 Compound: T-6342 Lab No.:CY7316 MetabolicActivatio-nS:9 NEGATIVE CONTROL SOLVENT CONTROL TEST ARTICLE Treatment PPMI 1640 Water MetabolicActivatio+nS:9 % mitotic Index 5.9 10.0A[/mi 6.8 0.167pg/ml 7.4 0.500pglml 4.4 1.67;Lg/mi 6.4 5.00ligtml 4.2 16.7pglml 5.8 50.0;Lg/mi 7.2 167gg/mi 7.1 500Ag/mi 5.6 1670jig/mi 2.9 5000ggiml* 0.0 NEGATIVE CONTROL SOLVENT CONTROL TEST ARTICLE Treatment PPMI 1640 Water 10.0pi/mi 0.167 gg/mi 0.500pg/ml 1.67pg/mi 5.00gg/ml 16.7jig/mi 50.0gg/ml 167gg/ml 500pgtml 1670pglml io0ogg/ml* Mitotic Index 7.4 8.3 9.4 9.6 7.6 8.3 8.4 7.9 8.6 8.0 8.2 0.0 *ToxicdoselevelRIMI 1640 ('tjltmuer(elium CHV StudyNo.: 1707')-0-449('0 19 TABLE2 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES CellsFixed 22.0[loursAfterTreatnient Z Assay No.: 17073 Trial I Date:08/07/96 Lab CY8066 CoiiipotiiiTd-:6342 CONTROLS NEGATIVE: PPMI 1640 SOLVENR: Water IO.Opl/mi llosiI-vIi:: mmc 0.300 1&glml CELLS SCORED A 100 B 100 A+B 200 A 100 B 100 A4B 200 A 25 B 25 Atll50 NUNIII17RAND TYIIE 01:ABERRATION OF % 0/0 NOT ABERPA- CEI.1,S Cl-*I-I,S ...........S..I..K..I..I..II..I.....I..:..................................0.1.1.1.1.:.R.".I.'.I.O.N.S...W..i.l.'.i..l.W.i.l..'.il [ll:R ABERRA- ABERR) 'FG SG UC Tn en in s,R QR CR D R Cl DF GT CE'I,L TIONS ]"IONS 21 1 31 3 3 6 42 71 11 3 1 1 1431 5 2 4 5 16 35 0.01 1.0 0.0 0.00 0.0 0.0 0.01 0.5 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.36 32.0 4.0 0.44 44.0 0.0 0.40 38.0* 2.0 I'ES'I'ARl'ICI-E 127lig/ml**A+B 0 253pgtnil A 100 7 B 100 2 1 A+B 200 9 1 505 jig/III[ A 100 5 1 B 100 6 A+B 200 11 1 101ogg/ml A 100 11 100 3 1 12 1 AiB 200 3 1 22 1510 jig/iiii A 100 4 3 2 11 100 2 1 1 A+B 200 6 3 3 1 20IOpg/ml***A+B 0 l(IIN,1l6l40=Culiuremediuni MMC=Mitoiii5,cin C Signit-icangtrie)a,tetrhanthesolventcontrolsp,<0.01. c) ** Chromosome aberrationnsotanalyzeddue tohighdrdosesavailablfeoranalysis. 0.00 0.0 0.01 1.0 0.01 0.5 0.01 1.0 0.00 0.0 0.01 0.5 0.03 2.0 0.01 1.0 0.02 1.5 0.02 2.0 0.02 2.0 0.02 2.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 ().0 0.5 0.0 00 0.0 Chromosome aberrationsotanalyzeddue toexcessivetoxi TABLE3 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES CellsFixed 22.1 Hours AfterTreatment Assay No.: 17073 Trial 2 Date:08/14/96 Lab CY8166 Compound: T-6342 NUMBEit AND TYPE 01'-ABERRATION CONTROLS NEGATIVE: SOLVENT: POSII*IVE: PPMI 1640 Water IO.Opi/mi mmc 0.300pgtml CELLS SCORED A 100 B too A+B 200 A 100 B 100 A+B 200 A 23 B 25 A+B 50 NOT OF % ABI-'RPA.C..O.M.P.U.T.I.'.D......S..[..N..t...l..l..[..,...E....................C.(.).M.l.l.:.l.....[...:..x.....................O.I.I.I.ER 'I'IONS CELI.S CELI.S Wl'i'llWl'I'll> ... .......... PER ABERRA-ABERRA TG SG :UC TB@ SB 11)::TR QR: CR D R Cl @DF GT CELL TIONS TIONS 2 1 3 2 2 5 11 61 1 1 2 624 32 3 94 7 21 1 31 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.01 1.0 0.0 0.01 1.0 0.0 0.01 1.0 0.0 0.60 40.0 20.0 0.36 32.0 4.0 0.48 36.00 12.0* TEST ARTICLE 125 pg/mi A 100 6 B 100 3 1 A+B 200 9 1 250pgtmi A 100 B 100 A+B 200 500 pg/mi A 100 6 1 B 100 3 A+B 200 9 1 900lig/nil A 100 3 1 B 100 4 A+B 200 7 1 1200pgtml**A+B 0 1600pg/ml**A+B 0 20OOpgtml*$ A+B 0 1 1 1 1 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.01 1.0 0.0 0.01 0.5 0.0 0.00 0,0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.01 1.0 0.0 0.01 0.5 o@o l@IIN,1l6l40 =Culture niedium MMC = MitoiTi),cCin * Signiricantglryeatetrhanthesolventcontrolsp,<0.01. Cliroiiiosonaibcerrationnsotanalyzeddue toexcessivetoxicity. TABLE4 CHROMOSOME ABERRATIONS IN UIUMAN LYMPHOCYTES z CellsFixed 46.0 Hours AfterTreatiiient Assa),No.: 17073 Trial4: 2 Date:08/14/96 Lab CY8166 --4 IL:oiiipouiTi-d6:342 1 NUMIIER AND TYPE OF ABERRATION r) ('ON,rROI.S NEGA IIVE: SOLVENT: RPMI 1640 Water CELLS SCOPIL OF ABERRA- @2MPNUoTTED ................C.O.M..PL.E.X..................................T.I.O.NS .... ................ PER TG SG UC B S]B ID @TRiQR:CR: D R ci DF GT CELL A 100 1 3 0.03 B 100 2 11 0.02 A+B 200 3 41 0.03 IO.Opl/mi A 100 2 0.02 B 100 2 11 0.02 A+B 200 2 31 0.02 % % CELLS CELLS Wl'ril WITH>L ABERPA- ABERRA- TIONS TIONS 2.0 1.0 2.0 0.0 2.0 0.5 2.0 0.0 2.0 0.0 2.0 0.0 'I'ES'I'ARl'ICLE 62.Spg/ml*A+B 0 125pg/mi A 100 5 B 100 6 A+B 200 11 250pg/nil A 100 5 B 100 4 A+13 200 9 500 pp,/ml A 100 4 13 100 5 1 A+B 200 9 1 900 pg/mi A 100 4 1 B 100 4 A+B 200 8 1 1200pglnil*A*+B 0 16001ig/inlA*+*n 0 2000pg/nil*$A+B 0 1 1 11 11 1 1 0.01 1.0 0.0 0.00 0.0 0.0 0.01 0.5 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.02 2.0 0.0 0.00 0.0 ()@o 0.01 1.0 0.0 0.01 1.0 0.0 0.00 0.0 0.0 0.01 0.5 0.0 ltl'hl1l040 ('tilltiiriediiiiii (,'Iiioiiiosaobieirireationnsotanalyzeddue it)Iiiglideorsesavailablfeorallilysis. . Cliroiiiosoiaibieerrationnsotanalyzeddue it)e.\cessivfeoxicil) CZ Assay No.: 17073 Compound: T-6342 Trial I TABLE5 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES CellsFixed22.0Hours AfterTreatnieiit Date:08/07/96 Lab CY8066 NIJMBI.'.RAN[) TYPE OF All[-.RRA'I'ION 4CONTROLS NEGATIVE: SOLVENT: floslIIVI-': CELLS SCORED PPMI 1640 A 100 B 100 Water IO.Opl/ml A+B 200 A 100 B 100 A+B 200 cil 50.0pg/ini, A 25 B 25 A+B 50 (,OkN,O'llpt Lj'l*[.'II)NII ... ............ TG SG UC TSB 'SB 11 11 3 31 61 4 45 85 2 2 71 14 1 21 2 OF % % C()Nflll,l:x ABERRA- CEI.1,S CELL ...................................O..'..l.l..l..[..;.R.'.I.'I.O.N.S Wl'i'llWl'i'll ID ::'rR QR CR D R Cl "DF GIGTT PER ABERRA-ADER CELL TIONS TION 23 23 2 462 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.02 2.0 0.0 0.00 0.0 0.0 0.01 1.0 0.0 0.52 44.0 9.0 0.88 52.0 28.0 0.70 48.0* 18.0* I'ESI'AR'FICLE 253pg/ml** A+B 0 505jig/nil A 100 2 B 100 2 A+n 200 4 10]Opg/nil A 100 B 100 6 A+B 200 6 1510pgtml A 100 4 1 3 11 100 7 1 1 A+ll 200 11 2 4 2010 pg/mi A 100 2 1 B 100 7 2 1 A+B 200 9 2 2 2510pg/nil**A*+B 0 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.03 2.0 1.0 0.01 1.0 0.0 0.02 1.5 0.5 0.01 I.o 0.0 0.01 1.0 0.0 0.01 1.0 0.0 ltl'NI1I640 CultureiiieditiiCiiil= Cyclolilit)spi,iiii,itic Sigiiii-icaigirtelayterthan thesolventcontrolsp,<0.01. * ('hrojiiosoniisebcrrationnostanalyzeddue tohigh@rdosesavailablfeoranalysis. Chromosome I)uctotoxicityo,nly83 metaphasesfrom the14culturewere analyzedforpolyploidyand endoreduplication. aberrationnsotanalyzeddue toexcessivetoxi TABLE6 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES CellsFixed 22.1 Hours AfterTreatment Assa),No.: 17073 Cojiil)otii1i'd-:6342 Trial 2 Date:08/14/96 Lab CY8166 NIJMIII-R ANI) l'YPEOF A13ERRATION 4@, Ic CON'I'ROI-S NO OF ('UI,LS.........I.:.'..[..)..S...I..M..I.'.L....E................CI.O.M.P.I..E.X..... ...........)..I...l.i.r.A.RBT.EI.OR.NRSA- SCORED 'I'G SG UC TB::SB- ID :TR:QR:CRI D R Cl PER GT CELL T.i. % % CELI.S CELI, Wil'il WI'Flf> ABFRRA-ABERK TIONS TIONS Ni:(iA'I'IVE:RPM] 1640 A 100 8 B 100 6 1 0.00 0.0 0.0 0.01 1.0 0.0 SOLVENT: Water A+B 200 14 10.0111/mi A 100 3 B 100 5 2 1 2 0.01 0.5 0.0 1 0.03 3.0 0.0 0.00 0.0 0.0 POSITIVE: CP A+B 200 8 2 2 50.Opg/ml$** A 25 4 2 7 2 1 B 25 5 1 62 1 1 0.02 1.5 0.0 0.40 32.0 8.0 0.36 36.0 0.0 A+B 50 9 3 13 2 2 2 0.39 34.0* 4.0 I'ESI'AR'riCLE 250 pg/mi A 100 5 1 1 It100 2 1 A+B 200 7 2 1 500tig/mi A 100 3 1 B 100 5 1 2 A+B 200 8 1 3 1000pg/Bil A 100 7 1 13 100 7 1 A+B 200 14 2 1500pgtml A 100 9 2 B 100 10 21 4 A+B 200 19 2 61 20OOpg/nil** Aill0 25OOpg/ivil** A4t) 0 30OOpgtml** A+B 0 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.01 1.0 0.0 0.02 2.0 0.0 0.02 1.5 0.0 0.01 1.0 0.0 0.01 1.0 0.0 0.01 1.0 0.0 0.03 2.0 1.0 0.04 4.0 0.0 0.04 3.0 0.5 RPMI 1640 Culturemedium CP = Cyclophosphamide Si@,iiit-ic-g,rieiaitie[rtvhan the solventcontrols.p<0.0 1. (*Iii-oiiiosaobieirireationnsotanalyzeddue it)excetsivetoxicity. Due to toxicityo,nly 56 metaphascsfrom theA cultureand 36 metaphasesfrom theB culturewere analyzedforpolyploidyand endoreduplication. TABLE7 CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES CellsFixed 46.0 Hours AfterTreatment Z Assay No.: 17073 Trial 2 Date:08/14/96 Lab CY8166 CojiipouiidT:-6342 4'4- C) CONTROLS NEGATIVE: PPMI 1640 SOI.VENT: Water 10.0Pt/all NUMBER AND TYPE OF ABERRATION NOT COMPUTED SIMPI.E CEI.1,S NO.T............. SCORED TG SG UCC TB SB COMPLEX ID :TR::QR::CR:: D R Cl A 100 3 B 100 2 1 A+B 200 3 1 A 100 1 1 1 B 100 3 1 A+B 200 4 1 2 #OF % % ABERRA- CELLS CELLS OTIIER TIONS Wl'fli WITtf>l PER ABERRA- ABERPA DF GT CELL TIONS TIONS 0.00 0.0 0.0 0.01 1.0 0.0 0.01 0.5 0.0 0.01 1.0 0.0 0.01 1.0 0.0 0.01 1.0 0.0 I'LS'ATR-l'ICLE 2SOpg/ml** A+B 0 500 tag/ml A 100 3 B 100 1 1 Ai-B200 4 1 loootig/mi A 100 3 B 100 2 A+B 200 5 1500 jig/mi A 100 4 B too 9 1 A+B 200 13 1 2000 pg/ml* A 52 2 2 B 148 2 A4-B 200 4 2 25OOpgtml*$* A+B 0 3000lig/lill@*%*i*l0l 1 1 1 1 11 2 31 11 11 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.00 0.0 0.0 0.01 1.0 0.0 0.01 0.5 0.0 o@oo 0.0 0.0 0.01 1.0 0.0 0.01 0.5 0.0 0.04 3.8 0.0 0.03 2.0 0.7 0.03 2.5 o@5 RPNII 1640 Culturemedium Signii-icantglryeaterthanthesolventcontrolsp,<0.01. Chromosome aberrationnsotanalyzeddue ti)higherdosesavailablfeoranalysis. Chromosome aberrationnsotanalyzeddue toexcessivetoxi [)tiict)it)xicitoyn,ly54 niciaphasefsrom litAe ctilluarned 58 iiiettiphafsreosntlhc13culturewere analyzedforpolyploidyand endoreduplica(ion. ,it TABLES CONTROL DATA OF CHROMOSOME ABERRATIONS IN HUMAN LYMPHOCYTES 4-95 THROUGH 12-95 NegativeControl SolventControl PositivCeontrol MitomycinC NegativeControl SolventControl PositivCeontrol Cyclophosphamide Activation Without MIN MAX AVG N Without MIN MAX AVG N Without MIN MAX AVG N with MIN MAX AVG N with MIN MAX AVG N With MIN MAX AVG N Of Aberrations PerCell % Of Cells With Aberrations % Of Cells With >1 Aberration % Polyploidy % Endore- % duplicated Mitotic Cells Index 0.00 0.0 0.06 2.5 0.012 0.72 Is is 0.0 0.0 0.0 0.4 0.5 1.0 1.0 7.1 0.06 0.18 0.03 3.57 19 17 35 18 0.00 0.0 0.0 0.0 0.0 0.5 0.06 2.5 0.5 1.0 2@O 10.9 0.011 0.79 0.05 0.14 0.10 4.52 29 29 29 Is 35 29 0.02 0.6() 0.267 19 1.5 39.5 22.22 19 0.0 0.0 0.0 0.1 15.0 0.0 0.0 6.6 3.63 0.00 0.00 2.46 19 11 31 19 0.00 0.0 0.0 0.0 0.0 0.6 0.03 2.0 0.5 1.0 0.5 9.6 0.010 0.75 0.06 0.28 0.02 4.68 19 is 18 17 27 Is 0.00 0.0 0.02 2.0 0.009 0.66 29 28 0.0 0.0 0.0 0.5 0.5 2.0 0:0 10.7 0.05 0.25 0.00 6.13 28 17 27 28 0.28 2.52 0.647 17 21.7 40.0 31.45 17 0.0 0.0 0.0 0.1 40.0 1.0 0.0 5.9 12.90 0.22 0.00 1.35 17 9 23 17 CHV Study No.: 17073-0-449CO 26 CORNINGHazieton 15.0 DEFINITIONOSF CHRONfOSO.\,AfBERRATIONSFOR GIEMSASTAINED CELLS SIMPLE TB ChromatidBreak: An achromatircegioninone chromatidl,argerthanthe ,.%idtohfa chromatid.The associatefdragmentmay be partiallo,r,c-ompletelydisplaced. SB Chromosome Break: Chromosome hasa clearbreak,formingan abnormal (deletedc)hromosome withan acentrifcragmentthatis dislocatedT.hisclassificatniown includestheacentric fragment(AF).An AF isdifferefnrtom a SB onlyin thatitcan notberelatetdoany specificchromosome. DM "Double Minute"Fragment: These aresmalldoubledots,which may represent terminaolr interstitdiealletionso,r even smallrings. These possibloeriginasrenotdistinguishable. COMPLEX ID InterstitDiealletion: Lengthof chromatid"cutout"from midregionof a chromatidr,esultinigna smallfragmentorringlying besidea shortenedchromatidora gap inthechromatid. TR Triradial: An exchangebetweentwo chromosomes,orone chromosome and an acentrifcragmen@ which resultisn a three-armecdonfiguration. QR Quadriradial: An exchangelikea triradiablu,tresultinigna fourarmed configuration. CR Complex Rearrangement: An exchangeamong more thantwo chromosomes or fragmentsc,ausedby theinductionofseveralbreaks. D Dicentric: An exchangebetweentwo chromosomes which results ina chromosome withtwo centromeres.Thisisoften associatewdithan acentrifcragmentinwhich caseitis classifieads DF. DF Dicentriwcithfragment. CHV StudyNo.: 17073-0-449CO 27 C:ORNING Hazleton COMPLEX (Continued) TC Tricentric: An exchange involvingthreechromosomes and resulting ina chromosome withthreecentromeres.Often associatewdithtwo tothreeAF. Such exchangescan involvemany chromosomes and arenamed as follows: QC Quadricentric: PC Pentacentric: HC Hexacentric: fourcentromeresu,p tofourAF fivecentromeresu,p tofiveAF sixcentromeresu,p tosixAF R Ring: A chromosome which forms a circlecontaininga centromere.This isoftenassociatedwith an acentric fragment,inwhich caseitisclassedas RF. RC Ring Chromatid: Singlechromatidring(acentric). RF Ring with associateadcentricfragment. ci Chromosome Intrachange: Exchange withina chromosome; e.g.,a fingthatdoes riotincludetheentirechromosome. T Translocation: Obvious transfeorf materialbetween two chromosomes resultinigntwo abnormalchromosomes. When identifiablsec,oredas "T" not"2AB". AB Abnormal: Abnormal monocentficchromosome. This isa chromosome whose morphology isabnormal forthe karyotype,and oftentheresultof a translocation, pericentricnversione,tc.Classificatiuosned if abnormalitycannotbe ascribedto,e.g.,a reciprocal translocation. CHV Stud),No.: 1707'3-0-449CO 28 OTHER GT Greaterthan Ten: pp PolyploidCell: E Endoreduplication: NOT COMPUTED TG Chromatid Gap: SG Chromosome Gap: UC UncoiledChromosome: CORNINGHazleton Greaterthan 10 aberrationsA: cellwhich containsmore than10 aberrationsH.eavilydamaged cellswillbe analyzedtoidentiftyhetypesofaberrationbsecause multiplefi-agments,uch as thosefound associatedwith a tricentridco,notcountas independentaberrations. A cellcontaininmgultiplecopiesof thehaploidnumber (n)ofchromosomes. A failuroefchromosomes toseparater,esultinigna 4n cell. ("tigdap").An achromatic(unstainedr)egioninone chromatidt,hesizeof which isequaltoor smallerthan thewidthof a chromatid.These arenotedbutnot usuallyincludedinfinaltotalosf aberrationass they may notallbe truebreaks. ("isochromatigdap,IG").Same as chromatidgap butat thesame locusinbothsistecrhromatids. Failureofchromatinpacking.Probablynota true aberration. CHV StudyNo.: 17073-0-449CO 29