Document 6wX68vban2R1drgkGGY9Yrnj4

CORNINGHazleton MUTAGENICITY TEST ON T6358 MEASURRNG CHROMOSOMAL ABERRATIONS IN CHINESE HAMSTER OVARY (CHO) CELLS FRNAL REPORT AUTHOR Hemalatha Murli,Ph.D. PERFORMING LABORATORY Coming HazletonInc.(CHV) 9200 LeesburgPike Vienna,Virginia22182 LABORATORY PROJECT IDENTIFICATION CHV StudyNo.: 17388-0-437 SUBMITTED TO 3M 3M Center St.Paul,Minnesota 55144-1000 STUDY COMPLETION DATF April25, 1996 CHV StudyNo.: 17388-0437 1 of 24 CORNING Hazleton QUALITY ASSURANCE STATEMENT ProjecTtitle:Chromosomal AberrationisnChineseHamster Ovary (CHO) Cells ProjectNo.: 20990 Assay No.: 17388 ProtocolNo.: 437 EditionNo.: 15 Qualit,@A,ssurance inspectionsof the study and review of the finalreportof the above referenced projectwere conducted according to the Standard Operating Procedures of the Quality Assurance Unit and according to thegeneralrequirementsof the appropriateGood Laboratory Practice regulations.Findings from the inspectionsand finalreportreview were reportedto management and to the study directoron thefollowing dates: Inspection/Date Findini!sReported Auditor Harvest/02/14/1996 02/14/1995 S. Ballenger Draft Report Review/04/20,21/1996 FinalReport Review/04/25/1996 04/22/1996 04/25/1996 C. Orantes C. Orantes QualityAssurance Unit Date Released CHV Study No.: 17388-0-437 2 CORNING STUDY COMPLIANCE AND CERTIFICATION Hazleton The stud@,was conductedin compliance with theGood LaboratoryPracticeregulationass set forthintheFood and Drug Administratio(nFDA) Title21 oftheU.S.Code of Federal RegulationsPart58.issuedDecember 22. 1978.(effectivJeune 20. 1979) with anN,applicable amendments. There were no significandte%,iationfsrom theaforementionedregulationsor the signedprotocolthatwould affectthe integn'tovf thestudy or theinterpretationf the testresults. The raw datahave been reviewed by the Study Director.who certifietshattheevaluationof the testarticlaes presentedhereinrepresentsan appropriateconclusionwithinthecontextof the studydesignand evaluationcr-iteria. All testand controlresultsin thisreportaresupportedby an expen*mcntaldatarecordand this recordhas been reviewed by theStudy Director.All raw data,docLLinentatiorne.cords,protocol and a copy of the finalreportgeneratedas a resultof thisstudywillbe archivedin the storage facilitioefsComing HazletonInc.foratleastone yearfollowingsubmissionofthefinalreportto the Sponsor. Aftertheone yearperiod,the Sponsor may electto have the aforementioned materialsretainedinthestoragefacilitioefsComing HazletonInc.foran additionalperiodof time,or senttoa storagefacilitdyesignatedby the Sponsor. Submitted by: Study Director: Hemalatha Murli.Ph.D. Mammalian Cytogenetics Department of Geneticand CellularToxicology -,/Sq-.4 Stu@y,com,pletion Date CHV StudyNo.: 17388-0437 3 CORIYING Hazleton TABLE OF CONTENTS PageNo. 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 StudyDirector 6.2 LaboratorySupervisor 7.0 OBJECTIVE ..........................................7 ............. 8.0 RATIONALE ..........................................8............. 9.0 EXPERIMENTAL DESIGN .................................8............ 10.0 MATERIALS AND METHODS ......................................9 ... 10.1 IndicatoCrells 10.2 CellCultureMedium 10.3 Negativeand SolventControls 10.4 PositiveControlAgents 10.5 RangefindingAssays 10.6 AberrationAsssay WithoutMetabolicActivation CHV Study No.: 17388-0-437 4 CORNING Hazleton 11.0 12.0 13.0 14.0 15.0 10.7 10.8 10.9 10.10 AberrationsAssav With Metabolic Activation Harvest Procedure SlidePreparationand Staining AberrationsAnalysis and Assay Evaluation RESULTS 11.1 11.2 11.3 11.4 11.5 ..................................................I ....... Solubilityand Dose Determination Rangefinding Assay Without Metabolic Activation Rangefinding Assay With Metabolic Activation Chromosomal AberrationsAssay Without Metabolic Activation Chromosomal AberrationsAssay With Metabolic Activation CONCLUSION .............................................15......... REFERENCES .............................................15......... EXPERIMENTAL DATA TABLES ................................17...... DEFINITIONS OF CHROMOSOME ABERRATIONS FOR GIEMSA STAINED CELLS .....................................................2.2. CHV Study No.: 17388-0437 5 CORNING Hazleton ABSTRACT The objectiveof thIisip_yilm assay was to evaluatethe ab*IiI'tvof T6358 to induce chromosomal aberrationsin Chinese hamster ovary (CHO) cellswith and without metabolic activation. For the dose rangefindingassays with and without metabolic activation.the testarticle%%-as dissolvedin sterildeeionizedwater at a concentrationof 501 mg/ml. Concentrationsof 0.167, 0.501,1.67.5.01, 16.7,50.1. 167,501, 1670. and 50 10 pglmi were testedin the dose rangefindingassavs.All dosing was achieved usinga dosing volume of I% (10.0pi/mi), Complete cytotoxicitywas observed in the culturesdosed with 501. 1670, and 5010 pg/ml in the nonactivationassay and with 1670 and 5010 pg/ml inthe assay with metabolicactivation.Venfew metaphases were availablein the culturetreatedwith 167 pg/ml and a severe cellcycle delay was observed. No cellcycle delay was observed in theculturesanalyzed in the metabolic activationassay. Based on thedata from the dose rangefindingassay,replicateculturesof CHO cellswere incubatedwith 12.5.25.0,50.0.75.0, 100, 150, and 200 pg/mi in a 20.0 hour aberrationsassay without metabolic activation.Replicatecultureswere treatedwith 125, 250, 500, 750, 1000, and 1500 gg/mi with a 20.0 hour aberrationsassay with metabolicactivation.Cultures treatedwith 75.0, 100, 150,and 200 gg/mi from the assay without metabolicactivationand culturestreated with 125,250, 500. and 750 pg/mi from the assay with metabolic activationwere analyzed for chromosomal aberrations.No significanitncreaseincellswith chromosomal aberrationswas observed at the concentrationsanalyzed. The testarticleT,6358, was considered negativeforinducingchromosomal aberrationsin CHO cellswith and without metabolic activation. CHV Study No.: 17388-0-437 6 CORNING Hazleton Chromosomal AberrationsinChinese Hamster Ovan, (CHO) Cells With T6358 1.0 SPONSOR: 3M -),o MATERIAL (TEST ARTICLE): 2.1 Client'IsdentificatioTn6:358 Date Received: January 16. 1996 2.3 PhvsicalDescriptionW:hite powder 2,4 GeneticsAssay No.: 17388 3.0 TYPE OF ASSAY: Chromosomal AberrationsinChinese Hamster Ovary (CHO) Cells 4.0 PROTOCOL NO.: 437, Edition15 5.0 STUDY DATES: 5.1 InitiatioDnate: -January18,1996 5.2 ExperimentalStartDate: January 30. 1996 5.3 ExperimentalTerminationDate:March 20, 1996 6.0 SUPERVISORY PERSONNEL: 6.1 Study Director:Hemalatha Murli,Ph.D. 6.2 LaboratorySupervisor:CarolS.Spicer,B.S. 7.0 OBJECTIVE: The objectiveofthisinyltm assaywas toevaluatetheabilityof thetestarticleT,6358, to inducechromosomal aberrationisnChinese hainsterovary (CHO) cells,withand without metabolicactivation. CHV Study No.: 17388-0-437 7 CORNING Hazleton 8.0 RATIONALE: The assayisdesignedtoestabliswhhetherthetestarticloer itsmetabolitescan interact withcellstoinducechromosome breaks.Chemicalivinducedlesionmsa-,,resulitn breaksinchromatinthatareeitherrepairedby thecellinsuch a way as tobe undetectable orresultin .-isibdlaemage. Aberrationasrea consequenceof failuroer mistakesinrepair processessuch thatbreaksdo notrejoinor rejoininabnormalconfiguration(sEvans. 1962). The assayisdesignedtoexamine cellsinthefirsmtitosisafterinitiatiofntreatmentwith a chemical.This designlimitslossof aberrantcellsduringthedivisionprocessor conversionintocomplex derivativedsuringsubsequentcellcycles.Inthecaseof CHO cellsm.ost dividingcellsexamined 8-12hoursaftertreatmentareinthefirsmtitosis(M, cells).Many testarticlecsauseseveredelayof progressiotnhroughthecellcycle.and the assayhas been designedtodetectthisdelayand allow forslowergrowth of damaged cellsby adjustmentsinthetime between treatmentand cellfixation. 9.0 EXPERIMENTAL DESIGN: Resultsfrom therangefindinagssaywere usedtodeterminethedose rangeto be used in thechromosomal aberrationasssayand todeterminetheoptimaltimeof harvestof the dosed cellsso thatprimarilymetaphasecellswhich were inthefirsmtetaphase since exposuretothetestarticlweould be analyzedforchromosomal aberrationsI.nthe rangefindinagssay,theculturewsere incubatedfor25-26 hourswith 5-bromo-2'deoxvuridine(BrdUrd).Thisenabledthemajorityofcellstoprogressthroughabouttwo celldivisionst,husprovidinga fullassessmentofcellcyclekineticsA. summary of the treatmentschedulefortherangefindingassayisgiven below. SummwZ@ ofRan2efindingAssay TreatmentSchedule inHours Test TestArticle Wash BrdUrd Wash Coicemid' Fixation - S9 0 - 2.2 25.4 25.7 l@7.7 -s9 0 2 2.2 - 25.7 27.7 Inthechromosomal aberrationasssays,replicatceulturewsere usedateach dose level. Singlecultureswere used forthenegativecontrols,olventcontrola,nd ateach oftwo dosesof thepositivceontrol.Intheaberrationasssaywith and withoutmetabolic activation2.0 hour harvestswere conducted.Chromosomal aberrationwsere analyzed from theculturestreateadtthefourhighestdose levelsand from one of thepositive controldoses.A summary of thetreatmentscheduleforthechromosomal aberrations assaysisgiven below. CHV StudyNo.: 17388-0-437 8 CORNING Hazleton Summarv ofChromosomal AberrationsAssa,@T-reatment Schedule inHours Test - S9 -+.-s9 TestArticle 0 0 Wash 17.6 2 Coicemid' 18 18 Fixation 20 20 10.0 MATERIALS AND METHODS: 10.1 IndicatoCrells: The Chinesehamsterovarycells(CHO-WBL) usedinthisassavwere from a permanentcelllineand were originalloybtainedfrom the laboratoroyf Dr. S. Wolff,Universityof CaliforniaS.an Francisco.The cellshave sincebeen recionedtomaintainkaryotypicstabilitTyh.iscelllinehas an averagecycletime of 12 to 14 hourswith a modal chromosome number of2 1. 10.7- CellCultureMedium: The CHO cellswere grown inMcCoy's 5a culturemedium which was supplementedwith 10 % fetalbovineserum (FBS),I% L-glutaminea.nd I% penicillaind streptomycina,tapproximately37*C, inan atmosphereofabout 5% CO, inair. 10.3 Negativeand SolventControls: Inthenonactivatioanssays,negativecontrolswere cultureswhich containonly cellsand culturemedium. Solventcontrolswere culturescontainingthesolvent forthetestarticles,terildeelonizedwater,atthehighestconcentratiounsed intest cultures(1%, i.e.1,0 gumi). Intheactivatioanssays,thenegativeand solvent controlswere the same as describedinthenonactivatioanssaysbutwiththeS9 activatiomnix included. 10.4 PositiveControlAgents: The positivceontrolagentswhich were used intheassayswere mitomycin C (MMC) forthenonactivatiosneriesand cyclophospharnid(eCP) inthemetabolic activatiosneries.Mitomycin C (CAS# 50-07-7,Sigma,Lot # 25HO619) isa clastogenthatdoesnot requiremetabolicactivationC.yclophosphamide (CAS 6055-19-2,Sigma, Lot # 67FO 155)does notactdirectlbyut must be convertedto CHV StudyNo.: 17388-0437 9 CORNINGHazieton activeintermediatebsy microsomalenzvmes. Intherangefindinagssan,t.wo concentrationsof MMC (0.25and 0.50 pglml) and CP (15.0and 20.0 4giml). In the chromosomal aberrationsassavs.two concentrationsof @VIMC (0.08 and 0.10 pg/mi) and CP (10.0and 15.0 pg/mi) were used to induce chromosomal aberrationsin theCHO cells.One of the dose levelswas ana]N,zedineach of the aberrationassays. Both MMC and CP were dissolvedin water. IO.-@ Rangefinding Assays: In theseassays.the cellswere culturedforapproximately 24 hours prior to treatmentby seeding approximately 0.3 x 10'per 25 cm2 flaskinto5 ml of complete McCoy's 5a culturemedium. 10.5.1 Assay Without Metabolic Activation: The cultureswere dosed with thetestarticlefor2.2 hours when 5-bromo2'-deoxyuridine(BrdUrd) was added at a finalconcentrationof 10 pM. The cultureswere washed 23.3 hours laterwith phosphate buffered saline and freshcomplete medium containingBrdUrd ata finalconcentrationof 10 gM, and Coicemid" (finalconcentration0.1 pg/mi) was added. The cultureswere then harvestedtwo hours laterand differentialtsvtainedfor the analysisof cellcycle delay using a modified fluorescence-plus-Giemsa (FPG) technique (See Sectionson Harvest and SlidePreparationand Staining). 10.5.2 Assay With Metabolic Activation: In thisassay,the CHO cellswere exposed to thetestarticlefortwo hours at =37*C inthe presence of a ratliverS9 reactionmixture (S9 15 pi/mi, NADP 1.5mg/ml, and isocitriaccid2.7 mg/mi). The S9 fraction (MolecularToxicology,Inc.,Lot #0583) was derivedfrom the liverof male Sprague-Dawley ratswhich had been previouslytreatedwith Aroclor 1254 to induce the mixed functionoxidase enzymes which are capable of metabolizingchemicals to more activeforms. The two hour incubation time was used because prolonged exposure to the S9 mixture might be toxicto the cellsand the enzyme activityof S9 islostrapidlyat =37*C. The mediuxn did not have FBS during theexposure period to avoid possibleinactivatioonf shortlivedand highlyreactiveintermediates produced by the S9 enzymes by binding to serum proteins. CHV Study No.: 17388-0437 10 CORNING Hazleton Aftertheexposureperiodthecellswere washed twicewith buffered saline.Complete NIcCov's5a medium containinBgrdUrd (final concentratio1n0 4M) was added tothecultureswhich were then incubatedfor25.5hourswithCoicemid'(finacloncentratio0n.1 pg/mi) added forthelast2.0hours. The culturewsere thenhar%,estefdi.xed.and slideswere preparedand stainedas was describedforthenonacti%,ation rangefindingassay. 10.5.3Assay Evaluation: One hundred consecutivemetaphasesfrom threedose levelswith metaphasesfrom theassaywithoutand with metabolicactivatio(n103 from thesolventcontrolfrom theactivatioanssay)were assessedforthe number ofcellcyclesthroughwhich thecellshad progressedwhileinthe presenceof BrdUrd. 10.6 AberrationAsssay WithoutMetabolicActivation: Cultureswere initiatebdy seedingapproximately1.2x 10'cellsper75 CM2 flask into10 ml of completeMcCoy*s 5a medium. One day aftercultureinitiatiotnh,e cellswere incubatedat=37*C with thetestarticlaetpredetermineddosesfor17.8 hours.The culturewsere thenwashed with bufferedsalineand complete McCoy's 5a medium containing0.1 pg/mi Coicemid" was placedbackontothe cells.Two hourslatert,hecellswere harvestedand airdriedslideswere made. The slideswere then stainedin5 % Glemsa solutionfortheanalysisof chromosomal aberrations. 10.7 AberrationAsssay With MetabolicActivation: Cultureswere initiatebdy seedingapproximately1.2x 10'cellsper75 cm2 flask into10 ml of completeMcCoy's 5a medium. One day afterculturienitiatiotnh,e culturewsere incubatedat =37'C for2 hoursinthepresenceof thetestarticlaend theS9 reactionmixtureinMcCoy's 5a medium withoutFBS. Afterthe2 hour exposureperiod,thecellswere washed twicewithbufferedsalineand thecells were refedwithcompleteMcCoy's 5a medium. The cellswere incubatedforthe restof thecultureperiodup tothetimeof harvestwith0.1 4g/mi Coicemid' presentduringthelast2.0hoursof incubation.The metaphase cellswere then harvestedand preparedforcytogeneticanalysis. CHV Study No.: 17388-0-437 11 CORNING Hazleton 10.8 HarvestProcedure: Priortotheharvestoftheculturesv.isualobservationosftoxicitwyere made. These obser-,,atioinscludedan assessmentofthepercentconfluenceofthecell monolaverwithinthecultureflasks.The culturewsere alsoevaluatedforthe presenceofmitotic(largeroundedcellso)rdead cellsfloatinignthemedium. The metaphasecellswere collectebdy a mitoticshake-off(Terasimaand Tolmach. 1961)and were treatedwith0.075 M KCI hvpotonicsolutionT.histreatment helpstoswellthecellsand thusdispersethechromosomes. The cultureswere thenfixedwithan absolutemethanol:glaciaalceticacid(3:1,v:v)fixativaend were washed severaltimesbeforeair-driesdlideswere prepared. 10.9 SlidePreparatioannd Staining: Slideswere preparedby droppingtheharvestedcultureosn cleanslides.The slidesfrom therangefindinagssayswere differentialsltyainedusinga modified fluorescence-plus-Giem(sFaPG) technique(aftePrerryand Wolff,1974;Goto,et al.,1978).The slidewsere stainedinHoechst33258 staine.xposedtoultraviolet lighta,nd thenstainedwith Giemsa Azure B stain.The slidespreparedfrom the aberrationasssaywere stainedwith5% Giemsa solutionfortheanalysisof mitoticchromosomal aberrationsA.ll slidewsere thenair-drieadnd coverslipped usingDepex' rrrountinmgedium. 10.10 AberrationsAnalysisand Assay Evaluation: Cellswere selectefdorgood morphology and onlycellswith thenumber of centromeresequaltothemodal number 21 :12:(range19-23)were analyzed. One hundredcellsfrom each replicatceultureatfourdose levelsofthetestarticle, and from thenegativeand solventcontrolculturewsere analyzedforthedifferent typesofchromosomal aberration(sEvans,1962;See Section15.0).At least25 cellswere analyzedforchromosomal aberrationfsrom one of thepositivecontrol cultures.For controlof bias,allslidesexceptforthepositivceontrolswere coded priortoanalysis.Cellswith aberrationwsere recordedon thedatasheetsby the microscope stagelocation. The followingfactorwsere takenintoaccountintheevaluationofthe chromosomal aberrationdsata: I The overallchromosomal aberratiofnrequencies. CHV StudyNo.: 17388-0437 12 CORNING Hazlcton ii.-IPangefindingAssay WithoutMetabolicActivation Floatinpd-ebrisn,o cellmonoiavers.and no visiblmeitoticcellswere observedin theculturedsosed with 1670 and 5010 @ig/ml.Unhealthycellmonolan-erf.loating debris.= 15% reductioninthecellmonolaverconfluencea.nd no visiblmeitotic cellswere observedintheculturetreatewdith 501 pg/ml. Unhealthycell monolayer.floatindgebris.= 15% reductioninthecellmonolaverconfluencea,nd severereductionsinthenumber of visiblmeitoticellswere observedinthe culturetreatewdith 167 @ig/mi.Toxicitywas manifestedon theslidesprepared from thiscultureby theavailabiliotfyonly89 metaphases.Cellcyclekinetics were evaluatedintheculturetsreatedwith 16.7,50.1,and 167 pg/ml (Table1). Severecellcycledelaywas observedintheculturedosed with 167 pg/ml. Based on theseresultsa,20 hour harvestwas selectedfortestingconcentrationosf 12.5. 25.0.50.0,75.0,100, 150,and 200 @ig/miinthenonactivatioanberrationasssay. 11.3 RangefindingAssay With MetabolicActivation Floatingdebrisn,o cellmonolayers,and no visiblmeitoticcellswere observedin theculturedsosed with 1670and 5010 gg/ml. No visualsignsof toxicitwyere observedinany oftheothercultures.Cellcyclekineticwsere evaluatedinthe culturetsreatewdith50.1,167,and 501 pg/mi (Table1).No cellcycledelaywas observedintheculturesanalyzed.Based on theseresultsa,20 hour harvestwas selectedfortestincgoncentrationosf 125,250,500, 750, 1000,and 1500 pg/ml in theaberrationasssaywith metabolicactivation. 11.4 Chromosomal AberrationsAssay WithoutMetabolicActivation Unhealthycellmonolayers,=30% reductioninthecellmonolayerconfluencea,nd severereductionsinthenumber of visiblmeitoticcellswere observedinthe culturetreatedwith200 ;ig/ml.Toxicitywas manifestedon theslidesprepared from theseculturebsy thepresenceof many interphasceellsand sparsenumbers of metaphases.Unhealthycellmonolayers,= 15% reductioninthecellmonolayer confluencea,nd reductionsinthenuznberofvisiblmeitoticcellswere observedin theculturetreatedwith 150 pg/ml. Slightlyunhealthycellmonolavers,slight reductionsinthenumbers of visiblmeitoticcellsa,nd = 15% reductioninthecell monolayer confluencewere observedintheculturetreatewdith 100 pg/ml. Chromosomal aberrationwsere evaluatedfrom theculturestreatedwith 75.0, 100,150,and 200 @Lg/ml(Table2).No s*tgnificaintcreaseincellswith chromosomal aberrationwsas observedattheconcentrationasnalyzed. CHV StudyNo.: 17389-0437 14 CORNING Hazleton Thesensitiovfitthyecelclultufroerinductioofnchromosomaalberratiiosns shown by theincreasedfrequencyofaberrationisnthecellsexposed to mitomvcin C, thepositivecontrolagent.The testarticliesconsideredneizative forinducingchromosomal aberrationusndernonactivatiocnonditions. 11.5 Chromosomal AberrationsAssay With MetabolicActivation No cellmonolayersand no visiblmeitoticcellswere observedinthecultures dosed with 1500 pLg/ml.Dead cellmonolayers,no visiblemitoticcellsa.nd <50/6 celImonotaver confluencewere observedintheculturedsosed with 1000 pg/ml. Slightluynhealthycellmonolayers,floatindgead cellsa,nd z 15% reductioninthe cellmonolayerconfluencewere observedintheculturetreatedwith 750 pp'mi. Toxicitywas manifestedon theslidespreparedfrom theseculturesby the availabiliotfysparsenumbers ofmetaphases.Chromosomal aberrationwsere evaluatedftom theculturestreatewdith 125,250,500.and 750 pgiml (Table3). No significanitncreaseincellswith chromosomal aberrationwsas observedatthe concentrationasnalyzed. The sensitiviotfythecellcultureforinductioonf chromosomal aberrationiss shown by theincreasedfrequencyofaberrationisnthecellsexposed to cyclophospharnidet,hepositivecontrolagent.The testarticliesconsidered negativeforinducingchromosomal aberrationusnderconditionsofmetabolic activation. 12.0 CONCLUSION: The testarticleT,6358, was considerednegativeforinducingchromosomal aberrationisn CHO cellswithand withoutmetabolicactivation. 13.0 REFERENCES: Armitage,P.StatisticMaelthods inMedicalResearch,John Wiley & Sons,Inc.N,ew York, NY, 1971. Evans,H.J.:Chromosomal aberrationpsroducedby ionizingradiation.International Review of Cytology,U:221-321,1962. Goto,K.,Maeda, S.,Kano, Y.,and Sugiyama@T.:Factorsinvolvedindifferential Giemsa-stainingofsistecrhromatids.Chromosoma, 0:351-359, 1978. CHV Study No.-.17388-0437 15 CORNING Hazleton Pem, P.and Wolff.S.:New Giemsa method forthedifferentisatlaininogf sister chromatids.Nature.151:156-158.1974. Sokal.R.R..and Rohif.F.J.:Biometn,,Ed. 2,@X'.hF.reeman and Compan@..Ne%k York. 1981. Terasima,T. And Tolmach. L.J.:changesinX-raysensitiviotfvHeLa cellsdurinpt-he divisiocnycle.Nature,190:1210-1211,1961. CHV StudyNo.: 17388-0437 16 CORNING Hazleton 14.0 EXPERIMENTAL DATA TABLES CHV Study No.: 17388-0-437 17 CORNING Hazleton TABLE I RANGEFINDINCI ASSAY FOR ASSESSING TOXICITY Assay No.: 17388 TrialNo.: I Date: 0 1l,03!96 Lab No..CY 1296 Compound: T-6358 Metabolic Activation@-S9 Treatment POSITIVE CONTROL NEGATIVE CONTROL SOLVENT CONTROL TEST ARTICLE mmc McCoy's Sa Water MetabolicActivation:-S9 0.250 Aig/ml 10.0plimi 16.7 pg/mi 50.1 pg/ml 167 pgymll* 501 pglml* 1670 pg/mll Confluence m I M I e M2 % Solvent ContToi 51 43 6 loo 0 3 97 100 1 0 99 [Do 0 3 97 100 0 10 go 100 72 15 13 96 - - 86 0 Treatment onfluence % Solvent m I M I iM2 Control POSITIVE CONTROL CP 15,0pgiml 49 49 2 IDO NEGATIVE CONTROL McCoy's 5a 0 0 100 100 SOLVENT CONTROL Water 10.0pl/ml*** 1 2 97 loo TEST ARTICLE 50@l pg/mi 0 5 95 100 167 pglmi 1 2 97 100 501 pg/mi 0 1 99 1()0 1670 pg/ml* 0 'Thisendpointisbasedupon visualobservationswhich aremade priortotheharvestof the metaphasecells.Actualcellcountsarenottakenand any hypenrophy of theattachedcells cannotbe evaluated.At thetimeof theconfluenccobservationtheflasksarealsoevaluatedfor theappcwwcc of floatinmgitoticellsand dead cells. Toxicdose level. Toxic dose levelo.nly 89 cellsavailablfeoranalysis. 103 metaphases analyzed. CHV Study No.: 17388-0-437 18 CORNING Hazleton LL: < LL; r4 z < z < Z r,4 < ....... ........ L3 -E E 7i@b cc z < 0L)Z CHV StudyNo.: 17388-0-437 E c7) 19 CORNING Hazleton F5 - - -- - -- - @l< > < L'@7 u < L) <z vzi rq r-4 < .1@ iu mu < ........ ........ . ........ z ........ -- - -- - -- -- < cc < cc < CD-E Z:: -.b =L Z: ZL ZL :L Go 00 m r- z E <u . 4n Lw _j > wi Z > < L" < z z Ln LU CHV StudyNo.: 17388-0-437 CL .2 c EE 20 TABLE4 CORNING Hazleton CONTROL DATA OF CHROMOSOME ABERRATIONS IN CHINESE HAMSTER 8/95 THROUGH 12!95 OVARY CELLS '@egativCeontrol l,olvenCtonLrol PositivCeontrol %litomvcinC 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 0 Of Aberrations Per Cell O.DO O@04 0,012 36 0.00 005 0012 36 0.24 2.96 0770 27 O'DO 0.03 0.017 31 o@oo O@ 12 0023 29 040 8 88 1 728 24 % Of Cells With Aberrations 0.0 4.0 1 D4 36 0.0 50 093 36 20,0 94@O 39,70 27 00 2.5 1.40 31 00 70 178 29 32.0 1000 57.17 24 Of Cells With >1 Aberrations o@o 05 001 36 00 05 o@01 36 0,0 60.0 16.52 27 00 1.0 010 31 00 4@O 0.26 29 8,0 100.0 3293 24 CHV StudyNo.: 17388-0-437 21 CORNING Hazicton 15.0 DEFINITIONSOF CHRONIOSOME ABERRATIONS FOR GIEMSA STAINED CELLS NOT COMPUTED TG Chromatid gap: SG Chromosome gap: UC Uncoiledchromosome: PP Polyploidcell: E Endoreduplication: SIMPLE TB Chromaticbreak: SB Chromosome break: DM "DoubleMinute" fragment: CHV Study No.: 17388-0-437 ("tigdap").An achromatic(unstainedr)egionin one chromatic,thesizeof which isequaltoor smallerthanthewidthof a chromatic.These are notedbutnotusuallyincludedinfinaltotalosf aberrationasstheymay notallbe truebreaks. ("isochromatigdap,IG"). Same as chromatidgap butatthesame locusin bothsistecrhromatids. Failureofchromatinpacking.Probablynota true aberration. A cellcontainingmultiplecopiesofthehaploid number (n)ofchromosomes. Not countedinthe cellsscoredforaberrations. 4n cellinwhich separationof chromosome pairs has failed.Not countedinthecellscoredfor aberrations. An achromaticregioninone chromatid,larger thanthewidthofa chromatid.The associated ftagmentmay be partialloyr completely displaced. Chromosome has a clearbreak,formingan abnormal(deletedc)hromosome withan acentric fragmentthatisdislocated. These aresmalldouble dots,some of which are terminaldeletionasnd some interstitdiealetions and probablysmallrings.Theiroriginsarenot distinguishable. 22 COMPLEX ID Interstitdiealletion: TR Triradial: QR Quadriradial: D Dicentn'c: DF TC Tricentric: QC Quadricentric: PC Pentacentric: HC Hexacentric: R Ring RC Ring Chromatid: RF CHV StudyNo.: 17388-0437 CORNINGHazieton Lengthof chromatid"cutout"from midregionof a chromatidresultinigna smallfragmentof ring lyingbesidea shortenedchromatidof a gap inthe chromatid. An exchange between two chromosomes. or one chromosome and an acentrifcragment,which resultisna three-arinecdonfiguration. As triradiablu,tresultinigna four-ar7ned configuration. An exchange between two chromosomes which resultisna chromosome withtwo centromeres. This isoftenassociatewdith an acentricfragment inwhich case itisclassifieads DF. Dicentricwith fragment. An exchange between two chromosomes which resultisna chromosome with threecentromeres. Oftenassociatedwithtwo tothreeacentricfragment (AF). Such exchangescan involvemany chromosomes and arenamed as follows: fourcentromeres,up to fourAF fivecentromeresu,p to fiveAF sixcentromeres,up tosixAF A chromosome which formsa circlecontaininga centromere.This isoftenassociatedwith an acentrifcragmentinwhich case itisclassedas P,F. Singlechromatidring(acentric). Ring with associateadcentricfragment. 23 Cl Chromosomientrachange: T Translocation: AB OTHER GT/> CORNING Hazleton Exchange withina chromosome. e.g.,a ringthat does notincludetheentirechromosome. Obvious transfeorf materialbetween two chromosomes resultinigntwo abnormal chromosomes. When identifiablsec,oredas "T" not "2Ab." Abnormal monocentricchromosome. This isa chromosome whose morphology isabnormal for thekarvotype,and oftentheresulotf a translocatiopne,ricentriicnversione,tc. Classificatiuosned ifabnormalitycannotbe ascribedto,e.g.,a reciprocatlranslocation. A cellwhich containsmore than 10 aberrations. A heavilydamaged cellshouldbe analyzedto identiftyhetypesof aberrationasnd may not actuallyhave >10,e.g.,multiplefragmentssuch as thosefound associatewdith a tricentric. CHV StudyNo.: 17388-0437 24 CHV STUDY NO. PROTOCOL NO. 437, SDITION 15 CHROMOSOMAL ABERRATIONS IN CHINESE OVARY (CEO) Cry-T. CORNINGHazleton HAMSTER Corning Hazleton Inc. (CHV) will conduct this study in compliance with Good Laboratory Practice (GLP) Regulations. This protocol, critical phase(s) of the work in progress and the final report will be subject to audit by Quality Assurance in accordance with SOPs at Corning Hazleton Inc. The study will be conducted by CHV at 9200 Leesburg Pike. Vienna, Virginia 22182. PART 1. SPONSOR INFORMATION AND APPROVALS I. SPONSOR IDENTIFICATION Company Name: Address: O/K - 5,f . P4.1@ m /v Il. TEST ARTICLE IDENTIFICATION: 5-E3 III. TEST ARTICLE ANALYSIS Determination of the test article stability and the test article characteristics as defined in the GLP regulations is the responsibility of the Sponsor. IV. NOTIFICATION OF REGULATORY SUBMISSION In order to comply, with the CLP regulations, consulting laboratories must be notified if all or part of a study is intended for regulatory subm.ission. CHV maintains a master schedule of studies which fall under regulator-y review. Please indicate which agency, if any, might receive the results of this study: Undetermined ril@ FDA r--@n EPA-TSCA EPA-FIFPA MAFF MORW OECD OTHER 4/95 1 of 18 Protocol No. 437, Edition 15 V. STUDY DATES Proposed Experimental Start Date: Proposed Experimental Termination Date: VI. APPROVAL OF STUDY PROTOCOL Study Director: sponsor: E. Murli, Ph.D. CORNINGHa2lecon Date. Date: 4/95 2 of 18 CORNINGHazleton Protocol No. 437, Edition 15 PART 2 - STUDY PROTOCOL CHROMOSOMAL ABERRATIONS IN CHINESE OVARY (CEO) CR-TI HAMSTER I. OBJECTIVE The objective test article (CHO) cells of this in virro assay is to evaluate the ability of a to induce chromosomal aberrations in Chinese hamster ovary with and without an exogenous metabolic activation system. II. DEFINITIONS Structural changes and rearrangements result from damage to the genetic material and can be analyzed in cultured cells. Chromosomal breaks are mutations; many carcinogens cause chromosomal damage and chromosomal changes have been associated with cancer. Chromosomal aberrations are therefore a relevant test for potential mutagens and carcinogens. Numerical are not detected aberrations scored in a in an assay or variations from the normal chromosomal number continuous cell line such as CHO cells, and are not such as this in which cells pass through only one division cycle before analysis. Chromosomal aberration: the same locus, or the one chromosome. damage exchange expressed between in both sister both chromatids chromatids at of more than Chromatid aberration: between chromatids. damage seen in a single chromatid, or exchange Ill. RATIONALE The assay aims to establish whether the test article or its metabolites can interact with cells to induce chromosomal breaks. Chemically induced lesions may result in breaks in chromatin that are either repaired by the cell in such a way as to be undetectable or result in visible damage. Aberrations are a consequence of failure or mistakes in repair processes such that breaks do not rejoin or rejoin in abnormal configurations (reviewed by Evans, 1962). Aberrations are examined after chemical exposure. process or be converted cycles. In the case of when cells enter mitosis before they can be lost into complex derivatives the CHO cells used here, for the first time during the division during subsequent cell most dividing 4195 3 of 18 ProtocolNo. 437, Edition15 CORNINGHazieton cells examined 8 to 12 hours after treatmentare in their first mitosis (Ml cells). However, many test articles cause severe delay of progression through the cell cycle, and this assay has been designed to detect this delay and allow for slower growth of damaged cells by adjustments in the time between treatmentand cell fixation (see Section V.B.). IV. MATERIALS A. Test Cells The CHO cell line was derived from an ovarian biopsy of a Chinese hamster. Cells to be used in this assay (CHO-WBL) are a subclone originally obtainedfrom Dr. S. Wolff, Universityof California, San Francisco. The cells were subsequentlyrecloned in this laboratory to maintain karyotypic stability. The CHO-WBL subclone is a permanent cell line with an average cycle time of 12 to 14 hours with a modal chromosome number of 21. B. Cell Culture Conditions CHO cells will be grown in McCoy's 5a culture medium supplemented with approximately101 fetal calf serum, L-glutamine (ZmM), penicillin (100 units/ml) and streptomycin(100 @kg/ml). Cultures will be incubated with loose caps in a humidified incubator at 37'C - 2'C in an atmost)hereof 5Z 1.5Z COZ in air. C. Test For Mvcoplasma Contamination Mycoplasma testing is performed routinely by examination of slides stained with Hoechst 33ZSS and observed under UV microscopy. Mycoplasma are detected as small, intensely fluorescent specks over the cytoplasmic region of the cells. Any contaminationwould be rapidly recognized both from the fluorescent-stainedappearance and from a decreased uptake of BrdUrd in the cell cycle delay test (SectionV.B.2.e.). D- Karyotype S ability Karyotype stability is under constant scrutiny because the endpoint of the assay is cytogenetic analysis. To ensure maximum stability, cells will be used that have been cloned (CHO-WBL) and subsequently stored in liquid nitrogen. These stocks have undergone 5-8 transfers in culture before cryopreservation. Samples will be thawed periodically, recultured twice a week, and discarded after about eight weeks of continuous culture. 4/95 4 of 18 ProtocolNo. 437, Edition 15 CORNING H,-Lziecon E. Test Article Solid or liquid test articlesare suitablefor this assay. The test article is dissolved in an appropriate solvent such as serumfree culture medium, water. dimethyl sulfaxide (DHSO), ethanol, or acetone. Serial dilutionswill be carried out so as to achieve desired final concentrationsby addition of 0.05 ml per 5 ml culture medium, unless insolubilityrequires a larger volume. The maxi.mumfinal concentrationsof solvent in the culture will be, unless otherwise specified by the Sponsor, up to -80Z culture medium, up to 1OZ water, 1Z DMSO or ethanol, or 0.5Z acetone. The test compound solution will be prepared immediately before use. F. ControlArticles 1. Negative and Solvent Controls Without activation: Negative controls will be cultureswhich contain only cells and culture medium. Solvent controls will be cultures containingthe solvent for the test article at the same concentrationused in test cultures. With activation: Negative and solvent controls,as defined above. will be treated with Sg activation mix in the same way as the test cultures. 2. Positive Controls Known mutagenic and chromosome breaking agents will be used at two concentrations to ensure adequate results, but only one concentration will be chosen for aberration analysis. Without activation: Mitom,7cinC (Mt4C)is a clastogen that does not require metabolic activation. MKC will be dissolved in water and used at final concentrations of 0.2 to 0.5 @Lglml in the rangefinding assay, 0.5 to 1.0 @Lg/mlfor fixation at 8-12 hours, and 0.040 to 0.100 @Lg/mlfor later fixation. With activation: hany mutagens do not act directly but must be converted to active intermediates by enz)rmesfound in microsomes. An example is cyclophosphamide (C?). used here as the positive control to demonstrate the activity of the Sg metabolic activation system. CP will be dissolved in water and used at final concentrations of 10 to 30 @Lgimlin the rangefinding assay. 10 to 50 @Lgjmlfor fixation at 8 to 12 hours, and 10 to 25 @Lg/mlfor later fixation. 4/95 5 of 18 Protocol No. 437, Edition 15 CORNINGHazleton G. The Metabolic Activation System The in vitro metabolic activation system consists of rat liver enzymes and an energy producing system necessary for their function (NADP and isocitric acid). Enzymes are contained in a preparation of liver micros=es (Sg fraction) from rats treated with Aroclor 1254 to induce enzymes capable of transforming chemicals to more active forms. The S9 fraction, prepared in sucrose or in potassium chloride from male Sprague Dawley rats, is purchased from commercial suppliers and is retained frozen at approximately -80*C until use. Aliquots of this S9 fraction will be thawed immediately before use and added to a 'core" reaction mixture to form the activation system described below: Component Concentration in Cultures NADP (sodium salt) Isocitric acid Homogenate (S9 fraction) V. EXPERIMENTAL DESIGN 1.5 mg/ml (1.8 mm) 2.7 mgiml (10.5 mM) 15.0 @Ll/ml* A. Solubility and Dose Determination The maximum dose will be determined on a case by case basis taking into account both solubility and any relevant cytotoxicity information available on the test article. The highest dose tested will be 5 mg/ml, unless higher doses are specified by the sponsor. A range of doses covering five orders of magnitude in a half-log series will be tested in the preliminary rangefinding test unless a dose range is specified by the Sponsor. If no solubility information has been provided by the Sponsor, a preliminary solubility test will be carried out with sterile deionized water, serum-free culture medium. DMSO, ethanol, or acetone. Stock solutions in DMSO or ethanol will be diluted onehundred-fold in culture medium in a precipitate test. Stock solutions in acetone will be diluted 1:200 in culture medium in a precipitate test. The solvent selected will be the one which gives the best solubility after dilution in medium. 4/95 The amount of S9 homogenate per culture will depend on tLe lot of S9 in use at any time. Each lot of S9 homogenate is tested when purchased before use in the assay. Because the protein content of S9 homogenate varies among lots, Sg at various concentrations is tested against reference chemicals, such as cyclophosphanlide, benzo(a)pyrene or dimethylnitrosamine. The optimum S9 concentration is selected. based on induction of S9 is used in 7ter chromatid exchanges in CHO cells, and this amount of subsequent assays with that particular lot of S9. 6 of 18 Protocol No. 437, Edition 15 CORNINGHazlecon 4/95 In some cases, test articles are apparently, insoluble in solvents that are compatible with tissue culture. In such cases, the vehicle chosen and the highest dose tested will be that in which an evenly dispersed suspension can be prepared. The top dose tested for liquid test articles in the rangefinding assay will be 5 mg/ml, which may also be calculated from the specific gravity. Solubility testing will be the same as described above. B. Rangefinding and Determination of Fixation Time 1. Rationale Most known chemical clastogens (chromosome breaking agents) require a period of DNA synthesis to convert initial DNA damage into chromosome alterations visible at mitosis. Many cells bearing aberrations may not complete the division process successfully and may die. The time of fixation for chromosome preparation is thus selected to allow cells to go through DNA synthesis (S-phase) and reach the first mitosis after treatment. Since under normal circumstances the cell cycle of CHO-WBL cells is about 13 hours, fixation 8 to 11 hours After addition of test article will collect cells in the first mitosis after the beginning of treatment. However, many clastogens in.,iib4-ptrogression of cells through the cycle, and damaged cells which recover enough to progress to mitosis will not be collected at an 8-11 hour fixation time. As an alternative to carrying out several tests in which cells are fixed at a series of times in order to avoid missing a "peak' of aberration yield, a preliminary assessment of cell cycle delay will be made by differential staining. Cells will be grown in the presence of the base analogue 5bromo-2'-deoxyuridine (BrdUrd); after two cell cycles, onehalf of a chromosome (sister chromatid) will be more heavily substituted with BrdUrd than the other. This chemical difference causes the chromatids to differ in the reaction with the stain. The characteristic staining pattern of a cell that has undergone two complete cell cycles with BrdUrd (M2 cells) is a clear differentiation between chromatids along the complete length of every chromosome. In the CHO-WBL cell system, approximately 85Z-IOOZ of cells fixed after 26 hours exposure to BrdUrd show this M2 staining pattern. After only one cell cycle, chromatids are evenly stained (MI cells). After more than one cycle but less than two complete cycles. inte=ediate patterns are found in which only parts of the chromosomes are differentially stained (M,+). Thus, if the 7 of 18 Protocol CORNINGHazleton No. 437, Edition 15 cell cycle is slowed dawn by the test article, both M, and Ml+ cells will appear in the preparations made after 26 hours in BrdUrd. An estimate of the degree of cell cycle delay may be made from the frequencies of M, cells. The fixation time for the subsequent chromosome aberration test will then be selected to ensure that most cells are in their first metaphase after treatment, allowing the progression of damaged cells to the division stage. 2. Preliminary Test for Toxicity and Cell Cycle Delay Cultures are seeded at about 0.3 X 106 cells per 25 cm2 plastic flasks in 5 ml of fresh culture medium. one flask will be initiated for each treatment (10 dose levels or the Sponsor requested doses, solvent, negative and positive controls). a. Test without metabolic activation The day after culture initiation, solvent or test article will be added to the cultures. Incubation at about 37*C will be continued for about 2.0 to 2.5 hours when BrdUrd (final concentration 10 @LK)will be added and the cultures will be returned to the incubator for a total of about 25.5 to 26.5 hours before fixation. Approximately 2.0 to 2.5 hours before fixation, the cell monolayers will be washed with phosphate buffered saline to remove the test article, and fresh medi= will be added which contains Colcemid(t at 0.1 @Lg/mland BrdUrd at 10 pM. Incubation with Colcemid(Dwill continue for about 1.0 to 2.0 hours. b. Test with the metabolic activation system One day after culture initiation, culture niediumwill be removed and the cells will be washed once with phosphate buffered saline to remove serum. Cells will then be incubated at about 37'C for about 2 hours in the presence of the test article. the S9 reaction mixture. and growth medium without fetal calf serum (FCS). The short incubation time is used because prolonged exposure to the S9 mixture is tox4-cto cells; also enzyme activity is lost rapidly at 37*C. Serum is omitted to avoid possible inactivation, by binding to serum proteins. of shortlived. highly reactive intermediates produced by S9 enzymes. After the 2 hour exposure period, the cells will be washed at least twice with buffered saline. and 4/95 8 of 18 CORNINGHazleton ProtocolNo. 437.Edition15 normal growth medi= containing 1OZ FCS and 10 @LmBrdUrd will be added. Incubation will be continued for about 25.5 to 26.5 hours, with ColcemidS (0.1 @Lg/ml)added for the last 1.0 to Z.0 hours of incubation. c. Sun=rT of Treatment Schedule* Test -s9 +s9 Chemical -2.25 -2.25 SCHEDUL(EHO S) Wash BrdUrd - 0 -0.25 0 Wash 24-25 - Coicemid Fixation 24.5 24.5 25.5-26.5 25.5-26.5 *Timesgiven are approximateand may vary accordingto the size of the assay. d. Modificationfor testarticlesthat are volatileand/or alter I)H For test articlesthat are known to be markedlyvolatile, the cultureflaskswill be incubatedwith tightlyclosed caps until the articleis washed off. To compensate for lack of C02 circulation,HEPES buffer (25 mM final concentration)will be includedin the medium. If marked pH changesare noticed (i.e., color changes in medium) during the solubilitytest, HEPES bufferwill be includedin the medi= to attempt to counteractthe pH change. Furtherneutralization(e.g.,with HC1 or NAOH) will be performedto maintainnormal culture pH range (approximately6.8 to 7.4) only after consultationwith the Sponsor. e. Cell fixation,staining,and scoringfor cell cycle delay Before fixation,flaskswill be examinedunder an inverted microscope to estimate cytotoxicityexpressed as growth inhibitionand mitotic suppression. Observations of the degree of confluencyand apparent availabilityof mitotic cells (large.rounded.healthycells on the surface of the monolayeror floatingin the medium)will be made. Cells will be harvested from the highest dose level expected to yield any metaphases and from at least 2 lower dose levels. Metaphasecells will be collected by mitotic shake-off(Terasimaand Tolmach, 1961), swollen with 75 mM KC1. fixed in methanol- glacial acetic acid (3:1,v/v),droppedonto glass slides,and air dried. 4/95 9 of 18 Protocol No. 437, Edition 15 CORNINGHazleton Differential staining is accomplished by a modified fluorescence-plus-Giemsa (FPG) technique (after Perry and Wolff, 1974; Goto, et al., 1978). Slides willbe stained for at least 10 minutes with Soechst 33258 (5 @Lglml) in phosphate buffer (pH 6.8). mounted in the same buffer, and exposed at 55*-65*C to 'black-light, from 15 watt tubes for the amount of time required for differentiation between chromatids (3-20 minutes). Finally. slides will be stained with 5Z Giemsa for 3 to 20 minutes and air dried. The slides from the highest dose levels "har-vested* will be examined for presence of delayed cells. An attempt will be made to scan and classify one hundred metaphase cells as MI. M, + or 2:M2. 3. Determination Test of Dose and Fixation Time for the Aberration The doses selected for the assay will be based on the observations of toxicity (confluence reduction and cell appearance) and cell cycle delay from the preliminary test, and the maximum dose selected will be the highest dose at which analyzable metaphase cells are expected to be obtained. In the absence of observed cell cycle delay, a fixation time 8 to 11 hours after addition of chemical will be used. If a cell cycle delay is observed, a later fixation time such as 18 hours or 24 hours will be selected based on the estimated time to give a minimum of 50Z cells in M, at the lowest dose. Two different fixation times will be used if necessary (e.g., if the top dose causes delay but the lower doses do not). C. Chromosome Aberration Test Cultures will be seeded at about 1.0 to 1.5 X 106 cells per 75 cm2 plastic flask in 10 nil of fresh culture medium. A minimum of five doses will be used, depending on the results of the rangefinding test (Section V.B). The doses may be more closely spaced than half-log dilutions depending on the pattern of toxicity observations. Duplicate flasks will be used for each dose level. Single flasks will be used for solvent and negative controls and for each of two doses of positive control compounds. 4195 10 of 18 Protocol No. 437, Edition 15 CORNINGHazieton 1. Assay without the metabolic activation system One day after culture initiation, the culture medi= will be replaced with fresh medium and the cells will be treated with the test article. The test article will be left in cultures for the entire period, the length of which will be determined from the prelinlinary test (usually 8 to 30 hours). About 2.0 hours before fixation, cultures will be washed with phosphate buffered saline, and fresh medium will be added which contains ColcemidO (0.1 @Lglml final concentration). Presence of ColcemidO helps to accumulate the dividing cells. 2. Assay with the metabolic activation system One day after culture initiation, the culture medi= will be removed and the cells will be washed once with saline. The cells will then be incubated at about 37*C for about 2 hours in the presence of the test article, S9 reaction alixture, and growth medium without fetal calf serum (FCS). After the 2 hour exposure period, cells will be washed at least twice with phosphate buffered saline, and normal growth medium containing 1OZ FCS will be added. Incubation will be continued for a further period of approximately 6 to 28 hours with Colcemidg (0-1 @Lg/ml final concentration) present during the last 1.0 to 2.0 hours to accumulate the dividing cells. 3. Cell stainina and scoring Metaphase cells will be subsequently collected and fixed as in section V.B.2.e. Those flasks with excessive toxicity will not be harvested. The slides will be air dried and stained with Giemsa. After drying, the slides will be rinsed in xylene and mounted permanently. For control of bias. the slides will be coded for analysis except for the positive controls, which will be checked first to make sure the aberration frequency is adequate. Normally, two hundred cells per dose level (100 from each of the duplicate flasks), if available, will be read from each of the top four doses. If two hundred cells were not available from each of these doses, lower doses may be evaluated. One hundred cells will be read from each of the negative and solvent controls. At least twenty-five cells will be read from one positive control and from those cultures that have greater than 5OZ of cells with one or more aberrations. The complete number of 100 cells per culture may not be available due to toxicity or quality of preparation. Cells will be 4/95 11 of 18 Protocol No. 437, Edition 15 CORNINGHa2leton selected for scoring on the basis of good morphology, and only cells with the number of centromeres equal to the modal number 21 t 2 (range of 19 to 23) will be analyzed. Standard forms will be used to record analyses. For each cell bearing an aberration, the microscope stage location will be noted so the cell may be relocated if necessary. The complete list of aberrations scored and their definitions is attached to this protocol (pages 16-18). Vi. DATA A. Data Presentation anri Evaluation Data will be summarized in tables showing the numbers of cells analyzed, types of aberrations found, frequencies of aberrations per cell, and percentages of cells bearing aberrations. Chromatid and isochromatid gaps will be noted but will not be added into the totals for aberration assessment since they may not be true breaks. B. ASsav Accer)tance Criteria Ar. assay will be considered accepted for evaluation of test results only if all of the following criteria are satisfied. Activation and nonactivation sections of the aberrations assay form independent units and will be repeated independently, as needed, to satisfy the acceptance criteria. a Unsatisfactorv Controls The assay will be repeated if: I. The negative or the solvent control is more than twice the upper limit of the range of historical control values (upper lialit-5: or more of cells with aberrations). 2. The positive control result is not significantly higher (p < 0.01) than the pooled negative and solvent controls. If the positive control result in the test with S9 is adequate in an assay 'where activation and nonactivation assays are run concurrently but the positive control in the nonactivat;-on assay ;.:a;-Ist,he test need not be repeated as the activation pos*-ti-vecontrol demonstrates the sensitivity of the cells. 4/95 12 of le 103675 Protocol No. 437. Edition 15 CORNINGH2zleton Lack of Toxicity If the aberration results are negative and there is no significant reduction in confluence or delay in cell cycle progression, the test may be repeated at higher doses if higher doses can be achieved (i.e. not limited by solubility or not greater than 5 mg/ml). Excess Toxicity The assay will be repeated if results are not available for at least three dose levels. 0 Sporadic Increase If a significant increase is seen at one or more levels but not in consecutive doses and if there clear evidence for a positive dose response, the be repeated to verify the significance. dose is no assay will If the assay has to be repeated because of equivocal results, only three dose levels will be analyzed, instead of four, in the second assay. C. Assay Evaluation The following factors are taken into account in evaluat4-on of the test article data: Overall aberration .crequencies. Percentage of cells with aberrations. Percentage of cells with more than one aberration. Evidence for increasing amounts of damage with increasing dose. i.e., a dose related increase in aberrations. Cells with complex aberrations may be considered to have more significance than simple aberrations. Statistical analysis employs the Fisher's Exact Test to compare the percentage of cells with aberrations in treated cells with pooled results from solvent and negative controls (Sokal and Rohlf. 1981). A linear trend test of increasing number of cells with aberrations with increasing dose (Armitage, 1971) will also be performed. The difference is considered significant when p<0.01. The final evaluation of the test article will be based upon scientific judgment. 4/95 13 of 18 Protocol No. 437, Edition 15 VII. REFEUNCES CORNINGHazleton I. Armitage, P. Statistical Methods in Medical Research, JobLn Wiley & Sons, Inc.. New York, NY, 1971. 2. Evans, E.J.: Chromosomal aberrations produced by ionizing radiation. International Review of Cytology, 13:221-321, 1962. 3. Goto, K., Maeda, S., Kano, Y.. and Sugiyma, T.: Factors involved in differential Giemsa-staining of sister chromatids. 66:351-359. 1978. Chromosoma. 4. Perry, P. and Wolff, S.: New Giemsa method for the differential staining of sister chromatids. Nature, 251:156-158, 1974. 5. Sokal, R.R. and Rohlf, F.J.: Biometry. Freeman. 1981. 6. Terasima, T. and Tolmach L.J.: Changes in X-ray sensitivity of HeLa cells during the division cycle. Nature, 190:1210-1211, 1961. VIII. REPORT FORMAT CHV employs a standard report format for each assay design. report will provide the following information. The final Sponsor identification. Quality Assurance statement. Statement of GLP Compliance. Signature of study director. Test article identification and CHV Study Number. A physical description of the test article and date of receipt will be included in this section. Type of assay and protocol number. Dates of study initiation and completion. Study director and senior technician. Methods. Evaluation criteria. Interpretation of results. Conclusions. References. Test results presented in tabular form. IX. CHANGES OR REVISIONS .Any changes or revisions of this approved protocol will be documented, signed by the Study Director, dated, and maintained with this protocol. 4/95 14 of 18 Protocol No. 437, Edition 15 CORNINGHazieton X. RECORDS TO BE MAINTAIN= All raw data, documentation. records, protocols, and final reports generated as a result of this study will be archived in the storage facilities of Corning Hazleton Inc. for at least one year following submission of the final report to the sponsor. After the one year period. the sponsor may elect to have the aforementioned materials retained in the storage facilities of Corning Hazleton Inc. for an additional period of time or sent to a storage facility designated by the sponsor. 4195 15 of 18 ProtocolNo. 437, Edition15 DEFINITIONS OF CIMOMOSOM&L ABEpauTIONS CORNINGH2zleton FOR GIEKSA STAINED Cr-r-T- NOT COMPUTED TG Chromatid Gap: SG ChromosomeGap: Uc Uncoiled chromosome: pp Polyplaidcell: E Endoreduplication: (*tidgap'). An achromatic(unstained)region in one chromatid,the size of which is equal to or smallerthan the width of a chromatid. These are noted but not usually included in final totals of aberrationsas they may not all be true breaks. ("isochromatidgap, IG*). Same as chromatid gap but at the same locus in both sister chromatids. Failure of chromatin packing. Probably not a true aberration. A cell containingmultiple copies of the haploid number (n) of chromosomes. only indexed if very common. 4n cell in which separation of chromosome pairs has failed. Only indexed if very common. iimp-LE TB Chromatid Break: SB Chromosome Break: DM 'Double Minute" fragment: 4/95 An achromatic region in one chromatid, larger than the width of a chromatid. The associated fragment may be partially or completely displaced. Chromosome has a clear break, forming an ab- normal (deleted) chromosome with an acentric fragment that is dislocated. This classification now includes the acentric fragment (AF). The AF was different from the SB only in that it was not apparently related to any specific chromosome. These are small double dots, some of which are terulinaldeletions and some interstitial deletions and probably small rings. Their origins are not distinguishable. 16 of 18 Protocol No. 437, Edition 15 CORNINGHazieton COMPLEX ID Interstitial Deletion: Length of chromatid "cut out' from midregion of a chromatirdesulting in a small fragment or ring lying beside a shortened chromatid or a gap in the chromatid. TR Triradial: An exchange between two chromosomes, or one chromosome and an acentric fragment. which results in a three-armed configuration. QR Quadriradial: As triradial, but resulting in a four-armed configuration. CR Complex Rearrangement: An exchange among more than two chromosomes or fragments which is the result of several breaks. D Dicentric: DF TC Tricentric: QC Quadricentric: PC Pentacentric: HC Hexacentric: R Ring: An exchange between two chromosomes which results in a chromosome with two centromeres. This is often associated with an acentric fragment in which case it is classified as DF. Dicentric with fragment. An exchange involving three chromosomes and resulting in a chromosome with three centromeres. Often associated with two to three AF. Such exchanges can involve many chromosomes and are named as follows: four centromeres, up to four AF five centromeres. up to five AF six centromeres, up to six AF A chromosome which forms a circle containing a centromere. This is often associated with an acentric fragment in which case it is classed as RF. RC Ring Chromatid: RF ci Chromosome Intrachange: T Translocation: Single chromatid ring (acentric). Ring with associated acentric fragment. Exchange within a chromosome; e.g., a ring that does not include the entire chromosome. Obvious transfer of material between two chromosomes resulting in two abnor-Al chromosomes. When identifiable, scored as *T* not *2AB'. 4/95 17 of 18 Protocol No. 437, Edition 15 COMPLEX (Continued) AB CORNINGHazieton Abnoriml monocentric chromosome. This is a chromosome whose morphology is abnormal for the karyotype, and often the result of a translocation, pericentric inversion. etc. Classification used if abnormality cannot be ascribed to; e.g., a reciprocal translocation. OTHER GT Greater than 10 aberrations: A cell which contains more than 10 aberrations. A heavily damaged cell should be analyzed to identify the types of aberrations and may not actually have >10, e.g., multiple fragments such as those found associated with a tricentric. 4/95 18 of 18