482-Genetic Toxicology DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells in vitro

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1、482Adopted:OECDGUIDELINEFORTESTINGOFCHEMICALS23Oct1986"GeneticToxicology:DNADamageandRepair/UnscheduledDNASynthesisinMammalianCellsinvitro"1.INTRODUCTORYINFORMATION•Prerequisites–Solid,liquid,vapourorgaseoustestsubstance–Chemicalidentificationoftestsub

2、stance–Purity(impurities)oftestsubstance–Solubilitycharacteristics–Meltingpoint/boilingpoint–pH(whereappropriate)–Vapourpressuredata(ifavailable)•StandarddocumentsTherearenorelevantinternationalstandards.2.METHODA.INTRODUCTIONTheguidelineforUnscheduled

3、DNASynthesis(UDS)inmammaliancellsinvitrodescribesproceduresutilizingprimarymammaliancellsorcontinuouscelllinestodetectDNArepairsynthesis.TheendpointofunscheduledDNAsynthesisismeasuredbytheuptakeof3radioactivelabelednucleotidee.g.H-TdRusingautoradiograp

4、hicorliquidscintillationcounting(LSC)procedures.UDSmayalsobemeasuredusinginvivosystems.•PrincipleofthemethodTheUDStestmeasurestheDNArepairsynthesisafterexcisionandremovalofastretchofDNAcontainingtheregionofdamageinducedbychemicalorphysicalagents.Thetes

5、tis3basedcommonlyontheincorporationoftritium-labelledthymidine(H-TdR)intotheDNAof3mammaliancellswhicharenotintheS-phaseofthecellcycle.TheuptakeofH-TdRmaybedeterminedbyautoradiographyorbyLSCofDNAfromthetreatedcells.Mammaliancellsinculture,unlessprimaryr

6、athepatocytesareused,aretreatedwiththetestsubstancewithandwithoutexogenousmammalianmetabolicactivation.UsersofthisTestGuidelineshouldconsultthePreface,inparticularparagraphs3,4,7and8.482page2"GeneticToxicology:DNADamageandRepair/UnscheduledDNASynthesis

7、inMammalianCellsinvitro"B.DESCRIPTIONOFTHETESTMETHOD•PreparationsTestsubstanceThetestsubstanceandcontrolsubstancesshouldbepreparedingrowthmedium,ordissolvedinanappropriatevehicleandthenfurtherdilutedingrowthmedium,foruseintheassay.Thefinalconcentration

8、ofthevehicleshouldnotaffectcellviability.CellsandcultureconditionsPrimarycultures(e.g.rathepatocytes),humanlymphocytesorestablishedcelllines(e.g.humandiploidfibroblasts)maybeusedintheassay.Appropriategrowthmedia,CO2concentration,tempera