ASTM_E_1280_-_97_2003.pdf

Designation:E 1280 97(Reapproved 2003)Standard Guide forPerforming the Mouse Lymphoma Assay for MammalianCell Mutagenicity1This standard is issued under the fixed designation E 1280;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon(e)indicates an editorial change since the last revision or reapproval.INTRODUCTIONThis guide was developed at the request of ASTM Subcommittee E47.09 on Biomarkers in orderto aid toxicologists,geneticists,biochemists,other researchers,and interested persons in theunderstanding,performance,and analysis of the mammalian cell mutagenicity test that uses theTK+/-3.7.2C strain of L5178Y mouse lymphoma cells.In this rapidly changing area of toxicology,itis not intended for this guide to replace,alter,or diminish the usefulness of presently availableprotocols and procedures.1.Scope1.1 The purpose and scope of this guide is to presentbackground material and to establish criteria by which proto-cols and procedures for conducting the L5178Y/TK+/-3.7.2Cmouse lymphoma mutagenicity assay(commonly referred to asthe mouse lymphoma assay,(MLA)can be properly under-stood and evaluated.This guide is also intended to aidresearchers and others to gain a better understanding of thecritical elements involved with mammalian cell mutagenicitytesting.More specifically,this guide is intended to provide forresearchers the accomplishment of the following goals:1.1.1 Provide an understanding of the critical procedures(steps)in the performance of this mammalian cell mutagenicitytest.1.1.2 Provide generalized criteria by which researchers canevaluate if they are properly performing,utilizing,and inter-preting this assay.1.1.3 Provide criteria by which individuals responsible forevaluating MLA data can determine if the experiments havebeen properly performed and interpreted.1.1.4 Provide a basis from which new procedures anddevelopments in testing procedures can be evaluated.1.1.5 Provide an understanding of the types of geneticdamage(that is,gene and chromosome mutation)that may bedetected in this mammalian cell mutagenicity test.2.Terminology2.1 Definitions:2.1.1 clastogenany agent that is capable of inducingchromosome breaks.2.1.2 gene mutationany heritable change whose physicalextent is restricted to the limits of a single gene.2.1.3 mutagenany physical or chemical agent capable ofinducing a mutation.2.1.4 mutationany heritable change in the genetic mate-rial,not caused by genetic segregation or genetic recombina-tion,and that is transmitted to daughter cells.2.2 Definitions of Terms Specific to This Standard:2.2.1 chromosome mutationa mutation resulting from astructural change to a chromosome involving the gain,loss,orrelocation of chromosome segments.Chromosome mutationscan be either intrachromosomal or interchromosomal.2.2.2 relative suspension growth(RSG)used to measurethe cytotoxicity of a given treatment based on the growth ofcells in suspension culture relative to the untreated or solventcontrol(s).RSG is calculated according to the method of Cliveand Spector(1).22.2.3 relative total growth(RTG)used as a means tomeasure the relative toxicity to cells(survival)followingtreatment in the mouse lymphoma assay.RTG is calculatedaccording to the method of Clive and Spector(1)and includesRSG as well as the ability to form colonies in the clonal phaseof the assay.2.3 Symbols:2.3.1 BrUdR5-bromo-28-deoxyuridine.2.3.2 BUdRbromouracil deoxyriboside.2.3.3 CASchemical abstract service.2.3.4 DMSOdimethylsulfoxide.2.3.5 MLAmouse lymphoma assay.1This guide is under the jurisdiction of Committee F04on Medical and SurgicalMaterials and Devices and is the direct responsibility of Subcommittee F04.16 onBiocompatibility Test Methods.Current edition approved Sept.10,2003.Published September 2003.Originallyapproved in 1989.Last previous edition approved in 1997 as E 1280 97.2The boldface numbers in parentheses refer to the list of references at the end ofthis guide.1Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959,United States.2.3.6 NADPnicotinamide-adenine dinucleotide phos-phate.2.3.7 TFTtrifluorothymidine.2.3.8 THMGthymidine+hypoxanthine+methotrexate+glycine.2.3.9 VCviable count(s).3.Significance and Use3.1 This guide is limited to procedures used solely for thetesting of substances to determine their mutagenicity and doesnot apply to other methods and uses such as exploringmechanisms of mutation.3.2 Recent evidence suggests that this assay measures a dualgenetic end point;therefore,some discussion of the relation-ships between mammalian cell mutagenicity testing results andthe results observed both in pure gene mutational assays and incytogenetic assays is necessary.However,it is not the intent ofthis guide to discuss other relationships between this mamma-lian cell mutagenicity testing results and the r