1、Designation:E 1263 97(Reapproved 2003)Standard Guide forConduct of Micronucleus Assays in Mammalian BoneMarrow Erythrocytes1This standard is issued under the fixed designation E 1263;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,th
2、e 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.1.Scope1.1 This guide provides recommended guidelines for per-forming the mammalian in vivo bone marrow micronucleusassay.U
3、nder appropriate test conditions,measurement of thefrequency of newly formed micronucleated erythrocytes inbone marrow provides a convenient index of chromosomaldamage in nucleated erythrocyte precursor cells.The rationalefor the occurrence of micronuclei in conjunction with chromo-somal damage has
4、been described previously(1).2This guidedescribes conditions under which the frequency of micronucle-ated erythrocytes in mammalian bone marrow is an appropriatemeasure of in vivo chromosomal damage,and provides guide-lines for the design and technical execution of assays employ-ing this endpoint.1.
5、2 The following guidelines for mammalian bone marrowerythrocyte micronucleus assays have been published by orga-nizations concerned with the evaluation of genotoxicity testdata.These references should be consulted for recommenda-tions on details not covered in depth by this guide and forrequirements
6、 of specific organizations or government agencies(2-6).1.3 This standard does not purport to address all of thesafety concerns,if any,associated with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of
7、 regulatory limitations prior to use.2.Summary of Guide2.1 Animals are exposed either acutely or chronically to atest substance.At predetermined times after or during expo-sure,animals are sacrificed and the bone marrow is extracted,spread on slides,and stained.The frequency of micronucleatedcells a
8、mong the newly-formed(RNA-containing)erythrocytesis determined,and this frequency is compared among treatmentgroups.The newly-formed erythrocytes are identified by stain-ing the residual RNA which remains in the newly-formed cellsfor about 2 days after enucleation.Cells that stain uniformlypositive
9、for RNA are referred to as polychromatic,or poly-chromatophilic,erythrocytes(PCEs).Cells that do not stainpositively for RNA are referred to as normochromatic eryth-rocytes(NCEs).An increase in the frequency of micronucle-ated PCEs relative to the vehicle control group indicates thatthe test substan
10、ce induced structural chromosomal damage orlagging chromosomes aneuploidy in the nucleated erythrocyticcells.3.Significance and Use3.1 This guide provides guidelines for the selection ofanimal species,dosage and sampling conditions,sampling andscoring methods,statistical design,and analysis of genot
11、oxic-ity assays in which the endpoint measured is the frequency ofmicronucleated erythrocytes in mammalian bone marrow.4.Animal Selection and Care4.1 Laboratory species that are suitable for use in this assayinclude the mouse(Mus musculus),rat(Rattus rattus),andChinese hamster(Cricetulus griseus)(1)
12、.Other species prob-ably are equally suitable.If species or strains not previouslyused are employed,it must be established that the preparationprocedure adequately visualizes RNA-containing erythrocytesand micronuclei,that potential artifacts such as aggregatedRNA and mast cell granules do not inter
13、fere with the identifi-cation of micronuclei under the conditions employed,and thatthe micronucleus frequency is responsive to known clastogensand aneuploidy-inducing agents in that species and strain.4.2 In choosing the species and strain of test animal,consideration should be given both to the ava
14、ilability ofhistorical data on the response of that species and strain toknown genotoxins and to the availability of other toxicity dataon the same test material in the species and strain chosen.Choice of the same strain to be used in other genotoxicityassays of the same test material,or in long-ter
15、m toxicity orcarcinogenicity bioassays,has the advantage that the micro-nucleus frequency can be directly compared with other end-points.The species for which the largest data base on knowngenotoxins is available is the mouse(1).1This guide is under the jurisdiction of ASTM Committee F04 on Medical
16、andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Sept.10,2003.Published September 2003.Originallyapproved in 1988.Last previous edition approved in 1997 as E 1263 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.4.3 Animals should be obtained fr
