1、Designation:E126288(Reapproved 2013)Standard Guide forPerformance of Chinese Hamster Ovary Cell/HypoxanthineGuanine Phosphoribosyl Transferase Gene Mutation Assay1This standard is issued under the fixed designation E1262;the number immediately following the designation indicates the year oforiginal
2、adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This guide highlights some of the more relevant bio-logical concepts as the
3、y are currently understood,and summa-rizes the critical technical aspects for acceptable bioassayperformances as they currently are perceived and practiced.The Chinese hamster ovary cell/hypoxanthine guanine phos-phoribosyl transferase(CHO/HGPRT)assay(1)2has beenwidely applied to the toxicological e
4、valuation of industrial andenvironmental chemicals.1.2 This guide concentrates on the practical aspects of cellculture,mutagenesis procedures,data analysis,quality control,and testing strategy.The suggested approach represents aconsensus of the panel members for the performance of theassay.It is to
5、be understood,however,that these are merelygeneral guidelines and are not to be followed without the useof sound scientific judgement.Users of the assay shouldevaluate their approach based on the properties of the sub-stances to be tested and the questions to be answered.1.3 Deviation from the guide
6、lines based on sound scientificjudgement should by no means invalidate the results obtained.1.4 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.1.5 This standard does not purport to address all of thesafety concerns,if any,associ
7、ated 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 regulatory limitations prior to use.2.Significance and Use2.1 The CHO/HGPRT assay detects forward mutations ofthe X-linked hypoxanthine-gua
8、nine phosphoribosyl transferase(hgprt)locus(coding for the enzyme,HGPRT)in Chinesehamster ovary(CHO)cells.Cells originally derived fromChinese hamster ovary tissue are exposed to a test article and,following an appropriate cell culture regimen,descendants ofthe original treated population are monito
9、red for the loss offunctional HGPRT,presumably due to mutations.Resistance toa purine analogue,6-thioguanine(6TG)(or less desirably,8-azaguanine(8AG),is employed as the genetic marker.HGPRT catalyzes the conversion of the nontoxic 6TG to itstoxic ribophosphorylated derivative.Loss of the enzyme or i
10、tsactivity therefore leads to cells resistant to 6TG.2.2 Because HGPRT is an enzyme of the purine nucleotidesalvage pathway,loss of the enzyme is not a lethal event.Different types of mutational events(base substitutions,frameshifts,deletions,some chromosomal type lesions,and soforth)should theoreti
11、cally be detectable at the hgprt locus.TheCHO/HGPRT assay has been used to study a wide range ofmutagens,including radiations(2-4),and a wide variety ofchemicals(1),and complex chemical mixtures(5).3.Characteristics of CHO Cells3.1 Different CHO cell lines/subclones are appropriate forthe CHO/HGPRT
12、assay.The CHO-K1-BH4 cell line developedand extensively characterized by(6)is probably the mostwidely employed.The CHO(WT)cell line and its derivative,CHO-AT3-2,are used to monitor mutations at other gene lociin addition to hgprt(7,8).While there are differences amongthe cell lines employed,a number
13、 of general characteristics arecritical for the performance of the assay:3.1.1 The cloning efficiency(CE)of the stock culturesshould not be less than 70%.The CE of untreated or solventcontrol experimental cultures should not be less than 50%.3.1.2 Cultures in logarithmic phase of growth should have
14、apopulation doubling time of 12 to 16 h.3.1.3 The modal chromosome number should be 20 or 21,as is characteristic of the particular cell line/subclone used.3.1.4 Cultures should be free from microbial and myco-plasma contamination.3.2 The cell properties that are critical for the assay shouldbe rout
15、inely monitored as part of the quality control regimen.Routine quality control procedures should include testing of1This guide is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devicesand is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test M
16、ethods.Current edition approved Oct.1,2013.Published October 2013.Originallyapproved in 1988.Last previous edition approved in 2008 as E1262 88(2008).DOI:10.1520/E1262-88R13.2The boldface numbers in parentheses refer to the list of references at the end ofthis guide.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 serum and media for each new purchase,as well as myco-plasma and karyotype checks at least once yearly,preferablyonce every
