1、Designation: E 1262 88 (Reapproved 2003)Standard Guide forPerformance of Chinese Hamster Ovary Cell/HypoxanthineGuanine Phosphoribosyl Transferase Gene Mutation Assay1This standard is issued under the fixed designation E 1262; the number immediately following the designation indicates the year ofori
2、ginal 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.1. Scope1.1 This guide highlights some of the more relevant bio-logical c
3、oncepts as they 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 t
4、he toxicological evaluation 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
5、 of theassay. It is to 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.
6、3 Deviation from the guidelines based on sound scientificjudgement should by no means invalidate the results obtained.1.4 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-pria
7、te 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-guanine phosphoribosyl transferase(hgprt) locus (coding for the enzyme, HGPRT) in Chinesehamster
8、 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 monitored for the loss offunctional HGPRT, presumably due to mutations. Resistance toa puri
9、ne 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 itsactivity therefore leads to cells resistant to 6TG.2.2 Because HGPRT is an
10、enzyme of the purine nucleotidesalvage pathway, loss of the enzyme is not a lethal event.Different types of mutational events (base substitutions, frame-shifts, deletions, some chromosomal type lesions, and so forth)should theoretically be detectable at the hgprt locus. TheCHO/HGPRT assay has been u
11、sed 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 assay. The CHO-K1-BH4 cell line developedand extensively char
12、acterized 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 of general characteristics arecritical for the per
13、formance 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 apopulation doubling time of 12 to 16 h.3.1.3
14、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 routinely monitored as part of the quality control
15、 regimen.Routine quality control procedures should include testing ofserum and media for each new purchase, as well as myco-plasma and karyotype checks at least once yearly, preferablyonce every three months.1This guide is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials
16、 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 1996 as E 1262 88 (1996).2The boldface numbers in parentheses refer to
17、 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. Mutagenesis Procedures4.1 The mutagenesis protocol can be divided into threephases: mutagen treatment, expression, and selection.4.2 M
18、utagen Treatment:4.2.1 Cell PlatingCells should be in exponential phasewhen plated for treatment. Several media (for example, HamsF12, alpha-MEM) that are known to be optimal for cell growthcan be used. Cells should be seeded at an appropriate celldensity to allow exponential growth as well as quant
19、itation ofinduced responses. A common practice is to plate 0.5 3 106cells in a 25cm2flask, or 1.5 3 106cells in a 75cm2flask, onthe day before treatment.4.2.2 Chemical HandlingThe solubility of the test articlein an appropriate medium should be determined before treat-ment. Commonly used solvents ar
20、e, in the order of preference,medium, water, dimethylsulfoxide, ethanol, and acetone. Gen-erally, the nonaqueous solvent concentration should not exceed1 % and should be constant for all samples. As part of thesolubility test, an aliquot of the test chemical should be addedto the treatment medium to
21、 note any pH changes, the presenceof any chemical precipitation, and any apparent reaction of thechemical or solvent with the culture vessel. The solvent ofchoice should not have any undesirable reactions with the testarticle, culture vessel, or cells.4.2.3 Addition of Test Article to CellsStock sol
22、utions ofthe test samples are prepared and aliquots are added to eachflask. Dilutions of the test article should be such that theconcentration of solvent remains constant for all samples. Cellsare generally treated with the test article for at least 3 h. Fortreatment times of 3 to 5 h, serumfree med
23、ium can be used. Asserum is required to maintain cell division, medium containingserum should be used for a prolonged treatment period (forexample, 16 h or longer). Serum requirement for treatmentperiods between 5 and 16 h should be determined on acase-by-case basis.4.2.4 Exogenous Activation System
24、sAroclor 1254-induced rat liver homogenate (S9) is the most commonly usedexogenous metabolic activating system for the assay. When S9is used, cofactors for the mixed function monooxygenasesshould be present. Calcium chloride (CaCl2), which enhancesthe mutagenicity of nitrosamines and polycyclic hydr
25、ocarbons(9, 10), appears to be another useful addition. However, theneed for CaCl2has yet to be documented for a wide variety ofchemicals. A commonly used cofactor mixture consists ofsodium phosphate (50 mM, pH 7.0 to 8.0), NADP (4 mM),glucose-6-phosphate (5 mM), potassium chloride (30 mM),magnesium
26、 chloride (10 mM), and CaCl2(10 mM). S9 is addeddirectly to the cofactor mixture. One volume of the S9/cofactormixture is added to 4 volumes of the treatment medium. Otherexogenous systems (for example, hepatocytes, S9 from otheranimal species or produced using different enzyme inductionconditions,
27、and other cofactor mixtures) can also be useddepending on the intent of the experiment.4.2.5 Estimation of CytotoxicityPlating CHO cells imme-diately after treatment for cytotoxicity determination is gener-ally expected to yield the most accurate results. Otherwise,cytotoxicity can be estimated on t
28、he day after treatment.Aliquots of the cells are plated to allow for colony develop-ment. Cytotoxicity is usually expressed as relative CE which isthe ratio of the CE of the treated cells to that of the solventcontrol. Viability determination should take into account anyloss of cells during the trea
29、tment period, cell trypsinizationprocedures, and the overnight incubation period.4.2.6 Positive and Solvent ControlsAn appropriate nega-tive control is treatment of cells with the solvent used for thetest article. Positive controls, both direct-acting and indirect-acting, should also be included to
30、demonstrate the adequacy ofthe experimental conditions to detect known mutagens. Anuntreated control may also be included to evaluate the effectsof the solvent on mutagenicity. Commonly used positivecontrols are ethyl methane sulfonate (EMS) and N-methyl-N8-nitro-N-nitrosoguanidine (MNNG) as direct-
31、acting mutagens,and benzo(a)pyrene (BaP) and dimethylnitrosamine (DMN) aspromutagens that require metabolic activation.4.3 Expression of Induced Mutations:4.3.1 After mutation at the hgprt locus, the mutant pheno-type requires a period of time before it is completely expressed(expression requires th
32、e loss of pre-existing enzyme activity).Phenotypic expression is presumably achieved by dilution ofthe pre-existing HGPRT enzyme and mRNA through celldivision and macromolecular turnover. At the normal popula-tion doubling times of 12 to 16 h for CHO cells, an expressionperiod of 7 to 9 days is gene
33、rally adequate (11, 12).4.3.2 The most widely employed method for phenotypicexpression allows exponential growth of the cells for a definedtime period after mutagen treatment. CHO cells can besubcultured with 0.05 % trypsin with or without EDTA.Aliquots of 1 3 106cells are subcultured at 2 or 3 day
34、intervalsin 100mm diameter tissue culture dishes or 75 cm2t-flasks.Either complete medium or hypoxanthine-free medium can beemployed, with either dialyzed or nondialyzed serum. It isimportant to ensure that the medium employed will allow apopulation doubling time of 12 to 16 h.4.3.3 Besides the norm
35、al growth of cells as monolayercultures, alternative methods of subculturing involving suspen-sion (8), unattached (13), and division arrested (14) cultureshave also been successful. The use of a particular subcultureregimen in the expression period should be substantiated bydata demonstrating the a
36、chievement of optimal expression.4.4 Mutant Selection:4.4.1 Conditions for the selection of mutants must bedefined to ensure that only mutant cells are able to formcolonies and that there is no significant reduction in the abilityof mutant cells to form colonies. In general, cells are plated intissu
37、e culture dishes for attached colony growth (11), or in agarfor suspended colony growth (16). An advantage of the formeris that after the colonies are fixed and stained, the plates can becounted at a later date. An advantage of the latter is thatmetabolic cooperation between wild type and mutant cel
38、ls isreduced, allowing selection of a higher cell number per plate.For attached colonies, the cells are in general cultured for aperiod of 6 to 8 days and the number of colonies counted afterfixing (for example, with 10 % formalin or 70 % methanol),and staining (for example, with 10 % Giemsa or crys
39、tal violet).Soft agar colonies are usually counted in situ after a culturingperiod of 10 to 14 days.E 1262 88 (2003)24.4.2 Reliable selection has been established inhypoxanthine-free medium containing dialyzed serum and 10M 6TG. Fetal bovine serum, newborn bovine serum, or calfserum can be used, pro
40、viding that the serum has been ad-equately tested and shown to support the desirable character-istics of CHO cells as described here. Dialyzed serum isusually necessary to eliminate the competition between 6TGand purine bases in the serum. It has been found that a selectioncell density of 2 3 105or
41、fewer cells per 100 mm dish forattached colony growth (14, 15) and 106or fewer cells per 100mm dish (in 30 mL of agar) for agar colony growth (16) allowsessentially 100 % recovery of mutant cells.5. Data Presentation5.1 Results from the assay should include the followingexperimental data:5.1.1 Conce
42、ntrations and solvents used for the test articleand positive controls.5.1.2 Absolute and relative cloning efficiencies (CE) in theconcurrent cytotoxicity assay.5.1.2.1 Absolute CEAbsolute CE equals the number ofcolonies formed divided by the number of cells plated.5.1.2.2 Relative CERelative CE equa
43、ls CE (treatment)divided by CE (solvent control).5.1.3 Actual number of mutant colonies observed for eachtreatment condition.5.1.4 Absolute CE at selection for each treatment condition.5.1.5 Mutant frequency (MF) values, expressed as mutantsper 106cells.5.1.5.1 Mutant Frequency (MF) ValuesMF values
44、equalthe number of mutant colonies divided by the number ofclonable cells.5.1.5.2 Number of Clonable CellsThe number of clonablecells equals the cells plated multiplied by the absolute CE atselection.6. Criteria for Data Acceptability6.1 Generally, for the data of a given assay to be acceptable,the
45、following criteria should be met:6.1.1 The absolute CE of the negative controls should not beless than 50 %. Absolute CE values lower than 50 % wouldindicate suboptimal culturing conditions for the cells.6.1.2 The mean mutant frequency of the solvent controls ineach experiment should fall within the
46、 range from 0 to 20mutants per 106clonable cells. A higher mutant frequency maypreclude detection of weak mutagens. Under such conditionsdata acceptability should be evaluated on a casebycase basis.6.1.3 The positive control must induce a statistically signifi-cant response at a magnitude appropriat
47、e for the mutagen underthe chosen experimental conditions.6.1.4 The highest test article concentration should, if pos-sible, result in a significant cytotoxic response (for example,10 % to 30 % survival, where survival is the percent of thetreated population that is viable after treatment). This isp
48、articularly important if the response is negative. For noncy-totoxic test articles, the highest concentration has generallybeen 1 to 10 mg/mL, or to the limit of solubility.7. Data Analysis7.1 Due to the possibility of stochastic fluctuation, onlysamples with no fewer than 100 000 viable cells after
49、 treat-ment should be used for data analysis. Judgement on mutage-nicity should be made based on the following information:7.1.1 Dose response relationship.7.1.2 Significance of response (in comparison to the nega-tive control).7.1.3 Reproducibility of the results.7.2 Exact statistical analysis is difficult because the distri-bution of the number of mutant colonies depends on thecomplex processes of cell growth and death after mutagentreatment. While other appropriate methods can be used, thefollowing two approximate methods are used commonly:7.2.1 Weighted Regressio
