Aaron C S, Bolcsfoldi G, Glatt H R, Moore M, Nishi Y, Stankowski L, Theiss J, Thompson E
Upjohn Company, Kalamazoo, MI 49001.
Mutat Res. 1994 Jun;312(3):235-9. doi: 10.1016/0165-1161(94)90038-8.
As part of the International Workshop on Standardization of Genotoxicity Test Procedures, in Melbourne, 27-28 February 1993, various international guidelines were examined with respect to protocol issues in the area of mammalian cell gene mutation assays. The working group on mammalian cell gene mutation assays discussed a wide range of protocol issues related to study design; in most cases the recommendations are reasonably consistent with existing guidelines. Agreement was reached on several issues as follows. The upper limit of concentration for testing non-toxic substances should be 10 mM or 5 mg/ml, whichever is lower. For testing toxic substances the criteria of an acceptable upper limit of concentration should yield 10-20% survival. Any of several established mammalian cell mutation assays (L5178Y TK+/-, CHO/HPRT, AS52/XPRT, V79/HPRT) can be used to evaluate mutagenesis in mammalian cells; the ouabain (Na/K-ATPase) system is not an acceptable mutation assay for routine evaluation of mutagenesis in mammalian cells. Ability to recover small colonies must be convincingly demonstrated when using the L5178Y TK+/- mouse lymphoma assay. In the mouse lymphoma assay (L5178Y TK+/-), colonies in positive controls and at least two (if available) representative positive doses of the test compound should be sized if a positive response is seen; in the event of a negative response due to the test compound, colony sizing of the positive control is necessary to validate the conduct of the assay. Testing both in the presence and absence of S9 metabolic activation is necessary. It was not possible to come to a firm conclusion about the length of treatment. There was a general agreement that extended treatment times (> 2 cell cycles) often bear more disadvantages than advantages and should only be used with adequate justification. It is not necessary to repeat clear positive or clear negative tests when the assay has been adequately performed; this recommendation differs significantly from the UK guidelines. If treatment groups are not replicated, the numbers of doses tested should be increased; this recommendation differs significantly from the UK guidelines. Each laboratory should establish a historical database for the performance of a given assay in that laboratory.
作为1993年2月27日至28日在墨尔本举行的遗传毒性试验程序标准化国际研讨会的一部分，对哺乳动物细胞基因突变试验领域方案问题的各种国际准则进行了审查。哺乳动物细胞基因突变试验工作组讨论了与研究设计相关的广泛方案问题；在大多数情况下，这些建议与现有准则相当一致。就以下几个问题达成了一致意见。测试无毒物质的浓度上限应为10 mM或5 mg/ml，以较低者为准。对于测试有毒物质，可接受的浓度上限标准应产生10%-20%的存活率。几种已确立的哺乳动物细胞突变试验（L5178Y TK+/-、CHO/HPRT、AS52/XPRT、V79/HPRT）中的任何一种都可用于评估哺乳动物细胞中的诱变作用；哇巴因（Na/K-ATP酶）系统不是用于常规评估哺乳动物细胞诱变作用的可接受突变试验。使用L5178Y TK+/-小鼠淋巴瘤试验时，必须令人信服地证明能够回收小菌落。在小鼠淋巴瘤试验（L5178Y TK+/-）中，如果观察到阳性反应，阳性对照和至少两个（如有）测试化合物的代表性阳性剂量的菌落应进行大小测定；如果由于测试化合物出现阴性反应，则需要对阳性对照进行菌落大小测定以验证试验的进行情况。必须在有和没有S9代谢活化的情况下进行测试。关于处理时间的长短无法得出明确结论。普遍认为延长处理时间（>2个细胞周期）往往弊大于利，仅应在有充分理由时使用。当试验已充分进行时，无需重复明确的阳性或阴性试验；这一建议与英国准则有很大不同。如果处理组未重复设置，则应增加测试的剂量数量；这一建议与英国准则有很大不同。每个实验室应为该实验室中特定试验的性能建立一个历史数据库。
