Safechem Consultants, 7 Cedar Mount, Lyndhurst SO43 7ED, UK.
Mutagenesis. 2010 Nov;25(6):531-8. doi: 10.1093/mutage/geq046. Epub 2010 Aug 18.
The ability of the in vitro mammalian cell tests currently used to identify genotoxins has been shown to be limited by a high rate of false-positive results, triggering further unnecessary testing in vivo. During an European Centre for the Validation of Alternative Methods workshop on how to improve the specificity of these assays, testing at high concentrations was identified as one possible source of false positives. Thus far, Organisation for Economic Co-operation and Development genotoxicity test guidelines have required testing of chemicals using mammalian cells in vitro should be undertaken to concentrations as high as 10 mM (5000 μg/ml). Recently, a draft revision of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use genotoxicity test guidelines has recommended that testing concentrations should be reduced to 1 mM (500 μg/ml). To assess the impact that this lowering would have on the outcome of in vitro genotoxicity testing, we established a database of 384 chemicals classified as rodent carcinogens and reported Ames test results and the test concentrations that produced positive results in the mouse lymphoma assay (MLA), in vitro chromosome aberration (CA) assay and in vitro micronucleus test. Genotoxicity testing results were illustrated for 229 and 338 compounds in the MLA and in vitro CA assay, respectively. Of these test compounds, 62.5% produced positive results in the MLA, of which 20.3% required testing between 1 and 10 mM. A total of 58.0% produced positive results in in vitro CA assays, of which 25.0% required testing between 1 and 10 mM. If the testing concentration limit for mammalian cell assays was reduced to 1 mM, 24 (6.25%) potential carcinogens would not be detected in any part of the standard in vitro genotoxicity test battery (Ames test, MLA and in vitro CA assay). Further re-evaluation and/or retest of these compounds by Kirkland and Fowler [Kirkland, D. and Fowler, P. (2010) Further analysis of Ames-negative rodent carcinogens that are only genotoxic in mammalian cells in vitro at concentrations exceeding 1 mM, including retesting of compounds of concern. Mutagenesis 25, 539-553] suggest that the current 10 mM top concentration can be reduced without any loss of sensitivity in detecting rodent carcinogens.
目前用于鉴定遗传毒物的体外哺乳动物细胞测试的能力已被证明受到高假阳性率的限制，这导致进一步进行不必要的体内测试。在欧洲替代方法验证中心关于如何提高这些测定法特异性的研讨会上，高浓度测试被确定为可能产生假阳性的一个来源。到目前为止，经济合作与发展组织的遗传毒性测试指南要求使用体外哺乳动物细胞对化学品进行测试，测试浓度应高达 10 mM（5000 μg/ml）。最近，国际人用药品注册技术协调会（ICH）遗传毒性测试指南修订草案建议，测试浓度应降低至 1 mM（500 μg/ml）。为了评估降低测试浓度对体外遗传毒性测试结果的影响，我们建立了一个数据库，其中包含 384 种被归类为啮齿动物致癌物的化学品，并报告了 Ames 测试结果以及在小鼠淋巴瘤测定（MLA）、体外染色体畸变（CA）测定和体外微核试验中产生阳性结果的测试浓度。MLA 和体外 CA 测定分别对 229 种和 338 种化合物的遗传毒性测试结果进行了说明。在这些测试化合物中，62.5%在 MLA 中产生阳性结果，其中 20.3%需要在 1 至 10 mM 之间进行测试。总共 58.0%在体外 CA 测定中产生阳性结果，其中 25.0%需要在 1 至 10 mM 之间进行测试。如果将哺乳动物细胞测定的测试浓度限制降低至 1 mM，则在标准体外遗传毒性测试组合（Ames 测试、MLA 和体外 CA 测定）的任何部分都不会检测到 24 种（6.25%）潜在的致癌物质。对这些化合物的进一步重新评估和/或由 Kirkland 和 Fowler [Kirkland，D. and Fowler，P.（2010）对在浓度超过 1 mM 时仅在体外哺乳动物细胞中具有遗传毒性的 Ames 阴性啮齿动物致癌物的进一步分析，包括对关注化合物的重新测试。诱变 25,539-553]进行的测试建议，可以在不降低检测啮齿动物致癌物的敏感性的情况下降低当前的 10 mM 最高浓度。
