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首页> 外文期刊>Mutation Research: International Journal on Mutagenesis, Chromosome Breakage and Related Subjects >How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop.
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How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop.

机译：进行体外遗传毒性测试时如何减少假阳性结果，从而避免不必要的后续动物测试：ECVAM研讨会报告。

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Workshop participants agreed that genotoxicity tests in mammalian cells in vitro produce a remarkably high and unacceptable occurrence of irrelevant positive results (e.g. when compared with rodent carcinogenicity). As reported in several recent reviews, the rate of irrelevant positives (i.e. low specificity) for some studies using in vitro methods (when compared to this "gold standard") means that an increased number of test articles are subjected to additional in vivo genotoxicity testing, in many cases before, e.g. the efficacy (in the case of pharmaceuticals) of the compound has been evaluated. If in vitro tests were more predictive for in vivo genotoxicity and carcinogenicity (i.e. fewer false positives) then there would be a significant reduction in the number of animals used. Beyond animal (or human) carcinogenicity as the "gold standard", it is acknowledged that genotoxicity tests provide much information about cellular behaviour, cell division processes and cellular fate to a (geno)toxic insult. Since the disease impact of these effects is seldom known, and a verification of relevant toxicity is normally also the subject of (sub)chronic animal studies, the prediction of in vivo relevant results from in vitro genotoxicity tests is also important for aspects that may not have a direct impact on carcinogenesis as the ultimate endpoint of concern. In order to address the high rate of in vitro false positive results, a 2-day workshop was held at the European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy in April 2006. More than 20 genotoxicity experts from academia, government and industry were invited to review data from the currently available cell systems, to discuss whether there exist cells and test systems that have a reduced tendency to false positive results, to review potential modifications to existing protocols and cell systems that might result in improved specificity, and to review the performance of some new test systems that show promise of improved specificity without sacrificing sensitivity. It was concluded that better guidance on the likely mechanisms resulting in positive results that are not biologically relevant for human health, and how to obtain evidence for those mechanisms, is needed both for practitioners and regulatory reviewers. Participants discussed the fact that cell lines commonly used for genotoxicity testing have a number of deficiencies that may contribute to the high false positive rate. These include, amongst others, lack of normal metabolism leading to reliance on exogenous metabolic activation systems (e.g. Aroclor-induced rat S9), impaired p53 function and altered DNA repair capability. The high concentrations of test chemicals (i.e. 10 mM or 5000 microg/ml, unless precluded by solubility or excessive toxicity) and the high levels of cytotoxicity currently required in mammalian cell genotoxicity tests were discussed as further potential sources of false positive results. Even if the goal is to detect carcinogens with short in vitro tests under more or less acute conditions, it does not seem logical to exceed the capabilities of cellular metabolic turnover, activation and defence processes. The concept of "promiscuous activation" was discussed. For numerous mutagens, the decisive in vivo enzymes are missing in vitro. However, if the substrate concentration is increased sufficiently, some other enzymes (that are unimportant in vivo) may take over the activation-leading to the same or a different active metabolite. Since we often do not use the right enzyme systems for positive controls in vitro, we have to rely on their promiscuous activation, i.e. to use excessive concentrations to get an empirical correlation between genotoxicity and carcinogenicity. A thorough review of published and industry data is urgently needed to determine whether the currently required limit concentration of 10mM or 5000 microg/ml, and high levels of cytotoxicity, are necessary for t
机译：参加研讨班的人一致认为，在体外哺乳动物细胞中进行遗传毒性测试会产生不相关的阳性结果（例如，与啮齿类动物的致癌性相比）时，出现的异常高得多且令人无法接受。正如最近的一些评论所报道的那样，使用体外方法进行某些研究的无关阳性率（即低特异性）（与该“金标准”相比）意味着越来越多的受试品接受了额外的体内基因毒性测试，以前很多情况下，例如已经评估了该化合物的功效（在药物的情况下）。如果体外试验更能预测体内的遗传毒性和致癌性（即假阳性更少），那么所用动物的数量将大大减少。除了作为“金标准”的动物（或人类）致癌性外，公认的遗传毒性测试还提供了有关细胞行为，细胞分裂过程和细胞命运（遗传毒性）的许多信息。由于这些作用对疾病的影响鲜为人知，并且相关毒性的验证通常也是（亚）慢性动物研究的主题，因此从体外遗传毒性测试中预测体内相关结果对于那些可能不了解的方面也很重要。作为致癌的最终终点，对癌变有直接影响。为了解决体外假阳性率高的问题，2006年4月在意大利伊斯普拉的欧洲替代方法验证中心（ECVAM）举行了为期2天的研讨会。来自学术界的20多位遗传毒性专家邀请政府和工业界审查来自当前可用细胞系统的数据，讨论是否存在降低假阳性结果趋势的细胞和测试系统，审查对现有方案和细胞系统的潜在修改，这些修改可能导致特异性提高，并审查了一些有望在不牺牲灵敏度的情况下提高特异性的新测试系统的性能。得出的结论是，从业者和监管审查者都需要对可能导致积极结果（与人类健康没有生物学关联）的可能机制的更好指导，以及如何获取这些机制的证据。与会者讨论了一个事实，即通常用于基因毒性测试的细胞系存在许多缺陷，可能会导致假阳性率高。这些包括：缺乏正常代谢，导致依赖于外源性代谢激活系统（例如，Aroclor诱导的大鼠S9），p53功能受损和DNA修复能力改变。讨论了高浓度的测试化学品（即10 mM或5000 microg / ml，除非由于溶解度或过度毒性而排除）和目前哺乳动物细胞遗传毒性测试中所需的高水平细胞毒性是假阳性结果的其他潜在来源。即使目标是在或多或少的急性条件下通过简短的体外试验来检测致癌物，似乎也无法超出细胞代谢代谢，激活和防御过程的能力。讨论了“混杂激活”的概念。对于许多诱变剂而言，体外缺乏决定性的体内酶。但是，如果底物浓度充分增加，则某些其他酶（在体内不重要）可能会取代激活物，导致相同或不同的活性代谢物。由于我们通常在体外不使用正确的酶系统作为阳性对照，因此我们不得不依靠它们的混杂激活，即使用过量的浓度来获得遗传毒性和致癌性之间的经验相关性。迫切需要对已发表的和行业数据进行彻底的审查，以确定当前必需的10mM或5000 microg / ml的极限浓度以及高水平的细胞毒性是否对t

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