Speit Günter, Kojima Hajime, Burlinson Brian, Collins Andrew R, Kasper Peter, Plappert-Helbig Ulla, Uno Yoshifumi, Vasquez Marie, Beevers Carol, De Boeck Marlies, Escobar Patricia A, Kitamoto Sachiko, Pant Kamala, Pfuhler Stefan, Tanaka Jin, Levy Dan D
Ulm University, Institute of Human Genetics, Ulm, Germany.
Japanese Center for the Validation of Alternative Methods, National Institute of Health Sciences, Tokyo, Japan.
Mutat Res Genet Toxicol Environ Mutagen. 2015 May 1;783:6-12. doi: 10.1016/j.mrgentox.2014.09.006. Epub 2014 Sep 22.
As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use.
作为第六届国际遗传毒性测试研讨会（IWGT）的一部分，彗星试验专家工作组评估了与体内彗星试验在监管遗传毒性测试中的应用相关的关键主题。涵盖的领域包括：适用性范围的确定和监管认可、方案关键参数的确定及试验标准化的尝试、与其他体内研究联合和整合的经验、实验室能力验证、所用方案的灵敏度和效能、不同组织的使用、样品冷冻以及细胞毒性适当测量方法的选择。标准方案可检测多种类型的DNA损伤，但并非能检测所有类型的DNA损害。标准方案的修改可用于检测其他特定类型的DNA损伤（如交联、大分子加合物、氧化碱基）。此外，工作组确定了关键参数，每个已发表的研究方案都应仔细控制并详细描述这些参数。如果体内彗星试验结果是在已证明具备能力的实验室获得的，则会更可靠。这包括对溶剂对照有充分反应以及对所检测的每个组织的阳性对照有充分反应的证明。普遍认为样品冷冻是一种选择，但需要更多数据以建立普遍接受的方案。关于组织毒性，工作组得出结论，细胞毒性可能是彗星试验结果的一个混杂因素。建议查看多个参数，如组织病理学观察、器官特异性临床化学以及组织炎症指标，以确定化合物特异性毒性是否可能影响结果。专家工作组得出结论，碱性体内彗星试验是一种成熟的遗传毒性评估试验，可推荐给监管机构使用。
