Kirsch-Volders Micheline, Sofuni Toshio, Aardema Marilyn, Albertini Silvio, Eastmond David, Fenech Michael, Ishidate Motoi, Kirchner Stephan, Lorge Elisabeth, Morita Takeshi, Norppa Hannu, Surrallés Jordi, Vanhauwaert Annelies, Wakata Akihiro
Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel, Pleinlaan 2, Brussels 1050, Belgium.
Mutat Res. 2003 Oct 7;540(2):153-63. doi: 10.1016/j.mrgentox.2003.07.005.
At the Washington "2nd International Workshop on Genotoxicity Testing" (25-26 March 1999) current methodologies and data for the in vitro micronucleus test were reviewed. As a result, guidelines for the conduct of specific aspects of the protocol were developed. Agreement was achieved on the following topics: choice of cells, slide preparation, analysis of micronuclei, toxicity, use of cytochalasin-B, number of doses, and treatment/harvest times [Environ. Mol. Mutagen. 35 (2000) 167]. Because there were a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at that time. These studies have now been completed and the data were reviewed at the Plymouth "3rd International Workshop on Genotoxicity Testing" (28-29 June 2002). Data from studies coordinated by the French Society of Genetic Toxicology, Japanese collaborative studies, European pharmaceutical industry validation studies, along with data from Lilly Research Laboratories were used to prepare conclusions on the main aspects of the in vitro micronucleus protocol. In this paper, the consensus agreements on the protocol for performing the in vitro micronucleus assay are presented. The major recommendations concern: 1. Demonstration of cell proliferation: both cell lines and lymphocytes can be used, but demonstration of cell proliferation in both control and treated cells is compulsory for the acceptance of the test. 2. Assessment of toxicity and dose range finding: assessment of toxicity should be performed by determining cell proliferation, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; and by determining other markers for cytotoxicity (confluency, apoptosis, necrosis) which can provide valuable additional information. 3. Treatment schedules for cell lines and lymphocytes. 4. Choice of positive controls: without S9-mix both a clastogen (e.g. mitomycin C or bleomycin) and an aneugen (e.g. colchicine) should be included as positive controls and a clastogen that requires S9 for activity when S9-mix is used (e.g. dimethylnitrosamine, or cyclophosphamide in those cell types that cannot activate this agent directly). 5. Duplicate cultures and number of cells to be scored. 6. Repeat experiments: in lymphocytes, for each experiment blood from 2 different healthy young and non-smoking donors should be compared. In cell lines, the experiments need only to be repeated if the first one is negative. 7.
statistical significance should not be the sole factor for determining positive results. Biological meaning should serve as a guideline. Examples of statistical analyses are given.
在华盛顿“第二届遗传毒性检测国际研讨会”（1999年3月25 - 26日）上，对体外微核试验的现行方法和数据进行了回顾。结果，制定了该试验方案特定方面的实施指南。就以下主题达成了共识：细胞选择、玻片制备、微核分析、毒性、细胞松弛素B的使用、剂量数量以及处理/收获时间[《环境与分子突变》35(2000)167]。由于当时有许多重要的体外微核验证研究正在进行中，所以当时无法设计出一个明确的、国际统一的试验方案。这些研究现已完成，并在普利茅斯“第三届遗传毒性检测国际研讨会”（2002年6月28 - 29日）上对数据进行了回顾。来自法国遗传毒理学协会协调的研究、日本合作研究、欧洲制药行业验证研究以及礼来研究实验室的数据被用于就体外微核试验方案的主要方面得出结论。本文介绍了关于进行体外微核试验方案的共识意见。主要建议涉及：1. 细胞增殖的证明：细胞系和淋巴细胞均可使用，但为了使试验结果可接受，必须证明对照细胞和处理细胞中均有细胞增殖。2. 毒性评估和剂量范围确定：毒性评估应通过测定细胞增殖来进行，例如在无细胞松弛素B时增加细胞计数(CC)或群体倍增(PD)，或者在有细胞松弛素B时测定胞质分裂阻滞增殖指数；并且通过测定其他细胞毒性标志物（汇合度、凋亡、坏死），这些标志物可提供有价值的额外信息。3. 细胞系和淋巴细胞的处理方案。4. 阳性对照的选择：在无S9混合液时，应同时包含一种断裂剂（如丝裂霉素C或博来霉素）和一种非整倍体诱导剂（如秋水仙碱）作为阳性对照，以及在使用S9混合液时需要S9激活活性的断裂剂（如二甲基亚硝胺，或在不能直接激活该试剂的细胞类型中的环磷酰胺）。5. 重复培养和要计数的细胞数量。6. 重复实验：在淋巴细胞中，对于每个实验，应比较来自2名不同健康年轻且不吸烟供体的血液。在细胞系中，只有当第一个实验结果为阴性时才需要重复实验。7.
统计学显著性不应是确定阳性结果的唯一因素。生物学意义应作为指导原则。给出了统计分析的示例。
