Report from the in vitro micronucleus assay working group
Report from the in vitro micronucleus assay working group
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DOI：
10.1016/j.mrgentox.2003.07.005
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发表时间：
2003-10-07
影响因子：
1.9
通讯作者：
Wakata, A
中科院分区：
文献类型：
--
作者：
Kirsch-Volders, M;Sofuni, T;Wakata, A
在华盛顿“第二届基因毒性测试国际研讨会”（1999 年 3 月 25-26 日）上，对当前体外微核测试的方法和数据进行了审查。因此，制定了该协议特定方面的实施指南。就以下主题达成了一致：细胞的选择、玻片制备、微核分析、毒性、细胞松弛素-B 的使用、剂量数和处理/收获时间 [Environ.摩尔。诱变剂。 35（2000）167]。由于许多重要的体外微核验证研究正在进行中，当时不可能设计出明确的、国际统一的方案。这些研究现已完成，数据已在普利茅斯“第三届基因毒性测试国际研讨会”（2002 年 6 月 28-29 日）上进行了审查。来自法国遗传毒理学会协调的研究数据、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来准备关于体外微核方案主要方面的结论。在本文中，提出了关于进行体外微核测定的协议的共识。主要建议涉及： 1．细胞增殖证明：细胞系和淋巴细胞都可以使用，但必须在对照和处理细胞中证明细胞增殖才能接受测试。2．毒性评估和剂量范围发现：毒性评估应通过确定细胞增殖来进行，例如细胞增殖。在没有细胞松弛素-B 的情况下增加细胞计数 (CC) 或群体倍增 (PD)，或者例如使用细胞松弛素-B 抑制胞质分裂增殖指数；并通过确定其他细胞毒性标志物（汇合、凋亡、坏死），这可以提供有价值的附加信息。3．细胞系和淋巴细胞的治疗方案。4．阳性对照的选择：在没有 S9-mix 的情况下，应将断裂剂（例如丝裂霉素 C 或博莱霉素）和非纽金（例如秋水仙碱）作为阳性对照，并且在使用 S9-mix 时需要使用需要 S9 才能发挥活性的断裂剂（例如，二甲基亚硝胺或无法直接激活该试剂的细胞类型中的环磷酰胺）。 5.重复培养物和要评分的细胞数量。6。重复实验：在淋巴细胞中，对于每个实验，应比较来自 2 个不同的健康年轻和非吸烟捐献者的血液。在细胞系中，如果第一次实验结果为阴性，则只需重复实验。7．统计：统计显着性不应成为确定阳性结果的唯一因素。生物学意义应作为指导。给出了统计分析的例子。 (C) 2003 Elsevier B.V 保留所有权利。
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. Statistics: 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. (C) 2003 Elsevier B.V All rights reserved.