Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig-a gene mutation assay, and (2) utilize flow cytometry-based methods to score each of these genotoxicity biomarkers. Proof-of-principle experiments were performed with 4-week-old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2-dimethylhydrazine, dimethylnitrosamine, 2,6-dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3-day exposure cycle, and three 3-day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday-Friday workweek. Key findings are that even these well-studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig-a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit-for-purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc.

中文翻译：

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3R 友好的研究设计促进了大鼠肝脏和血液微核分析以及 Pig-a 基因突变评估：使用 13 种参考化学品进行概念验证。

监管指导文件强调在评估化学品的体内遗传毒性潜力时评估多个组织中最合适的终点的价值。然而，进行多项独立研究以评估多个终点和/或组织隔室是资源密集型的。此外，当依赖于视觉检测时，对基因毒性终点进行评分的传统方法可能会比理想方法缓慢、乏味且不够客观。为了解决当前实践中的这些问题，我们尝试 (1) 设计与肝脏和血液微核终点以及 Pig-a 基因突变测定相兼容的资源节约治疗和收获计划，以及 (2) 利用基于流式细胞术的对这些基因毒性生物标志物进行评分的方法。对 4 周大的雄性和雌性 Crl:CD(SD) 大鼠进行了原理验证实验，这些大鼠暴露于马兜铃酸 I/II、苯并 [a] 芘、顺铂、环磷酰胺、二乙基亚硝胺、1,2-二甲基肼、二甲基亚硝胺、2,6-二硝基甲苯、羟基脲、美法仑、替莫唑胺、喹啉或长春碱。这 13 种化学品分别在两种治疗方案中进行了测试：一个 3 天暴露周期和三个 3 天暴露周期。每次暴露、血液采集和肝脏收获都是在标准的周一至周五工作周内完成的。主要发现是，即使这些经过充分研究的、相对有效的基因毒物在两种组织和所有检测中都没有活性（实际上，在所有三种检测中，只有顺铂明显呈阳性）；而 Pig-a 检测的灵敏度明显受益于三个对一个治疗周期，微核分析在两种研究设计中产生了性质相似的结果。总的来说，这些结果表明，通过使用适合目的的研究设计结合流式细胞术评分方法，同时评估三个终点和两个重要的组织区室，可以显着减少动物和其他资源需求，同时改善体内遗传毒性潜力的评估。环境。摩尔。Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc. 这些结果表明，通过使用适合目的的研究设计并结合流式细胞术评分方法，同时评估三个终点和两个重要的组织区室，可以显着减少动物和其他资源需求，同时改善对体内遗传毒性潜力的评估。环境。摩尔。Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc. 这些结果表明，通过使用适合目的的研究设计并结合流式细胞术评分方法，同时评估三个终点和两个重要的组织区室，可以显着减少动物和其他资源需求，同时改善对体内遗传毒性潜力的评估。环境。摩尔。Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc.