Preclinical Safety, Novartis Institutes for Biomedical Research, Novartis Pharma AG, WKL-135.2.25, Basel, Switzerland.
Litron Laboratories, 3500 Winton Place, Rochester, NY, USA.
Mutagenesis. 2020 Sep 12;35(4):349-358. doi: 10.1093/mutage/geaa014.
The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organisation for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the details that will need to be articulated in an eventual guideline are recommended treatment and harvest schedules. With this in mind, experiments reported herein were performed with Wistar Han rats exposed to aristolochic acid I (AA), 1,3-propane sultone, chlorambucil, thiotepa or melphalan using each of two commonly used treatment schedules: 3 or 28 consecutive days. In the case of the 3-day studies, blood was collected for Pig-a analysis on days 15 or 16 and 29 or 30. For the 28-day studies blood was collected on day 29 or 30. The effect of treatment on mutant reticulocytes and mutant erythrocytes was evaluated with parametric pair-wise tests. While each of the five mutagens increased mutant phenotype cell frequencies irrespective of study design, statistical significance was consistently achieved at lower dose levels when the 28-day format was used (e.g. 2.75 vs 20 mg/kg/bw for AA). To more thoroughly investigate the dose-response relationships, benchmark dose (BMD) analyses were performed with PROAST software. These results corroborate the pair-wise testing results in that lower BMD values were obtained with the 28-day design. Finally, mutagenic potency, as measured by BMD analyses, most consistently correlated with the mutagens' tumorigenic dose 50 values when the lengthier treatment schedule was used. Collectively, these results suggest that both 3- and 28-day treatment schedules have merit in hazard identification-type studies. That being said, for the purpose of regulatory safety assessments, there are clear advantages to study designs that utilise protracted exposures.
体内 Pig-a 检测法被用于安全性研究，以评估化学物质诱发体细胞基因突变的潜能。目前正在进行的工作旨在制定经合组织（OECD）测试指南，以支持常规用于监管目的（OECD 项目编号 4.93）。在最终指南中需要阐明的细节之一是建议的处理和收获时间表。考虑到这一点，本文报道的实验使用 Wistar Han 大鼠进行，这些大鼠暴露于马兜铃酸 I（AA）、1,3-丙烷磺内酯、苯丁酸氮芥、噻替派或美法仑，使用两种常用的处理方案之一：连续 3 天或 28 天。在 3 天研究中，在第 15 或 16 天和第 29 或 30 天采集血液进行 Pig-a 分析。对于 28 天的研究，在第 29 或 30 天采集血液。使用参数成对检验评估处理对突变性网织红细胞和突变性红细胞的影响。尽管五种诱变剂中的每一种都增加了突变表型细胞频率，无论研究设计如何，但当使用 28 天方案时，始终在较低剂量水平上达到统计学意义（例如，AA 为 2.75 与 20 mg/kg/bw）。为了更彻底地研究剂量-反应关系，使用 PROAST 软件进行了基准剂量（BMD）分析。这些结果与成对检验结果一致，即 28 天设计获得了较低的 BMD 值。最后，使用较长的处理方案时，BMD 分析测量的诱变剂效力与突变剂的肿瘤形成剂量 50 值最一致相关。总之，这些结果表明，3 天和 28 天的处理方案在危害识别类型的研究中都有价值。话虽如此，对于监管安全评估的目的，使用延长暴露的研究设计具有明显的优势。
