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 Organization for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the requirements for OECD approval are demonstrations of assay reliability, including reproducibility within and among laboratories. Experiments reported herein address the reproducibility of the rat blood Pig‐a assay using the reference mutagens chlorambucil and melphalan. These agents were evaluated for their ability to induce Pig‐a mutant erythrocytes in three separate studies conducted across two laboratories. Each of the studies utilized a common treatment schedule: 28 consecutive days of exposure via oral gavage. Whereas one laboratory studied Crl:CD(SD) rats, the other laboratory used Wistar Han rats. One or two days after cessation of treatment blood samples were collected for mutant reticulocyte and mutant erythrocyte measurements that were accomplished with the same analytical technique whereby samples were depleted of wildtype erythrocytes via immunomagnetic separation followed by flow cytometric enumeration of mutant phenotype cells (MutaFlow®). Dunnett's test results showed similar qualitative outcomes within and between laboratories, that is, each chemical and each study demonstrated statistically significant, dose‐related increases in mutant reticulocyte and erythrocyte frequencies. Benchmark dose analysis (PROAST software) provided a means to quantitatively analyze the results, and the relatively tight, overlapping benchmark dose confidence intervals observed for each of the two chemicals indicate that within and between laboratory reproducibility of the Pig‐a assay are high, adding further support for the development of an OECD test guideline.

中文翻译：

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大鼠血液 Pig-a 基因突变测定的实验室内和实验室间重现性。


体内Pig-a测定法正用于安全性研究，以评估化学品诱导体细胞基因突变的潜力。正在进行的工作旨在制定经济合作与发展组织 (OECD) 测试指南，以支持监管目的的常规使用（OECD 项目编号 4.93）。经合组织批准的要求之一是证明测​​定的可靠性，包括实验室内部和实验室之间的可重复性。本文报道的实验解决了使用参考诱变剂苯丁酸氮芥和美法仑进行大鼠血液Pig-a测定的再现性。在两个实验室进行的三项独立研究中，评估了这些药物诱导Pig-a突变红细胞的能力。每项研究均采用共同的治疗方案：通过口服强饲法连续 28 天暴露。一个实验室研究 Crl:CD(SD) 大鼠，而另一个实验室则使用 Wistar Han 大鼠。停止治疗一两天后，收集血样进行突变网织红细胞和突变红细胞测量，测量采用相同的分析技术完成，即通过免疫磁性分离去除样品中的野生型红细胞，然后对突变表型细胞进行流式细胞术计数 (MutaFlow®) 。 Dunnett 的测试结果显示实验室内部和实验室之间的定性结果相似，即每种化学物质和每项研究都表明突变网织红细胞和红细胞频率具有统计显着性、与剂量相关的增加。 基准剂量分析（PROAST 软件）提供了一种定量分析结果的方法，两种化学物质观察到的相对紧密、重叠的基准剂量置信区间表明Pig-a测定的实验室内和实验室之间的重现性很高，并补充道进一步支持经合组织测试指南的制定。