Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters.

IF 2.5 4区 医学 Q3 GENETICS & HEREDITY Mutagenesis Pub Date : 2024-03-12 DOI:10.1093/mutage/gead033
Dean N Thomas, John W Wills, Helen Tracey, Sandy J Baldwin, Mark Burman, Abbie N Williams, Danielle S G Harte, Ruby A Buckley, Anthony M Lynch
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Abstract

The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.

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亚硝胺致突变试验的艾姆斯试验研究设计:关键试验参数的定性和定量分析。
对具有基因毒性的 N-亚硝胺(NA)杂质进行严格控制是制药行业的一项重要安全考虑因素,尤其是考虑到最近药物产品的撤出。由于 N-亚硝胺具有诱变和致癌作用,因此属于遗传毒性杂质 "关注人群 "名单(ICH M7)。此外,由于艾姆斯试验的结果历来不一致,监管机构对艾姆斯试验预测 NAs 致癌潜力的能力也存在担忧。为解释这些不一致数据而提出的理由一般都与艾姆斯试验研究设计有关。这些方面包括载体溶剂的选择、肝脏 S9 的种类、细菌菌株、化合物浓度以及使用预孵育法与平板结合法。其中许多问题都源于统一埃姆斯试验指南之前产生的历史数据。因此,我们研究了各种埃姆斯试验检测参数,并使用定性分析和定量基准剂量模型来确定哪些组合提供了诱变效力方面最敏感的条件。我们研究了两种烷基亚硝胺,即 N-亚硝基二甲胺(NDMA)和 N-亚硝基二乙胺(NDEA)。在艾姆斯试验中很容易检测到 NDMA 和 NDEA 的诱变性,并确定了影响试验灵敏度排名的关键试验参数。预孵育法(30 分钟孵育)、适当的载体(水或甲醇)和仓鼠诱导肝 S9,以及鼠伤寒沙门氏菌菌株 TA100 和 TA1535 和大肠杆菌菌株 WP2uvrA(pKM101),为阿姆斯试验中 NDMA 和 NDEA 诱变效力提供了最灵敏的检测参数组合。利用这些参数和进一步的定量基准剂量模型,我们表明 N-亚硝基甲基乙胺(NMEA)在艾姆斯试验中呈阳性,因此不应再被视为历史上不一致的 NA。本文介绍的结果确定了一种灵敏的艾姆斯试验设计,可用于评估 NA,以支持可靠的杂质鉴定。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Mutagenesis
Mutagenesis 生物-毒理学
CiteScore
5.90
自引率
3.70%
发文量
22
审稿时长
6-12 weeks
期刊介绍: Mutagenesis is an international multi-disciplinary journal designed to bring together research aimed at the identification, characterization and elucidation of the mechanisms of action of physical, chemical and biological agents capable of producing genetic change in living organisms and the study of the consequences of such changes.
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