Chen Lizao, Li Nan, Liu Yanfeng, Faquet Brigitte, Alépée Nathalie, Ding Chunmei, Eilstein Joan, Zhong Lingyan, Peng Zhengang, Ma Jie, Cai Zhenzi, Ouedraogo Gladys
Advanced Research, L'Oréal Research and Innovation China, Shanghai, China.
Advanced Research, L'Oréal Research and Innovation, Aulnay-Sous-Bois, France.
Mutagenesis. 2021 Apr 28;36(1):51-61. doi: 10.1093/mutage/geaa003.
The European Regulation on Cosmetics (no. 1223/2009) has prohibited the use of animals in safety testing since March 2009 for ingredients used in cosmetics. Irreversible events at the chromosome level (clastogenesis and aneugenesis) are commonly evaluated by scoring either micronuclei or chromosome aberrations using cell-based genotoxicity assays. Like most in vitro genotoxicity assays, the 2D in vitro micronucleus assay exhibits a poor specificity and does not mimic the dermal route. To address these limitations, the current project aims to develop and validate a 3D micronucleus assay using the EpiSkin™ model. This project is scientifically supported by the Cosmetics Europe Genotoxicity Task Force. In a first step, two key criteria for the development of micronucleus assay, namely, the sufficient yield of cells from the EpiSkin™ model and an acceptable proliferation rate of the basal layer, were assessed and demonstrated. Subsequently, six chemicals (vinblastine, n-ethylnitrosourea, β-butyrolactone, 2-acetylaminofluorene, 2,4-dichlorophenoland d-limonene) were evaluated in the EpiSkin™ Micronucleus Assay. At least two independent experiments using 48- and 72-h incubations were performed for each chemical. Results showed good inter-experimental reproducibility, as well as the correct identification of all six tested chemicals. The metabolism of 2-acetylaminofluorene on the EpiSkin™ model was also investigated and confirmed by the formation of an intermediate metabolite (2-aminofluorene). These preliminary results from the EpiSkin™ Micronucleus Assay indicate that it is a promising in vitro assay for assessing genotoxicity. The availability and suitability of this test method contribute significantly to the development of non-animal testing methods in China and its impact on the worldwide field.
自2009年3月起，欧盟化妆品法规（第1223/2009号）禁止在化妆品成分安全测试中使用动物。染色体水平的不可逆事件（断裂发生和非整倍体发生）通常通过基于细胞的遗传毒性试验，对微核或染色体畸变进行评分来评估。与大多数体外遗传毒性试验一样，二维体外微核试验特异性较差，且不能模拟皮肤途径。为解决这些局限性，当前项目旨在开发并验证一种使用EpiSkin™模型的三维微核试验。该项目得到了欧洲化妆品协会遗传毒性特别工作组的科学支持。第一步，评估并证明了微核试验开发的两个关键标准，即从EpiSkin™模型获得足够的细胞产量以及基底层可接受的增殖率。随后，在EpiSkin™微核试验中对六种化学物质（长春碱、N-乙基亚硝基脲、β-丁内酯、2-乙酰氨基芴、2,4-二氯苯酚和d-柠檬烯）进行了评估。每种化学物质至少进行了两项独立实验，分别孵育48小时和72小时。结果显示实验间具有良好的可重复性，并且正确识别了所有六种测试化学物质。还对EpiSkin™模型上2-乙酰氨基芴的代谢进行了研究，并通过中间代谢产物（2-氨基芴）的形成得到了证实。EpiSkin™微核试验的这些初步结果表明，它是一种有前景的评估遗传毒性的体外试验方法。该测试方法的可用性和适用性对中国非动物测试方法的发展及其在全球领域的影响有重大贡献。
