OFI - Austrian Research Institute for Chemistry and Technology, Brehmstraße 14 A, 1110, Vienna, Austria.
Department of Applied Life Sciences, University of Applied Sciences, FH Campus Wien, Helmut-Qualtinger-Gasse 2, 1030, Vienna, Austria.
Mol Biol Rep. 2019 Oct;46(5):5089-5102. doi: 10.1007/s11033-019-04964-8. Epub 2019 Jul 30.
According to standing regulations animal tests are still state of the art for the evaluation of the sensitization potential of medical devices. The aim of our study was to develop an in vitro method that can be used for testing of extracts of medical devices. The novel MDA-ARE assay is a cell based reporter gene assay focused on the ARE-Nrf2 pathway, which is involved in the dermal sensitization process. Optimization of the reporter construct and the cell line resulted in an improvement of the detection limit and a reduction of the incubation time to 6 h, which lowers cytotoxic side effects of the extracts on the cells. Using the assay, 21 out of 22 pure chemicals were identified correctly as skin sensitizers or non-sensitizers. All sensitizers could be detected at far lower concentrations compared to the local lymph node assay, the state-of-the-art animal test. To evaluate the assay's suitability for the testing of medical devices, medical grade silicone containing 0.1% of known skin sensitizers was prepared as positive controls and extracts of these positive controls were tested in comparison to extracts from pure silicone samples. All silicone samples were correctly and reproducibly identified as sensitizing or non-sensitizing demonstrating that the MDA-ARE assay is a sensitive and reliable tool for the detection of skin sensitizers in extracts of medical devices. The developed and validated test protocol was used for medical device extracts and showed its applicability for real samples and thus can contribute to reduce or even to replace the need for animal tests.
根据现行规定，动物试验仍然是评估医疗器械致敏潜力的最先进方法。我们的研究目的是开发一种可用于测试医疗器械提取物的体外方法。新型 MDA-ARE 测定法是一种基于细胞的报告基因测定法，侧重于参与皮肤致敏过程的 ARE-Nrf2 途径。报告构建体和细胞系的优化导致检测限提高，孵育时间缩短至 6 小时，从而降低了提取物对细胞的细胞毒性副作用。使用该测定法，22 种纯化学物质中的 21 种被正确识别为皮肤致敏剂或非致敏剂。与最先进的动物试验局部淋巴结试验相比，所有的致敏剂都可以在低得多的浓度下被检测到。为了评估该测定法用于测试医疗器械的适用性，制备了含有 0.1%已知皮肤致敏剂的医用级硅酮作为阳性对照，并将这些阳性对照的提取物与纯硅酮样品的提取物进行了测试。所有硅酮样品均被正确且可重复地鉴定为致敏或非致敏，表明 MDA-ARE 测定法是一种用于检测医疗器械提取物中皮肤致敏剂的敏感可靠工具。所开发和验证的测试方案用于医疗器械提取物，并显示其对实际样品的适用性，因此有助于减少甚至替代动物试验的需求。
