A dual luciferase assay for evaluation of skin sensitizing potential of medical devices.

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.