Evaluation of an in-vitro test protocol using a three-dimensional human buccal oral mucosal model to assess the oral medical devices

This study establishes an in vitro protocol using the EpiOral three-dimensional human buccal oral mucosal model to assess potential irritation from oral medical devices and their leachables. EpiOral tissues were exposed to test substances (e.g., methyl acetate, Triton X-100, sodium lauryl sulfate (SLS), phosphoric acid, benzalkonium chloride) at concentrations relevant to dental materials, with exposure time for 50% tissue viability (ET-50) calculated from viability assays at 1, 4, and 18 h. The application volumes of the test substances (40 μL and 100 μL), vehicle controls (saline, sesame oil), and a 50% viability cut-off were validated using linear mixed models (LMM), post-hoc comparisons, and ROC analysis with Youden’s index. ET-50 assays correctly classified the test irritants: strong (e.g. SLS, phosphoric acid, ET-50 < 1 h), moderate (hydrogen peroxide, 1.12 h), and non-irritants (e.g., glycerol, polyethylene glycol >18 h). No significant viability differences occurred between 40 μL and 100 μL for most substances (p > 0.05), except hydrogen peroxide. Using a 50% viability cut-off for irritancy classification, all tested substances were correctly classified in this exploratory dataset (sensitivity/specificity = 1.000). The in vitro testing protocol using the EpiOral model provides a structured experimental approach for oral mucosal irritation assessment. Under the conditions evaluated in this study, a 100 μL application volume and a 50% viability threshold were found to be appropriate.