Household chemicals used daily are often combined, leading to inhalation exposure to mixtures. However, methods for assessing their toxic effects are limited. This study proposes an in vitro assay strategy for evaluating household chemical mixtures using benzalkonium chloride (BKC) and didecyldimethylammonium chloride (DDAC), a common disinfectant. Our approach utilizes the mode of action (MOA) of chemicals by applying toxicity units (TU) to assess the key events related to lung disease, such as reactive oxygen species (ROS) production and cell death. The TU (EC50) values for BKC and DDAC were 3.97 µg/mL and 1.89 µg/mL, respectively, from cytotoxicity results. The TU value of the mixture (5:5 ratio of BKC to DDAC) was calculated as 2.56 µg/mL. Using the OpenMRA platform, the TU values were predicted as 2.37 µg/mL with the concentration addition (CA) model and 11.26 µg/mL with the independent action (IA) model, indicating that the mixture effects were additive and closer to that predicted using the CA model. Both BKC and DDAC induced apoptosis and ROS production in human epithelial cells in a dose-dependent manner, suggesting similar modes of action in promoting cell death. Our results suggested that BKC and DDAC exhibited additive toxicity when combined. Our results demonstrate the utility of the TU-based approach, which combines cytotoxicity, ROS induction, and apoptosis measurements to evaluate mixture toxicity. This approach may be beneficial for assessing early key events relevant to lung diseases and offers a practical strategy for evaluating the inhalation toxicity of household chemical mixtures.
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