Developing a Novel In Vitro Toxicity Assay for Predicting Inhalation Toxicity in Rats

The development of alternative in vitro methods for assessing acute inhalation toxicity is a critical step toward reducing animal testing and aligns with the principles of the 3Rs (replacement, reduction, and refinement). In this study, we developed and optimized a neutral red uptake (NRU) assay using human lung adenocarcinoma cells (A549) as a predictive model (A549-NRU) for acute inhalation toxicity. To improve assay efficiency and robustness, we introduced two key modifications: the incubation time was reduced to 15 minutes to enable rapid and high-throughput screening, and for chemicals reactive with polystyrene 6-well glass plates were used to prevent chemical-induced degradation and ensure assay consistency. LC ₅₀ values were determined for 49 chemicals and compared with reported LC ₅₀ values from 4-hour rat inhalation studies. A significant positive correlation was observed between A549-NRU-derived LC ₅₀ values and in vivo LC ₅₀ values for water-soluble compounds and chemicals containing aldehyde, ketone, alcohol, ether, and epoxide functional groups, suggesting that in vivo LC ₅₀ values may be predictable using the A549-NRU assay. Additionally, A549-NRU LC ₅₀ values showed significant negative correlations with molecular weight and octanol–water partition coefficients, indicating that chemicals with higher values tended to be less cytotoxic in vitro . Importantly, the A549-NRU assay demonstrated stronger correlation with in vivo LC ₅₀ values than the conventional NRU assay using mouse 3T3 fibroblast cells. These findings support the use of the A549-NRU assay to estimate starting doses for in vivo studies, and potentially as an in vitro alternative for predicting acute inhalation toxicity.