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 essential to reduce animal testing and aligns with the 3Rs principles (replacement, reduction, refinement). In this study, we developed a neutral red uptake (NRU) assay using human lung adenocarcinoma cells (A549) as a predictive model (A549-NRU) for acute inhalation toxicity. The assay incorporates two key features: a 15-minute incubation time to simulate the transient contact of inhaled chemicals with the airway surface under acute inhalation conditions, and the use of both polystyrene plates and glass plates for chemicals reactive with polystyrene. LC₅₀ values were determined for 49 chemicals and compared with reported LC₅₀ values from 4-hour rat inhalation studies. A significant positive correlation was observed between A549-NRU-derived LC₅₀ values and in vivo LC₅₀ values for water-soluble compounds (r = 0.4632, p = 0.0197) as well as chemicals containing aldehyde and ketone (r = 0.9339, p = 0.0007), and alcohol, ether, and epoxide (r = 0.7668, p = 0.0159) functional groups, suggesting that in vivo LC₅₀ values may be predictable using the A549-NRU assay. Importantly, the A549-NRU assay (r = 0.8879, p = 0.1121) demonstrated a stronger correlation with in vivo LC₅₀ values than the conventional NRU assay using mouse 3T3 fibroblast cells (r = 0.4524, p = 0.5476). These findings support the A549-NRU assay as an alternative for predicting acute inhalation toxicity and for estimating starting doses for confirmatory in vivo studies.