Comparison of Air-Liquid Interface and Direct Liquid Application Exposures for Toxicological Screening of Didecyldimethylammonium Chloride (DDAC) in Human Bronchial Epithelial Cells
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New Approach Methods (NAMs) are increasingly used to assess the toxicity of inhalable substances, highlighting the need to optimize exposure techniques for airway cell cultures. While air-liquid interface (ALI) exposure systems preserve the physicochemical properties of inhalable chemicals and allow direct cell-toxicant interaction, they are often costly, low-throughput, and require specialized expertise. Direct liquid application (DLA) of chemicals to airway cells cultured at ALI has been proposed as a cost-effective alternative, but recent findings suggest DLA may negatively affect differentiated primary human bronchial epithelial cells (dpHBEC), even in the absence of chemical (Mallek et al., Front. Toxicol., 2023). Further characterization is required to determine whether this impacts chemical screening.
To assess whether exposure method alters points of departure (PODs) following chemical exposure, we exposed organotypic (MatTek EpiAirway, dpHBEC) and immortalized human bronchial epithelial (16HBE) cells to didecyldimethylammonium chloride (DDAC), a quaternary ammonium compound, using both ALI and DLA approaches. Toxicological endpoints included trans-epithelial electrical resistance (TEER), viability, cytotoxicity, and pro-inflammatory cytokine release. Benchmark dose modeling was performed with BMDS to derive PODs. Multiple liquid application volumes (10, 30, 50 µL) and timepoints (6h, 24h) were tested. In summary, PODs from DLA exposures to DDAC were higher than ALI exposures across all cell models tested at both timepoints. In MatTek EpiAirway cells, ALI exposures produced lower PODs for all endpoints except TEER, but epithelial resistance was significantly reduced in DLA vehicle controls in a volume-dependent manner which suggests that higher volumes lead to greater vehicle toxicities. ALI exposures to DDAC resulted in lower, more sensitive PODs in all endpoints for dpHBEC and 16HBE cell cultures. DLA vehicle toxicities were observed for several endpoints with dpHBECs but not 16HBE cells.
Overall, these findings suggest that DLA-derived PODs may require additional uncertainty factors in inhalation risk assessment. We recommend: (1) applying the minimum volume that provides uniform cell coverage in DLA studies, and (2) reporting PODs in mass per surface area regardless of exposure method. Further research is needed to quantify cellular uptake to determine whether differences in PODs between exposure methods are due to different cell uptake rates and whether in vitro dosimetry models could be applied to improve DLA dose metrics.