Effect of aerosolized benzalkonium chloride exposure in in vitro cell models using air-liquid interface culture for...

Pesticides, biocides, and disinfectants used in veterinary medicine are frequently applied in spray form, necessitating inhalation toxicity testing. This study aimed to cultivate cells in an air-liquid interface (ALI) system for in vitro inhalation toxicity screening as an alternative to animal testing and to evaluate the effects of benzalkonium chloride (BKC) exposure on cells. A549 and Calu-3 cells were cultured under ALI conditions for three weeks, with transepithelial electrical resistance (TEER) and hematoxylin and eosin (H&E) staining confirming cell proliferation, integrity, and multilayered epithelial formation. The cells were exposed to aerosolized BKC (2-16.5 mg/mL) using the VITROCELL® cloud system. Toxicity was assessed through WST-1, lactate dehydrogenase (LDH), and reactive oxygen species (ROS) assays, revealing concentration-dependent cytotoxicity. IC₅₀ values ranged from 4.5 to 19 mg/mL in A549 cells and 3.6 to 6.7 mg/mL in Calu-3 cells, indicating the latter's higher sensitivity to BKC exposure. LDH and ROS assays further demonstrated significant cell damage following exposure, supporting the use of Calu-3 cells for detecting toxicity indicators in inhalation studies. The results highlight the importance of minimizing aerosol exposure to protect respiratory health and demonstrate the potential of ALI systems for reliable inhalation toxicity assessments. However, additional studies are needed to evaluate the reproducibility of the cell models and incorporate more comprehensive toxicity mechanism indicators to enhance accuracy and reliability in inhalation toxicity evaluation.