DOI: 10.1007/s43188-025-00315-x
Ji-Hyun Bang¹˒², Siu Lee¹, Nam-Ju Kim¹, Ji-Yeon Kim¹, Hyun-Ok Ku¹, Byung-Suk Jeon³, Je-Won Ko², Tae-Won Kim², Hee Yi¹
¹ Veterinary Drugs and Biologics Division, Animal and Plant Quarantine Agency (APQA), Gimcheon-si, Republic of Korea
² College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea
³ College of Korean Medicine, Dongshin University, Naju-si, Republic of Korea
Pesticides, biocides, and disinfectants used in veterinary medicine are often applied as sprays, which makes inhalation toxicity assessment particularly relevant. This study evaluates air–liquid interface (ALI) cultures as an in vitro approach for inhalation toxicity screening and investigates the effects of aerosolized benzalkonium chloride (BKC) on respiratory epithelial cell models.
A549 and Calu-3 cells were maintained under ALI conditions for three weeks. Transepithelial electrical resistance (TEER) measurements and hematoxylin and eosin staining confirmed barrier formation, cellular integrity, and multilayered epithelial organization. The cultures were then exposed to aerosolized BKC (2 to 16.5 mg/mL) using the VITROCELL® cloud system. Toxicity was quantified with WST-1, lactate dehydrogenase (LDH), and reactive oxygen species (ROS) assays, demonstrating concentration-dependent cytotoxicity. IC50 values indicated a higher sensitivity of Calu-3 cells compared with A549 cells, and LDH and ROS results supported substantial membrane damage and oxidative stress at higher doses.
Overall, the findings support ALI-based models as a promising platform for inhalation toxicity screening and highlight Calu-3 cells as a particularly responsive system for detecting BKC-induced injury. The authors also note that further work is needed to confirm reproducibility and to expand mechanistic endpoints to strengthen the predictive value of this approach.
