David H. Brandwein, F. Adam Bettmann, Michael P. DeLorme, Alan T. Eveland, Lawrence M. Milchak
3M Corporate Toxicology and Environmental Science, St. Paul, MN
The STL is working to develop an in vitro screening ALI model to assess the acute respiratory irritation potential for new chemicals. These experiments examined multiple aspects of the model, including different cell culture systems (A549 and EpiAirway), different exposure methods (dynamic vapor and liquid phase), and different post exposure periods, all using acrolein as a model respiratory irritant. The goal was to better understand the critical parameters of the cell systems and exposure methods to enable the development of a consistent screening model, while gaining clarity of the dosimetry.
The 3M Strategic Toxicology Laboratory (STL) is an internal corporate resource that emphasizes the use of in vitro methodology when providing support to 3M businesses. The STL is investigating vapor and liquid phase exposure to cell cultures at the air-liquid interface (ALI) as an animal alternative assay to help assess respiratory toxicity. This study describes the use of acrolein, a potent respiratory toxicant, as a model test substance. Vapor atmospheres were generated using a Vitrocell® 12/12 system. A549 cells were seeded into Transwell® inserts, grown submerged for 1 week then raised to the ALI for an additional 7-14 days, creating cultures robust to the clean-air negative control conditions of the Vitrocell apparatus. EpiAirway™ tissues were purchased from MatTek. All exposures were carried out for three hours and viability assessed immediately or 20 – 24 hours post exposure. At the vapor phase, A549 cell viability was reduced to 58% (14.6 ppm) and 91% (4.6 ppm) compared with the Vitrocell clean-air control group, with a further decrease in each case of approximately 20% following the post-exposure period. Cell viability was not affected at the 1.46 ppm level. In the liquid phase, EpiAirway cultures were exposed to acrolein in corn oil ranging from 0.01 - 3,000 ug/mL. Viability decreased at all concentrations greater than 10 ug/mL. At 1,000 ug/mL, viability was reduced to 48% immediately and 4% 20 hours post exposure. A549 cultures were also exposed to acrolein dilutions in DPBS in the same range as EpiAirway and followed a similar viability profile. Both modes of exposure, vapor and liquid, demonstrated dose dependent effects on viability. However, the concentrations of acrolein required to decrease viability were much lower with vapor exposure than with direct liquid exposure. The liquid exposure level (1000 ug/ml) which produced approximately 50% reduction in viability (EC50) converts to over 400,000 ppm in air, whereas 14.6 ppm vapor exposure reduced viability to approximately 56%. Further experiments are required to better understand the discrepancy in the liquid phase and vapor phase levels required to achieve the EC50 and to identify the most appropriate comparison method, such as concentration or mass/surface area. Understanding these differences will be critical to achieve the best utilization of these in vitro methods as a tool for respiratory toxicity assessment.