An in vitro air-liquid interface inhalation platform for petroleum substances and constituents

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Sandra Verstraelen
An Jacobs
Jo Van Laer
Karen Hollanders
Masha Van Deun
Diane Bertels
Rob Brabers
Hilda Witters
Sylvie Remy
Lieve Geerts
Lize Deferme
Evelien Frijns


The goal is to optimize and show the validity of an in vitro method for inhalation testing of petroleum substances and its constituents at the air-liquid interface (ALI). The approach is demonstrated in a pilot study with ethylbenzene (EB), a mono-constituent petroleum substance using a human alveolar epithelial cell line model. This included the development and validation of a generation facility to obtain EB vapors and the optimization of an exposure system for a negative control (clean air, CA), positive control (nitrogen dioxide), and EB vapors. The optimal settings for the VITROCELL® 24/48 system were defined. Cytotoxicity, cell viability, inflammation, and oxidative stress were assessed in A549 after exposure to EB vapors. A concentration-dependent significant decrease in mean cell viability was observed after exposure, which was confirmed by a cytotoxicity test. The oxidative stress marker superoxide dismutase 2 was significantly increased, but no concentration-response was observed. A concentration-dependent significant increase in pro-inflammatory markers C-C motif chemokine ligand 2, interleukin (IL)6, and IL8 was observed for EB-exposed A549 cells compared to CA. The data demonstrated consistency between in vivo air concentrations at which adverse respiratory effects were observed and ALI-concentrations affecting cell viability, provided that the actual measured in vitro delivery efficiency of the compound were included. It can be concluded that extrapolating in vitro air concentrations (adjusted for delivery efficiency and absorption characteristics and applied for testing cell viability) to simulate in vivo air concentrations may be a promising method to screen for acute inhalation toxicity.

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Verstraelen, S., Jacobs, A., Van Laer, J., Hollanders, K., Van Deun, M., Bertels, D., Brabers, R., Witters, H., Remy, S., Geerts, L., Deferme, L. and Frijns, E. (2021) “An in vitro air-liquid interface inhalation platform for petroleum substances and constituents”, ALTEX - Alternatives to animal experimentation. doi: 10.14573/altex.2010211.

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