Advanced in vitro exposure systems.

An In Vitro Air-Liquid Interface Inhalation Platform for Petroleum Substances and Constituents

20. Apr. 2021

DOI: 10.14573/altex.2010211 

Sandra Verstraelen1, An Jacobs1, Jo Van Laer1, Karen Hollanders1, Masha Van Deun2, Diane Bertels2, Rob Brabers1, Hilda Witters1, Sylvie Remy1,3, Lieve Geerts1, Lize Deferme4 and Evelien Frijns1
1VITO NV (Flemish Institute for Technological Research), Unit HEALTH, Mol, Belgium; 
2VITO NV, Unit Separation and Conversion Technology (SCT), Mol, Belgium; 
3University of Antwerp, Department of Biomedical Sciences, Antwerp, Belgium; 
4ExxonMobil Petroleum and Chemical BV, Machelen, Belgium

This pilot study exposed a frequently used in vitro model (A549 cells) at the ALI to assess inhalation toxicity of the single compound EB. Experimental conditions using the VITROCELL® 24/48 exposure system were optimized to achieve a (low) delivery efficiency that resulted in dose-dependent biological changes. The data demonstrate consistency in effect levels when comparing cell viability in the ALI experiments with known in vivo non-lethal effects in humans. It can be concluded that QIVIVE from in vitro air concentrations applied for testing cell viability to in vivo air concentrations may be a promising method for screening for acute inhalation toxicity. 


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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