Alternative air-liquid interface method for inhalation toxicity testing of a petroleum-derived substance

Abstract

In vitro-based new approach methodologies (NAMs) provide a pragmatic solution to animal testing of petroleum substances and their constituents. A previous study exposed an in vitro model (A549 cells) at the air-liquid interface (ALI) to assess inhalation toxicity of a single compound, ethylbenzene. Experimental conditions using VITROCELL^Ⓡ 24/48 exposure system were optimized to achieve a deposition efficiency that resulted in dose-dependent biological changes. The feasibility of this set-up was evaluated for testing the complex substance gasoline, which, at only high concentrations, can induce mild respiratory irritation in animals and cough in humans.•Results showed that perpendicular ALI exposure flow systems (VITROCELL® 6/4 and 24/48) may not be appropriate for testing gasoline because it was not possible to achieve enough deposition onto the cells and in the culture medium to measure dose and to determine dose-dependent biological changes (more information can be found in 'Supplementary material and/or Additional information' section).•Structural features (e.g. aromatic or saturated hydrocarbon structure) and high hydrophobicity, together with the low concentrations of individual components in gasoline, may have caused the low deposition.•To achieve a higher deposition on the cells, A549 cells were exposed to gasoline at the ALI by passive dosing.The results demonstrate that the presented methodology is a promising NAM for inhalation toxicity testing of (semi-)volatile complex substances with low aqueous solubility.

Keywords: Gasoline; In vitro inhalation testing; Passive dosing.

Graphical abstract

Fig. 1

Passive exposure of A549 cells using 2 Petri dishes filled with 6 ml gasoline each in a humidified desiccator in a climatic chamber at 37 °C.

Fig. 2

Set up for sorbent tube (arrow) sampling of desiccator headspace.

Fig. 3

Stainless steel inserts in stainless steel well plate in desiccator in climatic chamber for deposited dose determination.

Fig. 4

Change in cell viability (as % compared to CA) of A549 cells after 1 h passive exposure to gasoline (glass vial, 1 Petri dish, 2 Petri dishes, filled with 6 ml gasoline each) based on 5 runs (of which 3 independent experiments). Box and whisker plots visualizing the range of the individual data points per condition. The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the inter-quartile range, or distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Data beyond the end of the whiskers are called "outlying" points and are plotted with an “x”; other individual data points are overlayed and plotted with filled dots. The mean is indicated by a square.