Air-liquid interface exposures for the assessment of aerosol and mixture toxicity

Toxicity testing of inhaled materials has typically involved in vivo animal exposures and/or in vitro exposures of submerged lung cell models. While such experimental work has provided important insight into hazard and mechanisms underlying adverse effects, these models have some significant limitations. For in vivo studies, these include the challenge of extrapolating across species, ethical questions, and cost. For conventional in vitro exposures, the submerged nature of the exposures complicates interpretation of dose and response, as it is often unclear how much material reaches the cells through the overlaying media, and submerged cells may respond differently due to the non-physiologically-relevant environment.

Recent advances are providing opportunities to conduct more physiologically-relevant exposures of human lung models. In air-liquid interface exposures, cells are cultured on an insert; they are fed from below by cell culture media, and can be exposed directly through the air. This enables delivery of airborne materials directly to lung models, facilitating determination of dose, and providing an exposure context that better mimics lung-environment interactions. However, delivery of test materials via the air and establishment and maintenance of conditions is technically challenging and requires considerable development and optimisation.

Here we provide an overview of our recent efforts to establish air-liquid interface exposure systems to assess the toxicity of inhaled particles, gases, and more complex mixtures. We show that air-liquid interface exposures enabled 1) detection of differential sensitivity to nanosized and micron-sized particles vs. little difference observed in submerged cell exposures; 2) assessment of ozone toxicity, vs. no effect in submerged cell exposures; and 3) evaluation of co-exposures to particles and gases indicating potential synergistic effects. Importantly, we established approaches to determine dose deposition, and show that real-time tracking of temperature, humidity, exposure concentrations, and dose delivered to the cells, enables dose-response relationships to be rigorously assessed under more physiologically-relevant conditions.

Collectively, assessing the strengths and limitations of these new approach methods for inhalation toxicity testing will increase confidence in their application to generate evidence in support of risk assessment initiatives.