Human in vitro airway models to assess point-of-contact respiratory toxicity of surfactants: development and evaluation of a repeated-exposure study design

Repeated-dose exposure studies can provide insights into the toxicity of test substances, including dose-response relationships, cumulative effects, delayed responses, and reversibility of effects. A limited number of in vitro studies have assessed the effects of repeated exposure of respiratory cells to chemicals applied as liquids (via direct pipetting). The work presented in this paper builds upon these published in vitro repeated-dose studies to develop and evaluate a repeat-exposure study design for use with reconstructed human respiratory epithelium (RHRE). To this end, a bronchial RHRE (MucilAir, Epithelix) was exposed to 30 µL (91 µL/cm²) of two surfactants-oleoyl sarcosine and Triton X-100-for 4 hours on up to 4 consecutive days. The results showed that the repeat-exposure protocol can be used to detect toxic effects, including differences in the responses caused by each surfactant. More specifically, while the effects of oleoyl sarcosine exposure increased over 4 days, the effects of Triton X-100 were the strongest on day 1 and decreased over the course of the experiment. This work helped refine a repeated-dose protocol that can be adapted to assess other chemistries when the effects of repeated exposure are of interest, and underscores the importance of key parameters, such as endpoint selection, timepoints, exposure duration, and washing schedules.