Quantification of freely dissolved effect concentrations in in vitro cell-based bioassays

Improved understanding of chemical exposure in in vitro bioassays is required for quantitative in vitro-in vivo extrapolation (QIVIVE). In this study, we quantified freely dissolved concentrations in medium sampled from in vitro cell-based bioassays (C_free,medium) for nine chemicals with different hydrophobicity and speciation at the time point of dosing and after an incubation period of 24 h using solid-phase microextraction. The chemicals were tested in two reporter gene assays, the AREc32 assay indicative of the oxidative stress response and the PPARγ-GeneBLAzer assay that responds to chemicals which bind to the peroxisome proliferator-activated receptor gamma. For seven of the nine chemicals, C_free,medium did not change significantly over time in both assays and the experimentally determined C_free,medium generally agreed well with predictions of a mass balance model that describes the partitioning between proteinaceous and lipidous medium constituents, cells and the aqueous phase. Two chemicals showed a decrease of C_free,medium in the AREc32 assay over time that was probably caused by cellular metabolism. Furthermore, C_free,medium of the acidic chemical diclofenac deviated from the model predictions by more than a factor of 10 at higher concentrations, which indicates nonlinear binding and saturation of the medium proteins. Bioassay results are typically reported as nominal effect concentrations (EC_nom), although it is established that freely dissolved effect concentrations (EC_free) are a better measure for the bioavailable dose and the method developed here provides a simple experimental approach to measure and model EC_free in in vitro bioassay for improved QIVIVE models.