Publication: Influence of in Vitro Assay Setup on the Apparent Cytotoxic Potency of Benzalkonium Chlorides

The nominal concentration, i.e. the theoretical concentration based on amount of test chemical added to culture medium, is generally used to express concentration-effect relationships in in vitro cytotoxicity assays. However, the nominal concentration does not necessarily directly represent the exposure concentration responsible for the observed effect at the target site in cells. Surfactants are expected to sorb to components in in vitro assays, yet there is limited knowledge about their sorption characteristics and how this might influence the apparent toxicity. This study aimed to demonstrate the effect of sorption to in vitro components on the observed cytotoxic potency of benzalkonium chlorides (BAQ) varying in alkyl chain length (6-18 carbon atoms) in a basal cytotoxicity assay with the rainbow trout gill cell line (RTgill-W1). Cells were exposed in 96 microtiter plates using serum-free exposure medium, or medium containing either Bovine Serum Albumin (BSA) (60 µM) or 10% fetal bovine serum (FBS). Before and after exposure, BAQ concentrations in exposure medium were analytically measured. Based on freely dissolved concentrations at the end of the test, longer alkyl chain length caused lower median effect concentrations (EC50) for C6-C14 benzalkonium compounds but EC50 values levelled off for C16-C18 benzalkonium compounds. For BAQ with ≥C12, calculated EC50 using measured concentrations in protein free medium were up to 25-fold lower when compared with EC50 calculated using nominal concentrations. When BSA or FBS was added to the medium, a decrease in cytotoxic potency was observed already for C8-benzalkonium and ranged up to 22-fold for long alkyl chain benzalkonium chlorides. Based on the results of this study, we recommend to quantify the in vitro toxic readout using a dose metric that is least influenced by assay setup, such as the measured free or cell-associated concentration, to facilitate the comparison of in vitro effect potencies from different assays and in vitro in vivo extrapolation.

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