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

Abstract

The nominal concentration is generally used to express concentration–effect relationships in in vitro toxicity assays. However, the nominal concentration does not necessarily represent the exposure concentration responsible for the observed effect. Surfactants accumulate at interphases and likely sorb to in vitro system components such as serum protein and well plate plastic. The extent of sorption and the consequences of this sorption on in vitro readouts is largely unknown for these chemicals. The aim of this study was to demonstrate the effect of sorption to in vitro components on the observed cytotoxic potency of benzalkonium chlorides (BAC) varying in alkyl chain length (6–18 carbon atoms, C6–18) in a basal cytotoxicity assay with the rainbow trout gill cell line (RTgill-W1). Cells were exposed for 48 h in 96-well plates to increasing concentration of BACs in exposure medium containing 0, 60 μM bovine serum albumin (BSA) or 10% fetal bovine serum (FBS). Before and after exposure, BAC concentrations in exposure medium were analytically determined. Based on freely dissolved concentrations at the end of the exposure, median effect concentrations (EC50) decreased with increasing alkyl chain length up to 14 carbons. For BAC with alkyl chains of 12 or more carbons, EC50’s based on measured concentrations after exposure in supplement-free medium were up to 25-times lower than EC50’s calculated using nominal concentrations. When BSA or FBS was added to the medium, a decrease in cytotoxic potency of up to 22 times was observed for BAC with alkyl chains of eight or more carbons. The results of this study emphasize the importance of expressing the in vitro readouts as a function of a dose metric that is least influenced by assay setup to compare assay sensitivities and chemical potencies.

Biotransformation Potential of Cationic Surfactants in Fish Assessed with Rainbow Trout Liver S9 Fractions

“…Similar calculations for T14, P16, S16, T16, T18, Q14, DDAC, and C 12‐16 ‐benzalkonium yield BCF baseline estimates that exceed empirical BCFs and BAFs by factors of 3.1, 70, 49, 42, 128, 33, 420, and 420, respectively. These lower‐than‐predicted empirical BCFs and BAFs may be due to problems with the measurements themselves, including issues associated with field sampling, such as the movements of sampled animals and overestimated bioavailability of aqueous chemical concentrations (Chen et al, 2014; Groothuis et al, 2019). Limitations on diffusive flux of permanently charged QACs across fish gills also may prolong the time to steady state or prevent systemic uptake entirely (Kierkegaard et al, 2020).…”

Characterization of dermal sensitization potential for industrial or agricultural chemicals with EpiSensA

“…In addition, EC200 and CV75 were highly correlated with methacrylate esters (Figure 1). It has been reported that alkyl chain length can correlate with cytotoxicity of chemicals based on data with benzalkonium chloride with various alkyl chain length (Groothuis et al, 2019). From this information, the positive response of h‐CLAT in this study may be influenced by cytotoxicity derived from alkyl chain length of methacrylate esters.…”

IVIVE: Facilitating the Use of In Vitro Toxicity Data in Risk Assessment and Decision Making

“…Non-specific migration to plastics and binding to media constituents (for example, serum proteins, lipids) have been documented, and test chemicals may evaporate, degrade, or be metabolized. All these factors can result in an underestimation of potency [ 121 ]. This information can be obtained by measuring chemical distribution in different tissue compartments or by mathematical model predictions.…”