Serum-free in vitro assessment of receptor-mediated endocrine activity including Phase-1 metabolism

Endocrine disruptors, which pose a risk to organisms and entire ecosystems even at low concentrations, can be detected by standardized in vitro methods according to OECD test guidelines 455 and 458. However, these methods have disadvantages of containing undefined animal supplements like fetal bovine serum (FBS), possibly leading to less reproducible results and ethical concerns, and lacking metabolic representation of whole organisms, which could lead to an over- or underestimation of the samples’ endocrine activity. We addressed both aspects by developing a simple, cost-effective FBS-free medium for long-term culture of the cell lines ERα-CALUX® and AR-CALUX® and by integrating S9-homogenates (rat-S9, human S9, and their animal-free alternatives ewoS9R and ewoS9H) into OECD test procedures to simulate phase I mammalian metabolism. Both OECD test procedures could be adapted to FBS-free conditions, yielding results similar to the FBS-containing assays, though some of the assessed model compounds were only metabolized in FBS-free or FBS-containing medium. Additionally, we found that rat- and human S9 metabolized most of the compounds assessed, whereas ewoS9R and ewoS9H transformed fewer substances and thus do not yet represent equivalent alternatives. Maximum changes in the biologically equivalent concentrations of a 296-fold bioactivation and a 1,540-fold detoxification were observed, which can make a substantial difference for risk assessment. Overall, our study demonstrated that OECD TG 455 and 458 can be performed under FBS-free conditions and with S9-homogenates of different origins, enabling endocrine activity testing using less or no animal components while enhancing the prediction of the in vivo situation. Plain language summary Hormonally active substances can be detected by standardized cell-based experiments. However, these methods use undefined and animal-derived supplements like fetal bovine serum (FBS) and whole living body processes (like biotransformation of substances to higher or less hormonally active compounds) are not reflected. To address both aspects, we adapted two cell-based assays to FBS-free conditions and integrated products containing biotransformation enzymes (rat-S9, human S9, and their animal-free alternatives ewoS9R and ewoS9H). We found that test procedures conducted with and without FBS yielded similar results, though some of the assessed test chemicals were only biotransformed in FBS-free or FBS-containing medium. Additionally, we found that rat and human S9 transformed most of the compounds assessed, whereas ewoS9R and ewoS9H changed the hormonal activity of fewer substances. Overall, our study demonstrated that two standardized hormone assays were conductible using less or no animal components while also including processes happening in whole organisms.