Endocrine disruptors, which pose a risk to organisms and entire ecosystems even at low concentrations, can be detected by using standardized in vitro methods according to OECD test guidelines 455 and 458. However, these methods have disadvantages of (1) containing undefined animal supplements like fetal bovine serum (FBS) possibly leading to less reproducible results and ethical concerns and (2) lacking metabolic representation of whole organisms which could lead to an over- or underestimation of the samples' endocrine activity. In our study, we addressed both aspects by developing a simple, cost-effective FBS-free medium for culturing the OECD-relevant 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 mammalian metabolism. We found that 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 changed the endocrine activity of fewer substances. 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 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 also enhancing the prediction to the in vivo situation.
Reichstein et al. (Thu,) studied this question.