Serum-free in vitro assessment of receptor-mediated endocrine activity including Phase I 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 require the undefined animal-derived supplement fetal bovine serum (FBS), which is associated with ethical concerns and may lead to variable results, and they lack representation of metabolic processes, which can result in the over- or underestimation of a chemical’s 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 phase I mammalian metabolism by including S9-homogenates (rat-S9, human S9) or their animal-free alternatives (ewoS9R and ewoS9H) into test procedures. Both OECD test procedures could be adapted to FBS-free conditions, yielding similar results to the FBS-containing assays, though some of the assessed test compounds were only metabolized in FBS-free or FBS-containing medium. 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. The maximum changes in the biologically equivalent concentrations of 296-fold bioactivation and 1,540-fold detoxification indicate that including metabolism can make a substantial difference for risk assessment. Overall, our study demonstrates 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 fewer or no animal components while improving the prediction of the in vivo activity.