Replacing animal-derived components in in vitro test guidelines OECD 455 and 487

The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly rel-evant for human-and environment-related risk assessment. To examine the effects on a mechanism-specific level, stan-dardized cell-based in vitro methods are widely applied. However, these methods include animal-derived components like fetal bovine serum (FBS) or rat-derived liver homogenate fractions (S9-mixes), which are a source of variability, reduced assay reproducibility and ethical concerns.In our study, we evaluated the adaptation of the cell-based in vitro OECD test guidelines TG 487 (assessment of geno-toxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ER alpha-CALUX (R) and GeneBLAzer'degrees ER alpha-UAS-bla GripTite'degrees (for OECD TG 455) were investigated for growth in a chemically defined medium without the addition of FBS. Secondly, the biotechnological S9-mix ewoS9R was implemented in comparison to the induced rat liver S9 to simulate in vivo metabolism capacities in both OECD test guidelines. As a model compound, Benzo[a]pyrene was used due to its increased genotoxicity and endocrine activity after metabolization. The metabolization of Benzo[a]Pyrene by S9-mixes was examined via chem-ical analysis.All cell lines (A549, ER alpha-CALUX (R) and GeneBLAzer'degrees Er alpha-UAS-bla GripTite'degrees) were successfully cultivated in chemi-cally defined media without FBS. The micronucleus assay could not be conducted in chemically defined medium due to formation of cell clusters. The methods for endocrine activity assessment could be conducted in chemically de-fined media or reduced FBS content, but with decreased assay sensitivity. The biotechnological ewoS9R showed potential to replace rat liver S9 in the micronucleus in FBS-medium with A549 cells and in the ER alpha-CALUX (R) assay in FBS-and chemically defined medium.Our study showed promising steps towards an animal-component free toxicity testing. After further improvements, the new methodology could lead to more reproducible and reliable results for risk assessment.

Automated summary

The evaluation of genotoxic or estrogenic potential in substances or samples is important for risk assessment. This study explored the use of animal-component-free methods to assess this potential, showing promising results and potential for more reproducible and reliable risk assessment.