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Human biomonitoring studies typically capture only a small and unknown fraction of the entire chemical universe. We combined chemical analysis with a high-throughput in vitro assay for neurotoxicity to capture complex mixtures of organic chemicals in blood. Plasma samples of 624 pregnant women from the German LiNA cohort were extracted with a nonselective extraction method for organic chemicals. 294 of >1000 target analytes were detected and quantified. Many of the detected chemicals as well as the whole extracts interfered with neurite development. Experimental testing of simulated complex mixtures of detected chemicals in the neurotoxicity assay confirmed additive mixture effects at concentrations less than individual chemicals' effect thresholds. The use of high-throughput target screening combined with bioassays has the potential to improve human biomonitoring and provide a new approach to including mixture effects in epidemiological studies.

The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly relevant for human- and environment-related risk assessment. To examine the effects on a mechanism-specific level, standardized 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 genotoxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ERα-CALUX® and GeneBLAzer™ ERα-UAS-bla GripTite™ (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 chemical analysis. All cell lines (A549, ERα-CALUX® and GeneBLAzer™ Erα-UAS-bla GripTite™) were successfully cultivated in chemically 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 defined 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 ERα-CALUX® 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., Competing Interests: Declaration of competing interest The corresponding author declares that Andreas Schiwy, Beat Thalmann and Henner Hollert are co-founders of EWOMIS GmbH, a company aimed to commercialise biotechnological metabolisation systems. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Discussions are ongoing on which dose metric should be used for quantitative in vitro -to- in vivo extrapolation (QIVIVE) of in vitro bioassay data. The nominal concentration of the test chemicals is most commonly used and easily accessible, while the concentration freely dissolved in the assay medium is considered to better reflect the bioavailable concentration but is tedious to measure. The aim of this study was to elucidate how much QIVIVE results will differ when using either nominal or freely dissolved concentrations. QIVIVE nom and QIVIVE free ratios, that is, the ratios of plasma concentrations divided by in vitro effect concentrations, were calculated for 10 pharmaceuticals using previously published nominal and freely dissolved effect concentrations for the activation of the peroxisome proliferator-activated receptor gamma (PPARγ) and the activation of oxidative stress response. The QIVIVE nom ratios were higher than QIVIVE free ratios by up to a factor of 60. The risk of in vivo effects was classified as being high or low for four chemicals using the QIVIVE nom and for three chemicals using QIVIVE free ratios. Unambiguous classification was possible for nine chemicals by combining the QIVIVE nom or QIVIVE free ratios with the respective specificity ratios (SR nom or SR free ) of the in vitro effect data, which helps to identify whether the specific effect was influenced by cytotoxicity. QIVIVE free models should be preferred as they account for differences in bioavailability between in vitro and in vivo , but QIVIVE nom may still be useful for screening the effects of large numbers of chemicals because it is generally more conservative. The use of SR of the in vitro effect data as a second classification factor is recommended for QIVIVE nom and QIVIVE free models because a clearer picture can be obtained with respect to the likelihood that a biological effect will occur and that it is not caused by nonspecific cytotoxicity.