Complex Distribution Phenomena and Plastic Binding of Test Chemicals in Cell Culture Experiments: Exemplification by Tebufenpyrad.

Biokinetic complexities (plastic sorption, protein binding, and cellular accumulation) may cause large discrepancies between nominal and biologically effective concentrations of test compounds assessed by new approach methods (NAMs). This case study was performed to explore a generally applicable workflow that addresses biokinetic complexities in the context of NAM-based hazard testing for next-generation risk assessment (NGRA). The pesticide tebufenpyrad (TEBU) is a challenging test compound, as it (i) is hydrophobic, (ii) has an intracellular target (mitochondrial respiration), and (iii) is acting at low concentrations (susceptible to biokinetic complexities). In the newly established NeuriTox-M neurotoxicity assay, based on human dopaminergic (LUHMES) neuron cultures, TEBU showed toxic effects at 20 nM. Mass spectrometric analyses of various experimental setups showed that a large fraction (75% to >90%) of TEBU was adsorbed to plastic. This effect was strongly attenuated by albumin in the medium. Cells, cultured on plastic, were considered unsuitable to assess cellular uptake. Therefore, alternatives were explored: when cells were used as suspension cultures (3%