From in vitro permeability to in vivo brain exposure: continued validation of a human iPSC-derived blood-brain barrier model with pesticides and stimulants

• Human blood-brain barrier (BBB) model advances prediction of in vivo permeability • High correlation of in vitro permeability to clinical stimulant brain penetration • BBB model displays in vivo -like P-gp functionality • Predictive BBB model reveals novel in vivo brain penetration data for pesticides Central nervous system exposure is a key condition for compounds to trigger neurotoxicity in the human brain. Consequently, the blood-brain barrier (BBB) is a critical determinant for brain penetration of substances and their potential to be neurotoxic. For the emerging integration of new approach methodologies (NAM) into regulatory risk assessment, in vitro BBB permeability has been derived as an important proxy for in vivo brain exposure. As until today no consensus BBB NAM is available, the European Partnership for the Assessment of Risks from Chemicals (PARC) has highlighted the need for a first-generation model for adult neurotoxicity. Therefore, the first goal of this study was to continue the characterization of our previously published human iPSC-derived BBB model and permeability testing method with a specific focus on carbohydrate metabolism and P-gp functionality. The second goal was to investigate the permeability of pesticides and stimulants to promote in vivo-in vitro model validation. Furthermore, novel brain penetration information on substances with public neurotoxicity implications should be derived. Based on new stimulant data, the results indicated an advanced suitability of the BBB model to predict human in vivo brain penetration (R 2 = 0.9327). With thousands of chemicals being marketed despite unknown brain penetration, this underlines a much-needed application domain of our model in future regulatory neurotoxicity assessment. In this context, applying the model to selected pesticides provided new insights into the potential brain exposure of neurotoxic compounds such as Atrazine, for which no human brain penetration data had previously been available. From in vitro permeability to in vivo brain exposure: Continued validation of a human iPSC-derived blood-brain barrier model with pesticides and stimulants. Created with Biorender.com