Implementing the 3R initiative to reduce animal experiments in brain penetration prediction for CNS-targeting drugs requires more predictive in vitro and in silico models. However, animal studies are still indispensable to obtaining brain concentration and determining the prediction performance of in vitro models. To reveal species differences and provide reliable data for IVIVE, in vitro models are required. Systems overexpressing MDR1 and BCRP are widely used to predict BBB penetration, highlighting the impact of the in vitro system on predictive performance. In this study, endogenous Abcb1 knock-out MDCKII cells overexpressing MDR1 of human, mouse, rat or cynomolgus monkey origin were used. Good correlations between ERs of 83 drugs determined in each cell line suggest limited species specificities. All cell lines differentiated CNS-penetrating compounds based on ERs with high efficiency and sensitivity. The correlation between in vivo and predicted K_p,uu,brain was the highest using total ER of human MDR1 and BCRP and optimized scaling factors. MDR1 interactors were tested on all MDR1 orthologs using digoxin and quinidine as substrates. We found several examples of inhibition dependent on either substrate or transporter abundance. In summary, this assay system has the potential for early-stage brain penetration screening. IC₅₀ comparison between orthologs is complex; correlation with transporter abundance data is not necessarily proportional and requires the understanding of modes of transporter inhibition.

Veterinary ectoparasiticides widely used on companion animals often contain synthetic insecticides whose agricultural applications have been restricted in the European Union because of environmental concerns. These neurotoxic compounds can persist on animal fur and enter surrounding environments, potentially exposing non-target organisms. The Great Tit (Parus major), a common passerine in both urban and forest habitats, frequently incorporates animal fur in nest linings, thereby creating a potential exposure route for breeding adults and nestlings. We analysed fur-containing nest material collected from 63 Great Tit nests in artificial nest boxes at an urban site and a nearby protected forest in Hungary during the 2025 breeding season, sampling at the mid-nestling stage and also after fledging. Using HPLC-MS/MS and GC-MS, we detected several veterinary ectoparasiticides in nest materials, including fipronil, fipronil sulfone, imidacloprid, and permethrin. Acetamiprid was found only in urban nests, indicating additional, non-veterinary environmental sources. Multiple insecticides were present in nest material, with higher contamination levels and a greater number of detected compounds in urban compared to forest nests. Residues were present at both sampling times but declined over the course of the breeding cycle. Although contamination was not associated with the measured reproductive parameters of Great Tits, our findings show that veterinary ectoparasiticides can contaminate wild bird nests, including those in protected forest ecosystems. This highlights a previously under-recognised pathway linking companion animal treatments to wildlife exposure and underscores the need to assess the ecological risks and trade-offs associated with widespread veterinary insecticide use.