Characterization, modification and applicability of an in vitro placental transfer assay for screening purposes P92 Caroline Gomes*; Catharina W. van Dongen; Barbara Birk; Eric Fabian; Julian Doersam; Bennard van Ravenzwaay; Robert Landsiedel Experimental Toxicology and Ecology, BASF SE *Contact: caroline.a.gomes@basf.com Ø Alternative methods to animal testing are being developed to address developmental toxicity and the placental transfer is essential to determine the potential embryo toxic effects of a substance. Ø The objective of this study was to improve and characterize the methodology of this model, and to assess the placental transfer of 7 substances to assess the model applicability. Introduction Figure 1. Schematic overview of in vitro BeWo transfer assay. Ø An in vitro placental transfer assay using the human trophoblastic cell line, BeWo b30 clone, has been developed using a transwell system (Figure 1) and showed good correlations with ex vivo data1,2 Methodology BeWo cells b30 clone (Addexbio, USA) were cultivated in the “regular condition” which refers to the methodology described in literature1. An optimization was tested to avoid refreshing the cell culture medium every day (Figure 2). The parameters used to check the barrier integrity, cell growth or cytotoxicity of the control substances are summarized in Figure 2. The Papp values were calculated by the equation: !"## (%&/() = ∆,/∆- .∗01 where ΔQ is the amount of compound entered in the basolateral compartment (nmol), Δt is the time (seconds) of the transfer experiment, A is the cell surface area in cm2 of the insert and C0 the exposure concentration (µM). Figure 2. Methodology scheme of the in vitro placental transfer using the BeWo b30 cell line. The parameters assessed overtime or at the testing transfer day (day 6) are numbered from 1 to 4. Fluorescein was measured by spectrophotometer. Amoxicillin and antipyrine were determined by LC-MS analysis at Pharmacelsus GmbH, we are grateful to Dr. Ursula Müller-Vieira. TEER: trans-epithelial electrical resistance. WPI: World Precision Instruments (USA). Results: characterization and optimization Figure 3. TEER measurements of BeWo b30 cell layer from day 3 to day 7 after seeding (A). Fluorescein transfer in the in vitro placental transfer model from day 3 to 7 after seeding (B). Data are shown as mean ± standard deviation of triplicates per experiment. Substance Ex vivo placental transfer index In vitro placental transfer Relative Papp Papp (10-6 cm/s) Recovery (%) Acyclovir 0.327 0.41 18 ± 1.2 128 Caffeine 0.958 1.34 59 ± 11 103 Cimetidine 0.469 0.30 13 ± 0.9 109 0 10 20 30 40 50 60 70 2 3 4 5 6 7 8 TEER (Ω·cm2) Days (post-seeding) 0 2 4 6 8 10 12 14 16 18 20 2 4 6 8 Papp value (x10-6 cm/s) Days (post-seeding) Run 1 - Regular Run 1 - Optimized Run 2 - Regular Run 2 Optimized Run 3 - Optimized 15 25 35 45 55 65 Papp value (x10-6 cm/s) Antipyrine Li et al., 2013 (1) Li et al., 2015 (3) Li et al., 2016 (4) Kloet et al., 2015 (5) Strikwold et al., 2017 (6) Run 1 - Regular Run 1 - Optimized Run 2 - Regular Run 2 - Optimized Run 3 - Optimized Results: model applicability Methodology The applicability of the model was assessed by: a) Comparing the in vitro relative Papp values of 3 substances (caffeine, acyclovir and cimetidine) with data from the ex vivo human placental transfer assay in the optimized condition b) Assessing the in vitro placental transfer of 4 azoles (difenoconazole, flusilazole, miconazole and triadimefon) in the regular condition R² = 0,9913 R² = 0,5991 R² = 0,9945 R² = 0,9742 0 2 4 6 8 10 0 20 40 60 80 100 Basolateral amount (nmol) Time (minutes) Flusilazole Miconazole Difenoconazole Triadimefon 0 5 10 Papp value (x10-6 cm/s) Amoxicillin Regular Optimized Whole insert Diverse layers Uniform layers Figure 4. Papp values of the permeability controls, Antipyrine and Amoxicillin, determined at day 6 (at 60 minutes) in the in vitro placental transfer model compared to literature. Data are shown as mea