Phenytoin

Methacrylic acid (MAA), N, N’-methylenebisacrylamide (MBAA) and acrylamide (AAm), ethylene glycol dimethacrylate (EDMA), vancomycin hydrochloride, phenobarbital sodium salt, phenytoin, caffeine anhydrous, theophylline, meropenem trihydrate, water-soluble carbodiimide (WSC), anhydrous sodium sulphate and sodium chloride were bought from Wako Pure Chemical Industry (Osaka, Japan). Methanol and N, N-dimethylformamide (DMF) were purchased from Kanto Chemical Co., Ltd. (Tokyo, Japan). The 3-Ferrocenoylpropyonic acid and allylamine were purchased from Tokyo Chemical Industry (Tokyo, Japan). Imipenem monohydrate was obtained from Combi-blocks (San Diego, CA, USA), and teicoplanin was obtained from Biodivision (Tokyo, Japan). Bovine blood for testing was bought from the Tokyo Shibaura Zoki Corporation (Tokyo, Japan) (5 g/L sodium citrate was added into the blood as an anticoagulant.) MAA and EDMA were used after purification by reduced-pressure distillation. Spherical graphite particles, 8 µm in diameter (SG-BH8), were donated by Ito Graphite Co., Ltd. (Kuwana, Japan).

Test compounds used in this study are listed in Table 1. These tested compounds are known to be teratogens inducing cleft palate in mammals and have been classified into various categories as a result of being tested in zebrafish experiments or chemical safety assays (Hillegass et al., 2008 (link); Selderslaghs et al., 2009 (link); Ito and Handa, 2012 (link); Lee et al., 2012 (link); Teixido et al., 2013 (link); Yamashita et al., 2014 (link); Inoue et al., 2016 (link); Martinez et al., 2018 (link); Cassar et al., 2019 (link)). The test compounds and exposure concentrations were determined based on Liu et al., 2020 (link). The exposure concentrations were as follows: hydroxyurea (1 mM, Sigma-Aldrich), valproic acid (7.5–30 μM, Wako), salicylic acid (100–400 μM, Wako), boric acid (1 mM, Wako), and caffeine (0.5–2 mM, Wako), which were diluted from stock solutions prepared with distilled water (Life Technologies), and imatinib (250 μM, Tokyo Chemical Industry), retinoic acid (10–50 nM, Tokyo Chemical Industry), thalidomide (400 μM, Tocris Bioscience), methotrexate (50–200 μM, Wako), warfarin (15–60 μM, Wako), phenytoin (1 mM, Wako), dexamethasone (1 mM, Wako), 5-fluorouracil (1 mM, Wako), and isoniazid (1 mM, LKT Laboratories), which were diluted from stock solutions prepared with dimethyl sulfoxide (DMSO, Wako).