Development of an in vitro reproductive screening assay for novel pharmaceutical compounds

An in vitro reproductive cell‐based toxicity assay was developed using MLTC‐1 (murine Leydig tumour cell line) in order to examine the reproductive toxicity of two novel nanopharmaceutical compounds, namely ethylene glycol mono allyl ether and poly(ethylene glycol) octa‐functionalized polyhedral oligomeric silsesquioxane. Three commonly used cytotoxicity assays, namely the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H ‐tetrazolium bromide], MTS [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H ‐tetrazolium] and Crystal Violet assays, were compared, and the MTT assay proved to be the most accurate and reproducible for the MLTC‐1 cell line. The doubling rate of the MLTC‐1 cells was 30±3.5 h and the optimal seeding density for the MTT assay was 20000 cells per well, and the optimized MTT assay utilized a 4 h cell adherence followed by incubation with 0.5 mg/ml MTT for 1 h. The intra‐ and inter‐assay CV (coefficient of variation) values were 12.3 and 11% respectively. MLTC‐1 cells only produce the reproductive hormone progesterone in response to hCG (human chorionic gonadotropin), which stimulated progesterone production dose‐dependently from 0 to 100 m.i.u. (milliinternational units)/ml (2706±1118 ng/ml). H2O2 as a negative control killed 100% of cells at 1000 μg/ml. The two nanopharmaceutical compounds were cytotoxic at concentrations ≥0.1 μg/ml, but hCG decreased cytotoxicity to ≥1000 μg/ml (P