Analysis of in vitro hepatotoxicity mixtures data after chronic exposure to cosmetic related compounds

Purpose: With the ban of cosmetic products tested on animals in march 2013, the cosmetic industry has to put additional efforts in the development of alternatives to animal testing to assess the safety of its ingredients. Hepatotoxicity is a major concern as it is the main organ affected by repeated exposure to xenobiotics (Bitsch et al., 2006). It can be evaluated by cytotoxicity studies. These studies are traditionally quantified at a final time point after exposure. We propose to move to real time cell viability data measurements by impedance metrics. This permits to assess the temporal dimension of the appearance of effects, which improves the relevance of in vitro - in vivo extrapolations through toxicokinetics modeling. Here, we propose toxicokinetics/toxicodynamics (TK/TD) models to analyze real time data for HepaRG cells exposed repatedly to mixtures of hepatotoxic cosmetic related compounds. Methods: HepaRG cells were exposed for 4 weeks to the binary and ternary mixtures of coumarin, isoeugenol and benzophenone-2. The single compounds cell viabilities were first analyzed using TK/TD models. Then, the estimated parameters from these models were implemented in the mixture TK/TD models by either considering concentration additivity or independent action approaches. Finally, in order to extrapolate the in vitro cytotoxicity to the in vivo hepatotoxicity, human toxicokinetic models with parameters either predicted by QSAR models or referenced in literature were coupled with the in vitro TD models. Results and conclusions: We showed no interaction between the tested compounds relative to effects but interactions relative to metabolism, which influences the fate of the compounds in the exposure system. We also showed the relevance of the coupling of the human toxicokinetic models and the TK/TD models to assess risk under repeated exposure to mixtures of cosmetic related compounds.