Abstract
While in vitro testing is used to identify hazards of chemicals, nominal in vitro assay concentrations may misrepresent potential in vivo effects and do not provide dose–response data which can be used for a risk assessment. We used reverse dosimetry to compare in vitro effect concentrations-to-in vivo doses causing toxic effects related to endocrine disruption. Ten compounds (acetaminophen, bisphenol A, caffeine, 17α-ethinylestradiol, fenarimol, flutamide, genistein, ketoconazole, methyltestosterone, and trenbolone) have been tested in the yeast estrogen screening (YES) or yeast androgen-screening (YAS) assays for estrogen and androgen receptor binding, as well as the H295R assay (OECD test guideline no. 456) for potential interaction with steroidogenesis. With the assumption of comparable concentration–response ratios of these effects in the applied in vitro systems and the in vivo environment, the lowest observed effect concentrations from these assays were extrapolated to oral doses (LOELs) by reverse dosimetry. For extrapolation, an eight-compartment Physiologically Based Toxicokinetic (PBTK) rat model based on in vitro and in silico input data was used. The predicted LOEL was then compared to the LOEL actually observed in corresponding in vivo studies (YES/YAS assay versus uterotrophic or Hershberger assay and steroidogenesis assay versus pubertal assay or generation studies). This evaluation resulted in 6 out of 10 compounds for which the predicted LOELs were in the same order of magnitude as the actual in vivo LOELs. For four compounds, the predicted LOELs differed by more than tenfold from the actual in vivo LOELs. In conclusion, these data demonstrate the applicability of reverse dosimetry using a simple PBTK model to serve in vitro–in silico-based risk assessment, but also identified cases and test substance were the applied methods are insufficient.
Highlights
The introduction of the “3Rs” principle, reduction, refinement and replacement, by Russell and Burch (1959), was instrumental in the development of alternative methods to animal experimentation
Test compounds were selected from a panel of compounds previously tested in steroidogenesis and yeast estrogen screening (YES)/yeast androgen-screening (YAS) in vitro assays to detect the potential for endocrine disruption (Kolle et al 2012)
Results from Physiologically Based Toxicokinetic (PBTK) modelling and lowest observed effect concentration (LOEC) from in vitro experiments were used for in vivo extrapolation (IVIVE)
Summary
The introduction of the “3Rs” principle, reduction, refinement and replacement, by Russell and Burch (1959), was instrumental in the development of alternative methods to animal experimentation. One of the goals is to replace animal testing for toxicological hazard assessment and ultimate for risk assessment. The overall toxicity of a compound in an in vivo organism is unlikely to be accurately reflected in a single stand-alone replacement model; rather, a battery of tests is required, such as those in the recently regulatory adopted strategy for skin sensitization (Bauch et al 2011). Knowledge of physiological and toxicological pathways has allowed the development of adverse outcome pathways (AOPs), which describe the biological key events leading to an adverse outcome in vivo (Vinken 2013)
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