Next generation risk assessment of human exposure to estrogens using safe comparator compound values based on in vitro bioactivity assays.

In next generation risk assessment (NGRA), the Dietary Comparator Ratio (DCR) can be used to assess the safety of chemical exposures to humans in a 3R compliant approach. The DCR compares the Exposure Activity Ratio (EAR) for exposure to a compound of interest (EARtest) to the EAR for an established safe exposure level to a comparator compound (EARcomparator), acting by the same mode of action. It can be concluded that the exposure to a test compound is safe at a corresponding DCR ≤ 1. In this study, genistein (GEN) was selected as a comparator compound by comparison of reported safe internal exposures to GEN to its BMCL05, as no effect level, the latter determined in the in vitro estrogenic MCF7/Bos proliferation, T47D ER-CALUX, and U2OS ERα-CALUX assay. The EARcomparator was defined using the BMCL05 and EC50 values from the 3 in vitro assays and subsequently used to calculate the DCRs for exposures to 14 test compounds, predicting the (absence of) estrogenicity. The predictions were evaluated by comparison to reported in vivo estrogenicity in humans for these exposures. The results obtained support in the DCR approach as an important animal-free new approach methodology (NAM) in NGRA and show how in vitro assays can be used to define DCR values.

Individual and combined developmental toxicity assessment of bisphenol A and genistein using the embryonic stem cell test in vitro.

The potential developmental toxicity of environmental estrogenic endocrine disruptors have become a great concern in recent years. In this study, two typical environmental oestrogen, namely, bisphenol A (BPA) and genistein (GEN) were investigated for potential embryotoxicity using the embryonic stem cell test model. Afterwards, a 4×4 full factorial design and the estimated marginal means plot were performed to assess the combined effects of these two compounds. According to the linear discriminant functions and classification criteria, bisphenol A and genistein were classified as weakly embryotoxic and strongly embryotoxic respectively. As for combined effects, the overall interaction between BPA and GEN on embryonic stem cells (ESCs) differentiation was synergistic at low dosages, however, on ESCs and 3T3 cell proliferation, the predominate action was additive. Considering the actual daily intake of these chemicals, it is concluded that BPA alone might not have adverse reproductive or developmental effects on human being. However, given that BPA and GEN do have synergistic effect at low concentration, they may disturb normal embryo development together, which could result in birth defect and behavioral alterations later in life.

Validation study of OECD rodent uterotrophic assay for the assessment of estrogenic activity in Sprague-Dawley immature female rats.

The Organization for Economic Cooperation and Development (OECD) is developing a screening and testing method to identify estrogenic/antiestrogenic compounds. Based on these demands, phase 1 study for OECD uterotrophic assay was undertaken. The OECD is in the process of validating the assay results from international participating laboratories, which carried out this study with established environmental estrogenic compounds using designed protocols. The aim of this study was to provide data for validating the OECD uterotrophic assay using Sprague-Dawley immature female rats when testing with weak or partial estrogenic compounds. Ethinyl estradiol (EE) at 0.3 or 1 microg/kg/d, a positive control used in the present study, significantly increased both uterine wet and blotted weights. In the case of weak estrogenic compounds, the uterine wet weights were significantly increased by bisphenol A (BPA) at 300 mg/kg/d, nonylphenol (NP) at 80 mg/kg/d, genistein (GN) at 35 mg/kg/d, and methoxychlor (MXC) at 500 mg/kg/d. In addition, the increase in uterine blotted weights also showed a similar pattern to that of uterine wet weights. However, both 1,1,1-trichloro-2,2-bis(p-chlorphenyl)ethane (o,p-DDT) and dibutyl phthalate (DBP) did not affect uterus (wet and blotted) weights at doses of 100 and 500 mg/kg/d. These results suggest that the increase in uterine weights should be considered useful as a sensitive endpoint for detecting weak estrogenic compounds in 3-d rodent uterotrophic assay. However, further combination studies using surrogate biomarkers may be needed to improve the sensitivity of this assay for the detection of weak estrogenic compounds, such as o,p-DDT.