The endocrine-disrupting activity of the synthesized bisphenols was assessed with two in vitro assays employing the MCF-7 and VM7LucE2 human mammary adenocarcinoma cell lines, expressing both human ER forms, ERα and ERβ (Figures and). The MCF-7 proliferation assay gauges the ability of evaluated bisphenols to mimic the activity of native 17β-estradiol (E2) in stimulating cell proliferation, while VM7LucE2 assay evaluates the affinity of bisphenols to the estrogen receptors relative to E2. In the first of the assays, the MCF-7 cells were treated with E2, BPA or the targeted bisphenols for 72 h at the concentrations covering the median bisphenols exposure levels in the population to 10 -4 M), and the estrogenic activities were determined as half-maximal effective concentration (EC50) for each of the evaluated compounds (Figure ). Additionally, a qualitative assessment of the influence of the tested bisphenols on cells proliferation and viability was used to visualize the endocrinedisrupting activity at each concentration (Figure ). Six of the evaluated bisphenols had minimal effect on cell proliferation (p,p'- and), while the others displayed EC50 values that were either higher or on par with BPA, that is promoting significant estrogen-driven cell proliferation. Subsequently, MCF7-derived VM7Luc4E2 cells, stably transfected with a human ER-responsive luciferase reporter gene, were used to evaluate binding and activation of ERα and ERβ by the bisphenol candidates. The estrogenic activity of bisphenols was calculated as the relative maximum %E2 (%RME2, a measure of response amplitude), i.e., the relative luminescence observed with the tested compound at a specific concentration relative to the maximum luminescence observed with E2 at the same concentration. According to the OECD test guideline No. 455, the %RME2 values less than 20% were considered insignificant. In this assay, BPA demonstrated a detectable response at almost all tested concentrations, whereas the majority of bisphenol candidates displayed lower estrogenic activity. Notably, three bisphenols displayed estrogenic activity at any concentration: p,p'-3, p,p'-5 and m,p'-5 (Figure ). An overlay of estrogenic activities from in silico and in vitro studies with the VM7LucE2 assay (using averaged values over the assessed concentration range) demonstrated that 18 out of 20 synthesized bisphenols were both predicted and experimentally measured to have lower activity compared to BPA (Figure ). This excellent correlation clearly demonstrates the capacity of the in silico model to provide valid predictions for this compound class, despite being a general model trained on a dataset with 6754 compounds of wide structural diversity including only 60 bisphenols. Only two bisphenolsp,p'-2 and p,p'-9stood out as outliers, demonstrating higher and lower predicted activities relative to BPA, respectively, while displaying opposite trend for activities in the in vitro assay.