## 2 Materials and Methods
Briefly, MCF-7 cloned cancer cells were cultured for routine maintenance in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS) (BioWittaker, Walkersville, MD, USA) in a 5% CO\({}_{2}\)/95% air atmosphere under saturated humidity at 37 \({}^{\circ}\)C. We subcultured cells at weekly intervals using a mix of 0.05% trypsin and 0.01% EDTA. Sex steroids were eliminated using charcoal and dextran extraction from the serum. For this purpose, a 5% charcoal suspension (Norit A, Sigma Chemical Co., St. Louis, MO, USA) was prepared with 0.5% dextran T-70 (Pharmacia-LKB, Uppsala, Sweden). Similar volume aliquots of the charcoal and dextran suspension were centrifuged at 1000\(\times\)\(g\) for 10 min. The supernatant was removed, and the serum aliquots were mixed with the charcoal granules. This charcoal/serum mixture was kept in suspension by rolling for six cycles min\({}^{-1}\) at 37 \({}^{\circ}\)C for 1 h. The suspension was then centrifuged at 1000\(\times\)\(g\) for 20 min, and the supernatant was filtered through a 0.22 mm filter (Millipore, Billerica, MA, USA). Bovine and human sera treated with carbon dextran (CDFBS and CDHuS, respectively) were stored at \(-\)20 \({}^{\circ}\)C until needed; MCF7 cells were used for the estrogenicity test following a slight modification of the original technique [53]. Briefly, cells were trypsinized and seeded in 24-well plates (Limbro, McLean, VA, USA) at initial concentrations of 0.22 mm and 0.22 mm (Millerpore, Billerica, Billerica, MA, USA). At initial concentrations of 20,000 cells per well in 5% FBS in DME, the cells were allowed to settle for 24 h, and then the seeding medium was replaced by DME without phenol red supplemented with 10% CDFBS or CDHuS. Various concentrations of phytoestrogen products were added, and the test was terminated after 144 h by withdrawing the medium from the wells, fixing the cells, and staining with sulphorhodamine-B (SRB), as previously described elsewhere [17; 45]. The Linearity of the SRB test according to cell number was checked before the cell growth experiments. The 100% proliferative effect (PE) was estimated as the relationship between the greatest cell yield achieved with 50 pM estradiol and the proliferation of control cells without hormones. Each phytoestrogen was tested in triplicate with a negative (vehicle) and positive (50 pM estradiol) control on each plaque. The PE of the standard phytoestrogen was referenced to the maximum PE achieved with estradiol, converted to estradiol equivalent units (Eeq) and expressed as nM concentration reading from a dose-response curve generated with estradiol (concentration range = 0.1 pM to 10 nM). The average cell number did not differ significantly from that of the steroid-free controls at concentrations <1 pM estradiol, equivalent to 1 fmol in 1 mL of culture medium. Thus, 1 fmol of estradiol per well was the minimum detectable estrogen level in this test. Concerning cell proliferation in the E-screen bioassay for the listed compounds, MCF-7 cells were prepared with these compounds at the indicated concentrations. Results are reported as the highest cell multiplication rate caused by the test sample (proliferative effect). This was measured as the ratio between the maximal proliferation rate obtained for the test sample and the multiplication rate achieved by the negative control. SPSS v.22.0 (IBM, Chicago, IL, USA) was applied to the statistical analysis. A descriptive analysis was performed to obtain means, standard deviations (SDs), medians, maximum and minimum values, and stepwise regression. \(p\) < 0.050 was assumed to be significant. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement was used throughout the study to guide the writing of this article.