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Methylparaben was tested for its estrogenic activity using several in vitro assays:

- Methylparaben was assessed for its estrogenic activity by using the yeast two-hybrid assay incorporating either the human or medaka estrogen receptorαand by using hERαcompetitive enzyme-linked immunosorbent assay (ER-ELISA). Methylparaben did not show any estrogenic properties in the yeast two-hybrid assay (up to 10,000 nM) and ER-ELISA (up to 38,000 nM).

- The estrogenic activity of Methylparaben towards etstrogen receptorsαand ß was measured by using three reporter cell lines HELN, HELN ERαand HELN ERß. Methylparaben did not show any estrogenic activity when applied to HELN, HELN ERα and HELN ERß cells up to 10 µM.

- A validated estrogen receptor competitive-binding assay to determine the estrogen receptor (ER) affinity for Methylparaben was utilized. Uteri from ovariectomized Sprague-Dawley rats were the ER source for the competitive binding assay. Methylparaben was assayed using a wide range of concentrations (10 nM to 0.1 mM) to determine the relative binding affinity value (RBA). Methylparaben exhibited a weak binding to the ER (Relative Binding Affinity: 0.0004% of 17ß-Estradiol Binding Affinity). The calculated IC50(50% inhibition of the 17ß-Estradiol binding) was 0.25 mM compared to an IC50 of 0.9 nM for 17ß-Estradiol.

- The effect (competitive inhibition of [3H]Estradiol binding, expression of oestrogen-regulated genes) of Methylparaben on MCF7 human breast cancer cells was investigated. The binding of Methylparaben to the ER was rather weak, requiring a minimum concentration of 500,000-fold molar excess over 17ß-Estradiol. Where 17ß-Estradiol acts maximally between 10-10and 10-8M in MCF7 cells Methylparaben acts in 10-4M range. Methylparaben gave a very weak effect on cell proliferation at 10-4M. No significant antagonist properties of Methylparaben were found on cell proliferation stimulated by 10-10M 17ß-Estradiol for concentrations of Methylparaben in up to 105molar excess.

- MCF7 human breast cancer cells were grown for 7 days under conditions of oestrogen deprivation, sufficient time to deplete the oestrogen memory without development of loss of response. Gene expression was studied after a further 7 days with 0.5 mM Methylparaben or 17ß-Estradiol (10 nM, positive control). Methylparaben increased the cell growth. However, the extent of overlap in identity of the genes up- or downregulated by Methylparaben did in majority not follow the same pattern of regulation as under 17ß-Estradiol.

 Taking into account all above mentioned results the estrogenic properties of Methylparaben are negligible. The binding to the estrogenic receptor is very weak and was shown at 106molar excess compared to ß-Estradiol. This is an artificial concentration and very unlikely to occur within the organism since Methylparaben is demonstrated to be metabolized and excreted rapidly (please refer to the toxicokinetic results). Methylparaben was not found to be a 17ß-Estradiol antagonist. Therefore, no concern is arising from Methylparaben with reference to the estrogenic activity.