Comprehensive estrogenicity assessment of 4-methylbenzophenone via in vivo, in vitro, and in silico approaches within an integrated testing strategy framework.

4-Methylbenzophenone (4-MBP) is extensively used as a photoinitiator in ultraviolet-cured printing inks and food packaging. This study comprehensively evaluated its estrogenic activity using an Integrated Testing Strategy (ITS) incorporating in vivo, in vitro, and in silico methodologies. Repeated oral administration of 4-MBP at 300 mg/kg (lowest observed effect concentration, LOEC) in ovariectomized rats significantly increased relative uterine weight (0.11 ± 0.01 % vs. 0.05 ± 0.00 % in controls) and serum estradiol levels elevated serum estradiol levels (4-fold over vehicle control). Histological analysis confirmed estrogenic alterations, including epithelial thickening, glandular degeneration, and stromal inflammation. Although estrogen receptor α (ERα) expression remained unchanged, aromatase (CYP19) was significantly upregulated in uterine tissue, suggesting that enhanced estrogen biosynthesis plays a key role in the effects elicited by 4-MBP. In vitro assays showed that 4-MBP activated ER transcription in HeLa9903 cells with a maximum relative proliferative capacity (RPCmax) of about 200 % compared with 1 nM 17β-estradiol, and significantly induced MCF-7 cell proliferation at 10⁻⁵ M, coinciding with peak CYP19 mRNA expression. CYP19 expression was also increased at the mRNA and protein levels, and evidence of post-transcriptional regulation was observed at higher concentrations (10 µM). In silico molecular docking and dynamic simulations corroborated these findings demonstrating strong binding affinities of 4-MBP to ERα (docking score as low as -8.7 kcal/mol) and ERβ. Our results indicate that 4-MBP exerts estrogenic effects by elevating estrogen synthesis and inducing direct ER transcriptional activation. These findings highlight the utility of ITS for evaluating endocrine-disrupting chemicals and emphasize the need for regulatory consideration of 4-MBP and structurally related compounds in consumer products.