Inability to confirm estrogenicity of the heterocyclic amine PhIP in two in vitro assays

2-Amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) is a heterocyclic amine which is found in food after cooking and which is a known mutagen. Reports from several laboratories have proposed that PhIP has estrogenic activity, which would classify PhIP as a xenoestrogen with human exposure via food. We tested PhIP in two cell-based assays for estrogenicity, both based on human cell lines but utilising different outcome measures: ERLUX (reporter-gene activation) and ESCREEN (cell proliferation).PhIP was inactive in both assays at concentrations spanning the picomolar to micromolar range. To eliminate supplier differences as an explanation for the disparity between these results and positive findings in the literature, we purchased PhIP from three suppliers and found no detectable estrogenic activity in any batch. 1H NMR spectroscopy confirmed the chemical identity of the tested stock solutions.Correct assay performance was confirmed by including positive and vehicle controls on every assay plate, and by demonstrating the expected responses to a panel of known estrogens (estradiol, bisphenol A, and genistein). Our results differ from those in the literature and, whilst the exact reason for this is unknown, we discuss possible explanations of the disparity. Our results provide no in vitro evidence for the classification of PhIP as an estrogen.