Estrogen receptor alpha (ERα) is often a primary target of endocrine disrupting chemicals (EDCs) and therefore several biochemical and cell-based assays for the detection of chemicals with... Show moreEstrogen receptor alpha (ERα) is often a primary target of endocrine disrupting chemicals (EDCs) and therefore several biochemical and cell-based assays for the detection of chemicals with estrogenic properties have been developed in the past. However, the current approaches are not suitable for the monitoring of pathway activation dynamics, and they are mostly based on expression constructs that lack physiological promoter regulation. We recently developed MCF7 fluorescent reporter cell lines of 3 different green fluorescent protein (GFP)-tagged ERα target genes: GREB1, PGR and TFF1. These reporters are under control of the full physiological promoter region and allow the monitoring of dynamic pro-proliferative pathway activation on a single cell level using a live-cell imaging set-up. In this study, we systematically characterized the response of these reporters to a full reference compound set of known estrogenic and non-estrogenic chemicals as defined by the Organization for Economic Co-Operation and Development (OECD). We linked activation of the pro-proliferative ERα pathway to a potential adverse outcome by additionally monitoring cell cycle progression and proliferation. The correct classification of the OECD reference compounds showed that our reporter platform has the same sensitivity and specificity as other validated artificial ERα pathway reporters, such as the ERα CALUX and VM7 Luc ER TA assay. By monitoring several key events (i.e. ER target activation, cell cycle progression and proliferation), and subsequently determining Point-of-Departure (POD) values, our reporter panel can be used in high-throughput testing for a physiologically more relevant, quantitative temporal endocrine modulation analysis to improve human carcinogen risk assessment. Show less