title: Towards an integrated in vitro strategy for estrogenicity testing.
abstract: In order to define an in vitro integrated testing strategy (ITS) for estrogenicity, a set of 23 reference compounds representing diverse chemical classes were tested in a series of in vitro assays including proliferation and reporter gene assays. Outcomes of these assays were combined with published results for estrogen receptor (ER) binding assays and the OECD validated BG1Luc ER transcriptional activation (TA) assay and compared with the outcomes of the in vivo uterotrophic assay to investigate which assays most accurately predict the in vivo uterotrophic effect and to identify discrepancies between the in vitro assays and the in vivo uterotrophic assay. All in vitro assays used revealed a reasonable to good correlation (R(2)  = 0.62-0.87) with the in vivo uterotrophic assay but the combination of the yeast estrogen bioassay with the U2OS ERα-CALUX assay seems most promising for an ITS for in vitro estrogenicity testing. The main outliers identified when correlating data from the different in vitro assays and the in vivo uterotrophic assay were 4-hydroxytamoxifen, testosterone and to a lesser extent apigenin, tamoxifen and kepone. Based on the modes of action possibly underlying these discrepancies it becomes evident that to further improve the ITS and ultimately replace animal testing for (anti-)estrogenic effects, the selected bioassays have to be combined with other types of in vitro assays, including for instance in vitro models for digestion, bioavailability and metabolism of the compounds under investigation.
authors.affiliation: Division of Toxicology, Wageningen University and Research Centre, Tuinlaan 5, 6703 HE, Wageningen, The Netherlands; Business Unit of Toxicology & Bioassays, RIKILT - Institute of Food Safety, Wageningen University and Research Centre, Akkermaalsbos 2, 6708 WB, Wageningen, The Netherlands.
authors.forename: Si
authors.initials: S
authors.lastname: Wang
authors.forename: Jac M M J G
authors.initials: JM
authors.lastname: Aarts
authors.forename: Laura H J
authors.initials: LH
authors.lastname: de Haan
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Argyriou
authors.forename: Ad A C M
authors.initials: AA
authors.lastname: Peijnenburg
authors.forename: Ivonne M C M
authors.initials: IM
authors.lastname: Rietjens
authors.forename: Toine F H
authors.initials: TF
authors.lastname: Bovee
mesh_terms: D000826:Animal Testing Alternatives; D001681:Biological Assay / Q000379:methods; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D052244:Endocrine Disruptors / Q000633:toxicity; D023381:Endpoint Determination; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D004967:Estrogens / Q000494:pharmacology*; D017930:Genes, Reporter; D006801:Humans; D061986:MCF-7 Cells; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D015533:Transcriptional Activation
chemical_list: D002352:Carrier Proteins; C484899:DNTTIP2 protein, human; D052244:Endocrine Disruptors; D047628:Estrogen Receptor alpha; D004967:Estrogens; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins
keywords: correlation; estrogenicity; integrated testing strategy; reporter gene assays; uterotrophic assay