Developing In Vitro Assays for Endocrine-Related Activities to Guide Risk Assessment
Michelle Miller, Pergentino Balbuena, Susan Ross, Rebecca Clewell. ScitoVation.
Developing strategies for in vitro screening and prioritization of compounds with endocrine activity has become a major strategic focus of the US EPA and the European Commission. Currently, the EPA’s Endocrine Disruptor Screening Program (EDSP) contains suites of assays to cover estrogen, androgen and thyroid hormone signaling in a two tiered approach. Tier 1 includes in vitro receptor activation assays and short‐term in vivo assays. Chemicals that yield positive responses in Tier 1 are then prioritized for testing in Tier 2, a process that includes more comprehensive, in‐depth in vivo studies. The estrogen pathway is represented by 16 ToxCastTM assays that provide a useful screen for hits, but do not use a biologically relevant cell model for uterine response in women. While the prescribed in vitro tests do measure several key events within the endocrine receptor signaling cascade, these assays are limited in their scope and do not account for tissue specific effects of estrogenic compounds. The current gold standard for in vivo estrogen activity is the Rat Uterotrophic Assay (RUA), but the existing in vitro assays do not include responses specific to the uterus despite demonstrated tissue-dependent effects in the human breast and uterus. The current paradigm recommends the use of in vitro assays for prioritization, but still requires movement into in vivo systems for risk assessment. In order to move beyond prioritization to in vitro-based safety assessment decisions, the in vitro systems must incorporate sufficient biology to accurately predict not only hazard, but also the dose-response for chemical effect. There remains a critical need for in vitro assays that reflect the relevant biology of the uterine estrogen response pathway and are capable of supporting chemical safety predictions without relying on additional in vivo experiments.
We use Ishikawa (IKA) cells as an in vitro model for examining dose-response for uterine effects of potential endocrine disrupting chemicals (EDCs). Estrogen signaling in the uterus involves a network of response pathways that ultimately lead to proliferation, changes in cell survival, transcriptional responses, and altered cell-cell interactions. We confirmed that the IKA cell model recapitulates in vivo protein, gene and proliferative responses to estrogens. Further, we compared the concentration response for these cells with human in vivo data to native ligand (estradiol) and the human drugs tamoxifen and ethinyl estradiol. The IKA proliferation model was highly sensitive and responded to these compounds at similar doses to normal adult women. To support screening activities, we have scaled this assay up to a higher throughput format. We have also successfully multiplexed the proliferation assay with viability markers and a readout for estrogen receptor mediated transcription. We have performed preliminary validation studies with a set of EDSP21 compounds to compare the accuracy of our methods to the assays now in the ToxCast™ repertoire and will be expanding to a larger set of compounds.
Implications: Preliminary results indicate that this assay has higher sensitivity, in terms of activating dose, than current HTS assays. Development of a validated fit-for-purpose functional assay that adequately accounts for key uterine biology will support risk based decisions for chemicals with estrogenic activity.
Key words: estrogen, endocrine disruption, EDSP
Project start and end dates: January 2015 – December 2016
Peer-reviewed publication(s): None to date
Other publication(s): None to date
Abstract creation date: February 2016
This abstract was prepared by the principal investigator for the project. Please see for more information about the LRI.