Evaluation of a rapid in vitro androgen receptor transcriptional activation assay using AR-EcoScreen™ cells

An accurate and reliable in vitro assay system has been needed for first tier screening of endocrine disrupting chemicals. For the purpose, we previously developed stable AR-EcoScreen™ cell lines to assess androgen receptor (AR)-mediated transcriptional activation. In this report, we evaluated AR-EcoScreen™ cell lines as the phase I of prevalidation study by determining the intra-laboratory reproducibility, assay stability, and overall protocol performance of AR-EcoScreen™ assays. Forty compounds recommended by the ICCVAM were tested for AR agonist and antagonist activity. The mean coefficient of variation (CV) for intra-assay reproducibility in the AR agonist assay was 4.35% for 5α-dihydrotestosterone (DHT), and that for the antagonist assay was 5.51% for hydroxyflutamide. The detection limit of the agonist assay was 2.3 × 10−11 M for 5α-dihydrotestosterone. Furthermore, we examined the overall performance of the method by comparing the predicted result with the ICCVAM classification. Thus, the overall sensitivity, specificity, and accuracy of the agonist assay were 89%, 94%, and 91%, respectively. For the antagonist assay, these values were 94%, 100%, and 96%, respectively. In summary, we concluded that AR-EcoScreen™ method was ready to proceed to the phase II prevalidation study to asses the inter-laboratory variability and transfer of the protocol.