U.S. Environmental Protection Agency Office of Research and Development EPA www.epa.gov ►Comparison of the HT-H295R screening data analyzed by ANOVA with the summary results available from the OECD validation study suggests that the HT-H295R assay predicted T and E2 effects well. ►The maxmMd is a reproducible and quantitative metric to determine the magnitude of chemical perturbation of steroidogenesis in HT-H295R, representing a data-driven option for evaluating lists of chemicals. ►The number of steroid hormone significantly perturbed in the HT-H295R assay does not indicate the magnitude of effect on steroidogenesis, whereas the maxmMd can differentiate strong and weak modulators of steroidogenesis. ►Selectivity scoring is a useful method to filter HT-H295R pathway positives for additional screening. ►Future work will include more comprehensive benchmarking of the maxmMd metric, and further adapting the HT- H295R kinetic model based on time-course experiments. We use Mahalanobis distance to quantify disruption of steroidogenesis across 11 steroid hormone measurements. Mahalanobis distance incorporates the effect size for each steroid hormone measure after adjusting for covariance between the steroid hormone measures. Evaluating Perturbation of In Vitro Steroidogenesis Using a High-throughput H295R Assay Derik E. Haggard1,2, Agnes L. Karmaus1,2,3, Matt T. Martin2,4, Richard S. Judson2, R. Woodrow Setzer2, Katie Paul Friedman2 1ORISE Postdoctoral Research Participant 2National Center for Computational Toxicology, Office of Research and Development, U.S. Environmental Protection Agency 3Currently at Integrated Laboratory Systems, Inc 4Currently at Drug Safety Research and Development, Global Investigative Toxicology, Pfizer Derik E. Haggard l haggard.derik@epa.gov l 919-541-0745 Background: Endocrine disruption is a toxicity of both physiological and regulatory importance; as steroid hormones regulate reproduction, development, and other biological processes, it is a priority to identify chemicals that may interact with production of these hormones. A high-throughput H295R assay (HT-H295R) was developed as part of the U.S. EPA’s ToxCast program that includes measurement of 11 hormones across the steroid hormone biosynthesis pathway expressed in H295R cells, including progestagens, corticosteroids, androgens, and estrogens. HT-H295R Screening: 2012 chemicals in single concentration screening, 656 in concentration-response. Objectives: ►Demonstrate that the HT-H295R assay may be used to predict testosterone (T) and estradiol (E2) production via comparison of the 25 reference chemicals in the OECD-validated H295R assay. ►Develop a statistical analysis that integrates data from 11 steroid hormones into a single numeric value that indicates the magnitude of effect on steroidogenesis in the HT-H295R assay. ►Begin development of a pathway-based kinetic model of steroidogenesis in HT-H295R. This poster does not reflect US EPA policy. D.E.H. was supported by appointment to the Research Participation Program of the U.S. Environmental Protection Agency, Office of Research and Development, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. EPA. ►A total of 107 chemicals were replicated across multiple blocks; of these, 94 chemicals (87.9%) had concordant maxmMd pathway responses. ►maxmMd values were compared to the sum of steroid hormone hit counts, as measured by ANOVA. ►The maxmMd provides a quantitative indicator of activity that can distinguish chemicals that exert different magnitudes of effect on steroidogenesis but have the same hit count. Effect Sensitivity Specificity Accuracy Testosterone up 1.00 0.89 0.90 Testosterone dn 0.67 0.92 0.83 Estradiol up 0.75 0.83 0.80 Estradiol dn 0.80 1.00 0.95 Left: The residual covariances of many hormone measures in HT-H295R are highly correlated. Middle: Example of Euclidean distance for correlated