Evaluation of EPA’s Tier 1 Endocrine Screening Battery and recommendations for improving the interpretation of screening results Christopher J. Borgert a,b,⇑ , Ellen M. Mihaich c , Terry F. Quill d , M.S. Marty e , Steven L. Levine f , Richard A. Becker g a Applied Pharmacology and Toxicology, Inc., 2250 NW 24th Avenue, Gainesville, FL 32605, USA b Center for Environmental and Human Toxicology, Dept. of Physiological Sciences, University of Florida College of Veterinary Medicine, Gainesville, FL 32610, USA c Environmental and Regulatory Resources, LLC, Durham, NC, USA d Quill Law Group, LLC, Washington, DC, USA e The Dow Chemical Company, Midland, MI, USA f Monsanto Company, St. Louis, MO, USA g American Chemistry Council, Washington, DC, USA article info Article history: Received 31 August 2010 Available online 18 January 2011 Keywords: Endocrine disrupter EDSP screening Screening assays Tier 1 assays Endocrine Screening Battery Estrogen assay Thyroid assay Androgen assay Pubertal assay Validation abstract EPA’s Endocrine Disruptor Screening Program (EDSP) was implemented in 2009–2010 with the issuance of test orders requiring manufacturers and registrants of 58 pesticide active ingredients and nine pesti- cide inert/high production volume chemicals to evaluate the potential of these chemicals to interact with the estrogen, androgen and thyroid hormone systems. The required endocrine screening will be con- ducted over the next 2–3 years. Based on estimates of the impacted sectors, costs are at least $750,000–$1,000,000 per substance if all of the Tier 1 assays must be conducted. The screening will entail evaluation of responses in EPA’s Tier 1 Endocrine Screening Battery (EDSP ESB), consisting of 11 distinct in vitro and in vivo assays. We reviewed the details of each test method and describe the critical factors integral to the design and conduct of the EDSP ESB assays as well as the limitations related to specificity and sensitivity. We discuss challenges to evaluating each assay, identify significant shortcomings, and make recommendations to enhance interpretation of results. Factors that affect the length of time neces- sary to complete the EDSP ESB for any particular substance are presented, and based on the overall anal- ysis, we recommend a sequence for running the EDSP ESB assays. It is imperative that a structured, systematic weight of evidence framework is promptly developed, subjected to peer review and adopted. This will help to ensure an objective analysis of the results of the required EDSP screening, consistent integration of results across the EDSP ESB assays, and consistent decision making as to whether subse- quent testing for adverse effects is needed. Based upon the limitations of the current EPA EDSP ESB, we concur with the Agency’s Scientific Advisory Panel’s recommendation that after the initial set of sub- stances has been screened, the EDSP ESB should pause so that the results can be fully analyzed to deter- mine the value of the existing assays. After this analysis, assays that are unnecessarily redundant or that lack endocrine specificity should be eliminated and if necessary, replaced by new or revised screens that are more mechanistically specific, rapid, reliable, and cost effective. Ó 2011 Published by Elsevier Inc. 1. Introduction In response to the public concern that certain environmental chemicals may interfere with endocrine processes in humans, the US Congress enacted Section 408(p) of the 1996 Food Quality Pro- tection Act (FQPA or ‘‘the Act’’) (US EPA, 1996) directing the US Environmental Protection Agency (EPA) to develop and implement a screening program using ‘‘validated test systems’’ to investigate the potential of chemicals to induce adverse health effects through endocrine pathways. Relevant assays available in 1996 varied sig- nificantly in their degree of development and validation and a screening battery had not yet been identified or adopted by EPA (EDSTAC, 1998). Several screens had an extensive history, e.g., the uterotrophic and the Hershberger screens, but others were only partially developed or were only hypothetically useful as screens, e.g., the amphibian developmental screen and the fish gonadal recrudescence screen. Developing and validating endocrine assays and developing a screening program for endocrine-related mechanistic responses have proven to be difficult and time-consuming endeavors for 0273-2300/$ - see front matter Ó 2011 Published by Elsevier Inc. doi:10.1016/j.yrtph.2011.01.003 ⇑ Corresponding author at: Applied Pharmacology and Toxicology, Inc., 2250 NW 24th Avenue, Gainesville, FL 32605, USA. E-mail address: cjborgert@apt-pharmatox.com (C.J. Borgert). Regulatory Toxicology and Pharmacology 59 (2011) 397–411 Contents lists available at ScienceDirect Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph