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ENDOCRINE DISRUPTOR SCREENING PROGRAM. Weight-of-Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing

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ENDOCRINE DISRUPTOR SCREENING PROGRAM DRAFT 6/29/2011 Weight-of-Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing Office of Chemical Safety and Pollution Prevention
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ENDOCRINE DISRUPTOR SCREENING PROGRAM DRAFT 6/29/2011 Weight-of-Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing Office of Chemical Safety and Pollution Prevention US Environmental Protection Agency Washington DC September 14, PREFACE The Agency submitted a draft of the Weight-of-Evidence (WoE) document for public review and comment as described in a Federal Register Notice issued November 4, 2010 (75 FR 67963). Submitted public comments were compiled and grouped according to the commonality among individual submissions so that they could be more readily and fully considered by EPA during revision of the WoE document. The WoE approach that has been revised and described herein is expected to provide general guidance to EPA staff and managers who will be reviewing data submitted in response to Orders for Tier 1 screening that began October 29, 2009 under the Endocrine Disruptor Screening Program (EDSP). Additionally, outside parties submitting data may be interested to know how the results from Tier 1 screening are being evaluated. This paper provides general guidance and is not binding on either EPA or any outside parties. The use of language such as will, is, may, can, or should in this paper does not connote any requirement for either EPA or any outside parties. As such, EPA may depart from the guidance where circumstances warrant and without prior notice. Application of WoE analysis is an integrative and interpretive process routinely used by EPA to evaluate health (USEPA 1991; 1996; 2002a; 2005) and ecological (USEPA, 1998) toxicity in a manner that takes into account all relevant scientific and technical information. The principles and criteria for weighing and integrating different lines of evidence articulated in existing EPA documents are considered generally applicable to evaluating data from the EDSP Tier 1 battery. It should be recognized that significant advances in both computational and molecularbased technologies are enabling a more rapid identification of markers for evaluating toxicity pathways since EPA began work on developing and implementing the EDSP in In 2007, the National Research Council Report Toxicity Testing in the 21st Century: A Vision and a Strategy (NRC, 2007) acknowledged these advances and recommended that the Agency develop a strategy to use modern in silico, computational models and molecular-based in vitro high-throughput screening assays to 2 increase the efficiency of, and reduce and ultimately replace reliance on, whole-animal toxicity testing. Currently, there are ongoing efforts within and outside the Agency to use endocrine screening as a prototype for applying these contemporary methods as proposed by the NRC. A key objective of the work is to improve the speed, reliability, cost effectiveness, and mechanistic specificity of the EDSP. In acknowledging this ongoing research, it should be stressed that the Agency s risk assessment guidance documents are typically viewed as living documents that is, they are open to periodic updates and revisions to reflect advances in the science and technology. Although the general principles and criteria articulated in this document for using a WoE approach apply to any study type, this policy is open to periodic updates to incorporate important new scientific and technical knowledge as it becomes available. 3 TABLE OF CONTENTS Abbreviations PURPOSE AND SCOPE OF DOCUMENT ENDOCRINE DISRUPTOR SCREENING PROGRAM (EDSP) OVERVIEW EDSP Tier 1 Battery of Screening Assays Assays for Detecting the Effect of Chemicals on the Estrogen Hormonal Pathway Assays for Detecting the Effect of Chemicals on the Androgen Hormonal Pathway Assays for Detecting the Effect of Chemicals on the Steroidogenic Pathway Assays for Detecting the Effect of Chemicals on the HPG Axis Assays for Detecting the Effect of Chemicals on the HPT Axis EDSP Tier 2 Testing SOURCES OF SCIENTIFIC AND TECHNICAL INFORMATION Test Guidelines EDSP Tier 1 Screening Studies Scientifically Relevant Information Test Guidelines Health and Ecological Effects Studies Published or Publically Available Peer-reviewed Studies QUALITY OF SCIENTIFIC AND TECHNICAL INFORMATION General Assessment Factors (GAF) Soundness Applicability and Utility Clarity and Completeness Uncertainty and Variability Evaluation and Review Standard Evaluation Procedure (SEP) and Data Evaluation Record (DER) WEIGHT-OF-EVIDENCE APPROACH Assembling and Evaluating the Individual Studies Integrating the Different Lines of Evidence Weight-of-Evidence Narrative/Characterization EDSP Tier 2 Testing Recommendations SUMMARY REFERENCES Abbreviations Abbreviation A AR DER EDSTAC EDSP E ER FIFRA FFDCA FQPA GAF GLP HPG HPT MoA OCSPP OECD PND SAB SAP SEP T WoE Terminology Androgen (hormonal pathway) Androgen Receptor Data Evaluation Record Endocrine Disruptor Screening and Testing Advisory Committee Endocrine Disruptor Screening Program Estrogen (hormonal pathway) Estrogen Receptor Federal Insecticide, Fungicide, Rodenticide Act Federal Food, Drug, and Cosmetic Act Food Quality Protection Act General Assessment Factors Good Laboratory Practices Hypothalamic-Pituitary-Gonadal Axis Hypothalamic-Pituitary-Thyroidal Axis Mode of Action Office of Chemical Safety Pollution and Prevention Organization for Economic Co-Operation and Development Postnatal Day Science Advisory Board Scientific Advisory Panel Standard Evaluation Procedure Thyroid (hormonal pathway) Weight-of-Evidence 5 1. PURPOSE AND SCOPE OF DOCUMENT This guidance document provides basic principles and criteria for using a weight-ofevidence (WoE) approach for evaluation and interpretation of EDSP Tier 1 screening, which includes Tier 1 assay results and other information to identify candidate chemicals for Tier 2 testing. General guidance is also provided on the considerations that will inform the tests and information that may be needed for Tier 2 testing. The purpose of the EDSP Tier 1 battery of screening assays is to identify chemicals that have the potential to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal pathways. Currently, the battery consists of 11 assays that have been developed and validated through a collaborative effort involving EPA program and research offices and published as harmonized test guidelines by the Office of Chemical Safety Pollution and Prevention (OCSPP 890 Guideline Series, Table 1). EPA intends to evaluate the results of the Tier 1 screening assays using a WoE approach to determine whether or not a chemical has the potential to interact with E, A, or T hormonal pathways and to assess the need for Tier 2 testing. The purpose of Tier 2 testing is to further characterize the effects on E, A, or T identified through Tier 1 screening by using Tier 2 in vivo studies that establish dose-response relationships for any potential adverse effects for risk assessment. EPA refers to the WoE approach as a collective evaluation of all pertinent information so that the full impact of biological plausibility and coherence is adequately considered. (USEPA, 1999). In its recommendations to EPA, the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) referred to the WoE approach as a process by which trained professionals judge the strengths and weaknesses of a collection of information to render an overall conclusion that may not be evident from consideration of the individual data (EDSTAC, 1998). The WoE approach for Tier 1 screening is discussed in Section 5 which is preceded by introductory and supportive information, including a brief historical overview of the 6 EDSP two-tiered screening and testing paradigm (Section 2), sources of scientific and technical information (Section 3), and general guidance for determining the quality and relevance of scientific and technical information (Section 4). 2. ENDOCRINE DISRUPTOR SCREENING PROGRAM (EDSP) OVERVIEW A detailed history of the program can be found at the EDSP website (http://epa.gov/endo/) and in other documents or websites referenced in this document. In 1996, amendments to the Federal Food, Drug, and Cosmetic Act (FFDCA) required EPA to: develop a screening program, using appropriate validated test systems and other scientifically relevant information, to determine whether certain substances may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or other such endocrine effect as the Administrator may designate [21 U.S.C. 346a(p)]. (http://www.epa.gov/pesticides/regulating/laws/fqpa/) Pursuant to the Administrator s discretionary authority, EPA adopted a two-tiered screening and testing strategy and expanded the EDSP to include the androgen and thyroid hormonal pathways and ecological effects: In 1998, subsequent to the EDSTAC recommendations (EDSTAC, 1998), EPA notified the public of a proposed EDSP as described in a Federal Register Notice issued December 28, 1998 (63 FR 71542). EPA submitted the proposal for review by the Agency s Science Advisory Board (SAB) and FIFRA Scientific Advisory Panel (SAP). A final report of the joint peer review is available (SAB/SAP, 1999). In 2008, after an extensive validation process (USEPA, 2007), including peer review of individual assays, EPA notified the public of the EDSP proposed Tier 1 battery of screening assays in a Federal Register Notice issued January 24, 2008 (73 FR 4216). EPA submitted the proposed battery for peer review by FIFRA SAP. A final report of the peer review is available (SAP, 2008). 7 In 2009, EPA notified the public of the current EDSP Tier 1 screening battery (Table 1) and availability of harmonized test guidelines (OCSPP 890 Guideline Series) for each of the assays in a Federal Register Notice issued October 21, 2009 (74 FR 54416). In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing Test Orders for Tier 1 screening was made available in a Federal Register Notice issued October 21, 2009 (74 FR 54422) EDSP Tier 1 Battery of Screening Assays The current EDSP Tier 1 battery consists of 11 diverse yet complementary in vitro and in vivo screening assays as recommended by the FIFRA SAP (SAP, 2008) and is indicated in Table 1. The battery of assays was designed to be conducted as a whole to maximize sensitivity and reliability for determining the potential of a chemical to interact with the E, A, or T hormonal pathways (EDSTAC, 1998). Various factors contributed to selecting the Tier 1 screen that generally included the potential of the assays to evaluate: E, A, or T hormonal pathway effects in different taxa, estrogen- and androgen-mediated effects via receptor binding (agonism and antagonism), estrogen-mediated gene transactivation, enzyme inhibition involving the reproductive steroidogenesis pathway, interactions with gonadal estrogen and androgen production that may alter feedback mechanisms involving the hypothalamic-pituitary-gonadal (HPG) axis, androgen and estrogen influenced endpoints within an assay that are complementary among the assays, and interactions with thyroid hormone production or function and associated alterations in feedback relationships involving the hypothalamic-pituitary-thyroid (HPT) axis. The robustness of the Tier 1 battery is based on the strengths of each individual assay and the complementary endpoints within the battery. Thus, the value of each 8 individual assay cannot be considered in isolation from other assays in the battery, as they have been combined in a manner such that limitations of one assay are complemented by the strengths of another (EDSTAC, 1998). Table 1. The EDSP Tier 1 screening assays encompass key endpoints within a MoA (e.g., receptor binding) and along endocrine pathways (e.g., steroidogenesis, effects on hypothalamic-pituitary-gonadal and thyroid axes) through which a chemical has the potential to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal pathways.* In vitro Screening Assay ER Binding (Rat uterine cytosol) ERα Transcriptional Activation (Human cell line HeLa-9903) AR Binding (Rat prostate cytosol) Steroidogenesis (Human Cell Line H295R) Aromatase (Human target tissue or cell-line microsomes) In vivo Uterotrophic (Rat) Hershberger (Rat) Pubertal Male (Rat) Pubertal Female (Rat) Fish Short-term Reproduction Amphibian Metamorphosis (Frog) Test Guideline OCSPP OCSPP OECD 455 OCSPP OCSPP OCSPP OCSPP OECD 440 OCSPP OECD 441 OCSPP OCSPP OCSPP OECD 229 OCSPP Receptor Binding Steroidogenesis HPG E Anti-E A Anti-A E A 1 Axis HPT Axis OECD 231 *Complementary endpoints across assays are indicated (solid black box) within each column reductase inhibition only. 9 In addition to the test guidelines, a detailed characterization of each Tier 1 screening assay, including its development, validation, strengths, and limitations, can be found in EPA Integrated Summary Reports or OECD Final Reports for individual assays at the EDSP website (http://epa.gov/endo/pubs/assayvalidation/index.htm). However, for the purposes of this document, an overview of the distinctive characteristics of each assay and their complementary endpoints within the battery of assays is provided in the next sections Assays for Detecting the Effect of Chemicals on the Estrogen Hormonal Pathway Five Tier 1 assays are capable of detecting chemicals with estrogenic and antiestrogenic activity (Table 1): 1. ER binding (rat uterine cytosol), 2. ER transcriptional activation (human cell line HeLa-9903), 3. Uterotrophic (rat), 4. Pubertal female (rat), and 5. Fish short-term reproduction. The in vitro ER binding assay examines the potential of a chemical to bind estrogen receptors (i.e., ER or ER ) isolated from the cytosol of excised rat uterine tissue. However, binding alone cannot distinguish whether the chemical is an estrogen agonist or antagonist. The in vitro ER transcriptional activation assay examines the potential of an estrogen agonist to activate ER- (i.e., ER ) mediated gene transcription in cells derived from a human cervical tumor. In vivo assays used to evaluate the estrogen pathway involve different routes of exposure to a chemical such as subcutaneous injection (uterotrophic), oral gavage (pubertal), and water (fish). The uterotrophic assay is conducted using adult ovariectomized or sexually immature intact female rats and has the potential to detect estrogen agonist activity based on: 10 an increase in uterine weight, and optional histology of uterus and vagina. The pubertal female assay is conducted in rats post-weaning and has the potential to detect both estrogen agonist and antagonist activity based on multiple endpoints: sexual developmental characteristics (age at vaginal opening and estrous cyclicity, length and percent of animals cycling), and weight and histology of reproductive organs (ovaries with oviducts and uterus with fluid, and pituitary gland). The fish short-term reproduction assay is conducted using mature male and female fathead minnows and has the potential to detect both estrogen agonist and antagonist activity based on multiple endpoints: behavior, fecundity, fertilization success, secondary sex characteristics (number and size of nuptial tubercles and dorsal nape pad in males), survival, body weight and length, gonadal size (gonado-somatic index) and histopathology, assay of plasma concentrations of vitellogenin in females and males, and optional assay of plasma concentrations of estradiol in females Assays for Detecting the Effect of Chemicals on the Androgen Hormonal Pathway Four Tier 1 assays are capable of detecting an androgenic or anti-androgenic effect of a chemical (Table 1): 1. AR binding (rat prostate cytosol), 2. Hershberger (rat), 3. Pubertal male (rat), and 11 4. Fish short-term reproduction. The in vitro AR binding assay examines the potential of a chemical to bind to the androgen receptor isolated from the cytosol of rat prostate tissue. However, binding alone cannot distinguish whether the chemical is an androgen agonist or antagonist. In vivo assays used to evaluate the androgen pathway involve different routes of exposure such as subcutaneous injection or oral gavage (Hershberger), oral gavage (pubertal), and water (fish). The Hershberger assay is conducted using castrated male peripubertal rats and has the potential to detect androgen agonist and antagonist activity as well as 5 -reductase inhibitors (i.e., inhibition of the conversion of exogenous testosterone to dihydrotestosterone) based on multiple endpoints involving changes (increase or decrease) in weight of androgen-dependent organs or tissues: ventral prostate, seminal vesicle plus coagulating gland with fluid, levator ani plus bulbocavernous muscle complex, paired Cowper s gland, and glans penis. The pubertal male assay is conducted in rats post-weaning and has the potential to detect both androgen agonist and antagonist activity based on multiple endpoints: sexual developmental characteristic (age at preputial separation), weight and histology of reproductive organs or tissues (testes, epididymides, ventral and dorsolateral prostate, seminal vesicle plus coagulating gland with fluid, levator ani plus bulbocavernous muscle complex, and pituitary gland), and assay of total serum concentrations of testosterone. The fish short-term reproduction assay is conducted using mature male and female fathead minnows and has the potential to detect both androgen agonist and antagonist activity based on multiple endpoints as already indicated in Section 2.1.1, including optional assay of plasma concentrations of testosterone in males. 12 Assays for Detecting the Effect of Chemicals on the Steroidogenic Pathway Six Tier 1 assays are capable of detecting disruption in the steroidogenic pathway (Table 1): 1. Steroidogenesis (human cell line H295R), 2. Aromatase (human target tissue or cell-line microsomes), 3. Hershberger (rat), 4. Pubertal male (rat), 5. Pubertal female (rat), and 6. Fish short-term reproduction. The in vitro steroidogenesis assay uses a human cell line (H295R) to examine the potential of a chemical to interact with the steroidogenic pathway based on the change (increase or decrease) in production of testosterone and estradiol. The in vitro aromatase assay uses human microsomes from various target tissues or cell lines to detect the inhibition of aromatase activity and the conversion of androgen to estrogen. The assay is not well suited to detect induction of aromatase activity. In regard to the in vivo assays and corresponding endpoints presented in preceding sections, an apparent effect on steroidogenesis (i.e., gonadal production of estrogen or androgen) may be observed based on changes in respective endpoints even though there are no apparent corroborating steroidogenic effects observed in vitro. It is possible that a chemical can disrupt steroidogenesis indirectly by acting directly on gonadotropin synthesis or secretion or by eliciting feedback responses along the HPG axis. For example, exposure to a chemical with androgen or estrogen activity may cause reductions in endogenous androgen or estrogen concentrations as a result of negative feedback along the HPG axis. The implication for an effect at the hypothalamic or pituitary levels is further explored in the next section Assays for Detecting the Effect of Chemicals on the HPG Axis In general, regulation of the HPG axis involves a complex array of positive and negative feedback mechanisms. Gonadal estrogen or androgens bind to corresponding 13 receptors in the hypothalamus or pituitary to regulate gonadotropic-releasing hormone and gonadotropic hormones, respectively, which in turn regulate ovarian and testicular steroidogenesis. The current Tier 1 screening battery does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of gonadal estrogen or androgen hormone production but does include three in vivo assays
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