10/18/2016. Endocrine Disruption and the Environment. Endocrine Policy Forum US EPA S LEGISLATIVE MANDATES (1996)

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1 Endocrine Disruption and the Environment Ellen Mihaich, Ph.D., DABT PCPC Environmental Safety Workshop October 25, Endocrine Policy Forum Consortium of List 1 test order recipients and other interested/involved parties Self-funded Represents >95% of List 1 test order recipients Additional stakeholders include CLA, ACC, ACI, CSPA, API, and consulting companies Objective to address regulatory and policy issues, technical guidance and science advocacy To undertake collaborations to tackle common areas of interest 2 US EPA S LEGISLATIVE MANDATES (1996) Food Quality Protection Act o Must screen pesticides (including inerts) for possible estrogenic effects that may affect human health o Must use appropriate validated test systems or other scientifically relevant information o Can include other endocrine effects Safe Drinking Water Act (SDWA) o Can screen drinking water contaminants to which substantial numbers of persons are exposed Substantial numbers and may be found in sources of drinking water not defined 3 1

2 EDSP HISTORY: 1996 TO TODAY Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) o EPA multi-stakeholder advisory committee evaluates methods for priority setting, screening, testing, and communications as key program elements 1999 to Today: Endocrine Disruptor Screening Program (EDSP) o EPA adopts EDSTAC recommendations, creates EDSP, and in coordination with OECD, works to standardize/validate screening and test protocols and guidelines st Tier 1 List finalized: contained 67 pesticides and pesticide inerts 2013 Second Tier 1 List finalized; 109 chemicals (41 pesticides) o Office of Management and Budget has not approved the Information Collection Request (ICR) from EPA o No test orders can be released until OMB approval of the ICR Work is ongoing to prioritize the list of 10,000 chemicals for screening so current List 2 chemicals may not be chosen ultimately 4 EDSTAC Conceptual Framework Tier 1 Screening Eleven In Vitro and In Vivo Assays Identify chemicals that can potentially interact with the endocrine system (Estrogen, Androgen, and Thyroid pathways) Maximize sensitivity to minimize false negatives A battery of screening assays, with deliberate redundancy Tier 2 Testing: Multigen studies in a range of species Confirm endocrine activity detected in Tier 1 and characterize adverse effects and Establish NOAELs and LOAELs for risk assessment for regulation of the substance 5 OECD Conceptual Framework Compendium of data / test methods useful for the identification and assessment of potential endocrine disrupters Five levels with increasing information Entering at all levels and exiting at all levels is possible Conceptual framework is not a test strategy Enter and exit at any level 6 2

3 OECD Conceptual Framework Level 1 Existing Data and Non-Test Information Mammalian and Non-Mammalian Toxicology Physical & chemical properties, e.g. MW, reactivity, volatility, biodegradability All available (eco)toxicological data from standardized or nonstandardized tests Read across, chemical categories, QSARs and other in silico predictions, and ADME model predictions Level 2 In vitro assays providing data about selected endocrine mechanism(s)/ pathway(s) (Mammalian and nonmammalian methods) Estrogen or androgen receptor binding affinity Estrogen receptor transactivation (OECD TG 455 [OECD TG 457]) Androgen or thyroid transactivation (If/when TGs are available) Steroidogenesis in vitro (OECD TG 456) MCF-7 cell proliferation assays (ER ant/agonist) Other assays as appropriate 7 OECD Conceptual Framework Level 3 In vivo assays providing data about selected endocrine mechanism(s) / pathway(s) Mammalian Toxicology Uterotrophic assay (OECD TG 440) Hershberger assay (OECD TG 441) Non-Mammalian Toxicology Xenopus embryo thyroid signalling assay (When/if TG is available) Amphibian metamorphosis assay (OECD TG 231) Fish Reproductive Screening Assay (OECD TG229) Fish Screening Assay (OECD TG 230) Androgenized female stickleback screen (GD 140) Level 4 In vivo assays providing data on adverse effects on endocrine relevant endpoints Repeated dose 28-day study (OECD TG 407) Repeated dose 90-day study (OECD TG 408) 1-generation assay (OECD TG 415) Male pubertal assay Female pubertal assay Intact adult male endocrine screening assay Prenatal developmental toxicity study (OECD TG 414) Chronic toxicity and carcinogenicity studies (OECD TG 451-3) Reproductive screening test (OECD TG 421 if enhanced) Combined 28-day/reproductive screening assay (OECD TG 422 if enhanced) Developmental neurotoxicity (TG 426) Fish sexual development test (OECD TG 234) Fish Reproduction Partial Lifecycle Test (when/if TG is Available) Larval Amphibian Growth & Development Assay (OECD 241) Avian Reproduction Assay (OECD TG 206) Mollusc Partial Lifecycle Assays (TGs 242 and 243) Chironomid Toxicity Test (TG ) Daphnia Reproduction Test (with male induction) (OECD TG 211) Earthworm Reproduction Test (OECD TG 222) Enchytraeid Reproduction Test (OECD TG 220) Sediment Water Lumbriculus Toxicity Test Using Spiked Sediment (OECD TG 225) Predatory mite reproduction test in soil (OECD TG 226) Collembolan Reproduction Test in Soil (TG OECD 232) 8 OECD Conceptual Framework Level 5 In vivo assays providing more comprehensive data on adverse effects on endocrine relevant endpoints over more extensive parts of the life cycle of the organism Mammalian Toxicology Extended one-generation reproductive Toxicity Study (OECD TG 443) 2-Generation assay (OECD TG 416 most recent update) Non-Mammalian Toxicology FLCTT (Fish LifeCycle Toxicity Test) (when TG is available) Medaka Extended One-Generation Test (MEORGT) (TG 240) Avian 2 generation reproductive toxicity assay (OCSPP ) Mysid Life Cycle Toxicity Test (when TG is available) Copepod Reproduction and Development Test (when TG is available) Sediment Water Chironomid Life Cycle Toxicity Test (TG 233) 9 3

4 Current EDSP Test Guidelines Tier 1 Tier 2 In vitro ER binding ER transcriptional activation AR binding Steroidogenesis, H295R Aromatase, recombinant In vivo (toxicology) Uterotrophic assay Hershberger assay 2-generation rat reproduction study (or extended 1-generation study) Pubertal male assay Pubertal female assay In vivo (ecotoxicology) Fish short-term screening assay Amphibian metamorphosis assay Medaka extended 1-generation study Larval amphibian growth and development test Avian 2-generation reproductive test 10 Tier 1 screens cost $750,000 to $1 million per chemical List 1 Results 52 chemicals (primarily pesticides) were screened in most, if not all, of the tier 1 screens o EPA evaluated all of the data and wrote weight of evidence reports All reports and study summaries are publically available on the EPA website There was no evidence for potential interaction with any of the endocrine pathways for 20 chemicals For 14 chemicals that showed potential interaction with one or more pathways, EPA already has enough information to conclude that they do not pose risks. Of the remaining 18 chemicals, all 18 showed potential interaction with the thyroid pathway, 17 with the androgen pathway, and 14 with the estrogen pathway. 11 EPA Overall Conclusions For several of the chemicals displaying activity in the Tier 1 screening assays, EPA determined they have enough information to conclude that they do not pose risks. EPA recommended the following higher tier studies: o A comparative thyroid assay for 4 chemicals that showed interaction with the thyroid pathway in mammals (not one of the validated Tier 2 tests) o A medaka extended one-generation reproductive test (MEOGRT) for 13 chemicals that showed potential interaction with the estrogen or androgen pathways in wildlife o A larval amphibian growth and development assay (LAGDA) for 5 chemicals that showed potential interaction with the thyroid pathway in wildlife 12 4

5 A Tier 2 Quandary Tier 2 is for determining adverse effects and to provide data for risk assessment In regulatory toxicology we test at levels that achieve some effect How will we determine if that effect is specifically endocrine related? Not as much of a problem in the US where regulations are based on risk but, In Europe, deciding if the effect is endocrine or may have implications for authorization based solely on hazard 13 Ecotoxicological Hazard and Risk Assessment SETAC Pellston Workshop 48 global experts brought together to address the controversy over hazard and risk Some scientists believe that EDS can be reliably assessed using the standard risk assessment paradigm, Others do not believe this is sufficiently precautionary and propose risk management on the basis of hazard alone 6 case studies evaluated Cross-cutting issues identified Conclusion: Provided that environmental exposure, relevant taxa and life-stages, delayed effects, and concentrationresponse relationships are adequately characterized then conducting environmental risk assessment of EDC is scientifically sound. 14 A Cross-Cutting Issue: Challenges in Assigning an Endocrine MOA Use of Weight of Evidence Establishing Biological Plausibility Computational/in vitro approaches In vivo concordance Essentiality of Key Events Adverse Outcome Pathway (AOP) Empirical Evidence Do the pieces fit?? Confounding Factors Systemic/overt toxicity Stress Infection Organ specific toxicity Nutritional status 15 5

6 Increasing power 10/18/2016 WHO/IPCS Definition WHO IPCS 2002: An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations. 16 In silico In vitro In vivo screening In vivo definitive Increasing power Weight of Evidence + = Endocrine disruptor Mechanism Adverse Effect 17 The Dilemma of Many Endpoints Chance of a Clean Chemical Screening Clean (1- p) n Tier 1 Endocrine Screening Battery: 89 endpoints; if all independent (0.95) 89 = 0.01 (0.99) 89 = endpoints; if every 4 th independent: (0.95) 22 = 0.32 (0.99) 22 =

7 If Only WoE Was This Easy! 19 Courtesy of Tim Ward WOE Conceptual Framework 1. Define Explicit Hypotheses 8 Total for Tier 1 Potential E+ / E- / A+ / A- / T+ / T- /Steroidogenesis induction/steroidogenesis inhibition 2. Systematic Literature Review 3. Evaluate Data Quality 4. Weight Endpoints Quantitatively or Rank-Ordered based on Explicit Criteria and Data 5. Weight Results within the context of known positives and negatives 6. Develop Narrative Interpretation listing all assumptions 20 Framework: Borgert et al Reg Tox Pharm 61: Relevance weightings: Borgert et al. 2014, Birth Defects Research, Part B, 101: Ranking Endpoints No hypothesis can be decided on the results of a single assay Battery Approach Rank the relevance (Wrel) of each endpoint for deciding each hypothesis Rank #1: specific & sensitive for the hypothesis; interpretable without other endpoints; in vivo endpoints rarely confounded by artifacts or non-specific activity. Rank #2: specific & sensitive for the hypothesis; interpretable alone, but less informative than Rank #1, often due to potential confounding; in vitro assays, many in vivo endpoints Rank #3: Relevant but only when corroborative of Rank #1 and #2 endpoints; many apical in vivo endpoints. No Response (NR) 21 7

8 Relevance Rankings By Hypothesis: Estrogen Agonist Estrogen Agonist Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints FSTRA ERTA ER binding Vitellogenin: increased in males ER agonism ER competitive binding Uterotrophic Assay FSTRA FSTRA Increased uterine weight (wet/blotted) Secondary sexual characteristics: decreased tubercle score: males Fecundity Gonad histopathology: males Behavior: males Pubertal Female Assay Female behavior GSI: males, females Gonad histopathology: Follicular atresia opening: reduced Fertilization success 22 Ovary weight: reduced Age at first estrous: reduced Pubertal Male Assay Testes weight Testes histopathology: atrophy Estradiol Testosterone Pubertal Female Assay Growth Estrous cyclicity Pubertal Male Assay Growth Ventral prostate weight Epididymides histopathology Steroidogenesis Assay Estradiol levels Relevance Rankings By Hypothesis: Estrogen Antagonist Estrogen Antagonist Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints No rank 1 endpoints identified ER Binding ER competitive binding Pubertal Female Assay opening: increased Age at first estrous FSTRA Vitellogenin: reduced in females Gonad histopathology: females Aromatase Aromatase inhibition Steroidogenesis Estradiol levels Pubertal Female Assay Estrous cyclicity Ovary histopathology: atrophy Ovary weight: reduced, with atrophy FSTRA Fecundity GSI Behavior Fertilization success Estradiol Testosterone 23 Relevance Rankings By Hypothesis: Androgen Agonist Androgen Agonist Hypothesis Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints Hershberger Pubertal Male Aromatase Weights of Cowper's gland, seminal separation: if Aromatase inhibition vesicle, LAB, glans penis, ventral accelerated prostate: concordance of all endpoints FSTRA Seminal vesicle + coagulating gland FSTRA weight (wet/blotted) Secondary sexual characteristics: Ventral prostate weight Fecundity decreased tubercle score: females Dorsolateral prostate weight Testosterone and estradiol levels LABC muscle complex weight GSI Epididymis weight Behavior Testes weight Fertilization success Testes histopathology Pubertal Female Epididymides histopathology Growth FSTRA opening Vitellogenin: reduced in females Uterus weight Gonad histopathology Ovaries weight 24 AR Binding Adrenals weight Competitive binding - NO Uterus histopathology Hershberger assay Ovary histopathology Concordance of two or more endpoints Pubertal Male (see rank 1) Growth Testosterone levels Steroidogenesis Testosterone levels Hershberger assay Only one of five endpoints respond (see Rank 1) 8

9 Relevance Rank-FSTRA Endpoints Endpoint E+ E- A+ A- T+ T- Steroid. Steroid. induct. inhib. Vitellogenin R1; m R2; f R2; f R2; f NR NR NR R2; f 2 o Sex R2; m NR R1: f R2; m NR NR NR NR Characteristic tubercle tubercle tubercle s score score score Fecundity R3 R3 R3 R3 NR NR NR R3 Estradiol Opt.R3 Opt.R3 Opt.R3 Opt.R3; m NR NR NR Opt.R3 Testosterone Opt.R3 Opt.R3 Opt.R3 Opt.R3; f NR NR NR Opt.R3 Gonad Histopatholog y GSI Behavior Fertilization Success R2 m R2; f R2 R2 NR NR NR R2; m R3; m R3 R3 R3 NR NR NR R3 f R2 m R3 R3 R3 NR NR NR R3 R3 f R3 R3 R3 R3 NR NR NR R3 Adult Survival NR NR NR NR NR NR NR NR 25 Evaluating the Responses Positive or negative responses in Rank 1 Preliminary indication of support for or against hypothesis Consistent positive or negative responses in Ranks 1 and 2 Sufficient support for or against the hypothesis Consult Rank 3 for positive, not needed for negative Positive responses in Rank 2, but not Rank 1 Strength of response and corroboration with Rank 3 critical, caseby-case evaluation needed 26 Hypothesis-Based WoE Framework Uses explicit relevance rankings of endpoints derived a priori hypothesis-based versus hypothesis-generating Transparent, objective and consistent interpretation of results for each hypothesis Systematic method to resolve inconsistencies in the data and focus on the most definitive information Can be applied to Tier 2 and other MoA as well Can be updated and altered with new information 27 9

10 Vtg (mg/ml) E2 (ng/ml) Control Fadrozole (µg/l) T ra n s p o rte r S te ro id h o rm o n e P X R P ro te a s e P R N u c le a r re c e p to r M R G R G P C R E R D N A b in d in g D e v e lo p m e n t C y to k in e C y p C e ll c y c le C e ll a d h e s io n m o le c u le s A R Cumulative Number of Eggs (Thousands) Fadrozole (ug/l) Control Exposure (d) 1 lo w e r lim it o f c y to to x ic ity C y to t o x ic it y p o in t 10/18/2016 Helping Establish Biological Plausibility: High-Throughput Methodologies Toxicity Testing in the 21 st Century o Criticism of EPA for how long the EDSP has taken and an interest in reducing animal testing has prompted new technologies and processes o ToxCast program at the heart of EPA screening methodology for bioactivity Battery of more than 700 in vitro, high-throughput screens Approximately 15% are directly relevant to EDSP Over 1,800 chemicals already screened and data in the public domain (actor.epa.gov/dashboard) EE2 in the ToxCast Battery oexpocast to be used with the bioactivity screens in order to consider exposure Currently only for human health assessment A C 5 0 v a lu e s ( M ) 1 0 Understanding Essentiality of Key Events: Generalized AOP Daniel L. Villeneuve et al. Toxicol. Sci. 2014;142: Adverse Outcome Pathway Adverse Outcome Pathway Toxicant Macro-Molecular Interactions Cellular Responses Organ Responses Individual Responses Population Responses Chemical Property Profile Receptor/Ligand Interaction DNA Binding Protein Oxidation Gene Activation Protein Production Altered Signaling Protein Depletion Altered Physiology Disrupted Homeostasis Altered Tissue Development or Function Lethality Impaired Development Impaired Reproduction Cancer Structure Recruitment Extinction N N Aromatase inhibition Reduced E2, Vtg synthesis Impaired vitellogenesis Reduced fecundity CN Molecular initiating event Key events or predictive relationships spanning levels of biological organization Adverse outcome relevant to risk assessment 30 Courtesy of G. Ankley 10

11 Possible Adverse Outcome Pathways Leading to a Reduction in Fecundity in Fish Exposed to Propiconazole Inhibition Aromatase Activation CAR/PXR Dysfunction Mitochondrial Reduction E2 synthesis Increase Cytochrome P450 expression Increase Oxidative stress Reduction E2 levels Increase E2 biotransformation Reduction Vitellogenin levels Decrease Transcription/Translation 31 Reduction Fecundity; filial ELS survival Decline Population trajectory Legend Key Event Relationships Strong Evidence Moderate Evidence Weak Evidence Comparative Evidence Key Events Direct PPZ evidence in fish Comparative PPZ evidence in mammals Comparative PPZ high-throughput evidence No evidence, potential KE A Line of Empirical Evidence: The Acute to Chronic Ratio The acute endpoint (e.g. LC50) represents nonspecific baseline toxicity. The chronic endpoint typically reflects a more specific mode of action. The more potent an endocrine disrupting agent, the higher will be the acute to chronic ratio (ACR). Ethinyl estradiol ACR = 5,700,000 Most industrial chemical ACR = Most aquatic BPA ACRs range from 1.4 to 100 (highest = 290; F2 hatchability in fathead study) Average = 25, median = 8 32 Calculating ACR Acute and chronic aquatic toxicity studies were assembled from published studies and government reports Scientifically valid studies data quality important Acute LC50 (mortality) and chronic NOEC for survival, growth and development, and reproductive endpoints were recorded Acute to chronic ratios were determined Comparisons made between same species All endpoints, endpoints minus chronic survival, and reproduction only endpoints All species combined and then separated by fish, invertebrate, and aquatic plant/algae 33 11

12 lo w e r lim it o f cyto to xicity C y to t o x ic it y p o in t 10/18/2016 ACR Based on Taxa Fish Invertebrate Algae 34 Now, Does It Make Sense? EE2 estrogenic bioactivity much lower than other bioactivity Propiconazole aromatase inhibiting, E and A activity in the same range (or higher) as other non-endocrine bioactivity Propiconazole in the ToxCast Battery EE2 in the ToxCast Battery T ra n s p o rte r S te ro id h o rm o n e P X R P ro te a s e P R N u clear recep to r M R G R G P C R E R D N A b in d in g D e v e lo p m e n t C y to k in e C y p C ell cycle C ell ad h esio n m o lecu les A R A C 5 0 v a lu e s ( M ) E s t r o g e n R e c e p t o r X e n o b io t ic m e t a b o lis m C yt o k in e G - P r o t e in R e c e p t o r M a tr ix M e ta llo p r o te in a s e P P AR 0 Toxcast Results for B PA A C 5 0 v a lu e s ( M ) Bioactivity screen indicates multiple MoA for BPA in the same concentration range 5 EPA Developing Reference Chemicals Tiers 1 and 2 studies being conducted to broaden in vivo ecotox data set 5 FSTRAs, 5 AMAs, 3 MEORGTs, 3 LAGDAs Additional FSTRAs are planned with fathead minnow, zebrafish, and medaka, to evaluate interspecies responses Reference chemicals: bisphenol A, dibutyl phthalate, triclosan, 4-nonylphenol, ethyl paraben Data will be used to compare to highthroughput and computational responses and to help build AOPs 36 12

13 What to Expect in 2016 (and beyond) Tier 2 test orders o EPA needs to provide OMB with updated ICR to include other possible tier 2 studies o OMB needs to approve ICR o Anticipated sometime in 2016 but will likely slip to 2017 Adoption of draft criteria in Europe o 1 st half 2017 ToxCast/ExpoCast prioritization Science Advisory Panel o Scope uncertain but likely focused on androgen and thyroid highthroughput screens and exposure (ExpoCast). o 2017 List 2 screening o Anticipated sometime after the SAP and possibly not until SAP provides EPA with report (they have 90 days to complete) o Anticipated first half Questions? 38 13