State of the Science Evaluation:

State of the Science Evaluation: Nonmonotonic Dose Responses as They Apply to Estrogen, Androgen, and Thyroid Pathways and EPA Testing and Assessment Procedures Photo image area measures 2 H x 6.93 W and can be masked by a collage strip of one, two or three images. The photo image area is located 3.19 from left and 3.81 from top of page. Each image used in collage should be reduced or cropped to a maximum of 2 high, stroked with a 1.5 pt white frame and positioned edge-to-edge with accompanying images. October 1, 2013 DRSG Webinar Office of Research and Development

Disclaimer The views expressed in this presentation are those of the author and do not represent the policy of the U.S. EPA. 1

2 EDCs, Low Dose and Controversy

The Low Dose Hypothesis Elements of the Low-Dose hypothesis EDCs have effects, especially on reproduction and development, at low doses Effects observed in exposed animals are occurring at doses similar to human exposures (i.e. at doses that are thought to be safe) Humans environmentally exposed to EDCs are affected by low doses L. Vandenberg presentation to EPA 11/06/12

2012 Endocrine Society Position Statement Basic scientists should be involved in evaluating the weight -of-evidence of EDC studies, as well as in the design and interpretation of studies that inform the regulation of EDCs. State-of-the-art molecular and cellular techniques, and highly sensitive model systems, need to be built into current testing, in consultation with the appropriate system experts. Testing needs to include models of developmental exposure during critical life periods when organisms may be most vulnerable to even very low -dose exposures. Endocrine principles should be incorporated into programs by the U.S. Environmental Protection Agency (EPA) and other agencies charged with evaluating chemicals for endocrine-disrupting potential. Environmental chemicals that interfere with any aspect of hormone action 4 should be presumed to cause adverse effects. Zoeller et al. 2012

What about NMDR? These examples, and the examples of low-dose effects in less wellstudied chemicals (Table 3), provide evidence that low-dose effects are common in EDC research and may be the default expectation for all chemicals with endocrine activity. These mechanisms are well understood, and by providing detailed biological insights at the molecular level into the etiology of NMDRCs, they strongly negate the presumption that has been central to regulatory toxicology that dose-response curves are by default monotonic. For both hormones and EDCs, NMDRCs should be the default assumption absent sufficient data to indicate otherwise. 5 Vandenberg et al. 2012

Nonmonotonicity and Low-dose Effects Some reports indicate that dose response curves are nonmonotonic in the lowdose range for endocrine disrupting chemicals. That is, effects observed at low doses may not be observed or may not be intensified at higher doses. If non-monotonic dose responses in human systems are demonstrated to be common for endocrine active agents, it could have significant implications for chemical assessment by regulatory agencies. 6

Hormones have non-linear, and often non-monotonic responses Welshons et al. 2003

Scope: NMDRs Rather than Low Dose Low dose effect = Biological changes that occur below NOAEL, or in the range of human exposures Non-monotonicity = Change in the sign of the slope of the dose response curve, which can occur over any range of exposure levels Max Tolerated Intake RfD LOAEL NOAEL

1993 - Convened international research needs workshop 1990s - Co-chaired CENR working group in mid 1990s 1998 - Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Dose selection for studies used in risk assessment must include attention to setting a low dose Range-finding studies should include sensitive endpoints that could detect effects at low exposure concentrations 2000 EPA/NIEHS Endocrine Disruptor Low Dose Peer Review Workshop www.epa.gov/endo/pubs/edmvs/lowdosepeerfinalrpt.pdf Defined Low dose a biological change, not limited to adverse effects, which occur either at human exposure levels or at doses below those routinely used in toxicity testing Key conclusions of Report Sufficient evidence for low-dose reproductive and developmental effects Some estrogens exhibit NMDRCs Implications for toxicity testing and risk assessment not resolved 2011 EPA Low Dose Workshop EPA scientists from Programs (OCSPP, ORD, OW) evaluate the science and implications for dose setting 9 Office of Research and Development EPA Has a Long History with Endocrine Disruptors

EPA s Statutory Authority Food Quality Protection Act (FQPA) August 3, 1996 Amended the Federal Food, Drug, and Cosmetic Act (FFDCA) Requires EPA to develop a screening program (EDSP) 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. 10 3

EPA Has a Long History with Endocrine Disruptors 1993 - Convened international research needs workshop 1990s - Co-chaired CENR working group in mid 1990s 1998 - Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Dose selection for studies used in risk assessment must include attention to setting a low dose Range-finding studies should include sensitive endpoints that could detect effects at low exposure concentrations 2000 EPA/NIEHS Endocrine Disruptor Low Dose Peer Review Workshop www.epa.gov/endo/pubs/edmvs/lowdosepeerfinalrpt.pdf Defined Low dose a biological change, not limited to adverse effects, which occur either at human exposure levels or at doses below those routinely used in toxicity testing Key conclusions of Report Sufficient evidence for low-dose reproductive and developmental effects Some estrogens exhibit NMDRCs Implications for toxicity testing and risk assessment not resolved 2011 EPA Low Dose Workshop EPA scientists from Programs (OCSPP, ORD, OW) evaluate the science and implications for dose setting 11

12 Scope of 2013 EPA Review on NMDR EPA is interested in all potential NMDR impacts, but current analysis focus is on Endocrine disrupting chemicals (EDCs), Acting through the estrogen, androgen, or thyroid hormone systems Not intended to be a definitive review (or assessment) of any particular chemical Because of the limitations of the literature, document is focused on single chemical exposures This is not intended to be a policy document After peer review document will be revised After revision, document may be considered by EPA to inform our decision making

How EPA Developed the Review Established a cross program and cross agency working group. FDA (CDER, CFSAN), NIEHS (NTP), NIH/ NICHD But this is an EPA document Sub groups of authors worked independently More than 1000 papers reviewed for mammalian E and A 270 relevant papers evaluated in detail NMDR 28 model chemicals were selected for E and A evaluation of aquatic species with 20 papers determined to be directly relevant for the current evaluation More than 1400 papers reviewed for mammalian T 68 papers identified relevant for aquatic species T 13

Literature Reviews Varied approaches to searching and reviewing the literature Methodologies described for each data section e.g. for mammalian thyroid, two approaches In depth review of 3 chemicals with well characterized MOA: perchlorate (NIS symporter inhibition), propylthiourea (TPO inhibition), and polyhalogenated aromatic hydrocarbons (NR mediated hepatic enzyme induction) Systematic review and criteria for study selection 31 studies with NMDR identified (30 in vivo, 1 in vitro) Table 5 (from Appendix C.3). Reference filters used to compile the final list of published papers searched for any evidence of NMDRC for thyroid-related endpoints. All studies rated as relevant (i.e., primary publication of chemical-specific information on thyroid endpoints) were further divided into chemical-studies (see text for definitions). Total Number of References 1153 Numbers of reference that were reviews or otherwise not relevant 339 Total number of relevant references with data from chemical exposures 814 14 Total Number of Chemical-Studies from relevant references 2060 Number of Non-Mammalian Chemical-Studies 229 Number of Mammalian In Vivo & All In Vitro Chemical-Studies 1831

Consensus Definitions for the Review Non Monotonic Dose Responses (NMDRs) measured biological effects with dose response curves that contain a point of inflection where the slope of the curve changes sign at one or more points within the tested range. Low Dose Effect a biological change occurring in the range of typical human exposures or at doses lower than those typically used in standard testing protocols. NTP (2001) Endocrine Disrupting Chemical (EDC) 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. WHO (2002) Adverse Effect a measured endpoint that displays a change in morphology, physiology, growth, development, reproduction, or life span of a cell or organism, system, or population that results in an impairment of functional capacity, an impairment of the capacity to compensate for additional stress, or an increase in susceptibility to other influences. Keller et al. (2010) 15

Overarching Science Question Are modifications to standard test guidelines for toxicity testing used in risk assessment needed to detect and characterize nonmonotonic adverse effects for chemicals, particularly those that are mediated by effects on the endocrine system? Office of Research and Development 16

Key Scientific Questions 1. Do non-monotonic dose responses (NMDR) exist for chemicals and if so under what conditions do they occur? 2. Do NMDRs result in adverse effects that are not captured using our current chemical testing strategies? 3. Do NMDRs provide key information that would alter EPA s current weight of evidence conclusions and risk assessment determinations, either qualitatively or quantitatively? *Questions will be addressed as they pertain to endocrine disruption via estrogen, androgen, or thyroid pathways. Office of Research and Development 17

How We Developed the Review MOA guided data evaluation for identification of NMDR Focus on E, A, and T hormone systems and the relationship of disruption to adverse effect. Data evaluated based on modified Hill criteria; biological plausibility, strength, consistency, experimental design, and statistical evaluation and interpretation. Role of pharmacokinetics important in interpretation Weight of Evidence Versus strength of evidence 18

Example WOE 22% 16% 45% 17% Input for the REACH-review in 2013 on endocrine disrupters DANISH CENTRE ON ENDOCRINE DISRUPTERS In Vitro studies from Vandenberg et al. 2012 review described as showing NMDR allocated into four groups. (N=80) 19

Anogenital Distance (mm) Example Statistics and Design Issues Did not re-do published statistics, but commented on applicability of tests e.g. failure to account for multiple comparisons by adjusting the p-value required for statistical significance in studies with large numbers of endpoints and dosage levels (Appendix A.1.d.1). Design 20 Limitations in experimental design, in particular the number of animals in each dose group, can lead to inaccurate interpretation of the data. Difficulty of conducting low dose studies of ubiquitous contaminants. 2.0 1.5 1.0 0.0 0.5 1.0 5.0 Possible Dietary Phthalate levels 21.0 214.0 500.0 50000.0 500000.0 DEHP micrograms per kg per day

Document Structure 1. Introduction 2. General Biological Concepts and Statistical Considerations 3. NMDRs from in vitro Studies 4. NMDRs from in vivo Studies 4.1 Aquatic Models 4.2 Mammalian Models 4.3 Human Studies and Epidemiology 5. Conclusions 6. Bibliography 7. Appendices 21

22 Results and Conclusions

What We Found 23 NMDR from exposure to xenobiotics, do occur in biological systems but are generally not common. For the majority of chemicals, current testing identified those with potential endocrine activity. This was consistent with the goal of chemical testing to identify the potential for hazard after exposure to the xenobiotic of concern, not to identify and describe 100% of all the possible biological effects. The current testing approaches perform this function successfully and, based on the current evaluation, are highly unlikely to mischaracterize a chemical that has the potential to adversely perturb the endocrine system due to an NMDR.

What We Found For E and A, endpoints closest to the molecular initiating event more likely to identify point of inflection than downstream effects (including the apical adverse outcomes). e.g. in vitro assays measuring E and A receptor signaling pathways. may result from assay artifacts, cytotoxicity, or improperly controlled experimental conditions. e.g. In fathead minnows NMDRs seen in short-term time course studies -- fadrozole, prochloraz and trenbolone for gene expression and steroid endpoints. In 21-d studies, no NMDRs for apical endpoints (i.e. changes in secondary sex characteristics, growth, fertility or fecundity) 24

1. Do nonmonotonic dose responses (NMDRs) exist for chemicals and if so under what conditions do they occur? Yes, exposures to chemicals can result in NMDRs for specific endpoints. 25

1. Do nonmonotonic dose responses (NMDRs) exist for chemicals and if so under what conditions do they occur? 26 NMDRs arise from complex relationships between the dose of toxicant at its target site and the effect of interest. NMDRs are biologically plausible when at least two activities act in opposition to each other. Assays that provide data at a lower level of biological organization are more likely to identify NMDRs. Reproducibility of NMDRs is important in establishing plausibility of a response. NMDRs were more commonly identified in in vitro studies, high-dose range-finding studies, and large in vivo studies with numerous endpoints.

1. NMDRs were more commonly identified in in vitro studies, high-dose range studies, and shortterm studies. in vitro studies with hormone sensitive cell lines, see NMDR when two (or more) monotonic responses affect a common endpoint (e.g. cell proliferation). Frequency of effects may peak in the mid-dose range, then reverse direction at a higher dose As one effect is replaced by another (more biologically important?) effect. e.g. Decreased glans penis weight identified only at mid dose; penis malformations identified only at high dose. In studies where hundreds of endpoints are evaluated, may see NMDR (e.g. multi-generation exposures in rodents) 27

2. Do NMDRs capture adverse effects that are not captured using our current chemical testing strategies (i.e., false negatives)? There are certainly adverse biological changes that may occur in a nonmonotonic manner that would not be captured using current testing strategies. No testing strategy is able to assess all potential adverse effects, for all biological systems, in all tissues, for all species, in all developmental time points. (Executive Summary) Question 2 was further expanded for clarification. 28

2a. Are there adverse effects with NMDRs that are not being identified using the current chemical testing strategies? Assessment of the adequacy of the current testing assays concluded that a number of standardized short- and longterm assays are sensitive in detecting chemicals that interfere with the E, A, and T signaling pathways. The EDSP screening battery can detect disruption of these pathways using combined in vitro and in vivo assays in mammalian and aquatic models. Standard multigenerational guideline tests have measures that are sensitive to disruption of the E and A signaling pathways. 29

Response (Tumor or Nontumor Data) 2a. Are there adverse effects with NMDRs that are not being identified using the current chemical testing strategies? The objective of USEPA s chemical testing strategy is not to identify all possible adverse effects, but rather, to identify sensitive endpoints relevant to human or ecological health, providing confidence that adverse effects are not being induced at dose levels below what was determined to be a NOAEL. Dose response assessment is not the focus of screening assays Environmental Exposure Levels of Interest x (Lowest 95% Confidence Limit on Dose) (Central Estimate) Empirical Range of Observation 30 10% x 0% Linear Default x x LED 10 ED 10 x Range of Extrapolation 0 UF Nonlinear Default Dose x NOAEL x LOAEL

2b. Are there NMDRs for adverse effects below the no observed adverse effect levels (NOAELS) or benchmark doses (BMD) derived from the current testing strategies? Emphasis of the review was on effect of NMDR on hazard identification. But for E, A, or T we saw little evidence of NMDRs for adverse effects below the NOAELS or BMD derived from the current testing strategies. It appears that data from current testing approaches support determination of appropriate NOAELS in the low dose range of exposure, even if not all effects for every chemical are identified. Reproducible NMDRs for adverse effects occur in the high dose region of the dose response curve. 31

2b. Are there NMDRs for adverse effects below the no observed adverse effect levels (NOAELS) or benchmark doses (BMD) derived from the current testing strategies? Modifications to current strategies, which could lead to a more clearly defined dose response characterization and could increase the statistical power to detect low dose effects may be appropriate in specific instances. 32

2c. Do EPA chemical testing strategies detect relevant adverse effects for chemicals which produce NMDR for specific endpoints? Standardized short and long term assays are sensitive in detecting chemicals that interfere with the E, A, and T pathways. USEPA testing strategies are reviewed periodically to assure they are incorporating the most sensitive and biologically relevant endpoints. 33 If an objective of testing is to define the shape of the dose response curve and to identify potential NMDRs, then the three treated groups and a control group used in current guideline studies may not be sufficient for this purpose.

3. Do NMDRs provide key information that would alter EPA s current weight of evidence conclusions and risk assessment determinations, either qualitatively or quantitatively? NMDR (nor other dose response curves) should not be assumed to be irrelevant or spurious Data from studies in which NMDRs are identified may be biologically relevant and as such should be evaluated in context with the totality of the available scientific data in WoE conclusions and risk assessment determinations. 34

3. Do NMDRs provide key information that would alter EPA s current weight of evidence conclusions and risk assessment determinations, either qualitatively or quantitatively? These data should be considered and analyzed, as all data are, and factored into the WoE based on standard criteria including, but not limited to, conduct of the studies, representation of biological processes that are relevant to the evaluation, biological plausibility, and reproducibility. 35 NMDRs can have impact on both qualitative and quantitative risk assessments, but cannot be considered in isolation from other data for the chemical and biological response being considered.

Conclusions 1. NMDRs do occur in estrogen, androgen, and thyroid systems in ecological and mammalian studies. 2. NMDRs are not unexpected in vitro, particularly when evaluating high dose levels and/or lower-order biological endpoints in estrogen androgen or thyroid systems. 3. NMDRs are not commonly identified in estrogen, androgen, or thyroid systems in vivo and are rarely seen in apical endpoints after low-dose and/or long-term exposure. 4. The nature of a dose response will vary over time, and nonmonotonicity due to compensation may be observed. Office of Research and Development 36

Conclusions 5. NMDRs observed in endocrine endpoints may be biologically relevant and should be evaluated in context with the totality of the available scientific data, including epidemiologic and human studies. 6. There is currently no reproducible evidence that the early key events involved in the expression of NMDRs that are identified at low dose are predictive of adverse outcomes that may be seen in humans or wildlife populations for estrogen, androgen or thyroid endpoints 7. Therefore, current testing strategies are unlikely to mischaracterize, as a consequence of NMDR, a chemical that has the potential for adverse perturbations of the estrogen, androgen or thyroid pathways. Office of Research and Development 37

National Academy of Sciences, Board on Environmental Studies and Toxicology External Peer Review Charge Questions: Is the State of the Science document scientifically sound and of high quality? For this evaluation, has EPA selected studies of suitable breadth, relevance, and quality? Has EPA fairly and soundly evaluated and integrated the weight of evidence from the diversity of studies (epidemiological, mode-of-action, animal testing)? Are the assumptions valid and reasonable? Are the conclusions valid? Are there potential limitations or data gaps that would substantially impact the conclusions? Office of Research and Development 38

National Academy of Sciences, Board on Environmental Studies and Toxicology External Peer Review David Savitz - Chair Andrea Baccarrelli Robert Chapin Richard Corley George Daston Russ Hauser Amy Herring Andreas Kortenkam p Heather Patisaul Elizabeth Pearce Tracey Woodruff Lauren Zeise Yilian g Zhu *Comments on panel selection being accepted thru 20 May 2013 Office of Research and Development 39

Next Steps 40 NRC External peer review Public meeting 07/23/13 EPA input to committee as requested 2 nd and 3 rd meetings closed Report in early 2014 Public comment Through NRC Response to peer review, revision of document Final document delivered to EPA Programs for their consideration in decision making

Nature News Highlights 09/24/13 Journal editors trade blows over toxicology Debate flares around European regulation of bisphenol A and other endocrine disrupters Plastic bottles and many other household items contain endocrine disrupters such as bisphenol A. ISTOCK/THINKSTOCK 41

Info about the report http://epa.gov/ncct/edr/non-monotonic.html To download the report http://epa.gov/ncct/download_files/edr/nmdr.pdf To submit public comments http://www8.nationalacademies.org/cp/projectview. aspx?key=49537 42