Speaker abstracts (in alphabetical order)

Gerald Ankley (US EPA, USA)

Possible strategies for EDC testing in the future: exploring roles of pathway-based in silico, in vitro and in vivo methods.

Current methods for screening, testing and monitoring endocrine-disrupting chemicals (EDCs) rely relatively substantially upon moderate- to long-term assays that can, in some instances, require significant numbers of animals. Recent developments in the areas of in vitro testing (e.g., high through-put technologies), in vitro/in vivo genomics, and computational biology (including bioinformatics) could provide the basis for efficiently detecting chemicals with the potential to perturb specific biological pathways within the hypothalamic-pituitary-gonadal and -thyroidal axes. These approaches, while not yet capable of wholly replacing longer-term whole-organism tests, nonetheless could fill roles related to screening/prioritization of large numbers of test chemicals for endocrine activities, or rapid broad-scale environmental monitoring of EDCs. This presentation will explore some of the newer technologies being considered for EDC testing and monitoring, including their potential advantages and drawbacks.

John Carter (Huntingdon Life Sciences, UK)

Contract research experience: Validation, conduct and reporting of in vivo mammalian and in vitro EDSP assays

A conventional perception of ‘screening’ assays might reasonably encompass a set of rapid, cost effective tests that are readily performed and succinctly reportable. In reality, our experience with each of the EPA EDSP Tier 1 screening battery tests presented an array of scientific, resource and even ethical issues that ‘en masse’ constituted  a significant challenge to delivery of a comprehensive contract ‘screening’ service.  In a multi-disciplinary organisation, shared experiences facilitated a standard approach to validation, problem solving, operational conduct and reporting of the EDSP assays including production of DER documentation for each completed screen.  

However, validation of the in vitro ‘analytical’ assays was found to require more than simple adherence to the published OPPTS guideline methods.  Multiple acceptance criteria and multiple reference chemicals, not least in the ER Transcriptional Activation assay, required adoption of a pragmatic approach to assessing validity of replicate assays together with sufficient and appropriate statistical support.  Prolonged attempts to consistently achieve the 2-fold increase in response to Forskolin as stipulated in the Steroidogenesis assay guideline were later acknowledged by EPA to be common to sufficient numbers of laboratories to warrant lowering of the acceptable Forskolin response to 1.5 fold. Successful validation of assays was tempered by the sober need to maintain sufficient validated ‘analysts’, whilst the relevance of somewhat ‘traditional’ ex-vivo receptor binding assays was considered questionable in the presence of alternative and specific  human cell line assays.

Operational refinements were introduced where possible - logistical consideration of the in vivo mammalian male and female pubertal assessments led to a single, combined study design enabling lower animal usage and reduced reporting time whilst robotic automation of the in vitro aromatase assay led to increased reproducibility.

Anne Gourmelon (OECD, FR)

What EDC-sensitive in vitro, in vivo mammalian and in vivo wildlife screens and tests are now standardised and published?

In 2002, the OECD Task Force on Endocrine Disrupter Testing and Assessment launched a Conceptual Framework structuring available test methods in a logical fashion, following information they provide on the endocrine activity of tested chemicals.  Followed a long journey to optimise, validate and finally adopt several Test Guidelines that are now used in several OECD countries to screen and test chemicals in vitro /in vivo for human health and the environment. Where possible, existing OECD Test Guidelines have been updated to include new endocrine-related endpoints, otherwise completely new Test Guidelines have been developed. In addition, about one third of publications in OECD Series on Testing and Assessment include validation reports, guidance and other documents aiming to support the application and interpretation of results from ED-related Test Guidelines.  In 2012, an OECD guidance document was drafted to support regulatory authorities in evaluating the evidence that chemicals are, or not, endocrine disrupters, based on the mass of information available.  Undoubtedly, there is still room to improve and combine more efficiently the tools available to authorities to support decision-making.

Tom Hutchinson

Although concern about reproductive health impacts of environmental contaminants goes back several decades in terms of wildlife populations, it was not until the 1990’s that wider awareness of this issue grew beyond the research community.  In the 1990’s, the US passed the Food Quality Protection Act (1996) and amended the Safe Drinking Water Act (1996) which mandated the US Environmental Protection Agency to develop an endocrine disruptor screening program (EDSP).  Similarly, the Japanese government established in 1998 the Strategic Program on Environmental Endocrine Disruptors, with similar initiatives taking place in many European countries and through the European Union Framework Research Programmes.  In the same period, in November 1996 the OECD took the initiative at the 25th Joint Meeting of the OECD Environmental Health and Safety programme to establish a special activity on Endocrine Disrupter Testing and Assessment (EDTA).  This initiative was made at the request of OECD member countries and international industry to ensure that testing and assessment approaches would not differ substantially among nations.  The focus of the EDTA activity was agreed to: (1) provide information on and coordinate national and regional activities concerning endocrine disrupter assessment; (2) develop new, and revise existing OECD test guidelines to detect endocrine disrupters; and (3) harmonize hazard and risk assessment approaches for endocrine disrupters.  Since 1996, a number of new OECD test guidelines have been successfully developed and validated for mammals, amphibians and fish.  Reflecting the priority given to the reproductive and developmental processes studied in research studies, the OECD test guidelines have prioritised an assessment of androgenic, oestrogenic, steroidogenic disruption and thyroid-related endpoints in model species.  At the same time, the OECD has developed a Conceptual Framework to provide guidance concerning the newly adopted test guidelines or those which might need to be developed in the future and what types of information they are provide with respect to endocrine disrupter assessment. Originally devised at the 6th OECD EDTA meeting in 2002, the Framework is intended to apply to both new and existing substances in different chemical sectors such as industrial chemicals, pharmaceuticals and plant protection products.  The Conceptual Framework was updated in 2011 to take into account the progress made in validating new mammalian and non-mammalian (environmental) test guidelines over the past several years.  Using ethinylestradiol, octylphenol and vinclozolin as case studies, the presentation will illustrate examples of how diverse types of in vitro and in vivo data can be evaluated in the revised OECD Conceptual Framework to illustrate its structure and utility.

Gerald A. LeBlanc (North Carolina State University, USA)

Novel Modes of Endocrine Action and Disruption

Reported increased incidents of reproductive dysfunction and population declines in wildlife populations have raised concern that disruption of key endocrine-signaling pathways by exposure to environmental chemicals may be involved. Efforts, to date, to identify endocrine disrupting activities of chemicals have focused largely upon the estrogen, androgen, and thyroid hormone signaling pathways. However, other endocrine pathways may be susceptible to environmental chemical-induced disruption in a manner that could contribute to increasing incidents of some disorders in wildlife populations. Candidate endocrine pathways include the hypothalamus:pituitary:adrenocortical (HPA) axis, the retinoid signaling pathway, the vitamin D signaling pathway, and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Potential targets of disruption along putative adverse outcome pathways associated with the signaling pathways were identified, along with assays that show promise in evaluating the target in a screening and testing program. Disruption of the HPA signaling pathway could alter processes that contribute to population sustainability, and this adverse outcome pathway has been well defined. Retinoid signaling via the retinoid X receptor cross-talks with several other signaling pathways through dimerization of the RXR with other hormone receptors.  Thus, disruption of retinoid signaling can result in multiple adverse outcomes.  Vitamin D signaling is involved in the development, maintenance, and function of many tissues and chemicals that disrupt vitamin D signaling, thus far identified, act primarily by altering Vitamin D metabolism.  PPARs are involved in lipid and glucose homeostasis, inflammation, and aspects of development. The adverse outcome pathway for PPARg is well established. In conclusion, several endocrine signaling pathways in addition to estrogen, androgen, and thyroid hormone pathways contribute to the maintenance of wildlife populations and may be susceptible to environmental endocrine disruptors.  OECD test guidelines can be modified to include new assays or the incorporation of novel endpoints into existing assays that would expand the repertoire of endocrine signaling pathways included in the screening and testing regimen.

Peter Matthiessen (Consultant ecotoxicology, UK)

What ecotoxicity tests for EDCs are still needed but have not yet been internationally standardised?

Great strides have been made by OECD in the last decade to develop and validate in vivo ecotoxicity tests with sensitivity to so-called EATS endocrine disrupting chemicals (those interacting with the estrogenic, androgenic, throid and steroidogenic systems). They include screening assays with fish and amphibians, and higher level tests with insects and fish. However, the 'toolbox' is far from complete, especially at the higher tiers required to follow up alerts raised at the screening stage, and the range of taxa available for testing is still limited. This presentation will provide information on ecotoxicity tests still being developed by OECD, including those with crustaceans, molluscs, fish, amphibians and birds. It will also discuss the need for yet other tests which are not currently part of the OECD validation programme, but which may be needed in future, including those for endocrine modalities not yet under consideration (e.g. interactions with the corticosteroid system).

Ellen Mihaich (Environmental and Regulatory Resources, USA)

The Law of Unintended Consequences: The Endocrine Regulations

In 1996, in response to public concern that some chemicals may interact with the endocrine system in humans, regulations were adopted in the US directing the US EPA to establish a screening program using "validated test systems" to investigate the potential for adverse health effects to be induced through endocrine pathways.  While the charge sounded simple enough, it neglected to consider the complex system being targeted for evaluation.  Policies and procedures had to be put in place, screens and tests had to be developed and validated, and significant resources had to be earmarked in order to comply with the program.  The US Endocrine Disruptor Screening Program (EDSP) has taken more than 10 years and millions of dollars to reach the point of initiating test orders for the performance of the screening battery for the first 67 chemicals in the program.  Now, US EPA is evaluating the screening data from this first list of chemicals and making decisions on what, if any, additional testing is required.   We look at the challenges faced in this complex regulatory program.  There were costs and impacts, expected and unexpected.   It is important to consider the progress made and any lessons learned as the program evolves to consider high-throughput screens and adverse outcome pathway.  It is also relevant to consider the impact testing results may have outside of the US as various regions around the globe work to implement their own definitions and criteria for endocrine disruption.

Sharon Munn (EC - JRC)

Feedback on progress with development of EU regulatory criteria for EDCs.

  • Underlying any regulatory controls of endocrine disrupting chemicals should be sound consistent scientifically-based criteria for the identification of EDCs.
  • A group of experts nominated by Member States, EU Agencies and relevant industry and non-governmental organisations, and led by the Joint Research Centre's Institute for Health and Consumer Protection (JRC-IHCP), have been tasked with providing advice to the European Commission on the development of such scientific criteria.
  • The presentation will focus on the progress of the discussions within this group.

Tinka Murk (Wageningen University, NL)

Novel in vitro and genomics methods in development as alternatives for vertebrate assays; the case of thyroid homone disrupting compounds (THDC).

THDC have many potential mechanisms of action, including altered hormone production, transport, metabolism, receptor activation and disruption of several feed-back mechanisms. Therefore it often is stated that it is not possible to replace in vivo by in vitro testing because effects may be missed or exaggerated  For several, but not all, of these endpoints in vitro assays are available. Modern -omics technologies applied to in vivo studies can help to reveal relevant and possibly new endpoints to include in a targeted THDC in vitro test battery. To be able to translate the in vitro to in vivo effects, kinetics of the compounds can be estimated using in vitro or in silico approaches to predict effective chemical concentrations in the blood following exposure. This presentation will briefly review known mechanisms for THD and in vitro THD tests currently available or under development for these endpoints, recommend a battery of rationally chosen test methods for a tiered approach with triggers for prioritization of compounds for further hazard and risk assessment, and indicate priority research gaps and needs.

Lisa Ortego (Bayer CropScience, USA)

How Hard Can Tier 1 Screens Be?  The Reality of EDSP Test Order Compliance

In 2012, the US EPA's Endocrine Disruptor Screening Program (EDSP) will be engaged in the evaluation of tier 1 screening data generated over the last two years for pesticides and other chemicals by industry test order recipients.  Data from the eleven in vitro and in vivo tier 1 screening battery are to be used in a weight of evidence evaluation to identify substances with the potential to interact with the estrogen, androgen, or thyroid hormone systems. As is to be expected in the launch of any testing program of this size, there have been challenges in the execution of the assays.  Adding to the complexity is the fact that the screens were developed to be highly sensitive in order to minimize the potential for false negatives.  However, they also include measurement of effects on several apical endpoints which are not specific to an endocrine mode of action.  The screens have been challenging to perform and the interpretation phase is also complex as the battery is to be interpreted as a whole, including any additional relevant information.  The interpretation will require transparency and scientific rigor, given that the outcome of the screening battery is to decide if any tier 2 chronic or multigenerational studies are required to determine risk.

Catherine Pepper (HSE, United Kingdom)

Regulatory view of new tests and their interpretation

The adoption and validation of new tests for endocrine disruption present a novel challenge for regulatory specialists. Traditionally, ecotoxicological studies have been evaluated with a view to using them in a risk assessment and determining whether the proposed use poses an acceptable risk to the environment.  Under new regulations, there is a need to identify endocrine disrupting chemicals.  This will be done via the use of assays and a hazard assessment, as opposed to a risk assessment.  These studies will be internationally accepted studies and the determination of whether a chemical is an endocrine disruptor or not will be via recognised definitions and criteria. This presentation will highlight some of the issues and challenges faced by regulatory specialists regarding the interpretation and use of these studies in the field of environmental toxicology.

Frauke Stock (UBA, DE)

Development of weight of evidence schemes for the assessment of endocrine disruptors - current scientific discussion

Different substance regulations stipulate that substances having endocrine disrupting properties might be regulated based on their intrinsic dangerous properties. However in order to distinguish between substances needing a hazard based assessment and those subject to normal risk assessment, criteria are need which describe the intrinsic properties in more detail. Guidance is needed on how to judged that a substance does indeed shows these properties.

The WHO/IPCS definitions is suggested by many experts to be a good starting point for developing criteria: “An endocrine disrupters 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”

But how much evidence is needed to conclude that the adverse effect is a consequence of an endocrine mode of action? And should exemptions be made? How could a weight of evidence approach look like?Suggestions by different member states are summarized and critical points arising when developing a weight  of evidence approach are discussed.

Dick Vethaak (VU University Amsterdam, NL)

The impacts of endocrine disrupting chemicals on the environment: state of knowledge and implications for testing and assessment

In the last decades, field and laboratory studies have shown that the exposure to endocrine disrupting chemicals (EDCs) can cause developmental, reproductive, neural, immune, and other problems in a range of wildlife taxa, and possibly humans. The list of proven or suspected EDCs is now very long and includes natural hormones, pharmaceuticals, some metals, pesticides, personal care products and other industrial chemicals.

This introductory talk will provide background information on the issue of EDCs in the environment with focus on new findings and developments. An overview of the (potential) role of EDCs in receptor and non-receptor mediated pathways is provided. The evidence for adverse effects of EDCs and commonalities in observed effects among various wildlife taxa is presented and discussed. Relevant issues in the study of EDCs (e.g. low dose effects, nonmonotonic dose-response curves, mixture effects, sensitive life stages, ecological relevance) are addressed. Finally, implications of the available research for the regulation of synthetic substances and for the protection of the environment are considered.

The scope and magnitude of harm to wildlife populations and possibly to humans by EDCs is becoming increasingly apparent, as our knowledge and understanding increases, and demonstrates the need for prompt policy actions and the need for regulation and testing of EDCs.

James Wheeler (Syngenta, UK)

Hazard, risk and the need for a scientific approach – criteria and weight of evidence

In recent years, endocrine disruption has become a topic of increasing public and regulatory concern. This presentation will outline some of the current approaches being taken by European regulatory agencies, US-EPA and industry working groups in order to devise suitable criteria for the identification and management of endocrine disrupting chemicals. Plant protection products are likely to be impacted by recent changes which will create hazard based cut-off (ineligibility for registration) criteria for endocrine disrupting properties and so are used here to illustrate the approaches under development. A key component of all the schemes is the establishment of weight of evidence methodologies. These aim to provide an independent assessment of the data’s reliability/repeatability and relevance to inform the question of significance. Then an overall weight of evidence, incorporating all available information, can be used to conclude whether a substance has sufficient evidence to be judged against specified criteria or if further testing is required.