[MUSIC] Hello, this is David Dix. I'm with the Office of Chemical Safety and Pollution Prevention at the US EPA. And today I'll be talking about US Environment and Protection Agency's Endocrine Disruptor Screening Program. Endocrine Disruptor Screening Program or EDSP was established in response to legislation passed in 1996. It was the Food Quality Protection Act to included amendments to FIFRA, essentially the pesticide laws in the US. FFDCA, the Federal Food Drug and Cosmetic Act which also covers pesticide chemicals, and a complementary amendment to the Safe Drinking Water Act. And what these amendments, what the Food Quality Protection Act required was that the agency established a screening mechanism to detect activity in chemicals like natural estrogens. So estrogenic activity as well as other hormone or endocrine like activity at the discretion of the administrator for all pesticide chemicals, pesticidal actives as well as pesticidal inert chemicals. And any other substance that may be found in sources of drinking water, and that wherein a substantial population may be exposed. In the definition of these amendments and these acts, we had the establishment the endocrine disruptor screening programming, as well as a scoping of the universe of chemicals relevant for the program. The agency set up a Federal Advisory Committee beginning in 1996 and the advisory committee functioned for several years. And in 1998, provided advice on how to establish the EDSP, the Endocrine Disruptor Screening Program, setting it up in three stages. Prioritization of chemicals, screening of chemicals for endocrine bioactivity, and then the chemicals that show relevant bioactivity and meet other requirements of the acts, further testing as necessary to establish dose response and adversity related to that endocrine bioactivity. And so, the program was established from the very beginning prioritizing chemicals using, ideally, high throughput and computational methods, and then rapidly screening the thousands of relevant chemicals to identify endocrine bioactivity. And taking the number of chemicals that show relevant endocrine bioactivity into more intense testing. And if you look at the three-part figure here, it lists the number of types of data sources in these boxes below, prioritization, screening, and testing, from quantitative structure activity relationships to high throughput screening and high throughput exposure tools such as EPA's ToxCast and ExpoCast programs. And these are readily described on EPA websites, as well as other monitoring data and other scientifically relevant information, OSRI, O-S-R-I. So that's also listed in this slide. And then it also lists the potential for Tier 1 and Tier 2 data at the screening and testing levels, so EDSP Tier 1 data and EDSP Tier 2. And I'll talk future sections of this presentation at some length about some of these types of data that come into play at the prioritization, screening and testing phase of the endocrine disruptor screening program. So over the course of the first ten years or so of the program, so from 1998 until 2008, a lot of energy was put into establishing these tiers of testing. And on the slide it lays out, in a very simple diagrammatic form, the span of biology of EPA EDSP Tier 1 test which measure molecular interactions between the chemical and biological targets, endocrine targets, leading to cellular responses and organ responses in either cell-based or animal-based screening systems. It also lays out the span of EPA EDSP Tier 2 test running from cellular responses and organ and organismal responses relating to potential population effects. And what we're looking to do in the screening and testing program is to identify and link endocrine bioactivity to adversity in the right portion of this diagram. And it's particularly through the EPA EDSP Tier 2 test to link this endocrine bioactivity to potential adversity in either a toxicity pathway or in a more extensive adverse outcome pathway. And I'll get back to these concepts later in the presentation, concepts of toxicity pathway and an adverse outcome pathway. But the way the screening and testing program, the EDSP was established, was along endocrine biological pathways. Estrogen, androgen and thyroid leading from endocrine bioactivity to adversity. And so the EPA's Endocrine Disruptor Screening Program was set up to test for various steps in these pathways leading from endocrine activity to adversity. Now we'll be talking about high throughput screening technologies and solutions that are being applied in the Endocrine Disruptor Screening Program. And the motivation for making use of high throughput screening technologies was a driver behind a pivot in the program over the past year in 2015. From a low-throughput approach that was allowing us to look at in the case of EDSP List 1, 67 chemicals, or in EDSP List 2, 107 chemicals, to a high-throughput approach that allows us to look at thousands of chemicals. And quickly address the 10,000 chemicals that are relevant to the Endocrine Disruptor Screening Program. Between pesticidal chemicals and nonpesticidal chemicals, potential drinking water contaminants, there are approximately 10,000 chemicals that we would like to prioritize per screening, and then rapidly screen a large portion of those 10,000 chemicals for endocrine activity. To do that we need to have high throughput solutions, predictive models, computational approaches that can efficiently allow us to work through these thousands of chemicals in the course of years, as opposed to decades or even longer. In the current approach of EDSP Tier 1 testing which is very animal dependent would literally take us decades. So EDSP list 1, we began working at that list of chemicals those 67 chemicals in 2009. And it took a little over five years to generate the data using the low throughput animal intensive tier 1 tests or screens to screen those 67 chemicals. So we needed to pivot the program to the high throughput and predictive models, computational models and we accomplished that in 2015. EDSP universe of chemical that are relevant for screening and testing include about over 800 conventional pesticidal active ingredients, over 300 antimicrobial active ingredients, 287 biological pesticide active ingredients. And then over 2,200 non food use, pesticidal formulation inert ingredients and over 1,500 food use inert ingredients. Additionally, there's over 1,500 fragrances which are also included as inert ingredients, pesticide formulations. And on top of those thousands of pesticidal actives and inert chemicals, over 3,000 chemicals as a minimum that are potential Safe Drinking Water Act relevant chemicals, potential contaminants of sources of drinking water here in the US. So at a minimum, there's over 10,000 chemicals that are relevant to the EDSP that we'd like to prioritize, and to a large degree, screen for potential endocrine activity. As far as the pesticidal actives and inerts, all of those many thousands of chemicals need to be screened for endocrine activity. So this was the driver for the pivot in the program to the use of high throughput assays and computational tools. We presented the first set of these tools in June of 2015 in the Federal Register asking for public comment as well as explaining the use of these high throughput assays and computational tools and the Endocrine Disruptor Screening Program. We're using those tools both to contribute to the weight-of-evidence evaluation of a chemical's potential bioactivity. But also and more significantly, to provide alternative data for specific endpoints relevant to the EDSP Tier 1 battery, to provide alternatives to the assays in the EDSP Tier 1 battery. We started developing these tools in the estrogen pathway. And those are what are presented in the Federal Register notice from June of 2015. But we also are developing complementary alternatives in the androgen and thyroid pathways. This table lays out the EDSP tier 1 battery of assays, as well as the EDSP tier tests. So I mentioned these earlier, tier 1 battery for identifying endocrine activity, and tier 2 tests for defining dose response and linking endocrine activity to adversity in reproduction or development. And you can look across this table and see that for the estrogen receptor binding assay, the estrogen receptor trans-activation assay and the in vivo or animal based uterotrophic assay. We've developed an alternative, the estrogen receptor model which is a collection of 18 high throughput screening assays, and a predictive model incorporating or integrating those data as an alternative to these first three EDSP Tier 1 assays. And this is exactly what was presented in the Federal Register notice in June of 2015. But the estrogen receptor model, the 18 ToxCast assays and the predictive model integrating those data as an alternative for the three tier 1 capacities, the Binding, the Transactivation, and the uterotrophic assay. So this is the first 3 out of the 11 tier 1 battery of assays that we've provided an alternative set of high-throughput and predictive model that could be utilized in lieu of the low throughput animal intensive Tier 1 assays. Working down the table, you can see we intend in the near future to provide similar androgen receptor models, AR model, alternatives for the AR binding and Hershberger assays. The Hershberger is another animal based, rat based assay. And in addition, steroidogenesis models, STR models using high throughput assays and predicted pathway models, and then combinations of this models, the The steroidogenesis, the AR and the thyroid, THY model. Again based in high throughput screening assays and predictive pathway models to provide alternatives to all the rest of the Tier 1 battery including the in vivo or in-life rat pubertal assays, fish short-term reproduction assays, and amphibian metamorphosis assays. So these high throughput screening, assays and computational or predictive models will, if successful over the course of the coming year or two, provide alternatives to the full EDSP Tier 1 battery of assays which are very low throughput and very animal and cost intensive. Moving beyond just the Tier 1 screening assays, we think that there's some potential for the high throughput assays in computational or predictive models to potentially provide alternatives to the EDSP Tier 2 tests. This is a longer term aspect of the project. We'll likely involve additional assays, additional modeling beyond what's necessary for the Tier 1 screening level alternative development. The Endocrine Disruptor Screening and Testing Program, as I mentioned earlier, was designed to test three different endocrine pathways, the estrogen pathway, the androgen pathway, and the thyroid pathway. That's represented in this slide as estrogenic or estrogen active, E+, antiestrogenic or anti is E-, and then androgen active or androgenic is A+, and antiandrogenic is A-. And then the thyroid axis is represented as HPT indicating the hypothalamic-pituitary-thyroid axis. And that's indicated relevant to the Tier 1 assays and the the Tier 2 assays in this diagram. You can see then, for example, the Binding, the estrogen receptor binding assay is relevant to the two estrogen pathways, positive and negative estrogenic and anti estrogenic, since something that binds the estrogen receptor pathway could be either. In comparison, the Transactivation assay, the second assay from Tier 1 is specific and only right in estrogenic not the anti-estrogenic mode. You see the AR binding assay relevant to the A+ and A- pathways. Steroidogenesis and then in vivo assays all relevant to specific estrogenic, androgenic or in the case of, for example, the amphibian metamorphosis assay specific to the HPT or thyroid pathway. Other assays are relevant to both estrogen and androgen, for example the fish short term reproduction. And then the pubertal female for her estrogen, pubertal male for androgen and in both cases they also are sensitive and detect effects mediated through the thyroid pathway. So you can infer from this the difficulty or complexity in interpreting some of these in vivo test results. The four Tier 2 tests in rat or the mammalian tests, the EOGRTS in fish in medoca, the MEOGRT assay, in frog, in Xenopus, the LAGDA assay. The amphibian assay, the inferred or Japanese quail, the JQTT assay are all extended one generation or multi-generational Tier 2 tests which will detect a variety of developmental and reproductive effects that could be mediated through any of the three relevant pathways to the program. The estrogen, androgen or the thyroid pathway. So at this point in time, we've made significant progress in developing high throughput alternatives to the EDSP Tier 1 battery of assays. For the estrogen receptor pathway, we have data coming online for close to 3,000 chemicals in the near term. For the androgen receptor pathway, it's a similar state. They have in hand data on close to 2,000 chemicals for both the estrogen receptor, androgen receptor and steroidogenesis data. The thyroid pathway we have data on about 1,000 chemicals, and some related metabolic data relating to hepatic clearance on about half of those. And so, you can see a growing body of data that will allow us to address the thousands of chemicals relevant to the Endocrine Disruptor Screening Program in a way that's much more efficient than the EDSP Tier 1 battery. EDSP Tier 1 battery, I have mentioned took about a decade to develop and validate, and over five years to generate data on just the first 52 chemicals. So we need a faster alternative and a high throughput and computational tools coming from ToxCast and Tox 21 are providing that. We'll have data on thousands of chemicals for the estrogen receptor, estrogen pathway, for the androgen receptor, the androgen pathway as well as steroidogenesis and thyroid in the coming years. And we can use these data as an alternative way to screen for endocrine activity that could lead to a higher risk for endocrine disruption from those chemicals. This ends this section introducing the high throughput and predictive pathway models as alternatives to the Endocrine Disruptor Screening Program, and we'll now be moving on to talk more specifically about one of those models, the estrogen receptor bioactivity model.
