By Lara A. Hall, MS, RQAP-GLP, Jane S. Vergnes, Ph.D., DABT®, and Lisa M. Campbell
On Tuesday, August 25, 2015, in a Federal Register notice, the U.S. Environmental Protection Agency (EPA) announced the addition of three Office of Chemical Safety and Pollution Prevention (OCSPP) final test guidelines to its 890 Series, entitled “Endocrine Disruptor Screening Program Test Guidelines,” as follows:
These test guidelines are part of a series of test guidelines established by OCSPP for use in developing data on potentially adverse effects of pesticides and chemical substances on the endocrine system for submission to EPA under the Federal Food, Drug, and Cosmetic Act (FFDCA) section 408 (21 U.S.C. 346a), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.). These final guidelines have been revised based on public comments received following the release of draft test guidelines in January 2015, existing EPA test guidelines, and concurrent Organisation for Economic Co-operation and Development (OECD) test guidelines.
EPA worked with OECD to harmonize test guidelines for MEOGRT and LAGDA. The specific OECD Guidelines for the Testing of Chemicals, Section 2, that apply to MEOGRT and LAGDA, are available here. Substantive changes reflected in the final OCSPP MEOGRT and LAGDA test guidelines include:
1. The test will end following hatching of the second generation (F2) offspring. The option for extending the MEOGRT through reproduction by the F2 generation has been removed from the final test guideline pending additional data. This is consistent with the decision made in the draft OECD test guideline for MEOGRT. This test guideline may be updated as new information and data are considered. For example, guidance on extending the F2 generation through reproduction may be potentially useful under certain circumstances (e.g., chemicals with high bioconcentration potential or indications of trans-generational effects in other taxa).
2. The mean water temperature over the duration of the MEOGRT has been changed to 25 ± 2 °C to be consistent with the analogous OECD test guideline.
3. The LAGDA developmental stage terminology has been clarified to avoid confusion with what is meant by complete metamorphosis.
4. An effort was made to clarify and provide more explicit guidance as to what specific histopathology is appropriate based on the results of the study, e.g., the conduct of liver and kidney histopathology in the MEOGRT and LAGDA test guidelines with respect to overt toxicity.
5. The rationale for use of solvent control only, dilution water control only, or pooled controls in the statistical analyses for the MEOGRT and LAGDA was clarified.
6. The guidelines have been modified to address commenters' concerns that they be more flexible and less prescriptive. Examples have been provided as appropriate to add clarity.
The JQTT draft test guideline (OCSPP 890.2100) was revised to address comments provided by the public, the draft OECD test guideline for the avian two-generation toxicity test in the Japanese quail, as well as the existing EPA test guidelines and OECD test guidelines for avian one-generation toxicity tests.
EPA revised the terminology, procedures, endpoints measured, figures, tables, and appendices in the JQTT test guideline to clarify specific points raised by public commenters, and to address commenters' concerns that they be more flexible and less prescriptive, as follows:
1. The revised test guideline includes fewer endpoints. For example, the revisions eliminated behavioral endpoints to reduce the overall numbers of birds required for the study; eliminated endpoints that are difficult to obtain (i.e., hormone levels measured in embryo blood samples); and eliminated redundant endpoints and statistical analyses.
2. For clarity, the test terminates with measurement of the 14-day survival of the F2 generation chicks. This is the minimum length of the study necessary to evaluate and measure a chemical's effect on the first generation’s (F1) reproductive performance. If delayed reproduction is observed in F1 birds, a decision to extend the F2 generation may be made. If extended, the test should be terminated when F2 birds are approximately 6 weeks old, when 90 percent of control animals have reached sexual maturity. The decision to limit the length of the JQTT is consistent with EPA's efforts to move to extended one-generation reproduction test protocols for Tier 2 testing rather than require multigenerational studies. Extended one-generation reproduction tests are scientifically justified in many cases, reduce the use of animals in testing, and reduce costs.
Electronic access to OCSPP test methods and guidelines is available here.
The release of these final testing guidelines marks another significant step in the overall Endocrine Disruptor Screening Program (EDSP), making way for the anticipated Tier 2 testing phase with the List 1 chemicals. EPA recently released Tier 1 weight-of-evidence assessments for List 1 substances in the EDSP and registrants are now receiving the associated data evaluation records (DER) for the Tier 1 screening studies. The purpose of the Tier 1 screening was to identify potential interactions with three hormonal pathways (estrogen, androgen and thyroid) in the endocrine system. As a result of the potential interactions with one or more of these pathways observed, EPA has recommended Tier 2, multigenerational studies across various species for 18 of the 52 List 1 chemicals, including the MEOGRTS (13 chemicals) and LAGDA (5 chemicals). Test Orders for Tier 2 studies will be issued following completion of the Information Collection Request (ICR) process within the Office of Management and Budget (OMB). The public review and comment period for this ICR concludes on September 2, 2015, with a response from OMB to follow in early October 2015. In the interim, registrants are closely reviewing their respective assessments and DERs, and industry groups are assessing the approach employed by EPA in these Tier 1 assessments, including but not limited to, the statistical reanalysis of study data and conclusions drawn by EPA.
By Lara A. Hall, Lisa M. Campbell, and Jane S. Vergnes, Ph.D.
The release of the first Tier 1 assessments in the Endocrine Disruptor Screening Program (EDSP) by the U.S. Environmental Protection Agency (EPA) on June 30, 2015, is a significant bench-mark in the program since the original List 1 test orders were initially issued in October 2009. Fifteen of the original 67 List 1 chemicals were subsequently cancelled or discontinued by the respective pesticide registrants. The intended purpose of the Tier 1 screening was to identify potential interactions of the remaining 52 chemicals with three hormonal pathways (estrogen, androgen and thyroid) in the endocrine system. EPA evaluated the data from 11 screening assays (five in vitro and six in vivo), along with other scientifically relevant information (OSRI) comprised of existing toxicology studies and peer reviewed published literature, and drew preliminary conclusions about the potential of these 52 chemicals to disrupt endocrine functions. Importantly, EPA noted on the EDSP webpage that “a result indicating potential should not be construed as meaning that EPA has concluded that the chemical is an endocrine disruptor.”
The individually published weight-of-evidence (WoE) assessments and anticipated data evaluation records (DER) can be accessed online for the 52 chemicals involved in the Tier 1 screening assessment. EPA summarized its conclusions from EPA’s Tier 1 WoE assessments as follows:
- No evidence of potential interaction with any of the endocrine pathways was identified for 20 chemicals.
- Fourteen chemicals showed potential interaction with one or more pathways, but based on the available information, do not pose a risk for endocrine disruption.
- Eighteen chemicals showed potential interaction with the thyroid pathway, 17 of which also showed potential interaction with the androgen pathway, and 14 of which showed potential interaction with the estrogen pathway.
To explore further any potential adverse effects on the endocrine system that may be caused by the 18 chemicals that EPA categorized in the third group noted above, EPA has recommended the following Tier 2, multigenerational studies across various species for them:
- A comparative thyroid assay for four chemicals that EPA found to have potential interaction with the thyroid pathway in mammals;
- The Medaka Extended One Generation Reproduction Test, MEOGRT (Draft Test Guideline OCSPP 890.2200), for 13 chemicals that EPA found to have potential interaction with the estrogen or androgen pathways in wildlife; and
- The Larval Amphibian Growth and Development Assay, LAGDA (Draft Test Guideline OCSPP 890.2300), for five chemicals that EPA found to have potential interaction with the thyroid pathway in wildlife.
EPA has not yet issued its final Tier 2 non-mammalian Office of Chemical Safety and Pollution Prevention (OCSPP) test guidelines (890 Series). Public comment on the proposed guidelines closed on March 31, 2015. It is expected that the release of these remaining guidelines will signal the approach of the Tier 2 test orders, which EPA is likely to issue in 2016. Although a formal public comment period is not expected to be opened for the Tier 1 assessments, affected registrants should have the opportunity to respond directly to EPA regarding WoE assessments and forthcoming DERs.
This release of the Tier 1 WoE assessments, the anticipated release of Tier 2 test guidelines, along with EPA’s commitment to further the development of high-throughput (HTP) assays and computational tools will significantly influence the prioritization of List 2 chemicals in the EDSP and the timing of the List 2 test orders. The revised List 2 includes 109 chemicals for Tier 1 screening. As with List 1, List 2 candidates reportedly were selected based on EPA’s review concerning their possible presence in public drinking water and/or registration review status within EPA, and not because of their potential to interfere with the endocrine systems of humans or other species.
By Lisa M. Campbell and Jane S. Vergnes, Ph.D.
On June 19, 2015, the U.S. Environmental Protection Agency (EPA) announced a plan for incorporating validated high throughput assays and a computational model into the Endocrine Disruptor Screening Program (EDSP) to screen chemicals for their ability to interact with the endocrine system. These proposed new methods would serve as an alternative for three of the eleven current assays in the EDSP Tier 1 screening battery, specifically the estrogen receptor binding (ER), estrogen receptor transactivation (ERTA), and uterotrophic assays.
These computational high-throughput (HTP) tools will have the potential to impact significantly the prioritization for testing and will have a significant impact on List 2 test orders. EPA states that use of these alternative methods will accelerate the pace of screening, decrease costs, and reduce animal testing. In addition, this approach advances the goal of providing sensitive, specific, quantitative, and efficient screening using alternative test methods to some assays in the Tier 1 battery to protect human health and the environment. EPA has stated its commitment to the development of HTP and computational tools to improve regulatory science, as recommended in the 2007 National Research Council report, “Toxicity Testing in the 21st Century: A Vision and a Strategy,” and to meet its statutory obligations in the face of challenging budget constraints.
Comments are due by August 18, 2015. EPA specifically seeks comment on the following issues, which are related to its stated intention to use the scientific tools discussed as alternatives to some of the current EDSP Tier 1 screening assays:
- The use of the ToxCastTM “ER Model” for bioactivity as an alternative method for the current ER binding and ERTA Tier 1 screening assays.
- The use of the ToxCastTM “ER Model” for bioactivity as an alternative method for the current uterotrophic Tier 1 screening assay.
- The use of results from the ToxCastTM “ER Model” for bioactivity on over 1800 chemicals as partial screening for the estrogen receptor pathway.
EPA concludes that the ToxCastTM “ER Model” meets the criteria for use as “other scientifically relevant information” to satisfy Tier 1 for the ER, ERTA, and uterotrophic assays. EPA’s conclusion is based upon the findings of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) peer review held in December, 2014 that endorsed the ToxCastTM “ER Model” as a replacement for the ER and ERTA assays, and additional data developed by EPA to address the SAP’s concerns regarding the uterotrophic assay. Recipients of EDSP Tier 1 test orders would have three options for addressing the Tier 1 requirements with respect to the three EDSP Tier 1 endpoints that EPA considers validated:
- Cite existing ToxCastTM “ER Model” data, if it is applicable.
- Generate new data using the 18 ER HTP assays and the ToxCastTM “ER Model.”
- Generate Tier 1 data using the validated methods for the ER, ERTA, and uterotrophic endpoints in the traditional EDSP Tier 1.
EPA is careful to note that activity in the ToxCastTM “ER Model” is not a determination that a chemical causes endocrine disruption, only that is has the potential to do so, and that further testing (Tier 2) would be needed to make a determination regarding the ability to cause “adverse effects in an intact organism or its progeny, or subpopulations,” as stated in the World Health Organization International Programme on Chemical Safety definition.
More detailed information on the Endocrine Disruptor Screening Program and its use of computational tools is available at: http://www.epa.gov/endo/ or http://www.epa.gov/endo/pubs/pivot.htm.
By Lynn L. Bergeson
On January 30, 2015, the U.S. Environmental Protection Agency (EPA) released drafts of new guidelines for animal testing of the endocrine disrupting effects of pesticides and other chemicals. The proposed guidelines outline how scientists can use Japanese quail, medaka fish, or amphibian larvae to conduct various endocrine tests. EPA reportedly also considered including mysid crustaceans on its list of non-mammals acceptable to use in endocrine testing, but did not because the data were not deemed "fully reliable" across all endpoints. Comments are due March 31, 2015.