June 12, Via USPS and to: Dear Administrator Jones:

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1 June 12, 2014 James Jones, Assistant Administrator U.S. Environmental Protection Agency Office of Chemical Safety and Pollution Prevention (7101M) 1200 Pennsylvania Avenue, N.W. Washington, D.C Re: Use of the fish embryo acute toxicity test as a replacement for the fish acute toxicity test in satisfying ecotoxicity data requirements under FIFRA and TSCA Via USPS and to: Jones.jim@Epa.gov. Dear Administrator Jones: People for the Ethical Treatment of Animals (PETA) and the Physicians Committee for Responsible Medicine (PCRM) represent more than three million members and supporters who are concerned about the suffering of animals used in regulatory testing, particularly in cases where scientifically valid alternative methods exist that provide advancements in animal welfare and progress towards achieving the 3 Rs (refinement, reduction and replacement of animal tests). We would like to call your attention to an alternative to the acute fish toxicity (AFT) test (OPPTS /OECD 203) that is required under 40 CFR for pesticide registration as well as under 40 CFR for evaluation of chemical safety. The AFT is not compatible with current animal welfare objectives because mortality is the primary endpoint and evidence continues to mount that shows fish suffer distress and pain (e.g., Braithwaite, ; Sneddon, ; Chandroo et al ). A strong body of work now exists that demonstrates the suitability of the fish embryo acute toxicity (FET) test (OECD 236) as a valid replacement for the AFT. Instead of the juvenile fish used in the AFT, the FET test involves exposure of recently fertilized embryos (<1 hour) to a series of chemical concentrations for a total duration of up to 96 hours. (The test was originally run for 48 hours, but due to prior concerns about some large molecular weight chemicals not being able to pass the chorion, and therefore potentially resulting in an under-prediction of toxicity, the test is now recommended to continue for 96 hours to avoid 1 Braithwaite, V Do fish feel pain? Oxford Univ. Press. 256 pp. 2 Sneddon, LU The evidence for pain in fish: the use of morphine as an analgesic. Appl.. Animal Behav. Sci. 83: Chandroo, KP et al Can fish suffer? Perspectives on sentience, pain, fear and stress. Appl. Animal Behav. Sci. 86:

2 this issue). The most commonly researched species has been the zebrafish (Danio rerio) although work is now being pursued with fathead minnows (Pimephales promelas), medaka (Oryzias latipes), and others. While the test still involves the use of animals, the embryos are in a life stage that lacks a fully functioning nervous system and, thus, are presumed to exhibit a reduced reaction to pain and distress. The FET test has received considerable attention over the last decade. It has been a standard component of effluent testing in Germany since A comprehensive review conducted for the German Federal Environmental Agency (Braunbeck and Lammer 2006) 4 determined that sufficient information existed to develop standard guidance for conduct of the FET test. This was brought forward to the OECD, which then convened an ad hoc expert group in 2006 to begin development of a test guideline for the FET method using zebrafish and initiated an inter-laboratory validation study. The German guideline was standardized at an international level with production of an International Organization for Standardization (ISO) method to determine acute toxicity of wastewater effluents using fish embryos (ISO 2007) 5. An International Life Sciences Institute/Health and Environmental Sciences Institute (ISLI-HESI) workshop was held in 2008 to explore the FET test as a model for predicting acute toxicity in fish, the results of which were summarized by Embry et al. (2010) 6. Conclusions from this workshop were that the FET test was a viable alternative to the AFT but that several issues needed to be overcome before there could be global acceptance of the assay, in particular the regional bias towards the use of some species versus others. Around the same time as the workshop Lammer et al. (2009) 7 compared results of testing 142 chemical compounds using both the FET and the AFT methods and found a very strong predictive relationship between the two. Regardless of the fish species used in the AFT test, the FET test appeared to be capable of accurately predicting the outcome. Currently, the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) is finalizing a recommendation document for the FET test that describes its validity, regulatory applicability, and proper scientific use (available at: The document references the 2012 OECD validation study, which was coordinated by EURL ECVAM with the support of a Validation Management Group established by OECD and supervised by the OECD ad hoc Expert Group on the Fish Embryo Toxicity Test. It also references a parallel study conducted by Belanger and colleagues 4 Braunbeck, T., Lammer, E., Draft detailed review paper on fish embryo toxicity assays. UBA Contract Number Umweltbundesamt German Federal Environment Agency, Dessau, Germany. 136 pp. 5 ISO, Water Quality Determination of the Acute Toxicity of Waste Water to Zebrafish Eggs (Danio rerio). ISO 15088: Embry MR et al The fish embryo toxicity test as an animal alternative method in hazard and risk assessment and scientific research. Aquat Toxicol 97: Lammer E et al Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C 149:

3 ( ), which evaluated the predictive capacity of the zebrafish FET tests for acute fish toxicity by comparing data from acute FET tests and juvenile or adult AFT tests. The EURL ECVAM document concludes that: 1. The OECD validation study showed that the FET is transferable and reproducible within and between laboratories. The retrospective analysis demonstrated, on the basis of data on 144 chemicals, a strong correlation (r = 0.9) between AFT data (96 h; five freshwater species recommended in OECD TG 203) and FET data ( h exposure; mainly zebrafish). 2. The FET test can provide information on acute toxicity to fish comparable to that derived from standard tests (e.g. OECD 203). 3. The chemicals evaluated in the retrospective analysis covered a broad range of physico-chemical properties, toxicological modes of action, and functional use, e.g. industrial chemicals (77), plant protection products (21), surfactants (15), pharmaceuticals (8), and biocides (5) thereby indicating a wide applicability domain of the FET test. 4. The FET test should be used for generating information on acute fish toxicity, where appropriate. In cases where it cannot be used, fish toxicity should be derived with the threshold approach following OECD Guidance Document 126 (OECD ). 5. The use of the FET test will result in an overall reduction of the numbers of juvenile and adult fish required for aquatic toxicity testing. A recently published study by Belanger et al. (2013) 10 provides an extensive review of 985 FET studies on 229 compounds and 1531 AFT studies on 151 compounds. The authors performed FET/AFT regressions to determine relationships based on physico-chemical properties, species choices, exposure duration, chemical classes, chemical uses, and modes of action. FET/AFT relationships were shown to be very robust across nine orders of magnitude of potency. The authors conclude that if the FET test is used as an alternative to the AFT test, it will provide nearly equivalent predictions of hazard while improving overall animal welfare. Personal communication with the lead author, who chairs the ILSI-HESI Animal Alternative Needs in Environmental Risk Assessment Technical Committee, reveals that he and others have another paper in peer review on effluent testing alternatives that contains additional information on the use of FET test. Much of this recent work has been done with the knowledge and 8 Belanger S.E., Rawlings J.M., Carr G.J An Update to the Fish Embryo Toxicity-Acute Fish 382 Toxicity Relationship and Prospects for Support of the Use of the FET as an Animal Alternative Document Developed for the February, 2012 Meeting of the OECD ad hoc Expert Group on the 384 Fish Embryo Test and provided to EURL ECVAM as part of the ESAC peer review of of the OECD 385 validation study of the Zebrafish Embryo Toxicity Test. 9 OECD 2010 Short Guidance on the Threshold Approach for Acute Fish Toxicity Testing. Series on 489 Testing and Assessment No Available at: 10 Belanger S et al. (2013) Use of fish embryo toxicity tests for the prediction of acute fish toxicity to chemicals. Environ Toxicol Chem 32(8):

4 participation of USEPA ORD Duluth lab. We have enclosed a copy of this article for your perusal. The FET test has the potential to significantly reduce the number of fully-formed fish used in acute toxicity testing while providing effective ecological hazard identification. There appears to be no reason to continue using OPPTS when the embryo test has been repeatedly shown to accurately predict outcomes in juvenile and adult fish. With the goal of minimizing the use of vertebrate animals that can experience pain and distress as well as harmonizing testing strategies with the EU, we request that you review the results of recent work on the FET test, take steps to implement its use as a replacement for the AFT test, and issue a policy statement that the method is acceptable to EPA. We have also contacted EPA s Water Permits Office requesting the AFT be replaced with the FET test in guidance on whole effluent testing. A copy of this letter will be sent to the Director of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), Warren Casey, and EPA s ICCVAM representative, Anna Lowit, for their consideration as well. In the interest of promoting animal welfare, we ask that you take steps now to review the evidence that supports the appropriateness of the FET test as a replacement to current fish acute toxicity tests for pesticide registration and chemical safety evaluation. Toxicological science is constantly evolving and it is essential that regulatory agencies take advantage of every opportunity to reduce animal testing until the use of animals can eventually be completely eliminated. Thank you for your time and consideration of our comments. We look forward to a positive response from your office and Patricia can be reached at or by at PatriciaB@peta.org if you have any questions. Kind regards, Patricia L. Bishop, MS Research Scientist Regulatory Testing Division People for the Ethical Treatment of Animals 4

5 Kristie Sullivan, MPH Director, Regulatory Testing Issues Physicians Committee for Responsible Medicine cc: Jack Housenger, Director, Office of Pesticide Programs Donald Brady, Director, Environmental Fate & Effects Division, OPP Wendy Cleland-Hamnett, Director, Office of Pollution Prevention & Toxics Maria Doa, Director, Chemical Control Division, OPPT Tala Henry, Director, Risk Assessment Division, OPPT Warren Casey, ICCVAM Anna Lowit, Health Effects Division, OPP 5