Endocrine Disruptor Screening Program (EDSP) Tier 2 Ecotoxicity Tests meeting. 2 The comments of PETA, PCRM and HSUS can be found at

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1 March 31, 2015 Sharlene Matten, Ph.D. Office of Pollution Prevention and Toxics (7407M) Environmental Protection Agency 1200 Pennsylvania Ave., N.W. Washington, DC Dear Dr. Matten: RE: Draft Test Guidelines; Endocrine Disruptor Screening Program Test Guidelines (Series 890); Three Tier 2 Non-Mammalian Tests; Docket ID EPA HQ OPPT These comments are submitted on behalf of People for the Ethical Treatment of Animals (PETA), the Physicians Committee for Responsible Medicine (PCRM), and the Humane Society of the United States (HSUS) national animal protection and scientific advocacy organizations with a combined constituency of more than 13 million Americans who share the common goal of promoting reliable and relevant regulatory testing methods and strategies that protect human health and the environment while reducing, and ultimately eliminating, the use of animals. We appreciate the opportunity to comment on the three draft non-mammalian Tier 2 test guidelines proposed for the Endocrine Disruptor Screening Program (EDSP). These include the Japanese quail two-generation reproduction test (JQTT) [OCSPP Test Guideline ], the Medaka extended one-generation reproduction test (MEOGRT) [OCSPP Test Guideline ], and the larval amphibian growth and development assay (LAGDA) [OCSPP Test Guideline ]. General Comments The validation of these tests was the subject of a FIFRA SAP meeting in June of At that time, PETA, PCRM and HSUS ( We ) had identified, along with other commenters, significant technical issues with the complexity and duration of these tests as well as their reliability in correctly demonstrating dose-response for endocrine-mediated adverse effects. 2 Numerous areas existed for introduction of error and variability that could confound the interpretation of findings. It appears from these revised protocols that much more guidance has been provided on the proper preparation of histopathologic samples and interpretation of results, and many visual examples are now given. Also, additional guidance on relevant statistical methods and interpretation of data is included. In all three draft Test Guidelines, it appears that additional guidance has been provided on ways to avoid chemical concentrations that cause overt toxicity and can lead to confounding of test results. However, the fact remains that only the most experienced labs will likely be able to successfully conduct these tests and the potential for 1 See for more information on the Endocrine Disruptor Screening Program (EDSP) Tier 2 Ecotoxicity Tests meeting. 2 The comments of PETA, PCRM and HSUS can be found at HQ OPP Page 1 of 6

2 inexperienced labs to generate poor data quality still exists. In many cases, the difficulty in selecting doses that inform endocrine-mediated adverse effects yet do not lead to overt toxicity will lead to unnecessary range finding studies that will result in yet more animal use. It should be noted that statistics on the use of animals for range-finding studies (for any repeat-dose study used by EPA) is usually not well reported. Stakeholders have yet to receive any indication as to what criteria the EPA will use to trigger the requirement to conduct Tier 2 testing. We are particularly concerned regarding what information will be used to trigger the JQTT as there is no Tier 1 test that might suggest potential vulnerability of birds to a particular chemical (and help define the doses to be used in a longer multi-generation test). While we are not advocating for an avian Tier 1 test, there should be environmental monitoring data available that would strongly suggest the likelihood for wild bird populations to be exposure to the chemical in question before the JQTT is required. Guidance from EPA on what triggers any Tier 2 testing, with an aim to minimizing animal use, is warranted. Animal Use All three of these vertebrate ecotoxicity tests use very large numbers of animals; we have previously published some information regarding the number of animals needed for the in vivo assays found in the EDSP Tier 1 battery. 3 However these revised guidelines make it difficult to determine the total number of animals required for each test. For the purpose of transparency, these numbers should be clearly stated and should include those animals used in the actual test as well as those used in range-finding experiments and those culled during implementation of the study protocol. In addition, the number of animals used per concentration or dose should be minimized based on the power of the test, in which acknowledging the absolute numbers is critical. In our previous comments, we had estimated that the JQTT would require the use of about 1,500 birds through the F2 generation. This requirement appears to be unchanged. Similarly, the total number of amphibians used in the LAGDA appears to be the same. The replacement of the original two-generation Medaka test with the MEOGRT is a step in the right direction and appears to have reduced the number of fish being used, but when adding in culled fish and those used in range-finding tests, the number is still quite high. The EPA should investigate the use of fit-for-purpose Integrated Approaches to Testing and Assessment (IATA) as published by EPA staff and others since this 2013 SAP. IATA allows for the maximal use of existing information once this information is organized into some mode of action framework (such as the increasingly popular Adverse Outcome Pathway approach) by identifying what key events along the causal pathway leading to some adverse regulatory outcome of concern. It may be the case that assays measuring discrete responses at the cellular or organ/tissue level can satisfy the needs of EDSP Tier 2, rather than conducting several multigenerational studies using animals. One such fit-for-purpose integrated approach calls for initial collection of non-animal data surrounding the tentative mode-of-action, such as in vitro screens 3 See Bishop, P.L. and Willett, C.E. (2014). The use and acceptance of other scientifically relevant information (OSRI) in the US Environmental Protection Agency (EPA) Endocrine Disruptor Screening Program. Birth Defects Res (Part B) 101: Page 2 of 6

3 for genotoxicity and bioactivity assisted by toxicogenomics approaches. 4 Such an approach could be facilitated by the suite of assays that have been incorporated into EPA s Toxicity Forecaster ( ToxCast ), which now includes over 1800 chemicals and 800 assay endpoints. 5 In fact, such an approach has been suggested by other stakeholders and referred to as a Tier 1.5 battery, in that additional (non-multi-generational in vivo data) data could be collected in a fitfor-purpose integrated fashion that can answer the Tier 2 data needs. Specific Comments A. Japanese Quail Two-generation Test (JQTT) 1. There has been a worldwide effort to move away from multi-generation reproduction protocols as demonstrated by development of the mammalian Extended One Generation Reproductive Toxicity Study (EOGRTS) 6, and now the MEOGRT discussed here. In fact, the EPA has used these newer one-generation studies to satisfy the requirements of the EDSP (specifically, the Tier 1 data package for 2,4-D included an acceptable EOGRTS), which could also be used for dose-response and thus satisfy the mammalian Tier 2 requirement. In the last review of the Tier 2 tests at the June 2013 FIFRA SAP meeting, evidence was presented that indicated there was little value added by addition of the F2 generation in the JQTT and the recommendation was to also modify this assay to an extended one-generation test. 7 We continue to support this recommendation and suggest that an F2 generation only be triggered based on a specific set of transparent criteria written into the Test Guideline. If no effects are seen in F0 and F1 birds, it appears unlikely that effects will be seen in F2 birds and not allowing chicks from the F2 generation to hatch has the potential to save up to 600 animals (likely more due to culling). If EPA does not feel enough data exist to support the change at this time, then we strongly urge the agency to reconsider this option after a certain number of two-generation tests are completed and the determination can be made as to when, if ever, the F2 data contributed significantly to hazard assessment. 2. In reviewing the guidance for housing conditions as provided in Table 1 (page 5), the minimum floor areas per bird at various weeks of age would appear to create extremely crowded and stressful conditions. For example, chicks one week old need only be given 50 cm 2 (7.75 in 2 ) each, which amounts to less than a 3 inch by 3 inch area. Birds four weeks of age and older get a minimum floor area of See Thomas et al. (2013). Incorporating new technologies into toxicity testing and risk assessment: Moving from 21 st Century vision to a data driven framework. Tox. Sci. 136(1): See, for example, Filer et al. (2014). Test driving ToxCast: endocrine profiling for 1858 chemicals included in phase II. Curr. Opin. Pharmacol. 19: Organization for Economic Cooperation and Development Test No. 443: Extended One Generation Reproductive Toxicity Study. Available at: ilibrary.org/environment/test no 443 extended one generation reproductive toxicity study_ en. 7 USAPHC Toxicology study No. 87 XE 0DUZ 11. Two generation test for endocrine disruption and reproductive toxicity in Japanese quail using trenbolone. Page 3 of 6

4 cm 2 (97 in 2 ) or a roughly 10 inch by 10 inch area. A note at the bottom of Table 1 indicates that the acceptability of housing conditions of adults will be evaluated on the basis of results of reproductive performance. This would seem to recognize crowding and subsequent stress as a factor in reproductive performance and a potential confounding factor when attempting to determine a chemical s effect on this endpoint. Therefore, it follows that birds should be housed in a manner that provides adequate space and minimizes stress from overcrowding. In addition, the test guideline does not provide any discussion of enrichment techniques for the birds, which could also contribute to decreased stress levels. Comments from the Panel during the 2013 SAP also focused on the effects of stress in animals during experiments and the need to limit controllable sources to the greatest extent possible. B. Medaka Extended One-Generation Reproduction Test (MEOGRT) 1. We are pleased to see that the EPA has attempted to reduce the number of animals required by modifying this test from the original two-generation to an extended one-generation test. However, it is still quite animal intensive as the F2 generation is still bred and allowed to hatch according to the current study protocol. We recommend review of this requirement and a determination be made as to whether the study could be terminated when the F1 generation lay their eggs. 2. The current protocol requires 6 replicates in the test concentrations and 12 in the controls. While we recognize that the number of controls has been increased compared to the number used in the validation study to increase the power of the study, we strongly suggest that this requirement be reviewed once more experience has been gained with the study to determine if this high number of replicates is necessary. 3. It appears that more detailed guidance has been added to the test protocol on the proper sexing of fish, which will hopefully reduce the incidence of phenotypic/genotypic sex mismatching and attendant skewing of results. 4. A flow-through exposure system is recommended as the preferred assay system on page 9. We suggest the MEOGRT protocol recommend consulting the same references as those given in the LAGDA test guideline for guidance on testing difficult substances and mixtures in flow-through systems (i.e., Organization for Economic Cooperation and Development (OECD) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures 8 ) and for conducting aquatic toxicity testing in general (i.e., USEPA Guidance Document on Special Considerations for Conducting Aquatic Laboratory Studies 9 ). 5. We are pleased to see that specific performance criteria for the test are listed and described starting on page 22. This should provide adequate feedback guidance to 8 OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD Series on Testing and Assessment, No. 23, OECD Publishing, Paris. DOI: / en. 9 OCSPP Page 4 of 6

5 labs at various points in the test as to whether or not the test is progressing acceptably or should be terminated due to failure in meeting certain criteria. C. Larval Amphibian Growth and Development Assay (LAGDA) 1. It appears that more detailed guidance has been included on feeding regimes, which should enhance reproducibility and reduce the incidence of bent tail syndrome. However, we note that Coady et al (2014) 10 reported that bent tail syndrome of 10-30% across entire tadpole spawns can occur for unknown reasons and are not related to chemical exposure or feeding regimes. 2. With respect to euthanasia procedures as described on page 12, the recommended protocol given is immersion in a 0.02% MS-222 solution (0.2 g/l) and the animal is considered properly euthanized and ready for necropsy when it is unresponsive to external stimuli such as pinching the hind limb with a pair of forceps. A study of euthanasia methods for Xenopus laevis evaluated use of MS- 222 and reported on the time to loss of consciousness and the reliability of achieving death with various dose levels 11. The results indicated that immersion for at least one hour in a 5 g/l (0.5 %) buffered solution of MS-222 was necessary to euthanize adult X. laevis and ensure complete cessation of heartbeat without recovery within 3 hours after removal from the solution. Immersion in lower concentrations did not ensure cessation of heartbeat in all frogs and some recovered complete mobility after removal from the solution. While the study did not evaluate tadpoles, only adult frogs, we suspect the 0.02% solution recommended in the LAGDA protocol is too low to ensure euthanasia of juvenile animals prior to necropsy. Therefore, we recommend review of this article and adjustment of the protocol to reflect use of higher concentrations of MS-222 to ensure that necropsy is not initiated prior to death occurring. 3. The LAGDA is not ideal for satisfying the dose-response needs of EDSP Tier 2, as the output of the assay (LC50) falls short of the toxicity reference value needed for an accurate quantitative ecological risk assessment. In general, EPA needs to establish a no effect concentration (NOEC) in rats, birds and/or fish to establish protective goals for both terrestrial and aquatic animals. Using an LC50 in place of NOEC, should this be needed, introduces an unacceptable level of uncertainty in what should be a more certain determination (i.e., defining the concentration at which no adverse effects occur). 4. We suggest EPA review the useful lessons learned from other amphibian assays, such as the Tier 1 Amphibian Metamorphosis Assay (OCSPP ), as there are important implications for selection and maintenance of test concentrations in flow-through systems as well as rearing amphibians under 10 Coady, KK, Lehman, CM, Currie, RJ, and Marino, TA Challenges and approaches to conducting and interpreting the amphibian metamorphosis assay and the fish short term reproduction assay. Birth Defects Research (Part B) 101: Torreilles, SL, McClure, CE, Green SL Evaluation and refinement of euthanasia methods for Xenopus laevis. J Am Assoc Lab Anim Sci Sep; 48(5): Page 5 of 6

6 experimental conditions. Coady et al. (2014) 10 again provides some very useful insight in this area. We conclude by reiterating our concerns over the use of these three vertebrate Tier 2 ecotoxicity tests. They are lengthy, complex and costly new protocols that kill thousands of animals and require a high level of expertise to properly carry out. It remains our belief that due to the lack of reproducible significant effects amongst labs participating in recent inter-laboratory ring studies, validation has not been adequately demonstrated for these tests to be used in the EDSP. Sincerely, Patricia L. Bishop, MS Research Scientist Regulatory Testing Division People for the Ethical Treatment of Animals Kristie Sullivan, MPH Director, Regulatory Testing Issues Physicians Committee for Responsible Medicine Catherine Willett, PhD Director, Regulatory Toxicology, Risk Assessment and Alternatives The Humane Society of the United States Page 6 of 6