Amphibian Metamorphosis Assay OCSPP Guideline 890.1100 Standard Evaluation Procedure (SEP) ENDOCRINE DISRUPTOR SCREENING PROGRAM U.S. Environmental Protection Agency Washington, DC 20460 30 September 2011 Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100 Table of Contents I. INTRODUCTION..................................................................................................... 1 II. THE AMPHIBIAN METAMORPHOSIS ASSAY ................................................ 2 A. Purpose of the Assay ............................................................................................ 2 B. Study Design ........................................................................................................ 2 III. EVALUATION OF STUDY CONDUCT ............................................................... 2 A. Test Species .......................................................................................................... 3 B. Equipment and Supplies ....................................................................................... 3 C. Chemical Testability ............................................................................................ 3 D. Exposure System .................................................................................................. 3 1. System Description ........................................................................................... 3 2. Water Quality ................................................................................................... 3 3. Iodide Concentration in Test Water .................................................................. 4 E. Holding of Animals .............................................................................................. 4 1. Adult Care and Breeding .................................................................................. 4 2. Embryo Selection ............................................................................................. 4 3. Larval Culture and Feeding .............................................................................. 4 F. Analytical Chemistry............................................................................................ 5 G. Selection of Test Concentrations.......................................................................... 5 1. Establishing the High Test Concentration ........................................................ 5 2. Test Concentration Range ................................................................................ 5 H. Test Procedure ...................................................................................................... 6 1. Day 0 (Test Initiation) ...................................................................................... 6 2. Day 7................................................................................................................. 7 3. Day 21 (Test Termination) ............................................................................... 7 I. Determination of Biological Endpoints................................................................ 8 1. Mortality and Clinical Signs ............................................................................. 9 2. Developmental Stage ........................................................................................ 9 3. Hind Limb Length (HLL) ................................................................................. 9 4. Snout-Vent Length (SVL) and Body Weight ................................................. 10 5. Thyroid Gland Histopathology ....................................................................... 10 6. Specimens Archival ........................................................................................ 11 7. Data Reporting and Completeness ................................................................. 11 IV. STUDY INTERPRETATION ............................................................................... 11 A. Test Validity Criteria .......................................................................................... 11 B. Performance Criteria .......................................................................................... 12 Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100 C. General Analysis ................................................................................................ 13 1. Statistical Analyses ......................................................................................... 13 2. Trends ............................................................................................................. 14 3. Histological Findings ...................................................................................... 15 D. Endpoint Interpretation ...................................................................................... 15 1. Advanced Development (Developmental Stage and Normalized HLL) ........ 15 2. Asynchronous Development (Unable to Stage) ............................................. 16 3. Histopathology................................................................................................ 17 4. Developmental Delay (Developmental Stage and Normalized HLL) ............ 18 5. Growth (SVL and Body Weight).................................................................... 18 E. Special Data Analysis Considerations................................................................ 18 1. Use of Compromised Treatment Levels ......................................................... 18 2. Solvent Controls ............................................................................................. 19 3. Treatment Groups Achieving NF Developmental Stage 60 and Above ........ 19 4. Histological Analyses and Developmental Stage ........................................... 19 V. DATA EVALUATION RECORD ......................................................................... 19 VI. REFERENCES ........................................................................................................ 20 Appendices Appendix 1: Staging ......................................................................................................... 22 Appendix 2: Length Measurement.................................................................................... 23 Appendix 3: Decision Logic for the Conduct and Interpretation of the AMA ................. 24 Appendix 4: Recommendations for Statistical Analyses Based on OECD 2006 ............. 25 Appendix 5: Expanded Bibliography................................................................................ 28 Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100 Page 1 of 51 I. Introduction This document was developed by EPA to provide guidance to EPA staff who will be reviewing the data submitted in response to Tier 1 Orders issued under the Endocrine Disruptor Screening Program (EDSP). This document provides general guidance and is not binding on either EPA or any outside parties. The use of language such as "will," "is," "may," "can" or "should" in this document does not connote any requirement for either EPA or any outside parties. As such, EPA may depart from the guidance where circumstances warrant and without prior notice. The SEPs are intended to be used in conjunction with the EDSP Test Guideline Series 890 and the Corrections and Clarifications document available on the EDSP web page. This Standard Evaluation Procedure (SEP) provides guidance on how EPA generally intends to review studies conducted using the OCSPP Guideline 890.1100 Amphibian Metamorphosis (Frog) Assay (AMA) that are submitted to support requirements imposed under the U.S. Environmental Protection Agency’s Endocrine Disruptor Screening Program (EDSP). The objective of EDSP Tier 1 assays is to characterize the potential of a chemical to interact with the endocrine system. The product of the review will be a Data Evaluation Record (DER) that reflects how well the study conforms to the Guideline, evaluates how well the study and analyses were performed, and provides the conclusions supported by the data. The DER will include, for example, a list of any significant deviations from the guideline and their potential impacts, a list of significant information missing from the study report, a description of how the statistical analyses were performed and whether they were performed according to the guideline, and any other information about the performance of the study that affects interpretation of the data within the context of the EDSP. The DER should record details on all endpoints required by the guideline. The DER is intended to contain enough information to provide EPA with the ability to determine whether the study is scientifically valid and provides the necessary information. The guideline recommends the critical materials, methods, and analyses that lead to successful performance of the assay. If a particular material, method, or analysis is specified in the guideline, it is usually because other materials, methods, or analyses are either known to be inappropriate, or at least have not been validated and there is concern for their potential influence on results. The Agency has posted Corrections and Clarifications on Technical Aspects of the EDSP Tier 1 Assays (OCSPP Test Guideline Series 890) in the docket; the link to this document may be found by way of the EDSP web page (http://www.epa.gov/endo/). It is therefore important to note deviations from specific materials, methods, or analyses in the DER, and provide the Agency’s opinion on whether the deviation/deficiency has an impact on the performance and results of the study or the acceptability of the study. Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100 Page 2 of 51 II. The Amphibian Metamorphosis Assay A. Purpose of the Assay The AMA is a screening assay intended to empirically identify substances which may interfere with the normal function of the Hypothalamus-Pituitary-Thyroid (HPT) axis (Fort et al., 2007). The AMA represents a generalized vertebrate model to the extent that it is based on the conserved structures and functions of the HPT axis. It is an important assay because amphibian metamorphosis provides a well-studied, thyroid- dependent process which responds to substances known to be active along the HPT axis, and it is the only assay in the EDSP Tier 1 battery that detects thyroid activity in an animal undergoing morphological development. It is intended to be included in a battery of in vitro and in vivo tests to identify substances with potential to interact with the endocrine system. B. Study Design The general experimental design entails exposing Nieuwkoop-Faber (NF) stage 51 African clawed frog (Xenopus laevis) tadpoles, in separate treatment groups, to a minimum of three respective concentrations of a test chemical or a negative (clean water) control for 21 days. There are four replicates of each test treatment. Larval density at test initiation is 20 tadpoles per test tank (replicate) for all treatment groups (80 larvae/treatment). The observational endpoints are hind limb length (HLL), snout-to-vent length (SVL), developmental stage, body weight, thyroid histopathology, and daily observations of mortality and clinical signs. In addition, whole thyroid tissue and plasma samples may be collected for analysis of thyroxine (T4) (OECD 2009), although this is not specified in the OCSPP 890.1100 test guideline. III. Evaluation of Study Conduct This section provides a summary description of the information that would generally be expected to be obtained from a study that had been conducted following the recommendations in the Test Guidelines. As described in this section, the DER reviewer is responsible for summarizing how the study was conducted, the extent to which that is consistent with the Guidelines, and how, if at all, that affected the validity of the study. This information will factor into the Agency’s interpretations of the data contained in the study report. Specific points that are important for the DER to address are highlighted in the individual sections below, as appropriate. The summary in this section is offered as a general outline to aid in preparation of the DER. The purpose of this section is not to serve as substitute for the Test Guidelines, nor to provide any guidance on how the study should be conducted. Rather, the summary is intended to provide context and examples illustrating to the individual preparing the DER what the DER would be expected to contain. Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100 Page 3 of 51 A. Test Species African clawed frog (Xenopus laevis) is the recommended species. The assay method was validated and OCSPP 890.1100 is explicit for this species. However, the Corrections and Clarifications document (EPA 2011) states that the method may also be applicable to Silurana (Xenopus) tropicalis, as demonstrated by Mitsui et al. (2006). If an alternate species is used, the DER should detail the relevant or significant deviations in the method in order to accommodate the alternate species (e.g., S. tropicalis). The DER should include a discussion of whatever evidence was used in the study report for the performance criteria that were used to support the reliability of the test. B. Equipment and Supplies The list of equipment and supplies from the test guideline is provided only as a non-exhaustive recommendation of what is typically needed to conduct a successful test. If equipment and supplies are used that differ from those identified in the test guideline, it is recommended that the DER identify the differences and state whether and how they may have affected the performance or outcome of the study. C. Chemical Testability The DER should summarize the results of any tests conducted to evaluate the extent to which concentration and stability of the test chemical in the exposure system were verified. The AMA is based upon an aqueous exposure protocol whereby the test chemical is introduced into the test chambers via a flow-through system. If a successful test is not possible for the chemical using a flow-through test system, a static renewal system is recommended. If neither system is capable of accommodating the test chemical even using approved co-solvents, then the default is to not test the chemical using this guideline. D. Exposure System 1. System Description A flow-through diluter system is preferred, when possible, over a static renewal system (OECD 2009). The system components should be described in the DER as well as the extent to which they generally comport with the exposure system description in the guideline and are capable of maintaining the experimental conditions recommended in Appendix 1 of the guideline. 2. Water Quality It is recommended that a description of the source water and chemical analysis results be provided in the DER, in addition to evidence that the water can support normal growth and development of X. laevis. Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100 Page 4 of 51 3. Iodide Concentration in Test Water Based on the available data from the validation studies, the assay has been demonstrated to work well when test water iodide (I-) concentrations ranged between 0.5 and 10µg/L. This is in addition to dietary iodide exposure at levels present in the  recommended feeding regime using Sera Micron diet. Ideally, the minimum iodide concentration in the test water would be 0.5 μg/L. Iodide supplementation is recommended if the concentration is naturally below 0.5 μg/L in the test water; then, the it is recommended that iodide supplementation not exceed 2 μg/L. For example, if the test water is reconstituted from deionized water, it is recommended that iodide be added at a minimum concentration of 0.5μg/L. Any supplementation of the test water with iodide or other salts would be relevant information for inclusion in the DER. In addition, any measurements of iodide concentrations in the test water (and diet, if applicable) should be reported in the DER. E. Holding of Animals 1. Adult Care and Breeding One option for adult care and breeding of X. laevis is to conduct these activities in accordance with existing guidance for the frog teratogenesis assay (ASTM 2004). A full description of any alternative animal care and breeding used would also be relevant information to be described in the DER, along with whether deviations from the recommended method may have had any significant impact on the study performance or interpretation, using performance criteria (Section IV-B of the SEP) as a guide. 2. Embryo Selection The guideline recommends that 2-3 of the best individual spawns be retained to evaluate the quality of the spawns. From these 2-3 spawns, it is recommended that the test organisms originate from the best single spawn, based upon embryo viability/appearance and the presence of an adequate number (> 1,500) of embryos. The guideline recommends against co-mixing spawns because this has been demonstrated to increase variability and decrease the statistical power of the test (OECD 2007a). It is useful for the DER to report how many spawns were evaluated, along with details of the evaluation method (e.g., timing of evaluation and number of eggs evaluated per sample), and whether the organisms selected for the definitive test originated from the best single spawn. 3. Larval Culture and Feeding The test guideline provides recommendations for handling the embryos and larvae (tadpoles) during the pre-exposure phase. If no tadpoles develop to NF stage 51 within Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100 Page 5 of 51 17 days after fertilization, then inappropriate environmental conditions, disease, or other stressors may be potential culprit(s). The test guideline recommends tadpoles be fed Sera Micron® (Sera GmbH, Heinsberg, Germany), or other diet that has demonstrated to allow equal performance of the AMA (OECD 2009), throughout the pre-exposure period (after NF stage 45/46) and during the entire test period of 21 days. It is recommended that the feeding regime during both the pre-exposure period and the test follow the recommendations in the guideline; the recommended regime (and associated nutrient and dietary iodide availability) resulted in adequate performance (i.e., growth and development of tadpoles) of the AMA during validation (OECD 2007a). The DER should document the feeding regime used. F. Analytical Chemistry The guideline recommends test solutions from each replicate tank at each concentration be sampled for analytical chemistry analyses at test initiation (day 0), and weekly during the test for a minimum of four samples. It is also recommended that each test concentration be analyzed during system preparation, prior to test initiation, to verify system performance. In addition, it is recommended that stock solutions be analyzed when they are changed, especially if the volume of the stock solution does not provide adequate amounts of chemical to span the duration of routine sampling periods. The sampling schedule and analyses performed should be provided in the DER. G. Selection of Test Concentrations 1. Establishing the High Test Concentration The test guideline recommends that the high test concentration be set as the lowest of the following values: (1) 100 mg/L, (2) the solubility limit of the test substance, or (3) the highest test concentration of the chemical which results in less than 10% acute mortality. The justification for using the highest test concentration that results in less than 10% mortality (if this is the lowest value of the three options above) is that this demonstrates that the chemical is tested up to a level where overt toxicity is observed, and thus adequately challenges the organism, without compromising the validity of the test. The DER should describe any basis for selecting the highest test concentration included in the study. 2. Test Concentration Range The guideline-specified minimum number of test concentrations is three plus a negative (clean water) control, plus a solvent/vehicle control if necessary. The recommended minimum differential between the highest and lowest test concentrations is approximately one order of magnitude [lowest = 0.1X(highest)]. The minimum recommended separation between individual concentrations is a factor 0.33X and the Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100 Page 6 of 51 maximum is a factor of 0.1X (OECD, 2008). The DER should identify the range of test concentrations and include any justification provided in the study report, if the test employed a range different from the recommended guidance. H. Test Procedure 1. Day 0 (Test Initiation) It is recommended that the DER identify the selection method for tadpoles used in the definitive test, along with the age (days post-fertilization), Nieuwkoop and Faber (1994) stage, and size range (if determined) of the tadpoles selected, as described in this section. The exposure is initiated when a sufficient number of tadpoles in the pre- exposure stock population, which are less than or equal to 17 days of age post- fertilization, have reached developmental stage 51 (Appendix 1) according to Nieuwkoop and Faber (1994). The test guideline includes additional guidance on optional size selection, based on total length (not snout-vent length), that complements the stage selection. The optional addition of size selection further reduces variability by ensuring that the NF stage 51 tadpoles fall within a specified size range. For selection of test animals, healthy and normal looking tadpoles of the stock population are pooled in a single vessel containing an appropriate volume of dilution water. For developmental stage determination, the Guidelines recommend that the tadpoles be individually removed from the pooling tank using a small net or strainer and transferred to a transparent measurement chamber (e.g., 100 mm Petri dish) containing dilution water. The developmental stage of the animals is determined using a binocular dissection microscope. Total length, in millimeters (mm), may also be determined at this time. To reduce variability associated with measurement (staging) error, it is important that staging be conducted as accurately as possible. It is preferred not to use anesthesia; however, it is possible to individually anesthetize the tadpoles using an appropriate method [e.g., 100 mg/L tricaine methanesulfonate (MS-222), appropriately buffered with sodium bicarbonate (pH 7.0)], prior to initial staging. Animals are carefully handled during transfer in order to minimize handling stress and to avoid any injury. If anaesthesia is used, the DER should document the method. While the complete Nieuwkoop and Faber (1994) guide may be consulted for comprehensive information on staging tadpoles, one can reliably determine NF stage using prominent morphological landmarks. For test initiation, the guidelines recommend the tadpoles selected should be at NF stage 51. The most prominent morphological staging landmark at NF stage 51 is hind limb morphology, where the hind limb bud is conical in shape and is 1.5X as long as it wide. Tadpoles that meet the stage criteria are held in a tank of clean culture water until the staging process is completed. Tadpoles exhibiting grossly visible malformations or injuries should be excluded from the assay. Once the staging is completed, the larvae are randomly distributed to exposure treatment Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100 Page 7 of 51 tanks until each tank contains 20 larvae. Each treatment tank is then inspected for animals with abnormal appearance (e.g., injuries, abnormal swimming behavior, etc.). Tadpoles that appear unhealthy are typically removed from the treatment tanks and replaced with larvae newly selected from the pooling tank. The DER should report any observations of abnormal appearance or behavior and whether tadpoles with the specified abnormality/ies were excluded from the assay. 2. Day 7 The guidelines recommend on day 7 of exposure, five randomly chosen tadpoles per replicate are removed from each test tank. The procedure used gives each test organism an equal probability of being selected; therefore, each tadpole is netted. Any randomization method is scientifically appropriate. Tadpoles not selected for day 7 measurements are returned to the tank of origin. The five tadpoles selected for measurements are humanely euthanized (e.g., in 150 to 200 mg/L MS-222, buffered with bicarbonate to pH 7.0). The euthanized tadpoles are rinsed in water and blotted dry. Body weight (also referred to as wet weight or blotted dry weight) is recorded to the nearest milligram (mg). Hind-limb length (HLL) and snout-vent length (SVL) are recorded to the nearest millimeter (mm). NF developmental stage is determined using a binocular dissection microscope. This assay does not track individuals (i.e., tadpoles are not marked) and thus it would not be scientifically appropriate to pair individual Day 7 observations with individual observations (e.g., size) at test initiation. It is recommended that the DER identify which measurements and observations were recorded on Day 7 of the assay and briefly describe the method used to subsample tadpoles (i.e., whether the selection method was consistent with the randomization procedure recommended in the guideline). 3. Day 21 (Test Termination) The guideline recommends that at test termination (day 21), the remaining tadpoles are removed from the test tanks and humanely euthanized, as described above. Tadpoles are rinsed in water and blotted dry. Body weight (mg), HLL and SVL (mm), and NF developmental stage are recorded for each individual. The guideline recommends that all larvae be placed in Davidson’s fixative for 48 to 72 hours (OECD 2007b), either as whole body samples or as trimmed head tissue samples containing the lower jaw. (Whole body samples may be preferable, since this preserves the association between individual observations of morphology and thyroid histopathology at test termination.) To maximize the viability of samples for potential future reference, it is recommended that all specimens be preserved using this method. However, the guideline recommends that a subset of only five tadpoles from each replicate tank be selected for thyroid histopathology in the AMA. The DER should identify which measurements and observations were recorded on Day 21 of the assay, along with the number of tadpoles selected for thyroid pathology and the methods of selection and preservation for these and any other archived specimens. More detailed information for the evaluation of study conduct with respect to biological endpoints is provided in section (III)(I) of this SEP.