United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 740-C-09-002 October 2009 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1100: Amphibian Metamorphosis (Frog) Page 1 NOTICE This guideline is one of a series of test guidelines established by the Office of Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection Agency for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a). The OPPTS test guidelines serve as a compendium of accepted scientific methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test, and is also used by EPA, the public, and the companies that are subject to data submission requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these guidelines are not binding on either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances warrant and without prior notice. The procedures contained in this guideline are strongly recommended for generating the data that are the subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. You may propose alternatives to the recommendations described in these guidelines, and the Agency will assess them for appropriateness on a case-by-case basis. For additional information about OPPTS harmonized test guidelines and to access the guidelines electronically, please go to http:/www.epa.gov/oppts and select “Test Methods the shipping of larval stages for use in the test is unacceptable. (2) Equipment and Supplies. The following equipment and supplies are needed for the conduct of this assay: (i) Exposure system (see description below); (ii) Glass or stainless steel aquaria (see description below); (iii) Breeding tanks (iv) Temperature controlling apparatus (e.g., heaters or coolers (adjustable to 22 1C); (v) Thermometer; (vi) Binocular dissection microscope; Page 4 (vii) Digital camera with at least 4 megapixel resolution and micro function; (ix) Image digitizing software; (x) Petri dish (e.g. 100 x 15 mm) or transparent plastic chamber of comparable size; (xi) Analytical balance capable of measuring to 3 decimal places (mg); (xii) Dissolved oxygen meter; (xiii) pH meter; (xiv) Light intensity meter capable of measuring in lux units; (xv) Miscellaneous laboratory glassware and tools; (xvi) Adjustable pipetters (10 to 5,000 L) or assorted pipettes of equivalent sizes; (xvii) Test chemical in sufficient quantities to conduct the study, preferably of one lot #; and (xviii) Analytical instrumentation appropriate for the chemical on test or contracted analytical services. (3) Chemical Testability. The AMA is based upon an aqueous exposure protocol whereby test chemical is introduced into the test chambers via a flow-through system. Flow-through methods however, introduce constraints on the types of chemicals that can be tested, as determined by the physicochemical properties of the compound. Therefore, prior to using this protocol, baseline information about the chemical should be obtained that is relevant to determining the testability, and the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures should be consulted (Ref. 5). Characteristics which indicate that the chemical may be difficult to test in aquatic systems include: high octanol water partitioning coefficients (log Kow), high volatility, susceptibility to hydrolysis, and susceptibility to photolysis under ambient laboratory lighting conditions. Other factors may also be relevant to determining testability and should be determined on a case by case basis. If a successful test is not possible for the chemical using a flow-through test system, a static renewal system may be employed. If neither system is capable of accommodating the test chemical, then the default is to not test it using this protocol. (4) Exposure System. A flow-through diluter system is preferred, when possible, over a static renewal system. If physical and/or chemical properties of any of the test substances are not amenable to a flow- through diluter system, then an alternative exposure system (e.g., static- renewal) can be employed. The system components should have water-contact components of glass, stainless steel, and/or Teflon. However, suitable plastics can be utilized if they do not compromise the study. Exposure tanks should be glass or stainless steel aquaria, equipped with standpipes that result in an appro