1、Designation: E1439 12Standard Guide forConducting the Frog Embryo Teratogenesis Assay-Xenopus(FETAX)1This standard is issued under the fixed designation E1439; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revisi
2、on. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide covers procedures for obtaining laboratorydata concerning the developmental toxicity of a test material.The test utili
3、zes embryos of the African clawed frog, Xenopuslaevis and is called FETAX (Frog Embryo TeratogenesisAssay-Xenopus) (1).2Some of these procedures will be usefulfor conducting developmental toxicity tests with other speciesof frogs although numerous modifications might be necessary.A list of alternati
4、ve anurans is presented in Appendix X1.1.2 A renewal exposure regimen and the collection of therequired mortality, malformation, and growth-inhibition dataare described. Special needs or circumstances might requiredifferent types of exposure and data concerning other effects.Some of these modificati
5、ons are listed in Appendix X2although other modifications might also be necessary. When-ever these procedures are altered or other species used, theresults of tests might not be comparable between modified andunmodified procedures. Any test that is conducted usingmodified procedures should be report
6、ed as having deviatedfrom the guide.1.3 These procedures are applicable to all chemicals eitherindividually or in formulations, commercial products or mix-tures that can be measured accurately at the necessary concen-trations in water. With appropriate modification these proce-dures can be used to c
7、onduct tests on the effects of temperature,dissolved oxygen, pH, physical agents, and on materials suchas aqueous effluents (see Guide E1192), surface and groundwaters, leachates, aqueous and solid phase extracts, and solidphase samples, such as soils and sediments, particulate matter,sediment, and
8、whole bulk soils and sediment.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitation
9、s prior to use.1.5 This guide is arranged as follows:SectionReferenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Safety Precautions 6Apparatus 7Water for Culturing Xenopus adults 8Requirements 8.1Source 8.2Treatment 8.3Characterization 8.4FETAX Solution Water 9Requirements 9.1F
10、ormulation 9.2Test Material 10General 10.1Stock Solution 10.2Test Organisms 11Species 11.1Source 11.2Adults 11.3Breeding 11.4Embryos 11.5Procedure 12Experimental Design 12.1Temperature and pH Requirements 12.2Beginning the Test 12.3Renewal 12.4Duration of Test 12.5Exogenous Metabolic Activation Syst
11、em (MAS) 12.6Biological Data 12.7Analytical Methodology 13Acceptability of the Test 14Documentation 15Keywords 16Appendixes 17X1. List of Alternative Species Appendix X1X2. Additional Endpoints and Alternative Exposures Appendix X2X3. Concentration Steps for Range-Finding Tests Appendix X3X4. Micros
12、ome Isolation Reagents and NADPH GeneratingSystem Components,Appendix X4References1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fateand is the direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology. A standard guide is a
13、document,developed using the consensus mechanisms of ASTM, that provides guidance forthe selection of procedures to accomplish a specific test but which does not stipulatespecific procedures.Current edition approved Dec. 1, 2012 Published January 2013. Originallyapproved in 1991. Last previous editi
14、on approved in 2004 as E1439 98 (2004).DOI: 10.1520/E1439-12.2The boldface numbers in parentheses refer to the list of references at the end ofthe text.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States12. Referenced Documents2.1 ASTM St
15、andards:3D1193 Specification for Reagent WaterE729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE943 Terminology Relating to Biological Effects and Envi-ronmental FateE1023 Guide for Assessing the Hazard of a Material toAquatic Organisms
16、and Their UsesE1192 Guide for Conducting Acute Toxicity Tests on Aque-ous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and AmphibiansE1391 Guide for Collection, Storage, Characterization, andManipulation of Sediments for Toxicological Testing andfor Selection of Samplers Used to Col
17、lect Benthic Inver-tebratesE1525 Guide for Designing Biological Tests with SedimentsE1706 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater InvertebratesIEEE/ASTM SI 10 American National Standard for Use ofthe International System of Units (SI): The Modern Me
18、tricSystem3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 The words “must,” “should,” “may,”“ can,” and“might,” have very specific meanings in this guide. “Must” isused to express an absolute requirement, that is, to state that thetest ought to be designed to satisfy the speci
19、fied condition,unless the purpose of the test requires a different design.“Must” is only used in connection with factors that directlyrelate to the acceptability of the test (see Section 14). “Should”is used to state that the specified condition is recommended andought to be met if possible.Although
20、 violation of one “should”is rarely a serious matter, violation of several will often renderthe results questionable. Terms such as “is desirable,” “is oftendesirable,” and “might be desirable” are used in connectionwith less important factors. “May” is used to mean “is (are)allowed to,”“ can” is us
21、ed to mean “is (are) able to,” and“might” is used to mean “could possibly.” Thus the classicdistinction between “may” and “can” is preserved, and “might”is never used as a synonym for either “may” or “can.”3.1.2 Adevelopmental toxicant is a test material that affectsany developmental process. Theref
22、ore, a developmental toxi-cant affects embryo mortality and malformation, and causesgrowth inhibition. A teratogen is a test material that causesabnormal morphogenesis (malformation). The Teratogenic In-dex or TI is a measure of potential developmental hazard (1).TI values higher than 1.5 signify la
23、rger separation of themortality and malformation concentration ranges and,therefore, a greater potential for all embryos to be malformedin the absence of significant embryo mortality. The TI isdefined as the ratio of the 96-h LC50 and the 96-h EC50(malformation).3.1.3 For definitions of other terms
24、used in this guide, referto Guides E729 and E1023, also Terminology E943. For anexplanation of units and symbols, refer to IEEE/ASTM SI 10.4. Summary of Guide4.1 In FETAX, range-finding and definitive tests are per-formed on each test material. A control in which no testmaterial has been added is us
25、ed to provide 1) a measure of theacceptability of the test by indicating the quality of embryosand the suitability of the FETAX solution, test conditions andhandling procedures, and 2) a basis for interpreting data fromother treatments. Each test consists of several different con-centrations of test
26、 material with at least two replicates of eachconcentration. Each of the three tests is conducted usingembryos from a different male/female pair of Xenopus laevis.A reference toxicant (6-aminonicotinamide) should be used asa quality control measure. The 96-h LC50 and 96-h EC50(malformation) are dete
27、rmined by an appropriate statisticalanalysis and the TI (Teratogenic Index) is calculated bydividing the 96-h LC50 by the 96-h EC50. Growth inhibition isdetermined by measuring the head-tail length of each embryoand determining whether growth at a particular concentration issignificantly different f
28、rom that of the control. Other usefuldata can be collected (for example, pigmentation, locomotion,and hatchability) to expand the utility of the test.5. Significance and Use5.1 FETAX is a rapid test for identifying potential develop-mental toxicity. Data may be extrapolated to other speciesincluding
29、 mammals. FETAX might be used to prioritizesamples for further tests which use mammals. Validationstudies using compounds with known mammalian or humandevelopmental toxicity, or both, suggest that the predictiveaccuracy will exceed 85 % (2). When evaluating a test materialfor mammalian developmental
30、 toxicity, FETAX must be usedwith and without a metabolic activation system (MAS). Use ofthis exogenous MAS should increase the predictive accuracy ofthe assay to approximately 95 %. The accuracy rate comparesfavorably with other currently available “ in vitro teratogenesisscreening assays” (3). Any
31、 assay employing cells, parts ofembryos, or whole embryos other than in vivo mammalianembryos is considered to be an in vitro assay.5.2 It is important to measure developmental toxicity be-cause embryo mortality, malformation, and growth inhibitioncan often occur at concentrations far less than thos
32、e required toaffect adult organisms.5.3 Because of the sensitivity of embryonic and early lifestages, FETAX provides information that might be useful inestimating the chronic toxicity of a test material to aquaticorganisms.5.4 Results from FETAX might be useful when derivingwater quality criteria fo
33、r aquatic organisms (4).5.5 FETAX results might be useful for studying structure-activity relationships between test materials and for studyingbioavailability.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of
34、ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.E1439 1226. Safety Precautions6.1 Many materials can affect humans adversely if precau-tions are inadequate. Therefore, skin contact with all testmaterials and solutions of them should be minimized by s
35、uchmeans as wearing appropriate protective gloves (especiallywhen washing equipment or putting hands in test solutions),laboratory coats, aprons, and safety glasses, and using pipets toremove organisms from test solutions. Special precautions,such as covering test chambers and ventilating the areasu
36、rrounding the chambers and the use of fume hoods, shouldbe taken when conducting tests on volatile materials. Informa-tion provided in Material Safety Data Sheets on toxicity tohumans (5), recommended handling procedures (6), andchemical and physical properties of the test material should bestudied
37、before a test is begun. Special procedures might benecessary with radiolabeled test materials (7) and with testmaterials that are, or are suspected of being, carcinogenic (8).6.2 Although disposal of stock solutions, test solutions, andtest organisms poses no special problems in most cases, healthan
38、d safety precautions and applicable regulations should beconsidered before beginning a test. Removal or degradation oftest material might be desirable before disposal of stock andtest solutions.6.3 Cleaning of equipment with a volatile solvent such asacetone should be performed only in a fume hood.6
39、.4 To prepare dilute acid solutions, concentrated acidshould be added to water, not vice versa. Opening a bottle ofconcentrated acid and adding concentrated acid to water shouldbe performed only in a fume hood.6.5 Because FETAX solution and test solutions are usuallygood conductors of electricity, u
40、se of ground fault systems andleak detectors should be considered to help avoid electricalshocks.7. Apparatus7.1 Facilities for Maintaining and Breeding XenopusAdults should be kept in an animal room that is isolated fromextraneous light which might interfere with a consistentphotoperiod of 12-h day
41、/12-h night. The role that circadianrhythm plays in Xenopus reproduction has not been investi-gated. A consistent photoperiod is therefore recommended sothat Xenopus can be bred year-round. Adults can be kept inlarge aquaria or in fiberglass or stainless steel raceways atdensities of 4 to 6 per 1800
42、 cm2of water surface area. Thesides of tanks should be opaque and at least 30 cm high. Thewater depth should be between 7 and 14 cm. Water temperaturefor adults should be 21 6 3C.7.1.1 Two types of breeding aquaria have been used suc-cessfully. A 5 or 10-gal aquarium may be used if fitted with a1-cm
43、 mesh suspended about 3-cm from the bottom of theaquarium so that deposited eggs will lie undisturbed on thebottom of the aquarium. Hardware cloth or other metal meshmust not be used. Nylon or plastic mesh is recommended. Thesides of the breeding aquarium should be opaque and anoptional bubbler may
44、be fitted to oxygenate the water. The topof the aquarium should be covered with an opaque porousmaterial such as a fiberglass furnace filter. Alternatively, anadequate breeding tank can be constructed from two plasticdish pans (at least 38 by 38 cm) stacked one in the other. Thefloor of the topmost
45、pan is perforated.Acork borer can be usedto create 1.5-cm holes for the eggs to fall through.7.2 Facilities for Conducting FETAXA constant tempera-ture room or a suitable incubator for embryos is requiredalthough a photoperiod is unnecessary. The incubator must becapable of holding 23 6 1C. Abnormal
46、 development willoccur at temperatures greater than 26C. Covered 60-mm glassPetri dishes should be used as test chambers except thatdisposable 55-mm polystyrene Petri dishes should be used if asubstantial amount of the test material binds to glass but not topolystyrene. A binocular dissection micros
47、cope capable ofmagnifications up to 30 is required to count and evaluateabnormal embryos. A digital camera with adequate zoom isused to enlarge embryo images two to three times for head-taillength measurements. It is also possible to measure embryolength through the use of a map measurer or an ocula
48、rmicrometer. However, the process is greatly facilitated byusing a digitizer interfaced to a microcomputer.7.3 Construction MaterialsEquipment and facilities thatcontact stock solutions, test solutions, or water in whichembryos will be placed should not contain substances that canbe leached or disso
49、lved by aqueous solutions in amounts thatwould adversely affect embryo growth or development.Additionally, items that contact stock solutions or test solutionsshould be chosen to minimize sorption of most test materialsfrom water. Glass, Type 316 stainless steel, nylon, and fluoro-carbon plastic should be used whenever possible to minimizedissolution, leaching, and sorption. Rigid plastics may be usedfor holding, acclimation, and in the water supply system, butthey should be soaked for a week before use in water used foradult maintenance.7.3.1 FETAX solution, stock solutions,
