1、Designation: E 1439 98 (Reapproved 2004)Standard Guide forConducting the Frog Embryo Teratogenesis Assay-Xenopus(FETAX)1This standard is issued under the fixed designation E 1439; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the
2、 year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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 ma
3、terial.The test utilizes embryos of the South African clawed frog,Xenopus laevis and is called FETAX (Frog Embryo Teratoge-nesis Assay-Xenopus) (1).2Some of these procedures will beuseful for conducting developmental toxicity tests with otherspecies of frogs although numerous modifications might ben
4、ecessary. A list of alternative anurans is presented in Appen-dix 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 ef
5、fects.Some of these modifications 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 usingmodifi
6、ed procedures should be reported 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 thes
7、e proce-dures can be used to conduct tests on the effects of temperature,dissolved oxygen, pH, physical agents, and on materials suchas aqueous effluents (see Guide E 1192), surface and groundwaters, leachates, aqueous extracts of water-insoluble materi-als, and solid phase samples, such as soils an
8、d sediments,particulate matter, sediment, and whole bulk soils and sedi-ment.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 determ
9、ine the applica-bility of regulatory limitations 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 8.3Characteriza
10、tion 8.4FETAX Solution Water 9Requirements 9.1Formulation 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
11、of Test 12.5Exogenous Metabolic Activation System (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 Step
12、s for Range-Finding Tests Appendix X3X4. Microsome Isolation Reagents and NADPH GeneratingSystem Components,Appendix X4References1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.01 on Aquatic A
13、ssessment and Toxicology. A standard guide is a 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 April 1, 2004. Published April 2004.
14、 Originallyapproved in 1991. Last previous edition approved in 1998 as E 1439 98.2The boldface numbers in parentheses refer to the list of references at the end ofthe text.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.2. Referenced
15、 Documents2.1 ASTM Standards:3D 1193 Specification for Reagent WaterE 380 Practice for Use of the International System of Units(SI) (the Modernized Metric System)4E 729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates andAmphibiansE 943 Terminology Relating
16、to Biological Effects and En-vironmental FateE 1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE 1192 Guide for Conducting Acute Toxicity Tests onAqueous Ambient Samples and Effluents with Fishes,Macroinvertebrates and AmphibiansE 1391 Guide for Collection, Storag
17、e, Characterization, andManipulation of Sediments for Toxicological Testing, andfor Selection of Samplers Used to Collect Benthic Inver-tebratesE 1525 Guide for Designing Biological Tests with Sedi-mentsE 1706 Test Methods for Measuring the Toxicity ofSediment-Associated Contaminants with Freshwater
18、 Inver-tebrates3. 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
19、specified 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.Alt
20、hough 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”
21、is used 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 A developmental toxicant is a test material that affectsany developmental process.
22、Therefore, 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 developmental hazard (1). TI valueshigher than 1.5 signify larger
23、 separation of the mortality andmalformation concentration ranges and, therefore, a greaterpotential for all embryos to be malformed in the absence ofsignificant embryo mortality. The TI is defined as the 96-hLC50 divided by the 96-h EC50 (malformation).3.1.3 For definitions of other terms used in t
24、his guide, referto Guides E 729 and E 1023, also Terminology E 943. For anexplanation of units and symbols, refer to Practice E 380.4. Summary of Guide4.1 In FETAX, range-finding and three replicate tests areperformed on each test material. A control in which no testmaterial has been added is used t
25、o 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 mat
26、erial with two replicate dishes 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 determined
27、by probit analysis and the TI(Teratogenic Index) is calculated by dividing the 96-h LC50 bythe 96-h EC50. Growth inhibition is determined by measuringthe head-tail length of each embryo and determining whethergrowth at a particular concentration is significantly differentfrom that of the control. Ot
28、her useful data can be collected (forexample, pigmentation, locomotion, and hatchability) to ex-pand the utility of the test.5. Significance and Use5.1 FETAX is a rapid test for identifying developmentaltoxicants. Data may be extrapolated to other species includingmammals. FETAX might be used to pri
29、oritize samples forfurther tests which use mammals. Validation studies usingcompounds with known mammalian or human developmentaltoxicity, or both, suggest that the predictive accuracy willexceed 85 % (2). When evaluating a test material for mamma-lian developmental toxicity, FETAX must be used with
30、 andwithout a metabolic activation system (MAS). Use of thisexogenous MAS should increase the predictive accuracy of theassay to approximately 95 %. The accuracy rate comparesfavorably with other currently available “ in vitro teratogenesisscreening assays” (3). Any assay employing cells, parts ofem
31、bryos, 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 those required toaffect adult organism
32、s.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 for aquatic organisms (4).5.5 FETAX
33、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 ASTMStandards volume information,
34、refer to the standards Document Summary page onthe ASTM website.4Withdrawn.E 1439 98 (2004)26. 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 suchmeans as wea
35、ring 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 areasurrounding the c
36、hambers 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 before a test i
37、s 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, healthand safety precau
38、tions 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.6.4 To prepare d
39、ilute 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, use of ground fa
40、ult 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/12-h night. Th
41、e 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 cm2of water su
42、rface 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 23 6 3C.7.1.1 Two types of breeding aquaria have been used suc-cessfully. A 5 or 10-gal glass aquarium may be used if fittedwith a 1-cm mesh sus
43、pended about 3-cm from the bottom ofthe aquarium 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 be fitted
44、 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 pan is pe
45、rforated.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 24 6 2C. Abnormal developm
46、ent 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 microscope capa
47、ble ofmagnifications up to 303 is required to count and evaluateabnormal embryos. A simple darkroom enlarger is used toenlarge embryo images two to three times for head-tail lengthmeasurements. It is also possible to measure embryo lengththrough the use of a map measurer or an ocular micrometer.Howe
48、ver, the process is greatly facilitated by using a digitizerinterfaced to a microcomputer. The microcomputer is also usedin data analysis.7.3 Construction MaterialsEquipment and facilities thatcontact stock solutions, test solutions, or water in whichembryos will be placed should not contain substan
49、ces that canbe leached or dissolved by aqueous solutions in amounts thatwould adversely affect embryo growth or development. Addi-tionally, 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 F
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