ASTM E1439-12(2019)
(Guide)Standard Guide for Conducting the Frog Embryo Teratogenesis Assay-Xenopus (FETAX)
Standard Guide for Conducting the Frog Embryo Teratogenesis Assay-Xenopus (FETAX)
SIGNIFICANCE AND USE
5.1 FETAX is a rapid test for identifying potential developmental toxicity. Data may be extrapolated to other species including mammals. FETAX might be used to prioritize samples for further tests which use mammals. Validation studies using compounds with known mammalian or human developmental toxicity, or both, suggest that the predictive accuracy will exceed 85 % (2) . When evaluating a test material for mammalian developmental toxicity, FETAX must be used with and without a metabolic activation system (MAS). Use of this exogenous MAS should increase the predictive accuracy of the assay to approximately 95 %. The accuracy rate compares favorably with other currently available “ in vitro teratogenesis screening assays” (3). Any assay employing cells, parts of embryos, or whole embryos other than in vivo mammalian embryos is considered to be an in vitro assay.
5.2 It is important to measure developmental toxicity because embryo mortality, malformation, and growth inhibition can often occur at concentrations far less than those required to affect adult organisms.
5.3 Because of the sensitivity of embryonic and early life stages, FETAX provides information that might be useful in estimating the chronic toxicity of a test material to aquatic organisms.
5.4 Results from FETAX might be useful when deriving water quality criteria for aquatic organisms (4).
5.5 FETAX results might be useful for studying structure-activity relationships between test materials and for studying bioavailability.
SCOPE
1.1 This guide covers procedures for obtaining laboratory data concerning the developmental toxicity of a test material. The test utilizes embryos of the African clawed frog, Xenopus laevis and is called FETAX (Frog Embryo Teratogenesis Assay-Xenopus) (1).2 Some of these procedures will be useful for conducting developmental toxicity tests with other species of frogs although numerous modifications might be necessary. A list of alternative anurans is presented in Appendix X1.
1.2 A renewal exposure regimen and the collection of the required mortality, malformation, and growth-inhibition data are described. Special needs or circumstances might require different types of exposure and data concerning other effects. Some of these modifications are listed in Appendix X2 although other modifications might also be necessary. Whenever these procedures are altered or other species used, the results of tests might not be comparable between modified and unmodified procedures. Any test that is conducted using modified procedures should be reported as having deviated from the guide.
1.3 These procedures are applicable to all chemicals either individually or in formulations, commercial products or mixtures that can be measured accurately at the necessary concentrations in water. With appropriate modification these procedures can be used to conduct tests on the effects of temperature, dissolved oxygen, pH, physical agents, and on materials such as aqueous effluents (see Guide E1192), surface and ground waters, leachates, aqueous and solid phase extracts, and solid phase samples, such as soils and sediments, particulate matter, sediment, and whole bulk soils and sediment.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.5 This guide is arranged as follows:
Section
Referenced Documents
2
Terminology
3
Summary of Guide
4
Significance and Use
5
Safety Precautions
6
Apparatus
7
Water for Culturing Xenopus adults
8
Requirements
8.1
Source
8.2
Treatment
8.3
Characterization
8.4
FETAX Solution Water
9
Requirements
9.1
Formulation
9.2 ...
General Information
Relations
Standards Content (Sample)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E1439 − 12 (Reapproved 2019)
Standard Guide for
Conducting the Frog Embryo Teratogenesis Assay-Xenopus
(FETAX)
This standard is issued under the fixed designation E1439; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter-
1.1 This guide covers procedures for obtaining laboratory
mine the applicability of regulatory limitations prior to use.
data concerning the developmental toxicity of a test material.
1.5 This guide is arranged as follows:
The test utilizes embryos of theAfrican clawed frog, Xenopus
laevis and is called FETAX (Frog Embryo Teratogenesis
Section
Referenced Documents 2
Assay-Xenopus) (1). Some of these procedures will be useful
Terminology 3
for conducting developmental toxicity tests with other species
Summary of Guide 4
of frogs although numerous modifications might be necessary. Significance and Use 5
Safety Precautions 6
A list of alternative anurans is presented in Appendix X1.
Apparatus 7
1.2 A renewal exposure regimen and the collection of the Water for Culturing Xenopus adults 8
Requirements 8.1
required mortality, malformation, and growth-inhibition data
Source 8.2
are described. Special needs or circumstances might require
Treatment 8.3
Characterization 8.4
different types of exposure and data concerning other effects.
FETAX Solution Water 9
Some of these modifications are listed in Appendix X2
Requirements 9.1
although other modifications might also be necessary. When-
Formulation 9.2
Test Material 10
ever these procedures are altered or other species used, the
General 10.1
results of tests might not be comparable between modified and
Stock Solution 10.2
unmodified procedures. Any test that is conducted using
Test Organisms 11
Species 11.1
modified procedures should be reported as having deviated
Source 11.2
from the guide.
Adults 11.3
Breeding 11.4
1.3 These procedures are applicable to all chemicals either
Embryos 11.5
individually or in formulations, commercial products or mix-
Procedure 12
tures that can be measured accurately at the necessary concen-
Experimental Design 12.1
Temperature and pH Requirements 12.2
trations in water. With appropriate modification these proce-
Beginning the Test 12.3
durescanbeusedtoconducttestsontheeffectsoftemperature,
Renewal 12.4
dissolved oxygen, pH, physical agents, and on materials such
Duration of Test 12.5
Exogenous Metabolic Activation System (MAS) 12.6
as aqueous effluents (see Guide E1192), surface and ground
Biological Data 12.7
waters, leachates, aqueous and solid phase extracts, and solid
Analytical Methodology 13
phase samples, such as soils and sediments, particulate matter, Acceptability of the Test 14
Documentation 15
sediment, and whole bulk soils and sediment.
Keywords 16
1.4 This standard does not purport to address all of the
Appendixes 17
X1. List of Alternative Species Appendix X1
safety concerns, if any, associated with its use. It is the
X2. Additional Endpoints and Alternative Exposures Appendix X2
X3. Concentration Steps for Range-Finding Tests Appendix X3
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
X4. Microsome Isolation Reagents and NADPH Generating Appendix X4
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
System Components,
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate. A References
standard guide is a document, developed using the consensus mechanisms of
1.6 This international standard was developed in accor-
ASTM, that provides guidance for the selection of procedures to accomplish a
dance with internationally recognized principles on standard-
specific test but which does not stipulate specific procedures.
Current edition approved Feb. 1, 2019 Published February 2019. Originally
ization established in the Decision on Principles for the
approved in 1991. Last previous edition approved in 2012 as E1439–12. DOI:
Development of International Standards, Guides and Recom-
10.1520/E1439-12R19.
2 mendations issued by the World Trade Organization Technical
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
the text. Barriers to Trade (TBT) Committee.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1439 − 12 (2019)
2. Referenced Documents therefore, a greater potential for all embryos to be malformed
3 in the absence of significant embryo mortality. The TI is
2.1 ASTM Standards:
defined as the ratio of the 96-h LC50 and the 96-h EC50
D1193Specification for Reagent Water
(malformation).
E729Guide for Conducting Acute Toxicity Tests on Test
3.1.3 For definitions of other terms used in this guide, refer
Materials with Fishes, Macroinvertebrates, and Amphib-
to Guides E729 and E1023, also Terminology E943. For an
ians
explanation of units and symbols, refer to IEEE/ASTM SI 10.
E943Terminology Relating to Biological Effects and Envi-
ronmental Fate
4. Summary of Guide
E1023Guide for Assessing the Hazard of a Material to
Aquatic Organisms and Their Uses
4.1 In FETAX, range-finding and definitive tests are per-
E1192Guide for ConductingAcute Toxicity Tests onAque-
formed on each test material. A control in which no test
ous Ambient Samples and Effluents with Fishes,
material has been added is used to provide 1) a measure of the
Macroinvertebrates, and Amphibians
acceptability of the test by indicating the quality of embryos
E1391Guide for Collection, Storage, Characterization, and
and the suitability of the FETAX solution, test conditions and
Manipulation of Sediments for Toxicological Testing and
handling procedures, and 2) a basis for interpreting data from
for Selection of Samplers Used to Collect Benthic Inver-
other treatments. Each test consists of several different con-
tebrates
centrations of test material with at least two replicates of each
E1525GuideforDesigningBiologicalTestswithSediments
concentration. Each of the three tests is conducted using
E1706TestMethodforMeasuringtheToxicityofSediment-
embryos from a different male/female pair of Xenopus laevis.
Associated Contaminants with Freshwater Invertebrates
Areference toxicant (6-aminonicotinamide) should be used as
(Withdrawn 2019)
a quality control measure. The 96-h LC50 and 96-h EC50
IEEE/ASTM SI 10American National Standard for Use of
(malformation) are determined by an appropriate statistical
theInternationalSystemofUnits(SI):TheModernMetric
analysis and the TI (Teratogenic Index) is calculated by
System
dividingthe96-hLC50bythe96-hEC50.Growthinhibitionis
determined by measuring the head-tail length of each embryo
3. Terminology
anddeterminingwhethergrowthataparticularconcentrationis
3.1 Definitions of Terms Specific to This Standard:
significantly different from that of the control. Other useful
3.1.1 The words “must,” “should,” “may,”“ can,” and
data can be collected (for example, pigmentation, locomotion,
“might,” have very specific meanings in this guide. “Must” is
and hatchability) to expand the utility of the test.
usedtoexpressanabsoluterequirement,thatis,tostatethatthe
test ought to be designed to satisfy the specified condition,
5. Significance and Use
unless the purpose of the test requires a different design.
5.1 FETAX is a rapid test for identifying potential develop-
“Must” is only used in connection with factors that directly
mental toxicity. Data may be extrapolated to other species
relatetotheacceptabilityofthetest(seeSection14).“Should”
including mammals. FETAX might be used to prioritize
isusedtostatethatthespecifiedconditionisrecommendedand
samples for further tests which use mammals. Validation
oughttobemetifpossible.Althoughviolationofone“should”
studies using compounds with known mammalian or human
is rarely a serious matter, violation of several will often render
developmental toxicity, or both, suggest that the predictive
the results questionable.Terms such as “is desirable,” “is often
accuracywillexceed85% (2).Whenevaluatingatestmaterial
desirable,” and “might be desirable” are used in connection
for mammalian developmental toxicity, FETAX must be used
with less important factors. “May” is used to mean “is (are)
with and without a metabolic activation system (MAS). Use of
allowed to,”“ can” is used to mean “is (are) able to,” and
thisexogenousMASshouldincreasethepredictiveaccuracyof
“might” is used to mean “could possibly.” Thus the classic
the assay to approximately 95%. The accuracy rate compares
distinctionbetween“may”and“can”ispreserved,and“might”
favorablywithothercurrentlyavailable“ in vitroteratogenesis
is never used as a synonym for either “may” or “can.”
screening assays” (3). Any assay employing cells, parts of
3.1.2 Adevelopmental toxicant is a test material that affects
embryos, or whole embryos other than in vivo mammalian
any developmental process. Therefore, a developmental toxi-
embryos is considered to be an in vitro assay.
cant affects embryo mortality and malformation, and causes
growth inhibition. A teratogen is a test material that causes
5.2 It is important to measure developmental toxicity be-
abnormal morphogenesis (malformation). The Teratogenic In-
cause embryo mortality, malformation, and growth inhibition
dex or TI is a measure of potential developmental hazard (1).
canoftenoccuratconcentrationsfarlessthanthoserequiredto
TI values higher than 1.5 signify larger separation of the
affect adult organisms.
mortality and malformation concentration ranges and,
5.3 Because of the sensitivity of embryonic and early life
stages, FETAX provides information that might be useful in
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
estimating the chronic toxicity of a test material to aquatic
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
organisms.
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
5.4 Results from FETAX might be useful when deriving
The last approved version of this historical standard is referenced on
www.astm.org. water quality criteria for aquatic organisms (4).
E1439 − 12 (2019)
5.5 FETAX results might be useful for studying structure- sides of the breeding aquarium should be opaque and an
activity relationships between test materials and for studying optional bubbler may be fitted to oxygenate the water. The top
bioavailability. of the aquarium should be covered with an opaque porous
material such as a fiberglass furnace filter. Alternatively, an
6. Safety Precautions
adequate breeding tank can be constructed from two plastic
dish pans (at least 38 by 38 cm) stacked one in the other. The
6.1 Many materials can affect humans adversely if precau-
floorofthetopmostpanisperforated.Acorkborercanbeused
tions are inadequate. Therefore, skin contact with all test
to create 1.5-cm holes for the eggs to fall through.
materials and solutions of them should be minimized by such
means as wearing appropriate protective gloves (especially
7.2 Facilities for Conducting FETAX—Aconstant tempera-
when washing equipment or putting hands in test solutions),
ture room or a suitable incubator for embryos is required
laboratorycoats,aprons,andsafetyglasses,andusingpipetsto
although a photoperiod is unnecessary. The incubator must be
remove organisms from test solutions. Special precautions,
capable of holding 23 6 1°C. Abnormal development will
such as covering test chambers and ventilating the area
occurattemperaturesgreaterthan26°C.Covered60-mmglass
surrounding the chambers and the use of fume hoods, should
Petri dishes should be used as test chambers except that
be taken when conducting tests on volatile materials. Informa-
disposable 55-mm polystyrene Petri dishes should be used if a
tion provided in Material Safety Data Sheets on toxicity to
substantial amount of the test material binds to glass but not to
humans (5), recommended handling procedures (6), and
polystyrene. A binocular dissection microscope capable of
chemical and physical properties of the test material should be
magnifications up to 30× is required to count and evaluate
studied before a test is begun. Special procedures might be
abnormal embryos. A digital camera with adequate zoom is
necessary with radiolabeled test materials (7) and with test
used to enlarge embryo images two to three times for head-tail
materials that are, or are suspected of being, carcinogenic (8).
length measurements. It is also possible to measure embryo
6.2 Although disposal of stock solutions, test solutions, and
length through the use of a map measurer or an ocular
test organisms poses no special problems in most cases, health
micrometer. However, the process is greatly facilitated by
and safety precautions and applicable regulations should be
using a digitizer interfaced to a microcomputer.
considered before beginning a test. Removal or degradation of
7.3 Construction Materials—Equipment and facilities that
test material might be desirable before disposal of stock and
contact stock solutions, test solutions, or water in which
test solutions.
embryos will be placed should not contain substances that can
6.3 Cleaning of equipment with a volatile solvent such as
be leached or dissolved by aqueous solutions in amounts that
acetone should be performed only in a fume hood.
would adversely affect embryo growth or development.
6.4 To prepare dilute acid solutions, concentrated acid Additionally,itemsthatcontactstocksolutionsortestsolutions
should be added to water, not vice versa. Opening a bottle of should be chosen to minimize sorption of most test materials
concentratedacidandaddingconcentratedacidtowatershould from water. Glass, Type 316 stainless steel, nylon, and fluoro-
be performed only in a fume hood. carbon plastic should be used whenever possible to minimize
dissolution, leaching, and sorption. Rigid plastics may be used
6.5 Because FETAX solution and test solutions are usually
for holding, acclimation, and in the water supply system, but
good conductors of electricity, use of ground fault systems and
they should be soaked for a week before use in water used for
leak detectors should be considered to help avoid electrical
adult maintenance.
shocks.
7.3.1 FETAX solution, stock solutions, or test solutions
should not c
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