ASTM E1439-12
(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 and health 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
Test Materia...
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Designation: E1439 − 12
Standard Guide for
Conducting the Frog Embryo Teratogenesis Assay-Xenopus
1
(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 1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 This guide covers procedures for obtaining laboratory
responsibility of the user of this standard to establish appro-
data concerning the developmental toxicity of a test material.
priate safety and health practices and determine the applica-
The test utilizes embryos of theAfrican clawed frog, Xenopus
bility of regulatory limitations prior to use.
laevis and is called FETAX (Frog Embryo Teratogenesis
2
Assay-Xenopus) (1). Some of these procedures will be useful
1.5 This guide is arranged as follows:
for conducting developmental toxicity tests with other species
Section
of frogs although numerous modifications might be necessary. Referenced Documents 2
Terminology 3
A list of alternative anurans is presented in Appendix X1.
Summary of Guide 4
Significance and Use 5
1.2 A renewal exposure regimen and the collection of the
Safety Precautions 6
required mortality, malformation, and growth-inhibition data
Apparatus 7
are described. Special needs or circumstances might require
Water for Culturing Xenopus adults 8
Requirements 8.1
different types of exposure and data concerning other effects.
Source 8.2
Some of these modifications are listed in Appendix X2
Treatment 8.3
although other modifications might also be necessary. When- Characterization 8.4
FETAX Solution Water 9
ever these procedures are altered or other species used, the
Requirements 9.1
results of tests might not be comparable between modified and
Formulation 9.2
unmodified procedures. Any test that is conducted using
Test Material 10
General 10.1
modified procedures should be reported as having deviated
Stock Solution 10.2
from the guide.
Test Organisms 11
Species 11.1
1.3 These procedures are applicable to all chemicals either
Source 11.2
individually or in formulations, commercial products or mix-
Adults 11.3
tures that can be measured accurately at the necessary concen- Breeding 11.4
Embryos 11.5
trations in water. With appropriate modification these proce-
Procedure 12
durescanbeusedtoconducttestsontheeffectsoftemperature,
Experimental Design 12.1
Temperature and pH Requirements 12.2
dissolved oxygen, pH, physical agents, and on materials such
Beginning the Test 12.3
as aqueous effluents (see Guide E1192), surface and ground
Renewal 12.4
waters, leachates, aqueous and solid phase extracts, and solid
Duration of Test 12.5
Exogenous Metabolic Activation System (MAS) 12.6
phase samples, such as soils and sediments, particulate matter,
Biological Data 12.7
sediment, and whole bulk soils and sediment.
Analytical Methodology 13
Acceptability of the Test 14
Documentation 15
1
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Keywords 16
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
Appendixes 17
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate. A
X1. List of Alternative Species Appendix X1
standard guide is a document, developed using the consensus mechanisms of
X2. Additional Endpoints and Alternative Exposures Appendix X2
ASTM, that provides guidance for the selection of procedures to accomplish a
X3. Concentration Steps for Range-Finding Tests Appendix X3
specific test but which does not stipulate specific procedures.
X4. Microsome Isolation Reagents and NADPH Generating Appendix X4
Current edition approved Dec. 1, 2012 Published January 2013. Originally System Components,
approved in 1991. Last previous edition approved in 2004 as E1439–98 (2004). References
DOI: 10.1520/E1439-12.
2
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
the text.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
E1439 − 12
2. Referenced Documents in the absence of significant embryo mortality. The TI is
defined as the ratio of the 96-h LC50 and the 96-h EC50
3
2.1 ASTM Standards:
(malformation).
D1193Specification for Reagent Water
3.1.3 For definitions of other terms used in this guide, refer
E729Guide for Conducting Acute Toxicity Tests on Test
to Guides E729 and E1023, also Terminology E943. For an
Materials with Fishes, Macroinvertebrates, and Amphib-
explanation of units and symbols, refer to IEEE/ASTM SI 10.
ians
E943Termi
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E1439 − 98 (Reapproved 2004) E1439 − 12
Standard Guide for
Conducting the Frog Embryo Teratogenesis Assay-Xenopus
1
(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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. 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 South African clawed frog, Xenopus laevis and is called FETAX (Frog Embryo Teratogenesis
2
Assay-Xenopus) (1). 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 extracts of water-insoluble materials,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 and health 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
Test Material 10
General 10.1
Stock Solution 10.2
Test Organisms 11
Species 11.1
Source 11.2
Adults 11.3
1
This guide is under the jurisdiction of ASTM Committee E47 on Biological Effects and Environmental Fateand is the direct responsibility of Subcommittee E47.01 on
Aquatic Assessment and Toxicology. A standard guide is a document, developed using the consensus mechanisms of ASTM, that provides guidance for the selection of
procedures to accomplish a specific test but which does not stipulate specific procedures.
Current edition approved April 1, 2004Dec. 1, 2012 Published April 2004January 2013. Originally approved in 1991. Last previous edition approved in 19982004 as
E1439 – 98.E1439 – 98 (2004). DOI: 10.1520/E1439-98R04.10.1520/E1439-12.
2
The boldface numbers in parentheses refer to the list of references at the end of the text.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
E1439 − 12
Breeding 11.4
Embryos 11.5
Procedure 12
Experimental Design 12.1
Temperature and pH Requirements 12.2
Beginning the Test 12.3
Renewal 12.4
Duration of Test 12.5
Exogenous Metabolic Activation System (MAS) 12.6
Biological Data 12.7
Analytical Methodology 13
Acceptability of the Test 14
Documentation 15
Keywords 16
Appendixes 17
X1. List of Alternative Species Appendix X1
X2. Additional Endpoints and Alternative Exposures Appendix X2
X3. Concentration Steps for Range-Finding Tests Appendix X3
X4. Microsome Isolation Reagents and NADPH Generating Appendix X4
System Components,
References
2
---------------------- Page: 2 ----------------------
E1439 − 12
2. Referenced Documents
3
2.1 AS
...
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