ASTM E1439-12(2019)

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 contact brass, copper, lead, galvanized metal, or
7. Apparatus
natural rubber before or during the test. Items made of
7.1 Facilities for Maintaining and Breeding Xenopus—
neoprenerubberorothermaterialsnotmentionedaboveshould
Adults should be kept in an animal room that is isolated from
not be used unless it has been shown that their use will not
extraneous light which might interfere with a consistent
adversely affect either survival or growth of the embryos and
photoperiod of 12-h day/12-h night. The role that circadian
larvae of the test species.
rhythm plays in Xenopus reproduction has not been investi-
gated. A consistent photoperiod is therefore recommended so
7.4 Cleaning—At the end of each test, all glass dishes and
that Xenopus can be bred year-round. Adults can be kept in
other glassware that are to be used again should be immedi-
large aquaria or in fiberglass or stainless steel raceways at
ately emptied, rinsed with water, and cleaned by the following
densities of 4 to 6 per 1800 cm of water surface area. The
procedure.
sides of tanks should be opaque and at least 30 cm high. The
7.4.1 Glassware Washing Procedure:
waterdepthshouldbebetween7and14cm.Watertemperature
7.4.1.1 Soak 15 min, and scrub with tissue culture compat-
for adults should be 21 6 3°C.
ible detergent in tap water.
7.1.1 Two types of breeding aquaria have been used suc-
7.4.1.2 Rinse twice with tap water.
cessfully. A 5 or 10-gal aquarium may be used if fitted with a
7.4.1.3 Rinse once with fresh, dilute (10%, v⁄v) hydro-
1-cm mesh suspended about 3-cm from the bottom of the
chloric acid to remove scale, metals, and bases.
aquarium so that deposited eggs will lie undisturbed on the
bottom of the aquarium. Hardware cloth or other metal mesh 7.4.1.4 RinsetwicewithwaterconformingtoTypeIIASTM
must not be used. Nylon or plastic mesh is recommended. The water (Specification D1193).
E1439 − 12 (2019)
7.4.1.5 Rinseoncewithfullstrengthreagent-grade acetone contaminatedwithoilorwatercontainingrustorsludge.Some
to remove organic compounds. compressed air supplies might also have a high level of carbon
7.4.1.6 Rinse well with hot ASTM Type II water. monoxide.Alow-pressure blower will provide high-quality air
7.4.1.7 Rinse well with ASTM Type I water or FETAX without the problems associated with a high-pressure air
solution. supply as long as its air supply is uncontaminated. Adequate
7.4.1.8 Heat the glassware in an oven at 150° C for3hto aeration will stabilize pH, bring concentrations of dissolved
drive off any residual acetone. Toxicity problems have oc- oxygenandothergasesintoequilibriumwithair,andminimize
curred in experiments when this glassware washing procedure oxygen demand and concentrations of volatiles. However, it is
was omitted. notabsolutelynecessarytoaeratethewaterfor Xenopusadults
(12).
7.5 Acceptability—Before FETAX is conducted in new test
8.3.2 Filtration through bag, sand, sock, or depth-type
facilities it is desirable to conduct a “non-toxicant” test, in
cartridge filters may be used to keep the concentration of
whichalltestchamberscontainFETAXsolutionwithnoadded
particulate matter acceptably low and as a pretreatment before
test material. The embryos should grow, develop, and survive
filtration through a finer filter. Organics may be removed by
in numbers consistent with an acceptable test (see 14.1). The
filtration through activated carbon filtration. Carbon filters
magnitude of the chamber-to-chamber variation should be
should be changed on a monthly basis, or when residual
evaluated.
chlorine is detected.
8.3.3 Water that might be contaminated with facultative
8. Water for Culturing Xenopus Adults
pathogens may be passed through a properly maintained
8.1 Requirements—Besides being available in adequate
ultraviolet sterilizer (13) equipped with an intensity meter and
supply, the water should allow satisfactory survival and repro-
flowcontrols,orpassedthroughafilterwithaporesizeof0.45
ductionoftheadults,beofuniformquality,andnotnecessarily
µm or less.
affect results of the test.
8.4 Characterization:
8.2 Source:
8.4.1 The following items should be measured at least
8.2.1 Natural water is preferred for adult culture. It should
annually:pH,totaldissolvedsolids(TDS),totalorganiccarbon
be obtained from an uncontaminated source that provides
(TOC), organophosphorus pesticides, organic chlorine (or
uniform quality. The quality of water from a well or spring is
organochlorine pesticides plus PCBs), chlorinated phenoxy
usually more uniform than that of a surface water. If a surface
herbicides, ammonia, bromide, beryllium, cadmium,
water is used as a source of fresh water, the intake should be
chromium, copper, iron, lead, manganese, mercury, nickel,
positioned to minimize fluctuations in quality and the possi-
selenium, silver, and zinc. For each method used the detection
bility of contamination and to maximize the concentration of
limit should be below the concentration in the water or the
dissolved oxygen to help ensure low concentrations of sulfide
lowest concentration that has been shown to adversely affect
and iron. FETAX solution is acceptable for adult culture. The
the test species.
cost and formulation time make it suitable only for small
8.4.2 Physical and chemical limits on water: pH should be
colonies. Water temperature should be adjusted to 21 6 3°C
between6.5and9 (14).TheTOCshouldbelessthan10mg/L,
before being used to culture adults.
while alkalinity and hardness both should be between 16 and
8.2.2 Dechlorinated water can be used to culture adults as
400 mg/L as CaCO (15). Table 1 shows the recommended
long as residual chlorine and its oxidants are reduced to levels
maximum concentrations for some contaminants that have
thatdonotaffectsurvivalandreproduction.Sodiumbisulfiteis
often been found to be in excess concentration in laboratory
probably better for dechlorinating water than sodium sulfite
water supplies. The values reported are one tenth of the
and both are more reliable than carbon filters, especially for
minimum concentration that inhibits growth. While these data
removing chloramines (9). Fluorides can be removed by
are not indicative of the effect of long-term exposure of adults
passage over activated alumina columns (10). In addition to
onreproductivesuccess,they,nonetheless,serveasaguidefor
residual chlorine, chloramines, and fluoride, municipal drink-
limitingadultexposuretothesemetals.Themaximumquantity
ing water often contains unacceptably high concentrations of
of the other contaminants listed in 8.4.1 should meet EPA
copper, lead, and zinc, and quality is often rather variable.
freshwater chronic water quality criteria (14).
Excessive concentrations of most metals can usually be re-
moved with a chelating resin (11).
TABLE 1 Recommended Maximum Concentrations of Some
8.3 Treatment:
Metals
8.3.1 A continuous flow system for culturing adults is
Recommended Maximum Concentration
A
recommended although a static system has proven successful.
Metal
(µg/L)
Water for culturing adults should be aerated by the use of air
Cadmium (2) 10.0
stones or surface aerators.Air used for aeration should be free
Lead (2) 5.0
Mercury (2) 0.144
of fumes, oil, and water. Compressed air supplies might be
Nickel (2) 25.0
Selenium (unpublished) 140.0
5 Zinc (2) 70.0
“Reagent Chemicals,American Chemical Society Specifications,”Am. Chemi-
A
cal Soc., Washington, DC. For suggestions on the testing of reagents not listed by
TestedinFETAXat100mg/LhardnessasCaCO .Valuesreportedareonetenth
the American Chemical Society, see “Analar Standards for Laboratory U.K.
of the minimum concentration to inhibit growth.
Chemicals,” BDH Ltd., Poole, Dorset, and the “United States Pharmacopeia.”
E1439 − 12 (2019)
9. FETAX Solution Water or organic acids, and chloride or nitrate salts of metals, might
affect the pH more than the use of minimum necessary amount
9.1 Requirements—FETAX solution should be used for
of a strong acid or base. Any adjustments of pH can send the
static renewal assays. FETAX solution should also be used for
test material through a transition to affect changes in such
flow-through experiments whenever possible. However, larger
properties as solubility or degree and type of dissociation, or
volumes need for breeding or flow-through exposure may
both. Prior to testing, as much chemical and physical data as
require the use of natural water sources, such as well water for
are available on the test material should be obtained and
dechlorinated tap water. Should the need for a large volume
considered prior to making decisions on pH adjustments.
preclude the use of FETAX solution, then water conforming to
10.2.2.1 If a solvent other than FETAX solution is used, its
the specifications listed in Section 8 may be used. The water
concentration in test solutions should be kept to a minimum
must allow embryonic growth at the same rate as FETAX
and should be low enough that it does not affect Xenopus
solution and there should be no differences between control
embryo growth and survival. Because of its low toxicity, low
mortality and malformation rates.
volatility, and high ability to dissolve many organic chemicals,
9.2 Formulation—FETAX solution is composed of 625 mg
triethylene glycol is often a good organic solvent for preparing
NaCl, 96 mg NaHCO , 30 mg KCl, 15 mg CaCl,60mg
3 2
stock solutions. Other water-miscible organic solvents such as
CaSO ·2H O, and 75 mg MgSO per litre of deionized or
4 2 4
dimethyl sulfoxide and acetone also may be used as solvents.
distilled water. The pH of the final solution should be 7.6 to
Concentrations of triethylene glycol, dimethyl sulfoxide, and
7.9. All chemicals should be reagent-grade or better. Deion-
acetone in test solutions should be <1.6%, <1.1%, and,
izedor distilled water must conform to Type I ASTM water
<1.1%v/v,respectively.Theseconcentrationshavebeenfound
(Specification D1193).
not to cause any adverse effects in FETAX (16). At times,
concentrations approaching 1% solvent are necessary to keep
10. Test Material
test materials in solution for FETAX. This is often the case
when the assay is used in testing pure compounds for the
10.1 General—The test material should be reagent-grade
or better unless a specific test involves an unknown complex purpose of comparing test results with mammalian data. If
possible, it is desirable to perform the test using two different
mixture, formulation, commercial product, or technical-grade
or use-grade material. Before a test is begun, the following solvents and compare the results. This will help in identifying
possible interactions between a solvent and test material.
should be known about the test material:
10.1.1 Identities and concentrations of major ingredients 10.2.2.2 Ethanol is not recommended because its terato-
and major impurities, for example, impurities constituting
genicindex(TI)inFETAXisapproximately1.4.Methanolhas
more than about 1% of the material. hightoxicityinFETAX.Acetonemightstimulatethegrowthof
10.1.2 Solubility and stability in water. microorganisms and is quite volatile. If an organic solvent is
10.1.3 Estimate of toxicity to humans. used it should be reagent-grade or better.Asurfactant should
10.1.4 Recommended handling procedures (see Section 6). not be used in the preparation of a stock solution because it
mightaffecttheformandtoxicityofthetestmaterialinthetest
10.1.5 For unknown samples much of the information
solutions.
specified in 10.1.1 – 10.1.4 will be lacking, but the pH,
hardness, alkalinity, and conductivity of the sample should be
10.2.3 If a solvent other than dilution-water or FETAX
measured. solutionisused,atleastonesolventcontrol,usingsolventfrom
the same batch used to make the stock solution, must be
10.2 Stock Solution:
included in the test and a dilution-water or FETAX solution
10.2.1 If the test material can not be directly added to the
control should be included in the test. If no solvent other than
test vessel, a stock solution should be prepared. Various
dilution-waterorFETAXsolutionisused,thenadilution-water
dilutions of the stock solution should be prepared in a separate
or FETAX solution control must be included in the test.
vesselpriortointroductiontothePetridishsoastoexposethe
10.2.3.1 The concentration of solvent must be the same in
embryos to a homogeneous mixture without concentration
all test solutions that contain test material and the solvent
gradients. If a stock solution is used, the concentration and
control must contain the same concentration of solvent.
stability of the test material in it should be determined before
10.2.3.2 If the test contains both a dilution-water or a
the beginning of the test. Stock solutions should be prepared
FETAX-solution control and a solvent control, the mortality,
daily unless analytical data indicate the solution is stable with
malformation,andgrowthinhibitionshouldbecomparedusing
time. If the test material is subject to photolysis, the stock
a two-tailed t-test. If a statistically significant difference in
solution should be shielded from light.
either mortality, malformation, or growth inhibition is detected
10.2.2 Except possibly for tests on hydrolyzable,
betweenthetwocontrols,onlythesolventcontrolmaybeused
oxidizable, and reducible materials, the preferred solvent is
as the basis for comparison in the calculation of results.
FETAXsolution.Alternatively,dechlorinatedtapwaterorwell
water may be used if adequately characterized. The minimum 10.2.3.3 If a solvent other than dilution-water or FETAX
necessary amount of a strong acid or base may be used in the solution is used to prepare a stock solution, it might be
preparation of an aqueous stock solution, but such acid or base desirable to conduct simultaneous tests using chemically un-
mightaffectthepHoftestsolutionsappreciably.Useofamore related solvents or two different concentrations of the same
soluble form of the test material, such as chloride or sulfate solvent to obtain information concerning possible effects of
salts of organic amines, sodium or potassium salts of phenols solvents on results of the test.
E1439 − 12 (2019)
11. Test Organisms to be conducted. Water temperature should be held at 20 6
2°C. To induce breeding, the male and the female should
11.1 Species—FETAX is designed to use embryos of the
receive 150 to 350 and 350 to 700 IU, respectively, of human
African clawed frog Xenopus laevis (Daudin). Information
chorionic gonadotropin by way of injection into the dorsal
regarding the basic biology and development of this species
lymph sac. The hormone concentration should be 1000 IU/mL
has been reported by Deuchar (17, 18). However, use of the
in sterile 0.9% NaCl. A 1-mL tuberculin syringe fitted with a
South African clawed frog, Silurana tropicalis is also accept-
⁄2-in. long, 26-gage needle should be used to make the
able. (Appendix X1). Appendix X1 also lists other North
injection.Largerboreneedlesmightallowleakageofhormone
Americanspeciesthatcanbeusedinsituationswhere Xenopus
from the injection site. The amount of human chorionic
laevis or Silurana tropicalis cannot, although there will be
gonadotropin injected depends on the time of year and condi-
differences in the rate of development and the method of
tion of the adults. Lower doses are usually used in spring and
inducing breeding. Many anurans only breed in a specific
higher doses in fall.Amplexus normally ensues within 2 to 6 h
season during the year. The length of exposure might have to
and egg deposition about 9 to 12 h after injection. The eggs
be altered to allow proper organogenesis.
should be immediately inspected for fertility and quality. The
11.2 Source—For breeding, adult frogs may be obtained
fertility rate should be >75%. Eggs laid in “strings” or not
from various supply houses or independent suppliers. Proven
perfectly round should not be used because they develop
breeders should be requested from the supplier. Each animal
abnormally.
shouldbethoroughlyexamineduponarrivalforskinlesionsor
11.5 Embryos:
red patches on the ventral surfaces. Skin lesions are indicative
11.5.1 Removal of Jelly Coat—De-jellying of embryos
of nematode infection while the red patches indicate Aeromo-
should begin immediately following the end of egg laying.
nasinfection.Careshouldbetakentoensurethatonlyhealthy,
De-jellyingofembryosshouldbecarriedoutbygentleswirling
sexually mature frogs are placed in the colony.
for1to3minina2%w/vL-cysteine(CAS#52-90-4)solution
11.3 Adults:
prepared in FETAX solution. The cysteine solution should be
11.3.1 Selection—Xenopus males should be 7.5 to 10 cm in
adjusted to pH 8.1 with 1 N NaOH. The solution should be
crown-rump length and at least two years of age. Males have
made up immediately prior to use. De-jellying should be
dark arm pads on the underside of each forearm and lack
monitored continuously and the process stopped just after all
cloacal lips. Females should be 10 to 12.5 cm in length and at
jellyisremoved.Careshouldbetakennottotreattheembryos
leastthreeyearsold.Femalesarealwayslargerthanmalesand
too long because survival will be reduced.
easily identified by the presence of fleshy cloacal lips.
11.5.2 Staging of Embryos—Nieuwkoop and Faber must be
11.3.2 Diet—The minimum recommended diet for adults
used in all staging of embryos (19).
should be three feedings per week of ground adult beef liver.
11.5.3 Embryo Selection—Normallycleavingembryosmust
Finely diced liver is an acceptable diet, especially for small
be selected for use in testing. The “Atlas of Abnormalities”
colonies. Liquid multiple vitamins should be added to the
should be consulted in order to determine which embryos are
ground beef liver. The concentration of vitamins is shown in
normal (19). It is best to use two levels of selection. In double
Table 2. Concentrations of vitamins from 0.05 to 0.075 cm /5
selection, normally cleaving embryos are first sorted into
g liver are appropriate. Alternatively, #3 size trout or salmon
dishes containing fresh FETAX solution. After a short period
pelletsobtainedcommerciallymaybeused.Allfoodshouldbe
during which cleavage continues, embryos are again sorted
screened for the test material if the test material is present in
ensuring that only normal embryos are selected. Abnormal
the environment.All liver beef must meet USDAstandards for
pigmentationshouldbeviewedasanindicatorofbadembryos.
human consumption.
Either Nieuwkoop and Faber (19) or the “Atlas ofAbnormali-
11.3.3 Temperature—Adults should be kept at 21 6 3°C.
ties” can be used as a reference to determine whether the
11.3.4 Circadian Rhythm—Adults should be kept on a 12-h
cleavage pattern is normal. Mid blastula (stage 8) to early
day/12-h night cycle. The role that circadian rhythm plays in
gastrula (stage 11) must be used to start the test. Embryos
Xenopus reproduction has not been investigated. Thus, a
chosen prior to stage 8 might develop abnormal cleavage
consistent photoperiod should be maintained.
patterns later whereas embryos selected after stage 11 have
11.4 Breeding—Males and females are bred as a single pair. commenced organogenesis. A large bore blood bank Pasteur
pipet can be used to transfer embryos at this stage without
The frogs should be bred in the same water in which the test is
harm. The sorting should be done in 100-mm Petri dishes.
A
TABLE 2 Recommended Concentration of Vitamins
12. Procedure
Vitamin A, IU 1500.
12.1 Experimental Design—FETAX is a 96-h renewal
Vitamin D, IU 400.
whole embryo assay that can be used to evaluate the develop-
Vitamin E, IU 5.
Vitamin C, mg 35.
mental toxicity of a test material. Exposure is continuous
Thiamine, mg 0.5
throughout the test. For each concentration two vessels each
Riboflavin, mg 0.6
containing a maximum of 25 embryos and 10 mL of test
Niacin, mg 8.
Vitamin B ,mg 0.4
Vitamin B , mcg 2.
A
Available from John A. Bantle, Dept. of Zoology, 430 LSW, Oklahoma State
Per millilitre of solution.
University, Stillwater, OK 74078.
E1439 − 12 (2019)
solutionareused.Foreachcontrol,fourvesselsofamaximum repeatability (see 12.3.2). Prior testing suggests that intrabora-
of 25 embryos each are used. Embryos must be randomly tory variability should yield a coefficient of variation less than
assigned to test vessels except when a forced air incubator is 100%.
used, in which there are no hot or cold locations. Test vessels
12.3.1.2 Selection of Concentrations—Concentration selec-
must be randomly assigned to their positions in the incubator.
tion is a multistep process depending on the nature of the test
Inordertoproperlyevaluatedevelopmentaltoxicity,mortality,
material and the results of the first test in this series. The first
malformation, and growth-inhibition, data must be collected.
test simply consists of a series of at least seven concentrations
In most tests it will be possible to generate concentration-
that differ by a factor of ten. If a metabolic activation system
response curves for mortality, malformation, and growth inhi-
(MAS) is to be used to assess possible effects on mammals or
bition.Themortalityandmalformationconcentration-response
for human health hazard assessment, all tests should be
curves should then be used to estimate the concentration that
performed with and without the metabolic activation system.
wouldaffect50%oftheexposedembryos.Atleast90%ofthe
Thisisusuallyadequatetodelineatetheconcentrationrangeto
FETAX-solution controls must have attained stage 46 at 96 h
establish the approximate MAS and No MAS 96-h LC50 and
(19).
EC50values.Thesecondrange-findingtestseriesisperformed
usingtheslidingscaleofconcentrationspresentedinAppendix
12.2 Temperature and pH Requirements:
X2.Thetablepresentsconcentrationvaluesfrom0.001to100;
12.2.1 Temperature—22 6 2°C must be maintained
in steps of 0.0005 between 0.001 and 0.1, steps of 0.05
throughout the 96-h test.Temperatures higher than 26°C cause
between 0.1 and 1, in steps of 0.5 between 1 and 10, and in
malformation whereas low temperatures prevent the controls
steps of 5 between 10 and 100. Using the sliding scale, the
from reaching stage 46 in 96 h.
value closest to the MAS and No MAS 96-h LC50 should be
12.2.2 pH—ThepHofthestockandtestsolutionsshouldbe
identified and then three values immediately below and three
7.7 and must be between 6.5 and 9.0 (14).The pH of a control
values immediately above the LC50 point chosen. The same
vessel and the pH of the highest test concentration should be
method should be used to estimate concentrations surrounding
measured at the beginning of the test and every 24 h thereafter
the 96-h EC50.Atest is performed and the data collected. The
to determine if they have changed.
96-hLC50andEC50valueswithconfidencelimitsshouldthen
be calculated. From the data obtained above, the 96-h LC5,
12.3 Beginning the Test:
LC16, LC50, LC84 and LC95 and the EC5, EC16, EC50,
12.3.1 Recommended Protocol for Testing New Materials—
EC84, and EC95 may be calculated. By determining these
Thefollowingsequenceshouldbefollowedwhentestinganew
values, the concentrations to be tested in the definitive tests
test material to determine the 96-h LC50 and the 96-h EC50
below are established and the slopes of the concentration-
(malformation)(termed the 96-h EC50). This procedure will
response curves are taken into consideration. Additional con-
guide initial range-finding experiments and help reduce repli-
centrations between the EC16 and EC84 are highly recom-
cate test variability. The procedure is iterative and designed to
mended to ensure obtaining a 96-h LC50 and EC50 values.
produce test concentrations for definitive experiments that will
However, the same concentrations must be used for each
yield96-hLC50and96-hEC50valueswithnarrowconfidence
replicate (definitive) test. Interlaboratory studies indicated a
intervals. This should be accomplished by defining several
reduction in intralaboratory test variability when the above
concentrations between the 16 and 84% effect concentrations
procedures were used. For some test materials it may be
(at least 3, preferably 5).The procedure is designed to account
necessary to use the results of the first definitive experiment as
for different slopes of concentration-response curves.
another range-finder and readjust the test concentrations again.
12.3.1.1 Goal of Range-Finding and Definitive Tests—
12.3.2 Replicate-Definitive Tests:
Range-findingtestsaretobeusedwheneverpossibletofindthe
12.3.2.1 Number of Tests and Data Collection—Atleasttwo
best approximation of the 96-h LC50 and EC50 for definitive
replicate-definitive tests are then performed with and without
testing. Once the data are collected from the range-finding
an exogenous MAS each with a separate clutch of embryos
tests, the expected 96-h LC50 and EC50 are estimated using
(see 12.3.3.2). The minimum five concentrations for each
probit analysis, trimmed Spearman-Karber analysis, or the
endpoint determined above are used with and without MAS.
two-pointgraphicalmethod.Thegraphicalmethodisusedonly
The experiments should yield acceptable MAS and NO MAS
when regular statistical analyses fail to generate useful data. If
96-hLC50andEC50values.Iftheydonot,thetestsshouldbe
the data allow probit analysis or trimmed Spearman-Karber
repeated. In some cases where test variability is extremely
methodstobeused,thenprobitanalysismaybeusedwhenthe
high, it may be necessary to determine whether the test
data meet normal distribution and homogeneity of variance
material is rapidly degrading, salting out or volatilizing out of
assumptions.TrimmedSpearman-Karberisusedwhenthedata
solution. Remember that MAS is only used when assessing
failtomeettheseassumptions.Range-findingtestsmaybypass
mammalian developmental toxicity.
the homogeneity of variance requirements here but not in
definitive tests discussed in 12.3.2. Definitive test data are 12.3.2.2 Ensuring Adequate Embryo Supply and Mainte-
analyzed similarly but may not bypass homogeneity of vari- nance of Separate Clutches—To ensure an adequate supply of
ance requirements. Growth inhibition data are not collected normal embryos for each test, three mating pairs should be
from range-finding tests. Once the definitive test concentra- induced and clutches harvested separately. Embryos should be
tions are selected, three definitive tests are performed that will sorted to ensure viability prior to testing. Each test uses early
yield 96-h LC50 and EC50 information with acceptable embryosderivedfromasinglematingpair;ifthecontrolsfrom
E1439 − 12 (2019)
a particular mating pair indicate a problem with fertility or stage 46. Deviations from this standard exposure time must be
viabilityofearlyembryos,thetestwillbeunacceptableforthat reported as deviating from standard FETAX conditions.
particular clutch. Each individual test will yield data that will
12.6 Exogenous Metabolic Activation System (MAS)—An
be used to generate concentration-response curves for
exogenous MAS must be used when FETAX is used to
mortality, malformation, and growth inhibition. It is necessary
evaluate developmental toxicity for human health hazard
to keep clutches separate because embryos from a particular
assessment.TheMASiscomposedofratlivermicrosomesand
matingpairmightdeveloppoorlyalthoughtheyinitiallyappear
a nicotinamide adenine dinucleotide (reduced form) [NADPH]
acceptable.Thiswouldcausealltheembryostobediscardedif
generator system which simulates mammalian metabolism.
embryos are mixed from different mating pairs.
Since early Xenopus embryos have limited xenobiotic meta-
Each test should be performed with embryos derived from a
bolic capabilities, particularly cytochrome (CYP) P-450, the
singlematingpairregardlessofthenumberofreplicatedishes.
incorporation of the exogenous system into the standard assay
The selection of experimental design and statistical methods
protocol is warranted. Aroclor 1254 may be used as a broad-
required to evaluate mortality, malformation, and growth-
spectruminducingagentandusedinthemajorityofsituations.
inhibition data, should consider the type of compound or
chemical mixture being evaluated and the limitations that Isoniazid induction or uninduced microsomes may be used in
sample or time availability might imply as far as appropriate those cases whereAroclor 1254 induction may repress specific
statistical techniques (20). P-450 isozymes. The nature of the test material may suggest
12.3.3 Reference Toxicant—For a positive control or refer- which inducing system to use. In cases where limited informa-
ence toxicant, 6-aminonicotinamide presents a mortality and tion is available concerning test material biotransformation, a
malformation database convenient for reference purposes.
setofAroclor1254andisoniazid-inducedratlivermicrosomes
Commercial sources for the 6-aminonicotinamide (CAS #329-
mixed in equivalent activity ratios may be used.
89-5; formula weight, 137.14) should specify the physico-
12.6.1 Sterile plastic Petri dishes should be used to mini-
chemicaldataandthepurityforthecompoundwhichensureits
mize bacterial contamination, although the volume is only 8
being comparable to that readily available to other laboratories
mL instead of the 10 mL in the glass dishes. Antibiotics are
(for example, UV spectroscopic characterization: at A and
requiredtoinhibitbacterialgrowthandthesemayinteractwith
pH 1.8, a 9.74 µg/mL solution of 6-aminonicotinamide has an
the test substance. Microsomal protein can slow growth and
absorbance of 1 and an absorbance ratio (A /A ) of 2.28).
257 302
development at concentrations greater than 60 µg/mL.
The purity should be >99%. From this published database, the
NADPH, which is required for microsomal activity, can also
96 h LC50 is approximately 2,230 mg/L and the 96 h EC50
cause abnormal development and it
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