draft oecd guideline for the testing of chemicals · 74 guideline is used for the testing of a...
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DRAFT OECD GUIDELINE FOR THE TESTING OF CHEMICALS 1
The Xenopus Embryonic Thyroid Assay (XETA) 2
1. INTRODUCTION 3
1. The need to develop and validate an assay capable of detecting substances active in the thyroid 4
system of vertebrate species originates from concerns that environmental levels of chemicals may cause 5
adverse effects in both humans and wildlife. 6
2. The Xenopus Embryonic Thyroid Assay (XETA) test guideline describes an aqueous assay that 7
utilizes free-living Xenopus laevis (X. Laevis) embryos at stage 45 (according to Nieuwkoop and Faber (1) 8
in a multi-well format to detect thyroid active chemicals. The XETA was designed as a screening assay to 9
provide a medium throughput and short-term assay to measure the response of embryonic stage tadpoles 10
to potential thyroid active chemical (2). The XETA is intended to be an amphibian screen classifying the 11
chemicals into potentially thyroid active or inactive but the XETA is not intended to give a NOEC or an 12
ECx for risk assessment. The XETA is placed at the level 3 of the OECD conceptual framework for the 13
testing of endocrine disrupters (3). The OECD GD 150 provides further guidance on the interpretation and 14
extrapolation between taxa of the results of the XETA (3). 15
3. The assay is transcription-based and uses a transgenic tadpole line harbouring the THbZIP-GFP 16
genetic construct. This transgenic line is commercially available. These Xenopus laevis embryos harbour 17
a genetic construct comprising of the promoter of the TH/bZIP gene, which is a marker for amphibian 18
metamorphosis, coupled to a fluorescent reporter gene (GFP). The TH/bZIP gene code a transcription 19
factor implicated in the phenomenon of metamorphosis, a process controlled by thyroid hormones. The 20
expression of the TH/bZIP gene is regulated directly at the moment of metamorphosis by thyroid hormone 21
(4). TH/bZIP expression is a trigger for metamorphosis and in part controls its timing. Altered timing of 22
metamorphosis is considered an adverse physiological outcome. The use of this gene as a biomarker allows 23
the detection of potential modulations of the thyroid activity induced by the test product. Before performing 24
the XETA the laboratory should verify that it has the certifications that may be required by the local 25
regulation on the use of level 1 transgenic organisms. The XETA should be performed using the THbZIP-26
GFP transgenic line used for the test guideline development. The use of another transgenic line based on 27
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the THbZIP promoter driving the expression of GFP or another reporter gene requires a complete OCDE 28
validation process to adapt the validation criteria, statistical analysis and fluorescence thresholds used in 29
the decision logic. 30
4. The assay measures the ability of a chemical to activate or inhibit transcription of the genetic 31
construct, whether directly through binding to the thyroid receptor (TR; see Annex 1 for abbreviations) or 32
modifying the binding of thyroid hormones (TH) to the TR, or indirectly by modifying the amount of TH 33
available to activate the TR and thereby transcription of the TH/bZIP-GFP construct. To date the XETA 34
has been shown to detect chemicals acting through various mechanisms of action including TH receptors, 35
agonists (e.g. T4, TRIAC) and antagonists (e.g. NH3 (a pharmacological antagonist of the TRs)), 36
modulators of TH clearance (including UDPGT (UDP-glucuronosyltransferase) modulators (e.g. 37
Phenobarbital)) and modulators of TH metabolism (including deiodinase inhibitors (e.g. iopanoic acid)) 38
(2)(5). In addition, the XETA potentially detects modulators of TH transport via interaction with TH 39
plasma binding proteins and inhibitors of TH transmembrane transporters. As Xenopus NF45 stage 40
embryos are not synthesising their own TH, inhibitors of TH synthesis are not intended to be detected by 41
the XETA. The XETA not allows to distinguish between the different modes of action but is informative 42
to know if the chemical act as a global activator or inhibitor of the thyroid signalling in the xenopus tadpole 43
at this developmental stage. As the transcription of the TH/bZIP-GFP construct implies the direct action 44
of the TR on the TH/bZIP promotor, chemical affecting the thyroid hormones action through alternative 45
signalling pathways that do not require direct interaction between thyroid hormone receptors and DNA (i.e 46
“non- genomic actions”) are not intended to be detected by the XETA. 47
5. This guideline proposal is based on two international ring studies conducted in 2012-2017 (5). The 48
test has been validated in 6 laboratories with 9 mono-constituent test substances. 49
6. The endpoint measured is fluorescence of tadpoles. When transcription of the genetic construct is 50
activated or inhibited following chemical exposure, tadpoles express more or less GFP (Green Fluorescent 51
Protein) and therefore emit more or less fluorescence compared to unexposed tadpoles where fluorescence 52
remains at the basal level. 53
7. The test chemical is tested in the presence and absence of 3.25 µg/L of the thyroid hormone T3 54
(triiodothyronine). As TH concentration remains very low at this larval stage, adding T3 to the test medium 55
allows the detection of substances affecting T3 availability or antagonising TR. 56
2. INITIAL CONSIDERATIONS AND LIMITATIONS 57
8. Before starting the assay, it is mandatory to have performed a series of experiments to determine 58
the appropriate methods for the fluorescence quantification and to demonstrate technical proficiency by 59
testing and correctly categorising proficiency substances into thyroid active or inactive chemicals (see 60
Table 1). 61
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62
9. This test guideline relies on the quantification of fluorescence in the tadpole body. A limitation of 63
this test guideline is that it should not be used for test chemicals emitting fluorescence between 500 and 64
550 nm when excited at wavelengths between 450 and 500 nm and able to accumulate in the tadpole body. 65
Test chemicals sharing these two properties may induce a fluorescence which could be interpreted as GFP 66
signal, leading to the test chemical being incorrectly identified as thyroid active. A simple protocol to 67
determine if the test chemical emits fluorescence is proposed in section 7.5. 68
69
70
10. When considering testing of mixtures, difficult-to-test chemicals (e.g. unstable), or test chemicals 71
not clearly within the applicability domain described in this Guideline, upfront consideration should be 72
given to whether the results of such testing will yield results that are scientifically meaningful. If the Test 73
Guideline is used for the testing of a mixture, a UVCB or a multi constituent substance, its composition 74
should, as far as possible, be characterized, e.g. by the chemical identity of its constituents, their 75
quantitative occurrence and their substance-specific properties. Recommendations about the testing of 76
difficult substances (e.g. mixture, UVCB or multi-constituent substance) are given in Guidance Document 77
No. 23 (6). 78
3. PRINCIPLE OF THE TEST 79
3.1. General experimental design 80
81
11. The general experimental design entails exposing stage NF45 transgenic THbZIP-GFP X. laevis 82
tadpoles in 6-well plates to a test chemical in the presence “spiked mode” and absence “unspiked mode” 83
of a cotreatment with 3.25 µg/L of T3. It is recommended to use a minimum of three concentrations plus 84
controls. The test uses 20 tadpoles distributed in two wells (10 tadpoles per well) per test concentration, 85
under semi-static conditions. The exposure duration is 72h with a daily renewal (i.e. after 24h and 48h) of 86
the exposure solutions. Three independent runs have to be performed for each assay. The assay measures 87
GFP fluorescence in the transgenic THbZIP-GFP tadpoles by way of a spectrofluorimeter or fluorescence 88
imaging that transforms the fluorescence signal to a numerical format. A detailed overview of test 89
conditions can be found in Annex 2. 90
91
3.2. Controls 92
12. The XETA requires the following control groups: 93
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Test medium control: 2 x 10 tadpoles/well are exposed to test medium only. This control 94
defines the basal fluorescence level of the tadpoles in the test medium. 95
T3 control: 2 x 10 tadpoles/well are exposed to 3.25 µg/l of T3. This control establishes 96
the fluorescence level for a T3 concentration of 3.25 µg/l. This concentration is equivalent 97
to the plasma T3 hormone concentration during tadpole metamorphosis and is known to 98
induce morphological changes and TH target genes modulation in premetamorphic 99
tadpoles (7,8). This control serves as a positive control for the groups without T3 100
cotreatment and a reference control for the group receiving a T3 cotreatment. 101
T4 control (saturation control): 2 x 10 tadpoles/well are exposed to 10 mg/l of T4. This 102
control establishes the maximal fluorescence level that could be quantified in the 103
experiment. This control serves as a positive control for the groups with T3 cotreatment 104
and together with the test medium control define the dynamic range of fluorescence for a 105
given experiment. 106
13. If a solvent is used, the test medium control, T3 control and T4 control should receive an equal 107
concentration of solvent (see also §35). 108
109
3.1. Replication 110
14. One test is composed of three independent and valid runs using 20 tadpoles/ treatment group (see 111
figure 1). Each run must be performed using independent solutions and spawn. The raw data for a given 112
test chemical is obtained by pooling together the data from the three runs to obtain n=60 fluorescence 113
values in each experimental group. 114
115
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116
Figure 1: Overview of the XETA. (“+/- T3” refers to unspiked and spiked groups) 117
118
4. INFORMATION ON THE TEST CHEMICAL 119
15. Information on the test chemical, should be reported (see section 8.4) and includes e.g. the 120
structural formula, purity and, if available, stability in light, stability under the test conditions, pKa, Pow, 121
information on the fate of the substance and on its potential for being rapidly degraded in the test system 122
e.g. results of a ready biodegradability test (see OECD TG 301 and 310 (9); (10)). 123
16. The water solubility of the test chemical in the test medium should be known and a validated 124
analytical method, of known accuracy, precision, and sensitivity, should be available for the quantification 125
of the test chemical in the test solutions with reported recovery efficiency and limit of quantification, 126
guidance for the validation of quantitative analytical methods could be found in the GD204 (11). Analytical 127
determination of the test chemical concentration should be performed before and after renewal. 128
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5. DEMONSTRATION OF PROFICIENCY 129
5.1. Fluorescence quantification 130
17. The XETA relies on the quantification of the fluorescence emitted by each tadpole. To ensure that 131
a proper and accurate quantification can be achieved, preliminary experiments should be conducted. These 132
experiments are performed to calibrate the spectrofluorimeter and to ensure that a suitable dynamic range 133
of fluorescence measurements can be read by the equipment. These experiments are detailed in Annex 3. 134
Alternatively, the fluorescence emitted by the tadpoles can be quantified by imaging using a fluorescence 135
microscope equipped with an appropriate camera (5). In this case the amplitude of fluorescence induction 136
obtained with a range of concentrations of T3 should meet the same criteria as for the spectrofluorimeter; 137
optimisation of image acquisition and image analysis parameters is recommended using the same 138
procedure as that detailed for spectrofluorimeters in Annex 3. 139
140
5.2. Proficiency substances 141
18. Prior to routine use of this Test Guideline, laboratories should demonstrate technical proficiency 142
by correctly categorising the four Proficiency Substances listed in Table 1. 143
Substance CASNR Category Concentrations to test Sensitivity range
T4 51-48-9 Active 0.01; 0.1; 1; 2; 5 and 2 mg/L 0.01; 0.1 mg/L
PTU 51-52-5 Active 1; 3; 10; 30; 100 mg/L 3 to 100 mg/L
Abamectine 67-64-1 Inert 0.1; 0.5; 1; 5 and 10 mg/L Inert
Methomyl 16752-77-5 Inert 18; 36; 56; 112; 168 µg/L Inert
Table 1. Proficiency Substances. T4 (thyroxine), PTU (Propylthiouracil) 144
145
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6. VALIDITY OF THE TEST 146
19. For the test to be valid, the following criteria should be met for each run and for the pool of the 147
three runs: 148
-A statistically significant induction of fluorescence should be measured between the test medium 149
control group and the T3 control group. The mean fluorescence of the T3 group should be at least 150
20% higher than mean of fluorescence of the test medium control group. 151
-A statistically significant induction of fluorescence of at least 70% should be present between T4 152
control group and the test medium control. 153
-The coefficient of variation of the fluorescence intensity measured for the test medium control 154
should not exceed 30%. 155
-The exposure should have been done at 21 ± 1°C for 72h +-2h. 156
-The initial pH of the exposure solution should be between 6.5 and 8.5. The inter-replicate and the 157
inter-treatment differential should not exceed 0.5. 158
- The mortality should not exceed 20% in each control groups. 159
In addition, for the test to be valid, at least three treatment levels should be uncompromised. A treatment 160
level with a mortality exceeding 20% in a run or in the pool of the three runs is compromised. 161
7. DESCRIPTION OF THE METHOD 162
7.1. Apparatus 163
20. Normal laboratory equipment and especially the following: 164
-laboratory incubator or any adequate apparatus for temperature and light control; 165
-transparent cell culture grade 6-well plates made of chemically inert material; 166
-conical bottom black 96-well plates certified for fluorescence quantification; 167
-pH meter; 168
-stereomicroscope equipped with a light source (for embryo and tadpole sorting); 169
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-spectrofluorimeter (96-well plate reader) or fluorescent microscope equipped for GFP 170
fluorescence quantification (5); 171
- analytical instrumentation appropriate for the chemical on test or contracted analytical services. 172
173
7.2. Test organism 174
21. The test organisms for the XETA are heterozygous X. laevis tadpoles of the THbZIP-GFP 175
transgenic line. These tadpoles should be produced by mating a homozygous THbZIP-GFP Xenopus and 176
a wild-type Xenopus. The THbZIP-GFP transgenic line is maintained in several laboratories (5) and can 177
be obtained upon subscribing a licence agreement. 178
22. The South African clawed frog, X. laevis, is the test species selected for the XETA. Xenopus laevis 179
is a relevant amphibian model because its early development and thyroid hormone dependent 180
metamorphosis have been extensively studied. In addition, amphibians and higher vertebrates, including 181
mammals, share high genetic homology (12) as well as similar biotransformation systems and homologous 182
endocrine pathways (13), allowing the XETA assay to provide information that may be extrapolated to 183
other taxa. This species is also utilized in the two OECD Test Guidelines using amphibians (AMA; OECD 184
TG 231 (14) and LAGDA OECD TG 241(15)). 185
23. In a given run, all tadpoles used as test organisms should be derived from the same spawn. The 186
exposure phase of the test is initiated with stage NF45 tadpoles (after 7 days post fertilisation at 21°C one-187
week post fertilisation at 21°C). Tadpoles should be ideally bred within the laboratory from stock animals. 188
Alternatively, tadpoles could be shipped from another laboratory and received the earliest as possible to 189
let the longest possible recovering period before the beginning of the test. Acclimation and batch 190
acceptance criteria are outlined in Annex 6. 191
24. Housing and care of X. laevis are described by Reed (16). Appropriate care and breeding of X. 192
laevis are described by the ASTM standardized guideline for the FETAX assay (17). A complete breeding 193
protocol is outlined in Annex 7. 194
195
7.3. Test Medium 196
25. The test medium could be any water permitting normal growth and development of X. laevis 197
tadpoles including glass bottled still mineral water, springwater, well water and charcoal-filtered tap water. 198
Because local water quality can differ substantially from one area to another, analysis of water quality 199
should be undertaken to screen for potential contaminant (including heavy metals) and chemicals likely to 200
interfere with the assay, particularly if historical data on the appropriateness of the water for raising 201
Xenopus is not available. Special attention should be given that the water is free of copper, chlorine and 202
chloramine; all of which are toxic to tadpoles, and free of known thyroid active substances. Results from 203
analysis of water quality should be reported. 204
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26. The following water quality standards are suitable for X. laevis (16). However, any medium that 205
supports the normal growth and development of X. laevis and meets the test validity criteria is suitable as 206
test medium. 207
Alkalinity 10 – 250 mg/L Ca CO3 208
Hardness = 75-150 mg/L 209
pH = 6.5-8.5 (the inter-replicate and the inter-treatment differential should not exceed 0.5) 210
Salinity = 0.4 mg/L 211
Conductivity = 50 -2000 μS/cm 212
Non-ionised ammonia (NH3) < 0.02 mg/L 213
Nitrite (NO2-) < 1 mg/L 214
Nitrate (NO3-) < 50 mg/L 215
Residual Chlorine <10 μg/LL 216
Dissolved oxygen content > 80% saturation 217
CO2 < 5 mg/L 218
219
7.4. Feeding 220
27. Tadpoles between developmental stages NF45 (beginning of the test) and NF47 (end of the test) 221
are used for this test. They are not fed before and during the test as yolk is still present in the intestine from 222
stage NF45 to stage NF47 and is used as the source of energy for the development of the tadpole (1). 223
224
7.5. Determining potential fluorescence of the test chemical 225
28. This test guideline should not be used for test chemicals emitting fluorescence between 500 and 226
550 nm when excited at wavelengths between 450 and 500 nm and able to accumulate in the tadpole body. 227
Test chemicals sharing these two properties may induce a fluorescence which could be interpreted as GFP 228
signal, leading to the test chemical being incorrectly identified as thyroid active. A simple protocol to 229
determine if the test chemical emits fluorescence at these wavelengths is to place 200 µL of a solution of 230
the test chemical at its solubility limit and 200 µL of test medium into two wells of a 96-well plate and to 231
quantify the fluorescence using the same apparatus and settings as for the quantification of tadpole 232
fluorescence. If a fluorescent chemical is identified, 20 wild type X. Laevis tadpoles could be exposed at 233
21°C for 72h with a daily renewal to the highest concentration intended to be tested in the XETA and the 234
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fluorescence should be quantified and compared to the fluorescence of a group of 20 wild type tadpoles 235
exposed to the test medium only in the same conditions. If a significant difference in fluorescence is 236
present, the chemical is fluorescent and accumulate in the tadpole body and should not be tested using the 237
XETA. 238
239
7.6. Selection of test concentrations 240
7.6.1. Establishing the maximum test concentration 241
29. For the purposes of this test, the maximum test concentration should be set by the solubility limit 242
of the test chemical in the test medium; the maximum tolerated concentration (MTC) for acutely toxic 243
chemicals; or 100 mg/L, whichever is lowest. 244
30. The MTC is defined as the highest test concentration of the chemical which results in less than 245
20% acute mortality and in less than 20% malformed tadpoles. Using this approach assumes existing 246
empirical acute mortality data from which the MTC can be estimated. Estimating the MTC can be inexact 247
and typically requires some professional judgment. Although the use of regression models may be the most 248
technically sound approach to estimating the MTC, a useful approximation of the MTC can be derived 249
from existing acute data by using 1/3 of the acute LC50 value. However, acute toxicity data may be lacking 250
for the test species. If species specific acute toxicity data are not available, then a 72-hours LC50 test can 251
be completed at 21°C with tadpoles that are representative (i.e., same stage) of those used in the XETA. 252
Optionally, if data from other aquatic species are available (e.g. LC50 studies in fish or other amphibian 253
species), then professional judgment may be used to estimate a likely MTC based on inter-species 254
extrapolation. It should be noted that acute toxicity studies are normally performed with adults, therefore, 255
the potential differences in sensitivity between life stages should be accounted for in the definition of the 256
MTC. 257
31. Alternatively, if the chemical is not acutely toxic and is soluble above 100 mg/L, then 100 mg/L 258
should be considered the highest test concentration, as this concentration is typically considered 259
“practically non-toxic”. 260
32. As a semi-static renewal method is used, then the stability of the test chemical concentration should 261
be documented. The stability of the test chemical should ideally allow the exposure concentration to remain 262
within 20% of the nominal concentration in a 24h time frame. Chemical analysis during the test shall be 263
performed to provide this information. This shall be performed at 0 h (fresh solution), 24 h (fresh solution 264
and solution that was in contact with the tadpoles), 48 h (fresh solution and solution that was in contact 265
with the tadpoles) and 72 h (solution that was in contact with the tadpoles) for each test concentration. If 266
the deviation from the nominal concentrations is greater than 20%, results should be based on the measured 267
concentrations. Twenty-four hours -+2h renewal periods are the longest periods accepted. If concentrations 268
cannot be maintained within +-20% in the test system, shortened renewal periods could be used. Use of 269
mean measured concentrations is allowed for substances that degrade giving a concentration with a greater 270
than 20% reduction compared to nominal despite shortened renewal periods. 271
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7.6.2. Test Concentration Range 272
33. There is a required minimum of three test concentrations and a test medium control (and vehicle 273
control if necessary). Generally, a concentration separation (spacing factor) of 3.2 to 10-fold is 274
recommended. 275
276
7.7. Test solutions 277
34. Test solutions of the chosen concentrations are usually prepared by dilution of a stock solution. 278
The pH of each test solution should be adjusted to a pH comprised between 6.5 and 8.5. Stock solutions 279
should be prepared by dissolving the test chemical in the test water using mechanical means such as 280
agitation, stirring or ultrasonication, or other appropriate methods. If possible, the use of solvents should 281
be avoided. For difficult test chemicals, including volatile or absorptive chemicals, the OECD Guidance 282
Document n°23 on aqueous-phase aquatic toxicity testing of difficult test chemical should be consulted 283
(6). 284
35. If a solvent is required in order to produce a suitably concentrated and homogeneous stock solution, 285
maximum solvent level should be at least one order of magnitude below the appropriate no-observed effect 286
concentration (NOEC) and in any case should not exceed 100 μl/L or 100 mg/L (6). It is recommended to 287
keep solvent concentration as low as possible (e.g, < 20 μl/L) to avoid potential effects of the solvent on 288
endpoints measured (18). 289
36. The concentration of solvent should be equal in all test concentrations and in all controls. Both 290
solvent control and test medium control, and both T3 control and T3 solvent control, should be used if the 291
solvent was not tested previously and shown to be negative using the XETA. The selection of an 292
appropriate solvent depends on the physico-chemical properties of the test chemical and on the sensitivity 293
of X. laevis, which should preferably be determined in a previous study. The following solvents have been 294
successfully used in the XETA: dimethylsulfoxyde (DMSO), ethanol, methanol, acetone, acetonitrile. 295
Please note that if DMSO have been successfully used in the XETA, it should not be preferentially used 296
as it increases the entrance of the chemical in the organism. To determine if the presence a solvent not 297
previously used for the XETA could hamper the reliability of the test results, a dose response experiment 298
adapted from Annex 3 (part 2) using T3 in the presence and absence of solvent should be undertaken to 299
determine if the solvent had any effect on the LOEC or on the amplitude of fluorescence induction. Ideally, 300
a XETA should be performed with the solvent as a test chemical to determine if it is positive in the XETA 301
and potentially determine a NOEC. 302
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8. PROCEDURE 303
8.1. Exposure conditions 304
37. The tadpoles are exposed in chemically inert cell culture grade 6-well plates (typically wells of 305
34mm internal diameter and 20mm height). Each well should contain ten tadpoles in 8 ml of solution. In a 306
run, 20 tadpoles are exposed to each test concentration. If plastic well plates are not appropriate for a given 307
test chemical, alternative glass vessels (i.e. small diameter petri dishes) should be used. 308
38. Tadpoles are maintained in an incubator for 72h ± 2h at 21 ± 1°C in constant dark throughout the 309
test. 310
311
8.2. Test Initiation and Conduct 312
Day 0 313
39. The exposure should be initiated when the tadpoles reach developmental stage NF45 (7 days post 314
fertilisation at 21°C; Annex 10). 315
40. All the tadpoles used for each run should originate from a single reproduction (i.e., spawn from 316
different females should not be mixed). For selection of test animals, tadpoles should be observed under a 317
binocular dissection microscope and tadpoles that exhibit grossly visible malformations, abnormal 318
pigmentation, or physical injury (e.g., damage of the tail, oedema, scoliosis) should be excluded from the 319
assay (Annex 8 and 9). Healthy and normal looking tadpoles of the stock population should be pooled in 320
a single vessel containing an appropriate volume of test medium. The selected tadpoles should be 321
homogenous in size, tadpoles presenting an obvious difference in size should be removed. Spawn that 322
contain less than 80% of normal and healthy tadpoles at stage NF45 should not be used for the test. 323
41. For developmental stage determination, tadpoles should be removed from the pooling tank using a 324
transfer pipette and isolated into a drop of dilution water in a transparent Petri dish. For stage determination, 325
it is preferential to not use anaesthetics. If used, methodology for appropriately using an anaesthetic such 326
as MS-222 (tricaine methanesulfonate) should be obtained from experienced laboratories and reported with 327
the test results. Typically, MS222 is used at 0.1g/l and buffed to pH 7-8. If the tadpoles are anesthetised, 328
the anaesthetic treatment should be applied in the same condition to all tadpoles and all tadpole groups. 329
Animals should be carefully handled during this transfer in order to minimize handling stress and to avoid 330
any injury. 331
42. The developmental stage of the animals is determined using a binocular dissection microscope. 332
According to Nieuwkoop and Faber (1), the primary developmental landmark for selecting stage NF45 333
organisms is intestinal morphology. At stage NF45, the intestinal spirals reach one and a half rotations 334
(Annex 10). The morphological characteristics of the intestine should be examined under the microscope. 335
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While the complete Nieuwkoop and Faber (1) guide should be consulted for comprehensive information 336
on staging tadpoles, one can reliably determine stage using this prominent morphological landmark. 337
43. Developmental stage distribution in X. laevis is homogenous around stage NF45 (19).Therefore, it 338
is recommended to stage 10% of the total of tadpoles required for the study and consider starting the 339
exposure to the test chemical when at least 80% of the staged tadpoles have reached stage NF45. Typically, 340
180 tadpoles are required for a run. It is recommended that approximately 18 tadpoles from the test batch 341
are staged. If at least 15 of these tadpoles have reached stage NF45 the batch is suitable to initiate a test. 342
44. To start the experiment, 10 tadpoles/well should be placed into 6-well plates in drops of test 343
medium (using a transfer pipet with the tip cut off to avoid damaging the tadpoles). The test medium should 344
be removed and the test chemical solutions added for the first time. One should pay attention to work with 345
one plate at a time to avoid drying out the tadpoles. An example of 6-well plate set up is shown in Annex 346
11. 347
348
Day 1 and day 2 349
45. The test chemical solutions and the controls solutions should be renewed at 24 and 48 hours ideally 350
± 1 h or ± 2 h. Each well is inspected for animals with abnormal appearance (injuries, abnormal swimming 351
behaviour, etc..). Dead tadpoles or those exhibiting grossly visible malformations (Annex 8) or injuries 352
should be removed and the latter be euthanised as prescribed in paragraph 46. All observations should be 353
recorded. Tadpoles exhibiting grossly visible malformations or injuries should be counted as dead tadpoles 354
to calculate the mortality. If >20% mortality is encountered in one of the control groups or leaving less 355
than three uncompromised treatment levels, then the on-going independent run is stopped and the source 356
of the mortality should be identified. 357
Day 3 Fluorescence quantification 358
46. The fluorescence of each tadpole is quantified after 72 hours ± 2 hours of exposure. Tadpoles 359
should be transferred individually (i.e. one tadpole per well) into wells of black 96-well plates with conical 360
bottoms suitable for fluorescence quantification. For this purpose, the test solutions should be first renewed 361
with 8 ml of test medium and dead tadpoles or those exhibiting grossly visible malformations should be 362
removed. All observations should be recorded. Tadpoles should then be anesthetized by adding 2 ml of 363
1g/L buffered MS222 into the wells. To avoid excessive anesthesia, only the number of tadpoles to fill one 364
96-well plate at a time are anesthesized. After complete anaesthesia (2 to 5 minutes), the tadpoles are 365
placed individually in each well of the 96-well plate. It is recommended to place the tadpoles from the 366
same well of the 6-well in the same row of the 96-well plate (i.e., each row of the 96-well plate is equal to 367
one well of the 6-well plate). An example of 96-well plate is shown in Annex 12. Each tadpole is placed 368
on its back using a thin transfer pipet (aspirating the medium back and forth will help to turn the tadpole) 369
and most of the medium is removed while maintaining enough moisture under the tadpole. The tadpoles 370
are placed separately in the wells with the head in the centre of the well and the dorsal surface in contact 371
with the plate. The tail of the tadpole is placed around the tadpole’s head. An image of tadpole in the 372
correct position is shown in Annex 12. The 96-well plate containing the tadpoles is then placed in the 373
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spectrofluorimeter or fluorescent microscope to quantify the fluorescence using the parameters identified 374
during the calibration. Please note that once the tadpoles have been placed on the back and the MS222 375
removed it is important to proceed quickly to the fluorescence quantification in order to prevent the 376
tadpoles from drying out At the end of the test, the tadpoles are usually at stage NF47. At stage NF47, 377
the intestinal spirals reach two and a half to three and a half rotations, xanthophores appears on the 378
abdomen and hind limb buds become distinct (Annex 10). 379
380
Terminating experiment 381
47. After reading the fluorescence, each well of the 96-well plate is filled with a solution of buffered 382
MS222 (1 g/L) using a squeeze bottle to euthanize the tadpoles. The 96-well plates are disposed as required 383
by local laboratory safety protocols. 384
385
8.3. Analysis of data / Evaluation of test results 386
8.3.1. Data analysis considerations 387
48. A trimming could be performed prior statistical analysis by omitting in each run the highest and lowest 388
10% of the fluorescence values for each control group and each concentration of T3-spiked and unspiked 389
test concentration (i.e. omitting the two highest and two lowest values of each group of 20 values). This 390
trim is intended to removed values that arise in the XETA from several events including missorted tadpoles 391
(abnormal size or pigmentation), misplaced tadpoles in the 96-well plate (tadpoles upside down or on their 392
side), tadpoles dying after anaesthesia and wells containing no tadpoles. Alternatively, if a fluorescent 393
microscope is used for fluorescence quantification, an image quality control should replace the 10% trim 394
to remove the values corresponding to these events before the statistical analysis. 395
49. Data from the three independent runs are regrouped to obtain 32 to 60 fluorescence values for each 396
concentration and controls. If a solvent was used in the experiment, an evaluation of the potential effects 397
of the solvent should be performed. This is done through a statistical comparison of the solvent control 398
group and the test medium control group. If statistically significant differences are detected between the 399
test medium only control and solvent control group, determine the study endpoints for the response 400
measures using the solvent control. If there is no statistically significant difference between the clean water 401
control and solvent control use the pooled test medium only and solvent controls. 402
403
8.3.2. Statistical analysis 404
50. Appropriate statistical methods should be used according to OECD Document 54 on the Current 405 Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application (20). In general, 406 effects on the fluorescence of the test chemical compared to the control are investigated using one-tailed 407 hypothesis testing at p <0.05. 408
│ 15
51. Step-down trend tests are recommended to determine whether there are significant differences (p < 409
0.05) between the control(s) and the various test chemical concentrations (20). Comparisons of the 410
concentration-response of tadpoles should be done either with Williams’ test or Dunnett’s test to determine 411
any statistical difference (see details in Annex 14). 412
413
8.3.3. Decision logic 414
52. Decision logic was developed for the XETA to provide logical assistance in the conduct and 415
interpretation of the result of the bioassay (see flow chart in figure 2). This decision logic is based on three 416
valid runs pooled for statistical analysis (see figure 1 in §14). A test chemical is considered to give a positive 417
result in the XETA if at least one concentration tested including the highest is active in T3-spiked and/or 418
unspiked mode. 419
In unspiked mode an active concentration is defined as a concentration giving a 420
statistically significant fluorescence increase of 12% or greater compared to the 421
test medium control. 422
In T3-spiked mode an active concentration is defined as a concentration giving a 423
statistically significant fluorescence increase or decrease of 12% or greater 424
compared to the T3 control. 425
53. Fluorescence decreases in unspiked mode are not expected as the tadpole does not synthetize its own 426
thyroid hormone at this development stage. If a statistically significant fluorescence decrease >12% is 427
observed in unspiked mode, it should be considered to repeated the XETA using a lower dose range or 428
performing another test. 429
430
16 │
431
432
Figure 2. Decision logic for the conduct of the XETA. 433
434
8.4. Test report 435
54. The test report should include the following information: 436
8.4.1. Test chemical: 437
- Mono-constituent substance: 438
│ 17
physical appearance, water solubility, and additional relevant physico-chemical properties; chemical 439
identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, 440
purity, chemical identity of impurities as appropriate and practically feasible, etc. (including the organic 441
carbon content, if appropriate). Information on biodegradation if available. 442
- Multi-constituent substance, UVCBs and mixtures: characterised as far as possible by chemical identity 443
(see above), quantitative occurrence and relevant physico-chemical properties of the constituents. 444
-Analytical method for quantification of the test chemical. 445
-Results from any preliminary studies on the stability or solubility of the test chemical. 446
-Results on lack of fluorescence emission at wavelength of 450 and 500 nm; as well on as lack of 447
accumulation in tadpole body for substances shown to be fluorescent at these wavelengths. 448
449
8.4.2. Test species: 450
-Scientific name, transgenic line, supplier or source, and culture conditions. 451
452
8.4.3. Test conditions: 453
-Test procedure used (e.g., concentrations tested, temperature, duration, semi-static, volume, number of 454
tadpoles per ml). 455
-Details of test medium characteristics (reference of mineral water or spring water, description of tap water 456
treatment (e.g. charcoal filtration…) and any measurements made. 457
-Method of preparation of stock solutions and frequency of renewal (the solvent and its concentration 458
should be given, when used). 459
-Brand and references of 6-well and 96-well plates used for exposure and fluorescence quantification. 460
-References and settings of the spectrofluorimeter or fluorescence microscope used for quantification. The 461
method used for image analysis should also be provided for this latter case. 462
463
8.4.4. Results: 464
-Results from any preliminary studies on the LC50 or MTC of the test chemical; 465
-The nominal test concentrations and results of all chemical analyses to determine the concentration of the 466
test chemical in the test vessels; the measured exposure concentration as an appropriate statistical average 467
(e.g. arithmetic mean, time-weighted mean etc)” where appropriate; the recovery efficiency of the 468
analytical method and the limit of quantification should also be reported. 469
18 │
-The numbers of dead and malformed tadpoles in each replicate and the day on which they occurred. 470
-Fluorescence quantification raw data (e.g. individual tadpole’s fluorescence raw data). 471
-Approach for the statistical analysis and treatment of data including statistical test used. 472
-Evidence that the controls meet the validity criteria. 473
-Evidence that all groups meet the validity criteria in regards to survival. 474
-The means of fluorescence of each experimental group including all control and test chemical 475
concentrations and their SEM (Standard Error of the Mean) should be presented both by a graphical 476
representation and in a table. 477
-The percentage of fluorescence increase or decrease for each concentration compared to its respective 478
control in spiked and unspiked modes. 479
-Other observed biological effects or measurements: report any other biological effects which were 480
observed or measured (e.g., abnormal behaviour, or malformations or abnormal pigmentation). 481
-An explanation for any deviation from the Test Guideline and deviation from the acceptance criteria, and 482
considerations of potential consequences on the outcome of the test. 483
-Where appropriate, a discussion presenting the list of concentrations found active in spiked and unspiked 484
mode. 485
-A conclusion presenting if the test chemical is found thyroid active or inactive. 486
487
│ 19
9. LITERATURE 488
(1) Nieuwkoop, P. D., Faber, J. (1994). Normal Table of Xenopus laevis (Daudin). Garland Publishing 489
Inc, New York ISBN 0-8153-1896-0. 490
(2) Fini, J.B., Le Mevel, S., Turque, N., Palmier, K., Zalko, D., Cravedi, J.P., Demeneix, B.A. (2007). An 491
in vivo multiwell-based fluorescent screen for monitoring vertebrate thyroid hormone disruption. 492
Environ. Sci. Technol., 41, 5908–5914. 493
(3) OECD (2018), Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating 494
Chemicals for Endocrine Disruption, OECD Series on Testing and Assessment, OECD Publishing, Paris. 495
https://doi.org/10.1787/9789264304741-en 496
(4) Furlow JD, Brown DD. In vitro and in vivo analysis of the regulation of a transcription factor gene 497
by thyroid hormone during Xenopus laevis metamorphosis. Mol Endocrinol. 1999 Dec;13(12):2076-89 498
(5) OCDE. Validation of the xenopus embryonic thyroid signaling assay (XETA) for the detection of 499
thyroid active substances. Draft 500
(6) OECD (2018). guidance document on aqueous-phase aquatic toxicity testing of difficult test 501
chemicals series on testing and assessment no. 23 (second edition). OECD, Paris 502
(7) Leloup, J., Buscaglia, M. (1977). Triiodothyronine, hormone of amphibian metamorphosis. 503
Comptes Rendus Hebdomadaires des Séances de l’Académie des Sciences Serie D, 284, 2261–2263. 504
(8) Shi YB. Amphibian metamorphosis, From morphology to molecular biology, Whiley-liss 2000 505
(9) OECD (1992). Test No. 301: Ready Biodegradability. OECD Guidelines for the Testing of 506
Chemicals. OECD, Paris. 507
(10) OECD (2014). Guidance document for single laboratory validation of quantitative analytical 508
methods - guidance used in support of pre-and-post-registration data requirements for plant protection 509
and biocidal products. Series on testing and assessment no. 204. OECD, Paris. 510
(11) OECD (2014). Test No. 310: Ready Biodegradability - CO2 in sealed vessels (Headspace Test). 511
OECD Guidelines for the Testing of Chemicals. OECD, Paris. 512
(12) Hellsten, U., Harland, R.M, Gilchrist, M.J., Hendrix, D., Jurka, J., Kapitonov, V., Ovcharenko, I., 513
Putnam, N.H., Shu, S., Taher, L., Blitz, I.L., Blumberg, B., Dichmann, D.S., Dubchak, I., Amaya, E., Detter, 514
J.C., Fletcher, R., Gerhard, D.S., Goodstein, D., Graves, T., Grigoriev, I,V., Grimwood, J., Kawashima, T., 515
Lindquist, E., Lucas, S.M., Mead, P.E., Mitros, T., Ogino, H., Ohta, Y., Poliakov, A.V., Pollet, N., Robert J,, 516
Salamov, A., Sater, A.K., Schmutz, J., Terry, A., Vize, P.D., Warren, W.C., Wells, D., Wills, A., Wilson, R.K., 517
20 │
Zimmerman, L.B., Zorn, A.M., Grainger, R., Grammer, T., Khokha, M.K., Richardson, P.M., Rokhsar, D.S. 518
(2010). The genome of the Western clawed frog Xenopus tropicalis. Science, 328(5978), 633-636. 519
(13) Fini, J.B., Rui, A., Debrauwer, L., Hillenweck, A., Le Mevel, S., Chevolleau, S., Boulahtouf, A., 520
Palmier, K., Balaguer, P., Cravedi, J.P., Demeneix, A., Zalko, D. (2012). Parallel biotransformation of 521
tetrabromobisphenol A in Xenopus laevis and mammals: Xenopus as a model for endocrine 522
perturbation studies. Tox. Sci., 125(2): 359-367. 523
(14) OCDE (2009), Test No. 231: Amphibian Metamorphosis Assay, OECD Guidelines for the Testing of 524
Chemicals, Section 2, Éditions OCDE, Paris, https://doi.org/10.1787/9789264076242-en. 525
(15) OCDE (2015), Test No. 241: The Larval Amphibian Growth and Development Assay (LAGDA), 526
OECD Guidelines for the Testing of Chemicals, Section 2, Éditions OCDE, Paris, 527
https://doi.org/10.1787/9789264242340- en. 528
(16) Reed, B.T. (2005). Guidance on the housing and care of the African clawed frog Xenopus laevis. 529
Research Animals Department. RSCPA. 530
(17) ASTM E1439-12. (2012). Standard Guide for Conducting the Frog Embryo Teratogenesis Assay-531
Xenopus (FETAX), ASTM International, West Conshohocken, PA, www.astm.org 532
(18) Hutchinson TH, Shillabeer N, Winter MJ and Pickford DB. (2006). Acute and Chronic Effects of 533
Carrier Solvents in Aquatic Organisms: A Critical Review. Review. Aquatic Toxicology 76: 69–92. 534
(19) Mengeling BJ, Wei Y, Dobrawa LN, Streekstra M, Louisse J, Singh V, Singh L, Lein PJ, Wulff H, 535
Murk AJ, Furlow JD. A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of 536
thyroid hormone signaling. Aquat Toxicol. 2017 Sep;190:1-10. 537
(20) OECD (2006). Hypothesis Testing, in Current Approaches in the Statistical Analysis of Ecotoxicity 538
Data: A Guidance to Application, Chapter 5 539
http://www.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2006)18&doclangu540
age=en 541
542
│ 21
ANNEX 1: ABREVIATIONS AND DEFINITIONS 543
544
GFP : Green Fluorescent Protein 545
LC50: Median Lethal Concentration is the concentration of a test chemical that is estimated to be lethal 546
to 50% of the test organisms within the test duration 547
LOEC: The Lowest Observed Effect Concentration is the lowest tested concentration at which the test 548
chemical is observed to have a statistically significant effect (at p < 0.05). All test concentrations above 549
the LOEC should have an effect equal to or greater than those observed at the LOEC. When these two 550
conditions cannot be satisfied, a full explanation should be given for how the LOEC (and hence the 551
NOEC) has been selected. 552
MS-222: (tricaine methanesulfonate; CAS: 886-86-2 ) 553
MTC: Maximum tolerated concentration. MTC is defined as the highest test concentration of the 554
chemical which results in less than10% acute mortality 555
NOEC: The No Observed Effect Concentration is the tested concentration immediately below the 556
LOEC. 557
PTU : Propylthiouracil (CAS: 51-52-5) 558
SEM: Standard Error of the Mean 559
SMILES: Simplified Molecular Input Line Entry Specification. 560
Spiked mode: Part of the XETA run in the presence of 3.25µg/l of T3 561
T3: Triiodothyronine (CAS 6893-02-3) 562
T4: thyroxine (CAS : 51-48-9) 563
TR: thyroid receptor 564
TH: thyroid hormones 565
THbZIP : Thyroid hormone beta zip transcription factor 566
Unspiked mode: Part of the XETA run in the absence of T3 567
UVCB: Substances of unknown or variable composition, complex reaction products or biological 568
materials. 569
22 │
ANNEX 2: Conditions of the Xenopus Embryonic Thyroid Signalling Assay 570
Test Animal THbZIP-GFP Xenopus laevis embryo
Endpoint Total fluorescence of individual tadpole
Exposure period Stage NF45 (beginning of the test) to stage NF47 (end of
the test)
Exposure duration 72h ± 2h
Exposure regime Renewal after 24 h and 48 h. No feeding.
pH 6.5 to 8.5
Incubation conditions during exposure 21 ± 1oC, dark
Tadpoles per concentration 10 tadpoles per well (6-well plate) x 2 wells (total of 20
tadpoles per concentration)
Volume of test medium 8 mL per well
Test medium Water permitting normal growth and development of X.
laevis tadpoles (refer to paragraph 26).
Number of Experiments Experiments are run 3 times for each test chemical with
different spawn and freshly prepared solutions.
Criteria for selecting test individuals Developmental stage (NF45), health of animal (alive and
no malformations), homogenous pigmentation.
Validity criteria
pH 6.5-8.5, temperature 21 ± 1, mortality ≤20% in all
control groups. Fluorescence induction >20% in the T3
control and >70% in the T4 control. CV<30% in the test
medium control. At least three uncompromised treatment
levels.
Test chemical concentration standard
Test chemical concentration should remain within 20% of
nominal throughout 24 hours otherwise the result should
be considered using the determined concentrations.
Controls
Controls Test medium (basal fluorescence)
T3 Control T3 (3.25 µg/L)
T4 Control T4 (10 mg/L) (saturation of the fluorescence signal)
571
│ 23
ANNEX 3: Calibration: Finding the optimal spectrofluorimeter settings 572
573
The goal of the calibration step is to ensure that an optimal amplitude of response in simple conditions 574 could be achieved using the equipment. It is crucial to ensure that the fluorescence readers allow a suitable 575 dynamic concentration response to be obtained with T3 before testing chemicals. 576
The calibration will require two steps: 577
1) Finding the best settings for the spectrofluorimeter to obtain a satisfactory amplitude of GFP 578 induction with a concentration of 3.25 µg/L T3. 579
2) Apply these settings for the quantitation of three concentration-response experiments with T3 to 580 check the amplitude of induction using increasing concentrations of T3 and the lowest 581 concentration of T3 that elicits a detectable GFP response detectable concentration. 582
583
1-Finding the suitable settings for the spectrofluorimeter 584
585
Before starting the experiment: 586 587
-Identify the parameters that could be set on the selected spectrofluorimeter model. 588
-Choose a first combination of settings based on the ones used during the interlaboratory 589 validation of the XETA (See XETA validation report (5), annex 2) or/and on any previous 590 experiment with the chosen spectrofluorimeter. Take into account that the settings must 591 lead to a 96-well plate reading time under 30 min. 592
-Identify the parameter to optimize first. 593
594
Prepare T3 and T4 stock solutions: 595 596
o T3 STOCK SOLUTION(10-2M) (6.51 G/L) 597 598
o This stock solution can be store 1 year at -20°C. 599 o Weigh 100 mg 3, 3’, 5-Triiodo-L-thyronine in a weigh boat and gently pour into to a 600
20 mL volumetric flask. 601 o Rinse the weigh boat with 4.47 ml of NaOH 1N and gently add to the 20ml volumetric 602
flask. 603 o Mix gently for 1 hour with a stir bar; make sure the solution is limpid. 604 o Transfer 4.1 ml of the solution into a new 20 ml volumetric flask; add 9.9 ml of 605
ultrapure water. 606 o Agitate 1 hour with a stir bar. 607 o Make enough aliquots of 30µl in 0.5ml eppendorf tubes to fill a freezer storage box. 608
24 │
o Put any remaining solution in several tubes of 1.5 ml. 609 o Storage: -20°C for 1 year. 610
611 o T4 0,8 G/L STOCK SOLUTION 612
Weigh 40 mg of T4 in a weigh boat and gently pour into to a 50 mL volumetric 613 flask. 614
Rinse the weigh boat with 50 ml of ultrapure water and gently add to the 615 volumetric flask. 616
Add 0,1ml of NaOH 1N. 617 Mix gently for 1 hour with a stir bar; make sure the solution is limpid. 618 Make 1ml aliquots and store at -20°C for 6 months maximum 619
620 Prepare the following solutions 621
-Exposure medium 622
o Place 1L of test medium in a 1L glass bottle 623
-Exposure medium+T3 3.25 µg/L: 624
o Place 1L of test medium in a 1L glass bottle 625 o Add 1ml of T3 at 3.25 mg/l 626
-Exposure medium +T4 10 mg/L 627
Prepare 300 ml of T4 (10 mg/L): 628
o Place 296.25 mL of test medium in a 500 ml bottle. 629 o Add 3.75 ml of T4 at 0.8 g/L. 630 o Mix by agitation of the bottle. 631 o Store at 4°C in the dark 632
633
Expose for 72h exposure 5 groups of 20 controls, 5 groups of T3 controls and 5 groups ofT4 634 controls tadpoles (in 6-well plate, 10 tadpoles per well) with a daily renewal as described in the 635 test guideline. 636
After 72 h, proceed to the anaesthesia and placement in one 96-well plate of the tadpoles 637 Use the spectrofluorimeter with a first set of parameters to quantify the fluorescence of the 638
tadpoles. Make several consecutive reads with the modification of only one parameter at a time. 639
Do not keep the same tadpoles anesthetised for more than 45 minutes. Longer times will induce tadpole 640 death leading to fluorescence artifacts. To continue the calibration after 45 minutes, anesthetise three new 641 groups of tadpoles and load a new 96-well plate. 642
After the acquisition of the data, calculate the mean fluorescence and the CV (coefficient of 643 variation) in each group. Calculate the percentage of fluorescence induction in the T3 group and 644 T4 groups. 645
646
-Percentage of induction T3 group (RFU = relative fluorescence units) 647
100*(mean RFU T3 group - mean RFU control group)/ mean RFU control group 648
│ 25
649
-Percentage of induction T4 group = 650
100*(mean RFU T4 group - mean RFU control group)/ mean RFU control group 651
652
Each parameter should to be optimised to: 653
-Obtain fluorescence induction between the control and the T3 control group as well as between 654 T3 controls and T4 controls tadpoles close to the ones observed during the validation of the XETA: 655
- Induction of fluorescence between the control and the T3 control group ranging from 656 30 to 75% with a mean of 45%. 657
- Induction of fluorescence between the control and the T4 control group ranging from 658 70 to 190% with a mean of 105%. 659
-Ensure that spectrofluorimeter saturation is never reached in the T4 controls tadpoles. 660
-Obtain a total 96-well plate reading time under 30 min, ideally 20 min. 661
When an optimised set of parameters has been determined, repeat the experiment on another spawn to 662 confirm the results. 663
2- Determination of the amplitude of fluorescence induction and the LOEC using 664
increasing concentrations of T3 665
666
This test should be repeated three times on three separate spawns. 667
Concentrations to test are: 0 mg/L (untreated control), 26 µg/L; 13 µg/L; 6.5 µg/L; 3.25 µg/L; 2 µg/L; 1.5 668 µg/L; 1 µg/L; 0.65 µg/L. 669
Preparation of solutions 670
Preparation of T3 3.25 mg/L solution: 671
T3-Intermediate Solution (0.65 g/L) 672
Remove an aliquot of Stock T3 at 6.51g/L (10-2M) from -20°C 673 Make an intermediate solution of T3 at 0.65g/L (250µL): 674
o Put 225µL of Test medium in an Eppendorf 1.5ml 675 o Add 25 µL of T3 at 6.51g/L 676 o Vortex the solution 677
T3 solution at 3.25 mg/L 678
Make a 3.25 mg/L (20ml): 679
26 │
o Put 19.9 ml of Test medium in a 50 ml falcon tube 680 o Add 100 µl of T3 solution at 0.65 g/L 681 o Vortex the solution 682
683
Preparation of T3 test solutions: 684
Solution
Name
Final
Concentration
(µg/L)
Intermediary
volume to
prepare (mL)
Volume stock solution
Volume Test
medium
(mL)
Final
Volume
(mL)
T3_26 26 130.0 1.04 mL of T3 3.25mg/L 129.0 65.0
T3_13 13 130.0 65 mL of T3_26 65.0 55.0
T3_6.5 6.5 150.0 75 mL of T3_13 75.0 65.0
T3_3.25 3.25 170.0 85 mL of T3_6.5 85.0 65.4
T3_2 2 170.0 105 mL of T3_3.25 65.4 57.5
T3_1.5 1.5 150.0 113 mL of T3_2 37.5 63.3
T3_1 1 130.0 87 mL of T3_1.5 43.3 65.0
T3_0.65 0.65 100.0 65 mL of T3_1 35.0 100.0
685
Store the dilutions at 4°C in the dark. Use the dilutions for the exposure and the two renewals. 686
Preparation of T4 687
Solutions 688
689
o Place 60 mL of test medium in 100 mL bottle 690 o Remove and discard 0.75 mL 691 o Add 0.75 ml of T4 0.8 g/L 692 o Mix by agitation 693 o Store at 4°C in the dark 694
695
Exposure and renewals 696
Exposure: 697
Prepare the test solutions of T3 as described. 698 Place sorted tadpoles into the 6-well plates in test medium with 10 tadpoles per well using a plastic 699
transfer pipet with the tip cut off to avoid damaging the tadpoles. Note: pay attention to include 700 only healthy-looking tadpoles homogenous in size and pigmentation. 701
Begin exposure by carefully aspirating the test medium solution on the tadpoles using a plastic 702 transfer pipet. Replace test medium solution with 8 ml of the appropriate solution. 703
│ 27
Renewal: 704
Test solutions are renewed at 24 and 48 h ± 1 h 705
Take the relevant stock solutions out of the fridge and place them at 21 ± 1°C for at least 706 one hour before the renewal. 707
Remove any dead or abnormal embryos. 708 Aspirate carefully approx. 90 % of the medium with a 3 mL pipet. 709 Fill each well with 8 ml of the appropriate renewal solution. 710 Place the plates in an incubator at 21 ± 1°C in the dark. 711
712
Measuring Fluorescence 713
After 72 h ± 2 h, proceed to the anaesthesia and placement of the tadpoles in 96 well plates as described 714 in the test guideline and measure the fluorescence. Proceed to data treatment and statistical analysis to 715 obtain a LOEC as described in the test guideline. 716
Interpreting the results: 717
As for the experiment described above the results should show: 718
- A fluorescence induction between the control and the T3 control group as well as between T3 controls 719 and T4 controls tadpoles close to the ones observed during the validation (see above). 720
-That the spectrofluorimeter saturation is never reached in the T4 controls tadpoles. 721
The LOEC found for T3 should be 3.25 µg/L or lower. 722
If the results are not in accordance with these three points, the spectrofluorimeter settings must be 723 refined. 724
725
726
28 │
ANNEX 4: Receiving embryos: acclimation and batch acceptance 727
Embryos should be received no later than 3 days before the test begins to allow a 728
proper recovery and acclimation of the tadpoles. 729
Batches should be accepted only if dead or abnormal embryos represent less than 730
20% 731
732
Guidance for embryos received three days before the start of the XETA: 733
Do not mix embryos from different spawns. 734 Sort embryos to remove dead and abnormal embryos, these embryos should represent less 735
than 20% otherwise the batch should not be use to perform the XETA. 736 Transfer only the living and normal healthy embryos of the same spawn to a 10-liter aquaria 737
containing test medium. 738 The maximum density per 10-liter aquarium tank is 800 embryos. 739 Determine the stage of development 740 The temperature of the tank from this point on will influence the stage of development of the 741
embryos on the day of the experiment. The embryos are expected to be received at stage 33-742 42 upon reception, use these guidelines to control the rate of growth : 743
o If at stages 30-32 upon reception : incubate at 23oC +/- 1oC ; 744 o If at stages 33-42 upon reception : incubate at 21oC +/- 1oC ; 745 o If at stages >/=43 upon reception : incubate at 18oC +/- 1oC 746
Incubate tadpoles without illumination (in the dark). 747 After the step above, do not renew medium in the tank until the start of the experiment when 748
the tadpoles are sorted. 749
750
│ 29
ANNEX 5: Breeding tadpoles 751
This section describes how laboratories could breed their own tadpoles. 752
The materials specifically needed for this procedure include: 753
Binocular microscope 754 Bleach solution (100%) 755 Gentamycin solution 756 2% Cysteine solution, pH 7.5-8 757 (des-gly, D-ala, pro-NHEt) LHRH acetate salt, Store at -20 °C 758 Human chorionic gonadotrophin (HCG), Store at 4°C 759 MMR (Marc’s Modified Ringer’s) 0.1X solution + Gentamycin 50 µg/L 760 Syringes, 5 mL and 1 mL 761 Syringe needle (Pink needle); G 18 1 ½; 762 Syringe needle (Orange needle); G 25 5/8 763 250 mL beaker to contain the eggs (blastula stage) 764
To prepare the tadpoles to be ready for use the following Monday or Tuesday, breeding must begin on the 765 Monday the week prior to the start of the experiment. 766
Sample Breeding Schedule (beginning on the Monday prior to the start of the experiment) 767
Day Activity Details
Monday prior
to start of
experiment
Mate male THbZIP-GFP and female wild
type adult Xenopus laevis.
Inject hormones to both sexes
(LHRH for male and HCG for
female) to induce breeding. Place
the Xenopus together in the same
tank.
Tuesday Cysteine and sort healthy embryos (at
blastula stage) and incubate in the dark at
21± 1 °C.
Keep spawns separate if more than
one spawn obtained
Wednesday Sort healthy embryos and refresh medium
with MMR + Gentamycin, Incubate in the
dark at 21± 1 °C.
Thursday Sort and refresh medium as above,
Incubate for the weekend in the dark at
21± 1 °C.
Following
Monday
Start experiment Embryos must be at stage NF45.
768
30 │
Preparation of solutions required for the breeding: 769
MMR (20X) Stock solution 770
For 2L: 771
234 g NaCl 772 6 g KCl 773 3.8 g MgCl2 774
11.76 g CaCl2 775
48 g HEPES 776 Place the powders in a 2L glass bottle 777
Add distilled water QSP 2L 778
Adjust pH with NaOH 1N to be 7.5-8 779
Storage at 4°C 2months max 780
MMR (0.1X) from 20X MMR 781
For 10 L: 782
Pour 50 ml of MMR 20X into a 10 L container 783 Fill the container to 10L with distilled water 784 Adjust the pH between to 7.5-8 using 1N NaOH 785 Storage at room temperature during use(2 weeks max) 786
MMR (0.1X) + gentamycin 50 µg/L 787
Place 2 mL of gentamycin 50 mg/L in a 2 L volumetric flask 788 Fill to 2L with MMR 0.1X 789 Cover the flask with parafilm 790 Mix by inverting several times 791
Storage at room temperature during use (2 weeks max) 792 793
Cysteine 2%, pH 7.5-8 794
For 100 mL 795 Weigh 2g of cysteine 796 Add 90 mL of MMR 0.1X 797 Adjust the pH to 7.5-7.7 using 10N NaOH, then 1N 798 Complete to 100 mL with MMR 0.1X 799 Use only on the day of the preparation 800
│ 31
Preparation of the GnRH (LHRH acetate salt) solution: 801
Remove the tube of LHRH acetate salt powder (GnRH) provided by the supplier from the -20°C 802 freezer 803
Resuspend the powder directly (5mg) in the tube above using 720 µL of sterile water (stock 804 solution: 6.94 µg/µL); Dilute 6 µL of the stock solution in 1 mL of sterile water (final solution 805 41.64 µg/ml) or for a larger volume, dilute 90 µL of stock solution in 15 mL of sterile water. 806
Make 1 mL aliquots in sterile 1.5 mL Eppendorf tubes. 807 Store aliquots at -20 oC. 808
Preparation of the HCG solution: 809
Obtain a vial of 5000 units of HGC in powder form from the supplier. 810 Using a sterile syringe, inject 5 mL of sodium chloride 0.9% into the tube of HCG in order to 811
obtain a concentration of 1 unit per µL. 812 Mix by manual agitation. 813 Any solution that remains unused can be stored at 4oC for two weeks maximum. 814
815
BREEDING PROCEDURE 816
Monday: Perform hormonal injections to induce the mating of the male and female adult X. laevis. 817
The Xenopus male could be injected either with GnRH or with HCG: 818
Injection of males with GnRH (LHRH acetate): 819
For reference, read: p126 &127 of The Laboratory XENOPUS sp. From Sherill L. Green. A 820 subcutaneous injection in dorsal lymph sac can be viewed at 821 http://www.jove.com/index/Details.stp?ID=890 (Cross and Powers 2008) 822
Remove an aliquot of GnRH (41.64 µg/mL) from the -20oC freezer and let it thaw at room 823 temperature. 824
To cause minimal distress, immobilize the frog by covering its eyes. 825 Using a 25 gauge (orange) needle and a 1ml syringe containing 0.120 mL of the thawed GnRH 826
solution, perform a dorsal subcutaneous injection. 827
Injection of males with HCG: 828
Using a 1 mL syringe and a 25 gauge (orange) needle, aspirate 10-25 units of HCG (depending on 829 the size of the frog). 830
Immobilise the frog on a wet paper towel on a bench and mask its eyes as described in the picture 831 below. 832
Perform a dorsal subcutaneous injection at the level of the lymphatic bags. Follow the same 833 instructions as above for the injection. 834
835
32 │
836
From The Laboratory XENOPUS sp. From Sherill L. Green 837
Injection of females with HCG: 838
Using a 1 mL syringe and a 25 gauge (orange) needle, aspirate 500-700 units of HCG (depending 839 on the size of the frog). Note: inject 500 units for a frog under 9 cm, and 700 units for a frog 9 cm 840 or above. 841
Immobilize the frog and mask its eyes. See scheme above. 842 Perform a dorsal subcutaneous injection at the level of the lymphatic bags. Follow the same 843
instructions as above for the injection. 844
Tuesday: (sort and incubate) 845
Harvest and dejelly the eggs: 846 o Harvest the eggs in 250 mL beaker, remove the excess of water 847 o Add enough cysteine 2% to cover the eggs, mix the eggs with cysteine by rotating the 848
beaker for 2 or 3 min until eggs are dissociated. It is important to be careful not to leave 849 the cysteine too long, otherwise it will affect the survival. This step is critical and has to 850 be performed with care. Cysteine solution should be acclimated to ~21°C. Temperature 851 impacts the effectiveness of the cysteine treatment and can also affect the health of the 852 embryos if they experience temperature shock. 853
o Stop the action of cysteine by adding MMR 0.1X in the beaker (almost to the top). 854 o Remove the liquid by pouring it slowly into a beaker of bleach solution and rinse again 3 855
times with MMR 0.1X. 856 o Place the embryos into large 100 mL annotated Petri plates by gently pouring from the 857
beaker. 858 859
Sort the embryos (dead versus alive), most of the embryos should be at the blastula stage: 860 861
o Place the dish under a binocular microscope and using a transfer pipet which tip is cut off 862 to avoid damaging the eggs, isolate each fertilized egg in a new 100 ml Petri dish 863 containing MMR 0.1X + gentamycin 50 µg/L. For description of eggs that can be found 864 in the dish, see Annex 7_sorting fertilized eggs. 865
Annotate the plates: line number, mating date, number of eggs per dish. Incubate plates in the dark at 21 ± 866 1°C. Wednesday: (sort and change medium) 867
Remove plates from incubator. 868 Sort the embryos, removing dead ones as on previous day using a transfer pipet with a cut off tip. 869
│ 33
Renew half of the medium, and resupply with MMR 0.1X + gentamycin 50 µg/L. 870 Incubate the plates at 21oC in the dark at 21 ± 1°C. 871
Thursday: (prepare for experiment or ship tadpoles to another laboratory) 872
Option 1: continue the culture until the start of the experiment 873
Fill ¼ of an aquarium with MMR 0.1X. 874 Annotate the aquarium with line number, date of reproduction. 875 Gently pour the embryos into the aquarium without mixing spawns, place 200 embryos/L 876
maximum. 877 Place the aquarium in a 21oC incubator in the dark for the weekend. 878
879
Option 2: Ship the embryos to another laboratory 880
Materials needed for shipping: 881
o Sterile 100 mL container (e.g., tissue culture flask with screw cap lid) 882 o Transfer pipets with the tips cut off 883 o Pipets (50 mL) 884 o Pipetaid 885
Please note that the embryos should be packed just before the shipment to reduce the stress. 886
Annotate the 100 mL container (both the cap and the container itself) with: 887 « Name of line » 888 « Date of reproduction » 889 « Number of embryos/container » 890
Add 50 mL of test medium to the container. 891 Place 100 live embryos in the container. 892 Close the container only when it is ready to be sent. 893 Place the container in a sealable freezer bag. 894 Close the bag. 895 Place the container in a polystyrene box and firmly secure it inside the box so that it does not move. 896 Close and tape the box carefully. 897 Mail to receiving laboratory by overnight mail. 898
899
34 │
ANNEX 6: Photographic guidance for identification of normal versus abnormal 900
tadpoles 901
902
Normal tadpole (A). Abnormal tadpoles: pigmentation and microcephaly (B), oedema and several 903
malformations (C and D). 904
905
│ 35
ANNEX 7: SORTING TADPOLES: ABNORMAL PIGMENTATION 906
907
Tadpoles presenting abnormal pigmentation are often found in Xenopus spawns and should be discarded 908
from the test organisms as the pigmentation could interfere with the fluorescence quantification. Such 909
embryos should be euthanised using the procedure described in paragraph 46. 910
911
912
Picture A is an example of a heterogeneous clutch of tadpole in terms of pigmentation. A tadpole presenting 913
an abnormal pigmentation (arrow). Picture B shows an example of a clutch homogenous for pigmentation. 914
915
A B
36 │
ANNEX 8: Staging stage NF45 and NF47 tadpoles 916
917
918
Stage NF45 tadpole: Dorsal view (A), ventral view showing the intestine spirals (B), lateral view (C) and 919
drawing of a ventral view (D). http://wiki.xenbase.org/xenwiki/index.php/Stage_45. Bar =2.5 mm 920
921
922
Stage NF47 tadpole: lateral view showing the xanthophores appearing on abdomen (A), ), ventral view 923
showing the intestine spirals (B) and drawings of lateral and ventral views (C and D). 924
http://wiki.xenbase.org/xenwiki/index.php/Stage_47 925
│ 37
ANNEX 9: Sample six-well plate set-up 926
Sample layout of 6-well plate exposure treatment for one run: 927
• 10 Embryos Per Well 928
• 3 Plates Total Required to Test One Test Chemical 929
Control Plate (1 plate): Three controls 930
931
932
Test Chemical (TC) 1 plate – one chemical, 2 x 3 concentrations) 933
934
935
TC
Conc A
TC
Conc A
TC
Conc B
TC
Conc B
TC
Conc C
TC
Conc C
Test
Medium
T4
Control
T3
Control
T3
Control
T4
Control
Test
Medium
38 │
936
Test Chemical + T3 (1 plate – one chemical + T3, 2 x 5 concentrations) 937
938
939
TC + T3
Conc A
TC + T3
Conc A
TC + T3
Conc B
TC + T3
Conc B
TC + T3
Conc C
TC + T3
Conc C
│ 39
ANNEX 10: Scheme of 96-well plates for fluorescence reading 940
941
942
943
944
Test chemical : Conc C1 (+ T3)
Test chemical : Conc C2 (+ T3)
40 │
ANNEX 11: Tadpole positioning 945
946
The figure below is showing the expected positioning of the tadpole in the well of the 96-well plate. 947
948
949
950
Image of a THbZIP-GFP tadpole using GFP fluorescence imaging in a well of a 96-well plate. 951
952
│ 41
ANNEX 12: METHODS FOR THE STATISTICAL ANALYSIS OF XETA DATA 953
954
This Annex describes a possible way for statistical analyses of the data obtained in the XETA. 955
956
Perform a 10% trim of each treatment group, omitting the highest 10% and lowest 10% of the 957
fluorescence values from each group. A trim less than 10% may be used based on expert judgement 958
959
Do an ANOVA with variance component for replicate, replicate-by-treatment, and well nested within 960
replicate by treatment or concentration as the only fixed factor and replicate as the only specified random 961
factor. Calculate the residuals from this ANOVA and output them to a new dataset. 962
963
Construct a histogram or QQ-plot or stem-and-leaf plot of the residuals and examine them for visual 964
consistency with a normal distribution and outlier identification. Ideally, a histogram of the residuals 965
should resemble a bell-shaped curve, symmetric around zero and tapering off in both directions. A QQ 966
plot should roughly follow a straight line. Moderate deviation from these ideals is acceptable. 967
Alternatively, compute the Shapiro-Wilk or Anderson-Darling test for normality at the 0.01 significance 968
level. Be aware that a large dataset may give rise to a significant test, i.e. indicate non-normality, even 969
though there is no need for concern. 970
971
Plot residuals vs. treatment to identify possible variance heterogeneity or give further indication of 972
outliers. Alternatively, compute Levene’s test (or some alternative) at the 0.01 significance level. 973
974
If the plots or formal tests indicate non-normality or variance heterogeneity that cannot be eliminated by 975
omitting a few outliers, then repeat the above steps with a log- or square-root transform of the data. 976
977
Once a normalizing, variance stabilizing transformation has been found, do Williams’ test for increasing 978
trend and separately for a decreasing trend based on the ANOVA indicated in the third paragraph of this 979
Annex 980
Examine the pooled treatment means, if any, from Williams’ test. If 3 or more adjacent treatment groups 981
are combined by the PAVA process underlying Williams’ test, examine the (un-amalgamated) treatment 982
42 │
means for consistency with a monotone concentration-response. If the data are not consistent with 983
monotonicity, then Williams’ test is not appropriate and Dunnett’s test should be used. 984
If Dunnett’s test is used, then the indicated ANOVA should include Dunnett’s test to compare treatments 985
to control. A 2-sided test should be used unless there is scientific justification to expect only a change in 986
one direction. Note: Dunnett’s test is done at the 0.05 significance level regardless of the ANOVA F-test. 987
988
Use the Tukey outlier rule to identify outliers from the ANOVA. If outliers are found and there is 989
evidence of non-normality or variance heterogeneity, repeat the ANOVA and Dunnett test with outliers 990
omitted. If the normality and variance heterogeneity issues disappear and Dunnett’s test identifies the 991
same NOEC as with the full data, then accept the NOEC. 992
Figure 1 captures the broad outline of this flow chart. 993
Figure 1. Flow Chart for Fluorescence Statistical Analysis 994
995
996