DRAFT OECD GUIDELINE FOR THE TESTING OF CHEMICALS · 74 Guideline is used for the testing of a mixture, a UVCB or a multi constituent substance, its composition ... 123 e.g. results

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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