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1 DRAFT NEW TEST GUIDELINE

2 Determination of relative metal/ release using a simple 3 simulated gastric fluid (0.032 M HCl)

4 INTRODUCTION

5 1. There are several laboratory methods to determine metal/metalloid releases in synthetic 6 fluids from various materials including art materials and toys, but also soils [e.g. (1)(2)(3)]. The 7 US EPA Validated Test Method 1340 (one of SW-846 guidance methods) corresponds to the 8 “in vitro bioaccessibility assay for in soil" (4), the validation of an “In Vitro 9 Bioaccessibility Test Method for Estimation of Bioavailability of arsenic from soil and 10 sediment” has also been conducted (5)(6). Some of the in vitro methods have been applied to 11 refine the assessments of human exposures to metals/ in soils and dusts [e.g. 12 (7)(8)(9)(10)]. Yet, until adoption of this Test Guideline, there was no internationally 13 harmonised protocol (e.g., OECD Test Guideline) on how to conduct a cell-free in vitro method 14 using a simulated gastric fluid to generate relative metal/metalloid release data for a broad 15 number of metals and metalloids. However, this Test Guideline does not discuss the regulatory 16 use of the data obtained with the test method. Relevant Competent Authorities may be consulted 17 for that purpose. 18 19 2. This Test Guideline describes how to measure material-specific metal/metalloid release 20 data and calculate relative metal/metalloid releases from materials such as metals and 21 metalloids, inorganic metal compounds and other inorganic complex metal(metalloid)- 22 containing materials [e.g., alloys, pigments, and materials of Unknown or Variable 23 composition, Complex reaction products and Biological materials (UVCBs)] in a simple 24 simulated gastric fluid composed of 0.032 M HCl, pH 1.5 ± 0.1. The main interest of the method 25 resides in comparing metal releases between two or more materials of the same metal/metalloid, 26 i.e., a test and a reference material. The way to select reference materials, and to measure and 27 compare metal releases of a test material relative to the one(s) from (a) reference material(s) is 28 described. This Test Guideline builds upon the experience gained from a round robin study 29 examining the performance of the method when applied to a variety of metal(metalloid)- 30 containing materials (11). 31 32 3. Definitions are provided in Annex 1.

33

34 INITIAL CONSIDERATIONS AND LIMITATIONS

35 4. The relative metal/metalloid release method was evaluated by EURL ECVAM and peer 36 reviewed by its Scientific Advisory Committee (ESAC) (12). The review concluded that the 37 fluid and experimental conditions used are representative of the gastric compartment. This 38 method is not intended to address the full gastrointestinal tract. Rather, it is a simple, 39 reproducible approach intended to maximise the release of metal/metalloid ions for the majority

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40 of the tested materials and to allow for the relative assessment of metal release between two or 41 more materials. ESAC considered the method appropriate to assess if a matrix effect occurs in 42 alloys (i.e. whether there is an increased or a decreased relative release of the metal ions from 43 the alloy compared to what is expected from the pure ingredient). The method is applicable to 44 many metals but it should not be used as stand-alone to predict absolute bioavailability after 45 oral intake. All relevant documents regarding the validation and peer review of the method are 46 available in the EURL ECVAM Tracking System on Alternative Methods (TSAR) 47 (https://tsar.jrc.ec.europa.eu/test-method/tm2016-02). 48 49 5. The relative metal/metalloid release method applies to test materials such as certain 50 metals and metalloids, inorganic metal compounds, or complex metal(metalloid)-containing 51 materials (e.g. alloys, UVBCs, pigments), in massive form (≥ 1mm diameter) or powder form 52 (≥ 0.1µm - < 1mm diameter). It can be applied to substances and mixtures. Importantly, this 53 Test Guideline does not apply to nanomaterial1. For nanomaterials, the relative metal/metalloid 54 release method in its current form may not allow for a complete separation of released metal 55 ions from the undissolved to be achieved. 56 57 6. For some metals such as mercury (Hg) and (Ag), or certain chemical forms of 58 metals such as antimony (Sb) trichloride (SbCl3) and Sb pentachloride (SbCl5), the results in 59 simulated gastric fluids of low pH (e.g., 0.032 M HCl, pH 1.5 ± 0.1) are unreliable due to 60 precipitation. In the case of Sb, this was observed with the two chemical forms of Sb that are 61 corrosive (SbCl3 and SbCl5), but not with other chemical forms (e.g. Sb metal, Sb ). In 62 general, any chemical forms of a metal/metalloid showing precipitation in 0.032 M HCl, are 63 outside the technical applicability domain of this Test Guideline. 64 65 7. The following metals (metalloids) and their compounds have shown that releases in 66 simulated gastric fluid can be measured and are within the technical applicability domain of 67 this Test Guideline: arsenic (As), gold (Au), boron (B), (Cd), (Co), 68 (Cr), (Cu), (Fe), germanium (Ge), indium (In), (Mn), molybdenum 69 (Mo), (Ni), lead (Pb), palladium (Pd), platinum (Pt), rhenium (Re), rhodium (Rh), 70 ruthenium (Ru), selenium (Se), (Si), antimony (Sb) non-corrosive compounds, 71 (Ti), (V), tungsten (W), (Zn) and zirconium (Zr). Data are not yet available for 72 beryllium (Be).

73 8. Relative metal/metalloid releases between test material and reference material can be 74 used to compare different materials of the same metal/metalloid and to rank them. It can also 75 be used to assess the presence of matrix effects in test materials (e.g., alloys), as matrix effects 76 can affect the expected metal/metalloid release in 0.032 M HCl compared to that expected from 77 the pure metal ingredients. It is required that the exposed surface of the materials or the specific 78 surface area (SSA) be reported since it strongly influences metal release. 79 80 9. For most of the metals and their chemical forms in the technical applicability domain 81 (see paragraph 7), fluids with pH ~1.5 may lead to higher metal ion releases compared to fluids 82 of neutral pH. However, for some of the metals/metalloids within the technical applicability

1 Nanomaterials: natural, incidental or manufactured materials containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm, as defined by the EU Commission in 2011 (13).

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83 domain, releases in 0.032 M HCl (simulated gastric fluid) may be lower than releases in other 84 simulated oral fluids with higher pH, such as intestinal fluid. It is important to be aware of the 85 known cases where metal/metalloid release in this Test Guideline may not be the highest. In 86 the case of Mo, Fe, Si, W and Sb, the release from some of their compounds and alloys may 87 not be the highest in 0.032 M HCl (e.g., Mo from MoO3 and some Mo alloys; Fe from FeMo 88 alloy, Si from FeSi alloy and Al silicate). For these elements, metal/metalloid release tests in a 89 second fluid of neutral pH (or water solubility) can provide information on whether simulated 90 gastric fluid could be an acceptable fluid to generate relative releases for the intended purpose 91 (14, 15, 16, 17, 18). Due to the relative nature of the method, even if acidic conditions provide 92 lower absolute releases, as long as the relative releases from 2 or more compounds in a given 93 fluid are compared (and metal release can be accurately measured), the final outcome may not 94 be significantly impacted from what can be predicted based on more neutral pH fluids. Thus, 95 this set of metals/metalloids can still fall within the applicability domain of the simple simulated 96 gastric fluid (0.032 M HCl) even if the acidic conditions do not represent a worst-case-scenario 97 for metal releases.

98 10. This method determines the release of the metal/metalloid as the concentration of the 99 element. However, the applied analytical method does not determine the form in which the 100 metal is released. For many elements, a single oxidation state will be present at pH 1.5 0.032M 101 HCl. For some elements, for which different oxidation or complexation states may be suspected 102 depending on the exact material, additional information is required to justify whether the metal 103 form released from the test material is the same as for the reference material. Examples of 104 analytical methods to assess speciation are described in paragraph 45.

105

106 PRINCIPLE OF THE TEST

107 11. The method involves the measurement of metal release from powder or massive forms 108 of test and reference materials in a solution of 0.032 M HCl, pH 1.5 ± 0.1 at 37 ± 1 °C, after 109 two hours incubation. The test medium and exposure conditions are chosen to mimic 110 metal/metalloid release in gastric fluid after oral ingestion in humans.

111 Main steps: Samples of the test or the reference materials are added in triplicate (i.e., each 112 sample is added to 3 independent vessels) to a solution of 0.032 M HCl at two different loadings 113 (ratio of volume of test medium to mass of sample, see paragraph 38) and incubated for two 114 hours before separating the metal ion released from the original sample by filtration. For each 115 loading (0.2 and 2 g/L) the test is performed in triplicate. Tests with negative/positive controls 116 are also conducted in parallel (see Annex 3).

117 The analytical measurements are reported as mass of metal ion released per volume of test 118 medium (at 2 different loadings) and are converted to other metrics such as mass of metal 119 released per mass of sample (see paragraphs 53-58). The values obtained for test and reference 120 materials are then compared.

121

122 INFORMATION ON THE TEST MATERIAL

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123 12. Test materials should be metals and metalloids in elemental form, inorganic 124 metal/metalloid compounds, or complex metal/metalloid-containing materials like e.g., alloys, 125 UVCBs and pigments. Test materials can be in powder or massive form and should be tested 126 in the form that is placed on the market or in which they can reasonably be expected to be used2. 127 Depending on the type of test material and physical form, specific sample preparation and test 128 execution applies.

129 13. When test materials on the market are present in micron-size powder form (defined as 130 particles < 1 mm diameter, but above 100 nm diameter), testing the subfraction of particles ≤ 131 100 µm in diameter is recommended where possible as it is also relevant to the fraction of the 132 inhalation exposure that becomes bioavailable through the oral route. While particles in the 133 range of 150-250 µm are often used for measuring bioaccessibility after oral intake, testing 134 finer particles (≤ 100 µm) provides a worst case scenario for absorption via the oral route and 135 also covers the inhaled particles that are deposited in the mid to upper regions of the respiratory 136 tract which are swallowed and then passed into the gastrointestinal tract.

137 14. If the test material on the market is present and only used as a massive form, this form 138 should be tested. The massive form of the test material (e.g., metal or alloy) can be tested as it 139 is or tested as an epoxy embedded sample. Annex 2 provides a description of the epoxy 140 embedding process and the advantages and disadvantages that it confers to the massive sample.

141 For massive samples, care must be taken since grinding and polishing operations during sample 142 preparation may cause the exposed surface to no longer be representative of the as received 143 material (this may be overcome by allowing the polished surface to be oxidized for a specified 144 period of time). In addition, care must be taken to not introduce crevices that promote enhanced 145 /metal release. The surfaces of the reference materials and the test materials are to be 146 treated in the same way to maximise comparability of results. Embedding samples in epoxy 147 resin may facilitate sample handling and provide a standardised exposed surface.

148 15. If particles are expected to be generated from the massive forms during normal 149 handling and use, then representative particles should be tested. In order to obtain the 150 representative particle size, potential methodologies for sample preparation include crushing, 151 grinding, milling, and/or sieving. Of these methods, sieving is always preferred, as this method 152 does not impart any physical or chemical changes to the sample (e.g., possible surface 153 oxidation/passivation). Crushing, grinding, and milling operations can have a significant effect 154 on metal ion release from some alloys and should only be considered when sieving is not 155 feasible or when these operations correspond to the foreseeable uses of the material. Under 156 these circumstances, it will be important to test the metal ion release from the material both as 157 initially received and after being crushed/ground/milled in order to establish whether the 158 crushing/grinding/milling operation has had an impact on the release properties of the test 159 material. Characterisation of the surface chemistry after crushing/grinding/milling operations, 160 can be important to interpret the metal release results before and after

2 For example, Article 5 of the EU CLP states: The information shall relate to the forms or physical states in which the substance is placed on the market and in which it can reasonably be expected to be used. The ECHA CLP Guidance (2017) (19) indicates: “Samples offered for testing must in all aspects be representative of the substance or mixture to be classified.” And it goes on to state that “In some cases, additional parameters like (e.g.) physical condition, particle size and shape, specific surface area, density, crystal structure, may influence the test result. Therefore, these properties should be mentioned in the test report. The tests must be performed on the substance or mixture in the appropriate physical form where changes in that form may influence the outcome of the test (see also Articles 5 and 6 of EU CLP).

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161 crushing/grinding/milling, as this is the interface that governs the release process. Any such 162 change of surface properties (including its reactivity) must be recorded and reported. Consider 163 the common or reasonable uses and collect sample of particles reflecting that use. This 164 information should normally be provided by the applicant/study sponsor.

165 16. For reasons of traceability, safe handling and storage of the materials, a test material 166 has to be accompanied by documentation (e.g., certificate of analysis), such as Material Safety 167 Data Sheets (MSDS) or equivalent. The information needed on the test material is described in 168 section Test report.

169 17. It is important to perform a visual inspection of the test material to assess its 170 homogeneity and to follow any storage instructions provided by the manufacturer or supplier 171 (e.g., powders should be stored under a nitrogen or an inert atmosphere, stored in a sealed 172 container, etc.). In the absence of storage instructions, the test material should be stored at room 173 temperature in an appropriate container, in a dry place and at dark conditions to protect it from 174 moisture and further surface oxidation.

175 18. Regarding test material handling, powders should be weighed in dry, acid-cleaned, 176 material-resistant or polymeric test vessels by using metal/metalloid free spatulas or 177 cleaned spoons. For massive forms of the test material that are not epoxy embedded (e.g., in 178 the form of discs, sheets, etc.), the weight, geometry, surface finish and the geometric surface 179 area of the sample should be recorded.

180

181 DEMONSTRATION OF PROFICIENCY Commented [EC1]: Comment to WNT

A physical repository containing proficiency materials will be set up 182 19. Laboratories should demonstrate technical proficiency prior to routine use of the to make these materials available to all interested laboratories at no 183 method. For a laboratory to demonstrate proficiency in testing both powders and massive forms, cost (please see Annex 4). A public database containing the repeated measurements of these materials (Shewhart charts) will also be made 184 proficiency materials in both these physical forms should be tested, although they do not need available. 185 to have the same composition. The four proficiency materials listed in Table 1 should be tested In addition to proficiency materials, the physical repository will 186 as received when the relative metal/metalloid release method is first established in the contain reference materials for alloys when using the method to calculate %RBC. Results obtained for these reference materials will 187 laboratory and the testing should be repeated periodically (at least once a year with at least one also be part of the public database (see below under reference 188 powder and one massive from Table 1). Laboratories should consult the values included in the materials). 189 database (link to be provided) and define the acceptance release ranges for each of the four 190 proficiency materials on the basis of a 95% Confidence Interval.

191 Table 1. Proficiency materials 192 Chemical name CAS or composition Physical state Accepted release ranges Commented [EC2]: Comment to WNT

Acceptance criteria for the proficiency materials will be provided on SS316 sheets: 68.1% Fe (balance), massive To be determined a public website once the materials have been tested as proposed in 16.2% Cr, 11.2% Ni, once the material Annex 4. This last column in Table 1 is kept in this draft TG for the purposes of the discussions at WNT/Expert Group but will be deleted 2.27% Mo, 1.10% Mn, has been tested as in the final version of the TG. 0.65% Si, 0.25% Cu, proposed in ≤0.18% Co, 0.12% Al. Annex 4 Brass Cu63/Zn37 discs: 63% copper, 37% zinc massive To be determined once the material has been tested as

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proposed in Annex 4 Cobalt metal 7440-48-4 powder To be determined once the material has been tested as proposed in Annex 4 Lead metal (CAS) 7439-92-1 powder To be determined once the material has been tested as proposed in Annex 4 193

194 20. The proficiency materials were selected to fulfil the following criteria:

195 o be easily (and foreseeably) available in the same physical form and quality as the test 196 material and at a low cost 197 o have well characterised metal/metalloid release data 198 o be stable under ambient conditions 199 o not pose a health risk during handling when the usual protective clothing, dust mask, 200 and gloves are worn.

201 21. In powder form, the proficiency materials have been characterised in terms of particle 202 size distribution or particle size range, purity and specific surface area (m²/g) measured using 203 Brunauer-Emmett-Teller (BET) analysis, transmission electron microscopy (TEM) or an 204 equivalent technique.

205

206 DESCRIPTION OF THE METHOD

207 Test preparation

208 22. The test system is the whole of the chosen test vessel with the appropriate volume of 209 0.032M HCl test medium (a minimum of 50 mL).

210 Test vessels

211 23. Inert, chemical-resistant Erlenmeyer flasks (e.g., Polyethylene Terephthalate Glycol 212 (PETG) or borosilicate glass) of 250 mL or larger vessels in case of testing massive materials 213 should be used for testing (up to two litre flasks). The vessels should be properly covered (e.g., 214 metal/metalloid free screw caps, silicon or rubber stoppers) to avoid (cross) contamination or 215 evaporation of the test medium. 216 217 24. Care should be taken to ensure that the test vessels do not interact with the test material, 218 either by causing abrasion or releasing metal ions to the test medium. Abrasion has been 219 observed with some materials like slags in borosilicate glass. Borosilicate can also contribute 220 Si and B to the test medium and should be avoided when Si or B are elements of interest (e.g., 221 silicates). In case where release of gas can be expected (e.g., carbonate salts brought in an acidic 222 environment), care needs to be taken that no pressure builds up in the vessel (avoid too tight-

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223 fitting caps). All test vessels, whether new or used, should be cleaned in an appropriate way to 224 reduce possible background levels of the elements of interest before use and to be suitable for 225 working with metals at trace levels.

226 Cleaning procedure for new and re-used test vessels: acid soak for 24h in 10% HNO3, then rinse 227 at least 4x in ultrapure water (e.g., MilliQ water 18.2 MΩ·cm) and dry (by air at room 228 temperature or in low temperature, < 50 °C oven).

229 25. For the handling of powders, prior to each use, metal/metalloid free spatulas or spoons 230 should, as a minimum, be carefully cleaned in ultrapure water and isopropyl alcohol or, 231 preferably, acid-cleaned in the manner described above. Do not use the same spatula or spoon 232 on different test samples unless it has undergone cleaning after the previous transfer process.

233 Test Medium

234 26. The test medium is 0.032 M HCl solution at 37 ± 1 °C and pH 1.5 ± 0.1. The chemical 235 composition is shown in Table 2; it simulates a simple gastric fluid and is based on the standards 236 ASTM D5517 (1), ASTM F963 (20), and BS EN 71-3 (2). The solution of the simple simulated 237 gastric fluid has to be prepared freshly every day. Once the temperature of the solution is stable 238 at 37 ± 1 °C, confirm that the pH of the final solution is within 1.5 ± 0.1 units.

239 Table 2. Chemical composition of simple simulated gastric fluid (0.032 M HCl).

Chemical Amount

HCl solution Start with 316 mL of a titrated 0.1 M HCl solution at 37 ± 1 °C. This titrated (analytical grade or 0.1 M HCl acid solution can be purchased or prepared. better)

Ultrapure water Add 684 mL of Ultrapure water at 37 ± 1 °C. (18.2 MΩ cm)

The temperature of the simulated gastric fluid should be kept at 37 ± 1 °C (e.g., water bath)

240 241 Negative and Positive Controls

242 27. Negative control: A test vessel with test medium only, with or without a clear resin in 243 the case of embedded massive samples. A negative control run performed in parallel helps to 244 detect background values of the elements of interest in the test fluid and helps rule out 245 contamination. The detected amounts of the elements of interest in the concurrent negative 246 control should be < 20 x SD of negative controls for the laboratory (based on 10 or more 247 replicate measurements). Higher values would indicate possible contaminations of the test 248 system, glassware, etc.

249 28. Positive control: A test vessel with test medium that contains a certain amount of a 250 well-soluble form of the element(s) of interest that is (are) measured. The positive control is 251 prepared by spiking a known amount of a Certified Standard Metal solution (containing the 252 element(s) of interest) into the test medium. A concentration in the range that can be expected

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253 to be released from the test material at one of the 2 loadings should be added to the test medium 254 at time = 0 and run through the protocol. When there is no information about the possible 255 behaviour of the test material, a concentration that to values that can be accurately 256 measured should be chosen. Recovery of 100 ± 10% is acceptable, based on the mean value of 257 10 or more replicate measurements of the positive control obtained in the laboratory. The 258 purpose of the positive control is to assure that the relative metal/metalloid release method 259 produces repeatable results over time. It provides evidence that the test system is responsive 260 under the actual conditions of the assay. The positive control is run in parallel with the test 261 materials of that element. The positive control can also be used to assess the recovery of the 262 metal ions of interest through the test (mixing with test medium, incubation, filtering, sampling, 263 etc.) and show eventual interaction with test medium.

264 Reference Materials

265 29. Reference materials: Reference materials should always be run in parallel with the 266 test materials to ensure comparability and reliability. The reference material(s) may span a 267 range of water solubilities. Examples of reference materials can be the water-soluble chemical 268 form of the metal/metalloid, the metal in its elemental state (zero valence), a representative 269 metal oxide, etc. The exact materials (i.e., size, form and surfaces treatments) used as reference 270 material(s) will depend on the exact question that is being addressed (see below).

271 30. Selection of reference materials: The selection of the reference material(s) will 272 depend on each metal (metalloid) of interest, the physical state of the test material and the 273 purpose of the test. It is not possible to predetermine what reference (‘source’) material(s) 274 should be selected when the method is to be used for the purpose of assessing how the 275 metal/metalloid release (bioaccessibility) of different materials of the same metal compare to 276 reference materials for which e.g., toxicological information and/or oral reference values 277 already exist. In this case, the testing laboratory/sponsor of the study should select (an) 278 appropriate reference material(s) depending on the testing material under assessment. For the 279 calculation of the relative bioaccessible concentration (%RBC) of a metal in an alloy to assess 280 the presence of a matrix effect affecting metal release, it is the pure metal ingredients of the 281 alloy that are used as reference materials and will be run in parallel with the test alloy materials. 282 The rationale behind the choice of reference material should be clearly documented and 283 information on particle size and specific surface area provided.

284 Equipment

285 31. Agitation equipment. Thermostated linear (horizontal) or orbital shaker (37 ± 1 °C, 286 100 rpm; stroke length=1 inch) can be used to provide agitation. Agitation at 100 rpm is selected 287 as a means to provide a gentle agitation that is sufficient to maintain the flow of the aqueous 288 medium over the test materials, while maintaining the integrity of the surface of the test 289 materials and any solid reaction product coatings formed during the test. Abrasion of particles 290 or sample surfaces will in this way be minimized.

291 32. Thermometer. Calibrated thermometer, readable to 0.1 °C.

292 33. pH meter. Calibrated pH meter readable to 0.01 pH units.

293 34. Balance. A calibrated microbalance at least readable to 0.01 mg; controlled with 294 standard weights daily before and after each use. Two decimal numbers should be reported.

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295 35. Filtration equipment. The use of filtration to separate undissolved from dissolved 296 metal ions is in line with ASTM 5517. Disposable 0.2 µm membrane filters, e.g., Whatman 297 UNIFLO syringe filters (Polytetrafluoroethylene (PTFE) membrane), Pall Acrodisc syringe 298 filters (Polyethersulfone (PES) membrane), or equivalent filter system should be used, as these 299 filters do not adsorb or release metals in any significant amount. Appropriate single-use latex- 300 and oil-free (e.g., disposable polypropylene) syringes (volume depending on the amount 301 required for the analysis technique used) should be used. Appropriate high-density 302 polypropylene sample tubes known not to release or adsorb significant amounts of the 303 metals/metalloids of interest should be used.

304 Note 1: The effectiveness of the filter to separate dissolved from undissolved metal/metalloid 305 for a particular test material can be confirmed by visual inspection and/or light scattering or 306 equivalent method. Any particles <0.2 µm can in theory pass the filter and will be counted as 307 soluble ions.

308 Test procedure

309 36. Sample loadings. Two loadings (0.2 and 2 g /L) are tested for each test material so that 310 materials that release low amounts of ions can be reliably assessed. The physical form of the 311 test material (i.e., powders, non- epoxy embedded massive samples, epoxy embedded massive 312 samples) determines how loading is defined (see Test setup).

313 Note 2: It is up to the test material provider to provide the testing laboratory with sufficient 314 quantities of a representative and homogeneous sample of the test material to complete all 315 testing at appropriate loadings, together with a complete sample characterisation (as described 316 in section Test Report). For powders, after the initial mixing of the test material, further mixing 317 of the test material in between weighing the three replicate samples, is not recommended.

318 Test setup

319 37. The test set up depends on the physical form of the test material.

320 38. At least eighteen pre-heated vessels should be set up for the test and reference materials 321 (eight vessels for each material, i.e., four vessels for each of the two loadings for an analysis in 322 triplicates plus one vessel for pH measurement) and positive and negative controls (at least 1 323 each) (see Annex 3). The size of the vessels or the amount of sample and volume of solution 324 added to these vessels will be determined for each of the two loadings by the sample 325 characteristics and analytical needs. A minimum of 50 mL of simulated gastric fluid is needed 326 with a liquid volume to headspace volume ratio of about 1:4. This flexibility is needed to make 327 sure that enough fluid is available to cover all the surfaces of massive samples that come in 328 different shapes (whether epoxy embedded or not).

329 Note 3: For every additional material tested (be it another test material or another reference 330 material), eight additional vessels will be needed (i.e., two loadings (0.2 and 2 g/L) with 331 triplicate samples plus pH measurement).

332 39. Test Material and reference material in powder form

333 At least 18 pre-heated Erlenmeyer flasks should be set up as follows:

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334  At least 1 negative control vessel that will contain only the test medium HCl 0.032M 335 pH 1.5 ± 0.1 at 37 ± 1 °C. 336  At least 1 positive control vessel that will contain a solution of the element(s) of interest 337 added to the test medium, (preferably) at a concentration within the range that can be 338 expected to be released from the test material. 339  Eight material vessels at 0.2 g/L loading (4 vessels for each test and reference material): 340 approximately 10 mg (± 0.05 mg) test material should be weighed into each of the 3 341 replica test material vessels and 1 extra test material vessel (for pH measurement at the 342 start of the test). The exact weight should be noted. 343  Eight material vessels at 2 g/L loading (4 vessels for each test and reference material): 344 approximately 100 mg (± 0.5 mg) test material should be weighed into each of the 3 345 replica test material vessels and 1 extra test material vessel (for pH measurement at the 346 start of the test). The exact weight should be noted.

347 A volume of 0.032 M HCl consistent with the desired loading (e.g., 50 mL in the examples 348 mentioned above) at pH of 1.5 ± 0.1, preheated to 37 ± 1 °C should be added to the negative 349 control vessel, to the positive control vessel, and to each of the eight material vessels. Swirling 350 is not recommended for powders, as they may adhere to the vessel walls.

351 All vessels should then be covered as soon as possible (within one minute of mixing) and placed 352 in a thermostatic shaker (37 ± 1 °C) for 2 hours: 1 hour at an agitation rate of 100 rpm followed 353 by 1 hour without agitation. The test should be performed in the dark.

354 The temperature and pH of the extra vessels (with the test and reference materials) should be 355 measured and recorded (t = 0 hour values). These flasks and their content can then be discarded.

356 40. Test Material and reference material in Massive Non-Epoxy-Embedded Form

357 These materials include e.g., non-embedded coupons or sheets, or massive materials placed on 358 the market as coarse pellets or granules (≥ 1 mm diameter).

359 At least 18 pre-heated Erlenmeyer flasks should be set up as described for powders but with 360 the following additional considerations:

361  Eight material vessels at 0.2 g/L loading (4 vessels for each test and reference material): 362 one or more pieces of test/reference material should be weighed into each of the 3 363 replica test/reference material vessels and 1 extra vessel (for pH measurement at the 364 start of the test). The exact weight should be noted. 365  Eight material vessels at 2 g/L loading (4 vessels for each test and reference material): 366 one or more pieces of test/reference material should be weighed into each of the 3 367 replica test/reference material vessels and 1 extra vessel (for pH measurement at the 368 start of the test). The exact weight should be noted.

369 Note 4: For non-epoxy-embedded massive samples (e.g., sheets, ≥ 1 mm diameter pellets or 370 granules), the weight of the sample can be used to modify the volume of fluid and comply with 371 the desired loading. [A surface equivalent loading can also be calculated for metal/metalloid 372 and alloy samples using the geometric exposed surface area of the sample.]

373 A volume of simulated gastric fluid consistent with the desired loading (e.g., 50 mL in above 374 examples) at pH of 1.5 ± 0.1, preheated to 37 ± 1 °C should be added to the negative control

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375 vessel, to the positive control vessel and to each of the eight material vessels. The vessels should 376 be gently swirled and visually inspected to ensure that the sample is covered by fluid and/or the 377 coarse pellets are dispersed in the solution.

378 41. Test Material and reference material in Massive Epoxy-Embedded Form (surface area 379 loading)

380 At least 18 pre-heated suitable sized vessels should be set up as described for massive non- 381 embedded samples (above) with the following additional considerations:

382  At least 1 negative control vessel that will contain a blank piece of epoxy resin, similar 383 in size to that used in the test and reference samples, and the test medium. 384  At least 1 positive control vessel that will contain a blank piece of epoxy resin + a 385 solution of the element(s) of interest added to the test medium at a concentration that 386 is within the range that can be expected to be released from the test material. 387  Eight material vessels at 0.2 g/L surface equivalent loading (4 vessels for each test and 388 reference material): pieces of epoxy resin with test/reference material with a known 389 exposed metal/metalloid surface should be introduced into each of the 3 test/reference 390 material vessels and 1 extra vessel (for pH measurement at the start of the test). The 391 exact weight should be noted. 392  Eight material vessels at 2 g/L surface equivalent loading (4 vessels for each test and 393 reference material): pieces of epoxy resin with test/reference material with a known 394 exposed metal/metalloid surface should be introduced into each of the 3 test/reference 395 material vessels and 1 extra vessel (for pH measurement at the start of the test). The 396 exact weight should be noted. 397 398 Note 5: The loading of epoxy-embedded massive samples (2 g/L or 0.2 g/L) can be expressed 399 as surface area equivalent. The surface equivalent loading calculation is described in Annex 2. 400 The metal release from these materials will be reported in two ways: 1) expressed per unit of 401 volume of liquid or per unit of mass, and 2) expressed per unit of surface area.

402 Observations, sampling and measurements at the end of the test

403 42. Any visual observations of e.g., precipitation, colouration, decolouration that differ 404 between replicate test vessels or are consistent for all three replicates, should be recorded as 405 they can help explain the metal release results.

406 43. The following sampling procedure should be used to collect and preserve the samples 407 for elemental analysis.

408  At the end of the 2 hours incubation period, two aliquots (e.g., 15 mL, dependent upon 409 the analytical set-up of each laboratory) should be removed from each vessel (at least 410 one negative control, at least one positive control, and 3 replicate test/reference material 411 vessels at each of 2 loadings) at a depth of 2/3 up from the bottom of the liquid (at least 412 28 analytical samples), filtered through a 0.2 µm filter (e.g., syringe), and transferred 413 to uniquely labelled (e.g., 15 mL polypropylene) sample tubes for analysis. Swirling of 414 the sample is recommended before taking the 2 aliquots for metal analysis. The filter 415 should be prewetted with 1 mL of the test solution before filtration of the test material 416 to avoid metal adsorption to the filter.

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417  The samples should be preserved with 1% HNO3 (100 µL HNO3 concentrated/10 mL 418 sample to keep the pH of the samples < 2. 419  The samples should be covered (to avoid evaporation and increased concentration) and 420 stored at room temperature in the dark until analysis; up to 1 month after sampling. 421  After sampling, the temperature and pH of all test vessels including the negative control 422 should be measured and recorded. 423  Sampling procedures are expected to be conducted in a timely manner (no longer than 424 10 minutes per test/reference material to process the samples at the end of the 425 incubation period) in order to e.g., keep variation between replica vessels as low as 426 possible.

427 Sample analysis

428 44. The concentrations of released elements of interest are measured with an appropriate 429 (validated) measurement method. In order to guarantee the basic quality (use of a validated 430 measuring method, involvement of appropriately trained staff, traceability of the 431 measurements, data processing and archiving, etc.) the sample analyses should be performed 432 in a laboratory that works according to a standardised quality system (e.g., GLP, GMP, 433 ISO17025).

434 45. Examples of universal and commonly used methods for measuring dissolved element 435 concentrations (and thus recommended) are listed below. However, other validated analysis 436 methods can also be applied (such as graphite furnace or flame atomic absorption 437 spectrophotometry, AAS).

438  ICP-MS (inductively coupled plasma-mass spectrometry); well documented in ISO 17294- 439 1 Water quality - Application of inductively coupled plasma mass spectrometry (ICP-MS) 440 - Part 1: General guidelines and ISO 17294-2 Water quality - Application of inductively 441 coupled plasma mass spectrometry (ICP-MS) - Part 2: Determination of selected elements 442 including uranium isotopes. 443  ICP-AES (inductively coupled plasma-atomic emission spectrometry); well documented in 444 ISO 11885 Water quality - Determination of selected elements by inductively coupled 445 plasma optical emission spectrometry (ICP-OES) 446  Speciation needs to be assessed when different oxidation states are suspected at pH 1.5. 447 (see paragraph 10). Examples of analytical methods that can be used for that purpose 448 include Ion Chromatography-Inductively Coupled Plasma Mass Spectrometry (IC-ICP- 449 MS), High-performance liquid chromatography coupled to inductively coupled plasma – 450 Mass spectrometry (HPLC-ICP-MS), and electrochemical tools such as stripping 451 voltammetry, ion selective electrodes. 452 453 46. Only 1 measurement result is reported for each analytical sample and element (in µg/L 454 or mg/L), for at least 28 analytical samples as described in paragraph 43.

455 47. In addition to the measurement results of the test/reference material samples, an 456 experimentally derived reporting limit is always provided for each element of interest (e.g., 457 limit of quantification (LOQ), limit of detection (LOD), see glossary for definitions, or other 458 reporting limits derived in the laboratories according to standard use). Other analytical 459 performance characteristics of the analysis (measuring range, repeatability, reproducibility, 460 accuracy, measurement uncertainty) should also be reported.

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461

462 DATA AND REPORTING

463 Data

464 48. Results should be reported as measured element release (μg metal release per L 465 simulated gastric fluid or mg metal release per L simulated gastric fluid). The absolute releases 466 need to be corrected by subtracting the negative control values in the case where they exceeded 467 the LOQ. All data should be corrected based on the actual loading for each test run.

468 49. Results should also be presented as:

469  absolute release per g test/reference material (mg/g)

470  percent of total element in the test/reference sample, released (%)

471 element released per unit surface area (mg/m2) in the case of metals (zero valence) and 472 alloys.

473 The absolute release per g test/reference material (mg/g) is calculated taking into account the 474 actual weighed amounts of test/reference item per test vessel. The percent of total element 475 released (%) is calculated as the ratio of the average measured amount of element released in 476 the test solution to the total amount of exposed element in the test solution.

477 % Element Released = element measured in extract (mg) /element in material sample 478 (mg) x 100

479 where element measured in extract (mg) = concentration of element in extract (mg/L) x vol 480 extract (L); and element in test material sample (mg) = element concentration in sample (mg/g) 481 x mass sample (g).

482 In the case of metal or alloy powders, the element released per unit surface area (mg/m2) is also 483 calculated as the measured amount of element in the test solution per exposed surface area 484 (usually based on the BET measurement). The element released per unit surface area (mg/m2) 485 should also be reported in the case of massive material.

486 50. An average element release is calculated first from the two analytical sample 487 measurements from each replicate test material sample. After subtracting the negative control 488 value (if applicable), the average element release, the standard deviation (SD) and the 489 coefficient of variation (CV) based on all three replicates of a test sample are calculated for 490 each loading. The between vessel CV is then used to check against the data acceptance criteria.

491 Data Acceptance Criteria

492 51. A test using the 0.032 M HCl Method can be assumed valid (i.e., satisfies quality 493 criteria of test system) if: 494  The values of pH and temperature of the test medium with test material at the start of 495 the test fall within the set boundary conditions, i.e., pH 1.5 ± 0.1 and temperature at 37 496 ± 1 °C.

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497  The values of pH and temperature of the negative control at the end of the test fall 498 within the set boundary conditions, i.e., pH 1.5 ± 0.1 and temperature at 37 ± 1 °C.

499  The values of pH among the three test material sample replicates at the end of the test 500 differ by no more than 10%. If the pH values among the three replicates differ by more 501 than 10%, contamination could be suspected, and the test should be repeated.

502 Note 6: At the end of the test, the pH of the triplicate test material vessels may be higher than 503 1.5, depending on the nature of the test material (e.g., ); this deviation should be recorded. 504 Where the pH values of the triplicate test material samples are significantly higher than 1.5, it 505 is recommended to compare the changes in pH between the high and low loadings. The increase 506 in pH can happen when the metal ion released has alkaline properties. At the low loading, the 507 buffering capacity of the test fluid will be less exhausted than at the high loading and it is likely 508 to yield pH values closer to 1.5. In this case, the recommendation is to use the data generated 509 from the low loading to calculate relative metal/metalloid release.

510 52. The test results are considered acceptable if the following criteria are met:

511  A LOQ or other reporting limit is available for each element of interest.

512  Results for the two analytical samples from each test material vessel (within vessel) do 513 not differ by more than 20% in ≤ LOQ – 10x LOQ range, or by 10% or less for 514 measurements in 10x LOQ – end of measuring range.

515  The between test material vessels’ Coefficient of Variation (CV) (see paragraph 50) is 516 ≤ 40% for each element of interest in ≤ LOQ – 10x LOQ range, or CV ≤ 20% for 517 measurements in 10x LOQ – end of measuring range.

518  Accurate element measurements (± 10% of reported values for Certified metal solution 519 selected as positive control) are obtained from a positive control certified solution that 520 contains the element of interest.

521 Note 7: When the CV values of test materials exceed the recommended values (leading to data 522 rejection), a thorough review of the sample characteristics should be conducted before it is 523 attempted to repeat the study; in some instances, the large variability in metal/metalloid release 524 could be linked to the intrinsic nature of the sample. Some materials (e.g., UVCBs) are very 525 complex and can, despite being from the same batch, have a wide range in the composition and 526 also in particle size, which will result in significant variations within a sample and between 527 samples. Large variations will therefore provide important information on the test material, but 528 this information needs to be combined with proper physical-material characterisation of the test 529 material, e.g., SEM-EDS, XPS, IR, BET, size distribution, etc.

530 Note 8: The average negative control measurements that exceed the LOQ should be subtracted 531 from test material sample values. Contamination is suspected when concurrent negative control 532 values are > 20 x SD of the mean negative control value for the laboratory (based on 10 or more 533 replicate measurements). In the latter case, a thorough review of all the test data should be 534 conducted (including ratio of test sample value to negative control value) before deciding 535 whether the study should be repeated.

536 Interpretation of results

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537 53. The metal/metalloid releases of a given element (metal or metalloid) released in the 538 simple simulated gastric fluid from different materials containing this element (test material 539 and reference material) can be compared to produce relative metal release values. Relative 540 metal release values can be used to compare two materials or to assess the matrix effect in an 541 alloy.

542 54. The initially measured values are reported as µg metal/L or mg metal/L. The calculated 543 values (expressed as mg metal/g sample, mg metal/cm2 sample, or as percent of metal content) 544 can be compared to those from reference materials. If the test and reference materials have been 545 tested at both of the recommended same two loadings (2 g/L and 0.2 g/L), then the results 546 obtained at the same (high or low) loading should be compared (e.g., test material mg metal/g 547 sample divided by reference material mg metal/g sample x 100).

548 55. In many cases, the relative in vitro bioaccessibility values using data from either 549 loading are likely to be comparable. If this is not the case, it is recommended to use the data 550 from the loading that yields the more reproducible data (lowest SD). A deviation from this 551 approach can be justified if for one of the samples, quantitative measurements are only obtained 552 at one loading (e.g., when the low loading yields values < LOQ, the use of the high loading 553 data only would be justified). In the case of massive metal samples, a correction by surface area 554 needs to be applied (mg metal/cm2 sample) to compare releases from samples that differ in 555 exposed surface area. Although this can also be applied to metallic powders, the release by 556 mass of sample remains in this case the first level of calculation when a mass loading is used. 557 Correction by surface area should not be applied when testing chemical forms of metals that 558 are not in the elemental (valence of zero) form. In some applications, it may be useful to 559 calculate the percent of metal content in the sample that is released (mg metal released/mg 560 metal in sample). This can be done with knowledge of the material formula, purity, and/or bulk 561 concentration.

562 56. In vitro bioaccessibility data generated from a test material alloy (in powder or massive 563 form) and from a reference material can also be compared as mentioned above. In most cases, 564 the reference material is a pure metal ingredient of the alloy, in matching powder or massive 565 form, at high and low loadings. In this case, the ratio of bioaccessibility values multiplied by 566 100 corresponds to the Relative Bioaccessible metal Concentration (%RBC) of the metal in the 567 alloy and can be calculated using the following equation 1.

푚푔 푚푒푡푎푙 푖표푛 푟푒푙푒푎푠푒푑 (푚푒푎푠푢푟푒푑 푖푛 푒푥푡푟푎푐푡)/푔 푎푙푙표푦 푠푎푚푝푙푒 푡푒푠푡푒푑 568 푅퐵퐶 (%) = 푥 100 푚푔 푚푒푡푎푙 푖표푛 푟푒푙푒푎푠푒푑 (푚푒푎푠푢푟푒푑 푖푛 푒푥푡푟푎푐푡)/푔 푝푢푟푒 푚푒푡푎푙 푠푎푚푝푙푒 푡푒푠푡푒푑

569

570 In many cases, the releases (mg metal/g) from the pure metal and the alloy can differ by orders 571 of magnitude. To assess matrix effects and calculate the %RBC, it is recommended to consider 572 the data collected at the low and high loading, separately, using Equation 1 above.

573 The most conservative estimate of the % RBC of the metal in an alloy can then be identified 574 and selected. The calculated %RBC of a metal in an alloy may be equal, higher or lower than 575 the %bulk concentration of that metal in the alloy, and in some cases, it may exceed 100%.

576  The % RBC will equal the %bulk concentration when there is no matrix effect in the 577 alloy; in other words, the alloy behaves as a simple mixture.

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578  The % RBC will be lower than the %bulk concentration when there is a matrix effect 579 in the alloy that lowers the effective release of that metal; in this case, the alloy behaves 580 as if it had a lower %bulk concentration of the metal.

581  The % RBC will be higher than the %bulk concentration when there is a matrix effect 582 in the alloy that enhances or promotes the effective release of the metal; in this case, 583 the alloy behaves as if it has a higher %bulk concentration of the metal.

584 57. Once the relative bioaccessibility or the %RBC are calculated, the 95%-confidence 585 interval (CI) can be calculated. The approach described by Fieller (21) for obtaining the 586 approximate 95% CI around the ratio of metal bioaccessibility values (i.e., %RBC) can be 587 applied. The formula involves calculation of the standard error of the ratio (quotient) by error 588 propagation, and then multiplying this by the appropriate quantile and add/subtract to/from the 589 ratio of the means, as per equation 2 below.

590 591 Where SEQ: Standard error of the quotient

592 Q: quotient of A/B

593 t*: 0.975-quantile of the t-distribution with n-2 degrees of freedom, where n is the number of 594 replicates for A plus the number of replicates for B

595 A: bioaccessibility of metal/metalloid from alloy

596 B: bioaccessibility of metal/metalloid from pure element

597 SEM: standard error of the mean of A or B

598 58. There are several software tools that allow the calculation of the CI. A free online 599 calculator for Fieller's approach for n < 20 can be found at 600 https://www.graphpad.com/quickcalcs/ErrorProp1.cfm (25). The numerator bioaccessibility 601 value needs to be multiplied by a suitable factor in order to get the required number of 602 significant digits. The number of significant digits needed may vary depending on the value of 603 the relative bioaccessibility or the %RBC, and the level of precision needed.

604 Test report

605 59. A test report is prepared for each material and should include (but is not limited to) the 606 following information:

607  Information on the laboratory/study

608 Identification of the study, the sponsors, the test facility, and study director;

609  Description of the test and reference materials:

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610 - Name, including identifiers such as CAS/EINECS numbers when relevant 611 - Batch number and/or sampling date 612 - Expiration date, when applicable 613 - Details of supplier(s)/manufacturer/importer (e.g., address, contact information) 614 - Information available on composition (e.g., Certificate of Analysis), at least on all 615 substances > 0.1 w% to assess purity of the sample and nature of impurities (% or ppm). 616  Information available on method of manufacture (particularly for powder forms), if 617 available, indicating if atomization (water or air), material or grinding method was 618 used. 619  Information on processing, preparation, surface finish, transport and storage, if 620 available (e.g., heat treatment/fabrication history; metallurgical microstructure 621 particularly for massive forms). 622  A justification of sample preparation, if a representative powder sample is generated 623 from a massive form (see paragraph 15). 624  Information on surface charge (particles), surface composition, and other surface 625 properties (e.g., surface oxidation for both massive and powder forms; surface finish 626 particularly for massive forms), if available. 627  Particle size distribution (powders only) using parameters such as d0.1, d0.5, and d0.9. 628 Report the method of assessment and if values are based on volume or number 629 distribution and in which media (e.g., composition, ionic strength, pH) the size 630 distribution was measured (e.g., air, water). E.g., size distribution may be determined 631 by sieve analysis or laser diffraction. 632  If surface charge information is available, include the medium used to measure the 633 surface charge (e.g., composition, ionic strength, pH). 634  Information on morphology (shape of the particles) and metallurgical structure, if 635 available, e.g., the morphology of particles may be determined by examination using a 636 Scanning Electron Microscope (SEM). 637  Information on particle agglomeration behaviour in solution, if available. For example, 638 agglomeration can be assessed by comparing size distribution measurements (using 639 laser diffraction techniques) in Phosphate Buffered Saline (PBS) to the size distribution 640 in air. 641  Specific surface area in m2/g (powders only) of metal/metalloid (or their compounds if 642 available). E.g., particle surface area can be determined by BET analysis (absorption 643 of nitrogen or krypton at cryogenic conditions) method (22). 644  Surface area (massive only). Measure the geometric exposed area of massive materials 645 and embedded test samples with a Vernier caliper or other suitable graduated 646 measuring device e.g., measuring projector. 647  If the sample (powder) was sieved, information about the original sample could also be 648 provided.

649  Description of the test system and loading: the following information should be 650 provided.

651 - Test/reference material weight, final loading and final volume 652 - pH and temperature at the start and the end of the test 653 - Detailed descriptions of the test apparatus and procedure, including analytical 654 instruments used

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655 - Surface area to volume ratio for massive materials 656 657  Results from the analyses of metal concentrations should include: 658 - Date of test 659 - LOQ, LOD, or other reporting limit 660 - Tabulation: metal release (µg/L or mg/L) (measured element concentrations in the 661 samples using one of the methods described in paragraph 45, including negative and 662 positive controls, test material samples, reference materials and/or proficiency material 663 samples). 664 - Mean metal release per g material (calculated mg/g) 665 - Mean percent of total element released (calculated %) 666 - Mean element release per unit surface area for metals and alloys (calculated mg/m2) 667 - Relative bioaccessibility or RBC

668  Description of quality assurance programme applied

669  Deviations from this Standard Operating Procedure, if any and reason(s) for such

670  Any circumstances that may have affected the results (i.e. description of other 671 effects observed, e.g., precipitation)

672  Reference to the records and all raw data necessary to reconstruct the study.

673

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674 LITERATURE

675 1. ASTM (American Society for Testing and Materials). (2014). Standard test method for 676 determining extractability of metals from art materials. In: ASTM, Annual Book of ASTM 677 Standards, vol. 06. 02, D5517–14. ASTM, Philadelphia, US. 678 2. BS (British Standard) EN 71–3. (2013). Safety of toys. Migration of certain elements. Cordeiro, 679 F., Baer, I, Robouch, P., Emteborg, H., Got, J.C., Kortsen, B., de la Calle, B. (2012). IMEP-34: 680 in toys according to EN 71–3:1994; Interlaboratory Comparison Report. JRC 681 Scientific and Policy Reports. EUR 25380 EN. 682 3. BARGE. (2016). The BARGE unified bioaccessibility method. The Bioaccessibility Research 683 Group of Europe. https://www.bgs.ac.uk/barge/ubm.html. 684 4. US EPA. 2017. SW-846 Test Method 1340: In Vitro Bioaccessibility Assay for Lead in Soil, 685 https://www.epa.gov/hw-sw846/sw-846-test-method-1340-vitro-bioaccessibility-assay-lead- 686 soil 687 5. ESTCP. (2012). Validation of an In Vitro Bioaccessibility Test Method for the Estimation of 688 the Bioavailability of Arsenic from Soil and Sediment Cost and Performance Report 689 Environmental Security Technology Certification Program U.S. Department of Defense (ER- 690 200916). 691 6. Brattin, W., Drexler, J., Lowney, Y., Griffin, S., Diamond, G., Woodbury, L. (2013). An in 692 vitro method for estimation of arsenic relative bioavailability in soil. J Toxicol Environ Health 693 76, 458-478. 694 7. Ruby, M.V., Schoof, R., Brattin, W., Goldade, M., Post, G., Harnois, M., Mosby, D.E., Casteel, 695 S.W., Berti, W., Carpenter, M., Edwards, D., Cragin, D., Chappell, W. (1999). Advances in 696 Evaluating the Oral Bioavailability of Inorganics in Soil for Use in Human Health Risk 697 Assessment. Env Sci Technol 21, 3697-3705. 698 8. Suh, M., Casteel, S., Dunsmore, M., Ring, C., Verwiel, A., Proctor, D.M. (2019). 699 Bioaccessibility and relative oral bioavailability of cobalt and nickel in residential soil and dust 700 affected by metal grinding operations. Sci Tot Environ 660, 677-689. 701 9. Whitacre, S.D., Basta, N.T., Stevens, B.N., Hanley, V., Anderson, R.H., Scheckel, K.G., Foster, 702 A.L. (2017). Modification of an existing in vitro method to predict relative bioavailable arsenic 703 in soil. Chemosphere 180, 545-552. 704 10. Wragg, J., Cave, M., Basta, N., Brandon, E., Casteel, S., Denys, S., Gron, C., Oomen, A., 705 Reimer, K., Tack, K., Van de Wiele, T. (2011). An inter-laboratory trial of the unified BARGE 706 bioaccessibility method for arsenic, cadmium and lead in soil. Sci Total Environ 409, 4016- 707 4030. 708 11. Henderson, R.G., Verougstraete, V., Anderson, K., Arbildua, J.J., Brock, T.O., Brouwers, T., 709 Cappellini, D., Delbeke, K., Herting, G., Hixon, G., Odnevall Wallinder, I., Rodriguez, P.H., 710 Van Assche, F., Wilrich, P., Oller, A.R. (2014). Interlaboratory Validation of Bioaccessibility 711 Testing for Metals. Regul Toxicol and Pharmacol 70(1), 170-181. 712 12. Eskes, C., Corsini, E., Hanley, V., Karadjova, I., Kopp-Schneider, A., Suh, M., Alves, P., 713 Clewell, R., Cotgreave, I., Navas, J., Piersma, A., Westmoreland, C. (2020). ESAC Opinion on 714 the Scientific Validity of the Bioelution Test Method, Viegas Barroso, J. editor(s), EUR 30281 715 EN, Publications Office of the European Union, Luxembourg, 2020, ISBN 978-92-76-20004- 716 8, doi:10.2760/023005, JRC121143. Available at: 717 http://publications.jrc.ec.europa.eu/repository/handle/JRC121143. 718 13. EU Commission: definition of nanomaterials: Commission Recommendation of 18 October 719 2011 on the definition of nanomaterial (2011/696/EU). OJ L 275, 20.10.2011, p. 38–40, 720 http://data.europa.eu/eli/reco/2011/696/oj.

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721 14. Mörsdorf, A., Odnevall Wallinder, I., Hedberg, Y. (2015). Bioaccessibility of micron‐sized 722 powder particles of molybdenum metal, iron metal, molybdenum and ferromolybdenum 723 ‐ Importance of surface oxides. Regulatory Toxicology and Pharmacology 72(3), 447‐457. 724 15. Herting, G., Jiang T., Sjöstedt, C., Odnevall Wallinder, I. (2014). Release of Si from silicon 725 metalloids, a FeSi alloy and a silicate mineral in simulated biological media. PLoS ONE 9(9). 726 16. Stefaniak, A.B., Virji, M.A., Harvey, C.J., Sbarra, D.C., Day, G.A., Hoover, M.D. (2010). 727 Influence of artificial gastric juice composition on bioaccessibility of cobalt- and tungsten- 728 containing powders. Int J Hyg Environ Health 213, 107–115. 729 17. IITRI. (2010). IIT Research Institute, Life Sciences group, Chicago IL, at 730 https://www.itia.info/assets/files/ACGIH/21_IITRI_Bioaccessibility_Studies_5_Tungsten_Co 731 mpounds.pdf 732 18. Hedberg, Y., Jiang, T., Odnevall-Wallinder, I. (2010). Bioaccessibility of antimony released 733 from four different antimony compounds in synthetic biological media. Commissioned by 734 International Antimony Association (i2a). 735 19. EU CLP. REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND 736 OF THE COUNCIL of 16 December 2008 on classification, labelling and packaging of 737 substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and 738 amending Regulation (EC) No 1907/2006, https://eur-lex.europa.eu/legal- 739 content/EN/TXT/?uri=CELEX:02008R1272-20200501 740 20. ASTM F963 ASTM (American Society for Testing and Materials). (2017). Standard Consumer 741 Safety Specification for Toy Safety F963-17. In: ASTM International, 100 Barr Harbor Drive, 742 PO Box C700, West Conshohocken, PA 19428-2959, US. 743 21. Fieller, E.C. (1954). Some problems in interval estimation. Journal of the Royal Statistical 744 Society, Series B 16(2), 175-185. 745 22. Brunauer, S., Emmett, P.H., Teller, E. (1938). Adsorption of Gases in Multimolecular 746 Layers. Journal of the American Material Society 60(2), 309-319. 747 23. OECD. (2015) Guidance on selecting a strategy for assessing the ecological risk of 748 organometallic and organic metal substances based on their environmental fate. Series on 749 Testing & Assessment 212. 750 http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2 751 015)2&doclanguage=en 752 24. Thompson, M., Ellison S.L.R., Wood, R. (2002). Harmonised Guidelines for single laboratory 753 validation of methods of analysis. Pure Appl. Chem., 74(5): 835-855 754 25. ECHA. (2016). Forum methodology for recommending analytical methods to check 755 compliance with REACH Annex XVII restrictions. ECHA-16-B-04-EN

756

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757 ANNEX 1: Definitions 758 759  Bioavailability: In vivo bioavailability is defined as the extent to which a substance is taken up by 760 an organism and is available for metabolism and interaction at target organ/sites (e.g. kidney, skin). 761  Bioaccessibility: In vitro bioaccessibility can be defined as the fraction of a substance that dissolves 762 under surrogate physiological conditions and is potentially available for absorption into systemic 763 circulation. Bioaccessibility corresponds to the metal ion released in in vitro tests that mimic 764 physiological fluids (e.g., oral bioaccessibility based on fluids simulating gastrointestinal tract; 765 dermal bioaccessibility based on fluid mimicking perspiration). In vitro bioaccessibility has also 766 been called IVBA in many publications. 767  BET: Brunauer, Emmett and Teller method to measure the specific surface area of a sample 768  CAS number: Chemical Abstracts Service number 769  Certified Standard Metal solution: standard solutions containing the metal(s) of interest that are 770 used for analytical calibration and is used to prepare the positive controls. 771  CV: Coefficient of variation 772  EINECS number: European Inventory of Existing Commercial Chemical Substances number 773  GLP: Good laboratory Practices 774  GMP: Good Manufacturing Practices 775  ICP AES: inductively coupled plasma-atomic emission spectrometry 776  ICP MS: inductively coupled plasma-mass spectrometry 777  Inorganics: Compounds that are not organometallics as defined in the OECD Guidance 212 (23), 778 i.e. compounds identified as coordination complexes where the metal or metalloid has covalent- 779 character bonds with , nitrogen, sulphur and/or phosphorus belonging to an organic moiety. 780 Dissociation of the metal or metalloid is generally considered to be negligible. 781  IR: Infrared 782  Loading: can be expressed in terms of mass or surface area per volume of test medium. Mass 783 loading is the ratio of test sample (mass) to test medium (volume) (e.g. 2 g/L). Surface area loading 784 is the ratio of mass equivalent surface area (area) to test medium (volume) (e.g. 10 cm2/L). 785  LOD (limit of detection): corresponds to the smallest amount or concentration of an analyte in the 786 test sample that can be reliably distinguished from zero (Thompson et al. 2002 (24); ECHA 2016 787 (29)). An experimentally-derived LOD for each metal can be calculated as three times the standard 788 deviation of the mean measured concentration of the test medium (i.e., negative control for the 789 laboratory that corresponds to the average of a minimum of 10 measurements). 790  LOQ (limit of quantification): corresponds to the concentration below which the analytical method 791 cannot operate with acceptable precision for the given matrix (25). An experimentally-derived LOQ 792 for each element can be calculated as six times the standard deviation of the mean measured 793 concentration of the test medium (i.e., negative control for the laboratory that corresponds to the 794 average of a minimum of 10 measurements). 795  Massive: metals with a particle size ≥ 1 mm diameter 796  MSDS: Material Safety Data Sheets 797  Negative control: test medium run in parallel with test material. 798  Negative control values for the laboratory: mean values from test medium only vessels collected at 799 the laboratory when the SOP was set up (10 values minimum) or representing the collective 800 database of all test medium-only values based on initial tests plus further tests. 801  Positive control: The positive control is prepared by spiking certain amounts of the Certified 802 Standard Metal solution into the test medium. Its purpose is to assure that the method using 0.032

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803 M HCl as simulated gastric fluid produces repeatable results. It provides evidence that the test 804 system is responsive under the actual conditions of the assay. The positive control is run in parallel 805 with the test materials of that metal or element. 806  Proficiency materials: are well characterised and defined materials (e.g. regarding elemental 807 composition and particle size) that may or may not include the metal of interest but need to match 808 the physical form of the test materials (e.g. samples of powder metals or metal compounds, 809 embedded form and non-embedded massive forms of alloys). These materials are tested initially by 810 the laboratory when establishing the SOP and are run periodically afterwards (at least once a year). 811  Reference materials: materials containing the same metal or element as the test material; the 812 reference material test results are used to calculate relative metal release (bioaccessibility) to 813 compare two similar materials or the relative bioaccessible concentration to compare an alloy to a 814 reference material to assess the matrix effect. For the latter case, the reference material and the test 815 material should have similar physical form. The selection of the exact reference material to use in 816 each case, depends on the application and on the characteristics of the test materials. 817  Relative bioaccessible concentration (%RBC): in the context of assessing the presence of a matrix 818 effect in alloys that affect metal release, the relative bioaccessible concentration (%RBC) can be 819 calculated and compared to the %bulk concentration. 푚푔 푚푒푡푎푙 푖표푛 푟푒푙푒푎푠푒푑 (푚푒푎푠푢푟푒푑 푖푛 푒푥푡푟푎푐푡)/푔 푎푙푙표푦 푠푎푚푝푙푒 푡푒푠푡푒푑 820 푅퐵퐶 (%) = 푥 100 푚푔 푚푒푡푎푙 푖표푛 푟푒푙푒푎푠푒푑 (푚푒푎푠푢푟푒푑 푖푛 푒푥푡푟푎푐푡)/푔 푝푢푟푒 푚푒푡푎푙 푠푎푚푝푙푒 푡푒푠푡푒푑

821  Relative metal bioaccessibility: results from the comparison of the metal release results of a given 822 metal released from different compounds of the same metal or from different materials containing 823 this metal. 824  SEM-EDS: Scanning Electron Microscopy / Energy Dispersive X-Ray Spectroscopy 825  SD: Standard deviation 826  SiC: 827  Test material: materials (e.g. substances, mixtures) for which generation of metal bioaccessibility 828 data are needed. Their bioaccessibility is usually compared to the bioaccessibility of a reference 829 (source) material (see also relative metal bioaccessibility). 830  Test materials should be metals and metalloids, inorganic metal compounds, or complex 831 metal(metalloids)-containing materials like e.g. alloys, UVCBs, pigments. 832  Test medium: simulated gastric fluid (HCl 0.032 M, pH 1.5 ± 0.1). 833  Test system: test medium + test vessels. 834

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835 ANNEX 2

836 Massive Samples

837 Massive forms of the test material can be tested as present on the market or as epoxy embedded samples. 838 For massive forms of the test material such as discs, rods, sheets, balls, pellets, etc., the weight, 839 geometry, surface finish and the geometric surface area of the sample should be recorded. Massive test 840 material samples should, generally be tested as close as possible to their as received condition (i.e., in 841 the form, and with the surface properties, in which they are placed on the market or reflecting conditions 842 of foreseeable use).

843 However, there are cases when massive samples need to be modified before testing. If the volumes of 844 medium required to achieve the required loadings of a massive sample (2 g/L and 0.2 g/L) are too large 845 to be feasible (maximum volume = 1250 mL for a two litre flask), two options are possible: 1) cut the 846 sample down to a smaller size and/or 2) reduce its exposed surface by epoxy embedding it.

847

848 Option 1: Cutting massive samples to smaller size (with or without subsequent embedding)

849 When smaller test material samples are prepared from the original -as received- material, cut edges 850 should be wet ground to 1200 grit (SiC paper)3 FEPA "P" grit sizes or equivalent in order to provide 851 flat surfaces to aid area measurements and to prevent lacerations from sharp edges. All wet grinding 852 should be followed immediately by mild ultrasonic cleaning in ethanol for 10 min and subsequently in 853 isopropyl alcohol for 10 minutes. If, however, the as received surface of the massive material has been 854 damaged by the sampling operation (e.g. scratched), the test surface and cut edges should be prepared 855 for testing by wet grinding with silicon carbide papers from coarse to fine grit, until a uniform surface 856 finish with 1200 grit is attained (this will also provide a flat surface to aid with area measurements). 857 Grinding should be followed immediately by mild ultrasonic cleaning in ethanol for 858 10 minutes and subsequently in isopropyl alcohol for 10 minutes, with a final rinse with isopropyl 859 alcohol. Afterwards, the samples should be dried under a stream of cold (not hot) nitrogen gas and 860 stored in a desiccator for a minimum of 168 hours prior to the start of the test. Cut pieces need to be 861 measured to the 0.1 or 0.01 mm scale using the appropriate measuring device. During the testing, care 862 should be taken to avoid the creation of crevices (not normally present in the sample) that may lead to 863 increased corrosion and release of metals. Cutting of soft metals and alloys should be carried out with 864 care in order to avoid damaging the test surface. Further special polish techniques may be required, 865 however, to provide a scratch-free surface. For example, polishing with a proprietary polishing fluid on 866 a selvyt cloth.

867

868 Option 2: Epoxy embedding massive samples (with or without cutting them first)

3 While there are several scales for grit papers, two of the most commonly used are the ANSI/CAMI in USA and the FEPA in Europe. For example, a FEPA P-grade 1200 has a median diameter of 15.3 µm and is equivalent to an ANSI grit 600 with a median diameter of 14.5 µm. An example of a comparison table can be found at https://www.buehler.com/grinding-and- polishing-guide.php

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869 When the sample cannot be cut to a smaller size without altering the physico-chemical properties of its 870 surface and hence possibly affecting the metal release, epoxy embedding (as described below) is an 871 option. Epoxy embedding decreases the effective surface area (i.e., the surface area available for 872 interaction with the medium) and presents some benefits. The surface properties of the embedded 873 sample can reflect the material as it is placed on the market or the surface can be treated by polishing 874 when making comparisons across similarly processed samples. When the sample is cut to a smaller size 875 before embedding, any fresh-cut surfaces can be shielded with the epoxy.

876 In addition to decreasing the volume of fluid needed for the test, embedding has the advantage of 877 avoiding abrasion during testing, and yielding a regular shape, therefore the exposed surface area can 878 be accurately determined.

879 Polishing an embedded sample has the advantage of creating a standardised, uniform, and well-defined 880 surface (for example by standardisation of the passivation time) which may increase reproducibility. 881 The disadvantage is that the polished surface no longer reflects the surface of the material as it is placed 882 on the market.

883 Polishing is not recommended for metal or alloy massive samples that:

884  Do not have a sufficiently homogeneous microstructure. 885  Have a porous structure. Openings may be created and filled with metal dust during polishing 886  Have a loosely grained structure, such that pieces break off during polishing 887  Have a low melting point. Polishing causes the temperature to rise locally despite water cooling 888  React strongly with the solvents (water, isopropanol) that are commonly used during sample 889 preparation. 890  Soft metals and alloys require special attention. The polishing needs to be adjusted for soft 891 materials, the sandpaper may need to be softened with wax or material polishing, and/or a final 892 etching step may be recommended. 893

894 Epoxy embedding process

895 The preparation of epoxy embedded samples is briefly described below and may occur in up to three 896 steps. The first step is cutting the massive metal, when this is needed. The second step is the epoxy 897 embedding itself. A third step, surface preparation, can be skipped when samples that reflect the surface 898 properties of the material as placed on the market are desired.

899 a. Cutting (optional): typically, coupons are cut from the massive metal by suitable means (e.g., water- 900 cutting, laser cutting, hacksaw, shears, etc.) depending on the size and shape of the sample (see 901 further details under Option 1 above). It is important that whatever technique is selected, it does not 902 alter the properties of the surface that will be exposed to the medium after embedding. The samples 903 are degreased with ethanol before embedding in epoxy resin.

904 b. Embedding: for embedding the samples one can use EpoFix Kit from Struers or equivalent. The 905 polymerisation process is done as directed by the resin supplier, e.g., Struers, as follows: to prepare 906 the resin fluid an amount of hardener is mixed into the correct amount of resin (25 parts of resin / 907 3 parts of hardener by weight). The mixture is mixed well for approximately 2 minutes without 908 introducing too many air bubbles. The mixture is left to rest for 2 minutes (pot life of this mixture: 909 about 30 minutes). The epoxy is carefully poured over the specimen in an achieved mould. A

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910 vacuum system can be used to reduce air bubbles. The embedded samples are left at room 911 temperature for hardening during at least 12 hours.

912 c. Surface preparation (optional): the surface preparation may depend on the nature and hardness of 913 the test material. This is best done by following the instructions of the manufacturer of the used 914 polishing machine (e.g., Struers). Ultimately, a uniform and scratch-free surface should be obtained. 915 The followed polishing step must be well documented and subsequently reported. The following 916 procedure was for example well applicable for medium hard materials:

917 Each epoxy sample is wet ground with 400 grit, 800 grit, 1200 grit and finally 2000 grit SiC paper in 918 sequence. In order to avoid maintaining a memory imprint of the grinding scratches deeper into the bulk 919 material matrix, it is important to apply only a very gentle pressure during the grinding. Each sample is 920 ground only for approximately one minute on each grit size paper with very gentle pressure, recognising 921 that hard and soft surfaces may require different grinding times. After wet grinding, the samples are 922 rinsed with ethanol and dried with nitrogen gas to avoid corrosion during the sample preparation. 923 Polishing is performed using 3, 1 and 0.25 μm diamond paste (DP-Paste M) in turn with ethanol as a 924 lubricant, and with utmost care. To obtain smooth scratch-free surfaces, the samples are held very gently 925 against the polishing cloth with very little applied pressure. Plenty of ethanol is used as a lubricant to 926 obtain a well-defined surface. Each polishing step with diamond paste takes about 5 to 10 minutes, 927 depending on the type of material. After polishing, the samples are cleaned with ethanol and quickly 928 dried with cold nitrogen gas. To remove any residual particles left on the surface after grinding and 929 polishing, all samples are cleaned ultrasonically first in ethanol for approximately 10 minutes followed 930 by ultrasonic cleaning in isopropyl alcohol for another 10 minutes, and a final rinsing with isopropyl 931 alcohol. Isopropyl alcohol is used as the last cleaning medium. After the ultrasonic cleaning, all samples 932 are dried quickly again with cold nitrogen gas.

933 The polished surface is checked by means of a microscope. The metal surfaces should be free from 934 scratches and spots, otherwise, re-polishing is necessary. The samples are stored for a minimum of 168 935 hours in a desiccator before testing. While in most cases the average free metal surface is about 100 936 mm², the exact exposed surface should always be measured and reported. After testing, the surface of 937 massive samples should be again looked at under a microscope to assess if surface alterations were 938 triggered by the testing conditions.

939 Figure 1. Example of polished surfaces embedded in epoxy resins

940

941

942 Surface Equivalent Loading for embedded materials

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943 Steps involved in the calculation of the surface equivalent loading for test materials in massive epoxy 944 embedded form:

945 1. Starting from a selected mass loading (e.g., 2 g/L) and with knowledge of the test material’s density, 946 a surface equivalent loading can be calculated by considering the massive material as a 1 mm 947 diameter sphere. 1 mm is the particle diameter which is the generally accepted to be the cut-off 948 between powders and massives (e.g. for aquatic toxicity classification under ECHA CLP Guidance 949 section IV.5.5 rev. 5 2017 or in Annex 9 of the UN GHS A9.7.5.4.3 GHS rev. 6 2015). 950 2. A spherical particle with a diameter of 1 mm has a surface area of 3.14 mm² (4·π·r²) and a volume 951 of 0.524 mm³ (⁴₃·π·r³). Using the information on density of the material (e.g. 8.38 mg/mm³), it can 952 be calculated that a Ø 1 mm spherical reference particle has a mass (mg) = density (mg/mm3) x 953 volume (mm3). In the example: 8.38 mg/mm³ x 0.524 mm³ = 4.39 mg per particle. 954 3. A loading of e.g. 2 g/L means that 2000 mg can be divided by 4.39 mg (the weight of a particle in 955 this example) to calculate the number of particles needed per litre of fluid (i.e. 456 particles/L in 956 the example above). 957 4. Since each particle has a surface area of 3.14 mm², a surface loading (e.g. in mm2/L) can be 958 calculated. In the example, a 2 g/L loading corresponds to 456 reference spheres and a total free 959 metal surface loading of 1432 mm²/L (456 particles x 3.14 mm2). 960 5. So, to test a massive embedded test material with a density of 8.38 mg/mm3, an exposed metal 961 surface of 1432 mm²/L is required. For a standard test volume of 50 mL this means an exposed 962 metal surface area of 71.65 mm² (1433 mm²/L x 0.05 L = 71.65 mm²) is needed. The loading for 963 other available surface areas can be adjusted by adding the corresponding amount of test medium 964 (within the vessel volume limits of minimum 50 mL and maximum 1250 mL). 965  If the massive embedded test material sample has, for example, 50 mm2 exposed surface per 966 piece, 29 pieces per litre would be needed for a 2 g/L loading (1432 mm²/L ÷ 50 mm2/piece 967 massive representative sample = 29 pieces/L). Depending on the flask size and considering the 968 volume limits of minimum 50 mL and maximum 1250 mL, 2 pieces with 70 mL medium, or 969 35 pieces with 1225 mL medium could be set up for testing. 970

971

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972 ANNEX 3: Test setup

973

974

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975

976 ANNEX 4: PROPOSED Sample Repository of Proficiency and Reference Materials 977

978 It is proposed to set up a “physical repository” that will include:

979  proficiency materials and 980  reference materials for alloys (i.e. pure metals in massive and powder form)

981 A Contractor who could provide such an archive facility in a central/defined location will be identified. 982 Contractor will be asked to properly login, maintain and ship out materials to researchers and facilities 983 worldwide. In this manner, investigators who want to perform the HCl 0.032M method will have access 984 to the same reference/proficiency materials.

985 Specific tasks of the Contractor;

986  The Contractor might coordinate -if he has the capacity to do so- some physical testing of the 987 samples on site or at another facility if some of this information is missing for the Repository 988 samples. The Contractor needs to possess and have expertise with the appropriate equipment to 989 conduct such activities as aliquoting the samples; storing them in a temperature and humidity- 990 controlled environment, and purging them with nitrogen gas in cases where surface oxidation 991 of the samples needs to be minimized. The Contractor will distribute the materials according to 992 the Classification and Labelling and Packaging Regulation, the UN Globally Harmonized 993 System of Classification and Labelling of Chemicals (GHS) requirements and regional 994 Transport Regulations. 995  The Contractor will provide the Test labs with clear instructions for how to store the materials 996  The Contractor will also send a report 2x/year to the Repository Sponsor (to be defined), 997 consisting in an inventory of repository materials and records of transactions that have taken 998 place over the last six months. The report will also include a financial statement showing 999 cumulative project expenses for the current reporting period, state possible problem areas or 1000 departures from the Scope of Work and/or budget, and measures undertaken to correct such 1001 problems or departures. 1002  The “physical repository” activity will be associated with the development of a publicly 1003 available database housing repeated measurements of these materials (with Shewhart charts). 1004 This could either be coordinated by the Contractor based on the samples present in the 1005 repository. Two or more test method capable laboratories will be asked to run aliquots of the 1006 Repository samples using the Test Guideline and to enter the results into the database. A 1007 minimum dataset for each sample would include triplicate measurements at two different 1008 loadings conducted at two independent laboratories. As more laboratories test the reference and 1009 proficiency materials, the new measurements will be added to the database.

1010 Samples:

1011  Manufacturing companies of the identified materials will be asked to provide a certain amount 1012 of powder (kilograms) or number of massive units (e.g. 100-200 of the same sample (e.g. same 1013 batch or lot number). The sample should correspond as much as possible to the form that is 1014 placed on the market or in which they can reasonably be expected to be used.

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1015  In the case of massive samples, it would also be appropriate to conduct the tests on polished 1016 surfaces. This would enable the Test laboratory to reuse the reference material, there would be 1017 less material to be discarded after use, i.e. lower cost, and less material that has to be stored at 1018 the physical repository; i.e. a more sustainable handling of resources. Likewise, powders should 1019 be stable and if the test lab follows the storage instructions provided by the Contractor, the 1020 powders could be stored at the test facility for future testing.

1021 For each sample the following information will be available:

1022 • Release data: A minimum dataset for each sample would include triplicate measurements at 1023 two different loadings (as per current Test Guideline) conducted at two independent 1024 laboratories. 1025 • Composition: actual chemical composition of the material as well as that specified in the 1026 Material Standard to which it conforms. This information should also include the level and 1027 type of impurities. 1028 • Information on the specific surface area (powders only), or geometric surface area (massives) 1029 and how they were measured. 1030 • Particle size distribution (powders only) [required information] using parameters such as 1031 d0.1, d0.5, and d0.9. Report method of assessment and if values are based on volume or 1032 number distribution and in which media (e.g. composition, ionic strength, pH) the size 1033 distribution was measured (e.g. air, water). 1034 • Information on morphology (shape of the particles) and metallurgical structure. 1035 • Information on manufacturing method (particularly for powder forms) with record of the 1036 mechanical and thermal history (indicating e.g. if atomization (water or air), chemical or 1037 grinding method was used). 1038 • Information on processing, preparation, surface finish, transport and storage, if available (e.g. 1039 heat treatment/fabrication history; metallurgical microstructure particularly for massive 1040 forms). 1041 • Information on surface charge (particles), surface composition, and other surface properties 1042 (e.g. surface oxidation for both massive and powder forms; surface finish particularly for 1043 massive forms), if available. If surface charge information is available, include the medium 1044 used to measure the surface charge (e.g. composition, ionic strength, pH). 1045 • Information on particle agglomeration behaviour in specified solutions and loadings, if 1046 available. 1047 • The surfaces of the reference materials and the test materials are to be treated in the same 1048 way to enable the calculation of relative metal release (bioaccessibility).

1049 Operational aspects:

1050 • The industry sector will pay a setup and maintenance fee for the storage of the samples 1051 • The samples would be shipped to the storage place by the commodity of the metal supplying 1052 the reference and/or proficiency materials. 1053 • Initial characterization of the material as well as initial 0.032M HCl tests at two separate 1054 laboratories to be covered by the commodity supplying the samples. 1055 • Access to the samples from interested sponsors/testing labs is free but they would pay the 1056 shipping costs.

1057 Timing:

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1058 • Identification of specific sample sources is ongoing and will continue with input from 1059 relevant experts. 1060 • Testing of repository samples will be conducted with the Test Guideline

1061 Materials that could be part of the repository include:

1062 Proficiency Materials:

1063 Proficiency materials are well characterized and defined materials (e.g. regarding elemental 1064 composition and particle size) that may or may not include the metal of interest but need to match the 1065 physical form of the test materials (e.g. samples of powder metals or metal compounds, embedded form 1066 and non-embedded massive forms of alloys).

1067  Massive 1068 o SS316 sheets: 68.1% Fe (balance), 16.2% Cr, 11.2% Ni, 2.27% Mo, 1.10% Mn, 0.65% 1069 Si, 0.25% Cu, ≤0.18% Co, 0.12% Al. 1070 o Brass Cu63/Zn37 discs: (63% copper, 37% zinc) 1071 1072 1073  Powders4 1074 o Cobalt metal (CAS 7440-48-4) 1075 o Lead metal powder (CAS 7439-92-1)

1076

1077 Reference Materials

1078 Reference materials are materials that contain the same metal as the test material (content >99%), with 1079 the selection of the most appropriate reference material(s) being dependent on the application and the 1080 characteristics of the test material.

1081

4 The exact samples, purity and particle size characteristics remain to be determined. These samples could also be used as a reference material for alloys.

30