{
  "url": "https://openlegislation.uk/eu-regulation-2009-761/annex-III/appendix",
  "json_url": "https://openlegislation.uk/eu-regulation-2009-761/annex-III/appendix.json",
  "legislation": "eur/2009/761",
  "legislation_title": "Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)",
  "legislation_url": "https://openlegislation.uk/eu-regulation-2009-761",
  "path": "annex/III/appendix",
  "kind": "appendix",
  "number": null,
  "heading": "Assessment of the performance characteristics of proposed in vitro reconstructed human epidermis models for skin irritation",
  "text": "INTRODUCTION\nProcedures proposed for use under this Test Method should be evaluated to determine their reliability and accuracy using substances representing the full range of Draize irritancy scores. When evaluated using the 20 recommended reference substances (Table 2), the proposed procedure should have reliability and accuracy values which are comparable to that of the validated reference method 1 (Table 3) (1). The accuracy and reliability standards that should be achieved are provided under II and III below. Non-classified and classified (UN GHS category 2) substances, representing relevant chemical classes are included, so that the reliability and performance (sensitivity, specificity, false negative rates, and false positive rates and accuracy) of the proposed test method can be compared to that of the validated reference method 1. The reliability of the test method, as well as its ability to correctly identify UN GHS category 2 irritant substances, should be determined prior to its use for testing new substances.\nPERFORMANCE STANDARDS\nThe Performance Standards comprise the following three elements I) Essential Test Method Components, II) Reference Substances and III) Defined Accuracy and Reliability Values (2). These Performance Standards are based on the Performance Standards defined after the completion of the ECVAM skin irritation validation study (3).\n(I)\nEssential Test Method Components\nGeneral model conditions\nNormal human keratinocytes should be used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) should be present under a functional stratum corneum. Stratum corneum should be multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic marker substances, e.g. SDS or Triton X-100. The barrier function may be assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50 % (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50 % (ET50) upon application of the marker substance at a specified, fixed concentration. The containment properties of the model should prevent the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure. The skin model should be free of contamination by bacteria, viruses, mycoplasma, or fungi.\nFunctional model conditions\nViability\nThe preferred assay for determining the magnitude of viability is the MTT (4). The OD of the extracted (solubilised) dye from the tissue treated with NC should be at least 20 fold greater than the OD of the extraction solvent alone. It should be documented that the tissue treated with NC is stable in culture (provide similar viability measurements) for the duration of the test exposure period.\nBarrier function\nThe stratum corneum and its lipid composition should be sufficient to resist the rapid penetration of cytotoxic marker substances, e.g. SDS or Triton X-100, as estimated by IC50 or ET50.\nMorphology\nHistological examination of the reconstructed skin/epidermis should be performed by appropriately qualified personnel demonstrating human skin/epidermis-like structure (including multilayered stratum corneum).\nReproducibility\nThe results of the method using a specific model should demonstrate reproducibility over time, preferably by an appropriate batch control (benchmark) substance (see definitions in section 1.2).\nQuality controls (QC) of the model\nEach batch of the epidermal model used should meet defined production release criteria, among which those for viability and for barrier function are the most relevant. An acceptability range (upper and lower limit) for the IC50 or the ET50 should be established by the skin model supplier (or investigator when using an in-house model). The barrier properties of the tissues should be verified by the laboratory after receipt of the tissues. Only results produced with qualified tissues can be accepted for reliable prediction of irritation effects. As an example, the acceptability ranges for the validated reference methods are given below.\nTable 1\nExamples of QC batch release criteria\nlower acceptance limit\nmean of acceptance range\nupper acceptance limit\nValidated reference method 1 (18 hours treatment with SDS)\nIC50 = 1,0 mg/ml\nIC50 = 2,32 mg/ml\nIC50 = 3,0 mg/ml\nValidated reference method 2 (1 % Triton X-100)\nET50 = 4,8 hr\nET50 = 6,7 hr\nET50 = 8,7 hr\n(II)\nReference Substances\nReference substances are used to determine if the reliability and accuracy of a proposed novel in vitro reconstructed human epidermis test method, proven to be structurally and functionally sufficiently similar to the validated reference methods, or representing a minor modification of a validated reference method, shows comparable performance to that of the validated reference method 1 (1). The 20 reference substances listed in Table 2 include substances representing different chemical classes of interest, as well as substances in UN GHS category 2. The substances included in this list comprise 10 UN GHS category 2 substances, 3 UN GHS optional category 3 substances and 7 non-categorised substances. Under this Test Method, the optional category 3 is considered as no category. These reference substances represent the minimum number of substances that should be used to evaluate the accuracy and reliability of a proposed reconstructed human epidermis test method for skin irritation. In situations where a listed substance is unavailable, other substances for which adequate in vivo reference data are available could be used. If desired, additional substances representing other chemical classes and for which adequate in vivo reference data are available may be added to the minimum list of reference substances to further evaluate the accuracy of the proposed test method.\nTable 2\nReference Substances for Determination of Accuracy and Reliability Values for Reconstructed Human Epidermis Skin Irritation Models\nThe 20 reference substances comprise a representative selection from the 58 substances which were originally used to validate reference method 1 (EpiSkin™). A complete list of test substances and the criteria for their selection are available (5).\nSubstance\nCAS No\nEINECS No\nPhysical state\nIn vivo score\nGHS in vitro cat.\nGHS in vivo cat.\n1-bromo-4-chlorobutane\n6940-78-9\n230-089-3\nL\n0\nCat. 2\nNo Cat.\nDiethyl phthalate\n84-66-2\n201-550-6\nL\n0\nNo Cat.\nNo Cat.\nNaphthalene acetic acid\n86-87-3\n201-705-8\nS\n0\nNo Cat.\nNo Cat.\nAllyl phenoxy-acetate\n7493-74-5\n231-335-2\nL\n0,3\nNo Cat.\nNo Cat.\nIsopropanol\n67-63-0\n200-661-7\nL\n0,3\nNo Cat.\nNo Cat.\n4-methyl-thio-benzaldehyde\n3446-89-7\n222-365-7\nL\n1\nCat. 2\nNo Cat.\nMethyl stearate\n112-61-8\n203-990-4\nS\n1\nNo Cat.\nNo Cat.\nHeptyl butyrate\n5870-93-9\n227-526-5\nL\n1,7\nNo Cat.\nOptional Cat. 3\nHexyl salicylate\n6259-76-3\n228-408-6\nL\n2\nNo Cat.\nOptional Cat. 3\nTri-isobutyl phosphate\n126-71-6\n204-798-3\nL\n2\nCat. 2\nOptional Cat. 3\n1-decanol\n112-30-1\n203-956-9\nL\n2,3\nCat. 2\nCat. 2\nCyclamen aldehyde\n103-95-7\n203-161-7\nL\n2,3\nCat. 2\nCat. 2\n1-bromohexane\n111-25-1\n203-850-2\nL\n2,7\nCat. 2\nCat. 2\n2-chloromethyl-3,5-dimethyl-4-methoxypyridine hydrochloride\n86604-75-3\n434-680-9\nS\n2,7\nCat. 2\nCat. 2\na-terpineol\n98-55-5\n202-680-6\nL\n2,7\nCat. 2\nCat. 2\ndi-n-propyl disulphide\n629-19-6\n211-079-8\nL\n3\nNo Cat.\nCat. 2\nButyl methacrylate\n97-88-1\n202-615-1\nL\n3\nCat. 2\nCat. 2\nBenzenethiol, 5-(1,1-dimethylethyl)-2-methyl\n7340-90-1\n438-520-9\nL\n3,3\nCat. 2\nCat. 2\n1-methyl-3-phenyl-1-piperazine\n5271-27-2\n431-180-2\nS\n3,3\nCat. 2\nCat. 2\nHeptanal\n111-71-7\n203-898-4\nL\n4\nCat. 2\nCat. 2\nThe substances listed in Table 2 provide a representative distribution of the 58 substances used in the ECVAM international skin irritation validation study (1). Their selection is based on the following criteria:\nthe substances are commercially available,\nthey are representative of the full range of Draize irritancy scores (from non-irritant to strong irritant),\nthey have a well-defined chemical structure,\nthey are representative of the validated method’s reproducibility and predictive capacity as determined in the ECVAM validation study,\nthey are representative of the chemical functionality used in the validation process,\nthey are not associated with an extremely toxic profile (e.g. carcinogenic or toxic to the reproductive system) and they are not associated with prohibitive disposal costs.\n(III)\nDefined Accuracy and Reliability Values\nThe performance (sensitivity, specificity, false negative rate, false positive rate and accuracy) of the proposed test method should be comparable to that of the validated reference method 1 (Table 3), i.e. sensitivity should be equal or higher (≥) than 80 %, specificity should be equal or higher (≥) than 70 %, and accuracy should be equal or higher (≥) than 75 %. The calculation of the performance should be done using all classifications obtained for the 20 substances in the different participating laboratories. The classification for each substance in each laboratory should be obtained by using the mean value of viability over the different runs performed (minimum three valid runs).\nTable 3\nPerformance of the Validated Reference Method 1\nTable 3 provides the performance of the validated reference method 1, with regard to its ability to correctly identify irritant substances (UN GHS category 2) and non-classified substances (no category including optional category 3) for the 58 and 20 Reference Substances (Table 2), respectively.\nEpiSkin™\nBased on 13 GHS cat. 2 irritants.\nBased on 45 GHS cat. 3 irritants or GHS no category chemicals.\nTest method\nNo. of Substances\nSensitivity\nSpecificity\nFalse Negative Rate\nFalse Positive rate\nAccuracy\nValidated Reference Method 1\n58\n87,2 %\n71,1 %\n12,8 %\n29,9 %\n74,7 %\nValidated Reference Method 1\n20\n90 %\n73,3 %\n10 %\n26,7 %\n81,7 %\nThe reliability of the proposed test method should be comparable to that of the validated reference methods.\nWithin-laboratory reproducibility\nAn assessment of within-laboratory variability should show a concordance of classifications (category 2/no category) obtained in different, independent test runs of the 20 Reference Substances within one single laboratory equal or higher (≥) than 90 %.\nBetween-laboratory reproducibility\nAn assessment of between-laboratory reproducibility is not essential if the proposed test method is to be used in one laboratory only. For methods to be transferred between laboratories, the concordance of classifications (category 2/no category) obtained in different, independent test runs of the 20 Reference Substances between preferentially a minimum of three laboratories should be equal or higher (≥) than 80 %.\nREFERENCES\n1.. Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet, R., Cotovió, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kandárová, H., Gamer, A., Remmele, M., Curren, R., Raabe, H., Cockshott, A., Gerner, I. and Zuang, V. (2007) The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Test. ATLA 35, 559-601.\n2.. OECD (2005) Guidance Document No. 34 on the validation and international acceptance of new or updated test methods for hazard assessment. OECD, Paris.\n3.. ECVAM (2007) Performance Standards for applying human skin models to in vitro skin irritation. Available under Download Study Documents, at http://ecvam.jrc.ec.europa.eu. Accessed on 27.10.2008.\n4.. Mosman, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55-63.\n5.. Eskes, C., Cole, T., Hoffmann, S., Worth, A., Cockshott, A., Gerner, I. & Zuang. V (2007) ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Selection of Test Chemicals. ATLA 35, 603-619.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "footnote",
      "label": "f1",
      "text": "The 20 reference substances comprise a representative selection from the 58 substances which were originally used to validate reference method 1 (EpiSkin™). A complete list of test substances and the criteria for their selection are available (5)."
    },
    {
      "type": "footnote",
      "label": "f2",
      "text": "Table 3 provides the performance of the validated reference method 1, with regard to its ability to correctly identify irritant substances (UN GHS category 2) and non-classified substances (no category including optional category 3) for the 58 and 20 Reference Substances (Table 2), respectively."
    },
    {
      "type": "footnote",
      "label": "f3",
      "text": "EpiSkin™"
    },
    {
      "type": "footnote",
      "label": "f4",
      "text": "Based on 13 GHS cat. 2 irritants."
    },
    {
      "type": "footnote",
      "label": "f5",
      "text": "Based on 45 GHS cat. 3 irritants or GHS no category chemicals."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eur/2009/761/annex/III/appendix",
    "retrieved_at": "2026-11-01T14:51:19.625001+00:00"
  },
  "licence": "Open Government Licence v3.0",
  "licence_url": "https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/",
  "attribution": "Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives."
}