ANNEX IIISupplementary provisions
ANNEX III B.46. IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST 1. METHOD 1.1. INTRODUCTION Skin irritation refers to the production of reversible damage to the skin following the application of a test substance for up to 4 hours [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS)](1). This Test Method provides an in vitro procedure that, depending on information requirements, may allow determining the skin irritancy of substances as a stand-alone replacement test within a testing strategy, in a weight of evidence approach (2). The assessment of skin irritation has typically involved the use of laboratory animals (See Method B.4)(3). In relation to animal welfare concerns, method B.4 allows for the determination of skin corrosion/irritation by applying a sequential testing strategy, using validated in vitro and ex vivo methods, thus avoiding pain and suffering of animals. Three validated in vitro Test Methods or Test Guidelines, B.40, B.40bis and TG 435 (4, 5, 6), are useful for the corrosivity part of the sequential testing strategy of B.4. This Test Method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermis keratinocytes as cell source, representative tissue and cytoarchitecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The procedure described under this Test Method allows the hazard identification of irritant substances in accordance with UN GHS category 2. This Test Method also includes a set of performance standards for the assessment of similar and modified reconstructed human epidermis based test methods (7). Prevalidation, optimisation and validation studies have been completed for two in vitro test methods (8, 9, 10, 11, 12, 13, 14, 15, 16, 17), commercially available as EpiSkin™ and EpiDerm™, using reconstructed human epidermis models. These references were based on R 38. Certain aspects of recalculation for the purposes of GHS are addressed in reference 25. Methods with a performance equivalent to EpiSkin™ (validated reference method 1) are recommended as a stand alone replacement test method for the rabbit in vivo test for classifying GHS category 2 irritant substances. Methods with a performance equivalent to EpiDerm™ (validated reference method 2) are only recommended as a screen test method, or as part of a sequential testing strategy in a weight of evidence approach, for classifying GHS category 2 irritant substances. Before a proposed in vitro reconstructed human epidermis model test for skin irritation can be used for regulatory purposes, its reliability, relevance (accuracy), and limitations for its proposed use should be determined to ensure that it is comparable to that of the validated reference method 1, in accordance with the performance standards set out in this Test Method (Appendix). Two other in vitro reconstructed human epidermis test methods, have been validated in accordance with the requirements under this Test Method, and show similar results as the validated reference method 1 (18). These are the modified EpiDerm™ test method (modified reference method 2) and the SkinEthic RHE™ test method (me-too method 1). 1.2. DEFINITIONS The following definitions are applied within this Test Method: Accuracy : The closeness of agreement between test method results and accepted reference values. It is a measure of test method performance and one aspect of relevance. The term is often used interchangeably with ‘concordance’ to mean the proportion of correct outcomes of a test method. Batch control substance : Benchmark substance producing a mid-range cell viability response of the tissue. Cell viability : Parameter measuring total activity of a cell population e.g. as ability of cellular mitochondrial dehydrogenases to reduce the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue;), which, depending on the endpoint measured and the test design used, correlates with the total number and/or vitality of living cells. ET 50 : The exposure time required to reduce cell viability by 50 % upon application of the marker substance at a specified, fixed concentration, see also IC 50 . False negative rate : The proportion of all positive substances falsely identified by a test method as negative. It is one indicator of test method performance. False positive rate : The proportion of all negative (non-active) substances that are falsely identified as positive. It is one indicator of test method performance. Infinite dose : Amount of test substance applied to the skin exceeding the amount required to completely and uniformly cover the skin surface. GHS (Globally Harmonized System of Classification and Labelling of Chemicals) : A system proposing the classification of substances and mixtures according to standardised types and levels of physical, health and environmental hazards, and addressing corresponding communication elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data sheets, so that to convey information on their adverse effects with a view to protect people (including employers, workers, transporters, consumers and emergency responders) and the environment (1) and implemented in the EU in Regulation (EC) No 1272/2008. IC 50 : The concentration at which a marker substance reduces the viability of the tissues by 50 % (IC 50 ) after a fixed exposure time, see also ET 50 . Performance standards : Standards, based on a validated reference method, that provide a basis for evaluating the comparability of a proposed test method that is mechanistically and functionally similar. Included are (I) essential test method components; (II) a minimum list of reference substances selected from among the substances used to demonstrate the acceptable performance of the validated reference method; and (III) the comparable levels of accuracy and reliability, based on what was obtained for the validated reference method, that the proposed test method should demonstrate when evaluated using the minimum list of reference substances. Reliability : Measures of the extent that a test method can be performed reproducibly within and between laboratories over time, when performed using the same protocol. It is assessed by calculating intra- and inter-laboratory reproducibility. Sensitivity : The proportion of all positive/active substances that are correctly classified by the test. It is a measure of accuracy for a test method that produces categorical results, and is an important consideration in assessing the relevance of a test method. Specificity : The proportion of all negative/inactive substances that are correctly classified by the test. It is a measure of accuracy for a test method that produces categorical results and is an important consideration in assessing the relevance of a test method. Skin irritation : The production of reversible damage to the skin following the application of a test substance for up to 4 hours. Skin irritation is a locally arising, non-immunogenic reaction, which appears shortly after stimulation (24). Its main characteristic is its reversible process involving inflammatory reactions and most of the clinical characteristic signs of irritation (erythema, oedema, itching and pain) related to an inflammatory process. 1.3. SCOPE AND LIMITATIONS A limitation of the reconstructed human epidermis tests falling under this Test Method is that they only classify substances as skin irritants according to UN GHS category 2. As they do not allow the classification of substances to the optional category 3 as defined in the UN GHS, all remaining substances will not be classified (no category). Depending on regulatory needs and possible future inclusion of new endpoints, improvements or development of new me-too tests, this Test Method may have to be revised. This Test method allows the hazard identification of irritant mono-constituent substances (19), but it does not provide adequate information on skin corrosion. Gases and aerosols cannot be tested, while mixtures have not been assessed yet in a validation study. 1.4. PRINCIPLE OF THE TEST The test substance is applied topically to a three-dimensional reconstructed human epidermis model, comprised of normal, human-derived epidermal keratinocytes, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo . The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability is measured by dehydrogenase conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; EINECS number 206-069-5, CAS number 298-93-1)], into a blue formazan salt that is quantitatively measured after extraction from tissues (20). Irritant substances are identified by their ability to decrease cell viability below defined threshold levels (i.e. ≤ 50 %, for UN GHS category 2 irritants). Substances that produce cell viabilities above the defined threshold level, will not be classified (i.e. > 50 %, no category). The reconstructed human epidermis model systems may be used to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water. Whenever possible, solids should be tested as a fine powder. Since 58 carefully selected substances, representing a wide spectrum of chemical classes, were included in the validation of the reconstructed human epidermis model test systems, the methods are expected to be generally applicable across chemical classes (16). The validation includes 13 GHS Cat. 2 irritants. It should be noted that non-corrosive acids, bases, salts and other inorganic substances were not included in the validation and some known classes of organic irritants such as hydroperoxides, phenols and surfactants were not included or were only included to a limited extent. 1.5. DEMONSTRATION OF PROFICIENCY Prior to routine use of a validated method that adheres to this Test Method, laboratories may wish to demonstrate technical proficiency, using the ten substances recommended in Table 1. Under this Test Method, the UN GHS optional category 3 is considered as no category. For novel similar (me-too) test methods developed under this Test Method that are structurally and functionally similar to the validated reference methods or for modifications of validated methods, the performance standards described in the Appendix to this Test Method should be used to demonstrate comparable reliability and accuracy of the new test method prior to its use for regulatory testing. Table 1 Proficiency Substances which are a subset of the Reference Substances listed in the Appendix Substance CAS Number In vivo score Physical state GHS category naphthalene acetic acid 86-87-3 0 S No Cat. isopropanol 67-63-0 0,3 L No Cat. methyl stearate 112-61-8 1 S No Cat. heptyl butyrate 5870-93-9 1,7 L Optional Cat. 3 hexyl salicylate 6259-76-3 2 L Optional Cat. 3 cyclamen aldehyde 103-95-7 2,3 L Cat. 2 1-bromohexane 111-25-1 2,7 L Cat. 2 butyl methacrylate 97-88-1 3 L Cat. 2 1-methyl-3-phenyl-1-piperazine 5271-27-2 3,3 S Cat. 2 Heptanal 111-71-7 4 L Cat. 2 1.6. DESCRIPTION OF THE METHOD The following is a description of the components and procedures of a reconstructed human epidermis model test for skin irritation assessment. A reconstructed human epidermis model can be constructed, prepared or obtained commercially (e.g. EpiSkin™, EpiDerm™ and SkinEthic RHE™). Standard test method protocols for EpiSkin™, EpiDerm™ and SkinEthic RHE™ can be obtained at [http://ecvam.jrc.ec.europa.eu](21, 22, 23). Testing should be performed according to the following: 1.6.1. Reconstructed Human Epidermis Model Components 1.6.1.1. General model conditions Normal 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. sodium dodecyl sulphate (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 % (IC 50 ) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50 % (ET 50 ) 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. 1.6.1.2. Functional model conditions 1.6.1.2.1. Viability The preferred assay for determining the magnitude of viability is the MTT (20). The optical density (OD) of the extracted (solubilised) dye from the tissue treated with the negative control (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. 1.6.1.2.2. Barrier function The 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 IC 50 or ET 50 . 1.6.1.2.3. Morphology Histological examination of the reconstructed skin/epidermis should be performed by appropriately qualified personnel demonstrating human skin/epidermis-like structure (including multilayered stratum corneum). 1.6.1.2.4. Reproducibility The results of the method using a specific model should demonstrate reproducibility over time, preferably by an appropriate batch control (benchmark) substance (see Appendix). 1.6.1.2.5. Quality controls (QC) of the model Each batch of the epidermal model used should meet defined production release criteria, among which those for viability (paragraph 1.6.1.2.1) and for barrier function (paragraph 1.6.1.2.2) are the most relevant. An acceptability range (upper and lower limit) for the IC 50 or the ET 50 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. Table 2 Examples of QC batch release criteria Lower acceptance limit Mean of acceptance range Upper acceptance limit Validated reference method 1 (18 hours treatment with SDS) IC 50 = 1,0 mg/ml IC 50 = 2,32 mg/ml IC 50 = 3,0 mg/ml Validated reference method 2 (1 % Triton X-100) ET 50 = 4,8 hr ET 50 = 6,7 hr ET 50 = 8,7 hr 1.6.1.3. Application of the Test and Control Substances A sufficient number of tissue replicates should be used for each treatment and for controls (at least three replicates per run). For liquid as well as solid substances, sufficient amount of test substance should be applied to uniformly cover the skin surface while avoiding an infinite dose (see 1.2 Definitions), i.e. a minimum of 25 μL/cm 2 or 25 mg/cm 2 should be used. For solid substances, the epidermis surface should be moistened with deionised or distilled water before application, to ensure good contact with the skin. Whenever possible, solids should be tested as a fine powder. At the end of the exposure period, the test substance should be carefully washed from the skin surface with aqueous buffer, or 0,9 % NaCl. Depending on the reconstructed human epidermis model used, the exposure period may vary between 15 to 60 minutes, and the incubation temperature between 20 and 37 °C. For details, see the Standard Operating Procedures for the three methods (21, 22, 23). Concurrent NC and positive controls (PC) should be used for each study to demonstrate that viability (NC), barrier function and resulting tissue sensitivity (PC) of the tissues are within a defined historical acceptance range. The suggested PC substance is 5 % aqueous SDS. The suggested NC substances are water or phosphate buffered saline (PBS). 1.6.1.4. Cell Viability Measurements The most important element of the test procedure is that viability measurements are not performed immediately after the exposure to the test substances, but after a sufficiently long post-treatment incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects. During the test optimisation phase (9, 10, 11, 12, 13), a 42 hours post-treatment incubation period proved to be optimal and was therefore used in the validation of the reference test methods. The MTT conversion assay is a quantitative validated method which should be used to measure cell viability. It is compatible with use in a three-dimensional tissue construct. The skin sample is placed in MTT solution of appropriate concentration (e.g. 0,3-1 mg/mL) for 3 hours. The precipitated blue formazan product is then extracted from the tissue using a solvent (e.g. isopropanol, acidic isopropanol), and the concentration of formazan is measured by determining the OD at 570 nm using a bandpass of maximum ±30 nm. Optical properties of the test substance or its chemical action on the MTT may interfere with the assay leading to a false estimate of viability (because the test substance may prevent or reverse the colour generation as well as cause it). This may occur when a specific test substance is not completely removed from the skin by rinsing or when it penetrates the epidermis. If the test substance acts directly on the MTT, is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement technique. For detailed description of how to test direct MTT reduction, please consult the test method protocol for the validated reference methods (21, 22, 23). Non-specific colour (NSC) due to these interferences should not exceed 30 % of NC (for corrections). If NSC > 30 %, the test substance is considered as incompatible with the test. 1.6.1.5. Assay Acceptability Criteria For each assay using valid batches (see paragraph 1.6.1.2.5), tissues treated with the NC should exhibit OD reflecting the quality of the tissues that followed all shipment and receipt steps and all the irritation protocol process. The OD values of controls should not be below historical established lower boundaries. Similarly, tissues treated with the PC, i.e. 5 % aqueous SDS, should reflect the sensitivity retained by tissues and their ability to respond to an irritant substance in the conditions of each individual assay (e.g. viability ≤ 40 % for the validated reference method 1, and ≤ 20 % for the validated reference method 2). Associated and appropriate measures of variability between tissue replicates should be defined (e.g. if standard deviations are used they should be ≤ 18 %). 2. DATA 2.1. DATA For each treatment, data from individual replicate test samples (e.g. OD values and calculated percentage cell viability data for each test substance, including classification) should be reported in tabular form, including data from repeat experiments as appropriate. In addition means ± standard deviation for each trial should be reported. Observed interactions with MTT reagent and coloured test substances should be reported for each tested substance. 2.2. INTERPRETATION OF RESULTS The OD values obtained with each test sample can be used to calculate the percentage of viability compared to NC, which is set at 100 %. The cut-off value of percentage cell viability distinguishing irritant from non-classified test substances and the statistical procedure(s) used to evaluate the results and identify irritant substances, should be clearly defined and documented, and proven to be appropriate. The cut-off values for the prediction of irritation associated with the validated reference methods is given below: The test substance is considered to be irritant to skin in accordance with UN GHS category 2: (i) if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %. The test substance is considered to have no category: (ii) if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %. 3. REPORTING 3.1. TEST REPORT The test report should include the following information: Test and Control Substances: — chemical name(s) such as IUPAC or CAS name and CAS number, if known, — purity and composition of the substance (in percentage(s) by weight), — physical-chemical properties relevant to the conduct of the study (e.g. physical state, stability and volatility, pH, water solubility if known), — treatment of the test/control substances prior to testing, if applicable (e.g. warming, grinding), — storage conditions. Justification of the skin model and protocol used. Test Conditions: — cell system used, — calibration information for measuring device, and bandpass used for measuring cell viability (e.g. spectrophotometer), — complete supporting information for the specific skin model used including its performance. This should include, but is not limited to: (i) viability; (ii) barrier function; (iii) morphology; (iv) reproducibility and predictivity; (v) quality controls (QC) of the model; — details of the test procedure used, — test doses used, duration of exposure and post treatment incubation period, — description of any modifications of the test procedure, — reference to historical data of the model. This should include, but is not limited to: (i) acceptability of the QC data with reference to historical batch data; (ii) acceptability of the positive and negative control values with reference to positive and negative control means and ranges, — description of evaluation criteria used including the justification for the selection of the cut-off point(s) for the prediction model. Results: — tabulation of data from individual test samples, — description of other effects observed. Discussion of the results. Conclusions. 4. REFERENCES 1. United Nations (UN) (2007). 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