OECD Dermal 402

OECD/OCDE439 Adopted: 22 July 2010© OECD, (2010) You are free to use this material for personal, non-commercial purposes without seeking prior consent from the OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written permission from the OECD.OECD GUIDELINE FOR THE TESTING OF CHEMICALSIn Vitro Skin Irritation: Reconstructed Human Epidermis Test MethodINTRODUCTION1. Skin irritation refers to the production of reversible damage to the skin following the application of a test chemical 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 Guideline (TG) provides an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with UN GHS and EU CLP Category 2 (1) (2) (3). For member countries or regions that do not adopt the optional UN GHS Category 3 (mild irritants), this Test Guideline can also be used to identify non-classified chemicals, i.e. UN GHS and EU CLP “No Category”(1)(3). Depending on the regulatory framework and the classification system in use, this test method may be used to determine the skin irritancy of chemicals as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy (4).2. The assessment of skin irritation has typically involved the use of laboratory animals [OECD TG 404; adopted in 1981 and revised in 1992 and 2002](4). In relation to animal welfare concerns, TG 404 was revised in 2002, allowing for the determination of skin corrosion/irritation by applying a tiered testing strategy, using validated in vitro or ex vivo test methods, thus avoiding pain and suffering of animals. Three validated in vitro test methods have been adopted as OECD TGs 430, 431 and 435 (5) (6) (7), to be used for the corrosivity part of the tiered testing strategy of TG 404 (4).3. This Test Guideline addresses the human health endpoint skin irritation. It is based on reconstructed human epidermis (RhE), which in its overall design (the use of human derived non-transformed epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis . This Test Guideline also includes a set of Performance Standards (PS)(Annex 2) for the assessment of similar and modified RhE-based test methods developed by EC-ECVAM (8), in accordance with the principles of Guidance Document No. 34 (9).4. There are three validated test methods that adhere to this Test Guideline. Prevalidation, optimisation and validation studies have been completed for an in vitro test method (10) (11) (12) (13) (14)(15) (16) (17) (18) (19) (20), using a RhE model, commercially available as EpiSkin™ (designated the Validated Reference Method – VRM). Two other commercially available in vitro skin irritation RhE test methods have shown similar results to the VRM according to PS-based validation (21), and these are the EpiDerm™ SIT (EPI-200) and the SkinEthic™ RHE test methods (22).5. Before a proposed similar or modified in vitro RhE test method other than the VRM, EpiDerm™ SIT (EPI-200) or SkinEthic™ RHE test methods can be used for regulatory purposes, its reliability, relevance (accuracy), and limitations for its proposed use should be determined in order to ensure that it can be regarded as similar to that of the VRM, in accordance with the requirements of the PS set out in this Test Guideline (Annex 2).439OECD/OCDE6. Definitions used are provided in Annex 1.INITIAL CONSIDERATIONS AND LIMITATIONS7. A limitation of the Test Guideline, as demonstrated by the validation study (16), is that it does not allow the classification of chemicals to the optional UN GHS Category 3 (mild irritants) (1). Thus, the regulatory framework in member countries will decide how this Test Guideline will be used. When used as a partial replacement test, follow-up in vivo testing may be required to fully characterize skin irritation potential (4). It is recognized that the use of human skin is subject to national and international ethical considerations and conditions.8. This Test Guideline addresses the in vitro skin irritation component of the tiered testing strategy of TG 404 on dermal corrosion/irritation (4). While this Test Guideline does not provide adequate information on skin corrosion, it should be noted that OECD TG 431 on skin corrosion is based on the same RhE test system, though using another protocol (6). This Test Guideline is based on RhE-models using human keratinocytes, which therefore represent in vitro the target organ of the species of interest. It moreover directly covers the initial step of the inflammatory cascade/mechanism of action (cell damage and tissue damage resulting in localised trauma) that occurs during irritation in vivo. A wide range of chemicals has been tested in the validation underlying this Test Guideline and the empirical database of the validation study amounted to 58 chemicals in total (16)(18)(23). The Test Guideline is applicable to 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 ground to a fine powder before application; no other pre-treatment of the sample is required. Gases and aerosols have not been assessed yet in a validation study (24). While it is conceivable that these can be tested using RhE technology, the current Test Guideline does not allow testing of gases and aerosols. It should also be noted that highly coloured chemicals may interfere with the cell viability measurements and need the use of adapted controls for corrections (see paragraphs 24-26).9. A single testing run composed of three replicate tissues should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5%, a second run should be considered, as well as a third one in case of discordant results between the first two runs.PRINCIPLE OF THE TEST10. The test chemical is applied topically to a three-dimensional RhE model, comprised of non-transformed human-derived epidermal keratinocytes, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.11. Chemical-induced skin irritation, manifested by erythema and oedema, is the result of a cascade of events beginning with penetration of the stratum corneum and damage to the underlying layers of keratinocytes. The dying keratinocytes release mediators that begin the inflammatory cascade which acts on the cells in the dermis, particularly the stromal and endothelial cells. It is the dilation and increased permeability of the endothelial cells that produce the observed erythema and oedema (24). The RhE-based test methods measure the initiating events in the cascade.12. Cell viability in RhE models is measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1], into a © OCDE, (2010) 2OECD/OCDE439 blue formazan salt that is quantitatively measured after extraction from tissues (25). Irritant chemicals areidentified by their ability to decrease cell viability below defined threshold levels (i.e.≤ 50%, for UN GHSCategory 2). Depending on the regulatory framework and applicability of the Test Guideline, chemicalsthat produce cell viabilities above the defined threshold level, may be considered non-irritants (i.e. > 50%,No Category).DEMONSTRATION OF PROFICIENCY13. Prior to routine use of any of the three validated test methods that adhere to this Test Guideline, laboratories should demonstrate technical proficiency, using the ten Proficiency Chemicals listed in Table1. For similar test methods developed under this Test Guideline or for modifications of any of the threevalidated test methods, the PS requirements described in Annex 2 of this Test Guideline should be metprior to using the test method for regulatory testing.14. As part of the proficiency exercise, it is recommended that the user verifies the barrier propertiesof the tissues after receipt as specified by the RhE model producer. This is particularly important if tissuesare shipped over long distance/time periods. Once a test method has been successfully established and proficiency in its use has been demonstrated, such verification will not be necessary on a routine basis. However, when using a test method routinely, it is recommended to continue to assess the barrier properties in regular intervals.The Proficiency Chemicals are a subset of the chemicals used in the validation study.2In vivo score in accordance with the OECD Test Guideline 404 (4).3Under this Test Guideline, the UN GHS optional Category 3 (mild irritants) (1) is considered as No Category.PROCEDURE3© OECD, (2010)439OECD/OCDE15. The following is a description of the components and procedures of a RhE test method for skin irritation assessment. A RhE model should be reconstructed, and can be in-house-prepared or obtained commercially. Standard Operating Procedures (SOPs) for the EpiSkin™, EpiDerm™ SIT (EPI-200) and SkinEthic™ RHE are available (26)(27)(28). Testing should be performed according to the following:R H E TEST METHOD COMPONENTSGeneral conditions16. Non-transformed human keratinocytes should be used to reconstruct 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 chemicals, e.g.sodium dodecyl sulphate (SDS) or Triton X-100. The barrier function should be demonstrated and may be assessed either by determination of the concentration at which a marker chemical 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 chemical at a specified, fixed concentration. The containment properties of the RhE 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 RhE model should be free of contamination by bacteria, viruses, mycoplasma, or fungi. Functional conditionsViability17. The assay used for determining the magnitude of viability is the MTT-assay (25). The RhE model users should ensure that each batch of the RhE model used meets defined criteria for the negative control (NC). The optical density (OD) of the extraction solvent alone should be sufficiently small, i.e. OD<0.1. An acceptability range (upper and lower limit) for the negative control OD values (In the Skin Irritation Test Method conditions) are established by the RhE model developer/supplier, and the acceptability ranges for the 3 validated test methods are given in Table 2. It should be documented that the tissues treated with NC are stable in culture (provide similar viability measurements) for the duration of the test exposure period.Table 2:Acceptability ranges for negative control OD valuesBarrier function18. The stratum corneum and its lipid composition should be sufficient to resist the rapid penetration of cytotoxic marker chemicals, e.g. SDS or Triton X-100, as estimated by IC50 or ET50 (Table 3).© OCDE, (2010) 4OECD/OCDE439Morphology19. Histological examination of the RhE model should be performed demonstrating human epidermis-like structure (including multilayered stratum corneum).Reproducibility20. The results of the positive and negative controls of the test method should demonstrate reproducibility over time.Quality control (QC)21. The RhE model developer/supplier should ensure and demonstrate that each batch of the RhEmodel used meets defined production release criteria, among which those for viability (paragraph 17),barrier function (paragraph 18) and morphology (paragraph 19) are the most relevant. These data shouldbe provided to the test method users, so that they are able to include this information in the test report. An acceptability range (upper and lower limit) for the IC50 or the ET50 should be established by the RhE modeldeveloper/supplier (or investigator when using an in-house model). Only results produced with qualifiedtissues can be accepted for reliable prediction of irritation classification. As an example, the acceptabilityranges for the three validated test methods are given in Table 3.Table 3:Examples of QC batch release criteriaApplication of the Test and Control Chemicals22. At least three replicates should be used for each test chemical and for the controls in each run.For liquid as well as solid chemicals, sufficient amount of test chemical should be applied to uniformlycover the epidermis surface while avoiding an infinite dose, i.e.a minimum of 25 μL/cm2 or 25 mg/cm2should be used. For solid chemicals, the epidermis surface should be moistened with deionised or distilledwater before application, to improve contact between the test chemical and the epidermis surface.Whenever possible, solids should be tested as a fine powder. At the end of the exposure period, the testchemical should be carefully washed from the epidermis surface with aqueous buffer, or 0.9% NaCl. Depending on which of the three validated RhE test methods is used, the exposure period varies between15 and 60 minutes, and the incubation temperature between 20 and 37°C. These exposure periods and temperatures are optimized for each RhE test method and represent the different intrinsic properties of thetest methods, for details, see the Standard Operating Protocols (SOPs) for the test methods (26)(27)(28).5© OECD, (2010)439OECD/OCDE23. Concurrent NC and positive controls (PC) should be used in each run to demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) of the tissues are within a defined historical acceptance range. The suggested PC chemical is 5% aqueous SDS. The suggested NC chemicals are water or phosphate buffered saline (PBS).Cell Viability Measurements24. The most important element of the test procedure is that viability measurements are not performed immediately after the exposure to the test chemicals, but after a sufficiently long post-treatment incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects. The test optimisation phase (11) (12) (13) (14) (15) demonstrated that a 42 hours post-treatment incubation period was optimal.25. The MTT assay is a validated quantitative method which should be used to measure cell viability under this Test Guideline. It is compatible with use in a three-dimensional tissue construct. The tissue 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 filter band pass of maximum ± 30 nm.26.Optical properties of the test chemical or its chemical action on the MTT may interfere with the assay leading to a false estimate of viability (because the test chemical may prevent or reverse the colour generation as well as cause it). This may occur when a specific test chemical is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If a test chemical acts directly on the MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test chemical interference with the viability measurement technique. Detailed description of how to correct direct MTT reduction and interferences by colouring agents is available in the SOPs for the three validated test methods (26)(27)(28).Acceptability Criteria27. For each test method using valid RhE model batches (see paragraph 21), tissues treated with the NC should exhibit OD reflecting the quality of the tissues that followed shipment, receipt steps and all protocol processes. Control OD values should not be below historically established boundaries. Similarly, tissues treated with the PC, i.e.5% aqueous SDS, should reflect their ability to respond to an irritant chemical under the conditions of the test method (26) (27) (28). Associated and appropriate measures of variability between tissue replicates should be defined (e.g.if standard deviations (SD) are used they should be within the 1-sided 95% tolerance interval calculated from historical data; for the VRM SD < 18%).Interpretation of Results and Prediction Model28. The OD values obtained with each test chemical can be used to calculate the percentage of viability normalised to NC, which is set to 100%. The cut-off value of percentage cell viability distinguishing irritant from non-classified test chemicals and the statistical procedure(s) used to evaluate the results and identify irritant chemicals should be clearly defined, documented, and proven to be appropriate. The cut-off values for the prediction of irritation are given below:The test chemical is considered to be irritant to skin in accordance with UN GHS Category 2 ifthe tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.© OCDE, (2010) 6OECD/OCDE4397© OECD, (2010)Depending on the regulatory framework in member countries, the test chemical may be considered as non-irritant DATA AND REPORTINGto skin in accordance with UN GHS No Category if the tissue viabilityafter exposure and post-treatment incubation is more than (>) 50%.Data29. For each run, data from individual replicate tissues (e.g. OD values and calculated percentage cell viability data for each test chemical, including classification) should be reported in tabular form, including data from repeat experiments as appropriate. In addition means ± SD for each run should be reported. Observed interactions with MTT reagent and coloured test chemicals should be reported for each tested chemical.Test Report30.The test report should include the following information: Test and Control Chemicals:- C hemical name(s) such as CAS name and number, if known;- P urity and composition of the chemical (in percentage(s) by weight);-Physical-chemical properties relevant to the conduct of the study (e.g. physical state, stability,volatility, pH and water solubility if known);-Treatment of the test/control chemicals prior to testing, if applicable (e.g. warming,grinding);- S torage conditions;Justification of the RhE model and protocol used Test Conditions:- C ell system used;- C omplete supporting information for the specific RhE model used including its performance.This should include, but is not limited to;i) viabilityii) barrier functioniii) morphologyiv) reproducibility and predictivityv) Quality controls (QC) of the model- D etails of the test procedure used;- T est doses used, duration of exposure and post treatment incubation period;- D escription of any modifications of the test procedure;- R eference 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 dataii) acceptability of the positive and negative control values with reference to positive andnegative control means and ranges439OECD/OCDE-D escription of evaluation criteria used including the justification for the selection of the cut-off point(s) for the prediction model;-Reference to historical control data;Results:-T abulation of data from individual test chemicals for each run and each replicatemeasurement;-I ndication of controls used for direct MTT-reducers and/or colouring test chemicals;-D escription of other effects observed;Discussion of the resultsConclusion© OCDE, (2010)8OECD/OCDE439LITERATURE1.UN (2009), United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Third revised edition, UN New York and Geneva. Available at:[/trans/danger/publi/ghs/ghs_rev03/03files_e.html]2.EC-ECVAM (2009), Statement on the “Performance under UN GHS of three in vitro assays forskin irritation testing and the adaptation of the Reference Chemicals and Defined Accuracy Values of theECVAM skin irritation Performance Standards”, issued by the ECVAM Scientific Advisory Committee(ESAC30), 9 April 2009. Available at: [http://ecvam.jrc.ec.europa.eu]3.EC (2008), REGULATION (EC) No 1272/2008 of the European Parliament and of the Councilof 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending andrepealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006. OfficialJournal of the European Union L353, 1-1355.4.OECD (2004), Acute Dermal Irritation/Corrosion, OECD Guideline for the Testing of Chemicals No. 404, OECD, Paris. Available at: [/env/testguidelines]5.OECD (2004), In Vitro Skin Corrosion: Transcutaneous Electrical Resistance (TER),OECDGuideline for the Testing of Chemicals No. 430, OECD, Paris. Available at:[/env/testguidelines]6.OECD (2004), In Vitro Skin Corrosion: Human Skin Model Test, OECD Guideline for theTesting of Chemicals No. 431, OECD, Paris. Available at: [/env/testguidelines]7.OECD (2006), In Vitro Membrane Barrier Test Method for Skin Corrosion, OECD Guideline forthe Testing of Chemicals No. 435, OECD, Paris. Available at: [/env/testguidelines]8.EC-ECVAM (2009), Performance Standards for in vitro skin irritation test methods based on Reconstructed human Epidermis (RhE)? Available at: [http://ecvam.jrc.ec.europa.eu]9.OECD (2005), Guidance Document on the Validation and International Acceptance of New orUpdated Test Methods for Hazard Assessment, OECD Series on Testing and Assessment No. 34, OECD,Paris. Available at: [/env/testguidelines]10.Fentem, J.H., Briggs, D., Chesné, C., Elliot, G.R., Harbell, J.W., Heylings, J.R., Portes, P.,Roguet, R., van de Sandt, J.J. M. and Botham, P. (2001), A prevalidation study on in vitro tests for acuteskin irritation, Results and evaluation by the Management Team, Toxicol. in Vitro 15, 57-93.11.Portes, P., Grandidier, M.-H., Cohen, C. and Roguet, R. (2002), Refinement of the EPISKINprotocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidationstudy, Toxicol. in Vitro 16, 765–770.12.Kandárová, H., Liebsch, M., Genschow, E., Gerner, I., Traue, D., Slawik, B. and Spielmann, H. (2004), Optimisation of the EpiDerm test protocol for the upcoming ECVAM validation study on in vitroskin irritation tests, ALTEX 21, 107–114.13.Kandárová, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, D. and Spielmann, H. (2005), The EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritationtests – An assessment of the performance of the optimised test, ATLA 33, 351-367.9© OECD, (2010)439OECD/OCDE14.Cotovio, J., Grandidier, M.-H., Portes, P., Roguet, R. and Rubinsteen, G. (2005), The in vitro acute skin irritation of chemicals: optimisation of the EPISKIN prediction model within the framework of the ECVAM validation process, ATLA 33, 329-349.15.Zuang, V., Balls, M., Botham, P.A., Coquette, A., Corsini, E., Curren, R.D., Elliot, G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., van De Sandt, J.J.M., Wiemann, C. and Worth, A. (2002), Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation, The European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2, ATLA 30, 109-129.16.Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet, R., Cotovio, 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, 559-601.17.Hoffmann, S. (2006), ECVAM skin irritation validation study phase II: Analysis of the primary endpoint MTT and the secondary endpoint IL1-α. Available at: [http://ecvam.jrc.ec.europa.eu]18.Eskes, C., Cole, T., Hoffmann, S., Worth, A., Cockshott, A., Gerner, I. and Zuang, V. (2007), The ECVAM international validation study on in vitro tests for acute skin irritation: selection of test chemicals, ATLA 35, 603-619.19.Cotovio, J., Grandidier, M.-H., Lelièvre, D., Roguet, R., Tinois-Tessonneaud, E. and Leclaire, J. (2007), In vitro acute skin irritancy of chemicals using the validated EPISKIN model in a tiered strategy - Results and performances with 184 cosmetic ingredients, AATEX, 14, 351-358.20.EC-ECVAM (2007), Statement on the validity of in vitro tests for skin irritation, issued by the ECVAM Scientific Advisory Committee (ESAC26), 27 April 2007. Available at: [http://ecvam.jrc.ec.europa.eu]21.EC-ECVAM (2007), Performance Standards for applying human skin models to in vitro skin irritation testing. Available at: [http://ecvam.jrc.ec.europa.eu]22.EC-ECVAM (2008), Statement on the scientific validity of in vitro tests for skin irritation testing, issued by the ECVAM Scientific Advisory Committee (ESAC29), 5 November 2008. Available at: [http://ecvam.jrc.ec.europa.eu]23.OECD (20xx), Explanatory background document to the OECD draft Test Guideline on in vitro skin irritation testing. To be published in OECD Series on Testing and Assessment, No. 1XX, OECD, Paris. Available at:24.Welss, T., Basketter, D.A. and Schröder, K.R. (2004), In vitro skin irritation: fact and future. State of the art review of mechanisms and models, Toxicol. in Vitro 18, 231-243.25.Mosmann, T. (1983), Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65, 55-63.26.EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals. Available at: [http://ecvam.jrc.ec.europa.eu]© OCDE, (2010)1027.EpiDerm™ SOP, Version 7.0 (Revised March 2009), Protocol for: In vitro EpiDerm™ skin irritation test (EPI-200-SIT), For use with MatTek Corporation's reconstructed human epidermal model EpiDerm (EPI-200). Available at: [http://ecvam.jrc.ec.europa.eu]28.SkinEthic™ RHE SOP, Version 2.0 (February 2009), SkinEthic skin irritation test-42bis test method for the prediction of acute skin irritation of chemicals: 42 minutes application + 42 hours post-incubation. Available at: [http://ecvam.jrc.ec.europa.eu]29.Harvell, J.D., Lamminstausta, K., and Maibach, H.I. (1995), Irritant contact dermatitis, In: Practical Contact Dermatitis, pp 7-18, (Ed. Guin J. D.). Mc Graw-Hill, New York.30.EC (2001), Commission Directive 2001/59/EC of 6 August 2001 adapting to technical progress for the 28th time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances, Official Journal of the European Union L225, 1-333.31.Basketter, D.A., York, M., McFadden, J.P. and Robinson, M.K. (2004), Determination of skin irritation potential in the human 4-h patch test. Contact Dermatitis 51, 1-4.32.Jirova, D., Liebsch, M., Basketter, D., Spiller, E., Kejlova, K., Bendova, H., Marriott, M. and Kandarova, H. (2007), Comparison of human skin irritation and photo-irritation patch test data with cellular in vitro assays and animal in vivo data, AATEX, 14, 359-365.33.Jírová, D., Basketter, D., Liebsch, M., Bendová, H., Kejlová, K., Marriott, M. and Kandárová, H. (2010), Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data, Contact Dermatitis, 62, 109-116.11。

D2-40D2-40: An Overview of a Unique Immunohistochemical MarkerIntroductionImmunohistochemistry (IHC) is a widely used technique in the field of pathology that allows for the visualization of specific target antigens within tissues. One such marker is D2-40, which has gained significant attention due to its unique properties and potential diagnostic implications. In this article, we will discuss the characteristics of D2-40, its applications, and its relevance in various pathological conditions.D2-40: A Brief BackgroundD2-40, also known as Podoplanin, is a protein encoded by the PDPN gene. Initially identified as a podocyte membrane antigen, it was shown to play a crucial role in the development of lymphatic vessels. D2-40 is characterized by its expression in endothelial cells of lymphatic vessels, certain subsets of macrophages, and some neoplastic cells of mesothelial and epithelial origin.Expression Patterns and LocalizationD2-40 has shown a consistent pattern of expression in lymphatic endothelial cells throughout various organs and tissues. By utilizing D2-40 immunohistochemistry, these lymphatic vessels can be easily identified and distinguished from blood vessels, which are negative for D2-40 expression. The use of D2-40 has proven to be particularly helpful in studying lymphatic metastasis and lymphatic invasion in various cancers, such as breast, lung, and colorectal carcinomas.Applications in Tumor Diagnosis and PrognosisOne of the most significant clinical applications of D2-40 is in tumor diagnosis and prognosis. Research has shown that D2-40 expression is associated with lymphatic vessel density and lymph node metastasis in several types of cancers. In breast cancer, for example, the presence of D2-40-positive tumor cells in the lymphatic vessels has been correlated with an increased risk of nodal metastasis. Similarly, in lung cancer, D2-40 expression has been associated with an unfavorable prognosis.D2-40 as a Diagnostic MarkerThe distinctive expression pattern of D2-40 has made it a valuable diagnostic marker, especially in tumors where lymphatic invasion is an important factor. In malignant mesothelioma, D2-40 has been shown to be highly sensitive and specific for differentiating it from reactive mesothelial hyperplasia. The absence of D2-40 staining in reactive mesothelial cells helps in ruling out the possibility of malignancy, making it a useful tool for accurate diagnosis.Distinctive Features of D2-40Apart from its diagnostic significance, D2-40 also possesses some unique features that make it an interesting marker to study. Research has shown that D2-40 is involved in the regulation of cell migration, invasion, and metastasis. It has been demonstrated that D2-40 interacts with the C-type lectin-like receptor 2 (CLEC-2), leading to platelet aggregation and lymphatic vessel remodeling. These findings suggest potential therapeutic implications for targeting D2-40 in metastatic disease.ConclusionD2-40, or Podoplanin, is a unique immunohistochemical marker with specific expression in lymphatic endothelial cells and some neoplastic cells. It has become an important diagnostic tool in various cancers, aiding in the identification of lymphatic invasion and predicting prognosis. Additionally, its distinct features and involvement in cellular processes make D2-40 an intriguing marker with potential therapeutic applications. Further research and studies are needed to fully explore the role of D2-40 in various pathological conditions and its potential as a therapeutic target.。

Date: 12 April 2010 NANOCYL TM NC7000 / MSDS / Page 1/6 Revision N°7 Supersedes: 31 August 2009NANOCYL TM NC7000 Material Safety Data Sheet1. Identification of the product and the companyTrade Name: NANOCYL TM NC7000Type of product: Carbon nanotubes powderCompany: Nanocyl saRue de l’Essor, 4B-5060 SambrevilleBelgiumPhone: + 32 71 750 380 (office hour)Fax: + 32 71 750 390E-mail: info@2. Hazards identificationThis product is not dangerous product for health and environment3. Composition/information on ingredientsComponents N° CAS % (wt.) Classification Synthetic graphite (Carbon nanotubes) 7782-42-5 90 % /Cobalt oxides 1308-06-1 < 1% Xi R42/43Others Proprietary 9% /4. First aid measuresAfter skin contact: Remove contaminated clothing immediately. Wash contacted skin areas with plenty of cold to lukewarm water and soap. If irritation develops, consult a physician.Eye contact: Hold the eyes open and rinse with water for a sufficiently long period of time (at least10 minutes). Obtain medical attention if pain, blurred vision, swelling, burning orredness persist.After inhalation: If dusts are inhaled, move the person into fresh air, keep warm and allow to rest. If breathing is difficult, oxygen may be administered and medical attention should beobtained.Date: 12 April 2010 NANOCYL TM NC7000 / MSDS / Page 2/6 Revision N°7 Supersedes: 31 August 2009Ingestion: Do not induce vomiting. If vomiting occurs spontaneously, keep head belowhips to prevent aspiration. Obtain medical attention.5. Fire-fighting measuresSuitable extinguishing media: Water fog, Foam, Carbon Dioxide, Dry Chemical.Specific hazards: /Protective equipments: Wear self-contained breathing apparatus. Wear suitable protective clothing. Combustion products: May form toxic fumes, carbon monoxide, carbon dioxide, metal oxides.6. Accidental release measuresPersonal precautions: Equip cleanup crew with proper protection (see chapter 8). Ensure adequateventilation/exhaust extraction. Prevent formation of explosive dust-airmixture.Environmental precautions: Collect for disposal. Avoid discharge to natural waters, sewers and biologicalwaste water treatment plants.Spill procedures: Collect the spill material using a vacuum with Hepa filter or damp sweep.Avoid formation of dust. Use sealable dedicated containers.7. Handling and storageHandling: Good ventilation of the workplace is required. Avoid formation of dust.Contact with skin and eyes and inhalation of dust must be avoided under allcircumstances. Handle in accordance with good industrial hygiene andsafety procedures. Wash with soap and water before eating, drinking,smoking, applying cosmetics, or using toilet facilities.Storage: Keep only in the original closed container in a dedicated place. Keepcontainers in a cool, dry place with adequate ventilation. Keep away fromopen flames and high temperature.Date: 12 April 2010 NANOCYL TM NC7000 / MSDS / Page 3/6 Revision N°7 Supersedes: 31 August 20098. Exposure controls / personal protectionPersonal protective equipment:Hand protection: Protecting gloves, for example in nitrile rubber.Skin and body protection: Wear gloves and other clothing as required to avoid contact.Respiratory protection: Wear suitable respiratory equipment with high efficiency dust cartridge at leasta P2 filter when directly exposed or handling the powder.Eye protection: Chemical goggles or safety glasses.In case of contact, ensure prompt removal from eyes, skin and clothing. Wash hands and other exposed areas with mild soap and water before eat, drink or smoke and leaving work. Facilities storing or utilizing this material should be equipped with an eyewash facility. Change contaminated clothing immediately.9. Physical and chemical propertiesForm and Color: black powderOdor: odorlessMelting point/range: not applicableBoiling point/range: not applicableVapor pressure: not applicableBulk density: ~ 60 g/LSolubility in water: insolubleViscosity: not applicablepH: not applicableExplosibility class: ST1 (VDI 2263)Explosive properties: Kst=42 bar.m/s (VDI 2263)Smouldering temperature: > 400°C (EN 50281-2-1)Minimum Ignition Energy: > 10J (VDI 2263)NOTE: The physical data presented above are typical values and should not be construed as a specification.Date: 12 April 2010 NANOCYL TM NC7000 / MSDS / Page 4/6 Revision N°7 Supersedes: 31 August 200910. Stability and reactivityStability: Stable under normal handling and storage conditions.Materials to avoid: Strong oxidizing and reducing agents.11. Toxicological informationAcute toxicity, oral: No toxicity.Method: Modified OECD 420Results based on a similar product: LD50 rat > 5,000 mg/kg.Method: OECD 423Acute toxicity, dermal: No toxicity.Method: Modified OECD 431Results based on a similar product: LD50 rat > 2,000 mg/kg.Method: OECD 402Inhalation: Concentration dependant effects.Method: Modified OECD 403 and OECD 413Skin irritation: No irritation and no corrosion.Method: Modified OECD 431Ingestion: No effect.Method: Modified OECD 420Cytotoxicity: No cytotoxicity.Method: Modified OECD 432Carcinogenicity: No sign of carcinogenicity.Method: Cell Transformation AssayDate: 12 April 2010 NANOCYL TM NC7000 / MSDS / Page 5/6 Revision N°7 Supersedes: 31 August 2009No carcinogenic response.Method: 2-year bioassay in the peritoneal cavity of the rat12. Ecological informationBiodegradability: Results based on a similar product: 0 %Method: OECD 301FToxicity to fish: Results based on a similar product: LC50 > 100 mg/l.Method: OECD 203Toxicity for daphnia: Results based on a similar product: EC50 > 100 mg/l.Method: Directive 92/69/EECToxicity for algae: Results based on a similar product: EC50 > 100 mg/l.Method: OECD 201Environmental precautions: Do not allow to enter ground soil, sewage, drains.13. Disposal considerationsProduct: Dispose in accordance with applicable international, national and local laws,ordinances and statutes. For disposal within the EC, use the appropriate codeaccording to the European Waste Catalogue (EWC).Container: Empty containers can be landfilled after have been emptied as thoroughly aspossible, when in compliance with the Environmental Protection Regulationand with local, state and federal regulations.14. Transport informationAccording to national and international guidelines, which regulate the road-, rail-, air-, and sea transport, this product is classified as not dangerous.Date: 12 April 2010 NANOCYL TM NC7000 / MSDS / Page 6/6 Revision N°7 Supersedes: 31 August 200915. Regulatory informationLabelling according to EEC DirectiveSymbol(s) : /R Phrase(s) : /S Phrase(s) : S7Keep container tightly closed.S22 Do not breathe dust.S29Do not empty into drains.S36/37/39 Wear suitable protective clothing, gloves and eye/faceprotection.This material is listed on the US Toxic Substances Control Act (TSCA) Inventory and the following chemical inventories: Canadian Domestic Substances List (DSL), European Inventory of Existing Commercial Chemical Substances (EINECS), Korean Existing Chemicals List (ECL), Australian Inventory of Chemical Substances (AICS), the Philippines Inventory of Chemicals and Chemical Substances (PICCS), and the Swiss Giftliste 1 Inventory of Notified New Substances. In addition, this substance is not regulated in Japan and excluded from the Japanese Chemical Substances Control Law according to the Japanese Ministry of Economy, Trade and Industry, formerly the Ministry of International Trade and Industry (MITI).16. Other informationR Phrase(s) components:R42/43 May cause sensitisation by inhalation and skin contact.The contents and format of this MSDS are in accordance with EEC Regulation 1907/2006 art. 31. Disclaimer: Nanocyl s.a. provides the information contained herein in good faith and makes no representation as to its comprehensiveness or accuracy. This document is intended only as a guide to the appropriate precautionary handling of the material by a properly trained person using this product. Individuals receiving the information must exercise their independent judgment in determining its appropriateness for a particular purpose. As the conditions or methods of use are beyond our control, we do not assume any responsibility and expressly disclaim any liability for any use of this material.。

SAFETY DATA SHEETIssuing Date: 26-Mar-2015Revision Date: 14-May-2018Version 21. IDENTIFICATIONProduct Name Ultra TideÒ Laundry DetergentProduct Code(s)5-80Product Identifier96733935_PROF_NGProduct Type:Finished Product - Professional Use OnlyRecommended Use Laundry Care.Restrictions on Use Use only as directed on label.Details of the supplier of the safety data sheet Procter & Gamble Professional 1 P&G PlazaCincinnati, Ohio 45202 Procter & Gamble Inc.P.O. Box 355, Station A Toronto, ON M5W 1C51-800-332-7787E-mail AddressEmergency Telephone Transportation (24 HR)CHEMTREC - 1-800-424-9300(U.S./ Canada) or 1-703-527-3887Mexico toll free in country: 800-681-95312. HAZARD IDENTIFICATIONThis product is classified under 29CFR 1910.1200(d) and the Canadian Hazardous Products Regulation as follows:. Hazard CategoryAcute toxicity - Oral Category 4Eye Damage / Irritation Category 2BSignal Word WARNINGHazard Statements Causes eye irritationHarmful if swallowedHazard pictogramsPrecautionary Statements Wash hands thoroughly after handling3. COMPOSITION/INFORMATION ON INGREDIENTSIngredients are listed according to 29CFR 1910.1200 Appendix D and the Canadian Hazardous Products Regulation Additional informationActual concentrations withheld as trade secret in accordance with Canadian HPR4. FIRST AID MEASURESFirst aid measures for different exposure routes Eye contactRinse with plenty of water. Get medical attention immediately if irritation persists.Do not eat, drink or smoke when using this productPrecautionary Statements -ResponseIF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, ifpresent and easy to do. Continue rinsingIf eye irritation persists: Get medical advice/attention IF SWALLOWED:Rinse with plenty of water Drink 1 or 2 glasses of waterCall a POISON CENTER or doctor/physician if you feel unwellPrecautionary Statements - Storage NonePrecautionary Statements - Disposal Dispose of contents/container in accordance with local regulation Hazards not otherwise classified (HNOC)NoneChemical Name SynonymsTrade SecretCAS-No Weight-%Sodium carbonateCarbonic acid sodiumsalt (1:2)Yes497-19-830 - 60Silicic acid, aluminum sodium salt Silicic acid, aluminumsodium saltYes 1344-00-910 - 30Benzenesulfonic acid,mono-C10-16-alkyl derivs., sodium salts Benzenesulfonic acid,mono-C10-16-alkylderivs., sodium saltsYes 68081-81-2 5 - 10Carbonic acid disodium salt, compd.with hydrogen peroxide Carbonic acid disodiumsalt, compd. withhydrogen peroxideYes 15630-89-4 3 - 7Silicic acid, sodium salt Silicic acid, sodium salt Yes 1344-09-8 1 - 5Sulfuric acid, mono-C14-18-alkyl esters, sodium salts Sulfuric acid,mono-C14-18-alkylesters, sodium saltsYes 68081-98-1 1 - 5Sodium 2-(nonanoyloxy)benzenesulfonate Nonanoic acid,sulfophenyl ester,sodium salt (1:1)Yes 91125-43-8 1 - 5Heptadecanol, hydrogen sulfate,branched, sodium salt Heptadecanol, hydrogensulfate, branched,sodium saltYes 343978-15-4 1 - 5Poly(oxy-1,2-ethanediyl),alpha-sulfo-omega-hydroxy-,C10-16-alkyl ethers, sodium salts Poly(oxy-1,2-ethanediyl),alpha-sulfo-omega-hydro xy-, C10-16-alkyl ethers,sodium saltsYes68585-34-21 - 5Hexadecanol, hydrogen sulfate,branched, sodium salt Hexadecanol, hydrogensulfate, branched,sodium saltYes 343978-24-50.5 - 1.5Glycine,N,N-bis[2-[bis(carboxymethyl)amino]et hyl]-, sodium salt (1:5)Glycine,N,N-bis[2-[bis(carboxymethyl)amino]ethyl]-,sodium salt (1:5)Yes140-01-20.1 - 1Skin contact Rinse with plenty of water. Get medical attention if irritation develops and persists. Ingestion Rinse mouth. Drink 1 or 2 glasses of water. Do NOT induce vomiting. Get medical attentionimmediately if symptoms occur.Inhalation Move to fresh air. If symptoms persist, call a physician.Most important symptoms/effects,acute and delayedNone under normal use conditions.F. Indication of immediate medical attention and special treatment needed, if necessaryNotes to Physician Treat symptomatically.5. FIRE-FIGHTING MEASURESSuitable extinguishing media Dry chemical, CO 2, alcohol-resistant foam or water spray.Unsuitable Extinguishing Media None.Special hazard None known.Special protective equipment for fire-fighters As in any fire, wear self-contained breathing apparatus pressure-demand, MSHA/NIOSH (approved or equivalent) and full protective gear.Specific hazards arising from thechemicalNone.6. ACCIDENTAL RELEASE MEASURESPersonal precautions, protective equipment and emergency proceduresPersonal precautions Use personal protective equipment. Do not get in eyes, on skin, or on clothing.Advice for emergency responders Use personal protective equipment as required.Methods and materials for containment and cleaning upMethods for containment Prevent dust cloud. Prevent further leakage or spillage if safe to do so.Methods for cleaning up Sweep up and shovel into suitable containers for disposal. Dispose of in accordance withlocal regulations.7. HANDLING AND STORAGEPrecautions for safe handlingAdvice on safe handling Use personal protective equipment as required. Keep container closed when not in use.Never return spills in original containers for re-use. Keep out of the reach of children. Conditions for safe storage, including any incompatibilitiesStorage Conditions Keep containers tightly closed in a dry, cool and well-ventilated place.Incompatible products None known.8. EXPOSURE CONTROLS/PERSONAL PROTECTIONControl parametersExposure Guidelines.Chemical Name CAS-No Alberta Quebec Ontario TWAEV British Columbia1344-00-9TWA: 1 mg/m3TWA: 1.0 mg/m3 Silicic acid, aluminum sodiumsaltNo relevant exposure guidelines for other ingredientsExposure controlsEngineering Measures Distribution, Workplace and Household Settings:Ensure adequate ventilationProduct Manufacturing Plant (needed at Product-Producing Plant ONLY):Where reasonably practicable this should be achieved by the use of local exhaustventilation and good general extractionPersonal Protective EquipmentEye Protection Distribution, Workplace and Household Settings:No special protective equipment requiredProduct Manufacturing Plant (needed at Product-Producing Plant ONLY):Use appropriate eye protectionHand Protection Distribution, Workplace and Household Settings:No special protective equipment requiredProduct Manufacturing Plant (needed at Product-Producing Plant ONLY):Protective glovesSkin and Body Protection Distribution, Workplace and Household Settings:No special protective equipment requiredProduct Manufacturing Plant (needed at Product-Producing Plant ONLY):Wear suitable protective clothingRespiratory Protection Distribution, Workplace and Household Settings:No special protective equipment requiredProduct Manufacturing Plant (needed at Product-Producing Plant ONLY):In case of inadequate ventilation wear respiratory protection9. PHYSICAL AND CHEMICAL PROPERTIESPhysical State @20°C SolidAppearance white powderOdor PerfumeOdor threshold No information availableProperty Values NotepH value10 - 11.5(as 1% solution)Melting Point / Freezing Point No information availableBoiling point / boiling range No information availableFlash point No information available.Evaporation rate No information availableFlammability (solid, gas)No information availableVOC Content (%)Products comply with US state and federal regulations for VOC content in consumer products.10. STABILITY AND REACTIVITYReactivity None under normal use conditions.StabilityStable under normal conditions.Hazardous polymerization Hazardous polymerization does not occur.Hazardous Reactions None under normal processing.Conditions to Avoid None under normal processing.Incompatible materialsNone in particular.Hazardous Decomposition Products None under normal use conditions.11. TOXICOLOGICAL INFORMATIONProduct InformationInformation on likely routes of exposure Inhalation No known effect.Skin contact No known effect.Ingestion May be harmful if swallowed.Eye contactIrritating to eyes.Delayed and immediate effects as well as chronic effects from short and long-term exposure Acute toxicityMay be harmful if swallowed.Skin corrosion/irritationNo known effect.Serious eye damage/eye irritation Irritating to eyes.Skin sensitizationNo known effect.Respiratory sensitization No known effect.Germ cell mutagenicity No known effect.Neurological Effects No known effect.Reproductive toxicity No known effect.Developmental toxicity No known effect.TeratogenicityNo known effect.STOT - single exposure No known effect.STOT - repeated exposure No known effect.Target Organ Effects No known effect.Aspiration hazard No known effect.CarcinogenicityNo known effect.Component InformationChemical Name CAS-No Oral LD50Dermal LD50Inhalation LC50Sodium carbonate497-19-82800 mg/kg bw > 2000 mg/kg bw (US EPA 16 CFR 1500.40)-Silicic acid, aluminum sodium salt1344-00-9> 10000 mg/kg bw> 5000 mg/kg bw (//OECD > 2.08 mg/L air (//OECDFlammability Limits in AirUpper flammability limitNo information available Lower Flammability LimitNo information available Vapor pressureNo information available Vapor densityNo information available Relative density0.55Water solubility100%Partition coefficient: n-octanol/water No information availableAutoignition temperatureNo information available .Decomposition temperatureNo information available .ViscosityNo information available(//OECD 401)402)403)Carbonic acid disodium salt, compd.with hydrogen peroxide 15630-89-4893 mg/kg bw (US EPA1984)> 2000 mg/kg bw (USEPA)-Silicic acid, sodium salt1344-09-83400 mg/kg bw (OECD401)> 5000 mg/kg bw-Poly(oxy-1,2-ethanediyl),alpha-sulfo-omega-hydroxy-,C10-16-alkyl ethers, sodium salts68585-34-2>2001 mg/kg--Glycine,N,N-bis[2-[bis(carboxymethyl)amino ]ethyl]-, sodium salt (1:5)140-01-2> 5000 mg/kg bw (OECD401)> 2000 mg/kg bw (OECD402)-12. ECOLOGICAL INFORMATIONEcotoxicityThe product is not expected to be hazardous to the environment.Persistence and degradability No information available.Bioaccumulative potential No information available.Mobility No information available.Other adverse effects No information available.13. DISPOSAL CONSIDERATIONS Waste treatment methodsWaste from Residues/Unused Products Disposal should be in accordance with applicable regional, national and local laws and regulations.Contaminated packaging Disposal should be in accordance with applicable regional, national and local laws andregulations.California Hazardous Waste Codes(non-household setting)33114. TRANSPORT INFORMATIONDOT Not regulatedIMDG Not regulatedIATA Not regulated15. REGULATORY INFORMATIONU.S. Federal RegulationsSARA 313Section 313 of Title III of the Superfund Amendments and Reauthorization Act of 1986 (SARA). This product does not contain any chemicals which are subject to the reporting requirements of the Act and Title 40 of the Code of Federal Regulations, Part 372CERCLAThis material, as supplied, contains one or more substances regulated as a hazardous substance under the Comprehensive Environmental Response Compensation and Liability Act (CERCLA) (40 CFR 302):.Chemical Name CAS-No HazardousSubstances RQs Extremely HazardousSubstances RQsCERCLA/SARA 302TPQSodium hydroxide1310-73-21000 lb-Clean Air Act, Section 112 Hazardous Air Pollutants (HAPs) (see 40 CFR 61)This product does not contain any substances regulated as hazardous air pollutants (HAPS) under Section 112 of the Clean Air Act Amendments of 1990.Clean Water ActThis product contains the following substances which are regulated pollutants pursuant to the Clean Water Act (40 CFR 122.21 and 40 CFR 122.42):.Chemical Name CAS-No CWA - ReportableQuantities CWA - ToxicPollutantsCWA - PriorityPollutantsCWA - HazardousSubstancesSodium hydroxide1310-73-21000 lb--X California Proposition 65This product is not subject to warning labeling under California Proposition 65.U.S. State Regulations (RTK).Chemical Name CAS-No Massachusetts Sulfuric acid sodium salt (1:2)7757-82-6XChemical Name CAS-No Pennsylvania Sulfuric acid sodium salt (1:2)7757-82-6XSodium hydroxide1310-73-2XInternational InventoriesUnited StatesAll intentionally-added components of this product(s) are listed on the US TSCA Inventory.CanadaThis product is in compliance with CEPA for import by P&G.LegendUnited States Toxic Substances Control Act Section 8(b) Inventory (TSCA)CEPA - Canadian Environmental Protection Act16. OTHER INFORMATIONHMIS Ratings HMIS RatingsHealth hazard1Flammability1Physical hazard0Health hazard1Flammability1Instability0Issuing Date:26-Mar-2015Revision Date:14-May-2018Disclaimer.The information provided in this Safety Data Sheet is correct to the best of our knowledge, information and belief at the date of its publication. The information given is designed only as a guidance for safe handling, use, processing, storage, transportation, disposal and release and is not to be considered a warranty or quality specification. The information relates only to the specific material designated and may not be valid for such material used in combination with any other materials or in any process, unlessspecified in the textEnd of SDS。