Skin Irritation Hazard of Chemicals Assessed by the EpiSkin™ In Vitro Test Method

  • Nathalie AlépéeEmail author
  • Marie Hélène Grandidier
  • José Cotovio


The EpiSkin™ skin irritation method is an internationally accepted test method used to detect the skin irritation properties of chemicals. The EpiSkin™ skin irritation test method has been shown to be robust and transferable. The reproducibility and predictivity of the EpiSkin™ assay have been evaluated in validation studies organized with three laboratories, and with a large set of 122 chemicals through a systematic in vivo Draize comparative evaluation for safety assessment, that delivered results that matched very well with corresponding in vivo data showing 75% for specificity (n = 65), 94% for sensitivity (n = 57) and 84% for accuracy (n = 122). It could be regarded as a primary source of information concerning the skin irritation properties of the tested chemical and be used in combination with a battery of assays, such as the EpiSkin™ skin corrosion method, in any initiative aimed at developing integrated testing strategies for full evaluation of local skin effects.


Skin irritation In vitro Reconstructed human epidermis EpiSkin™ OECD TG 439 


  1. 1.
    EC. REGULATION (EC) No. 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No. 793/93 and Commission Regulation (EC) No. 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. Off J Eur Union. 2006;L396:1–1355.Google Scholar
  2. 2.
    EC. Regulation (EC) No. 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products. Off J Eur Union. 2009;L342:59–209. Google Scholar
  3. 3.
    Draize JH, Woodard G, Calvery HO. Methods for the study of irritation and toxicity of substances applied directly on the skin and mucous membranes. J Pharmacol Exp Ther. 1944;82:377–90.Google Scholar
  4. 4.
    OECD. Guideline for the testing of chemicals, No. 431. In vitro skin corrosion: human skin model test, 8pp. Paris, France: OECD. 2015. Available at
  5. 5.
    OECD. Explanatory background document to the OECD draft Test Guideline on in vitro skin irritation testing. Published in OECD Series on Testing and Assessment, No. 137, OECD, Paris. 2010. Available at
  6. 6.
    Weil CS, Scala RA. Study of intra-and interlaboratory variability in the results of rabbit eye and skin irritation tests. Toxicol Appl Pharmacol. 1971;19:276–360.CrossRefPubMedGoogle Scholar
  7. 7.
    EC. Regulation (EC). Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. Off J Eur Union. 1986;L358:0001–28.Google Scholar
  8. 8.
    EC. Regulation (EC). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes Text with EEA relevance. Off J Eur Union. 2010;L276:33–79.Google Scholar
  9. 9.
    United Nations (UN). Globally harmonized system of classification and labelling of chemicals (GHS). 6th revised ed; 2015. UN New York and Geneva, 2013Google Scholar
  10. 10.
    EC. REGULATION (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No. 1907/2006. Off J Eur Union. 2008;L353:1–1355.Google Scholar
  11. 11.
    Emmett EA. Toxic responses of the skin. In: Klaassen CD, Amdur MO, Doull J, editors. Casarett and Dull’s toxicology. New York, NY, USA: Macmillan; 1986. p. 412–31.Google Scholar
  12. 12.
    Eskes C, Detappe V, Koëter H, Kreysa J, Liebsch M, Zuang V, Amcoff P, Barroso J, Cotovio J, Guest R, Hermann M, Hoffmann S, Masson P, Alépée N, Arce LA, Brüschweiler B, Catone T, Cihak R, Clouzeau J, D'Abrosca F, Delveaux C, Derouette JP, Engelking O, Facchini D, Fröhlicher M, Hofmann M, Hopf N, Molinari J, Oberli A, Ott M, Peter R, Sá-Rocha VM, Schenk D, Tomicic C, Vanparys P, Verdon B, Wallenhorst T, Winkler GC, Depallens O. Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations. Regul Toxicol Pharmacol. 2012;62:393–403.CrossRefPubMedGoogle Scholar
  13. 13.
    OECD. In vitro skin irritation: reconstructed human epidermis test method. OECD guideline for the testing of chemicals No. 439, OECD, Paris. Originally adopted in 2010. 2015. Available at
  14. 14.
    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, Zuang V. 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. 2007;35:559–601.PubMedPubMedCentralGoogle Scholar
  15. 15.
    EURL ECVAM Statement on the validity of in vitro tests for skin irritation, issued by the ECVAM Scientific Advisory Committee (ESAC26), 27 Apr 2007. Available at
  16. 16.
    EURL-ECVAM Statement on the “Performance under UN GHS of three in vitro assays for skin irritation testing and the adaptation of the Reference Chemicals and Defined Accuracy Values of the ECVAM skin irritation Performance Standards”, issued by the ECVAM Scientific Advisory Committee (ESAC30), 9 Apr 2009. Available at
  17. 17.
    Basketter DA, York M, McFadden JP, Robinson MK. Determination of skin irritation potential in the human 4-h patch test. Contact Dermatitis. 2004;51:1–4.CrossRefPubMedGoogle Scholar
  18. 18.
    Jirova D, Liebsch M, Basketter D, Spiller E, Kejlova K, Bendova H, Marriott M, Kandarova H. Comparison of human skin irritation and photo-irritation patch test data with cellular in vitro assays and animal in vivo data. AATEX. 2007;14(Special Issue):359–65.Google Scholar
  19. 19.
    Basketter DA, Whittle E, Griffiths HA, York M. The identification and classification of skin irritation hazard by human patch test. Food Chem Toxicol. 1994;32:769–75.CrossRefPubMedGoogle Scholar
  20. 20.
    Basketter DA, Whittle E, Chamberlain M. Identification of irritation and corrosion hazards to skin: an alternative strategy to animal testing. Food Chem Toxicol. 1994;32:539–42.CrossRefPubMedGoogle Scholar
  21. 21.
    Basketter DA, Chamberlain M, Griffiths HA, York M. The classification of skin irritants by human patch test. Food Chem Toxicol. 1997;35:845–52.CrossRefPubMedGoogle Scholar
  22. 22.
    Basketter DA, Jirova J, Kandarova H. Review of skin irritation/corrosion Hazards on the basis of human data: a regulatory perspective. Interdiscip Toxicol. 2012;5(2):98–104.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Robinson MK, McFadden JP, Basketter DA. Validity and ethics of the human 4 hour patch test as an alternative method to assess acute skin irritation potential. Contact Dermatitis. 2001;45:1–12.CrossRefPubMedGoogle Scholar
  24. 24.
    Jirova D, Basketter D, Liebsch M, Bendova H, Keilova K, Marriott M, Kandarova H. Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data. Contact Dermatitis. 2010;6:109–16.CrossRefGoogle Scholar
  25. 25.
    Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63.CrossRefGoogle Scholar
  26. 26.
    EpiSkin™ SOP, Version 1.8, ECVAM skin irritation validation study: validation of the EpiSkin™ test method 15 min–42 hours for the prediction of acute skin irritation of chemicals. 2009. Available at
  27. 27.
    OECD. In vitro skin corrosion: reconstructed human epidermis (RHE) test method. OECD guideline for the testing of chemicals. Originally adopted in 2004, OECD, Paris. 2016. Available at
  28. 28.
    OECD. Guidance document on integrated approaches to testing and assessment of skin irritation/corrosion, series on testing and assessment, No. 203, OECD, Paris. 2014. Available at
  29. 29.
    Alépée N, Grandidier MH, Cotovio J. Sub-categorisation of skin corrosive chemicals by the EpiSkin™ reconstructed human epidermis skin corrosion test method according to UN GHS: Revision of OECD Test Guideline 431. Toxicol In Vitro. 2014;28:131–45.CrossRefPubMedGoogle Scholar
  30. 30.
    FDA. Guidance for industry: bioanalytical method validation. U.S. Department of Health and Human Services, Food and Drug Administration. May 2001. Available at
  31. 31.
    Alépée N, Barroso J, De Smedt A, De Wever B, Hibatallah J, Klaric M, Mewes KR, Millet M, Pfannenbecker U, Tailhardat M, Templier M, McNamee P. Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro reconstructed human tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals. Toxicol In Vitro. 2015;29:741–61.CrossRefPubMedGoogle Scholar
  32. 32.
    Scientific Committee on Consumer Safety (SCCS), 2010. Memorandum (addendum) on the in vitro test EPISKIN™ for skin irritation testing. SCCS/1392/10SCCS/1392/10. Available at
  33. 33.
    Alépée N, Hibatallah J, Klaric M, Mewes KR, Pfannenbecker U, McNamee P. Assessment of cosmetic ingredients in the in vitro reconstructed human epidermis test method EpiSkin™ using HPLC/UPLC-spectrophotometry in the MTT-reduction assay. Toxicol In Vitro. 2016;33:105–17.CrossRefPubMedGoogle Scholar
  34. 34.
    Scientific Committee on Consumer Safety (SCCS). The sccs notes of guidance for the testing of cosmetic ingredients and their safety evaluation 9th revision. SCCS/1564/15 Revised version of 25 Apr 2016. Available at
  35. 35.
    Nickoloff BJ, Turka LA. Keratinocytes: key immunocytes of the integument. Am J Pathol. 1993;143:325–31.PubMedPubMedCentralGoogle Scholar
  36. 36.
    Williams IR, Kupper TS. Immunity at the surface: Homeostatic mechanisms of the skin immune system. Life Sci. 1996;58:1485–507.CrossRefPubMedGoogle Scholar
  37. 37.
    Cohen C, Selvi-Bignon C, Barbuer A, Rougier A, Lacheretz F, Roguet R. Measurement of pro inflammatory mediators production by cultured keratinocytes: a predictive assessment of cutaneous irritation. In Alternative Methods in Toxicology, Vol. 10. In: Rougier A, Goldberg AM, Maibach HI, editors. Vitro Skin Toxicology. New York, NY, USA: Mary Ann Liebert; 1994. p. 83–96.Google Scholar
  38. 38.
    Roguet, R., and Cotovió, J.. Interleukin 1 alpha (IL-1α). Rationale and use. Role in inflammation/irritation processes. Measurement conditions specificities. Document provided on 30 March 2007 for consideration by the SIVS Management Team and Peer Review Panel. 2007Google Scholar
  39. 39.
    Molinari J, Eskes C, Andres E, Remoué N, Sá-Rocha VM, Hurtado SP. Improved procedures for in vitro skin irritation testing of sticky and greasy natural botanicals. Toxicol In Vitro. 2013;27(1):441–50.CrossRefPubMedGoogle Scholar
  40. 40.
    Groux H, Lelievre D, Cottrez F, Auriault C, Alépée N, Meunier JR, Cotovio J. Evaluation of IRR-IS®, an EpiSkin™ based model for quantifying chemical irritation potency. Oral Communication No. 1.5, ESTIV 2012, 17th Oct 2012, Lisbon, Portugal. In: Poster presented at 51st Annual Meeting of the Society of Toxicology, Mar 11–15, 2012, San Francisco, USA. The Toxicologist, p. 286. 2012. Available at
  41. 41.
    Boelsma E, Ponec M. Basics (guidelines) on cell culture testing for topical/dermatological drugs/products and cosmetics with regard to efficacy and safety of the preparations. In:Dermatopharmacology of topical preparations, vol. 2000. Heidelberg: Springer; 2000. p. 37–57.CrossRefGoogle Scholar
  42. 42.
    Welss T, Basketter DA, Schröder KR. In vitro skin irritation: facts and future. State of the art review of mechanisms and models. Toxicol In Vitro. 2004;18(3):231–43.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Nathalie Alépée
    • 1
    Email author
  • Marie Hélène Grandidier
    • 1
  • José Cotovio
    • 1
  1. 1.L’Oréal Research and InnovationAulnay-sous-Bois CedexFrance

Personalised recommendations