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An In Vitro Skin Irritation Test Using the SkinEthic™ Reconstructed Human Epidermal (RHE) Model

  • Nathalie Alépée
  • Marie Hélène Grandidier
  • Carine Tornier
  • José Cotovio
Chapter

Abstract

The SkinEthic™ Reconstructed Human Epidermal (RHE) skin irritation method is an internationally accepted OECD test method used to detect the skin irritation properties of chemicals (OECD TG 439). The SkinEthic™ RHE skin irritation test method has been shown to be robust and transferable. The reproducibility and predictivity of the SkinEthic™ RHE assay have been evaluated in validation studies organized with three laboratories, and with a set of 20 chemicals through a systematic and in vivo Draize comparative evaluation for safety assessment, delivering results that matched very well with corresponding in vivo data, showing 69% specificity, 100% sensitivity and 80% accuracy under GHS classification system. 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 SkinEthic™ RHE skin corrosion method, in any initiative aimed at developing integrated testing strategies for full evaluation of local skin effects.

Keywords

Hazard identification Irritation Skin tissue models Validation EURL-ECVAM Full replacement 

References

  1. 1.
    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
  2. 2.
    Nickoloff BJ, Turka LA. Keratinocytes: key immunocytes of the integument. Am J Pathol. 1993;143:325–31.PubMedPubMedCentralGoogle Scholar
  3. 3.
    Fluhr JW, Darlenski R, Angelova-Fischer I, Tsankov N, Basketter D. Skin irritation and sensitization: mechanisms and new approaches for risk assessment. 1. Skin irritation. Skin Pharmacol Physiol. 2008;21(3):124–35.CrossRefPubMedGoogle Scholar
  4. 4.
    Kindt TJ, Osborne BA, Goldsby RA. Immunology. 6th ed. New York: Freeman and Company; 2006.Google Scholar
  5. 5.
    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
  6. 6.
    OECD. OECD Guideline for the testing of chemicals, no. 404: acute dermal irritation/corrosion. Organisation for economic cooperation and development, Paris, France; 2002.Google Scholar
  7. 7.
    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
  8. 8.
    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
  9. 9.
    Worth AP, Cronin MT. The use of bootstrap resampling to assess the variability of Draize tissue scores. ATLA. 2001;29(5):557–73.PubMedGoogle Scholar
  10. 10.
    Campbell RL, Bruce RD. Comparative dermatotoxicology. I. Direct comparison of rabbit and human primary skin irritation responses to isopropylmyristate. Toxicol Appl Pharmacol. 1981;59:555–63.CrossRefPubMedGoogle Scholar
  11. 11.
    Robinson MK, Perkins MA, Basketter DA. Application of a four hour human patch test method for comparative and investigative assessment of skin irritation. Contact Dermatitis. 1998;38:194–202.CrossRefPubMedGoogle Scholar
  12. 12.
    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
  13. 13.
    Hartung T, Bremer S, Casati S, Coecke S, Corvi R, Fortaner S, Gribaldo L, Halder M, Janusch Roi A, Prieto P, Sabbioni E, Worth A, Zuang V. ECVAM’s response to the changing political environment for alternatives: consequences of the European union chemicals and cosmetics policies. ATLA. 2003;31:473–81.PubMedGoogle Scholar
  14. 14.
    Van De Sandt J, Roguet R, Cohen C, Esdaile D, Ponec M, Corsini E, Barker C, Fusenig N, Liebsch M, Benford D, De Brugerolle De Fraissinette A, Fartasch M. The use of human keratinocytes and human skin models for predicting skin irritation. ATLA. 1999;27:723–43.Google Scholar
  15. 15.
    Zuang V, Rona C, Archer G, Berardesca E. Detection of skin irritation potential of cosmetics by non-invasive measurements. Skin Pharmacol Appl Ski Physiol. 2000;13:358–71.CrossRefGoogle Scholar
  16. 16.
    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: Rougier A, Goldberg AM, Maibach HI, editors. Alternative methods in toxicology, In vitro skin toxicology, vol. Vol. 10. New York, NY, USA: Mary Ann Liebert; 1994. p. 83–96.Google Scholar
  17. 17.
    Faller C, Bracher M. Reconstructed skin kits: reproducibility of cutaneous irritancy testing. Skin Pharmacol Appl Ski Physiol. 2002;15:74–91.CrossRefGoogle Scholar
  18. 18.
    Perkins MA, Osborne R, Rana FR, Ghassemi A, Robinson MK. Comparison of in vitro and in vivo human skin responses to consumer products and ingredients with a range of irritancy potential. Toxicol Sci. 1999;48:218–29.CrossRefPubMedGoogle Scholar
  19. 19.
    Roguet R. Use of skin cells culture for in vitro assessment of corrosion and cutaneous irritancy. Cell Biol Toxicol. 1999;15:63–75.Google Scholar
  20. 20.
    Alépée N, Tornier C, Robert C, Amsellem C, Roux MH, Doucet O, Pachot J, Meloni M, de Brugerolle de Fraissinette A. A catch-up validation study on reconstructed human epidermis (SkinEthic™ RHE) for full replacement of the Draize skin irritation test. Toxicol In Vitro. 2010;24:257–66.CrossRefPubMedGoogle Scholar
  21. 21.
    Kojima H, Ando Y, Idehara K, Katoh M, Kosaka T, Miyaoka E, Shinoda S, Suzuki T, Yamaguchi Y, Yoshimura I, Yuasa A, Watanabe Y, Omori T. Validation study of the in vitro skin irritation test with the LabCyte EPI-MODEL24. Altern Lab Anim. 2012;40:33–50.Google Scholar
  22. 22.
    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
  23. 23.
    Prunerias M, Régnier M, Woodley D. Methods for cultivation of keratinocytes with an air-liquid interface. J Investig Dermatol. 1983;81(sup n°1):28s–33s.CrossRefGoogle Scholar
  24. 24.
    Rosdy M, Clauss LC. Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air–liquid interface. J Invest Dermatol. 1990;95:409–14.CrossRefPubMedGoogle Scholar
  25. 25.
    Doucet O, Robert C, Zastrow L. Use of a serum-free reconstituted epidermis as a skin pharmacological model. Toxicol In Vitro. 1996;10:305–13.CrossRefPubMedGoogle Scholar
  26. 26.
    Kandárová H, Liebsch M, Spielmann H, Genschow E, Schmidt E, Guest R, Whittingham A, Warren N, Gamer A, Remmele M, Kaufmann T, Wittmer E, De Wever B, Rosdy M. Assessment of the SkinEthic reconstituted human epidermis for skin corrosion testing according to OECD guideline 431. Toxicol In Vitro. 2006;20:547–59.Google Scholar
  27. 27.
    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: http://www.oecd.org/env/ehs/testing/seriesontestingandassessmenttestingforhumanhealth.htm.
  28. 28.
    ECVAM. Statement of the ECVAM Scientific Advisory Committee (ESAC) on the scientific validity of in vitro tests for skin irritation testing. 2008. Available at: http://ecvam.jrc.it.
  29. 29.
    United Nations Economic Commission for Europe (UNECE). UN globally harmonised system of classification and labelling of chemicals. 2nd revised ed; 2008. http://www.unece.org/trans/danger/publi/ghs/ghs_rev02/02files_e.html. (as of April 2009)Google Scholar
  30. 30.
    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 April 2009; 2009. Available at: http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing.
  31. 31.
    Griesinger C., Barroso J., and Zuang V.. ECVAM background document on the recent adaptations of the ECVAM performance standards for in vitro skin irritation testing in the context of the drafting process of an EU Test Method and an OECD draft Test Guideline. Ispra, November 13, 2008; 2008. Available at: http://ecvam.jrc.ec.europa.eu/.
  32. 32.
    OECD. In vitro skin irritation: Reconstructed Human Epidermis Test Method. OECD Guideline for the Testing of Chemicals No. 439, OECD, Paris; 2015. Available at: http://www.oecd.org/env/testguidelines.
  33. 33.
    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: http://www.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2010)36&doclanguage=en.
  34. 34.
    United Nations (UN). Globally harmonized system of classification and labelling of chemicals (GHS), 2nd revised edn, UN New York and Geneva, 2013; 2013. Available at: http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_e.html.
  35. 35.
    Alépée N, Grandidier MH, Tornier C, Cotovio J. An integrated testing strategy for in vitro skin corrosion and irritation assessment using SkinEthic™ reconstructed human epidermis. Toxicol In Vitro. 2015;29:1779–92.CrossRefPubMedGoogle Scholar
  36. 36.
    ECVAM. Statement on the validity of in vitro tests for skin irritation, issued by the ECVAM Scientific Advisory Committee (ESAC26), 27 April 2007; 2007. Available at: http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing.
  37. 37.
    Zuang V, Eskes C, Worth A, Cole T, Hoffmann S, Saliner AG, Netzeva T, Patlewicz G, Cockshott A, Gerner I. Report from the chemical selection sub-committee to the management team on potential reasons for the misclassification of chemicals in the EpiSkin and EpiDerm assays. Available under “Validation Study Documents”; 2007. Available at: http://ecvam.jrc.ec.europa.eu/.
  38. 38.
    Tornier C, Amsellem C, Fraissinette Ade Brugerolle A, Alépée N. Assessment of the optimized SkinEthic™ reconstructed human epidermis (RHE) 42 bis skin irritation protocol over 39 test substances. Toxicol In Vitro. 2010;24(1):245–56.CrossRefPubMedGoogle Scholar
  39. 39.
    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
  40. 40.
    EC. 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. Off J Eur Union. 2001;L225:1–333.Google Scholar
  41. 41.
    OECD. In vitro skin irritation: reconstructed human epidermis test method. Revised OECD Guideline TG 439 version originally adopted in 2010, OECD, Paris; 2013. Available at: http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-4-health-effects_20745788.
  42. 42.
    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
  43. 43.
    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
  44. 44.
    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
  45. 45.
    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
  46. 46.
    Robinson MK, Whittle E, Basketter DA. A two center study of the development of acute irritation responses to fatty acids. Am J Contact Dermatol. 1999;10:136–45.CrossRefGoogle Scholar
  47. 47.
    Jirova D, Basketter D, Liebsch M, Bendova H, Keilova K, Marriott M, Kandárová H. Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data. Contact Dermatitis. 2010;6:109–16.CrossRefPubMedGoogle Scholar
  48. 48.
    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
  49. 49.
    SkinEthic™ RHE SOP, Version 2.0. SkinEthic skin irritation test-42bis test method for the prediction of acute skin irritation of chemicals: 42 minutes application +42 hours post-incubation; 2009. Available at: http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing.
  50. 50.
    OECD. In vitro skin corrosion: reconstructed human epidermis (RHE) test method. OECD Guideline for the Testing of Chemicals. Revised version originally adopted in 2004, OECD, Paris; 2014. Available at: http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-4-health-effects_20745788.
  51. 51.
    Alépée N, Robert C, Tornier C, Cotovio J. The usefulness of the validated SkinEthic™ RHE test method to identify skin corrosive UN GHS subcategories. Toxicol In Vitro. 2014;28:616–25.CrossRefPubMedGoogle Scholar
  52. 52.
    Groux, H., Lelievre, D., Cottrez, F., Auriault, C., Alépée, N., Meunier, J.R., and Cotovio, J.. Evaluation of IRR-IS®, an EpiSkin™ based model for quantifying chemical irritation potency. Oral Communication N°1.5, ESTIV2012, 17th October 2012, Lisbon, Portugal. Poster presented at 51st Annual Meeting of the Society of Toxicology, March 11–15, 2012, San Francisco, USA. The Toxicologist; 2012. p. 286. Available at: http://www.toxicology.org/AI/Pub/Tox/2012Tox.pdf.
  53. 53.
    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. Available at: http://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32009R1223 Google Scholar
  54. 54.
    EC. Regulation (EC) No 528/2012 of the European Parliament and of the Council of 22 May 2012 concerning the making available on the market and use of biocidal products Text with EEA relevance. Off J Eur Union. 2012;L167:1–123Google Scholar
  55. 55.
    Molinari J, Eskes C, Andres E, Remoué N, Sá-Rocha VM, Hurtado SP, Barrichello C. Improved procedures for in vitro skin irritation testing of sticky and greasy natural botanicals. Toxicol In Vitro. 2013;27(1):441–50.CrossRefPubMedGoogle Scholar
  56. 56.
    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
  57. 57.
    Kolle SN, Sullivan KM, Mehling A, van Ravenzwaay B, Landsiedel R. Applicability of in vitro tests for skin irritation and corrosion to regulatory classification schemes: substantiating test strategies with data from routine studies. Regul Toxicol Pharmacol. 2012;64(3):402–14.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Nathalie Alépée
    • 1
  • Marie Hélène Grandidier
    • 1
  • Carine Tornier
    • 2
  • José Cotovio
    • 1
  1. 1.L’Oréal Research and InnovationAulnay-sous-Bois CedexFrance
  2. 2.EPISKINLyonFrance

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