The SenCeeTox® Assay

  • Donald Keller
  • Caroline Bauch
  • Prakash Patel


Chemical sensitization is a complex biological process initiated by electrophilic substances and several assaults to different cell types. One prominent intracellular pathway involved is the Nrf2/Keap1 signaling pathway which is triggered and expression of genes containing the Electrophilic/Aromatic Response Element (EpRE/ARE) in their promotor region is activated. As a cellular defense mechanism the products of these genes act to reduce the oxidative stress and to detoxify the xenobiotic. The SenCeeTox assay can be performed on a monolayer culture of a keratinocyte cell line, HaCaT, or can be applied in a more advanced reconstituted epidermis model, which allows the testing of finished products, extracts. Changes in gene expression profiles of 11 genes containing the EpRE/ARE in their promotors are monitored and the protein reactivity of the test articles based on their direct reaction with glutathione is measured. Molecular descriptors are taken into account to allow the calculation of a predicted LLNA EC3 value in mM using a pattern recognition model and by combining the data of protein reactivity with the gene expression and cell viability data.


  1. 1.
    Thyssen JP, Linneberg A, Johansen JD. The epidemiology of contact allergy in the general population-prevalence and main findings. Contact Dermatitis. 2007;57:287–99.CrossRefPubMedGoogle Scholar
  2. 2.
    Zug KA, Warshaw EM, Fowler JF Jr, Maibach HI, Belsito DL, Pratt MD, Sasseville D, Storrs FJ, Taylor JS, Mathias CG. Patch test results of the North American contact dermatitis group 2005-2006. Dermatitis. 2009;20:149–60.PubMedGoogle Scholar
  3. 3.
    Kimber I, Basketter DA, Gerberick GF, Dearman RJ. Allergic contact dermatitis. Int Immunopharmacol. 2002;2:201–11.CrossRefPubMedGoogle Scholar
  4. 4.
    2003/15/EC. Directive 2003/15/EC of the European Parliament and the Council of 27 February 2003 amending Council Directive 76/768/EEC on the approximations of laws of the Member States relating to cosmetic products. Off J Eur Union. 2003;L66:26–35.Google Scholar
  5. 5.
    EU 1223/2009. Regulation (EC) No 1223/2009 of the European parliament and of the council. Off J Eur Union. 2009;L342:59–209.Google Scholar
  6. 6.
    Jaworska J, Harol A, Kern PS, Gerberick GF. Integrating non-animal test information into an adaptive testing strategy – skin sensitization proof of concept case. ALTEX. 2011;28:211–25.CrossRefPubMedGoogle Scholar
  7. 7.
    Jowsey IR, Basketter DA, Westmoreland C, Kimber I. A future approach to measuring relative skin sensitising potency: a proposal. J Appl Toxicol. 2006;26:341–50.CrossRefPubMedGoogle Scholar
  8. 8.
    Mehling A, Eriksson T, Eltze T, Kolle SN, Ramirez T, Teubner W, van Ravenzwaay B, Landsiedel R. Non-animal test methods for predicting skin sensitization potentials. Arch Toxicol. 2012;86:1273–95.CrossRefPubMedGoogle Scholar
  9. 9.
    Dupuis G, Benezra C. Allergic contact dermatitis to simple chemicals: a molecular approach. New York: Marcel Dekker; 1982.Google Scholar
  10. 10.
    Landsteiner K, Jacobs J. Studies on the sensitization of animals with simple chemical compounds. J Exp Med. 1936;64:625–39.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Weltzien HU, Moulon C, Martin SF, Padovan E, Hartmann U, Kohler J. T cell immune response to haptens. Structural models for allergic and autoimmune reaction. Toxicology. 1996;107:141–51.CrossRefPubMedGoogle Scholar
  12. 12.
    Roberts DW, Lepoittevin JP. Hapten-protein interactions. In: Lepoittevin JP, Basketter DA, Goossens A, Karlbert A-T, editors. Allergic contact dermatitis: the molecular basis. Berlin: Springer; 1998. p. 81–111.CrossRefGoogle Scholar
  13. 13.
    Kim BS, Miyagawa F, Cho Y-H, Bennett CL, Clausen BE, Katz SI. Keratinocytes function as accessory cells for presentation of endogenous antigen expressed in the epidermis. J Investig Dermatol. 2009;129:2805–17.CrossRefPubMedGoogle Scholar
  14. 14.
    Kimber I, Cumberbatch M. Dendritic cells in cutaneous immune response to chemical allergens. Food Chem Toxicol. 1992;117:137–46.Google Scholar
  15. 15.
    Aiba S, Terunuma A, Manome H, Tagami H. Dendritic cells differently respond to haptens and irritants by their production of cytokines and expression of co-stimulatory molecules. Eur J Immunol. 1997;27:3031–8.CrossRefPubMedGoogle Scholar
  16. 16.
    Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392(6673):245–52.CrossRefPubMedGoogle Scholar
  17. 17.
    Banchereau J, Briere F, Caus C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18:767–811.CrossRefGoogle Scholar
  18. 18.
    McKim JM Jr, Keller DJ, Gorski JR. A new in vitro method for identifying chemical sensitizers combining peptide binding with ARE/EpRE-mediated gene expression in human skin cells. Cutan Ocul Toxicol. 2010;29(3):170–97.CrossRefGoogle Scholar
  19. 19.
    Natsch A, Emter R. Skin sensitizers induce antioxidant response element dependent genes: application to the in vitro testing of the sensitization potential of chemicals. Toxicol Sci. 2008;102(1):110–9.CrossRefPubMedGoogle Scholar
  20. 20.
    Dinkova-Kostova AT, Holtzclaw WD, Kensler TW. The role of Keap1 in cellular protective responses. Chem Res Toxicol. 2005;18(12):1779–91.CrossRefPubMedGoogle Scholar
  21. 21.
    Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang MI, Kobayashi A, Yamamoto M, Kensler TW, Talalay P. Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers. Proc Natl Acad Sci U S A. 2004;101(7):2040–5.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Wang W, Jaiswal AK. Nuclear factor Nrf2 and antioxidant response element regulate NRH:quinone oxidoreductase 2 (NQO2) gene expression and antioxidant induction. Free Radic Biol Med. 2006;40(7):1119–30.CrossRefPubMedGoogle Scholar
  23. 23.
    McKim JM Jr, Keller DJ, Gorski JR. An in vitro method for detecting chemical sensitization using human reconstructed skin models and its applicability to cosmetic, pharmaceutical and medical device safety testing. Cutan Ocul Toxicol. 2012;31(4):292–305.CrossRefPubMedGoogle Scholar
  24. 24.
    Kimber I, Basketter DA, Butler M, Gamer A, Garrigue JL, Gerberick GF, Newsome C, Steiling W, Vohr HW. Classification of contact allergens according to potency: proposals. Food Chem Toxicol. 2003;41(12):1799–809.CrossRefPubMedGoogle Scholar
  25. 25.
    Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin J-P. Development of a peptide reactivity assay for screening contact allergens. Toxicol Sci. 2004;81:332–43.CrossRefPubMedGoogle Scholar
  26. 26.
    Griffith OW. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem. 1980;106(1):207–12.CrossRefPubMedGoogle Scholar
  27. 27.
    Chein CW, Chiang MC, Ho IC, Lee TC. Association of chromosomal alterations with arsenite-induced tumorigenicity of human HaCaT keratinocytes in nude mice. Environ Health Perspect. 2004;112(17):1704–10.CrossRefGoogle Scholar
  28. 28.
    Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;16:55–63.CrossRefGoogle Scholar
  29. 29.
    Krstajic D, Buturovic LJ, Leahy DE, Thomas S. Cross-validation pitfalls when selecting and assessing regression and classification models. J Chem. 2014;6(1):10.CrossRefGoogle Scholar
  30. 30.
    Clippinger A, Keller D, McKim JM, Witters H, Van Rompay AR. Inter-laboratory validation of an in vitro method to classify skin sensitizers. In: Society of toxicology 53rd annual meeting. Society of toxicology. Phoenix Convention Center, Phoenix. 24–27 Mar 2014. Poster presentation. 2014.Google Scholar
  31. 31.
    Basketter DA, Lea LJ, Cooper K, Stocks J, Dickens A, Pate I, Dearman RJ, Kimber I. Threshold for classification as a skin sensitizer in the local lymph node assay: a statistical evaluation. Food Chem Toxicol. 1999;37(12):1167–74.CrossRefPubMedGoogle Scholar
  32. 32.
    Natsch A, Emter R, Ellis G. Filling the concept with data: integrating data from different in vitro and in silico assays on skin sensitizers to explore the battery approach for animal-free skin sensitization testing. Toxicol Sci. 2009;107(1):106–21.CrossRefPubMedGoogle Scholar
  33. 33.
    Gerberick GF, Ryan CA, Kern PS, Schlatter H, Dearman RJ, Kimber I, Patlewicz GY, Basketter DA. Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods. Dermatitis. 2005;16(4):157–202.PubMedGoogle Scholar
  34. 34.
    Reisinger K, Hoffmann S, Alépée N, Ashikaga T, Barroso J, Elcombe C, Gellatly N, Galbiati V, Gibbs S, Groux H, Hibatallah J, Keller D, Kern P, Klaric M, Kolle S, Kuehnl J, Lambrechts N, Lindstedt M, Millet M, Martinozzi-Teissier S, Natsch A, Petersohn D, Pike I, Sakaguchi H, Schepky A, Tailhardat M, Templier M, van Vliet E, Maxwell G. Systematic evaluation of non-animal test methods for skin sensitisation safety assessment. Toxicol In Vitro. 2015;29(1):259–70.CrossRefPubMedGoogle Scholar
  35. 35.
    Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R. Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol. 2012;63(3):489–504.CrossRefPubMedGoogle Scholar
  36. 36.
    Bauch C, Walker P, Patel P, Thomas S, Keller D, Dilworth C. Comparison and combination of in silico and in vitro skin sensitization assays to predict skin sensitization potential and potency. In: Poster session at: in vitro toxicology society, annual IVTS meeting, Nov 10–11, AstraZeneca, Alderley Park, Cheshire, UK. 2014.Google Scholar
  37. 37.
    Bauch C, Jamin A, Willoughby JA, Keller DJ, Patel P, Thomas S, Dilworth C, Walker P. Combination of cheminformatics and in vitro assays to predict skin sensitization potential and potency. In: session at: Society of toxicology, 54th annual meeting and ToxExpo™, Mar 22–26, San Diego, California, USA. 2015.Google Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.CyprotexKalamazooUSA

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