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The h-CLAT Method

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Alternatives for Dermal Toxicity Testing
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Abstract

The h-CLAT method, which was developed in Japan, is based on the augmentation of surface marker CD86 and/or CD54 expression in THP-1 cells. After 24 h treatment with a test chemical, the expression of CD86 and CD54 of THP-1 cells is analysed using flow cytometry. The main prerequisite for the method is the solubility of the test chemical in the culture medium. Many publications have shown the usefulness of the h-CLAT method for the prediction of the skin sensitization potential of targeted chemicals. Moreover, after formal validation of the h-CLAT method by EURL ECVAM, the OECD has recently adopted a new TG No. 442E for the h-CLAT method[1]. Because h-CLAT will not be sufficient as a stand-alone method to cover the endpoint of skin sensitization, data generated with the h-CLAT method should be considered in the context of integrated approaches. The test developers strongly believe that the h-CLAT method will play an important role in in vitro skin sensitization testing.

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References

  1. OECD. OECD Guideline for the testing of chemicals, for a new test guideline, Test No. 442E: in vitro skin sensitization: human Cell Line Activation Test (h-CLAT). 2016. http://www.oecd-ilibrary.org/environment/test-no-442e-in-vitro-skin-sensitisation_9789264264359-en;jsessionid=2711x8ivdmkft.x-oecd-live-03. Accessed 29July 2016.

  2. Basketter DA, Casati S, Gerberick GF, Griem P, Philips B, Worth A. Skin sensitisation. Altern Lab Anim. 2005;33:83–103.

    PubMed  CAS  Google Scholar 

  3. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245–52.

    Article  CAS  PubMed  Google Scholar 

  4. Hart DNJ. Dendritic cells: unique leukocyte population which control the primary immune response. Blood. 1997;90:3245–87.

    PubMed  CAS  Google Scholar 

  5. Aiba S, Katz SI. Phenotypic and functional characteristics of in vivo-activated Langerhans cells. J Immunol. 1990;145:2791–6.

    PubMed  CAS  Google Scholar 

  6. Ozawa H, Nakagawa S, Tagami H, Aiba S. Interleukin-1 beta and granulocyte macrophage colony-stimulating factor mediate Langerhans cell maturation differently. J Invest Dermatol. 1996;106:441–5.

    Article  CAS  PubMed  Google Scholar 

  7. Aiba S, Terunuma A, Manome H, Tagami H. Dendritic cells differently responded to haptens and irritants by their production of cytokines and expression of co-stimulatory molecules. Eur J Immunol. 1997;27:3031–8.

    Article  CAS  PubMed  Google Scholar 

  8. Rougier N, Redziniak G, Mougin D, Schmitt D, Vincent C. In vitro evaluation of the sensitizaiton potential of weak contact allergens using Langerhans-like dendritic cells and autologous T cells. Toxicology. 2000;145:73–82.

    Article  CAS  PubMed  Google Scholar 

  9. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer. 1980;26:171–6.

    Article  CAS  PubMed  Google Scholar 

  10. Tsuchiya S, Kobayashi Y, Goto Y, Okumura H, Nakae S, Konno T, Tada K. Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester. Cancer Res. 1982;42:1530–6.

    PubMed  CAS  Google Scholar 

  11. Ade N, Leon F, Pallardy M, Peiffer JL, Kerdine-Romer S, Tissier MH, Bonnet PA, Fabre I, Ourlin JC. HMOX1 and NQO1 genes are up-regulated in response to contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway. Toxicol Sci. 2009;107:451–60.

    Article  CAS  PubMed  Google Scholar 

  12. Ashikaga T, Hoya M, Itagaki H, Katumura Y, Aiba S. Evaluation of CD86 expression and MHC class II molecule internalization in THP-1 human monocyte cells as predictive endpoints for contact sensitizers. Toxicol In Vitro. 2002;16:711–6.

    Article  CAS  PubMed  Google Scholar 

  13. Tietze C, Blömeke B. Sensitization assays: monocyte-derived dendritic cells versus a monocytic cell line (THP-1). J Toxicol Environ Health A. 2008;71:965–8.

    Article  CAS  PubMed  Google Scholar 

  14. Yoshida Y, Sakaguchi H, Ito Y, Okuda M, Suzuki H. Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naïve THP-1 cell line. Toxicol In Vitro. 2003;17:221–2.

    Article  CAS  PubMed  Google Scholar 

  15. Ashikaga T, Yoshida Y, Hirota M, Yoneyama K, Itagaki H, Sakaguchi H, Miyazawa M, Ito Y, Suzuki H, Toyoda H. Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol. Toxicol In Vitro. 2006;20:767–73.

    Article  CAS  PubMed  Google Scholar 

  16. Mitjans M, Viviani B, Lucchi L, Galli CL, Marinovich M, Corsini E. Role of p38 MAPK in the selective release of IL-8 induced by chemical allergen in naive THp-1 cells. Toxicol In Vitro. 2008;22:386–95.

    Article  CAS  PubMed  Google Scholar 

  17. Sakaguchi H, Ashikaga T, Miyazawa M, Yoshida Y, Ito Y, Yoneyama K, Hirota M, Itagaki H, Toyoda H, Suzuki H. Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT. Toxicol In Vitro. 2006;20:774–84.

    Article  CAS  PubMed  Google Scholar 

  18. Miyazawa M, Ito Y, Yoshida Y, Sakaguchi H, Suzuki H. Phenotypic alterations and cytokine production inTHP-1 cells in response to allergens. Toxicol In Vitro. 2007;21:428–37.

    Article  CAS  PubMed  Google Scholar 

  19. Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. A comparative evaluation of in vitro skin sensitisation tests: the human cell-lineactivation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 2010;38:275–84.

    PubMed  CAS  Google Scholar 

  20. Ashikaga T, Sakaguchi H, Okamoto K, Mizuno M, Sato J, Yamada T, Yoshida M, Ota N, Hasegawa S, Kodama T, Okamoto Y, Kuwahara H, Kosaka N, Sono S, Ohno Y. Assessment of the human Cell Line Activation Test (h-CLAT) for skin sensitization; results of the first Japanese inter-laboratory study. Altern Anim Test Exp. 2008;13:27–35.

    Google Scholar 

  21. Sakaguchi H, Ryan C, Ovigne JM, Schroeder KR, Ashikaga T. Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials. Toxicol In Vitro. 2010;24:1810–20.

    Article  CAS  PubMed  Google Scholar 

  22. EC EURL-ECVAM. Recommendation on the human Cell Line Activation Test (h-CLAT) for skin sensitisation testing. 2013. Accessible at: https://eurl-ecvam.jrc.ec.europa.eu/eurl-ecvam-recommendations.

  23. Takenouchi O, Miyazawa M, Saito K, Ashikaga T, Sakaguchi H. Predictive performance of the human Cell Line Activation Test (h-CLAT) for lipophilic chemicals with high octanol-water partition coefficients. J Toxcol Sci. 2013;38:599–609.

    Article  CAS  Google Scholar 

  24. Nukada Y, Ashikaga T, Sakaguchi H, Sono S, Mugita N, Hirota M, Miyazawa M, Ito Y, Sasa H, Nishiyama N. Predictive performance for human skin sensitizing potential of the Human Cell Line Activation Test (h-CLAT). Contact Dermatitis. 2011;65:343–53.

    Article  CAS  PubMed  Google Scholar 

  25. DB-ALM (IVITTOX). Protocol 158: human Cell Line Activation Test (h-CLAT). 2014. http://ecvam-dbalm.jrc.ec.europa.eu/. Accessed 6 Jan 2015.

  26. National Toxicology Program. Development of integrated testing strategies. 2014. http://ntp.niehs.nih.gov/pubhealth/evalatm/integrated-testing-strategies/index.html. Accessed 7 Jan 2015.

  27. Takenouchi O, Fukui S, Okamoto K, Kurotani S, Imai N, Fujishiro M, Kyotani D, Kato Y, Kasahara T, Fujita M, Toyoda A, Sekiya D, Watanabe S, Seto H, Hirota M, Ashikaga T, Miyazawa M. Test battery with the human cell line activation test, direct peptide reactivity assay and DEREK based on a 139 chemical data set for predicting skin sensitizing potential and potency of chemicals. J Appl Toxicol. 2014;35:1318–32.

    Article  CAS  Google Scholar 

  28. Tsujita-Inoue K, Hirota M, Ashikaga T, Atobe T, Kouzuki H, Aiba S. Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays. Toxicol In Vitro. 2014;28:626–39.

    Article  CAS  PubMed  Google Scholar 

  29. Urbisch D, Mehling A, Guth K, Ramirez T, Honarvar N, Kolle S, Landsiedel R, Jaworska J, Kern PS, Gerberick F, Natsch A, Emter R, Ashikaga T, Miyazawa M, Sakaguchi H. Assessing skin sensitization hazard in mice and men using non-animal test methods. Regul Toxicol Pharmacol. 2014; doi:10.1016/j.yrtph.2014.12.008.

    Article  PubMed  CAS  Google Scholar 

  30. Van der Veen JW, Rorije E, Emter R, Natch A, van Loveren H, Ezendam J. Evaluating the performance of integrated approaches for hazard identification of skin sensitizing chemicals. Regul Toxicol Pharmacol. 2014;69:371–9.

    Article  CAS  PubMed  Google Scholar 

  31. Jaworska J, Natsch A, Ryan C, Strickland J, Ashikaga T, Miyazawa M, Sakaguchi H. Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment: a decision support system for quantitative weight of evidence and adaptive testing strategy. Arch Toxicol. 2015;89:2355–83.

    Article  CAS  PubMed  Google Scholar 

  32. Mizuno M, Yoshida M, Kodama T, Kosaka N, Okamoto K, Sono S, Yamada T, Hasegawa S, Ashikaga T, Kuwahara H, Sakaguchi H, Sato J, Ota N, Okamoto Y, Ohno Y. Effects of pre-culture conditions on the human Cell Line Activation Test (h-CLAT) results: results of the 4th Japanese inter-laboratory study. Altern Anim Test Exp. 2008;13:70–82.

    Google Scholar 

  33. Kosaka M, Okamoto K, Mizuno M, Yamada T, Yoshida M, Kodama T, Sono S, Ashikaga T, Sato J, Ota N, Hasegawa S, Okamoto Y, Kuwahara H, Sakaguchi H, Ohno Y. A study of the criteria for selection of THP-1 cells in the human Cell Line Activation Test (h-CLAT): results of 2nd Japanese inter-laboratory study. Altern Anim Test Exp. 2008;13:55–62.

    Google Scholar 

  34. Nukada Y, Miyazawa M, Kosaka N, Ito Y, Sakaguchi H, Nishiyama N. Production of IL-8 in THP-1 cells following contact allergen stimulation via mitogen-activated protein kinase activation or tumor necrosis factor-alpha production. J Toxicol Sci. 2008;33:175–85.

    Article  CAS  PubMed  Google Scholar 

  35. Suzuki M, Hirota M, Hagino S, Itagaki H, Aiba S. Evaluation of changes of cell-surface thiols as a new biomarker for in vitro sensitization test. Toxicol In Vitro. 2009;23:687–96.

    Article  CAS  PubMed  Google Scholar 

  36. Hoya M, Hirota M, Suzuki M, Hagino S, Itagaki H, Aiba S. Development of an in vitro photosensitization assay using human monocyte-derived cells. Toxicol In Vitro. 2009;23:911–8.

    Article  CAS  PubMed  Google Scholar 

  37. Hennen J, Aeby P, Goebel C, Schettgen T, Oberli A, Kalmes M, Blömeke B. Cross talk between keratinocytes and dendritic cells: impact on the prediction of sensitization. Toxicol Sci. 2011;123:501–10.

    Article  CAS  PubMed  Google Scholar 

  38. Tamura A, Fukumoto I, Yui N, Matsumura M, Miura H. Increasing the repeating units of ethylene glycol-based dimethacrylates directed toward reduced oxidative stress and co-stimulatory factors expression in human monocytic cells. J Biomed Mater Res. 2014; doi:10.1002/jbm.a.35251.

    Article  PubMed  CAS  Google Scholar 

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Sakaguchi, H., Ashikaga, T. (2017). The h-CLAT Method. In: Eskes, C., van Vliet, E., Maibach, H. (eds) Alternatives for Dermal Toxicity Testing. Springer, Cham. https://doi.org/10.1007/978-3-319-50353-0_21

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  • DOI: https://doi.org/10.1007/978-3-319-50353-0_21

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