Abstract
Cosmetic ingredients must be toxicologically assessed to determine their skin sensitizing potential. The in vitro human cell line activation test (h-CLAT; OECD TG 442E) addresses the activation of dermal dendritic cells by analyzing specific protein expression after exposure of THP-1 cells to the test chemical. According to the protocol, FITC-labeled antibodies are used for protein detection. However, some chemicals show strong autofluorescence at FITC-specific wavelengths so that antibody-specific signals cannot be distinguished appropriately from autofluorescence background. This leads to inconclusive or false-negative predictions. Alternative fluorochromes can be used if their equivalence with the FITC-labeled antibodies is proven. In the current paper we describe the results of a proficiency exercise, based on the proficiency chemicals listed in the guideline, with FITC-labeled antibodies as the benchmark and APC-labeled antibodies as an alternative detection system. APC emits fluorescence at longer wavelengths, thus avoiding interference in the FITC spectrum. Irrespective of the employed fluorochrome, all chemicals were classified correctly, and the EC150 and 200 values were in the same order of magnitude. Hence, the equivalence in performance of FITC- and APC-labeled antibodies was demonstrated, and the respective demand of the guideline was fulfilled. In a case study, we then tested a proprietary oxidative hair dye using both fluorochromes. Using APC-labeled antibodies, the hair dye was unambiguously identified as a sensitizer, whereas no classification could be made with the FITC-labeled antibodies. With APC, fluorescence interference can be circumvented and the applicability domain of the h-CLAT extended to include autofluorescent chemicals.
Highlights
Allergic contact dermatitis (ACD) is a widespread adverse health condition throughout the human population. 8 to 30% of Europeans develop an ACD during their lifetime depending, e.g., on their country of origin, ethnicity and on the allergens to which they are exposed
Non-treated THP-1 cells as negative control (NC) and cells exposed to DNCB as positive control (PC) were analyzed in each run, as indicated in OECD testing guidelines (TG) 442E
The mean fluorescence intensity (MFI) for CD86 was higher than the MFI for CD54 with the FITC-labeled antibodies, whereas CD54 was higher than CD86 with APC
Summary
Allergic contact dermatitis (ACD) is a widespread adverse health condition throughout the human population. 8 to 30% of Europeans develop an ACD during their lifetime depending, e.g., on their country of origin, ethnicity and on the allergens to which they are exposed. Allergic contact dermatitis (ACD) is a widespread adverse health condition throughout the human population. In order to protect people from exposure to a potentially sensitizing agent, all ingredients of cosmetics must be toxicologically assessed with respect to their skin-sensitizing potential. To be in line with the European Cosmetics Directive 1223/2009/EU (EU, 2009), the toxicological assessment of chemicals must be conducted by completely animal-free methodologies, relying solely on in vitro, in chemico and in silico approaches. The physiological mechanisms leading to allergic contact dermatitis (ACD) after exposure to an allergen are described in the adverse outcome pathway (AOP) for skin sensitization (OECD, 2014, 2018). Langerhans cells within the skin become activated and migrate out of the epidermis to the adjacent draining lymph node (KE3). KE4 comprises T cell activation and proliferation in the lymph node
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