Extending the applicability domain of the human cell line activation test (h-CLAT).

Cosmetic ingredients must be toxicologically assessed due of their skin sensitizing potential. The in vitro human cell line activation test (h-CLAT), an OECD-approved test method (TG442E), addresses the activation of dermal dendritic cells by analyzing specific protein expression after chemical exposure of THP-1 cells. According to the protocol, FITC-labelled antibodies are used for protein detection. However, some chemicals show strong autofluorescence at the FITC-specific wavelengths, so that antibody-specific signals and autofluorescence background cannot be distinguished appropriately. This leads to inconclusive or false negative predictions. Alternative fluorochromes are covered by the TG442E, but only if their equivalence with the FITC-labelled antibodies are proven. In the current paper we describe the results of a proficiency exercise, based on the proficiency chemicals listed in TG442E, with APC as an alternative fluorochrome and FITC as benchmark. APC emits fluorescence at longer wavelengths, thus avoiding fluorescence interference in the FITC spectrum. Irrespective of the employed fluorochrome, all chemicals were correctly classified, and the EC150 and 200 values were in the same order of magnitude. Hence, the equivalence in performance of FITC- and APC-labelled antibodies was demonstrated and the respective demand of the TG442E fulfilled. In a case study we tested a proprietary oxidative hair dye with both fluorochromes. With APC, the hair dye was unambiguously identified as a sensitizer, whereas with the FITC-labelled antibodies no classification could be made. With APC, fluorescence interference can be circumvented and the applicability domain of the h-CLAT extended for autofluorescent chemicals.