Evaluating animal-free approaches to the testing of skin sensitisers

Abstract

With the implementation of the 7th Amendment of the EU Cosmetics Directive in 2013, Local lymph node assay (LLNA) assay can no longer be used for testing cosmetic ingredients. The current thesis focused on three aspects for further understanding and elucidating the sensitisation process in order to contribute to the reduction, refinement and replacement (“3Rs”) of the animal models with alternative non-animal approaches. The performed quantitative whole human skin proteome analysis was established and identified total of 260 proteins. The applied GeLC-MS/MS identified proteins with diverse biological functions, sub-cellular locations and involvement of toxicological pathways at cellular and molecular levels. An in chemico approach, utilising Top-down MS technique, was applied, using Apomyglobin as recombinant protein. Results have shown a shift of the m/z (mass/ charge) ratio towards a higher value after treating with an extreme sensitiser, 1-chloro-2,4-dinitrobenzene (DNCB), indicating haptenisation . Further results have shown more binding sites on Apomyglobin by DNCB at a physiological condition. An in vitro model utilizing qRT-PCR (real time quantitative polymerase chain reaction) was used to investigate the effect of chemicals with different potencies and binding mechanisms: DNCB (2,4- dinitrochlorobenzen) and Phthalic anhydride (PA) on GST Pi gene expression in HaCaT cells. Experimental data showed DNCB to be a more potent Nrf2 activator in comparison to PA.