A test substance is considered to be sensitiser if it increases the expression (compared to the solvent control) of at least 7 genes measured by qPCR in either the “SENS-IS” or the “ARE” gene sets. To take into account non-specific gene over-expression due to cell stress, the induction of more than 20 genes in the irritation gene check details set, classifies a result as inconclusive and the test substance is re-analysed at a lower concentration. Similarly to the LLNA, potency

is classified according to the concentration of test material needed to induce a positive response: positive at 0.1%: extreme; positive at 1%: strong; positive at 10%: moderate; positive at 50%: weak. Sens-IS is considered to mainly address key event 2 from the skin sensitisation AOP, but may, as ARE-activated genes are included, also provide information on protein reactivity of a test chemical. The SenCeeTox method is a test battery of three independent

assays addressing several key events to provide information on the skin sensitisation potential of substances and to assign them to GW-572016 research buy a certain subset of potency categories (McKim et al., 2012). Protein reactivity is evaluated in a cell-free manner by measurement of the concentration of free glutathione (GSH) after incubation with the test substance for 24 h at room temperature. The amount of free GSH is determined by a colorimetric assay with 5,5′-Dithio-bis(2-nitrobenzoic acid (DTNB) in relation to the vehicle control. An epidermal

skin equivalent (EpiDerm™, MatTek, MA) is used for gene expression analysis and cytotoxicity determination. Viability of skin tissues is measured by assaying for lactose dehydrogenase (LDH) activity. Expression of four housekeeping and seven target genes (NADPH-quinone oxidoreductase 1, Aldoketoreductase 1C2, Interleukin 8, Cytochrome P450 1A1, Aldehyde dehydrogenase 3A, Heme-oxygenase 1, Glutamate cysteine ligase catalytic subunit C) is monitored after topical exposure of the model skin tissues to the test substances at a range of six concentrations (0.1, 5, 100, 250, 500, and 2500 μM) for 24 h. Concentrations, which result in cell viability before of less than 50% compared to the vehicle control, are disregarded for the determination of the sensitising potential/potency. Finally, a gated algorithm is used to transform the viability, gene induction and glutathione reactivity data into a toxicity index for each substance. This method covers key event 1 (in terms of protein reactivity) and 2 (in terms of keratinocyte activation) in the skin sensitisation AOP. The GARD assay uses proliferating MUTZ-3 cells (a human myeloid leukemia-derived cell line) to measure gene expression induced by test substances.