The 2nd key event in the skin sensitisation AOP considers whether a single chemical, formulation or mixture has the ability to induce activation of keratinocytes and the associated inflammatory response, following exposure to the skin.

Using a luciferase reporter linked to induction of the ARE-response pathway, our GLP OECD 442D KeratinoSens™ assay service allows safety assessments of chemicals against the AOP for skin sensitisation.

You can find out more information on this service here: https://gentronix.co.uk/services/skin-sensitisation/keratinosens

The OECD 442D KeratinoSens™ assay enables safety assessment of chemicals (mono- or multi-constituent), products, or formulation against the second key event (KE2) in the skin sensitisation AOP: the activation of the ARE-NRF2 pathway. This pathway is triggered as part of the immune and inflammatory response to haptens forming (covalent skin peptide binding by chemicals detected using OCED 442C DPRA). Such activation following chemical exposure has the potential to trigger the activation of dendritic cells and T-cells; leading to the exposed individual generating an immune response that could be allergenic on subsequent exposure.

As such, the OECD 442D assay presents a complementary toxicology endpoint to both the OECD 442C DPRA assay, by confirming observed peptide depletion can trigger its downstream consequence, and also OECD 442E h-CLAT, showing that dendritic cell activation is or is not driven through inflammatory pathway activation.

The GLP OECD 442D KeratinoSens™ assay is operated in a 96-well microplate format, using a keratinocyte cell line engineered to express a luciferase reporter, with an increase in luciferase protein linked to activation of the ARE-NRF2 pathway. Chemicals are exposed to the modified HaCat cell line across a concentration series, and ARE-NRF2 driven luciferase signal measured, alongside MTT based cell viability. Our laboratory has established this method in line with the OECD 442D guideline and demonstrated proficiency against all test chemicals with expected sensitiser and non-sensitiser outcomes confirmed. More information can be found here: https://gentronix.co.uk/services/skin-sensitisation/keratinosens/

When used in combination with the OECD 442C DPRA and/or OECD 442E h-CLAT endpoints, the KeratinoSens™ provides important data covering KE2 of the AOP. This is in terms of both classification of a chemical as presenting a skin sensitisation safety risk, but also mechanistic information to determine if such risk is a result of alterations in gene expression of oxidative stress response pathways, which then leads to local inflammatory response and wider immune cell activation.