KeratinoSens Assay for Identifying Skin Sensitizers
Skin Sensitization Overview
Many animal based tests for skin sensitization exist, such as the Guinea Pig Maximization Test (GPMT) and the Local Lymph Node Assay (LLNA). IIVS is proud to offer a non-animal, cell based alternative assay for detecting skin sensitizers called the KeratinoSens assay. Developed by Givaudan, the KeratinoSens assay is based on a stable reporter construct consisting of a luciferase gene under the control of the antioxidant response element (ARE) in a HaCaT (immortalized keratinocyte) cell line. The luciferase gene allows for the detection of sensitization potential based on the bioluminescent activity of ATP in which light is produced after the breakdown of the protein luciferin by luciferase. These inserted genes allow for the exploitation of the signaling pathway of Keap1-Nrf2, which has previously been shown to be activated during skin sensitization events (Natsch & Emter, 2008; Natsch et al., 2009).
As the deadline approaches to end animal testing to assess skin sensitization in the European Union, three alternative tests (the human cell line activation test (h-CLAT), the myeloid U937 skin sensitization test (MUSST), and the direct peptide reactivity assay (DPRA)) are currently undergoing phase three validation by ECVAM. In addition, the KeratinoSens assay has demonstrated great potential as a non-animal approach to assessing the skin sensition potential of materials. The KeratinoSens assay has undergone a multi-laboratory ring trial, led by Givaudan and the published results are currently being reviewed by ECVAM along with the previously mentioned alternative methods.
A common feature of skin sensitizers is their intrinsic electrophilicity, or their potential to be transformed or metabolized to electrophilic chemicals. Within the KeratinoSens cells, the charged sensitizer binds with, and induces conformational changes in the Keap1 sensor, thereby breaking the Keap1-Nrf2 complex, allowing the Nrf2 to bind to the ARE element in the nucleus, and thus initiating DNA transcription. The transcription of the luciferase gene results in the production of luciferase enzyme. This enzyme breaks down the reagent substrate luciferin which produces light in the presence of ATP, and this luminescence can be measured using a luminometer. In addition to sensitization potential, the cytotoxicity of each test material is determined by evaluation with the vital dye MTT. For specific assay procedures, please see Step-By-Step
Assay Design: Quick Facts
Assay Model: KeratinoSens cells seeded into 96-well plates: 3 plates for luciferase induction and 1 clear bottomed plate for cytotoxicity analysis
Endpoints: Cytotoxicity - IC50 (the concentration of the test material that causes a 50% decrease in viability relative to solvent controls) Luciferase Induction - Imax (the maximum fold induction as compared to the solvent controls) - EC1.5 value (the concentration in which luciferase gene induction is 1.5 times above threshold)
Each plate includes positive and solvent controls. For more information about testing your materials using this assay, please see Applications. Specialized protocols may be prepared as requested through consultation with an IIVS Study Director.