In Vitro and In Chemico Skin Sensitization Assays
Skin Sensitization Overview
Determination of skin sensitization potential is a critical toxicological endpoint in the safety assessment of novel chemicals. Although the Guinea Pig Maximization Test (GPMT) and in vivo Local Lymph node assay (LLNA) have traditionally been used to assess skin sensitization, recent activity has focused on the development of novel alternative assays for the endpoint.
As a culmination of these efforts, the Organization for Economic Cooperation and Development (OECD) published the following test guidelines for non-animal skin sensitization testing on 5 February 2015: ARE-Nrf2 Luciferase Test Method (also referred to as the KeratinoSensTM Assay) (OECD TG 442D) and Direct Peptide Reactivity Assay (DPRA) (OECD TG 442C). Most recently, on July 29, 2016, the OECD published a test guideline for the Human Cell Line Activation Test (h-CLAT) (OECD TG 442E).
IIVS offers all three OECD-approved methods for non-animal skin sensitization testing as part of our GLP-compliant laboratory testing services.
Given the complex cascade of events leading to skin sensitization, it is generally thought that an integrated testing approach combining multiple assays and in silico predictive tools will be needed to fully replace the animal based methods. There is a significant effort underway to ascertain how the non-animal assays may be combined to both qualitatively and quantitatively assess skin sensitization most effectively.
We participated in the validation of the LuSens assay and the KeratinoSensTM assay, which are ARE-reporter gene assays, and recently added the h-CLAT assay as a commercial service in response to the July 2016 publication of the OECD TG. Read press release.
Please contact us for information on these skin sensitization assays and how they may be used as part of an integrated testing strategy within your existing testing program.
The KeratinoSensTM assay is a cell-based reporter gene assay which identifies skin sensitizers by measuring the induction of luciferase under the control of the antioxidant response element (ARE) derived from the human AKR1C2 gene. In the proposed Adverse Outcome Pathway (AOP) leading to skin sensitization, this method addresses the second key event, gene expression in keratinocytes associated with the antioxidant/electrophile response element (ARE)-dependent pathway. The IIVS laboratory participated in the validation studies led by Givaudan to assess the KeratinoSensTM assay which demonstrated that the assay was transferable, reproducible, and predictive as compared to the historical animal data. Following extensive review of the data by EURL ECVAM, the assay was endorsed by ECVAM, and the OECD followed with a test guideline (OECD 442D).
Direct Peptide Reactivity Assay
The DPRA is an in chemico assay that identifies dermal sensitizers based on their reactivity with synthetic peptides containing either lysine or cysteine. The assay models the first key event, protein reactivity, in the skin sensitization AOP. Through collaboration with the method developers at Procter & Gamble, IIVS scientists received training, successfully demonstrated regulatory-required proficiency, and are offering the assay as a commercial service. IIVS is currently exploring the use of the OECD approved DPRA method (OECD 442C) in combination with other assays, including the KeratinoSensTM assay, to improve predictions.
Human Cell Line Activation Test (h-CLAT)
The human Cell Line Activation Test (h-CLAT) is cell-based method which identifies skin sensitizers by examining changes in the expression of cell surface markers (CD54 and CD86). Following exposure of the THP-1 human monocyte cell line to the test substance, expression levels of CD54 and CD86 are quantified by flow cytometry and compared to controls. The h-CLAT method addresses the third key event, dendritic cell activation, of the skin sensitization AOP. The method has been reviewed by EURL-ECVAM and has been recommended as a useful tool in an integrated testing strategy for identification of skin sensitizers. The assay has an OECD-approved guideline and is included among our other sensitization assays, KeratinoSensTM and DPRA.
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View our latest poster on skin sensitization presented at the 2016 SOT meeting.