Regulatory h-CLAT Skin Sensitisation Test

WHY CONDUCT h-CLAT SKIN SENSITISATION TESTING?

Of the three human skin sensitisation tests that reflect Key Events in the Adverse Outcome Pathway (AOP), the h-CLAT test represents Key Event 3, the activation of the local immune response via dendritic cells in the epidermis. Test items are classified based on a “two out of three” approach.

OVERVIEW

Skin sensitisation is defined as an allergic response following skin contact with the tested chemical, as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).

The test described here is fully accepted at a regulatory level to support discrimination of Skin Sensitisers and Non-Sensitisers in accordance with the UN GHS. It is appropriate for compliance with a range of legislation including REACH (EC 1907/2006), the CLP Regulation (EC 1272/2008), the Biocidal Products Regulation (EC 528/2012), the Plant Protection Products Regulation (EC 1107/2009) and the Cosmetics Regulation (EC 1223/2009). The h-CLAT test also provides a useful screen at non-regulatory stages of product development.

The adverse outcome pathway leading to skin sensitisation involves a number of key events. This test method addresses Key Event 3 – activation of Dendritic Cells (DC). Skin sensitisers have been reported to induce the expression of cell membrane markers (CD54 and CD86) associated with DC activation. These surface molecules are typical markers of DC activation that play a critical role in T-cell priming by DCs, activating an immune response.

The h-CLAT method is an in vitro test that quantifies changes of cell surface marker expression (CD54 and CD86) following 24 hour exposure to a test item using a human monocytic leukaemia cell line (THP-1) that mimics DC activation. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle controls are calculated and used in the prediction model to support the discrimination between Sensitisers (positive for cell surface marker up-regulation) and Non-Sensitisers (negative for cell surface marker upregulation).

Data generated using the h-CLAT method should be considered in the context of an integrated approach i.e. in combination with other in vitro and in chemico skin sensitisation models.

The standard h-CLAT test includes the use of animal-derived components in the test system, such as serum and antibodies. In order to provide our clients with a truly animal-free test, XCellR8 has adapted the test to animal product-free conditions. Proficiency testing as per OECD Test Guideline 442e has demonstrated our successful classification of a panel of reference chemicals in terms of skin sensitisation potential. The adapted test is suitable for submissions under REACH and, following discussion at the OECD Expert Working Group on skin sensitisation, is currently under consideration by the WNT (National Co-ordinators’ Committee) for listing in OECD TG 442e. A full technical report on the Proficiency Testing and further regulatory details are available on request.

TEST SYSTEM

The test system consists of THP-1 cells that are incubated in vitro with a test item for 24 hours followed by detection of fluorochrome-tagged anti-CD54 and anti-CD86 antibodies by flow cytometry.

SUMMARY OF THE TEST METHOD

Dose Finding Assay (CV75)

• THP-1 cells are pre-cultured for either 48 or 72 hours.
• At the end of the THP-1 pre-culture period, a stock solution of the test item is prepared in an appropriate solvent and diluted over an 8 dose range.
• Vehicle (solvent) controls are prepared.
• The diluted test item and the vehicle control are incubated with THP-1 cells in a 96 well plate for 24 hours.
• After the 24 hour exposure period, THP-1 cells are washed and then stained with propidium iodide.
• Propidium iodide is taken up into dead cells and can therefore be used to separate the live cell population from the dead cell population.
• The % of live cells is determined using flow cytometry and the data used to derive the test item concentration at which 75% of the cells are live (CV75)
• A range of acceptance criteria must be satisfied in order for the test to be considered valid.
• Two independent runs are required to derive the CV75

Measurement of CD54 and CD86 expression

• THP-1 cells are pre-cultured for either 48 or 72 hours.
• At the end of the THP-1 pre-culture period, a stock solution of the test item is prepared in the previously determined solvent and diluted over an8 dose range around the CV75 value.
• Vehicle (solvent) controls and a positive control that is known to increase expression of CD54 and CD86 are prepared.
• The diluted test item, the vehicle control and the positive control are incubated with THP-1 cells in a 24 well plate for 24 hours.
• After the 24 hour exposure period, THP-1 cells are washed and then stained with propidium iodide and either the anti-CD54, anti-CD86 or negative control antibodies.
• Expression of CD54 and CD86 is measured in 10,000 live cells from eachtest condition using flow cytometry
• The THP-1 cell expression of CD54 and CD86 after exposure to the test item is calculated relative to the vehicle control
• If the expression level crosses the pre-set threshold for one or both of the cell surface markers at one of the test item concentrations where the cell viability is above 50% the test item is considered to be positive i.e. a sensitiser
• A range of acceptance criteria must be satisfied in order for the test to be considered valid
• Two concordant independent runs are required to derive the final h-CLAT prediction

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