Human Cell Line Activation Test (h-CLAT)

In Vitro Skin Sensitisation Assay

The in vitro h-CLAT addresses the third molecular key event of the adverse outcome pathway (AOP) of skin sensitization.¹ The UN GHS (United Nations Globally Harmonized System of Classification and Labelling of Chemicals) defines a skin sensitiser as a substance that will cause an allergic response after skin contact.²

The h-CLAT is validated by the EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing) and is performed in accordance with the OECD guidance OECD 442E at Eurofins BioPharma Product Testing Munich GmbH ^{1, 3} with chemicals, cosmetics or personal care products and pharmaceuticals.

The h-CLAT is one of three test methods (DPRA and KeratinoSens^TM) for the assessment of skin sensitisation potential.

Assessment of Skin Sensitisation Potential with the h-CLAT

• The third molecular key event of skin sensitisation addresses the activation of dendritic cells (DC), which is typically accompanied by expression of specific cell surface markers, chemokines and cytokines.
• The surface molecules CD86 and CD54 are typical markers of DC activation and play a critical role in T-cell priming by DCs, activating an immune response.
• The human monocytic leukemia cell line (THP-1) is used to quantify the expression of the two cell surface markers CD86 and CD54 using fluorescence-activated cell sorting (FACS). The changes in the expression level reflect the activation of DCs triggered by sensitisers.
• The cytotoxicity is also measured to conduct whether the upregulation of the surface markers CD86 and CD54 occurs at sub-cytotoxic concentrations.

"The third key event is the activation of dendritic cells (DC), typically assessed by expression of specific cell surface markers. These surface molecules CD86 and CD54 are typical markers of monocytic THP-1 cell activation and play a critical role in T-cell priming." 1 The elevation is detected with fluorescence-activated cell sorting (FACS) of the two markers CD86 and CD54.

Procedure

Principle of the h-CLAT

Data

Eurofins Data for demonstrating technical proficiency of the h-CLAT

n.a.: not specified in the guideline    EF = Eurofins Munich GmbH

Table 1: Eurofins h-CLAT data of nine tested proficiency chemicals compared to the data of the OECD guideline.¹

In Table 1 the obtained data from the h-CLAT of four non-sensitising and five sensitising chemicals are shown. The prediction of all tested chemicals was correct in comparison to the classification of the OECD guideline.

References

1. OECD Guidelines for Testing of Chemicals, No. 442E: “In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 09 October 2017.
2. UN (2015), United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Sixth revised edition, UN New York and Geneva.
3. EC EURL-ECVAM (2015). Recommendation on the human Cell Line Activation Test (h-CLAT) for skin sensitisation testing.

Kontakt

Direct Peptide Reactivity Assay (DPRA)

KeratinoSens^TM

Human Cell Line Activation Test (h-CLAT)

Genomic Allergen Rapid Detection (GARD TM)

in vitro Skin Irritaion

in vitro Skin Irritation of Medical Devices

Skin Corrosion

Genotoxicity Testing

'Genetic toxicology: choosing the right testing strategy'

Chromosome Aberration Test

HPRT Assay