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Skin Sensitisation: the human Cell Line Activation Test (h-CLAT)

The human Cell Line Activation Test (h-CLAT) is used to contribute to the assessment of the skin sensitisation potential of chemicals.

The method addresses the third key event (KE3) of the skin sensitisation Adverse Outcome Pathway (AOP) by quantifying changes in the expression of cell surface markers associated with the process of activation of monocytes and dendritic cells.

The h-CLAT method was jointly developed by Shiseido and Kao Corporation in Japan and has been evaluated in a EURL ECVAM coordinated validation study and subsequently underwent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC).

The method is adopted by the OECD as TG 442E.

The results of the validation study, the peer review of ESAC and our recommendations can be found on TSAR, the Tracking System for Alternative methods towards Regulatory acceptance.

Read more about the human Cell Line Activation Test (h-CLAT) on TSAR

Skin sensitisation

[collapsed]Skin sensitisation is the regulatory endpoint aiming at the identification of chemicals able to elicit an allergic response in susceptible individuals.

Following repeated exposure to a sensitising agent, the adverse health effect of allergic contact dermatitis (ACD) may be provoked. Thus the development of ACD is characterised by two distinct phases:

a) the induction of specialised immunological memory following the initial exposure to an allergen, called sensitisation and

b) elicitation of the clinical allergic response following subsequent exposure to the allergen.[/collapse]

The human Cell Line Activation Test (h-CLAT)

[collapsed]The h-CLAT measures the expression of the CD86 and CD54 membrane markers in THP-1 cells, a human monocytic leukemia cell line used as a surrogate model for dendritic cells. The expression levels of the markers are measured by flow cytometry following 24 hours of exposure to eight serial concentrations of test chemical selected on the basis of a pre-determined CV75 (i.e. the concentration of test chemical that allows 75% of cell survival).

The h-CLAT test method is designed to discriminate between sensitising and non-sensitising chemicals whereby chemicals are classified as sensitisers if the relative fluorescence intensity (RFI) of either CD86 and/or CD54 exceeds a defined threshold (i.e. RFI CD86≥150 and RFI CD54≥200) compared to the vehicle control wells at any tested concentration, in at least two out of three independent measurements (i.e. repetitions).

Cell viability is measured concurrently by Propidium Iodide (PI) staining and RFI values are considered for the prediction only if cell viability is above 50%.[/collapse]

Animal testing replacement

[collapsed]The test method addresses only one mechanisms of the cascade of events that leads to the development of skin sensitisation.

On its own the method is unlikely to replace any one particular animal test method. Rather, it is expected to be used in conjunction with other methods to be able to replace the current animal tests. Such complimentary information may come from other testing methods that address other key events involved in skin sensitisation.

Examples of such non-animal, alternative methods might include the Direct Peptide Reactivity Assay (DPRA) or the Keratinosens assay.

Currently skin sensitisation tests in animals are described here.[/collapse]

Validation study


In 2008, EURL ECVAM received a joint submission from Kao Corporation and Shiseido relating to h-CLAT. The method had previously been subjected to a variety of evaluation studies. On the basis of the information submitted the method was judged to be sufficiently mature enough to enter the EURL ECVAM validation process.

The validation of the method was conducted in the period November 2009 to November 2012.

The validation study was designed to evaluate the transferability and reliability of the method when challenged with 24 coded (blinded) chemicals. Secondary objectives included a preliminary evaluation of the ability of the method to reliably discriminate skin sensitising and non-sensitising chemicals.

The experimental design foresaw the testing of 24 coded test substances in three independent laboratories for the assessment of between-laboratory reproducibility. A subset of the test items were selected for additional testing to assess within-laboratory reproducibility.

Transferability was also assessed in the process of establishing the methods in each of the laboratories that took part.

In addition, it was foreseen that the generated data could be used to give only a preliminary assessment of predictive capacity since the DPRA cannot be used as one-to-one replacement of the animal tests.[/collapse]

Validation study outcomes

[collapsed]It was found that the information generated by h-CLAT was relevant to the assessment of the skin sensitisation potential of chemicals.

The method is transferable to laboratories experienced with cell culture techniques and flow cytometry. In terms of reproducibility we found that the within and between laboratories reproducibility were both in the order of 80%.

The accuracy of h-CLAT in terms of discriminating the sensitisers and non-sensitisers tested in the validation study was in the region of 76%.[/collapse]

EURL ECVAM recommendations

[collapsed]On the basis of the validation study performed by EURL ECVAM, and reports from the scientific literature, the following main recommendations have been made:

  • Data generated with the h-CLAT method should prove valuable as part of integrated approaches to testing and assessment (IATA) together with complementary information (e.g. other in vitro data, QSAR and/or read-across predictions).
  • The method also generates concentration-response information that may contribute to the assessment of skin sensitisation potency e.g. by supporting, within an integrated approach, the subcategorisation of sensitisers. However, more work is needed to determine to which extent h-CLAT results can contribute to potency prediction.
  • According to a subsequent study performed by EURL ECVAM and a group of experts, the method is able to correctly identify the majority of tested chemicals requiring activation to act as sensitisers.
  • EURL ECVAM fully support the development of an OECD test guideline for this method.
  • h-CLAT data should be considered before any animal experiments are performed and that such data should be used in the context of weight-of-evidence judgements.[/collapse]