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The KeratinoSens assay is used to contribute to the assessment of the skin sensitisation potential of chemicals. The method addresses the second key event (KE2) of the skin sensitisation Adverse Outcome Pathway (AOP) by measuring the activation of a cytoprotective (the Keap1-Nrf2-ARE) pathway in keratinocytes.

The KeratinoSens was developed by Givaudan, a producer of fragrances and flavours. Between 2009 and 2010, the company conducted a validation study that focussed on the transferability and reproducibility of the method.

Upon completion of the study, Givaudan submitted to EURL ECVAM all the relevant information for formal evaluation. The method was then peer-reviewed by the EURL ECVAM Scientific Advisory Committee (ESAC).

The method is adopted by the OECD as TG 442D and by the EU as TM B60.

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 KeratinoSens assay on TSAR

Skin sensitization

[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.[/collapsed]

KeratinoSens assay for skin sensitisation testing

[collapsed]The KeratinoSens is a reporter gene assay which uses an immortalised adherent cell line derived from an expanded clone of HaCaT human keratinocytes. The cell line is transfected with a selectable plasmid which contains the luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from the AKR1C2 gene.

Measurement of the luciferase gene induction, using well established light producing luciferase substrates, is used as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic chemicals. The pathway is considered a major regulator of responses to oxidative stress. Since most skin sensitisers are electrophiles and therefore capable of inducing this type of stress in cells, the pathway is regarded as relevant for skin sensitisation.

In the KeratinoSens chemicals are classified as sensitisers if they induce a statistically significant induction of the luciferase gene above a given threshold in two out of three experiments performed on different days. This is established in parallel to cytotoxicity measurements to assess gene induction levels at sub-cytotoxic concentrations.

Since cells are exposed to 12 concentrations of the test chemicals, the concentration needed for a statistically significant luciferase gene induction above the threshold (EC1.5 value) can be extrapolated from the dose response curve. In addition, the maximal fold induction of the luciferase gene over solvent control (Imax) is determined.[/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 human cell line activation test (h-CLAT).

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

Validation study

[collapsed]Givaudan coordinated a validation study of the KeratinoSens test method in 2009 and 2010 with a focus on its transferability and reproducibility within and between laboratories.

Once that study was completed, Givaudan submitted the data to us for further evaluation. Subsequently we requested further information on the within laboratory reproducibility of the method and together all that data was then independently peer-reviewed by ESAC.

Their review was finalised in December 2012. Based on the data received and the opinion of ESAC, EURL ECVAM published the recommendation on the method in February 2014.[/collapse]

Validation study outcomes

[collapsed]Based on data supplied, the test method proved to be transferable to laboratories experienced in cell culture and the within- and between-laboratories reproducibility for positive/negative predictions were both 86%.

The Givaudan-coordinated validation study generated preliminary information on the test method's predictive capacity and it was found that the accuracy of the KeratinoSen to discriminate skin sensitisers from non-sensitisers was 90% (sensitivity 87%, specificity 100%; n=21).

The accuracy calculated for an additional set of chemicals (77 sensitisers and 104 non-sensitisers) tested in-house by Givaudan was 75%.

Finally, there were indications that the method could provide concentration-response information that may contribute to assessment of sensitising potency.[/collapse]

EURL ECVAM recommendation

[collapsed] On the basis of the validation study that was performed by Givaudan, subsequent data they provided and reports from the scientific literature, EURL ECVAM made the following main recommendations:

  • The data produced by the KeratinoSens test method should prove valuable as part of a integrated approaches to testing and assessment (IATA) together with other with complementary information to replace relevant animal-based studies for skin sensitisation.
  • To support such development of integrated approaches, the applicability of the KeratinoSenstest method to other type of chemicals than those already tested, should be further characterised. Based on the data either received or requested, the assay seems to be nevertheless applicable to a wide range of chemicals. However, negative results should be interpreted with some caution because of the specific cysteine-dependent mechanism of activation of the signalling pathway and because some chemicals requiring biotransformation by P450 enzymes to act as sensitisers are not correctly detected. While a variety of pre-haptens (i.e. chemicals activated by auto-oxidation) are correctly detected by the assay, those with a slow oxidation rate may also provide false negative results.
  • Data from the KeratinoSens test method 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]