In vitro skin sensitisation evaluation: Spotlight on the SENS-IS

Thanks to its design, the SENS-IS is a test of choice for the assessment of the sensitising potential of cosmetics. It can be used for many types of cosmetic ingredients even those that are poorly soluble, it allows to estimate the sensitising potency from weak sensitiser to extreme sensitiser, it is particularly adapted to formulations and represents a promising candidate for HRIP replacement.

To understand why SENSIS is a very good test, let's go back to the fundamentals of in vitro skin sensitisation evaluation.

The determination of skin sensitisation potential is one of the most important toxicological endpoint in the development and evaluation of ingredients used in fragrance, cosmetic and personal care products.

Since the total animal testing ban in March 2013, in vitro skin sensitisation tests are still one of the current hot topics in the world of cosmetic ingredients evaluation. During the last few years numerous of in vitro tests have been developed.

The approach used for in vitro sensitisation tests development is based on the AOP concept (Adverse Outcome Pathway1) summarised below:

AOP concept relies with the analysis of every step of the mechanistic reactions involved in the sensitisation phenomenon, from the chemical structure and properties of the potential sensitiser, to the organism response.

It allows to determine 4 major events names “Key Event”, numbered from 1 to 4. The sensitisation mechanism cannot be summarized with only one Key Event, the aim is to cover the maximum of Key Event.

Among the in vitro alternative methods developed we can cite the followings:

  • KE1: DPRA
  • KE2: KeratinoSens, LuSens, SENS-IS, IL18 assessment
  • KE3: h-CLAT, U-SENS, GARD, VitoSens, IL8-Luc assay
  • KE4 being at the level of organ, there is no in vitro test for this step.

Five test are fully validated and are covered by an OCDE Guideline: DPRA (OECD 442C), KeratinoSens and LuSens (OCDE 442D) and h-CLAT, U-SENS and IL8-Luc assay (OECD 442E).

For SENS‑IS and GARD an external validation has been completed and is under review at the ESAC.

Among them, one is of particular interest: the SENS-IS.

This test has been developed by the company Immunosearch (Grasse – France) and the overall procedure is described on the following scheme.

The test is based on the analysis of the expression of a set of genes involved in sensitisation pathway on reconstructed human epidermis models (Episkin) using RT-qPCR method.

To address all the aspects of complexity of skin sensitisation and to take into account the variety of different types of chemical sensitisers, SENS-IS uses a set of 62 biomarkers split into 3 groups: a group of 24 genes to measure skin irritation, a group of 17 genes involved antioxidant pathways (ARE genes) and a group of 21 genes involved in sensitisation (SENS-IS genes).

A molecule is classified as positive if at least 7/17 genes in ARE genes group and/or - 7/21 genes in SENS-IS genes group are significantly induced2.

A ring-study was performed that showed good sensitivity, specificity and accuracy (respectively 97.7%, 95.2% and 96.6%) against LLNA data3.

Due to his design, the SENS-IS exhibits some characteristics that make it a test of choice for the assessment of the sensitising potential of cosmetics.

  • We can consider that it is addressing the 3 first key events of the AOP: the KE1 through the expression of genes under the control of « sensor » proteins, the KE2 through the expression of genes indicating keratinocytes activation and the KE3 through the expression of genes implicated in dendritic cells activation and maturation

  • It can be used for many types of cosmetic ingredients even those that are poorly soluble. And due to the topical application, it is particularly adapted to formulations and represents a promising candidate for HRIP replacement

  • The test item is applied on the skin model at 4 different concentrations (50%, 10%; 1% and 0.1%). According to the positivity observed at the different concentration, it is therefore possible to estimate the sensitizing potency from weak sensitiser if positive at 50% to extreme sensitiser if positive at 0.1%. It is therefore a real alternative to LLNA for the evaluation of fragrance ingredients’ NESIL.

However, due to its complexity and the fact that no specific step has been identified for the determination of the potency of an allergen, the sensitisation mechanism cannot be summarized with only one KE.

And despite of the clear advantages of the SENS-IS in regards with other tests, an Integrated Testing Strategy (ITS) is needed to cover the maximum of KE for the skin sensitisation hazard identification and classification.

There is no official or recommended ITS and many individual strategies are described in the annex I of the OECD No 256 document 4.

Ideally, the ITS should target the KE1, KE2 and KE3, which is theoretically the case for the SENS-IS.

The approach often used is a "2 out of 3" strategy. 3 tests, i.e. DPRA, KeratinoSens and h-CLAT are usually performed together in combination and 2 of 3 tests must be matching for rating.

The SENS-IS, already used for sensitisation in vitro test by major cosmetic companies, is now a fourth tests complementing this battery.

The 3 tests can be performed sequentially and some companies adopted the strategy to stop at the first one if the result is positive. The SENS-IS is often used as the first test.

In addition, due to the potential complexity of samples the DPRA is often not applicable and a fourth test should be used. In that case the most currently used test is the SENS-IS, still with a "2 out of 3" approach.

Whatever your project or your product is, the sensitisation potential of an ingredient is a critical element of the safety assessment and IDEA Lab can help you to establish the right testing strategy according to the type of sample and in combination with the other toxicological tests.

Frédéric NUNZI, PhD
Doctor in Cell Biology and Microbiology / Head of IDEA Lab
Eurotox Expert (Regulatory and in vitro Toxicology)
f.nunzi@groupeideatests.com


References

1. McKay, C. et al., (2013) From pathways to people: applying the Adverse Outcome Pathway (AOP) for skin sensitisation to risk assessment. Altex 30, p473-486
2. Cottrez, F. et al, (2015) Genes specifically modulated in sensitized skins allow the detection of sensitizers in a reconstructed human skin model. Development of the SENS-IS assay. Toxicology in Vitro 29 (2015) 787–802
3. Cottrez, F. et al, (2016) SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitisation potency: Reproducibility and predictivity results from an inter-laboratory study. Toxicology in Vitro 32 (2016) 248-260
4. OECD No 256 – Series on Testing and Assessment - Annex I: Case Studies to the Guidance Document on the Reporting of Defined Approaches and Individual Information Sources to be used within Integrated Approaches to testing and Assessment (IATA) for Skin Sensitization. Oct. 2016.

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