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KeratinoSens (TM)

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In Vitro Skin Sensitisation Assay (ARE-Nrf2 Luciferase Test Method)

The in vitro KeratinoSensTM assay addresses the second molecular key event of the adverse outcome pathway (AOP) of skin sensitisatio.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 KeratinoSensTM assay 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 442D at Eurofins BioPharma Product Testing Munich GmbH1, 3 with chemicals, cosmetics or personal care products and pharmaceuticals.

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

 

Assessment of Skin Sensitisation Potential with the KeratinoSensTM

  • The second molecular key event of skin sensitisation addresses the induction of cyto-protective signaling pathways like the Keap1-Nrf2-ARE pathway in keratinocytes in responds to electrophiles and oxidative stress.
  • The effect on the antioxidant response element (ARE) dependent pathway is assessed by measuring the induction of the luciferase gene, an ARE-dependent gene product, with luminescence detection.
  • The luciferase signal reflects the activation of endogenous Nrf2-dependent genes which are triggered by sensitisers.1, 3, 4
  • An MTT assay is performed, too, to see if the cells are stressed or if the elevation of the luminescence is due to sensitising properties of the test item.

 

Eurofins KeratinoSens Assay

"Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). Small electrophilic substances such as skin sensitisers can act on the sensor protein Keap1, by e.g. covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2. The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes."1 As a result, a higher luciferase activity is present and the cells are viable >70%.

 

Ostern

Procedure

Principle of the KeratinoSensTM

Protocol

Cell line

KeratinoSensTM cell line

Immortalised adherent human keratinocytes (HaCaT)
stably transfected with a selectable plasmid

Analysis

Induction of luciferase reporter gene expression measured by luminescence
Cell viability determination

Concentrations

Triplicates of 12 stock solutions

Final concentrations range from 0.98 to 2000 µM
Alternative concentrations possible (e.g. bad solubility)

Exposure time

48 h

Quality controls

Positive control: cinnamic aldehyde (4 to 64 µM)

Solvent control: 1% dimethylsulfoxid, medium, 1% tetrahydrofuran

Solvents of test chemical

Dimethylsulfoxid

(Sterile) water

Tetrahydrofuran

Application

Two independently performed experiments

Equivocal results require a third repetition

Data delivery

Maximal fold induction (Imax)

EC1.5 value of luciferase activity

IC30 and IC50 values of cell viability

Dose response curves for luciferase activity induction and cell viability

Positive prediction

Significant luciferase activity induction >1.5 fold

EC1.5 is <1000 µM

Cell viability >70%

Dose response for luciferase induction

 

Data

Eurofins data for demonstration technical proficiency of the KeratinoSensTM Assay

Chemical

EC1.5*
(OECD)

IC50# (OECD)

Prediction (OECD)

EC1.5* (EF)

IC50#
(EF)

Prediction (EF)

Non-sensitising Chemicals

Isopropanol

>1000

>1000

negative

>2000

>2000

negative

Salicylic acid

>1000

>1000

negative

>2000

>2000

negative

Lactic acid

>1000

>1000

negative

>2000

>2000

negative

Glycerol

>1000

>1000

negative

>2000

>2000

negative

Sensitising Chemicals

Cinnamyl alcohol

25-175

>1000

positive

139.01

>2000

positive

Ethylene glycol dimethacrylate

5-125

>500

positive

35.53

1811.39

positive

2-Mercapthobenzothiazole

25-250

>500

positive

91.92

>2000

positive

Methyldibromo glutaronitrile

<20

20-100

positive

9.74

28.46

positive

4-Methylaminophenol sulfate

<12.5

20-200

positive

8.07

25.12

positive

2,4-Dinitro-chlorbenzene

<12.5

5-20

positive

3.19

12.16

positive

* = µg/mL; # = µM                                     EF = Eurofins Munich GmbH

Table 1: Eurofins data of the KeratinoSensTM – ten tested proficiency chemicals compared to the data of the OECD guideline.1

 

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

 KeratinoSens_Cinnamic Aldehyde

 

 KeratinoSens_Glycerol

 

Figures 1 and 2: Eurofins KeratinoSens™ data of the fold induction and the viability of the sensitiser cinnamic aldehyde (Figure 1) and the non-sensitiser glycerol (Figure 2).

The graphs show the induction of the luciferase gene expression (blue line) and MTT data (grey line) used to determine cytotoxicity of the chemicals cinnamic aldehyde and glycerol. The red line represents the threshold of luciferase induction, at which chemicals are classified as sensitisers.

 

References

  1. OECD Guidelines for Testing of Chemicals, number 442d “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method” (adopted: June 25, 2018).
  2. UN (2015), United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Sixth revised edition, UN New York and Geneva.
  3. EURL-ECVAM (2014). Recommendation on the KeratinoSens™ assay for skin sensitization testing, 42 pp.
  4. Emter R., Ellis G., Natsch A.(2010). Performance of a novel keratinocyte-based reporter cell lineto screen skin sensitizers in vitro. Toxicology and Applied Pharmacology 245, 281-290.