Jojoba, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Test material

Test material information

Constituent 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Constituent 2

Reference substance name:

Jojoba Esters

Cas Number:

61789-91-1

IUPAC Name:

Jojoba Esters

Constituent 3

Reference substance name:

Alcohols, C14-22 and C16-22-unsatd.

EC Number:

268-107-7

EC Name:

Alcohols, C14-22 and C16-22-unsatd.

Cas Number:

68002-95-9

IUPAC Name:

Alcohols, C14-22 and C16-22 unsaturated

Constituent 4

Reference substance name:

Fatty acids, C14-22 and C16-22-unsatd., potassium salts

EC Number:

286-079-4

EC Name:

Fatty acids, C14-22 and C16-22-unsatd., potassium salts

Cas Number:

85186-93-2

IUPAC Name:

Fatty acids, C14-22 and C16-22 unsaturated, potassium salts

Test material form:

semi-solid (amorphous): gel

Specific details on test material used for the study:

the test product was applied diluted in the range from 0,98 to 2000 μM or from 0,196 to 400 μg/ml.

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

The test was performed by using the instaCELL® KeratinoSens® assay kit protocol (Accelerate, Cat N° SF220-01) in accordance with DB-ALM Protocol n° 155: KeratinoSens™ Skin Sensitisation & Allergic Contact Dermatitis and OECD Test No. 442D: In Vitro Skin Sensitisation assays addressing the AOP key event on keratinocyte activation.
The KeratinoSens® cells have been developed by stable transfection of a human keratinocyte cell line (HaCaT) with a luciferase reporter gene (pGL2, Promega) controlled by a Nrf2 responsive enhancer element from the AKR1C2 gene. Under nonstressed conditions, the Nrf2 transcription factor is covalently bound to Keap1 in the cytoplasm. This binding leads to ubiquitination and degradation of Nrf2 and therefore to inhibition of the antioxidant response. Under conditions of electrophilic stress, sensitizers covalently bind to Keap1, and Nrf2 is released into the nucleus. In the nucleus, Nrf2 binds to the promoter
region of the antioxidant response element (ARE) and initiates the expression of cytoprotective enzymes. The expression of luciferase in stressed KeratinoSens® cells is proportional to the activation of the Nrf2/Keap1 pathway activated by sensitizers.
KeratinoSens® cells has been validated by the ECVAM and adopted by the OECD TG 422D.
Cells were seeded in a 96 well plate at 37°C, 5% CO2 until cultures reach confluency.
Medium was replaced with new medium and the test product was applied diluted in the range from 0,98 to 2000 μM or from 0,196 to 400 μg/ml. The test product exposition last for 48 hours.
Resauzurin was added to each well for 4 hours at 37°C, then fluorescence at 540EX/590EM was measured to determine the viability of the cells

Cells were then washed with PBS and One-GloTM reagent was dispensed to each well. The plate was incubated again at room temperature in the dark for 20 min and luminescence was measured with an integration time of 1 s/ well. Test was performed in three trials.
Ethylene glycol dimethylacrylate (EGDMA) was tested as a positive control.
Assay buffer, containing 1% DMSO was used as negative control.

Vehicle / solvent control:

water

Negative control:

other: Assay buffer, containing 1% DMSO was used as negative control

Positive control:

EGDMA (120 M) [442D]

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

12.4 µg/mL

Cell viability:

valid

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

127.95 µg/mL

Cell viability:

Valid

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test material is a skin sensitizer