Star anise, Illicium verum, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

adopted February 2015

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: DRAFT KEY EVENT BASED TEST GUIDELINES 442D “In Vitro Skin Sensitisation assays addressing the AOP Key Event on: Keratinocyte activation”

Version / remarks:

Revised TG 442D – Draft v. 7 July 2017

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD. (22. May 2015). PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS. ENV/JM/MONO(2015)6.

Version / remarks:

22. May 2015

Deviations:

not specified

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EU-Method B.60 of the Commission Regulation (EU) No. 2017/735 adopted 14. Feb. 2017: “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method”

Version / remarks:

adopted 14. Feb. 2017

Deviations:

not specified

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ESAC Opinion No. 2016-04 of 24 June 2016: ESAC Opinion on the BASF coordinat-ed Performance Standards based validation of the LuSens test method for skin sensi-tisation testing

Version / remarks:

Opinion No. 2016-04 of 24 June 2016

Deviations:

not specified

Test guideline 6

Qualifier:

according to guideline

Guideline:

other: ASF SE: Protocol LuSens Assay, Last update: 16. May 2014 (reported by Ramirez et al. 2016)

Version / remarks:

Last update: 16. May 2014

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This in vitro study was performed to assess the potential of the test item Essential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillation to activate the Nrf2 transcription factor by using the genetically modified keratinocyte cell-line “LuSens” (Bauch et al. 2012).

Test material

Test material information

Constituent 1

Reference substance name:

(E)-anethole

EC Number:

224-052-0

EC Name:

(E)-anethole

Cas Number:

4180-23-8

Molecular formula:

C10H12O

IUPAC Name:

1-methoxy-4-prop-1-en-1-ylbenzene

Constituent 2

Reference substance name:

4-allylanisole

EC Number:

205-427-8

EC Name:

4-allylanisole

Cas Number:

140-67-0

Molecular formula:

C10H12O

IUPAC Name:

1-allyl-4-methoxybenzene

1

Reference substance name:

Caryophyllene

EC Number:

201-746-1

EC Name:

Caryophyllene

Cas Number:

87-44-5

Molecular formula:

C15H24

IUPAC Name:

4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene

Constituent 3

Reference substance name:

2,6-dimethyl-6-(4-methyl-3-pentenyl)bicyclo[3.1.1]hept-2-ene

EC Number:

241-702-9

EC Name:

2,6-dimethyl-6-(4-methyl-3-pentenyl)bicyclo[3.1.1]hept-2-ene

Cas Number:

17699-05-7

Molecular formula:

C15H24

IUPAC Name:

2,6-dimethyl-6-(4-methylpent-3-en-1-yl)bicyclo[3.1.1]hept-2-ene

Test material form:

liquid

In vitro test system

Details on the study design:

-Solvent: dimethyl sulfoxide (DMSO).
-Negative control: DL-Lactic acid.
-Positive control: EGDMA (Ethylene glycol dimethylacrylate)
-Cell line: LuSens cell line (BASF SE), are stored in liquid nitrogen in the cell bank of LAUS GmbH.
-Cell cultivate condition: In DMEM (9 % FCS (Fetal calf serum) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
-Cell viability measurement: MTT
-Chemicals and Media: Ca2+/Mg2+-Solution for PBS, EDTA Solution (250 g/L), FCS (Fetal Calf Serum) Superior, Lysepuffer Glo Lysis Buffer 1X.
-Test vessels: 96-well plates.


PERFORMANCE OF THE STUDY:
Cytotoxicity Range Finder Test:
Method: measuring the cell viability with MTT.
Exposure time: 48h
Nominal concentrations of the test item: 0.98 µg/mL, 1.95 µg/mL, 3.91 µg/mL, 7.81 µg/mL, 15.63 µg/mL, 31.25 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL, 1000 µg/mL, 2000 µg/mL.

Dose Selection for Experiment I and II:
the following 12 nominal concentrations were chosen for experiment I and II:
16.8 µg/mL, 20.2 µg/mL, 24.2 µg/mL, 29.1 µg/mL, 34.9 µg/mL, 41.9 µg/mL, 50.2 µg/mL, 60.3 µg/mL, 72.3 µg/mL, 86.8 µg/mL, 104.2 µg/mL, 125 µg/mL。

Results and discussion

Positive control results:

Experiment I: Induced a clear effect with an induction value of 4.0 fold in comparison to the solvent control.
Experiment II: Induced a clear effect with an induction value of 5.9 fold in comparison to the solvent control.

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

In vitro / in chemico

Results

Key result

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: luciferase induction ≥ 1.5 fold.

Remarks:

luciferase induction ≥ 1.5 fold.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test item, Essential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillation, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

Executive summary:

This in vitro study evaluates the potential of the test itemEssential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillationtoactivate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay).Since July 2017 a reviewed version of the OECD 442D is available, which includes the LuSens test. Therefore this study is performed in accordance to this draft OECD 442D with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on: Keratinocyte activation”.

The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (125 µg/mL) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.

A statistically significant and reproducible dose-dependent increase in luciferase induction≥1.5 fold in more than two non-cytotoxic consecutive test item concentrations was observed in both experiments.