Star anise, Illicium verum, ext.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Version 439, adopted 28. July 2015

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted 06. Jul. 2012

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

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

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Brati-slava.

Details on animal used as source of test system:

The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissues are cultured on specially prepared cell culture inserts.
Origin: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Brati-slava.
Designation of the kit: EPI-200-SIT
Day of delivery: 12. Jun. 2018
Batch no.: 28623

Justification for test system used:

The test consists of a topical exposure of the neat test item to a human reconstructed epi-dermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreated nega-tive controls is used to predict the skin irritation potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 2
DYE BINDING METHOD
- Dye used in the dye-binding assay: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT)
- Spectrophotometer: Plate spectrophotometer
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 (Pre-Tests, Pre-Incubation of Tissues and Treatment)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL test item+ nylon mesh

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS buffer + nylon mesh

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% SDS solution + nylon mesh

Duration of treatment / exposure:

23 hours and 20 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2

Duration of post-treatment incubation (if applicable):

19 hours and 10 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

58.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 58.3 %+-4.4%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Essential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillation is considered non-irritant to skin in the Reconstructed human Epi-dermis (RhE) Test Method.

Executive summary:

One valid experiment was performed.

Three tissues of the human skin model EpiDerm^TMwere treated withEssential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillationfor 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63  cm²; as indicated by the supplier).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 <= mean OD <= 2.8, OD was 1.5. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.8% (required: <=20%).

The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: <= 18%).

 

After the treatment with the test item, the mean value of relative tissue viability was reduced to 58.3 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non- irritant to skin.