Star anise, Illicium verum, ext.

Eye irritation

Administrative data

Endpoint:

eye 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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 09. Oct. 2017

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD 160: “GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLEC-TION OF DATA ON NON-SEVERE IRRITANTS”; 25. Oct. 2011

Version / remarks:

25. Oct. 2011

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

adopted 14. Feb. 2017

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

Test animals / tissue source

Species:

cattle

Strain:

other: Bos primigenius Taurus (fresh bovine corneas)

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt So-lution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour 30 minutes.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Test item: 750 µL
Positive control: 750 µL
Negative control: 750 µL

- Concentration (if solution):
Test item: Tested directly, without dilution or preparation of a solution
Positive control: Dimethylformamide, DMF, CAS-No. 68-12-2, undiluted, batch no.: 475235719
Negative control: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. wa-ter (1:10), batch no.: 20180503

Duration of treatment / exposure:

10 minutes at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 hours at 32 ± 1 °C

Number of animals or in vitro replicates:

3

Details on study design:

-SELECTION AND PREPARATION OF CORNEAS:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt So-lution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour 30 minutes.
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.

-QUALITY CHECK OF THE ISOLATED CORNEAS:
The corneas were examined and only corneas which were free from damages were used

-NUMBER OF REPLICATES: 3

-NEGATIVE CONTROL USED: Yes (HBSS)

-POSITIVE CONTROL USED: Yes (Dimethylformamide (undiluted))

-APPLICATION DOSE AND EXPOSURE TIME: 750 µL, 10min

-TREATMENT METHOD: Closed chamber

-POST-INCUBATION PERIOD: Yes (2 hours at 32 ± 1 °C)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
Rinsing the anterior chamber with cMEM with phenol red and final rinsing with cMEM without phenol red.
- POST-EXPOSURE INCUBATION:
90 minutes at 32 ± 1 °C.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

2.95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	0.93	1.01	70.18%
	1.76
	0.35
Test Item Essential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillation	3.64	2.95	20.99%
	2.75
	2.45
Positive Control DMF undiluted	102.78	100.81	3.37%
	96.89
	102.75

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Executive summary:

Two experiments were performed, but the first experiment was aborted, because of a technical problem at the opacitometer. The raw data are kept in the GLP-archive of the test facility but are not reported. The second experiment performed was valid.

 

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test itemEssential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillationwas brought onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In VitroIrritancy Score) is 1.01.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 100.81.

 

Under the conditions of this study, the test itemEssential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillationshowed no effects on the cornea of the bovine eye. The calculated IVIS is 2.95.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.