Elemol-rich fraction of essential oil of Canarium commune (Burseraceae) obtained from exudate by fractional distillation

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-26 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 without any deviation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 28 July 2015

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(1S,2S,4R)-(-)-α,α-dimethyl-1-vinyl-o-menth-8-ene-4-methanol

EC Number:

211-360-5

EC Name:

(1S,2S,4R)-(-)-α,α-dimethyl-1-vinyl-o-menth-8-ene-4-methanol

Cas Number:

639-99-6

Molecular formula:

C15H26O

IUPAC Name:

2-[(1R,3S,4S)-4-ethenyl-4-methyl-3-prop-1-en-2-ylcyclohexyl]propan-2-ol

Constituent 2

Reference substance name:

5-allyl-1,2,3-trimethoxybenzene

EC Number:

207-649-0

EC Name:

5-allyl-1,2,3-trimethoxybenzene

Cas Number:

487-11-6

Molecular formula:

C12H16O3

IUPAC Name:

1,2,3-trimethoxy-5-prop-2-enylbenzene

Constituent 3

Reference substance name:

Dipentene

EC Number:

205-341-0

EC Name:

Dipentene

Cas Number:

138-86-3

Molecular formula:

C10H16

IUPAC Name:

4-isopropenyl-1-methylcyclohexene

Constituent 4

Reference substance name:

p-menth-1-en-8-ol

EC Number:

202-680-6

EC Name:

p-menth-1-en-8-ol

Cas Number:

98-55-5

Molecular formula:

C10H18O

IUPAC Name:

alpha,alpha-4-trimethyl-3-cyclohexene-1-methanol

Constituent 5

Reference substance name:

p-mentha-1,4(8)-diene

EC Number:

209-578-0

EC Name:

p-mentha-1,4(8)-diene

Cas Number:

586-62-9

Molecular formula:

C10H16

IUPAC Name:

4-isopropylidene-1-methylcyclohexene

Constituent 6

Reference substance name:

p-mentha-1,5-diene

EC Number:

202-792-5

EC Name:

p-mentha-1,5-diene

Cas Number:

99-83-2

Molecular formula:

C10H16

IUPAC Name:

5-isopropyl-2-methylcyclohexa-1,3-diene

Constituent 7

Reference substance name:

4-allylveratrole

EC Number:

202-223-0

EC Name:

4-allylveratrole

Cas Number:

93-15-2

Molecular formula:

C11H14O2

IUPAC Name:

1,2-dimethoxy-4-(prop-2-en-1-yl)benzene

Constituent 8

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

Constituent 9

Reference substance name:

Thuj-4(10)-ene

EC Number:

222-212-4

EC Name:

Thuj-4(10)-ene

Cas Number:

3387-41-5

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylenebicyclo[3.1.0]hexane

Test material form:

liquid

Details on test material:

Test item: Fraction of natural essential oil (Canarium commune)
CAS No.: 97675-63-3
EC No.: 307-636-0
Batch No.: *0617/1
Appearance: yellowish liquid
Storage conditions: Room temperature (10 °C - 30 °C), dark, dry, in tightly closed original container (aluminum bottle)
Expiry date: July 20, 2019

Test animals / tissue source

Species:

other: Reconstructed human Cornea-like Epithelia

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation]

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration: Undiluted

Duration of treatment / exposure:

30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)

Duration of post- treatment incubation (in vitro):

- Post-exposure immersion period: 12 minutes at room temperature

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues.

Details on study design:

MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the tissues in their well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 20 hours and 34 minutes at standard culture conditions.
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- Treatment and post-treatment incubation of the tissues:
The test item was applied, as supplied, at the dose of 50 μL, to 2 living PBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable RhCE tissue replicates during 30 minutes at standard culture conditions.
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for a 2-hour post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.
- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 2 hours and 45 minutes at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 17 hours and 56 minutes at 6±3°C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD measurement of formazan extracts at 570 nm was measured in triplicate samples using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Results and discussion

In vitro

Results

Irritation parameter:

other: % mean viability of the tissues

Run / experiment:

Main test

Value:

103.82

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test item was103.82% versus 21.76% in the positive control (Methyl acetate). Refer to Table 7.3.2/1 for more details.

Any other information on results incl. tables

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

	Tissue	OD	Mean OD/disc (#)	Mean OD/product	Viability %	Mean viability %	Difference of viability %
Negative control	1	0.969	0.955	0.981	97.35	100.00	5.30
		0.924
		0.974
	2	0.950	1.007		102.65
		1.031
		1.040
Positive control	1	0.195	0.214	0.214	21.81	21.76	0.10
		0.231
		0.216
	2	0.213	0.213		21.71
		0.215
		0.211
Test item	1	1.158	1.053	1.019	107.34	103.82	7.03
		1.019
		0.983
	2	1.139	0.984		100.31
		0.903
		0.910

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was identified as not requiring classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and no signal word are required.

Executive summary:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test item.

The test item was applied, as supplied, at the dose of 50 μL, to 2 living PBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 12-minute post-exposure immersion period at room temperature and a 2-hour post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

The mean percent tissue viability of the RhCE replicates treated with the test item was 103.82% versus 21.76% in the positive control (Methyl acetate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was identified as not requiring classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and no signal word are required.