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

Endpoint summary

Description of key information

- Skin irritation/corrosion: irritating, based on the rules of the CLP Regulation for classification of mixtures
- Eye irritation: non-irritant (OECD 492, GLP, K, Rel. 1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion, other

Remarks:

Classification based on calculation rules for mixtures of the CLP Regulation

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

no guideline required

Principles of method if other than guideline:

Classification based on calculation rules for mixtures of the CLP Regulation

Irritation parameter:

other: classification

Remarks on result:

other: skin irritant category 2

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Executive summary:

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the skin irritation/corrosion hazard of the registered substance. The decision of classification as skin irritant was based on existing data on constituents (additivity principles): the registered substance has more than 10% of its constituents classified as Skin irritant Category 2 (limonene, alpha-terpineol, alpha-phellandrene, beta-eudesmol and p-cymene); therefore, it should be classified as a skin irritant Category 2 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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]

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.

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the skin irritation/corrosion hazard of the registered substance. The decision of classification as skin irritant was based on existing data on constituents (additivity principles):the registered substance has more than 10% of its constituents classified as Skin irritant Category 2 (limonene, alpha-terpineol, alpha-phellandrene, beta-eudesmol and p-cymene); therefore, it should be classified as a skin irritant Category 2 without further testingaccording to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

Eye irritation:

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.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information and typical composition provided by the Lead Registrant, the registered substance has more than 10% of its constituents classified as Skin irritant Category 2 (limonene, alpha-terpineol, alpha-phellandrene, beta-eudesmol and p-cymene); therefore, it should be classified as a skin irritant Category 2 (H315) without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

According to the results obtained in a guideline in vitro eye irritation study on the registered substance, it does not need t be classified for eye irritation according to Regulation No 1272/2008 (CLP) and GHS.

No information was available regarding respiratory irritation.