Essential oil of Boswellia carterii (Burseraceae) obtained from gum by distillation

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 - 26 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 492 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

05 March 2015

Test material

Test material information

1

Reference substance name:

1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene; 2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

EC Number:

946-037-4

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene; 2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Olibanum essential oil
- Batch no.: 0018454
- Analytical purity: 100% wt UVCB substance
- CAS No.: 8016-36-2
- EINECS-No.: 289-620-2
- Appearance: Pale yellow liquid
- Homogeneity: Homogeneous
- Storage condition of test material: Room temperature 20 ± 5 °C), keep away from light/ humidity; test item was stored in a closed aluminium vessel at 15.2-20.9 °C away from light and humidity.

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

Species: Reconstructed human Cornea-like Epithelium (tissues).
Supplier: MatTek, Bratislava, Slovak Republic.
Selection: at receipt, the tissues were inspected for obvious defects as they could have been rejected based on blistering, excess fluid or air bubbles below the tissue insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
Storage conditions: at receipt, the living EpiOcular™ tissues were stored as described in the Pre-incubation of the tissues on their day of arrival.

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): 50 μL
- Concentration (if solution): Undiiluted

Duration of treatment / exposure:

Test item, negative and positive controls were applied topically on duplicate tissues and incubated at 37 °C, 5% CO2 in a humidified incubator for 30 minutes (± 2 minutes).

Duration of post- treatment incubation (in vitro):

At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes (± 2 minutes) at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for another 120 minutes (± 15 minutes) at 37 °C, 5% CO2 in a humidified incubator.

Number of animals or in vitro replicates:

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

Details on study design:

RhCE tissue construct used: The EpiOcular™ model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinized epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotypic 3D cornea-like model. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue. The EpiOcular tissues were used within 72 h of their production.

Preliminary tests: Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its coloring potential.
- Test for direct MTT reduction with the test item: 50 μL of the test item or negative control were added to 1 mL of a 1.0 mg/mL MTT solution, then incubated in darkness at 37 °C for 3 h (± 10 minutes) and color of the solutions obtained was evaluated.
- Test for the detection of the coloring potential of the test item: The maximum amount of test item, 50 μL was added to 1 mL of water and incubated for at least 1 hour in the dark at 37 °C, 5% CO2 and 2 mL of isopropanol, incubated in a 6-well plate and placed on an orbital plate shaker for 2 to 3 h at room temperature. After that, the presence and intensity of the coloration were evaluated.

Main test:
- Doses of test chemical and control substances used: 50 μL
- Pre-incubation of the tissues: Tissues were equilibrated to room temperature for at least 15 minutes. The tissue inserts were transferred aseptically into the 6-well plate and pre-incubated at 37 °C, 5% CO2 in a humidified incubator for 1 h. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24 h) at 37 °C, 5% CO2 in a humidified incubator.
Treatment of tissues
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Test item and both negative and positive controls were applied topically on duplicate tissues and incubated at 37 °C, 5% CO2 for 30 minutes (± 2 minutes). At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes (± 2 minutes) at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for another 120 minutes (± 15 minutes) at 37 °C, 5% CO2 in a humidified incubator.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): One 6-well plate was used for the first test item-treated tissues. Positive and negative controls were placed on separate 6-well plates (one plate for each).Test item, negative and positive controls were applied on duplicate tissues.
- MTT viability assay: Following the post-treatment incubation, the cell viability was assessed by means of the colorimetric MTT reduction assay. Tissues were incubated with MTT solution in 24-well plates for 3 h (± 10 minutes) at 37 °C, 5% CO2 in a humidified incubator. At the end of the 3 h incubation period, tissues were blotted on absorbent paper and the degree of MTT staining was evaluated. For the test item, negative and positive control-treated tissues, the inserts were transferred to new wells of the 24-well plate containing 2 mL of isopropanol per well. Formazan extraction was performed overnight at 2-8 °C and protected from light.
- Optical Density measurements: At the end of the formazan extraction period, tissues (test item, negative and positive control-treated tissues) were pierced. The extract solution was mixed and two 200 μL aliquots were transferred to the pre-labeled 96-well plate. For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 μL of isopropanol only was used as the blank. The OD was measured at 570 nm using a plate reader.
Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

Results and discussion

In vitro

Results

Irritation parameter:

other: relative mean viability of the tissues (%)

Run / experiment:

30 minutes

Value:

98

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:

PRELIMINARY TESTS
Test for direct MTT reduction with the test item: The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.
Test for the detection of the colouring potential of the test item: During this test, as both water and isopropanol solutions containing the test item did not change colour, the test item was found not to have a colouring potential. As a result, no additional controls were used in parallel to the main test.

MAIN TEST
Evaluation of the colouration of tissues at the end of the MTT incubation period: Test item-treated tissues appeared to be blue which was considered indicative for viable tissues. Negative control tissues appeared to be blue and positive control tissues were white/blue (with blue colouration at the periphery of tissues only) which was considered to be indicative of viable and semi-viable tissues, respectively.
Evaluation of the MTT results: The relative mean viability of the tissues treated with the test item was 98% with a standard deviation of 3%. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.

ACCEPTANCE OF RESULTS:
- All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the study was considered to be valid.

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

 

Group	Exposure duration	Tissue No.	OD570 nm measurements		Mean blank	cOD570 nm measurements		Mean cOD570 nm	Viability (%)
			1st	2nd		1st	2nd
Negative control	30 min	1	1.950	1.889	0.037	1.913	1.852	1.883	106
		2	1.719	1.699		1.682	1.662	1.672	94
Positive control	30 min	1	0.893	0.873	0.037	0.856	0.836	0.846	48
		2	0.960	0.915		0.923	0.878	0.901	51
Test item	30 min	1	1.851	1.778	0.037	1.814	1.741	1.777	100
		2	1.740	1.721		1.703	1.684	1.693	95

 

Table 7.3.2/2: Main test - Mean tissue viability and standard deviations for the test item, the negative and positive controls

 

Group	Exposure duration	cOD570 nm		Viability (%)
		Mean	SD	Mean	SD	Difference (%)
Negative control	30 min	1.777	0.149	100	8	12
Positive control	30 min	0.873	0.039	49	2	3
Test item	30 min	1.735	0.059	98	3	5

 

OD = optical density

cOD = blank corrected optical density

SD = standard deviation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, OLIBANUM ESSENTIAL OIL, is considered to be non-irritant to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be: no category (GHS 2013 and Regulation (EC) No. 1272/2008).

Executive summary:

An in vitro eye irritation test on the EpiOcular^TM cornea epithelial 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.

 

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the eye irritation potential of the test item was assessed in the main test. The test item and both the negative and positive controls were applied topically on duplicate tissues and incubated at 37 °C for 30 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for another 120 minutes at 37 °C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

 

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.

 

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

The relative mean viability of the tissues treated with the test item was 98% with a standard deviation of 3%. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.

 

Under the experimental conditions of this study, the test item, OLIBANUM ESSENTIAL OIL, is considered to be non-irritant to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be: no category (GHS 2013 and Regulation (EC) No. 1272/2008).