Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10-20 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)

Test material

Constituent 1
Reference substance name:
Essential oil of Canarium commune (Burseraceae) obtained from gum by steam distillation
EC Number:
945-898-3
Cas Number:
97675-63-3
Molecular formula:
not relevant for a UVCB substance
IUPAC Name:
Essential oil of Canarium commune (Burseraceae) obtained from gum by steam distillation
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Elemi oil
- Batch no.: 15/01509
- Analytical purity: 100% wt UVCB substance
- CAS No.: 8023-89-0
- EINECS-No.: 232-557-2
- Expiry date: Nov. 2016
- Date of Receipt: 16. Oct. 2015
- Appearance: courless to pale yellow liquid
- Homogeneity: Homogeneous
- Storage condition of test material: Room temperature 20 ± 5 °C, keep away from light/ humidity, heep under inert gas. The test item was stored in a closed aluminium vessel at 15.2-21 °C away from light and under inert gas.

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 at +5°C until pre-incubation.
- Description: The EpiOcular™ model consists of an airlifted, living, multi-layered 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.
- Expiry date: The EpiOcular tissues were used within 72 hours of their production.

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): Undiluted
Duration of treatment / exposure:
30±2 min at +37°C, 5% CO2 in a humidified incubator
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion : 12±2 min at room temperature
- Post-exposure incubation period: 120±15 min 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:
PRELIMINARY TESTS
- Test for direct MTT reduction with the test item:
50 μL of the test item were added to 1 mL of a 1.0 mg/mL freshly prepared MTT solution. A negative control was tested concurrently by adding 50 μL of sterile deionized water to 1 mL of a 1.0 mg/mL freshly prepared MTT solution. Both mixtures were incubated in darkness at 37°C for 3 hours (± 10 minutes) and the colour of the solutions obtained was evaluated.
- Test for the detection of the colouring potential of the test item:
50 μL of the test item 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 hours at room temperature. After that, the presence and intensity of the colouration were evaluated.

MAIN TEST
- Pre-incubation of the tissues: Pre-incubation was performed the day before treatment. For pre-incubation, tissues were equilibrated (in the 24-well shipping container) 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 hour. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at +37°C, 5% CO2 in a humidified incubator.
- Treatment and post-treatment incubation of the tissues: During each main test, the test item and both negative and positive controls were applied topically on duplicate tissues and incubated at 37°C 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 120 minutes (± 15 minutes) at 37°C, 5% CO2 in a humidified incubator.
- MTT viability assay: Following the post-treatment incubation, a volume of 0.3 mL of a freshly prepared MTT solution (1.0 mg/mL) was added into new wells of pre-labelled 24-well plates. At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were then transferred to the MTT pre-filled wells and incubated for 3 hours (± 10 minutes) at 37°C, 5% CO2 in a humidified incubator. At the end of the 3-hour incubation period, the underside of each tissue was blotted on absorbent paper to dry. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated. For the 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 so that isopropanol was flowing into the insert on the tissue surface. As the test item was a non-colourant liquid, the inserts were treated in the same way. Plates were covered with a sealer to prevent evaporation. 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 using a pipette and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate. One 96-well plate was used for the negative and positive controls (placed at opposite end of the plate), and a separate 96-well plate was used for test item-treated tissues.
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.

Results and discussion

In vitro

Results
Irritation parameter:
other: relative mean % viability of the tissues
Run / experiment:
Main test
Value:
88
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/white (with white discolouration in the centre of tissues) which was considered to be indicative of semi-viable tissue. 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 MTT results: The relative mean viability of the tissues treated with the test item was 88% 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. Refer Tables 7.3.2/1 and 7.3.2/2 for more details.

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.

OD (570 nm) measurements

Mean blank

cOD (570 nm) measurements

Mean cOD (570 nm)

Viability (%)

1st

2nd

1st

2nd

Negative control

30 min

1

1.786

1.814

0.039

1.747

1.775

1.761

105

2

1.611

1.629

1.572

1.590

1.761

95

Positive control

30 min

1

0.878

0.887

0.039

0.839

0.848

0.844

50

2

0.839

0.805

0.800

0.766

0.783

47

Test item

30 min

1

1.553

1.545

0.037

1.516

1.508

1.512

90

2

1.483

1.482

1.446

1.445

1.445

86

OD = Optical density

cOD = blank corrected optical density

 

 

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

cOD (570 nm)

Viability (%)

Mean

SD

Mean

SD

Difference (%)

Negative control

30 min

1.671

0.127

100

8

11

Positive control

30 min

0.813

0.043

49

3

4

Test item

30 min

1.479

0.047

88

3

4

cOD = blank corrected optical density

SD = Standard deviation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test substance is considered to be non-irritant to Reconstructed Human Cornea-like Epithelium; therefore it is not classified according to the Regulation (EC) N° 1272-2008 and to the GHS.
Executive summary:

An in vitro eye irritation test using the EpiOcular™ 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 substance.

 

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 test substance and both the negative and positive controls were applied topically on duplicate tissues of Reconstructed Human Cornea-like Epithelium, and incubated at +37°C for 30 min. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 min at room temperature to remove any remaining test substance absorbed into the tissue, blotted on absorbent material, and then incubated for another 120 min at 37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric 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 substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the relative mean viability of the tissues treated with the test substance was 88% 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.

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

 

Under the test conditions, the test substance is considered to be non-irritant to Reconstructed Human Cornea-like Epithelium; therefore it is not classified according to the Regulation (EC) N° 1272-2008 and to the GHS.