Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 January to 21 April 2016
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:
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. certificate)
Remarks:
05 March 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): SAUGE SCLAREE ESS. / CLARY SAGE OIL
- Other name Salvia sclarea oil
- CAS No.: 8016-63-5
- EINECS-No.: 283-911-8
- Appearance: Pale yellow liquid
- Expiry date: Sep. 2018
- Date of Receipt: 18. Dec. 2015
- Batch no.: EE 86453
- Analytical purity: 100% wt UVCB substance
- Homogeneity: Homogeneous
- Storage condition of test material: Room temperature 20 ± 5 °C; test item was stored in a closed aluminium vessel at 15.2-20.9 °C away from light and humidity under inert gas.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Linalool consortium / EE 86453
- Physical state: Clear pale yellow liquid
- Date of receipt: 22 December 2015
- Expiration date of the lot/batch: December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature, protected from light and under nitrogen atmosphere

Test animals / tissue source

Species:
other: reconstructed 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 Preincubation 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:
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).
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 2 hours (± 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, including batch number: The EpiOcularTM (OCL-200, OCL-212) 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 hours of their production. Batch numbers 21590 and 23706 were used for the first and second main tests, respectively.

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 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 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: (i) 1 mL of water and incubated for at least 1 hour in the dark at 37 °C, 5% CO2 and (ii) 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 coloration were evaluated.

Main test:
- Doses of test chemical and control substances used: 50 μL

- Pre-incubation of the tissues: On the day before treatment, 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 (16-24 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: 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 another 2 hours (± 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 hours (± 10 minutes) at 37 °C, 5% CO2 in a humidified incubator. At the end of the 3-hour 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, the plates were placed under orbital shaking at room temperature for at least 15 minutes before using them. Then, 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). Two main tests were performed in this study and the final conclusion is based on results of both main tests.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: relative mean viability of the tissues (%)
Run / experiment:
First main test
Value:
76
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: relative mean viability of the tissues (%)
Run / experiment:
Second main test
Value:
70
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 tests.
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 tests.

MAIN TESTS
Evaluation of the colouration of tissues at the end of the MTT incubation period: In the first main test, all test item-treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue. In the second main test, the test item-treated tissue No. 1 appeared blue which was considered to be indicative of viable tissue and the tissue No. 2 was blue/white with some white spots at the center of tissue which was considered to be indicative of semi-viable tissue.
Evaluation of the MTT results:
First main test: With one exception (i.e. the relative mean viability of the positive control was 51% instead of < 50%), the acceptance criteria were fulfilled, therefore this first main test was considered to be valid. The relative mean viability of the tissues treated with the test item was 76% with a % difference of 8% between duplicate tissues.
Second main test: All acceptance criteria were fulfilled, therefore this second main test was considered to be valid. The relative mean viability of the tissues treated with the test item was 70% with a % difference of 0% between duplicate tissues. As the mean viability was > 60% after the MTT reduction in both runs, the results met the criteria for a non-irritant response.

Any other information on results incl. tables

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

 

Group

Exposure duration

Tissue No.

OD570 nmmeasurements

 

Mean blank

cOD570 nmmeasurements

 

Mean

cOD570 nm

Viability (%)

1st

2nd

1st

2nd

First main test

Negative control

30 min

1

1.522

1.497

0.037

1.485

1.460

1.473

92

2

1.750

1.751

1.713

1.714

1.714

108

Positive control

30 min

1

0.860

0.826

0.037

0.823

0.789

0.806

51

2

0.862

0.846

0.825

0.809

0.817

51

Test item

30 min

1

1.323

1.300

0.037

1.286

1.263

1.275

80

2

1.185

1.185

1.148

1.148

1.148

72

Second main test

Negative control

30 min

1

2.166

2.164

0.037

2.129

2.127

2.128

104

2

2.019

2.021

1.982

1.984

1.983

96

Positive control

30 min

1

0.905

0.909

0.037

0.868

0.872

0.870

42

2

0.830

0.839

0.793

0.802

0.798

39

Test item

30 min

1

1.438

1.506

0.037

1.401

1.469

1.435

70

2

1.467

1.463

1.430

1.426

1.428

69

 

Table 7.3.2/2: Main tests - 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 (%)

First main test

Negative control

30 min

1.593

0.170

100

11

15

Positive control

30 min

0.812

0.008

51

0

1

Test item

30 min

1.212

0.089

76

6

8

Second main test

Negative control

30 min

2.056

0.103

100

5

7

Positive control

30 min

0.834

0.051

41

2

4

Test item

30 min

1.432

0.005

70

0

0

 

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 and based on the two independent main tests, the test item SAUGE SCLAREE ESS. / CLARY SAGE OIL is considered to be non-irritating 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 teston the EpiOcularTM 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 coloring potential. Following the preliminary tests, the eye irritation potential of the test item was assessed in main tests. 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. 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 2 hours 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).Two main tests were performed in this study and the final conclusion is based on results of both main tests.

 

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

 

First main test: With one exception in the first main test (i.e.the relative mean viability of the positive control was > 50% (i.e.51%)), the acceptance criteria were fulfilled. This main test was therefore considered to be valid.

The relative mean viability of the tissues treated with the test item was 76% with a % difference of 8% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.

Second main test: Due to results of the positive control obtained just above the positivity threshold in the first main test and due to modification steps in the EpiOcular procedure, it was agreed with the Sponsor to perform a second main test in order to confirm the first ones. All acceptance criteria were fulfilled, therefore this second main test is considered to be valid. 

The relative mean viability of the tissues treated with the test item was 70% with a % difference of 0% between duplicate tissues. 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 and based on the two independent main tests, the test item SAUGE SCLAREE ESS. / CLARY SAGE OIL is considered to be non-irritating 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).