Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January to 04 August 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 deviations.

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
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name: Lavandin Essential oil
- Other name: Lavandin Grosso; Lavandin Grosso France; Lavandula x hybrida, Lavandula hybrida oil; lavender, lavandula hybrida, ext.; Huile essentielle de lavandin.
- Batch no.: 150095
- CAS No.: 91722-69-9
- EINECS-No.: 294-470-6
- Appearance: clear, yellow liquid
- Purity: 100% wt-UVCB substance
- Homogeneity: homogeneous
- Expiry date: 7 october. 2018
- Storage: Fridge (2-8°C), keep away from light, keep under inert gas.The test item was stored in a closed aluminium vessel at 2.5 – 5.0 °C, away from light, under inert gas.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 150095
- Physical state: Transparent liquid
- Dates of receipt: 15 January 2016 (used for the preliminary and invalidated first main test); 09 February 2016 (used for the validated first main test); 23 May 2016 (used for the second main test); 04 July 2016 (used from the third main test)
- Expiration date of the lot/batch: 30 September 2018
- Purity test date: 11 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in the refrigerator set at 5 °C, protected from light, under nitrogen atmosphere

Test animals / tissue source

Species:
other: Reconstructed Human cornea-like epithelium
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:
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 23700; 23711 and 23722 were used for the first, second and third 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 of treatment for the first main test or on the day before treatment for the second and third main tests, 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).

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: relative mean viability of the tissues
Run / experiment:
First main test
Value:
64
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.
Irritation parameter:
other: relative mean viability of the tissues
Run / experiment:
Second main test
Value:
53
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
non concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.
Irritation parameter:
other: relative mean viability of the tissues
Run / experiment:
Third main test
Value:
59
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissue led to non-concordant classification, this was considered as a borderline result.
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 coloring potential of the test item: During this test, as both water and isopropanol solutions containing the test item did not change color, the test item was found not to have a coloring potential. As a result, no additional controls were used in parallel to the main test.

MAIN TESTS
Evaluation of the coloration of tissues at the end of the MTT incubation period: All test item-treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue.
Evaluation of the MTT results:
First main test: The relative mean viability of the tissues treated with the test item was 64% with a difference of 1% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response. However, since a mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.
Second main test: The relative mean viability of the tissues treated with the test item was 53% with a difference of 3% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However, since non concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.
Third main test: The relative mean viability of the tissues treated with the test item was 59% with a difference of 13% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However since mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissue led to non-concordant classification, this was considered as a borderline result.
Two out of three main tests gave irritant responses (with one of them with borderline results, i.e. mean % viability equal to 60 ± 5%). When tissues (from all main tests) are considered individually: 50% gave irritant responses (i.e. below 60% viability) and 50% non-irritant response. Even if two out of three main tests gave borderline results, a fourth main test would not be necessary. When all main tests are considered together, the mean %viability tends to borderline results with positive classification (irritant), indeed a significant decrease of viability is obtained. As a result and under our experimental conditions, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium.

ACCEPTANCE OF RESULTS:
- All of the acceptance criteria for the negative and positive controls were fulfilled, therefore each main test was considered as valid.

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

1.847

1.850

0.037

1.810

1.813

1.812

96

2

2.001

1.993

1.964

1.956

1.960

104

Positive control

30 min

1

0.686

0.695

0.037

0.649

0.658

0.654

35

2

0.774

0.780

0.737

0.743

0.740

39

Test item

30 min

1

1.245

1.236

0.038

1.207

1.198

1.202

64

2

1.261

1.255

1.223

1.217

1.220

65

Second main test

Negative control

30 min

1

1.992

1.936

0.041

1.952

1.896

1.924

95

2

2.154

2.172

2.114

2.132

2.123

105

Positive control

30 min

1

0.947

0.953

0.041

0.907

0.913

0.910

45

2

0.775

0.773

0.735

0.733

0.734

36

Test item

30 min

1

1.145

1.143

0.037

1.108

1.106

1.107

55

2

1.091

1.084

1.054

1.047

1.050

52

Third main test

Negative control

30 min

1

1.992

1.990

0.043

1.949

1.947

1.948

102

2

1.909

1.924

1.866

1.881

1.873

98

Positive control

30 min

1

0.679

0.677

0.043

0.636

0.634

0.635

33

2

0.631

0.632

0.588

0.589

0.588

31

Test item

30 min

1

1.040

1.039

0.042

0.998

0.997

0.997

52

2

1.290

1.289

1.248

1.247

1.247

65

 

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

1.886

0.105

100

6

8

Positive control

30 min

0.697

0.061

37

3

5

Test item

30 min

1.211

0.012

64

1

1

Second main test

Negative control

30 min

2.023

0.141

100

7

10

Positive control

30 min

0.822

0.124

41

6

9

Test item

30 min

1.079

0.040

53

2

3

Third main test

Negative control

30 min

1.911

0.053

100

3

4

Positive control

30 min

0.612

0.033

32

2

2

Test item

30 min

1.122

0.177

59

9

13

 

OD = optical density

cOD = blank corrected optical density

SD = Standard deviation

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under the experimental conditions of this study and based on results of three independent and validated main tests, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be:category 2 (GHS 2013) and category 2 (H319) (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).

 

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

 

First main test: The relative mean viability of the tissues treated with the test item was 64% with a difference of 1% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response. However, since a mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.

Second main test: The relative mean viability of the tissues treated with the test item was 53% with a difference of 3% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However, since non-concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.

Third main test: The relative mean viability of the tissues treated with the test item was 59% with a difference of 13% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However since mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissues led to non-concordant classification, this was considered as a borderline result.

Two out of three main tests gave irritant responses (with one of them with borderline results,i.e.mean % viability equal to 60 ± 5%). When tissues (from all main tests) are considered individually: 50% gave irritant responses (i.e.below 60% viability) and 50% non-irritant response. Even if two out of three main tests gave borderline results, a fourth main test would not be necessary. When all main tests are considered together, the mean % viability tends to borderline results with positive classification (irritant), indeed a significant decrease of viability is obtained. As a result and under our experimental conditions, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium.

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

 

Under the experimental conditions of this study and based on results of three independent and validated main tests, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be: category 2 (GHS 2013) and category 2 (H319) (Regulation (EC) No. 1272/2008).