Registration Dossier

Administrative data

Description of key information

- Skin irritation/corrosion: irritating (OECD 439 and OECD 431 , GLP, Rel. 1)

- Eye irritation:not classified (OECD TG 492, GLP, Rel. 1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: reconstituted epidermis
Cell type:
other: reconstituted epidermis (epiCS®, CellSystems®)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 0.60 cm2 reconstituted epidermis (epiCS®)

EXPOSURE
- The test item has been applied to the epidermal surface of 2 human skin model, during 3 minutes and during 1 hour.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 3 minutes and 1 hour after the test item application, the human epidermis was washed20 times with 20 mL of DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability is quantified by measurement of the cellular mitochondrial dehydrogenases activity. These enzymes are responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; EINECS number 206-069-5, CAS number 298-93-1)] reduction into blue formazan in the viable cells. The skin sample is placed in MTT solution of appropriate concentration (e.g. 0.3 or 1 mg/mL) for 2 hours and 55 minutes at 37°C ± 1°C. The precipitated blue formazan product is then extracted using a solvent (e.g. isopropanol), and the concentration of formazan is measured by determining the Optical Density (OD) at a wavelength between 540 and 600 nm (preferably 570 nm). The measured absorbances are proportional to the number of living cells.
The measurement of OD was performed using the ELx800 absorbance microplate reader supplied by BioTek and the validated software Gens ELISA V1.05.11 supplied by BioTek.

NUMBER OF REPLICATE TISSUES:
Duplicate skin tissues for test item, negative and positive controls

VIABILITY
Viability = (OD test item / OD negative control) x 100
For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, should be reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
3 minutes and 1 hour
Number of replicates:
Duplicate skin tissues for test item, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
main test (3 minutes)
Value:
96.99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not specified
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test (1 hour)
Value:
80.21
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
VIABILITY
- 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 96.99% and 80.21%, respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes; 3 minutes and 1 hour after the negative control application, the viability of the human skin model has been 100%.
- Acceptance criteria met for positive control: Yes; 1 hour after the positive control application, the viability of the human skin model has been 1.18%.

Deviation to the study plan

Acceptability criteria:
The mean OD of negative control tissues for the treatment of 3 minutes and for the treatment of 1 hour were respectively 1.246 and 1.282 instead of ≥ 0.3 and ≤ 0.9 as initially scheduled.

Considering the results obtained and the fact that this values remain in the range of our historical negative control data (see appendices 2 & 3), this deviation is considered as without impact on the conclusion of the study.

Table 7.3.1/1: Skin corrosion assay: Results

 

Skin

OD

Mean OD / disc (#)

Mean OD / product

Viability %

Mean viability %

Viability difference between replicates %

Treatment: 3 min

Negative control

11

0.672

0.710

0.730

97.26

100

5.5

0.744

0.712

12

0.752

0.750

102.74

0.755

0.741

Positive control

13

0.640

0.562

0.491

76.99

67.26

19.5

0.515

0.529

14

0.464

0.420

57.33

0.408

0.529

Test item

17

0.646

0.654

0.708

89.59

96.99

14.8

0.692

0.623

18

0.890

0.762

104.38

0.704

0.692

Treatment: 1 hour

Negative control

1

0.909

0.793

0.723

109.76

100

19.5

0.775

0.695

2

0.680

0.652

90.24

0.630

0.646

Positive control

3

0.010

0.009

0.009

1.25

1.18

0.1

0.008

0.008

4

0.006

0.008

1.11

0.008

0.008

Test item

7

0.586

0.581

0.580

80.42

80.21

0.4

0.572

0.584

8

0.687

0.578

80.00

0.535

0.511

#: mean of 3 values

OD: optical density

Note:

30 minutes exposure: If the viability obtained for the test substance is greater than 50%, then it is non-corrosive.

1 hour exposure: If the viability obtained for the test substance is greater than15%, then it is non-corrosive.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”.
The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.
Executive summary:

The test item was applied as supplied, at the dose of 50 μL, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 431 dated 28 July 2015 and the method B.40bis of the Council Regulation No. 440/2008.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 96.99% and 80.21% versus 67.26% and 1.18%, respectively, with the

positive control item (potassium hydroxide 8N).

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”.

The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2017 - february 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 439 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied
Test system:
human skin model
Source species:
other: reconstructed epidermises
Cell type:
non-transformed keratinocytes
Cell source:
other: foreskin
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17) were received, and on the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA) during 2 hours and 50 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA).

TREATMENT
- The test item was applied as supplied, at the approximate dose of 16 µL, on the epidermal surface of 3 living human skin models during 42 minutes at room temperature.
- In the same experimental conditions, a positive control (5% SDS) and a negative control (DPBS) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the human epidermises were washed with 25 x 1 mL of DPBS. The rinsed tissues were checked for any coloration and noted to be slightly brown instead of being whitish as for the coloration of the negative control tissues. Residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse. They were incubated for a 43 hours post-treatment incubation period in fresh medium between 35.4 and 38.6 °C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD were proportional to the number of living cells.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours between 35.4 and 38.6 °C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measurement of OD was performed 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.

VIABILITY CALCULATION:
- The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1.

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.
- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is "non-corrosive". In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.
- The test item is considered to be irritant or corrosive to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤50% and in absence of information on a skin corrosion test. In accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion test, the item has to be classified in Category 2 "Irritant" or in Category 1 "Corrosive". The corresponding hazard statement is respectively, "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger".
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
42 minutes at room temperature
Duration of post-treatment incubation (if applicable):
43 hours between 35.4 and 38.6 °C,
Number of replicates:
3 living human skin models
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
2.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
2.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
2.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues was 2.3%, versus 0.9% in the positive control (5% Sodium Dodecyl Sulfate).

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

 

Skin tissues

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Standard deviation (SD)

Negative control

1

1.130

1.206

1.123

107.4

100

6.8

1.250

1.239 

2

1.106

1.109

98.7

1.100 

1.120 

3

1.045

1.055

93.9

1.050

1.070

Positive control

4

0.009

0.010

0.010

0.9

0.9

0.1

0.010 

0.010 

5

0.011

0.011

1.0

0.010

0.011

6

0.010 

0.010

0.9

0.011 

0.010

Test item

10

0.025 

0.025

0.026

2.2

2.3

0.1

0.025 

0.024

11

0.027

0.027

2.4

0.028

0.027 

12

0.025 

0.025

2.2

0.024

0.025 

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Interpretation of results:
other: Category 2 (irritating to skin) or Category 1 (corrosive) based on GHS criteria
Conclusions:
Under the test conditions and in accordance with Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item has to be classified in Category 2 "Irritating to skin" or in Category 1 "Corrosive". The hazard statement "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger" are required.
Executive summary:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

The test item was applied as supplied, at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 43 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The mean percent viability of the treated tissues was 2.3%, versus 0.9% in the positive control (5% Sodium Dodecyl Sulfate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item has to be classified in Category 2 "Irritating to skin" or in Category 1 "Corrosive". The hazard statement "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger" are required.

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:
December 2017 - February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was used as supplied in the study
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
- Amount(s) applied (volume or weight with unit): 50 µL
- 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
- Post-exposure incubation period: 2 hours at standard culture conditions
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 12 tissues in their 24-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 55 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 (50 µL) to 2 living RhCE (EpiOcularTM 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 3 hours 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 18 hours and 50 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: Viability %
Run / experiment:
1
Value:
75.01
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability %
Run / experiment:
2
Value:
89.32
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 82.17% versus 18.36% in the positive control (Methyl acetate). Refer Tables 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

0.920

0.950

0.951

99.95

100.00

0.22

0.952

0.978

2

0.933

0.951

100.05

0.954

0.967

Positive control

3

0.023

0.025

0.175

2.63

18.36

31.46

0.026

0.027

4

0.318

0.324

34.09

0.321

0.334

Test item

9

0.685

0.713

0.781

75.01

82.17

14.31

0.725

0.730

10

0.830

0.849

89.32

0.846

0.873

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Acceptability criteria:

The difference of viability between the 2 tissues treated with the positive control was 31.46%, instead of 20% at the maximum as initially scheduled.

Considering the results obtained, this deviation is considered as without impact on the conclusion of the study.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance was not identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).
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 substance.

The test substance (50 µL) was applied to two 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 a MTT assay.

The mean percent tissue viability of the RhCE replicates treated with the test substance was 82.17% versus 18.36% in the positive control (Methyl acetate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance was not identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

The test item was applied as supplied, at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 43 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The mean percent viability of the treated tissues was 2.3%, versus 0.9% in the positive control (5% Sodium Dodecyl Sulfate).

Skin corrosion:

The test item was applied as supplied, at the dose of 50 μL, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 431 dated 28 July 2015 and the method B.40bis of the Council Regulation No. 440/2008.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 96.99% and 80.21% versus 67.26% and 1.18%, respectively, with the

positive control item (potassium hydroxide 8N).

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 substance.

The test substance (50 µL) was applied to two 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 a MTT assay.

The mean percent tissue viability of the RhCE replicates treated with the test substance was 82.17% versus 18.36% in the positive control (Methyl acetate).

Justification for classification or non-classification

Self - classification :

According to results from OECD 439 and OECD 431 study, the registered substance is classified as category 2 for skin irritation (H315) according to Regulation EC N° 1272/2008 (CLP) and GHS.

According to the results of OECD TG 492, the registered substance should not be classified according to Regulation EC N° 1272/2008 (CLP) and GHS.