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Administrative data

Description of key information

In an in vitro skin irritation study (OECD 439) test item was non irritant to skin (GLP, Rel. 1)

In an in vitro eye irritation and corrosion studies (OECD 492) test item was not considered as irritant to the eyes (GLP, Rel. 1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July - 24 August 2017
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)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
dated 23 July 2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
27 April 2017
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 1003398942
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 35 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 after powder reduction, at the approximate dose of 16 mg, 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 41 hours and 25 minutes post-treatment incubation period in fresh medium at 37°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 at 37°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 mg
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
42 minutes at room temperature
Duration of post-treatment incubation (if applicable):
41 hours and 25 minutes post-incubation period at 37°C, 5% CO2
Number of replicates:
3 living human skin models
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
mean value
Value:
119.7
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:
MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues was 119.7% (considered as 100%), versus 1.6% 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

OD

Mean OD / disc

(#)

Mean OD / product

Viability

%

Mean viability

%

Standard deviation

(SD)

Negative control

1

0.794

0.817

0.779

104.9

100.0

7.4

0.832

0.826

2

0.831

0.807

103.6

0.782

0.807

3

0.720

0.713

91.5

0.695

0.724

Positive control

1

0.013

0.013

0.013

1.7

1.6

0.2

0.013

0.013

2

0.011

0.011

1.4

0.011

0.011

3

0.015

0.014

1.8

0.014

0.014

Test item

1

0.748

0.740

0.932

95.0

119.7

22.2

0.721

0.752

2

1.154

1.076

138.1

1.128

0.947

3

0.988

0.981

125.9

0.894

1.061

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

OD: optical density

The difference of viability between the 3 tissues treated with the test item was 22.2%, instead of 18% at the maximum as initially scheduled.

 As the test item remains clearly classified as “Non irritant to the skin”, even when considering each replicate separately, this deviation has no impact on the conclusion and the validity 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 item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is 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 after powder reduction, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 41 hours and 25 minutes 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 corrected percent viability of the treated tissues was 119.7% (considered as 100%), versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
18-20 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 492 without any deviation
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)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 1003398942

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item was reduced in fine powder and applied directly to RhCE tissues.
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
- Concentration: Undiluted; test item was reduced in fine powder and applied directly to RhCE tissues.
Duration of treatment / exposure:
6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion period: 25 minutes at room temperature
- Post-exposure incubation period: 17 hours and 55 minutes 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 21 hours and 05 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:
As the test item was a paste considered as a solid, it was administered after being directly applied on a nylon mesh in order to cover the entire surface of the epidermis (corresponding to 50 mg). The test item was applied to 2 DPBS pre-treated RhCE (EpiOcularTM tissue model) during 6 hours 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 25-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 17-hour and 55-minute 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 17 hours and 05 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: % mean viability of the tissues
Run / experiment:
Main test
Value:
101.18
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:
MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 101.18% versus 26.37% 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

0.802

0.769

0.848

90.74

100.00

18.53

0.746

0.758

2

1.130

0.926

109.26

0.827

0.821

Positive control

1

0.237

0.244

0.224

28.79

26.37

4.84

0.238

0.256

2

0.201

0.203

23.95

0.202

0.206

Test item

1

0.883

0.877

0.858

103.48

101.18

4.60

0.880

0.866

2

0.850

0.838

98.88

0.840

0.824

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

OD: optical density

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 identified as not requiring classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and no signal word are required.
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 was reduced in fine powder and applied to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 25-minute post-exposure immersion period at room temperature and a 17-hour and 55-minute 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 101.18% versus 26.37% in the positive control (Methyl acetate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance was identified as not requiring classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and no signal word are required.

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 after powder reduction, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 41 hours and 25 minutes 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 corrected percent viability of the treated tissues was 119.7% (considered as 100%), versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

 

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

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 was reduced in fine powder and applied to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 25-minute post-exposure immersion period at room temperature and a 17-hour and 55-minute 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 101.18% versus 26.37% in the positive control (Methyl acetate).

 

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance was identified as not requiring classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and no signal word are required.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, the registered substance is not classified as skin irritant, and not classified as eye irritant according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).