Registration Dossier

Administrative data

Description of key information

- Skin Corrosion/ irritation : irritating based available results: (OECD 439, GLP, Rel.1 and Read across for OECD 431, GLP, Rel1)

- Seriou eye damage/ eye irritant: Eye damage category 1, based on the rules of the CLP Regulation for classification of mixtures

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-14 September 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:
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 HUMAN EPIDERMIS (RHE) TISSUE
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 10 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 including two RHE models for the non-specific colour (NSC) control.
- Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction. Moreover, 2 living and 2 killed Human skin model surfaces were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate non-specific living and killed colour controls.
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.
- 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 42 hours 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 except for the 2 living and 2 killed Human skin model surfaces for the non-specific colour control which were placed in the maintenance medium . 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
- As the test item is identified as producing both direct MTT reduction and colour interference:
True viability % = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT + OD of killed tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 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):
42 hours 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:
main test (duration of exposure: 42 minutes)
Value:
2.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues was 2.3%, versus 2.4% 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

0.939

1.008

0.841

119.8

100

17.3

1.050

1.035

2

0.729

0.738

87.7

0.776

0.711

3

0.819

0.778

92.5

0.742

0.775

Positive control

1

0.019

0.019

0.02

2.3

2.4

0.9

0.020

0.019

2

0.013

0.013

1.5

0.014

0.012

3

0.041

0.028

3.3

0.021

0.023

Test item

1

0.161

0.166

0.134

19.7

16

6.4

0.165

0.172

2

0.166

0.165

19.6

0.164

0.165

3

0.073

0.072

8.6

0.074

0.071

Test item (NSC control) living tissues

1

0.028

0.029

0.029

3.4

3.4

0

0.031

0.028

2

0.030

0.029

3.4

0.029

0.028

Test item (NSC control) killed tissues

1

0.028

0.01

0.009

1.2

1.1

0.2

0.031

0.028

2

0.030

0.008

1

0.029

0.028

Test item (NSC control) killed tissues MTT control

1

0.105

0.118

0.095

14

11.3

3.9

0.120

0.130

2

0.078

0.072

8.6

0.069

0.071

Test item corrected

2.3

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 42 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 was2.3%, versus 2.4% 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:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
07 September - 04 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 431 without any deviation.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
dated 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: method B.40bis of the Council Regulation No. 440/2008
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
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:
95.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test (1 hour)
Value:
17.01
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosion
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 95.10% and 17.01%, 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 0.62%.

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

1

1.288

1.245

1.246

99.96

100

0.1

1.25

1.197

2

1.274

1.246

100.04

1.236

1.227

Positive control

3

0.433

0.428

0.72

34.36

57.77

46.8

0.423

0.427

4

1.069

1.011

81.17

0.967

0.997

Test item

7

1.25

1.225

1.185

98.35

95.1

6.5

1.212

1.213

8

1.165

1.144

91.85

1.147

1.12

Treatment: 1 hour

Negative control

11

1.443

1.231

1.282

96.06

100

7.9

1.113

1.137

12

1.341

1.332

103.94

1.314

1.34

Positive control

13

0.002

0.003

0.008

0.23

0.62

0.8

0.003

0.004

14

0.014

0.013

1.01

0.013

0.013

Test item

17

0.231

0.225

0.218

17.56

17.01

1.1

0.22

0.224

18

0.221

0.211

16.47

0.207

0.204

#: 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:
Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. No hazard statement or signal word are required.
Executive summary:

An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

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, 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.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 95.10% and 17.01% versus 57.77% and 0.62%, respectively, with the positive control item (potassium hydroxide 8N).

Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. No hazard statement or signal word are required.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
07 September - 04 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 431 without any deviation.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See attached document in section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
dated 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: method B.40bis of the Council Regulation No. 440/2008
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test (3 minutes)
Value:
95.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test (1 hour)
Value:
17.01
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosion
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 95.10% and 17.01%, 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 0.62%.

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

1

1.288

1.245

1.246

99.96

100

0.1

1.25

1.197

2

1.274

1.246

100.04

1.236

1.227

Positive control

3

0.433

0.428

0.72

34.36

57.77

46.8

0.423

0.427

4

1.069

1.011

81.17

0.967

0.997

Test item

7

1.25

1.225

1.185

98.35

95.1

6.5

1.212

1.213

8

1.165

1.144

91.85

1.147

1.12

Treatment: 1 hour

Negative control

11

1.443

1.231

1.282

96.06

100

7.9

1.113

1.137

12

1.341

1.332

103.94

1.314

1.34

Positive control

13

0.002

0.003

0.008

0.23

0.62

0.8

0.003

0.004

14

0.014

0.013

1.01

0.013

0.013

Test item

17

0.231

0.225

0.218

17.56

17.01

1.1

0.22

0.224

18

0.221

0.211

16.47

0.207

0.204

#: 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:
Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. No hazard statement or signal word are required. Therefore the registered substance is considered as non corrosive to the skin.
Executive summary:

An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

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, 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.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 95.10% and 17.01% versus 57.77% and 0.62%, respectively, with the positive control item (potassium hydroxide 8N).

Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. No hazard statement or signal word are required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation, other
Remarks:
Classification based on calculation rules for mixtures of the CLP Regulation
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
accepted calculation method
Qualifier:
no guideline required
Principles of method if other than guideline:
Classification based on calculation rules for mixtures of the CLP Regulation
Irritation parameter:
other: Classification
Remarks on result:
other: Eye damage category 1
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Executive summary:

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation. The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the serious eye damage/ eye irritant hazard of the registered substance. The decision of classification as eye Damage category 1 was based on existing data on constituents (additivity principles) the registered substance has a constituent classified as Eye Damage Category 1 present at more than 3% and should be classified as a Eye damage category 1 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

Constituent

 

CAS

Classification

Source

Benzoic acid

65 -85 -0

H318

ECHA and C&L inventoryself classification

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2017 - February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 438 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
dated 26 July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
dated 08 December 2010
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.
- Characteristics of donor animals (e.g. age, sex, weight): The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
- Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 11 Ocotber 2017 at 8:25 am.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Phycher on 11 October 2017 at 9:55 am.
- Indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: None
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): The test item was applied, as supplied, at the dose of 30 μL, to 3 enucleated chicken eyes, during 10 seconds.
Duration of treatment / exposure:
10 seconds
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
- The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus were at a controlled temperature between 32.9 °C and 33.5 °C.
- After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
- Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 48 and 60 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
- 1, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: 5% Benzalkonium chloride

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus and placed in a horizontal position. The test item was applied, as supplied, at the dose of 30 μL, to 3 enucleated chicken eyes, during 10 seconds.

REMOVAL OF TEST SUBSTANCE
- After exposure, the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
- Treated corneas were evaluated before the pre-treatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. 1. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm.
- The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after exposure to the test item) were determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
The mean percentage of corneal swelling for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for the test item.
- Mean maximum opacity score: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris clearly visible
2: Easily discernible translucent area; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention value for all tested eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA:
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for the test item.
Once each endpoint was evaluated, ICE classes were assigned based on a predetermined range. Interpretation of corneal thickness, opacity, and fluorescein retention using four ICE classes was done according to the table 7.3.2/1, 7.3.2/2, 7.3.2/3.
Irritation parameter:
cornea opacity score
Run / experiment:
mean of three runs
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
mean of three runs
Value:
1.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
mean of three runs
Value:
4
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:
OCULAR REACTIONS:
The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 0.2, corresponding to ICE class I;
- mean score of fluorescein retention: 1.7, corresponding to ICE class III;
- maximal mean corneal swelling: 4%, corresponding to ICE class I.
The combination of the three endpoints for the test item was 2 x I, 1 x III.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

Table 7.3.2/5: Individual and average values for evaluation of corneal lesions after treatment

Endpoint measured

Eye No.

Time (min)

0

30

75

120

180

240

Corneal opacity

4

0.00

0.00

0.00

0.00

0.00

0.50

5

0.00

0.00

0.00

0.00

0.00

0.00

6

0.00

0.00

0.00

0.00

0.00

0.00

Mean

0.00

0.00

0.00

0.00

0.00

0.20

ICE class

I

Fluorescein retention

4

0.50

1.00

-

-

-

-

5

0.50

2.00

-

-

-

-

6

0.50

2.00

-

-

-

-

Mean

0.50

1.70

-

-

-

-

ICE class

III

Corneal thickness

4

0.60

0.60

0.61

0.61

0.61

0.61

5

0.58

0.60

0.62

0.62

0.64

0.64

6

0.56

0.56

0.56

0.56

0.56

0.56

Corneal swelling (%)

4

-

0.00

2.00

2.00

2.00

2.00

5

-

3.00

7.00

7.00

10.00

10.00

6

-

0.00

0.00

0.00

0.00

0.00

Mean

-

1.00

3.00

3.00

4.00

4.00

ICE class

I

Combination of the 3 Endpoints

2 x I, 1 x III

CLASSIFICATION

No prediction can be made

Interpretation of results:
other: no prediction can be made
Conclusions:
Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained lead to the category “no prediction can be made”, as defined by the OECD guideline No. 438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.
Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes. The test item was applied, as supplied, at the dose of 30 μL, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.2, corresponding to ICE class I;

- mean score of fluorescein retention: 1.7, corresponding to ICE class III;

- maximal mean corneal swelling: 4%, corresponding to ICE class I.

The combination of the three endpoints for the test item was 2 x I, 1 x III.

The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained lead to the category “no prediction can be made”, as defined by the OECD guideline No. 438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the Eye and skin irritation/corrosion hazard of the registered substance. The decision of classification as eye corrosive was based on existing data on constituents (additivity principles) the registered substance has more than 3 % classified as Eye damage Category 1 and should be classified as serious eye damage without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008

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 42 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 was2.3%, versus 2.4% 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.

Skin corrosion:

An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

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, 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.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 95.10% and 17.01% versus 57.77% and 0.62%, respectively, with the positive control item (potassium hydroxide 8N).

Under the test conditions, in accordance with the Regulation (EC) No. 1272/2008, the results obtained enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. No hazard statement or signal word are required. Therefore the registered substance is not considered as corrosive to the skin

Eye irritation

Constituent

 

CAS

Classification

Source

Benzoic acid

65 -85 -0

H318

ECHA and C&L inventoryself classification

Source: ECHA disseminated dossiersor self classification

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 and typical composition provided by the Lead Registrant, the registered substance is classified as skin irritant: Skin Irritant Category 2 (H315: Causes skin irritation) and Eye irritant category 1 (H318) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).