Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion: Bottom-up approach:

- SkinEthic: Category 1 or 2 (OECD 439, GLP, K, rel. 1);

- EpiCs: not corrosive to skin (OECD 431, GLP, K, rel.1).

CONCLUSION: Irritating (Category 2).


Eye irritation: Bottom-up approach:

- EpiOcular: Category 1 or 2 (OECD 492, GLP, K, rel.1);

- ICE: No category (OECD 438, GLP, K, rel.1)

CONCLUSION: Irritating (Category 2)

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:
31 August - 07 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
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: foreskin
Justification for test system used:
Following the REACH bottom-up strategy described in the ECHA R.7a guidance, the SkinEthic™ Reconstructed Human Epidermis Model method was used to assess skin irritation.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ Reconstructed Human Epidermis Model, SkinEthic Laboratories, Lyon, France (RHE/S/17)
- Tissue batch number(s): 17-RHE-091
- Expiration date: September 11, 2017
- Date of testing: between 31 August 2017 and 07 September 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 42 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 41 hours and 15 minutes at 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS (Dutscher - Batch No. 9130617).
- Observable damage in the tissue due to washing: none, but residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours at 37°C
- Spectrophotometer: 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.
- Wavelength: 570 nm
The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol : the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.2 (CV = 1.9%) (O.D. > 0.7)
- Barrier function: 4.8 h (4.0h < ET50 < 10.0 h)
- Morphology: 6 cells layers (> 4). Absence of significant histological abnormalities. Satisfactory (Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum)
- Contamination: absence of HIV1 & 2 antibodies, hepatitis C and antibodies, hepatitis C and antigen HBsp on blood; absence of mycoplasma on epidermal cells
- Reproducibility: All of the values for the negative and positive control groups fell within or very close to the historical ranges of the testing laboratory obtained in the previous year. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The direct interaction of MTT with the test item was checked by adding approximately 50 mg of the test item applied on a nylon mesh to 1 mL of solution of MTT at 1 mg/mL. A yellow to brown solution was observed after 3 hours of incubation between 36.5°C and 37.8°C, 5% CO2.
> Therefore, there is no direct interaction between the test item and MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is < 50% and in the absence of information on a skin corrosion test.
- The test substance is considered to be non-irritant to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5 % in distilled water
Duration of treatment / exposure:
42 minutes at room temperature
Duration of post-treatment incubation (if applicable):
41 hours and 15 minutes post-incubation period at 37°C, 5% CO2
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
42 minutes
Value:
10.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.6%
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 10.4%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

- OTHER EFFECTS:
- Visible damage on test system: none but residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse
- Direct-MTT reduction: none.
- Colour interference with MTT: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control OD of the 3 replicates is > 0.4 and < 1.5 (values between 0.573 and 0.860). [The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol : the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.]
- Acceptance criteria met for positive control: yes, the positive control is classified as irritant
- Acceptance criteria met for variability between replicate measurements: yes, the SD values of the % viability are ≤ 18% (values between 0.1 and 16.7%).
- Range of historical values if different from the ones specified in the test guideline: The positive control OD were within the historical control range. The negative control OD of one of the replicate were below the historical control range. However, this deviation is not considered to have affected the integrity of the study or the overall conclusion.

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

0.573

0.613

0.759

80.8

100.0

16.7

0.626

0.641

2

0.818

0.824

108.6

0.827

0.826

3

0.826

0.839

110.6

0.831

0.860

Positive control

1

0.012

0.013

0.012

1.7

1.6

0.1

0.013

0.013

2

0.011

0.011

1.4

0.011

0.011

3

0.013

0.012

1.8

0.011

0.011

Test item

1

0.039

0.042

0.079

5.5

10.4

6.3

0.043

0.043

2

0.130

0.133

17.5

0.133

0.136

3

0.061

0.061

8.0

0.061

0.060

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

OD: optical density

Interpretation of results:
other: Category 1 (corrosive) or Category 2 (irritant) base don GHS criteria
Conclusions:
Under the experimental conditions of this study, in the absence of information on skin corrosion, the test substance has to be classified in Category 2 "Irritating to skin" or Category 1 "Corrosive" according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the SkinEthic reconstructed human epidermis model.

Due to its nature, the test item LABDANUM ABS SUPER - 953418 was administered after being directly applied on a nylon mesh (corresponding to 16 mg) in order to cover the entire surface of 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 41 hours 15 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.

Residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse.

The mean percent viability of the treated tissues was 10.4%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

Concerning acceptability criteria:

- The negative control OD of the 3 replicates is > 0.4 and < 1.5 (values between 0.573 and 0.860).

- The SD values of the % viability are ≤ 18% (values between 0.1 and 16.7%).

- The positive control is classified as irritant.

- The 3 replicates of the test item gave concordant classification.

The quality criteria required for acceptance of results in the test were satisfied.

The positive control OD were within the historical control range. The negative control OD of one of the replicate were below the historical control range. However, this deviation is not considered to have affected the integrity of the study or the overall conclusion.

 

Under the experimental conditions of this study, in the absence of information on skin corrosion, the test substance has to be classified in Category 2 "Irritating to skin" or Category 1 "Corrosive" according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August - 25 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 431 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
(bis)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
27 April 2017
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: foreskin
Justification for test system used:
Following the REACH bottom-up strategy described in the ECHA R.7a guidance, after the positive result obtained in the EpiSkin™ RHE Model method for skin irritation, the EpiCS™ RHE Model method was used to assess skin corrosion.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiCS™, supplied by CellSystems
- Tissue batch number(s): To be completed
- Expiration date: To be completed
- Date of testing: To be completed

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes (room temperature) and 1 hour (37°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 mL DPBS, 20 times
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours at 37°C
- Spectrophotometer: 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.
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: To be completed
- Barrier function: To be completed
- Morphology: To be completed
- Contamination: To be completed
- Reproducibility: To be completed

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The direct interaction of MTT with the test item was checked by adding approximately 50 mg of the test item applied on a nylon mesh to 1 mL of solution of MTT at 1 mg/mL. A yellow to brown solution was observed after 3 hours of incubation between 36.5°C and 37.8°C, 5% CO2.
> Therefore, there is no direct interaction between the test item and MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- Step 1: The test substance is considered to be corrosive to skin if the viability is:
< 50% after 3 min exposure
≥ 50% after 3 min exposure AND < 15% after 60 min exposure
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure
- Step 2: The test substance is subcategorised:
1A if viability is < 15% after 3 min exposure
1B/1C if viability is ≥ 15% after 3 min exposure
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): to be completed
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
3 minutes
Value:
74.08
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
11.10%
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
1 hour
Value:
87.57
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
0.32 %
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 10.4%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

- OTHER EFFECTS:
- Visible damage on test system: none but residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse
- Direct-MTT reduction: none.
- Colour interference with MTT: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control OD of the 2 replicates is > 0.8 and < 2.8 for every exposure time (values between 1.043 and 1.290).
- Acceptance criteria met for positive control: yes, the mean tissue viability of the 2 replicates exposed for 1 hour are < 20% (0.39 %)
- Acceptance criteria met for variability between replicate measurements: yes, in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates was < 30% (values between 0.1 and 28.9%).
- Range of historical values if different from the ones specified in the test guideline: To be completed

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

 

Tissue

Exposure Period

OD

Mean OD/ disc

Mean OD / Product

Viability (%)

Mean viability (%)

Viability difference between replicates

(%)

Negative Control

3 Minutes

1.276

1.290

1.261

1.276

1.248

102.24

100

4.5

1.212

1.196

1.252

1.220

97.76

60 Minutes

1.126

1.170

1.050

1.115

1.102

101.18

100

2.4

1.107

1.043

1.118

1.089

98.82

Positive Control

3 Minutes

0.174

0.162

0.154

0.163

0.139

13.06

11.10

3.9

0.122

0.113

0.108

0.114

9.13

60 Minutes

0.004

0.004

0.004

0.004

0.004

0.36

0.32

0.1

0.002

0.004

0.003

0.003

0.27

Test Item

3 Minutes

1.056

1.066

1.194

1.105

0.925

88.54

74.08

28.9

0.738

0.718

0.777

0.744

59.62

60 Minutes

0.787

0.892

0.878

0.852

0.965

77.31

87.57

20.5

1.029

1.081

1.124

1.078

97.82

OD: optical density

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the experimental conditions of this study, the test substance is not a classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 (H315: Causes skin irritation) according to CLP and GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiCS reconstructed human epidermis model.

As the test item LABDANUM ABS SUPER - 953418 was a paste considered as a solid, it was administered after being directly applied on a nylon mesh (corresponding to 25 mg) in order to cover the entire surface of 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 74.08 % and 87.57%, versus 11.10% and 0.32%, respectively, with the positive control item (potassium hydroxide 8N).

Concerning acceptability criteria:

- Acceptance criteria met for negative control: yes, the negative control OD of the 2 replicates is > 0.8 and < 2.8 for every exposure time (values between 1.043 and 1.290).

- Acceptance criteria met for positive control: yes, the mean tissue viability of the 2 replicates exposed for 1 hour are < 20% (0.39 %)

- Acceptance criteria met for variability between replicate measurements: yes, in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates was < 30% (values between 0.1 and 28.9%).

The quality criteria required for acceptance of results in the test were satisfied.

Under the experimental conditions of this study, the test substance is not a classified for skin corrosion according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS. Based on the result of the in vitro skin irritation study, the test substance is therefore classified as Skin irritant Category 2 (H315: Causes skin irritation) according to CLP and GHS.

This study is considered as acceptable and satisfies the requirement for skin corrosion endpoint.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 August - 21 September 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)
Remarks:
27 April 2017
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: the OECD 492 (adopted in 2015), is validated and has regulatory acceptance. This test guideline is applicable to solid and waxes, so is considered to be applicable to the test item.

RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM (RhCE)
- Model used: EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation
- Tissue batch number(s): 27005
- Keratinocyte strain: 4F1188

FUNCTIONAL MODEL CONDITIONS
- Tissue viability: 1.419, within the acceptance criteria (1.1-3.0)
- Barrier function: 15.97 min, within the acceptance criteria (12.2-37.5)
- Sterility: Sterile
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 mg
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): undiluted
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: 18 hours at standard culture conditions
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used: method for solids (not pipetteable), as described in the OECD TG 492
- RhCE tissue construct used, including batch number: EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 27005
- Doses of test chemical and control substances used: 50 mg / 50 µL
- Duration and temperature of
Exposure: 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Post-exposure immersion: 25 minutes at room temperature
Post-exposure incubation periods: 18 hours at standard culture conditions
- Description of any modifications to the test procedure: None
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): not applicable.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan, measuring device (e.g. spectrophotometer): 570 nm, 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.
- Description of the method used to quantify MTT formazan
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 2 hours and 52 minutes 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 2 hours at 6±3°C in the dark.
The concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extractions in isopropanol (1:2).
The OD at 570 nm was measured in triplicate samples of formazan extracts.
The measured OD are proportional to the number of living cells.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test substance is considered to be corrosive or irritant to eyes if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60%. When the final mean percent tissue viability is ≤ 60%, further testing with other test methods will be required because the RhCE test method shows a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.
The test substance is considered to be non-irritant to eyes if the mean percent tissue viability after exposure and post-exposure incubation is > 60%.
- Complete supporting information for the specific RhCE tissue construct used
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: included in the report, follows the OECD TG 492.
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
1
Value:
29.05
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
40.34%
Remarks on result:
positive indication of irritation
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
2
Value:
8.73
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
14.96 %
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
MAIN TEST
- MTT assay results - First run: The mean percent tissue viability of the RhCE replicates treated with the test substance was 29.05 versus 40.34% in the positive control (Methyl acetate).
- MTT assay results - Second run: The mean percent tissue viability of the RhCE replicates treated with the test substance was 8.73 versus 14.96% in the positive control (Methyl acetate).

OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
FIRST RUN
- Acceptance criteria met for negative control: yes, the negative control OD is > 0.4 and < 1.25 (values between 0.528 and 0.700). [The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol : the acceptability criteria should be in the range ≥ 0.4 and ≤1.25 for the negative control.]
- Acceptance criteria met for positive control: no, the percentage of viability of 1/2 epidermis of the positive control (Methyl acetate) was higher than 50%
<=> a second run was performed
SECOND RUN:
- Acceptance criteria met for negative control: yes, the negative control OD is > 0.4 and < 1.25 (values between 0.528 and 0.700).
- Acceptance criteria met for positive control: yes, the mean relative viability of the positive control is below 50% of the negative control viability (14.96%).
- Acceptance criteria met for variability between replicate measurements: yes, the difference of viability between the two relating tissues of the test item and the positive control are < 20% (1.83% and 2.66%, respectively). The difference of viability between the two relating tissues is slightly above the threshold of 20% for the negative control (21.11%). However, this deviation is not considered to have affected the integrity of the study or the overall conclusion.
- Range of historical values if different from the ones specified in the test guideline: The positive and negative control OD were within the historical control ranges

Table 7.3.2/1: First run - 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

0.861

0.858

0.992

86.54

100.00

26.93

0.855

0.858

2

1.185

1.125

113.46

1.092

1.098

Positive control

1

0.502

0.497

0.400

50.13

40.34

19.57

0.486

0.502

2

0.305

0.303

30.56

0.308

0.295

Test item

1

0.181

0.182

0.228

18.36

29.05

21.38

0.182

0.182

2

0.395

0.394

39.75

0.393

0.395

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

OD: optical density

Table 7.3.2/2: Second run - 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

0.700

0.665

0.602

110.56

100.00

21.11

0.640

0.657

2

0.550

0.538

89.44

0.537

0.528

Positive control

1

0.111

0.098

0.090

16.29

14.96

2.66

0.094

0.091

2

0.076

0.082

13.62

0.087

0.085

Test item

1

0.047

0.047

0.053

7.81

8.73

1.83

0.048

0.048

2

0.058

0.058

9.64

0.059

0.058

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

OD: optical density

Interpretation of results:
other: Category 1 (irreversible effects on the eye) or Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
With a percentage of tissue viability < 60%, the test item requires classification as irritating or corrosive to eyes according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.
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.

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 itemLABDANUM ABS SUPER - 953418 was applied, to 2 DPBS pre-treated RhCE (EpiOcularTMtissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 25 minutes post-exposure immersion period at room temperature and an 18 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. The experimental protocol was established in accordance withO.E.C.D. Test Guideline No. 492 adopted 28 July 2015.

 

During the first run, the percentage of viability of 1/2 epidermis of the positive control (Methyl acetate) was higher than 50%. It was not possible to conclude for the test item because the acceptability criteria defined in OECD 439 was not respected. A second run was necessary.

 

In the second run, the mean percent tissue viability of the RhCE replicates treated with the test item LABDANUM ABS SUPER - 953418 was 8.73%, versus 14.96% in the positive control (Methyl acetate).

 

In the second run, the quality criteria required for acceptance of results in the test were satisfied:

- Acceptance criteria met for negative control: yes, the negative control OD is > 0.4 and < 1.25 (values between 0.528 and 0.700).

- Acceptance criteria met for positive control: yes, the mean relative viability of the positive control is below 50% of the negative control viability (14.96%).

- Acceptance criteria met for variability between replicate measurements: yes, the difference of viability between the two relating tissues of the test item and the positive control are < 20% (1.83% and 2.66%, respectively).  The difference of viability between the two relating tissues is slightly above the threshold of 20% for the negative control (21.11%). However, this deviation is not considered to have affected the integrity of the study or the overall conclusion.

 

The positive and negative control OD were within the historical control ranges.

With a percentage of tissue viability < 60%, the test item requires classification as irritating or corrosive to eyes according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for eye irritation endpoint.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2017
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 29 November 2017 at 8:30 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 29 November 2017 at 10:10 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): In order to cover the entire surface of the cornea test item was applied on three gauzes at an approximate dose of 260 mg. The gauzes were subsequently applied 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 33 °C and 33.7 °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 45 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 controls and test item, respectively.

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: Sodium hydroxide

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. A gauze impregnated with 0.3127 g, 0.3210 g or 0.3276 g of the test item was applied for 10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item.

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
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:
0.8
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:
0
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, corresponding to ICE class I;
- mean score of fluorescein retention: 0.8, corresponding to ICE class II;
- maximal mean corneal swelling: 0%, corresponding to ICE class I.

The combination of the three endpoints for the test item was 3 x I.


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, Sodium hydroxide, 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

1

0

0

0

0

0

0

2

0

0

0

0

0

0

3

0

0

0

0

0

0

Mean

0

0

0

0

0

0

ICE class

I

Fluorescein retention

1

0.5

0.5

-

-

-

-

2

0.5

1

-

-

-

-

3

0.5

1

-

-

-

-

Mean

0.5

0.8

-

-

-

-

ICE class

II

Corneal thickness

1

0.63

0.63

0.63

0.63

0.63

0.63

2

0.62

0.62

0.62

0.62

0.62

0.62

3

0.60

0.60

0.60

0.60

0.60

0.60

Corneal swelling (%)

1

-

0

0

0

0

0

2

-

0

0

0

0

0

3

-

0

0

0

0

0

Mean

-

0

0

0

0

0

ICE class

I

Combination of the 3 Endpoints

2 x I, 1 x II

CLASSIFICATION

No category

Interpretation of results:
GHS criteria not met
Conclusions:
In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to “no category”, as defined by OECD guideline No.438. Therefore, test item is predicted as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test.
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.

In order to cover the entire surface of the cornea test item was applied on three gauzes at an approximate dose of 260 mg. The gauzes were subsequently applied to 3 enucleated chicken eyes during 10 seconds. Then the eyes were rinsed four times 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, corresponding to ICE class I;

- mean score of fluorescein retention: 0.8, corresponding to ICE class II;

- maximal mean corneal swelling: 0%, corresponding to ICE class I.

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

The combination of the three endpoints for the positive control, Sodium hydroxide, 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.

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to “no category”, as defined by OECD guideline No.438. Therefore, test item is predicted as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2017), was used to evaluate the skin corrosion/irritation potential of the registered substance:

 

Element

Information

Conclusion

Comments

Existing data on physico
- chemical properties

1a

Is the substance spontaneously flammable in air or in contact with water or moisture at room temperature?

NO

 

1b

Is the substance an organic hydroperoxide or an organic peroxide?

NO

 

1c

Is the pH of the substance ≤ 2.0 or ≥ 11.5?

NO

 

1d

Are there other physical or chemical properties that indicate that the substance is corrosive/irritant?

NO

 

Existing human data

2

Are there adequate existing human data which provide evidence that the substance is a corrosive
or irritant?

NO

 

Existing animal data from corrosion/irritation studies

3

Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?

NO

(at the initiation of the dossier, no test was available)

Existing data from general toxicity studies via the dermal route and from sensitisation studies

4a

Is the substance classified as acutely toxic by the dermal route (Category 1)?

NO

 

4b

Has the substance proven to be a corrosive, irritant or non-irritant in a suitable acute dermal toxicity test?

NO

 

4c

Has the substance proven to be a corrosive or an irritant in sensitisation studies or after repeated
exposure?

NO

(at the initiation of the dossier, no test was available)

Existing/new (Q)SAR data and read
-across

5a

Are there structurally related substances (suitable “read-across” or grouping), which are classified as corrosive to the skin (Skin Corrosive Cat. 1), or do suitable (Q)SAR methods indicate corrosion
potential of the substance?

NO

Not applicable, due to the mostly unknown composition (UVCB Type II substance)

5b

Are there structurally related substances (suitable “read-across” or grouping), which are classified as irritant to the skin (Skin Irritant Cat. 2), or indicating that the substance is non-irritant, or do suitable (Q)SAR methods indicate irritant or non-irritant potential of the substance?

NO

Not applicable, due to the mostly unknown composition (UVCB Type II substance)

Existing in vitro data

6a

Has the substance demonstrated corrosive properties in an EU/OECD adopted in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but
are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met

NO

(at the initiation of the dossier, no test was available)

6b

Has the substance demonstrated irritant or non-irritant properties in an EU/OECD adopted
in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but
are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are
met.

NO

(at the initiation of the dossier, no test was available)

6c

Are there data from a non-validated suitable in vitro test(s), which provide sound conclusive evidence that the substance is corrosive/ irritant?

NO

 

Weight-of- Evidence analysis

7

The “elements” described above may be arranged as appropriate. Taking all available existing and
relevant data mentioned above (Elements 1-6) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and –if so –how to classify and label?

NO

 

New in vitro test for corrosivity

8

Does the substance demonstrate corrosive properties in (an) EU/OECD adopted in vitro test(s) for skin corrosion?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

NO

(at the initiation of the dossier, no test was available).
An in vitro test for corrosion (OECD TG 431) was performed after the test for irritation according to the bottom-up approach. Mean tissues viability = 74.08% after 3minutes, 87.57% after 1 hour <=> is not corrosive.

New in vitro test for irritation

9

Does the substance demonstrate irritating or non-irritating properties in (an) EU/OECD adopted in vitro test(s) for skin irritation?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

YES

Following the bottom-up approach, an OECD TG 439 (SkinEthic) study was performed. Mean tissue viability = 10,4 % <=> Irritant or Corrosive to skin
<=> an in vitro test for corrosion was performed.

New in vivo test for corrosion/irritation

10

To be used only as a last resort

NO

 

Following the REACH bottom-up approach, an in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the SkinEthic reconstructed human epidermis model.

The relative mean viability of the test item treated tissues was 10.4%.

The quality criteria required for acceptance of results in the test were satisfied. With a tissue viability < 50%, the test material was considered to be irritant or corrosive to skin.

Therefore, an in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiCS reconstructed human epidermis model.

The relative mean viability of the test item treated tissues was 74.08% after 3 minutes and 87.57% after 1 hour.

The quality criteria required for acceptance of results in the test were satisfied. With a tissue viability > 50%, the test material was considered to be not corrosive to skin.

Based on the results of the in vitro skin irritation and skin corrosion studies, the registered substance is classified as irritant to skin.

Eye irritation:

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2017), was used to evaluate the eye damage/irritation potential of the registered substance:

 

Element

Information

Conclusion

Comments

Conclusion of the information strategy on skin corrosion/irritation

0

Is the substance classified as a skin corrosive?

NO

 

Existing data on physico
- chemical properties

1a

Is the substance spontaneously flammable in air or in contact with water or moisture at room temperature?

NO

 

1b

Is the substance an organic hydroperoxide or an organic peroxide?

NO

 

1c

Is the pH of the substance ≤ 2.0 or ≥ 11.5?

NO

 

1d

Are there other physical or chemical properties that indicate that the substance causes serious eye damage or eye irritation?

NO

 

Existing human data

2

Are there adequate existing human data which provide evidence that the substance has the potential to cause serious eye damage or eye irritation?

NO

 

Existing animal data from corrosion/irritation studies

3

Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?

NO

(at the initiation of the dossier, no test was available)

Existing/new (Q)SAR data and read-across

4

Are there structurally related substances (suitable “read-across” or grouping), which are classified as causing serious eye damage/eye irritation, or indicating that the substance is non-irritant, or do valid (Q)SAR methods indicate serious eye damage/eye irritation or non-irritation of the substance?

NO

Not applicable, due to the mostly unknown composition (UVCB Type II substance)

Existing in vitro data

5a

Has the substance demonstrated serious eye damage, eye irritation or non-irritating properties in an EU/OECD adopted in vitro test?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.

NO

(at the initiation of the dossier, no test was available)

5b

Are there acceptable data from (a) non-validated suitable in vitro test(s), which provide sound evidence that the substance causes serious eye damage/eye irritation?

NO

 

Weight-of- Evidence analysis

6

The “elements” described above may be arranged as appropriate. Taking all available existing and relevant data mentioned above (Elements 0 – 5) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and – if so – how to classify and label?

NO

 

New in vitro tests for serious eye damage/eye irritation (Annex VII to the REACH Regulation)

7a

Does the substance demonstrate serious eye damage, eye irritation or non-irritant properties in (an) EU/OECD adopted in vitro test(s) for the eye hazard charaterisation?
Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions of Annex XI are met.

NO

(at the initiation of the dossier, no test was available).
An in vitro test for corrosion (OECD TG 438) was planned after the test for irritation according to the bottom-up approach. The study is on-going at the time of dossier submission.

7b

Does the substance demonstrate serious eye damage or eye irritant properties in (a) non-validated suitable in vitro test(s) for serious eye damage/eye irritation?

YES

Following the bottom-up approach, an OECD TG 492 (EpiOcular) study was performed. Mean tissue viability = 8,73% <=> Irritant or Corrosive to eyes.
<=> an in vitro test for corrosion was performed.

New in vivo test for serious eye damage/eye irritation as a last resort (Annex VIII to the REACH Regulation)

8b

Does the substance demonstrate serious eye damage or eye irritation in an OECD adopted in vivo test?

NO

 

Following the REACH bottom-up approach, an in vitro eye irritation study (Envigo, 2017, Rel.1) was performed according to the OECD Guideline 492 and in compliance with GLP, using the EpiOcular Reconstructed human Cornea-like Epithelium.

The relative mean viability of the test item treated tissues was 8.73%.

The quality criteria required for acceptance of results in the test were satisfied. With a tissue viability < 60%, the test material was considered to be irritant or corrosive to eyes.

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

- maximal mean score of corneal opacity: 0, corresponding to ICE class I;

- mean score of fluorescein retention: 0.8, corresponding to ICE class II;

- maximal mean corneal swelling: 0%, corresponding to ICE class I.

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

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to “no category”, as defined by OECD guideline No.438. Therefore, test item is predicted as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test.

Therefore the registered substance is classified as irritating to eyes.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

Based on the available information, the substance should be classified as skin irritant Category 2 (H315: Causes skin irritation) and eye irritant Category 2 (H319: Causes serious eye irritation) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No data was available regarding respiratory irritation.