Registration Dossier

Administrative data

Description of key information

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

- Eye irritation:irritating (OECD TG 492 and OECD 438, GLP, Rel. 1)

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:
September - November 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 epidermises
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 : 41 hours and 15 minutes at 37°C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- 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


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2.
- 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 µl
- 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
Value:
3.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.6%
Other effects / acceptance of results:
MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues was 3.6%, 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

0.040

0.037

0.028

4.9

3.6

1.1

0.036

0.036

2

0.020

0.020

2.6

0.020

0.020

3

0.027

0.026

3.4

0.026

0.026

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

OD: optical density

Interpretation of results:
other: Category 1 (corrosive) or Category 2 (irritant) based on 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.

The test item was administered as supplied, at the dose of 16µl, to 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.

The mean percent viability of the treated tissues was 3.6%, 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:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: reconstituted epidermis
Cell type:
other: reconstituted epidermis (epiCS®, CellSystems®)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 0.60 cm2 reconstituted epidermis (epiCS®)

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

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

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

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

VIABILITY
Viability = (OD test item / OD negative control) x 100
For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, should be reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
3 minutes and 1 hour
Number of replicates:
Duplicate skin tissues for test item, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
at 3 minutes
Value:
89.43
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
at 1 hour
Value:
70.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Treatment 3 minutes

 

 

 

S kin

 

OD

 

Mean OD / disc(#)

 

Mean OD / product

Viability

%

Meanviability

%

Viability difference between replicates %

 

 

Negative control

 

1

0.669

0.688

0.701

 

0.686

 

 

0.691

 

99.35

 

 

100.00

 

 

1.3

 

2

0.715

0.652

0.717

 

0.695

 

100.65

 

 

 

Positive control

 

3

0.025

0.025

0.024

 

0.025

 

 

 

0.042

 

3.62

 

 

 

6.08

 

 

 

4.9

 

4

0.059

0.058

0.059

 

0.059

 

8.54

 

 

Test itemPH-17/0329

 

5

0.682

0.644

0.646

 

0.658

 

 

 

0.618

 

95.29

 

 

 

89.43

 

 

 

11.7

 

6

0.580

0.570

0.579

 

0.577

 

83.56

 

 

 

Treatment 1 hour

 

 

 

S kin

 

OD

 

Mean OD / disc(#)

 

Mean OD / product

Viability

%

Meanviability

%

Viability difference between replicates %

 

 

Negative control

 

15

0.856

0.834

0.835

 

0.842

 

 

0.771

 

109.28

 

 

100.00

 

 

18.6

 

16

0.710

676.000

0.711

 

0.699

 

90.72

 

 

Positive control

 

17

0.004

0.004

0.004

 

0.004

 

 

0.003

 

0.52

 

 

0.39

 

 

0.3

 

18

0.002

0.002

0.002

 

0.002

 

0.26

 

Test item PH- 17/0329

 

19

0.696

0.659

0.658

 

0.671

 

 

0.546

 

87.09

 

 

70.80

 

 

32.6

 

20

0.435

0.408

0.416

 

0.420

 

54.51

#: 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 89.43% and 70.80% versus 6.08% and 0.39%, 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: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Auguste - November 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:
other: Reconstructed human Cornea-like Epithelia
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, liquids, semi solids 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): 27002

FUNCTIONAL MODEL CONDITIONS
- Tissue viability: 1.269, within the acceptance criteria (1.1-3.0)
- Barrier function: 24.36 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 µl undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
-

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
Duration of treatment / exposure:
30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion period: 12 minutes at room temperature
- Post-exposure incubation period: 2 hours and 03 minutes at standard culture conditions
Number of animals or in vitro replicates:
2
Details on study design:
MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the tissues in their well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 20 hours and 10 minutes at standard culture conditions.
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- Treatment and post-treatment incubation of the tissues:
The test item was applied, as supplied, at the dose of 50 μL, to 2 living PBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable RhCE tissue replicates during 30 minutes at standard culture conditions.
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for a 2-hour and 03 minute post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.
- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 2 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 5 hours and 25 minutes at 6±3°C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD measurement of formazan extracts at 570 nm was measured in triplicate samples using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
1
Value:
44.79
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
24.44%
Other effects / acceptance of results:
MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 44.79 versus 24.44% 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 0.835 and 0.855).
- Acceptance criteria met for positive control: yes, the mean relative viability of the positive control is below 50% of the negative control viability (24.44%).
- Acceptance criteria met for variability between replicate measurements: yes, the difference of viability between the two relating tissues of the negative and positive control are < 20% (1.42% and 1.78%, respectively). The difference of viability between the two relating tissues is above the threshold of 20% for the test item (28.05%). In addition, one epithelium showed a percentage of viability of 58.82% which is considered to be a bordeline value (60%+/- 5%) according to OECD Test Guideline No 492. However, the two replicates showed concordant positive results, therefore a second test was not deemed necessary.
- 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: 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

0.846

0.839

0.845

99.29

100.00

1.42

0.835

0.836

2

0.845

0.851

100.71

0.855

0.854

Positive control

1

0.227

0.214

0.207

25.33

24.44

1.78

0.211

0.205

2

0.202

0.199

23.55

0.198

0.198

Test item

1

0.257

0.260

0.379

30.77

44.79

28.05

0.264

0.258

2

0.494

0.497

58.82

0.503

0.495

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

OD: optical density

Concerning acceptability criteria:

- The negative control OD is > 0.4 and < 1.25 (values between 0.835 and 0.855).

- The mean relative viability of the positive control is below 50% of the negative control viability (24.44%).

- The difference of viability between the two relating tissues of the negative and positive controls are < 20% (1.42% and 1.78%, respectively).The difference of viability between the two relating tissues is above the threshold of 20% for the test item (28.05%). In addition, one epidermis showed a percentage of viability of 58.82%, which is considered to be a borderline value (60% ± 5%) according to O.E.C.D. Test Guideline No. 492. However, the two replicates showed concordant positive results, therefore a second test was not deemed necessary

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.

The test item was applied, as supplied at the dose of 50 µl, to 2 living DPBS pre-treated RhCE (EpiOcularTMtissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and an 2 hours and 03 minutes 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.

 

The mean percent tissue viability of the RhCE replicates treated with the test item Labdanum absolute was 44.79%, versus 24.44% in the positive control (Methyl acetate).

 

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 0.835 and 0.855).

- Acceptance criteria met for positive control: yes, the mean relative viability of the positive control is below 50% of the negative control viability (24.44%).

- Acceptance criteria met for variability between replicate measurements: yes, the difference of viability between the two relating tissues of the negative and positive control are < 20% (1.42% and 1.78%, respectively).  The difference of viability between the two relating tissues is above the threshold of 20% for the test item (28.05%). In addition, one epithelium showed a percentage of viability of 58.82%, which is considered to be a borderline value (60% +/- 5%) according to OECD Test Guideline No. 492. However, the two replicates showed concordant positive results, therefore a second test was not deemed necessary.

 

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:
April - August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
The test item was used as supplied in the study
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 04 June 2018 at 8:25 a.m..
- 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 04 June 2018 at 10:15 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): 30 μL
- Concentration (if solution): Test item was used as supplied
Duration of treatment / exposure:
Test item was applied for 10 seconds to the cornea
Number of animals or in vitro replicates:
1, 3 and 3 eyes for negative & positive control and test item, respectively.
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 32.0 °C and 32.2 °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, the eyes were incubated between 45 and 64 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, placed in a horizontal position, and 30 μL of the test item was applied, as supplied, to the cornea for 10 seconds such that the entire surface of the cornea was evenly covered with the test item.

REMOVAL OF TEST SUBSTANCE
- After exposure, test item was rinsed from the eye with 20 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. I. 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:
maximal mean score
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
mean score
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: corneal swelling percentage
Run / experiment:
maximal mean
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:

OCULAR REACTIONS:
- maximal mean score of corneal opacity: 1.3, corresponding to ICE class II;
- mean score of fluorescein retention: 3, corresponding to ICE class IV;
- maximal mean corneal swelling: 1%, corresponding to ICE class I.
The combination of the three endpoints for test item was 1 x IV, 1 x II, 1 x I.

ACCEPTANCE OF RESULTS:
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

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

 

Endpoint measured

Eye No.

Time (minutes)

0

30

75

120

180

240

Corneal opacity

13

0

1

1

1

1

1

14

0

1

1

1

1

1

15

0

2

2

2

2

2

Mean

0.0

1.3

1.3

1.3

1.3

1.3

ICE class

         II

Fluorescein retention

13

0.5

3

-

-

-

-

14

0.5

3

-

-

-

-

15

0.5

3

-

-

-

-

Mean

0.5

3.0

 

-

-

-

ICE class

                       IV

Corneal thickness

13

0.59

0.59

0.60

0.60

0.60

0.60

14

0.61

0.61

0.61

0.61

0.61

0.61

15

0.62

0.63

0.63

0.63

0.63

0.63

Corneal swelling (%)

13

-

0

2

2

2

2

14

-

0

0

0

0

0

15

-

2

2

2

2

2

Mean

-

1

1

1

1

1

ICE class

      I

Combination of the three endpoints

1 x IV, 1 x II , 1 x I

Classification

No prediction can be made

Note:

No morphological effects were noted, whatever the examination time

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by OECD guideline No.438.
Therefore, test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) in the Isolated Chicken Eye test.
Based on the results from the previous study OECD 439 the test item can be classified as skin irritant (Category 2).
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.

 

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: 1.3, corresponding to ICE class II;

- mean score of fluorescein retention: 3, corresponding to ICE class IV;

- maximal mean corneal swelling: 1%, corresponding to ICE class I.

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

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.

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by OECD guideline No.438.

Therefore, test item  is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) in 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:

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 test item was administered as supplied, at the dose of 16µl, to 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.

The mean percent viability of the treated tissues was 3.6%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

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.

 

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 89.43% and 70.80% versus 6.08% and 0.39%, respectively, with the positive control item (potassium hydroxide 8N).

Eye irritation:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

The test item was applied, as supplied at the dose of 50 µl, to 2 living DPBS pre-treated RhCE (EpiOcularTMtissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and an 2 hours and 03 minutes 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.

 The mean percent tissue viability of the RhCE replicates treated with the test item Labdanum absolute was 44.79%, versus 24.44% in the positive control (Methyl acetate).

 

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

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.

Eye corrosion

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.

 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: 1.3, corresponding to ICE class II;

- mean score of fluorescein retention: 3, corresponding to ICE class IV;

- maximal mean corneal swelling: 1%, corresponding to ICE class I.

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

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.

Justification for classification or non-classification

Self - classification :

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

According to the results of OECD TG 492, and OECD 439 the registered substance is classified as Eye irritant Category 2 (H319) according to Regulation EC N° 1272/2008 (CLP) and GHS.