Registration Dossier

Administrative data

Description of key information

Based on the results of an OECD 437 study, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (UN GHS no Category) (reference 7.3.2 -1).

Based on the results of an OECD 492 study the test item is requiring classification for eye damaging potential (UN GHS Category 1 or Category 2) (reference 7.3.2 -2)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 August 2017 - 16 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: reconstructed human epidermis
Cell type:
non-transformed keratinocytes
Justification for test system used:
The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis, and has been validated by the ECVAM in 2008.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number(s): 17-RHE-094
- Date of initiation of testing: On day of receipt the pre-incubation phase of the tissues started.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with minimum 25 mL DPBS; excess DPBS was removed by shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin category 2 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 mg

NEGATIVE CONTROL
- Amount applied: 16 µL

POSITIVE CONTROL
- Amount applied: 16 µL
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
Tissue 1
Run / experiment:
1
Value:
81.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
Tissue 2
Run / experiment:
1
Value:
96.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
Tissue 3
Run / experiment:
1
Value:
101.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item is considered as non-irritant to skin (UN GHS: No Category).
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test item the tissue viability was 93.2% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
weight of evidence
Study period:
14 August 2017 - 9 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:
2017
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age of donor animals: 5-131 months
- Croneal diameter: 26 - 27 mm
- Storage, temperature and transport conditions of ocular tissue: cooled on ice
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL of the dissolved test item (i.e. 150 mg/750 µL)
- Concentration (of solution): 20 % (w/v)

VEHICLE
- Amount applied: 750 uL
- Concentration: 0.9% sodium chloride solution
- Lot/batch no.: 16455011
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 corneas per group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
After delivery all eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
At the end of the incubation period, the basal opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). The light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values). Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: solvent control used

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of the dissolved test item (i.e. 150 mg/750 µL), 240 min

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE:
After the incubation period the corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The light transmission through the corneas was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany).
- Corneal permeability: The amount of fluorescein that crossed the cornea was measured spectrophotometrically with a microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS) Calculation:
The following formula (referring to OECD Guideline 437) was used to determine the In Vitro Irritancy Score (IVIS) of the negative control:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The following formula was used to determine the In Vitro Irritancy Score (IVIS) of the positive control and the test item:
IVIS = corrected opacity value + (15 x corrected permeability OD490 value)
The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values.

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage, Category 1 (according to GHS)
Irritation parameter:
in vitro irritation score
Remarks:
Tissue 1
Run / experiment:
1
Value:
2.815
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
Tissue 2
Run / experiment:
1
Value:
3.385
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
Tissue 1
Run / experiment:
1
Value:
4.445
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes
Interpretation of results:
other: Not Category 1 (irreversible effects on the eye) based on GHS
Conclusions:
Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.
Executive summary:

The objective of the present study was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) solution in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the dissolved test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (MS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.4 (study acceptance criteria range: -1.4 - 3.2). Treatment with the positive control (20% imidazole) revealed an IVIS of 107.4 (study acceptance criteria range: 81.8 - 132.7). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 3.5 and, thus higher than 3 and lower than 55. Therefore, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (UN GHS no Category).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14 August 2017 - 9 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: reconstructed human cornea-like epithelium
Details on test animals or tissues and environmental conditions:
Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Characterisation of the test system:
- Supplier: MatTek In Vitro Life Science Laboratories
- Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
- Lot No.: 27002
- Keratinocyte strain: 4F1188
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg
Duration of treatment / exposure:
6 hours (± 15 minutes)
Duration of post- treatment incubation (in vitro):
25 minutes (± 2 minutes) at room temperature and 18 hours (± 15 minutes) at 37°C
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used:

Preparation:
On day of receipt, the tissues were equilibrated in their 24-well shipping container to room temperature for about 15 minutes. Afterwards the tissues were removed from the shipping container using sterile forceps and transferred to 6-well plates containing 1 mL pre-warmed (37°C) assay medium. Any agarose adhering to the inserts was removed by gentle blotting on gauze or paper towel. Afterwards, the tissues were incubated at 37°C and 5% CO2 overnight (16-24 hours) without medium exchange.

Pre-Treatment:
After the overnight incubation, the tissues were pre-wetted with 20 µL DPBS and incubated at 37°C and 5% CO2 for 30 minutes (± 2 minutes).

Exposure and Post-Treatment:
After the 30 minute DPBS pre-treatment, the liquid test item, the negative and the positive control were tested by applying 50 mg topically on the EpiOcular™ tissues. The tissues were placed back into the culture medium after dosing and incubated at 37°C and 5% CO2 for 6 hours (± 15 minutes). At the end of the 6 hours treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS. Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the two tissues per group were rinsed simultaneously by holding the replicate inserts together by their collars using forceps. The test item or control articles were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed at least two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS at least three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 25 minutes (± 2 minutes) at room temperature. After the 25 minutes incubation, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert were blotted on absorbent material and transferred in 6-well plates filled with 1 mL of pre-warmed (37°C) assay medium for 18 hours (± 15 minutes) at 37°C and 5% CO2.

- RhCE tissue construct used, including batch number:

Supplier: MatTek In Vitro Life Science Laboratories
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27002
Keratinocyte strain: 4F1188

- Doses of test chemical and control substances used: test chemical: 50 mg, control substances: 50 µL

- Duration and temperature of exposure, post-exposure: exposure: 6 hours (± 15 minutes).
at 37 °C; post-exposure: 25 minutes (± 2 minutes) at room temperature and 18 hours (± 15 minutes) at 37°C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength used for quantifying MTT formazan: 570 nm

- Description of the method used to quantify MTT formazan:

After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2.
The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically.
The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:

If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is <60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptance Criteria:
The results are acceptable if:
1. The negative control OD >0.8 and <2.5,
2. The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: below 50% of control viability
3. Acceptable variability between tissue replicates: < 20 %
Irritation parameter:
other: Viability (%)
Remarks:
Tissue 1
Run / experiment:
1
Value:
3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability (%)
Remarks:
Tissue 2
Run / experiment:
1
Value:
3.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Direct MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1: Results

Group

Tissue 1

Tissue 2

Mean

SD

Difference
between tissue
replicates

 

OD

Viability

OD

Viability

OD

Viability

Negative
Control

1.186

100.1%

1.184

99.9%

1.185

100.0%

0.14

0.2%

Positive
Control

0.213

18.0%

0.195

16.5%

0.204

17.3%

1.06

1.5%

Test item

0.036

3.0%

0.042

3.5%

0.039

3.3%

0.35

0.5%

Interpretation of results:
other: Category 1 or 2 based on GHS criteria
Conclusions:
Following treatment with the test item, the mean tissue viability was 3.3% and, thus, lower than 60%, i.e. according to OECD 492 the test item is identified as requiring classification and labelling according to UN GHS (Category 1 or Category 2).
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 1.185 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 17.3% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the mean tissue viability was 3.3% and, thus, lower than 60%, i.e. according to OECD 492 the test item is identified as requiring classification and labelling according to UN GHS (Category 1 or Category 2).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

OECD 439

The objective of the study according to OECD guideline 439 was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test item the tissue viability was 93.2% and, thus, higher than 50%,i.e.according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Eye irritation

OECD 437

The objective of the study according to OECD guideline 437 was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) solution in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the dissolved test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (MS). After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.4 (study acceptance criteria range: -1.4 - 3.2). Treatment with the positive control (20% imidazole) revealed an IVIS of 107.4 (study acceptance criteria range: 81.8 - 132.7). Therefore, the study fulfilled the acceptance criteria.The IVIS obtained after treatment with the test item was 3.5 and, thus higher than 3 and lower than 55. Therefore, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (UN GHS no Category).

OECD 492

The objective of the study according to OECD guideline 492 was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. After treatment with the negative control (sterile deionized water) the mean OD was 1.185 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 17.3% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the mean tissue viability was 3.3% and, thus, lower than 60%, i.e. according to OECD 492 the test item is identified as requiring classification and labelling according to UN GHS (Category 1 or Category 2).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substance is not considered to be classified for skin irritation and considered to be classified for eye damaging potential (UN GHS Category 2, H319) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.