Registration Dossier

Administrative data

Description of key information

Skin irritation:

Based on the results of an OECD 439, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category; reference 7.3.1-1).

Eye irritation:

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)

Conclusion: Based on a weight of evidence approach the test item should be classified for eye irritation as UN GHS Cat. 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:
2019-02-13 to 2019-02-15
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:
human
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: 19-RHE-021

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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 5 minutes)
- Spectrophotometer: microplate reader
- 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 or Category 1 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin (no Category) if the viability is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 mg ± 2 mg per tissue

NEGATIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue

POSITIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue
Duration of treatment / exposure:
42 minutes (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 1
Value:
104.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 2
Value:
98.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 3
Value:
96.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
100.1
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: 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

The results obtained after treatment of the reconstructed human epidermis model with the test item are given in the following table.

 Table 1: Results

Group

Tissue 1

Tissue 2

Tissue 3

Mean

SD

OD

viability

OD

viability

OD

viability

OD

viability

viability

Negative Control

1.801

101.3%

1.764

99.2%

1.770

99.6%

1.778

100.0%

1.1%

Positive Control

0.024

1.3%

0.023

1.3%

0.027

1.5%

0.025

1.4%

7.1%

Test item

1.859

104.6%

1.759

98.9%

1.721

96.8%

1.780

100.1%

4.0%

 

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).
Executive summary:

The potential of the test item to induce skin irritation was investigated in an in vitro human skin model according to OECD guideline 439.

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 100.1% 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).
Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2019-04-02 to 2019-04-04
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.

Characterization of the Test System:
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27098
Keratinocyte strain: 4F118 8
Supplier: MatTek In Vitro Life Science Laboratories
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg per tissue
Duration of treatment / exposure:
6 hours (± 15 minutes)
Duration of post- treatment incubation (in vitro):
25 minutes (± 2 minutes)
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 pL DPBS and incubated at 37 °C and 5% C02 for 30 minutes (± 2 minutes).

Treatment:
After the 30-minute DPBS pre-treatment, the negative and the positive control were tested by applying 50 µL topically on the EpiOcular™ tissues. The solid test item was tested by evenly 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.

Post-Treatment Incubation
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% C02.

- RhCE tissue construct used, including batch number :
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27098
Keratinocyte strain: 4F118 8
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used : Test item: 50 mg per tissue; control substances: 50 µL per tissue

- 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

- 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 plate 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 :
The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60%. In this case no further testing in other test methods is required.
If the mean percent tissue viability is less than or equal 60%, no prediction can be made. In this case, further testing with other test methods will be required because RhCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 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 (treatment of liquid test items): below 50% of control viability
b) 6-hour exposure (treatment of solid test items): below 50% of control viability
3. The difference of viability between the two relating tissues of a single chemical is <20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: Viability (%)
Remarks:
Tissue 1
Run / experiment:
1
Value:
1.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability (%)
Remarks:
Tissue 2
Run / experiment:
2
Value:
1.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability (%)
Run / experiment:
Mean (tissue 1 and 2)
Value:
1.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid

The results obtained after treatment of the reconstructed human cornea-like epithelium (RhCE) model with the test item are given in the following table:

Table 1: Results

Group

Tissue 1

Tissue 2

Mean

SD

Difference between tissue replicates

OD

Viability

OD

Viability

OD

Viability

Viability

Negative Control

1.732

97.8%

1.809

102.1%

1.771

100.0%

3.04

4.3%

Positive Control

0.564

31.8%

0.608

34.3%

0.586

33.1%

1.77

2.5%

Test item

0.028

1.6%

0.021

1.2%

0.025

1.4%

0.28

0.4%

Following treatment with the test item, the tissue viability was 1.4% and, thus, lower than 60%. i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Interpretation of results:
other: Category 1 or 2 based on GHS criteria
Conclusions:
Following treatment with the test item, the tissue viability was 1.4% and, thus, lower than 60%. i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.
Executive summary:

The potential of the test item to induce eye irritation was investigated in an in vitro human cornea model according to OECD guideline 492.

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 1.771 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 33.1% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 1.4% and, thus, lower than 60%, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

OECD 439

The potential of the test item to induce skin irritation was investigated in an in vitro human skin model according to OECD guideline 439 (reference 7.3.1-1).

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 100.1% 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).
Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

Eye irritation

OECD 437

The potential of the test item to induce serious eye damage was examined in the BCOP assay according to OECD guideline 437 (reference 7.3.2-1). 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 (IVIS). After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.0 (study acceptance criteria range: -1.4 - 3.1). Treatment with the positive control (20% Imidazole) revealed an IVIS of 111.7 (study acceptance criteria range: 82.7 - 132.3). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 23.1 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

OECD 492:

As no prediction can be made from the BCOP Test a second in vitro test was performed in a top-down approach as recommended by OECD (IATA for serious eye damage and eye irritation, 2017).

The potential of the test item to induce eye irritation was investigated in an in vitro human cornea model according to OECD guideline 492 (reference 7.3.2-2). 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 1.771 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 33.1% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 1.4% and, thus, lower than 60%, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

The test item was evaluated for serious eye effects (OECD 437) for eye irritation (OECD 492) in validated in vitro test methods using a top-down approach. No prediction can be made based on both test systems therefore a weight of evidence approach is used to come to a conclusion. In the two in vitro tests adverse effects on cell viability and opacity of the cornea are observed but the substance is not identified as UN GHS Cat.1.
The mean IVIS in the Bovine Corneal Opacity and Permeability method (OECD 437) was 23.1 and thus below to the threshold of 55 for UN GHS Category 1. Furthermore, the mean cell viability obtained in the Eye Irritation Test (OECD 492) is 1.4 % and thus far below the threshold of 60 % which triggers “No Category”. In conclusion, the test item should be classified for eye irritation as UN GHS Cat.2. Therefore, no further testing is necessary.

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 twelfth time in Regulation (EU) 2019/521.