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Administrative data

Description of key information

Skin irritation

In a study according OECD TG 439, the test item is not identified as requiring classification and labelling according to UN GHS, as the mean percentage tissue viability was higher than 50% of the negative control (reference 7.3.1-1).

Eye irritation

In a study according OECD TG 492 using the EpiOcular™ model, the tissue viability was 69.1% and the test item is identified as not requiring classification and labeling according to UN GHS (No Category) (reference 7.3.2 -1).

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:
June 12,2019 - July 9,2019
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:
July 28, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The SkinEthic™ RHE-model RHE/S/17 was obtained from Episkin/SkinEthic Laboratories, Lyon, France.
- Tissue batch number(s): 19-RHE-110

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: 25 mL DPBS
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 5 minutes)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
- Viability: OD 1.3(CV= 1.7%)
- Barrier function: ET50=5.9h
- Morphology: Multi-layered, highly differentiated epidermis consisting of basal, spinous and granular layers, and a multi-layered stratum corneum.

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 50%.
A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is < 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 16 mg ± 2 mg per tissue

NEGATIVE CONTROL
- Amount(s) applied: 16 µL ± 0.5 uL per tissue

POSITIVE CONTROL
- Amount(s) applied: 16 µL ± 0.5 uL 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:
1st experiment
Value:
64.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
1.3%
Other effects / acceptance of results:
- Visible damage on test system: No
- 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

Table 1: Results

Group

Tissue 1

Tissue 2

Tissue 3

Mean

SD

 

 

OD

viability

OD

viability

OD

viability

OD

viability

viability

Negative
Control

1.845

1057%

1.753

100.5%

1.638

93.9%

1.745

100.0%

5.9%

Positive
Control

0.023

1.3%

0.025

1.4%

0.021

1.2%

0.023

1.3%

7.7%

Test item

1.098

62.9%

1.087

62.3%

1.199

68.7%

1.128

64.6%

5.4%

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.
Executive summary:

A study was performed according to the OECD TG 439 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 64.6% 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
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 29 , 2019 - July 4, 2019
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:
October 9, 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 mg


Duration of treatment / exposure:
6 hours (± 15 minutes)
Duration of post- treatment incubation (in vitro):
18 hours (± 15 minutes)
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used
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.

- RhCE tissue construct used, including batch number
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 30609 Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used
Solid test item: 50 mg per tissue
Negative control: 50 µL per tissue
Positive control: 50 µL per tissue

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
Exposure: 6 hours (± 15 minutes) at 37°C and 5% CO2
Post-exposure immersion: 25 minutes (± 2 minutes) at room temperature
Post-exposure incubation: 18 hours (± 15 minutes) at 37°C and 5% CO2

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
A 1.0 mg/mL MTT solution (in DMEM) was prepared. 50 mg of the test item were added to 1 mL of the MTT solution in a 6-well plate and the mixture was incubated at 37 ± 1°C and 5% CO2 and protected from light for 3 hours (± 5 minutes). Sterile deionised water (50 µL) was used as negative control concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
The non-colored test item was tested for its ability to become colorant after contact with water or isopropanol. For this purpose, 50 mg of the test item were added to 1.0 mL of water in a 6-well plate and the mixture was incubated for 1 hour at 37 ± 1°C and 5% CO2 protected from light. Furthermore, 50 mg were added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in 6-well plates for 2 to 3 hours at room temperature.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled)
The test item as well as the positive and negative control were tested in batch-duplicates. Therefore, a total number of six tissues was used in this study.

- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
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. The results of the functional test and the printout of the measurement were included in the raw data of the study.

- 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 pL 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.

- 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.

- Acceptable variability between tissue replicates for positive and negative controls
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

- Acceptable variability between tissue replicates for the test chemical
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
Run / experiment:
1st experiment
Value:
69.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
31.4%
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: 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.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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

Viability

 

 

Negative
Control

1.190

95.8%

1.294

104.2%

1.242

100.0%

5.94

8.4%

Positive Control

0.420

33.8%

0.360

29 0%

0.390

31.4%

3.39

4.8%

Test item

0.879

70.8%

0.837

67.4%

0.858

69.1%

2 40

3.4%

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item did not show an eye irritating potential.
Executive summary:

A study was performed according to OECD TG 492 and the MatTek Protocol "EpiOcular™ Eye Irritation Test (OCL-200-EIT)" to investigate the eye irritation potential of the test material.

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 1.242 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 31.4% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 69.1% and, thus, higher than 60%, i.e. according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

A study was performed according to the OECD TG 439 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 64.6% 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

A study was performed according to OECD TG 492 and the MatTek Protocol "EpiOcular™ Eye Irritation Test (OCL-200-EIT)" to investigate the eye irritation potential of the test material.

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 1.242 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 31.4% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 69.1% and, thus, higher than 60%, i.e. according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category).

Justification for classification or non-classification

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

The available data for skin and eye irritation are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is not considered to be classified for skin and eye irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.