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Diss Factsheets

Administrative data

Description of key information

Skin Irritation: In a study according to OECD TG 439 the test item is not identified to possess an irritant potential to skin (reference 7.3.1 -1).

Eye Irritation: In a study according to OECD TG 492 the test item is identified as not requiring classification and labeling according to UN GHS (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:
October 10, 2018 - October 12, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
artificial membrane barrier model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE-model RHE/S/17 from Episkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 18-RHE-118

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: once gently rinsed with a min. of 25 µL 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 at 37°C and 5% CO2
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
see table 1

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
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.

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The OD values for the negative control shall be in the range of ≥ 0.8 and ≤ 3.0 as given in OECD Guideline 439.
Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
The negative control data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm. The standard deviation value is considered valid if ≤ 18% of group mean-value.
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is < 40%. The standard deviation value is considered valid if ≤ 18% of group mean-value.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 ± 2 mg per tissue

NEGATIVE CONTROL
- Amount applied (volume or weight): 16 ± 0.5 µL per tissue

POSITIVE CONTROL
- Amount applied (volume or weight): 16 ± 0.5 µL per tissue
Duration of treatment / exposure:
42 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st
Value:
85.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
1%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
The pre-test for direct MTT-reducing capacity of the test item did not result in blue colour, i.e. the test item is not a direct MTT reducer.
- Colour interference with MTT: no
In the pre-test, medium colouration by the test item was observed, but no tissues were stained during the study. Therefore, no additional tissues for colour control were treated according to the SOP Skinethic Skin Irritation Test (2009).

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 3: Results

Group

Tissue 1

Tissue 2

Tissue 3

Mean

SD

 

 

OD

viability

OD

viability

OD

viability

OD

viability

viability

Negative Control

2.071

106.7%

1.787

92.1%

1.965

101.2%

1.941

100.00

7.4%

Positive Control

0.020

1.0%

0.019

1.0%

0.020

1.0%

0.020

1.0%

0.0%

Test item

1.595

82.2%

1.651

85.1%

1.729

89.1%

1.658

85.5%

4.1%

Table 4: Acceptability of the Test

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

 

Acceptance Criterion

Result

Negative control OD

≥ 0.8 and ≤3.0

1.787 to 2.071

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

 

Acceptance Criterion

Result

Mean OD negative control

≥ 1.2

1.941

Mean viability positive control

<40%

1.0%

SD of group-mean value

 

≤ 18%

0.0% (positive control)

7.4% (negative control)

 

 

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

 

Acceptance Criterion

Result

Mean OD negative control

≥ 1.420

1.941

Mean viability positive control

≤ 2.82%

1.0%

Test Item Data Acceptance Criteria:

 

Acceptance Criterion

Result

SD of group-mean value

≤ 18%

4.1%

The study met all acceptance criteria.

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 according OECD TG 439 was conducted 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 deionized 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 85.5% 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:
November 13, 2018 - November 15, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
human
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.
- Description of the cell system used: The EpiOcular™-model is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg per tissue


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:
- RhCE tissue construct used, including batch number :
The EpiOcular™ Tissues (OCL-200, OCL-212) was obtained from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.
Lot No.: 27079
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:
incubated at 37°C and 5% CO2 for 6 hours (±15 minutes), post-treatment: 18 hours (±15 minutes) at 37°C and 5% CO2.

- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2

- Wavelength and band pass used for quantifying MTT formazan:
The OD was read using a spectrophotometer at 570 nm wavelength.

- 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.

- 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.

- Complete supporting information for the specific RhCE tissue construct used : see table 1

- 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. 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:
1
Value:
90.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
25.4%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

In the assessment of coloured or staining test items the OD of the test item solution (after subtraction of the OD for isopropanol) was 0.0045 and, thus, < 0.08. Therefore, the test item was not considered as possibly interacting with the MTT measurement and no additional test on colourant controls according to the protocol provided by the supplier (MatTek In Vitro Life Science Laboratories) was performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
The negative control OD is >0.8 and <2.5 (2.049 and 2.148).
- Acceptance criteria met for positive control: yes
The mean relative viability of the positive control is below 50% of the negative control viability (25.4%). The difference of viability between the two relating tissues of a single chemical is <20%
(values between 4.7% to 9.8%) in the same run (for positive and negative control tissues and tissues of single chemicals).
The study met all acceptance criteria.

Table 2: Results

Group

Tissue 1

Tissue 2

Mean

SD

Difference
between tissue
replicates

 

 

 

 

OD

Viability

OD

viability

OD

Viability

Viability

Negative
Control

2.148

102.3%

2.049

97.6%

2.099

100.0%

3.32

4.7%

 

Positive Control

0.636

30.3%

0.430

20.5%

0.533

25.4%

6.93

9.8%

Test item

1.965

93.6%

1.834

87.4%

1.900

90.5%

4.38

6.2%

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is identified as not requiring classification and labeling according to UN GHS (No Category).
Executive summary:

A study according OECD TG 492 was conducted 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 alter 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 2.099 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 25.4% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 90.5% and, thus, higher than 60%, i.e. according to ORCD 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 according OECD TG 439 was conducted 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 deionized 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 85.5% and thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (reference 7.3.1 -1).

Eye Irritation

A study according OECD TG 492 was conducted 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 alter 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 2.099 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 25.4% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 90.5% and, thus, higher than 60%, i.e. according to ORCD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category) (reference 7.3.2 -1).

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.