Registration Dossier

Administrative data

Description of key information

Based on the result of the OECD 439 study, the test item is considered to be irritant to the skin (Cat. 2).

Based on the result of the OECD 431 study, the test item is not considered to be corrosive to the skin.

Based on the result of the OECD 437 study, the test item is not considered to be irritant to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 April 2017 - 30 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: B.40 bis (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: EpiDermTM Kit
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
Justification for test system used:
The EpiDermTM is an in vitro system recommended by the corresponding guideline.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 Kit
- Tissue batch number: 25811
- Date of initiation of testing: 2017-05-10

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 3 minutes; room temperature for 60 minutes

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 20 times
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax Molecular Devices, Softmax Pro
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA
The test substance is considered to be corrosive to skin if:
- < 50% after 3 minutes exposure
- ≥ 50% after 3 minutes exposure AND < 15% after 60 minutes exposure
The test substance is considered to be non-corrosive to skin if:
- ≥ 50% after 3 minutes exposure AND ≥ 15% after 60 minutes exposure
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL
Duration of treatment / exposure:
3 min and 60 min
Number of replicates:
2
Irritation / corrosion parameter:
other: Relative Absorbance (% of Negative Control)
Run / experiment:
1_Exposure: 3 min
Value:
103.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: Relative Absorbance (% of Negative Control)
Run / experiment:
2_Exposure: 3 min
Value:
107.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: Relative Absorbance (% of Negative Control)
Run / experiment:
1_Exposure: 1 h
Value:
123.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: Relative Absorbance (% of Negative Control)
Run / experiment:
2_Exposure: 1h
Value:
126.5
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: none
- Colour interference with MTT: none

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:
The test item is non corrosive to skin according to EU CLP and UN GHS.
Executive summary:

An in vitro study was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item passed the pre-test for colour interference and for direct MTT reduction. Consequently, additional tests were not necessary.

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. After exposure of the tissues to the test item the relative absorbance value did not decrease (105.7%) after 3 minutes exposure. After 1 hour exposure the relative absorbance value was also not reduced (124.9%). Both values did not affect the threshold for corrosiveness which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. The required acceptability criteria were met. The test item is not considered to be corrosive.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February 2015
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France
Vehicle:
unchanged (no vehicle)
Details on test system:
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Twenty five μl of the undiluted test substance was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 µL
Duration of treatment / exposure:
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
Mean tissue viability
Run / experiment:
Repeat experiment
Value:
18
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test item is considered to be irritant.
Executive summary:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 18% (individual values of 25, 19 and 10%). Since the mean relative tissue viability for the test item was below or equal to 50% it is considered to be irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
09 December, 2014
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
(2013)
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:
(2010)
Deviations:
no
Principles of method if other than guideline:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes
Details on test animals or tissues and environmental conditions:
Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount applied: approx. 750 µL per cornea

NEGATIVE CONTROL
- Amount applied: 750 µL of physiological saline per cornea

POSITIVE CONTROL
Amount applied: 750 µL per cornea
Concentration: 10% (w/v) Benzalkonium Chloride
Duration of treatment / exposure:
10 minutes
Number of animals or in vitro replicates:
Three corneas / substance or control.
Details on study design:
TEST SITE
- Isolated bovine cornea, incubation at 32°C.

REMOVAL OF TEST SUBSTANCE
- Washing: yes
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed (possible pH effects of the test substance on the corneas were recorded) by incubation for 120 minutesat 32°C. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated (incubation 90 minutes at 32°C).

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).
Opacity and permeability values were also evaluated independently.

TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≤ 3 is defined as non irritant, IVIS > 3 ≤ 55 mild to moderate and IVIS ≥ 55 is defined as a corrosive or severe irritant

ACCEPTABILITY OF THE ASSAY:
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within the laboratory historical mean value.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean_time point: 10 min
Value:
-1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

The corneas treated with the test item showed opacity values ranging from -3 to 0 and permeability value was -0.002 for all three corneas. The corneas were clear after the 10 minutes of treatment with the test item. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -3.0 to 0.0 after 10 minutes of treatment with the test item.

Interpretation of results:
not irritating
Conclusions:
The test item is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test performed according to OECD 437 guideline and GLP principles, as the test item induced an IVIS ≤ 3.
Executive summary:

The test item was screened for the eye irritation and corrosion potential using the Bovine Corneal Opacity and Permeability test performed according to OECD 437 guideline and GLP principles.

Adequate negative and positive controls were included and it was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test item did not induce ocular irritation through both endpoints, opacity and permeability, resulting in a mean in vitro irritancy score of -1.0 after 10 minutes of treatment. Based on the results, it is concluded that the test item is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test. Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to Regulation (EC) 1272/2008.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 18% (individual values of 25, 19 and 10%). Since the mean relative tissue viability for the test item was below or equal to 50% it is considered to be irritant.

Skin corrosion;

An in vitro study was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™ tissues models. The test item passed the pre-test for colour interference and for direct MTT reduction. Consequently, additional tests were not necessary. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. After exposure of the tissues to the test item the relative absorbance value did not decrease (105.7%) after 3 minutes exposure. After 1 hour exposure the relative absorbance value was also not reduced (124.9%). Both values did not affect the threshold for corrosiveness which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. The required acceptability criteria were met. The test item is not considered to be corrosive.

Eye irritation:

The test item was screened for the eye irritation and corrosion potential using the Bovine Corneal Opacity and Permeability test performed according to OECD 437 guideline and GLP principles. Adequate negative and positive controls were included and it was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, opacity and permeability, resulting in a meanin vitroirritancy score of -1.0 after 10 minutes of treatment. Based on the results, it is concluded that the test item is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test. Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to Regulation (EC) 1272/2008.

Justification for classification or non-classification

Classification, Labelling, 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 considered to be irritant to the skin (Cat. 2, H315) and is not considered to be irritant to the eye under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.