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Description of key information

In an in vitro study according OECD TG 431 (2004) the test item is not considered to possess a corrosive potential (reference 7.3.1 -1).

In an in vitro study according to B.46 of Council Regulation (EC) No. 761/2009 the test item is considered to possess an irritant potential (reference 7.3.1 -2).

Based on the results of both studies the test substance is considered to have the potential to cause skin irritation but not skin corrosion.

 

In an in vitro study according OECD TG 437 the test item is not considered to possess a corrosive potential (reference 7.3.2 -1).

In an in vitro study according to OECD TG 492 the test item is determined to not require classification for eye irritation (reference 7.3.2 -2).

Based on the results of both studies the test substance is considered to have no potential to cause eye irritation or serious damage to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 17, 2010 - June 19,2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes
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: vitro skin model RHE by SkinEthic Laboratories

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: At the end of the treatment interval, the inserts were removed immediately from the 24-well plate. Using a multi pipette the tissues were gently rinsed with a minimum volume of 25 mL PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/L
- Incubation time: 3 hours
- 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 if the viability is less or equal than 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.

ACCEPTABILITY OF THE ASSAY
Negative control (NC acceptance criteria: The NC data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm according to the historical database. The Standard Deviation value is considered as valid if it is ≤ 18 %, according to the Performance Standards (ECVAM SIVS, 2007).Positive control (PO acceptance criteria: The PC data meet the acceptance criteria if the mean viability, expressed as % of the NC, is < 40 % and the Standard Deviation value is ≤ 18 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration: 5% in PBS
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 run
Value:
36
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
2.64%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: no

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

The results obtained after treatment are given in the following table:

Dose Group Treatment
Interval
Optical Density
Tissue 1
Optical Density
Tissue 2
Optical Density
Tissue 3
Mean relative
Viability [%]
neg. control 42 min 1.361 1.200 1.298 100.0
pos. control 42 min 0.033 0.036 0.034 2.64
test item 42 min 0.446 0.451 0.493 36.00


Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system.

After treatment with the negative control the absorbance values reached the required acceptability criterion of an OD >1.2 for the treatment interval thus showing the quality of the tissues.

The mean relative tissue viability after treatment with the test item was significantly decreased after treatment (mean relative viability = 36.00 %). Therefore, the test item is considered to possess an potential for skin irritation.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the experimental conditions reported, the test item is irritant to skin.
Executive summary:

A study according study according B.46 of Council Regulation (EC) No. 761/2009 was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test consisted of a topical exposure of the test item to a human reconstructed model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt, that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential.

Triplicates of the human skin model RHE were treated either with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (PBS-buffer) or the positive control (5 % Sodiumdodecylsulphat-solution) were applied to each tissue. Before adding the test item, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to each tissue.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system. After treatment with the negative control the absorbance values reached the required acceptability criterion of mean OD > 1.2 for the treatment interval thus showing the quality of the tissues. The tissue viability after treatment with the test item was significantly decreased (mean viability: 36.00 %). Therefore, the test item is considered to possess an irritant potential.

Under the experimental conditions reported, the test item is irritant to skin.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 08, 2010 - April 09, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes
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: vitro skin model RHE by SkinEthic Laboratories

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the treatment interval, the inserts were removed immediately from the 24-well plate. Using a multi pipette the tissues were gently rinsed with a minimum volume of 25 mL PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/L
- Incubation time: 3 hours
- 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 corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

ACCEPTABILITY OF THE ASSAY
Negative control (NC acceptance criteria: The NC data meet the acceptance criteria if the mean OD value of the 3 tissues is > 0.8 at 570 nm.
Positive control (PC) acceptance criteria: The PC data meet the acceptance criteria if it is classified as corrosive.
Test substance data acceptance criteria: Coefficient of variation of identically treated tissues has to be less than 30 %, with the exception of cases with OD below 0.3.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL
Duration of treatment / exposure:
3 min and 1hours
Duration of post-treatment incubation (if applicable):
None
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st run (3 min exposure)
Value:
116.04
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
18.6%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2nd run (1 hour exposure)
Value:
82.47
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
18.3%
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: no

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

The results obtained after treatment are given in the following table:

dose group treatment interval mean optical density mean relative viability [%]
neg. control 3 min 1.668 100.0
1 h 2.045 100.0
pos. control 3 min 0.310 18.60
1 h 0.374 18.30
test item 3 min 1.935 116.04
1 h 1.687 82.47


Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system.

After treatment with the negative control the absorbance values reached the required acceptability criterion of an OD >0.8 for the treatment interval thus showing the quality of the tissues.

The mean relative tissue viability after treatment with the test item was not significantly decreased after treatment. Therefore, the test item is not considered to possess a corrosive potential.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not considered to possess a corrosive potential.
Executive summary:

A study according OECD TG 431 (2004) was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test. The test consisted of a topical exposure of the test item to a human reconstructed model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin corrosion.

Triplicates of the human skin model RHE were treated either with the test item, negative or the positive control for 3 minutes and 1 hour. 40 µL of the negative control (distilled water) or 40 µL of the positive control (Potassium hydroxide, 8N) were applied to each tissue. Before adding the test item, 20 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis. Afterwards, 20 mg of the test item was applied to each tissue.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system. After treatment with the negative control the absorbance values reached the required acceptability criterion of an optical density (OD) >0.8 for the treatment interval thus showing the quality of the tissues. The mean relative tissue viability after treatment with the test item was not significantly decreased. Therefore, the test item is not considered to possess a corrosive potential.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
November 19,2019 - November 21, 2019
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:
June 18, 2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.69 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:
31 July 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
human
Details on test animals or tissues and environmental conditions:
- Description of the cell system used: 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.
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):
18 hours (± 15 minutes)
Number of animals or in vitro replicates:
3
Details on study design:
- RhCE tissue construct used:
The EpiOcular™ Tissues (OCL-200, OCL-212) was obtained from MatTek In Vitro Life Science Laboratories. Bratislava. Slovakia.

- 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:
The tissues were incubated at 37°C and 5% CO2 for 6 hours (± 15 minutes). Post-treatment Incubation was for 18 hours (± 15 minutes) at 37°C and 5% CO2.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
For correct interpretation of results, it is necessary to assess the ability of a test item to directly reduce MTT. For this purpose, 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 uL) was used as negative control concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

- Number of tissue replicates used per test chemical and controls: 3

- 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.

- 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 PdiCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

- Acceptance Criteria:
1. The negative control OD is >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
Run / experiment:
1st run
Value:
113.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
34.1%
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.

ACCEPTANCE OF RESULTS:
1. The negative control OD is >0.8 and <2.5 (1.924 and 2.060).
2. The mean relative viability of the positive control is below 50% of the negative control viability (34.1%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 3.7% to 6.8%) in the same run (for positive and negative control tissues and tissues of single chemicals).
The study met all acceptance criteria.

Table 1: Results

Group

Tissue 1

Tissue 2

Mean

SD

Difference
between tissue
replicates

 

OD

Viability

OD

Viability

OD

Viability

Negative
Control

1.924

96.6%

2.060

103.4%

1.992

100.0%

4.81

6.8%

Positive Control

0.633

31.8%

0.725

36.4%

0.679

34.1%

3.25

4.6%

Test item

2.217

111.3%

2.290

115.0%

2.254

113.2%

2.62

3.7%

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item did not show an eye irritating potential in the EpiOcular™-model.
Executive summary:

A study according OECD TG 492 was conducted to investigate the potential of the test 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 OD was 1.924 and 2.060 of the two treated tissue, respectively (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 34.1% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 113.2% 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:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May 04 , 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany

- Number of animals: no data

- Characteristics of donor animals (e.g. age, sex, weight): no data

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
The isolated eyes were transported to the laboratory in Hank's BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 (+ Earle's salts, + L-Glutamine, + 2.2 g/L sodium carbonate, - Phenol Red, Invitrogen GmbH, 64293 Darmstadt) supplemented with 2 µg/mL Taurine, and stored in the refrigerator at 2 - 8 °C until the following day. Shortly before use, 50 µg/mL Dextran (Sigma, 89555 Steinheim, Germany) was added to the medium.

- indication of any existing defects or lesions in ocular tissue samples:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in the experiment were collected in Hank's BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects listed above.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Concentration: 20% (w/v) in saline

VEHICLE
- Concentration: 0.9% NaCl (w/v) in deionised water
Duration of treatment / exposure:
240 ± 5 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Each cornea was mounted in a cornea holder 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 but stretching was avoided. After 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 complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 2 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED
0.9%(w/v) NaCl in deionised water

POSITIVE CONTROL USED
10% (w/v) Benzalconium chloride in 0.9% (w/v) NaCl (saline, produced in-house)

APPLICATION DOSE AND EXPOSURE TIME
Prior to the application the test item was suspended (20% (w/v)) in saline (0.9% NaCl (w/v) in deionised water). For this reason, 800 mg of the test item were introduced in 4 mL saline. Fresh cMEM was placed in the posterior compartment, while the anterior compartment received the test item or negative or positive control. The controls were applied at a volume of 0.75 mL ensuring sufficient coverage of the epithelial surface of the corneae and the chamber was incubated at 32 ± 2 °C in the water-bath in a horizontal position. The incubation time lasted 240 ± 5 minutes.

TREATMENT METHOD: open chamber

REMOVAL OF TEST SUBSTANCE
After the test item or control items, respectively, were rinsed off from the application side by changing cMEM three times, fresh cMEM was replaced in both compartments and opacity was measured (t240). In the second step of the assay, permeability of the cornea, was determined.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was measured with an opacitometer resulting in a numerical opacity value.
- Corneal permeability: After the opacity measurement, the cMEM was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. The corneae were incubated again in a horizontal position for about 90 minutes in a water-bath at 32 ± 2 °C. cMEM from the posterior compartment was removed with a 5 mL-syringe, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the in vitro irritation score of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro scores of the positive control and of the test item, respectively:
In vitro Irritation Score = (opacity value - opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score value of each treated group was calculated from the individual in vitro irritation scores.

In vitro Score:
0 - 3 Non eye irritant
3.1 - 25 Mild eye irritant
25.1 - 55 Moderate eye irritant
55.1 - 80 Severe eye irritant
> 80 Very severe eye irritant

CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable since the positive control caused an at least moderate effect (in vitro irritation score was 191.42, what means very severe irritant) on the corneae, and since the negative control did not cause any effect (irritation score was 1.31) on the corneae.
Irritation parameter:
in vitro irritation score
Run / experiment:
1st run
Value:
11.21
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
1.31
Positive controls validity:
valid
Remarks:
191.42
Other effects / acceptance of results:
The test item caused weak opacity but no permeability of the corneae compared with the results of the negative control. The calculated in vitro score was 11.21 and therefore, the test item was classified as mild eye irritant.

Table 1: Results

Test Group

Opacity value =
Difference (t240-t0)
of Opacity *

Permeability at
490 nm (OD490)*

In vitro
Score

Mean in
vitro Score

Proposed
in vitro
Irritation
Scale

 

 

Mean

 

Mean

 

 

 

Negative
Control

0

0.33

0.070

0.065

1.05

1.31

Non eye

irritant

 

 

1

 

 

0.060

 

 

1.90

 

 

 

 

 

 

0

 

 

0.065

 

 

0.98

 

 

 

 

Positive
Control

187.67*

0.106*

189.26

191.42

Very severe
eye irritant

 

 

182.67*

0.046*

183.36

 

 

 

 

 

 

198.67*

0.198*

201.64

 

 

 

 

Test item

1.67*

- 0.003*

1.62

11.21

Mild eye
irritant

 

 

26.67*

-0.013*

26.47

 

 

 

 

 

 

5.67*

- 0.008*

5.55

 

 

 

 

*Corrected values

 

Interpretation of results:
GHS criteria not met
Conclusions:
The study performed shows that the test item is considered to be a mild eye irritant in vitro by means of the BCOP assay.
Executive summary:

An in vitro study according OECD TG 437 was performed to assess the corneal irritation and damage potential of the test item by means of the BCOP assay using fresh bovine corneae.Damage is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitometer and a visible light spectrophotometer, respectively. Both measurements are used to calculate an in vitro irritation score, which is used to assign an in vitro irritancy category. After a first opacity measurement of the fresh bovine corneae (t0), the test item/saline (0.9 % (w/v) NaCl in deionised water) stock, the positive, or the negative controls were applied to the epithelial surface of three corneae each and incubated for 240 minutes at 32 +/- 2 °C. After the incubation phase, the cornea were each rinsed and opacity was measured again (t240). Thereafter the permeability of the corneae was determined. Therefore, 1 mL of a fluorescein solution was added to the anterior chamber of the corneal holder and after 90 min incubation in a horizontal position at 32 +/- 2 °C, the permeability of the corneae was measured quantitatively using the increase in fluorescence in the posterior chamber medium at 490 nm.

The negative control (0.9 % NaCl solution) yields no increase of opacity nor permeability of the corneae, while the the positive control (10 % (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae, thus demonstrating the validity of the assay. The test item caused weak opacity, but no permeability of the corneae. The calculated mean in vitro score was 11.21. Based on the irritation schema, the test item shows mild mild eye irritant properties in vitro.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

To determine the corrosion or irritation potential of the test item for the skin a stepwise weight of evidence approach was done. A skin corrosion assay (RhE) according to OECD Guideline 431 (version 2004) was conducted resulting in no classification in Category 1. Therefore, a skin irritation study (RhE) according to B.46 (version of Council Regulation (EC) No. 761/2009) was conducted.

 

Studies

OECD 431 (reference 7.3.1 -1)

A study according OECD TG 431 (2004) was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test. The test consisted of a topical exposure of the test item to a human reconstructed model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin corrosion. Triplicates of the human skin model RHE were treated either with the test item, negative or the positive control for 3 minutes and 1 hour. 40 µL of the negative control (distilled water) or 40 µL of the positive control (Potassium hydroxide, 8N) were applied to each tissue. Before adding the test item, 20 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis. Afterwards, 20 mg of the test item was applied to each tissue. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system. After treatment with the negative control the absorbance values reached the required acceptability criterion of an optical density (OD) >0.8 for the treatment interval thus showing the quality of the tissues. The mean relative tissue viability after treatment with the test item was not significantly decreased. Therefore, the test item is not considered to possess a corrosive potential.

 

EU B.46 (reference 7.3.1 -2)

A study according to B.46 of Council Regulation (EC) No. 761/2009 was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test consisted of a topical exposure of the test item to a human reconstructed model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential. Triplicates of the human skin model RHE were treated either with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (PBS-buffer) or the positive control (5 % Sodiumdodecylsulphat-solution) were applied to each tissue. Before adding the test item, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to each tissue. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system. After treatment with the negative control the absorbance values reached the required acceptability criterion of mean OD > 1.2 for the treatment interval thus showing the quality of the tissues. The tissue viability after treatment with the test item was significantly decreased (mean viability: 36.00 %). Therefore, the test item is considered to possess an irritant potential. Under the experimental conditions reported, the test item is irritant to skin.

 

Conclusion

Using the weight of evidence approach the results of the two in vitro studies on skin corrosion (OEDC 431) and skin irritation (B.46) demonstrate that the test item is not considered as requiring classification for skin corrosion (UN GHS Category 1). However, it is considered to be irritant to skin (UN GHS Category 2). Therefore, the test item is determined to require classification for skin irritation Cat.2 (H315: Causes skin irritation.).

 

 

Eye irritation

To determine the corrosion or irritation potential of the test substance for the eye a stepwise weight of evidence approach was done. A Bovine Corneal Opacity and Permeability Test according to OECD 437 was conducted resulting in a mean IVIS score of 11.21 or “mild irritant” according to the study report. The IVIS score according to OECD 437 (2009) did not lead to identification as corrosive or severe irritation for the substance. As outlined in the guideline “if the test substance is not identified as an ocular corrosive or severe irritant, additional testing should be conducted for classification and labeling purposes”. Therefore an eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 was conducted.

Studies

OECD 437 (reference 7.3.2 -1)

An in vitro study according OECD TG 437 was performed to assess the corneal irritation and damage potential of the test item by means of the BCOP assay using fresh bovine cornea. Damage is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitometer and a visible light spectrophotometer, respectively. Both measurements are used to calculate an in vitro irritation score, which is used to assign an in vitro irritancy category. After a first opacity measurement of the fresh bovine cornea (t0), the test item/saline (0.9 % (w/v) NaCl in deionised water) stock, the positive, or the negative controls were applied to the epithelial surface of three cornea each and incubated for 240 minutes at 32 +/- 2 °C. After the incubation phase, the cornea were each rinsed and opacity was measured again (t240). Thereafter the permeability of the cornea was determined. Therefore, 1 mL of a fluorescein solution was added to the anterior chamber of the corneal holder and after 90 min incubation in a horizontal position at 32 +/- 2 °C, the permeability of the cornea was measured quantitatively using the increase in fluorescence in the posterior chamber medium at 490 nm. The negative control (0.9 % NaCl solution) yields no increase of opacity nor permeability of the cornea, while the positive control (10 % (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the cornea, thus demonstrating the validity of the assay. The test item caused weak opacity, but no permeability of the cornea. The calculated mean in vitro score was 11.21. Based on the irritation schema, the test item shows mild mild eye irritant properties in vitro.

 

OECD 492 (reference 7.3.2 -2)

A study according OECD TG 492 was conducted to investigate the potential of the test 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 OD was 1.924 and 2.060 of the two treated tissue, respectively (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 34.1 % (study acceptance criterion: <50 %). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 113.2 % 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).

 

Conclusion

Based on the result from the Bovine Corneal Opacity and Permeability Test according to OECD 437 a categorization into Category 1 was excluded. The second in vitro study, the eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492, determined that the test item does not require classification and labeling according to UN GHS (No Category). The weight of evidence with the two conducted studies determined that the test substance is not to be classified as 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 considered to be classified for skin irritation Cat.2 (H315: Causes skin irritation) but not for eye irritation under Regulation (EC) No 1272/2008, as amended for the fourteenth time in Regulation (EU) 2020/217.