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Description of key information

Skin irritation / corrosion:

Two in vitro studies are available.

One in vitro skin corrosion study was performed in accordance with OECD Guideline 431. The test item is not corrosive.

Another in vitro skin irritation study was performed in accordance with OECD Guideline 439. The test item is non-irritant.

Eye irritation:

The study was performed in accordance with OECD Guideline 437. The test item induced an IVIS ≤ 3, so no classification is required for eye irritation or serious eye damage.

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:
From 2019-04-01 to 2019-04-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: A03-18-0081
Purity: ≥99.9%
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 30311 Kit F

TEMPERATURE USED FOR TEST SYSTEM
- Temperature: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 36 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.8 - 37.9 °C).

REMOVAL OF TEST MATERIAL AND CONTROLS : After the exposure period, the tissues washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0.8 - 2.8

NUMBER OF REPLICATE TISSUES: two tissues for 3-minute exposure and two for a 1-hour exposure, 2 tissues for the negative and positive controls for both the 3-minute and 1-hour time point

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%.]
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: applied undiluted (50 μL)

NEGATIVE CONTROL
- Amount(s) applied: 50 μL Milli-Q water

POSITIVE CONTROL
- Amount(s) applied: 50 μL 8N KOH
Duration of treatment / exposure:
3 minutes and 1 hour
Number of replicates:
two tissues for 3-minute exposure and two for a 1-hour exposure, 2 tissues for the negative and positive controls for both the 3-minute and 1-hour time point
Irritation / corrosion parameter:
% tissue viability
Value:
84
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The mean relative tissue viability after 3-minute treatment
Irritation / corrosion parameter:
% tissue viability
Value:
92
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The mean relative tissue viability after 1-hour treatment
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: the test item did not interfere with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean relative tissue viability of 9.8% after the 1-hour exposure.
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 15%
Interpretation of results:
other: unclassified
Conclusions:
The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) according to OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method).

The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour. The test item was applied undiluted (50 μL) directly on top of the skin tissue.

 

The positive control had a mean relative tissue viability of 9.8% after the 1-hour exposure.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 15%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 84% and 92%, respectively.

Because the mean relative tissue viability for Vinyl ethylene carbonate was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

 

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2019-04-24 to 2019-04-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
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)
Specific details on test material used for the study:
Batch No.: A03-18-0081
Purity: ≥99.9%
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SMTM
- Tissue batch number(s): 19-EKIN-017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 15 ± 0.5 minutes at room temperature.
- Temperature of post-treatment incubation (if applicable): incubated for 42 hours at 37 °C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with phosphate buffered saline to remove residual test item. 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: final concentration 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per test item together with negative and positive controls.

DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three in dividual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is < 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): applied undiluted (25 μL)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL PBS (Phosphate buffered saline)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL 5% SDS (Sodium dodecyl sulfate)
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
107
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: No color changes were observed it was concluded that the test item did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 13%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was < 9%, indicating that the test system functioned properly.
Interpretation of results:
other: unclassified
Conclusions:
The test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

The objective of this study was to evaluate the test item for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)) according to OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method).

The possible skin irritation potential of the test item was tested through topical application for 15 minutes. The test item was applied undiluted (25 μL), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test item.

 

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 107%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 13% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 9%, indicating that the test system functioned properly.

 

In conclusion, the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

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:
2019-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: A03-18-0081
Purity: ≥99.9%
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: 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.
- Transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
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): 750 μL
Duration of treatment / exposure:
10 min
Duration of post- treatment incubation (in vitro):
120 ± 10 minutes
Number of animals or in vitro replicates:
Three corneas for each treatment group.
Details on study design:
PREPARATION OF CORNEAS :
The isolated corneas were stored in a petri dish with cMEM (Earle's Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.

SELECTION OF CORNEAS:
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

QUALITY CHECK OF THE ISOLATED CORNEAS :
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

NUMBER OF REPLICATES :
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED : Yes, physiological saline.

POSITIVE CONTROL USED : Yes, ethanol.

APPLICATION DOSE AND EXPOSURE TIME : 750 μL; 10 min

POST-INCUBATION PERIOD: yes, incubated for 120 ± 10 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed with MEM with phenol red (Earle's Minimum Essential Medium) and thereafter with cMEM.
- POST-EXPOSURE INCUBATION: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.
Irritation parameter:
in vitro irritation score
Value:
-0.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: A mean in vitro irritation score
Other effects / acceptance of results:
OTHER EFFECTS:
The corneas were clear after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: Yes. The mean in vitro irritancy score of the positive control (Ethanol) was 48 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The individual in vitro irritancy scores for the negative controls ranged from -0.7 to 0.2. The corneas treated with the negative control item were clear after the 10 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 33 to 60. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from -1.5 to -0.5 and permeability values ranging from -0.004 to 0.001. The corneas were clear after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.5 to -0.5 after 10 minutes of treatment with the test item.

Interpretation of results:
other: unclassified
Conclusions:
The test item induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage).

The eye damage of the test item was tested through topical application for 10 minutes. The test item was applied as it is (750 μL) directly on top of the corneas.

 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (Ethanol) was 48 and was within two standard deviations of the current historical positive control mean. 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, resulting in a mean in vitro irritancy score of -0.9 after 10 minutes of treatment.

In conclusion, since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion:

The objective of this study was to evaluate the test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) according to OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method).

The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour. The test item was applied undiluted (50 μL) directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 9.8% after the 1-hour exposure.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 15%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 84% and 92%, respectively.

Because the mean relative tissue viability for Vinyl ethylene carbonate was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Skin irritation:

The objective of this study was to evaluate the test item for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)) according to OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method).

The possible skin irritation potential of the test item was tested through topical application for 15 minutes. The test item was applied undiluted (25 μL), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test item.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 107%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 13% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 9%, indicating that the test system functioned properly.

In conclusion, the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Eye irritation:

The objective of this study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage).

The eye damage of the test item was tested through topical application for 10 minutes. The test item was applied as it is (750 μL) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (Ethanol) was 48 and was within two standard deviations of the current historical positive control mean. 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, resulting in a mean in vitro irritancy score of -0.9 after 10 minutes of treatment.

In conclusion, since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Justification for classification or non-classification

Skin irritation / corrosion:

The test item is not corrosive in the in vitro skin corrosion test and non-irritant in the in vitro skin irritation test.

According to Regulation (EC) No 1272/2008, section 3.2.2.1, this substance should not be classified for this endpoint.

Serious eye damage/eye irritation:

The test item did not induce ocular irritation and induced an IVIS ≤ 3.

According to Regulation (EC) No 1272/2008, section 3.3.2.1, this substance is not classified for this endpoint.