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Description of key information

Skin irritation / corrosion:

Two in vitro studies are available.

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

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

Eye irritation:

The study was performed in accordance with OECD Guideline 437. The test substance induces 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 2018-12-17 to 2018-12-21
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
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a
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): 29674 Kit K and L

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature
- Temperature of post-treatment incubation (if applicable): 36.8 - 37.6 °C (actual range)

REMOVAL OF TEST MATERIAL AND CONTROLS: washed with phosphate buffered saline to remove residual test item, 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: 5 mg/mL diluted (1:5)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® 1200 Pro Plate Reader.
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0.8 - 2.8

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 20 - 100%

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 [complete, e.g. 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 [complete, e.g. 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 (volume or weight with unit): 27.0 - 32.3 mg

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 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:
93
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean tissue viability at 3-minute application
Irritation / corrosion parameter:
% tissue viability
Value:
53
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean tissue viability at 1-hour application
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 6.6% 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 ≤13%
Interpretation of results:
other: unclassified
Conclusions:
In conclusion, the test substance 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 substance for its ability to induce skin corrosion in accordance with OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method).

The possible corrosive potential of the test substance was tested through topical application for 3 minutes and 1 hour. Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of the test substance was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 6.6% 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 ≤ 13%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 93% and 53%, respectively. The mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment.

In conclusion, the test substance 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 2019-02-12 to 2019-02-18
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
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a
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 Small Model TM
- Tissue batch number(s): 19-EKIN-007

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature
- Temperature of post-treatment incubation (if applicable): actual range 37.0 - 37.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS : 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

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control

NUMBER OF REPLICATE TISSUES: three tissues each for test item treatment, negative and positive control

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- A test item is considered irritant in the skin irritation test 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.
- A test item is considered non-irritant in the in vitro skin irritation test 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): 16.8 to 18.9 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 tissues per test item together with negative and positive controls.
Irritation / corrosion parameter:
% tissue viability
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean tissue viability after 15 ± 0.5 minutes 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 of the negative control tissues was within the laboratory historical control data rang
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.3%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was ≤ 7.0%, indicating that the test system functioned properly.
Interpretation of results:
other: unclassified
Conclusions:
In conclusion, the test substance is non-irritant in the in vitro skin irritation test under the experimental conditions.
Executive summary:

The objective of this study was to evaluate the test substance for its ability to induce skin irritation according to 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. 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 positive control had a mean cell viability of 7.3% 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 ≤ 7.0%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 100%.

In conclusion, the test substance is non-irritant in the in vitro skin irritation test under the experimental conditions.

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:
From 2018-12-17 to 2018-12-18
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
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a
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.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
Storage: 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) Fetal Bovine Serum).
Transport: 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): 352.5 to 383.3 mg
Duration of treatment / exposure:
240 ± 10 minutes
Number of animals or in vitro replicates:
three corneas for each treatment group.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS :
- 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) Fetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) 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.
- Cornea Selection: 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. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

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 for each treatment group

NEGATIVE CONTROL USED : physiological saline

POSITIVE CONTROL USED : 20% (w/v) Imidazole solution prepared in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
- Dose: 352.5 to 383.3 mg of the test item; 750 μl of the negative control and 20% (w/v) Imidazole solution (positive control)
- Exposure time: 240 ± 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red

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 microtiter plate 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:
251
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
271
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
297
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean opacity and mean permeability for the negative controls are 0.5 and 0.014, respectively, which are less than the upper limits of the laboratory historical range.
- Acceptance criteria met for positive control: The mean in vitro irritancy score for the positive controls is 138 that falls within two standard deviations of the current historical mean.

The individual in vitro irritancy scores for the negative controls ranged from -0.4 to 1.7. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 118 to 149. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from 242 to 288 and permeability values ranging from 0.166 to 0.599. The corneas were turbid after the 240 minutes of treatment with the test item. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 251 to 297 after 240 minutes of treatment with the test item.

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In conclusion, since the test substance induced an IVIS ≥55, it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test substance 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).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for approximately 240 minutes.

 

The individual in vitro irritancy scores for the negative controls ranged from -0.4 to 1.7. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 118 to 149. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from 242 to 288 and permeability values ranging from 0.166 to 0.599. The corneas were turbid after the 240 minutes of treatment with the test item. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 251 to 297 after 240 minutes of treatment with the test item. The test item induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 273 after 4 hours of treatment.

 

In conclusion, since the test substance induced an IVIS55, it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion:

The objective of this study was to evaluate the test substance for its ability to induce skin corrosion in accordance with OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method).

The possible corrosive potential of the test substance was tested through topical application for 3 minutes and 1 hour. Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of the test substance was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 6.6% 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 ≤ 13%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 93% and 53%, respectively. The mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment.

In conclusion, the test substance 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 substance for its ability to induce skin irritation according to 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. 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 positive control had a mean cell viability of 7.3% 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 ≤ 7.0%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 100%.

In conclusion, the test substance is non-irritant in the in vitro skin irritation test under the experimental conditions.

Eye irritation:

The objective of this study was to evaluate the eye hazard potential of the test substance 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).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for approximately 240 minutes.

 

The individual in vitro irritancy scores for the negative controls ranged from -0.4 to 1.7. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 118 to 149. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from 242 to 288 and permeability values ranging from 0.166 to 0.599. The corneas were turbid after the 240 minutes of treatment with the test item. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 251 to 297 after 240 minutes of treatment with the test item. The test item induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 273 after 4 hours of treatment.

 

In conclusion, since the test substance induced an IVIS55, it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

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:

Since the test substance induced an IVIS55, it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test.

According to Regulation (EC) No 1272/2008, section 3.3.2.1, this substance should be classified as category 1 for this endpoint.