Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
In vitro skin corrosion test with EpiDerm (EPI-200))
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 26, 2017 to July 27, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted 29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Remarks:
EpiDerm Skin Model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts. Recommended test system in international guidelines (OECD and EC)
Vehicle:
unchanged (no vehicle)
Details on test system:
Test system specifications: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum was supplied by MatTek In Vitro Life ScienceLaboratories, Bratislava, Slovak Republic.

System conditions: Human keratinocytes are used to construct the epithelium. Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model was supplied free of contamination with bacteria, mycoplasma and fungi.

Assessment of MTT Interacting substances: In order to assess the potential non-specific reduction of the test substance, 50 μL of test substance was added to 1 mL of 1.0 mg/mL MTT and the colour change was assessedcafter incubation for 60 minutes at 37±1°C, 5±1% CO2, 95% RH. There was no change in colour therefore the test substance did not interact with MTT.

Assessment for colour interference: 50 μL of test substance was added to both 0.3 mL deionized water and isopropanol, before being incubated for 60 minutes at 37±1°C, 5±1% CO2, 95% RH. The solutions did not become coloured, therefore the test substance was deemed not to have the potential to stain the tissue.

Application of test and control substances: On the day of receipt EpiDermTM tissues were transferred to refrigerator at 2 to 8°C and stored overnight. The next day, approximately 1 hour before starting the assay, the tissues were prepared for treatment in labelled 6-well plates. The test was performed on a total of four tissues per test substance, negative and positive control, out of which two were used for a 3 minute application and two tissues were used for a 1 hour application. A volume of 50 μL of the undiluted test substance was applied to the tissue. Further tissues were concurrently treated with 50 μL distilled water (negative control) and with 50 μL 8M potassium hydroxide (positive control). After the 3 minute or 1-hour contact periods, the tissues were washed with phosphate buffered saline (PBS) to remove residual material. The rinsed tissues were kept in 24-well plates (holding plates) until all tissues were dosed and rinsed. The positive control material was a direct MTT reducer, therefore additional positive controls were performed. The positive control substance was applied to two freeze-killed tissues (thawed on the day of treatment) per exposure time.

Cell viability measurements: 1 mg/mL MTT-medium and were incubated for 3 hours (37±1°C, 5±1% CO2, 95% RH). After incubation any resultant colour was extracted with 2 mL isopropanol overnight. The optical density of each resultant extract was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test substance was classified according to the remaining cell viability obtained after test substance treatment with either of the two treatment times.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL
Duration of treatment / exposure:
3 minutes and 1 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Remarks:
cytotoxicity
Run / experiment:
3 minutes treatment
Value:
ca. 82
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
cytotoxicity
Run / experiment:
1 h treatment
Value:
ca. 73.9
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
-The test substance was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test substance did not interfere with the MTT endpoint.

The mean absorption at 570 nm measured after treatment with the test substance and controls were as follows:
1) Negative control 1.100 (3 minute application) and 1.211 (1 h application)
2) Test substance 0.905 (3 minute application) and 0.894 (1 h application)
3) Positive control 0.294 (3 minute application) and 0.210 (1 h application)

- The mean tissue viability obtained after 3 minute and 1 h treatments with the test substance compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after the 3 minute and 1 h treatments with the test substance compared to the negative control tissues was 82% and 73.9% respectively. Because the mean relative tissue viability for the test substance was not below 50% after 3 minutes treatment and not below 15% after 1 h treatment the test substance is considered to be not corrosive.

- The OD values for the negative controls were between 0.8 and 2.8, and were within observed historical ranges. The mean relative tissue viability following the 1 h exposure to the positive control was 4.4%.

- In the range of 20 - 100% viability the coefficient of variation between tissue replicates was <30%, indicating that the test system functioned properly. The coefficient of variation between tissue replicates treated with the test substance was 5.9% for the 3-minute treatment, which is above acceptance criteria. Since all individual viabilities were >50%, the test outcome was considered valid.
Interpretation of results:
other: CLP criteria not met
Conclusions:
Under the study conditions, the test substance was determined to be non corrosive to skin based on human three dimensional epidermal model (EpiDerm (EPI-200)).
Executive summary:

An in vitro study was conducted to determine the skin corrosion potential of the substance according to OECD Guideline 431 and EU Method B.40 bis in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Duplicate tissue sample were exposed to 50 µL test substance for 3 min and 1 h. Post treatment period, a determination of the cytotoxic effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin corrosion was expressed as the remaining cell viability after exposure to the test substance. The positive control had a mean relative tissue viability of 4.4% after the 1-h exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 and the laboratory historical control data range. In the range of 20 - 100% viability the coefficient of variation between tissue replicates was <30%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 3 min and 1 h treatments with the test substance compared to the negative control tissues was 82% and 73.9%, respectively. Because the mean relative tissue viability for the test substance was above 50 and 15% after 3 min and 1 h treatment respectively, so the test substance is considered to be non corrosive. Under the study conditions, the test substance was determined to be non-corrosive to skin based on human three dimensional epidermal model (EpiDerm (EPI-200)) (Payne, 2017).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 04, 2017 to December 08, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
The standard deviation for test substance treated tissues was >18%. It was considered that the deviation did not affect the integrity or outcome of the study as 2 out of the 3 replicates gave unequivocal negative results.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
The standard deviation for test substance treated tissues was >18%. It was considered that the deviation did not affect the integrity or outcome of the study as 2 out of the 3 replicates gave unequivocal negative results.
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Justification for test system used:
Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum Human keratinocytes are used to construct the epithelium. Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model is supplied free of contamination with bacteria, mycoplasma and fungi. Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
Specification: EpiDermTM SIT (EPI-200) three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum, supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic.

General model conditions: Human keratinocytes are used to construct the epithelium. Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model is supplied free of contamination with bacteria, mycoplasma and fungi.

Functional Model Conditions: The magnitude of viability is quantified using MTT and measuring the optical density (OD) of the extracted (solubilised) dye from each tissue. The negative control tissue has been shown to be stable in culture for the duration of the test exposure period. The stratum corneum is sufficiently robust to resist the rapid penetration of certain cytotoxic marker chemicals (e.g. 5% SDS). The tissue has been shown to demonstrate reproducibility over time.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30 µL
Duration of treatment / exposure:
1 h
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Triplicate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 h
Value:
57.1
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Cell viability measurement:

Substance Tissue Replicate OD570 Corrected Mean % Relative Survival Standard Deviation Coefficient of Variance
Aliquot 1 Aliquot 2 Mean Tissue Mean
Negative control A 0.962 1.111 1.036 1.036 96.4 100 3.77 3.77
B 1.122 1.113 1.117 1.117 103.9
C 1.039 1.107 1.073 1.073 99.8
Test substance A 0.044 0.049 0.047 0.047 4.3 57.1 46.18 80.85
B 0.993 0.945 0.969 0.969 90.1
C 0.856 0.798 0.827 0.827 76.9
Positive control A 0.065 0.051 0.058 0.058 5.4 4.2 1.01 23.83
B 0.039 0.044 0.042 0.042 3.9
C 0.037 0.037 0.037 0.037 3.5
Blank 0 0
Interpretation of results:
other: CLP criteria not met
Conclusions:
Under the study conditions, based on the percentage of viability of 57.1%, the test substance was concluded to be non-irritant to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the substance according to OECD Guideline 439, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative control by application of 30 µL for 60 min (25 min at room temperature and 35 min at 37°C, 5% CO2) and 42 h post incubation period. PBS was used as negative control and 5% of sodium dodecyl sulphate solution in PBS was used as positive control. Subsequently, viability of the tissues was assessed and compared to the negative control. The absolute mean of the positive and negative control tissues was within the acceptance limits of Guideline and the laboratory historical control data range indicating that the test system functioned properly. The viability of the incubated tissues in the test item treated groups was found to be 57.1%. Under the study conditions, the test substance was concluded to be non-irritant to skin (Dreher, 2018).

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
key study
Study period:
From October 16, 2017 to October 19, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
Deviations:
no
Principles of method if other than guideline:
This study was performed in order to evaluate the potential of test substancein a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study. The EpiOcularTM Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of irreversible tissue damage caused by cytotoxic effects in the human cornea model. Strategic combinations of other accepted test methods with OECD Guideline 492 can be used as replacement of the in vivo Draize test. It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcularTM EIT is intended to differentiate substances that are “not eye irritant” from those that require labelling as either GHS category 1 or 2 for serious eye damage or eye irritation potential. Eye irritant substances are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-iphenyltetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison with untreated negative controls is used to predict the eye irritation potential. The test was performed according to the protocol provided from MatTek Corporation.
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: human-derived keratinocytes
Details on test animals or tissues and environmental conditions:
The EpiOcular tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
1) Tissue 51.7 mg
2) Tissue 49.8 mg
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
18 h
Number of animals or in vitro replicates:
Duplicates
Irritation parameter:
other: % relative tissue viability
Run / experiment:
6 h
Value:
8.8
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

% Viability positive control and test substance:

Designation Positive Control Test substance
% Viability (Tissue 1) 39.90% 7.30%
% Viability (Tissue 2) 43.00% 10.30%
% Viability Mean 41.40% 8.80%
Interpretation of results:
other: CLP criteria not met (Inconclusive)
Conclusions:
Under the study conditions, based on the percentage viability of 8.8%, the test substance wasconsidered either eye irritant or inducing serious eye damage to the human eye.
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the substance according to OECD Guideline 492, in compliance with GLP. Two tissues of the EpiOcular tissue model were treated with the test substance, positive control and negative control. Tissues were pre-wetted with 20 μL of PBS (Sterile Phosphate Buffered Saline) prior to topical application of approximately 50±2 mg of neat test substance. Sterile demineralised water was used as negative control and methyl acetate as positive control. After 6 h exposure on the surface of EpiOcular reconstructed ocular epithelium and 18 h post-incubation time, the viability of the tissues was assessed and compared to a negative control. The MTT viability test readings were performed at 570 nm without reference filter. Under the study conditions, based on the percentage viability of 8.8%, the test substance was concluded to be irritant to the human eye. However further GHS classification Category 1 or 2 cannot be concluded based on the result. Under the study conditions, based on the percentage viability of 8.8%, the test substance wasconsidered either eye irritant or inducing serious eye damage to the human eye (Andres, 2018).

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
The Bovine Corneal Opacity and Permeability Assay (BCOP)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 08, 2017 to August 08, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes
Species:
other: Bovine
Details on test animals or tissues and environmental conditions:
Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

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: Eyes were collected after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL) in a suitably sized container and transported on the same day to the testing facility.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL at 32°C
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
-
Duration of post- treatment incubation (in vitro):
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.
Number of animals or in vitro replicates:
3
Details on study design:
Preparation of Corneas:
On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

Cornea Selection and Opacity Reading:
Upon arrival at the test facility, the corneas were excised from the eyes and loaded onto the specifically designed holders. Both chambers of each holder were filled with pre-warmed Minimal Essential Medium (MEM), with the posterior chamber filled first, ensuring that no bubbles were formed. The holders were incubated at 32±1°C for 1 hour. After the incubation, the media was removed from both the anterior and posterior chambers. Fresh media was added to the posterior chamber first and then the anterior chamber (this media replacement order ensured the cornea retained its natural curvature as much as possible). The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

Treatment of Corneas and Opacity Measurements:
A volume of 750 μL of the test substance was applied to each of three corneas followed by a 10 minute incubation at 32°C. After this incubation, each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test substance had been removed successfully). The corneas were then washed once in media without phenol red, the holders re filled. The corneas were incubated for 2 h after which, corneal opacity
was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1.5 h. Following this period, the media in the posterior chamber was removed and held in a labelled tube. Three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490) using a spectrophotometer. A volume of 750 μL of the negative or positive control was similarly applied to further groups of three corneas.
Irritation parameter:
in vitro irritation score
Run / experiment:
10 minutes exposure
Value:
29.08
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Remarks on result:
other: Inconcluisve
Other effects / acceptance of results:
The mean corrected opacity reading for the test substance was 15.0, and the mean corrected opacity reading for the positive control was 54.3. The mean group corrected optical density for the test substance was 0.939 and the mean group corrected optical density for the positive control was 0.569. The mean in vitro irritancy score of the positive control (Dimethylformamide) was 62.86 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 substance produced IVIS score of 29.08 after 10 minutes of treatment.

Corneal opacity:

Test chemical Cornea number Initial opacity Post incubation opacity Change in opacity Mean change in opacity Corrected opacity Mean corrected opacity
Negative control 13 0 0 0 0.67 -0.7 0
Negative control 14 -1 0 1 0.3
Negative control 15 0 1 1 0.3
Test substance 16 -1 25 26 NA 25.3 15
Test substance 18 0 7 7 6.3
Test substance 20 0 14 14 13.3
Positive control 21 0 56 56 NA 55.3 54.3
Positive control 23 0 50 50 49.3
Positive control 24 0 59 59 58.3

Corneal permeability:

Test chemical Cornea number Mean blank OD490 OD490 Corrected OD490 Mean corrected OD490 Final corrected OD490 Mean
Group Corrected OD490
Negative control 13 0.038 0.048 0.01 0.011 -0.001 0
Negative control 14 0.052 0.014 0.003
Negative control 15 0.048 0.01 -0.001
Test substance 16 NA 1.378 1.34 NA 1.328 0.939
Test substance 18 0.669 0.631 0.62
Test substance 20 0.918 0.88 0.868
Positive control 21 NA 0.614 0.576 NA 0.564 0.569
Positive control 23 0.547 0.51 0.498
Positive control 24 0.692 0.655 0.643

Calculated IVIS:

Test chemical Mean Opacity Mean Permeability IVIS (Mean Opacity + (15 x Mean Permeability))
Negative control 0 0 0
Test substance 15 0.939 29.08
Positive control 54.3 0.569 62.86

NA: Not applicable

Interpretation of results:
other: CLP criteria not met (Inconclusive)
Conclusions:
Under the study conditions, eye irritation potential of the test substance was determined to be inconclusive based on bovine corneal opacity and permeability test (IVIS score - 29.08).
Executive summary:

An in vitro study was conducted to determine the eye damage potential of the substance according to OECD Guideline 437 in compliance with GLP. The eye damage potential of the test substance was tested through topical application for 10 min. The test substance was applied as is (750 µL) directly on top of the freshly isolated bovine cornea sample. The negative control responses for opacity and permeability was less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritation of the corneas. The mean corrected opacity reading for the test substance was 15.0, and the mean corrected opacity reading for the positive control was 54.3. The mean group corrected optical density for the test substance was 0.939 and the mean group corrected optical density for the positive control was 0.569. The mean in vitro irritancy score of the positive control (Dimethylformamide) was 62.86 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 substance produced IVIS score of 29.08 after 10 min of exposure. Under the study conditions, eye irritation potential of the test substance was determined to be inconclusive based on bovine corneal opacity and permeability test (Payne, 2017).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Study 1: An in vitro study was conducted to determine the skin corrosion potential of the substance according to OECD Guideline 431 and EU Method B.40 bis in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Duplicate tissue sample were exposed to 50 µL test substance for 3 min and 1 h. Post treatment period, a determination of the cytotoxic effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin corrosion was expressed as the remaining cell viability after exposure to the test substance. The positive control had a mean relative tissue viability of 4.4% after the 1-h exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 and the laboratory historical control data range. In the range of 20 - 100% viability the coefficient of variation between tissue replicates was <30%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 3 min and 1 h treatments with the test substance compared to the negative control tissues was 82% and 73.9%, respectively. Because the mean relative tissue viability for the test substance was above 50 and 15% after 3 min and 1 h treatment respectively, so the test substance is considered to be non corrosive. Under the study conditions, the test substance was determined to be non-corrosive to skin based on human three dimensional epidermal model (EpiDerm (EPI-200)) (Payne, 2017).

 

Study 2: An in vitro study was conducted to determine the skin irritation potential of the substance according to OECD Guideline 439, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative control by application of 30 µL for 60 min (25 min at room temperature and 35 min at 37°C, 5% CO2) and 42 h post incubation period. PBS was used as negative control and 5% of sodium dodecyl sulphate solution in PBS was used as positive control. Subsequently, viability of the tissues was assessed and compared to the negative control. The absolute mean of the positive and negative control tissues was within the acceptance limits of Guideline and the laboratory historical control data range indicating that the test system functioned properly. The viability of the incubated tissues in the test item treated groups was found to be 57.1%. Under the study conditions, the test substance was concluded to be non-irritant to skin (Dreher, 2018).

 

Eye irritation

Study 1: An in vitro study was conducted to determine the eye damage potential of the substance according to OECD Guideline 437 in compliance with GLP. The eye damage potential of the test substance was tested through topical application for 10 min. The test substance was applied as is (750 µL) directly on top of the freshly isolated bovine cornea sample. The negative control responses for opacity and permeability was less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritation of the corneas. The mean corrected opacity reading for the test substance was 15.0, and the mean corrected opacity reading for the positive control was 54.3. The mean group corrected optical density for the test substance was 0.939 and the mean group corrected optical density for the positive control was 0.569. The mean in vitro irritancy score of the positive control (Dimethylformamide) was 62.86 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 substance produced IVIS score of 29.08 after 10 min of exposure. Under the study conditions, eye irritation potential of the test substance was determined to be inconclusive based on bovine corneal opacity and permeability test (Payne, 2017).

 

Study 2: An in vitro study was conducted to determine the eye irritation potential of the substance according to OECD Guideline 492, in compliance with GLP. Two tissues of the EpiOcular tissue model were treated with the test substance, positive control and negative control. Tissues were pre-wetted with 20 μL of PBS (Sterile Phosphate Buffered Saline) prior to topical application of approximately 50±2 mg of neat test substance. Sterile demineralised water was used as negative control and methyl acetate as positive control. After 6 h exposure on the surface of EpiOcular reconstructed ocular epithelium and 18 h post-incubation time, the viability of the tissues was assessed and compared to a negative control. The MTT viability test readings were performed at 570 nm without reference filter. Under the study conditions, based on the percentage viability of 8.8%, the test substance was concluded to be irritant to the human eye. However further GHS classification Category 1 or 2 cannot be concluded based on the result. Under the study conditions, based on the percentage viability of 8.8%, the test substance wasconsidered either eye irritant or inducing serious eye damage to the human eye (Andres, 2018).

Justification for classification or non-classification

Skin irritation:

Based on the results of the in vitro skin irritation studies, the substance does not warrant classification for skin irritation according to EU CLP (1272/2008/EC) criteria.

Eye irritation:

Considering the results of the in vitro eye irritation assays as weight of evidence, the substance is considered to be irritant to eyes and warrants a classification as Eye Irritant 2 (H319: Causes serious eye irritation according to EU CLP (1272/2008/EC) criteria.