Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion

The test item was demonstrated to be non-irritant in an in vitro study performed according to OECD guideline 439 (Reconstructed Human Epidermis Test method, EPISKIN) and conform GLP requirements (Kanizsai, 2016). This study was considered reliable without restrictions (Klimisch 1) and is considered as the key study for endpoint coverage.

Eye irritation

The test item was demonstrated to be non-irritant to eyes in an in vitro study performed according to OECD guideline 492 (Reconstructed Human Cornea-like Epithelium test method, EpiOcular study) and conform GLP requirements (Valin, 2016b). This study was considered reliable without restrictions (Klimisch 1) and is considered as the key study for endpoint coverage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-05-02 to 2016-08-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin(TM) SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin(TM) test method 15 min - 42 h for the prediction of acute skin irritation of chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was applied in its original form, no formulation was required.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified (adult)
Source strain:
other: not applicable
Justification for test system used:
The EPISKINTM(SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439). Therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN (SM) (0.38 cm²), SkinEthic, France
- Tissue batch number(s): 16-EKIN-018
- Expiry date: May 09, 2016
- Date of initiation of testing: May 04, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25.1-27.2°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C
- All incubations were carried out in a humid atmosphere (> 95%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C. Temperature and humidity were continuously monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the 15 minutes incubation time, the EPISKIN (SM) units were removed and rinsed thoroughly with phosphate buffered saline (PBS) to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: yes, plate reader, not further specified
- 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 item is considered to be non-irritant to skin if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than 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): approximately 20.5 mg

NEGATIVE CONTROL (Phosphate Buffered Saline)
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL (Sodium Dodecyl Sulphate solution)
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 5% (w/v)
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 h)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 replicates
Value:
79.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues was in the recommended range (0.851). Standard deviation of the viability results for negative control samples was 6.6.
- Acceptance criteria met for positive control: Yes. The positive control treated tissues showed 4.8% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.7.
- Acceptance criteria met for variability between replicate measurements: Yes. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 3.2.
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Optical density (OD) and the calculated non-specific colour % of the additional control tissue

 Additional control Optical Density (OD)        NSC% 
     Measured  Blanc corrected  
 Treated with 1  0.052  0.005  
 gadolinium oxalate  0.055  0.008  0.7
  mean   -  0.006  

Notes: Mean blank value was 0.047. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Optical density (OD) and the calculated relative viability % of the samples

 Substance  Optical density (OD)        Viability
     Measured  Blank corrected (% RV) 
 Negative control:  1  0.952  0.904  106.2
 Phosphate Buffered Saline

 2

 0.905  0.857  100.7
   3  0.839  0.792  93.0
   mean  -  0.851  100.0
 Positive control:  1  0.085  0.038  4.4
 5% (w/v) SDS solution  2  0.085  0.038  4.4
   3  0.095  0.048  5.6
   mean  -  0.041  4.8
 Test item:  1  0.733  0.685  80.5
 gadolinium oxalate  2  0.749  0.702  82.5
   3  0.696  0.648  76.2
   mean  -  0.679  79.7
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study (in vitro EPISKIN model test according to OECD guideline 439), the test item was determined to be non-irritant to skin. Based on these results, the test item is considered not classified according to the CLP Regulation.
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 2016-06-03 to 2016-11-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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)
Version / remarks:
adopted 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- No correction factor applied in this study
- The test item was used in its original form, as supplied by the Sponsor.
Species:
human
Strain:
other: Reconstructed human cornea-like epithelium (tissues)
Details on test animals or tissues and environmental conditions:
- Source: MatTek, Bratislava, Slovak Republic
- Expiry date: The EpiOcular tissues were used within 72 hours of their production.
- Selection: At receipt, the tissues were inspected for obvious defects as they could have been rejected based on blistering, excess fluid or air bubbles below the tissue insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
- Storage conditions: At receipt, the living EpiOcular tissues were stored on their day of arrival, at 37°C, 5% CO2, in a humidified incubator.
- Description of the cell system used: EpiOcular living tissue consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinised epithelium which models the corneal epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotype 3D cornea-like model. The 3D tissue consist of highly organised cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.
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): 51 mg
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular tissue, MatTek, Bratislava, Slovak Republic. Batch number documented in a certificate of analysis archived in the study files.
- Doses of test chemical and control substances used: 51 mg of test item, 50 µL of deinonised water for negative control, 50 µL methyl acetate for positive control
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Exposure for 6 hours at 37°C, then soaked in assay medium for 25 minutes at room temperature, blotted, and then incubated for 18 hours at 37°C.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: As the test item was found in the preliminary test not to have any colouring potential and any direct MTT reducing properties, no additional controls were run during the main test.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: MTT formazan precipicates are extracted using isopropanol and quantified using spectrophotometry. Formazan extraction was performed overnight at +2-8°C and protected from light. The OD was measured at 570 nm using a plate reader.
- Acceptable variability between tissue replicates for positive and negative controls: Negative control acceptance criteria: mean cOD between 0.8 and 2.5. Positive control acceptance criteria: relative mean viability of the positive control is < 50% of the relative mean viability of the negative control.
- Acceptable variability between tissue replicates for the test chemical: Acceptable if the difference of viability between the two tissue replicates is < 20%.
- Data interpretation: A test substance is predicted as ocular irritant, if the mean relative tissue viability (%) of two tissues exposed to the test item is ≤ 60%.
Irritation parameter:
other: relative viability in %
Run / experiment:
mean of duplicate tissues
Value:
104
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Mean cOD for the two replicate tissues was 1.817 and 1.592.
- Acceptance criteria met for positive control: Yes. 22% viability was observed for both tissue replicates in the positive control, whereas 93 and 107 % viability was observed for the two tissue replicates in the negative control. The relative mean viability in the positive control is hence < 50% of that in the negative control.
- Acceptable variability between replicate tissues treated with test item: Yes. A difference of only 3% was obtained between the replicate tissues.
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was tested for its eye irritation potential in a Reconstructed Human Cornea-like Epithelium assay performed according to OECD guideline 492. Since mean viability in the treated tissues after MTT reduction was 104%, which is higher than the cut-off level of 60%, the test results meet the criteria for a non-irritant response. Therefore, the test substance is not to be classified for eye irritation under the CLP Regulation.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from 2016-05-10 to 2016-08-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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:
yes
Remarks:
The negative control response resulted in a mean OD490 value higher than the established upper limit for background permeability for bovine corneas treated with the negative control substance (0.9% physiological saline, established upper limit = 0.0296).
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Treatment of test material prior to testing: The test item was tested undiluted (in its original form).
- Correction factor: No correction factor was applied for this type of study.
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, France.
- Characteristics of donor animals (e.g. age, sex, weight): bovine cattle were up to 12 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to CiToxLAB France at ambient temperature in a cool box, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.
- Time interval prior to initiating testing: Maximum 24 hours, stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C.
- Indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final).
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 mg (± 75 mg)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL (± 8 µL)
- Concentration (if solution): 0.9%

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL (± 8 µL)
- Concentration (if solution): 20%
Duration of treatment / exposure:
4 hours (± 5 minutes)
Duration of post- treatment incubation (in vitro):
90 minutes (± 5 minutes)
Number of animals or in vitro replicates:
The test item, the negative and the positive controls were tested on three corneas each.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- Macroscopic examination was performed on all eyes to detect the presence of any unacceptable defects (opacity, scratches, pigmentation, neovascularisation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light. The tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out.
- The corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used within a maximum of 24 hours. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.
- The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded. The corneas were then mounted in the corneal holders (one cornea per holder) with the endothelial side against the O-ring of the posterior chamber. The anterior half of the holder was then positioned on top of the cornea and tightened with screws.
- For pre-incubation, both chambers of the corneal holder were filled to overflowing with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. The posterior chamber was always filled first to maintain the natural concave shape of the cornea. After making sure that no air bubbles were present within the holder, it was immersed in a water bath, horizontally (cornea positioned vertically), up to approximately three quarters of its height. The holders were pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C (± 1°C).
- At the end of the pre-incubation period, the medium was removed from both chambers of the holder using a metal gavage tube attached to a vacuum pump to ensure complete evacuation. They were refilled with fresh cMEM (previously heated to +32°C), starting with the posterior chamber and taking care that no air bubbles were present. The chambers were re-sealed and the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0.

QUALITY CHECK OF THE ISOLATED CORNEAS: Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED
- 0.9% physiological saline (0.9% sodium chloride (NaCl))

POSITIVE CONTROL USED
- 20% imidazole solution in 0.9% NaCl (w/v)

APPLICATION DOSE AND EXPOSURE TIME: 750 mg (± 75 mg) of test item, and 750 μL (± 8 μL) of both positive and negative controls. Total treatment time was 4 h.

TREATMENT METHOD
- Closed chamber method for negative and positive controls.
- Open chamber method for test item treatment.
- Test item application: The window-locking ring and glass window from the anterior chamber were removed. The test item was gently applied onto the epithelium of the cornea, as uniformly as possible in order to ensure that it covers the whole epithelial surface. The glass window of the anterior chamber was then replaced (without the window-locking ring).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times with pre-warmed cMEM containing phenol red, then the corneas were finally rinsed with pre-warmed cMEM without phenol red.

POST-EXPOSURE INCUBATION: Yes, 90 min in 5 mg/mL fluorescein stain at +32°C.

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

ACCEPTANCE CRITERIA:
For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean;
- the mean opacity of the negative control corneas should be < 4.4;
- the mean OD490 nm of the negative control corneas should be < 0.0296.

DECISION CRITERIA:
The IVIS cut-off values for identifying the test item as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are the following:
IVIS UN GHS
If the test item induces an IVIS ≤ 3 No category
If the test item induces an 3 < IVIS ≤ 55 No prediction can be made
If the test item induces an IVIS > 55 Category 1

A single experiment composed of at least three corneas is sufficient for a test item when the resulting classification is unequivocal.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3
Value:
12
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: range = 8-15
Other effects / acceptance of results:
FURTHER DETAILS ON RESULTS
Mean opacity scores (range):
- negative control: 2 (-1-6)
- positive control (corrected corneal opacity): 117 (117-118)
- test item (corrected corneal opacity): 11.7 (8-15)

Mean permeability scores (range):
- negative control: 0.040 (0.011-0.057)
- positive control (corrected optical density): 2.878 (2.732-3.536)
- test item (corrected corneal opacity): -0.015 (-0.013 to -0.018)

Mean in vitro irritancy score (range):
- positive control: 160 (153-170)
- test item: 12 (8-15)

Study plan deviation:
The mean OD490 nm of the negative control corneas was found to be 0.040 instead of below 0.0296, as specified in the study plan. This deviation would lead to an over-correction of the cOD490 nm. The mean test item OD490 nm is lower than the negative control OD490 nm, therefore the cOD490 nm is negative and not taken into account in the calculation of the IVIS. Consequently, the IVIS of the test item is only based on opacity cOPT and the opacity acceptance criterion for the negative control was fulfilled (i.e. < 4.4). Therefore and based on the obtained results, this deviation is considered not to have any impact on the conclusion of the study. Indeed, even with this over-correction, acceptance criteria of positive control are met and results of test item-treated corneas are unequivocal.

Visible damage on test system: Residual amounts of test item were observed on the corneas treated with the test item. No notable opaque spots or irregularities were observed on negative control-treated corneas. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

The irritating potential of the test item is based on the calculation of an IVIS (In Vitro Irritancy Score) which combines permeability and opacity values for each cornea. In the present study, the IVIS obtained for the test item was only based on the opacity value. As residual test item was noted over the corneas after the rinsing phase, it is therefore not possible to determine if the high corneal opacity values noted were due to the test item reacting with cell structures (i.e. protein precipitation, etc.) or only to these residual amounts of test item which were stuck to the cornea.

ACCEPTANCE OF RESULTS
With one exception (mean OD490 nm of the negative control corneas was 0.04 instead of < 0.0296, see above), all acceptance criteria were met. The deviation was not considered to have any impact on the conclusion of the study. The study was therefore considered as valid.
Interpretation of results:
study cannot be used for classification
Conclusions:
Under the experimental conditions of this study, the irritation potential of the test item, gadolinium oxalate, could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to OECD Guideline 437, further testing will be conducted for classification and labeling purposes (i.e. CiToxLAB France/EpiOcular study No. 43941 TIO).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

One reliable (Klimisch 1) in vitro study is available (Kanizsai, 2016). This study was performed according to OECD guideline 439 (Reconstructed Human Epidermis Test method, EPISKIN) and conform GLP requirements. The mean % tissue viability in the three tissue replicates exposed to the test item was 79.7%. A test item is considered to be non-irritant to skin if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post-incubation is more than 50% of the mean viability of the negative controls. Since the mean viability in the negative controls was 100%, the test item was concluded to be non-irritant under the conditions of the study. Based on these results, the test item is considered not classified according to the CLP Regulation. The study of Kanizsai (2016) is considered as the key study for endpoint coverage.

Eye irritation

Two reliable (Klimisch 1) in vitro studies are available.

The first study (Valin, 2016a) was performed according to OECD guideline 437 (Bovine Corneal Opacity and Permeability test method) and conform GLP requirements. In such study, the irritating potential of the test item is based on the calculation of an IVIS (In Vitro Irritancy Score) which combines permeability and opacity values for each cornea. The mean IVIS for the three replicates exposed to the test item was 12. Since the IVIS was > 3 and <= 55, it could not be concluded whether or not the test item should be classified or not for eye irritancy. It should be noted that the IVIS obtained for the test item was only based on the opacity value in this study. This was due to the fact that the acceptance criterium for the mean OD490 nm of the negative control was not met. The OD490 nm should have been < 0.0296 but was found to be 0.040. This deviation would have led to an over-correction of the cOD490 nm. The mean test item OD490 nm was lower than the negative control OD490 nm, therefore the cOD490 nm was negative and not taken into account in the calculation of the IVIS. This deviation was not considered to have had any impact on the conclusion of the study. Indeed, even with this over-correction, acceptance criteria of the positive control were met and results of the test-item treated corneas were unequivocal. Because after the rinsing phase, residual amounts of test item were observed on the corneas treated with the test item, it was not possible to determine if the high corneal opacity values noted were due to the test item reacting with cell structures (i.e. protein precipitation, etc.) or only to these residual amounts of test item which were stuck to the cornea. Because of the inconclusive results of this test, further testing was conducted for classification and labelling purposes.

The test substance was consequently tested for its eye irritation potential in a Reconstructed Human Cornea-like Epithelium assay performed according to OECD guideline 492 and conform GLP requirements (Valin, 2016b). Since mean viability in the treated tissues after MTT reduction was 104%, which is higher than the cut-off level of 60%, the test results met the criteria for a non-irritant response. Therefore, the test substance could be concluded not to be classified for eye irritation under the CLP Regulation. This study is considered the key study for endpoint coverage.

Justification for classification or non-classification

Skin irritation/corrosion

The test item was demonstrated not to be irritant to skin in an in vitro study performed according to OECD guideline 439 and is therefore not to be classified according to the CLP regulation.

Eye irritation

The test item was demonstrated not to be irritant to eyes in an in vitro study performed according to OECD guideline 492 and is therefore not to be classified according to the CLP Regulation.