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 (Orovecz, 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 (Richez, 2017). 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 2016-07-06 to 2016-09-09
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)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
6 July 2012
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)
Specific details on test material used for the study:
- Treatment of test material prior to testing: None, the test item was applied as supplied.
- No correction for purity of the test item was applied.
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 EPISKIN TM(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 TM(SM) (0.38 cm2), SkinEthic, France
- Tissue batch number(s): 16-EKIN-027
- Expiry date: 2016-07-11
- Date of initiation of testing: 2016-07-06

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (23.4-27.4°C)
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C
- All incubations were carried out in a humid atmosphere (80-100%) 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 15 min 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
- Prior to treatment, the test item was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test items had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test items to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritant to skin if the relative mean 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 variability 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): 20 mg

NEGATIVE CONTROL (PBS)
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL (SDS)
- 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 (± 1h)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 replicates
Value:
90.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
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: As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was 0.021, consequently, Non Specific Colour % was calculated to be 3.0%. This value was below 5%, therefore additional data calculation was not necessary.

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 (0.699). Standard deviation of the viability results for negative control samples was 4.1.
- Acceptance criteria met for positive control: Yes. The positive control treated tissues showed 6.5% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.9.
- 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 2.2.
- The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissue

 Additional control

Optical Density (OD)

 NSC%

     Measured  Blank corrected

       

3.0

 Treated with  1  0.071  0.025
 diytterbium trioxide  2  0.064  0.018
   Mean  ----  0.021

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

 Substance

Optical Density (OD)       

 Viability

     Measured Blank corrected 

 (%RV)

 Negative Control:  0.718  0.672  96.1
 Phosphate buffered saline  2  0.775  0.729  104.2
   3  0.743  0.697  99.7
   Mean  ---- 0.699   100.0
 Positive Control:  1  0.087  0.041  5.8
 5% (w/v) SDS solution  0.089  0.043  6.1
   0.099  0.053  7.5
   Mean  ----  0.045  6.5
 Test Item:  1  0.673  0.627  89.7
 diytterbium trioxide  2  0.697  0.651  93.1
   3  0.669  0.623 89.1 
   Mean  ----  0.633  90.6
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
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-06-20 to 2017-08-07
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)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: The EpiOcularTM model 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 cornea 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 consists of highly organised cell layers similar to those 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 (± 1 mg)

NEGATIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 µL

POSITIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:
6 hours (± 15 minutes)
Duration of post- treatment incubation (in vitro):
18 hours (± 15 minutes)
Number of animals or in vitro replicates:
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 during 2 to 3 hours at room temperature by placing the plates on an orbital plate shaker. 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:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Test for direct MTT reduction with the test item: The MTT solution containing the test item did not turn blue/purple compared to the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.
- Test for the detection of the colouring potential of the test item: During this test, as both water and isopropanol solutions containing the test item did not change colour, the test item was found not to have a colouring potential. As a result, no additional controls were used in parallel to the main test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Mean cOD for the two replicate tissues was 2.117.
- Acceptance criteria met for positive control: Yes. Mean viability observed in the positive control was 14%, whereas a mean viability of 100% was observed for the two tissue replicates in the negative control. The relative mean viability in the positive control was 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 99%, 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 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 (Orovecz, 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 90.6%. 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 Orovecz (2016) is considered as the key study for endpoint coverage.

Eye irritation

One reliable (Klimisch 1) in vitro study is available (Richez, 2017). In this study, the test substance was tested for its eye irritation potential in a Reconstructed Human Cornea-like Epithelium assay performed according to OECD guideline 492 and conform GLP requirements. Since mean viability in the treated tissues after MTT reduction was 99%, 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.