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Administrative data

Description of key information

Skin irritation (OECD 439): not irritating

Eye irritation (OECD 492): not irritating

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 18 Jun. 2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Comission Regulation (EU) No. 640/2012 amending Regulation (EC) No. 761/2009, Annex III, EU method B.46 'In vitro skin irritation: reconstructed human epidermis model test'
Version / remarks:
adopted 06. Jul. 2012
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Mainz, Germany
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM (EPI-200-SIT)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200-SIT) (MatTek In Vitro Life Science Laboratories, Bratislava)
- Tissue batch number(s): 30808
- Delivery date: 23. Jul. 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: M icrotiter plate photometer Anthos Reader 2010 Flexi, Anthos Microsysteme GmbH
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 1.841 ± 0.032 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.26 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce EpiDerm™ tissue are screened for potential biological contaminants. Tests for the following potential biological contaminant were performed: HIV-1 virus (Oligonucleotide-direct amplification), Hepatitis B virus (Oligonucleotide-direct amplification), Hepatitis C virus (Oligonucleotide-direct amplification), Bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture); no contaminations detected.
- Reproducibility: given

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Direct MTT reduction by the test item had not taken place and no data correction was necessary.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive / irritant to skin if the tissue viability after 1 h is equal or less than 50%
- The test substance is considered to be non-irritant to skin if the tissue viability after 1 h exposure is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 µL

NEGATIVE CONTROL
- Amount(s) applied: 30 µL

POSITIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
35 min at 37°C and 25 min at RT
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
triplicates for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
1 h exposure
Value:
106.4
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:
- Direct-MTT reduction: Direct MTT reduction by the test item had not taken place and no data correction was necessary.
- Colour interference with MTT: The solution was colourless, therefore no binding capacity had to be tested.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.429 and 1.658).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour was ≤ 20% compared to the negative control (3.1%).
- Acceptance criteria met for variability between replicate measurements: The SD of mean viability of the tissue replicates is ≤ 18% (values between 0.1% and 9.8%).
- Range of historical values if different from the ones specified in the test guideline: The mean OD of the tissue replicates treated with the negative control of all experiments up to 03. Jul. 2019 was 0.476 - 2.471 (mean: 1.787, SD 0.317).

Table 1: MTT assay after 60 min exposure

  Negative control Positive control Test item
Tissue sample 1 2 3 1 2 3 1 2 3
OD570 1.585 1.702 1.443 0.080 0.081 0.082 1.523 1.770 1.752
1.586 1.682 1.482 0.080 0.081 0.084 1.519 1.760 1.749
OD570(mean-blank) 1.552 1.658 1.429 0.046 0.047 0.049 1.487 1.731 1.717
OD570(mean of 3 tissues) 1.546 0.047 1.645
Viability (%) 100 3.1 106.4
SD - 0.1 8.9
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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 25 Jun. 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Mainz, Germany
Species:
human
Strain:
other: EpiOcular™
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability : Commercially available EpiOcular™ kit
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 23. Jul. 2019
Batch no.: 30618
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 EpiOcular™ 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.
The certificate of analysis is included in the study report. No contaminants were detected. Tissue viability and the barrier function tests were within the acceptable ranges and indicated appropriate formation of the mucosal barrier and a viable basal cell layer.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:
28 min at 37 ± 1°C
Duration of post- treatment incubation (in vitro):
120 min at 37 ± 1°C
Number of animals or in vitro replicates:
in duplicates for each treatment and control group
Details on study design:
- Details of the test procedure used
Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 1 h. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 16 hours and 58 minutes.
Exposure and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and then incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 30 minutes. After that, 50 µL of the controls and the test item were applied in duplicate in one-minute- intervals. This was done in such a fashion that the upper surface of the tissue was covered. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue of each plate, the plate was transferred into the incubator for 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity. At the end of the exposure time, the inserts were removed from the plates in one-minute intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred to and immersed in 5 mL of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes post soak at room temperature. After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95%
relative humidity.
After the post-treatment incubation, the MTT assay was performed.
- Description of the method used to quantify MTT formazan :
A 24-well-plate was prepared with 300 µL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity. At last, the test item inserts were thoroughly dried and set into the empty 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The inserts of the controls were set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plates were firmly sealed to avoid evaporation of the solvent and then stored in the refrigerator overnight. On the next day the plate was shaken for 2 hours at room temperature, protected from light. The inserts of the test item were pierced with an injection needle, taking care that all colour is extracted and the inserts were then discarded.
The inserts of the controls were discarded without piercing the tissues, as they were extracted in a 6-well-plate. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 µL solution (each) were pipetted into a 96-wellplate. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectrophotometer (Anthos Reader 2010 Flexi, Anthos Microsysteme GmbH) at 570 nm. The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).
Calculation
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the two relating tissues for controls and test item
To calculate the relative tissue viability, the following equation was used:
% Viability = [(OD corrected of test item or positive control) / (OD corrected of mean negative control)]
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :
Eye hazard potential is assessed using the following criteria (according to guideline):
% Viability > 60%: non eye irritant / UN GHS classification: no category
% Viability ≤ 60%: at least eye irritant / UN GHS classification: No prediction can be made (category 1 or 2)
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : The values for negative control and for positive control were within the range of historical data of the test facility.
- Complete supporting information for the specific RhCE tissue construct used
- Reference to historical data of the RhCE tissue construct : yes, historical data available and included in the report
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals :The validity of the EpiOcular test at LAUS GmbH was demonstrated in a proficiency study. For this purpose, 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested.
- Positive and negative control means and acceptance ranges based on historical data :
Optical density negative control: mean 1.870, SD 0.272, range: 1.167 - 2.437
Relative tissue viability positive control: mean 32.1%, SD 7.3%, range: 12.4 - 57.2%
- Acceptable variability between tissue replicates for positive and negative controls : < 20%
- Acceptable variability between tissue replicates for the test chemical: <20%
Irritation parameter:
other: % tissue viability
Remarks:
mean value of two tissues (tissue 1 / tissue 2)
Run / experiment:
28 min exposure
Value:
97.5
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: not detected

DEMONSTRATION OF TECHNICAL PROFICIENCY: All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcular test was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes; Mean OD of negative control found = 2.0
- Acceptance criteria met for positive control: yes ; % mean relative viability of positive control found = 41.2%
- Variation within replicates: negative control 1.7%, positive control 3.3%, test item 6.2%;
The values for negative control, positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Table 1: Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate 1 2 3 4 5 6 7 8 Mean
Absorbance 0.033 0.034 0.034 0.033 0.034 0.034 0.034 0.035 0.034

Table 2: Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designa
tion
Measure
ment 
Negative Control  Positive Control Fatty acids, C18-
unsaturated, 1,6
Hexanediol Diester
Tissue 1  1 2.053 0.882 2.001
2 1.973 0.868 2.016
Tissue 2  1 1.965 0.818 1.880
2 1.996 0.802 1.892

Table 3: Mean Absorbance Negative Control, Positive Control and Test Item (corrected with mean absorbance value of isopropanol)

Designation  Negative Control  Positive Control Fatty acids, C18-
unsaturated, 1,6
Hexanediol Diester
Mean – blank
(Tissue 1) 
1.979 0.841 1.975
Mean – blank
(Tissue 2) 
1.947 0.776 1.852

Table 4: % Viability Positive Control and Test Item

Designation  Positive Control  Fatty acids, C18-unsaturated,
1,6 Hexanediol Diester
% Viability (Tissue 1)  42.8 100.6
% Viability (Tissue 2)  39.5 94.3
% Viability Mean  41.2 97.5

Table 5: Historical Data

Parameter  Optical Density
Negative Control
Relative Tissue Viability
Positive Control
Demineralised H2O  Demineralised H2O  Methyl acetate
Exposure time  30 minutes
Mean  1.870 32.1%
Standard deviation  0.272 7.3%
Range  1.167 - 2.437  12.4 - 57.2%
Study 19062801G891  1.963 41.2
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
Under the conditions of the conducted test, the test substance did not possess irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

The skin irritation properties of the test substance were tested in an in vitro skin irritation study according to OECD 439 and in compliance with GLP (Dako AG, 2019, key). Three tissues of the human skin model EpiDermTMwere treated with the test item for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2). Dulbecco’s Phosphate Buffered Saline (DPBS)-buffer was used as negative control and 5% Sodium dodecyl sulphate (SDS) solution was used as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean optical density (OD) ≤ 2.8, OD was 1.5. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 3.1% (required:≤20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). After the treatment with the test item, the mean value of relative tissue viability was increased to 106.4% compared to the negative control. This value is above the threshold for a skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin. Therefore, the test item is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

 

Eye irritation

The eye irritation potential of the test substance was determined by an in vitro eye irritation test according to OECD 492 and in compliance with GLP (Dako AG, 2019, key). The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 28 minutes. 50 µL of the liquid test item was applied to two tissue replicates. After treatment, the substance was rinsed from the tissue. Subsequently the cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a formazan. The formazan formation was evaluated by measuring the optical density (OD) of the resulting solution at the wavelength of 570 nm. Demineralised water was used as negative control and methyl acetate was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 2.0. The positive control showed clear eye irritating effects; the mean value of the relative tissue viability was 41.2% (< 50%). The variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 97.5 %. This value is above the threshold for an eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant. Under the conditions of the test, the test substance is thus considered non-eye irritant in the EpiOcularTMEye Irritation Test.

Justification for classification or non-classification

The available data on irritation do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.