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Administrative data

Description of key information

kin irritation/corrosion:


OECD 439 (RL1): The test item is not irritating to skin. 


Eye irritation/corrosion


OECD 492 (RL1): The test item is not irritating to the eyes.


 

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:
October 9 to 11, 2019
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:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: skin model obtained from Episkin/SkinEthic Laboratories, Lyon, France
Justification for test system used:
stansard model
Vehicle:
unchanged (no vehicle)
Details on test system:
CELL CULTURE
- Supplier: Episkin/SkinEthic Laboratories, Lyon, France
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 19-RHE-146


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)
Duration of treatment / exposure:
42 min (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
run 1, mean of three tissues
Value:
95.3
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
- Direct-MTT reduction: none
- Colour interference with MTT: none

- Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The OD values for the negative control shall be in the range of ≥ 0.8 and ≤ 3.0 as given in OECD Guideline 439. The values obtained were: 1.714 to 1.779

- Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
The negative control data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm. The standard deviation value is considered valid if ≤ 18% of group mean-value.
The results obtained were:
Mean OD = 1.757, SD =2.1%

The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is < 40%. The standard deviation value is considered valid if ≤ 18% of group mean-value.
Mean OD = 0.026, SD =6.7%, mean viability: 1.5 %

 





























 GroupTime / [min] Mean OD Mean Relative viability / [%]
 Negative Control42 1.757100 
 Positive Control42

0.026



1.5



 Test Material



42



1.675



95.3


Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).
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:
October 15 to 17, 2019
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)
Deviations:
yes
Remarks:
No information on historical data given.
GLP compliance:
yes
Species:
human
Strain:
other: NA
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace in vivo animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The EpiOcularTM human cell construct (OCL-200, OCL-212, MatTek Corporation) is used in this assay. It is a nonkeratinized epithelium prepared from normal human keratinocytes. All biological components of the EpiOcularTM tissue and the kit culture medium have been tested and are free of contamination. An analysis for tissue functionality and quality was performed: the barrier function, cell viability were meeting the acceptance criteria as well as the histological appearance of the cells.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 mg
- Concentration: undiluted
Duration of treatment / exposure:
6 h ± 15 min
Duration of post- treatment incubation (in vitro):
25 ± 2 min (Post-exposure immersion incubation)
18 h ± 15 min (Post-treatment incubation)
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used
The cytotoxicity of the test item (and thus the ocular irritation potential) is evaluated by measuring the relative viability of the treated RhCE tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT in control and test item-treated cultures.

- RhCE tissue construct used, including batch number
The EpiOcularTM human cell construct (OCL-200 and OCL-212, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot number: 30630)
- Doses of test chemical and control substances used
50 mg test item; 50 μL positive control (methyl acetate) and 50 μL negative control (sterile deionized water)
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan
Inserts were removed from the 24-well plate after the incubation time in MTT solution (3 h ± 10 min), the bottom of the insert was blotted on absorbent material and transferred to a 6-well plate containing 2 mL isopropanol per well in a manner avoiding the isopropanol to flow into the insert. The plate was sealed with standard plate sealer. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2-3 h at room temperature. 200 μL samples from each tube were placed into the wells of a 96-well plate and the absorbance/optical density was measured in a 96-well plate spectrophotometer to determine cell viability.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test item requires classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60% of the negative control.
The test item requires no classification and labelling according to UN GHS (No Category), if the mean percent tissue viability after exposure and post-exposure incubation is > 60%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
No information is given on historical data, but the results obtained for the negative and positive control meet the acceptance criteria given in the OECD guideline 492.

- Complete supporting information for the specific RhCE tissue construct used
The barrier function integrity test resulted in an ET50 of 4.6, cell viability was determined to have an OD of 1.3, the RHE model was tested to be a well differentiated epidermis with 5.5 cell layers without any significant histological abnormality, thus meeting the acceptance criteria.


- Positive and negative control means and acceptance ranges based on TG OECD 492
negative control mean OD: 1.869 (acceptance range: 0.8 - 2.5)
positive control mean viablity: 2.19% (acceptance criteria: < 50% of the negative control)

- Acceptable variability between tissue replicates for positive and negative controls
The variability between tissue replicates for positive and negative controls meets the acceptance criteria of < 20%.a

- Acceptable variability between tissue replicates for the test chemical
The variability between tissue replicates for the test chemical meets the acceptance criteria of < 20%.
Irritation parameter:
percent tissue viability 
Run / experiment:
run 1, mean of three tissues
Value:
99.9
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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, as the mean OD value (1.869) of the two negative control tissues lies between 0.8 and 2.5.
- Acceptance criteria met for positive control: Yes, as the mean percentage viability for the positive control (26.5%) lies below 50% of the control viability.
- The variability between tissue replicates for positive, negative controls and test chemical were 2.19%, 4.74% and 0.42 % respectively, thus meeting the acceptance criteria of < 20%.

The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer. In the pre-test medium coloration by the test item was observed, but no tissues were stained during the study. Therefore, no additional tissues for color control were treated.

Table 1: MTT results of eye irritation study


















































































Controls



 



Optical Density (OD)



Viability (%)



Δ%



Negative Control: Sterile deionized water



1



1.806



96.6



17.4



2



1.931



103.3



mean



1.869



100.0



-



standard deviation (SD)



4.74



 



Positive Control:
Methyl acetate



1



0.524



28



2.19



2



0.466



24.9



mean



0.495



26.5



-



standard deviation (SD)



1.34



 



Test item



1



1.873



100.2



0.42



2



1.861



99.6



mean



1.867



67.5



-



standard deviation (SD)



99.9



-


Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item did not show an eye hazard potential.
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. 


Duplicates of the EpiOcular-model were treated with the test item, the negative or the positive control for 6 hours (+/- 15 min). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. The validity criteria were met. Following treatment with the test item, the tissue viability was 99.9% and, thus, higher than 60%, i.e. according to OECD 492 the test item did not show an eye hazard potential. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin and eye irritation/corrosion, the test item is not considered to be classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.