[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 AUG 2021 - 27 SEP 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

bis(2,2,6,6-tetramethyl-1-(1,3-benzothiazol-2-ylsulfanyl)piperidin-4-yl)carbonate

Cas Number:

2311845-49-3

Molecular formula:

C33H42N4O3S4

IUPAC Name:

bis(2,2,6,6-tetramethyl-1-(1,3-benzothiazol-2-ylsulfanyl)piperidin-4-yl)carbonate

Test material form:

solid: particulate/powder

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

EpiOcular™ (OCL-200-EIT)
The EpiOcular™ human cell construct (MatTek In Vitro Life Science Laboratories) is used in this assay. This three-dimensional human cornea model allows the identification of test items with the potential to induce eye irritation or serious eye damage by assessing cell viability after treatment.
- Source: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava, Slovakia
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The preparation of tissues for treatment was started after arrival in TOXI-COOP ZRT.’s Laboratory. Therefore, the storage of EpiOcular™ (OCL-200-EIT) units was not necessary. The assay medium supplied with the kits was stored in refrigerator (2-8 °C).
- Justification for selection : The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.
- Quality check of the isolated corneas: The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 μL of 0.3 % (v/v) Triton X-100). A certificate of quality as provided by the supplier is annexed to this report.

Test system

Vehicle:

other: MTT stock solution dissolved to a final concentration of 5 mg/mL in Ca++ and Mg++ free Dulbecco's phosphate buffered saline (DPBS).

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 50 mg test item was applied topically onto the EpiOcular™ tissues, approximately 83.3 mg/cm2 application.

VEHICLE
- Amount(s) applied (volume or weight with unit): Approximately 50 mg test item was added to 1 mL of 1 mg/mL MTT solution in a tube
- Lot/batch no. (if required): MKCL1832

Duration of treatment / exposure:

The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere).

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes at standard culture conditions (Post-exposure Incubation)

Number of animals or in vitro replicates:

In this assay 2 replicates per test item, 2 replicates of the negative control and 2 replicates of the positive control were used.

Details on study design:

NUMBER OF REPLICATES: In this assay 2 replicates per test item, 2 replicates of the negative control and 2 replicates of the positive control were used.

NEGATIVE CONTROL USED: Sterile deionized water, Ultrapure water (prepared in the testing laboratory): Type: Synergy Smart UV HF ASTM Type 1: F8JA80461C, Batch Number: 20210811
Expiry date: August 18, 2021, Storage: Room temperature

POSITIVE CONTROL USED: Methyl Acetate (MA), 99.7% purity, Supplier: MatTek, Lot No.: 012621MJA, CAS No: 79-20-9, Expiration Date: January 26, 2022, Storage condition: Room temperature and dry place

APPLICATION DOSE AND EXPOSURE TIME: The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere).

POST-INCUBATION PERIOD: After the incubation time the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS. After rinsing, the tissues were transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25±2 minutes immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the Post-Soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37 °C) Assay Medium.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation time the EpiOcular™ units were removed and rinsed thoroughly.
- POST-EXPOSURE INCUBATION: The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-exposure Incubation). After the post-exposure incubation the EpiOcular™ units were transferred into the 24-well plate filled with MTT ready to use solution (300 μL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 15 min) at 37±2 °C in an incubator with 5±1 % CO2, protected from light, ≥ 95 % humidified atmosphere.

METHODS FOR MEASURED ENDPOINTS:
In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item.

DECISION CRITERIA:
Assay Acceptance Criteria
-The mean OD value of the two negative control tissues should be >0.8 and <2.8
- The acceptable percentage viability for positive control (mean of two tissues) is: 6 hours exposure: below 50 % of negative control viability (solids)
- The difference of viability between the two relating tissues of a single chemical should be < 20 % in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

mean percent tissue viability 

Run / experiment:

mean

Value:

105

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

percent tissue viability 

Run / experiment:

1/ replicate 1

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

percent tissue viability 

Run / experiment:

1/ replicate 2

Value:

108

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method TOXI-COOP ZRT. demonstrated the technical proficiency in a separate study (Study No.: 392-492-1722) using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the two negative control tissues was: 2.098
- Acceptance criteria met for positive control: The positive control result showed 3 % viability after 6 hours exposure.
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 105 % relative to the negative control). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, this in vitro eye irritation test, using the EpiOcular™ Model, indicated that the test item reveals no eye irritation potential under the applied testing conditions and is considered as non-irritant to eye (UN GHS No Category).

Executive summary:

The In Vitro Eye Irritation test was performed to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro, according to the OECD guideline 492 and under GLP compliance.

Before the treatment the tissues were pre-wetted with 20 μL of Ca++Mg++ Free- Dulbecco's phosphate buffered saline (DPBS) and incubated at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere) for 30±2 minutes. Disks of EpiOcular™ (two units) were treated with the test item (approx. 50 mg, equal to 83.3 mg/cm²) and incubated for 6 hours (± 15 min) at standard culture conditions.

Exposure to the test item was terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 25±2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, the test item-treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-exposure incubation). Fresh Assay Medium was used during the Post-Soak and Post-exposure incubation. Subsequently, the viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37±2 °C in an incubator with 5±1 % CO2, protected from light, ≥ 95 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes. The post-exposure procedure was conducted identically to the test item-treated tissues, as described above.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 105 % relative to the negative control). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control.

The positive and negative controls showed the expected values within acceptable limits. The experiment is considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ Model, indicated that the test item reveals no eye irritation potential under the applied testing conditions and is considered as non-irritant to eye (UN GHS No Category).