1H-Indene-1,3(2H)-dione, 2-(2-quinolinyl)-, sulfonated, sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 10th, 2020 to June 22nd, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

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 June 18th, 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EpiOcularTM Eye irritation test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals for use with MatTek Corporation’s Reconstructed Human EpiOcularTM Model. MatTek Protocol.

Version / remarks:

Adopted October 2nd, 2017

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1H-Indene-1,3(2H)-dione, 2-(2-quinolinyl)-, sulfonated, sodium salts

EC Number:

305-897-5

EC Name:

1H-Indene-1,3(2H)-dione, 2-(2-quinolinyl)-, sulfonated, sodium salts

Cas Number:

95193-83-2

Molecular formula:

C18H11NO5S to C18H8NO11S3.3Na

IUPAC Name:

trisodium hydrogen bis(2-(1,3-dioxo-5-sulfonato-2,3-dihydro-1H-inden-2-yl)quinoline-8-sulfonate)

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

Test system: Reconstructed ocular tissue model OCL EpiOcular™ tissues 0,6cm2, MatTek containing normal human keratinocytes (both for first and second run).

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test item: 50 mg (minimum of 33 mg/cm2)
Negative control: 50 µl
Positive control: 50 µl

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25 minutes at room temperature in assay medium. After that the tissues were further incubated in a plate with 1ml of assay medium for 18 hours + 5 minutes at 37°C, 5% CO2.

Number of animals or in vitro replicates:

3

Details on study design:

The test item was preliminary tested to evaluate its interference with MTT: 50mg of the test item were put in contact with 1ml of MTT and incubated for 180 minutes at 37 °C and 5% CO2. A negative control (50 µl of sterile deionized water) was used. The MTT solution with the test item did not change the color so it had not reduced the MTT.
The test item was also preliminary tested to evaluate if it became colorant material after application to the tissues and, therefore, if it could interfere with the endpoint measurement. 50mg of the test item were put in contact with 2ml of isopropanol and incubated for 3 hours under stirring. At the same time 50 mg of the test item were put in contact with 1ml of water and incubated for 1 hours at 37°C and 5% CO2. Two 200µl aliquots of isopropanol solutions and of pure isopropanol or water solution and pure water were transferred to a 96-well plate and the absorbance was measured with a plate reader equipped with the MTT measurement wavelength filter. The OD of the test item solution was < 0.08 after subtraction of the OD for isopropanol or water, so the material was not considered as possibly interacting with the MTT measurement.

Definitive test:
The tissues were pre-wetted with 20µl of Ca and Mg free-PBS for 30 minutes at 37 °C and 5% CO2. After that about 50 mg (minimum of 33 mg/cm2) of the test items and 50 µl of the controls were applied on each epithelium unit in three replicates. The exposition were carried out for 6 hours at 37 °C, 5% CO2. At the end of the exposure period, the test item and controls were removed with multiple washing with Ca and Mg free-PBS and the epithelium units were immersed in assay medium and incubated at room temperature for 25 minutes. After that the tissues were further incubated in a plate with 1 ml of assay medium for 18 hours + 5 minutes at 37 °C, 5% CO2.
At the end of the incubation, the viability assay was performed to evaluate the cell survival in the ocular epithelium units. Epithelium units were treated with 300 µl of 1 mg/ml MTT solution (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide) for 3 h at 37 °C, 5% CO2. The solution was then removed and replaced with 2 ml isopropanol, with further 2 hours incubation at room temperature under speed shaking. 2 aliquots of every sample were transferred to a 96 well plate for the reading. The absorbance of the three replicates was read with a microplate reader equipped with a wavelength of 570 nm filter. The absorbance values were corrected by subtracting the reading of the blanks (diluent only).

After subtraction of the mean OD blanks, the mean OD of all negative control tissues corresponds to 100% viability. The results are expressed in terms of viability:
% of cell viability = [mean OD (570 nm) test item per tissue / mean OD (570 nm) negative control] x 100.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: In vitro cell viability (%)

Run / experiment:

First

Value:

66.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Borderline results (non-concordant replicates measurement). Therefore, a second run was performed.

Results 2

Irritation parameter:

other: in vitro cell viability (%)

Run / experiment:

Second

Value:

82.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS (first and second runs):
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No.

ACCEPTANCE OF RESULTS FIRS RUN:
- Acceptance criteria met for negative control: yes. OD = 1.6; standard deviation = 8.9%
- Acceptance criteria met for positive control: yes. Mean viability = 5.1 %; standard deviation = 1.8
- Test item: yes. Standard deviation = 13.5 %
- Blank (isopropanol): yes. OD < 0.1.

ACCEPTANCE OF RESULTS SECOND RUN:
- Acceptance criteria met for negative control: yes. OD = 1.8; standard deviation = 4.8%
- Acceptance criteria met for positive control: yes. Mean viability = 2.1 %; standard deviation = 0.3
- Test item: yes. Standard deviation = 9.3 %
- Blank (isopropanol): yes. OD < 0.1.

Any other information on results incl. tables

The test item resulted to be not eye irritant under the conditions of the study because its mean cell viability is higher than 60% in in both experimental runs.

Applicant's summary and conclusion

Interpretation of results:

other: Not eye irritant according to the CLP Regulation (EC) No. 1272/2008

Conclusions:

Under the conditions of the test, the test item resulted to be not eye irritant.

Executive summary:

The potential of the test item to induce eye irritation has been evaluated in a in vitro test according to the OECD Guideline 492 using the commercial reconstructed human cornea-like epithelium (RhCE) EpiOcular. Two runs with three replicates each have been performed because of borderline results (non-concordant replicate measurement) in the first run.

The test item showed a mean cell survival of 66.5% (±13.5) in the first run and 82.2% (±9.3) in the second run, so it resulted to be not an eye irritant because its cell viability is higher than 60% . All the acceptance criteria were passed.