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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 2nd to March 31st, 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Adopted June 18th, 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol n°135: SkinEthicTM Skin Irritation Test

Version / remarks:

Adopted April 5th, 2018

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

In vitro test system

Test system:

human skin model

Remarks:

SkinEthic RHE/Human Epidermis (RHE/S/17)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Normal human keratinocytes were grown on inert polycarbonate filters in chemically defined medium for 17 days. The product was prepared and packaged using aseptic techniques. Store in incubator at 37 °C, 5% CO2 with saturated humidity.
- Quality control for skin discs: Exposure time inducing 50% viability using Triton X-100 1% = 5.3h (required: 4.0h ≤ ET50 ≤ 10.0h).

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Multiple washing with PBS

CELL VIABILITY ASSAY: MTT assay.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test material: 16 ± 2 mg
Negative control (PBS without Ca and Mg): 16 ± 2 µl
Positive control (SDS 5%): 16 ± 2 µl

Duration of treatment / exposure:

42 minutes (room temperature)

Duration of post-treatment incubation (if applicable):

42 hours (37°C, 5% CO2)

Number of replicates:

3

Test system

Details on study design:

The test item was preliminary tested to evaluate its interference with MTT endopoint. 16 mg ± 2 mg of the test item were put in contact with 0.3 ml of MTT and incubated for 180 ± 15 minutes at 37 °C and 5% CO2. A negative control (16 µl of sterile deionized water) was run concurrently. MTT solution color did not change its color into blue/purple, indicating that the test item was not reducing the MTT.

Furthermore the test item was preliminary tested to evaluate its colorant properties. 10 mg ± 1 mg of the test item were put in contact with 0.09 ml of water and incubated for 30 minutes at room temperature after shaking. A negative control (10 µl of sterile deionized water) was run concurrently. The absorbance was measured with a plate reader equipped with the MTT measurement wavelength filter. After subtraction of the OD of negative control, the OD of the test item solution was < 0.1 (0.0357), indicating that the test item does not interact with the MTT measurement.

In the definitive test, after over-night incubation in the growth medium, the epidermis units were treated with the test item. 16 mg ± 2 mg of test item, and 16 µl ± 2 µl of positive (SDS 5%) and negative (PBS without Ca and Mg) controls were applied on epidermis units in three replicates. The exposition was carried out for 42 minutes at room temperature. At the end of the exposure period the samples were removed with multiple washing with PBS and the tissues were further incubated in 2 ml of medium at 37°C, 5% CO2 for 42 h. At the end of the exposure, the viability assay was performed to evaluate the cell survival in the epidermis units. Epidermis units were treated with 0.3 ml 1 mg/ml MTT solution (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) for 3 h at 37°C. The solution was then removed and replaced with isopropanol, with further 2 h incubation at room temperature under solution speed shaking. 2 aliquots of isopropanol were sampled for every epidermis units and transferred to a 96 well plate for the reading (6 readings for each units of test item, positive and negative controls). The absorbance was read with a microplate reader equipped with the 570 nm. The absorbance values were corrected by subtracting the reading of the blanks (diluent only).

After the blanks subtraction the results are expressed in terms of cell viability expressed as percentage:
Cell viability = [OD(570 nm) test item per tissue /mean OD(570 nm) negative control] x 100.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

106.74

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. OD = 2.10; standard deviation = 2.73%
- Acceptance criteria met for positive control: yes. Mean viability = 0.87; standard deviation = 0.87%
- Test item: yes. Standard deviation = 5.10 %
- Blank (isopropanol): yes. OD < 0.1.

Any other information on results incl. tables

The test item results is not irritant to the skin, because its mean cell viability is higher than 50%.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

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

Executive summary:

The skin irritation potential of the test item on Reconstructed Human Epidermis (RHE SkinEthic) was investigated according to the OECD Guideline 439 in triplicates. The test item, under the conditions of the test, showed a mean cell survival of 106.74% (±5.10) and therefore it resulted to be non skin irritant as its cell viability is higher than 50%. All the acceptance criteria were passed.