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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 7th to July 15th, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the testing of chemicals. No. 442E. In vitro skin sensitization assays addressing the key event of activation of dendritic cells on the adverse outcome pathway for skin sensitisation.

Version / remarks:

Adopted June 30th, 2022

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: IL-8 Luc assay protocol ver 024.2, Department of Dermatology, Tohoku University Graduate School of Medicine.

Version / remarks:

Adopted March 10th, 2017

Deviations:

not specified

GLP compliance:

yes

Type of study:

activation of dendritic cells

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

Details of test system:

THP-G8 cell line [442E]

Details on the study design:

TEST SYSTEM:
Human macrophage-like THP-1-derived IL8 reporter cell line THP-G8 was used. This cell line harbours the Stable Luciferase Orange (SLO) and Stable Luciferase Red (SLR) luciferase genes under the control of the IL-8 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoters, respectively. The cell line was established at Tohoku University School of Medicine. Batch 170809III31/25.


TEST PHASES:
Prior to use for testing, the cells were qualified by conducting a reactivity check using positive control 4-Nitrobenzyl bromide and the negative control, lactic acid. Cells were used after 3 passages after thawing. All the acceptance criteria were passed.
First run: this test was performed to determine the cytotoxic concentrations and to examine the skin sensitizing potential of the test item. The CV05 (Minimum concentration at which test item shows less than 0.05 of Inh-GAPLA*) was calculated.
Second: the tested concentrations started by the 4 times higher than the CV05. This run provided the final sensitizing prediction about the test item.

(*Inh-GAPLA: Mean (n = 4) GAPLA of THP-G8 cells treated with test item / Mean (n = 4) GAPLA of untreated cells. It is used as indicator of cytotoxicity).

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item resulted to be soluble at 20 mg/ml concentration in X-VIVO15 (stock solutions)
- Preparation of the test chemical serial dilutions: Successively, using X-VIVO15, serial dilutions at a dilution factor of 2 were prepared using a 96-well assay block.
- Preparation of the positive controls: the positive control (4-Nitrobenzyl bromide) is insoluble in X-VIVO15 and the 20 mg/ml suspension was vortexed and shaken in a rotor at 9 rpm for 2 hours and then centrifuged at 15.000 rpm for 5 minutes; the resulting supernatant was used for the test. The supernatant was diluted 1:4 for the first tested concentration and further diluted at factor of 2 and 4 (final concentrations tested on the cells were: dilution 1:8; 1:16 and 1:32).
- Preparation of the negative controls: The negative control (lactic acid) was dissolved at 20 mg/ml concentration in X-VIVO15. The first stock concentration was 4 mg/ml and then two serial dilutions of 1:2 were prepared. The final concentrations tested on the cells were: 2 mg/ml, 1 mg/ml, 0.5 mg/ml.
- Log Kow of the test chemical: -3.04

FIRST RUN:
- 50µl/well of THP-G8 cells (passage 24, four passages after thawing) were seeded at a density of 1∙10^6 cells/ml in a 96-well plate. 3 different plates were prepared for test item, positive and negative controls. Wells with only medium without the cells were used as blanks. The test item was preliminary tested to evaluate its solubility in X-VIVO15 medium. The test item resulted to be soluble at 20 mg/ml concentration in X-VIVO15 (stock solutions). Successively, using X-VIVO15, serial dilutions at a dilution factor of 2 were prepared using a 96-well assay block. Next 50 µl/well of each diluted solution was added to 50 µl of the cell suspension in the 96-well flat bottom black plate. For the test item the final concentrations of the test item ranged from 0.002 to 2 mg/ml. Each concentration was tested in 4 replicates.

SECOND AND THIRD RUN:
These runs were performed on a different day with fresh X-VIVO15 stock solutions of test item and independently harvested cells. Cells came from the same thawed vials cells and were used at passage 25 (five passages after thawing). The X-VIVO15 stock solution should be made at the concentration 4 times higher than the concentration of the cell viability 05 (CV05; the lowest concentration at which the Inh-GAPLA becomes < 0.05) in the first experiment. Nevertheless, the Inh-GAPLA did not decrease below 0.05 in the first run, so the X-VIVO15 stock solution was made at the first run highest concentration, 20 mg/ml. Serial dilutions of the X-VIVO15 second stock solutions were made at a dilution factor of 1.5 (from 0.03 to 2 mg/ml) using a 96-well assay block. Next, 50 µl/well of each diluted solution were added to 50µl of the cell suspension in the wells of a 96-well flat-bottom black plate. Each concentration of the test item was tested in four replicates. Cells were incubated for 16 hours at 37 °C and 5% CO2 after which the luciferase activity was measured.


LUCIFERASE ACTIVITY MEASUREMENTS
- Luminescence was measured using a 96-well microplate luminometer equipped with optical filter 600 nm LP. 100 µl of pre-warmed Tripluc® Luciferase assay reagent was transferred to each well of the plate containing the cell suspension treated with or without test item. After plate shaking bioluminescence was measured for 3 seconds in the absence and in the presence of the optical filter.

EXPRESSION OF THE RESULTS AND INTERPRETATION
The results are expressed in terms of:
- GAPLA: SLR Luciferase activity reflecting GADPH promoter activity
- IL8LA: SLO Luciferase activity reflecting IL-8 promoter activity
- nIL8LA: IL8LA/ GAPLA
- Ind- IL8LA: nIL8LA of THP-G8 cells treated with test item / nIL8LA of untreated cells
- Inh- GAPLA: GAPLA of THP-G8 cells treated with test item / GAPLA of untreated cells
- CV05: Minimum concentration at which test item shows less than 0.05 of Inh- GAPLA.

For each concentration mean ± SD of IL8LA, mean ± SD of GAPLA, mean ± SD of nIL8LA, mean ± SD of Ind- IL8LA, mean ± SD of Inh- GAPLA and the 95% confidence interval of Ind- IL8LA were calculated.

The interpretation of the results is carried out according to the following criteria:
- An IL-8 Luc assay prediction is judged positive if the test item has a Ind-IL8LA ≥ 1.4 and the lower limit of the 95% confidence interval of Ind-IL8LA is ≥ 1.0.
- An IL-8 Luc assay prediction is judged negative if the test item has a Ind-IL8LA < 1.4 and the lower limit of the 95% confidence interval of Ind-IL8LA is < 1.0.

Test items that provide two positive results from among the 1st, 2nd, 3rd, or 4th runs are identified as positive whereas those that give three negative results from among 1st,2nd, 3rd, or 4th runs are identified as supposed negative.

ACCEPTANCE CRITERIA
For reactivity check of the cells:
- Positive control has to produce a positive response to Ind-IL8LA (≥1.4) in at least one tested concentration.
- Negative control has to produce a negative response to Ind-IL8LA (<1.4) in all the tested concentration

For each experimental run:
- Ind- IL8LA has to be more than 5.0 at least in one concentration of the positive control
- Ind- IL8LA has to be less than 1.4 at any concentration of the negative control
- Data from plates for which the GAPLA of control wells with cells and Tripluc but without test item is less than 5 times of that of well containing test medium only have to be rejected.
- Data from plates for which the Inh-GAPLA of all concentrations of the test item or control chemicals is less than 0.05 have to be rejected. In this case the first run has to be repeated so the highest final concentration of the repeated test is the lowest final concentration of the previous test.

Vehicle / solvent control:

X-VIVO 15 [442E]

Negative control:

DL-Lactic acid

Positive control:

4-nitrobenzyl bromide (4-NBB) [442E]

Results and discussion

Positive control results:

Reactivity test: Ind-IL8LA = 5.82, 5.04 and 0.81 (Ind-IL8LA (≥1.4) in at least one tested concentration, acceptance criterion passed).
First run: Ind-IL8LA = 11.27, 7.13, 0.60 (Ind-IL8LA > 5.0 in at least one tested concentration, acceptance criterion passed).
Second run: Ind-IL8LA = 5.67, 3.98, 0.01 (Ind-IL8LA > 5.0 in at least one tested concentration, acceptance criterion passed).
Third run: Ind-IL8LA = 8.60, 6.17, 2.84 (Ind-IL8LA > 5.0 in at least one tested concentration, acceptance criterion passed).

In vitro / in chemico

Results

Results 1

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Ind-IL8LA [442E]

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The Ind-IL8LA value was < 1.4 in all teste concentrations.

Results 2

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Ind-IL8LA [442E]

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The Ind-IL8LA value was < 1.4 in all tested concentrations.

Results 3

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

Ind-IL8LA [442E]

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The Ind-IL8LA value was < 1.4 in all tested concentrations.

Other effects / acceptance of results:

All acceptance criteria were met.

Applicant's summary and conclusion

Interpretation of results:

other: According to the CLP Regulation (EC) No. 1272/2008, the test item shall not be classified as Skin Sensitizer.

Conclusions:

Under the conditions of the test, the test item resulted to be non-sensitizer to the skin bacause in three different experimental runs the Ind-IL8A value was < 1.4 in all tested concentrations.

Executive summary:

The potential of the test item to induce IL-8 activation in THP-1-derived IL8 reporter cell line THP-G8 was investigated with the IL-8 Luc assay, performed in accordance to the OECD Guideline 442e. The test item showed, under the conditions of the test, an Ind-IL8LA < 1.4 for all the tested concentrations in three experimental runs. All the acceptance criteria were passed. Under the conditions of the test, the test item shall not be classified as skin sensitizer according to the CLP regulation (EC) No. 1272/2008. The results obtained by the IL-8 Luc assay should not be used on their own to predict potency for safety assessment decisions but should be instead used within Integrated Approaches to Testing and Assessment (IATA).