5',5'''-[ethylenebis(p-phenyleneazo)]bis[1',2'-dihydro-6'-hydroxy-4'-methyl-2'-oxo-1,3'-bipyridinium] dilactate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 16th to 27th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

adopted February 04, 2015

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Basic Yellow 094

IUPAC Name:

Basic Yellow 094

In vitro test system

Details on the study design:

SUBSTANCES
- Test item: the test item was tested at 12 concentrations, according to a geometric progression of ratio 2 from 0.2 μg/ml to 400 μg/ml.
- Negative control: 6 wells of solvent control (1 % DMSO in treatment medium) with cells and 1 well of solvent control without cell by culture plate.
- Positive control: 5 concentrations of cinnamaldehyde on each culture plate. The concentration varies from 4 to 64 μM according to a geometric progression of ratio 2.

REPETITIONS
The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.

PARAMETERS MEASURED
Two parameters are measured: the luciferase induction and the cytotoxicity.

CELLS
- Cells: KeratinoSensTM (Givaudan).
- Culturing conditions: cells were cultured in maintenance medium at 37 °C, 5 % CO2.
- Mycoplasma: cells are exempt of mycoplasma. Assessment of mycoplasma was performed according to standardized protocol.

PROCEDURES
CELL SEEDING (day 01)
The cells were trypsinized; cells suspension were adjusted to a density of 8 x 10^4 cells/ml in seeding medium.
125 μl of the cell suspension at 8 x10^4 cells/ml (i.e. 10^4 cells per well) were distributed in three white plates for the induction measurement and two transparent plates to assess the cytotoxicity. The seeded plates were incubated 24 hours ± 1 hour at 37 °C, 5 % CO2.
Note: the H12 wells were left without cells and allowed the measurement of blanks.

PREPARATION OF THE TEST ITEM DILUTIONS (day 02)
Preparation of the 100 X plate
A 100-fold concentrated dilutions series was prepared in 96-well plate.

Test item
The test item stock solution was diluted in sterile water. The stock solution was prepared at 410 mg/ml (i.e. 4 %).
The test item was placed in one of the rows B to F. 100 μl of treatment culture medium 4 % DMSO were distributed from columns 1 to 11. 200 μl of the 1.6 mg/ml stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 μl of the column 12 in the column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.

Positive control
The positive control stock solution was prepared at 200 mM in DMSO according to the formula below, then diluted to the concentration of 6.4 mM.
Then, 100 μl of DMSO were distributed in row G from columns 7 to 10.200 μl of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 μl of the column 11 in the column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.

Negative control
100 μl of DMSO were distributed in row G columns l to 6 and 12 and in the well H12.

Preparation of the 4 X dilution plate
The 100 X DMSO plate was diluted 25 fold ina new plate (4 X). For the test item, the level of the DMSO is adjusted to 1 % final.

CONTACT BETWEEN CELLS AND TEST ITEM/CONTROLS (day 02)
In the 5 seeded plates, the medium was aspirated and replaced with 150 μl of treatment medium.
Then the 4 X plate was replicated 5 times: 50 pl from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37 °C, 5 % CO2).

LUCITERASE ACTIVITY (day 04)
After 48 hours, the medium was aspirated and each well was gently washed once with 200 μl of PBS. Then 100 μl of luciferase substrate (luciferine + ATP + lysing agent) were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis. The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

CELL VIABILITY ASSESSMENT WITH MTT METHOD (day 04)
Afier 48 hours, the medium was aspirated and each well was gently washed once with 200 μl of PBS.
Then, 225 μl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5 mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37 °C, 5 % CO2).
Afier this contact time, the staining solution was eliminated and the cells were treated with 200 μl of 10 % SDS one night in the dark (37 °C, 5 % CO2). Afier a 10 minutes homogenization, the absorbances were measured at 540 nm.

VALIDATION
To validate the test, it is essential to check the validity criteria for the test.

Positive Control
- the gene induction must be statistically significant above the threshold of 1.5 in at least one dose,
- the EC1.5 value should be between testing laboratory historical data: mean EC1.5 value ± 2 SD and the average induction, in each repetition, for cinnamaldehyde at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde should be carefully checked and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.

Negative control
For each repetition, the coefficient of variation of the solvent controls (3 × 6 wells) must be less than 20 %.
lf for one repetition the validity criteria are not met, a third repetition should be considered.

Results and discussion

Positive control results:

The EC1.5 value was between historical data, i.e. 3.73 for cinnamaldehyde at 64 μM.

In vitro / in chemico

Results

Run / experiment:

other: mean of 2 runs

Parameter:

other: Imax

Value:

1.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The Imax is a ratio between 2 luminescences. It is a parameter without unit

Other effects / acceptance of results:

The I max resulted to be lower than 1.5, thus no EC1.5 can be determined.

VALIDATION
Positive control: the gene induction resulted to be statistically significant above the threshold of 1.5 in at least one dose; in addition, the EC1.5 value was between historical data, i.e. 3.73 for cinnamaldehyde at 64 μM.

Negative control: the coefficient of variation of the solvent controls (3 × 6 wells) was be less than 20 %.

Any other information on results incl. tables

TEST ITEM

	Viability, C170 µg/ml	Induction
		Imax	EC1.5 linear µg/ml	EC1.5 lin/log µg/ml
Replicate 1	> 400	1.15	-	-
Replicate 2	> 400	1.46	-	-
Mean	-	1.3	-	-
Geom mean	> 400	-	-	-

CONTROLS

Cinnamaldehyde

	4 µM	8 µM	16 µM	32 µM	64 µM	EC 1.5	Imax
Replicate 1	1.02	1.13	1.42	1.78	3.7	19.39	3.7
Replicate 2	1.18	1.34	1.99	2.49	3.77	9.93	3.77
Mean	1.1	1.24	1.71	2.14	3.73	13.87*	3.73

*geometric mean

	CV % control solvent
Replicate 1	9.4
Replicate 2	11.8

Applicant's summary and conclusion

Interpretation of results:

other: data used as part of a global assessment, including several experimental evidences.

Conclusions:

The substance resulted to not activate the Keap1-Nrf2-ARE, under the tested conditions.

Executive summary:

The skin sensitisation potential of test substance was investigated by Keratinocytes test based on the signaling pathway Keap1-Nrf2-ARE coupled to the luciferase reporter gene. The experiment was conducted following the method and procedures outlined into the OECD guideline 442D. The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.

During both the repetitions performed, the Imax resulted to be lower than 1.5, thus no EC1.5 can be determined. All the validity criteria resulted to be satified. The two repetitions were considered as negative.

Conclusion

The substance results to not activate the Keap1-Nrf2-ARE, under the tested conditions