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

Endpoint summary

Description of key information

Not skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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.

Positive control results:

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

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 %.

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

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

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 7th to December 21st, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome pathway for Skin Sensitisation)

Version / remarks:

9 October, 2017

GLP compliance:

yes

Type of study:

activation of dendritic cells

Details on the study design:

- Concentration range: 2.35 to 10 μg/ml in the main test; 0.39 to 50 μg/ml in the preliminary cytotoxicity test.
- Solvent: DMSO (Dimethysulfoxide).

Run / experiment:

other: all runs

Parameter:

other: RFI of CD86 (cell viability > 50 %)

Value:

150

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: all runs

Parameter:

other: RFI of CD54 (cell viability > 50 %)

Value:

200

Remarks on result:

no indication of skin sensitisation

Test h-CLAT results

	Concentration range*	Viability	CD54	CD86	MIT (μg/ml)
Run 1	2.35 to 10 μg/ml	> 50 % (except for the highest concentration	RFI < 200	RFI < 150	-
Run 1		> 50 %	RFI < 200	RFI < 150
Run 3		> 50 %	RFI < 200	RFI < 150

Preliminary cytotoxicity results

- Concentration range: 0.39 to 50 µg/ml

- CV75 4.3 µg/ml

- Conclusion: cytotoxic, modification of concentration range for h-CLAT tests

Interpretation of results:

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

Conclusions:

The test quantifing changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line resulted to be negative.

Executive summary:

The substance was tested for skin sensitisation potential, following the method and procedures described into the OECD guideline 442E. DMSO (Dimethysulfoxide) was used as an intermediate solvent. Based on the preliminary cytotoxicity test results, the substance resulted to be cytotoxic, thus the concentration range tested in the main experiment was 2.35 to 10 µg/ml.

Experiment was conducted in triplicate.

The RFI of CD86 resulted to be lower than 150 % at any tested concentration, in all runs; the RFI of CD54 resulted to be lower than 200 % at any tested concentration, in any run.

Therefore, under the test conditions of the experimental human Cell Line Activation Test, the test item has been judged to be a non-sensitizer.

 

Conclusion

The test quantifing changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line resulted to be negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

An in vivo test on Basic Yellow 094 was available, according to Magnusson and Kilgman method. As it was not possible to inject the product intradermally, it was applied epicutaneously, after skin scratching (abrading). The first application was made in presence of Freund's adjuvant 50 %, administered by intradermal injection at the level of the cutaneous application. The substance was then applied epicutaneously at 1 %.

None of the test animals presented positive reactions during the challenge. However, due to the low purity of tested sample, test results were not considered as suitable for classification.

To evaluate the skin sensitisation potential of Basic Yellow 094, three in vitro skin sensitisation studies were proposed. Specifically, these three studies examine the ability of the test item to induce peptide/protein bonding (Adverse Outcome Pathway (AOP) Key Event 1, in a DPRA), a keratinocyte response (AOP Key Event 2, in an ARE Nrf2 LuSens assay) and a monocytic/dendritic cell response (AOP Key Event 3, in a h-CLAT). Together, these data can be used to interpret the skin sensitisation potential of a substance. The following experimental findings are reported:

(1) Potential to induce peptide/protein bonding can be evaluated in a Direct Peptide Reactivity Assay (DPRA), according to OECD guideline 442C (2015). However, during the preparation of reaction samples, solubility issues were encountered and it was not possible to continue with the assay.

(2) The potential of Basic Yellow 094 to activate the Nrf2 transcription factor was assessed in the genetically modified keratinocyte cell-line ARE Nrf2 Luciferase “LuSens” Assay, according to OECD guideline 442D. No substantial, reproducible, dose-dependent increase in luciferase induction above 1.5-fold was observed in either of the two repetitions of the experiment performed. Therefore, the assay was negative and the test item is not considered to have the potential to activate Nrf2 transcription factor and does not induce a keratinocyte response.

(3) A Human Cell Line Activation Test (h-CLAT) was performed on the human monocytic leukaemia cell line (THP-1 cells), according to OECD guideline 442E. Based on the preliminary cytotoxicity test results, the substance resulted cytotoxic, therefore the concentration range tested in the main experiment was 2.35 to 10 µg/ml. The relative fluorescence intensity (RFI) of the CD86 marker resulted to be lower than 150 % at any tested concentration, in all runs; the RFI of CD54 resulted to be lower than 200 % at any tested concentration, in any run. Therefore, the test item is not considered to activate a dendritic cell response.

An available experimental study on a structural analogue of Basic Yellow 094, Similar Substance 02, was also taken into consideration. Details on the read-across approach are reported in IUCLID section 13.

The skin sensitisation potential of Similar Substance 02 was evaluated in an experimental study according to OECD guideline 406. The induction phase was performed by intradermal injection of 0.1 % w/w test item followed by an epidermal application of 25 % w/w. The first challenge was performed 2 weeks after induction by epidermal application at 25 % w/w; a second challenge was then performed by the same route and concentration. No differences between the test group and the vehicle-treated controls were evident after epidermal challenge applications. Therefore, the substance is considered to possess no skin sensitising potential in albino guidea pigs. After the challenge application, no positive results were observed in the animals of the test article treated group: readings taken at both 24 and 48 hours resulted to be 0 % in both cases. No toxic symptoms were evident in the guinea pigs of either the control or test groups. No death occurred.

Based on the consistently negative results of the two in vitro skin sensitisation studies and the GPMT performed using low substance content, as well as the negative GPMT study performed on the structural analogue (Similar Substance 02), Basic Yellow 094 is considered as not sensitising.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), a skin sensitiser means a substance that will lead to an allergic response following skin contact. Effects seen in either humans or animals will normally justify classification in a weight of evidence approach for skin sensitisers.

The substance tested negative in 2 in vitro studies; no posiitve response was seen in a GPMT after an intradermal induction dose of 0.1 % w/w.

On these bases, Basic Yellow 094 is considered as devoid of a skin sentising potential and is not classified within the CLP Regulation (EC 1272/2008).