2,5-dichloro-4-[[2-(dibutylamino)-4-methyl-6-[[2-(4-sulphophenyl)ethyl]amino]-5-pyrimidinyl]azo]benzenesulphonic acid, sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 04 to November 13, 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:

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

Test material

Test material information

Constituent 1

Reference substance name:

Acid Yelow 236

IUPAC Name:

Acid Yelow 236

In vitro test system

Details on the study design:

CELLS
- Source: THP-1 (monocytic leukeamia cell line) cells provided from American Type Culture Collection.
- Storage: cells were stored in liquid nitrogen and the assays were performed thanks to a master bank supplied by Biopredic International (Saint Gregoire - France).
- Culturing: cryopreserved cells have been thawed. Cells were cultured, at 37 °C under 5 % CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10 % Foetal Calf Serum, 100 units/ml penicillin and 100 pg/ml streptomycin.
- Seeding: THP-1 were routinely seeded every 3-4 days at the density of 0.15 to 0.2 x10^6 cells/ml.
- Preparation of test incolumun: for testing, THP-1 cells were seeded at a density of 0.2 x 10^6 and pre-cultured in culture flasks for 72 hours.
- Viability: the quality of each batch of THP-1 cells should be checked. Viability of the cells must be above 90 %.

MAIN EXPERIMENT
Test item
- Concentration range: 7.81 to 100 μg/ml. The maximum concentration tested was 100 μg/ml, depending on its poor solubility.
- Solvent: the test item was tested in a medium containing 0.2 % DMSO. (Dimethysulfoxide).

Procedures
Based on the cytotoxlcity assay the eight final test item concentrations were selected.
ln case of non-toxic concentration for the top dose used in preliminary test, the maximum concentration selected for activation test did not exceed 1000 µg/ml when the test item was dissolved or stably dispersed in Ethanol or DMSO, and 5000 µg/ml when the test item was dissolved in a saline vehicle.
Each experiment of activation test was performed on eight concentrations.
THP-1 cells were plated at 1 x 10^6 cells/ml/well in 24 well plates and treated for 24:05 hours with selected test item concentrations. After treatment cells were washed twice with FACS buffer. Then cells were stained for 30 min at 4 °C with the following fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (mAbs): anti-human CD54, anti-human CD86; FITC labelled-mouse lgG1. Using the manufacturer's recommended dilutions, cells were incubated with above mAbs at 6 µl/3 x 10^5 cells /50 µl for the anti-human CD86 mAb, and 3 µl/3 x 10^§5 cells /50 µl for the anti-human CD54 mAb. FITC labelled-mouse lgG1 was used as an isotype control at a dilution of 3 µl/3 x 10^5 cells /50 µl. Then, the cells were stained also with 7-AAD for 30 min at 4 °C. After washing and resuspension with FACS buffer, the fluorescence intensities of the THP-1 cell surface markers were then analysed by flow cytometry using GUAVA and InCyte software, on 10000 living cells.

CONTROLS
Vehicle control: as the maximal dose for test item was obtained in the DMSO, the vehicle control for all of the assays in the study was the DMSO.
Negative control: Lactic Acid (LA) produced a negative response for both markers.
Reference controls: 2,4-dinitrochlorobenzene (DNCB), nickel sulfate (NiSO4) produced a positive response for both CD86 and CD54.

PRELIMINARY CYTOTOXICITY
The cytotoxicity of the test item was evaluated in order to select at least 4-5 concentrations able to induce cytotoxicity, around 50 %, for the highest one. Assessment of cell toxicity was performed by determining cell viability on THP-1 cells, using the 7-AAD inclusion methods.
Eight concentrations of test item have been prepared by a two-fold serial dilution from a maximum final concentration of 1000 µg/ml or lower, depending on its solubility limit.
In the day of testing, cells harvested from culture flask were suspended with fresh culture medium at 2 x 10^6 cells/ml. Then, THP-1 cells were distributed into a 24 well flat-bottom plate with 500 µl (1 x 10^6 cells/well) with various concentrations of test item (1:1 ratio) for 24±0.5 hours at 37 “C under 5 % CO2. After treatment cells were washed twice with phosphate-buffered containing 0.1 % (w/v) bovine serum albumin identified as FACS buffer. The cells were stained with 7-AAD (5 µg/ml final concentration). Then cells were analysed with flow cytometry using GUAVA and InCyte software to measure cell viability. The living cells (7-AAD-) gate was set in the 7-AAD negative area. 10^4 7-AAD- cells were counted as the living population.
According to the results the dose levels for the main study were selected.

VALIDITY OF THE STUDY
Acceptability criteria for evaluating results induced by the positive control, the vehicles controls and the test item, were based on previous studies cited above and the guideline for h-CLAT (OECD 442E, 2016).
The study is considered valid if the following criteria are fully met:
- in the positive control (DNCB) RFl values of both CD86 and CD54 should be over the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %).
- in the positive control the cell viability should be more than 50 %
- in the vehicle control cell viability should be more than 90 %
- in the vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %) compared to the medium control
- in the vehicle control the MFI ratio of both CD86 and CD54 to isotype control should be > 105 %
- in the test item cell viability should be more than 50 % in at least four tested concentrations in each run.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: both two runs

Parameter:

other: % RFI of CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

cell viability > 50 %

Results 2

Run / experiment:

other: both two runs

Parameter:

other: % RFI of CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

cell viability > 50 %

Other effects / acceptance of results:

No "increase" of the CD54/86 expression compared with the negative control was noticed, for any dose of test item.
In both experiments, an absence of any increase of the CD54/86 expression was observed with the test item. None of the tested doses induced a 1.5 or 2 fold increase of CD86 and CD54 expression respectively compared to the negative control.

No contamination was noticed during the study.

PRELIMINARY CYTOTOXICITY
Based on the solubility test, the doses range for cytotoxicity test was the following (with two fold dilution factor): from 0.78 to 100 µg/ml.

VALIDITY OF THE STUDY
Test system was validated as all criteria were fully accepted.
Negative controls (DMSO) showed cell viability values acceptable regarding the acceptance criteria.
Positive controls showed an increase of CD54/86 expression (RFI ≥ 200/150 respectively) compared to the negative control.
These results validated the experimental conditions.

Any other information on results incl. tables

	Concentration range*	Viability	CD54	CD86	MIT (μg/ml)
Run 1	7.81 to 100 μg/ml	> 50 %	RFI < 200	RFI < 150	-
Run 2		> 50 %	RFI < 200	RFI < 150

RFI of CD54/86 expression

Sample	Experiment 1			Experiment 2
	CD54	CD86	CV (%)	CD54	CD86	CV (%)
RPMI	100	100	98	100	100	98
DMSO	108	118	98	106	105	98
DNCB, 4 µg/ml	1301	517	77	914	410	81
Test iten (vehicle 0.2 % DMSO), 0.78 µg/ml	98	138	97	98	134	97
Test iten (vehicle 0.2 % DMSO), 1.56 µg/ml	88	132	97	91	126	97
Test iten (vehicle 0.2 % DMSO), 3.13 µg/ml	90	144	97	85	137	97
Test iten (vehicle 0.2 % DMSO), 6.25 µg/ml	88	122	97	90	145	97
Test iten (vehicle 0.2 % DMSO), 12.5 µg/ml	89	135	97	40	105	98
Test iten (vehicle 0.2 % DMSO), 25 µg/ml	74	118	98	50	109	98
Test iten (vehicle 0.2 % DMSO), 50 µg/ml	79	123	97	51	90	98
Test iten (vehicle 0.2 % DMSO), 100 µg/ml	58	89	97	53	71	98

PRELIMINARY CYTOTOXICITY

Dose level µg/ml	% cell viability
100	97
50.0	97
25.0	97
12.5	98
6.25	98
3.13	98
1.56	98
0.78	98

QUALITY CONTROL OF THE TEST SYSTEM

CV and ratio of both markers CD54 and CD86 to isotype control obtained for vehicles and controls

	CV (%)		Ratio CD54/IgG		Ratio CD86/IgG
Sample	Criteria	Result	Criteria	Result	Criteria	Result
RPMI	> 90 %	98	> 105	124	> 105	183
0.9 % NaCl	> 90 %	98	> 105	124	> 105	194
DMSO	> 90 %	98	> 105	128	> 105	206
NiSO4	> 50 %	81
DNCB	> 50 %	77
LA	> 50 %	98

RFI values obtained for vehicles and controls

Sample	RFI
	Criteria	Result CD54	Result CD86
0.9 % NaCl	CD54 ≤ 200 and CD86≤150 %	100	110
DMSO	CD54 ≤ 200 and CD86≤150 %	108	118
NiSO4	CD54 ≥ 200 % and CD86 2≥ 150 %	4984	365
DNCB	CD54 ≥ 200 % and CD86 2≥ 150 %	1301	517
LA	CD54 ≤ 200 and CD86≤150 %	193	116

Applicant's summary and conclusion

Interpretation of results:

other: 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

Conclusions:

The test quantifing changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell lineresulted 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; the maximum concentration tested was 100 μg/ml, depending on its poor solubility. Experiment was conducted in duplicate.

The RFI of CD86 resulted to be lower than 150 % at any tested concentration (with cell viability ≥ 50 %), in both runs; the RFI of CD54 resulted to be lower than 200 % at any tested concentration (with cell viability ≥ 50 %), in both runs.

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 lineresulted to be negative.