Sodium 2-[[[2,5-dichloro-4-[(2-methyl-1H-indol-3-yl)azo]phenyl]sulphonyl]amino]ethanesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 August 2017 (Seeding of the cells, 1st experiment) - 09 January 2018 (Experimental completion date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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: human Cell Line Activation Test (h-CLAT))

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The OECD TG 442 E may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-[[[2,5-dichloro-4-[(2-methyl-1H-indol-3-yl)azo]phenyl]sulphonyl]amino]ethanesulphonate

EC Number:

273-402-9

EC Name:

Sodium 2-[[[2,5-dichloro-4-[(2-methyl-1H-indol-3-yl)azo]phenyl]sulphonyl]amino]ethanesulphonate

Cas Number:

68959-19-3

Molecular formula:

C17H16Cl2N4O5S2.Na

IUPAC Name:

sodium 2-[[[2,5-dichloro-4-[(2-methyl-1H-indol-3-yl)azo]phenyl]sulphonyl]amino]ethanesulphonate

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

Cell line: THP-1 cells
The human monocytic leukemia cell line was obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB202).

Results and discussion

Positive control results:

The positive control used was 1- chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7), 4.0 µg/mL in 0.2% DMSO in culture medium. The positive and negative and vehicle control data is comparable to the historical data. The results can be seen in Table 2.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Relative fluorescence intensity (RFI) %

Remarks:

RFI CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative activation of dendritic cells according to OECD 442E at concentrations of 49-174 µg/mL

Remarks:

viability ≥ 50%

Results 2

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Relative fluorescence intensity (RFI) %

Remarks:

RFI CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Activation of dendritic cells positive according to OECD 442E of ≥ 200% at concentrations of 101-174 µg/mL. Activation of ≥ 150% at concentrations of 49-84 µg/mL.

Remarks:

viability ≥ 50%

Results 3

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: Relative fluorescence intensity (RFI) %

Remarks:

RDI CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative activation of dendritic cells according to OECD 442E at concentrations of 49-174 µg/mL

Remarks:

viability ≥ 50%

Results 4

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: Relative fluorescence intensity (RFI) %

Remarks:

RFI CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Activation of dendritic cells positive according to OECD 442E at concentrations of 49-174 µg/mL

Remarks:

viability ≥ 99%

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Acceptance criteria
- A tested concentration is not to be further evaluated when relative viability is less than 50%.
- Cell viability of vehicle control cells must yield at least 90%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%.
- In the negative control (LA), RFI values of both CD86 and CD54 should not exceed the positive criteria (RFI CD86< 150% and RFI CD54 < 200%) and cell viability should be ≥ 50%.
- For all vehicle controls, the MFI ratio of both CD86 and CD54 to isotype controls should be ≥ 105%.  
- The reactivity check of new thawed cells should produce the following result: - Positive response in CD86 and CD54 for NiSO4 and DNCB - Negative response in CD86 and CD54 for LA.
- In addition, positive, negative and vehicle control data should lie within the range of the historic data

Evaluation of results
- A test substance is predicted to activate monocytic THP-1 cells when CD86 expression is increased ≥ 150% and/or CD54 expression increased ≥ 200% at any concentration in relation to vehicle control that do not reduce viability below 50% and reproduced in the same cell surface marker in at least two independent experiments.
- A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 5000 µg/mL for the vehicle culture medium or 1000 µg/mL for 0.2% DMSO in culture medium) or up to the cytotoxicity limit (viability less than 90% at the highest concentration tested).  
- To be relevant for evaluation, the cell viability must be more than 50% in at least four tested concentrations of an experiment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Please refer to the historical control data in the 'Attached background material' section

Any other information on results incl. tables

Preliminary test

- The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve of the 3rd pre-test to be 145 µg/mL^3.

- The results of the 1st pre-test are not included in the report as the pre-test is considered invalid due to differing results with the 1st main experiment.

Experiment 1

- The concentration selection of the 1st experiment was performed based on a 1st pre-test.

- The 1st experiment is not useful for evaluation since there was no cytotoxicity below 50% and no positive response (RFI CD 86 ≥ 150% and/or CD54 ≥ 200%) was observed.

- The 1st pre-test is not included in this report as this pre-test is considered to be invalid due to the differeing results of the 1st main experiment.

Experiment 2 and 3

- These experiments are considered to be invalid due to technical problems with the flow cytometer and are not included in this report.

Experiment 4

- The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated by linear regression from the results of the 84 µg/mL and the 101 µg/mL concentration to be 96 µg/mL.

- The calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable.

Experiment 5

- The calculation of an EC200% was not applicable as fold inductions above 200% were obtained in all tested concentrations.

- The calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable.

Table 2: Summary of h-CLAT Main Experiments: mean values of RFI CD86, RFI CD54 and relative viability. 2 valid and evaluable experiments (4^thand 5^th) were performed. The 2^ndexperiment was not evaluable due to technical error and is not included in the report.
4^thexperiment				5^thexperiment
Concentration (test substance)   [µg/mL)	RFI CD86   Mean [%]	RFI CD54   Mean [%]	Viability   Relative viability [%]	Concentration (test substance)   [µg/mL)	RFI CD86   Mean  [%]	RFI CD54   Mean [%]	Viability   Relative viability [%]
49	76	180	100	49	89	579	100
58	67	151	100	58	42	693	100
70	79	190	100	70	79	571	100
84	62	182	100	84	67	657	100
101	56	207	100	101	61	828	99
121	40	362	96	121	48	867	98
145	43	469	91	145	42	1599	92
174	38	725	85	174	37	1827	73
VC	100	100	100	VC	100	100	100
LA 1000 µg/mL	74	108	100	LA 1000 µg/mL	83	139	100
DNCB 4 µG/mL	303	1153	75	DNCB 4 µG/mL	275	496	85
RFI above 150% (CD86) or 200% (CD54) with relative viability ≥50% are indicated in bold. VC: vehicle (culture medium); LA: lactic acid, negative control; DNCB: 1 -chloro-2, 4 -dinitrobenzene, positive control

None of the experiments met the ''borderline criteria'' and therefore the results were considered to be unambiguous.

Applicant's summary and conclusion

Interpretation of results:

other: positive prediction of skin sensitisation in h-CLAT assay

Conclusions:

Based on the observed results and applying the evaluation criteria in accordance with OECD Guideline 422 E, Acid Yellow 203 induces dendritic cell activation and is predicted to be a skin sensitiser.

Executive summary:

The skin sensitising potential of the test substance, Acid Yellow 230, was determined via the in vitro Human Cell Line Activation Test (h-CLAT) that evaluates that ability of Acid Yellow 230 to induce the expression of cell membrane markers (CD86 and CD54) and thus activate dendritic cells. The study was conducted following the OECD Guideline for Testing of Chemicals TG. 442E, adopted July 2016 (‘In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)’)

 

Main Assay

The test substance, Acid Yellow 230 was weighed and topped up with culture medium to achieve the required 2x concentration of the high concentration, Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution. The test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry. 

In order to determine the concentrations suitable for the main experiment, pre-tests (non-GLP) were performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value was determined by linear regression from the concentration response curve of the 3rd pre-test to be 145 µg/mL^3.

In the main test after 24-hour exposure THP-1 cells were stained with FITC labeled anti-humanCD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 2 valid and evaluable experiments were performed. The 1st experiment was not useful for evaluation as the concentrations selected were too low. The 2nd and 3rd experiment are considered to be invalid due to technical problems with the flow cytometer and are not included in the report. At concentrations used in the 4th and 5th experiments, the test substance was not soluble in DMSO (500 x stock preparations) and in 0.2% DMSO in culture medium (final concentrations) at the time of application. Visual observations conducted after 24 -hours showed that concentrations of 121 µg/mL and below were solutions.

The test substance was tested at a concentration range of 49 -174 µg/mL. The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated to be 96 µg/mL. The calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable as positive criteria were not met. None of the experiments met the ''borderline criteria'' and therefore the results were considered to be unambiguous.

 

In accordance with the evaluation of results, in the 4th experiments, Acid Yellow 230 induced CD86 expression < 150% and CD54 expression ≥ 200% at 101 -174 µg/mL and ≥ 150% at 49 -84 µg/mL, with a relative viability of ≥ 50% at all concentrations tested. Similarly, in the 3rd experiment, CD86 expression was <150% and CD54 expression was induced > 200% at all concentrations tested, with a relative viability of ≥ 50%. This thus indicates that the test substance is predicted to activate monocytic THP-1 cells. The results are also relevant for evaluation, since the relative cell viability results are more than 90% in more than four of the test concentrations in the 1st and 3rd experiments.

The acceptance criteria were met in all experiments and the results for the positive, negative and vehicle controls are comparable with historic data.

 

Conclusion

Based on the observed results and taking into account the evaluation criteria, it can be concluded that after 24 hours of exposure to the test substance, Acid Yellow 230, CD86 and CD54 expression was induced in THP-1 cells with at least 50% viability in at least two independent experiments. Therefore, it can be concluded that Acid Yellow 230 induces dendritic cell activation and is predicted to be a skin sensitiser.