[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium methyl sulphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 March 2018 (Seeding of the cells,1st experiment) - 12 April 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:

[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium methyl sulphate

EC Number:

248-957-5

EC Name:

[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium methyl sulphate

Cas Number:

28313-51-1

Molecular formula:

C22H29N6S.CH3O4S

IUPAC Name:

{2-[ethyl({3-methyl-4-[(E)-2-(3-phenyl-1,2,4-thiadiazol-5-yl)diazen-1-yl]phenyl})amino]ethyl}trimethylazanium methyl sulfate

Constituent 2

Reference substance name:

[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium sulphate

EC Number:

286-603-1

EC Name:

[2-[ethyl[3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)azo]phenyl]amino]ethyl]trimethylammonium sulphate

Cas Number:

85283-76-7

Molecular formula:

C22H29N6S.1/2O4S

IUPAC Name:

bis[2-(ethyl{3-methyl-4-[(3-phenyl-1,2,4-thiadiazol-5-yl)diazenyl]phenyl}amino)-N,N,N-trimethylethanaminium] sulfate

Test material form:

solid: particulate/powder

Remarks:

Grey

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test-substance No.: 17/0373-1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: ambient, under light exclusion
- Storage stability: the stability under storage conditions over the exposure period was guaranteed by the sponsor, and the sponsor hold this responsibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test substance was weighed and topped up with the chosen vehicle (culture medium) to achieve the required 2x concentration of the highest concentration (stock solution).
- Culture medium was chosen as the vehicle, since good homogeneity of the preparation was achieved.
- Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution according to the planned concentrations in culture medium.
- The test-substance preparations were prepared by stirring and ultra-sonication.
- Visual inspection of each dilution step was performed

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).

Historical control data:
- A reactivity check is performed with each new-thawed cells prior to the use of the cells for a study, as proposed in the OECD test guideline, using nickel (II) sulphate hexahydrate as well as lactic acid and 1-chloro-2, 4-dinitrobenzene.

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 (attached below). The results can be seen in Table 3.

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: No activation of dendritic cells according to OECD 442E at any of the concentrations tested (RFI <150%)

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 at concentrations of 1.7-5.1 µg/mL

Remarks:

viability ≥50%

Results 3

Key result

Run / experiment:

other: Experiment 2

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: Activation of dendritic cells positive according to OECD 442E at concentrations of 0.8-1.2 µg/mL

Remarks:

viability ≥50%

Results 4

Key result

Run / experiment:

other: Experiment 2

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 - RFI >200% at 0.8-1.7 µg/mL and 2.5 µg/mL.

Remarks:

viability ≥50%

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 cytotoxicity assessment

Table 2: h-CLAT. Results of preliminary cytotoxicity assessment
Concentration (final test substance [µg/mL])	Concentration (test substance [µg/mL])	% PI negative cells Replicate 1	% P1 negative cells Replicate 2	Mean viability of duplicates	Relative viability mean [%]
VC	VC	95	95	95	100
3.9	4.4	82	76	79	83
7.8	8.8	51	53	52	55
16	18	34	32	33	35
31	35	-	-	-	-
63	71	-	-	-	-
125	142	-	-	-	-
250	283	-	-	-	-
500	566	-	-	-	-
1000	1133	-	-	-	-
2500	2831	-	-	-	-
5000	5663	-	-	-	-
- = Due to insolubility of the test substance, no measurement is available. The final test substance concentrations were calculated considering the purity/contents of 88.3%.

- A positive response (CD54 ≥ 200%) was observed in all non-cytotoxic concentrations (6 of 8 concentrations), but the relative viability at the lowest concentration was 73%.

- In order to confirm the results of the 1st experiment and to achieve a concentration with higher viability, another experiment was conducted using lower concentrations.

- The CV75 value was determined by linear regression from the concentration-response curve to be 5.1 µg/mL^3.

Main Experiments

Table 3: Summary of h-CLAT Main Experiments: mean values of RFI CD86, RFI CD54 and relative viability.
Experiment 1				Experiment 2
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 [%]
1.7	142	243	73	0.8	189	270	82
2.0	104	214	65	1.0	174	221	75
2.5	104	236	60	1.2	180	235	63
2.9	78	211	69	1.4	139	220	61
3.5	80	224	74	1.7	117	226	64
4.2	76	273	66	2.0	106	179	59
5.1	65	252	50	2.5	92	217	56
6.1	55	254	39	2.9	79	163	57
VC	100	100	100	VC	100	100	100
LA 1000 µg/mL	78	100	100	LA 1000 µg/mL	81	100	100
DNCB 4 µG/mL	292	1255	80	DNCB 4 µG/mL	317	1443	77
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

In Experiment 1, the h-CLAT CD86 met the ''borderline'' criteria, but the results of both h-CLAT experiments produced unambiguous positive results.

Experiment 1

Calculation of an EC150% (the concentration resulting in a RFI of 150% for CD86 was not applicable as positive criteria were not met and an EC200% (the concentration resulting in a RF1 of 200%) for CD54 was not applicable as fold inductions above 200% were observed at the lowest concentration.

Experiment 2

Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 and an EC200% (the concentration resulting in a RFI of 200%) for CD54 was not applicable as fold inductions above 200% were observed at the lowest concentration.

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, Basic Red 23 induces dendritic cell activation and is predicted to be a skin sensitiser.

Executive summary:

The skin sensitising potential of the test substance, Basic Red 29, was determined via the in vitro Human Cell Line Activation Test (h-CLAT) that evaluates that ability of Basic Red 23 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, Basic Red 23 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, a pre-test (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 was determined via linear regression from the concentration-response curve to be 5.1 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. At concentrations used in the main experiment the test substance was soluble in culture medium (2 x stock preparations and final concentrations) and no precipitates were observed at any concentration after 24 hours.

A concentration range of 1.7 -6.1 µg/mL was employed based on the pre-test. In Experiment 1, the calculation of an EC150% (the concentration resulting in a RFI of 150% for CD86 was not applicable as positive criteria were not met and an EC200% (the concentration resulting in a RF1 of 200%) for CD54 was not applicable as fold inductions above 200% were observed at the lowest concentration. In Experiment 2, the calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 and an EC200% (the concentration resulting in a RFI of 200%) for CD54 was not applicable as fold inductions above 200% were observed at the lowest concentration. In Experiment 1, the h-CLAT CD86 met the ''borderline'' criteria, but the results of both h-CLAT experiments produced unambiguous positive results. 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, Basic Red 23, 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 Basic Red 23 induces dendritic cell activation and is predicted to be a skin sensitiser.