Tetrasodium 3,3'-[carbonylbis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

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:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Deviations:

no

Principles of method if other than guideline:

OECD Guidelines for Testing of Chemicals Method 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

Test material

Test material information

1

Reference substance name:

Tetrasodium 3,3'-[carbonylbis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

EC Number:

221-728-7

EC Name:

Tetrasodium 3,3'-[carbonylbis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

Cas Number:

3214-47-9

Molecular formula:

C35H28N6O13S4.4Na

IUPAC Name:

tetrasodium 3,3'-[carbonylbis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

Dose Finding Assay
A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control.
The test article was dissolved at 250 mg/mL in DMSO then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent. The stock solutions were then further diluted 250-fold in culture medium (working solutions). The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to obtain a final range of concentrations in the plate of 3.9 - 500 µg/mL.
The test article was prepared shortly before testing. Preparation was conducted under subdued lighting with the aid of vortex mixing and ultrasonication for approximately 30 minutes.


CD86/CD54 Expression Measurement
As there was no effect on viability in the dose finding assay and no CV75 was obtained, the maximum attainable concentration of 250 mg/mL was used for the expression measurement. Eight stock solutions of test article were prepared by 1.2 fold serial dilutions to give eight concentrations, and the stock solutions were then further diluted 250-fold into the culture medium (working solutions). The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate. Final test concentrations in a range from 139.54 to 500.00 µg/mL (after dilution in medium) were used for the expression measurements.
The test article was prepared shortly before testing. Preparation was conducted under subdued lighting with the aid of vortex mixing, ultrasonication for approximately 5 minutes and warming at 37°C.


Cell Culture Maintenance
THP-1 cells were cultured, 37±1ºC under 5±1% CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin.
The cells were passaged every 2-3 days at a density of 0.1 to 0.2 x 106 cells/mL and maintained at a density from 0.1 x 106 to 0.8 x 106 cells/mL. Cell density did not exceed 1 x 106 cells/mL.

Reactivity Check
This was performed using DNCB (CAS no. 97-00-7, ≥99% purity), nickel sulphate (CAS no. 10101-97-0, 99% purity) and lactic acid (CAS no. 50-21-5, 85% purity) two weeks after thawing.
DNCB and nickel sulphate should produce a positive response of both CD86 and CD54 and lactic acid should produce a negative response of both CD86 and CD54. Only cells which passed the reactivity check were used for the assay.

Plate Preparation
THP-1 cells were pre-cultured in culture flasks either at a density of 0.2 x 106 cells/mL for 48 hours or at a density of 0.1 x 106 cells/ml for 72 hours. On the days of testing, cells were harvested from the flasks and were re-suspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 24 well flat-bottomed plate (500 µL/well) (expression assay) or a 96-well flat-bottomed plate (80 µL/1.6 x 105 cells per well) (DRF assay(s)).

Results and discussion

Positive control results:

The positive control was 2,4-dinitrochlorobenzene (DNCB) prepared in DMSO (2 mg/mL stock), diluted in culture medium to obtain a working solution of 8 µg/mL, and final treatment concentration of 4 µg/mL in the plate.

For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.

In vitro / in chemico

Results

Key result

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Analysis of Results
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for the positive control cells and test article-treated cells were calculated according to the following equation:
RFI = MFI of chemical-treated cells – MFI of chemical-treated isotype control cells x100
MFI of solvent/vehicle-treated cells – MFI of solvent/vehicle-treated isotype control cells

The cell viability from the isotype control cells (stained with mouse IgG1 antibodies) was calculated using the following equation:
Cell Viability = number of living cells x100
total number of acquired cells

Prediction Model
An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h CLAT prediction is considered NEGATIVE:
• The RFI of CD86 is ≥150% at any tested concentration (with cell viability ≥50%)
• The RFI of CD54 is ≥200% at any tested concentration (with cell viability ≥50%).

Calculation of Effective Concentration (EC) Values
For test articles predicted as POSITIVE, two EC values, the EC150 for CD86 and the EC200 for CD54, are calculated as follows:
EC150 (for CD86) = Bconcentration + [(150 – BRFI) / (ARFI – BRFI) x (Aconcentration – Bconcentration)]
EC200 (for CD54) = Bconcentration + [(200 – BRFI) / (ARFI – BRFI) x (Aconcentration– Bconcentration)]
Where:
Aconcentration is the lowest concentration in µg/mL with RFI >150 (CD86) or 200 (CD54)
Bconcentration is the highest concentration in µg/mL with RFI <150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI >150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI <150 (CD86) or 200 (CD54).

Assay Acceptance Criteria
• The cell viabilities of medium and solvent control should be higher than 90%
• In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%)
• For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability should be more than 50%
• For the test article, the cell viability should be more than 50% in at least four tested doses in each run.

Negative Results
Negative results are acceptable only for test articles exhibiting a cell viability of less than 90% at 1.2 x CV75 (highest concentration). If the cell viability at 1.2 x CV75 is equal to or greater than 90% the negative results should be discarded and the dose selection should be refined by repeating the CV75 determination.
When 5000 µg/mL in saline (or medium), 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of the test article, a negative result is acceptable even if the cell viability is above 90%.

Any other information on results incl. tables

Dose Finding Assay

No effect on viability was noted, therefore no CV75 value could be calculated.

 CD86/CD54 Expression Results

In both experiments the RFI values for CD86 were <150% and the RFI values for CD54 were <200% at all concentrations. The test article therefore gave a negative prediction in the assay.

At concentrations of 241.13 and 347.22µg/mL there were unusually low RFI CD86 and RFI CD54 values as a result of high isotype MFI values (Experiment 2). This was considered not to have affected the outcome of the study, as there were clearly negative results at all other concentrations.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test article was considered to be negative in the human Cell Line Activation Test, when tested up to the maximum attainable concentration.

Executive summary:

The study was conducted to investigate the potential of the test item to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data 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.

The human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E.

For the dose finding assay, the test article was dissolved inanhydrous analytical grade dimethyl sulfoxide (DMSO)at the maximum attainable concentration of 250 mg/mL giving a maximum test concentration of 500 µg/mL. No reduction in viability was observed.

For the expression measurements, test concentrations in a range from 139.54 to 500.00 μg/mL (after dilution in medium) were used.

Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 10⁶cells per well.

After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes.

The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

In both Experiments the RFI values for CD86 were <150% and the RFI values for CD54 were <200% at all concentrations. The test article therefore gave a negative prediction in the assay.

All acceptance criteria of the h-CLAT assay parameters were met in each experiment.

The test article was considered to be negative in the human Cell Line Activation Test, when tested up to the maximum attainable concentration.