[2-[4-(dihexylamino)-2-methylbenzylidene]benzo[b]thien-3(2H)-ylidene]malononitrile S,S-dioxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

other company data

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)

Deviations:

yes

Remarks:

The 2-mercaptoethanol was not used in the culture medium because this toxic compound is not necessary for the culture of THP-1

GLP compliance:

yes

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Disperse Blue 354

IUPAC Name:

Disperse Blue 354

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

DMSO (Dimethysulfoxide) was used as an intermediate solvent.
Concentration range: 3.91 to 500 µg/ml

METHODS
Preliminary study: Cytotoxicity assays
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 (Fb). The cells were stained with 7-AAD (5 µg/mL final concentration). Then cells were analysed with flow cytometry using GUAVA (Merck Millipore, France) and lnCyte software to measure cell viability. The living cells (7-AAD) gate was set in the 7-AAD negative area. 104 7-AAD- cells were counted as the living population.
According to the results the dose levels for the main study were selected.

Main study: Activation test
Based on the cytotoxicity assay the eight final test item concentrations were selected. In 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. The range of concentrations has been adjusted for activation test 2 and 3 (if necessary) depending on results obtained in previous experiments, to calculate the minimum induction threshold. Each experiment of activation test was performed on eight concentrations.
THP-1 cells were plated at 1*106 cells/mL/well in 24 well plates and treated for 24 ± 0.5 hours with selected test item concentrations. After treatment cells were washed twice with Fb. 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/3x10‸5 cells /50 µL for the anti-human CD86 mAb, and 3 µL/3x105 cells /50 µL for the anti—human CD54 mAb. FITC labelled-mouse IgG1 was used as an isotype control at a dilution of 3 µL/3x10‸5 cells /50 µL. Then, the cells were stained also with 7—AAD for 30 min at 4°C. After washing and resuspension with Fb, the fluorescence intensities of the THP-1 cell surface markers were then analysed by flow cytometry using GUAVA (Merck Millipore, France) and lnCyte software, on 10000 living cells.

Validation 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 (OECD442E, 2016).
This study is considered valid if the following criteria are fully met:
- In the positive control (DNCB):
RFI values of both CD86 and CD54 should be over the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
The cell viability Should be more than 50%
- In the vehicle control (medium, 0.9% NaCl, DMSO, Ethanol etc):
Cell viability Should be more than 90%
In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) compared to the medium 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

Run / experiment:

other: mean of two runs

Parameter:

other: Minimum induction threshold (MIT)

Value:

79.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

	Concentration range*	Viability	CD54	CD86	MIT (μg/ml)
Run 1	3.91 to 500 μg/ml	> 50 %	RFI > 200	RFI < 150	79.6
Run 2		> 50 %	RFI > 200	RFI < 150

Applicant's summary and conclusion

Interpretation of results:

other: Skin sensitizer

Remarks:

according to the criteria set up on the OECD guideline 442E

Conclusions:

Skin sensitising

Executive summary:

The test substance was evaluated for its potential to induce skin sensitisation according to the procedure described in the OECD gudeline 442E.

No cytotoxicity was induced on THP-1 cells by the test item. In the assay conditions, a reproducible "increase" of the CD54 expression compared with the negative control at least for two dose-levels of Disperse Blue 354 was noticed.

In the first experiment, a dose-response relationship was noticed only for CD54 marker with an increase of 2.01 to 14.5 fold of expression compared to the negative control.

In the second experiment, a dose-response relationship was also observed for the CD54 marker with an increase of 6.12 to 7.77 fold of expression compared to the negative control. A Slight increase of expression for CD86 marker (fold increase 1.58) was only observed for one close, but without no-dose effect.

Conclusion

Based on these results, the test item demonstrated an in vitro sensitizing potential with a Minimum Induction Threshold (MIT) of 79.6 µg/mL (in active substance) in condition of the experimental human Cell Line Activation Test, during this study.