Octasodium 7,7'-[(2,2'-disulphonato[1,1'-biphenyl]-4,4'-diyl)bis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino[2-(carbamoylamino)]-4,1-phenylene]azo]]bis(naphthalene-1,3,6-trisulphonate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From November 13 to 24, 2017

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

Version / remarks:

2016

GLP compliance:

yes

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Reactive Yellow 084

IUPAC Name:

Reactive Yellow 084

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

TEST SYSTEM
Specifications
Human monocytic leukemia cell line, THP-1 (an immortalised human monocytic leukemia cell line, used as a surrogate for dendritic cells), supplied by American Type Culture Collection (ATCC), Manassas, VA 20110 USA.

Identification
Test system was suitably labelled to clearly identify the study number, test article, test article concentration, positive and vehicle controls.

Cell culture maintenance
THP-1 cells were cultured, at 37 ºC under 5 % CO2 and humidified atmosphere, in RPMI0-1640 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 0.2E+06 cells/ml and maintained at a density from 0.1E+06 to 0.8E+06 cells/ml. Cell density did not exceed 1E+06 cells/ml.

Reactivity check
This was performed using DNCB, nickel sulphate and lactic acid 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 0.2E+06 cells/ml for 48 hours or at a density of 0.1E+06 cells/ml for 72 hours. On the days of testing, cells were harvested from the flasks and were resuspended with fresh culture medium at 2E+06 cells/ml. The cells were then distributed into a 96-well flat-bottom plate (80 µl/1.6E+05 cells per well).

STUDY DESIGN
Dose finding assay
The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24 hours at 37 °C, 5 % CO2. After the 24-hour incubation period, all cells from a 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 μl of phosphate buffered saline containing 0.1 % bovine serum albumin (FACS buffer) and re-suspended in 190 μl of FACS buffer. 10 μl of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 μg/ml).
PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10000 viable cells were acquired.
Cell viability was calculated using the following equation:
Cell Viability = number of living cells / total number of acquired cells × 100
The CV75 value, i.e. a concentration showing 75 % of THP-1 cell survival (25 % cytotoxicity), was calculated by log-linear interpolation using the following equation:
Log CV75 = (75 - c) × log (b) - (75-a) × log (d) / (a - c)
where:
a was the minimum value of cell viability over 75 % in testing groups
c was the maximum value of cell viability below 75 % in testing groups
b and d were the concentrations showing the value of cell viability a and c, respectively

CD86/CD54 expression measurement
One experiment (consisting of two independent runs) was needed to drive a prediction. Each independent run was performed on different days. On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2E+06 cells/ml. The cells were then distributed into a 24-well plate (500 μl/1E+06 cells per well).
The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. Plates were sealed and then incubated for 24 hours ± 20 minutes at 37 °C, 5 % CO2.
After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 ml of FACS buffer. After washing, the cells were blocked with 600 μl of blocking solution (FACS buffer containing 0.01 % (w/v) globulin) at 4 ºC for 15 minutes.
After blocking, the cells were split into three aliquots of 180 μl into a 96-well plate and centrifuged (ca. 250 g, 3 minutes).
After centrifugation, the cells were stained with 50 μl of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4 ºC for 30 minutes.
The stained cells were washed three times with an excess of FACS buffer, resuspended in FACS buffer and 12.5 μg/ml PI solution was added (to give a final PI concentration of 0.625 μg/ml).
The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Results and discussion

Positive control results:

RFI values were ≥ 150 % for CD86 and ≥ 200 % for CD54 in all each independent runs. Cell viability was > 50 % in each independent run.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: exp.1 and 2

Parameter:

other: RFI CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

other: exp.1 and 2

Parameter:

other: RFI CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Cell viabilities of medium and solvent control were higher than 90 % in each independent run.
In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %).
For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control was > 105 % on all occasions.
For the positive control (DNCB), RFI values were ≥ 150 % for CD86 and ≥ 200 % for CD54 in all each independent runs. Cell viability was > 50 % in each independent run.
For test substance, cell viability was more than 50 % in all tested concentrations in each independent run.

Any other information on results incl. tables

Dose range finding assay

No toxicity was noted at the maximum concentration, thus CV75 value for test article was > 1000 µg/ml.

CD86 / CD54 expression results

conc. µg/ml	RFI (CD86)		RFI (CD54)
	exp. 1	exp. 2	exp. 1	exp. 2
279.08	25	28	102	97
334.90	17	33	96	112
401.88	18	27	79	95
482.25	21	33	81	90
578.70	22	35	95	85
694.44	16	30	81	86
833.33	18	31	82	89
1000.0	21	32	85	97
solvent control	100	100	100	100
positive control	246	401	601	900

Applicant's summary and conclusion

Interpretation of results:

other: study is part of a global assessment on skin sensitisation

Conclusions:

Positive in human Cell Line Activation Test due to:
RFI (CD86) < 150 % in exp. 1 and 2
RFI (CD54) < 200 % in exp. 1 and 2

Executive summary:

Method

Human Cell Line Activation Test (hCLAT) was conducted according to OECD guideline 442E.

Test substance was dissolved in saline and a dose finding assay was conducted to determine a concentration showing 75 % THP-1 cell survival (CV75).

Test substance was dissolved at 100 mg/ml in saline, then 8 stock solutions of each test article were prepared by 1.2 -fold serial dilutions using saline.

Aliquots of 500 μl of each working solutions were mixed 1:1 with cell suspensions at 1.0E+06 cells per well. Each plate was sealed using a plate sealer and then incubated at 37±1 °C, 5 % (v/v) CO₂ in air, in a humidified environment for 24 ± 0.5 hours.

After the 24-hour incubation period, cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 μl of blocking solution (FACS buffer containing 0.01 % w/v globulin) at 4 °C for 15 minutes.

After blocking, the cells were split into three aliquots of 180 μl into a 96-well plate (or sample tubes), centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4 °C for 30 minutes.

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

For dose finding assay, test substance was dissolved in saline at a concentration of 100 mg/ml (orange/brown suspension) giving a maximum test concentration of 1000 µg/ml. No effect on viability was noted in the dose finding assay, thus final test concentrations in a range from 279 to 1000 µg/ml after dilution in medium were used for expression measurements.

Results

RFI values < 150 % for CD86 and < 200 % for CD54 were found in both experiment 1 and 2. Accordingly, test substance was considered as negative in the hCLAT.