Reaction products of diazotised 2-amino-4,6-dinitrophenol coupled with 4-Naphthalenesulfonic acid, 2-hydroxy-6-[(sulfomethyl)amino]-, monosodium salt, chelated with chromium (3+), sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 442E (In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

GLP compliance:

yes

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Acid Black 071 - Similar Substance 01

IUPAC Name:

Acid Black 071 - Similar Substance 01

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

Test System
1. 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.
2. Identification
The test system was suitably labelled to clearly identify the study number, test article, test article concentration, positive and vehicle controls.
3. 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 0.2 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.
4. 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.
5. 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 resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 96-well flat-bottomed plate (80 μL/1.6 x 105 cells per well).

Study Design
1. 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 90 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 10,000 viable cells were acquired.

CD86/CD54 Expression Measurement
One experiment (consisting of three independent runs) was needed to drive a prediction. The positive control data from the initial second run failed to meet the acceptance criteria. The data was invalidated and a repeat run conducted. Each independent run was performed on a different day.
On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 24-well plate (500 μL/1 x 106 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. The plates were sealed and then incubated for 24 hours 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 (approximately 250 g, 5 minutes).
After centrifugation, the cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4ºC for 30 minutes.
The stained cells were washed three times with an excess of FACS buffer, re-suspended 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.

Test Article Formulation
1. Dose Finding Assay
The test article was dissolved at 100 mg/mL or 500 mg/mL in saline, then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent.
The stock solutions were then further diluted 50-fold in culture medium (working solutions).
2. CD86/CD54 Expression Measurement
The test article was dissolved at 500 mg/mL in saline, then eight stock solutions of each test article were prepared by 1.2-fold serial dilutions using saline. The stock solutions were then further diluted 50-fold into the culture medium (working solutions).

Results and discussion

In vitro / in chemico

Results

Results 1

Parameter:

other: RFI for CD86

Value:

150

Remarks on result:

other: in all independent runs

Results 2

Parameter:

other: RFI for CD54

Value:

200

Remarks on result:

other: in 2 out of 3 independent runs

Results 3

Parameter:

other: Cell viability

Value:

90

Remarks on result:

other: in all 3 independant runs

Applicant's summary and conclusion

Interpretation of results:

other: Not skin sensitising

Remarks:

according to the criteria described in the OECD guideline 442E

Conclusions:

Not skin sensitising

Executive summary:

Method

The study was conducted to investigate the potential of the test substance 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 test article was dissolved in saline and a dose finding assay was conducted to determine a concentration showing 75 % THP-1 cell survival (CV75).

The test article was dissolved at 500 mg/mL in saline, then eight stock solutions of each test article were prepared by 1.2-fold serial dilutions using saline. The stock solutions were then further diluted 50-fold into the culture medium (working solutions).

Aliquots of 500 μL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 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, the 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, 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 solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Results

No toxicity was noted in the dose finding assay at the maximum concentration, therefore the CV75 value for the test article was >5000 μg/mL.

In the main study, the RFI for CD86 was <150 % in all 3 independent runs, the RFI for CD54 was < 200% in 2 out of 3 independent runs and cell viability was > 90 % in all 3 independent runs. All assay acceptance criteria were met.

The test article was considered to be negative in the human Cell Line Activation Test.