Reaction products of 6-amino-4-naphthol-2-sulfonic acid with formaldehyde and sodium metabisulfite, coupled with diazotised 5-amino-2,4-dinitrophenol, 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:

2018

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:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Acid Black 071:2 - Similar Substance 02

IUPAC Name:

Acid Black 071:2 - Similar Substance 02

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design
1. Treatment Plate Preparation
The cells were 80 - 90 % confluent. On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated at 37 ± 1 °C, 5 % (v/v) CO2, for 24 ± 1 hours.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.
2. Treatment
At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added.
One well per plate was left empty (no cells and no treatment) to assess background values.
Aliquots of 50 µL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed and incubated at 37 ± 1°C, 5 % (v/v) CO2 in air, in a humidified environment for 48 ± 1 hours.
For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration.
Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from the same passage.
3. Cytotoxicity Assessment
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).The plate was sealed and incubated for 4 hours at 37 ± 1 °C, 5 % (v/v) CO2.
The MTT medium was removed and SDS (at 10 % w/v) added per well. The plate was sealed and placed into an incubator at 37 ± 1 °C, 5 % (v/v) CO2 in air and left overnight.
After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.
4. Luciferase Activity Measurements
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25 ± 2°C, loaded into the luminescence plate reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Experment 1

Parameter:

other: I max

Value:

1.03

Results 2

Run / experiment:

other: Experiment 2

Parameter:

other: I max

Value:

1.27

Any other information on results incl. tables

Viability

The cell viability measurement was not applicable as there were no EC_1.5 determining concentrations in Experiments 1 or 2.

The IC₅₀values could not be calculated for Experiments 1 and 2 as the minimum viability did not drop below 50 %.

The IC₃₀value was 389.84µg/mL in Experiment 1. This value could not be calculated for Experiment 2 as all viability results were above 70 %.

Assay Acceptance

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 µM in Experiment 1 and at concentrations of 32 to 64 µM in Experiment 2.

The EC_1.5values for the positive control were 8.08 and 18.37 µM in Experiments 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM were 16.90 and 3.30 in Experiments 1 and 2, respectively.

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 8.20 % and 5.68 % in Experiments 1 and 2, respectively.

All acceptance criteria were met, with the exception that in Experiment 1, the EC_1.5for the positive control was above the range specified in the protocol at 16.90. However, luciferase induction values showed a clear dose response, therefore we consider that this was a valid experiment.

The assay was considered valid as the results in Experiment 1 were replicated in Experiment 2.

Applicant's summary and conclusion

Interpretation of results:

other: Not skin sensitizer

Remarks:

according to the criteria described in the OECD guideline 442D

Conclusions:

Not skin sensitizer

Executive summary:

Method

The study was conducted to investigate the potential of the test substance to induce genes that are regulated by the antioxidant response element (ARE). 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 test article was dissolved in dimethyl sulfoxide (DMSO) to the final stock concentration (40 mg/mL). Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article (0.020, 0.039, 0.078, 0.156, 0.313, 0.63, 1.25, 2.5, 5, 10, 20 and 40 mg/mL).

The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations ranged from 0.2 to 400 µg/mL.

Aliquots of 50 µL of each of the final concentrationswere transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37 ± 1 °C, 5 % (v/v) CO₂in air, in a humidified environmentfor 48 ± 1 hours.

After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The plate was sealed and incubated for 4 hours at 37 ± 1 °C, 5 % (v/v) CO₂. The MTT medium was then removed and SDS (at 10 % w/v) added per well. The plate was sealed and placed into an incubator at 37 ± 1 °C, 5 % (v/v) CO₂in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25 ± 2 °C, loaded into the luminescence plate reader and read.

Results

Criteria	Experiment 1	Experiment 2
Imax	1.03	1.27
Cell Viability	Not applicable	Not applicable
EC1.5	Not calculated	Not calculated
Dose Response	No	No

All acceptance criteria were met, with the exception that in Experiment 1, the EC_1.5for the positive control was above the range specified in the protocol at 16.90. However, luciferase induction values showed a clear dose response, therefore we consider that this was a valid experiment.

As the I_max for the test article was below 1.5-fold in each experiment, The test article was considered to be negative in the ARE-Nrf2 Luciferase Test.