Disodium naphthalene-1,5-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of February 2015

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Disodium naphthalene-1,5-disulphonate

EC Number:

216-732-0

EC Name:

Disodium naphthalene-1,5-disulphonate

Cas Number:

1655-29-4

Molecular formula:

C10H8O6S2.2Na

IUPAC Name:

disodium naphthalene-1,5-disulphonate

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

dihydrogen oxide

Test material form:

solid: crystalline

Details on test material:

Test item: Armstrongsäure-di-Natriumsalz Monohydrat
CAS name: 1,5-Naphthalenedisulfonic acid, sodium salt (1 :2) Monohydrat
CAS number: 207569-02-6
Empirical formula: C10 H6 O6 S2 * 2Na * H2O
Molecular mass: 350.3 g/mol
Batch number: CHNHFC1602
Content monohydrat-form: 99.8 %
Corresponds to 94,7 % anhydrous form, CAS: 1655-29-4

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CHNHFC1602
Calculations of all test article concentrations stated in this report include a correction for content (purity) using a factor of 1.056.

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The study was conducted to investigate the potential of the test item 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 ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

Specifications:
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland as specified in OECD Test Guideline 442D (batch number 42, passage 12 for Experiment 1 and batch number 45, passage 12 for Experiment 2).

Preparation of Cultures:
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing 9% foetal bovine serum and 1% Geneticin.

Treatment:
In 96-well plates, incubated at 37±1°C, 5% (v/v) CO2, for 48±1 hours in medium with serum but without Geneticin. 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.

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.

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.

Positive controls:
Cinnamic aldehyde (CAS No. 14371-10-9), supplied by Sigma Aldrich Chemical Co. Ltd. was used as the positive control.

Negative controls:
The negative control was diluted into culture medium containing serum so that the final concentration was 1%. An aqueous solvent was used, therefore DMSO was added to the treatment solutions at 1% (final concentration).

Results and discussion

Positive control results:

The assay aceptance criteria for the positive controls were met: 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 Experiments 1 and 2.
The EC1.5 values for the positive control were 8.51 and 8.50 µM in Experiments 1 and 2, respectively. The average induction for the positive control at 64 µM was 4.97 in Experiment 1 and 17.47 in Experiment 2. Although the latter value was above the range specified in the protocol, the assay was considered valid as there was a clear dose response with increasing luciferase activity induction.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: experiment 1

Parameter:

other: Imax

Value:

1.19

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: experiment 2

Parameter:

other: Imax

Value:

1.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

other: experiment 1 and 2

Parameter:

other: EC1.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

no statistically significant increases

Results 4

Run / experiment:

other: experiment 1 and 2

Parameter:

other: dose response for luciferase induction

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

no apparent overall dose response observed

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: proven

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 9.44% and 15.84% in Experiments 1 and 2, respectively.

Any other information on results incl. tables

All assay acceptance criteria were met.

Since the Imax in both experiments was less than 1.5-fold, the test article was considered to be negative in the ARE-Nrf2 Luciferase Test.

Applicant's summary and conclusion

Executive summary:

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed acording to OECD 442D. The test article was dissolved in water to the final concentration of the stock solution (200 mM) with further dilution in water and cell culture medium to obtain final concentrations of 0.98 to 2000 µM. The assay acceptance criteria for the positive control cinnamic aldehyde and the negative control DMSO were met in this test. Treatment of the cell for 48 hours with the test item led to the following results: The maximal average fold increases (Imax) were 1.19 and 1.10 for experiments 1 and 2, respectively. There were no EC1.5 values for Experiments 1 or 2 determinable as there were no statistically significant increases in induction. In Experiments 1 and 2, there was no apparent overall dose response for luciferase and the dose response curves were not biphasic.

All assay acceptance criteria were met. Since the Imax in both experiments was less than 1.5-fold, the test article was considered to be negative in the ARE-Nrf2 Luciferase Test.