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Diss Factsheets

Administrative data

Description of key information

The test article, Bayscript Magenta BB, was considered to be negative in the Luciferase Test ARE-Nrf2 cells derived from HaCaT human keratinocytes. In the DPRA test the cysteine 1:10 prediction model was applied to the test item. A depletion of cysteine peptide became obvious in the DPRA (100%). According to the cysteine 1:10 prediction model “high reactivity” was derived for the test item in water, leading to a DPRA prediction of “positive“.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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).

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.
Run / experiment:
other: experiment 1
Parameter:
other: Imax
Value:
1.2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: experiment 2
Parameter:
other: Imax
Value:
3.94
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: experiment 1 and 3
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
Run / experiment:
other: experiment 1 and 3
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
Run / experiment:
other: 2
Parameter:
other: EC1.5
Value:
8.66
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 3
Parameter:
other: Imax
Value:
2.09
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Not statistically significant
Run / experiment:
other: experiment 2
Parameter:
other: dose response for luciferase induction
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Yes until toxic concentrations reached
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 10.32%, 11.85% and 7.82% in Experiments 1, 2 and 3, respectively.

All acceptance criteria were met in each experiment, with the exception that in Experiment 1 the EC1.5 value for the positive control was 3.25 and the average induction at 64 μM was 8.25. However, the assay was considered valid as there was a clear dose response with increasing luciferase activity induction with the positive control.

Executive summary:

The study was conducted to investigate the potential of Bayscript Magenta BB to induce genes that are regulated by the antioxidant response element (ARE).

The test article was dissolved in dimethyl sulfoxide (DMSO) to the final concentration of the stock solution (200 mM). Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM).

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 romide (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) 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.

The acceptance criteria were met in each experiment, with the exception that in Experiment 1 the EC1.5 for the positive control was 3.25 and the average induction for the positive control at 64 μM was 8.25. However, the assay was considered valid as there was a clear dose response with increasing luciferase activity induction with the positive control.

None of the four criteria specified in the prediction model for a positive prediction (Imax > 1.5-fold, cell viability > 70%, EC1.5 value < 1000 μM, dose response) were met in Experiment 1, whereas all four criteria were met in Experiment 2 and only one was met in Experiment 3. It is therefore considered that the test article is predicted as negative in the assay based on 2 out of the 3 experiments giving negative results.

The test article, Bayscript Magenta BB, was considered to be negative in the Luciferase Test ARE-Nrf2 cells derived from HaCaT human keratinocytes.

Justification for classification or non-classification

Based on the positive result in the in vitro DPRA test and the negative result in the in vitro Keratino-Sens(TM) test a third in vitro study, the h-Clat (OECD TG 442E), was initiated and will be reported in a dossier update. Based on the result of this in vitro test additional in vivo testing might be initiated to gain information on potency of potential in vivo sensititization activities. No information on skin sensitization potency can be derived from skin sensitization hazard reported in in vitro studies.