[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2017 - 01 December 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:

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

Version / remarks:

Adopted February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

batch number: 616F-2072 (NBK-003072-796)

In vitro test system

Details on the study design:

MATERIALS AND METHODS
- Vehicle of the test item: Dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).
- Negative control: vehicle of the test item
- Positive control: Ethylene dimethacrylate glycol, cas n 97-90-5
- blank: no cells and no treatment
- Item solutions preparations: the test substance was first dissolved in DMSO at 10 mg/mL concentration. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay
- tested concentrations: 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.098 and 0.049 µg/mL (final concentration DMSO of 1%). All formulations were clear soilutions
- number of tests: all concentrations were tested in triplicate

PARAMETERS
- Imax = The maximal average fold induction of luciferase activity value observed at any concentration of the tested chemical and positive control
- EC1.5 = the value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold
- IC30 = concentration values for 30% reduction of cellular viability.
- IC50 = concentration values for 50% reduction of cellular viability

TEST SYSTEM
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

SUBCULTURING
Cells were subcultured upon reaching 80-90% confluency.

ENVIROMENTAL CONDITIONS
- humid atmosphere: 80 - 100% (actual range 72 – 100 %)
- CO2: 5.0 ± 0.5% CO2 in air
- light conditions: dark
- temperature: 37.0 ± 1.0°C (actual range 36.0 – 37.0°C).
Temperature and humidity were continuously monitored throughout the experiment.
The CO2 percentage was monitored once on each working day.

EXPERIMENTAL DESIGN
Plating of Cells:
- For testing, cells were 80-90% confluent.
- One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium.
- For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay.
- The cells were incubated overnight in the incubator.
- The passage number used was p+8 in experiment 1 and p+6 in experiment 2.
Treatment of Cells:
- The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added.
- The treated plates were then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2.
Luciferase Activity Measurement
- The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together.
- The assay plates were removed from the incubator and the medium is removed.
- Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well.
- The plates were shaken for at least 3 minutes at room temperature.
- Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
Cytotoxicity Assessment
- For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2.
- The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well.
- After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

NUMBER OF EXPERIMENTS
- Initially, experiment 2 did not pass all the acceptability criteria and therefore this part of the study was repeated. In total 2 valid experiments were performed

DEVIATIONS
Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Results and discussion

Positive control results:

The positive control was Ethylene dimethacrylate glycol.
Two indipendent experiments were performed.

- The luciferase activity induction obtained with the positive control was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 µM (43 µM and 28 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.18-fold and 4.41-fold in experiment 1 and 2, respectively).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: first experiment

Parameter:

other: I max

Remarks:

Maximum Luciferase Activity Induction

Value:

1.48

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: inconclusive

Remarks:

Imax experiment 1 = 1.48 fold

Results 2

Key result

Run / experiment:

other: In both independent experiments

Parameter:

other: induction of luciferase acticity

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: inconclusive

Remarks:

No EC1.5 value was measured at any of the concentrations in both experiments

Results 3

Key result

Run / experiment:

other: first experiment

Parameter:

other: IC30

Remarks:

value in µg/mL

Value:

9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item showed toxicity

Results 4

Key result

Run / experiment:

other: second experiment

Parameter:

other: IC30

Remarks:

value in µg/mL

Value:

10

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item showed toxicity

Results 5

Key result

Run / experiment:

other: first experiment

Parameter:

other: IC50

Remarks:

value in µg/mL

Value:

10

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item showed toxicity

Results 6

Key result

Run / experiment:

other: second experiment

Parameter:

other: IC50

Remarks:

value in µg/mL

Value:

11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item showed toxicity

Results 7

Key result

Run / experiment:

other: second experiment

Parameter:

other: Imax

Remarks:

Maximum Luciferase Activity Induction

Value:

1.35

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: inconclusive

Remarks:

Imax experiment 2 = 1.35 fold

Applicant's summary and conclusion

Interpretation of results:

other: inconclusive

Conclusions:

Under the applied experimental conditions, the test substance is classified as inconclusive in the KeratinoSensTMassay since negative results (<1.5-fold induction) were observed at test concentrations < 200µg/mL.

Executive summary:

The ability of the test substance to induce skin sensitization was evaluated according to the OECD guideline 442D (In Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method), adopted in February 2015

Two independent experiments were performed. In these experiments, the cells were incubated for 48 hours with the following concentrations of the test item: 0.049; 0.098; 0.20; 0.39, 0.78; 1.6; 3.1; 6.3; 13; 25; 50; 100 µg/mL. The esperiments were performed in triplicate

The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

In both the experiments:

- the test item showed toxicity (IC₃₀values of 9.0 µg/mLand 10 µg/mL and IC₅₀values of 10 µg/mL and 11 µg/mL)

- No biologically relevant induction of the luciferase activity (no EC_1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 1.48-fold and 1.35-fold

The test item is classified as inconclusive in the KeratinoSens^TMassay since negative results (<1.5-fold induction) were observed at tested concentrations under the applied experimental conditions.