[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-03-13 to 2019-05-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

Adopted 06-2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

The KeratinoSens test (ARE-Nrf2 luciferase reporter assay) is part of an in vitro/in chemico testing strategy (OECD guidelines 442C-E) addressing The second Adverse Outcome Pathway Key Event of inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways. Mechanistically-based in chemico and in vitro test methods addressing the first three key events of the skin sensitisation AOP have been adopted for contributing to the evaluation of the skin sensitisation hazard potential of chemicals.

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of diethyl (E)-2-methylbut-2-enedioate, diethyl (Z)-2-methylbut-2-enedioate and diethyl 2-methylenebutanedioate

Cas Number:

2245967-74-0

Molecular formula:

C9H14O4

IUPAC Name:

reaction mass of diethyl (E)-2-methylbut-2-enedioate, diethyl (Z)-2-methylbut-2-enedioate and diethyl 2-methylenebutanedioate

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item was dissolved in DMSO to a final concentration of 200 mM
- Preparation of the test chemical serial dilutions: From the 200 mM stock solution 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 with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%).
- Preparation of the positive controls: Ethylene dimethacrylate glycol was used as positive control. A 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted in exposure medium so that the final concentration of the positive control ranged from 7.8 to 250 μM (final concentration DMSO of 1%).
- Preparation of the vehicle control: The vehicle control was 1% DMSO in exposure medium.
- Stable dispersion obtained: Yes, no precipitation occured.

DOSE RANGE FINDING ASSAY:
- Solubility in solvents: The test item was dissolved in DMSO to a final concentration of 200 mM (clear colorless solution).
- Solubility in incubation medium: The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax formed a homogeneous solution (no precipitation).
- Cytotoxicity assessment performed: No.
- Final concentration range selected on basis of: 2000 µM were selected as highest concentration for the main assay based on the highest dose required in the current guideline.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: 3
- Test chemical concentrations: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM
- Application procedure: 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. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0°C in the presence of 5% CO2.
- Exposure time: 48 hours ± 1
- Study evaluation and decision criteria used: See "Any other information on material and methods incl. tables" and "Attached background material"
- Description on study acceptance criteria: See "Acceptance criteria" in "Any other information on material and methods incl. tables"

SEEDING AND INCUBATION
- Seeding conditions: 10,000 cells/well, passage 6 (experiment 1), passage 8 (experiment 2), passage 10 (experiment 3)
- Incubation conditions: At 37±1.0°C in the presence of 5% CO2
- Precipitation noted: No

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer: TECAN Infinite® M200 Pro Plate Reader to (integration time two seconds).
- Plate used: 96-well plates, manufacturer or wells type not indicated
- Lysate preparation: By addition of Steady-Glo Luciferase substrate solution (prior to addition, it was mixed 1:1 with exposure medium)

DATA EVALUATION
- 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 and cells were incubated for 3 - 4 hours at 37°C± 1.0°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.
- Prediction model used: See "Data interpretation" in "Any other information on material and methods incl. tables".

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

EGDMA (120 M) [442D]

Results and discussion

Positive control results:

The fold luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was 2.49 (experiment 1), 3.24 (experiment 2) and 2.49 (experiment 3)

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Value:

947 %

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

128 %

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

723 %

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

13 µM

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 5

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 6

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

21 µM

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not indicated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.

Applicant's summary and conclusion

Interpretation of results:

other: Activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes

Conclusions:

In conclusion, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this GLP compliant study according to OECD guideline 442D was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The study procedures described in this report were based on the most recent OECD guideline. The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed.

All experiments passed the acceptance criteria:

• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.


• The EC1.5 of the positive control was within two standard deviations of the historical mean (41, 31 and 50 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.49-fold, 3.24-fold and 2.49-fold in experiment 1, 2 and 3, respectively).


• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (12%, 12% and 9.6% in experiment 1, 2 and 3, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. In the first experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 21 μM) was measured. The maximum luciferase activity induction (Imax) was 7.23-fold leading to an individual run conclusion of positive.

In the second experiment, the test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.28-fold leading to an individual run conclusion of negative.

Since the first two experiments were not concordant a third experiment was performed to provide a final conclusion.

In the third experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 13 μM) was measured. The maximum luciferase activity induction (Imax) was 9.47-fold leading to an individual run conclusion of positive.

The test item is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments. In conclusion, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.