Indene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 Jul 2018 - 10 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The existing knowledge of the chemical and biological mechanisms associated with skin sensitisation has been summarized in the form of an Adverse Outcome Pathway (AOP). The second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways. The KeratinoSensTM test (an ARE-Nrf2 luciferase reporter assay) is performed to address this second key event. This test is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in vitro test to be performed as part of the testing strategy for the skin sensitisation endpoint.

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:

Feb 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Indene

EC Number:

202-393-6

EC Name:

Indene

Cas Number:

95-13-6

Molecular formula:

C9H8

IUPAC Name:

indene

Test material form:

liquid

Details on test material:

Identification: Indene

In vitro test system

Details on the study design:

DOSE FORMULATION
- A correction factor according to correct for the purity (96.6%) was applied in this study.
- A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM. This concentration was selected as the highest concentration for the main assay and it is the highest dose required in the current guideline.

MAIN EXPERIMENT
- The test item was dissolved in dimethyl sulfoxide (DMSO) at 200mM. 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 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%). All concentrations of the test item were tested in triplicate.
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- Test item concentrations were used within 2.5 hours after preparation.

CONTROL ITEMS
- Vehicle: dimethyl sulfoxide (DMSO)
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

- Positive control: Ethylene dimethacrylate glycol
Preparation of the positive control: 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO. The final concentration ranges from 7.8 to 250 μM (final concentration DMSO of 1%).
All concentrations of the positive control were tested in triplicate.

- Blank control: On each plate three blank wells were tested (no cells and no treatment).

TEST DESIGN
- 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). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.
- 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.
- 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. 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°C in the presence of 5% CO2. In total 2 valid experiments were performed.
- Luciferase activity measurement: 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 KeratinoSens™ 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 - 4 hours at 37°C, 5% CO2. The MTT medium was then removed and cells were lysed, subsequently the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

DATA ANALYSIS

Interpretation
The following parameters are calculated in the KeratinoSensTM test method:
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control.
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained.
- The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Acceptance 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 vehicle (negative) 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:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.70 and the EC 1.5 was 55 μM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.35 and the EC 1.5 was 18 μM.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.47

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: EC1.5: not possible to calculate

Results 2

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.03

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: EC1.5: not possible to calculate

Other effects / acceptance of results:

ACCEPTANCE: both tests 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 between 5 and 125 μM (55 μM and 18 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.70-fold and 2.35-fold in experiment 1 and 2, respectively).
- The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (18% and 13% in experiment 1 and 2, respectively).

CYTOTOXICITY: yes

Experiment 1:
IC30:671 μM
IC50: 813 μM.

Experiment 2:
IC30: 785 μM
IC50: 993 μM.

PRECIPITATION: no
No precipitation was observed at the start and end of the incubation period.

Applicant's summary and conclusion

Interpretation of results:

other: Study cannot be used for classification independently, but in a WoE for the end point Skin Sensitisation.

Conclusions:

A KeratinoSens™assay was performed according to OECD 442D guideline and following GLP principles to assess the skin sensitisation hazard potential. Indene was classified as negative in the Keratinosens assay since no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes was observed.

Executive summary:

A KeratinoSens™assay was performed according to OECD 442D guideline and following GLP principles to assess the skin sensitisation hazard potential of Indene. Indene was dissolved in dimethyl sulfoxide and tested at 4-fold dilution series from 0.98 µM to 2000 µM. Two independent experiments were performed. Both experiments passed the acceptance criteria and the test conditions were adequate, implying that the test system functioned properly. Indene showed toxicity (IC30 values of 671μM and 785μM and IC50 values of 813μM and 993μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. In conclusion, Indene was found to be negative in the Keratinosens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000μM.