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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
06 Mar 2017 to 17 Mar 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:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February, 2015
GLP compliance:
yes
Type of study:
other: in vitro ARE-Nrf2 luciferase test method (KeratinoSensTM)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Zschimmer and Schwarz GmbH + Co. KG.
- Identification: 1739 MIPA-CITRAT
- Batch: 207459-01
- Purity/Composition: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Test item storage: At room temperature
- Stable under storage conditions until: 13 January 2018 (expiry date)

OTHER SPECIFICS
- Appearance: Clear yellow liquid
- Purity/Composition correction factor: Yes, according to the purity: 1.49

In vitro test system

Details on study design:
CONTROLS
- vehicle control:1% DMSO
- positive control: Ethylene dimethacrylate glycol (CAS 97-90-5)

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- A correction was made for the purity/composition of the test item. A correction factor of 1.49 was used. The test item solutions were prepared based on the molecular weight specified in gram dry matter/mol (288.78 dry weight/mol).
- A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (clear colourless solution). The 100-fold dilution in DMEM of 200 mM showed no precipitation (final concentration 2000 µM). This concentration was selected as highest concentration for the main assay.
- In the main assay a 200 mM stock solution in DMSO was prepared. From the stock 11 spike solution were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.977 µM (final concentration DMSO of 1%; study plan deviation).
- All concentrations of the test item were tested in triplicate.
- All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates (MTT-assay; study plan deviation).

PREPARATION OF THE POSITIVE CONTROL
For ethylene dimethacrylate glycol (CAS 97-90-5) a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted such that the final concentration of the positive control ranges from 7.81 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

SOLVENT CONTROL
The solvent control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

BLANK
Three blank wells were tested on a separate plate (no cells and no treatment; study plan deviation).

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. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE
- Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
- Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 μg/mL).
- Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
- Environmental conditions: All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 51 – 86 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 – 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. 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.
- Subculturing: Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages (actual passage number used 22 and 25).

EXPERIMENTAL DESIGN
- Two independent experiments were performed.
- 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 then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2. In total 2 experiments were performed.
- 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 luminometer to assess the quantity of luciferase (integration time one second).
- 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 with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
- 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 7.81 to 250 μM).
- 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.
- 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.

DATA ANALYSIS
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.

DATA INTERPRETATION
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
- The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s ttest)
- The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
- The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no defined MW
- There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 μM or 200 μg/mL should be considered as inconclusive.

Results and discussion

Positive control results:
EXPERIMENT 1
- Imax: 11.13 μM
- EC1.5: 7.3 μM

EXPERIMENT 2
- Imax: 4.28 μM
- EC1.5: 37.6 μM

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: viablity (%)
Run / experiment:
Experiment 1 and 2
Value:
> 70
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: no toxicity
Key result
Parameter:
other: Imax (µM)
Remarks:
The maximal average fold induction of luciferase activity
Run / experiment:
Experiment 1
Value:
1.41
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Remarks:
no EC1.5 could be calculated
Key result
Parameter:
other: Imax (µM)
Remarks:
The maximal average fold induction of luciferase
Run / experiment:
Experiment 2
Value:
1.29
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Remarks:
no EC1.5 could be calculated
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
In combination with DPRA assay (OECD 442C)