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Toxicological information

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:
2017-01-23 to 2017-02-17
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:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2015
Qualifier:
according to guideline
Guideline:
other: KeratinoSens(TM), EURL ECVAM DB-ALM Protocol No 155,
Version / remarks:
2015
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
This test method is able to detect chemicals that have sensitisation potential by addressing the second molecular key event of the adverse outcome pathway, namely the activation of keratinocytes, and allows for hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the adverse outcome pathway.

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane Hydrobromide
EC Number:
928-779-0
Molecular formula:
C18-H21-N-O4 x HBr
IUPAC Name:
1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane Hydrobromide
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:TH 1165 Ketone I BR (Batch No: 600CC)
- Expiration date of the lot/batch: 28 February 2017
- Purity test date:No details reported.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions:stable at storgae conditions
- Solubility and stability of the test substance in the solvent/vehicle: The test item was prepared immediatlely prior to use.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No details reported.
- Preliminary purification step (if any):No details reported.
- Final dilution of a dissolved solid, stock liquid or gel:No details reported.
- Final preparation of a solid:No details reported.

In vitro test system

Details on the study design:
TEST SYSTEM
- Cell line: transgenic cell line KeratinoSens™ (Givaudan, Switzerland) derived from human keratinocytes (HaCaT)

TEST-SUBSTANCE PREPARATION
- Concentrations: 2000, 1000, 500, 250, 125, 61.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
- Stock: 4x concentration of the highest concentration (diluted 1:4 when incubated with the cells)
- Vehicle: DMSO (1% (v/v)

CONTROLS
- Positive control (PC): Cinnamic in 1% DMSO: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
- Negative control : DMSO: 1% (v/v) in test item exposure medium
- Blank control: Culture medium without cells

MEDIUM
- Maintenance Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885- 025) with 1.0 g/L D-glucose and Na-Pyruvate + 10% fetal bovine calf serum + 1% geneticin (500 µg/mL)

- Assay Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025) with 1.0 g/L D-glucose and Na-Pyruvate + 10% fetal bovine calf serum

- Test Item Exposure Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885- 025) with 1.0 g/L D-glucose and Na-Pyruvate + 1% fetal bovine calf serum

Experimental Procedure
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent
96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

Cell viability:
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at lambda = 600 nm.

ANALYSIS
- Calculation of Cell Viability
- Calculation of the Maximal Induction of the Luciferase Activity (Imax)
- Calculation of the EC1.5
- Calculation of IC50 and IC

ACCEPTANCE CRITERIA
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.


EVALUATION CRITERIA
The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repitition, all of the three first conditions are met but a clear dose respone for the luciferase induction cannot be observed, the result of that repitition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 uM is considered inconclusive.

Results and discussion

Positive control results:
Refer to "Any other information on result incl. tables".

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
3.64
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: at 500 µM
Run / experiment:
other: 1
Parameter:
other: Cell viability (%)
Value:
25.6
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 1
Parameter:
other: EC 1.5 (µM)
Value:
307.69
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
1.2
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: Cell viability (%)
Value:
32.5
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.80 (experiment 1); 3.42 (experiment 2)). The calculated EC1.5 was between 7 and 30 µM (23.16 µM (experiment 1); 19.42 µm (experiment 2)). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (7.5% (experiment 1); 15.8% (experiment 2)).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, based on the results of this study, the test item can be considered as a "non sensitiser".
Executive summary:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely the activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

The test item was was dissolved in DMSO. Based on a molecular weight of 396.29 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 61.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 3.64 was determined at a test item concentration of 500 µM. The corresponding cell viability was 25.6%. Only at a test item concentration of 500 µM a significant luciferase induction >1.5 was found.The calculated EC1.5was < 1000 µM (307.69 µM).

In the second experiment, a max luciferase activity (Imax) induction of 1.20 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 32.5%. No significant luciferase induction >1.5 was found. Therefore, noEC1.5could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.Under the condition of this study the test item is therefore considered as a "non sensitiser". The controls confirmed the validity of the study.

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, based on the results of this study, the test item can be considered as a "non sensitiser". The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.