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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:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes

Test material

Constituent 1
Chemical structure
Reference substance name:
(3E)-4-[(1S,3aS,4R,7aS)-1,7a-dimethyloctahydro-4H-1,4-methanoinden-4-yl]pent-3-en-2-one
EC Number:
948-498-7
Cas Number:
143785-33-5
Molecular formula:
C17H26O
IUPAC Name:
(3E)-4-[(1S,3aS,4R,7aS)-1,7a-dimethyloctahydro-4H-1,4-methanoinden-4-yl]pent-3-en-2-one
Test material form:
liquid
Specific details on test material used for the study:
Expiry Date: 20 December 2020
Storage Conditions: at room temperature, protection from light
Description: Colourless to yellow liquid

In vitro test system

Details on the study design:
PREPARATION OF THE TEST ITEM:
- The test item was freshly prepared immediately prior to use. The test item was dissolved in Tetrahydrofuran (THF, CAS No.: 109-99-9, purity ≥99%; Merck; Lot No.: 17C091195; I864407; Sigma; Lot No.: STBW3566). A stock solution of 200 mM was prepared for experiment 1 and 2. In the third experiment a stock solution of 5 mM was prepared by pre-weighing the test material into a glass vial. A factor of 1.22 to correct for the purity of the test item was used. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2 for experiment 1 and 2 and a constant dilution factor of 1:1.33 for experiment 3. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. Since the test item was dissolved in THF, DMSO was added at a final concentration of 4% (v/v). These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.
CONTROLS:
- A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.
- Blank: A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
- Negative Control: DMSO (AppliChem; Lot No.: 0001055932; 00001179895; 0001336139) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
- Solvent Control: THF (Sigma; Lot No.: STBW3566) at a final concentration of 1% (v/v) in test item exposure medium was used as solvent control. Six wells were included in every testing plate.
- Positive Control: Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Alfa Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001055932; 00001179895; 0001336139) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.
CELL LINE
- The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 11 in experiment 1; P 02 in experiment 2; P 11 in experiment 3) were used. Cells were cultured in 75 cm 2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.
COMPOSITION OF MEDIA
- Maintenance Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with 10% fetal bovine calf serum and 1% geneticin (final concentration: 500 μg/mL)
- Assay Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with 10% fetal bovine calf serum
- Test Item Exposure Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the 1% fetal bovine calf serum.
DOSE GROUPS
1. Negative Control: 1% (v/v) DMSO in test item exposure medium and 1% (v/v) THF; 2. Positive Control: CA: 4 μM, 8 μM, 16 μM; 32 μM; 64 μM; 3. Test Item: 12 concentrations of the test item (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM in experiment 1 and 2 and 50, 37.51, 28.14, 21.11, 15.84, 11.88, 8.91, 6.69, 5.02, 3.76, 2.82, 2.12 µM in experiment 3). Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run.
EXPERIMENTAL PROCEDURE
- A cell suspension of 8E4 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1E4 cells were dispensed in each well, except for the blank. 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 crosscontamination 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 sec. before assessing the luciferase activity for 2 sec. 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% CO 2 . 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% CO 2 overnight (experiment 2) or over the weekend (experiment 1 and 3). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.
DATA ANALYSIS
- For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run is performed. The following parameters were calculated: Cell Viability, Maximal Induction of the Luciferase Activity (Imax), EC1.5, IC50 and IC30. For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells. The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.
PREDICTION MODEL
- The test item is considered positive if the following conditions were met in at least two independently prepared test repetitions: 1) Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control; 2) cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5; 3) EC1.5 value is <1000 μM; 4) an apparent overall dose-response for luciferase induction. If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 μM is considered as inconclusive. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.
ACCEPTANCE CRITERIA
- The test meets acceptance criteria if: 1) 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; 2) the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8; 3) the EC1.5 value of the positive control is within two standard deviations of the historical mean 4) 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.

Results and discussion

Positive control results:
EC1.5 was determined to be 12.24, 13.22, and 13.50 for experiment 1, 2, and 3, respectively.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5
Value:
22.3
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: EC1.5
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
1.03
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
2.22
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: Imax
Value:
1.37
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.
In the second experiment, a max luciferase activity (Imax) induction of 2.22 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 111.1%. No further significant luciferase induction >1.5 was found in the tested concentration range. The calculated EC1.5 was <1000 µM (22.30 µM).
In the third experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could 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 non-sensitiser.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met