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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
6.1 Test Item
Designation in Test Facility: 17092503G
Date of Receipt: 25. Sep. 2017
Condition at Receipt: Room temperature, in proper conditions
6.1.1 Specification
The following information concerning identity and composition of the test item was provid-ed by the sponsor.
Name gamma-Dodecalactone
Batch no. 050170905
Appearance clear, colorless liquid
Composition gamma-Dodecalactone
Purity 99.02%
Homogeneity homogeneous
Expiry date 18. Sep. 2018
Storage Room Temperature: (20 ± 5°C)
The following information was provided by the sponsor as well:
CAS No. 2305-05-7
EINECS-No. 218-971-6
Molecular formula C12H22O2
Molecular weight 198.3 g/mol
Vapour pressure unknown
Stability H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: unknown; EtOH: 0.1 - 1 g/L; acetone: unknown; CH3CN: unknown; DMSO: unknown
Production date 19. Sep. 2017
6.1.2 Storage
The test item was stored in the test facility at room temperature (20 ± 5°C).
6.1.3 Preparation
The solubility of the test item was determined in a non-GLP pre-test in dimethyl sulfoxide (DMSO) and medium (DMEM). The test item forms a suspension in DMEM but is soluble in DMSO at the required concentration (200 mM). Therefore, DMSO was used as solvent.
Since the final concentration of the solvent during treatment is limited to 1 %, a stock solution containing 200 mM (CRFT) and 250 mM (experiment I and II) test item in DMSO was prepared. In experiment I and II this stock solution was first 1:10 diluted. Subsequent dilution to 1% finally yielded a maximum concentration of 2000 µM in the pre-test and 250 µM in the experiments.
For that, the stock solution was first used to prepare a geometric series of solutions (CRFT: factor 2; main experiments: factor 1.2) on a master plate. Afterwards all concentrations were further diluted (1:25) in medium no. 3 on a dilution plate. Another 1:4 dilution was achieved by adding 50 µL of each concentration of the dilution plate to the corresponding wells of the test plate containing the cells as well as 150 µL medium no. 3. In the end, the total dilution factor was 1:100. The stock solution as well as the dilutions were freshly pre-pared on the day of treatment.

In vitro test system

Details on the study design:
6.3 Test System
6.3.1 Reasons for the Choice of the LuSens Cell Line
The LuSens cell line was specially designed for this test system by the BASF SE (Lud-wigshafen, Germany). It employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).
6.3.2 Cell Cultures
For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitro-gen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 14 were used. For both main experiments cells of passage 6 were used. After thawing the cells were cultivated in DMEM (9 % FCS) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Results and discussion

Positive control results:
The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.

Found in experiment I
Positive control Fold induction: 7.7
Relative viability: 90.3 %

Found in experiment II
Positive control Fold induction: 6.0
Relative viability: 89.4 %

In vitro / in chemico

Key result
Run / experiment:
other: Fold induction
Fold induction = [(Lsample - Lblank) / (Lsolvent - Lblank)]
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
In conclusion, it can be stated that under the experimental conditions of this study, the test item, gamma-Dodecalactone, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).