Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-09-05 to 2018-02-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
yes
Remarks:
see box "Principles of method if other than guideline"
Principles of method if other than guideline:
Deviations from the Guideline:

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is only based on the OECD 442D Guideline.
The deviations from the OECD 442D are given below:
1. For the test the LuSens cell line was used. This cell line was developed by the BASF SE.
2. In order to determine the concentration range applicable for experiment I and II a Cytotoxicity Range Finder Test (CRFT) was performed. This test was performed in accordance to the protocol of the BASF SE.
3. The dilution factor in the experiments is 1.2-fold.
4. The controls are tested at only one concentration.
5. As positive control EGDMA (Ethylene glycol dimethylacrylate) was used.
6. During the experimental performance the luciferase induction is measured at a second 96-well plate in accordance to the protocol of the BASF SE.
7. Regarding the acceptance criteria, the positive control must induce a luciferase induction of a minimum of 2.5-fold in comparison to the solvent control. In addition, the viability must be ≥ 70%. The negative control must induce a luciferase induction of < 1.5-fold and a viability of ≥ 70%. Regarding the test item, a minimum of 3 test item concentrations has to be analysable (viability: ≥ 70%).
GLP compliance:
yes
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name: Vinylcyclohexane
- CAS No.: 695-12-5
- Batch No.: VCH/7/17K1
- Expiry date: 30 March 2019
- Purity: 99.8 %
- Appearance: Clear, colourless liquid
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: A final dilution to 1% DMSO as a solvent yielded a maximum test substance concentration of 2000µM

In vitro test system

Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:

- A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the con-centration range applicable for the main experiments. In the CRFT the following 12 nominal concentrations of the test item were tested: 0.98 µM, 1.95 µM, 3.91 µM, 7.81 µM, 15.63 µM, 31.25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM, 1000 µM, 2000 µM
- Since a cytotoxic reaction was observed in the CRFT the following 12 nominal concentrations were chosen for experiment I and II: 34 µM, 40 µM, 48 µM, 58 µM, 70 µM, 84 µM, 100 µM, 121 µM, 145 µM, 174 µM, 208 µM, 250 µM. Experiment II served only to confirm the results of experiment I

- Cell Seeding: The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium no. 2 was added. After centrifugation (5 min at 380 g), the supernatant was discarded and the cells were resuspended in medium no. 2. After quantification, the cell suspension was adjusted to 83000 (± 10%) cells per mL. 120 µL of the cell suspension (ca. 10000 cells) were seeded in two clear flat bottom 96 well plates. Two plates each for Experiment I and II were prepared, one for viability and the other for luciferase induction measurement. Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5% CO2 in a humidified atmosphere for 24 h and 30 min in Experiment I and 24 h and 45 min in Experiment II.

- Treatment Procedure: After the incubation time the medium was removed from the cells and 150 µL medium no. 3 were added to each well. Afterwards 50 µL of each single test item concentration and the controls were added to the cells in triplicates (test item concentrations). 24 wells were used for solvent control, 12 wells were used for growth control (cells + medium no. 3), 6 wells were used for negative control, 5 wells for positive control and 1 well for blank. The plates were sealed with breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards the plates were incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.

- Evaluation of Viability: The MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 µL MTT working solution were added to each well. The plates were incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5% CO2 in a humidified atmosphere. Afterwards the solution was re-moved and 100 µL of lysis buffer were added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer and color change was measured.

- Evaluation of luciferase activity: All solutions were removed from the wells and the cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Afterwards 100 µL per well of a Lysis buffer were added to the cells and incubated for 5 min at room temperature. During this process, the plate was slightly moved. Afterwards 100 µL Steady-Glo® Rea-gent were added to each well and the plate was shaken again slowly for 5 min at room temperature. Then, 160 µL per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometer.

Results and discussion

Positive control results:
- In Experiment I, the fold induction was 6.5 and the relative viability was measured to be 93.9%
- In Experiment II, the fold induction was 5.9 and the relative viability was measured to be 84.9%
- Acceptance criteria: Fold induction: ≥ 2.5 fold and relative viability: at least 70%

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Fold induction
Run / experiment:
Experiment I and Experiment II
Value:
< 1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Relative Viability
Run / experiment:
Experiment I and Experiment II
Value:
> 0.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that under the experimental conditions of this study, the test item Vinylcyclohexane was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor (no sensitizing potential).
Executive summary:

In a dermal sensitization study conducted according to OECD 442D with Vinylcyclohexane (99.8% purity) in DMSO, the sensitization potential of the test item was assessed on the basis of the activation of keratinocytes using the genetically modified keratinocyte cell line 'LuSens'. Cells were incubated with the test item for 48 h at different concentrations (34 µM, 40 µM, 48 µM, 58 µM, 70 µM, 84 µM, 100 µM, 121 µM, 145 µM, 174 µM, 208 µM, 250 µM) and later checked for luciferase activity.

Sensitization was scored by measuring the luciferase activity induction and relative cell viability. For both experiment I and II, the fold induction was less than a 1.5 and the cell viability was greater than 70%. Therefore, in this study, the test item is not considered to be a skin sensitizer under UN GHS guidelines.