Vinylcyclohexane

Endpoint summary

Description of key information

The dermal sensitisation potential of Vinylcyclohexane was tested in two in vitro skin sensitisation studies conducted according to OECD 442C and 442D. In both in vitro studies the target substance showed no skin sensitising potential. Based on the results, Vinylcyclohexane can be considered as not skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

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

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

Details on the 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.

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%

Key result

Run / experiment:

other: Experiment I and Experiment II

Parameter:

other: Fold induction

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

Run / experiment:

other: Experiment I and Experiment II

Parameter:

other: Relative Viability

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-11-02 to 2018-04-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

Adopted 4th February, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

N.A.

Details on the study design:

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

Dissolution of the test item:
Two batches of 100 mM test item solutions were prepared:
- 33.1 mg test item were dissolved in 3.00 mL acetonitrile (batch 20171108, used for Lys-peptide assay)
- 33.4 mg test item were dissolved in 3.03 mL acetonitrile (batch 20171109, used for Cys-peptide assay)
The test item stock solution was freshly prepared for each assay.

Test solutions
Dilution buffers:
- 2 mL Acetonitrile were mixed with 8 mL phosphate buffer, pH 7.5 (Peptide dilution buffer C)
- 2 mL Acetonitrile were mixed with 8 mL ammonium acetate buffer, pH 10.2 (Peptide dilution buffer K)

Peptide stock solutions:
The peptide stock solutions were freshly prepared for each assay.
- 0.667 mM Cys-Peptide solution was prepared by dissolving 15.0 mg of the peptide in 30 mL phosphate buffer, pH 7.5.
- 0.667 mM Lys-Peptide solution is prepared by dissolving 15.5 mg of the peptide in 30.0 mL ammonium acetate buffer, pH 10.2.

Peptide calibration standards:
From each peptide stock solution the following calibration standards were prepared in the appropriate dilution buffer: 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017 mM Peptide.
Calibration samples were analysed before the samples containing the test item. Blank dilution buffer was also measured.

Test item samples:
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were pre-pared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions. A final volume of 1 mL per sample was prepared for each sample.

Incubation:
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 +/- 2.5 °C for 24 +/- 2 h. None of the samples were turbid after incubation.

Measurements: Measurements were performed using the HPLC method

Positive control results:

The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0% (75.41%) and ≤ 14.9% (1.9), respectively for the Cys-peptide. The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0% (36.66%) and ≤ 11.6% (2.7), respectively, for the Lys-peptide.

Key result

Run / experiment:

other: Mean of calculated peptide depletion values for Lys- and Cys-Peptide

Parameter:

other: Mean peptide depletion [%]

Value:

1.27

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: mean of triplicates

Parameter:

other: Mean peptide depletion for the Lys-peptide [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: mean of triplicates

Parameter:

other: Mean peptide depletion for the Cys-peptide [%]

Value:

2.54

Vehicle controls validity:

valid

Negative controls validity:

not examined

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:
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results obtained from a direct peptide reactivity assay (OECD 442C), the test item Vinylcyclohexane can be considered as not sensitizing to the skin.

Executive summary:

In a dermal sensitization study conducted according to OECD guideline 442C, the test item (99.8% purity) in acetonitrile was tested using the direct peptide reactivity assay. 100 mM test item in acetonitrile was incubated 24 ± 2 h at 25 °C together with cysteine and lysine peptides, respectively and the peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide. All acceptance criteria were fulfilled and the test was considered as valid. The Cys-peptide depletion was 2.54%, the Lys-peptide depletion was 0.00% and the resulting mean peptide depletion was 1.27%. Based on the results, the DPRA prediction for the test item Vinylcyclohexane was negative with reactivity class minimal according to the Cysteine 1:10/Lysine 1:50 prediction model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

The sensitisation potential of Vinylcyclohexane was assessed in two in vitro skin sensitisation studies conducted according to OECD testing guideline 442C and 442D.

In the study conducted according to OECD 442D the sensitisation 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 and later checked for luciferase activity. Sensitisation 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.

In the study conducted according to OECD 442C the sensitisation potential of the test item was assessed on the basis of the reactivity of the test item towards cysteine and lysine containing peptides. 100 mM test item in acetonitrile was incubated 24 ± 2 h at 25 °C together with cysteine and lysine peptides, respectively and the peptide concentration after the incubation was measured using HPLC-UV. All acceptance criteria were fulfilled and the test was considered valid. The DPRA prediction for the test item Vinylcyclohexane was negative with reactivity class minimal according to the Cysteine 1:10/Lysine 1:50 prediction model.

Justification for classification or non-classification

In two in vitro skin sensitisation studies conducted according to OECD 442C and 442D, Vinylcyclohexane was tested negative for skin sensitisation. Based on the results, no classification of Vinylcyclohexane is warranted.