(1α,2α,5α)-2,2,6-trimethylbicyclo[3.1.1]heptan-2-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-01-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

2018-06

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

U937 cell line activation test (U-SENS™)

Test material

Test material information

Constituent 1

Reference substance name:

(1α,2α,5α)-2,2,6-trimethylbicyclo[3.1.1]heptan-2-ol

EC Number:

225-591-4

EC Name:

(1α,2α,5α)-2,2,6-trimethylbicyclo[3.1.1]heptan-2-ol

Cas Number:

4948-28-1

Molecular formula:

C10H18O

IUPAC Name:

(1R,2S,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-2-ol

In vitro test system

Details of test system:

U-937 cell line [442E]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: On the day of the experiment, the test item was dissolved in DMSO.
- Preparation of the test chemical serial dilutions: Dilutions in DMSO were prepared from the stock solution and further diluted with culture medium. By mixing the dilutions (100 µL) with the cell suspension, the final treatment concentrations were achieved.
- Preparation of the positive controls: Picryl sulfonic acid (TNBS), (1 M in water) was diluted with culture medium to a final concentration 50 μg/mL. Each volume (100 μL) of the dilutions oof the positive control was added to the cells according to the plate template.
- Preparation of the solvent, vehicle and negative controls: The negative control Lactic acid was diluted with culture medium to a final concentration 200 μg/mL. Culture medium was used as medium control. Each volume (100 μL) of the dilutions of the negative control and the medium control was added to the cells according to the plate template.
- Stable dispersion obtained: Yes

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: The dilutions of the test item were tested in two replicates (one labelled with anti-CD86 and one with mouse IgG1). The control groups were tested in six replicates (three labelled with anti-CD86 and three with mouse IgG1).
- Number of repetitions: The test item was tested in two independent runs.
- Test chemical concentrations: 1, 10, 20, 50, 100 and 200 µg/mL
- Application procedure: Each volume (100 μL) of the dilutions of the test item, culture medium, positive, negative and solvent control was added to the cells according to the plate template. The treated U937 cells were incubated for 45 ± 3 h at 37 ± 1.5 °C and 5.0 ± 0.5% CO2 atmosphere. Prior to incubation, plates are sealed with semi-permeable membrane, to avoid evaporation of volatile test items and cross-contamination between cells treated with the test item. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
- Exposure time: 45 ± 3 hours
- Study evaluation and decision criteria used: See "Any other information on materials and methods incl. tables"
- Description on study acceptance criteria: See "Any other information on materials and methods incl. tables"

SEEDING AND INCUBATION
- Seeding conditions: 0.49-0.51 × 10^4 U937 cells/well were seeded in each corresponding well of a 96-well flat bottom plate. Cells were used up to seven weeks after thawing (i.e. 21 passages).
- Incubation conditions: The cells are sub-cultured twice or three times weekly. Cell seeding was carried out at a cell density of 0.15-0.3 × 10^6 cells/mL in culture medium. The cell density should not exceed 2 × 10^6 cells/mL. The U937 cell suspension was incubated at 37 ± 1.5 °C and 5.0 ± 0.5% CO2 atmosphere. RPMI 1640 Medium, GlutaMAXTM supplement including 25 mM HEPES, supplemented with 10% FBS (v/v), 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) was used to culture the cells and during the assay.
- Washing conditions: After incubation, all cells were collected by centrifugation (approx. 200 × g, 5 min) and then washed with approx. 100 μL of staining buffer (PBS with 5% (w/v) FBS).

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used: FACSCalibur, Becton Dickinson GmbH, software Cellquest Pro 6.0.
- Plate used: 96-well flat bottom plates
- Propidium iodide staining/cytotoxicity measurements: 7-AAD stain was used for cytotoxicity staining
- Preparation for CD54 and/or CD86 expression measurements/cell staining: All cells were transferred according to the plate template in a v-shape 96-well plate, collected by centrifugation (approx. 200 × g, 5 min) and then washed with approx. 100 μL of staining buffer (PBS with 5% (w/v) FBS). Thereafter, the cells were centrifuged, and the cell pellets were re-suspended in 100 μL staining buffer. The cells were stained with FITC-labelled anti-CD86 and mouse IgG1 (isotype control) and incubated light protected for 30 ± 5 min on ice. After staining with the antibodies, the cells were washed twice with 100 μL of staining buffer and once with 100 μL ice cold PBS. The cells were re-suspended in 100 μL ice cold PBS and transferred into microtubes (300 μL PBS/tube). At least 10 min before the flow cytometry acquisition, 5 μL of a 7-AAD solution/tube was added. For measurement each microtube was placed in a proper cytometer tube and vortexed.

DATA EVALUATION
- Cytotoxicity assessment: See "Any other information on materials and methods incl. tables"
- Prediction model used: See "Any other information on materials and methods incl. tables"

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

picrylsulfonic acid/2,4,6-trinitro-benzene-sulfonic acid (TNBS) [442E]

Results and discussion

Positive control results:

The positive control (Picryl sulfonic acid, final concentration 50 μg/mL) showed an approx. 3- to 4-fold induction in both experiments and no cytotoxicity. Thus, the acceptance criteria for the positive control were met.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Cell viability:

Cytotoxicity was observed at the highest tested concentration (200 µg/mL)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Not determinable as als tested concentrations gave a positive response > 150% of the control.

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

CV70 [442E]

Value:

181 µg/mL

Cell viability:

Cytotoxicity was observed at the highest tested concentration (200 µg/mL)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

CV70 [442E]

Cell viability:

No cytotoxicity observed up to the highest tested concentration

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

61.6 µg/mL

Cell viability:

No cytotoxicity observed up to the highest tested concentration

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Technical proficiency was demonstrated using the suggested proficiency chemicals in OECD 442E.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

The study can be used within the integrated approach adressing key events of the skin sensitisation AOP.

Conclusions:

In an in vitro skin sensitisation test (U-SENS™) according to OECD 442, the test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation adverse outcome pathway.

Executive summary:

An in vitro skin sensitization test (U-SENS™) according to OECD 442E und GLP was performed to assess the skin sensitization potential of the test item dissolved in DMSO when administered to U937 cells for 45 ± 3 h. This U-SENS™ test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), and ARE-Nrf2 (luciferase test method)) based on the OECD Adverse Outcome Pathway (AOP) for the assessment of the skin sensitisation potential of chemicals. The highest test item concentration was 200 μg/mL in accordance to the OECD guideline 442E. For CD86 expression measurement the following concentrations of the test item were tested in two main experiments (U-SENS™):

1, 10, 20, 50, 100 and 200 μg/mL in first experiment and

1, 20, 50, 70, 100 and 200 μg/mL in the second experiment.

In the first and second experiment, all acceptance criteria including the CD86 stimulation index (S.I.) of the positive and negative controls were met. In the first experiment, no cytotoxic effects (cell viability <70%) were observed following incubation with the test item. In the second experiment, cytotoxic effects were observed following incubation with the test item with the highest test item concentration of 200 μg/mL. Therefore, the highest concentration was excluded from the assessment. The CV70 value could be calculated as 181.0 μg/mL in the second experiment. In the first experiment, the CD86 stimulation index (S.I.) was higher or equal to 150% after treatment with the test item concentration 100 μg/mL and 200 μg/mL. In the second experiment, the CD86 stimulation index (S.I.) was higher or equal to 150% after treatment with the test item at all concentrations. In conclusion, the test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation adverse outcome pathway.