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

Endpoint summary

Description of key information

The three key events of the skin sensitisation AOP were tested experimentally make a final decision on the skin sensitising potential of the test substance.

 

In a Direct Peptide Reactivity Assay (DPRA) according to OECD guideline 442C, an overall depletion value of 4.82% in the first experiment and 5.87% in the second run, places the test item in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer.

 

In a the ARE-Nrf2 Luciferase Test according to OECD guideline 442D, the test item did not activate the LuSens cells up to a concentration of 145 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP.

 

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.

 

Conclusion: As 2 out of 3 tests reflecting key events of the skin sensitisation AOP yielded negative results, in was concluded that the test substance in not 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:

2022-01-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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™)

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]

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.

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.

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

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

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-07-04

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 KeratinoSens™ test method)

Version / remarks:

2018-06

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase LuSens test method

Details of test system:

Lusens transgenic cell line [442D]

Details on the study design:

442D test method:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: On the day of the experiment, the test item was dissolved or stably dispersed in DMSO.
- Preparation of the test chemical serial dilutions: For the MTT test (dose finding assay) twelve concentrations of the test item were prepared by 1:2 serial dilutions from the highest soluble concentration. The highest test concentration for the dose finding assay was 2000 μM in accordance to the OECD guideline 442D. For the treatment of the cells in the main experiments, a stock solution of the test item and the controls were prepared. It was subsequently diluted 1:25 in treatment medium. For treatment 50 μL of the test item and control dilutions and the medium control were added into the corresponding wells each distributed with 150 µL treatment medium (1:4 dilution).
- Preparation of the positive controls: EGDMA (final concentration 120 μM) was prepared in Treatment medium including 1% (v/v) DMSO
- Preparation of the solvent, vehicle and negative controls: DMSO solvent control (final concentration 1% (v/v)) was prepared in treatment medium. Lactic acid negative control (final concentration 5000 μM) was prepared in Treatment medium including 1% (v/v) DMSO.
- Stable dispersion obtained: Yes

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 2000 µM (in accordance to the OECD guideline 442D)
- Solubility in solvents: The test item was soluble in DMSO
- Solubility in incubation medium: The test item was soluble in culture medium at the tested concentrations
- Cytotoxicity assessment performed: Yes
- Final concentration range selected on basis of: At least six test item concentrations were tested in the main experiments. The highest concentration used was CV75 × 1.2 in the first main experiment. At least five further test item dilutions were prepared by serial dilution with a dilution factor of 1.2.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 24 replicates (solvent control DMSO), five replicates (the positive control), six replicates (the negative control), twelve replicates (treatment medium control) as well as three replicates for each test item concentration
- Number of repetitions: The test item was tested in three independent main experiments.
- Test chemical concentrations: 58.3, 70.0, 83.9, 101, 121, 145 μM
- Application procedure: 24 h ± 30 min after seeding of the cells, seeding medium were removed and 150 μL of treatment medium were distributed in each well. Thereafter, 50 μL of the test item and control dilutions and the medium control were added into the corresponding wells
- Exposure time: 48 ± 1 hours under standard cell culture conditions
- 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: 9000 to 11000 LuSens cells per well. The passage numbers of the used LuSens cells were 11 in the cytotoxicity test and 13, 5 and 7 in the LuSens test for the main experiments 1, 2 and 3, respectively.
- Incubation conditions: 37 ± 1.5 °C and 5.0 ± 0.5 % CO2 (standard cell culture conditions)
- Washing conditions: At the end of the incubation period, treatment medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+
- Precipitation noted: No

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminomete: The absorption and luminescence measurement of the LuSens samples were conducted with a Multimode Reader (TriStar2 LB 942) by Berthold Technologies GmbH Co KG, Germany
- Plate used: 96 well microtiter plate
- Lysate preparation: Steady-Glo® mix was be prepared by adding Steady-Glo® buffer to one bottle of Steady-Glo® substrate and mixing by inversion until the substrate was dissolved. The Steady-Glo® working solution was prepared by mixing one part of DPBS (without Ca2+/Mg2+) with one part of Steady- Glo® mix. 200 μL of the Steady-Glo® working solution was added in each well. After slowly shaking of the microtiter plate for at least 10 min in the dark, the plate was transferred to a microplate reader.

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:

EGDMA (120 M) [442D]

Positive control results:

The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (Experiment 1: 4.62; Experiment 2: 8.82; Experiment 3: 21.34).

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

No increase above 1.5 of the fold luciferase induction was observed.

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

No increase above 1.5 of the fold luciferase induction was observed.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Cell viability:

The cell viability was above 70% at all tested concentrations.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

No EC1.5 could be determined as the fold induction was above 1.5 at all concentrations and no dose-dependence could be observed. The fold induction was between 1.7 and 3.42 at all concentrations.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (experiment 1: 0.94; experiment 2: 1.32; experiment 3: 1.03 was <1.5-fold as compared to the average solvent control.
- Acceptance criteria met for positive control: The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (experiment 1: 4.62; experiment 2: 8.82; experiment 3: 21.34).
- Acceptance criteria met for variability between replicate measurements: Yes. The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (experiment 1: 11.7%; experiment 2: 10.4%; experiment 3: 9.7).
- Range of historical values: See "Attached background material"

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 a the ARE-Nrf2 Luciferase Test according to OECD guideline 442D, the test item did not activate the LuSens cells up to a concentration of 145 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP.

Executive summary:

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of the skin sensitization Adverse Outcome Pathway (AOP)) of the test item. In the dose finding assay, cytotoxic effects were observed following incubation with the test item starting with the concentration of 500 μM up to the highest tested concentration of 2000 μM (threshold of cytotoxicity: <70% cell viability). The CV75 value of the cytotoxicity test was calculated as 120.8 μM. The test item was tested in 3 independent main experiments. The following concentrations of the test item were tested in the main experiments: 58.3, 70.0, 83.9, 101, 121, 145 μM In the first main experiment, after treatment with the test item for 48 ± 1 h the luciferase induction is ≥ 1.5-fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥70%) and at least 3 tested concentrations are non-cytotoxic. Therefore, the LuSens prediction for the first main experiment is considered positive. In the second and third main experiment, after treatment with the test item for 48 ± 1 h the luciferase induction is <1.5-fold compared to the solvent control in all tested concentrations, but 101 μM in the third main experiment. Since this effect was only observed in one concentration in the third main experiment and all other test item concentrations resulted in no luciferase induction >1.5 in the second and third main experiment, the LuSens prediction is considered negative.

Since a negative outcome was observed in 2 out of 3 main experiments, the overall prediction for the LuSens test is negative.

 

The acceptance criteria were met:

• The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 62; ME 2: 8.82; ME 3: 21.34).


• The positive control had a relative cell viability ≥70% as compared to the solvent control (ME 1: 117.44%; ME 2: 105.50%; ME 3: 111.15%).


• The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 0.94; ME 2: 1.32; ME 3: 1.03), as well as the basal expression of untreated cells, medium control (ME 1: 1.03; ME 2: 1.32; ME 3: 1.16), was <1.5-fold as compared to the average solvent control.


• The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (ME 1: 11.7%; ME 2: 10.4%; ME 3: 9.7%).


• At least three test item concentrations had a cell viability of at least 70% relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability <70%.

 

In conclusion, the test item did not activate the LuSens cells up to a concentration of 145 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-02-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

2020-06-26

Deviations:

yes

Remarks:

Parallel dilution instead of serial dilution calibration standards were conducted. This was considered to have no impact on the quality of the study.

Principles of method if other than guideline:

Parallel dilution instead of serial dilution calibration standards were conducted. This was considered to have no impact on the quality of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

other: Cysteine peptide (Ac-RFAACAA-OH) / Lysine peptide (Ac-RFAAKAA-OH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of aliquots of the appropriate peptide in approximately 20 mL aliquots of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).
- Preparation of the test chemical solutions: The test item was weighed into reaction tube and dissolved immediately before testing in acetonitrile to prepare a 100 mM stock solution.
- Preparation of the positive control: The positive control chemical (trans-cinnamaldehyde) was prepared at a concentration of 100 mM in acetonitrile.
- Preparation of reference controls and co-elution controls:
Reference control A: For the verification of the HPLC system suitability (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=1 with 3-fold injections.
Reference control B: For the stability of the reference controls over time (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=6.
Reference control C1: Peptide stability control for the solvent used to dissolve the test item and the positive control (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=3.
Co-elution control: Sample prepared of the respective peptide buffer and the test item or the positive control without peptide. n=1, each.

The reference control A, B and C1 samples of both peptides were prepared at a concentration of 500 μM in acetonitrile.

INCUBATION
- Incubation conditions: The reaction solutions were incubated in the dark at 22.5-30 ºC for 24 ± 2 h prior to initiation of the analysis run.
- Precipitation noted: The appearance of the test item and positive control samples in the HPLC vials was visually inspected and documented after preparation and prior to initiation of the HPLC run. No precipitation was noted in the study report.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys
- Verification of the suitability of the HPLC for test chemical and control substances: Analysis was performed according to the proficiency study 1841800 conducted at the laboratory.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm and 258 nm

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

mean lysine depletion

Value:

1.17 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

mean cystein depletion

Value:

10.6 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean lysine depletion

Value:

0.133 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean cystein depletion

Value:

9.51 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Analysis was performed according to the proficiency study conducted in the laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for reference controls A to C: Yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values: See "Attachments"

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 a Direct Peptide Reactivity Assay (DPRA) according to OECD guideline 442C, an overall depletion value of 4.82% in the first experiment and 5.87% in the second run, places the test item in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer.

Executive summary:

The purpose of this study (based on the OECD guidelines for the testing of chemicals: test guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item according to GLP. This direct peptide reactivity assay can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), ARE-Nrf2 luciferase test method) based on the OECD adverse Outcome Pathway (AOP) for the assessment of the skin sensitisation potential of chemicals. The test item was dissolved in acetonitrile when incubated for 24 ± 2 h in the range between 22.5 and 30 °C. Solutions of the test item were analysed by the DPRA method in both the cysteine and lysine containing synthetic peptides. There were no co-elution peaks in either the cysteine or lysine assays observed. With no to minimal mean depletion of both peptides (4.82%) in the presence of the test item, is therefore predicted by DPRA as negative and not to be a potential skin sensitizer based on this assay. According to the OECD TG 442C this value is in the borderline range of the cysteine 1:10/lysine 1:50 prediction model (3% to 10% depletion) and therefore the HPLC analysis was repeated. In the repetition of the experiment the mean depletion of both peptides in the presence of the test item was 5.87% and confirmed the first experiment. The DPRA prediction is therefore negative with the reactivity class “No or minimal”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

WoE, in chemico skin sensitisation (DPRA), RL1

The purpose of this study (based on the OECD guidelines for the testing of chemicals: test guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item according to GLP. This direct peptide reactivity assay can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), ARE-Nrf2 luciferase test method) based on the OECD adverse Outcome Pathway (AOP) for the assessment of the skin sensitisation potential of chemicals. The test item was dissolved in acetonitrile when incubated for 24 ± 2 h in the range between 22.5 and 30 °C. Solutions of the test item were analysed by the DPRA method in both the cysteine and lysine containing synthetic peptides. There were no co-elution peaks in either the cysteine or lysine assays observed. With no to minimal mean depletion of both peptides (4.82%) in the presence of the test item, is therefore predicted by DPRA as negative and not to be a potential skin sensitizer based on this assay. According to the OECD TG 442C this value is in the borderline range of the cysteine 1:10/lysine 1:50 prediction model (3% to 10% depletion) and therefore the HPLC analysis was repeated. In the repetition of the experiment the mean depletion of both peptides in the presence of the test item was 5.87% and confirmed the first experiment. The DPRA prediction is therefore negative with the reactivity class “No or minimal”.

 

 

WoE, in vitro skin sensitisation (LuSens), RL1

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of the skin sensitization Adverse Outcome Pathway (AOP)) of the test item. In the dose finding assay, cytotoxic effects were observed following incubation with the test item starting with the concentration of 500 μM up to the highest tested concentration of 2000 μM (threshold of cytotoxicity: <70% cell viability). The CV75 value of the cytotoxicity test was calculated as 120.8 μM. The test item was tested in 3 independent main experiments. The following concentrations of the test item were tested in the main experiments: 58.3, 70.0, 83.9, 101, 121, 145 μM In the first main experiment, after treatment with the test item for 48 ± 1 h the luciferase induction is ≥ 1.5-fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥70%) and at least 3 tested concentrations are non-cytotoxic. Therefore, the LuSens prediction for the first main experiment is considered positive. In the second and third main experiment, after treatment with the test item for 48 ± 1 h the luciferase induction is <1.5-fold compared to the solvent control in all tested concentrations, but 101 μM in the third main experiment. Since this effect was only observed in one concentration in the third main experiment and all other test item concentrations resulted in no luciferase induction >1.5 in the second and third main experiment, the LuSens prediction is considered negative.

Since a negative outcome was observed in 2 out of 3 main experiments, the overall prediction for the LuSens test is negative.

 

The acceptance criteria were met:

• The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 62; ME 2: 8.82; ME 3: 21.34).


• The positive control had a relative cell viability ≥70% as compared to the solvent control (ME 1: 117.44%; ME 2: 105.50%; ME 3: 111.15%).


• The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 0.94; ME 2: 1.32; ME 3: 1.03), as well as the basal expression of untreated cells, medium control (ME 1: 1.03; ME 2: 1.32; ME 3: 1.16), was <1.5-fold as compared to the average solvent control.


• The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (ME 1: 11.7%; ME 2: 10.4%; ME 3: 9.7%).


• At least three test item concentrations had a cell viability of at least 70% relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability <70%.

 

In conclusion, the test item did not activate the LuSens cells up to a concentration of 145 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP.

 

 

WoE, in vitro skin sensitisation (USens), RL1

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.

 

Overall conclusion: As 2 out of 3 tests reflecting key events of the skin sensitisation AOP yielded negative results, in was concluded that the test substance in not sensitising. 

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is not classified and labelled for skin sensitisation according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.