Registration Dossier

Administrative data

Description of key information

Using Toxtree no skin sensitization potential was identified.

A test strategy will be performed in accordance with OECD TG 442C, OECD TG 442D and OECD TG 442E in order to determine the skin sensitization potential of the test item. The study was initiated in January 2020 but was not completed by the CRO in due time to complete the registration dossier. The study record will be updated as soon as the results will become available.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
Synthetic peptides:
Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, are used.
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 775.9 g/mol)

Instruments and Devices:
Analytical and precision scales
Volumetric measurement tools
pH-meter
Standard laboratory glassware
Incubation chamber capable of holding 25.0 ± 2.5 °C

Chemicals:
Water for chromatography
H2O, HPLC grade
Demineralised water
H2O, from ion exchange cartridge. Total organic carbon (TOC) < 1 ppm
Acetonitrile for chromatography
CH3CN, ACN, HPLC grade
Trifluoroacetic acid
TFA, for spectroscopy, Merck
Isopropanol
CH3CHOHCH3, p.a.

Buffers:
25 mM Phosphate buffer
2.23 g disodium hydrogen phosphate dihydrate are dissolved in demineralised water, pH is adjusted to 7.5 with NaOH solution or H3PO4 (final volume 500 mL).
5 mM Ammonium acetate
964 mg ammonium acetate (anhydrous) are dissolved in demineralised water, pH is ad-justed to 10.2 with ammonia or glacial acetic acid (final volume 500 mL).

Positive control:
Cinnamaldehyde (CAS 104-55-2, food grade ≥95 %),100 mM solution in acetonitrile for the cysteine peptide
2,3-Butanedione (CAS 431-03-8, >97 %), 100 mM solution in ace-tonitrile for the lysine peptide

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution are prepared in triplicate (Sets A, B1, B2 and C, total 12 samples per peptide). Set A is analysed together with the peptide calibration standards, sets B1 and B2 are analysed at the start and end of the analysis sequence and are used as stability control for the peptide over the total analysis time. Set C is incubated and analysed together with the samples and is used for calculation of the peptide depletion.

Co-elution control
Sample prepared from the respective peptide buffer and the test item, but without peptide.

Peptide stock solutions
The peptide stock solutions are freshly prepared for each assay.
0.667 mM Cys-Peptide solution is prepared by dissolving an appropriate amount of the peptide in 25 mM phosphate buffer, pH 7.5.
0.667 mM Lys-Peptide solution is prepared by dissolving an appropriate amount of the peptide in 25 mM ammonium acetate buffer, pH 10.2.

Test item stock solution:
The test item stock solution is freshly prepared for each assay.
100 mM test item solution is prepared was prepared.
Peptide calibration standards:
From each peptide stock solution the following calibration standards will be prepared in the appropriate dilution buffer: 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017 mM peptide. Blank dilution buffer is also measured. Calibration samples are analysed before the samples containing the test item.

Test item samples:
Samples are prepared in triplicate for each peptide. The Cys-peptide samples are 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 described. A final volume of 1 mL per sample is prepared for each sample

Incubation:
The positive control, solvent control and test item samples are incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 24 ± 2 h. Samples appearing turbid or where precipitation is visible by the unaided eye are centri-fuged (benchtop centrifuge, 10 min at 4500 rpm) and only the clear supernatant is used for measurement.


Measurements
HPLC system with UV/VIS-Detector


Evaluation of results:
Evaluation criteria of results according to the cysteine 1:10 / lysine 1:50 prediction model.
Mean peptide depletion [%] Reactivity Evaluation
> 42.47 high reactivity positive
> 22.62 ≤ 42.47 moderate reactivity positive
> 6.38 ≤ 22.62 low reactivity positive
0- ≤ 6.385 minimal or no reactivity negative

Evaluation criteria of results according to the cysteine 1:10 prediction model.
mean Cys peptide depletion [%] Reactivity Evaluation
> 98.24 - ≤ 100 high reactivity positive
> 23.09 ≤ 98.24 moderate reactivity positive
> 13.89 ≤ 23.09 low reactivity positive
0 - ≤ 13.89 minimal or no reactivity negative

Acceptance criteria
The standard calibration curve should have an r² > 0.99
The measured values of solvent control samples should be 0.50 ± 0.05 mM
The variation coefficient (relative standard deviation, RSD) of measured values of the nine samples from sets B1, B2 and C should be < 15 %
The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
The mean peptide depletion value for the positive control 2,3-butanedione should be 10 % - 45 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
The standard deviation (SD) of the peptide depletion should be < 14.9 % for the per cent Cys-peptide and < 11.6 % for the per cent Lys-peptide, respectively
The mean peptide concentration of the three reference controls C in the solvent used for the test item stock solution should be 0.50 ± 0.05 mM.
If one of the acceptance criteria is not fulfilled, the test is repeated. If the result is unam-biguous even though one of the criteria is not met, the test may be considered valid by the study director without repetition of the test, but justification must be given.
If the mean percent depletion falls in the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run should be considered, as well as a third one in case of discordant results between the first two runs.







Key result
Parameter:
other: mean peptide depletion of both peptides (%)
Remarks on result:
other: results not yet available
Conclusions:
The skin sensitisation potential of the test item will be determined.
Executive summary:

A study will be performed in accordance with OECD TG 442C in order to determine the skin sensitization potential of the test item. The study was initiated in January 2020 but was not completed by the CRO in due time to complete the registration dossier. The study record will be updated as soon as the results will become available.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline for Testing of Chemicals TG. 442E (“In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT”)
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Details on study design:
Skin sensitisation (In vitro test system)
Cell line used: THP-1 cells, human monocytic leukemia cell line
Material and conditions:
- For cell culture standard laboratory equipment was used.
- Flow cytometer
- Culture medium: RPMI 1640: with L-glutamine, 25mM HEPES + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol
- Buffer: Phosphate Buffered Saline (DPBS) without Ca2+ / Mg2+ + 0.1% BSA
- Blocking Solution 0.01% Globulins Cohn fraction II,III
- Antibodies: FITC anti-human CD54 (DAKO/DAK-F714301); FITC mouse IgG1 (DAKO/DAKX092701); FITC anti-human CD86 (BD Pharmingen 555657)
- Antibody working solutions: for CD86 6 μL Antibody + 44 μL Buffer; for CD54 and lgG1 3 μL Antibody + 47 μL Buffer
- Reagent for cytotoxicity test: Propidium iodide

Controls used:
- Negative control: Lactic acid, 1000 μg/mL in culture medium
- Vehicle control: culture medium
- Positive control: 1- chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7), 4.0 μg/mL in 0.2% DMSO in culture medium
- Isotype control: In order to help distinguish non-specific staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1.

Selection of concentrations
In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test-substance preparation (0.5 μg/mL up to 500 μg/mL) corresponding to final test substance concentrations of 0.5 μg/mL up to 5000 μg/mL (taking the purity into account) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance was determined by linear regression from the concentration response curve .

Experimental procedure
Two independent, valid experiments were performed. In each experiment, duplicates of each test substance concentration were tested.
- Cell preparation:
THP-1 cells from the working cell bank were thawed and cultured in suspension using the cell medium under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) until for 5 passages but not longer than passage 30 prior to testing. For substance incubation, cells were seeded in 24-well plates (500 μL of 2.0 x 10^6 cells/mL cell suspensions).

- Test substance application for MTT and luciferase assay:
Treatment was performed by adding 500 μL of test-substance preparation to the cells, thus diluting the 2x concentrated test substance preparations to their final concentration and the cells to 1.0 x 10^6 cells/mL. After test substance application the plates were sealed with semipermeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 24 hours.
Each test substance concentration was visually inspected directly after application and after the exposure period of 24 hours in order to detect test-substance precipitates.

- Cell staining and flow cytometric analysis:
After visual inspection the cells werecollected by centrifugation and washed twice with 1 mL buffer. Cells were incubated with 600 μL of 0.01% Globulins Chon fraction II,III at 4°C for 15 min to block FC receptors (FcR). After FcR blocking, cells of each treatment condition were divided into 3 aliquotes in 96-well microtiter plates. Cells were centrifuged, supernatant was discarded and 50 μL working antibody solution was added to each pellet. Cell staining was performed at 4°C for 30 min in the dark. After staining the cells were washed twice with 200 μL buffer and finally re-suspended in 200 μL buffer. Before analysis in flow cytometer the cells were stained with 5 μL of PI.

Calculation and data evaluation:
The CV75-value (relative survival rate) was calculated by linear regression. The relative cell viability (PI staining) was calculated as a mean of the independent replicates. Analysis of the membrane markers was performed in 10,000 living cells, determined by PI staining. Concentrations inducing
viability less than 50% were not considered for further assessment of dendritic cell activation. Data evaluation is performed with mean fluorescence intensity (MFI) of chemical treated cells among the viable cells, with systematic isotype control use to quantify and remove non-specific antibody binding.

Acceptance criteria:
- A tested concentration is not to be further evaluated when relative viability is less than 50%.
- Cell viability of vehicle control cells must yield at least 90%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%.
- In the negative control (LA), RFI values of both CD86 and CD54 should not exceed the positive criteria (RFI CD86< 150% and RFI CD54 < 200%) and cell viability should be ≥ 50%.
- For all vehicle controls, the MFI ratio of both CD86 and CD54 to isotype controls should be ≥ 105%.
- The reactivity check of new thawed cells should produce a positive response in CD86 and CD54 for NiSO4 and DNCB and a negative response in CD86 and CD54 for LA.
- Positive, negative and vehicle control data should lie within the range of the historic data.

Evaluation of results:
The test substance is considered positive for activation of monocytic THP-1 cells when CD86 expression is increased ≥ 150% and/or CD54 expression increased ≥ 200% at any concentration in relation to vehicle control that do not reduce viability below 50% and reproduced in the same cell surface marker in at least two independent experiments and at least one concentration must lie above the borderline area (> 170% (CD86) or 220% (CD54)).






Key result
Parameter:
other: EC200% (CD54) (the concentration resulting in a RFI of 200%)
Remarks on result:
other: results not yet available
Conclusions:
The skin sensitisation potential of the test item will be determined.
Executive summary:

A study will be performed in accordance with OECD TG 442E in order to determine the skin sensitization potential of the test item. The study was initiated in January 2020 but was not completed by the CRO in due time to complete the registration dossier. The study record will be updated as soon as the results will become available.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
In accordance with the OECD guideline 442D, the maximum final test item concentration should be 2000 μM. For a test chemical which has no defined molecular weight, the final test item concentration 400 μg/mL can also be used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility).

Experimental Performance:
At the time of seeding the cells were 80 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05% EDTA. 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 83 000 (±10 %) cells/mL. 120 μL of the cell suspension (≙ 10 000 cells) were seeded in two clear flat bottom 96 well plates (one for viability and one 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. The treatment procedure was performed on both 96 well plates identically: 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.

For the evaluation of the viability, one of the plates was used:
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 removed 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. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5- Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow color) to its insoluble formazan (purple color) in living cells and therefore indicates the amount of living cells. After the measurement of the color change, the values were transferred in a validated spreadsheet for the calculation of the viability.

For the evaluation of the Luciferase induction, the second plate was used:
For the evaluation of the Luciferase expression 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® Reagent 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 luminometerFor calculation of the luciferase induction as well as the relative viability a validated Microsoft Excel® file was used.

Key result
Parameter:
other: fold Luciferase induction
Remarks on result:
other: results not yet available
Conclusions:
The skin sensitisation potential of the test item will be determined.
Executive summary:

A study will be performed in accordance with OECD TG 442D in order to determine the skin sensitization potential of the test item. The study was initiated in January 2020 but was not completed by the CRO in due time to complete the registration dossier. The study record will be updated as soon as the results will become available.

Justification for classification or non-classification