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Description of key information

In an integrated approach adressing key events of the skin sensitisation AOP, results from an in chemico skin sensitisation study according to OECD guideline 442 C (DPRA, negative), from an in vitro ARE-Nrf2 Luciferase Test according to OECD guideline 442 D (KeratinoSens, negative) and an in vitro human Cell Line Activation Test according to OECD guideline 442E (h-CLAT, positive) were combined and predicted a non-skin sensitising potential of the test item.

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:
weight of evidence
Study period:
2020-07-20 to 2020-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: EU method B.59: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)
Version / remarks:
2017-02-14
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2020-06-26
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
The 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 for the assessment of the skin sensitisation potential of chemicals.
Details on the study design:
TEST CHEMICALS
Synthetic peptides:
- Peptides with 95.8 % (cysteine) and 93.84 % (lysine) purity
- Sequence cysteine peptides: Ac-RFAACAA-OH (MW = 750.9 g/mol)
- Sequence lysine-Peptide: Ac-RFAAKAA-OH (MW = 775.9 g/mol)

Buffers:
- Phosphate buffer pH 7.5 ± 0.05
- Ammonium acetate buffer pH 10.2 ± 0.05

Peptide Stock Solutions:
- Cysteine peptide stock solution: 0.667 mM, 0.501 mg/mL
- Lysine peptide stock solution: 0.667 mM, 0.518 mg/mL

Control samples:
- Reference control A (system suitability): Peptide stock solutions combined with acetonitrile, 3 replicates
- Reference control B (stability of the peptides): Peptide stock solutions combined with acetonitrile and measured before and after the reaction, 3 replicates
- Reference control C (solvent control): Peptide stock solutions combined with the respective solvent of the test item (and acetonitrile in case of cysteine peptide), 3 replicates
- Positive control (cinnamaldehyde): Peptide stock solutions combined with positive control stock solution (100 mM) (and acetonitrile in case of cysteine peptide), 3 replicates
- Co-elution controls (test item and peptide coelution): Test item stock solution (and acetonitrile in case of cysteine peptide) combined with the respective buffer solutions in each run

Calibration solutions:
Six calibration standard points were prepared by serial dilution of the peptide stock solutions with the following nominal molarities: STD 1 = 0.534 mM, STD 2 = 0.267 mM, STD 3 = 0.1335 mM, STD 4 = 0.0667 mM, STD 5 = 0.0334 mM and STD 6 = 0.0167 mM. As dilution buffer a 20% acetonitrile buffer solution (phosphate or ammonium acetate) was used. For the zero standard point (STD 7 = 0 mM) dilution buffer was used.

TREATMENT
Peptide stock solutions (750 µL) were combined with test item/positive control/solvent stock solutions (50 µL for cysteine peptides or 250 µL for lysine peptides) were combined and placed to the HPLC autosampler for 24 ± 2 h incubation at 22.5 - 30 °C in the dark. HPLC analysis of the batch of reaction samples started 24 ± 2 h hours after the test chemical was added to the peptide solution. The batches consisted of 2 parts: one part with the A reference controls, the calibration standards and the co-elution controls. These samples could be run before the 24 ± 2 h incubation time ends and right before the other part started or right after the other part. The other part contained the B and C reference controls, the positive controls and the reaction samples and these samples were run right after the 24 ± 2 h incubation time ended. The total HPLC analysis time was less than 21 hours. 3 replicates of reaction samples were measured.

TECHNICAL EQUIPMENT
- HPLC: Shimadzu LC-2030i Prominence (serial number L21445402951AE)
- Detector: D2 lamp (220 nm)
- Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 μm)
- Column temperature: 30°C
- Sample temperature: 25°C
- Injection volume: 7 µL
- System equilibration: running mobile phase A and mobile phase B in a ratio of 1:1 for 2 hours at 30°C column temperature and running the gradient twice before injecting the first sample
- Run time: 20 min
- Mobile Phase A: 0.100 % (v/v) trifluoroacetic acid in ultrapure water
- Mobile Phase B: 0.085 % (v/v) trifluoroacetic acid in acetonitrile
Positive control results:
The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 65.49 % ± 0.31 % and a mean lysine peptide depletion value of 52.71 % ± 0.87 %.
Key result
Run / experiment:
other: 1
Parameter:
other: mean peptide depletion (%) for cysteine
Value:
9.4
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1
Parameter:
other: mean peptide depletion (%) for lysine
Value:
1.27
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1
Parameter:
other: mean peptide depletion of both peptides (%)
Value:
5.34
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

Co-elution: The test chemical did not absorb at 220 nm significantly (since the peak of co-elution control did not reach 10 % compared to the respective reference control) when tested with the cysteine and lysine peptides. Therefore, no co-elution was observed with either of the peptides. The range of retention time for cysteine peptide was between 8.229 and 8.458 and the range of retention time for lysine peptide was between 6.126 and 6.283.


 


System suitability: Reference control A replicates were included in the HPLC run sequence to verify the HPLC system suitability prior to analysis. The mean peptide concentration of reference control A sample replicates was 0.50 mM for the cysteine and 0.49 mM for the lysine peptide.


 


Table 2: Cysteine peptide depletion values for the positive control and the test item















































Sample



Peptide conc. Calculated (mM)



Mean peptide conc. (mM)



Peptide depletion (%)



Standard deviation (SD)


for peptide depletion (%)



CINNAMALDEHYDE,


rep I



0.16



 


 


0.16



65.72 %



 


 


0.31 %



CINNAMALDEHYDE,


rep II



0.16



65.14 %



CINNAMALDEHYDE,


rep III



0.16



65.62 %



Test item, rep I



0.46



 


0.44



4.82 %



 


7.27 %



Test item, rep II



0.40



17.78 %



Test item, rep III



0.46



5.59 %



 


Table 3: Lysine peptide depletion values for the positive control and the test item















































Sample



Peptide conc. Calculated (mM)



Mean peptide conc. (mM)



Peptide depletion (%)



Standard deviation (SD)


for peptide depletion (%)



CINNAMALDEHYDE,


rep I



0.23



 


 


0.23



53.68 %



 


 


0.87 %



CINNAMALDEHYDE,


rep II



0.24



51.99 %



CINNAMALDEHYDE,


rep III



0.23



52.46 %



Test item, rep I



0.48



 


0.49



2.09 %



 


0.71 %



Test item, rep II



0.49



0.82 %



Test item, rep III



0.49



0.91 %



 

Interpretation of results:
other: No peptide reactivity
Conclusions:
Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item showed no or minimal reactivity towards the synthetic peptides; thus is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.
Executive summary:

In the course of this study, the skin sensitisation potential of the test item was studied using the Direct Peptide Reactivity Assay method (DPRA Method). For the test chemical and positive control substance, in order to derive a prediction, two independent tests were conducted, one with cysteine and lysine peptides each. The results of the two valid runs were used for the classification of the test item. Peptide depletion which resulted from the positive control cinnamaldehyde was 65.49 % ± 0.31 % for cysteine peptides and 52.71 % ± 0.87 % for lysine peptides, fulfilling all acceptance criteria for the positive control. The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for cysteine (0.48 – 0.50 mM) and lysine peptides (0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 1.9 % and 0.4 % percentages for cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 9.40 % ± 7.27 % while the percent lysine peptide depletion was 1.27 % ± 0.71 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 5.34 %, which did not exceed the 6.38 % threshold of the applicable prediction model and fell into the no or minimal reactivity class.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-07-14 to 2020-09-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: B.71 COMMISSION REGULATION (EU) 2019/1390 of 31 July 2019 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008
Version / remarks:
2019-07-31
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for Testing of Chemicals TG. 442E (“In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT”)
Version / remarks:
2018-06-25
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
The human Cell Line Activation Test (h-CLAT) can be used as part of a testing battery (including e.g. the Direct Peptide Reactivity assay and the ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.
Details on the study design:
TEST SYSTEM
- Cell line used: THP-1 cells, human monocytic leukemia cell line

TECHNICAL EQUIPMENT
Beckman Coulter Flow Cytometer, CytoFLEX System B4-R0-V0, blue laser (488 nm), FSC, SSC, FL1 (FITC, 525/40 BP) and FL3 (PC5.5, 690/50 BP) filters

CHEMICALS, BUFFER and MEDIA
- Cell culture medium: RPMI-1640 modified medium (with 25 mM HEPES buffer) supplemented with 10 (v/v) % fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2.05 mM L-glutamine solution and 0.05 mM 2-mercaptoethanol
- Buffer: Phosphate Buffered Saline (DPBS) + 0.1% BSA
- Blocking Solution: 0.01 (w/v) % globulin Cohn fraction solution in buffer
- Staining solution: 0.0125 mg/mL of propidium iodide solution in DPBS
- Antibodies: FITC anti-human CD54 (Dako, Batch: 20067597); FITC mouse IgG1 (Dako, Batch: 20069190); FITC anti-human CD86 (BD Pharmingen, Batch: 9322850)
- Antibody working solutions: for CD86 6 μL Antibody + 44 μL Buffer; for CD54 and lgG1 3 μL Antibody + 47 μL Buffer

CONTROLS
- Negative control: Cell culture medium
- Solvent control: 0.2% DMSO (solvent for positive control), NaCl solution (for test item)
- Positive control: 4.0 μg/mL 1-chloro-2,4-dinitrobenzene

PRE-EXPERIMENT: In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 8 final test substance concentrations of 7.8 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 .

MAIN EXPERIMENT:
- Replicates: Three independent, valid experiments were performed. In each experiment, duplicates of each test substance concentration were tested.
- Concentrations: The test item was first diluted to the concentration corresponding to the CV75 × 1.2 value (1260.3 μg/mL) determined in the dose finding assays. 8 concentrations were used for exposure: 351.6, 422.0, 506.4, 607.6, 729.2, 875.0, 1050.0 and 1260.0 µL/mL
- Test item exposure: Test item and control substances were mixed with 500 μL of suspended cells (1 × 106 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24±0.5 hours at at approximately 37° C and 5 % CO2.
- FITC staining: After 24 ± 0.5 hours of exposure, cells were transferred from the 24-well plate into sample tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by centrifugation (300 g, 5 min, 4 ºC). The washing step was repeated once more with 1 mL of FACS buffer. After washing, cells were blocked with 600 μL of 1 × blocking solution and incubated at approximately 4°C for 15 min. After blocking, cells were split in three aliquots of 200 μL into sample tubes. After centrifugation (300 g, 5 min, 4 ºC), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at approximately 4° C for 30 min. After washing twice with 150 μL of FACS buffer, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.
- RFI determination: The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1. A total of 10,000 living cells (PI negative) were acquired (when the cell viability was low, up to 30,000 cells including dead cells could be acquired). Alternatively, data could be acquired for one minute after the initiation of the analysis. The cell viability was automatically calculated by the cytometer analysis program. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 marked positive control (ctrl) and chemical-treated cells were calculated according to the following equation: RFI = (MFI of chemical-treated cells (CD86 / CD54) – MFI of chemical- treated isotype control cells) / (MFI of solvent-treated control cells (CD86 / CD54) – MFI of solvent-treated isotype control cells) × 100
The cell viability from the isotype control (ctrl) cells (which are stained with mouse IgG1 (isotype) antibodies) was also noted.
Key result
Run / experiment:
other: median of 3 runs
Parameter:
other: EC150% (CD86) (the concentration resulting in a RFI of 150%) in µg/mL
Remarks:
median of 3 runs
Value:
551
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: median of 3 runs
Parameter:
other: EC200% (CD54) (the concentration resulting in a RFI of 200%) in µg/mL
Remarks:
median of 3 runs
Value:
709.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Run 3
Parameter:
other: EC150% (CD86) (the concentration resulting in a RFI of 150%) in µg/mL
Value:
394.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Run 2
Parameter:
other: EC150% (CD86) (the concentration resulting in a RFI of 150%) in µg/mL
Value:
561.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Run 1
Parameter:
other: EC150% (CD86) (the concentration resulting in a RFI of 150%) in µg/mL
Value:
551
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Run 3
Parameter:
other: EC200% (CD54) (the concentration resulting in a RFI of 200%) in µg/mL
Value:
403.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Run 2
Parameter:
other: EC200% (CD54) (the concentration resulting in a RFI of 200%) in µg/mL
Value:
761.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Run 1
Parameter:
other: EC200% (CD54) (the concentration resulting in a RFI of 200%) in µg/mL
Value:
709.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in APPENDIX II of the Test Guideline 442E, the laboratory demonstrated technical proficiency by correctly obtaining the expected h-CLAT prediction for the 10 proficiency substances recommended in the OECD TG 442E and by obtaining CV75, EC150 and EC200 values that fell within the respective reference ranges. Moreover, a historical database of data generated with the reactivity checks and with the positive and solvent/vehicle controls is maintained over time to confirm the reproducibility of the test method in the laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
Interpretation of results:
other: h-CLAT activation observed
Conclusions:
Based on these results and the h-CLAT prediction model, the test item demonstrated an in vitro sensitizing potential under the experimental conditions of the human Cell Line Activation Test.
Executive summary:

In the course of this study, the skin sensitisation potential of the test item was studied using the human Cell Line Activation Test (h-CLAT). The extent of cytotoxicity induced in THP-1 cells by the test item was studied in five dose finding tests. Since no cytotoxicity was observed in the first run, the concentration range was shifted to start from the highest concentration recommended by the OECD test guideline 442E (5000 μg/mL). Since in the second run cytotoxicity was observed at the higher tested concentrations, lower concentration range was used in further dose finding tests in order to be able to determine CV75 value more precisely. Based on the overall results of the dose finding tests, eight doses between 1260.3 μg/mL and 351.7 μg/mL (nominal concentrations) were used for the main test in four independent runs out of which three runs were concluded valid. The increase in CD86 marker expression (RFI) was greater than 150 % at several tested doses (with >50 % of cell viability) compared to the respective negative controls in all independent valid runs. Based on the three positive results out of three valid runs, CD86 marker expression was concluded to be concordantly positive. Thus, the effective concentration for CD86 expression (EC150) was determined by linear interpolation, the EC150 value for CD86 expression was 551.0 μg/mL. The increase of CD54 marker expression (RFI) was greater than 200 % compared to the respective negative controls at several tested concentrations (with >50 % of cell viability) in all independent valid runs. Based on the concordant results of the three valid individual runs for CD54 expression, the prediction was concluded positive. Thus, the effective concentration for CD54 expression (EC200) was determined by linear interpolation, the EC200 value for CD54 expression was
709.5 μg/mL. Since the CD86 and CD54 markers gave positive results in all independent valid runs, the overall h-CLAT prediction was concluded to be positive, as well.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-07-13 to 2020-09-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: EU-Method B.60 of the Commission Regulation (EU) No. 2017/735 adopted 14. Feb. 2017: “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method”
Version / remarks:
2017-02-14
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2018-06-25
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The ARE-Nrf2 luciferase test method can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), direct peptide reactivity assay (DPRA)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.
Details on the study design:
MEDIA
- Maintenance (culture) medium: DMEM supplemented with 9.0 (v/v) % fetal bovine serum (FBS) and ~ 500 μg/mL G418.
- Thawing medium: DMEM containing 9.1 (v/v) % FBS without G418
- Exposure medium: DMEM containing 1 (v/v) % FBS without G418

CONTROLS
- Positive control: Trans-Cinnamaldehyd (4 to 64 μM)
- Negative/solvent control: 1% DMSO

EXPERIMENTAL PROCEDURE
- Replicates: 2 individual tests, three replicates per test for luciferase activity and one for cell viability

- Test concentrations: For the test item, twelve doses ranging from 2000 μM to 0.98 μM were used in two independent tests.

- Preparation of cells: At the time of seeding, the cells were 80 % confluent. After quantification, ≙ 10 000 cells per well were seeded in thawing medium in 96 well plates. Cells were grown for 24 ± 0.5 hours at 37 ± 1 °C in the presence of 5 % CO2.

- Exposure: 50 μL of 4× test item/PK/NK master solution and 150 μL of exposure medium was added to each well. The plates were sealed with breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2.

- Luciferase activity measurements: After the 48-hour exposure time with the test item and control substances, cells were washed with DPBS (270 μL). 1× lysis buffer (20 μL) was added to each well for 20 minutes at room temperature. Plates with the cell lysate were then placed in the luminometer for reading. First, the luciferase substrate (50 μL) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.

- Cytotoxicity: After 48 h, incubation medium was replaced with MTT working solution (200 μL, approx. 595 µg/mL) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilised by the addition of isopropanol (50 μL). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer. 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.
Positive control results:
The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at several concentrations in both tests. The EC1.5 values of the positive control fell between 7 μM and 30 μM (13 μM and 9 μM in the first and second tests respectively). The average inductions in the parallel plates for Trans-Cinnamaldehyde at 64 μM were 8.90 fold and 38.56 fold in the first and second tests, respectively. In both tests the luciferase activity induction was outside of the 2 – 8-fold induction range, but there was a clear dose response relationship in the luciferase activity induction for the positive control, therefore both tests were accepted as valid. There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the tested concentrations in either tests.
Key result
Run / experiment:
other: Second run
Parameter:
other: fold Luciferase induction
Value:
0.99
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: First run
Parameter:
other: fold Luciferase induction
Value:
0.93
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:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in the OECD Test Guideline 442D, the laboratory demonstrated technical proficiency, using the 10 Proficiency Substances listed in APPENDIX IA - ANNEX 1 of TG 442D. Moreover, a historical database of data generated with the positive control is maintained over time to confirm the reproducibility of the test method in the laboratory.
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:
other: no activation of keratinocytes
Conclusions:
Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative under the experimental conditions of the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).
Executive summary:

In the course of this study, the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method). For the test item and positive control substance, in order to derive a prediction, two independent tests were conducted. Since the results of the two runs were concordant and met the acceptance criteria, a third run was not needed to derive a conclusion. The luciferase activity induction obtained with the positive control, Trans- Cinnamaldehyde, was statistically significant above the threshold of 1.5- fold in both tests. For the test item, twelve doses ranging from 2000 μM to 0.98 μM were used in both tests. The test item induced no cytotoxicity (viability < 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, IC30 and IC50 values were calculated in none of the tests. Both tests were concluded negative, meaning that the induction values of the test item did not exceed the 1.5-fold threshold, therefore EC1.5 values could not be determined. Moreover, no dose response could be observed in any of the tests. Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

DPRA, WoE


In the course of this study, the skin sensitisation potential of the test item was studied using the Direct Peptide Reactivity Assay method (OECD guideline 442C). For the test chemical and positive control substance, in order to derive a prediction, two independent tests were conducted, one with cysteine and lysine peptides each. The results of the two valid runs were used for the classification of the test item. Peptide depletion which resulted from the positive control cinnamaldehyde was 65.49 % ± 0.31 % for cysteine peptides and 52.71 % ± 0.87 % for lysine peptides, fulfilling all acceptance criteria for the positive control. The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for cysteine (0.48 – 0.50 mM) and lysine peptides (0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 1.9 % and 0.4 % percentages for cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 9.40 % ± 7.27 % while the percent lysine peptide depletion was 1.27 % ± 0.71 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 5.34 %, which did not exceed the 6.38 % threshold of the applicable prediction model and fell into the no or minimal reactivity class.


 


ARE-Nrf2 Luciferase Test Method, WoE


In the course of this study, the skin sensitization potential of the test item was studied using the KeratinoSens™ method (OECD guideline 442D). For the test item and positive control substance, in order to derive a prediction, two independent tests were conducted. Since the results of the two runs were concordant and met the acceptance criteria, a third run was not needed to derive a conclusion. The luciferase activity induction obtained with the positive control, Trans- Cinnamaldehyde, was statistically significant above the threshold of 1.5- fold in both tests. For the test item, twelve doses ranging from 2000 μM to 0.98 μM were used in both tests. The test item induced no cytotoxicity (viability < 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, IC30 and IC50 values were calculated in none of the tests. Both tests were concluded negative, meaning that the induction values of the test item did not exceed the 1.5-fold threshold, therefore EC1.5 values could not be determined. Moreover, no dose response could be observed in any of the tests. Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).


 


h-CLAT, WoE


In the course of this study, the skin sensitisation potential of the test item was studied using the human Cell Line Activation Test (OECD guideline 442E). The extent of cytotoxicity induced in THP-1 cells by the test item was studied in five dose finding tests. Since no cytotoxicity was observed in the first run, the concentration range was shifted to start from at the highest concentration recommended by the OECD test guideline 442E (5000 μg/mL). Since in the second run cytotoxicity was observed at the higher tested concentrations, lower concentration range was used in further dose finding tests in order to be able to determine CV75 value more precisely. Based on the overall results of the dose finding tests, eight doses between 1260.3 μg/mL and 351.7 μg/mL (nominal concentrations) were used for the main test in four independent runs out of which three runs were concluded valid. The increase in CD86 marker expression (RFI) was greater than 150 % at several tested doses (with >50 % of cell viability) compared to the respective negative controls in all independent valid runs. Based on the three positive results out of three valid runs, CD86 marker expression was concluded to be concordantly positive. Thus, the effective concentration for CD86 expression (EC150) was determined by linear interpolation, the EC150 value for CD86 expression was 551.0 μg/mL. The increase of CD54 marker expression (RFI) was greater than 200 % compared to the respective negative controls at several tested concentrations (with >50 % of cell viability) in all independent valid runs. Based on the concordant results of the three valid individual runs for CD54 expression, the prediction was concluded positive. Thus, the effective concentration for CD54 expression (EC200) was determined by linear interpolation, the EC200 value for CD54 expression was 709.5 μg/mL. Since the CD86 and CD54 markers gave positive results in all independent valid runs, the overall h-CLAT prediction was concluded to be positive, as well.


 


Conclusion: In summary, it can be concluded that the test substance has no skin sensitising properties.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, it is concluded that the test item does not have skin sensitising potential and therefore shall not be classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/1182.