Registration Dossier
Registration Dossier
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EC number: 287-722-1 | CAS number: 85567-10-8
Endpoint summary
Administrative data
Description of key information
For the endpoint of skin sensitisation, an in chemico study, the direct peptide reactivity assay (DPRA) and two in vitro studies, the human cell line activation test (h-CLAT) and the KeratinoSens™ assay were perfomed according to the respective OECD TG 442C, E and D and under GLP. A WoE approach was applied.
DPRA
In the first in chemico Direct Peptide Reactive Assay (DPRA) the test item showed high reactivity towards the peptides.
h-CLAT
In the second study the test item did not upregulate the cell surface marker in at least two independent runs.
KeratinoSens™
In the third study, the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 05.10-2017.-26-01-2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD guideline 442E (In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
- Justification for non-LLNA method:
- In accordance with Annex VII of REACH regulation
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: yes
- Solubility and stability of the test substance in the solvent/vehicle: stable - Details on the study design:
- TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line (ATCC TIB-202)
TEST-SUBSTANCE PREPARATION
- Concentration dose finding assay 1: 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.81 µg/mL
- Concentration dose finding assay 2: 5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 µg/mL
- Concentrations: experiment 1 and 2: 5000, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49, 1395.41 µg/mL.
- Vehicle: 0.9% NaCl
CONTROLS
- Solvent control: 0.2% DMSO (v/v) in cell culture medium
- Positive control: 1-chloro-2-4-dinitrobenzene (DNCB) at a final conc. of 4.0 μg/mL
MEDIUM
- Culture medium: RPMI 1640: with 2 mM L-glutamine, 25mM HEPES + 10% FBS + 100 U/ml Penicillin/100 µg/mL Streptomycin + 0.05 mM 2-mercaptoethanol
- FACS Buffer: Phosphate Buffered Saline (DPBS) + 0.1% BSA
- Blocking buffer: FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction)
- Reagent for cytotoxicity test: Propidium iodide (Sigma)
EXPERIMENTAL PROCEDURE
- Replicates: 1
- Experiments: 2
- Exposure period: 24 ± 0.5 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin and 2 -mercaptethanol
ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 ± 0.5 hours after exposure
DATA EVALUATION
- CV75 calculation: Relative survival rate is calculated by linear extrapolation. This value is the substance concentration at which cell viability is 75% compared to the vehicle control.
- Relative cell viability: % relative cell viability = (number of living cells / total number of aquired cells)* 100
- Relative fluorescence intensity: RFI (%) = ( MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) / (MFI of vehicle control cells - MFI of vehicle isotype control cells) * 100
EVALUATION RESULTS
- Positive result: A test is considered to be positive when the dendritic cells are activated meaning that CD86 expression is increased ≥150% and/or CD54 expression increased ≥ 200% in relation to vehicle control in at least 2 independent experiments (cell vialbility ≥ 50%)
- Negative result: A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%.
ACCEPTANCE CRITERIA
The test meets acceptance criteria if:
- the cell viability of the solvent controls is >90%,
- the cell viability of at least four tested doses of the test item in each run is >50%,
- the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
- the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
- the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%. - Positive control results:
- The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments.
The threshold of 150% for CD86 (415% experiment 1; 376% experiment 2) and 200% for CD54 (312% experiment 1; 476% experiment 2) were clearly exceeded. - Key result
- Run / experiment:
- other: experiment 1
- Parameter:
- other: CD86 expression (%)
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: experiment 2
- Parameter:
- other: CD86 expression (%)
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: experiment 1
- Parameter:
- other: CD54 expression (%)
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: experiment 2
- Parameter:
- other: CD54 expression (%)
- Value:
- 200
- 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:
- The controls confirmed the validity of the study.
The viability of the solvent control was > 90% (96.3-98.3% experiment 1; 97.1-97.69% experiment 2).
The number of tested test item concentrations with cell viability > 50% was ≥ 4 (8 in experiment 1 and 8 in experiment 2).
The RFI for CD86 and CD54 of cells treated with the solvent DMSO was < 150% (131% experiment 1; 117% experiment 2) and ≤ 200% (113% experiment 1; 88% experiment 2).
The MFI ratio of the medium control and isotype IgG1control was ≥ 105% for CD86 (149% experiment 1; 206% experiment 2) and CD54 (129% experiment 1; 171% experiment 2).
The MFI ratio of the solvent control (DMSO) and isotype IgG1 control was ≥ 105% for CD86 (173% experiment 1; 2256% experiment 2) and CD54 (137% experiment 1; 163% experiment 2). - Interpretation of results:
- other: The study alone cannot be used for the classification purpose but in a WoE approach
- Conclusions:
- The test item did not upregulate the cell surface marker in at least two independant runs.
Therefore, the test item might be considered as non-sensitiser. - Executive summary:
In the current study the skin sensitisation potential of the test item was assessed according to the new OECD 442E TG and in compliance to GLP.
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.
Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.
The test item was dissolved in 0.9% NaCl. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. No CV75 was derived in the dose finding assay.
The main experiment was performed covering the following concentration steps:
5000, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49 and 1395.41 µg/mL
Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
No cytotoxicity effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 92.3% (CD86), 93.1% (CD54) and 92.1% (isotype IgG1 control) in the first experiment. and to 92.1% (CD86), 90.7% (CD54) and 89.8% (isotype IgG1 control).
The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item might be considered as non-sensitiser.
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (404% experiment 1; 317% experiment 2) and 200% for CD54 (476% experiment 1; 410% experiment 2) were clearly exceeded.
In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore, the test item might be considered as non-sensitiser.
The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in a weight of evidence approach in which minimally 2 key events of the adverse outcome pathway (AOP) are tested.
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Deviations:
- no
- Principles of method if other than guideline:
- - Principle of test: DPRA is supposed to address the molecular initiating event to the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic model peptides containing either lysine or cysteine. The percentage of depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between sensitizers and non sensitizers.
- The method is design to predict and classify the skin sensitising potential of a chemical by assessment of its reactivity towards a synthetic cysteine and lysine containing peptide, by measuring the depletion using HPLC. - GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Justification for non-LLNA method:
- In accordance with Annex VII of REACH regulation
- Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage.
The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared (solvent water). - Details on the study design:
- 1. Preparation of Peptides:
- 19.33 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (37.535 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 19.85 mg lysine peptide with an amino acid sequence of Ac RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (37.083 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.
2. Preparation of controls:
- Reference controls:
- Reference control A (undiluted) was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run
- Reference control B (undiluted) was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run
- Reference control C (undiluted) was set up for the test item and the positive control. RC C for the test item was prepared using the respective solvent used to solubilize the test item. RC C for the positive control was prepared using acetonitrile. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.
- Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution.. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
- Positive control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetronitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
3. Dose group:
- Reference Control C: undiluted
- Test item: 100 mM stock solution
- Positive control: 100 mM stock solution
4. Pre-experiment:
Solubiluty of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100mM).
The test item was dissolved in dist. water.
5. Experimental procedure:
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. - Key result
- Run / experiment:
- other: 1
- Parameter:
- other: Mean peptide depletion
- Value:
- 97.17
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes - Interpretation of results:
- other: The study alone cannot be used for the classification purpose but in a WoE approach
- Conclusions:
- In this study under the given conditions the test item showed high reactivity towards the peptides. Therefore, the test item is considered to have sensitising potential.
The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA - Executive summary:
In this in chemico study Direct Peptide Reactive Assay (DPRA), the skin sensitisation potential of the test item Reactive Yellow 42 was assessed according to the OECD 442C and under GLP without significant deviations.
Following a pre-experiment to determine the solubility of the test item in different solvent, the test item (in triplicate) was incubated at a concentration of 100 mM (65.248 mg/mL) with the peptides containing either cystiene or lysine for 24 +/- 2h at 25 +/- 2.5 °C.
After the incubation periode, the samples were analysed by HPLC in order to quantify the percentage of the mean peptide depletion.
No co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).
The mean depletion of both peptides was 97.17% showed a high reactivity towards the synthetic peptides. Based on the prediction model 1, the test item can be considered as a potential skin sensitiser. Although precipitation was observed prior to the HPLC analysis within the cysteine run, the results are considered as valid as the depletion is 97.17% and no underestimation of the test item reactivity is possible. Due to a minor co-elution in the lysine peptide experiment the true lysine and overall peptide depletion is suspected to be higher.
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of positive control on both peptides was 71.95%.
In conclusion, in this study under the given conditions the test item showed high reactivity towards the peptides. Therefore, the test item is considered to have sensitising potential.
The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in a weight of evidence approach in which minimally 2 key events of the adverse outcome pathway (AOP) are tested.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2018-04-04-2018-04-16
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Version / remarks:
- February 04, 2015
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Details on the study design:
- CELL LINE:
transgenic cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct.
- Source: KeratinoSens™ (Givaudan, Switzerland)
TEST-SUBSTANCE PREPARATION:
- Concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
- Stock: 200 mM
- Vehicle: dist. water
CONTROLS:
- Negative control: DMSO at a final concentration of 1% (v/v) in test item exposure medium
- Blank: blank well with no seeded cells
- Positive control: Cinnamic aldehyde dissolved in DMSO
MEDIUM:
- Maintenance Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
--10% fetal bovine calf serum
-- 1% geneticin (final concentration: 500 µg/mL)
- Assay Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
-- 10% fetal bovine calf serum
- Test Item Exposure Medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
-- 1% fetal bovine calf serum
EXPERIMENTAL PROCEDURE:
- Replicates: 3
- Experiments: 2
- Exposure period: 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2
- Luciferase assay:
-- Washing cells: once with DPBS
-- Incubation: 20 min at room temperature in the absence of light
- MTT assay:
-- 4 h at 37 °C ± 1 °C and 5% CO2
-- incubation overnight (experiment 1) or over the weekend (experiment 2) at 37 °C ± 1 °C and 5% CO2
ANALYSIS:
- Absorbance measurement: OD at λ = 600 nm
DATA EVALUATION
- two independent repetitions with three replicates for every concentration
- Calculation of Cell Viability:
Cell Viability [%] = ((V_sample-V_blank))/((V_solvent-V_blank))×100
Vsample = MTT absorbance reading in the test chemical well
Vblank = MTT absorbance reading in the blank well containing no cells and no treatment
Vsolvent = average MTT absorbance reading in the wells containing cells and solvent (negative) control
- Calculation of the Maximal Induction of the Luciferase Activity (Imax):
The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test item was calculated
Fold Induction = ((L_sample-L_blank))/((L_solvent-L_blank))
Lsample = luminescence reading in the test chemical well
Lblank = luminescence reading in the blank well containing no cells and no treatment
Lsolvent = luminescence reading in the wells containing cells and solvent (negative) control
- Calculation of the EC1.5:
The EC1.5 will be calculated by linear extrapolation and the overall EC1.5 was calculated as the geometric mean of the individual repetitions.
EC1.5 = (C_b-C_a )×((1.5-I_a)/(I_b-I_a ))+C_a
Ca = lowest concentration in µM with >1.5 fold induction
Cb = highest concentration in µM with <1.5 fold induction
Ia = fold-induction measured at the lowest concentration with >1.5 fold induction (mean of three replicate wells)
Ib = fold-induction measured at the highest concentration with <1.5 fold induction (mean of three replicate wells)
- Calculation of IC50 and IC30:
The IC50 and IC30 were calculated by linear extrapolation and the overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions.
ICx = (C_b-C_a )×(((100-x)-Va))/(Vb-Va ))+Ca
X=% reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca = the lowest concentration in µM with >X% reduction in cell viability
Cb = highest concentration in µM with
Vb = % viability at the highest concentration with
PREDICTION MODEL:
KeratinoSens™ prediction is considered positive if the following conditions will be met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.
ACCEPTANCE CRITERIA:
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition. - Positive control results:
- The positive controls passed the acceptance criteria
- Run / experiment:
- other: Experiment 1
- Parameter:
- other: lowest tested concentration (µM ) with a significant luciferase induction >1.5 (1.61)
- Value:
- 31.25
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: Experiment 2
- Parameter:
- other: lowest tested concentration (µM ) with a significant luciferase induction >1.5 (1.52)
- Value:
- 62.5
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Other effects / acceptance of results:
- The controls confirmed the validity of the study
- Interpretation of results:
- other: The study alone cannot be used for the classification purpose but in a WoE approach
- Conclusions:
- In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item might be a sensitiser.
- Executive summary:
In the current study the skin sensitising potential of the test item was assessed according to OECD 442D and in compliance with GLP.
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.
A set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:
2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.
In the first experiment, a max luciferase activity (Imax) induction of 31.56 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 65.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.61) was found to be 31.25 µM. The corresponding cell viability was >70% (117.4%).The calculated EC1.5was<1000 µM (18.95 µM).
In the second experiment, a max luciferase activity (Imax) induction of 21.17 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 41.7%. The lowest tested concentration with a significant luciferase induction >1.5 (1.52) was found to be 62.50 µM. The corresponding cell viability was >70% (90.4%).The calculated EC1.5was<1000 µM (60.23 µM).
A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as sensitiser.
The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in a weight of evidence approach in which minimally 2 key events of the adverse outcome pathway (AOP) are tested.
The controls confirmed the validity of the study.
Referenceopen allclose all
CD54 and CD86 Expression Experiment 1
| Sample | Conc (mg/ml) | Cell Viability (%) CD86 | Cell Viability (%) CD54 | RFI (%) CD 86 | RFI (%) CD54 |
| Medium control | 98.3 | 97.7 | 100 | 100 | |
| Solvent Control | 0.20% | 96.4 | 96.3 | 131 | 113 |
| DNCB | 4.0 | 82.0 | 80.4 | 404 | 476 |
| Test item | 5000 | 92.3 | 93.1 | -7 | 142 |
| 4166.67 | 93.4 | 92.7 | 2 | 163 | |
| 3472.22 | 93.0 | 93.5 | 13 | 168 | |
| 2893.52 | 93.5 | 92.1 | 12 | 175 | |
| 2411.27 | 93.3 | 93.9 | 18 | 119 | |
| 2009.39 | 94.5 | 93.3 | 21 | 99 | |
| 1674.49 | 94.4 | 95.0 | 22 | 88 | |
| 1395.41 | 93.8 | 93.5 | 32 | 92 |
CD54 and CD86 Expression Experiment 2
| Sample | Conc (mg/ml) | Cell Viability (%) CD86 | Cell Viability (%) CD54 | RFI (%) CD 86 | RFI (%) CD54 |
| Medium control | 97.2 | 97.4 | 100 | 100 | |
| Solvent Control | 0.20% | 97.6 | 97.4 | 117 | 88 |
| DNCB | 4.0 | 80.2 | 81.1 | 317 | 410 |
| test item | 5000 | 92.1 | 90.7 | -4 | 55 |
| 4166.67 | 93.2 | 92.5 | 3 | 96 | |
| 3472.22 | 93.1 | 92.6 | 7 | 105 | |
| 2893.52 | 93.1 | 93.3 | 18 | 95 | |
| 2411.27 | 94.6 | 95.0 | 11 | 69 | |
| 2009.39 | 95.0 | 94.7 | -2 | 24 | |
| 1674.49 | 95.2 | 94.6 | 28 | 65 | |
| 1395.41 | 95.4 | 95.3 | 28 | 60 |
1. Pre-experiments:
Solubility of the test item was determined. The test item was not soluble in acetonitrile but completely soluble in water.
No turbidity, precipitation and phase separation but an orange coloration of the solution was observed for the test item solutions. All test item preparations of the main experiment were prepared using water.
2. Precipitation and Phase separations:
All test item solutions were freshly prepared immediately prior to use.
A slight precipitation but not turbidity or phase separation was observed for the samples of the positive control. Samples were not centrifuged prior HPLC analysis, since the acceptance criteria for the depletion range of the positive control were fulfiled, the observed precipitations and phase separatios were regarded as insignificant.
3. Co-elution with the peptide peaks:
No relevant co-elution of the test item with the peptide peaks observed.
4. Results Calibration Curve: Cysteine and Lysine values of the Calibration curve:
| Sample | Cysteine peptide | Lysine peptide | ||
| Peak Area 220 nm | Peptide Concentration mM | Peak Area 220 nm | Peptide concentration mM | |
| STD1 | 4833.4658 | 0.5340 | 4509.2012 | 0.5340 |
| STD2 | 2490.8552 | 0.2670 | 2271.9509 | 0.2670 |
| STD3 | 1243.9002 | 0.1335 | 1158.2763 | 0.1335 |
| STD4 |
613.3855 | 0.0667 | 566.7153 | 0.0667 |
| STD5 | 307.0688 | 0.0334 | 284.8300 | 0.0334 |
| STD6 | 148.4497 | 0.0167 | 142.8151 | 0.0167 |
| STD7 | 0.0000 | 0.0000 | 0.0000 | 0.0000 |
Cysteine Peptide Calibration Curve: y = 9083.23x+12.56 ; R² = 0.9998
Lysine Peptide Calibration Curve: y = 8447.13x+7.62 ; R² = 0.9999
5. Results of Cysteine Peptide Depletion:
| Sample | Peak Area 220 nm | Peptide concentration (mM) | Peptide Depletion (%) | Mean Peptide Depletion (%) | SD of Peptide Depletion (%) | CV of Peptide Depletion (%) |
| Positive Control | 1325.0602 | 0.1445 | 71.28 | 71.95 | 0.69 | 0.01 |
| 1261.7949 | 0.1375 | 72.65 | ||||
| 1295.4968 | 0.1412 | 71.92 | ||||
| Test Item | 0 | - | 100.00 | 100.00 | 0.00 | 0.00 |
| 0 | - | 100.00 | ||||
| 0 | - | 100.00 |
6. Results of Lysine Peptide Depletion:
| Sample | Peak Area 220 nm | Peptide concentration (mM) | Peptide Depletion (%) | Mean Peptide Depletion (%) | SD of Peptide Depletion (%) | CV of Peptide Depletion (%) |
| Positive Control | 1766.7614 | 0.2083 | 56.65 | 56.76 | 1.06 | 0.02 |
| 1715.1394 | 0.2021 | 57.91 | ||||
| 1801.3400 | 0.2123 | 55.80 | ||||
| Test Item | 226.3366* | - | 100.00 | 100.00 | 0.00 | 0.00 |
| 222.4073* | - | 100.00 | ||||
| 234.6890* | - | 100.00 |
* Due to a (minor) co-elution in the range of the lysine peptide peak, these values are not considered as reflecting the actual remaining lysine peptide content. Therefore, the true peptde depletion is suspected to be higher.
7. Categorization of the Test item
| Prediction Model | Prediction Model 1 (Cyteine and Lysine Peptide/ Ration 1:10 and 1:50) | ||
| Test substance | Mean Peptide Depletion (%) | Reactivity Category | Prediction |
| Test item | 97.17* | High reactivity | Sensitiser |
| Positive Control | 64.37 | High reactivity | Sensitiser |
* Due to the above mentioned co-elution in the lysine peptide experiment the true peptide depletion is suspected to be higher.
Induction of Luciferase Activity – Overall Induction
| Concentration (microM) | Fold induction | ||||
| Experiment 1 | Experiment 2 | Mean | SD | ||
| Solvent Control | 1.00 | 1.00 | 1.00 | 0.00 | |
| Positive Control | 4.0 | 1.12 | 1.08 | 1.10 | 0.03 |
| 8.0 | 1.13 | 1.17 | 1.15 | 0.03 | |
| 16.0 | 1.29 | 1.44 | 1.37 | 0.10 | |
| 32.0 | 1.77 | 1.55 | 1.66* | 0.15 | |
| 64.0 | 3.42 | 2.26 | 2.84 | 0.83 | |
| Test Item | 0.98 | 1.43 | 1.07 | 1.25 | 0.25 |
| 1.95 | 1.47 | 0.90 | 1.18 | 0.40 | |
| 3.91 | 1.34 | 0.93 | 1.14 | 0.29 | |
| 7.81 | 1.45 | 1.00 | 1.22 | 0.32 | |
| 15.63 | 1.47 | 1.04 | 1.26 | 0.30 | |
| 31.25 | 1.61 | 1.26 | 1.44 | 0.25 | |
| 62.50 | 1.89 | 1.52 | 1.70 | 0.26 | |
| 125.00 | 1.57 | 1.56 | 1.57* | 0.00 | |
| 250.00 | 2.11 | 1.69 | 1.9* | 0.29 | |
| 500.00 | 3.17 | 4.03 | 3.6* | 0.61 | |
| 1000.00 | 12.47 | 21.17 | 16.82 | 6.15 | |
| 2000.00 | 31.56 | overflow | 31.56 | - | |
* = significant induction according to Student’s t-test, p<0.05
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
- Additional information:
DPRA
The test item (in triplicate) was incubated at a concentration of 100 mM (65.248 mg/mL) with the peptides containing either cysteine or lysine for 24 +/- 2h at 25 +/- 2.5 °C. After the incubation periode, the samples were analysed by HPLC in order to quantify the percentage of the mean peptide depletion. Sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control.
The mean depletion of both peptides was 97.17% showed a high reactivity towards the synthetic peptides. Based on the prediction model 1, the test item can be considered as a potential skin sensitiser.
h-CLAT
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.
The main experiment was performed covering the following concentration steps: 5000, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49 and 1395.41 µg/mL. Cells were incubated with the test item for 24h at 37°C.
Relative cell viability at the highest test item concentration was reduced to 92.3% (CD86), 93.1% (CD54) and 92.1% (isotype IgG1 control) in the first experiment and to 92.1% (CD86), 90.7% (CD54) and 89.8% (isotype IgG1 control) in a second experiment.
The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item might be considered as non-sensitiser.
KeratinoSens™
The in vitro KeratinoSens™ assay enables detection of the sensitising potential by measuring the activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.
The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.
In the first experiment, a max luciferase activity (Imax) induction of 31.56 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 65.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.61) was found to be 31.25 µM. The corresponding cell viability was >70% (117.4%).The calculated EC1.5was<1000 µM (18.95 µM). In the second experiment, a max luciferase activity (Imax) induction of 21.17 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 41.7%. The lowest tested concentration with a significant luciferase induction >1.5 (1.52) was found to be 62.50 µM. The corresponding cell viability was >70% (90.4%).The calculated EC1.5was<1000 µM (60.23 µM).
A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as sensitiser.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Skin sensitisation
In an in chemico Direct Peptide Reactive Assay (DPRA), sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control.
The mean depletion of both peptides was 97.17% showed a high reactivity towards the synthetic peptides. Based on the prediction model 1, the test item can be considered as a potential skin sensitiser.
In a second study, the skin sensitisation potential of the test item was assessed in the in vitro human cell line activation test (h-CLAT), which enables detection of the sensitising potential of a test item by measuring the dendritic cell activation by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.
In this study the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore, based on this study the test item is considered as a non-sensitiser.
In a third study, the sensitising potential was assessed in vitro by measuring the activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as sensitiser.
WoE approach
According to Regulation (EC) 1272/2008 (CLP), these in vitro/in chemico assays cannot be considered as stand alone tests, but the results from such tests can be used together with other data in a weight of evidence assessment.
Regarding the classification of the substance for skin sensitisation, the results of the three individual assays of the in chemico/in vitro skin sensitisation test battery (DPRA, h-CLAT and KeratinoSens) need to be taken together as they reflect three key events in the adverse outcome pathway (AOP) leading to skin sensitisation. For this reason, a WoE approach is applied for the test battery stating that: 'any two of the three tests determine the overall results, i.e. any two positive results drive the prediction of a sensitizer, while any two negative results drive the prediction of a test substance to be a non-sensitizer'.
Two positive results were obtained, in the DPRA and in the KeratinoSens, leading to the conclusion that the substance is probably a skin sensitizer.
In conclusion and based on this WoE approach, the substance should be considered to be sensitizer according to Regulation No 1272/2008, section 3.4.
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