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EC number: 680-413-6 | CAS number: 217437-44-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 28 Jun - 06 Jul 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Version / remarks:
- Adopted: 4 February 2015
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- 2-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]ethyl 2-methylprop-2-enoate
- EC Number:
- 680-413-6
- Cas Number:
- 217437-44-0
- Molecular formula:
- C12H17N3O3
- IUPAC Name:
- 2-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]ethyl 2-methylprop-2-enoate
Constituent 1
In chemico test system
- Details on the study design:
- TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.
TEST SYSTEM
- Supplier: JPT Peptide Technologies GmbH
- Synthetic cysteine-containing peptide: Ac-RFAACAA-COOH
- Synthetic lysine-containing peptide: Ac-RFAAKAA-COOH
SOLVENT CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Solvent: acetonitrile (ACN)
- For both the cysteine and lysine reactivity assay: 40.92 mg of test item was solved in 1628 μL ACN (100 mM solution). Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved.
POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Concentration: 100 mM in ACN
CONTROLS
- Reference control, calibration solution and co-elution control were prepared.
PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM
INCUBATION CONDITIONS OF THE TEST SUBSTANCE WITH THE PEPTIDE SOLUTIONS
- Peptide to test substance ratios: Cysteine-containing peptide: 1:10; Lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: at least 22.5 ± 0.5 h prior to initiation of the analysis run
NUMBER OF REPLICATES
for each peptide in triplicates
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: HPLC-PDA Method
- Column: Zorbax SB-C18, 100 mm x 2.1 mm, df = 3.5 μm (Agilent Technologies, Santa Clara, CA, USA)
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in Milli-Q water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Cysteine %B: 10, 25, 90, 90, 10, 10
Lysine %B: 10, 20, 90, 90, 10, 10
Time (min): 0, 10, 11, 13, 13.5, 20
- Detector Wavelength: Photodiode array detection, monitoring at 220 and 258 nm
- Calibration standard concentrations of both peptides: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 and 0.000 mM
- Column temperature: 30 °C
- Injection volume: 10 µL
Results and discussion
- Positive control results:
- - The mean percent cysteine-containing peptide depletion for the positive control cinnamic aldehyde was 71.2% ± 0.5%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
- The mean percent lysine-containing peptide depletion for the positive control cinnamic aldehyde was 48.5% ± 1.2%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).
In vitro / in chemico
Results
- Key result
- Run / experiment:
- other: Cysteine 1:10 / Lysine 1:50 Prediction Model
- Parameter:
- other: % depletion
- Remarks:
- mean of cysteine- and lysine-containg peptide
- Value:
- 27.1
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- moderate reactivity
- Other effects / acceptance of results:
- OTHER EFFECTS:
A phase separation was observed after the incubation period for lysine-containing peptide, one cannot be sure how much test item remained in the solution to react with the peptide. Consequently, it might be possible that the reactivity class for lysine-containing peptide is underestimated. No precipitate or phase separation was observed after the incubation of the synthetic peptide containing cysteine with the test item sample.
ACCEPTANCE OF RESULTS:
Acceptability of the Cysteine Reactivity Assay
- The correlation coefficient (r2) of the cysteine-containing peptide standard calibration curve was 0.995. Since the r2 was >0.99, the standard calibration curve was accepted.
- The mean peptide concentration of reference controls A was 0.533 ± 0.009 mM while the mean peptide concentration of reference controls C was 0.536 ± 0.011 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the percent cysteine-containing peptide depletion.
- The coefficient of variation (CV) of the peptide areas for the nine reference controls B and C was 3.3%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the reference control samples was 16.90. The mean A220/A258 ratio ± 10% range was 15.21-18.59. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The percent cysteine-containing peptide depletion was calculated versus the mean cysteine-containing peptide peak area of reference controls C. The mean percent cysteine-containing peptide depletion for the positive control cinnamic aldehyde was 71.2% ± 0.5%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
Acceptability of the Lysine Reactivity Assay
- The correlation coefficient (r2) of the lysine-containing peptide standard calibration curve was 0.995. Since the r2 was >0.99, the lysine-containing peptide standard calibration curve was accepted.
- The mean peptide concentration of reference controls A was 0.501 ± 0.005 mM while the mean peptide concentration of reference controls C was 0.465 ± 0.008 mM. The means of reference control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent lysine-containing peptide depletion.
- The CV of the peptide areas for the nine reference controls B and C was 3.4%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the reference control samples was 14.51. The mean A220/A258 ratio ± 10% range was 13.06-15.96. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The percent lysine-containing peptide depletion was calculated versus the mean lysine-containing peptide peak area of reference controls C. The mean percent lysine-containing peptide depletion for the positive control cinnamic aldehyde was 48.5% ± 1.2%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).
Any other information on results incl. tables
Table : DPRA Prediction and Reactivity Classification for the Test Item
|
% Cysteine-containing peptide depletion |
% Lysine-containing peptide depletion |
Mean of SPCC and SPCL depletion |
DPRA prediction and reactivity classification |
||
|
Mean |
SD |
Mean |
SD |
|
Cysteine 1:10 / Lysine 1:50 prediction model |
Test item |
51.3 |
1.7 |
2.9 |
4.6 |
27.1 |
Positive: Moderate reactivity |
SD = Standard Deviation
Applicant's summary and conclusion
- Interpretation of results:
- other: DPRA prediction: skin sensitising potential based on the key event “protein reactivity”.
- Conclusions:
- Under the conditions of the Direct Peptide Reactivity Assay the test substance showed moderate peptide reactivity. Skin sensitising potential was observed based on the key event “protein reactivity”.
- Executive summary:
The test substance gave a positive outcome in the DPRA and the KeratinoSensTM assay. These results indicate that it is likely that the test substance will interact with protein moieties in vivo and that the substance is able to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes. Both properties can potentially lead to skin sensitization in humans. This conclusion is supported by the positive DEREK assessment, which concludes on strong sensitizing properties related to the presence of an alpha,beta-unsaturated ester. Taken all data together it is concluded that it is sufficiently demonstrated that the test substance has skin sensitizing properties. The test substance should therefore be classified as a skin sensitizer (cat. 1). Currently, no conclusion on sub-category can be made based on in vitro studies. Please also refer to attachment in Section 13: Weight of Evidence for Skin Sensitisation (statement 2018).
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