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EC number: 812-549-0 | CAS number: 881539-89-5
- 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:
- 17 Oct - 09 Nov 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
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 in 2019
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt Rheinland-Pfalz, Mainz, Germany
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- disodium 4-[(1-hydroxyprop-2-en-1-yl)amino]-4'-(prop-2-enamido)-[1,1'-biphenyl]-2,2'-disulfonate
- EC Number:
- 812-549-0
- Cas Number:
- 881539-89-5
- Molecular formula:
- C18H14N2Na2O8S2
- IUPAC Name:
- disodium 4-[(1-hydroxyprop-2-en-1-yl)amino]-4'-(prop-2-enamido)-[1,1'-biphenyl]-2,2'-disulfonate
1
In chemico test system
- Details on the study design:
- Skin sensitisation (in chemico test system) - Details on study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test substance by quantifying the reactivity (covalent binding) of the test substance towards synthetic peptides containing either lysine or cysteine.
TEST METHOD
The DPRA is an in chemico procedure that addresses the molecular initiating event leading to skin sensitization, namely covalent protein binding, by quantifying the reactivity of test substances towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity. The result may be used to discriminate between skin sensitizers and non-sensitizers, as part of a weight of evidence approach.
TEST SYSTEM
- Supplier of synthetic peptides: RS synthesis, Louisville, USA
- Peptide stock solution preparation: Stock solutions of cysteine and lysine peptides were freshly prepared for each assay.
Cysteine-containing peptide:
- Concentration: 0.667 mM Cys-Peptide stock solution was prepared by dissolving 20 mg of the peptide in 40 mL phosphate buffer, pH 7.5.
Lysine-containing peptide:
- Concentration: 0.667 mM Lys-Peptide stock solution was prepared by dissolving 24.8 mg of the peptide in 48 mL ammonium acetate buffer, pH 10.2.
VEHICLE CONTROL
- Substance: Water for chromatography, H2O, Honeywell, HPLC grade (referred to as HPLC grade water)
- Justification for selecting vehicle: In a non-GLP pre-test the solubility of the test item was determined in (in descending order, until it was dissolved completely): 1) Acetonitrile (ACN); 2) HPLC grade water. Based on this non-GLP pre-test, water was found to be a suitable vehicle.
POSITIVE CONTROL
- Substance:
1) Cinnamaldehyde (CAS 104-55-2, food grade ≥ 95%, batch no. MKBT8955V, expiration date: Feb 2020) was used as a 100 mM (± 10%) solution in acetonitrile for the Cys-peptide.
2) 2,3-Butanedione (CAS 431-03-8, ≥ 99%, batch no. BCBW1565, expiration date: Oct 2020) was used as a 100 mM (± 10%) solution in acetonitrile for the Lys-peptide.
- Preparation: Positive controls were treated identically to the test substance. As cinnamaldehyde mixed with the Lys-peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-butanedione was used as positive control showing mid-range depletion for the Lys-peptide.
CO-ELUTION CONTROL
Sample prepared from the respective peptide buffer (100 mM Phosphate buffer, pH 7.5 or 100 mM ammonium acetate buffer, pH 10.2) and the test substance, but without peptide. It was included in the assay to detect possible co-elution of the test substance with either the lysine or the cysteine peptide.
REFERENCE CONTROLS
For both peptides, four sets of solvent controls using acetonitrile instead of test substance stock solution were prepared in triplicate (sets A, B1, B2, and C, in total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was used to calculate of the peptide depletion. Additionally, a solvent control containing HPLC grade water instead of test substance solution was prepared in triplicate and also incubated and analysed together with the samples and was used to calculate the depletion of the test substance.
TEST SUBSTANCE PREPARATION
The Cys-peptide samples were prepared in 1:10 molar ratio (0.5 mM peptide with 5 mM test substance solution).
The Lys-peptide samples were prepared in 1:50 molar ratio (0.5 mM peptide with 25 mM test substance solution) using the stock solutions described above. A final volume of 1 mL per sample was prepared for each sample.
The 100 mM test substance solution was prepared by dissolving 151.6 mg (Cys-peptide assay) and 151.9 mg (Lys-peptide assay) test substance in 3 mL HPLC grade water. The solution was vortexed until the test substance was dissolved completely.
INCUBATION CONDITIONS
- Temperature used during incubation: 25.0 °C
- Duration of incubation: 22 h
NUMBER OF REPLICATES
All sample preparations (including controls) were carried out in triplicate in one valid run of the assay.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
HPLC components: Solvent Rack SRD3400, Quaternary pump HPG-3400SD, Autosampler WPS-3000SL, Column compartment TCC-3000SD, Photometer VWD-3400RS (Thermo Fisher Scientific)
Column: An ACE Excel SuperC18 150x3 mm column with 3 μm particles and pre-column Phenomenex SecurityGuard C18, 4x3 mm was used. This column was selected because it delivers substantially better peak shape for the peptides than the Agilent Zorbax SB-C18 column recommended in the guideline OECD 442C.
Flow rate: 0.55 mL/min
Injection volume: 7 μL
Column temperature: 30 °C
Wavelength: 220 nm for quantitation and 258 nm as indicator for co-elution.
Results and discussion
- Positive control results:
- - The mean peptide depletion with 79.50% and a standard deviation (SD) of 0.52% of the 3 replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0% and SD < 14.9%, respectively, for the Cys-peptide.
- The mean peptide depletion with 23.78% and a SD of 1.60% of 3 replicates of the positive control 2,3-butanedione were in the acceptable range of 10.0 – 45.0% and SD < 11.6%, respectively, for the Lys-peptide.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: Cysteine/Lysine
- Parameter:
- other: mean peptide depletion (%)
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Remarks:
- due to co-elution of the test substance with the cysteine and lysine peptides
- Key result
- Run / experiment:
- other: Cysteine
- Parameter:
- other: mean peptide depletion (%)
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Remarks:
- due to co-elution of the test substance with the cysteine peptide
- Key result
- Run / experiment:
- other: Lysine
- Parameter:
- other: mean peptide depletion (%)
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Remarks:
- due to co-elution of the test substance with the lysine peptide
- Other effects / acceptance of results:
- Due to co-elution of the test substance with the model peptides, no percent peptide-depletion could be calculated for the test substance of both peptide-assays.
DEMONSTRATION OF TECHNICAL PROFICIENCY:
The ten proficiency chemicals listed in the guideline OECD 442C were tested.
All ten proficiency chemicals showed the expected DPRA prediction for both peptide assays. For the Cys-peptide assay nine of the ten chemicals and for the Lys-peptide assay all ten chemicals showed depletion values consistent with the classification reported in the OECD guideline.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control:
The mean peptide concentration of all solvent controls (Reference A and Reference C) were with 0.52 mM, 0.50 mM, and 0.49 mM for the Cys-peptide and 0.51 mM, 0.53 mM, and 0.52 mM for the Lys-peptide, respectively, in the acceptable range of 0.50 ± 0.05 mM.
The variation coefficients (RSD) of the measured values of Reference controls B and C in acetonitrile were in the acceptable range with 3.0% for the Cys-peptide and 0.9% for the Lys-peptide, respectively.
Hence, the acceptance criteria were met.
- Acceptance criteria met for positive control:
The mean peptide depletion with 79.50% and a standard deviation (SD) of 0.52% of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0% and SD < 14.9% for the Cys-peptide.
The mean peptide depletion with 23.78% and a SD of 1.60% of three replicates of the positive control 2,3-butanedione were in the acceptable range of 10.0 – 45.0% and SD < 11.6% for the Lys-peptide.
Hence, the acceptance criteria were met.
- Range of historical control data:
Positive control Cys-peptide depletion range: 67.09 – 98.37%
Positive control Lys-peptide depletion range: 13.66 – 41.94%
Applicant's summary and conclusion
- Interpretation of results:
- other: skin sensitising potential based on the key event “protein reactivity” not determinable
- Conclusions:
- Under the conditions of the present study, the test substance co-eluted with both synthetic model peptides. Thus, the DPRA prediction for the test substance is ”inconclusive” according to the test guideline OECD 442C.
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