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EC number: 200-389-9 | CAS number: 58-61-7
- 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:
- 20 - 24 October 2017
- 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:
- 2015
- Deviations:
- yes
- Remarks:
- Due to the nature of the test item and its incompatibility with any solvents known to be suitable for cysteine testing, this DPRA study was terminated prior to its completion.
- GLP compliance:
- no
- Remarks:
- Due to the nature of the test item and its incompatibility with any solvents known to be suitable for cysteine testing, this DPRA study was terminated prior to its completion.
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- Adenosine
- EC Number:
- 200-389-9
- EC Name:
- Adenosine
- Cas Number:
- 58-61-7
- Molecular formula:
- C10H13N5O4
- IUPAC Name:
- adenosine
- Reference substance name:
- Water
- EC Number:
- 231-791-2
- EC Name:
- Water
- Cas Number:
- 7732-18-5
- Molecular formula:
- H2O
- IUPAC Name:
- Oxidane
- Test material form:
- solid: particulate/powder
- Details on test material:
- Batch No.: 10004539
Storage at room temperature approx. 20°C
Constituent 1
impurity 1
In chemico test system
- Details on the study design:
- SUMMARY: DPRA measures the reaction of the test item with synthetic peptides containing cysteine (Ac-RFAACAA-COOH) or lysine (Ac-RFAAKAA-COOH). The custom peptides contained cysteine or lysine as the nucleophilic reaction centres and phenylalanine to facilitate HPLC detection. Test item and peptide were combined and incubated together for 24 h at room temperature. Following this incubation, the concentration of free (i.e. unreacted) peptide remaining was measured by HPLC immediately prior to the lysine peptide assay.
EXPERIMENTAL PROCEDURES
PEPTIDES:
Source: RS Synthesis
Batch:
- Lysine: Catalogue No. 110716-2, Batch No. P161108-LC107617, purity 98.14%
BUFFERS USED:
- Ammonium acetate buffer: ca 100 mM, pH 10.2
SOLUBILITY ASSESSMENT:
- NMP:DMSO (1:1, v/v) was selected as the most suitable solvent for the test material
PREPARATION PEPTIDE STOCK SOLUTIONS:
- LYSINE: stock solution of 0.667 mM in ammonium acetate buffer
LYSINE PEPTIDE ASSAY:
-PREPARATION: Adenosine was dissolved in NMP:DMSO (1:1, v/v) at a final concentration of 101 mM (101% of target). Cinnamic aldehyde was dissolved in acetonitrile at a final concentration of 101 mM (101% of target). All solutions were prepared immediately prior to use.
- PREPARATION OF THE STANDARD CURVE: Dilution buffer was prepared by mixing ammonium acetate buffer (pH 10.2, 8 mL) with acetonitrile (2 mL). Standard 1 (STD1-Lys) was prepared by mixing lysine peptide stock solution (1600 µL) with acetonitrile (400 µL). Serial dilutions (1:1, v/v) were prepared from this to make a standard curve (STD1-Lys to STD6-Lys). An additional sample containing only dilution buffer was included as a blank (0 mM) standard. The standard curve was analyzed by HPLC.
- REFERENCE CONTROL:Acetonitrile (250 µL) was mixed with lysine peptide stock solution (750 µL) Three replicates of this were produced for Reference Control A, which were used to determine the accuracy of the lysine peptide calibration curve.. Six replicates of Reference Control B were prepared as described for Reference Control A. Three replicates were analysed at the beginning of the testing run, and three at the end of the testing run, to demonstrate peptide stability over the analysis time. Two groups of Reference Control C samples were prepared containing either acetonitrile (250 µL) and lysine peptide stock (750 µL), or NMP:DMSO (1:1, v/v, 250 µL) and lysine peptide stock (750 µL). Three replicates of each were distributed throughout the run. These samples were included in the assay run together with the samples containing each test item to verify that the solvent does not impact upon peptide stability during the assay, and to calculate percentage peptide depletion.
- PEPTIDE ASSAY METHOD: TThe lysine assay contained a 1:50 molar ratio of peptide to test item. Cinnamic aldehyde (positive control) or test item (250 µL) were mixed with lysine peptide stock (750 µL). The vials were mixed by inversion. The co-elution control was prepared by mixing together ammonium acetate buffer (750 µL) and test item (250 µL). All vials were stored in the dark at ambient temperature for 24-36 h until analysed by HPLC.
CHROMATOGRAPHIC AND DETECTOR PARAMETERS
- Column: Phenomenex Luna C18 (2) (2 x 100 mm, 3 µm)
- Run Time: 20 min
- Mobile Phase Conditions: Mobile Phase A: trifluoracetic acid (0.1%, v/v) in Milli-Q H2O
Mobile Phase B: trifluoracetic acid (0.085%, v/v) in acetonitrile
- Flow Rate: 0.35 mL/min
- Column Temperature: 30°C
- Auto Sampler Temperature: Room temperature
- Injection Volume: 7 µL
- UV Wavelength: 220 nm
- HPLC Gradient: see below
CALCULATIONS:
The concentration of peptide remaining in each sample following incubation was calculated from integrated peak area, with reference to the peptide standard curve. Percent peptide depletion was calculated from the following formula:
Peptide Depletion (%) = 1 – ( Peak Area (Sample) / Mean Peak Area (Reference Control C)) x 100
Results and discussion
- Positive control results:
- The % lysine depletion value for the positive control was 48.6%
In vitro / in chemico
Results
- Run / experiment:
- other: DPRA cysteine and lysine prediction model
- Parameter:
- other: % Lysisne Peptide Depletion
- Value:
- -1.2
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks:
- Cinnamic aldehyde
- Remarks on result:
- not determinable
- Other effects / acceptance of results:
- System Suitability for the Lysine Assay:
The calibration linearity, r2, for the lysine standard curve was 0.9998. This met the acceptance criteria for r2 which was >0.990.
The mean peptide concentration of Reference Control A was 0.501 ± 0.009 mM (mean ± SD).
The calculated peptide concentration in the Reference Control C samples in acetonitrile was 0.499 ± 0.005 mM. The calculated peptide concentration in the Reference Control C samples in NMP:DMSO was 0.494 ± 0.007 mM. These controls met the acceptance criteria (0.5 ± 0.05 mM).
For the six Reference Control B and three Reference Control C samples in acetonitrile, the coefficient of variation (CV) was 1.1% (acceptance criteria for CV was <15%).
The mean percentage peptide depletion value of the three replicates for cinnamic aldehyde fell within the lower bound and upper bound values of 40.2% and 69.0% for lysine, with the SD <11.6%. The actual percentage peptide depletion value reported for cinnamic aldehyde was 48.6% ± 2.4% (mean ± SD).
Finally, the standard deviation of replicate test item samples was <11.6% for the test item (actual SD was 1.2%).
The time between sample preparation and the last injection of a sequence was not greater than 48 h.
Guideline deviations:
Due to the nature of the test item and its incompatibility with any solvents known to be suitable for cysteine testing, this DPRA study was terminated prior to its completion.
Adenosine binding with the lysine peptide resulted in -1.2% ± 1.2% peptide depletion and no co-elution of the test item with the lysine peptide was observed. Due to the nature of the test item and its incompatibility with any solvents known to be suitable for cysteine testing, this DPRA study was terminated prior to its completion.
Any other information on results incl. tables
Test Item | % Peptide Depletion Lysine (Mean ± SD) |
Adenosine |
-1.2 ± 1.2 |
Positive control (cinnamic aldehyde) |
48.6 ± 2.4 |
Classification cannot be determined by the lysine assay alone and, therefore, reactivity of Adenosine (CAS No. 58-61-7) was inconclusive.
Applicant's summary and conclusion
- Interpretation of results:
- other: Classification cannot be determined by the lysine assay alone and was inconclusive.
- Conclusions:
- In conclusion, the reactivity of Adenosine (CAS No. 58-61-7) was inconclusive due to the incompatibility of the test item.
- Executive summary:
Skin sensitisation is a type IV (delayed) hypersensitivity reaction that results from the interaction of a sensitising agent with host proteins to form an immunogenic complex.
Small molecules that can interact with proteins in this way are referred to as haptens, and are generally not immunogenic in isolation. Hapten-modified proteins are recognised as foreign by antigen presenting cells, leading to T-cell activation and localised inflammation at the site of all subsequent exposures to the hapten.
Most skin sensitising agents are electrophiles, i.e. will accept an electron pair from a nucleophile to form a covalent bond. The amino acids, cysteine and lysine, are thought to be the nucleophiles most frequently modified in proteins during sensitisation, and the ability of small molecules to react with these amino acids forms the basis of the Direct Peptide Reactivity Assay (DPRA).
The objective of this study was to assess the peptide binding capabilities of Adenosine (CAS No. 58-61-7) using synthetic cysteine and lysine peptides and to classify the test item to one of the four reactivity classes leading to a DPRA prediction according to the prediction model.
Due to the nature of the test item and its incompatibility with any solvents known to be suitable for cysteine testing, this DPRA study was terminated prior to its completion.
The solubility of the test item was assessed and 1 methyl-2-pyrrolidinone (NMP): dimethyl sulfoxide (DMSO), (1:1, v/v) chosen as the appropriate solvent. Test item and lysine peptide were combined and incubated together for ca 24-36 h at room temperature. Following this incubation, the concentration of free (i.e. unreacted) peptide remaining was measured by HPLC. The percent lysine peptide depletion values calculated for the test item are presented in the table below. All acceptance criteria for the lysine assay were met, however, classification cannot be determined by the lysine assay alone and, therefore, reactivity of Adenosine (CAS No. 58-61-7) was inconclusive.
Test Item
% Lysine Peptide Depletion (Mean ± SD) Adenosine -1.2 ± 1.2 Cinnamic Aldehyde (Positive Control) 48.6 ± 2.4 In conclusion, the reactivity of Adenosine (CAS No. 58-61-7) was inconclusive due to the incompatibility of the test item.
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