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EC number: 845-622-0 | CAS number: 2050038-84-9
- 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:
- key study
- Study period:
- October 12, 2018 - April 9, 2019
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- GLP compliance:
- no
- Remarks:
- The procedures used here were adapted from OECD guideline 442C (In ChemicoSkin Sensitisation: Direct Peptide Reactivity Assay (DPRA)).
- Type of study:
- other: Direct Peptide Reactivity Assay (DPRA)
- Justification for non-LLNA method:
- The Direct Peptide Reactivity Assay was used to assess the skin sensitizing potential of the test articles. Synthetic peptides containing cysteine or lysine were reacted with the test articles for 24± 2 hours. After the incubation period, the extent of peptide depletion was analyzed using High Performance Liquid Chromatography (HPLC) coupled with ultra-violet (UV) spectrometric detection.
Test material
- Reference substance name:
- 12-[(3R,4S)-4-ethylpyrrolidin-3-yl]-1,5,7,10-tetraazatricyclo[7.3.0.0²,⁶]dodeca-2(6),3,7,9,11-pentaene dihydrochloride
- EC Number:
- 845-622-0
- Cas Number:
- 2050038-84-9
- Molecular formula:
- C14H19Cl2N5
- IUPAC Name:
- 12-[(3R,4S)-4-ethylpyrrolidin-3-yl]-1,5,7,10-tetraazatricyclo[7.3.0.0²,⁶]dodeca-2(6),3,7,9,11-pentaene dihydrochloride
- Test material form:
- solid: particulate/powder
Constituent 1
In chemico test system
- Details on the study design:
- Test Article Preparation:
The test articles were prepared at 100 mM concentrations in an appropriate solvent. Calculations using the molecular weight and purity of the test article were performed to determine the appropriate amount of test article to weigh out in order to achieve approximately 3 mL of the 100 mM sample. On the day of testing, the test articles were weighed into prelabeled glass vials and stored at room temperature until time to perform the assay.
Test Article Solubility Test:
A solubility test was performed for the test articles in order to determine an appropriate solvent that completely dissolved each test article at a 100 mM concentration.The test articles was found to be soluble in water with brief vortexing.
Peptide Preparation:
Custom synthetic peptides containing cysteine or lysine as the reactive centers (with phenylalanine to aid in detection) were used in this assay. The purity of each peptide was at least 90%. Peptide samples were newly prepared for the sample set, and a single preparation of the peptide was used throughout the sample set. The cysteine peptide was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 7.5±0.1 phosphate buffer. The actual pH of the phosphate buffer on the day of testing was 7.5. The lysine peptide was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 10.2±0.1 acetate buffer. The actual pH of the acetate buffer on the day of testing was 10.1. The peptide solutions were gently mixed on the shaker.
Peptide Standards:
A set of serially diluted standards were prepared for each peptide. The top stock of the standards (0.534 mM) was prepared from the 0.667 mM peptide solution in acetonitrile. The remaining standards were prepared by serial dilution in dilution buffer (20% acetonitrile in phosphate buffer for the cysteine peptide or acetate buffer for the lysine peptide). Six standards were prepared at concentrations of 0.534-0.017 mM. A seventh standard was prepared containing only dilution buffer. Approximately 1 mL of each standard was pipetted into the appropriate prelabeled autosampler vials.
Controls:
The positive control used in this assay was cinnamic aldehyde prepared at a concentration of 100 mM. The positive control was reacted with the peptides in the same fashion as the test articles. There were three sets of reference controls of acetonitrile run at different points throughout the assay (reference controls A-C). Triplicate reference controls were also prepared for each solvent used in the assay. These controls consist of the solvent (acetonitrileor isopropanol) reacted with the peptide in the absence of test article. A coelution control was also prepared for the test article.The coelution control consisted of the test article without the peptide. The purpose of the coelution control was to determine if the test article elution from the HPLC column overlapped with the peptide elution.
Reaction Mixture Preparation:
Prior to testing, the test article was diluted in the appropriate solvent to yield a 100 mM test article concentration. The test article dilutions were mixed as determined during the solubility test (vortexing). The final dosing solutions was prepared for the test article, positive control, and reference control in the pre-labeled auto sampler vials. Triplicate samples were prepared for the test articles and controls. A single sample was prepared for each coelution control.
HPLC Set-up and Operation:
The separations module used in this assay was a Waters 2695 HPLC system. This system consisted of a solvent management system for the mobile phases and a sample management system for the test articles and controls. The HPLC system was coupled to a photodiode array detector set at 220 nm. The column used was a Zorbax SB-C18 column (Agilent) with dimensions of 2.1 mm x 100 mm x 3.5 micron. The column was primed for at least two hours before the start of the assay. To prime the column, equal parts of mobile phase A (0.1% trifluoroacetic acid in HPLC grade water) and mobile phase B (0.08% trifluoroacetic acid in HPLC grade acetonitrile) were passed through the column.
Once the column was primed for at least two hours, and the samples were prepared, the autosampler vials were placed into the designated locations of the separations module carousels. The samples were incubated in the dark at room temperature for 24±2 hours.
A gradient elution was used in this assay. The mobile phase changed from 10-25% acetonitrile over a 10 minute period to allow for sample separation and gradually elute most of the sample from the column. This was followed by a rapid increase to 90% acetonitrile to remove anything remaining on the column. The column was allowed to equilibrate back to initial specs for 7 minutes between injections.
The Empower3software was used to convert the absorbance data from the UV detector into chromatograms of intensity versus retention time for the samples and controls. At the end of the run, each chromatogram was integrated in order for the software to calculate the area under the peptide peak. Cysteine and lysine elute from the column at known times, so it was possible to determine which peaks in the chromatograms represented the peptides and use the areas under those peaks for the subsequent calculations.
Results and discussion
- Positive control results:
- The positive control results for cinnamic aldehyde were a % mean peptide depletion of cysteine of 74.56 and lysine of 67.46.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Parameter:
- other: % Mean Peptide Depletion of Cysteine
- Value:
- 10.51
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Parameter:
- other: % Mean Peptide Depletion of Lysine
- Value:
- 13.08
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Parameter:
- other: % Mean Peptide Depletion of Cysteine and Lysine
- Value:
- 11.79
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
Applicant's summary and conclusion
- Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- According to the results from the direct peptide reactivity assay, the test material was considered to be a sensitizer and therefore, met the GHS criteria for category 1 classification.
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