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EC number: 300-491-4 | CAS number: 93940-93-3
- 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:
- 2017-10-11 to 2017-10-27
- 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:
- 04 February 2015
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper
- EC Number:
- 300-491-4
- EC Name:
- Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper
- Cas Number:
- 93940-93-3
- Molecular formula:
- C10H20CuN2O6
- IUPAC Name:
- bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper
- Test material form:
- solid: particulate/powder
Constituent 1
In chemico test system
- Details on the study design:
- TEST-SUBSTANCE PREPARATION
- Stock solution: 84.5 mM
- Vehicle: water
- Reason for the vehicle: The test substance is soluble in water
CONTROLS
- Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
- Co-elution control: buffer and test substance without the peptide
- Positive control: Cinnamic aldehyde in acetonitrile
PEPTIDES
- Synthetic peptides:
-- Cysteine- (C-) containing peptide: Ac-RFAACAA
-- Lysine- (K-) containing peptide: Ac-RFAAKAA
- Stock solution:
-- C-containing peptide: 0.667 mM of peptide in pH 7.5 phosphate buffer
-- K-containing peptide: 0.667 mM of peptide in pH 10.2 ammonium acetate buffer
EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Determination remaining non-depleted peptide concentration: HPLC at 220nm: HPLC analysis started 22 to 26 hours after sample preparation and the analysis time was less than 30 hours.
- Calibration samples: samples of a known peptide concentration are measured in parallel
PREPARATIONS SAMPLES
- Calibration sample was prepared from the peptide stock solution in 20% acetonitrile in the respective buffer using serial concentration: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 or 0.000 mM peptide
- Test-substance samples: samples were incubated at 25°C +/- 2.5°C in the dark for 24 +/- 2 hours and visually investigated for any precipitate that may occur during the exposure period.
- Reference controls, co-elution controls as well as the positive control were set up in parallel (see table below)
MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC/DAD Agilent 1200 series with Chemstation
Wavelength: 220 nm for quantification and 258 nm as indicator of co-elution
Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 µm Agilent
Pre-Column: Phenomenex AJO 4286, 4 x 3 mm
Colunm temperature: 30°C
Sample temperature: 25°C
Run time: 20 min
Injection volume: 10 µL
- Detector: UV detector
DATA EVALUATION
- Calculation of the peptide concentrations: peptide concentration (mM) = [peak area (mAU * s) - b] /m
with b: axis intercept and m: slope
- The percent peptide depletion (PPD) was calculated according to the following formula:
PPD = [ 1 – ( Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in the Reference Control C)] * 100
ACCEPTANCE CRITERIA
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.
EVALUATION RESULTS
- Chemical reactivity was determined by mean peptide depletion [%] and was rated as
-- high: mean peptide depletion > 42.47
-- moderate: mean peptide depletion > 22.62 ≤ 42.47
-- low: mean peptide depletion > 6.38 ≤ 22.62
-- minimal: mean peptide depletion ≤ 6.38
High, moderate and low reactivity are evaluated as positive.
- In case the mean peptide depletion cannot be determined due to invalid K-peptide depletion the
evaluation is performed as follows:
-- high: mean peptide depletion > 98.24
-- moderate: mean peptide depletion > 23.09 ≤ 98.24
-- low: mean peptide depletion > 13.89 ≤ 23.09
-- minimal: mean peptide depletion ≤ 13.89
High, moderate and low reactivity are evaluated as positive.
Results and discussion
- Positive control results:
- Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation was regarded as insignificant.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: cysteine
- Parameter:
- other: mean depletion % of peptide
- Value:
- 100
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: lysine
- Parameter:
- other: mean depletion % of peptide
- Value:
- 2.66
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: lysine and cysteine
- Parameter:
- other: mean depletion % of peptide
- Value:
- 51.33
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- The mean peptide depletion of the positive control was 75.35% (cysteine) and 56.98% (lysine)
Any other information on results incl. tables
Results of the Cysteine Peptide Depletion
Cysteine Peptide |
||||||
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
809.6354 |
0.1341 |
74.93 |
75.35 |
0.40 |
0.53 |
794.7175 |
0.1317 |
75.39 |
||||
783.8397 |
0.1299 |
75.72 |
||||
Test Item |
0.0000 |
0.0036 |
100.00 |
100.00 |
0.00 |
0.00 |
0.0000 |
0.0036 |
100.00 |
||||
0.0000 |
0.0036 |
100.00 |
Results of the Lysine Peptide Depletion
Lysine Peptide |
||||||
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1768.9691 |
0.2117 |
57.97 |
56.98 |
1.07 |
1.87 |
1804.7944 |
0.2160 |
57.12 |
||||
1858.1759 |
0.2224 |
55.85 |
||||
Test Item |
4077.2800 |
0.4880 |
1.87 |
2.66 |
0.84 |
31.54 |
4007.8833 |
0.4797 |
3.54 |
||||
4048.2310 |
0.4846 |
2.57 |
Categorization of the Test Item
Prediction Model |
Prediction Model 1 |
Prediction Model 2 |
||||
Test Substance |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Test Item |
51.33 |
High Reactivity |
sensitiser |
100.00 |
High Reactivity |
sensitizer |
Positive Control |
66.16 |
High Reactivity |
sensitizer |
75.35 |
Moderate Reactivity |
sensitizer |
Applicant's summary and conclusion
- Interpretation of results:
- other:
- Remarks:
- The study alone cannot be used for the classification purpose but in a WoE approach of minimally 2 in vitro assessment representing the key events in AOP
- Conclusions:
- In a DPRA study, the test item showed high reactivity towards the cysteine peptide. Based on these results the test item might be considered to have a sensitising potential.
- Executive summary:
In the current study the skin sensitisation effect of the test item was assessed in an in chemico assay according to OECD 442C and in compliance to GLP. The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
In the present study the test item was dissolved in water, based on the results of the pre-experiments. A blue- violet solution was obtained.
Based on a molecular weight of 327.82 g/mol a 84.5 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.
For all experiments no turbidity, precipitation or phase separation was observed when diluted with the cysteine or lysine peptide solution respectively.
After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any samples. Precipitation was observed for the samples of the positive control and co-elution of the positive control. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation was regarded as insignificant.
No co-elution of test item with the peptide peaks was observed. Sensitising 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 Cwater).
The 84.5 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 51.33 %, which is > 6.38% cut-off. According to the evaluation criteria in the guideline, if a test chemical is tested in concentration < 100 mM, a positive result can still be used to support the identification of the test chemical as a skin sensitiser. Based on the prediction model 1 the test item can be considered as sensitiser.
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.16%.
In conclusion, in this study, under the given conditions the test item was shown to have skin 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.
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