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EC number: 225-403-0 | CAS number: 4826-71-5
- 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:
- January 16, 2017 - January 18, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Protocol no.154 ECVAM DB ALM
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- calcium tetrahydrate chloride [2-(trimethylazaniumyl)ethyl] phosphate
- Cas Number:
- 72556-74-2
- Molecular formula:
- C5H21CaClNO8P
- IUPAC Name:
- calcium tetrahydrate chloride [2-(trimethylazaniumyl)ethyl] phosphate
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
In chemico test system
- Details on the study design:
- - Positive control: 100 mM cinnamaldehyde (CAS 104-55-2, Sigma Aldrich batch no. MKBT8955V) (i.e. 5 or 25 mM as final concentration in the reaction mixtures, respectively with cysteine and lysine).
- Co-elution control: 100 mM test item in the appropiate buffer
- 4 references made with 0.500 mM (final concentration in the reaction mixtures) peptides solutions in acetonitrile or in the test item solvent were included in the analysis:
1) Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy,
2) Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time.
3) Reference control C: prepared with acetonitrile, the positive control solvent, in order to check its influence on the peptide stability.
4) Reference control C': prepared with water, the test item solvent, in order to check its influence on the peptide stability.
- Test system: Cysteine peptide (RS Synthesis, LLC; ref. Ac RFAACAA-COOH; batch no. 160113; purity 95%) and Lysine peptide (RS Synthesis, LLC; ref. Ac RFAACAA-COOH; batch no. 160113; purity 91.2%).
Analytical method:
- Waters e2695 HPLC (Waters 2489 UV detector), cortecs column C18 2.7 µm, dimensions 2.1 x 100 mm
The HPLC column was installed and equilibrated at 30ºC with 50 % of phase A and 50% of phase B, for at least 20 minutes before use. The gradient was performed at least one time before to use the column.
- Conditions:
Mobile phase A: trifluoroacetic acid at 0.1% (v/v) in water; flow rate: 0.21 mL/min; injection volume: 7 μL; wavelength: 220 nm; sequence: 0, 10, 11, 13, 13.5 and 20 minutes.
Mobile phase B: trifluoroacetic acid at 0.085% (v/v) in acetonitrile; flow rate: 0.21 mL/min; injection volume: 7 μL; wavelength: 220 nm; sequence: 0, 10, 11, 13, 13.5 and 20 minutes.
- Method validation parameters: Linearity (R2 ≥ 0.999586) meet the criterion of approval (R2 ≥ 0.99).
A complete sequence was performed for each sample.
The column was re-equilibrated to initial conditions (90% Phase A and 10% of phase B) at least 4 minutes between each injection.
Sequence of the analysis:
The analysis was programmed according to the following principles:
- The reference controls B were placed at the beginning and the end of the analysis (3 repetitions).
- The reference controls C were placed at the beginning of each repetition.
- The standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence.
Interpretation of the results:
- 0.00 % ≤ mean of cysteine and lysine % depletion ≤ 6.38 % = No or minimal reactivity = Negative DPRA Prediction
- 6.38 % ≤ mean of cysteine and lysine % depletion ≤ 22.62 % = Low reactivity = Positive DPRA Prediction
- 22.62 % ≤ mean of cysteine and lysine % depletion ≤ 42.47 % = Moderate reactivity = Positive DPRA Prediction
- 42.47 % ≤ mean of cysteine and lysine % depletion ≤ 100 % = High reactivity = Positive DPRA Prediction
Results and discussion
- Positive control results:
- The depletion mean of cinnamaldehyde was 51.08 for Lysine and 76.99 for Cystine.
In vitro / in chemico
Resultsopen allclose all
- Run / experiment:
- mean
- Parameter:
- other: % Lysine peptide depletion
- Value:
- 0.3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- mean
- Parameter:
- other: % Cystine peptide depletion
- Value:
- 0.68
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- mean
- Parameter:
- other: % depletion
- Value:
- 0.42
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the expected DPRA prediction for the 10 proficiency substances was obtained. The resulted cysteine and lysine depletion values fall within the respective reference range for 9 out of the 10 proficiency substances for each peptide (8 out of 10 expected in the OECD guide line).
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference control: The mean concentration of the peptides for the Reference control A and for the Reference control C met the concentration validity criteria mM: 0.500 ± 0.05 mM.
Reference control B/C the CV of the 9 controls B and C were less than 15% (%CV lysine= 1.03, %CV cystine= 1.60)
reference control C the mean
- Acceptance criteria met for positive control: yes, the depletion mean was 51.08 for Lysine (validity criteria between 40.2% and 69.4%) and 76.99 for Cystine (validity criteria between 60.8% and 100%)
- Acceptance criteria met for variability between replicate measurements: the SD of measurements were 0.28 for Lysine (SD valitidy criteria <11.6%) and 0.68 for Cysteine (SD validity criteria >14.9%)
Any other information on results incl. tables
The depletion rate was calculated for each type of peptide according to the following formula:
Depletion % = [1- (Peptide peak area with the replicate injection/mean peptide peak area with the C base control)] x 100
Table 1. Reference control results.
|
Lysine Peptide |
Cysteine Peptide |
|
|
Concentration (nM) |
Concentration (nM) |
Concentration validity criteria (mM) |
Reference A |
0.508 |
0.500 |
0.500±0.050 |
* Reference C |
0.510 |
0.476 |
|
* Reference C’ |
0.490 |
0.472 |
* Reference C: positive control diluant, i.e. acetonitrile
* Reference C’: test item diluent, i.e. water
Table 2. Coefficient of variation between reference controls B and C.
|
CV % |
CV % |
CV validity criteria |
Reference B/C |
1.03 |
1.60 |
< 15% |
Table 3. Positive control results.
Cinnamaldehyde |
Depletion in Lysine Peptide % |
Depletion in Cysteine Peptide % |
Repetition 1 |
47.43 |
79.53 |
Repetition 1 |
54.48 |
76.39 |
Repetition 1 |
51.35 |
75.04 |
Mean |
51.08 |
76.99 |
Depletion validity criteria |
40.2 < Depletion > 69.4 |
60.8 < Depletion > 100 |
Table 4. Test item results.
|
Depletion in Lysine Peptide % |
Depletion in Cysteine Peptide % |
Mean Depletion % |
Repetition 1 |
0.36 |
0.30 |
|
Repetition 1 |
0.55 |
0 |
|
Repetition 1 |
0 |
1.29 |
|
Mean |
0.30 |
0.53 |
0.42 |
SD |
0.28 |
0.68 |
|
SD Validity criteria |
< 11.6% |
< 14.9% |
|
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item showed mean depletion of 0.30% for Lysine and 0.53% for Cysteine, i.e. an overall average of 0.42%, reflecting no or minimal reactivity, therefore a negative prediction of DPRA.
- Executive summary:
To support the identification of the sensitization potential of the test item the method DPRA has been performed according to OECD 442C, following GLP. The method is based on the interaction between the test item and lysine or cysteine rich peptides detected with a high performance liquid chromatography (HPLC). Samples were dissolved immediately before use. Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively. The remaining concentration of peptides is measured after 24 hours of incubating with the test item at 25ºC. It is measured with the UV detector of the HPLC system, after gradient elution, at 220 nm. The test item showed mean depletion of 0.30% for Lysine and 0.53% for Cysteine, i.e. an overall average of 0.42%, reflecting no or minimal reactivity, therefore a negative prediction of DPRA.
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