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EC number: 695-735-2 | CAS number: 68489-14-5
- 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:
- key study
- Study period:
- 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 guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of study:
- direct peptide reactivity assay (DPRA)
- Justification for non-LLNA method:
- The OECD TG 442 C may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Test material
- Reference substance name:
- ethyl 2-{[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]formamido}acetate
- EC Number:
- 695-735-2
- Cas Number:
- 68489-14-5
- Molecular formula:
- C15H27NO3
- IUPAC Name:
- ethyl 2-{[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]formamido}acetate
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: white crystalline powder, WS-5, batch no.: 80100039
- Expiration date of the batch: 16 January 2019
- Purity test date: 99.57%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry ambient temperature
The test article, WS-5 produced a visually clear solution at a concentration of 100 mM acetonitrile, which was the first of the listed vehicles specified in the protocol.
Formulations were prepared shortly before testing.
In chemico test system
- Details on the study design:
- Skin sensitisation (In chemico test system) - Details on study design:
Test Article Incubation:
Each test solution was prepared at ratios of 1:10 and 1:50 with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25°C for 24±2 hours, in the dark. At the end of the incubation period the samples were visually inspected for precipitate formation. Samples were centrifuged at 400 g for 5
minutes.
Analytical Method:
The remaining concentration of cysteine- or lysine-containing peptides following the 24-hour incubation period was measured by high performance liquid chromatography
(HPLC) with gradient elution and UV detection at 220 nm.
Reference and Co-elution Controls:
Reference controls were prepared for each peptide.
Reference Control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference Control B (six replicates) was used to verify the
stability of the reference controls over time and Reference Control C (in triplicate) was used to verify that acetonitrile did not impact the percent peptide depletion.
Co-elution controls were prepared to detect possible co-elution of the test article with the peptides.
Calibration Curves for Peptides:
Calibration curves were prepared for each peptide using concentrations of 0.0167, 0.0334, 0.0667, 0.1335, 0.267 and 0.534 mM (Standards 1 to 6)
Results and discussion
- Positive control results:
- Cinnamaldehyde (CAS No. 104-55-2, batch number MKBT8955V, purity 98.5%, expiry 29 February 2020) was used as the positive control. The positive control was dissolved in acetonitrile at a concentration of 100 mM. For results, please see table 2 and 3.
In vitro / in chemico
Results
- Key result
- Run / experiment:
- other: 3
- Parameter:
- other: The mean of cysteine and lysine percentage depletion
- Value:
- 3.73
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: DPRA prediction: negative (no or minimal reactivity) according to OECD 442C
- Remarks:
- Negative prediction in the Direct Peptide Reactivity Assay
- Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
The mean percent cysteine and percent lysine depletion value was calculated. Negative depletion was considered as “0” when calculating the mean. By using the
cysteine 1:10/lysine 1:50 prediction model below, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an IATA.
The percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%.
ACCEPTANCE OF RESULTS:
The following criteria should be met for a run to be considered valid:
- The standard calibration curve should have a r2 >0.99.
The r value for the standard calibration curve was 1.000 and as 0.9963 for lysine and cyteine depletion, respectively.
- The mean peptide concentration for reference controls A should be 0.50±0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference
controls B and C should be <15.0%.
For lysine and cysteine depletion, the mean Peptide Concentration (mM) for the Reference Controls A and C were as follows:
Reference Controls A - 0.50
- The mean PPD value of the three replicates for the positive control and maximum standard deviation (SD) must fall within the ranges:
Peptide Mean PPD Values (%) Lower Bound Mean PPD Values (%) Upper Bound SD
Cysteine 60.8 100 <14.9
Lysine 40.2 69.0 <11.6
- The maximum standard deviation for the test article replicates should be <14.9 for the percent cysteine depletion and <11.6 for the percent lysine depletion.
The SD for Mean PPD for lysine and cysteine depletion were 0.81 and 2.72 , respectively.
Any other information on results incl. tables
Table 1. Cysteine 1:10/lysine 1:50 Prediction Model used in this testing to discriminate between skin sensitisers and non-sensitisers in the framework of an IATA
Mean of Cysteine and Lysine % Depletion |
Reactivity Class |
DPRA Prediction |
0% ≤ mean % depletion ≤6.38% |
No or minimal reactivity |
Negative |
6.38% < mean % depletion ≤22.62% |
Low reactivity |
Positive |
22.62% < mean % depletion ≤42.47% |
Moderate reactivity |
|
42.47% < mean % depletion ≤100% |
High reactivity |
Table 2. The percentage peptide depletion (PPD) for lysine
Substance |
Replicate Peptide Peak Areas |
Reference Control C |
PPD |
Mean PPD |
SD |
WS-5 Test Article |
37.49 |
37.26 |
-0.62 # |
0.30 |
0.81 |
37.41 |
-0.40 # |
||||
36.93 |
0.89 |
||||
Cinnamaldehyde Positive Control |
17.63 |
37.26 |
52.68 |
50.35 |
4.61 |
17.39 |
53.33 |
||||
20.48 |
45.03 |
#negative value was considered as “0” when calculating the mean
Table 3. The percentage peptide depletion (PPD) for cysteine
Substance |
Replicate Peptide Peak Areas |
Reference Control C |
PPD |
Mean PPD |
SD |
WS-5 Test Article |
22.13 |
23.06 |
4.03 |
7.16 |
2.72 |
20.99 |
8.98 |
||||
21.11 |
8.46 |
||||
Cinnamaldehyde Positive Control |
8.15 |
23.06 |
64.66 |
65.50 |
0.74 |
7.89 |
65.78 |
||||
7.83 |
66.05 |
Applicant's summary and conclusion
- Interpretation of results:
- other: negative prediction for skin sensitisation
- Conclusions:
- The percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%. The test article, WS-5, was therefore considered to have no or minimal reactivity and gave a negative prediction in the Direct Peptide Reactivity Assay, performed according to OECD Guidelines for Testing of Chemicals Method 442C.
- Executive summary:
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptides following incubation with the test article.
The test article, WS-5 was dissolved in acetonitrile at a concentration of 100 mM. The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours in glass autosampler vials, protected from light and set at 25°C.
Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with UV detection.
Cysteine and lysine peptide percent depletion (PPD) values were then calculated and used in a prediction model which allows assigning the test article to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.
In presence of the test substance, WS-5, the percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%.
All analytical acceptance criteria for each peptide run were met and the positive control proved the validity of the test.
Conclusion:
The test article, WS-5, was considered to have no or minimal reactivity and gave a negative prediction in the Direct Peptide Reactivity Assay according to OECD TG 442 C.
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