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EC number: 253-379-1 | CAS number: 37172-53-5
- 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
- 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 guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Version / remarks:
- Adopted 4 February 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
- Version / remarks:
- 12 January 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Justification for non-LLNA method:
- The DPRA has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an IATA to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Test material
- Reference substance name:
- Methyl 2-hexyl-3-oxocyclopentanecarboxylate
- EC Number:
- 253-379-1
- EC Name:
- Methyl 2-hexyl-3-oxocyclopentanecarboxylate
- Cas Number:
- 37172-53-5
- Molecular formula:
- C13H22O3
- IUPAC Name:
- methyl 2-hexyl-3-oxocyclopentane-1-carboxylate
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- Description: Colourless to pale yellow liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020
In chemico test system
- Details on the study design:
- TEST SYSTEM
- Test system: Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA).
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Source: JPT Peptide Technologies GmbH
- Batch SPCC: > 95%; Lot. No.: 111016HS_MHeW1017
- Batch SPCL: > 95%; Lot. No.: 120514HSDW_W1117
- Storage: All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.
TEST ITEM PREPARATION
- The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage.
- The test item was pre-weighed into a glass vial and was dissolved in acetonitrile, an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.
- Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
- Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
EXPERIMENTAL DESIGN
- The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Samples were prepared according to the scheme described in Table 1 in ‘Any other information on materials and methods incl. tables’.
- Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400x g) to force precipitates to the bottom of the vial. After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using a HPLC procedure.
PREPARATION OF SOLUTIONS FOR CYSTEINE REACTIVITY ASSAY
- Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: 20.07 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.92 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- SPCC Reference Control Solutions: Three SPCC reference control (RC) solutions (RC A, RC B and RC C) were prepared according to Table 1 in ‘Any other information on materials and methods incl. tables’.
- SPCC Calibration Curve: A SPCC calibration curve was prepared as described in Table 2 under ‘Any other information on materials and methods incl. tables’.
- Co-elution Control, Test Item and Positive Control Samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in Table 1 under ‘Any other information on materials and methods incl. tables’.
PREPARATION OF SOLUTIONS FOR LYSINE REACTIVITY ASSAY
- 20.42 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (38.78 mL) to reach a concentration of 0.667 mM.
- SPCL Reference Control Solutions: Three SPCL reference control (RC) solutions (RC A, RC B and RC C) were prepared according to Table 1 in ‘Any other information on materials and methods incl. tables’.
- SPCL Calibration Curve: A SPCL peptide calibration curve was prepared as described in Table 2 under ‘Any other information on materials and methods incl. tables’.
- Co-elution Control, Test Item and Positive Control Samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in Table 1 under ‘Any other information on materials and methods incl. tables’.
HPLC-PDA ANALYSIS
- Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
- HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day.
- The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.
ACCEPTABILITY 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.
ANALYSIS
DATA EVALUATION
- The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions.
- The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was checked for consistency between reference control and test item samples. If this ratio was not consistent a co-elution was assumed and the evaluation would be adjusted accordingly.
- Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser, if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as “0” when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM.
DATA INTERPRETATION
- By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) shown in Table 3 in ‘Any other information materials and methods incl. tables’, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate or high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA. In the framework of an IATA the test substance may be considered as non-sensitiser to skin, if the mean depletion of both peptides is below 6.38%. See Table 3 in ‘Any other information on materials and methods incl. tables’.
- In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible. If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive". In cases where the co-elution occurs only with the lysine peptide prediction model 2 can be applied (cysteine 1:10 prediction model) see Table 4 in ‘Any other information materials and methods incl. tables’.
Results and discussion
- Positive control results:
- CYSTEINE REACTIVITY ASSAY (Table 1 and 3 in 'Any other information on results incl. tables')
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 69.51% ± 0.61%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
LYSINE REACTIVITY ASSAY (Table 2 and 4 in 'Any other information on results incl. tables')
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 61.12% ± 1.28%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).
In vitro / in chemico
Resultsopen allclose all
- Key result
- Parameter:
- other: Mean of SPCC and SPCL depletion (%)
- Value:
- 0
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Remarks on result:
- no indication of skin sensitisation
- Parameter:
- other: Cysteine reactivity
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Parameter:
- other: Lysine reactivity
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Other effects / acceptance of results:
- PRE-EXPERIMENT
Solubility of the test item was determined prior to the main experiment. The test item was soluble in acetonitrile. No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using acetonitrile. All test item solutions were freshly prepared immediately prior to use.
MAIN EXPERIMENT
- Precipitation: For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. 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 of the samples.
- Phase separation: For the 100 mM stock solution of the test item phase separation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the test item and the positive control (including the co-elution control).
- Co-elution with peptide peaks: No co-elution of the test item with any of the peptide peaks was observed.
- Depletion of the Cysteine and Lysine Peptides: The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. See Table 1 and 2 in ‘Any other information on results incl. tables’.
Any other information on results incl. tables
Table 1. Depletion of the Cysteine Peptide
Cysteine Peptide |
||||||
Sample |
Peak Area at 220 nm |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1471.0265 |
0.1596 |
69.33 |
|
|
|
1486.4678 |
0.1613 |
69.01 |
69.51 |
0.61 |
0.88 |
|
1429.7356 |
0.1551 |
70.19 |
|
|
|
|
Test Item |
4806.7178 |
0.5218 |
0.00 |
|
|
|
4824.4810 |
0.5238 |
0.00 |
0.00 |
0.00 |
- |
|
4837.3389 |
0.5252 |
0.00 |
|
|
|
Table 2. Depletion of the Lysine Peptide
Lysine Peptide |
||||||
Sample |
Peak Area at 220 nm |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1548.8514 |
0.1877 |
62.22 |
|
|
|
1581.2108 |
0.1916 |
61.43 |
61.12 |
1.28 |
2.09 |
|
1651.4639 |
0.2002 |
59.72 |
|
|
|
|
Test Item |
4150.5811 |
0.5047 |
0.00 |
|
|
|
4159.9512 |
0.5058 |
0.00 |
0.00 |
0.00 |
- |
|
4154.1421 |
0.5051 |
0.00 |
|
|
|
Table 3. Results of the Reference Controls for the Cysteine Peptide
Cysteine Peptide Run |
||||||||
Sample |
Peptide Peak Area |
Peptide Concentration [mM] |
||||||
PA |
Mean |
SD |
CV [%] |
Peptide Concentration |
Mean |
SD |
CV [%] |
|
Reference A 1 |
4794.1187 |
4762.6523 |
74.2400 |
1.56 |
0.5205 |
0.5170 |
0.0081 |
1.56 |
Reference A 2 |
4815.9771 |
0.5228 |
||||||
Reference A 3 |
4677.8613 |
0.5078 |
||||||
Reference B 1 |
4733.2197 |
4788.7638 |
81.7780 |
1.71 |
0.5138 |
0.5199 |
0.0089 |
1.71 |
Reference B 2 |
4766.4439 |
0.5175 |
||||||
Reference B 3 |
4777.8418 |
0.5187 |
||||||
Reference B 4 |
4763.3130 |
0.5171 |
||||||
Reference B 5 |
4739.6685 |
0.5145 |
||||||
Reference B 6 |
4952.0962 |
0.5376 |
||||||
Reference C 1 (PC solvent) |
4813.2861 |
4796.4543 |
14.5915 |
0.30 |
0.5225 |
0.5207 |
0.0016 |
0.30 |
Reference C 2 (PC solvent) |
4788.6934 |
0.5199 |
||||||
Reference C 3 (PC solvent) |
4787.3833 |
0.5197 |
||||||
Reference C 1 (TI solvent) |
4813.2861 |
4796.4543 |
14.5915 |
0.30 |
0.5225 |
0.5207 |
0.0016 |
0.30 |
Reference C 2 (TI solvent) |
4788.6934 |
0.5199 |
||||||
Reference C 3 (TI solvent) |
4787.3833 |
0.5197 |
Table 4. Results of the Reference Controls for the Lysine Peptide
Lysine Peptide Run |
||||||||
Sample |
Peptide Peak Area |
Peptide Concentration [mM] |
||||||
PA |
Mean |
SD |
CV [%] |
Peptide Concentration |
Mean |
SD |
CV [%] |
|
Reference A 1 |
4151.8887 |
4142.8311 |
11.9519 |
0.29 |
0.5049 |
0.5038 |
0.0015 |
0.29 |
Reference A 2 |
4147.3198 |
0.5043 |
||||||
Reference A 3 |
4129.2847 |
0.5021 |
||||||
Reference B 1 |
4154.4273 |
4131.4318 |
14.6986 |
0.36 |
0.5052 |
0.5024 |
0.0018 |
0.36 |
Reference B 2 |
4127.5840 |
0.5019 |
||||||
Reference B 3 |
4110.1455 |
0.4998 |
||||||
Reference B 4 |
4139.1934 |
0.5033 |
||||||
Reference B 5 |
4130.5562 |
0.5023 |
||||||
Reference B 6 |
4126.6846 |
0.5018 |
||||||
Reference C 1 (PC solvent) |
4123.4346 |
4099.5856 |
34.5874 |
0.84 |
0.5014 |
0.4985 |
0.0042 |
0.85 |
Reference C 2 (PC solvent) |
4115.4048 |
0.5004 |
||||||
Reference C 3 (PC solvent) |
4059.9175 |
0.4937 |
||||||
Reference C 1 (TI solvent) |
4123.4346 |
4099.5856 |
34.5874 |
0.84 |
0.5014 |
0.4985 |
0.0042 |
0.85 |
Reference C 2 (TI solvent) |
4115.4048 |
0.5004 |
||||||
Reference C 3 (TI solvent) |
4059.9175 |
0.4937 |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Remarks:
- In combination with KeratinoSens assay (OECD 442D)
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