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EC number: 287-673-6 | CAS number: 85566-63-8
- 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:
- 31 October 2017- 23 November 2017
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Results were very weakly positive with low reactivity
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))
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- direct peptide reactivity assay (DPRA)
- Justification for non-LLNA method:
- Regulation 1223/2009, Article 18, restricts the use of in vivo studies on cosmetic raw materials (the sole use of this substance), therefore a recognised in chemico test was conducted on the test material.
Test material
- Reference substance name:
- Dioctyl maleate, branched
- EC Number:
- 287-673-6
- EC Name:
- Dioctyl maleate, branched
- Cas Number:
- 85566-63-8
- Molecular formula:
- C20H36O4
- IUPAC Name:
- 1,4-bis(octan-2-yl) (2Z)-but-2-enedioate
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- Test Article Bernel Ester DCM
CAS Number 85566-63-8
Storage 15 to 25˚C, protected from light
Purity 92.78%*
* Assumed 100% for testing
In chemico test system
- Details on the study design:
- Test Article Formulation
The test article was dissolved in acetonitrile. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 100 mM.
The positive control was dissolved in acetonitrile at a concentration of 100 mM.
A stock solution containing cysteine at approximately 0.667 mM was prepared in 100 mM Phosphate Buffer pH 7.5 and a stock solution containing lysine at approximately 0.667 mM was prepared in 100 mM ammonium acetate buffer pH 10.2.
Formulations were prepared shortly before testing.
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. At the end of the incubation period the samples were visually inspected for precipitate formation.
Analytical Method
The following HPLC conditions were applied:
Column: Agilent Zorbax SB-C18 2.1 mm x 100 mm, 3.5 µm or equivalent
Wavelength: 220 nm
Guard column: Phenomenex Security Guard c18 4 mm x 2 mm
Flow rate: 0.35 mL/min
Oven temperature: 30°C
Sample temperature: 25°C
Injection volume: 7 µL
Mobile Phase:
Phase A: 0.1% (v/v) of trifluoroacetic acid in MilliQ water
Phase B: 0.085% (v/v) of trifluoroacetic acid in acetonitrile
Gradient: Time (min) Phase A Phase B
0 90 10
10 75 25
11 10 90
13 10 90
13.5 90 10
20 90 10
Reference and Co-elution Controls
Reference controls were prepared for each peptide.
Reference Control A and B for each peptide were prepared by adding 750 µL of peptide stock solution to 250 µL of acetonitrile.
Reference Control C for cysteine was prepared by adding 750 µL of peptide stock solution to 200 µL of acetonitrile and 50 µL vehicle.
Reference Control C for lysine was prepared by adding 750 µL of peptide stock solution to 250 µL vehicle.
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. A mixture of 750 µL of 100 mM Phosphate Buffer pH 7.5, 200 µL of acetonitrile and 50 µL of test article solution was used to detect possible co-elution of the test article with cysteine. A mixture of 750 µL of 100 mM ammonium acetate buffer pH 10.2 and 250 µL of test article solution was used to detect possible co elution of the test article with lysine.
Calibration Curves for Peptides
Calibration curves were prepared for each peptide using a range of concentrations from approximately 0.534 mM to 0.0167 mM (Standards 1 to 6).
Standard 1 was prepared at approximatively 0.534 mM by dilution of 1600 µL of the peptide stock solution (0.667 mM) with 400 µL of acetonitrile.
Standards 2 to 6 for cysteine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM Phosphate Buffer pH 7.5).
Standards 2 to 6 for lysine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM ammonium acetate buffer pH 10.2).
Samples of dilution buffer alone were also prepared.
Sample Analysis Sequence
The analysis sequence for each peptide was as follows:
System suitability Standard 1
Dilution buffer
Calibration standards and reference controls Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
Standard 6
Dilution Buffer
Reference Control A, rep 1
Reference Control A, rep 2
Reference Control A, rep 3
Co-elution controls Co-elution control for test article
Reference controls Reference Control B, rep 1
Reference Control B, rep 2
Reference Control B, rep 3
First set of replicates Reference Control C, rep 1
Positive Control, rep 1
Test sample, rep 1
Second set of replicates Reference Control C, rep 2
Positive Control, rep 2
Test sample, rep 2
Third set of replicates Reference Control C, rep3
Positive Control, rep 3
Test sample, rep 3
Reference controls Reference Control B, rep 4
Reference Control B, rep 5
Reference Control B, rep 6
DATA EVALUATION
Data Analysis and Calculations
The chromatographic data were collected and processed using Chromeleon, a validated data capture system.
A calibration curve was generated based on the concentration of standards and the peak area. All the peaks of the samples were integrated (standards, samples and controls) “valley to valley” (when possible) and Cysteine and Lysine peptides concentrations were determined from absorbance at 220 nm using the respective calibration curves. Areas were determined for all the samples.
The PPD of each peptide was determined in each sample with the following formula:
PPD = (1-(Peptide peak area in replicate injection/Mean peptide peak area in reference controls C) x 100
The mean peptide peak areas for the nine Reference Controls B and C, Standard Deviation (SD) and Coefficient of Variation (CV) were calculated.
The mean peptide peak areas and the mean peptide concentrations (mM) for the three Reference Controls C were calculated.
The chromatograms of Co-elution Controls were compared to the chromatograms of Reference Controls C.
For the positive control and for the test article, the PPD in each replicate was calculated from the peptide area of the replicate injection and the mean peptide peak for Reference Control C. The PPD of every injected positive control and test chemical replicate was calculated. The mean PPD of the three replicate determinations and SD were calculated.
The mean percent cysteine and percent lysine depletion values were calculated for each test chemical (negative depletion is considered as “0” when calculating the mean).
Assay Acceptance Criteria
The following criteria should be met for a run to be considered valid:
• The standard calibration curve should have a r2>0.99.
• 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%.
• The mean PPD value of the three replicates for the positive control and maximum standard deviation (SD) must fall within the ranges in the following table:
Peptide Mean PPD Values (%)
Lower Bound Upper Bound SD
Cysteine 60.8 100 <14.9
Lysine 40.2 69.0 <11.6
The following criteria should be met for the test article’s result to be considered valid:
• 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 mean peptide concentration of the three reference controls C in the appropriate should be 0.50±0.05 mM.
#Prediction Model
The mean percent cysteine and percent lysine depletion value was calculated. 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.
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
22.62% < mean % depletion ≤42.47% Moderate reactivity Positive
42.47% < mean % depletion ≤100% High reactivity
The mean percent depletion fell in the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model, therefore a second run was required.
Results and discussion
- Positive control results:
- Cinnamic aldehyde (CAS No. 104-55-2, batch number MKBT8955V purity 99.1%, expiry 29 February 2020) was used as the positive control.
The mean percentage peptide depletion (PPD) values for the positive control were:
- 62.06for lysine
- 51.04 for cysteine
In vitro / in chemico
Resultsopen allclose all
- Run / experiment:
- other: 1-3
- Parameter:
- other: % peptide depletion (PPD) lysine
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 1-3
- Parameter:
- other: % peptide depletion (PPD) cysteine
- Value:
- 6.29
- 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:
- All acceptance criteria were met in both Experiments, with the exception that in Experiment 2, the mean peptide concentration for Reference Control A was just below the range specified, at 0.43 mM and 0.44 mM for cysteine and lysine, respectively. As the routine system suitability test prior to sample analysis was satisfactory, the results were considered valid.
Any other information on results incl. tables
Lysine Depletion, Experiment 1
The percentage peptide depletion values were as follows:
Substance | Replicate Peptide Peak Areas | Reference Control C Mean Peptide Peak Area | PPD | Mean PPD | SD |
Test Article | 33.86 | 38.04 | 10.99 | 8.02 | 2.68 |
35.84 | 5.78 | ||||
35.27 | 7.28 | ||||
Positive Control | 13.16 | 38.04 | 65.4 | 62.06 | 3.41 |
15.75 | 58.6 | ||||
14.39 | 62.17 |
The r2 value for the standard calibration curve was 0.99995.
The peptide concentrations for the Reference Controls A and C were as follows:
Reference Control | Peptide Concentration (mM) | |||
Replicate 1 | Replicate 2 | Replicate 3 | Mean | |
A | 0.46 | 0.47 | 0.48 | 0.47 |
C | 0.47 | 0.48 | 0.48 | 0.48 |
The peak area results for Reference Controls B and C were as follows:
Reference Control | Replicate | Peptide Peak Area |
B | 1 | 37.49 |
2 | 36.67 | |
3 | 40.15 | |
4 | 38.58 | |
5 | 39.19 | |
6 | 39.42 | |
C | 1 | 37.28 |
2 | 38.43 | |
3 | 38.41 | |
Mean | 38.4 | |
SD | 1.11 | |
CV | 2.89 |
Lysine Depletion, Experiment 2
The percentage peptide depletion values were as follows:
Substance | Replicate Peptide Peak Areas | Reference Control C Mean Peptide Peak Area | PPD | Mean PPD | SD |
Test Article | 33.85 | 28.84 | 0 | 1.24 | 1.25 |
28.12 | 2.5 | ||||
28.49 | 1.21 | ||||
Positive Control | 13.75 | 28.84 | 52.32 | 51.04 | 9.89 |
17.14 | 40.57 | ||||
11.47 | 60.23 |
The r2 value for the standard calibration curve was 0.99994.
The peptide concentrations for the Reference Controls A and C were as follows:
Reference Control | Peptide Concentration (mM) | |||
Replicate 1 | Replicate 2 | Replicate 3 | Mean | |
A | 0.44 | 0.43 | 0.44 | 0.44 |
C | 0.45 | 0.45 | 0.46 | 0.45 |
The peak area results for Reference Controls B and C were as follows:
Reference Control | Replicate | Peptide Peak Area |
B | 1 | 33.42 |
2 | 27.95 | |
3 | 27.71 | |
4 | 28.5 | |
5 | 31.77 | |
6 | 28.03 | |
C | 1 | 28.72 |
2 | 28.37 | |
3 | 29.45 | |
Mean | 29.32 | |
SD | 1.97 | |
CV | 6.7 |
Cysteine Depletion, Experiment 1
The percentage peptide depletion values were as follows:
Substance | Replicate Peptide Peak Areas | Reference Control C Mean Peptide Peak Area | PPD | Mean PPD | SD |
Test Article | 24.75 | 26.41 | 6.29 | 7.03 | 0.65 |
24.45 | 7.42 | ||||
24.46 | 7.38 | ||||
Positive Control | 6.34 | 26.41 | 75.99 | 76.47 | 1.46 |
6.52 | 75.31 | ||||
5.78 | 78.11 |
The r2 value for the standard calibration curve was 0.99865.
The peptide concentrations for the Reference Controls A and C were as follows:
Reference Control | Peptide Concentration (mM) | |||
Replicate 1 | Replicate 2 | Replicate 3 | Mean | |
A | 0.44 | 0.49 | 0.5 | 0.48 |
C | 0.49 | 0.48 | 0.49 | 0.49 |
The peak area results for Reference Controls B and C were as follows:
Reference Control | Replicate | Peptide Peak Area |
B | 1 | 26.53 |
2 | 26.61 | |
3 | 27.12 | |
4 | 25.89 | |
5 | 25.58 | |
6 | 25.94 | |
C | 1 | 26.86 |
2 | 25.87 | |
3 | 26.48 | |
Mean | 26.32 | |
SD | 0.52 | |
CV | 1.98 |
Cysteine Depletion, Experiment 2
The percentage peptide depletion values were as follows:
Substance | Replicate Peptide Peak Areas | Reference Control C Mean Peptide Peak Area | PPD | Mean PPD | SD |
Test Article | 15.46 | 17.2 | 10.12 | 12.29 | 7.17 |
13.71 | 20.29 | ||||
16.09 | 6.45 | ||||
Positive Control | 6.11 | 17.2 | 64.48 | 65.72 | 4.66 |
6.57 | 61.8 | ||||
5.01 | 70.87 |
The r2 value for the standard calibration curve was 0.99874.
The peptide concentrations for the Reference Controls A and C were as follows:
Reference Control | Peptide Concentration (mM) | |||
Replicate 1 | Replicate 2 | Replicate 3 | Mean | |
A | 0.41 | 0.44 | 0.42 | 0.43 |
C | 0.44 | 0.46 | 0.44 | 0.45 |
The peak area results for Reference Controls B and C were as follows:
Reference Control | Replicate | Peptide Peak Area |
B | 1 | 16.55 |
2 | 16.9 | |
3 | 15.05 | |
4 | 14.04 | |
5 | 14.16 | |
6 | 14.89 | |
C | 1 | 17.13 |
2 | 17.7 | |
3 | 16.78 | |
Mean | 15.91 | |
SD | 1.38 | |
CV | 8.66 |
Applicant's summary and conclusion
- Interpretation of results:
- study cannot be used for classification
- Conclusions:
- The test article, Bernel Ester DCM, was considered to be weakly positive (low reactivity) in the Direct Peptide Reactivity Assay.
- Executive summary:
The study was conducted to quantify the reactivity of Bernel Ester DCM towards model synthetic peptides containing either lysine or cysteine. The data 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.
The test article was dissolved in acetonitrileat 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.
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.
In Experiment 1, the cysteine depletion value was 7.03%, the lysine depletion value was 8.02% and the mean of the cysteine and lysine depletion values was 7.52%. As the results fell within the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model, a further run was conducted.
In Experiment 2, the cysteine depletion value was 12.29%, the lysine depletion value was 1.24% and the mean of the cysteine and lysine depletion values was 6.76%. These results were concordant with those of Experiment 1.
All acceptance criteria were met in both Experiments, with the exception that in Experiment 2, the mean peptide concentration for Reference Control A was just below the range specified, at 0.43 mM and 0.44 mM for cysteine and lysine, respectively. As the routine system suitability test prior to sample analysis was satisfactory, the results were considered valid.
The test article, Bernel Ester DCM, was considered to be weakly positive with low reactivity in the Direct Peptide Reactivity Assay.
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