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EC number: 909-017-6 | CAS number: -
- 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:
- 11 March 2020 - 08 April 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Version / remarks:
- 18 June 2019
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- Reaction mass of 1-hydroxydecan-3-one and 3-(hydroxymethyl)nonan-2-one and nonan-2-one
- EC Number:
- 909-017-6
- Molecular formula:
- C10H20O2 C9H28O
- IUPAC Name:
- Reaction mass of 1-hydroxydecan-3-one and 3-(hydroxymethyl)nonan-2-one and nonan-2-one
- Test material form:
- liquid
1
In chemico test system
- Details on the study design:
- Preparation of the Test Item
- 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 an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. A factor of 1.04 was used to correct for the purity of the test item.
Controls
- Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
- Positive Control: 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 Control: 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.
- Reference Control: Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run. Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run. Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run. Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.
Test System
- HPLC System: HPLC/DAD: Agilent Infinity 1260 II with Chromeleon 7.2 SR5; Detection: 220 nm signal for quantitation, 258 nm signal used as indicator for co-elution; Analytical Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 μm, Agilent Art. Nr. 861753-902; Pre-Column: Phenomenex, AJO-4286, 4.0 x 2.0 mm; Column Temperature: 30°C; Sample Temperature: 20-25°C; Run Time: 20 minutes; Gradient: Flow: 0.35 mL/min; Time%A/%B: 0 min/90/10; 10 min/75/25; 11 min/10/90; 13 min10/90; 13.5 min/90/10; Injection Volume: 4 μL
- HPLC Mobile Phase: HPLC Mobile Phase A: 0.1% ( v/v) trifluoroacetic acid in water; HPLC Mobile Phase B: 0.085% ( v/v) trifluoroacetic acid in acetonitrile
- Peptides: 18.18 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (35.25 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM; 20.13 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 (37.78 mL) to reach a concentration of 0.667 mM; All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.
Pre-Experiments
- Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated in the following solvents suitable for the test: acetonitrile, dist. water, dist. water : acetonitrile 1:1 (v/v), isopropanol. The test item was not soluble in acetonitrile, dist. water and dist. water : acetonitrile 1:1 (v/v). The test item was completely soluble in isopropanol, therefore, isopropanol was chosen as suitable vehicle for the main experiments.
Experimental Procedure
- Incubation of the Test Item with the Cysteine and Lysine Peptide: 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. 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 the following HPLC procedure.
- Preparation of the HPLC Standard Calibration Curve: A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions (0.534, 0.267, 0.134, 0.067, 0.033, 0.017, 0.000 mM).
- 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.
Data analysis
- 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 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 Reference Control C))*100. 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 different from 100 mM. By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) 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 (See Any other information on materials and methods) 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%.
- 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 Any other information on materials and methods). A single HPLC analysis for both the cysteine and the lysine peptide should be sufficient for a test chemical when the result is unequivocal. However, in cases of results close to the threshold used to discriminate between positive and negative results (i.e. borderline results), additional testing may be necessary. In situations where the mean percent depletion falls in the range of 3% to 10% for the cysteine, 1:10/lysine, 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run should be considered, as well as a third one in case of discordant results between the first two runs.
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.
Results and discussion
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- run/experiment 1
- Parameter:
- mean cystein depletion
- Value:
- 36.01 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- run/experiment 1
- Parameter:
- mean lysine depletion
- Value:
- 2.98 %
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- Pre-Experiments
- Solubility of the test item was determined prior to the main experiment. The test item was soluble in isopropanol. No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using isopropanol. All test item solutions were freshly prepared immediately prior to use.
Precipitation and Phase Separation
- All test item solutions were freshly prepared immediately prior to use. 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. For the 100 mM stock solution of the test item no turbidity or precipitation 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. No precipitation, turbidity or phase separation was observed for any of the samples.
Co-elution with the Peptide Peaks
- No relevant co-elution of the test item with any of the peptide peaks was observed. To detect a co-elution of the test item with the peptide peak, a ratio of the 220 nm peak area and the 258 nm peak area was calculated too (peak purity indication). If the ratio of the control samples and the test item samples do not differ more than 10% from each other, no sign for a co-elution is given. For the cysteine measurement the peak purity indication was < 10% (5.8 % test item replicate 1; 6.0 % test item replicate 2; 1.3 % test item replicate 3; mean: 4.37 %) and for the lysine measurement the peak purity indication was < 10% (0.3 % test item replicate 1; 2.0 % test item replicate 2; 0.1 % test item replicate 3; mean: 0.8 %).
See Tables in "any other information on results incl. tables" for acceptability criteria.
Any other information on results incl. tables
Table 3: Depletion of the Cysteine Peptide
Sample |
Peak Area at 220 nm |
Peptide Concentration |
Peptide Depletion |
Mean Peptide Depletion |
SD of Peptide Depletion |
CV of depletion |
mAU |
mM |
% |
% |
% |
% |
|
Positive control |
4.8870 |
0.1599 |
68.73 |
69.27 |
0.92 |
1.33 |
4.8860 |
0.1598 |
68.74 |
||||
4.6380 |
0.1518 |
70.33 |
||||
Test item |
9.9700 |
0.3255 |
36.04 |
36.01 |
0.65 |
1.80 |
10.0780 |
0.3291 |
35.35 |
||||
9.8760 |
0.3225 |
36.64 |
Table 4: Depletion of the Cysteine Peptide
Sample |
Peak Area at 220 nm |
Peptide Concentration |
Peptide Depletion |
Mean Peptide Depletion |
SD of Peptide Depletion |
CV of depletion |
mAU |
mM |
% |
% |
% |
% |
|
Positive control |
3.7970 |
0.1668 |
66.62 |
66.30 |
0.65 |
0.97 |
3.7870 |
0.1663 |
66.71 |
||||
3.9190 |
0.1721 |
65.55 |
||||
Test item |
11.0580 |
0.4853 |
3.82 |
2.98 |
0.78 |
26.21 |
11.1690 |
0.4902 |
2.86 |
||||
11.2360 |
0.4932 |
2.27 |
Table 5: Acceptance Criteria for Cysteine Peptide
Acceptance Criterion |
Range |
Value |
pass/fail |
coefficient of determination |
R² >0.99 |
1.000 |
Pass |
mean peptide concentration of RC A |
0.45 ≤ x ≤ 0.55 mM |
0.5229 |
Pass |
mean peptide concentration of RC C (PC) |
0.45 ≤ x ≤ 0.55 mM |
0.5100 |
Pass |
mean peptide concentration of RC C (TI) |
0.45 ≤ x ≤ 0.55 mM |
0.5086 |
Pass |
CV of the peak area of RC B |
<15% |
0.58 |
Pass |
CV of the peak area of RC C (PC) |
<15% |
0.15 |
Pass |
CV of the peak area of RC C (TI) |
<15% |
0.77 |
Pass |
mean peptide depletion of the PC |
60.8% < x < 100% |
69.27 |
Pass |
SD of peptide depletion of the PC replicates |
<14.9% |
0.92 |
Pass |
SD of peptide depletion of the TI replicates |
<14.9% |
0.65 |
Pass |
Table 6: Acceptance Criteria for Cysteine Peptide
Acceptance Criterion |
Range |
Value |
pass/fail |
coefficient of determination |
R² >0.99 |
1.000 |
Pass |
mean peptide concentration of RC A |
0.45 ≤ x ≤ 0.55 mM |
0.4987 |
Pass |
mean peptide concentration of RC C (PC) |
0.45 ≤ x ≤ 0.55 mM |
0.4993 |
Pass |
mean peptide concentration of RC C (TI) |
0.45 ≤ x ≤ 0.55 mM |
0.5046 |
Pass |
CV of the peak area of RC B |
<15% |
0.82 |
Pass |
CV of the peak area of RC C (PC) |
<15% |
0.36 |
Pass |
CV of the peak area of RC C (TI) |
<15% |
1.93 |
Pass |
mean peptide depletion of the PC |
40.2% < x < 69.0% |
66.30 |
Pass |
SD of peptide depletion of the PC replicates |
<11.6% |
0.65 |
Pass |
SD of peptide depletion of the TI replicates |
<11.6% |
0.78 |
Pass |
Applicant's summary and conclusion
- Interpretation of results:
- other: DPRA was positive
- Conclusions:
- The substance was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
- Executive summary:
In a GLP-compliant OECD guideline 442C study, the Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. The test substance was dissolved in isopropanol, based on the results of the pre-experiments. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution and the 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 of the samples. No co-elution of the 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. The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (19.50%). Based on the prediction model 1, the substance was positive in the DPRA and was classified in the “low reactivity class”.
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