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EC number: 232-107-5 | CAS number: 7787-20-4
- 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:
- 05 February 2018- 02 March 2018
- 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 guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- 1,3,3-trimethylnorbornan-2-one
- EC Number:
- 232-107-5
- EC Name:
- 1,3,3-trimethylnorbornan-2-one
- Cas Number:
- 7787-20-4
- Molecular formula:
- C10H16O
- IUPAC Name:
- 1,3,3-trimethylnorbornan-2-one
- Test material form:
- liquid
Constituent 1
In chemico test system
- Details on the study design:
- Details on study design:
ITEM SOLVENT
Initial election of test item solvent: solubility of the test item in an appropriate solvent was assessed before performing the assay. The chosen solvent was acetonitrile.
TEST SYSTEM
Cysteine peptide (supplier RS Synthesis, Louisville KY, USA; Ref. Ac RFAACAA-COOH; batch no 170809, 171229)
Lysine peptide (supplier RS Synthesis, Louisville KY, USA; Ref. Ac-RFAAKAA-COOH; batch no 170809, 161121)
CONTROLS
Positive control: Cinnamic Aldehyde (CAS 104-55-2; batch no MKBX8146V, Sigma Aldrich, purity 98.9%)
Co-elution control: test item in phosphate buffer (100 mM; pH 7.5) for cysteine peptide and ammonium acetate buffer (100 mM; pH 10.2) for lysine peptide.
Reference control A, B and C were prepared by adding 250 µL of acetonitrile to 750 µL of 0.667 mM Lysine peptide prepared in 100 mM Ammonium acetate buffer or 0.667 mM cysteine peptide prepared in 100 mM phosphate buffer.
Reference control A was used to verify the accuracy of the calibration curve for peptide quantification. Reference control A was injected in triplicates.
Reference control B was injected in triplicates each both in the beginning and in the end of the experimental run to verify the stability of the peptide over the analysis time.
Reference control C was included in every assay run together with the samples in triplicates. They were used to verify that the solvent does not have any impact on the percent peptide depletion. The Reference Controls C was used to calculate Percent Peptide Depletion by the test item.
PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item and positive control were dispensed using micropipette into clean and dry glass vials. 100 mM stock solution of test item and positive control (Cinnamic aldehyde) was prepared fresh, immediately before use.
Test item solution: Test item stock solution of 100 mM was prepared by dispensing 35.3 µL of test item into a vial and dissolved by adding 1964.7 µL of acetonitrile and mixed well.
Positive control solution: Positive control of 100 mM stock solution was prepared by dispensing 26.5 µL into a vial followed by adding 1973.5 µL of acetonitrile.
PREPARATION OF THE PEPTIDE SOLUTIONS
Peptide solutions were prepared at 0.667 mM:
Cysteine solution: Cysteine peptide Ac-RFAACAA-COOH, 0.667mM, 0.501 mg/mL: Using an electronic balance, Cysteine peptide was weighed and dissolved in appropriate volume of phosphate buffer.
Lysine solution: Lysine peptide Ac-RFAAKAA-COOH, 0.667mM, 0.518 mg/mL: Using an electronic balance, Lysine peptide was weighed and dissolved in appropriate volume of ammonium acetate buffer.
TEST SOLUTIONS
For each run, test item stock and positive control solutions were prepared before the start of sample preparation for analysis.
Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively.
Test samples were prepared in triplicates for both peptides.
Both cysteine and lysine peptides were prepared on the same day once and remaining experiments peptides were tested individually.
All the replicates analysed in the same run were prepared with the same peptide stock solutions.
1 ml of each solution was prepared according to the following quantities:
Cysteine test solution (0.5 mM Peptide, 5 mM test chemical): 750 µl of cysteine peptide solution (buffer only to check co-elution) + 50 µl of test chemical solution or solvent for Reference controls + 200 µl of acetonitrile.
Lysine test solution (0.5 mM Peptide, 25 mM test chemical): 750 µl of lysine peptide solution (buffer only to check co-elution) + 250 µl of test chemical solution or solvent for Reference controls.
Vials were capped, vortexed to mix and placed in (dark) at 25ºC for 24 hours. HPLC analysis of the batch of samples was started after 24 hours the test item addition to the peptide solution.
HPLC ANALYSIS
Apparatus:
Column: Agilent Zorbax SB-C18 2.1 mm X 100 mm X 3.5 micron
Detector: Photodiode Array detector or Fixed Wavelength Absorbance detector with 220 nm signal for quantification.
Calibration curve:
Six peptides standards solutions between 0.534 mM and 0.0167 mM (dilution factor 2) were prepared in 20% acetonitrile in phosphate buffer for cysteine peptide and ammonium acetate for lysine peptide. The dilution buffer was also included as blank in the standard calibration curve.
Equilibration of the column: not specified.
Volume injected: 10 µl of each sample
Flow rate: 0.35 mL/min
Sequence (gradient): see table 1 on “Any other information on materials and methods incl. tables”
Duration of re-equilibration to the initial conditions between injections: Not specified.
Analysis sequence:
Two separate analysis sequences were prepared as below:
Analysis sequence 1: Calibration standards (Standard curve), Reference Controls A and Co-elution Controls.
Analysis sequence 2: Reference Controls B and sets of replicates of Reference Controls C, Positive Control and test item.
The first analysis sequence was timed to complete prior to the end of the 24 hour incubation and the second analysis sequence was timed to assure that the first sample starts 24 ± 2 hours after the test item was mixed with the peptide solution.
The difference between the first replicate analysis and the last replicate analysis was not more than 30 hours.
DEVIATIONS FROM OECD GUIDELINE: No.
DEVIATIONS FROM OECD GUIDELINE: No.
The column used has 2.7 µm particle size when the column described in the OECD 442C has 3.5 µm particle size. According to the guideline, the set-up parameters were adjusted to guarantee an appropriate elution and integration of the cysteine and lysine peptides. Proficiency substances recommended in the OECD guideline were performed in these conditions.
Results and discussion
- Positive control results:
- The depletion mean rate was 71.18% for cysteine peptide and 45.60% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.0% for the lysine.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: mean of 3 repetitions
- Parameter:
- other: %depletion in lysine peptide
- Value:
- 1.38
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: Mean of 3 repetitions
- Parameter:
- other: %depletion in cysteine peptide
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: Mean of lysine and cysteine peptides
- Parameter:
- other: mean %depletion in peptides
- Value:
- 0.69
- Vehicle controls validity:
- valid
- 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 10 proficiency substances recommended in the OECD guideline 442C were performed, obtaining values that fall within the respective reference range for 8 out of the 10 for each peptide.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for calibration curve: yes, coefficient r2 was higher than 0.99 for lysine and cysteine.
- Acceptance criteria met for reference control A: yes, the mean peptide concentration of reference controls A was 0.47 mM for both cysteine and lysine peptides which are equal to 0.50± 0.05 mM.
- Acceptance criteria met for reference controls B and C: yes, the mean peptide concentration of the three Reference Controls C was 0.46 mM and 0.52 mM for cysteine and lysine peptides respectively which are equal to 0.50± 0.05 mM. The CV (coefficient of variation) of the nine controls B and C was 1.46% and 5.67% for cysteine and lysine peptide which is lower than 15%.
- Acceptance criteria met for positive control: Yes, the depletion mean rate was 71.18% for cysteine peptide and 45.60% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine. The SD of the 3 repetitions was 1.64% (limit < 14.9%) for the percent cysteine depletion and 19.34% (limit <11.6%) for the percent lysine depletion. However, this criterion is considered valid as the third replicate of percent lysine depletion is an outlier based on the results of Grubb’s test and the mean percent lysine peptide depletion of the positive control is 45.60% which is within the range as suggested by OECD 442C test guideline.
- Acceptance criteria met for variability between replicate measurements: Yes, SD of the 3 repetitions of the test item for each peptide was 4.78% for cysteine and 8.32% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively.
Any other information on results incl. tables
Table 3: Mean and SD of Percent Peptide depletion for Cysteine and Lysine
For Cysteine peptide |
||||
Sample ID |
Peak area of cysteine peptide |
% peptide depletion |
Mean % Peptide Depletion |
SD |
Cinnamic aldehyde-replicate 1 |
972449.0 |
71.35 |
71.18 |
1.64 |
Cinnamic aldehyde-replicate 2 |
925851 |
72.72 |
||
Cinnamic aldehyde-replicate 3 |
1036665 |
69.46 |
||
D027-01 replicate 1 |
3439003.0 |
-1.31 |
-0.64 (Considered as 0) |
4.78 |
D027-01 replicate 2 |
3243768.0 |
4.44 |
||
D027-01 replicate 3 |
3565975 |
-5.05 |
For Lysine peptide |
||||
Sample ID |
Peak area of Lysine peptide |
% peptide depletion |
Mean % Peptide Depletion |
SD |
Cinnamic aldehyde-replicate 1 |
1731217 |
57.01 |
45.60 |
19.34 |
Cinnamic aldehyde-replicate 2 |
1750371 |
56.53 |
||
*Cinnamic aldehyde-replicate 3 |
3089854 |
23.26 |
||
D027-01 replicate 1 |
4357614 |
-8.22 |
1.38 |
8.32 |
D027-01 replicate 2 |
3790534 |
5.86 |
||
D027-01 replicate 3 |
3765346 |
6.49 |
D027-01: 3,3-Dimethyl-8,9-Dinorbornan-2-one
* replicate 3 is an outlier
Table 4: Reactivity Class Classification of Test Item and Positive Control as per Cysteine 1:10/Lysine 1:50 Prediction Model
Sample ID/ Code |
Name of the chemical |
Mean % of Cysteine Peptide Depletion |
Mean % of Lysine Peptide Depletion |
Mean % peptide depletion |
Reactivity class |
DPRA prediction |
CA |
Cinnamic aldehyde |
71.18 |
45.60 |
58.39 |
High Reactivity |
Positive |
D027-01 |
3,3-Dimethyl-8,9-Dinorbornan-2-one |
0 |
1.38 |
0.69 |
No or Minimal Reactivity |
Negative |
Applicant's summary and conclusion
- Interpretation of results:
- other: DPRA test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442C.
- Conclusions:
- The test item shows mean depletion of cysteine and lysine peptides of 0.69%, reflecting no or minimal reactivity and thus a negative prediction of DPRA skin sensitization test.
- Executive summary:
A DPRA skin sensitization test was performed for test item as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item and Cinnamaldehyde (positive control) were prepared at 100 mM in acetonitrile as solvent. Reference controls A, B and C were prepared with acetonitrile in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item, positive control and reference controls were incubated for 24 hours ± 2 hours at 25°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, and then analyzed with HPLC to measure the peptide depletion. Test samples were prepared in triplicates for both peptides. All validity criteria were fulfilled. The mean of cysteine and lysine peptide depletion by the positive control was 58.39% which shows that it is a sensitizer with high reactivity confirming the sensitivity of the assay. The mean of depletion by the test item was 0.69% which shows that the test item has no or minimal reactivity on the skin sensitisation potential. Under the testing conditions, the test item was concluded as negative or non-sensitiser with no or minimal reactivity in Direct Peptide Reactivity Assay (DPRA).
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