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EC number: 243-497-1 | CAS number: 20073-13-6
- 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 vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental Start Date: 6th July 2021
Experimental Completion Date: 23rd July 2021 - Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
- Report date:
- 2021
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- ARE-Nrf2 luciferase KeratinoSens™ test method
Test material
- Reference substance name:
- Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate
- EC Number:
- 243-497-1
- EC Name:
- Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate
- Cas Number:
- 20073-13-6
- Molecular formula:
- C13H20O3
- IUPAC Name:
- methyl 2-[(1S,2S)-3-oxo-2-[(2Z)-pent-2-en-1-yl]cyclopentyl]acetate
Constituent 1
- Specific details on test material used for the study:
- Methyl Jasmonate, also known as Jasmoneige
Chemical name Methyl 3-oxo-2-(pent-2-enyl)cyclopentaneacetate
CAS number 39924-52-2,
Lot number 0900175
Description: colourless to pale yellow liquid.
Received: 03 June 2021 and stored at 2-8°C, protected from light.
Expiry date: 25 March 2023 (2.5 years after production; production date 24 September 2020)
Purity: 99.03% (purity sum of three isomers); 6.19% trans-trans isomer, 87.80% trans-cis isomer,
5.03% cis-cis isomer
In vitro test system
- Details on the study design:
- Test Article Formulation
Preliminary solubility data indicated that Methyl Jasmonate was soluble in dimethyl sulfoxide (DMSO) up to at least 50.89 mg/mL, which was in excess of the target concentration 200 mM (equivalent to 44.86 mg/mL).
Test formulations were prepared using DMSO. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 200 mM.
Serial dilutions were made (from the 200 mM stock) using the vehicle to obtain 12 master concentrations ranging from 0.098 to 200 mM.
The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations range from 0.98 to 2000 μM.
Treatment Plate Preparation
The cells were 80-90% confluent. On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated for 24±1 hours an incubator set to 37°C, 5% CO2.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.
Treatment
At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added.
On each plate, one well per replicate was left empty (no cells and no treatment) to assess background values.
Aliquots of 50 μL of each of the final concentrations were transferred to give three luciferase replicates on a white-walled plate and a single viability replicate on a clear-walled plate. Each plate was sealed and incubated for 48±1 hours in a humidified incubator set to 37°C, 5% CO2.
For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of two independent repetitions each containing three replicates of each concentration.
Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from different batches.
It may be noted that initial Experiment 2 treatments were terminated due to a technical error, and the data are not reported. These treatments were repeated in order to provide the Experiment 2 data which are presented in this report.
Cytotoxicity Assessment
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The plate was sealed and placed in an incubator set to 37°C, 5% CO2 for 4 hours. The MTT medium was removed and 200 μL SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO2, and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.
Luciferase Activity Measurements
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read using the following parameters: 100 μL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time. - Vehicle / solvent control:
- DMSO
- Negative control:
- other: DMSO. The negative control was diluted into culture medium containing serum so that the final concentration was 1%.
- Positive control:
- cinnamic aldehyde [442D]
Results and discussion
- Positive control results:
- The EC1.5 value for the positive control were 5.96 and 9.17 μM in Experiments 1 and 2, respectively. The average induction in the three replicates for the positive control at 64 μM were 1.18 and 5.86 in Experiments 1 and 2, respectively. The average induction for the positive control at 64 μM in Experiment 1 was outside the range specified in the Protocol. As however the EC1.5 was within the range seen at these laboratories and a clear positive dose response was observed for luciferase induction, these data were considered to be acceptable.
In vitro / in chemico
Resultsopen allclose all
- Run / experiment:
- mean
- Parameter:
- Imax [442D]
- Value:
- 1.16
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- IC50 [442D]
- Value:
- 2 000 µM
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- IC30 [442D]
- Value:
- 2 000 µM
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- IC30 [442D]
- Value:
- 1 208.46 µM
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- IC50 [442D]
- Value:
- 865.72 µM
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- Imax [442D]
- Value:
- 1.19
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- Imax [442D]
- Value:
- 1.13
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Other effects / acceptance of results:
- Calculation of Maximal Average Fold Induction and Determination of Luciferase Activity
There were no EC1.5 values for Experiments 1 or 2 as there were no statistically significant increases in induction.
Viability
The cell viability measurement was not applicable as there were no EC1.5 determining concentrations in Experiments 1 or 2.
An anomalous toxicity response which was not in line with any other treatment concentration was noted on the viability plate at 7.81 mM in Experiment 1. The unusual response was noted following treatment and incubation, and a viability of -0.06% was subsequently returned following MTT assay. The preceding three and subsequent eight test concentrations in Experiment 1, and also all test concentrations in Experiment 2 had no reduction in viability (all values >70%). It was therefore considered that a technical error likely occurred on the viability plate at 7.81 mM in
Experiment 1 which resulted in this anomalous toxicity, and accordingly, the obtained viability value was disregarded and is presented in Table 9.3 for information only.
As all viability results for Experiment 2 were above 70%, the IC50 and IC30 values for this experiment were >2000 μM and could not be calculated. The geometric mean IC50 and IC30 values of Experiments 1 and 2 could therefore not be calculated.
Assay Acceptance
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 8 to 64 μM in Experiment 1 and at concentrations of 16 to 64 μM in Experiment 2.
The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 11.33% and 12.74% in Experiments 1 and 2, respectively.
Any other information on results incl. tables
Luminescence Readings for Experiment 1
|
| Concentration (µM) |
|
|
|
|
|
|
|
|
| ||
Substance |
| 0.98 | 1.95 | 3.91 | 7.81 | 15.63 | 31.25 | 62.5 | 125 | 250 | 500 | 1000 | 2000 |
| Plate 1 | 402359 | 482958 | 388531 | 463364 | 535506 | 447711 | 545741 | 467357 | 467398 | 412005 | 431378 | 563452 |
| Plate 2 | 459264 | 441075 | 486799 | 541909 | 453109 | 507595 | 486831 | 505190 | 479749 | 464852 | 496811 | 502881 |
Methyl Jasmonate | Plate 3 | 488889 | 465327 | 470398 | 477052 | 460814 | 475381 | 464764 | 459837 | 452389 | 486306 | 450089 | 607077 |
| Mean fold |
|
|
|
|
|
|
|
|
|
|
|
|
| Induction | 0.92 | 0.94 | 0.91 | 1.01 | 0.99 | 0.97 | 1.02 | 0.97 | 0.95 | 0.92 | 0.94 | 1.13* |
Substance | Individual Values | ||||||
Negative control | Plate 1 | 418088 | 463819 | 504379 | 515926 | 530726 | 457400 |
Plate 2 | 386501 | 453527 | 455478 | 607793 | 469996 | 474925 | |
Plate 3 | 480423 | 474502 | 501564 | 510936 | 531838 | 603037 |
Concentration (µM) | ||||||
Substance |
| 4 | 8 | 16 | 32 | 64 |
| Plate 1 | 718252 | 840496 | 1057104 | 2074031 | 5435230 |
Positive control | Plate 2 | 637787 | 751442 | 1090331 | 1986846 | 5763708 |
| Plate 3 | 683427 | 788623 | 1097392 | 1968326 | 5194634 |
| Mean fold Induction | 1.39 | 1.62# | 2.21# | 4.11# | 11.18# |
* Imax
# Luciferase activity induction statistically significant above the threshold of 1.5
Luminescence Readings for Experiment 2
Concentration (µM) | |||||||||||||
Substance |
| 0.98 | 1.95 | 3.91 | 7.81 | 15.63 | 31.25 | 62.5 | 125 | 250 | 500 | 1000 | 2000 |
| Plate 1 | 606307 | 482318 | 473109 | 523432 | 492419 | 435818 | 524813 | 431267 | 613633 | 531213 | 439794 | 491825 |
| Plate 2 | 491285 | 534258 | 497339 | 541145 | 541341 | 558171 | 550899 | 499604 | 662367 | 557156 | 497099 | 535143 |
Methyl Jasmonate | Plate 3 | 466586 | 429721 | 581515 | 520107 | 522527 | 627890 | 510305 | 480316 | 596500 | 490784 | 453397 | 465583 |
| Mean fold |
|
|
|
|
|
|
|
|
|
|
|
|
| Induction | 0.99 | 0.92 | 0.98 | 1.00 | 0.98 | 1.02 | 1.00 | 0.89 | 1.19* | 1.00 | 0.88 | 0.94 |
Substance | Individual Values | ||||||
Negative control | Plate 1 | 604784 | 463981 | 502707 | 502184 | 498945 | 479730 |
Plate 2 | 495566 | 552454 | 480448 | 525347 | 594084 | 598658 | |
Plate 3 | 515906 | 419065 | 458233 | 501998 | 593701 | 692315 |
| Concentration (µM) | |||||
Substance |
| 4 | 8 | 16 | 32 | 64 |
Positive control | Plate 1 | 729083 | 702254 | 961233 | 1575379 | 2960198 |
Plate 2 | 851784 | 802070 | 1139438 | 1593390 | 3528909 | |
Plate 3 | 752865 | 729654 | 1065849 | 1742544 | 2761048 | |
Mean fold Induction | 1.48 | 1.41 | 2.00# | 3.11# | 5.86# |
* Imax
# Luciferase activity induction statistically significant above the threshold of 1.
MTT-Absorbance Readings
| concentration | (µM) |
|
|
|
|
|
|
|
|
|
|
Substance | 0.98 | 1.95 | 3.91 | 7.81 | 15.63 | 31.25 | 62.5 | 125 | 250 | 500 | 1000 | 2000 |
Methyl Jasmonate Experiment 1 | 0.474 | 0.471 | 0.453 | 0.000 | 0.459 | 0.509 | 0.483 | 0.498 | 0.555 | 0.640 | 0.649 | 0.556 |
Experiment 2 | 0.668 | 0.599 | 0.578 | 0.644 | 0.608 | 0.614 | 0.568 | 0.709 | 0.766 | 0.856 | 0.897 | 0.686 |
Viability (%) Experiment 1 | 99.08 | 98.41 | 94.57 | -0.06* | 95.91 | 106.27 | 100.81 | 103.99 | 115.90 | 133.63 | 135.51 | 116.19 |
Experiment 2 | 101.80 | 91.40 | 88.18 | 98.15 | 92.76 | 93.67 | 86.59 | 108.17 | 116.75 | 130.52 | 136.86 | 104.59 |
*Data considered anomalous and disregarded
The Imax values were calculated as follows:
Parameter | Experiment 1 | Experiment 2 | Mean |
Imax | 1.13 | 1.19 | 1.16 |
There were no EC1.5 values for Experiments 1 or 2 as there were no statistically significant increases in induction.
Applicant's summary and conclusion
- Interpretation of results:
- study cannot be used for classification
- Conclusions:
- The Imax values were less than 1.5 in both experiments, therefore Methyl Jasmonate was considered to be negative in the ARE-Nrf2 Luciferase Test.
- Executive summary:
The study was conducted to investigate the potential of Methyl Jasmonate to induce genes that are regulated by the antioxidant response element (ARE). 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 dimethyl sulfoxide (DMSO) to the final stock concentration (200 mM). Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article in the range 0.098 to 200 mM.
The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations ranged from 0.98 to 2000 μM.
Aliquots of 50 μL of each of the final concentrations were transferred to give three luciferase replicates on a white-walled plate and a single viability replicate on a clear-walled plate. Each plate was sealed using a plate sealer and then incubated for 48±1 hours in a humidified incubator set to 37°C, 5% CO2.
After the 48-hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours in an incubator set to 37°C, 5% CO2. The MTT medium was then removed and 200 μL SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO2, and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.
After the 48-hour exposure period, the cells in the luciferase plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read.
The Imax value in both experiments was less than 1.5.
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