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EC number: - | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
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- Density
- Particle size distribution (Granulometry)
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- 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
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- Biotransformation and kinetics
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- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Genetic toxicity
- Carcinogenicity
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- Specific investigations
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- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 November 2020 - 17 December 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Substance information is used to read across from
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 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- June 2020
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- (Z)‐ethyl 2‐methylpent‐3‐enoate
- EC Number:
- 854-058-4
- Cas Number:
- 58625-89-1
- Molecular formula:
- C8H14O2
- IUPAC Name:
- (Z)‐ethyl 2‐methylpent‐3‐enoate
- Test material form:
- liquid
Constituent 1
Method
- Target gene:
- Salmonella typhimurium: histidine locus
Escherichia coli: tryptophan locus
Species / strainopen allclose all
- Species / strain / cell type:
- E. coli WP2 uvr A
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal enzymes (S9 homogenate) were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized.
- concentration S9 in the S9-mix: 5% (v/v) S9-fraction - Test concentrations with justification for top dose:
- Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
First experiment: 52, 164, 512, 1600 and 5000 μg/plate.
Second experiment: 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (without S9-mix); 52, 164, 512, 1600 and 5000 μg/plate (with S9-mix) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- With metabolic activation
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: ICR-191
- Remarks:
- Without metabolic activation
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^9 cells/mL
- Test substance added in agar (plate incorporation); preincubation
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period (experiment 2): 30 ± 2 minutes by 70 rpm at 37 ± 1°C
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0°C (experiment 1); 48 ± 4 h at 37.0 ± 1.0°C (experiment 2, after preincubation)
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope. - Evaluation criteria:
- No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1: No cytotoxicity Experiment 2: Cytotoxicity in the absence and presence of S9-mix
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1: No cytotoxicity Experiment 2: Cytotoxicity in the absence and presence of S9-mix
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1: No cytotoxicity Experiment 2: Cytotoxicity in the absence and presence of S9-mix
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1: No cytotoxicity Experiment 2: Cytotoxicity in the absence and presence of S9-mix
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1: No cytotoxicity Experiment 2: Cytotoxicity in the absence and presence of S9-mix
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Experiment 1:
- Precipitate: Precipitation of the test item on the plates was not observed in any tester strain.
- Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the
number of revertants were observed.
- Mutagenicity: In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
Experiment 2:
- Precipitation of the test item on the plates was not observed any tester strain.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
- Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
Acceptability
All bacterial strains showed negative responses over the entire dose-range, i.e. no dose-related increase in the number of revertants in two independently repeated experiments.
The negative control values were within the laboratory historical control data ranges.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA98 in the presence of S9-mix in the first experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was above the historical control data range, this deviation in the mean plate count of the positive control had no effect on the results of the study.
Any other information on results incl. tables
Table 1. Dose-Range Finding Test: Mutagenic Response of Ethyl 2-methyl-3- pentenoate in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay
Direct Plate Assay
(µg/plate) |
| ||
|
|
|
|
Without S9-mix
Positive control | 799 | ± | 113 |
| 1746 | ± | 122 |
|
|
|
|
|
Solvent control | 92 | ± | 5 |
| 16 | ± | 4 |
|
|
|
|
|
1.7 | 102 | ± | 7 |
| 18 | ± | 3 |
|
|
|
|
|
5.4 | 119 | ± | 6 |
| 19 | ± | 4 |
|
|
|
|
|
17 | 97 | ± | 6 |
| 16 | ± | 2 |
|
|
|
|
|
52 | 91 | ± | 11 |
| 15 | ± | 1 |
|
|
|
|
|
164 | 94 | ± | 19 |
| 19 | ± | 5 |
|
|
|
|
|
512 | 80 | ± | 6 |
| 15 | ± | 8 |
|
|
|
|
|
1600 | 87 | ± | 7 |
| 20 | ± | 2 |
|
|
|
|
|
5000 | 80 | ± | 7 | n NP | 12 | ± | 3 | n NP |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
With S9-mix
Positive control | 1367 | ± | 44 |
| 383 | ± | 7 |
|
|
|
|
|
Solvent control | 85 | ± | 12 |
| 14 | ± | 4 |
|
|
|
|
|
1.7 | 90 | ± | 6 |
| 16 | ± | 7 |
|
|
|
|
|
5.4 | 96 | ± | 7 |
| 13 | ± | 5 |
|
|
|
|
|
17 | 95 | ± | 24 |
| 12 | ± | 7 |
|
|
|
|
|
52 | 83 | ± | 10 |
| 16 | ± | 2 |
|
|
|
|
|
164 | 84 | ± | 2 |
| 15 | ± | 11 |
|
|
|
|
|
512 | 86 | ± | 10 |
| 12 | ± | 6 |
|
|
|
|
|
1600 | 70 | ± | 3 |
| 15 | ± | 3 |
|
|
|
|
|
5000 | 80 | ± | 12 | n NP | 21 | ± | 11 | n NP |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
NP | No precipitate |
n | Normal bacterial background lawn |
Table 2. Experiment 1: Mutagenic Response of Ethyl 2-methyl-3-pentenoate in the Salmonella typhimurium Reverse Mutation Assay
Direct Plate Assay
(µg/plate) |
| ||
|
|
|
|
Without S9-mix
Positive control | 972 | ± | 39 |
| 1135 | ± | 85 |
| 1795 | ± | 78 |
|
Solvent control | 8 | ± | 7 |
| 3 | ± | 1 |
| 15 | ± | 2 |
|
52 | 10 | ± | 2 |
| 4 | ± | 3 |
| 11 | ± | 4 |
|
164 | 8 | ± | 4 |
| 6 | ± | 4 |
| 16 | ± | 3 |
|
512 | 8 | ± | 3 |
| 4 | ± | 4 |
| 15 | ± | 5 |
|
1600 | 6 | ± | 4 |
| 3 | ± | 2 |
| 15 | ± | 7 |
|
5000 | 11 | ± | 3 | n NP | 2 | ± | 2 | n NP | 12 | ± | 3 | n NP |
|
|
|
|
|
|
|
|
|
|
|
|
|
With S9-mix
Positive control | 306 | ± | 8 |
| 243 | ± | 12 |
| 2334 | ± | 77 |
|
Solvent control | 8 | ± | 3 |
| 5 | ± | 2 |
| 17 | ± | 3 |
|
52 | 12 | ± | 3 |
| 5 | ± | 5 |
| 18 | ± | 5 |
|
164 | 9 | ± | 4 |
| 3 | ± | 2 |
| 17 | ± | 3 |
|
512 | 7 | ± | 4 |
| 3 | ± | 0 |
| 16 | ± | 6 |
|
1600 | 15 | ± | 5 |
| 2 | ± | 2 |
| 13 | ± | 1 |
|
5000 | 7 | ± | 1 | n NP | 4 | ± | 3 | n NP | 14 | ± | 7 | n NP |
|
|
|
|
|
|
|
|
|
|
|
|
|
NP | No precipitate |
n | Normal bacterial background lawn |
Table 3. Experiment 2: Mutagenic Response of Ethyl 2-methyl-3-pentenoate in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay
Pre-incubation Assay
(µg/plate) |
| ||||
|
|
|
|
|
|
Without S9-mix
Positive control | 1139 | ± | 89 |
| 164 | ± | 15 |
| 1896 | ± | 106 |
| 838 | ± | 59 |
| 1501 | ± | 71 |
|
Solvent control | 8 | ± | 3 |
| 3 | ± | 2 |
| 6 | ± | 4 |
| 102 | ± | 17 |
| 17 | ± | 6 |
|
Positive control 1 | 1023 | ± | 45 |
| 118 | ± | 11 |
| 1988 | ± | 44 |
| 627 | ± | 23 |
| 1251 | ± | 236 |
|
DMSO 1 | 11 | ± | 5 |
| 2 | ± | 1 |
| 11 | ± | 4 |
| 81 | ± | 15 |
| 16 | ± | 5 |
|
5.4 1 | 10 | ± | 3 |
| 3 | ± | 1 |
| 9 | ± | 4 |
| 76 | ± | 12 |
| 12 | ± | 2 |
|
17 1 | 6 | ± | 1 |
| 3 | ± | 2 |
| 9 | ± | 4 |
| 86 | ± | 10 |
| 14 | ± | 7 |
|
52 | 6 | ± | 5 |
| 1 | ± | 0 |
| 10 | ± | 2 |
| 106 | ± | 12 |
| 22 | ± | 6 |
|
164 | 9 | ± | 1 | n | 3 | ± | 1 | n | 11 | ± | 7 | n | 109 | ± | 12 | n | 17 | ± | 3 |
|
512 | 6 | ± | 3 | s | 4 | ± | 3 | s | 9 | ± | 4 | s | 99 | ± | 11 | s | 26 | ± | 4 | n |
1600 |
|
|
| e MC |
|
|
| e MC |
|
|
| e MC |
|
|
| e MC |
|
|
| e MC |
5000 |
|
|
| e NP MC |
|
|
| e NP MC |
|
|
| e NP MC |
|
|
| e NP MC |
|
|
| e NP MC |
With S9-mix
Positive control | 250 | ± | 24 |
| 153 | ± | 29 |
| 661 | ± | 27 |
| 2185 | ± | 48 |
| 425 | ± | 74 |
|
Solvent control | 9 | ± | 3 |
| 3 | ± | 1 |
| 14 | ± | 6 |
| 95 | ± | 8 |
| 34 | ± | 4 |
|
52 | 5 | ± | 0 |
| 3 | ± | 2 |
| 16 | ± | 4 |
| 96 | ± | 4 |
| 22 | ± | 7 |
|
164 | 8 | ± | 4 |
| 3 | ± | 2 |
| 13 | ± | 3 |
| 106 | ± | 13 |
| 35 | ± | 4 |
|
512 | 7 | ± | 5 | n | 4 | ± | 1 | n | 15 | ± | 5 | n | 83 | ± | 1 | n | 32 | ± | 2 |
|
1600 | 5 | ± | 2 | m | 1 | ± | 2 | m | 7 | ± | 8 | m | 70 | ± | 8 | m | 25 | ± | 3 | n |
5000 |
|
| e NP MC |
|
| e NP MC |
|
| e NP MC |
|
| e NP MC |
|
| e NP MC |
MC | Microcolonies |
NP | No precipitate |
e | Bacterial background lawn extremely reduced |
m | Bacterial background lawn moderately reduced |
n | Normal bacterial background lawn |
s | Bacterial background lawn slightly reduced |
1 | Data from an additional experiment |
Applicant's summary and conclusion
- Conclusions:
- The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and the Escherichia coli Reverse Mutation Assay (Plate Incorporation and Pre-Incubation Methods) performed according to OECD 471.
- Executive summary:
The substance is tested in the Ames test according to OECD TG 471 and following GLP. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. The following concentrations were tested: 52, 164, 512, 1600 and 5000 μg/plate in experiment 1; 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (without S9-mix); 52, 164, 512, 1600 and 5000 μg/plate (with S9-mix) in experiment 2. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a dose-related increase in the number of revertant colonies in each of the five tester strains (TA1535, TA1537, TA98 and TA100 WP2uvrA) both in the absence and presence of S9-metabolic activation. Based on this the substance is not mutagenic in this test.
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