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EC number: 203-380-8 | CAS number: 106-27-4
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- March - May 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Guidelines: “Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: gene mutation assay in mammalian cells
Test material
- Reference substance name:
- 3-methylbutyl isovalerate
- EC Number:
- 211-536-1
- EC Name:
- 3-methylbutyl isovalerate
- Cas Number:
- 659-70-1
- Molecular formula:
- C10H20O2
- IUPAC Name:
- 3-methylbutyl 3-methylbutanoate
Constituent 1
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- other: Chinese hamster (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Suitability of cells: Yes
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10 % FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. Cell culture incubation: humidified atmosphere with 1.5 % CO2 at 37 °C.
- Properly maintained: [yes]
- Checked for Mycoplasma contamination: [yes]
- Checked for karyotype stability: [yes)
- "Cleansed' against high spontaneous background: [yes]
- Metabolic activation:
- with and without
- Metabolic activation system:
- mammalian microsomal fraction S9 mix
- Test concentrations with justification for top dose:
- Doses applied in the gene mutation assay (concentrations marked with ¹ were chosen for the mutation rate analysis):
Experiment I (without S9, 4 h exposure): 1.7, 3.4 ¹, 6.8 ¹, 13.5 ¹, 27.0 ¹, 54.0 ¹, 108.0 µg/mL
Experiment II (with S9, 4 h exposure): 26.9 ¹, 53.8 ¹, 107.5 ¹, 215.0 ¹, 430.0* ¹, 860.0* µg/mL
* indicates Phase separation observed
The cultures at the lowest concentration without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines. The cultures at the highest concentration without metabolic activation were not continued based on exceedingly severe cytotoxicity. The cultures at the highest concentration with metabolic activation were not continued to avoid analysis of too many insoluble concentrations. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 d
- Selection time (if incubation with a selection agent): 8 d
SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)
NUMBER OF REPLICATIONS: 5 to determine mutation (culture flasks with medium containing 6-TG per concentration)
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Stained with 10 % methylene blue in 0.01 % KOH solution
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: The experimental part of the main experiment without metabolic activation was prematurely terminated as exceedingly severe cytotoxicity occurred already at low concentrations. This experimental part was repeated as experiment IA in a lower concentration range. Experiment IA was again terminated due to severe cytotoxicity and repeated as experiment IB with even lower concentrations. The data of experiment IB are reported as first experiment without metabolic activation. - Evaluation criteria:
- A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares). - Statistics:
- Statistical Analysis: A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
experimental group p-value
experiment I, culture I without S9 mix, p-value: 0.492
experiment I, culture II without S9 mix, p-value: 0.025 S
experiment I, culture I with S9 mix, p-value: 0.453
experiment I, culture II with S9 mix, p-value: 0.855
S = significant trend
Results and discussion
Test results
- Key result
- Species / strain:
- other: Chinese hamster (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No relevant and reproducible increase in mutant colony numbers/1E6 cells was observed in the main experiment up to the maximum concentration. The 95 % confidence interval was not exceeded.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the second culture without metabolic activation. This trend however, was judged as irrelevant as all of the absolute values of the mutation frequency remained within the 95 % confidence interval.
In the main experiment with and without S9 mix the range of the solvent controls was from 13.0 up to 18.1 mutants per 1E6 cells; the range of the groups treated with the test item was from 9.3 up to 26.9 mutants per 1E6 cells.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
Applicant's summary and conclusion
- Conclusions:
- The test item did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic in this HPRT assay.
- Executive summary:
The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (1723 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiment was limited by the cytotoxicity and the solubility of the test item.
The experimental part of the main experiment without metabolic activation was prematurely terminated as exceedingly severe cytotoxicity occurred already at low concentrations. This experimental part was repeated as experiment IA in a lower concentration range. Experiment IA was again terminated due to severe cytotoxicity and repeated as experiment IB with even lower concentrations. The data of experiment IB are reported as the first experiment without metabolic activation.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
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