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EC number: 271-272-8 | CAS number: 68527-63-9
- 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:
- 2021-06-28 to 2022-01-03
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
- Report date:
- 2022
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Version / remarks:
- adopted 29th July 2016
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Reference substance name:
- Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1)
- EC Number:
- 271-272-8
- EC Name:
- Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1)
- Cas Number:
- 68527-63-9
- Molecular formula:
- C22H42N2O.C4H10O4S
- IUPAC Name:
- 2-{2-[(8E)-heptadec-8-en-1-yl]-4,5-dihydro-1H-imidazol-1-yl}ethan-1-ol diethyl sulfate
- Test material form:
- liquid
- Details on test material:
- Test item formulations were found to be homogeneous and stable up to 24 hour in vehicle corn oil.
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- batch number of test material: 10419
- Purity, including information on contaminants, isomers, etc.: > 93 %
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry and dark at ambient room temperature (20 – 30 °C)
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- CHO-K1 cell
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- concentration or volume of S9 mix and S9 in the final culture medium: 1% v/v - Test concentrations with justification for top dose:
- The main study was performed with test item concentrations of 0.488281, 0.976563, 1.953125 and 3.90625 µg/ml in the absence and 0.976563 , 1.953125, 3.90625 and 7.8125 µg/ml in the presence of metabolic activation system along with vehicle, negative and positive controls.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: test item was found to be insoluble in distilled water, while it was found to be soluble in dimethyl sulfoxide. Hence, dimethyl sulfoxide was selected as vehicle for this study.
- Justification for percentage of solvent in the final culture medium: no precipitation was observed at the concentrations of 2000, 1000 and 500 µg/ml. Based on these results 2000 µg/ml was selected as a highest concentration for preliminary cytotoxicity assay.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Media and Culture conditions
- appropriate culture medium and incubation conditions (culture vessels, humidified atmosphere of 5 % C0 2, and incubation temperature of 37± 2 °C) was used for maintaining the cells
- a-MEM medium (supplemented with nucleosides) with 10 % Fetal Bovine Serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) for maintaining CHO-K1 cell lines was used
Maintenance of Cell line
- cell cultures cleansed of pre-existing mutant cells, e.g. by culturing in HAT medium (medium containing Hypoxanthine, Aminopterin, and Thymidine) were used for HPRT test
Test ltem Solubility
- solubility, precipitation, pH and osmolality of the test item in media was determined before the cytotoxicity test
- pH and Osmolality of the test item in media was checked at 0 hour and at 4 hour after incubation in CO2 incubator
Preliminary Cytotoxicity Study
- 200 mg of test item was made up to 1 ml in dimethyl aulfoxideto get a final concentration 200 mg/ml. From this stock concentration, 0.5 ml was aspirated and added to 0.5 ml of DMSO to get a stock concentration of 100 mg/ml. The subsequent serial dilutions were made to get concentrations of 50, 25, 12.5 and 6.25 mg/ml with dimethyl sulfoxide using a spacing factor of 2
Test ltem Dilution Preparation of Preliminary Cytotoxicity Assay (II)
- evaluation of cytotoxicity was done to define the concentrations to be used in the main experiment. Cytotoxicity was evaluated using RS, i.e. cloning efficiency (CE) of cells plated immediately after treatment adjusted by any lass of cells during treatment as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100 %). - Evaluation criteria:
- Criteria for a Positive Response
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- the increase is concentration-related when evaluated with a trend test.
- any of the results are outside the distribution of the historical negative control data.
- when all the above criteria are met, the test item is then considered able to induce gene mutations in cultured mammalian cells in this test system (CHO-K1 cell line).
Criteria for a Negative Response
- none of the test concentrations exhibit a statistically significant increase compared with the concurrent negative control.
- there is no concentration-related increase when evaluated with a trend test.
- when the above criteria are met, the test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system (CHO-K1 cell line). - Statistics:
- Non-parametric statistics was performed to assess a possible dose dependent increase of mutant frequencies using validated statistics software. The number of mutant colonies obtained for the groups treated with the test item was comparable to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both biological relevance and statistical significance was considered together.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In Preliminary Cytotoxicity Assay (1) cytotoxicity was observed at the lowest tested concentration of 62.5 µg/ml (relative survival (RS): 2.32 %) in the absence and (RS: 1.69 %) in the presence of metabolic activation system as compared to vehicle control
Applicant's summary and conclusion
- Conclusions:
- Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5- dihydro-1H-imidazole-1-ethanol (1:1) did not induce gene mutations in the CHO-K1 cell line, in the presence and absence of exogenous metabolic activation system (S9), respectively.
- Executive summary:
In vitro Mammalian Cell Gene Mutation Test (HPRT assay) for test item Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) in CHO-K1 cells has been performed as per OECD guideline No. 476 & B.17.
The test item was found to be insoluble in distilled water, while it was found to be soluble in dimethyl sulfoxide.
According to the results of the Cytotoxicity Assay II, the main study was performed at the concentrations of 0.488281, 0.976563, 1.953125 and 3.90625 µg/ml in the absence and 0.976563, 1.953125, 3.90625 and 7.8125 µg/ml in the presence (1 % v/v S9 Mix) of metabolic activation system. In Phase 1, the RS was 86.37 (0.488281 µg/ml ), 70.75 (0.976563 µg/ml), 75.69 (1.953125µg/ml) and 64.62 (3.90625µg/ml) in the absence and 89.30 (0.976563 µg/ml), 76.44 (1.953125 µg/ml ), 67.48 (3.90625 µg/ml ) and 61.74 (7.8125 µg/ml) in the presence (1 % v/v S9 Mix) of metabolic activation system.The observed mean mutant frequency was 8.44, 8.38, 8.93 and 9.66 at 0.488281, 0.976563, 1.953125 and 3.90625 µg/ml, respectively in absence and 8.10, 8.71, 9.03 and 9.52 at0.976563, 1.953125, 3.90625 and 7.8125 µg/ml, respectively in presence (1 % v/v S9 Mix), as compared to vehicle control.
On the basis of the results of this study Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5- dihydro-1H-imidazole-1-ethanol (1:1) did not induce gene mutations in the CHO-K1 cell line, in the presence and absence of exogenous metabolic activation system (S9), respectively under the specified experimental condition.
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