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EC number: 245-950-9 | CAS number: 23949-66-8
- 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
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011-08-30 until 2011-10-25
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP study conducted according to international guidelines. No relevant deviations have been reported so that the study is considered reliable.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 011
- Report date:
- 2011
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
- Principles of method if other than guideline:
- first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- N-(2-ethoxyphenyl)-N'-(2-ethylphenyl)oxamide
- EC Number:
- 245-950-9
- EC Name:
- N-(2-ethoxyphenyl)-N'-(2-ethylphenyl)oxamide
- Cas Number:
- 23949-66-8
- Molecular formula:
- C18H20N2O3
- IUPAC Name:
- N-(2-ethoxyphenyl)-N'-(2-ethylphenyl)ethanediamide
- Details on test material:
- - Molecular weight (if other than submission substance):312 g/mol
- Physical state: Solid, white
- Analytical purity: 99.23 g/mol
- Purity: 99.23 %
- Purity test date 2010-09-01:
- Lot/batch No.: CHA0054786
- Expiration date of the lot/batch: 2013-12-03
- Stability under test conditions: Not indicated by the sponsor
- Storage condition of test material: Room temperature
Constituent 1
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without metabolic activation: 6.3; 12.5; 25.0; 50.0; 100; 200; 400 µg/mL
with metabolic activation: 6.3; 12.5; 25.0; 50.0; 100; 3200 µg/mL
Experiment II:
without metabolic activation: 6.3; 12.5; 25.0; 50.0; 100; 200; 3200 µg/mL
with metabolic activation: 6.3; 12.5; 25.0; 50.0; 100; 200 µg/mL
In the first experiment the concentration of 6.3 µg/mL with metabolic activation was not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures at 100 µg/mL and above in experiment I without metabolic activa-tion were not continued to avoid analysis of too many precipitating concentrations. In the second experiment the concentration of 100 and 200 µg/mL without metabolic activation and at 100 µg/mL with metabolic activation were not continued for the same reason. - Vehicle / solvent:
- DMSO
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5x10exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points 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 reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was 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 relevance and statistical significance were considered together.
Results and discussion
Test resultsopen allclose all
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected
- Effects of osmolality: Not increased
- Evaporation from medium: Not examined
- Water solubility: Not indicated by the sponsor
- Precipitation: Precipitation was observed in the first experiment: at 12.5 to 400 µg/mL without metabolic activation; at 50.0 to 3200 µg/mL with metabolic activation; in the second experiment: at 25.0 to 3200 µg/mL without metabolic activation; at 50.0 to 200 µg/mL with metabolic activation.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
In the range finding pre-experiment test item concentrations between 25 and 3200 µg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant toxic effects oc-curred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 50 µg/mL and above in the presence and absence of metabolic activation (4 and 24 hours treatment).
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The basic level of osmolarity however, was relatively high even at the solvent control based on a DMSO concentration of 1%. DMSO interferes with the freezing point depression technique used to measure osmolarity.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed into the lower range. Larger spacing was used at high concentrations to avoid analysis of too many precipitating concentrations.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or a relative cell density below 50% of the corresponding solvent control occurred in the first experiment without metabolic activation at 50 µg/mL and above. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Summary Table
relative | relative | relative | mutant | relative | relative | relative | mutant | ||||||
conc. | P | S9 | cloning | cell | cloning | colonies/ | induction | cloning | cell | cloning | colonies/ | induction | |
µg/mL | mix | efficiency I | density | efficiency II | 106cells | factor | efficiency I | density | efficiency II | 106cells | factor | ||
% | % | % | % | % | % | ||||||||
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 |
Experiment I / 4 h treatment | culture I | culture II | |||||||||||
Solvent control with DMSO | - | 100.0 | 100.0 | 100.0 | 17.7 | 1.0 | 100.0 | 100.0 | 100.0 | 5.5 | 1.0 | ||
Positive control (EMS) | 150.0 | - | 71.1 | 102.0 | 103.8 | 84.4 | 4.8 | 73.5 | 150.2 | 93.9 | 93.0 | 16.9 | |
Test item | 6.3 | - | 79.0 | 115.2 | 97.5 | 8.7 | 0.5 | 81.5 | 154.1 | 96.0 | 21.8 | 4.0 | |
Test item | 12.5 | P | - | 69.9 | 95.3 | 103.6 | 3.5 | 0.2 | 71.1 | 168.5 | 97.7 | 8.8 | 1.6 |
Test item | 25.0 | P | - | 67.6 | 109.7 | 106.7 | 25.1 | 1.4 | 70.4 | 85.9 | 96.2 | 17.7 | 3.2 |
Test item | 50.0 | P | - | 48.3 | 90.7 | 105.6 | 6.3 | 0.4 | 47.1 | 116.6 | 92.0 | 18.3 | 3.3 |
Test item | 100.0 | P | - | 41.6 | culture was not continued# | 31.1 | culture was not continued# | ||||||
Test item | 200.0 | P | - | 30.0 | culture was not continued# | 31.7 | culture was not continued# | ||||||
Test item | 400.0 | P | - | 29.2 | culture was not continued# | 40.4 | culture was not continued# | ||||||
Solvent control with DMSO | + | 100.0 | 100.0 | 100.0 | 21.0 | 1.0 | 100.0 | 100.0 | 100.0 | 14.3 | 1.0 | ||
Positive control (DMBA) | 1.1 | + | 41.6 | 55.3 | 86.4 | 647.1 | 30.8 | 49.2 | 49.4 | 100.1 | 862.1 | 60.4 | |
Test item | 6.3 | + | 103.7 | culture was not continued## | 94.8 | culture was not continued## | |||||||
Test item | 12.5 | + | 100.8 | 119.7 | 99.6 | 7.4 | 0.4 | 100.3 | 98.5 | 102.8 | 19.6 | 1.4 | |
Test item | 25.0 | + | 102.2 | 98.9 | 102.7 | 14.2 | 0.7 | 91.8 | 93.2 | 93.5 | 17.9 | 1.3 | |
Test item | 50.0 | P | + | 103.9 | 91.5 | 96.7 | 20.2 | 1.0 | 92.5 | 83.3 | 106.6 | 13.8 | 1.0 |
Test item | 100.0 | P | + | 81.5 | 79.0 | 82.7 | 22.2 | 1.1 | 91.6 | 82.5 | 110.7 | 11.6 | 0.8 |
Test item | 3200.0 | P | + | 101.2 | 95.2 | 91.5 | 14.9 | 0.7 | 86.5 | 97.3 | 112.5 | 14.0 | 1.0 |
Experiment II / 24 h treatment | culture I | culture II | |||||||||||
Solvent control with DMSO | - | 100.0 | 100.0 | 100.0 | 6.0 | 1.0 | 100.0 | 100.0 | 100.0 | 7.4 | 1.0 | ||
Positive control (EMS) | 150.0 | - | 98.7 | 86.9 | 91.7 | 221.2 | 36.6 | 102.3 | 86.8 | 108.6 | 225.1 | 30.4 | |
Test item | 6.3 | - | 100.1 | 91.2 | 94.8 | 6.3 | 1.0 | 99.8 | 89.5 | 100.7 | 9.9 | 1.3 | |
Test item | 12.5 | - | 103.4 | 119.2 | 87.9 | 15.2 | 2.5 | 110.6 | 96.4 | 94.2 | 14.2 | 1.9 | |
Test item | 25.0 | P | - | 102.7 | 81.8 | 101.7 | 10.1 | 1.7 | 103.9 | 75.1 | 107.0 | 11.8 | 1.6 |
Test item | 50.0 | P | - | 96.4 | 93.6 | 85.8 | 6.2 | 1.0 | 103.1 | 73.5 | 116.7 | 4.3 | 0.6 |
Test item | 100.0 | P | - | 96.7 | culture was not continued# | 102.0 | culture was not continued# | ||||||
Test item | 200.0 | P | - | 92.0 | culture was not continued# | 104.0 | culture was not continued# | ||||||
Test item | 3200.0 | P | - | 96.9 | 69.8 | 88.9 | 11.8 | 2.0 | 102.6 | 80.1 | 120.0 | 10.0 | 1.3 |
Experiment II / 4 h treatment | |||||||||||||
Solvent control with DMSO | + | 100.0 | 100.0 | 100.0 | 4.8 | 1.0 | 100.0 | 100.0 | 100.0 | 9.8 | 1.0 | ||
Positive control (DMBA) | 1.1 | + | 47.1 | 36.1 | 67.8 | 352.6 | 73.1 | 74.1 | 44.3 | 87.3 | 589.4 | 60.4 | |
Test item | 6.3 | + | 86.2 | 70.6 | 74.9 | 7.0 | 1.4 | 99.4 | 116.6 | 78.2 | 13.5 | 1.4 | |
Test item | 12.5 | + | 94.6 | 81.3 | 81.7 | 12.6 | 2.6 | 94.8 | 102.3 | 76.7 | 7.1 | 0.7 | |
Test item | 25.0 | + | 91.0 | 92.8 | 80.5 | 9.6 | 2.0 | 93.9 | 115.8 | 88.8 | 13.0 | 1.3 | |
Test item | 50.0 | P | + | 91.0 | 75.3 | 66.4 | 10.5 | 2.2 | 91.6 | 106.6 | 70.5 | 10.2 | 1.0 |
Test item | 100.0 | P | + | 86.4 | culture was not continued# | 98.9 | culture was not continued# | ||||||
Test item | 200.0 | P | + | 84.3 | 107.9 | 73.1 | 5.0 | 1.0 | 105.8 | 135.9 | 96.2 | 6.4 | 0.7 |
# culture was not continued to avoid analysis of too many precipitating concentrations
## culture was not continued since a minimum of only four analysable concentrations is required
P precipitation
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
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. - Executive summary:
The test item Hostavin VSU P was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The maximum concentration of the test item equals a molar concentration of 10 mM, with respect to the purity of the test item (99.23 %).
Precipitation at the end of treatment, visible to the unaided eye occurred at 50.0 µg/mL and above with and without metabolic activation in the pre-experiment. In experiment I precipitation was noted at 12.5 µg/mL and above without metabolic activation and at 50.0 µg/mL and above with metabolic activation. In experiment II precipitation was observed at 25.0 µg/mL and above without metabolic activation and at 50.0 µg/mL and above with metabolic activation.
Relevant cytotoxic effects indicated by a relative cloning efficiency I or a relative cell density below 50% of the corresponding solvent control occurred in the first experiment without metabolic activation at 50 µg/mL and above.
No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The induction factor reached the threshold of three times the corresponding solvent control in the second culture of experiment I without metabolic activation at 6.3, 50, and 100 µg/mL (4 hours treatment). This effect however, was judged as based upon the rather low solvent control of just 5.5 mutant colonies/106cells. The absolute values of the mutation frequencies (21.8, 17.7, and 18.3 mutant colonies per 106cells) remained well within the historical range of solvent controls.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 4.8 up to 21.0 mutants per 106cells; the range of the groups treated with the test item was from 3.5 up to 25.1 mutants per 106cells.
(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
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