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EC number: 266-719-9 | CAS number: 67564-91-4
- 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
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17. Jan. 1994 - 03. Mar.1994
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 994
- Report date:
- 1994
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 1984
- Deviations:
- no
- 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:
- cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine
- EC Number:
- 266-719-9
- EC Name:
- cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine
- Cas Number:
- 67564-91-4
- Molecular formula:
- C20H33NO
- IUPAC Name:
- (2R,6S)-4-[3-(4-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine
Constituent 1
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Chinese Hamster Ovary Cells (CHO) K 1; ICN Flow Labs., Meckenheim, FRG
- Suitability of cells: Recommended by guideline
For cell lines:
- Absence of Mycoplasma contamination: Not specified
- Methods for maintenance in cell culture: Cells were routinely grown in monolayers at approx. 37°C in an incubator. For each subculture cells were trypsinized and reseeded at a density of approx. 100,000 or 300,000— 500,000 cells in 25 cm2 or 80 cm2 flasks respectively and covered with medium (approx.5ml medium for small flasks, approx. 10 ml for 80 cm2 flasks).
-Subcultivations: 2 subcultivations were performed per week Incubation conditions: approx. 37°C; approx.90% humidity; approx. 5% CO2
- Modal number of chromosomes: 18-22
- Periodically checked for karyotype stability: no
- Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
1. Ham's F12 medium, supplemented with 1% v/v glutamine (200 mM) and 10% v/v fetal calf serum (FCS); During exposure to the test substance Ham's F12 medium was used without FCS supplementation
2. Selection medium A ("HAT—medium"): Glutamine—and FCS-supplemented Ham's F12 medium containing per ml: 3.88x 10exp3 mg Thymidine13.61x 10exp3 mg Hypoxanthine 0.19x10exp3 mg Aminopterine
3.Selection medium B ("TG—medium"): Glutamine—and FCS—supplemented, Hypoxanthine—free Ham's F12 medium with 6-Thioguanine (TG) at a final concentration of 10 pg/ml. All media were supplemented with Penicillin (50IU/ml) and Strepto-mycin (50ug/ml).
37°C humidified atmosphere containing approx. 5% carbondioxide (incubator)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: 5 male Sprague-Dawley Rats
- method of preparation of S9 mix: 500 mg Aroclor1254 solution in cornoil (20%w/v) per kg body weight was administered once intraperitoneally to 5 male Spraque—Dawley rats 5 days before sacrifice. At the 5th day the animals were sacrificed and the livers prepared. All the preparation steps for recovering the microsome enzymes were carried out using sterile solvents and vessels at a temperature of +4°C. The livers were weighed and washed in an equivalent volume of a 150mM CaCl2-solution (approx.1 ml per g wet liver), then cut into small pieces and homogenized in a 3-fold volume of 150 mM CaCl2 solution. After the homogenate had been centrifuged at 9,000g for 10 minutes at +4°C, the supernatant (S-9fraction) was deep frozen in 3 ml portions in dry ice and stored for a maximum of 12 months at -70 to -80°C
A sufficient amount of S-9Fraction was thawed to room temperature prior to each experiment. 3 parts of S-9 fraction were mixed with 7 parts of S-9 supplement of a stock solution
- concentration or volume of S9 mix and S9 in the final culture medium: 2ml S9-Mix + 7ml cell culture medium+ 1ml ml test substance - Test concentrations with justification for top dose:
- Experiment 1 (without rat liver S-9 mix): 0;
0.1;
0.215;
0.464;
1;
2.15;
4.64;
10 µg/ml
Experiment1 (with rat liver S-9mix):0;
1;
2.15;
4.64;
10;
21.5;
46.4;
100µg/ml
Experiment2 (without rat liver S-9 mix):0;
0.25;
0.5;
0.75;
1;
2.5;
5;
7.5µg/ml
Experiment 2 (with rat liver S—9 mix):0;
10;
25;
50;
75;
100;
150;
200 µg/ml
In a range—finding cytotoxicity test cells were treated with Fenpropimorph at concentrations ranging from 0.05 to 100 µg/ml (without S-9 mix) and from 0.1 to 500 µg/ml (with S-9 mix). This resulted in marked cytotoxicity at concentrations > 5 µg/ml in the absence and > 100µg/ml in the presence of S-9 mix. - Vehicle / solvent:
- 1 % Ethanol
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 500,000
- Test substance added in: medium
TREATMENT AND HARVEST SCHEDULE:
- Pretreatment of cells: During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated from the stock cultures by growing the cells for 3 - 4 days in HAT medium
- Exposure duration/duration of treatment: 4 hours
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 1 week
- Selection time (if incubation with a selective agent): 1 week
- At the end of the expression period, 6 x 300,000 cells from each treatment group were seeded in six flasks with selection medium containing 6-thioguanine (10µg/ml) for 1 week
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 6 x ~300,000, A Coulter counter was used. The numbers obtained in the untreated controls were cross—checked in a Bürker chamber. Colony counting: Giemsa-stained colonies were scored visually
-Fixing and staining: Ethanol642/Giemsa
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cloning efficiency - Rationale for test conditions:
- In a range—finding cytotoxicity test cells were treated with Fenpropimorph at concentrations ranging from 0.05 to 100 µg/ml (withoutS-9 mix) and from 0.1 to 500 µg/ml (with S-9 mix). This resulted in marked cytotoxicity at concentrations > 5 µg/ml in the´absence and > 100µg/ml in the presence of S-9mix.
- Evaluation criteria:
- The criteria for a positive response are: Increases of the corrected mutation frequencies over the concurrent control values and over 15 mutants per 10exp6 clonable cells and/or the evidence of a dose—response relationship in the increase in mutant frequencies. Evidence of reproducibility of any increase in mutant frequencies. A statistically significant increase in mutantfrequenciesandtheevidenceofa dose-response relationship. Isolated increases of mutant frequencies above 15 mutants per 10exp6 clonable cells or isolated statistically significant increases in the lower concentrations without a dose relationship may indicate a biological effect, but are not regarded as sufficient evidence for mutagenicity.
- Statistics:
- The number of mutant colonies in each flask of the dose groups, the positive controls and the untreated controls was compared to that of the solvent control groups using the Fisher-Pitman-Test for the hypothesis of equal means. The independence of the 6 flasks within each group was assumed for this test. If the results were significant, labels (* for p < 0.05;** for p < 0.01) were printed with the group values (uncorrected mutant frequency) in the tables. The test was performed one—sided.
Results and discussion
Test results
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Reduced cell densities and reduced cloning efficiencies were found 18 - 20 hours after exposure to the test substance at concentrations >5 µg/ml in the experiments without S—9 mix and concentrations >100µg/ml in the presence of S—9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Applicant's summary and conclusion
- Conclusions:
- It was concluded that Fenpropimorph did not demonstrate a mutagenic potential under the conditions used in this invitro gene mutation assay.
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