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EC number: 918-205-7 | CAS number: -
- 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
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- 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
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- Endpoint summary
- Stability
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- 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 bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1998-10-09 to 1998-10-19
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 998
- Report date:
- 1998
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted July 21, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted December, 1992
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Ethyltriphenylfosfonium bromide
- EC Number:
- 216-223-3
- EC Name:
- Ethyltriphenylfosfonium bromide
- Cas Number:
- 1530-32-1
- Molecular formula:
- C20H20P.Br
- IUPAC Name:
- bromo(ethyl)triphenylphosphorane
Constituent 1
Method
- Target gene:
- Salmonella strain Histidin mutation Mutation type
TA98 hisD3052/R-factor* Frameshifts
TA100 hisG46/R-factor* Base-pair substitution
TA1535 hisG46 Base-pair substitution
TA1537 hisC3076 Frameshifts
*: R-factor = plasmid pKM101 (increase error-prone DNA repair)
E.coli stain WP2uvrA DNA targets: A˙T (trpE65, lysT) and G˙C (glnU, tyrT, tyrU) base pairs. For further characterization of Trp+reversions in Escherichia coli strain WP2uvrA please refer to Toshihiro Ohta et al., Mutagenesis (2002) 17 (4): 313-316
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix (rat)
- Test concentrations with justification for top dose:
- 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg / plate
- Vehicle / solvent:
- Milli-Q water
Controlsopen allclose all
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- saline
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 1 µg/plate
- Positive control substance:
- sodium azide
- Remarks:
- TA1535 without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- saline
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 60 µg/plate
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA1537 without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- saline
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 4 µg/plate
- Positive control substance:
- other: daunomycine (DM)
- Remarks:
- TA98 without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 650 µg/plate
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- TA100 without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 10 µg/plate
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- WP2uvrA without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 2.5 µg/plate
- Positive control substance:
- other: 2-aminoanthracene (2AA)
- Remarks:
- TA1537 with metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 1 µg/plate
- Positive control substance:
- other: 2-aminoanthracene (2AA)
- Remarks:
- TA1535, TA98, TA100 and E.coli WP2uvrA with metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 5 µg/plate
- Positive control substance:
- other: 2-aminoanthracene (2AA)
- Remarks:
- WP2uvrA with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: n.a.
- Exposure duration: 48h
- Expression time (cells in growth medium): n.a.
- Selection time (if incubation with a selection agent): n.a.
- Fixation time (start of exposure up to fixation or harvest of cells): n.a.
SELECTION AGENT (mutation assays): histidine (Salmonella typhimurium strains), tryptophan (Escherichia coli WP2uvrA)
SPINDLE INHIBITOR (cytogenetic assays): n.a.
STAIN (for cytogenetic assays): n.a.
NUMBER OF REPLICATIONS: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: n.a.
NUMBER OF CELLS EVALUATED: n.a.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n.a.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n.a.
DETERMINATION OF CYTOTOXICITY
- Method: determination of inhibited bacterial growth
- Any supplementary information relevant to cytotoxicity: n.a.
OTHER EXAMINATIONS:
- Determination of polyploidy: n.a.
- Determination of endoreplication: n.a
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): n.a.
- OTHER:
Preparation of S9-fraction:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany. The animals were housed at the test facility in a special room under standard laboratory conditions, as described in the SOP's. The rats were injected intraperitoneally with a solution (20% (w/v)) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C) and identified by the day of preparation.
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml or 5.0 mL aqua bidest (first or second experiment respectively); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 MMgC1 2 solution; 1 mL 0.33 MKCl solution. The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.
EXPERIMENTAL PROCEDURE
Dose range finding test
Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9mix.
Eight concentrations were tested in triplicate. This dose range finding test was reported as apart of the first experiment of the mutation assay. The highest concentration of Ethyltriphenylfosfonium bromide used in the subsequent mutation assay was 5 mg/plate or the level at which the test substance inhibited bacterial growth.
Mutation assay
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments. Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test substance in Milli-Q water and either 0.5 mL S9mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37°C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Colony counting
The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present.
ACCEPTABILITY OF ASSAY
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance.
c) The selected dose range should include a clearly toxic concentration or should be exhibit by limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate. - Evaluation criteria:
- A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- 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
- 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
- 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
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- DOSE RANGE FINDING TEST
Ethyltriphenylfosfonium bromide was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
This dose range finding test is reported as a part of the first experiment of the mutation test.
Based on the dose range finding data, the following dose range was selected for the first mutation assay with the Salmonella typhimurium strains TA1535, TA1537 and TA98:
Without S9-mix: 33, 100, 333, 1000 and 3330 µg/plate.
With S9-mix : 33, 100, 333, 1000 and 3330 µg/plate.
Based on the toxicity data of the first experiment, the following dose range was tested in the second experiment with the Salmonella typhimurium strains:
Without S9-mix: 33, 100, 333, 1000 and 3330 µg/plate.
With S9-mix : 33, 100, 333, 1000 and 3330 µg/plate.
Based on the toxicity data of the dose range finding study, the following dose range was tested in the second experiment with the Escherichia coli strain:
Without S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate.
With S9-mix : 100, 333, 1000, 3330 and 5000 µg/plate.
Precipitate
The test substance did not precipitate in the top agar. Precipitation of Ethyltriphenylfosfonium bromide on the plates was not observed at the start or at the end of the incubation period in tester strain TA100 and WP2uvrA.
Toxicity
To determine the toxicity of Ethyltriphenylfosfonium bromide, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
In strain TA100, in the absence of S9-mix, an extreme reduction of the bacterial background lawn and an increase in the size of microcolonies were observed at test substance concentrations of 3330 and 5000 µg/plate. In the presence of S9-mix, a moderate reduction of the bacterial background lawn and an extreme reduction in the number of His+ revertants was observed at the concentration of 3330 µg/plate. An extreme reduction of the bacterial background lawn and an increase in the size of microcolonies were observed at the test substance concentration of 5000 µg/plate.
In strain WP2uvrA, a slight reduction of the bacterial background lawn was observed at the concentration of 3330 µg/plate. A moderate reduction of the bacterial background lawn was observed at the concentration of 5000 µg/plate. An extreme reduction in the number of Trp+ revertants was observed at the concentration of 5000 µg/plate, however the mean plate count of revertants was not below the historical control data range.
MUTATION ASSAY
Number of revertants
All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
CONCLUSION
Based on the results of this study it is concluded that Ethyltriphenylfosfonium bromide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. - Remarks on result:
- other: toxicity (reduction of the bacterial background lawn) at 3330 µg/plate
Applicant's summary and conclusion
- Conclusions:
- Based on the results of this study it is concluded that Ethyltriphenylfosfonium bromide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
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