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EC number: 284-698-4 | CAS number: 84962-05-0
- 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
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Test material form:
- other: waxy solid
- Details on test material:
- None
Constituent 1
- Specific details on test material used for the study:
- Identification: FAT 93580/A
Batch: 0022357000
Purity: 94.3 %
Description: Extremely pale yellow waxy solid
Expiry Date: 15 December 2015
Storage Conditions: Room temperature in the dark.
Method
- Target gene:
- Histidine gene
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- The Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally, due to the "deep rough" (rfa) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TAlOO, the R factor plasmid pKMlOl enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000)
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Pre-Incubation Method: 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in tetrahydrofuran at 200 mg/mL in solubility checks performed in-house. Tetrahydrofuran was therefore selected as the vehicle.
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9-mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2- Aminoanthracene, Benzo(a)pyrene
- Remarks:
- with S9-mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) - Experiment 1
NUMBER OF REPLICATIONS: 3
For each strain and dose level, including the controls, a minimum of three plates were used.
Experiment 1: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 0.025 mL of test item or vehicle or 0.1 mL of positive control.
- 2 mL of trace amino-acid supplemented media (at approximately 45 °C) containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer.
- 0.5 mL of S9-mix (for experiments with metabolic activation).
METHOD OF APPLICATION: preincubation - Experiment 2
NUMBER OF REPLICATIONS: 3
For each strain and dose level, including the controls, a minimum of three plates were used.
- 0.1 mL of the appropriate bacterial strain culture
- 0.5 mL of phosphate buffer and 0.025 mL of the test item formulation or solvent or 0.1 mL of appropriate positive control
- 2 mL of amino-acid supplemented media
- 0.5 mL of S9-mix (for experiments with metabolic activation).
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h - Evaluation criteria:
- 1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Acceptance Criteria:
All tester strain cultures should be in the range of 0.9 to 9x10^9 bacteria per mL.
There should be a minimum of four non-toxic test item dose levels. - Statistics:
- Statistical analysis of the data as determined by UKEMS (Mahon et al. 1989)
Results and discussion
Test results
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- at and above 1500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (particulate in appearance) was observed above 1500 µg/plate (under an inverted microscope at 500 µg/plate in Experiment 2), this observation did not prevent the scoring of revertant colonies.
No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. Small, statistically significant increases in TA98 revertant colony frequency were observed in Experiment 2 at 50, 150, 500, 1500 µg/plate (absence of S9-mix) and 1500 µg/plate (presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.6 times the concurrent vehicle controls.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Any other information on results incl. tables
Table 1 Experiment 1 without metabolic activation
Test period | from 2013-04-04 (2013-04-11/16) | to 2013-04-07 (2013-04-14/19) | |||||||||
S9-Mix (-) | Dose level per plate (µg) | Number of revertants | |||||||||
Base-pair substitutions strains | Frameshift strains | ||||||||||
TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
mean | SD | mean | SD | mean | SD | mean | SD | mean | SD | ||
solvent control | 93 | 17 | 18 | 3 | 32 | 10 | 25 | 9 | 10 | 2 | |
1.5 | 87 | 21 | 14 | 4 | 38 | 6 | 19 | 7 | 9 | 6 | |
5 | 81 | 7 | 12 | 2 | 32 | 7 | 21 | 6 | 11 | 6 | |
15 | 99 | 4 | 17 | 1 | 34 | 5 | 20 | 7 | 11 | 4 | |
50 | 81 | 17 | 18 | 2 | 32 | 6 | 20 | 8 | 5 | 2 | |
150 | 79 | 11 | 16 | 5 | 37 | 2 | 21 | 5 | 10 | 5 | |
500 | 76 | 9 | 20 | 6 | 37 | 0 | 18 | 2 | 15 | 6 | |
1500 | 101 | 2 | 21 | 4 | 34 | 7 | 23 | 6 | 11 | 5 | |
5000 | 73 | 11 | 19 | 4 | 31 | 7 | 19 | 6 | 12 | 2 | |
Positive control | ENNG | ENNG | ENNG | 4NQO | 9AA | ||||||
Dose level (µg) | 3 | 5 | 2 | 0.2 | 80 | ||||||
No. revertants | 364 | 36 | 977 | 339 | 567 | 75 | 166 | 28 | 377 | 48 |
Table 2 Experiment 1 with metabolic activation
Test period | from 2013-04-04 (2013-04-11) | to 2013-04-07 (2013-04-14) | |||||||||
S9-Mix (+) | Dose level per plate (µg) | Number of revertants | |||||||||
Base-pair substitutions strains | Frameshift strains | ||||||||||
TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
mean | SD | mean | SD | mean | SD | mean | SD | mean | SD | ||
solvent control | 105 | 15 | 16 | 3 | 33 | 6 | 29 | 8 | 15 | 1 | |
1.5 | 105 | 20 | 12 | 3 | 38 | 2 | 28 | 4 | 11 | 5 | |
5 | 99 | 14 | 12 | 5 | 35 | 5 | 21 | 5 | 15 | 4 | |
15 | 101 | 9 | 10 | 2 | 37 | 3 | 19 | 5 | 15 | 3 | |
50 | 97 | 10 | 15 | 2 | 31 | 12 | 32 | 2 | 14 | 6 | |
150 | 96 | 9 | 12 | 4 | 35 | 11 | 18 | 1 | 10 | 3 | |
500 | 95 | 3 | 15 | 2 | 31 | 6 | 25 | 5 | 13 | 4 | |
1500 | 97 | 2 | 13 | 4 | 31 | 6 | 24 | 8 | 11 | 2 | |
5000 | 102 | 7 | 14 | 3 | 38 | 4 | 21 | 4 | 16 | 4 | |
Positive control | 2AA | 2AA | 2AA | BP | 2AA | ||||||
Dose level (µg) | 1 | 2 | 10 | 5 | 2 | ||||||
No. revertants | 644 | 110 | 178 | 10 | 306 | 27 | 299 | 3 | 231 | 25 |
Table 3 Experiment 2 without metabolic activation
Test period | from 2013-04-23 | to 2013-04-26 | |||||||||
S9-Mix (-) | Dose level per plate (µg) | Number of revertants | |||||||||
Base-pair substitutions strains | Frameshift strains | ||||||||||
TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
mean | SD | mean | SD | mean | SD | mean | SD | mean | SD | ||
solvent control | 105 | 22 | 14 | 6 | 38 | 6 | 14 | 3 | 16 | 6 | |
50 | 111 | 18 | 18 | 3 | 34 | 10 | 23 ** | 2 | 16 | 8 | |
150 | 101 | 5 | 21 | 5 | 44 | 3 | 21 * | 3 | 21 | 7 | |
500 | 109 | 8 | 18 | 8 | 34 | 6 | 21 * | 4 | 14 | 6 | |
1500 | 106 | 13 | 17 | 2 | 31 | 5 | 23 ** | 2 | 13 | 5 | |
5000 | 80 | 8 | 11 | 2 | 30 | 4 | 20 | 3 | 13 | 8 | |
Positive control | ENNG | ENNG | ENNG | 4NQO | 9AA | ||||||
Dose level (µg) | 3 | 5 | 2 | 0.2 | 80 | ||||||
No. revertants | 973 | 11 | 1298 | 37 | 910 | 59 | 232 | 6 | 983 | 226 |
* p<0.05; ** p<0.01
Table 4 Experiment 2 with metabolic activation
Test period | from 2013-04-23 | to 2013-04-26 | |||||||||
S9-Mix (+) | Dose level per plate (µg) | Number of revertants | |||||||||
Base-pair substitutions strains | Frameshift strains | ||||||||||
TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
mean | SD | mean | SD | mean | SD | mean | SD | mean | SD | ||
solvent control | 88 | 9 | 9 | 2 | 39 | 9 | 20 | 3 | 18 | 6 | |
50 | 90 | 2 | 12 | 3 | 44 | 4 | 23 | 2 | 17 | 3 | |
150 | 88 | 8 | 12 | 4 | 44 | 15 | 25 | 3 | 14 | 2 | |
500 | 90 | 4 | 11 | 4 | 47 | 7 | 28 | 6 | 14 | 2 | |
1500 | 87 | 1 | 14 | 8 | 34 | 1 | 30 * | 3 | 13 | 2 | |
5000 | 75 | 6 | 11 | 3 | 28 | 6 | 23 | 6 | 8 | 1 | |
Positive control | 2AA | 2AA | 2AA | BP | 2AA | ||||||
Dose level (µg) | 1 | 2 | 10 | 5 | 2 | ||||||
No. revertants | 1166 | 34 | 267 | 7 | 207 | 30 | 158 | 6 | 261 | 51 |
* p<0.05
Applicant's summary and conclusion
- Conclusions:
- FAT#: 93580/A was considered to be non-mutagenic under the conditions of this test.
- Executive summary:
FAT 93580/A was tested according to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 44012008 of 30 May 2008 and the USA, EPA (TSCA) OCSPP harmonized guidelines.
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and preincubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors).
The dose range for Experiment 1 was pre-determined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 50 to 5000 µg/plate.
The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (particulate in appearance) was observed by eye at and above 1500 µg/plate (under an inverted microscope at 500 µg/plate in Experiment 2), this observation did not prevent the scoring of revertant colonies.
No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. Small, statistically significant increases in TA98 revertant colony frequency were observed in Experiment 2 at 50, 150, 500, 1500 µg/plate (absence of S9-mix) and 1500 µg/plate (presence of S9-mix).
These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.6 times the concurrent vehicle controls. FAT #: 93580/A was considered to be non-mutagenic under the conditions of this test.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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