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EC number: 947-478-5 | 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
- 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
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- 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:
- The experimental phases of the study were performed between 13 July 2011 and 09 September 2011.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
- 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:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine for Salmonella.
Tryptophan for E.Coli - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/betanaphthoflavone induced rat liver, S9
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1:
Salmonella strains (with and without S9 mix): 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
E.coli WP2uvrA (with and without S9 mix: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2:
0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulpoxide was therefore selected as the vehicle. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 10 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation for Experiment 2.
DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
NUMBER OF REPLICATIONS: Triplicate plating.
DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn. - Evaluation criteria:
- Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
-All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
-All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per ml.
-Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
-There should be a minimum of four non-toxic test item dose levels.
-There should be no evidence of excessive contamination.
Evaluation Criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
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).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. - Statistics:
- Standard Deviation
Statistical analysis of data as determined by UKEMS - Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECFIC CONFOUNDING FACTORS:
- Precipitation: A test item precipitate (globular in appearance) was observed at 5000 µg/plate.
RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item exhibited toxicity at 5000 (µg/plate to TA100 and WP2uvrA in the presence of S9-mix only. No toxicity was noted to either strain in the absence of S9-mix. The test item formulation and S9-mix used in this experiment were both shown to be sterile.
COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix. In the main test (preincubation), the test item again induced a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially noted from 500 and 1500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with and without S9-mix and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Under the study conditions, the test substance was considered to be non-mutagenic.
- Executive summary:
A study was conducted to determine the in vitro mutagenic potential of the test substance according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS guidelines, in compliance with GLP. Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with the test item,Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered), using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day (pre-incubation method) using a dose range of 5 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test item formulations.
Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item.The vehicle (dimethyl sulphoxide) 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.In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix. In the main test (pre-incubation), the test item again induced a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially noted from 500 and 1500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with and without S9-mix and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (globular in appearance) was observed at 5000 µg/plate.No 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. Under the study conditions, the test substance was considered to be non-mutagenic (Bowles and Thompson, 2012).
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- The experimental phases of the study were performed between 13 July 2011 and 09 September 2011.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
- Justification for type of information:
- Refer to the section 13 of IUCLID dataset for details on the read across justification. The in vitro genetic toxicity study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
- Reason / purpose for cross-reference:
- read-across source
- 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:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine for Salmonella.
Tryptophan for E.Coli - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/betanaphthoflavone induced rat liver, S9
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1:
Salmonella strains (with and without S9 mix): 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
E.coli WP2uvrA (with and without S9 mix: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2:
0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulpoxide was therefore selected as the vehicle. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 1 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 2 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene: 10 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation for Experiment 2.
DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
NUMBER OF REPLICATIONS: Triplicate plating.
DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn. - Evaluation criteria:
- Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
-All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
-All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per ml.
-Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
-There should be a minimum of four non-toxic test item dose levels.
-There should be no evidence of excessive contamination.
Evaluation Criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
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).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. - Statistics:
- Standard Deviation
Statistical analysis of data as determined by UKEMS - Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECFIC CONFOUNDING FACTORS:
- Precipitation: A test item precipitate (globular in appearance) was observed at 5000 µg/plate.
RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item exhibited toxicity at 5000 (µg/plate to TA100 and WP2uvrA in the presence of S9-mix only. No toxicity was noted to either strain in the absence of S9-mix. The test item formulation and S9-mix used in this experiment were both shown to be sterile.
COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix. In the main test (preincubation), the test item again induced a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially noted from 500 and 1500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with and without S9-mix and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The test substance was considered to be non-mutagenic under the conditions of this study.
- Executive summary:
A study was conducted to determine the in vitro mutagenic potential of the read-across substance according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS guidelines, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered), using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day (pre-incubation method) using a dose range of 5 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test item formulations.
Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item. The vehicle (dimethyl sulphoxide) 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. In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix. In the main test (pre-incubation), the test item again induced a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially noted from 500 and 1500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with and without S9-mix and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (globular in appearance) was observed at 5000 µg/plate. No 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. Under the study conditions, the test substance was considered to be non-mutagenic (Bowles and Thompson, 2012).
Referenceopen allclose all
Preliminary Toxicity Test
The test item exhibited toxicity at 5000 µg/plate to TA100 and WP2uvrA in the presence of S9-mix only. No toxicity was noted to either strain in the absence of S9-mix. The test item formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay were:
With (+) or without (-) S9‑mix |
Strain |
Dose (µg/plate) |
||||||||||
0 |
0.15 |
0.5 |
1.5 |
5 |
15 |
50 |
150 |
500 |
1500 |
5000 |
||
- |
TA100 |
89 |
110 |
92 |
73 |
84 |
76 |
82 |
66 |
67 |
63 |
61 |
+ |
TA100 |
82 |
79 |
73 |
74 |
102 |
85 |
74 |
79 |
69 |
67 |
56* |
- |
WP2uvrA |
25 |
28 |
31 |
25 |
34 |
20 |
31 |
31 |
24 |
27 |
16 |
+ |
WP2uvrA |
34 |
19 |
26 |
20 |
29 |
29 |
19 |
32 |
28 |
17 |
8* |
* : Partial absence of bacterial background lawn
Mutation Test
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4
and Table 5 for Experiment 2. The results are also expressed graphically in Figure 1 to Figure 4 (see attached background material for Tables and Figures).
A history profile of untreated/vehicle and positive controls (reference items) is presented in attached background material.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.
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 59-mix and the sensitivity of the bacterial strains.
Table 1: Spontaneous Mutation Rates (Concurrent Negative Control)
Experiment 1 (Range-finding Test)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
114 |
|
21 |
|
26 |
|
22 |
|
8 |
|
108 |
(110) |
29 |
(25) |
31 |
(30) |
21 |
(21) |
14 |
(11) |
108 |
|
25 |
|
33 |
|
20 |
|
11 |
|
Experiment 2 (Main Test)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
104 |
|
21 |
|
25 |
34 |
|
14 |
|
|
73 |
(90) |
29 |
(24) |
26 |
(31) |
37 |
(36) |
15 |
(14) |
93 |
|
23 |
41 |
33 |
|
36 |
|
14 |
|
Preliminary Toxicity Test
The test item exhibited toxicity at 5000 µg/plate to TA100 and WP2uvrA in the presence of S9-mix only. No toxicity was noted to either strain in the absence of S9-mix. The test item formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay were:
With (+) or without (-) S9‑mix |
Strain |
Dose (µg/plate) |
||||||||||
0 |
0.15 |
0.5 |
1.5 |
5 |
15 |
50 |
150 |
500 |
1500 |
5000 |
||
- |
TA100 |
89 |
110 |
92 |
73 |
84 |
76 |
82 |
66 |
67 |
63 |
61 |
+ |
TA100 |
82 |
79 |
73 |
74 |
102 |
85 |
74 |
79 |
69 |
67 |
56* |
- |
WP2uvrA |
25 |
28 |
31 |
25 |
34 |
20 |
31 |
31 |
24 |
27 |
16 |
+ |
WP2uvrA |
34 |
19 |
26 |
20 |
29 |
29 |
19 |
32 |
28 |
17 |
8* |
* : Partial absence of bacterial background lawn
Mutation Test
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4
and Table 5 for Experiment 2. The results are also expressed graphically in Figure 1 to Figure 4 (see attached background material for Tables and Figures).
A history profile of untreated/vehicle and positive controls (reference items) is presented in attached background material.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.
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 59-mix and the sensitivity of the bacterial strains.
Table 1: Spontaneous Mutation Rates (Concurrent Negative Control)
Experiment 1 (Range-finding Test)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
114 |
|
21 |
|
26 |
|
22 |
|
8 |
|
108 |
(110) |
29 |
(25) |
31 |
(30) |
21 |
(21) |
14 |
(11) |
108 |
|
25 |
|
33 |
|
20 |
|
11 |
|
Experiment 2 (Main Test)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
104 |
|
21 |
|
25 |
34 |
|
14 |
|
|
73 |
(90) |
29 |
(24) |
26 |
(31) |
37 |
(36) |
15 |
(14) |
93 |
|
23 |
41 |
33 |
|
36 |
|
14 |
|
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
A study was conducted to determine the in vitro mutagenic potential of the read-across substance according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS guidelines, in compliance with GLP. Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with the test item,Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered), using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day (pre-incubation method) using a dose range of 5 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test item formulations.
Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item.The vehicle (dimethyl sulphoxide) 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.In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix. In the main test (pre-incubation), the test item again induced a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially noted from 500 and 1500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with and without S9-mix and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (globular in appearance) was observed at 5000 µg/plate.No 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. Under the study conditions, the test substancewas considered to be non-mutagenic (Bowles and Thompson, 2012).
Justification for classification or non-classification
Based on the in vitro genetic toxicity study with the read-across substance, no conclusion on classification for genetic toxicity could be derived according to EU CLP (1272/2008) criteria.
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