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EC number: 230-896-0 | CAS number: 7360-38-5
- 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
Description of key information
Gene mutation in bacteria (OECD 471, Ames test): S. typhimurium TA 1535, TA 1537, TA 98, and TA 100: negative with and without metabolic activation
Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): primary rat hepatocytes: negative
Gene mutation in mammalian cells (OECD 476, HGPRT Test): Chinese hamster lung fibroblasts (V79): negative with and without metabolic activation
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 Jan - 4 Feb 1990
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- only four strains tested
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Dose range finding test: 5, 50, 500 and 5000 µg/plate with and without metabolic activation
Main test: 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 9-Aminoacridine (80 µg/pltae ) for TA1537; N-Ethyl-N'-nitrosoguanidine (3 µg/plate) for TA100, (5 µg/plate) for TA1535; 2-Nitrofluorene (1 µg/plate) for TA98.
- Remarks:
- Without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: With S9: 2-Aminoanthracene (0.5 µg/plate) for TA98, (1 µg/plate) for TA100, (2 µg/plate) for TA1535 and TA1537
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
- Preincubation period: 1 h, 37 °C
- Exposure duration: 24 h (range finding test), 72 h (main test), 37 °C
NUMBER OF REPLICATIONS: triplicates, each in two experiments
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
OTHER:
- S9: added at 3% (main test) and 10% (range finding test and main test). In a rerun S9 of the main test S9 was added at 10% and 30%, respectively - Evaluation criteria:
- The test substance is considered to show evidence of mutagenic activity, if treatment with a test material produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix.
The test substance is considered to show no evidence of mutagenic activity, if treatment with a test material does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain. - Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 5000µg/plate for all strains in the presence and absence of S9 - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 26 Feb - 2 May 1990
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured human lymphocytes
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 with 20% Foetal Calf Serum (Gibco) and 2% phytohaemagglutinin
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 and 4000 µg/mL with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Without S9: Ethylmethane sulphonate (Sigma London Chemical Company Ltd.) at 750 µg/mL in DMSO; with S9: Cyclophosphamide (Sigma London Chemical Company Ltd.) at 20 µg/mL in distilled water
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 22
SPINDLE INHIBITOR (cytogenetic assays): cholchicine, 0.25 µg/mL
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: in duplicates
NUMBER OF CELLS EVALUATED: 100 from each culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER
S9: two hours after dosing sulture medium with S9 was replaced with fresh medium - Statistics:
- Fisher's test
- Key result
- Species / strain:
- lymphocytes: cultured human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test compound was miscible with ethanol at a concentration of 470 mg/mL, the highest tested. However, the final concentrations above 4000 µg/mL were immiscible with aqueous tissue culture medium, when dosed at 1% v/v. 4000 µg/mL was regarded and referred to as the maximum achievable concentration in the test.
COMPARISON WITH HISTORICAL CONTROL DATA:
The aberration frequency of 1.5% at a concentration of 1000 µg/mL in the presence of metabolic activation fell well within the historical control ranges observed in the laboratory - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 Jul - 26 Sep 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted 29 Jul 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Medicines and Healthcare Products Regulatory Agency, Department of Health, London, United Kingdom
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, dark - Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Harlan CCR
- Suitability of cells: Cell type selected is listed as one of the recommended cell types in OECD guideline 476
- Methods for maintenance in cell culture: Freshly thawed cells from stock cultures were maintained in 225 cm2 culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C incubation temperature.
- Cell cycle length, doubling time or proliferation index: 12 - 16 h
- Modal number of chromosomes: 22
MEDIA USED
- Type and identity of media: Eagles Minimal Essential (MEM) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered, including pen/strep and 10% fetal bovine serum (FBS), 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/b-naphtho flavone
- Test concentrations with justification for top dose:
- Without metabolic activation: 0.25, 0.5, 1, 2, 4, 8, 16, 32 µg/mL (4h)
With metabolic activation: 0.25, 0.5, 1, 2, 4, 8, 16, 32 µg/mL (4h)
The selection of the concentrations used in the main experiments was based on data from the range-finding experiment. Precipitation was observed at and above 15.63 µg/mL during the 4 h treatment with and without metabolic activation. The maximum concentrations selected for the main mutagenicity experiment were therefore limited by the onset of the test item precipitate, in both, the asbsence and presence of metabolic activation. - Vehicle / solvent:
- Acetone
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 1 x 10E7 cells per 225 cm2 flask
DURATION :
- Exposure duration: 4 h exposure with and without S9 mix
- Expression time (cells in growth medium): At the end of the treatment period the cells were plated for determination of the cloning efficiency and the mutation frequency and incubated for 6 or 7 days.
- Selection time: 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 - 14 days
SELECTION AGENT: 11 µg/mL 6-Thioguanine (6-TG)
STAIN: Giemsa
NUMBER OF REPLICATIONS: triplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY :
- Method: relative survival and cloning efficiecy - Evaluation criteria:
- A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentration exhibits a statistically significant increae compared with the concurrent vehicle control
- the increase is considered to be concentration-related
- the results are outside the range of the historical negative control data for the test item concentrations
A test item is considered to be clearly negative if, in all of the experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
- there is no concentration related increase
- the results for the test item concentration are within the range of the historical negative control data
In case the response is neither clearly negative nor clearly positive or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. - Statistics:
- The Student's t-test was applied to compare the mutation data of each individual concentration with the vehicle control.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm at the concentration levels investigated
- Precipitation: Preicipitation of the test item was observed at and above 15.63 µg/mL in both the presence and absence of metabolic activation
RANGE-FINDING/SCREENING STUDIES:
A concentration range of 0.49 to 125 µg/mL was used in a preliminary cytotoxicity test. At the end of the exposure period, precipitation of the test item was observed at and above 15.63 µg/mL in both the absence and presence of metabolic activation. There was no evidence of any marked concentration related-reductions in relative survival in either the presence or absence of metabolic activation.
HISTORICAL CONTROL DATA
- Positive historical control data: The mean number of mutants with metabolic activation is 334.40 ± 114.17 (range: 209 - 774, n=30) for 1 µg/mL DMBA and 484.97 ± 174.83 (range: 103 - 925, n=30) for 2 µg/mL DMBA. The mean number of mutants without metabolic activation is 261.30 ± 36.82 (range: 171 - 346, n=30) for 500 µg/mL EMS and 417.54 ± 63.80 (range: 291 - 574, n=28) for 750 µg/mL (see Table 3 under "Any other information results incl tables"). The results of the test fall within the historical control data range.
- Vehicle historical control data: The mean number of mutants with metabolic activation is 12.07 ± 4.44 (range: 5 - 23, n=30). The mean number of mutants without metabolic acativation is 12.47 ± 3.95 (range: 5 - 24, n=30) (see Table 4 under "Any other information results incl tables"). The results of the test fall within the historical control data range. - Conclusions:
- Interpretation of results: negative
Referenceopen allclose all
Results main experiment:
Experiment 1:
Dose Level (µg/plate) | Liver S-9 mix | TA 1535 | TA1537 | TA98 | TA100 | ||||
mean | SD | mean | SD | mean | SD | mean | SD | ||
5000 | - | 10 | 1.0 | 10 | 2.1 | 19 | 0.0 | 64 | 5.6 |
1500 | - | 11 | 1.7 | 9 | 0.6 | 23 | 3.0 | 90 | 1.0 |
500 | - | 14 | 2.5 | 10 | 2.1 | 20 | 1.5 | 96 | 2.5 |
150 | - | 12 | 2.0 | 9 | 1.5 | 22 | 3.1 | 102 | 2.6 |
50 | - | 11 | 1.0 | 9 | 0.6 | 21 | 2.0 | 101 | 3.1 |
0 | - | 17 | 1.0 | 10 | 0.6 | 24 | 5.0 | 104 | 4.5 |
Solvent | - | 15 | 1.0 | 10 | 1.0 | 26 | 4.0 | 112 | 2.5 |
ENNG (5 µg/plate) | - | 458 | 67.1 | ||||||
9-AC (80 µg/plate) | - | 1108 | 51.9 | ||||||
NF (1 µg/plate) | - | 85 | 9.0 | ||||||
ENNG (3 µg/plate) | - | 349 | 44.3 | ||||||
5000 | 3% | 12 | 1.5 | 10 | 0.6 | 24 | 2.1 | 72 | 1.5 |
1500 | 3% | 11 | 4.4 | 10 | 1.2 | 21 | 2.1 | 92 | 2.0 |
500 | 3% | 14 | 1.0 | 9 | 1.0 | 19 | 1.0 | 99 | 6.1 |
150 | 3% | 11 | 1.0 | 8 | 1.0 | 22 | 2.6 | 101 | 5.0 |
50 | 3% | 13 | 1.0 | 10 | 1.2 | 20 | 0.6 | 100 | 2.0 |
0 | 3% | 18 | 1.7 | 10 | 2.5 | 17 | 2.1 | 99 | 6.4 |
Solvent | 3% | 11 | 2.5 | 9 | 1.0 | 20 | 1.2 | 102 | 4.5 |
AA (0.5 µg/plate) | 3% | 161 | 20.0 | ||||||
AA (1 µg/plate) | 3% | 295 | 21.6 | ||||||
AA (2 µg/plate) | 3% | 56 | 3.8 | 80 | 11.5 | ||||
5000 | 10% | 12 | 1.5 | 10 | 2.1 | 22 | 1.5 | 73 | 1.0 |
1500 | 10% | 11 | 1.2 | 9 | 1.0 | 20 | 1.0 | 93 | 4.2 |
500 | 10% | 14 | 2.5 | 12 | 2.1 | 20 | 3.8 | 97 | 7.6 |
150 | 10% | 15 | 2.8 | 10 | 1.5 | 22 | 3.2 | 101 | 2.3 |
50 | 10% | 11 | 2.5 | 10 | 1.5 | 21 | 1.4 | 108 | 2.0 |
0 | 10% | 14 | 1.0 | 8 | 1.5 | 23 | 3.6 | 104 | 10.2 |
Solvent | 10% | 14 | 1.5 | 11 | 1.5 | 24 | 3.2 | 101 | 2.3 |
AA (0.5 µg/plate) | 10% | 143 | 11.0 | ||||||
AA (1 µg/plate) | 10% | 353 | 10.1 | ||||||
AA (2 µg/plate) | 10% | 57 | 7.6 | 70 | 8.0 |
Experiment 2:
Dose Level (µg/plate) | Liver S-9 mix | TA 1535 | TA1537 | TA98 | TA100 | ||||
mean | SD | mean | SD | mean | SD | mean | SD | ||
5000 | - | 10 | 3.2 | 10 | 1.0 | 19 | 3.1 | 82 | 6.1 |
1500 | - | 10 | 1.0 | 14 | 0.6 | 21 | 0.0 | 104 | 7.5 |
500 | - | 9 | 0.6 | 10 | 2.5 | 21 | 2.5 | 100 | 11.2 |
150 | - | 12 | 1.0 | 11 | 2.6 | 19 | 1.0 | 95 | 5.6 |
50 | - | 10 | 1.0 | 14 | 1.5 | 22 | 0.6 | 100 | 2.5 |
0 | - | 9 | 0.6 | 11 | 1.2 | 23 | 3.0 | 89 | 7.6 |
Solvent | - | 11 | 0.6 | 12 | 3.2 | 22 | 1.2 | 100 | 14.5 |
ENNG (5 µg/plate) | - | 439 | 48.8 | ||||||
9-AC (80 µg/plate) | - | x | x | ||||||
NF (1 µg/plate) | - | 80 | 8.0 | ||||||
ENNG (3 µg/plate) | - | 424 | 56.0 | ||||||
5000 | 10% | 11 | 2.0 | 17 | 3.0 | 19 | 2.1 | 92 | 4.0 |
1500 | 10% | 14 | 2.0 | 16 | 0.6 | 22 | 2.5 | 100 | 12.7 |
500 | 10% | 11 | 2.1 | 13 | 1.2 | 20 | 0.0 | 107 | 3.1 |
150 | 10% | 10 | 0.6 | 15 | 1.5 | 22 | 1.0 | 108 | 4.0 |
50 | 10% | 12 | 2.5 | 12 | 2.1 | 23 | 1.5 | 107 | 3.1 |
0 | 10% | 10 | 2.0 | 16 | 4.2 | 20 | 2.0 | 100 | 10.4 |
Solvent | 10% | 10 | 1.7 | 15 | 2.5 | 22 | 2.5 | 105 | 10.5 |
AA (0.5 µg/plate) | 10% | 87 | 1.2 | ||||||
AA (1 µg/plate) | 10% | 358 | 48.0 | ||||||
AA (2 µg/plate) | 10% | 71 | 18.8 | 68 | 12.8 | ||||
5000 | 30% | 9 | 0.6 | 17 | 1.2 | 19 | 3.5 | 101 | 7.6 |
1500 | 30% | 9 | 0.6 | 16 | 2.1 | 23 | 2.0 | 106 | 5.3 |
500 | 30% | 10 | 1.5 | 14 | 2.0 | 21 | 3.1 | 107 | 4.5 |
150 | 30% | 11 | 0.6 | 14 | 2.3 | 24 | 0.0 | 110 | 3.0 |
50 | 30% | 12 | 1.7 | 14 | 2.9 | 27 | 1.0 | 110 | 9.0 |
0 | 30% | 12 | 1.0 | 16 | 0.6 | 21 | 2.1 | 118 | 8.6 |
Solvent | 30% | 12 | 2.1 | 17 | 2.5 | 21 | 1.2 | 110 | 10.7 |
AA (0.5 µg/plate) | 30% | 83 | 6.1 | ||||||
AA (1 µg/plate) | 30% | 299 | 30.2 | ||||||
AA (2 µg/plate) | 30% | 74 | 9.3 | 61 | 8.5 |
SD: standard deviation
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
9 -AC: 9 -aminoacridine
AA: 2 -aminoanthracene
NF: 2 -nitrofluorene
x: too many colonies to count accurately
Results:
Metabolic activation |
Test item |
Concentration (µg/mL) |
No. Of analyzed cells |
Aberrant Cells (mean %) |
Relative mitotic index (%) |
|
with gaps |
without gaps |
|||||
- |
Ethanol |
10 µl/mL |
100 |
0 |
0 |
100 |
- |
Glycerol trioctanoate |
7.8 |
- |
- |
- |
121 |
- |
15.6 |
- |
- |
- |
100 |
|
- |
31.3 |
- |
- |
- |
125 |
|
- |
62.5 |
- |
- |
- |
104 |
|
- |
125 |
- |
- |
- |
121 |
|
- |
250 |
- |
- |
- |
129 |
|
- |
500 |
100 |
0.5 |
0.5 |
118 |
|
- |
1000 |
100 |
0.5 |
0.5 |
104 |
|
- |
2000 |
100 |
1.0 |
1.0 |
150 |
|
- |
4000 |
100 |
0.0 |
0.0 |
125 |
|
- |
EMS |
750 |
100 |
15.5*** |
14.5*** |
- |
+ |
Ethanol |
10 µl/mL |
100 |
0.0 |
0.0 |
100 |
+ |
|
7.8 |
- |
- |
- |
82 |
+ |
|
15.6 |
- |
- |
- |
94 |
+ |
|
31.3 |
- |
- |
- |
97 |
+ |
|
62.5 |
- |
- |
- |
0 |
+ |
|
125 |
- |
- |
- |
90 |
+ |
|
250 |
- |
- |
- |
85 |
+ |
Glycerol trioctanoate |
500 |
100 |
1.0 |
1.0 |
91 |
+ |
1000 |
100 |
1.5* |
1.5* |
94 |
|
+ |
2000 |
100 |
0.0 |
0.0 |
89 |
|
+ |
4000 |
100 |
0.0 |
0.0 |
61 |
|
+ |
CP |
750 |
100 |
16.0*** |
16.0*** |
- |
* p<0.05; *** p<0.001
EMS: Ethylmethane sulphonate
CP: Cyclophosphamide
mean%: total abberant cells *100 / total cells scored
Table 1: Main mutation experiment, 4 hour exposure without metabolic activation (S9)
|
|
Day 0 Viability |
Day 7 Viability |
Day 7 Mutant |
||||||||||||||||||||||||
Dose (µg/mL) |
|
Colonies/flask (200 cells plated/flask) |
|
Adjusted CE |
|
|
Colonies/flask (200 cells plated/flask) |
|
|
Mean % Control |
|
|
|
|
Group MFS 10-6 |
|||||||||||||
% CE |
RS |
Mean RS |
% CE |
% Control |
Colonies/flask (2x105 cells plated/flask) |
MF |
MFS 10-6 |
SD |
||||||||||||||||||||
0 |
A |
139 |
122 |
166 |
71.2 |
57.5 |
100 |
100 |
137 |
156 |
170 |
77.2 |
100 |
100 |
0 |
6 |
3 |
0 |
2 |
5 |
2 |
2 |
0 |
5 |
12.5 |
16.2 |
1.93 |
15 |
B |
158 |
153 |
176 |
81.2 |
63.3 |
100 |
149 |
168 |
154 |
78.5 |
100 |
3 |
4 |
0 |
4 |
4 |
0 |
2 |
2 |
3 |
0 |
11 |
14 |
|||||
0.25 |
A |
Not plated, surplus to requirements |
|
Not plated, surplus to requirements |
|
Not plated, surplus to requirements |
|
|
||||||||||||||||||||
B |
Not plated, surplus to requirements |
Not plated, surplus to requirements |
Not plated, surplus to requirements |
|||||||||||||||||||||||||
0.5 |
A |
142 |
162 |
166 |
78.3 |
63.3 |
110 |
103 |
133 |
163 |
159 |
75.8 |
98.3 |
97 |
1 |
2 |
3 |
3 |
5 |
0 |
0 |
3 |
3 |
0 |
10 |
13.2 |
1.5 |
10 |
B |
162 |
158 |
150 |
78.3 |
61.1 |
96.5 |
160 |
137 |
151 |
74.7 |
95.1 |
0 |
0 |
1 |
1 |
1 |
0 |
3 |
3 |
0 |
1 |
5 |
6.7 |
|||||
1 |
A |
160 |
171 |
173 |
84 |
67.9 |
118 |
105 |
154 |
148 |
156 |
76.3 |
98.9 |
99 |
0 |
0 |
4 |
5 |
3 |
3 |
1 |
4 |
2 |
0 |
11 |
14.4 |
1.56 |
11 |
B |
142 |
154 |
149 |
74.2 |
57.9 |
91.4 |
153 |
149 |
162 |
77.3 |
98.5 |
0 |
0 |
2 |
1 |
2 |
3 |
1 |
2 |
0 |
1 |
6 |
7.8 |
|||||
2 |
A |
130 |
145 |
155 |
71.7 |
57.9 |
101 |
110 |
140 |
123 |
153 |
69.3 |
89.8 |
94 |
2 |
2 |
3 |
5 |
0 |
4 |
3 |
2 |
2 |
3 |
13 |
18.8 |
2.02 |
19 |
B |
190 |
172 |
216 |
96.3 |
75.1 |
119 |
150 |
160 |
155 |
77.5 |
98.7 |
6 |
7 |
0 |
1 |
3 |
1 |
3 |
3 |
6 |
0 |
15 |
19.4 |
|||||
4 |
A |
197 |
150 |
171 |
86.3 |
69.8 |
121 |
113 |
148 |
145 |
148 |
73.5 |
95.2 |
96 |
4 |
2 |
0 |
3 |
5 |
0 |
4 |
1 |
1 |
4 |
12 |
16.3 |
2.08 |
18 |
B |
166 |
173 |
168 |
84.5 |
65.9 |
104 |
161 |
141 |
152 |
75.7 |
96.4 |
3 |
4 |
4 |
0 |
4 |
7 |
2 |
0 |
5 |
0 |
14.5 |
19.2 |
|||||
8 |
A |
165 |
153 |
148 |
77.7 |
62.8 |
109 |
103 |
149 |
187 |
168 |
84 |
108.9 |
106 |
4 |
4 |
1 |
0 |
2 |
2 |
4 |
1 |
2 |
1 |
10.5 |
12.5 |
1.79 |
13 |
B |
168 |
140 |
159 |
77.8 |
60.7 |
95.9 |
170 |
150 |
162 |
80.3 |
102.3 |
3 |
4 |
1 |
0 |
5 |
1 |
1 |
1 |
6 |
0 |
11 |
13.7 |
|||||
16 P |
A |
155 |
161 |
167 |
80.5 |
65.1 |
113 |
112 |
136 |
161 |
192 |
81.5 |
105.6 |
102 |
4 |
4 |
3 |
1 |
2 |
4 |
5 |
0 |
2 |
3 |
14 |
17.2 |
1.24 |
16 |
B |
166 |
196 |
175 |
89.5 |
69.8 |
110 |
137 |
160 |
170 |
77.8 |
99.2 |
3 |
1 |
2 |
1 |
3 |
3 |
2 |
2 |
2 |
3 |
11 |
14.1 |
|||||
32 P |
A |
Not plated due to precipitate |
|
Not plated due to precipitate |
|
Not plated due to precipitate |
|
|
||||||||||||||||||||
B |
Not plated due to precipitate |
Not plated due to precipitate |
Not plated due to precipitate |
|||||||||||||||||||||||||
EMS 500 |
A |
110 |
114 |
122 |
57.7 |
46.6 |
81 |
84 |
179 |
148 |
132 |
76.5 |
99.1 |
80 |
55 |
35 |
28 |
40 |
53 |
32 |
36 |
42 |
54 |
30 |
203 |
264.7 |
8.51 |
352 |
B |
151 |
134 |
143 |
71.3 |
55.6 |
87.9 |
100 |
74 |
111 |
47.5 |
60.5 |
45 |
36 |
46 |
32 |
38 |
56 |
36 |
42 |
40 |
46 |
209 |
438.9 |
|||||
EMS 750 |
A |
84 |
73 |
107 |
44 |
35.6 |
61.8 |
64 |
132 |
142 |
130 |
67.3 |
87.3 |
74 |
72 |
54 |
44 |
57 |
61 |
40 |
50 |
56 |
41 |
50 |
263 |
389.9 |
12.1 |
562 |
B |
86 |
131 |
109 |
54.3 |
42.4 |
66.9 |
74 |
108 |
101 |
47.2 |
60.1 |
73 |
63 |
84 |
63 |
58 |
70 |
66 |
70 |
77 |
69 |
347 |
734.6 |
DMBA = Dimethyl benzanthracene
CE = Cloning efficiency
RS = Relative survival
MF = Mutant frequency
MFS = Mutant frequency per survivor
SD = Standard deviation
Table 2: Main mutation experiment, 4 hour exposure with metabolic activation (S9)
|
|
Day 0 Viability |
Day 7 Viability |
Day 7 Mutant |
|||||||||||||||||||||||||||
|
|
Colonies/flask (200 cells plated/flask) |
|
|
|
|
Colonies/flask (200 cells plated/flask) |
|
|
|
|
|
|
|
Group MFS 10-6 |
||||||||||||||||
Dose (µg/mL) |
% CE |
Adjusted CE |
RS |
Mean RS |
% CE |
% Control |
Mean % Control |
Colonies/flask (2x105 cells plated/flask) |
MF |
MFS 10-6 |
SD |
||||||||||||||||||||
0 |
A |
181 |
145 |
100 |
71 |
48.3 |
100 |
100 |
186 |
179 |
174 |
89.8 |
100 |
100 |
3 |
1 |
2 |
1 |
0 |
0 |
4 |
3 |
3 |
3 |
10 |
11.1 |
1.64 |
15 |
|||
B |
181 |
137 |
150 |
78 |
58.8 |
100 |
169 |
150 |
164 |
80.5 |
100 |
3 |
3 |
0 |
5 |
0 |
3 |
4 |
5 |
2 |
4 |
14.5 |
18 |
||||||||
0.25 |
A |
Not plated, surplus to requirements |
|
Not plated, surplus to requirements |
|
Not plated, surplus to requirements |
|
|
|||||||||||||||||||||||
B |
Not plated, surplus to requirements |
Not plated, surplus to requirements |
Not plated, surplus to requirements |
||||||||||||||||||||||||||||
0.5 |
A |
142 |
126 |
126 |
65.7 |
44.7 |
92.5 |
93 |
203 |
166 |
178 |
91.2 |
101.5 |
98 |
1 |
3 |
0 |
4 |
4 |
2 |
3 |
2 |
4 |
3 |
13 |
14.3 |
1.18 |
14 |
|||
B |
147 |
146 |
140 |
72.2 |
54.4 |
92.5 |
148 |
162 |
149 |
76.5 |
95 |
0 |
2 |
3 |
2 |
2 |
3 |
3 |
3 |
2 |
1 |
10.5 |
13.7 |
||||||||
1 |
A |
182 |
129 |
152 |
77.2 |
52.5 |
108.7 |
105 |
163 |
188 |
174 |
87.5 |
97.4 |
98 |
1 |
0 |
4 |
2 |
2 |
5 |
2 |
2 |
1 |
4 |
11.5 |
13.1 |
1.5 |
15 |
|||
B |
172 |
160 |
141 |
78.8 |
59.4 |
101.1 |
168 |
171 |
142 |
80.2 |
99.6 |
1 |
3 |
2 |
4 |
3 |
0 |
3 |
4 |
2 |
5 |
13.5 |
16.8 |
||||||||
2 |
A |
122 |
120 |
130 |
62 |
42.2 |
87.3 |
100 |
202 |
189 |
207 |
99.7 |
110.9 |
105 |
2 |
3 |
4 |
0 |
5 |
0 |
4 |
3 |
3 |
4 |
14 |
14 |
1.66 |
15 |
|||
B |
184 |
171 |
170 |
87.5 |
65.9 |
112.2 |
170 |
149 |
159 |
79.7 |
99 |
2 |
0 |
3 |
4 |
2 |
4 |
0 |
4 |
5 |
2 |
13 |
16.3 |
||||||||
4 |
A |
126 |
146 |
160 |
72 |
49 |
101.4 |
109 |
176 |
172 |
174 |
87 |
96.8 |
98 |
4 |
1 |
4 |
0 |
4 |
1 |
2 |
3 |
2 |
3 |
12 |
13.8 |
1.4 |
15 |
|||
B |
184 |
178 |
183 |
90.8 |
68.5 |
116.5 |
168 |
149 |
160 |
79.5 |
98.8 |
2 |
4 |
2 |
3 |
2 |
0 |
5 |
4 |
2 |
2 |
13 |
16.4 |
||||||||
8 |
A |
149 |
126 |
115 |
65 |
44.3 |
91.5 |
95 |
202 |
184 |
186 |
95.3 |
106.1 |
104 |
2 |
4 |
3 |
0 |
3 |
2 |
3 |
4 |
4 |
1 |
13 |
13.6 |
1.28 |
14 |
|||
B |
159 |
172 |
132 |
77.2 |
58.2 |
98.9 |
170 |
168 |
150 |
81.3 |
101 |
0 |
3 |
2 |
3 |
4 |
2 |
4 |
3 |
2 |
1 |
12 |
14.8 |
||||||||
16 |
A |
140 |
130 |
139 |
68.2 |
46.4 |
96 |
100 |
182 |
168 |
154 |
84 |
93.5 |
97 |
4 |
2 |
5 |
0 |
2 |
2 |
4 |
3 |
2 |
2 |
13 |
15.5 |
1.45 |
16 |
|||
B |
170 |
155 |
160 |
80.8 |
60.9 |
103.6 |
172 |
152 |
163 |
81.2 |
100.8 |
5 |
2 |
2 |
3 |
4 |
1 |
0 |
4 |
3 |
4 |
14 |
17.2 |
||||||||
32 |
A |
Not plated due to precipitate |
|
Not plated due to precipitate |
|
Not plated due to precipitate |
|
|
|||||||||||||||||||||||
B |
Not plated due to precipitate |
Not plated due to precipitate |
Not plated due to precipitate |
||||||||||||||||||||||||||||
DMBA 1 |
A |
52 |
60 |
69 |
30.2 |
20.5 |
42.5 |
74 |
189 |
180 |
186 |
92.5 |
103 |
100 |
66 |
76 |
61 |
63 |
63 |
61 |
48 |
58 |
53 |
72 |
311 |
335.7 |
13.43 |
441 |
|||
B |
192 |
169 |
137 |
83 |
62.6 |
106.4 |
147 |
157 |
160 |
77.3 |
96.1 |
90 |
76 |
83 |
91 |
86 |
84 |
77 |
91 |
89 |
78 |
423 |
546.3 |
||||||||
DMBA 2 |
A |
40 |
35 |
30 |
17.5 |
11.9 |
24.6 |
38 |
160 |
178 |
157 |
82.5 |
91.8 |
86 |
83 |
66 |
59 |
62 |
46 |
56 |
80 |
68 |
73 |
62 |
328 |
397 |
17.8 |
532 |
|||
B |
64 |
99 |
79 |
40.3 |
30.4 |
51.7 |
120 |
140 |
132 |
65.3 |
81.2 |
74 |
81 |
99 |
110 |
101 |
101 |
75 |
93 |
54 |
84 |
436 |
667.3 |
DMBA = Dimethyl benzanthracene
CE = Cloning efficiency
RS = Relative survival
MF = Mutant frequency
MFS = Mutant frequency per survivor
SD = Standard deviation
Table 3: Historical control ranges for vehicle control cultures
Vehicle Control Mutant Frequencies per survivor x 10-6 | ||
4-Hour Without S9 | 4-Hour With S9 | |
Minimum | 5 | 5 |
Maximum | 23 | 24 |
Mean | 12.07 | 12.47 |
Standard Deviation | 4.44 | 3.95 |
Number of Experiments | 30 | 30 |
Table 4: Historical control ranges for positive control cultures
Positive Control Mutant Frequencies per survivor x 10-6 |
||||
|
4-Hour Without S9 (EMS) |
4-Hour Without S9 (EMS) |
4-Hour With S9 |
4-Hour With S9 |
500 µg/mL |
750 µg/mL |
1 µg/mL |
2 µg/mL |
|
Minimum |
171 |
291 |
209 |
103 |
Maximum |
346 |
574 |
774 |
925 |
Mean |
261.3 |
417.54 |
334.4 |
484.97 |
Standard Deviation |
36.82 |
63.8 |
114.17 |
174.83 |
Number of Experiments |
30 |
28 |
30 |
30 |
DMBA = Dimethyl benzanthracene
EMS = Ethyl methane sulphonate
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro Gene mutation in bacteria
Gene mutation in bacteria by propane-1,2,3-triyl- 2-ethylhexanoate was analysed in an Ames test performed according to GLP and OECD guideline 471, with the deviation that only four bacteria strains were used (Huntingdon, 1990a). Bacteria strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were treated with the test substance (diluted in DMSO) at concentrations of 5, 50, 500 and 5000 µg/plate in the dose-range finding test and 50, 150, 500, 1500, 5000 µg/plate in the main test, with and without metabolic activation. As no cytotoxicity and no increase in revertant colonies was noted in any strain and concentration tested, propane-1,2,3-triyl- 2-ethylhexanoate was considered to be not mutagenic in bacteria under the condition of this test.
In vitro cytogenicity in mammalian cells
In vitro cytogenicity was evaluated in an in vitro mammalian chromosome aberration test performed according to GLP and OECD guideline 473 (Huntingdon, 1990b). Cultured human lymphocytes were treated with diluted test substance (vehicle: ethanol) at concentration of 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 and 4000 µg/mL, with and without metabolic activation (S9 mix). Since no increase in the number of aberrant cells was noted at any concentration tested, propane-1,2,3-triyl- 2-ethylhexanoate was considered to be not clastogenic under the conditions of this study.
In vitro gene mutation in mammalian cells
The test item was analysed for its mutagenic potential in mammalian cells according to OECD guideline 476 and in compliance with GLP (Envigo, 2017). Gene mutation in the HPRT locus were investigated in Chinese Hamster Lung Fibroblasts (V79 cells) in the presence and absence of metabolic activation (S9-mix from rats treated with phenobarbital/b-naphtoflavone). A preliminary range-finding study revealed no concentration-related effects up to a concentration of 125 µg/mL with and without metabolic activation. However, precipitation of the test item was observed at and above 15.63 µg/mL. In two independent experiments, the cells were exposed to the test item at eight different concentrations (range 0.25 to 32 µg/mL) for four hours with and without S9-mix. Concurrent vehicle controls and positive control substances were included. Relative survival and mutation frequencies in the absence and presence of metabolic activation were investigated.
The vehicle control values were within the range of historical control data. The positive control substances (ethyl metahane sulphonate - S9-mix and dimethyl benzanthracene + S9-mix) markedly increased mutant frequencies, demonstrating the sensitivity of the test and the activity of the metabolic activation system. Treatment with the test item did not significantly induce the number of forward mutations at the HPRT locus of V79 cells, neither in the presence, nor in the absence of metabolic activation. Under the conditions of the study, propane-1,2,3-triyl 2-ethylhexanoate revealed no mutagenic potential in Chinese Hamster V79 cells in vitro.
Conclusion
In conclusion, assessment of the available experimental data on gene mutation in bacteria, gene mutation in mammalian cells and chromosome aberration in mammalian cells suggests that propane-1,2,3-triyl- 2-ethylhexanoate is neither mutagenic nor clastogenic in vitro.
Justification for classification or non-classification
The available data on gene mutation do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.
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