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EC number: 223-445-4 | CAS number: 3896-11-5
- 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
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- Nanomaterial pour density
- Nanomaterial photocatalytic activity
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- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 006
- Report date:
- 2006
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- Bumetrizole
- EC Number:
- 223-445-4
- EC Name:
- Bumetrizole
- Cas Number:
- 3896-11-5
- Molecular formula:
- C17 H18 Cl N3 O
- IUPAC Name:
- 2-tert-butyl-6-(5-chloro-2H-benzotriazol-2-yl)-4-methylphenol
- Test material form:
- solid
Constituent 1
- Specific details on test material used for the study:
- - Name of test substance (as cited in study report): Bumetrizole
- Analytical purity: 99.9 % w/w
- Lot/batch No.: 01721IW4
- Stability under test conditions: stable
- Storage condition of test material: stored sealed in a cabinet at 20.0 - 25.3 °C
- Other: Supplier: Ciba Specialty Chemicals, Osaka, Japan
Method
- Target gene:
- not applicable
Species / strain
- Species / strain / cell type:
- mammalian cell line, other: CHL/IU
- Details on mammalian cell type (if applicable):
- - Periodically checked for Mycoplasma contamination: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver induced with phenobarbital and 5,6-benzoflavone (see table 1)
- Test concentrations with justification for top dose:
- Cell growth inhibition test:
Short treatment test (6 hr test/18 hr recovery): 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 or 3200 µg/mL
Continuous treatment test (24 hour test/0 hour recovery): 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 or 3200 µg/mL
Chromosome aberration test:
Short treatment test (6 hr test/18 hr recovery): 150, 300, 600, 1200 or 2400 µg/mL
Continuous treatment test (24 hour test/0 hour recovery): 75, 150, 300, 600 or 1200 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is commonly used for the reverse mutagenicity test on bacteria and it forms a good suspension.
Controls
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: see table 2
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Pulse treatment: 6 hours; Continuous treatment: 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200 in total per dose
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes - Evaluation criteria:
- The test result was considered negative when the occurrence rate of cells with numerical or structural aberrations was below 5% and false positive for a rate between 5-10 % and positive for above 10 % with a dose related increase. The occurence rate of cells with structural aberrations was calculated with/without gaps and judged by the occurrence rate without gaps.
- Statistics:
- none
Results and discussion
Test results
- Species / strain:
- mammalian cell line, other: CHL/IU
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- from 1200 µg/plate (pulse treatment), > 600 µg/plate (continuous treatment)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
Any other information on results incl. tables
1. Short Treatment Method (results of the tests are shown in Tables 3 and 4.)
The rate of cells with numerical aberrations was below 1.5% for all concentrations with and without S9 mix. The rate of cells with structural aberrations was below 1.0% in all concentrations with and without S9 mix.
The cell survival rate in plates with S9 mix was more than 90% for 150 and 300 µg/mL. At concentrations above 600 µg/mL, survival rate decreased proportionally to concentrations, reaching 34% at the maximum concentration of 2400 µg/mL. The cell survival rate without S9 mix was more than 90% for 150 and 300 µg/mL. At concentrations above 600 µg/mL the value decreased in proportion to higher concentration, attaining 34% at the maximum concentration of 2400 µg/mL.
A white oil filmy precipitate and white impalpable precipitates were observed in all concentrations.
In the negative control, 0% and 0.5% of cells carried numerical aberrations in the absence of S9 mix and presence of S9 mix, respectively. The occurrence rate of cells with structural aberrations was 0% in the presence of S9 mix and 0.5% in the absence of S9 mix. In the positive controls, the occurrence rate of cells with numerical aberrations with (DMN; 500 µg/mL) and without S9 mix (MMC;0.1 µg/mL) was 0%. The occurrence rate of cells with structural aberrations was 72.5% with S9 mix and 52.5% without S9 mix. In conclusion, the induction rate of chromosomal aberrations in the negative and positive control groups was within the range of background thus satisfying the crieria of a valid test.
2. Continuous Treatment Method (test results are shown in Table 5)
The occurrence rate of cells with numerical aberrations in the bumetrizole treatment group was below 1.5% for all concentrations. Likewise, the occurrence rate of cells with structural aberrations was below 1.0% for all concentrations.
The survival rate of cells was above 90% for 75 and 150 µg/mL. However, above 300 µg/mL, survival decreases in proportion to higher concentration, and was 34% for the maximum concentration of 1200 µg/mL.
White filmy oil precipitates and white impalpable precipitates were observed for all concentrations when dosing preparations were added to culture medium in the beginning and also at the end of treatment.
In the negative control, the percentage of cells with numerical aberrations and structural aberrations was 0% and 0.5%, respectively. In the positive controls, the percentage of cells with numerical aberrations and structural aberrations (MMC: 0.05 µg/mL) was 0%, and 43.5%, respectively. Hence, the induction rate of chromosomal aberrations in negative and positive control groups was within the range of background, thus satisfying the criteria of a valid test.
Table 3: Chromosome aberration test results (short term treatment) -S9
Treatment period (h) |
S9 mix |
Dosage of test substance (mg/mL) |
Number of cells showing structural chromosome aberration (incidence, %) |
Number of gap appearances |
Cell growth index (%) |
Number of cells showing numerical chromosome aberration (incidence, %) |
|||||||||
Number of cells observed |
Chromatid break |
Chromatid exchange |
Chromosome break |
Chromosome exchange |
Other |
Total number of aberrations (%) |
Number of cells observed |
Polyploid |
Others |
Total numberof aberrant cells (%) |
|||||
6-18 |
- |
Negative control (DMSO ) |
200 |
1 |
0 |
0 |
0 |
0 |
1 (0.5) |
0 |
100 |
200 |
0 |
0 |
0 (0) |
6-18 |
- |
0.150 * |
200 |
0 |
0 |
0 |
0 |
0 |
0 (0) |
0 |
95 |
200 |
1 |
0 |
1 (0.5) |
6-18 |
- |
0.300 * |
200 |
1 |
0 |
0 |
0 |
0 |
1 (0.5) |
0 |
95 |
200 |
0 |
0 |
0 (0) |
6-18 |
- |
0.600 * |
200 |
0 |
0 |
0 |
0 |
0 |
0 (0) |
0 |
89 |
200 |
0 |
0 |
0 (0) |
6-18 |
- |
1.200 * |
200 |
1 |
0 |
0 |
0 |
0 |
1 (0.5) |
0 |
48 |
200 |
3 |
0 |
3 (1.5) |
6-18 |
- |
2.400 * |
200 |
0 |
1 |
0 |
0 |
0 |
1 (0.5) |
0 |
34 |
200 |
2 |
0 |
2 (1.0) |
6-18 |
- |
Positive control (MMC) 0.0001 |
200 |
53 |
75 |
0 |
0 |
0 |
105 (52.5) |
0 |
88 |
200 |
0 |
0 |
0 (0) |
*: Precipitate observed
Table 4: Chromosome aberration test results (short term treatment) +S9
Treatment period (h) |
S9 mix |
Dosage of test substance (mg/ml) |
Number of cells showing structural chromosome aberration (incidence, %) |
Number of gap appearances |
Cell growth index (%) |
Number of cells showing numerical chromosome aberration (incidence, %) |
|||||||||
Number of cells observed |
Chromatid break |
Chromatid exchange |
Chromosome break |
Chromosome exchange |
Other |
Total number of aberrations (%) |
Number of cells observed |
Polyploid |
Others |
Total numberof aberrant cells (%) |
|||||
6-18 |
+ |
Negative control (DMSO ) |
200 |
0 |
0 |
0 |
0 |
0 |
0 (0) |
0 |
100 |
200 |
1 |
0 |
1 (0.5) |
6-18 |
+ |
0.150 * |
200 |
0 |
0 |
0 |
0 |
0 |
0 (0) |
0 |
96 |
200 |
1 |
0 |
1 (0.5) |
6-18 |
+ |
0.300 * |
200 |
1 |
1 |
0 |
0 |
0 |
2 (1.0) |
0 |
91 |
200 |
0 |
0 |
0 (0) |
6-18 |
+ |
0.600 * |
200 |
1 |
0 |
0 |
0 |
0 |
1 (0.5) |
0 |
85 |
200 |
0 |
0 |
0 (0) |
6-18 |
+ |
1.200 * |
200 |
0 |
0 |
0 |
0 |
0 |
0 (0) |
0 |
51 |
200 |
3 |
0 |
3 (1.5) |
6-18 |
+ |
2.400 * |
200 |
0 |
0 |
0 |
0 |
0 |
0 (0) |
0 |
34 |
200 |
3 |
0 |
1 (1.5) |
6-18 |
+ |
Positive control (DMN) 0.500 |
200 |
66 |
124 |
0 |
2 |
0 |
145 (72.5) |
0 |
85 |
200 |
0 |
0 |
0 (0) |
*: Precipitate observed
Table 5: Chromosome aberration test results (continuous treatment)
Treatment period (h) |
Dosage of test substance (mg/ml) |
Number of cells showing structural chromosome aberration (incidence, %) |
Number of gap appearances |
Cell growth index (%) |
Number of cells showing numerical chromosome aberration (incidence, %) |
|||||||||
Number of cells observed |
Chromatid break |
Chromatid exchange |
Chromosome break |
Chromosome exchange |
Other |
Total number of aberrations (%) |
Number of cells observed |
Polyploid |
Others |
Total numberof aberrant cells (%) |
||||
24-0 |
Negative control (DMSO ) |
200 |
0 |
1 |
0 |
0 |
0 |
1 (0.5) |
0 |
100 |
200 |
0 |
0 |
0 (0) |
24-0 |
0.075* |
200 |
0 |
0 |
0 |
0 |
0 |
0 (0) |
0 |
97 |
200 |
0 |
0 |
0 (0) |
24-0 |
0.150* |
200 |
1 |
0 |
0 |
1 |
0 |
2 (1.0) |
0 |
92 |
200 |
2 |
0 |
2 (1.0) |
24-0 |
0.300* |
200 |
0 |
1 |
0 |
0 |
0 |
1 (0.5) |
0 |
88 |
200 |
1 |
0 |
1 (0.5) |
24-0 |
0.600* |
200 |
1 |
0 |
0 |
0 |
0 |
1 (0.5) |
0 |
62 |
200 |
3 |
0 |
3(1.5) |
24-0 |
1.200* |
200 |
0 |
0 |
0 |
0 |
0 |
0 (0) |
0 |
34 |
200 |
2 |
0 |
2 (1.0) |
24-0 |
Positive control (MMC) 0.00005 |
200 |
40 |
56 |
0 |
0 |
0 |
87 (43.5) |
0 |
88 |
200 |
0 |
0 |
0 (0) |
*: Precipitate observed
Applicant's summary and conclusion
- Conclusions:
- In conclusion, the in vitro chromosome aberration assay resulted in the test substance being negative (with and without metabolic activation) for inducing genotoxicity and thus, is considered to be non-clastogenic.
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