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EC number: 944-461-4 | 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
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- Flash point
- Auto flammability
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- Oxidising properties
- Oxidation reduction potential
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- Storage stability and reactivity towards container material
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- Additional physico-chemical information
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- Endpoint summary
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
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- Toxicological Summary
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Genetic toxicity in vitro:
Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 100 and TA 98, and E. coli WP2 uvr A , with and without metabolic activation
Chromosome aberration in mammalian cells (OECD 473): negative in Chinese hamster lung fibroblasts (CHL), without metabolic activation
Gene mutation in mammalian cells (OECD 476): negative in Chinese hamster lung fibroblasts, with and without metabolic activation
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:
- 21 Dec 2009 - 01 Feb 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted Jul 1997
- 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 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The Department of Health of the Government of the United Kingdom, UK
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (S. typhimurium strains)
trp operon (E. coli strain) - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 80 mg/kg bw/day phenobarbitone and 100 mg/kg bw/day beta-naphthoflavone
- Test concentrations with justification for top dose:
- Experiment 1 and 2: 50, 150, 500, 1500 and 5000 μg/plate, with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was soluble in DMSO at 50 mg/mL - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-aminoanthracene
- Remarks:
- See 'Details on test system and conditions' for concentrations and strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation, experiment 1) and preincubation (experiment 2)
DURATION
- Preincubation period: 20 min (experiment 2)
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 replication each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: reduction in growth of bacterial background lawn
OTHER:
- positive control substances used: N-ethyl-N'-nitro-N-nitrosoguanidine (2 μg/plate in DMSO, -S9, WP 2urvA-; 3 μg/plate, -S9, TA 100; 5 μg/plate, -S9, TA 1535); 9-aminoacridine (80 μg/plate in DMSO, -S9, TA 1537); 4-nitroquinoline-1-oxide (0.2 μg/plate in DMSO, -S9, TA 98); 2-aminoanthracene (1 μg/plate in DMSO, +S9, TA 100; 2 μg/plate, +S9 TA 1535 and TA 1537; 10 μg/plate, +S9, WP 2urvA-); benzo-a-pyrene (5 μg/plate in DMSO, +S9, TA 98) - Evaluation criteria:
- There are several criteria for determining a positive result, such as the dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal. - 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, but tested up to precipitating concentrations
- Remarks:
- precipitation was observed from 1500 μg/plate in experiment 1 and from 500 μg/plate in experiment 2, with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- precipitation was observed from 1500 μg/plate in experiment 1 and from 500 μg/plate in experiment 2, with and without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitate was observed at concentrations at and above 1500 μg/plate in experiment 1 and concentrations at and above 500 μg/plate in experiment 2, with and without metabolic activation, respectively. This did not affect the scoring of revertant colonies.
RANGE-FINDING/SCREENING STUDIES: A toxicity screening study was performed to determine the toxicity of the test substance. 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate test substance was tested on TA 100 and E. coli WP2 uvrA using the plate incorporation method. Precipitation of the test material was observed at 5000 μg/plate. A range-finding study (plate incorporation method) was performed on all strains using dose levels of 50 - 5000 μg/plate. As the results for all the tested dose levels were valid, the results were used as part of the main study (experiment 1).
COMPARISON WITH HISTORICAL CONTROL DATA: yes, the results of the vehicle and positive controls fell within the range of the historical control data (see Table 3 under 'Any other information on results incl. tables')
ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed at any dose level. - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 May - 14 Sep 2016
- 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 Jul 2015
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: in vitro gene mutation study in mammalian cells
- 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: JCRB Cell Bank, Japan
- Cell cycle length, doubling time or proliferation index: 13 h doubling time
- Modal number of chromosomes: 22
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: EMEM supplemented with 10% fetal calf serum, 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- 33.0, 65.0, 130 and 260 µg/mL (4 h), with and without metabolic activation. Precipitation of the test substance was observed in the culture medium at 260 µg/mL, which was selected as the highest dose level.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was insoluble in water and acetone, and soluble in DMSO at concentrations up to 26 mg/mL. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h exposure with and without metabolic activation
- Expression time (cells in growth medium): 7-8 days
- Selection time (if incubation with a selection agent): 9 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: four dishes in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: for the cytotoxicity evaluation, cells were fixed with formaldehyde and stained with crystal violet, after which the cells were washed and relative cell survival measured. - Evaluation criteria:
- When the mutation frequency (MF) in each test article treatment group satisfied all criteria described below, the results were judged to be positive. When any criterion was not satisfied, the results were judged to be negative. When different results between cultures (dish 1 and dish 2) were obtained, the results were comprehensively evaluated by considering their differences.
The result was judged to be positive:
1) When the average MFs in the test article treatment groups were three times or more than that of the concurrent negative (solvent) control group. When the MFs in the concurrent negative (solvent) control are zero, MFs in the test article treatment groups were three times or more than the mean MFs of the historical data of the negative control group.
2) When the average MFs in treatment groups were over ranges of the historical data of the negative control group. - 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
- Precipitation: In the main study, precipitation was observed at 260 µg/mLwith and without metabolic activation at the beginning and at the end of the treatment period, and at 130 µg/mL with metabolic activation at the end of the treatment period. This was not considered to affect the results.
RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentration range to be used in the main study. Precipitation of the test substance was observed in the culture medium at 260 µg/mL, which was selected as the highest dose level. The cytotoxicity test was performed using 6 concentrations in the range 8.1 - 260 µg/mL. No cytotoxicity was observed at any concentration level and the highest concentration was therefore selected to be the highest concentration in the main study.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The results of the positive controls fell within the range of the historical control data with and without metabolic activation, and the results of the negative control fell within the range of the historical control data without metabolic activation (see table 4 under "Any other information on results incl. tables"). The mutation frequency in the negative control group with metabolic activation was higher than the historical control data range and the experiment was therefore repeated. In the 2nd experiment, the mutation frequency in the negative control group with metabolic activation fell within the range of the historical control data.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: A preliminary cytotoxicity test was perfomed at concentrations of 8.1, 16.0, 33.0, 65.0, 130 and 260 µg/mL. The relative cell survival was shown to be comparable between the test substance and the solvent control. - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- Guideline study with acceptable restrictions. The sampling time for 6 h treatment is not equivalent to 1.5 normal cell cycle length, no concurrent measures of cytotoxicity for all cultures in the main experiment.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted Sep 2014
- Deviations:
- yes
- Remarks:
- The sampling time for 6 h treatment is not equivalent to 1.5 normal cell cycle length, no concurrent measures of cytotoxicity for all cultures in the main experiment.
- Qualifier:
- according to guideline
- Guideline:
- other: The Guidelines for the Testing of Chemicals (State Environmental Protection Administration of China) 2004.5
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- other: Chinese hamster lung fibroblasts (CHL)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 medium, supplemented with 10% fetal bovine serum, 1000 U/mL penicillin, 100 µg/mL streptomycin, 2 mM/L L-glutamine
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- 6 h treatment with and without metabolic activation: 156, 312 and 625 µg/mL
24 h treatment, without metabolic activation: 156, 312 and 625 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- 10 µg/mL mitomycin C in physiological saline, -S9; 1 mg/mL cyclophosphamide in physiological saline, +S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 6 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 6 h treatment: 6 h; 24 h treatment: 24 h
SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colchicine, treatment 4 h prior to harvesting
STAIN (for cytogenetic assays): 10% Giemsa
NUMBER OF REPLICATIONS: 3
NUMBER OF CELLS EVALUATED: 200 per culture
DETERMINATION OF CYTOTOXICITY
- Method: MTT cytotoxicity assay
OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no - Evaluation criteria:
- The tested concentrations were considered positive when statistically significant increases in total alterations frequency exceeded the historical control mean.
- Statistics:
- The data was analysed using the chi-square test with the software SPSS 11.5 for Windows.
- Key result
- Species / strain:
- other: Chinese hamster lung fibroblasts (CHL)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: the IC50 was calculated to be 0.640 mg/mL in the preliminary study
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: An MTT cytotoxicity assay was performed to select suitable dose levels for the main study. The concentrations 0.078, 0.156, 0.312, 0.625, 1.25, 2.50 and 5.0 mg/mL were used (no metabolic activation reported). Four replications per dose level were used. The IC50 was calculated using the SPSS16.0 software, resulting in an IC50 of 0.640 mg/mL (See table 2 under ' Any other information on results incl. tables').
COMPARISON WITH HISTORICAL CONTROL DATA: No
Referenceopen allclose all
Table 1. Test results of experiment 1 (plate incorporation method)
With or without S9-mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvR |
TA98 |
TA1537 |
||
– |
DMSO |
134 ± 5.7 |
27 ± 2.3 |
28 ± 0.6 |
26 ± 2.0 |
11 ± 5.0 |
– |
50 |
136 ± 4.4 |
25 ± 0.0 |
26 ± 2.5 |
27 ± 0.6 |
14 ± 1.5 |
– |
150 |
131 ± 6.4 |
29 ± 3.8 |
25 ± 6.7 |
24 ± 4.5 |
11 ± 3.8 |
– |
500 |
115 ± 21.7 |
28 ± 1.5 |
27 ± 1.2 |
25 ± 1.0 |
17 ± 1.0 |
– |
1500* |
106 ± 18.0 |
25 ± 4.2 |
22 ± 4.7 |
22 ± 3.6 |
10 ± 2.6 |
– |
5000* |
116 ± 5.1 |
32 ± 2.1 |
16 ± 1.7 |
19 ± 1.2 |
10 ± 2.1 |
Positive controls, -S9 |
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
Concentrations (μg/plate) |
3 |
5 |
2 |
0.2 |
80 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
429 ± 43.7 |
514 ± 43.1 |
726 ± 77.5 |
133 ± 11.0 |
884 ± 92.2 |
|
+ |
DMSO |
102 ± 11.6 |
12 ± 2.5 |
25 ± 3.5 |
28 ± 2.3 |
12 ± 3.2 |
+ |
50 |
109 ± 10.4 |
12 ± 1.5 |
27 ± 0.6 |
28 ± 3.5 |
15 ± 1.0 |
+ |
150 |
106 ± 3.2 |
12 ± 1.0 |
29 ± 0.6 |
28 ± 1.2 |
11 ± 4.0 |
+ |
500 |
97 ± 1.5 |
14 ± 1.7 |
26 ± 5.0 |
25 ± 2.1 |
13 ± 2.6 |
+ |
1500* |
101 ± 8.1 |
14 ± 1.2 |
24 ± 3.5 |
26 ± 2.6 |
11 ± 2.0 |
+ |
5000* |
93 ± 3.0 |
13 ± 2.6 |
26 ± 2.5 |
27 ± 1.5 |
9 ± 2.6 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
BaP |
2AA |
Concentrations (μg/plate) |
1 |
2 |
10 |
5 |
2 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1547 ± 34.6 |
223 ± 24.5 |
367 ± 41.1 |
218 ± 74.1 |
299 ± 29.5 |
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-nitroquinoline N-oxide
9AA:9-aminocridine
2AA: 2-amino-anthracene
BaP: benzo-a-pyrene
*: precipitation
Table 2. Test results of experiment 2 (plate incorporation method)
With or without S9-mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvR |
TA98 |
TA1537 |
||
– |
DMSO |
102 ± 12.7 |
24 ± 2.5 |
22 ± 6.1 |
18 ± 4.4 |
16 ± 2.5 |
– |
50 |
85 ± 12.4 |
24 ± 5.0 |
21 ± 2.6 |
15 ± 2.3 |
13 ± 1.2 |
– |
150 |
94 ± 7.1 |
24 ± 2.9 |
18 ± 2.5 |
17 ± 5.1 |
13 ± 1.7 |
– |
500* |
90 ± 10.0 |
23 ± 4.0 |
21 ± 0.6 |
20 ± 2.1 |
15 ± 6.1 |
– |
1500* |
94 ± 5.1 |
26 ± 3.5 |
22 ± 1.5 |
20 ± 2.6 |
14 ± 1.5 |
– |
5000* |
96 ± 4.5 |
24 ± 2.3 |
23 ± 3.5 |
20 ± 2.0 |
14 ± 2.0 |
Positive controls, -S9 |
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
Concentrations (μg/plate) |
3 |
5 |
2 |
0.2 |
80 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
613 ± 32.2 |
578 ± 58.0 |
861 ± 68.6 |
144 ± 9.5 |
354 ± 120.8 |
|
+ |
DMSO |
99 ± 8.5 |
16 ± 2.1 |
28 ± 5.1 |
27 ± 6.7 |
14 ± 3.2 |
+ |
50 |
90 ± 4.2 |
15 ± 1.0 |
29 ± 1.5 |
25 ± 4.0 |
10 ± 1.5 |
+ |
150 |
94 ± 6.7 |
14 ± 1.5 |
25 ± 3.6 |
25 ± 8.2 |
14 ± 1.5 |
+ |
500* |
98 ± 12.7 |
15 ± 1.2 |
24 ± 3.1 |
28 ± 1.7 |
14 ± 2.0 |
+ |
1500* |
98 ± 2.1 |
16 ± 3.0 |
25 ± 3.6 |
26 ± 4.4 |
16 ± 2.5 |
+ |
5000* |
97 ± 4.7 |
12 ± 1.5 |
26 ± 1.5 |
25 ± 2.3 |
12 ± 1.5 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
BaP |
2AA |
Concentrations (μg/plate) |
1 |
2 |
10 |
5 |
2 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
586 ± 96.2 |
235 ± 12.0 |
259 ± 36.7 |
387 ± 51.7 |
165 ± 8.4 |
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-nitroquinoline N-oxide
9AA:9-aminocridine
2AA: 2-amino-anthracene
BaP: benzo-a-pyrene
*: precipitation
Table 3. Historical controls
Strain |
Control |
without S9-mix |
with S9-mix |
Year |
|
|
Mean ± SD |
Mean ± SD |
|
TA100 |
Solvent/untreated |
94 ± 16.0 |
87 ± 14.8 |
2007 |
TA100 |
Positive |
558 ± 313.5 |
1231 ± 651.1 |
2007 |
TA1535 |
Solvent/untreated |
21 ± 5.2 |
13 ± 3.6 |
2007 |
TA1535 |
Positive |
343 ± 864.2 |
231 ± 476.5 |
2007 |
WP2 uvR |
Solvent/untreated |
26 ± 5.8 |
28 ± 6.4 |
2007 |
WP2 uvR |
Positive |
652 ± 707.9 |
505 ± 535.2 |
2007 |
TA98 |
Solvent/untreated |
21 ± 5.5 |
26 ± 6.0 |
2007 |
TA98 |
Positive |
256 ± 289.7 |
308 ± 280.5 |
2007 |
TA1537 |
Solvent/untreated |
11 ± 3.8 |
13 ± 4.1 |
2007 |
TA1537 |
Positive |
1697 ± 1193.6 |
339 ± 254.4 |
2007 |
TA100 |
Solvent/untreated |
105 ± 18.7 |
101 ± 18.5 |
2008 |
TA100 |
Positive |
487 ± 184.7 |
1138 ± 517.2 |
2008 |
TA1535 |
Solvent/untreated |
23 ± 5.3 |
15 ± 4.8 |
2008 |
TA1535 |
Positive |
210 ± 107.7 |
272 ± 94.9 |
2008 |
WP2 uvR |
Solvent/untreated |
26 ± 5.9 |
29 ± 7.2 |
2008 |
WP2 uvR |
Positive |
548 ± 231.5 |
426 ± 273.9 |
2008 |
TA98 |
Solvent/untreated |
19 ± 4.6 |
26 ± 5.3 |
2008 |
TA98 |
Positive |
155 ± 70.8 |
247 ± 178.2 |
2008 |
TA1537 |
Solvent/untreated |
13 ± 3.7 |
13 ± 3.8 |
2008 |
TA1537 |
Positive |
930 ± 476.2 |
279 ± 101.7 |
2008 |
Table 1: test results for 4-h treatment without metabolic activation
Group |
Concentration (µg/mL) |
Cytotoxicity |
Mutation |
|
|
|
Average in % |
6-TG resistant colonies/dish (mean) |
Mutation frequency (10-6) |
DMSO |
1 vol% |
100.0 |
1.3 |
15.2 |
Test substance |
33.0 |
114.0 |
0.8 |
7.8 |
Test substance |
65.0 |
105.0 |
0.9 |
9.8 |
Test substance |
130 |
97.7 |
0.9 |
10.7 |
Test substance |
260* |
103.6 |
1.2 |
12.9 |
EMS |
1000 |
77.8 |
91.4 |
1413 |
EMS: ethylmethanesulphonate
*precipitation observed
Table 2: test results for 4-h treatment with metabolic activation (1st experiment)
Group |
Concentration (µg/mL) |
Cytotoxicity |
Mutation |
|
|
|
Average in % |
6-TG resistant colonies/dish (mean) |
Mutation frequency (10-6) |
DMSO |
1 vol% |
100.0 |
1.3 |
14.8** |
Test substance |
33.0 |
98.5 |
0.6 |
7.1 |
Test substance |
65.0 |
101.0 |
0.4 |
4.6 |
Test substance |
130* |
106.7 |
1.3 |
14.2** |
Test substance |
260* |
97.6 |
1.4 |
16.8** |
NDMA |
1000 |
68.4 |
37.8 |
642.2 |
NDMA: N-dimethylnitrosamine
*precipitation observed
**The mutation frequency was higher than the upper limit of the historical control data range
Table 3: test results for 4-h treatment with metabolic activation (2nd experiment)
Group |
Concentration (µg/mL) |
Cytotoxicity |
Mutation |
|
|
|
Average in % |
6-TG resistant colonies/dish (mean) |
Mutation frequency (10-6) |
DMSO |
1 vol% |
100.0 |
0.7 |
6.6 |
Test substance |
33.0 |
95.8 |
0.3 |
2.6 |
Test substance |
65.0 |
98.2 |
0.3 |
3.1 |
Test substance |
130* |
99.6 |
0.4 |
3.6 |
Test substance |
260* |
97.4 |
0.6 |
5.7 |
NDMA |
1000 |
73.5 |
39.0 |
532.0 |
NDMA: N-dimethylnitrosamine
*precipitation observed
Table 4: Historical control data of gene mutation test (4-hour treatment) using V79 cells
S9-mix |
Control |
Mutation frequency (10-6) |
|||
|
|
No. of data |
Mean ± SD |
Minimum |
Maximum |
- |
Negative control |
38 |
3.9 ± 4.9 |
0 |
21.0 |
- |
EMS (1 mg/mL) |
27 |
1002 ± 249 |
588 |
1530 |
+ |
Negative control |
44 |
3.6 ± 2.7 |
0 |
10.2 |
+ |
NDMA (2 mg/mL) |
26 |
601 ± 359 |
229 |
1453 |
SD: standard deviation
NDMA: N-dimethylnitrosamine
EMS: ethylmethanesulphonate
Table 1: Chromosome aberrations, summarised data
Metabolic activation |
Test substance concentration (μg/mL) |
Total number of metaphases analysed |
Chromosome aberration cell count |
Chromosome aberration type* |
6 h treatment, 6h harvesting time |
||||
- |
Vehicle control (DMSO) |
200 |
4 |
b/p |
- |
156 |
200 |
3 |
b |
- |
312 |
200 |
3 |
b |
- |
625 |
200 |
3 |
b |
- |
mitomycin C |
200 |
45 |
b/p |
24 h treatment, 24h harvesting time |
||||
- |
Vehicle control (DMSO) |
200 |
4 |
b/p |
- |
156 |
200 |
4 |
b/p |
- |
312 |
200 |
5 |
b/p |
- |
625 |
200 |
6 |
b/p |
- |
mitomycin C |
200 |
54 |
b/p/l |
6 h treatment, 6h harvesting time |
||||
+ |
Vehicle control (DMSO) |
200 |
3 |
b/p |
+ |
156 |
200 |
2 |
b |
+ |
312 |
200 |
4 |
b |
+ |
625 |
200 |
4 |
b |
+ |
cyclophosphamide |
200 |
51 |
b/p/l |
* b: break, d: deletion, t: translocation, r: ring, f: fragment, other: dicentric, triradial, quadriradial etc.
Table 2: Cytotoxicity
Dose mg/mL |
Number of wells |
Optical Density |
DMSO |
4 |
1.013 ± 0.0387 |
0.078 |
4 |
0.978 ± 0.0318 |
0.156 |
4 |
0.908 v 0.0331 |
0.312 |
4 |
0.683 ± 0.0849 |
0.625 |
4 |
0.486 ± 0.0319 |
1.25 |
4 |
0.283 ± 0.0427 |
2.50 |
4 |
0.161 ± 0.0134 |
5.0 |
4 |
0.091 ± 0.0142 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Micronucleus test (OECD 474): negative in mouse bone marrow cells
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- No historical negative or positive control data were reported, dose volume was 20 mL/kg bw instead of max. 10 mL/kg bw, 2000 immature erythrocytes/animal were scored instead of 4000.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- adopted Sep 2014
- Deviations:
- yes
- Remarks:
- No historical negative or positive control data were reported, dose volume 20 mL/kg bw, 2000 immature erythrocytes/ animal were scored
- Qualifier:
- according to guideline
- Guideline:
- other: The Guidelines for the Testing of Chemicals (State Environmental Protection Administration of China) 2004.5
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- other: The Guidelines for the Hazard Evaluation of New Chemical Substances (HJ/T 154-2004)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: Kunming (SPF grade)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Vital River Laboratory Animal Technology Co. Ltd., Beijing, China
- Weight at study initiation: 25 - 28 g (females, main study), 26 - 28 g (males, main study); 18.1 - 20.4 (females, preliminary study), 18.7 - 20.1 (males, preliminary study)
- Fasting period before study: from 12 h before dosing until 3 h after (preliminary study only)
- Housing: the animals were housed in clear plastic cages with stainless steel tops
- Diet: feed (SCXK (JIN) 2012-0001, Tianjin Huarong Laboratory Animal Science and Technology Co., Ltd., Tianijin, China), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: vegetable oil
- Justification for choice of solvent/vehicle: the test substance formed a dosable and apparently miscible suspension in vegetable oil
- Concentration of test material in vehicle: approximately 100 mg/mL; administered as 2 mL/100 g bw - Duration of treatment / exposure:
- 2 days
- Frequency of treatment:
- daily (2 doses in total)
- Post exposure period:
- 24 h
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 40 mg/kg bw - Tissues and cell types examined:
- Tissue: bone marrow
Cell type: bone marrow cells - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: No mortality or signs of toxicity were noted in mice administered a single dose of 5000 mg/kg bw in the range-finding study. The recommended limit dose according the OECD guideline 474 is 2000 mg/kg bw for administration periods of less than 14 days. Therefore the limit dose of 2000 mg/kg bw/day was selected.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): the bone marrow cells were harvested 24 h after the last test substance administration.
DETAILS OF SLIDE PREPARATION: Slides were fixed with methanol and stained with Giemsa solution.
METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCE) were scored per animal to determine the frequency of micronucleated polychromatic erythrocytes (MNPCE). The ratio of polychromatic to monochromatic erythrocytes (PCE/NCE) was determined. - Statistics:
- The statistical significance between groups was analysed using ANOVA. All results were expressed as mean ± standard deviation; values were considered significant at p < 0.05.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw
- Clinical signs of toxicity in test animals: none
- Rationale for exposure: the range-finding study was performed to establish the highest dose level at which no or low systemic toxicity was observed.
- Other: 5 mice/sex were administered 5000 mg/kg bw by gavage as a single dose. There was no mortality during the 14-day observation period. No adverse clinical signs were observed and the body weight gain was within the expected range for this species and strain. The histopathological examination did not show any abnormal results.
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): the induction of micronuclei was not significantly increased in the treatment group, compared with the control group (see Table 1 - 3 under 'Any other information on results incl. tables').
- Ratio of PCE/NCE (for Micronucleus assay): For females, the ratio of PCE/NCE was 1.06, 1.06 and 1.05 for the vehicle, treatment and positive control group, respectively. For males, the ratio of PCE/NCE was 1.05, 1.03 and 1.02 for the vehicle, treatment and positive control group, respectively.
- Appropriateness of dose levels and route: the route and dose level was suitable under the conditions of the study.
- Statistical evaluation: the difference between the vehicle control and test substance group was not significant.
OTHER:
No effects on body weight were observed in any of the control or treatment groups.
Reference
Table 1: Results of the in vivo micronucleus assay in male animals
|
|
|
Total micronuclei per 2000 PCEs at sampling time |
Exp group |
Number of animals/sex |
Dose [mg/kg bw] |
24 h |
Vehicle control (vegetable oil) |
5 |
0 |
7 |
Test substance
|
5 |
2000 |
11 |
Positive control (cyclophosphamide) |
5 |
40 |
192* |
*statistically significant (p<0.05);
Table 2: Results of the in vivo micronucleus assay in female animals.
|
|
|
Total micronuclei per 2000 PCEs at sampling time |
Exp group |
Number of animals/sex |
Dose [mg/kg bw] |
24 h |
Vehicle control (vegetable oil) |
5 |
0 |
10 |
Test substance
|
5 |
2000 |
8 |
Positive control (cyclophosphamide) |
5 |
40 |
205* |
*statistically significant (p<0.05);
Table 3: Results of the in vivo micronucleus assay
Treatment group |
Dose [mg/kg bw] |
Sampling time [h] |
Mean frequency of PCE with MN
|
PCE/NCE ratio |
Females |
||||
Vehicle control (vegetable oil) |
0 |
24 |
0.01 |
1.06 ± 0.04 |
Test substance |
2000 |
24 |
0.01 |
1.06 ± 0.04 |
Positive control (cyclophosphamide) |
40 |
24 |
2.05 |
1.05 ± 0.02 |
Males |
||||
Vehicle control (vegetable oil) |
0 |
24 |
0.07 |
1.05 ± 0.03 |
Test substance |
2000 |
24 |
0.08 |
1.03 ± 0.03 |
Positive control (cyclophosphamide) |
40 |
24 |
1.92 |
1.02 ± 0.04 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Genetic toxicity in vitro
An in vitro bacterial reverse mutation assay (Ames test) was performed with Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene (Sanders, 2010). The study was performed according to OECD guideline 471 and under GLP conditions using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvR at concentrations up to 5000 µg/plate. Experiment 1 was carried out as a plate incorporation assay, while experiment 2 was performed as a pre-incubation assay. No cytotoxicity was observed. Precipitation was noted from 500 µg/plate in experiment 1 and from 1500 µg/plate in experiment 2, respectively, with and without metabolic activation. This did not affect the scoring of revertant colonies. The positive and vehicle (DMSO) controls were valid. The test substance did not induce reversions in any of the S. typhimurium or E. coli strains with or without metabolic activation.
The potential of Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene to induce chromosomal aberrations was tested in an assay with Chinese hamster lung fibroblasts (CHL), performed according to OECD 473 (Zhang, 2014). CHL cells were exposed to test substance concentrations of 156, 312 and 625 µg/mL. The upper concentration was determined in a range-finding study, in which an IC50 value of 0.640 was calculated. A short-term treatment (6 hours) was performed with a 6-hour harvest time, with and without metabolic activation. In addition, a 24-hour treatment with 24-hour harvest time without metabolic activation was performed. There were no increases in the number of chromosome aberrations at any dose level. The positive controls induced a significant increase in the number of chromosomal aberrations. Therefore the test substance is not considered to cause cytogenetic effects under the conditions of this test. As the results of the test are only valid for the 24-hour exposure duration without metabolic activation, the study cannot be used for classification purposes.
An in vitro gene mutation test was performed with Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene in Chinese lung fibroblasts (V79) (Yamakage, 2016). The study was conducted according to OECD guideline 476 and GLP. The cells were treated with 33.0, 65.0, 130 and 260 µg/mL for 4 h, with and without metabolic activation. Precipitation of the test substance was observed from the start of the treatment at 260 µg/mL with and without metabolic activation, and at 130 µg/mL with metabolic activation at the end of the treatment. The precipitation was not considered to affect the results. No cytotoxicity was observed at any concentration. The results of the positive controls with and without metabolic activation, and the results of the negative control without metabolic activation fell within the range of the historical control data. The mutation frequency in the negative control group with metabolic activation was higher than the historical control data range and the experiment was therefore repeated. In the 2nd experiment, the mutation frequency in the negative control group with metabolic activation fell within the range of the historical control data. The test substance did not cause an increase in mutation frequency with and without metabolic activation.
Genetic toxicity in vivo
The potential for in vivo genetic toxicity of Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene was assessed in a micronucleus test performed according to the Guidelines for the Testing of Chemicals (State Environmental Protection Administration of China) 2004.5 and similar to OECD guideline 474 (Li, 2015). 5 Kunming mice/sex/dose were administered 2000 mg/kg bw/day test substance in vegetable oil by gavage for 2 days. The treatment and vehicle animals were sacrificed 24 hours after dosing. Bone marrow cells from the femur were extracted, and slides were prepared and stained with a Giemsa solution. No effects on body weight were observed in any of the control or treatment groups. The vehicle control group and positive control group were shown to be valid. For the treatment group, no increase in the frequency of micronucleated polychromatic erythrocytes was observed in the isolated polychromatic erythrocytes and no decrease in the ratio of polychromatic to normochromatic erythrocytes was noted, compared with the vehicle control group.
Overall conclusion for genetic toxicity
Based on the available data, Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene is considered to be not mutagenic in vitro, and not clastogenic in vivo.
Justification for classification or non-classification
Based on the available data, the results on genetic toxicity for the test substance does not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.
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