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EC number: 449-360-4 | CAS number: 647828-16-8
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 April 2003 to 22 May 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 003
- Report date:
- 2003
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- -
- EC Number:
- 449-360-4
- EC Name:
- -
- Cas Number:
- 647828-16-8
- Molecular formula:
- C18H32O
- IUPAC Name:
- decahydro-2,2,6,6,7,8,8-heptamethyl-2H-Indeno[4,5-b] furan
- Test material form:
- liquid
Constituent 1
Method
- Target gene:
- Histidine for Salmonella
Tryptophan for E.Coli
Species / strainopen allclose all
- 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:
- Phenobarbitone and betanaphthoflavone induced rat liver (S9-mix)
- 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.
Main experiment 1 and 2: 50, 150, 500, 1500, 5000 µg/plate. - Vehicle / solvent:
- Dimethyl sulphoxide
Controlsopen allclose all
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
In agar (plate incorporation)
DURATION:
- Pre-incubation period for bacterial strains: 10 hours
- Exposure duration: 48 - 72 hours
- Expression time: Not applicable
- Selection time: 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:
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain should be confirmed, e.g., rfa cell-wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 1 to 9.9 X 109 bacteria per mL.
- Each mean positive control value should be at least 2 times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of 4 non-toxic test substance dose levels.
- There should not be an excessive loss of plates due to contamination.
Evaluation Criteria:
- When the test substance has induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least 1 strain of bacteria, it is considered to be a positive result. - Statistics:
- The mean and standard deviation were calculated. Dunnetts linear regression analysis was performed.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Remarks:
- The number of revertant colonies per plate for the concurrent vehicle control was inside the historical control range of the laboratory.
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks:
- The positive control induced a number of revertant colonies per plate that demonstrated the effective performance of the assay.
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Remarks:
- The number of revertant colonies per plate for the concurrent vehicle control was inside the historical control range of the laboratory.
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks:
- The positive control induced a number of revertant colonies per plate that demonstrated the effective performance of the assay.
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Remarks:
- The number of revertant colonies per plate for the concurrent vehicle control was inside the historical control range of the laboratory.
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks:
- The positive control induced a number of revertant colonies per plate that demonstrated the effective performance of the assay.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Remarks:
- The number of revertant colonies per plate for the concurrent vehicle control was inside the historical control range of the laboratory.
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks:
- The positive control induced a number of revertant colonies per plate that demonstrated the effective performance of the assay.
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Remarks:
- The number of revertant colonies per plate for the concurrent vehicle control was inside the historical control range of the laboratory.
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks:
- The positive control induced a number of revertant colonies per plate that demonstrated the effective performance of the assay.
- Additional information on results:
- Preliminary Toxicity Test
- The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.
Mutation Test
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The S9-mix used in both experiments of the main test was shown to be sterile.
- Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
- The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2.
- A history profile of vehicle and positive control values is presented in Table 3.
- The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- 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.
- All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Any other information on results incl. tables
Preliminary Toxicity Test:
The test substance was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test substance formulation and S9-mix used in this experiment were both shown to be effectively sterile.
The number 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 | 74 | 56 | 61 | 73 | 62 | 67 | 66 | 63 | 61 | 70 | 93 |
+ | TA100 | 89 | 110 | 92 | 118 | 105 | 67 | 82 | 89 | 102 | 96 | 101 |
- | WP2uvrA- | 15 | 14 | 15 | 19 | 12 | 26 | 17 | 13 | 15 | 14 | 15 |
+ | WP2uvrA- | 13 | 11 | 15 | 9 | 16 | 14 | 9 | 14 | 18 | 14 | 24 |
Mutation Test:
Table 1. Spontaneous Mutation Rates (Concurrent Negative Control).
Experiment 1
Number of revertants (mean number of colonies per plate) | |||||||||
Base-pair Substitution Type | Frameshift Type | ||||||||
TA100 | TA1535 | WPAuvrA | TA98 | TA1537 | |||||
106 | (91) | 13 | (11) | 18 | (20) | 24 | (18) | 8 | (7) |
86 | 11 | 19 | 12 | 9 | |||||
80 | 9 | 22 | 17 | 4 |
Experiment 2
Number of revertants (mean number of colonies per plate) | |||||||||
Base-pair Substitution Type | Frameshift Type | ||||||||
TA100 | TA1535 | WPAuvrA | TA98 | TA1537 | |||||
81 | (82) | 32 | (27) | 26 | (26) | 23 | (19) | 5 | (8) |
81 | 21 | 24 | 17 | 9 | |||||
83 | 29 | 28 | 18 | 10 |
Table 2a: Range-finding test – Without Metabolic Activation: Mean value and SD
| TA100 | TA1535 | WP2uvrA- | TA98 | TA1537 |
0 | 70 (7.5) | 13 (1.2) | 20 (5.5) | 23 (5.0) | 8 (5.1) |
50 | 70 (6.7) | 16 (0.6) | 16 (2.9) | 19 (3.8) | 7 (0.6) |
150 | 56 (6.7) | 14 (4.0) | 20 (3.8) | 21 (5.8) | 8 (4.2) |
500 | 52 (9.5) | 12 (1.2) | 16 (4.0) | 18 (5.8) | 9 (0.6) |
1500 | 55 (4.4) | 13 (4.4) | 19 (1.2) | 31 (2.1) | 7 (4.6) |
5000 | 66 (5.8) | 14 (5.8) | 22 (3.8) | 23 (2.0) | 12 (4.6) |
Positive control | ENNG | ENNG | ENNG | 4NQO | 9AA |
| 365 (112.8) | 258 (9.6) | 762 (43.8) | 277 (29.5) | 1263 (131.8) |
Table 2a: Range-finding test – With Metabolic Activation: Mean value and SD
| TA100 | TA1535 | WP2uvrA- | TA98 | TA1537 |
0 | 96 (6.4) | 15 (1.5) | 32 (0.6) | 26 (3.1) | 15 (2.5) |
50 | 97 (7.2) | 13 (2.9) | 24 (0.6) | 32 (5.7) | 11 (2.9) |
150 | 79 (0.6) | 11 (0.6) | 24 (3.0) | 32 (2.5) | 7 (1.5) |
500 | 85 (4.7) | 12 (6.1) | 27 (5.3) | 24 (5.8) | 12 (1.2) |
1500 | 88 (2.1) | 16 (3.1) | 24 (5.6) | 29 (0.0) | 5 (0.6) |
5000 | 74 (17.5) | 14 (4.0) | 26 (5.5) | 22 (0.0) | 9 (4.6) |
Positive control | 2AA | 2AA | 2AA | BP | 2AA |
| 1749 (242) | 221 (7.8) | 637 (172.8) | 326 (22.6) | 341 (14.7) |
Table 2c: Main test – Without Metabolic Activation: Mean value and SD
| TA100 | TA1535 | WP2uvrA- | TA98 | TA1537 |
0 | 108 (11.1) | 15 (1.5) | 14 (3.5) | 17 (1.0) | 8 (2.5) |
50 | 86 (7.8) | 12 (3.5) | 17 (4.5) | 15 (4.0) | 8 (0.6) |
150 | 88 (11.0) | 12 (4.5) | 13 (2.3) | 22 (5.6) | 6 (1.7) |
500 | 96 (18.8) | 10 (0.6) | 14 (2.0) | 16 (3.2) | 7 (1.0) |
1500 | 81 (5.0) | 10 (1.7) | 13 (5.6) | 19 (8.0) | 7 (1.2) |
5000 | 80 (13.4) | 15 (4.2) | 18 (3.0) | 28 (8.4) | 7 (2.5) |
Positive control | ENNG | ENNG | ENNG | 4NQO | 9AA |
| 361 (80.9) | 176 (32.1) | 717 (37.4) | 109 (8.4) | 759 (20.2) |
Table 2d: Main test – With Metabolic Activation: Mean value and SD
| TA100 | TA1535 | WP2uvrA- | TA98 | TA1537 |
0 | 106 (12.9) | 13 (6.1) | 19 (2.3) | 20 (2.5) | 10 (0.0) |
50 | 86 (2.1) | 8 (3.1) | 22 (1.5) | 21 (4.2) | 7 (2.5) |
150 | 95 (11.6) | 7 (2.3) | 17 (3.6) | 29 (6.6) | 4 (0.6) |
500 | 87 (8.0) | 14 (9.6) | 14 (1.0) | 26 (2.6) | 7 (1.7) |
1500 | 74 (10.1) | 16 (10.2) | 12 (1.7) | 24 (3.0) | 4 (1.0) |
5000 | 85 (16.5) | 12 (1.7) | 21 (3.1) | 22 (4.9) | 10 (3.6) |
Positive control | 2AA | 2AA | 2AA | BP | 2AA |
| 1363 (49.7) | 239 (22.7) | 362 (4.6) | 202 (16.3) | 288 (48.8) |
ENNG: N-ethyl-N’-nitro-N-nitrosoguanidine; 4NQO: 4-Nitroquinoline-1-oxide; 9AA: 9-Aminoacridine
Table 3a: Historical Control Data: Combined vehicle and untreated control: 2001
| TA100 |
| TA1535 |
| WP2uvrA- |
| TA98 |
| TA1537 |
|
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Mean | 106 | 116 | 16 | 15 | 22 | 26 | 23 | 32 | 11 | 13 |
SD | 23 | 23 | 4 | 4 | 5 | 6 | 7 | 8 | 4 | 4 |
Min | 58 | 62 | 8 | 7 | 11 | 13 | 10 | 13 | 2 | 4 |
Max | 178 | 178 | 37 | 37 | 41 | 52 | 56 | 58 | 23 | 29 |
N | 817 | 658 | 790 | 621 | 656 | 501 | 814 | 662 | 793 | 630 |
Table 3b: Historical Control Data: Combined vehicle and untreated control: 2002
| TA100 |
| TA1535 |
| WP2uvrA- |
| TA98 |
| TA1537 |
|
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Mean | 91 | 100 | 20 | 17 | 23 | 27 | 22 | 35 | 12 | 17 |
SD | 16.6 | 18.0 | 5.5 | 4.3 | 4.8 | 5.3 | 5.2 | 6.8 | 4 | 4.7 |
Min | 61 | 68 | 8 | 8 | 10 | 14 | 11 | 13 | 4 | 5 |
Max | 160 | 162 | 38 | 37 | 47 | 45 | 44 | 66 | 29 | 38 |
N | 939 | 742 | 912 | 718 | 685 | 521 | 946 | 749 | 918 | 718 |
Table 3c: Historical Control Data: Positive control: 2001
| TA100 |
| TA1535 |
| WP2uvrA- |
| TA98 |
| TA1537 |
|
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Mean | 505 | 1582 | 431 | 336 | 685 | 756 | 140 | 303 | 1415 | 428 |
SD | 190 | 541 | 412 | 110 | 323 | 243 | 43 | 96 | 713 | 146 |
Min | 259 | 454 | 98 | 113 | 248 | 242 | 72 | 136 | 197 | 139 |
Max | 1829 | 2738 | 2039 | 990 | 1960 | 1340 | 362 | 679 | 3616 | 792 |
N | 165 | 163 | 163 | 161 | 156 | 155 | 166 | 164 | 162 | 160 |
Table 3d: Historical Control Data: Positive control: 2002
| TA100 |
| TA1535 |
| WP2uvrA- |
| TA98 |
| TA1537 |
|
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
Mean | 460 | 1880 | 347 | 310 | 621 | 866 | 136 | 239 | 1953 | 453 |
SD | 118.2 | 594.3 | 204.0 | 119.7 | 235.5 | 350.5 | 38.2 | 74.7 | 803.1 | 145.9 |
Min | 235 | 499 | 80 | 91 | 185 | 210 | 66 | 91 | 486 | 140 |
Max | 952 | 3397 | 1385 | 810 | 1295 | 3406 | 323 | 507 | 4622 | 1365 |
N | 190 | 190 | 188 | 186 | 169 | 168 | 192 | 192 | 188 | 184 |
Applicant's summary and conclusion
- Conclusions:
- The test substance was considered to be non-mutagenic under the conditions of this Bacterial Reverse Mutation Test (Ames) that was performed in accordance with OECD TG 471.
- Executive summary:
A Bacterial Reverse Mutation Test (Ames) was performed in accordance with OECD TG 471 and under GLP conditions to determine the mutagenic potential of the test substance. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9-mix). The dose range of the main test was determined in a preliminary toxicity assay using TA100 and WP2uvrA- and was determined to be 50 to 5000 µg/plate. Both main tests were performed at 50, 150, 500, 1500, and 5000 µg/plate. 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test substance was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of the S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation. Based on the observed results, the test substance was considered to be non-mutagenic under the conditions of this test.
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