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EC number: 946-877-1 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
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- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
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- 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:
- From 14 June to 1 July, 2002
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 002
- Report date:
- 2002
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted July 21, 1997
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Acid Green 025
- IUPAC Name:
- Acid Green 025
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Salmonella typhimurium TA 98 and TA 1535 were obtained from Prof. B. Ames, Berkeley, USA. Strain TA 100 was obtained from Dr. M. Schüpbach, Hoffmann-La Roche Limited, Basel, Switzerland. Strain TA 1537 was obtained from Dr. S. Albertini, Hoffmann-La Roche Limited, Basel, Switzerland. Escherichia coli (WP2 uvrA) was obtained from the National Collection of lndustrial Bacteria, Aberdeen, Scotland.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + DMSO (10ml + 1ml) in a deep freezer at about -80 °C.
- Precultures: aliquots from frozen stocks were grown in liquid nutrient broth medium for 8 hours and then used for the experiment. The bacterial cultures were incubated in a time-temperature controlled incubater at about 37 °C.
MAINTAINANCE
- Periodically characteristic check: the strains were checked every two to six months. The presence of the rfa character was assayed by the sensitivity for crystal-violet. The deletion of the uvrB gene was demonstrated by the sensitivity for UV-light.
- Periodically 'cleansed' against high spontaneous background: yes
- Periodically ampicillin resistance check: the Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance.
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat-liver post mitochondrial supernatant (S9 fraction)
- Test concentrations with justification for top dose:
- Main experiments: 187.5, 375.0, 750.0, 1500.0 and 3000.0 µg/plate
- Vehicle / solvent:
- - Vehicle: bidistilled water.
- Test solution: on day of experiment test item was dissolved in bidistilled water after warming up to 50 °C in a water bath and strongly shaken. The test item was soluble up to the concentration of 30 mg/ml.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- cyclophosphamide
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- PLATES TEST
The following materials were mixed in a test tube and poured onto the minimal agar plates: 100 µl of test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control); 500 µl 59 mix (for test with metabolic activation) or 59 mix substitution buffer (for test without metabolic activation); 100 µl bacteria suspension (cf. test system, pre-culture of the strains), 2000 µl overlay agar.
PREINCUBATION TEST
In the pre-incubation assay 100 µl test solution,500 µl S9 mix and 100 µl bacterial suspension were mixed in a test tube and shaken at about 37 °C for 30 minutes. After pre-incubation 2.0 ml overlay agar (about 45 °C) was added to each tube and well mixed. The mixture was poured on minimal agar plates. In addition, the respective controls (solvent) and positive controls were run together with each strain.
INCUBATION :after solidification the plates were incubated upside down for at least 48 hours at 37 °C ± 2 °C in the dark.
NUMBER OF REPLICATIONS: three replicates x concentration x strain.
MAMMALIAN MICROSOMAL FRACTION S9 MIX
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male rats (HanBrl:WIST SPF). The animals were treated with Aroclor 1254, 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content of the S9 fraction was 42.5 mg/ml.
On day of experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % vlv in the mixture. Cofactors were added to the 59 mix to reach the following concentrations in the 59 mix: 8 mM MgCl2; 33 mM KCI; 5 mM Glucose-6-Phosphate; 4 mM NADP, in 100 mM sodium phosphate-buffer, pH7.4.
Before starting the experiment the S9 mix was sterile filtered and stored in a refrigerator.
PRE-EXPERIMENT FOR TOTOXICITY - range finding test
- Strains: S. typhimurium TA 100 and E. coli WP2 uvrA
- Metabolic activation: with and without
- Concentrations: 12.3, 37.0,111.1, 333.3, 1000.0 and 3000.0 µg/plate
- Incubation: plates were inverted and incubated for about 48 hours at 37 ± 2 °C in darkness. - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment.
However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- A statistical analysis was not required.
Results and discussion
Test resultsopen allclose all
- Species / strain:
- S. typhimurium, other: TA 100, TA 1535, TA 98 and TA 1537
- 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
- Species / strain:
- E. coli WP2 uvr A
- 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:
- The plates incubated with the test item showed normal background growth up to 3000 µg/plate with and without S9 mix in both experiments. No reduction in the growth of the bacterial background lawn was observed. No precipitation of the test item was visible.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any concentration level, neither in the presence nor in the absence of an metabolic activation system.
In the first mutagenicity test without metabolic activation on strain E. coliWP2 uvrA, a concentration-dependent increase in the number of revertant colonies was observed at higher concentrations. Since this effect did not reach the threshold of twice of the corresponding solvent control and could not be repeated in the second experiment, it is attributed to occasionally occurring fluctuations in bacterial growth.
ln both experiments on strain TA 98 without metabolic activation, the number of revertant colonies in the positive control were somewhat below the expected value. This effect is considered to be based upon biological irrelevant fluctuations in the number of revertant colonies and has no impact on the outcome of the study.
CONTROLS
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
PRE-EXPERIMENT FOR TOTOXICITY - range finding test
Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.
From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 3000.0 µg/plate with and without metabolic activation.
Any other information on results incl. tables
Summary of the First Mutagenicity Test - Experiment with metabolic activation
Concentration (µg/plate) | Mean | ||||
TA 100 | TA 1535 | WP2 uvrA | TA 98 | TA 1537 | |
Bidist. Water | 143 | 13 | 32 | 39 | 14 |
Test item 187.5 | 154 | 24 | 34 | 34 | 't7 |
Test item 375 | 167 | 18 | 37 | 34 | 15 |
Test item 750 | 161 | 22 | 33 | 29 | 18 |
Test item 1500 | 160 | 17 | 41 | 34 | 15 |
Test item 3000 | 150 | 19 | 32 | 38 | 16 |
2-A-anthracene 1.5 | 1803 | - | - | 1373 | 281 |
2-A-anthracene 20.0 | - | - | 947 | - | - |
CPA 200.0 | - | 272 | - | - | - |
Summary of the First Mutagenicity Test - Experiment without metabolic activation
Concentration (µg/plate) | Mean | ||||
TA 100 | TA 1535 | WP2 uvrA | TA 98 | TA 1537 | |
Bidist. Water | 135 | 14 | 24 | 17 | 15 |
Test item 187.5 | 141 | 14 | 26 | 30 | 12 |
Test item 375 | 161 | 17 | 27 | 29 | 10 |
Test item 750 | 165 | 21 | 29 | 24 | 15 |
Test item 1500 | 167 | 15 | 39 | 22 | 14 |
Test item 3000 | 158 | 17 | 41 | 24 | 12 |
2-Nitrofluorene 5.0 | - | - | - | 261 | - |
4-Nitroquinoline 2.0 | - | - | 446 | - | - |
9-A-acridine 80.0 | - | - | - | - | 962 |
Sodium azide 2.0 | 676 | 427 | - | - | - |
Summary of the Second Mutagenicity Test - Experiment with metabolic activation
Concentration (µg/plate) | Mean | ||||
TA 100 | TA 1535 | WP2 uvrA | TA 98 | TA 1537 | |
Bidist. Water | 142 | 21 | 50 | 33 | 15 |
Test item 187.5 | 163 | 21 | 50 | 40 | 16 |
Test item 375 | 155 | 18 | 58 | 42 | 27 |
Test item 750 | 137 | 20 | 55 | 32 | 26 |
Test item 1500 | 149 | 20 | 40 | 36 | 19 |
Test item 3000 | 134 | 18 | 49 | 28 | 13 |
2-A-anthracene 1.5 | 1992 | - | - | 834 | 203 |
2-A-anthracene 20.0 | - | - | 587 | - | - |
CPA 200.0 | - | 477 | - | - | - |
Summary of the Second Mutagenicity Test - Experiment without metabolic activation
Concentration (µg/plate) | Mean | ||||
TA 100 | TA 1535 | WP2 uvrA | TA 98 | TA 1537 | |
Bidist. Water | 132 | 17 | 38 | 22 | 13 |
Test item 187.5 | 143 | 21 | 46 | 29 | 13 |
Test item 375 | 125 | 17 | 38 | 34 | 14 |
Test item 750 | 140 | 19 | 43 | 31 | 18 |
Test item 1500 | 156 | 19 | 45 | 21 | 12 |
Test item 3000 | 151 | 20 | 47 | 21 | 12 |
2-Nitrofluorene 5.0 | - | - | - | 335 | - |
4-Nitroquinoline 2.0 | - | - | 314 | - | - |
9-A-acridine 80.0 | - | - | - | - | 900 |
Sodium azide 2.0 | 793 | 482 | - | - | - |
Range-finding test
Strain | TA 100 | WP2 uvrA | ||||||
Concentration (µg/plate) | Revertants per plate | Factor | Revertants per plate | Factor | ||||
-S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | |
Bidist. Water | 118 | 178 | 1.0 | 1.0 | 38 | 35 | 1.0 | 1.0 |
12.3 | 125 | 125 | 1.1 | 0.7 | 30 | 36 | 0.8 | 1.0 |
37.0 | 128 | 148 | 1.1 | 0.8 | 34 | 41 | 0.9 | 1.2 |
111.1 | 143 | 151 | 1.2 | 0.9 | 49 | 46 | 1.3 | 1.3 |
333.3 | 150 | 151 | 1.3 | 0.9 | 43 | 35 | 1.1 | 1.0 |
1000.0 | 144 | 163 | 1.2 | 0.9 | 32 | 35 | 0.8 | 1.0 |
3000.0 | 149 | 154 | 1.3 | 0.9 | 43 | 53 | 1.1 | 1.5 |
Applicant's summary and conclusion
- Conclusions:
- Under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used.
Therefore, test substance is considered to be non-mutagenic in Salmonella typhimurium and Escherichia coli reverse mutation assay. - Executive summary:
The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 100, TA 1535, TA 98, TA 1537 and E. coliWP2 uvrA. The test was performed in two independent experiments both with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The test item was dissolved in bidistilled water and tested at five concentrations: 187.5, 375.0, 750.0, 1500.0 and 3000.0 µg/plate.
In order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations used in the first experiment. The test with metabolic activation was carried out as pre-incubation assay.
Bidistilled water was selected to be the best solvent compared with the usual organic solvents. However, the limit of solubility was at the concentration of 3000 µg/plate.
Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation with the concentrations of 12.3, 37.0,1 1 1 .1 , 333.3, 1000.0 and 3000.0 µg/plate.
Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.
From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 3000.0 µg/plate with and without metabolic activation.
In the first mutagenicity test performed with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with test item at any concentrations. In the second mutagenicity test carried out with and without metabolic activation, again treatment of the tester strains with the test item did not lead to an increase in the number of revertant colonies at any concentrations. In both mutagenicity tests normal background growth was observed with all strains at all concentrations. The number of revertant colonies was not reduced.
The test item did not precipitate on the surface of the agar plates.
Negative (solvent) and positive controls were found to be within acceptable ranges.
Conclusion
Under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used.
Therefore, test substance is considered to be non-mutagenic in Salmonella typhimurium and Escherichia coli reverse mutation assay.
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