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EC number: 277-459-0 | CAS number: 73398-89-7
- 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
- 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
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- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
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- Endpoint summary
- Stability
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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
- 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:
- 03 July 2016 to 20 July 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 3,6-bis(diethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthylium tetrachlorozincate
- EC Number:
- 277-459-0
- EC Name:
- 3,6-bis(diethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthylium tetrachlorozincate
- Cas Number:
- 73398-89-7
- Molecular formula:
- C29H33N2O3.1/2Cl4Zn
- IUPAC Name:
- bis{3,6-bis(diethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthenium} tetrachlorozincate(2-)
- Test material form:
- solid: particulate/powder
- Details on test material:
- - Appearance: Greenish powder
- Storage conditions of test material: At room temperature
- Stable until: 10 June 2021 (expiry date)
Constituent 1
Method
- Target gene:
- - Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix (rat liver S9-mix induced by Aroclor 1254)
- Test concentrations with justification for top dose:
- - Dose range finding study (TA100 and WP2uvrA only): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 1 (TA1535, TA1537 and TA98): 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate (absence and presence of S9-mix)
- Experiment 2 (Salmonella strains): 4.7, 8.4, 15, 27, 48, 86 and 154 μg/plate (absence of S9-mix)
- Experiment 2 (Salmonella strains): 8.4, 15, 27, 48, 86 and 154 μg/plate (presence of S9-mix)
- Experiment 2 (WP2uvrA): 4.7, 8.4, 15, 27, 48, 86, 154 and 275 μg/plate (absence of S9-mix)
- Experiment 2 (WP2uvrA): 8.4, 15, 27, 48, 86, 154 and 275 μg/plate (presence of S9-mix) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Chosen following a solubility test
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: ICR-191and 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DOSE RANGE FINDING TEST/ MUTATION ASSAY
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5 % (v/v) S9-mix and reported as part of the first mutation experiment. The highest concentration of test material used in the subsequent mutation assay was the level at which the test material inhibited bacterial growth.
MUTATION ASSAY
At least five different doses (increasing with approximately half-log steps) of the test material were tested in each strain both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test material was tested both in the absence and presence of 10 % (v/v) S9-mix in all tester strains.
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
NUMBER OF REPLICATIONS: Testing was performed in triplicate
COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. - Evaluation criteria:
- ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the testing facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
DATA EVALUATION
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment. - Statistics:
- No formal hypothesis testing was done.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium, other: TA1535, TA1537 and TA98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- DOSE RANGE FINDING TEST/FIRST MUTATION EXPERIMENT
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain.
- Toxicity: Cytotoxicity, as evidenced by a reduction in the bacterial background lawn and/or decrease in the number of revertants was observed in all tester strains.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested. Although at a concentration of 5.4 μg/plate in tester strain TA100 (presence of S9-mix), the number of revertants was above the historical control data range, the increase was only 1.7-fold and therefore did not meet the criteria of a positive response.
MUTATION EXPERIMENT 2
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period.
- Toxicity: In the second mutation assay, cytotoxicity, as evidenced by a reduction in the bacterial background lawn and/or decrease in the number of revertants was observed in all tester strains with the exception of tester strain WP2uvrA.
- Mutagenicity: In the second mutation assay, no increase in the number of revertants was observed upon treatment with test material in tester strains TA1537, TA98 and WP2uvrA. In tester strain TA1535 in the absence of S9-mix an up to 4.4-fold increase was observed. In addition in tester strain TA100 up to 2.1- and 2.0-fold increases were observed in the absence and presence of S9-mix, respectively. In addition, the number of revertants (tester strain TA100) was outside the historical control data ranges.
DISCUSSION
Increases in the number of revertants were observed in tester strain TA100 (both mutation assays) and in tester strain TA1535 in the absence of S9-mix in the second mutation assay only. Although the 4.4-fold increase in TA1535 showed no dose relation and was not outside the historical control data range, the increases in tester strain TA100 were 2-fold or more and outside the historical control data range in the second mutation assay. Therefore these increases were considered biologically relevant. No increase in the number of revertants was observed upon treatment with the test material in tester strains TA1537, TA98 and WP2uvrA.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Any other information on results incl. tables
Table 1: Dose Range-finder and Experiment 1 (Plate incorporation assay 5 % S9)
+/- S9 Mix |
Concentration (µg/plate) |
Mean number of colonies/plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
- |
PC DMSO 0.55 1.7 5.4 17 52 164 512 1600 5000 |
1002 95 - 84 147 115 31 n m 0 a 0 a 0 a NP |
887 8 11 6 21 23 23 n 0 s NP - - - |
1483 35 - 32 32 32 22 8 n 0 a 0 a 0 a NP |
1334 15 11 16 21 16 8 0 n NP - - - |
692 5 5 6 5 10 n 0 s 0 a NP - - - |
+ |
PC DMSO 0.55 1.7 5.4 17 52 164 512 1600 5000 |
1108 108 - 95 184 143 113 n 0 m 0 a 0 a 0 a NP |
195 10 9 15 28 21 16 n 1 s NP - - - |
424 39 - 32 44 39 32 21 n 0 a 0 a 0 a NP |
1396 19 15 23 27 26 23 0 n NP - - - |
655 8 6 5 8 9 2 n e NP MC - - - |
Mean number of revertant colonies/3 replicate plates
PC = Positive control
MC = Microcolonies
NP = No precipitate
a = Bacterial background lawn absent
e = Bacterial background lawn extremely reduced
m = Bacterial background lawn moderately reduced
n = Normal bacterial background lawn
s = Bacterial background lawn slightly reduced
Table 2: Experiment 2 (Plate incorporation assay 10 % S9)
+/- S9 Mix |
Concentration (µg/plate) |
Mean number of colonies/plate |
||||
Base-pair Substitution Type |
Frameshift Type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
- |
PC DMSO 4.7 8.4 15 27 48 86 154 275 |
868 91 151 117 160 188 63 n 2 m 1 m NP - |
921 8* 22 19 31 35 21 n 21 s 2 s NP - |
1201 28 - - 21 20 16 16 18 17 n NP |
1372 15 21 18 21 20 22 9 1 n NP - |
1077 7 6 7 5 6 4 n 3 s 3 m NP - |
+ |
PC DMSO 8.4 15 27 48 86 154 275 |
709 91 122 150 179 111 n 86 s 1 m NP - |
150 11 22 29 29 22 n 8 s 3 m NP - |
350 45 - 34 28 36 22 19 23 n NP |
380 24 24 28 36 24 19 10 n NP - |
127 9 8 8 8 4 n 6 s 1 m NP - |
Mean number of revertant colonies/3 replicate plates
PC = Positive control
NP = No precipitate
m = Bacterial background lawn moderately reduced
n = Normal bacterial background lawn
s = Bacterial background lawn slightly reduced
*Plate infected, mean of 2 plates
Applicant's summary and conclusion
- Conclusions:
- Under the conditions of the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.
- Executive summary:
The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.
The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).
In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn was observed in both strains. Results were reported as part of the first mutation assay.
Based on the results of the dose range finding test, the test material was tested in the first mutation assay at a concentration range of 0.55 to 164 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment of the assay with additional parameters, the test material was tested up to a dose level of 154 μg/plate in the absence and presence of 10 % (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and up to a dose level of 275 μg/plate in tester strain WP2uvrA. Cytotoxicity, as evidenced by a reduction in the bacterial background lawn and/or decrease in the number of revertants, was observed in all tester strains with the exception of WP2uvrA in the second mutation assay.
Increases in the number of revertants were observed in tester strain TA100 (both mutation assays) and in tester strain TA1535 in the absence of S9-mix in the second mutation assay only. Although the 4.4-fold increase in TA1535 showed no dose relation and was not outside the historical control data range, the increases in tester strain TA100 were 2-fold or more and outside the historical control data range in the second mutation assay. Therefore these increases were considered biologically relevant. No increase in the number of revertants was observed upon treatment with the test material in tester strains TA1537, TA98 and WP2uvrA.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Under the conditions of the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.
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