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EC number: 288-914-8 | CAS number: 85940-25-6
- 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:
- 17 July 2017 - 29 November 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
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
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.
- EC Number:
- 288-914-8
- EC Name:
- Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.
- Cas Number:
- 85940-25-6
- Molecular formula:
- Molecular formula is not available
- IUPAC Name:
- Reaction product of phenol, 4-(9H-carbazol-3-ylamino)- with sodium polysulfide, leuco derivatives
- Test material form:
- solid: particulate/powder
- Details on test material:
- Test Item: Leuco Sulfur Blue 20P
Appearance: Black powder
Constituent 1
Method
- Target gene:
- In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair.
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/-naphthoflavone-induced rats.
- Test concentrations with justification for top dose:
- 1600, 1000, 500, 250, 160, 100, 50, 16 and 5 µg/plate
- Vehicle / solvent:
- dimethyl sulfoxide (DMSO)
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-Nitro-1,2-phenylenediamine, 2-aminoanthracene
- Details on test system and experimental conditions:
- Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures.
Storage of Tester Strains
The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.
Confirmation of Phenotypes of Tester Strains
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.
Spontaneous Reversion of Tester Strains
Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Procedure for Bacterial Cultures
The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 11-13 hours in a 37 oC Benchtop Incubator Shaker.
Viability and the Cell Count of the Testing Bacterial Cultures
The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates . The viable cell number of the cultures was determined by manual colony counting.
Metabolic Activation System
The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).
Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA). - Rationale for test conditions:
- Selection of the concentration range for the initial mutation test was done on the basis of solubility test and concentration range finding test. The investigated concentration range for the confirmatory mutation test was chosen based on the results of the initial mutation test.At the concentration choice the non-toxicityof the test item and the appearance of precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye.To confirm and to investigate the reproducibility of the positive result of the initial mutation test the following nine concentration levels were investigated in the confirmatory mutation test:±S9 mix: 1600, 1000, 500, 250, 160, 100, 50, 16 and 5 µg/plate.
When evaluated by the naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains down to and including the concentration level of 500 µg/plate in the absence and down to and including the concentration level of 250 µg/plate in the presence of exogenous metabolic activation following the plate incorporation procedures.In the initial mutation test an inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain in the absence (in the concentration range of 1600-160 µg/plate) and also in the presence (at the concentrations of 1600 and 500 µg/plate) of exogenous metabolic activation. In confirmatory mutation test the inhibitory effect was indicated by absent or decreased revertant colony counts and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 160 µg/plate was considered as lowest concentration showing cytotoxicity.
The revertant colony numbers of solvent control (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. - Evaluation criteria:
- The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined visually by manual counting, and the mean values, standard deviations and the mutation rates were calculated.
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a negative response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In general, 160 µg/plate was considered as lowest concentration showing cytotoxicity.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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
- Key result
- Species / strain:
- S. typhimurium TA 100
- 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
- Key result
- 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:
- Validity of the Performed Experiments
The tester strains used in this study demonstrated the specific phenotype characteristics , were in line with the corresponding historical control data ranges , and showed the adequate strain culture titer. Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system .
Each of the investigated reference mutagens showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective solvent control in all main experimental phases and the number of revertants in most cases fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in the main experimental phases , in the tester strains. The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates showed characteristic mean numbers agreed with the actual historical control data ranges in the examined strains in both main experimental phases. Seven concentration levels were investigated in the initial mutation test and nine in the confirmatory mutation test. In the performed main experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain. All criteria for the validity of the performed experiments have therefore been met.
Controls
In the performed initial and confirmatory mutation test multiple test items were tested with reference values from the common parallel controls. In the initial and confirmatory mutation tests the revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains . In the Initial Mutation Test, in the case of Salmonella typhimurium TA98 the revertant colony numbers of 2-aminoanthracene (2AA) were above the corresponding historical control data range; however the higher counts were considered as acceptable without any effect on the final conclusion of the study. The revertant colony numbers of the untreated and ultrapure water control plates in different experimental phases were slightly higher or lower than the DMSO control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges .
In summary, the actual values of untreated, solvent and positive controls were in line with the criteria for validity of the assay.
Initial Mutation Test (Plate Incorporation Test)
In this test negative mutagenicity results were obtained in Salmonella typhimurium TA98, TA100, TA1535 and Escherichia coli WP2 uvrA strains in the absence and also in the presence of exogenous metabolic activation (±S9 mix). Unequivocal positive results were noticed following treatment with the test item in the investigated Salmonella typhimurium TA1537 strain (±S9 mix). The obtained revertant colony number increases were clearly above the corresponding historical control data range and the relevant genotoxicological threshold for being positive at the concentrations of 50, 16 and 5 µg/plate (-S9 mix) and at the concentration range of 1600-16 µg/plate (+S9 mix) (with exception of the concentration level of 500 µg/plate, where the obtained increase was above the historical control data range, but remained below the threshold for being positive). The higher revertant colony numbers (when compared to the revertant colony numbers of the solvent control) were above the corresponding historical control data range; however remained below the threshold, for being positive in S. typhimurium TA98 at the concentration range of 1600-50 µg/plate (-S9 mix). Under the experimental conditions applied, the test item induced gene mutations by frameshifts in the genome of the Salmonella typhimurium TA1537 tester strain examined. Inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain in the absence (in the concentration range of 1600-160 µg/plate) and also in the presence (at the concentrations of 1600 and 500 µg/plate) of exogenous metabolic activation. The inhibitory effect was indicated by absent or decreased revertant colony counts (compared to the revertant colony numbers of the DMSO control) and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 160 µg/plate was considered as lowest concentration showing cytotoxicity. When evaluated by the naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at 1600 and 500 µg/plate in the absence and also in the presence of exogenous metabolic activation. To confirm and to investigate the reproducibility of this positive result a confirmatory mutation test was performed with S. typhimurium TA1537 in the absence and presence of exogenous metabolic activation (±S9 mix). The strains Salmonella typhimurium TA98, TA100, TA1535 and Escherichia coli WP2 uvrA were not further investigated.
Confirmatory Mutation Test (Plate Incorporation Test)
The positive results already noticed in the initial mutation test in the investigated Salmonella typhimurium TA1537 strain (±S9 mix) were successfully confirmed. The revertant colony number increases were above the corresponding historical control data range and the relevant genotoxicological threshold for being positive at the concentrations of 100, 50, 16 and 5 µg/plate (-S9 mix) and at the concentration range of 500-50 µg/plate (+S9 mix). The revertant colony number increase was above the historical control data range and followed a dose-relationship, but remained below the threshold for being positive at 16 µg/plate (+S9 mix). All of the obtained increased tendencies followed clear dose-relationship. The inhibitory tendencies were similar to that already observed in the initial mutation test. An inhibitory effect of the test item was observed in the concentration range of 1600-160 µg/plate in the absence and at the concentrations of 1600, 1000 and 500 µg/plate in the presence of exogenous metabolic activation. The inhibitory effect was indicated by decreased revertant colony counts (compared to the revertant colony numbers of the DMSO control) and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 160 µg/plate was considered as lowest concentration showing cytotoxicity. Non-interfering test item precipitate was noticed on the plates at 1600, 1000 and 500 µg/plate in the absence and at 1600-250 µg/plate, in the presence of exogenous metabolic activation.
Any other information on results incl. tables
Summary Table of the Results of the Concentration Range Finding Test
Concentration Range Finding Test (Informatory Toxicity Test) |
|
||||||||
Concentrations (mg/plate) |
Salmonella typhimurium tester strains |
||||||||
TA 98 |
TA 100 |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
||||||
Mean values of revertants per plate and |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
|
Untreated Control |
16.3 |
0.92 |
18.3 |
0.82 |
85.7 |
0.94 |
120.0 |
1.10 |
|
DMSO Control |
17.7 |
1.00 |
22.3 |
1.00 |
90.7 |
1.00 |
108.7 |
1.00 |
|
Ultrapure Water Control |
– |
– |
– |
– |
86.0 |
1.00 |
– |
– |
|
5000 |
20.3 |
1.15 |
13.7 |
0.61 |
85.3 |
0.94 |
88.7 |
0.82 |
|
1600 |
22.0 |
1.25 |
18.7 |
0.84 |
93.0 |
1.03 |
94.0 |
0.87 |
|
500 |
16.3 |
0.92 |
23.7 |
1.06 |
110.0 |
1.21 |
105.3 |
0.97 |
|
160 |
13.0 |
0.74 |
26.7 |
1.19 |
97.7 |
1.08 |
119.7 |
1.10 |
|
50 |
14.3 |
0.81 |
29.3 |
1.31 |
96.7 |
1.07 |
113.0 |
1.04 |
|
16 |
20.0 |
1.13 |
36.7 |
1.64 |
99.7 |
1.10 |
101.7 |
0.94 |
|
5 |
20.0 |
1.13 |
33.7 |
1.51 |
92.7 |
1.02 |
104.0 |
0.96 |
|
NPD (4mg) |
193.3 |
10.94 |
– |
– |
– |
– |
– |
– |
|
SAZ (2mg) |
– |
– |
– |
– |
776.0 |
9.02 |
– |
– |
|
2AA (2mg) |
– |
– |
1066.7 |
47.76 |
– |
– |
2017.3 |
18.56 |
|
MR:Mutation
Rate; NPD:4-Nitro-1,2-phenylenediamine;SAZ:
Sodium azide;
2AA: 2-aminoanthracene
Remarks:DMSO was applied as solvent of the test item and positive control substances: NPD and 2AA and the ultrapure water was applied as solvent for the SAZ. The mutation rate of the test item, the untreated control the NPD and 2AA is given referring to the DMSO. The mutation rate of the SAZ positive control is given referring to the ultrapure water.
Summary Table of the Results of the Initial Mutation Test
Initial Mutation Test (Plate Incorporation Test) |
||||||||||||||||||||
Concentrations (mg/plate) |
Salmonella typhimuriumtester strains |
Escherichiacoli |
||||||||||||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2uvrA |
||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||||||||
Mean values of revertants per plate Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
17.3 |
0.96 |
23.3 |
0.93 |
88.7 |
1.08 |
110.7 |
1.09 |
9.7 |
1.00 |
12.3 |
0.97 |
6.3 |
0.90 |
7.0 |
0.84 |
33.0 |
1.19 |
37.7 |
1.03 |
DMSO Control |
18.0 |
1.00 |
25.0 |
1.00 |
82.3 |
1.00 |
101.3 |
1.00 |
9.7 |
1.00 |
12.7 |
1.00 |
7.0 |
1.00 |
8.3 |
1.00 |
27.7 |
1.00 |
36.7 |
1.00 |
Ultrapure Water Control |
– |
– |
– |
– |
81.7 |
1.00 |
– |
– |
8.7 |
1.00 |
– |
– |
– |
– |
– |
– |
32.0 |
1.00 |
– |
– |
1600 |
41.7 |
2.31 |
25.7 |
1.03 |
99.0 |
1.20 |
139.0 |
1.37 |
10.0 |
1.03 |
11.0 |
0.87 |
0.0 |
0.00 |
52.3 |
6.28 |
28.3 |
1.02 |
31.3 |
0.85 |
500 |
40.0 |
2.22 |
26.7 |
1.07 |
83.3 |
1.01 |
113.7 |
1.12 |
11.0 |
1.14 |
11.0 |
0.87 |
1.0 |
0.14 |
24.0 |
2.88 |
34.7 |
1.25 |
35.0 |
0.95 |
160 |
47.3 |
2.63 |
32.0 |
1.28 |
85.0 |
1.03 |
139.0 |
1.37 |
10.3 |
1.07 |
14.3 |
1.13 |
13.3 |
1.90 |
141.3 |
16.96 |
36.7 |
1.33 |
42.0 |
1.15 |
50 |
50.7 |
2.81 |
29.3 |
1.17 |
81.3 |
0.99 |
116.0 |
1.14 |
8.7 |
0.90 |
10.7 |
0.84 |
58.3 |
8.33 |
68.7 |
8.24 |
34.0 |
1.23 |
40.3 |
1.10 |
16 |
31.7 |
1.76 |
26.0 |
1.04 |
93.7 |
1.14 |
103.7 |
1.02 |
10.0 |
1.03 |
11.0 |
0.87 |
37.3 |
5.33 |
32.3 |
3.88 |
41.3 |
1.49 |
38.0 |
1.04 |
5 |
24.0 |
1.33 |
22.7 |
0.91 |
92.0 |
1.12 |
96.0 |
0.95 |
9.3 |
0.97 |
10.7 |
0.84 |
27.7 |
3.95 |
13.0 |
1.56 |
28.3 |
1.02 |
34.0 |
0.93 |
1.6 |
24.7 |
1.37 |
25.7 |
1.03 |
81.3 |
0.99 |
97.0 |
0.96 |
10.0 |
1.03 |
10.0 |
0.79 |
13.7 |
1.95 |
12.0 |
1.44 |
33.0 |
1.19 |
28.0 |
0.76 |
NPD (4mg) |
199.3 |
11.07 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2mg) |
– |
– |
– |
– |
944.0 |
11.56 |
– |
– |
1377.3 |
158.92 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
9AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
960.3 |
137.19 |
– |
– |
– |
– |
– |
– |
MMS (2mL) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
889.3 |
27.79 |
– |
– |
2AA (2mg) |
– |
– |
2757.3 |
110.29 |
– |
– |
2526.7 |
24.93 |
– |
– |
193.7 |
15.29 |
– |
– |
311.7 |
37.40 |
– |
– |
– |
– |
2AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
181.0 |
4.94 |
MR:Mutation Rate; NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene
Remarks: DMSO was applied as solvent of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as solvent for the SAZ and MMS. The mutation rate of the test item and the untreated control is given referring to the DMSO. The mutation rate of the NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of the SAZ and MMS positive control is given referring to the ultrapure water.
Summary Table of the Results of the Confirmatory Mutation Test
Confirmatory Mutation Test (Plate Incorporation Test) |
||||
Concentrations (mg/plate) |
Salmonella typhimurium |
|||
TA 1537 |
||||
-S9 |
+S9 |
|||
Mean values of revertants per plate and |
Mean |
MR |
Mean |
MR |
Untreated Control |
9.7 |
1.53 |
11.3 |
1.00 |
DMSO Control |
6.3 |
1.00 |
11.3 |
1.00 |
1600 |
10.7 |
1.68 |
21.0 |
1.85 |
1000 |
10.7 |
1.68 |
27.3 |
2.41 |
500 |
3.3 |
0.53 |
57.3 |
5.06 |
250 |
12.7 |
2.00 |
101.7 |
8.97 |
160 |
14.3 |
2.26 |
102.0 |
9.00 |
100 |
42.0 |
6.63 |
83.3 |
7.35 |
50 |
26.3 |
4.16 |
42.0 |
3.71 |
16 |
36.3 |
5.74 |
26.7 |
2.35 |
5 |
53.7 |
8.47 |
12.3 |
1.09 |
9AA (50mg) |
622.7 |
98.32 |
– |
– |
2AA (2mg) |
– |
– |
183.3 |
16.18 |
MR:Mutation Rate;9AA:9-Aminoacridine;2AA: 2-aminoanthracene
Remarks:DMSO was applied as solvent of the test item and positive control substances: 9AA and 2AA. The mutation rate of the test item, the untreated control, the 9AA and the 9AA is given referring to the DMSO.
Results of the Concentration Range Finding Test inSalmonella typhimuriumTA98
Test Item: |
Leuco Sulfur Blue 20P |
||||||||
Date of Experiment: |
May 23-25, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA98 |
||||||||
Cell count (Overnight culture): |
2.54 x 109CFU/mL |
||||||||
|
|
||||||||
Without Exogenous Metabolic Activation (-S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
19 |
14 |
16 |
16.3 |
‑ |
2.52 |
0.92 |
|
DMSO Control |
|
16 |
18 |
19 |
17.7 |
‑ |
1.53 |
1.00 |
|
5000 |
|
18 |
19 |
24 |
20.3 |
P |
3.21 |
1.15 |
|
1600 |
|
28 |
14 |
24 |
22.0 |
P |
7.21 |
1.25 |
|
500 |
|
16 |
17 |
16 |
16.3 |
SP |
0.58 |
0.92 |
|
160 |
|
13 |
12 |
14 |
13.0 |
SP |
1.00 |
0.74 |
|
50 |
|
14 |
19 |
10 |
14.3 |
‑ |
4.51 |
0.81 |
|
16 |
|
15 |
18 |
27 |
20.0 |
‑ |
6.24 |
1.13 |
|
5 |
|
24 |
22 |
14 |
20.0 |
‑ |
5.29 |
1.13 |
|
Positive reference control (NPD)(4 µg/plate) |
|
204 |
222 |
154 |
193.3 |
‑ |
35.23 |
10.94 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
17 |
24 |
14 |
18.3 |
‑ |
5.13 |
0.82 |
|
DMSO Control |
|
15 |
22 |
30 |
22.3 |
‑ |
7.51 |
1.00 |
|
5000 |
|
12 |
19 |
10 |
13.7 |
P |
4.73 |
0.61 |
|
1600 |
|
17 |
18 |
21 |
18.7 |
P |
2.08 |
0.84 |
|
500 |
|
36 |
15 |
20 |
23.7 |
SP |
10.97 |
1.06 |
|
160 |
|
26 |
33 |
21 |
26.7 |
‑ |
6.03 |
1.19 |
|
50 |
|
26 |
29 |
33 |
29.3 |
‑ |
3.51 |
1.31 |
|
16 |
|
32 |
43 |
35 |
36.7 |
‑ |
5.69 |
1.64 |
|
5 |
|
37 |
34 |
30 |
33.7 |
‑ |
3.51 |
1.51 |
|
Positive reference control (2AA)(2 µg/plate) |
|
640 |
1360 |
1200 |
1066.7 |
‑ |
378.07 |
47.76 |
|
Obs : Observation (made by naked eye) P : Precipitate
SD : Standard Deviation SP :Slight precipitate
MR : Mutation Rate ‑ : Normal background lawn development, no precipitate
NPD:4-Nitro-1,2-phenylenediamine
2AA:2-aminoanthracene
Remark: DMSO was applied as solvent of the test item and the positive control substances NPD and 2AA. The mutation rate of the test item, the untreated control the NPD and 2AA is given referring to the DMSO.
Results of the Concentration Range Finding Test inSalmonella typhimuriumTA100
Test Item: |
Leuco Sulfur Blue 20P |
||||||||
Date of Experiment: |
May 23-25, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA100 |
||||||||
Cell count (Overnight culture): |
2.41 x 109CFU/mL |
||||||||
|
|
||||||||
Without Exogenous Metabolic Activation (-S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
77 |
90 |
90 |
85.7 |
‑ |
7.51 |
0.94 |
|
DMSO Control |
|
89 |
87 |
96 |
90.7 |
‑ |
4.73 |
1.00 |
|
Ultrapure Water Control |
|
77 |
83 |
98 |
86.0 |
‑ |
10.82 |
1.00 |
|
5000 |
|
89 |
88 |
79 |
85.3 |
P |
5.51 |
0.94 |
|
1600 |
|
89 |
85 |
105 |
93.0 |
P |
10.58 |
1.03 |
|
500 |
|
117 |
101 |
112 |
110.0 |
SP |
8.19 |
1.21 |
|
160 |
|
102 |
94 |
97 |
97.7 |
SP |
4.04 |
1.08 |
|
50 |
|
106 |
84 |
100 |
96.7 |
‑ |
11.37 |
1.07 |
|
16 |
|
95 |
111 |
93 |
99.7 |
‑ |
9.87 |
1.10 |
|
5 |
|
96 |
86 |
96 |
92.7 |
‑ |
5.77 |
1.02 |
|
Positive reference control (SAZ)(2 µg/plate) |
|
648 |
600 |
1080 |
776.0 |
‑ |
264.36 |
9.02 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
121 |
109 |
130 |
120.0 |
‑ |
10.54 |
1.10 |
|
DMSO Control |
|
105 |
109 |
112 |
108.7 |
‑ |
3.51 |
1.00 |
|
5000 |
|
86 |
94 |
86 |
88.7 |
P |
4.62 |
0.82 |
|
1600 |
|
100 |
90 |
92 |
94.0 |
P |
5.29 |
0.87 |
|
500 |
|
108 |
108 |
100 |
105.3 |
SP |
4.62 |
0.97 |
|
160 |
|
121 |
118 |
120 |
119.7 |
‑ |
1.53 |
1.10 |
|
50 |
|
110 |
107 |
122 |
113.0 |
‑ |
7.94 |
1.04 |
|
16 |
|
90 |
100 |
115 |
101.7 |
‑ |
12.58 |
0.94 |
|
5 |
|
96 |
106 |
110 |
104.0 |
‑ |
7.21 |
0.96 |
|
Positive reference control (2AA)(2 µg/plate) |
|
2328 |
2008 |
1716 |
2017.3 |
‑ |
306.11 |
18.56 |
|
Obs : Observation (made by naked eye) P : Precipitate
SD : Standard Deviation SP :Slight precipitate
MR : Mutation Rate ‑ : Normal background lawn development, no precipitate
SAZ:Sodium azide
2AA:2-aminoanthracene
Remark: DMSO was applied as solvent of the test item and the positive control substance 2AA. The ultrapure water was applied as solvent of the positive control substance SAZ. The mutation rate of the test item the untreated control and 2AA is given referring to the DMSO, the mutation rate of SAZ is given referring to ultrapure water.
Results of the Initial Mutation Test inSalmonella typhimuriumTA98
Test Item: |
Leuco Sulfur Blue 20P |
||||||||
Date of Experiment: |
July 19 – 21, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA98 |
||||||||
Cell count (Overnight culture): |
1.73 x 109CFU/mL |
||||||||
|
|
||||||||
Without Exogenous Metabolic Activation (-S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
13 |
20 |
19 |
17.3 |
‑ |
3.79 |
0.96 |
|
DMSO Control |
|
16 |
18 |
20 |
18.0 |
‑ |
2.00 |
1.00 |
|
1600 |
|
41 |
38 |
46 |
41.7 |
P |
4.04 |
2.31 |
|
500 |
|
41 |
34 |
45 |
40.0 |
P |
5.57 |
2.22 |
|
160 |
|
37 |
53 |
52 |
47.3 |
‑ |
8.96 |
2.63 |
|
50 |
|
44 |
53 |
55 |
50.7 |
‑ |
5.86 |
2.81 |
|
16 |
|
37 |
27 |
31 |
31.7 |
‑ |
5.03 |
1.76 |
|
5 |
|
23 |
25 |
24 |
24.0 |
‑ |
1.00 |
1.33 |
|
1.6 |
|
29 |
29 |
16 |
24.7 |
‑ |
7.51 |
1.37 |
|
Positive reference control (NPD)(4 µg/plate) |
|
206 |
170 |
222 |
199.3 |
‑ |
26.63 |
11.07 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
22 |
25 |
23 |
23.3 |
‑ |
1.53 |
0.93 |
|
DMSO Control |
|
24 |
26 |
25 |
25.0 |
‑ |
1.00 |
1.00 |
|
1600 |
|
27 |
24 |
26 |
25.7 |
P |
1.53 |
1.03 |
|
500 |
|
37 |
18 |
25 |
26.7 |
P |
9.61 |
1.07 |
|
160 |
|
39 |
26 |
31 |
32.0 |
‑ |
6.56 |
1.28 |
|
50 |
|
31 |
26 |
31 |
29.3 |
‑ |
2.89 |
1.17 |
|
16 |
|
22 |
28 |
28 |
26.0 |
‑ |
3.46 |
1.04 |
|
5 |
|
21 |
29 |
18 |
22.7 |
‑ |
5.69 |
0.91 |
|
1.6 |
|
28 |
25 |
24 |
25.7 |
‑ |
2.08 |
1.03 |
|
Positive reference control (2AA)(2 µg/plate) |
|
3024 |
2664 |
2584 |
2757.3 |
‑ |
234.38 |
110.29 |
|
Obs : Observation (made by naked eye) P : Precipitate
SD : Standard Deviation ‑ : Normal background lawn development, no precipitate
MR : Mutation Rate
NPD:4-Nitro-1,2-phenylenediamine
2AA:2-aminoanthracene
Remark: DMSO was applied as solvent of the test item and the positive control substances NPD and 2AA. The mutation rate of the test item, the untreated control the NPD and 2AA is given referring to the DMSO.
Results of the Initial Mutation Test inSalmonella typhimuriumTA100
Test Item: |
Leuco Sulfur Blue 20P |
||||||||
Date of Experiment: |
July 19 – 21, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA100 |
||||||||
Cell count (Overnight culture): |
1.69 x 109CFU/mL |
||||||||
|
|
||||||||
Without Exogenous Metabolic Activation (-S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
98 |
86 |
82 |
88.7 |
‑ |
8.33 |
1.08 |
|
DMSO Control |
|
83 |
81 |
83 |
82.3 |
‑ |
1.15 |
1.00 |
|
Ultrapure Water Control |
|
82 |
84 |
79 |
81.7 |
‑ |
2.52 |
1.00 |
|
1600 |
|
100 |
116 |
81 |
99.0 |
P |
17.52 |
1.20 |
|
500 |
|
96 |
82 |
72 |
83.3 |
P |
12.06 |
1.01 |
|
160 |
|
91 |
83 |
81 |
85.0 |
‑ |
5.29 |
1.03 |
|
50 |
|
85 |
88 |
71 |
81.3 |
‑ |
9.07 |
0.99 |
|
16 |
|
104 |
90 |
87 |
93.7 |
‑ |
9.07 |
1.14 |
|
5 |
|
97 |
84 |
95 |
92.0 |
‑ |
7.00 |
1.12 |
|
1.6 |
|
81 |
92 |
71 |
81.3 |
‑ |
10.50 |
0.99 |
|
Positive reference control (SAZ)(2 µg/plate) |
|
872 |
1016 |
944 |
944.0 |
‑ |
72.00 |
11.56 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
105 |
96 |
131 |
110.7 |
‑ |
18.18 |
1.09 |
|
DMSO Control |
|
94 |
95 |
115 |
101.3 |
‑ |
11.85 |
1.00 |
|
1600 |
|
114 |
152 |
151 |
139.0 |
P |
21.66 |
1.37 |
|
500 |
|
115 |
103 |
123 |
113.7 |
P |
10.07 |
1.12 |
|
160 |
|
149 |
140 |
128 |
139.0 |
‑ |
10.54 |
1.37 |
|
50 |
|
111 |
111 |
126 |
116.0 |
‑ |
8.66 |
1.14 |
|
16 |
|
107 |
101 |
103 |
103.7 |
‑ |
3.06 |
1.02 |
|
5 |
|
106 |
92 |
90 |
96.0 |
‑ |
8.72 |
0.95 |
|
1.6 |
|
99 |
94 |
98 |
97.0 |
‑ |
2.65 |
0.96 |
|
Positive reference control (2AA)(2 µg/plate) |
|
2600 |
2512 |
2468 |
2526.7 |
‑ |
67.21 |
24.93 |
|
Obs : Observation (made by naked eye) P : Precipitate
SD : Standard Deviation ‑ : Normal background lawn development, no precipitate
MR : Mutation Rate
SAZ:Sodium azide
2AA:2-aminoanthracene
Remark: DMSO was applied as solvent of the test item and the positive control substance 2AA. The ultrapure water was applied as solvent of the positive control substance SAZ. The mutation rate of the test item the untreated control and 2AA is given referring to the DMSO, the mutation rate of SAZ is given referring to ultrapure water.
Results of the Initial Mutation Test inSalmonella typhimuriumTA1535
Test Item: |
Leuco Sulfur Blue 20P |
||||||||
Date of Experiment: |
July 19 – 21, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA1535 |
||||||||
Cell count (Overnight culture): |
2.09 x 109CFU/mL |
||||||||
|
|
||||||||
Without Exogenous Metabolic Activation (-S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
6 |
10 |
13 |
9.7 |
‑ |
3.51 |
1.00 |
|
DMSO Control |
|
9 |
11 |
9 |
9.7 |
‑ |
1.15 |
1.00 |
|
Ultrapure Water Control |
|
8 |
8 |
10 |
8.7 |
‑ |
1.15 |
1.00 |
|
1600 |
|
7 |
8 |
15 |
10.0 |
P |
4.36 |
1.03 |
|
500 |
|
12 |
11 |
10 |
11.0 |
P |
1.00 |
1.14 |
|
160 |
|
10 |
14 |
7 |
10.3 |
‑ |
3.51 |
1.07 |
|
50 |
|
6 |
13 |
7 |
8.7 |
‑ |
3.79 |
0.90 |
|
16 |
|
12 |
8 |
10 |
10.0 |
‑ |
2.00 |
1.03 |
|
5 |
|
10 |
11 |
7 |
9.3 |
‑ |
2.08 |
0.97 |
|
1.6 |
|
11 |
8 |
11 |
10.0 |
‑ |
1.73 |
1.03 |
|
Positive reference control (SAZ)(2 µg/plate) |
|
1320 |
1392 |
1420 |
1377.3 |
‑ |
51.59 |
158.92 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
9 |
14 |
14 |
12.3 |
‑ |
2.89 |
0.97 |
|
DMSO Control |
|
19 |
10 |
9 |
12.7 |
‑ |
5.51 |
1.00 |
|
1600 |
|
8 |
11 |
14 |
11.0 |
P |
3.00 |
0.87 |
|
500 |
|
14 |
9 |
10 |
11.0 |
P |
2.65 |
0.87 |
|
160 |
|
14 |
18 |
11 |
14.3 |
‑ |
3.51 |
1.13 |
|
50 |
|
15 |
10 |
7 |
10.7 |
‑ |
4.04 |
0.84 |
|
16 |
|
15 |
8 |
10 |
11.0 |
‑ |
3.61 |
0.87 |
|
5 |
|
11 |
12 |
9 |
10.7 |
‑ |
1.53 |
0.84 |
|
1.6 |
|
12 |
6 |
12 |
10.0 |
‑ |
3.46 |
0.79 |
|
Positive reference control (2AA)(2 µg/plate) |
|
118 |
242 |
221 |
193.7 |
‑ |
66.37 |
15.29 |
|
Obs : Observation (made by naked eye) P : Precipitate
SD : Standard Deviation ‑ : Normal background lawn development, no precipitate
MR : Mutation Rate
SAZ:Sodium azide
2AA:2-aminoanthracene
Remark: DMSO was applied as solvent of the test item and the positive control substance 2AA. The ultrapure water was applied as solvent of the positive control substance SAZ. The mutation rate of the test item the untreated control and 2AA is given referring to the DMSO, the mutation rate of SAZ is given referring to ultrapure water.
Results of the Initial Mutation Test inSalmonella typhimurium TA1537
Test Item: |
Leuco Sulfur Blue 20P |
||||||||
Date of Experiment: |
July 19 – 21, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA1537 |
||||||||
Cell count (Overnight culture): |
1.24 x 109CFU/mL |
||||||||
|
|
||||||||
Without Exogenous Metabolic Activation (-S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
5 |
6 |
8 |
6.3 |
‑ |
1.53 |
0.90 |
|
DMSO Control |
|
7 |
6 |
8 |
7.0 |
‑ |
1.00 |
1.00 |
|
1600 |
|
0 |
0 |
0 |
0.0 |
B, P |
0.00 |
0.00 |
|
500 |
|
0 |
2 |
1 |
1.0 |
B, P |
1.00 |
0.14 |
|
160 |
|
6 |
20 |
14 |
13.3 |
SB |
7.02 |
1.90 |
|
50 |
|
55 |
55 |
65 |
58.3 |
‑ |
5.77 |
8.33 |
|
16 |
|
40 |
32 |
40 |
37.3 |
‑ |
4.62 |
5.33 |
|
5 |
|
29 |
33 |
21 |
27.7 |
‑ |
6.11 |
3.95 |
|
1.6 |
|
16 |
8 |
17 |
13.7 |
‑ |
4.93 |
1.95 |
|
Positive reference control (9AA)(50 µg/plate) |
|
847 |
758 |
1276 |
960.3 |
‑ |
276.97 |
137.19 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
4 |
11 |
6 |
7.0 |
‑ |
3.61 |
0.84 |
|
DMSO Control |
|
8 |
10 |
7 |
8.3 |
‑ |
1.53 |
1.00 |
|
1600 |
|
55 |
59 |
43 |
52.3 |
SB, P |
8.33 |
6.28 |
|
500 |
|
24 |
24 |
24 |
24.0 |
SB, P |
0.00 |
2.88 |
|
160 |
|
134 |
160 |
130 |
141.3 |
‑ |
16.29 |
16.96 |
|
50 |
|
64 |
70 |
72 |
68.7 |
‑ |
4.16 |
8.24 |
|
16 |
|
29 |
31 |
37 |
32.3 |
‑ |
4.16 |
3.88 |
|
5 |
|
15 |
12 |
12 |
13.0 |
‑ |
1.73 |
1.56 |
|
1.6 |
|
16 |
10 |
10 |
12.0 |
‑ |
3.46 |
1.44 |
|
Positive reference control (2AA)(2 µg/plate) |
|
287 |
323 |
325 |
311.7 |
‑ |
21.39 |
37.40 |
|
Obs : Observation (made by naked eye) P : Precipitate
SD : Standard Deviation B :Reduced background lawn development
MR : Mutation Rate SB : Slightly reduced background lawn development
9AA:9-Aminoacridine ‑ : Normal background lawn development, no precipitate
2AA:2-aminoanthracene
Remark: DMSO was applied as solvent of the test item and the positive control substances 9AA and 2AA. The mutation rate of the test item, the untreated control the 9AA and 2AA is given referring to the DMSO.
Results of the Initial Mutation Test inEscherichia coliWP2uvrA
Test Item: |
Leuco Sulfur Blue 20P |
||||||||
Date of Experiment: |
July 19 – 21, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Escherichia coliWP2uvrA |
||||||||
Cell count (Overnight culture): |
3.67 x 109CFU/mL |
||||||||
|
|
||||||||
Without Exogenous Metabolic Activation (-S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
42 |
29 |
28 |
33.0 |
‑ |
7.81 |
1.19 |
|
DMSO Control |
|
20 |
33 |
30 |
27.7 |
‑ |
6.81 |
1.00 |
|
Ultrapure Water Control |
|
32 |
30 |
34 |
32.0 |
‑ |
2.00 |
1.00 |
|
1600 |
|
33 |
25 |
27 |
28.3 |
P |
4.16 |
1.02 |
|
500 |
|
33 |
34 |
37 |
34.7 |
P |
2.08 |
1.25 |
|
160 |
|
35 |
29 |
46 |
36.7 |
‑ |
8.62 |
1.33 |
|
50 |
|
28 |
34 |
40 |
34.0 |
‑ |
6.00 |
1.23 |
|
16 |
|
35 |
47 |
42 |
41.3 |
‑ |
6.03 |
1.49 |
|
5 |
|
23 |
29 |
33 |
28.3 |
‑ |
5.03 |
1.02 |
|
1.6 |
|
28 |
41 |
30 |
33.0 |
‑ |
7.00 |
1.19 |
|
Positive reference control (MMS)(2 µL/plate) |
|
988 |
888 |
792 |
889.3 |
‑ |
98.01 |
27.79 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
40 |
37 |
36 |
37.7 |
‑ |
2.08 |
1.03 |
|
DMSO Control |
|
36 |
30 |
44 |
36.7 |
‑ |
7.02 |
1.00 |
|
1600 |
|
33 |
30 |
31 |
31.3 |
P |
1.53 |
0.85 |
|
500 |
|
41 |
37 |
27 |
35.0 |
P |
7.21 |
0.95 |
|
160 |
|
44 |
38 |
44 |
42.0 |
‑ |
3.46 |
1.15 |
|
50 |
|
47 |
41 |
33 |
40.3 |
‑ |
7.02 |
1.10 |
|
16 |
|
36 |
38 |
40 |
38.0 |
‑ |
2.00 |
1.04 |
|
5 |
|
32 |
34 |
36 |
34.0 |
‑ |
2.00 |
0.93 |
|
1.6 |
|
22 |
34 |
28 |
28.0 |
‑ |
6.00 |
0.76 |
|
Positive reference control (2AA)(50 µg/plate) |
|
184 |
169 |
190 |
181.0 |
‑ |
10.82 |
4.94 |
|
Obs : Observation (made by naked eye) P : Precipitate
SD : Standard Deviation ‑ : Normal background lawn development, no precipitate
MR : Mutation Rate
MMS:Methyl methanesulfonate
2AA:2-aminoanthracene
Remark: DMSO was applied as solvent of the test item and the positive control substance 2AA. The ultrapure water was applied as solvent of the positive control substance MMS. The mutation rate of the test item the untreated control and 2AA is given referring to the DMSO, the mutation rate of MMS is given referring to ultrapure water.
Results of the Confirmatory Mutation Test inSalmonella typhimuriumTA1537
Test Item: |
Leuco Sulfur Blue 20P |
||||||||
Date of Experiment: |
October 18 – 20, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA1537 |
||||||||
Cell count (Overnight culture): |
1.67 x 109CFU/mL |
||||||||
|
|
||||||||
Without Exogenous Metabolic Activation (-S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
14 |
6 |
9 |
9.7 |
‑ |
4.04 |
1.53 |
|
DMSO Control |
|
4 |
11 |
4 |
6.3 |
‑ |
4.04 |
1.00 |
|
1600 |
|
11 |
6 |
15 |
10.7 |
B, P |
4.51 |
1.68 |
|
1000 |
|
6 |
13 |
13 |
10.7 |
B, P |
4.04 |
1.68 |
|
500 |
|
0 |
10 |
0 |
3.3 |
B, SP |
5.77 |
0.53 |
|
250 |
|
10 |
15 |
13 |
12.7 |
B |
2.52 |
2.00 |
|
160 |
|
18 |
11 |
14 |
14.3 |
SB |
3.51 |
2.26 |
|
100 |
|
34 |
44 |
48 |
42.0 |
‑ |
7.21 |
6.63 |
|
50 |
|
30 |
27 |
22 |
26.3 |
‑ |
4.04 |
4.16 |
|
16 |
|
39 |
33 |
37 |
36.3 |
‑ |
3.06 |
5.74 |
|
5 |
|
51 |
56 |
54 |
53.7 |
‑ |
2.52 |
8.47 |
|
Positive reference control (9AA)(50 µg/plate) |
|
512 |
636 |
720 |
622.7 |
‑ |
104.64 |
98.32 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
13 |
13 |
8 |
11.3 |
‑ |
2.89 |
1.00 |
|
DMSO Control |
|
8 |
17 |
9 |
11.3 |
‑ |
4.93 |
1.00 |
|
1600 |
|
19 |
28 |
16 |
21.0 |
SB, P |
6.24 |
1.85 |
|
1000 |
|
24 |
29 |
29 |
27.3 |
SB, P |
2.89 |
2.41 |
|
500 |
|
68 |
42 |
62 |
57.3 |
SB, P |
13.61 |
5.06 |
|
250 |
|
98 |
111 |
96 |
101.7 |
SP |
8.14 |
8.97 |
|
160 |
|
96 |
104 |
106 |
102.0 |
‑ |
5.29 |
9.00 |
|
100 |
|
105 |
55 |
90 |
83.3 |
‑ |
25.66 |
7.35 |
|
50 |
|
38 |
47 |
41 |
42.0 |
‑ |
4.58 |
3.71 |
|
16 |
|
24 |
24 |
32 |
26.7 |
‑ |
4.62 |
2.35 |
|
5 |
|
14 |
12 |
11 |
12.3 |
‑ |
1.53 |
1.09 |
|
Positive reference control (2AA)(2 µg/plate) |
|
202 |
164 |
184 |
183.3 |
‑ |
19.01 |
16.18 |
|
Obs : Observation (made by naked eye) P :Precipitate
SD : Standard Deviation SP :Slight precipitate
MR : Mutation Rate B :Reduced background lawn development
9AA:9-Aminoacridine SB :Slightly reduced background lawn development
2AA:2-aminoanthracene ‑ : Normal background lawn development, no precipitate
Remark: DMSO was applied as solvent of the test item and the positive control substances 9AA and 2AA. The mutation rate of the test item, the untreated control the 9AA and 2AA is given referring to the DMSO.
Historical Control Values for Revertants/Plate (for the Period of 2008-2016)
|
Bacterial strains |
||||||
Historical control data of untreated control |
‑S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
21.0 |
105.0 |
10.5 |
8.1 |
25.4 |
||
SD |
3.7 |
25.7 |
1.4 |
2.3 |
5.2 |
||
Minimum |
9 |
66 |
3 |
2 |
11 |
||
Maximum |
39 |
155 |
23 |
19 |
45 |
||
n |
226 |
236 |
216 |
214 |
215 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
27.5 |
117.1 |
11.8 |
9.0 |
33.9 |
||
SD |
4.3 |
18.1 |
1.4 |
1.9 |
5.2 |
||
Minimum |
12 |
75 |
4 |
2 |
17 |
||
Maximum |
46 |
166 |
23 |
20 |
56 |
||
n |
226 |
236 |
216 |
214 |
215 |
||
|
Bacterial strains |
||||||
Historical control data of DMSO control |
‑S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
20.4 |
100.1 |
10.3 |
7.9 |
24.7 |
||
SD |
3.6 |
24.8 |
1.3 |
2.4 |
4.6 |
||
Minimum |
10 |
64 |
3 |
2 |
11 |
||
Maximum |
38 |
147 |
23 |
20 |
45 |
||
n |
226 |
236 |
216 |
214 |
215 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
26.5 |
113.8 |
11.8 |
8.8 |
33.7 |
||
SD |
4.1 |
18.3 |
1.5 |
1.9 |
5.0 |
||
Minimum |
15 |
71 |
3 |
3 |
16 |
||
Maximum |
47 |
162 |
25 |
20 |
57 |
||
n |
226 |
236 |
216 |
214 |
215 |
||
|
Bacterial strains |
||||||
Historical control data of Water control |
‑S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
21.9 |
104.7 |
10.5 |
7.6 |
26.1 |
||
SD |
3.7 |
25.9 |
1.5 |
2.2 |
5.5 |
||
Minimum |
12 |
68 |
3 |
2 |
12 |
||
Maximum |
35 |
154 |
24 |
16 |
48 |
||
n |
89 |
236 |
216 |
89 |
215 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
27.4 |
117.3 |
11.4 |
8.7 |
34.9 |
||
SD |
4.0 |
18.5 |
1.3 |
2.2 |
4.9 |
||
Minimum |
15 |
83 |
4 |
3 |
18 |
||
Maximum |
43 |
167 |
22 |
16 |
57 |
||
n |
89 |
152 |
149 |
89 |
148 |
Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2uvrA
SD: Standard deviation; DMSO: Dimethyl sulfoxide; n: number of studies
Historical Control Values for Revertants/Plate (for the Period of 2008-2016) (continued)
|
Bacterial strains |
||||||
Historical control data of positive controls |
‑S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Average |
260.1 |
977.2 |
847.3 |
478.6 |
724.5 |
||
SD |
31.8 |
150.6 |
126.3 |
104.5 |
65.0 |
||
Minimum |
123 |
521 |
359 |
110 |
320 |
||
Maximum |
664 |
1970 |
1855 |
1601 |
1313 |
||
n |
226 |
236 |
216 |
214 |
215 |
||
+S9 |
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Average |
1222.7 |
1436.4 |
164.1 |
147.0 |
257.7 |
||
SD |
274.9 |
318.3 |
33.1 |
20.1 |
72.5 |
||
Minimum |
386 |
583 |
85 |
69 |
140 |
||
Maximum |
2676 |
2988 |
498 |
399 |
477 |
||
n |
226 |
236 |
216 |
214 |
215 |
Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535,
TA1537;E. coli:Escherichia coliWP2uvrA
SD: Standard deviation; DMSO: Dimethyl sulfoxide; n: number of studies
Applicant's summary and conclusion
- Conclusions:
- The test item showed a mutagenic activity on Salmonella typhimurium TA1537 carrying frameshift mutation in the absence and presence of exogenous metabolic activation, under the test conditions used in this study.
- Executive summary:
The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The study included preliminary solubility tests, preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (repeated plate incorporation test). The initial mutation test was carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. The confirmatory mutation test was carried out using Salmonella typhimurium TA1537. Based on the results of the solubility test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content (92.5 %) was made in the experiments. Based on the results of the preliminary concentration range finding test (informatory toxicity test) the following concentrations of the test item were prepared and investigated in the initial mutation test: ±S9 mix:1600; 500; 160; 50; 16; 5 and 1.6 µg/plate. The selection of the concentration range was based on the recommendations in OECD 471 guideline. At the concentration choice the non-toxicity of the test item and the appearance of precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye. To confirm and to investigate the reproducibility of the positive result of the initial mutation test the following nine concentration levels were investigated in the confirmatory mutation test: ±S9 mix: 1600, 1000, 500, 250, 160, 100, 50, 16 and 5 µg/plate.
When evaluated by the naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains down to and including the concentration level of 500 µg/plate in the absence and down to and including the concentration level of 250 µg/plate in the presence of exogenous metabolic activation following the plate incorporation procedures. In the initial mutation test an inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain in the absence (in the concentration range of 1600-160 µg/plate) and also in the presence (at the concentrations of 1600 and 500 µg/plate) of exogenous metabolic activation. The confirmatory mutation test repeated the initial mutation test results. In both experimental phases the inhibitory effect was indicated by absent or decreased revertant colony counts (compared to the revertant colony numbers of the DMSO control) and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 160 µg/plate was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of solvent control (DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected biologically relevant increases (more than 3-fold increase)in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges (or even were above the historical control data range), thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. Unequivocal positive results, confirmed by a repeat of the experiment, were noticed following the plate incorporation procedures (initial and confirmatory mutation tests) in the investigated Salmonella typhimurium TA1537 strain (±S9 Mix). The obtained revertant colony number increases were clearly above the corresponding historical control data range and the relevant genotoxicological threshold for being positive in both experiments at the concentration range of 50‑5 µg/plate (-S9 mix) and at the concentrations range of 160-50 µg/plate (+S9 mix). The obtained increased tendencies followed clear dose-relationship. Under the experimental conditions applied, the test item induced gene mutations by frameshifts in the genome of the Salmonella typhimurium TA1537 tester strain examined. In conclusion, the test item showed a mutagenic activity on Salmonella typhimurium TA1537 carrying frameshift mutation in the absence and presence of exogenous metabolic activation, under the test conditions used in this study.
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