Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 291-833-0 | CAS number: 90480-94-7
- 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
- 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:
- 20 October 2017 - 10 January 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
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-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))
- EC Number:
- 291-833-0
- EC Name:
- Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))
- Cas Number:
- 90480-94-7
- Molecular formula:
- not applicable
- IUPAC Name:
- Reaction product of 4-aminophenol with 4-(2-naphthalenylamino)phenol and sodium polysulfide
- Test material form:
- solid
- Details on test material:
- Test item: Leuco Sulphur Black 11
Appearance: black solid
CAS No: 90480-94-7
EC No: 291-833-0
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:
- 5000; 1600; 500; 160; 50 and 16 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:
- Origin of the Bacterial Strains
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 10-13 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:
- Justification of concentrations:
Selection of the concentration range for the initial mutation test was done on the basis of the referred OECD Guideline 471, on the basis of the solubility test
and concentration range finding test (informatory toxicity 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-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 same concentration levels were investigated
in
the confirmatory mutation test:
±S9 mix: 5000; 1600; 500; 160; 50 and 16 µg/plate.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98,
TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
In the informatory toxicity test the revertant colony numbers of the solvent control plates with and without S9 mix were in line with the corresponding
historical control data ranges. The positive control treatments showed the expected biological relevant increases in induced revertant colonies in both tester
strains. - 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 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- 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:
- positive
- 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 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
- 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:
- 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 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 concentration range finding test and six concentration levels were investigated in the initial mutation test and 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. 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 the Salmonella typhimurium TA98, TA1537 and Escherichia coli WP2 uvrA strains in the absence and also in the presence of an exogenous metabolic activation system (±S9 mix).Unequivocal positive results were noticed following treatment with the test item in the investigated Salmonella typhimurium TA100 and TA1535 strains (±S9 mix). The obtained revertant colony number increases were clearly above the corresponding historical control data ranges and the relevant genotoxicological threshold for being positive in TA100 at the concentrations of 5000 and 1600 µg/plate (±S9 mix); in TA1535 in the concentration range of 5000-500 µg/plate (±S9 mix). The obtained increased tendencies followed clear dose-relationship. Furthermore significantly increased revertant colony numbers (above the corresponding historical control data ranges; however below the genotoxicological threshold for being positive) were noticed in Escherichia coli WP2 uvrA (±S9 mix). The increased revertant colony numbers were above the corresponding historical control data range; however below the genotoxicological threshold for being positive in S. typhimurium TA1535 at 160 µg/plate (-S9 mix), in E. coli WP2 uvrA 5000, 1600 and 500 µg/plate (-S9 mix), and at 5000 and 1600 µg/plate (+S9 mix); furthermore in S. typhimurium TA1537 at 5000 µg/plate (+S9 mix). The obtained increased tendencies in E. coli WP2 uvrA followed clear dose-relationship. No substantial increases were observed in revertant colony numbers of S. typhimurium TA98 (±S9 mix) and (with exception of the unique increase at 5000 µg/plate (+S9 mix)) S. typhimurium TA1537 (±S9 mix). In this test inhibitory, cytotoxic effects of the test item were not observed. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) were considered to be within the biological variability range of the applied test system. When evaluated by naked eye, non-interfering test item precipitate (test item particles) was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 µg/plate in the absence and presence of an exogenous metabolic activation (±S9 mix).
To confirm and to investigate the reproducibility of this positive result a confirmatory mutation test was performed with S. typhimurium TA100 and TA1535 strains in the absence and presence of exogenous metabolic activation (±S9 mix). The strains Salmonella typhimurium TA98, TA1537 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 TA100 and TA1535 strains (±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 in strain TA100 at the highest examined concentration of 5000 µg/plate (±S9 mix); and in strain TA1535 in the concentration range of 5000-500 µg/plate (±S9 mix). The obtained increased tendencies, especially in the latter case, followed a clear dose-relationship.The increased revertant colony numbers remained below the corresponding historical control data range and below the threshold for being positive; however followed a dose-relationship in Salmonella typhimurium TA100, at 1600 µg/plate (±S9 mix) and in TA1535 at 160 and 50 µg/plate (±S9 mix). Similarly to the initial mutation test results, in the confirmatory mutation test an inhibitory, cytotoxic effect of the test item was not observed. When evaluated by naked eye, non-interfering test item precipitate (test item particles) was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 µg/plate in the absence and presence of an exogenous metabolic activation (±S9 mix).
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 typhimuriumtester 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 |
20.7 |
1.00 |
26.3 |
1.05 |
86.3 |
1.10 |
105.0 |
1.16 |
|
DMSO Control |
20.7 |
1.00 |
25.0 |
1.00 |
78.7 |
1.00 |
90.3 |
1.00 |
|
Ultrapure Water Control |
– |
– |
– |
– |
84.7 |
1.00 |
– |
– |
|
5000 |
21.3 |
1.03 |
16.7 |
0.67 |
120.3 |
1.53 |
123.3 |
1.37 |
|
1600 |
16.3 |
0.79 |
15.0 |
0.60 |
82.7 |
1.05 |
92.0 |
1.02 |
|
500 |
17.7 |
0.85 |
16.0 |
0.64 |
77.3 |
0.98 |
85.0 |
0.94 |
|
160 |
20.3 |
0.98 |
22.7 |
0.91 |
67.7 |
0.86 |
82.3 |
0.91 |
|
50 |
10.0 |
0.48 |
25.7 |
1.03 |
70.7 |
0.90 |
77.0 |
0.85 |
|
16 |
18.7 |
0.90 |
22.3 |
0.89 |
82.0 |
1.04 |
78.0 |
0.86 |
|
5 |
19.0 |
0.92 |
19.7 |
0.79 |
77.3 |
0.98 |
98.0 |
1.08 |
|
NPD (4mg) |
223.0 |
10.79 |
– |
– |
– |
– |
– |
– |
|
SAZ (2mg) |
– |
– |
– |
– |
949.3 |
11.21 |
– |
– |
|
2AA (2mg) |
– |
– |
1421.3 |
56.85 |
– |
– |
2406.7 |
26.64 |
|
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 |
29.0 |
1.67 |
28.0 |
0.97 |
92.3 |
1.26 |
88.7 |
1.09 |
10.0 |
0.79 |
11.3 |
1.21 |
12.3 |
1.16 |
9.7 |
0.88 |
29.7 |
0.96 |
42.0 |
0.85 |
DMSO Control |
17.3 |
1.00 |
29.0 |
1.00 |
73.0 |
1.00 |
81.0 |
1.00 |
12.7 |
1.00 |
9.3 |
1.00 |
10.7 |
1.00 |
11.0 |
1.00 |
31.0 |
1.00 |
49.3 |
1.00 |
Ultrapure Water Control |
– |
– |
– |
– |
81.0 |
1.00 |
– |
– |
9.7 |
1.00 |
– |
– |
– |
– |
– |
– |
45.7 |
1.00 |
– |
– |
5000 |
17.3 |
1.00 |
25.7 |
0.89 |
207.3 |
2.84 |
252.3 |
3.12 |
297.3 |
23.47 |
213.7 |
22.89 |
13.7 |
1.28 |
20.7 |
1.88 |
84.0 |
2.71 |
76.7 |
1.55 |
1600 |
21.3 |
1.23 |
31.0 |
1.07 |
147.7 |
2.02 |
189.0 |
2.33 |
163.0 |
12.87 |
128.3 |
13.75 |
14.7 |
1.38 |
8.0 |
0.73 |
74.3 |
2.40 |
65.3 |
1.32 |
500 |
23.7 |
1.37 |
28.0 |
0.97 |
115.7 |
1.58 |
123.3 |
1.52 |
62.3 |
4.92 |
52.3 |
5.61 |
13.0 |
1.22 |
11.3 |
1.03 |
50.0 |
1.61 |
43.3 |
0.88 |
160 |
21.7 |
1.25 |
32.7 |
1.13 |
107.3 |
1.47 |
110.3 |
1.36 |
33.3 |
2.63 |
18.7 |
2.00 |
14.3 |
1.34 |
13.7 |
1.24 |
39.7 |
1.28 |
37.3 |
0.76 |
50 |
19.0 |
1.10 |
31.3 |
1.08 |
100.7 |
1.38 |
94.0 |
1.16 |
14.0 |
1.11 |
13.0 |
1.39 |
11.0 |
1.03 |
11.3 |
1.03 |
41.3 |
1.33 |
40.0 |
0.81 |
16 |
25.0 |
1.44 |
32.3 |
1.11 |
104.3 |
1.43 |
108.0 |
1.33 |
17.3 |
1.37 |
11.7 |
1.25 |
10.3 |
0.97 |
11.7 |
1.06 |
30.0 |
0.97 |
47.7 |
0.97 |
NPD (4mg) |
371.3 |
21.42 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2mg) |
– |
– |
– |
– |
1058.7 |
13.07 |
– |
– |
1088.7 |
112.62 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
9AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
925.3 |
86.75 |
– |
– |
– |
– |
– |
– |
MMS (2mL) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
575.3 |
12.60 |
– |
– |
2AA (2mg) |
– |
– |
1718.7 |
59.26 |
– |
– |
1952.0 |
24.10 |
– |
– |
202.0 |
21.64 |
– |
– |
188.0 |
17.09 |
– |
– |
– |
– |
2AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
244.7 |
4.96 |
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 typhimuriumtester strains |
|||||||
TA 100 |
TA 1535 |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
|||||
Mean values of revertants per plate Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
96.3 |
1.08 |
101.3 |
0.97 |
8.7 |
0.84 |
12.7 |
1.19 |
DMSO Control |
89.0 |
1.00 |
104.3 |
1.00 |
10.3 |
1.00 |
10.7 |
1.00 |
Ultrapure Water Control |
92.7 |
1.00 |
– |
– |
9.0 |
1.00 |
– |
– |
5000 |
224.7 |
2.52 |
261.0 |
2.50 |
185.3 |
17.94 |
248.3 |
23.28 |
1600 |
140.0 |
1.57 |
161.3 |
1.55 |
84.7 |
8.19 |
100.0 |
9.38 |
500 |
96.0 |
1.08 |
110.0 |
1.05 |
41.7 |
4.03 |
46.0 |
4.31 |
160 |
86.0 |
0.97 |
95.3 |
0.91 |
15.3 |
1.48 |
20.0 |
1.88 |
50 |
84.0 |
0.94 |
94.3 |
0.90 |
13.7 |
1.32 |
17.3 |
1.63 |
16 |
97.0 |
1.09 |
91.3 |
0.88 |
11.0 |
1.06 |
11.7 |
1.09 |
SAZ (2mg) |
1173.3 |
12.66 |
– |
– |
880.0 |
97.78 |
– |
– |
2AA (2mg) |
– |
– |
1834.7 |
17.58 |
– |
– |
256.7 |
24.06 |
MR:Mutation Rate;SAZ: Sodium azide;2AA: 2-aminoanthracene
Remarks: DMSO was applied as solvent of the test item and positive control substance: 2AA and the ultrapure water was applied as solvent for the SAZ. The mutation rate of the test item, the untreated control, and the 2AA is given referring to the DMSO, the mutation rate of the SAZ positive control is given referring to the ultrapure water.
Results of the Concentration Range Finding Test inSalmonella typhimuriumTA98
Test Item: |
Leuco Sulphur Black 11 |
||||||||
Date of Experiment: |
July 26-28, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA98 |
||||||||
Cell count (Overnight culture): |
1.10 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 |
|
21 |
20 |
21 |
20.7 |
‑ |
0.58 |
1.00 |
|
DMSO Control |
|
19 |
23 |
20 |
20.7 |
‑ |
2.08 |
1.00 |
|
5000 |
|
23 |
19 |
22 |
21.3 |
P |
2.08 |
1.03 |
|
1600 |
|
13 |
21 |
15 |
16.3 |
SP |
4.16 |
0.79 |
|
500 |
|
17 |
18 |
18 |
17.7 |
‑ |
0.58 |
0.85 |
|
160 |
|
18 |
24 |
19 |
20.3 |
‑ |
3.21 |
0.98 |
|
50 |
|
10 |
10 |
10 |
10.0 |
‑ |
0.00 |
0.48 |
|
16 |
|
21 |
17 |
18 |
18.7 |
‑ |
2.08 |
0.90 |
|
5 |
|
21 |
14 |
22 |
19.0 |
‑ |
4.36 |
0.92 |
|
Positive reference control (NPD)(4 µg/plate) |
|
245 |
203 |
221 |
223.0 |
‑ |
21.07 |
10.79 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
24 |
28 |
27 |
26.3 |
‑ |
2.08 |
1.05 |
|
DMSO Control |
|
23 |
20 |
32 |
25.0 |
‑ |
6.24 |
1.00 |
|
5000 |
|
16 |
14 |
20 |
16.7 |
P |
3.06 |
0.67 |
|
1600 |
|
17 |
12 |
16 |
15.0 |
SP |
2.65 |
0.60 |
|
500 |
|
15 |
15 |
18 |
16.0 |
‑ |
1.73 |
0.64 |
|
160 |
|
23 |
21 |
24 |
22.7 |
‑ |
1.53 |
0.91 |
|
50 |
|
31 |
24 |
22 |
25.7 |
‑ |
4.73 |
1.03 |
|
16 |
|
22 |
22 |
23 |
22.3 |
‑ |
0.58 |
0.89 |
|
5 |
|
19 |
22 |
18 |
19.7 |
‑ |
2.08 |
0.79 |
|
Positive reference control (2AA)(2 µg/plate) |
|
1712 |
1416 |
1136 |
1421.3 |
‑ |
288.04 |
56.85 |
|
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 Sulphur Black 11 |
||||||||
Date of Experiment: |
July 26-28, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA100 |
||||||||
Cell count (Overnight culture): |
1.47 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 |
|
87 |
82 |
90 |
86.3 |
‑ |
4.04 |
1.10 |
|
DMSO Control |
|
84 |
69 |
83 |
78.7 |
‑ |
8.39 |
1.00 |
|
Ultrapure Water Control |
|
87 |
80 |
87 |
84.7 |
‑ |
4.04 |
1.00 |
|
5000 |
|
103 |
130 |
128 |
120.3 |
P |
15.04 |
1.53 |
|
1600 |
|
77 |
83 |
88 |
82.7 |
SP |
5.51 |
1.05 |
|
500 |
|
73 |
76 |
83 |
77.3 |
‑ |
5.13 |
0.98 |
|
160 |
|
63 |
66 |
74 |
67.7 |
‑ |
5.69 |
0.86 |
|
50 |
|
74 |
84 |
54 |
70.7 |
‑ |
15.28 |
0.90 |
|
16 |
|
77 |
73 |
96 |
82.0 |
‑ |
12.29 |
1.04 |
|
5 |
|
90 |
73 |
69 |
77.3 |
‑ |
11.15 |
0.98 |
|
Positive reference control (SAZ)(2 µg/plate) |
|
928 |
976 |
944 |
949.3 |
‑ |
24.44 |
11.21 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
95 |
113 |
107 |
105.0 |
‑ |
9.17 |
1.16 |
|
DMSO Control |
|
95 |
87 |
89 |
90.3 |
‑ |
4.16 |
1.00 |
|
5000 |
|
112 |
147 |
111 |
123.3 |
P |
20.50 |
1.37 |
|
1600 |
|
91 |
92 |
93 |
92.0 |
SP |
1.00 |
1.02 |
|
500 |
|
74 |
93 |
88 |
85.0 |
‑ |
9.85 |
0.94 |
|
160 |
|
86 |
85 |
76 |
82.3 |
‑ |
5.51 |
0.91 |
|
50 |
|
84 |
81 |
66 |
77.0 |
‑ |
9.64 |
0.85 |
|
16 |
|
87 |
77 |
70 |
78.0 |
‑ |
8.54 |
0.86 |
|
5 |
|
112 |
94 |
88 |
98.0 |
‑ |
12.49 |
1.08 |
|
Positive reference control (2AA)(2 µg/plate) |
|
2480 |
2410 |
2330 |
2406.7 |
‑ |
75.06 |
26.64 |
|
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 Sulphur Black 11 |
||||||||
Date of Experiment: |
October 24 – 26, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA98 |
||||||||
Cell count (Overnight culture): |
1.84 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 |
|
27 |
30 |
30 |
29.0 |
‑ |
1.73 |
1.67 |
|
DMSO Control |
|
15 |
18 |
19 |
17.3 |
‑ |
2.08 |
1.00 |
|
5000 |
|
20 |
15 |
17 |
17.3 |
TIP |
2.52 |
1.00 |
|
1600 |
|
22 |
15 |
27 |
21.3 |
TIP |
6.03 |
1.23 |
|
500 |
|
25 |
22 |
24 |
23.7 |
‑ |
1.53 |
1.37 |
|
160 |
|
20 |
25 |
20 |
21.7 |
‑ |
2.89 |
1.25 |
|
50 |
|
17 |
22 |
18 |
19.0 |
‑ |
2.65 |
1.10 |
|
16 |
|
19 |
18 |
38 |
25.0 |
‑ |
11.27 |
1.44 |
|
Positive reference control (NPD)(4 µg/plate) |
|
412 |
382 |
320 |
371.3 |
‑ |
46.92 |
21.42 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
30 |
31 |
23 |
28.0 |
‑ |
4.36 |
0.97 |
|
DMSO Control |
|
29 |
31 |
27 |
29.0 |
‑ |
2.00 |
1.00 |
|
5000 |
|
24 |
29 |
24 |
25.7 |
TIP |
2.89 |
0.89 |
|
1600 |
|
32 |
32 |
29 |
31.0 |
TIP |
1.73 |
1.07 |
|
500 |
|
34 |
23 |
27 |
28.0 |
‑ |
5.57 |
0.97 |
|
160 |
|
31 |
31 |
36 |
32.7 |
‑ |
2.89 |
1.13 |
|
50 |
|
24 |
33 |
37 |
31.3 |
‑ |
6.66 |
1.08 |
|
16 |
|
26 |
36 |
35 |
32.3 |
‑ |
5.51 |
1.11 |
|
Positive reference control (2AA)(2 µg/plate) |
|
1704 |
1828 |
1624 |
1718.7 |
‑ |
102.79 |
59.26 |
|
Obs : Observation (made by naked eye) TIP : Test Item Particles
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 Sulphur Black 11 |
||||||||
Date of Experiment: |
October 24 – 26, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA100 |
||||||||
Cell count (Overnight culture): |
1.40 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 |
|
94 |
89 |
94 |
92.3 |
‑ |
2.89 |
1.26 |
|
DMSO Control |
|
70 |
73 |
76 |
73.0 |
‑ |
3.00 |
1.00 |
|
Ultrapure Water Control |
|
73 |
80 |
90 |
81.0 |
‑ |
8.54 |
1.00 |
|
5000 |
|
197 |
205 |
220 |
207.3 |
TIP |
11.68 |
2.84 |
|
1600 |
|
155 |
146 |
142 |
147.7 |
TIP |
6.66 |
2.02 |
|
500 |
|
110 |
107 |
130 |
115.7 |
‑ |
12.50 |
1.58 |
|
160 |
|
101 |
103 |
118 |
107.3 |
‑ |
9.29 |
1.47 |
|
50 |
|
104 |
96 |
102 |
100.7 |
‑ |
4.16 |
1.38 |
|
16 |
|
109 |
106 |
98 |
104.3 |
‑ |
5.69 |
1.43 |
|
Positive reference control (SAZ)(2 µg/plate) |
|
1040 |
1080 |
1056 |
1058.7 |
‑ |
20.13 |
13.07 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
82 |
98 |
86 |
88.7 |
‑ |
8.33 |
1.09 |
|
DMSO Control |
|
77 |
79 |
87 |
81.0 |
‑ |
5.29 |
1.00 |
|
5000 |
|
292 |
236 |
229 |
252.3 |
TIP |
34.53 |
3.12 |
|
1600 |
|
201 |
175 |
191 |
189.0 |
TIP |
13.11 |
2.33 |
|
500 |
|
122 |
113 |
135 |
123.3 |
‑ |
11.06 |
1.52 |
|
160 |
|
118 |
113 |
100 |
110.3 |
‑ |
9.29 |
1.36 |
|
50 |
|
99 |
95 |
88 |
94.0 |
‑ |
5.57 |
1.16 |
|
16 |
|
107 |
117 |
100 |
108.0 |
‑ |
8.54 |
1.33 |
|
Positive reference control (2AA)(2 µg/plate) |
|
2000 |
2016 |
1840 |
1952.0 |
‑ |
97.32 |
24.10 |
|
Obs : Observation (made by naked eye) TIP : Test Item Particles
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 Sulphur Black 11 |
||||||||
Date of Experiment: |
October 24 – 26, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA1535 |
||||||||
Cell count (Overnight culture): |
2.15 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 |
|
15 |
7 |
8 |
10.0 |
‑ |
4.36 |
0.79 |
|
DMSO Control |
|
12 |
10 |
16 |
12.7 |
‑ |
3.06 |
1.00 |
|
Ultrapure Water Control |
|
10 |
14 |
5 |
9.7 |
‑ |
4.51 |
1.00 |
|
5000 |
|
312 |
284 |
296 |
297.3 |
TIP |
14.05 |
23.47 |
|
1600 |
|
145 |
172 |
172 |
163.0 |
TIP |
15.59 |
12.87 |
|
500 |
|
57 |
70 |
60 |
62.3 |
‑ |
6.81 |
4.92 |
|
160 |
|
28 |
32 |
40 |
33.3 |
‑ |
6.11 |
2.63 |
|
50 |
|
15 |
15 |
12 |
14.0 |
‑ |
1.73 |
1.11 |
|
16 |
|
18 |
19 |
15 |
17.3 |
‑ |
2.08 |
1.37 |
|
Positive reference control (SAZ)(2 µg/plate) |
|
1136 |
1000 |
1130 |
1088.7 |
‑ |
76.85 |
112.62 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
13 |
9 |
12 |
11.3 |
‑ |
2.08 |
1.21 |
|
DMSO Control |
|
11 |
13 |
4 |
9.3 |
‑ |
4.73 |
1.00 |
|
5000 |
|
229 |
198 |
214 |
213.7 |
TIP |
15.50 |
22.89 |
|
1600 |
|
144 |
109 |
132 |
128.3 |
TIP |
17.79 |
13.75 |
|
500 |
|
48 |
46 |
63 |
52.3 |
‑ |
9.29 |
5.61 |
|
160 |
|
17 |
13 |
26 |
18.7 |
‑ |
6.66 |
2.00 |
|
50 |
|
12 |
12 |
15 |
13.0 |
‑ |
1.73 |
1.39 |
|
16 |
|
12 |
11 |
12 |
11.7 |
‑ |
0.58 |
1.25 |
|
Positive reference control (2AA)(2 µg/plate) |
|
204 |
182 |
220 |
202.0 |
‑ |
19.08 |
21.64 |
|
Obs : Observation (made by naked eye) TIP : Test Item Particles
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 typhimuriumTA1537
Test Item: |
Leuco Sulphur Black 11 |
||||||||
Date of Experiment: |
October 24 – 26, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA1537 |
||||||||
Cell count (Overnight culture): |
1.97 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 |
|
18 |
8 |
11 |
12.3 |
‑ |
5.13 |
1.16 |
|
DMSO Control |
|
16 |
8 |
8 |
10.7 |
‑ |
4.62 |
1.00 |
|
5000 |
|
16 |
8 |
17 |
13.7 |
TIP |
4.93 |
1.28 |
|
1600 |
|
14 |
12 |
18 |
14.7 |
TIP |
3.06 |
1.38 |
|
500 |
|
14 |
13 |
12 |
13.0 |
‑ |
1.00 |
1.22 |
|
160 |
|
15 |
8 |
20 |
14.3 |
‑ |
6.03 |
1.34 |
|
50 |
|
7 |
12 |
14 |
11.0 |
‑ |
3.61 |
1.03 |
|
16 |
|
12 |
7 |
12 |
10.3 |
‑ |
2.89 |
0.97 |
|
Positive reference control (9AA)(50 µg/plate) |
|
920 |
968 |
888 |
925.3 |
‑ |
40.27 |
86.75 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
14 |
7 |
8 |
9.7 |
‑ |
3.79 |
0.88 |
|
DMSO Control |
|
15 |
11 |
7 |
11.0 |
‑ |
4.00 |
1.00 |
|
5000 |
|
22 |
18 |
22 |
20.7 |
TIP |
2.31 |
1.88 |
|
1600 |
|
11 |
7 |
6 |
8.0 |
TIP |
2.65 |
0.73 |
|
500 |
|
11 |
9 |
14 |
11.3 |
‑ |
2.52 |
1.03 |
|
160 |
|
15 |
10 |
16 |
13.7 |
‑ |
3.21 |
1.24 |
|
50 |
|
9 |
15 |
10 |
11.3 |
‑ |
3.21 |
1.03 |
|
16 |
|
10 |
12 |
13 |
11.7 |
‑ |
1.53 |
1.06 |
|
Positive reference control (2AA)(2 µg/plate) |
|
192 |
171 |
201 |
188.0 |
‑ |
15.39 |
17.09 |
|
Obs : Observation (made by naked eye) TIP : Test Item Particles
SD : Standard Deviation ‑ : Normal background lawn development, no precipitate
MR : Mutation Rate
9AA:9-Aminoacridine
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 Sulphur Black 11 |
||||||||
Date of Experiment: |
October 24 – 26, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Escherichia coliWP2uvrA |
||||||||
Cell count (Overnight culture): |
3.23 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 |
|
27 |
36 |
26 |
29.7 |
‑ |
5.51 |
0.96 |
|
DMSO Control |
|
27 |
37 |
29 |
31.0 |
‑ |
5.29 |
1.00 |
|
Ultrapure Water Control |
|
46 |
49 |
42 |
45.7 |
‑ |
3.51 |
1.00 |
|
5000 |
|
87 |
80 |
85 |
84.0 |
TIP |
3.61 |
2.71 |
|
1600 |
|
73 |
70 |
80 |
74.3 |
TIP |
5.13 |
2.40 |
|
500 |
|
45 |
48 |
57 |
50.0 |
‑ |
6.24 |
1.61 |
|
160 |
|
39 |
39 |
41 |
39.7 |
‑ |
1.15 |
1.28 |
|
50 |
|
36 |
49 |
39 |
41.3 |
‑ |
6.81 |
1.33 |
|
16 |
|
31 |
33 |
26 |
30.0 |
‑ |
3.61 |
0.97 |
|
Positive reference control (MMS)(2 µL/plate) |
|
645 |
549 |
532 |
575.3 |
‑ |
60.93 |
12.60 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
44 |
35 |
47 |
42.0 |
‑ |
6.24 |
0.85 |
|
DMSO Control |
|
45 |
50 |
53 |
49.3 |
‑ |
4.04 |
1.00 |
|
5000 |
|
83 |
74 |
73 |
76.7 |
TIP |
5.51 |
1.55 |
|
1600 |
|
76 |
63 |
57 |
65.3 |
TIP |
9.71 |
1.32 |
|
500 |
|
46 |
46 |
38 |
43.3 |
‑ |
4.62 |
0.88 |
|
160 |
|
40 |
33 |
39 |
37.3 |
‑ |
3.79 |
0.76 |
|
50 |
|
32 |
48 |
40 |
40.0 |
‑ |
8.00 |
0.81 |
|
16 |
|
50 |
47 |
46 |
47.7 |
‑ |
2.08 |
0.97 |
|
Positive reference control (2AA)(50 µg/plate) |
|
255 |
238 |
241 |
244.7 |
‑ |
9.07 |
4.96 |
|
Obs : Observation (made by naked eye) TIP : Test Item Particles
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 typhimuriumTA100
Test Item: |
Leuco Sulphur Black 11 |
||||||||
Date of Experiment: |
November 15 – 17, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA100 |
||||||||
Cell count (Overnight culture): |
1.32 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 |
|
97 |
99 |
93 |
96.3 |
‑ |
3.06 |
1.08 |
|
DMSO Control |
|
87 |
90 |
90 |
89.0 |
‑ |
1.73 |
1.00 |
|
Ultrapure Water Control |
|
101 |
86 |
91 |
92.7 |
‑ |
7.64 |
1.00 |
|
5000 |
|
228 |
236 |
210 |
224.7 |
TIP |
13.32 |
2.52 |
|
1600 |
|
128 |
136 |
156 |
140.0 |
TIP |
14.42 |
1.57 |
|
500 |
|
94 |
94 |
100 |
96.0 |
‑ |
3.46 |
1.08 |
|
160 |
|
72 |
94 |
92 |
86.0 |
‑ |
12.17 |
0.97 |
|
50 |
|
84 |
82 |
86 |
84.0 |
‑ |
2.00 |
0.94 |
|
16 |
|
87 |
106 |
98 |
97.0 |
‑ |
9.54 |
1.09 |
|
Positive reference control (SAZ)(2 µg/plate) |
|
1192 |
1160 |
1168 |
1173.3 |
‑ |
16.65 |
12.66 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
116 |
109 |
79 |
101.3 |
‑ |
19.66 |
0.97 |
|
DMSO Control |
|
104 |
109 |
100 |
104.3 |
‑ |
4.51 |
1.00 |
|
5000 |
|
222 |
261 |
300 |
261.0 |
TIP |
39.00 |
2.50 |
|
1600 |
|
170 |
146 |
168 |
161.3 |
TIP |
13.32 |
1.55 |
|
500 |
|
112 |
105 |
113 |
110.0 |
‑ |
4.36 |
1.05 |
|
160 |
|
88 |
104 |
94 |
95.3 |
‑ |
8.08 |
0.91 |
|
50 |
|
90 |
106 |
87 |
94.3 |
‑ |
10.21 |
0.90 |
|
16 |
|
94 |
82 |
98 |
91.3 |
‑ |
8.33 |
0.88 |
|
Positive reference control (2AA)(2 µg/plate) |
|
1624 |
2128 |
1752 |
1834.7 |
‑ |
261.97 |
17.58 |
|
Obs : Observation (made by naked eye) TIP : Test Item Particles
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 Confirmatory Mutation Test inSalmonella typhimuriumTA1535
Test Item: |
Leuco Sulphur Black 11 |
||||||||
Date of Experiment: |
November 15 – 17, 2017 |
||||||||
Applied Method: |
Plate Incorporation |
||||||||
Strain: |
Salmonella typhimuriumTA1535 |
||||||||
Cell count (Overnight culture): |
1.40 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 |
|
8 |
12 |
6 |
8.7 |
‑ |
3.06 |
0.84 |
|
DMSO Control |
|
12 |
8 |
11 |
10.3 |
‑ |
2.08 |
1.00 |
|
Ultrapure Water Control |
|
13 |
7 |
7 |
9.0 |
‑ |
3.46 |
1.00 |
|
5000 |
|
167 |
182 |
207 |
185.3 |
TIP |
20.21 |
17.94 |
|
1600 |
|
75 |
89 |
90 |
84.7 |
TIP |
8.39 |
8.19 |
|
500 |
|
36 |
53 |
36 |
41.7 |
‑ |
9.81 |
4.03 |
|
160 |
|
18 |
19 |
9 |
15.3 |
‑ |
5.51 |
1.48 |
|
50 |
|
14 |
14 |
13 |
13.7 |
‑ |
0.58 |
1.32 |
|
16 |
|
4 |
19 |
10 |
11.0 |
‑ |
7.55 |
1.06 |
|
Positive reference control (SAZ)(2 µg/plate) |
|
824 |
880 |
936 |
880.0 |
‑ |
56.00 |
97.78 |
|
With Exogenous Metabolic Activation (+S9 mix) |
|||||||||
Concentration (µg/plate) |
|
Revertant per Plate |
Mean |
Obs |
SD |
MR |
|||
Parallel: |
1 |
2 |
3 |
||||||
Untreated Control |
|
10 |
13 |
15 |
12.7 |
‑ |
2.52 |
1.19 |
|
DMSO Control |
|
8 |
14 |
10 |
10.7 |
‑ |
3.06 |
1.00 |
|
5000 |
|
239 |
252 |
254 |
248.3 |
TIP |
8.14 |
23.28 |
|
1600 |
|
89 |
109 |
102 |
100.0 |
TIP |
10.15 |
9.38 |
|
500 |
|
42 |
54 |
42 |
46.0 |
‑ |
6.93 |
4.31 |
|
160 |
|
23 |
21 |
16 |
20.0 |
‑ |
3.61 |
1.88 |
|
50 |
|
19 |
18 |
15 |
17.3 |
‑ |
2.08 |
1.63 |
|
16 |
|
9 |
13 |
13 |
11.7 |
‑ |
2.31 |
1.09 |
|
Positive reference control (2AA)(2 µg/plate) |
|
278 |
260 |
232 |
256.7 |
‑ |
23.18 |
24.06 |
|
Obs : Observation (made by naked eye) TIP : Test Item Particles
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.
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 typhimuriumTA98, TA100, TA1535,
TA1537;E. coli:Escherichia coliWP2uvrA
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 was determined to be mutagenic in Salmonella typhimurium TA100 and TA1535 strains in the absence and presence of an exogenous metabolic activation system.
- Executive summary:
The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD guideline 471. The initial mutation test was carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) 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 TA100 and TA1535. The study included preliminary solubility tests, a preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (repeated plate incorporation test).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 a correction of the concentrations for the active component content (77.31 %) 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: 5000;1600; 500; 160; 50 and 16 µg/plate. The selection of the concentration range was based on the recommendations in the OECD guideline 471. 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 same concentration levels were investigated in the confirmatory mutation test: ±S9 mix: 5000; 1600; 500; 160; 50 and 16 µg/plate. When evaluated by naked eye, non-interfering test item precipitate (test item particles) was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 µg/plate in the absence and presence of an exogenous metabolic activation (±S9 mix) system. The test item did not show unequivocal inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) were considered to be within the biological variability range of the applied test system. The revertant colony numbers of solvent control plates (DMSO) 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, 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 test) in the investigated Salmonella typhimuriumTA100 and TA1535 strains in the absence and presence of the exogenous metabolic activation system (±S9 mix). The obtained revertant colony number increases were clearly above the corresponding historical control data ranges and the relevant genotoxicological threshold for being positive in both experiments. The increases were observed in strain TA100 at the highest examined concentration of 5000 µg/plate (±S9 mix) and in strain TA1535 in the concentration range of 5000-500 µg/plate (±S9 mix). The obtained increased tendencies, especially in the latter case, followed a clear dose-relationship. Therefore, the test item was determined to be mutagenic in Salmonella typhimurium TA100 and TA1535 strains carrying base pair substitutions, in the absence and presence of an exogenous metabolic activation system, under the test conditions used in this study.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.