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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 288-896-1 | CAS number: 85940-08-5 This substance is identified in the Colour Index by Colour Index Constitution Number, C.I. 53015.
- 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
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test item showed a mutagenic activity on Salmonella typhimurium TA98, TA100 and TA1535 strains in the absence and presence of an exogenous metabolic activation system. However, it is assumed that the test item is not mutagenic in vivo. The substance is a high molecular weight UVCB compound with number-average molecular weight of 155 738 g/mol and weight-average molecular weight of 1 553 053 314 g/mol and no fraction with a molecular weight below 1000 Da. Thus, it is highly unlikely that the substance can pass intact cell membranes. In contrast to mammalian cells the S. typhimurium strains used in the bacterial reverse mutation assay have a rfa mutation which enhances the permeability of the cell membrane. It is assumed that the mutagenic result obtained with the test substance in tester strain TA98, TA100 and TA1535 is a result of the impaired barrier function of the bacterial cell wall and not relevant for intact organisms. In order to confirm the assumption further experiments will be initiated and the IUCLID dossier will be updated as soon as the results become available.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09 November 2017 - 19 February 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- 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
- 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 / 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; 16 and 5 µg/plate
- Vehicle / solvent:
- dimethyl sulfoxide (DMSO)
- 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 10-14 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 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.
The study included a preliminary solubility test, 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).
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; 16 and 5 µg/plate.
In the initial mutation test an unequivocal inhibitory, cytotoxic effect of the test item was observed in S. typhimurium TA1537 strain at 5000 and 1600 µg/plate, in the absence and also in the presence of exogenous metabolic activation (±S9 mix). The cytotoxicity was indicated by decreased revertant colony counts and/or affected background lawn development: reduced or slightly reduced background lawn. The revertant colony numbers of the 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 fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. - Evaluation criteria:
- Evaluation of Experimental Data
The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined visually by manual counting
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.
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- 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
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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 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
- 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 (according to the provided Certificates, Appendix VIII) 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 initial and confirmatory mutation tests. 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 Salmonella typhimurium 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 TA98, TA100 and TA1535 strains (±S9 mix). The increased revertant colony numbers were above the threshold for being positive in S. typhimurium TA98 at the concentration of 5000 µg/plate (-S9 mix) and in the range of 5000-50 µg/plate (+S9 mix), in TA100 at the concentrations of 5000 and 1600 µg/plate ( S9 mix) and in the concentration range of 5000-500 µg/plate (+S9 mix); furthermore in TA1535 at 5000 µg/plate (±S9 mix). The increased revertant colony numbers were above the corresponding historical control data range, however, they were below the genotoxicological threshold for being positive in S. typhimurium TA98 at 1600, 500 and 160 µg/plate (-S9 mix). The significant revertant colony number increases showed clear dose-related tendency in S. typhimurium TA98, TA100 and TA1535 strains in the absence and presence of exogenous metabolic activation (± S9 mix). In this test an unequivocal inhibitory, cytotoxic effect of the test item was observed in S. typhimurium TA1537 strain at 5000 and 1600 µg/plate, in the absence and also in the presence of exogenous metabolic activation (±S9 mix). The cytotoxicity was indicated by decreased revertant colony counts and/or affected background lawn development: reduced or slightly reduced background lawn. 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 5000 and 1600 µg/plate in the absence and also in the presence of an exogenous metabolic activation system (±S9 mix). To confirm and to investigate the reproducibility of this positive result a confirmatory mutation test was performed with the S. typhimurium strain TA98, TA100 and TA1535 strains in the absence and presence of an exogenous metabolic activation system (±S9 mix). The strains Salmonella typhimurium
TA1537 and Escherichia coli WP2 uvrA were not further investigated.
Confirmatory Mutation Test (Plate Incorporation Test)
While the positive results already noticed in the initial mutation test in the investigated Salmonella typhimurium TA98 strain (±S9 mix), TA100 strain (±S9 mix) and TA1535 strain (+S9 mix) were successfully confirmed, the revertant colony number increases remained just below the threshold for being positive (borderline results) in TA1535 in the absence of an exogenous metabolic activation system (-S9 mix). The increased revertant colony numbers were above the threshold for being positive in S. typhimurium TA98 at the concentrations of 5000 and 1600 µg/plate (-S9 mix) and in the range of 5000-50 µg/plate (+S9 mix), in TA100 at the concentrations of 5000 and 1600 µg/plate ( S9 mix) and in the concentration range of 5000-500 µg/plate (+S9 mix); furthermore in TA1535 at 5000 µg/plate (+S9 mix). The increased revertant colony numbers were above the corresponding historical control data range; however just below the genotoxicological threshold for being positive (borderline results) in S. typhimurium TA1535 at 5000 µg/plate (-S9 mix). Similarly to the initial mutation test results the significantly increased revertant colony number showed clear dose-related tendency in absence and also in the presence of exogenous metabolic activation (± S9 mix). Non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at 5000 and 1600 µg/plate in the absence and also in the presence of an exogenous metabolic activation system (±S9 mix). - Conclusions:
- In conclusion, the test item showed a mutagenic activity on Salmonella typhimurium TA98, TA100 and TA1535 strains carrying frameshift and base-pair substitution in the absence and presence of an exogenous metabolic activation system, 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 according to OECD guideline 471. 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 TA98, TA100 and TA1535. The study included a preliminary solubility test, 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 (74.99 %) 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; 16 and 5 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 from the initial mutation test in the confirmatory mutation test the same concentration levels were investigated (±S9 mix: 5000; 1600; 500; 160; 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 at 5000 and 1600 µg/plate in the absence and also in the presence of an exogenous metabolic activation system (±S9 mix). In the initial mutation test an unequivocal inhibitory, cytotoxic effect of the test item was observed in S. typhimurium TA1537 strain at 5000 and 1600 µg/plate, in the absence and also in the presence of exogenous metabolic activation (±S9 mix). The cytotoxicity was indicated by decreased revertant colony counts and/or affected background lawn development: reduced or slightly reduced background lawn. The revertant colony numbers of the 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 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 typhimurium TA98, TA100 strains (±S9 mix) and in TA1535 strain (+S9 mix). The positive results obtained in the initial mutation test were not confirmed in the repeated plate incorporation procedure in case of TA1535 in the absence of exogenous metabolic activation (-S9 mix). The increased revertant colony counts remained just below the threshold for being positive (borderline results). However, the obtained increased tendencies followed a clear dose-relationship in all above listed cases. The positive mutagenicity results were obtained at higher concentration levels, mostly at precipitating concentrations (5000 and 1600 µg/plate); however unequivocal, confirmed positive results were noticed at non-precipitated concentrations in S. typhimurium TA98 at the concentrations of 500, 160 and 50 µg/plate (+S9 mix) and in TA100 at 500 µg/plate (+S9 mix). The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item induced gene mutations by frameshifts and base pair substitutions in the genome of the Salmonella typhimurium TA98, TA100 and TA1535 tester strains examined. The unequivocal positive results were confirmed by a repeat of the plate incorporation procedures (initial and confirmatory mutation tests) in the absence and presence of exogenous metabolic activation (±S9 mix). In conclusion, the test item showed a mutagenic activity on Salmonella typhimurium TA98, TA100 and TA1535 strains carrying frameshift and base-pair substitution in the absence and presence of an exogenous metabolic activation system, under the test conditions used in this study.
Reference
SUMMARY TABLES OF THE EXPERIMENTS
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 |
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 |
36.0 |
1.74 |
79.7 |
3.19 |
292.3 |
3.72 |
464.0 |
5.14 |
|
1600 |
52.0 |
2.52 |
133.3 |
5.33 |
165.3 |
2.10 |
442.7 |
4.90 |
|
500 |
37.7 |
1.82 |
174.7 |
6.99 |
123.0 |
1.56 |
246.0 |
2.72 |
|
160 |
38.7 |
1.87 |
164.7 |
6.59 |
105.0 |
1.33 |
167.3 |
1.85 |
|
50 |
19.3 |
0.94 |
78.0 |
3.12 |
81.0 |
1.03 |
100.7 |
1.11 |
|
16 |
27.7 |
1.34 |
36.0 |
1.44 |
75.3 |
0.96 |
103.3 |
1.14 |
|
5 |
17.7 |
0.85 |
23.0 |
0.92 |
75.0 |
0.95 |
98.7 |
1.09 |
|
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 typhimurium tester 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 |
23.7 |
1.37 |
26.7 |
1.36 |
99.7 |
1.17 |
105.7 |
1.17 |
9.3 |
0.82 |
10.0 |
1.03 |
9.3 |
1.22 |
10.7 |
1.14 |
37.0 |
1.10 |
41.0 |
0.95 |
DMSO Control |
17.3 |
1.00 |
19.7 |
1.00 |
85.3 |
1.00 |
90.0 |
1.00 |
11.3 |
1.00 |
9.7 |
1.00 |
7.7 |
1.00 |
9.3 |
1.00 |
33.7 |
1.00 |
43.3 |
1.00 |
Ultrapure Water Control |
– |
– |
– |
– |
85.3 |
1.00 |
– |
– |
10.3 |
1.00 |
– |
– |
– |
– |
– |
– |
37.0 |
1.00 |
– |
– |
5000 |
86.7 |
5.00 |
122.7 |
6.24 |
268.0 |
3.14 |
335.3 |
3.73 |
40.0 |
3.53 |
50.3 |
5.21 |
4.0 |
0.52 |
10.0 |
1.07 |
31.3 |
0.93 |
42.3 |
0.98 |
1600 |
45.0 |
2.60 |
110.7 |
5.63 |
197.7 |
2.32 |
289.3 |
3.21 |
16.0 |
1.41 |
21.7 |
2.24 |
0.7 |
0.09 |
2.0 |
0.21 |
26.7 |
0.79 |
52.0 |
1.20 |
500 |
41.0 |
2.37 |
190.0 |
9.66 |
127.0 |
1.49 |
248.3 |
2.76 |
12.3 |
1.09 |
12.0 |
1.24 |
7.3 |
0.96 |
14.7 |
1.57 |
32.0 |
0.95 |
54.0 |
1.25 |
160 |
40.0 |
2.31 |
140.0 |
7.12 |
103.3 |
1.21 |
158.0 |
1.76 |
14.7 |
1.29 |
12.3 |
1.28 |
8.7 |
1.13 |
8.0 |
0.86 |
37.3 |
1.11 |
42.3 |
0.98 |
50 |
21.0 |
1.21 |
78.7 |
4.00 |
83.7 |
0.98 |
100.3 |
1.11 |
10.3 |
0.91 |
8.7 |
0.90 |
8.3 |
1.09 |
9.0 |
0.96 |
36.0 |
1.07 |
36.7 |
0.85 |
16 |
22.7 |
1.31 |
42.7 |
2.17 |
86.3 |
1.01 |
97.7 |
1.09 |
11.0 |
0.97 |
14.3 |
1.48 |
9.0 |
1.17 |
9.7 |
1.04 |
39.3 |
1.17 |
39.3 |
0.91 |
5 |
19.7 |
1.13 |
31.7 |
1.61 |
77.3 |
0.91 |
81.7 |
0.91 |
9.3 |
0.82 |
9.0 |
0.93 |
5.7 |
0.74 |
9.7 |
1.04 |
31.3 |
0.93 |
44.3 |
1.02 |
NPD (4mg) |
283.3 |
16.35 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2mg) |
– |
– |
– |
– |
1397.3 |
16.38 |
– |
– |
1141.3 |
110.45 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
9AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
602.7 |
78.61 |
– |
– |
– |
– |
– |
– |
MMS (2mL) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
980.0 |
26.49 |
– |
– |
2AA (2mg) |
– |
– |
2376.0 |
120.81 |
– |
– |
1920.0 |
21.33 |
– |
– |
175.7 |
18.17 |
– |
– |
182.7 |
19.57 |
– |
– |
– |
– |
2AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
230.3 |
5.32 |
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 tester strains |
|||||||||||
TA 98 |
TA 100 |
TA 1535 |
||||||||||
-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 |
Untreated Control |
16.3 |
1.17 |
18.7 |
0.84 |
97.7 |
0.99 |
105.0 |
1.05 |
10.7 |
1.03 |
10.7 |
0.97 |
DMSO Control |
14.0 |
1.00 |
22.3 |
1.00 |
99.0 |
1.00 |
99.7 |
1.00 |
10.3 |
1.00 |
11.0 |
1.00 |
Ultrapure Water Control |
– |
– |
– |
– |
99.7 |
1.00 |
– |
– |
10.0 |
1.00 |
– |
– |
5000 |
46.0 |
3.29 |
107.3 |
4.81 |
215.7 |
2.18 |
297.0 |
2.98 |
30.3 |
2.94 |
43.7 |
3.97 |
1600 |
44.3 |
3.17 |
153.3 |
6.87 |
201.3 |
2.03 |
245.0 |
2.46 |
20.3 |
1.97 |
26.0 |
2.36 |
500 |
34.7 |
2.48 |
231.0 |
10.34 |
132.0 |
1.33 |
224.3 |
2.25 |
12.7 |
1.23 |
15.3 |
1.39 |
160 |
24.7 |
1.76 |
154.0 |
6.90 |
112.3 |
1.13 |
152.3 |
1.53 |
11.7 |
1.13 |
11.3 |
1.03 |
50 |
15.0 |
1.07 |
92.3 |
4.13 |
81.0 |
0.82 |
100.7 |
1.01 |
12.7 |
1.23 |
12.3 |
1.12 |
16 |
14.7 |
1.05 |
36.7 |
1.64 |
88.0 |
0.89 |
93.7 |
0.94 |
8.3 |
0.81 |
12.0 |
1.09 |
5 |
15.7 |
1.12 |
36.7 |
1.64 |
84.7 |
0.86 |
95.7 |
0.96 |
6.7 |
0.65 |
10.3 |
0.94 |
NPD (4mg) |
368.7 |
26.33 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2mg) |
– |
– |
– |
– |
1805.3 |
18.11 |
– |
– |
1157.3 |
115.73 |
– |
– |
2AA (2mg) |
– |
– |
1240.7 |
55.55 |
– |
– |
1730.7 |
17.36 |
– |
– |
166.0 |
15.09 |
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 and the untreated control is given referring to the DMSO. The mutation rate of the NPD and 2AA is given referring to the DMSO and the mutation rate of the SAZ positive control is given referring to the 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
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
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 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 TA98, TA100 and TA1535. The study included a preliminary solubility test, 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 (74.99 %) 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; 16 and 5 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 from the initial mutation test in the confirmatory mutation test the same concentration levels were investigated (±S9 mix: 5000; 1600; 500; 160; 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 at 5000 and 1600 µg/plate in the absence and also in the presence of an exogenous metabolic activation system (±S9 mix). In the initial mutation test an unequivocal inhibitory, cytotoxic effect of the test item was observed in S. typhimurium TA1537 strain at 5000 and 1600 µg/plate, in the absence and also in the presence of exogenous metabolic activation (±S9 mix). The cytotoxicity was indicated by decreased revertant colony counts and/or affected background lawn development: reduced or slightly reduced background lawn. The revertant colony numbers of the 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 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 typhimurium TA98, TA100 strains (±S9 mix) and in TA1535 strain (+S9 mix). The positive results obtained in the initial mutation test were not confirmed in the repeated plate incorporation procedure in case of TA1535 in the absence of exogenous metabolic activation (-S9 mix). The increased revertant colony counts remained just below the threshold for being positive (borderline results). However, the obtained increased tendencies followed a clear dose-relationship in all above listed cases. The positive mutagenicity results were obtained at higher concentration levels, mostly at precipitating concentrations (5000 and 1600 µg/plate); however unequivocal, confirmed positive results were noticed at non-precipitated concentrations in S. typhimurium TA98 at the concentrations of 500, 160 and 50 µg/plate (+S9 mix) and in TA100 at 500 µg/plate (+S9 mix). The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item induced gene mutations by frameshifts and base pair substitutions in the genome of the Salmonella typhimurium TA98, TA100 and TA1535 tester strains examined. The unequivocal positive results were confirmed by a repeat of the plate incorporation procedures (initial and confirmatory mutation tests) in the absence and presence of exogenous metabolic activation (±S9 mix). In conclusion, the test item showed a mutagenic activity on Salmonella typhimurium TA98, TA100 and TA1535 strains carrying frameshift and base-pair substitution in the absence and presence of an exogenous metabolic activation system, under the test conditions used in this study.
Justification for classification or non-classification
Classification, Labeling, and Packaging Regulation (EC) No 1272/2008
Based on available data on genetic toxicity, a final conclusion on C&L is currently not possible. Further experiments will be initiated to confirm this classfication. The IUCLID dossier will be updated as soon as the results become available.
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.