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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 225-004-1 | CAS number: 4602-84-0
- 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
Genetic toxicity in vitro: The genotoxicity potential of the test item was assessed in bacterial gene mutation, human lymphocyte chromosome aberration and mouse lymphoma cell mutation assays. Negative results were observed in all in vitro assays, therefore there is no evidence of genotoxicity of the test item.
Genetic toxicity in vivo: No positive results were observed in any of the in vitro genotoxicity studies required for the Annex VII or VIII information requirements under REACH, therefore in vivo data for genetic toxicity is not required.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted according to OECD 471 and GLP. The test included five strains of Salmonella typhimurium, with TA102 in place of an Escherichia coli strain.
- 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:
- 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine reversion his- to his+
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Additional strain / cell type characteristics:
- nitroreductase deficient
- Remarks:
- All strains: rfa- mutation; strains TA 1535, TA 1537, TA 98 and TA 100: uvrB- mutation; strains TA 98, TA 100 and TA102: R-factor mutation; strain TA 102: hisG428 mutation
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/beta-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- 3, 10, 33, 100. 333. 1000. 2500 and 5000 µg/plate for pre-experiment (main experiment I) and main experiment II
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO; purity >99%
- Justification for choice of solvent/vehicle: solubility properties and relative non-toxicity to the bacteria - Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-ortho-phenylene -diamine, 2-aminoanthracene
- Details on test system and experimental conditions:
- EXPERIMENT I and EXPERIMENT 1A (repeat) -
In Experiment I strain TA 1537 without S9 mix showed a reduction in the number of revertants below the indication factor of 0.5 - indication of toxicity at nearly all concentrations, this part was repeated under identical conditions and reported as EXPERIMENT 1A
METHOD OF APPLICATION: In agar (plate incorporation);
DURATION
Exposure duration: - Exposure duration: at least 48 hours at 37C in the dark
NUMBER OF REPLICATIONS:Three
EXPERIMENT II
METHOD OF APPLICATION: ; pre incubation test
DURATION
- Preincubation period: 60min at 37C in the dark
- Exposure duration: at least 48 hours at 37C in the dark
-
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY determined in Experiment I
---------
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
SELECTION AGENT (mutation assays):
NUMBER OF REPLICATIONS: 3
NUMBER OF CELLS EVALUATED:
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth
- Evaluation criteria:
- Test system is considered mutagenic if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA 102) or thrice (TA 1535 and TA1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more that one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- Statistics:
- Not mandatory under OECD test guidleine 471
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No substantial increase in revertant colony numbers in any of the 5 tester strains was observed following treatment with the test item at any concentration level, neither in the presence or absence of metabolic activation (S9 mix) . There was also no tendency for higher mutation rates with increasing concentration below the range normally considered to be biologically significant.
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed in test tubes from 1000 to 5000 mcg/plate in both experiments - and on the incubated agar paltes from 2500 to 5000 mcg/plate
-
RANGE-FINDING/SCREENING STUDIES: As all plates were evaluable in the range finding study, this study was reported as Experiment I.
COMPARISON WITH HISTORICAL CONTROL DATA:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects were observed at the following concentrations (mcg/plate)
TA1535 : Experiment I plate test 2500-5000 +/- S9 ) ; Experiment II pre-incubation ; 2500-5000 +/- S9
TA1537 : Experiment I plate test several doses upto 5000 - S9 ;2500 +S9 ; (Experiment Ia plate test repeat) 333-5000mcg/plate -S9; Experiment II pre-incubation ; 1000 -5000 - S9 ; 333 -5000 +S9
TA98 : Experiment I plate test 5000 - S9 ;2500 +S9 ; Experiment II pre-incubation ; 100, 2500-5000 -S9 ;2500 -5000 +S9
TA100 : Experiment I plate test 333-5000 - S9 ;1000-5000 +S9 ; Experiment II pre-incubation ; 1000-5000 +/- S9
TA102 : Experiment I plate test 2500-5000+/ - S9 Experiment II pre-incubation 2500-5000+/ - S9 - Conclusions:
- No substantail increase in revertant colony numbers in any of the 5 tester strains was observed following treatment with the test item at any concentration level, neither in the presence or absence of metabolic activation (S9 mix) . There was also no tendency for higher mutation rates with increasing concentration below the range normally considered to be biologically significant. Under the experimental conditions, the test item did not induce gene mutations by base pair or frameshift in the genome of the species tested. The test item is not considered to be mutagenic in the Salmonella tyhimurium reverse transcription assay.
- Executive summary:
The study was performed to assess the potential of the test item to induce gene mutations according to the plate incorporation test (Experiment I) or the pre-incubation test (Experiment II) using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102. The assay was performed in two independent experiments with or without liver microsomal activation (S9 mix). Since results for TA1537 without metabolic activation showed a reduction of revertants (below the indication level of 0.5) at nearly all concentrations, this part of Experiment I was repeated under identical conditions as Experiment IA. In all experiments, test concentrations ranged from 3 to 5000 µg/plate, and toxic effects were observed at the higher concentrations tested. No substantial increase in revertant colony numbers in any of the 5 tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix) . There was also no tendency for higher mutation rates with increasing concentration below the range normally considered to be biologically significant. Appropriate mutagens were used as positive controls and showed a distinct increase in revertant colonies. Under the experimental conditions, the test item did not induce gene mutations by base pair or frameshift in the genome of the species tested. The test item is not considered to be mutagenic in the Salmonella typhimurium reverse transcription assay. This study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD 471.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Healthy donors not receiving medication
- Cell cycle length, doubling time or proliferation index: Mitotic activity began at about 40 hours after PHA stimulation and reached a maximum at around 3 days.
- Sex, age and number of blood donors if applicable: Female donor (46 years old) for Experiment I and a 45 year-old male and a 24 year-old male donor for Experiment II
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1) containing 10 % FCS (fetal calf serum) and supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL), the anticoagulant heparin (25,000 U.S.P.-U/mL) and HEPES (final concentration 10 mM). All incubations were done at 37°C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).
- Periodically checked for karyotype stability: The chromosome constitution remained diploid during short-term culture. - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity tests were performed in duplicate in order to determine test concentrations. Test item concentrations ranged between 14.7 and the highest concentration of 2270 µg/mL (approximately 10 mM). Precipitation of the test item was observed at 79.0 µg/mL and above in the absence of S9 and at 741.2 µg/mL in the presence of S9 mix. In addition, 22 hours treatment with >45.2 µg/mL in the absence of S9 induced strong toxic effects. Therefore, top concentrations of 139.9 µg/mL and 750 µg/mL were chosen for tests in the absence and presence of S9, respectively.
- EXPERIMENT I: 14.7, 25.8, 45.2, 79.0, 138.3, 242.0, 423.6, 741.2, 1297.1 and 2270.0 µg/mL were tested
- EXPERIMENT II without S9: 0.9, 1.6, 2.8, 4.9, 8.5, 14.9, 26.1, 45.7, 80.0, 139.9 µg/mL
- EXPERIMENT II with S9: 4.9, 8.5, 14.7, 14.9, 25.8, 26.1, 45.2, 45.7, 79.0, 80.0, 138.3, 139.9, 242.0, 244.9, 423.6, 428.6, 741.2, 750.0, 1297.1, 2270.0 µg/mL (test was repeated). - Vehicle / solvent:
- - Solvent (vehicle): Dimethyl sulfoxide (DMSO 99.5% purity)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
EXPOSURE
- The chromosomes were prepared for 22 hours (Experiment I) and 46 hours (Experiment II) after exposure initiation. Thus, exposure duration was 4, 22 and 46 hours.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis) was determined in addition to the number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
OTHER EXAMINATIONS:
- Analysis of metaphase cells: Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations.
- Metaphase plates (n=100) were scored for cytogenetic damage.
- Determination of chromosome aberrations: the number of induced structural chromosome aberrations - Evaluation criteria:
- CLASSIFICATIONS
- Non-mutagenic: the number of induced structural chromosome aberrations is in the range of testing facilities historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- Mutagenic: the number of induced structural chromosome aberrations is not in the range of the testing facilities historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) or a concentration-related increase is observed
- Aneugenic: the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 0.8 % polyploid cells). - Statistics:
- Statistical significance was confirmed by means of the Fisher's exact test (p<0.05).
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Clear cytotoxicity was observed at the high concentrations in Experiment I, while in Experiment II concentrations showing clear cytotoxic effects were not scorable for cytogenetic damage. In both experiments, in the absence and presence of an S9 metabolic activation system, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of cells after treatment (0.0 – 3.8% aberrant cells) were within historical values for controls (0.0 – 4.0% aberrant cells). Slight increases in the aberrant cells were noted, however, as these fell within the laboratory’s control data, the statistical significance and dose-dependency were regarded as biologically insignificant.
No biologically relevant increase in the rate of polyploid metaphases were detected after treatment (0.0 - 0.2%) in Experiment I and II, relative to solvent vehicle controls (0.0%). EMS (825, 660 or 770 µg/mL) or CPA (22.5 µg/mL) positive controls were successfully identified in the assay. - Conclusions:
- It can be concluded that, under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro in the absence or presence of metabolic activation.
- Executive summary:
The test item dissolved in DMSO was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro, either in the presence or absence of an exogenous S9 metabolic activation system. S9 liver fraction was prepared from Wistar Hanlbm rats exposed to phenobarbital (80 mg/kg bw) and β-naphthoflavone (80 mg/kg bw). The S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final concentration of 0.75 mg/mL in the short-term cultures of human lymphocytes stimulated to replicate. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix and 22 hours without S9 mix and chromosomes were prepared for 22 hours. In Experiment II, the exposure periods were 4 hours with S9 mix and 46 hours without S9 mix, with 46 hours’ chromosome preparation. The assay adhered to acceptability criteria for ethylmethane sulfonate (EMS) and cyclophosphamide (CPA) positive controls in the absence or presence of S9 mix, respectively. In both studiess the number of induced structural chromosome aberrations of the test item were within the range of historical control data (0.0 – 4.0% aberrant cells, exclusive gaps). Thus, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. This study is reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 473 and EU B.10.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase Locus (TK+/-)
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Laboratory culture
- Suitability of cells: The L5178Y cell line has successfully been used in in vitro experiments for many years. L5178Y cells are characterised by a high proliferation rate and cloning efficiencies of untreated cells of usually more than 50 % both necessary for the appropriate performance of the study. The cells have a stable karyotype with a near diploid chromosome number.
- Cell cycle length, doubling time or proliferation index: Doubling time 10 - 12 h
- Methods for maintenance in cell culture if applicable: The cells are subcultured two times prior to treatment.
- Modal number of chromosomes: 40 ± 2
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 complete culture medium. The cell cultures are incubated at 37 ± 1.5°C in a
humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: Yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/-Naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- EXPERIMENT 1:
- Without S9 mix: 1.3; 2.5; 5.0; 10.0; and 20.0 µg/mL
- With S9 mix: 4.4; 8.8; 17.5; 35.0; and 70.0 µg/mL
EXPERIMENT 2:
- Without S9 mix: 5.0; 10.0; 20.0; 27.5; and 35.0 µg/mL
- With S9 mix: 8.8; 17.5; 35.0; 52.8; and 70.0 µg/mL - Vehicle / solvent:
- -Solvent Vehicle: Dimethyl sulfoxide (DMSO)
- Immediately before treatment, the test item was dissolved in DMSO (99.5%) to achieve a final DMSO concentration in culture medium of 0.5% (v/v). - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Remarks:
- In addition, a metabolic activation positive control, cyclophosphamide (CPA) was also tested.
- Details on test system and experimental conditions:
- Cell cultures were selectively grown ('cleansed') in RPMI 1640-HAT medium supplemented with hypoxanthine, aminopterin and thymidine, and screened for mycoplasma contamination and karyotype stability.
In order to test the effect of metabolism on the in vitro system. S9 liver fraction was prepared from Wistar Hanlhm rats exposed to phenobarbital (80 mg/kg bw) and β-naphthoflavone (80 mg/kg bw). The S9 supernatant was thawed and mixed with S9 cofactor solution to achieve final concentrations of 28.3 mg/mL and 31.0 mg/mL in experiment I and II, respectively.
Forty-eight-hour toxicity was observed at concentrations of 27.5 and 35.0 µg/mL without metabolic activation and at 105.0 µg/mL with metabolic activation (experiment I).
Method of determination of toxicity:cloning efficiency; relative total growth - Evaluation criteria:
- A test item is classified as mutagenic if the induced mutation frequency reproducibility exceeds a threshold of 126 colonies per 10 million cells above the solvent control, in a dose-dependent manner. The mutagenic response is considered to be reproducible if it occurs in both parallel cultures. Clastogenic effects are indicated by a dose-dependent shift in the ratio of small versus large colonies.
- Statistics:
- A linear regression (least squares) was performed to assess a possible dose-dependent increase of mutant frequencies using SYSTAT Softare.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- No substantial and reproducible dose dependent increase of the mutation frequency was observed in experiments with and without metabolic activation.
- Conclusions:
- The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the L5178Y cell line, in the absence or presence of S9 liver fraction metabolic acitvation.
- Executive summary:
The study was performed to investigate the potential of the test item to induce mutation at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. This in vitro test is an assay for the detection of forward gene mutations at the autosomal thymidine kinase (TK) locus of heterozygous L5178Y/TK+/- cells to TK-/- mutants. In addition, evidence has been obtained that small TK-/- colonies may result from chromosomal damage to the TK locus and adjacent genes. Gene and chromosome mutations are considered as an initial step in the carcinogenic process. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. Following a 48-hour phenotypic expression time, treated cell populations were monitored for the loss of the functional TK enzyme. TK catalyses the conversion of trifluorothymidine (TFT) to cytostatic and cytotoxic trifluorothymidine-monophosphate derivative. Therefore, cells deficient in TK due to a forward mutation are resistant to TFT. No substantial and reproducible dose dependent increase of the mutation frequency was observed in experiments with and without metabolic activation. It can be stated that under the experimental conditions defined, the test item did not induce mutation in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. This study is considered to be reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 476 and EU B.17.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Study conducted according to GLP, however no guideline reported. One strain (TA102 or E.coli) has been omitted based on current guideline recommendations and the plate incorporation method was used for both experiments; no analytical data on purity of the test substance was included.
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- No guidelines reported, however experimental design was similar to OECD 471 and EU B.13/14. The study deviates from the OECD guideline method in that one strain (TA 102 or E. coli WP2) has been omitted and the plate incorporation method was used for both experiments.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver (S9)
- Test concentrations with justification for top dose:
- Range-finding study: 19.5, 39, 78, 156, 312, 625, 1250, 2500, 5000 and 10000 µg/plate; a mild bacteriotoxic effect was observed at the two highest concentrations.
Definitive study: 8, 40, 200, 1000 and 5000 µg/plate - Vehicle / solvent:
- Ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- sodium azide
- other: 9-aminoacridinehydrochloride, 4-nitro-1,2-phenylene diamine, 2-aminoanthracene
- Remarks:
- Positive controls sodium azide, 9-aminoacridinehydrochloride and 4-nitro-1,2-phenylene diamine were used only without S 9 mix, and the positive control 2-aminoanthracene only with S 9 mix.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: 16 hours
- Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 4 plates per strain and dose, both with and without S 9 mix
NUMBER OF CELLS EVALUATED: 0.1 mL of bacterial suspension at 10E6 cells/mL - Evaluation criteria:
- The following criteria were used for the acceptance of an assay:
- The negative controls have to be within the expected range as defined by literature data (Maron and Ames 1983).
- The positive controls have to show sufficient effects as defined by the laboratories' experience.
- The titer determination must yield a sufficient bacterial density in the suspension.
A reproducible and dose related increase of mutant counts for at least one strain is considered positive. For TA 100, TA 98 andn TA 1535 a twofold increase of revertants compared to the negative controls should be reached, whereas for TA 1537 a threefold increase should be reached. Otherwise the results are estimated as negative. - Statistics:
- Not mandatory under OECD test guidleine 471. The mean values and standard deviations were calculated for the number of revertants formed per plate.
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: A mild bacteriotoxic effect was observed at the two highest concentrations.
- Conclusions:
- None of the four tester strains used showed any dose related and relevant increase in mutant counts over those of the negative controls at any concentration level, neither in the tests with nor without S9 mix. There was no indication of a bacteriotoxic effect of the test item in all of the employed doses and inhibition of bacterial growth was not noted.
- Executive summary:
The potential of the test item to induce gene mutations was assessed in a plate incorporation test using Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98. A range-finding study at ten concentrations ranging from 19.5 to 10000 µg/plate found bacteriotoxic effects at 5000 and 10000 µg/plate, the two highest concentrations tested. The doses for the definitive test were set at 8, 40, 200, 1000 and 5000 µg/plate. The test systems were evaluated with and without rat liver microsomal activation (S9) mix after 48 hours of exposure. None of the tester strains used showed any dose related and relevant increase in mutant counts over those of the negative controls at any concentration level, neither in the tests with or without S9 mix. This study is reliable with restrictions (Klimisch 2) as the study was conducted according to GLP, however no guideline reported. One strain (TA102 or E.coli) has been omitted based on current OECD 471 recommendations and the plate incorporation method was used for both experiments; no analytical data on purity of the test substance was included.
Referenceopen allclose all
Table 1. Summary results of pre-experiment and Experiment I
Metabolic activation |
Test Group |
Dose Level (µg/plate) |
Revertant Colony Counts (Mean ± SD) |
||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
|||
Without activation |
DMSO |
|
13 ± 3 |
17 ± 3BM |
31 ± 5 |
104 ± 8BM |
526 ± 53 |
Untreated |
|
12 ± 2 |
14 ± 4BM |
32 ± 6 |
129 ± 7BM |
467 ± 24 |
|
Farnesol |
3 |
13 ± 0 |
15 ± 3BM |
29 ± 6 |
106 ± 10BM |
449 ± 21 |
|
|
10 |
15 ± 3 |
12 ± 1BM |
37 ± 12 |
12 ± 1BM |
471 ± 86 |
|
|
33 |
10 ± 6 |
6 ± 2BM |
27 ± 2 |
6 ± 2BM |
465 ± 36 |
|
|
100 |
16 ± 5MR |
6 ± 2BM |
27 ± 3 |
6 ± 2BM |
402 ± 25 |
|
|
333 |
10 ± 4RM |
7 ± 4BM |
26 ± 2 |
7 ± 4BM |
357 ± 11 |
|
|
1000 |
7 ± 1MR |
9 ± 5BM |
19 ± 0 |
9 ± 5BMR |
352 ± 21 |
|
|
2500 |
4 ± 2PMR |
6 ± 3PMB |
17 ± 4PM |
6 ± 3PMBR |
186 ± 13PM |
|
|
5000 |
4 ± 3PMR |
5 ± 2PMB |
13 ± 2PM |
5 ± 2PMB |
172 ± 16PM |
|
NaN3 |
10 |
2176 ± 87 |
|
|
2024 ± 85 |
|
|
4-NOPD |
10 |
|
|
432 ± 7BM |
|
|
|
4-NOPD |
50 |
|
117 ± 8BM |
|
|
|
|
MMS |
3.0 |
|
|
|
|
4424 ± 108 |
|
With activation |
DMSO |
|
24 ± 3 |
21 ± 6BM |
41 ± 6 |
109 ± 10BM |
551 ± 27 |
Untreated |
|
20 ± 6 |
17 ± 3BM |
35 ± 1 |
111 ± 10BM |
554 ± 21 |
|
Farnesol |
3 |
23 ± 12 |
17 ± 6BM |
36 ± 8 |
111 ± 10BM |
562 ± 74 |
|
|
10 |
24 ± 5 |
21 ± 4BM |
33 ± 10 |
103 ± 6BM |
532 ± 22 |
|
|
33 |
20 ± 6 |
16 ± 6BM |
49 ± 2 |
107 ± 12BM |
544 ± 23 |
|
|
100 |
20 ± 7 |
15 ± 6BM |
43 ± 4 |
63 ± 6BM |
526 ± 13 |
|
|
333 |
13 ± 2 |
17 ± 3BM |
28 ± 5 |
56 ± 7BM |
398 ± 39 |
|
|
1000 |
13 ± 1 |
13 ± 6BM |
30 ± 6 |
28 ± 2BMR |
379 ± 16 |
|
|
2500 |
5 ± 2PM |
7 ± 3PMB |
18 ± 1P |
21 ± 2PMBR |
215 ± 10PM |
|
|
5000 |
7 ± 1PM |
14 ± 1PMB |
19 ± 6P |
18 ± 2PMB |
223 ± 11PM |
|
2-AA |
2.5 |
308 ± 14 |
301 ± 11BM |
1686 ± 122 |
2383 ± 187 |
|
|
2-AA |
10.0 |
|
|
|
|
2214 ± 287 |
NaN3= Sodium azide
2-AA = 2-aminoanthracene
MMS = Methyl methane sulfonate
4-NOPD = 4-Nitro-o-phenylene-diamine
B = Extensive bacterial growth
M = Manual count
R = Reduced background growth
P = Precipitate
Table 2. Summary of Results Pre-Experiment and Experiment Ia
Metabolic activation |
Test Group |
Dose Level (µg/plate) |
Revertant Colony Counts (Mean ± SD) |
TA 1537 |
|||
Without activation |
DMSO |
|
15 ± 2 |
Untreated |
|
19 ± 2 |
|
Farnesol |
3 |
16 ± 4 |
|
|
10 |
13 ± 5 |
|
|
33 |
10 ± 2 |
|
|
100 |
8 ± 2 |
|
|
333 |
6 ± 3 |
|
|
1000 |
6 ± 2MR |
|
|
2500 |
3 ± 2MR |
|
|
5000 |
1 ± 1MR |
|
4-NOPD |
50 |
143 ± 17 |
4-NOPD = 4-Nitro-o-phenylene-diamine
M = Manual count
R = Reduced background death
Table 3. Summary of Results Experiment II
Metabolic activation |
Test Group |
Dose Level (µg/plate) |
Revertant Colony Counts (Mean ± SD) |
||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
|||
Without activation |
DMSO |
|
14 ± 1 |
11 ± 3 |
42 ± 4 |
118 ± 15 |
413 ± 9 |
Untreated |
|
13 ± 2 |
13 ± 8 |
24 ± 2 |
130 ± 10 |
485 ± 9 |
|
Farnesol |
3 |
13 ± 2 |
15 ± 4 |
25 ± 4 |
119 ± 5 |
437 ± 14 |
|
|
10 |
10 ± 2 |
13 ± 7 |
24 ± 3 |
99 ± 11 |
442 ± 23 |
|
|
33 |
15 ± 4 |
6 ± 6 |
19 ± 4 |
64 ± 7 |
406 ± 43 |
|
|
100 |
14 ± 2 |
13 ± 1 |
17 ± 5 |
71 ± 6 |
273 ± 13 |
|
|
333 |
3 ± 1MR |
6 ± 1MR |
25 ± 3MR |
59 ± 24MR |
379 ± 13MR |
|
|
1000 |
3 ± 2MR |
2 ± 3MR |
21 ± 3MR |
39 ± 8MR |
356 ± 41MR |
|
|
2500 |
0 ± 0MR |
0 ± 0MR |
11 ± 2MR |
22 ± 6MR |
123 ± 18MR |
|
|
5000 |
0 ± 1MR |
0 ± 0MR |
4 ± 2MR |
14 ± 1MR |
64 ± 8MR |
|
NaN3 |
10 |
2184 ± 27 |
|
|
2117 ± 79 |
|
|
4-NOPD |
10 |
|
|
514 ± 78 |
|
|
|
4-NOPD |
50 |
|
137 ± 27 |
|
|
|
|
MMS |
3.0 µL |
|
|
|
|
3301 ± 380 |
|
With activation |
DMSO |
|
13 ± 1 |
16 ± 1 |
30 ± 5 |
143 ± 17 |
607 ± 38 |
Untreated |
|
12 ± 1 |
20 ± 8 |
36 ± 1 |
148 ± 12 |
678 ± 42 |
|
Farnesol |
3 |
9 ± 4 |
18 ± 3 |
40 ± 9 |
148 ± 14 |
604 ± 19 |
|
|
10 |
10 ± 2 |
17 ± 8 |
43 ± 3 |
154 ± 11 |
568 ± 47 |
|
|
33 |
9 ± 2 |
16 ± 3 |
34 ± 3 |
151 ± 7 |
648 ± 23 |
|
|
100 |
8 ± 2 |
10 ± 4 |
30 ± 7 |
121 ± 13 |
588 ± 47 |
|
|
333 |
2 ± 1MR |
6 ±2MR |
34 ± 6MR |
88 ± 10MR |
523 ± 56MR |
|
|
1000 |
3 ± 3MR |
3 ± 1MR |
34 ± 1MR |
32 ± 4MR |
432 ± 67MR |
|
|
2500 |
2 ± 2MR |
1 ± 2MR |
13 ± 6MR |
22 ± 4MR |
174 ± 13MR |
|
|
5000 |
1 ± 1MR |
3 ± 3MR |
6 ± 1MR |
19 ± 2MR |
118 ± 18MR |
|
2-AA |
2.5 |
891 ± 30 |
266 ± 35 |
1858 ± 1207 |
2576 ± 61 |
|
|
2-AA |
10.0 |
|
|
|
|
2805 ± 70 |
NaN3= Sodium azide
2-AA = 2-aminoanthracene
MMS = Methyl methane sulfonate
4-NOPD = 4-Nitro-o-phenylene-diamine
B = Extensive bacterial growth
M = Manual count
R = Reduced background growth
P = Precipitate
Table 1. Summary of results of the chromosomal aberration study
|
* Inclusive cells carrying exchanges
** Evaluation of 200 metaphases per culture
S Aberration frequency statistically significant higher than corresponding control values
1 DMSO 0.5% (v/v)
2 EMS 825.0 µg/mL
3 EMS 660.0 µg/mL
4 EMS 770.0 µg/mL
5 CPA 22.5 µg/mL
Table 1. Summary of results
|
p = precipitation
Threshold = number of mutant colonies per 106cells of each solvent control plus 126
* Culture was not continued due to exceedingly severe toxic effects
** Culture was not continued since a minimum of four concentrations is required by the guidelines
The values printed in bold italics are judged as invalid, since the acceptance criteria are not met
|
Salmonella typhimurium TA 1535 |
|||||
Dose µg/plate |
S-9 mix |
Revertants/plate |
x |
SD |
Quotient |
In the absence of an S-9 metabolising system |
|||||
0 |
- |
13/21/14/20 |
17.00 |
4.10 |
1.00 |
8 |
- |
17/14/8/13 |
13.00 |
3.74 |
0.76 |
40 |
- |
11/6/8/11 |
9.00 |
2.45 |
0.52 |
200 |
- |
8/6/11/9 |
8.50 |
2.08 |
0.50 |
1000 |
- |
10/12/4/8 |
8.50 |
3.41 |
0.50 |
5000 |
- |
3/15/7/9 |
8.50 |
5.00 |
0.50 |
10µg Na azid |
- |
2240/2500/2300/2200 |
2310 |
133 |
136 |
Presence of an S-9 metabolising system |
|||||
0 |
+ |
20/17/10/20 |
16.75 |
4.71 |
1.00 |
8 |
+ |
19/21/19/15 |
18.50 |
2.51 |
1.05 |
40 |
+ |
18/30/18/20 |
21.50 |
5.74 |
1.23 |
200 |
+ |
26/15/16/25 |
20.50 |
5.80 |
1.17 |
1000 |
+ |
9/18/12/* |
13.00 |
4.58 |
0.76 |
5000 |
+ |
18/11/13/* |
14.00 |
3.60 |
0.82 |
2µg 2AA * unsterile |
+ |
160/152/198/* |
170 |
24.57 |
10.0 |
|
|
|
|
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Genetic toxicology assesses the interaction of agents with DNA to produce gene mutations or chromosome alterations. Whilst mutagenicity refers to the production of transmissible genetic alteration, genotoxicity covers a broader spectrum of endpoints, including unscheduled DNA synthesis (UDS), sister chromatid exchanges (SCEs) and DNA strand breaks as measures of genotoxicity.
Genetic toxicity in vitro: A total of four Annex VII and Annex VIII in vitro genetic toxicity studies have been conducted with the test item. In the key chromosome aberration test using human lymphocytes, and at a range of test item dose concentrations including cytotoxic doses, the number of induced structural chromosome aberrations was within the historical control data when tested in the presence and absence of an exogenous S9 metabolic activation system (2008). Thus no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. This study is reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 473 and EU B.10.
An in vitro mammalian cell gene mutation assay conducted according to OECD 476 showed no substantial or reproducible dose dependent increases in the mutation frequency in the absence and presence of metabolic activation (2008). Under the experimental conditions, the test item did not induce mutations at the mouse lymphoma thymidine kinase locus in the L5178Y cell line. This study is considered to be reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 476 and EU B.17.
A Bacterial Reverse Mutation Assay (Ames test) was conducted to evaluate the potential of the test item to induce gene mutations Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98 (2008). There was no indication of a bacteriotoxic effects in any of the employed doses, and inhibition of bacterial growth was not noted. Under the experimental conditions the test substance did not induce gene mutations by base pair or frameshift in the genome of the species tested. The test item is not considered to be mutagenic in the Salmonella typhimurium reverse transcription assay in the presence or absence of a metabolising system. This study is reliable without restrictions (Klimisch 1) as it is GLP-compliant and was conducted according to OECD 471 and EU B.13/14. This conclusion is supported by another Ames assay conducted with Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98 in a plate incorporation test (1989). None of the strains tested showed any dose related and relevant increase in mutant counts over those of the negative controls at any concentration level, neither in the tests with or without S9 mix. This study is reliable with restrictions (Klimisch 2) as the study was conducted according to GLP, however no guideline reported and there were minor restrictions in experimental design and reporting.
Genetic toxicity in vivo: No positive results were observed in any of the genotoxicity studies required for the Annex VII or VIII information requirements under REACH, therefore in vivo data for genetic toxicity is not required.
Justification for classification or non-classification
Since the results from all in vitro experimental studies were negative, it can be concluded that there is no evidence of relevant intrinsic genotoxic properties that would initiate classification. Consequently, no further testing to include in vivo studies was justified and the substance was not classifiable as mutagenic according to CLP Regulation (EC) No. 1272/2008.
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.