Registration Dossier
Registration Dossier
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EC number: 203-929-1 | CAS number: 112-03-8
- 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
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- Nanomaterial pour density
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- 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
Based on the results of the test substance and read across in vitro genotoxicity studies, the test substance is considered to have no genotoxic potential.
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:
- From December 07, 1994 to December 16, 1994
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- 0.8, 4, 20, 100, 500 and 2,500 µg/plate (for both with and without metabolic activation)
- Vehicle / solvent:
- Ethanol
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- Tested for TA 100, TA 1535 in the absence of S9-mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Tested for TA 1537 in the absence of S9-mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- Tested for TA 98 in the absence of S9-mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-amino anthracene
- Remarks:
- Tested for TA 100, TA 1535, TA 1537, TA 98 in presence of S9-mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In agar (plate incorporation)
DURATION:
- Preincubation period: 48h - Evaluation criteria:
- The test substance is classified as mutagenic if it has either of the following effects:
- 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn.
- Dose related increase in the mean number of revertants per plate of atleast one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in atleast two to three concentration of the test substance at complete bacterial background lawn. - Key result
- Species / strain:
- S. typhimurium TA 1535
- 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:
- 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:
- S. typhimurium TA 98
- 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:
- S. typhimurium TA 100
- 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
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- not determined
- Remarks:
- Conducted according to old OECD guideline
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Additional information on results:
- Solubility: Visible precipitation of the test substance on the plates was observed at 5,000 µg/plate in the first experiment.
Toxicity: The test substance was toxic to most of the bacterial strain at doses of 100 µg/plate and above. Thinning of the bacterial lawn and a reduction in the number of colonies were observed at this dose. - Conclusions:
- Under the study conditions, the test substance was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100 or TA1535, with or without metabolic activation.
- Executive summary:
A study was conducted to determine the in vitro genetic toxicity of the test substance, C18 TMAC (79.8% active) according to a method similar to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were treated with the test substance using the Ames plate incorporation method. Tests were carried out in triplicate, both with and without the addition ofmetabolic activation (S9-mix). The concentration range of the test substance was 0.8 to 2500 µg/plate. Cytotoxicity was observed at ≥100 µg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains at any of the test substance concentrations tested, either with or without metabolic activation. Under the study conditions, the test substance was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100 or TA1535, with or without metabolic activation (Muller, 1995).
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Study period:
- From 21 August, 2006 to 01 November, 2006
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study was conducted according to the OECD guideline 471 and Commission Directive 2000/32/EC, Annex-4D as well as in compliance with GLP. KL2 due to RA
- Justification for type of information:
- Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Directive 2000/32/EC, L1362000, Annex 4D
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- 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:
- S9 mix
- Test concentrations with justification for top dose:
- Experiment I (with and without S9 mix): 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/plate
Experiment II (with and without S9 mix): 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: Deionised water was chosen because of its solubility properties and its low toxicity to the bacteria. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Deionised water
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- 10 µg/plate for TA 1535 and TA 100 (Without metabolic activation)
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Deionised water
- Positive controls:
- other:
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine, 10 µg/plate for TA 98, 50 µg/plate for TA 1537 (Without metabolic activation)
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Deionised water
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- 3 µL/plate for WP2 uvrA (Without metabolic activation)
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Deionised water
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene, 2.5 µg/plate for TA 1535, TA 1537, TA 98, TA 100 and 10 µg/plate for WP2 uvrA (With metabolic activation)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: Triplicates - Evaluation criteria:
- A test item is considered as a mutagen if there is a biologically relevant increase in the number of revertants exceeding the threshold by twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)
- Vehicle 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:
- cytotoxicity
- Remarks:
- Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)
- Vehicle 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:
- cytotoxicity
- Remarks:
- Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Under the study conditions, the test substance was non-mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2 uvrA in the reverse mutation assay with and without metabolic activation.
- Executive summary:
An in vitro study was conducted to investigate the potential of read across substance, C16 TMAC (25% active in water) to induce gene mutations Salmonella typhimurium strains, according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA were treated with the read across substance using the Ames plate incorporation (pre-experiment and experiment I) and pre-incubation methods (experiment II). The assay was performed in three independent experiments both with and without metabolic activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The concentrations of the read across substance ranged from 3 to 5000 µg/plate in the pre-experiment, from 0.1 to 333 µg/plate in experiment I and from 0.01 to 333 µg/plate in experiment II. The plates incubated with the read across substance showed reduced background growth in all strains with and without metabolic activation in all experiments. Strong cytotoxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups with and without S9 mix. No substantial increase in revertant colony numbers in any of the five tester strains was observed following treatment with the read across substance either in the presence or absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. All positive control showed a distinct increase in the number of revertant colonies. Under the study conditions, the substance was determined to be non-mutagenic with and without metabolic activation in the reverse mutation assay (Sokolowski, 2006). Based on the results of the read across study, similar result can be expected for the test substance.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From April 17, 1989 to September 15, 1989
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Toxicity test guideline, Japan 1984
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM medium supplemented with 10% foetal calf Serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- without S9 mix: (7 hours: 1.0 µg/mL; 18 hours: 0.3, 1.0 and 3.0 µg/mL; 28 hours: 3.0 µg/mL)
with S9 mix: (7 hours: 10.0 µg/mL; 18 hours: 1.0, 3.0 and 10.0 µg/mL; 28 hours: 10.0 µg/mL) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- with metabolic activation
- Positive control substance:
- cyclophosphamide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h
- Fixation time (start of exposure up to fixation or harvest of cells): 7h (high dose), 18h (low, medium and high dose), and 28h (high dose)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (approx. 0.2 µg/mL/culture medium)
STAIN (for cytogenetic assays): Giemsa stains
NUMBER OF REPLICATIONS: Two
NUMBER OF CELLS EVALUATED: 100 cells of each cell culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Evaluation criteria:
- - A test article is classified as clastogenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
- A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-clastogenic in this system. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- 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:
- - In the pre-experiments for toxicity the colony forming ability of the V79 cells was totally reduced after treatment with 6.0 µg/mL. Accordingly, one (for 7 and 28h time point) and three concentrations (for 18h time point) were selected to evaluate metaphases for cytogenetic damage. Mitotic index was reduced after treatment with the highest dose levels in the absence and presence of S9 mix, in the main test.
- Mutation results:
There was no relevant increase in cells with structural aberrations after treatment with the test substance at any fixation interval either without or with metabolic activation by S9 mix. Positive controls showed distinct increases in cells with structural chromosome aberrations. The sensitivity of the test system and efficacy of the S9 mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
(COMPARISON WITH HISTORICAL CONTROL DATA: Yes) - Conclusions:
- Under the study conditions, the read across substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation.
- Executive summary:
An in vitro study was conducted to investigate the potential of test substance, C16 TMAC (24 -26% active in water) to induce chromosome aberrations in V79 Chinese hamster lung cells, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. The concentration range of the test substance was determined in a pre-experiment using the plating efficiency assay as an indicator of toxicity response. Cells were exposed for 7, 18 or 28 h at concentrations levels of 0.3 to 10.0 µg a.i./mL test substance with or without metabolic activation. Treatment with 3.0 µg/mL and 10.0 µg a.i./mL completely reduced the plating efficiency of the V79 cells. The mitotic index was reduced after treatment with the highest concentration at each fixation interval in the presence and absence of S9 mix. Positive controls showed a distinct increase in the number of cells with structural chromosome aberrations. There was no relevant increase in cells with structural aberrations after treatment with the test substance at any fixation interval either without or with S9 mix. Under the study conditions; the test substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation (Heidemann, 1989).
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Study period:
- From August 30, 2006 to March 12, 2007
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- KL2 due to RA
- Justification for type of information:
- Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Directive 2000/32/EC, L1362000, Annex 4E
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (minimal essential medium) supplemented with 10% foetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Experiment I: without S9 mix: 0.2, 0.4, 0.8, 1.5 and 2.3 µg/mL, with S9 mix: 1.6, 3.1, 6.3, 12.5 and 18.8 µg/mL
Experiment II:without S9 mix: 0.4, 0.8, 1.6, 3.1 and 4.7 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: deionised water
- Untreated negative controls:
- yes
- Remarks:
- Untreated control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Deionised water
- True negative controls:
- other: Untreated cells were cultivated without interruption throughout the assay and without addition of test item, in order to obtain the initial spontaneous mutation rate at the beginning of the experiments.
- Positive controls:
- yes
- Remarks:
- Without metabolic activation
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- yes
- Remarks:
- Untreated control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Deionised water
- True negative controls:
- other: Untreated cells were cultivated without interruption throughout the assay and without addition of test item, in order to obtain the initial spontaneous mutation rate at the beginning of the experiments.
- Positive controls:
- yes
- Remarks:
- With metabolic activation
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Seeded into plastic culture flasks
DURATION
- Preincubation period: 24h
- Exposure duration: 4h (in experiment I; with and without S9), 24h (in experiment II; without S9)
- Expression time (cells in growth medium): 7d
- Selection time (if incubation with a selection agent): Day 7
SELECTION AGENT (mutation assays): Thioguanine (6TG)
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- The test item was considered positive if (a) It reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment. (b) There is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants. (c) Survival of the responding dose group is at least 30%. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.
- Statistics:
- A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects
- Precipitation: No precipitation or phase separation of the test substance was observed up to the maximum concentration in both main experiments.
RANGE-FINDING/SCREENING STUDIES: Two range finding pre-tests were performed in the presence (4h treatment) and absence (4h and 24h treatment) of S9. In the first pre-test test substance concentrations between 25 and 3200 µg/mL (active substance) were used to evaluate toxicity. In this first pretest strong toxic effects were noted at all concentrations with and without metabolic activation. Therefore, a second pre-test was performed using concentrations of 0.2 to 25 µg/mL (active substance).
Following 4h treatment without S9 mix strong toxicity occurred at 1.58 µg/mL. The cell growth was completely stopped at the next higher concentration of 3.13 µg/mL and above. In the presence of S9 mix (4h treatment) strong toxicity was determined at the highest concentration of 25 µg/mL. After 24h of treatment a relevant toxic effect occurred at 3.13 µg/mL. At all higher concentrations the cell growth was also completely inhibited.
COMPARISON WITH HISTORICAL CONTROL DATA: Yes - Conclusions:
- Under the study conditions, the read across substance did not induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation.
- Executive summary:
An in vitro study was conducted to investigate the potential of C16 TMAC (25% active in water) to induce gene mutations at the HPRT locus in V79 Chinese hamster cells, according to OECD Guideline 476, in compliance with GLP. The assay was performed in two independent experiments. The cells were exposed to the test substance for 4 h in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The concentrations of the test substance in experiment I ranged from 1.6 to 5 µg a.i./mL and 0.2 to 3 µg a.i./mL with and without S9 mix respectively and from 0.4 to 6.3 µg a.i./mL without S9 mix in experiment II. All positive controls showed a distinct increase in the number of mutant colonies. No substantial and reproducible concentration-dependent increases in the mutation frequency at the HPRT locus, was seen in test substance-treatment cells either with or without metabolic activation. Under the study conditions, the test substance did not induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation (Wollny, 2007).
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The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of S-9 Mix. No dose dependent effect was obtained. The test was performed in two independent experiments. It was concluded that the test substance not mutagenic in these bacterial test systems either in the absence or in the presence of an exogenous metabolizing system.
The sensitivity of the test system and efficacy of the S9 mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
Mutation results:
Main experiment:
- No relevant and reproducible increase of the mutation frequency occurred at any concentration with and without metabolic activation. All mutant frequencies remained well within the historical range of negative and solvent controls.
- A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in both cultures of experiment I with metabolic activation. However, a small increase of the mutation frequency at toxic concentrations is common in this assay system and does not indicate a possible mutagenic potential provided that the mutation frequency does not exceed the threshold of 3 times above the corresponding solvent control. Since the mutation frequency neither exceeded the historical range of negative and solvent controls nor the threshold as indicated above, the statistical results were considered as biologically irrelevant.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Study 1: A study was conducted to determine thein vitrogenetic toxicity of the test substance, C18 TMAC (79.8% active) according to a method similar to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were treated with the test substance using the Ames plate incorporation method. Tests were carried out in triplicate, both with and without the addition ofmetabolic activation (S9-mix). The concentration range of the test substance was 0.8 to 2500 µg/plate. Cytotoxicity was observed at ≥100 µg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains at any of the test substance concentrations tested, either with or without metabolic activation. Under the study conditions, the test substance was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100 or TA1535, with or without metabolic activation (Muller, 1995).
Study 2: An in vitro study was conducted to investigate the potential of read across substance, C16 TMAC (25% active in water) to induce gene mutations Salmonella typhimurium strains, according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA were treated with the read across substance using the Ames plate incorporation (pre-experiment and experiment I) and pre-incubation methods (experiment II). The assay was performed in three independent experiments both with and without metabolic activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The concentrations of the read across substance ranged from 3 to 5000 µg/plate in the pre-experiment, from 0.1 to 333 µg/plate in experiment I and from 0.01 to 333 µg/plate in experiment II. The plates incubated with the read across substance showed reduced background growth in all strains with and without metabolic activation in all experiments. Strong cytotoxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups with and without S9 mix. No substantial increase in revertant colony numbers in any of the five tester strains was observed following treatment with the read across substance either in the presence or absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. All positive control showed a distinct increase in the number of revertant colonies. Under the study conditions, the substance was determined to be non-mutagenic with and without metabolic activation in the reverse mutation assay (Sokolowski, 2006). Based on the results of the read across study, similar result can be expected for the test substance.
Study 3: An in vitro study was conducted to investigate the potential of read across substance, C16 TMAC (24 -26% active in water) to induce chromosome aberrations in V79 Chinese hamster lung cells, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. The concentration range of the read across substance was determined in a pre-experiment using the plating efficiency assay as an indicator of toxicity response. Cells were exposed for 7, 18 or 28 h at concentrations levels of 0.3 to 10.0 µg a.i./mL read across substance with or without metabolic activation. Treatment with 3.0 µg/mL and 10.0 µg a.i./mL completely reduced the plating efficiency of the V79 cells. The mitotic index was reduced after treatment with the highest concentration at each fixation interval in the presence and absence of S9 mix. Positive controls showed a distinct increase in the number of cells with structural chromosome aberrations. There was no relevant increase in cells with structural aberrations after treatment with the read across substance at any fixation interval either without or with S9 mix. Under the study conditions, the read across substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation (Heidemann, 1989).
Study 4: An in vitro study was conducted to investigate the potential of C16 TMAC (25% active in water) to induce gene mutations at the HPRT locus in V79 Chinese hamster cells, according to OECD Guideline 476, in compliance with GLP. The assay was performed in two independent experiments. The cells were exposed to the read across substance for 4 h in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The concentrations of the read across substance in experiment I ranged from 1.6 to 5 µg a.i./mL and 0.2 to 3 µg a.i./mL with and without S9 mix respectively and from 0.4 to 6.3 µg a.i./mL without S9 mix in experiment II. All positive controls showed a distinct increase in the number of mutant colonies. No substantial and reproducible concentration-dependent increases in the mutation frequency at the HPRT locus, was seen in read across substance-treatment cells either with or without metabolic activation. Under the study conditions, the read across substance did not induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation (Wollny, 2007).
Based on the results of the test substance and read across in vitro genotoxicity studies, the test substance is considered to have no genotoxicity potential.
Justification for classification or non-classification
Based on the results of the test substance and read acrossin vitrogenotoxicity studies, the test substance does not warrant a classification for genotoxicity according to the EU CLP criteria (Regulation 1272/2008/EC).
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.
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