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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 209-294-7 | CAS number: 565-80-0
- 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
A bacterial reverse mutation assay was conducted GLP-compliant according to OECD Testguideline 471. The test was performed with and without metabolic activation in S. typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA strains. There was no mutagenic effect of test material in the presence or absence of metabolic activation in the Standard Plate Test, nor in the Preincubation Test with or without metabolic activation.
An in vitro mammalian cell micronucleus test was conducted GLP-compliant according to OECD Testguideline 487. The test was performed with and without metabolic activation in human lymphoblastoid cells (TK6). The test material induced micronuclei in the in vitro assay. As the test material contains 0.3 % of a substance with a potent structural alert, it is possible that the result applies to the impurity rather than the main component.
The potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus was investigated GLP-compliant in Chinese hamster ovary (CHO) cells with and without metabolic activation in vitro according to OECD Testguideline 476 (In Vitro Mammalian Cell Gene Mutation Test). No cytotoxicity was observed up to the highest concentrations evaluated for gene mutations. The test substance did not cause any biologically relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system.
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:
- Experimental starting date (1st administration of the test substance): 17 May 2017; Experimental completion date (evaluation last experiment): 23 Jun 2017
- 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:
- 21 Jul 1997
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- No L 142
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- Aug 1998
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Name of test substance (as cited in report): Diisopropyl Ketone; 2,4-Dimethyl-3-Pentanone
- Appearance: slightly yellowish liquid
- Batch Number: 151005
- Purity: 99.2%
- Water content: 0.1%
- Impurities: 0.3% 2,4-dimethylpent-1-en-3-one and 0.1% 2-methylpentan-3-one - Target gene:
- Histidine revertants (for Salmonella typhimurium strains)
tryptophan revertants (for Escherichia coli strain) - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 (rat liver), phenobarbital and ß-naphthoflavone induced
- Test concentrations with justification for top dose:
- 1st experiment: 0; 33; 100; 333; 1000; 2500 and 5000 µg/plate
2nd experiment: The doses, strains and test conditions were selected depending on the results of the first investigation. In case of negative results in the standard plate test the preincubation test will be carried out as 2nd Experiment. - Vehicle / solvent:
- Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (2-AA)
- Remarks:
- With S-9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- Remarks:
- Without S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylenediamine (NOPD)
- Remarks:
- Without S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- Remarks:
- Without S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- Without S9
- Details on test system and experimental conditions:
- STANDARD PLATE TEST:
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al.
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCI) and 10 mL amino acid solution wiere kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation) or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his+ / trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive lnstruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Number of plates: 3 test plates per dose or per control
PREINCUBATION TEST: In case of negative findings in the standard plate test, a repeat experiment following the preincubation method was performed to confirm the data.
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al.
0.1 mL test solution or vehicle (negative control), 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) was incubated at 37 °C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
DETERMINATION OF CYTOTOXICITY:
Toxicity detected by a;
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth) was recorded for all test groups both with and without S9 mix in all experiments and was indicated in the tables. Single values with a factor ≤. 0.6 were not been detected as toxicity in low dose groups.
SOLUBILITY:
If precipitation of the test substance occured, it was recorded and indicated in the tables. As long as precipitation does not interfere with the colony scoring, 5 mg/plate, regarding the purity of the test substance, were generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/ substantiation. - Evaluation criteria:
- Mutagenicity
lndividual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative controls in all experiments. 6 doses of the test substance were tested with a maximum of 5 mg/plate regarding the purity of the test substance. Triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.
Acceptance criteria
The experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls has to be within the range of the historical negative control data for each tester strain.
• The sterility controls has to reveal no indication of bacterial contamination.
• The positive control substances both with and without S9 mix have to induce a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture has to be approx. 10E+9 cells per mL.
Assessment criteria
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA98, TA100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA1535 and TA1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains is within the historical negative control data range under all experimental conditions in at least 2 experiments carried out independently of each other. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test substance is insoluble in water; therefore, DMSO was used as vehicle
- Precipitation: No test substance precipitation was found with and without S9 mix.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Without S9 mix
TA 1535 (MNNG): 3967 (1541 - 6171, SD 1253.9)
TA 100 (MNNG): 3298 (1126 - 5557, SD 1109.6)
TA 1537 (AAC): 1044 (253 - 2190, SD 404.3)
TA 98 (NOPD): 863 (324 - 1746, SD 206.8)
E.coli (4-NQO): 860 (164 - 1721, SD 431.9)
With S9 mix
TA 1535 (2-AA): 197 (105 - 520, SD 56.3)
TA 100 (2-AA): 1748 (272 - 3021, SD 587.2)
TA 1537 (2-AA): 141 (50 - 399, SD 50.3)
TA 98 (2-AA): 1524 (493 - 3096, SD 529.1)
E.coli (2-AA): 133 (61 - 537, SD 61.8)
- Negative (solvent/vehicle) historical control data:
Without S9 mix
TA 1535: 10 (7 - 16, SD 2.0)
TA 100: 100 (71 - 132, SD 11.4)
TA 1537: 8 (5 - 13, SD 1.7)
TA 98: 21 (14 - 34, SD 3.3)
E.coli: 24 (15 - 34, SD 3.9)
With S9 mix
TA 1535: 10 (6 - 18, SD 2.0)
TA 100: 107 (70 - 147, SD 13.7)
TA 1537: 9 (5 - 16, SD 2.1)
TA 98: 28 (12 - 38, SD 4.5)
E.coli: 25 (17 - 36, SD 4.4) - Conclusions:
- Under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.
Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA
Dose range: 33 - 5000 µg/plate (SPT and PIT)
Test conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).
Solubility: No precipitation of the test substance was found with and without S9 mix.
Toxicity: No bacteriotoxic effect was observed.
Mutagenicity: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental starting date: 21 April 2017 ; Experimental completion date: 02 June 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2016
- Deviations:
- yes
- Remarks:
- A second 3+27 hour treatment in the presence of S-9 was performed
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- - Name of test substance (as cited in report): Diisopropyl Ketone; 2,4-Dimethyl-3-Pentanone
- Appearance: slightly yellowish liquid
- Batch Number: 151005
- Purity: 99.2%
- Water content: 0.1%
- Impurities: 0.3% 2,4-dimethylpent-1-en-3-one and 0.1% 2-methylpentan-3-one - Species / strain / cell type:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 Fraction, Aroclor induced
- Test concentrations with justification for top dose:
- Preliminary solubility assessments were made, a vehicle chosen and dilutions arranged that allow maximum exposure up to the solubility limit, 10 mM or 2000 μg/mL, whichever is lower. For freely soluble test articles, where the molecular weight is unknown, the highest concentration tested is 2000 μg/mL (OECD, 2016).
If there is no information to satisfactorily define the concentrations to be tested in the Micronucleus Experiment, a preliminary toxicity Range-Finder Experiment was performed, using a series of concentrations separated by approximately two-fold intervals, ranging down from the upper limit. All concentration ranges are detailed in the raw data. For freely soluble non-toxic test articles in aqueous solution, where the molecular weight is unknown, the following concentration ranges may be tested.
Range finder experiment:
Concentration of treatment solution (mg/mL) 0.7256, 0.1209, 0.2016, 0.3359, 0.5599, 0.9331, 1.555, 2.592, 4.32, 7.2, 12, 20
Final Concentration (μg/mL): 7.256, 12.09, 20.16, 33.59, 55.99, 93.31, 155.5, 259.2, 432, 720, 1200, 2000
Micronucleus Experiment(s):
To be determined, based on range finding experiment. Concentrations are 10-fold more than those stated to achieve the indicated final concentrations if the vehicle is organic. An appropriate number of test article concentrations was selected for the Micronucleus Experiments, in order that a suitable range of cytotoxicity was achieved. - Vehicle / solvent:
- Vehicle controls comprised treatments with the chosen vehicle diluted to the same extent as the test article solutions. The preferred vehicles are water or dimethyl sulphoxide (DMSO). The choice of vehicle is confirmed in the raw data.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Noscapine
- Details on test system and experimental conditions:
- Cell treatment and harvest times
To provide an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment and to ensure that only cells that divided during or after treatment are microscopically scored cytochalasin B is added to the cultures.
The proposed treatment condidtions are as follows:
+ S9 short treament: Cells are exposed for 3 hours. Upon ending of the exposure duration S9 and the test substance are removed and Cytochalasin B added. 27 hours later cells are harvested.
- S9 short treament: Cells are exposed for 3 hours. Upon ending of the exposure duration the test substance is removed and Cytochalasin B added. 27 hours later cells are harvested.
- S9 extended treament: Cells are exposed for 30 hours in the presence of Cytochalasin B. After the exposure period cells are harvested
+ S9 extended treament: Cells are exposed for 30 hours in the presence of S9 and Cytochalasin B. After the exposure period cells are harvested.
Osmolality and pH:
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit can give rise to chromosome aberrations (Scott et al., 1991; Brusick, 1986). Therefore, osmolality and the effect of the test article on the pH of the culture medium, are assessed during the study. Osmolality and pH are initially measured on post-treatment media in the cytotoxicity Range-Finder Experiment. If marked changes are observed, further measurements may be made in the Micronucleus Experiment(s).
Harvesting:
At the defined sampling time, cultures were:
• Centrifuged at approx. 200 g, 5 minutes
• Resuspended in 4 mL (hypotonic) 0.075 M KCl at 37°C for 4 minutes
• Fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (7:1 v/v), centrifuged (approx. 200 g, 5 minutes) and resuspended, followed by further centrifugation and fixation (approx. 1250 g, 2-3 minutes) until the cell pellets are clean
• Stored in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours.
Slide Preparation:
• Cells centrifuged (approx. 1250 g, 2-3 minutes) and resuspended in a minimal amount of fresh fixative (if required)
• Several drops of suspension gently spread onto multiple clean, dry microscope slides
• Slides air-dried then stored protected from light at room temperature prior to staining
• Slides stained by immersion in 12.5 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes, following by a wash with PBS (with agitation) for a few seconds
• The quality of the stain will be checked. If slides appear over-stained (leaching of orange colouration), slides will be returned to the PBS bath for a further wash and the check repeated
• Stained slides will be air-dried and stored protected from light at room temperature prior to analysis
• Immediately prior to analysis 1-2 drops of PBS will be added to the slides before mounting with glass coverslips. - Evaluation criteria:
- For valid data, the test article is considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations is observed
2. An incidence of cells with micronuclei at such a concentration that exceeds the normal range in both replicates is observed
3. A concentration-related increase in the proportion of cells with micronuclei is observed (positive trend test).
The test article is considered positive in this assay if all of the above criteria are met.
The test article is considered negative in this assay if none of the above criteria are met.
Results which only partially satisfy the above criteria are dealt with on a case-by-case basis. - Species / strain:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- 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 marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (1150 μg/mL), compared to the concurrent vehicle controls
- Remarks on result:
- other: after 30h exposure
- Conclusions:
- The test material induced micronuclei in the in-vitro assay. As the test material contains 0.3% of a substance with a potent structural alert, it is possible that the result applies to the impurity rather than the main component.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- 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:
- 2016
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- 1998
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
- Type of assay:
- other: Gene Mutation Assay in Mammalian Cells in vitro (HPRT)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source of test material: BASF SE
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: No test substance precipitation in culture medium occured up to the highest applied concentration in the absence and presence of S9 mix at the end of treatment.
- Solubility and stability of the test substance in the solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was emulsified in culture medium (Ham's F12). The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve homogeneity of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted from according to the planned doses. All test substance formulations were prepared immediately before administration. To keep the test substance homogeneously in the vehicle, the test substance preparation was carefully pipetted before removal. - Target gene:
- hprt
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Suitability of cells: The CHO cell line is a permanent cell line derived from the Chinese hamster and has a high proliferation rate, high plating efficiency (about 90 %) and karyotype with a modal number of 20 chromosomes.
- Cell cycle length, doubling time or proliferation index: 12 - 16 hours
- Number of passages if applicable: At least 2 passages were performed before cells were taken for the experiment. A further passage was also necessary in order to prepare test cultures.
- Methods for maintenance in cell culture if applicable: For cell cultivation, deep-frozen cell suspensions were thawed at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm² plastic flasks containing about 5 mL Ham's F12 medium including 10 % (v/v) FCS. Cells were grown with 5 % (v/v) CO2 at 37 °C and ≥ 90 % relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 12 - 16 hours
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10 % (v/v) fetal calf serum (FCS), 1 % (v/v) penicillin / streptomycin (stock solution: 10000 IU / 10000 µg/mL) and 1 % (v/v) amphotericine B (stock solution: 250 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no - Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital and ß-naphthoflavone induced rat liver S9 fraction
- Test concentrations with justification for top dose:
- According to an initial range-finding cytotoxicity test for the determination of the experimental doses the top concentration was determined to be the limit concentration of 10 mM (1150 µg/mL). In the main experiments the following concentrations were tested.
1st experiment
without S9 mix
0; 71.9; 143.8; 287.5; 575.0; 1150.0 µg/mL
with S9 mix
0; 71.9; 143.8; 287.5; 575.0; 1150.0 µg/mL
2nd experiment
without S9 mix
0; 100.0; 200.0; 400.0; 800.0; 1150.0 µg/mL
with S9 mix
0; 100.0; 200.0; 400.0; 800.0; 1150.0 µg/mL
All test groups except for 100.0 µg/mL in the 2nd experiment were evaluated for gene mutations. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: aqueous culture medium (Ham's F12)
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 20 x 10^6 cells in 40 mL
DURATION
- Preincubation period: about 20 - 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 16 days
SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): Giemsa
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.
NUMBER OF CELLS EVALUATED: 200 cells per concentration
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative survival after treatment; mutant frequency - Evaluation criteria:
- The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative controls should not be less than 50 % (with and without S9 mix).
- The background mutant frequency in the negative controls should be within the historical negative control data range (95 % control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not "under control".
- The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range.
A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation Frequencies exceeds both the concurrent negative / vehicle control value and the range of the laboratory's historical negative control data (95 % control limit).
Isolated increases of mutant frequencies abouve the historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of the laboratory's historical negative control data (95 % control limit). - Statistics:
- An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
If the results of these tests were statistically significant compared with the respective vehicle control, labels are printed in the tables.
However, both, biological and statistical significance are considered together. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: In this study, in the absence and presence of S9 mix, no precipitation in culture medium was observed macroscopically up to the highest applied test substance concentration.
RANGE-FINDING/SCREENING STUDIES: In the pretest for toxicity based on the molecular weight of the test substance 1150 µg/mL (approx. 10 mM) was used as top concentration both with and without S9 mix 4-hour exposure time. In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations tested. The highest applied concentration of 1150.0 µg/mL was a homogeneous emulsion in the vehicle (culture medium). No test substance precipitation in culture medium occured up to the highest applied concentration in the absence and presence of S9 mix at the end of treatment. After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced RS of about or below 20 % of control up to the highest applied concentration.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: without S9 mix: MF(corr.): 42.47 - 419.90 per 10^6 cells; with S9 mix: MF(corr.): 21.52 - 270.48 per 10^6 cells)
- Negative (solvent/vehicle) historical control data: MF(corr.): 0.00 - 6.84 per 10^6 cells) - Conclusions:
- In the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
- Executive summary:
The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and ß-naphthoflavone induced rats (exogenous metabolic activation).
According to an initial range-finding cytotoxicity test for the determination of the experimental doses the top concentration was determined to be the limit concentration of 10 mM (1150 µg/mL). In the main experiments the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:
1st Experiment
without S9 mix
0; 71.9; 143.8; 287.5; 575.0; 1150.0 µg/mL
with S9 mix
0; 71.9; 143.8; 287.5; 575.0; 1150.0 µg/mL
2nd Experiment
without S9 mix
0; 100.0; 200.0; 400.0; 800.0; 1150.0 µg/mL
with S9 mix
0; 100.0; 200.0; 400.0; 800.0; 1150.0 µg/mL
Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6 -thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.
The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12 -dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.
In this study, in both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.
Based on the results of the present study, the test substance did not cause any biologically relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.
Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Referenceopen allclose all
Table 1: Standard plate test without metabolic activation
Strain |
Test group |
Dose [µg/plate] |
Mean revertants per plate |
Standard deviation |
Factor |
Individual revertant colony counts |
TA 1535 |
DMSO Test item
MNNG |
- 33 100 333 1000 2500 5000 5.0 |
9.3 11.0 8.7 10.7 7.0 7.0 10.0 5654.7 |
3.1 2.0 2.3 2.1 1.0 1.0 5.2 494.0 |
- 1.2 0.9 1.1 0.8 0.8 1.1 605.9 |
6, 12, 10 13, 9, 11 10, 6, 10 9, 13, 10 6, 7, 8 8, 7, 6 16, 7, 7 5472, 6214, 5278 |
TA 100 |
DMSO Test item
MNNG |
- 33 100 333 1000 2500 5000 5.0 |
86.0 93.7 86.7 91.7 95.7 97.0 101.0 4315.7 |
1.0 10.6 22.9 12.7 2.3 2.0 8.7 619.0 |
- 1.1 1.0 1.1 1.1 1.1 1.2 50.2 |
87, 86, 85 92, 105, 84 76, 71, 113 100, 77, 98 93, 97, 97 97, 95, 99 91, 107, 105 5022, 4057, 3868 |
TA 1537 |
DMSO Test item
AAC |
- 33 100 333 1000 2500 5000 100 |
8.0 11.0 8.7 9.0 8.7 10.0 7.7 403.0 |
1.0 2.6 4.2 2.6 4.7 2.6 1.5 61.5 |
- 1.4 1.1 1.1 1.1 1.3 1.0 50.4 |
8, 7, 9 12, 8, 13 10, 4, 12 6, 10, 11 14, 7, 5 9, 8, 13 9, 8, 6 368, 474, 367 |
TA 98 |
DMSO Test item
NOPD |
- 33 100 333 1000 2500 5000 10 |
20.3 18.3 17.0 25.3 19.7 19.7 18.7 606.0 |
2.9 3.5 5.3 3.2 2.1 8.1 6.4 33.5 |
- 0.9 0.8 1.2 1.0 1.0 0.9 29.8 |
17, 22, 22 15, 22, 18 19, 11, 21 23, 24, 29 22, 19, 18 21, 11, 27 16, 26, 14 640, 573, 605 |
E.coli |
DMSO Test item
4-NQO |
- 33 100 333 1000 2500 5000 5 |
24.7 25.3 25.3 28.7 23.3 24.3 25.3 1096.3 |
5.9 4.0 4.0 5.0 5.5 4.0 5.5 62.7 |
- 1.0 1.0 1.2 0.9 1.0 1.0 44.4 |
27, 29, 18 29, 21, 26 29, 26, 21 24, 34, 28 27, 17, 26 28, 25, 20 25, 31, 20 1107, 1029, 1153 |
Table 2: Standard plate test with metabolic activation
Strain |
Test group |
Dose [µg/plate] |
Mean revertants per plate |
Standard deviation |
Factor |
Individual revertant colony counts |
TA 1535 |
DMSO Test item
2-AA |
- 33 100 333 1000 2500 5000 2.5 |
9.7 11.3 10.0 11.0 7.0 10.3 12.0 240.3 |
3.8 1.5 2.0 2.6 2.0 3.1 3.0 45.1 |
- 1.2 1.0 1.1 0.7 1.1 1.2 24.9 |
7, 8, 14 13, 10, 11 10, 12, 8 12, 8, 13 9, 7, 5 11, 13, 7 9, 12, 15 284, 243, 194 |
TA 100 |
DMSO Test item
2-AA |
- 33 100 333 1000 2500 5000 2.5 |
108.0 97.0 110.7 105.7 106.7 108.0 112.7 2813.0 |
18.1 7.5 19.1 5.5 3.2 12.1 5.0 37.4 |
- 0.9 1.0 1.0 1.0 1.0 1.0 26.0 |
110, 89, 125 90, 105, 96 89, 118, 125 103, 112, 102 109, 103, 108 119, 110, 95 118, 108, 112 2807, 2853, 2779 |
TA 1537 |
DMSO Test item
2-AA |
- 33 100 333 1000 2500 5000 2.5 |
6.7 8.7 9.0 5.0 6.7 7.7 7.0 169.0 |
1.5 1.5 2.6 1.0 0.6 6.0 1.0 23.8 |
- 1.3 1.4 0.8 1.0 1.2 1.1 25.4 |
8, 7, 5 9, 10, 7 11, 10, 6 4, 6, 5 7, 7, 6 14, 2, 7 6, 7, 8 178, 187, 142 |
TA 98 |
DMSO Test item
2-AA |
- 33 100 333 1000 2500 5000 2.5 |
31.0 26.0 33.7 29.0 23.3 27.7 33.3 1726.7 |
2.0 7.5 6.7 6.6 2.5 1.5 7.2 208.0 |
- 0.8 1.1 0.9 0.8 0.9 1.1 55.7 |
29, 33, 31 19, 25, 34 26, 37, 38 30, 35, 22 21, 23, 26 26, 29, 28 25, 37, 38 1964, 1576, 1640 |
E.coli |
DMSO Test item
2-AA |
- 33 100 333 1000 2500 5000 60 |
21.7 19.3 21.0 27.0 27.0 25.3 28.0 109.3 |
3.5 3.2 2.0 5.2 7.0 7.5 7.0 14.7 |
- 0.9 1.0 1.2 1.2 1.2 1.3 5.0 |
22, 18, 25 17, 23, 18 21, 23, 19 24, 24, 33 20, 27, 34 33, 18, 25 31, 20, 33 98, 126, 104 |
Table 3: Preincubation test without metabolic activation
Strain |
Test group |
Dose [µg/plate] |
Mean revertants per plate |
Standard deviation |
Factor |
Individual revertant colony counts |
TA 1535 |
DMSO Test item
MNNG |
- 33 100 333 1000 2500 5000 5.0 |
13.3 12.7 11.3 16.3 13.7 15.3 14.0 2754.0 |
2.3 4.0 2.5 2.3 2.3 4.0 3.5 71.5 |
- 1.0 0.9 1.2 1.0 1.2 1.1 206.6 |
12, 12, 16 8, 15, 15 14, 11, 9 15, 19, 15 15, 11, 15 20, 13, 13 10, 16, 16 2683, 2826, 2753 |
TA 100 |
DMSO Test item
MNNG |
- 33 100 333 1000 2500 5000 5.0 |
106.0 102.0 97.3 91.3 125.0 102.0 88.0 2623.0 |
12.0 8.5 5.0 6.0 12.2 2.6 10.1 243.4 |
- 1.0 0.9 0.9 1.2 1.0 0.8 24.7 |
106, 94, 118 111, 94, 101 92, 98, 102 85, 92, 97 117, 139, 119 100, 105, 101 86, 99, 79 2342, 2759, 2768 |
TA 1537 |
DMSO Test item
AAC |
- 33 100 333 1000 2500 5000 100 |
9.0 11.3 11.7 9.3 10.0 9.7 8.7 737.3 |
1.7 2.5 2.1 3.1 1.0 3.2 3.8 103.2 |
- 1.3 1.3 1.0 1.1 1.1 1.0 81.9 |
10, 7, 10 11, 14, 9 14, 10, 11 12, 10, 6 11, 10, 9 11, 6, 12 6, 7, 13 784, 619, 809 |
TA 98 |
DMSO Test item
NOPD |
- 33 100 333 1000 2500 5000 10 |
17.0 19.7 20.0 20.0 19.3 17.7 17.3 490.3 |
3.5 6.0 3.0 4.4 2.1 1.5 4.9 10.0 |
- 1.2 1.2 1.2 1.1 1.0 1.0 28.8 |
15, 21, 15 14, 19, 26 23, 20, 17 23, 22, 15 20, 21, 17 18, 16, 19 15, 14, 23 491, 480, 500 |
E.coli |
DMSO Test item
4-NQO |
- 33 100 333 1000 2500 5000 5 |
23.3 22.7 19.0 19.3 21.7 18.3 23.0 196.3 |
2.5 5.9 5.2 4.9 8.0 2.5 2.6 9.1 |
- 1.0 0.8 0.8 0.9 0.8 1.0 8.4 |
23, 21, 26 16, 25, 27 25, 16, 16 25, 17, 16 30, 21, 14 16, 21, 18 26, 22, 21 188, 195, 206 |
Table 4: Preincubation test with metabolic activation
Strain |
Test group |
Dose [µg/plate] |
Mean revertants per plate |
Standard deviation |
Factor |
Individual revertant colony counts |
TA 1535 |
DMSO Test item
2-AA |
- 33 100 333 1000 2500 5000 2.5 |
8.7 11.3 9.3 7.3 11.3 9.7 12.0 254.0 |
2.1 1.2 2.3 2.1 2.5 4.6 6.1 17.4 |
- 1.3 1.1 0.8 1.3 1.1 1.4 29.3 |
8, 7, 11 12, 12, 10 12, 8, 8 8, 5, 9 9, 11, 14 15, 7, 7 15, 5, 16 274, 246, 242 |
TA 100 |
DMSO Test item
2-AA |
- 33 100 333 1000 2500 5000 2.5 |
99.0 104.0 90.7 92.0 99.0 94.3 100.0 2640.3 |
7.9 11.3 9.0 2.6 8.9 5.5 11.3 32.7 |
- 1.1 0.9 0.9 1.0 1.0 1.0 26.7 |
102, 105, 90 117, 98, 97 101, 85, 86 95, 90, 91 96, 109, 92 100, 89, 94 93, 94, 113 2644, 2606, 2671 |
TA 1537 |
DMSO Test item
2-AA |
- 33 100 333 1000 2500 5000 2.5 |
11.0 12.0 11.3 11.7 8.3 6.0 7.0 63.7 |
4.6 3.0 2.1 2.5 2.9 1.0 1.0 9.9 |
- 1.1 1.0 1.1 0.8 0.5 0.6 5.8 |
15, 12, 6 9, 12, 15 12, 9, 13 14, 12, 9 10, 5, 10 6, 7, 5 8, 7, 6 57, 59, 75 |
TA 98 |
DMSO Test item
2-AA |
- 33 100 333 1000 2500 5000 2.5 |
28.7 28.3 25.3 30.3 23.0 27.3 16.0 1587.0 |
3.5 8.1 1.2 4.2 5.3 9.0 5.2 226.0 |
- 1.0 0.9 1.1 0.8 1.0 0.6 55.4 |
29, 32, 25 34, 19, 32 26, 26, 24 27, 29, 35 19, 29, 21 17, 33, 32 22, 13, 13 1512, 1408, 1841 |
E.coli |
DMSO Test item
2-AA |
- 33 100 333 1000 2500 5000 60 |
21.3 19.7 22.3 23.7 25.7 23.0 23.0 84.0 |
5.5 5.7 4.0 2.1 7.1 5.0 5.0 20.5 |
- 0.9 1.0 1.1 1.2 1.1 1.1 3.9 |
27, 21, 16 15, 26, 18 20, 20, 27 26, 23, 22 32, 27, 18 23, 18, 28 28, 18, 23 64, 105, 83 |
Data for 3+27 Hour Treatments -S-9, Micronucleus Experiment, Trial 2
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
48 |
397 |
55 |
500 |
1.01 |
|
|
B |
65 |
381 |
54 |
500 |
0.98 |
|
|
C |
61 |
376 |
63 |
500 |
1.00 |
|
|
D |
69 |
398 |
33 |
500 |
0.93 |
|
|
Total |
243 |
1552 |
205 |
2000 |
0.98 |
- |
200.0 |
A |
64 |
374 |
62 |
500 |
1.00 |
|
|
B |
61 |
388 |
51 |
500 |
0.98 |
|
|
Total |
125 |
762 |
113 |
1000 |
0.99 |
0 |
400.0 |
A |
98 |
366 |
36 |
500 |
0.88 |
|
|
B |
111 |
349 |
40 |
500 |
0.86 |
|
|
Total |
209 |
715 |
76 |
1000 |
0.87 |
12 # |
600.0 |
A |
165 |
321 |
14 |
500 |
0.70 |
|
|
B |
156 |
324 |
20 |
500 |
0.73 |
|
|
Total |
321 |
645 |
34 |
1000 |
0.71 |
27 # |
800.0 |
A |
265 |
233 |
2 |
500 |
0.47 |
|
|
B |
210 |
280 |
10 |
500 |
0.60 |
|
|
Total |
475 |
513 |
12 |
1000 |
0.54 |
45 |
900.0 |
A |
236 |
258 |
7 |
501 |
0.54 |
|
|
B |
193 |
299 |
8 |
500 |
0.63 |
|
|
Total |
429 |
557 |
15 |
1001 |
0.59 |
40 # |
950.0 |
A |
284 |
214 |
2 |
500 |
0.44 |
|
|
B |
282 |
213 |
5 |
500 |
0.45 |
|
|
Total |
566 |
427 |
7 |
1000 |
0.44 |
55 |
1000 |
A |
234 |
265 |
1 |
500 |
0.53 |
|
|
B |
218 |
276 |
6 |
500 |
0.58 |
|
|
Total |
452 |
541 |
7 |
1000 |
0.56 |
43 |
1025 |
A |
232 |
265 |
3 |
500 |
0.54 |
|
|
B |
204 |
283 |
13 |
500 |
0.62 |
|
|
Total |
436 |
548 |
16 |
1000 |
0.58 |
41 |
1050 |
A |
206 |
289 |
5 |
500 |
0.60 |
|
|
B |
152 |
333 |
15 |
500 |
0.73 |
|
|
Total |
358 |
622 |
20 |
1000 |
0.66 |
33 |
1075 |
A |
240 |
252 |
8 |
500 |
0.54 |
|
|
B |
297 |
201 |
2 |
500 |
0.41 |
|
|
Total |
537 |
453 |
10 |
1000 |
0.47 |
52 # |
1100 |
A |
448 |
51 |
1 |
500 |
0.11 |
|
|
B |
427 |
73 |
0 |
500 |
0.15 |
|
|
Total |
875 |
124 |
1 |
1000 |
0.13 |
87 |
1150 |
A |
238 |
259 |
3 |
500 |
0.53 |
|
|
B |
297 |
201 |
2 |
500 |
0.41 |
|
|
Total |
535 |
460 |
5 |
1000 |
0.47 |
52 # |
MMC, 0.10 |
A |
206 |
289 |
5 |
500 |
0.60 |
|
|
B |
291 |
209 |
0 |
500 |
0.42 |
|
|
Total |
497 |
498 |
5 |
1000 |
0.51 |
48 # |
MMC, 0.20 |
A |
262 |
237 |
1 |
500 |
0.48 |
|
|
B |
298 |
198 |
4 |
500 |
0.41 |
|
|
Total |
560 |
435 |
5 |
1000 |
0.45 |
55 # |
Mono = Mononucleate Bi = Binucleate Multi = Multinucleate RI = Replication index
# Highlighted concentrations selected for analysis
Data for 3+27 Hour Treatments +S-9, Micronucleus Experiment, Trial 3
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicit y Based on RI (%) |
Vehicle |
A |
73 |
295 |
132 |
500 |
1.12 |
|
|
B |
48 |
332 |
120 |
500 |
1.14 |
|
|
C |
67 |
324 |
109 |
500 |
1.08 |
|
|
D |
60 |
339 |
101 |
500 |
1.08 |
|
|
Total |
248 |
1290 |
462 |
2000 |
1.11 |
- |
200.0 |
A |
57 |
349 |
94 |
500 |
1.07 |
|
|
B |
55 |
344 |
101 |
500 |
1.09 |
|
|
Total |
112 |
693 |
195 |
1000 |
1.08 |
2 |
400.0 |
A |
48 |
366 |
86 |
500 |
1.08 |
|
|
B |
83 |
392 |
25 |
500 |
0.88 |
|
|
Total |
131 |
758 |
111 |
1000 |
0.98 |
11 # |
600.0 |
A |
75 |
382 |
43 |
500 |
0.94 |
|
|
B |
91 |
376 |
33 |
500 |
0.88 |
|
|
Total |
166 |
758 |
76 |
1000 |
0.91 |
18 |
700.0 |
A |
95 |
350 |
55 |
500 |
0.92 |
|
|
B |
135 |
325 |
40 |
500 |
0.81 |
|
|
Total |
230 |
675 |
95 |
1000 |
0.87 |
22 |
750.0 |
A |
96 |
388 |
16 |
500 |
0.84 |
|
|
B |
89 |
355 |
56 |
500 |
0.93 |
|
|
Total |
185 |
743 |
72 |
1000 |
0.89 |
20 |
800.0 |
A |
91 |
376 |
33 |
500 |
0.88 |
|
|
B |
77 |
386 |
37 |
500 |
0.92 |
|
|
Total |
168 |
762 |
70 |
1000 |
0.90 |
19 |
850.0 |
A |
85 |
371 |
44 |
500 |
0.92 |
|
|
B |
127 |
327 |
46 |
500 |
0.84 |
|
|
Total |
212 |
698 |
90 |
1000 |
0.88 |
21 |
900.0 |
A |
130 |
346 |
24 |
500 |
0.79 |
|
|
B |
119 |
362 |
19 |
500 |
0.80 |
|
|
Total |
249 |
708 |
43 |
1000 |
0.79 |
28 # |
950.0 |
A |
111 |
356 |
33 |
500 |
0.84 |
|
|
B |
132 |
340 |
28 |
500 |
0.79 |
|
|
Total |
243 |
696 |
61 |
1000 |
0.82 |
26 |
1000 |
A |
161 |
326 |
13 |
500 |
0.70 |
|
|
B |
125 |
331 |
44 |
500 |
0.84 |
|
|
Total |
286 |
657 |
57 |
1000 |
0.77 |
30 |
1050 |
A |
152 |
312 |
36 |
500 |
0.77 |
|
|
B |
169 |
308 |
23 |
500 |
0.71 |
|
|
Total |
321 |
620 |
59 |
1000 |
0.74 |
33 |
1150 |
A |
200 |
294 |
6 |
500 |
0.61 |
|
|
B |
205 |
285 |
10 |
500 |
0.61 |
|
|
Total |
405 |
579 |
16 |
1000 |
0.61 |
45 # |
CPA, 3.00 |
A |
157 |
329 |
14 |
500 |
0.71 |
|
|
B |
176 |
311 |
13 |
500 |
0.67 |
|
|
Total |
333 |
640 |
27 |
1000 |
0.69 |
37 |
CPA, 5.00 |
A |
205 |
284 |
11 |
500 |
0.61 |
|
|
B |
179 |
316 |
5 |
500 |
0.65 |
|
|
Total |
384 |
600 |
16 |
1000 |
0.63 |
43 # |
Mono = Mononucleate Bi = Binucleate Multi = Multinucleate RI = Replication index
# Highlighted concentrations selected for analysis
Data for 30+0 Hour Treatments -S-9, Micronucleus Experiment, Trial 1
Treatment (µg/mL) |
Replicate |
Mono |
Bi |
Multi |
Total |
RI |
Cytotoxicity Based on RI (%) |
Vehicle |
A |
57 |
302 |
141 |
500 |
1.17 |
|
|
B |
50 |
341 |
109 |
500 |
1.12 |
|
|
C |
48 |
337 |
115 |
500 |
1.13 |
|
|
D |
57 |
312 |
131 |
500 |
1.15 |
|
|
Total |
212 |
1292 |
496 |
2000 |
1.14 |
- |
100.0 |
A |
43 |
327 |
130 |
500 |
1.17 |
|
|
B |
44 |
336 |
120 |
500 |
1.15 |
|
|
Total |
87 |
663 |
250 |
1000 |
1.16 |
0 |
200.0 |
A |
48 |
365 |
87 |
500 |
1.08 |
|
|
B |
69 |
329 |
102 |
500 |
1.07 |
|
|
Total |
117 |
694 |
189 |
1000 |
1.07 |
6 |
300.0 |
A |
65 |
387 |
48 |
500 |
0.97 |
|
|
B |
60 |
347 |
93 |
500 |
1.07 |
|
|
Total |
125 |
734 |
141 |
1000 |
1.02 |
11 |
400.0 |
A |
57 |
378 |
65 |
500 |
1.02 |
|
|
B |
42 |
355 |
103 |
500 |
1.12 |
|
|
Total |
99 |
733 |
168 |
1000 |
1.07 |
6 |
450.0 |
A |
55 |
381 |
64 |
500 |
1.02 |
|
|
B |
44 |
401 |
55 |
500 |
1.02 |
|
|
Total |
99 |
782 |
119 |
1000 |
1.02 |
11 |
500.0 |
A |
73 |
388 |
39 |
500 |
0.93 |
|
|
B |
80 |
351 |
69 |
500 |
0.98 |
|
|
Total |
153 |
739 |
108 |
1000 |
0.96 |
16 |
550.0 |
A |
58 |
399 |
43 |
500 |
0.97 |
|
|
B |
53 |
385 |
62 |
500 |
1.02 |
|
|
Total |
111 |
784 |
105 |
1000 |
0.99 |
13 |
600.0 |
A |
62 |
387 |
51 |
500 |
0.98 |
|
|
B |
70 |
365 |
65 |
500 |
0.99 |
|
|
Total |
132 |
752 |
116 |
1000 |
0.98 |
14 # |
650.0 |
A |
131 |
356 |
13 |
500 |
0.76 |
|
|
B |
127 |
356 |
17 |
500 |
0.78 |
|
|
Total |
258 |
712 |
30 |
1000 |
0.77 |
32 |
700.0 |
A |
87 |
387 |
26 |
500 |
0.88 |
|
|
B |
96 |
364 |
40 |
500 |
0.89 |
|
|
Total |
183 |
751 |
66 |
1000 |
0.88 |
23 |
750.0 |
A |
286 |
211 |
3 |
500 |
0.43 |
|
|
B |
123 |
359 |
18 |
500 |
0.79 |
|
|
Total |
409 |
570 |
21 |
1000 |
0.61 |
46 |
800.0 |
A |
167 |
327 |
6 |
500 |
0.68 |
|
|
B |
115 |
362 |
23 |
500 |
0.82 |
|
|
Total |
282 |
689 |
29 |
1000 |
0.75 |
35 # |
1000 |
A |
265 |
230 |
5 |
500 |
0.48 |
|
|
B |
242 |
255 |
3 |
500 |
0.52 |
|
|
Total |
507 |
485 |
8 |
1000 |
0.50 |
56 # |
NOS, 10.0 |
A |
89 |
370 |
41 |
500 |
0.90 |
|
|
B |
86 |
378 |
36 |
500 |
0.90 |
|
|
Total |
175 |
748 |
77 |
1000 |
0.90 |
21 |
NOS, 20.0 |
A |
111 |
301 |
88 |
500 |
0.95 |
|
|
B |
118 |
277 |
105 |
500 |
0.97 |
|
|
Total |
229 |
578 |
193 |
1000 |
0.96 |
16 # |
Mono = Mononucleate Bi = Binucleate Multi = Multinucleate RI = Replication index
# Highlighted concentrations selected for analysis
Table 1: Summary of results - experimental parts without and with S9 mix
Experiment |
Exposure period [h] |
Test groups [µg/mL] |
S9 mix |
Prec.* |
Genotoxicity** MFcorr.[per 106cells] |
Cytotoxicity*** |
|
RS [%] |
CE2[%] |
||||||
1 |
4 |
Negative control1 71.9 143.8 287.5 575.0 1150.0 Positive control2 |
- - - - - - - |
n.d. - - - - - n.d. |
5.97 1.07 3.55 2.51 2.72 1.80 178.95s |
100.0 95.3 107.2 120.8 101.7 93.4 70.1 |
100.0 146.9 106.3 112.9 115.4 105.0 83.6 |
2 |
4 |
Negative control1 100.0 200.0 400.0 800.0 1150.0 Positive control2 |
- - - - - - - |
n.d. - - - - - n.d. |
3.46 n.c.1 0.29 1.34 1.20 2.66 125.48s |
100.0 99.6 93.6 92.7 96.6 82.4 75.2 |
100.0 n.c.1 106.9 117.0 105.0 106.3 82.7 |
1 |
4 |
Negative control1 71.9 143.8 287.5 575.0 1150.0 Positive control3 |
+ + + + + + + |
n.d. - - - - - n.d. |
2.28 1.71 0.74 0.00 1.21 3.97 162.80s |
100.0 85.6 109.3 116.2 122.8 100.6 88.2 |
100.0 95.1 87.6 89.9 80.5 82.1 67.4 |
2 |
4 |
Negative control1 100.0 200.0 400.0 800.0 1150.0 Positive control3 |
+ + + + + + + |
n.d. - - - - - n.d. |
4.57 n.c.1 6.95 3.91 0.30 3.17 102.70s |
100.0 108.0 100.4 98.0 92.2 92.5 94.7 |
100.0 n.c.1 100.9 93.6 101.5 96.0 67.7 |
*: Macroscopically visible precipitation in culture medium at the end of exposure period
**: Mutant frequency MFcorr.: mutant colonies per 106cells corrected with the CE2value
***: Cloning efficiency related to the respective vehicle control
s: Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)
n.c.1: Culture was not continued since a minimum of only four analyzable concentrations is required
n.d.: Not determined
1: Medium
2: EMS 400 µg/mL
3: DMBA 1.25 µg/mL
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
The positive result in the in-vitro study would normally require a testing proposal for an in-vivo study. In this case, the substance is handled at an industrial site under strictly controlled conditions. Therefore, no further testing is proposed.
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames Test
The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.
Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA
Dose range: 33 - 5000 µg/plate (SPT and PIT)
Test conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).
Solubility: No precipitation of the test substance was found with and without S9 mix.
Toxicity: No bacteriotoxic effect was observed.
Mutagenicity: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.
Micronucleus Test
An in vitro mammalian cell micronucleus test was performed with and without metabolic activation (Aroclor induced S9 fraction) using human lymphoblastoid cells (TK6).
To provide an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment and to ensure that only cells that divided during or after treatment are microscopically scored Cytochalasin B is added to the cultures.
The proposed treatment conditions are as follows:
+ S9 short treatment: Cells are exposed for 3 hours. Upon ending of the exposure duration S9 and the test substance are removed and Cytochalasin B added. 27 hours later cells are harvested.
- S9 short treatment: Cells are exposed for 3 hours. Upon ending of the exposure duration the test substance is removed and Cytochalasin B added. 27 hours later cells are harvested.
- S9 extended treatment: Cells are exposed for 30 hours in the presence of Cytochalasin B. After the exposure period cells are harvested.
+ S9 extended treatment: Cells are exposed for 30 hours in the presence of S9 and Cytochalasin B. After the exposure period cells are harvested.
Changes in osmolality and of more than 50 mOsm/kg and fluctuations in pH of more than one unit can give rise to chromosome aberrations (Scott et al., 1991; Brusick, 1986). Therefore, osmolality and the effect of the test article on the pH of the culture medium are assessed during the study. Osmolality and pH are initially measured on post-treatment media in the cytotoxicity Range-Finder Experiment. If marked changes are observed, further measurements may be made in the Micronucleus Experiment(s).
Harvesting: At the defined sampling time, cultures were
- centrifuged at approx. 200 g, 5 minutes
- resuspended in 4 mL (hypotonic) 0.075 M KCl at 37 °C for 4 minutes
- fixed by dropping the KCl suspension into fresh, cold methanol / glacial acetic acid (7:1 v/v), centrifuged (approx. 200 g, 5 minutes) and resuspended, followed by further centrifugation and fixation (approx. 1250 g, 2 - 3 minutes) until the cell pellets are clean
- stored in fixative at 2 - 8 °C prior to slide preparation for a minimum of 3 hours.
Slide preparation:
- cells centrifuged (approx. 1250 g, 2 - 3 minutes) and resuspended in a minimal amount of fresh fixative (if required)
- several drops of suspension gently spread onto multiple clean, dry microscope slides
- slides air-dried then stored protected from light at room temperature prior to staining
- slides stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes, following by a wash with PBS (with agitation) for a few seconds
- the quality of the stain will be checked. If slides appear over-stained (leaching of orange colouration), slides will be returned to the PBS bath for a further wash and the check repeated
- stained slides will be air-dried and stored protected from light at room temperature prior to analysis
- immediately prior to analysis 1 - 2 drops of PBS will be added to the slides before mounting with glass coverslips.
The test material induced micronuclei in the in vitro assay. As the test material contains 0.3 % of a substance with a potent structural alert, it is possible that the result applies to the impurity rather than the main component.
HPRT
The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and ß-naphthoflavone induced rats (exogenous metabolic activation).
According to an initial range-finding cytotoxicity test for the determination of the experimental doses the top concentration was determined to be the limit concentration of 10 mM (1150 µg/mL). In the main experiments the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:
1st Experiment
without S9 mix
0; 71.9; 143.8; 287.5; 575.0; 1150.0µg/mL
with S9 mix
0; 71.9; 143.8; 287.5; 575.0; 1150.0µg/mL
2nd Experiment
without S9 mix
0;100.0;200.0; 400.0; 800.0; 1150.0µg/mL
with S9 mix
0;100.0;200.0; 400.0; 800.0; 1150.0µg/mL
Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6 -thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.
The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12 -dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.
In this study, in both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.
Based on the results of the present study, the test substance did not cause any biologically relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.
Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames Test (OECD 471, GLP) and in the HPRT Test (OECD 476, GLP). As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No. 2015/1221.
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.