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EC number: 224-632-3 | CAS number: 4432-31-9
- 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
In a study according to OECD TG 471 under GLP conditions, the substance was not mutagenic in five strains (TA98, TA100, TA1535, TA1537 (all Salmonella typhimurium) and WP2 uvrA (E. coli)) with and without metabolic activation (S9) (reference 7.6.1 -1).
The genotoxicity of the substance was investigated in an in vitro micronucleus test according to OECD TG 487 under GLP-conditions. The substance was considered as not genotoxic under the test conditions (reference 7.6.1 -2).
The mutagenicity of the substance was investigated in an in vitro mammalian cell gene mutation test using the HPRT genes according to OECD TG 476 under GLP-conditions using the Chinese hamster cells V79. The substance was considered as not mutagenic under the conditions of this test (reference 7.6.1 -3).
Same results are expected for the anhydrous form (CAS 4432-31-9).
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-08-01 to 2017-09-22
- 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:
- July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- May 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: CLS (Eppelheim, Germany)
- Suitability of cells: analysis of modal chromosome number
- Cell cycle length, doubling time or proliferation index: cell cycle time was determined in experiment and within the normal range; doubling time: 10-12 h
- Modal number of chromosomes: 22
MEDIA USED
- Type and identity of media including CO2 concentration : DMEM (from Biochrom AG) was used as a basis for complete culture medium with medium DMEM with 5% HS (5% horse serum, 1% Penicillin/Streptomycin, 1% phytohaemagglutinin solution) and selection medium (5% horse serum, 1% Penicillin/Streptomycin, 2 µg/mL 6-thioguanine (1 mg/mL))
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system
- source of S9 : S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from a specialized company (Trinova Biochem GmbH, Gießen), produced from the livers of male Sprague-Dawley rats
which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of the S9 fraction during treatment is 0.4 %. - Test concentrations with justification for top dose:
- 0.31, 0.63, 1.25, 2.5, 5, 10 mM
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMEM
- Justification for choice of solvent/vehicle: no effects on cell viability and good solubility of test substance - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- with metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1000000/dish (10 cm diameter)
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Experiment 1: 4h (with and without metabolic activation)
Experiment 2: 24h (without metabolic activation)
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): at least 168 h
- Selection time: 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-15 days
- Selection agent: 6-thioguanine
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency - Rationale for test conditions:
- The conditions of experiment 1 are according to the recommendations of the OECD TG 476.
The second experiment was performed to confirm the findings of experiment 1. - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Chi-square test with a significance level of 5%
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
-
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Water solubility: the substance was highly soluble
- Precipitation: no
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES:
- cytotoxicity pre-test: highest recommended concentration (10 mM) not cytotoxic, determined by cloning efficiency) and not precipitating after 4h exposure with and without metabolic activation
STUDY RESULTS
- Results from cytotoxicity measurements: see attached results
- Genotoxicity results: see attached results
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (number of mutant colonies per 1000000 cells):
With S9 (4h treatment): DMBA: range: 100 - 461; mean: 2431; sd: 72.2
Without S9 (4h treatment): EMS: range: 71 - 207; mean: 127; sd: 33.4
Without S9 (24h treatment): EMS: range: 35 - 517; mean: 237; sd: 97.0
- Negative (solvent/vehicle) historical control data(number of mutant colonies per 1000000 cells):
With S9 (4h treatment): DMEM: range: 4 - 35; mean: 17; sd: 9.3
With S9 (4h treatment): DMSO: range: 2 - 39; mean: 15; sd: 9.2
Without S9 (4h treatment): DMEM: range: 2- 32; mean: 15; sd: 9.0
Without S9 (24h treatment): DMEM: range: 4 - 27; mean: 12; sd: 6.6
- Conclusions:
- In conclusion, it can be stated that under the experimental conditions of this study the test substance did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test item is considered to be non-mutagenic under the conditions of the HPRT assay.
- Executive summary:
A study according OECD TG 476 was performed to investigate the potential of the substance to induce mutations at the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79).
The assay comprised a pre-test and two independent experiments (experiment I and II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.
The first main experiment (experiment I) was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. The second experiment (experiment II) was performed with a treatment period of 24 hours without metabolic activation. The highest nominal concentration (10 mM) applied was chosen based on the good solubility of the test item in organic solvents and aqueous media and on the absence of cytotoxicity. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.
No substantial and reproducible dose-dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration (10 mM) of the test item.
In conclusion, it can be stated that under the experimental conditions of this study the test item did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-08-29 to 2016-09-08
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
- Version / remarks:
- 2012
- 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
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system
- source of S9 : The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2.; D-35394 Giessen, Germany)
- concentration or volume of S9 mix and S9 in the final culture medium : 10% S9 mix in final culture medium
- quality controls of S9: enzymatic activity, sterility, metabolic capability - Test concentrations with justification for top dose:
- First and second experiment: 16, 50, 160, 500, 1600, 5000 µg/plate, where 5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances
- Vehicle / solvent:
- - Vehicle/solvent used: ultrapure water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitro-1,2-phenylenediamine
- Remarks:
- without S9 mix, TA98
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with S9 mix, S. typhimurium strains and E. coli WP2 uvrA
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- without S9 mix, TA100 and TA1535
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix, TA1537
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without S9 mix, E.coli WP2 uvrA
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation); preincubation
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 hours
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: number of revertant colonies (with TA98 and TA100) - Evaluation criteria:
- A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
The pH of the test item stock solution (prepared for the highest concentration) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at the highest concentration of 50 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (50 mg/mL) were 3.50 and 3.52. The pH of the different overlays without test item stock solution was in the range of 7.12 - 7.30, and the pH of overlays completed with test item stock solution was ~7 in all cases.
Extremes of pH could have influencing effect on the mutagenicity results; however in this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the result interpretation.
- Water solubility: 50 mg/mL
- Precipitation and time of the determination: no
RANGE-FINDING/SCREENING STUDIES:
Based on the solubility test, a stock solution with a concentration of 50 mg/mL was prepared in ultrapure water and diluted in 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 μg/plate of the test item.
The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
STUDY RESULTS
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
Sporadically increased revertant colony numbers were noticed in the performed experiments; these increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the Initial Mutation Test (Plate Incorporation Test) in S. typhimurium TA1537 strain at 16 μg/plate, in presence of metabolic activation (+S9). This higher value however remained in the range of the corresponding vehicle historical control data. The mutation rate was 1.81, which was far below the genotoxicological threshold for being positive, was a unique value without any biological significance.
- Signs of toxicity : no cytotoxicity observed
- Individual plate counts : see attached results
- Mean number of revertant colonies per plate and standard deviation : see attached results
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see attached results
- Negative (solvent/vehicle) historical control data: see attached results - Conclusions:
- The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.
- Executive summary:
A study according OECD TG 471 was performed with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).
Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity.
Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:
5000; 1600; 500; 160; 50 and 16 μg/plate.
In the preliminary experiments the pH of the aqueous test item solution (50 mg/mL) was found as 3.47. Extremes of pH could have influencing effect on the mutagenicity results, therefore in the Initial and Confirmatory Mutation Tests the pH of the test item stock solution (prepared for the highest concentration of 50 mg/mL) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at 50 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (50 mg/mL) were 3.50 and 3.52. The pH of the test item containing overlays was ~7 in both experiments. In this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the mutagenicity result interpretation.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.
The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-06-12 to 2017-08-22
- 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:
- July 29, 2016
- Deviations:
- yes
- Remarks:
- Determination of cytotoxicity in experiment II: Only 499 cells were evaluated in solvent control of positive control. The deviation is uncritical since it is only marginal and has no significant effect on the end result of the positive control.
- Qualifier:
- according to guideline
- Guideline:
- other: EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”
- Version / remarks:
- February 2017
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: young, non-smoking donors (25 year old male and 31 year old male)
- Suitability of cells: human peripheral lymphocytes are suitable due to their low and stable background rate of micronuclei and as they eliminate inter-species differences
- Cell cycle length, doubling time or proliferation index: Cytokinesis-block proliferation index (CBPI): approx. 1.65
- Sex, age and number of blood donors: 25 year old male and 31 year old male
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: 12h
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 (from Biochrom AG) wad used as a basis for complete culture medium (15% FCS, 1% Penicillin/Streptomycin, 1% phytohaemagglutinin solution) and minimal culture medium (MCM) (1% Penicillin/Streptomycin, 2% phytohaemagglutinin solution)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no information
- Periodically checked for karyotype stability: not applicable
- Periodically 'cleansed' against high spontaneous background: not applicable - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system
- source of S9 : S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from Trinova Biochem GmbH, Gießen; produced from the livers of male Sprague-Dawley rats which
were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- method of preparation of S9 mix:
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) - Test concentrations with justification for top dose:
- - test concentrations (in the experiment): 0.12, 0.24, 0.49, 0.98, 1.95 mg/mL (max. concentration corresponding to 10 mM)
- concentrations scored for micronuclei: 0.49, 0.98, 1.95 mg/mL
- Top dose as recommended in OECD TG 487, i.e. 10 mM - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: minimal culture medium, i.e. RPMI 1640 (Biochrom AG, Berlin, Germany) with 1% Penicillin/streptomycin and 2% phytohaemagglutin solution
- Justification for choice of solvent/vehicle: no solvent effect on cell viability and the test substance was completely soluble - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclohexylamine
- Remarks:
- with S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- colchicine
- Remarks:
- without S9 mix
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment 1: 4h; Experiment 2: 23.5h
- Expression time (cells in growth medium): Experiment 1: 19h; Experiment 2: none
- Harvest time after the end of treatment (sampling/recovery times): Experiment 1: 23h; Experiment 2: 23.5h
FOR MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: cytochalasin B (final concentration 5 μg/mL) was added after exposure and the cells were incubated at 37±1°C in a humidified atmosphere with 5.0 ± 0.5 % CO2 until preparation
- Methods of slide preparation and staining technique used including the stain used:
Each cell culture was harvested and processed separately. The cells were spun down by gentle centrifugation (10 min, 500 * g). The supernatant was discarded and the cells were re-suspended in 12 ml hypotonic KCl solution. The cell suspension was allowed to stand for 15 min at room temperature (20 ± 5°C). After removal of the hypotonic solution by centrifugation (10 min, 500 * g), the cell pellet was fixated with a mixture of methanol and glacial acetic acid (3:1). After fixation at 2 – 8 °C for minimum 30 min, the cell suspension was spun down by gentle centrifugation (10 min, 500 * g), the supernatant was discarded and the cell pellet was re-suspended in fixative again. The washing procedures were repeated until the cell pellet was white.
The slides were prepared by dropping the cell suspension onto a clean microscope slide. The cells were then stained with a 10% solution of Giemsa. All slides were independently coded before microscopic analysis.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): At least 1000 binucleated cells per culture were scored for micronuclei. Only cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included in the analysis
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index - Rationale for test conditions:
- According to recommendation in the OECD TG 487 for lymphocytes, i.e. for the initial experiment 3-6h and for the subsequent experiment 1.5-2 cell cycles (18-24h)
- Evaluation criteria:
- The test item is considered to have no genotoxic effects if it meets all of the following criteria:
- Neither a statistically significant nor a concentration-related increase of the number of micronucleated cells in the evaluated test concentrations is observed.
- The obtained results lie within the range of the historical laboratory control data for solvent controls, considering also e.g 95.5% control limits where appropriate
The test item is considered to have genotoxic effects if, in any of the experimental conditions, it meets all of the following criteria:
- At least one test concentration shows a statistically significant increase of micronucleated cells compared to the concurrent solvent control.
- In at least one experimental condition a dose-related increase of micronucleated cells can be observed.
- Any of the results lies outside the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5% control limits where appropriate.
When all of these criteria are met, the test chemical is considered to be able to induce chromosomal breaks and/or gain or loss of genetic material in this test system. - Statistics:
- Fisher's exact test with a significance level of 5% (for each treatment-control comparison)
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Water solubility: the substance was highly soluble
- Precipitation: no
- Definition of acceptable cells for analysis: binucleated cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES: experiment 1 was initially the range-finding experiment, but was also a valid of a full experiment
STUDY RESULTS
- Results from cytotoxicity measurements: see attached results
- Genotoxicity results: see attached results
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Without S9: 0.3 µg/mL MMC (short exposure): range: 1.08 - 7.67; mean: 3.37; sd: 1.73
0.3 µg/mL MMC (extended exposure): range: 1.33 - 7.75; mean: 3.85; sd: 1.67
Colchicine: range: 1.24 - 10.32; mean: 5.74; sd: 2.64
With S9: CPA: range: 1.25 - 5.14; mean: 2.63; sd: 0.81
- Negative (solvent/vehicle) historical control data:
Without S9: medium: range: 0.05- 1.06; mean: 0.44; sd: 0.27
NaCl (0.9%): range: 0.05 - 1.24; mean: 0.47; sd: 0.30
With S9: medium: range: 0.10- 0.83; mean: 0.32; sd: 0.16
NaCl (0.9%): range: 0.05 - 1.11; mean: 0.36; sd: 0.22 - Conclusions:
- In conclusion, under the experimental conditions reported, the test item does not induce the formation of micronuclei in human lymphocytes in vitro. Therefore, the test item is considered as not genotoxic.
- Executive summary:
A study according OECD TG 487 was performed to assess the genotoxic potential of the test item to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).
The test item was dissolved in minimal culture medium to prepare a stock solution with a concentration of 100 mM corresponding to 10 mM as highest concentration in the test. In addition, a geometric series of dilutions was prepared from the stock solution.
Two independent and valid experiments were performed.
Human peripheral blood lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item or positive control, respectively. After the culture harvest time, the cells were harvested and slides were prepared. Cytotoxicity and level of micronuclei were determined.
In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test item to cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all cultures treated with solvent control, positive control and test item. On the basis of these data, the concentrations to be scored for micronuclei were selected. No cytotoxic effect was detected in any of the tested concentrations in both experiments.
Therefore, the three highest test item concentrations were evaluated for micronuclei.
Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations was observed. All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.
In conclusion, under the experimental conditions reported, the substance does not induce the formation of micronuclei in human lymphocytes in vitro.
Referenceopen allclose all
Table 1: Mutagenicity in Experiment I with Metabolic Activation
|
Conc. |
Culture |
Cells seeded |
Number of colonies per dish |
CE mut |
MF |
MF |
Mean |
||||
|
[mM] |
|
total |
I |
II |
III |
IV |
V |
|
|
Per 10E6 cells |
|
Solvent Control Test Item |
- |
A |
2503264 |
2 |
8 |
4 |
5 |
1 |
0.00001 |
0.00002 |
20 |
18 |
- |
B |
2500521 |
5 |
3 |
3 |
0 |
4 |
0.00001 |
0.00002 |
15 |
||
Solvent Control DMBA |
|
A |
2497798 |
12 |
4 |
6 |
8 |
6 |
0.00001 |
0.00003 |
34 |
25 |
- |
B |
2498400 |
0 |
1 |
3 |
5 |
4 |
0.00001 |
0.00002 |
17 |
||
Positive Control (DMBA) |
1.5 µg/mL |
A |
2499294 |
34 |
36 |
36 |
37 |
37 |
0.00007 |
0.00020 |
196 |
239 |
B |
2502737 |
47 |
65 |
51 |
43 |
50 |
0.00010 |
0.00028 |
282 |
|||
Test item |
10 |
A |
2499351 |
4 |
4 |
3 |
4 |
3 |
0.00001 |
0.00002 |
19 |
26 |
10 |
B |
2499539 |
9 |
10 |
5 |
3 |
7 |
0.00001 |
0.00003 |
34 |
||
Test item |
5 |
A |
2496838 |
2 |
3 |
4 |
2 |
4 |
0.00001 |
0.00002 |
18 |
12 |
5 |
B |
2501022 |
0 |
2 |
2 |
1 |
1 |
0.00000 |
0.00001 |
6 |
||
Test Item |
2.5 |
A |
2500169 |
3 |
6 |
3 |
4 |
7 |
0.00001 |
0.00003 |
26 |
21 |
2.5 |
B |
2495657 |
2 |
3 |
2 |
5 |
2 |
0.00001 |
0.00002 |
16 |
||
Test Item |
1.25 |
A |
2501957 |
1 |
0 |
1 |
1 |
1 |
0.00000 |
0.00000 |
4 |
9 |
1.25 |
B |
2501145 |
4 |
4 |
2 |
1 |
2 |
0.00001 |
0.00001 |
14 |
||
Test Item |
0.63 |
A |
2500115 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.63 |
B |
2500176 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
||
Test item |
0.31 |
A |
2498270 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.31 |
B |
2498015 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a = not analysed because the OECD 476 guideline requires only 4 concentrations
Table 2: Mutagenicity in Experiment I without Metabolic Activation
|
Conc. |
Culture |
Cells seeded |
Number of colonies per dish |
CE mut |
MF |
MF |
Mean |
||||
|
[mM] |
|
total |
I |
II |
III |
IV |
V |
|
|
Per 10E6 cells |
|
Solvent Control Test Item |
- |
A |
2497286 |
4 |
4 |
5 |
4 |
7 |
0.00001 |
0.00002 |
23 |
26 |
- |
B |
2498413 |
5 |
7 |
6 |
5 |
5 |
0.00001 |
0.00003 |
30 |
||
Solvent Control DMBA |
|
A |
2501274 |
0 |
6 |
2 |
2 |
5 |
0.00001 |
0.00002 |
1 |
11 |
- |
B |
2496879 |
0 |
3 |
1 |
0 |
2 |
0.00000 |
0.00001 |
6 |
||
Positive Control (DMBA) |
300 µg/mL |
A |
2496689 |
33 |
29 |
27 |
31 |
25 |
0.00006 |
0.00014 |
137 |
145 |
B |
2500960 |
26 |
23 |
24 |
31 |
23 |
0.00005 |
0.00015 |
153 |
|||
Test item |
10 |
A |
2497166 |
6 |
8 |
9 |
8 |
7 |
0.00002 |
0.00004 |
35 |
23 |
10 |
B |
2496330 |
2 |
3 |
3 |
1 |
1 |
0.00000 |
0.00001 |
10 |
||
Test item |
5 |
A |
2498978 |
3 |
5 |
4 |
1 |
7 |
0.00001 |
0.00002 |
21 |
21 |
5 |
B |
2497623 |
1 |
3 |
6 |
6 |
4 |
0.00001 |
0.00002 |
20 |
||
Test Item |
2.5 |
A |
2500349 |
3 |
5 |
2 |
3 |
3 |
0.00001 |
0.00002 |
16 |
20 |
2.5 |
B |
2503188 |
9 |
6 |
5 |
3 |
1 |
0.00001 |
0.00002 |
24 |
||
Test Item |
1.25 |
A |
2499500 |
4 |
5 |
4 |
2 |
5 |
0.00001 |
0.00002 |
21 |
21 |
1.25 |
B |
2502812 |
3 |
3 |
3 |
6 |
5 |
0.00001 |
0.00002 |
21 |
||
Test Item |
0.63 |
A |
2500478 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.63 |
B |
2503155 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
||
Test item |
0.31 |
A |
2497970 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.31 |
B |
2499536 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
Table 3: Mutagenicity in Experiment II without Metabolic Activation
|
Conc. |
Culture |
Cells seeded |
Number of colonies per dish |
CE mut |
MF |
MF |
Mean |
||||
|
[mM] |
|
total |
I |
II |
III |
IV |
V |
|
|
Per 106 cells |
|
Solvent Control Test Item |
- |
A |
2500933 |
0 |
0 |
3 |
1 |
1 |
0.00000 |
0.00001 |
7 |
11 |
- |
B |
2496490 |
1 |
3 |
2 |
2 |
1 |
0.00000 |
0.00001 |
14 |
||
Solvent Control DMBA |
|
A |
2497077 |
4 |
3 |
0 |
2 |
4 |
0.00001 |
0.00002 |
21 |
25 |
- |
B |
2501950 |
3 |
1 |
3 |
5 |
3 |
0.00001 |
0.00003 |
29 |
||
Positive Control (DMBA) |
300 µg/mL |
A |
2497340 |
22 |
25 |
23 |
21 |
24 |
0.00005 |
0.00027 |
270 |
280 |
B |
2503958 |
22 |
38 |
30 |
35 |
40 |
0.00007 |
0.00029 |
289 |
|||
Test item |
10 |
A |
2501018 |
1 |
2 |
0 |
2 |
0 |
0.00000 |
0.00001 |
7 |
12 |
10 |
B |
2498673 |
3 |
3 |
5 |
1 |
1 |
0.00001 |
0.00002 |
17 |
||
Test item |
5 |
A |
2505206 |
1 |
3 |
2 |
1 |
3 |
0.00000 |
0.00001 |
9 |
18 |
5 |
B |
2496744 |
5 |
3 |
3 |
1 |
2 |
0.00001 |
0.00003 |
27 |
||
Test Item |
2.5 |
A |
2499913 |
2 |
6 |
3 |
6 |
1 |
0.00001 |
0.00003 |
30 |
31 |
2.5 |
B |
2499050 |
3 |
4 |
6 |
0 |
4 |
0.00001 |
0.00003 |
32 |
||
Test Item |
1.25 |
A |
2501820 |
1 |
1 |
1 |
0 |
0 |
0.00000 |
0.00000 |
5 |
19 |
1.25 |
B |
2499000 |
8 |
1 |
3 |
2 |
4 |
0.00001 |
0.00003 |
34 |
||
Test Item |
0.63 |
A |
2498840 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.63 |
B |
2503972 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
||
Test item |
0.31 |
A |
2497800 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
0.31 |
B |
2498438 |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a |
n/a = not analysed because the OECD 476 guideline requires only 4 concentrations
Table 4: Regression Parameters
Treatment |
Correlation coefficient |r| |
p-value |
Exp. I with metabolic activation |
0.718 |
0.282 |
Exp. I without metabolic activation |
0.911 |
0.089 |
Exp. II without metabolic activation |
0.305 |
0.695 |
Table 5: Statistical Significance, pooled replicates (arithmetic means)
Treatment |
p-Values |
||
experiment I |
experiment II |
||
with S9 |
without S9 |
without S9 |
|
Positive control DMBA |
< 0.01 |
- |
- |
Positive control EMS |
- |
< 0.01 |
< 0.01 |
Test item 10 mM |
0.209 |
n/c |
0.487 |
Test item 5 mM |
n/c |
n/c |
0.182 |
Test item 2.5 mM |
0.435 |
n/c |
0.002* |
Test item 1.25 mM |
n/c |
n/c |
0.139 |
n.c.: not calculated because the MF is lower than or equal to the solvent control
* = statistically significantly increased in comparison to the solvent control.
Table 6: Statistical Significance, individual cultures (Comparison of values of replicate A with corresponding solvent control replicate of A and comparison of values of replicate B with corresponding solvent control replicate of B)
Treatment |
Culture |
p-Values |
||
experiment I |
experiment II |
|||
with S9 |
without S9 |
without S9 |
||
Positive control DMBA |
A |
< 0.01 |
- |
- |
B |
< 0.01 |
- |
- |
|
Positive control EMS |
A |
- |
< 0.01 |
< 0.01 |
B |
- |
< 0.01 |
< 0.01 |
|
Test item 10 mM |
A |
n/c |
0.113 |
n/c |
B |
0.006* |
n/c |
0.420 |
|
Test item 5 mM |
A |
n/c |
n/c |
0.430 |
B |
n/c |
n/c |
0.042* |
|
Test item 2.5 mM |
A |
0.313 |
n/c |
< 0.000* |
B |
0.490 |
n/c |
0.007* |
|
Test item 1.25 mM |
A |
n/c |
n/c |
n/c |
B |
n/c |
n/c |
0.003* |
n/c: not calculated because the MF is lower than or equal to the solvent control
* = statistically significantly increased in comparison to the solvent control.
Table 7: Historical Data of the Solvent Controls
|
Number of mutant colonies per 10E6 cells |
||
4 h treatment / with metabolic activation |
|||
|
Solvent control |
Positive control (DMBA) |
|
|
DMEM |
DMSO |
|
Mean value |
16 |
15 |
238 |
Standard deviation |
8.8 |
9.6 |
74.6 |
Range |
4 - 35 |
2 - 44 |
96 - 461 |
95 % confindence interval |
0* - 34 |
0* - 34 |
89 - 388 |
Number of experiments |
18 |
60 |
41 |
Study no.: 17050301G865 |
18 |
25 |
239 |
4 h treatment / with metabolic activation |
|||
|
Solvent control |
Positive control (DMBA) |
|
|
DMEM |
||
Mean value |
16 |
136 |
|
Standard deviation |
10.5 |
97.7 |
|
Range |
2 - 48 |
71 - 294 |
|
95 % confindence interval |
0* - 37 |
46 – 227 |
|
Number of experiments |
52 |
39 |
|
Study no.: 17050301G865 |
26, 11 |
145 |
|
24 h treatment / with metabolic activation |
|||
|
Solvent control |
Positive control (DMBA) |
|
|
DMEM |
||
Mean value |
12 |
232 |
|
Standard deviation |
6.6 |
97.7 |
|
Range |
4 - 27 |
35 – 517 |
|
95 % confindence interval |
0* - 25 |
36 – 427 |
|
Number of experiments |
40 |
34 |
|
Study no.: 17050301G865 |
11, 25 |
280 |
Table 1: Summary Table of the Results of the Concentration Range Finding Test
Range finding Test (Informatory Toxicity Test) |
||||||||
Concentration (µg/plate) |
Salmonella typhimurium tester strains |
|||||||
TA 98 |
TA 100 |
|||||||
-S9 |
+ S9 |
-S9 |
+S9 |
|||||
Mean values of revertants per plate and Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
16.7 |
0.88 |
21.3 |
1.02 |
95.7 |
1.03 |
115.0 |
0.93 |
DMSO Control |
19.0 |
1.00 |
23.0 |
1.00 |
- |
- |
108.7 |
1.00 |
Ultrapure Water Control |
19.0 |
1.00 |
21.0 |
1.00 |
93.3 |
1.00 |
123.3 |
1.00 |
5000 |
18.0 |
0.95 |
25.3 |
1.21 |
107.3 |
1.15 |
129.7 |
1.05 |
1600 |
19.3 |
1.02 |
30.7 |
1.46 |
100.0 |
1.07 |
112.7 |
0.91 |
500 |
20.7 |
1.09 |
26.7 |
1.27 |
131.3 |
1.41 |
122.0 |
0.99 |
160 |
19.3 |
1.02 |
22.3 |
1.06 |
94.0 |
1.01 |
120.0 |
0.97 |
50 |
20.3 |
1.07 |
29.7 |
1.41 |
102.7 |
1.10 |
126.7 |
1.03 |
16 |
19.0 |
1.00 |
21.7 |
1.03 |
96.0 |
1.03 |
120.7 |
0.98 |
5 |
15.7 |
0.82 |
29.7 |
1.41 |
104.0 |
1.11 |
114.7 |
0.93 |
NPD (4 µg) |
283.3 |
14.91 |
- |
- |
- |
- |
- |
- |
SAZ (2 µg) |
- |
- |
- |
- |
12240 |
13.11 |
- |
- |
2 AA (2 µg) |
- |
- |
1114.7 |
48.46 |
- |
- |
17387 |
6.00 |
MR: Mutation Rate
Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and the DMSO was applied as vehicle for positive control substances: NPD and 2AA. The mutation rate of the test item, SAZ and untreated control is given referring to the ultrapure water; the mutation rate of NPD and 2AA is given referring to DMSO.
Table 2: Summary Table of the Results of the Initial Mutation Test
Confirmatory Mutation Test (Pre-Incubation Test) |
||||||||||||||||||||
Concentration (µg/plate) |
Salmonella typhimurium tester strains |
Escherichia coli WP2uvrA |
||||||||||||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
|||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||||||||
Mean values of revertants per plate Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
17.0 |
0.98 |
20.3 |
0.94 |
88.7 |
0.81 |
144.7 |
1.17 |
9.0 |
0.96 |
11.7 |
1.09 |
8.7 |
1.63 |
5.3 |
1.00 |
22.3 |
1.24 |
24.3 |
1.00 |
DMSO Control |
17.3 |
1.00 |
22.3 |
1.00 |
- |
- |
109.7 |
1.00 |
- |
- |
9.0 |
1.00 |
7.7 |
1.00 |
7.7 |
1.00 |
- |
- |
25.3 |
1.00 |
Ultrapure Water Control |
17.3 |
1.00 |
21.7 |
1.00 |
110.0 |
1.00 |
123.7 |
1.00 |
9.3 |
1.00 |
10.7 |
1.00 |
5.3 |
1.00 |
5.3 |
1.00 |
18.0 |
1.00 |
24.3 |
1.00 |
5000 |
24.7 |
1.42 |
28.7 |
1.32 |
105.0 |
0.95 |
114.7 |
0.93 |
8.3 |
0.89 |
13.3 |
1.25 |
5.3 |
1.00 |
6.7 |
1.25 |
23.7 |
1.31 |
28.0 |
1.15 |
1600 |
25.7 |
1.48 |
29.7 |
1.37 |
99.7 |
0.91 |
111.0 |
0.90 |
10.3 |
1.11 |
10.3 |
0.97 |
5.7 |
1.06 |
7.3 |
1.38 |
25.0 |
1.39 |
31.0 |
1.27 |
500 |
19.3 |
1.12 |
21.0 |
0.97 |
105.3 |
0.96 |
100.3 |
0.81 |
10.7 |
1.14 |
10.3 |
0.97 |
4.7 |
0.88 |
6.0 |
1.13 |
21.7 |
1.20 |
27.3 |
1.12 |
160 |
20.0 |
1.15 |
27.7 |
1.28 |
96.0 |
0.87 |
91.0 |
0.74 |
10.3 |
1.11 |
13.3 |
1.25 |
6.0 |
1.13 |
7.0 |
1.31 |
20.3 |
1.13 |
24.0 |
0.99 |
50 |
18.0 |
1.04 |
17.7 |
0.82 |
88.7 |
0.81 |
104.3 |
0.84 |
10.3 |
1.11 |
10.3 |
0.97 |
9.0 |
1.69 |
5.7 |
1.06 |
22.0 |
1.22 |
25.0 |
1.03 |
16 |
22.3 |
1.29 |
32.7 |
1.51 |
91.0 |
0.83 |
96.3 |
0.78 |
8.7 |
0.93 |
11.7 |
1.09 |
7.3 |
1.38 |
9.7 |
1.81 |
16.0 |
0.89 |
25.0 |
1.03 |
NPD (4 µg) |
470.0 |
27.12 |
- |
|
|
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
SAZ (2 µg) |
- |
- |
- |
- |
1549.3 |
-14.08 |
- |
- |
979.3 |
104.93 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
9AA (50 µg) |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
860.0 |
112.27 |
- |
- |
- |
- |
- |
- |
MMS (2 µL) |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
2AA (2 µg) |
- |
- |
1176.0 |
52.66 |
- |
- |
1008.0 |
9.19 |
- |
- |
179.3 |
19.93 |
- |
- |
169.0 |
22.04 |
- |
- |
- |
- |
2AA (50 µg) |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
342.0 |
13.50 |
MR: Mutation Rate
Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.
Table 3: Historical Control Values for Revertants/Plate (for the Period of 2008-2015)
|
Bacterial strains |
||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|||
Historical control data of untreated control |
-S9 |
Average |
21.4 |
106.0 |
10.4 |
8.1 |
25.6 |
SD |
3.7 |
27.3 |
1.5 |
2.5 |
5.5 |
||
Minimum |
9 |
65 |
3 |
2 |
11 |
||
Maximum |
39 |
157 |
23 |
19 |
45 |
||
+S9 |
Average |
28.0 |
117.1 |
11.9 |
9.0 |
34.3 |
|
SD |
4.2 |
19.4 |
1.5 |
2.0 |
5.4 |
||
Minimum |
12 |
75 |
4 |
3 |
18 |
||
Maximum |
48 |
166 |
24 |
20 |
56 |
||
Historical control data of DMSO control |
-S9 |
Average |
20.9 |
101.4 |
10.3 |
7.9 |
24.9 |
SD |
3.5 |
26.2 |
1.4 |
2.5 |
4.9 |
||
Minimum |
10 |
65 |
3 |
2 |
11 |
||
Maximum |
39 |
150 |
23 |
20 |
44 |
||
+S9 |
Average |
27.1 |
114.7 |
12.0 |
8.8 |
34.2 |
|
SD |
4.0 |
19.3 |
1.5 |
2.1 |
5.2 |
||
Minimum |
15 |
71 |
4 |
3 |
16 |
||
Maximum |
48 |
161 |
24 |
20 |
56 |
||
Historical control data of Water control |
-S9 |
Average |
22.4 |
105.5 |
10.4 |
7.5 |
26.3 |
SD |
3.6 |
27.6 |
1.6 |
2.3 |
5.9 |
||
Minimum |
12 |
67 |
3 |
2 |
13 |
||
Maximum |
36 |
156 |
24 |
15 |
47 |
||
+S9 |
Average |
28.0 |
117.4 |
11.5 |
8.7 |
35.2 |
|
SD |
4.0 |
19.8 |
1.4 |
2.3 |
5.2 |
||
Minimum |
15 |
83 |
4 |
4 |
18 |
||
Maximum |
43 |
166 |
22 |
16 |
56 |
||
Historical control data of positive controls |
-S9 |
Average |
255.6 |
958.9 |
842.1 |
467.4 |
712.3 |
SD |
30.7 |
149.9 |
134.0 |
105.7 |
57.5 |
||
Minimum |
123 |
522 |
354 |
109 |
320 |
||
Maximum |
647 |
1927 |
1871 |
1498 |
1283 |
||
+S9 |
Average |
1224.8 |
1431.9 |
165.4 |
148.0 |
264.7 |
|
SD |
293.8 |
339.9 |
35.1 |
21.3 |
74.2 |
||
Minimum |
409 |
581 |
85 |
68 |
141 |
||
Maximum |
2587 |
2923 |
507 |
407 |
487 |
Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2 uvrA
SD: Standard deviation;
DMSO: Dimethyl sulfoxide
Table 1: Results of Cytotoxicity Test Experiment I without Metabolic Activation
Treatment |
Precipitation |
Haemolysis |
Mean CBPI |
Mean % Cytotoxicity |
Solvent control MCM |
no |
no |
1.680 |
- |
Solvent control 0.9% NaCl 0.5%v/v |
no |
moderate |
1.60 |
- |
Positive control MMC 0.15 µg/mL |
no |
moderate |
1.548 |
3.5 |
Positive control MMC 0.3 µg/mL |
no |
slight |
1.560 |
2.7 |
Test item 1.95 mg/mL |
no |
slight |
1.657 |
1.4 |
Test item 0.98 mg/mL |
no |
slight |
1.716 |
-2.2 |
Test item 0.49 mg/mL |
no |
slight |
1.703 |
-1.3 |
Test item 0.24 mg/mL |
no |
slight |
1.643 |
2.2 |
Test item 0.12 mg/mL |
no |
slight |
1.653 |
1.6 |
Table 2: Results of Cytotoxicity Test Experiment I with Metabolic Activation
Treatment |
Precipitation |
Haemolysis |
Mean CBPI |
Mean % Cytotoxicity |
Solvent control MCM |
no |
no |
1.674 |
- |
Solvent control 0.9% NaCl 0.5%v/v |
no |
no |
1.607 |
- |
Positive control CPA 20 µg/mL |
no |
no |
1.316 |
18.1 |
Positive control CPA 30 µg/mL |
no |
no |
1.246 |
22.5 |
Test item 1.95 mg/mL |
no |
slight |
1.648 |
1.6 |
Test item 0.98 mg/mL |
no |
slight |
1.661 |
0.8 |
Test item 0.49 mg/mL |
no |
slight |
1.648 |
1.6 |
Test item 0.24 mg/mL |
no |
slight |
1.618 |
3.3 |
Test item 0.12 mg/mL |
no |
slight |
1.587 |
5.2 |
Table 3: Genotoxicity Results Experiment I
Treatment |
Average CBPI |
Cytotoxicity (%) |
Total No. of BINC examined |
Total No. of MBNC |
% MBNC |
Without metabolic activation |
|||||
Solvent control MCM |
1.680 |
- |
2004 |
9 |
0.45 |
Solvent control 0.9% NaCl 0.5%v/v |
1.604 |
- |
2000 |
10 |
0.50 |
Positive control MMC 0.3 µg/mL |
1.560 |
2.7 |
2002 |
52 |
2.60** |
Test item 1.95 mg/mL |
1.657 |
1.4 |
2000 |
5 |
0.25 |
Test item 0.98 mg/mL |
1.716 |
-2.2 |
2001 |
11 |
0.55 |
Test item 0.49 mg/mL |
1.703 |
-1.3 |
2000 |
8 |
0.40 |
With metabolic activation |
|||||
Solvent control MCM |
1.674 |
- |
2000 |
6 |
0.30 |
Solvent control 0.9% NaCl 0.5%v/v |
1.607 |
- |
2000 |
10 |
0.50 |
Positive control CPA 30 µg/mL |
1.246 |
22.5 |
2002 |
36 |
1.80** |
Test item 1.95 mg/mL |
1.648 |
1.6 |
2000 |
9 |
0.45 |
Test item 0.98 mg/mL |
1.661 |
0.8 |
2000 |
6 |
0.30 |
Test item 0.49 mg/mL |
1.648 |
1.6 |
2000 |
12 |
0.60 |
Asterisks indicate statistically significant differences to solvent control, with ** p < 0.01
Table 4: Results of Cytotoxicity Test Experiment II without Metabolic Activation
Treatment |
Precipitation |
Haemolysis |
Mean CBPI |
Mean % Cytotoxicity |
Solvent control MCM |
no |
no |
1.673 |
|
Solvent control 0.9% NaCl 0.5%v/v |
no |
no |
1.776 |
- |
Positive control MMC 0.3 µg/mL |
no |
no |
1.498 |
15.6 |
Positive control Colchicine 0.03 µg/mL |
no |
no |
1.216 |
31.5 |
Positive control Colchicine 0.0035 µg/mL |
no |
no |
1.208 |
32.0 |
Test item 1.95 mg/mL |
no |
no |
1.884 |
-12.6 |
Test item 0.98 mg/mL |
no |
no |
2.009 |
-20.1 |
Test item 0.49 mg/mL |
no |
no |
1.886 |
-12.7 |
Test item 0.24 mg/mL |
no |
no |
1.991 |
-19.0 |
Test item 0.12 mg/mL |
no |
no |
1.901 |
-13.6 |
For abbreviations see Annex 2 – Glossary
Table 5: Results Experiment II
Treatment |
Average CBPI |
Cytotoxicity (%) |
Total No. of BINC examined |
Total No. of MBNC |
% MBNC |
Solvent control MCM |
1.673 |
- |
2000 |
21 |
1.05 |
Solvent control 0.9% NaCl 0.5%v/v |
1.776 |
- |
2000 |
7 |
0.35 |
Positive control MMC 0.3 µg/mL |
1.498 |
15.6 |
2000 |
47 |
2.35** |
Positive control Colchicine 0.035 µg/mL |
1.208 |
32.0 |
2000 |
88 |
4.40** |
Test item 1.95 mg/mL |
1.884 |
-12.6 |
2000 |
14 |
0.70 |
Test item 0.98 mg/mL |
2.009 |
-20.1 |
2000 |
12 |
0.60 |
Test item 0.49 mg/mL |
1.886 |
-12.7 |
2000 |
9 |
0.45 |
Asterisks indicate statistically significant differences to solvent control, with ** p < 0.01
Table 6: Statistical Significance
Treatment |
p-Values Experiment I |
p-Values Experiment II |
|
without S9 |
with S9 |
without S9 |
|
Positive control MMC 0.3 µg/mL |
p < 0.01 |
- |
p < 0.01 |
Positive control CPA 30 µg/mL |
- |
p < 0.01 |
- |
Positive control Colchicine 0.035 µg/mL |
- |
- |
p < 0.01 |
Test item 1.95 mg/mL |
n.c. |
0.227 |
n.c. |
Test item 0.98 mg/mL |
0.330 |
n.c. |
n.c. |
Test item 0.49 mg/mL |
n.c. |
0.083 |
n.c. |
n.c.: not calculated because the ratio of MBNC is lower than or equal to the solvent control
Table 7: Historical Data without metabolic activation
Parameter |
% of MBNC |
||||
Substance |
Medium |
NaCl 0.9% |
MMC (short exposure) |
MMC (extended exposure) |
Colchicine |
Mean |
0.44 |
0.48 |
3.42 |
3.85 |
5.74 |
Standard Deviation |
0.27 |
0.28 |
1.94 |
1.67 |
2.64 |
Range (min – max) |
0.05 - 1.06 |
0.10 - 1.19 |
1.08 - 7.67 |
1.33 - 7.75 |
1.24 - 10.32 |
95% confidence interval |
0* - 0.98 |
0* - 1.04 |
0* - 7.29 |
0.52 – 7.19 |
0.47 - 11.02 |
Number of experiments |
35 |
30 |
29 |
29 |
14 |
Number of BINCs evaluated |
70831 |
61290 |
59691 |
59156 |
24008 |
Study 17050301G860 Experiment I |
0.45 |
0.50 |
2.6 |
- |
- |
Study 17050301G860 Experiment II |
1.05** |
0.35 |
- |
2.35 |
4.40 |
* calculated values are < 0. Since these values have no biological relevance, they are set equal to 0.
** Value is outside the 95.5 % confidence interval but still within the complete range of the historical
data of this solvent control.
Table 8: Historical Data with metabolic activation
Parameter |
% of MBNC |
||
Substance |
Medium |
NaCl 0.9% 0.5% v/v |
CPA |
Mean |
0.31 |
0.34 |
2.94 |
Standard Deviation |
0.14 |
0.21 |
0.92 |
Range (min – max) |
0.10 - 0.63 |
0.05 - 0.86 |
1.79 - 5.14 |
95% confidence interval |
0.04 – 0.58 |
0* - 0.75 |
1.10 – 4.77 |
Number of experiments |
28 |
28 |
28 |
Number of BINCs evaluated |
57349 |
57220 |
56922 |
Study 17050301G860 Experiment I |
0.30 |
0.50 |
1.80 |
* calculated values are < 0. Since these values have no biological relevance, they are set equal to 0
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames test
A study according OECD TG 471 was performed with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test). Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity. Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:
5000; 1600; 500; 160; 50 and 16 μg/plate. In the preliminary experiments the pH of the aqueous test item solution (50 mg/mL) was found as 3.47. Extremes of pH could have influencing effect on the mutagenicity results, therefore in the Initial and Confirmatory Mutation Tests thepH of the test item stock solution (prepared for the highest concentration of 50 mg/mL) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at 50 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (50 mg/mL) were 3.50 and 3.52. The pH of the test item containing overlays was ~7 in both experiments. In this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the mutagenicity result interpretation.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.
The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
Micronucleus test
A study according OECD TG 487 was performed to assess the genotoxic potential of the test item to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).
The test item was dissolved in minimal culture medium to prepare a stock solution with a concentration of 100 mM corresponding to 10 mM as highest concentration in the test. In addition, a geometric series of dilutions was prepared from the stock solution.
Two independent and valid experiments were performed. Human peripheral blood lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item or positive control, respectively. After the culture harvest time, the cells were harvested and slides were prepared. Cytotoxicity and level of micronuclei were determined.
In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test item to cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all cultures treated with solvent control, positive control and test item. On the basis of these data, the concentrations to be scored for micronuclei were selected. No cytotoxic effect was detected in any of the tested concentrations in both experiments.
Therefore, the three highest test item concentrations were evaluated for micronuclei.
Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations was observed. All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system. In conclusion, under the experimental conditions reported, the substance does not induce the formation of micronuclei in human lymphocytes in vitro.
HPRT test
A study according OECD TG 476 was performed to investigate the potential of the substance to induce mutations at the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79).
The assay comprised a pre-test and two independent experiments (experiment I and II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined. The first main experiment (experiment I) was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. The second experiment (experiment II) was performed with a treatment period of 24 hours without metabolic activation. The highest nominal concentration (10 mM) applied was chosen based on the good solubility of the test item in organic solvents and aqueous media and on the absence of cytotoxicity. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system. No substantial and reproducible dose-dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration (10 mM) of the test item.
In conclusion, it can be stated that under the experimental conditions of this study the test item did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.
Justification for classification or non-classification
Classification, Labeling, and Packaging Regulation (EC) No 1272/2008
The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, amended for the fifteenth time in Regulation (EU) 2020/1182.
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