Registration Dossier
Registration Dossier
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EC number: 205-748-3 | CAS number: 149-87-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames Test (Read-across from structurally similar substance)
Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), both in the presence and in the absence of metabolic activation.
Chromosome Aberration Study (Read-across from structurally similar substance)
Under the conditions of the study, the test material did not induce reproducible, statistically significant increases in chromosome aberrations in either assay, with or without metabolic activation. Therefore, the test material is considered not clastogenic in this test system.
Gene Mutation Study in Mammalian Cells (Read-across from structurally similar substance)
Treatment with the test material did not result in a statistically and biologically significant increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9 fraction) in the Mouse Lymphoma Assay. The test material was therefore considered to have no mutagenic potential.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 March 2012 to 30 March 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine and tryptophan operon.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Nutrient Broth No.2, 25.0 g per 1000 mL. Sterilization was performed at 121 °C in an autoclave.
- Properly maintained: Yes, all strains were stored at -80 ± 10 ºC. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent. Additional strain characteristics were checked regularly. Growing cultures were prepared form the frozen bacterial cultures, thawed at room temperature. 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a water bath shaker.
- Periodically "cleansed" against high spontaneous background: Yes. - Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Nutrient Broth No.2, 25.0 g per 1000 mL. Sterilization was performed at 121 °C in an autoclave.
- Properly maintained: Yes, all strains were stored at -80 ± 10 ºC. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent. Additional strain characteristics were checked regularly. Growing cultures were prepared form the frozen bacterial cultures, thawed at room temperature. 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a water bath shaker.
- Periodically "cleansed" against high spontaneous background: Yes. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction
- Test concentrations with justification for top dose:
- Main test: 5000, 2500, 1250, 625, 312.5, 156.25 and 78.125 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The solvent was chosen based on a preliminary solubility test. The test material was soluble in distilled water at 100 mg/mL concentration. Therefore, distilled water was chosen as the solvent for this study. The obtained formulations (50 μL) with the solution of top agar (2 mL) and phosphate buffer (500 µL) were examined in a test tube without test bacterium suspension and found to form a clear solution at 5000 µg/tube. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water and DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-1,2-phenylene-diamine; 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Preincubation method.
Before the overlaying, 50 μL of the test solution (or solvent or reference control), 100 μL of the overnight culture of the tester strains (cultured in Nutrient Broth No.2.) and 0.5 mL of the S9 mix or phosphate buffer (pH 7.4) were added into appropriate tubes to provide direct contact between bacteria and the test material. These tubes were then gently mixed and then incubated for 20 minutes at 37 ºC in a shaking incubator. After the pre-incubation period, 2 mL of molten top agar (kept at 45 °C) were added to the tubes; the content was mixed and poured on the surface of minimal glucose agar plates. After preparation, the plates were incubated at 37 °C.
DURATION
- Exposure duration: 48 hours.
NUMBER OF REPLICATIONS: Each concentration was performed in triplicate. A second test was performed at the same dosing range to confirm the results of the first assay.
OBSERVATIONS
The colony numbers on the untreated / negative / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported.
RANGE FINDING TEST:
- Test concentrations: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate, with and without metabolic activation.
- Tester strains: TA98 and TA100. - Evaluation criteria:
- The study was considered valid if:
- The number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests.
- At least five analyzable (non-cytotoxic) concentrations were presented in all strains of the main tests.
A positive (mutagenic) response:
- A dose related increase in the number of revertants occurred and/or,
- A reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- In all strains: the number of reversion more than twice higher than the reversion rate of the solvent control
A negative (non-mutagenic) response:
The test material was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test material and for the controls using a spreadsheet.
Mutation factor (MF) = mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate. - Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- MAIN TESTS
Higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Test in some sporadic cases. However, no dose-dependence was observed and they were below the biologically relevant threshold value and were non-reproducible. The numbers of revertant colonies were within the historical control range in all cases, so they were considered as reflecting the biological variability of the test.
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None observed in either test with or without metabolic activation.
RANGE-FINDING/SCREENING STUDIES
- Results used for determination of the concentration range for the definitive test: The observed numbers of revertant colonies were mostly in the normal range. Slightly higher or slightly lower colony counts compared to the solvent control were observed in some cases; however, they were without biological significance and in the historical control range in all cases. Therefore, they were considered to reflect the biological variability of the test system. No precipitate or sign of cytotoxicity were observed in the preliminary experiment. Based on the observed result of the preliminary experiment, 5000 μg/plate concentration was selected as the highest examined dose in the main test and lower concentrations will be evenly spaced by a factor of two. - Conclusions:
- Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2 uvrA both in the presence and in the absence of metabolic activation.
- Executive summary:
The mutagenic potential of the test material was assessed in an Ames test conducted under GLP conditions and in line with the standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100. S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2uvrA were exposed to the test material at concentrations of 5 000, 2500, 1 250, 625, 312.5, 156.25 and 78.125 µg/plate using the preincubation method. Positive and solvent controls were run concurrently for comparison.
Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA both in the presence and in the absence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted on read-across material
- Justification for type of information:
- Read-across from a structurally similar substance. Please refer to the RAAF report (section 13) for further information/ justification.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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:
- not examined
- Positive controls validity:
- valid
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 29 August 2012 to 16 December 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Dulbecco’s Modified Eagle’s Medium.
- Properly maintained: Yes, stocks were kept in a freezer at -80 ± 10 °C. The laboratory cultures were maintained in 150 cm² plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air.
- Periodically checked for Mycoplasma contamination: Yes, no infection was noted. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Assay 1 and 2 test concentrations: 5000, 2500, 1250, 625 and 312.5 μg/mL (with and without metabolic activation).
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Distilled water.
- Justification for choice of solvent/vehicle: Based on the results of a short preliminary solubility test, the test item was soluble in Distilled water at 500 mg/mL. Therefore, Distilled water was selected as solvent for the study. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- Positive control solutions were prepared immediately before the treatment of the cells and filtered sterile using a 0.22 μm syringe filter before use.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium.
Cells were treated with test material solutions, negative (solvent) or positive control solution (treatment volume: 100 μL/dish in all cases) for the given period of time at 37°C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with Dulbecco’s Modified Eagle’s Medium (supplemented with 2 mM L-glutamine and 1 v/v% Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
DURATION
- Exposure duration:
> Assay 1: 3 hours with and without S9-mix.
> Assay 2: 20 hours without S9-mix and 3 hours with S9-mix.
- Expression time:
> Assay 1: Cells were harvested 20 hours after the beginning of treatment (approximately 1.5 normal cell cycles).
> Assay 2: Cells were harvested 28 hours after the beginning of treatment (approximately 2 normal cell cycles).
SPINDLE INHIBITOR: Colchicine (0.2 μg/mL) was added to cultures 2 to 2.5 hours prior to harvesting.
The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (Methanol:Acetic acid 3:1 (v:v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried.
STAIN: The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted.
NUMBER OF REPLICATIONS: Two replications were performed.
NUMBER OF CELLS EVALUATED: At least one hundred metaphases with 22±2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Examination of slides from a culture was halted when 15 or more metaphases with aberrations (excluding gaps) had been recorded for that culture. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
DETERMINATION OF CYTOTOXICITY
- Method: For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (solvent) control as % relative survival.
OTHER EXAMINATIONS:
The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. - Evaluation criteria:
- The assay is considered valid, if the following criteria are met:
- The solvent control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.
A test material is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.
The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.
A test material is concluded to have given a negative response if no reproducible, statistically significant increases are observed. - Statistics:
- For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.
- 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 examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No large changes in the pH were observed.
- Effects of osmolality: A slightly higher than usual difference was observed in osmolality was observed at the highest concentration (5000 μg/mL) in both experiments of Assays 1 and 2.
- Water solubility: No insolubility was detected at the end of the treatment period in the final treatment medium for either Assay 1 or 2.
RANGE-FINDING/SCREENING STUDIES: A total of eight test concentrations between 5000 and 2.29 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.
Cells survival relative to the negative solvent control ranged from (exposure time/harvest time):
> Assay A: 88 – 114 % (3/20 without S9-mix) and 84 – 123 % (3/20 with S9-mix).
> Assay B: 77 – 127 % (20/28 without S9-mix) and 88 – 116 % (3/28 with S9-mix).
All observations were normal throughout the study, pH measured 7.4 in all samples and osmolarity ranged from 321 to 402 mmol/kg.
COMPARISON WITH HISTORICAL CONTROL DATA: The negative (solvent) control data were within the laboratory’s normal range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed in both experiment in Assays 1 and 2.
CHROMOSOME ADERRATION EVALUATION
Concentrations of 5000, 2500 and 1250 μg/mL (a total of three) were chosen for evaluation in both cases in Assays 1 and 2. Based on the observed results, an additional concentration (625 μg/mL) was evaluated in Assay 1 in the experiment with metabolic activation.
None of the treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in either assay without metabolic activation or in Assay 2 with metabolic activation. Following the initial analysis of three concentrations, a significant increase in chromosome aberrations at 1250 μg/mL in Assay 1 with metabolic activation was observed. No increase was found at two higher concentrations (2500 and 5000 μg/mL). A further concentration, 625 μg/mL, was then analysed and no increase in aberrations was observed. Therefore the increase was not repeatable either at other concentrations within the assay or between assays.
POLYPLOID AND ENDOREDUPLICATED METAPHASES
The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid metaphases (1-3) were found in some cases in the negative (solvent) control or test material treated samples in the performed experiments. No endoreduplicated metaphases were found in the main tests. - Conclusions:
- Under the conditions of the study, the test material did not induce reproducible, statistically significant increases in chromosome aberrations in either assay, with or without metabolic activation. Therefore, the test material is considered not clastogenic in this test system.
- Executive summary:
The clastogenic potential of the test material was determined in an in vitro mammalian cell chromosome aberration assay performed under GLP conditions and in line with the standardised guideline OECD 473, EU Method B.10 and EPA OPPTS 870.5375.
Cultures of Chinese hamster lung fibroblasts (V79) cells were exposed to the test material at concentrations of 5000, 2500, 1250, 625 and 312.5 μg/mL, in two assays, with and without metabolic activation. Assay 1 was performed with two experiments both with a three hour exposure period and 20 hours harvesting time. Both experiments were performed in the presence or absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats. Assay 2 was performed either in the presence of S9-mix with a three hour exposure period, or in the absence of S9-mix with a 20 hour exposure period. Both experiments were conducted with a harvesting time of 28 hours.
Treatment with the test material did not result in a statistically and biologically significant, repeatable, dose-dependent increase in the frequency of the cells with structural chromosome aberrations either in the presence or absence of a metabolic activation system. Additionally there was no indication of polyploidy or endoreplication.
The test material is therefore considered not to be clastogenic in this test system.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted on read-across material
- Justification for type of information:
- Read-across from a structurally similar substance. Please refer to the RAAF report (section 13) for further information/ justification.
- Reason / purpose for cross-reference:
- read-across source
- 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 examined
- Positive controls validity:
- valid
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 October 2012 to 4 February 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- tk+/- (thymidine kinase) locus.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI-10 medium.
- Properly maintained: Yes, cells were stored as frozen stocks in liquid nitrogen. For each experiment, one or more vials were thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study).
- Periodically checked for Mycoplasma contamination: Yes.
- Periodically "cleansed" against high spontaneous background: Yes. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 5000, 1666.7, 555.6, 185.2, 61.73 and 20.58 μg/mL, with and without metabolic activation in both assay 1 and 2.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Distilled water.
- Justification for choice of solvent/vehicle: Based on the available information (CiToxLAB study code: 12/073-020C), the test item is soluble in Distilled water at 500 mg/mL concentration. As this solvent is compatible with the test system, it was selected for vehicle (solvent) of the study. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Distilled water and DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Remarks:
- DMSO was used as the solvent of the positive control chemicals.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium.
A suitable volume (0.2 mL) of RPMI-5 medium, vehicle, test material formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or of 150 mM KCl (in case of 3-hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, at least 10^7 cells were placed in each of a series of 75 cm² sterile flasks. For the 24-hour treatment, at least 4x10^6 cells were placed in each of a series of 25 cm² sterile flasks. The treatment medium contained a reduced serum level of 5% (v/v) (RPMI-5).
TEST SYSTEM
- Exposure duration: During the treatment period, cultures were incubated at 37 °C ± 1 °C (approximately 5% CO2 in air). Gentle shaking was used during the 3-hour treatments. After the treatment period, cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in 20 mL RPMI-10.
> Assay 1: 3-hours in the presence and absence of S9 mix.
> Assay 2: 3-hours in the presence of S9 mix and for 24-hours in the absence of S9 mix.
- Expression time: Three days, during which subculturing was performed daily.
- Selection time: Two weeks.
SELECTION AGENT: Trifuorothymidine.
NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.
DETERMINATION OF CYTOTOXICITY
- Method: Relative survival.
OTHER EXAMINATIONS
- Cultures were visually examined at the beginning and end of treatments.
- Measurement of pH and osmolality was also performed after the treatment period. - Evaluation criteria:
- ASSAY ACCEPTANCE CRITERIA
The assay was considered valid if all of the following criteria were met:
- The mutant frequency in the negative (vehicle) control cultures fell within the normal range (50-170 mutants per 10^6 viable cells).
- The positive control chemicals induced a statistically significant increase in the mutant frequency.
- The plating efficiency (PE viability) of the negative (vehicle) controls was within the range of 65% to 120% at the end of the expression period.
- At least four test concentrations were present, where the highest concentration produced approximately 80-90 % toxicity (measured by %RS or RTG), resulted in precipitation, or was 5 mg/mL, 5 μL/mL or 0.01 M (whichever is the lowest), or it was the highest practical concentration.
EVALUATION CRITERIA
The test material was considered to be mutagenic in this assay if all the following criteria were met:
- The assay was valid.
- Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency were observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
- The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
- There was a significant concentration-relationship as indicated by a linear trend analysis (p < 0.05).
- The mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 10^6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis. - Statistics:
- Statistical significance of mutant frequencies (total wells with clones) was performed using computer software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
- 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 examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in pH after treatment in either assay.
- Effects of osmolality: There were no large changes in osmolality after treatment in either assay.
RANGE-FINDING/SCREENING STUDIES:
Treatment concentrations for the mutation assay were selected based on the results of two short Preliminary Toxicity Tests. 3-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test material concentrations to determine toxicity immediately after the treatments. The highest concentration tested in the preliminary experiment was 5000 μg/mL.
No insolubility and no cytotoxicity was observed in the preliminary experiments. Therefore, the concentration selected for the main experiments were expected to cover the concentration range from the maximum cytotoxicity (resulting approximately 10-20 % relative survival) to little or no cytotoxicity. The selected highest concentration was 5000 μg/mL. The lower test concentrations were generally separated by factor of three. Six test concentrations were selected for the main experiments.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity of the test material was observed in either assay 1 or 2.
- Water solubility: No insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments.
MUTATION ASSAY EVALUATION
Assay 1: No significant increase in the mutation frequency was observed at any of the six evaluated concentrations, which had been exposed to the test material for 3 hours both in the absence and presence of metabolic activation. Furthermore no dose response to the treatment was indicated by the linear trend analysis. This assay was considered as negative.
Assay 2: An evaluation was made using data of all the six examined concentrations. No statistically significant increases in the mutation frequency were observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis. This assay was considered as negative, thus confirmed the results of the first main test.
Some minor increases in the mutation frequency were observed sporadically in Assays 1 and 2; however, they were without any statistical significance and the difference between the observed values and the relevant solvent control value did not exceed the global evaluation factor, so they was considered as biologically not relevant increases, just showing the biological variability of the test system. - Conclusions:
- Treatment with the test material did not result in a statistically and biologically significant increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9 fraction) in the Mouse Lymphoma Assay. The test material was therefore considered to have no mutagenic potential.
- Executive summary:
The mutagenic potential of the test material was determined in an in vitro mouse lymphoma assay, conducted under GLP conditions and in line with the standardised guidelines OECD 476 and EU Method B.17.
Cultures of mouse lymphoma L5178Y cells were exposed to the test material at concentrations of 5000, 1666.7, 555.6, 185.2, 61.73 and 20.58 μg/mL, in two assays, both with and without the addition of a rat metabolic activation system (S9 fraction). Assay 1 was performed with two experiments, both with a three hour exposure period; however the first was performed in the presence of metabolic activation and the second without. Assay 2 was performed again with two experiments. The first was performed with metabolic activation and an exposure period of 3 hours, whereas the second experiment was performed without metabolic activation with a 24 hour exposure period. All experiments were conducted with an expression time of 3 days and a selection time of 2 weeks, with trifuorothymidine as the selection agent.
Treatment with the test material did not result in a statistically and biologically significant increase in mutation frequencies either in the presence or absence of metabolic activation system in the Mouse Lymphoma Assay. The test material was therefore considered to have no mutagenic potential under the conditions of this test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted on read-across material
- Justification for type of information:
- Read-across from a structurally similar substance. Please refer to the RAAF report (section 13) for further information/ justification.
- Reason / purpose for cross-reference:
- read-across source
- 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 examined
- Positive controls validity:
- valid
Referenceopen allclose all
Table 1: Summary of Initial Mutation Test
Concentration (µg/plate) |
S. typhimurium tester strains |
E. coli |
|||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2uvrA |
|||||||
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
||
Untreated Control |
Mean |
28.0 |
33.7 |
98.3 |
110.3 |
10.3 |
11.3 |
9.7 |
11.3 |
29.3 |
41.3 |
MF |
1.29 |
0.90 |
0.99 |
0.94 |
1.00 |
0.92 |
1.00 |
0.83 |
0.77 |
0.82 |
|
Distilled Water Control |
Mean |
21.7 |
37.3 |
99.3 |
117.3 |
10.3 |
12.3 |
9.7 |
13.7 |
38.0 |
50.7 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
DMSO Control |
Mean |
23.3 |
32.7 |
- |
121.3 |
- |
16.3 |
11.3 |
13.7 |
- |
35.3 |
MF |
1.08 |
0.88 |
- |
1.03 |
- |
1.32 |
1.17 |
1.00 |
- |
0.70 |
|
5000 |
Mean |
29.7 |
32.3 |
93.7 |
112.7 |
8.0 |
11.0 |
13.0 |
11.7 |
33.3 |
44.3 |
MF |
1.37 |
0.87 |
0.94 |
0.96 |
0.77 |
0.89 |
1.34 |
0.85 |
0.88 |
0.88 |
|
2500 |
Mean |
24.7 |
34.3 |
108.7 |
118.0 |
12.7 |
13.0 |
10.7 |
12.3 |
30.0 |
42.3 |
MF |
1.14 |
0.92 |
1.09 |
1.01 |
1.23 |
1.05 |
1.10 |
0.90 |
0.79 |
0.84 |
|
1250 |
Mean |
26.7 |
32.7 |
106.0 |
130.7 |
9.7 |
10.0 |
11.0 |
12.0 |
37.3 |
46.3 |
MF |
1.23 |
0.88 |
1.07 |
1.11 |
0.94 |
0.81 |
1.14 |
0.88 |
0.98 |
0.91 |
|
625 |
Mean |
24.3 |
32.7 |
99.7 |
129.0 |
11.3 |
11.0 |
9.0 |
14.7 |
38.7 |
44.7 |
MF |
1.12 |
0.88 |
1.00 |
1.10 |
1.10 |
0.89 |
0.93 |
1.07 |
1.02 |
0.88 |
|
312.5 |
Mean |
26.0 |
23.0 |
114.3 |
123.0 |
11.3 |
15.0 |
9.7 |
12.0 |
37.7 |
48.0 |
MF |
1.20 |
0.62 |
1.15 |
1.05 |
1.10 |
1.22 |
1.00 |
0.88 |
0.99 |
0.95 |
|
156.25 |
Mean |
33.0 |
28.3 |
100.0 |
134.7 |
6.7 |
11.7 |
8.0 |
13.3 |
36.0 |
41.3 |
MF |
1.52 |
0.76 |
1.01 |
1.15 |
0.65 |
0.95 |
0.83 |
0.98 |
0.95 |
0.82 |
|
78.125 |
Mean |
22.7 |
33.0 |
103.0 |
123.7 |
4.3 |
14.0 |
8.0 |
13.3 |
22.3 |
45.7 |
MF |
1.05 |
0.88 |
1.04 |
1.05 |
0.42 |
1.14 |
0.83 |
0.98 |
0.59 |
0.90 |
|
Table 2: Summary of Confirmatory Mutation Test
Concentration (µg/plate) |
S. typhimurium tester strains |
E. coli |
|||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2uvrA |
|||||||
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
||
Untreated Control |
Mean |
22.0 |
32.3 |
110.3 |
123.3 |
7.0 |
9.7 |
8.3 |
5.7 |
27.3 |
37.3 |
MF |
0.65 |
0.96 |
0.96 |
1.00 |
0.81 |
1.07 |
1.14 |
0.61 |
1.11 |
0.97 |
|
Distilled Water Control |
Mean |
33.7 |
33.7 |
115.3 |
123.3 |
8.7 |
9.0 |
7.3 |
9.3 |
24.7 |
38.7 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
DMSO Control |
Mean |
31.0 |
32.7 |
- |
123.7 |
- |
10.0 |
7.0 |
6.7 |
- |
37.7 |
MF |
0.92 |
0.97 |
- |
1.00 |
- |
1.11 |
0.95 |
0.71 |
- |
0.97 |
|
5000 |
Mean |
28.0 |
28.3 |
97.7 |
114.0 |
6.3 |
9.0 |
7.7 |
9.3 |
31.7 |
42.0 |
MF |
0.83 |
0.84 |
0.85 |
0.92 |
0.73 |
1.00 |
1.05 |
1.00 |
1.28 |
1.09 |
|
2500 |
Mean |
34.7 |
35.3 |
102.7 |
130.3 |
7.0 |
16.0 |
7.0 |
8.3 |
33.0 |
43.7 |
MF |
1.03 |
1.05 |
0.89 |
1.06 |
0.81 |
1.78 |
0.95 |
0.89 |
1.34 |
1.13 |
|
1250 |
Mean |
28.7 |
29.0 |
108.0 |
119.7 |
5.3 |
8.7 |
6.0 |
9.3 |
27.7 |
47.7 |
MF |
0.85 |
0.86 |
0.94 |
0.97 |
0.62 |
0.96 |
0.82 |
1.00 |
1.12 |
1.23 |
|
625 |
Mean |
29.3 |
33.3 |
116.3 |
101.0 |
6.0 |
11.3 |
7.7 |
7.7 |
32.7 |
36.3 |
MF |
0.87 |
0.99 |
1.01 |
0.82 |
0.69 |
1.26 |
1.05 |
0.82 |
1.32 |
0.94 |
|
312.5 |
Mean |
24.7 |
39.3 |
101.0 |
133.7 |
6.7 |
10.7 |
5.7 |
6.7 |
31.0 |
43.7 |
MF |
0.73 |
1.17 |
0.88 |
1.08 |
0.77 |
1.19 |
0.77 |
0.71 |
1.26 |
1.13 |
|
156.25 |
Mean |
25.3 |
33.3 |
114.3 |
124.0 |
6.3 |
13.3 |
6.0 |
5.7 |
34.0 |
40.7 |
MF |
0.75 |
0.99 |
0.99 |
1.01 |
0.73 |
1.48 |
0.82 |
0.61 |
1.38 |
1.05 |
|
78.125 |
Mean |
24.0 |
34.7 |
104.7 |
111.0 |
7.0 |
10.7 |
8.0 |
7.3 |
29.0 |
39.3 |
MF |
0.71 |
1.03 |
0.91 |
0.90 |
0.81 |
1.19 |
1.09 |
0.79 |
1.18 |
1.02 |
|
Table 1: Summary Table of Chromosome Aberration Assay 1
S9-mix |
Concentration (µg/mL) |
Exposure/Harvest Time |
Relative Survival (%) # |
Mean % Aberrant Cells ## |
- |
Negative (solvent) Control |
3h/20h |
100 |
0.5 |
5000 |
3h/20h |
95 |
0.0 |
|
2500 |
3h/20h |
115 |
0.5 |
|
1250 |
3h/20h |
109 |
2.0 |
|
625 |
3h/20h |
83 |
NE |
|
312.5 |
3h/20h |
101 |
NE |
|
Positive Control |
3h/20h |
80 |
10.5*** |
|
+ |
Negative (solvent) Control |
3h/20h |
100 |
1.0 |
5000 |
3h/20h |
90 |
0.0 |
|
2500 |
3h/20h |
96 |
3.5 |
|
1250 |
3h/20h |
92 |
6.5** |
|
625 |
3h/20h |
102 |
3.0 |
|
312.5 |
3h/20h |
100 |
NE |
|
Positive Control |
3h/20h |
59 |
100.0*** |
Negative (solvent) control: 1% (v/v) Distilled water
Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL
Positive control (+S9): Cyclophosphamide, 6 μg/mL
NE: not evaluated
#: compared to the negative (solvent) control
##: excluding gaps
**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control.
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control.
Table 2: Summary Table of Chromosome Aberration Assay 2
S9-mix |
Concentration (µg/mL) |
Exposure/Harvest Time |
Relative Survival (%) # |
Mean % Aberrant Cells ## |
- |
Negative (solvent) Control |
20h/28h |
100 |
2.0 |
5000 |
20h/28h |
81 |
2.0 |
|
2500 |
20h/28h |
87 |
0.5 |
|
1250 |
20h/28h |
85 |
0.5 |
|
625 |
20h/28h |
95 |
NE |
|
312.5 |
20h/28h |
83 |
NE |
|
Positive Control |
20h/28h |
62 |
30.4*** |
|
+ |
Negative (solvent) Control |
3h/28h |
100 |
1.5 |
5000 |
3h/28h |
85 |
2.5 |
|
2500 |
3h/28h |
85 |
1.5 |
|
1250 |
3h/28h |
83 |
2.0 |
|
625 |
3h/28h |
93 |
NE |
|
312.5 |
3h/28h |
89 |
NE |
|
Positive Control |
3h/28h |
35 |
100.0*** |
Negative (solvent) control: 1% (v/v) Distilled water
Positive control (-S9): Ethyl methanesulfonate, 0.4 μL/mL
Positive control (+S9): Cyclophosphamide, 6 μg/mL
NE: not evaluated
#: compared to the negative (solvent) control
##: excluding gaps
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control.
Table 3: Summary Table of Polyploidy and Endoreduplicated Cells Assay 1
S9-mix |
Concentration (µg/mL) |
Exposure/Harvest Time |
No. of Cells Observed |
|
Polyploid |
Endoreduplicated |
|||
- |
Negative (solvent) Control |
3h/20h |
0 |
0 |
1250 |
3h/20h |
2 |
0 |
|
2500 |
3h/20h |
0 |
0 |
|
5000 |
3h/20h |
3 |
0 |
|
Positive Control |
3h/20h |
0 |
0 |
|
+ |
Negative (solvent) Control |
3h/20h |
0 |
0 |
625 |
3h/20h |
0 |
0 |
|
1250 |
3h/20h |
1 |
0 |
|
2500 |
3h/20h |
2 |
0 |
|
5000 |
3h/20h |
1 |
0 |
|
Positive Control |
3h/20h |
0 |
0 |
|
Table 4: Summary Table of Polyploidy and Endoreduplicated Cells Assay 2
S9-mix |
Concentration (µg/mL) |
Exposure/Harvest Time |
No. of Cells Observed |
|
Polyploid |
Endoreduplicated |
|||
- |
Negative (solvent) Control |
20h/28h |
1 |
0 |
1250 |
20h/28h |
0 |
0 |
|
2500 |
20h/28h |
2 |
0 |
|
5000 |
20h/28h |
0 |
0 |
|
Positive Control |
20h/28h |
0 |
0 |
|
+ |
Negative (solvent) Control |
3h/28h |
0 |
0 |
1250 |
3h/28h |
0 |
0 |
|
2500 |
3h/28h |
0 |
0 |
|
5000 |
3h/28h |
2 |
0 |
|
Positive Control |
3h/28h |
0 |
0 |
|
Table 1: Summary of Results for Assay 1
S9-mix |
Treatment Period (hours) |
Sample Concentration (µg/mL) |
Survival Results |
Viability |
Mutagenicity |
|
PE % |
Harmonised %RS § |
PE % |
MF 10^6 viable cells |
|||
+ |
3 |
5000 |
71.15 |
90 |
70.65 |
95.6 |
166.7 |
70.15 |
104 |
75.36 |
69.1 |
||
555.6 |
77.01 |
110 |
77.01 |
88.6 |
||
185.2 |
70.15 |
108 |
70.65 |
100.9 |
||
61.73 |
76.45 |
114 |
70.15 |
116.7 |
||
20.58 |
76.36 |
104 |
71.66 |
91.1 |
||
Vehicle Control |
70.15 |
100 |
68.18 |
121.2 |
||
Vehicle Control for CP (4 µg/mL) |
73.75 |
104 |
67.69 |
97.5 |
||
Untreated Control |
71.15 |
91 |
65.80 |
102.6 |
||
Positive Control CP |
37.13 |
54 |
42.03 |
865.5* |
||
- |
3 |
5000 |
81.04 |
96 |
70.15 |
109.1 |
166.7 |
76.45 |
100 |
70.15 |
103.7 |
||
555.6 |
72.70 |
95 |
56.30 |
121.2 |
||
185.2 |
74.82 |
104 |
61.30 |
124.8 |
||
61.73 |
73.22 |
96 |
58.75 |
116.2 |
||
20.58 |
74.82 |
92 |
69.15 |
107.4 |
||
Vehicle Control |
73.22 |
100 |
75.90 |
100.8 |
||
Vehicle Control for NQO (0.15 µg/mL) |
82.85 |
104 |
71.15 |
98.0 |
||
Untreated Control |
76.45 |
103 |
63.96 |
111.4 |
||
Positive Control NQO |
52.04 |
63 |
59.17 |
635.6* |
||
CP = Cyclophosphamide; NQO = 4-Nitroquinoline-N-oxide; PE % = Plating Efficiency; %RS = percentage relative survival; MF = mutation frequency
Vehicle control = 1% (v/v) Distilled water
Vehicle control for CP and NQO = 1% (v/v) DMSO
§ = Relative survival values (%) corrected with the post treatment cell concentrations.
* Statistically significant.
Table 2: Summary of Results for Assay 2
S9-mix |
Treatment Period (hours) |
Sample Concentration (µg/mL) |
Survival Results |
Viability |
Mutagenicity |
|
PE % |
Harmonised %RS § |
PE % |
MF 10^6 viable cells |
|||
+ |
3 |
5000 |
77.57 |
104 |
74.28 |
73.1 |
166.7 |
74.82 |
103 |
68.18 |
94.7 |
||
555.6 |
72.70 |
106 |
72.18 |
106.0 |
||
185.2 |
68.66 |
94 |
75.90 |
83.1 |
||
61.73 |
71.15 |
108 |
66.74 |
82.4 |
||
20.58 |
69.65 |
107 |
69.15 |
96.5 |
||
Vehicle Control |
73.22 |
100 |
66.27 |
108.7 |
||
Vehicle Control for CP (4 µg/mL) |
74.28 |
109 |
66.27 |
104.1 |
||
Untreated Control |
75.36 |
103 |
65.80 |
108.3 |
||
Positive Control CP |
39.84 |
29 |
49.12 |
1130.4* |
||
- |
24 |
5000 |
65.34 |
115 |
63.06 |
133.5 |
166.7 |
66.27 |
102 |
66.74 |
112.4 |
||
555.6 |
71.15 |
121 |
70.65 |
96.6 |
||
185.2 |
65.34 |
108 |
71.15 |
118.3 |
||
61.73 |
67.22 |
111 |
68.66 |
124.8 |
||
20.58 |
73.22 |
129 |
64.42 |
99.0 |
||
Vehicle Control |
67.69 |
100 |
68.66 |
88.6 |
||
Vehicle Control for NQO (0.15 µg/mL) |
71.15 |
120 |
72.18 |
111.3 |
||
Untreated Control |
68.18 |
100 |
62.62 |
136.9 |
||
Positive Control NQO |
27.58 |
42 |
62.62 |
686.7* |
||
CP = Cyclophosphamide; NQO = 4-Nitroquinoline-N-oxide; PE % = Plating Efficiency; %RS = percentage relative survival; MF = mutation frequency
Vehicle control = 1% (v/v) Distilled water
Vehicle control for CP and NQO = 1% (v/v) DMSO
§ = Relative survival values (%) corrected with the post treatment cell concentrations.
* Statistically significant.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Ames Test (Read-across from structurally similar substance)
The mutagenic potential of the test material was assessed in an Ames test conducted under GLP conditions and in line with standardised guidelines.
S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2uvrA were exposed to the test material at concentrations of 5 000, 2 500, 1 250, 625, 312.5, 156.25 and 78.125 µg/plate using the pre-incubation method. Positive and solvent controls were run concurrently for comparison.
Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA both in the presence and in the absence of metabolic activation.
Chromosome Aberration Study (Read-across from structurally similar substance)
The clastogenic potential of the test material was determined in an in vitro mammalian cell chromosome aberration assay performed under GLP conditions and in line with the standardised guideline OECD 473, EU Method B.10 and EPA OPPTS 870.5375.
Cultures of Chinese hamster lung fibroblasts (V79) cells were exposed to the test material at concentrations of 5000, 2500, 1250, 625 and 312.5 μg/mL, in two assays, with and without metabolic activation. Assay 1 was performed with two experiments both with a three hour exposure period and 20 hours harvesting time. Both experiments were performed in the presence or absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats. Assay 2 was performed either in the presence of S9-mix with a three hour exposure period, or in the absence of S9-mix with a 20 hour exposure period. Both experiments were conducted with a harvesting time of 28 hours.
Treatment with the test material did not result in a statistically and biologically significant, repeatable, dose-dependent increase in the frequency of the cells with structural chromosome aberrations either in the presence or absence of a metabolic activation system. Additionally there was no indication of polyploidy or endoreplication.
The test material is therefore considered not to be clastogenic in this test system.
Gene Mutation Study in Mammalian Cells (Read-across from structurally similar substance)
The mutagenic potential of the test material was determined in an in vitro mouse lymphoma assay, conducted under GLP conditions and in line with the standardised guidelines OECD 476 and EU Method B.17.
Cultures of mouse lymphoma L5178Y cells were exposed to the test material at concentrations of 5000, 1666.7, 555.6, 185.2, 61.73 and 20.58 μg/mL, in two assays, both with and without the addition of a rat metabolic activation system (S9 fraction). Assay 1 was performed with two experiments, both with a three hour exposure period; however the first was performed in the presence of metabolic activation and the second without. Assay 2 was performed again with two experiments. The first was performed with metabolic activation and an exposure period of 3 hours, whereas the second experiment was performed without metabolic activation with a 24 hour exposure period. All experiments were conducted with an expression time of 3 days and a selection time of 2 weeks, with trifuorothymidine as the selection agent.
Treatment with the test material did not result in a statistically and biologically significant increase in mutation frequencies either in the presence or absence of metabolic activation system in the Mouse Lymphoma Assay. The test material was therefore considered to have no mutagenic potential under the conditions of this test.
Justification for classification or non-classification
Based on the submitted in vitro data, testing does not indicate any evidence of genetic toxicity of the test material. In accordance with criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material does not require classification for genetic toxicity.
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