Registration Dossier
Registration Dossier
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EC number: 700-673-7 | CAS number: 132638-45-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2012
negative, in vitro chromosome aberration test with human lymphocytes (with and without S-9 activation), OECD TG 473, 2012
negative, in vitro gene mutation to , TK +/-, locus of the L5178Y mouse lymphoma cell line (with and without S-9 activation), OECD TG 490, 2016
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 28-09-2011 to 17-11-2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- 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
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: July 2011; signature: August 2011
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine or tryptophan locus
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbitone/B-Naphthoflavone induced rat liver S9.
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test (plate incorporation method): 0, 0.15, 0.50, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1 (plate incorporation method): 0, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 0, 50, 150, 500, 1500 and 5000 µg/plate
Dose levels were selected based on the results of the Preliminary Toxicity Test. The top dose was the guideline specified limit dose level selected due to absence of cytotoxicity and solubility of the test item in the solvent/vehicle. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed. Dimethyl sulphoxide was selected as the vehicle. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without metabolic activation S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- With metabolic activation S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)
DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Experiment 2. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media. Subsequently, the procedure for incubation and duration was the same as in Experiment 1.
SELECTION AGENT (mutation assays): histidine-deficient agar
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal. - Statistics:
- Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- See table 1 to table 4
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY: cytotoxicity was not evident at any concentration
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation. - Executive summary:
The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using both the Ames plate incorporation and pre incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. Five test item dose levels were again selected in Experiment 2 and was 50 to 5000 µg/plate. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial lawn in all strains up to 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9‑mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06-01-2012 to 24-05-2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- 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:
- equivalent or similar to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- : Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: July 2011; signature: August 2011
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable (chromosome aberration test)
- Species / strain / cell type:
- lymphocytes: human
- Test concentrations with justification for top dose:
- The maximum dose level was 1962 µg/mL, calculated to be equivalent to 10 mM concentration, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al ., 1991) within the 0 to 1962 μg/mL range (full results recorded in the full study report).
I. Preliminary toxicity test: 0 (control), 7.66, 15.33, 30.66, 61.31, 122.63, 245.255, 490.5, 981 and 1962 μg/mL
Within three exposure groups:
i) 4-hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
II. Main Test:
Experiment 1
4(20)-hour without S9: 0*, 250, 500, 750*, 1000*, 1250*, 1500* μg/ml and MMC 0.4*
4(20)-hour with S9: 0*, 250, 500*, 750, 1000*, 1250*, 1500* μg/ml and CP 5*
Experiment 2
4(20)-hour with S9: 0*, 500, 750, 1000*, 1125*, 1250*, 1375*, 1500, 1625 μg/ml and CP 5*
24-hour without S9: 0*, 15.63, 31.25*, 62.5*, 125, 220*, 375, 500 μg/mL and MMC 0.2*
where:
* = dose levels selected for metaphase analysis
MMC= Mitomycin C
CP = Cyclophosphamide - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in Minimal Essential Medium (MEM) at 19.62 mg/mL but was soluble in DMSO at 196.2 mg/mL in solubility checks performed. The maximum dose level (determined prior to the test based on molecular weight) was 1962 µg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1962 μg/mL range (full results recorded in the full study report). The test item was formulated within two hours of it being applied to the test system. - Untreated negative controls:
- other:
- Remarks:
- Vehicle control served as the negative control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without metabolic activation, 0.4 and 0.2 μg/ml experiment 1 and 2 respectively.
- Untreated negative controls:
- other:
- Remarks:
- Vehicle control served as the negative control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation 5 μg/ml, experiment 1 and 2 respectively
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Other:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 9.05 mL MEM, 10% (FBS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.75 mL heparinized whole blood
DURATION
- Preincubation period: Not reported.
- Exposure duration:
The preliminary toxicity test was performed using both of the exposure conditions as described for both experiments (below) in the absence of metabolic activation only.
I. With Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of the Main Experiments. After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.
II. Without Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.
In the 24-hour exposure in the absence of S9, the exposure was continuous. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 μg/ml)
NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.
NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes. Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) including endoreduplicated cells, reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range was reported in the full study report.
- Other: Scoring: Where possible, the first 100 consecutive well-spread metaphases from each concentration (200 per duplicate) were assessed for observations, if the cell had 44 to 48 chromosomes, any breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines. Where the analysis of the slide resulted in a large frequency of aberrant cells then the analysis was terminated after a total of 50 cells where there were 30 to 50% with aberrations any gaps, breaks or rearrangements were recorded. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. - Evaluation criteria:
- Positive response criteria
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration-related or a statistically significant increase is exhibited in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
Negative response criteria
A test item can be classified as non-genotoxic if:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment.
Statistical analysis is also performed (see: ‘Statistics’). Biological relevance of the results are to be considered first. Statistical methods are used to analyze the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response. - Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. Analysis of data from in vitro cytogenetic assays. In Statistical Evaluation of mutagenicity test data: UKEMS sub-committee on guidelines for mutagenicity testing. Report Part III (Ed: Kirkland, D.J.), Cambridge University Press (1989)
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 1250 μg/ml (+/- S9, 4 hr exposure); 250 μg/ml (-S9, 24 hr exposure).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: here was no significant change osmolality (did not increase by more than 50 mOsm) when the test item was dosed into media
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation:
In the preliminary test: A precipitate of the test item was observed in the parallel blood-free cultures at 1962 µg/mL in the 4(20)-hour exposure groups in the presence of S9. A precipitate of the test item was observed in the parallel blood-free cultures at or above 981 µg/mL in the 4(20)-hour and 24-hour exposure groups absence of S9.
Main test: Experiment 1: The qualitative assessment of the slides determined that the toxicity and precipitate was similar to that observed in the Preliminary Toxicity Test. Precipitate observations were made at the end of exposure in blood-free cultures and greasy/oily precipitate was noted at or above 1250 µg/mL in the presence or absence of S9. The maximum dose level selected for metaphase analysis was based on toxicity for both
exposure groups, 4(20)-hours and was 1500 μg/ml in the presence and absence of S9. In Experiment 2: a precipitate of the test item in the pellet was observed at the end of the treatment period at and above 1375 μg/ml in the presence of S9. No precipitate was observed at the end of the exposure period in the absence of S9 at up to 500 μg/ml in the 24-hour continuous exposure group.
- Other confounding effects: None reported.
RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 1962 μg/mL. The maximum dose was the maximum recommended dose level. The selection of the maximum dose level was based on toxicity for the main test.
COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations. (Within the Historic Control Data range presented in the full study report).
ADDITIONAL INFORMATION ON CYTOTOXICITY: See Table 1 and 2. - Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study, the test substance was considered to be non-clastogenic to human lymphocytes in vitro. - Executive summary:
The study was performed to the requirements of OECD TG 473 and EU Method B.10 under GLP conditions to assess the potential chromosomal mutagenicity of the test substance, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The exposure conditions were divided between experiment 1 which was 4(20)-hour with and without S9 activation, and experiment 2 which was 4(20)-hour with S9 activation and 24-hour exposure without S9 activation. The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test (Cell Growth Inhibition Test) where the results indicated that the maximum concentration should be limited to the lowest precipitating dose level with toxicity also being taken into account in the dose selection. The dose levels selected for the Main Test were as follows, experiment 1: 4(20)-hour with and without S9-Mix (2%): 0, 250, 500, 750, 1000, 1250, 1500 μg/mL. For experiment 2: 4(20)-hour with S9 mix (2%): 0, 500, 750, 1000, 1125, 1250, 1375, μg/mL. In the 24-hour continuous exposure without S9 mix activation the doses levels were: 0, 15.63, 31.25, 62.5, 125, 250, 375, 500 μg/mL. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. In Experiment 1, small but statistically significant increases in the frequency of cells with aberrations were observed, predominately in the presence of metabolic activation. The maximum response observed in the presence of metabolic activation was associated with a dose level that induced approximately 50% mitotic inhibition. In the absence of metabolic activation the response was observed at a dose level that exceeded the optimum target level of 50% toxicity. In both cases the response was not clearly dose-related. In Experiment 2, the response observed in the presence of S9 was much weaker and was associated with a dose level that slightly exceeded the approximate 50% mitotic inhibition. It should also be noted that it only marginally exceeded the in-house historical vehicle control upper limit of 2.0% for with-S9 exposures but was the same as the overall historical maximum for all exposure conditions (2.5%). A small but statistically significant response was observed in the 24-hour continuous exposure at 250 μg/ml, however the aberrations observed were all in the ‘A’ culture and were all break type aberrations. The response was therefore, considered to be of little biological relevance. It was considered that the small but statistically significant increase in the frequency of aberrant cells observed in experiment 1 in the presence of S9 was not toxicologically significant. The response was not clearly dose-related and not reproduced in a repeat experiment using the same experimental conditions, and dose levels that induced similar levels of toxicity. It was therefore concluded that the clastogenic activity was occurring at or around the onset of excessive toxicity giving some doubt to its biological relevance and, therefore, toxicological significance. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10-09-2015 to 06-10-2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- Japanese MITI/MHW guidelines: `Kanpoan No. 287 - Environment Protection Agency`; `Eisei No. 127 - Ministry of Health and Welfare` and `Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry`
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: July 2015; signature: September 2015
- Type of assay:
- in vitro mammalian cell transformation assay
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble at 1962 μg/mL, equivalent to 10 mM in DMSO
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item formulated in DMSO at μg/mL. Serial dilutions prepared from stock.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: Stock prepared at 1962 μg/mL
- Final preparation of a solid: Not applicable.
FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid formulations in vehicle, of the solid test item.
OTHER SPECIFICS: Not applicable - Target gene:
- Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Recognised supplier (documented in full study report).
- Suitability of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line has been used successfully in in vitro experiments for many years and is guideline specified (OECD TG 490). The test system has been extensively validated.
- Cell cycle length, doubling time or proliferation index: Doubling time ca. 12 hours. Determined under normal growth conditions.
- Sex, age and number of blood donors if applicable: Not applicable.
- Whether whole blood or separated lymphocytes were used if applicable: Not applicable.
- Number of passages if applicable: Not applicable.
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: The karyotype for the cell line has been published and the modal chromosome number is 40 (ref: OECD TG 490).
- Normal (negative control) cell cycle time: Doubling time ca. 12 hours.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with
Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Properly maintained: Yes.
- Periodically checked for Mycoplasma contamination: Yes. Master stocks of cells were tested and found to be free of
mycoplasma.
- Periodically checked for karyotype stability: Yes.
- Periodically 'cleansed' against high spontaneous background: Yes. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL),
Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix. S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0. 20% S9-mix (i.e. 2% final concentration of S9) was added to the subsequent testing and control cultures.
- Test concentrations with justification for top dose:
- The maximum dose level was 1962 µg/mL, calculated to be equivalent to 10 mM concentration, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1962 μg/mL range (full results recorded in the full study report). Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Specifically, in the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival.
I. Preliminary toxicity test: 0 (control), 7.66, 15.33, 30.66, 61.31, 122.63, 245.25, 490.5, 981 and 1962 μg/mL
Within three exposure groups:
i) 4-hours exposure to the test item without S9-mix
ii) 4-hours exposure to the test item with S9-mix (2%)
iii) 24-hour exposure to the test item without S9-mix
There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. The toxicity curve was very steep in all three of the exposure groups. Precipitate of the test item was observed at and above 981 μg/mL.
II. Main Test:
4-hour without S9: 0 (control), 7.81, 15.63, 31.25 62.5, 125, 250, 375, 500, 625, 750 μg/ml and EMS 400 μg/ml
4-hour with S9: 0 (control), 7.81, 15.63, 31.25 62.5, 125, 250, 375, 500, 625, 750 μg/ml and CP 1.5 μg/ml
24-hour without S9: 0 (control), 1.25, 2.5, 5, 10, 15, 20, 30, 40, 50, 60 μg/mL and EMS 150 μg/ml
where:
EMS = Ethylmethanesulphonate
CP = Cyclophosphamide
The cultures were then incubated for 24 hours, and sub-cultured subject to acceptable limits of mean cell count. Following a further 24 hours the cultures were counted and discarded. Giving total of 48 hours for expression period. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was soluble in DMSO at 196.2 mg/mL in solubility checks performed. The maximum dose level (determined prior to the test based on molecular weight) was 1962 µg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1962 μg/mL range (full results recorded in the full study report). The test item was formulated within two hours of it being applied to the test system.
Applicant assessment indicates: The test item had been demonstrated to be insoluble in Minimal Essential Medium (MEM) in previous mammalian cell line testing. DMSO is a guideline accepted vehicle with an available laboratory historic control data set. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- Full details of the positive control substances, is available in the full study report
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation with test item.
- Cell density at seeding (if applicable): Preliminary Test: 4-hour exposure: 5 x10^5 cells/mL ; 24-hour exposure: 1.5 x10^5 cells/mL ; Definitive Test: 4-hour exposure: 5 x10^5 cells/mL; 24-hour exposure: 1.5 x10^5 cells/mL
DURATION
- Preincubation period: Preliminary Test: 4-hour exposure: 45 minutes ; 24-hour exposure: 45 minutes ; Definitive Test: 4-hour exposure: 55 minutes ; 24-hour exposure: 55 minutes
- Exposure duration: Treatment was for 4 hours at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air, post exposure (total time 12 to 14 days from exposure start). To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells.
SELECTION AGENT (mutation assays): 5-Trifluorothymidine at 4 μg/mL
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): Not applicable.
NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air, post exposure. Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air, post exposure with two hours incubation.
NUMBER OF CELLS EVALUATED: 2000 cells/well were seeded with selection agent. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) analysis. Scoring of plates daily for %RSG and %V to obtain RTG. Mutation scoring of plates was ultimately performed for the presence of mutant colonies; large and small colonies analyses was additionally conducted.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Not applicable.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not applicable.
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG) and cloning efficiency via viability (%)
- Any supplementary information relevant to cytotoxicity: percentage relative suspension growth (%RSG) (post exposure toxicity during the expression period); viability (%) from non-selective medium cultures.
OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable.
- Determination of endoreplication: Not applicable.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable. - Rationale for test conditions:
- See tables. The rationale for selection of concentrations was based on preliminary toxicity testing and number of cultures was in accordance with the guideline (duplicate). Optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved.
- Evaluation criteria:
- An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered
unable to induce mutations in this test system.
Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance. - Statistics:
- Statistical guidelines recommended by the UKEMS (Robinson W. D. et al., Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp 102-140). The statistical package used indicates the presence of statistically significant increases and linear-trend events.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: Precipitate was observed at or above 981 μg/mL in the preliminary toxicity test.
- Conclusions:
- Interpretation of results:
Negative
Under the conditions of this study, the test substance was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro. - Executive summary:
The study was performed to the requirements of OECD TG 490 and EU Method B.17, US EPA OPPTS 870.5300 and Japan guidelines for screening mutagenicity testing of chemicals under GLP conditions to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary cytotoxicity test at up to 1260 μg/mL concentration, a definitive test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at ten dose levels in duplicate, together with vehicle (DMSO), and positive controls. Exposures were conducted using a 4-hour exposure with and without metabolic activation (2% S9), and a 24-hour exposure without metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item exhibited dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were: 4-hour with and without S9 (2%): 0, 31.25, 62.5, 125, 250, 375, 500, 625, 750 μg/mL and24- without S9 (2%): 0, 1.25, 2.5, 5, 10, 15, 20, 30, 40 μg/mL. Additional concentrations were excluded, as applicable. The maximum dose level used in the definite test was limited by test item-induced toxicity. No precipitate of the test item was observed at any of the dose levels used in the definitive test. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolism activation system. The test item did not induce any increases in mutant frequency above the Global Evaluation Factor (GEF). Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.
Referenceopen allclose all
Table 1 Test Results: Range-Finding Test – Without Metabolic Activation
Test Substance Concentration |
Mean number of colonies per plate (SD) |
||||
Base-pair substitution type |
Frame-shift type |
||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
0 |
75 (14.5) |
12 (1.5) |
27 (4.2) |
19 (7.0) |
12 (3.5) |
50 |
83 (4.0) |
13 (4.2) |
30 (1.0) |
15 (1.0) |
13 (1.0) |
150 |
75 (1.0) |
15 (1.5) |
24 (2.1) |
11 (1.7) |
13 (1.5) |
500 |
84 (3.2) |
15 (3.2) |
29 (0.6) |
15 (2.6) |
14 (0.6) |
1500 |
72 (4.6) |
15 (0.6) |
24 (2.1) |
12 (3.8) |
11 (2.1) |
5000 |
77 (0.6) |
16 (2.5) |
19 (6.8) |
15 (3.8) |
9 (2.1) |
Positive Controls |
|
|
|
|
|
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
Concentration |
3 |
5 |
2 |
0.2 |
80 |
Mean number of colonies per plate (SD) |
415 |
101 |
723 |
110 |
925 |
ENNG
N-ethyl-N’-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
Table 2 Test Results: Range-Finding Test – With Metabolic Activation
Test Substance Concentration |
Mean number of colonies per plate (SD) |
||||
Base-pair substitution type |
Frame-shift type |
||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
0 |
73 (10.2) |
12 (1.2) |
34 (2.3) |
25 (1.2) |
12 (5.2) |
50 |
70 (5.6) |
12 (3.1) |
32 (0.6) |
18 (3.1) |
10 (1.5) |
150 |
80 (5.9) |
11 (3.5) |
32 (2.5) |
18 (0.0) |
10 (0.0) |
500 |
87 (20.8) |
13 (1.5) |
26 (1.0) |
14 (0.6) |
6 (0.6) |
1500 |
77 (3.5) |
12 (1.2) |
24 (5.1) |
11 (3.1) |
15 (1.0) |
5000 |
78 (10.7) |
9 (3.5) |
23 (3.2) |
13 (2.3) |
9 (2.1) |
Positive Controls |
|
|
|
|
|
Name |
2AA |
2AA |
2AA |
BP |
2AA |
Concentration |
1 |
2 |
10 |
5 |
2 |
Mean number of colonies per plate (SD) |
720 |
336 |
269 |
295 |
256 |
BP
Benzo(a)pyrene
2AA 2-Aminoanthracene
Table 3 Test Results: Main Test – Without Metabolic Activation
Test Substance Concentration |
Mean number of colonies per plate (SD) |
||||
Base-pair substitution type |
Frame-shift type |
||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
0 |
75 (13.5) |
20 (8.5) |
24 (4.5) |
28 (2.5) |
14 (4.0) |
50 |
68 (7.0) |
22 (8.7) |
15 (2.0) |
22 (4.5) |
13 (4.6) |
150 |
72 (3.2) |
18 (3.2) |
18 (6.9) |
22 (1.5) |
12 (3.0) |
500 |
79 (11.6) |
19 (5.0) |
20 (2.1) |
16 (2.0) |
13 (2.1) |
1500 |
65 (1.0) |
16 (3.5) |
14 (1.7) |
23 (3.1) |
7 (1.2) |
5000 |
74 (16.1) |
20 (6.1) |
14 (3.0) |
25 (9.0) |
11 (4.5) |
Positive Controls |
|
|
|
|
|
Name |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
Concentration |
3 |
5 |
2 |
0.2 |
80 |
Mean number of colonies per plate (SD) |
379 |
162 |
721 |
113 |
1393 |
ENNG
N-ethyl-N’-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
Table 4 Test Results: Main Test – With Metabolic Activation
Test Substance Concentration |
Mean number of colonies per plate (SD) |
||||
Base-pair substitution type |
Frame-shift type |
||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
0 |
99 (10.2) |
14 (3.8) |
32 (5.6) |
13 (3.2) |
15 (3.1) |
50 |
77 (12.3) |
19 (4.5) |
33 (3.0) |
14 (3.1) |
16 (2.5) |
150 |
93 (10.3) |
17 (8.5) |
32 (5.1) |
13 (1.7) |
13 (2.9) |
500 |
76 (6.6) |
19 (2.1) |
30 (0.6) |
18 (7.1) |
12 (3.6) |
1500 |
88 (6.9) |
16 (4.4) |
25 (5.5) |
15 (5.0) |
15 (3.1) |
5000 |
73 (14.8) |
21 (1.7) |
22 (10.4) |
16 (2.9) |
9 (1.0) |
Positive Controls |
|
|
|
|
|
Name |
2AA |
2AA |
2AA |
BP |
2AA |
Concentration |
1 |
2 |
10 |
5 |
2 |
Mean number of colonies per plate (SD) |
707 |
389 |
198 |
248 |
247 |
BP
Benzo(a)pyrene
2AA 2-Aminoanthracene
Table 1: Results of preliminary cytotoxicity study
4(20)-hour without metabolic activation |
4(20)-hour with metabolic activation |
||
Dose Level (μg/ml) |
Mitotic Index (%) |
Dose Level (μg/ml) |
Mitotic Index (%) |
0 |
100 |
0 |
100 |
7.66 |
- |
7.66 |
- |
15.33 |
- |
15.33 |
- |
30.66 |
- |
30.66 |
- |
61.31 |
- |
61.31 |
- |
122.63 |
- |
122.63 |
- |
245.25 |
104 |
245.25 |
98 |
490.5 |
75 |
490.5 |
116 |
981 |
90 P |
981 |
59 |
1962 |
6 P |
1962 |
16 P |
P Precipitate observed
Table 2: Results of cytogenicity study, Experiments 1 and 2
Concentration |
Cells observed |
Percentage of cells showing chromosome aberrations % |
g |
Mitotic Index % |
Number and percentage of cells showing numerical aberrations (%) |
||||||||
ctb |
cte |
csb |
sce |
others |
Total |
Observed |
Polyploids |
Others |
Total (%) |
||||
Experiment 1: 4 hour treatment, without metabolic activation |
|||||||||||||
01 |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
200 |
0 |
0 |
0 |
750 |
200 |
1.5 |
0.5 |
0.5 |
0 |
0 |
2.5* |
0.4 |
93 |
200 |
0 |
0 |
0 |
1000 |
200 |
0 |
0 |
0.5 |
0 |
0 |
0.5 |
1.0 |
87 |
200 |
0 |
0 |
0 |
1250 |
100 |
0.5 |
0 |
0.5 |
0 |
0 |
1.0 |
0.5 |
66 |
201 |
1 |
0 |
1 (0.5) |
1500 |
200 |
3.5 |
1.0 |
0 |
0 |
0 |
4.5** |
1.0 |
35 |
200 |
0 |
0 |
0 |
Positive control |
1502 |
16.0 |
13.3 |
0.7 |
0 |
0 |
28.7*** |
2.0 |
66 |
150 |
0 |
0 |
0 |
Experiment 1: 4 hour treatment, with metabolic activation |
|||||||||||||
01 |
100 |
0.5 |
0 |
0 |
0 |
0 |
0.5 |
0 |
100 |
200 |
0 |
0 |
0 |
500 |
200 |
2.5 |
0 |
1.0 |
0 |
0 |
3.5* |
1.5 |
89 |
200 |
0 |
0 |
0 |
1000 |
200 |
1.0 |
0 |
1.0 |
0.5 |
0 |
2.0 |
2.5 |
62 |
201 |
1 |
0 |
1 (0.5) |
1250 |
200 |
3.5 |
1.0 |
0 |
0 |
0 |
4.5*** |
2 |
68 |
208 |
8 |
0 |
8** (3.8) |
1500 |
200 |
4.5 |
1.0 |
1.0 |
0 |
0 |
6.5*** |
3.0 |
44 |
204 |
4 |
0 |
4 (2.0) |
Positive control |
1002 |
27 |
10 |
7 |
1 |
0 |
41*** |
4.0 |
31 |
100 |
0 |
0 |
0 |
Experiment 2: 24 hour treatment, without metabolic activation |
|||||||||||||
01 |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
200 |
0 |
0 |
0 |
31.25 |
200 |
0 |
0 |
1 |
0 |
0 |
1 |
0 |
112 |
200 |
0 |
0 |
0 |
62.5 |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
76 |
200 |
0 |
0 |
0 |
250 |
200 |
3 |
0 |
0 |
0 |
0 |
3* |
1.0 |
54 |
200 |
0 |
0 |
0 |
Positive control |
200 |
5.0 |
4.5 |
0 |
0 |
0 |
9.0*** |
0 |
60 |
200 |
0 |
0 |
0 |
Experiment 2: 4 hour treatment, with metabolic activation |
|||||||||||||
01 |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
4.90/2.20 100% |
200 |
0 |
0 |
0 |
1000 |
200 |
0 |
0 |
0.5 |
0.5 |
0 |
1.0 |
0 |
89 |
203 |
3 |
0 |
1.5 |
1250 |
200 |
1 |
0.5 |
0.5 |
0 |
0 |
2.0 |
0 |
59 |
201 |
1 |
0 |
1 (0.5) |
1375 |
200 |
1.5 |
1 |
0 |
0 |
0 |
2.5* |
0.5 |
41 |
203 |
3 |
0 |
3 (1.5) |
Positive control |
200 |
13.0 |
4.5 |
0.5 |
0 |
0 |
16.5*** |
2.5 |
15 |
200 |
0 |
0 |
0 |
1Solvent control with dimethyl sulfoxide
2One or both positive control cultures: slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.
ctb : Chromatid breaks
cte : Chromatid exchanges
csb : Chromosome breaks
cse : Chromosome exchanges
g : Gaps
* p < 0.05
** p < 0.01
*** p < 0.001
Table 1: Results of preliminary cytotoxicity test
Concentration (μg/mL) |
% RSG (-S9) 4-Hour Exposure |
% RSG (+S9) 4-Hour Exposure |
% RSG (-S9) 24-Hour Exposure |
0 |
100 |
100 |
100 |
7.66 |
94 |
88 |
90 |
15.33 |
90 |
79 |
46 |
30.66 |
87 |
86 |
26 |
61.31 |
79 |
75 |
8 |
122.63 |
79 |
57 |
3 |
245.25 |
61 |
36 |
2 |
490.5 |
31 |
21 |
1 |
981.0 |
2 |
8 |
0 |
1962.0 |
1 |
1 |
0 |
|
|
|
|
Table 2: Definitive Test - summary of results: 4-hour exposure - with and without S9
4-hours –S9 |
4-hours +S9 |
||||||||
Concentration (μg/mL) |
Notes |
%RSG |
RTG |
MF§ |
Concentration (μg/mL) |
Notes |
%RSG |
RTG |
MF§ |
0 |
|
100 |
1.0 |
136.09 |
0 |
|
100 |
1.00 |
169.61 |
7.81 |
Ø |
85 |
|
|
7.81 |
Ø |
92 |
|
|
15.63 |
Ø |
77 |
|
|
15.63 |
Ø |
79 |
|
|
31.25 |
|
82 |
0.78 |
118.00 |
31.25 |
|
88 |
0.87 |
174.58 |
62.5 |
|
76 |
0.74 |
138.76 |
62.5 |
|
42 |
0.49 |
187.50 |
125 |
$$ |
62 |
0.79 |
147.18 |
125 |
|
35 |
0.31 |
182.95 |
250 |
|
53 |
0.49 |
201.95 |
250 |
|
34 |
0.28 |
237.95 |
375 |
|
33 |
0.31 |
177.08 |
375 |
X |
17 |
0.08 |
244.43 |
500 |
|
24 |
0.22 |
198.92 |
500 |
X |
18 |
0.08 |
292.85 |
625 |
|
16 |
0.10 |
204.29 |
625 |
X |
14 |
0.10 |
213.91 |
750 |
X |
8 |
0.05 |
197.45 |
750 |
X |
10 |
0.07 |
297.12 |
|
|
|
|
|
|
|
|
|
|
MF positive criteria |
|
262.09 |
MF positive criteria |
|
295.61 |
||||
|
|
|
|
|
|
|
|
|
|
PC |
|
|
|
|
PC |
|
|
|
|
EMS 400 |
|
60 |
0.38 |
873.64 |
CP 1.5 |
|
69 |
0.51 |
972.07 |
|
|
|
|
|
|
|
|
|
|
$ = Cell counts (x1^05 cells/mL). Set up on previous day to 2 x 10^5 cells/mL unless otherwise stated in parenthesis.
%RSG = Relative Suspension Growth
RTG = Relative Total Growth
CP = Cyclophosphamide
EMS = Ethylmethanesulphonate
MF§ = 5-TFT resistant mutants/10^6 viable cells 2 days after exposure
$$ = Evidence of heterogeneity (poor correlation between A and B cultures)
Ø = Not plated for viability or 5-TFT resistance
X = Excluded due to excessive toxicity
Table 3: Definitive Test - summary of results: 24-hour exposure – without S9
24-hours –S9 |
||||
Concentration (μg/mL) |
Notes |
%RSG |
RTG |
MF§ |
0 |
|
100 |
1.00 |
164.62 |
1.25 |
|
80 |
0.65 |
173.04 |
2.5 |
|
66 |
0.78 |
130.57 |
5 |
|
71 |
0.74 |
191.23 |
10 |
|
56 |
0.75 |
122.97 |
15 |
|
39 |
0.46 |
165.93 |
20 |
|
33 |
0.43 |
166.20 |
30 |
|
23 |
0.29 |
184.95 |
40 |
|
16 |
0.20 |
226.58 |
50 |
Ø |
9 |
|
|
60 |
Ø |
7 |
|
|
|
|
|
|
|
MF positive criteria |
|
262.09 |
||
|
|
|
|
|
PC |
|
|
|
|
EMS 400 |
|
40 |
0.37 |
860.09 |
|
|
|
|
|
MF§ = 5-TFT resistant mutants/10^6 viable cells 2 days after exposure
Ø = Not plated for viability or 5-TFT resistance
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
OECD TG 471, 2012 - The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using both the Ames plate incorporation and pre incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. Five test item dose levels were again selected in Experiment 2 and was 50 to 5000 µg/plate. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial lawn in all strains up to 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9‑mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.
OECD TG 473, 2012 - The study was performed to the requirements of OECD TG 473 and EU Method B.10 under GLP conditions to assess the potential chromosomal mutagenicity of the test substance, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The exposure conditions were divided between experiment 1 which was 4(20)-hour with and without S9 activation, and experiment 2 which was 4(20)-hour with S9 activation and 24-hour exposure without S9 activation. The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test (Cell Growth Inhibition Test) where the results indicated that the maximum concentration should be limited to the lowest precipitating dose level with toxicity also being taken into account in the dose selection. The dose levels selected for the Main Test were as follows, experiment 1: 4(20)-hour with and without S9-Mix (2%): 0, 250, 500, 750, 1000, 1250, 1500 μg/mL. For experiment 2: 4(20)-hour with S9 mix (2%): 0, 500, 750, 1000, 1125, 1250, 1375, μg/mL. In the 24-hour continuous exposure without S9 mix activation the doses levels were: 0, 15.63, 31.25, 62.5, 125, 250, 375, 500 μg/mL. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. In Experiment 1, small but statistically significant increases in the frequency of cells with aberrations were observed, predominately in the presence of metabolic activation. The maximum response observed in the presence of metabolic activation was associated with a dose level that induced approximately 50% mitotic inhibition. In the absence of metabolic activation the response was observed at a dose level that exceeded the optimum target level of 50% toxicity. In both cases the response was not clearly dose-related. In Experiment 2, the response observed in the presence of S9 was much weaker and was associated with a dose level that slightly exceeded the approximate 50% mitotic inhibition. It should also be noted that it only marginally exceeded the in-house historical vehicle control upper limit of 2.0% for with-S9 exposures but was the same as the overall historical maximum for all exposure conditions (2.5%). A small but statistically significant response was observed in the 24-hour continuous exposure at 250 μg/ml, however the aberrations observed were all in the ‘A’ culture and were all break type aberrations. The response was therefore, considered to be of little biological relevance. It was considered that the small but statistically significant increase in the frequency of aberrant cells observed in experiment 1 in the presence of S9 was not toxicologically significant. The response was not clearly dose-related and not reproduced in a repeat experiment using the same experimental conditions, and dose levels that induced similar levels of toxicity. It was therefore concluded that the clastogenic activity was occurring at or around the onset of excessive toxicity giving some doubt to its biological relevance and, therefore, toxicological significance. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
OECD TG 490, 2016: The study was performed to the requirements of OECD TG 490 and EU Method B.17, US EPA OPPTS 870.5300 and Japan guidelines for screening mutagenicity testing of chemicals under GLP conditions to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary cytotoxicity test at up to 1260 μg/mL concentration, a definitive test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at ten dose levels in duplicate, together with vehicle (DMSO), and positive controls. Exposures were conducted using a 4-hour exposure with and without metabolic activation (2% S9), and a 24-hour exposure without metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item exhibited dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were: 4-hour with and without S9 (2%): 0, 31.25, 62.5, 125, 250, 375, 500, 625, 750 μg/mL and24- without S9 (2%): 0, 1.25, 2.5, 5, 10, 15, 20, 30, 40 μg/mL. Additional concentrations were excluded, as applicable. The maximum dose level used in the definite test was limited by test item-induced toxicity. No precipitate of the test item was observed at any of the dose levels used in the definitive test. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolism activation system. The test item did not induce any increases in mutant frequency above the Global Evaluation Factor (GEF). Under the conditions of this study, the test item was considered to be non-mutagenic at the TK +/- locus to mouse L5178Y lymphoma cells in vitro.
Justification for classification or non-classification
The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity
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