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EC number: 943-495-7 | CAS number: 689-65-6
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, 2017
negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2017
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:
- 19-11-2015 to 08-12-2015
- 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 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: June 2015; signature: September 2015
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9
- Test concentrations with justification for top dose:
- Experiment 1 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Part of the first mutation test was repeated due to excessive toxicity (TA1535 and TA1537 dosed in the absence of S9-mix) employing an amended test item dose range of 0.05 to 150 µg/plate.
Experiment 2 (pre-incubation method): Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item following the change in test methodology. The dose levels were selected based on the results of Experiment 1.
Salmonella strains TA1535 and TA1537 (absence of S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate.
Salmonella strains TA100 and TA98 and E.coli strain WP2uvrA (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Salmonella strains TA1535 and TA1537 and E.coli strain WP2uvrA (presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strain TA100 (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strain TA98 (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate. - 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
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Experiment 1. in medium; in agar (pre-incubation) ; Experiment 2. in medium; in agar (pre-incubation).
The choice of application was due to the test item to either have unknown volatility or was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls.
DURATION
- Exposure duration:
Experiment 1. All of the plates were pre-incubated in sealed, small volume containers, by application of 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 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media. All of the plates were sealed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure (37 ± 3 ºC for approximately 48 hours) to minimize potential losses of the test item from the plates. After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were performed, predominantly due to colonies spreading, colonies on the edge of the plates and artefacts on the plates, thus distorting the actual plate count.
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 ± 3 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.
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 (Cariello and Piegorsch, 1996)).
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:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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:
- cytotoxicity
- Remarks:
- See table 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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 material 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 the Ames pre incubation method at up to eight 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 and was 1.5 to 5000 µg/plate. Part of the first mutation test was repeated due to excessive toxicity (TA1535 and TA1537 dosed in the absence of S9-mix) employing an amended test item dose range of 0.05 to 150 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels were again selected in Experiment 2 in order to achieve a minimum of four non-toxic dose levels and the toxic limit of the test item. The dose range was amended following the results of Experiment 1 and ranged between 0.015 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or the toxic limit of the test item depending on the strain type and presence of S9-mix.In the presence S9-mix weakened bacterial background lawns were noted from 150 µg/plate (TA1535), 500 µg/plate (TA1537 and WP2uvrA), 1500 µg/plate (TA100) and at 5000 µg/plate (TA98). Consequently for the second mutation test the same maximum dose level or toxic limit was used, depending on bacterial strain type and presence or absence of S9-mix.Results from the second mutation test confirmed the toxicity previously noted with weakened bacterial background lawns noted in the absence of S9-mix from 15 µg/plate (TA1535 and TA1537), 50 µg/plate (WP2uvrA) and 150 µg/plate (TA100 and TA98). In the presence of S9-mix weakened bacterial background lawns were noted from 150 µg/plate (TA1535 and TA1537), 500 µg/plate (WP2uvrA), 1500 µg/plate (TA100) and at 5000 µg/plate (TA98). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9‑mix. 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. 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.A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9-mix at 1.5 µg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 1.5 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control.It was concluded that, under the conditions of this assay, the test substance 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:
- 21-12-2015 to 11-02-2015
- 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 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according 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
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: July 2016; signature: October 2016
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable (chromosome aberration test)
- Species / strain / cell type:
- lymphocytes: Human lymphocytes
- Details on mammalian cell type (if applicable):
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours. Further details on the donors is available in the full study report.Lot No.
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Microsomal fraction: PB/βNF S9 23/08/15
- Test concentrations with justification for top dose:
- The maximum dose level was 1823 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 1823 μg/mL range (full results recorded in the full study report).
I. Preliminary toxicity test: 0 (control) , 7.12, 14.24, 28.48, 56.97, 113.94, 227.88, 455.75, 911.5 and 1823 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:
4(20)-hour without S9: 0*, 0.88, 1.75, 3.5*, 7.0*, 10.5*, 14.0*, 17.5, 21.0 μg/mL and MMC 0.1*
4(20)-hour with S9: 0*, 1.75, 3.5, 7.0, 14.0*, 17.5*, 28.0*, 42.0*, 56.0 μg/mL and CP 2*
24-hour without S9: 0*, 1.75, 3.5, 7.0*, 14.0*, 17.5*, 28.0*, 42.0, 56.0 μg/mL and MMC 0.1*
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 immiscible in aqueous media at 18.23 mg/mL but was miscible in dimethyl sulphoxide at 182.3 mg/mL in solubility checks performed. The maximum dose level (determined prior to the test based on molecular weight) was 1823 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 1823 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: Vehicle control served as the negative control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- Full details on the positive controls is reported in the full study report.
- 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 non-vented 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 0.05 mL (50 μL) 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. 1mL 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 Experiment. 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 0.05 mL (50 μL) 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. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. 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.
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 (%) 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, 300 consecutive well-spread metaphases from each concentration (150 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), ISCN (1985). Where the analysis of the slide resulted in a large frequency of aberrant cells then the analysis was terminated after a total of 15 metaphases with aberrations (excluding gaps) 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 exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.
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.
3) There is no concentration-related increase at any dose level.
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. A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis. - 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 lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 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 the end of the exposure at and above 113.94 µg/mL in all of the exposure groups.
Main test: The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Cell Growth Inhibition Test and that there were metaphases suitable for scoring present up to the maximum dose level of test item in the 4(20)-hour exposure groups and up to 42 µg/mL in the 24-hour continuous exposure group. No precipitate was observed at the end of exposure in blood-free cultures in any of the exposure groups.
- Other confounding effects: In the preliminary test: Hemolysis was observed following exposure to the test item at and above 28.48 µg/mL in the 4(20)-hour exposure groups and at and above 56.97 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. In the main test: Hemolysis was observed at and above 28 µg/mL in the presence of S9 only.
RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 1823 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 ‘other confounding effects’ listed above. - 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 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 Japan METI guidelines 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 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 on toxicity for all exposure groups for dose selection. The dose levels selected for the Main Test were as follows: 4(20)-hour without S9-Mix (2%) : 0, 0.88, 1.75, 3.5, 7, 10.5, 14, 17.5, 21 μg/mL and 4(20)-hour with S9-Mix (2%) and 24-hour continuous exposure without S9: 0, 1.75, 3.5, 7, 14, 17.5, 28, 42, 56 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. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced approximately optimum toxicity as defined by the guideline. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
Referenceopen allclose all
Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535† |
WP2uvrA |
TA98 |
TA1537† |
|||||||
Solvent Control (DMSO) |
98 100 103 |
(100) 2.5# |
17 22 16 |
(18) 3.2 |
21 19 C |
(20) 1.4 |
20 21 30 |
(24) 5.5 |
10 8 14 |
(11) 3.1 |
|
0.05 µg |
N/T |
20 12 15 |
(16) 4.0 |
N/T |
N/T |
14 16 5 |
(12) 5.9 |
||||
0.15 µg |
N/T |
12 11 14 |
(12) 1.5 |
N/T |
N/T |
8 18 13 |
(13) 5.0 |
||||
0.5 µg |
N/T |
9 14 9 |
(11) 2.9 |
N/T |
N/T |
7 4 21 |
(11) 9.1 |
||||
1.5 µg |
98 72 81 |
(84) 13.2 |
11 26 18 |
(18) 7.5 |
20 22 14 |
(19) 4.2 |
29 25 23 |
(26) 3.1 |
16 17 10 |
(14) 3.8 |
|
5 µg |
68 64 100 |
(77) 19.7 |
16 8 11 |
(12) 4.0 |
25 8 20 |
(18) 8.7 |
30 30 31 |
(30) 0.6 |
13 9 23 |
(15) 7.2 |
|
15 µg |
86 81 69 |
(79) 8.7 |
6 S 8 S 6 S |
(7) 1.2 |
26 17 20 |
(21) 4.6 |
16 16 20 |
(17) 2.3 |
17 S 6 S 3 S |
(9) 7.4 |
|
50 µg |
82 60 55 |
(66) 14.4 |
7 S 3 S 9 S |
(6) 3.1 |
21 13 14 |
(16) 4.4 |
25 22 16 |
(21) 4.6 |
4 S 3 S 5 S |
(4) 1.0 |
|
150 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
21 S 13 S 8 S |
(14) 6.6 |
14 S 10 S 16 S |
(13) 3.1 |
0 V 0 V 0 V |
(0) 0.0 |
|
500 µg |
0 V 0 V 0 V |
(0) 0.0 |
N/T |
9 S 7 S 7 S |
(8) 1.2 |
23 S 16 S 17 S |
(19) 3.8 |
N/T |
|||
1500 µg |
0 V 0 V 0 V |
(0) 0.0 |
N/T |
0 V 0 V 0 V |
(0) 0.0 |
13 S 18 S 17 S |
(16) 2.6 |
N/T |
|||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
N/T |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
N/T |
|||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
|||||||
300 302 289 |
(297) 7.0 |
221 195 139 |
(185) 41.9 |
525 955 858 |
(779) 225.5 |
330 341 315 |
(329) 13.1 |
933 367 346 |
(549) 333.0 |
||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
115 115 104 |
(111) 6.4# |
8 15 9 |
(11) 3.8 |
19 22 27 |
(23) 4.0 |
35 17 36 |
(29) 10.7 |
12 13 17 |
(14) 2.6 |
|
1.5 µg |
133 129 125 |
* (129) 4.0 |
20 7 15 |
(14) 6.6 |
36 14 35 |
(28) 12.4 |
40 29 33 |
(34) 5.6 |
22 10 14 |
(15) 6.1 |
|
5 µg |
100 111 99 |
(103) 6.7 |
12 18 18 |
(16) 3.5 |
20 21 26 |
(22) 3.2 |
29 22 30 |
(27) 4.4 |
12 17 20 |
(16) 4.0 |
|
15 µg |
117 116 115 |
(116) 1.0 |
14 9 18 |
(14) 4.5 |
40 39 23 |
(34) 9.5 |
31 29 27 |
(29) 2.0 |
22 16 21 |
(20) 3.2 |
|
50 µg |
117 112 112 |
(114) 2.9 |
5 9 8 |
(7) 2.1 |
34 38 35 |
(36) 2.1 |
32 29 31 |
(31) 1.5 |
10 14 8 |
(11) 3.1 |
|
150 µg |
107 100 96 |
(101) 5.6 |
10 S 12 S 12 S |
(11) 1.2 |
18 13 17 |
(16) 2.6 |
25 29 25 |
(26) 2.3 |
5 4 10 |
(6) 3.2 |
|
500 µg |
72 68 81 |
(74) 6.7 |
0 V 0 V 0 V |
(0) 0.0 |
8 S 16 S 23 S |
(16) 7.5 |
27 21 26 |
(25) 3.2 |
5 S 4 S 7 S |
(5) 1.5 |
|
1500 µg |
63 S 76 S 82 S |
(74) 9.7 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
17 21 29 |
(22) 6.1 |
0 V 0 V 0 V |
(0) 0.0 |
|
5000 µg |
56 S 45 S 40 S |
(47) 8.2 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
26 S 18 S 30 S |
(25) 6.1 |
0 V 0 V 0 V |
(0) 0.0 |
|
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
|||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
|||||||
1043 1075 1060 |
(1059) 16.0 |
290 276 258 |
(275) 16.0 |
171 242 175 |
(196) 39.9 |
141 148 158 |
(149) 8.5 |
225 275 220 |
(240) 30.4 |
||
†: Experimental procedure repeated at a later date (reported in full study report); due to toxicity in the original test
C : Contaminated
#: Standard deviation
* : p≤0.05
Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
65 77 64 |
(69) 7.2# |
16 19 17 |
(17) 1.5 |
15 23 12 |
(17) 5.7 |
23 24 32 |
(26) 4.9 |
11 3 7 |
(7) 4.0 |
|
0.015 µg |
N/T |
|
12 12 13 |
(12) 0.6 |
N/T |
|
N/T |
|
11 7 16 |
(11) 4.5 |
|
0.05 µg |
62 56 55 |
(58) 3.8 |
17 17 12 |
(15) 2.9 |
23 17 19 |
(20) 3.1 |
20 32 18 |
(23) 7.6 |
15 7 5 |
(9) 5.3 |
|
0.15 µg |
71 83 57 |
(70) 13.0 |
14 7 13 |
(11) 3.8 |
6 24 15 |
(15) 9.0 |
27 27 25 |
(26) 1.2 |
7 14 14 |
(12) 4.0 |
|
0.5 µg |
60 73 68 |
(67) 6.6 |
16 15 12 |
(14) 2.1 |
14 13 14 |
(14) 0.6 |
13 29 32 |
(25) 10.2 |
1 6 6 |
(4) 2.9 |
|
1.5 µg |
75 76 74 |
(75) 1.0 |
14 16 12 |
(14) 2.0 |
16 16 21 |
(18) 2.9 |
27 26 11 |
(21) 9.0 |
10 5 6 |
(7) 2.6 |
|
5 µg |
58 56 50 |
(55) 4.2 |
25 16 17 |
(19) 4.9 |
19 8 12 |
(13) 5.6 |
16 14 15 |
(15) 1.0 |
7 5 8 |
(7) 1.5 |
|
15 µg |
51 55 43 |
(50) 6.1 |
14 S 16 S 16 S |
(15) 1.2 |
7 11 16 |
(11) 4.5 |
27 24 17 |
(23) 5.1 |
5 S 2 S 4 S |
(4) 1.5 |
|
50 µg |
51 59 56 |
(55) 4.0 |
7 S 7 S 9 S |
(8) 1.2 |
14 S 6 S 6 S |
(9) 4.6 |
22 20 14 |
(19) 4.2 |
0 V 0 V 0 V |
(0) 0.0 |
|
150 µg |
44 S 52 S 57 S |
(51) 6.6 |
N/T |
|
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
N/T |
|
|
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
|||||||
1322 1173 1346 |
(1280) 93.7 |
1082 1153 1183 |
(1139) 51.9 |
322 347 264 |
(311) 42.6 |
227 253 256 |
(245) 15.9 |
211 319 624 |
(385) 214.2 |
||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||
Solvent Control (DMSO) |
69 70 65 |
(68) 2.6# |
8 7 8 |
(8) 0.6 |
15 21 17 |
(18) 3.1 |
30 22 17 |
(23) 6.6 |
13 13 8 |
(11) 2.9 |
|
0.15 µg |
N/T |
|
9 8 7 |
(8) 1.0 |
27 16 16 |
(20) 6.4 |
N/T |
|
10 3 9 |
(7) 3.8 |
|
0.5 µg |
64 66 69 |
(66) 2.5 |
10 8 8 |
(9) 1.2 |
24 18 14 |
(19) 5.0 |
N/T |
|
12 4 9 |
(8) 4.0 |
|
1.5 µg |
65 69 73 |
(69) 4.0 |
5 9 4 |
(6) 2.6 |
15 8 23 |
(15) 7.5 |
21 18 29 |
(23) 5.7 |
7 11 16 |
(11) 4.5 |
|
5 µg |
75 59 60 |
(65) 9.0 |
8 11 12 |
(10) 2.1 |
18 16 20 |
(18) 2.0 |
16 20 28 |
(21) 6.1 |
16 6 6 |
(9) 5.8 |
|
15 µg |
45 61 46 |
(51) 9.0 |
6 9 5 |
(7) 2.1 |
17 14 25 |
(19) 5.7 |
17 10 16 |
(14) 3.8 |
7 4 14 |
(8) 5.1 |
|
50 µg |
64 65 59 |
(63) 3.2 |
14 9 7 |
(10) 3.6 |
15 23 24 |
(21) 4.9 |
18 13 11 |
(14) 3.6 |
4 5 9 |
(6) 2.6 |
|
150 µg |
75 69 58 |
(67) 8.6 |
0 V 0 V 0 V |
(0) 0.0 |
20 18 20 |
(19) 1.2 |
15 12 18 |
(15) 3.0 |
10 S 5 S 7 S |
(7) 2.5 |
|
500 µg |
71 63 54 |
(63) 8.5 |
0 V 0 V 0 V |
(0) 0.0 |
8 S 16 S 9 S |
(11) 4.4 |
12 22 22 |
(19) 5.8 |
0 V 0 V 0 V |
(0) 0.0 |
|
1500 µg |
44 S 52 S 55 S |
(50) 5.7 |
N/T |
|
N/T |
|
19 21 18 |
(19) 1.5 |
N/T |
|
|
5000 µg |
N/T |
|
N/T |
|
N/T |
|
14 S 5 S 7 S |
(9) 4.7 |
N/T |
|
|
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
|||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
|||||||
395 390 387 |
(391) 4.0 |
130 129 143 |
(134) 7.8 |
110 105 103 |
(106) 3.6 |
122 131 107 |
(120) 12.1 |
222 198 211 |
(210) 12.0 |
||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
N/T: Not tested at this dose level
S: Sparse bacterial background lawn
T: Toxic, no bacterial background lawn
V: Very weak bacterial background lawn
#: Standard deviation
All positive and vehicle and negative controls were within laboratory historic values.
Table 3. Spontaneous Mutation Rates (Concurrent Negative Controls): Experiment 1 and 2, respectively
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
82 |
|
8 |
(16) |
25 |
(25) |
30 |
(32) |
12 |
(11) |
108 |
(97) |
18 |
23 |
30 |
8 |
||||
100 |
|
22 |
|
27 |
|
35 |
|
13 |
|
|
|
|
|
|
|
|
|
|
|
|
|
29 |
(25)† |
|
|
|
|
13 |
(12)† |
|
|
20 |
|
|
|
|
|
10 |
|
|
|
26 |
|
|
|
|
|
12 |
|
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
88 |
(89) |
16 |
(14) |
23 |
(22) |
20 |
(19) |
11 |
(7) |
79 |
11 |
27 |
16 |
3 |
|||||
101 |
|
14 |
|
15 |
|
21 |
|
6 |
|
†: Experimental procedure repeated at a later date (without S9-mix) due to toxicity in the original test
1. Chromosome Aberration Test – Main Test
The dose levels of the controls and the test item are given below:
4(20)-hour without S9: 0*, 0.88, 1.75, 3.5*, 7.0*, 10.5*, 14.0*, 17.5, 21.0 μg/mL and MMC 0.1*
4(20)-hour with S9: 0*, 1.75, 3.5, 7.0, 14.0*, 17.5*, 28.0*, 42.0*, 56.0 μg/mL and CP 2*
24-hour without S9: 0*, 1.75, 3.5, 7.0*, 14.0*, 17.5*, 28.0*, 42.0, 56.0 μg/mL and MMC 0.1*
where: * = dose levels selected for metaphase analysis ; MMC= Mitomycin C and CP = Cyclophosphamide
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Cell Growth Inhibition Test. There were metaphases suitable for scoring present up to 42 µg/mL in the 4(20)-hour exposure groups in the presence and absence of S9 and up to 42 µg/mL in the 24-hour exposure group. The qualitative observations indicated a dose-related inhibition of mitotic index was observed in all exposure groups. In the 4(20)-hour exposure group in the absence of S9, 11% and 55% mitotic inhibition was achieved at 10.5 and 14 μg/mL, respectively. In the presence of S9, 39% and 72% inhibition of mitotic index was observed at 42 and 56 μg/mL, respectively whereas an inhibition of mitotic index of 90% was noted at 42 μg/mL in the 24-hour continuous exposure group. Additionally, the mitotic inhibition in this exposure group varied where 17%, 52% and 17% mitotic inhibition was observed at 14, 17.5 and 28 μg/mL, respectively, meaning optimum toxicity was observed at 17.5 μg/mL. Consequently, the maximum dose level selected for metaphase analysis was 14 μg/mL, 42 μg/mL and 28 μg/mL in the 4(20)-hour exposure group in the absence of S9, 4(20)-hour
exposure group in the presence of S9 and 24-hour continuous exposure group, respectively. No precipitate was observed at the end of exposure in blood-free cultures in any of the exposure groups. Hemolysis was observed at and above 28 μg/mL in the presence of S9 only.
- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.
- 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.
- The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
- The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups. There were two indications of endoreduplication noted.
2. Discussion:
- In the 4(20)-hour exposure group in the absence of S9, 11% and 55% mitotic inhibition was achieved at 10.5 and 14 μg/mL, respectively. Therefore, the maximum dose level assessed for mitotic index was optimum toxicity as defined by the OECD TG 473 guideline (55±5%).
- In the 4(20)-hour exposure group in the presence of S9, 39% and 72% inhibition of mitotic index was observed at 42 and 56 μg/mL, respectively. Consequently, 56 μg/mL was not assessed for metaphase analysis due to being excessive toxic as define by the test guideline. Therefore, the maximum concentration assessed for metaphase analysis had a mitotic inhibition of 39% which is approaching optimum toxicity. Additionally, hemolysis was observed at and above 28 μg/mL confirming that cell death was occurring in the cultures. Increases in mitotic index was observed at 14 and 17.5 μg/mL which matches the observations and conclusions made in the preliminary toxicity test.
- In the 24-hour exposure group an inhibition of mitotic index of 90% was noted at 42 μg/mL. Additionally, the mitotic inhibition in this exposure group varied where 17%, 52% and 17% mitotic inhibition was observed at 14, 17.5 and 28 μg/mL, respectively, meaning optimum toxicity was observed at 17.5 μg/mL.
- Optimum toxicity was achieved in the cultures in the absence of S9. In the presence of S9 the steep cytotoxic response coupled with increases in mitotic index and cell death made achieving optimal toxicity difficult. This was evident from the preliminary toxicity test where there were substantial increases in mitotic index prior to observed excessive toxicity in the 4(20)-hour exposure group and presence of S9. This was considered indicative of cell cycle delay and consequentially, due to repair processes more cells entering metaphase at just below excessive cytotoxicity. Under the conditions of the study, the test item was considered to have been adequately tested as the test item was tested up to cytotoxic conditions and scored at dose levels close to and below, excessive cytotoxicity. No significant increases in chromosome aberrations was observed at any dose level or exposure group tested.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Key study : OECD TG 471, 2017 : 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 the Ames pre incubation method at up to eight 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 and was 1.5 to 5000 µg/plate. Part of the first mutation test was repeated due to excessive toxicity (TA1535 and TA1537 dosed in the absence of S9-mix) employing an amended test item dose range of 0.05 to 150 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels were again selected in Experiment 2 in order to achieve a minimum of four non-toxic dose levels and the toxic limit of the test item. The dose range was amended following the results of Experiment 1 and ranged between 0.015 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or the toxic limit of the test item depending on the strain type and presence of S9-mix.In the presence S9-mix weakened bacterial background lawns were noted from 150 µg/plate (TA1535), 500 µg/plate (TA1537 and WP2uvrA), 1500 µg/plate (TA100) and at 5000 µg/plate (TA98). Consequently for the second mutation test the same maximum dose level or toxic limit was used, depending on bacterial strain type and presence or absence of S9-mix.Results from the second mutation test confirmed the toxicity previously noted with weakened bacterial background lawns noted in the absence of S9-mix from 15 µg/plate (TA1535 and TA1537), 50 µg/plate (WP2uvrA) and 150 µg/plate (TA100 and TA98). In the presence of S9-mix weakened bacterial background lawns were noted from 150 µg/plate (TA1535 and TA1537), 500 µg/plate (WP2uvrA), 1500 µg/plate (TA100) and at 5000 µg/plate (TA98). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9‑mix. 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. 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.A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9-mix at 1.5 µg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 1.5 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control. It was concluded that, under the conditions of this assay, the test substance 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.
Key study : OECD TG 473, 2017 : The study was performed to the requirements of OECD TG 473 and Japan METI guidelines 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 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 on toxicity for all exposure groups for dose selection. The dose levels selected for the Main Test were as follows: 4(20)-hour without S9-Mix (2%) : 0, 0.88, 1.75, 3.5, 7, 10.5, 14, 17.5, 21 μg/mL and 4(20)-hour with S9-Mix (2%) and 24-hour continuous exposure without S9: 0, 1.75, 3.5, 7, 14, 17.5, 28, 42, 56 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. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced approximately optimum toxicity as defined by the guideline. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes 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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