Registration Dossier
Registration Dossier
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EC number: 942-422-6 | CAS number: -
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, 2015
negative, in vitro HPRT locus gene mutation (with and without S-9 activation), OECD TG 476, 2018
non-clastogenic and non-aneugenic, in vitro mammalian cell micronucleus (with and without S-9 activation), OECD TG 487, 2018
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 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: March 2014; signature: May 2014
- 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 - Range Finding Test (plate incorporation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method):
Salmonella strains with and without S9-mix - TA100, TA1535, TA1537: 0, 1.5, 5, 15, 50, 150, 500 µg/plate
Salmonella strains with S9 mix - TA98: 0, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
Salmonella strain without S9-mix - TA98: 0, 1.5, 5, 15, 50, 150, 500 µg/plate
E.coli strain with and without S9-mix - WP2uvrA: 0, 15, 50, 150, 500, 1500 µg/plate
One bacterial strain (Ecoli - WP2uvrA dosed in the absence of S9-mix) exhibited excessive toxicity and required a repeat experiment using the following dose range: 0, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Up to seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item following the change in test methodology. - 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 therefore selected as the vehicle.
- Other: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No correction was made for purity as the test item is a UVCB. All formulations were used within four hours of preparation and were assumed to be stable for this period. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10-4 microns. - 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 (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)
DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 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 ± 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. Several manual counts were required, predominantly due to interference in the base agar e.g. minor precipitation of salts/dust particles and marks on the base of the plates slightly distorting the counts.
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
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- 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 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. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.5 and 5000 µg/plate depending on bacterial tester strain and absence or presence of S9-mix. Up to seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item following the change in test methodology. 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 sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (light and globular in appearance) was noted under an inverted microscope at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies. 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 gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: July 2017; signature: November 2017
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the Chinese hamster V79 cell line
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type.
- Cell cycle length, doubling time or proliferation index: Doubling time 12 - 16 h in stock cultures.
- 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: Laboratory stock cell cultures will be periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen. Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.
- Modal number of chromosomes: The cells have a stable karyotype with a modal chromosome number of 22 (Reference: Howard-Flanders, 1981).
- Normal (negative control) cell cycle time: The average absolute cloning efficiency of the Day 7 negative controls should exceed 50%.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For use, a sample of cells was removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10 % fetal bovine serum (FBS)) at approximately 37 °C with 5 % CO2 in humidified air.
- Properly maintained: Yes.
- Periodically checked for Mycoplasma contamination: Yes.
- Periodically checked for karyotype stability: Yes.
- Periodically 'cleansed' against high spontaneous background: Yes. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix: Prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G-6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration.
- Test concentrations with justification for top dose:
- The maximum recommended dose level was 5000 µg/mL, on the basis of the UVCB designation of the substance. 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 5000 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. Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended concentration (10 mM, 2 mg/mL or 2 µL/mL whichever is lower, or 5 mg/mL for a UVCB*), or include the lowest precipitating concentration. Where a test item is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic concentration should be used.
I. Preliminary Test:
Due to the high levels of precipitate observed in the solubility test, the concentration range of test item used was 2.44 to 625 µg/mL.
0 (control), 2.44, 4.88, 9.77, 19.53, 39.06, 78.13, 156.24, 312.5 and 625 μg/mL (limited by test item precipitation).
II. Main Test:
Main Test (with S9-mix): 0 (control)*, 10*, 20*, 40*, 80*, 100*, 120*, 140, 160, DMBA:1.0* and 2.0* µg/mL
Main Test (without S9-mix): 0 (control)*, 5*, 10*, 20*, 40*, 50*, 60, 70, 80, EMS: 500* and 750* µg/mL
* concentration levels plated for cloning efficiency and mutant frequency
Plated concentrations were based on cytotoxicity, as applicable. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Following solubility checks in a preceding Micronucleus, in vitro assay (details provided in the full study report): the test item was fully miscible in DMSO at 500 mg/mL. The maximum recommended dose level was 5000 µg/mL, on the basis of the UVCB designation of the substance. 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 5000 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: DMSO is a guideline accepted vehicle with an available laboratory historic control data set. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Pre-incubation with test item
- Cell density at seeding (if applicable): For mutation expression = 2,000,000 cells per 225 cm2 flask; for cloning efficiency = 200 cells per 25 cm2 flask.
DURATION
- Preincubation period: Cells were seeded at 1 x 10^7 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items.
- Exposure duration: Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.
SELECTION AGENT (mutation assays): Not applicable.
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): Not applicable. See ‘Methods of Slide Preparation and Staining Technique Used’ for further information on staining technique.
NUMBER OF REPLICATIONS: 1 for growth and mutation expression and 3 for cloning efficiency.
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes
NUMBER OF CELLS EVALUATED: 2 x 10^5 cells/petri dish (ten replicates per group) to determine mutant frequency; triplicate at 200 cells/25 cm2 flask to determine cloning efficiency
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: Cloning Efficiency (CE)
- Any supplementary information relevant to cytotoxicity: Cloning Efficiency (CE), % control, mutant plate counts, mutant frequency/10^6 (MF10^-6) and mutant frequency/10^6 survival rate (MFSV) were calculated.
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.
- OTHER: Not applicable. - Evaluation criteria:
- Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- When there is an indication of any increases in mutant frequency at any concentration, comparisons are made between the appropriate vehicle control value and each individual concentration, using Student’s t-test.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 for further information
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects determined.
- Effects of osmolality: No effect determined.
- Evaporation from medium: Tissue culture flasks were sealed due to the potential volatility of the test item
- Water solubility: Formulated in DMSO
- Precipitation: Yes, above cytotoxic dose levels. Not plated when observed (see Table 1 for more information)
- Definition of acceptable cells for analysis: Reported and within specification. Fresh cultures used for each test.
- Other confounding effects: Not applicable.
RANGE-FINDING/SCREENING STUDIES: A range finding (preliminary test was performed and was used to determine concentrations, employed in the main test).
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: Not applicable.
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Not applicable.
- Indication whether binucleate or mononucleate where appropriate: Not applicable.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: All Positive Controls gave marked increases in mutant frequency in the current HCD range (except the PC: 2 µg/mL DMBA in the 4-hour exposure with S9, which exceeded the HCD range, but which gave a value considered acceptable for entry into the historical control data). This indicates the test and the metabolic activation system were operating as expected. The purpose of the PC is to verify the functioning of the test system. Full details are provided in the full study report on the PC HCD.
- Negative (solvent/vehicle) historical control data: All vehicle controls indicated background mutant frequencies per survivor (MFS) in the current HCD range. Full details are provided in the full study report on the VC HCD. - 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 TG 476 under GLP conditions to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. Following a check for solubility which indicated precipitation above 625 μg/mL in DMSO solvent, a preliminary test for cytotoxicity within the range 2.44 to 625 μg/mL concentration was performed. Subsequently, Chinese hamster (V79) cells were treated in a main test with the test item at eight concentrations, in duplicate, together with vehicle (DMSO) and positive controls in both the absence and presence of metabolic activation. The dose levels were selected from using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by a combination of toxicity and the onset of test item precipitate in the absence of metabolic activation, and toxicity in the presence of metabolic activity. The concentrations employed including vehicle and positive controls were, respectively: (i) with S9-mix (2%): 0 (control), 10, 20, 40, 80, 100, 120, 140, 160, DMBA: 1.0 and 2.0 µg/mL and (ii) without S9-mix: 0 (control), 5, 10, 20, 40, 50, 60, 70, 80, EMS: 500 and 750 µg/mL. The test item exhibited dose-related toxicity to the cells. The dose levels plated for cloning efficiency and expression of mutant colonies were: 4-hour with S9 (2%): 5, 10, 20, 40, 50 μg/mL and 4-hour without S9: 10, 20, 40, 80, 100, 120 μg/mL, respectively. Concurrent vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The concurrent 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 metabolizing system. Based on the Day 0 cloning efficiencies, optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. There was no evidence of any reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test using a dose range that included the lowest precipitating dose level, and achieved optimum levels of toxicity. Under the conditions of this study, the test item was considered to be non-mutagenic on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line in vitro.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- 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 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: July 2017; signature: November 2017
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable (in vitro micronucleus test of: clastogenic and aneugenic potential)
- Species / strain / cell type:
- 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.
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Cytochalasin B (4.5 μg/mL)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Lot Numbers: PB/βNF S9 31/03/17 and PB/βNF S9 30/06/17
- Test concentrations with justification for top dose:
- The maximum recommended dose level was 5000 µg/mL, on the basis of the UVCB designation of the substance. 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 5000 μg/mL range (full results recorded in the full study report). The preliminary test used a range of test item concentrations of: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL.
I. Preliminary Test:
0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 625 μg/mL in the 4-hour exposure group in the absence of metabolic activation (S9) and at and above 312.5 μg/mL in the presence of S9-mix and the 24-hour continuous exposure group. A reduced cell pellet was observed at and above 312.5 μg/mL in the 4-hour exposure groups and at and above 156.25 μg/mL in the 24-hour exposure group. Erythrocyte hemolysis was observed following exposure to the test item at and above 78.13 μg/mL in all three exposure groups.
Results from the preliminary toxicity test were used to set the test item dose levels for the main experiments.
II. Main Test:
4(20)-hour without S9: 0*, 5, 10, 20, 40*, 80*, 100*, 120, 160 and MMC 0.2*
4(20)-hour with S9 (2%): 0*, 10, 20, 40, 80, 100*, 120*, 160*, 240 and CP 5*
24-hour without S9: 0*, 10, 20, 40*, 80*, 120*, 160, 240, 320 and DC 0.075*
where:
* = dose levels selected for analysis of MN frequency in binucleate cells
MMC= Mitomycin C
CP = Cyclophosphamide
DC = Demecolcine - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Following solubility checks, the test item was fully miscible in DMSO at 500 mg/mL. The maximum recommended dose level was 5000 µg/mL, on the basis of the UVCB designation of the substance. 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 5000 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: DMSO is a guideline accepted vehicle with an available laboratory historic control data set. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Demecolcine
- 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:
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 (100 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. The nominal total volume of each culture was 10 mL.
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 reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
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 (100 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, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
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. The tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
SELECTION AGENT (mutation assays): Not applicable.
SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data. When the slides were dry, they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.
NUMBER OF CELLS EVALUATED: The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls.
DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)
The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis and estimate test item cytotoxicity.
OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable.
- Other: Scoring: Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors. The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter. - Evaluation criteria:
- Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are substantially outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test item l is then considered unable to induce chromosome breaks and/or gain or loss in this test system. - Statistics:
- The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., (2003), In vitro micronucleus test. In: Encyclopedia of Biopharmaceutical Statistics, 2nd edition. S Chow ed. Marcel Dekker, Inc. New York, NY, pp. 463 – 467.). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.
- Species / strain:
- lymphocytes: Human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table 1 for further information
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: There 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: Formulated in DMSO.
- 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 625 μg/mL in the 4-hour exposure group in the absence of metabolic activation (S9) and at and above 312.5 μg/mL in the presence of S9-mix and the 24-hour continuous exposure group. Main test: Precipitate observations were made at the end of exposure in blood-free cultures and no precipitate was observed in all three exposure groups (up to 160 and 240 µg/mL in the 4(20)-hour groups without and with S9 and/or 320 µg/mL in the 24-hour exposure groups, respectively.
- Other confounding effects: In the preliminary test and the main test: Haemolysis was observed in the 4(20)-hour exposure groups, at and above 78.13 µg/mL in all three exposure groups. Additionally, a reduced cell pellet was observed at and above 312.5 μg/mL in the 4-hour exposure groups and at and above 156.25 μg/mL in the 24-hour exposure group. In the definitive test: hemolysis was observed following exposure to the test item at and above 80 μg/mL in 4-hour exposure groups and at and above 120 μg/mL in the 24-hour exposure group. Additionally, a reduced cell pellet was observed at and above 160 μg/mL in the 4-hour exposure group in the presence of S9 only and in the 24-hour exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- Definition of acceptable cells for analysis: The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.
RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 5000 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.
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: See table 1
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: See table 1
- Indication whether binucleate or mononucleate where appropriate: See table 1
COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (DMSO) controls had frequencies of cells with micronuclei 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 micronuclei. (Within the Historic Control Data range presented in the full study report).
ADDITIONAL INFORMATION ON CYTOTOXICITY: See ‘other confounding effects’ listed above.
- Measurement of cytotoxicity used: CBPI
- Other observations when applicable: frequency of binucleated cells - Conclusions:
- Interpretation of results:
negative
Under the conditions of this study the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in the presence and absence of S9 activation. - Executive summary:
The study was performed to the requirements of OECD TG 487 under GLP conditions to assess the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4 hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test with a dose range of 19.53 to 5000 µg/mL in DMSO vehicle which indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows: 4-hour without S9-Mix (2%): 0, 5, 10, 40, 80, 100, 120, 160 μg/mL, with S9-Mix (2%): 0, 10, 20, 40, 80, 100, 120, 160, 240 μg/mL and 24-hour without S9: 0, 10, 20, 40, 80, 120, 160, 240, 320 μg/mL, respectively. All vehicle (DMSO)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with micronuclei, 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 micronuclei, using a dose range that included a dose level that induced approximately or exceeded 50% cytostasis. Under the conditions of this study, the test item was considered to be non-clastogenic and non- aneugenic 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) |
94 69 82 |
(82) 12.5# |
12 20 11 |
(14) 4.9 |
17 28 28 |
(24) 6.4 |
25 21 29 |
(25) 4.0 |
12 13 9 |
(11) 2.1 |
|
1.5 µg |
87 82 80 |
(83) 3.6 |
19 15 7 |
(14) 6.1 |
24 17 12 |
(18) 6.0 |
17 21 19 |
(19) 2.0 |
8 5 12 |
(8) 3.5 |
|
5 µg |
91 69 79 |
(80) 11.0 |
11 9 15 |
(12) 3.1 |
17 13 20 |
(17) 3.5 |
20 25 15 |
(20) 5.0 |
5 19 9 |
(11) 7.2 |
|
15 µg |
91 104 80 |
(92) 12.0 |
17 17 20 |
(18) 1.7 |
19 8 20 |
(16) 6.7 |
19 15 23 |
(19) 4.0 |
13 13 9 |
(12) 2.3 |
|
50 µg |
92 83 71 |
(82) 10.5 |
12 12 12 |
(12) 0.0 |
16 20 19 |
(18) 2.1 |
19 24 21 |
(21) 2.5 |
8 8 11 |
(9) 1.7 |
|
150 µg |
72 64 87 |
(74) 11.7 |
16 9 7 |
(11) 4.7 |
25 25 20 |
(23) 2.9 |
15 15 20 |
(17) 2.9 |
3 12 5 |
(7) 4.7 |
|
500 µg |
12 S 24 S 17 S |
(18) 6.0 |
0 V 0 V 0 V |
(0) 0.0 |
28 17 19 |
(21) 5.9 |
15 S 13 S 13 S |
(14) 1.2 |
0 V 0 V 0 V |
(0) 0.0 |
|
1500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
17 15 21 |
(18) 3.1 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
|
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
21 9 21 |
(17) 6.9 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
|
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 |
|||||||
505 613 470 |
(529) 74.5 |
176 176 170 |
(174) 3.5 |
106 147 95 |
(116) 27.4 |
172 146 124 |
(147) 24.0 |
2401 2685 1867 |
(2318) 415.3 |
||
|
|||||||||||
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) |
100 79 104 |
(94) 13.4# |
9 12 9 |
(10) 1.7 |
32 37 20 |
(30) 8.7 |
28 24 20 |
(24) 4.0 |
21 12 16 |
(16) 4.5 |
|
1.5 µg |
96 95 99 |
(97) 2.1 |
9 17 13 |
(13) 4.0 |
27 17 17 |
(20) 5.8 |
16 17 31 |
(21) 8.4 |
20 15 20 |
(18) 2.9 |
|
5 µg |
111 99 76 |
(95) 17.8 |
13 13 12 |
(13) 0.6 |
28 24 35 |
(29) 5.6 |
25 21 17 |
(21) 4.0 |
23 19 16 |
(19) 3.5 |
|
15 µg |
87 115 102 |
(101) 14.0 |
12 9 21 |
(14) 6.2 |
24 31 29 |
(28) 3.6 |
13 17 20 |
(17) 3.5 |
19 21 12 |
(17) 4.7 |
|
50 µg |
91 82 86 |
(86) 4.5 |
12 12 7 |
(10) 2.9 |
35 33 40 |
(36) 3.6 |
17 23 23 |
(21) 3.5 |
16 9 7 |
(11) 4.7 |
|
150 µg |
78 108 67 |
(84) 21.2 |
9 13 21 |
(14) 6.1 |
31 21 20 |
(24) 6.1 |
21 19 25 |
(22) 3.1 |
9 11 12 |
(11) 1.5 |
|
500 µg |
56 S 57 S 49 S |
(54) 4.4 |
11 S 11 S 9 S |
(10) 1.2 |
31 27 8 |
(22) 12.3 |
25 25 19 |
(23) 3.5 |
8 S 11 S 16 S |
(12) 4.0 |
|
1500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
13 23 21 |
(19) 5.3 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
|
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
11 11 16 |
(13) 2.9 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(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 |
|||||||
2087 1918 1703 |
(1903) 192.5 |
175 196 204 |
(192) 15.0 |
221 182 230 |
(211) 25.5 |
180 235 204 |
(206) 27.6 |
251 358 362 |
(324) 63.0 |
||
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) |
71 88 86 |
(82) 9.3# |
11 15 17 |
(14) 3.1 |
21 27 15 |
(21) 6.0 |
8 23 9 |
(13) 8.4 |
17 16 16 |
(16) 0.6 |
||
0.5 µg |
79 82 83 |
(81) 2.1 |
13 19 9 |
(14) 5.0 |
N/T |
20 13 21 |
(18) 4.4 |
15 20 16 |
(17) 2.6 |
|||
1.5 µg |
78 75 92 |
(82) 9.1 |
24 13 12 |
(16) 6.7 |
16 27 13 |
(19) 7.4 |
13 11 19 |
(14) 4.2 |
13 21 20 |
(18) 4.4 |
||
5 µg |
67 74 95 |
(79) 14.6 |
21 8 8 |
(12) 7.5 |
16 24 31 |
(24) 7.5 |
20 15 9 |
(15) 5.5 |
20 17 17 |
(18) 1.7 |
||
15 µg |
84 75 74 |
(78) 5.5 |
13 16 17 |
(15) 2.1 |
16 24 13 |
(18) 5.7 |
11 15 12 |
(13) 2.1 |
16 11 12 |
(13) 2.6 |
||
50 µg |
88 75 74 |
(79) 7.8 |
12 11 14 |
(12) 1.5 |
19 23 27 |
(23) 4.0 |
8 8 11 |
(9) 1.7 |
8 8 12 |
(9) 2.3 |
||
150 µg |
94 S 69 S 79 S |
(81) 12.6 |
11 S 13 S 11 S |
(12) 1.2 |
15 12 16 |
(14) 2.1 |
9 15 15 |
(13) 3.5 |
0 V 0 V 0 V |
(0) 0.0 |
||
500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
20 S 20 S 20 S |
(20) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
1500 µg |
N/T |
N/T |
23 S 19 S 15 S |
(19) 4.0 |
N/T |
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 |
||||||||
601 552 611 |
(588) 31.6 |
1588 1009 611 |
(1069) 491.3 |
1036 619 687 |
(781) 223.7 |
204 203 210 |
(206) 3.8 |
731 552 528 |
(604) 110.9 |
|||
|
||||||||||||
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) |
64 69 84 |
(72) 10.4# |
13 7 16 |
(12) 4.6 |
21 29 35 |
(28) 7.0 |
12 7 25 |
(15) 9.3 |
23 11 4 |
(13) 9.6 |
||
0.5 µg |
83 74 78 |
(78) 4.5 |
12 20 11 |
(14) 4.9 |
N/T |
N/T |
11 12 8 |
(10) 2.1 |
||||
1.5 µg |
112 68 86 |
(89) 22.1 |
12 16 7 |
(12) 4.5 |
N/T |
18 13 11 |
(14) 3.6 |
9 9 20 |
(13) 6.4 |
|||
5 µg |
100 83 75 |
(86) 12.8 |
7 9 9 |
(8) 1.2 |
N/T |
16 15 20 |
(17) 2.6 |
16 12 12 |
(13) 2.3 |
|||
15 µg |
87 72 80 |
(80) 7.5 |
8 8 9 |
(8) 0.6 |
39 31 32 |
(34) 4.4 |
25 16 20 |
(20) 4.5 |
12 8 4 |
(8) 4.0 |
||
50 µg |
103 78 72 |
(84) 16.4 |
9 10 12 |
(10) 1.5 |
29 32 24 |
(28) 4.0 |
19 15 19 |
(18) 2.3 |
13 5 12 |
(10) 4.4 |
||
150 µg |
99 72 110 |
(94) 19.6 |
9 8 8 |
(8) 0.6 |
23 15 20 |
(19) 4.0 |
9 9 15 |
(11) 3.5 |
5 23 7 |
(12) 9.9 |
||
500 µg |
79 S 65 S 60 S |
(68) 9.8 |
9 S 12 S 9 S |
(10) 1.7 |
15 21 17 |
(18) 3.1 |
31 13 17 |
(20) 9.5 |
9 S 1 S 4 S |
(5) 4.0 |
||
1500 µg |
N/T |
N/T |
24 17 19 |
(20) 3.6 |
0 V 0 V 0 V |
(0) 0.0 |
N/T |
|||||
5000 µg |
N/T |
N/T |
16 S 28 S 21 S |
(22) 6.0 |
N/T |
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 |
||||||||
964 981 1028 |
(991) 33.2 |
139 166 174 |
(160) 18.3 |
103 99 123 |
(108) 12.9 |
79 114 124 |
(106) 23.6 |
72 128 131 |
(110) 33.2 |
|||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
P: Test item precipitate
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
T: Toxic, no bacterial background lawn
#: Standard deviation
N/T: Not tested at this dose level
Table 3. Spontaneous Mutation Rates (Concurrent Negative Controls)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
124 |
|
24 |
|
37 |
|
25 |
|
5 |
|
80 |
(94) |
25 |
(23) |
24 |
(29) |
20 |
(23) |
4 |
(6) |
78 |
|
20 |
|
27 |
|
25 |
|
9 |
|
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
112 |
|
8 |
|
32 |
|
21 |
|
12 |
|
99 |
(106) |
9 |
(13) |
37 |
(31) |
25 |
(19) |
8 |
(12) |
108 |
|
23 |
|
23 |
|
11 |
|
16 |
|
|
15 |
|
|
||||||
28 |
(22)† |
||||||||
24 |
|
||||||||
† Experimental procedure repeated at a later date (without S9-mix) due to toxicity in the original test.
All positive and vehicle and negative controls were within laboratory historic values.
Table 1. Main test: 4-hour exposure without and with Metabolic Activation (S9)
4-hour exposure without S9 |
||||||||||||||||||||||||||||
|
|
|
Day 0 Viability |
Day 7 Viability |
Day 7 Mutant |
|||||||||||||||||||||||
Dose (µg/mL) |
|
|
Colonies / flask (200 cells in plated flask) |
% CE |
% Control |
Mean % Control |
Colonies / flask (200 cells in plated flask) |
% CE |
% Control |
Mean % Control |
Colonies / flask (2x10^5 cells plated/flask) |
MF |
MSF 10^-6 |
SD |
Group MFS 10^-6 |
|||||||||||||
0 |
|
A |
125 |
124 |
116 |
60.8 |
100 |
|
166 |
179 |
174 |
86.5 |
100 |
|
3 |
3 |
5 |
2 |
4 |
2 |
0 |
5 |
2 |
5 |
15.5 |
17.9 |
|
|
|
|
B |
152 |
163 |
161 |
79.3 |
100 |
100 |
144 |
158 |
170 |
78.7 |
100 |
100 |
1 |
1 |
2 |
2 |
2 |
1 |
5 |
1 |
2 |
1 |
9 |
11.4 |
1.57 |
15 |
5 |
|
A |
119 |
121 |
136 |
62.7 |
103.0 |
|
174 |
151 |
175 |
83.3 |
96.3 |
|
2 |
1 |
3 |
3 |
0 |
0 |
3 |
1 |
1 |
3 |
8.5 |
10.2 |
|
|
|
|
B |
146 |
154 |
153 |
75.5 |
95.2 |
99 |
176 |
150 |
161 |
81.2 |
103.2 |
100 |
1 |
5 |
4 |
2 |
1 |
1 |
4 |
3 |
3 |
4 |
14 |
17.2 |
1.45 |
14 |
10 |
|
A |
126 |
121 |
136 |
63.8 |
104.9 |
|
159 |
168 |
151 |
79.7 |
92.1 |
|
3 |
6 |
1 |
3 |
6 |
1 |
2 |
1 |
2 |
5 |
15 |
18.8 |
|
|
|
|
B |
179 |
192 |
203 |
95.7 |
120.6 |
113 |
159 |
155 |
160 |
79.0 |
100.4 |
96 |
5 |
5 |
4 |
3 |
6 |
0 |
2 |
4 |
2 |
1 |
16 |
20.3 |
1.92 |
20 |
20 |
|
A |
128 |
133 |
125 |
64.3 |
105.8 |
|
172 |
176 |
154 |
83.7 |
96.7 |
|
0 |
5 |
1 |
1 |
3 |
4 |
2 |
4 |
0 |
4 |
12 |
14.3 |
|
|
|
|
B |
168 |
173 |
154 |
82.5 |
104.0 |
105 |
149 |
147 |
155 |
75.2 |
95.6 |
96 |
2 |
6 |
5 |
4 |
3 |
1 |
1 |
5 |
3 |
5 |
17.5 |
23.3 |
1.85 |
19 |
40 |
|
A |
114 |
123 |
112 |
58.2 |
95.6 |
|
171 |
194 |
165 |
88.3 |
102.1 |
|
3 |
3 |
3 |
4 |
2 |
1 |
1 |
2 |
7 |
1 |
13.5 |
15.3 |
|
|
|
|
B |
184 |
200 |
169 |
92.2 |
116.2 |
106 |
149 |
160 |
151 |
76.7 |
97.5 |
100 |
1 |
1 |
2 |
0 |
1 |
2 |
2 |
1 |
1 |
3 |
7 |
9.1 |
1.54 |
12 |
50 |
P |
A |
8 |
12 |
16 |
6.0 |
9.9 |
|
153 |
172 |
143 |
78.0 |
90.2 |
|
1 |
2 |
1 |
0 |
3 |
1 |
1 |
2 |
1 |
0 |
6 |
7.7 |
|
|
|
|
B |
37 |
34 |
44 |
19.2 |
24.2 |
17 |
154 |
168 |
152 |
79.0 |
100.4 |
95 |
2 |
1 |
0 |
2 |
1 |
5 |
5 |
5 |
1 |
1 |
11.5 |
14.6 |
1.59 |
11 |
60 |
P |
A |
Not plated due to toxicity / precipitate |
|
Not plated due to toxicity / precipitate |
|
Not plated due to toxicity / precipitate |
- |
|
|
||||||||||||||||||
|
|
B |
Not plated due to toxicity / precipitate |
- |
Not plated due to toxicity / precipitate |
- |
Not plated due to toxicity / precipitate |
- |
- |
- |
||||||||||||||||||
70 |
P |
A |
Not plated due to toxicity / precipitate |
|
Not plated due to toxicity / precipitate |
|
Not plated due to toxicity / precipitate |
- |
|
|
||||||||||||||||||
|
|
B |
Not plated due to toxicity / precipitate |
- |
Not plated due to toxicity / precipitate |
- |
Not plated due to toxicity / precipitate |
- |
- |
- |
||||||||||||||||||
80 |
P |
A |
Not plated due to toxicity / precipitate |
|
Not plated due to toxicity / precipitate |
|
Not plated due to toxicity / precipitate |
- |
|
|
||||||||||||||||||
|
|
B |
Not plated due to toxicity / precipitate |
- |
Not plated due to toxicity / precipitate |
- |
Not plated due to toxicity / precipitate |
- |
- |
- |
||||||||||||||||||
EMS |
|
A |
132 |
127 |
144 |
67.2 |
110.4 |
|
164 |
159 |
166 |
81.5 |
94.2 |
|
40 |
52 |
38 |
37 |
45 |
59 |
41 |
44 |
49 |
63 |
234 |
287.1 |
|
|
500 |
|
B |
175 |
184 |
177 |
89.3 |
112.6 |
112 |
168 |
132 |
150 |
75.0 |
95.3 |
95 |
42 |
44 |
38 |
52 |
38 |
27 |
37 |
38 |
37 |
35 |
194 |
258.7 |
8.59 |
273 |
EMS |
|
A |
123 |
113 |
86 |
53.7 |
88.2 |
|
145 |
141 |
140 |
71.0 |
82.1 |
|
67 |
49 |
81 |
74 |
58 |
79 |
74 |
72 |
62 |
63 |
339.5 |
478.2 |
|
|
750 |
|
B |
203 |
216 |
213 |
105.3 |
132.8 |
110 |
161 |
129 |
150 |
73.3 |
93.2 |
88 |
53 |
52 |
60 |
73 |
77 |
61 |
53 |
72 |
71 |
57 |
314.5 |
428.9 |
9.83 |
454 |
4-hour exposure with S9 |
||||||||||||||||||||||||||||
|
4-hour exposure with S9 |
|||||||||||||||||||||||||||
|
|
Day 0 Viability |
Day 7 Viability |
Day 7 Mutant |
||||||||||||||||||||||||
Dose (µg/mL) |
|
|
Colonies / flask (200 cells in plated flask) |
% CE |
% Control |
Mean % Control |
Colonies / flask (200 cells in plated flask) |
% CE |
% Control |
Mean % Control |
Colonies / flask (2x10^5 cells plated/flask) |
MF |
MSF 10^-6 |
SD |
Group MFS 10^-6 |
|||||||||||||
0 |
|
A |
168 |
162 |
158 |
81.3 |
100 |
|
150 |
166 |
164 |
80.0 |
100 |
|
4 |
1 |
1 |
1 |
0 |
4 |
0 |
0 |
2 |
1 |
7 |
8.8 |
|
|
|
|
B |
160 |
162 |
160 |
80.3 |
100 |
100 |
157 |
142 |
154 |
75.5 |
100 |
100 |
0 |
5 |
2 |
1 |
1 |
2 |
5 |
0 |
0 |
3 |
9.5 |
12.6 |
1.69 |
11 |
10 |
|
A |
187 |
177 |
185 |
91.5 |
112.5 |
|
143 |
155 |
154 |
75.3 |
94.2 |
|
3 |
4 |
0 |
0 |
0 |
1 |
4 |
1 |
0 |
1 |
7 |
9.3 |
|
|
|
|
B |
196 |
202 |
195 |
98.8 |
123.0 |
118 |
148 |
138 |
186 |
78.7 |
104.2 |
99 |
1 |
0 |
0 |
3 |
3 |
0 |
0 |
1 |
1 |
0 |
4.5 |
5.7 |
1.42 |
8 |
20 |
|
A |
195 |
178 |
196 |
94.8 |
116.6 |
|
134 |
149 |
166 |
74.8 |
93.5 |
|
1 |
2 |
2 |
3 |
4 |
3 |
1 |
1 |
4 |
2 |
11.5 |
15.4 |
|
|
|
|
B |
202 |
189 |
176 |
94.5 |
117.6 |
117 |
132 |
141 |
148 |
70.2 |
92.9 |
93 |
0 |
1 |
2 |
1 |
3 |
5 |
2 |
1 |
1 |
1 |
8.5 |
12.1 |
1.30 |
14 |
40 |
|
A |
191 |
176 |
177 |
90.7 |
111.5 |
|
148 |
162 |
146 |
76.0 |
95.0 |
|
2 |
2 |
1 |
2 |
3 |
3 |
2 |
1 |
2 |
3 |
10.5 |
13.8 |
|
|
|
|
B |
190 |
186 |
190 |
94.3 |
117.4 |
114 |
161 |
168 |
127 |
76.0 |
100.7 |
98 |
0 |
2 |
2 |
0 |
0 |
1 |
4 |
3 |
4 |
3 |
9.5 |
12.5 |
1.21 |
13 |
80 |
|
A |
171 |
188 |
191 |
91.7 |
112.7 |
|
163 |
156 |
151 |
78.3 |
97.9 |
|
0 |
1 |
2 |
4 |
1 |
2 |
3 |
2 |
2 |
0 |
8.5 |
10.9 |
|
|
|
|
B |
147 |
148 |
149 |
74.0 |
92.1 |
102 |
163 |
150 |
147 |
76.7 |
101.5 |
100 |
0 |
1 |
0 |
2 |
3 |
2 |
1 |
0 |
2 |
2 |
6.5 |
8.5 |
1.15 |
10 |
100 |
|
A |
136 |
138 |
162 |
72.7 |
89.3 |
|
162 |
146 |
155 |
77.2 |
96.5 |
|
2 |
1 |
1 |
2 |
0 |
2 |
3 |
2 |
0 |
0 |
6.5 |
8.4 |
|
|
|
|
B |
153 |
151 |
162 |
77.7 |
96.7 |
93 |
173 |
166 |
157 |
82.7 |
109.5 |
103 |
3 |
2 |
1 |
2 |
0 |
2 |
2 |
2 |
3 |
1 |
9 |
10.9 |
1.00 |
10 |
120 |
|
A |
29 |
22 |
28 |
13.2 |
16.2 |
|
160 |
140 |
145 |
74.2 |
92.7 |
|
1 |
0 |
0 |
1 |
1 |
2 |
2 |
2 |
2 |
1 |
6 |
8.1 |
|
|
|
|
B |
31 |
22 |
32 |
14.2 |
17.6 |
17 |
160 |
150 |
149 |
76.5 |
101.3 |
97 |
0 |
1 |
2 |
2 |
2 |
2 |
2 |
4 |
2 |
2 |
9.5 |
12.4 |
0.94 |
10 |
140 |
|
A |
0 |
0 |
0 |
0.0 |
0.0 |
|
Not plated due to toxicity |
|
Not plated due to toxicity |
|
|
|||||||||||||||
|
|
B |
6 |
2 |
2 |
1.7 |
2.1 |
1 |
Not plated due to toxicity |
- |
Not plated due to toxicity |
|
- |
|||||||||||||||
160 |
|
A |
Not plated due to toxicity |
|
Not plated due to toxicity |
|
Not plated due to toxicity |
|
|
|||||||||||||||||||
|
|
B |
Not plated due to toxicity |
- |
Not plated due to toxicity |
- |
Not plated due to toxicity |
|
- |
|||||||||||||||||||
DMBA |
|
A |
133 |
139 |
141 |
68.8 |
84.6 |
|
113 |
123 |
122 |
59.7 |
74.6 |
|
64 |
69 |
59 |
60 |
48 |
69 |
73 |
76 |
51 |
59 |
314 |
526.3 |
|
|
1 |
|
B |
150 |
147 |
149 |
74.3 |
92.5 |
89 |
175 |
151 |
143 |
78.2 |
103.5 |
89 |
69 |
65 |
71 |
44 |
64 |
67 |
56 |
83 |
85 |
71 |
337.5 |
431.8 |
10.61 |
479 |
DMBA |
|
A |
129 |
123 |
132 |
64.0 |
78.7 |
|
96 |
95 |
101 |
48.7 |
60.8 |
|
121 |
126 |
101 |
102 |
115 |
119 |
112 |
87 |
86 |
121 |
545 |
1119.9 |
|
|
2 |
|
B |
105 |
110 |
98 |
52.2 |
64.9 |
72 |
159 |
157 |
165 |
80.2 |
106.2 |
84 |
126 |
124 |
115 |
109 |
125 |
106 |
126 |
110 |
102 |
129 |
586 |
731.0 |
12.70 |
925 |
EMS = Ethyl methane sulphonate
DMBA = Dimethyl benzanthracene
CE = Cloning efficiency
MF = Mutant frequency
MFS = Mutant frequency per survivor
SD = Standard deviation
P = Precipitate present at the end of exposure period
Table 1. Definitive test: 4-hour exposure without and with S9 (2%) and 24-hour exposure without S9: CBPI and micronucleus data
Exposure |
Dose Level |
Notes |
Replicate |
Nucleate cells /500 cells |
CBPI |
Mean CBPI |
% cytostasis |
MN per 1000 Binucleate cells |
% Binucleate Cells with MN |
Mean % Binucleate Cells with MN |
||||
Mono |
Bi |
Multi |
1 MN |
2 MN |
> 2 MN |
|||||||||
4-hours (without S9) |
0 |
|
A |
148 |
282 |
70 |
1.84 |
|
|
2 |
0 |
0 |
0.20 |
|
|
|
B |
272 |
235 |
50 |
1.60 |
1.72 |
0 |
15 |
3 |
1 |
1.90 |
1.05 |
|
5 |
|
A |
- |
- |
- |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
10 |
|
A |
- |
- |
- |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
20 |
|
A |
- |
- |
- |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
40 |
|
A |
273 |
192 |
35 |
1.52 |
|
|
1 |
0 |
0 |
0.10 |
|
|
|
|
B |
224 |
236 |
40 |
1.63 |
1.58 |
20 |
3 |
0 |
0 |
0.30 |
0.20 |
|
80 |
H |
A |
253 |
221 |
26 |
1.55 |
|
|
3 |
0 |
0 |
0.30 |
|
|
|
|
B |
244 |
231 |
25 |
1.56 |
1.56 |
23 |
6 |
0 |
1 |
0.70 |
0.50 |
|
100 |
H |
A |
258 |
232 |
11 |
1.51 |
|
|
4 |
0 |
0 |
0.40 |
|
|
|
|
B |
267 |
225 |
8 |
1.48 |
1.50 |
31 |
3 |
2 |
0 |
0.50 |
0.45 |
|
120 |
H |
A |
499 |
83 |
3 |
1.15 |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
435 |
64 |
1 |
1.13 |
1.14 |
81 |
- |
- |
- |
- |
- |
|
160 |
H |
A |
NB |
NB |
NB |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
NB |
NB |
NB |
- |
- |
- |
- |
- |
- |
- |
- |
|
MMC |
|
A |
284 |
208 |
8 |
1.45 |
|
|
36 |
1 |
0 |
3.70 |
|
|
0.2 |
|
B |
256 |
234 |
10 |
1.51 |
1.48 |
33 |
43 |
4 |
0 |
4.70 |
4.20 *** |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
4-hours (with S9 2%) |
0 |
|
A |
172 |
276 |
52 |
1.76 |
|
|
4 |
0 |
0 |
0.40 |
|
|
|
B |
168 |
291 |
41 |
1.75 |
1.76 |
0 |
4 |
1 |
0 |
0.50 |
0.45 |
|
10 |
|
A |
- |
- |
- |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
20 |
|
A |
- |
- |
- |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
40 |
|
A |
- |
- |
- |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
80 |
H |
A |
169 |
262 |
69 |
1.80 |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
189 |
268 |
49 |
1.72 |
1.76 |
0‡ |
- |
- |
- |
- |
- |
|
100 |
H |
A |
151 |
293 |
56 |
1.81 |
|
|
7 |
1 |
0 |
0.80 |
|
|
|
|
B |
190 |
281 |
29 |
1.68 |
1.75 |
1 |
3 |
0 |
0 |
0.30 |
0.55 |
|
120 |
H |
A |
209 |
257 |
34 |
1.65 |
|
|
2 |
2 |
0 |
0.40 |
|
|
|
|
B |
220 |
254 |
26 |
1.61 |
1.63 |
17 |
5 |
1 |
0 |
0.60 |
0.50 |
|
160 |
H R |
A |
422 |
90 |
2 |
1.18 |
|
|
2 |
0 |
0 |
0.45 |
|
|
|
|
B |
376 |
137 |
8 |
1.29 |
1.24 |
69 |
12 |
0 |
0 |
1.20 |
0.83 |
|
240 |
H R |
A |
NB |
NB |
NB |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
NB |
NB |
NB |
- |
- |
- |
- |
- |
- |
- |
- |
|
CP |
|
A |
350 |
147 |
3 |
1.31 |
|
|
16 |
1 |
0 |
1.70 |
||
5 |
|
B |
359 |
137 |
4 |
1.29 |
1.30 |
60 |
42 |
3 |
1 |
4.60 |
3.15 *** |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
24-hours (without S9) |
0 |
|
A |
108 |
301 |
91 |
1.97 |
|
|
8 |
0 |
0 |
0.80 |
|
|
|
B |
114 |
297 |
91 |
1.95 |
1.96 |
0 |
6 |
1 |
1 |
0.80 |
0.80 |
|
10 |
|
A |
- |
- |
- |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
20 |
|
A |
- |
- |
- |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
40 |
|
A |
171 |
272 |
57 |
1.77 |
|
|
3 |
0 |
0 |
0.30 |
|
|
|
|
B |
132 |
304 |
70 |
1.88 |
1.83 |
14 |
8 |
0 |
0 |
0.80 |
0.55 |
|
80 |
H |
A |
206 |
275 |
19 |
1.63 |
|
|
5 |
0 |
0 |
0.50 |
|
|
|
|
B |
212 |
266 |
22 |
1.62 |
1.63 |
35 |
8 |
2 |
0 |
1.00 |
0.75 |
|
120 |
H |
A |
283 |
211 |
6 |
1.45 |
|
|
11 |
1 |
0 |
1.20 |
|
|
|
|
B |
279 |
214 |
7 |
1.46 |
1.46 |
53 |
6 |
0 |
1 |
0.70 |
0.95 |
|
160 |
H R |
A |
307 |
190 |
3 |
1.39 |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
347 |
153 |
0 |
1.31 |
1.35 |
64 |
- |
- |
- |
- |
- |
|
240 |
H R |
A |
NB |
NB |
NB |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
NB |
NB |
NB |
- |
- |
- |
- |
- |
- |
- |
- |
|
320 |
H R |
A |
NB |
NB |
NB |
- |
|
|
- |
- |
- |
- |
- |
|
|
|
B |
NB |
NB |
NB |
- |
- |
- |
- |
- |
- |
- |
- |
|
DC |
|
A |
263 |
203 |
38 |
1.55 |
|
|
38 |
11 |
4 |
5.30 |
|
|
0.075 |
|
B |
269 |
194 |
37 |
1.54 |
1.55 |
43 |
31 |
11 |
2 |
4.40 |
4.85 *** |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
MMC = Mitomycin C
CP = Cyclophosphamide
DC = Demecolcine
***= P<0.001
- = Not selected for scoring
NB = No binucleate cells or insufficient binucleate cells for scoring
H = Hemolysis observed at the end of exposure
R = Reduced cell pellet
‡= Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
Not applicable.
Additional information
OECD TG 471, 2015 - 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 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. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.5 and 5000 µg/plate depending on bacterial tester strain and absence or presence of S9-mix. Up to seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item following the change in test methodology. 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 sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (light and globular in appearance) was noted under an inverted microscope at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies. 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.
OECD TG 476, 2018 - The study was performed to the requirements of OECD TG 476 under GLP conditions to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. Following a check for solubility which indicated precipitation above 625 μg/mL in DMSO solvent, a preliminary test for cytotoxicity within the range 2.44 to 625 μg/mL concentration was performed. Subsequently, Chinese hamster (V79) cells were treated in a main test with the test item at eight concentrations, in duplicate, together with vehicle (DMSO) and positive controls in both the absence and presence of metabolic activation. The dose levels were selected from using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by a combination of toxicity and the onset of test item precipitate in the absence of metabolic activation, and toxicity in the presence of metabolic activity. The concentrations employed including vehicle and positive controls were, respectively: (i) with S9-mix (2%): 0 (control), 10, 20, 40, 80, 100, 120, 140, 160, DMBA: 1.0 and 2.0 µg/mL and (ii) without S9-mix: 0 (control), 5, 10, 20, 40, 50, 60, 70, 80, EMS: 500 and 750 µg/mL. The test item exhibited dose-related toxicity to the cells. The dose levels plated for cloning efficiency and expression of mutant colonies were: 4-hour with S9 (2%): 5, 10, 20, 40, 50 μg/mL and 4-hour without S9: 10, 20, 40, 80, 100, 120 μg/mL, respectively. Concurrent vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The concurrent 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 metabolizing system. Based on the Day 0 cloning efficiencies, optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. There was no evidence of any reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test using a dose range that included the lowest precipitating dose level, and achieved optimum levels of toxicity. Under the conditions of this study, the test item was considered to be non-mutagenic on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line in vitro.
OECD TG 487, 2018 - The study was performed to the requirements of OECD TG 487 under GLP conditions to assess the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4 hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test with a dose range of 19.53 to 5000 µg/mL in DMSO vehicle which indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows: 4-hour without S9-Mix (2%): 0, 5, 10, 40, 80, 100, 120, 160 μg/mL, with S9-Mix (2%): 0, 10, 20, 40, 80, 100, 120, 160, 240 μg/mL and 24-hour without S9: 0, 10, 20, 40, 80, 120, 160, 240, 320 μg/mL, respectively. All vehicle (DMSO)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with micronuclei, 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 micronuclei, using a dose range that included a dose level that induced approximately or exceeded 50% cytostasis. Under the conditions of this study, the test item was considered to be non-clastogenic and non- aneugenic 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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