Registration Dossier
Registration Dossier
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EC number: 293-766-2 | CAS number: 91082-52-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro genotoxicity studies
OECD TG 471: negative with and without S9 mix
OECD TG 476: negative with and without S9 mix
OECD TG 486: negative with and without S9 mix
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:
- Experimental start: 05 May 2016, Experimental completion: 20 May 2016; Final Report: 01 September 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from male Sprague-Dawley derived rats
- Test concentrations with justification for top dose:
- 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the absence and presence of metabolic activation. The maximum concentration was selected based on the standard limit concentration recommended in the regulatory guidelines that the assay follows.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO, ACS reagent grade
- Justification for choice of solvent/vehicle: the test substances dissolved completely in the vehicle at the highest dose tested - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable): at least 10^9 per mL at test begin
DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): 10 hours
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: toxicity was observed as a thin background lawn of non-revertant colonies and/or a reduction in the number of revertant colonies
NUMBER OF REPLICATIONS: 3 - Rationale for test conditions:
- Following relevant test guideline
- Evaluation criteria:
- Criteria for valid test:
- the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory (maintained as a rolling record over two years or a minimum of 20 data sets)
- the positive control compounds must induce an increase in mean revertant colony numbers of at least twice that of the concurrent vehicle control
- mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9 per mL
- a minimum of five analysable concentrations must be present with at least four showing no signs of toxic effects, evident as bacterial inhibition and/or a reduction in the number of revertants below the indication factor of 0.5
If exposure to a test substance produces a reproducible increase in mean revertant colony numbers of at least twice that of the vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic effects.
If exposure to a test substance does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained. - Statistics:
- The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance.
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no fluctuations in pH of the medium were observed at 2000 μg/mL of more than 1.0 unit compared with the vehicle control
- Effects of osmolality: no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control
- Evaporation from medium: not reported
- Precipitation: no precipitation was observed
RANGE-FINDING/SCREENING STUDIES:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- See under any other information on results - Conclusions:
- The test substance showed no evidence of mutagenic activity in the tested strains of S. typhimurium and E. coli in the absence or presence of metabolic activation.
- Executive summary:
The mutagenic potential of the substance was studied in an in vitro bacterial reverse mutation (Ames) study in accordance with OECD TG 471 (1997) under GLP. The experiment is considered relevant, adequate and conclusive.
Experiments were conducted with the four histidine-dependent auxotrophic mutants of Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and in addition with the tryptophan dependent mutant of Escherichia coli strain WP2 uvrA (pKM101) that were exposed to the test substance dissolved in dimethyl sulfoxide (DMSO).
Two independent mutation experiments were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first experiment was a standard plate incorporation assay; the second experiment included a pre-incubation stage. Concentrations of up to 5000 µg/plate were tested, which is the standard limit concentration recommended in the current test guideline. In the first experiment, toxicity occurred, observed as a thin background lawn of non-revertant colonies and/or a reduction in the number of revertant colonies, following exposure to the test substance in strain TA98 at 50 µg/plate and above in the absence of S9 mix and at 1500 µg/plate and above in the presence of S9 mix. Strain TA98, in the absence of S9 mix, did not fulfil the criteria of a valid experiment and an additional plain incorporation experiment was therefore with this strain. In the second experiment, a pre-incubation test, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers) was obtained in strain TA98 following exposure to 500 µg/plate and above in the absence and presence of S9 mix. No precipitate was observed on plates following exposure to the test substance in both experiments. The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the S9 mix. The mean revertant colony numbers for the vehicle controls were within or close to the historical control range for the lab. The concurrent sterility controls demonstrated the absence of microbial contamination of the S9 mix, buffer or test substance formulation. No evidence of mutagenic activity of the test substance was observed at any tested concentration in either experiments in the absence or presence of S9 mix, and it was concluded that the test substance was not mutagenic in this Ames test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental start: 26 July 2016; Experimental completion: 2 September 2016; Final report: 12 January 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro HPRT mutation test
- Target gene:
- functionally hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: European Collection of Cell Cultures
- Suitability of cells: yes
MEDIA USED
- Type and identity of media including CO2 concentration:
H0, Ham's Nutrient Mixture F12, supplemented wiht 2 mM L-glutamine and 50 µg/mL gentamicin
H10, H0 medium supplemented with 10% heat inactivated foetal calf serum
All cell cultures were maintained in an atmosphere of 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Preliminary toxicity experiment: 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
Main experiment without S9 mix: 62.5, 125, 250, 500, 1000 and 2000 µg/mL
Main experiment with S9 mix: 62.5, 125, 250, 500, 1000 and 2000 µg/mL
In the absence of limiting cytotoxicity or precipitate, the maximum test concentration should correspond to 10 mM, 2 mg/mL or 2 µL/mL, whichever is the lowest. - Vehicle / solvent:
- - Vehicle: DMSO, ACS reagent grade
- Justification for choice of solvent/vehicle: the test substance was soluble in the vehicle and DMSO is a standard vehicle recommended in the test guideline - Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium H10
- Cell density at seeding (if applicable): Two flasks were prepared to determine average cell density across all flasks at the beginning of the treatment period to ensure a minimum of 20 x 10^6 cells being present during treatment
DURATION
- Preincubation period: approximated 20 hours at 34 to 39 °C, in an atmosphere of 5% CO2 in air
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days at 34 to 39 °C, in an atmosphere of 5% CO2 in humidified air
- Selection time (if incubation with a selection agent): 7 days at 34 to 39 °C, in an atmosphere of 5% CO2 in humidified air
SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)
NUMBER OF REPLICATIONS: two cultures for each test concentration and four cultures for each vehicle control
METHODS FOR FIXATION AND STAINING: colonies growing in the flasks for fixed and stained in a methanol:Giemsa solution (4:1 v/v) at the end of incubation
DETERMINATION OF CYTOTOXICITY
- Cytotoxicity was measured as Day 1 relative survival (RS) in comparison with the vehicle control - Rationale for test conditions:
- The test conditions were as required by the regulatory guideline.
- Evaluation criteria:
- Tests were accepted when there were no confounding technical probems such as contamination, excessive numbers of outliers and excessive toxicity.
The acceptance criteria for the tests with the test substance were:
- the selection of the top dose concentration of the test substance was in accordance with the guidance
The acceptance criteria for the tests with the vehicle controls were:
- the mean vehicle control value for mutant frequency was between 1 and 20 x 10^-6
- the mean cloning/plating efficiency was between 65 and 120%
- obvious outliers were excluded, with at least two vehicle controls remaining
- vehicle controls of both parallel cultures were remaining within or close to the conrol limit of the laboratory historical control data range
The acceptance criteria for the tests with the positive controls were:
- positive controls showed statistically significant increase in the mean total MF above the mean concurrent vehicle control MF and within, or close to, the range of the historical control data
A substance is considered to show a positive, genotoxic potential, if all acceptability criteria and the all the following criteria are met:
- at least one of the test concentrations exhibited a statistically significant increase in the mutant frequency compared with the concurrent negative control
- the increase was concentration-dependent when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical negative control data
A substance is considered to not show a genotoxic potential, if all acceptability criteria and:
- none of the test concentrations exhibited a statistically significant increase in the mutant frequency compared with the concurrent negative control
- there was no concentration-dependent increase when evaluated with an appropriate trend test
- all results were inside the distribution of the historical negative control data - Statistics:
- The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Experiments were conducted for a linear concentration-response relationship of the test item, for non-linearity and for the comparison of positive control and treated groups to solvent control.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- 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: No fluctuations in pH of the medium were observed at 2000 µg/mL of more than 1.0 unit compared with the vehicle control
- Effects of osmolality: The osmolality of the test item in medium was tested at 2000 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Evaporation from medium: not reported
- Precipitation: precipitation at the top dose of 2000 µg/mL was observed at the end of the treatment in the preliminary toxicity study, but not in the main study
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data: see below under any other information on results
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity was measured as Day 1 relative survival - Conclusions:
- The test substance did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.
- Executive summary:
The test substance was tested for mutagenic potential in an in vitro mammalian cell mutation assay in accordance with OECD TG 476 (2015) and under GLP. The experiment is considered relevant, adequate and conclusive. The test system is designed to detect and quantify forward mutation at the functionally hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells. Two independent tests in the absence and presence of exogenous metabolic activation (S9 mix) were conducted.
The vehicle used in the tests was dimethyl sulfoxide (DMSO), in which the test substance dissolved at up to 200 mg/mL. The highest final concentration used in the preliminary toxicity test was 2000 µg/mL, which is the standard limit concentration within the test system as recommended in the regulatory guideline. Precipitate was observed by eye at least at the end of treatment at 2000 µg/mL. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure to the test substance at concentrations in the range from 15.63 to 2000 µg/mL, RS values ranged from 93 to 59% and from 103 to 76%, in the absence and presence of S9 mix, respectively.
In the main mutation experiment in the absence of S9 mix, cells were exposed to concentrations of the test substance in the range from 62.5 to 2000 µg/mL. No precipitation was observed by eye at the end of the treatment. RS values ranged from 98 to 65% relative to the vehicle control. The test substance did not induce a statistically significant increase in mutant frequency. The positive control, ethyl methanesulphonate, induced a significant increase in mutant frequency demonstrating the correct functioning of the assay.
In the main mutation experiment in the presence of S9 mix, cells were exposed to concentrations of the test substances in the range from 62.5 to 2000 µg/mL. No precipiation was observed by eye at the end of the treatment. RS values ranged from 104 to 88% relative to the vehicle control. The test substance did not induce a statistically significant increase in mutant frequency. The positive control substance, 3-methylcholantrene, induced a significant increase in the mutant frequency demonstrating the correct functioning of the assay.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental start: 09 May 2016; Experimental completion: 31 May 2016; Final report: 12 September 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: obtained from blood collected from two healthy, non-smoking adult donors
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: human blood
- Suitability of cells: yes
- Sex, age and number of blood donors: in total two female or male, healthy, non-smoking, adult (18-35 years old) donors
- Whether whole blood or separated lymphocytes were used: whole blood
- Methods for maintenance in cell culture if applicable: lymphocytes were stimulated to undergo cell division by the addition of phytohaemagglutinin (PHA), cultures were established from pooled samples and dispensed as 5 mL aliquots in sterile universal containers so that each culture contained 0.4 mL of blood, 4.5 mL of HML media and 0.1 mL of PHA solution, and all cultures were incubated at 37 °C and resuspended twice daily by gentle inversion
MEDIA USED
- Type and identity of media: HML Media, RPMI 1640, supplemented with 10% foetoal calf serum, 0.2 IU/mL penicillin/ 20 µg/mL streptomycin and 2.0 mM L-glutamine
- Properly maintained: yes - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Preliminary test: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
Main test (3 hours, no S9 mix): 250, 500, 1000 and 2000 µg/mL
Main test (3 hours, with S9 mix): 250, 500, 1000 and 2000 µg/mL
Main test (20 hours, no S9 mix): 25, 250, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 µg/mL
The highest concentration was intended to be that which caused a depression in the cytokinesis-block proliferative index (CBPI) equivalent to 55±5% cytostasis (approximately) when compared with the concurrent vehicle control or, where no cytostasis was observed, the maximum concentration as recommended in the test guidelines or the limit of solubility. The top dose of 2000 µg/mL was based on solubility of the test substance in DMSO, which was found to be 400 mg/mL, giving a dose of 2000 µg/mL when dosed at 0.5% v/v. - Vehicle / solvent:
- - Vehicle used: DMSO, ACS reagent grade
- Justification for choice of vehicle: DMSO is recommended as vehicle in the current guideline, and the test substance was soluble in the vehicle - Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- other: cyclophosphamide (CPP), colchicine (CC)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium, HML Media RPMI 1640, supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin, 20 µg/mL streptomycin and 2.0 mM L-glutamine
DURATION
- Preincubation period: lymphocyte cultures were incubated for approximately 48 hours following stimulation with PHA, before addition of the test substance
- Exposure duration: 3 hours or 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 37 °C, cultures were then incubated for 3 minutes at 37 °C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1 v/v methanol: acetic acid) was added slowly onto the culture surface and the cultures were slowly inverted to mix. The cultures were centrifuged at 500 g for five minutes. The supernatant was removed, and the cell pellet re-suspended. A further 4 mL of fresh fixative was then added and the cells stored at 4 °C until slide preparation.
SLIDE PREPARATION: cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared. Pre-cleaned microscope slides were prepared for each culture by aliquoting the re-suspended cells onto the slides, and allowing the slides to air-dry. One slide was prepared from each culture. The remaining cell cultures were stored at approximately 4 °C until slide analysis was complete.
STAIN (for cytogenetic assays): slide were rinsed in purified water, stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes, washed in purified water for 5 minutes, rinsed in cold tap water for 2 minutes and stored at room temperature protected from light until scoring under a microscope.
NUMBER OF REPLICATIONS: duplicate cultures were prepared for each treatment level and positive control cultures; quadruplicate cultures were prepared for vehicle controls; two slides were prepared from each culture.
NUMBER OF CELLS EVALUATED: the incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible from at least 2000 binucleate cells per concentration (4000 for vehicle controls)
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell, and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The colour of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.
DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test item on the cells may lead either to a reduction in cell replication (cytostasis) or to cell death. Cytokinesis-block proliferative index (CBPI) values significantly less than the concurrent vehicle control values are indicative of cytostasis. Furthermore, also the cell integrity was analysed.
OTHER EXAMINATIONS:
- The presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells and debris, if any, was also noted. - Rationale for test conditions:
- The test conditions were as recommended in the current guideline.
- Evaluation criteria:
- The acceptance criteria were that:
- the concurrent negative control must be considered acceptable for addition to the laboratory's historical negative control database
- the concurrent postive control substances must induce responses that are compatible with the laboratory's historical positive control database and produce statistically significant increases compared to the concurrent negative control
- the criteria for selection of the top dose concentration are consistent with those outlined in the study plan
If all the following criteria were met, a positive genotoxic potential of the test substance was considered:
- the acceptance criteria must have been met
- at least one of the test concentrations exhibited a statistically significant increase in the frequency of micronucleated cells compared with the concurrent negative control
- the increase in the frequency of micronucleated cells was dose related when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical negative control data
If all of the follwing criteria were met, a negative genotoxic potential of the test substance was considered:
- the acceptance criteria must have been met
- none of the test concentrations exhibited a statistically significant increase in the frequency of micronucleated cells compared with the concurrent negative control
- there was no concentration-related increase when evaluated with an appropriate trend test
all results were inside the distribution of the historical negative control data - Statistics:
- The analysis assumed that the replicate was the experimental unit. An arcsine square-root transformation was used to transform the data. CA3490A treated groups were then compared to control using Williams' tests (Williams 1971, 1972). Positive controls were compared to control using t-tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups. Statistical significance was declared at the 5% level for all tests.
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- 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: No fluctuations in pH of the medium were observed at 2000 μg/mL of more than 1.0 unit compared with the vehicle control
- Effects of osmolality: The osmolality of the test item in medium was tested at 2000 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control
- Evaporation from medium: not reported
- Definition of acceptable cells for analysis: Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell, and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The color of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.
- Other confounding effects:
RANGE-FINDING/SCREENING STUDIES:
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- See under any other information
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells] - Remarks on result:
- other: No cytotoxicity, but tested up to limit concentrations
- Conclusions:
- The test substance did no increase the induction of micronuclei in cultured human lymphocytes in this in vitro micronucleus assay.
- Executive summary:
An in vitro micronucleus study was conducted to test the potential of the substance to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in accordance with OECD TG 487 (2014) and under GLP. The experiment is considered relevant, adequate and conclusive.
The study consisted of a preliminary toxicity test and a main micronucleus test. Human lymphocytes in whole blood cultures were exposed to the test substance for 3 hours in the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. The maximum final concentration to which cells were exposed was 2000 µg/mL. The concentration was chosen with regard to the purity of the test substance and with respect to the current guideline. Vehicle (dimethyl sulfoxide, DMSO) and positive controls were included in the studies.
Three test concentrations were assessed for determination of induction of micronuclei. Following 3-hour treatment in the absence and presence of S9 mix, the highest concentration of 2000 µg/mL was chosen as the highest test concentration. There were no reductions in CBPI obtained with the test substance at any concentration tested, and the concentrations selected for the micronucleus analysis were 500, 1000 and 2000 µg/mL. Following 20-hour treatment in the absence of S9 mix, a reduction of CBPI equivalent to 53.4% cytostasis was obtained with the test substance at 950 µg/mL. Concentrations selected for the micronucleus analysis were 25, 800 and 950 µg/mL. The test substance did not cause a statistically significant increase in the number of binucleated cells containing micronuclei when compared with the vehicle control in the absence or presence of S9 mix folliwng an exposure period of 3 hours, or in the absence of S9 mix after 20 hours of exposure. The positive control substances caused significant increase in the number of binucleated cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system. It was concluded that the test substance did no increase the induction of micronuclei in cultured human lymphocytes in this in vitro micronucleus assay.
Referenceopen allclose all
Table 1: Results of Experiment 1, plate incorporation
Experiment 1, plate incorporation, no metabolic activation |
||||||||||||||||||||||||
Strain |
Addition |
Concentration per plate [µg] |
Mean revertant number per plate |
Standard deviation |
Fold increase over vehicle |
Individual revertant counts |
||||||||||||||||||
TA98 |
DMSO |
|
31.0 |
1.7 |
|
32 |
29 |
32 |
||||||||||||||||
Test substance |
5 |
26.7 |
6.5 |
0.9 |
27 |
33 |
20 |
|||||||||||||||||
15 |
24.3 |
1.5 |
0.8 |
24 |
23 |
26 |
||||||||||||||||||
50 |
12.7 |
4.6 |
0.4 |
18 |
10 |
10 |
||||||||||||||||||
150 |
7.7 |
2.3 |
0.2 |
9 |
9 |
5 |
||||||||||||||||||
500 |
9.0 |
4.4 |
0.3 |
4 |
12 |
11 |
||||||||||||||||||
1500 |
12.7 |
2.1 |
0.4 |
15S |
11S |
12S |
||||||||||||||||||
5000 |
8.7 |
2.1 |
0.3 |
7S |
11S |
8S |
||||||||||||||||||
|
||||||||||||||||||||||||
TA100 |
DMSO |
|
141.0 |
6.9 |
|
137 |
149 |
137 |
||||||||||||||||
Test substance |
5 |
133.0 |
11.4 |
0.9 |
120 |
138 |
141 |
|||||||||||||||||
15 |
149.7 |
2.5 |
1.1 |
152 |
150 |
147 |
||||||||||||||||||
50 |
107.3 |
27.6 |
0.8 |
139 |
88 |
95 |
||||||||||||||||||
150 |
104.7 |
5.8 |
0.7 |
108 |
98 |
108 |
||||||||||||||||||
500 |
93.0 |
11.8 |
0.7 |
106 |
90 |
83 |
||||||||||||||||||
1500 |
114.7 |
4.0 |
0.8 |
117 |
110 |
117 |
||||||||||||||||||
5000 |
111.7 |
7.0 |
0.8 |
119 |
105 |
111 |
||||||||||||||||||
|
||||||||||||||||||||||||
TA1535 |
DMSO |
|
22.3 |
9.5 |
|
13 |
32 |
22 |
||||||||||||||||
Test substance |
5 |
21.0 |
1.0 |
0.9 |
21 |
22 |
20 |
|||||||||||||||||
15 |
18.0 |
3.5 |
0.8 |
16 |
16 |
22 |
||||||||||||||||||
50 |
18.0 |
8.0 |
0.8 |
26 |
10 |
18 |
||||||||||||||||||
150 |
14.0 |
6.1 |
0.6 |
11 |
10 |
21 |
||||||||||||||||||
500 |
18.7 |
4.7 |
0.8 |
17 |
15 |
24 |
||||||||||||||||||
1500 |
17.0 |
4.6 |
0.8 |
21 |
12 |
18 |
||||||||||||||||||
5000 |
21.7 |
7.8 |
1.0 |
24 |
28 |
13 |
||||||||||||||||||
|
||||||||||||||||||||||||
TA1537 |
DMSO |
|
17.7 |
4.5 |
|
13 |
18 |
22 |
||||||||||||||||
Test substance |
5 |
16.7 |
7.1 |
0.9 |
18 |
9 |
23 |
|||||||||||||||||
15 |
19.0 |
5.6 |
1.1 |
24 |
13 |
20 |
||||||||||||||||||
50 |
12.3 |
3.1 |
0.7 |
15 |
9 |
13 |
||||||||||||||||||
150 |
12.3 |
3.2 |
0.7 |
11 |
10 |
16 |
||||||||||||||||||
500 |
12.7 |
2.1 |
0.7 |
12 |
15 |
11 |
||||||||||||||||||
1500 |
11.7 |
1.5 |
0.7 |
10 |
13 |
12 |
||||||||||||||||||
5000 |
17.7 |
9.0 |
1.0 |
13 |
28 |
12 |
||||||||||||||||||
|
||||||||||||||||||||||||
WP2 uvrA (pKM101) |
DMSO |
|
156.7 |
17.7 |
|
177 |
148 |
145 |
||||||||||||||||
Test substance |
5 |
161.3 |
9.7 |
1.0 |
153 |
159 |
172 |
|||||||||||||||||
15 |
164.0 |
5.6 |
1.0 |
169 |
165 |
158 |
||||||||||||||||||
50 |
128.0 |
13.0 |
0.8 |
143 |
120 |
121 |
||||||||||||||||||
150 |
121.7 |
11.2 |
0.8 |
109 |
130 |
126 |
||||||||||||||||||
500 |
125.7 |
9.1 |
0.8 |
116 |
127 |
134 |
||||||||||||||||||
1500 |
162.0 |
10.6 |
1.0 |
174 |
158 |
154 |
||||||||||||||||||
5000 |
124.3 |
16.3 |
0.8 |
142 |
110 |
121 |
||||||||||||||||||
S: slight thinning of background lawn |
||||||||||||||||||||||||
Experiment 1, plate incorporation, no metabolic activation, positive controls |
||||||||||||||||||||||||
TA98 |
2-nitrofluorene |
2 |
243.3 |
9.2 |
7.8 |
238 |
238 |
254 |
||||||||||||||||
TA100 |
Sodium azide |
2 |
540.0 |
6.2 |
3.8 |
542 |
533 |
545 |
||||||||||||||||
TA1535 |
Sodium azide |
2 |
828.3 |
44.5 |
37.1 |
821 |
876 |
788 |
||||||||||||||||
TA1537 |
9-aminoacridine |
50 |
202.0 |
19.5 |
11.4 |
182 |
221 |
203 |
||||||||||||||||
WP2 uvrA |
4-nitroquinoline-1-oxide |
2 |
847.7 |
95.1 |
5.4 |
740 |
920 |
883 |
||||||||||||||||
Experiment 1, plate incorporation, viability |
||||||||||||||||||||||||
|
|
Strain |
|
Mean counts per plate |
Standard deviation |
Individual counts (100 µL aliquots of 10^-6 dilution of 10-hour culture) |
||||||||||||||||||
|
|
TA98 |
Viability |
244.7 |
17.0 |
225 |
255 |
254 |
||||||||||||||||
|
|
TA100 |
Viability |
327.0 |
12.3 |
332 |
313 |
336 |
||||||||||||||||
|
|
TA1535 |
Viability |
351.0 |
89.7 |
331 |
449 |
273 |
||||||||||||||||
|
|
TA1537 |
Viability |
242.7 |
9.1 |
251 |
233 |
244 |
||||||||||||||||
|
|
WP2 uvrA |
Viability |
330.0 |
13.2 |
345 |
320 |
324 |
||||||||||||||||
Experiment 1, plate incorporation, with metabolic activation |
||||||||||||||||||||||||
Strain |
Addition |
Concentration per plate [µg] |
Mean revertant number per plate |
Standard deviation |
Fold increase over vehicle |
Individual revertant counts |
||||||||||||||||||
TA98 |
DMSO |
|
31.7 |
6.4 |
|
39 |
27 |
29 |
||||||||||||||||
Test substance |
5 |
20.0 |
3.6 |
0.6 |
23 |
21 |
16 |
|||||||||||||||||
15 |
19.3 |
3.1 |
0.6 |
22 |
20 |
16 |
||||||||||||||||||
50 |
20.3 |
3.8 |
0.6 |
16 |
23 |
22 |
||||||||||||||||||
150 |
21.3 |
0.6 |
0.7 |
22 |
21 |
21 |
||||||||||||||||||
500 |
26.3 |
2.1 |
0.8 |
27 |
28 |
24 |
||||||||||||||||||
1500 |
21.7 |
2.5 |
0.7 |
24S |
22S |
19S |
||||||||||||||||||
5000 |
22.7 |
3.8 |
0.7 |
21S |
20S |
27S |
||||||||||||||||||
|
||||||||||||||||||||||||
TA100 |
DMSO |
|
150.0 |
14.9 |
|
133 |
161 |
156 |
||||||||||||||||
Test substance |
5 |
109.3 |
9.2 |
0.7 |
104 |
120 |
104 |
|||||||||||||||||
15 |
108.0 |
8.0 |
0.7 |
116 |
108 |
100 |
||||||||||||||||||
50 |
110.0 |
6.6 |
0.7 |
111 |
116 |
103 |
||||||||||||||||||
150 |
108.0 |
15.9 |
0.7 |
114 |
90 |
120 |
||||||||||||||||||
500 |
126.3 |
10.2 |
0.8 |
138 |
119 |
122 |
||||||||||||||||||
1500 |
131.3 |
5.0 |
0.9 |
136 |
126 |
132 |
||||||||||||||||||
5000 |
134.7 |
9.0 |
0.9 |
134 |
126 |
144 |
||||||||||||||||||
|
||||||||||||||||||||||||
TA1535 |
DMSO |
|
14.0 |
2.6 |
|
15 |
16 |
11 |
||||||||||||||||
Test substance |
5 |
14.0 |
3.5 |
1.0 |
12 |
12 |
18 |
|||||||||||||||||
15 |
10.7 |
4.5 |
0.8 |
11 |
15 |
6 |
||||||||||||||||||
50 |
14.7 |
7.5 |
1.0 |
7 |
22 |
15 |
||||||||||||||||||
150 |
9.3 |
3.8 |
0.7 |
12 |
11 |
5 |
||||||||||||||||||
500 |
16.7 |
4.5 |
1.2 |
12 |
21 |
17 |
||||||||||||||||||
1500 |
20.3 |
2.9 |
1.5 |
22 |
17 |
22 |
||||||||||||||||||
5000 |
13.3 |
8.1 |
1.0 |
12 |
22 |
6 |
||||||||||||||||||
|
||||||||||||||||||||||||
TA1537 |
DMSO |
|
19.7 |
6.7 |
|
23 |
24 |
12 |
||||||||||||||||
Test substance |
5 |
17.0 |
6.9 |
0.9 |
21 |
21 |
9 |
|||||||||||||||||
15 |
14.7 |
1.5 |
0.7 |
13 |
15 |
16 |
||||||||||||||||||
50 |
15.0 |
4.4 |
0.8 |
18 |
10 |
17 |
||||||||||||||||||
150 |
15.7 |
6.4 |
0.8 |
23 |
12 |
12 |
||||||||||||||||||
500 |
14.3 |
3.8 |
0.7 |
10 |
16 |
17 |
||||||||||||||||||
1500 |
18.0 |
5.0 |
0.9 |
13 |
23 |
18 |
||||||||||||||||||
5000 |
17.0 |
5.3 |
0.9 |
13 |
15 |
23 |
||||||||||||||||||
|
||||||||||||||||||||||||
WP2 uvrA (pKM101) |
DMSO |
|
199.0 |
14.0 |
|
189 |
215 |
193 |
||||||||||||||||
Test substance |
5 |
152.3 |
4.7 |
0.8 |
154 |
147 |
156 |
|||||||||||||||||
15 |
167.7 |
17.8 |
0.8 |
160 |
155 |
188 |
||||||||||||||||||
50 |
184.0 |
19.0 |
0.9 |
189 |
200 |
163 |
||||||||||||||||||
150 |
174.3 |
3.8 |
0.9 |
176 |
170 |
177 |
||||||||||||||||||
500 |
182.3 |
5.9 |
0.9 |
178 |
180 |
189 |
||||||||||||||||||
1500 |
199.7 |
20.6 |
1.0 |
178 |
202 |
219 |
||||||||||||||||||
5000 |
152.3 |
22.3 |
0.8 |
178 |
138 |
141 |
||||||||||||||||||
S: slight thinning of background lawn |
||||||||||||||||||||||||
Experiment 1, plate incorporation, with metabolic activation, positive control |
||||||||||||||||||||||||
TA98 |
Benzo[a]pyrene |
5 |
139.0 |
7.2 |
4.4 |
141 |
131 |
145 |
||||||||||||||||
TA100 |
2-aminoanthracene |
5 |
817.7 |
388.6 |
5.5 |
1266 |
577 |
610 |
||||||||||||||||
TA1535 |
2-aminoanthracene |
5 |
542.0 |
50.9 |
38.7 |
488 |
589 |
549 |
||||||||||||||||
TA1537 |
Benzo[a]pyrene |
5 |
737 |
2.9 |
3.7 |
77 |
72 |
72 |
||||||||||||||||
WP2 uvrA |
2-aminoanthracene |
10 |
834.0 |
35.0 |
4.2 |
874 |
819 |
809 |
||||||||||||||||
Additional experiment 1, plate incorporation, no metabolic activation |
||||||||||||||||||||||||
Strain |
Addition |
Concentration per plate [µg] |
Mean revertant number per plate |
Standard deviation |
Fold increase over vehicle |
Individual revertant counts |
||||||||||||||||||
TA98 |
DMSO |
|
21.7 |
0.6 |
|
22 |
21 |
22 |
||||||||||||||||
Test substance |
0.5 |
17.3 |
0.6 |
0.8 |
17 |
17 |
18 |
|||||||||||||||||
1.0 |
17.3 |
3.2 |
0.8 |
15 |
16 |
21 |
||||||||||||||||||
5 |
12.0 |
1.7 |
0.6 |
10 |
13 |
13 |
||||||||||||||||||
15 |
19.3 |
3.1 |
0.9 |
22 |
16 |
20 |
||||||||||||||||||
50 |
21.0 |
0.0 |
1.0 |
21 |
21 |
21 |
||||||||||||||||||
150 |
20.0 |
7.0 |
0.9 |
17 |
28 |
15 |
||||||||||||||||||
500 |
13.3 |
3.5 |
0.6 |
13 |
10 |
17 |
||||||||||||||||||
1500 |
8.3 |
0.6 |
0.4 |
8S |
9S |
8S |
||||||||||||||||||
5000 |
6.0 |
1.0 |
0.3 |
6S |
7S |
5S |
||||||||||||||||||
S: slight thinning of background lawn |
||||||||||||||||||||||||
Additional experiment 1, plate incorporation, no metabolic activation, positive control |
||||||||||||||||||||||||
TA98 |
2-nitrofluorene |
2 |
262.7 |
16.9 |
12.1 |
255 |
282 |
251 |
||||||||||||||||
Additional experiment 1, plate incorporation, no metabolic activation, viability |
||||||||||||||||||||||||
Strain |
|
Mean counts per plate |
Standard deviation |
Individual counts (100 µL aliquots of 10^-6 dilution of 10-hour culture) |
||||||||||||||||||||
TA98 |
Viability |
211.0 |
29.5 |
177 |
226 |
230 |
||||||||||||||||||
Table 2: Results of Experiment 2, pre-incubation
Experiment 2, pre-incubation, no metabolic activation |
||||||||
Strain |
Addition |
Concentration per plate [µg] |
Mean revertant number per plate |
Standard deviation |
Fold increase over vehicle |
Individual revertant counts |
||
TA98 |
DMSO |
|
50.3 |
12.7 |
|
65 |
43 |
43 |
Test substance |
0.5 |
39.7 |
2.9 |
0.8 |
38 |
38 |
43 |
|
1.5 |
41.0 |
3.5 |
0.8 |
45 |
39 |
39 |
||
5 |
45.7 |
9.3 |
0.9 |
38 |
43 |
56 |
||
15 |
52.0 |
8.9 |
1.0 |
45 |
62 |
49 |
||
50 |
43.0 |
0.0 |
0.9 |
53 |
43 |
43 |
||
150 |
33.3 |
5.5 |
0.7 |
39 |
28 |
33 |
||
500 |
19.3 |
1.2 |
0.4 |
20S |
15S |
20S |
||
1500 |
19.7 |
1.5 |
0.4 |
21T |
20T |
18T |
||
5000 |
20.3 |
3.1 |
0.4 |
17T |
23T |
21T |
||
|
||||||||
TA100 |
DMSO |
|
146.0 |
9.5 |
|
137 |
156 |
145 |
Test substance |
5 |
152.7 |
5.5 |
1.0 |
158 |
147 |
153 |
|
15 |
151.7 |
23.4 |
1.0 |
169 |
161 |
125 |
||
50 |
109.7 |
26.1 |
0.8 |
139 |
89 |
101 |
||
150 |
109.0 |
10.6 |
0.7 |
101 |
121 |
105 |
||
500 |
107.0 |
5.6 |
0.7 |
108 |
101 |
112 |
||
1500 |
111.7 |
13.5 |
0.8 |
98 |
112 |
125 |
||
5000 |
121.7 |
24.6 |
0.8 |
94 |
141 |
130 |
||
|
||||||||
TA1535 |
DMSO |
|
16.3 |
5.7 |
|
21 |
18 |
10 |
Test substance |
5 |
13.3 |
2.5 |
0.8 |
11 |
13 |
16 |
|
15 |
20.7 |
12.9 |
1.3 |
10 |
17 |
35 |
||
50 |
14.3 |
5.8 |
0.9 |
21 |
11 |
11 |
||
150 |
12.0 |
1.7 |
0.7 |
13 |
10 |
13 |
||
500 |
13.3 |
2.3 |
0.8 |
16 |
12 |
12 |
||
1500 |
18.3 |
2.5 |
1.1 |
21 |
16 |
18 |
||
5000 |
15.3 |
5.8 |
0.9 |
22 |
12 |
12 |
||
|
||||||||
TA1537 |
DMSO |
|
10.0 |
2.6 |
|
11 |
12 |
7 |
Test substance |
5 |
13.7 |
8.5 |
1.4 |
17 |
4 |
20 |
|
15 |
11.0 |
6.2 |
1.1 |
6 |
18 |
9 |
||
50 |
6.0 |
3.5 |
0.6 |
4 |
10 |
4 |
||
150 |
11.7 |
1.2 |
1.2 |
11 |
13 |
11 |
||
500 |
6.0 |
3.6 |
0.6 |
2 |
7 |
9 |
||
1500 |
6.7 |
2.5 |
0.7 |
4 |
7 |
9 |
||
5000 |
5.7 |
2.9 |
0.6 |
4 |
9 |
4 |
||
|
||||||||
WP2 uvrA (pKM101) |
DMSO |
|
163.7 |
24.0 |
|
177 |
136 |
178 |
Test substance |
5 |
168.0 |
6.6 |
1.0 |
161 |
174 |
169 |
|
15 |
193.7 |
25.0 |
1.2 |
172 |
221 |
188 |
||
50 |
135.0 |
17.4 |
0.8 |
155 |
123 |
127 |
||
150 |
137.7 |
6.7 |
0.8 |
142 |
130 |
141 |
||
500 |
138.3 |
10.2 |
0.8 |
134 |
131 |
150 |
||
1500 |
136.0 |
3.0 |
0.8 |
136 |
139 |
133 |
||
5000 |
103.7 |
9.2 |
0.6 |
93 |
109 |
109 |
||
Experiment 2, pre-incubation, no metabolic activation, positive control |
||||||||
TA98 |
2-nitrofluorene |
2 |
237.2 |
42.9 |
4.7 |
189 |
271 |
252 |
TA100 |
Sodium azide |
2 |
652.7 |
31.8 |
4.5 |
630 |
689 |
639 |
TA1535 |
Sodium azide |
2 |
672.7 |
58.5 |
41.2 |
732 |
671 |
615 |
TA1537 |
9-aminoacridine |
50 |
191.3 |
28.9 |
28.9 |
209 |
207 |
158 |
WP2 uvrA |
4-nitroquinoline-1-oxide |
2 |
1028.7 |
12.1 |
6.3 |
1038 |
1033 |
1015 |
S: slight thinning of background lawn; T: thinning of background lawn |
||||||||
Experiment 2, pre-incubation, with metabolic activation |
||||||||
Strain |
Addition |
Concentration per plate [µg] |
Mean revertant number per plate |
Standard deviation |
Fold increase over vehicle |
Individual revertant counts |
||
TA98 |
DMSO |
|
30.0 |
1.7 |
|
32 |
29 |
29 |
Test substance |
0.5 |
24.3 |
7.5 |
0.8 |
32 |
17 |
24 |
|
1.5 |
25.0 |
5.2 |
0.8 |
22 |
22 |
31 |
||
5 |
32.0 |
1.7 |
1.1 |
31 |
31 |
34 |
||
15 |
33.0 |
3.5 |
1.1 |
31 |
37 |
31 |
||
50 |
21.3 |
5.8 |
0.7 |
18 |
18 |
28 |
||
150 |
21.0 |
0.0 |
1.7 |
21 |
21 |
21 |
||
500 |
28.3 |
3.2 |
0.9 |
27S |
32S |
26S |
||
1500 |
24.0 |
2.0 |
0.8 |
26T |
22T |
24T |
||
5000 |
18.0 |
2.6 |
0.6 |
16T |
17T |
21T |
||
|
||||||||
TA100 |
DMSO |
|
142.0 |
10.0 |
|
142 |
132 |
152 |
Test substance |
5 |
100.7 |
7.6 |
0.7 |
104 |
92 |
106 |
|
15 |
110.0 |
4.6 |
0.8 |
106 |
109 |
115 |
||
50 |
114.3 |
11.6 |
0.8 |
101 |
122 |
120 |
||
150 |
124.3 |
13.1 |
0.9 |
138 |
112 |
123 |
||
500 |
132.3 |
13.3 |
0.9 |
117 |
141 |
139 |
||
1500 |
141.7 |
2.5 |
1.0 |
139 |
142 |
144 |
||
5000 |
145.0 |
12.8 |
1.0 |
148 |
156 |
131 |
||
|
||||||||
TA1535 |
DMSO |
|
15.0 |
3.0 |
|
12 |
15 |
18 |
Test substance |
5 |
10.7 |
4.7 |
0.7 |
16 |
9 |
7 |
|
15 |
13.7 |
1.2 |
0.9 |
15 |
13 |
13 |
||
50 |
11.0 |
6.2 |
0.7 |
6 |
9 |
18 |
||
150 |
12.3 |
7.1 |
0.8 |
6 |
11 |
20 |
||
500 |
16.0 |
8.8 |
1.1 |
10 |
26 |
12 |
||
1500 |
10.7 |
8.1 |
0.7 |
18 |
2 |
12 |
||
5000 |
11.0 |
1.7 |
0.7 |
10 |
13 |
10 |
||
|
||||||||
TA1537 |
DMSO |
|
16.7 |
4.5 |
|
21 |
12 |
17 |
Test substance |
5 |
10.3 |
1.5 |
0.6 |
12 |
9 |
10 |
|
15 |
10.7 |
4.5 |
0.6 |
15 |
11 |
6 |
||
50 |
12.0 |
0.0 |
0.7 |
12 |
12 |
12 |
||
150 |
13.7 |
2.3 |
0.8 |
15 |
15 |
11 |
||
500 |
16.3 |
3.5 |
1.0 |
13 |
20 |
16 |
||
1500 |
12.0 |
1.7 |
0.7 |
13 |
10 |
13 |
||
5000 |
12.7 |
3.8 |
0.8 |
11 |
10 |
17 |
||
|
||||||||
WP2 uvrA (pKM101) |
DMSO |
|
200.7 |
15.0 |
|
215 |
185 |
202 |
Test substance |
5 |
177.0 |
39.1 |
0.9 |
132 |
202 |
197 |
|
15 |
173.3 |
15.4 |
0.9 |
166 |
191 |
163 |
||
50 |
166.0 |
10.5 |
0.8 |
156 |
177 |
165 |
||
150 |
176.3 |
11.7 |
0.9 |
163 |
185 |
181 |
||
500 |
164.7 |
5.5 |
0.8 |
165 |
159 |
170 |
||
1500 |
157.7 |
7.4 |
0.8 |
155 |
166 |
152 |
||
5000 |
143.0 |
5.3 |
0.7 |
139 |
149 |
141 |
||
S: slight thinning of background lawn; T: thinning of background lawn |
||||||||
Experiment 2, pre-incubation, with metabolic activation, positive control |
||||||||
TA98 |
Benzo[a]pyrene |
5 |
175.7 |
1.2 |
5.9 |
175 |
177 |
175 |
TA100 |
2-aminoanthracene |
5 |
2141.7 |
377.8 |
15.1 |
1711 |
2297 |
2417 |
TA1535 |
2-aminoanthracene |
5 |
361.0 |
19.5 |
24.1 |
346 |
354 |
383 |
TA1537 |
Benzo[a]pyrene |
5 |
159.3 |
1.2 |
9.6 |
158 |
160 |
160 |
WP2 uvrA |
2-aminoanthracene |
10 |
1038.0 |
71.5 |
5.2 |
1109 |
1039 |
966 |
Experiment 2, pre-incubation, viability |
||||||||
|
|
Strain |
|
Mean counts per plate |
Standard deviation |
Individual colony counts (100 µL aliquots of 10^-6 dilution of 10-hour culture) |
||
|
|
TA98 |
Viability |
150.0 |
9.8 |
142 |
147 |
161 |
|
|
TA100 |
Viability |
138.3 |
8.4 |
148 |
133 |
134 |
|
|
TA1535 |
Viability |
136.3 |
9.3 |
132 |
130 |
147 |
|
|
TA1537 |
Viability |
111.7 |
21.1 |
99 |
100 |
136 |
|
|
WP2 uvrA (pKM101) |
Viability |
188.7 |
23.1 |
167 |
213 |
186 |
Table 3: Historical control data
DMSO |
||||||||||
|
TA100 |
TA1535 |
WP2 uvrA (pKM101) |
TA98 |
TA1537 |
|||||
S9Mix |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
Max |
234 |
237 |
47 |
55 |
221 |
280 |
78 |
84 |
63 |
50 |
Min |
103 |
91 |
11 |
11 |
54 |
56 |
24 |
27 |
8 |
15 |
Mean |
158 |
168 |
26 |
23 |
193 |
193 |
42 |
55 |
21 |
32 |
No. of values |
147 |
145 |
147 |
141 |
131 |
131 |
153 |
149 |
146 |
143 |
St.dev. |
24 |
27 |
6 |
7 |
37 |
37 |
9 |
12 |
7 |
7 |
Upper 95% limit |
206 |
222 |
38 |
36 |
266 |
266 |
59 |
80 |
35 |
46 |
Lower 95% limit |
110 |
114 |
14 |
10 |
121 |
121 |
24 |
31 |
6 |
18 |
Positive controls |
||||||||||
|
TA100 |
TA1535 |
WP2 uvrA (pKM101) |
TA98 |
TA1537 |
|||||
|
NaN3 |
AAN |
NaN3 |
AAN |
NQO |
AAN |
2NF |
B[a]P |
AAC |
B[a]P |
S9Mix |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
Conc. (µg/plate) |
2 |
5 |
2 |
5 |
2 |
10 |
2 |
5 |
50 |
5 |
Max |
2776 |
4210 |
1255 |
716 |
3730 |
1923 |
802 |
648 |
2181 |
609 |
Min |
396 |
425 |
209 |
147 |
639 |
352 |
82 |
90 |
87 |
84 |
Mean |
1017 |
2017 |
799 |
378 |
2192 |
974 |
246 |
270 |
370 |
159 |
No. of values |
198 |
196 |
199 |
194 |
189 |
186 |
205 |
202 |
196 |
193 |
St.dev. |
292 |
763 |
219 |
127 |
651 |
368 |
123 |
94 |
270 |
58 |
NaN3: Sodium azide; 2NF: 2-nitrofluorene; AAC: 9-aminoacridine; B[a]P: Benzo[a]pyrene; AAN: 2-aminoanthracene |
||||||||||
Table 1: Day 1 Relative Survival (RS), preliminary toxicity test
3-hour treatment in the absence of S9 mix, preliminary toxicity test |
||||||||
Concentration of test substance (µg/mL) |
Cell count Day 1 (x 10^6/mL) |
Number of colonies on plate |
Total # of colonies |
Cloning efficiency (%) |
Adjusted cloning efficiency (%) |
Relative survival (%) |
||
Plate 1 |
Plate 2 |
Plate 3 |
||||||
Vehicle control: DMSO (1% v/v) |
1.15 |
120 |
132 |
128 |
380 |
63 |
63 |
100 |
Vehicle control: DMSO (1% v/v) |
1.03 |
132 |
128 |
119 |
379 |
|
|
|
15.63 |
1.03 |
120 |
117 |
120 |
357 |
60 |
56 |
89 |
31.25 |
1.00 |
109 |
111 |
97 |
317 |
53 |
48 |
77 |
62.5 |
0.93 |
128 |
137 |
118 |
383 |
64 |
54 |
86 |
125 |
1.05 |
123 |
103 |
142 |
368 |
61 |
59 |
93 |
250 |
0.97 |
118 |
109 |
116 |
343 |
57 |
51 |
80 |
500 |
0.94 |
89 |
104 |
141 |
334 |
56 |
48 |
76 |
1000 |
0.92 |
104 |
115 |
118 |
337 |
56 |
47 |
75 |
2000P |
0.89 |
96 |
90 |
90 |
276 |
46 |
37 |
59 |
P: precipitate observed by eye at the end of treatment; cell count pre-treatment = 1.1 x 10^6 cells/mL |
||||||||
3-hour treatment in the presence of S9 mix, preliminary toxicity test |
||||||||
Concentration of test substance (µg/mL) |
Cell count Day 1 (x 10^6/mL) |
Number of colonies on plate |
Total # of colonies |
Cloning efficiency (%) |
Adjusted cloning efficiency (%) |
Relative survival (%) |
||
Plate 1 |
Plate 2 |
Plate 3 |
||||||
Vehicle control: DMSO (1% v/v) |
0.88 |
147 |
132 |
137 |
416 |
65 |
55 |
100 |
Vehicle control: DMSO (1% v/v) |
0.97 |
143 |
126 |
99 |
368 |
|
|
|
15.63 |
0.98 |
116 |
129 |
120 |
365 |
61 |
55 |
99 |
31.25 |
0.90 |
133 |
105 |
97 |
335 |
56 |
46 |
83 |
62.5 |
0.93 |
106 |
100 |
106 |
312 |
52 |
44 |
80 |
125 |
0.89 |
114 |
103 |
93 |
310 |
52 |
42 |
76 |
250 |
0.93 |
152 |
129 |
121 |
402 |
67 |
57 |
103 |
500 |
0.98 |
119 |
110 |
109 |
338 |
56 |
50 |
91 |
1000 |
0.93 |
119 |
111 |
89 |
319 |
53 |
45 |
82 |
2000P |
0.96 |
111 |
107 |
103 |
321 |
54 |
47 |
85 |
P: precipitate observed by eye at the end of treatment; cell count pre-treatment = 1.1 x 10^6 cells/mL |
||||||||
Table 2: Day 1 Relative Survival (RS), main test
3-hour treatment in the absence of S9 mix, main test |
|||||||||
Concentration of test substance (µg/mL) |
Cell count Day 1 (x 10^6/mL) |
Number of colonies on plate |
Total # of colonies |
Cloning efficiency (%) |
Adjusted cloning efficiency (%) |
Relative survival (%) |
Mean RS (%) |
||
Plate 1 |
Plate 2 |
Plate 3 |
|||||||
Vehicle control: DMSO (1% v/v) |
1.47 1.56 1.75 1.63 |
192 180 121 129 |
196 168 150 136 |
188 171 137 123 |
576 519 408 388 |
79 |
90 |
100 |
100 |
62.5 |
1.62 1.60 |
125 135 |
134 101 |
127 125 |
386 361 |
64 60 |
74 69 |
83 76 |
79 |
125 |
1.60 1.63 |
95 114 |
97 106 |
130 125 |
322 348 |
54 58 |
61 67 |
68 75 |
72 |
250 |
1.75 1.60 |
151 141 |
157 165 |
111 168 |
419 474 |
70 79 |
87 90 |
97 100 |
98 |
500 |
1.55 1.56 |
168 163 |
150 115 |
145 137 |
463 415 |
77 69 |
85 77 |
95 86 |
90 |
1000 |
1.63 1.54 |
120 107 |
88 145 |
99 153 |
307 405 |
51 68 |
60 74 |
66 82 |
74 |
2000 |
1.61 1.61 |
110 109 |
102 99 |
101 93 |
313 301 |
52 50 |
60 57 |
66 64 |
65 |
|
Ethyl methanesulphonate – Positive control |
||||||||
250 |
1.65 |
86 |
81 |
89 |
256 |
43 |
50 |
56 |
67 |
1.55 |
109 |
132 |
142 |
383 |
64 |
70 |
78 |
||
Cell count pre-treatment = 1.4 x 10^6 cells/mL |
|||||||||
3-hour treatment in the presence of S9 mix, main test |
|||||||||
Concentration of test substance (µg/mL) |
Cell count Day 1 (x 10^6/mL) |
Number of colonies on plate |
Total # of colonies |
Cloning efficiency (%) |
Adjusted cloning efficiency (%) |
Relative survival (%) |
Mean RS (%) |
||
Plate 1 |
Plate 2 |
Plate 3 |
|||||||
Vehicle control: DMSO (1% v/v) |
1.94 1.93 1.89 1.67 |
114 137 148 119 |
115 154 129 117 |
133 151 139 122 |
362 442 416 358 |
66 |
76 |
100 |
100 |
62.5 |
1.80 1.77 |
120 142 |
123 128 |
155 141 |
398 411 |
66 69 |
74 75 |
98 99 |
98 |
125 |
1.90 1.74 |
98 99 |
118 148 |
115 130 |
331 377 |
55 63 |
65 68 |
86 90 |
88 |
250 |
1.88 1.83 |
107 107 |
119 162 |
132 157 |
358 426 |
60 71 |
70 81 |
92 106 |
99 |
500 |
1.98 1.84 |
122 121 |
131 154 |
167 86 |
420 361 |
70 60 |
86 69 |
113 90 |
102 |
1000 |
1.92 1.90 |
120 115 |
145 140 |
128 153 |
393 408 |
66 68 |
78 80 |
103 106 |
104 |
2000 |
2.00 1.87 |
110 120 |
117 132 |
131 152 |
358 404 |
60 67 |
74 79 |
97 103 |
100 |
|
3-methylcholanthrene – Positive control |
||||||||
5 |
1.90 |
118 |
105 |
140 |
363 |
61 |
72 |
94 |
101 |
1.86 |
162 |
135 |
124 |
421 |
70 |
81 |
107 |
||
Cell count pre-treatment = 1.6 x 10^6 cells/mL |
|||||||||
Table 3: Day 8 Cloning Efficiency
3-hour treatment in the absence of S9 mix, main test |
|||||
Concentration of test substance (µg/mL) |
Number of colonies on plate |
Total # of colonies |
Cloning efficiency in non-selective medium (%) |
||
Plate 1 |
Plate 2 |
Plate 3 |
|||
Vehicle control: DMSO (1% v/v) |
176 181 151 172 |
188 171 147 189 |
159 193 174 160 |
523 545 472 521 |
87 91 79 87 |
62.5 |
172 164 |
161 180 |
176 165 |
509 509 |
85 85 |
125 |
175 182 |
146 173 |
171 184 |
492 539 |
82 90 |
250 |
152 176 |
148 145 |
156 165 |
456 486 |
76 81 |
500 |
145 121 |
118 127 |
135 155 |
398 403 |
66 67 |
1000 |
123 154 |
138 136 |
148 106 |
409 396 |
68 66 |
2000 |
177 144 |
158 143 |
151 131 |
486 418 |
81 70 |
|
Ethyl methanesulphonate – Positive control |
||||
250 |
139 |
126 |
106 |
371 |
62 |
149 |
128 |
148 |
425 |
71 |
|
3-hour treatment in the presence of S9 mix, main test |
|||||
Concentration of test substance (µg/mL) |
Number of colonies on plate |
Total # of colonies |
Cloning efficiency in non-selective medium (%) |
||
Plate 1 |
Plate 2 |
Plate 3 |
|||
Vehicle control: DMSO (1% v/v) |
136 173 152 166 |
133 144 154 152 |
133 163 150 152 |
402 480 456 470 |
67 80 76 78 |
62.5 |
168 157 |
163 169 |
154 162 |
485 488 |
81 81 |
125 |
154 139 |
165 142 |
161 131 |
480 412 |
80 69 |
250 |
159 143 |
163 127 |
168 136 |
490 406 |
82 68 |
500 |
132 132 |
137 137 |
139 138 |
408 407 |
68 68 |
1000 |
137 123 |
135 117 |
124 134 |
396 374 |
66 62 |
2000 |
119 117 |
124 110 |
113 121 |
356 348 |
59 58 |
|
3-methylcholanthrene – Positive control |
||||
5 |
134 |
137 |
138 |
409 |
68 |
126 |
127 |
143 |
396 |
66 |
|
Table 4: Day 8 Mutant Frequency
3-hour treatment in the absence of S9 mix, main test |
|||||||||
Concentration of test substance (µg/mL) |
Number of colonies on plate |
Total # of colonies |
Cloning efficiency in selective medium (%) |
Mutant frequency per 10^6 viable cells |
Mean mutant frequency per 10^6 viable cells |
||||
Plate 1 |
Plate 2 |
Plate 3 |
Plate 4 |
Plate 5 |
|||||
Vehicle control: DMSO (1% v/v) |
1 0 1 0 |
0 2 0 0 |
1 2 0 0 |
0 0 0 1 |
0 0 0 0 |
2 4 1 1 |
0.00008 0.00016 0.00014 0.00004 |
0.92 1.76 0.51 0.46 |
0.91 |
62.5 |
0 0 |
0 0 |
1 0 |
0 0 |
1 0 |
2 0 |
0.00008 0.00000 |
0.94 0.00 |
0.47 |
125 |
2 1 |
0 1 |
1 0 |
1 0 |
0 1 |
4 3 |
0.00016 0.00012 |
1.95 1.34 |
1.64 |
250 |
0 1 |
0 0 |
0 0 |
0 0 |
0 1 |
0 2 |
0.00000 0.00008 |
0.00 0.99 |
0.49 |
500 |
0 0 |
1 0 |
0 0 |
0 0 |
1 0 |
2 0 |
0.00008 0.00000 |
1.21 0.00 |
0.60 |
1000 |
1 2 |
1 0 |
1 0 |
0 0 |
0 0 |
3 2 |
0.00012 0.00008 |
1.76 1.21 |
1.49 |
2000 |
0 1 |
0 0 |
0 1 |
1 0 |
1 0 |
2 2 |
0.00008 0.00008 |
0.99 1.15 |
1.07 |
|
Ethyl methanesulphonate – Positive control |
||||||||
250 |
16 |
15 |
14 |
21 |
16 |
82 |
0.00328 |
53.05 |
51.09*** |
20 |
19 |
15 |
14 |
19 |
87 |
0.00348 |
49.13 |
||
*** p<0.001, statistically significant increase over concurrent vehicle control mutant frequency |
|||||||||
3-hour treatment in the presence of S9 mix, main test |
|||||||||
Concentration of test substance (µg/mL) |
Number of colonies on plate |
Total # of colonies |
Cloning efficiency in selective medium (%) |
Mutant frequency per 10^6 viable cells |
Mean mutant frequency per 10^6 viable cells |
||||
Plate 1 |
Plate 2 |
Plate 3 |
Plate 4 |
Plate 5 |
|||||
Vehicle control: DMSO (1% v/v) |
0 0 0 1 |
0 0 0 0 |
2 0 1 0 |
0 0 2 0 |
1 1 1 0 |
3 1 4 1 |
0.00012 0.00004 0.00016 0.00004 |
1.79 0.50 2.11 0.51 |
1.23 |
62.5 |
1 0 |
0 0 |
0 0 |
1 0 |
2 0 |
4 0 |
0.00016 0.00000 |
1.98 0.00 |
0.99 |
125 |
0 0 |
0 0 |
0 2 |
1 0 |
0 0 |
1 2 |
0.00004 0.00008 |
0.50 1.17 |
0.83 |
250 |
1 1 |
0 0 |
0 1 |
0 2 |
0 2 |
1 6 |
0.00004 0.00024 |
0.49 3.55 |
2.02 |
500 |
3 1 |
2 0 |
1 0 |
4 0 |
0 0 |
10 1 |
0.00040 0.00004 |
5.88 0.59 |
3.24 |
1000 |
0 1 |
0 0 |
0 1 |
0 0 |
1 0 |
1 2 |
0.00004 0.00008 |
0.61 1.28 |
0.94 |
2000 |
0 1 |
0 0 |
0 0 |
0 0 |
1 0 |
1 1 |
0.00004 0.00004 |
0.67 0.69 |
0.68 |
|
3-methylcholanthrene – Positive control |
||||||||
5 |
28 |
26 |
27 |
29 |
24 |
134 |
0.00536 |
78.63 |
80.53*** |
28 |
27 |
31 |
28 |
22 |
136 |
0.00544 |
82.42 |
||
*** p<0.001, statistically significant increase over concurrent vehicle control mutant frequency |
|||||||||
Table 5: Historical control data
In the absence of S9 mix |
|||
|
|
Mean Day 1 Cloning Efficiency (%) |
Mean Mutant Frequency (x10^-6) |
Vehicle controls |
Mean |
72 |
7.0 |
Maximum |
95 |
19.3 |
|
Standard deviation |
0.1 |
5.9 |
|
Ethyl methanesulphonate 250µg/mL |
Mean |
- |
301.7 |
Maximum |
- |
72.1 |
|
Standard deviation |
- |
132 |
|
Upper control limit for vehicle controls: 11.5 x 10^-6; Number of experiments: 26 |
|||
In the presence of S9 mix |
|||
|
|
Mean Day 1 Cloning Efficiency (%) |
Mean Mutant Frequency (x10^-6) |
Vehicle controls |
Mean |
74 |
7.6 |
Maximum |
88 |
20.5 |
|
Standard deviation |
0.1 |
6.1 |
|
3-methylcholanthrene 5µg/mL |
Mean |
- |
380.5 |
Maximum |
- |
62.6 |
|
Standard deviation |
- |
173 |
|
Upper control limit for vehicle controls: 11.9 x 10^-6; Number of experiments: 27 |
|||
Table 1: results obtained in the main study
3-hour treatment without S9-mix |
|||||||||||||||||||||||
Concentration (µg/mL) |
Mononucleate cells |
Binucleate cells |
Polynucleate cells |
CBPI |
Mean CBPI |
Mean cytostasis (%) |
Binucleated cells containing micronuclei |
||||||||||||||||
Per 1000 cells |
Mean |
p-valuea |
Trend test p-valueb |
||||||||||||||||||||
Vehicle: DMSO |
84 89 96 68 |
377 397 374 393 |
42 28 34 45 |
1.92 1.88 1.88 1.95 |
1.91 |
0 |
4 5 12 6 |
6.8 |
|
|
|||||||||||||
Test item 250 |
58 79 |
394 403 |
50 38 |
1.98 1.92 |
1.95 |
-5 |
NA NA |
|
|
|
|||||||||||||
Test item 500 |
86 79 |
388 402 |
54 49 |
1.94 1.95 |
1.95 |
-4.3 |
6 3 |
4.5 |
0.807 |
|
|||||||||||||
Test item 1000 |
72 87 |
410 392 |
42 25 |
1.94 1.88 |
1.91 |
-0.3 |
5 5 |
5.0 |
0.807 |
0.656 |
|||||||||||||
Test item 2000 |
107 82 |
375 398 |
19 21 |
1.82 1.88 |
1.85 |
6.2 |
7 4 |
5.5 |
0.807 |
0.817 |
|||||||||||||
Mitomycin C 0.3 |
120 122 |
367 364 |
15 14 |
1.79 1.78 |
1.79 |
13.2 |
23 16 |
19.5 |
0.021* |
|
|||||||||||||
Colchicine 0.07 |
122 209 |
355 282 |
29 15 |
1.82 1.62 |
1.72 |
21.0 |
15 37 |
26.0 |
0.005* |
|
|||||||||||||
a) p-values are for comparisons to control using Williams’ test for the test substance and t-test otherwise; b) trend test p-values are for the linear contrast including the control group and lower concentrations of the same compound; * p<0.05; ** p>0.01; CBPI: cytokinesis block proliferative index; NA: not analysed for micronucleus frequency |
|||||||||||||||||||||||
3-hour treatment with S9-mix |
|||||||||||||||||||||||
Concentration (µg/mL) |
Mononucleate cells |
Binucleate cells |
Polynucleate cells |
CBPI |
Mean CBPI |
Mean cytostasis (%) |
Binucleated cells containing micronuclei |
||||||||||||||||
Per 1000 cells |
Mean |
p-valuea |
Trend test p-valueb |
||||||||||||||||||||
Vehicle: DMSO |
71 82 66 92 |
393 397 408 391 |
36 21 26 17 |
1.93 1.88 1.92 1.85 |
1.89 |
0 |
8 5 7 9 |
7.3 |
|
|
|||||||||||||
Test item 250 |
64 83 |
401 407 |
35 14 |
1.94 1.86 |
1.90 |
-0.9 |
NA NA |
|
|
|
|||||||||||||
Test item 500 |
65 55 |
412 422 |
24 23 |
1.92 1.94 |
1.93 |
-3.6 |
5 6 |
5.5 |
1.000 |
|
|||||||||||||
Test item 1000 |
61 51 |
412 430 |
31 19 |
1.94 1.94 |
1.94 |
-4.9 |
6 5 |
5.5 |
1.000 |
0.238 |
|||||||||||||
Test item 2000 |
92 67 |
391 416 |
17 17 |
1.85 1.90 |
1.88 |
2.2 |
10 7 |
8.5 |
0.525 |
0.442 |
|||||||||||||
Cyclophosphamide 5 |
180 199 |
324 300 |
0 1 |
1.64 1.60 |
1.62 |
30.3 |
18 14 |
16.0 |
0.001** |
|
|||||||||||||
a) p-values are for comparisons to control using Williams’ test for the test substance and t-test otherwise; b) trend test p-values are for the linear contrast including the control group and lower concentrations of the same compound; * p<0.05; ** p>0.01; CBPI: cytokinesis block proliferative index; NA: not analysed for micronucleus frequency |
|||||||||||||||||||||||
20-hour treatment without S9-mix |
|||||||||||||||||||||||
Concentration (µg/mL) |
Mononucleate cells |
Binucleate cells |
Polynucleate cells |
CBPI |
Mean CBPI |
Mean cytostasis (%) |
Binucleated cells containing micronuclei |
||||||||||||||||
Per 1000 cells |
Mean |
p-valuea |
Trend test p-valueb |
||||||||||||||||||||
Vehicle: DMSO |
48 89 85 75 |
402 381 356 356 |
50 40 59 69 |
2.00 1.90 1.95 1.99 |
1.96 |
0 |
11 3 7 6 |
6.8 |
|
|
|||||||||||||
Test item 25 |
60 66 |
398 383 |
47 52 |
1.97 1.97 |
1.97 |
-1.3 |
5 6 |
5.5 |
0.834 |
|
|||||||||||||
Test item 250 |
102 99 |
401 398 |
33 11 |
1.87 1.83 |
1.85 |
11.7 |
NA NA |
|
|
|
|||||||||||||
Test item 500 |
199 132 |
307 366 |
1 2 |
1.61 1.74 |
1.67 |
29.8 |
NA NA |
|
|
|
|||||||||||||
Test item 550 |
191 158 |
324 315 |
19 27 |
1.68 1.74 |
1.71 |
26.3 |
NA NA |
|
|
|
|||||||||||||
Test item 600 |
151 118 |
334 372 |
17 9 |
1.73 1.78 |
1.76 |
21.2 |
NA NA |
|
|
|
|||||||||||||
Test item 650 |
125 102 |
349 374 |
26 24 |
1.80 1.84 |
1.82 |
14.4 |
NA NA |
|
|
|
|||||||||||||
Test item 700 |
135 136 |
356 363 |
9 12 |
1.75 1.76 |
1.75 |
21.7 |
NA NA |
|
|
|
|||||||||||||
Test item 750 |
158 169 |
337 324 |
5 7 |
1.69 1.68 |
1.69 |
28.7 |
NA NA |
|
|
|
|||||||||||||
Test item 800 |
165 147 |
328 350 |
7 3 |
1.68 1.71 |
1.70 |
27.4 |
4 8 |
6.0 |
0.834 |
0.767 |
|||||||||||||
Test item 850 |
166 172 |
330 325 |
4 3 |
1.68 1.66 |
1.67 |
30.4 |
NA NA |
|
|
|
|||||||||||||
Test item 900 |
190 209 |
310 291 |
0 0 |
1.62 1.58 |
1.60 |
37.5 |
NA NA |
|
|
|
|||||||||||||
Test item 950 |
287 265 |
213 235 |
0 0 |
1.43 1.47 |
1.45 |
53.4 |
5 6 |
5.5 |
0.813 |
0.701 |
|||||||||||||
Test item 1000 |
472 481 |
28 19 |
0 0 |
1.06 1.04 |
1.05 |
95.1 |
NA NA |
|
|
|
|||||||||||||
Mitomycin C 0.1 |
102 95 |
401 405 |
2 9 |
1.80 1.83 |
1.82 |
15.0 |
17 19 |
18.0 |
0.003** |
|
|||||||||||||
Colchicine 0.02 |
272 289 |
239 221 |
3 6 |
1,48 1.45 |
1.46 |
51.7 |
16 16 |
16.0 |
0.006** |
|
|||||||||||||
a) p-values are for comparisons to control using Williams’ test for the test substance and t-test otherwise; b) trend test p-values are for the linear contrast including the control group and lower concentrations of the same compound; * p<0.05; ** p>0.01; CBPI: cytokinesis block proliferative index; NA: not analysed for micronucleus frequency |
|||||||||||||||||||||||
Table 2: Historical control data
Historical control data without S9 mix, 3 hours (number of tests: 22) |
||||||
|
Vehicle control |
Mitomycin C (0.2 or 0.3 µg/mL) |
Colchicine (0.05, 0.06 or 0.07 µg/mL) |
|||
|
Binucleate individual MN/1000 cells |
Binucleate Group MN |
Binucleate individual MN/1000 cells |
Binucleate Group MN |
Binucleate individual MN/1000 cells |
Binucleate Group MN |
Minimum |
10 |
25 |
180 |
205 |
160 |
175 |
Maximum |
110 |
98 |
670 |
645 |
430 |
380 |
Mean |
64 |
64 |
331 |
331 |
240 |
240 |
Standard deviation |
24 |
17 |
98 |
94 |
63 |
59 |
Upper control limit |
|
92 |
|
|
|
|
Historical control data with S9 mix, 3 hours (number of tests: 23) |
||||||
|
Vehicle control |
Mitomycin C (0.2 or 0.3 µg/mL) |
|
|||
|
Binucleate individual MN/1000 cells |
Binucleate Group MN |
Binucleate individual MN/1000 cells |
Binucleate Group MN |
|
|
Minimum |
00 |
28 |
120 |
130 |
|
|
Maximum |
130 |
105 |
280 |
270 |
|
|
Mean |
64 |
64 |
188 |
188 |
|
|
Standard deviation |
27 |
19 |
37 |
34 |
|
|
Upper control limit |
|
97 |
|
|
|
|
Historical control data without S9 mix, 20 hours (number of tests: 23) |
||||||
|
Vehicle control |
Mitomycin C (0.2 or 0.3 µg/mL) |
Colchicine (0.05, 0.06 or 0.07 µg/mL) |
|||
|
Binucleate individual MN/1000 cells |
Binucleate Group MN |
Binucleate individual MN/1000 cells |
Binucleate Group MN |
Binucleate individual MN/1000 cells |
Binucleate Group MN |
Minimum |
20 |
33 |
150 |
160 |
140 |
140 |
Maximum |
120 |
105 |
410 |
375 |
230 |
215 |
Mean |
74 |
74 |
232 |
232 |
181 |
181 |
Standard deviation |
26 |
22 |
66 |
61 |
22 |
19 |
Upper control limit |
|
96 |
|
|
|
|
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Three in vitro genetic toxicity studies are available, which are reliable, relevant and adequate for the derivation of a classification of the test substance. All studies were negative with and without metabolic activation (S9 mix). Based on these results, the substance is considered to be non-genotoxic. A classification for genotoxicity under the CLP Regulation (EC) No. 1272/2008 is not warranted. No further testing of the substance for genotoxicity in vivo is required.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
