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EC number: - | CAS number: -
- Life Cycle description
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- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
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- Ecotoxicological Summary
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- Short-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
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Endpoint summary
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Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
2 Ames tests are available on two different qualities of the registered substance (Legal entity compositions A and B). Both showed negative results with and without metabolic activation.
The in vitro micronucleus performed on the registered substance showed negative results with and without metabolic activation
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:
- 17/07/2019 to 12/08/2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)
- Version / remarks:
- June 2012
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 30 May 2008
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella strains: histidine locus
E. coli strain: Tryptophan locus - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: rfa- (increase permeability of bacterial cells to larger molecules); uvrB- (inactivation of the excision repair system) for all salmonella strains, and plasmid pKM101 R factor for TA98 and TA100 (to enhance chemical and UV induced mutagenesis)
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- other: uvrA- : DNA repair deficiency which enhances its sensitivity to some mutagenic compounds
- Cytokinesis block (if used):
- Not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
Rat liver S9
- source of S9
: purchased from Moltox, Lot No. 4061 (Exp 1) and 4123 (Exp 2)
- method of preparation of S9 mix :
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9: 5.0 mL; 1.65 M KCl/0.4 M MgCl2: 1.0 mL; 0.1 M Glucose-6-phosphate: 2.5 mL; 0.1 M NADP 2.0 mL; 0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL; Sterile distilled water: 14.5 mL.
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
- concentration: 10% of S9 in the S9-mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 Quality Control and Production Certificates are available in the study report. - Test concentrations with justification for top dose:
- Experiment 1: The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were originally assayed in triplicate against each tester strain.
However, the test item induced excessive toxicity (resulting in an insufficient number of non-toxic dose concentrations) to all of the tester strains dosed in the absence of metabolic activation and, therefore, part of the experiment was repeated using an amended dose range of 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate.
Experiment 2:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
All tester strains (absence of S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 μg/plate.
All tester strains (presence of S9 mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide (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 in-house. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-aminoanthracene (2AA)
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments
: 2
METHOD OF TREATMENT/ EXPOSURE:
The test item was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls. For volatile substances, the pre-incubation method may increase exposure of the bacteria to the test item in comparison to the standard plate incorporation method.
TREATMENT SCHEDULE:
- Preincubation period: 20 minutes
- Exposure duration/duration of treatment:
the plates were incubated in sealed anaerobic jars or bags at 37 ± 3 °C for between 48 and 72 hours
SELECTIVE AGENT: histidine, Tryptophan deficient agar
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction of the growth of the bacterial background lawn by microscopically assessment of the plate's thinning
METHODS FOR MEASUREMENTS OF GENOTOXICIY
: presence of revertant colonies using an automated colony counting system. - Rationale for test conditions:
- In accordance with the OECD TG 471 guidelines.
- 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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- see tables 3 to 6
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA98 and TA100 rfa, uvrB, pKM101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- see tables 3 to 6
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA1535 and TA1537, rfa, uvrB
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- see tables 3 to 6
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No test item precipitate was
observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: Results for the negative controls (spontaneous mutation rates) were considered to be acceptable (see tables 1 and 2 below). 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.
Ames test:
- Signs of toxicity: in the experiment 1, the maximum dose level of the test item was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. However, the test item induced excessive toxicity to all of the tester strains dosed in the absence of metabolic activation (S9-mix) and, therefore, part of the experiment was repeated using an amended dose range of 0.015 to 50 μg/plate.
The test item induced a toxic response with weakened bacterial background lawns noted to all of the tester strains dosed in the absence of metabolic activation (S9-mix) at and above 15 μg/plate. In the presence of metabolic activation (S9-mix), weakened bacterial background lawns were initially noted from 150 μg/plate (all of the Salmonella strains) and 500 μg/plate (WP2uvrA).
In the experiment 2, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of metabolic activation (S9-mix) from 15 μg/plate (all of the Salmonella strains) and 50 μg/plate (WP2uvrA). In the presence of metabolic activation (S9-mix), weakened bacterial background lawns were noted from 150 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535,TA98 and WP2uvrA).
- Mean number of revertant colonies per plate and standard deviation: There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in both experiments.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, as well as the number of data)
- Positive historical control data: the current positive HCD dataset is presented in the full study report
- Negative (solvent/vehicle) historical control data: The combined Vehicle and untreated control HCD dataset is presented in the full study report - Conclusions:
- 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:
ST 08 C 19 was tested to evaluate its potential mutagenicity in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA-. The study was performed according to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing and under GLP conditions.
Bacteria were exposed to the test item diluted in DMSO vehicle using the pre-incubation method in two independent experiments 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).
Due to excessive toxicity observed without metabolic activation in the experiment 1, the concentration selected for the first experiment were in the range of 0.015 to 50 µg/plate without metabolic activation and 1.5 to 5000 µg/plate with metabolic activation. The concentration range for Experiment 2 was amended following the results of Experiment 1 and was 0.015 to 50 µg/plate without metabolic activation and 0.15 to 500 μg/plate with metabolic activation. Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve four non toxic dose levels and to achieve the toxic limit of the test item.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. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Cytotoxicity was observed in all tester strains with and without metbaolic activation in both experiments (visible reduction in the growth of the bacterial background lawns).
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in both experiments.
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 bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 31-03-2021 to 11-05-2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: ICH S2(R1)
- Version / remarks:
- Guideline adopted June 2012 (ICH S2(R1) Federal Register
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- 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:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine or tryptophan locus
- Species / strain / cell type:
- E. coli WP2 uvr A
- Remarks:
- bacteria
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Remarks:
- bacteria
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: Rat liver S9 (10% liver S9 in standard co-factors)
- source of S9: Purchased from recognised supplier (dates within full study report); S9 Microsomal fractions: Lot No.’s 4370 (Experiment 1) and 4359 (Experiment 2)
- method of preparation of S9 mix: Documented in the full study report. The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A Certificate of S9 QC and Production certificate including Activity is presented in the full study report. - Test concentrations with justification for top dose:
- Experiment 1 (pre-incubation method): 0; 1.5; 5; 15; 50; 150; 500; 1500 and 5000 µg/plate.
Experiment 1-repeat (pre-incubation method): 0; 0.005; 0.015; 0.05; 0.15; 0.5; 1.5; 5; 15; 50 and 150 µg/plate.
Experiment 2 (pre-incubation method): 0; 0.005; 0.015; 0.05; 0.15; 0.5; 1.5; 5; 15; 50 and 150 µg/plate.
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were originally assayed in triplicate against each tester strain. However, the test item induced excessive toxicity (resulting in an insufficient number of non-toxic dose concentrations) to four of the tester strains dosed in the absence of S9-mix and, therefore, part of the experiment was repeated using an amended dose range as follows:
TA100: 0.005, 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 µg/plate.
WP2uvrA and TA98: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate.
TA1537: 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (dimethylsulphoxide)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed in house. DMSO was therefore selected as the vehicle.
- Other: Formulated concentrations were adjusted/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-Aminoanthracene (2AA)
- Remarks:
- With metabolic activation S9
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without metabolic activation S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Experiment 1. in medium; in agar (pre-incubation) ; Experiment 2. in medium; in agar (pre-incubation).
The test item was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls. For volatile substances, the pre-incubation method may increase exposure of the bacteria to the test item in comparison to the standard plate incorporation method.
The test item was accurately weighed and, on the day of each experiment, approximate
half-log dilutions prepared in high purity DMSO by mixing on a vortex mixer. Formulated concentrations were adjusted to allow for the stated water/impurity content (2.7%) of the test item. All test item preparation and dosing was performed under yellow safety lighting.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine (for S. typhimurium strains) or tryptophan (for E.coli strain) supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
DURATION:
Exposure duration:
Experiment 1 - All of the plates were pre-incubated in sealed, small volume containers, by application of 0.1 mL of the appropriate bacterial strain culture, 0.5 ml of phosphate buffer OR S9-mix (as appropriate) and 0.1 ml of the appropriate concentration of 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 trace amino-acid supplemented media. All of the plates were sealed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure (37 ± 3 ºC for between 48 and 72 hours) to minimize potential losses of the test item from the plates. After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A single manual count was performed due to revertant colonies spreading slightly which prevented an accurate automated count. Manual counts are considered to be equivalent to the machine counts.
Experiment 2 - As the result of Experiment 1 was considered negative, Experiment 2 was again performed using the pre-incubation method in the presence and absence of metabolic activation (S9 mix).
SELECTION AGENT (mutation assays): histidine-deficient agar
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Rationale for test conditions:
- In accordance with the OECD TG 471 guidelines.
- 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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal. - Statistics:
- Mean & Standard deviation.
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See Tables 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See Tables 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See Tables 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See Tables 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See Tables 1 and 2
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The current Positive HCD dataset is presented in the full study report.
- Negative (solvent/vehicle) historical control data: The current background spontaneous revertant counts in concurrent untreated controls and/or or vehicle controls ; historic negative controls are presented in the full study report. - Conclusions:
- In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test ST 04 C 21 was considered to be non-mutagenic.
- Executive summary:
Introduction:
The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MOE, the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, 21 July 1997 as updated in 2020, Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.
Methods:
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames pre-incubation method at 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 original dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. However, the test item induced excessive toxicity to four of the tester strains dosed in the absence of metabolic activation (S9-mix) and, therefore, part of the experiment was repeated using an amended dose range between 0.005 and 150 µg/plate, depending on tester strain type. The dose range was amended, following the results of Experiment 1, and ranged between 0.005 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non‑toxic dose levels as required by the test guideline and were selected based on the cytotoxicity noted in Experiment 1. Both experiments were performed using the pre-incubation methodology because the volatility of the test item was considered to be incompatible with the plate incorporation method. Each test was performed using fresh cultures of the bacterial strains and fresh test item formulations.
Results:
The number of revertant counts for the solvent (dimethyl sulphoxide (DMSO)) control plates were 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the metabolic activation (S9-mix) were validated.
Depending on bacterial strain type and presence or absence of S9-mix, the maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate or the toxic limit. In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawn of all of the tester strains, initially from 5 and 150 µg/plate in the absence and presence of S9‑mix, respectively.
Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) or the toxic limit was employed in the second mutation test. The test item once again induced a toxic response with a visible reduction in the growth of the bacterial background lawns noted to all of the tester strains, initially from 15 and 150 µg/plate in the absence and presence of S9‑mix, respectively.
No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix in Experiments 1 and 2.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 1.
Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 2.
Under the conditions of this test, the test item was considered to be non-mutagenic.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22-07-2019 to 31-10-2019
- 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:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: Whole blood cultures from Human male volunteer (non-smoking, 18-35y old). The volunteer has been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly
- Cytokinesis block (if used):
- Cytochalasin B at a final concentration of 4.5 %g/mL
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: MOLTOX from Trinova Biochem - from liver tissue of males Sprague-Dawley rats (5-6 weeks old), lot No 4061 expiry date 14 feb 2021
- method of preparation of S9 mix: The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 was pre-tested for acceptability by the supplier prior to purchase and was supplied with a relevant “Quality Control & Production Certificate” which is presented in the annex of the study report. - Test concentrations with justification for top dose:
- The selection of the maximum dose level for the Main Experiment (Mutation Test) was based on toxicity for all of the exposure groups in the presence and absence of S9.
test concentrations chosen for the main test:
- 4h without S9 mix: 0 to 64 µg/mL (0, 8, 16, 32, 40, 48, 56 and 64 μg/mL)
- 4h with S9 mix: 0 to 128 µg/mL (0, 16, 32, 64, 72, 80, 88, 96 and 128 μg/mL)
- 24h continuous exposure without S9 mix: 0 to 64 µg/mL (0, 8, 16, 32, 40, 48, 56 and 64 μg/mL)
The 24-hour continuous exposure group was repeated due to not meeting the maximum permissible dose level stated in the OECD 487 Test Guideline.
The dose range of test item used was 0, 16, 32, 48, 56, 64, 72, 96, 112 and 128 μg/mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in aqueous media at 20 mg/mL but was fully miscible in DMSO at 200 mg/mL in solubility checks performed in-house. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Demecolcine (DEME-C)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate for cultures treated with the test item, quadruplicate for the vehicle,
- Number of independent experiments: the results obtained in the 4h exposure without metabolic action were repeated with a second experiment in increasing the exposure time to 24h. Part of the main experiment was repeated on a separate day due not meeting the OECD Guideline requirements for determining the maximum dose level analyzed for micronuclei. Therefore, the 24-hour continuous exposure group was repeated where the maximum concentration should be limited by toxicity.
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable
- Test substance added in medium: 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: not applicable
- Exposure duration/duration of treatment: 4 or 24h
- Harvest time after the end of treatment (sampling/recovery times): after exposure with test item (or vehicle or positive control) 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.
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: cytochalasine B, used at 4.5 µg/mL, cells are treated at the end of the exposure period with the test item and during 24h
- Methods of slide preparation and staining technique used including the stain used (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 with gentle warming. Each slide was permanently labeled 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 cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
The micronucleus frequency in 1000 binucleated cells was analyzed per culture (2000 binucleated cells per concentration for the test item and positive control and 4000 binucleated cells for the vehicle controls). Cells with 1, 2 or more micronuclei were recorded and included in the total.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): 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.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- cytokinesis-block proliferation index. A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI (Cytokinesis Block Proliferation Index) value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. - Rationale for test conditions:
- In accordance with OECD 487 Test guideline.
- Evaluation criteria:
- Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase when evaluated with an appropriate trend test.
3. The results in all evaluated dose groups are within the range of the laboratory historical control data.
The test system is then considered to be unable to induce chromosome breaks and/or gain or loss.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response. - 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). 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.
The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level. - Species / strain:
- lymphocytes: From Human blood donor
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There was no significant change in pH when the test item was dosed into media
- Data on osmolality: the osmolality did not increase by more than 50 mOsm.
- Precipitation and time of the determination: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at 128 μg/mL in the presence of S9 only.
- Definition of acceptable cells for analysis: The qualitative assessment of the slides determined that the toxicity and precipitate was similar to that observed in the Cell Growth Inhibition Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item in the absence of S9 only (64 μg/mL). In the presence of S9, the maximum dose level of the test item with binucleate cells suitable for scoring was 96 μg/mL.
- Other confounding effects:
RANGE-FINDING/SCREENING STUDIES (if applicable):
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 125 μg/mL and 250 μg/mL in the 4-hour exposure groups in the absence and presence of metabolic activation (S9), respectively and at and above 62.5 μg/mL in the 24-hour continuous exposure group.
Hemolysis was observed following exposure to the test item at and above 125 μg/mL in the 4-hour exposure groups and at and above 250 μg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 31.25 μg/mL in the exposure groups in the absence of S9 and up to 62.5 μg/mL in the presence of S9. The test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment (Mutation Test) was based on toxicity for all of the exposure groups in the presence and absence of S9.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: See tables.
For all test methods and criteria for data analysis and interpretation: See tables. Also see above “Evaluation” and “Statistics” fields.
- Concentration-response relationship where possible: See tables.
- Statistical analysis: See tables.
Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: See tables.
o In the case of the cytokinesis-block method: CBPI and/or ; distribution of mono-, bi- and multi-nucleated cells : see tables.
o Other observations when applicable (complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells): see tables.
- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate: see tables
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- All the positive control items produced a statistically significant increase in the number of binucleated cells with micronuclei (statistically significant p ≤ 0.01 or were within the 95% Historic Control Data limit range presented in the full study report).
- Negative (solvent/vehicle) historical control data: All vehicle (DMSO) controls had frequencies of mono- and binucleated cells with micronuclei within the range expected for normal human lymphocytes. (Within the 95% Historic Control Data limit range presented in the full study report). - Conclusions:
- Under the conditions of this study, the registered substance was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
- Executive summary:
An in vitro micronucleus test was performed on test item ST 08 C 19 according to the requirements of OECD TG 487 under GLP conditions to assess the test item ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (1.8% S9-mix).
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 where the results indicated that the maximum concentration should be limited by toxicity for the short-term exposure groups and by precipitate in the case of the 24-hour continuous exposure group. The dose levels selected for the Main Experiment were as follows:- 4-hour without S9: 0, 8, 16, 32, 40, 48, 56, 64 µg/mL
- 4-hour with S9 (2%): 0, 16, 32, 64, 72, 80, 88, 96, 128 µg/mL
- 24-hour without S9: 0, 8, 16, 32, 40, 48, 56, 64 µg/mL
Part of the main experiment was repeated on a separate day due not meeting the OECD Guideline requirements for determining the maximum dose level analyzed for micronuclei. Therefore, the 24-hour continuous exposure group was repeated where the maximum concentration should be limited by toxicity with the following dose range:
- 24-hour without S9: 0, 16, 32, 48, 56, 64, 72, 96, 112, 128 µg/mL
All vehicle (dimethyl sulphoxide (DMSO)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, 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 and biologically relevant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that either induced approximately 50% reduction in CBPI or was the lowest precipitating dose level, depending on exposure group.
The test item, ST 08 C 19 was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro with and without metabolic activation.
Referenceopen allclose all
Table 1: Spontaneous Mutation Rates (Concurrent Negative Controls) in Experiment 1
Number of revertants (mean number of colonies per plate) | |||||
Base-pair substitution type | Frameshift type | ||||
TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |
96 | 29 | 34 | 26 | 29 | |
103 (104) | 23 (23) | 34 (35) | 39 | (34) | 25 (25) |
112 | 16 | 38 | 36 | 21 | |
147 | 21 | 38 | 44 | 15 | |
120 (135)† | 15 (20)† | 36 (38)† | 31 | (35)† | 13 (13)† |
138 | 24 | 40 | 30 | 10 |
Table 2: Spontaneous Mutation Rates (Concurrent Negative Controls) in Experiment 2
Number of revertants (mean number of colonies per plate) | |||||||
Base-pair substitution type | Frameshift type | ||||||
TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||
131 | 9 | 23 | 27 | 15 | |||
112 (120) | 14 | (14) | 28 (26) | 24 | (23) | 9 | (11) |
116 | 20 | 27 | 18 | 9 |
Table 3: Results of Experiment 1 - without metabolic activation (pre-incubation method)
Test Period | From: 01 August 2019 | To: 04 August 2019 | |||||
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) | 130 (133) 131 4.9# 139 | 17 (16) 15 1.0 16 | 44 (33) 27 9.3 29 | 24 (27) 30 3.1 26 | 13 (14) 14 0.6 14 | ||
0.015 µg | 141 134 (141) 148 7.0 | 13 16 (15) 15 1.5 | 29 40 (33) 29 6.4 | 25 30 (28) 29 2.6 | 13 13 (16) 23 5.8 | ||
0.05 µg | 146 127 (134) 130 10.2 | 14 17 (16) 18 2.1 | 25 17 (21) 22 4.0 | 29 24 (25) 22 3.6 | 9 10 (11) 15 3.2 | ||
0.15 µg | 126 164 (153) 168 23.2 | 15 18 (16) 16 1.5 | 31 34 (32) 32 1.5 | 27 33 (30) 31 3.1 | 19 13 (15) 13 3.5 | ||
0.5 µg | 147 154 (146) 138 8.0 | 8 22 (13) 10 7.6 | 37 33 (33) 30 3.5 | 29 28 (28) 26 1.5 | 16 22 (17) 12 5.0 | ||
1.5 µg | 162 (148) 135 13.5 148 | 12 (14) 17 2.5 14 | 48 (41) 28 11.5 48 | 35 (32) 27 4.6 35 | 18 (14) 11 3.6 13 | ||
5 µg | 160 (153) 143 8.9 156 | 14 (15) 22 6.1 10 | 34 (34) 31 3.0 37 | 36 (32) 35 6.7 24 | 12 (15) 10 6.4 22 | ||
15 µg | 143 S 158 S (158) 172 S 14.5 | 11 S 17 S (14) 15 S 3.1 | 31 S 37 S (33) 31 S 3.5 | 38 S 39 S (35) 29 S 5.5 | 16 S 14 S (15) 14 S 1.2 | ||
50 µg | 0 V 0 V (0) 0 V 0.0 | 17 S 11 S (14) 13 S 3.1 | 10 V 12 V (13) 16 V 3.1 | 26 S 33 S (30) 32 S 3.8 | 0 T 0 T (0) 0 T 0.0 |
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation
Table 4: Results of Experiment 1 - with metabolic activation (pre-incubation method)
Test Period | From: 25 July 2019 | To: 28 July 2019 | |||||
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) | 95 (91) 86 4.5# 91 | 15 (13) 9 3.8 16 | 40 (41) 44 2.3 40 | 26 (31) 30 6.1 38 | 21 (22) 21 1.2 23 | ||
1.5 µg | 92 83 (87) 85 4.7 | 17 16 (14) 8 4.9 | 46 31 (36) 30 9.0 | 36 40 (35) 28 6.1 | 24 16 (19) 16 4.6 | ||
5 µg | 96 98 (95) 90 4.2 | 29 17 (20) 13 8.3 | 55 37 (48) 52 9.6 | 32 37 (32) 27 5.0 | 26 21 (24) 25 2.6 | ||
15 µg | 90 107 (101) 106 9.5 | 21 24 (20) 16 4.0 | 56 45 (48) 42 7.4 | 36 29 (32) 31 3.6 | 25 24 (25) 25 0.6 | ||
50 µg | 91 103 (94) 88 7.9 | 16 27 (19) 14 7.0 | 44 42 (43) 44 1.2 | 33 34 (31) 25 4.9 | 22 32 (26) 24 5.3 | ||
150 µg | 0 V (0) 0 V 0.0 0 V | 18 S (18) 23 S 4.5 14 S | 41 (37) 37 3.5 34 | 32 S (29) 28 S 2.6 27 S | 15 S (19) 27 S 6.9 15 S | ||
500 µg | 0 T (0) 0 T 0.0 0 T | 0 T (0) 0 T 0.0 0 T | 37 S (40) 47 S 5.8 37 S | 0 V (0) 0 V 0.0 0 V | 0 V (0) 0 V 0.0 0 V | ||
1500 µg | 0 T 0 T (0) 0 T 0.0 | 0 T 0 T (0) 0 T 0.0 | 35 S 45 S (37) 32 S 6.8 | 0 V 0 V (0) 0 V 0.0 | 0 T 0 T (0) 0 T 0.0 | ||
5000 µg | 0 T 0 T (0) 0 T 0.0 | 0 T 0 T (0) 0 T 0.0 | 56 S 57 S (55) 52 S 2.6 | 0 T 0 T (0) 0 T 0.0 | 0 T 0 T (0) 0 T 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 | |||
1571 (1494) 1522 93.6 1390 | 241 (273) 260 40.1 318 | 138 (148) 155 9.1 152 | 121 (116) 101 13.6 127 | 165 (172) 170 7.6 180 |
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation
Table 5: Results of Experiment 2 - without metabolic activation (pre-incubation method)
Test Period | From: 08 August 2019 | To: 11 August 2019 | |||||
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) | 113 (113) 122 9.5# 103 | 11 (11) 9 2.0 13 | 23 (25) 23 4.0 30 | 26 (25) 17 8.0 33 | 16 (12) 10 3.5 10 | ||
0.015 µg | 113 110 (110) 106 3.5 | 13 8 (12) 16 4.0 | 35 17 (29) 35 10.4 | 19 17 (20) 25 4.2 | 12 6 (9) 9 3.0 | ||
0.05 µg | 129 119 (121) 115 7.2 | 12 11 (11) 10 1.0 | 29 23 (31) 41 9.2 | 27 31 (25) 17 7.2 | 4 7 (7) 11 3.5 | ||
0.15 µg | 100 123 (112) 113 11.5 | 11 6 (9) 11 2.9 | 38 24 (31) 30 7.0 | 25 29 (25) 20 4.5 | 11 6 (9) 11 2.9 | ||
0.5 µg | 108 98 (103) 102 5.0 | 5 15 (8) 5 5.8 | 23 23 (24) 26 1.7 | 13 13 (19) 32 11.0 | 18 20 (16) 11 4.7 | ||
1.5 µg | 114 (111) 109 2.5 111 | 10 (10) 13 2.5 8 | 30 (29) 31 3.2 25 | 25 (27) 16 12.1 40 | 12 (12) 9 3.5 16 | ||
5 µg | 111 (111) 101 9.5 120 | 14 (10) 8 3.5 8 | 24 (20) 21 4.0 16 | 20 (20) 21 1.0 19 | 14 (12) 11 1.7 11 | ||
15 µg | 86 S 86 S (85) 83 S 1.7 | 11 S 9 S (11) 13 S 2.0 | 31 25 (27) 26 3.2 | 15 S 33 S (23) 21 S 9.2 | 7 S 9 S (8) 8 S 1.0 | ||
50 µg | 89 S 70 S (84) 93 S 12.3 | 10 S 6 S (8) 9 S 2.1 | 15 S 29 S (26) 35 S 10.3 | 0 V 0 V (0) 0 V 0.0 | 0 V 0 V (0) 0 V 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 | |||
376 (344) 258 75.3 398 | 769 (1341) 2145 716.6 1110 | 517 (498) 499 19.5 478 | 156 (181) 186 23.4 202 | 211 (187) 201 33.3 149 |
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
S Sparse bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation
Table 6: Results of Experiment 2 - with metabolic activation (pre-incubation method)
Test Period | From: 08 August 2019 | To: 11 August 2019 | |||||
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 (104) 101 6.7# 112 | 14 (13) 14 2.3 10 | 31 (41) 48 8.7 43 | 26 (27) 29 1.5 27 | 18 (14) 11 3.6 13 | ||
0.15 µg | 115 106 (104) 92 11.6 | 6 14 (10) 11 4.0 | 36 46 (42) 45 5.5 | 30 25 (25) 21 4.5 | 12 10 (11) 10 1.2 | ||
0.5 µg | 94 112 (101) 98 9.5 | 14 15 (13) 11 2.1 | 29 40 (39) 49 10.0 | 27 31 (26) 19 6.1 | 14 7 (10) 8 3.8 | ||
1.5 µg | 80 102 (105) 133 26.6 | 18 12 (16) 17 3.2 | 41 47 (48) 57 8.1 | 21 23 (22) 21 1.2 | 15 12 (13) 12 1.7 | ||
5 µg | 108 130 (117) 113 11.5 | 6 8 (9) 13 3.6 | 47 42 (46) 48 3.2 | 17 28 (27) 35 9.1 | 17 18 (18) 19 1.0 | ||
15 µg | 116 (118) 119 1.5 118 | 9 (7) 7 2.5 4 | 50 (45) 39 5.6 46 | 33 (29) 27 3.5 27 | 9 (12) 14 2.6 13 | ||
50 µg | 98 (110) 122 12.0 111 | 21 (13) 11 7.6 6 | 32 (39) 43 5.9 41 | 29 (25) 19 5.1 26 | 17 (12) 13 5.6 6 | ||
150 µg | 64 S 67 S (66) 66 S 1.5 | 11 9 (13) 18 4.7 | 34 42 (37) 35 4.4 | 35 34 (31) 24 6.1 | 6 S 7 S (7) 9 S 1.5 | ||
500 µg | 0 T 0 T (0) 0 T 0.0 | 11 S 6 S (7) 5 S 3.2 | 40 S 32 S (35) 33 S 4.4 | 0 V 0 V (0) 0 V 0.0 | 0 V 0 V (0) 0 V 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 | |||
1306 (1366) 1397 52.3 1396 | 202 (217) 226 13.3 224 | 110 (110) 110 0.0 110 | 154 (159) 151 11.4 172 | 131 (147) 145 17.6 166 |
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation
Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) | Dose Level | Number of revertants (rounded mean) +/- SD | |||||||||
Per Plate | Base-pair substitution strains | Frameshift strains | |||||||||
TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
Solvent Control | 89 | -84 | 9 | -11 | 17 | -15 | 19 | -19 | 18 | -15 | |
(DMSO) | 90 | 9.0# | 10 | 2.1 | 12 | 2.5 | 23 | 4.5 | 12 | 3.0 | |
74 | 13 | 15 | 14 | 15 | |||||||
1.5 µg | 98 | -81 | 13 | -13 | 16 | -17 | 20 | -18 | 12 | -13 | |
76 | 14.7 | 18 | 4.5 | 19 | 1.7 | 18 | 2.0 | 8 | 6.1 | ||
70 | 9 | 16 | 16 | 20 | |||||||
5 µg | 58 S | -63 | 18 | -16 | 22 | -22 | 22 | -22 | 7 | -8 | |
68 S | 5.0 | 18 | 2.9 | 20 | 2.5 | 22 | 0.0 | 8 | 1.5 | ||
63 S | 13 | 25 | 22 | 10 | |||||||
15 µg | 65 S | -66 | 12 | -12 | 12 S | -18 | 17 S | -16 | 6 | -8 | |
67 S | 1.0 | 16 | 4.5 | 24 S | 6.0 | 14 S | 2.1 | 12 | 3.2 | ||
66 S | 7 | 19 S | 18 S | 7 | |||||||
50 µg | 41 S | -43 | 17 | -10 | 9 S | -12 | 10 S | -14 | 5 S | -7 | |
43 S | 1.5 | 8 | 6.2 | 10 S | 4.9 | 14 S | 4.0 | 12 S | 4.0 | ||
44 S | 5 | 18 S | 18 S | 5 S | |||||||
150 µg | 0 T | 0 | 0 V | 0 | 0 V | 0 | 0 V | 0 | 0 V | 0 | |
0 T | 0.0 | 0 V | 0.0 | 0 V | 0.0 | 0 V | 0.0 | 0 V | 0.0 | ||
0 T | 0 V | 0 V | 0 V | 0 V | |||||||
500 µg | 0 T | 0 | 0 T | 0 | 0 V | 0 | 0 T | 0 | 0 T | 0 | |
0 T | 0.0 | 0 T | 0.0 | 0 V | 0.0 | 0 T | 0.0 | 0 T | 0.0 | ||
0 T | 0 T | 0 V | 0 T | 0 T | |||||||
1500 µg | 0 T | 0 | 0 T | 0 | 0 V | 0 | 0 T | 0 | 0 T | 0 | |
0 T | 0.0 | 0 T | 0.0 | 0 V | 0.0 | 0 T | 0.0 | 0 T | 0.0 | ||
0 T | 0 T | 0 V | 0 T | 0 T | |||||||
5000 µg | 0 T | 0 | 0 T | 0 | 0 V | 0 | 0 T | 0 | 0 T | 0 | |
0 T | 0.0 | 0 T | 0.0 | 0 V | 0.0 | 0 T | 0.0 | 0 T | 0.0 | ||
0 T | 0 T | 0 V | 0 T | 0 T | |||||||
Positive controls | Name | ENNG | ENNG | ENNG | 4NQO | 9AA | |||||
S9-Mix (-) | Dose Level | 3 µg | 5 µg | 2 µg | 0.2 µg | 80 µg | |||||
No. of Revertants | 547 | -527 | 592 | -1184 | 548 | -483 | 238 | -219 | 131 | -145 | |
539 | 28.6 | 1575 | 521.2 | 377 | 92.4 | 200 | 19.0 | 150 | 12.7 | ||
494 | 1384 | 523 | 218 | 155 | |||||||
S9-Mix (+) | Dose Level | Number of revertants (rounded mean) +/- SD | |||||||||
Per Plate | Base-pair substitution strains | Frameshift strains | |||||||||
TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
Solvent Control | 88 | -87 | 11 | -11 | 20 | -20 | 30 | -25 | 18 | -12 | |
(DMSO) | 87 | 1.5# | 11 | 0.6 | 17 | 2.5 | 24 | 5.0 | 9 | 4.9 | |
85 | 12 | 22 | 20 | 10 | |||||||
1.5 µg | 125 | -106 | 10 | -9 | 22 | -23 | 27 | -22 | 15 | -16 | |
96 | 16.2 | 8 | 1.2 | 31 | 7.1 | 20 | 4.7 | 19 | 2.6 | ||
98 | 10 | 17 | 18 | 14 | |||||||
5 µg | 106 | -111 | 10 | -8 | 21 | -19 | 19 | -18 | 11 | -14 | |
117 | 5.7 | 9 | 2.1 | 19 | 2.0 | 22 | 4.0 | 15 | 3.1 | ||
109 | 6 | 17 | 14 | 17 | |||||||
15 µg | 115 | -100 | 16 | -12 | 31 | -23 | 26 | -23 | 14 | -13 | |
99 | 14.0 | 9 | 3.6 | 22 | 7.5 | 27 | 6.7 | 14 | 1.7 | ||
87 | 11 | 16 | 15 | 11 | |||||||
50 µg | 96 | -107 | 9 | -9 | 28 | -27 | 21 | -23 | 16 | -13 | |
120 | 12.1 | 11 | 1.5 | 37 | 10.5 | 28 | 4.0 | 10 | 3.0 | ||
106 | 8 | 16 | 21 | 13 | |||||||
150 µg | 0 T | 0 | 7 | -7 | 18 | -20 | 21 S | -19 | 17 | -12 | |
0 T | 0.0 | 5 | 1.5 | 25 | 4.0 | 23 S | 5.3 | 11 | 4.2 | ||
0 T | 8 | 18 | 13 S | 9 | |||||||
500 µg | 0 T | 0 | 9 | -9 | 15 S | -21 | 14 S | -17 | 0 T | 0 | |
0 T | 0.0 | 8 | 0.6 | 28 S | 6.7 | 17 S | 3.5 | 0 T | 0.0 | ||
0 T | 9 | 19 S | 21 S | 0 T | |||||||
1500 µg | 0 T | 0 | 12 S | -8 | 0 V | 0 | 0 V | 0 | 0 T | 0 | |
0 T | 0.0 | 6 S | 3.5 | 0 V | 0.0 | 0 V | 0.0 | 0 T | 0.0 | ||
0 T | 6 S | 0 V | 0 V | 0 T | |||||||
5000 µg | 0 T | 0 | 0 T | 0 | 0 V | 0 | 0 T | 0 | 0 T | 0 | |
0 T | 0.0 | 0 T | 0.0 | 0 V | 0.0 | 0 T | 0.0 | 0 T | 0.0 | ||
0 T | 0 T | 0 V | 0 T | 0 T | |||||||
Positive controls | Name | 2AA | 2AA | 2AA | BP | 2AA | |||||
S9-Mix (+) | Dose Level | 1 µg | 2 µg | 10 µg | 5 µg | 2 µg | |||||
No. of Revertants | 1932 | -1530 | 259 | -244 | 204 | -208 | 113 | -128 | 301 | -296 | |
1194 | 373.5 | 235 | 13.3 | 213 | 4.7 | 148 | 18.0 | 316 | 22.4 | ||
1463 | 237 | 206 | 123 | 272 |
Table 1' - Test Results: Experiment 1 (repeat) without metabolic activation and results of concurrent positive controls
S9-Mix (-) | Dose Level | Number of revertants (rounded mean) +/- SD | |||||||
Per Plate | Base-pair substitution strains | Frameshift strains | |||||||
TA100 | WP2uvrA | TA98 | TA1537 | ||||||
Solvent Control | 80 | -90 | 20 | -22 | 21 | -23 | 6 | -12 | |
(DMSO) | 113 | 19.7# | 22 | 1.5 | 22 | 2.1 | 15 | 5.5 | |
78 | 23 | 25 | 16 | ||||||
0.005 µg | 79 | -82 | N/T | N/T | N/T | ||||
84 | 2.5 | ||||||||
82 | |||||||||
0.015 µg | 79 | -87 | 26 | -23 | 13 | -13 | N/T | ||
94 | 7.6 | 20 | 3.1 | 14 | 1.0 | ||||
89 | 22 | 12 | |||||||
0.05 µg | 80 | -82 | 11 | -19 | 12 | -13 | 8 | -8 | |
96 | 12.7 | 26 | 7.5 | 12 | 1.7 | 8 | 0.6 | ||
71 | 19 | 15 | 7 | ||||||
0.15 µg | 85 | -89 | 25 | -21 | 18 | -14 | 16 | -8 | |
88 | 4.6 | 15 | 5.5 | 8 | 5.3 | 4 | 6.9 | ||
94 | 24 | 16 | 4 | ||||||
0.5 µg | 98 | -100 | 12 | -18 | 13 | -16 | 6 | -10 | |
92 | 8.6 | 21 | 5.2 | 17 | 2.3 | 6 | 6.4 | ||
109 | 21 | 17 | 17 | ||||||
1.5 µg | 97 | -93 | 14 | -19 | 15 | -16 | 8 | -8 | |
100 | 10.2 | 19 | 5.0 | 10 | 6.6 | 6 | 1.5 | ||
81 | 24 | 23 | 9 | ||||||
5 µg | 93 S | -92 | 17 | -25 | 13 | -15 | 10 | -12 | |
94 S | 2.6 | 23 | 8.6 | 24 | 8.6 | 11 | 2.1 | ||
89 S | 34 | 7 | 14 | ||||||
15 µg | 85 S | -80 | 25 S | -23 | 16 S | -13 | 3 | -13 | |
77 S | 4.2 | 26 S | 3.8 | 12 S | 2.6 | 15 | 8.7 | ||
79 S | 19 S | 11 S | 20 | ||||||
50 µg | N/T | 19 S | -16 | 13 S | -16 | 9 S | -11 | ||
15 S | 3.1 | 16 S | 3.5 | 5 S | 7.2 | ||||
13 S | 20 S | 19 S | |||||||
150 µg | N/T | N/T | N/T | 0 V | 0 | ||||
0 V | 0.0 | ||||||||
0 V | |||||||||
Positive controls | Name | ENNG | ENNG | 4NQO | 9AA | ||||
S9-Mix (-) | Dose Level | 3 µg | 2 µg | 0.2 µg | 80 µg | ||||
No. of Revertants | 466 | -484 | 518 | -450 | 191 | -204 | 104 | -105 | |
500 | 17.0 | 420 | 59.0 | 197 | 17.6 | 88 | 17.0 | ||
485 | 412 | 224 | 122 |
The original dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. However, the test item induced excessive toxicity to four of the tester strains dosed in the absence of metabolic activation (S9-mix) and, therefore, part of the experiment was repeated (Table 1') using an amended dose range between 0.005 and 150 µg/plate, depending on tester strain type. The dose range was amended, following the results of Experiment 1, and ranged between 0.005 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix.
Table 2.- Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls
S9-Mix (-) | Dose Level | Number of revertants (rounded mean) +/- SD | ||||||||||
Per Plate | Base-pair substitution strains | Frameshift strains | ||||||||||
TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | ||||||||
Solvent Control | 134 | -120 | 17 | -17 | 18 | -21 | 26 | -19 | 8 | -11 | ||
(DMSO) | 104 | 15.0# | 10 | 6.5 | 23 | 2.9 | 18 | 6.1 | 17 | 5.5 | ||
121 | 23 | 23 | 14 | 7 | ||||||||
0.005 µg | 149 | -133 | N/T | N/T | N/T | N/T | ||||||
131 | 15.1 | |||||||||||
119 | ||||||||||||
0.015 µg | 126 | -122 | N/T | 19 | -14 | 11 | -14 | N/T | ||||
119 | 3.6 | 12 | 4.0 | 11 | 4.6 | |||||||
121 | 12 | 19 | ||||||||||
0.05 µg | 108 | -115 | 23 | -18 | 13 | -16 | 13 | -18 | 6 | -9 | ||
118 | 5.8 | 14 | 4.7 | 18 | 2.9 | 20 | 4.0 | 13 | 3.8 | |||
118 | 16 | 18 | 20 | 7 | ||||||||
0.15 µg | 117 | -100 | 15 | -17 | 20 | -19 | 15 | -19 | 8 | -11 | ||
88 | 15.3 | 17 | 1.5 | 16 | 2.6 | 24 | 4.7 | 12 | 3.1 | |||
94 | 18 | 21 | 17 | 14 | ||||||||
0.5 µg | 120 | -133 | 12 | -14 | 26 | -21 | 27 | -21 | 11 | -10 | ||
111 | 31.2 | 22 | 6.8 | 14 | 6.1 | 24 | 7.4 | 8 | 2.1 | |||
169 | 9 | 22 | 13 | 12 | ||||||||
1.5 µg | 137 | -110 | 14 | -16 | 29 | -23 | 16 | -12 | 9 | -7 | ||
88 | 25.0 | 12 | 4.7 | 15 | 7.1 | 12 | 3.5 | 7 | 1.5 | |||
104 | 21 | 24 | 9 | 6 | ||||||||
5 µg | 112 | -107 | 13 | -13 | 29 | -19 | 8 | -20 | 7 | -9 | ||
112 | 8.7 | 12 | 1.0 | 13 | 9.0 | 27 | 10.4 | 9 | 1.5 | |||
97 | 14 | 14 | 25 | 10 | ||||||||
15 µg | 86 S | -99 | 13 S | -12 | 29 | -19 | 10 S | -13 | 6 | -6 | ||
90 S | 19.7 | 12 S | 0.6 | 11 | 9.2 | 21 S | 7.0 | 5 | 0.6 | |||
122 S | 12 S | 17 | 8 S | 6 | ||||||||
50 µg | N/T | 14 S | -12 | 25 S | -23 | 0 V | 0 | 0 V | 0 | |||
11 S | 1.7 | 19 S | 3.5 | 0 V | 0.0 | 0 V | 0.0 | |||||
11 S | 25 S | 0 V | 0 V | |||||||||
150 µg | N/T | 0 V | 0 | N/T | N/T | 0 V | 0 | |||||
0 V | 0.0 | 0 V | 0.0 | |||||||||
0 V | 0 V | |||||||||||
Positive controls | Name | ENNG | ENNG | ENNG | 4NQO | 9AA | ||||||
S9-Mix (-) | Dose Level | 3 µg | 5 µg | 2 µg | 0.2 µg | 80 µg | ||||||
No. of Revertants | 679 | -700 | 1451 | -1287 | 347 | -280 | 168 | -199 | 77 | -77 | ||
659 | 55.2 | 1080 | 189.3 | 355 | 123.6 | 202 | 30.1 | 78 | 0.6 | |||
763 | 1331 | 137 | 228 | 77 | ||||||||
S9-Mix (+) | Dose Level | Number of revertants (rounded mean) +/- SD | ||||||||||
Per Plate | Base-pair substitution strains | Frameshift strains | ||||||||||
TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | ||||||||
Solvent Control | 125 | -120 | 12 | -13 | 19 | -23 | 20 | -19 | 8 | -8 | ||
(DMSO) | 120 | 5.5# | 15 | 1.5 | 29 | 5.1 | 14 | 5.0 | 6 | 2.5 | ||
114 | 13 | 22 | 24 | 11 | ||||||||
0.05 µg | 130 | -118 | N/T | N/T | N/T | N/T | ||||||
121 | 13.2 | |||||||||||
104 | ||||||||||||
0.15 µg | 158 | -139 | N/T | N/T | 25 | -23 | 10 | -11 | ||||
125 | 17.2 | 21 | 2.1 | 11 | 1.0 | |||||||
133 | 22 | 12 | ||||||||||
0.5 µg | 149 | -136 | N/T | 21 | -23 | 32 | -27 | 11 | -11 | |||
130 | 11.3 | 23 | 2.0 | 25 | 4.4 | 11 | 0.0 | |||||
129 | 25 | 24 | 11 | |||||||||
1.5 µg | 131 | -124 | 11 | -12 | 23 | -24 | 19 | -19 | 9 | -11 | ||
128 | 10.2 | 13 | 1.2 | 23 | 1.7 | 16 | 2.5 | 9 | 2.9 | |||
112 | 13 | 26 | 21 | 14 | ||||||||
5 µg | 113 | -113 | 14 | -14 | 31 | -30 | 32 | -27 | 16 | -13 | ||
117 | 4.0 | 10 | 4.0 | 24 | 5.6 | 22 | 5.0 | 6 | 6.1 | |||
109 | 18 | 35 | 26 | 17 | ||||||||
15 µg | 120 | -113 | 11 | -11 | 22 | -29 | 19 | -19 | 10 | -11 | ||
115 | 8.7 | 10 | 1.5 | 37 | 7.5 | 15 | 3.5 | 12 | 1.2 | |||
103 | 13 | 28 | 22 | 12 | ||||||||
50 µg | 106 | -111 | 12 | -10 | 30 | -25 | 36 | -24 | 7 | -9 | ||
131 | 18.0 | 10 | 2.0 | 25 | 4.5 | 18 | 10.7 | 9 | 2.5 | |||
96 | 8 | 21 | 17 | 12 | ||||||||
150 µg | 162 S | -120 | 8 S | -6 | 21 | -24 | 22 S | -23 | 9 S | -12 | ||
105 S | 36.5 | 5 S | 2.1 | 25 | 3.1 | 26 S | 2.6 | 15 S | 3.1 | |||
94 S | 4 S | 27 | 21 S | 11 S | ||||||||
500 µg | N/T | 0 V | 0 | 17 S | -16 | 0 V | 0 | 0 T | 0 | |||
0 V | 0.0 | 14 S | 1.7 | 0 V | 0.0 | 0 T | 0.0 | |||||
0 V | 17 S | 0 V | 0 T | |||||||||
1500 µg | N/T | 0 T | 0 | 0 V | 0 | N/T | N/T | |||||
0 T | 0.0 | 0 V | 0.0 | |||||||||
0 T | 0 V | |||||||||||
5000 µg | N/T | 0 T | 0 | N/T | N/T | N/T | ||||||
0 T | 0.0 | |||||||||||
0 T | ||||||||||||
Positive controls | Name | 2AA | 2AA | 2AA | BP | 2AA | ||||||
S9-Mix (+) | Dose Level | 1 µg | 2 µg | 10 µg | 5 µg | 2 µg | ||||||
No. of Revertants | 1086 | -1220 | 179 | -185 | 214 | -241 | 143 | -127 | 211 | -197 | ||
1121 | 202.5 | 182 | 7.9 | 203 | 57.1 | 126 | 15.5 | 181 | 15.1 | |||
1453 | 194 | 307 | 112 | 199 |
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
N/T Not tested at this dose level
S Sparse bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation
Table 1. CBPI Data - Preliminary Toxicity Test - 4-hour exposure without Metabolic Activation (S9)
Treatment/ Concentration (µg/mL) | Mononucleate Cells | Binucleate Cells | Multinucleate Cells | CBPIc | Cytostasis (% ) | ||||||||||||||||||||||||||||||||||||||||
Vehicle (DMSO) | 264 | 219 | 17 | 1.48 | 0 | ||||||||||||||||||||||||||||||||||||||||
293 | 190 | 17 | |||||||||||||||||||||||||||||||||||||||||||
7.81 | 263 | 226 | 11 | 1.50 | 0‡ | ||||||||||||||||||||||||||||||||||||||||
15.63 | 245 | 240 | 15 | 1.54 | 0‡ | ||||||||||||||||||||||||||||||||||||||||
31.25 | 314 | 180 | 6 | 1.38 | 21 | ||||||||||||||||||||||||||||||||||||||||
62.5 | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||||
125 P H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||||
250 P H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||||
500 P H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||||
1000 P H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||||
2000 P H | NB | NB | NB | - | - |
c Mean value for vehicle
- Not selected for scoring
NB No binucleate cells or insufficient binucleate cells for scoring
P Precipitate observed at end of exposure period in blood-free cultures
H Hemolysis observed at the end of exposure in blood cultures
‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control
DMSO Dimethyl sulphoxide
Table 2: CBPI Data - Preliminary Toxicity Test - 4-hour exposure with Metabolic Activation (S9)
Treatment / Concentration (µg/mL) | Mononucleate Cells | Binucleate Cells | Multinucleate Cells | CBPIc | Cytostasis (% ) | ||||||||||||||||||||||||||||||||||||||||
Vehicle (DM SO) | 225 | 227 | 48 | 1.63 | 0 | ||||||||||||||||||||||||||||||||||||||||
235 | 231 | 34 | |||||||||||||||||||||||||||||||||||||||||||
7.81 | 211 | 244 | 45 | 1.67 | 0‡ | ||||||||||||||||||||||||||||||||||||||||
15.63 | 247 | 219 | 34 | 1.57 | 9 | ||||||||||||||||||||||||||||||||||||||||
31.25 | 205 | 266 | 29 | 1.65 | 0‡ | ||||||||||||||||||||||||||||||||||||||||
62.5 | 259 | 238 | 3 | 1.49 | 22 | ||||||||||||||||||||||||||||||||||||||||
125 H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||||
250 P H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||||
500 P H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||||
1000 P H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||||
2000 P H | NB | NB | NB | - | - |
c Mean value for vehicle
- Not selected for scoring
NB No binucleate cells or insufficient binucleate cells for scoring
P Precipitate observed at end of exposure period in blood-free cultures
H Hemolysis observed at the end of exposure in blood cultures
‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control
DMSO Dimethyl sulphoxide
Table 3: CBPI Data - Preliminary Toxicity Test - 24-hour exposure without Metabolic Activation (S9)
Treatment / Concentration (µg/mL) | Mononucleate Cells | Binucleate Cells | Multinucleate Cells | CBPIc | Cytostasis (% ) | ||||||||||||||||||||||||||||||||||||||
Vehicle (DM SO) | 144 | 274 | 82 | 1.79 | 0 | ||||||||||||||||||||||||||||||||||||||
194 | 261 | 45 | |||||||||||||||||||||||||||||||||||||||||
7.81 | 175 | 259 | 66 | 1.78 | 1 | ||||||||||||||||||||||||||||||||||||||
15.63 | 150 | 270 | 80 | 1.86 | 0‡ | ||||||||||||||||||||||||||||||||||||||
31.25 | 162 | 296 | 42 | 1.76 | 4 | ||||||||||||||||||||||||||||||||||||||
62.5 P | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||
125 P | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||
250 P H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||
500 P H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||
1000 P H | NB | NB | NB | - | - | ||||||||||||||||||||||||||||||||||||||
2000 P H | NB | NB | NB | - | - |
c Mean value for vehicle
- Not selected for scoring
NB No binucleate cells or insufficient binucleate cells for scoring
P Precipitate observed at end of exposure period in blood-free cultures
H Hemolysis observed at the end of exposure in blood cultures
‡ Cytostasis reported as 0 as the CBPI value is equal to or higher than the solvent control
DMSO Dimethyl sulphoxide
Table 4: CBPI Data – Main Experiment - 4-Hour Exposure without Metabolic Activation (S9)
Treatment/ Concentration (µg/mL) | Replicate | Mononucleate Cells | Binucleate Cells | Multinucleate Cells | CBPI | Mean CBPI | Mean Cytostasis (% ) | |||||||||||||||||||||||||||||||||||||||||||||||||
Vehicle (DMSO) | A1 | 214 | 249 | 37 | 1.65 | 1.59 | 0 | |||||||||||||||||||||||||||||||||||||||||||||||||
A2 | 240 | 234 | 26 | 1.57 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
B1 | 274 | 187 | 39 | 1.53 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
B2 | 240 | 224 | 36 | 1.59 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
8 | A | - | - | - | - | - | - | |||||||||||||||||||||||||||||||||||||||||||||||||
B | - | - | - | - | ||||||||||||||||||||||||||||||||||||||||||||||||||||
16 | A | 254 | 212 | 34 | 1.56 | 1.55 | 7 | |||||||||||||||||||||||||||||||||||||||||||||||||
B | 266 | 204 | 30 | 1.53 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
32 | A | 273 | 206 | 21 | 1.50 | 1.48 | 18 | |||||||||||||||||||||||||||||||||||||||||||||||||
B | 288 | 195 | 17 | 1.46 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
40 | A | 291 | 201 | 8 | 1.43 | 1.44 | 25 | |||||||||||||||||||||||||||||||||||||||||||||||||
B | 289 | 196 | 15 | 1.45 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
48 | A | 346 | 151 | 3 | 1.31 | 1.29 | 51 | |||||||||||||||||||||||||||||||||||||||||||||||||
B | 371 | 126 | 3 | 1.26 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
56 | A | 364 | 133 | 3 | 1.28 | 1.25 | 57 | |||||||||||||||||||||||||||||||||||||||||||||||||
B | 390 | 110 | 0 | 1.22 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
64 | A | FB | FB | FB | - | - | - | |||||||||||||||||||||||||||||||||||||||||||||||||
B | FB | FB | FB | - | ||||||||||||||||||||||||||||||||||||||||||||||||||||
MMC 0.2 | A | 296 | 194 | 10 | 1.43 | 1.47 | 20 | |||||||||||||||||||||||||||||||||||||||||||||||||
B | 250 | 248 | 2 | 1.50 |
- Not selected for scoring
FB Too few binucleate cells available for scoring
MMC Mitomycin C
DMSO Dimethyl sulphoxide
Table 5: CBPI Data – Main Experiment - 4-Hour Exposure with Metabolic Activation (S9)
Treatment/ Concentration (µg/mL) | Replicate | Mononucleate Cells | Binucleate Cells | Multinucleate Cells | CBPI | Mean CBPI | Mean Cytostasis (% ) | |||||||||||||||||||||||||||||||||||||||||||
Vehicle (DMSO) | A1 | 243 | 237 | 24 | 1.57 | 1.57 | 0 | |||||||||||||||||||||||||||||||||||||||||||
A2 | 205 | 256 | 41 | 1.67 | ||||||||||||||||||||||||||||||||||||||||||||||
B1 | 257 | 217 | 26 | 1.54 | ||||||||||||||||||||||||||||||||||||||||||||||
B2 | 288 | 217 | 22 | 1.50 | ||||||||||||||||||||||||||||||||||||||||||||||
16 | A | - | - | - | - | - | - | |||||||||||||||||||||||||||||||||||||||||||
B | - | - | - | - | ||||||||||||||||||||||||||||||||||||||||||||||
32 | A | - | - | - | - | - | - | |||||||||||||||||||||||||||||||||||||||||||
B | - | - | - | - | ||||||||||||||||||||||||||||||||||||||||||||||
64 | A | - | - | - | - | - | - | |||||||||||||||||||||||||||||||||||||||||||
B | - | - | - | - | ||||||||||||||||||||||||||||||||||||||||||||||
72 | A | 271 | 219 | 10 | 1.48 | 1.43 | 25 | |||||||||||||||||||||||||||||||||||||||||||
B | 316 | 181 | 3 | 1.37 | ||||||||||||||||||||||||||||||||||||||||||||||
80 | A | 266 | 230 | 4 | 1.48 | 1.44 | 23 | |||||||||||||||||||||||||||||||||||||||||||
B | 311 | 186 | 7 | 1.40 | ||||||||||||||||||||||||||||||||||||||||||||||
88 | A | 373 | 126 | 1 | 1.26 | 1.32 | 44 | |||||||||||||||||||||||||||||||||||||||||||
B | 313 | 184 | 3 | 1.38 | ||||||||||||||||||||||||||||||||||||||||||||||
96 | A | 358 | 142 | 0 | 1.28 | 1.26 | 55 | |||||||||||||||||||||||||||||||||||||||||||
B | 390 | 118 | 0 | 1.23 | ||||||||||||||||||||||||||||||||||||||||||||||
128 P | A | NB | NB | NB | - | - | - | |||||||||||||||||||||||||||||||||||||||||||
B | NB | NB | NB | - | ||||||||||||||||||||||||||||||||||||||||||||||
CP 5 | A | 345 | 157 | 0 | 1.31 | 1.30 | 47 | |||||||||||||||||||||||||||||||||||||||||||
B | 359 | 139 | 2 | 1.29 |
- Not selected for scoring
P Precipitate observed at the end of exposure period in blood-free cultures
NB No binucleates cells available for scoring
CP Cyclophosphamide
DMSO Dimethyl sulphoxide
Table 6: CBPI Data – Main Experiment - 24-Hour Exposure without Metabolic Activation (S9)
Treatment/ Concentration (µg/mL) | Replicate | Mononucleate Cells | Binucleate Cells | Multinucleate Cells | CBPI | Mean CBPI | Mean Cytostasis (% ) | ||||||||||||||||||||||||||||||||||||||||||
Vehicle (DMSO) | A1 | 196 | 287 | 17 | 1.64 | 1.72 | 0 | ||||||||||||||||||||||||||||||||||||||||||
A2 | 142 | 342 | 16 | 1.75 | |||||||||||||||||||||||||||||||||||||||||||||
B1 | 171 | 315 | 14 | 1.69 | |||||||||||||||||||||||||||||||||||||||||||||
B2 | 123 | 354 | 23 | 1.80 | |||||||||||||||||||||||||||||||||||||||||||||
8 | A | - | - | - | - | - | - | ||||||||||||||||||||||||||||||||||||||||||
B | - | - | - | - | |||||||||||||||||||||||||||||||||||||||||||||
16 | A | - | - | - | - | - | - | ||||||||||||||||||||||||||||||||||||||||||
B | - | - | - | - | |||||||||||||||||||||||||||||||||||||||||||||
32 | A | - | - | - | - | - | - | ||||||||||||||||||||||||||||||||||||||||||
B | - | - | - | - | |||||||||||||||||||||||||||||||||||||||||||||
40 | A | - | - | - | - | - | - | ||||||||||||||||||||||||||||||||||||||||||
B | - | - | - | - | |||||||||||||||||||||||||||||||||||||||||||||
48 | A | 201 | 281 | 18 | 1.63 | 1.68 | 6 | ||||||||||||||||||||||||||||||||||||||||||
B | 156 | 328 | 16 | 1.72 | |||||||||||||||||||||||||||||||||||||||||||||
56 | A | 195 | 301 | 4 | 1.62 | 1.65 | 10 | ||||||||||||||||||||||||||||||||||||||||||
B | 180 | 310 | 12 | 1.67 | |||||||||||||||||||||||||||||||||||||||||||||
64 | A | 244 | 256 | 3 | 1.52 | 1.52 | 28 | ||||||||||||||||||||||||||||||||||||||||||
B | 247 | 250 | 3 | 1.51 | |||||||||||||||||||||||||||||||||||||||||||||
DC 0.075 | A | 330 | 167 | 3 | 1.35 | 1.37 | 49 | ||||||||||||||||||||||||||||||||||||||||||
B | 319 | 172 | 9 | 1.38 |
- Not selected for scoring
DC Demecolcine
DMSO Dimethyl sulphoxide
Table 7: CBPI Data – Main Experiment Repeat - 24-Hour Exposure without Metabolic Activation (S9)
Treatment/ Concentration (µg/mL) | Replicate | Mononucleate Cells | Binucleate Cells | Multinucleate Cells | CBPI | Mean CBPI | Mean Cytostasis (% ) | |||||||||||||||||||||||||||||||||||||||||
Vehicle (DMSO) | A1 | 165 | 235 | 100 | 1.87 | 1.80 | 0 | |||||||||||||||||||||||||||||||||||||||||
A2 | 220 | 214 | 66 | 1.69 | ||||||||||||||||||||||||||||||||||||||||||||
B1 | 160 | 245 | 95 | 1.87 | ||||||||||||||||||||||||||||||||||||||||||||
B2 | 193 | 227 | 80 | 1.77 | ||||||||||||||||||||||||||||||||||||||||||||
16 | A | - | - | - | - | - | - | |||||||||||||||||||||||||||||||||||||||||
B | - | - | - | - | ||||||||||||||||||||||||||||||||||||||||||||
32 | A | - | - | - | - | - | - | |||||||||||||||||||||||||||||||||||||||||
B | - | - | - | - | ||||||||||||||||||||||||||||||||||||||||||||
48 | A | 201 | 247 | 52 | 1.70 | 1.68 | 15 | |||||||||||||||||||||||||||||||||||||||||
B | 223 | 224 | 53 | 1.66 | ||||||||||||||||||||||||||||||||||||||||||||
56 | A | 234 | 232 | 34 | 1.60 | 1.60 | 25 | |||||||||||||||||||||||||||||||||||||||||
B | 235 | 231 | 34 | 1.60 | ||||||||||||||||||||||||||||||||||||||||||||
64 | A | 277 | 194 | 29 | 1.50 | 1.49 | 39 | |||||||||||||||||||||||||||||||||||||||||
B | 278 | 204 | 18 | 1.48 | ||||||||||||||||||||||||||||||||||||||||||||
72 H | A | 330 | 168 | 2 | 1.34 | 1.35 | 56 | |||||||||||||||||||||||||||||||||||||||||
B | 321 | 176 | 3 | 1.36 | ||||||||||||||||||||||||||||||||||||||||||||
96 H | A | 411 | 88 | 1 | 1.18 | 1.16 | 81 | |||||||||||||||||||||||||||||||||||||||||
B | 437 | 63 | 0 | 1.13 | ||||||||||||||||||||||||||||||||||||||||||||
112 H | A | FB | FB | FB | - | - | - | |||||||||||||||||||||||||||||||||||||||||
B | FB | FB | FB | - | ||||||||||||||||||||||||||||||||||||||||||||
128 H | A | NB | NB | NB | - | - | - | |||||||||||||||||||||||||||||||||||||||||
B | NB | NB | NB | - | ||||||||||||||||||||||||||||||||||||||||||||
DC 0.075 | A | 322 | 155 | 23 | 1.40 | 1.40 | 50 | |||||||||||||||||||||||||||||||||||||||||
B | 334 | 132 | 34 | 1.40 |
- Not selected for scoring
H Haemolysis observed at the end of exposure period
FB Few binucleate cells available for scoring
NB No binucleate cells available for scoring
DC Demecolcine
DMSO Dimethyl sulphoxide
Table 8: Cytostasis and Micronucleus Data – Main Experiment - 4-Hour Exposure without Metabolic Activation (S9)
Treatment/ Concentration (μg/mL) | Replicate | Mean Cytostasis (% ) | Binucleated cells containing micronuclei | |||||||||||||||||||||||||||||||||||||||||||
% | Mean | p -valueb | Trend test p -valued | |||||||||||||||||||||||||||||||||||||||||||
Vehicle (DMSO) | A1 | 0 | 0.30 | 0.33 | - | 0.451 | ||||||||||||||||||||||||||||||||||||||||
A2 | 0.40 | |||||||||||||||||||||||||||||||||||||||||||||
B1 | 0.40 | |||||||||||||||||||||||||||||||||||||||||||||
B2 | 0.20 | |||||||||||||||||||||||||||||||||||||||||||||
40 | A | 25 | 0.00 | 0.35 | - | |||||||||||||||||||||||||||||||||||||||||
B | 0.70 | |||||||||||||||||||||||||||||||||||||||||||||
48 | A | 51 | 0.20 | 0.25 | - | |||||||||||||||||||||||||||||||||||||||||
B | 0.30 | |||||||||||||||||||||||||||||||||||||||||||||
56 | A | 57 | 0.00 | 0.25 | - | |||||||||||||||||||||||||||||||||||||||||
B | 0.50 | |||||||||||||||||||||||||||||||||||||||||||||
MMC 0.2 | A | 20 | 3.00 | 3.10 | 7.49E-20 *** | |||||||||||||||||||||||||||||||||||||||||
B | 3.20 |
b p-values are for comparison with the control using Chi-square test
d Linear Regression value calculated from individual Arcsin values
MMC Mitomycin C
*** P<0.001
DMSO Dimethyl sulphoxide
Table 9: Cytostasis and Micronucleus Data – Main Experiment - 4-Hour Exposure with Metabolic Activation (S9)
Treatment/ Concentration (μg/mL) | Replicate | % Cytostasis | Binucleated cells containing micronuclei | |||||||||||||||||||||||||||||||||||||||||||||
% | Mean | p -valueb | Trend test p -valued | |||||||||||||||||||||||||||||||||||||||||||||
Vehicle (DMSO) | A1 | 0 | 0.00 | 0.28 | - | 0.238 | ||||||||||||||||||||||||||||||||||||||||||
A2 | 0.60 | |||||||||||||||||||||||||||||||||||||||||||||||
B1 | 0.30 | |||||||||||||||||||||||||||||||||||||||||||||||
B2 | 0.20 | |||||||||||||||||||||||||||||||||||||||||||||||
80 | A | 23 | 0.70 | 0.65 | 0.0301* | |||||||||||||||||||||||||||||||||||||||||||
B | 0.60 | |||||||||||||||||||||||||||||||||||||||||||||||
88 | A | 44 | 0.50 | 0.45 | 0.2676 | |||||||||||||||||||||||||||||||||||||||||||
B | 0.40 | |||||||||||||||||||||||||||||||||||||||||||||||
96 | A | 55 | 0.10 | 0.35 | 0.6165 | |||||||||||||||||||||||||||||||||||||||||||
B | 0.60 | |||||||||||||||||||||||||||||||||||||||||||||||
CP 5 | A | 47 | 2.90 | 2.65 | 3.12E-17 *** | |||||||||||||||||||||||||||||||||||||||||||
B | 2.40 |
b p-values are for comparison with the control using Chi-square test
d Linear Regression value calculated from individual Arcsin values
CP Cyclophosphamide
* P<0.05
*** P<0.001
DMSO Dimethyl sulphoxide
Table 10: Cytostasis and Micronucleus Data – Main Experiment - 24-Hour Exposure without Metabolic Activation (S9)
Treatment/ Concentration (μg/mL) | Replicate | Mean Cytostasis (% ) | Binucleated cells containing micronuclei | |||||||||||||||||||||||||||||||||||||||||
% | Mean | p -valueb | Trend test p -valued | |||||||||||||||||||||||||||||||||||||||||
Vehicle (DMSO) | A1 | 0 | 0.40 | 0.43 | - | 0.563 | ||||||||||||||||||||||||||||||||||||||
A2 | 0.40 | |||||||||||||||||||||||||||||||||||||||||||
B1 | 0.40 | |||||||||||||||||||||||||||||||||||||||||||
B2 | 0.50 | |||||||||||||||||||||||||||||||||||||||||||
48 | A | 6 | 0.20 | 0.55 | - | |||||||||||||||||||||||||||||||||||||||
B | 0.90 | |||||||||||||||||||||||||||||||||||||||||||
56 | A | 10 | 0.20 | 0.15 | - | |||||||||||||||||||||||||||||||||||||||
B | 0.10 | |||||||||||||||||||||||||||||||||||||||||||
64 P | A | 28 | 0.20 | 0.45 | - | |||||||||||||||||||||||||||||||||||||||
B | 0.70 | |||||||||||||||||||||||||||||||||||||||||||
DC 0.075 | A | 49 | 3.90 | 4.40 | 1.77E-28 *** | |||||||||||||||||||||||||||||||||||||||
B | 4.90 |
b p-values are for comparison with the control using Chi-square test
d Trend test p-values using Linear regression model applied to control and test item concentrations
P Precipitate
DC Demecolcine
DMSO Dimethyl sulphoxide
*** P<0.001
Table 11: Cytostasis and Micronucleus Data – Main Experiment Repeat- 24-Hour Exposure without Metabolic Activation (S9)
Treatment/ Concentration (μg/mL) | Replicate | Mean Cytostasis (% ) | Binucleated cells containing micronuclei | |||||||||||||||||||||||||||||||||||||||||||||
% | Mean % | p -valueb | Trend test p -valued | |||||||||||||||||||||||||||||||||||||||||||||
Vehicle (DMSO) | A1 | 0 | 0.20 | 0.35 | - | 0.222 | ||||||||||||||||||||||||||||||||||||||||||
A2 | 0.20 | |||||||||||||||||||||||||||||||||||||||||||||||
B1 | 0.90 | |||||||||||||||||||||||||||||||||||||||||||||||
B2 | 0.10 | |||||||||||||||||||||||||||||||||||||||||||||||
56 | A | 25 | 0.20 | 0.65 | 0.1017 | |||||||||||||||||||||||||||||||||||||||||||
B | 1.10 | |||||||||||||||||||||||||||||||||||||||||||||||
64 | A | 39 | 0.60 | 0.60 | 0.1646 | |||||||||||||||||||||||||||||||||||||||||||
B | 0.60 | |||||||||||||||||||||||||||||||||||||||||||||||
72 | A | 56 | 0.20 | 0.35 | - | |||||||||||||||||||||||||||||||||||||||||||
B | 0.50 | |||||||||||||||||||||||||||||||||||||||||||||||
DC 0.075 | A | 50 | 7.20 | 5.95 | 7.37E-44 *** | - | ||||||||||||||||||||||||||||||||||||||||||
B | 4.70 |
b p-values are for comparison with the control using Chi-square test
d Linear Regression value calculated from individual Arcsin values
DC Demecolcine
DMSO Dimethyl sulphoxide
*** P<0.001
Table 12: Summary of Results
Exposure Condition | Treatment/ Concentration (μg/mL) | Cytostasis (% ) | % Binucleated cells containing micronucleia | ||||||||||||||||||||||||||||||||||||||
Mean | p -valueb | Trend test p -valued | |||||||||||||||||||||||||||||||||||||||
4-hour -S9 | DMSO | 0 | 0.33 | - | 0.451 | ||||||||||||||||||||||||||||||||||||
40 | 25 | 0.35 | - | ||||||||||||||||||||||||||||||||||||||
48 | 51 | 0.25 | - | ||||||||||||||||||||||||||||||||||||||
56 | 57 | 0.25 | - | ||||||||||||||||||||||||||||||||||||||
MMC 0.2 | 20 | 3.10 | 7.49E-20*** | - | |||||||||||||||||||||||||||||||||||||
4-hour +S9 | DMSO | 0 | 0.28 | - | 0.238 | ||||||||||||||||||||||||||||||||||||
80 | 23 | 0.65 | 0.0301* | ||||||||||||||||||||||||||||||||||||||
88 | 44 | 0.45 | 0.2676 | ||||||||||||||||||||||||||||||||||||||
96 | 55 | 0.35 | 0.6165 | ||||||||||||||||||||||||||||||||||||||
CP 5 | 47 | 2.65 | 3.12E-17*** | - | |||||||||||||||||||||||||||||||||||||
24-hour -S9 | DMSO | 0 | 0.43 | - | 0.563 | ||||||||||||||||||||||||||||||||||||
48 | 6 | 0.46 | - | ||||||||||||||||||||||||||||||||||||||
56 | 10 | 0.15 | - | ||||||||||||||||||||||||||||||||||||||
64 | 28 | 0.45 | - | ||||||||||||||||||||||||||||||||||||||
DC 0.075 | 49 | 4.40 | 1.77E-28 *** | - | |||||||||||||||||||||||||||||||||||||
24-hour -S9 Repeat | DMSO | 0 | 0.35 | - | 0.222 | ||||||||||||||||||||||||||||||||||||
56 | 25 | 0.65 | 0.1017 | ||||||||||||||||||||||||||||||||||||||
64 | 39 | 0.60 | 0.1646 | ||||||||||||||||||||||||||||||||||||||
72 | 56 | 0.35 | - | ||||||||||||||||||||||||||||||||||||||
DC 0.075 | 50 | 5.95 | 7.37E-44*** | - |
a The number of micronucleated cells determined in a sample of 2000 binucleate cells (4000 for vehicle)
b p-values are for comparison with the control using Chi-square test
d Linear Regression value calculated from individual Arcsin values
P Precipitate observed at end of exposure period in blood-free cultures
MMC Mitomycin C
CP Cyclophosphamide
DC Demecolcine
* P<0.05
*** P<0.001
DMSO Dimethyl sulphoxide
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
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