Registration Dossier
Registration Dossier
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EC number: 601-472-6 | CAS number: 117314-20-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Clearly negative in vitro studies - both with and without metabolic activation.
Gene mutation (Bacterial reverse mutation assay / Ames test, in vitro) -
(GLP, OECD TG 471, EU method B13/14) : S. typhimurium TA 100, TA98,
TA1535, TA1537 and E. coli WP2uvrA: negative with and without metabolic
activation, 9 concentrations (0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000
µg/plate)
Mouse lymphoma assay (in vitro) - (GLP, OECD 476, EU Method B17):
negative with and without metabolic activation (TK +/- locus in mouse
lymphoma L5178Y cells), 7 concentrations (0, 156.25, 312.5, 625, 1250,
2500 and 5000 μg/mL)
Chromosome aberration test (human lymphocytes, in vitro) - (GLP, OECD 473) : negative with and without metabolic activation, 8 concentrations (0, 0.09, 0.188, 0.375, 0.75, 1.25, 2.5 and 5 µg/ml).
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 8.10.2014-6.11.2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The study was performed according to the recommended Guidelines (OECD Guidelines for Testing of Chemicals No. 471 (1997) "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, USA, EPA OCSPP harmonized guideline 870.5100 - Bacterial Reverse Mutation Test, Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries) and GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- yes
- Remarks:
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- (EC) 440/2008 of 30 May 2008
- Deviations:
- yes
- Remarks:
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Certificate is included as an attachment to the study report.
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Type of mutations indicated: frame shift mutations, base-pair substitutions
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- other: Genotype: trp-; uvrA-:; Type of mutations indicated: base-pair substitution
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: Genotype: his C 3076; rfa-; uvrB-: /his D 3052; rfa-; uvrB-;R-factor: /his G 46; rfa-; uvrB-: /his G 46; rfa-; uvrB-;R-factor; Type of mutations indicated: frame shift mutations/base-pair substitutions
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- The test item was tested using the following method. The maximum concentration was 5000 ug/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Sterile, distilled water
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in sterile distilled water, therefore, this solvent was selected as the vehicle. - Untreated negative controls:
- yes
- Remarks:
- Negative (untreated) controls were used in parallel with the test item.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Culture with vehicle (sterile, distilled water)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- 2-Aminoanthracene and benzo(a)pyrene were used in the series of plates with S9-mix
- Details on test system and experimental conditions:
- Experiment 1.
DOSES: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate were assayed in triplicate against each tester strain, using the direct plate incorporation method.
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h (incubation at 37 C± 3 C)
NUMBER OF REPLICATIONS: 3
OTHER EXAMINATIONS:
All of 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).
Experiment 2.
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
METHOD OF APPLICATION: preincubation
DOSES: 50, 150, 500, 1500 and 5000 ug/plate were assayed in triplicate against each tester strain, using the pre-incubation method.
DURATION
- Exposure duration: 20 min preincubation (37 C± 3 C with shaking), 48 h incubation (37 C± 3 C)
NUMBER OF REPLICATIONS: 3
OTHER EXAMINATIONS:
All of 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). - Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item 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:
- The individual plate counts, the mean number of revertant colonies and the standard deviations were determined for the test item, positive and vehicle controls, both with and without metabolic activation.
The analyses were appropriate to determine the mutagenicity of the test item.
A history profile of vehicle, untreated and positive control values (reference items) are also presented. - Species / strain:
- other: S. typhimurium strains TA1535, TA1537, TA98 and TA100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative 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:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Insoluble in water. However, the test item formed the best doseable suspension in sterile distilled water, therefore, this solvent was selected as the vehicle.
- Precipitation: test item precipitate (greasy in appearance) was observed at and above 1500 ug/plate. This observation did not prevent the scoring of revertant colonies.
COMPARISON WITH HISTORICAL CONTROL DATA: See table 1 - Remarks on result:
- other: all strains tested
- Conclusions:
- Interpretation of results: negative. Not classified according to the CLP Regulation based on the Reverse Mutation Assay 'Ames Test' using Salmonella
typhimurium and Escherichia coli. The test item was considered to be non-mutagenic under the conditions of the test. - Executive summary:
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 ug/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 50 to 5000 μg/plate.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of S9-mix, in the second mutation test (pre-incubation method). A test item precipitate (greasy in appearance) was observed at and above 1500 g/plate, this observation did not prevent the scoring of revertant colonies.
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 S9-mix in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant
colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 2 (pre-incubation method).
The substance was considered to be non-mutagenic under the conditions of the test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 31.3.2015-9.6.2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The study was performed following the recommended Guidelines (OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Test" and Method B17 of Commission Regulation (EC) No. 440/2008) and GLP.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- (EC) No. 440/2008 of 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Adopted 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Cerificate is included in the study report as an attachment
- Type of assay:
- other: In vitro gene mutation study in mammalian cells
- Target gene:
- Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Suitability of cells: The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al., 1980; Jotz and Mitchell, 1981).
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours and were subcultured accordingly.
MEDIA USED
- Type and identity of media: The stocks of cells are routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- The molecular weight of the test item was 843, therefore the maximum proposed dose level was 5000 μg/mL, the maximum recommended dose.
Experiment 1 (Concentration of test item (μg/mL) plated for mutant frequency): 156.25, 312.5, 625, 1250, 2500, 5000
Experiment 2 (Concentration of test item (μg/mL) plated for mutant frequency): 156.25, 312.5, 625, 1250, 2500, 5000 - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: R0 (RPMI 1640 without serum)
- Solvent (R0 medium) treatment groups were used as the vehicle controls - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- R0
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium (microtitre plates)
- Cell density at the beginning (if applicable): 1x 10^6 cells/mL and 0.3 x 10^6 cells/mL
DURATION
- Exposure duration: 4 h or 24 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 - 14 days
SELECTION AGENT (mutation assays): trifluorothymidine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: mutant frequency: 2000 cells/well; viability: 2 cells/well - Rationale for test conditions:
- A preliminary toxicity test was performed on the cell cultures. The dose range used in the preliminary toxicity test was 19.53 to 5000 μg/mL for all three of the exposure groups. During the course of the study dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/mL or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002). - Evaluation criteria:
- For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10^-6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
- Statistics:
- During the course of the study dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are usually the primary factor to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values markedly less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.
The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). Small significant increases designated by the UKEMS statistical package will be reviewed using
the evaluation criteria, and may be disregarded at the Study Director’s discretion.
Statistical analysis: Viability (%V), Suspension Growth, Relative Suspension Growth (%RSG), Relative Total Growth, 5-TFT resistant mutants, cell count.
The Reviewer considers the analyses used appropriate. - Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- L5178Y TK+/- 3.7.2c mouse lymphoma cell line
- Metabolic activation:
- with and without
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- The test item did not induce any toxicologically significant dose related increases in the mutant frequency at any of the dose levels (including the maximum recommended dose), in either the absence or presence of metabolic activation
- 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 effect. There was no marked change in pH when the test item was dosed into media.
- Effects of osmolality: No effect. The osmolality did not increase by more than 50 mOsm when the test item was dosed into media.
- Water solubility: Test item not soluble in water
- Precipitation: Precipitate of the test item was observed at all of the dose levels.
RANGE-FINDING/SCREENING STUDIES:
For results of the Preliminary Cytotoxicity Test, see Table 1 in "Any other information on results incl. tables".
COMPARISON WITH HISTORICAL CONTROL DATA:
For historical control data, see Tables 15 and 16 in "Any other information on results incl. tables".
ADDITIONAL INFORMATION ON CYTOTOXICITY:
See Table 2 in "Any other information on results incl. tables". - Conclusions:
- Interpretation of results: negative. The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells. The substance does not fulfill the classification criteria as mutagenic according to the CLP Regulation (EC No 1272/2008).
- Executive summary:
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests" adopted 21 July 1997, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.
Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.
The maximum dose level used in the Mutagenicity Test was the maximum recommended dose of 5000 μg/mL. Precipitate of the test item was observed at and above 39.06 μg/mL in the Mutagenicity Test. The vehicle controls had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.
As the test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells, the substance does not fulfill the classification criteria as mutagenic according to the CLP Regulation (EC No 1272/2008).
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09.4.2015 - 21.5.2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The study was performed according to recommended Guidelines (OECD Guidelines for Testing of Chemicals No. 473 " In Vitro Mammalian Chromosome Aberration Test", adopted 26 September 2014) and GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- Adopted 26 September 2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Certificate is attached to the study report
- Type of assay:
- other: In vitro mammalian chromosome aberration test
- Target gene:
- Structural chromosomal aberrations
- Species / strain / cell type:
- lymphocytes: metaphase cells of normal human lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability.
- Suitability of cells: Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test. The volunteers of the cells had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Cell cycle length, doubling time or proliferation index: The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.
- Sex, age and number of blood donors if applicable: The details of the donors used are:
Preliminary Toxicity Test: male, aged 34 years
Main Experiment: male, aged 29 years
- Whether whole blood or separated lymphocytes were used if applicable: heparinized whole blood
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin,
amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- 0, 0.09, 0.188, 0.375, 0.75, 1.25, 2.5, 5 μg test item/mL
The molecular weight of the test item was supplied as 843; therefore, the maximum dose level was 2000 μg/mL, which was the maximum recommended dose level. The dose range for the Preliminary Toxicity Test was 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and was 5 μg/mL for both the 4(20)-hour exposure groups and for the continuous exposure group. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Minimal Essential Medium
- Justification for choice of solvent/vehicle: The test item was suspended in Minimal Essential Medium (MEM) at 20 mg/mL as it did form a suspension suitable for dosing at this concentration. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Minimal Essential Medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 48 hours
- Exposure duration:
4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest.
4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest.
24-hour continuous exposure to the test item without S9-mix prior to cell harvest.
- Expression time (cells in growth medium): 20 hours
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to
slide preparation.
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently 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 EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Where possible the first 150 consecutive well-spread metaphases from each culture were counted, where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including endoreduplicated cells) is reported
- Other: Qualitative Slide Assessment - Rationale for test conditions:
- Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test. The test methods of the study are designed to be compatible with the procedures indicated by the OECD Guidelines for Testing of Chemicals No. 473 " In Vitro Mammalian Chromosome Aberration Test".
- Evaluation criteria:
- The following criteria were used to determine a valid assay:
- The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
- All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
- The required number of cells and concentrations were analyzed. - Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis. - Species / strain:
- lymphocytes: metaphase cells of normal human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effect. There was no significant change in pH when the test item was dosed into media.
- Effects of osmolality: No effect. When the test item was dosed into media, the osmolality did not increase by more than 50 mOsm.
- Water solubility: The test item is not soluble in water
- Precipitation: Precipitate observations were made at the end of exposure in the parallel blood-free cultures and was noted at 5 μg/mL level in all three of the exposure groups in the parallel blood-free cultures.
RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum dose was the maximum recommended dose level.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 31.25 μg/mL in the 4(20)-hour exposure groups and at all test item dose levels in the 24-hour exposure group. A precipitate in the pellet of the blood cultures of the4(20)-hour exposure groups at the end of exposure was noted when the cultures were centrifuged and washed at all test item dose levels. Precipitate of the test item was also observed on the slides of the 4(20)-hour exposure groups in the absence and presence of S9 mix at and above 15.63 μg/mL, and in the 24-hour exposure group at and above 7.81 μg/mL.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 500 μg/mL in all three of the exposure groups. The mitotic index data are presented in the study. The test item induced some modest evidence of toxicity but the dose selection for the main experiment was limited to include the lowest precipitating dose level as directed by the OECD 473 test guideline.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and was 5 μg/mL for both the 4(20)-hour exposure groups and for the continuous exposure group.
COMPARISON WITH HISTORICAL CONTROL DATA:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix. For Historical Aberration Ranges, see Tables 8 and 9 in "Any other information on results incl. tables". - Conclusions:
- The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
The substance does not fulfill the classification criteria as mutagenic according to the CLP Regulation (EC No 1272/2008). - Executive summary:
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. In this study, the main experiment was performed using three exposure conditions; 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.
The dose levels selected for the main experiment were based on the results of the preliminary toxicity test and were limited to include the lowest precipitating dose level.
All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.
All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.
The test item was considered to be non-clastogenic to human lymphocytes in vitro. The substance does not fulfil the criteria as mutagenic according to the CLP Regulation (EC No 1272/2008).
Referenceopen allclose all
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SD Standard deviation
Min Minimum value
Max Maximum value
† Number of mean values used to create dataset
Table 2 Spontaneous Mutation Rates (Concurrent Negative Controls).
Experiment 1
| Number of revertants (mean number of colonies per plate) | |||||||||
| Base-pair substitution type | Frameshift type | ||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||
| 71 | (81) | 8 | (15) | 23 | (26) | 17 | (14) | 7 | (10) |
| 87 | 13 | 31 | 12 | 12 | |||||
| 84 | 23 | 23 | 12 | 11 | |||||
Experiment 2
| Number of revertants (mean number of colonies per plate) | |||||||||
| Base-pair substitution type | Frameshift type | ||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||
| 103 | (100) | 21 | (15) | 11 | (17) | 11 | (14) | 11 | (9) |
| 95 | 11 | 23 | 12 | 7 | |||||
| 103 | 12 | 17 | 20 | 9 | |||||
Table 3. Test Results:
Experiment 1 – Without Metabolic Activation
| Test Period From: 20 October 2014 To: 23 October 2014 | |||||||||||
| S9-Mix (-) |
Dose Level Per Plate | Number of revertants (mean) +/- SD | |||||||||
| Base-pair substitution strains | Frameshift strains | ||||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
| Solvent Control (WATER) |
87 | (80) | 16 | (13) | 27 | (23) | 19 | (20) | 9 | (11) | |
| 76 | 6.4+ | 11 | 2.5 | 20 | 3.5 | 19 | 1.2 | 13 | 2.1 | ||
| 76 | 13 | 23 | 21 | 12 | |||||||
| 1.5 ug | 82 | (82) | 8 | (11) | 28 | (26) | 20 | (24) | 9 | (10) | |
| 83 | 0.6 | 16 | 4.6 | 20 | 4.9 | 27 | 3.5 | 13 | 2.6 | ||
| 82 | 8 | 29 | 24 | 8 | |||||||
| 5 ug | 75 | (73) | 12 | (15) | 28 | (27) | 17 | (15) | 5 | (7) | |
| 79 | 7.2 | 19 | 3.8 | 36 | 9.5 | 12 | 2.5 | 5 | 4.0 | ||
| 65 | 13 | 17 | 15 | 12 | |||||||
| 15 ug | 107 | (91) | 12 | (10) | 24 | (23) | 15 | (15) | 8 | (7) | |
| 82 | 14.2 | 11 | 2.6 | 29 | 6.0 | 17 | 2.5 | 8 | 1.7 | ||
| 83 | 7 | 17 | 12 | 5 | |||||||
| 50 ug | 84 | (76) | 20 | (14) | 19 | (21) | 12 | (15) | 11 | (10) | |
| 60 | 13.6 | 15 | 6.0 | 12 | 10.7 | 12 | 5.2 | 12 | 2.6 | ||
| 83 | 8 | 33 | 21 | 7 | |||||||
| 150 ug | 83 | (86) | 15 | (12) | 11 | (16) | 13 | (16) | 9 | (10) | |
| 91 | 4.6 | 15 | 4.6 | 21 | 5.0 | 24 | 7.0 | 5 | 5.0 | ||
| 83 | 7 | 17 | 11 | 15 | |||||||
| 500 ug | 86 | (83) | 12 | (14) | 12 | (20) | 13 | (15) | 5 | (11) | |
| 72 | 9.5 | 20 | 5.7 | 28 | 8.0 | 15 | 1.5 | 16 | 5.6 | ||
| 90 | 9 | 20 | 16 | 12 | |||||||
| 1500 ug | 76P | (80) | 12P | (12) | 21P | (17) | 11P | (16) | 11P | (12) | |
| 83P | 3.5 | 13P | 1.0 | 15P | 3.5 | 20P | 4.6 | 16P | 4.0 | ||
| 80P | 11P | 15P | 17P | 8P | |||||||
| 5000 ug | 100P | (89) | 11P | (11) | 15P | (21) | 11P | (10) | 15P | (10) | |
| 92P | 12.2 | 8P | 3.5 | 21P | 6.5 | 8P | 1.7 | 7P | 4.2 | ||
| 76P | 15P | 28P | 11P | 9P | |||||||
| Positive controls S9-Mix (-) | Name Dose Level No. of Revertants | ENNG | ENNG | ENNG | 4NQO | 9AA | |||||
| 3 ug | 5 ug | 2 ug | 0.2 ug | 80 ug | |||||||
| 648 | (632) | 859 | (852) | 1121 | (1162) | 158 | (178) | 970 | (1186) | ||
| 539 | 85.7 | 841 | 9.9 | 1276 | 100.4 | 219 | 35.8 | 1056 | 302.1 | ||
| 708 | 857 | 1088 | 156 | 1531 | |||||||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
P Test item precipitate
+ Standard deviation
Experiment 1 – With Metabolic Activation
| Test Period From: 20 October 2014 To: 23 October 2014 | |||||||||||
| S9-Mix (+) |
Dose Level Per Plate | Number of revertants (mean) +/- SD | |||||||||
| Base-pair substitution strains | Frameshift strains | ||||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
| Solvent Control (WATER) |
98 | (87) | 17 | (13) | 21 | (19) | 16 | (20) | 19 | (12) | |
| 79 | 10.0+ | 12 | 3.2 | 21 | 2.9 | 24 | 4.0 | 9 | 5.8 | ||
| 83 | 11 | 16 | 21 | 9 | |||||||
| 1.5 ug | 74 | (74) | 9 | (11) | 20 | (24) | 16 | (19) | 11 | (13) | |
| 76 | 2.0 | 9 | 3.5 | 24 | 3.5 | 29 | 9.3 | 15 | 2.1 | ||
| 72 | 15 | 27 | 11 | 12 | |||||||
| 5 ug | 79 | (77) | 11 | (15) | 24 | (21) | 16 | (16) | 15 | (10) | |
| 86 | 10.7 | 11 | 7.5 | 20 | 2.6 | 16 | 0.6 | 7 | 4.2 | ||
| 65 | 24 | 19 | 15 | 9 | |||||||
| 15 ug | 71 | (78) | 11 | (12) | 24 | (23) | 19 | (20) | 16 | (14) | |
| 72 | 11.3 | 9 | 3.6 | 25 | 2.6 | 23 | 2.3 | 16 | 4.0 | ||
| 91 | 16 | 20 | 19 | 9 | |||||||
| 50 ug | 95 | (93) | 12 | (13) | 17 | (19) | 12 | (18) | 19 | (12) | |
| 82 | 10.1 | 11 | 2.1 | 20 | 1.5 | 17 | 6.6 | 11 | 6.1 | ||
| 102 | 15 | 19 | 25 | 7 | |||||||
| 150 ug | 69 | (81) | 12 | (12) | 29 | (21) | 15 | (21) | 15 | (15) | |
| 107 | 22.2 | 11 | 0.6 | 13 | 8.0 | 23 | 4.9 | 19 | 3.5 | ||
| 68 | 12 | 20 | 24 | 12 | |||||||
| 500 ug | 79 | (91) | 13 | (10) | 24 | (23) | 21 | (24) | 9 | (11) | |
| 83 | 20.8 | 7 | 3.1 | 19 | 3.2 | 27 | 3.0 | 15 | 3.8 | ||
| 115 | 11 | 25 | 24 | 8 | |||||||
| 1500 ug | 98P | (82) | 16P | (14) | 25P | (23 | 17P | (19) | 20P | (16) | |
| 64P | 17.0 | 13P | 2.1 | 23P | 2.5 | 17P | 3.5 | 16P | 4.5 | ||
| 83P | 12P | 20p | 23P | 11P | |||||||
| 5000 ug | 86P | (82) | 7P | (8) | 19P | (16) | 15P | (17) | 8P | (10) | |
| 76P | 53. | 8P | 1.0 | 13P | 3.1 | 25P | 6.8 | 5P | 5.7 | ||
| 84P | 9P | 15P | 12P | 16P | |||||||
| Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants | 2AA | 2AA | 2AA | BP | 2AA | |||||
| 3 ug | 5 ug | 2 ug | 5 ug | 80 ug | |||||||
| 1431 | (1544) | 188 | (216) | 237 | (237) | 275 | (261) | 674 | (677) | ||
| 1612 | 98.5 | 211 | 30.8 | 237 | 0.0 | 257 | 12.5 | 659 | 19.7 | ||
| 1589 | 249 | 237 | 251 | 698 | |||||||
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
P Test item precipitate
+ Standard deviation
Experiment 2 – Without Metabolic Activation
| Test Period From: 3 November 2014 To: 6 November 2014 | |||||||||||
| S9-Mix (-) |
Dose Level Per Plate | Number of revertants (mean) +/- SD | |||||||||
| Base-pair substitution strains | Frameshift strains | ||||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
| Solvent Control (WATER) |
92 | (101) | 11 | (13) | 25 | (21) | 12 | (15) | 9 | (11) | |
| 100 | 9.0+ | 11 | 3.5 | 23 | 5.3 | 15 | 2.5 | 11 | 2.0 | ||
| 110 | 17 | 15 | 17 | 13 | |||||||
| 50 ug | 91 | (93) | 9 | (13) | 13 | (14) | 16 | (13) | 9 | (12) | |
| 84 | 9.6 | 16 | 3.8 | 17 | 2.3 | 12 | 2.3 | 15 | 3.1 | ||
| 103 | 15 | 13 | 12 | 13 | |||||||
| 150 ug | 106 | (100) | 9 | (9) | 13 | (14) | 24 | (16) | 9 | (11) | |
| 87 | 11.3 | 9 | 0 | 11 | 4.2 | 11 | 7.8 | 8 | 4.9 | ||
| 107 | 9 | 19 | 12 | 17 | |||||||
| 500 ug | 104 | (103) | 15 | (13) | 11 | (15) | 15 | (16) | 16 | (9) | |
| 96 | 7.0 | 12 | 2.1 | 13 | 4.7 | 9 | 3.8 | 3 | 6.6 | ||
| 110 | 11 | 20 | 16 | 8 | |||||||
| 1500 ug | 107P | (92) | 13P | (12) | 20P | (17) | 11P | (10) | 9P | (10) | |
| 88P | 13.1 | 15P | 3.1 | 20P | 5.2 | 9P | 1.2 | 8P | 2.1 | ||
| 82P | 9P | 11P | 11P | 12P | |||||||
| 5000 ug | 79P | (80) | 13P | (16) | 13P | (16) | 16P | (15) | 7P | (8) | |
| 87P | 6.1 | 20P | 3.6 | 19P | 3.0 | 11P | 4.0 | 5P | 4.2 | ||
| 75P | 15P | 16P | 19P | 13P | |||||||
| Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants | ENNG | ENNG | ENNG | 4NQO | 9AA | |||||
| 3 ug | 5 ug | 2 ug | 0.2 ug | 80 ug | |||||||
| 763 | (660) | 186 | (171) | 664 | (644) | 198 | (211) | 517 | (441) | ||
| 664 | 105.1 | 114 | 51.6 | 631 | 17.8 | 227 | 14.7 | 429 | 71.2 | ||
| 553 | 214 | 636 | 208 | 376 | |||||||
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
P Test item precipitate
+ Standard deviation
Experiment 2 – With Metabolic Activation
| Test Period From: 3 November 2014 To: 6 November 2014 | |||||||||||
| S9-Mix (+) |
Dose Level Per Plate | Number of revertants (mean) +/- SD | |||||||||
| Base-pair substitution strains | Frameshift strains | ||||||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||||||
| Solvent Control (WATER) |
107 | (103) | 9 | (11) | 20 | (21) | 23 | (20) | 8 | (9) | |
| 96 | 6.4+ | 12 | 1.7 | 17 | 4.0 | 16 | 3.5 | 11 | 1.7 | ||
| 107 | 12 | 25 | 20 | 8 | |||||||
| 50 ug | 118 | (111) | 11 | (13) | 17 | (18) | 20 | (18) | 13 | (10) | |
| 108 | 6.4 | 12 | 2.1 | 15 | 3.1 | 16 | 2.1 | 5 | 4.4 | ||
| 106 | 15 | 21 | 17 | 12 | |||||||
| 150 ug | 100 | (93) | 19 | (14) | 19 | (20) | 32 | (21) | 8 | (9) | |
| 100 | 12.1 | 12 | 4.4 | 24 | 4.0 | 17 | 3.8 | 12 | 2.3 | ||
| 79 | 11 | 16 | 24 | 8 | |||||||
| 500 ug | 116 | (104) | 9 | (10) | 25 | (23) | 25 | (20) | 19 | (15) | |
| 92 | 12.1 | 9 | 1.2 | 19 | 3.2 | 27 | 10.4 | 15 | 3.5 | ||
| 104 | 11 | 24 | 8 | 12 | |||||||
| 1500 ug | 86P | (93) | 11P | (11) | 25P | (20) | 23P | (18) | 8P | (9) | |
| 88P | 12.0 | 12P | 0.6 | 21P | 5.0 | 13P | 5.0 | 7P | 2.6 | ||
| 104P | 11P | 15P | 19P | 12P | |||||||
| 5000 ug | 100P | (102) | 11P | (11) | 23P | (20) | 21P | (20) | 9P | (14) | |
| 94P | 8.6 | 11P | 0.6 | 25P | 6.4 | 23P | 3.1 | 13P | 5.0 | ||
| 111P | 12P | 13P | 17P | 19P | |||||||
| Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants | 2AA | 2AA | 2AA | BP | 2AA | |||||
| 1 ug | 2 ug | 10 ug | 5 ug | 2 ug | |||||||
| 3626 | (3505) | 261 | (279) | 178 | (187) | 106 | (123) | 535 | (562) | ||
| 3417 | 108.8 | 287 | 15.9 | 178 | 16.2 | 126 | 16.2 | 516 | 64.5 | ||
| 3473 | 290 | 206 | 138 | 636 | |||||||
BP Benzo(a)pyrene
2AA 2-Aminoanthracene
P Test item precipitate
+ Standard deviation
Key to tables 1 to 16:
$ = Cell counts (x10^5 cells/mL). Set up on previous day to 2 x 10^5 cells/mL unless otherwise stated in parenthesis.
%RSG = Relative Suspension Growth
RTG = Relative Total Growth
%V = Viability Day 2
SG = Suspension growth
§ or # = Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis
A,B = Replicate cultures
CP = Cyclophosphamide
EMS = Ethylmethanesulphonate
MF§ = 5-TFT resistant mutants/10^6 viable cells 2 days after treatment
NP = Not plated, surplus to requirements
Ø = Not plated for viability or 5-TFT resistance
Nv = Number of wells scored, viability plates
Yv = Number of wells without colonies, viability plates
Ym = Number of wells without colonies, mutation plates
Nm = Number of wells scored, mutation plates
Table 1. Preliminary Cytotoxicity Test
The dose range of the test item used in the preliminary toxicity test was 19.53 to 5000 μg/mL.
The results for the Relative Suspension Growth (%RSG) were as follows:
| Dose (ug/ml) | % RSG (-S9) 4-hour exposure | % RSG (+S9) 4-hour exposure | % RSG (-9) 24-hour exposure |
| 0 | 100 | 100 | 100 |
| 19.53 | 99 | 103 | 117 |
| 39.06 | 98 | 102 | 118 |
| 78.13 | 97 | 102 | 115 |
| 156.25 | 98 | 97 | 118 |
| 312.5 | 78 | 47 | 93 |
| 625 | 61 | 58 | 79 |
| 1250 | 49 | 58 | 78 |
| 2500 | 50 | 49 | 57 |
| 5000 | 38 | 34 | 41 |
There was evidence of modest reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. Precipitate of the test item was observed at all of the dose levels. Based on the %RSG values observed, the maximum dose level in the subsequent Mutagenicity Test was the maximum recommended dose of 5000 μg/mL.
Table 2. Mutagenicity Test - Summary of results
Experiment 1:
| Treatment (μg/ml) | 4-Hours-S9 | Treatment (μg/ml) |
4-Hours+S9 | ||||
| %RSG | RTG | MF§ | %RSG | RTG | MF§ | ||
| 0 | 100 | 1 | 105.50 | 0 | 100 | 1 | 127.25 |
| 39.06 Ø | 96 | 39.06 Ø | 99 | ||||
| 78.13 Ø | 101 | 78.13 Ø | 102 | ||||
| 156.25 | 94 | 1.01 | 103.45 | 156.25 | 107 | 1.06 | 112.66 |
| 312.5 | 89 | 0.97 | 111.70 | 312.5 | 93 | 0.98 | 106.08 |
| 625 | 84 | 0.89 | 116.36 | 625 | 72 | 1.01 | 88.03 |
| 1250 | 64 | 0.92 | 99.28 | 1250 | 69 | 1.04 | 89.16 |
| 2500 | 59 | 0.78 | 111.22 | 2500 | 60 | 0.73 | 106.06 |
| 5000 | 43 | 0.64 | 104.09 | 5000 | 44 | 0.60 | 124.27 |
| Linear trend | NS |
Linear trend | NS |
||||
| EMS | CP | ||||||
| 400 | 68 | 0.53 | 971.56 | 1.5 | 71 | 0.50 | 1007.24 |
Experiment 2:
| Treatment (μg/ml) | 24-Hours-S9 | Treatment (μg/ml) |
4-Hours+S9 | ||||
| %RSG | RTG | MF§ | %RSG | RTG | MF§ | ||
| 0 | 100 | 1 | 144.22 | 0 | 100 | 1 | 125.12 |
| 39.06 Ø | 91 | 39.06 Ø | 98 | ||||
| 78.13 Ø | 95 | 78.13 Ø | 94 | ||||
| 156.25 | 93 | 1.10 | 100.89 | 156.25 | 102 | 1.09 | 133.06 |
| 312.5 | 105 | 1.09 | 123.21 | 312.5 | 88 | 1.09 | 96.46 |
| 625 | 41 | 0.83 | 116.68 | 625 | 61 | 0.71 | 113.45 |
| 1250 | 36 | 0.89 | 104.09 | 1250 | 59 | 0.90 | 98.16 |
| 2500 | 33 | 0.81 | 97.72 | 2500 | 54 | 0.91 | 89.16 |
| 5000 | 7 | 0.27 | 130.27 | 5000 | 42 | 0.62 | 117.62 |
| Linear trend | NS |
Linear trend | NS |
||||
| EMS | CP | ||||||
| 150 | 34 | 0.36 | 1137.01 | 1.5 | 71 | 0.58 | 705.30 |
Table 3. Cell and 96-Well Plate Counts: Experiment 1 (-S9) 4-Hour Exposure
| Treatment | Cell counts $ | Viability § | Resistant mutants § | |||||||||
| (μg/ml) | after day 2 | after day 2 | ||||||||||
| 2 cells/well | 2000 cells/well | |||||||||||
| 0 h | 24 h | 48 h | ||||||||||
| 0 | A | 9.64 | 6.05 | 7.59 | 73 | 76 | 81 | 75 | 16 | 15 | 13 | 13 |
| B | 9.39 | 6.02 | 7.62 | 70 | 73 | 83 | 79 | 14 | 17 | 15 | 15 | |
| 39.06 | A | 9.87 | 6.36 | 7.02 | NP | NP | NP | NP | ||||
| B | 9.4 | 6.12 | 6.98 | NP | NP | NP | NP | |||||
| 78.13 | A | 9.69 | 6.39 | 7.46 | NP | NP | NP | NP | ||||
| B | 9.77 | 6.27 | 6.88 | NP | NP | NP | NP | |||||
| 156.25 | A | 9.58 | 5.97 | 7.05 | 77 | 80 | 17 | 10 | ||||
| B | 9.29 | 6.54 | 6.89 | 82 | 75 | 19 | 16 | |||||
| 312.5 | A | 9.71 | 6.02 | 6.78 | 80 | 75 | 18 | 18 | ||||
| B | 9.89 | 6.07 | 6.38 | 83 | 77 | 15 | 16 | |||||
| 625 | A | 7.75 | 7.7 | 5.95 | 80 | 80 | 17 | 18 | ||||
| B | 8.5 | 7.35 | 6 | 78 | 76 | 17 | 17 | |||||
| 1250 | A | 7.25 | 6.2 | 6.05 | 85 | 83 | 19 | 21 | ||||
| B | 8.15 | 5.6 | 6.2 | 91 | 86 | 20 | 18 | |||||
| 2500 | A | 7.25 | 5.65 | 6.3 | 83 | 81 | 23 | 19 | ||||
| B | 7.2 | 5.85 | 6.1 | 85 | 88 | 17 | 21 | |||||
| 5000 | A | 6 | 5.5 | 5.85 | 90 | 85 | 23 | 20 | ||||
| B | 6 | 5.6 | 5.5 | 85 | 87 | 17 | 23 | |||||
Positive control EMS (μg/ml) |
||||||||||||
| 400 | A | 9.05 | 5.19 | 6.25 | 66 | 63 | 72 | 65 | ||||
| B | 9.06 | 5.22 | 6.42 | 73 | 69 | 65 | 65 | |||||
Table 4. Statistical Analysis: Experiment 1 (-S9) 4-Hour Exposure
| Treatment | SG | %RSG | %V | RTG | MF§ | |
| (μg/ml) | ||||||
| 0 | 11.47 | 100 | 79.06 | 1 | 105.5 | |
| 39.06 | Ø | 10.92 | 96 | |||
| 78.13 | Ø | 11.35 | 101 | |||
| 156.25 | 10.9 | 94 | 85.11 | 1.01 | 103.45 | |
| 312.5 | 9.94 | 89 | 85.83 | 0.97 | 111.7 | |
| 625 | 11.24 | 84 | 85.11 | 0.89 | 116.36 | |
| 1250 | 9.03 | 64 | 114.35 | 0.92 | 99.28 | |
| 2500 | 8.91 | 59 | 105.02 | 0.78 | 111.22 | |
| 5000 | 7.87 | 43 | 116.99 | 0.64 | 104.09 | |
| Positive control EMS | ||||||
| Treatment | SG | %RSG | %V | RTG | MF§ | |
| (μg/ml) | ||||||
| 400 | 8.24 | 68 | 61.16 | 0.53 | 971.56 | |
Test for linear trend
| Slope | -3.73E-10 |
| Variance | 6.91E-18 |
| b²/Sb | 0.02 |
Table 5. Large and Small Colonies Analysis: Experiment 1 (-S9) 4-Hour Exposure
| Treatment | Viability # | Small colonies # | Large colonies # | ||||||||||
| (μg/ml) | after day 2 | after day 2 | after day 2 | ||||||||||
| 0 | A | 73 | 76 | 81 | 75 | 7 | 8 | 7 | 9 | 9 | 7 | 6 | 4 |
| B | 70 | 73 | 83 | 79 | 7 | 9 | 4 | 7 | 7 | 8 | 11 | 8 | |
| 156.25 | A | 77 | 80 | 6 | 8 | 11 | 2 | ||||||
| B | 82 | 75 | 10 | 7 | 9 | 9 | |||||||
| 312.5 | A | 80 | 75 | 8 | 8 | 10 | 10 | ||||||
| B | 83 | 77 | 6 | 7 | 9 | 9 | |||||||
| 625 | A | 80 | 80 | 9 | 7 | 8 | 11 | ||||||
| B | 78 | 76 | 7 | 7 | 10 | 10 | |||||||
| 1250 | A | 85 | 83 | 8 | 8 | 11 | 13 | ||||||
| B | 91 | 86 | 9 | 10 | 11 | 8 | |||||||
| 2500 | A | 83 | 81 | 8 | 8 | 15 | 11 | ||||||
| B | 85 | 88 | 9 | 7 | 8 | 14 | |||||||
| 5000 | A | 90 | 85 | 9 | 8 | 14 | 12 | ||||||
| B | 85 | 87 | 9 | 7 | 8 | 16 | |||||||
| 400 EMS | A | 66 | 63 | 32 | 26 | 40 | 39 | ||||||
| B | 73 | 69 | 20 | 36 | 45 | 29 | |||||||
Mutation frequencies
| Treatment | Viable | Small colonies | Large colonies | Proportion small colony mutants | |||||
| (μg/ml) | |||||||||
| Mutants | Mutants | ||||||||
| Yv | Nv | Ym | Nm | MF§ | Ym | Nm | MF§ | ||
| 0 | 158 | 768 | 710 | 768 | 49.7 | 708 | 768 | 51.4 | 0.49 |
| 156.25 | 70 | 384 | 353 | 384 | 49.5 | 353 | 384 | 49.5 | 0.5 |
| 312.5 | 69 | 384 | 355 | 384 | 45.7 | 346 | 384 | 60.7 | 0.43 |
| 625 | 70 | 384 | 354 | 384 | 47.8 | 345 | 384 | 62.9 | 0.43 |
| 1250 | 39 | 384 | 349 | 384 | 41.8 | 341 | 384 | 51.9 | 0.45 |
| 2500 | 47 | 384 | 352 | 384 | 41.4 | 336 | 384 | 63.6 | 0.4 |
| 5000 | 37 | 384 | 351 | 384 | 38.4 | 334 | 384 | 59.6 | 0.4 |
| 400 EMS | 113 | 384 | 270 | 384 | 287.9 | 231 | 384 | 415.5 | 0.43 |
Table 6. Cell and 96-Well Plate Counts: Experiment 1 (+S9) 4-Hour Exposure
| Treatment | Cell counts $ | Viability § | Resistant mutants § | |||||||||
| (μg/ml) | after day 2 | after day 2 | ||||||||||
| 2 cells/well | 2000 cells/well | |||||||||||
| 0 h | 24 h | 48 h | ||||||||||
| 0 | A | 9.53 | 6.3 | 7.58 | 70 | 79 | 82 | 78 | 17 | 18 | 23 | 19 |
| B | 9.98 | 5.56 | 7.44 | 78 | 79 | 82 | 79 | 17 | 19 | 18 | 18 | |
| 39.06 | A | 9.3 | 6.11 | 7.36 | NP | NP | NP | NP | ||||
| B | 9.56 | 6.1 | 7.6 | NP | NP | NP | NP | |||||
| 78.13 | A | 8.95 | 6.59 | 7.89 | NP | NP | NP | NP | ||||
| B | 9.12 | 5.83 | 7.83 | NP | NP | NP | NP | |||||
| 156.25 | A | 8.63 | 6.86 | 7.76 | 82 | 79 | 19 | 14 | ||||
| B | 9.13 | 6.19 | 8.25 | 75 | 76 | 17 | 16 | |||||
| 312.5 | A | 8.5 | 6.38 | 7.78 | 84 | 81 | 16 | 17 | ||||
| B | 8.54 | 5.68 | 7.91 | 79 | 77 | 18 | 16 | |||||
| 625 | A | 8.3 | 6.4 | 5.75 | 91 | 83 | 15 | 19 | ||||
| B | 8.55 | 5.7 | 6.5 | 91 | 84 | 22 | 17 | |||||
| 1250 | A | 8.5 | 5.9 | 5.95 | 91 | 89 | 20 | 18 | ||||
| B | 7.9 | 5.4 | 6.95 | 87 | 88 | 20 | 21 | |||||
| 2500 | A | 6.25 | 6.6 | 6.35 | 87 | 87 | 19 | 21 | ||||
| B | 6.9 | 5.2 | 7.15 | 83 | 79 | 19 | 17 | |||||
| 5000 | A | 4.3 | 5.85 | 6.25 | 88 | 88 | 28 | 27 | ||||
| B | 5.45 | 6.05 | 6.95 | 83 | 86 | 20 | 20 | |||||
Positive control CP (μg/ml) |
||||||||||||
| 1.5 | A | 9.74 | 4.25 | 7.45 | 63 | 64 | 62 | 67 | ||||
| B | 9.75 | 4.86 | 6.37 | 74 | 68 | 66 | 75 | |||||
Table 7. Statistical Analysis: Experiment 1 (+S9) 4-Hour Exposure
| Treatment | SG | %RSG | %V | RTG | MF§ | |
| (μg/ml) | ||||||
| 0 | 11.13 | 100 | 84.75 | 1 | 127.25 | |
| 39.06 | Ø | 11.42 | 99 | |||
| 78.13 | Ø | 12.2 | 102 | |||
| 156.25 | 13.06 | 107 | 83.7 | 1.06 | 112.66 | |
| 312.5 | 11.83 | 93 | 90.38 | 0.98 | 106.08 | |
| 625 | 9.26 | 72 | 119.76 | 1.01 | 88.03 | |
| 1250 | 9.11 | 69 | 129.17 | 1.04 | 89.16 | |
| 2500 | 9.96 | 60 | 103.97 | 0.73 | 106.06 | |
| 5000 | 9.82 | 44 | 114.35 | 0.6 | 124.27 | |
Positive control EMS |
||||||
| Treatment | SG | %RSG | %V | RTG | MF§ | |
| (μg/ml) | ||||||
| 1.5 | 7.87 | 71 | 60.29 | 0.5 | 1007.24 | |
Test for linear trend
| Slope | 1.64E-09 |
| Variance | 8.88E-18 |
| b²/Sb | 0.302 |
Table 8. Large and Small Colonies Analysis: Experiment 1 (+S9) 4-Hour Exposure
| Treatment | Viability # | Small colonies # | Large colonies # | ||||||||||
| (μg/ml) | after day 2 | after day 2 | after day 2 | ||||||||||
| 0 | A | 70 | 79 | 82 | 78 | 7 | 10 | 14 | 10 | 10 | 8 | 9 | 9 |
| B | 78 | 79 | 82 | 79 | 7 | 10 | 8 | 8 | 10 | 9 | 10 | 10 | |
| 156.25 | A | 82 | 79 | 9 | 8 | 10 | 6 | ||||||
| B | 75 | 76 | 9 | 9 | 8 | 7 | |||||||
| 312.5 | A | 84 | 81 | 8 | 10 | 8 | 7 | ||||||
| B | 79 | 77 | 10 | 8 | 8 | 8 | |||||||
| 625 | A | 91 | 83 | 11 | 10 | 4 | 9 | ||||||
| B | 91 | 84 | 9 | 9 | 13 | 8 | |||||||
| 1250 | A | 91 | 89 | 7 | 10 | 13 | 8 | ||||||
| B | 87 | 88 | 13 | 10 | 7 | 11 | |||||||
| 2500 | A | 87 | 87 | 9 | 12 | 10 | 9 | ||||||
| B | 83 | 79 | 11 | 7 | 8 | 10 | |||||||
| 5000 | A | 88 | 88 | 12 | 13 | 16 | 14 | ||||||
| B | 83 | 86 | 12 | 9 | 8 | 11 | |||||||
| 1.5 CP | A | 63 | 64 | 47 | 46 | 15 | 21 | ||||||
| B | 74 | 68 | 40 | 49 | 26 | 26 | |||||||
Mutation frequencies
| Treatment | Viable | Small colonies | Large colonies | Proportion small colony mutants | |||||
| (μg/ml) | |||||||||
| Mutants | Mutants | ||||||||
| Yv | Nv | Ym | Nm | MF§ | Ym | Nm | MF§ | ||
| 0 | 141 | 768 | 694 | 768 | 59.8 | 693 | 768 | 60.6 | 0.5 |
| 156.25 | 72 | 384 | 349 | 384 | 57.1 | 353 | 384 | 50.3 | 0.53 |
| 312.5 | 63 | 384 | 348 | 384 | 54.5 | 353 | 384 | 46.6 | 0.54 |
| 625 | 35 | 384 | 345 | 384 | 44.7 | 350 | 384 | 38.7 | 0.53 |
| 1250 | 29 | 384 | 344 | 384 | 42.6 | 345 | 384 | 41.5 | 0.51 |
| 2500 | 48 | 384 | 345 | 384 | 51.5 | 347 | 384 | 48.7 | 0.51 |
| 5000 | 39 | 384 | 338 | 384 | 55.8 | 335 | 384 | 59.7 | 0.48 |
| 1.5 CP | 115 | 384 | 202 | 384 | 532.8 | 296 | 384 | 215.9 | 0.67 |
Table 9. Cell and 96-Well Plate Counts: Experiment 2 (-S9) 24-Hour Exposure
| Treatment | Cell counts $ | Viability § | Resistant mutants § | |||||||||
| (μg/ml) | after day 2 | after day 2 | ||||||||||
| 2 cells/well | 2000 cells/well | |||||||||||
| 0 h | 24 h | 48 h | ||||||||||
| 0 | A | 8.02 | 6.92 | 7.86 | 72 | 75 | 70 | 77 | 18 | 18 | 20 | 18 |
| B | 9.22 | 6.32 | 7.52 | 70 | 77 | 75 | 70 | 20 | 19 | 17 | 14 | |
| 39.06 | A | 7.85 | 8.13 | 6.36 | NP | NP | NP | NP | ||||
| B | 8.44 | 7.38 | 6.98 | NP | NP | NP | NP | |||||
| 78.13 | A | 9.34 | 6.82 | 6.98 | NP | NP | NP | NP | ||||
| B | 8 | 7.77 | 6.14 | NP | NP | NP | NP | |||||
| 156.25 | A | 9.19 | 6.58 | 7.03 | 81 | 81 | 15 | 20 | ||||
| B | 8.6 | 5.68 | 7.41 | 79 | 78 | 14 | 14 | |||||
| 312.5 | A | 9.37 | 6.35 | 7.47 | 78 | 79 | 16 | 20 | ||||
| B | 8.47 | 6.63 | 7.85 | 73 | 73 | 16 | 15 | |||||
| 625 | A | 6.05 | 4.95 | 9.45 | 82 | 85 | 22 | 16 | ||||
| B | 4.9 | 7.8 | 6.8 | 84 | 75 | 19 | 19 | |||||
| 1250 | A | 4.3 | 7.9 | 6.55 | 85 | 84 | 22 | 17 | ||||
| B | 6.9 | 4.9 | 7 | 90 | 88 | 22 | 22 | |||||
| 2500 | A | 6.35 | 5.85 | 5.7 | 89 | 86 | 21 | 21 | ||||
| B | 5.45 | 5.45 | 7.15 | 88 | 87 | 18 | 21 | |||||
| 5000 | A | 3.8 | 5.2 | 4.9 | 86 | 78 | 22 | 24 | ||||
| B | 3.15 | 3.75 | 4.85 | 88 | 92 | 27 | 25 | |||||
Positive control EMS (μg/ml) |
||||||||||||
| 150 | A | 5.66 | 5.76 | 6.12 | 69 | 54 | 79 | 73 | ||||
| B | 6.19 | 6.4 | 5.85 | 67 | 60 | 56 | 60 | |||||
Table 10. Statistical Analysis: Experiment 2 (-S9) 24-Hour Exposure
| Treatment | SG | %RSG | %V | RTG | MF§ | |
| (μg/ml) | ||||||
| 0 | 73.14 | 100 | 71.99 | 1 | 144.22 | |
| 39.06 | Ø | 70.22 | 91 | |||
| 78.13 | Ø | 69.15 | 95 | |||
| 156.25 | 65.61 | 93 | 88.81 | 1.1 | 100.89 | |
| 312.5 | 73.91 | 105 | 77.81 | 1.09 | 123.21 | |
| 625 | 47.26 | 41 | 94.51 | 0.83 | 116.68 | |
| 1250 | 40.47 | 36 | 116.99 | 0.89 | 104.09 | |
| 2500 | 35.7 | 33 | 121.21 | 0.81 | 97.72 | |
| 5000 | 12.63 | 7 | 113.09 | 0.27 | 130.27 | |
| Positive control EMS | ||||||
| Treatment | SG | %RSG | %V | RTG | MF§ | |
| (μg/ml) | ||||||
| 150 | 35.93 | 34 | 52.64 | 0.36 | 1137.01 | |
Test for linear trend
| Slope | -7.94E-10 |
| Variance | 9.33E-18 |
| b²/Sb | 0.068 |
Table 11. Large and Small Colonies Analysis: Experiment 2 (-S9) 24-Hour Exposure
| Treatment | Viability # | Small colonies # | Large colonies # | ||||||||||
| (μg/ml) | after day 2 | after day 2 | after day 2 | ||||||||||
| 0 | A | 72 | 75 | 70 | 77 | 2 | 7 | 12 | 9 | 16 | 11 | 8 | 9 |
| B | 70 | 77 | 75 | 70 | 7 | 8 | 8 | 7 | 13 | 11 | 9 | 7 | |
| 156.25 | A | 81 | 81 | 6 | 9 | 9 | 11 | ||||||
| B | 79 | 78 | 7 | 8 | 7 | 6 | |||||||
| 312.5 | A | 78 | 79 | 6 | 9 | 10 | 11 | ||||||
| B | 73 | 73 | 9 | 8 | 7 | 7 | |||||||
| 625 | A | 82 | 85 | 7 | 6 | 15 | 10 | ||||||
| B | 84 | 75 | 10 | 4 | 9 | 15 | |||||||
| 1250 | A | 85 | 84 | 7 | 7 | 15 | 10 | ||||||
| B | 90 | 88 | 11 | 13 | 11 | 9 | |||||||
| 2500 | A | 89 | 86 | 9 | 10 | 12 | 11 | ||||||
| B | 88 | 87 | 9 | 12 | 9 | 9 | |||||||
| 5000 | A | 86 | 78 | 9 | 10 | 13 | 14 | ||||||
| B | 88 | 92 | 11 | 10 | 16 | 15 | |||||||
| 150 EMS | A | 69 | 54 | 34 | 34 | 45 | 39 | ||||||
| B | 67 | 60 | 20 | 29 | 36 | 31 | |||||||
Mutation frequencies
| Treatment | Viable | Small colonies | Large colonies | Proportion small colony mutants | |||||
| (μg/ml) | |||||||||
| Mutants | Mutants | ||||||||
| Yv | Nv | Ym | Nm | MF§ | Ym | Nm | MF§ | ||
| 0 | 182 | 768 | 708 | 768 | 56.5 | 684 | 768 | 80.5 | 0.42 |
| 156.25 | 65 | 384 | 354 | 384 | 45.8 | 351 | 384 | 50.6 | 0.48 |
| 312.5 | 81 | 384 | 352 | 384 | 55.9 | 349 | 384 | 61.4 | 0.48 |
| 625 | 58 | 384 | 357 | 384 | 38.6 | 335 | 384 | 72.2 | 0.36 |
| 1250 | 37 | 384 | 346 | 384 | 44.5 | 339 | 384 | 53.3 | 0.46 |
| 2500 | 34 | 384 | 344 | 384 | 45.4 | 343 | 384 | 46.6 | 0.49 |
| 5000 | 40 | 384 | 344 | 384 | 48.6 | 326 | 384 | 72.4 | 0.41 |
| 150 EMS | 134 | 384 | 267 | 384 | 345.2 | 233 | 384 | 474.5 | 0.44 |
Table 12. Cell and 96 -Well Plate Counts: Experiment 2 (+S9) 4 -Hour Exposure
| Treatment | Cell counts $ | Viability § | Resistant mutants § | |||||||||
| (μg/ml) | after day 2 | after day 2 | ||||||||||
| 2 cells/well | 2000 cells/well | |||||||||||
| 0 h | 24 h | 48 h | ||||||||||
| 0 | A | 8.67 | 7.26 | 7.31 | 75 | 76 | 81 | 73 | 17 | 18 | 16 | 16 |
| B | 8.77 | 6.69 | 7.26 | 74 | 74 | 70 | 77 | 16 | 14 | 19 | 17 | |
| 39.06 | A | 8.54 | 7.46 | 6.80 | NP | NP | NP | NP | ||||
| B | 8.35 | 7.19 | 7.27 | NP | NP | NP | NP | |||||
| 78.13 | A | 7.61 | 7.62 | 7.30 |
NP | NP | NP | NP | ||||
| B | 8.75 | 6.99 | 6.63 | NP | NP | NP | NP | |||||
| 156.25 | A | 8.10 | 7.56 | 7.33 | 77 | 80 | 16 | 21 | ||||
| B | 8.01 | 8.11 | 6.98 | 79 | 73 | 18 | 20 | |||||
| 312.5 | A | 8.33 | 7.80 | 5.76 | 76 | 82 | 16 | 15 | ||||
| B | 8.36 | 7.26 | 6.69 | 87 | 81 | 18 | 15 | |||||
| 625 | A | 6.10 | 7.60 | 7.15 | 77 | 75 | 18 | 15 | ||||
| B | 5.50 | 6.35 | 6.25 | 85 | 80 | 17 | 19 | |||||
| 1250 | A | 5.95 | 5.85 | 6.70 | 87 | 87 | 21 | 22 | ||||
| B | 5.90 | 6.75 | 7.30 | 85 | 87 | 17 | 18 | |||||
| 2500 | A | 7.10 | 5.10 | 6.60 | 92 | 88 | 17 | 22 | ||||
| B | 6.20 | 4.95 | 7.65 | 89 | 86 | 24 | 16 | |||||
| 5000 | A | 4.50 | 6.60 | 7.20 | 87 | 79 | 21 | 20 | ||||
| B | 4.55 | 5.30 | 6.75 | 89 | 87 | 24 | 23 | |||||
| Positive control CP (μg/ml) | ||||||||||||
| 1.5 | A | 8.25 | 6.82 | 5.94 | 74 | 66 | 67 | 52 | ||||
| B | 8.35 | 6.07 | 5.81 | 74 | 60 | 56 | 50 | |||||
Table 13. Statistical Analysis: Experiment 2 (+S9) 4-Hour Exposure
| Treatment | SG | %RSG | %V | RTG | MF§ | |
| (μg/ml) | ||||||
| 0 | 12.7 | 100 | 75.99 | 1 | 125.12 | |
| 39.06 | Ø | 12.88 | 98 | |||
| 78.13 | Ø | 12.72 | 94 | |||
| 156.25 | 14.01 | 102 | 81.66 | 1.09 | 133.06 | |
| 312.5 | 11.72 | 88 | 94.51 | 1.09 | 96.46 | |
| 625 | 11.68 | 61 | 87.3 | 0.71 | 113.45 | |
| 1250 | 11.03 | 59 | 115.65 | 0.9 | 98.16 | |
| 2500 | 8.95 | 54 | 129.17 | 0.91 | 89.16 | |
| 5000 | 10.38 | 42 | 110.65 | 0.62 | 117.62 | |
| Positive control EMS | ||||||
| Treatment | SG | %RSG | %V | RTG | MF§ | |
| (μg/ml) | ||||||
| 1.5 | 9.47 | 71 | 62.51 | 0.58 | 705.3 | |
Test for linear trend
| Slope | -2.07E-09 |
| Variance | 8.32E-18 |
| b²/Sb | 0.513 |
Table 14. Large and Small Colonies Analysis: Experiment 2 (+S9) 4 -Hour Exposure
| Treatment | Viability # | Small colonies # | Large colonies # | ||||||||||
| (μg/ml) | after day 2 | after day 2 | after day 2 | ||||||||||
| 0 | A | 75 | 76 | 81 | 73 | 8 | 7 | 8 | 6 | 9 | 11 | 8 | 10 |
| B | 74 | 74 | 70 | 77 | 7 | 8 | 11 | 9 | 9 | 6 | 8 | 8 | |
| 156.25 | A | 77 | 80 | 7 | 9 | 9 | 12 | ||||||
| B | 79 | 73 | 10 | 9 | 8 | 11 | |||||||
| 312.5 | A | 76 | 82 | 7 | 7 | 9 | 8 | ||||||
| B | 87 | 81 | 8 | 9 | 10 | 6 | |||||||
| 625 | A | 77 | 75 | 5 | 7 | 13 | 8 | ||||||
| B | 85 | 80 | 6 | 9 | 11 | 10 | |||||||
| 1250 | A | 87 | 87 | 10 | 9 | 11 | 13 | ||||||
| B | 85 | 87 | 8 | 10 | 9 | 8 | |||||||
| 2500 | A | 92 | 88 | 9 | 5 | 8 | 17 | ||||||
| B | 89 | 86 | 11 | 6 | 13 | 10 | |||||||
| 5000 | A | 87 | 79 | 9 | 7 | 12 | 13 | ||||||
| B | 89 | 87 | 10 | 8 | 14 | 15 | |||||||
| 1.5 CP | A | 74 | 66 | 47 | 38 | 20 | 14 | ||||||
| B | 74 | 60 | 40 | 37 | 16 | 13 | |||||||
Mutation frequencies
| Treatment | Viable | Small colonies | Large colonies | Proportion small colony mutants | |||||
| (μg/ml) | |||||||||
| Mutants | Mutants | ||||||||
| Yv | Nv | Ym | Nm | MF§ | Ym | Nm | MF§ | ||
| 0 | 168 | 768 | 704 | 768 | 57.3 | 699 | 768 | 61.9 | 0.48 |
| 156.25 | 75 | 384 | 349 | 384 | 58.5 | 344 | 384 | 67.4 | 0.47 |
| 312.5 | 58 | 384 | 353 | 384 | 44.5 | 351 | 384 | 47.5 | 0.48 |
| 625 | 67 | 384 | 357 | 384 | 41.8 | 342 | 384 | 66.3 | 0.39 |
| 1250 | 38 | 384 | 347 | 384 | 43.8 | 343 | 384 | 48.8 | 0.47 |
| 2500 | 29 | 384 | 353 | 384 | 32.6 | 336 | 384 | 51.7 | 0.39 |
| 5000 | 42 | 384 | 350 | 384 | 41.9 | 330 | 384 | 68.5 | 0.39 |
| 1.5 CP | 110 | 384 | 222 | 384 | 438.3 | 321 | 384 | 143.3 | 0.72 |
Table 15. Historical Vehicle and Positive Control Mutation Frequencies
Experiments -S9
| Vehicle control | Positive control |
| MF | MF |
| 145.91 | 950.8 |
| 120.76 | 1112.08 |
| 143.66 | 896.5 |
| 162.69 | 1229.56 |
| 158.66 | 1390.11 |
| 144.88 | 1458.54 |
| 105.22 | 1119.99 |
| 95.59 | 1090.89 |
| 114.37 | 1030.44 |
| 140.14 | 1014.32 |
| 99.14 | 1219.66 |
| 110.92 | 861.66 |
| 135.32 | 880.2 |
| 165.68 | 1305.45 |
| 125.21 | 905.45 |
| 118.81 | 1456.13 |
| 132.29 | 971.93 |
| 125.59 | 783.41 |
| 130.09 | 1021.61 |
| 160.24 | 674.85 |
Table 16. Historical Vehicle and Positive Control Mutation Frequencies
Experiments +S9
| Vehicle control | Positive control |
| MF | MF |
| 96.54 | 1073.42 |
| 120.03 | 1221.59 |
| 151.9 | 830.77 |
| 140.46 | 1320.32 |
| 126.59 | 742.1 |
| 175.99 | 1137.22 |
| 179.16 | 1474.77 |
| 110.11 | 931.18 |
| 107.82 | 892.85 |
| 133.99 | 1335.45 |
| 118.75 | 796.22 |
| 127.35 | 613.66 |
| 134.47 | 1089.84 |
| 131.07 | 905.61 |
| 118.37 | 1427.97 |
| 108.9 | 963.74 |
| 141.92 | 1000 |
| 109.28 | 724.93 |
| 127.28 | 1120.01 |
| 156.35 | 695.46 |
Preliminary Toxicity Test
Table 1. Mitotic Index - Preliminary Toxicity Test
| Dose Level (μg/mL) | 4(20)-Hour Without S9 | 4(20)-Hour With S9 | 24-Hour Without S9 | |||
| Mitotic Index | % of Control | Mitotic Index | % of Control | Mitotic Index | % of Control | |
| 0 | 6.3 | 100 | 6 | 100 | 4.05 | 100 |
| 7.81 | 3.65 | 58 | 3.7 | 62 | 3.45P‡ | 85 |
| 15.63 | 3.8P‡ | 60 | 5.15P‡ | 86 | 3.45P‡ | 85 |
| 31.25 | -P‡ | - | -P‡ | - | -P‡ | - |
| 62.5 | -P‡ | - | -P‡ | - | -P‡ | - |
| 125 | -P‡ | - | -P‡ | - | -P‡ | - |
| 250 | -P‡ | - | -P‡ | - | -P‡ | - |
| 500 | -P‡ | - | -P‡ | - | -P‡ | - |
| 1000 | -P‡ | - | -P‡ | - | NM P‡ | NM |
| 2000 | -P‡ | - | -P‡ | - | NM P‡ | NM |
- = Not assessed for mitotic index
NM = No metaphases or insufficient metaphases suitable for scoring
P = Precipitate observed at end of exposure period in blood-free cultures
‡ = Precipitate observed on the slides
Chromosome Aberration Test – Main Experiment
Table 2. Mitotic Index – Main Experiment (4(20)-hour Exposure Groups)
| Dose Level (μg/mL) | 4(20)-Hour Without S9 | 4(20)-Hour With S9 | ||||||
| A | B | Mean | % of Control | A | B | Mean | % of Control | |
| 0 | 3.5 | 6.55 | 5.03 | 100 | 5.5 | 4.8 | 5.15 | 100 |
| 0.09 | - | - | - | - | - | - | - | - |
| 0.188 | - | - | - | - | - | - | - | - |
| 0.375 | - | - | - | - | - | - | - | - |
| 0.75 | - | - | - | - | - | - | - | - |
| 1.25 | 7.05 | 5.1 | 6.08 | 121 | 4.6 | 4.2 | 4.4 | 85 |
| 2.5 | 4.7 | 4 | 4.35 | 87 | 7.95 | 4 | 5.98 | 116 |
| 5 | 6.45P | 3.6P | 5.03 | 100 | 6.55P | 7.4P | 6.98 | 135 |
| MMC 0.3 | 2.8 | 2.25 | 2.53 | 50 | NA | NA | NA | NA |
| CP 3 | NA | NA | NA | NA | 1.55 | 1.4 | 1.48 | 29 |
MMC = Mitomycin C
CP = Cyclophosphamide
NA = Not applicable
- = Not assessed for mitotic index
P = Precipitate observed at end of exposure period in blood-free cultures
Table 3. Mitotic Index – Main Experiment (24-hour Exposure Group)
| Dose Level (μg/mL) | 4(20)-Hour Without S9 | |||
| A | B | Mean | % of Control | |
| 0 | 6.75 | 5.5 | 6.13 | 100 |
| 0.09 | - | - | - | - |
| 0.188 | - | - | - | - |
| 0.375 | - | - | - | - |
| 0.75 | - | - | - | - |
| 1.25 | 3.8 | 3.65 | 3.73 | 61 |
| 2.5 | 5.1 | 5.2 | 5.15 | 84 |
| 5 | 5.05P | 4.6P | 4.83 | 79 |
| MMC 0.1 | 1.65 | 2.05 | 1.85 | 30 |
MMC = Mitomycin C
- = Not assessed for mitotic index
P = Precipitate observed at end of exposure period in blood-free cultures
Table 4. Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure Without Metabolic Activation (S9)
| Treatment Group | Replicate | Mitotic Index (%) |
Number of Cells Scored |
Number of Aberrations | Total Number of Aberrations | Frequency of Aberrant Cells | |||||||
| Gaps | Chromatid | Chromosome | Others | ||||||||||
| Breaks | Exchanges | Breaks | Exchanges | X | (+ Gaps) | (-Gaps) | (+ Gaps) | (-Gaps) | |||||
| Vehicle Control (MEM) |
A | 3.5 | 150 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 |
| B | 6.55 | 150 | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | |
| Total | (100) | 300 | 1 | 1 | 0 | 0 | 0 | 0 | 2 | 1 | 2 (0.7) | 1 (0.3) | |
| 1.25 ug/mL | A | 7.05 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 5.1 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Total | (121) | 300 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 (0.0) | 0 (0.0) | |
| 2.5 ug/mL | A | 4.7 | 150 | 0 | 2 | 0 | 1 | 1 | 0 | 4 | 4 | 4 | 4 |
| B | 4 | 150 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 1 | |
| Total | (87) | 300 | 0 | 2 | 0 | 1 | 2 | 0 | 5 | 5 | 5 (1.7) | 5 (1.7) | |
| 5 ug/mL | A | 6.45 | 150 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 |
| B | 3.6 | 150 | 2 | 0 | 0 | 1 | 0 | 0 | 3 | 1 | 3 | 1 | |
| Total | (100) | 300 | 3 | 0 | 0 | 1 | 0 | 0 | 4 | 1 | 4 (1.3) | 1 (0.3) | |
| Positive Control MMC 0.3 ug/mL | A | 2.8 | 44a | 3 | 11 | 5 | 1 | 0 | 0 | 20 | 17 | 16 | 15 |
| B | 2.25 | 44a | 3 | 10 | 14 | 5 | 1 | 0 | 33 | 30 | 17 | 15 | |
| Total | (50) | 88 | 6 | 21 | 19 | 6 | 1 | 0 | 53 | 47 | 33 (37.5) | 30*** (34.1) | |
MMC = Mitomycin C
a Slide evaluation terminated when 15 metaphases with aberrations (excluding gaps) had been observed
*** P < 0.001
MEM Minimal Essential Medium
Table 5. Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure With Metabolic Activation (2% S9)
| Treatment Group | Replicate | Mitotic Index (%) |
Number of Cells Scored |
Number of Aberrations | Total Number of Aberrations | Frequency of Aberrant Cells | |||||||
| Gaps | Chromatid | Chromosome | Others | ||||||||||
| Breaks | Exchanges | Breaks | Exchanges | X | (+ Gaps) | (-Gaps) | (+ Gaps) | (-Gaps) | |||||
| Vehicle Control (MEM) |
A | 5.5 | 150 | 2 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 2 | 0 |
| B | 4.8 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Total | (100) | 300 | 2 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 2 (0.7) | 0 (0.0) | |
| 1.25 ug/mL | A | 4.6 | 150 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 |
| B | 4.2 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Total | (85) | 300 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 (0.3) | 0 (0.0) | |
| 2.5 ug/mL | A | 7.95 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 4 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Total | (116) | 300 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 (0.0) | 0 (0.0) | |
| 5 ug/mL | A | 6.55 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 7.4 | 150 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | |
| Total | (135) | 300 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 (0.3) | 0 (0.0) | |
| Positive Control CP 3 ug/mL | A | 1.55 | 70a | 1 | 14 | 1 | 0 | 0 | 0 | 16 | 15 | 15 | 15 |
| B | 1.4 | 73a | 2 | 13 | 2 | 2 | 0 | 0 | 19 | 17 | 16 | 15 | |
| Total | (29) | 143 | 3 | 27 | 3 | 2 | 0 | 0 | 35 | 32 | 31 (21.7) | 30*** (21.0) | |
CP = Cyclophosphamide
a Slide evaluation terminated when 15 metaphases with aberrations (excluding gaps) had been observed
*** P < 0.001
MEM Minimal Essential Medium
Table 6. Results of Chromosome Aberration Test – Main Experiment 24-hour Continuous Exposure
Without Metabolic Activation (S9)
| Treatment Group | Replicate | Mitotic Index (%) |
Number of Cells Scored |
Number of Aberrations | Total Number of Aberrations | Frequency of Aberrant Cells | |||||||
| Gaps | Chromatid | Chromosome | Others | ||||||||||
| Breaks | Exchanges | Breaks | Exchanges | X | (+ Gaps) | (-Gaps) | (+ Gaps) | (-Gaps) | |||||
| Vehicle Control (MEM) |
A | 6.75 | 150 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 |
| B | 5.5 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Total | (100) | 300 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 (0.3) | 0 (0.0) | |
| 1.25 ug/mL | A | 3.8 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 3.65 | 150 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | |
| Total | (61) | 300 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 (0.3) | 0 (0.0) | |
| 2.5 ug/mL | A | 5.1 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 5.2 | 150 | 2 | 1 | 0 | 0 | 0 | 0 | 3 | 1 | 3 | 1 | |
| Total | (84) | 300 | 2 | 1 | 0 | 0 | 0 | 0 | 3 | 1 | 3 (1.0) | 1 (0.3) | |
| 5 ug/mL | A | 5.05 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B | 4.6 | 150 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Total | (79) | 300 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 (0.0) | 0 (0.0) | |
| Positive Control CP 3 ug/mL | A | 1.65 | 29a | 0 | 11 | 4 | 4 | 0 | 0 | 19 | 19 | 15 | 15 |
| B | 2.05 | 125a | 0 | 13 | 5 | 0 | 0 | 0 | 18 | 18 | 15 | 15 | |
| Total | (30) | 154 | 0 | 24 | 9 | 4 | 0 | 0 | 37 | 37 | 30 (19.5) | 30*** (19.5) | |
MMC = Mitomycin C
a Slide evaluation terminated when 15 metaphases with aberrations (excluding gaps) had been observed
*** P < 0.001
MEM Minimal Essential Medium
Table 7. Mean Frequency of Polyploid Cells (%),Main Experiment
| Dose Level (μg/mL) |
Exposure Group | ||
| 4(20)-Hour Without S9 | 4(20)-Hour With S9 | 24-Hour Without S9 | |
| 0 | 0 | 0 | 0 |
| 1.25 | 0 | 0 | 0 |
| 2.5 | 0 | 0.3 | 0 |
| 5 | 0 | 0 | 0 |
| MMC 0.4 | 0 | NA | NA |
| MMC 0.1 | NA | NA | 0 |
| CP 3 | NA | 0 | NA |
MMC = Mitomycin C
CP = Cyclophosphamide
NA = Not applicable
Table 8. Historical Aberration Ranges for Vehicle Control Cultures
| 4(20)-hour exposure without S9 |
4(20)-hour exposure with S9 (1%) |
24-hour exposure without S9 |
4(20)-hour exposure with S9 (2%) |
|||||
| % cells with aberrations (-gaps) |
% cells with polyploids |
% cells with aberrations (-gaps) |
% cells with polyploids |
% cells with aberrations (-gaps) |
% cells with polyploids |
% cells with aberrations (-gaps) |
% cells with polyploids |
|
| Minimum | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Maximum | 3 | 1 | 2 | 0.5 | 2.5 | 1 | 2 | 1 |
| Mean | 0.48 | 0.05 | 0.4 | 0.02 | 0.44 | 0.07 | 0.53 | 0.09 |
| Standard Deviation | 0.56 | 0.17 | 0.48 | 0.11 | 0.52 | 0.22 | 0.56 | 0.24 |
| Number | 88 | 88 | 87 | 87 | 86 | 86 | 87 | 87 |
Table 9. Historical Aberration Range for Positive Control Cultures
| 4(20)-hour exposure without S9 |
4(20)-hour exposure with S9 (1%) |
24-hour exposure without S9 |
4(20)-hour exposure with S9 (2%) |
|||||
| % cells with aberrations (-gaps) |
% cells with polyploids |
% cells with aberrations (-gaps) |
% cells with polyploids |
% cells with aberrations (-gaps) |
% cells with polyploids |
% cells with aberrations (-gaps) |
% cells with polyploids |
|
| Minimum | 14 | 0 | 10 | 0 | 8.7 | 0 | 7.3 | 0 |
| Maximum | 72 | 1 | 53 | 1 | 68 | 1 | 54 | 1 |
| Mean | 39.86 | 0.02 | 30.66 | 0.02 | 39.09 | 0.02 | 25.32 | 0.06 |
| Standard Deviation | 14.04 | 0.13 | 9.65 | 0.12 | 14.57 | 0.013 | 10.09 | 0.21 |
| Number | 88 | 88 | 87 | 87 | 87 | 87 | 88 | 88 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Justification for selection of genetic toxicity endpoint
No study was selected, since all three in vitro studies were
negative.
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
There is no reason to believe that the negative results of all the three in vitro tests in genetic toxicity would not be relevant to humans. The substance is not classified as mutagenic.
Additional information
All the in vitro studies were GLP compliant guideline studies and
negative in terms of adverse effects observed. There are no data gaps in
genetic toxicity. Since all three in vitro studies were negative, there
is no need to perform in vivo studies on genetic toxicity. Therefore,
there is no reason to believe that these results would not be relevant
to humans.
Short description of key information:
Clearly negative in vitro studies - both with and without metabolic
activation.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
The substance does not fulfill the classification criteria as mutagenic according to the CLP Regulation (EC No 1272/2008). All three in vitro studies were negative.
Reverse Mutation Assay 'Ames Test' using Salmonella typhimurium and Escherichia coli: the test item was considered to be non-mutagenic under the conditions of the test.
L5178Y TK +/- Mouse Lymphoma Assay: the test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.
Chromosome Aberration Test in Human Lymphocytes in vitro: the test item was considered to be non-clastogenic to human lymphocytes in vitro.
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