Registration Dossier
Registration Dossier
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EC number: 701-186-2 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro bacterial reverse mutation test: Key study: Test method according to the OECD Guideline 471 with GLP study. The test item was found non-mutagenic on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) tester strains up to the highest tested concentration of 5 mg/plate both in presence and absence of metabolic activation.
In vitro mammalian chromosomal aberration test: Key study: Test method according to the OECD Guideline 473 with GLP study. The test item was found as non-clastogenic up to the concentration of 0.25 mg/mL both in the presence and absence of metabolic activation under the test conditions.
In vitro mammalian cell gene mutation test: Key study: Test method according to the OECD Guideline 476 with GLP study. The test item was found as non-mutagenic up to the concentration of 0.5 mg/mL, both in the presence and absence of metabolic activation under the test conditions.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19 July 2020 to 20 August 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: CHO AA8 cells, Batch No.5000062 procured from American Type Culture Collection (ATCC)
- Suitability of cells: The CHO AA8 cells are one of the recommended test systems by regulatory agencies for conducting in vitro mammalian gene mutation test.
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Alpha Minimal Essential Medium (MEM) without ribonucleosides containing 10% Fetal Bovine Serum (FBS) and antibiotics (1% Penicillin and streptomycin) were used. The pH of the culture medium was 7.32 to 7.33. The media was stored at 2 to 8ºC till use thawed to room temperature before use. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: liver of male Wistar rats.
- method of preparation of S9 mix: The S9 homogenate was prepared from male wistar rats induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. One mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.32 and 7.33.
- concentration or volume of S9 mix and S9 in the final culture medium:
1 mL, 10% (v/v) S9 mix and 0.1 mL, 1% (v/v) S9.
- quality controls of S9: Batch of S9 homogenate was assessed for sterility, protein content and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain. - Test concentrations with justification for top dose:
- 0.0625, 0.125, 0.25 and 0.5 mg/mL.
The top dose was selected based on preliminary solubility and precipitation tests as well as an initial cytotoxicity test. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on the solubility results.
- Percentage of solvent in the final culture medium: 1% v/v. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 (cytotoxicity and cloning efficiency in non-selective medium) and 5 (cloning efficiency of mutant colonies in selective medium)
- Number of independent experiments: 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approx. 2 x 10^6 cells/culture flask (Initial cytotoxicity test and Gene mutation test).
- Test substance added in medium.
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours and 2 minutes at 37±1ºC with 5±1% CO2.
- Harvest time after the end of treatment (sampling/recovery times): 7 days
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 8 days
- Selection time (if incubation with a selective agent): 8 days.
- Method used: monolayer cultures.
- Selective agent: 10 μM of 6-Thioguanine. Flasks were incubated at 37±1°C with 5±1% CO2 for 8 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 200 x 3 per group (cytotoxicity and cloning efficiency in non-selective medium) and 4x10^5 x 5 per group (cloning efficiency of mutant colonies in selective medium). Flasks were incubated at 37±1°C with 5±1% CO2 for 8 days. Post incubation period, medium from each dish was aspirated and stained with 5% Giemsa stain, number of colonies formed were counted manually.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)
- Any supplementary information relevant to cytotoxicity: An initial cytotoxicity test was carried out at five different concentrations (0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL of the test item). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival according to formulas included in annex 2 of OECD TG 476. - Rationale for test conditions:
- In preliminary solubility and precipitation tests, heavy precipitations were observed at 1 and 2 mg/mL while only slight precipitation was observed at 0.5 mg/mL which was selected as highest dose in the initial cytotoxicity test. At 0.5 mg/mL, the Relative Survival was greater than 10%. Therefore 0.5 mg/mL was selected as the highest concentration for testing in the gene mutation test.
- Evaluation criteria:
- A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
1) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2) The increase is concentration-related when evaluated with an appropriate trend test.
3) Any of the results are outside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
A test chemical is considered clearly negative if, in all experimental conditions examined:
1) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2) There is no concentration-related increase when evaluated with an appropriate trend test.
3) All results are inside the distribution of the historical negative/vehicle control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formula: Y=(X+A)eB, where Y = transformed mutant frequency, X = observed mutant frequency and A, B = constants (viz. A = 1 and B = 0.15). Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0.
The statistical significances are designated by the superscripts as given below:
* Statistically significant (p<0.05) change than the vehicle control group. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- CHO AA8 Cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No change in pH was observed in any of the test concentrations.
- Possibility of evaporation from medium: no
- Precipitation and time of the determination: slight precipitation was observed at 0.5 mg/mL, heavy precipitation was observed at 1 and 2 mg/mL.
RANGE-FINDING/SCREENING STUDIES (if applicable):
In the initial cytotoxicity test, there was no evidence of excessive cytotoxicity (˂10% RS) up to 0.5 mg/mL in both presence of metabolic activation and absence of metabolic activation when compared to vehicle control. In the presence of metabolic activation, the RS values ranged from 50.41 % to 90.08 % and in the absence of metabolic activation, the RS values ranged from 52.54 % to 90.68 % at the concentrations of 0.03125, 0.0625, 0.125, 0.25, 0.5 mg/mL.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: mutant frequencies of 22.58 and 24.73 per 2×10^6 cells were observed in the vehicle control, with and without metabolic activation, respectively. Positive controls Benzo (a) pyrene and 4-Nitroquinoline N-oxide gave mutant frequencies of 268.13 and 264.13 per 2×10^6 cells, respectively.
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative survival (RS): There was no evidence of excessive cytotoxicity (˂10% RS) at any of the concentrations both in presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 50.43 to 84.35 % and in the absence of metabolic activation the RS values ranged from 51.72 to 83.62 % respectively.
- Genotoxicity results:
The test item, resulted in mutant frequencies of 22.99 to 23.60 per 2×10^6 cells in the presence of metabolic activation and mutant frequencies of 25.00 to 25.29 per 2×10^6 cells in the absence of metabolic activation.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see Table 7
- Negative (solvent/vehicle) historical control data: see Table 7 - Conclusions:
- In an in vitro HPRT gene mutation test using CHO AA8 cells, the test item is considered as non-mutagenic up to the concentration of 0.5 mg/mL, both in presence and absence of metabolic activation.
- Executive summary:
A gene mutation study at HPRT gene using CHO AA8 cells was performed for the test item, with and without metabolic activation (±S9), according to OECD 476 Guideline (GLP study). Based on the results of the solubility, pH and precipitation tests, the test item was formulated in DMSO as vehicle and 0.5 mg/mL was selected as the highest concentration in an initial cytotoxicity test. This test was conducted at concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL. There was no evidence of excessive cytotoxicity (˂10% RS) at and up to 0.5 mg/mL in both presence and absence of metabolic activation when compared to vehicle control. Therefore, 0.5 mg/mL was selected as the highest concentration in the gene mutation test. The main test was conducted at the concentrations of 0.0625, 0.125, 0.25 and 0.5 mg/mL with an exposure time of 3 h and 2 min both in the presence and absence of metabolic activation and 6-Thioguanine as the selective agent. DMSO alone was tested as solvent control and Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls. Parallel cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival. There was no statistically significant increase in number of mutant colonies at any of the concentrations tested when compared with the vehicle control. All results were inside the distribution of the historical vehicle control data. Positive controls resulted in mutant frequencies, which were statistically significant when compared with the vehicle control and within the historical positive control data. Based on these results, the test item is considered as non-mutagenic up to the concentration of 0.5 mg/mL, both in the presence and absence of metabolic activation under the tested conditions.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19 June 2020 to 10 September 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: Human Peripheral Blood Lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Human Peripheral Blood Lymphocytes from blood bank (Sanjeevini Blood Bank, Vidyanagar, Tumkur)
- Suitability of cells: as per the regulatory requirements the human peripheral blood lymphocytes is one of the recommended test systems
For lymphocytes:
- Sex, age and number of blood donors: 23 year-old male, non-smoking, no known illnesses or recent exposure to genotoxic chemicals or radiation was used. Blood was collected from a single donor.
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Roswell Park Memorial Institute (RPMI) media supplemented with 10% FBS and antibiotics (1% Penicillin-Streptomycin) was used. The pH of the culture medium used was in the range of 7.30 to 7.35. The media was stored at 2 to 8ºC till use and was thawed to room temperature before use. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: liver of male Wistar rats.
- method of preparation of S9 mix: The S9 homogenate was prepared from male Wistar rats induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL, respectively, for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. 1 mL of S9 homogenate was thawed immediately before use and mixed with the 9 mL of co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.31 for initial cytotoxicity test and pH 7.34 for chromosomal aberration test.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL, 10% (v/v) S9 mix and 0.05 mL, 1% (v/v) S9.
- quality controls of S9: Batch of S9 homogenate was assessed for sterility, protein content and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain. - Test concentrations with justification for top dose:
- 0.0625, 0.125 and 0.25 mg/mL (the highest dose with a percentage reduction in mitotic index (MI) no more than 45±5% in the initial cytotoxicity test).
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (50 μL per 5 mL medium)
- Justification for choice of solvent/vehicle: it forms a uniform suspension with the test item - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 44 to 48 hours, with phytohaemagglutinin (PHA) to induce cell division prior to exposure.
- Exposure duration/duration of treatment: 3 h and 45 min (short term treatment, +S9 and -S9); 21 h and 20 min (long term treatment, -S9)
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colchicine, 0.3 μg/mL, 1 to 3 hours incubation.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
After treatment, cell pellet was mixed with 4 mL of freshly prepared warm 0.56% Potassium chloride. Cell suspension was incubated for 10 min at RT and later it was centrifuged at 1800 rpm for 10 min. Supernatant was discarded and cell pellet was mixed with 4 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 min at RT and later suspension was centrifuged at 2200 rpm for 10 min. The procedure was repeated twice by adding 4 mL of cold acetic acid: methanol fixative (1:3).
Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension were aspirated and dropped onto the chilled slide. The slides were air dried. Minimum of 3 slides were prepared for each treatment replicate. Slides were stained using 5% Giemsa stain for 15 min.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 300 (150 per replicate).
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate (300 well spread metaphases per concentration). Aberrations identified were gaps, breaks, exchanges, fragments, ring, deletion, dicentric. Gaps were recorded separately and reported but not included in the total aberration frequency.
- Determination of polyploidy: yes
- Determination of endoreplication: yes
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: For each replicate minimum of 500 cells were scored. Cytotoxicity was determined by calculating percentage reduction in mitotic index by using the formula:
(%) Reduction in MI= [(Percentage MI of VC - Percentage MI of treated)/Percentage MI of VC] ×100; VC: Vehicle Control, MI: Mitotic Index. - Evaluation criteria:
- Positive result if, in any of the experimental conditions examined:
*At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
*The increase is dose-related when evaluated with an appropriate trend test.
*Any of the results are outside the distribution of the historical negative control data.
Negative result if, in all experimental conditions examined:
*None of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
*There is no concentration-related increase when evaluated with an appropriate trend test.
*All results are inside the distribution of the historical vehicle control data. - Statistics:
- Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p<0.05).
- Key result
- Species / strain:
- lymphocytes: Human Peripheral Blood Lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (see Table 2)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES (if applicable):
In a precipitation test conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL, after 24 h no precipitation was observed at 0.03125, 0.0625, 0.125 and 0.25 mg/mL, slight precipitation was observed at 0.5 mg/mL and heavy precipitation at 1 and 2 mg/mL.
Based on these results an initial cytotoxicity study up to 0.5 mg/mL was conducted. Reduction in % MI at 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL was 10.18, 20.10, 31.70, 40.08 and 66.24 in presence of metabolic activation (3 to 6 hours), 9.93, 21.31, 36.21, 41.83 and 66.93 in absence of metabolic activation (3 to 6 hours) and 14.62, 24.36, 30.00, 43.72 and 70.00 in absence of metabolic activation (20 to 24 hours) respectively.
Based on these results, the chromosomal aberration test was conducted up to 0.25 mg/mL.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: see Tables 2 and 4.
Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI): The reduction in mitotic index observed in test item concentration at the highest tested dose (0.25 mg/mL) was 38.1, 37.47 and 39.44 in the presence of metabolic activation (3 to 6 hours), in the absence of metabolic activation (3 to 6 hours) and in the absence of metabolic activation system (20 to 24 hours) respectively.
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps: see Table 4
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps: see Table 4
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes): Not seen.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: See Table 5
- Negative (solvent/vehicle) historical control data: See Table 5 - Conclusions:
- In an in vitro mammalian chromosomal aberration test in human lymphocytes, the test item was found as non-clastogenic up to the concentration of 0.25 mg/mL, both in presence and absence of metabolic activation.
- Executive summary:
The test substance was evaluated for chromosomal aberrations in human lymphocytes according to OECD Guideline 473, following the Principles of GLP. Based on the results of the solubility and precipitation tests, the test item was formulated in DMSO as vehicle and 0.5 mg/mL was selected as highest concentration in an initial cytotoxicity test. This test was conducted at concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL. The percentage reduction in Mitotic Index (MI) was in the range of 66.24 to 70.00 at 0.5 mg/mL and 9.93 to 43.72 at 0.03125, 0.0625, 0.125 and 0.25 mg/mL. The dose of 0.25 mg/mL was selected as the highest concentration for the chromosomal aberration test as the reduction in % MI was not more than 45±5%. Cultured human peripheral blood lymphocytes previously incubated for 44 -48 h with PHA in RPMI medium were treated with test item in DMSO at the concentrations of 0.0625, 0.125 and 0.25 mg/mL. The treatment was carried out in duplicates for the short term period (3 h and 45 min) both in the presence and absence of metabolic activation (S9 mix) and for the long term period (21 h and 20 min) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at 10 µg/mL and Mytomycin-C at 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls. Colchicine was used as the metaphase-arresting substance. There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentration levels tested. The mean reduction in MI at the highest dose of 0.25 mg/mL was 37.47 – 39.44 % for all the treatments. The concurrent vehicle and positive control values were within the 95% control limits of the distribution of the laboratory’s historical vehicle and positive control database. All acceptability criteria were met. Based on these results, the test item is considered as non-clastogenic up to the concentration of 0.25 mg/mL both in the presence and absence of metabolic activation under the presented test conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 April 2020 to 20 April 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- 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
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: liver of male Wistar rats.
- method of preparation of S9 mix: The S9 homogenate was prepared from male Wistar rats induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL, respectively, for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. A volume of 1 mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM Nicotinamide Adenine Dinucleotide Phosphate (NADP) disodium salt, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.30 for initial cytotoxicity, plate incorporation and preincubation method.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL, 10% (v/v) S9 mix and 0.05 mL, 1% (v/v) S9.
- quality controls of S9: Each batch of S9 homogenate was assessed for sterility by streaking the supernatant fluid on Nutrient Agar plates and incubated at 37±1ºC for 24 hours. It was found sterile and was further evaluated for its protein content (Modified Lowry Assay, Sword and Thomson, 1980) and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain. The results were found to be acceptable for the tested parameters. - Test concentrations with justification for top dose:
- 0.05, 0.16, 0.5, 1.6 and 5 mg/plate were selected (with half-log dose interval) for plate incorporation method and preincubation method. Top dose of 5 mg/plate was set after initial cytotoxicity test in which no cytotoxicity was found up to 5 mg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was found insoluble in distilled water, ethanol, acetone and formed suspension in dimethyl sulphoxide and dimethyl formamide at a concentration of 50 mg/mL. Dimethyl sulphoxide was selected as vehicle for the study. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2 (plate incorporation and preincubation methods).
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 18E8 cells/mL.
- Test substance added in agar (plate incorporation, first trial); preincubation (second trial).
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 23 minutes (preincubation method)
- Exposure duration/duration of treatment: 64 hours and 3 minutes (plate incorporation method) and 64 hours and 41 minutes (preincubation method)
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Bacterial Background lawn (see Table 1).
- Any supplementary information relevant to cytotoxicity: The initial cytotoxicity test was conducted for the selection of test concentration for the mutation assay. S. typhimurium TA100 tester strain was exposed to concentrations of 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 5 mg/plate of test item in triplicate, both in the presence and absence of metabolic activation along with concurrent vehicle control (DMSO). Each concentration of test item was mixed with soft agar containing histidine and biotin, S9 mix (for presence of metabolic activation), PBS (-S9), Salmonella typhimurium TA100 of cell density approximately 18×108 cells/mL overlaid on to pre-labeled minimal glucose agar plates. The plates were incubated at 37±1ºC for 48 hours and 16 minutes.
METHODS FOR MEASUREMENT OF GENOTOXICITY
The bacterial suspension of each tester strain was diluted up to 10-7 in Phosphate Buffer Saline and 1000 μL of the diluted suspension from each tester strain was plated onto nutrient agar plates in triplicate. The plates were incubated at 37±1ºC for approx. 64 hours for plate incorporation and for preincubation method. After incubation, the number of colonies in each plate were counted manually and expressed as number of Colony Forming Units per mL (CFU/mL) of the bacterial suspension. - Rationale for test conditions:
- Precipitation test: The test item resulted mild precipitation at 5 mg/plate, slight precipitation at 3.2 mg/plate and no precipitation at 0.1, 0.2, 0.4, 0.8, 1.6 mg/plate tested concentration.
Initial cytotoxicity test: The Test item showed no cytotoxicity at 0.00625 to 5 mg/plate tested concentrations with lawn intensity 4+ (Thick lawn) when compared to vehicle control. - Evaluation criteria:
- The conditions necessary for determining a positive result were established as follows:
There should be a concentration related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and Escherichia coli WP2 uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537. - Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle 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:
- no cytotoxicity
- Vehicle 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:
- no cytotoxicity
- Vehicle 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- STUDY RESULTS
- Concurrent vehicle negative and positive control data: Plate incorporation method: The specific positive controls tested simultaneously produced approximately 3.3 to 13.8 fold increase in mean number of revertants as compared to the vehicle control. Preincubation method: The specific positive controls tested simultaneously produced approximately 3.5 to 15.3 fold increase in mean number of revertants as compared to the vehicle control.
Ames test:
- Signs of toxicity: no
- Individual plate counts: yes
- Mean number of revertant colonies per plate and standard deviation: see Tables 2 and 3.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: See Table 4
- Negative (solvent/vehicle) historical control data: See Table 4 - Conclusions:
- In an in vitro bacterial reverse mutation test on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) tester strains, the test item is considered as non-mutagenic up to the highest tested concentration of 5 mg/plate, both in presence and absence of metabolic activation.
- Executive summary:
A bacterial reverse mutation study was performed for the test item according to OECD 471 Guideline (GLP study). After the initial precipitation and cytotoxicity tests, concentrations of 0.05, 0.16, 0.5, 1.6 and 5 mg/plate were chosen for both plate incorporation method and for preincubation method in the presence and absence of metabolic activation system (S9) using dimethyl sulphoxide as vehicle. Positives 2-nitrofluorene, sodium azide, 9-Aminoacridine and 4-nitroquinoline N-oxide for -S9 trials and 2-Aminoanthracene for +S9 trials were tested simultaneously.
The mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both trials. The number of revertant colonies in the positive controls resulted in 3.3 to 15.3 fold increase under identical conditions.
Therefore, based on the obtained results from this in vitro bacterial reverse mutation test on S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101) tester strains, the test item is considered as non-mutagenic up to the highest tested concentration of 5 mg/plate, both in presence and absence of metabolic activation, under the tested laboratory conditions.
Referenceopen allclose all
Table 1. Summary of initial cytotoxicity test.
| Set No. | Treatment | Concentration (mg/mL) | Average Colony Count ± SD | Cloning Efficiency (CE) | Adjusted Cloning Efficiency (ACE) | Relative Survival (RS) (%) |
| Set 1 +S9 | Vehicle Control (DMSO) | - | 187.33±5.13 | 0.94 | 1.21 | - |
| Ultramarine Violet Pigment Violet 15 CI 77007 | 0.03125 | 182.67±3.06 | 0.91 | 1.09 | 90.08 | |
| 0.0625 | 183.00±3.61 | 0.92 | 1.03 | 85.12 | ||
| 0.125 | 181.33±3.79 | 0.91 | 0.95 | 78.51 | ||
| 0.25 | 169.33±4.73 | 0.85 | 0.79 | 65.29 | ||
| 0.5 | 153.67±5.69 | 0.77 | 0.61 | 50.41 | ||
| Set 2 -S9 | Vehicle Control (DMSO) | - | 185.33±5.51 | 0.93 | 1.18 | - |
| Ultramarine Violet Pigment Violet 15 CI 77007 | 0.03125 | 179.00±3.00 | 0.90 | 1.07 | 90.68 | |
| 0.0625 | 175.00±5.57 | 0.88 | 1.01 | 85.59 | ||
| 0.125 | 171.33±3.21 | 0.86 | 0.91 | 77.12 | ||
| 0.25 | 165.67±8.33 | 0.83 | 0.80 | 67.80 | ||
| 0.5 | 156.00±7.94 | 0.78 | 0.62 | 52.54 |
+S9: with metabolic activation; -S9: without metabolic activation;
*Note: Cloning Efficiency = 200 cells plated for each replicate.
Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.
RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.
CE = Number of colonies/Number of cells plated.
Table 2. Summary of parallel cytotoxicity test-gene mutation test.
| Set No. | Treatment | Concentration (mg/mL) | Average Colony Count ± SD | Cloning Efficiency (CE) | Adjusted Cloning Efficiency (ACE) | Relative Survival (RS) (%) |
| Set 1 +S9 | Vehicle Control (DMSO) | - | 186.00±6.93 | 0.93 | 1.15 | - |
| Ultramarine Violet Pigment Violet 15 CI 77007 | 0.0625 | 174.00±4.00 | 0.87 | 0.97 | 84.35 | |
| 0.125 | 165.67±8.50 | 0.83 | 0.84 | 73.04 | ||
| 0.25 | 165.67±4.93 | 0.83 | 0.74 | 64.35 | ||
| 0.5 | 155.33±6.11 | 0.78 | 0.58 | 50.43 | ||
| Benzo(a)pyrene (Positive Control) | 3 μg/mL | 177.33±3.06 | 0.89 | 1.05 | 91.30 | |
| Set 2 -S9 | Vehicle Control (DMSO) | - | 187.00±3.61 | 0.94 | 1.16 | - |
| Ultramarine Violet Pigment Violet 15 CI 77007 | 0.0625 | 178.33±4.04 | 0.89 | 0.97 | 83.62 | |
| 0.125 | 168.00±10.58 | 0.84 | 0.88 | 75.86 | ||
| 0.25 | 168.00±5.29 | 0.84 | 0.74 | 63.79 | ||
| 0.5 | 155.67±4.51 | 0.78 | 0.60 | 51.72 | ||
| 4 Nitroquinoline N-oxide (Positive Control) | 1 μg/mL | 182.00±9.85 | 0.91 | 1.07 | 92.24 |
+S9: with metabolic activation; -S9: without metabolic activation;
*Note: Cloning Efficiency = 200 cells plated for each replicate.
RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.
CE = Number of colonies/Number of cells plated.
Adjusted CE = CE × Number of cells at the end of treatment/number of cells at the beginning of treatment.
Table 3. Summary of gene mutation test.
| Set No. | Treatment | Concentration (mg/mL) | *Average Colony Count ± SD | Cloning Efficiency in selective media | Cloning Efficiency in non-selective media | Total number of mutant colonies / 2x106 cells | Mutant Frequency/ 2x106 cells |
| Set 1 +S9 | Vehicle Control (DMSO) | - | 186.67±1.15 | 0.0000105 | 0.93 | 21 | 22.58 |
| Ultramarine Violet Pigment Violet 15 CI 77007 | 0.0625 | 182.00±2.00 | 0.0000105 | 0.91 | 21 | 23.08 | |
| 0.125 | 174.67±4.16 | 0.0000100 | 0.87 | 20 | 22.99 | ||
| 0.25 | 177.67±2.52 | 0.0000105 | 0.89 | 21 | 23.60 | ||
| 0.5 | 171.00±3.61 | 0.0000100 | 0.86 | 20 | 23.26 | ||
| Benzo(a)pyrene (Positive Control) | 3 μg/mL | 182.67±7.57 | 0.0001220 | 0.91 | 244 | 268.13** | |
| Set 2 -S9 | Vehicle Control (DMSO) | - | 186.00±4.58 | 0.0000115 | 0.93 | 23 | 24.73 |
| Ultramarine Violet Pigment Violet 15 CI 77007 | 0.0625 | 181.33±4.16 | 0.0000115 | 0.91 | 23 | 25.27 | |
| 0.125 | 175.00±5.00 | 0.0000110 | 0.88 | 22 | 25.00 | ||
| 0.25 | 175.67±5.09 | 0.0000110 | 0.88 | 22 | 25.00 | ||
| 0.5 | 174.67±7.57 | 0.0000110 | 0.87 | 22 | 25.29 | ||
| 4 Nitroquinoline N-oxide (Positive Control) | 1 μg/mL | 183.33±5.86 | 0.0000115 | 0.92 | 243 | 264.13** |
+S9: with metabolic activation; -S9: without metabolic activation; *Note: Cloning efficiency = 200 cells plated for each replicate.
**: Statistically significant (p˂0.05).
Mutant Frequency = Cloning efficiency of mutant colonies in selective medium/Cloning efficiency in non- selective medium.
Table 4. Individual data of initial cytotoxicity test.
Set No. | Treatment | Concentration (mg/mL) | Replicate | No. of Colonies/200 Cells | ||
R1 | R2 | R3 | ||||
Set 1 +S9 | Vehicle Control (DMSO) | - | 3 | 193 | 186 | 183 |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.03125 | 3 | 180 | 186 | 182 | |
0.0625 | 3 | 184 | 186 | 179 | ||
0.125 | 3 | 177 | 184 | 183 | ||
0.25 | 3 | 173 | 164 | 171 | ||
0.5 | 3 | 149 | 152 | 160 | ||
Set 2 -S9 | Vehicle Control (DMSO) | - | 3 | 191 | 180 | 185 |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.03125 | 3 | 182 | 179 | 176 | |
0.0625 | 3 | 176 | 180 | 169 | ||
0.125 | 3 | 175 | 169 | 170 | ||
0.25 | 3 | 159 | 163 | 175 | ||
0.5 | 3 | 159 | 162 | 147 | ||
Set No. | Treatment | Concentration (mg/mL) | Cell count (×106) | ||
Set 1 +S9 | End of Treatment | Vehicle Control (DMSO) | 27.60 | ||
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.03125 | 25.80 | |||
0.0625 | 24.00 | ||||
0.125 | 22.50 | ||||
0.25 | 20.10 | ||||
0.5 | 16.95 | ||||
Set 2 -S9 | End of Treatment | Vehicle Control (DMSO) | 27.30 | ||
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.03125 | 25.50 | |||
0.0625 | 24.60 | ||||
0.125 | 22.65 | ||||
0.25 | 20.70 | ||||
0.5 | 17.10 | ||||
Beginning of the Treatment | 21.50 | ||||
Table 5. Individual data of parallel cytotoxicity test.
Set No. | Treatment | Concentration (mg/mL) | Replicate | No. of Colonies/200 Cells | ||
R1 | R2 | R3 | ||||
Set 1 +S9 | Vehicle Control (DMSO) | - | 3 | 190 | 190 | 178 |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.0625 | 3 | 170 | 174 | 178 | |
0.125 | 3 | 169 | 172 | 156 | ||
0.25 | 3 | 160 | 168 | 169 | ||
0.5 | 3 | 154 | 150 | 162 | ||
Benzo(a)pyrene (Positive Control) | 3 µg/mL | 3 | 180 | 178 | 174 | |
Set 2 -S9 | Vehicle Control (DMSO) | - | 3 | 190 | 188 | 183 |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.0625 | 3 | 179 | 182 | 174 | |
0.125 | 3 | 176 | 172 | 156 | ||
0.25 | 3 | 170 | 162 | 172 | ||
0.5 | 3 | 151 | 156 | 160 | ||
4 Nitroquinoline N-oxide (Positive Control) | 1 µg/mL | 3 | 190 | 185 | 171 | |
Set No. | Treatment | Concentration (mg/mL) | Cell Count (×106) | |
Set 1 +S9 | End of Treatment | Vehicle Control (DMSO) | 27.30 | |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.0625 | 24.45 | ||
0.125 | 22.35 | |||
0.25 | 19.65 | |||
0.5 | 16.35 | |||
Positive Control Benzo(a)pyrene (3 µg/mL) | 25.95 | |||
Set 2 -S9 | End of Treatment | Vehicle Control (DMSO) | 27.15 | |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.0625 | 24.00 | ||
0.125 | 22.95 | |||
0.25 | 19.50 | |||
0.5 | 16.80 | |||
Positive Control 4 Nitroquinoline N-oxide (1 µg/mL) | 25.80 | |||
Beginning of the Treatment | 22.00 | |||
Table 6. Individual data of mutant phenotype
Set No. | Treatment | Concentration (mg/mL) | No. of Colonies/200 Cells | No. of Mutant Colonies/2×106Cells | ||||||
Replicate 1 | Replicate 2 | Replicate 3 | Replicate 1 | Replicate 2 | Replicate 3 | Replicate 4 | Replicate 5 | |||
Set 1 | Vehicle Control (DMSO) | - | 186 | 188 | 186 | 5 | 4 | 4 | 6 | 2 |
Ultramarine Violet Pigment Violet 15 CI 77007 | 0.0625 | 180 | 184 | 182 | 4 | 4 | 2 | 8 | 3 | |
0.125 | 176 | 170 | 178 | 0 | 6 | 5 | 4 | 5 | ||
0.25 | 180 | 178 | 175 | 3 | 8 | 2 | 6 | 2 | ||
0.5 | 175 | 170 | 168 | 2 | 2 | 10 | 1 | 5 | ||
Benzo(a) pyrene (Positive Control) | 3 µg/mL | 188 | 186 | 174 | 43 | 48 | 52 | 55 | 46 | |
Set 2 | Vehicle Control (DMSO) | - | 185 | 191 | 182 | 8 | 3 | 5 | 5 | 2 |
Ultramarine Violet Pigment Violet 15 CI 77007 | 0.0625 | 178 | 186 | 180 | 5 | 4 | 3 | 7 | 4 | |
0.125 | 180 | 170 | 175 | 2 | 1 | 7 | 9 | 3 | ||
0.25 | 176 | 179 | 175 | 4 | 6 | 5 | 2 | 5 | ||
0.5 | 180 | 178 | 166 | 9 | 1 | 7 | 0 | 5 | ||
4 Nitroquinoline N-oxide (Positive Control) | 1 µg/mL | 190 | 179 | 181 | 52 | 47 | 43 | 49 | 52 | |
Note: For cloning efficiency 200 cells were plated for each replicate and the No. of Mutant Colonies mentioned in the table is obtained from 2 x106 cells (that is from 5 replicates).
Table 7. Historical data.
| Vehicle-DMSO | With Metabolic Activation (3 to 6 hours) | Without Metabolic Activation (3 to 6 hours) |
| Mean Data of Mutant Frequency/2x106 Cells | 24.51 | 25.43 |
| Standard Deviation | 2.81 | 1.89 |
| Margin of Error | 1.95 | 1.31 |
| Upper bound | 26.46 | 26.74 |
| Lower bound | 22.56 | 24.12 |
| Positive Control- Benzo(a)pyrene and 4- Nitroquinoline N-oxide | With Metabolic Activation (3 to 6 hours) [Benzo(a)pyrene] | Without Metabolic Activation (3 to 6 hours)[4 Nitroquinoline N-oxide] |
| Mean Data of Mutant Frequency/2x106 Cells | 261.94 | 264.60 |
| Standard Deviation | 27.28 | 18.52 |
| Margin of Error | 17.82 | 12.10 |
| Upper bound | 279.76 | 276.70 |
| Lower bound | 244.12 | 252.50 |
Table 1. Summary of percentage mitotic index for initial cytotoxicity test
Set No. | Treatment | Concentrations (mg/mL) | Average % Mitotic Index | % Reduction of Mitotic Index |
Set 1 (+S9) (3 to 6 hours) | Vehicle Control | - | 7.76 | - |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.03125 | 6.97 | 10.18 | |
0.0625 | 6.20 | 20.10 | ||
0.125 | 5.30 | 31.70 | ||
0.25 | 4.65 | 40.08 | ||
0.5 | 2.62 | 66.24 | ||
Set 2 (-S9) (3 to 6 hours) | Vehicle Control | - | 7.65 | - |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.03125 | 6.89 | 9.93 | |
0.0625 | 6.02 | 21.31 | ||
0.125 | 4.88 | 36.21 | ||
0.25 | 4.45 | 41.83 | ||
0.5 | 2.53 | 66.93 | ||
Set 3 (-S9) (20 to 24 hours) | Vehicle Control | - | 7.80 | - |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.03125 | 6.66 | 14.62 | |
0.0625 | 5.90 | 24.36 | ||
0.125 | 5.46 | 30.00 | ||
0.25 | 4.39 | 43.72 | ||
0.5 | 2.34 | 70.00 |
Table 2. Summary of chromosomal aberrations and mitotic index
Set No. | Treatment | Concentrations (mg/mL) | Mean % MI | Mean % Reduction in MI | Mean of Total Aberrations with Gaps | Mean of Total Aberrations without Gaps | Mean of Total Aberrant cells without Gaps | Mean of Percentage Aberrated Cells |
Set 1 (+S9) (3 to 6 hours) | Vehicle Control | - | 7.90 | NA | 2.0 | 2.0 | 2.0 | 1.33 |
Positive Control (Cyclophosphamide monohydrate) | 10 µg/mL | 7.07 | 10.51 | 18.5 | 18.5 | 16.5 | 11.00* | |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.0625 | 6.44 | 18.48 | 2.0 | 2.0 | 1.5 | 1.00 | |
0.125 | 5.72 | 27.59 | 2.5 | 2.5 | 2.0 | 1.33 | ||
0.25 | 4.89 | 38.1 | 2.0 | 2.0 | 2.0 | 1.33 |
Set No. | Treatment | Concentrations (mg/mL) | Mean % MI | Mean % Reduction in MI | Mean of Total Aberrations with Gaps | Mean of Total Aberrations without Gaps | Mean of Total Aberrant cells without Gaps | Mean of Percentage Aberrated Cells |
Set 2 (-S9) (3 to 6 hours) | Vehicle Control | - | 7.74 | NA | 2 | 2 | 2.0 | 1.33 |
Positive Control (Mitomycin-C) | 0.05 µg/mL | 7.21 | 6.85 | 16.5 | 16.5 | 15.5 | 10.33* | |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.0625 | 6.29 | 18.73 | 2 | 2 | 2 | 1.33 | |
0.125 | 5.42 | 29.97 | 1.5 | 1.5 | 1.5 | 1.00 | ||
0.25 | 4.84 | 37.47 | 2.5 | 2.5 | 2.5 | 1.67 |
Set No. | Treatment | Concentrations (mg/mL) | Mean % MI | Mean % Reduction in MI | Mean of Total Aberrations with Gaps | Mean of Total Aberrations without Gaps | Mean of Total Aberrant cells without Gaps | Mean of Percentage Aberrated Cells |
Set 3 (-S9) (20 to 24 hours) | Vehicle Control | - | 7.91 | NA | 2.5 | 2.5 | 2.0 | 1.33 |
Positive Control (Mitomycin-C) | 0.05 µg/mL | 7.06 | 10.75 | 16 | 16 | 15.5 | 10.33* | |
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.0625 | 6.31 | 20.23 | 3 | 2.5 | 2.0 | 1.33 | |
0.125 | 5.61 | 29.08 | 2.5 | 2.5 | 2.5 | 1.67 | ||
0.25 | 4.79 | 39.44 | 3.5 | 3.5 | 2.5 | 1.67 |
MI: Mitotic Index; *: Statistically significant; +S9: With metabolic activation; -S9: Without metabolic activation
Table 3. Individual data of percentage mitotic index for initial cytotoxicity test
Set No. | Treatment | Concentrations (mg/mL) | Replicate | Total No. of Cells | No. of Mitotic Cells | % Mitotic Index | Mean% Mitotic Index |
Set 1 (+S9) (3 to 6 hours) | Vehicle Control | 0 | R1 | 523 | 39 | 7.46 | 7.76 |
R2 | 521 | 42 | 8.06 | ||||
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.03125 | R1 | 523 | 36 | 6.88 | 6.97 | |
R2 | 525 | 37 | 7.05 | ||||
0.0625 | R1 | 514 | 33 | 6.42 | 6.20 | ||
R2 | 519 | 31 | 5.97 | ||||
0.125 | R1 | 521 | 28 | 5.37 | 5.30 | ||
R2 | 516 | 27 | 5.23 | ||||
0.25 | R1 | 514 | 25 | 4.86 | 4.65 | ||
R2 | 518 | 23 | 4.44 | ||||
0.5 | R1 | 509 | 12 | 2.36 | 2.62 | ||
R2 | 520 | 15 | 2.88 | ||||
Set 2 (-S9) (3 to 6 hours)
| Vehicle Control | 0 | R1 | 523 | 38 | 7.27 | 7.65 |
R2 | 524 | 42 | 8.02 | ||||
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.03125 | R1 | 516 | 36 | 6.98 | 6.89 | |
R2 | 515 | 35 | 6.80 | ||||
0.0625 | R1 | 521 | 32 | 6.14 | 6.02 | ||
R2 | 509 | 30 | 5.89 | ||||
0.125 | R1 | 514 | 26 | 5.06 | 4.88 | ||
R2 | 512 | 24 | 4.69 | ||||
0.25 | R1 | 509 | 23 | 4.52 | 4.45 | ||
R2 | 504 | 22 | 4.37 | ||||
0.5 | R1 | 509 | 12 | 2.36 | 2.53 | ||
R2 | 520 | 14 | 2.69 | ||||
Set 3 (-S9) (20 to 24 hours) | Vehicle Control | 0 | R1 | 514 | 39 | 7.59 | 7.80 |
R2 | 512 | 41 | 8.01 | ||||
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.03125 | R1 | 518 | 36 | 6.95 | 6.66 | |
R2 | 519 | 33 | 6.36 | ||||
0.0625 | R1 | 513 | 29 | 5.65 | 5.90 | ||
R2 | 520 | 32 | 6.15 | ||||
0.125 | R1 | 514 | 26 | 5.06 | 5.46 | ||
R2 | 512 | 30 | 5.86 | ||||
0.25 | R1 | 513 | 23 | 4.48 | 4.39 | ||
R2 | 512 | 22 | 4.30 | ||||
0.5 | R1 | 510 | 13 | 2.55 | 2.34 | ||
R2 | 517 | 11 | 2.13 |
Table 4. Individual data of chromosomal aberrations and mitotic index
Set No. | Treatment | Concentrations (mg/mL) | Replicate | Mitotic Index | Aberrations | Total No. of Aberrations | Total No. of Aberrations without Gaps | Total no. of Aberrant cells | Total % of Aberrated cells | ||||||||||||
Gaps | Breaks | Exchanges | Frag ments | Ring | Deletion | Di centric | |||||||||||||||
Total No. of Cells | Total No. of MC | Mitotic Index | Percentage of MI | Chro matid | Chromo some | Chro matid | Chromo some | Chro matid | Chromo some | ||||||||||||
Set 1 (+S9) (3 to 6 hours) | Vehicle Control | - | R1 | 523 | 40 | 0.0765 | 7.65 | - | - | - | 1 | 1 | - |
| - | - | - | 2 | 2 | 2 | 1.33 |
R2 | 516 | 42 | 0.0814 | 8.14 | - | - | - | 2 | - | - |
| - | - | - | 2 | 2 | 2 | 1.33 | |||
Positive Control (Cyclophosphamide Monohydrate)
| 10 µg/mL | R1 | 514 | 36 | 0.0700 | 7.00 | - | - | 2 | 14 | - | 1 | 1 | - | - | - | 18 | 18 | 16 | 10.67 | |
R2 | 519 | 37 | 0.0713 | 7.13 | - | - | 5 | 9 | 1 | - | 3 | - | - | - | 18/ | 18 | 17 | 11.33 | |||
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.0625 | R1 | 520 | 33 | 0.0635 | 6.35 | - | - | 1 | 1 | - | - | - | - | - | - | 2 | 2 | 1 | 0.67 | |
R2 | 521 | 34 | 0.0653 | 6.53 | - | - | - | 2 | - | - | - | - | - | - | 2 | 2 | 2 | 1.33 | |||
0.125 | R1 | 513 | 30 | 0.0585 | 5.85 | - | - | - | 2 | - | 1 | - | - | - | - | 3 | 3 | 2 | 1.33 | ||
R2 | 520 | 29 | 0.0558 | 5.58 | - | - | - | 2 | - | - | - | - | - | - | 2 | 2 | 2 | 1.33 | |||
0.25 | R1 | 523 | 26 | 0.0497 | 4.97 | - | - | - | 1 | - | 1 | - | - | - | - | 2 | 2 | 2 | 1.33 | ||
R2 | 521 | 25 | 0.0480 | 4.80 | - | - | - | 2 | - | - | - | - | - | - | 2 | 2 | 2 | 1.33 | |||
Set No. | Treatment | Concentrations (mg/mL) | Replicate | Mitotic Index | Aberrations | Total No. of Aberrations | Total No. of Aberrations without Gaps | Total no. of Aberrant cells | Total % of Aberrated cells | ||||||||||||
Gaps | Breaks | Exchanges | Frag ments | Ring | Deletion | Di centric | |||||||||||||||
Total No. of Cells | Total No. of MC | Mitotic Index | Percentage of MI | Chro matid | Chromo some | Chro matid | Chromo some | Chro matid | Chromo some | ||||||||||||
Set 2 (-S9) (3 to 6 hours) | Vehicle Control | - | R1 | 520 | 42 | 0.0808 | 8.08 | - | - | - | 2 | - | - | - | - | - | - | 2 | 2 | 2 | 1.33 |
R2 | 514 | 28 | 0.0739 | 7.39 | - | - | - | 2 | - | - | - | - | - | - | 2 | 2 | 2 | 1.33 | |||
Positive Control (Mitomycin C) | 0.05 µg/mL | R1 | 506 | 36 | 0.0711 | 7.11 | - | - | 5 | 9 | 1 | 1 | 3 | - | - | - | 19 | 19 | 17 | 11.33 | |
R2 | 520 | 38 | 0.0731 | 7.31 | - | - | 1 | 10 | - | - | 3 | - | - | - | 14 | 14 | 14 | 9.33 | |||
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.0625 | R1 | 513 | 33 | 0.0643 | 6.43 | - | - | - | 2 | - | - | - | - | - | - | 2 | 2 | 2 | 1.33 | |
R2 | 520 | 32 | 0.0615 | 6.15 | - | - | - | 2 | - | - | - | - | - | - | 2 | 2 | 2 | 1.33 | |||
0.125 | R1 | 516 | 28 | 0.0543 | 5.43 | - | - | - | 2 | - | - | - | - | - | - | 2 | 2 | 2 | 1.33 | ||
R2 | 518 | 28 | 0.0541 | 5.41 | - | - | - | 1 | - | - | - | - | - | - | 1 | 1 | 1 | 0.67 | |||
0.25 | R1 | 520 | 24 | 0.0462 | 4.62 | - | - | - | 3 | - | - | - | - | - | - | 3 | 3 | 3 | 2.00 | ||
R2 | 514 | 26 | 0.0506 | 5.06 | - | - | - | 2 | - | - | - | - | - | - | 2 | 2 | 2 | 1.33 | |||
Set No. | Treatment | Concentrations (mg/mL) | Replicate | Mitotic Index | Aberrations | Total No. of Aberrations | Total No. of Aberrations without Gaps | Total no. of Aberrant cells | Total % of Aberrated cells | ||||||||||||
Gaps | Breaks | Exchanges | Frag ments | Ring | Deletion | Di centric | |||||||||||||||
Total No. of Cells | Total No. of MC | Mitotic Index | Percentage of MI | Chro matid | Chromo some | Chro matid | Chromo some | Chro matid | Chromo some | ||||||||||||
Set 3 (-S9) (20 to 24 hours) | Vehicle Control | - | R1 | 512 | 39 | 0.0762 | 7.62 | - | - | 1 | 1 | - | - | 1 | - | - | - | 3 | 3 | 2 | 1.33 |
R2 | 512 | 42 | 0.0820 | 8.20 | - | - | - | 2 | - | - |
| - | - | - | 2 | 2 | 2 | 1.33 | |||
Positive Control (Mitomycin C) | 0.05 µg/mL | R1 | 516 | 37 | 0.0717 | 7.17 | - | - | 2 | 8 | - | - | 4 | - | - | - | 14 | 14 | 14 | 9.33 | |
R2 | 518 | 36 | 0.0695 | 6.95 | - | - | 1 | 13 | - | - | 3 | - | 1 | - | 18 | 18 | 17 | 11.33 | |||
Ultramarine Violet Pigment Violet 15 CI 77007
| 0.0625 | R1 | 510 | 32 | 0.0627 | 6.27 | - | - | - | 1 | - | - | 1 | - | - | - | 2 | 2 | 2 | 1.33 | |
R2 | 520 | 33 | 0.0635 | 6.35 | - | 1 | 2 | - | - | - | 3 | - | - | - | 4 | 3 | 2 | 1.33 | |||
0.125 | R1 | 512 | 30 | 0.0586 | 5.86 | - | - | 1 | - | - | - | - | - | - | - | 3 | 3 | 3 | 2.00 | ||
R2 | 523 | 28 | 0.0535 | 5.35 | - | - | - | 1 | - | - | - | - | 1 | - | 2 | 2 | 2 | 1.33 | |||
0.25 | R1 | 509 | 24 | 0.0472 | 4.72 | - | - | 1 | - | - | - | 3 | - | - | - | 4 | 4 | 3 | 2.00 | ||
R2 | 514 | 25 | 0.0486 | 4.86 | - | - | - | 2 | - | - | 1 | - | - | - | 3 | 3 | 2 | 1.33 | |||
MI: Mitotic Index, MC: Mitotic cells, R1: Replicate 1, R2: Replicate 2, +S9: With metabolic activation, -S9: Without metabolic activation. 150 metaphases evaluated per replicate.
Table 5. Historical data
Vehicle control - DMSO
95% Confidence level | |||
| With S9 (3 to 6 hours) | Without S9 (3 to 6 hours) | Without S9 (20 to 24 hours) |
Average | 1.07 | 1.03 | 1.03 |
Standard Deviation | 0.34 | 0.43 | 0.38 |
Sample Size | 25.00 | 25.00 | 25.00 |
Margin of Error | 0.13 | 0.17 | 0.15 |
Upper Bound | 1.20 | 1.19 | 1.18 |
Lower Bound | 0.93 | 0.86 | 0.88 |
95% Confidence level | 1.96 | 1.96 | 1.96 |
Max | 2.00 | 2.00 | 2.00 |
Min | 0.67 | 0.00 | 0.00 |
Positive control
95% Confidence level | |||
| With S9 (3 to 6 hours) Cyclophosphamide monohydrate | Without S9 (3 to 6 hours) Mitomycin-C | Without S9 (20 to 24 hours) Mitomycin-C |
Average | 10.10 | 10.20 | 10.22 |
Standard Deviation | 0.78 | 0.59 | 0.60 |
Sample Size | 34.00 | 34.00 | 34.00 |
Margin of Error | 0.26 | 0.20 | 0.20 |
Upper Bound | 10.36 | 10.39 | 10.42 |
Lower Bound | 9.84 | 10.00 | 10.01 |
95% Confidence level | 1.96 | 1.96 | 1.96 |
Max | 11.33 | 11.33 | 12.00 |
Min | 8.67 | 8.67 | 9.33 |
Table 2. Summary of colony counts of revertants- trial 1. Plate incorporation method.
| Treatment | Test Concentration (mg/plate) | No. of revertants (mean of 3 plates) | ||||||||||
| With S9 | Without S9 | |||||||||||
| Salmonella typhimurium | E.coli WP2 uvrA pKM 101 | Salmonella typhimurium | E.coli WP2 uvrA pKM 101 | |||||||||
| TA 98 | TA 100 | TA 1535 | TA 1537 | TA 98 | TA 100 | TA 1535 | TA 1537 | |||||
| Vehicle Control | 100 μL of Dimethyl Sulphoxide | Mean | 31.3 | 120.0 | 20.0 | 10.0 | 79.0 | 29.0 | 107.0 | 19.0 | 9.0 | 75.3 |
| ±SD | 3.1 | 3.0 | 2.0 | 2.0 | 3.6 | 1.0 | 2.6 | 1.0 | 2.0 | 4.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| Test item | 0.05 | Mean | 31.7 | 108.3 | 20.0 | 8.3 | 78.7 | 29.7 | 110.0 | 18.0 | 8.3 | 74.0 |
| ±SD | 1.5 | 7.5 | 1.0 | 1.5 | 2.5 | 1.5 | 6.6 | 2.0 | 1.2 | 3.5 | ||
| Fold Increase | 1.0 | 0.9 | 1.0 | 0.8 | 1.0 | 1.0 | 1.0 | 0.9 | 0.9 | 1.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| 0.16 | Mean | 32.0 | 112.0 | 19.0 | 9.0 | 78.3 | 28.7 | 105.0 | 17.3 | 8.0 | 76.0 | |
| ±SD | 2.6 | 3.0 | 1.7 | 2.0 | 2.1 | 1.5 | 3.6 | 1.5 | 1.0 | 5.6 | ||
| Fold Increase | 1.0 | 0.9 | 1.0 | 0.9 | 1.0 | 1.0 | 1.0 | 0.9 | 0.9 | 1.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| 0.5 | Mean | 31.7 | 103.0 | 18.7 | 8.3 | 78.7 | 29.7 | 103.3 | 17.7 | 8.0 | 77.3 | |
| ±SD | 1.5 | 4.4 | 1.5 | 1.5 | 2.5 | 1.5 | 2.5 | 1.5 | 1.0 | 2.5 | ||
| Fold Increase | 1.0 | 0.9 | 0.9 | 0.8 | 1.0 | 1.0 | 1.0 | 0.9 | 0.9 | 1.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| 1.6 | Mean | 30.3 | 101.3 | 18.0 | 8.0 | 77.7 | 28.0 | 93.7 | 17.3 | 7.7 | 76.7 | |
| ±SD | 1.5 | 3.5 | 1.0 | 1.0 | 2.1 | 1.0 | 3.1 | 0.6 | 0.6 | 2.1 | ||
| Fold Increase | 1.0 | 0.8 | 0.9 | 0.8 | 1.0 | 1.0 | 0.9 | 0.9 | 0.9 | 1.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| 5 | Mean | 27.0 | 93.0 | 16.0 | 7.0 | 74.3 | 25.3 | 90.3 | 14.7 | 6.7 | 71.0 | |
| ±SD | 1.0 | 2.0 | 1.0 | 1.0 | 2.1 | 0.6 | 1.5 | 0.6 | 0.6 | 1.0 | ||
| Fold Increase | 0.9 | 0.8 | 0.8 | 0.7 | 0.9 | 0.9 | 0.8 | 0.8 | 0.7 | 0.9 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| Positive Control | Mean | 369.3 | 400.3 | 145.0 | 132.7 | 396.7 | 359.0 | 389.0 | 133.7 | 124.0 | 387.0 | |
| ±SD | 13.0 | 8.6 | 7.0 | 7.0 | 11.0 | 8.2 | 6.6 | 7.1 | 9.8 | 11.4 | ||
| Fold Increase | 11.8 | 3.3 | 7.3 | 13.3 | 5.0 | 12.4 | 3.6 | 7.0 | 13.8 | 5.1 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
Lawn intensity:
4+= Thick lawns: Distinguished by a healthy (Normal) background lawn comparable to vehicle control plates.
Values of Revertants are in Mean±SD
Positive controls:
With S9:
For Salmonella typhimurium TA98, TA100, TA1535 and TA1537 = 4 μg/plate of 2-Aminoanthracene
For E.coli uvrA pKM 101 = 30 μg/plate of 2-Aminoanthracene
Without S9:
For TA98: 2 μg/plate of 2-Nitrofluorene
For TA100 and TA1535: 1 μg/plate of Sodium azide
For TA1537: 50 μg/plate of 9-Aminoacridine
For E.coli uvrA pKM 101: 5 μg/plate of 4 Nitroquinoline N-oxide
Table 3. Summary of colony counts of revertants- trial 2. Preincubation method.
| Treatment | Test Concentration (mg/plate) | No. of revertants (mean of 3 plates) | ||||||||||
| With S9 | Without S9 | |||||||||||
| Salmonella typhimurium | E.coli WP2 uvrA pKM 101 | Salmonella typhimurium | E.coli WP2 uvrA pKM 101 | |||||||||
| TA 98 | TA 100 | TA 1535 | TA 1537 | TA 98 | TA 100 | TA 1535 | TA 1537 | |||||
| Vehicle Control | 100 μL of Dimethyl Sulphoxide | Mean | 30.3 | 115.0 | 21.3 | 10.3 | 78.7 | 27.3 | 105.7 | 18.3 | 8.3 | 76.0 |
| ±SD | 2.5 | 6.6 | 1.5 | 1.2 | 2.5 | 1.5 | 4.2 | 2.5 | 1.5 | 4.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| Test item | 0.05 | Mean | 29.3 | 116.7 | 18.3 | 9.7 | 77.7 | 27.0 | 114.3 | 18.3 | 8.3 | 72.3 |
| ±SD | 1.2 | 3.1 | 1.5 | 2.1 | 2.1 | 1.7 | 2.1 | 2.1 | 1.5 | 3.2 | ||
| Fold Increase | 1.0 | 1.0 | 0.9 | 0.9 | 1.0 | 1.0 | 1.1 | 1.0 | 1.0 | 1.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| 0.16 | Mean | 29.0 | 113.0 | 18.0 | 9.0 | 76.0 | 26.7 | 106.3 | 16.3 | 8.0 | 73.7 | |
| ±SD | 2.0 | 3.6 | 2.6 | 1.0 | 4.4 | 1.5 | 5.7 | 1.5 | 1.0 | 3.1 | ||
| Fold Increase | 1.0 | 1.0 | 0.8 | 0.9 | 1.0 | 1.0 | 1.0 | 0.9 | 1.0 | 1.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| 0.5 | Mean | 30.0 | 107.7 | 18.3 | 9.7 | 77.3 | 26.7 | 106.3 | 16.3 | 8.0 | 73.7 | |
| ±SD | 2.0 | 4.0 | 2.1 | 1.5 | 4.2 | 1.5 | 5.7 | 1.5 | 1.0 | 3.1 | ||
| Fold Increase | 1.0 | 0.9 | 0.9 | 0.9 | 1.0 | 1.0 | 1.0 | 0.9 | 1.0 | 1.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| 1.6 | Mean | 29.0 | 95.3 | 17.0 | 8.7 | 78.7 | 26.3 | 92.7 | 15.7 | 7.3 | 73.0 | |
| ±SD | 1.0 | 4.5 | 1.0 | 0.6 | 3.5 | 1.2 | 3.1 | 0.6 | 0.6 | 2.6 | ||
| Fold Increase | 1.0 | 0.8 | 0.8 | 0.8 | 1.0 | 1.0 | 0.9 | 0.9 | 0.9 | 1.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| 5 | Mean | 25.7 | 92.3 | 15.7 | 6.7 | 73.3 | 24.3 | 90.3 | 14.0 | 6.3 | 72.7 | |
| ±SD | 1.2 | 3.2 | 1.5 | 0.6 | 3.1 | 1.5 | 1.5 | 1.0 | 0.6 | 2.5 | ||
| Fold Increase | 0.8 | 0.8 | 0.7 | 0.6 | 0.9 | 0.9 | 0.9 | 0.8 | 0.8 | 1.0 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
| Positive Control | Mean | 369.7 | 407.0 | 150.3 | 132.7 | 399.7 | 354.7 | 400.3 | 143.7 | 127.7 | 393.0 | |
| ±SD | 6.5 | 10.6 | 6.7 | 8.7 | 9.1 | 6.5 | 8.5 | 6.0 | 6.5 | 9.8 | ||
| Fold Increase | 12.2 | 3.5 | 7.0 | 12.8 | 5.1 | 13.0 | 3.8 | 7.8 | 15.3 | 5.2 | ||
| Lawn Intensity | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | 4+ | ||
Lawn intensity: 4+= Thick lawns: Distinguished by a healthy (Normal) background lawn comparable to vehicle control plates.
Values of Revertants are in Mean±SD
Positive controls:
With S9:
For Salmonella typhimurium TA98, TA100, TA1535 and TA1537 = 4 μg/plate of 2-Aminoanthracene
For E.coli uvrA pKM 101 = 30 μg/plate of 2-Aminoanthracene
Without S9:
For TA98: 2 μg/plate of 2-Nitrofluorene
For TA100 and TA1535: 1 μg/plate of Sodium azide
For TA1537: 50 μg/plate of 9-Aminoacridine
For E.coli uvrA pKM 101: 5 μg/plate of 4 Nitroquinoline N-oxide
Table 4. Historical data.
| Plate incorporation method | |||||||||||
| Metabolic activation | With Metabolic Activation | Without Metabolic Activation | |||||||||
| Tester strain | Salmonella typhimurium | E.coli uvrA pKM 101 | Salmonella typhimurium | E.coli uvrA pKM 101 | |||||||
| TA 98 | TA 100 | TA 1535 | TA 1537 | TA 98 | TA 100 | TA 1535 | TA 1537 | ||||
| Vehicle Control (Dimethyl sulphoxide) | Mean | 23.4 | 105.6 | 21.3 | 9.5 | 170.9 | 21.8 | 103.3 | 20.6 | 9.0 | 170.1 |
| ±SD | 4.4 | 8.2 | 2.6 | 2.2 | 7.9 | 3.5 | 7.2 | 2.6 | 2.1 | 7.6 | |
| Min | 16 | 83 | 13 | 4 | 152 | 15 | 73 | 14 | 5 | 150 | |
| Max | 40 | 130 | 29 | 16 | 191 | 34 | 126 | 27 | 15 | 191 | |
| Positive Control | Mean | 385.9 | 392.3 | 140.1 | 117.8 | 392.5 | 375.7 | 386.3 | 137.6 | 116.2 | 389.3 |
| ±SD | 25.0 | 17.6 | 10.2 | 8.9 | 15.3 | 29.7 | 17.6 | 12.0 | 9.8 | 13.1 | |
| Min | 256 | 246 | 92 | 87 | 320 | 271 | 270 | 94 | 81 | 321 | |
| Max | 440 | 434 | 182 | 149 | 433 | 430 | 446 | 190 | 160 | 441 | |
Preincubation method | |||||||||||
| Metabolic activation | With Metabolic Activation | Without Metabolic Activation | |||||||||
| Tester strain | Salmonella typhimurium | E.coli uvrA pKM 101 | Salmonella typhimurium | E.coli uvrA pKM 101 | |||||||
| TA 98 | TA 100 | TA 1535 | TA 1537 | TA 98 | TA 100 | TA 1535 | TA 1537 | ||||
| Vehicle Control (Dimethyl sulphoxide) | Mean | 23.4 | 105.5 | 21.2 | 9.7 | 172.0 | 21.8 | 102.5 | 20.7 | 9.2 | 169.8 |
| ±SD | 4.4 | 8.0 | 2.6 | 2.1 | 7.4 | 3.5 | 6.8 | 2.5 | 2.4 | 7.8 | |
| Min | 16 | 78 | 14 | 4 | 153 | 15 | 79 | 14 | 4 | 150 | |
| Max | 40 | 128 | 28 | 17 | 198 | 35 | 129 | 28 | 23 | 198 | |
| Positive Control | Mean | 385.8 | 393.2 | 140.4 | 117.6 | 391.9 | 375.8 | 388.0 | 137.2 | 115.6 | 389.5 |
| ±SD | 23.1 | 17.1 | 10.7 | 8.7 | 15.5 | 30.2 | 16.3 | 12.6 | 9.0 | 13.5 | |
| Min | 290 | 293 | 100 | 90 | 330 | 242 | 312 | 91 | 86 | 321 | |
| Max | 429 | 436 | 187 | 151 | 432 | 428 | 450 | 200 | 173 | 426 | |
Min: Minimum no. of colonies, Max: Maximum no. of colonies, SD: Standard Deviation
Note: Number of E.coli uvrA pKM 101 revertant and vehicle colonies not matching with historical data range. Strains used for the study are from new batch and numbers of colonies are as per COA send along with the strains.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Based on the available information, the substance is considered to be negative for genetic toxicity, and therefore the substance is not classified in accordance with CLP Regulation (EC) no 1272/2008.
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