Registration Dossier
Registration Dossier
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EC number: 213-879-2 | CAS number: 1047-16-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Pigment Violet 19 (nano form) was fond not mutagenic nor clastogenic in bacteria (Ames) and mammalian cells.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Mammalian Cell Gene Mutation Tests
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 July 2021 to 04 November 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted on 29th July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes
- Target gene:
- CHO AA8 cells
- Species / strain / cell type:
- other: CHO AA8 cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection (ATCC)
- Cell doubling time : Approximately 12 hours
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Alpha MEM CULTURE MEDIA with 10% FBS and 1% penstrep and 5±1% CO2
- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Doubling time model chromosome number cells were checked. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 homogenate
- Test concentrations with justification for top dose:
- 15.625, 31.25, 62.5 and 125 µg/mL based on initial cytotoxicity test
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2×107
DURATION
- Exposure duration: 3 hours and 38 minutes
- Expression time (cells in growth medium): 7 days for mutant phenotype expression
- Selection time (if incubation with a selection agent):8 days
SELECTION AGENT (mutation assays): 10 µM of 6-Thioguanine
STAIN : 5% giemsa
NUMBER OF REPLICATIONS: 5
METHODS STAINING TECHNIQUE USED: Post incubation period, medium from each culture flask was aspirated and stained with 5% Giemsa stain. Number of colonies formed was counted manually.
DETERMINATION OF CYTOTOXICITY
- Method:cloning efficiency - Rationale for test conditions:
- Acceptance of a test is based on the following criteria:
•The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
•Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
•Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
•Adequate number of cells and concentrations are analysable (according to OECD guidelines for testing of chemicals, No. 476).
•The criteria for the selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476. - Evaluation criteria:
- Colony counts in selective media and non-selective media
- Statistics:
- yes, Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0.
- Key result
- Species / strain:
- other: CHO AA8 cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- 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 change in pH was observed in any of the test concentrations
- Water solubility: Insoluble
- Precipitation: The precipitation and pH were tested at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL concentrations. Post 3 hours of incubation, no precipitation was observed at the tested concentrations of 0.03125, 0.0625 mg/mL, moderate precipitation was observed at 0.125 and 0.25 mg/mL, heavy precipitation was observed at 0.5, 1 and 2 mg/mL
RANGE-FINDING/SCREENING STUDIES: Yes
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
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
- Negative (solvent/vehicle) historical control data: Negative controls were maintained.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relative survivality - Conclusions:
- The test item is considered as non-mutagenic at and up to the concentration of 125 µg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.
- Executive summary:
The test item was evaluated for gene mutation test in CHO AA8 cells.
The test item formed a suspension in acetone at concentration of 200 mg/mL. After 3 hours of incubation, no precipitation was observed at the tested concentrations of 0.03125 and 0.0625 mg/mL, moderate precipitation was observed at 0.125 and 0.25 mg/mL, and heavy precipitation was observed at 0.5, 1 and 2 mg/mL. No change in pH was observed in any of the test concentrations.
On the basis of precipitation results, 0.125 mg/mL (125 µg/mL) was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 7.8125, 15.625, 31.25, 62.5 and 125 µg/mL using acetone as a vehicle in four flasks/group in the presence and absence of metabolic activation (4 hours and 5 minutes). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test.
The results of the initial cytotoxicity test indicated that the Relative Survival was greater than 10% (62.61 % in presence of metabolic activation and64.60 % in absence of metabolic activation) at 125 µg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 125 µg/mL was selected as highest concentration for gene mutation test.
The gene mutation test was conducted at the concentrations of 125, 62.5, 31.25 and 15.625 µg/mL using acetone as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 38 minutes).Benzo(a) pyrene and4 Nitroquinoline N-oxidewere used aspositive controlsfor the gene mutation test.
Cytotoxicity as Relative Survival was 64.10 % in presence of metabolic activation and63.48 % in absence of metabolic activationat the highest tested concentration of 125µg/mL. Relative Survival of negative control was 100.00 in presence of metabolic activation and 103.48 in absence of metabolic activation.There was no statistically significant increase in mutant frequencies at any of the concentrations tested and negative control when compared with the vehicle control.
There was statistically significant increase in mutant frequenciesfor positive controlswhen compared with the vehicle controlin bothmetabolic activation andabsence of metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 January 2021 to 06 May 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- OECD 473
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted on 29th July 2016.
- Deviations:
- no
- Principles of method if other than guideline:
- - Principle of test:
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle: acetone - Target gene:
- Human Peripheral Blood Lymphocytes were used
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- Human Peripheral Blood Lymphocytes were used
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 homogenate
- Test concentrations with justification for top dose:
- 0.015625, 0.03125 and 0.0625 mg/mL, top dose selected based on initial cytotoxicity results
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: formed suspension in 200 mg/mL - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- 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 - RPMI
DURATION
- Exposure duration: 3 to 6 hours and 24 hours
SELECTION AGENT (mutation assays): RPMI 1640 MEDIUM
STAIN : Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Pellets were mixed with 4 to 5 mL of freshly prepared warm 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. 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 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. 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 pre labeled with study number, with (+S9) or without metabolic activation (-S9), treatment/group and slide number. The slides were air dried
NUMBER OF CELLS EVALUATED: 150 metaphase
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Rationale for test conditions:
- • Three analyzable concentrations were used for chromosomal aberration test.
• Initial cytotoxicity was conducted at concentrations of 0.015625, 0.03125, 0.0625 and 0.125 mg/mL. The percentage reduction in Mitotic Index was in the range of 10.00 to 45.61 at 0.0625 mg/mL. As the percentage reduction in MI was not more than 45±5% at 0.0625 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.015625 and 0.03125 mg/mL - Evaluation criteria:
- Percentage mitotic index
- Statistics:
- yes
- Key result
- Species / strain:
- lymphocytes: Human Peripheral Blood Lymphocytes
- 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
- Effects of pH: No change in pH was observed in any of the concentrations tested upto 2 mg/mL
- Water solubility: insoluble
- Precipitation: Precipitation test was conducted at 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 24 hours of incubation, heavy precipitation was observed at 0.5, 1 and 2 mg/mL, moderate precipitation was observed at 0.125, 0.25 mg/mL and mild precipitation was observed at 0.0625 mg/mL. No precipitation was observed at 0.03125 and 0.015625 mg/mL
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: NA
- Negative (solvent/vehicle) historical control data: NEGATIVE CONTROLS MAINTAINED. - Conclusions:
- Based on the results obtained, the test item is considered as non-clastogenic upto the concentration of 0.0625 mg/mL both in the presence and absence of metabolic activation under the presented test conditions.
- Executive summary:
The test item was evaluated for chromosomal aberrations in human lymphocytes.
The test item formed suspension in acetone at 200 mg/mL. Precipitation and pH test was conducted at 0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 24 hours of incubation, heavy precipitation was observed at 0.5, 1 and 2 mg/mL, moderate precipitation was observed at 0.25, 0.125 mg/mL, mild precipitation was observed at 0.625 mg/mL. No precipitation was observed at 0.03125 and 0.015625 mg/mL. No change in pH was observed in any of the concentrations tested upto 2 mg/mL. Hence, 0.125 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.015625, 0.03125 and 0.0625 mg/mL of test item.
In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 10.00 to 45.61 at 0.015625, 0.03125 and 0.0625mg/mL (as there were colored particles and precipitation was interfering with the cells, the slides could not be evaluated at 0.125 mg/mL) in presence of metabolic activation (short term), in absence of metabolic activation (short term) in absence of metabolic activation (long term), respectively. As the percentage reduction in mitotic index was not more than 45±5% at 0.0625 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. other concentrations tested were 0.015625 and 0.03125 mg/mL.
In the chromosomal aberration test, the cells were treated with test item at the concentrations of 0.015625, 0.03125 and 0.0625mg/mLusing acetone as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mitomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.
The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, thecells were treated with a metaphase-arresting substance (colchicine), stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.
There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the test item concentration levels tested.Positive controls induced statistically significant aberrant cells when compared to vehicle control.
Theconcurrent vehicle control values were within the 95% control limits of the distribution of the laboratory’s historical vehicle control database.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 15 DEC 2004 to 10 JAN 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD 471)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- in accordance to German Chemikaliengesetz and Directive 88/320/EEC
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- induced rat liver S9 (phenobarbital/ß-naphtoflavone used for induction)
- Test concentrations with justification for top dose:
- Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II (preincubation test): 33, 100, 333, 1000, 2500, and 5000 µg/plate - Vehicle / solvent:
- - Vehicle used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide (TA 1535, TA 100), 4-nitro-o-phenylene-diamine (TA1537, TA 98), methyl methan sulfonate (WP2uvrA)
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (all strains)
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment I: plate incorporation
Experiment II: preincubation
DURATION
- Preincubation period: only Experiment II: 60 minutes at 37 °C
- Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including controls - Evaluation criteria:
- test item considerer as a mutagen, if:
- number of revertants exceeding a threshold of twice (TA 98, TA 100 and WP2uvrA) or
- number of revertants exceeding a threshold of thrice (TA 1535, TA 1537)
the colony count of the corresponding solvent control
- a dose dependent increase is considered relevant if the threshold is exceeded more than once
- increase in threshold at only one concentration is judged relevant if reproduced in a second independent experiment
-dose dependent increase below the threshold is regarded as indication of a mutagenic potential if reproduced in a second independent experiment, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered relevant - Species / strain:
- other: S. typhimurium TA 1535, TA 98, TA 100 and TA 1537, E. coli WP2uvrA
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- minor effect in plate incorporation assay at 5000 µg/plate in TA1537 (reduction in the number of revertants to ~50% of control plates)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible in Experiment II without S9 at a concentration of 5000 µg/plate and with S9-Mix at concentrations of 1000 µg/plate and above
COMPARISON WITH HISTORICAL CONTROL DATA:
- historical range of positive controls was exceeded with metabolic activation in strains TA 98 and TA 1537 (Experiment I) and in strain TA 100 (Experiment/ and II), this effect indicates the sensitivity of the strains rather than compromising the assay
- Strain WP2uvrA (ExperimentI) with metabolic activation historical control range of negative and solvent controls was slightly exceeded
- Strain WP2uvrA and TA 1535 (ExperimentII) with and without metabolic activation historical control range of negative and solvent controls was slightly exceeded
- Strain TA 1535 and TA 98 (ExperimentII) with metabolic activation historical control range of negative and solvent controls was slightly exceeded
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- TA 1537: plate incorporation assay without metabolic activation minor effect at 5000 µg/plate - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and preincubation assay) with and without metabolic activation. - Executive summary:
Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and Escherichia coli strain WP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate using the plate incorporation assay. Additionally, a preincubation assay with or without metabolic activation was performed using the concentrations 33, 100, 333, 1000, 2500, and 5000 µg/plate.
The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.
Referenceopen allclose all
TABLE 1. SUMMARY OF INITIAL CYTOTOXICITY TEST
Set No. | Treatment | Concentration (µg/mL) | Average Colony Count± SD | Cloning Efficiency (CE) | Adjusted Cloning Efficiency (ACE) | Relative Survival (RS) (%) | ||
Set 1 +S9 | Vehicle Control (Acetone) | - | 189.67 | ± | 2.08 | 0.95 | 1.15 | - |
test item | 7.8125 | 185.67 | ± | 1.53 | 0.93 | 1.10 | 95.65 | |
15.625 | 180.67 | ± | 5.03 | 0.90 | 1.06 | 92.17 | ||
31.25 | 175.33 | ± | 3.51 | 0.88 | 1.00 | 86.96 | ||
62.5 | 164.33 | ± | 4.04 | 0.82 | 0.88 | 76.52 | ||
125 | 144.00 | ± | 5.20 | 0.72 | 0.72 | 62.61 | ||
Set 2 -S9 | Vehicle Control (Acetone) | - | 187.67 | ± | 2.52 | 0.94 | 1.13 | - |
test item | 7.8125 | 182.33 | ± | 2.52 | 0.91 | 1.06 | 93.81 | |
15.625 | 182.00 | ± | 3.00 | 0.91 | 1.04 | 92.04 | ||
31.25 | 178.00 | ± | 2.65 | 0.89 | 0.99 | 87.61 | ||
62.5 | 165.00 | ± | 4.58 | 0.83 | 0.90 | 79.65 | ||
125 | 142.67 | ± | 9.45 | 0.71 | 0.73 | 64.60 | ||
+S9: with metabolic activation; -S9: without metabolic activation;
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 1. SUMMARY OF PARALLEL CYTOTOXICITY TEST- GENE MUTATION TEST
Set No. | Treatment | Concentration (µg/mL) | Average Colony Count ± SD | Cloning Efficiency (CE) | Adjusted Cloning Efficiency (ACE) | Relative Survival (RS) (%) | ||
Set 1 +S9 | Vehicle Control (Acetone) | - | 190.33 | ± | 1.53 | 0.95 | 1.17 | - |
Negative control | - | 190.67 | ± | 1.15 | 0.95 | 1.17 | 100.00 | |
test item | 15.625 | 182.00 | ± | 2.00 | 0.91 | 1.09 | 93.16 | |
31.25 | 172.67 | ± | 4.51 | 0.86 | 0.99 | 84.62 | ||
62.5 | 168.33 | ± | 2.08 | 0.84 | 0.90 | 76.92 | ||
125 | 147.67 | ± | 2.52 | 0.74 | 0.75 | 64.10 | ||
Benzo(a)pyrene (Positive Control) | 3 | 180.00 | ± | 7.81 | 0.90 | 1.07 | 91.45 | |
Set 2 | Vehicle Control (Acetone) | - | 188.67 | ± | 5.03 | 0.94 | 1.15 | - |
Negative control | - | 189.33 | ± | 2.08 | 0.95 | 1.19 | 103.48 | |
test item | 15.625 | 178.67 | ± | 1.15 | 0.89 | 1.07 | 93.04 | |
31.25 | 174.33 | ± | 5.03 | 0.87 | 1.01 | 87.83 | ||
62.5 | 167.33 | ± | 5.51 | 0.84 | 0.88 | 76.52 | ||
125 | 143.00 | ± | 4.58 | 0.72 | 0.73 | 63.48 | ||
4 Nitroquinoline N-oxide (Positive Control) | 1 | 179.33 | ± | 4.51 | 0.90 | 1.09 | 94.78 | |
+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.
Set No. | Treatment | Concentration (µg/mL) | *Average Colony Count ± SD | Cloning Efficiency in selective media | Cloning Efficiency in non-selective media* | Total number of Mutant Colonies/ 2×106cells | Mutant Frequency/ 2×106cells | ||
Set 1 +S9 | Vehicle Control (Acetone) | - | 186.67 | ± | 4.04 | 0.0000105 | 0.93 | 21 | 22.58 |
Negative control | - | 187.00 | ± | 2.65 | 0.0000110 | 0.94 | 22 | 23.40 | |
15.625 | 186.00 | ± | 1.00 | 0.0000110 | 0.93 | 22 | 23.66 | ||
31.25 | 183.33 | ± | 3.79 | 0.0000110 | 0.92 | 22 | 23.91 | ||
62.5 | 178.67 | ± | 2.31 | 0.0000110 | 0.89 | 22 | 24.72 | ||
125 | 175.67 | ± | 3.51 | 0.0000105 | 0.88 | 21 | 23.86 | ||
Benzo(a)pyrene (Positive Control) | 3 | 181.67 | ± | 1.53 | 0.0001140 | 0.91 | 228 | 250.55** | |
Set 2 -S9 | Vehicle Control (Acetone) | - | 188.33 | ± | 2.08 | 0.0000115 | 0.94 | 23 | 24.47 |
Negative control | - | 187.33 | ± | 4.04 | 0.0000115 | 0.94 | 23 | 24.47 | |
test item | 15.625 | 184.67 | ± | 5.51 | 0.0000115 | 0.92 | 23 | 25.00 | |
31.25 | 183.00 | ± | 5.57 | 0.0000120 | 0.92 | 24 | 26.09 | ||
62.5 | 182.33 | ± | 6.03 | 0.0000110 | 0.91 | 22 | 24.18 | ||
125 | 174.33 | ± | 5.03 | 0.0000115 | 0.87 | 23 | 26.44 | ||
4 Nitroquinoline N-oxide (Positive Control) | 1 | 183.33 | ± | 4.51 | 0.0001170 | 0.92 | 234 | 254.35** | |
TABLE 1. SUMMARY OF GENE MUTATION TEST
+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.
Pre study:
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 | 0 | 7.50 | - | ||||||
Test item | NC | 7.27 | 3.07 | |||||||
0.015625 | 6.75 | 10.00 | ||||||||
0.03125 | 5.99 | 20.13 | ||||||||
0.0625 | 4.30 | 42.67 | ||||||||
0.125 | - | - | ||||||||
Set 2 (-S9) (3 to 6 hours) | Vehicle Control | 0 | 7.51 | - | ||||||
Test item | NC | 7.17 | 4.53 | |||||||
0.015625 | 6.74 | 10.25 | ||||||||
0.03125 | 6.11 | 18.64 | ||||||||
0.0625 | 4.34 | 42.21 | ||||||||
0.125 | - | - | ||||||||
Set 3 (-S9) (20 to 24 hours) | Vehicle Control | 0 | 7.85 | - | ||||||
Test item | NC | 7.52 | 4.20 | |||||||
0.015625 | 6.69 | 14.78 | ||||||||
0.03125 | 6.03 | 23.18 | ||||||||
0.0625 | 4.27 | 45.61 | ||||||||
0.125 | - | - |
+S9: With metabolic activation, -S9: Without metabolic activation.
Main study :
TABLE 1. SUMMARY OFCHROMOSOMAL ABERRATIONSAND 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 | 0 | 7.65 | NA | 2.0 | 2.0 | 2.0 | 1.33 |
Positive Control (Cyclophosphamide monohydrate) | 10 (µg/mL) | 7.26 | 5.1 | 17.0 | 17.0 | 15.5 | 10.34* | |
Test item | NC | 7.36 | 3.79 | 2.0 | 2.0 | 1.5 | 1.00 | |
0.015625 | 6.47 | 15.42 | 1.5 | 1.5 | 1.5 | 1.00 | ||
0.03125 | 5.85 | 23.53 | 2.5 | 2.5 | 2.0 | 1.33 | ||
0.0625 | 4.16 | 45.62 | 2.5 | 2.5 | 2.5 | 1.67 |
MI: Mitotic Index; *: Statistically significant; +S9: With metabolic activation.
TABLE 2 (Contd…). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX
Refer Appendix 2
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 | 0 | 7.67 | NA | 2 | 2 | 2 | 1.33 |
Positive Control (Mitomycin-C) | 0.05 (µg/mL) | 6.97 | 9.13 | 18.5 | 18 | 15 | 10.00* | |
Test item | NC | 7.29 | 4.95 | 1.5 | 1.5 | 1.5 | 1.00 | |
0.015625 | 6.72 | 12.39 | 1.5 | 1.5 | 1.5 | 1.00 | ||
0.03125 | 6.24 | 18.64 | 2.2 | 2.5 | 2 | 1.33 | ||
0.0625 | 4.44 | 42.11 | 2.5 | 2.5 | 2 | 1.33 |
MI: Mitotic Index; *: Statistically significant; -S9: Without metabolic activation.
TABLE 2 (Contd…).SUMMARY OFCHROMOSOMAL ABERRATIONSAND 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 3 (-S9) (20 to 24 hours) | Vehicle Control | 0 | 7.81 | NA | 2.00 | 2.00 | 2.00 | 1.33 |
Positive Control (Mitomycin-C) | 0.05 (µg/mL) | 7.17 | 8.19 | 19.50 | 19.00 | 16.00 | 10.67* | |
Test item | NC | 7.41 | 5.12 | 2.50 | 2.50 | 2.00 | 1.33 | |
0.015625 | 6.50 | 16.77 | 1.50 | 1.50 | 1.50 | 1.00 | ||
0.03125 | 5.84 | 25.22 | 2.50 | 2.50 | 2.00 | 1.33 | ||
0.0625 | 4.220 | 45.97 | 3 | 3 | 2.5 | 1.67 |
MI: Mitotic Index; *: Statistically significant; -S9: Without metabolic activation.
The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
not applicable
Additional information
Justification for classification or non-classification
No classification
The registration substance does not likely cause mutagenic or clastogenic effects in bacteria, mammalian cell
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