Registration Dossier
Registration Dossier
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EC number: 888-364-4 | CAS number: 146569-48-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The muta- and clastogenic potential of the test item was evaluated within three GLP conform studies according to OECD TG (471, 476, 473).
Within OECD TG 471, the test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test. The test item resulted in cytotoxicity from 0.8 to 5 mg/plate in the presence and absence of metabolic activation system when compared to vehicle control. Based on the results of initial cytotoxicity test, concentrations of0.008, 0.025, 0.08,0.25 and 0.8 mg/plate were selected for testing in the mutation test. The test item was assessed for its mutagenic effects using Salmonella typhimurium tester strains: TA98, TA100, TA1535, TA1537 and Escherichiacoli WP2 uvrA (pKM101). Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the 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 the trials.
The test item was assessed for its potential to induce gene mutations in mammalian cells according to OECD TG 476 investigating mutations at the Hprt locus of CHO AA8 cells of the Chinese Hamster. The results of the initial cytotoxicity test indicated that the Relative Survival was greater than 10% (69.30 % in presence of metabolic activation and 69.57 % in absence of metabolic activation) at 1 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results 1 mg/mL was selected as highest concentration for gene mutation test. The gene mutation test was conducted at the concentrations 0.125, 0.25, 0.5 and 1mg/mL using distilled water as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 1 minute). There was no statistically significant increase in mutant frequencies at any of the concentrations tested, in presence and absence of metabolic activation, when compared with the vehicle control. Moreover, treatment with the test item resulted in mutant frequencies which fell within acceptable ranges with regard to historical controls.
The test item was evaluated for chromosomal aberrations in human lymphocytes according to OECD TG 473. In initial cytotoxicity test, the percentage reduction in mitotic index was in the range of 66.09 to 96.17 at 0.25 to 1 mg/mL. The percentage reduction in mitotic index was in the range of 10.09 to 47.83 at 0.0625 to 0.125 mg/mL, as the percentage reduction in MI was not more than 45±5% at 0.125 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. other concentrations tested were 0.03125 and 0.0625 mg/mL. 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 regardless of metabolic activation.
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:
- 27 August 2021 to 20 December 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
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
-Batch No.of test material:EX.14402.600
- Expiration date of the lot/batch:No change of properties know over time (endless)
[Unlimited shelf-life (no changes of properties over time known)]
- Purity test date:87.6 %
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29ºC)
- Solubility of test substance in the vehicle: Distilled water
- Reactivity of the test substance with the vehicle of the cell culture medium: NA - Target gene:
- HPRT
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- AA8
- 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:
- 0.125, 0.25, 0.5 and 1 mg/mL.
• Since the test item did not precipitate at 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL, moderate precipitation was observed at 1 mg/mL and heavy precipitations was observed at 2 mg/mL. Hence, 1 mg/mL was considered as the highest concentration in the initial cytotoxicity test.
• At a concentration of 1 mg/mL, the Relative Survival was greater than 10%. Therefore 1 mg/mL was selected as the highest concentration for testing in gene mutation test.
• Based on the results of initial cytotoxicity test, four concentrations i.e. 0.125, 0.25, 0.5 and 1 mg/mL were selected for gene mutation test. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: found soluble in distilled water at 200 mg/mL - Untreated negative controls:
- no
- 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): 2×107METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2×107
DURATION
- Exposure duration: 3 hours and 1 minute
- Expression time (cells in growth medium): 8 days for mutant phenotype expression
- Selection time (if incubation with a selection agent):7 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:
- 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:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- 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: yes
- Precipitation: Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL to determine the ability of the test item to cause precipitation in the medium. 100 µL of test item (3.125, 6.25, 12.5, 25, 50, 100 and 200 mg/mL) was mixed with 10 mL of culture media and incubated at 37±1ºC with 5±1% CO2 for 3 hours and 30 minutes. Post incubation, results were recorded for change in pH and signs of precipitation.
RANGE-FINDING: yes
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
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
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relative survivality - Conclusions:
- Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 1 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.
- Executive summary:
The test item was assessed for its potential to induce gene mutations at the Hprt locus of CHO AA8 cells of the Chinese Hamster.
The test item found soluble in distilled water at 200 mg/mL concentration. Post 3 hours of and 30 minutes of incubation, no precipitation was observed at the tested concentrations of 0.03125, 0.0625, 0.125, 0.25, 0.5 mg/mL, moderate precipitation was observed at 1 mg/mL, and heavy precipitation was observed at 2 mg/mL. No change in pH was observed in any of the test concentrations.
On the basis of precipitation results, 1 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL using distilled water as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 hours and 17 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% (69.30 % in presence of metabolic activation and 69.57 % in absence of metabolic activation) at 1 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results 1 mg/mL was selected as highest concentration for gene mutation test.
The gene mutation test was conducted at the concentrations 0.125, 0.25, 0.5 and 1mg/mL using distilled water as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 1 minute).
Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls for the gene mutation test.Cytotoxicity as Relative Survival was 67.83 % in presence of metabolic activation and66.39 % in absence of metabolic activationat the highest tested concentration of 1 mg/mL.
There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control. Moreover, treatment with the test item resulted in mutant frequencies which fell within acceptable ranges with regard to historical controls.
There was statistically significant increase in mutant frequenciesfor positive controlswhen compared with the vehicle controlin both metabolic activation and absence 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:
- 08 October 2020 to 12 January 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 29th July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- 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.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Liver Homogentae
- Test concentrations with justification for top dose:
- 0.03125, 0.0625 and 0.125 mg/mL concentrations selected as per initial cytotoxicity test results. The percentage reduction in Mitotic Indexat0.0625, 0.125, 0.25, 0.5 and 1 mg/mLwas 10.77, 45.48, 66.09, 72.34and 93.48in presence of metabolic activation (short term),14.54, 47.83, 67.98, 77.42 and 96.17in absence of metabolic activation (short term)and 10.09, 46.61, 68.92, 72.64 and 93.49in absence of metabolic activation (long term)respectively.
- Vehicle / solvent:
- Distilled water
- Untreated negative controls:
- no
- 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:
Slide Evaluation
• Coding of slides was not carried out for initial cytotoxicity test. For each replicate minimum of 500 cells were scored.
• Percent mitotic index (MI %) was determined by the following formula
Mitotic Index
MI% = Number of Mitotic cells × 100
Total number of cells scored
Percent reduction in mitotic index was obtained by using the formula:
= [(Percentage MI of VC - Percentage MI of treated)/Percentage MI of VC] ×100
VC: Vehicle Control, MI: Mitotic Index.
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 3
METHOD OF TREATMENT/ EXPOSURE:
• Whole blood of volume 0.5 mL was added to each tube containing culture media of volume 4.4 mL of media + 100 µL of phytohaemagglutinin (PHA) and incubated for 44 to 48 hours at 37±1ºC and 5±1% CO2.
• Post 44 to 48 hours of incubation with PHA, cells from tubes were centrifuged at 1500 rpm for 10 minutes, supernatant was discarded.
• Cell pellet was resuspended with 2 mL fresh media.
•For tubes with metabolic activation (+S9) - set 1, cell suspension was mixed with 50 µL each of the respective test concentrations/vehicle, 0.5 mL of S9 mix and volume was made up to 5 mL with culture media.
•For tubes without metabolic activation (-S9) - set 2 and 3, cell suspension was mixed with 50 µL each of the respective test concentrations/vehicle and the volume was made up to 5 mL with culture media.
• All the test item concentrations and controls were maintained in duplicates.
• Cells were incubated both with metabolic activation (+S9) - set 1 for 3 to 6 hours, without metabolic activation (-S9) - set 2 for 3 to 6 hours and without metabolic activation (-S9) for 20 to 24 hours (1.5 cell cycle length) for set 3 at 37±1ºC and 5±1% CO2 as mentioned in the table.
• The treatment for set 1 and 2 tubes were terminated post 3 to 6 hours of incubation, by centrifugation at 1500 rpm for 10 minutes.
• Supernatant was discarded and cell pellet was mixed with 5 mL of culture medium and incubated further to complete 20 to 24 hours starting from the start of treatment.
• The treatment for set 3 tubes were terminated post 20 to 24 hours of incubation, by centrifugation at 1500 rpm for 10 minutes.
• Before 1 to 3 hours of harvesting, colchicine of concentration 0.3 µg/mL was added to all the tubes of set 1, 2 and 3. Post incubation of 1 to 3 hours with colchicine, cell suspension was collected to pre labeled tubes and centrifuged for 10 minutes at 1500 rpm.
Slide Preparation
Pellets were mixed with 4 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.
Minimum of 3 slides were prepared for each treatment replicate. Slides were stained using 5% Giemsa stain for 15 minutes.
FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): colchicine, 1-3 h
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): All slides including vehicle control, treatment and positive controls of chromosomal aberration test were coded before evaluation. Cytotoxicity was determined by calculating percentage reduction in mitotic index(%).Concurrent measures of mitotic index for all treated and vehicle control cultures were determined. The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the metaphases with aberrations were recorded in raw data. Gaps were recorded separately and reported but not included in the total aberration frequency.- Rationale for test conditions:
- •The concurrent vehicle control is considered acceptable for addition to the laboratory historical negative control database.
•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 control.
•All three experimental conditions should be tested unless one resulted in positive results.
•Adequate number of cells (at least 300 well spread metaphases per concentration) and concentrations (at least three analyzable concentrations) should be analyzed. - Evaluation criteria:
- •All slides including vehicle control, treatment and positive controls of chromosomal aberration test were coded before evaluation.
•Cytotoxicity was determined by calculating percentage reduction in mitotic index (%).
•Concurrent measures of mitotic index for all treated and vehicle control cultures were determined.
•The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the metaphases with aberrations were recorded in raw data.
•Gaps were recorded separately and reported but not included in the total aberration frequency. - 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) and the statistical significance was designated by the superscripts through out the report as stated below:
* Statistically significant (p<0.05) change than the vehicle control group. - Key result
- Species / strain:
- lymphocytes: primary culture
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Water solubility: Test item was soluble in distilled water at 200 mg/mL.
Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 24 hours 30 minutes of incubation, heavy precipitation was observed at 2 mg/mL. Mild and moderate precipitation was observed at 0.5 and 1 mg/mL. No precipitation was observed upto 0.03125 mg/mL. No change in pH was observed in any of the concentrations tested upto 2 mg/mL. Hence, 1 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.0625, 0.125, 0.25 and 0.5 mg/mL of test item.
In initial cytotoxicity test, the percentage reduction in mitotic index was in the range of 66.09 to 96.17 at 0.25 to 1 mg/mL. The percentage reduction in mitotic index was in the range of 10.09 to 47.83 at 0.0625 to 0.125 mg/mL, as the percentage reduction in MI was not more than 45±5% at 0.125 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. other concentrations tested were 0.03125 and 0.0625 mg/mL. - Conclusions:
- Based on the results obtained, the test item, the test item is considered as non-clastogenic up to the concentration of 0.125 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.
Test item was soluble in distilled water at 200 mg/mL. Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 24 hours 30 minutes of incubation, heavy precipitation was observed at 2 mg/mL. Mild and moderate precipitation was observed at 0.5 and 1mg/mL. No precipitation was observed upto 0.03125 mg/mL. No change in pH was observed in any of the concentrations tested upto 2 mg/mL. Hence, 1 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.0625, 0.125, 0.25 and 0.5 mg/mL of test item.
In initial cytotoxicity test, the percentage reductionin mitotic index was in the range of 66.09 to 96.17 at 0.25 to 1 mg/mL. The percentage reduction in mitotic index was in the range of 10.09 to 47.83 at 0.0625 to 0.125 mg/mL, as the percentage reduction in MI was not more than 45±5% at 0.125 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. other concentrations tested were 0.03125 and0.0625 mg/mL.
In the chromosomal aberration test, the cells were treated with test item at the concentrations of 0.03125, 0.0625 and 0.125mg/mLusing distilled water 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), harvested, 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.
The concurrent 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
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09 October 2020 TO 10 December 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- data waiving: supporting information
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted on 26th June 2020
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: EX.14402.600
- Expiration date of the batch: No change of properties known over time
(enless)
- Purity: 87.6 %
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29ºC)
- Solubility of the test substance in the solvent/vehicle: Distilled water - Target gene:
- At the histidine locus of Salmonella typhimurium and at the tryptophan locus of Escherichia coli tester strains
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 homogenate
- Test concentrations with justification for top dose:
- Based on the results of initial cytotoxicity test 0.008, 0.025, 0.08, 0.25 and 0.8 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Found soluble in distilled water at the concentration of 50 mg/mL - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-aminoantracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation;
- Tester strain density : 1 to 2 ×108 cells/mL
DURATION
- Preincubation period: 20 to 25 minutes
SELECTION AGENT (mutation assays): Minimal glucose agar plates
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Lawn intensity and number of revertant colonies - Rationale for test conditions:
- The test item resulted in noprecipitation from 0.1 mg/plate to 5 mg/plate at the tested concentrations. Based on the results of precipitation test, the 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 were selected for the initial cytotoxicity test.The test item resultedincytotoxicity at 3.2mg/plate and 5mg/plate with lawn intensity 1+ (very thin lawn), at 1.6 mg/plate with lawn intensity 2+ (thin lawn), at 0.8 mg/plate with lawn intensity 3+ (slightly thin lawn) and no cytotoxicity was observed from 0.00625 to 0.4 mg/plate with lawn intensity 4+ (thick lawn) in the presence and absence of metabolic activation system when compared to vehicle control 4+ (thick lawn) (see Table 1).
- Evaluation criteria:
- The conditions necessary for determining a positive result are, there should be a dose related increase in the mean revertants per plate of at least that one tester strain over a minimum of two increasing doses 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 limit dose tested 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.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Based on the above interpretation, the test item can be evaluated as negative.
The Bacterial reverse mutation test was considered acceptable as it meets the following criteria:
• The positive control substances produced a significant increase in mutant colony frequencies.
• The spontaneous reversion rates in the solvent control are in the range of in house historical control data and preferably within the range reported in the literature and as per new batch of certificate of analysis received by the vendor.
• The tester strains must met all required genetic characterization.
• Tester Strains used were in the approximate range of 1×109 to 2×109 cells/mL.
• Minimum of five analyzable test item dose concentrations were used to evaluate the mutation assay and none of these must show any signs of contamination.
• Cytotoxicity was defined as greater than 50% reduction in mean revertants per concentration relative to the mean - Statistics:
- Not applicable
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- 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:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Plate Incorporation Method:
Mean 383.1 402.9 141.0 120.0 394.7 363.9 393.5 132.1 112.4 387.8
±SD 11.8 10.3 8.5 8.7 11.7 13.2 10.0 11.2 8.2 11.7
Min 320 370 112 96 356 310 348 94 91 352
Max 416 425 165 142 421 398 416 159 143 412
Preincubation Method:
Positive Control Mean 381.9 402.7 140.1 119.2 396.1 365.7 394.6 131.6 111.4 389.1
±SD 11.1 10.2 9.5 7.9 11.8 12.0 9.8 11.9 8.2 12.2
Min 351 370 107 93 348 329 365 100 90 352
Max 415 430 163 140 421 402 426 158 134 415
- Solvent/vehicle historical control data:
Plate Incorporation Method:
Vehicle Control (Distilled water) Mean 30.0 109.7 18.6 9.2 71.5 27.7 103.7 17.3 8.1 68.7
±SD 5.6 6.4 2.6 1.7 5.3 5.5 5.5 2.4 1.6 4.6
Min 17 98 14 6 61 16 90 12 5 57
Max 43 121 25 13 84 41 114 22 12 79
Preincubation Method:
Vehicle Control (Distilled water) Mean 29.7 110.4 19.1 9.6 72.8 27.1 105.5 17.8 8.5 68.8
±SD 5.7 6.3 3.0 1.9 4.8 5.5 4.6 2.7 1.7 5.5
Min 16 97 14 5 64 17 94 13 6 56
Max 42 123 26 14 84 40 116 24 13 82
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Lawn intensity and number of revertant colonies - Conclusions:
- Based on the results of the study it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.8 mg/plate under the test conditions when tested on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) tester strains.
- Executive summary:
The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test.
The test itemfound solublein distilled water at a concentration of 50mg/mL and resulted in no precipitation from 0.1 to 5mg/plate at the tested concentrations, hence 5mg/platewas selected as the highest test concentration for initial cytotoxicity test.
In the initial cytotoxicity test, Salmonella typhimurium TA100 tester strain was exposed to vehicle control and range of test item concentrations from 0.00625 to 5mg/plate.
The test item resulted in cytotoxicity from 0.8 to 5 mg/plate in the presence and absence of metabolic activation system when compared to vehicle control.
Based on the results of initial cytotoxicity test, concentrations of0.008, 0.025, 0.08,0.25 and 0.8mg/plate were selected for testing in the mutation test.
The test item was assessed for its mutagenic effects using Salmonella typhimurium tester strains: TA98, TA100, TA1535, TA1537 and Escherichiacoli WP2 uvrA (pKM101). The test item was tested for plate incorporation method (trail I) and for preincubation method (trail II) in the presence and absence of metabolic activation system using distilled waterasvehicle and appropriate positive controls (2-nitrofluorene, sodium azide, 9-Aminoacridine and4-nitroquinoline N-oxidefor trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. The trials were carried out in triplicate.
Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the 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 the trials. The number of revertant colonies in the positive controls resulted in 3.7 to 13.4 fold increase under identical 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 (distilled water) | - | 185.33 | ± | 6.51 | 0.93 | 1.14 | - |
test item | 0.0625 | 184.00 | ± | 2.00 | 0.92 | 1.11 | 97.37 | |
0.125 | 183.33 | ± | 5.51 | 0.92 | 1.10 | 96.49 | ||
0.25 | 182.00 | ± | 6.00 | 0.91 | 1.08 | 94.74 | ||
0.5 | 169.67 | ± | 4.51 | 0.85 | 0.98 | 85.96 | ||
1 | 158.33 | ± | 2.52 | 0.79 | 0.79 | 69.30 | ||
Set 2 -S9 | Vehicle Control (distilled water) | - | 187.00 | ± | 6.08 | 0.94 | 1.15 | - |
test item | 0.0625 | 185.00 | ± | 4.58 | 0.93 | 1.13 | 98.26 | |
0.125 | 183.33 | ± | 3.06 | 0.92 | 1.10 | 95.65 | ||
0.25 | 182.33 | ± | 5.86 | 0.91 | 1.07 | 93.04 | ||
0.5 | 173.33 | ± | 6.11 | 0.87 | 1.00 | 86.96 | ||
1 | 157.00 | ± | 7.55 | 0.79 | 0.80 | 69.57 | ||
+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 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 (distilled water) | - | 189.67 | ± | 4.16 | 0.95 | 1.15 | - |
test item | 0.125 | 183.67 | ± | 5.51 | 0.92 | 1.09 | 94.78 | |
0.25 | 182.33 | ± | 3.06 | 0.91 | 1.07 | 93.04 | ||
0.5 | 172.33 | ± | 7.37 | 0.86 | 1.00 | 86.96 | ||
1 | 157.33 | ± | 9.07 | 0.79 | 0.78 | 67.83 | ||
Benzo(a)pyrene (Positive Control) | 3 µg/mL | 181.33 | ± | 6.81 | 0.91 | 1.09 | 94.78 | |
Set 2 | Vehicle Control (distilled water) | - | 188.00 | ± | 3.61 | 0.94 | 1.19 | - |
test item | 0.125 | 187.67 | ± | 1.53 | 0.94 | 1.13 | 94.96 | |
0.25 | 186.00 | ± | 3.61 | 0.93 | 1.10 | 92.44 | ||
0.5 | 176.33 | ± | 5.51 | 0.88 | 1.01 | 84.87 | ||
1 | 155.33 | ± | 5.03 | 0.78 | 0.79 | 66.39 | ||
4 Nitroquinoline N-oxide (Positive Control) | 1 µg/mL | 186.67 | ± | 3.06 | 0.93 | 1.11 | 93.28 | |
+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/ 2×106cells | Mutant Frequency/ 2×106cells | ||
Set 1 +S9 | Vehicle Control (distilled water) | - | 188.33 | ± | 3.79 | 0.0000122 | 0.94 | 23 | 24.47 |
test item | 0.125 | 187.33 | ± | 7.02 | 0.0000122 | 0.94 | 23 | 24.47 | |
0.25 | 187.00 | ± | 2.65 | 0.0000117 | 0.94 | 22 | 23.40 | ||
0.5 | 185.00 | ± | 5.29 | 0.0000124 | 0.93 | 23 | 24.73 | ||
1 | 184.00 | ± | 2.00 | 0.0000120 | 0.92 | 22 | 23.91 | ||
Benzo(a)pyrene (Positive Control) | 3 µg/mL | 186.33 | ± | 2.52 | 0.0001290 | 0.93 | 240 | 258.06** | |
Set 2 -S9 | Vehicle Control (distilled water) | - | 186.33 | ± | 2.08 | 0.0000124 | 0.93 | 23 | 24.73 |
test item | 0.125 | 186.00 | ± | 3.00 | 0.0000129 | 0.93 | 24 | 25.81 | |
0.25 | 185.00 | ± | 3.00 | 0.0000129 | 0.93 | 24 | 25.81 | ||
0.5 | 184.33 | ± | 1.53 | 0.0000125 | 0.92 | 23 | 25.00 | ||
1 | 183.67 | ± | 3.06 | 0.0000125 | 0.92 | 23 | 25.00 | ||
4 Nitroquinoline N-oxide (Positive Control) | 1 µg/mL | 185.67 | ± | 1.15 | 0.0001328 | 0.93 | 247 | 265.59** | |
+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 2. 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 1 (+S9) (3 to 6 hours) | Vehicle Control | 0 | 7.62 | NA | 2.0 | 2.0 | 2.0 | 1.33 | |||||||||
Positive Control (Cyclophosphamide monohydrate) | 10 (µg/mL) | 6.96 | 8.66 | 19.5 | 18.0 | 15.0 | 10.00* | ||||||||||
Test item | 0.03125 | 6.99 | 8.27 | 2.5 | 1.5 | 1.5 | 1.00 | ||||||||||
0.0625 | 6.67 | 12.47 | 2.0 | 1.5 | 1.5 | 1.00 | |||||||||||
0.125 | 4.12 | 45.93 | 3.0 | 3.0 | 2.0 | 1.33 |
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 | 7.70 | NA | 1.5 | 1.5 | 1.5 | 1.00 |
Positive Control (Mitomycin-C) | 0.05 (µg/mL) | 7.00 | 9.09 | 24 | 21.0 | 15.5 | 10.34* | |
Test item | 0.03125 | 7.12 | 7.53 | 1.5 | 1.5 | 1.5 | 1.00 | |
0.0625 | 6.63 | 13.9 | 1.5 | 1.5 | 1.5 | 1.00 | ||
0.125 | 4.15 | 46.10 | 3 | 2.5 | 2.0 | 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 | 7.72 | NA | 1.5 | 1.5 | 1.5 | 1.00 |
Positive Control (Mitomycin-C) | 0.05 (µg/mL) | 7.06 | 8.55 | 21 | 18.5 | 15 | 10.00* | |
Test item | 0.03125 | 7.09 | 8.16 | 3 | 2.5 | 2 | 1.33 | |
0.0625 | 6.74 | 12.69 | 2 | 1.5 | 1.5 | 1.00 | ||
0.125 | 4.08 | 47.15 | 2.5 | 2.5 | 2 | 1.34 |
Refer Appendix 2
MI: Mitotic Index; *: Statistically significant; -S9: Without metabolic activation.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
Based on the results of the OECD TG 471 study it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.8 mg/plate under the test conditions when tested on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) tester strains.
Based on the results obtained wihtin the OECD TG 476, the test item is considered as non-mutagenic in mammalian cells at and up to the concentration of 1 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.
Based on the results of the OECD TG 473, the test item is considered as non-clastogenic up to the concentration of 0.125 mg/mL both in the presence and absence of metabolic activation under the presented test conditions.
All in all, the test item did now show mutagenic or genotoxic potential and therefore there is no justification for a classification.
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