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EC number: 270-192-0 | CAS number: 68412-48-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Genetic toxicity in vitro - Bacterial Reverse Mutation Assay
No mutagenic activity in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of a post mitochondrial supernatant (S9 fraction).
Genetic toxicity in vitro - Mammalian Chromosome Aberration Test
Not clastogenic in Chinese hamster V79 cells with and without metabolic activation.
In vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay)
Not mutagenic in Mouse Lymphoma Assay on L5178Y TK +/- 3.7.2 C cells with and without metabolic activation.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23 June 2017 to 24 August 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial Reverse Mutation Test”, adopted 21st July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Commission Regulation (EC) No. 440/2008, B.13/14. “Mutagenicity: Reverse Mutation Test Using Bacteria”, 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- EPA Health Effects Test Guidelines, OPPTS 870.5100 “Bacterial Reverse Mutation Test”, EPA 712-C-98-247, August 1998
EPA Health Effects Test Guidelines, OPPTS 870.5100 “Escherichia coli WP2 and WP2 uvrA Reverse Mutation Assays”, EPA 712-C-96-247, June 1996 (Public Draft) - Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- histidine and tryptophan
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-Mix
- Test concentrations with justification for top dose:
- Based on the results of the Compatibility Test and available information, the test item formulated in Acetone. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 g test item/plate and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. Examined concentrations in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate in Salmonella typhimurium strains without metabolic activation and 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate in Escherichia coli WP2 uvrA strain without metabolic activation.
- Vehicle / solvent:
- Acetone was used as vehicle to prepare the stock formulation of the test material.
Acetone:
Supplier: SIGMA-ALDRICH / VWR
Batch No.: 15J060514
Expiry date: 31 October 2020 - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-1,2-phenylene-diamine (NPD); 2-aminoanthracene (2-AA)
- Details on test system and experimental conditions:
- The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test, a Confirmatory Mutation Test and a Complementary Confirmatory Mutation Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, the pre-incubation method was used.
Concentrations
Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test different concentrations were used.
Preliminary Compatibility Test
The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and Acetone. Test item was insoluble at 100 mg/mL concentration using Distilled water. Partial dissolution was observed at the same concentration using DMSO. The test item was soluble at this concentration using Acetone, therefore Acetone was selected as vehicle (solvent) for the study.
The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.
Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, 100 mg/mL stock formulation was prepared in Acetone which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.
Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test)
Based on the results of the preliminary tests, 100 mg/mL stock formulation was prepared from the test item with Acetone, which was diluted by serial dilutions in at last five steps to obtain at last six dosing formulations. The maximum test concentration was 5000 μg test item/plate in the main tests.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate in Salmonella typhimurium strains without metabolic activation and 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate in Escherichia coli WP2 uvrA strain without metabolic activation.
Control Groups Used in the Tests
Strain-specific positive and negative (vehicle/solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen vehicle (solvent) induced no deleterious or mutagenic effects.
Procedure for Exposure in the Initial Mutation Test
A standard plate incorporation procedure was performed, as an Initial Mutation Test. Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45 °C).
The content of the tubes:
top agar 2000 μL
vehicle (solvent) or test item formulation (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hours.
Procedure for Exposure in the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test and Complementary Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent).
The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hours. - Rationale for test conditions:
- The experimental methods were conducted according to the methods described by Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, OECD Guideline No. 471, 1997, Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996, and according to the relevant SOPs of CiToxLAB Hungary Ltd.
- Evaluation criteria:
- The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitate was observed in all tester strains at 5000 μg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitate was observed in all tester strains at 5000 μg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitate was observed in all tester strains at 5000 μg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitate was observed in all tester strains at 5000 μg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitate was observed in all tester strains at 5000 μg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test each sample (including the controls) was tested in triplicate.
In the Range Finding Test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
The observed number of revertant colonies was in the normal range. Minor differences compared to the solvent control numbers were observed. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
Precipitate / slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000 and 2500 μg/plate. Precipitate did not interfere with the colony counting or the background lawn growth evaluation.
Inhibitory, cytotoxic effects of the test item (slightly reduced background lawn development) were observed in both tester strains with and without metabolic activation at the concentration of 5000 μg/plate in the Preliminary Range Finding Test.
INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test, the pre-incubation method was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain.
The Initial Mutation Test and Confirmatory Mutation Test were performed in the presence and absence of metabolic activation system (±S9 mix). Each test was performed with appropriate untreated, negative (vehicle/solvent) and positive controls.
In the main tests each sample (including the controls) was tested in triplicate.
In the Confirmatory Mutation Test using the pre-incubation method, excessive cytotoxicity was observed in all bacterial strains without metabolic activation at several concentrations. In this case, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, an additional experiment (Complementary Confirmatory Mutation Test) was performed in these strains in an additional experimental period (Experimental Period III) to complete the data. The experimental conditions were the same as in the Confirmatory Mutation Test.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate in Salmonella typhimurium strains without metabolic activation and 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate in Escherichia coli WP2 uvrA strain without metabolic activation.
In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends.
In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain without metabolic activation at the concentrations of 1581 μg/plate. The mutation factor value at each concentration was 1.65. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
In the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 5 μg/plate concentration with metabolic activation. The mutation factor value was 1.46. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
Higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Tests in some other cases as well.
However, no dose-dependence was observed and they were below the biologically relevant threshold value and were within the historical control range, they were considered as reflecting the biological variability of the test.
Slightly lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Tests at some non-cytotoxic concentrations. However, the mean numbers of revertant colonies were within the historical control range, thus they were considered as biological variability of the test system.
Inhibitory, cytotoxic effects of the test item (reduced / slightly reduced background lawn development) were observed in the Initial Mutation Test without metabolic activation at 5000 μg/plate concentration in Salmonella typhimurium TA100, in the Confirmatory Mutation Tests with metabolic activation at 5000 and 1581 μg/plate concentrations in all examined bacterial strains and in Complementary Confirmatory Mutation Test without metabolic activation at 50 μg/plate concentration in Salmonella typhimurium strains; at 500 μg/plate concentrations in Escherichia coli WP2 uvrA strain.
Precipitate was observed in the Initial Mutation Test in all tester strains at 5000 μg/plate concentrations with and without metabolic activation and Confirmatory Mutation Tests in all tester strains at 5000 μg/plate concentrations with metabolic activation. Precipitate did not interfere with the colony counting or the background lawn growth evaluation.
VALIDITY OF THE TESTS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (vehicle/solvent) and positive control plates were within the historical control range. At least five analysable concentrations were presented in all strains of the main tests.
The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid. - Conclusions:
- The test item AMINOX® was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test, a Confirmatory Mutation Test and a Complementary Confirmatory Mutation Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, the preincubation method was used.
The reported data of the mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item AMINOX® had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study. - Executive summary:
The test item AMINOX® was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method).
Based on the results of the Compatibility Test and available information, the test item formulated in Acetone. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. Examined concentrations in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate in Salmonella typhimurium strains without metabolic activation and 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate in Escherichia coli WP2 uvrA strain without metabolic activation.
In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value when compared to the solvent control and were within the normal biological variability of the test system. There were no dose-related trends and no indication of any treatment effect.
Inhibitory, cytotoxic effects of the test item were observed in the Initial Mutation Test in Salmonella typhimurium TA100 strain without metabolic activation at 5000 μg test item/plate and in the Confirmatory Mutation Test in all examined strains with metabolic activation at 5000 and 1581 μg test item/plate and Complementary Confirmatory Mutation Test in all Salmonella typhimurium strains without metabolic activation at 50 μg/plate concentrations and in Escherichia coli WP2 uvrA strain without metabolic activation at 500 μg/plate.
Precipitate was detected on the plates in the Initial Mutation Test and Confirmatory Mutation Test in all examined strains with or without metabolic activation at 5000 μg concentration. Precipitate did not interfere with the colony counting or the background lawn growth evaluation.
The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid.
The reported data of the mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item AMINOX® had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 September 2018 - 18 October 2018
- 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
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Name: AMINOX®
Chemical name: 2-Propanone, reaction products with diphenylamine
CAS number: 68412-48-6
Batch/Lot Number: T7C16001
Description: Brown flakes
Purity: 100% (as a UVCB)
Expiry date: 14 March 2019
Storage condition: Controlled room temperature (15-25 ºC, below 70 RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety. - Target gene:
- Structural chromosome aberrations.
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- V79: Chinese hamster lung, male
ECACC Cat. No.: 86041102
Lot No.: 10H016
Date of working lot: 31 July 2015
Supplier: ECACC (European Collection of Cells Cultures)
Morphology: Fibroblast
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10°C (for short-term storage) or in liquid nitrogen (long-term storage). The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.
Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1% (v/v) Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10% (v/v) heat-inactivated fetal bovine serum (DMEM-10, culture medium). When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study). During the treatments, the serum content of the medium was reduced to 5% (v/v) (DMEM-5). - Additional strain / cell type characteristics:
- other: not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Citoxlab Hungary Ltd. according to Ames et al. [4] and Maron and Ames [5]. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly
Preparation of Rat Liver Homogenate S9 Fraction
On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.
Homogenates were centrifuged for 10 minutes at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fraction for this study was 14 September 2017 (Citoxlab code: E12713, Expiry date: 14 September 2019).
The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The protein concentration of the S9 fraction used in the study was determined to be 28.9 g/L. The sterility of the preparation was confirmed.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.
Preparation of S9-mix
The complete S9-mix was freshly prepared on the day of use according to the following ratio:
S9 fraction 3 mL
HEPES 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL
Prior to addition to the culture medium the S9-mix was kept in an ice bath.
For all cultures treated in the presence of S9-mix, a 0.5 mL aliquot of the mix was added to each cell culture (final volume: 10 mL). The final concentration of the liver homogenate in the test system was 1.5%. - Test concentrations with justification for top dose:
- A total of ten test concentrations between 2000 and 3.906 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.
Toxicity and Concentration Selection
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
In this Preliminary Toxicity Test, the cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time and for 20 hours in the absence of S9-mix with a 20-hour harvesting time.
The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.
At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts). - Vehicle / solvent:
- Negative (vehicle) control (DMSO) was run concurrently with treatment groups. Based on the trial formulations, the test item was soluble in DMSO.
Negative (vehicle) and positive controls were included in the experiments. Furthermore, untreated controls were also included in the preliminary experiments and main tests.
Positive Controls
Without metabolic activation
Name (Abbreviation): Ethyl methanesulfonate (EMS)
Ethyl methanesulfonate, a known mutagen and clastogen, was dissolved in DMEM and was used as a positive control for the non-activation experiments at a final concentration of 0.4 μL/mL or 1.0 μL/mL.
With metabolic activation
Name (Abbreviation): Cyclophosphamide monohydrate (CP) - Untreated negative controls:
- yes
- Remarks:
- (Vehicle)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- INDICATOR CELLS
V79: Chinese hamster lung, male
ECACC Cat. No.: 86041102
Lot No.: 10H016
Date of working lot: 31 July 2015
Supplier: ECACC (European Collection of Cells Cultures)
Morphology: Fibroblast
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10°C (for short-term storage) or in liquid nitrogen (long-term storage). The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.
Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1% (v/v) Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10% (v/v) heat-inactivated fetal bovine serum (DMEM-10, culture medium). When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study). During the treatments, the serum content of the medium was reduced to 5% (v/v) (DMEM-5).
Induction of Rat Liver Enzymes
Male Wistar rats (321-412 g animals were 9 weeks old at the initiation) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Initiation dates of the induction of liver enzymes used for preparation S9 used in this study was 11 September 2017.
Treatment of the Cells
For the cytogenetic experiments, 1-3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 105 cells/dish concentration and incubated for approximately 24 hours at 37°C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test item concentration or controls.
After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.4 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.
Cells were treated with different concentration test item solutions, untreated, negative (vehicle) or positive control solution (treatment volume was 100 μL/dish ) for the given period of time at 37°C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1% (v/v) Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) from the beginning of treatment.
Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality was also performed at the end of the treatment period in the preliminary and both main tests.
For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts).
Chromosome Aberration Assays
The Chromosome Aberration Assays were conducted as two independent experiments (Assay 1 and Assay 2) in the presence and in the absence of metabolic activation. In Assay 1, 3-hour treatment was performed with and without metabolic activation (in the presence and absence of S9 mix); cells were harvested 20-hour after the beginning of the treatment.
In Assay 2, a 3-hour treatment was performed with metabolic activation (in the presence of S9 mix) and 20-hour without metabolic activation (in the absence of S9 mix) in duplicate cultures; cells were harvested 20-hour after the beginning of the treatment.
Preparation of Chromosomes
2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 μg/mL). The cells were swollen with 0.075 M KCl hypotonic solution for 4 minutes, then were washed in fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells* was dropped onto clean microscope slides and air-dried. The slides were stained with 5% Giemsa solution, air-dried and coverslips were mounted. At least three slides were prepared for each culture.
*Note: Fixed cells were stored frozen in case if any additional action was required (as documented in the raw data and reported). After the finalization of the report, the remaining frozen cell suspension samples will be discarded.
Examination of Slides
The stained slides were given random unique code numbers at the Test Facility by a person who was not involved in the metaphase analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.
At least 150* metaphases with 22±2 chromosomes (centromeres) from each culture (replicate) were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. - Rationale for test conditions:
- In accordance with the test guidelines
- Evaluation criteria:
- EVALUATION OF THE RESULTS
The assay is considered valid, if the following criteria are met:
- The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.
The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolality of the treated cultures.
Evidence of a dose-response relationship (if any) was considered to support the conclusion.
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed. - Statistics:
- For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- VEHICLE AND CONCENTRATION SELECTION
Based on the available solubility information (trial formulations of the test item performed at the Test Facility), DMSO was selected for vehicle (solvent) of the study. The highest examined concentration in the preliminary test was 2000 μg/mL.
Concentration Selection Cytotoxicity Assays (3-hour treatment with and without metabolic activation, 20-hour harvesting time; and 20-hour treatment without metabolic activation, 20-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays.
A total of ten test concentrations between 2000 and 3.906 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay. Treatment concentrations for the chromosome aberration assays were selected on the basis of results of the performed Concentration Selection Cytotoxicity Assays according to the OECD guideline instructions (up to the solubility limit or cytotoxicity limit).
CHROMOSOME ABERRATION ASSAYS
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 125, 100, 75, 50, 25 and 12.5 μg/mL (experiment with and without metabolic activation).
In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 125-50 μg/mL concentration range with and without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay (for more details see Table 5 and Table 6 of Appendix 4). Therefore, concentrations of 50, 25 and 12.5 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic.
In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 125, 100, 75, 50, 25 and 12.5 μg/mL (experiment with metabolic activation) and 200, 150, 125, 100, 75, 50 and 25 μg/mL (experiment without metabolic activation).
In Assay 2, insolubility/minimal amount of insolubility was detected at the end of the treatment period in the final treatment medium in the 125-25 μg/mL concentration range with metabolic activation and in the 200-25 μg/mL concentrations without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment without metabolic activation (RICC value of the highest evaluated concentration (200 μg/mL) without metabolic activation was 12%). No cytotoxicity was observed in the experiment with metabolic activation. Therefore, concentrations of 50, 25 and 12.5 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 75, 50 and 25 μg/mL (a total of three) were evaluated in the experiment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.
Polyploid metaphases (1-8) were found in all cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with AMINOX®. No endoreduplicated metaphases were detected in the performed experiments.
VALIDITY OF THE STUDY
The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility was detected in all experiments with and without metabolic activation; while marked cytotoxicity was detected in Assay 2 without metabolic activation. The evaluated concentration ranges of Assay 1 and Assay 2 were considered to be adequate, as they covered the range from insolubility to little insolubility and/or from toxicity to no or little toxicity.
Three test item concentrations were evaluated in each experiment.
The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were within the acceptable range. Historical control data are presented in Appendix 6.
In the performed experiments, the positive control substances (Cyclophosphamide (CP) in the experiments with metabolic activation and Ethyl methanesulfonate (EMS) in the experiments without metabolic activation) caused the expected statistically significant increase in the number of cells with structural chromosome aberrations (Tables 9-12 of Appendix 5) demonstrating the sensitivity of the test system in each assay.
The study was considered to be valid. - Conclusions:
- The test item AMINOX® was tested for potential clastogenic activity using the Chromosome Aberration Assay. The study included Concentration Selection Cytotoxicity Assay and two Chromosome Aberration Assays.
The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.
Treatment with the test item did not result in a statistically and biologically significant, reproducible, dose-dependent increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats.
In conclusion, AMINOX® did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, AMINOX® was considered as not clastogenic in this test system. - Executive summary:
AMINOX® was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in DMSO and it was examined up to cytotoxic concentrations according to the OECD guideline recommendations. In independent Chromosome Aberration Assays using duplicate cultures, at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each evaluated test item treated, negative (vehicle) and positive control sample.
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 125, 100, 75, 50, 25 and 12.5 μg/mL (experiment with and without metabolic activation).
In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 125-50 μg/mL concentration range with and without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 50, 25 and 12.5 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic.
In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 125, 100, 75, 50, 25 and 12.5 μg/mL (experiment with metabolic activation) and 200, 150, 125, 100, 75, 50 and 25 μg/mL (experiment without metabolic activation).
In Assay 2, insolubility/minimal insolubility was detected at the end of the treatment period in the final treatment medium in the 125-25 μg/mL concentration range with metabolic activation and in the 200-25 μg/mL concentration range without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment without metabolic activation (RICC value of the highest evaluated concentration (200 μg/mL) without metabolic activation was 12%). No cytotoxicity was observed in the experiment with metabolic activation. Therefore, concentrations of 50, 25 and 12.5 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 75, 50 and 25 μg/mL (a total of three) were evaluated in the experiment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.
Polyploid metaphases were found in all cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with AMINOX®. No endoreduplicated metaphases were detected in the performed experiments.
The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.
In conclusion, AMINOX® did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, AMINOX® was considered as not clastogenic in this test system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 October 2018 to 19 November 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Thymidine kinase (tk)
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Product No.: CRL-9518
Lot No.: 60797977
Supplier: American Type Culture Collection
(Manassas, Virginia, USA)
Date of receipt: 27 February 2014
Date of working lot: 17 May 2017 (MP14)
The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than three times before used in the main assays. - Metabolic activation:
- with and without
- Metabolic activation system:
- The post-mitochondrial fraction (S9 fraction) was prepared from rat liver [3, 4] by the Microbiological Laboratory of Citoxlab Hungary Ltd. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived annually.
Induction of Liver Enzymes:
Male Wistar rats (321-412 g, animals were 9 weeks old at initiation) were treated with Phenobarbital (PB) and -naphthoflavone (BNF) at 80 mg/kg/day by oral gavage (for both inducers) for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before euthanasia when food was removed. Euthanasia was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used in the preparation of S9 fraction used in this study was 11 September 2017.
Preparation of Rat Liver Homogenate S9 Fraction:
On Day 4, the rats were euthanized and the livers removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 minutes at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was distributed in 1-5 mL portions, frozen quickly and stored at -80 10ºC. Sterility of the preparation was confirmed.
The protein concentration was determined by colorimetric test by chemical analyser at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The protein concentration of the S9 fraction used was determined to be 28.9 g/L. The date of preparation of S9 fraction for this study was 14 September 2017 (Citoxlab code: E12713, Expiry date: 14 September 2019).
The biological activity of each batch of S9 was characterized in the Salmonella assay using 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study was found active under the test conditions.
For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%. Cultures treated in the absence of S9-mix received 1 mL of 150 mM KCl (except for the 24-hour treatment). Prior to addition to the culture medium, the S9-mix was kept in an ice bath. - Test concentrations with justification for top dose:
- Treatment concentrations for the mutation assays were selected on the basis of the result of a short preliminary toxicity test. Three-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test item concentrations to determine toxicity immediately after the treatments.
The highest concentration tested in the preliminary test was 2000 µg/mL (the recommended maximum concentration). Treatment of cell cultures was made as described in the next section for the main mutation assays. However, single cultures were only used and positive controls were not included. After the treatment period, cell concentrations were determined using a haemocytometer. Cells were transferred for the expression period for two extra days and repeated cell counting was performed. Visual examination for precipitation of test item in the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed after the treatment period.
Insolubility and cytotoxicity were detected in the preliminary experiment. The concentrations were selected for the main assays according to the OECD No. 490 guideline instructions. At least six concentrations with and without metabolic activation were selected for Assays 1-2. - Vehicle / solvent:
- Based on the available information (trial formulation of the test item performed in another in vivo study at the Test Facility [17/174-001P]), DMSO is selected for vehicle (solvent) of this study. The test item was soluble at 200 mg/mL concentration using Dimethyl sulfoxide (DMSO) as vehicle. This vehicle (solvent) is compatible with the survival of the cells and the S9 activity.
For the treatments in the study, stock formulations (200 mg/mL was used in the preliminary experiment and in the main assays) were prepared in the testing laboratory as follows. The necessary amount of the test item was weighed into a calibrated volumetric flask; approximately 80% of the required volume of the vehicle (solvent) was added and stirred by a vortex until homogeneity was reached. Then, it was filled up to the final volume with the vehicle (solvent) to form a stock formulation. Five minutes ultrasonic bath was used for better dissolution. From the stock formulation several dilutions were prepared using the selected vehicle for dosing formulations. In each case, the vehicle was filtered sterile before the formulation step using a 0.22 µm syringe filter (Supplier: Millipore, Lot No.: R8DA52451, Expiry date: April 2021 (preliminary experiment and Assays 1-2); before the preparation of the dosing formulations. The stock formulation was not filtered sterile. The stock formulations and all the dilutions were prepared immediately before the treatment of the cells in a sterile hood.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Details on test system and experimental conditions:
- In Assay 1, cells were treated for 3 hours in the presence and absence of S9-mix. In Assay 2, cells were treated for 3 hours in the presence of S9-mix and for 24 hours in the absence of S9-mix.
A suitable volume (0.2 mL for a final volume of 20 mL (10 µL/mL) of RPMI-5 medium, vehicles (solvents), test item formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or 1.0 mL of 150 mM KCl (in case of 3-hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, 107 cells were placed in each of a series of 75 cm2 sterile flasks. For the 24-hour treatment, 6x106 cells were placed in each of a series of 25 cm2 sterile flasks. The treatment medium contained a reduced serum level of 5% (v/v) RPMI-5.
Duplicate cultures were used for each treatment. Cultures were visually examined at the beginning and end of treatments. During the treatment period, cultures were incubated at 37°C ± 1°C (approximately 5% CO2 in air). Gentle shaking was used during the treatments. Measurement of pH and osmolality was also performed after the treatment period.
Then cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in at least 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer. Where sufficient cells survived, cell density was adjusted to a concentration of 2x105 cells/mL (if possible). Cells were transferred to flasks for growth through the expression period (maximum 30 mL of suspension) or diluted to be plated for survival. - Rationale for test conditions:
- In accordance with test guidelines
- Evaluation criteria:
- The test item was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:
1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative (vehicle) control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).
2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.
The test item was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test item was considered unable to induce mutations in this test system.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. Caution was exercised with positive results obtained at levels of cytotoxicity lower than 10% (as measured by RTG). - Statistics:
- Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software.
The negative (vehicle/solvent) control log mutant frequency (LMF) was compared to the LMF of each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY EXPERIMENT
Treatment concentrations for the mutation assay were selected based on the results of a short Preliminary Toxicity Test. 3-hour treatment in the presence and absence of metabolic activation system (S9-mix) and 24-hour treatment in the absence of metabolic activation system were performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest concentration tested in the preliminary experiment using DMSO as vehicle was 2000 µg/mL (the recommended maximum concentration).
In the preliminary experiment cytotoxicity was detected at higher concentrations. Insolubility / minimal amount of insolubility was observed with and without metabolic activation at concentration range of 2000-125 µg/mL with and without (short treatment) metabolic activation and 2000-62.5 µg/mL without (long treatment) metabolic activation.
Concentrations were selected for the main experiments according to the instructions of the relevant OECD No. 490 guideline. At least six concentrations were selected for the main experiments in each assay.
MUTATION ASSAYS
In the mutation assays, cells were exposed to the test item for 3 hours with or without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The cells were plated for determination of survival data and in parallel
subcultured without test item for approximately 2 days to allow expression of the genetic changes. At the end of the expression period, cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.
Assay 1
In Assay 1, a 3-hour treatment with metabolic activation (in the presence of
S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 2000, 666.67, 222.22, 74.07, 24.69 and 8.23 µg/mL with and without metabolic activation.
In Assay 1, there were no large changes in pH or osmolality after treatment.
In Assay 1, insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment with and without metabolic activation at concentration range of 2000-24.69 µg/mL concentrations.
Presence of S9-mix (3-hour treatment)
In the presence of S9-mix (3-hour treatment), cytotoxicity of the test item was observed at concentration range of (2000-222.22 µg/mL). No cytotoxicity or significant cytotoxicity was observed at lower concentrations. The Relative Total Growth (RTG) value of the highest evaluated concentration was 14%. The evaluation was made using data of all six concentrations (concentration range of 2000-8.23 µg/mL). There was statistically significant increase in the mutation frequency values in the two highest evaluated concentrations in Assay 1 and concentration related increase was also indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant. Therefore, this experiment was considered as being negative.
Note: The observed precipitate made the counting of colonies on the mutagenicity plates very difficult especially at 2000 µg/mL concentration, and it made the counting difficult at 666.67 µg/mL concentration. This fact had no effect on the evaluation of the plates.
Absence of S9-mix (3-hour treatment)
In the absence of S9-mix (3-hour treatment), cytotoxicity of the test item was observed at concentration range of (2000-222.22 µg/mL). No cytotoxicity or significant cytotoxicity was observed at lower concentrations. The Relative Total Growth (RTG) value of the highest evaluated concentration was 24%. An evaluation was made using data of all six concentrations (concentration range of 2000-8.23 µg/mL). There was statistically significant increase in the mutation frequency values in the highest evaluated concentration, but it was statistically borderline. Concentration related increase was also indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant. Therefore, this experiment was considered as being negative.
Note: The observed precipitate made the counting of colonies on the mutagenicity plates very difficult at 2000 µg/mL concentration. This fact had no effect on the evaluation of the plates.
Assay 2
In Assay 2, a 3-hour treatment with metabolic activation (in the presence of
S9-mix) and a 24-hour treatment without metabolic activation (in the absence of
S9-mix) were performed. Treatment concentrations were 2000, 1333.33, 666.67, 222.22, 74.07, 24.69 and 8.23 µg/mL with metabolic activation and 1000, 750, 500, 333.33, 111.11, 37.04 and 12.35 µg/mL without metabolic activation.
In Assay 2, there were no large changes in pH or osmolality after treatment.
In Assay 2, insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment with metabolic activation at concentration range of 2000-24.69 µg/mL and without metabolic activation at concentration range of 1000-111.11 µg/mL.
Presence of S9-mix (3-hour treatment)
In the presence of S9-mix (3-hour treatment), cytotoxicity of the test item was observed at concentration range of (2000-222.22 µg/mL). No cytotoxicity or significant cytotoxicity was observed at lower concentrations. The Relative Total Growth (RTG) value of the highest evaluated concentration was 14%. The evaluation was made using data of all seven concentrations (concentration range of 2000-8.23 µg/mL). There was statistically significant increase in the mutation frequency values in the two highest evaluated concentrations in Assay 2 and concentration related increase was also indicated by the linear trend analysis.
However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant. This experiment was considered as being negative and confirmed the negative result of the first test with metabolic activation.
Note: The observed precipitate made the counting of colonies on the mutagenicity plates very difficult at 2000 and 1333.33 µg/mL concentrations, and significant precipitate was also observed at 666.67 µg/mL concentration. This fact had no effect on the evaluation of the plates.
Absence of S9-mix (24-hour treatment)
In the absence of S9-mix (24-hour treatment), cytotoxicity of the test item was observed at concentration range of 1000-37.04 µg/mL. Excessive cytotoxicity was observed at 1000 and 750 µg/mL concentrations. No significant cytotoxicity was observed at 12.35 µg/mL concentration. Thus, the evaluation was made using data of five concentrations (concentration range of 500-12.35 µg/mL). The Relative Total Growth (RTG) value of the highest evaluated concentration was 7%.
No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being negative.
Notes:
1. In case of the 12.35 µg/mL signs of infection was observed in certain wells of replicate “A” on the mutagenicity plates, therefore only replicate “B” was used for mutagenicity evaluation. The data used for this concentration were consistent with the overall results from other concentrations, so the use of the one replicate in data analysis is considered to be valid.
2. The observed precipitate made the counting of colonies on the mutagenicity plates difficult at 1000 µg/mL concentration. This fact had no effect on the evaluation of the plates.
VALIDITY OF THE MUTATION ASSAYS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently in the study. The spontaneous mutation frequency of the negative (vehicle/solvent) and untreated controls were in the recommended range (50-170 x 10-6) in all cases.
The positive controls (Cyclophosphamide in the presence of metabolic activation and
4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays. All of the positive control samples in the performed experiments fulfilled at least one of the relevant OECD No. 490 criteria.
The plating efficiencies for the negative (vehicle/solvent) controls of the test item and positive control item as well as the untreated control samples at the end of the expression period (PEviability) were acceptable in all assays.
The number of test concentrations evaluated was at least five in Assay 1 and Assay 2, which met the acceptance criteria about the minimum number of evaluated concentrations.
The tested concentration range in the study was considered to be adequate as the highest evaluated concentration was the recommended maximum concentration (2000 µg/mL) in case of with metabolic activation and short treatment without metabolic activation. The tested concentration range in the study was considered to be adequate as the highest evaluated concentration showed proper degree of cytotoxicity (approximately 80-90%, i.e. approximately 10-20 relative total growth*) in case of long treatment without metabolic activation. Lower test concentrations were usually spaced by a factor of two or three, but more closely spaced concentration were used in the expected cytotoxic range in all cases in an attempt to obtain values in the range of 10-20% viability.
*Note: In Assay 2 without metabolic activation the relative total growth of the highest evaluated concentration (500 µg/mL) was 7%, which was slightly below the target range according to the OECD guideline, however the relative total growth value of the next evaluated concentration (333.33 µg/mL) was within the target range (RTG was 11%). The 500 µg/mL was evaluated in order to properly cover concentrations from cytotoxicity to no significant cytotoxicity. Thus, the results were considered to cover appropriate concentrations, being acceptable to justify the study for the exposures without metabolic activation (long treatment).
Suspension growth value of the untreated and negative (vehicle/solvent) control samples were in line with the recommended range in all cases (i.e. 8-32 fold in case of short treatments and 32-180 fold in case of long treatment).
The overall study was considered to be valid. - Conclusions:
- The Mouse Lymphoma Assay on L5178Y TK +/- 3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.
In this Mouse Lymphoma Assay, treatment with the test item did not result in any reproducible increases in the mutation frequency in the presence or absence of a rat metabolic activation system (S9 fraction) in Assay 1 or Assay 2. Statistical differences were not supported by any results above the GEF. The observed results were repeatable within and between assays. Overall the test item was considered to be negative with and without metabolic activation.
In conclusion, no mutagenic effect of AMINOX® was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. - Executive summary:
An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/-3.7.2 C cells at the tk locus to test the potential ofAMINOX®test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). The design of this study was based on the OECDNo. 490 guideline,and the study was performed in compliance with Citoxlab Hungary Ltd. standard operating procedures and with the OECD Principles of Good Laboratory Practice.
Dimethyl sulfoxide was used as vehicle of the test item in this study. Based on the preliminary toxicity test and in agreement with the Sponsor, the following test item concentrations were examined in the main mutation assays:
Assay 1, 3-hour treatment with metabolic activation: 2000, 666.67, 222.22, 74.07, 24.69 and 8.23 µg/mL,
Assay 1, 3-hour treatment without metabolic activation:2000, 666.67, 222.22, 74.07, 24.69 and 8.23 µg/mL,
Assay 2, 3-hour treatment with metabolic activation: 2000,1333.33,666.67, 222.22, 74.07, 24.69 and 8.23 µg/mL,
Assay 2, 24-hour treatment without metabolic activation: 1000, 750, 500, 333.33, 111.11, 37.04 and 12.35 µg/mL.
In Assays 1-2, there were no large changes in pH or osmolality after treatment. Insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment in Assays 1-2 with and without metabolic activation at several concentrations.
In Assay 1,following a 3-hour treatment with metabolic activation, cytotoxicity of the test item was observed at concentration range of (2000-222.22 µg/mL). No cytotoxicity or significant cytotoxicity was observed at lower concentrations.The Relative Total Growth (RTG) value of the highest evaluated concentration was 14%. The evaluation was made using data of all six concentrations (concentration range of 2000-8.23 µg/mL). There was statistically significant increase in the mutation frequency values in the two highest evaluated concentrations in Assay 1 and concentration related increase was also indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant. Therefore,this experiment was considered as being negative.
In Assay 1,following a 3-hour treatment without metabolic activation, cytotoxicity of the test item was observed at concentration range of (2000-222.22 µg/mL).No cytotoxicity or significant cytotoxicity was observed at lower concentrations.The Relative Total Growth (RTG) value of the highest evaluated concentration was 24%. An evaluation was made using data of all six concentrations (concentration range of 2000-8.23 µg/mL). There was statistically significant increase in the mutation frequency values in the highest evaluated concentration, but it was statistically borderline. Concentration related increase was also indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant. Therefore,this experiment was considered as being negative.
In Assay 2,following a 3-hour treatment with metabolic activation, cytotoxicity of the test item was observed at concentration range of (2000-222.22 µg/mL).No cytotoxicity or significant cytotoxicity was observed at lower concentrations.The Relative Total Growth (RTG) value of the highest evaluated concentration was 14%. The evaluation was made using data of all seven concentrations (concentration range of 2000-8.23 µg/mL). There was statistically significant increase in the mutation frequency values in the two highest evaluated concentrations in Assay 2 and concentration related increase was also indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant.This experiment was considered as being negative andconfirmed the negative result of the first test with metabolic activation.
In Assay 2,following a 24-hour treatment without metabolic activation, cytotoxicity of the test item was observed at concentration range of 1000-37.04 µg/mL.Excessive cytotoxicity was observed at 1000 and 750 µg/mL concentrations.No significant cytotoxicity was observed at 12.35 µg/mL concentration.Thus,the evaluation was made using data of five concentrations (concentration range of 500-12.35 µg/mL). The Relative Total Growth (RTG) value of the highest evaluated concentration was 7%. No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis.This experiment was considered as being negative.
The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.
In conclusion, no mutagenic effect of AMINOX®was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Referenceopen allclose all
Summary Table of the Range Finding Test
Concentrations (μg/plate) |
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimurium strains |
|||
TA98 |
TA100 |
||||
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
22.0 |
23.3 |
110.0 |
114.3 |
MF |
0.99 |
1.00 |
1.01 |
1.01 |
|
Distilled water control |
Mean |
- |
- |
108.3 |
- |
MF |
- |
- |
0.99 |
- |
|
DMSO control |
Mean |
23.7 |
23.0 |
- |
111.3 |
MF |
1.06 |
0.99 |
- |
0.99 |
|
Acetone control |
Mean |
22.3 |
27.0 |
51.7 |
71.0 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
24.3 |
27.0 |
51.7 |
71.0 |
MF |
1.09 |
1.16 |
0.47 |
0.63 |
|
2500 |
Mean |
27.3 |
26.7 |
115.3 |
114.7 |
MF |
1.22 |
1.14 |
1.05 |
1.02 |
|
1000 |
Mean |
24.3 |
26.0 |
119.3 |
112.0 |
MF |
1.09 |
1.11 |
1.09 |
0.99 |
|
316 |
Mean |
23.3 |
28.0 |
114.3 |
124.0 |
MF |
1.04 |
1.20 |
1.05 |
1.10 |
|
100 |
Mean |
21.0 |
26.7 |
122.7 |
133.0 |
MF |
0.94 |
1.14 |
1.12 |
1.18 |
|
31.6 |
Mean |
23.3 |
26.0 |
125.0 |
121.0 |
MF |
1.04 |
1.11 |
1.14 |
1.07 |
|
10 |
Mean |
22.3 |
23.0 |
117.7 |
120.7 |
MF |
1.00 |
0.99 |
1.08 |
1.07 |
|
NPD (4μg) |
Mean |
412.0 |
- |
- |
- |
MF |
17.41 |
- |
- |
- |
|
2AA (2μg) |
Mean |
- |
2426.7 |
- |
2373.3 |
MF |
- |
105.51 |
- |
21.32 |
|
SAZ (2μg) |
Mean |
- |
- |
1205.3 |
- |
MF |
- |
- |
11.13 |
- |
|
-: Not applicable
Summary Table of the Initial Mutation Test
Concentrations (μg/plate) |
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimurium tester strains |
Escherichia coli |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
15.36 |
27.7 |
85.7 |
77.3 |
10.3 |
8.3 |
13.3 |
16.3 |
48.3 |
48.0 |
MF |
0.71 |
1.26 |
0.96 |
1.08 |
1.19 |
0.89 |
0.98 |
1.04 |
0.98 |
1.04 |
|
Distilled water control |
Mean |
- |
- |
81.0 |
- |
10.7 |
- |
- |
- |
46.0 |
- |
MF |
- |
- |
0.91 |
- |
1.23 |
- |
- |
- |
0.93 |
- |
|
DMSO control |
Mean |
14.7 |
20.3 |
- |
83.3 |
- |
11.3 |
14.0 |
14.7 |
- |
53.0 |
MF |
0.68 |
0.92 |
- |
1.16 |
- |
1.21 |
1.02 |
0.94 |
- |
1.15 |
|
Acetone control |
Mean |
21.7 |
22.0 |
89.0 |
71.7 |
8.7 |
9.3 |
13.7 |
15.7 |
49.3 |
46.0 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
17.0 |
18.0 |
52.3 |
64.3 |
13.7 |
9.3 |
8.7 |
14.0 |
42.3 |
49.0 |
MF |
0.78 |
0.82 |
0.59 |
0.90 |
1.58 |
1.00 |
0.63 |
0.89 |
0.86 |
1.07 |
|
1581 |
Mean |
21.0 |
20.0 |
95.3 |
69.3 |
14.3 |
13.0 |
13.7 |
10.3 |
44.0 |
52.0 |
MF |
0.97 |
0.91 |
1.07 |
0.97 |
1.65 |
1.39 |
1.00 |
0.66 |
0.89 |
1.13 |
|
500 |
Mean |
18.7 |
20.7 |
85.0 |
60.7 |
12.3 |
12.3 |
15.0 |
14.7 |
46.0 |
44.7 |
MF |
0.86 |
0.94 |
0.96 |
0..85 |
1.42 |
1.32 |
1.10 |
0.94 |
0.93 |
0.97 |
|
158.1 |
Mean |
22.7 |
21.7 |
83.0 |
68.0 |
11.3 |
9.0 |
14.7 |
16.0 |
36.7 |
47.7 |
MF |
1.05 |
0.98 |
0.93 |
0.95 |
1.31 |
0.96 |
1.07 |
1.02 |
0.74 |
1.04 |
|
50 |
Mean |
21.3 |
21.3 |
83.7 |
73.3 |
11.7 |
10.0 |
17.3 |
13.7 |
40.3 |
46.7 |
MF |
0.98 |
0.97 |
0.94 |
1.02 |
1.35 |
1.07 |
1.27 |
0.87 |
0.82 |
1.01 |
|
15.81 |
Mean |
21.0 |
24.0 |
84.0 |
79.3 |
13.7 |
9.7 |
10.7 |
13.0 |
45.3 |
42.3 |
MF |
0.97 |
1.09 |
0.94 |
1.11 |
1.58 |
1.04 |
0.78 |
0.83 |
0.92 |
0.92 |
|
NPD (4μg) |
Mean |
404.7 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
MF |
27.59 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
2AA (2μg) |
Mean |
- |
2474.7 |
- |
2405.3 |
- |
206.7 |
- |
207.3 |
- |
- |
MF |
- |
121.70 |
- |
28.86 |
- |
18.24 |
- |
14.14 |
- |
- |
|
2AA (50μg) |
Mean |
- |
- |
- |
- |
- |
- |
- |
- |
- |
253.7 |
MF |
- |
- |
- |
- |
- |
- |
- |
- |
- |
4.79 |
|
SAZ (2μg) |
Mean |
- |
- |
1205.3 |
- |
1214.0 |
- |
- |
- |
- |
- |
MF |
- |
- |
14.88 |
- |
113.81 |
- |
- |
- |
- |
- |
|
9AA (50μg) |
Mean |
- |
- |
- |
- |
- |
- |
418.7 |
- |
- |
- |
MF |
- |
- |
- |
- |
- |
- |
29.90 |
- |
- |
- |
|
MMS (2μL) |
Mean |
- |
- |
- |
- |
- |
- |
- |
- |
990.7 |
- |
MF |
- |
- |
- |
- |
- |
- |
- |
- |
21.54 |
- |
|
-: Not applicable
Summary Table of the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test
Concentrations (μg/plate) |
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimurium tester strains |
Escherichia coli |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
20.0 |
27.3 |
98.7 |
104.0 |
11.0 |
10.0 |
10.3 |
13.0 |
66.7 |
67.7 |
MF |
1.22 |
1.15 |
1.02 |
1.13 |
0.80 |
1.07 |
1.19 |
1.08 |
1.09 |
1.05 |
|
Distilled water control |
Mean |
- |
- |
94.3 |
- |
12.3 |
- |
- |
- |
62.7 |
- |
MF |
- |
- |
0.97 |
- |
0.90 |
- |
- |
- |
1.03 |
- |
|
DMSO control |
Mean |
17.3 |
27.0 |
- |
87.3 |
- |
11.0 |
8.3 |
15.3 |
- |
71.0 |
MF |
0.96 |
1.14 |
- |
0.95 |
- |
1.18 |
0.96 |
1.28 |
- |
1.10 |
|
Acetone control |
Mean |
18.0 |
23.7 |
97.0 |
92.3 |
13.7 |
9.3 |
8.7 |
12.0 |
61.0 |
64.7 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
- |
15.7 |
- |
66.3 |
- |
5.7 |
- |
5.7 |
- |
54.7 |
MF |
- |
0.66 |
- |
0.72 |
- |
0.61 |
- |
0.47 |
- |
0.85 |
|
1581 |
Mean |
- |
15.3 |
- |
54.3 |
- |
9.3 |
- |
6.3 |
- |
56.0 |
MF |
- |
0.65 |
- |
0.59 |
- |
1.00 |
- |
0.53 |
- |
0.87 |
|
500 |
Mean |
- |
27.3 |
- |
77.3 |
- |
11.7 |
- |
11.3 |
29.3 |
54.7 |
MF |
- |
1.15 |
- |
0.84 |
- |
1.25 |
- |
0.94 |
0.48 |
0.85 |
|
158.1 |
Mean |
- |
29.7 |
- |
96.3 |
- |
11.3 |
- |
16.0 |
50.7 |
56.7 |
MF |
- |
1.25 |
- |
1.04 |
- |
1.21 |
- |
1.33 |
0.83 |
0.88 |
|
50 |
Mean |
14.0 |
28.7 |
60.3 |
94.7 |
5.3 |
8.0 |
2.7 |
13.7 |
59.3 |
59.7 |
MF |
0.78 |
1.21 |
0.63 |
1.03 |
0.39 |
0.86 |
0.31 |
1.14 |
0.97 |
0.92 |
|
15.81 |
Mean |
16.7 |
28.3 |
82.0 |
93.7 |
11.7 |
12.7 |
6.3 |
13.7 |
65.7 |
68.3 |
MF |
0.93 |
1.20 |
0.85 |
1.01 |
0.85 |
1.36 |
0.73 |
1.14 |
1.08 |
1.06 |
|
5 |
Mean |
21.7 |
24.3 |
94.0 |
102.3 |
11.3 |
13.7 |
7.3 |
13.7 |
68.3 |
67.7 |
MF |
1.20 |
1.03 |
0.97 |
1.11 |
0.83 |
1.46 |
0.85 |
1.14 |
1.12 |
1.05 |
|
1.581 |
Mean |
19.7 |
- |
96.7 |
- |
9.3 |
- |
9.7 |
- |
68.0 |
- |
MF |
1.09 |
- |
1.00 |
- |
0.68 |
- |
1.12 |
- |
1.11 |
- |
|
0.5 |
Mean |
21.3 |
- |
94.7 |
- |
9.7 |
- |
8.3 |
- |
- |
- |
MF |
1.19 |
- |
0.98 |
- |
0.71 |
- |
0.96 |
- |
- |
- |
|
0.1581 |
Mean |
20.0 |
- |
92.0 |
- |
10.7 |
- |
8.3 |
- |
- |
- |
MF |
1.11 |
- |
0.95 |
- |
0.78 |
- |
0.96 |
- |
- |
- |
|
NPD (4μg) |
Mean |
338.7 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
MF |
19.54 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
2AA (2μg) |
Mean |
- |
2325.3 |
- |
2373.3 |
- |
226.7 |
- |
205.3 |
- |
- |
MF |
- |
86.12 |
- |
27.18 |
- |
20.61 |
- |
13.39 |
- |
- |
|
2AA (50μg) |
Mean |
- |
- |
- |
- |
- |
- |
- |
- |
- |
286.0 |
MF |
- |
- |
- |
- |
- |
- |
- |
- |
- |
4.03 |
|
SAZ (2μg) |
Mean |
- |
- |
1134.7 |
- |
1068.0 |
- |
- |
- |
- |
- |
MF |
- |
- |
12.03 |
- |
86.59 |
- |
- |
- |
- |
- |
|
9AA (50μg) |
Mean |
- |
- |
- |
- |
- |
- |
476.0 |
- |
- |
- |
MF |
- |
- |
- |
- |
- |
- |
57.12 |
- |
- |
- |
|
MMS (2μL) |
Mean |
- |
- |
- |
- |
- |
- |
- |
- |
1084.0 |
- |
MF |
- |
- |
- |
- |
- |
- |
- |
- |
17.30 |
- |
|
-: Not applicable
Historical Control Data
(Period of 2011 – 2016)
Untreated control data |
||||||||||
|
Without metabolic activation (-S9 Mix) |
With metabolic activation (+S9 Mix) |
||||||||
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Mean |
22.7 |
103.6 |
11.8 |
7.2 |
33.4 |
29.7 |
111.7 |
11.5 |
8.9 |
39.1 |
St. dev. |
5.8 |
21.4 |
5.1 |
3.3 |
9.7 |
6.8 |
19.6 |
3.9 |
3.8 |
9.9 |
Range |
9-50 |
54-210 |
1-46 |
1-24 |
11-82 |
10-56 |
65-204 |
1-39 |
1-29 |
19-89 |
n |
1371 |
1357 |
1365 |
1371 |
1374 |
1377 |
1365 |
1373 |
1380 |
1371 |
DMSO control data |
||||||||||
|
Without metabolic activation (-S9 Mix) |
With metabolic activation (+S9 Mix) |
||||||||
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1536 |
E. coli |
Mean |
21.7 |
98.9 |
12.0 |
7.1 |
32.3 |
28.7 |
109.5 |
11.3 |
8.7 |
38.1 |
St. dev. |
5.7 |
20.7 |
5.0 |
3.3 |
9.6 |
7.0 |
20.7 |
3.8 |
3.7 |
9.7 |
Range |
6-55 |
40-217 |
1-43 |
1-25 |
7-81 |
11-67 |
53-229 |
2-33 |
1-29 |
9-85 |
n |
1482 |
1473 |
1479 |
1485 |
1482 |
1487 |
1476 |
1487 |
1491 |
1482 |
Distilled water control data |
||||||||||
|
Without metabolic activation (-S9 Mix) |
With metabolic activation (+S9 Mix) |
||||||||
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Mean |
23.5 |
103.2 |
11.9 |
7.8 |
34.5 |
30.8 |
112.2 |
11.3 |
9.3 |
40.3 |
St. dev. |
6.0 |
22.4 |
4.9 |
3.4 |
9.8 |
7.1 |
21.8 |
3.7 |
3.7 |
10.0 |
Range |
11-45 |
45-215 |
2-47 |
2-24 |
12-84 |
10-53 |
64-222 |
3-39 |
1-24 |
13-91 |
n |
267 |
1359 |
1365 |
270 |
1392 |
267 |
1371 |
1380 |
267 |
1383 |
DMF control data |
||||||||||
|
Without metabolic activation (-S9 Mix) |
With metabolic activation (+S9 Mix) |
||||||||
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Mean |
20.4 |
89.9 |
11.2 |
6.9 |
34.7 |
28.1 |
100.3 |
11.0 |
8.0 |
38.0 |
St. dev. |
5.6 |
17.8 |
4.7 |
3.1 |
12.3 |
7.0 |
19.2 |
3.6 |
3.1 |
10.2 |
Range |
8-38 |
54-152 |
1-34 |
1-19 |
16-99 |
13-49 |
60-156 |
3-21 |
1-23 |
17-76 |
n |
216 |
216 |
216 |
216 |
207 |
216 |
216 |
216 |
213 |
207 |
Acetone control data |
||||||||||
|
Without metabolic activation (-S9 Mix) |
With metabolic activation (+S9 Mix) |
||||||||
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Mean |
22.6 |
98.1 |
12.1 |
7.4 |
35.0 |
29.1 |
108.1 |
11.1 |
8.6 |
40.5 |
St. dev. |
5.1 |
15.4 |
5.8 |
2.9 |
9.3 |
6.7 |
14.2 |
3.4 |
3.3 |
9.0 |
Range |
11-39 |
62-160 |
4-49 |
1-17 |
17-62 |
15-52 |
66-177 |
4-22 |
1-19 |
17-69 |
n |
278 |
279 |
279 |
279 |
276 |
279 |
279 |
282 |
279 |
279 |
Positive reference control data |
||||||||||
|
Without metabolic activation (-S9 Mix) |
With metabolic activation (+S9 Mix) |
||||||||
|
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
Mean |
357.2 |
1229.3 |
1169.8 |
454.1 |
1034.3 |
2410.2 |
2429.6 |
235.1 |
221.3 |
257.4 |
St. dev. |
113.8 |
207.5 |
204.2 |
169.7 |
141.7 |
317.7 |
291.6 |
135.9 |
56.2 |
113.4 |
Range |
152-2336 |
536-2120 |
208-2440 |
149-2104 |
488-1708 |
312-4918 |
1192-5240 |
101-2216 |
117-838 |
125-2512 |
n |
1371 |
1359 |
1365 |
1371 |
1377 |
1378 |
1365 |
1377 |
1380 |
1371 |
TA98: Salmonella typhimurium TA98, TA100: Salmonella typhimurium TA100, TA1535: Salmonella typhimurium TA1535, TA1537: Salmonella typhimurium TA1537, E.coli: Escherichia coli WP2 uvrA, n: number of cases.
Summarized results of the Concentration Selection Cytotoxicity without metabolic activation (3h/20h)
Test group |
Dose (μg/mL) |
S9-mix |
Treatment/ sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
|
Untreated control |
– |
– |
3/20 |
3.28E+06 |
118 |
normal / normal (pH: 8.0; osm: 340 mmol/kg) |
|
Negative (vehicle) control |
– |
– |
3/20 |
2.98E+06 |
100 |
normal / normal (pH: 8.0; osm: 491 mmol/kg) |
|
AMINOX® |
2000 |
– |
3/20 |
2.58E+06 |
76 |
precipitate / discoloured medium# , precipitate (pH: 8.0; osm: 466 mmol/kg) |
|
1000 |
– |
3/20 |
2.48E+06 |
69 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 481 mmol/kg) |
||
500 |
– |
3/20 |
2.50E+06 |
71 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 488 mmol/kg) |
||
250 |
– |
3/20 |
2.88E+06 |
94 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 488 mmol/kg) |
||
125 |
– |
3/20 |
2.85E+06 |
92 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 489 mmol/kg) |
||
62.5 |
– |
3/20 |
2.70E+06 |
83 |
discoloured medium#, precipitate / discoloured medium#, precipitate# (pH: 8.0; osm: 494 mmol/kg) |
||
31.25 |
– |
3/20 |
2.65E+06 |
80 |
precipitate # / precipitate # (pH: 8.0; osm: 489 mmol/kg) |
||
15.625 |
– |
3/20 |
2.90E+06 |
95 |
normal / normal (pH: 8.0; osm: 491 mmol/kg) |
||
7.813 |
– |
3/20 |
3.08E+06 |
106 |
normal / normal (pH: 8.0; osm: 495 mmol/kg) |
||
3.906 |
– |
3/20 |
2.98E+06 |
100 |
normal / normal (pH: 8.0; osm: 498 mmol/kg) |
||
*: compared to the negative (vehicle) control (1% (v/v) DMSO) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolality
Note:
Duplicate counts were performed at each counting.
Summarized results of the Concentration Selection Cytotoxicity with metabolic activation (3h/20h)
Test group |
Dose (μg/mL) |
S9-mix |
Treatment/ sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
– |
+ |
3/20 |
3.08E+06 |
128 |
normal / normal (pH: 8.0; osm: 336 mmol/kg) |
Negative (vehicle) control |
– |
+ |
3/20 |
2.70E+06 |
100 |
normal / normal (pH: 8.0; osm: 488 mmol/kg) |
AMINOX® |
2000 |
+ |
3/20 |
2.53E+06 |
87 |
precipitate / discoloured medium# , precipitate (pH: 8.0; osm: 458 mmol/kg) |
1000 |
+ |
3/20 |
2.28E+06 |
69 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 473 mmol/kg) |
|
500 |
+ |
3/20 |
2.03E+06 |
50 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 481 mmol/kg) |
|
250 |
+ |
3/20 |
3.08E+06 |
128 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 490 mmol/kg) |
|
125 |
+ |
3/20 |
2.95E+06 |
118 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 485 mmol/kg) |
|
62.5 |
+ |
3/20 |
3.08E+06 |
128 |
discoloured medium#, precipitate / discoloured medium#, precipitate# (pH: 8.0; osm: 482 mmol/kg) |
|
31.25 |
+ |
3/20 |
2.68E+06 |
98 |
precipitate # / normal (pH: 8.0; osm: 487 mmol/kg) |
|
15.625 |
+ |
3/20 |
3.28E+06 |
142 |
normal / normal (pH: 8.0; osm: 484 mmol/kg) |
|
7.813 |
+ |
3/20 |
3.28E+06 |
142 |
normal / normal (pH: 8.0; osm: 487 mmol/kg) |
|
3.906 |
+ |
3/20 |
3.20E+06 |
137 |
normal / normal (pH: 8.0; osm: 507 mmol/kg) |
*: compared to the negative (vehicle) control (1% (v/v) DMSO) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolality
Note: Duplicate counts were performed at each counting.
Summarized results of the Concentration Selection Cytotoxicity without metabolic activation (20h/20h)
Test group |
Dose (μg/mL) |
S9-mix |
Treatment/ sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
– |
– |
20/20 |
3.73E+06 |
120 |
normal / normal (pH: 8.0; osm: 343 mmol/kg) |
Negative (vehicle) control |
– |
– |
20/20 |
3.33E+06 |
100 |
normal / normal (pH: 8.0; osm: 483 mmol/kg) |
AMINOX® |
2000 |
– |
20/20 |
1.88E+06 |
27 |
precipitate / discoloured medium# , precipitate (pH: 8.0; osm: 483 mmol/kg) |
1000 |
– |
20/20 |
4.00E+05 |
not applicable |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 472 mmol/kg) |
|
500 |
– |
20/20 |
4.75E+05 |
not applicable |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 480 mmol/kg) |
|
250 |
– |
20/20 |
1.18E+06 |
not applicable |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 495 mmol/kg) |
|
125 |
– |
20/20 |
1.90E+06 |
28 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 487 mmol/kg) |
|
62.5 |
– |
20/20 |
2.03E+06 |
35 |
discoloured medium#, precipitate / discoloured medium#, precipitate (pH: 8.0; osm: 492 mmol/kg) |
|
31.25 |
– |
20/20 |
2.78E+06 |
72 |
precipitate # / precipitate # (pH: 8.0; osm: 489 mmol/kg) |
|
15.625 |
– |
20/20 |
3.45E+06 |
106 |
normal / normal (pH: 8.0; osm: 490 mmol/kg) |
|
7.813 |
– |
20/20 |
3.38E+06 |
103 |
normal / normal (pH: 8.0; osm: 489 mmol/kg) |
|
3.906 |
– |
20/20 |
3.20E+06 |
94 |
normal / normal (pH: 8.0; osm: 492 mmol/kg) |
*: compared to the negative (vehicle) control (1% (v/v) DMSO) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolality
Note: Duplicate counts were performed at each counting.
Cytotoxicity Results of Chromosome Aberration Assay 1 experiment without metabolic activation
Test group |
Dose (μg/mL) |
S9-mix |
Treatment/ sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
|
Untreated control |
– |
– |
3/20 |
6.25E+06 |
116 |
normal / normal (pH: 7.7; osm: 349 mmol/kg) |
|
Negative (vehicle) control |
– |
– |
3/20 |
5.65E+06 |
100 |
normal / normal (pH: 7.7; osm: 501 mmol/kg) |
|
AMINOX® |
125 |
– |
3/20 |
4.80E+06 |
78 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 7.7; osm: 482 mmol/kg) |
|
100 |
– |
3/20 |
5.00E+06 |
83 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 7.7; osm: 489 mmol/kg) |
||
75 |
– |
3/20 |
5.00E+06 |
83 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 7.7; osm: 493 mmol/kg) |
||
50 |
– |
3/20 |
5.20E+06 |
88 |
discoloured medium#, precipitate / discoloured medium, precipitate (pH: 7.7; osm: 498 mmol/kg) |
||
25 |
– |
3/20 |
5.00E+06 |
83 |
normal / normal (pH: 7.7; osm: 490 mmol/kg) |
||
12.5 |
– |
3/20 |
5.40E+06 |
94 |
normal / normal (pH: 7.7; osm: 494 mmol/kg) |
||
Positive control (1 μL/mL EMS) |
- |
– |
3/20 |
3.90E+06 |
55 |
normal / normal (pH: 7.7; osm: 361 mmol/kg) |
|
*: compared to the negative (vehicle) control (1% (v/v) DMSO) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolality
Note: Duplicate counts were performed at each counting.
Cytotoxicity Results of Chromosome Aberration Assay 1 experiment with metabolic activation
Test group |
Dose (μg/mL) |
S9-mix |
Treatment/ sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
– |
+ |
3/20 |
5.50E+06 |
97 |
normal / normal (pH: 7.7; osm: 350 mmol/kg) |
Negative (vehicle) control |
– |
+ |
3/20 |
5.60E+06 |
100 |
normal / normal (pH: 7.4; osm: 494 mmol/kg) |
AMINOX® |
125 |
+ |
3/20 |
5.05E+06 |
86 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 7.4; osm: 489 mmol/kg) |
100 |
+ |
3/20 |
5.05E+06 |
86 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 7.4; osm: 528 mmol/kg) |
|
75 |
+ |
3/20 |
5.05E+06 |
86 |
discoloured medium#, precipitate / discoloured medium, precipitate (pH: 7.4; osm: 490 mmol/kg) |
|
50 |
+ |
3/20 |
4.85E+06 |
80 |
discoloured medium#, precipitate / discoloured medium#, precipitate (pH: 7.4; osm: 504 mmol/kg) |
|
25 |
+ |
3/20 |
5.25E+06 |
91 |
normal / normal (pH: 7.4; osm: 483 mmol/kg) |
|
12.5 |
+ |
3/20 |
5.70E+06 |
103 |
normal / normal (pH: 7.4; osm: 491 mmol/kg) |
|
Positive control (6 μg/mL CP) |
– |
+ |
3/20 |
3.25E+06 |
38 |
normal / normal (pH: 7.4; osm: 358 mmol/kg) |
*: compared to the negative (vehicle) control (1% (v/v) DMSO) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolality
Note: Duplicate counts were performed at each counting.
Cytotoxicity Results of Chromosome Aberration Assay 2 experiment without metabolic activation
Test group |
Dose μg/mL |
S9-mix |
Treatment/ sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
– |
– |
20/20 |
5.10E+06 |
82 |
normal / normal (pH: 8.0; osm: 348 mmol/kg) |
Negative (vehicle) control |
– |
– |
20/20 |
5.90E+06 |
100 |
normal / normal (pH: 8.0; osm: 494 mmol/kg) |
AMINOX® |
200 |
– |
20/20 |
2.05E+06 |
12 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 489 mmol/kg) |
150 |
– |
20/20 |
2.45E+06 |
21 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 494 mmol/kg) |
|
125 |
– |
20/20 |
2.85E+06 |
30 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 491 mmol/kg) |
|
100 |
– |
20/20 |
2.95E+06 |
32 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 501 mmol/kg) |
|
75 |
– |
20/20 |
3.85E+06 |
53 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 505 mmol/kg) |
|
50 |
– |
20/20 |
3.70E+06 |
50 |
discoloured medium#, precipitate / discoloured medium#, precipitate (pH: 8.0; osm: 493 mmol/kg) |
|
25 |
– |
20/20 |
5.10E+06 |
82 |
precipitate# / precipitate# (pH: 8.0; osm: 497 mmol/kg) |
|
Positive control (0.4 μL/mL EMS) |
- |
– |
20/20 |
3.65E+06 |
48 |
normal / normal (pH: 8.0; osm: 363 mmol/kg) |
*: compared to the negative (vehicle) control (1% (v/v) DMSO) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolality
Note: Duplicate counts were performed at each counting.
Cytotoxicity Results of Chromosome Aberration Assay 2 experiment with metabolic activation
Test group |
Dose (μg/mL) |
S9-mix |
Treatment/ sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
– |
+ |
3/20 |
4.95E+06 |
106 |
normal / normal (pH: 8.0; osm: 349 mmol/kg) |
Negative (vehicle) control |
– |
+ |
3/20 |
4.75E+06 |
100 |
normal / normal (pH: 8.0; osm: 489 mmol/kg) |
AMINOX® |
125 |
+ |
3/20 |
4.90E+06 |
105 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 502 mmol/kg) |
100 |
+ |
3/20 |
4.60E+06 |
95 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 484 mmol/kg) |
|
75 |
+ |
3/20 |
5.00E+06 |
108 |
discoloured medium, precipitate / discoloured medium, precipitate (pH: 8.0; osm: 490 mmol/kg) |
|
50 |
+ |
3/20 |
5.05E+06 |
109 |
discoloured medium#, precipitate / discoloured medium#, precipitate (pH: 8.0; osm: 503 mmol/kg) |
|
25 |
+ |
3/20 |
4.75E+06 |
100 |
discoloured medium#, precipitate# / discoloured medium#, precipitate# (pH: 8.0; osm: 484 mmol/kg) |
|
12.5 |
+ |
3/20 |
5.00E+06 |
108 |
normal / normal (pH: 8.0; osm: 488 mmol/kg) |
|
Positive control (6 μg/mL CP) |
– |
+ |
3/20 |
2.45E+06 |
28 |
normal / normal (pH: 8.0; osm: 354 mmol/kg) |
*: compared to the negative (vehicle) control (1% (v/v) DMSO) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolality
Note: Duplicate counts were performed at each counting.
Summary table of Chromosome Aberration Assay 1 without metabolic activation
Concentration (µg/mL) [Number of analyzed |
Time of treatment / sampling |
Sampling RICC# |
Insolubility ## |
Mean % aberrant cells ### |
AMINOX® without metabolic activation (-S9) |
||||
Untreated control |
3 h / 20 h |
116 |
- |
NE |
Negative (vehicle) control [300] |
3 h / 20 h |
100 |
- |
2.7 |
125 |
3 h / 20 h |
78 |
+b |
NE |
100 |
3 h / 20 h |
83 |
+b |
NE |
75 |
3 h / 20 h |
83 |
+b |
NE |
50 [300] |
3 h / 20 h |
88 |
+b |
3.0 |
25 [300] |
3 h / 20 h |
83 |
- |
1.7 |
12.5 [300] |
3 h / 20 h |
94 |
- |
1.7 |
Positive control [305] |
3 h / 20 h |
55 |
- |
16.4*** |
16.4***
Negative (vehicle) control: (1% (v/v) DMSO)
Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
b: discoloured medium
#: compared to the negative (vehicle) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative.
Summary table of Chromosome Aberration Assay 1 with metabolic activation
Concentration (µg/mL) [Number of analyzed |
Time of treatment / sampling |
Sampling RICC# |
Insolubility ## |
Mean % aberrant cells ### |
AMINOX® with metabolic activation (+S9) |
||||
Untreated control |
3 h / 20 h |
97 |
- |
NE |
Negative (vehicle) control [300] |
3 h / 20 h |
100 |
- |
2.7 |
125 |
3 h / 20 h |
86 |
+b |
NE |
100 |
3 h / 20 h |
86 |
+b |
NE |
75 |
3 h / 20 h |
86 |
+b |
NE |
50 [300] |
3 h / 20 h |
80 |
+b |
5.0 |
25 [300] |
3 h / 20 h |
91 |
- |
2.7 |
12.5 [300] |
3 h / 20 h |
103 |
- |
3.7 |
Positive control [77] |
3 h / 20 h |
38 |
- |
64.9*** |
Negative (vehicle) control: (1% (v/v) DMSO)
Positive control (+S9): Cyclophosphamide, 6 μg/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
#: compared to the negative (vehicle) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
b: discoloured medium
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative
Summary table of Chromosome Aberration Assay 2 without metabolic activation
Concentration (µg/mL) [Number of analyzed |
Time of treatment / sampling |
Sampling RICC# |
Insolubility ## |
Mean % aberrant cells ### |
AMINOX® without metabolic activation (-S9) |
||||
Untreated control |
20 h / 20 h |
82 |
- |
NE |
Negative (vehicle) control [300] |
20 h / 20 h |
100 |
- |
4.0 |
200 |
20 h / 20 h |
12 |
+b |
NE |
150 |
20 h / 20 h |
21 |
+b |
NE |
125 |
20 h / 20 h |
30 |
+b |
NE |
100 |
20 h / 20 h |
32 |
+b |
NE |
75 [300] |
20 h / 20 h |
53 |
+b |
4.0 |
50 [300] |
20 h / 20 h |
50 |
+b |
3.0 |
25 [300] |
20 h / 20 h |
82 |
+b |
3.0 |
Positive control [305] |
20 h / 20 h |
48 |
- |
32.5 |
Negative (vehicle) control: (1% (v/v) DMSO)
Positive control (-S9): Ethyl methanesulfonate, 0.4 μL/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
#: compared to the negative (vehicle) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
a: minimal amount
b: discoloured medium
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative
Summary table of Chromosome Aberration Assay 2 with metabolic activation
Concentration (µg/mL) [Number of analyzed |
Time of treatment / sampling |
Sampling RICC# |
Insolubility ## |
Mean % aberrant cells ### |
AMINOX® with metabolic activation (+S9) |
||||
Untreated control |
3 h / 20 h |
106 |
- |
NE |
Negative (vehicle) control [300] |
3 h / 20 h |
100 |
- |
5.0 |
125 |
3 h / 20 h |
105 |
+b |
NE |
100 |
3 h / 20 h |
95 |
+b |
NE |
75 |
3 h / 20 h |
108 |
+b |
NE |
50 [300] |
3 h / 20 h |
109 |
+b |
3.3 |
25 [300] |
3 h / 20 h |
100 |
- |
2.7 |
12.5 [300] |
3 h / 20 h |
108 |
- |
3.7 |
Positive control [77] |
3 h / 20 h |
28 |
- |
90.9*** |
Negative (vehicle) control: (1% (v/v) DMSO)
Positive control (+S9): Cyclophosphamide, 6 μg/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
#: compared to the negative (vehicle) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
a: minimal amount
b: discoloured medium
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative
Historical control data: 3/20h treatment/sampling time without S9-mix
|
aberration rate (phases with aberration in %) |
|||
|
negative control |
Positive control (EMS) |
||
|
Incl. Gaps |
Excl.Gaps |
Incl. Gaps |
Excl.Gaps |
Mean |
2.71 |
1.20 |
22.67 |
18.02 |
SD |
1.65 |
0.82 |
12.43 |
8.16 |
Range |
0-7 |
0-3 |
4-63 |
4-40 |
N |
46 |
46 |
40 |
40 |
SD = standard deviation
Range = min.-max. values
n = number of experiments
EMS = Ethyl methanesulfonate
CP = Cyclophosphamide
Historical control data:3h/20h treatment/sampling time with S9-mix
|
aberration rate (phases with aberration in %) |
|||
|
negative control |
Positive control (EMS) |
||
|
Incl. Gaps |
Excl.Gaps |
Incl. Gaps |
Excl.Gaps |
Mean |
3.24 |
1.45 |
73.00 |
69.50 |
SD |
1.57 |
0.95 |
23.51 |
25.64 |
Range |
0-8 |
0-4 |
21-100 |
21-100 |
n |
43 |
43 |
21 |
21 |
SD = standard deviation
Range = min.-max. values
n = number of experiments
EMS = Ethyl methanesulfonate
CP = Cyclophosphamide
Historical control data: 20h/28h treatment/sampling time without S9-mix
|
aberration rate (phases with aberration in %) |
|||
|
negative control |
Positive control (EMS) |
||
|
Incl. Gaps |
Excl.Gaps |
Incl. Gaps |
Excl.Gaps |
Mean |
2.58 |
1.13 |
34.97 |
30.96 |
SD |
1.81 |
0.86 |
10.94 |
9.80 |
Range |
0-8 |
0-4 |
5-76 |
5-68.2 |
n |
43 |
43 |
43 |
43 |
SD = standard deviation
Range = min.-max. values
n = number of experiments
EMS = Ethyl methanesulfonate
CP = Cyclophosphamide
Historical control data:3h/28h treatment/sampling time with S9-mix
|
aberration rate (phases with aberration in %) |
|||
|
negative control |
Positive control (EMS) |
||
|
Incl. Gaps |
Excl.Gaps |
Incl. Gaps |
Excl.Gaps |
Mean |
3.07 |
1.47 |
52.63 |
48.80 |
SD |
1.64 |
1.07 |
23.98 |
24.49 |
Range |
0-8 |
0-5 |
7-93.8 |
6-93.8 |
n |
43 |
43 |
21 |
21 |
SD = standard deviation
Range = min.-max. values
n = number of experiments
EMS = Ethyl methanesulfonate
CP = Cyclophosphamide
Results of V29 chromosome aberration assay – assay 1
Activation test condition |
Relative increase in cell counts (%) |
No. scored metaphases (cells) |
No of aberrant cells |
Aberration rate (aberrant cells / 100 cells) |
Number of aberrations |
Aberrations |
||||||||
Gap + |
Gap - |
Gap - |
Gap + |
Gap - |
Chromatid |
Chromosome |
||||||||
Gap |
Break |
Exchange |
Gap |
Break |
Exchange |
Multiple aberrations |
||||||||
Solvent control A |
100 |
150 |
9 |
7 |
0.047 |
9 |
7 |
2 |
4 |
2 |
0 |
0 |
1 |
0 |
Solvent control B |
150 |
4 |
1 |
0.007 |
5 |
1 |
4 |
1 |
0 |
0 |
0 |
0 |
0 |
|
Mean |
|
4.3 |
2.7 |
0.027 |
4.7 |
2.7 |
|
|
|
|
|
|
|
|
Positive control A |
|
145 |
30 |
25 |
0.172 |
35 |
29 |
6 |
14 |
10 |
0 |
0 |
5 |
0 |
Positive control B |
|
160 |
30 |
25 |
0.156 |
40 |
35 |
5 |
17 |
17 |
0 |
0 |
0 |
1 |
Total |
55 |
|
60 |
50 |
0.164*** |
75 |
64 |
|
|
|
|
|
|
|
Test item 1 |
||||||||||||||
12.5 µg/ml A |
|
150 |
5 |
2 |
0.013 |
5 |
2 |
3 |
1 |
1 |
0 |
0 |
0 |
0 |
12.5 µg/ml B |
|
150 |
7 |
3 |
0.020 |
7 |
3 |
4 |
1 |
0 |
0 |
0 |
2 |
0 |
Mean |
94 |
|
4.0 |
1.7 |
0.017 |
4.0 |
1.7 |
|
|
|
|
|
|
|
25 µg/ml A |
|
150 |
5 |
2 |
0.013 |
5 |
2 |
3 |
2 |
0 |
0 |
0 |
0 |
0 |
25 µg/ml B |
|
150 |
6 |
3 |
0.020 |
6 |
3 |
3 |
3 |
0 |
0 |
0 |
0 |
0 |
Mean |
83 |
|
3.7 |
1.7 |
0.017 |
3.7 |
1.7 |
|
|
|
|
|
|
|
50 µg/ml A |
|
150 |
7 |
3 |
0.040 |
7 |
6 |
1 |
4 |
0 |
0 |
0 |
2 |
0 |
50 µg/ml B |
|
150 |
4 |
6 |
0.020 |
4 |
3 |
1 |
1 |
0 |
0 |
0 |
2 |
0 |
Mean |
88 |
|
3.7 |
3.0 |
0.030 |
3.7 |
3.0 |
|
|
|
|
|
|
|
Solvent: DMSO
Positive control: Ethyl methansulphonate 1.0µl/ml
Harvesting time: 20 hours from the treatment
Duplicate cultures: A, B
*** p <0.001 fishers exact test comparing the number of aberrant cells excluding gaps with corresponding negative control
Results of V29 chromosome aberration assay – assay 1
Activation test condition |
Relative increase in cell counts (%) |
No. scored metaphases (cells) |
No of aberrant cells |
Aberration rate (aberrant cells / 100 cells) |
Number of aberrations |
Aberrations |
||||||||
Gap + |
Gap - |
Gap - |
Gap + |
Gap - |
Chromatid |
Chromosome |
||||||||
Gap |
Break |
Exchange |
Gap |
Break |
Exchange |
Multiple aberrations |
||||||||
Solvent control A |
100 |
150 |
6 |
3 |
0.020 |
6 |
3 |
3 |
3 |
0 |
0 |
0 |
0 |
0 |
Solvent control B |
150 |
7 |
5 |
0.033 |
8 |
6 |
2 |
3 |
1 |
0 |
0 |
2 |
0 |
|
Mean |
|
4.3 |
2.7 |
0.027 |
4.7 |
3.0 |
|
|
|
|
|
|
|
|
Positive control A |
|
38 |
25 |
25 |
0.658 |
39 |
38 |
1 |
9 |
26 |
0 |
0 |
2 |
1 |
Positive control B |
|
39 |
25 |
25 |
0.641 |
38 |
38 |
0 |
16 |
21 |
0 |
0 |
1 |
0 |
Total |
38 |
77 |
50 |
50 |
0.649*** |
77 |
76 |
|
|
|
|
|
|
|
Test Item |
||||||||||||||
12.5 µg/ml A |
|
150 |
3 |
1 |
0.007 |
3 |
1 |
2 |
1 |
0 |
0 |
0 |
0 |
0 |
12.5 µg/ml B |
|
150 |
14 |
10 |
0.067 |
15 |
11 |
4 |
6 |
2 |
0 |
1 |
1 |
1 |
Mean |
103 |
|
5.7 |
3.7 |
0.037 |
6.0 |
4.0 |
|
|
|
|
|
|
|
25 µg/ml A |
|
150 |
8 |
7 |
0.047 |
8 |
7 |
1 |
4 |
1 |
0 |
0 |
2 |
0 |
25 µg/ml B |
|
150 |
2 |
1 |
0.007 |
2 |
1 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
Mean |
91 |
|
3.3 |
2.7 |
0.027 |
3.3 |
2.7 |
|
|
|
|
|
|
|
50 µg/ml A |
|
150 |
13 |
10 |
0.067 |
13 |
10 |
3 |
7 |
3 |
0 |
0 |
0 |
0 |
50 µg/ml B |
|
150 |
8 |
5 |
0.033 |
8 |
5 |
3 |
3 |
1 |
0 |
0 |
1 |
0 |
Mean |
80 |
|
7.0 |
5.0 |
0.050 |
7.0 |
5.0 |
|
|
|
|
|
|
|
Solvent: DMSO
Positive control: Cyclophosphamide 6.0 µg/ml
Harvesting time: 20 hours from the treatment
Duplicate cultures: A, B
*** p <0.001 fishers exact test comparing the number of aberrant cells excluding gaps with corresponding negative control
Results of V79 chromosome aberration assay – assay 2
Activation test condition |
Relative increase in cell counts (%) |
No. scored metaphases (cells) |
No of aberrant cells |
Aberration rate (aberrant cells / 100 cells) |
Number of aberrations |
Aberrations |
||||||||
Gap + |
Gap - |
Gap - |
Gap + |
Gap - |
Chromatid |
Chromosome |
||||||||
Gap |
Break |
Exchange |
Gap |
Break |
Exchange |
Multiple aberrations |
||||||||
Solvent control A |
100 |
150 |
15 |
10 |
0.067 |
19 |
10 |
7 |
7 |
1 |
2 |
1 |
1 |
0 |
Solvent control B |
150 |
9 |
2 |
0.013 |
9 |
2 |
5 |
1 |
0 |
2 |
0 |
1 |
0 |
|
Mean |
|
8.0 |
4.0 |
0.040 |
9.3 |
4.0 |
|
|
|
|
|
|
|
|
Positive control A |
|
77 |
27 |
25 |
0.325 |
37 |
29 |
8 |
11 |
18 |
0 |
0 |
0 |
0 |
Positive control B |
|
77 |
27 |
25 |
0.325 |
36 |
29 |
6 |
11 |
16 |
1 |
1 |
1 |
0 |
Total |
48 |
154 |
54 |
50 |
0.325*** |
73 |
58 |
|
|
|
|
|
|
|
Test item |
||||||||||||||
25 µg/ml A |
|
150 |
9 |
7 |
0.047 |
9 |
7 |
2 |
2 |
2 |
0 |
0 |
3 |
0 |
25 µg/ml B |
|
150 |
7 |
2 |
0.013 |
7 |
2 |
5 |
0 |
1 |
0 |
0 |
1 |
0 |
Mean |
82 |
|
5.3 |
3.0 |
0.030 |
5.3 |
3.0 |
|
|
|
|
|
|
|
50 µg/ml A |
|
150 |
9 |
5 |
0.033 |
12 |
5 |
7 |
4 |
0 |
0 |
0 |
1 |
0 |
50 µg/ml B |
|
150 |
6 |
4 |
0.027 |
8 |
4 |
4 |
0 |
3 |
0 |
0 |
1 |
0 |
Mean |
50 |
|
5.0 |
3.0 |
0.030 |
6.7 |
3.0 |
|
|
|
|
|
|
|
75 µg/ml A |
|
150 |
8 |
5 |
0.033 |
8 |
5 |
3 |
2 |
2 |
0 |
0 |
1 |
0 |
75 µg/ml B |
|
150 |
13 |
7 |
0.047 |
15 |
7 |
8 |
3 |
1 |
0 |
1 |
1 |
1 |
Mean |
53 |
|
7.0 |
4.0 |
0.040 |
7.7 |
4.0 |
|
|
|
|
|
|
|
Solvent: DMSO
Positive control: Ethyl methanesulphonate 1.0 µg/ml
Harvesting time: 20 hours from the treatment
Duplicate cultures: A, B
*** p <0.001 fishers exact test comparing the number of aberrant cells excluding gaps with corresponding negative control
Results of V79 chromosome aberration assay – assay 2
Activation test condition |
Relative increase in cell counts (%) |
No. scored metaphases (cells) |
No of aberrant cells |
Aberration rate (aberrant cells / 100 cells) |
Number of aberrations |
Aberrations |
||||||||
Gap + |
Gap - |
Gap - |
Gap + |
Gap - |
Chromatid |
Chromosome |
||||||||
Gap |
Break |
Exchange |
Gap |
Break |
Exchange |
Multiple aberrations |
||||||||
Solvent control A |
100 |
150 |
6 |
1 |
0.007 |
6 |
1 |
5 |
0 |
0 |
0 |
0 |
1 |
0 |
Solvent control B |
150 |
19 |
14 |
0.093 |
26 |
19 |
6 |
8 |
8 |
1 |
2 |
1 |
0 |
|
Mean |
|
8.3 |
5.0 |
0.050 |
10.7 |
6.7 |
|
|
|
|
|
|
|
|
Positive control A |
|
28 |
25 |
25 |
0.893 |
47 |
44 |
3 |
10 |
23 |
0 |
0 |
0 |
11 |
Positive control B |
|
27 |
25 |
25 |
0.926 |
51 |
49 |
2 |
7 |
32 |
0 |
0 |
1 |
9 |
Total |
28 |
55 |
50 |
50 |
0.909*** |
98 |
93 |
|
|
|
|
|
|
|
Test item |
||||||||||||||
12.5 µg/ml A |
|
150 |
15 |
5 |
0.033 |
18 |
7 |
10 |
6 |
0 |
1 |
0 |
0 |
1 |
12.5 µg/ml B |
|
150 |
12 |
3 |
0.020 |
14 |
3 |
10 |
3 |
0 |
1 |
0 |
0 |
0 |
Mean |
108 |
|
9.0 |
2.7 |
0.027 |
10.7 |
3.3 |
|
|
|
|
|
|
|
25 µg/ml A |
|
150 |
11 |
6 |
0.040 |
16 |
9 |
6 |
4 |
4 |
1 |
0 |
1 |
0 |
25 µg/ml B |
|
150 |
10 |
2 |
0.013 |
12 |
4 |
8 |
0 |
1 |
0 |
1 |
2 |
0 |
Mean |
100 |
|
7.0 |
2.7 |
0.027 |
9.3 |
4.3 |
|
|
|
|
|
|
|
50 µg/ml A |
|
150 |
10 |
4 |
0.027 |
11 |
5 |
6 |
3 |
2 |
0 |
0 |
0 |
0 |
50 µg/ml B |
|
150 |
13 |
6 |
0.040 |
15 |
6 |
9 |
6 |
0 |
0 |
0 |
0 |
0 |
Mean |
109 |
|
7.7 |
3.3 |
0.033 |
8.7 |
3.7 |
|
|
|
|
|
|
|
Solvent: DMSO
Positive control: Cyclophosphamide 6.0 µg/ml
Harvesting time: 20 hours from the treatment
Duplicate cultures: A, B
*** p <0.001 fishers exact test comparing the number of aberrant cells excluding gaps with corresponding negative control
Assay 1 –Number of polyploid and endoreduplicated cells
Concentration µg/ml |
Treatment time |
Harvesting time |
No of cells observed |
|
Polyploid |
Endoreduplicated |
|||
Without metabolic activation (-S9) |
||||
Solvent control |
3h |
20h |
1 |
0 |
12.5 |
3h |
20h |
2 |
0 |
25 |
3h |
20h |
2 |
0 |
50 |
3h |
20h |
2 |
0 |
Positive control (305) |
3h |
20h |
4 |
0 |
With metabolic activation (+S9) |
||||
Solvent control |
3h |
20h |
5 |
0 |
12.5 |
3h |
20h |
1 |
0 |
25 |
3h |
20h |
1 |
0 |
50 |
3h |
20h |
4 |
0 |
Positive control (77) |
3h |
20h |
1 |
0 |
Solvent control: DMSO
Positive control (-S9): etheyl methanesulphonate 1.0µl/ml
Positive control (+S9): cyclophosphamide 6.0 µg/ml
The number of polyploid and endoredulicated cells were determined in 300 cells of each test group except shown in brackets
Assay 2 –Number of polyploid and endoreduplicated cells
Concentration µg/ml |
Treatment time |
Harvesting time |
No of cells observed |
|
Polyploid |
Endoreduplicated |
|||
Without metabolic activation (-S9) |
||||
Solvent control |
20h |
20h |
5 |
0 |
25 |
20h |
20h |
7 |
0 |
50 |
20h |
20h |
5 |
0 |
75 |
20h |
20h |
2 |
0 |
Positive control (154) |
20h |
20h |
1 |
0 |
With metabolic activation (+S9) |
||||
Solvent control |
3h |
20h |
8 |
0 |
12.5 |
3h |
20h |
4 |
0 |
25 |
3h |
20h |
3 |
0 |
50 |
3h |
20h |
3 |
0 |
Positive control (55) |
3h |
20h |
1 |
0 |
Solvent control: DMSO
Positive control (-S9): etheyl methanesulphonate 0.4µl/ml
Positive control (+S9): cyclophosphamide 6.0 µg/ml
The number of polyploid and endoredulicated cells were determined in 300 cells of each test group except shown in brackets
Results of the Preliminary Toxicity Test
(3-hour treatment in the presence of metabolic activation)
Test item (or solvent) concentration |
Total cell number, |
Total cell number, (Relative Survival*, %) |
Total cell number, |
Observations at the beginning / after treatment** |
Untreated control |
6.40E+06 (83) |
1.94E+07 (111) |
2.18E+07 (89) |
B: normal A: normal (pH: 7.0, osmolality: 314 mmol/kg) |
Negative (vehicle) control |
7.70E+06 (100) |
1.74E+07 (100) |
2.46E+07 (100) |
B: normal A: normal (pH: 7.0, osmolality: 471 mmol/kg) |
2000 μg/mL |
5.20E+06 (68) |
5.03E+06 (29) |
9.20E+06 (37) |
B: discoloured medium, precipitate A: discoloured medium, precipitate (pH: 7.0, osmolality: 437 mmol/kg) |
1000 μg/mL |
6.30E+06 (82) |
4.65E+06 (27) |
1.68E+07 (68) |
B: discoloured medium, precipitate A: discoloured medium, precipitate (pH: 7.0, osmolality: 458 mmol/kg) |
500 μg/mL |
5.70E+06 (74) |
6.45E+06 (37) |
1.86E+07 (76) |
B: discoloured medium, precipitate A: discoloured medium, precipitate (pH: 7.0, osmolality: 462 mmol/kg) |
250 μg/mL |
5.80E+06 (75) |
8.85E+06 (51) |
1.74E+07 (71) |
B: discoloured medium, precipitate A: discoloured medium, precipitate (pH: 7.0, osmolality: 462 mmol/kg) |
125 μg/mL |
6.45E+06 (84) |
1.76E+07 (101) |
2.14E+07 (87) |
B: discoloured medium, precipitate A: discoloured medium, precipitate (pH: 7.0, osmolality: 464 mmol/kg) |
62.5 μg/mL |
6.45E+06 (84) |
1.41E+07 (81) |
2.88E+07 (117) |
B: discoloured medium#, precipitate# A: normal (pH: 7.0, osmolality: 467 mmol/kg) |
31.25 μg/mL |
6.85E+06 (89) |
1.50E+07 (86) |
2.58E+07 (105) |
B: precipitate# A: normal (pH: 7.0, osmolality: 463 mmol/kg) |
15.625 μg/mL |
6.00E+06 (78) |
1.58E+07 (91) |
2.36E+07 (96) |
B: precipitate# A: normal (pH: 7.0, osmolality: 466 mmol/kg) |
7.813 μg/mL |
6.15E+06 (80) |
1.17E+07 (67) |
2.56E+07 (104) |
B: normal A: normal (pH: 7.0, osmolality: 474 mmol/kg) |
3.906 μg/mL |
7.70E+06 (100) |
1.64E+07 (94) |
2.54E+07 (103) |
B: normal A: normal (pH: 7.0, osmolality: 468 mmol/kg) |
*: compared to the relevant negative (vehicle) control (1% (v/v) DMSO)
**: B: at the beginning of the treatment, A: at the end of the treatment
#: minimal amount
Results of the Preliminary Toxicity Test
(3-hour treatment in the absence of metabolic activation)
Test item (or solvent) concentration |
Total cell number, |
Total cell number, (Relative Survival*, %) |
Total cell number, |
Observations at the beginning / after treatment** |
Untreated control |
1.22E+07 (103) |
1.62E+07 (127) |
2.80E+07 (96) |
B: normal A: normal (pH: 7.0, osmolality: 308 mmol/kg) |
Negative (vehicle) control |
1.18E+07 (100) |
1.28E+07 (100) |
2.92E+07 (100) |
B: normal A: normal (pH: 7.0, osmolality: 463 mmol/kg) |
2000 μg/mL |
5.65E+06 (48) |
5.63E+06 (44) |
2.70E+06 (9) |
B: discoloured medium, precipitate A: discoloured medium, precipitate (pH: 7.0, osmolality: 433 mmol/kg) |
1000 μg/mL |
6.70E+06 (57) |
8.10E+06 (64) |
1.42E+07 (49) |
B: discoloured medium, precipitate A: discoloured medium, precipitate (pH: 7.0, osmolality: 449 mmol/kg) |
500 μg/mL |
6.60E+06 (56) |
1.01E+07 (79) |
1.66E+07 (57) |
B: discoloured medium, precipitate A: discoloured medium, precipitate (pH: 7.0, osmolality: 457 mmol/kg) |
250 μg/mL |
8.15E+06 (69) |
1.23E+07 (96) |
2.14E+07 (73) |
B: discoloured medium, precipitate A: discoloured medium, precipitate (pH: 7.0, osmolality: 461 mmol/kg) |
125 μg/mL |
9.05E+06 (77) |
1.71E+07 (134) |
2.14E+07 (73) |
B: discoloured medium, precipitate A: discoloured medium, precipitate (pH: 7.0, osmolality: 458 mmol/kg) |
62.5 μg/mL |
8.70E+06 (74) |
1.56E+07 (122) |
1.94E+07 (66) |
B: discoloured medium#, precipitate# A: discoloured medium#(pH: 7.0, osmolality: 459 mmol/kg) |
31.25 μg/mL |
1.00E+07 (85) |
1.58E+07 (124) |
2.06E+07 (71) |
B: precipitate# A: normal (pH: 7.0, osmolality: 460 mmol/kg) |
15.625 μg/mL |
1.06E+07 (90) |
1.47E+07 (115) |
2.36E+07 (81) |
B: precipitate# A: normal (pH: 7.0, osmolality: 464 mmol/kg) |
7.813 μg/mL |
1.05E+07 (89) |
1.59E+07 (125) |
2.28E+07 (78) |
B: normal A: normal (pH: 7.0, osmolality: 463 mmol/kg) |
3.906 μg/mL |
1.15E+07 (97) |
1.64E+07 (128) |
2.06E+07 (71) |
B: normal A: normal (pH: 7.0, osmolality: 464 mmol/kg) |
*: compared to the relevant negative (vehicle) control (1% (v/v) DMSO)
#: minimal amount
**: B: at the beginning of the treatment, A: at the end of the treatment
Results of the Preliminary Toxicity Test
24-hour treatment in the absence of metabolic activation
Test item (or solvent) concentration |
Total cell number, |
Total cell number, (Relative Survival*, %) |
Total cell number, |
Observations at the beginning / after treatment** |
Untreated control |
1.86E+07 (106) |
2.22E+07 (137) |
2.08E+07 (116) |
B: normal A: normal (pH: 7.0, osmolality: 312 mmol/kg) |
Negative (vehicle) control |
1.75E+07 (100) |
1.62E+07 (100) |
1.80E+07 (100) |
B: normal A: normal (pH: 7.0, osmolality: 465 mmol/kg) |
2000 μg/mL |
2.40E+06 (14) |
3.00E+05 (2) |
0.00E+00 (0) |
B: discoloured medium, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 440 mmol/kg) |
1000 μg/mL |
3.15E+06 (18) |
6.00E+05 (4) |
1.40E+06 (8) |
B: discoloured medium, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 454 mmol/kg) |
500 μg/mL |
4.45E+06 (26) |
2.25E+06 (14) |
6.30E+06 (35) |
B: discoloured medium, precipitate A: discoloured medium#, precipitate(pH: 7.0, osmolality: 463 mmol/kg) |
250 μg/mL |
4.10E+06 (23) |
6.23E+06 (38) |
1.16E+07 (64) |
B: discoloured medium, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 460 mmol/kg) |
125 μg/mL |
5.65E+06 (32) |
9.45E+06 (58) |
1.74E+07 (97) |
B: discoloured medium, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 462 mmol/kg) |
62.5 μg/mL |
8.85E+06 (51) |
1.17E+07 (72) |
1.90E+07 (106) |
B: discoloured medium#, precipitate# A: discoloured medium#, precipitate#(pH: 7.0, osmolality: 464 mmol/kg) |
31.25 μg/mL |
1.10E+07 (63) |
1.50E+07 (93) |
1.80E+07 (100) |
B: normal A: discoloured medium#(pH: 7.0, osmolality: 467 mmol/kg) |
15.625 μg/mL |
1.51E+07 (87) |
1.47E+07 (91) |
1.90E+07 (106) |
B: normal A: normal (pH: 7.0, osmolality: 469 mmol/kg) |
7.813 μg/mL |
1.47E+07 (84) |
1.70E+07 (105) |
2.28E+07 (127) |
B: normal A: normal (pH: 7.0, osmolality: 462 mmol/kg) |
3.906 μg/mL |
1.46E+07 (83) |
1.62E+07 (100) |
1.98E+07 (110) |
B: normal A: normal (pH: 7.0, osmolality: 461 mmol/kg) |
*: compared to the relevant negative (vehicle) control (1% (v/v) DMSO)
#: minimal amount
**: B: at the beginning of the treatment, A: at the end of the treatment
Survival Results of Assay 1 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating |
+ |
3 |
A1 |
2000 µg/mL |
86/384 |
0.935 |
666.67 µg/mL |
79/384 |
0.988 |
|||
222.22 µg/mL |
108/384 |
0.793 |
|||
74.07 µg/mL |
101/384 |
0.835 |
|||
24.69 µg/mL |
102/384 |
0.829 |
|||
8.23 µg/mL |
89/384 |
0.914 |
|||
Vehicle control |
87/384 |
0.928 |
|||
Untreated control |
99/384 |
0.847 |
|||
Positive control (CP) |
292/384 |
0.171 |
A1 = Assay 1
+ = in the presence of S9-mix
Negative (vehicle) control = 1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 μg/mL)
Survival Results of Assay 1 (II.)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating |
- |
3 |
A1 |
2000 µg/mL |
73/384 |
1.038 |
666.67 µg/mL |
43/384 |
1.368 |
|||
222.22 µg/mL |
78/384 |
0.996 |
|||
74.07 µg/mL |
73/384 |
1.038 |
|||
24.69 µg/mL |
75/384 |
1.021 |
|||
8.23 µg/mL |
78/384 |
0.996 |
|||
Vehicle control |
65/384 |
1.110 |
|||
Untreated control |
55/384 |
1.215 |
|||
Positive control (NQO) |
83/384 |
0.957 |
A1 = Assay 1
- = in the absence of S9-mix
Negative (vehicle) control = 1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.15 μg/mL)
Survival Results – Assay 2 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating |
+ |
3 |
A2 |
2000 µg/mL |
67/384 |
1.091 |
1333.33 µg/mL |
91/384 |
0.900 |
|||
666.67 µg/mL |
77/384 |
1.004 |
|||
222.22 µg/mL |
107/384 |
0.799 |
|||
74.07 µg/mL |
123/384 |
0.712 |
|||
24.69 µg/mL |
98/384 |
0.854 |
|||
8.23 µg/mL |
64/384 |
1.120 |
|||
Vehicle control |
99/384 |
0.847 |
|||
Untreated control |
121/384 |
0.722 |
|||
Positive control (CP) |
214/384 |
0.365 |
A2 = Assay 2
+ = in the presence of S9-mix
Negative (vehicle) control = 1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 μg/mL)
Survival Results – Assay 2 (II.)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating |
- |
24 |
A2 |
1000 µg/mL |
251/384 |
0.266 |
750 µg/mL |
174/384 |
0.495 |
|||
500 µg/mL |
117/384 |
0.743 |
|||
333.33 µg/mL |
113/384 |
0.765 |
|||
111.11 µg/mL |
113/384 |
0.765 |
|||
37.04 µg/mL |
90/384 |
0.907 |
|||
12.35 µg/mL |
110/384 |
0.781 |
|||
Vehicle control |
121/384 |
0.722 |
|||
Untreated control |
104/384 |
0.816 |
|||
Positive control (NQO) |
207/384 |
0.386 |
A2 = Assay 2
- = in the absence of S9-mix
Negative (vehicle) control = 1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.1 μg/mL)
Viability Results of Assay 1 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency |
Relative |
+ |
3 |
A1 |
2000 µg/mL |
101/384 |
0.835 |
14 |
666.67 µg/mL |
83/384 |
0.957 |
45 |
|||
222.22 µg/mL |
117/384 |
0.743 |
31 |
|||
74.07 µg/mL |
93/384 |
0.886 |
78 |
|||
24.69 µg/mL |
91/384 |
0.900 |
73 |
|||
8.23 µg/mL |
93/384 |
0.886 |
116 |
|||
Vehicle control |
97/384 |
0.860 |
100 |
|||
Untreated control |
93/384 |
0.886 |
135 |
|||
Positive control (CP) |
249/384 |
0.835 |
16 |
A1 = Assay 1
+ = in the presence of S9-mix
Negative (vehicle) control = 1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 μg/mL)
Viability Results of Assay 1 (II.)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency |
Relative |
- |
3 |
A1 |
2000 µg/mL |
53/384 |
1.238 |
24 |
666.67 µg/mL |
64/384 |
1.120 |
50 |
|||
222.22 µg/mL |
77/384 |
1.004 |
51 |
|||
74.07 µg/mL |
74/384 |
1.029 |
74 |
|||
24.69 µg/mL |
73/384 |
1.038 |
80 |
|||
8.23 µg/mL |
88/384 |
0.921 |
90 |
|||
Vehicle control |
68/384 |
1.082 |
100 |
|||
Untreated control |
68/384 |
1.082 |
112 |
|||
Positive control (NQO) |
97/384 |
0.860 |
65 |
A1 = Assay 1
- = in the absence of S9-mix
Negative (vehicle) control = 1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.15 μg/mL)
Viability Results of Assay 2 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency |
Relative |
+ |
3 |
A2 |
2000 µg/mL |
40/384 |
1.414 |
14 |
1333.33 µg/mL |
78/384 |
0.996 |
17 |
|||
666.67 µg/mL |
58/384 |
1.181 |
32 |
|||
222.22 µg/mL |
74/384 |
1.029 |
27 |
|||
74.07 µg/mL |
78/384 |
0.996 |
65 |
|||
24.69 µg/mL |
80/384 |
0.980 |
88 |
|||
8.23 µg/mL |
68/384 |
1.082 |
130 |
|||
Vehicle control |
98/384 |
0.854 |
100 |
|||
Untreated control |
100/384 |
0.841 |
108 |
|||
Positive control (CP) |
188/384 |
0.446 |
30 |
A2 = Assay 2
+ = in the presence of S9-mix
Negative (vehicle) control = 1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 μg/mL)
Viability Results of Assay 2 (IL)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency |
Relative |
|
- |
24 |
A2 |
1000 µg/mL |
147/384 |
0.600 |
1 |
|
750 µg/mL |
110/384 |
0.781 |
3 |
||||
500 µg/mL |
86/384 |
0.935 |
7 |
||||
333.33 µg/mL |
87/384 |
0.928 |
11 |
||||
111.11 µg/mL |
116/384 |
0.748 |
19 |
||||
37.04 µg/mL |
103/384 |
0.822 |
36 |
||||
12.35. µg/mL |
98/384 |
0.854 |
67 |
||||
Vehicle control |
109/384 |
0.787 |
100 |
||||
Untreated control |
113/384 |
0.765 |
111 |
||||
Positive control (NQO) |
114/384 |
0.759 |
39 |
||||
A2 = Assay 2
- = in the absence of S9-mix
Negative (vehicle) control = 1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.1 μg/mL)
Mutagenicity Results of Assay 1 (I.)
(3-hour treatment in the presence of S9-mix)
S9 mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/ total number of wells |
Dn2/var(Dn)¨ |
Mutation frequency |
+ |
3 |
2000 µg/mL |
563/768 |
65/768 |
140/768 |
7.064 |
186.0* |
666.67 µg/mL |
542/768 |
76/768 |
150/768 |
6.733 |
182.0* |
||
222.22 µg/mL |
602/768 |
54/768 |
112/768 |
4.089 |
163.9 |
||
74.07 µg/mL |
627/768 |
73/768 |
68/768 |
0.192 |
114.4 |
||
24.69 µg/mL |
635/768 |
70/768 |
63/768 |
0.009 |
105.7 |
||
8.23 µg/mL |
615/768 |
70/768 |
83/768 |
0.703 |
125.3 |
||
Vehicle control |
643/768 |
51/768 |
74/768 |
-- |
103.3 |
||
Untreated control |
634/768 |
63/768 |
71/768 |
-- |
108.2 |
||
Positive control |
217/768 |
124/768 |
427/768 |
¨¨ |
2334.1* |
In linear trend analysisβ2/var (β) = 9.50,significant (at p<0.01)
* = Statistically significant
¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >5.24(at p<0.05).
¨¨= Evaluated by T-test for independent samples (compared to the DMSO vehicle control).
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dnβ = slope of the curve var(β) = variance of the slope
+ = in the presence of S9-mix
Negative (vehicle) control =1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 1 (II.)
(3-hour treatment in the absence of S9-mix)
S9 mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/ total number of wells |
Dn2/var(Dn)¨ |
Mutation frequency |
- |
3 |
2000 µg/mL |
517/768 |
90/768 |
161/768 |
5.350 |
159.9* |
666.67 µg/mL |
565/768 |
68/768 |
135/768 |
2.415 |
137.1 |
||
222.22 µg/mL |
586/768 |
76/768 |
106/768 |
2.107 |
134.7 |
||
74.07 µg/mL |
618/768 |
65/768 |
85/768 |
0.122 |
105.6 |
||
24.69 µg/mL |
589/768 |
97/768 |
82/768 |
1.479 |
127.9 |
||
8.23 µg/mL |
605/768 |
59/768 |
104/768 |
1.576 |
129.5 |
||
Vehicle control |
622/768 |
61/768 |
85/768 |
-- |
97.4 |
||
Untreated control |
608/768 |
64/768 |
96/768 |
-- |
108.0 |
||
Positive control |
231/768 |
244/768 |
293/768 |
¨¨ |
698.5* |
In linear trend analysisβ2/var (β) = 4.24,significant (at p<0.01)
* = Statistically significant
¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >5.24(at p<0.05).
¨¨= Evaluated by T-test for independent samples (compared to the DMSO vehicle control).
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn β = slope of the curve var(β) = variance of the slope
- = in the absence of S9-mix
Negative (vehicle) control =1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 2 (I.)
(3-hour treatment in the presence of S9-mix)
S9 mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/ total number of wells |
Dn2/var(Dn)¨ |
Mutation frequency |
+ |
3 |
2000 µg/mL |
440/768 |
148/768 |
180/768 |
8.415 |
197.0* |
1333.33 µg/mL |
531/768 |
92/768 |
145/768 |
6.651 |
185.2* |
||
666.67 µg/mL |
516/768 |
98/768 |
154/768 |
4.834 |
168.3 |
||
222.22 µg/mL |
585/768 |
88/768 |
95/768 |
1.351 |
132.2 |
||
74.07 µg/mL |
560/768 |
102/768 |
96/768 |
2.556 |
147.0 |
||
24.69 µg/mL |
570/768 |
111/768 |
87/768 |
3.007 |
152.1 |
||
8.23 µg/mL |
591/768 |
92/768 |
85/768 |
0.654 |
121.1 |
||
Vehicle control |
649/768 |
64/768 |
55/768 |
-- |
98.6 |
||
Untreated control |
632/768 |
62/768 |
74/768 |
-- |
115.9 |
||
Positive control |
86/768 |
209/768 |
473/768 |
¨¨ |
2452.5* |
In linear trend analysisβ2/var (β) = 9.70,significant (at p<0.001)
* = Statistically significant
¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >5.48(at p<0.05).
¨¨= Evaluated by T-test for independent samples (compared to the DMSO vehicle control).
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn β = slope of the curve var(β) = variance of the slope
+ = in the presence of S9-mix
Negative (vehicle) control =1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 2 (II.)
(24-hour treatment in the absence of S9-mix)
S9 mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/ total number of wells |
Dn2/var(Dn)¨ |
Mutation frequency |
- |
24 |
500 µg/mL |
636/768 |
58/768 |
74/768 |
0.068 |
100.8 |
333.33 µg/mL |
654/768 |
39/768 |
75/768 |
0.108 |
86.6 |
||
111.11 µg/mL |
654/768 |
48/768 |
66/768 |
0.244 |
107.4 |
||
37.04 µg/mL |
658/768 |
43/768 |
67/768 |
0.0002 |
94.0 |
||
12.35 µg/mLN |
338/384 |
14/384 |
32/384 |
-- |
75.9 |
||
Vehicle control |
662/768 |
47/768 |
59/768 |
-- |
94.4 |
||
Untreated control |
645/768 |
41/768 |
82/768 |
-- |
114.1 |
||
Positive control |
267/768 |
223/768 |
278/768 |
¨¨ |
696.0* |
In linear trend analysisβ2/var (β) = 0.001, notsignificant
* = Statistically significant
¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >4.67(at p<0.05).
¨¨= Evaluated by T-test for independent samples (compared to the DMSO vehicle control).
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn β = slope of the curvevar(β) = variance of the slope
- = in the absence of S9-mix
Negative (vehicle) control = 1% (v/v) DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: Mutation frequency refers to 106viable cells
N: In case of the 12.35 µg/mL signs of infection were observed in certain wells of replicate “A” on the mutagenicity plates, thus only replicate “B” was used for mutagenicity evaluation. Therefore, theDn2/var(Dn) could not be determined for this concentration and this concentration could not be considered in the linear trend analysis. However,the data used for this concentration were consistent with the overall results from other concentrations, so the use of the one replicate in data analysis is considered to be valid withno impact on the results or integrity of the study, the result is considered to be negative.
Individual Survival Data of Assay 1
Concentration |
Number of empty wells |
|
Assay 1 |
||
3-hour treatment, S9+ |
||
A |
B |
|
Untreated control |
31 |
23 |
23 |
22 |
|
Negative (solvent) control |
27 |
20 |
21 |
19 |
|
2000 µg/mL |
18 |
27 |
21 |
20 |
|
666.67 µg/mL |
25 |
19 |
16 |
19 |
|
222.22 µg/mL |
24 |
28 |
25 |
31 |
|
74.07 µg/mL |
27 |
25 |
24 |
25 |
|
24.69 µg/mL |
30 |
22 |
27 |
23 |
|
8.23 µg/mL |
27 |
19 |
29 |
14 |
|
Positive control |
72 |
73 |
75 |
72 |
|
S9+ = in the presence of S9-mix
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: A and B were duplicate cultures.
Individual Survival Data of Assay 1
Concentration |
Number of empty wells |
|
Assay 1 |
||
3-hour treatment, S9- |
||
A |
B |
|
Untreated control |
14 |
17 |
10 |
14 |
|
Negative (solvent) control |
13 |
15 |
19 |
18 |
|
2000 µg/mL |
21 |
10 |
28 |
14 |
|
666.67 µg/mL |
8 |
11 |
12 |
12 |
|
222.22 µg/mL |
20 |
27 |
15 |
16 |
|
74.07 µg/mL |
26 |
18 |
15 |
14 |
|
24.69 µg/mL |
10 |
25 |
18 |
22 |
|
8.23 µg/mL |
10 |
35 |
11 |
22 |
|
Positive control |
18 |
24 |
18 |
23 |
|
S9- = in the absence of S9-mix
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: A and B were duplicate cultures.
Individual Survival Data of Assay 2
Concentration |
Number of empty wells |
|
Assay 2 |
||
3-hour treatment, S9+ |
||
A |
B |
|
Untreated control |
26 |
33 |
30 |
32 |
|
Negative (solvent) control |
14 |
30 |
19 |
36 |
|
2000 µg/mL |
17 |
15 |
16 |
19 |
|
1333.33 µg/mL |
29 |
12 |
28 |
22 |
|
666.67 µg/mL |
18 |
13 |
22 |
24 |
|
222.22 µg/mL |
30 |
26 |
27 |
24 |
|
74.07 µg/mL |
29 |
38 |
27 |
29 |
|
24.69 µg/mL |
22 |
21 |
23 |
32 |
|
8.23 µg/mL |
17 |
17 |
14 |
16 |
|
Positive control |
54 |
55 |
40 |
65 |
|
S9+ = in the presence of S9-mix
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: A and B were duplicate cultures.
Individual Survival Data of Assay 2
Concentration |
Number of empty wells |
|
Assay 2 |
||
24-hour treatment, S9- |
||
A |
B |
|
Untreated control |
31 |
34 |
18 |
21 |
|
Negative (solvent) control |
27 |
33 |
29 |
32 |
|
1000 µg/mL |
64 |
63 |
65 |
59 |
|
750 µg/mL |
42 |
49 |
38 |
45 |
|
500 µg/mL |
27 |
31 |
28 |
31 |
|
333.33 µg/mL |
22 |
36 |
20 |
35 |
|
111.11 µg/mL |
33 |
31 |
24 |
25 |
|
37.04 µg/mL |
17 |
31 |
20 |
22 |
|
12.35 µg/mL |
21 |
25 |
28 |
36 |
|
Positive control |
46 |
51 |
52 |
58 |
|
S9- = in the absence of S9-mix
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: A and B were duplicate cultures.
Individual Viability Data of Assay 1
Concentration |
Number of empty wells |
|
Assay 1 |
||
3-hour treatment, S9+ |
||
A |
B |
|
Untreated control |
27 |
28 |
16 |
22 |
|
Negative (solvent) control |
18 |
32 |
29 |
18 |
|
2000 µg/mL |
29 |
26 |
22 |
24 |
|
666.67 µg/mL |
16 |
22 |
20 |
25 |
|
222.22 µg/mL |
23 |
44 |
28 |
22 |
|
74.07 µg/mL |
17 |
29 |
16 |
31 |
|
24.69 µg/mL |
16 |
29 |
25 |
21 |
|
8.23 µg/mL |
23 |
30 |
17 |
23 |
|
Positive control |
63 |
65 |
66 |
55 |
|
S9+ = in the presence of S9-mix
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: A and B were duplicate cultures.
Individual Viability Data of Assay 1
Concentration |
Number of empty wells |
|
Assay 1 |
||
3-hour treatment, S9- |
||
A |
B |
|
Untreated control |
11 |
16 |
22 |
19 |
|
Negative (solvent) control |
17 |
13 |
20 |
18 |
|
2000 µg/mL |
12 |
19 |
10 |
12 |
|
666.67 µg/mL |
12 |
22 |
10 |
20 |
|
222.22 µg/mL |
25 |
15 |
23 |
14 |
|
74.07 µg/mL |
25 |
9 |
23 |
17 |
|
24.69 µg/mL |
12 |
22 |
15 |
24 |
|
8.23 µg/mL |
26 |
18 |
24 |
20 |
|
Positive control |
20 |
19 |
28 |
30 |
|
S9- = in the absence of S9-mix
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: A and B were duplicate cultures.
Individual Viability Data of Assay 2
Concentration |
Number of empty wells |
|
Assay 2 |
||
3-hour treatment, S9+ |
||
A |
B |
|
Untreated control |
23 |
32 |
21 |
24 |
|
Negative (solvent) control |
15 |
26 |
22 |
35 |
|
2000 µg/mL |
3 |
13 |
12 |
12 |
|
1333.33 µg/mL |
25 |
14 |
18 |
21 |
|
666.67 µg/mL |
4 |
25 |
7 |
22 |
|
222.22 µg/mL |
23 |
10 |
25 |
16 |
|
74.07 µg/mL |
18 |
20 |
24 |
16 |
|
24.69 µg/mL |
28 |
12 |
29 |
11 |
|
8.23 µg/mL |
22 |
23 |
8 |
15 |
|
Positive control |
49 |
51 |
45 |
43 |
|
S9+ = in the presence of S9-mix
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: A and B were duplicate cultures.
Individual Viability Data of Assay 2
Concentration |
Number of empty wells |
|
Assay 2 |
||
24-hour treatment, S9- |
||
A |
B |
|
Untreated control |
32 |
26 |
25 |
30 |
|
Negative (solvent) control |
22 |
29 |
32 |
26 |
|
1000 µg/mL |
32 |
50 |
25 |
40 |
|
750 µg/mL |
28 |
32 |
25 |
25 |
|
500 µg/mL |
19 |
26 |
27 |
14 |
|
333.33 µg/mL |
22 |
14 |
35 |
16 |
|
111.11 µg/mL |
30 |
26 |
29 |
31 |
|
37.04 µg/mL |
28 |
25 |
21 |
29 |
|
12.35 µg/mL |
24 |
27 |
24 |
23 |
|
Positive control |
31 |
29 |
37 |
17 |
|
S9- = in the absence of S9-mix
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: A and B were duplicate cultures.
Individual Mutagenicity Data of Assay 1 (number of empty wells)
Concentration |
Number of empty wells |
Concentration |
Number of empty wells |
||
Assay 1 |
Assay 1 |
||||
3-hour treatment, S9+ |
3-hour treatment, S9+ |
||||
A |
B |
A |
B |
||
Untreated control |
78 |
80 |
8.23 µg/mL |
80 |
77 |
80 |
78 |
70 |
76 |
||
75 |
82 |
82 |
76 |
||
84 |
77 |
73 |
81 |
||
Negative (solvent) control |
83 |
82 |
Positive control (4 μg/mL CP) |
26 |
32 |
81 |
84 |
28 |
24 |
||
75 |
81 |
31 |
28 |
||
82 |
75 |
21 |
27 |
||
2000µg/mL |
74 |
64 |
- |
- |
- |
74 |
71 |
- |
- |
||
67 |
70 |
- |
- |
||
71 |
72 |
- |
- |
||
666.67µg/mL |
71 |
70 |
- |
- |
- |
57 |
63 |
- |
- |
||
71 |
68 |
- |
- |
||
73 |
69 |
- |
- |
||
222.22 µg/mL |
77 |
74 |
- |
- |
- |
75 |
77 |
- |
- |
||
72 |
78 |
- |
- |
||
72 |
77 |
- |
- |
||
74.07 µg/mL |
81 |
77 |
- |
- |
- |
72 |
85 |
- |
- |
||
76 |
82 |
- |
- |
||
79 |
75 |
- |
- |
||
24.69 µg/mL |
74 |
81 |
- |
- |
- |
82 |
84 |
- |
- |
||
74 |
82 |
- |
- |
||
77 |
81 |
- |
- |
||
S9+ = in the presence of S9-mix
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: A and B were duplicate cultures.
Individual Mutagenicity Data of Assay 1 (number of empty wells)
Concentration |
Number of empty wells |
Concentration |
Number of empty wells |
||
Assay 1 |
Assay 1 |
||||
3-hour treatment, S9- |
3-hour treatment, S9- |
||||
A |
B |
A |
B |
||
Untreated control |
77 |
63 |
8.23 µg/mL |
75 |
79 |
79 |
80 |
70 |
78 |
||
79 |
82 |
79 |
74 |
||
77 |
71 |
73 |
77 |
||
Negative (solvent) control |
75 |
85 |
Positive control (0.15 μg/mL NQO) |
27 |
30 |
78 |
79 |
30 |
34 |
||
73 |
79 |
28 |
30 |
||
75 |
78 |
26 |
26 |
||
2000µg/mL |
57 |
73 |
- |
- |
- |
54 |
70 |
- |
- |
||
54 |
66 |
- |
- |
||
68 |
75 |
- |
- |
||
666.67µg/mL |
66 |
73 |
- |
- |
- |
63 |
74 |
- |
- |
||
73 |
72 |
- |
- |
||
76 |
68 |
- |
- |
||
222.22 µg/mL |
79 |
68 |
- |
- |
- |
78 |
78 |
- |
- |
||
71 |
67 |
- |
- |
||
72 |
72 |
- |
- |
||
74.07 µg/mL |
80 |
79 |
- |
- |
- |
78 |
71 |
- |
- |
||
78 |
78 |
- |
- |
||
78 |
76 |
- |
- |
||
24.69 µg/mL |
74 |
72 |
- |
- |
- |
73 |
76 |
- |
- |
||
71 |
76 |
- |
- |
||
71 |
76 |
- |
- |
||
S9- = in the absence of S9-mix
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: A and B were duplicate cultures.
Individual Mutagenicity Data of Assay 2 (number of empty wells)
Concentration |
Number of empty wells |
Concentration |
Number of empty wells |
||
Assay 2 |
Assay 2 |
||||
3-hour treatment, S9+ |
3-hour treatment, S9+ |
||||
A |
B |
A |
B |
||
Untreated control |
78 |
82 |
Positive control |
14 |
9 |
78 |
79 |
13 |
15 |
||
79 |
77 |
8 |
11 |
||
82 |
77 |
6 |
10 |
||
Negative (solvent) control |
80 |
81 |
- |
- |
- |
72 |
83 |
- |
- |
||
85 |
84 |
- |
- |
||
79 |
85 |
- |
- |
||
2000 µg/mL |
45 |
53 |
- |
- |
- |
42 |
60 |
- |
- |
||
56 |
53 |
- |
- |
||
69 |
62 |
- |
- |
||
1333.33 µg/mL |
77 |
62 |
- |
- |
- |
71 |
61 |
- |
- |
||
76 |
54 |
- |
- |
||
70 |
60 |
- |
- |
||
666.67 µg/mL |
62 |
64 |
- |
- |
- |
64 |
68 |
- |
- |
||
61 |
62 |
- |
- |
||
68 |
67 |
- |
- |
||
222.22 µg/mL |
76 |
66 |
- |
- |
- |
81 |
70 |
- |
- |
||
76 |
73 |
- |
- |
||
69 |
74 |
- |
- |
||
74.07 µg/mL |
72 |
72 |
- |
- |
- |
68 |
69 |
- |
- |
||
66 |
67 |
- |
- |
||
73 |
73 |
- |
- |
||
24.69 µg/mL |
73 |
67 |
- |
- |
- |
77 |
66 |
- |
- |
||
73 |
77 |
- |
- |
||
69 |
68 |
- |
- |
||
8.23 µg/mL |
74 |
72 |
- |
- |
- |
76 |
75 |
- |
- |
||
78 |
74 |
- |
- |
||
74 |
68 |
- |
- |
||
S9+ = in the presence of S9-mix
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: A and B were duplicate cultures.
Individual Mutagenicity Data of Assay 2 (number of empty wells)
Concentration |
Number of empty wells |
Concentration |
Number of empty wells |
||
Assay 2 |
Assay 2 |
||||
24-hour treatment, S9- |
24-hour treatment, S9- |
||||
A |
B |
A |
B |
||
Untreated control |
87 |
77 |
Positive control |
40 |
21 |
86 |
76 |
47 |
24 |
||
82 |
76 |
39 |
22 |
||
83 |
78 |
46 |
28 |
||
Negative (solvent) control |
84 |
86 |
- |
- |
- |
79 |
85 |
- |
- |
||
80 |
82 |
- |
- |
||
81 |
85 |
- |
- |
||
1000 µg/mL |
78 |
72 |
- |
- |
- |
74 |
76 |
- |
- |
||
79 |
78 |
- |
- |
||
75 |
81 |
- |
- |
||
750 µg/mL |
74 |
77 |
- |
- |
- |
73 |
78 |
- |
- |
||
78 |
72 |
- |
- |
||
86 |
68 |
- |
- |
||
500 µg/mL |
80 |
76 |
- |
- |
- |
87 |
79 |
- |
- |
||
80 |
83 |
- |
- |
||
82 |
69 |
- |
- |
||
333.33 µg/mL |
79 |
85 |
- |
- |
- |
80 |
78 |
- |
- |
||
78 |
85 |
- |
- |
||
85 |
84 |
- |
- |
||
111.11 µg/mL |
84 |
88 |
- |
- |
- |
80 |
78 |
- |
- |
||
75 |
86 |
- |
- |
||
79 |
84 |
- |
- |
||
37.04 µg/mL |
83 |
77 |
- |
- |
- |
79 |
86 |
- |
- |
||
81 |
83 |
- |
- |
||
82 |
87 |
- |
- |
||
12.35 µg/mL |
N |
87 |
- |
- |
- |
N |
88 |
- |
- |
||
N |
80 |
- |
- |
||
N |
83 |
- |
- |
||
S9- = in the absence of S9-mix
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: A and B were duplicate cultures.
N: Only replicate “B” was used for mutagenicity evaluation.
Individual Mutagenicity Data of Assay 1 (number of large and small colonies)
Concentration |
Number of colonies |
Concentration |
Number of colonies |
||||||
Assay 1 |
Assay 1 |
||||||||
3-hour treatment, S9+ |
3-hour treatment, S9+ |
||||||||
A |
B |
A |
B |
||||||
L |
S |
L |
S |
L |
S |
L |
S |
||
Untreated control |
5 |
13 |
6 |
10 |
8.23 µg/mL |
11 |
5 |
7 |
12 |
8 |
8 |
6 |
12 |
12 |
14 |
7 |
13 |
||
13 |
8 |
7 |
7 |
6 |
8 |
8 |
12 |
||
8 |
4 |
10 |
9 |
10 |
13 |
9 |
6 |
||
Negative (solvent) control |
7 |
6 |
7 |
7 |
Positive control (4 μg/mL CP) |
19 |
51 |
12 |
52 |
7 |
8 |
7 |
5 |
13 |
55 |
13 |
59 |
||
9 |
12 |
6 |
9 |
18 |
47 |
12 |
56 |
||
4 |
10 |
4 |
17 |
22 |
53 |
15 |
54 |
||
2000µg/mL |
9 |
13 |
8 |
24 |
- |
- |
- |
- |
- |
5 |
17 |
7 |
18 |
- |
- |
- |
- |
||
10 |
19 |
11 |
15 |
- |
- |
- |
- |
||
9 |
16 |
6 |
18 |
- |
- |
- |
- |
||
666.67µg/mL |
9 |
16 |
12 |
14 |
- |
- |
- |
- |
- |
14 |
25 |
10 |
23 |
- |
- |
- |
- |
||
6 |
19 |
6 |
22 |
- |
- |
- |
- |
||
15 |
8 |
4 |
23 |
- |
- |
- |
- |
||
222.22 µg/mL |
7 |
12 |
6 |
16 |
- |
- |
- |
- |
- |
7 |
14 |
5 |
14 |
- |
- |
- |
- |
||
10 |
14 |
5 |
13 |
- |
- |
- |
- |
||
9 |
15 |
5 |
14 |
- |
- |
- |
- |
||
74.07 µg/mL |
11 |
4 |
9 |
10 |
- |
- |
- |
- |
- |
10 |
14 |
4 |
7 |
- |
- |
- |
- |
||
12 |
8 |
7 |
7 |
- |
- |
- |
- |
||
12 |
5 |
8 |
13 |
- |
- |
- |
- |
||
24.69 µg/mL |
16 |
6 |
8 |
7 |
- |
- |
- |
- |
- |
8 |
6 |
6 |
6 |
- |
- |
- |
- |
||
10 |
12 |
4 |
10 |
- |
- |
- |
- |
||
11 |
8 |
7 |
8 |
- |
- |
- |
- |
||
S9+ = in the presence of S9-mix
L/S = large / small colonies
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: A and B were duplicate cultures.
Individual Mutagenicity Data of Assay 1 (number of large and small colonies)
Concentration |
Number of colonies |
Concentration |
Number of colonies |
||||||
Assay 1 |
Assay 1 |
||||||||
3-hour treatment, S9- |
3-hour treatment, S9- |
||||||||
A |
B |
A |
B |
||||||
L |
S |
L |
S |
L |
S |
L |
S |
||
Untreated control |
9 |
10 |
9 |
24 |
8.23 µg/mL |
5 |
16 |
4 |
13 |
6 |
11 |
9 |
7 |
10 |
16 |
6 |
12 |
||
6 |
11 |
7 |
7 |
6 |
11 |
10 |
12 |
||
5 |
14 |
13 |
12 |
12 |
11 |
6 |
13 |
||
Negative (solvent) control |
8 |
13 |
2 |
9 |
Positive control (0.15 μg/mL NQO) |
28 |
41 |
28 |
38 |
11 |
7 |
9 |
8 |
28 |
38 |
30 |
32 |
||
10 |
13 |
8 |
9 |
33 |
35 |
36 |
30 |
||
6 |
15 |
7 |
11 |
29 |
41 |
32 |
38 |
||
2000µg/mL |
12 |
27 |
11 |
12 |
- |
- |
- |
- |
- |
17 |
25 |
8 |
18 |
- |
- |
- |
- |
||
19 |
23 |
8 |
22 |
- |
- |
- |
- |
||
8 |
20 |
7 |
14 |
- |
- |
- |
- |
||
666.67µg/mL |
15 |
15 |
7 |
16 |
- |
- |
- |
- |
- |
6 |
27 |
7 |
15 |
- |
- |
- |
- |
||
7 |
16 |
8 |
16 |
- |
- |
- |
- |
||
10 |
10 |
8 |
20 |
- |
- |
- |
- |
||
222.22 µg/mL |
6 |
11 |
13 |
15 |
- |
- |
- |
- |
- |
7 |
11 |
9 |
9 |
- |
- |
- |
- |
||
11 |
14 |
15 |
14 |
- |
- |
- |
- |
||
7 |
16 |
8 |
16 |
- |
- |
- |
- |
||
74.07 µg/mL |
9 |
7 |
7 |
10 |
- |
- |
- |
- |
- |
9 |
9 |
7 |
18 |
- |
- |
- |
- |
||
10 |
8 |
8 |
10 |
- |
- |
- |
- |
||
5 |
13 |
10 |
10 |
- |
- |
- |
- |
||
24.69 µg/mL |
13 |
9 |
13 |
11 |
- |
- |
- |
- |
- |
15 |
8 |
7 |
13 |
- |
- |
- |
- |
||
13 |
12 |
12 |
8 |
- |
- |
- |
- |
||
14 |
11 |
10 |
10 |
- |
- |
- |
- |
||
S9- = in the absenceof S9-mix
L/S = large / small colonies
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: A and B were duplicate cultures.
Individual Mutagenicity Data of Assay 2 (number of large and small colonies)
Concentration |
Number of colonies |
Concentration |
Number of colonies |
||||||
Assay 2 |
Assay 2 |
||||||||
3-hour treatment, S9+ |
3-hour treatment, S9+ |
||||||||
A |
B |
A |
B |
||||||
L |
S |
L |
S |
L |
S |
L |
S |
||
Untreated control |
7 |
11 |
7 |
7 |
Positive control |
27 |
55 |
31 |
56 |
8 |
10 |
6 |
11 |
21 |
62 |
26 |
55 |
||
8 |
9 |
10 |
9 |
26 |
62 |
26 |
59 |
||
9 |
5 |
7 |
12 |
28 |
62 |
24 |
62 |
||
Negative (solvent) control |
9 |
7 |
7 |
8 |
- |
- |
- |
- |
- |
13 |
11 |
7 |
6 |
- |
- |
- |
- |
||
9 |
2 |
7 |
5 |
- |
- |
- |
- |
||
6 |
11 |
6 |
5 |
- |
- |
- |
- |
||
2000 µg/mL |
22 |
29 |
17 |
26 |
- |
- |
- |
- |
- |
22 |
32 |
15 |
21 |
- |
- |
- |
- |
||
19 |
21 |
24 |
19 |
- |
- |
- |
- |
||
14 |
13 |
15 |
19 |
- |
- |
- |
- |
||
1333.33 µg/mL |
6 |
13 |
18 |
16 |
- |
- |
- |
- |
- |
9 |
16 |
15 |
20 |
- |
- |
- |
- |
||
7 |
13 |
16 |
26 |
- |
- |
- |
- |
||
10 |
16 |
11 |
25 |
- |
- |
- |
- |
||
666.67 µg/mL |
15 |
19 |
12 |
20 |
- |
- |
- |
- |
- |
10 |
22 |
14 |
14 |
- |
- |
- |
- |
||
14 |
21 |
14 |
20 |
- |
- |
- |
- |
||
9 |
19 |
10 |
19 |
- |
- |
- |
- |
||
222.22 µg/mL |
8 |
12 |
13 |
17 |
- |
- |
- |
- |
- |
6 |
9 |
12 |
14 |
- |
- |
- |
- |
||
9 |
11 |
11 |
12 |
- |
- |
- |
- |
||
13 |
14 |
16 |
6 |
- |
- |
- |
- |
||
74.07 µg/mL |
10 |
14 |
9 |
15 |
- |
- |
- |
- |
- |
7 |
11 |
12 |
15 |
- |
- |
- |
- |
||
13 |
17 |
20 |
9 |
- |
- |
- |
- |
||
17 |
6 |
14 |
9 |
- |
- |
- |
- |
||
24.69 µg/mL |
12 |
11 |
16 |
13 |
- |
- |
- |
- |
- |
9 |
10 |
21 |
9 |
- |
- |
- |
- |
||
14 |
9 |
7 |
12 |
- |
- |
- |
- |
||
18 |
9 |
14 |
14 |
- |
- |
- |
- |
||
8.23 µg/mL |
12 |
10 |
11 |
13 |
- |
- |
- |
- |
- |
13 |
7 |
11 |
10 |
- |
- |
- |
- |
||
7 |
11 |
11 |
11 |
- |
- |
- |
- |
||
15 |
7 |
12 |
16 |
- |
- |
- |
- |
||
S9+ = in the presence of S9-mix
L/S = large / small colonies
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: A and B were duplicate cultures.
Individual Mutagenicity Data of Assay 2 (number of large and small colonies)
Concentration |
Number of colonies |
Concentration |
Number of colonies |
||||||
Assay 2 |
Assay 2 |
||||||||
24-hour treatment, S9- |
24-hour treatment, S9- |
||||||||
A |
B |
A |
B |
||||||
L |
S |
L |
S |
L |
S |
L |
S |
||
Untreated control |
1 |
8 |
5 |
14 |
Positive control |
21 |
35 |
34 |
41 |
1 |
9 |
9 |
11 |
18 |
31 |
34 |
38 |
||
4 |
10 |
6 |
14 |
19 |
38 |
41 |
33 |
||
7 |
6 |
8 |
10 |
19 |
31 |
37 |
31 |
||
Negative (solvent) control |
5 |
7 |
7 |
3 |
- |
- |
- |
- |
- |
8 |
9 |
6 |
5 |
- |
- |
- |
- |
||
8 |
8 |
7 |
7 |
- |
- |
- |
- |
||
2 |
13 |
4 |
7 |
- |
- |
- |
- |
||
1000 µg/mL |
6 |
12 |
6 |
18 |
- |
- |
- |
- |
- |
9 |
13 |
11 |
9 |
- |
- |
- |
- |
||
5 |
12 |
5 |
13 |
- |
- |
- |
- |
||
8 |
13 |
5 |
10 |
- |
- |
- |
- |
||
750 µg/mL |
14 |
8 |
12 |
7 |
- |
- |
- |
- |
- |
12 |
11 |
7 |
11 |
- |
- |
- |
- |
||
7 |
11 |
5 |
19 |
- |
- |
- |
- |
||
9 |
1 |
16 |
12 |
- |
- |
- |
- |
||
500 µg/mL |
8 |
8 |
8 |
12 |
- |
- |
- |
- |
- |
4 |
5 |
9 |
8 |
- |
- |
- |
- |
||
5 |
11 |
6 |
7 |
- |
- |
- |
- |
||
6 |
8 |
12 |
15 |
- |
- |
- |
- |
||
333.33 µg/mL |
7 |
10 |
4 |
7 |
- |
- |
- |
- |
- |
7 |
9 |
6 |
12 |
- |
- |
- |
- |
||
6 |
12 |
2 |
9 |
- |
- |
- |
- |
||
5 |
6 |
2 |
10 |
- |
- |
- |
- |
||
111.11 µg/mL |
5 |
7 |
1 |
7 |
- |
- |
- |
- |
- |
7 |
9 |
9 |
9 |
- |
- |
- |
- |
||
12 |
9 |
4 |
6 |
- |
- |
- |
- |
||
5 |
12 |
5 |
7 |
- |
- |
- |
- |
||
37.04 µg/mL |
3 |
10 |
10 |
9 |
- |
- |
- |
- |
- |
6 |
11 |
5 |
5 |
- |
- |
- |
- |
||
2 |
13 |
5 |
8 |
- |
- |
- |
- |
||
5 |
9 |
7 |
2 |
- |
- |
- |
- |
||
12.35 µg/mL |
7 |
3 |
1 |
8 |
- |
- |
- |
- |
- |
5 |
2 |
3 |
5 |
- |
- |
- |
- |
||
3 |
2 |
7 |
9 |
- |
- |
- |
- |
||
9 |
3 |
3 |
10 |
- |
- |
- |
- |
||
S9- = in the absence of S9-mix
L/S = large / small colonies
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: A and B were duplicate cultures.
Observations in the Main Tests
Assay 1: 3-hour treatment in the presence of metabolic activation
Test item (or vehicle) concentration |
pH |
Osmolality |
Observations |
||
Untreated control |
7.0 |
7.0 |
307 |
307 |
normal / normal |
Negative (solvent) control |
7.0 |
7.0 |
461 |
458 |
normal / normal |
2000 µg/mL |
7.0 |
7.0 |
432 |
428 |
discoloured medium#, precipitate / discoloured medium#,precipitate |
666.67 µg/mL |
7.0 |
7.0 |
445 |
447 |
discoloured medium, precipitate / discoloured medium,precipitate |
222.22 µg/mL |
7.0 |
7.0 |
453 |
456 |
discoloured medium, precipitate / discoloured medium, precipitate |
74.07 µg/mL |
7.0 |
7.0 |
462 |
456 |
discoloured medium, precipitate / discoloured medium, precipitate |
24.69 µg/mL |
7.0 |
7.0 |
459 |
458 |
discoloured medium#, precipitate#, / discoloured medium#,precipitate#, |
8.23 µg/mL |
7.0 |
7.0 |
457 |
451 |
normal / normal |
Positive control |
7.0 |
7.0 |
454 |
460 |
normal / normal |
DMSO = Dimethyl sulfoxide #minimal amount
CP = Cyclophosphamide
Observations in the Main Tests
Assay 1: 3-hour treatment in the absence of metabolic activation
Test item (or vehicle) concentration |
pH |
Osmolality |
Observations |
||
Untreated control |
7.0 |
7.0 |
304 |
300 |
normal / normal |
Negative (solvent) control |
7.0 |
7.0 |
457 |
454 |
normal / normal |
2000 µg/mL |
7.0 |
7.0 |
427 |
421 |
discoloured medium, precipitate / discoloured medium,precipitate |
666.67 µg/mL |
7.0 |
7.0 |
485 |
458 |
discoloured medium, precipitate / discoloured medium,precipitate |
222.22 µg/mL |
7.0 |
7.0 |
448 |
458 |
discoloured medium, precipitate / discoloured medium, precipitate |
74.07 µg/mL |
7.0 |
7.0 |
453 |
459 |
discoloured medium, precipitate / discoloured medium, precipitate |
24.69 µg/mL |
7.0 |
7.0 |
452 |
453 |
discoloured medium#, precipitate#, / discoloured medium#,precipitate#, |
8.23 µg/mL |
7.0 |
7.0 |
451 |
451 |
normal / normal |
Positive control |
7.0 |
7.0 |
451 |
458 |
normal / normal |
DMSO = Dimethyl sulfoxide #minimal amount
NQO = 4-Nitroquinoline-N-oxide
Observations in the Main Tests
Assay 2: 3-hour treatment in the presence of metabolic activation
Test item (or vehicle) concentration |
pH |
Osmolality |
Observations |
||
Untreated control |
7.0 |
7.0 |
300 |
294 |
normal / normal |
Negative (solvent) control |
7.0 |
7.0 |
450 |
446 |
normal / normal |
2000 µg/mL |
7.0 |
7.0 |
418 |
419 |
discoloured medium#, precipitate / discoloured medium,precipitate |
1333.33 µg/mL |
7.0 |
7.0 |
427 |
427 |
discoloured medium, precipitate / discoloured medium,precipitate |
666.67 µg/mL |
7.0 |
7.0 |
436 |
439 |
discoloured medium, precipitate / discoloured medium, precipitate |
222.22 µg/mL |
7.0 |
7.0 |
442 |
446 |
discoloured medium, precipitate / discoloured medium, precipitate |
74.07 µg/mL |
7.0 |
7.0 |
444 |
446 |
discoloured medium, precipitate / discoloured medium#, precipitate |
24.69 µg/mL |
7.0 |
7.0 |
447 |
448 |
discoloured medium#, precipitate#, / discoloured medium#,precipitate# |
8.23 µg/mL |
7.0 |
7.0 |
445 |
444 |
normal / normal |
Positive control |
7.0 |
7.0 |
452 |
453 |
normal / normal |
DMSO = Dimethyl sulfoxide #minimal amount
CP = Cyclophosphamide
Observations in the Main Tests
Assay 2: 24-hour treatment in the absence of metabolic activation
Test item (or vehicle) concentration |
pH |
Osmolality |
Observations |
||
Untreated control |
7.0 |
7.0 |
298 |
294 |
normal / normal |
Negative (solvent) control |
7.0 |
7.0 |
459 |
454 |
normal / normal |
1000 µg/mL |
7.0 |
7.0 |
444 |
443 |
discoloured medium, precipitate / discoloured medium,precipitate |
750 µg/mL |
7.0 |
7.0 |
448 |
442 |
discoloured medium, precipitate / discoloured medium,precipitate |
500 µg/mL |
7.0 |
7.0 |
451 |
443 |
discoloured medium, precipitate / discoloured medium, precipitate |
333.33 µg/mL |
7.0 |
7.0 |
452 |
448 |
discoloured medium, precipitate / discoloured medium, precipitate |
111.11 µg/mL |
7.0 |
7.0 |
453 |
452 |
discoloured medium#, precipitate / discoloured medium,precipitate |
37.04 µg/mL |
7.0 |
7.0 |
455 |
455 |
normal /discoloured medium# |
12.35 µg/mL |
7.0 |
7.0 |
453 |
453 |
normal /discoloured medium# |
Positive control |
7.0 |
7.0 |
452 |
453 |
normal / normal |
DMSO = Dimethyl sulfoxideminimal amount #:minimal amount
NQO = 4-Nitroquinoline-N-oxide
Cell Counting Results of the Main Tests
Assay 1: 3-hour treatment in the presence of metabolic activation
Test item (or vehicle) concentration |
Total cell number |
Total cell number |
Total cell number |
Suspension |
|||
Untreated control |
7.25E+06 |
5.75E+06 |
2.42E+07 |
1.79E+07 |
2.78E+07 |
1.88E+07 |
13.6 |
Negative (vehicle) control (1% (v/v) DMSO) |
6.30E+06 |
5.65E+06 |
2.00E+07 |
1.71E+07 |
1.99E+07 |
2.02E+07 |
10.4 |
2000 µg/mL |
4.75E+06 |
4.80E+06 |
4.88E+06 |
4.65E+06 |
7.30E+06 |
6.70E+06 |
1.5 |
666.67 µg/mL |
6.15E+06 |
5.20E+06 |
6.45E+06 |
6.30E+06 |
2.16E+07 |
2.28E+07 |
4.2 |
222.22 µg/mL |
5.00E+06 |
3.60E+06 |
6.00E+06 |
5.10E+06 |
1.73E+07 |
1.51E+07 |
3.8 |
74.07 µg/mL |
4.45E+06 |
4.10E+06 |
1.01E+07 |
8.40E+06 |
2.41E+07 |
1.93E+07 |
7.8 |
24.69 µg/mL |
3.55E+06 |
3.60E+06 |
9.00E+06 |
7.80E+06 |
1.45E+07 |
2.25E+07 |
7.2 |
8.23 µg/mL |
5.50E+06 |
4.95E+06 |
1.64E+07 |
1.23E+07 |
2.20E+07 |
2.92E+07 |
11.7 |
Positive control (4 μg/mL CP) |
3.10E+06 |
5.30E+06 |
6.30E+06 |
1.07E+07 |
1.04E+07 |
1.25E+07 |
3.9 |
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Cell Counting Results of the Main Tests
Assay 1: 3-hour treatment in the absence of metabolic activation
Test item (or vehicle) concentration |
Total cell number |
Total cell number |
Total cell number |
Suspension |
|||
Untreated control |
1.03E+07 |
1.08E+07 |
2.37E+07 |
2.06E+07 |
2.49E+07 |
2.81E+07 |
16.3 |
Negative (vehicle) control (1% (v/v) DMSO) |
9.40E+06 |
9.20E+06 |
2.13E+07 |
2.15E+07 |
2.38E+07 |
2.53E+07 |
14.6 |
2000 µg/mL |
3.50E+06 |
3.45E+06 |
3.98E+06 |
4.35E+06 |
7.60E+06 |
1.34E+07 |
3.0 |
666.67 µg/mL |
4.15E+06 |
4.05E+06 |
9.30E+06 |
8.10E+06 |
1.80E+07 |
2.17E+07 |
7.0 |
222.22 µg/mL |
6.15E+06 |
6.55E+06 |
1.23E+07 |
1.53E+07 |
2.14E+07 |
2.05E+07 |
8.0 |
74.07 µg/mL |
5.45E+06 |
7.55E+06 |
1.64E+07 |
1.73E+07 |
2.40E+07 |
2.47E+07 |
11.4 |
24.69 µg/mL |
6.90E+06 |
8.85E+06 |
1.98E+07 |
1.89E+07 |
2.32E+07 |
2.22E+07 |
12.2 |
8.23 µg/mL |
8.55E+06 |
9.40E+06 |
2.36E+07 |
1.95E+07 |
2.67E+07 |
2.47E+07 |
15.4 |
Positive control (0.15 μg/mL NQO) |
8.50E+06 |
9.55E+06 |
2.01E+07 |
1.68E+07 |
2.51E+07 |
2.17E+07 |
12.0 |
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Cell Counting Results of the Main Test
Assay 2: 3-hour treatment in the presence of metabolic activation
Test item (or vehicle) concentration |
Total cell number |
Total cell number |
Total cell number |
Suspension |
|||
Untreated control |
6.50E+06 |
6.55E+06 |
1.86E+07 |
1.74E+07 |
2.31E+07 |
2.15E+07 |
11.2 |
Negative (vehicle) control (1% (v/v) DMSO) |
6.70E+06 |
5.30E+06 |
1.80E+07 |
1.62E+07 |
2.52E+07 |
1.78E+07 |
10.2 |
2000 µg/mL |
2.45E+06 |
3.10E+06 |
2.03E+06 |
1.50E+06 |
3.20E+06 |
1.50E+06 |
0.8 |
1333.33 µg/mL |
4.00E+06 |
4.60E+06 |
3.45E+06 |
3.38E+06 |
5.00E+06 |
7.50E+06 |
1.5 |
666.67 µg/mL |
4.15E+06 |
3.70E+06 |
5.18E+06 |
4.13E+06 |
9.20E+06 |
9.30E+06 |
2.4 |
222.22 µg/mL |
3.10E+06 |
2.75E+06 |
3.83E+06 |
3.98E+06 |
7.50E+06 |
5.90E+06 |
2.3 |
74.07 µg/mL |
4.05E+06 |
3.85E+06 |
7.05E+06 |
6.90E+06 |
2.18E+07 |
1.67E+07 |
5.7 |
24.69 µg/mL |
3.70E+06 |
3.45E+06 |
8.70E+06 |
8.40E+06 |
2.06E+07 |
1.85E+07 |
7.8 |
8.23 µg/mL |
4.40E+06 |
4.00E+06 |
1.25E+07 |
1.17E+07 |
2.28E+07 |
2.08E+07 |
10.4 |
Positive control (4 μg/mL CP) |
6.05E+06 |
6.05E+06 |
1.70E+07 |
1.25E+07 |
1.49E+07 |
1.42E+07 |
5.9 |
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Cell Counting Results of the Main Tests
Assay 2: 24-hour treatment in the absence of metabolic activation
Test item (or vehicle) concentration |
Total cell number |
Total cell number |
Total cell number |
Suspension |
|||
Untreated control |
2.13E+07 |
2.18E+07 |
2.15E+07 |
1.91E+07 |
2.52E+07 |
2.44E+07 |
50.0 |
Negative (vehicle) control (1% (v/v) DMSO) |
2.15E+07 |
2.33E+07 |
2.13E+07 |
1.80E+07 |
2.26E+07 |
2.04E+07 |
43.7 |
1000 µg/mL |
2.10E+06 |
1.55E+06 |
6.00E+05 |
3.00E+05 |
1.90E+06 |
1.70E+06 |
0.3 |
750 µg/mL |
3.20E+06 |
3.05E+06 |
1.88E+06 |
2.70E+06 |
6.20E+06 |
7.50E+06 |
1.1 |
500 µg/mL |
4.05E+06 |
4.40E+06 |
4.28E+06 |
5.10E+06 |
1.47E+07 |
1.70E+07 |
2.6 |
333.33 µg/mL |
4.50E+06 |
4.80E+06 |
7.80E+06 |
7.80E+06 |
1.70E+07 |
1.92E+07 |
3.9 |
111.11 µg/mL |
5.95E+06 |
6.20E+06 |
1.31E+07 |
1.50E+07 |
2.22E+07 |
2.25E+07 |
8.8 |
37.04 µg/mL |
7.05E+06 |
7.90E+06 |
1.71E+07 |
1.89E+07 |
2.52E+07 |
2.33E+07 |
15.1 |
12.35 µg/mL |
1.28E+07 |
1.44E+07 |
1.74E+07 |
1.95E+07 |
2.29E+07 |
2.40E+07 |
27.2 |
Positive control (0.1 μg/mL NQO) |
1.25E+07 |
1.54E+07 |
1.58E+07 |
1.71E+07 |
1.74E+07 |
1.58E+07 |
17.6 |
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Historical Control Data
Mutation Frequency of the Negative Controls (2006-2016) |
||||||
|
Culture medium |
Distilled water |
||||
Treatments |
3h,S9+ |
3h,S9- |
24h,S9- |
3h,S9+ |
3h,S9- |
24h,S9- |
Average |
94.3 |
103.6 |
106.4 |
90.4 |
96.6 |
96.3 |
SD |
26.9 |
35.3 |
27.4 |
22.7 |
19.0 |
24.6 |
Min. |
39.3 |
52.6 |
41.7 |
33.4 |
55.1 |
43.2 |
Max. |
198.5 |
235.6 |
179.1 |
121.8 |
125.0 |
141.1 |
n |
84 |
43 |
44 |
26 |
13 |
13 |
|
Dimethyl sulfoxide (DMSO) |
|
||||
Treatments |
3h,S9+ |
3h,S9- |
24h,S9- |
|
|
|
Average |
97.3 |
97.3 |
98.9 |
|
|
|
SD |
33.7 |
38.5 |
26.8 |
|
|
|
Min. |
44.2 |
33.7 |
47.1 |
|
|
|
Max. |
269.9 |
261.6 |
159.4 |
|
|
|
n |
101 |
57 |
50 |
|
|
|
Mutation Frequency of the Positive Controls (2006-2016) |
||||||
|
Cyclophosphamide |
4-Nitroquinoline-N-oxide |
||||
Treatments |
3h,S9+ |
|
|
|
3h,S9- |
24h,S9- |
Average |
1178.7 |
|
|
|
722.2 |
831.9 |
SD |
524.7 |
|
|
|
330.0 |
337.2 |
Min. |
196.1 |
|
|
|
223.5 |
245.0 |
Max. |
2642.5 |
|
|
|
1687.3 |
1577.6 |
n |
106 |
|
|
|
58 |
52 |
h = hour
SD = Standard Deviation
S9+ = experiment with metabolic activation
S9- = experiment without metabolic activation
n = number of case
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Genetic toxicity in vitro - Bacterial Reverse Mutation Assay
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method).
In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value when compared to the solvent control and were within the normal biological variability of the test system. There were no dose-related trends and no indication of any treatment effect.
Inhibitory, cytotoxic effects of the test item were observed in the Initial Mutation Test in Salmonella typhimurium TA100 strain without metabolic activation at 5000 μg test item/plate and in the Confirmatory Mutation Test in all examined strains with metabolic activation at 5000 and 1581 μg test item/plate and Complementary Confirmatory Mutation Test in all Salmonella typhimurium strains without metabolic activation at 50 μg/plate concentrations and in Escherichia coli WP2 uvrA strain without metabolic activation at 500 μg/plate.
Precipitate was detected on the plates in the Initial Mutation Test and Confirmatory Mutation Test in all examined strains with or without metabolic activation at 5000 μg concentration. Precipitate did not interfere with the colony counting or the background lawn growth evaluation.
The reported data of the mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item AMINOX® had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.
Genetic toxicity in vitro - Mammalian Chromosome Aberration Test
AMINOX® was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in DMSO and it was examined up to cytotoxic concentrations according to the OECD guideline recommendations. In independent Chromosome Aberration Assays using duplicate cultures, at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each evaluated test item treated, negative (vehicle) and positive control sample.
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 125, 100, 75, 50, 25 and 12.5 μg/mL (experiment with and without metabolic activation).
In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 125-50 μg/mL concentration range with and without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 50, 25 and 12.5 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic.
In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 125, 100, 75, 50, 25 and 12.5 μg/mL (experiment with metabolic activation) and 200, 150, 125, 100, 75, 50 and 25 μg/mL (experiment without metabolic activation).
In Assay 2, insolubility/minimal insolubility was detected at the end of the treatment period in the final treatment medium in the 125-25 μg/mL concentration range with metabolic activation and in the 200-25 μg/mL concentration range without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment without metabolic activation (RICC value of the highest evaluated concentration (200 μg/mL) without metabolic activation was 12%). No cytotoxicity was observed in the experiment with metabolic activation. Therefore, concentrations of 50, 25 and 12.5 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 75, 50 and 25 μg/mL (a total of three) were evaluated in the experiment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.
Polyploid metaphases were found in all cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with AMINOX®. No endoreduplicated metaphases were detected in the performed experiments.
The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.
In conclusion, AMINOX® did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, AMINOX® was considered as not clastogenic in this test system.
In vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay)
An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/-3.7.2 C cells at the tk locus to test the potential ofAMINOX®test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). The design of this study was based on the OECDNo. 490 guideline,and the study was performed in compliance with Citoxlab Hungary Ltd. standard operating procedures and with the OECD Principles of Good Laboratory Practice.
Dimethyl sulfoxide was used as vehicle of the test item in this study. Based on the preliminary toxicity test and in agreement with the Sponsor, the following test item concentrations were examined in the main mutation assays:
Assay 1, 3-hour treatment with metabolic activation: 2000, 666.67, 222.22, 74.07, 24.69 and 8.23 µg/mL,
Assay 1, 3-hour treatment without metabolic activation:2000, 666.67, 222.22, 74.07, 24.69 and 8.23 µg/mL,
Assay 2, 3-hour treatment with metabolic activation: 2000,1333.33,666.67, 222.22, 74.07, 24.69 and 8.23 µg/mL,
Assay 2, 24-hour treatment without metabolic activation: 1000, 750, 500, 333.33, 111.11, 37.04 and 12.35 µg/mL.
In Assays 1-2, there were no large changes in pH or osmolality after treatment. Insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment in Assays 1-2 with and without metabolic activation at several concentrations.
In Assay 1,following a 3-hour treatment with metabolic activation, cytotoxicity of the test item was observed at concentration range of (2000-222.22 µg/mL). No cytotoxicity or significant cytotoxicity was observed at lower concentrations.The Relative Total Growth (RTG) value of the highest evaluated concentration was 14%. The evaluation was made using data of all six concentrations (concentration range of 2000-8.23 µg/mL). There was statistically significant increase in the mutation frequency values in the two highest evaluated concentrations in Assay 1 and concentration related increase was also indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant. Therefore,this experiment was considered as being negative.
In Assay 1,following a 3-hour treatment without metabolic activation, cytotoxicity of the test item was observed at concentration range of (2000-222.22 µg/mL).No cytotoxicity or significant cytotoxicity was observed at lower concentrations.The Relative Total Growth (RTG) value of the highest evaluated concentration was 24%. An evaluation was made using data of all six concentrations (concentration range of 2000-8.23 µg/mL). There was statistically significant increase in the mutation frequency values in the highest evaluated concentration, but it was statistically borderline. Concentration related increase was also indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant. Therefore,this experiment was considered as being negative.
In Assay 2,following a 3-hour treatment with metabolic activation, cytotoxicity of the test item was observed at concentration range of (2000-222.22 µg/mL).No cytotoxicity or significant cytotoxicity was observed at lower concentrations.The Relative Total Growth (RTG) value of the highest evaluated concentration was 14%. The evaluation was made using data of all seven concentrations (concentration range of 2000-8.23 µg/mL). There was statistically significant increase in the mutation frequency values in the two highest evaluated concentrations in Assay 2 and concentration related increase was also indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant.This experiment was considered as being negative andconfirmed the negative result of the first test with metabolic activation.
In Assay 2,following a 24-hour treatment without metabolic activation, cytotoxicity of the test item was observed at concentration range of 1000-37.04 µg/mL.Excessive cytotoxicity was observed at 1000 and 750 µg/mL concentrations.No significant cytotoxicity was observed at 12.35 µg/mL concentration.Thus,the evaluation was made using data of five concentrations (concentration range of 500-12.35 µg/mL). The Relative Total Growth (RTG) value of the highest evaluated concentration was 7%. No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis.This experiment was considered as being negative.
The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.
In conclusion, no mutagenic effect of AMINOX®was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Justification for classification or non-classification
The substance has been determined not to be mutagenic or clastogenic in an in vitro Bacterial Reverse Mutation Assay and Mammalian Chromosome Aberration Test. The substance therefore does not require classifying as a mutagen according to Regulation 1272/2008.
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