Registration Dossier
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EC number: 485-230-3 | CAS number: 1455-42-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test substance was assessed according to internationally recognized guidelines in three in vitro tests. Firstly, an in vitro gene mutation study in bacteria (Ames test), an in vitro mammalian chromosome aberration study and an in vitro gene mutation study in mammalian cells. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation in the Ames test. In the chromosome aberration test, the test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the presence or absence of a liver enzyme metabolizing system, and was therefore considered non-clastogenic to human lymphocytes in vitro.
In the in vitro gene mutation study in mammalian cells the test substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.
Based on three negative results in vitro, the test item is considered non-mutagenic.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Testing was conducted between 17 May 2006 and 16 June 2006.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Test was performed according to guideline.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- S. typhimurium: Histidine
E. coli: Tryptophan - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S 9 mix; pretreated
- Test concentrations with justification for top dose:
- Preliminary toxicity test: Concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Mutation test experiments 1 and 2: Concentrations tested were 50, 150, 500, 1500 and 5000 µg/plate. - Vehicle / solvent:
- Solvent: Dimethyl sulphoxide
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- Preliminary toxicity test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing an aliquot of bacterial culture, 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test material formulation and 0. 5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel Bonner Minimal agar (30 mL/plate). Ten concentrations of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL molten trace histidine or tryptophan supplemented, top agar, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn.
Mutation test – Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots of one of the bacterial cultures were dispensed into sets of test tubes followed by2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL or S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed into the surface of Vogel Bonner Minimal agar plates (one tube per plate). This procedure was repeated in triplicate for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a colony counter.
Mutation test – Experiment 2
The second experiment was performed as above, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as experiment 1 (50 to 5000 µg/plate).
Acceptance criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell wall mutation and pKM101 plasmid R-factor.
All tester strain cultures should be in the approximate range of 1 to 9.9E09 bacteria per mL.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination. - Evaluation criteria:
- There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistically methods, as recommended by the UKEMS can also be used as an aid to evaluation, however statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal. - Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- (> 5000 µg/plate)
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- (> 5000 µg/plate)
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- (> 5000 µg/plate)
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- (> 5000 µg/plate)
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- (> 5000 µg/plate)
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary toxicity test results:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.
Mutation test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not provided in the report. The S9 mix and the amino-acid supplemented top agar used in both experiments were shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pale, particulate precipitate was observed at 5000 µg/plate which did not prevent the scoring of revertant colonies.
No significant increases in the frequency or revertant colonies were recorded for any of the strains of bacteria at any dose level, with or without metabolic activation.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test material was considered to be non-mutagenic under the conditions of the test both with and without metabolic activation. - Executive summary:
Introduction.
The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals no 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Methods.
Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % Liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test formulations.
Results.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pale, particulate precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
Conclusion.
The test material was considered to be non-mutagenic under the conditions of the test.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2006-05-16 to 2006-09-22
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Test was performed according to guideline.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: Human
- Details on mammalian cell type (if applicable):
- Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory had been determined to be approximately 17 hours under typical experimental exposure conditions.
Cell culture
Cells were grown in Eagle’s minimal essential medium with HEPES buffer (MEM) supplemented in-house with L-glutamine, penicillin/streptomycin, amphotericin B and 15 % foetal calf serum, at 37 °C with 5 % CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 µg/mL final concentration. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix from phenobarbitone, β-naphtoflavone induced rats
- Test concentrations with justification for top dose:
- Concentration range in the Experiment 1 without metabolic activation (4(20)-hour): 0, 95, 190, 380, 760, 1520, 3040 µg/mL, with 0, 380, 760 and 1520 µg/mL selected for metaphase analysis.
Concentration range in the Experiment 1 with metabolic activation (4(20)-hour): 0, 95, 190, 380, 760, 1520, 3040 µg/mL, with 0, 380, 760 and 1520 µg/mL selected for metaphase analysis.
Concentration range in the Experiment 2 without metabolic activation (24-hour): 0, 95, 190, 380, 760, 1140, 1520 µg/mL, with 0, 380, 760 and 1520 µg/mL selected for metaphase analysis.
Concentration range in the Experiment 2 withmetabolic activation (4(20)-hour): 0, 95, 190, 380, 760, 1520, 3040 µg/mL, with 0, 380, 760 and 1520 µg/mL selected for metaphase analysis. - Vehicle / solvent:
- Dimethyl sulphoxide (DMSO)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- With metabolic activation (S9) treatment
After approximately 48 hours incubation at 37 °C, 5 % CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20 % S9-mix (i.e. 2 % final concentration of S9 in standard co-factors) was added to the cultures of Preliminary Toxicity Test and of Experiment 1.
In experiment 2, 1 mL of 10 % S9-mix (i.e. 1 % final concentration of S9 in standard co-factors was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.
After 4 hours at 37 °C, the cultures were centrifuged, the treatment medium removed by suction and replaced with 8 mL wash of MEM culture medium. After a further centrifugation, the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 h at 37 °C in 5 % CO2 in humidified air.
Without metabolic activation (S9) treatment
In experiment 1, after approximately 48 hours incubation at 37 °C with 5 % CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then re-suspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test control solution or 0.1 mL if positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 h at 37 °C, the cultures were centrifuged, the treatment medium was removed by suction and replaced with and 8 mL wash of MEM culture medium. After a further centrifugation, the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 h.
In experiment 2, in the absence of metabolic activation the exposure was continuous for 24 h. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 h incubation, the cultures were removed from the incubator and dosed with 0.1 mL vehicle control, test material dose solution or 0.1 mL positive control positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at 37 °C for 24 h.
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period and a continuous exposure of 24 hours without metabolic activation. The dose range of test material used was 11.88 to 3040 µg/mL. Parallel flasks containing culture medium without whole blood, were establish for the three exposure conditions so that test material precipitate observation could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test material toxicity and for selection of the dose levels for the main study.
Experiment 1
4 –hour exposure to the test material without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest.
4-hour exposure to the test material with S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest.
Experiment 2
24 –hour exposure to the test material without S9-mix media prior to cell harvest.
4-hour exposure to the test material with S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest.
Cell harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded and the cells re-suspended in 0.075 M hypotonic KCL. After approximately 14 minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at 4 °C for at least four hours to ensure complete fixation.
Preparation of metaphase spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.
Staining
When the slides were dry they were stained in 5 % Gurrs Giemsa for 5 min, rinsed, dried and cover slipped using mounting medium.
Quantitative slide assessment
The slides were checked microscopically to determine the quality of the metaphase and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.
Coding
The slides were coded using a computerised random number generator.
Mitotic index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Scoring of chromosome damage
The metaphase analysis for the first experiment was subcontracted.
Where possible, the first 100 consecutive well-spread metaphases from each culture were counted and recorded. Where there were approximately 50 % cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44 – 48 chromosomes, any gaps, breaks or rearrangements were noted according to the International System for Chromosome Nomenclature (1985) as described by Scott et al and compatible and equitable to the simplified version of Savage (1976) recommended in the 1983 UKEMS guideline for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. - Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploidy cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test.
- Species / strain:
- lymphocytes: Human
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- (> 1520 µg/ml)
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- lymphocytes: Human
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- (> 1520 µg/ml)
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary toxicity test:
The does range for the Preliminary Toxicity Test was 11.88 to 3040 µg/mL. The maximum dose was based on the maximum recommended dose level, a 10 mM concentration. A precipitate of test material was observed in parallel blood-free cultures in all exposure groups at and above 760 µg/mL. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 3040 µg/mL all exposure groups. The test material induced no dose related evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level for testing in the main experiments was based on a 10 mM concentration and was 3040 µg/mL for both pulse exposure groups and 1520 µg/mL in the continuous exposure groups.
Chromosome aberration test – Experiment 1:
The qualitative assessment of the slides determined that there was no toxicity as was expected from the observations in the Preliminary Toxicity Test and that there were scorable metaphases present up to the maximum dose level of test material, 3040 µg/mL in both the absence and presence of metabolic activation (S9). Precipitate observations in the Preliminary Toxicity Test are considered to be representative for the study and a precipitate of test material was noted at and above 760 µg/mL in both exposure conditions.
The mitotic index data confirm the qualitative observations in that there was no dose-related inhibition of mitotic index observed in either the absence or presence of metabolic activation.
Therefore, as there was no toxicity observed the maximum dose level selected for metaphase analysis was based on the lowest two precipitating dose levels (1520 µg/mL and 760 µg/mL).
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The test material did not induce a statistically significant increase in the numbers of polyploidy cells at any dose level in either of the exposure groups.
Chromosome Aberration Test: Experiment 2:
The qualitative assessment of the slides determined that there were scorable metaphases present at the maximum dose levels tested in both the absence and presence of S9. Precipitate observations in the Preliminary Toxicity Test are considered to be representative for the study and a precipitate of test material was noted in blood-free cultures at and above 760 µg/mL, in both exposure conditions.
The mitotic index data confirms the qualitative observations in that there was no dose-related inhibition of mitotic index observed in the presence of metabolic activation up to the maximum dose level tested. There was a slight reduction in mitotic index at the maximum dose tested, 1520 µg/mL in the 24 h continuous exposure.
Therefore the maximum dose level selected for metaphase analysis was the same as Experiment 1, and was based on the two lowest precipitating dose levels (1520 and 760 µg/mL) for both exposure groups.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.
The test material did not induce a statistically significant increase in the numbers of polyploidy cells at any dose level in either of the exposure groups. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro. - Executive summary:
Introduction
This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 “Genetic Toxicology: Chromosome Aberration Test” and Method B10 of Commission Directive 200/32/EC. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for mutagenicity.
Methods
Duplicate cultures of human lymphocytes, treated with the test material were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used in the study, i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at 2 % final concentration with cell harvest after a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1 % final S9 concentration), whilst in the absence of metabolic activation, the exposure time was increased to 24 hours.
Results
All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.
All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and the activity of the metabolising system
The test material was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that induced a dose level that was a precipitating dose level.
Conclusion
The test material was considered to be non-clastogenic to human lymphocytes in vitro.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Remarks:
- TK+/- 3.7.2c
- Metabolic activation:
- with and without
- Metabolic activation system:
- The Rat S9 Microsomal fraction used during the course of the study was purchased from Moltox, Lot No. 4370 with an expiry date of 24 November 2024. The S9 was adjusted to a final protein content of 20 mg/mL before use. The S9 mix was prepared by mixing S9 with 100 mM phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% S9-mix concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.
- Test concentrations with justification for top dose:
- 4-hour without S9: 0, 15.63, 31.25, 62.5, 125, 250, 500
4-hour with S9: 0, 7.81, 15.63, 31.25, 62.5, 125, 250 - Vehicle / solvent:
- Dimethyl Sulphoxide (DMSO)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl Sulphoxide (DMSO)
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Cell Cleansing: The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen, they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24-hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e., THMG without Methotrexate) before being returned to R10 medium.
Test Item Preparation: The molecular weight of the test item was given as 304.4, therefore, the maximum concentration was 2000 μg/mL, which was the maximum recommended concentration. The purity of the test item was 98% and was accounted for in the test item formulations. The test item was insoluble in culture media at 20 mg/mL and DMSO and Acetone at 200 mg/mL but was soluble at 100 mg/mL in DMSO in solubility checks performed in-house. Prior to each experiment, the test item was accurately weighed, dissolved in DMSO and serial dilutions prepared. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
Preliminary Toxicity Test: A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9). The dose range used in the preliminary toxicity test was 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL. The maximum concentration was limited by precipitate observed in the solubility test. Following the exposure periods, the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL. The cultures were incubated at approximately 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24-hours, the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent solvent control performed to give a percentage Relative Suspension Growth (%RSG) value. Results from the preliminary toxicity test were used to set the test item concentrations for the mutagenicity experiments. Maximum concentrations were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB*) the upper concentration may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating concentrations will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 %. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).
Mutagenicity Test: Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight concentrations of the test item and solvent and positive controls. To each universal was added 2 mL of S9-mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL. Two exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix. The concentrations of test item used were 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%). The concentrations of test item used were 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL.
The exposure vessels were incubated at 37±2 °C for 4-hours with continuous shaking using an orbital shaker within an incubated hood. - Evaluation criteria:
- This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity).Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual concentration. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Concentrations that have RTG survival values markedly less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e., increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, i.e., the mutant frequency of the concurrent solvent control plus 126, which is based on the analysis of the distribution of the solvent control MF data from participating laboratories. Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system. Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system. - Statistics:
- The computer systems used on this study to acquire and/or quantify data were the following systems:
• Veeva QMS – Electric communication system. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test item, Spiroglycol, did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.
- Executive summary:
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016. One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight concentrations in duplicate, together with solvent (Dimethyl Sulphoxide), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (S9). The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The concentrations plated for cloning efficiency and expression of mutant colonies were as follows:
Group
Concentration of Spiroglycol (μg/mL) plated for cloning efficiency and mutant frequency
4-hour without S9
0, 15.63, 31.25, 62.5, 125, 250, 500
4-hour with S9
0, 7.81, 15.63, 31.25, 62.5, 125, 250
The maximum concentration used was 1000 μg/mL and was limited by the presence of precipitate. A precipitate of test item was observed at and above 500 μg/mL at the end of the exposure period in the absence of S9 and at and above 250 μg/mL in the presence of S9. The solvent control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test item did not induce any increases in the mutant frequency at any of the concentrations in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating concentration in both exposure groups, and at least four analysable concentrations in each exposure group, as recommended by the OECD 490 guideline. The results observed in both exposure groups were considered to fulfil the criteria for a clearly negative outcome. The test item, Spiroglycol, did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.
Referenceopen allclose all
Preliminary toxicity test
The number of revertant colonies for the toxicity assay were:
With (+) or without (-) S9-mix |
Strain |
Dose (µg/plate) |
||||||||||
0 |
0.15 |
0.5 |
1.5 |
5 |
15 |
50 |
150 |
500 |
1500 |
5000 |
||
- |
TA 100 |
110 |
99 |
87 |
102 |
104 |
106 |
98 |
113 |
107 |
104 |
89P |
+ |
TA 100 |
89 |
88 |
106 |
75 |
88 |
74 |
81 |
78 |
60 |
62 |
73P |
- |
WP3uvrA- |
21 |
21 |
23 |
31 |
24 |
13 |
30 |
24 |
13 |
18 |
23P |
+ |
WP3uvrA- |
26 |
24 |
46 |
26 |
31 |
25 |
36 |
30 |
27 |
21 |
21P |
Where P = Precipitate
Spontaneous mutation rates (concurrent negative controls)
Experiment 1:
Number of revertants (mean number of colonies per plate) |
|||||||||
Base pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||
98 |
(107) |
13 |
(11) |
22 |
(27) |
20 |
(18) |
19 |
(10) |
123 |
19 |
40 |
14 |
7 |
|||||
99 |
24 |
19 |
21 |
3 |
|||||
Experiment 2:
Number of revertants (mean number of colonies per plate) |
|||||||||
Base pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||
67 |
(81) |
15 |
(19) |
22 |
(25) |
29 |
(19) |
7 |
(11) |
96 |
19 |
26 |
14 |
14 |
|||||
81 |
24 |
26 |
14 |
11 |
|||||
Test results – Experiment 1 – Without metabolic activation
With or without S9-mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
|||||||||
Base pair substitution type |
Frameshift type |
||||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||||
- |
0 |
102 |
(91) 11.5# |
10 |
(11) 1.7 |
18 |
(21) 2.9 |
13 |
(19) 8.7 |
15 |
(19) 4.5 |
79 |
10 |
23 |
15 |
19 |
|||||||
91 |
13 |
23 |
29 |
24 |
|||||||
- |
50 |
84 |
(88) 4.0 |
14 |
(13) 2.1 |
21 |
(21) 0.0 |
25 |
(25) 2.5 |
14 |
(12) 3.5 |
89 |
11 |
21 |
27 |
8 |
|||||||
92 |
15 |
21 |
22 |
14 |
|||||||
- |
150 |
88 |
(89) 1.0 |
9 |
(10) 2.3 |
29 |
(21) 7.6 |
14 |
(20) 5.3 |
19 |
(13) 5.3 |
90 |
13 |
14 |
24 |
9 |
|||||||
89 |
9 |
19 |
22 |
11 |
|||||||
- |
500 |
104 |
(91) 11.7 |
10 |
(12) 2.5 |
18 |
(20) 1.5 |
20 |
(22) 4.0 |
12 |
(14) 2.1 |
89 |
12 |
21 |
27 |
16 |
|||||||
81 |
15 |
20 |
20 |
13 |
|||||||
- |
1500 |
80 |
(81) 3.1 |
15 |
(13) 2.5 |
20 |
(21) 0.6 |
19 |
(21) 7.2 |
15 |
(13) 3.5 |
78 |
13 |
21 |
15 |
9 |
|||||||
84 |
10 |
21 |
29 |
15 |
|||||||
- |
5000 |
85P |
(85) 0.6 |
19P |
(13) 4.9 |
25P |
(20) |
22P |
(19) 7.0 |
11P |
(9) 2.0 |
85P |
11P |
15P |
24P |
9P |
|||||||
86P |
10P |
20P |
11P |
7P |
|||||||
Positive controls S9-mix - |
Name |
EENG |
EENG |
EENG5.0 |
4NQO |
9AA |
|||||
Concentration (µ/plate) |
3 |
5 |
2 |
0.2 |
|
||||||
Number of colonies per plate |
558 |
(553) 4.2 |
676 |
(528) 166.2 |
749 |
(841) 90.1 |
172 |
(175) 47.1 |
2716 |
(2543) 229.0 |
|
550 |
559 |
846 |
130 |
2283 |
|||||||
552 |
348 |
929 |
224 |
2629 |
|||||||
Test results – Experiment 1 – With metabolic activation
With or without S9-mix |
Test substance concentration (µg/plate) |
Number of revertants (mean number of colonies per plate) |
|||||||||
Base pair substitution type |
Frameshift type |
||||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||||
+ |
0 |
86 |
(86) 1.0# |
13 |
(14) 1.2 |
25 |
(30) 7.2 |
33 |
(31) 4.4 |
9 |
(10) 0.6 |
87 |
15 |
26 |
34 |
10 |
|||||||
85 |
13 |
38 |
26 |
10 |
|||||||
+ |
50 |
80 |
(81) 0.6 |
11 |
(11) 0.6 |
31 |
(26) 4.7 |
30 |
(18) 10.1 |
7 |
(8) 1.2 |
81 |
12 |
14 |
12 |
9 |
|||||||
81 |
11 |
22 |
13 |
9 |
|||||||
+ |
150 |
77 |
(82) 8.4 |
16 |
(13) 2.5 |
41 |
(29) 10.1 |
26 |
(27) 6.6 |
8 |
(10) 2.6 |
78 |
11 |
23 |
21 |
13 |
|||||||
92 |
13 |
24 |
34 |
9 |
|||||||
+ |
500 |
79 |
(88) 10.8
|
11 |
(10) 1.0 |
26 |
(26) 4.0 |
32 |
(29) 3.6 |
12 |
(10) 2.1 |
100 |
10 |
22 |
30 |
9 |
|||||||
85 |
9 |
30 |
25 |
8 |
|||||||
+ |
1500 |
84 |
(87) 2.9 |
13 |
(12) 1.7 |
23 |
(25) 2.0 |
21 |
(23) 2.0 |
12 |
(8) 4.0 |
89 |
13 |
25 |
25 |
4 |
|||||||
89 |
10 |
27 |
23 |
8 |
|||||||
+ |
5000 |
76P |
(84) 9.7 |
10P |
(10) 1.5 |
26P |
(28) 4.0 |
29P |
(28) 2.3 |
10P |
(9) 2.1 |
95P |
12P |
26P |
25P |
7P |
|||||||
82P |
9P |
33P |
29P |
11P |
|||||||
Positive controls S9-mix + |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
|||||
Concentration (µ/plate) |
1 |
2 |
10 |
5 |
2 |
||||||
Number of colonies per plate |
1828 |
(1869) 53.7 |
230 |
(234) 6.1 |
300 |
(370) 62.0 |
213 |
(227) 12.7 |
591 |
(657) 106.6 |
|
1850 |
231 |
394 |
238 |
600 |
|||||||
1930 |
241 |
417 |
229 |
780 |
|||||||
P = Precipitate
# = Standard deviation
Mitotic index – Preliminary toxicity test
4-hour treatment, 20 hour recovery period – S9
4-hour treatment, 20 hour recovery period + S9
24 h treatment – S9
Concentration (µg/mL) |
4 (20)h without S9 |
4 (20)h with S9 |
24 h without S9 |
|||
Mitotic index |
% of control |
Mitotic index |
% of control |
Mitotic index |
% of control |
|
0 |
2.80 |
100 |
5.70 |
100 |
5.40 |
100 |
11.88 |
- |
- |
- |
- |
- |
- |
23.75 |
- |
- |
- |
- |
- |
- |
47.5 |
- |
- |
- |
- |
- |
- |
95 |
- |
- |
- |
- |
- |
- |
190 |
4.2 |
150 |
3.80 |
67 |
6.35 |
118 |
380 |
5.8 |
207 |
5.15 |
90 |
4.10 |
76 |
760 |
0.60P |
21 |
2.90P |
51 |
4.25P |
79 |
1520 |
-P |
- |
-P |
- |
-P |
- |
3040 |
-P |
- |
-P |
- |
-P |
- |
- = Not assessed for mitotic index
P = Precipitate observed at the end of the exposure period in blood-free cultures.
Mitotic index – Experiment 1
Dose level (µg/mL) |
4 h treatment without S9 |
4 h treatment with S9 |
||||||
A |
B |
Mean |
% of control |
A |
B |
Mean |
% of control |
|
0 |
4.60 |
4.45 |
4.53 |
100 |
3.15 |
5.60 |
4.38 |
100 |
190 |
- |
- |
- |
- |
- |
- |
- |
- |
380 |
3.80 |
4.60 |
4.20 |
93 |
3.10 |
3.65 |
3.38 |
77 |
760 |
8.30P |
5.15P |
6.73 |
149 |
4.20P |
4.05P |
4.13 |
94 |
1520 |
3.50P |
3.25P |
3.38 |
75 |
2.80P |
3.50P |
3.15 |
72 |
3040 |
3.15P |
3.55P |
3.35 |
74 |
4.70P |
5.05P |
4.88 |
111 |
MMC 0.4 |
1.50 |
2.40 |
1.95 |
43 |
NA |
NA |
NA |
NA |
CP 5 |
NA |
NA |
NA |
NA |
1.00 |
0.70 |
0.85 |
19 |
MMC = Mitomycin C
CP = Cyclophosphamide
NA = Not applicable
- = Not assessed for mitotic index
P = Precipitate of test material
Mitotic index – Experiment 2
Dose level (µg/mL) |
4 h treatment without S9 |
4 h treatment with S9 |
||||||
A |
B |
Mean |
% of control |
A |
B |
Mean |
% of control |
|
0 |
5.60 |
4.65 |
5.13 |
100 |
7.10 |
5.90 |
6.50 |
100 |
380 |
3.55 |
3.35 |
3.45 |
67 |
6.15 |
7.20 |
6.68 |
103 |
760 |
3.65P |
2.80P |
3.23 |
63 |
7.00P |
8.00P |
7.50 |
115 |
1140 |
-P |
-P |
- |
- |
NA |
NA |
NA |
NA |
1520 |
2.65P |
3.00P |
2.83 |
55 |
8.10P |
7.25P |
7.68 |
118 |
3040 |
NA |
NA |
NA |
- |
-P |
-P |
- |
- |
MMC 0.4 |
2.95 |
2.60 |
2.78 |
54 |
NA |
NA |
NA |
NA |
CP 5 |
NA |
NA |
NA |
NA |
3.15 |
2.00 |
2.58 |
40 |
MMC = Mitomycin C
CP = Cyclophosphamide
NA = Not applicable
- = Not assessed for mitotic index
P = Precipitate of test material
Results of chromosome aberration test - Experiment 1 without metabolic activation (S9)
Treatment group |
Replicate |
Mitotic index (%) |
No. of cells scored |
No. of aberrations |
Total no. of aberrations |
Frequency of aberrations |
|||||||
Gaps |
Chromatid |
Chromosome |
Others X |
(+gaps) |
(-gaps) |
(+gaps) |
(-gaps) |
||||||
Breaks |
Exchanges |
Breaks |
Exchanges |
||||||||||
Vehicle control |
A |
4.6 |
100 |
3 |
0 |
0 |
1 |
0 |
0 |
4 |
1 |
4 |
1 |
B |
4.45 |
100 |
2 |
2 |
0 |
1 |
0 |
0 |
5 |
3 |
5 |
3 |
|
Total |
(100) |
200 |
5 |
2 |
0 |
2 |
0 |
0 |
9 |
4 |
9 (4.5) |
4 (2.0) |
|
380 µg/mL |
A |
3.80 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
4.60 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
(93) |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 (0.0) |
0 (0.0) |
|
760 µg/mL |
A |
8.30 |
100 |
1 |
1 |
0 |
0 |
0 |
0 |
2 |
1 |
2 |
1 |
B |
5.15 |
100 |
4 |
0 |
0 |
4 |
0 |
0 |
8 |
4 |
7 |
3 |
|
Total |
(149) |
200 |
5 |
1 |
0 |
4 |
0 |
0 |
10 |
5 |
9 (4.5) |
4 (2.0) |
|
1520 µg/mL |
A |
3.50 |
100 |
5 |
4 |
0 |
0 |
0 |
0 |
9 |
4 |
8 |
4 |
B |
3.25 |
100 |
2 |
0 |
0 |
1 |
0 |
0 |
3 |
1 |
33 |
1 |
|
Total |
(75) |
200 |
7 |
4 |
0 |
1 |
0 |
0 |
12 |
5 |
11 (5.5) |
5 (2.5) |
|
Positive control MMC 0.4 µg/mL |
A |
1.50 |
50a |
4 |
30 |
22 |
3 |
0 |
0 |
59 |
55 |
29 |
28 |
B |
2.40 |
50a |
3 |
23 |
18 |
3 |
0 |
1 |
48 |
45 |
25 |
24 |
|
Total |
(43) |
100 |
7 |
53 |
40 |
6 |
0 |
1 |
107 |
100 |
54 (54.0) |
52*** (52) |
|
MMC = Mitomycin C
a = slide evaluation terminated at 50 cells because approximately 50 % cells with aberrations had been observed.
*** = P < 0.001
Results of chromosome aberration test - Experiment 1 with metabolic activation (S9)
Treatment group |
Replicate |
Mitotic index (%) |
No. of cells scored |
No. of aberrations |
Total no. of aberrations |
Frequency of aberrations |
|||||||
Gaps |
Chromatid |
Chromosome |
Others |
(+gaps) |
(-gaps) |
(+gaps) |
(-gaps) |
||||||
Breaks |
Exchanges |
Breaks |
Exchanges |
||||||||||
Vehicle control |
A |
3.15 |
100 |
4 |
0 |
0 |
1 |
0 |
0 |
5 |
1 |
4 |
1 |
B |
5.60 |
100 |
1 |
2 |
0 |
1 |
0 |
0 |
4 |
3 |
4 |
3 |
|
Total |
(100) |
200 |
5 |
2 |
0 |
2 |
0 |
0 |
9 |
4 |
8 (4.0) |
4 (2.0) |
|
380 µg/mL |
A |
3.10 |
100 |
2 |
0 |
0 |
2 |
0 |
0 |
4 |
2 |
4 |
2 |
B |
3.65 |
100 |
4 |
0 |
0 |
0 |
0 |
0 |
4 |
0 |
4 |
0 |
|
Total |
(77) |
200 |
6 |
0 |
0 |
2 |
0 |
0 |
8 |
2 |
8 (4.0) |
2 (1.0) |
|
760 µg/mL |
A |
4.20 |
100 |
1 |
1 |
0 |
0 |
0 |
0 |
2 |
1 |
2 |
1 |
B |
4.05 |
100 |
3 |
1 |
0 |
0 |
0 |
0 |
4 |
1 |
4 |
1 |
|
Total |
(94) |
200 |
4 |
2 |
0 |
0 |
0 |
0 |
6 |
2 |
6 (3.0) |
2 (1.0) |
|
1520 µg/mL |
A |
2.80 |
100 |
4 |
3 |
0 |
0 |
0 |
0 |
7 |
3 |
6 |
2 |
B |
3.50 |
100 |
3 |
0 |
0 |
0 |
0 |
0 |
3 |
0 |
3 |
0 |
|
Total |
(72) |
200 |
7 |
3 |
0 |
0 |
0 |
0 |
10 |
3 |
9 (4.5) |
2 (1.0) |
|
Positive control CP 5 µg/mL |
A |
1.00 |
100 |
8 |
37 |
9 |
1 |
0 |
0 |
55 |
47 |
38 |
33 |
B |
0.70 |
100 |
14 |
38 |
11 |
0 |
0 |
0 |
63 |
49 |
33 |
30 |
|
Total |
(19) |
200 |
22 |
75 |
20 |
1 |
0 |
0 |
118 |
96 |
71 (35.5) |
63*** (31.5) |
|
CP = Cyclophosphamide
*** = P < 0.001
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Additional information from genetic toxicity in vitro:
A mutation is a permanent change in the amount or structure of the genetic material in a cell. The term “mutation” applies to both heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known, including specific base pair changes and chromosomal translocations. The term “mutagenic” and “mutagen” are used for agents giving rise to an increased occurrence of mutations in populations of cells or organisms.
The more generic terms “genotoxic” and “genotoxicity” apply to agents or processes which alter the structure, information content or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non-physiological manner temporarily alter its replication. Genotoxicity test results are usually taken as indicators for mutagenic effects.
The in vitro gene mutation (Ames) study was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and his co-workers and Garner et al, in which mutagenic activity is assessed by exposing histidine auxotrops of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli to various concentrations of the test material. The method conforms to guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities, including METI, MHLW and Maff. This method also conforms with the OECD Guidelines for the Testing of Chemicals No. 471, Method B13/14 in EC Commission Directive 2000/32/EC and the USA EPA (TSCA) OPPTS 870.5100 guideline.
The Salmonella strains used in the test are incapable of synthesising histidine and are therefore dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic gent, these bacteria may undergo a reverse mutation to histidine independent form which are detected by their ability to grow on a histidine deficient medium. Using various strains of this organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional trains have been introduced. These include a deletion though the excision repair gene (uvrB- Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall. A mutant strain of E. coli (WP2uvrA-) which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence was used to complement the salmonella strains. This strain also has a deletion in the excision repair gene (uvrA-). Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell, the test material and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre-treated with a compound known to induce an elevated level of these enzymes.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. The test item was therefore considered to be non-mutagenic in vitro.
The purpose of the chromosome aberration study was to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocytes. The detection of structural chromosomal aberrations in cultured mammalian cells supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al., 1990)
The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytesin vitro.
Justification for selection of genetic toxicity endpoint
The test was performed to GLP according to internationally
recognised guidelines in appropriate test species.
Justification for classification or non-classification
This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.
To arrive at a classification, test results are considered from experiments determining mutagenic and genotoxic effects in germ and/or somatic cells of exposed animals and in in vitro tests.
The system is hazard based, classifying substances on the basis of their intrinsic ability to induce mutations in germs cells, and does not give a quantitative assessment of the risk.
To this end, the test substance has been assessed according to internationally recognized guidelines in three in vitro tests. An in vitro gene mutation study in bacteria (Ames test), an in vitro mammalian chromosome aberration study and in vitro gene mutation study in mammalian cells.
In the Ames test, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation.
In the chromosome aberration test, the test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the presence or absence of a liver enzyme metabolizing system, and was therefore considered non-clastogenic to human lymphocytes in vitro. The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF.
Based on three negative results in vitro, the test item is considered non-mutagenic.
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