Registration Dossier
Registration Dossier
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EC number: 215-214-1 | CAS number: 1313-97-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The substance did not show any mutagenic activity in a bacterial reverse
mutation test (conducted in accordance with OECD method 471) with
Salmonella typhimurium and Escherichia coli with and without metabolic
activation.
The substance has been shown to be non-mutagenic to CHO cells in an HPRT
study conducted in accordance with OECD method 476.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
- 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:
- 21 May 2007 - 3 August 2007
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Government revised Chemical Substance Law according to Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of the Ministry of International Trade and Industry, 9th December 1996.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- S. typhimurium: Histidine locus.
E. coli: Tryptophan locus. - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from rats induced with Aroclor 1254
- Test concentrations with justification for top dose:
- - 156.3, 312.5, 625, 1250 and 2500 µg/plate, for the TA 102 strain in the first experiment and all tester strains in the second experiment, either with or without S9 mix,
- 312.5, 625, 1250, 2500 and 5000 µg/plate, for all tester strains except for the TA 102 in the first experiment, either with or without S9 mix. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in most vehicles used for in vitro tests and so was prepared as an homogeneous suspension.
The test item was suspended at the following concentrations:
100 mg/mL for the preliminary test and the first experiment; 50 mg/mL for the second experiment. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-Anthramine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method.
DURATION
- Preincubation period: 60 minutes, 37 °C
- Exposure duration: 48 - 72 hours
NUMBER OF REPLICATES: three plates/dose-level
POSITIVE CONTROLS
The positive controls were dissolved in dimethylsulfoxide (except for Mitomycin C which was dissolved in distilled water).
Without S9 mix:
-sodium azide (NAN3)
-9-Aminoacridine (9AA)
-2-Nitrofluorene (2NF)
-Mitomycin C (MMC)
-4-Nitroquinoline 1-oxide (4NQO)
With S9 mix:
-2-Anthramine (2AM)
-Benzo(a)pyrene (BaP)
TEST SYSTEM
The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximately 6 mL of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours, to produce bacterial suspensions.
Metabolic activation system:
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route. The S9 fraction was preserved in sterile tubes at -80 °C, until use.
The S9 mix was prepared at +4 °C immediately before use and maintained at this temperature until added to the overlay agar.
The composition of S9 mix was as follows:
Glucose-6-phosphate 5 mM
NADP 4 mM
KCl 33 mM
MgCl2 8 mM
Sodium phosphate buffer pH 7.4 100 mM
S9 fraction, protein concentration: 36.4 mg/mL 10 % (v/v)
water to volume
EXPERIMENTAL DESIGN
Treatment:
The test material was tested in a preliminary test and two mutagenicity experiments.
The direct plate incorporation method was performed as follows: test material solution (0.05 mL), S9 mix when required (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45 °C). After rapid homogenisation, the mixture was overlaid onto a Petri plate containing minimum medium.
The preincubation method was performed as follows: test material solution (0.05 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37 °C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK). - Evaluation criteria:
- A reproducible 2-fold increase (for the TA 98, TA 100, TA 102 and WP2 uvrA strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-response was considered as a positive result. Reference to historical data, or other considerations of biological relevance were taken into account in the evaluation of the data obtained.
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A moderate to marked precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥156.3 µg/plate.
RANGE-FINDING/SCREENING STUDIES:
To assess the toxicity of the test material to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100, TA 102 and WP2 uvrA strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 100 µg/plate. At 5000 µg/plate the strong precipitate sometimes (with the TA 102 strain with and without S9 mix) interfered with scoring making the plates unreadable.
No noteworthy toxicity which could be considered as relevant was noted towards the four strains used, with and without S9 mix.
COMPARISON WITH HISTORICAL CONTROL DATA: The control data reported in these report are in the range of the historical control data observed in the laboratory. - Conclusions:
- Interpretation of results: Negative
Under the conditions of this study, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli. - Executive summary:
The potential of the test material to induce reverse gene mutations in Salmonella typhimurium and Escherichia coli was evaluated in a study performed according to the standardised guidelines OECD 471 and EU Method B13/14.
The test material was tested in two independent experiments, with and without a metabolic activation system (S9 mix, prepared from a liver post mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254).
Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 and one strain of Escherichia coli, WP2uvrA, were used. Each strain was exposed to 156.3 to 5000 µg/plate of the test material (three plates/dose-level). After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored. Solvent control (DMSO) and positive controls were used.
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
A moderate to marked precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 156.3 µg/plate. No toxicity was noted towards all the strains used either with or without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the six strains.
Under the conditions of the study, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 July 2012 - 19 March 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: The United Kingdom Environmental Mutagen Society (Cole et al, 1990)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus.
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Cell line: CHO-K1.
- Cell Culture: The stocks of cells were stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in Hams F12 medium, supplemented with 5 % foetal bovine serum (FBS) and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per mL) at 37 °C with 5 % CO₂ in air.
- Cell Cleansing: Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days (Hams F12 growth medium supplemented with Hypoxanthine (13.6 µg/mL, 100 µM), Aminopterin (0.0178 µg/mL, 0.4 µM) and Thymidine (3.85 µg/mL, 16 µM)). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Induced rat liver homogenate metabolising system (S9)
- Test concentrations with justification for top dose:
- Preliminary Cytotoxicity Test
- 0, 13.14, 26.29, 52.58, 105.16, 210.31, 420.63, 841.25, 1682.5 and 3365 µg/mL (with and without 2 % S9)
Mutagenicity Test
- 4 hour exposure (-S9): 0, 105.16, 210.31, 420.63, 841.25, 1682.5 and 3365 µg/mL
- 4 hour exposure (+2 % S9): 0, 26.29, 52.58, 105.16, 210.31, 420.63, 841.25, 1261.88 and 1682.5 µg/mL
- 24 hour exposure (-S9): 0, 1.64, 3.29, 6.57, 13.14, 26.29, 52.58, 105.16 and 210.31 µg/mL
- 4 hour exposure (+1 % S9): 0, 210.31, 420.63, 841.25, 1682.5, 2523.75 and 3365 µg/mL - Vehicle / solvent:
- Hams F12 culture medium. There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels investigated.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Hams F12 culture medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: dimethyl benzanthracene
- Remarks:
- Positive controls were dosed in DMSO at 1%.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.
MUTAGENICITY TEST
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. For the 4 hour exposure groups of Experiment 1, cells were seeded at 1.5 x 10⁶/75 cm² flask approximately 48 hours before being exposed to the test or control materials. In Experiment 2, cells were seeded approximately 48 hours before being exposed to the test or control materials at 1.0 x 10⁶/75 cm² flask for the 24 hour exposure group and at 1.5 x 10⁶/75 cm² flask for the 4 hour exposure group in the presence of S9.
Duplicate cultures were set up, both in the presence and absence of metabolic activation, with up to eight dose levels of test material, vehicle and positive controls. Treatment was for 4 hours in serum free media (Hams F12) or for 24 hours in Hams F12 with 1 % serum at 37 °C in an incubator with a humidified atmosphere of 5 % CO₂ in air.
At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in Hams F12 with 5 % FBS. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10⁶ cells/flask in a 225 cm² flask to allow growth and expression of induced mutants, and in triplicate in 25 cm² flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in Hams F12 with 5 % FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5 % CO₂ in air.
Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
During the 7 Day expression period the cultures were subcultured and maintained at 2 x 10⁶ cells/225 cm² flask on day 3 to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm² flask in 5 mL of Hams F12 with 5 % FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10⁵ cells/75 cm² flask (5 replicates per group) in Hams F12 with 5 % FBS, supplemented with 10 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at 37 °C in an incubator with humidified atmosphere of 5 % CO₂ in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.
The percentage of viability and mutation frequency per survivor were calculated for each dose group (as per the calculations defined in the field “Any other information on materials and methods incl. tables”).
Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10 % Giemsa solution for 5 minutes.
ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115 % with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70 % range may be accepted but this will be dependent on the scientific judgement of the Study Director. All assays below 50 % cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10^-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10^-6 will not be used for the evaluation of a test material.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test material clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test material must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test materials with little or no mutagenic activity should include an acceptable assay where concentrations of the test material have reduced the clonal survival to approximately 10 to 15 % of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/mL) or twice the solubility limit of the test material in culture medium. Where a test material is excessively toxic, with a steep response curve, a concentration that is at least 75 % of the toxic dose level should be used. There is no maximum toxicity requirement for test materials that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test material, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test material. - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- EXPERIMENT 1
A precipitate of the test material was seen at the end of exposure at and above 52.58 and 210.31 μg/mL in the absence and presence of S9 respectively.
There was a dose related reduction in Day 0 cloning efficiency in the absence of S9 slightly less to that seen in the Preliminary Cytotoxicity Test. A reduction of 71 % was achieved at 1682.5 μg/mL. The 4 hour exposure group in the presence of S9 demonstrated no reduction in the Day 0 cloning efficiency at the dose levels tested.
There was no marked reduction in the Day 7 cloning efficiencies of the 4 hour exposure group in the presence of S9. In the 4 hour exposure group in the absence of S9 there was a reduction in the Day 7 cloning efficiencies of 25 and 42 % at 1261.88 and 1682.5 μg/mL respectively. Due to prolonged toxicity during the expression period the ‘B’ replicate of the 1682.5 μg/mL dose level in the 4 hour exposure group in the absence of S9 was too toxic for plating more than 3 mutant flasks due to insufficient cell numbers. This would indicate that the test material was tested to the limit of acceptable toxicity.
There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10^-6 in the presence of S9. In the absence of S9 there was an increase in the mutation frequency over the vehicle control of more than 20 x 10^-6 at 841.25 μg/mL but since this was set against a low vehicle control value and it was not part of a dose related response it was considered to be of no toxicological significance.
The Day 0 and Day 7 vehicle control cloning efficiencies for the 4 hour exposure group in the presence of S9 did not achieve 70% cloning efficiency in all the replicates, however since they achieved at least 50 % this was considered to be acceptable.
The vehicle control value for the 4 hour exposure in the presence of S9 was within the maximum upper limit of 25 x 10^-6 mutants per viable cell, and the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.
EXPERIMENT 2
A precipitate of the test material was seen at the end of the exposure period in the absence of S9 at and above 26.29 μg/mL and at all test material dose levels in the presence of S9.
There was no marked toxicity observed in the presence of S9 at Day 0. The 24 hour exposure group demonstrated a dose related toxicity curve at Day 0 with the upper two doses of 105.16 and 210.31 μg/mL exceeding the ideal 80 % toxicity.
There was no marked reduction in the Day 7 cloning efficiencies of either exposure group.
The Day 7 vehicle control cloning efficiencies for the 24 hour exposure group and the Day 0 and Day 7 vehicle control cloning efficiencies for the 4 hour exposure group did not achieve 70 % cloning efficiency in all the replicates, however since they achieved at least 50 % this was considered to be acceptable.
An increase in mutant frequency of greater than 20 x 10^-6 was seen in the 4 hour exposure group in the presence of S9 at the maximum dose tested (3365 μg/mL) and at 1682.5 μg/mL. However, these responses were relatively modest, not part of a true dose-related response and were only marginally above the highest acceptable value for a vehicle control and were, therefore, considered to be of no toxicological significance. In the 24 hour exposure group a similar response was seen at 105.16 μg/mL which achieved close to the limit of acceptable toxicity at Day 0. The response was again considered to be of no toxicological significance and was considered to be related to toxicity. This was confirmed by the response seen at 210.31 μg/mL in the 24 hour exposure group which demonstrated a large increase in mutant frequency. However, due to prolonged toxicity during the expression period there were insufficient cells in the ‘A’ replicate to plate 5 mutant flasks and, therefore, this dose level can be excluded on the basis of toxicity. The response seen at this toxic dose level does indicate that the increase in mutant frequency is due to a cytotoxic rather than a mutagenic mechanism. There were also increases in mutant frequency of greater than 20 x 10^-6 seen in the 24 hour exposure group at 1.64 and 26.29 μg/mL but these increases were not dose related and were only marginally above the maximum acceptable upper limit for a vehicle control and were therefore considered to be of no toxicological significance.
The vehicle control values were all within the ideal maximum upper limit of 25 x 10^-6 mutants per viable cell, and the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected. - Conclusions:
- Interpretation of results: Negative
Under the conditions of this study, the test material is considered to be non-mutagenic to CHO cells at the HPRT locus. - Executive summary:
A study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells in vitro in accordance with the standardised guidelines OECD 476, EU Method B.17, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the EPA OPPTS 870.5300.
CHO cells were treated with the test material at up to eight dose levels, in duplicate, together with negative and positive controls. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.
Four treatment conditions were used for the test. In Experiment 1, a 4 hour exposure in the absence of metabolic activation (dose levels 0, 105.16, 210.31, 420.63, 841.25, 1682.5 and 3365 µg/mL) and a 4 hour exposure in the presence of 2 % S9 (dose levels 0, 26.29, 52.58, 105.16, 210.31, 420.63, 841.25, 1261.88 and 1682.5 µg/mL). In Experiment 2, the 4 hour exposure was repeated using a 1 % final S9 concentration (dose levels 0, 210.31, 420.63, 841.25, 1682.5, 2523.75 and 3365 µg/mL), whilst in the absence of metabolic activation the exposure time was increased to 24 hours (dose levels 0, 1.64, 3.29, 6.57, 13.14, 26.29, 52.58, 105.16 and 210.31 µg/mL).
The test material demonstrated no dose related increases in mutant frequency at any dose level, with or without metabolic activation in Experiment 1.
An increase in mutant frequency of greater than 20 x 10 -6 was seen in the 4 hour exposure group in the presence of S9 in Experiment 2 at the maximum dose tested (3365 μg/mL) and an interim dose level of 1682.5 μg/mL. However, these responses were relatively modest, not part of any true dose-related effect and were only marginally above the highest acceptable value for a vehicle control and were therefore considered to be of no toxicological significance. In the 24 hour exposure group a similar response was seen at 105.16 μg/mL which achieved close to the limit of acceptable toxicity at Day 0. The response was again considered to be of no toxicological significance and was considered to be related to toxicity. The dose level of 210.31 μg/mL in the 24 hour exposure group demonstrated a large increase in mutant frequency but there were insufficient cells due to poor recovery from post treatment toxicity in the ‘A’ replicate to plate 5 mutant flasks and so this dose level can be excluded on the basis of toxicity. However, the response seen at this toxic dose level does indicate that the increases in mutant frequency are due to cytotoxicity rather than a mutagenic mechanism.
The negative and positive controls gave mutant frequencies within the range expected, indicating the satisfactory performance of the test and of the metabolising system.
Under the conditions of this study, the test material is considered to be non-mutagenic to CHO cells at the HPRT locus.
Referenceopen allclose all
Table 2: First experiment (direct plate incorporation) -Mean revertant colony counts
|
TA 102 |
TA 1535 |
TA 1537 |
||||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
314 |
459 |
No |
11 |
21 |
No |
8 |
6 |
No |
156.3 |
279 |
413 |
No (Mp) |
- |
- |
- |
- |
- |
- |
312.5 |
340 |
478 |
No (Mp) |
11 |
19 |
No (Mp) |
8 |
7 |
No (Mp) |
625 |
292 |
560 |
No (Mp) |
12 |
18 |
No (Mp) |
4 |
7 |
No (Mp) |
1250 |
234 |
458 |
No (Sp) |
12 |
7 |
No (Mp/Sp) |
3 |
7 |
No (Sp) |
2500 |
64 |
302 |
No (Sp) |
6 |
10 |
No (Sp) |
2 |
4 |
No (Sp) |
5000 |
- |
- |
- |
U |
5 |
No (Sp) |
U |
3 |
No (Sp) |
Positive control |
1540 |
3709 |
No |
491 |
158 |
No |
448 |
85 |
No |
|
TA 98 |
TA 100 |
WP2uvrA |
||||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
20 |
33 |
No |
151 |
107 |
No |
27 |
41 |
No |
312.5 |
18 |
28 |
No (Mp) |
150 |
92 |
No (Mp) |
34 |
49 |
No (Mp) |
625 |
18 |
19 |
No (Mp) |
207 |
119 |
No (Sp/Mp) |
38 |
41 |
No (Mp) |
1250 |
19 |
22 |
No (Sp) |
215 |
119 |
No (Sp/Mp) |
30 |
38 |
No (Sp) |
2500 |
14 |
29 |
No (Sp) |
271 |
109 |
No (Sp) |
16 |
37 |
No (Sp) |
5000 |
U |
15 |
No (Sp) |
U |
88 |
No (Sp) |
U |
37 |
No (Sp) |
Positive control |
140 |
1090 |
No |
706 |
438 |
No |
1347 |
309 |
No |
*solvent control with DMSO
Mp : Moderate precipitate
Sp : Strong precipitate
U: unreadable
MA : metabolic activation
Table 3: Second experiment (direct plate incorporation without S9 mix and preincubation with S9 mix) -Mean revertant colony counts
|
TA 1535 |
TA 1537 |
TA 98 |
||||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
15 |
13 |
No |
4 |
7 |
No |
30 |
25 |
No |
156.3 |
14 |
13 |
No (Mp) |
8 |
5 |
No (Mp) |
25 |
23 |
No (Mp) |
312.5 |
12 |
13 |
No (Mp) |
6 |
9 |
No (Mp) |
22 |
20 |
No (Mp) |
625 |
11 |
10 |
No (Mp) |
7 |
7 |
No (Mp) |
23 |
23 |
No (Mp) |
1250 |
15 |
9 |
No (Sp) |
3 |
6 |
No (Sp) |
23 |
15 |
No (Sp) |
2500 |
10 |
4 |
No (Sp) |
2 |
3 |
No (Sp) |
18 |
16 |
No (Sp) |
Positive control |
577 |
184 |
No |
351 |
126 |
No |
192 |
1144 |
No |
|
TA 100 |
TA 102 |
WP2 uvrA |
||||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
99 |
138 |
No |
393 |
520 |
No |
27 |
24 |
No |
156.3 |
113 |
151 |
No (Mp) |
412 |
426 |
No (Mp) |
29 |
30 |
No (Mp) |
312.5 |
125 |
98 |
No (Mp) |
525 |
419 |
No (Mp) |
27 |
24 |
No (Mp) |
625 |
134 |
143 |
No (Mp) |
521 |
581 |
No (Mp) |
28 |
28 |
No (Mp) |
1250 |
98 |
104 |
No (Sp) |
486 |
562 |
No (Sp) |
27 |
27 |
No (Sp) |
2500 |
65 |
59 |
No (Sp) |
497 |
658 |
No (Sp) |
25 |
24 |
No (Sp) |
Positive control |
594 |
493 |
No |
2953 |
1660 |
No |
806 |
213 |
No |
*solvent control with DMSO
Mp : Moderate precipitate
Sp : Strong precipitate
U: unreadable
MA : metabolic activation
Table 1 Experiment 1
Exposure Time (hours) |
Dose (µg/mL) |
Replicate |
Day 7 Mutant |
|||
MF |
MFS 10-6 |
SD |
Group MFS 10-6 |
|||
4 (-S9) |
0 |
A |
13 |
12.9 |
1.32 |
9 |
B |
5 |
5.4 |
||||
26.29 |
A |
15 |
14.4 |
2.00 |
14 |
|
B |
12 |
13.8 |
||||
52.58 C |
A |
21 |
23.6 |
1.76 |
17 |
|
B |
9 |
10.3 |
||||
105.16 CP |
A |
16 |
18.3 |
1.48 |
21 |
|
B |
22 |
23.5 |
||||
210.31 CP |
A |
13 |
13.3 |
1.41 |
15 |
|
B |
17 |
17.6 |
||||
420.63 CP |
A |
22 |
22.6 |
2.26 |
22 |
|
B |
18 |
21.1 |
||||
841.25 CP |
A |
26 |
26.0 |
1.97 |
34 |
|
B |
35 |
41.8 |
||||
1261.88 CP |
A |
21 |
31.2 |
1.93 |
27 |
|
B |
17 |
22.0 |
||||
1682.5 CP |
A |
26 |
40.2 |
1.81 |
29 |
|
B |
9 |
18.4 |
||||
EMS 500 |
A |
129 |
178.3 |
5.48 |
170 |
|
B |
136 |
161.6 |
||||
EMS 750 |
A |
157 |
259.5 |
4.04 |
225 |
|
B |
154 |
191.3 |
||||
4 (+S9) |
0 |
A |
16 |
25.3 |
1.57 |
18 |
B |
7 |
9.9 |
||||
105.16 |
A |
9 |
13.5 |
1.55 |
13 |
|
B |
9 |
12.6 |
||||
210.31 CP |
A |
13 |
19.7 |
1.08 |
19 |
|
B |
12 |
17.4 |
||||
420.63 CP |
A |
11 |
20.8 |
0.99 |
18 |
|
B |
10 |
14.6 |
||||
841.25 CP |
A |
8 |
13.6 |
0.95 |
11 |
|
B |
5 |
8.3 |
||||
1682.5 CP |
A |
11 |
18.4 |
1.29 |
16 |
|
B |
8 |
12.6 |
||||
3365 CP |
A |
10 |
18.3 |
1.42 |
19 |
|
B |
13 |
19.5 |
||||
DMBA 0.5 |
A |
99 |
167.3 |
2.72 |
156 |
|
B |
86 |
144.1 |
||||
DMBA 1 |
A |
101 |
229.5 |
5.31 |
198 |
|
B |
89 |
166.9 |
||||
C = Cloudy precipitate
P = Precipitate
MF = Mutant frequency
MFS = Mutant frequency per survivor
SD = Standard deviation
EMS = Ethyl methane sulphonate
DMBA = Dimethyl benzanthracene
Table 2 Experiment 2
Exposure Time (hours) |
Dose (µg/mL) |
Replicate |
Day 7 Mutant |
|||
MF |
MFS 10-6 |
SD |
Group MFS 10-6 |
|||
24 (-S9) |
0 |
A |
12 |
15.4 |
1.32 |
20 |
B |
20 |
25.4 |
||||
1.64 |
A |
30 |
51.6 |
1.57 |
41 |
|
B |
23 |
29.4 |
||||
3.29 |
A |
28 |
38.6 |
1.49 |
37 |
|
B |
25 |
34.6 |
||||
6.57 |
A |
22 |
34.2 |
0.70 |
32 |
|
B |
24 |
30.3 |
||||
13.14 |
A |
20 |
35.7 |
1.34 |
36 |
|
B |
27 |
36.8 |
||||
26.29 CP |
A |
24 |
37.3 |
1.64 |
41 |
|
B |
33 |
45.1 |
||||
52.58 CP |
A |
9 |
12.4 |
2.53 |
29 |
|
B |
29 |
45.0 |
||||
105.16 CP |
A |
39 |
60.6 |
1.55 |
52 |
|
B |
29 |
44.2 |
||||
210.31 CP |
A |
13 |
433.3 |
1.39 |
230 |
|
B |
21 |
27.6 |
||||
EMS 200 |
A |
195 |
313.7 |
7.35 |
349 |
|
B |
223 |
383.4 |
||||
EMS 300 |
A |
296 |
574.8 |
13.03 |
471 |
|
B |
185 |
566.3 |
||||
4 (+S9) |
0 |
A |
11 |
14.5 |
1.42 |
19 |
B |
16 |
23.4 |
||||
210.31 CP |
A |
21 |
30.5 |
1.69 |
35 |
|
B |
31 |
39.4 |
||||
420.63 CP |
A |
23 |
34.5 |
1.34 |
29 |
|
B |
20 |
22.8 |
||||
841.25 CP |
A |
25 |
32.7 |
2.38 |
32 |
|
B |
24 |
30.4 |
||||
1682.5 CP |
A |
28 |
53.8 |
1.52 |
41 |
|
B |
21 |
28.7 |
||||
2523.75 CP |
A |
24 |
32.0 |
1.89 |
23 |
|
B |
9 |
13.1 |
||||
3365 CP |
A |
39 |
62.6 |
2.71 |
48 |
|
B |
24 |
33.0 |
||||
DMBA 0.5 |
A |
213 |
297.2 |
7.92 |
317 |
|
B |
210 |
336.9 |
||||
DMBA 1 |
A |
195 |
400.7 |
5.25 |
395 |
|
B |
188 |
389.0 |
||||
CP = Cloudy precipitate
MF = Mutant frequency
MFS = Mutant frequency per survivor
EMS = Ethyl methane sulphonate
DMBA = Dimethyl benzanthracene
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In a mouse micronucleus study conducted in accordance with OECD method 474 the substance did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells.
The results of a second in vivo study conducted in a method
equivalent to OECD Guideline 475 (Mammalian Bone Marrow Chromosome
Aberration Test) were disregarded on the basis that this study used an
unsuitable method of preparing the test material. The test material was
dissolved in concentrated hydrochloric acid before administration. This
would result in the conversion of the oxide form of the metal to the
chloride. As such, the result obtained in the study is not
representative of the substance to be registered.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23 September 2008 - 16 March 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- The CPA (positive control) was prepared in water instead of distilled water.
- Principles of method if other than guideline:
- There was a minor deviation from the study protocol which was not considered to have compromised the validity or integrity of the study in any way.
-The CPA (positive control) was prepared in water instead of distilled water. - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: Swiss Ico: OF1 (IOPS Caw)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: on the day of treatment, the animals were approximately 6 weeks old.
- Weight at study initiation: at the beginning of treatment the mean bogy weight was 31.5 g for males (ranging from 29.3 to 34.8 g) and 25.7 g for females (ranging from 23.5 to 28.2 g).
- Assigned to test groups randomly: yes, Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex. Subsequently, each group was assigned to a different treatment group. Identification: individual tail marking upon treatment.
- Fasting period before study: none
- Housing: The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust.
- Diet (e.g. ad libitum): All animals had free access to a pelleted maintenance diet. Each batch of food is analysed by the supplier for composition and contaminant levels.
- Water (e.g. ad libitum): Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum. Bacteriological and chemical analysis of water are performed regularly by external laboratories, These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines).
- Acclimation period: at least 5 days before the day of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C (During the main study the temperature in the animal room was sometimes slightly outside of this range (down to 19 °C).
- Humidity (%): 30 to 70 %,
- Air changes (per hr): at least 12 cycles/hour of filtered non-recycled fresh air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h (07:00 - 19:00)
IN-LIFE DATES: From: 4 NOVEMBER 2008 To: 06 NOVEMBER 2008 - Route of administration:
- intraperitoneal
- Vehicle:
- Name : 0.9 % NaCl,
Batch Nos.: WGP092/6 and WGP092/7,
Supplier: Laboratoire Fresenius Kabi, Sèvres, France.
Route and frequency of administration: intraperitoneal / two treatments separated by 24 hours
Volume of administration: 10 mL/kg - Details on exposure:
- A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.
In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice were given intraperitoneal administrations of the test material at dose-levels of 187.5, 375 and 750 mg/kg/day and 500, 1000 and 2000 mg/kg/day for males and females respectively, over a 2-day period.
One group of five males and five females received the vehicle (NaCl 0.9 %) under the same experimental conditions, and acted as control group.
One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.
The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. - Duration of treatment / exposure:
- 48 hours
- Frequency of treatment:
- two treatments separated by 24 hours
- Post exposure period:
- none
- Remarks:
- Doses / Concentrations:
187.5, 375 and 750 mg/kg/day and 500, 1000 and 2000 mg/kg/day for males and females respectively
Basis:
nominal conc. - No. of animals per sex per dose:
- Preliminary toxicity test: 9 male and 12 female mice were used.
Main cytogenetic test: 56 mice, 28 males and 28 females were used, i.e. 5 animals per sex per dose level.
Blood sampling: 6 mice; 3 males at dose-level 750 mg/kg/day and 3 females at dose-level 2000 mg/kg/day were used. - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- The positive control was Cyclophosphamide (CPA, CAS No. 50-18-0, Sigma, Saint-Quentin-Fallavier, France), batch No. 076K1050 dissolved in distilled water at a concentration of 50 mg/kg. The preparation was stored at -20 °C and thawed immediately before use.
Route and frequency of administration: oral / one treatment
Volume of administration: 10 mL/kg - Tissues and cell types examined:
- Erythrocytes in the bone marrow of mice
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.
In order to select the top dose-level for the cytogenetic study, 500, 750 and 1000 mg/kg were administered twice, 24 hours apart, to three males and 500, 1000, 1500 and 2000 mg/kg were administered twice, 24 hours apart, to three females.
The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines:
-since toxic effects were observed, the choice of the top dose-level was based on the level of toxicity, such that a higher dose-level was expected to induce lethality for males;
-since no severe toxic effects were observed, the top dose-level was 2000 mg/kg/day for females.
TREATMENT AND SAMPLING TIMES:
In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice were given intraperitoneal administrations of the test material at dose-levels of 187.5, 375 and 750 mg/kg/day and 500, 100 and 2000 mg/kg/day for males and females respectively, over a 2-day period.
One group of five males and five females received the vehicle (NaCl 0.9 %) under the same experimental conditions, and acted as control group.
One group of five males and five females received the positive control test material (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.
The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.
Blood samples for the determination of plasma levels of the test material were taken at the following time: 1 hour (three males and three females) following the second treatment with the high dose level.
DETAILS OF SLIDE PREPARATION:
At the time of sacrifice, all the animals were deeply anaesthetised by an intraperitoneal injection of sodium pentobarbital, then killed by exsanguination. The femurs were removed and the bone marrow was flushed out with foetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were re-suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa.
The slides were coded so that the scorer was unaware of the treatment group of the slide under evaluation ("blind" scoring).
METHOD OF ANALYSIS: For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
The analysis of the slides was performed at Microptic, cytogenetic services, 2 Langland Close Mumbles, Swansea SA3 4LY, UK, in compliance with GLP.
OTHER:
Blood samples for the determination of plasma levels of the test material were taken at the following time: 1 hour (three males and three females) following the second treatment with the high dose level.
Venous blood (approximately 1 mL) was taken into a tube containing lithium heparinate from the orbital sinus of the animals under light isoflurane anaesthesia.
After the blood sampling, the animals were deeply anaesthetised by an intraperitoneal injection of sodium pentobarbital, then killed by exsanguination and discarded without necropsy.
The blood was centrifuged (10 min at 4000 rpm, at +4 °C) and the plasma was kept frozen in individual tubes at -20 °C. - Evaluation criteria:
- For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
- Statistics:
- Normality and homogeneity of variances has been tested using a Kolmogorov Smirnov test and a Bartlett test.
If normality and homogeneity of variances were demonstrated, the statistical comparisons was performed using a Student t-test (two groups) or a one-way analysis of variance (> or = three groups) followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (> or = three groups) was performed followed by a Dunn test (if necessary).
All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05 for all tests. - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- A decrease in the PE/NE ratios was observed in males and females at all tested dose-levels compared to the vehicle control.
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
At 500 mg/kg/day (three males and three females), neither mortality nor clinical signs were noted in any animals.
At 750 mg/kg/day (three males), no mortality occurred, one out of the three males showed piloerection.
At 1000 mg/kg/day (three males and three females), one out of three males was found dead 24 hours after the second treatment. Pilorection, hypoactivity and half-closed eyes were noted prior to its death. Surviving animals (including females) showed piloerection and hypoactivity.
At 1500 mg/kg/day (three females), no mortality occurred, one out of three females showed piloerection.
At 2000 mg/kg/day (three females), no mortality occurred, all females showed piloerection, together with half-closed eyes for one animal.
No change in the body temperature which could be considered as excessive (increase of at least 1 °C or decrease of at least 3 °C for 5 or more hours) was recorded in the animals following either treatment, at any tested dose-levels.
RESULTS OF DEFINITIVE STUDY
No clinical signs and no mortality were observed in males given 187.5 mg/kg/day and in females given, 500, 1000 and 2000 mg/kg/day.
At 375 mg/kg/day (five males) and 750 mg/kg/day (eight males), no mortality occurred, but piloerection was observed following the second treatment in 2/5 males and 7/8 males, respectively.
The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with historical data. Cyclophosphamide induced significant increases (p<0.05 in males and p<0.01 in females) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered as valid.
The mean MPE values as well as the PE/NE ratios in the test material treated groups were similar to those of the vehicle group.
A decrease in the PE/NE ratios was observed at all tested dose-levels compared to the vehicle control. In females, this decrease was statistically significant (up to p<0.01), showing that the bone marrow cells were effectively exposed to the test material.
The determination of the plasma levels of the test material showed that at least 1/3 males and 2/3 females were exposed to the test material, since the concentrations in the samples taken 1 hour following the second treatment were 4.84, 12.1 and 14.7 ng/g, respectively.
Taking into account the clinical signs observed in some animals from the high-dose group, the test material concentrations found in the plasma samples and finally the decrease in the PE/NE ratios, the systemic exposure of the animals as well as the exposure of the bone marrow cells to the test material were clearly demonstrated. - Conclusions:
- Interpretation of results: Negative
Under the experimental conditions of this study, the test material did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 187.5, 375 and 750 mg/kg/day in males or at the dose-levels of 500, 1000 and 2000 mg/kg/day in females. - Executive summary:
The potential of the test material to induce structural or numerical damage in bone marrow cells of Swiss Ico: OF1 (IOPS Caw) mice was evaluated in a study performed according to the standardised guidelines OECD 474 and EU Method B.12.
A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female mice were administered the test material via the intraperitoneal route at dose-levels of 187.5, 375 and 750 mg/kg/day and 500, 100 and 2000 mg/kg/day for males and females respectively, over a 2-day period.
Three additional male and female mice were added in the high dose groups in order to determine the plasma level of the test material. These animals were killed 1 hour after the last treatment and their blood collected, centrifuged and frozen until determination of the test material level in the plasma.
An additional group received the vehicle under the same experimental conditions and acted as control and another group received a positive control test material (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
No clinical signs and no mortality were observed in males given 187.5 mg/kg/day and in females given 500, 1000 and 2000 mg/kg/day. At 375 and 750 mg/kg/day (male groups), no mortality occurred, but piloerection was observed in some animals following the second treatment. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with historical data. The study was therefore considered valid.
The mean MPE values as well as the PE/NE ratios in the test material treated groups were similar to those of the vehicle group indicating no effect of the test material.
A decrease in the PE/NE ratios was observed at all tested dose-levels compared to the vehicle control. In females, this decrease was statistically significant (up to p<0.01), showing that the bone marrow cells were effectively exposed to the test material.
The determination of the plasma levels of the test material showed that at least 1/3 males and 2/3 females were exposed to the test material, since the concentrations in the samples taken 1 hour following the second treatment were 4.84, 12.1 and 14.7 ng/g, respectively.
Taking into account the clinical signs observed in some animals from the high-dose group, the test material concentrations found in the plasma samples and finally the decrease in the PE/NE ratios, the systemic exposure of the animals, as well as the exposure of the bone marrow cells to the test material, were clearly demonstrated.
Under the conditions of this experiment, the test material did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- disregarded due to major methodological deficiencies
- Study period:
- Not reported
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- The study was disregarded due to the use of an unsuitable method of preparing the test material. The test material was dissolved in concentrated hydrochloric acid before administration. This would result in the conversion of the oxide form of the metal to the chloride. As such, the result obtained in the study is not representative of the test material.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
- Principles of method if other than guideline:
- The study was not conducted in accordance with a guideline, however the method followed was broadly similar to the principles of OECD 475.
- GLP compliance:
- not specified
- Type of assay:
- chromosome aberration assay
- Species:
- mouse
- Strain:
- Swiss
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 25 - 30 g - Route of administration:
- intraperitoneal
- Vehicle:
- Hydrochloric acid.
A primary stock solution of the test material was prepared by dissolving 1 g of the powder in 1 mL of concentrated HCl. The solution was then made up to its final volume of 30 mL by adding 29 mL of distilled water. - Frequency of treatment:
- Animals were injected with a single intraperitoneal dose.
- Post exposure period:
- Cells were analysed 6, 12 and 24 hours after treatment.
- Remarks:
- Doses / Concentrations:
5.30, 8.60, 17.20 and 43.0 mg/100 g bw
Basis:
no data - No. of animals per sex per dose:
- 5 animals (3 males and 2 females) were used for each dose at each sampling time.
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- 0.15 mg/100 g bw of mitomycin C was used.
- Tissues and cell types examined:
- Bone marrow from the femur.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: The selection of the maximum dose was based upon the depression in mitotic index (not more than 50 %) in the bone marrow cells, measured at the same sampling times as those used for chromosomal aberration assay.
TREATMENT AND SAMPLING TIMES: 2 hours before termination, each animal received a freshly prepared colchicine solution at the rate of 4 mg/kg bw through i.p. injection.
The animals were killed by cervical dislocation at 6, 12 and 24 hours after treatment. Bone marrow from both femurs was collected by flushing with 0.075 M potassium chloride (pre-warmed at 37 °C), incubated at 37 °C for 25 min, centrifuged and fixed in cold fixative (3:1 ethanol:glacial acetic acid).
DETAILS OF SLIDE PREPARATION: Samples were washed twice in fixative, dropped on clean chilled slides, flame dried and stained in buffered Giemsa.
METHOD OF ANALYSIS: The slides were coded. 100 metaphases/animal (500 metaphases/dose) were screened for the presence of chromosome aberrations. The number and type of aberrations were scored. - Evaluation criteria:
- Data recorded were evaluated as the number of chromosome aberrations per cell (excluding gaps, stickiness and pulverisations) and as the percent of aberrant cells (excluding gaps).
Chromatid and isochromatid gaps, chromatid and chromosome breaks, chromatid and chromosome exchanges, ring chromosomes, centric fusions, stickiness and pulverisation were recorded as chromosomal abnormalities. - Statistics:
- For statistical analysis, the one tailed trend test (Margolin et al., 1986) and the two-way ANOVA (Harter, 1960) were employed.
- Sex:
- male/female
- Genotoxicity:
- positive
- Toxicity:
- not specified
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- Table 1 shows the data on the induction of chromosome aberrations.
A single exposure to different doses significantly enhanced the frequency of chromosome aberrations at metaphase. The number of aberrations per cell and percent aberrant cells increased, when compared to the negative control, with an increase in dose. The frequencies also increased with the lapse of time, attaining a maximal at the 12 hour sampling time.
The decrease in the percentage of aberrant cells after 24 hours suggests reduction in the survival rate of the affected cells and their subsequent elimination.
The number of chromosome aberrations per cell and percent aberrant cells, as shown by a one-tailed trend test, showed a positive trend with the dose.
Two-way ANOVA (Table 2) showed significant differences between the treatments with regard to percent aberrant cells and chromosome aberrations cell.
Aberrations recorded were chromatid and chromosome breaks, gaps, exchanges, centric fusion, ring chromosomes, stickiness and pulverisations. Chromatid and chromosome breaks were present in higher frequencies than the other types and were induced by all doses. The frequencies of chromatid breaks were higher at the 6 hour sampling time whereas chromosome breaks were present in higher frequencies at the 12 hour sampling time. All other types were recorded only after treatment with the higher doses. - Conclusions:
- Interpretation of results: Positive
The results of this study demonstrate that the test material is clastogenic in vivo in the mouse. - Executive summary:
The potential of the test material to cause chromosome aberrations in bone marrow cells was investigated in an in vivo study in the Swiss albino mouse. No guideline was followed, however the method used was broadly similar to the OECD 475 guideline.
A stock solution was formed by dissolving 1 g of the test material in 1 mL of concentrated hydrochloric acid. This was then made to volume (30 mL) with 29 mL of distilled water.
Animals were injected with a single intraperitoneal dose at dose levels of 5.30, 8.60, 17.20 and 43.0 mg/100 g bw. Cells were analysed 6, 12 and 24 hours after treatment. Mitomycin C was used as the positive control.
A single exposure to different doses significantly enhanced the frequency of chromosome aberrations at metaphase. The number of aberrations per cell and percent aberrant cells increased, when compared to the negative control, with an increase in dose.
Aberrations recorded were chromatid and chromosome breaks, gaps, exchanges, centric fusion, ring chromosomes, stickiness and pulverisations. Chromatid and chromosome breaks were present in higher frequencies than the other types and were induced by all doses.
The results of this study demonstrate that the test material is clastogenic in vivo in the mouse. However, this study used an unsuitable method of preparing the test material. The test material was dissolved in concentrated hydrochloric acid before administration. This would result in the conversion of the oxide form of the metal to the chloride. As such, the result obtained in the study is not representative of the substance to be registered.
Referenceopen allclose all
Results of the cytogenetic test :
|
Group |
Doses
(mg/kg/day) |
MPE/1000PE |
PE/NE ratio |
Time of sacrifice after the last administration |
||
mean |
(sd) |
mean |
(sd) |
||||
Males |
|
|
|
|
|
|
24 h |
|
Vehicle |
- |
1.7 |
(0.9) |
0.8 |
(0.2) |
|
|
|
187.5 |
1.9 |
(1.1) |
0.4 |
(0.2) |
|
|
Test material |
375 |
1.0 |
(0.9) |
0.4 |
(0.1) |
|
|
|
750 |
1.7 |
(1.0) |
0.3 |
(0.1)* |
|
|
Cyclophosphamide |
50 |
25.1 |
(5.5)* |
1.0 |
(0.1) |
|
Females |
|
|
|
|
|
|
24 h |
|
Vehicle |
- |
1.2 |
(0.4) |
0.7 |
(0.1) |
|
|
|
500 |
0.6 |
(0.7) |
0.4 |
(0.1) |
|
|
Test material |
1000 |
1.2 |
(0.7) |
0.3 |
(0.2)** |
|
|
|
2000 |
0.9 |
(1.2) |
0.4 |
(0.2)* |
|
|
Cyclophosphamide |
50 |
17.5 |
(4.4)** |
0.7 |
(0.2) |
|
Five animals per group
MPE : Micronucleated Polychromatic Erythrocytes
PE: Polychromatic Erythrocytes
NE: Normochromatic Erythrocytes
Sd: standard deviation
Statistical significance:
* p <0.05
** p <0.01
Table 1: Chromosomal Aberrations Induced - 500 metaphases analysed per dose (100 per animal)
Sampling Time (h) |
Treatment |
Dose (mg/100 g bw) |
Gap* |
Total Chromosome Aberrations |
Chromosome Aberrations per Cell† |
% Abnormal Cells‡ |
|||||
CtB |
ChB |
CF |
RC |
Ex |
St + Pl |
||||||
6 |
Solvent Mitomycin C Test material |
- 0.15 5.30 8.60 17.20 43.00 |
6 58 10 12 14 18 |
5 28 6 8 13 15 |
- 27 4 4 10 10 |
- 6 - - - 2 |
- 18 - - 2 4 |
- 16 - - - 3 |
- - - - - - |
0.010 0.190 0.02 0.024 0.050 0.068 |
1.00 16.20 1.20 1.8 2.80 4.60 |
Trend test p<0.001 |
|||||||||||
12 |
Solvent Mitomycin C Test material |
- 0.15 5.30 8.60 17.20 43.00 |
7 65 10 15 16 22 |
7 40 8 9 10 12 |
- 55 12 14 19 18 |
- 8 - - - 2 |
- 22 - - 4 5 |
- 22 - - - 4 |
- - - - - 4 |
0.014 0.294 0.040 0.046 0.066 0.082 |
1.40 21.40 2.00 3.00 4.00 5.60 |
Trend test p<0.001 |
|||||||||||
24 |
Solvent Mitomycin C Test material |
- 0.15 5.30 8.60 17.20 43.00 |
7 49 6 9 10 14 |
10 22 6 8 7 5 |
- 28 8 6 7 10 |
- 2 - - - - |
- 19 - 2 2 3 |
- 11 - - - 1 |
- - - - - 5 |
0.020 0.164 0.028 0.032 0.032 0.038 |
1.80 14.60 2.00 2.40 2.60 3.60 |
Trend test p<0.001 |
|||||||||||
*Includes both chromatid and isochromatid gap.
†Excludes gaps and stickiness and pulverisation.
‡Includes stickiness and pulverisation whenever recorded.
CtB: chromatid break
ChB: chromosome break
CF: centric fusion
RC: ring chromosome
EX: exchange
St + Pl: stickiness and pulverisation
Table 2: Two-way ANOVA of Percent Aberrant Cells and Chromosome Aberrations per Cell
Sources of Variation |
df |
Mean Squares |
F-ratio |
% Aberrant Cells Duration Concentration Error |
2 4 8 |
1.17 4.87 0.28 |
4.17 19.39** |
Chromosome Aberrations per Cell Duration Concentration Error |
2 4 8 |
0.0055 0.0102 0.0012 |
4.58* 8.50* |
*Significant at 5 % level
**Significant at 1 % level
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Ames test (CIT, 2007)
The potential of the test material to induce reverse gene mutations in Salmonella typhimurium and Escherichia coli was evaluated in a study performed according to the standardised guidelines OECD 471 and EU Method B13/14.
The test material was tested in two independent experiments, with and without a metabolic activation system (S9 mix, prepared from a liver post mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254).
Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 and one strain of Escherichia coli, WP2uvrA, were used. Each strain was exposed to 156.3 to 5000 µg/plate of the test material (three plates/dose-level). After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored. Solvent control (DMSO) and positive controls were used.
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
A moderate to marked precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 156.3 µg/plate. No toxicity was noted towards all the strains used either with or without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the six strains.
Under the conditions of the study, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli with and without metabolic activation.
CHO HPRT (Harlan, 2013)
A study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells in vitro in accordance with the standardised guidelines OECD 476, EU Method B.17, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the EPA OPPTS 870.5300.
CHO cells were treated with the test material at up to eight dose levels, in duplicate, together with negative and positive controls. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.
Four treatment conditions were used for the test. In Experiment 1, a 4 hour exposure in the absence of metabolic activation (dose levels 0, 105.16, 210.31, 420.63, 841.25, 1682.5 and 3365 µg/mL) and a 4 hour exposure in the presence of 2 % S9
(dose levels 0, 26.29, 52.58, 105.16, 210.31, 420.63, 841.25, 1261.88 and 1682.5 µg/mL). In Experiment 2, the 4 hour exposure was repeated using a 1 % final S9 concentration (dose levels 0, 210.31, 420.63, 841.25, 1682.5, 2523.75 and 3365 µg/mL), whilst in the absence of metabolic activation the exposure time was increased to 24 hours (dose levels 0, 1.64, 3.29, 6.57, 13.14, 26.29, 52.58, 105.16 and 210.31 µg/mL).
The test material demonstrated no dose related increases in mutant frequency at any dose level, with or without metabolic activation in Experiment 1.
An increase in mutant frequency of greater than 20 x 10-6was seen in the 4 hour exposure group in the presence of S9 in Experiment 2 at the maximum dose tested (3365 μg/mL) and an interim dose level of 1682.5 μg/mL. However, these responses were relatively modest, not part of any true dose-related effect and were only marginally above the highest acceptable value for a vehicle control and were therefore considered to be of no toxicological significance. In the 24 hour exposure group a similar response was seen at 105.16 μg/mL which achieved close to the limit of acceptable toxicity at Day 0. The response was again considered to be of no toxicological significance and was considered to be related to toxicity. The dose level of 210.31 μg/mL in the 24 hour exposure group demonstrated a large increase in mutant frequency but there were insufficient cells due to poor recovery from post treatment toxicity in the ‘A’ replicate to plate 5 mutant flasks and so this dose level can be excluded on the basis of toxicity. However, the response seen at this toxic dose level does indicate that the increases in mutant frequency are due to cytotoxicity rather than a mutagenic mechanism.
The negative and positive controls gave mutant frequencies within the range expected, indicating the satisfactory performance of the test and of the metabolising system.
Under the conditions of this study, the test material is considered to be non-mutagenic to CHO cells at the HPRT locus.
In vivo mouse micronucleus test (CIT, 2011)
The potential of the test material to induce structural or numerical damage in bone marrow cells of Swiss Ico: OF1 (IOPS Caw) mice was evaluated in a study performed according to the standardised guidelines OECD 474 and EU Method B.12.
A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female mice were administered the test material via the intraperitoneal route at dose-levels of 187.5, 375 and 750 mg/kg/day and 500, 100 and 2000 mg/kg/day for males and females respectively, over a 2-day period.
Three additional male and female mice were added in the high dose groups in order to determine the plasma level of the test material. These animals were killed 1 hour after the last treatment and their blood collected, centrifuged and frozen until determination of the test material level in the plasma.
An additional group received the vehicle under the same experimental conditions and acted as control and another group received a positive control test material (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
No clinical signs and no mortality were observed in males given 187.5 mg/kg/day and in females given 500, 1000 and 2000 mg/kg/day. At 375 and 750 mg/kg/day (male groups), no mortality occurred, but piloerection was observed in some animals following the second treatment. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with historical data. The study was therefore considered valid.
The mean MPE values as well as the PE/NE ratios in the test material treated groups were similar to those of the vehicle group indicating no effect of the test material.
A decrease in the PE/NE ratios was observed at all tested dose-levels compared to the vehicle control. In females, this decrease was statistically significant (up to p<0.01), showing that the bone marrow cells were effectively exposed to the test material.
The determination of the plasma levels of the test material showed that at least 1/3 males and 2/3 females were exposed to the test material, since the concentrations in the samples taken 1 hour following the second treatment were 4.84, 12.1 and 14.7 ng/g, respectively.
Taking into account the clinical signs observed in some animals from the high-dose group, the test material concentrations found in the plasma samples and finally the decrease in the PE/NE ratios, the systemic exposure of the animals, as well as the exposure of the bone marrow cells to the test material, were clearly demonstrated.
Under the conditions of this experiment, the test material did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells.
In vivo chromosome aberration test (Jha and Singh, 1995) (disregarded)
The results of a second in vivo study conducted in a method equivalent to OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test) were disregarded on the basis that this study used an unsuitable method of preparing the test material. The test material was dissolved in concentrated hydrochloric acid before administration. This would result in the conversion of the oxide form of the metal to the chloride. As such, the result obtained in the study is not representative of the substance to be registered.
Justification for classification or non-classification
The substance has given negative results in two in vitro and one in vivo key mutagenicity studies. There is therefore no requirement to classify this substance.
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