Registration Dossier
Registration Dossier
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EC number: 234-857-9 | CAS number: 12037-29-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro gene mutation in bacteria (Ames): CIT (2007): Negative with and without metabolic activation.
In vitro cytogenicity in mammalian cells: Harlan (2013): Negative with and without metabolic activation.
In vitro gene mutation in mammalian cells: Harlan (2013): Positive with and without metabolic activation.
In vitro gene mutation in mammalian cells: CiToxLAB (2013): Negative with and without metabolic activation (read-across substance).
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:
- 22 May 2007 - 17 December 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
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- S. typhimurium: Histidine locus.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Additional strain / cell type characteristics:
- other: rfa mutation; uvrB mutation (except TA102); addition of plasmid pKM101 to TA98, TA100 and TA102.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from a liver post-mitochondrial fraction of rats induced with Aroclor 1254
- Test concentrations with justification for top dose:
- 312.5, 625, 1250, 2500 and 5000 µg/plate for the three experiments, with and without S9 mix.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test material is insoluble in most vehicles - formed homogeneous suspension to the naked eye in DMSO. Suspended at the concentration of 100 mg/mL.
- volume of vehicle/solvent in the medium: 0.05 mL per 2.60 mL medium - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-Anthramine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: All experiments were performed according to the direct plate incorporation method except for the second and third test with S9 mix, which were performed according to the preincubation method.
DURATION
- Preincubation period: 60 minutes, 37 °C
- Exposure duration: 48 to 72 hours
NUMBER OF REPLICATES: three plates/dose-level
OTHER: SCORING METHOD: automated - Evaluation criteria:
- A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 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.
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 312.5 µg/plate (the precipitate did not interfere with the scoring).
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 and TA 102 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. No noteworthy toxicity was noted towards the three strains used, either with or 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. The study was therefore considered valid.
Results
In the first experiment, no noteworthy toxicity was induced in any of the five tester strains with and without S9 mix. In the second experiment with S9 mix (preincubation method) with the TA 98 strain, up to 3.3-fold increase in the number of revertants were noted, without any clear evidence of dose-relationship. A third experiment was performed with this strain under the same experimental conditions in order to check the reliability of these slight increases. No confirmation of the increase in the number of revertants was observed in this third experiment. The test material did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the remaining tester strains. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results: negative
Under the experimental conditions, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium. - Executive summary:
The objective of this study was to evaluate the potential of the test material to induce reverse gene mutations in Salmonella typhimurium in accordance with the standardised guidelines OECD 471 and EU Method B13/14.
The test material was tested with and without a metabolic activation system (S9 mix, prepared from a liver post mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254).
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test material (three plates/dose-level) ranging from 312.5 to 5000 µg/plate. After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored. Solvent control (DMSO) and positive controls were used.
The numbers of revertants for the vehicle and positive controls were within the ranges specified in the acceptance criteria. The study was therefore considered valid.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 312.5 µg/plate. No noteworthy toxicity was induced in any of the five tester strains.
The test item did not induce any noteworthy increase in the number of revertants which could be considered as relevant, either with or without S9 mix, in any of the five tester strains.
Under these experimental conditions, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
- 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:
- 12 September 2012 - 14 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 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- 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 knowingly 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 the testing laboratory has been determined to be approximately 16 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 10 % foetal bovine serum (FBS), at approximately 37 °C with 5 % CO₂ in humidified air.
The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA). - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver homogenate metabolising system (S9)
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test:
0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL in both the absence and presence of metabolic activation.
Experiments 1 and 2:
0, 5, 10, 20, 40, 80 and 160 µg/mL in both the absence and presence of metabolic activation. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Eagle's minimal essential medium with HEPES buffer (MEM) at 50 mg/mL
- Preparation of test material in vehicle
The test material was accurately weighed, suspended in MEM and serial dilutions prepared.
There was no significant change in pH when the test item was dosed into MEM and the osmolality did not increase by more than 50 mOsm.
The test material was formulated within two hours of it being applied to the test system. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- MEM
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- CULTURE CONDITIONS
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.94 - 9.15 mL MEM, 10 % (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.65 - 0.75 mL heparinised whole blood
WITH METABOLIC ACTIVATION (S9) TREATMENT
After approximately 48 hours incubation at approximately 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 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 the 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 approximately 37 °C, 5 % CO₂ in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 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 hours at approximately 37 °C in 5 % CO₂ in humidified air.
WITHOUT METABOLIC ACTIVATION (S9) TREATMENT
In Experiment 1, after approximately 48 hours incubation at approximately 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 1 mL of the appropriate vehicle control, test material solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 °C, 5 % CO₂ in humidified air the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 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 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1 mL of vehicle control, test material dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 °C, 5 % CO₂ in humidified air for 24 hours.
Experiment 1
There were 2 exposure conditions conducted for Experiment 1:
i) 4 hour exposure to the test material without S9-mix followed by 20 hour culture in treatment-free media prior to cell harvest.
ii) 4 hour exposure to the test material with S9-mix (2 %) followed by 20 hour culture in treatment-free media prior to cell harvest.
Experiment 2
There were 2 exposure conditions conducted for Experiment 2:
i) 24 hour continuous exposure to the test material without S9-mix prior to cell harvest.
ii) 4 hour exposure to the test material with S9-mix (1 %) 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 fourteen 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 approximately 4 °C to ensure complete fixation.
SLIDE PREPARATION
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. When the slides were dry, they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
QUALITATIVE SLIDE ASSESSMENT
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose level for mitotic index evaluation.
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
Where possible, the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50 % of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44 - 48 chromosomes, any gaps, breaks or rearrangements were noted. In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. - Evaluation criteria:
- A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
- Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
- Key result
- Species / strain:
- lymphocytes: Human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY TOXICITY TEST
Precipitate observations were made from the parallel blood-free cultures at the end of the exposure. In the 4(20) hour exposure group in the absence of S9, precipitate was observed at and above 78.13 µg/mL with a black colour observed at and above 312.5 µg/mL. In the 4(20)-hour exposure group in the presence of S9, precipitate was observed at all dose levels tested with a black colour observed at and above 625 µg/mL.
However, due to the fine suspension of the test material, precipitate persisted on the slides at and above 78.13 µg/mL which became obscuring at and above 625 and 1250 µg/mL, with and without S9, respectively. In the 24 hour continuous exposure group, precipitate was observed at and above 39.06 µg/mL with a black colour observed at and above 312.5 µg/mL. Again, due to the fine suspension of the test material, precipitate persisted on the slides at and above 78.13 µg/mL which became obscuring at and above 625 µg/mL. Haemolysis was observed at 5000 µg/mL in the 24 hour exposure group only. In this instance, haemolysis is thought to be the result of disruption of the membranes of erythrocytes and not an indicator of toxicity to lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 µg/mL in all exposure groups but that excessive precipitate was obscuring the metaphases. There was no marked reduction in the Mitotic Index in any exposure group.
The selection of the maximum dose level for the main test was based on precipitate rather than the onset of toxicity in all exposure groups tested because it had persisted on to the slides at the end of exposure.
CHROMOSOME ABERRATION TEST - EXPERIMENT 1
The qualitative assessment of the slides determined that there was no toxicity present and that there were metaphases suitable for scoring present at the maximum test material dose level tested, 160 µg/mL, in both exposure groups. Precipitate observations were made at the end of exposure and precipitate in the pellet was noted at all dose levels tested in both exposure groups. Also, the precipitate persisted after exposure on the slides at and above 40 µg/mL. This difference is due to the fact that the test material was a suspension in MEM and even the lower dose levels would have suspended test material present which persisted on to the slides.
The mitotic index summary data are given in Table 1. These data show there was no dose-related reduction in Mitotic Index either with or without S9.
The maximum dose level selected for metaphase analysis was, therefore, 160 µg/mL in both the presence and absence of S9.
A summary of the chromosome aberration data is given in Table 2. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations, indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the exposure groups dosed in the presence or absence of S9, which included at least one precipitating dose level.
A summary of the polyploid cell frequency data is also given in Table 2. The test material did not induce a statistically significant increase in the numbers of polyploid 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 metaphases suitable for scoring present at the maximum test material dose level of 160 µg/mL in the absence and presence of S9. Precipitate observations were made at the end of exposure and precipitate was noted at all dose levels tested in the 24 hour exposure but only at and above 10 µg/mL in the 4(20) hour exposure group. Also, the precipitate persisted after exposure on the slides at and above 40 µg/mL and 80 µg/mL in the absence and presence of S9, respectively. This difference is due to the fact that the test material was a suspension in MEM and even the lower dose levels would have suspended test material present which persisted on to the slides.
The mitotic index data are given in Table 1. They confirm the qualitative observations in that no inhibition of mitotic index was observed.
The maximum dose level selected for metaphase analysis was, therefore, 160 µg/mL both in the absence and presence of S9.
A summary of the chromosome aberration data is given in Table 3. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the exposure groups dosed in the presence or absence of S9, which included at least one precipitating dose level.
A summary of the polyploid cell frequency data is also given in Table 3. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results: negative
Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic. - Executive summary:
The potential of the test material to induce chromosomal aberrations was investigated in vitro in accordance with the standardised guidelines OECD 473 and EU Method B.10.
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. The dose levels used in all the experiments were 0, 5, 10, 20, 40, 80 and 160 µg/mL.
Four treatment conditions were used for the study:
In Experiment 1, cells were exposed for 4 hours in the presence of an induced rat liver homogenate metabolising system (S9 at a 2 % final concentration) with cell harvest after a 20 hour expression period and a 4 hour exposure in the absence of metabolic activation with a 20 hour expression period.
In Experiment 2, the 4 hours exposure period 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.
All vehicle 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 that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the exposure groups dosed in the presence or absence of S9, which included at least one precipitating dose level.
Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.
- 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 - 29 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
- 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
- Type and identity of media: Hams F12 growth medium, supplemented with 5% foetal bovine serum (FBS) and antibiotics (Penicillin/Streptomycin at 100 units/100 μg per mL).
- Properly maintained: yes. 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.
- Periodically "cleansed" against high spontaneous background: yes. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Experiment 1:
39.06, 78.13, 156.3, 312.5, 625, 1250, 2500, 5000 µg/mL (without S9-mix)
156.25, 312.5, 625, 1250, 2500, 5000 (with 2 % S9-mix)
Experiment 2:
78.13, 156.25, 312.5, 625, 1250, 1875, 2500, 3750 (without S9-mix)
312.5, 625, 1250, 2500, 3750, 5000 (with 1 % S9-mix) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Hams F12 culture medium.
- 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 dissolved in DMSO and dosed at 1%.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in serum free medium (Ham F12).
DURATION
- Exposure duration:
> Experiment 1: 4 hours with and without 2 % S9-mix.
> Experiment 2: 4 hours with and without 1 % S9 mix.
- Expression time: 7 days.
- Selection time: 14 days.
SELECTION AGENT: 10 µg/mL 6-thioguanine (6-TG).
STAIN: Cultures were fixed with methanol and then stained with 10 % Giemsa solution for 5 minutes.
INCUBATION CONDITONS: In culture medium at 37 °C in an incubator with a humidified atmosphere of 5 % CO2 in air.
NUMBER OF REPLICATIONS: Duplicate cultures were prepared for all exposure groups and controls.
NUMBER OF CELLS EVALUATED:
Cloning efficiency: 200 cells plated per flask.
Mutation frequency: 2 x 10^5 cells plated per flask.
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency.
PRELIMINARY CYTOTOXICITY TEST
- Dose level: 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL.
- Test systems: 4 hour exposure (6-7 hour expression time) with and without metabolic activation (S9-mix), and 24 hour exposure (6-7 hour expression time) without metabolic activation.
- Evaluation: The total colony numbers were used to determine cloning efficiencies.
- The results were used for determination of the definitive test dosing range. - Statistics:
- The cloning efficiency (CE), mean plate counts (x), % control, mutant plate counts, mutant frequency/10^6 (MF) and mutant frequency/10^6 survival rate (MFSV) were calculated using the formulae given in the field “Any other information on materials and methods incl. tables”.
The calculations were performed using an spreadsheet which may result in minor variations in the calculated values when compared to manual calculations due to rounding up differences. - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: 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.
- Precipitation: A precipitate of the test material was seen at the end of exposure at all dose levels in both Experiments 1 and 2 with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES
Precipitation of the test material was observed at the end of exposure at all dose levels in all exposure groups.
Dose related toxicity seen in the 4-hour exposure group and in the 24-hour exposure group in the absence of metabolic activation (S9). There was no marked toxicity in the 4-hour exposure group in the presence of S9. Therefore it was considered justified to test beyond the onset of test material precipitate.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: A reduction in the cloning efficiency of 64% compared to the vehicle control was achieved at 1250 μg/mL in the absence of metabolic activation at Day 0. The dose levels of 2500 μg/mL and 5000 μg/mL were both too toxic for plating. At Day 7, dose related toxicity was observed with a 48% reduction in cloning efficiency compared to the vehicle control at 1250 μg/mL. This indicated residual toxicity was sustained during the expression period.
There was no marked reduction in cloning efficiency in the presence of metabolic activation.
Experiment 2: Similar to Experiment 1, dose related toxicity was demonstrated at Day 0 in the absence of metabolic activation. The dose level of 3750 μg/mL was at the limit of acceptable toxicity with 89% reduction in cloning efficiency compared to the vehicle control. The toxicity curve was seen to plateau at 1250 μg/mL, 1875 μg/mL, and 2500 μg/mL with reductions in cloning efficiency of 69%, 74% and 76% respectively compared to the vehicle control. At Day 7 a modest reduction in cloning efficiency was observed, 21% at 3750 μg/mL compared to the vehicle control.
There was no marked reduction in cloning efficiency in the presence of metabolic activation.
MUTATION FREQUENCY:
Increases in mutant frequency of greater than 20 x 10^-6 were seen in the 4-hour exposure group in the absence of S9 in Experiment 1 between 78.13 μg/mL and 1250 μg/mL. These increases in mutant frequency generally increased with increasing dose although there was a small decrease at 625 μg/mL. The Experiment 2 repeat of the 4-hour exposure in the absence of S9 also demonstrated increases in the mutant frequency greater than 20 x 10^-6 over the vehicle control and although the responses were not as great as those seen in Experiment 1 they were considered to be sufficient to confirm the positive result seen in Experiment 1 in the absence of S9.
The test material did not induce any significant or dose-related increases in mutant frequency per survivor in the presence of metabolic activation in Experiment 1 but did show a clear dose related response in Experiment 2 where a reduced S9 concentration was used. This difference is considered to be due to the S9 concentration being reduced with its protective effects in Experiment 2. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results: positive with and without metabolic activation
Increases in mutant frequency of greater than 20 x 10^-6 were seen in the 4-hour exposure group in the absence of S9 in Experiment 1 and Experiment 2. The test material did not induce any significant or dose-related increases in mutant frequency per survivor in the presence of metabolic activation (2 % S9) in Experiment 1 but did show a clear dose related response in Experiment 2 where a reduced S9 concentration (1%) was used.
Therefore under the conditions of the test, the test material was considered to be mutagenic to CHO cells at the HPRT locus in the presence and absence of metabolic activation. - Executive summary:
The genotoxic potential of the test material was assessed in an in vitro gene mutation assay with mammalian cells. The study was performed under GLP conditions and in line with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870. 5300. Chinese hamster Ovary (CHO-K1) cells were exposed to the test material in two separate experiments. Both experiments were conducted with a 4 hour exposure period and a 7 day expression period, with and without metabolic activation. In experiment 1, metabolic activation was provided by 2% S9 mix, whereas 1% was used in experiment 2. Cultures were exposed to the test material at concentrations up to 5000 µg/mL in experiment 1 ± S9 mix as well as in experiment 2 with S9, and up to 3750 µg/mL in experiment 2 without S9 mix.
Increases in mutant frequency of greater than 20 x 10-6were seen in the 4-hour exposure group in the absence of S9 in Experiment 1 and Experiment 2. The test material did not induce any significant or dose-related increases in mutant frequency per survivor in the presence of metabolic activation (2 % S9) in Experiment 1 but did show a clear dose related response in Experiment 2 where a reduced S9 concentration (1%) was used.
Significant cyctotoxic effects were observed at concentrations ≥ 1250 µg/mL in the absence of metabolic activation, producing reductions in cloning efficiency between 21 and 89%.
Therefore under the conditions of the test, the test material was considered to be mutagenic to CHO cells at the HPRT locus in the presence and absence of 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:
- supporting study
- Study period:
- 4 July 2013 to 12 August 2013
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- 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. Since the study was conducted with a reaction mass, containing the metal carbonate, rather than with the substance itself, it has been assigned a reliability score of 2 and used as supporting information.
- 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
- 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
- Type and identity of media: Hams F12-10 medium, supplemented with 10 % v/v foetal bovine serum (FBS), 0.01 mL/mL L-Glutamine and 0.01 mL/mL antibiotics (Penicillin/Streptomycin).
- Properly maintained: yes. The stocks of cells were stored in liquid nitrogen. Cells were routinely cultured in Hams F12-10 medium, at 37°C with 5% CO2 in air.
- Periodically checked for Mycoplasma contamination: yes.
- Periodically "cleansed" against high spontaneous background: yes. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Assay 1:
156.25, 312.5, 625, 1250, 2500, 5000 µg/mL (5 hour treatment with and without S9-mix)
Assay 2:
156.25, 312.5, 625, 1250, 2500, 5000 µg/mL (5 hour treatment with S9-mix)
156.25, 312.5, 625, 1250, 2500, 3750, 5000 µg/mL (24 hour treatment without S9-mix) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 1 % (w/v) methyl cellulose solution (in distilled water); DMSO was used as the vehicle in the positive controls.
- Justification for choice of solvent/vehicle: Based on the available information and the results of the performed Preliminary Solubility Test, no proper formulation was achieved at 500 mg/mL concentration using Distilled water, DMSO, Acetone or N,N-Dimethylformamide (DMF) as vehicle. However, the formulation of 500 mg/mL using 1 % (w/v) methyl cellulose solution was suitable for the test. As this vehicle is compatible with the survival of the mammalian cells and the metabolic activation system, it was selected for vehicle of the study. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 1% (w/v) methyl cellulose solution
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: 7,12-dimethylbenz[a]anthracene
- Remarks:
- Positive controls were dissolved in DMSO. EMS was used at a final concentration of 0.4 µL/mL; DMBA was used at a final concentration of 20 µg/mL. Positive control solutions were filtered sterile by using a 0.22 μm syringe filter before treatment.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- 5 hour treatments: 10^6 cells were placed in each of a series of sterile dishes (diameter approx. 10 mm) and incubated for about 24 hours before treatment at 37 °C (± 0.5 °C) in a humidified atmosphere of 5 % (± 0.3 %) CO2. On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 5-hour treatment contained 1 % (v/v) serum (F12-1, for treatment without metabolic activation) or 5 % (v/v) serum (F12-5, for treatment with metabolic activation).
A suitable volume (100 μL) of vehicle (solvent), test material solution or positive control solution was added to the 10-mL final volume. In case of experiment with metabolic activation, 1.0 mL of S9-mix was added to the cultures. After the 5-hour incubation period at 37 °C (± 0.5 °C) in a humidified atmosphere of 5 % (± 0.3 %) CO2, the cultures were washed thoroughly with F12-10 medium (culture medium). Then, dishes were covered with 10 mL fresh F12-10 medium and incubated for 19 hours at 37 °C (± 0.5 °C) in a humidified atmosphere of 5 % (± 0.3 %) CO2. After the 19-hour incubation period, cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer and a microscope. In samples where sufficient cells survived, cell number was adjusted to 2 × 10^5 cells/mL. Cells were transferred to dishes for growth through the expression period or diluted to be plated for survival.
- 24 hour treatments: 0.5 × 10^6 cells were placed in each of a series of sterile dishes (diameter approx. 10 mm) and incubated for approximately 24 hours before treatment at 37 °C (± 0.5 °C) in a humidified atmosphere of 5 % (± 0.3 %) CO2. On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 24-hour treatment contained 5 % serum (F12-5). A suitable volume (100 μL) of vehicle (solvent), test material solution or positive control solution was added to the 10 mL final volume. After the 24-hour incubation period at 37 °C (± 0.5 °C) in a humidified atmosphere of 5 % (± 0.3 %) CO2, cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer and a microscope. In samples where sufficient cells survived, cell number was adjusted to 2 × 10^5 cells/mL. Cells were transferred to dishes for growth through the expression period or diluted to be plated for survival.
DURATION
- Exposure duration:
> Assay 1: 5 hour treatment with and without S9-mix
> Assay 2: 5 hour treatment with S9-mix; 24 hours without S9-mix
- Expression time: 7 days (on day 8)
- Selection time: 7 days (on day 15)
SELECTION AGENT: 20 µg/mL 6-thioguanine (6-TG).
STAIN: Cultures were fixed with methanol and then stained with 10 % Giemsa solution for 30 minutes.
INCUBATION CONDITONS: In culture medium at 37 °C in an incubator with a humidified atmosphere of 5 % CO2 in air.
NUMBER OF REPLICATIONS: Duplicate cultures were prepared for all exposure groups and controls.
NUMBER OF CELLS EVALUATED:
Cloning efficiency: 200 cells plated per flask (3 per replicate).
Mutation frequency: 2 x 10^5 cells plated per flask (3 per replicate).
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, expressed as percentage of the total number of colonies divided by the total number of cells plated for colony growing.
PRELIMINARY CYTOTOXICITY TEST
- Dose level: 19.53 - 5000 μg/mL (single treatments)
- Test systems: 5 hour treatment in the presence and absence of S9-mix and a 24-hour treatment in the absence of S9-mix.
- Evaluation: Following treatments (as cytotoxicity was observed on Day 1), cell number in the samples was adjusted to 2 x 10^5 cells/mL after counting and cells were transferred to dishes for growth for 5 or 7 additional days (in case of short treatments and long treatment, respectively). After the incubation period, cell concentrations were determined using a haemocytometer and a microscope on Day 3, 6 and/or 8.
- The results were used for determination of the definitive test dosing range. - Evaluation criteria:
- The assay was considered valid if all the following criteria were met:
1. The mutant frequency in the negative (vehicle) control cultures was in accordance with the historical control data.
2. The positive control chemicals induced a clear increase in mutant frequency.
3. The cloning efficiency of the negative controls was between the range of 60 % to 140 % on Day 1 and 70 % to 130 % on Day 8.
4. At least four test material concentrations in duplicate cultures were presented.
The test material was considered to be mutagenic in this assay if the following criteria were met:
1. The assay was valid.
2. The mutant frequency at one or more doses was significantly greater than that of the relevant negative (vehicle) control (p < 0.05).
3. Increase of the mutant frequency was reproducible.
4. There was a dose-response relationship. - Statistics:
- The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was made. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Significant results with inter-group comparisons were further compared using Kruskal-Wallis and Mann-Whitney U-tests. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2003 software (R-squared values were calculated for the log concentration vs. the mutation frequency).
- Key result
- 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 examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There were no large changes in the pH or osmolality at any of the dose levels investigated, in either assay.
- Precipitation: Precipitation of the test material was detected in the treatment medium at the end of exposure in the 156.25 - 50000 µg/L treatment groups in both assays (with and without S9-mix).
GENOTOXICITY AND INFORMATION ON CYTOTOXICITY:
- Assay 1: In the presence of S9-mix (5-hour treatment), cytotoxicity of the test material was observed. At the end of the treatment period the relative survival value of the highest examined concentration of 5000 μg/mL was 18.7 %. No statistically significant increases or biologically relevant increases in the numerical value of the mutation frequency compared to the vehicle control were observed at any examined concentrations (a total of six concentrations were evaluated) and there was no dose response to the treatment (a trend analysis showed no effect of treatment).
In the absence of S9-mix (5-hour treatment), cytotoxicity of the test material was observed. At the end of the treatment period the relative survival value of the highest examined concentration of 5000 μg/mL was 16.6 %. All of the examined concentrations (a total of six) were evaluated and a statistically significant increase in the mutation frequency compared to the vehicle control was only observed at 5000 μg/mL. However, the observed mutation frequency value was within the historical control range; furthermore, it was not statistically significant when compared to the data of the DMSO (vehicle of the positive control) control samples. There was no dose response to the treatment (a trend analysis showed no effect of treatment). Therefore the numerically high value was considered to reflect the biological variability of the test. It is considered that there were no biologically significant effects in this assay.
- Assay 2: In the presence of S9-mix (5-hour treatment), cytotoxicity of the test material was observed. At the end of the treatment period the relative survival value of the highest examined concentration of 5000 μg/mL was 22.3 %. All of the examined concentrations (a total of six) were evaluated and a statistically significant increase in the mutant frequency compared to the vehicle control was observed at 5000 μg/mL, although the observed value was well within the historical control range. A slight dose response to the treatment was indicated by trend analysis. In the overall evaluation of this assay it is considered that there were no biologically significant effects.
In the absence of S9-mix (24-hour treatment), marked cytotoxicity was observed. At the end of the treatment period the relative survival value at the highest examined concentration of 5000 μg/mL was 7.4 %. An evaluation was made using the data of all the seven examined concentrations in this case. No statistically significant or biologically relevant increase in the mutation frequency was observed in this experiment. There was no dose response to the treatment (a trend analysis showed no effect of treatment).
DISCUSSION
The sporadic, statistically significant increases in mutation frequency were examined for consistency; none of them were repeatable when comparing Assay 1 and Assay 2. Additionally, they were observed only at cytotoxic concentrations (relative survival values were less than 25 %). The observed mutation frequency values were within the historical control range in all of those cases. Together with the lack of correlation with dose levels, this confirms that there were no biologically significant differences between treated samples and vehicle controls. - Conclusions:
- Interpretation of results: negative with and without metabolic activation
No mutagenic effect of the test material was observed either in the presence or absence of the metabolic activation system under the conditions of this HPRT assay. - Executive summary:
The genotoxic potential of the test material was assessed in an in vitro gene mutation assay with mammalian cells. The study was performed under GLP conditions and in line with the standardised guidelines OECD 476 and EU Method B.17.
Chinese hamster Ovary (CHO-K1) cells were exposed to the test material in two separate assays. Treatments were carried out for 5 hours with and without metabolic activation (± rat liver metabolising system, i.e. S9-mix) and for 24 hours without metabolic activation (-S9-mix). Due to the chemical characteristics of the test material, 1% (w/v) methyl cellulose solution was used as the vehicle of the test material in this study. Treatment concentrations for the mutation assay were selected for the main tests based on the results of a preliminary toxicity test.
The study was considered to be valid and to reflect the real potential of the test material to cause mutations in the cultured mammalian cells used in this study. Treatment with the test material did not result in a statistically or biologically significant, repeatable, dose-dependent increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9) in this study.
Therefore, under the conditions of the study, the test material was concluded not to be mutagenic either in the presence or absence of metabolic activation.
Referenceopen allclose all
Table 2: First experiment (direct plate incorporation) - Mean revertant colony counts
|
TA 1535 |
TA 1537 |
TA 98 |
||||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
28 |
22 |
No |
5 |
8 |
No |
25 |
38 |
No |
312.5 |
31 |
29 |
No (Mp) |
7 |
10 |
No (Mp) |
24 |
32 |
No (Mp) |
625 |
38 |
35 |
No (Mp) |
8 |
9 |
No (Mp) |
26 |
25 |
No (Mp) |
1250 |
32 |
24 |
No (Sp/Mp) |
9 |
8 |
No (Mp) |
27 |
29 |
No (Mp) |
2500 |
36 |
15 |
No (Sp) |
10 |
9 |
No (Sp) |
18 |
29 |
No (Sp) |
5000 |
34 |
19 |
No (Sp) |
4 |
6 |
No (Sp) |
16 |
29 |
No (Sp) |
Positive control |
668 |
188 |
No |
352 |
128 |
No |
161 |
1287 |
No |
|
TA 100 |
TA 102 |
||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
120 |
138 |
No |
388 |
480 |
No |
312.5 |
118 |
141 |
No (Mp) |
330 |
405 |
No (Mp) |
625 |
123 |
129 |
No (Mp) |
449 |
378 |
No (Mp) |
1250 |
104 |
115 |
No (Mp) |
405 |
380 |
No (Mp) |
2500 |
113 |
123 |
No (Sp) |
422 |
406 |
No (Sp) |
5000 |
126 |
109 |
No (Sp) |
497 |
208 |
No (Sp) |
Positive control |
604 |
567 |
No |
2129 |
3775 |
No |
*solvent control with DMSO
Mp : Moderate precipitate
Sp : Strong precipitate
MA : Metabolic activation
Table 3: Second experiment (direct plate incorporation without S9 mix and preincubation with S9 mix) - Mean revertant colony count
|
TA 1535 |
TA 1537 |
TA 98 |
||||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
11 |
7 |
No |
5 |
7 |
No |
25 |
21 |
No |
312.5 |
12 |
11 |
No (Mp) |
3 |
6 |
No (Mp) |
29 |
44 |
No (Mp) |
625 |
10 |
17 |
No (Mp) |
5 |
10 |
No (Mp) |
40 |
33 |
No (Mp) |
1250 |
10 |
18 |
No (Sp/Mp) |
4 |
10 |
No (Sp/Mp) |
29 |
39 |
No (Sp/Mp) |
2500 |
15 |
13 |
No (Sp) |
8 |
4 |
No (Sp) |
37 |
69 |
No (Sp) |
5000 |
9 |
13 |
No (Sp) |
5 |
6 |
No (Sp) |
21 |
21 |
No (Sp) |
Positive control |
593 |
153 |
No |
1443 |
148 |
No |
303 |
1377 |
No |
|
TA 100 |
TA 102 |
||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
127 |
136 |
No |
341 |
551 |
No |
312.5 |
123 |
150 |
No (Mp) |
275 |
454 |
No (Mp) |
625 |
140 |
164 |
No (Mp) |
362 |
569 |
No (Mp) |
1250 |
131 |
135 |
No (Sp/Mp) |
338 |
508 |
No (Sp/Mp) |
2500 |
152 |
108 |
No (Sp) |
344 |
523 |
No (Sp) |
5000 |
146 |
101 |
No (Sp) |
279 |
233 |
No (Sp) |
Positive control |
655 |
712 |
No |
1907 |
1682 |
No |
*solvent control with DMSO
Mp : Moderate precipitate
Sp : Strong precipitate
MA : Metabolic activation
Table 4: Third experiment (preincubation with S9 mix) - Mean revertant colony count
|
TA 98 |
||
Conc. |
- MA |
+ MA |
Cytotoxic |
0* |
- |
21 |
No |
312.5 |
- |
16 |
No (Mp) |
625 |
- |
22 |
No (Mp) |
1250 |
- |
21 |
No (Sp) |
2500 |
- |
23 |
No (Sp) |
5000 |
- |
23 |
No (Sp) |
Positive control |
- |
1342 |
No |
*solvent control with DMSO
Mp : Moderate precipitate
Sp : Strong precipitate
MA : Metabolic activation
Table 1: Summary of Mitotic Index Data from Experiments 1 and 2
Dose Level (µg/mL) |
Experiment 1 |
Experiment 2 |
||||||
4 Hour Treatment -S9 |
4 Hour Treatment +S9 |
24 Hour Treatment -S9 |
4 Hour Treatment +S9 |
|||||
Mean |
% of Control |
Mean |
% of Control |
Mean |
% of Control |
Mean |
% of Control |
|
0 5 10 20 40 80 160 |
6.15 - - 6.75 - 6.88 6.30 |
100 - - 110 - 109 102 |
4.95 - - 4.25 - 5.38 3.80 |
100 - - 86 - 109 77 |
4.90 - - 4.20 - 5.00 5.00 |
100 - - 86 - 102 102 |
4.40 - - 4.85 - 4.48 4.13 |
100 - - 110 - 102 94 |
Mitomycin C 0.4 |
2.93 |
48 |
- |
- |
1.55 |
32 |
- |
- |
Cyclophosphamide 5 |
- |
- |
1.63 |
33 |
- |
- |
1.75 |
40 |
Precipitate was present for all dose levels evaluated; precipitate was present on the slide at evaluation for both the 80 and 160 µg/mL dose levels.
Table 2: Summary of Chromosome Aberration Data for Experiment 1
Treatment Period (hours) |
Dose Level (µg/mL) |
|
No. and % Cells Showing Structural Aberrations†(%) |
No. and % Cells Showing Gaps (%) |
No. and % Cells Showing Numerical Aberrations‡(%) |
4 (-S9-mix) |
0 |
Total % |
1 0.5 |
0 0.0 |
0 0.0 |
20 |
Total % |
0 0.0 |
0 0.0 |
0 0.0 |
|
80 |
Total % |
2 1.0 |
0 0.0 |
0 0.0 |
|
160 |
Total % |
2 1.0 |
3 1.5 |
0 0.0 |
|
MMC 0.4 |
Total % |
56*** 56.0 |
4 4.0 |
1 1.0 |
|
4 (+S9-mix) |
0 |
Total % |
0 0.0 |
0 0.0 |
0 0.0 |
20 |
Total % |
0 0.0 |
0 0.0 |
0 0.0 |
|
80 |
Total % |
2 1.0 |
4 2.0 |
0 0.0 |
|
160 |
Total % |
0 0.0 |
0 0.0 |
0 0.0 |
|
CP 5 |
Total % |
33*** 33.0 |
8 8.0 |
0 0.0 |
Results are the mean values from two replicates.
MMC = Mitomycin C; CP = Cyclophosphamide.
†Includes chromatid and chromosome breaks, chromatid and chromosome exchanges.
‡Includes polyploids and other numerical chromosome aberrations.
***p = <0.001
Table 3: Summary of Chromosome Aberration Data for Experiment 2
Treatment Period (hours) |
Dose Level (µg/mL) |
|
No. and % Cells Showing Structural Aberrations†(%) |
No. and % Cells Showing Gaps (%) |
No. and % Cells Showing Numerical Aberrations‡(%) |
24 (-S9-mix) |
0 |
Total % |
0 0.0 |
2 1.0 |
0 0.0 |
20 |
Total % |
0 0.0 |
0 0.0 |
0 0.0 |
|
80 |
Total % |
0 0.0 |
0 0.0 |
0 0.0 |
|
160 |
Total % |
0 0.0 |
0 0.0 |
0 0.0 |
|
MMC 0.4 |
Total % |
34*** 34.0 |
7 7.0 |
0 0.0 |
|
4 (+S9-mix) |
0 |
Total % |
0 0.0 |
1 0.5 |
0 0.0 |
20 |
Total % |
1 0.5 |
2 1.0 |
1 0.5 |
|
80 |
Total % |
2 1.0 |
0 0.0 |
0 0.0 |
|
160 |
Total % |
0 0.0 |
0 0.0 |
0 0.0 |
|
CP 5 |
Total % |
33*** 16.5 |
14 7.0 |
1 0.5 |
Results are the mean values from two replicates.
MMC = Mitomycin C; CP = Cyclophosphamide.
†Includes chromatid and chromosome breaks, chromatid and chromosome exchanges.
‡Includes polyploids and other numerical chromosome aberrations.
***p = <0.001
Table 1: Results Experiment 1 – 4 Hour Exposure Without Metabolic Activation (S9)
Dose Group (µg/mL) |
Day 0 Viability; Mean CE % of Control |
Day 7 Viability; Mean CE % of Control |
Day 7 Group Mutant Frequency per Survivor ^-6 (SD) |
0.0 |
100 |
100 |
34 (0.63) |
39.06 * |
99 |
102 |
44 (2.31) |
78.13 * |
77 |
91 |
77 (3.44) |
156.25 * |
70 |
82 |
106 (3.26) |
312.5 * |
58 |
69 |
136 (3.37) |
625 * |
50 |
59 |
114 (4.23) |
1250 * |
36 |
52 |
156 (2.53) |
2500 * |
11 |
‡ |
‡ |
5000 * |
6 |
‡ |
‡ |
EMS 500 |
67 |
87 |
268 (8.60) |
EMS 750 |
38 |
71 |
408 (15.03) |
* = precipitation; ‡= too toxic for plating; CE = Cloning Efficiency; EMS = Ethyl methane sulphonate
Table 2: Results Experiment 1 – 4 Hour Exposure With Metabolic Activation (2 % S9)
Dose Group (µg/mL) |
Day 0 Viability; Mean CE % of Control |
Day 7 Viability; Mean CE % of Control |
Day 7 Group Mutant Frequency per Survivor ^-6 (SD) |
0.0 |
100 |
100 |
23 (1.78) |
156.25 * |
101 |
92 |
25 (1.58) |
312.5 * |
91 |
96 |
27 (1.40) |
625 * |
93 |
90 |
35 (2.50) |
1250 * |
88 |
85 |
30 (1.52) |
2500 * |
98 |
110 |
20 (1.57) |
5000 * |
86 |
102 |
31 (1.55) |
DMBA 0.5 |
74 |
96 |
105 (2.79) |
DMBA 1 |
29 |
64 |
242 (4.79) |
* = precipitation; CE = Cloning Efficiency; DMBA = Dimethyl benzanthracene
Table 3: Results Experiment 2 – 4 Hour Exposure Without Metabolic Activation (S9)
Dose Group (µg/mL) |
Day 0 Viability; Mean CE % of Control |
Day 7 Viability; Mean CE % of Control |
Day 7 Group Mutant Frequency per Survivor ^-6 (SD) |
0.0 |
100 |
100 |
23 (1.57) |
78.13 * |
75 |
96 |
48 (2.72) |
156.25 * |
70 |
87 |
61 (3.30) |
312.5 * |
73 |
101 |
54 (3.10) |
625 * |
47 |
93 |
92 (4.49) |
1250 * |
31 |
102 |
55 (3.83) |
1875 * |
26 |
94 |
62 (4.99) |
2500 * |
24 |
83 |
153 (6.75) |
3750 * |
11 |
79 |
32 (2.07) |
EMS 500 |
47 |
82 |
221 (5.12) |
EMS 750 |
24 |
73 |
226 (4.40) |
* = precipitation; CE = Cloning Efficiency; EMS = Ethyl methane sulphonate
Table 4: Results Experiment 2 – 4 Hour Exposure With Metabolic Activation (1 % S9)
Dose Group (µg/mL) |
Day 0 Viability; Mean CE % of Control |
Day 7 Viability; Mean CE % of Control |
Day 7 Group Mutant Frequency per Survivor ^-6 (SD) |
0.0 |
100 |
100 |
21 (1.43) |
312.5 * |
89 |
86 |
115 (9.09) |
625 * |
95 |
78 |
122 (7.59) |
1250 * |
84 |
85 |
142 (4.71) |
2500 * |
84 |
86 |
127 (6.24) |
3750 * |
92 |
80 |
150 (5.66) |
5000 * |
89 |
86 |
150 (8.31) |
DMBA 0.5 |
98 |
60 |
349 (6.66) |
DMBA 1 |
32 |
48 |
522 (3.92) |
* = precipitation; CE = Cloning Efficiency; DMBA = Dimethyl benzanthracene
Table 1: Summary of Results - Survival
S9-mix |
Treatment period |
Assay |
Treatment |
Total no. of colonies |
Cloning efficiency |
Relative survival (%) |
with |
5 |
1 |
5000 µg/mL |
198 |
16.5 |
18.7 |
2500 µg/mL |
391 |
32.6 |
37.0 |
|||
1250 µg/mL |
648 |
54.0 |
61.3 |
|||
625 µg/mL |
808 |
67.3 |
76.4 |
|||
312.5 µg/mL |
1023 |
85.3 |
96.8 |
|||
156.25 µg/mL |
1059 |
88.3 |
100.2 |
|||
Negative control |
1057 |
88.1 |
100.0 |
|||
Negative control for DMBA |
943 |
78.6 |
89.2 |
|||
Untreated control |
1143 |
95.3 |
108.1 |
|||
Positive control (DMBA) |
89 |
7.4 |
8.4 |
|||
without |
5 |
1 |
5000 µg/mL |
192 |
16.0 |
16.6 |
2500 µg/mL |
283 |
23.6 |
24.4 |
|||
1250 µg/mL |
485 |
40.4 |
41.8 |
|||
625 µg/mL |
685 |
57.1 |
59.1 |
|||
312.5 µg/mL |
1021 |
85.1 |
88.1 |
|||
156.25 µg/mL |
1191 |
99.3 |
102.8 |
|||
Negative control |
1159 |
96.6 |
100.0 |
|||
Negative control for EMS |
1064 |
88.7 |
91.8 |
|||
Untreated control |
1206 |
100.5 |
104.1 |
|||
Positive control (EMS) |
1002 |
83.5 |
86.5 |
|||
with |
5 |
2 |
5000 µg/mL |
239 |
19.9 |
22.3 |
2500 µg/mL |
463 |
38.6 |
43.2 |
|||
1250 µg/mL |
618 |
51.5 |
57.6 |
|||
625 µg/mL |
774 |
64.5 |
72.2 |
|||
312.5 µg/mL |
1066 |
88.8 |
99.4 |
|||
156.25 µg/mL |
1056 |
88.0 |
98.5 |
|||
Negative control |
1072 |
89.3 |
100.0 |
|||
Negative control for DMBA |
973 |
81.1 |
90.8 |
|||
Untreated control |
1148 |
95.7 |
107.1 |
|||
Positive control (DMBA) |
105 |
8.8 |
9.8 |
|||
without |
24 |
2 |
5000 µg/mL |
85 |
7.1 |
7.4 |
3750 µg/mL |
165 |
13.8 |
14.3 |
|||
2500 µg/mL |
235 |
19.6 |
20.3 |
|||
1250 µg/mL |
410 |
34.2 |
35.5 |
|||
625 µg/mL |
651 |
54.3 |
56.4 |
|||
312.5 µg/mL |
1034 |
86.2 |
89.5 |
|||
156.25 µg/mL |
1139 |
94.9 |
98.6 |
|||
Negative control |
1155 |
96.3 |
100.0 |
|||
Negative control for EMS |
1095 |
91.3 |
94.8 |
|||
Untreated control |
1201 |
100.1 |
104.0 |
|||
Positive control (EMS) |
415 |
34.6 |
35.9 |
DMBA: 7,12 -dimethylbenz[a]anthracene, 2 µg/mL
EMS: ethyl methanesulphonate, 0.4 µL/mL
Table 2: Summary of Results - Viability
S9-mix |
Treatment period |
Assay |
Treatment |
Total no. of colonies |
Cloning efficiency (%) |
with |
5 |
1 |
5000 µg/mL |
1080 |
90.0 |
2500 µg/mL |
1116 |
93.0 |
|||
1250 µg/mL |
1105 |
92.1 |
|||
625 µg/mL |
1051 |
87.6 |
|||
312.5 µg/mL |
1095 |
91.3 |
|||
156.25 µg/mL |
1106 |
92.2 |
|||
Negative control |
1013 |
84.4 |
|||
Negative control for DMBA |
1115 |
92.9 |
|||
Untreated control |
1201 |
100.1 |
|||
Positive control (DMBA) |
902 |
75.2 |
|||
without |
5 |
1 |
5000 µg/mL |
1036 |
86.3 |
2500 µg/mL |
1117 |
93.1 |
|||
1250 µg/mL |
1084 |
90.3 |
|||
625 µg/mL |
1110 |
92.5 |
|||
312.5 µg/mL |
1142 |
95.2 |
|||
156.25 µg/mL |
1138 |
94.8 |
|||
Negative control |
1148 |
95.7 |
|||
Negative control for EMS |
1093 |
91.1 |
|||
Untreated control |
1172 |
97.7 |
|||
Positive control (EMS) |
1032 |
86.0 |
|||
with |
5 |
2 |
5000 µg/mL |
1028 |
85.7 |
2500 µg/mL |
1121 |
93.4 |
|||
1250 µg/mL |
1129 |
94.1 |
|||
625 µg/mL |
1110 |
92.5 |
|||
312.5 µg/mL |
1171 |
97.6 |
|||
156.25 µg/mL |
1110 |
92.5 |
|||
Negative control |
1065 |
88.8 |
|||
Negative control for DMBA |
969 |
80.8 |
|||
Untreated control |
1152 |
96.0 |
|||
Positive control (DMBA) |
886 |
73.8 |
|||
without |
24 |
2 |
5000 µg/mL |
971 |
80.9 |
3750 µg/mL |
1041 |
86.8 |
|||
2500 µg/mL |
1122 |
93.5 |
|||
1250 µg/mL |
1105 |
92.1 |
|||
625 µg/mL |
1140 |
95.0 |
|||
312.5 µg/mL |
1127 |
93.9 |
|||
156.25 µg/mL |
1146 |
95.5 |
|||
Negative control |
1013 |
84.4 |
|||
Negative control for EMS |
980 |
81.7 |
|||
Untreated control |
1128 |
94.0 |
|||
Positive control (EMS) |
974 |
81.2 |
DMBA: 7,12 -dimethylbenz[a]anthracene, 2 µg/mL
EMS: ethyl methanesulphonate, 0.4 µL/mL
Table 3: Summary of Results - Mutagenicity
S9-mix |
Treatment period |
Assay |
Treatment |
Total no. of colonies |
Mutation frequency |
with |
5 |
1 |
5000 µg/mL |
23 |
12.8 |
2500 µg/mL |
20 |
10.7 |
|||
1250 µg/mL |
18 |
9.7 |
|||
625 µg/mL |
21 |
12.0 |
|||
312.5 µg/mL |
31 |
17.0 |
|||
156.25 µg/mL |
31 |
16.9 |
|||
Negative control |
16 |
9.5 |
|||
Negative control for DMBA |
33 |
17.5 |
|||
Untreated control |
19 |
9.4 |
|||
Positive control (DMBA) |
1328 |
883.4** |
|||
without |
5 |
1 |
5000 µg/mL |
31 |
19.7* |
2500 µg/mL |
20 |
10.7 |
|||
1250 µg/mL |
11 |
6.1 |
|||
625 µg/mL |
12 |
6.5 |
|||
312.5 µg/mL |
14 |
7.4 |
|||
156.25 µg/mL |
18 |
9.5 |
|||
Negative control |
20 |
10.6 |
|||
Negative control for EMS |
31 |
17.0* |
|||
Untreated control |
14 |
7.2 |
|||
Positive control (EMS) |
593 |
344.7** |
|||
with |
5 |
2 |
5000 µg/mL |
41 |
23.9* |
2500 µg/mL |
35 |
18.7 |
|||
1250 µg/mL |
28 |
14.9 |
|||
625 µg/mL |
23 |
12.5 |
|||
312.5 µg/mL |
29 |
14.8 |
|||
156.25 µg/mL |
22 |
11.9 |
|||
Negative control |
24 |
13.4 |
|||
Negative control for DMBA |
19 |
11.7 |
|||
Untreated control |
26 |
13.5 |
|||
Positive control (DMBA) |
1719 |
1165.0** |
|||
without |
24 |
2 |
5000 µg/mL |
32 |
19.9 |
3750 µg/mL |
56 |
32.5 |
|||
2500 µg/mL |
23 |
12.3 |
|||
1250 µg/mL |
34 |
18.4 |
|||
625 µg/mL |
22 |
11.5 |
|||
312.5 µg/mL |
28 |
15.0 |
|||
156.25 µg/mL |
30 |
15.7 |
|||
Negative control |
29 |
17.1 |
|||
Negative control for EMS |
22 |
13.5 |
|||
Untreated control |
24 |
12.8 |
|||
Positive control (EMS) |
1560 |
961.0** |
DMBA: 7,12 -dimethylbenz[a]anthracene, 2 µg/mL
EMS: ethyl methanesulphonate, 0.4 µL/mL
* Statistically significant at p < 0.05 compared to the relevant vehicle control
** Statistically significant at p < 0.01 compared to the relevant vehicle control
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
In vivo transgenic rodent mutation assay; Kasamoto (2018): Under the conditions in this study the test material did not induce gene mutation in either the liver or glandular stomach of transgenic mice.
Link to relevant study records
- 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.
- Dose / conc.:
- 5.13 other: mg/100 g bw
- Dose / conc.:
- 7.7 other: mg/100 g bw
- Dose / conc.:
- 15.4 other: mg/100 g bw
- Dose / conc.:
- 38.5 other: mg/100 g bw
- 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 the study was disregarded due to the use of an unsuitable method of preparing the test material. Indeed, 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.
- Endpoint:
- in vivo mammalian somatic and germ cell study: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 January 2018 to 30 July 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- transgenic rodent mutagenicity assay
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Under room temperature in the dark and sealed container
- Stability under test conditions: Stable
- Species:
- mouse
- Strain:
- other: CD2-LacZ80/HazfBR (Muta™Mouse) [SPF]
- Remarks:
- Transgenic mouse
- Details on species / strain selection:
- CD2-LacZ80/HazfBR mice are commonly used as transgenic animals, and animals of this strain are readily available in in vivo gene mutation assays.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 9 weeks of age at the time of assignment to groups.
- Weight at study initiation: 23.6 to 27.3 g
- Assigned to test groups randomly: Animals were assigned to groups based on their body weights on Day 1 using LATOX-F/V5 computer system package. Unassigned animals were excluded from the study on Day 1. The weight range of animals was within the mean weight ± 20 %.
- Housing: All animals were housed in a barrier system room under positive pressure. Two or three animals were housed in a plastic cage (W 18.2 × D 26.0 × H 12.8 cm) with bedding. Analytical results of levels of contaminants in the bedding were within the acceptable limits of the Japan Experimental Animal Feed Association. The animal cages, the beddings and the feeders were replaced on the day of grouping and once a week thereafter. The water bottles were replaced once every 2 or 3 days.
- Diet: Ad libitum access to pellet diet CRF-1. Food was replenished at the time of exchanging the feeders. Contaminant levels were within the acceptable limits proposed by the Japan Experimental Animal Feed Association.
- Water: Animals were provided access to tap water from water bottles ad libitum. Contaminant levels in the water were within the acceptable limits of the Tap Water Quality Standard. Examination also confirmed that no bacteria were detected in the water.
- Acclimation period: Upon arrival, each animal was monitored for their general condition and body weight gain. During the quarantine and acclimation period (Day -8 to Day 1), the animals were observed once a day. The body weight of each animal was measured on the day of receipt and at the end of the quarantine and acclimation period. None of the animals showed abnormalities in their general condition or body weight gain.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26 °C (actual values: 22.8 to 23.0 °C)
- Humidity (%): 35 to 70 %RH (actual values: 40 to 55 %RH)
- Air changes (per hr): 12 times or more/hour
- Photoperiod (hrs dark / hrs light): 12 hours (light on: 7:00, light off: 19:00) - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: Methylcellulose 400
- Lot/batch no.: WDP4521
- 0.5 % (w/v) MC, the vehicle to prepare test material formulations, was used as a negative control.
- Dose volume: 10 mL/kg
- Preparation of vehicle: An aqueous solution of methylcellulose 400 at 0.5 % (w/v) was used for the negative control. Methylcellulose 400 (4 g) was accurately weighed and transferred into a stoppered volumetric cylinder. A small volume of warmed (approximately 80 °C) water for injection was repeatedly added to dissolve it. After this solution was cooled to room temperature, it was adjusted to 800 mL with water for injection to make 0.5 w/v% solution. The prepared vehicle was stored in a refrigerator and was used within 14 days after preparation. - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- 2.5, 5.0 or 10 g of the test material for the 25, 50 and 100 mg/mL solutions respectively were weighed and transferred to a stoppered measuring cylinder and mixed with a little vehicle.
- Solutions were diluted to 100 mL with vehicle to prepare 25, 50 and 100 mg/mL formulations, respectively.
- The formulations were fully mixed by manual inversion and then stirred by magnetic stirrer until visibly homogeneous.
Within a concentration range of 10.0 to 200 mg/mL, the test material was confirmed to be stable and homogeneous in 0.5 % (w/v) methylcellulose 400 after storage for at least 8 days at ambient laboratory temperature and 2 to 8 °C in the dark (non-GLP validation study). In the same non-GLP validation study it was confirmed under non-GLP condition that the 10.0 and 100 mg/mL dosing formulations stored at 0 to 10 °C were stable and homogeneous in 0.5 w/v% MC for at least 15 days.
Dispensing and storage of the test material formulations
- Each test material formulation was divided into 5 or 7 portions (including 1 portion as reserve) for each administration under continuous stirring using a magnetic stirrer and stored in tight and light-resistant containers in a refrigerator (acceptable temperature range: 1 to 9 °C) until use. The actual temperature range was 3.6 to 7.6 °C during the storage period. The stored formulations were fully mixed by manual inversion and then stirred by a magnetic stirrer at least 30 minutes before use.
Analysis of test material formulations
- The analysis for the test material formulations was conducted at IDEA Consultants, Inc. under non-GLP condition.
- A portion (approximately 6 mL) of the test material formulations prepared on the first preparation day was submitted to IDEA Consultants, Inc. The concentration of each sample was measured using ICP-MS and the ratio to the nominal concentration was calculated from the mean concentrations. If the ratios of the mean concentration of the test material in each formulations to the nominal concentrations are within 10 % and the relative standard deviation (RSD) of the concentrations in three layers is 10 % or less, the test material formulations were judged to be appropriately prepared.
- As a result, the ratios to the nominal concentrations in the test material formulations were 108.0 to 109.8 %, and the RSD were 3.8 to 5.2 %. These results showed that the test material formulations had been appropriately prepared.
RATIONALE FOR SELECTION OF THE DOSE LEVEL
- In the acute oral toxicity study male and female Sprague-Dawley (SD) rats treated with 2000 mg/kg of test material in 0.5 % (w/v) methylcellulose, no clinical signs and no changes of the body weight gain of the animals were observed. Combined repeated dose and reproduction/developmental screening study using SD rats revealed that the no observed adverse effect level (NOAEL) was 1000 mg/kg/day.
-Toxicity to mice was not observed, in the dose range-finding study in CD2F1/Slc mice treated with 100, 300 or 1000 mg/kg of test material in 0.5 w/v% MC. Therefore, a dosage of 1000 mg/kg/day was selected as the high dose and 2 dosage levels including 250 and 500 mg/kg/day as lower doses were used in the present study.
ADMINISTRATION METHOD
- Before administration, the test material formulations were shaken by hand. Then, the individual dosing volumes of test material formulations were taken under continuous stirring using a magnetic stirrer for at least 30 minutes before use.
- The vehicle and the test material were administered orally according to the test guideline.
- The vehicle and the test material were administered to animals once daily for 28 consecutive days by using a disposable syringe with a Teflon sonde.
- The dosing volume (mL) was set at 0.1 mL per 10 g of body weight. The individual dosing volume (mL) was calculated on the basis of the most recent individual body weight - Duration of treatment / exposure:
- - The administration period was 4 weeks (28 days).
- To ensure that the data would be available for each 5 animals in a group for the main study and germ cell study, 12 animals each were assigned in all groups. At first, 5 animals were selected by the ascending order of animal ID No. and used for evaluation in the main study. The remaining animals were retained for approximately 7 weeks after the end of the dosing period in order to collect germ cells, for retention as a contigency in the event that germ cell assessments were necessary. - Frequency of treatment:
- Once daily
- Post exposure period:
- Manifestation time 3 days for animals assigned to somatic tissue assessments (main study); to enable adequate sperm maturation animals assigned to germ cell analysis had a manifestation time of 7 weeks (49 days).
- Dose / conc.:
- 250 mg/kg bw/day
- Remarks:
- Concentration of dosing formulation: 25 mg/mL
- Dose / conc.:
- 500 mg/kg bw/day
- Remarks:
- Concentration of dosing formulation: 50 mg/mL
- Dose / conc.:
- 1 000 mg/kg bw/day
- Remarks:
- Concentration of dosing formulation: 100 mg/mL
- No. of animals per sex per dose:
- 5 male mice per group in the main study.
7 male mice per group in the germ cell study. - Control animals:
- yes, concurrent vehicle
- yes, historical
- Positive control(s):
- N-ethyl-N-nitrosourea (ENU), contains 40 % water, 1.8 % AcOH.
- Justification for choice of positive control(s): Based on consideration of information in academic documents.
PREPARATION OF POSITIVE CONTROL SOLUTION
- 50 mg of ENU was weighed, transferred into a graduated test tube and diluted to 5 mL with 1/15 mol/L sodium-phosphate buffer (pH 6) to make a 10 mg/mL solution. The positive control solution was prepared just before use.
- The dose was 100 mg/kg.
- Concentration of dosing formulation: 10 mg/mL
- Dosing volume: 10 mL/kg
- The positive control material was administered intraperitoneally once daily for 2 consecutive days at about 24-hour intervals using a disposable syringe attaching a 25G needle. The dosing volume was set at 0.1 mL per 10 g of body weight and was calculated on the basis of the most recent individual body weight - Tissues and cell types examined:
- Main study: The liver, stomach, testes and vas deferens/cauda epididymis were removed from each animal.
Germ cell study: The testes and cauda epididymis were separately removed from each animal. - Details of tissue and slide preparation:
- TISSUE RETENTION
LIVER: Two samples were prepared from the left lateral lobe, Each sample was separately put into microtubes and frozen in liquid nitrogen (LN2). The other lobes were put into a storage bag, squashed and then frozen in LN2. The remaining part of left lateral lobe was fixed in an adequate volume of 10 vol% neutral buffered formalin solution.
STOMACH: The greater curvature of the stomach was incised. The stomach contents were removed by washing with physiological saline. The
stomach piece (included forestomach and glandular stomach) was cut to about 4 × 10 mm size. These parts were fixed in an adequate volume of 10 vol% neutral buffered formalin solution. The remaining part was separated into forestomach part and glandular stomach part. Glandular stomach (two pieces) and forestomach were put into each storage bag and frozen in LN2.
GERM CELLS (Main study): The testes and vas deferens/cauda epididymis were put into a storage bag, separately and squashed then frozen in LN2.
GERM CELLS (Germ cell study): The right and left testis and right and left cauda epididymides were put into a microtube separately and frozen in LN2.
All frozen tissues were stored in an ultra-low temperature freezer.
EXTRACTION OF GENOMIC DNA
- Extraction of genomic DNA in the liver and glandular stomach was conducted.
- In the germ cell, extraction of genomic DNA and examination for mutagenicity was not conducted.
- 3 mL of the buffer for tissue breakage (containing RNase) were poured into a Dounce-type homogenizer and cooled with ice. Each frozen tissue sample was put into the homogeniser and homogenised with a pestle. The homogenised tissue fragments were poured gently into an ice-cooled centrifuge tube containing 3 mL of 0.5 mol/L sucrose solution, and centrifuged at 3000 rotations/min (1710 G) for 10 minutes. The supernatant was removed with a dropper and 3 mL of cooled RNase-containing Dounce buffer were added to the tube and mixed well (nuclear/cell suspension).
- Then, 3 mL of proteinase K solution was added to the nuclear/cell suspension and gently mixed by inversion. This suspension was incubated at 50 °C for about 2 to 2.5 hours until it became clear. The same volume (about 6 mL) of Phenol/Chloroform (Ph/Cl) mixture was added to the solution and mixed by inversion a few times, mixed by using a rotator for 10 minutes, and finally centrifuged at 2500 rotations/min (1190 G) for 10 minutes. Next, the upper layer (water layer) was gently collected and transferred into another centrifuge tube by a transfer pipette. This procedure was repeated twice (volume of Ph/Cl mixture was same as the removed water layer). After removal of the water layer, the same volume of chloroform/isoamyl alcohol at a volume ratio of 24:1 was poured into the tube. The contents were mixed by inversion a few times, mixed by using a rotator for 10 minutes, and finally centrifuged at 2500 rotations/min (1190 G) for 10 minutes. Then, the water layer was transferred into another centrifuge tube. Genomic DNA was extracted by gradually adding ethanol in the tube. Extracted genomic DNA was transferred into a microtube containing 70 % ethanol and stood for about 10 minutes. The contents were centrifuged at 13,000 rotations/min (13240 G) for 10 minutes. After the supernatant was removed as much as possible using a micropipette, the tube was allowed to stand at room temperature to evaporate ethanol. Appropriate volume (100 μL) of TE buffer was added to the tube. The tube was allowed to stand overnight at room temperature to dissolve DNA residues. The DNA solution was stored in a refrigerator after preparation. The concentration of DNA in the genomic DNA solution was measured using a spectrophotometer and adjusted to about 200 to 600 μg/mL with the TE buffer.
PREPARATION OF TEST STRAINS
-30 mL of LB broth, 300 μL of maltose solution (200 mg/mL), 30 μL of ampicillin solution (50 mg/mL), and 30 μL of kanamycin solution (20 mg/mL) were poured into a 200-mL baffled Erlenmeyer flask. A suspension (50 μL) of Escherichia coli C (lacZ–, gal E–) that had been thawed after being frozen at -80 °C was inoculated into the flask. And it was incubated overnight at 37°C with a shaker at 120 strokes/min as the pre-incubation culture.
- 100 mL of LB broth and 1 mL of maltose solution (200 mg/mL) were poured into a 500-mL baffled Erlenmeyer flask. The pre-incubation culture (1 mL) was inoculated into the flask and it was incubated for about 2 hours (OD: 0.831 to 0.936) under the same conditions as the pre-incubation. Then, the bacterial suspension was centrifuged at 1000 rotations/min for 10 minutes. The supernatant was removed and the cells were suspended in LB broth containing 10 mmol/L magnesium sulfate (E. coli suspension).
PACKAGING OF GENOMIC DNA
- Packaging was conducted according to the instruction manual attached to Transpack packaging extract.
- One red tube of Transpack packaging extract was thawed. Using a pipette, 10 μL of genomic DNA solution was transferred to the red tube. The packaging reaction was mixed by pipetting and the tube was incubated at 30 °C for 90 minutes. Next, a blue tube of Transpack packaging extract was thawed, and 10 μL of it were transferred to the red tube containing a packaging reaction, and mixed in the same manner. It was incubated at 30 °C for another 90 minutes, diluted with 700 μL of SM buffer, and mixed (packaged DNA sample).
PLATING OF PACKAGING DNA
- 1 mL of E. coli suspension for calculating total number of plaques (for tittering) and 2 mL for calculating mutant frequency (for selection) were dispensed into each tube. Then, the entire volume of packaged DNA sample (about 700 μL) was added to E. coli suspension in the tube for selection (total of about 2700 μL) and mixed. The tube was incubated at room temperature for about 30 minutes to allow phage to infect E. coli. 30 μL of the content were diluted 10-fold with 270 μL of LB broth containing 10 mmol/L magnesium sulfate. 30 μL of it were transferred to the tube for tittering and stirred.
The magnesium sulfate solution (1 mol/L) was added to the agar solution at a volume ratio of 2:100 to make the top agar for tittering. The P-gal solution was added to the agar solution at a volume ratio of 2:100 to make the top agar for selection. Then, 17 mL of top agar was added to the tube for tittering and mixed. The contents were poured over a LB agar plate. To the tube for selection, 16 mL of top agar was added and the contents were poured over a LB agar plate in the same manner as the tube for tittering.
- The agar plate was incubated in an incubator at 37 °C overnight.
In the liver of animal ID No. 1105, the above packaging procedure was repeated twice until the total number of plaques per animal reached 300,000.
PLAQUE COUNTING
Calculation of total number of plaques:
- The number of plaques (N) in the plates for tittering was counted, and then the total number of plaques was calculated using the following equation.
Total number of plaques = N × (300 μL/30 μL) × (2 700 μL/30 μL)
= 900 × N
CALCULATION OF NUMBER OF MUTANT PLAQUES
- The number of plaques in the plates for selection was counted and recognized as mutant plaques.
CALCULATION OF MUTANT FREQUENCY
- The lacZ gene was selected as a reporter gene.
- The mutant frequency in a concerned organ was calculated by dividing the number of mutant plaques by the total number of plaques.
Mutant frequency = Number of mutant plaques / Total number of plaques - Evaluation criteria:
- The results were evaluated as positive if the mutant frequency for any tissue in any test substance-treated group was significantly different from that in the negative control group. Final judgment includes a comparison against the historical control data and was made in consideration of biological relevance under the test conditions.
- Statistics:
- The obtained data for in-life phase of this study were collected and analysed using the computer system (LATOX-F/V5).
MUTANT FREQUENCY
- The data on the mutant frequency from the negative control group and each test material-treated group were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity was determined (not significant on Bartlett’s test), then Dunnett’s multiple comparison test was performed to assess the statistical significance of differences between the negative control group and each test material-treated group (two-sided, familywise significance level of 0.05). If there was no homogeneity (significant on Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) was performed to analyse the differences.
-The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance was determined (not significant on F test), Student’s t test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t test (two-sided, significance level of 0.05) was performed to analyse the differences.
The results were evaluated as positive if the mutant frequency for any tissue in any test material-treated group was significantly different from that in the negative control group. Final judgment was include a comparison against the historical control data and was made in consideration of biological relevance under the test conditions. - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- LIVER
- In the negative control group, the mean ± SD of mutant frequency among the individuals was 26.9 ± 5.3 (×10^-6).
- The means (±SD) of mutant frequencies in the test material-treated groups, 250, 500 and 1 000 mg/kg/day, were 32.3 ± 8.1 (×10^-6), 30.6 ± 6.6 (×10^-6) and 31.3 ± 3.5 (×10^-6), respectively. All mutant frequencies for the test material-treated groups fell within the laboratory’s acceptable range (based on their historical data) and no statistically significant increase was observed between any of the test group and negative control group.
- The positive control exhibited high and statistically significant mutant frequencies [114.1 ± 14.1 (×10^-6)] compared to those in the negative control (p<0.05).
GLANDULAR STOMACH
- In the negative control group, the mean ± SD of mutant frequency among the individuals was 31.1 ± 9.2 (×10^-6).
- The means (±SD) of mutant frequencies in the test material-treated groups, 250, 500, and 1 000 mg/kg/day, were 30.9 ± 16.2 (×10^-6), 31.6 ± 13.0 (×10^-6) and 32.2 ± 8.6 (×10^-6), respectively. All mutant frequencies for the test material-treated groups fell within the laboratory’s acceptable range (based on their historical data) and no statistically significant increase was observed between any of the test group and negative control group.
- The positive control exhibited high and statistically significant mutant frequencies [287.3 ± 58.9 (×10^-6)] compared to those in the negative control (p<0.05).
BODY WEIGHT AND GENERAL CONDITIONS
- There were no statistically significant differences in body weights between the negative control group and each of the test material-treated groups during the administration period.
- There was no finding in the general condition in any of the test material-treated groups.
FOOD CONSUMPTION
- In the 250, 500 and 1 000 mg/kg/day group, the food consumption from Day 29 to 31 were significantly lower than that in the negative control group. However, these were judged to be not a test material-treatment effect because these were transient and slight changes and not dose-related.
ORGAN WEIGHT AND ABSOLUTE ORGAN WEIGHT / BODY WEIGHT RATIO
- In the 1000 mg/kg/day group, the stomach weight and relative stomach weight to body weight were significantly higher than that in the negative control group.
There were no statistically differences from the negative control group in the liver weight, testes weight or their relative weights to body weight.
GROSS NECROPSY FINDINGS
A cyst in right kidney was observed in the 500 mg/kg/day group. However, this change was considered not to be due to the test material treatment because it was observed in one animal in the middle treatment group only. - Conclusions:
- Under the conditions in this study the test material did not induce gene mutation in either the liver or glandular stomach of transgenic mice.
- Executive summary:
A gene mutation assay with transgenic male mice (Muta™ Mouse) was conducted to assess the potential of the test material to induce gene mutations (reporter gene: lacZ) in the liver and glandular stomach in accordance with the standardised guideline OECD 488.
In a dose-finding study CD2F1/Slc mice were treated for 14 days with 100, 300 and 1 000 mg/kg/day by oral gavage. No deaths or clinical signs of toxicity were observed in any of the dosed groups. Therefore, a limit dosage of 1 000 mg/kg was selected as the high dose and 500 and 250 mg/kg/day were selected as lower doses for the gene mutation assay with Muta™ Mouse.
The test material was administered to male transgenic mice (CD2-LacZ80/HazfBR (Muta™ Mouse)) orally for 28 consecutive days by gavage. In the main study, the liver and glandular stomach were removed after 3 days of manifestation period (Day 29 to 31), and the mutant frequencies were determined. As a result, there were no significant differences in the mutant frequencies in the liver and glandular stomach in any of the groups treated with the test material as compared to the negative control group. The mutant frequencies in the liver and glandular stomach in the positive control group treated with 2 doses of N-ethyl-N-nitrosourea (ENU, dosage level of 100 mg/kg, i.p.) at 24h intervals, were increased. These increases were statistically significant compared with those of the negative control group. No clinical signs, no significant decrease of body weights and no test material-related reduction of food consumption were observed in any groups. There were no test material-related changes noted at necropsy.
In the germ cell study, the testes and cauda epididymis were removed after 49 days of manifestation period (Day 29 to 77). Since no positive response was observed in somatic cells, the mutant frequencies in the germ cells were not evaluated.
It was, therefore, concluded that the test material did not induce gene mutation in the liver or glandular stomach of transgenic mice under the conditions in this study.
Referenceopen allclose all
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 5.13 7.70 15.40 38.50 |
6 58 7 8 11 9 |
5 28 8 13 11 15 |
- 27 2 7 9 12 |
- 6 - - 2 3 |
- 18 1 2 3 5 |
- 16 - - - 2 |
- - - - - - |
0.010 0.190 0.022 0.044 0.050 0.074 |
1.00 16.20 2.00 3.20 3.60 5.20 |
Trend test p<0.001 |
|||||||||||
12 |
Solvent Mitomycin C Test material
|
- 0.15 5.13 7.70 15.40 38.50 |
7 65 7 11 14 10 |
7 40 8 9 13 14 |
- 55 10 14 15 19 |
- 8 - - 3 3 |
- 22 - 4 6 6 |
- 22 - - 2 2 |
- - - - 2 4 |
0.014 0.294 0.036 0.054 0.078 0.088 |
1.40 21.40 2.40 3.20 5.60 6.40 |
Trend test p<0.001 |
|||||||||||
24 |
Solvent Mitomycin C Test material
|
- 0.15 5.13 7.70 15.40 38.50 |
7 49 4 5 13 7 |
10 22 5 8 9 9 |
- 28 5 9 11 13 |
- 2 - - 2 1 |
- 19 2 3 3 2 |
- 11 - - 2 3 |
- - - - 3 3 |
0.020 0.064 0.024 0.040 0.054 0.056 |
1.80 14.60 1.80 3.00 4.40 5.00 |
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 |
0.87 8.74 0.24 |
6.62 36.41** |
Chromosome Aberrations per Cell Duration Concentration Error |
2 4 8 |
0.0004 0.0017 0.00005 |
8.00* 34.00* |
*Significant at 5 % level
**Significant at 1 % level
Historical Control Data (Transgenic Rodent Gene Mutation Assay (Muta™Mouse:lacZ Assay))
Negative Control
Organ |
n |
Mutant Frequency [x 10^-6] |
Acceptable Range (Mean ± 3 x S.D.) |
||
Mean ± SD |
Min – Max |
Lower |
Upper |
||
Liver |
126 |
50.1 ± 15.7 |
19.0 - 95.0 |
3.0 |
97.2 |
Glandular stomach |
84 |
46.2 ± 11.5 |
24.0 – 84.7 |
11.7 |
80.7 |
Positive Control
Organ |
n |
Mutant Frequency [x 10^-6] |
|
Mean ± SD |
Min – Max |
||
Liver |
104 |
141.8 ± 38.4 |
84.8 - 285.8 |
Glandular stomach |
59 |
474.0 ± 123.9 |
182.4 - 785.3 |
Negative control: Including water for injection, 0.5 % methylcellulose, corn oil,etc.
Positive control: N-ethyl-N-nitrosourea (ENU, 100 mg/kg)
The above historical control data consists of those pooled from October 26, 2011 to October 13, 2017.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro gene mutation in bacteria (Ames)
An OECD 471 study was performed to evaluate the potential of the test material to induce reverse gene mutations in Salmonella typhimurium. The test material was tested with and without a metabolic activation system (S9 mix, prepared from a liver post mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254).
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test material (three plates/dose-level) ranging from 312.5 to 5000 µg/plate. After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored. Solvent control (DMSO) and positive controls were used.
The numbers of revertants for the vehicle and positive controls were within the ranges specified in the acceptance criteria. The study was therefore considered valid.
A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 312.5 µg/plate. No noteworthy toxicity was induced in any of the five tester strains.
The test item did not induce any noteworthy increase in the number of revertants which could be considered as relevant, either with or without S9 mix, in any of the five tester strains.
Under these experimental conditions, the test material did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
In vitro cytogenicity in mammalian cells
The potential of the test material to induce chromosomal aberrations was investigated in vitro in accordance with the standardised guidelines OECD 473 and EU Method B.10.
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. The dose levels used in all the experiments were 0, 5, 10, 20, 40, 80 and 160 µg/mL.
Four treatment conditions were used for the study:
In Experiment 1, cells were exposed for 4 hours in the presence of an induced rat liver homogenate metabolising system (S9 at a 2 % final concentration) with cell harvest after a 20 hour expression period and a 4 hour exposure in the absence of metabolic activation with a 20 hour expression period.
In Experiment 2, the 4 hours exposure period 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.
All vehicle 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 that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the exposure groups dosed in the presence or absence of S9, which included at least one precipitating dose level.
Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.
In vitro gene mutation in mammalian cells
The genotoxic potential of the test material was assessed in an in vitro gene mutation assay with mammalian cells. The study was performed under GLP conditions and in line with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870. 5300. Chinese hamster Ovary (CHO-K1) cells were exposed to the test material in two separate experiments. Both experiments were conducted with a 4 hour exposure period and a 7 day expression period, with and without metabolic activation. In experiment 1, metabolic activation was provided by 2% S9 mix, whereas 1% was used in experiment 2. Cultures were exposed to the test material at concentrations up to 5000 µg/mL in experiment 1 ± S9 mix as well as in experiment 2 with S9, and up to 3750 µg/mL in experiment 2 without S9 mix.
Increases in mutant frequency of greater than 20 x 10-6were seen in the 4-hour exposure group in the absence of S9 in Experiment 1 and Experiment 2. The test material did not induce any significant or dose-related increases in mutant frequency per survivor in the presence of metabolic activation (2 % S9) in Experiment 1 but did show a clear dose related response in Experiment 2 where a reduced S9 concentration (1%) was used.
Significant cyctotoxic effects were observed at concentrations ≥ 1250 µg/mL in the absence of metabolic activation, producing reductions in cloning efficiency between 21 and 89%.
Therefore under the conditions of the test, the test material was considered to be mutagenic to CHO cells at the HPRT locus in the presence and absence of metabolic activation.
In a further, supporting study, the genotoxic potential of a reaction mass of neodymium carbonate and praseodymium carbonate was assessed in an in vitro gene mutation assay with mammalian cells. The study was performed under GLP conditions and in line with the standardised guidelines OECD 476 and EU Method B.17. However, since the study was conducted with a reaction mass, containing the metal carbonate, rather than with the substance itself, it has been assigned a reliability score of 2 and used as supporting information.
During the study, Chinese hamster Ovary (CHO-K1) cells were exposed to the test material in two separate assays. Treatments were carried out for 5 hours with and without metabolic activation (± rat liver metabolising system, i.e. S9-mix) and for 24 hours without metabolic activation (-S9-mix). Due to the chemical characteristics of the test material, 1% (w/v) methyl cellulose solution was used as the vehicle of the test material in this study. Treatment concentrations for the mutation assay were selected for the main tests based on the results of a preliminary toxicity test.
The study was considered to be valid and to reflect the real potential of the test material to cause mutations in the cultured mammalian cells used in this study. Treatment with the test material did not result in a statistically or biologically significant, repeatable, dose-dependent increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9) in this study.
Therefore, under the conditions of the study, the test material was concluded not to be mutagenic either in the presence or absence of metabolic activation.
In vivo transgenic rodent mutation assay
A gene mutation assay with transgenic male mice (Muta™ Mouse) was conducted to assess the potential of the test material to induce gene mutations (reporter gene: lacZ) in the liver and glandular stomach in accordance with the standardised guideline OECD 488 under GLP conditions.
In a dose-finding study CD2F1/Slc mice were treated for 14 days with 100, 300 and 1 000 mg/kg/day by oral gavage. No deaths or clinical signs of toxicity were observed in any of the dosed groups. Therefore, a limit dosage of 1 000 mg/kg was selected as the high dose and 500 and 250 mg/kg/day were selected as lower doses for the gene mutation assay with Muta™ Mouse.
The test material was administered to male transgenic mice (CD2-LacZ80/HazfBR (Muta™ Mouse)) orally for 28 consecutive days by gavage. In the main study, the liver and glandular stomach were removed after 3 days of manifestation period (Day 29 to 31), and the mutant frequencies were determined. As a result, there were no significant differences in the mutant frequencies in the liver and glandular stomach in any of the groups treated with the test material as compared to the negative control group. The mutant frequencies in the liver and glandular stomach in the positive control group treated with 2 doses of N-ethyl-N-nitrosourea (ENU, dosage level of 100 mg/kg, i.p.) at 24h intervals, were increased. These increases were statistically significant compared with the negative control group. No clinical signs, no significant decrease of body weights and no test material-related reduction of food consumption were observed in any groups. There were no test material-related changes noted at necropsy.
In the germ cell study, the testes and cauda epididymis were removed after 49 days of manifestation period (Day 29 to 77). Since no positive response was observed in somatic cells, the mutant frequencies in the germ cells were not evaluated.
It was, therefore, concluded that the test material did not induce gene mutation in the liver or glandular stomach of transgenic mice under the conditions in this study.
In vivo mammalian bone marrow chromosome aberration test (DISREGARDED STUDY)
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.
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. Further investigation into the presence of the substance in the plasma is ongoing and the results, once available, will be reported in an update to the registration dossier.
Summary
Based on the results of the available studies, the genetic toxicity profile of the test substance contains some ambiguities. Negative results were obtained in two of the key studies; the Ames test and the in vitro chromosome aberration study in human lymphocytes. Similarly a negative result was seen in a CHO HPRT study in a similar substance. A positive result was, however, obtained in the in vitro gene mutation in mammalian cells study on the registered substance itself in both the absence and presence of metabolic activation. In view of this, an in vivo transgenic rodent study was conducted which produced negative results.
The weight of evidence indicates that the substance should not be classified with regards to its mutagenic potential.
Justification for selection of genetic toxicity endpoint
Four studies (in vitro gene mutation in bacteria (Ames) (CIT 2007), in vitro cytogenicity in mammalian cells (Harlan 2013, Chrom Ab), in vitro gene mutation in mammalian cells (Harlan 2013 CHO HPRT) and the in vivo transgenic rodent mutation assay (Kasamoto 2018) have been selected as key to address the genetic toxicity endpoint. All are well reported guideline studies, conducted to GLP standard and assigned reliability scores of 1 in accordance with Klimisch (1997). In combination they are considered to give a broad and comprehensive overview of the genotoxic potential of the substance.
Justification for classification or non-classification
Typically, classification of substances as a mutagen is based on positive results in in vivo mutagenicity studies. Whilst in some cases positive results in in vitro studies can be considered sufficient to warrant classification, they typically serve to highlight any potential need to conduct in vivo testing. In the studies performed on the substance a mixture of positive and negative results were produced, however the weight of evidence from both the in vitro testing and in particular thein vivo study in transgenic rodents is such that an overall negative result can be concluded.
As such in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is considered not to require classification with respect to genetic toxicity.
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